LIST OF ABBREVATIONS ATP: Adenosine triphosphate AT/ET: Angiotensin

Keywords: Antidepressants Boc, Di-tert-Butyl carbamate BuLi, Butyl lithium Cyclic Adenosine monophosphate Cbz, Benzyl carbamate

ACKNOWLEDGMENT

First of all, I would like to thank Professor Yves Troin, the director of my thesis, for accepting me to enroll as a PhD student to work under his direction. He was encouraging and was always ready to help when I faced some problems in chemistry. He provided me enough freedom in exploring the areas of my research. He encouraged me not just to confine in the day-to-day laboratory work but also to be autonomous in other areas of our research.

I would like to thank Doctor Isabelle Abrunhosa Thomas, without whom I would have had tough time not just in lab but also in day-to-day life. I learnt a lot of chemistry concepts from her during the course of these three years and she was always available for me when I need some advice or help in chemistry. She created an ambient environment for me to work these three years and she had atmost faith on my work. Apart from this, she was kind and took care of me in these three years not just in the lab but also outside the laboratory . I would also like to thank Professor Jean Suffert of Université de Strasbourg, Professor Damien Prim of Université de Versailles and Professor Maria M. M. Santos of Universidade de Lisboa for accepting as referees to judge my thesis. I would like to extend my thanks to Pr. Santos for accepting to be part of my jury even though she has to travel a long distance.

I would like to thank Professor Gérald Guillaumet of Université d'Orléans, Professor Pascale Moreau of Université Blaise Pascal, Professor Denis Ardid of Université d'Auvergne for accepting to be part of the jury for my thesis.

I would like to extend my thanks to Professor Denis Ardid, one of the principle investigator and collaborator of our project who has encouraged us to conduct the pharmacological activity of our molecules and helped in lot of ways to enhance this project according to the clinical setting. I would like to thank Miss. Julie Barbier, Laboratory Technician at Université d'Auvergne for accompanying me during the biological evaluation part of our project and the rapport & humour we shared at times. I would like to thank Professor Sylvie Ducki and Doctor Kasi Sankar who i consider as mentors for my scientific career. They encouraged me to pursue research as my career and whatever the aptitude i have developed towards science, is because of them. I would like to extend my thanks to the present and ex-members of CESMA, Dr. Khalil Bennis, Dr. Isabelle Ripoche, Dr. Pierre Chalard, Dr. Jean-Philippe Roblin and students like Ostache Cosmin, Marie Harel, Li Jie, Dr. Oana-Patriciu, Dr. Tomas Carillo Marquez, Romain Jouve, Dr. Naoual Bouzidi, Dr. Wahid-Bux Jatoi, Quentin Lenin, Ombeline Danton, Camille Monnier etc who all helped me in the course of these three years.

I would like to specially thank Doctor Delphine Vivier and Gary Vallon with whom i shared most of the time during my stay in CESMA. Since my arrival, Delphine and Gary helped me in uncountable number of aspects and never said no when i needed them.

I also would like to thank Safia Laid, Gaelle Framery and other members of ENSCCF for their help during these three years.

I would like to thank my parents K. L. Hemachandra and K. H. Indira without whom i would have never been in the stage where i am now. They supported and helped in all stages of my life.

LIST OF TABLES

I. PAIN

Pain, in broad-spectrum is not classified as a disease but a symptom associated with a disease or a disorder. According to International Association for Study of Pain (IASP), pain can be defined as "An unpleasant sensory and emotional experience associated with actual and potential tissue damage, or described in terms of such damage, or both [1]."

Even though pain is not the main reason for mortality, in most of the cases it is undeniably associated with a form of disease or a disorder (Cancer, HIV, Ischemic heart disease etc.) responsible for the major causes of death in the world [2]. Every year, around 7.2 million people suffering from pain originated due to HIV or cancer are prone to death worldwide (Figure .1). In 2012, not less than 2.4 million deaths are due to untreated pain [3]. And so, treating pain might not directly cure a disease but will surely diminish the effect of the associated disease or disorder and thus results in relatively better quality of life for the patient.

Some other forms of pain (migraine, Irritable Bowel Syndrome, rheumatoid arthritis) won't result in the death of a patient instead will decrease the quality of life [4]. Especially chronic pain, which is majorly responsible for many of such cases and treating it might be cumbersome as some patients as well as physicians can't different between diverse forms of pain and treat one instead of the other with an improper adjuvants and dosage regimen [5].

Thus, a proper understanding of different forms of pain is important which will help for better treatment of a disease or a disorder.

A. Classification:

Classifying pain into different forms is complicated as there are different rationales for sorting them. They are classified according to their time period (Figure .2), frequency, location, intensity and cause of pain attack [6,7]. 1.1. Acute pain: Pain which lasts for a period of 3-6 months duration is called as acute pain [9]. Acute pain is physiological pain and serves as protective function. Alongside this, acute pain has clearer and visible association with injury or disease. For example, postsurgical pain is a form of acute pain and the reason behind it is clear i.e. because of surgical wound. Nociceptive pain can be a better word and/synonymic for acute pain as nociceptive pain is caused by noxious stimulus due to thermal (Heat or cold), mechanical (cuts, crushing etc.) and chemical (chilli powder on cuts or eyes etc.) in whose case, the reason for pain is clear and expected to fade with the time period as the noxious stimuli diminishes.

Henceforth, it serves as a protective function by alarming the brain that something deleterious process is happening to the body and withdrawal is obligatory to protect the body from further damage [10].

Not just treating the cause of pain and/ injury, it is also equally important to treat the pain itself. By doing this, we can lessen the effect of disease or injury and also not to transform acute pain to chronic [11]. Depending on the intensity of pain, medication used for acute pain includes Acetaminophen, Codeine, Ibuprofen, Hydromorphone etc.

(Figure .3). 1.2 Chronic pain: It can be designated as more 'complicated' form of pain and lasts for a period of more than 6 months [10]. Even though it is also associated with some injury like acute pain, with the time period, the reason becomes less clear and persists for longer time than the injury. Along with the common NSAIDS used against pain, chronic pain treatment includes antidepressants, anticonvulsants, corticosteroids etc. (Figure 4). Chronic pain can be further classified based on the cause and/ origin of pain [12] i. Nociceptive pain: It is caused by the noxious stimuli as a result of activation of nociceptive receptors called as nociceptors. Based on its profundity, it is sorted out as a) Superficial pain is a response due to the activation of nociceptors distributed in skin or superficial tissues as in the case of first degree burns and minor cuts and bruises.

b) Deep pain occurs in tissues deep inside and is further classified into 1) Deep somatic pain occurs in the tendons, ligaments, bones, blood vessels etc. and is fading, vaguely localized pain as in the case of pain during broken bones, sprains.

2) Deep visceral pain occurs in the visceral organs and is generally localized but hard to locate as 'referred pain (pain response in region other than injured location)' might also be present in other visceral space (apart from the actually injured organ) due to the inflammatory response by the damaged tissue as in the case of Irritable Bowel Syndrome.

ii. Neuropathic pain: It is caused by the damage (due to lesions, injury etc.) of the nervous system, to be exact, somatosensory nervous system. It is co-associated with allodynia (pain from non-painful stimulus) or dysesthesia (abnormal sensations) and occurs as episodic or continuous.

a) Central neuropathic pain is found in multiple sclerosis and spinal cord injuries.

Amongst a cluster of theories resulting in central pain, central sensitization is more acceptable one. The dorsal horn neurons which forms the spinothalamic tract contributes the major ascending nociceptive pathway (Figure 5). Due to the ongoing spontaneous peripheral rise in activity, spinothalamic tract also develops increased background activity and responses to afferent impulses including normal touch stimuli. All these cascade reactions synergise to form a state called central sensitization.

b) Peripheral neuropathic pain is due to a lesion in the nerve ending which modulates the nerve activity at the injured space. These modulations include, abnormal excitability, increased sensitivity to physical, chemical stimuli. These cascades contribute to form the state of peripheral sensitization.

Along with the classification of pain using time frame as the rationale, there are other basis which includes frequency, location, cause of pain etc. But all give the same explanations which have been covered in the above classification.

Intensity is another classification criteria set by some clinicians and it shows some interesting facts as below [13].

2. Intensity: Based on intensity, pain is rated on a scale of 1 to 10, 1 being normal and 10 being in severe pain (Figure .6).

Figure 6.showing pain scaling and experience of a patient

There is ambiguity in this classification as most of the patients over time gets adapted to the pain experiences and cannot express the same pain anguish which they showed in the beginning. It lacks the consistency as the rating changes from one individual to the other. This kind of classification is least desirable by most of the scientific community but preferred by physicians in some countries.

Moving further, whatever the pain is, there is a means of transportation of signalling from the place of injury to the brain and vice versa for the modulation. Thus, a look into the pain origin, transformation and modulation will further help us to understand better physiology of pain and ultimately for better designing of drugs for pain.

II. PAIN ORIGIN, TRANSMISSION AND MODULATION

Pain is a role of the nervous system in alarming the body and cautioning of potential or actual injury. And hence, this complex pain signalling system has many elements involved and nociceptors are considered to be one of the important elements of all in pain pathway.

Before discussing about the nociceptors, a brief preface about the nervous system and their characteristics helps for a better understanding of the pain signalling pathways.

A. Nervous system

The nervous system can be divided into two segments i.e. central and peripheral nervous system. Central nervous system (CNS) consists of brain and spinal cord and this is the region of the nervous system where the processing of received information and subsequent influence on the resulting activity takes place. On the other hand, peripheral nervous system (PNS) consists of spinal and cranial nerves and acts as the bridge between CNS and rest of the body (Figure 7) [14]. These signalling pathways are mainly attributed to three kinds of neurons [15];

 Afferent neuron sends the signal from the place of stimuli to CNS. These neurons are distributed in PNS and consist of long axons and sensory receptor (for example, nociceptors) on one of their ends.

 Efferent neuron takes back the signal from CNS to effector organs. In PNS, it has long axon.

 Interneurons are confined completely to CNS and acts as a link between afferent and efferent neurons.

The sensory afferent nerve fibre helps in reception of various stimuli such as touch, pain, pressure, temperature changes etc. These stimuli conduction start from the receptors of the sensory afferent neurons and ends in the effector organ (Figure 8). This conduction of stimulus results in transmitting the concerned signal to the CNS for processing and for a subsequently resulted action which again passes from CNS to PNS and finally to motor efferent division through which the signal passes to the effector organ such as muscles/glands [16].

Thus, the review of nervous system as above can be useful in further study aspects involved in pain signalling pathways.

B. Pain pathway

Pain pathway and processing occurs in 4 different steps as follows;

1. Transduction

Transmission

Perception

Modulation

Each step has its own importance and variety of drugs acts on different stages of this pain signalling pathway. These stages can be summarized as follows;

Transduction:

Transduction can be defined in general as a process of conversion of one form of energy into another form by a transducer (for example, conversion of electrical energy into light energy by an electric bulb). In case of pain, the noxious stimuli like physical and chemical are converted into a pain signal by a transducer called as nociceptor. Nociceptors are distributed all along the body and exposed to different noxious stimuli.

Nociceptors: They are sensory receptors which have an ability to identify noxious stimuli and convert the stimuli into pain signal (to be exact, electrical signal) [16]. The nociceptors are formed by free nerve endings of A-δ and C fibres of the nervous system. They can be activated by thermal, mechanical and chemical stimuli [17]. Even some inflammatory mediators like bradykinins, prostaglandins, cytokines, Calcitonin Gene Related Peptide, Serotonin etc. can activate nociceptors. These mediators are also responsible for primary sensitization, where the activation threshold of the nociceptors becomes less.

Nociceptors are distributed in  Somatic structures like muscles, skin, bones, joints, connective tissue etc.

 Visceral structures like liver, intestine etc.

In conjunction with A-δ and C fibres, other primary afferent fibres like A-β exists but they carry non-noxious stimuli. Along with the formation of nociceptors, there are certain characteristics of them which make these fibres active in specific cases (

Receptor activation thresholds

Low High and low High

Sensation on stimulation

Light touch, nonnoxious Rapid, sharp, localized pain Slow, diffuse, dull pain Table 1 .Showing comparison of different primary afferent nerve fibres

A-δ and C fibres contribute majorly for carrying the pain nociception and they vary from each other in case of many aspects. C fibres conduct the pain signal slower than any A-δ fibre and this can be the reason when someone is injures, at first stance they feel a sharp and localized pain (due to A-δ fibre) and when the time passes, the pain becomes more dull and diffused (due to C fibres) [18]. But C fibres contribute 70% of nociceptors and hence it is the major fibre making up the volume for nociception.

Transmission

The transduced signal from previous step reaches the next step i.e. transmission.

Transmission is the process by which impulses are sent to the dorsal horn of the spinal cord and then along the sensory tracts to the brain (Figure 9) [19]. Transmission of the impulse to the CNS is also done by two major primary afferent neurons i.e. A-δ fibres and C fibres through three different types of transmission and the transmitted nerve impulse also called as 'action potential' is resulted from the activation of specific ionic channels like sodium channel.

Transmission occurs in three different stages as follows [20];

Peripheral transmission:

The noxious stimuli travel alongside within peripheral nerve fibres and terminate at dorsal horn of the spinal cord.

Synaptic transmission:

The signal reaching dorsal horn through peripheral nerve fibres synapse with the second order neurons.

Central transmission:

The signal after synaptic transmission further conducted through neurons which bridges spinal cord and rise to the thalamus and in turn branches to the brainstem.

Finally, the transmitted nociceptive signal reaches different areas of brain. This terminates the transmission step and advances to the next step i.e. perception.

Perception

The noxious stimuli which reach the brain is processed and in turn recognized as a pain. The precise region of brain involved in perception is unclear but there are certain areas of brain which can be labelled as prime regions where defining of pain process takes place [21]. In Figure 10, the involvement of numerous regions of brain in thermal pain clearly states a complex pain perception processes taking place in brain. The somatosensory cortex (both primary and secondary) is important for the localization and identification of form of pain as well as degree or intensity of it. And limbic system involves in emotional response for a pain signal. Other regions include insula, frontal lobes etc.

After the processing of pain signal, the next step involves the reaction of brain to the pain stimulus received in the form of modulation.

Modulation

The perceived noxious stimulus by brain is processed and an inhibitory pathway starts resulting in inhibition of nociceptive signal. During the modulation phase, the descending neurons from the brain release certain substances that inhibit the transmission of noxious stimuli [22]. There are many theories involved in pain modulation and among those, more accepted theories are as follows; descending modulation [24]. The above named regions have high concentrations of opioid receptors and endogenous opioids answering to the question, why opioids are potent pain killers. The pathway reaches dorsal horn of spinal cord and is monoaminergic, which uses serotonin and norepinephrine as prime neurotransmitters. Even though believed that the mechanism involves activation of descending pathway and release of opioids endogenously results in inhibition, the clear and complex mechanism is not well understood till date.

As we saw that the pain pathway is complex and interconnected, there are certain analgesics which act at different levels of this signalling pathway to inhibit the nociception.

Figure 12.Drugs acting at different levels as analgesics

Hence the drugs used against pain in day-to-day life can be summarized as above (Figure 12). Point to be noted, not just analgesics but other categories of drugs are also can be used to suppress the nociceptive stimuli. In the future chapters, some of the antidepressants acting as pain can be discussed in brief to validate this rationale.

A brief overview of pain origin and signalling along with drugs acting at different levels paves a path for the next important and hot topic, the pain and its sufferers worldwide.

C. Global data on pain

Pain is one of the most prevalent symptoms associated with many disease/disorders.

Among pain, acute pain does have sufficient medication as well as the analgesics currently available are efficacious and potent. On the contrary, the chronic pain and its current medication is one amongst most other disorders lacking potent treatments. As we know that the chronic patient has deprived quality of life, the global data poses some interesting facts (Figure 13). The data retrieved from Global Burden Data (GBD) [25] clearly depicts that chronic pain conditions like low back pain ranks 1 in most of the patients. It averages about Major depressive disorder (ranks 2, mean-63,239,300) is a condition which coexists with most of the chronic pain condition but even though vice versa is not factual.

Having a closer look on the deaths global data (Figure 14), shows the results as follows.

Figure 14.screenshot showing global ranking with respect to deaths

The ischemic heart disease caused by visceral inflammation as one of the reasons ranks 1 st with a mean of about 7,029,270 (6,577,160 to 7,431,130).

'Non-infective inflammatory bowel syndrome' is a chronic visceral inflammatory condition and a major group of diseases accounting for deaths and Disability-adjusted life years (DALYs) in recent years (Figure 15). This group of diseases includes Crohn's disease, Ulcerative colitis etc. And hence, the major problem when it comes to chronic pain includes visceral pain and migraine which turns our spotlight on to it.

III. MIGRAINE & VISCERAL INFLAMMATORY PAIN

A. Migraine

Migraine is a complex vasodilatative and incapacitating disorder occurring in almost 15% of the adults throughout the world among whom the female population are affected majorly. It is a periodic affliction characterised by one-sided, pulsating headache associated with photo & phonophobias [26]. Typical migraine attack ranges from 1.5 times a month and reaches its toll around an age of 40 years during patient's lifetime [27].

Key facts & figures

 ≈15% of world population suffers from migraine among which 70% are women.

 Migraine is a syndrome with a group of symptoms among which, all of them or some of them are shown during one migraine attack. A study among 4000 migraine patients have shown symptoms as follows [28];

Symptom Percentage

Pulsating ache 85

Photophobia 80

Phonophobia 76

Gastric related disturbances 73

One-sided pain 59

Visual disturbances 44

Aura 36  50% of the migraine in patients remains un-investigated and not properly treated of which half of the subjects remains unconsulted with the doctor [29].

 Migraine is ranked amongst top 20 disabilities throughout the world [30].

 In United Kingdom, 0.19 million migraine flare-ups each day [31].

 Depression is 3 times more often in migraine patients compared to the healthy volunteers.

 Most notably, migraine research is the least funded neurobiological disorder when equated with its financial impact on public [32].

Pathophysiology of migraine

The initiation of migraine even after years of research is still unclear. But many hypothetical theories are proposed by different scientists making it a difficult target to work on. One of the main theories which have an adequate theoretical basis is Cortical Spreading Depression (CSD) [33]. CSD onsets as a wave of electrical signal in cerebral cortex. The key event in unveiling and dissemination of CSD can be related to drastic decrease of neuronal membrane resistance in association with a series of events like extracellular increase of neurotransmitters & potassium (K + ) ions and intracellular decrease of sodium (Na + ) and calcium (Ca ++ ) ions [34]. The modulation of CSD threshold is influenced by several factors like environment, genetic makeup, age, hormones etc. However the recent preclinical & clinical investigations showed a clear coherence of migraine with Calcitonin Gene-Related Peptide (CGRP), a potent vasodilator. Hence most research groups working on migraine shifted their focus onto CGRP in the recent years.

Treatment of migraine

Drugs used for migraine therapy are majorly triptan class even though CGRP receptor antagonists are potent. This accounts for no CGRP blockers in market till date but still in the drug developmental stages. Apart from triptans, other NSAIDS such as acetaminophen, ibuprofen, and aspirin are used for mild migraines. Some more drugs such as opioids, antidepressants, corticosteroids, ergotamines proved to be effective but their usage is limited due to the side-effects shown by them. And hence, the drugs which are both effective and

showing least side effects are as follows;

5-HT 1B/1D receptor antagonists for migraine therapy; the triptans

The triptans class of the drugs are the efficacious, potent and mostly used drugs for migraine therapy even at present [35]. The name 'Triptans' indicates its core chemical moiety i.e. tryptamine. All the class of triptans have 5-Hydroxy tryptamine as their core moiety.

Some triptans are as follows (table 3);

Name

i. Complications of triptans

Alongside its efficacy, triptans have side effects like myocardial infarction, hypertension, tachycardia, angina pectoris, renal & hepatic failures, serious allergic reactions, dry mouth, gastro & heart related problems etc [36]. Increased doses of sumatriptan results in sulfhaemoglobinemia, a reversible state where blood turns into greenish-black due to the coordination of sulphur onto the haemoglobin moiety [37]. A life menacing medical condition called 'serotonin syndrome' is resulted due to drug-drug interaction of triptans with either mono amino oxidase class of anti-depressant drugs or serotonergic drugs [38].

Calcitonin Gene Related Peptide

Calcitonin Gene-Related peptide, a 37 amino acid neuropeptide which is involved in several physiological processes came into the lime-light after extensive studies on migraine has shown some interesting facts [39]. Among the migraine patients, the brain plasma showed abnormal increase in CGRP concentration during the attack which infers the involvement of CGRP in migraine [40]. This observation has shifted whole migraine research onto CGRP and its receptor antagonists.

There has been some extensive studies on the distribution of CGRP receptors in recent times stating that they are widely distributed in central and peripheral nervous system [41]. In central nervous system (CNS), it acts as a neurotransmitter. In peripheral nervous system (PNS) its action has been exploited for its importance in the dilation of smooth muscles which results not only in migraine but also in some neglected visceral disorders like Irritable Bowel Syndrome (IBS) and Crohn's disease (Figure 16). Ca ++ (Figure 17). Thus these alterations results in hyperpolarisation or vasodilatation [43,44].

In case of migraine, due to the vasodilatation or distension of the cranial blood vessels and resistance & limitation from the skull bone results in the pulsating and severe pain in head. In order to counteract this vasodilatation, the binding site of natural ligand is masked using antagonists [45] and the cascade of reactions can be turned off resulting in no vasodilatation step.

ii. Discovery of peptide antagonists for CGRP receptor

CGRP receptor was first antagonised by CGRP 8-37 , a peptide chain of 30 amino acid which was discovered from the intertwining of the natural ligand i.e. CGRP [46] (Figure 18). Further research at Boehringer Ingelheim has developed some small peptides derived from the C terminus of the natural ligand [47]. Later on by High Throughput Screening, a (R) Tyr (S) Lys peptide was identified as a lead. A series of modulations like rigidification of C & N terminus, bio-isosteric replacement keeping the peptide pharmacophore untouched resulted in the first potent CGRP receptor antagonist called as BIBN-4096 (Olcegepant® ® ) (Figure 19). Olcegepant® discriminates the rodent and primate CGRP receptors [47]. It has more affinity towards primate receptor with K i = 0.014 nM compared with that of rodent receptor showing K i = 3.4 nM. This is due to the replacement of Tryptophan with Lysine at 74 th position of the receptor in rodent species implying the importance of tryptophan interaction to have potency for small molecules. Even though BIBN4096 is potent and selective antagonist, the peptidic nature of the molecule has limited its usage due to poor lipophilicity.

iii. Non peptide CGRP antagonists

MK-0974 called as Telcagepant® ® (Figure 20) is orally active, potent antagonist developed by Merck & Co. Being a non-peptide, it is more lipophilic with equal potency as BIBN4096 hence advantageous over latter [48]. Like BIBN4096, it is 1500 folds more selective towards primate CGRP receptor. MK-0974 was terminated from its phase III clinical trials as there were cases showing increased serum transaminases, which inclines to hepatotoxicity [49]. BMS-927711 by Bristol Myers Squibb is another orally active, potent and selective with K i = 0.027 nM has recently finished 2 nd of the 3 tests for the approval into the market [50]. Adding to this list, some more non peptide antagonists are currently in their preclinical evaluation [51].

With the inspiration from development of potent CGRP antagonist in the form of Telcagepant®, Merck continued their research on this receptor and fabricated MK-3207. showing an efficacy better than many other triptans as well as CGRP antagonist counterparts for a dose of 200 mg. Also to consider, the clinical studies have been conducted on a small sample size of the dose group and hence gave a significant overlap between confidence intervals between each dose. And so, a possible conclusion on its efficacy can't be drawn as of now and has to be validated further.

The other candidate from Boehringer Ingelheim, BI-44370 was also evaluated clinically [51]. $825 million from labrys biologics [52]. Zecuity ® by NuPathe Inc, an advanced transdermal patch loaded with sumatriptan delivers a dose of 6.5mg over a period of four hours and demonstrated to be effective for migraine. Allergan's Botox ® usage is continued as a preventive treatment for chronic migraine. Another monoclonal antibody LY2951742

developed by Arteaus biotech is also used as a preventive medication. It has recently finished the phase II trials sponsored by Eli lilly and showed significant efficacy and tolerability [53].

ALD403 by Alder biopharmaceuticals finished phase II clinical trials and showed radical decrease of Migraine headache days by 75% [54].

Along with medication for treating migraine (chronic), there are certain class of drugs which now-a-days are prescribed as preventive [51]. But they are not as potent as above classes.

 Cardiovascular drugs like beta blockers (Propranolol, Metaprolo, Timolol) and calcium channel blockers (Verapamil) which are used for high blood pressure are prescribed as preventive medication for migraine.

 Tricyclic antidepressants like Amitriptyline, Venlafaxine.

 Antiepileptics like Valproate, Topiramate.

 OnabotulinumtoxinA i.e. botox also proved to be an efficient preventive medication.

Thus, migraine medication which currently used has more side-effects relative to their beneficial & desired therapeutic effect. Thus validating a new target for treating migraine seems to be inevitable.

After having a gaze at migraine, let us have a brief outlook about visceral pain which is also our target to act on.

B. Visceral pain

Visceral pain is a form of pain in viscera i.e. internal hollow organs and marks a prime grave problem when it comes to viscera [55]. It is one of the chronic pain conditions and is caused mainly due to the activation of nociceptors of abdominal, pelvic or thoracic viscera [68]. Point to be remembered that unlike in other cases of pain perception by nociceptors, the visceral pain nociceptor activation is mainly caused by stretching, inflammation, ischemic conditions etc. and relatively insensible to cuts and burns [56].

Neuropathic and other chronic pains might or might not happen during lifetime of an individual but visceral pain has become inevitable now-a-days like abdominal pain due to IBS, angina pectoris during ischemia etc.

Causes of visceral pain

 Nociception caused by direct injury of visceral organs as in the case of ischemia.

 Inflammation, either acute or chronic dilation of viscera as in IBS, Ulcerative colitis.

Crucial observation is not all the internal organs are prone to visceral pain. Solid organs like liver, kidneys are not prone to this form of pain [57]. Considering an example as in the case of lesions in liver/pancreas, they are not transmitted as pain signal to the CNS.

Instead they are discovered when there is other abnormal condition of liver as in like jaundice (which is painless) to identify a lesion or carcinomas of pancreas.

Clinical manifestation of visceral pain

Identifying a visceral pain is most difficult out of all in clinical setting. At the same time, it can't be ignored as they are sometimes associated with prime life threatening disease like ischemia.

Classifying a visceral pain is also cumbersome as explained earlier due to their vague symptoms and complex nature. But in a whole, they are classified as below (Figure 23); Even though referred pain is not a direct manifestation of visceral pain, it indirectly shows up the condition of the viscera [58].

True visceral pain

Poorly localized, diffused randomly and unclearly defined form of pain [59]. It typical place of exhibition includes anterior abdominal region, below the sternum and around the diaphragm and is manifested as heavy and compressed. It co-exhibited with severe perfusion, gastro-intestinal and psychological disturbances. E.g. Myocardial infarction.

Visceral hyperalgesia

It is also poorly localized and slow. Hyperalgesia is a condition in which the sensitivity towards the noxious stimuli increases [60]. Visceral hyperalgesia shows similar increased sensitivity to pain subsequent to an inflammation and with/without injury internally. E.g. IBS, chronic pelvic pain, fibromyalgia etc.

Viscera-viscero hyperalgesia

It occurs due to the sensory interactions between two visceral organs which shares same portion of afferent pathway [61]. As in the case of coronary heart disease and gallbladder calculosis, the patient experiences synergistically double the pain with these two conditions existing together because of same afferent pathways.

Referred pain without hyperalgesia

Referred pain can be defined as the condition in which the symptoms of pain occur in completely different place than that of the affected visceral organ. E.g. Pain during a heart attack. In the above said case, the referred pain occurs in the upper part of left arm and hand.

This happens due to the misinterpretation in processing of signals by the brain due to similar sensory signalling pathways.

Referred pain with hyperalgesia

A referred pain associated with hyperalgeisa occurs in superficial muscles. It is also termed as viscera-somatic hyperalgesia. This form of pain occurs due to central sensitization posed due to viscera-convergent neurons.

Thus, all forms of visceral pains are unclear, poorly localized, not easily detectable and complicated. All these reasons make visceral pain one of the gravest problems to treat.

Pathophysiology of visceral pain

Visceral pain in general is transmitted in two different mechanisms. They are classified according to their sensitization properties. Sensitization is a non-associative study process in which a repeated administration of stimuli results in increase of the shown response. This sensitization can be central or peripheral depending on which, visceral pain is classified as follows

Central sensitization

Central sensitization phenomenon happens due to the increased excitability of neurons in CNS which results in abnormal firing of responses even though the signal received is normal. This abnormal excitability is attributed to the nociceptors which alter the strength of connection of synapses between nociceptors and the neurons of spinal cord. Even though we feel that pain originates from periphery, it actually arises due to abnormality of processing in CNS. The tactile allodynia due to light touch happens due to central sensitization. For example, in migraine, slight touch of hair feels so painful so as the case after various surgeries.

Peripheral sensitization

It is a result of decreased threshold and henceforth increased responsiveness of the peripheral ends of the nociceptors [60]. For example, as in the case of sun burns, the feeling of heat increases even the burnt area is exposed to normal water. This sensitization is due to the action of released chemical messengers around the affected area. These messengers increase the transmission of noxious stimuli resulting in sensitization.

The release of prostaglandin PGE 2 is due to the activation of neutrophils which further releases the enzyme cyclooxygenase-2 (cox-2). This cox-2 is responsible for production of PGE 2. NSAIDS like aspirin acts by inhibiting cox-2 [62].

Medication for visceral pain

Almost all the drugs used against migraine are prescribed for treating visceral pain.

Most common drugs include NSAIDS, opioids, antidepressants, etc.

Recent studies have shown the distribution of CGRP receptors in visceral organs [63].

Due to its potent vasodilatative action and least side effects, the research groups are hitting on finding a target for this receptor.

IV. RATIONALE FOR THE PROJECT

The main rationale for the project can be attributed in simple words as acting at two different levels for an effective treatment of chronic pain. We nominated these two effects to be concurrent, synergistic and more practical to achieve. According to the study conducted by Burgess et al, [64] the recent identification and development of analgesics can be accounted as follows In their study, they stated that the design and synthesis of new analgesic molecules in recent times (blue region of pie chart) accounts for 7% of the drugs which entered the market.

Whereas, the reformulation (crimson red region of pie chart), where the pharmaceutical companies modulates the actual formulation of potent drugs to increase the bioavailability and in turn increase the drug efficacy accounts for 57% of the drugs in current market.

The remaining 37% (green region of pie chart) account for the drugs which are combinations of two varied mechanism of actions. And hence, the pharmaceutical companies play safe by choosing two varied and well know potent and efficacious moieties as leads and develop a new analgesic molecule from them. The pharmaceutical companies and research groups started using this particular rationale due to the reasons like  Due to synergistic activity, this method decreases the usage of dose of two drugs.

 It decrease the side effects posed by two different drugs.

 It decrease the cost of production of two different drugs

As a result, the dual activity drugs identification has been enrolled as an active area and a hot topic in current drug discovery and development of not just analgesics but many other drugs. Hence, a superficial outlook on dual activity drugs development in current market will be helpful for our better understanding.

A. Dual activity

Dual activity molecules tend to a single moiety possessing two pharmacological effects, which can be used to treat same disease or two different diseases [65]. The approach of incorporating two desired pharmacological effects in one chemical structure is not just a fashionable thematic, but also an important pharmaceutical application now-a-days. With this approach, the advantages include the following;

1. Applicability of combinatorial methodology by using already existing and efficacious drugs to profit a variety of new generation dual activity molecules.

2. Easier management of a disease with simplified drug and dose regimen for the patient.

3. Decrease the dose of two different drugs (In case the two desired effects are synergistic).

4. Decrease the side effects of two different drugs.

5.

In industrial point of view, decrease the cost of production of two drugs.

Currently, there are varieties of pharmacologically dual active chemical entities in the pharmaceutical market ranging from analgesics to anti-cancerous agents. A quick overview of those entities and developmental strategies are not just attention-grabbing, but also hints a lead for further research in pharmaceutical research and development. Thus the design of dual cyclooxygenase/5-lipoxygenase inhibitors has been proposed

Drugs for same diseases

and lime lighted in order to bestow a superior anti-inflammatory nature as well as decrease of side effects due to 5-LOX pathway. The antagonists used to design these kind of dual moieties are mostly already existing drugs. Thus, a brief over view about the dual active COX/5-LOX is discussed further.

Modified NSAIDs

Indomethacin is well-versed NSAIDs and is modified to a dual COX/5-LOX inhibitors by converting the carboxylic group to an N-hydroxyurea [70]. Licofelone is therapeutically applied for osteoarthritis. It showed a potent dual activity without showing any side effects [68].

N

Along with anti-inflammatory agents, there are other class of drugs which are used to treat other diseases/disorders as follows.

Dual Angiotensin II and Endothelin receptor antagonists: They act on both Angiotensin II and Endothelin I receptors which are implicated in hypertension [69]. These two endogenous potent vasoconstrictors acts in a positive feedback loop thus potentiating each other's release and action.

The rationale for the design of Dual Angiotensin II and Endothelin receptor antagonists was done keeping in mind the key pharmacophore scaffolds of both classes of drugs. In both class of these drugs, the biphenyl moiety is constant and positions series of modifications leads to compound 2 (Figure 29). BMS 193884 is a potent endothelin receptor antagonist and Irbesartan on the other hand is angiotensin receptor antagonist [69]. The combination of these two results in compound 2 which is potent dual active AT/ET antagonist.

Not just above stated dual active therapeutic agents, there are other diseases/disorders being treated. For example, dual topoisomearse I/II inhibitors uses dual active molecule strategy to treat cancer [66].

As presented at the beginning of the dual activity discussion, there is applicability of dual active therapeutics for different disease/disorders as shown below.

Drugs for different diseases/disorders:

On contrary to the first group of dual active drugs, there exists certain drugs which are combination of two different mechanisms/different targets and also implicates for treating two different disease or disorder. For example, Dual μ-opioid receptor and serotonin/norepinephrine reuptake inhibitors: They act on both opioid receptor and as a serotonin/norepinephrine reuptake inhibitor [68]. Thus the first action results in analgesic effect and the latter results for its antidepressant action. As a result, these dual opioid receptor and serotonin/ norepinephrine reuptake inhibitors are effectively used for chronic pain for its better management.

For example, Tramadol acts as opioid receptor and as a serotonin reuptake inhibitor and Tapentadol acts as opioid receptor and as a norepinephrine reuptake inhibitor. These two drugs not only treat pain but also can be used to combat depression.

Tramadol

Tramadol is dual active opioid receptor (central acting) and serotonin reuptake inhibitor [68]. Chemically it is 2-((dimethylamino)methyl)-1-(3-methoxyphenyl)cyclohexanol and occurs in both R-and S-stereoisomers but commercially, tramadol is marketed as a racemic mixture because of the complementary action of both isomers. Analgesic activity is accredited to low but efficient affinity for the µ-opioid receptor as well as reuptake inhibition of the monoamine, serotonin. It shows no selectivity between µ, k, δ-opioid and shows highest affinity with µ-opioid (K i 2.1 µM) but the active metabolite of tramadol, o-desmethyl tramadol shows higher affinity than tramadol itself. Along with opioid action for pain relief, it acts with a secondary pathway for relieving pain using descending inhibitory system (this part has been discussed in-depth in depression and its management chapter). Venlafaxine is a SNRI used to treat depression, whose structure is intimately related to tramadol.

As seen in the previous chapters, antidepressants act mainly by recapture of serotonin or norepinephrine. Thus, tramadol due to its descending inhibitory activity on monoaminergic system acts as a moderate antidepressant in treatment of chronic pain.

Tapentalol

It is another dual active opioid receptor and norepinephrine reuptake inhibitor which acts in a similar way as that of tramadol. Its analgesic property is more than tramadol but less than morphine. But its norepinephrine reuptake activity is poor compared to its preceded counterpart i.e. tramadol. Tapentadol's dual activity has been validated by studying the inhibition of norepinephrine reuptake with a Ki of 0.48 µM and serotonin reuptake with a K i = 2.37 µM, clearly showing its affinity towards norepinephrine than that of serotonin.

Along with above discussed analgesics, there are other categories of drugs which are applied with dual activity principle and thus can be used as a treatment for two different drugs/diseases.

With the above described rationale, we have designed the prototype of the project.

B. Design of the project

As presented earlier, the first target for acting on chronic conditions like migraine and visceral pain was CGRP receptor antagonism. For selecting the second target to achieve a dual active moiety, we have done some more literature investigation and presented the full prototype of the project as below We have chosen tricyclic antidepressant property to be our secondary effect to be incorporated into the dual active molecule along with CGRP antagonism. This has been proposed due to the effect of tricyclic antidepressants on chronic pain implication. As reported in the previous subsection [refer figure 12], tricyclic antidepressants along with its antidepressant property also have analgesic effect due to its action on descending pain modulation pathway.

Thus, we can sum-it-up and present that we wanted to incorporate CGRP antagonism and tricyclic antidepressant property into a single moiety to effectively treat chronic conditions like migraine and visceral pain.

As we have selected antidepressant property as our second target to achieve, we will have a brief introduction about the depression and effect of antidepressant on chronic pain.

C. Depression and its management

1. Depression: It is a state of low mood and aversion to the activity that can affect a person's behaviour, thoughts, feelings and sense of well-being. It is potentially a life-threatening disorder that affects millions of people around the world irrespective of the age group.

Symptoms includes mood swing (majorly depressed mood), low in energy, anhedonia, suicidal tendencies, psychomotor as well as gastrointestinal disturbances [71]. Depression is a complex phenomenon which shows fluctuating symptoms (depending on patient), all as well as few and mild to severe symptoms. Thus all the above ins and outs interpret for non-suitability of ADs against acute pain. There are definite points which show us the correlation between depression and chronic pain;

 Pain and major depression mostly overlap each other. A statistical study was done using depression and painful symptoms, which includes Headaches, Neck pain, abdominal pain, chest pain etc.

Figure 33.Showing correlation of pain and major depression

If we consider the case of chronic pain vs. depression, statistical data between some chronic pains with a tinge of depression like migraine, back pain and arthritis are as above [72].

 Depression and chronic pain shares a common neurobiology and neurochemistry i.e.

involvement of serotonin, norepinephrine along with similar Hypothalamic-Pituitary-Adrenal (HPA) axis, autonomic nervous system and inflammatory cytokine (Tissue necrosis factor-α and interleukin-6) disturbances.



Antidepressants can recover chronic pain symptoms with or without the existence of major depression in the patient. Even the vice versa condition applies i.e. antidepressants can recover major depression with or without the existence of chronic pain in the patient.

Chronic pain and depression:

Recent studies showed that patients suffering from pain have a risk of inclining towards depression 2 to 5 times more than a normal individual. But the assessment or identification of these two syndromes (pain and depression) is cumbersome as both of them have shared symptoms like gastrointestinal disturbances, fatigue, sleep disturbances etc. The comorbidity between pain and depression paves a rationale for using antidepressants again chronic pain.

Classification of depression

Minor depression:

It lasts for two weeks or longer and can't be categorized into major depression due to its symptoms. When left untreated, there is high risk for developing major depression.

Along with the above stated depressions, there are some other unclassified forms like Bipolar disorder, psychotic depression, postpartum depression, seasonal affective disorder etc.

which are rare and affects minority of population.

Biochemistry of depression:

Depression in a general context is due to the decreased monoamine neurotransmitter concentration in the synaptic cleft. All monoamine transmitters are biosynthesized from aromatic amino acids like phenylalanine, tryptophan, tyrosine and thyroid hormones by the enzymatic action of amino acid decarboxylase [74].

Table 4.showing different monoamine neurotransmitters

These monoamines play an important role in cognitive processes such as mood, emotions, memory etc. Thus the drugs used to increase the monoamines in the synaptic cleft are used as first line medication for depression and other related disorders.

Mechanism of action:

Transmission of signal from one neuron to other neuron involves a cascade of steps ranging from transport of amino acids from blood to the brain where the neurotransmitters are biosynthesized from their respective amino acids with the help of enzymes [74]. Thus formed

Treatment of depression [75]:

The treatment for depression can be classified based on various methods used to treat it. Out of all, the three most important and effective treatment methods include psychotherapy, electroconvulsive therapy and pharmacotherapy.

Pharmacotherapy is proved to me most effective compared to the rest and is considered as first line treatment for depression.

Pharmacotherapy:

It can be defined as the therapy to treat the depression using pharmaceutical drugs. It includes the drugs used to treat major depressive disorders like Obsessive Compulsive Disorder (OCD), chronic pain, Neuropathic pain, Migraine, Substance abuse, Eating and sleeping disorders etc.

i. Mechanism of action:

As discussed in the aetiology of depression, the main principles in anti-depressant drug design and target validation, the limelight is completely focused on two targets i.e. Monoamine reuptake and Monoamine Oxidase inhibition (MAOI) which tends to increase the intracellular concentration of monoamines. Anti-depressants acts by selectively binding to monoamine transporters like specific serotonin, norepinephrine or dopamine and thus directly increase the monoamine concentration in the synapses. On the contrary, the inhibition of Monoamine Oxidase (MAO) enzyme using anti-depressants also proved to be effective as it inhibits the monoamine metabolism by MAO in the presynapses after their reuptake. Hence it decreases the metabolism of monoamines and makes them available for the next cycle of signal transmission and thus indirectly participates in the increase of monoamines.

Based on their mechanism of action, the generation, structure, selectivity, anti-depressants are classified as follows;

Anti-depressant drugs

.Antidepressants based on their generation

All anti-depressants are equally effective and hence choosing one amongst solely depends on bearing in mind its side effects, drug interactions, cost etc.

Pharmacotherapeutic drugs:

TCAs

MOA inhibitors SSRIs SNRIs

Classification Chemical structure of the ligands Mechanism of action Selectivity towards receptor Selectivity towards receptor

Mechanism of action

Serotonin and Norepinephrine reuptake inhibition by blocking the reuptake sites.

Blocking the Monoamine oxidase mediated enzymatic metabolism of serotonin and norepinephrine

Serotonin reuptake inhibition by selectively blocking the reuptake sites.

Norepinephrine reuptake inhibition by selectively blocking the reuptake sites.

Place in therapy

Drug interactions

Examples

D. Depression vs chronic pain

Antidepressants (ADs) are one of the important adjuvant in the dosage regimen of pain management, especially chronic pain. Reasons for ADs as not an adjuvant for acute pain include [76];

 The chemical changes (neuroplasticity) in brain due to pain are shown only in chronic pain subjects but not in their counterpart i.e. acute pain subjects.  Among the two pain components i.e. sensory and affective, the affective component is not revealed in acute pain.

 Acute treatment of pain involves medication which are given in a single or multiple doses of small range (in days) but whereas ADs need to be given for long-term medication.

Biochemistry of antidepressants in chronic pain management:

As summarized above, antidepressants can be effectively used for treating pain symptoms even though there is no existence of major depression. This shows that the antidepressants alone possess analgesic effect. The analgesic effect of antidepressants can be shown at two levels i.e. at central and peripheral.

The central effect can be accounted because of their action on descending pain pathway through spinal cord [77]. Even though multiple mechanisms are proposed, the potentiation or synergistic action of the antidepressants in pain management is still unclear till date. Probably the most accepted theory with stronger support is their action on serotonin and norepinephrine receptors, in precise, on the descending pain pathway through spinal cord. Other theories include antidepressant's adjunctive therapeutic actions through histamine receptors and/ sodium channel modulation. Norepinephrine and serotonin's are the involved in the nociceptive signalling at multiple levels. In chronic patients, the activity of descending inhibitory bulbospinal pathway is compromised and ADs are considered to enhance their activity by blocking the 5HT 1A receptor. Not just with this, ADs manipulate several other areas of pain circuit (like locus coeruleus nucleus, the dorsal horn of spinal cord etc.). All these manipulations finally results in increased nociceptive threshold of the subject.

The peripheral effect is quite contrary to what happens in the central pathway. ADs can't act on serotonin or norepinephrine, as these neurotransmitters increases tends to enhance the nociceptive signalling instead of decreasing [77]. Therefore, competitive inhibition of serotonin, noradreno, histamine or muscarinic acetylcholine (ACH) receptors decreases the nociceptive signal transmission. ADs like amitriptyline, captivatingly, enhance the adenosine transmission by increasing extracellular adenosine.

Antidepressants and pain:

Most of the antidepressants can be used in the management of pain. But not all of them show same degree of effectiveness in treating the chronic pain. Some of the examples in different classes of antidepressants and they effectiveness towards chronic pain management are as follows [76];

2.1 Tricyclic antidepressants (TCAs) are the prime choice in the management of chronic pain. The dose to relieve or to treat chronic pain is relatively less than that used for its antidepressant action. To be accurate, tertiary amine TCAs are more efficacious when compared to Secondary amine TCAs. But their use is limited by weight gain, cardiovascular effects, overdose complications etc.

Selective Serotonin Reuptake Inhibitors (SSRIs)

shows moderate to no efficacy in chronic pain management. Even though they relatively show least side effects when compared with TCAs, they are not efficacious for chronic pain management. Paroxetine and citalopram showed moderate efficacy whereas fluoxetine showed no effectiveness in chronic pain relief. 2.4 Norepinephrine and dopamine modulators showed better efficacy than SSRIs but not more than TCAs & SNRIs. 300mg/day of bupropion showed similar efficacy as TCAs and considered to be optimal for chronic pain management. As discussed earlier, action on serotonin and norepinephrine is important for chronic pain management. Instead, bupropion proved it wrong by showing effectiveness in chronic pain management even though it acts on norepinephrine and dopamine (instead of serotonin). But their usage can be limited because of cases of seizures in the subjects.

Antidepressants

Hence, from the above proof based efficacy results, we can make out that the tricyclic antidepressants are deemed to be the best drug entities to treat chronic pain. They proved to be potent as well even on low doses. The other class of drugs, SNRIs also showed potential activity again chronic pain. This also proves that, to act on chronic pain, the drug molecules should act on both serotonin and norpinephrine which are the main neurotransmitters involved in both depression and pain signalling.

Henceforth, we had an outlook of both primary and secondary targets of our dual activity project. Now, let's sum-up all together and have a preface about the project.

V. CONTEXT OF THE PROJECT

Calcitonin Gene Related Peptide receptor is one such target which is implicated in treating chronic pain conditions like visceral pain and migraine. Telcagepant® ® is a prime molecule when it comes to CGRP antagonism.

Tricyclic antidepressants are drugs which are not just used for treating depression but also as an analgesic for treating chronic pain. Imipramine is one of the TCAs, which even in low doses showed potent analgesic property.

Thus the concept of the project was to create a hybrid molecule by combining Telcagepant® ® and Imipramine in a single moiety for a better treatment of chronic pain. We want to combine the Telcagepant® ® and Imipramine in a single moiety to recreate a hybrid structure which can be potentially used for treating some of the chronic pain conditions.

As we have made our first stage of drug discovery process by outlining the concept and approach towards the project, we proceeded to the second stage where the molecular modeling and docking studies have been performed. This will be explained in detail in the next chapter starting from design till validating the potential hits.

Take-home message from this chapter

In this chapter, we have seen 'pain' in broader context as one of the grave forms of illness in current generation to be treated. The data of global sufferers due to pain are more dynamic like never before.

Even among pain, acute pain does have improved and efficacious medication available in market. On contrary, even though chronic pain affects almost 36 % of Europeans and 43%

of Americans, there are still inefficacious pharmacotherapeutics available. And if potent and efficacious drugs are available, they are posing some potent side effects as well.

Migraine and visceral pain are two of the most dreaded conditions under the roof of chronic pain. On one hand, migraine causes diminished quality of life whereas in the other hand, visceral pain reduces quality of life and sometimes being fatal. Even in 2010, with the current technology in diagnosis and treatment methods, the mortality and years passed with disability rates are climbing rampantly.

Calcitonin Gene Related Peptide receptor is one such target which is implicated in treating chronic pain conditions like visceral pain and migraine. Telcagepant® is a prime molecule when it comes to CGRP antagonism.

Depression is associated in most of the cases with chronic pain and patient has to take multiple doses of medication to treat both chronic pain and depression individually. The doses of antidepressants used are also high ranging between 150-300 mg.

Hence dual active moieties which can use the synergistic principle of treating chronic pain are potent future generation molecules to be developed in treating them.

Tricyclic antidepressants are drugs which are not just used for treating depression but also as an analgesic for treating chronic pain. Imipramine is one of the TCAs, which even in low doses showed potent analgesic property.

Thus the concept of the project was to create a hybrid molecule by combining Telcagepant® and Imipramine in a single moiety for a better treatment of chronic pain.

Winding up with the concept derived from this chapter, we will look into the next stage of drug discovery i.e. crystal structure of protein and molecular modeling studies in the next chapter.

I. CALCITONIN GENE RELATED PEPTIDE RECEPTOR

Calcitonin Gene Related Peptide (CGRP) receptor is structurally a heterodimeric protein with CALCitonin Receptor Like receptor (CALCRL) and Receptor Activity Modifying Protein-1 (RAMP-1) as its subunits [1][2][3]. These receptors on activation by the endogenous natural ligand results in various physiological disorders like Migraine, Crohn's disease, Irritable Bowel Syndrome, Systemic Inflammatory Response Syndrome etc [4][5][6].

Among the two subunits involved, CALCRL belongs to the Class B (Figure 37 receptor respectively [11,12]. The notch up of protein-protein interaction of CALCRL with different partner proteins (Figure 37(d)) is as follows ( Like for the other GPCRs, hitting a hotspot of CGRP receptor is a challenging task due to the least reported and exploited binding sites on the receptor. Hence we will try to validate different aspects of the CGRP receptor with the aid of in silico tools to draw some possible hotspots of the receptor and craft a pathway in CGRP research.

A. DRUGGABILITY

Druggability of a protein can be defined as a virtual recognition or derivation of the pocket of a protein, which acts as a site of interaction for probable ligand [14]. From this recognition, it paves a pathway for drug discovery process of a disease or a disorder. This virtual screening of druggable site is considered as the foundation for a thriving drug discovery process [15]. On an average, only ≈15% of the total available proteins in humans is found to be modulators of a disorder or disease out of which only around 15% are druggable [16]. Hence finding a new target for newly developed drugs is least probable. However these figures can be increased if the number of protein-protein interactions can be disturbed by the ligands so that new vicinities of the protein are exposed increasing the chances of the druggable sites [17].

Druggability study of CGRP receptor

The Druggability of CGRP receptor was identified using DrugEBility ® online software by European Molecular Biology Laboratory-European Bioinformatics Institute [18]. To begin with, the crystal structure of the protein was retrieved from the Protein Data Bank (PDB) by Royal Collaboratory for Structural Bioinformatics with PDB ID 3N7S [19]. Initial study includes general druggability of the receptor's two subunits individually (Figure 38). Hence the proteins CALCRL and RAMP-1 preliminary impressions are as follows; Druggability score can be defined as likelihood of small molecules which satisfies the Lipinski's rule of five to have a good binding in the pocket. Tractability is similar to druggability but with more flexibility in some of the Lipinski's rules. Ensemble Druggability is mean of druggability score but calculated in various models (Table 9). Both the subunits have relatively same nature in their sites (Figure 38). They showed 0% of Druggability and Ensemble Druggability. However, interestingly they showed 50% of Tractability in their binding sites once again proving the hypothesis that relatively bulkier ligands might serve as a good hits for this receptor.

Score

Druggability validation of CGRP receptor

Following a brief study on the general druggability and tractability of CGRP receptor family in general, the individual druggability validation of the receptor is investigated using the DrugEBility ® online software. In this method, the DrugEBility ® software takes up the receptor replica via uploading manually the protein model or by entering the PDB code directly. After a run-through and scrutinizing the database, the software provides the top four By having a closer look at the 4 sites of the CALCRL & RAMP-1 subunits, there is a clear visualization of the probable binding sites on CALCRL showed relatively smaller druggable volumes (674.1, 498.2, 288.9 and 207.5Å 3 ) compared to that of RAMP-1(1468.5, 434.9, 394.8, 300.3Å 3 ). And furthermore, the site 1 of RAMP-1 is envisaged as a tractable site with a volume of 1468.5Å 3 . Thus designing an antagonist with CALCRL interacting fragment relatively smaller to that of RAMP-1 is more persuasive & reasonable theory. This hypothesis can be validated using a well known CGRP antagonist Olcegepant® and its binding inside the receptor pocket. Olcegepant® into the receptor is considered as inevitable and important interaction (Figure 39). Hence this fragment has been well thought-out as universal for all the CGRP antagonists from there on [20].

Whereas the uninteracted fragment of Olcegepant® in CALCRL is thought to be interacting with the associated protein RAMP-1. The site 1 of RAMP-1 (Table 10) shows the tractability score hence proves that (S)-2-amino-N-((R)-6-amino-1-oxo-1-(4-(pyridin-4yl)piperazin-1-yl)hexan-2-yl)-3-(3,5-dibromo-4-hydroxyphenyl) propanamide (the fragment not shown in the red dotted line of Olcegepant®) is having an interaction with it. The large volume of the site 1 (1468.5 Å 3 ) also proves that considerable bulky interacting fragment is required for a healthy fit as well as interactions.

B. DRUG-RECEPTOR INTERACTION STUDY:

The crystal structure of CGRP receptor with Olcegepant® as the ligand showed the significance of various interactions [19]. The fragment interacting with CALCRL protein showed some key interactions like Thr122, Asp94. Thr122, a polar residue acts as a backbone donor for the carbonyl oxygen of 3-(piperidin-4-yl)-3, 4-dihydroquinazolin-2(1H)-one and Asp-94, an acidic residue shows a side chain acceptor interaction with the nitrogen on the pyridine (Figure 40).

C. SPECIES SELECTIVIY DECISIVE RESIDUES

The importance of some amino acids apart from those listed above is studied using deletion or mutational investigation. The significance of various amino acid residues as well as the binding of CGRP antagonists specifically to CALCRL/RAMP-1 complex but not for the other protein-protein complexes like RAMP-2 or RAMP-3 has been documented using the genetic engineering methods.

The radioligand-isotope binding assay illustrated that rodent CGRP receptor (both Mice & Rats) showed less affinity for the CGRP antagonists compared to that of primate receptor. This decrease of affinity in rodent CGRP receptor is attributed to the fact that at 74 th position of the RAMP-1, the Tryptophan is replaced with Lysine. The crystal structure clearly showed the Trp74 has formed a hydrophobic roof in the RAMP-1 and this observation is found to be crucial to derive lot of important conclusions. Olcegepant® showed 100 times less affinity in rodent species [22] whereas Telcagepant® showed 1500 [23]. This variation of affinity for these antagonists might be endorsed due to the protein-ligand hydrophobic pocket in rodents has been decreased [24], the other functionalities of Olcegepant® like 4-(pyridin-4-yl)piperazin-1-yl, Amino hexyl moieties might act as steric hindrance and thus reducing the ligand-Trp74 interaction. Hence the replacement of Tryptophan with Lysine couldn't result in huge impact. In the case of Telcagepant®, the antagonist interacting with RAMP-1 is small and non-sterically hindered, thus the Tryptophan replacement can easily show the impact on affinity.

Not only with affinity facet, tryptophan 74 might also implicate in selectivity of CGRP antagonists towards CALCRL/RAMP-1 complex [25]. This Trp74 is expressed exclusively in RAMP-1. However, in RAMP-2 or RAMP-3 it is replaced by Glutamic acid. Hence

II. MOLECULAR MODELING STUDIES

With the innate knowledge from the previous chapter on the CGRP receptor, its druggability, hotspots and key residues, we further proceeded to the molecular modelling and docking studies using the crystal structure of CGRP receptor. This particular chapter can be discussed in three different subsets as; [26], using which the important aspects like solvation of the system and ligand interactions in the receptor have been visualized.

During the refinement step, we prepared our molecule ready for docking by removing the already interacting ligand (in our case, Olcegepant®), chains B and C as well as the water molecules leaving the following system i.e. HOH201A; HOH206D; HOH203D; HOH210D; HOH203A; HOH210A; HOH225D; HOH216A (Figure 41). . Finally, this model is minimized using molecular mechanics (refer appendix for details). Hence this energy minimization step results in a low energy and refined crystal structure which will be engaged in the docking simulation.

B. Modelling of the ligands

As described in general during the course of first chapter, we have recreated a hybrid structure which has been derived from Telcagepant® ® and Imipramine cross-over. The hybridization can be depicted as below The fragment coloured in red is carried as a privileged structure from CGRP receptor antagonists and the fragment in blue was designed according to the structure of the receptor.

During the design of the ligands, we kept in mind various important points. Hence, first we will look into the aspects and design of ligands for CGRP antagonism.

Structure based ligand designing

As stated, the crystal structure of CGRP receptor with highest possible resolution has been retrieved from PDB. Using this model, structure based ligand designing of the potential hits was done (Figure 43). During this process of designing the hits, we followed different set of medicinal chemistry rules for drug designing in order to provide an optimized potential hits. The region in white represents RAMP-1 where as in yellow is CALCRL. The fragment in our hits interacting with CALCRL is highly potent and is most common fragment of currently available CGRP antagonists.

Whereas, the most important region for CGRP antagonism is RAMP-1 and further in that, Tryptophan 74 is highly important for affinity towards the receptor. As this RAMP-1 requires hydrophobic interactions, the fragment of the hits interacting here has to be hydrophobic. And last but not the least, both the fragments can't be tightly bound as this may result in high crash and strain in the pocket and hence there has to be flexibility between both the fragments which can be obtained by using a linker to have easy rotation.

Ligand designing

The fragment designed is solely for the RAMP-1 region as we use optimized and already used privileged fragment for CALCRL part.

Points considered during designing:

 No same volume fragments: The RAMP-1 is big in volume and we can't design a fragment of same volume as they will be facing problem for drug transportation to reach the place of interaction. Even if it reaches, they can't fit because of strain and crash inside the pocket.

 Chiral carbons incorporation: If we can incorporate chiral carbons, depending on the interaction required, we can reach both sides of the plane of the molecule. Even if tough to reach the required interaction, homologation can help to fill the desired interactions (Figure 44). Thus along with the above points, we have chosen piperidine to be the core moiety as the laboratory has expertise in synthesis of chiral piperidines and indigenous developed methodologies for using different substituants.

Application of medicinal chemistry principles

Hydrophobic RAMP-1 fragment has been designed as shown below. In conclusion, all the families are derived using structure based ligand designing by using medicinal chemistry principles as described above.

And so, these designed molecules are taken to the next level i.e. docking studies.

All the ligands docked in this current study are modelled using same protocol as described in the appendix.

C. Docking

The designed ligands have been forwarded to a in silico interaction studies using docking procedure. This part of the study has been done with the help of 'Molecular modellization' team representing Mr. Lionel Nauton and Dr. Vincent Thery. This doking has been performed using the energy minimized receptor model and designed ligands. The procedure followed has been briefed in the appendix.

At the end of the docking simulation, all obtained results were combined in a single data file, in order to make statistical studies on obtained scores. Telcagepant® and Olcegepant® were also included in the docked ligands as they serve as markers or references.

These docked conformers are consistent with the crystallographic structures which we retrieved previously from PDB (Figure 46). Zone2: This area depicts a very strong or too many electrostatic interactions. Most potent CGRP antagonist, Olcegepant® lies here. Hence hits from this region can be interesting when it comes to considering electrostatic interaction scores alone.

Zone 3: This area can't be ignored either as the previous two. We have found the grip (good placement in the pocket) as we wanted. We can consider the hits in this zone as well.

Zone 4: This zone corresponds to the hits with desired interactions as well as total scores and we also found Telcagepant® in this region. After this initial screening, and after checking remaining solutions and applying different filters, we shortened the hits from 2225 to 36 possibilities with complete disappearance of several compounds of different families.

D. Identification and classification of potential hits

The docking results were further scrutinized using various filters (Figure 48). The most probable bioactive forms were hypothesised by the above discussed factors like electrostatic interactions, crash and strain (grouped together as 'Total score').

.showing the total scores of family 1 potential hits

Family-1 (Figure 49) docking scores along with their substituants and configurations in different chiral positions (Table 11) suggest the equality of both benzyl and trifluoro ethyl substituants for interactions in receptor. Family-3 (Figure 51) docking scores along with their substituants and configurations in different chiral positions (Table 13) gives an observation over the chirality as well as importance of substitutions at position 'Y' for new electrostatic interactions.

Thus the above docking results paved a rationale for identifying the potential hits for the CGRP antagonists. And in the next subunits, we can have a brief over view on some more aspects like pocket volume importance, drug-likeliness etc.

E. Receptor pocket volume Vs ligand volumes

During the preliminary stages of this chapter, we have studied the binding pocket along with their volumes. Thus the volume of the pocket suggests that mere 'flat and small molecules' are not sufficient for a proper drug-receptor interaction. Instead, the ligands have to be designed by considering the volume (to be precise, size) and amino acids in the vicinity of the pocket. Along with the volume aspect, we also have to note down the point that incorporating chiral carbons wherever required will increase the chances of effortless reaching of the substituants of the ligand towards the amino acids of the receptor. As we already saw the volume of the binding pockets, now let's see the volume of the ligands which we have considered as probable hits (Table 14). The pocket volume for CALCRL interacting fragment (198.81) vs pocket of CALCRL (674.1) is constant for all the molecules and hence the interacting hit's volumes inside the pocket pose no issues. On the other hand, the fragment of our hits interacting with RAMP-1 is relatively big when compared with their counterparts interacting with CALCRL. The RAMP-1 volume (1468.5) can play crucial role as we saw in the previous chapters about the importance of RAMP interaction with hits for a potent activity. And thus a bigger in volume fragments is designed for a better interaction.

RAMP-1;

With this, the molecular studies on the receptor and their ligands can be concluded.

There is one more aspect called drug-likeliness of molecules which play important part in pharmacokinetics which will be discussed in the next subset.

F. Drug-likeliness properties:

Drug-likeliness properties are ideal characteristics of a molecule to be qualified as a drug entity. Prof. Lipinski postulated a group of rules to quantify and validate the druglikeliness of a drug. Drugs satisfying these rules posses optimized pharmacokinetic properties.

These rules are called Lipinski's rule of 5 [29].

Lipinski's rule of 5

According to Lipinski's rule of 5, an ideal drug should have following characters  A molecular weight (MW) of not more than 500 daltons.

 Partition coefficient LogP not more than 5.

 No more than 10 hydrogen bond acceptors (HBA) like N, O.

 No more than 5 hydrogen bond donors (HBD) like NH, OH.

Note than even though there are only 4 rules, Lipinski's rule of 5 was named so because all the descriptors are multiples of 5.

Hence the drug-likeliness of the designed molecules in general is shown in Table 15. The potential hits studied for drug-likeliness properties by Molsoft ® showed that most of the molecules complies the Lipinski's rule of 5. Some observations noted from the results are as follows;

Ligand

 Molecular weights of the potential hits have slightly surpassed the proposed limit but comparing with the lead molecule Telcagepant®, they are in concordance.

 Hydrogen bond acceptors and donors of all the molecules are within the range of rule of 5.

 Partition coefficient LogP is a determining factor for both absorption and distribution of the drug molecule. If they are more than 5, they are hydrophobic and won't be completely soluble in body system and on the other hand, way less than 5 (around 0-1) results in hydrophilic molecules posing the same problem of solubility and bioavailability. All the hits designed showed optimized LogP values around 3-5.

 Polar surface area shows the sum of surface volume of all the polar atoms of the molecule. PSA value less than 90 is required to cross Blood-Brain-Barrier when they want to act as CNS drugs. All the designed hits are well within the range of rule of 5.

Along with that, the Molsoft ® online tool gives a score for each molecule submitted.

These are called model score. They provide a graph taking into consideration 5000 of marketed drugs from World Drug Index (positives) and 10000 of carefully selected non-drug compounds (negatives). And so on, the potential hits are having drug-likeliness as well as their scores are almost same as that of Telcagepant®. Hence all the hits are hypothesized to have good pharmacokinetic properties.

In conclusion, potential hits have been designed using structure based ligand designing and later validated for their interactions using docking simulation as well as all other possible

Insilco validation methods such as pocket vs ligand volumes, drug-likeliness properties etc.

finally providing a list of 36 potential Calcitonin Gene Related Peptide antagonists.

Thus obtained hits have to be synthesized to validate the hypothesized concept in living systems, which brings us to the next part of drug discovery process i.e. designing of synthetic methodology for the potential hits.

III. RETROSYNTHETIC DESIGN FOR SYNTHESIS OF HITS

The potential hits obtained through molecular modeling and docking studies have been initially scrutinized to propose a generalized retrosynthetic design. The following design seems to be legit of all to achieve the desired moiety. The prototypical design proposes that we can reach the potential hit starting from two different sources, either aspartic acid or methyl crotonate depending on the family to be synthesized. The core piperidine moieties can be achieved using a Aza-Michael intramolecular reaction to give a piperidine ring with desired configuration and substituants.

N N O X R 4 N N NH N O O R 1 R 2 N X R 4 N N NH N O O R 2 N N N R 1 N Y X R 4 N N NH N O O R 2 HN N NH N O + N Y R 4 R 2 Z O N R 4 R 3 O O R 6
Finally, a convergent synthesis can be followed to couple with the privileged structure in order to accomplish the potential dual active molecules.

Thus, in the next chapter, we will have an in-depth analysis and investigate about the piperidine synthesis proposed by different research groups and retrieve some of the available methods and optimize according to our desire to synthesize dual active molecules.

Take-home message from this chapter:

In the current chapter we tried to cross-examine all possible aspects of CGRP receptor ranging from basic information on the association of different proteins to form the receptor to more complex Druggability, Tractability and Drug-receptor interaction studies. With the aid of bioinformatics, we have put forth an outcome which proves to be more feasible to achieve the CGRP antagonists with increased affinity and potency. We have placed up front some key amino acids which are impeccable for an optimal drug-receptor interaction as well as crucial for species & receptor selectivity of ligands. Hence all these descriptors can be clubbed and publicized as the 'hotspots' and consequently showing how to proceed in targeting these 'hotspots' will be viable for providing an optimal drug interaction in CGRP receptor.

Along with the study of the receptor, molecular modeling studies have also been conducted to put forth a database 2225 molecules which were further engaged in docking simulation. Finally the prioritized list of molecules has been selected based on the benchmark i.e. score more than the 'lead' molecule Telcagepant®. These filters finally provided us with 36 'potential hits' which can be classified into three structural families.

From thereon, we saw some more important aspects such as pocket vs ligand volumes, drug-likeliness properties etc. through which the concept for designing the hits have been validated.

In final units we saw the retro synthetic design of synthetic methodology for the potential hits in general. Thus forwarding our drug discovery process to the next aspect i.e.

'chemistry as well as synthesis of the hits'.

I. LITERATURE INVESTIGATION

The piperidine moiety is a structural unit which is most common in skeleton of numerous alkaloids and biologically active molecules [1]. The key objective of many groups working on this skeleton includes modulation of the stereoselectivity of the skeleton [2].

Keeping in mind the large number of applied methodologies, we have cited in this chapter some of the most recent work in the synthesis of piperidines and in particular with the substitutions at 2, 4 and 6 positions with principle lead being the alkaloid (+)-241D, a compound isolated from an American frog Dendrobates speciosus [3] which showed potent inhibition of nicotinic acetylcholine receptor [4] and 4-hydroxy pipecolic acid which has been also obtained from leaves of Calliantra pittieri and Strophantus scandeus [5]. (2S,4R)-4hydroxy pipecolic acid and 6-substituted analogs are also the core of biologically important compouds as, in particular the Palinavir structure an HIV-protease inhibitor [6]. Due to their different structures and consequently to their synthetic approaches ( the amino acid functionality), this chapter is divided in two parts : the former is devoted to 2,6disubstituted 4-hydroxy piperidines and the second to 4-hydroxy pipecolic acid derivatives.

A. Synthesis of 2, 6-disubstituted-4-hydroxy piperidines 1. Synthesis by using metal catalysis

Majority of the methods followed for the formation of piperidine skeleton involves an intramolecular cyclization principle to form a C-N bond. This step determines and accomplish with the maximum stereoselectivity inclusion in the resulted piperidine ring with substitution at 2 and 6 positions. In the current chapter, we will deal with the methodologies and developments concerning the synthesis of 2, 4 and 6 substituted piperidines.

Application of Iridium complex:

The method developed by Helmchen and coll. [7] allows to access (+)-241D alkaloid relatively in limited steps compared with other methodologies developed in the recent times. Thus obtained piperidine 4 was transformed into (+)-241D alkaloid using optimized conditions like protection of amine & hydroxyl functionalities followed by homologation using metathesis with desired carbon chain, reduction and finally a saponification to give the desired compound (scheme 2). The same strategy has been applied for piperidine 5 to give (+)-6-epi 241D.

NH

Application of Gold complex:

Synthesis of (+)-241D alkaloid has also been described using an intramolecular cyclization reaction catalysed by gold complex [8]. This involves an alkylation of Weinreb's amide 6 in presence of butyllithium and undecyne giving an adduct 7. Finally, the cyclization occurs in presence of coordinated chlorine complexed with gold/triphenylphosphine associated with silver to obtain the cyclic enamine compound 8. Lastly, washing with acidic medium followed by reduction gives alkaloid (+)-241D.

Another synthetic method using gold complex for the synthesis of (+)-241D alkaloid has been recently described in the literature [9]. The first chiral centre has been introduced by alkylation of sulfinimine [10] to give compound 9 followed by deprotection of amine and later acylating using acid chloride gives corresponding amide 10. In presence of gold complex, an intermediate 11 has been derived using cyclization which was later selectively reduced to give α-amino ether 12 which spontaneously rearrange to give 4-piperidone 13 using the Petasis-Ferrier type mechanism [11]. Compound 14 was obtained from 13 using simple reduction. Based on the reducing agent used, they were able to modulate the stereochemistry at position 4.

An example to investigate the efficacy of this method has been applied to synthesize a natural compound as depicted below.

Miscellaneous metal complex:

The intramolecular reaction has also been studied using various alcohols and derivatives of allylic alcohols to synthesize stereoselective piperidines using other metal complexes like palladium [12], or Iron (III) chloride hexahydrate [13] etc.

Synthesis by intramolecular reductive amination

The formation of piperidine cycle through reductive amination remains one of the most exploited methods for the reason being impeccable control over the stereochemistry of the retrieved piperidines.



Chandrasekhar and coll. [14] used this method starting from homoallyl amine 15 which has been obtained by using decanal and valine as the starting materials. The compound 15 was transformed into amino aldehyde 16, which being unstable reacts spontaneously with D-proline through aldol condensation to give a ketone 17a and 17b

Decanal

with an overall yield of 56%.

The deprotection of the amine as well as intramolecular cyclization through reductive amination was carried as a two-step & one pot synthesis by using palladium on carbon as catalyst. This method, when applied to 17a gave (-)-241D alkaloid and 17b gave 4-epi-(-) isomer.

 Das and coll. [15] used a method which was mostly similar to that of the Chandrasekhar and coll., except varying the chirality on the final molecule.

Scheme 8.Showing the application of reductive amination by Das and coll.

In this case, decanal was treated with (S)-tert-butanesulfinamide for the synthesis of imine 18 which was later alkylated using allyl bromide in presence of Indium which gave the amine 19 with high stereoselectivity (96/4). For feasibility during the later stages of the reaction, protecting group was being replaced to give homoallyl amine 20. The oxidation of 20 to 21 was done using ruthenium chloride and aldol reaction was done in the presence of acetone and sodium hydroxide to recover a mixture of 22a and 22b in 1/1 and was finally cyclized according to the procedure cited above to give (+)-241D and 4-epi-(+)-241D alkaloids.



Following the same strategy, Rao and coll. [16] described a stereoselective synthesis of alkaloid (+)-241D. This method is almost of same which was described above and the only difference here is the formation of the first asymmetric centre Starting from the decanal, an enantiomeric allylation has been carried with complex of titanium and (S,S) BINOL and in presence of allyltributyltin [17] This was followed by activation of alcohol functionality of 23 with tosylate and later a nucleophilic substitution reaction using sodium azide to give compound 24 which was further reduced with lithium aluminium hydride. Thus formed amine was protected with benzyl carbamate to give 25. An asymmetric dihydroxylation of alkene was carried out using the protocol proposed by K.B. Sharpless [18] which gave diols 26 and 27 in 80/20 ratio. The major compound 26 was further treated with sodium hydride in presence of tosyl chloride at 0 °C to give an epoxide which was immediately engaged to react with vinyl cuprate to obtain homoallylic alcohol 28. The oxidation of 28 according to the protocol developed by Wacker gave 22b and this act as a substrate for reductive amination to give the alkaloid (+)-241D.

Synthesis by intramolecular nucleophilic displacement

An alternative and frequently used way to synthesize piperidine cycle consists of an intramolecular cyclization of an amine with a potent leaving group (SN 2 ).

Activation in the form of mesylate or tosylate

 Toyooka and coll. [19] described the synthesis of polyhydroxypiperidines as potential inhibitors of α-Lfucosidase. In this synthesis, the chirality appears from using the L-alanine and branching of the different carbon chains with asymmetric centres has been studied. According to the configuration required, change of epoxide and aziridine results in modulation of configurations at 2 and 6 positions as described in the following According to the cited experimental conditions, in both 2, 6 cis piperidine and trans, the selective deprotection of one or the other or two dithiane groups are possible (scheme 11). The choice of the reducing agent permits the formation of an alcohol with desired configuration and thus introducing another asymmetric carbon in the final molecule.

Activation through a Mitsunobu reaction

 Krische and coll. [22] has described recently the access of stereoselective 2, 4, 5 trisubstituted piperidines (scheme 12). The ultimate step for final cyclization uses the classic Mitsunobu reaction [23].

Miscellaneous methods

Asymmetric hetero Diels-Alder reactions

This reaction accounts as a method being exploited the most because of its control over regio and steroselectivities. This also allows for an amplification to give polysubstituted piperidine cycles. It is a well known fact that the imines are reactive compounds involved in Diels Alder reaction carried in presence of dienes like Danishefsky's diene(for example) [24]. On the other hand, this reaction had proved to be unpredictable and depends a great deal on the engaged substrates. The inoculation of a chiral centre in the final product is interesting if we can control the absolute configuration which can be done via the transition state of the reaction by using chiral inducer.

The selected way by He and coll. [25] consists of using a cyclic sulfonylimine in presence of a chiral inducer which was derived from natural Cinchona alkaloids.

+ N S O O O R 2 O F CO 2 H chiral catalyst N S O O O R 2 O dr >19/1 ee >90% N OMe NH 2 H chiral catalyst

Scheme 15.Showing the application of Diels Alder reaction

The overall yield of the reaction (>65%) was good with an excellent diastereomeric ratio and enantiomeric excess. Thus final steps include deprotection of the nitrogen atom, stereoselective reduction of ketone to alcohol which allows the access of 2, 4, 6 substituted and chiral piperidine.

Application of nitrones

Nitrones are the synthetic intermediates which can access heterocycles with nitrogen.

1, 3 dipolar cycloaddition of nitrones on alkenes permits for the formation of the isoxazolidines and also leads for formation of many other heterocyclic derivatives.

However, this method is limited by a weak diastereoselectivity owing to the nature of utilized alkene during the reaction.

Chattopadhyay and coll. [26] has used the methodology first described by Merino and coll. [27 ] for synthesizing two enantiomers of the alkaloid 241D. The reaction of (R)-2,3-Ocyclohexylidene glyceraldehyde with allylamine gave a corresponding imine 43 which later was alkylated using organozinc and organomagnesium to give 44 and 45 respectively. In a pretty similar way, 49 was used to obtain (2R, 4S, 6S)-(+)-241D alkaloid through intermediate 51.

Application of Aza-Michael type intramolecular cyclization

Intramolecular cyclization reaction between an amine and an α,β unsaturated ketone (Aza-Michael) is also a method of choice for the synthesis of piperidines. This particular methodology is mostly used to synthesize recemic mixture and few also describe to obtain them with stereoselectivity. Recently enantioselective synthesis using organocatalysts has been described [29] but the reported reactions mostly used the intramolecular reaction. This intramolecular reaction has been exploited a great number [30] and in this sub-section, we try to put forth a few of them.



Hong and coll. has described the preparation of (+)-myrtine and the (-)-epimyrtine starting from a precursor with an asymmetric centre in α-postition to the nitrogen [31].

Starting from an allylic alcohol 52, an oxidation with manganese dioxide gave the α,β This reaction was applied for the synthesis of (-)-epimyrtine 56 and (+)-myrtine 57 starting from 54 and 55 respectively. Del Pozo and coll. has described the synthesis of (+)-myrtine 57 by using same methodology as above. Commencing from aldehyde 58 in presence of benzoic acid and a chiral catalyst gave the piperidine 59 (63% yield and 94% ee) which on further reaction with allyl magnesium bromide gave the alcohol 60. Compound 60 was oxidized using Dess-Martin reagent to give the ketone 61 which on reconjugation gave 62. After the déprotection of protecting group in acidic condition, a new cyclization was carried out using Michael reaction to give (+)-myrtine 57 [32].

 Krishna and coll. [33] has described synthesis of (-)-andrachcinidine through Aza- Akiyama and coll. [34] has described that the Aza-Michael reaction can also be carried out without the protection of the amine. But using the chiral catalyst however is necessary to inoculate the asymmetric centres. In this case, they used a phosphoric acid derivative as the catalyst. The solvent used in this reaction proved to be crucial. Thus, the dihydro 2, 3-quinol-4-one 66 was prepared from the precursor 65. A systemic study on the parameters affecting the formation of major configuration was also conducted. This study has notable showed the importance of alcohol used during the reaction as shown in the below examples [36] In the first case, the cyclization of compound 70 in presence of methanol gave 2, 6 cis 72 isomer as major compound which was later transformed into (+)-241D alkaloid. Whereas in the second case, the cyclization of compound 73 driven in presence of ethylene glycol gave 2, 6 trans 74 in majority which subsequently was transformed into (-)-solenopsine A.

 Hurvois and coll. [37] has described a method to synthesize (+)-241D and (+)-myrtine Synthesis of (+)-myrtine 57 was done using 80a as a starting material. The cyano cuprate was obtained through metallation using s-BuLi as well as treatment with copper(I) cyanide and lithium chloride. The alkylation with iodo-4-chloro butane gave the desired 2, 6 disubstituted 81 which after transacetalation in acidic medium forwards the intramolecular cyclization to form (+)-myrtine 57.

Synthesis of alkaloid (+)-241D starts with the same compound 80a. The alkylation in presence of s-BuLi and DMF gave the piperidine 82, a mixture of cis/trans-80/20. The major isomer was separated through column chromatography and further engaged in Wittig reaction followed by reduction of the double bond which finally gave compound 83. An acidic treatment results in formation of ketone and freeing the amine simultaneously. This has been succeeded by a hydrogenation reaction to retrieve the alkaloid (+)-241D.



Dieter and coll. [38] reported the alkylation of N-Boc-4-pyridone 84 through Grignard reaction in presence of trimethylsilane chloride. This procedure avoided the use of copper salts customarily used to forward this transformation [39]. The dihydropyridine thus obtained depends on the nature of organomagnesium utilized during the reaction. However, the nature of the reagents employed has great influence on stereoselectivity of the recovered products. One of their application which gave 2, 6 cis majorly is as follows [40].

B. SYNTHESIS OF 4-HYDROXY-6-SUBSTITUTED PIPECOLIC ACID/ESTER

Natural amino acid, L-pipecolic acid 87 plays an important role as a biologically active moiety especially at CNS level of mammalians [41]. The oxygenated derivative at 4 th position, 4-oxo-pipecolic acid 88, the (2S,4R) and (2S,4S) 4-hydroxypipecolic acid 89 and 90 are also important as building blacks for pharmacologically active molecules [42]. They can also act as constrained amino acids and by this way they can replace natural peptides with the advantage of a greater metabolic stability [43]. In this context synthesis of constrained peptides is still an important challenge in medicinal chemistry [44].

For the above listed reasons, a number of research groups have committed themselves for the enantioselective synthesis for 88, 89, 90 and also their analogues either by resolution of the racemic intermediates or by using a chiral starting material. Recently, number of ways to reach the 4-substituted pipecolic acid and its analogues has been reported [45]. In this current subsection, we tried to present some of the synthetic strategies which have been developed in the past 10 years.

Synthesis using chiral auxiliaries from amino acids

The amino acids are the most utilized precursors in chiral synthesis. Indeed, the amino acids comprise a stereocenter at α position and there are several possible strategies to modulate and substitute the core are at hand.



Aitken and coll. The nitrile functionality was later transformed into amide in presence of hydrogen peroxide to give 95. A basic workup followed by an acid hydrolysis results in the formation of a lactone 96. This compound 96, on hydrogenation gave (S)-4-oxopipecolic acid 88.



According to one of the reviewed literature, Hou and coll. [47] used the chiral synthon 97 which was also obtained from phenylglycinol [48]. The alkylation of 97 with 2-(iodomethyl)allyl benzoate 98 resulted in the formation of compound 99. This has been followed by the deprotection of nitrogen and subsequent alkylation with allyl bromide giving compound 100.



According to the strategy proposed and developed by Seebach and coll. [49], Haufe and coll. used the imidazoline 104 [50] to synthesize the compound 108. The alkylation of the compound 104 using 2-fluoroallyltosylate 105 gave the alkylated imidazoline 106 with an excellent yield and high diastereoselectivity. Treatment of 106 in aqueous acidic medium resulted in the formation of 107. Thus formed compound 107was reduced and hydrolyzed to give 6-tert-butyl-4-hydroxy pipecolic acid 108.

Synthesis using chiral auxiliaries from chiral alcohols



Occhiato and coll. [51] has described the synthesis of (2S,4R)-4-hydroxy pipecolic acid 89 starting from (3S)-4-chloro- On the other hand, if the nitrogen deprotection was followed by a treatment in basic medium results in the formation of 2,6 cis isomer 139 through an Aza-Michael intramolecular reaction [54]. The compound 139 was then reduced stereoselectively in presence of a bulky reducing agent to retrieve (2S,4S,6S) 6-substituted-4-hydroxy methyl pipecolate 140 in good yields.

This method is found to be general and can be applied to access piperidines with variety of substitutions at 6 th position. This can be achieved by engaging the required aldehyde during the HWE reaction to incorporate requisite substitution on 6 th position.

C. CONCLUSION

Far from being exhaustive, this section shows the principle methods for the preparation of 2,6 disubstituted piperidines and pipecolic derivatives, with diversified approaches (asymmetric version, application to natural products synthesis, methodology,..) which render the diversity and richness of all the research which are done in this area.

Although a great number of syntheses permit the preparation of piperidines possessing one or two asymmetric centres, a few of them are able to control all the potential asymmetric centres of this class of heterocycles. So, as we have demonstrated that the synthetic route developed in our laboratory was relevant for the asymmetric synthesis of piperidines derivatives, we have choosen this route for compounds issued from family 3. On the other hand, for family 1,

we have used the procedure described by Sutherland since it seems to be the most applicable process for our purpose.

II. STRATEGY FOR THE SYNTHESIS OF FAMILY 1

The retro synthetic strategy for the access of family 1, with the core benzodiazepanone moiety can be framed as depicted in the scheme 41. This proposed scheme portray that the desired urea like molecule 141 can be obtained by pairing the fragment 142 with that of 143. Whereas in fragment 143, the stereochemistry is modulates as we saw in the previous chapter, incorporation of asymmetric carbons deemed to be fruitful in a better reach and interaction with the amino acids in the binding pocket of CGRP receptor. Therefore, stereochemistry is fixed according to the molecules prioritized based on the docking results. This fragment 143 with the desired stereochemistry on the piperidinic moiety can be obtained from the desired ketone intermediate 144 by using the Functional Group Interconversion (FGI) principle. The intermediate 144 is possible from a reaction between key synthons A and B.

Synthon A

To obtain the synthon A, the retro synthetic scheme proposed is as follows;

Synthon B

To obtain synthon B, the retro synthetic scheme designed is as follows; The proposed retro synthetic scheme shows that the synthon B can be derived from a simple reductive amination of the commercially available 2-bromobenzaldehyde and amine B' with desired substituants [56,57]. This substituent can either be a phenyl or CF 3 based on the desired substitution on the final potential hit molecule.

Imperative points derived from this synthetic strategy

 The synthesis of final potential hit is achieved by 'convergent synthesis'

A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME

In the previous section, the synthetic scheme to obtain the final hit molecule 141 has been successfully designed. In the current segment, the application of the designed scheme will be discussed step-by-step in detail.

Synthon A

The piperidine synthon A with substitution at 2 and 6, a methyl and an ester/acid functionality will be obtained in 5 steps. In the first step, the aspartic acid will be esterified followed by the N-protection. This N-protected compound will participate in nucleophilic substitution reaction with phosphonate followed by Horner-Wadsworth-Emmons reaction to give α, β-unsaturated ketone. The last step involves a 6-Endo-Trig-cyclization to form piperidine moiety. Now let's focus on each step in facet.

Synthesis of 6-methyl-4-oxopiperidine-2-carboxylic acid from aspartic acid

We have used the same methodology for both D-and L-aspartic acid until we reach the synthon A. Hence throughout the course of this discussion, we will exemplify the steps using D-aspartic acid.

Fischer-Speier esterification of aspartic acid

The first step involves the esterification of commercially available D-aspartic acid. We used the classical Fischer-Speier esterification method [58] because of its simple yet effective esterification to recover the desired product.

Protection of amine group in dimethyl ester of aspartic acid

In the literature, to orient the selectivity during nucleophilic attack of the dialkylmethyl phosphonate, there are two described procedures [59]. The ester 145 from previous step can be subjected to an N-protection by using rather bulky protecting groups like trityl chloride or di-tert-Butyl carbamate, which helps in regioselective reaction during the nucleophilic substitution step to access phosphonate 149 and thus prevents the side product formation. The following minimum energy conformers obtained through energy minimization molecular mechanics done using ChemDraw ® ultra shows that a single tert-Butyl carbamate protection results in exposure of both esters and hence it lacks in the regioselectivity during the next step. On the other hand, a di-tert-Butyl carbamate protected amine exposes only one ester leaving the other ester under the influence of steric hindrance [60].

Synthesis of β-keto phosphonate ester

Regioselective reaction of 146 with 4.5 equiv each of dimethyl methylphosphonate and n-BuLi (which subsequently gives 4.5 equiv of lithium anion of dimethyl methylphosphonate) gave exclusively 147 with a yield of 77%.

Scheme 46.showing preparation of β -keto phosphonate ester

When we tried the same protocol with diethyl methyl phosphonate as the anion source, the reaction gave the product 148 but relatively in poorer yield (28%) and purification was much difficult as described in the literature [59].

Synthesis of (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate

Thus formed β-keto phosphonate ester 147 has been subjected to Horner-Wadsworth-Emmons (HWE) reaction in a gentle condition, which was optimized in the laboratory itself to get E form of α, β unsaturated ketone 149 in majority [61].

Stereoselectivity of HWE

HWE reaction predominantly gives E-alkene. Systemic study conducted by Thompson

and Heathcock [62] showed that the following are key factors determining the formation of E alkene.

 Li > Na > K salts during the reaction.

 Increased steric hinderance or bulkiness of the aldehyde used.

 Higher reaction temperatures (23 °C over -78 °C).

 Preferring 1, 2-dimethoxyethane over tetrahydofuran.

Along with the above factors, another study showed that bulky phosphonate and bulky electron withdrawing groups results in E alkene over Z form.

Currently optimized condition gave better yields compared to other methods like Masamune and Roush or Rathke's methodology [63,64].

There are other modifications for HWE reaction which may result in formation of Z alkene predominantly. Still and Gennari [65] identified a methodology which produces Z:E in a ratio of 12:1 to 50:1. Use of phosphonates with electron withdrawing groups like trifluoroethyl along with strong bases like Potassium bis(trimethylsilyl)amide gave exclusively Z alkene.

Mechanism involved in HWE to give E-alkene

HWE reaction has many transition states during its course of formation of alkene.

Deprotonation of phosphonate by a base to produce carbanion of phosphonate accounts for the first step in HWE. In the next stage, nucleophilic addition of carbanion onto the carbonyl group of aldehyde results in the intermediates x and y. This step as considered as rate limiting step. Finally there is an elimination step which results in the formation of alkenes. The scheme of mechanism is as follows; Michael intramolecular reaction serves as the rationale for the synthesis of 6 from 5a.

As reported by Sutherland et al [53,54], this step involves 6-endo-trig cyclization of (R,E)methyl 4-oxo-2-(tritylamino)hept-5-enoate in accordance to Baldwin's rules [55]. They used this methodology for similar substrate as 149 but with different aliphatic substitutions like As the conditions they followed and their diastereomeric ratios were excellent, we went after them with similar conditions. Following the procedure stated above, there was multiplet. On the other hand, protons H 5e and H 3e shows similar splitting and coupling constants. The splitting goes like H 5e -H 5a which are geminal protons and hence shows J = 13.3 Hz, H 5e -H 3e which are characteristic W pattern splitting and shows J = 2.7 Hz and finally H 5e -H 6a which are vicinal protons and thus shows J = 2.7 Hz. The same kind of pattern applies for H 3e also, considering the fact that both H 5e and H 3e are having similar environment of protons. H 3a and H 5a also show similarity due to the same vicinity. The splitting pattern goes like H 5a -H 2a which are vicinal protons and hence shows, a transdiaxial splitting J = 13.2 Hz, H 5a -H 5e which are geminal protons and hence shows J = 13.2 Hz. Whereas, H 2a shows splitting pattern as H 2a -H 3a which are vicinal protons and hence shows, a transdiaxial splitting J = 12.2 Hz and H 2a -H 3e showing J = 2.8 Hz.

(2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (trans)

Trans piperidine shows mostly similar kind of splitting pattern and coupling constants as that of cis isomer. Protons like H 6 , H 5a , H 5e are absolutely in accordance with that of cis piperidine's coupling constants. But, the characteristic coupling constant was shown by H 2 proton. In case of trans, there is no transdiaxial splitting which we observed in case of cis (J 12.2 Hz, H 2a -H 3a ). Instead, there is appearance of transequatorial splitting between H 2e -H 3e .

The splitting pattern is as follows: From here, a phenomenon called rotamers has started posing hard time in the structural analysis of the formed compounds.

Rotamer: These are defined as isomers which interconvert into another form by rotation among its single bonds. They are in general called as conformational isomers or conformers and to be precise, rotamers [69]. If R' was a bulky substitution (i.e., tert.-Bu, Ph, O-Tol, etc.), the cis rotamer is dominant in deuterated chloroform during the NMR analysis [70].

For tertiary amides, the case is a bit staggering as there are also two forms, the second form being formed due to the lower apparent symmetry [71]. It can be explained as below;

H N CH 3 O CH 3 H N CH 3 O CH 3

Figure 63.showing amide rotamers for tertiary amides

The two forms as shown and their ratio in NMR depend completely on the solvent system as well as the temperature. Rotations along these single bonds are constrained by a 'rotational energy barrier' which ought to be crossed to interconvert one conformer to the other. Conformational isomerism takes place when the rotation around the single bond is relatively unhindered i.e with no bulky groups around. That is, the energy barrier should be less enough for the interconversion [72]. So, from the above 13 C NMR, we can make out that there are two carbonyl carbons coexisting at room temperature.

Compound

Hence, with this step, we have successfully reached the synthon A, an acid synthon. Now for obtaining a mono-peptide, we carried an amide bond formation with two different amines as discussed in the next segment.

Synthon B

Synthon II is an aryl amine synthon. As stated in the previous chapter, the amine synthon which later gets incorporated in the final molecule plays an important role for interactions. Depending on the amine used, they provide a hydrophobic interaction with tryptophan 74 of RAMP-1 or in some cases when there are fluorine substitution results in better bioavailability thus improving pharmacokinetic properties.

A generalized reductive amination has been followed to synthesize this fragment which was retrieved from the literature [56,57]. The reductive amination condition followed was depicted below: With this, two key synthons A & B were successfully optimized and synthesized in large scale. The next step involves an amide bond formation to obtain a monopeptide.

Precedent work and optimizations:

Initially, an exclusive study on the design and optimization of this step using another racemic substrate with a similar core piperidine ring but without analogous substituants (no methyl group on 6 th position) was done within the team of Prof. Yves Troin and Dr. Isabelle

Thomas.

The synthesis of 144 like synthon was first attempted by following the conditions proposed by Ma and co-workers [73]. They achieved the condensation of L-proline (similar to When these conditions were applied into pipecolic acid to verify whether they could be feasible to get a molecule similar to synthon 144, It was observed on GC-MS that although there was a small peak corresponding to the desired product, most of amine 160 did not react, and found being de-brominated, which suggested that the Ullmann cross-coupling might have worked well but the peptide bond formation was not achieved whatsoever, as shown in scheme 54. Neither addition of DCC or L-proline as ligand was able to push the reaction to improve the yield of the desired product. is the other way around, peptide bond formation to achieve E followed by intramolecular cyclization, which could be promoted by palladium or copper catalysis. The reactions was attempted to be carried out using palladium mediated catalysis. 10 mol% of Pd 2 (dba) 3 and BINAP as ligand were employed [77]. The reaction was performed in dry DMF at 110°C and again no trace of the desired product was observed. Of all the conditions, the Pd(OAc) 2 with Xantphos as ligand in distilled toluene reflux gave the desired compound F in the 80% of yield. Thus this deemed to be the useful catalytic system which can be applied to substrates like ours. To be noted, the reaction gave the desired product in non-polar solvent but not in polar solvents.

Application of the previously optimized conditions

Thus, learning the dos and don'ts from the optimized conditions from the past, we initially applied the same strategy and conditions for similar but not the same substrate we used. The initial design of the future steps is as follows; Following the above depicted scheme, the final benzodiazepanone moiety can be achieved from the monopeptide F which has been obtained using an acid synthon A and amine synthon B.

Peptide coupling for amide bond formation:

As per the previously optimized condition, we initially used the coupling agent N,N'dicyclohexylcarbodiimide (DCC) to obtain the desired monopeptide as depicted in the following scheme. We started the optimization with DCC as coupling agent not just because of the previous optimizations on racemic mixture, but also it is one of the coupling agent in market claimed to decrease the epimerization of formed peptides, as in the current case we used asymmetric substrates [78]. Unlike in the optimization stage, when we tried on our synthon A as the substrate, there was no desired product formation. Instead, there was only formation of side product during the DCC coupling reaction (Scheme 62). Even on repeating this reaction with addition of catalytic amounts of DMAP, to fasten-up the reaction process, the results were still not satisfactory.

Mechanism of DCC mediated amide bond formation

We investigated what precisely was the side product formed by studying the mechanistic approach of DCC mediated amide bond formation. The steps involved in the DCC coupling include an initial deprotonation of the N-protected pipecolic acid substrate A by DCC followed by a nucleophilic attack of the carboxylate onto the imide which consequently gives an O-acylurea. This O-acylurea is formed as an inevitable intermediate during the course of imide coupling agent mediated amide bond formation. Thus formed Oacylurea, can form 3 subsequent products as follows  On addition of amine, the desired mono-peptide can be formed due to the nucleophilic attack of the amine onto the activated N-protected pipecolic acid-DCC complex.

 On addition of one more equivalent of N-protected pipecolic acid, there occurs a formation of anhydride from O-acylurea. This anhydride is highly reactive which immediately reacts when an amine is added. This particular step finally gives a mono-peptide as well as one equivalent of the N-protected pipecolic acid. But mostly, no one follows this step since expensive N-protected acid substrate addition is not worthy.

All these steps are depicted in the following scheme. This can be confirmed by the peaks in the range of 24-32 ppm and these peaks being secondary/quaternary carbons. If there is formation of desired product, this area (region of alicyclics/aliphatics) can't produce these many peaks. For further confirmation, the tertiary carbons and the carbonyl carbons match perfectly with the undesired N-acylurea.

This may be attributed due to following reasons  Conformation achieved by the 2, 6 disubstituted pipecolic acid.

 The bulkiness of the amine synthon due to two aromatic rings.

 The electronic properties of the amine synthon as there are two aromatic rings which alters the nucleophilicity of the amine.

If the bulkiness and nucleophilicity of amine are sole reasons, this type of reaction when performed using 2-substituted pipecolic acid (as shown in scheme 58) shouldn't work either. As it worked with 2-substituted pipecolic acid but not for 2, 6 disubstituted pipecolic acid, it shows the conformation achieved by this substrate might have been the reason which posed issues during the coupling reaction with this kind of amine (may be the secondary reason).

After failing to synthesize the mono-peptide using DCC mediated coupling, we tried many other coupling agents as tabulated below. We tried a series of coupling agents based on literature [79]. We tried to optimize the condition suitable for both small scale and gram scale synthesis. The coupling agents we employed were as follows (Blue-works for both amines, Red-works for benzyl amine alone)

Coupling agent Additive

Coupling agents like EDC, HBTU, and COMU ® showed no desired product formation whatsoever. Thus, we tried to put forth the discussion only on the coupling agents which gave the desired mono-peptide F from now on.

Propane Phosphonic Acid Anhydride-T3P ®

T3P ® is chemically propane phosphonic acid anhydride developed by Euticals pharmaceuticals [80]. This coupling agent is prominent for being one of the best coupling agents available in the market due to its low epimerization (1.8%), high reaction selectivity and purity along with ease of handling [80] [START_REF] Garcia | For a brief review of the reagent[END_REF].

We applied T3P ® to synthesize the desired mono-peptide with the two amines 160 and 161. The reaction condition followed is as follows Even though the yields were not good, this coupling agent worked for both the amine substrates we used. To make a point, the same reaction when carried in DMF didn't forward and gave the starting materials as such. The mechanism with which it proceeds is as follows The reaction proceeds by an initial deprotonation by a base to form a carboxylate which attacks the cyclic propane phosphonic acid anhydride. This attack opens up the T3P ® and forms a highly activated group on the acid substrate. A final nucleophilic attack by the amine B results in yielding desired amide G with side product which can be easily washed-off through water workup. Thus this reaction gives relatively clean reaction mixture at the end of the reaction as the byproduct formed is of phosphoric acid which is thoroughly soluble in water. This reaction worked well when we used T3P ® /DCM 50 wt % solution. Whereas, when we tried the same reaction in T3P ® /DMF 50 wt % solution, there was no desired product but the starting materials were recovered as such.

As the yield of the reaction when used with T3P ® was not good, we tried another organophosphorous coupling agent, DEPBT.

3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one-DEPBT

DEPBT is an organophosphorous coupling agent belonging to the family of benzotriazine class of coupling agents [81]. It is another coupling agent which showed the best results for resisting the racemization of substrate as well as formed amides during the coupling reactions [82]. Hence, we applied DEPBT for the synthesis of our mono-peptide.

With the condition retrieved from the literature, we have applied the same on the synthesis of our mono-peptides. The synthetic condition followed was With DEPBT as coupling agent, we were able to synthesize the desired mono-peptide when the amine substrate was benzyl with an excellent yield. Whereas, if the amine substrate was CF 3 , the reaction wasn't going forward and the starting materials were recovered as such.

The reaction proceeds in a similar fashion as that of other benzotriazine class of coupling

agents. An initial deprotonation of acid substrate follows a nucleophilic attack of the carboxylate onto the phosphoryl group of DEPBT which finally forms an activated benzotriazine-acid substrate complex. This activated complex, on nucleophilic attack of amine B onto it results in the desired mono-peptide F. The DEPBT mediated coupling worked well with benzyl amine substrate and showed no product formation for CF 3 amine. Even in protecting groups, it showed partiality with ditert-Butyl carbamate giving major yield of 86% and benzyl carbamate giving 72%.

Note:

The analysis of the synthesized compounds was done with the help of Electrospray ionization Mass Spectroscopy (ESI-MS) from now on because of the problem posed during the interpretation of NMR spectra due to the occurrence of 4 rotamers.

Thus formed mono-peptides have been forwarded to the next step, deprotection of the PGs to facilitate a C-N cross coupling reaction.

Deprotection of mono-peptides

The mono-peptides achieved in the previous step have been subjected to deprotection of the protecting groups in order to carry out a C-N cross coupling in the future steps. Well documented and mostly used methodologies have been applied to obtain the deprotection of tert-Butyl carbamate and benzyl carbamate protecting groups [67].

For deprotection of both the protecting groups, we used conditions from literature which are used on similar amide substrates as these kinds of substrates are highly prone for hydrolysis in presence of acid/base along with small amount of water.

Deprotection of tert-Butyl carbamate

The deprotection of tert-Butyl carbamate was optimized earlier with the help of trifluoroacetic acid in dichloromethane with the help of small amount of water using racemic substrate without any substitution on 6 th position and cyclic acetal protection on 4 th position.

This method was followed with the similar condition as reported and optimized previously as below Following the same condition which has been optimized, we weren't able to reproduce the deprotection of tert-Butyl carbamate moiety. This might be due to two reasons;

 The absence of ketal at 4 th position as this may change the conformation of the piperidine chair.

 The substitution at 6 th position by methyl group which may be posing either a sterical hindrance for the attack or changing the conformation from that of the already optimized substrate.

The substitution of ketal at 4 th position changes the entire conformation of the piperidine has been studied by using 2, 6 cis piperidine ester. Thus, this perhaps may be one of the reasons that the deprotection of tert-Butyl carbamate from piperidine with cycle acetal in previously optimized condition was working and for ketone due to the conformational strain, the deprotection may not be going forward.

Hence we tried to reprotect the ketone to ketal. One of the easiest methods to obtain a methoxy ketal is to do Fischer-Speier esterification using thionyl chloride. But we can't follow this due to the presence of hydrolysable amide bond. Hence we tried other method retrieved from literature as follows [83] 178 Thus, due to the in-applicability of other harsh methods, we applied the above said condition in para toluene sulfonic acid with 1,3 propanediol with dichloromethane as solvent which gave no protection at all.

In another study, which will be discussed in family 3 of the same chapter, we showed that the 2,6 substituted piperidines are formed from a tert-Butyl carbamate α, β-unsaturated ketones via an intermediate ketone to methoxy ketal followed by cyclization into piperidine and finally deprotection of tert-Butyl carbamate to give a free amine by using p-TsOH in trimethylorthoformate [84,85].

Hence, this also confirms that the deprotection of tert-Butyl carbamate was done after the protection of ketone to ketal. Thus, we applied the same methodology here with the conditions as below With this step, we not just monitored the proceedings of the reaction but also, we wanted to know the effect of substitution on 4 th position during the C-N cross coupling reaction.

Deprotection of the benzylcarbamate on the piperidine

Benzyl carbamate protecting group can be deprotected by many conditions like saponification, acid/base hydrolysis, catalytic hydrogenation etc [67]. But the problem face again was due to the presence of amide bond in our structure which will be hydrolysed. On a simple note, we can also do a hydrogenation, but due to the presence of bromide on the aromatic ring, which gets debrominated during the hydrogenation we can't carry this type of deprotection as well.

Hence we chose trimethylsilyl iodide which in the literature has been applied for the similar kind of substrates [67,86] and we handled carefully to prevent hydrolysis. The condition we followed was as below Hence forth, we were able to protect the ketone in facile condition but we can't deprotect the benzyl carbamate. We took thus formed compound and engaged in deprotection using the TMSI with similar conditions as follows However, after the reaction, we were able to deprotect the ketal to form a ketone but we weren't able to deprotect the benzyl carbamate. Hence, we came to the same compound where we started i.e. a ketone.

Thus obtained product can't be cyclized due to the protection of amine and hence, it was engaged to synthesize the subclass of family 1 with an amide fragment as discussed earlier.

Whereas the compounds which were deprotected from tert-Butyl carbamate has been engaged in a C-N cross coupling reaction.

C-N cross coupling reaction

Thus formed mono-peptides from previous step have been engaged into a cyclization reaction by forming a C-N bond. From the optimizations done within the laboratory as presented in previous section, we chose Buchwald-Hartwig cross coupling reaction to be the principle in forming the C-N bond. Hence, we took the same conditions as optimized previously and applied onto our amide synthon C. The reaction was followed as below When we used the same condition developed initially, we weren't able to forward the reaction to get our desired product. Even on doubling the catalytic load, there was no formation of desired product. Thus, keeping the solvent constant, we optimized the reaction using another catalytic system as follows; As we can see, the distance between the aromatic halogen and amine is too long and this might affect the formation of C-N bond. And so, we weren't able to retrieve the cyclized form of trans.

Meanwhile, the rotamers which were posing the problem since the protection of amine and formation of peptide bond has totally gone astray once the cyclization has been done. As a result, a spotless and uncluttered spectrum has been recorded for this cyclized compound.

Figure 69.showing the disappearance of rotamers on cyclizing

Thus the cyclized as well as the N-protected mono-peptides has been forwarded to the next step where the deprotection of ketal to ketone has been performed.

Deprotection of ketal

Thus formed amides and a benzodiazepanone from previous reactions have to be finally deprotected from a ketal to a ketone at 4 th position. As presented in previous sections, this has been optimized within the laboratory and thus, we applied this condition to obtain the desired ketones. The generalized reaction is as follows We applied this condition on many substrates and their deprotection went smoothly with good yields. These deprotected ketones have been forwarded to the next step, where the Functional Group Interconversion (FGI) to an amine using a series of modulations. This FGI part has been conferred after the discussion of family 3 in general.

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III. STRATEGY FOR THE SYNTHESIS OF FAMILY 3

Candidate The intermediate 197 of each subclass can be obtained by using intra or intermolecular cross-coupling reaction to form C-N bond from the desired synthons II of respective subclass.

The synthons III of all subclasses can be obtained without any transformation from the corresponding piperidine III of each subclass or after a reduction or N-deprotection or coupling reaction. The various piperidine synthons III as presented during the bibliographic study will be synthesized using Aza-Michael intramolecular reaction [88,89,90]. Similar to family 1, the key intermediate reaction 'Horner-Wadsworth-Emmons' preceding with phosphonate insertion into the substrate was applied to transform 204 to V and finally to IV [91,92]. Synthon VI is mostly available commercially and sometimes synthesized by nucleophilic substitution reaction. Finally, synthon 204 is obtained by Aza-Michael insertion of Davies amine onto the commercially available methyl crotonate 205 [93]. The orientation/configuration of asymmetric carbon will be fixed at the first step with the help of Davies amine. The retro-synthetic design for key synthon III is crucial. The attribution of both stereocenters and the desired substituants are done during the synthesis of synthon III.

Synthon III is mostly kept constant and obtained in similar way for all the three subclasses of family 3.

Imperative points derived from this synthetic strategy

 The synthesis of final potential hit is achieved by 'convergent synthesis' same as we saw earlier in family 1. Above derived strategies for the synthesis of family 3 molecules are applied to obtain them with desired stereochemistry and substituants.

A. APPLICATION OF THE DESIGNED SYNTHETIC SCHEME

In previous discussion, the synthetic scheme to obtain the final hit molecule 194 has been successfully designed. In the current segment, the application of the designed scheme will be discussed step-by-step in detail.

Piperidine Synthon III

The synthon III has been obtained using asymmetric synthesis from commercially available starting material, methyl crotonate. The methodology has been designed and developed within the laboratory by using Michael reaction [90]. The first step in synthesis of synthon III is formation of (S)-methyl 3-(benzyl((R)-1-phenylethyl)amino)butanoate.

For all the three subclasses of family 3, the synthetic methodology remains alike until synthon III formation.

Synthesis of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate

The synthesis of family 3 molecules begins with this step which involves formation of (S)-methyl 3-(benzyl((R)-1-phenylethyl)amino)butanoate. This step results in attribution of a stereocenter at β position. Among the available methods in the literature [95,96], the following method is most facile, takes bare minimum steps to obtain the desired product and gives highest amount of diastereomeric ratio (94%).

Scheme 87.showing Aza-Michael addition of Davies amine to α,β-unsaturated ester

This step involves Aza-Michael reaction, where a conjugate addition of enantiopure lithium (S)-N-benzyl-1-phenylethanamine to an α,β-unsaturated ester i.e. methyl crotonate was done at -78 °C using anhydrous conditions to give compound 204.

The diastereomeric ratio is normally above 94% and this can be accredited to the structural conformation of Davies amine. The two phenyl groups in Davies amine attain a minimum energy conformation as a sandwiched model or a butterfly model where both of them lie parallel to each other. This is also called as π-stacking [95]. The nucleophilic attack of lone pair of electron on nitrogen onto the double bond of methyl crotonate can take place in two ways, above the plane and below the plane of the α,β-unsaturated ester (Figure 70). In route A, the compound 204 was engaged into hydrogenation to give 206 which was followed by protecting the amine functionality by suitable protecting group. In final step, phosphonation reaction was followed to obtain compound V. On contrary, in route B, compound 204 was allowed to undergo phosphonation as the fundamental step to obtain 207 and later applied with hydrogenation to recover a free amine in salt form 209. But the hydrogenation reaction from 207 gave 208 instead of 209 due to β-elimination and subsequent reduction of alkene. Even though variety of hydrogenation conditions like Pearlman's catalyst with 60 pound per square inch (PSI) in methanol or Pd/C in methanol and reflux with ammonium formate as a hydrogen donor, there occurs formation of 208 [97]. To present in general, whatsoever the condition is, the final compound ultimately deemed to be 208.

As the desired product V from route B can't be achieved, we have nominated route A as the prime way of synthesis of compound V. Route A starts with the N-debenzylation of compound 204 as discussed in the next section.

Synthesis of (S)-4-methoxy-4-oxobutan-2-aminium acetate

Compound 204 obtained in the previous step was engaged in hydrogenation reaction using Pearlman's catalyst, Pd(OH) 2 /C [98,99]. Among the available methods, the proposed method deemed to be more facile and gives desired product 211 with least by products (mono bezylated amine) [56].

showing hydrogenation of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate

Not just the catalyst, but also solvent and acid used plays a crucial role in obtaining the desired product. Methanol and ethanol are considered to be the best solvent to give compound 211 faster while other solvents increase the time of product formation [98]. Even the pressure employed plays determining factor in deriving the compound 211 without any side products [99].

We followed the heterogeneous catalytic method for the N-debenzylation of compound 204 using Parr apparatus. As obtained from the literature, we used Pd(OH) 2 as catalyst with methanol being the solvent and carried the reaction at 60 PSI pressure to derive compound 211 [100]. This method in general gives free amine but due to the presence of acetic acid, the acetic acid salt of amine 211 is obtained.

Thus obtained compound 211 without any purification was engaged in next step where the free amines have been protected using various protecting groups.

Protection of (S)-3-methoxy-3-oxobutan-1-aminium acetate

In order to prevent the side product formation in future steps, the amine functionality of compound 211 has been protected. The rationale for using protecting groups (PGs) depends We followed a generalized method for protection of compound 211 using carbamate PGs. This method has been retrieved from literature [101, 102, 103] which uses a facile condition as stated in the procedure. First time since the beginning of reaction, purification has been carried out and the yield was quantified. The overall percentage yields in all the cases were ranging in and around 50%. On the other hand, we used some PG like ethyl carbamate which is less bulky to overcome steric hindrance in further steps. Overall, the desired compounds were obtained in and around 52-58% in 6-8 h.

We used various PGs to study a range of factors affecting Michael cyclization reaction which we applied during later stages of the synthesis. Thus obtained N-protected amines VII were subjected to phosphonation reaction in the next stage.

Synthesis of ketophosphonates

A generalized phophonation condition has been applied using the guidelines from literature to give compound VIII [85]. Unlike in family 1, use of diethyl methylphosphonate for synthesizing ketophosphonates V didn't pose any obstacles during purification step. Excess of diethyl methylphosphonate used in the reaction can be easily removed by distillation at reduced pressures followed by column chromatography yields ketophosphonates in the range of 48-65%.

Similar to that of family 1, the synthesized ketophosphonates V has been used as substrates for the key intermediate Horner-Wadsworth-Emmons reaction.

Synthesis of α,β-unsaturated ketones: application of Horner-Wadsworth-

Emmons condition

The ketophosphonates synthesized in previous step has been treated with different aldehydes using Horner-Wadsworth-Emmons (HWE) reaction to get various α,β-unsaturated ketones with desired substituants. These substituants are the result of chosen aldehyde during the reaction. Most of the aldehydes we used were commercially available apart from some of them. However, they are typically aromatic aldehydes and can be synthesized using nucleophilic substitution reaction as followed [105].

i. Synthesis of biaryl aldehydes

Along with the commercially available aldehydes used to synthesize α,β-unsaturated ketones, we used some other biaryl aldehydes. These biaryl aldehydes were synthesized using facile nucleophilic substitution reaction on various substrates but from similar reagents and methodology retrieved from the literature [106].

.showing different biaryl aldehydes with their yield

Using the nucleophilic substitution reaction, compounds 218, 219 were synthesized in good yields.

Whereas in the case of 220, the yield is mere 20% in 1 H NMR integration and making the situation worse, on purification, perhaps due to the instability of aldehyde 220 we weren't able to isolate the desired product. Hence, we followed route B instead of route A. For route B, commercially available 2-nitrobenzaldehyde was used during HWE which can be followed by reduction from nitro to amine. The final tetracyclic skeleton 198 was achieved by a C-N cross coupling reaction. Hence, this route was found to be more feasible and thus employed to obtain 198 instead of route A.

Apart from the above discussed aldehydes, we used other aldehydes based on the subclass which we have to synthesize. They include 2-Bromobenzaldehyde, 2-Fluoro-5nitrobenzaldehyde. And thus, we retrieved all the required starting material for key reaction HWE.

ii. Horner-Wadsworth-Emmons

This key intermediate step will be determining factor as the aldehyde used during the course of this reaction results as substituants in the final molecule. We followed similar methodology as we discussed previously for family 1. We used facile and workup-friendly The general applicability of the condition we used for HWE has been studied with various aldehydes. As we can see, the reaction times and the yields are good for all the series of phosphonates we used. Hence, the retrieved compound IV was engaged in the next step i.e.

Aza-Michael intramolecular cyclization.

Aza-Michael intramolecular cyclization

The series synthesized during HWE was later subjected to Aza-Michael intramolecular cyclization. As shown during the bibliographical survey on 2,6 disubstituted piperidones, Aza-Michael reaction is prime choice for synthesis of 2,6 disubstituted piperidones with intramolecular type cyclization in chiral form.

We developed a simple but efficient reaction condition for Aza-Michael intramolecular reaction to obtain piperidines [85]. The reaction was carried in presence of ptoluenesulfonic acid, trimethylorthoformate and alcohol as shown below.  The quantity of the acid source used has also deemed to be important factor. The amount of the acid influence the kinetics of the reaction as well the cis to trans ratio of the formed piperidine. Thus, keeping in mind the above factors we designed our synthesis accordingly. For the synthesis of our piperidines, we used a generalized condition which we optimized to reach the 2,6-cis piperidine conformation as major compound. An initial addition of 0.2 equivalents of pTsOH with 5 to 10 equivalents of trimethylorthoformate at room temperature was followed. The methoxy substituants of the ketal were generated in situ by decomposition of the trimethylorthoformate in acidic conditions. This ketal was chosen in order to avoid a high steric hindrance due to the substituants in the ortho position of the aromatic ring.

Scheme 97.showing Michael intramolecular reaction in general context

We applied this on variety of E-alkenes for studying the applicability of the developed reaction. In general, this reaction gave satisfactory yields of desired product with cis/trans ratio fluctuating.

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Table 34.showing application of Michael intramolecular reaction for ethyl and benzyl carbamate PGs

As presented earlier, we tried on variety of substrates and studied the effect of substituants on the yield and ratios of isomers formed. When we tried the PG to be ethyl and benzyl carbamate, the reaction forwards by the protection of ketone to ketal and cyclization mediated by 0.2 equivalents of p-TsOH to form piperidines. Whereas, when the PG is tert-Butyl carbamate, there occurs protection of ketone to ketal and cyclization mediated by 0.2 equivalents of p-TsOH to form piperidine and further addition of 1 equivalent of p-TsOH deprotects the tert-Butyl carbamate in situ. This is attributed to the acid mediated deprotection of the tert-Butyl carbamate and for the other PGs we used, the deprotection condition is not the same. Another point to be noted that, when the piperidine formed is N-carbamate, the cis and trans can't be isolated using column chromatography. Hence, we used 1 H NMR to integrate and quantify the ratios of cis/trans.

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Henceforth, the piperidines formed using Michael intramolecular reaction were sorted and carried to the next step where the tetracyclic moieties were formed.

Formation of tetracyclic moiety

Thus formed piperidines in Aza-Michael intramolecular cyclization were categorized according to their applicability in each subclass of family 3. To brush up, we already showed that the family 3 was further classified into three subclasses. Their structures are as follows This family of molecules was classified into subclasses based on heteroatomic substitution in their core moiety. They were designed to study the hydrogen bond donor, acceptor and a homologated hydrogen bond donor effect towards the receptor interaction and consequently its biological activity.

Thus, to avoid the perplexity over the synthesis of each of them, from now on we will deal with individual subclass of family 3 until we reach core tetracyclic moiety.

Subclass A

Subclass A represents tetracyclic core moiety with a hydrogen bond donor (NH, in our case) as the heteroatomic substitution in it. Its synthetic design was as follows

Scheme 98.showing multiple approaches to reach subclass A

Route A: It starts with the reduction of compound 226 which was followed by a C-N cross coupling reaction. This cross coupling was done by using 1,2 dihalogenated benzene (in our case; 1,2 dibromobenzene) and this mono cross coupled compound was later deprotected from ethyl carbamate which exposes free amine group. The last step is to follow-up another C-N cross coupling to give desired compound. Overall, it takes 4 steps to retrieve the desired product.

Route B:

It starts with the deprotection of ethyl carbamate from compound 226 which was followed by reduction of nitro functionality to amine. To finish, the concluding step is a double C-N cross coupling reaction to form a cycle. Overall, it takes 3 steps to retrieve the desired product.

Route C:

Route C a : It starts with a piperidine moiety 239 with free amine functionality and a nitro group. Thus, we start from reduction of nitro group to amine Route C b : It starts with a piperidine moiety 237 with Cbz protection and a nitro group.

Hydrogenation was followed to do an in situ deprotection of Cbz as well as reduction of nitro to amine.

Both route C a & C b were later followed by a similar double C-N cross coupling reaction as discussed in route B. Overall, it takes just 2 steps to obtain the desired product.

Hence, amongst all the routes, route C was found to be more ergonomic and feasible which persuaded us to follow this route to obtain subclass A.

i. Application of designed route C

The designed route C a was applied for synthesizing the subclass of family Pd 2 (dba) 3 (5) BINAP (7.5) NaOtBu (1.2) 12 A(55), B+C ( 40) Toluene (110°C)

Pd 2 (dba) 3 (5) BINAP ( 15) NaOtBu (1.2) 24 A(60), B+C (35) Mesitylene (110°C) Pd(dba) 2 (10) BINAP ( 15) NaOtBu (1.2) 72 A(65), B+C( 30) Toluene (110°C)

Pd(dba) 2 (10) BINAP ( 15) NaOtBu (1.2) 24 A (quantitative) Toluene sealed tube (130°C)

Pd 2 (dba) 3 (5) BINAP ( 15) NaOtBu (1.2) 24 A (quantitative) Toluene sealed tube (130°C)

Pd 2 (dba) 3 (5) BINAP ( 15) NaOtBu (1.2) 24 A (quantitative) Dioxane sealed tube (120°C) As the results in table 37 clearly shows that using Bis(dibenzylideneacetone)palladium (Pd(dba) 2 ) catalyst with (2,2'-bis(diphenylphosphino)-1,1'-binaphthyl) (BINAP) gave the desired compound in quantitative yield. The reaction can be done in a polar or non polar solvent, and the best conditions were obtained when a sealed tube was used. Then to decrease the time of the reaction we wanted to optimize further in the future to try microwave condition and to use dioxane as a solvent for better activation.

The same condition was then applied on the piperidine 2,6-trans and it give exactly the same result in dioxane as the piperidine 2,6-cis. The yield of 80 % in toluene was attributed to the lower solubility of this diastereoisomer in this solvent.

On entries 2, 3 and 4 we came across side products B & C. To understand the whole mechanism of the cyclization, we wanted to know which of the two C-N cross couplings takes place first. Either the first C-N bond insertion was on the piperidine nitrogen or on the aniline.

Hence, we investigated on the piperidine 239 by trying the following method using the same catalytic system which gave us the desired product.

Scheme 102.showing investigation on first C-N cross coupling

After 24 hours, only 10 % (calculated through 1 H NMR integration) of desired compound X was observed. This concludes that the preferred formation of desired compound forwards in the following fashion. The first C-N bond formation takes place on the aniline moiety followed by the intramolecular C-N bond connection on the piperidine ring. This mechanism is similar to the formation of the benzodiazepanone family 1 ring. Merely no product was obtained if the first C-N bond was tried on the piperidine ring by intermolecular reaction.

This concludes that the formation of the 7 membered ring can be reached only by intramolecular C-N formation.

Then according to the above disclosed results, we can propose the following mechanism, in two cross coupling cycles to get the desired tetracyclic subclass A. In the first cycle, first step includes oxidative addition of 1,2 dibromobenzene to a Pd(0) species which will be followed by deprotonation, addition of amine to the oxidative addition complex and finally a reductive elimination to windup the first cycle to give a mono cross coupled product. The second cycle follows the same pattern but starts with the mono coupled product formed in the first cycle which finally ends up to give the tetracyclic subclass A. We tried to study the effect of 2, 6 cis and 2, 6 trans as described in the family 1. For this study, we used the following method In this cyclization, we were able to form either the desired product 199 or a secondary product 248 resulted due to beta-hydride elimination or catalyst decomposition as we showed in the catalytic cycle below as well as according to the results described in the literature. B Pd 2 (dba) 3 (5) BINAP ( 15 According this table, comparing the result between the bromide and iodide substrates, the cylization was observed only on the iodide substrate. The results also share the information that a lot of hydrogenated side product was generated. We weren't able to separate the 3 molecules (Starting material, cyclic, hydrogenated) by purification on a column chromatography as all the 3 products have the same R f on TLC. Then to precisely identify and confirm the hypothesis and to quantify using 1 H NMR & 13 C NMR, we had engaged the piperidine 202 in a hydrogenation reaction in presence of Pd(OH) 2 /C at 1 atmosphere of H 2 (scheme 108). As we saw in the earlier discussions, the aryl iodides form more stable complexes with the catalytic-ligand system and even if a reductive amination reaction takes place, it will be very slow. By this time, the side reaction called β-hydride elimination happens and results in de-halogenation of the substrate as seen in our case.

Contemplations on cross coupling reaction by Buchwald:

With the persistence of the problem created due to the β-hydride elimination, we did the review of literature to identify and sort-out the problem. Some of the key facts identified are as follows  There are no catalytic system which is universally applicable for all kinds of substrates  To sum it up, a simple guide for some commonly encountered problems with its

Inefficient reductive elimination

Use a ligand which gives faster reductive elimination

Table 40.Showing problems and how they can be solved according to Buchwald

Overall, Buchwald proposed that the ligand like Pd 2 (dba) 3 with bidentate ligand like BINAP, dppf, Xantphos with sodium tert-butoxide as the base and running the reaction in non-polar solvent, toluene might give desired product in good yields. Whereas, he also proposed that the reaction might not give the desired compound if the substrate is an aryl iodide and suggest that the reaction takes place if the monodentate ligands like Brettphos, XPhos and RuPhos are used for aryl iodides.

On this conclusion we decided to try the XPhos and Pd(OAc) 2 as the source of Palladium. We have also decided to investigate the influence of the concentration on the intramolecular reaction and the influence of the load of catalyst used. The results are reported below on the table 109. Reactions were performed on toluene, in sealed tube at 130 °C over a period of 72h and the base used was the NaOtBu or Cs 2 CO 3 . The first conclusion was to perform the reaction, as we did for the first subclass, in presence of bidentate ligand. The use of XPhos has not shown the formation of desired 7 member ring. The influence of the concentration was put in front in comparison with entries 2 and 4. More concentrated, the intermolecular palladium reaction following by the reductive animation was more efficient. The ratio of catalytic system also affects the formation of the desired product and unfavored the hydrogenated product. At present, we weren't able to optimize a perfect condition to obtain the desired product. The next trials will be changing the As a one last attempt, we tried another strategy based on the subclass A methodology.

In here, the corresponding piperidine 240 which possess a bromide group on the aromatic ring was used as a substrate to couple with the 1,2-bromophenol. The condition applied was toluene as solvent in presence of Pd 2 (dBba) 3 /BINAP as this deemed to be the best condition for the subclass A. But this also proved to be unfruitful. And hence, when, we tried to see if this type of cyclization works using 1,2 dibromoaniline which obviously gives the Subclass A, there was no product formation whatsoever. Hence, this shows that this type of cyclization can't be reached with our substrate.

Subclass C

Subclass C is an analogue of the Subclass A and it represents a tetracyclic core moiety with a hydrogen bond donor (OH) on the chain substituted to the nitrogen on the 7 membered ring. This chain will pave's a path to reach a hydrogen interaction with a Glycine on the RAMP-1 of the CGRP receptor. Another possible interaction with a meta substitution on one of the phenyl group to reach Asp 78 in the RAMP-1 part of the CGRP receptor was also identified during the molecular studies. Initially, we have prepared 300 and 303 molecule needed for our different routes.

Polarity : 2 Hydrogens bond interactions

Preparation of 303

Two different ways [35] were described in the literature to get the precursor of mono protected iodide 303. First method involves benzaldehyde, where the transformation of the carbonyl functions into the dioxolane Y 1 , which was subjected to react with the DIBAL-H to form the benzyloxyethanol Y 2 with an overall yield of 19 %. The second way investigated was the direct mono alkylation of the corresponding ethylene glycol in presence of benzyl bromide. According the yield, the second way was preferred. The mono alcohol was then transformed into the iodide derivative by reaction in presence of iodine and triphenyl phosphine under a "Wittig" type procedure with a yield of 53 % [122]. After condensation of the ethanolamine on phthalic anhydride, the desired phthalimide W 1 was obtained with a quantitative yield and O alkylated with the opted protecting group.

The free amine was then obtained after hydrazinolysis with an overall yield ranging around 30

% respectively for the O-MOM and O-Bn derivative.

Formation of the tetracore Route C: R2 = H

From the tetracore 198 and previous optimizations and through procedures already described in the literature on the 7 member ring skeleton [140]. We have tried to N-alkylate the aniline group, and the results are showed in the table 42.

Table 42.Showing the alkylation of subclass A with different conditions

As showed in the differents entries, various conditions were employed: N-alkylation by using a halogenated substrat or aldehyde subtract. None of the case, we could manage to transform the tetracore 200, certainly due to the low reactivity of the aniline fonction.

Formation of the tetracore Route A and B : R 2 = H, and NO 2

We shifted to route A and B as it involves the conditions of cross coupling reaction to form the 7-member ring 200 and as we optimized the conditions as presented earlier with a similar substrate, we thought of applying the same condition over here. Initially, precursor 297 and 298 were prepared to be engaged in the Buchwald cross coupling reaction.

The coupling reaction by using the optimized condition as described in the previous section to form a C-N bond with a yield of 88% has been followed by hydrogenation to free the amine using Pearlman's catalyst under 1 atm of H 2 304 was obtaining with an overall yield of 72%. In route B, the first step to prepare the precursor XX before cyclization involves a nucleophilic substitution (scheme X) reaction. The transformation was observed only when the halogen is a fluoride. Moreover, under those conditions, the nitrogen was deprotected in situ which had allowed us to reach the desired XX in one step with 65 % of yield. Three trails were conducted on this substrate, but this reaction wasn't going forward even on doubling the load and running the reaction for longer time. Only under microwave we were able to observe a mere transformation of 6% which was calculated through 1 H NMR.

We started optimizing these reactions at the end of my PhD and so, no further optimization methodologies were performed. In future, we would like to use another solvent like dioxane in microwave condition which could increase the energy to the catalytic system which might give us the desired product.

Deprotection of ketal

The ketals formed during the previous steps have been deprotected so that they can be transferred to the next step where the FGI takes place. The general deprotection methodology we followed was optimized within the team [refer 1 st chapter]. Hence, the method followed With this, we have successfully reached the ketone of both the subclass moieties, now let's proceed to the next step where the FGI occurs and finally these fragments will be converged to obtain the potential CGRP receptor antagonists.

IV. FUNCTIONAL GROUP INTERCONVERSION

The ketones obtained from ketal are then converted into amines using a series of Functional Group Interconversion (FGI) principle. We will present here a general structure representing all our synthesized molecules. The initial design to obtain the amines from ketones is as follows The ketones obtained from deprotection of ketals as we saw in previous chapter, can be reduced to obtain the alcohol. These alcohols will be chemically transformed into corresponding azide according to SN 2 process in one or two steps. At this stage, the stereocenter of the position 4 on the piperidine ring will be fixed in accordance with the docking calculations. Then, the azides will be reduced to form the desired amine fragment which would be advanced to the final compound on convergence with the privileged structure. This was our initial prototype to synthesize the amines from ketones. Now let us look into each individual steps in detail.

Reduction of ketone to alcohol

According the literature, differents reductives agent could be used to form alcohol from the corresponding ketone. This reduction can form a mixture of enantiomers as shown in scheme 121, and this selectivity is entirely dependent of the reducing agent used [123,124].

Scheme 121.Showing the reduction of piperidone using sodium borohydride and L-selectride

If the reducing agent is a bulky one like L-selectride, due to the 1,3-diaxial interaction, the hydride attack takes place from below the plane due to steric hindrance posed by the piperidone conformation. As a result, there is formation of axial alcohol in majority. On contrary, if the hydride source is small, the attack can occur from any side of the plane but it prefers the attack from above the plane to give the more stable equatorial alcohol. Hence, a similar strategy is followed for reduction of all the ketones and the yields in all the cases are quantitative. But the formed diastereoisomers can't be purified using column chromatography due to no difference in retardation factors. The diastereoisomer formation has been identified and confirmed by using 1 H NMR coupling constant (figure 76), and the ratios have been calculated using integration.

Thus, a general depiction of reduction from ketone to alcohol can be shown as follows

N°

Application of Mitsunobu reaction (direct S N 2) formation

The formed alcohols from previous steps were converted into azide directly with the help of Mitsunobu reaction [125,126]. Till now this condition has been tested for two of our substrates, one belonging to family 1 and the other for subclass A of family 3. In previous study on racemic structure belonging to family 1, the conditions we applied were as follows. This condition has been tried previously within the laboratory to convert the alcohols belonging to family 1 directly to an azide. But we weren't able to achieve this conversion even on using two variants of azodicarboxylate sources.

But when the same condition was applied for the synthesis of subclass A of family 3, the desired product was formed. The desired azide product thus formed without any purification was engaged into reduction from azide to amine to give the final fragment of subclass A belonging to family 3. The first step involves the Mitsunobu reaction mediated by DIAD to convert the alcohol to azide. This conversion is S N 2 type reaction and thus there is inversion of stereochemistry at 4 th position from equatorial to axial [127]. Thus formed desired product without any purification was engaged into the succeeding step to reduce the azide into amine.

The overall yield of this reaction was 62% after purification and occurs in a two-step process from the alcohol into the amine.

As the application of Mitsunobu failed for family 1, we have followed the previously designed step through the activation of alcohol for the formation of azides.

Activation of alcohol

The alcohols of family 1 obtained from ketones through reduction were then activated in order to obtain an intermediate with good leaving groups i.e. sulfonates [128]. In a previous study conducted within the laboratory, we tried two different sulfonates for the activation of alcohols as depicted below. Hence, the activation using mesylate gave the desired activated sulfonate whereas the tosylate activation gave no desired product [129]. Thus, we used this condition to apply for our synthesis. Thus activated alcohols using mesylate were converted into azides using a nucleophilic substitution reaction.

Nucleophilic substitution reaction to form azides and sunsequent amines

The active sulfonates formed from previous step were converted into corresponding azides using an S N 2 reaction, in presence of sodium azide in reflux of the DMF [130]. Due to this, an inversion of configuration happens at 4 th position. Thus, all the mesylates which were in equatorial position interchanges into axial azides. Thus formed azides, mostly gave clean reaction mixture. Hence, we didn't perform the purification step and converted the azides to amine in succeeding step by using Staudinger reaction. They were transformed into amines using triphenylphosphine mediated reduction reaction [131]. We chose this clause over the others due to the mild conditions followed during this step which won't remove the bromide on the aromatic ring. Along with this, triphenylphosphine mediated reduction reaction on these type of chiral substrates prevents racemization of starting materials as stated in the literature [132].

After optimized all the steps retrieved as well known in the literature [124, 131], we applied the best condition to an overall 3 steps process and purified after reduction into amine: An overall yield ranging from 62-76% was observed over a three-step process.

Thus formed amines are considered as the RAMP-1 fragments and carried onto the next step where it was coupled with a privileged structure using convergent synthetic methodology.

V. CONVERGENCE OF THE TWO FRAGMENTS

The amine fragments of the family 1& 3 as briefed in previous chapters have The results has been followed by another test using hot-plate method inorder to validate the results and even it showed that the fragment used in Telcagepant ® is more potent than that of the one used in Olcegepant ® with over 5 folds of difference. This part of the results and discussion has been put forwarded in the pharmacological tests of our antagonists in the next chapter.

These results made us to choose the fragment of Telcagepant ® and we carried further with our convergent synthesis to retrieve our antagonists. This 1-(Piperidin-4-yl)-1Himidazo[4,5-b]-pyridin-2(3H)-one fragment will be further called as 'privileged structure-PS'. Of all available routes designed and presented in figure 3, route B was reported to be the most feasible and yields the PS in minimal steps. Hence, we followed route B to synthesize compound 142. An optimization for reagents & reaction conditions used has been done by David K. L et al [134] showing the importance of using sodium triacetoxyborane as reducing agent and trifluoroacetic acid for reductive amination in ethyl acetate. All these factors plays important role in regioselectivity of reductive amination. When used the above said conditions, the ratio of desired product to that of side products was 56/1 with a reaction time decreasing from 24 hours to 1hour.

Synthesis of 1-(Piperidin

Thus obtained 279 has been engaged in deprotection step to yield the compound 142. Hence, the compound 142 has been retrieved with good yields in 3-step process. Thus synthesized fragment was engaged for a convergent and coupling step with that of the fragments synthesized in family 1 and 3.

Coupling of the fragments

For obtaining the CGRP antagonists, a final coupling reaction has to be done between the RAMP-1 antagonist fragment and CALCRL interacting fragment. This particular subset, for easy comprehension and depiction has been presented in the form of a generalized approach as follows. The strategy to synthesize BMS846372 has been proposed by Luo G et al. [138]. The first reaction involves the coupling of carbonyldiimidazole with the PS to form x. The compound y was obtained starting from 2-bromonicotinaldehyde over 10 steps. Thus synthesized y has been deprotonated by using potassium tert-butoxide to form an alcoholate anion which subsequently follows a nucleophilic attack over the carbonyl group of x. This leads to the formation of BMS846372 with a yield of 97%.

As the above proposed strategy looks facile with excellent yields, we took it as basis for us and designed the following routes for coupling the fragments The planned route A gives carbonate CGRP antagonist 11 obtained starting from 10

i.e. piperidin-4-ol starting material which was obtained by a reduction from piperid-4-one 9.

Hence, this route gave the final molecule in minimal 2 steps from the ketone 9. On contrary, route B takes 5 steps from the ketone 9. First step is reduction of piperid-4-one 9 to piperidin-4-ol 10 which was followed by an activation of alcohol to a mesylate 12. This compound 12 will be further transformed into an azide 13 using nucleophilic substitution reaction and later to an amine 14 by reduction. This compound 14 was used as a fragment for coupling to synthesize carbamate CGRP antagonist 15.

Therefore, due to its minimal steps to retrieve the compound 11, we initially chose route A.

ROUTE A: This route selection has been followed by the study of coupling agents used to couple the two fragments. We carried this study using a piperidin-4-ol analog, cyclohexane-4ol due to their similar conformations. As presented earlier, the synthesis of BMS846372 has been taken as methodological prototype. Thus, we applied the same to our compound of subclass A to couple it with the PS. On following the same condition, the reaction didn't proceed forward and gave the starting materials as such. We thought that the deprotonation of the alcohol by potassium tertbutoxide was not forwarding due to the electronic property of our molecule which hamper the

.showing the route A to obtain the carbonate CGRP antagonists

As tabulated above, all conditions to obtain the carbonate CGRP antagonists had failed. In some cases there is formation of product but as less as 1% (calculated through 1 H NMR). This may be accounted due to the electronic properties of the alcohol substrates we used. After deprotonation by using a base, the alcoholate anion has to attack the carbonyl group quickly which might not have been the case in our compounds. This might be a slow and rate limiting step in our substrates and hence there was no reaction or very less formation of desired compound.

The following 1 H NMR shows the doubled peak of the pyridine moiety and identified through their coupling constants. This signal corresponds to the proton right beside the nitrogen on pyridine. On integrating and quantifying, the desired carbonate was present in less than 1% overall yield.

Even the cyclohexanol substrate wasn't able to couple with the PS made us to believe that the reaction of alcoholate anion with this type of substrates are rate limiting and if proceeds, the reaction rate might have been really slow. And being a reactive anion, the alcoholate wasn't able to react with the substrate and instead gets protonated back and gives us the starting material as such.

ROUTE B:

We took the condition optimized in the literature and modulated with small changes in accordance with our substrate. This optimization was done within the team by using a similar substrate. The conditions of optimization were as follows In all the cases, the yields are satisfactory and give the desired product in over a period of 24h.

From the compounds obtained above, depending on the protecting group, a general methodology for corresponding PG has been applied to obtain the final compound.

Concluding scratch to give potential CGRP antagonists

The two fragments combined in the previous step have been subjected for final touchups to recover our potential CGRP antagonists. Mostly this last step includes deprotection and sometimes a dehalogenation step as well to retrieve the desired compound. Now let's take each case. The entry 1 is having a tert-Butyl carbamate protecting group which was deprotected using trifluoroaceticacid as presented in the previous chapters. The deprotected compound x was left in salt form as this will increase the solubility in aqueous phase of vehicle (5% Tween 80 in saline 0.9%) during the pharmacological evaluation. This easy solubility also increases the bioavailability of the tested compounds more readily in biological system.

Entries 2, 3, 4 and 5 were having benzyl carbamate as protecting groups and also we want to debrominate the compounds as bromine in biological system has proved to be mutagenic [139]. And thus, we did a hydrogenation reaction which deprotects the benzyl carbamate and dehaolgenates in situ. The hydrogenation as presented before is quantitative and gives the desired product in 24 hours.

And the last entry, compound 292 needs no further steps and hence qualifies as such to be tested in biological evaluation.

Take home message from this chapter

In this chapter, we illustrated the conception of organic skeletons based on multistep synthesis for two hybrid families (family 1 and 3, scheme 140) that I had to construct for my PhD project. Those structures have been handpicked based on the molecular modeling and docking studies made on the CGRP receptor. The best substituents have been selected to innoculate in each family and we had proposed a convergent synthesis limelighted on the preparation of a common structure: the 2,6-piperidin-4-one. Piperidine moieties were prepared in 6-steps form the corresponding methyl crotonate for family 3 and in 5-steps from the chiral aspartic acid for the family 1. The chirality for the family 3 has been introduced during the first step through an Aza-Michael insertion of the desired chiral Davies Amine. The piperidine skeleton was obtained for both families from an intramolecular reaction based on an Aza-Michael principle, methodologies that we had to optimize or which have been published by the laboratory.

N N O X R 4 N N NH N O O R 1 R 2 N Y X R 4 N N NH N O O R 2 HN N NH N O + N Y R 4 R 2 Z O N R 4 R 3 O O R 6
According to the family, various procedures were made to reach RAMP-1 fragment and none of them were known in the literature. Later, several ways to construct those fragments were investigated. For the family 1, the optimal procedure started after optimization of the synthesis for desired N-protected pipecolic acid. The benzodiazepanone 7 membered ring have been obtained in 4-step synthesis through an amide bond formation following by an in-situ ketone protection and N-deprotection followed by an intramolecular palladium catalysed Buchwald cross coupling reaction. Only the benzodiazepanone with R = benzyl could be reached and some peptides before cyclisation were also used as RAMP-1 fragment.

Scheme 141. showing design of family 1

For family 3, desired substituents on the piperidine ring for reaching the 7-membered skeleton were directed on the HWE reaction. Piperidine 2,6-cis and 2,6-trans were obtained in favor of the cis isomer majority. Only the cis configuration was used for the following steps. The 3 subsclass A, B and C 7 membered rings were investigated to be prepared by using an intramolecular palladium catalyzed reaction. Only candidate from the subclass A and B could be reached with an average to good yields, and concerned methods are still in optmisation progress.

BIOASSAY

The course of earlier chapters has introduced us the diverse structure of pain along with the aetiology implicated in it. Along with this, in the subsequent chapters, we have seen the design and synthesis of the potential CGRP antagonists. In the current section, we would have a detailed outlook about the application of the synthesized potential CGRP antagonists in pain models, to be precise on nociceptive pain replica.

As we have discussed in the preceding chapters, different type of pain stimulation exists. It includes nociceptive and neuropathic in general. Hence for the study of analgesic activity of the synthesised molecules, knowledge of which form of pain we are acting on is obligatory. For this reason, the choice of biological models for the analgesic tests depends on the pain initiation mechanism, feasibility, and animal ethics along with the pharmacologist who conducts the test.

We studied the pharamacological activity of our molecules using hot-plate method for analgesic property, actophotometry for sedative property and porsolt forced swimming test for antidepressant property.

A. Pain bioassay

Analgesic activity in biological systems can be assessed using various models.

Depending on the administered stimuli, the nociceptive pain models can be classified as below [1]

Figure 83.showing different nociceptive bioassays

The choice of the model depends on various factors like sensitivity, reproducibility and region of brain involved in response for stimuli [2,3,4].

Advantages of thermal stimuli administered nociceptive assay

Among the three stimulations which can be administered for the mice, electrical and chemical stimuli are more aggressive and may cause internal abrasion (in case of chemical stimuli) and thus obtained results are more prone to unreliability and not bring robust due to changes during the mice behavioural studies [5,6].

On the other hand, the thermal stimuli give more reliable and reproducible data [7].

Even amongst the thermal stimulus administered nociceptive assay, the hot plate method is found to be more reliable as the assay involves higher brain functions and is considered a supraspinal organized response [2]. Whereas, the tail flick and paw withdrawal methods involves spinal reflux to a noxious stimuli [3]. Hence, the nociceptive pain inhibitory data obtained through Hot plate method deemed to provide a deeper pain processing responsive study than that of a simple reflux as in case of other thermal stimulation tests.

As an outcome, we used the Hot plate method to be our essential principle test to envisage the nociceptive pain inhibitory properties of our designed and synthesized potential CGRP antagonists.

Hot plate method:

1.1 Rationale: Rodent species are characterised by thermo-sensitivity in paws for the non damaging heat changes. Especially, rodents are able to express the heat response by wiggling, jumping and licking of the paws particularly of hind leg. Hence crafting this as a basis, the hot plate method has been developed in mid 19 th century [8]. The time taken for showing the above said responses is increased (if the molecules tested are having analgesic property) in general.

General procedure:

The hot plate method was initially designed by Woolfee and McDonald in 1944. But the currently followed method is a result of series of modifications [9¸ 10]. And mostly followed method is as follows;

Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. The commercially available hot plate will be having a temperature controlled surface, a plastic cage for enclosing the mice along with the time and temperature display. The surface in general was maintained at 52°C and is made of copper. An initial habituation to the cage for individual mice was performed with the surface being at room temperature. Then the mice are placed on the heated surface (at 52°C) and using the stop watch, the time at which the mice expresses the pain response like wiggling, jumping or licking of hind paws are noted with a cut-off time of 30 sec above which was considered as error and restarted again. When it shows the above signs, the mouse is instantaneously taken out of the hot plate. The molecule to be tested for analgesic property was administered subcutaneously. After the administration, a latency period of 15 minutes was given and the mice was kept on the hot plate again at same temperature to see if there is any increase of time of perception of pain/expressing the pain responses due to the analgesic property induced by the molecule administered.

Assessment

In this particular pain model, the time of showing the response before and after the administration of the molecule can be regarded as the criteria for evaluation of analgesic property. Even the values of the control group and the experimental group can be used for statistical comparison using t-test.

Significant points to be noted

 Centrally acting analgesics will increase the time of showing the responses where as peripherally acting analgesic derivatives showed no response according to the literature [11].

 A hot plate method shows pseudo positive results when the analgesic moiety tested possesses some secondary activities like muscle relaxation, sedation or psychomimetic property [12].

 Even among rodents, the sensitivity for temperature difference from the hot plate to that of environment was detected higher in mice whereas the rats showed no difference [13].

Antinociceptive property of the synthesized CGRP antagonists

The designed and synthesized potential hits which were discussed in the previous sections have been tested for their antinociceptive activity using hot plate method. We carried the antinociceptive activity studies in two batches till now. The strategy for studying the analgesic property of molecules was as follows  We tested the fragments of the synthesized hits to facilitate the easy structure activity relationship (SAR) studies.

 Variety of designed RAMP-1 interacting fragments of family 1, 3 and privileged CALCRL interacting fragment has been tested individually as well as in coalition.

 We predominantly tried to validate the reliability of in silico scores obtained by using the results acquired through in vivo tests in biological system.

 We investigated various factors like the effect of the substituants, the configuration, the enantiomers etc. affecting the antinociceptive property.

Thus, the first consignment of in vivo results is as follows The normalization of the results to a percentage scale was done by using the following formula.

×100

During the continuation of study in identifying the lead, we have conducted nociceptive pain inhibitory property tests for another batch of potential CGRP antagonists.

Thus, the second consignment of in vivo results is as follows Thus obtained percentage pain inhibition values from two batches have been studied to understand the structure activity relationship. Some of the key identifications are as follows  A dose of 5 mg/kg showed significant amount of pain inhibition.

 The latency period of 15 minutes proves that the tested molecules are potent and shows the desired analgesic activity in less time.

 All the hits tested showed a potent nociceptive pain inhibitory activity  None of the tested molecules showed amplification of pain/induction of pain.

 We maintained privileged structure as 1A instead of 1B as 1A stands out to be the best with percentage pain inhibition of 72.

 Apart from privileged structure, 2A showed the best pain inhibition with a value of 55% and tops in the contender to be a potential lead.

 The racemic structures 4A & 7A, even though a mixture of two enantiomers showed good pain inhibitory property.

 The fragments belonging to family 3 also possessed antinociceptive properties and among which, the flexible fragment 5A showed better results than that of the rigid counterpart 6A. Hence, the proposed hypothesis (refer chapter 3, Family-3) on a flexible structure for this family has been justified. A final molecule coupled with privileged structure will be studied in the future tests.

 The fragment 7B showed 40% of pain inhibition and hence a final molecule coupled with privileged structure will be studied in the future tests.

 Ligand 2B showed 31% of pain inhibition whereas 3B showed 47% even though they both are similar structures with former being an S-enantiomer and latter being an Renantiomer.

 The ligand 5B showed a mere 20% of pain inhibition attributing the loss of analgesic property either due to the configuration or change of substitution (phenyl with CF 3 ) when compared with 3B.

Hence, during the course of above study, we envisaged several factors involved in gain or loss of nociceptive activity. We investigated the effect of substituants, the conformation, configuration etc. Most of the time, the obtained results are in correlation with the results retrieved from the molecular modelling studies.

With thus obtained pleasing results, a III rd batch of potential CGRP antagonists are currently under investigation and will be presented during the thesis defence.

To authenticate that the obtained analgesic activity is not due to the sedative property of the molecules, we performed the test for sedation using actimetry assay for locomotory activity.

B. Locomotory activity bioassay

Locomotion tends to the movement from one place to the other. In rodent species like mice and rats, the most important amongst the catalogue of impulsive activity is locomotion.

The exploration, a most common activity of rodent species along with locomotion are implicated in many behavioural utilities and are prejudiced by environmental conditions such as light, temperature, sound etc., circadian rhythm, food/drink deprivation, age, gender and many more factors [14,15]. These locomotory and exploration behaviour is mediated by neurotransmitters which can be modulated by many drugs such as epileptics, psychomimetics, analgesics, antidepressants etc [16,17].

Hence, we made this as a rationale to identify whether the pain inhibitory property of our molecules are solely due to its antinociceptive property or due to the sedative property.

Alongside this, we also want to envisage the sedative side effect our molecules which might be possessed in their tricyclic and tetracyclic moieties as we have structural resemblance with benzodiazepines.

There are various locomotor activity assessment tests available like actimeter, circadian locomotor activity, open field test for basal global activity, emergence test, novel object test, hole board test, treadmill test etc. Among all the available bioassays for locomotor activity, actimeter with photocell-based detection is one of the quickest, robust and gives reliable data [18].

Photoactometer-an infrared photocell-based detection

The actimeter used for assessing the locomotor activity of the rodents are mostly automated now-a-days. The structure of actimeter includes a rectangular box which has enough space for free movement of the rodents inside it. Two photo emitters are placed on one end of the box which on other side trespasses with a pair of photo detectors. These photobeams, when broken registers a count on a digital counter. In some variants, photocells are placed on top of the cage as well in order to identify the rearing along with locomotor activity.

General procedure

Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. For the preliminary testing of the equipment's condition, the instrument was switched on and by manual cut-off, all the photocells were tested. This was followed by a subcutaneous administration of the molecules to be tested. After a latency period of 15 mins, the mice were placed in the actimeter box and left for the locomotor activity assay for a period of 10 mins. During this time, the digital counter outside the box shows the count of cut-offs. Once the test was done, the cages were cleaned and disinfected to carry the next batch of testing.

Assessment

In this actimeter, the difference in count of cut-offs between the mice administered with molecules and the control can be regarded as the criteria for evaluation of locomotor activity. In simple words, if the molecules don't possess sedative property, it shows more or equivalent count as that of the control group.

Locomotory activity of the synthesized CGRP antagonists

The designed and synthesized potential CGRP antagonists were tested for their locomotor activity using photoactometer. Similar to the antinociceptive assay, the tests were conducted in two different batches.

The locomotor activity of first batch of compounds tested is as follows

.showing cut-off counts for administration of antagonists in 1st batch

Unlike in hot plate method, here the results of administered molecule will be compared with that of the control group. These results showed that there was no significant change in locomotory activity because of the administered molecules.

Further, the tests on second batch of potential CGRP antagonists were conducted and the results were as follows

.showing cut-off counts for administration of antagonists in 2nd batch

Summing it up, the results retrieved from the locomotory activity studies showed no significant sedation which made us to draw two important and significant conclusions as following  The antinociceptive property shown during the hot plate method was not because of sedative property of the administered molecules.

 Concurrently, we also saw that our molecules, even though has a tricyclic and tetracyclic core moiety like benzodiazepines, don't possess sedation as a side effect.

With these two studies, the main goal of our project has been investigated and validated with significant and pleasing results. As discussed in the preceding section, the bioassay for depression will be studied to authenticate the concept of dual activity.

C. Antidepressant bioassay

Depression can be defined clinically as a complex pathophysiological state, which also involves neuroendocrine symptoms which is highly difficult to replicate in animals models [19]. The foremost hurdle in identifying a novel antidepressant drug is lack of animal models which look a lot like depressive disorder as well as selectively responsive to clinically effectual antidepressant therapy [20]. During the course of period, various models like reserpine effects reversal test designed in 1960 is dated to be the first of its kind which led to the isolation of desipramine and express its antidepressant activity [21]. One of the models developed is Porsolt Forced Swimming Test (FST) by Porsalt.R.D in 1977 which is considered as most reliable, sensitive and mimics clinical treatment like response [22,23].

Porsolt Forced Swimming Test (FST)

Porsolt et al observed during a behavioural study of rodents in a maze that among the rats which are involved in the test, most of them found the exit within a period of 10 mins and some of them stayed floating passively. This led to the design of FST which is also called as behavioural despair in mice.

General procedure

Each group of 10-12 male Swiss albino mice weighing around 18-22 grams were used for each dosage of molecule. The testing apparatus is cheap and even a laboratory beaker can be used. The beaker was initially filled with water at a temperature of 25 °C and maintained the same for entire period of assay. The beaker used should be in such a depth that the mice can't touch the bottom of the beaker with its tail or feet. An ideal depth of the beaker is 30 cms.

Figure 90.showing FST

The molecules to be tested were administered subcutaneously and the mice was given a latency period of 15 mins. After which, each mice was placed in the beaker of water and calculated for the immobility of the mice due to loss of hope to escape from the beaker. The test runs for a time of 5 mins and immediately thereafter, the mice was taken off from the water and dried completely before placing back in the cage. The beaker in general was cleaned and disinfected during constant intervals of time.

Assessment

During the course of the FST test, two parameters were noted down. One being the first immobilization time and second was overall immobile time. The obtained results from the tested molecules were compared with that of the control group. In simple terms, the immobilization time for tested compounds should be less than that of the control group to have antidepressant property.

Similar to the other tests which we performed, we conducted FST in two batches. The results of 1 st batch are as follows We can consider either of the two immobilization times to predict the antidepressant property of the administered molecule. As shown in the above table, the immobilization times of all the molecules administered are less than that of the control group and hence, they showed some amount of antidepressant nature but only 2A when applied with statistics showed significance. We continued to search for a molecule with more significant antidepressant property than that of 2A. Hence we followed this study with another batch.

The results for the second batch are as follows Hence, even though the molecules tested in II nd batch possessed considerable amount of desired antidepressant property, they lack of significance.

Overall, the test results for antidepressant property identified through FST showed not excellent but definitely significant amount of desired property. Molecules like 2A showed good as well as significant results where as compounds like 5A, 2B, 4B, 5B and 7B showed good but lacked the significance in them.

Take home message from this chapter

The designed and synthesized molecules have been tested for their pharmacological properties. The prime focus was kept on antinociceptive activity and thus we did hot plate method to show that the molecules possess analgesic property. We even validated that the analgesic property showed by our molecules are not because of the sedation but due to their direct analgesic effect by conducting photoactometer test for locomotor activity. Molecules like 1A, 2A, 4A, 5A, 7A, 3B and 7B showed an excellent and significant amount of pain inhibition (>37%) for the mere amount of dose we used i.e. 5mg/kg. The other point to be kept in mind is to get the desired activity, it just took 15 minutes of time which gave us the above results. This demonstrates that our molecules possess potent analgesic property.

Photoactometer test was performed to show that our molecules don't possess sedation as a side effect and hence the analgesic property showed by our molecules is not because of sedation.

To validate our concept of dual activity to treat chronic pain, we also tested our molecules for antidepressant effect. This test showed that 2A is both significant and persuasive where as 5A, 2B, 4B, 5B and 7B have antidepressant property but aren't significant with their results.

Another batch of molecules is currently ready to be tested for all the above said tests and the results will be presented during the thesis defence.

"In conclusion, molecules 2A, 5A and 7B showed our desired dual activity whereas 1A, 2A, 4A, 5A, 7A, 3B and 7B showed potent antinociceptive effect. Hence, further structure activity relation (SAR) study is in process to take these as leads and make lead modifications for the amplification of the desired dual active molecules or to enhance the antinociceptive effect alone. This will be followed by in-depth mechanistic study of the identified lead to recognize how their pharmacodynamic and pharmacokinetic modulations are taking place within a biological system"

GENERAL CONCLUSION

During the course of my PhD, we tried to find a true answer towards the treatment of chronic pain (migraine and viscerals) and on the postulate based on the use of an hybrid molecule which could possess a dual activity : antagonist for the CGRP receptor and inhibitor for the recapture of monoamines.

Before starting the synthesis of antagonists for the CGRP receptor, we initially conducted a deep survey on the receptor and its properties for designing better and efficacious antagonists. During this course, we identified that unlike other receptors, to distort the action of this receptor, we need to design ligands which are 'out of box' or we should be linient about the conventional Lipinski's rule of 5. This may be attributed to the huge receptor pockets and not just one interaction but a group of interactions are important for antagonizing the CGRP receptor. And hence, we designed ligands directly inspired form two molecules known for there activities on the CGPR receptor (antagonist: Telcagepant) and as inhibitor for the recapture of monoamines (Imipramine) and conducted an in silico docking simulation and from its results, we identified potential hits from 2225 proposed molecules 39 structures which were distributed among three families (10.1 ≤ Score Total ≤ 12.9) which correspond with a total score up to the Telcagepant (Score Total = 10.08).

From then on, we were working on synthesizing the potential hits obtained from docking ranks and testing them in biological systems as well to validate our prototypes.

The preparation of those structures is based on a convergent synthesis from the corresponding piperidine well substituted RAMP-1 fragment to attach to the more potent CALCRL fragment. After a chapter of bibliography to put in front the best procedure to reach the desired disubtituted 2,6-piperidone, we have selected published and optimized, for the two families, an intramolecular Michael synthesis started from methyl crotonate or aspartic acid.

According to the family, and to access to the skeleton RAMP-1 fragment, we have to work out and optimized new process to reach the different 7-member ring heterocycle form family 1 and 3. Those procedures are focus on an intramolecular Buchwald "type" reaction in presence of palladium. Three new skeletons belonged to family 1 and family 3 have been successfully prepared. An optimization to improve yield is in progress in the laboratory.

The various piperidinone were then transformed into the corresponding amine to be further connected to the CALCLR fragment. Five finals molecules with fragment RAMP-1 and CALCLR were next tested for there potencies to act on pain. Seven other molecules, corresponding at some part of the final molecules were also tested.

Then till now, we have tested around twelve molecules in various tests to assay their pharmacological properties. All have shown antalgic effect, and for some (2A, 5A, 7B) a dual effect was observed. (SATT) and they are currently considering our project to take over. During this period, we want to take 2A as lead and modulate on R 1 , R 2 and R 3 as following: As depicted above, we wanted to design in the future, a new series of CGRP antagonists using the model proposed above. We wanted to study taking 2A as the lead and conduct a rigorous Structure Activity Relationship study. The results, with the help of SATT will be patented.

GENERAL REMARKS

Thin layer chromatography were performed on TLC pre-coated aluminium backed silica plates Kieselgel 60 F254 (Merck) or glass backed silica Duracil 25 UV254 (Macherey nagel).

Spots were visualized using UV light (254 nm) before using an ethanolic solution of paraanisaldehyde or phosphomolybdic acid (heating).

Purifications by column chromatography were carried out on silica gel Kiesgel 60 (0.073-0.230).

Melting points were measured by a Kofler Hot bench or Reichert plate-heating microscope.

Optical rotations were measured on a ADP 440 Bellingham polarimeter at the wavelength of sodium D ray (Ä = 589 nm).

1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance spectrometer at 400.13 and 100.61 MHz respectively. Chemical shifts δ were reported in ppm relative to solvent residual signals ( 1 H and 13 C). The coupling constants J were given in Hertz (Hz). The abbreviations used for signal descriptions are as:

-ax : axial -eq : equatorial Numbers placed in figures refer to NMR attributions, and were given independently from IUPAC nomenclature.

MOLECULAR MODELLING STUDIES

Docking studies have been done by Mr. Lionel Nauton and Dr. Vincent thery. Whereas the other modeling studies presented previously has been generated by myself using various online and offline tools as reported. Most of the structures were drawn using ChemDraw ® and sometimes using Marvinsketch. Various experimental methods followed during the study are as follows.

Modeling of the receptor:

Previously refined crystal structure was opened in 'Biopolymer' of sybylx2.0 to adjust the backbones in a correct way in order to add the hydrogen atoms on the receptor considering the hydrogen interactions. Finally, this model was minimized using molecular mechanics Hence this energy minimization step resulted in a low energy and refined crystal structure which will be engaged in the docking simulation.

Energy minimization of refined protein model

Method: conjugate gradient

cases, the injection was done 15 min before the behavioral assessment. The injections were done into the peritoneal cavity of the mice through subcutaneous route of administration.

Hot plate method: After acclimatizing for 5 min on hot plate at room temperature, mice were placed on the plate set at 52 °C until they started licking their forepaws (Latencies) (cut-off time: 30 s). After having obtained two consecutive stable latencies, treatment effects were assessed after 15 min. The results were expressed as: ×100

Photoactometer: Unlike Hot plate method, no acclimatization was done. The mice were treated with the testing compound and after 15 min, they were placed in the rectangular box and closed. After the test run of 10 min, the cut-offs of the photocell beam due to the movement of mice was noted which was recorded on the counter outside the box. The results were directly compared with that of control group to assess sedative activity.

Porsolt Forced Swimming Test: The mice were treated with the testing compound and after 15 min, they were placed in a beaker of water which was maintained at 25 °C. After the test run of 5 min, the 1 st time of immobilization and overall immobility times were recorded using a stop watch. The results were directly compared with that of control group to assess antidepressant activity. [α] D 25 = + 27. 9 (c 1.10, CHCl 3 ).

CAS Registry Number: 130622-08-1

The analysis is in accordance with the literature.

(R)-dimethyl 2-(bis(tert-butoxycarbonyl)amino)succinate 145b To a solution of (R)-dimethyl 2-(tert-butoxycarbonylamino)succinate (2.0 g, 5.53 mmol, 1.0 equiv) in tetrahydrofuran (30 mL) at 0 °C was added slowly sodium hydride ( 0.15 g, 6.08 mmol,1.1 equiv) and continued to stir at 0 °C for 30 min. This was followed by bringing the reaction to the room temperature and addition of di-tert-butyl-dicarbonate (1. Thionyl chloride (15 mL, 206.5 mmol, 0.9 equiv) was added dropwise over 10 min to a stirred mixture of L-aspartic acid (30 g, 225.4 mmol, 1.0 equiv) and Methanol (300 mL) at 0 °C. The mixture was set to reflux and stirred for 16 h. The reaction mixture was cooled down to room temperature and later concentrated in vacuo to give the methyl ester salt (44.0 g, 98% yield) as a white waxy solid.

Mp: 114-116 °C. Chemical Formula: C 27 H 30 NO To a solution of (S)-methyl 5-(dimethoxyphosphoryl)-4-oxo-2-(tritylamino)pentanoate (20.4 g, 33.2 mmol, 1.0 equiv) in tetrahydrofuran (91 mL) at room temperature was added Ba(OH) 2 .H 2 O (8.14 g, 43.17 mmol, 1.3 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of acetaldehyde (2.4 mL, 43.17 mmol, 1.3 equiv) diluted in tetrahydrofuran/water: 40/1 (91 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give (S,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate as a colorless oil (13.0 g, 95%). R f 0.37 (SiO 2 , cyclohexane/ethyl acetate: 8/2). To a solution of (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (0.2 g, 1.17 mmol, 1.0 equiv) in DCM (5 mL) and distilled H 2 O (0.5 mL) at 0 °C was added with trifluoroacetic acid TFA ( 0.27 mL, 2.34 mmol, 2.0 equiv) dropwise over a time of 2 min. The reaction mixture was continued to stir at room temperature for 2 h. The reaction mixture was basified using 1N NaOH till we reach a pH of 9-10 and later diluted with dichloromethane (10 mL) and washed using brine (3x10 mL), dried over MgSO 4 and finally concentrated in vacuo. To a solution of (R,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate (13 g, 31.46 mmol, 1.0 equiv) in methanol (260 mL) at room temperature was added dropwise 2M hydrochloric acid (158 mL, 314.6 mmol, 10.0 equiv) and left to stir for 60 minutes. The reaction mixture was diluted with distilled water (320 mL) followed by N, N-diisopropylethylamine (66 mL, 380 mmol, 12.0 equiv) until pH was adjusted to 10. The reaction mixture was continued to stir at room temperature for 12 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 7), washed with ethyl acetate (3x200 mL). pH of the aqueous phase was further adjusted to 14 by using 1M NaOH (200 mL), washed with ethyl acetate (3x200 mL) To a solution of sodium (2R)-6-methyl- [α] D 25 = + 15.9 (c 1.02, CHCl 3 ). To a solution of sodium (2R)-6-methyl-4-oxopiperidine-2-carboxylate (5.63 g, 31.46 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added dropwise benzyl chloroformate (6.7 mL, 47.2 mmol, 1.5 equiv. If the reaction mixture's pH is acidic, adjust the pH to basic using 1M NaOH followed by further addition of one more equivalent of benzyl chloroformate. The reaction mixture was continued to stir at room temperature for 16 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo.

HR-ESI-MS

The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 1/9) to give (2R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid as an orange-yellow oil (8.14 g, 86%) as yellow oil. R f 0.12 (SiO 2 , cyclohexane/ethyl acetate: 1/9).

Note:

The attribution of chemical shift for each proton was difficult due to the presence of 2 rotamers. Percentages of two rotamers were (66/33). To a solution of sodium (2S)-6-methyl-4-oxopiperidine-2-carboxylate (4.75 g, 26.5 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added dropwise benzyl chloroformate (5.65 mL, 39.7 mmol, 1.5 equiv. If the reaction mixture's pH is acidic, adjust the pH to basic using 1M NaOH followed by further addition of one more equivalent of benzyl chloroformate. The reaction mixture was continued to stir at room temperature for 16 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 1/9) to give (2S)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid as an orange-yellow oil (6.48 g, 84%) as yellow oil.

[α] D 25 = -5.1 (c 1.05, CHCl 3 ).

Apart from the optical activity, rest of the data remains same as that of (2R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid. To a solution of 2-bromobenzaldehyde (3 g, 16.22 mmol, 1.0 equiv) and benzylamine (2.66 mL, 24.32 mmol, 1.5 equiv) in methanol (100 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.92 g, 24.32 mmol, 1.5 equiv). The reaction mixture was continued to stir at room temperature for 12 h. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9.5/0.5) to give N-benzyl-1-(2bromophenyl)methanamine as a colorless oil (4.25 g, 95%). R f 0.15 (SiO 2 , 100% ethyl acetate). To a solution of 1-(tert-butoxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.8 g,

N-benzyl-1-(2-bromophenyl)methanamine 160

Method A

To a solution of (R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.2 g, 0.68 mmol, 1.0 equiv) in dichloromethane (5 mL) at 0 °C was added N,N-Diisopropylethylamine (0.24 mL , 1.37 mmol, 2.0 equiv) followed by (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (0.41 g, 1.37 mmol, 2.0 equiv). To (2R,6R)-tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1carboxylate (0.34 g, 0.66 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl orthoformate (0.36 mL, 3.30 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.14 g, 0.73 mmol, 1.1 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH (3x30 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 85/15) to give (2R,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6-methylpiperidine-2-carboxamide as a colorless viscous oil (0.29 g, 95%). The attribution of chemical shift for each proton is difficult due to the presence of rotamers.

Note

O O

The NMR spectra of trans isomer was not only rotamer prone but also badly resoluted. Hence it is neither predictable nor identifiable. Hence, we analyzed only using the following data. A 25 mL sealed tube was charged with tris(dibenzylideneacetone)dipalladium-Pd 2 (dba) 3 (0.042 g, 0.068 mmol, 0.15 equiv) and (2,2'-bis(diphenylphosphino)-1,1'-binaphthyl)-BINAP (0.074 g, 0.114 mmol, 0.25 equiv) which was later added with fresh anhydrous toluene (3 mL) and left for stirring at room temperature for 10 min. This has been followed by the addition of a solution of (2R,6R)-N-benzyl-N-(2-bromobenzyl)-4,4-dimethoxy-6methylpiperidine-2-carboxamide (0.210 g, 0.455 mmol, 1 equiv) in anhydrous toluene (1 mL) through cannula during which, the stirring was continued. Finally, sodium tert-butoxide NaotBu (0.053 g, 0.55 mmol, 1.2 equiv) was added and the sealed tube was stoppered tightly and continued to stir at 100 °C for 10 h. The resulting mixture has been filtered through Celite ® bed and thus collected filtrate was diluted with DCM (20 mL) and washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (1R,4aR)-6-benzyl-3,3-dimethoxy-1-methyl- Chemical Formula: C 20 H 24 N 2 O 2 A 25 mL sealed tube was charged with Pd(dba) 2 (0.040 g, 0.068 mmol, 0.1 equiv) and BINAP (0.064 g, 0.102 mmol, 0.15 equiv) which was later added with fresh anhydrous toluene (3 mL) and left for stirring at room temperature for 10 min. This has been followed by the addition of a solution of 2-((2S,6S)-4,4-dimethoxy-6-methylpiperidin-2-yl)aniline (0.17 g, 0.68 mmol, 1 equiv) in anhydrous toluene (1 mL) through cannula followed by 1, 2 dibromobenzene (0.17 mL, 0.75 mmol, 1.1 equiv) during which, the stirring was continued.

Finally, sodium tert-butoxide NaotBu (0.079 g, 0. Chemical Formula: C 20 H 23 NO 3 Molecular Weight: 325.40

Following the general method as stated for 198, we synthesized 199 R f 0.30 (SiO 2 , cyclohexane/ethyl acetate: 7/3).

Yield: 99% To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (11.4 g, 63.6 mmol, 1.0 equiv) in DCM (320 mL) and H 2 O (320 mL) at room temperature was added with sodium carbonate (13.5 g, 127 mmol, 2.0 equiv) and ethyl chloroformate (12.2 mL, 127 mmol, 2.0 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 3 h.

The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (7.4 g, 42.0 mmol, 1.0 equiv) in DCM (160 mL) and H 2 O (160 mL) at room temperature was added with sodium carbonate (11.2 g, 105 mmol, 2.5 equiv) and Di-tert-butyl dicarbonate (14.0 g, 63 mmol, 1.5 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 10 h.

The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. The crude product was purified using column chromatography on silica (cyclohexane/ethylacetate: 7/3) to give (S)-methyl 3-(tertbutoxycarbonylamino)butanoate as yellow oil. (57%) R f 0.40 (SiO 2 , cyclohexane/ethyl acetate: 7/3). To a solution of (S)-4-methoxy-4-oxobutan-2-aminium acetate (7.4 g, 42.0 mmol, 1.0 equiv) in DCM (160 mL) and H 2 O (160 mL) at room temperature was added with sodium carbonate (11.2 g, 105 mmol, 2.5 equiv) and benzyl chloroformate (9.0 mL, 63 mmol, 1.5 equiv) slowly over a period of 5 min. The resulting solution was stirred at room temperature for 10 h. The aqueous layer was extracted with DCM (3x200 mL), combined organic layers were dried over MgSO4 and then concentrated in vacuo. The crude product was purified using column chromatography on silica (cyclohexane/ethylacetate: 7/3) to give (S)-methyl 3-(benzyloxycarbonylamino)butanoate as yellow oil. (52%) R f 0.45 (SiO 2 , cyclohexane/ethyl acetate: 7/3). To a solution of diethyl methylphosphonate (4.25 mL, 29.1 mmol, 2.5 equiv) in anhydrous THF (60 mL) at -78 °C was added dropwise n-butyl lithium (11.6 mL, 1.6 M in hexane, 29.1 mmol, 2,5 equiv). After 20 min at -78 °C, a solution of (S)-methyl 3-(ethoxycarbonylamino)butanoate (2.2 g, 11.6 mmol, 1.0 equiv) in anhydrous THF (12 mL) was added dropwise. After addition, the temperature of the reaction was kept at -78 °C for 30 min, then allowed to reach 0 °C in 1 h, quenched with saturated solution of ammonium chloride, and extracted with ethyl acetate (3x100 mL). After drying over MgSO 4 and concentration in vacuo, the crude oil was first distillated at low pressure to remove excess diethyl methylphosphonate, and the residue purified using column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (S)-ethyl 5-(diethoxyphosphoryl)-4-oxopentan-2ylcarbamate as a yellow oil (3.3 g, 55% yield). R f 0.3 (SiO 2 , cyclohexane/ethyl acetate: 7/3). Chemical Formula: C 14 H 28 NO 6 P Molecular Weight: 337. 34 We followed the similar methodology as optimized for (S)-ethyl 5-(diethoxyphosphoryl)-4oxopentan-2-ylcarbamate to give (S)-tert-butyl (5-(diethoxyphosphoryl)-4-oxopentan-2yl)carbamate as a yellow oil with an yield of 63%. R f 0.4 (SiO 2 , cyclohexane/ethyl acetate: 7/3). We followed the similar methodology as optimized for (S)-ethyl 5-(diethoxyphosphoryl)- The analysis is in accordance with the literature.

GENERAL METHOD FOR HORNER-WADSWORTH-EMMONS REACTION

To a solution of dimethoxyphosphoryl (1.0 equiv) in tetrahydrofuran ( To a solution of 2-iodophenol (2 g, 9.1 mmol, 1.0 equiv) in DMF anhydride (16 mL) at room temperature was added with 2-fluorobenzaldehyde (1.128 g, 9.1 mmol, 1.0 equiv) and K 2 CO 3 (2.01 g, 14.54 mmol, 1.6 equiv). The reaction mixture was allowed to reflux and continued to stir for 7 h. The reaction mixture was cooled to room temperature and concentrated in vacuo.

The resulting residue was diluted with ethyl acetate (200 mL), washed with NH 4 Cl (2x200 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give 2-(2iodophenoxy)benzaldehyde as a brown oil (2.85 g, 97%). R f 0.74 (SiO 2 , cyclohexane/ethyl acetate: 7/3) 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 10.06 (1H, s, H 13 ), 7.99 (1H, dd, J = 7.8, 1.8 Hz, H 1 ), 7.93 (1H, dd, J = 7.9, 1.5 Hz, H 8 ), 7.52 (1H, ddd, J = 8.9, 7.4, 1.8 Hz, H 2 ), 7.39 (1H, td, J =

GENERAL METHOD FOR PIPERIDINE CORE FORMATION

To alkene (0.34 g, 0.66 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl orthoformate (0.36 mL, 3.30 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.14 g, 0.73 mmol, 1.1 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH 3.90 (m, 2H, H2), 3.03 (s, 3H, H7),2.80 (s, 3H, H8), 2.62 (dd, J = 14.5, 5.4 , 1H, H4), 2.33 (dd, J = 14.5, 5.8, 1H, H4), 2.05 (dd, J = 14.6, 6.2 , 1H, H5), 1.89 (m, 1H, H5), 1.57 (d, J = 6.9, 3H, H9), 0.91 (t, J = 6.9 , 3H, H1)

cis.

NMR 1 H (400 MHz, CDCl3) δ: 7.82 (dd, J=8.0,1.4, 1H, H13), 7.39 (dd, J=8.3,1.0, 1H, H10), 7.01 (td, J=7.6,1.1, 1H, H12), 6.75 (td, J=7.8,1.3, 1H, H11),4.32 (dd, J=11et2.6,1H, H3), 3.26 (s, 3H, H7,8), 3.14 (s, 3H, H7,8), 2.86-2.91 (m, 1H, H6), 2.58 (dt, J=13.7et2.7, 1H,H4e), 2.04 (dt, J=12.6,2.6, 1H, H5e), 1.53 (dd, J=12.2et12.3, 1H, ), 1.31 (dd, J=12.4et11.91,1H,), 0.96 (d, J=7.0, 3H, H9,). A generalized hydrogenation method as proposed for (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6methylpiperidine-2-carboxamide has been followed. (m, 2H, H2), 3.16 (s, 3H, H8), 2.89 (s, 3H, H7), 2.55 (dd, J = 14.5, 5.9, 1H, H4eq), 2.21 (dd, J = 14.5, 5.6,1H, H4ax), 2.00 (dd, J = 14.9, 3.5, 1H, H5eq), 1.95 (dd, J = 14.9, 6.0, 1H, H5ax),1.29 (d, J = 6.9, 3H, H9), 1.04 (t, J = 7.1, 3H, H1). NMR 13 C (101 MHz, CDCl 3 ) δ 165.1, 155.9, 149.7, 147.9, 138. 1, 133.8, 128.2, 125.4, 123.1, 121.6, 120.8, 114.3, 98.4, 88.9, 62.0, 52.3, 47.9, 47.6, 47.3, 37.4, 36.7, 22.8, 14.4. To a solution of (2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4oxopiperidine-1-carboxylate (0.400 g, 0.72 mmol, 1.0 equiv) in methanol (10 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.33 g, 0.86 mmol, 1.2 equiv).

Physical properties

The reaction mixture was continued to stir at room temperature for 30 min. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (50 mL), washed with water (3x50 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate as a yellow oil (0.39 g, 98%). R f 0.13 (SiO 2 , cyclohexane/ethyl acetate: 7/3). To a solution of (2S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4oxopiperidine-1-carboxylate (0.376 g, 0.68 mmol, 1.0 equiv) in methanol (9.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.30 g, 0.82 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (50 mL), washed with water (3x50 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: To a solution of (2R,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4oxopiperidine-1-carboxylate (0.265 g, 0.482 mmol, 1.0 equiv) in methanol (7.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.21 g, 0.578 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (20 mL), washed with water (3x20 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2R,4S,6S)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6methylpiperidine-1-carboxylate as a yellow oil (0.37 g, 97%). R f 0.17 (SiO 2 , cyclohexane/ethyl acetate: 7/3). To a solution of (2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6methylpiperidine-1-carboxylate ( 0.3 g, 0.54 mmol, 1.0 equiv) in methanol (5 mL) at room temperature was added with Pd(OH) 2 ( 0.015 g, 0.11 mmol, 0.20 equiv). The mixture was kept under hydrogen atmosphere and continued to stir for 12 h. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (30 mL), washed with water (3x30 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to yield (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6-methylpiperidine-2-carboxamide as yellow solid. R f 0.1 (SiO 2 , cyclohexane/ethyl acetate: 5/5).

HR-ESI-MS

HR-ESI-MS

Proton NMR is difficult to predict even now. And thus, we completely depend on carbon NMR. Tert-Butyl-4-(2-aminopyridin-3-ylamino)piperidine-1-carboxylate (5.0 g, 17.1 mmol, 1.0 equiv), and acetonitrile (50 mL) were treated with N,N-diisopropylethylamine (4.89 g, 37.8 mmol) followed by 1,1′-carbonyldiimidazole (4.16 g, 2.56 mol) at ambient temperature.

Rotamer

The mixture was stirred for 2 h. The reaction mixture was cooled to 0-5 °C and stirred for 30 min. The resulting slurry was filtered, and the cake was washed with acetonitrile (200 mL) and dried at 50 °C in vacuo to afford Tert-butyl 4-(2-oxo-2,3-dihydro-1H-imidazo [4,5b]pyridin-1-yl)piperidine-1-carboxylate (4.85 g, 82%) as a white solid. The analysis is in accordance with the literature.

tert-butyl 4-hydroxypiperidine-1-carboxylate 283 To a solution of tert-butyl 4-oxopiperidine-1-carboxylate (4 g, 20 mmol, 1 equiv) in methanol (100 ml) at 0°C was added powdered sodium borohydride (0.76 g, 20 mmol, 1 equiv) slowly.

The resulting solution was stirred at room temperature for 15 min and concentrated in vacuo.

The white solid residue was dissolved in ethyl acetate (100 mL) and extracted with brine solution (3x30 mL). The organic extracts were dried over MgSO 4 , filtered and concentrated in vacuo to give 4.213 g (quantitative yield) of tert-butyl 4-hydroxypiperidine-1-carboxylate as yellow-orange oil. R f 0.17 (cyclohexane/ethyl acetate : 7/3). We used generalized method as presented for 155'. R f 0.45 (SiO 2 , cyclohexane/ethyl acetate: 7/3).

Yield: 97%

1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 0.93 (1H, d, J = 6.2 Hz, H7), 1.03 (1H, t, J = 12.3 Hz, H5a), 1.48 (1H, m), 1.92 (1H, t, J = 12.2 Hz, H 3a ), 2.17 We followed a similar method as proposed for (2R,4R,6R)-benzyl 2-(benzyl(2bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. Yield is quantitative R f 0.25 (SiO 2 , cyclohexane/ethyl acetate: 5/5). We used generalized method as presented for 155' R f 0.25 (SiO 2 , cyclohexane/ethyl acetate: 7/3).

Yield: 98% We followed a similar method as proposed for (2R,4R,6R)-benzyl 2-(benzyl(2bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. Yield is quantitative R f 0.17 (SiO 2 , cyclohexane/ethyl acetate: 5/5). We followed the general method as 268.

  This work has been carried out in the laboratory of Conception Et Synthèse de Molécules Antalgiques (CESMA) affiliated to the Institut de Chimie de Clermont-Ferrand (ICCF-UMR 6296) of Université Blaise Pascal, Clermont-Ferrand under the supervision of Professor Yves Troin and Doctor Isabelle Abrunhosa Thomas.

Figure 1 .

 1 Figure 1.Deaths due to untreated pain associated with disease/disorder ...........................................

Figure 2 .

 2 Figure 2.Classification of pain based on time period of occurrence .....................................................

Figure 3 .

 3 Figure 3.Drugs used for acute pain .......................................................................................................

Figure 4 .

 4 Figure 4.Drugs used for chronic pain ....................................................................................................

Figure 5 .

 5 Figure 5.showing ascending pain pathway ...........................................................................................

Figure 6 .

 6 Figure 6.showing pain scaling and experience of a patient ..................................................................

Figure 7 .

 7 Figure 7.showing signalling pathways of CNS & PNS ............................................................................

Figure 8 .

 8 Figure 8.showing signalling pathways from receptor to the effector organ .........................................

Figure 9 .

 9 Figure 9.showing signalling pathways from receptor to spinal cord by transmission ..........................

Figure 10 .

 10 Figure 10.showing areas of brain implicated in thermal pain signalling ...............................................

Figure 11 .

 11 Figure 11.showing gate control theory of pain .....................................................................................

Figure 12 .

 12 Figure 12.Drugs acting at different levels as analgesics ........................................................................

Figure 13 .

 13 Figure 13.screenshot showing global ranking with respect to Years Lived with Disability (YLD) .........

Figure 14 .

 14 Figure 14.screenshot showing global ranking with respect to deaths ..................................................

Figure 15 .

 15 Figure 15.screenshot showing global deaths due to Non-infectious inflammatory bowel syndrome .

Figure 16 .

 16 Figure 16.Distribution & implications of CGRP receptor .......................................................................

Figure 17 .

 17 Figure 17.Molecular level cascade events of CGRP receptor activation ...............................................

Figure 18 .

 18 Figure 18.Peptide antagonists for CGRP receptor.................................................................................

Figure 19 .

 19 Figure 19.Sequence of modulations resulting in BIBN-4096 .................................................................

Figure 20 .

 20 Figure 20.Non peptide antagonists .......................................................................................................

Figure 21 .

 21 Figure 21.Non peptide antagonists by Merck .......................................................................................

Figure 22 .

 22 Figure 22.Non peptide antagonists by Boehringer Ingelheim ..............................................................

Figure 23 .

 23 Figure 23.showing various forms of visceral pain .................................................................................

Figure 24 .

 24 Figure 24.Showing the account of analgesics developed recently .......................................................

Figure 25 .

 25 Figure 25.Dual cyclooxygenase/5-lipoxygenase inhibitors ...................................................................

Figure 26 .

 26 Figure 26.Indomethacin derived dual COX/5-LOX inhibitors ................................................................

Figure 27 .

 27 Figure 27.Tepoxaline as dual COX/5-LOX inhibitors ..............................................................................

Figure 28 .

 28 Figure 28.Licofelone as dual COX/5-LOX inhibitors ...............................................................................

Figure 29 .

 29 Figure 29.Dual Angiotensin II and Endothelin I receptors antagonist development ............................

Figure 30 .

 30 Figure 30.Isomers of Tramadol .............................................................................................................

Figure 31 .

 31 Figure 31.Dual activity of Tapentalol.....................................................................................................

Figure 32 .

 32 Figure 32.Dual activity design for the project .......................................................................................

Figure 33 .

 33 Figure 33.Showing correlation of pain and major depression ..............................................................

Figure 34 .

 34 Figure 34.Serotonin and norepinephrine function ...............................................................................

Figure 35 .

 35 Figure 35.Biological action of monoamines ..........................................................................................

Figure 36 .

 36 Figure 36.context of the project ............................................................................................................

Figure 37 .

 37 Figure 37.(a).GPCR Tree (b) Class-B Secretin family (c) CGRP receptor (d) CALCRL-protein interactions ...............................................................................................................................................................

Figure 38 .Figure 39 .Figure 40 .

 383940 Figure 38.Depicting the Tractability, Druggability and Ensemble druggability of CALCLR and RAMP-173 Figure 39.showing the CALCRLs site 1 & the fragment of Olcegepant® embedded inside the pocket Figure 40.Crystal structure of CGRP receptor stabilized with Olcegepant® studied using Molecular Operating Environment® (MOE) software [21] .....................................................................................

Figure 41 .

 41 Figure 41.showing the important water molecules and its interactions ..............................................

Figure 42 .

 42 Figure 42.Showing the hybridization of two leads ................................................................................

Figure 43 .

 43 Figure 43.showing structure based ligand designing ............................................................................

Figure 44 .

 44 Figure 44.Showing importance of stereocenters ..................................................................................

Figure 45 .

 45 Figure 45.showing medicinal chemistry aspects applied for ligand design ..........................................

Figure 46 .

 46 Figure 46.showing the Olcegepant® and Telcagepant® in the docked site ..........................................

Figure 47 .

 47 Figure 47.showing the SARmap graph of potential hits & different zones with lead as reference ......

Figure 48 .

 48 Figure 48.showing the filers and descriptors applied to identify potential hits ...................................

Figure 49 .

 49 Figure 49.showing the general structure of family 1 ............................................................................

Figure 50 .

 50 Figure 50.showing the general structure of family 2 ............................................................................

Figure 51 .

 51 Figure 51.showing the general structure of family 3 ............................................................................

Figure 52 .

 52 Figure 52.showing the plot using drug-likeliness model score .............................................................

Figure 53 .

 53 Figure 53.Showing (+)-241D alkaloid, (2S,4R)4-hydroxy pipecolic acid and Palinavir ..........................

Figure 54 .

 54 Figure 54.Showing the L-pipecolic acid and its analogues ..................................................................

Figure 55 .

 55 Figure 55.showing steric hindrance due to mono and di-boc (diterbutylester) PG ............................

Figure 56 .

 56 Figure 56.showing protection of dimethyl ester of aspartic acid with trityl PG .................................

Figure 57 .

 57 Figure 57.showing preparation of energy states of intermediates during HWE ................................

Figure 58 .

 58 Figure 58.showing preparation of β -keto phosphonate ester as E isomer ........................................

Figure 59 .

 59 Figure 59.showing preparation of β -keto phosphonate ester as E isomer ........................................

Figure 60 .

 60 Figure 60.showing preparation of cis/trans piperidine with 75/25 ratio ............................................

Figure 61 .

 61 Figure 61.showing splitting pattern with coupling constants for H2e proton of trans piperidine .....

Figure 62 .

 62 Figure 62.showing amide rotamers for secondary amides .................................................................

Figure 63 .

 63 Figure 63.showing amide rotamers for tertiary amides......................................................................

Figure 64 .

 64 Figure 64.showing amide rotamers for compound 158 ......................................................................

Figure 65 .

 65 Figure 65.showing rotamer of cis/trans piperidine in DEPT NMR ......................................................

Figure 66 .

 66 Figure 66.showing the DEPT NMR showing N-acylurea ......................................................................

Figure 67 .

 67 Figure 67.showing the 1 H NMR showing piperidinone .......................................................................

Figure 68 .

 68 Figure 68.showing the 1H NMR showing piperidine with ketal at 4th position .................................

Figure 69 .

 69 Figure 69.showing the disappearance of rotamers on cyclizing .........................................................

Figure 70 .

 70 Figure 70.showing the diastereomeric selectivity for the formation of compound 1 ........................

Figure 71 .

 71 Figure 71.showing two routes to obtain compound V ........................................................................

Figure 72 .

 72 Figure 72.showing three subclasses of family 3 ..................................................................................

Figure 73 .

 73 Figure 73.Contributors for Buchwald-Hartwig C-N coupling ..............................................................

Figure 74 .

 74 Figure 74.Choice of different catalytic systems on Buchwald-Hartwig C-N coupling .........................

Figure 75 .

 75 Figure 75. Showing the conformation of sublcass C in CGRP pocket ..................................................

Figure 76 .

 76 Figure 76.showing the formation of equatorial and axial alcohols .....................................................

Figure 77 .

 77 Figure 77.Showing the CALCRL fragments used for potent CGRP antagonists ...................................

Figure 78 .

 78 Figure 78.Showing the privileged structure we used for CGRP antagonists .......................................

Figure 79 .

 79 Figure 79.showing the carbamate like antagonist synthesized by David K.L et al ..............................

Figure 80 .

 80 Figure 80.showing the carbamate like antagonist BMS846372 ..........................................................

Figure 81 .

 81 Figure 81.showing the 1H NMR of the carbonate CGRP antagonists .................................................

Figure 82 .

 82 Figure 82.showing different nociceptive bioassays ............................................................................

Figure 83 .

 83 Figure 83.Showing Hot plate device ....................................................................................................

Figure 84 .

 84 Figure 84.showing time of response before and after administration of antagonists in I st batch ......

Figure 85 .

 85 Figure 85.showing time of response before and after administration of antagonists in IInd batch ..

Figure 86 .

 86 Figure 86.showing photoactometer ....................................................................................................

Figure 87 .

 87 Figure 87.showing photoactometer results in Ist batch .....................................................................

Figure 88 .

 88 Figure 88.showing photoactometer results in IInd batch ...................................................................Figure 89.showing FST .........................................................................................................................

Figure 90 .

 90 Figure 90. showing first and total immobilization times in Ist batch of FST .......................................

Figure 91 .

 91 Figure 91.showing first and total immobilization times in IInd batch of FST ......................................

Figure 1 .

 1 Figure 1.Deaths due to untreated pain associated with disease/disorder

Figure 2 .

 2 Figure 2.Classification of pain based on time period of occurrence 1. Classification based on time period: Based on time course of existence, pain is classified into[8]

Figure 3 .

 3 Figure 3.Drugs used for acute pain

Figure 4 .

 4 Figure 4.Drugs used for chronic pain

Figure 5 .

 5 Figure 5.showing ascending pain pathway

Figure 7 .

 7 Figure 7.showing signalling pathways of CNS & PNS

Figure 8 .

 8 Figure 8.showing signalling pathways from receptor to the effector organ

Figure 9 .

 9 Figure 9.showing signalling pathways from receptor to spinal cord by transmission

Figure 10 .

 10 Figure 10.showing areas of brain implicated in thermal pain signalling

4. 1

 1 Gate control theory of pain: This inhibitory pathway theory for pain was proposed by Melzack and wall [23]. It states that by activation of A-α fibres by non-noxious and touch stimulus, results in activation of interneurons thus resulting in inhibition of noxious stimuli conducting through C fibres.

Figure 11 .showing gate control theory of pain 4 . 2

 1142 Figure 11.showing gate control theory of pain 4.2 Descending modulation: The received and processed noxious stimulus in brain is further answered as a reaction by the brain in the form of modulation. Periaqueductal grey in midbrain and rostral ventromedial medulla are two important regions of brain involved in

Figure 13 .

 13 Figure 13.screenshot showing global ranking with respect to Years Lived with Disability (YLD)

Figure 15 .

 15 Figure 15.screenshot showing global deaths due to Non-infectious inflammatory bowel syndrome Around 34,000 deaths and 2,875,360 DALYs have been reported in 2010 and making the situation worse, the rate is increasing rapidly since 1990.

  Initial 2 h relief, S = Sustained pain-free, C = Consistency, T = Tolerability) (= indicates equal, -indicates less & + indicates more activity when compared withSumatriptan 100 mg, N° of subjects -24089)[27]

Figure 16 .

 16 Figure 16.Distribution & implications of CGRP receptorTherefore, current migraine therapy rationale is based on its mechanism of dilation of cranial blood vessels.

Figure 17 .

 17 Figure 17.Molecular level cascade events of CGRP receptor activation

Figure 18 .

 18 Figure 18.Peptide antagonists for CGRP receptor

Figure 19 .

 19 Figure 19.Sequence of modulations resulting in BIBN-4096

Figure 20 .

 20 Figure 20.Non peptide antagonists

Figure 21 .

 21 Figure 21.Non peptide antagonists by MerckMK-3207 showed 50-100 folds increased potency than MK-0974 (Telcagepant®) in preclinical studies[51]. These positive results have been validated even on clinical studies

Figure 22 .

 22 Figure 22.Non peptide antagonists by Boehringer IngelheimIn phase II study, it showed efficacy on acute migraine and compared with placebo and eletriptan. 400 mg showed statistical significance over placebo with a response rate of 27.4% whereas 200 mg showed no statistical significance. The current development of BI-44370 is in uncertain as Boehringer Ingelheim announced no progress further from phase II.

Figure 23 .

 23 Figure 23.showing various forms of visceral pain

4 31 19 Figure 24 .

 1924 Figure 24.Showing the account of analgesics developed recently

Dual Cyclooxygenase/ 5 -

 5 /disorders: There exists certain drugs which are combination of two different mechanisms/different targets but implicates for treating the same disease or disorder. For example, Lipoxygenase inhibitors: They act on both cyclooxygenase and 5lipoxygenase enzymes which are responsible for synthesis of pro-inflammatory bodies[66]. It includes drugs like Tepoxaline, Licofelone, Flufenamic acid derivatives etc. Arachidonic acid is chemically polyunsaturated fatty acid and is abundant in phospholipid cell membrane. They are synthesised by the phospholipase-A2 mediated conversion of membrane phospholipids. Thus formed arachidonic acid can be metabolised by the influence of two enzymes cyclooxygenase (COX) and 5-lipoxygenase (5-LOX). The subsequent products from these two enzyme catalysed pathways are pro-inflammatory bodies prostaglandins and leukotrienes (Figure25).

Figure 25 .

 25 Figure 25.Dual cyclooxygenase/5-lipoxygenase inhibitors

Figure 26 .

 26 Figure 26.Indomethacin derived dual COX/5-LOX inhibitorsThis N-hydroxyurea, obtained from Zileuton (a 5-LOX inhibitor) plays as a chelator by complexing with the non-heme iron of 5-LOX. Thus resulted compound 1 is potent dual COX/5-LOX inhibitor.Another potent dual COX/5-LOX inhibitor is Tepoxaline which again incorporates two individual activities in same moiety (Figure27).

Figure 27 .Figure 28 .

 2728 Figure 27.Tepoxaline as dual COX/5-LOX inhibitors

Figure 29 .

 29 Figure 29.Dual Angiotensin II and Endothelin I receptors antagonist development

Figure 30 .

 30 Figure 30.Isomers of Tramadol

Figure 31 .

 31 Figure 31.Dual activity of Tapentalol

Figure 32 .

 32 Figure 32.Dual activity design for the project

Figure 34 .Serotonin and norepinephrine function 3 . 1

 3431 Figure 34.Serotonin and norepinephrine function

  amino acids are stored in the synaptic vesicles and later get released into the synaptic cleft when there is a Ca 2+ dependent process (which depends on the firing rate of neurons). After the release of the neurotransmitters from the presynapse, they won't cross the postsynapse instead activate the receptors on the surface of postsynapse to induce cascade of cellular response.

Figure 35 .

 35 Figure 35.Biological action of monoamines Once activating the receptors, the functioning of the neurotransmitters are terminated for this cycle and the neurotransmitters are either transported back to the presynapses from the synaptic cleft using specific monoamine transporters which are selective for each monoamine or simply metabolized by the enzyme Monoamine Oxidase (MAO) in presynapses. Thus these are the two probable mechanisms through which the concentration of the monoamines in the synaptic cleft are decreased and in turn results in variety of mood related disorders especially depression. Hence these are the potential targets for therapy of depression.

Table 6 .

 6 Antidepressants based on their mechanism

Figure 36 .

 36 Figure 36.context of the project

  (b)) of G-Protein Coupled Receptors (GPCR) (Figure37(a))[7]. Whereas RAMP is an activity modulator associated with different GPCRs like Calcitonin receptor or Calcitonin receptor like receptor to incarnate various dimeric receptors[8, 9]. There are three different RAMPs; RAMP-1, RAMP-2 and RAMP-3[10].

Figure 37 .

 37 Figure 37.(a).GPCR Tree (b) Class-B Secretin family (c) CGRP receptor (d) CALCRL-protein interactions CALCRL associating with RAMP-1 gives CGRP receptor (Figure 37(c)) while RAMP-2 and 3 results in Adrenomedullin receptor or CGRP/Adrenomedullin dual activating

Figure 38 .

 38 Figure 38.Depicting the Tractability, Druggability and Ensemble druggability of CALCLR and RAMP-1

Figure 39 .

 39 Figure 39.showing the CALCRLs site 1 & the fragment of Olcegepant® embedded inside the pocket The fraction of the ligand which was embedded in the site 1 of CALCRL is 3-(piperidin-4-yl)-3, 4-dihydroquinazolin-2(1H)-one. The binding of this fragment of

Figure 40 .

 40 Figure 40.Crystal structure of CGRP receptor stabilized with Olcegepant® studied using Molecular Operating Environment® (MOE) software [21] Whereas the (S)-2-amino-N-((R)-6-amino-1-oxo-1-(4-(pyridin-4-yl) piperazin-1-yl) hexan-2-yl)-3-(3, 5-dibromo-4-hydroxyphenyl) propanamide interacting with RAMP-1 illustrate key interactions with Asp71, Arg38 & Arg67. Asp71, an acidic residue acts as side chain acceptor from the amine of the pentan-1-amine and Arg38, Arg67, both being basic residues shows water dependent hydrogen bond with the oxygen of the 2, 6-dibromo-4ethylphenol.

Tryptophan 74 might

 74 play a crucial role both in selectivity and affinity of the antagonists. Alanine 70, Aspartic acid 71, Histidine 75, Phenylanlanine 83, Tryptophan 84 and Proline 85 also are other miscellaneous amino acids whose interactions can serve in answering a better CGRP antagonism. Henceforth, with the knowledge obtained through the study of CGRP receptor, we performed molecular modelling & docking studies in the next stage to identify potential hits for the CGRP receptor.

Figure 41 .

 41 Figure 41.showing the important water molecules and its interactions

Figure 42 .

 42 Figure 42.Showing the hybridization of two leads

Figure 43 .

 43 Figure 43.showing structure based ligand designing

Figure 44 .

 44 Figure 44.Showing importance of stereocenters  No flat molecules: For the same reason as stated in the point above, a flat molecule can't reach the desired interaction (Figure44). Instead, using conformational isomers might help in reaching the amino acids by modulating the substituants on them.

Figure 45 .showing medicinal chemistry aspects applied for ligand design 1 . 2 . 3 .

 45123 Figure 45.showing medicinal chemistry aspects applied for ligand design 1. Molecule I has been developed first from which rest of the molecules have been derived using various principles of drug design. Replacement with fluorine from I to II was done in order to increase the metabolic stability of the molecule, aqueous solubility, antidepressant property. Molecule III is obtained by ring opening principle from I with increased aqueous solubility, flexibility inside the pocket and increasing the hydrogen bond donor by exposing NH. These modifications gave us family 1.

Figure 46 .

 46 Figure 46.showing the Olcegepant® and Telcagepant® in the docked site Superposition of the crystal structure of Telcagepant® (the one available in PDB) with the result of docking;

Figure 47 .Zone 1 :

 471 Figure 47.showing the SARmap graph of potential hits & different zones with lead as reference Zone 1: This region corresponds to low electrostatic interactions between the ligand and the receptor. The hits found in this zone were expected to have least interaction with the pocket and consequently in the receptor.

Figure 48 .Figure 49 .

 4849 Figure 48.showing the filers and descriptors applied to identify potential hitsAlongside total score, the final prioritized list of '36 potential hits' was selected by fixing a benchmark of total score to be 10.5(score of Telcagepant® is 10.8). These 36 probable hits can be classified into 3 families based on their structures.

Figure 50 .

 50 Figure 50.showing the general structure of family 2

Figure 51 .showing the general structure of family 3 Table 13 .

 51313 Figure 51.showing the general structure of family 3

3 CALCRL;

 3 Top 4 binding site volumes are 1468.5, 434.9, 394.8, 300.3 Å Top 4 binding site volumes are 674.1, 498.2, 288.9 and 207.5Å3

Figure 52 .

 52 Figure 52.showing the plot using drug-likeliness model score The Gaussian distribution curve of drug vs non-drugs was plotted using the druglikeliness model scores. The blue plot has drug-like molecules and green plot gives nondrugs. The plot generated using our hit molecules showed that all of them are actually distributed in the mean area of the drug-like blue plot. And other point to be noted, these molecules are having a model score quite close to the lead molecule Telcagepant®.

Scheme 1 .

 1 Scheme 1.showing a generalized retrosynthetic design

Figure 53 .

 53 Figure 53.Showing (+)-241D alkaloid, (2S,4R)4-hydroxy pipecolic acid and Palinavir

Scheme 2 .Scheme 3 .

 23 Scheme 2.Showing the formation of piperidine using chiral ligand L

Scheme 4 .

 4 Scheme 4.Showing the synthesis of (+)-241D alkaloid using gold complex

Scheme 5 .

 5 Scheme 5.Showing the synthesis of (+)-241D alkaloid using gold complex

Scheme 6 .

 6 Scheme 6.Showing the application of gold complex for another example

Scheme 10 .Scheme 11 .

 1011 Scheme 10.Showing the application by Toyooka and coll.An anodic oxidation of the compound 29 gives 30 which was later reacted with allyltrimethylsilane to give 31. The double bond was later functionalized to obtain α-Lfucosidase inhibitor, 32 (IC 50 = 0.0005µM, K i = 0.0011 µM).

Scheme 12 .

 12 Scheme 12.Showing the inoculation of 3rd asymmetric centre

Scheme 13 .

 13 Scheme 13.Showing the synthesis of 2, 4, 5 trisubstituted piperidines

2 Scheme 14 .

 214 Scheme 14.Showing the synthesis of piperidine moiety using Diels Alder reaction

Scheme 16 .Scheme 17 .

 1617 Scheme 16.Showing application of dipolar cycloaddition reaction by Chattopadhyay and coll.On subsequent oxidation of 44 and 45 with the help of hydrogen peroxide gave the nitrenes 46 and 47 with a good yield. The key step i.e. 1, 3 dipolar cycloaddition of nitrenes

unsaturated aldehyde 53 .

 53 The Aza-Michael reaction was then carried in presence of a chiral inducer. The configuration of the inducer determines the absolute configuration on the final molecule giving 2, 6 cis (54) or 2, 6 trans (55).

Scheme 18 .

 18 Scheme 18.Showing the application of Aza-Michael cyclization by Hong and coll.

Scheme 19 .

 19 Scheme 19.Showing the synthesis of (-)-epimyrtine 56 and (+)-myrtine 57.

Scheme 22 .Scheme 23 .

 2223 Scheme 22.Showing the application of Aza-Michael by Akiyama and coll.Troin and coll. has also used Aza-Michael intramolecular reaction to synthesize substituted piperidines starting from compound like 67. In presence of ethylene glycol, paratoluene sulfonic acid and trimethylorthoformate, this reaction gave derivatives of 2, 6 trans in majority 68[35].

Scheme 25 .Scheme 26 .

 2526 Scheme 25.Showing the application of electrochemistry by Hurvois et alCompound 78 on reduction with sodium borohydride (with a iminium transition state) gave sterioisomers 79a,b which are separated through column chromatography using silica gel.The major isomer 79a was deprotected by hydrogenation and later reprotected to form N-Boc 80a. A similar methodology starting now from N-protected piperidone (S configuration) 76b was followed to give 80b.

Scheme 27 .Scheme 28 .

 2728 Scheme 27.Showing the substituted piperidine synthesis Dieter and coll.

Scheme 29 .

 29 Scheme 29.Showing the 2, 6 cis substituted piperidine

Figure 54 .

 54 Figure 54.Showing the L-pipecolic acid and its analogues

30 Scheme 30 .

 3030 Scheme 30.Showing the synthesis of compound 88 proposed by Aitken and coll.

Scheme 31 .

 31 Scheme 31.Showing the synthesis of compound ent-89 proposed by Hou and coll. This was followed with a metathesis reaction (GRUBBS 2 nd generation) which allowed to recover the bicyclic compound 101 with an excellent yield. The intermediate 102 was obtained by a palladium catalyzed reaction in presence of formic acid and a ligand which was finally transformed into 103 by an oxidative cleavage using Osmium tetraoxide. The compound 103 was finally transformed into enantiomer of 89.

5 Scheme 38 .

 538 Scheme 38.Showing the synthesis of compound 134 proposed by Merino and coll.Sutherland and coll.[53] has utilized the aspartic acid as the building blocks for the synthesis of 4-hydroxypipecolic acid derivatives. After the protection and activation, the ester 135 was transformed into β-ketophosphonate 136 which, during Horner-Wadsworth-Emmons with a range of alcohols gave corresponding enones 137 with good yields.

Scheme 39 .

 39 Scheme 39.Showing the synthesis of compound 138 proposed by Sutherland and coll. The compound 137 was treated with acidic medium (TFA), inducing the deprotection on nitrogen which reacts with benzaldehyde giving an imine intermediate which on reduction with sodium cyanoborohydride gave the compound 138 (2S,4S,6R) 6-substituted-4-hydroxy methyl pipecolate) which exclusively was trans . The compound 138 passes through a transition state during the 6-Endo-Trig cyclization.

Scheme 40 .

 40 Scheme 40.Showing the synthesis of compound 140 proposed by Sutherland et al.

  Scheme 41.showing retro synthetic scheme for family 1

Scheme 42 .

 42 Scheme 42.showing retro synthetic scheme for synthon ISynthon A is N-protected 6-methyl-4-oxopiperidine-2-carboxylic acid synthon. In previous chapter, various methodologies concerning how to synthesize the synthon A have been discussed in detail. According to the bibliographic conclusion from previous section, to reach the key piperidine skeleton of family 1, a pipecolic acid derivative, we have nominated the process proposed by Sutherland et al[53,54].

  Scheme 43.showing retro synthetic scheme for synthon II



  The Michael intramolecular reaction for obtaining the synthon A act as key step to introduce stereocenters on the final molecule in a single step.  Substituants on the final molecule 141 depends on the amine B' which we use in the formation of the synthon B.  Another key asymmetric center on the final molecule 141 is obtained by FGI from 144 to 143.

  Scheme 44.showing esterification of aspartic acid

Scheme 45 .

 45 Scheme 45.showing protection of dimethyl ester of aspartic acidIf the PG used was trityl, it was one-step process whereas in case of tert-Butyl carbamate protection, it was a two-step synthesis to do a double protection of nitrogen to duly induce selectivity on the nucleophilic attack of the phosphonate. The first protection of amine using tert-Butyl carbamate gave 82% of compound 147 which on further deprotonation by a strong base and reaction with another equivalent of di-tert-Butyl carbamate gave compound 148 with a yield of 72%. Compound 146 was obtained by a simple nucleophilic substitution reaction in one step.

Figure 55 .

 55 Figure 55.showing steric hindrance due to mono and di-boc (diterbutylester) PG

Figure 56 .

 56 Figure 56.showing protection of dimethyl ester of aspartic acid with trityl PG

Scheme 47 .

 47 Scheme 47.showing preparation of β -keto phosphonate esterThis HWE reaction is considered as the key intermediate reaction in whole synthesis as the substituent on the final molecule depends on which aldehyde we use during this step.

Scheme 48 .Figure 57 .Figure 58 .

 485758 Scheme 48.showing preparation of β -keto phosphonate esterIn the formation of alkenes through HWE, the oxaphosphetane (147a,b) and betaine (147a',b') were considered as the slowest and oxyphosphetane being the lowest energy compared to that of their corresponding betaines[66] has been confirmed by low temperature 31 P NMR studies. The energy states of the starting material and the transition intermediates are as follows;

Figure 59 . 1 . 5

 5915 Figure 59.showing preparation of β -keto phosphonate ester as E isomer

  isobutyl and propyl at 6 th position. The results for them were as follows

  Scheme 50.showing preparation of methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate.Condition2M HCl (Eq) DIPEA (Eq) Yield of 155/156 (%)

Figure 60 .

 60 Figure 60.showing preparation of cis/trans piperidine with 75/25 ratio Thus isolated (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (cis) and (2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate (trans) have different splitting patterns when it comes to proton NMR and they serve as basis for identification of the isomers.

  ppm, doublet of triplet 3H e Same splitting pattern as 5He; 2.34 ppm, doublet of triplet ppm, doublet of doublet Table 19.showing splitting pattern with coupling constants for cis piperidine Cis piperidine, as portrayed above has distinguishing coupling constants and splitting patterns. Protons H 6a shows H 6a -H 5a , H 6a -H 5e , H 6a -H CH3 which finally was shown as a J 5e-5a = 13.3 Hz J 5e-6a = 2.7 Hz J W = 2.7 Hz J 5a-5e = 13.2 Hz J 5a-6a = 11.7 Hz J 2a-3a = 12.2 Hz J 2a-3e = 2.8 Hz J 2a-3e = 2.8 Hz

Figure 61 .

 61 Figure 61.showing splitting pattern with coupling constants for H2e proton of trans piperidineAlong with the above depicted splitting, the H 3a -H 2a (J = 12.2 Hz) in cis became H 3a -H 2e (J = 3.4 Hz) as well as H 3e -H 2a (J = 2.7 Hz) in cis became H 3e -H 2e (J = 8.8 Hz).

J

  Scheme 51.showing protection of methyl compound 157

Figure 62 .

 62 Figure 62.showing amide rotamers for secondary amides

  92 kJ/mol (D 2 O) N,N-Dimethylacetamide 80 kJ/mol (D 2 O) N,N-Dimethylbenzamide 66 kJ/mol (CDCl 3 )

Figure 64 .

 64 Figure 64.showing amide rotamers for compound 158We even tried to change the temperature to 20, 30, 40, 50 & 60 °C and see the effect on rotational barrier energy which might result in better resolution. But we saw neither decrease of rotamers nor a better resolution. This might be perhaps because of non-interconvertable nature of the rotamers or may be due to high barrier energy.

Figure 65 .

 65 Figure 65.showing rotamer of cis/trans piperidine in DEPT NMR

  Scheme 52.showing reductive amination to synthesize compounds 160 and 161 N° Aldehyde Amine Condition product Yield (%)

synthon A)Scheme 53 .

 53 Scheme 53.Showing the optimization using L-proline

Scheme 54 .

 54 Scheme 54.showing application of optimized condition on our substrate

Scheme 55 .Scheme 56 .

 5556 Scheme 55.showing newly proposed design to obtain synthon C

Scheme 57 .

 57 Scheme 57.showing application of palladium in route A to obtain synthon DRoute B: Following this way, synthon 169 was coupled first to synthon 160 through a peptide bond formation (see scheme 58). First, pipecolic acid (synthon A with no substitution at 4 th position) was protected with a di-tert-Butyl carbamate group, which was achieved employing traditional condition[16]. Bromoamine 160 was then coupled with DCC coupling agent, which has been optimized as follows.

Table 25 .Scheme 60 .

 2560 Scheme 60.showing application of route B to obtain synthon C

Scheme 61 .

 61 Scheme 61.showing previously optimized condition to chiral substrate

Scheme 62 .

 62 Scheme 62.showing the peptide coupling using DCC/DMAP

FScheme 64 .Scheme 65 .

 6465 Scheme 63.showing the peptide coupling mechanism using DCC

Figure 66 .

 66 Figure 66.showing the DEPT NMR showing N-acylurea The above shown DEPT NMR of clearly showed the formation of N-acylurea 176.

Scheme 66 .

 66 Scheme 66.showing the general peptide coupling reaction

  Scheme 67.showing the T3P® peptide coupling giving desired compound

Scheme 68 .

 68 Scheme 68.showing the mechanism of T3P® peptide coupling

Scheme 69 .

 69 Scheme 69.showing the DEPBT peptide coupling giving desired compound

FTable 27 .

 27 Scheme 70.showing the mechanism of DEPBT peptide coupling

Scheme 71 .

 71 Scheme 71.showing the deprotection of tert-Butyl carbamate from the substrate

Figure 67 .showing the 1 H

 671 Figure 67.showing the 1 H NMR showing piperidinoneWhen there is ketone on 4 th , even though it still remains in a chair conformation, the strain of the chair varies. The splitting of both vicinal and geminal protons of both axial and equatorial protons on 3 rd and 5 th position changes considerably.

Figure 68 .

 68 Figure 68.showing the 1H NMR showing piperidine with ketal at 4th position

Scheme 72 .

 72 Scheme 72.showing the protection of ketone to cyclic ketal

Scheme 73 .Scheme 74 .

 7374 Scheme 73.showing the protection of ketone to methoxy ketal & in situ deprotection of tert-Butyl carbamate

3 H 75 .Scheme 76 .

 37576 Scheme 75.showing the deprotection of benzyl carbamate by TMSI

Scheme 77 .

 77 Scheme 77.showing the deprotection of benzyl carbamate with TMSI

  Scheme 78.showing the C-N cross coupling using Pd(OAc)2/Xantphos

Scheme 79 .ITable 28 .Scheme 81 .

 792881 Scheme 79.showing the C-N cross coupling using Pd2(dba)3/BINAP

Scheme 83 .

 83 Scheme 83.showing the deprotection of ketal to ketone

Scheme 84 .showing the retrosynthetic strategy for family 3

 843 Scheme 84.showing the retrosynthetic strategy for family 3This proposed scheme portray that the desired urea like molecule 194 can be obtained by uniting the fragment 142 with that of 195. Whereas in fragment 195, the stereochemistry is modulates as we saw in the previous chapter, incorporation of asymmetric carbons deemed to

  Scheme 86.showing retro synthetic scheme for synthon III



  Like family 1, there are no steps which induce 2 stereocenters in one single step.  The Aza-Michael reaction acts as key step to introduce stereocenters on the final molecule, once during conversion of 205 to 204 and again during IV to III.  Substituants on the final molecule 194 depends on the aldehyde VI which we use in the formation of the synthon IV.  Just by using corresponding aldehydes VI, all three subclasses of family 3 can be obtained. Another key asymmetric center on the final molecule 194 is obtained by FGI from 196 to 195.

Figure 70 .Figure 71 .

 7071 Figure 70.showing the diastereomeric selectivity for the formation of compound 1The stereochemistry of the methyl group on Davies amine plays crucial role in determining the position of attack taking place and subsequently the stereochemistry obtained in the compound 204. In our case, S-Davies amine, the methyl group lies below the plane and hence the attack of the Davies amine onto the α,β-unsaturated ester happens from below the plane. This is due to the steric hindrance posed by the methyl group on Davies amine.

  Scheme 88.showing hydrogenation of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate

  on several factors as listed below.  The PGs should give good quantity of desired compound  The PGs shouldn't pose any steric hindrance in the succeeding reactions.  The protection step shouldn't alter any other functionalities of the molecule.  The PGs should have facile cleavage step.  The deprotecting step shouldn't affect any other functionality changes of the molecule.These main reasons made us to selected di-tert-butyl dicarbonate ((Boc) 2 O), benzyl chloroformate (Cbz) and ethyl carbamate.

  Scheme 89.showing protection of (S)-4-methoxy-4-oxobutan-2-aminium acetate N° substrate PGs Time(h) Product Yield % (3 steps)

Scheme 90 .

 90 Scheme 90.showing synthesis of ketophosphonates

  Scheme 91.showing synthesis of biaryl aldehydesN°Nucleophile Aldehyde Time (hours) Product Yield (%)

  Scheme 92.showing routes to obtain compound 198

Scheme 93 .

 93 Scheme 93.showing Horner-Wadsworth-Emmons reaction

2 229

 2 Table 33.showing different alkenes with their yield

Scheme 94 .Scheme 95 .Scheme 96 .

 949596 Scheme 94.showing general approach for Aza-Michael intramolecular cyclization

Figure 72 .

 72 Figure 72.showing three subclasses of family 3

Thus obtained 2 , 6

 26 Scheme 100.showing route C to reach subclass A The compound 244 thus obtained acts as substrate for Buchwald-Hartwig cross coupling reaction. We considered the following points before commencing the cross coupling reaction [108].  Buchwald stated that monodentate phosphine ligands are ineffective with aryl iodides as they allow them to form more stable palladium-iodide dimers.  Pd-C and Pd-P rotation barriers established to be superior for bigger halides.

  Scheme 103.showing prime site for C-N cross coupling

  Scheme 104.Mechanism for Buchwald-Hartwig C-N coupling Thus the Scheme 104 showing prime site for C-N cross coupling predicts that the Buchwald-Hartwig reaction for subclass A compounds happens in above depicted mechanism. In the first cycle, first step includes oxidative addition of 1,2 dibromobenzene to

  Scheme 105.showing trans C-N cross couplingBut, in case of family 3, the trans position is obtained due to the changing of methyl from equatorial to axial but not the substitution at 2 nd position. As a result, we were easily able to cyclize the 2, 6 trans with same yields as that of 2,6 cis.

Figure 73 . 2 . 2

 7322 Figure 73. NMR showing the cyclized 198

  Scheme 107.Showing the catalytic cycle for subclass BThe cycle goes in a similar fashion as shown in the subclass A for the second cross coupling cycle. It starts by an oxidative addition resulting of the insertion of the palladium (0) on the R-X bond. The intermediate is obtained after deprotonation of the amine on the piperidine ring and coupling intramolecular connection with the palladium. At this stage, two reactions are possible, either the reductive elimination to reach the desired 7 member ring or the β-hydride elimination to give the de-halogenated product 248. To be pointed out here, this side product wasn't observed in the previous subclass A.

  Scheme 108.Hydrogenation of compound 248



  Figure 74.Contributors for Buchwald-Hartwig C-N coupling N° Factor Contribution Palladium source Impacts efficient formation of active catalyst species Ligand Choice depends on substrate combination Temperature Influences rate of cross-coupling and formation of by-products Solvent Determines solubility of substrates and base Base Choice of base influences rate, functional group tolerance and byproduct formation

Figure 75 .

 75 Figure 75.Choice of different catalytic systems on Buchwald-Hartwig C-N coupling

  Scheme 109.Optimization of catalytic systems for subclass B

  Scheme 110.protection of HY

  Scheme 111. Another way to achieve Subclass B

FamilyFigure 76 .

 76 Figure 76. Showing the conformation of sublcass C in CGRP pocketAccording to the docking scores and the results obtained for the subclass A, we have proposed 3 different ways to reach the desired molecules (scheme 112). Route A, B and C depending of the substitution (NO 2 or H) on the meta position of the aromatic ring. NO 2 will be the precursor of the NH 2 function that we targeted to do an interaction with the aspartic acid on the pocket.

  Scheme 113.Showing the synthesis of O-protected iodoethanol

  Scheme 115.showing the alkylation designed according to route C N° Alkylating agent Reactifs conditions Yield (%) 1

  Scheme 116.Showing route A

  Scheme 117. Showing route B

91 Table 44 .

 9144 Scheme 119.showing the general deprotection method

  Scheme 120.showing general synthetic scheme to obtain amine from ketone

  Scheme 122.Showing the configuration achieved after reduciton

Table 45 .

 45 scheme followed was as given below

Figure 77 .

 77 Figure 77.showing the formation of equatorial and axial alcoholsThe formation of equatorial alcohol as major product can be accredited because of the characteristic axial coupling constant mesure on the tdd signal at 3.38 ppm of proton at 4 th position with large coupling constants of 11.2. At this stage H 2 , H 4 and H 6 are three axial proton according theirs coupling constant on the piperiding ring. A de of 60 % in favor of the equatorial alcohol (2S, 4S, 6S) have been calculated. Both diastereoisomeres wasn't separable and used as a mixture in the following step.

  Scheme 124.showing the application of Mitsunobu condition for racemic structure

  Scheme 125.showing the application of Mitsunobu condition for subclass A of family 3

  Scheme 126.showing the activation of alcohols of family 1

97 Table 46 .

 9746 Scheme 127.showing the activation of alcohols using mesylate for family 1N°Substrate Product Yield (%)

62 Table 47 .

 6247 Scheme 128.. showing the activation of alcohols using mesylate for family 1 N° Substrate Product Yield (%)

Figure 78 .

 78 Figure 78.Showing the CALCRL fragments used for potent CGRP antagonistsDue to their discrepancy from one CALCRL interacting fragment in more potent Olcegepant ® to that of Telcagepant ® (refer the red-highlighted fragment), we tested both the fragments individually as well as a substructure for our antagonists (see chapter 4 : biological evaluation of molecules). Previous study within the group assayed using Writhing test in mice showed that 289 possessed potent activity than the other side chains when coupled with our RAMP-1 fragments.

Figure 79 .

 79 Figure 79.Showing the privileged structure we used for CGRP antagonistsDESIGN AND SYNTHESIS OF PRIVILEGED STRUCTURE

- 4 -

 4 Scheme 130.Showing the synthesis of compound 1a

  Scheme 131.Showing the synthesis of compound 1

Figure 80 .

 80 Scheme 132.Coupling of the fragmentsUrea like final molecules were obtained by a simple coupling reaction between the two fragments as stated above.

Figure 81 .

 81 Figure 81.showing the carbamate like antagonist BMS846372This encouraged us to follow the BMS846372 and synthesize a carbonate like moiety which also decreases the number of synthetic steps during the course and thus increasing overall yield.

  Scheme 134.showing the route A and B to obtain the CGRP antagonists

  Scheme 135.showing the route A to obtain the carbonate CGRP antagonists

  Scheme 136.showing the application of BMS846372 synthetic methodology

  deprotonation step. And hence, we tried a series of coupling agents and bases with different substrates. The conditions used are as follows;

Figure 82 .

 82 Figure 82.showing the 1H NMR of the carbonate CGRP antagonists Thus, we changed our route and continued to use the route B by means of the conditions described in the literature [138].

  Scheme 137.showing the route B to obtain the carbamate CGRP antagonistsThis optimized condition has been employed to our substrates to synthesize the carbamate CGRP antagonists.

  Scheme 138.showing deprotection of Boc in entry 1

3

 3 Scheme 139.showing deprotection and debromination in entries 2, 3, 4

  Scheme 140. General synthetic scheme

Scheme 142. showing design of family 3

 3 Scheme 142. showing design of family 3All the ketone obtained have been next transform into amine through a S N 2 procedure after activation of the corresponding alcohol and branched to the CALCRL optimize fragment. Overall, 6 final molecules containing RAMP-1 fragment as well as CALCLR fragment were tested and a lot of other fragments were also tested for their in vivo pharmacological activity.

Figure 84 .

 84 Figure 84.Showing Hot plate device

Figure 85 . 41 Table 50 .

 854150 Figure 85.showing time of response before and after administration of antagonists in I st batch (Vehicle-solution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

Figure 86 . 40 Table 51 .

 864051 Figure 86.showing time of response before and after administration of antagonists in IInd batch (Vehiclesolution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

Figure 87 .

 87 Figure 87.showing photoactometer

Figure 88 .

 88 Figure 88.showing photoactometer results in 1st batch (Vehicle-solution of 5% Tween 80 in saline)The first batch of potential CGRP antagonists showed no big difference in the cut-off count from the control group. The cut-off counts were as follows

Figure 89 .

 89 Figure 89.showing photoactometer results in 2nd batch (Vehicle-solution of 5% Tween 80 in saline)Akin to the first set of compounds tested for locomotory activity changes, even the second batch of our molecules showed no significant alterations of cut-off count from that of the control group.

Figure 91 .

 91 Figure 91. showing first and total immobilization times in Ist batch of FST (Vehiclesolution of 5% Tween 80 in saline) (***=p ≤ 0.001, **=p ≤ 0.01, *=p ≤ 0.05)

Figure 92 .

 92 Figure 92.showing first and total immobilization times in 2nd batch of FST (Vehicle-solution of 5% Tween 80 in saline)

3 2A

 3 Scheme 2 : result of the hybrid molecule 2 on hot plate and porsolt method by using a dose of 5mg/kg (SC)

Figure 93 .

 93 Figure 93. Showing the perspective of our project

  doublet of doublets of doublets High Resolution Electro-Spray Ionisation Mass Spectra (HR-ESI-MS) were obtained from the "Service de Spectrométrie de masse du service UBP-START (Université Blaise Pascal Clermont II).High Performance Liquid Chromatography (HPLC) analysis conditions used for progress of the reaction; column: Poroschell 120 EC-C18 2.7 µm (3.0 x 50 mm) , temperature column: 40 °C; injection: 10 µL, 1 mL/min; solvents used A-water with 0.1% formic acid and B-methanol (95:5 to 5:95, KBNUNO) method using UV using 254 nm and 280.5 nm.

Force fields:

  Tripos Charges: MMF94 (suitable for H interactions of protein) Gradient: 0.05 Dielectric constant: 4.0 (protein environment) Aggregates: The backbone and 'O' of the water molecule.

[α] D 20 =

 20 + 20.8 (c 1.01, CHCl 3 ). CAS Registry Number: 32213-95-9.The analysis is in accordance with the literature.

  and aqueous phase was concentrated in vacuo to obtain sodium (2R)-6-methyl-4oxopiperidine-2-carboxylate. The sodium salt thus obtained is further engaged in the succeeding steps without purification.Note: The attribution of chemical shift for each proton was difficult due to the presence of large amount of diisopropylethylamine hydrochloride salt with relatively less amount of desired product. of (S,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate (11 g, 26.55 mmol, 1.0 equiv) in methanol (220 mL) at room temperature was added dropwise 2M hydrochloric acid (133 mL, 265.7 mmol, 10.0 equiv) and left to stir for 60 minutes. The reaction mixture was diluted with distilled water (270 mL) followed by N, N-diisopropylethylamine (55 mL, 315.7 mmol, 12 equiv) until pH was adjusted to 10. The reaction mixture was continued to stir at room temperature for 12 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 7), washed with ethyl acetate (3x200 mL). pH of the aqueous phase was further adjusted to 14 by using 1M NaOH (180 mL), washed with ethyl acetate (3x200 mL) and aqueous phase was concentrated in vacuo to obtain sodium (2S)-6-methyl-4oxopiperidine-2-carboxylate. The sodium salt thus obtained is further engaged in the succeeding steps without purification.Note:The attribution of chemical shift for each proton was difficult due to the presence of large amount of diisopropylethylamine hydrochloride salt with relatively less amount of desired product.

: 8 9

 8 calculated for C 12 H 19 NO 5 (M+H -): 270.1341, found: 270.1369. Chemical Formula: C 15 H 17 NO 5 Molecular Weight: 291.29

[α] D 25 =

 25 + 10.9 (c 1.00, CHCl 3 ).HR-ESI-MS: calculated for C 15 H 17 NO 5 (M+H -): 290.1028, found: 290.1030.

Chemical

  Formula: C 14 H 14 BrN Molecular Weight: 276.17

3 . 27 R f 0. 27 ( 27 R f 0. 19 (

 327272719 11 mmol, 1.0 equiv) in dichloromethane (10 mL) at 0 °C was added N,N-Diisopropylethylamine (1.08 mL , 6.218 mmol, 2.0 equiv) followed by (3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one) (1.86 g, 6.218 mmol, 2.0 equiv).The reaction mixture was continued to stir at 0 °C for 60 min. The mixture was later added with a solution of N-benzyl-1-(2-bromophenyl)methanamine (0.86 g, 3.11 mmol, 1.0 equiv) in dichloromethane (5 mL) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with dichloromethane (150 mL), washed with saturated aqueous solution of NaHCO 3 (3x150 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (1.28 g, 72%).Note:Tert-butyl-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1carboxylate was obtained in 70/30 ration of cis/trans. SiO 2 , cyclohexane/ethyl acetate: 8/2) 1 HR-ESI-MS: calculated for C 26 H 31 BrN 2 O 4 (M+H + ): 515.1545, found: 515.1534. (2S,6R)-tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-SiO 2 , cyclohexane/ethyl acetate: 3/7)

30 R 30 R 45 35 To a solution of 1 - 25 FR 25 FR

 3030453512525 The reaction mixture was continued to stir at 0 °C for 60 min. The mixture was later added with a solution of N-benzyl-1-(2-bromophenyl)methanamine in dichloromethane (3 mL) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous solution of NaHCO 3 (3x50 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (0.27 g, 72%).Method BTo a solution of (R)-1-(benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (0.2 g, 0.68 mmol, 1.0 equiv) and N-benzyl-1-(2-bromophenyl)methanamine (0.21 g, 0.75 mmol, 1.1 equiv) in dichloromethane (8 mL) at 0 °C was added N,N-Diethylethanamine (0.29 mL , 2.06 mmol, 3.0 equiv) followed by 1-Propylphosphonic acid cyclic anhydride, 50% (w/w) solution in dichloromethane (3.27 mL, 6.86 mmol, 10.0 equiv). The reaction mixture was continued to stir at room temperature for 14 h. The resulting mixture was diluted with dichloromethane (50 mL), washed with saturated aqueous solution of NaHCO 3 (3x50 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (0.15 g, 40%). Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans. Note: Due to the presence of 4 rotamers, the structural elucidation of the synthesized compound was not possible. Even on changing the solvents, the spectra still remains unpredictable. Hence, we analyzed the compound using HR-ESI-MS alone.Even the performance of optical activity deemed to be difficult as there are 4 rotamers and the tested compounds optical activity were not reproducible and kept on fluctuating. f 0.35 (SiO 2 , cyclohexane/ethyl acetate: 3/7).HR-ESI-MS: calculated for C 29 H 29 BrN 2 O 4 (M+H + ): 549.1389, found: 549.1401. f 0.31 (SiO 2 , cyclohexane/ethyl acetate: 3/7). HR-ESI-MS: calculated for C 29 H 29 BrN 2 O 4 (M+H + ): 549.1389, found: 549.1365.We performed the reaction with the same quantity and procedure as briefed for Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate.All the obtained data are similar to that of (R)-Benzyl-2-(benzyl(2bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate. (benzyloxycarbonyl)-6-methyl-4-oxopiperidine-2-carboxylic acid (1.5 g, 5.15 mmol, 1.0 equiv) and N-(2-bromobenzyl)-2,2,2-trifluoroethanamine (1.52 mL, 5.66 mmol, 1.1 equiv) in dichloromethane (60 mL) at 0 °C was added N,N-Diethylethanamine (4.3 mL , 30.8 mmol, 6.0 equiv) followed by 1-Propylphosphonic acid cyclic anhydride, 50% (w/w) solution in dichloromethane (24.6 mL, 51.4 mmol, 10.0 equiv). The reaction mixture was continued to stir at room temperature for 14 h. The resulting mixture was diluted with dichloromethane (250 mL), washed with saturated aqueous solution of NaHCO 3 (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give benzyl 2-((2bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate as a colorless oil (1.37 g, 37%). Note: Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans. f 0.36 (SiO 2 , cyclohexane/ethyl acetate: 3/7). HR-ESI-MS: calculated for C 24 H 24 BrF 3 N 2 O 4 (M+H + ): 541.0950, found: 541.0998. (2S,6R)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4f 0.45 (SiO 2 , cyclohexane/ethyl acetate: 3/7) HR-ESI-MS: calculated for C 10 H 19 NO 4 (M+H + ): 541.0950, found: 541.0987. tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6-methylpiperidine-

:R

  Benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate was obtained in 70/30 ration of cis/trans. f 0.17 (SiO 2 , cyclohexane/ethyl acetate: 85/15).

R f 0. 10 (O

 10 SiO 2 , cyclohexane/ethyl acetate: 85/15) HR-ESI-MS: calculated for C 23 H 29 BrN 2 O 3 (M+H + ): 461.1440, found: 461.1454. Chemical Formula: C 23 H 28 N 2 O 3 Molecular Weight: 380.48

[α] D 25 =

 25 -11.1(c=1.05 CHCl 3 ) HR-ESI-MS: calculated for C 20 H 24 N 2 O 2 325.1916, found: 325.1907. (4S,14bS)-2,2-dimethoxy-4-methyl-2,3,4,14b-tetrahydro-1H-dibenzo[b,f]pyrido[1

  of 20% Pd(OH) 2 /C (3.18 g, 30 mmol, 0.5 equiv) in methanol (100 mL), a solution of (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate (19.8 g, 63.6 mmol, 1.0 equiv) in methanol (100 mL), H 2 O (50 mL), acetic acid (20 mL) was added and the mixture was placed on a Parr apparatus and stirred under a hydrogen atmosphere (60 psi) for 24 h. The catalyst was then removed by filtration on Celite. The residue was concentrated in vacuo to give (S)-4-methoxy-4-oxobutan-2-aminium acetate as a pale yellow oil. The crude product thus obtained was engaged into the next step without any purification.

8. 1 , 1 . 5

 115 Hz, H 3 ), 7.22 (1H, t, J = 7.5 Hz, H 7 ), 6.96-7.04 (2H, m, H 4,6 ), 6.77 (1H, dd, J = 8.3Hz, H 5 ).HR-ESI-MS: calculated for C 23 H 26 INO 4 (M+H + ): 323,9647, found: of 2-bromophenol (2 g, 11.56 mmol, 1.0 equiv) in DMF anhydride (16 mL) at room temperature was added with 2-fluorobenzaldehyde (1.43 g, 11.56 mmol, 1.0 equiv) and K 2 CO 3 (2.29 g, 17.34 mmol, 1.5 equiv). The reaction mixture was allowed to reflux and continued to stir for 7 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was diluted with ethyl acetate (200 mL), washed with NH 4 Cl (2x200 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give 2-(2-bromophenoxy)benzaldehyde as a brown oil (3.13 g, 98%). R f 0.74 (SiO 2 , cyclohexane/ethyl acetate: 7/3) 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 10.06 (1H, s, H 13 ), 7.96 (1H, dd, J = 7.8, 1.7 Hz, H 1 ), 7.69 (1H, dd, J = 8.0, 1.5 Hz, H 8 ), 7.50 (1H, ddd, J = 8.1, 7.3, 1.8 Hz, H 2 ), 7.35 (1H, ddd, J = 8.1, 7.5, 1.6 Hz, H 3 ), 7.17-7.23 (1H, m, H 7 ), 7.10-7.16 (1H, m, H 6 ), 7.09 (1H, dd, J = 8.1, 1.5 Hz, H 4 ), 6.75 (1H, dd, J = 8.4, 0.6 Hz, H 5 ). 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 189.93 (C=O, C 13 ), 135.86, 134.31, 129.11, 128.67, 126.37, 123.47, 121.82, 117.04 (Ar-CH), 159.60, 152.57 (Ar-C), 126.20 (C 12 ), 115.57 (Ar-Br, of (S)-tert-butyl 5-(diethoxyphosphoryl)-4-oxopentan-2-ylcarbamate (1.77 g, 5.25 mmol, 1.0 equiv) in THF (50 mL) at room temperature was added Ba(OH) 2 .H 2 O (1.25 g, 6.56 mmol, 1.25 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of 2-(2-iodophenoxy)benzaldehyde (1.7 g, 5.25 mmol, 1.0 equiv) diluted in THF/H 2 O: 40/1 (30 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (150 mL), washed with water (3x150 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (S,E)-tert-butyl 6-(2-(2-iodophenoxy)phenyl)-4oxohex-5-en-2-ylcarbamate (2.08 g, 83%). R f 0.43 (cyclohexane/ethyl acetate: 7/3) Yield: 83% 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 7.93 (1H, J = 16.4 Hz, H 6 ), 7.91 (1H, dd, J = 7.9, 1.6 Hz, H 8 ), 7.69 (1H, dd, J = 7.9, 1.5 Hz, H 17 ), 7.30-7.38 (2H, m, H 10, 16 ), 7.17 (1H, t, J = 7.2 Hz, H 15 ), 6.93 (1H, td, J = 7.6, 1.4 Hz, H 9 ), 6.87 (1H, dd, J = 8.1, 1.4 Hz, H 11 ), 6.85 (1H, d, J = 16.4 Hz, H 5 ), 6.79 (1H, dd, J = 8.3 Hz, 0.8Hz, H 14 ), 5.03 (1H, brs, NH), 4.02-4.17 (1H, m, H 2 ), 2.98 (1H, dd, J = 16.0, 4.8 Hz, H 3 ), 2.80 (1H, dd, J = 16.2, 6.4 Hz, H 3 ), 1.44 (9H, s, H 21 ), 1.24 (3H, d, J = 6.7 Hz, H 1 ). 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 10.72 (C 1 ), 14.75 (C 21 ), 44.20 (C 2 ), 45.76 (C 3 ), 53.61 (C 20 ), 118.21 (C 5 ), 125.57 (C 6 ), 119.71, 124.07, 126.02, 128.35, 128.90, 129.99, 131.91, 135.28, 137.77, 140.25 (Ar-C 11-18 ), 155.73 (C 12 ), 156.02 (C 13 ), 170.61 (C 19 ), 194.70 (C 4 ). HR-ESI-MS: calculated for C 23 H 26 INO 4 (M+H + ): 508.0985, found: 508.0993.To a solution of (S)-tert-butyl 5-(diethoxyphosphoryl)-4-oxopentan-2-ylcarbamate (4.44 g, 13.17 mmol, 1.0 equiv) in THF (80 mL) at room temperature was added Ba(OH) 2 .H 2 O (3.0 g, 16.47 mmol, 1.25 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of 2-(2-bromophenoxy)benzaldehyde (3.65 g, 13.171 mmol, 1.0 equiv) diluted in THF/H 2 O: 40/1 (60 mL) and continued to stir at room temperature for further 45 minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (S,E)-tert-butyl 6-(2-(2-bromophenoxy)phenyl)-4oxohex-5-en-2-ylcarbamate (5.15 g, 85%). R f 0.41 (cyclohexane/ethyl acetate: 7/3) Yield: 85%1 H NMR (400 MHz, C 6 D 6 ) δ (ppm) 8.06 (1H, J = 16.4 Hz, H 6 ), 7.34 (1H, dd, J = 8.0, 1.5 Hz, H 8 ), 7.26 (1H, dd, J = 7.9, 1.5 Hz, H 17 ), 6.83 (1H, ddd, J = 8.2, 7.4, 1.7 Hz, H 10 ), 6.70-6.77 (3H, m, H 9,15,16 ), 6.49-6.59 (2H, m, H 5,11 ), 6.46 (1H, dd, J = 8.2 Hz, 1.0 Hz, H 14 ), 4.85 (1H, brs, NH), 4.10-4.24 (1H, m, H 2 ), 2.57 (1H, dd, J = 16.1, 4.7 Hz, H 3 ), 2.35 (1H, dd, J = 16.1, 6.3 Hz, H 3 ), 1.44 (9H, s, H 21 ), 0.99 (3H, d, J = 6.7 Hz, H 1 ). 13 C NMR (100 MHz, C 6 D 6 ) δ (ppm) 20.50 (C 1 ), 28.55 (C 21 ), 44.03 (C 2 ), 45.06 (C 3 ), 76.57 (C 20 ), 115.43 (C 5 ), 125.99 (C 6 ), 117.84, 121.13, 123.85, 125.63, 128.84, 128.97, 131.51, 134.23, 136.99, 142.17 (Ar-C 11-18 ), 153.63 (C 12 ), 155.94 (C 13 ), 173.19 (C 19 ), 198.22 (C 4 ). HR-ESI-MS: calculated for C 23 H 26 INO 4 (M+H + ): 460.1123, found: 460.1143. [α] D 20 = -10.55 (c=1.00 CHCl 3 ).

(

  3x30 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 85/15) δ (ppm) 7.51 (dd, J = 7.7, 1.2 , 1H, Har),7.47 (dd, J = 7.7, 0.7 , 1H, Har), 7.09 (t, J = 7.7 , 1H, Har), 6.79 (t, J = 7.7, 1H, Har), 5.58 (dd, J = 12.4, 5.1, 1H, H3), 4.85 (qtd, J = 7.0, 3.7 ,1H, H6), 3.91-4.08 (m, 2H, H2), 3.15 (s, 3H, H7), 2.97 (s, 3H, H8), 2.57 (dd, J = 14.3,5.1, 1H, H4), 2.11 (dd, J = 14.1, 7.5 Hz, 1H, H5), 1.93 (dd, J = 14.3, 12.4, 1H, H4), 1.72 (dd, J = 14.1, 3.7 Hz, 1H, H5), 1.40 (d, J = 7,0 Hz, 3H, H9), 0.83(t, J = 7.1, 3H, H1). cis. NMR 1 H (400 MHz, C 6 D 6 ): 7.77 (d, J = 8.2, 1H, Har), 7.46 (dd, J = 7.7, 0.7 , 1H, Har), 7.46 (t, J = 7.7, 1H, Har), 6.76 (m, 1H, Har), 5.56(dd, J = 5.8, 5.4 , 1H, H3), 4.69 (m, 1H, H6),

[α] D 25 =( 2

 252 -11.3(c=1.0 CHCl 3 ) HRMS ESI (M + H) + ion: calc. C 17 H 24 NO 4 Br 386.0861, found. 386.0893 Physical properties : Brown oil Yield : 80%. Cis/Trans : 68/32.

NMR 1 H

 1 (400 MHz, CDCl3) δ :7.58 (dd, J=8.1,1.1, 1H, H13), 7.51 (dd, J=8.0,1.4, 1H, H10), 7.31 (td, J=6.8,0.8, 1H, H12), 7.11(td, J=6.9,1.0, 1H, H11), 4.54 (dd, J=11.4et2.5, 1H, H3), 3.52-3.56 (m, 1H, H6), 3.26 (s, 3H, H7,8), 3.18 (s, 3H, H7,8), 2.12 (dt ,J=13.1, 2.5, 1H, H4e), 1.93 (dt, J=13.1et2.5, 1H, H5e), 1.83 (dd, J=13.4et5.2, 1H, H4a), 1.57 (dd, J=12.1et13.3, 1H, H5a), 1.39 (d, J=6.7, 3H, H9).

  , CDCl3) δ: 7.61 (dd, J=8.2,1.0, 1H, H13), 7.52 (dd, J=8.0,1.4, 1H, H10), 7.29 (td, J=7.0,0.8, 1H, H12), 7.10 (td, J=7.6,1.6, 1H, H11), 4.53 (dd, J=11.2et2.3, 1H, H3), 3.52-3.56 (m, 1H, H6), 3.26 (s, 3H, H7,8), 3.18 (s, 3H, H7,8), 2.22 (dt , J=13.1et2.3, 1H, H4e), 2.03 (dt, J=12.9et2.3, 1H, H5e), 1.38 (dd, J=13.0,12.3, 1H, H4a), 1.24 (dd, J=12.4et13.1, 1H, H5a), 1.12-1.14 (d, J=6.1, 3H, H9).

13 C( 2

 132 NMR (CDCl 3 ): δ(ppm). 21.3(C-9), 40/41(C-7,8), 48.7(C-4), 49.4(C-5), 50.0(C-3), 59.8(C-6), 90.8(C-14), 142/127/126/124(C-ar). H) + ion: calc. C 14 H 20 NO 2 Br 314.0756, found. 314.0771 Physical property : Brown oil Yield : 65%. Cis/Trans : 62/38. Rf = 0.45 Cyclohexane/Ethyl acetate 70/30 trans. NMR 1 H (400 MHz, CDCl3) δ: 7.74(dd, J=8.04,1.1, 1H, H13), 7.38(dd, J=8.15,1.2, 1H, H10), 7.02 (m, 1H, H12), 6.75 (td, J=7.9,1.3, 1H, H11),4.73 (dd, J=11et2.5,1H, H3), 3.21 (s, 3H, H7,8), 3.07 (s, 3H, H7,8), 2.86-2.91 (m, 1H, H6), 2.42 (dt, J=13.4,3.4et1.6, 1H, H5e),1.78 (dt, J=13.0et2.6.2, 1H, H4e), 1.66 (dd, J=13.2et12.1, 1H, H4a), 1.49 (1H, m), 1.24 (d, J=7.0, 3H, H9).

NMR 13 C

 13 (CDCl 3 ): δ (ppm). 20.2(C-9), 36.2/37.8(C-7,8), 45.5(C-4), 47.4(C-5), 51.9(C-3), 59.8(C-6), 98.8(C-14), 118/127/126/147(C-aromatic). Cis: [α] D 25oC = + 27.9(c=1.02 CHCl 3 ) Trans: [α] D 25C = + 10.5(c=1.01 CHCl 3 ) HRMS ESI (M + H) + ion: calc. C 14 H 21 N2O 4 280.1501, found. 280.1511.

NMR 13 C 2

 132 100 MHz CDCl 3 : δ (ppm). 19.5(C-9), 36.2/36.9(C-7,8), 46.6(C-4), 47.2(C-5), 51.5(C-3), 99.8(C-14), 116/127/147(C-aromatic). Physical properties : Brown oil Yield : 86%. Proportion Cis/Trans : 62/38. Rf = 0.30 Cyclohexane/Ethyl acetate 70/30 cis : NMR 1 H (400 MHz, CDCl 3 ): δ7.78 (d, J=7.8, 1H, Har), 7.29 (d, J=7.6, 1H, Har), 7.22-7.01 (m, 4H, Har), 6.77 (m, 1H, Har), 5.17 (dd, J = 7.1, 6.4, 1H, H3), 4.37 (m, 1H, H6), 4.06-3.94

56 6 Chemical 2 Chemical 4

 56624 (400 MHz, CDCl 3 ): δ 7.73 (d, J=8.4, 1H, Har), 7.22-7.01 (m,, 4H, Har), 6.80-6.71 (m, 2H, Har), 5.14 (t, J = 5.3, 1H, H3), 4.53 (m, 1H, H6), 4.06-3.94 (m, 2H, H2), 3.11 (s, 3H, H8), 3.07 (s, 3H, H7), 2.85 (dd, J = 14.7, 4.6, 1H, H4eq), 2.15 (ddd, J = 14.7, 8.2, 1.01, 1H, We performed the reaction with the same quantity and procedure as briefed for (2R,4S,6S)benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. All the obtained data are similar to that of (2R,4S,6S)-benzyl 2-(benzyl(2bromobenzyl)carbamoyl)-4-hydroxy-6-methylpiperidine-1-carboxylate. (2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-Chemical Formula: C 25 H 28 BrF 3 N 2 O 6 S Molecular Weight: 621.46To a solution of (2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-4hydroxy-6-methylpiperidine-1-carboxylate (0.34 g, 0.629 mmol, 1.0 equiv) in DCM (5 mL) at room temperature was added with triéthylamine (0.175 mL, 1.26 mmol, 2.0 equiv) followed by a dropwise addition of methanesulfonyl chloride (0.07 mL, 0.944 mmol, 1.5 equiv) and continued to stir at room temperature for 5 h. The reaction mixture was diluted with DCM (20 mL) washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over MgSO 4 and concentrated in vacuo. This gives ((2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2trifluoroethyl)carbamoyl)-6-methyl-4-(methylsulfonyloxy)piperidine-1-carboxylate as colourless oil. Due to stability issues, without any purification, the crude was engaged into the next step. R f 0.20 (SiO 2 , cyclohexane/ethyl acetate: 5/5).HR-ESI-MS: calculated for C 25 H 28 BrF 3 N 2 O 6 S (M+H + ): 621.0882, found: 621.0931. Formula: C 11 H 21 NO 5 S Molecular Weight: 279.35 To a solution of tert-butyl 4-hydroxypiperidine-1-carboxylate (4.04 g, 20 mmol, 1 equiv) in anhydrous dichloromethane (100 mL) at 0°C was added methylsulfonyl chloride (2.32 mL, 30 mmol, 1.5 equiv), triethylamine (5.57 mL, 40 mmol, 2 equiv). The resulting mixture was stirred at room temperature for 3 h. Saturated NH 4 Cl and 1N HCl were added until pH 2-3 was reached and the aqueous layer is extracted with dichloromethane (3x20 mL). The organic layers were combined, dried over MgSO 4 , filtered and concentrated in vacuo to give 5.6 g (quantitative yield) of tert-butyl 4-(methylsulfonyloxy)piperidine-1-carboxylate as yellow solid. R f = 0.22 (cyclohexane/ethyl acetate : 7/3) 1 H NMR (400 MHz, CDC13) δ(ppm) 4.75-4.82 (m, 1H, H 3 ), 3.60-3.71 (m, 2H, H 2 ), 3.21-3.32 (m, 2H, H 4 ), 2.95(s, 3H, H 6 ), 1.78-1.85 (m, 2H, H 1 ), 1.67-1.78 (m, 2H, H 5 ), 1.35 (s, 9H, of (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-(methylsulfonyloxy)piperidine-1-carboxylate (0.43 g, 0.68 mmol, 1.0 equiv) in anhydrous DMF (3.2 mL) at room temperature was added with sodium azide (0.1 g, 1.37 mmol, 2.0 equiv) and continued to stir at 85 °C for 24 h. The reaction mixture was evaporated in vacuo and the resulting residue was dissolved in DCM (20 mL) and washed with water (2x20 mL), dried over MgSO 4 and finally evaporated in vacuo. This gives (2S,4R,6R)-benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methylpiperidine-1-carboxylate as pale yellow non viscous liquid. Due to stability issues, without any purification, the crude was engaged into the next step. R f 0.76 (SiO 2 , cyclohexane/ethyl acetate: 6/4). HR-ESI-MS: calculated for C 29 H 30 BrN 5 O 3 (M+H + ): 576,1610, found: 576.1671. Formula: C 29 H 32 BrN 3 O 3 Molecular Weight: 550.48To a solution of (2S,4R,6R)-benzyl 4-azido-2-(benzyl(2-bromobenzyl)carbamoyl)-6methylpiperidine-1-carboxylate (0.39 g, 0.68 mmol, 1.0 equiv) in THF (7.5 mL) and H 2 O yellow liquid. Due to stability issues, without any purification, the crude was engaged into the next step. R f 0.65 (SiO 2 , cyclohexane/ethyl acetate: 6/4). )-6-methylpiperidine-1-carboxylate (0.24 g, 0.80 mmol, 1.0 equiv) in THF (8 mL) and H 2 O (0.8 mL) at room temperature was added with triphenylphosphine (0.17 g, 0.83 mmol, 1.2 equiv) and continued to stir at room temperature for . The reaction mixture was evaporated in vacuo and the resulting residue was diluted with ethyl acetate and H 2 O (20 mL). The pH was adjusted to be acidic using 1N HCL (until TLC showed no traces of product) and discarded. The combined aqueous layer was taken to 0°C and the pH carefully was adjusted to 14 with NaOH. Then it was extracted with ethyl acetate (3x30 mL). The combined organic layer was dried over MgSO 4 and concentrated in vacuo. The crude thus obtained was purified using column chromatography on silica (cyclohexane/ethyl acetate: 5/5) to yield (2S,4R,6S)-benzyl 4-amino-2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6methylpiperidine-1-carboxylate. M.P: 62-64 °C HR-ESI-MS: calculated for C 24 H 27 BrF 3 N 3 O 3 (M+H + ): 542.1266, found: 542.1284. (2R,4R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-

:

  calculated for C 29 H 31 BrN 2 O 4 (M+H + ): 551.1545, found: 551.1579.

7/ 3 )

 3 to give (2S,4S,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4-hydroxy-6methylpiperidine-1-carboxylate as a yellow oil (0.37 g, 97%).R f 0.11 (SiO 2 , cyclohexane/ethyl acetate: 7/3). HR-ESI-MS: calculated for C 29 H 31 BrN 2 O 4 (M+H + ): 551.1545, found: 551.1526. of (2S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate (0.35 g, 0.647 mmol, 1.0 equiv) in methanol (8.5 mL) at 0 °C was added slowly in portion wise powdered sodium borohydride (0.29 g, 0.775 mmol, 1.2 equiv). The reaction mixture was continued to stir at room temperature for 30 mins. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (30 mL), washed with water (3x30 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (2S,4S,6S)-benzyl 2-((2-bromobenzyl)(2,2,2-trifluoroethyl)carbamoyl)-4hydroxy-6-methylpiperidine-1-carboxylate as a yellow oil (0.35 g, quantitative). R f 0.13 (SiO 2 , cyclohexane/ethyl acetate: 7/3). HR-ESI-MS: calculated for C 24 H 26 BrF 3 N 2 O 4 (M+H + ): 543.1106, found: 543.1159.

:

  calculated for C 29 H 31 BrN 2 O 4 (M+H + ): 551.1545, found: 551.1526. [α] D 25 = -4.7 (c 1.01, CHCl 3 ). (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6-methylpiperidine-2-carboxamide

1 H

 1 NMR (400 MHz, DMSO-d 6 ) δ (ppm) 1.44 (9H, s, 9H), 1.72 (2H, d, J = 10.4 Hz), 2.13 (2H, dq, J = 12.4 Hz, 4.4 Hz), 2.78-2.84 (2H, m), 4.10 (2H, J = 12.0 Hz, d), 4.32-4.38 (1H, m), 6.99 (1H, dd, J = 7.6 Hz, 5.2 Hz), 7.53 (1H, dd, J = 8.0 Hz, 0.8 Hz), 7.90 (1H, dd, J = 5.2 Hz, 1.2 Hz), 11.54 (1H, s). CAS Registry Number: 781649-87-4.The analysis is in accordance with the literature.1-(Piperidin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one dihydrochloride -4-(2-oxo-2,3-dihydro-1H-imidazo[4,5-b]pyridin-1-yl)piperidine-1-carboxylate(5.0 g, 15.7 mmol) and HCl (4.0 M in EtOH; 2.50 mL) were heated to 50-55 °C for 10 h. The reaction mixture was cooled to ambient temperature and the resulting slurry filtered. The cake was washed with ethanol (100 mL) and dried at 50 °C in vacuo to afford 4.28 g, 94% of 1-(Piperidin-4-yl)-1H-imidazo[4,5-b]pyridin-2(3H)-one dihydrochloride as an off-white solid. 1 H NMR (400 MHz, DMSO-d 6 ) δ 1.87 (2H, d, J = 12.4 Hz), 2.60-2.69 (2H, m), 3.09 (2H, q, J = 12.0 Hz,), 3.40 (2H, d, J = 12.4 Hz), 4.60 (1H, dt, J = 12.4 Hz, 3.6 Hz), 7.05 (1H, dd, J = 7.6 Hz, 5.2 Hz), 7.89-7.94 ( 3H, m), 9.14 (1H, brs), 9.48 (1H, brs),11.67 (1H, brs). CAS Registry Number: 1209456-28-9.

1 H

 1 NMR (CDCl 3 , 400 MHz) δ (ppm) 3.87-3.76 (m, 3H, H 3 , H 2 ), 3.02 -2.96 (m, 2H, H 4 ), 2.13 (br, 1H, H 6 ), 1.85 -1.79 (m, 2H, H 1 ), 1.47 -1.38 (m, 2H, H 5 ), 1.43 (s, 9H, H 7-9 ). CAS Registry Number: 109384-19-2.The analysis is in accordance with the literature.

  (1H, t, J = 16.3, 4.6 Hz ), 2.36 (1H, m), 2.57 (1H, m, H 6 ), 2.64 (1H, m), 3.63 (1H, dd, J = 15.3 Hz, H 2 ), 6.55-6.67 (1H, m, Har), 6.88-6.96 (1H, m, Har), 6.94-7.03 (1H, m, Har), 7.04-7.18 (1H, m, Har), 7.30 (1H, m, Har), 7.440 (1H, d, J = 8.0 Hz; Har), 7.50-7.69 (1H, m, Har).

[α] D 25 =

 25 -29.1(c=1.00 CHCl 3 ) HR-ESI-MS: calculated for C 18 H 18 N 2 O 279.1916, found: 279.1968.

1 H

 1 NMR (400 MHz, CDCl 3 ) δ (ppm) 1.04 (1H, m), 1.31 (1H, m), 1.73 (1H, m), 1.92 (2H, m), 3.38 (1H, J = 11.2 Hz, H4), 6.55-6.67 (1H, m, Har), 6.88-6.96 (1H, m, Har), 6.56 (1H, m, Har), 6.96 (1H, m, Har), 7.14 (1H, m, Har), 7.44 (1H, d, J = 8.0 Hz; Har), 7.56 (1H, m, Har).

13 C

 13 NMR (100 MHz, C 6 D 6 ) δ (ppm) 22.60 (C7), , 39.17 (C3), 43.63 (C5), 50.35 (C6), 59.30 (C2), 69.35 (C4), 112.93, 116.01, 119.39, 120.62, 121.41, 126.88, 127.94, 128.87, 132.22, 133.70, 142.31, 142.52(Car).

[α] D 25 = 20 O

 2520 -15.1(c=1.10 CHCl 3 ) HR-ESI-MS: calculated for C 18 H 20 N 2 O 281.2314, found: 281.2328.Chemical Formula: C 18 H 17NO 2

[α] D 25 =

 25 -9.6(c=1.00 CHCl 3 ) HR-ESI-MS: calculated for C 18 H 17 NO 2 280.5471, found: 280.5401.

1 H

 1 NMR (400 MHz, CDCl 3 ) δ (ppm) 1.16 (1H, m), 1.56 (1H, m), 1.88 (1H, m), 2.21 (2H, m), 3.66 (1H, J = 11.2 Hz, H4), 4.03 (1H, d, J = 11.5, 4.3 Hz, H2) 7.21 (2H, m, Har), 7.32 (2H, m, Har), 7.54 (1H, m, Har), 7.71 (1H, m, Har), 7.79 (1H, m, Har), 7.85 (1H, d, J = 8.0 Hz; Har).

[α] D 25 =

 25 -25.1(c=1.15 CHCl 3 ) HR-ESI-MS: calculated for C 18 H 19 NO 2 282.0017, found: 282.0178.

F 41 Similar

 41 Chemical Formula: C 35 H 37BrF 3 N 7 O 5 Molecular Weight: 772.61 HR-ESI-MS calcd for C 35 H 37 BrF 3 N 7 O 5 (M+H + )=773.0123, found=773.0238. tert-Butyl carbamate deprotection method as that of 155' 1 H NMR (400 MHz, CD 3 OD) δ (ppm) 1.77 (4H, m), 2.10 (2H, t, J = 15.5), 2.31 (2H, q, J = 12.2 Hz), 3.01 (5H, dt, J = 25.3, 12.3 Hz, 1H), 3.43 (2H, d, J = 12.7 Hz), 4.23 (2H, d, J = 13.2 Hz), 7.08 (1H, S, Har), 7.96 (1H, s), 8.90 (2H, s). HR-ESI-MS calcd for C 19 H 25 F 3 N 6 O 4 (M+H + )=345.2039, found=345.2039. Hydrogenation procedure as followed in (2R,4R,6R)-N,N-dibenzyl-4-hydroxy-6methylpiperidine-2-carboxamide. Physical property: white solid M.P: 163-165 °C.

O

  Chemical Formula: C 33 H 39 N 7 O 3

Table 1 .

 1 Showing comparison of different primary afferent nerve fibres ............................................

Table 2 .

 2 Symptoms in migraine patients ................................................................................................

Table 3 .

 3 Structure & activity of triptans in clinical stage ........................................................................

Table 4 .

 4 showing different monoamine neurotransmitters ..................................................................

Table 5 .

 5 Antidepressants based on their generation .............................................................................

Table 6 .

 6 Antidepressants based on their mechanism ............................................................................

Table 7 .

 7 Antidepressant's effectivity against chronic pain.....................................................................

Table 8 .

 8 CALCRL interaction with various proteins generated using STRING-8 software[13] ..............

Table 9 .

 9 Druggability and Tractability scoring criterion [18] ..................................................................

Table 10

 10

.Visualizing the druggable sites of CALCRL & RAMP-1 generated by DrugEBility® software [18] ............................................................................................................................................................... Table 11.showing the total scores of family 1 potential hits ................................................................ Table 12.showing the total scores of family 2 potential hits ................................................................ Table 13.showing the total scores of family 3 potential hits ................................................................ Table 14.Showing the volume of potential hits in comparison with the druggable & tractable sites of CGRP receptor [28] ................................................................................................................................ Table 15.showing the drug-likeliness of potential hits generated using Molsoft® ............................... Table 16.Showing the effect of configuration modulation from 38 to 39 .......................................... Table 17.Showing the reaction scale and substrates used by Sutherland et al. ................................. Table 18.showing optimization to form compound 7 ......................................................................... Table 19.showing splitting pattern with coupling constants for cis piperidine .................................. Table 20.Showing the effect of solvent on rotational barrier .............................................................

Table 21 .

 21 conditions to synthesize compounds 11 and 12 ..................................................................

Table 22 .

 22 Showing the optimization conditions used for coupling the synthon A & B ........................

Table 23 .

 23 showing application deprotection in route B to obtain synthon D ......................................

Table 24 .

 24 showing optimization of cross coupling reaction .................................................................

Table 25 .

 25 showing application of cross coupling reaction ...................................................................

Table 26 .

 26 showing the employed coupling agents (-not worked, *not tried) ......................................

Table 27

 27

.Showing the employed substrates for DEPBT mediated coupling ....................................... Table 28.showing the 4th position affecting C-N cross coupling ........................................................ Table 29.showing the deprotection of ketal to ketone .......................................................................

Table 30 .

 30 showing different PGs used with their yield ........................................................................

Table 31 .

 31 showing different ketophosphonates with their yield .........................................................

Table 32 .

 32 showing different biaryl aldehydes with their yield .............................................................

Table 33 .

 33 showing different alkenes with their yield ...........................................................................

Table 34 .

 34 showing application of Michael intramolecular reaction for ethyl and benzyl carbamate PGs .............................................................................................................................................................

Table 35 .

 35 showing application of Michael intramolecular reaction for tert-Butyl carbamate PG .......

Table 36 .

 36 Ratios of piperidine formed ..................................................................................................

Table 37 .

 37 showing different various Buchwald-Hartwig conditions ....................................................

Table 38 .

 38 Showing the variance of substrate .......................................................................................

Table 39

 39

.showing contributors for Buchwald-Hartwig C-N coupling ..................................................

Table 40 .

 40 Showing problems and how they can be solved according to Buchwald ............................

Table 41

 41

.showing different various Buchwald-Hartwig conditions we tried ...................................... Table 42.Showing the alkylation of subclass A with different conditions ........................................... Table 43.Cyclization optimization for subclass C ................................................................................ Table 44.showing the deprotection substrates and products ............................................................ Table 45.showing the activation of alcohols using mesylate for family 1 ........................................... Table 46.showing the conversion of mesylate to amine for family 1 ................................................. Table 47.showing the route A to obtain the carbonate CGRP antagonists.........................................

Table 48.showing the route B we used to obtain the carbamate CGRP antagonists ......................... Table 49.showing time of response before and after administration of antagonists in Ist batch ...... Table 50.showing time of response before and after administration of antagonists in IInd batch.... Table 51.showing cut-off counts for administration of antagonists in Ist batch ................................

Table 52 .

 52 showing cut-off counts for administration of antagonists in IInd batch ..............................

Table 53 .

 53 showing immobilization times on administration of antagonists in Ist batch .....................

Table 54 .

 54 showing immobilization times on administration of antagonists in IInd batch ...................

  Table.1).

	Conduction velocity	> 40 ms -1	5-15 ms -1	< 2 ms -1
	Character	A-β fibres	A-δ fibres	C fibres
	Function	Motor	Sensory	Autonomic
	Diameter	Large	Small 2-5µm	Smallest <2µm
	Myelinated	Highly	Thinly	Unmyelinated

Table 2 .Symptoms in migraine patients

 2

Table 3 .Structure & activity of triptans in clinical stage

 3

[73]: 2.1 Major depressive disorder or Major depression: It

	disables a person from
	functioning normally. It interferes in every aspect of patient's life like working, sleeping,
	eating, studying etc. Some people may experience an episodic form of major depression
	only once in their lifetime but mostly it recurs as multiple episodes in most of the patients.

2.2 Dysthymia or Dysthymic disorder: It

	won't disable a person from normal
	functioning and shows the symptoms of depression (mildly intense) for long-term i.e. 2
	years or more. People with dysthymia experiences one or more episodes of major depression
	in their life time.

Efficacy Evidence based support Dose Side effect loading

	Tertiary amine tricyclic			Low to
	antidepressants	Yes	+++	standard	+++
	(Amitriptyline, Imipramine)
	Venlafaxine	Yes	++	Standard	+
	Duloxetine	Yes	++	Standard	+
	Bupropion	Yes	+	Standard	+
	Secondary amine tricyclic	Yes	++	Low to	+++
	antidepressants(Desipramine,			Standard
	Nortriptyline)
	Paroxetine, citalopram	Modest	+	Standard	+
	Fluoxetine	No	--	Standard	+

Table 7 .Antidepressant's effectivity against chronic pain 2.3 Serotonin Norepinephrine Reuptake inhibitors (SNRIs) Showed

 7

	better efficacy

Table 8 )

 8 ;

	Partner protein	Score
	RAMP-1 (Receptor Activity Modifying Protein-1)	0.999
	CALCA (Calcitonin related peptide alpha)	0.995
	RAMP-2 (Receptor Activity Modifying Protein-2)	0.994

Table 8 .CALCRL interaction with various proteins generated using STRING-8 software [13]

 8

Molecular Weight Hydrogen Bond Donors Hydrogen Bond Acceptors Calculated LogP Number of rotatable bonds Others

	Tractability 200<MW<800	≤ 8	≤ 15	≤ 8	≤ 16	No metal,
						sugar,
	Druggability 100<MW<550	≤ 5	≤ 10	≤ 5	≤ 10	carbohydrates

Table 9 .Druggability and Tractability scoring criterion [18]

 9

Table 10 )

 10 , it shows quite clearly that Olcegepant® is interacting in the vicinity of the site 1 of CALCRL whereas RAMP-1 has a tractable site 1 which opens the port for persuading new and important interactions.

	probable binding sites of a protein. As CGRP receptor is a heterodimeric protein, two
	individual runs on both CALCRL & RAMP-1 is required.

CALCRL Site Site on protein V (Å 3 )

		4		300.3	0.00	0.96 0.00	0.86	-0.99
				(V-volume, D-druggable, C-confidence, T-tractable, E-ensemble)
					D	C	T	C	E
		1		674.1	0.00	0.73 0.00	0.71	-0.98
		2		498.2	0.00	0.96 0.00	0.86	-0.99
		3		288.9	0.00	0.96 0.00	0.86	-0.99
		4		207.5	0.00	0.96 0.00	0.86	-0.99
		1		1468.5	0.00	0.73 1.00	0.83	-0.98
	RAMP-1	2 3	RAMP-1	434.9 394.8	0.00 0.00	0.96 0.00 0.96 0.00	0.86 0.86	-0.99 -0.97

Table 10 .Visualizing the druggable sites of CALCRL & RAMP-1 generated by DrugEBility® software [18]

 10

Table 14 .Showing the volume of potential hits in comparison with the druggable & tractable sites of CGRP receptor [28]

 14

Table 15 .showing the drug-likeliness of potential hits generated using Molsoft®

 15

+ NH 2 CO 2 Me C 9 H 19 NHCbz OsO 4 NaIO 4 C 9 H 19 O NH 16 15 D-proline

				Cbz		Cbz
						NH	OH	O	NH	OH	O
							+
	Acetone	C	9 H	19		C	9 H 19
							17b
						17a
						OH
							H 2 , Pd/C
	H	2 , Pd/C
							17b
	17a
		95%	C 9 H 19	N	95%	C 9 H	N
							H
						H

19 OH (-)-241D (-)-epi 241D H Cbz Scheme 7.Showing the application of reductive amination by Chandrasekhar and coll.

2 , Pd/C N H C 9 H 19 OH H 2 , Pd/C N H C 9 H 19 OH (+)-241D (+)-epi-241D H 2 N S O (S)-tert-butane sulfinamide C 9 H 19 H

	O				O
	Decanal + 2. CbzCl, Na 2 CO 3 1. 4M HCl Acetone NaOH H N 22a S 18 C 9 H 19 NH Cbz C 9 H 19 NH Cbz OH 20	O	In, THF Br RuCl 3 NaIO 4 C 9 H 19 Cbz +	22b C 9 H 19 HN C 9 H 19 Cbz NH OH 19	S NH O 21	O	d.r : 96/4 H
	22b		22a

H 19 OH (+)-241D TiCl 4, (S,S)-BINOL 23 1. TsCl, pyridine Br d.r : 96/4 e.e : 98% 1. LiAlH 4 /THF 2. CbzCl, Na 2 CO 3 C 9 H 19

  which gave homoallylic alcohol 23 in an enantiomeric excess of 98%.

	Decanal	OH			N 3
		Ag 2 O, allyltributyltin C 9 H 19			C 9 H 19
						24
	26	H 2 , Pd/C C 9 H 19 NH Cbz O C 9 NH AD-mix-beta tBuOH-H 2 O OH 25 26 1. NaH/THF, 0 °C, TsCl Cbz NH Cbz 2. ,CuI MgBr C 9 H 19	OH 80% C 9 H	N H +	C 9 H 19 Cbz	NH 27	OH	OH 20%

19 NH Cbz OH 28 PdCl 2 , CuCl O 2 22b C 9 H 19 NH Cbz OH O Scheme 9.Showing the application of reductive amination by Rao and coll.

Table 16 .Showing the effect of configuration modulation from 38 to 39

 16 table.

	compound	R 1	R 2	Resultant molecule	Configuration Percentage
	38 (S,S)			39 (R,S)	2, 6 cis	87
	38 (R,R)	Methyl Benzyl	39 (S,R)	2, 6 cis	85
	38 (S,R)			39 (R,R)	2, 6 trans	55
	38 (R,S)			39 (S,S)	2, 6 trans	58

  Michael cyclization. In this case, the absolute configuration on asymmetric centres were controlled during the construction of the intermediate 63 which later was cyclised without any catalyst to give exclusively 2, 6 cis piperidine 64.

	Bn	Cbz
	O HN	O	1. TFA, i PrOH	OH	O
			2. H 2 ,Pd/C	N H
		63		64

Scheme 21.Showing the synthesis of (-)-andrachcinidine by Krishna and coll.

solenopsine A Scheme 24.Showing the application of Aza-Michael by Troin and coll.

						MeO	OMe
				MeO	OMe
	Cbz
	NH O
		MeOH
						+
	C 9 H 19	pTSOH			N	C 9 H 19	N	C 9 H 19
		CH(OCH 3 )	3	Cbz	Cbz
	70						72
					71
							71 / 72 : 15 / 85
					OH
	72
					N	C 9 H 19
					H
				(+)-241D
						O	O
				O	O
	Cbz
	NH O	(CH 2 OH)	2
						+
	C 11 H 23	pTSOH			N	C 11 H 23	N	C 11 H 23
		CH(OCH 3 ) 3	Cbz	Cbz
	73						75
					74
							74 / 75 : 82 / 18
	74
					N	C 11 H 23
					H
				(-)-

/ MeOH 2) 6N HCl, reflux Scheme 32.Showing the synthesis of compound 108 proposed by Haufe and coll.

	tBu	N N H 3 C H	H	O	N N Boc tBu 104 -HF F t Bu tBu O H 3 C	N N H N H O 107	LDA/THF F OTs 105 O F O NH 1) NaBH4 tBu Boc N N t Bu	O 106 N H F tBu F 95%, 99% e.e MeOH/H 2 O HCl 4N O NH t Bu N H OH O OH 108	F	N H	OH	O	NH

2 Et Cl OH 109 CO 2 Et NC OPMB 110 NaBH 4 NiCl 2 CO 2 Et OPMB H 2 N O N H O OPMB 111 1) nBuLi, ClCO 2 Me 2) KHMDS, PhNTf 2 N OTf OPMB 112 CO 2 Me Pd(OAc) 2 Ph 3 P, CO, MeOH N CO 2 Me OPMB CO 2 Me N CO 2 Me OTBDMS CO 2 Me 113 N H OH CO 2 H .HCl 89. HCl 1) H 2 , Pd/c 2) 2N HCl Scheme 33.Showing the synthesis of compound 89 proposed by Occhiato and coll.

  3-hydroxy-ethylbutanoate 109. Transformation of chloro functionality to a nitrile followed by protection of alcohol gave compound 110. The hydrolysis of the nitrile functionality to form an amide resulted in intramolecular cyclization

	N	O	N	CO 2 Me	N
	CO 2 Me	CO 2 Me	CO 2 Me
	114		115

giving away 2-piperidone 111. This compound 111 was later protected to form N-carbamate and then to an enol triflate which, by methoxy carbonylation reaction catalyzed by palladium salt, furnished compound 112. Changing of protection group of alcohol (e.g 113) succeeding by a hydrogenolysis of the double bond and deprotection gave the compound 89 in salt form.

CO

The same research group has proposed another method which allows accessing a new way to synthesize 4-hydroxy-pipecolic acid using biocatalysts

[52]

. Starting from N-protected 2-piperidone 114, and as described before, a palladium catalyzed methoxy carboxylation resulted in the formation of an enamine 115 which on subsequent allylic oxidation gave an allylic alcohol 116.

CO 2 Me OH 116 Scheme 34.Showing the synthesis of compound 116 proposed by Occhiato et al.

  Kinetic de-racemization of 116 in presence of the enzyme lipase (Candida antarctica) preferentially gave the isomer R with good enantiomeric excess.

			O
	OH		O	Me	OH
		Lipase
				+
	N	CO 2 Me	N	CO 2 Me	N	CO 2 Me
	CO 2 Me	CO 2 Me	CO 2 Me
	116		117		118
	Scheme 35.

Showing the synthesis of compounds 117 and 118 proposed by Occhiato et al.

  After the separation of mixture, the isomers 117 and 118 were transformed into corresponding pipecolate esters 120 and ent-120 in few steps.

	O
	O	OMe	OH		OH
		NaOMe
		MeOH
	N	CO 2 Me	N	CO 2 Me	N	CO	2 Me
					H
	CO 2 Me	CO 2 Me
					(2S, 4R)
	117		119		120
		OH		OH
		N	CO 2 Me	N	CO 2 Me
				H
		CO 2 Me
				(2R, 4S)
		118		ent-120

Scheme 36.Showing the synthesis of compound 120 proposed by Occhiato and coll.



Riera and coll. has used chiral epoxyde 121 to introduce the amine counterpart for providing compound 122 This was followed with a metathesis reaction (GRUBBS 2 nd generation) which allowed to recover compound 123. Oxydative cleavage with sodium periodate followed by iodolactonisation reaction gave 124 which dehalogenation reaction with tributyl tin provided bicyclic compound 125 Finally, basic hydrolysis and deprotection of the amino group gave (2S,4R)-4-hydroxy pipecolic acid 89

[53]

.

Scheme 37.Showing the synthesis of compound 89 proposed by Riera and coll.

	121	122		123
	4, THF/H 2 O	I 2 , KI, NaHCO 3
	2) NaClO 2 , t-BuOH/THF
		124		125
	Bu 3 SnH, AIBN	NaOH/H 2 O	TFA	NH
			HO	COOH
	126	127		89



Merino and coll. has used the dimethyl acetal of glyceraldehyde 128 as source of chirality. Condensation of benzyl hydroxylamine gave nitrone 129 which was alkylated with allyl magnesium bromide in the presence of ZnBr 2 , furnishing compound 130 in excellent yield.

Oxydation with MnO 2 gave a new nitrone 131 which cyclized in toluene to the oxazolidine 132. Functionnal group interconversion led to 133 which, by treatment with zinc in acidic medium provided the (2R,4S,6S) 6-phenyl-4-hydroxy pipecolic acid 134

[27]

.

Table 17 .Showing the reaction scale and substrates used by Sutherland et al.

 17

Table 18 .showing optimization to form compound 152 Hence

 18 Thus formed compounds 155 & 156 are cis/trans isomer and both of them are easily separable through normal phase column chromatography. In our case, we experienced relative amounts of formation of cis and trans isomers as 75/25 (calculated using 1 H NMR) and this can be attributed for cis being thermodynamically stable form where as trans being kinetically stable. And hence the initial formation of trans isomer slowly results in the conversion into cis form being thermodynamically stable. But this conversion depends on various factors like time, temperature, substituants etc. and hence results in not complete transformation into cis

conditions 2 & 3 are well optimized and reproducible compared to 1 even thought it gave compound 152 instead of desired products 150 & 151.

form, instead leaving some portion of trans also in the final mixture.

Table 20 .Showing the effect of solvent on rotational barrier

 20

Table 21 .conditions to synthesize compounds 11 and 12

 21

Table 22 .Showing the optimization conditions used for coupling the synthon 169 & 160

 22 The coupled synthon 170 has been forwarded to the next step where the deprotection of tert-butyl carbamate was carried. A variety of conditions have been used to optimize this step

	as follows
				Br				Br		O		Br
	O	O			O	O
		N Boc	O	N	Condition	N H	O	N	+	N H	O	N
		171				172				173

Thus developed condition has been applied onto the synthon 160 & 165 to synthesize 171.

Scheme 59.showing application deprotection in route B to obtain synthon E

	Conditions	Yield (%)	172	173
	TFA, dry DCM	90	4	1
	Bu 4 NF, THF	-	-	-
	BF 3 , dry THF	62	1	99

Table 23 .showing application deprotection in route B to obtain synthon E

 23 Thus, we selected the deprotection of the piperidine condition to be TFA/DCM (90%) was carried out prior to intramolecular coupling with 10 mol% of Pd(OAc) 2 and 30 mol% Xantphos. We tried a range of conditions to achieve the desired product

	Catalyst	Ligand	Base	Temperature Time (h)	Solvent	Yield (%)
						Dioxane	-
						DMSO	-
	Pd(OAc) 2 Xantphos Cs 2 CO 3	110 °C	48h	Toluene	54-63%
						Distille Toluene	80%
	CuI					Toluene	-

Table 24 .showing optimization of cross coupling reaction

 24

Table 26 .showing the employed coupling agents (-not worked, *not tried)

 26

Table 29 .showing the deprotection of ketal to ketone

 29

Table 30 .showing different PGs used with their yield

 30

Table 31 .showing different ketophosphonates with their yield

 31

Table 35 .showing application of Michael intramolecular reaction for tert-Butyl carbamate PG

 35

, H 2 MeOH 1 atm, RT Quantitativ e R R=H, Cbz N O O NO 2 R R=H, Cbz 225, 228 + N H O O NH 2 + IIIA 244 Scheme 99.Showing formation of amine

			O	O	O	O
	N Pd/CN° NO 2 Ketone Cyclisation Reduction	N H	NH 2 Total Yield(%)
			Yield (%)	Yield(%)
			(cis/trans)	(cis/trans)
	1	225	65 (67/33)	Quantitative (67/33)	65
	2	228	76 (72/28)	Quantitative (72/28)	76

3. It can be prepared in two-step process. The first step involves reduction and second step is a C-N Palladium cross-coupling reaction using 1,2-dibromobenzene [107]. The Buchwald-Hartwig reaction have been choosen, as the prime C-N bond forming reaction for all the molecules we

have synthesized because of its applicability on our substrate as portrayed during the optimization of family 1. When the protecting group is tert-Butyl carbamate, the starting material can be obtained in two-step process, a cyclisation and in situ deprotection followed by hydrogenation from nitro to amine.Whereas, if the PG is benzyl carbamate, after cyclization, hydrogenation can be performed to deprrotect and reduce nitro to amine in situ.

Table 36 .Ratios of piperidine formed

 36

Table 37 .showing different various Buchwald-Hartwig conditions

 37

Table 38 .Showing the variance of substrate

 38

Table 39 .showing contributors for Buchwald-Hartwig C-N coupling

 39

					No additives
				No additives required	Active species	required, reduction of Pd not required
					L	1 Pd0)	Pd	2 (dba) 3
		NH	2	Not applicable	dba can bind
	Pd			for all ligands	Pd and poison
	L	Cl			reaction
				Anilines and
	L=Monodentate ligands	amides needs exogenous	Economic
				reductants
				Pd(OAc)
					2

 Although Pd(OAc) 2 is attractive even on large scale amination, its reactivity poses some drawback for using on lot of substrates

[113]

. On contrary, preheating Pd 2 (dba) 3 , ligand and base in solvent prior to the addition of substrates is highly beneficial and thus highlights the formation of the L 1 Pd complex

[114]

.

Table 41 .showing different various Buchwald-Hartwig conditions we tried

 41

Table 43 .Cyclization optimization for subclass C

 43

Table 49 .showing the route B we used to obtain the carbamate CGRP antagonists

 49

Table 54 .showing immobilization times on administration of antagonists in 1st batch

 54

Table 55 .showing immobilization times on administration of antagonists in 2nd batch

 55

  was washed with water (3x100 mL) & dried over MgSO 4 and then concentrated in vacuo to give yellow viscous oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 7/3) to give (R)-dimethyl 2-(tert-butoxycarbonylamino)succinate as a white crystalline sticky solid (2.16 g, 82%).

	R f 0.12 (SiO 2 , cyclohexane/ethyl acetate: 7/3).
	Mp: 61-62 °C.
	1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1 .40 (9H, s, H 5, 6, 7 ), 2.70-3.10 (2H, m, H 3 ), 3.69 (3H,
	s, OCH 3 ), 3.76 (3H, s, OCH 3 ), 4.59 (1H, m, H 2 ).

13 C NMR (100 MHz, CDCl 3 )

  δ (ppm) 18.4 (C 6 ), 44.6 (C 3 ), 51.60 (C 1 ), 53.36 (C 2 ), 71.01 (C 7 ), 126.33, 127.68, 128.59, 131.90, 143.28 (CH ar ), 145.63 (C ar ), 174.20 (C 9 ), 197.04 (C 8 ).

	[α] D 25 = -8.7 (c 0.975, CHCl 3 ).
	HR-ESI-MS: calculated for C 27 H 28 NO 3 (M+H + ): 414.2069, found: 414.2088.
	(S,E)-methyl 4-oxo-2-(tritylamino)hept-5-enoate 149'
	6	5
		4	O	O	1
			3
				2	O
			NH
	Chemical Formula: C 27 H 27 NO 3
	Molecular Weight: 413.50

4-dimethoxy-6-methylpiperidine-2-carboxylate

  1313240-35-5.The analysis is in accordance with the literature. °C. The mixture was set to reflux and stirred for 16 h.The reaction mixture was cooled down to room temperature and later concentrated in vacuo.

	1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.92 (3H, dd, J = 6.8, 1.7 Hz, H 6 ), 2.69 (1H, dd, J = 15.4, 7.0 Hz, H 3 ), 2.82 (1H, dd, J = 15.3, 5.2 Hz, H 3 ), 2.89 (1H, J = 10 Hz, NH), 3.29 (3H, s, OCH 3 ), 3.72-3.80 (1H, m, H 2 ), 6.10 (1H, dq, J = 15.8, 1.6 Hz, H 4 ), 6.80 (1H, dq, J = 15.8, 6.8 Hz, H 5 ), 7.17-7.27 (5H, m, Ar-H), 7.29-7.36 (4H, m, Ar-H), 7.48-7.54 (6H, m, Ar-H). H O O O O Chemical Formula: C 10 H 19 NO 4 Molecular Weight: 217.26 Thionyl chloride (0.47 mL, 6.35 mmol, 0.9 equiv) was added dropwise over 10 min to a stirred mixture of sodium (6R)-6-methyl-4-oxopiperidine-2-carboxylate (1.1 g, 7.06 mmol, 1.0 equiv) and Methanol (10 mL) at 0 The resulting residue was diluted with dichloromethane (100 mL), washed with 1N NaOH (3x100 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 3/7) to give methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate as a brown oil (1.21 g, 91%). Note: Methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate gives 75/25 ration of cis/trans 1. (2R,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate 155 N H O O O O 1 2 3 4 5 6 7 8 9 10 11 R f 0.3 (SiO 2 , cyclohexane/ethyl acetate: 3/7). Hz, 3H, H 7 (CH 3 )), 1.37 (dd, J = 16.1, 9.2 Hz, 1H, H 3a ), 1.97 (dt, J = 13.3, 2.7 Hz, 1H, H 5e ), 2.34 (dt, J = 13.1, 2.8 Hz, 1H, H 3e ), 2.78-2.88 (m, 1H, H 6a ) 3.17 (3H, s, OCH 3 ), 3.21 (3H, s, O-CH 3 ), 3.55 (dd, J = 12.2, 2.8 Hz, H 2a ), 3.72(s, 3H, H 11 (CH 3 )). 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 22.15 (C 7 ), 35.78 (C 3 ), 40.61 (C 5 ), 47.52 (C 8 ), 48.03 (C 9 ), 52.13 (C 6 ), 55.95 (C 11 ), 57.96 (C 2 ), 99.27 (C 4 ), 173.30 (C 10 ). [α] D 20 = -2.6 (c 1.0, CHCl 3 ). HR-ESI-MS: calculated for C 10 H 19 NO 4 (M+H + ): 218.1392, found: 218.1402. 2. (2S,6R)-methyl 4,4-dimethoxy-6-methylpiperidine-2-carboxylate 156 N H O O O O 1 2 3 4 5 6 7 8 9 10 11 R f 0.16 (SiO 2 , cyclohexane/ethyl acetate: 3/7). 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.02 (dd, J = 13.0, 2.7 Hz, 1H, H 5a ), 1.11 (d, J = 6.5 Hz, 3H, H 7 (CH 3 )), 1.25 (dd, J = 16.0, 3.7 Hz, 1H, H 3a ), 1.85 (dt, J = 13.1, 2.8 Hz, 1H, H 5e ), 2.19 (dt, J = 13.2, 8.8 Hz, 1H, H 3e ), 2.98 (3H, s, OCH 3 ), 3.14 (3H, s, O-CH 3 ), 3.17-3.26 (m, 1H, H 6a ), H), 3.71 (s, 3H, H 11 (CH 3 )). 3.80 (dd, J = 6.2, 1.5 Hz, H 2e ) 13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 21.03 (C 7 ), 36.51 (C 3 ), 39.33 (C 5 ), 47.10 (C 8 ), 47.79 (C 9 ), 51.28 (C 6 ), 56.89 (C 11 ), 57.31 (C 2 ), 98.74 (C 4 ), 172.11 (C 10 ). [α] D 20 = + 3.1 (c 1.05, CHCl 3 ). HR-ESI-MS: calculated for C 10 H 19 NO 4 (M+H + ): 218.1392, found: 218.1398. (2R,6R)-methyl 6-methyl-4-oxopiperidine-2-carboxylate 155' N H O O 1 2 3 4 5 6 7 8 9 O Chemical Formula: C 8 H 13 NO 3 CAS Registry Number: Methyl 4,N 1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.08 (dd, J = 13.1, 2.8 Hz, 1H, H 5a ), 1.13 (d, J = 6.4 Molecular Weight: 171.19

  4-oxopiperidine-2-carboxylate (1.25 g, 7.01 mmol, 1.0 equiv) in methanol (250 mL) at room temperature was added with di-tert-butyl dicarbonate (2.29 g, 10.52 mmol, 1.5 equiv). The reaction mixture was continued to stir in refluxed condition for 16 h and concentrated in vacuo. The resulting residue was diluted with water (until pH 6), extracted with ethyl acetate (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica The attribution of chemical shift for each proton was difficult due to the presence of 2 rotamers. Percentages of two rotamers were (55/45).

	(cyclohexane/ethyl acetate: 1/9) to give (R)-1-(tert-butoxycarbonyl)-6-methyl-4-
	oxopiperidine-2-carboxylic acid (1.29 g, 72%) as yellow oil.
	R f 0.12 (SiO 2 , cyclohexane/ethyl acetate: 1/9).
	1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.22 (3H, d, J = 6.6 Hz, H 7 ), 1.28 (3H, d, J = 6.8 Hz,
	H 7 ), 1.41 (9H, s, H 10, 11, 12 ), 1.45 (9H, s, H 10, 11, 12 ), 2.29-2.43 (2H, m), 2.56-2.75 (2H, m),
	2.79-3.06 (4H, m), 4.30-4.67 (2H, m), 4.73-5.19 (2H, m).
	Rotamer A
	13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 20.82 (C 7 ), 22.63 (C 10, 11, 12 ), 36.49 (C 3 ), 44.75 (C 5 ),
	49.64 (C 6 ), 53.24 (C 2 ), 81.63 (C 9 ), 175.05 (C 8 ), 176.42 (C 1 ), 206.27 (C 4 ).
	Rotamer B
	13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 20.28 (C 7 ), 28.30 (C 10, 11, 12 ), 39.68 (C 3 ), 45.46 (C 5 ),
	49.64 (C 6 ), 53.39 (C 2 ), 81.83 (C 9 ), 175.51 (C 8 ), 177.17 (C 1 ), 206.53 (C 4 ).

Note:

1 H NMR (400 MHz, CDCl 3 ) δ

  (ppm) 1.18 (3H, d, J = 6.6 Hz, H 7 ), 1.25 (3H, d, J = 6.8 Hz, H 7 ), 2.50-3.02 (5H, m, H 6a, 3, 5 ), 4.33-4.69 (2H, m, H 2a ), 5.11 (4H, m, H 9 ), 7.25 (10H, m, H Ar ).

	Rotamer A
	13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 20.28 (C 7 ), 36.27 (C 3 ), 44.53 (C 5 ), 47.37 (C 6 ), 53.34
	(C 2 ), 67.30 (C 9 ), 127.85, 128.09, 128.30 (CH Ar ), 135.77 (C Ar ), 174.48 (C 8 ), 175.49 (C 1 )
	205.92 (C 4 ).

Rotamer B 13 C NMR (100 MHz, CDCl 3 ) δ

  (ppm) 20.72 (C 7 ), 39.63 (C 3 ), 45.40 (C 5 ), 48.52 (C 6 ), 53.41 (C 2 ), 68.23 (C 9 ), 127.99, 128.22, 128.57 (CH Ar ), 135.90 (C Ar ), 175.12 (C 8 ), 177.09 (C 1 ) 205.92 (C 4 ).

1 H NMR (400 MHz, CDCl 3 )

  δ (ppm) 1.73 (brs, 1H, NH), 3.83 (s, 2H, H 6 ), 3.92 (s, 2H, H 5 ), 7.15 (td, J = 7.7, 1.7 Hz, 1H, H 2 ), 7.28-7.45 (m, 7H, Ar-H), 7.57 (dd, J = 7.9, 1.1 Hz, 1H, H 4 ).

	CAS Registry Number: 65185-56-0.
	The analysis is in accordance with the literature.
	N-(

2-bromobenzyl)-2,2,2-trifluoroethanamine 161

  s, 2H, H 5 ), 7.18 (td, J = 7.8, 1.8 Hz, 1H, H 2 ), 7.33 (td, J = 7.5, 1.2 Hz, H 3 ), 7.41 (dd, J = 7.6, 1.5 Hz, H 1 )7.59 (dd, J = 8.0, 1.1 Hz, 1H, H 4 ).

	CAS Registry Number: 558483-94-6.
	The analysis is in accordance with the literature.
	tert-butyl 2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-carboxylate
			177
		O
		N		N
	O	O	O				Br
	Chemical Formula: C 26 H 31 BrN 2 O 4
	Molecular Weight: 515.43
	3 2	4 1	Br 5	N H	6	F F	F
	Chemical Formula: C 9 H 9 BrF 3 N
	Molecular Weight: 268.07
	To a solution of 2-bromobenzaldehyde (1.58 g, 8.57 mmol, 1.0 equiv) and 2,2,2
	trifluoroehtylamine (0.68 mL, 8.57 mmol, 1.0 equiv) in methanol (100 mL) at 0 °C was added
	slowly in portion wise acetic acid (1.47 mL, 25.7 mmol, 3.0 equiv) followed by sodium
	cyanoborohydride (2.153 g, 34.26 mmol, 4.0 equiv). The reaction mixture was continued to
	stir at room temperature for 12 h. The mixture was diluted with dichloromethane (50 mL),
	washed with saturated aqueous solution of NaHCO 3 (3x50 mL), dried over MgSO 4 and finally
	concentrated in vacuo. The crude product was purified by column chromatography on silica
	(cyclohexane/ethyl acetate: 7/3) to give N-benzyl-1-(2-bromophenyl)methanamine as a
	colorless oil (4.25 g, 95%).

R f 0.1 (SiO 2 , 100% ethyl acetate).

(

1 HR-ESI-MS: calculated

  for C 26 H 31 BrN 2 O 4 (M+H + ): 515.1545, found: 515.1569.

(R

)-Benzyl-2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1- carboxylate 179

	O
		N
	N
		O	Br
	O	O
	Chemical Formula: C 29 H 29 BrN 2 O 4
	Molecular Weight: 549.45

1 H NMR (400 MHz, C 6 D 6 ) δ

	Rotamer B
	(ppm) 0.86-0.96(4H, m, H 5a ), 0.98 (6H, d, J = 6.5 Hz, H 7 ), 1.02
	(6H, d, J = 6.5 Hz, H 7 ), 1.30-1.48 (8H, m, H 5e/3e+3a/6a ), 1.85-2.02 (8H, m, H 5e/3e+3a/6a ), 2.28
	(4H, ddt, J = 12.7, 9.9, 2.5 Hz, H 5e/3e ) 2.88 (6H, s, H 23/24 ), 2.89 (6H, s, H 23/24 ), 3.10 (6H, s,
	H 23/24 ), 3.13 (6H, s, H 23/24 ), 3.92 (2H, dd, J = 11.8, 2.7 Hz, H 2a ), 4.11 (2H, dd, J = 11.8, 2.7
	Hz, H 2a ), 4.33-5.15 (16H, m, H 10/16 ), 6.71-7.36 (32H, m, Ar-H), 7.40 (2H, d, J = 7.9 Hz, H 19 ),
	7.51 (2H, d, J = 7.6 Hz, H 19 ).
	Rotamer A
	13 C NMR (101 MHz, C 6 D 6 ) δ (ppm) 19.09 (C 7 ), 36.82 (C 3 ), 41.70 (C 5 ), 46.87 (C 24, 25 ), 48.39
	(C 6 ), 49.04 (C 9 ), 50.52 (C 16 ), 54.17 (C 2 ), 99.98 (C 4 ), 123.06 (C-Br), 126.81, 127.82, 128.06,
	128.30, 128.73, 129.05, 133.19, 136.19, 137.33(C Ar ).

13 C NMR (101 MHz, C 6 D 6 ) δ

  (ppm) 22.13 (C 7 ), 36.87 (C 3 ), 41.81 (C 5 ), 47.23 (C 24, 25 ), 48.71 (C 6 ), 49.15 (C 9 ), 50.65 (C 16 ), 54.35 (C 2 ), 100.10 (C 4 ), 124.10 (C-Br), 126.98, 127.89, 128.51, 128.59, 128.92, 129.59, 133.02, 137.12, 137.94 (C Ar ).

	HR-ESI-MS: calculated for C 23 H 29 BrN 2 O 3 (M+H + ): 461.1440, found: 461.1426.

NMR (101 MHz, C 6 D 6 ) δ

  (ppm) 21.34 (C 7 ), 39.32 (C 3 ), 41.49 (C 5 ), 47.43 (C 23 ), 47.57 (C 24 ), 48.40 (C 9 ), 49.43 (C 2 ), 52.14 (C 16 ), 68.27 (C 6 ), 98.06 (C 4 ), 122.32, 123.20, 127.20, 127.51, 128.55, 128.70, 128.79 (C Ar ), 137.42 (C 17 ), 138.22 (C 10 ), 148.39 (C 22 ), 172.27 (C 8 ).

		(2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6-
			methylpiperidine-1-carboxylate 190
			O	O
			N		N
			O	O	O	Br
			Chemical Formula: C 31 H 35 BrN 2 O 5
			Molecular Weight: 595.52
	To	(2R,6R)-benzyl	2-(benzyl(2-bromobenzyl)carbamoyl)-6-methyl-4-oxopiperidine-1-
	carboxylate (0.350 g, 0.64 mmol, 1.0 equiv) in a round bottom flask was added with trimethyl
	2,3,4,4a,6,7-hexahydrobenzo[f]pyrido[1,2-a][1,4]diazepin-5(1H)-one as a colorless oil (0.09 orthoformate (0.35 mL, 3.19 mmol, 5.0 equiv) and p-toluenesulphonic acid (0.03 g, 0.13
	g, 52%).
	R f 0.52 (SiO 2 , cyclohexane/ethyl acetate: 5/5).
	1 H NMR (400 MHz, C 6 D 6 ) δ (ppm) 0.85 (3H, d, J = 5.9 Hz, H 7 ), 1.45 (1H, dd, J = 13.1, 11.6
	Hz, H 5a ), 2.04-2.14 (2H, m, H 3e, 3a ), 3.03-3.06 (1H, m, H 5e ), 3.07 (3H, s, H 23 ), 3.18 (3H, s, (2R,6R)-benzyl 2-(benzyl(2-bromobenzyl)carbamoyl)-4,4-dimethoxy-6-methylpiperidine-1-
	H 24 ), 3.37 (1H, d, J = 13.4, H 16 ), 3.37-3.45 (1H, m, H 6a ), 4.02 (1H, dd, J = 12.4, 2.6 Hz, H 2a ), carboxylate as a colorless oil (0.35 g, 91%).
	4.58 (2H, d, J = 1.9 Hz, H 9 ), 5.06 (1H, d, J = 13.1 Hz, H 16 ), 6.54 (1H, dd, J = 7.4, 1.5 Hz, R f 0.45 (SiO 2 , cyclohexane/ethyl acetate: 7/3).
	H 21 ), 6.75 (1H, td, J = 7.4, 1.1 Hz, H 20 ), 6.84 (1H, d, J = 7.8 Hz, H 18 ), 7.01 (1H, td, J = 7.5,
	1.1 Hz, H 19 ), 7.05-7.14 (5H, m, H 11-15 ).
	HR-ESI-MS: calculated for C 23 H 28 N 2 O 3 (M+H + ): 381.2178, found: 381.2188.
	[α] D 20 = + 10.6 (c 1.05, CHCl 3 ).

13 C

mmol, 0.2 equiv) and continued to stir at room temperature for 10 h. The resulting mixture was diluted with ethyl acetate (30 mL) and washed with aqueous solution of 1.5M NaOH (3x30 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 70/30) to give HR-ESI-MS: calculated for C 31 H 35 BrN 2 O 5 (M+H + ): 596.1729, found: 596.1788.

  82 mmol, 1.2 equiv) was added and the sealed tube was stoppered tightly and continued to stir at 100 °C for 10 h. The resulting mixture has been filtered through Celite ® bed and thus collected filtrate was diluted with DCM (20 mL) and washed with saturated aqueous sodium bicarbonate solution (3x20 mL), dried over MgSO 4 and evaporated in vacuo. The crude product was purified by column

	chromatography on silica (cyclohexane/ethyl acetate: 5/5) to give (4S,14bS)-2,2-dimethoxy-
	4-methyl-1,2,3,4,10,14b-hexahydrodibenzo[b,f]pyrido[1,2-d][1,4]diazepine as a yellow oil
	(0.22 g, quantitative).
	1 H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.28 (2H, d, J = 6.0 Hz, H 7 ), 1.31-1.38 (1H, t, J = 12.6
	Hz, H 5a ), 1.95 (1H, t, J = 12.7 Hz, H 3a ), 2.11 (1H, dt, J = 13.1, 2.7 Hz, H 3e ), 3.07-3.18 (1H,
	m, H 6a ), 3.28 (3H, s, 0-CH 3 ), 3.29 (3H, s, 0-CH 3 ), 4.06 (1H, dd, J = 12.1, 2.6 Hz, H 2a ), 6.79
	(1H, td, J = 8.0 Hz, 1.4 Hz, H ar ), 6.99 (1h, J = 7.4 Hz, H ar ), 7.26 (3H, m, H ar ), 7.43 (1H, d, J =
	7.9 Hz, H ar ), 7.48 (1H, d, J = 7.9 Hz, H ar ), 7.63 (1H, dd, J = 7.9, 1.3 Hz, H ar ), 9.35 (1H, brs,
	NH).
	13 C NMR (100 MHz, CDCl 3 ) δ (ppm) 22.58 (C 7 ), 35.97 (C 3 ), 40.39 (C 5 ), 47.47 (C 21 ), 47.57
	(C 22 ), 53.52 (C 6 ), 57.76 (C 6 ), 99.78 (C 4 ), 112.96, 116.07, 118.54, 120.40, 121.04, 127.86,
	127.98, 128.57, 128.77, 131.30, 133.31, 141.74, 142.05 (Car).

R f 0.45 (SiO 2 , cyclohexane/ethyl acetate: 7/3).

400 MHz, CDCl 3 )

   (ppm) 1.01 (3H, d, J = 6.6 Hz, H 14 ), 1.25 (3H, d, J = 7 Hz, H 7 ), 2.00 (1H, dd, J = 14.2, 7.8 Hz, H 16' ), 2.25 (1H, dd, J = 14.2, 6.2 Hz, 1H, H 16 ), 3.30 (1H,

	R f 0.8 (SiO 2 , cyclohexane/ethyl acetate: 9/1)
	m, H 15 ), 3.35 (3H, s, H 17 ), 3.68 (1H, d, J = 14.6 Hz, H 8 ), 3.76 (1H, q, J = 7 Hz, H 6 ), 7.05-7.28
	(m, 10H, H ar ).
	1 H NMR (400 MHz, C 6 D 6 ) δ (ppm) 1.10 (1H, d, J = 6.2, H 7 ), 1.71 (1H, t, J = 12.6 Hz, H5a),
	1.94-2.07 (2H, m, H3a, 3e), 2.37 (1H, td, J = 13.3, 3.2 Hz, H5e), 2.54 (1H, dqd, J = 12.1, 6.1
	13 C NMR (100 MHz, C 6 D 6 ) δ (ppm) 21.58 (C7), 40.11 (C3), 41.52 (C5), 47.34 (C21,22),
	56.46 (C6), 60.17 (C2), 98.45 (C4), 112.11, 119.03, 120.87, 122.93, 123.89, 124.52, 125.04,
	126.06, 127.34, 129.57, 156.68 (Car).
	[α] D 25 = -10.8(c=1.05 CHCl 3 )
	HR-ESI-MS: calculated for C 20 H 23 NO 3 326.2413, found: 326.2407.
	(2S,6S)-ethyl2-(2-(2-iodophenoxy)phenyl)-4,4-dimethoxy-6-methylpiperidine-1-
	carboxylate 202
			17
	8	7
			I
				16
	O	O
	5
	4 3	O	15
			14
		2
	6
	N		13
	9
	H
		10	12
			11
			cis
	Chemical Formula: C 20 H 24 INO 3
	Exact Mass: 453.0801

Hz, 2.8 Hz, H6e), 3.02 (3H, s, O-Me), 3.18 (3H, s, O-Me), 7.11 (2H, tdd, J = 8.5, 7.4, 1.4 Hz, Har), 7.21 (1H, t, J = 7.6 Hz, Har), 7.36 (1H, dd, J = 7.2, 1.3 Hz, Har), 7.61 (1H, dd, J = 7.6, 1.1 Hz, Har). 7.68 (1H, dd, J = 6.3 Hz, 1.3 Hz, Har), 7.75 (1H, d, J = 7.4 Hz, Har).

1 H NMR (

1 H NMR (400 MHz, CDCl 3 ) 

	(ppm) 1.15-1.20 (6H, m), 1.33-1.21 (6H, m), 2.71 (1H, dd, J
	= 17.1, 5.7 Hz, H 4/4' ), 2.84 (1H, dd, J = 17.1, 6.0 Hz, H 4/4' ), 2.99 (1H, dd, J = 22.6, 14.0 Hz,
	H 8/8' ), 3.08 (1H, dd, J = 23.0, 14.0 Hz, H 8/8' ), 4.16-3.94 (7H, m), 5.03 (1H, brs, H 3 ).

1 H NMR (400 MHz, CDCl 3 ) δ

  (ppm) 1.20 (d, J=6.8, 3H, H 1 ), 1.33 (t, J=7.5, 6H, H 10, 12 ), 1.41 (s, 9H, H 5,6,7 ), 2.80 (dd, J= 17.3 and 5.6, 2H, H 4,4' ), 3.11 (dd, J= 23.0 and 13.1, 2H, H 8,8' ), 4.06-4.18 (m, 5H, H 2, 9, 11 ), 4.91 (m, 1H, H 3 ).

ethyl 6-(2-bromophenyl)-4-oxohex-5-en-2-ylcarbamate 222 O NH O O Br 1 2 3 4 5 6 7 8 11 12 9 10 14 13

	6.57 (d, J= 16.2, 1H, H5), 5.08 (se, 1H, H3), 4.15-3.88 (m, 3H, H6), 2.96 (dd, J = 16.4,4.8,
	1H, H4), 2.79 (dd, J = 16.3, 6.2, 1H, H4), 1.22 (d, J = 6.8, 3H, H1),1.16 (t, J = 7.1, 3H, H7)
	NMR 13 C (100 MHz CDCl 3 ): δ (ppm) 14.5 (C 7 ), 20.6 (C 1 ), 43,2 (C 4 ), 60.7 (C 6 ), 127,135,
	132, 127, 141 (C-8), 157.1 (C-14), 198.2 (C-13).
	[α] D 25 = -18.5(c 1.12 CHCl 3 )
	HRMS-ESI (M + Na), m/z: calcd. for C 15 H 18 BrNO 3 Na 362.0368 found 362.0371.
	80 mL) at room (S,E)-ethyl 6-(2-(2-iodophenoxy)phenyl)-4-oxohex-5-en-2-ylcarbamate 223
	temperature was added Ba(OH) 2 .H 2 O (1.3 equiv) and left for stirring. After 30 minutes, the reaction mixture was added with a solution of aldehyde (1.3 equiv) diluted in tetrahydrofuran/water: 40/1 (80 mL) and continued to stir at room temperature for further 45 O NH O O 1 2 6 3 4 5 7 8 9 10
	I	O	13	12	11
	16	15	14
	Chemical Formula: C21H22INO4
	Exact Mass: 479.0594
	Yield : 91%
	Rf = 0.27 Cyclohexane/Ethyl acetate 70/30.
	Chemical Formula: C 15 H 18 BrNO 3
	Molecular Weight: 340.21
	Physical property: Yellow solid
	M.P: 89-90 °C
	Yield : 88%
	R f 0.35 (SiO 2 , cyclohexane/ethyl acetate: 7/3).

minutes. The mixture was concentrated in vacuo. The resulting residue was diluted with ethyl acetate (250 mL), washed with water (3x250 mL), dried over MgSO 4 and finally concentrated in vacuo. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 8/2) to give E-alkene.

Now let's see each formation of alkene individually (S,E)-

NMR 1 H (400 MHz, CDCl 3 ): δ (ppm) 7.86 (d, J = 16.2, 1H, H8), 7.56 (d, J = 7.8, 1H, H12), 7.56 (d, J = 7.8,1H, H9,), 7.27 (t, J = 7.8 Hz, 1H, H10), 7.17 (td, J = 7.8, 1.5 Hz, 1H, H11),

NMR 1 H (400 MHz, CDCl 3 ):: 

	(S,E)-tert-butyl (6-(2-bromophenyl)-4-oxohex-5-en-2yl)carbamate 224
		6 13	O
	7	O	NH 3	O	8	Br
		1 2	4	5		12
						9	10	11
	Chemical Formula: C17H22BrNO3
		Exact Mass: 367.0783
	Physical property: Yellow solid
	Yield : 90%
	Rf = 0.45 Cyclohexane/ethyl acetate 70/30.
	NMR 1 H (400 MHz, CDCl 3 ):δ (ppm) 7.92 (d, J = 16 Hz, 2H, H8), 7.61 (td,J=8.2,1.4, 2H,
	H12,9), 7.33 (td,J=7.2,1.1, 1H, H11),7.23 (td, J=7.8,1.6, 1H, H10), 6.63 (d, J = 16 Hz, 1H,
	H5), 4.98 (m, 1H, H3), 4.09-4.12 (m, 1H, H2), 2.92 (dd, J=16, 2H, H4), 1.42 (s, 9H, H6,7,13),
	1.26 (d, J=7, 3H, H7).
	NMR 13 C 100 MHz CDCl 3 : δ (ppm). 20.7(C-1), 28.8(C-6,7,13), 46.3(C-2), 49(C-4), 67.7(C
	14), 127.5(C-5),131/133/127/120 (C-ar), 141(C-8).
	[α] D 25 = -25.3(c=1.0 CHCl 3 )
		7.89 (dd, J = 16 , 1H, H8), 7.81-7.90 (m, 1H, H16),7.66
	(dd, J=8.0,1.1, 2H, H19), 7.29-7.34 (m, 2H, H14,15), 7.15(t, J=7.6, 1H, H11), 6.9 (td,
	J=7.4,1.5, 1H, H10), 6.78-6.80 (dd,J=7.8, 1H, H12), 6.82 (d, J = 16 , 1H, H5), 6.77 (dd,
	J=8.24,1.1, 1H, H13), 5.18 (m, 1H, H3), 4.05-4.17 (m, 3H, H6,2), 2.82-2.97(m, 2H, H4),
	1.16-1.12 (m, 6H, H1,7).
	NMR 13 C (100 MHz, CDCl 3 ):  (ppm). 14.6 (C-7), 20.8 (C-1), 43 (C-2), 46 (C-4), 60.4 (C -
	6), 125.7(C-5), 88.7, 127.1, 128/165/123/132/137/122,140(C-8), 155.1(C-14), 193(C-13).
	[] D 25 = -22.5(c=1.02 CHCl 3 )
	HRMS ESI (M + H) + ion: calc. C 21 H 23 NO 4 I 480.0672, found. 480.0672

HRMS ESI (M + H) + ion: calc

  . C 17 H 23 NO 3 Br 368.0861, found. 368.

0893 (S,E)-tert-butyl (6-(2-nitrophenyl)-4-oxohex-5-en-2-yl)carbamate 225

	NMR 1 H (400 MHz, CDCl 3 ):: δ (ppm) 8.05-8.07 (m, 2H, H12,9), 8.01 (d, J = 16, 2H, H8), ,
	7.63-7.66 (m, 1H, H11),7.53-7.57 (m, 1H, H10), 6.59 (d, J = 16, 1H, H5), 4.92 (m, 1H, H3),
	4.08-4.10 (m, 1H, H2), 2.86 (2 dd, J=16,6, 2H, H4), 1.42 (s, 9H, H6,7,13), 1.26 (d, J=7, 3H,
	H7).
	NMR 13 C (CDCl 3 ): δ (ppm). 20.5(C-1), 27.4(C-6,7,13), 46.6(C-2), 46.4(C-4), 75.7(C-14),
	125.5(C-5),128/129/131/120 (C-ar), 138(C-8), 152.1(C-14),188.9(C-16).
	[α] D 25 = -30(c=1.12 CHCl 3 )
	HRMS ESI (M + H) + ion: calc. C 17 H 22 N 2 O 5 357.1426 found. 357.1405
	2-(2-iodophenoxy)benzaldehyde
		H 1 13 12	O 11	O	10	I	9	8
		2		4 5		7
			3				6
		Chemical Formula: C 13 H 9 IO 2
		Molecular Weight: 324.11
	6
		13	O
				3
	7	O	NH	O			NO
								2
								8
		1 2				12
				4		5
								11
								9
								10
	Chemical Formula: C17H22N2O5
		Exact Mass: 334.1529
	Physical property: Yellow solid
	Yield: 75%
	Rf = 0.35 Cyclohexane/Ethyl acetate 70/30.

  : Brown oil

	Yield : quatitative
	NMR 1 H (400 MHz, CDCl3) δ: 7.00-7.03(m, H10,1H, H13), 6.73-6.77(m, 2H,
	H11,H12), 4.20 (dd, J=12.5et2.6, 1H, H3), 3.49 (s, 3H, H7,8), 3.20 (s, 3H, H7,8), 2.86-2.91

(, m, 1H H6), 2.57 (dt, J=13.7et2.7, 1H, H4e), 2.01 (dt, J=12.2,2.7, 1H, H5e), 1.73 (dd, 1H, J=13.5et13.3), 1.37 (d, 3H, J=7.0, H9).

A 13 C NMR (100 MHz, CDCl 3 ) δ

  (ppm) 19.02 (C 7 ), 37.20 (C 3 ), 43.58 (C 5 ), 47.22 (C 6 ), 52.89 (C 2 ), 53.49 (C 9 ), 127.64, 128.33, 129.08, 129.96, 136.73, 137.44 (CH ar ), 170.33 (C 8 ).

	Rotamer B

13 C NMR (100 MHz, CDCl 3 )

  δ (ppm) 20.72 (C 7 ), 38.13 (C 3 ), 43.98 (C 5 ), 48.02 (C 6 ), 53.41 (C 2 ), 54.10 (C 9 ), 127.94, 128.74, 129.75, 133.01, 136.85, 137.60 (CH Ar ), 170.33 (C 8 ). calculated for C 21 H 26 N 2 O 2 (M+H + ): 339.2073, found: 339.2048.

	O	O
	N
	N
	O
	[α] D 25 = + 17.9 (c 1.10, CHCl 3 ). HR-ESI-MS: N Chemical Formula: C 16 H 22 N 4 O 3 N H Exact Mass: 318.16

13 C NMR (100 MHz, C 6 D 6 ) δ

  (ppm) 22.52 (C7), 30.17 (C3), 44.98 (C5), 49.61 (C6), 60.58 (C2), 113.0, 116.31, 119.13, 120.97, 121.42, 127.41, 127.97, 128.62, 128.96, 130.37, 133.61, 141.90 (Car), 206.18 (C=O).

100 MHz, C 6 D 6 )

  CDCl 3 ) δ (ppm) 1.00 (1H, d, J = 6.2, H 7 ), 1.12 (1H, t, J = 12.6 Hz, H5a), 1.72-1.99 (2H, m, H3a, 3e), 2.10 (1H, td, J = 13.3, 3.2 Hz, H5e), 2.15 (1H, dqd, J = 12.1, 6.1 Hz, 2.8 Hz, H6e), 7.18 (2H, tdd, J = 8.5, 7.4, 1.4 Hz, Har), 7.20 (1H, t, J = 7.6 Hz, Har), 7.29 (1H, dd, J = 7.2, 1.3 Hz, Har), 7.52 (1H, dd, J = 7.6, 1.1 Hz, Har). 7.60(1H, dd, J = 6.3 Hz, 1.3 Hz, Har), 7.69 (1H, d, J = 7.4 Hz, Har). δ (ppm) 21.71 (C7), 30.19 (C3), 39.39 (C5), 59.48 (C6), 63.37 (C2), 112.08, 117.77, 119.61, 120.99, 123.06, 123.99, 124.71, 125.71, 126.89, 127.57, 154.13, 156.51 (Car), 205.04 (C=O).

1 H NMR (400 MHz,

13

C NMR (

13 C NMR (100 MHz, C 6 D 6 )

  δ (ppm) 22.60 (C7), , 39.17 (C3), 43.63 (C5), 50.35 (C6), 59.30 (C2), 69.35 (C4), 112.93, 116.01, 119.39, 120.62, 121.41, 126.88, 127.94, 128.87, 132.22, 133.70, 142.31, 142.52(Car).

1 H NMR (400 MHz, CD 3 OD) δ

  (ppm) 1.47 (9H, s, H16, 17, 18), 1.87 (4H, ddd, J = 15.9, 12.3, 2.3 Hz, H 1, 5), 2.31 (2H, qd, J = 12.6, 4.3 Hz), 2.95 (2H, m), 4.09 (2H, d, J = 13.4 Hz), 4.25 (2H, d, J = 13.3 Hz), 4.94 (6H, s), 7.56 (1H, J = 7.9, 1.3 Hz), 7.72 (2H, s), 7.96 (1H, dd, J = 5.3, 1.2 Hz).

13 C NMR (

100 MHz, CD 3 OD

  ) δ (ppm) 28.67 (C16, 17, 18), 30.09, 44.64, 52.38 (C alicyclic), 81.06, 116.71, 118.00, 124.96, 141.11 (Car), 156.55, 159.10 (C=O). HR-ESI-MS calcd for C 22 H 34 N 6 O 4 (M+H + )=445.2563, found=445.2576. yl)piperidine-1-carboxamido)piperidine-1-carboxylate.

13 C NMR (400 MHz, CD 3 OD)

  δ (ppm) 21.03, 21.11, 30.12, 30.21, 30.80, 33.87, 37.41, 44.90, 46.48, 46.54, 47.82, 51.02, 51.02, 51.19, 52.23, 52.34, 53.20, 53.28, 116.75, 116.93, 118.14, 129.10, 129.77, 130.15, 137,18, 137.89, 141.11, 144.77, 155.27, 159.76, 173.81,

	173.86 (C=O)

HR-ESI-MS calcd

  for C 33 H 39 N 7 O 3 for 582.3193, found 582.3204.

Scheme 20.Showing the synthesis of (+)-myrtine by Del Pozo and coll.

H NMR (400 MHz, CDCl 3 ) δ (ppm) 1.87 (brs, 1H, NH), 3.20 (q, J = 9.4 Hz,

2H, H 6 ), 4.01

H NMR (400 MHz, CDCl 3 ) : δ (ppm) 1.16 (d, J= 6.7, 3H, H 1 ), 1.26 (t, J=6.2, 6H, H 10, 12 ),

Molecular Weight: 279.33

LIST OF SCHEMES

All the ligands docked in this current study were modeled using same protocol as described below. Initially molecules have been drawn in 2D version using Marvinsketch with a stress on the stereochemistry on the chiral carbons. Further, their geometry in 3D were obtained again using the same program i.e. Marvinsketch. To end with, the stereochemistry was ensured before recording the structures in MOL2 format. The ligands were then reopened in sybylx2.0 and any missing hydrogen atoms were added.

Conformational studies:

Each molecule thus modeled is subjected to random search geometry. The geometry of lowest energy conformation is then recovered and inserted in SDF format which later can be directly used for docking.

All these stages were passed on to reach the docking simulation where an in silico interactions was visualized.

BIOLOGICAL ASSAY

Animal models handling: Male swiss albino mice (18-22 g) were purchased from Elevage Janvier. Animals were housed under controlled environmental conditions (22 °C, 55% humidity) and kept under a 12/12 hours light/dark cycle with food and water ad libitum for a week before the commencement of the tests. The tests were performed as a double blind study in a quiet cabin by the same manipulator to minimize the errors as well as to avoid discomfort to the animals. Animals were randomly divided into groups of 10 mice each and each mouse was not treated with the same compound twice. This study was followed by euthanization of

Behavioral testing in mice

These experiments were carried out at Neuro-Dol by me and Julie Barbier following the ethical principles thoroughly.

Injection:

The tested compounds were dissolved in vehicle (5% Tween 80 in saline (0.9%), 0.5% DMSO), 0.5% DMSO was used to enhance the solubility of compounds. In all the [α] D 20 = -13.3 (c 1.00, CHCl 3 ).

The analysis is in accordance with the literature.

(R)-dimethyl 2-(tert-butoxycarbonylamino)succinate 145a To a solution of (R)-dimethyl 2-aminosuccinate hydrochloride (2.0 g, 10.12 mmol, 1.0 equiv) in methanol (50 mL) at room temperature under argon was added dropwise triethylamine (4.23 mL, 30.36 mmol, 3.0 equiv) followed by di-tert-butyl-dicarbonate (2.64 g, 12.14 mmol, 1.2 equiv). The reaction mixture was continued to stir for 6 h. The mixture To a solution of (R)-dimethyl 2-aminosuccinate hydrochloride (29.53 g, 150 mmol, 1.0 equiv) in dichloromethane (450 mL) at 0 °C under argon was added dropwise triethylamine (62.72 mL, 450 mmol, 3.0 equiv) followed by triphenylmethyl chloride (50 g, 180 mmol, 1.2 equiv).

The reaction mixture was allowed to come down to room temperature and stirred for 6 h. The mixture was washed with water (3x300 mL), dried over MgSO4 and then concentrated in vacuo to give yellow oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9/1) to give (R)-dimethyl 2-(tritylamino)succinate as a white crystalline solid (52.3 g, 86%). To a solution of (S)-dimethyl 2-aminosuccinate hydrochloride (29.64 g, 150 mmol, 1.0 equiv) in dichloromethane (450 mL) at 0 °C under argon was added dropwise triethylamine (62.76 mL, 450 mmol, 3.0 equiv) followed by triphenylmethyl chloride (50 g, 180 mmol, 1.2 equiv).

The reaction mixture was allowed to come down to room temperature and stirred for 6 h. The mixture was washed with water (3x300 mL) & dried (MgSO4) and then concentrated in vacuo to give yellow oil. The crude product was purified by column chromatography on silica (cyclohexane/ethyl acetate: 9/1) to give (S)-dimethyl 2-(tritylamino)succinate as a white crystalline solid (50.8 g, 84%). To a solution of (S)-N-benzyl-N-α-methyl benzylamine (13.4.0 mL, 63.6 mmol, 1.0 equiv) in dry THF (200 mL) at 0 °C was slowly added with n-butyl lithium (28 mL, 1.6 M in hexane, 70 mmol, 1.1 equiv). The resultant pink solution of lithium amide was stirred for 30 min at 0 °C and then cooled to -78 °C before dropwise addition of a solution of methyl crotonate (7.4 mL, 70 mmol, 1.1 equiv) in dry THF (60 mL). The mixture was stirred at -78 °C for 2 h.

Then, a saturated aqueous solution of ammonium chloride (80 mL) was added slowly, and the resulting solution was allowed to warm to room temperature. The solution was extracted with ethyl acetate (3x100 mL). Combined organic extracts were dried over MgSO 4 , filtered, and evaporated in vacuo to give (S)-methyl 3-(benzyl((S)-1-phenylethyl)amino)butanoate as yellow oil. The crude product thus obtained was engaged into the next step without any purification.

(0.75 mL) at room temperature was added with triphenylphosphine (0.27 g, 1.02 mmol, 1.5 equiv) and continued to stir at room temperature for . The reaction mixture was evaporated in vacuo and the resulting residue was diluted with ethyl acetate and H 2 O (20 mL). The pH was adjusted to be acidic using 1N HCL (until TLC showed no traces of product) and discarded.

The combined aqueous layer was taken to 0°C and the pH carefully was adjusted to 14 with NaOH. Then it was extracted with ethyl acetate (3x30 mL). The combined organic layer was dried over MgSO 4 and concentrated in vacuo. The crude thus obtained was purified using column chromatography on silica (cyclohexane/ethyl acetate: A 5mL one-neck round-bottomed flask was charged with N,N-carbonyldiimidazole (31mg, 0.19mmol) and flushed with argon, carefully pumped out at vacuum and refilled with argon. Such procedure was done three times. Then freshly distilled THF (2mL) was added and stirred. In the meantime, a 10mL one-neck round-bottomed flask was attached to a refrigerant and charged with 286 (50mg, 0.15mmol). Then, it was flushed with argon, the inner atmosphere carefully pumped out at vacuum and refilled with argon. Such procedure was done three times as well. Then freshly distilled THF (3.6mL) and TEA (0.022mL, 0.19mmol) were added via syringe and then stirred at r.t. a while under argon. Then, this solution was added drop-wise very slowly (within 30 min) via syringe to the previous one.

The resulting solution was stirred for 1 hour under argon.

Last, compound 142 (34mg, 0.19mmol) and TEA (0.051mL, 0.39mmol) were added and the solution stirred in one portion and the resulting solution stirred under argon at 65°C for overnight.

The following day it was left to cool down to r.t. and AcOEt was added. The organic solution was washed with NaOH 1M and brine, dried over anhydrous magnesium sulphate and solvent removed under reduced pressure. The resulting crude was purified by flash chromatography (AcOEt/methanol) to give 47mg of 19 (57% yield, rf= 0.54 in AcOEt/methanol 8/2).

( Exact Mass: 292. 37 2,3-Diamino pyridine (10.0 g, 91.6 mmol, 1.0 equiv), tert-butyl 4-oxopiperidine-1carboxylate (21.9 g, 110 mmol, 1.2 equiv) and ethyl acetate (100 mL) were cooled to 0-5 °C.

Trifluoroacetic acid (23.4 g, 205 mmol, 2.2 equiv) followed by sodium triacetoxyborohydride (29.1 g, 137 mmol, 1.5 equiv) were added at ≤10 °C. The reaction mixture was warmed to ambient temperature, stirred for 2 h, and then cooled to ≤10 °C. The reaction mixture was quenched with 10% sodium hydroxide solution (∼180 mL), and the pH was adjusted to 8.5-9.0 while maintaining the reaction temperature at ≤10 °C. The reaction mixture was diluted with ethyl acetate (200 mL), and the organic layer was separated and washed with water (100 mL), dried over MgSO 4 and concentrated in vacuo. Thus obtained brow coloured compound without any purification results in tert-butyl 4-((2-aminopyridin-3-yl)amino)piperidine-1carboxylate (24g, 86%).