Sylvain Routier

Pr Sylvie Chalon

Pr Manuela

Samuel Becket

FAIL BETTER"

To begin with, I would like to thank all the member of the jury who kindly accepted to judge and evaluated the work I have done in the past wonderful three years. You are all inspirational experts to look up to and I feel lucky to have crossed your paths

Preamble

Alzheimer's disease is complex neurodegenerative disease characterised by a progressive loss of memory and cognition. AD is the first cause of dementia of people over 75 years old and affects around 20% of people of more than 75 years old. With more than 4.6 million new cases of dementia per year in the world, finding a treatment against AD is real public health issue. We estimate that 46.8 million people were affected with dementia in 2015 and this number will grow to reach 135.5 million in 2050.

AD is a multifactorial disease with diverse metabolic consequences such as the apparition of "senile plaques" formed by the β-amyloid peptide (Amyloid cascade hypothesis), the neuroinflammation, the oxidative stress, the hyperphosphorylation of the Tau protein leading to neurofibrillary tangles, and finally a diminution of the levels of Acetylcholine (Cholinergic hypothesis).

Despite the apparition of biomarkers allowing an early diagnosis, there is no existing curative treatment of the disease to date. Indeed, actual treatments only have a palliative action, their goal is to improve the patient's life and to slow down the progression of AD.

In view of the complexity of AD, the therapeutic paradigm "one compound-one-target" has shown its limits in the treatment of AD. Thus, new strategies are emerging to overcome the lack of efficiency of the current pharmacotherapy in the last past decade.

My PhD subject consisted in designing and developing series of original multi-target directed ligands (MTDL). Indeed, this therapeutic strategy appears to be the most promising and efficient approach to obtain innovative drugs against CNS-related pathologies. Over the past few years, numerous MTDLs targeting AChE and another therapeutic target of AD such as kinases, MAO-A and B, oxidative stress and α7 nicotinic receptors were designed and synthetized by Professor Renard's team. In collaboration with the Professor Routier (ICOA, Orléans), we developed new combined MTDLs able to act simultaneously on two targets involved in AD: acetylcholinesterase and α7 nicotinic receptors. The in vitro affinities of the compounds toward α7 nicotinic receptors were assayed by Professor Chalon's team (CHU of Tours) who has a great expertise in the domain of nicotinic receptors, and the cholinesterases inhibition assays were performed by Professor Bartolini's team (University of Bologna). This manuscript is made up of a general introduction, three chapters, a conclusion and an experimental part. The introduction will be dedicated to general information concerning AD, its histopathology, the known therapeutic targets, the several approved or awaiting approval drugs, the multi-target directed ligand concept and a non-exhaustive bibliographic study of the various MTDLs designed in the past years. The first chapter will concern the design, synthesis and biological in vitro evaluation of a heterodimer linking a huprine fragment able to inhibit AChE and a capsaicin fragment with antioxidant properties whose in vivo elevations are ongoing in the laboratory of Pr. Inestrosa in Chile. Then, the chapter II will be dedicated to the in silico assays, design, synthesis and in vitro evaluation of a new family of combined MTDLs based on the Rivastigmine scaffold, an AChE inhibitor, combined to a quinuclidine fragment, an α7 nicotinic receptors agonist designed by our collaborators, the Professor Routier's team. Finally, the chapter III will expose our synthetic efforts in order to develop another new family of combined MTDLs targeting AChE and α7 nicotinic receptor by combining Donepezil's structure and the quinuclidine scaffold from the 7 nAChR ligands developed by our partner in Orleans.

Another part of my

Finally, in the conclusion, we will summarize the main results (syntheses and biological evaluations) and the perspectives of this project.

General introduction

1. History of Alzheimer's disease 1 In 1901, the German psychiatrist and neuropathologist Aloïs Alzheimer studied the case of a 51 years old woman. Auguste Deter was admitted in the Frankfort hospital for dementia. Her symptoms were various, she was suffering from memory troubles, mutism, disorientation and hallucinations. These symptoms were already well known in cases of dementia, but it was the first time they were observed in such a young patient.

Picture 1: Picture of Alois Alzheimer

After her death, in 1906, Aloïs Alzheimer performed an autopsy of her brain and discovered two major neuropathologic lesions: senile plaques and neurofibrillary deterioration.

He observed a cortex's atrophy and the loss of grey matter layers. This peripherical part of the brain is involved in memory, reflexion and language associated mechanisms. Moreover, he discovered, using a brain's slice coloured with silver salt observed with a microscope, the presence of two abnormal residues, one intracellular and another extracellular. Once again, these residues had already been noticed inside the brains of older patients 2 .

In 1910, the name "Alzheimer's disease" was given to the pathology by Emil Kraeplin, a professor in psychiatry and director of the Ludwig-Maximilians-Universität München psychiatry laboratory. Other scientists are not to be neglected in the discovery:

-Oskar Fischer, a Czech psychiatrist and neuropathologist, described the existence of senile plaques in the brains of 12 patients diagnosed with dementia 3 , 1 Historique de la maladie d'Alzheimer : découverte, identification ... Fondation Vaincre Alzheimer. 2 Alzheimer, A. über eigenartige Krankheitsfälle des späteren Alters. Z. Für Gesamte Neurol. Psychiatr. 1911, 4 (1), 356. [START_REF] Fischer | Miliare Nekrosen mit drusigen Wucherungen der Neurofibril-len, eine regelma ssige Vera nderung der Hirnrinde bei seniler[END_REF] Fischer, O. Miliare Nekrosen mit drusigen Wucherungen der Neurofibril-len, eine regelma ssige Vera nderung der Hirnrinde bei seniler. R DemenzMonatsschr Psychiat Neurol 1907, 22, 361-372. -Gaetano Perusini, collaborator of Aloïs Alzheimer, also helped a lot to the discovery of AD 4 .

In the 80's, thanks to the progress in medicine and particularly in the development of new analytical tools, the biological constituents of the AD's lesions were characterised:

-the beta-amyloid protein was described as the major constituent of the senile plaques in 1984 by George Glenner, an American pathologist [START_REF] Glenner | Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein[END_REF] , -in 1985, the Belgian Jean-Pierre Brion brought to light the implication of Tau protein hyperphosphorylation in the neurofibrillary deterioration 6 .

Worldwide impact and epidemiology

AD is the first cause of dementia in the elderly patients and affects at least 20% of the elders over 75 years of age. With more than 4.6 million of new cases of dementia each year worldwide, finding a treatment for AD is a global public health challenge. An estimate of 46.8 million people was diagnosed with dementia in 2015. Moreover, with an aging population, this number could raise to 135.5 million in 2050 [START_REF]The Global Impact of Dementia | Alzheimer's Disease International[END_REF] . Dementia is a general term which includes AD but also vascular dementia, fronto-temporal dementia and Lewi's body dementia. The prevalence of the various pathologies is substantially different, indeed, AD is diagnosed in 50% to 75% of cases whereas vascular dementia is diagnosed in 20% to 30% of cases, the average is 5% to 10% for the fronto-temporal dementia and less than 5% for the Lewi's body dementia.

Moreover, even if AD is a worldwide public health issue (Figure 1), it seems that people over 60 years of age in developing countries are less impacted by these disorders. 4 Perusini, G. U ber klinisch und histologisch eigenartige psychischeErkrankungen des spa ẗeren Lebensalters. Nissl-Alzheimers HistolHistopathol Arb Hirnr 1909, 3, 297-351. [START_REF] Glenner | Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein[END_REF] Glenner, G. G.; Wong, C. W. Alzheimer's Disease: Initial Report of the Purification and Characterization of a Novel Cerebrovascular Amyloid Protein. Biochem. Biophys. Res. Commun. 1984, 120 (3), 885-890. 6 Brion, J. P.; Passareiro, H.; Nunez, J.; Flament Durand, J. Mise en évidence immunologique de la protéine tau au niveau des lésions de dégénérescence neurofibrillaire de la maladie d 'Alzheimer. Arch. Biol. (Liege) 1985, 95, 229-235. 7 World Alzheimer Report 2015: The Global Impact of Dementia | Alzheimer's Disease International https://www.alz.co.uk/research/world-report-2015.

Figure 1: Worldwide prevalence of AD

Another issue needing to be raised is the economic impact associated with these pathologies. Indeed, in 2010, the cost of the patients' care was evaluated at 604 billion US dollars, which is comparable to 1% of the global GDP. As an example, 70% of global costs are represented by western Europe and USA.

These figures emphasize the fact that the patients' care is not the same internationally.

Disease symptoms 8

Concerning AD 's symptoms, they can be divided in two types:

-the cognitive symptoms, -the behavioural and psychological symptoms.

Being aware of these disruptions is a way to detect the pathology and diagnose its progress, but also to adapt the patient's care. [START_REF]Les symptômes de la maladie d'Alzheimer[END_REF] Les symptômes de la maladie d'Alzheimer https://www.francealzheimer.org/maladie-dalzheimer-vosquestions-nos-reponses/symptomes-de-maladie-dalzheimer/.

Cognitive symptoms

The memory loss is usually the first visible cognitive AD's symptom. At first, the episodic memory of the patient is lost, the person forgets recent events but not older memories. Because AD is a neurodegenerative disease, the symptoms are evolutionary, and at some point, the other types of memories are affected (work memory, loss of words, etc). Thus, it starts to be complex to remember immediate information, to learn new skills and to orient oneself in space.

After the memory symptoms, the other important troubles are related to the language and are called aphasia. The patient progressively loses the communicating, understanding and finally talking abilities.

First, the vocabulary is reduced, then, the person speaks with less and less words until he reaches a total muteness. Then, usual moves begin to be challenging to perform. It usually begins with complex actions like writing but ends with easy tasks like masticating or swallowing. Moreover, people with AD tend to forget faces they knew, it is called agnosia. After this, the executive functions' dysfunction will occur, and it is more and more complicated for the patient to plan, think and focus. The patient slowly abandons these kinds of tasks and relies on other people for each one of every day's life actions.

Behavioural and psychological symptoms of dementia

Behavioural symptoms can also occur in patients with AD, like rapid and violent mood change, anxiety, irrational fear, apathy or depression and negative thought. Therefore, patients need to be taken care of at almost every moment and are not independent anymore, which contributes to the high social costs for AD.

Diagnosis of AD 9

Establishing a diagnosis during the pathology's early stages is a significant challenge. Indeed, an early detection of the pathology allows a good patient's care, but differentiating AD's symptoms from normal ageing, especially during preclinical phase (Figure 2) is a major challenge.

The first warning usually comes from the patient himself or his family, they will describe a memory loss that will lead to a neuropsychological overview. The doctor will perform a series of tests to evaluate memory, but also, orientation in space and time, language, comprehension and attention.

The second way to perform AD diagnosis is the use of biochemical and imaging tools able to evaluate the different pathology biomarkers:

9 Diagnostic de la maladie d'Alzheimer. Fondation pour la Recherche sur Alzheimer.

-MRI imaging, -PET scan, -lumbar puncture.

MRI imaging

MRI study is a non-invasive and affordable technique allowing the doctors to visualize atrophies of certain parts of the brain like a cortical atrophy or a hippocampus atrophy. It is also used to discriminate AD from other pathologies, by visualizing a cerebral tumour for example 10 . For a few years, it has been employed to detect neuronal degeneration of AD patients and to follow the evolution of the disease in clinical trials [START_REF] Sperling | Amyloid-Related Imaging Abnormalities in Amyloid-Modifying Therapeutic Trials: Recommendations from the Alzheimer's Association Research Roundtable Workgroup[END_REF] .

As shown on Figure 3 [START_REF] Ledig | Structural Brain Imaging in Alzheimer's Disease and Mild Cognitive Impairment: Biomarker Analysis and Shared Morphometry Database[END_REF] , we can see the use of MRI to detect AD. As a matter of fact, there is a blatant difference between the healthy control brain and the AD subject's brain. MRI imaging also provides crucial information concerning the neuronal degeneration's evolution by monitoring the patient's brain.

PET scan

PET scan is a functional imaging process and is used to highlight an abnormally low effective functioning brain and to visualize neuronal lesions. Indeed, PET scan can be used to find early AD diagnosis markers [START_REF] Oukoloff | PET and SPECT Radiotracers for Alzheimer's Disease[END_REF] . Numerous brain systems can be monitored, like cerebral glucose metabolism, neurotransmitters systems, neuroinflammation and protein aggregates (beta amyloid deposits). [START_REF] Ledig | Structural Brain Imaging in Alzheimer's Disease and Mild Cognitive Impairment: Biomarker Analysis and Shared Morphometry Database[END_REF] And finally, a high [START_REF] Sperling | Amyloid-Related Imaging Abnormalities in Amyloid-Modifying Therapeutic Trials: Recommendations from the Alzheimer's Association Research Roundtable Workgroup[END_REF] C-PMP binding ((Figure 4 -d) leads to the conclusion that AChE activity is oddly high [START_REF] Nordberg | The Use of PET in Alzheimer Disease[END_REF] . The monitoring of all these markers allows the doctors to diagnose patients with AD and researchers to better understand the disease thanks to a "coloured map of the brain activity".

More recently, other radiotracers [START_REF] Schilling | Imaging Alzheimer's Disease Pathophysiology with PET[END_REF] such as Amivid [START_REF] Fischer | Alzheimer's Disease Neuroimaging Initiative. Altered Whole-Brain White Matter Networks in Preclinical Alzheimer's Disease[END_REF] , Neuraceq [START_REF] Syed | F]Florbetaben: A Review in β-Amyloid PET Imaging in Cognitive Impairment[END_REF] or Vizamyl [START_REF] Thal | F]Flutemetamol Amyloid Positron Emission Tomography in Preclinical and Symptomatic Alzheimer's Disease: Specific Detection of Advanced Phases of Amyloid-β Pathology[END_REF] have been developed

and FDA-approved to quantify Aβ plaques in brains of patients with AD with more accuracy (Figure 5). [START_REF] Nordberg | The Use of PET in Alzheimer Disease[END_REF] [START_REF] Syed | F]Florbetaben: A Review in β-Amyloid PET Imaging in Cognitive Impairment[END_REF] Syed, Y. Y.; Deeks, E. [( 18)F]Florbetaben: A Review in β-Amyloid PET Imaging in Cognitive Impairment. CNS Drugs 2015, 29 (7), 605-613. [START_REF] Thal | F]Flutemetamol Amyloid Positron Emission Tomography in Preclinical and Symptomatic Alzheimer's Disease: Specific Detection of Advanced Phases of Amyloid-β Pathology[END_REF] Thal, D. R.; Beach, T. G.; Zanette, M.; Heurling, K.; Chakrabarty, A.; Ismail, A.; Smith, A. P. L.; Buckley, C.

[( 18)F]Flutemetamol Amyloid Positron Emission Tomography in Preclinical and Symptomatic Alzheimer's Disease: Specific Detection of Advanced Phases of Amyloid-β Pathology. Alzheimers Dement. J. Alzheimers Assoc. 2015, 11 (8), 975-985. In addition, Tau-specific PET tracers are currently in early stages of clinical trials, Flortaucipir [START_REF] Schonhaut | 18F-Flortaucipir Tau PET Distinguishes Established Progressive Supranuclear Palsy from Controls and Parkinson's Disease: A Multicenter Study[END_REF] and THK5317 [START_REF] Chiotis | Imaging In-Vivo Tau Pathology in Alzheimer's Disease with THK5317 PET in a Multimodal Paradigm[END_REF] have displayed promising in vivo results as biomarkers for Tau pathology (Figure 6).

Lumbar puncture

The lumbar puncture is used with increasing frequency since it gives major information for the diagnosis of AD. This procedure begins with a sample collection of the cerebrospinal fluid, followed by a fluid's dosage to detect specific AD biomarkers levels such as hyperphosphorylated and normal Tau protein and beta-amyloid peptide. It helps the early stage diagnosis of the pathology, indeed, it has been observed that, rapidly, the Tau protein concentration raises to 2 or 3 times compared to a normal concentration and the hyperphosphorylated Tau protein is a major biomarker of the disease [START_REF]Having a lumbar puncture[END_REF] . [START_REF] Schonhaut | 18F-Flortaucipir Tau PET Distinguishes Established Progressive Supranuclear Palsy from Controls and Parkinson's Disease: A Multicenter Study[END_REF] [START_REF]Having a lumbar puncture[END_REF] Having a lumbar puncture https://www.alzheimers.org.uk/research/take-part-research/lumbar-puncture.

Histopathology of AD

AD histopathology is not fully known, nevertheless, we can observe 3 aspects of the neuropathology:

-the cholinergic system, -the amyloid cascade, -the neurofibrillary degeneration.

The neurotransmission

Mechanism of the neurotransmission

The neurotransmission's mechanism is a vital process for the CNS accurate functioning (Figure 7). ACh, is the most ubiquitous neurotransmitter of the human body and is synthesized by a single step reaction from choline and acetyl coenzyme A through choline acetyltransferase catalysis (Scheme 1) [START_REF] Colovic | Acetylcholinesterase Inhibitors: Pharmacology and Toxicology[END_REF] .

ACh is formed inside the neurones, then stocked within synaptic vesicles. Its transfer to the synaptic cleft is done by an energy-dependent pump acidifying the vesicle. The neurotransmitter's purpose is to activate the synapses through binding to nicotinic and muscarinic receptors on the post-synaptic cell [START_REF] Colovic | Acetylcholinesterase Inhibitors: Pharmacology and Toxicology[END_REF] once released into the synaptic cleft. Aiming at restoring the resting levels of neurotransmitter in the synaptic cleft, ACh must be hydrolysed by following a mechanism catalysed by a specific enzyme, named AChE (Scheme 1). [START_REF] Colovic | Acetylcholinesterase Inhibitors: Pharmacology and Toxicology[END_REF] Colovic, M. B.; Krstic, D. Z.; Lazarevic-Pasti, T. D.; Bondzic, A. M.; Vasic, V. M. Acetylcholinesterase Inhibitors: Pharmacology and Toxicology. Curr. Neuropharmacol. 2013, 11 (3), 315-335. [START_REF]Acetylcholine Transmitter[END_REF] Acetylcholine Transmitter | Physiology Plus http://physiologyplus.com/acetylcholine-transmitter/.

Scheme 1: Mechanism of ACh formation and hydrolysis

Cholinergic hypothesis

The cholinergic hypothesis was the first stated hypothesis to explain the mechanism of AD. Since 1976, numerous studies showed a misfunctioning of cholinergic system of patients with AD [START_REF] Bowen | NEUROTRANSMITTER-RELATED ENZYMES AND INDICES OF HYPOXIA IN SENILE DEMENTIA AND OTHER ABIOTROPHIES[END_REF] . It has been

shown that in patients with AD, ACh levels decrease in the CNS leading to a decrease of neuronal activity [START_REF] Sugimoto | Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors[END_REF] .

In 1983, Sims et al. [START_REF] Sims | Presynaptic Cholinergic Dysfunction in Patients with Dementia[END_REF] brought to light that ACh synthesis was significantly reduced in brain samples of patients with dementia and especially with AD (Figure 8-A). It has also been shown that choline acetyltransferase activity's decrease was linked to an increase of all kinds of dementia [START_REF] Ladner | Pharmacological Drug Treatment of Alzheimer Disease: The Cholinergic Hypothesis Revisited[END_REF] . Indeed, Sims et al. showed that choline acetyltransferase activity tends to decrease in patients with AD (Figure 8-B). [START_REF] Bowen | NEUROTRANSMITTER-RELATED ENZYMES AND INDICES OF HYPOXIA IN SENILE DEMENTIA AND OTHER ABIOTROPHIES[END_REF] 99 (3), 459-496. [START_REF] Sugimoto | Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors[END_REF] Sugimoto, H.; Yamanishi, Y.; Iimura, Y.; Kawakami, Y. Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors. Curr. Med. Chem. 2000, 7 (3), 303-339. [START_REF] Sims | Presynaptic Cholinergic Dysfunction in Patients with Dementia[END_REF] Sims, N. R.; Bowen, D. M.; Allen, S. J.; Smith, C. C. T.; Neary, D.; Thomas, D. J.; Davison, A. N. Presynaptic Cholinergic Dysfunction in Patients with Dementia. J. Neurochem. 1983, 40 (2), 503-509. [START_REF] Ladner | Pharmacological Drug Treatment of Alzheimer Disease: The Cholinergic Hypothesis Revisited[END_REF] Ladner, C. J.; Lee, J. M. Pharmacological Drug Treatment of Alzheimer Disease: The Cholinergic Hypothesis Revisited. J. Neuropathol. Exp. Neurol. 1998, 57 (8), 719-731.

Amyloid cascade

Since Dr. Alzheimer's first studies in 1906, it has been shown that senile plaques and neurofibrillary deterioration are involved in AD. In the past years, different groups have investigated the role of the Aβ extracellular peptides in AD pathogenesis [START_REF] Hardy | The Amyloid Hypothesis for Alzheimer's Disease: A Critical Reappraisal[END_REF] . The APP (Amyloid Precursor Protein) is a transmembrane glycoprotein which seems to play different roles [START_REF]Les plaques amyloïdes -Site des ressources d'ACCES pour enseigner la Science de la Vie et de la Terre[END_REF] :

-surface receptor, -interactions between the cells, -useful in the cytoskeleton formation, [START_REF] Hardy | The Amyloid Hypothesis for Alzheimer's Disease: A Critical Reappraisal[END_REF] Hardy, J. The Amyloid Hypothesis for Alzheimer's Disease: A Critical Reappraisal. J. Neurochem. 2009, 110 (4), 1129-1134. [START_REF]Les plaques amyloïdes -Site des ressources d'ACCES pour enseigner la Science de la Vie et de la Terre[END_REF] Les plaques amyloïdes -Site des ressources d'ACCES pour enseigner la Science de la Vie et de la Terre http://acces.ens-lyon.fr/acces/thematiques/neurosciences/actualisation-des-connaissances/maladies-ettraitements/alzheimer/la-maladie-dalzheimer-a-lechelle-cellulaire-et-moleculaire/les-palques. -useful in the intracellular Ca 2+ levels regulation, -synapses formation, -neurones protection against oxidative stress and interaction with the Tau protein, -neuronal plasticity and neurotransmission in the cholinergic system.

The APP is made of up of 770 amino acids coded by only one gene found in the chromosome 21 and is found in three major forms according to the number of amino acids constituting the protein:

-APP 695 which the most common form of the young and non-pathologic brain, -APP 751,

-APP 770 which is found in brains of premature child and in genetic forms of AD.

It has been found in 1999 that the protein is cleaved by three secretases (α, β and γ) to give amyloid peptides. The cleavage can follow two pathways: the non-amyloidogenic pathway and the amyloidogenic pathway [START_REF] De Strooper | Proteases and Proteolysis in Alzheimer Disease: A Multifactorial View on the Disease Process[END_REF] .

The non-amyloidogenic pathway

The cleavage is initiated by an α-secretase in the cytosolic domain at the 687 th amino acid of the chain to give a N-terminal peptide (APP α) and a C-terminal fragment (C83). In a second time, the γ-secretase cleaves the resulting C-terminal fragment (C83) producing a small and soluble peptide (p3) (Figure 9).

The amyloidogenic pathway

The amyloidogenic pathway's first step is the APP cleavage by the β-secretase at the 671 th amino acid to give a N-terminal peptide (APP β) and another C-terminal fragment (C99). Then, the residual fragment C99 is cleaved by the γ-secretase to give two types of Aβ peptides (Figure 10). The first one is the Aβ40 (40 amino acids) which represents normally 80-90% of the Aβ peptides in the brain and Aβ42

(42 amino acids), produced normally in small amounts. Unfortunately, this second peptide is insoluble and cytotoxic and tends to aggregate spontaneously in oligomers leading to fibrils and senile plaques (Figure 11) [START_REF] Ubhi | Alzheimer's Disease: Recent Advances and Future Perspectives[END_REF] . It is well known that, in young brains, Aβ concentrations are maintained in steady-state levels because an equilibrium between its production and its elimination has been observed, this equilibrium is regulated via a cascade of degradative enzymes. Nevertheless, in aging brains, Aβ formation and elimination are impaired, which leads to an accumulation of the peptide and senile plaques formation causing a neuronal activity alteration induced by the neuronal cell death [START_REF] Shankar | Alzheimer's Disease: Synaptic Dysfunction and Aβ[END_REF] . Moreover, studies have shown that there is a strong affinity between the Aβ peptide and the homomeric α7 nAChR facilitating [START_REF] Ubhi | Alzheimer's Disease: Recent Advances and Future Perspectives[END_REF] Ubhi, K.; Masliah, E. Alzheimer's Disease: Recent Advances and Future Perspectives. J. Alzheimers Dis. JAD 2013, 33 Suppl 1, S185-194. [START_REF] Schreck | Statistical Mechanical Treatments of Protein Amyloid Formation[END_REF] Schreck, J. S.; Yuan, J.-M. Statistical Mechanical Treatments of Protein Amyloid Formation. Int. J. Mol. Sci. 2013, 14 (9), 17420-17452. [START_REF] Shankar | Alzheimer's Disease: Synaptic Dysfunction and Aβ[END_REF] Shankar, G. M.; Walsh, D. M. Alzheimer's Disease: Synaptic Dysfunction and Aβ. Mol. Neurodegener. 2009, 4

(1), 48.

the peptide's aggregation and an inhibition of the neuroprotection signals [START_REF] Yu | Upregulation of Astrocytic Α7 Nicotinic Receptors in Alzheimer's Disease Brain-Possible Relevant to Amyloid Pathology[END_REF] . As shown on Figure 12, a normal low concentration of Aβ (picomolar range) does not affect the α7 nAChR activity. At higher concentration of Aβ (nanomolar range), the oligomerisation and aggregation of Aβ leads to the antagonization of nicotinic receptors.

Neurofibrillary degeneration

Tau proteins belong to the family of microtubule associated proteins (MAPs) and are found in many animal species. In the human case, they are mainly found in neurones where they play a major role in the formation of microtubules from tubular monomers. They also help the regulation of microtubules stability and connexion between the tubules and other elements of the cytoskeleton. They are part of a family of 6 isoforms going from 352 to 441 amino acids, these variants of the protein differ from each other by the presence of "repeat-regions" (3 or 4, R1-R4 Figure 13) in the C-terminal part of the protein.

Another variation is the presence or not of one or two inserts (29 or 58 amino acids, E2 or E2/E3 Figure [START_REF] Yu | Upregulation of Astrocytic Α7 Nicotinic Receptors in Alzheimer's Disease Brain-Possible Relevant to Amyloid Pathology[END_REF] Yu, W.; Mechawar, N.; Krantic, S.; Chabot, J.-G.; Quirion, R. Upregulation of Astrocytic Α7 Nicotinic Receptors in Alzheimer's Disease Brain-Possible Relevant to Amyloid Pathology. Mol. Neurodegener. 2012, 7 (1), O7. [START_REF] Sadigh-Eteghad | Beta-Amyloid Exhibits Antagonistic Effects on Alpha 7 Nicotinic Acetylcholine Receptors in Orchestrated Manner[END_REF] Sadigh-Eteghad, S.; Talebi, M.; Farhoudi, M.; Golzari, S. E. J.; Sabermarouf, B.; Mahmoudi, J. Beta-Amyloid Exhibits Antagonistic Effects on Alpha 7 Nicotinic Acetylcholine Receptors in Orchestrated Manner. J. Med. Hypotheses Ideas 2014, 8 (2), 49-52. 13) in the N-terminal part 36 . Because of all these differences, it might be rational to hypothesize that each isoform of the Tau protein has a different role and repartition in the body.

Figure 13: Schematic representation of a tau isoform 36 The tau protein (Figure 13) is composed of two main parts:

-a projection domain with an acidic region and a Proline-rich region. This part of the protein is useful to interact with cytoskeleton elements in order to determine spacing between microtubules in axons. Thanks to a phosphorylation of serine and threonine, it allows a regulation of the interaction between the microtubules, -a microtubule binding domain with a C-terminal part which regulates the rate of microtubules polymerisation and is also involved in the binding with proteins like protein phosphatase 2A or presenilin 1.

Tau proteins are highly hydrophilic and so, highly soluble and heat stable. In patients with AD, it has been observed that the protein is abnormally hyperphosphorylated. Indeed, Tau protein has approximately 79 sites of possible phosphorylation [START_REF] Morishima-Kawashima | Proline-Directed and Non-Proline-Directed Phosphorylation of PHF-Tau[END_REF] . Concerning patients with AD, it has been observed the presence of 3 isoforms of hyperphosphorylated Tau protein: Tau55, Tau64 and Tau69. The major consequence of these insoluble and pathological isoforms formation is the loss of affinity with the microtubules, weakening the cytoskeleton and leading to neuronal death [START_REF] Drechsel | Modulation of the Dynamic Instability of Tubulin Assembly by the Microtubule-Associated Protein Tau[END_REF] .

Moreover, the tau protein aggregates and polymerize to give paired helical filaments (PHF) and then neurofibrillary tangles (NFTs) (Figure 14). 36 Buée, L.; Bussière, T.; Buée-Scherrer, V.; Delacourte, A.; Hof, P. R. Tau Protein Isoforms, Phosphorylation and Role in Neurodegenerative Disorders11These Authors Contributed Equally to This Work. Brain Res. Rev. 2000, 33 (1), 95-130. 37 Morishima-Kawashima, M.; Hasegawa, M.; Takio, K.; Suzuki, M.; Yoshida, H.; Titani, K.; Ihara, Y. Proline-Directed and Non-Proline-Directed Phosphorylation of PHF-Tau. J. Biol. Chem. 1995, 270 (2), 823-829. [START_REF] Drechsel | Modulation of the Dynamic Instability of Tubulin Assembly by the Microtubule-Associated Protein Tau[END_REF] Beginning by the monomer form of the protein, it will assemble to give a dimer with a PHF core, then a soluble oligomer and then an insoluble granular tau oligomer (GTO) which is the direct precursor of PHFs and thus of NFTs 40 .

Therapeutic targets

Given the multifactorial nature of the pathology, a very large number of targets are studied to cure AD. Unfortunately, it would be too tedious to develop all the therapeutic targets. Hence, a choice to present the more advanced projects concerning the most widespread targets in the pharmaceuticals industries has been made. The choice was made according to the data given in Alzforum.org.

From this point of view, we observed that the main targets are:

-AChE inhibition, -cholinergic receptors activation, -amyloid-related strategy, -neurofibrillary degeneration strategy, -oxidative stress decrease.

Along with the development of new molecules related with these targets, another approach should not be disregarded, the repositioning of molecules already used in other pathologies. This approach allows the industries to gain time and money because the compounds already passed the early stages of clinical trials. [START_REF] Verwilst | Shedding Light on Tau Protein Aggregation: The Progress in Developing Highly Selective Fluorophores[END_REF]

AChE inhibition

Historically, the first studied target was AChE. Indeed, as it was explained in the beginning of this chapter, the first histopathological hypothesis was the cholinergic one. Since AChE hydrolyses ACh, the rational approach is to inhibit this enzyme to restore ACh levels in the CNS. Hence, in the 90's, the first AChE inhibitors (AChEI) were developed ( Figure 15). Today, 3 of them are still in the market (Tacrine was withdrawn because of its hepatotoxicity).

Figure 15: Commercially available AChE inhibitors

The first AChEI is Tacrine 41 described as a reversible AChEI in 1953 by Shaw and Bentley and developed by Pfizer in 1993 (and withdrawn in 2004). Tacrine causes allosteric reversible inhibition of the enzyme through its binding to a hydrophobic region near the anionic site on the enzyme's surface.

Pharmacokinetic studies showed the ability of the molecule to easily cross the BBB and to inhibit the cholinesterases in the CNS. Indeed, Tacrine is not selective for AChE as compared to BChE, on the contrary the Ki obtained for hAChE inhibition are of 424 nM whereas the results for hBChE are of 45.8 nM [START_REF] Bolognesi | Multi-Target-Directed Drug Design Strategy: From a Dual Binding Site Acetylcholinesterase Inhibitor to a Trifunctional Compound against Alzheimer's Disease[END_REF] . Even though clinical trials [START_REF] Arrieta | Results and Quality of Clinical Trials of Tacrine in the Treatment of Alzheimer's Disease: A Systematic Review of the Literature[END_REF][START_REF] Summers | Oral Tetrahydroaminoacridine in Long-Term Treatment of Senile Dementia, Alzheimer Type[END_REF] have shown that doses of 120 and 160 mg/day had beneficial effects on patients, they were only palliative and failed to induce any decline of the pathology.

Moreover, it has been proven that a treatment with Tacrine causes adverse effects and hepatotoxicity due to the formation of several hydroxyderivatives during the liver metabolization by microsomal cytochrome P450 enzymes family 45 .

The second well known commercially available reversible AChEI is Donepezil also developed by Pfizer, in 1996. It was a new class of AChEIs bearing an indanone moiety showing a longer and more selective activity toward AChE than Tacrine. Indeed, an IC50 for hAChE of 5.7 nM was found compared to an IC50 hBChE of 4100 nM [START_REF] Sugimoto | Donepezil Hydrochloride (E2020) and Other Acetylcholinesterase Inhibitors[END_REF] . Moreover, early clinical trials in 1996 have demonstrated that Donepezil at a dosage of 5 mg/day for 12 weeks improves cognitive functions in patients with AD. These studies have also shown that administration is not compromised by adverse events or hepatotoxicity, unlike

Tacrine 46 .

The third molecule is Galantamine commercialized in 2000 by Janssen. It is known to be a centrally acting, selective, reversible and competitive AChEI 47 . In vitro studies have brought to light that Galantamine has an IC50 of 0.8 μM toward hAChE which is ten times more effective than toward hBChE (IC50 = 7.3 μM). Moreover, it has shown an activity as an allosteric modulator of the neuronal nicotinic receptors nAChR or ACh 48 , allowing a better sensitivity for the ionic canal opening during the ACh fixation and also a slower receptors desensitization 49 .

The last commercially available AChEI is Rivastigmine commercialised in 1997 by the pharmaceutical company Novartis and as a patch since 2007. Because it generates significantly few gastrointestinal side effects 50 , it enables patients to receive high therapeutic doses. It is currently the most used drug against dementia in a galenic form. Rivastigmine has an indirect parasympathomimetic way of action, it increases the ACh levels inside the synaptic cleft, causes the same effects toward nicotinic and muscarinic receptors of the CNS than ACh and is a pseudo-irreversible cholinesterases inhibitor 51 .

Indeed, its mechanism of action is based on the presence of a carbamate in the structure, the carbamate function will lead to AChE inhibition by a slowly reversible carbamoylation of the enzyme's catalytic serine (Ser203).

The mechanism is the same for ACh and Rivastigmine, the first step is a deacylation (ACh) or decarbamoylation (Rivastigmine) by cholinesterase active serine residue, and a cleavage of the ligand.

In the Rivastigmine case, the carbamate transfer from the stigmine to the residual serine of AChE is a slow process, thus, the IC50 values of this inhibitor are low (hAChE IC50 = 48 μM and IC50 hBChE = 54 μM). However, where the deacylation step is fast in the case of ACh, for the stigmine, the dissociation of the AChE's carbamoyl group is a slow process, which leaves AChE inactive for a long time 51,[START_REF] Bar-On | Kinetic and Structural Studies on the Interaction of Cholinesterases with the Anti-Alzheimer Drug Rivastigmine[END_REF] (Figure 16). Indeed, studies on the human cerebrospinal fluid have shown that a dose of 3 mg of Rivastigmine leads to an effective AChE inhibition for 10 h [START_REF] Polinsky | Clinical Pharmacology of Rivastigmine: A New-Generation Acetylcholinesterase Inhibitor for the Treatment of Alzheimer's Disease[END_REF] . This long enzyme inactivation by Rivastigmine compensates for the low IC50 values toward the two cholinesterases. Besides these four molecules, other structures have been investigated to develop cholines inhibitors, especially AChE (Figure 17). Even if we can observe various structures, most of them failed during the clinical trials due to a lack of activity, efficiency or a high toxicity. For example, the Metrifonate developed by Bayer in 2006, was discontinued because of its side effects on neuromuscular transmission and respiratory paralysis.

Figure 17: Examples of other AChE inhibitors investigated

Yet, Memogain developed by Galantos Pharma, is still in phase I. Memogain (or GLN-1062) is an inactive prodrug of Galantamine. Developing a Galantamine prodrug enabled to increase the compound's lipophilicity as compared to the first-generation drug in order to facilitate its BBB crossing ability, and its capacity to be enzymatically cleaved in the brain and release the active drug. The development of a prodrug also helped reducing adverse effects [START_REF] Maelicke | a Pleasant, Highly Potent and Neuroprotective Novel Drug Treatment in Alzheimer's Disease[END_REF] . Indeed, studies on ferrets have proven that the prodrug's adverse effects are lower than the one induced by Galantamine (Figure 18 -A). This decrease allows to administrate higher drugs doses to patients of AD, the dose could be increased from 16-24 mg/day (for Galantamine) to 226 mg/day (for Memogain) [START_REF] Maelicke | Memogain Is a Galantamine Pro-Drug Having Dramatically Reduced Adverse Effects and Enhanced Efficacy[END_REF] , the performance recovery also seems to be improved with Memogain (Figure 18 -B). [START_REF] Maelicke | a Pleasant, Highly Potent and Neuroprotective Novel Drug Treatment in Alzheimer's Disease[END_REF]

Activation of the cholinergic receptors

Another approach to develop treatments against AD is to explore the synthesis of molecules able to activate acetylcholine receptors. They are ligand-activated neurotransmitter receptors composed of two major types:

-the metabotropic muscarinic receptors, -the ionotropic nicotinic receptors.

Nicotinic receptors activation

Nicotinic receptors are ionotropic receptors directly linked to ion channels. They are composed of five subunits symmetrically arranged around a central pore. Where muscle-type receptors are made up of α, β, γ, δ and ε subunits, neuronal-types are composed of only α and β subunits. Neuronal receptors are either homomeric (only α or only β) or heteromeric (different subunits with at least one α and one β) [START_REF] Zouridakis | Recent Advances in Understanding the Structure of Nicotinic Acetylcholine Receptors[END_REF] (Figure 19). [START_REF] Zouridakis | Recent Advances in Understanding the Structure of Nicotinic Acetylcholine Receptors[END_REF] Figure 19: Schematic view of homomeric and heteromeric nAChR [START_REF] Grando | Connections of Nicotine to Cancer[END_REF] When ACh binds to the nAChR, it leads to a conformation change of the structure which opens the ion channel leading to the cations crossing (K + , Ca 2+ and Na + ). The flux of entering Na + and exiting K + leads to a brief membrane depolarization and thus a nervous transmission or muscular contraction. The next step is the desensitization followed by a return of the receptor in a state of rest 58 (Figure 20). Because of the high number of different nAChRs (combinations of up to 12 different subunits), the nAChRs are not very well known. However, 17 vertebrate nAChRs have been discovered (12 neuronaltype and 5 muscle-type). Among these identified receptors, three are widely expressed in the CNS and are involved in the post and presynaptic excitation: α4β2, α3β4, α7 [START_REF] Graham | Immunohistochemical Localisation of Nicotinic Acetylcholine Receptor Subunits in Human Cerebellum[END_REF] .

In addition to having a strong affinity toward Aβ peptide leading to an acceleration of the senile plaques' formation, studies have shown that the number of α7 (and α4β2) receptors are reduced at the frontal cortex and hippocampus levels in brains of patients with AD [START_REF] Paterson | Neuronal Nicotinic Receptors in the Human Brain[END_REF] . For these two main reasons, it was rational to investigate new molecules with an agonist activity toward nAChRs (Figure 21).

Figure 21: Example of nAChRs agonists

Unfortunately, either α4β2 (Nefiracetam, ABT418, AZD1446) or α7 agonists (Nelonicline, RG3487) failed in advanced phases of clinical trials. The major issue in the nAChRs agonists development is the rapid desensitization of the receptors when these agonists are dispensed.

Promising nAChR agonists -The quinuclidine scaffold

In 2017, Yang et al. [START_REF] Yang | The Current Agonists and Positive Allosteric Modulators of α 7 NAChR for CNS Indications in Clinical Trials[END_REF] wrote a well-documented review about agonists and positive allosteric modulators of α7 nAChRs modulators in clinical trials for CNS related diseases. Among the different modulators like PAMs (Positive Allosteric Modulators) and ago-PAMs (Ago-Positive Allosteric Modulators, which are both orthosteric agonists and allosteric modulators), they developed the state of the art in the progress in the development of agonists of these receptors.

As shown on Figure 22, quinuclidine derivatives like spirooxazolidinones and quinuclidine ethers, amides and carbamates have been widely used in the research of full or partial α7 nAChR agonists.

Currently, most of the existing compounds are partial agonists; Unlike full agonists, they can only produce a small maximum current even if all the receptors are occupied. Indeed, since 1957, it has [START_REF] Paterson | Neuronal Nicotinic Receptors in the Human Brain[END_REF] been supposed that this small response is explained by their inefficiency at eliciting the conformational change between shut and open states of the ion channel in the case of a ligand-activated ion channel 63 .

One of the first developed α7 nAChR partial agonist bearing a quinuclidine was AZD0328 by AstraZeneca which is a derivative of an existing spirooxazolidinone series. It exhibits an EC50 of 338 nM and an efficacy of 65% on Xenopus oocytes expressing human α7 nAChR. In contrast, its maximal activities are only of 12% on h5HT3A, 4% on α4β2 nAChR and no activity on α3β4 nAChR 64 . Studies have also shown that the compound is stable and has good pharmacokinetic properties which suggests it could be a good compound for clinical trial 65 . As a matter of fact, a phase I clinical trial for AD was completed in 2008 and AZD0328 was repositioned as a drug for schizophrenia and went to phase II clinical trial. However, the company stopped the trials because the drug was "unlikely to meet the current target product profile" 66 .

Reported in 2016, another pirooxazolidinone derivative was developed, BMS-933043, by Bristol-Myers Squibb as a selective α7 nAChR partial agonist. Preclinical studies showed a cognition enhancement [START_REF] Bristow | The Novel, Nicotinic Alpha7 Receptor Partial Agonist, BMS-933043, Improves Cognition and Sensory Processing in Preclinical Models of Schizophrenia[END_REF] and a phase I clinical trial was started for schizophrenia.

The most famous example of α7 nAChR agonist is Encenicline developed by FORUM Pharmaceuticals Inc., and reached the phase III clinical trial for the treatment of AD. It is a partial selective agonist and as shown on Figure 22, Encenicline bears a quinuclidine fragment and has an amide bond. It has been developed for the treatment of cognitive deficits in AD but also in Schizophrenia. Rationally, selective α7 nAChR agonists like Encenicline enhance cognition without any side effects associated with an overactivation of other receptors like α4β2 nAChR or muscarinic AChRs 68 . It has been proven that Encenicline penetrates the BBB and it has shown improvement in the memory performances in rats, by potentiating the ACh response. Indeed, it enhances dopamine, glutamate and ACh efflux in the rat cortex [START_REF] Huang | The Novel Α7 Nicotinic Acetylcholine Receptor Agonist EVP-6124 Enhances Dopamine, Acetylcholine, and Glutamate Efflux in Rat Cortex and Nucleus Accumbens[END_REF][START_REF] Huang | The Alpha-7 Receptor Agonist EVP-6124 Increases Dopamine and Glutamate Efflux in Rat Medial Prefrontal Cortex and Nucleus Accumbens[END_REF] because it is also an antagonist at 5-HT3 receptors (IC50 < 10 nM) [START_REF] Prickaerts | EVP-6124, a Novel and Selective Α7 Nicotinic Acetylcholine Receptor Partial Agonist, Improves Memory Performance by Potentiating the Acetylcholine Response of Α7 Nicotinic Acetylcholine Receptors[END_REF] . It has been reported that this compound acts like a co-agonist with ACh and sensitizes the α7 nAChR receptor to its natural ligand. That is why lower doses of AChEIs, when administrated with Encenicline, are effective enough to restore the memory function in an object recognition task [START_REF] Prickaerts | EVP-6124, a Novel and Selective Α7 Nicotinic Acetylcholine Receptor Partial Agonist, Improves Memory Performance by Potentiating the Acetylcholine Response of Α7 Nicotinic Acetylcholine Receptors[END_REF] . However, the phase III clinical trials conducted from 2012 to 2016 for schizophrenia were not conclusive and the phase III clinical trial for AD was suspended in 2017. However, MEM3454 did not improve cognitive deficits in patients with schizophrenia but mild negative symptoms were improved 73 .

Then, TC-5619 (Bradanicline), an α7 nAChR full agonist, was developed in 2013 by Targacept. As shown on Figure 22, it has an excellent affinity and activity on α7 nAChRs 74 . On top of its good values of EC50, TC-5619 has shown promising properties during in vivo studies, like good pharmacokinetic profile and rapid BBB permeability. Nevertheless, during phase II clinical trial, TC-5619 failed to improve cognitive deficits and negative symptoms in schizophrenia 75,76 . However, the good safety profile of TC-5619 in the clinical trials for schizophrenia and hyperactivity disorder could justify its use for further clinical trials for other diseases like AD 77 .

Finally, a quinuclidine ether partial agonist of α7 nAChR has been recently developed by Novartis.

AQW051 has shown promising results 78 , a good pharmacokinetic profile and could also be useful in

Parkinson's disease. AQW051 has been in phase II clinical trial for schizophrenia, AD and Parkinson's disease but failed to reduce dyskinesia or parkinsonian severity 79 .

Even though the majority of developed α7 nAChR agonists failed to pass the high phases of clinical trials, we can notice that all of them seems to have good results toward the receptors (good Ki, good EC50, promising PK profiles, rapid CNS penetration) and they all have a common quinuclidine fragment.

Muscarinic receptors activation 80

mAChRs forms G protein-coupled receptor complexes in the membrane of neurons, G-proteins are used in their signalling mechanism. They are made up of seven transmembrane regions and 5 of these receptors have been identified: M1, M2, M3, M4 and M5. When M1, M3 and M5 couple preferentially to G-proteins Gq/G11 and activate phospholipases, M2 and M4 couple to Gi/G0 G-proteins and inhibit the adenylcyclase activity and modulate the ionic channels conductance [START_REF] Wess | Muscarinic Acetylcholine Receptors: Mutant Mice Provide New Insights for Drug Development[END_REF][START_REF] Conn | Subtype-Selective Allosteric Modulators of Muscarinic Receptors for the Treatment of CNS Disorders[END_REF] . Apart from M5, all the receptors are abundant in the brain, and therefore, important in CNS function like memory and learning activities. In the pathology of AD, mAChRs could be divided in two groups based on their effects:

-the "M2/M4" group,

-the "M1/M3" group.

Studies have shown a M2 mAChR decrease in patients with AD and it has been suggested that a M2 receptor stimulation decreases β-secretase expression [START_REF] Züchner | Beta-Secretase BACE1 Is Differentially Controlled through Muscarinic Acetylcholine Receptor Signaling[END_REF] . Therefore, a logical extrapolation could be that an impoverishment of the receptor could lead to an Aβ peptide increase. Besides, G proteincoupled receptor kinase 5 (GRK-5) deficiency impairs the "M2/M4" group and leads to a cholinergic hypofunction [START_REF] Liu | GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization[END_REF] .

Moreover, the "M1/M3" group has been involved in ACh-mediated cognition. Studies have suggested that stimulation of the two receptors promotes the APP cleavage by the non-amyloidogenic pathway 19564-19571. which decreases Aβ peptide levels. Another aspect is the neurons protection from Aβ peptide toxicity through a pathway involving GSK3 kinases regulation [START_REF] Farías | M1 Muscarinic Receptor Activation Protects Neurons from Beta-Amyloid Toxicity. A Role for Wnt Signaling Pathway[END_REF] .

Thus, there is a strong hypothesis that the Aβ peptide, Tau protein and mAChRs are linked in the pathology of AD (Figure 23).

Figure 23: Putative role of M1 mAChR in AD 86

For all these reasons, mAChRs modulators have been investigated in the treatment of AD (Figure 24).

The major targeted receptor is the M1 mAChR and among the recent developed compounds, only one seems to be promising (HTL0018318). Indeed, HTL0018318, a M1 mAChRs selective agonist, was developed in 2016 by Allergan plc. A phase I clinical trial was ran in 2016, but unfortunately in 2018, the phase II trial was temporary put on hold by the company because of toxicology results from an animal study in non-human primates 87 .

NMDA receptors antagonists

N-methyl-D-aspartate receptor (NMDA), found in nerve cells, is known to be an ion channel protein and part of the ionotropic glutamate receptor family with the AMPA and kainate receptors. NMDA is a heterodimeric complex made up of three different subunits (at least two GluN1 and GluN2 or GluN3 subunits). Moreover, GluN2 has four isoforms (GluN2A-D) and GluN3 has two (GluN3A and GluN3B).

This variety of isoforms and possible assembling leads to a various number of combinations for the receptor (Figure 25). NMDA receptors are expressed in the CNS and play an important role in synaptic functions like learning and memory activity or synaptic plasticity. Indeed, they allow the crossing of cations as Ca 2+ responsible for a fast synaptic transmission [START_REF] Gielen | Fonctionnement des récepteurs-canaux du glutamate -Des proteines responsables de la transmission synaptique excitatrice[END_REF] . For the channel to open and let the Ca 2+ cross, the NMDA receptor needs to be activated by L-glutamate (binding with a GluN2 subunit), bound to a co-agonist like glycine 87 Alzheimer Europe -News -Science watch -Tuesday 18 September 2018 -Sosei and Allergan suspend clinical activities of HTL0018318 in AD https://www.alzheimer-europe.org/News/Science-watch/Tuesday-18-September-2018-Sosei-and-Allergan-suspend-clinical-activities-of-HTL0018318-in-AD. [START_REF] Hansen | Structure, Function, and Allosteric Modulation of NMDA Receptors[END_REF] or D-serine (at the GluN1 subunit level) 90,91 but it also undergoes a depolarization of the dendrite (Figure 25).

Moreover, studies have brought to light that Aβ peptide's oligomers interact with the receptor by inhibiting the long-term potentiation 92 , which is a crucial function for the synaptic efficiency, leading to a loss of synapses and therefore a cognition deficit.

Anti-aggregates of Aβ

As it was explained before, the Aβ peptide spreading and aggregation is a key trigger of AD, therefore, finding a way to avoid the peptide assembly or induce its clearance in the organism could be a viable strategy. With this approach, small molecules have been developed in the past years, a combination of two already approved drugs, Cromolyn (ALZT-OP1a) and Ibuprofen (ALZT-OP1b) is currently in phase III (Figure 27). Recently, Ibuprofen was shown to help lowering the Aβ peptides effects in cell-based and animal models of AD 94,95 . Moreover, cromolyn, a mast cell stabilizer usually used to treat asthma, acts as an anti-inflammatory compound. In 2015, a study showed that Cromolyn inhibits Aβ aggregation in vitro and decreases soluble Aβ levels in the brain in vivo 96 .

Figure 27: Anti-aggregate "drug cocktail"

α-secretase activation

As it was explained before, α-secretase is involved in the APP cleavage in a non-amyloidogenic pathway giving small soluble and non-cytotoxic peptides. Thus, activating this secretase seems rational to promote the APPα formation and block the Aβ peptides development. Moreover, APPα has another role which is to increase the mechanisms of neuroprotection to reduce the Aβ peptide action [START_REF] Stein | Neutralization of Transthyretin Reverses the Neuroprotective Effects of Secreted Amyloid Precursor Protein (APP) in APPSw Mice Resulting in Tau Phosphorylation and Loss of Hippocampal Neurons: Support for the Amyloid Hypothesis[END_REF] .

Different promising molecules have been developed for this purpose (Figure 28) and are currently in phase II or III clinical trials. For instance, Acitretin was reported to reduce Aβ peptide levels and promote the non-amyloidogenic pathway of the APP cleavage in mice [START_REF] Tippmann | Up-Regulation of the Alpha-Secretase ADAM10 by Retinoic Acid Receptors and Acitretin[END_REF] and increase α-secretases activity in humans [START_REF] Endres | Increased CSF APPs-α Levels in Patients with Alzheimer Disease Treated with Acitretin[END_REF] . 24 (35), 7707-7717. [START_REF] Tippmann | Up-Regulation of the Alpha-Secretase ADAM10 by Retinoic Acid Receptors and Acitretin[END_REF] Tippmann, F.; Hundt, J.; Schneider, A.; Endres, K.; Fahrenholz, F. Up-Regulation of the Alpha-Secretase ADAM10 by Retinoic Acid Receptors and Acitretin. FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 2009, 23 (6), 1643-1654. [START_REF] Endres | Increased CSF APPs-α Levels in Patients with Alzheimer Disease Treated with Acitretin[END_REF] Like the α and β secretases, the γ-secretase plays a key role in the APP cleavage and is involved in the two different cleavage process. The first objective was to develop γ-secretase inhibitors, but the adverse effects were important. Indeed, the inhibitors were efficient toward the γ-secretase but also toward the Notch protein, which was leading to a strong toxicity and an increase of infections104 . That is why the second approach was to develop γ-secretase modulators and one is currently in clinical trial (Figure 32). As it was outlined in the beginning of this chapter, the Tau protein is modified in brains of patients with AD and this alteration is leading to the NFTs formation. Numerous kinases and phosphatases have been identified to regulate the protein phosphorylation. The kinases can be divided in three groups:

the proline-directed kinases made up of glycogen synthase kinase-3 (GSK-3), 5' adenosine monophosphate-activated protein kinase (AMPK) and the cyclin-dependent kinase 5 (CDK5),

-the non-proline-directed kinases like microtubule affinity-regulating kinases (MARKs), cyclic AMP-dependent protein kinase A (PKA) or casein kinase 1 (CK1),

-tyrosine kinases composed of Fyn, Abl and Syk.

Even if many of these enzymes play an important role in the Tau protein phosphorylation, the most important kinases in the hyperphosphorylation process are CDK5 and GSK3β, responsible of the phosphorylation of numerous tau protein's residues and promoting the insoluble oligomers formation 106 . Moreover, GSK-3 inhibition has been shown to lower deficits in long term potentiation 107 . This is probably why most of the drug developments in tau hyperphosphorylation focus on GSK-3 108 . But all the developed small molecules inhibiting GSK-3 have been discontinued, the latest molecule being Tideglusib (Figure 34).Indeed, studies on DN-GSK-3β have shown that an extensive GSK-3 inhibition was correlated to neuronal apoptosis and impaired motor coordination109 .

Figure 34: Structure of Tideglusib, a GSK3 inhibitor 6.5.2. Anti-aggregate of the Tau protein

Another approach is to inhibit the Tau protein oligomerisation by developing small molecules able to interact with the Tau protein core. Indeed, in cellulo and in vitro studies have shown that it is possible to inhibit the oligomers formation by interacting with the main protein residues (Tyr197, Tyr310, Phe346, Phe378 and Tyr394)110 . For that purpose, two compounds have been developed (Figure 35).

Figure 35: Anti-aggregate inhibitors

Firstly, Rember TM was tested in humans and showed good results concerning the cognition level improvements during a phase II clinical trial. Unfortunately, the main issue with this compound was the weak bioavailability. Rember TM was discontinued and the firm developed another molecule, LMTM, based on the same scaffold, able to act as a bioavailable prodrug of Rember TM. The compound is currently in phase III clinical trial.

Microtubules stabilisation

Another consequence of the Tau hyperphosphorylation is a destabilisation of the microtubules leading to neuronal death (Figure 36). Microtubules are formed via a heterodimeric polymerization of two globular protein, alpha and beta tubulin, into protofilaments (usually 13 protofilaments) creating a hollow pipe of approximatively 24 nm. In neurons, they are essential for structure maintenance, axonal transport and synaptic plasticity 111 .

Figure 36: Microtubule destabilisation 111

In CNS-related disorders, especially in AD, microtubule loss from axons and dendrites is a major contributor to neurodegeneration. One hypothesis is that abnormally phosphorylated Tau detaches from the microtubules and leads to a tubule's density decrease. Therefore, this loss of mass directly impacts the neuron capacity to maintain synaptic connections and axonal transport. However, there is very little information proving that the tubules simply disassembly by normal dynamic properties.

Another hypothesis includes a microtubule protecting effect of the Tau protein against various proteins able to disassembly the microtubules 112 . Developing molecules stabilising the microtubules could counteract the abnormal detachment of the Tau protein and help maintaining microtubules' organisation and dynamic. Numerous drugs like taxanes used in oncology could be able to stabilise the microtubules by binding to the β-tubulin 113 . Hence, anti-cancer analogues have been developed to this end (Figure 37).

Oxidative stress reduction

Oxidative stress is a key factor of AD. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) production appear to abnormally increase in patients with AD. Indeed, a mitochondria's malfunction leading to a significant production of superoxide ions could explain this behaviour 114 . The direct consequence is a hydrogen peroxide increase in the cytoplasm causing free radicals' formation via a Fenton reaction (Scheme 2) 115 . Studies have brought to light that because of a ferritin diminution in patients with AD, free iron ions are more present, therefore, free radicals' formation is promoted 116 . and its polymerization 117 and upregulate the production of both secretases (β-secretase and γsecretase) involved in the amyloidogenic pathway leading to an increased production of Aβ 118 . Hence, it would seem wise to take an interest in the development of antioxidant to counteract this aspect of the pathology by trapping the free radicals. Well known antioxidants have been studied and seem promising (Figure 38). Indeed, Resveratrol 119,120 , a polyphenol, can improve the patients' cognitive functions for mild to moderate stages of AD with a dose of 200 mg/day 121 . Regarding Melatonin, which is a sleeping hormone, it is currently in phase II and appears to help protecting the cells from oxidative stress by tackling free radicals.

Other strategies

We have presented various therapeutic targets and the development of numerous small molecules intended to overcome the main abnormal behaviour of CNS-related pathologies. Nevertheless, even though, some molecules seem promising, the majority fails to pass the late stage of clinical development because of their inactivity or adverse effects. In the past years, other strategies have been investigated, like immunotherapy (vaccines) or drug repositioning. As a conclusion, the multifactorial character of the pathology involving a large number of enzymes and receptors, could explain the high number of drugs that failed to pass all the clinical trials. The "one molecule for one target" does not seem to be a suitable solution for this kind of disorder. Therefore, in the past decade, besides the drug cocktail acting on different therapeutic targets, a new approach has emerged, the "multi-target directed ligand (MTDLs)" approach130 .

7. The "multi-target directed ligand" approach 7.1. Different therapeutic "multi-target" approaches

In view of the complexity of AD, the "one molecule for one target" concept, implying the use of a drug bearing only one pharmacophore acting selectively on a sole target, seems to have reached its limits.

Thus, for the past decade, other approaches are emerging and are both in the private and academic laboratories (Figure 41):

the « Drug cocktail » approach consists in the prescription of different drugs (usually 2 or

3) to the patient. This approach has proved successful for the treatment of AIDS for instance. This kind of therapy has two major issues, principally problems of therapeutic observance and the fact that it is a heavy treatment, especially for people with cognitive disorders, requiring to take each drug at a definite time depending on its pharmacokinetics and pharmacodynamics parameters.

the "single-pill drug combination" which only one medicine composed of different active substances in order to avoid the problems of therapeutic observance. However, this approach is very complicated because it is mandatory to know very well the reactivity of each compound to avoid or predict the secondary effects. Moreover, the active substances must have close pharmaceutical and pharmacodynamics profiles,

the "Multitarget directed ligands (MTDLs)". Here, the aim is to pair several pharmacophores acting on different enzymes or receptors involved in the disease. One of the advantages of the treatment "one molecule, several targets" is to reduce the secondary effects induced by the interactions between different active substances.

Moreover, it gives a less heavy treatment to the patients of AD.

MTDLs conception

There are five types of pharmacophores' combinations to obtain different MTDLs with a variable synthetic complexity (Figure 42). They can be divided in two groups: MTDLs with a distinct linker and MTDLs without a linker.

Figure 42: Different types of MTDLs

For the first group, there is major difference between the two types of MTDLs. Cleavable conjugates use a linker designed to be cleaved in the organism to release two independent ligands. Concerning the second group no linker is used to bind the two pharmacophores, the molecules are merged to some degree thanks to a common moiety. The blend's degree between the two pharmacophores will determine if the MTDLs is fused, combined or even integrated 131 to give MTDLs with lower molecular weights.

When developing MTDLs, specifications must be respected:

-the pharmacophores' choice must lead to a ligand with optimal pharmacokinetic and physicochemical properties, -affinities and/or efficacies toward the two targets must be similar. Indeed, unlike a "drug cocktail", a 1/1 stoichiometry of a MTDL does not allow a therapeutic doses adjustment, -Affinities of the MTDL toward each target must be in the same range of the observed affinities of each pharmacophore.

This kind of molecules in the treatment of multifactorial CNS-related pathologies like AD has numerous benefits:

-a better long-term efficacy of the treatment by limiting issues associated with low therapeutic observance, -a limitation of adverse effects, -simplified pharmacokinetic studies, -simplified clinical trials, -simplified drug formulation and production. A large variety of MTDLs have been described in the literature and used in various pathologies. activity. Most of the cleavable conjugated MTDLs reported bear an ester-based linker cleavable by plasma esterase to release two independent drugs like NO-aspirin (Figure 43 -1). Moreover, in order to obtain an opioid analgesic with few adverse effects, Montero et al., worked on the association of a guanidium group from the I2 ligand and the fentanyl, an opioid 133 (Figure 43 -3). MTDLs have also been investigated to develop combined drug able to bind and inhibit two proteases, in this example, a metalloprotease inhibitor and a cathepsin inhibitor (Figure 43 -4). Finally, the most complex and less developed MTDLs are the integrated ones. One example of this kind of ligands is a combination of a thromboxane synthase inhibitor pharmacophore and a thromboxane receptor antagonist (Figure 43 -5).

MTDLs designed for AD's treatment

Due to the rapid development of MTDLs synthesis in the past years and to the high number of publications on this topic, we made the choice to only take an interest on MTDLs designed against AD carrying an AChEI. More comprehensive reviews on MTDLs used for AD, have been published

recently 134,135,136,137 . Promising activities against all the targets were obtained but optimisation investigations might be necessary to increase these results.

MTDLs targeting kinases

Another field of research in the professor Renard's team has been the development of conjugated MTDLs inhibiting AChE and kinases. Dr. Killian Oukoloff designed a series of Tacrine -Valmerin (GSK-3 inhibitor) derivatives as part of his PhD work (Figure 49). Among all the compound he synthesized, 28

and 29 were the most promising ligands. Especially 28 gave promising results against AChE with a good selectivity compared to BChE. It also turned out to be selective of GSK-3α/β compared to CDK5/p25 162 . 162 Concerning all the previously described MTDLs, regardless of the therapeutic target, it could be noted that they were all designed with a linker, the direct consequence of it being that the molecular weight of this type of MTDLs is usually extremely high. When developing drugs against CNS-related pathologies, a high molecular weight could problematic because of issues to cross the BBB. In patients with AD, in vitro studies using radiolabelled ligands on brain slices have shown an important decrease of 5HT4R in the hippocampus and frontal cortex 163 suggesting that 5HT4R were involved in cognitive learning and memory processes. Hence, targeting 5HT4R in the development of anti-AD drugs appeared to be a promising approach. Indeed, 5HT4R combined with AChEIs have been proven to be capable of repairing cholinergic damages through a promotion of ACh release in the prefrontal cortex and hippocampus. Moreover, activating 5HT4R could promote the non-amyloidogenic pathway of the APP cleavage 164 , lowering the levels of Aβ in brains of AD patients.

In that context, combined MTDLs targeting simultaneously AChE and 5HT4R have been developed

(Figure 50). In 2014, Donecopride

Conclusion

It has been brought to light in this introduction that AD is highly complex CNS-related pathology with several biological mechanisms involved. Despite numerous attempts of the pharmaceutical companies to design new drugs, only four palliative drugs managed to pass all the clinical trials to be FDAapproved (Figure 51). During the last decades, it has been assumed that the "one compound for one target" was not the optimal solution against a multifactorial pathology like AD, and research groups decided to design MTDLs tackling two or more therapeutic targets of AD.

Figure 51: Approved AD drugs

For more than a decade, the professor Renard's team has been developing medicinal chemistry projects targeting AChE inhibition 170,171,172,173 and developing MTDLs in the treatment of AD. The therapeutic targets tackled by the group researchers were diverse as shown on Figure 52. Indeed, over the past few years, 41 MTDLs targeting AChE and another therapeutic target of AD were designed 174 .

Moreover, the research group gained a strong expertise in the use of a key reaction in medicinal chemistry, such as the use of the Huisgen cycloaddition "click reaction" either in a classical pathway 170,172 or in situ 175 . 170 Rivastigmine scaffold, an AChEI, combined to a quinuclidine fragment, an α7 nicotinic receptors agonist designed by our collaborators, the Professor Routier's team. Finally, in chapter III will be about another new family of combined MTDLs targeting AChE and α7 nicotinic receptor, the variation will concern the choice of the pharmacophore inhibiting AChE. Indeed, in the last chapter, the two combined fragments will be a quinuclidine, for the α7 nicotinic receptors part, and a Donepezil fragment, for the AChE inhibition part.

The main challenge of the two last projects was the design and synthesis of combined MTDLs with a common moiety in order to obtain innovative MTDLs with a small molecular weight. Moreover, it has been shown in this general introduction that the therapeutic targets of AD are numerous, however, working on MTDLs with α7 nAChR agonist properties is simultaneously a key challenge and an innovative way to design AD treatments. Indeed, the number of MTDLs combining an AChEI and an α7 nAChR agonist is small, nevertheless, as an example, Dr Monika Bouet designed a quinuclidine-Tacrine hybrid 33 (Figure 53) as part of her post-doctoral work. Conjugated MTDL 33 displayed a good inhibitory activity against AChE combined with a α7 nAChR partial agonist properties.

Aim of the project

As explained in the general introduction of this thesis, since AD is a complex multifactorial disease, the best approach to find innovative and efficient treatment against this pathology seems to be the Before presenting the synthesis of this MTDL and the in vitro results, the biological targets, namely AChE and BChE, their physiological roles and 3D structures will be exposed. Then, we'll present the structure of the huprine, its synthesis and the derivatives prepared by our group (e.g., selective AChE inhibitors, MTDLs, fluorescent probes, affinity chromatography for BChE purification). At the end, the interest of the use of antioxidant compounds in the treatment of AD and especially capsaicin will be discussed. Eventually, the optimized synthesis of 34 will be presented. Indeed, during the nerve impulse, ACh is released from the neuronal cholinergic synapses and diffused in the synaptic cleft, thus neurotransmitters' concentration in this area becomes particularly high (around 500 mM) 180 and ACh binds to mAChRs and nAChRs receptors known to be involved in the nerve impulse. In the synaptic cleft, ACh hydrolysis into acetic acid and choline is then rapidly achieved by AChE with a particularly high turnover number of approximatively 25 000 molecules of neurotransmitter every second,181 in order to end nerve impulse propagation AChE can thus be considered as the second fastest known enzyme, second to Carbonic Anhydrase182 .

Biological targets

Structural characteristic of AChE

Active site

The active site is made up of an acylation site where ACh is hydrolysed and a cation-π site in which the ACh quaternary ammonium is bound.

The acylation site can be divided in three sub-groups:

-a catalytic triad, -an oxyanion hole, -an acyl pocket.

Catalytic triad (Figure 58)

The triad plays a key role in the ACh hydrolysis and is formed by three residues: Glu334, Ser203 and His447. Ser203 and His447 are directly involved in the hydrolysis and used respectively as nucleophilic attacking group and general acid-base catalytic elements 193 . Indeed, Ser203's nucleophilic character is increased thanks to His447 and Glu334 assistance. After the acyl transfer from ACh to Ser203, stabilisation of the transition state is induced by Glu334 via electrostatic interactions 193 and via N-H amide residues contained in the oxyanion hole. Then, an acyl-enzyme intermediate is formed to release choline and the enzyme is regenerated through a reaction between the intermediate and a molecule of water (which nucleophilic character is once again activated by His447 and Glu334) 194 .

Figure 58: Catalytic cycle of ACh hydrolysis (extracted from Dr. Julien Renou's thesis)

Oxyanion hole

This part of the active site is made up of residues Gly121, Gly122 and Ala204 which allows the ACh's ester activation through H-bond formation between the N-H of their amide and the oxyanion. These residues are also used to stabilise the reaction intermediate 195 .

Acyl pocket

The acyl pocket made up of Trp236, Phe295 and Phe297 interacts with the ester part of ACh and stabilise it via Van der Waals interactions with the methyl's acyl group 196 . Biochemistry 1997, 36 (24), 7481-7489.

The cation-π site, made up of three aromatic residues (Trp86, Tyr337 and Phe338) which interact with the ACh ammonium group through cation-π bounds. The role of these interactions is to direct the ACh's acyl part to the catalytic triad and stabilize the substrate197 .

Hydrophobic gorge

The hydrophobic gorge has a depth of 20 Å and a width of 5 Å and is comprised of 14 aromatic residues.

It links the acylation site and the peripheral site and helps guiding and orientating the enzyme's substrates. Moreover, the aromatic residues are able to bind to ACh quaternary ammonium through cation-π interactions198 .

Peripheral "anionic" site (PAS)

The peripheral anionic site is found in the hydrophobic moiety's entry of AChE gorge, and, despite its name, is made up of uncharged residues, a majority of aromatic residues (for example: Trp286, Tyr72, Tyr124, Tyr341) but also of non-aromatic residues like Asp74. The role of this PAS as ACh binding unit before it enters the gorge, through affinity with the ammonium has been identified long time ago, leading to a false interpretation that this positive charge binding moiety should be constituted of anionic residues giving the "anionic" part of the name, before resolutions of crystal structures proved that this affinity was due to cation -π interactions 199 . Studies have brought to light the importance of the PAS aromatic residues which are able to perform a first recognition of the ligands approaching the enzyme by interacting with them in π -π stacking or cation-π types interactions 200 . The PAS has also a major role in ACh hydrolysis, indeed, when ACh is close to the enzyme, its trimethylammonium group binds to the peripheral Trp286 which leads to a position and conformational change of the Trp86 in the active site. Thereafter, if the ligand is found in low concentrations, the hydrolysis is fast whereas a high ACh concentration a substrate inhibition of the enzyme occurs slowing down the hydrolysis process 201 .

Structural characteristics

BChE structure (Figure 59) is close to AChE structure with 65% of sequence homology and the same disulphide bridges 209 . It has been observed that BChE active site is also made up of an acylation site and a cation -π site, however, its acylation site is larger (500 Å for BChE against 300 Å for AChE) 210 .

Moreover, BChE owns a catalytic triad with the same residues than the AChE triad, a serine (Ser198), a histidine (His438) and a glutamate (Glu325). The main difference between the two cholinesterases is found in the acyl pocket, indeed, where AChE owns two aromatic residues leading to the enzyme's specificity towards acetates BChE owns a leucine (Leu286) and a valine (Val288), two aliphatic residues giving BChE the opportunity to catalyse the hydrolysis of several choline-based esters 211 . Moreover, the BChE acyl pocket is larger than the AChE acyl pocket, due to the replacement of 6 aromatic residues (in the case of AChE) by aliphatic residues (in the case of BChE). This difference is known to be responsible of the poor selectivity of the enzyme as far as the acyl part is concerned, since it confers to larger molecules the possibility to enter and interact with the BChE active site 212 . Concerning the peripheral site, which is larger than the AChE peripheral site, only two residues (Asp70 and Tyr332) interact with the quaternary ammonium of choline-based esters. A H-bond between these two amino acids is also capable to control the functional architecture of BChE 214,215 . The inhibitory potency (in the nanomolar range) of huprine toward AChE could be explained through several strong interactions within the active site of the human enzyme. As showed in the X-ray crystal structure of hAChE in a complex with huprine W 216 , the central aromatic ring of the quinolinium is facing Trp286 while the lateral aromatic ring is facing Tyr337. Moreover, chlorine atom interacts strongly with Trp439 and an additional stabilization of the quinolinium structure is provided by a hydrogen bond between the aromatic nitrogen and the main chain carbonyl of His447 (Figure 61). Several structure-activity relationship (SAR) studies have been realized by Pr. Muñoz-Torrero's team and ours in order to optimize the structure of huprine's scaffold 217,218,219,220 . Structural modification studies have been performed in position 9, 12 and 13 and in the aromatic moiety of this structure.

Chosen structures

The first structural modifications were performed on position 13 by adding alkoxy groups, alkyl groups, a ketone or even by changing the (E) isomer of the ethylene group by the (Z) isomer. Nevertheless, as 216 opposed to the huperzine A, the presence of a substituent in the position 13 decreased dramatically the inhibition potency of huprine analogues 178 .

The second SAR study consisted in modifying the substituent in the position 9, Camps et al. replaced the methyl group of the huperzine A by different substituents such as alkyls groups, allyl, phenyl or a hydrogen atom. The various structural modifications brought to light that only the presence of a methyl or ethyl group was accepted in order to maintain good inhibitory activities against AChE 218,221 .

Finally, an addition of a methyl group or a chlorine atom 219 on the benzene ring moiety displayed promising results. Through the various structural studies, it was possible to better understand the role of each hybrid's constituents and design the first two most potent monomeric AChE inhibitors, huprine X bearing an ethyl group in position 9, and huprine Y bearing a methyl group in the same position (Figure 62) 222,223 .

Huprine functionalized in position 9

Other functionalisation studies of huprine scaffold have been performed by Pr. Renard's team to develop a series of original potent dimeric AChEIs by functionalising the position 9 of the huprine scaffold 231 . After developing an efficient synthesis route to the huprine analogues functionalised in position 9 217,232,233 ,Ronco et al. 190 have synthesised many huprine analogues and some of which are bearing a substituent containing a 1,2,3-triazole moiety (Figure 10). The incorporation of this triazole moiety allowed us to develop efficient and convergent synthetises of heterodimeric AChEIs by using the copper-catalysed alkyne-azide cycloaddition (CuAAC). Moreover, as observed in the crystal structure of mAChE in complex with tacrine based MTDL anti-TZ2-PA6 (PDB 1Q84) 234 reported by Marchot and Bourne, the introduction of the triazole ring branched to the aniline moiety of tacrine (position 12 in huprine) would allow formation of favourable hydrogen bond interactions with wellidentified residues (Tyr337, Tyr124, Tyr341) at the bottom of the gorge and within the active-site gorge of AChE. In vitro evaluations of their inhibitory activities against hAChE and the resolution of X-ray crystal structure of mAChE in complex with 43 (PDB 4A16) allowed us to rationally design potent AChE inhibitors at subnanomolar levels. Indeed, it was demonstrated that a linker possessing 4 carbon atoms (41, 42 and 43) was favourable for a good placement of the triazole ring inside the enzyme leading to derivatives almost 500 times more potent than functionalised huprines bearing a 2 carbon atoms linker (38, 39 and 40), and that the interaction occurred at another binding site situated at an upper place of the gorge closer to the PAS.

Others valorisation of huprine compounds

Based on these results, the bioorganic team has decided to develop several applications using huprine compound such as the preparation of an efficient resin (Hupresin® sold by a lab spin-off company ChemForase) for the purification of BChE and the preparation of a selective and sensitive near-infrared fluorescent probe for imaging AChE in the neuromuscular junction (NMJ) in muscles and in the brain.

Resin for the purification of BChE 235

By grafting a huprine compound functionalised in position 9 on a solid support named Sepharose (Scheme 4), the team of Pr. Renard has developed an efficient resin to purify BChE from human plasma or transgenic plants 233,236,,237,238,239 and HupNIR2 (Figure 66). These two probes exhibited a high affinity toward AChE versus BChE (Table 1) (IC50 is 400-fold higher for AChE than BChE). The confocal fluorescence imaging showed a specific labelling of AChE in the NMJ in the muscles (mice) and even in the brain region displaying a low AChE concentration as striatum.

Capsaicin -a well-known antioxidant

Capsaicin is the active substance of chili peppers obtained from the plants of genus Capsicum and is part of a chemical group called capsaicinoids. Capsicum's effects on human body have been studied

for more than a century, for example, Hogyes observed the burning sensation produced by an extract of capsicum when applied on human skin 244 . Indeed, over the years, Capsaicin and its analogues have been studied in the treatment of diverse pathologies including CNS-related diseases for its potent antioxidant properties 245 (Figure 68). Due to its well-known antioxidative properties, capsaicin has been evaluated for its biological properties against CNS-related pathologies and it has been shown that using capsaicin moiety could have important implications in the prevention or treatment of neurodegenerative diseases such as AD 246 . As an illustration of its antioxidant property, it has been proven that, at 60 μg/mL, Capsaicin is able to scavenge 91.9% of the DPPH free radicals 247 , it has been suggested that the C7-benzyl carbon was the important functional group responsible for the potent observed antioxidant properties. 244 Hence, vitamin E has been studied in the design of treatments against AD, it has been demonstrated that the molecule was capable to counteract Aβ-induced protein oxidation, ROS production and neurotoxicity 261 which might lead to an improvement of cognitive and memory deficit.

Moreover, "drug cocktail" approaches have been investigating to increase the activity of vitamin E by potentially having a synergic action with the additional compound. A combination of vitamin E and memantine 262 and a combination of vitamin E and selenium 263 have been put through phase III clinical trial in 2014 and 2017. Selenium was chosen due to studies correlating its decrease in elderly population's plasma and cognitive decline 264,265 . Concerning the first trial, no information following the trial has been displayed, and unfortunately, the vitamin E-Selenium cocktail was discontinued due to its inactivity to prevent dementia.

Moreover, because it has been shown that oxidative stress was linked to the rise of hyperphosphorylated Tau protein levels and Aβ aggregates levels 266 , combining an anti-AChE activity with antioxidant properties could lead to the synthesis of heterodimers able to act on several therapeutic targets involved in AD. During her PhD in our team, Dr. Sovy Chao designed such conjugated heterodimers by combining a huprine fragment to an antioxidant moiety using the CuAAC.

The lead compound of the series was HUP-RES (Figure 71), a hybrid conjugating huprine and a resveratrol moiety, indeed, it displayed promising inhibition properties against hAChE and hBChE combined to a good activity and selectivity against MAO-A compared to MAO-B. Studies of its inhibitory activity against Aβ auto-induced aggregation and ORAC determination are still ongoing.

Figure 71: Structure of HUP-RES

As a conclusion, this overview of the chosen targets and pharmacophores has brought to light that designing MTDLs targeting simultaneously AChE and oxidative stress could lead to potent MTDLs against AD. Indeed, despite the fact that antioxidants used alone did not display high potency against neurodegenerative pathologies, conjugating an antioxidant pharmacophore to an AChEI could have a synergic effect increasing the properties of the chosen structures. Capsaicin was chosen as the antioxidant fragment because of its simple structure and its ability to scavenge DPPH free radicals, while huprine was chosen because of its high potency against AChE and BChE combined to the great expertise concerning huprine synthesis in the Professor Renard's team.

Hybrid's synthesis

In this context, in collaboration with the Professor Muñoz-Torrero's team, 9 new MTDLs combining an

AChEI and an antioxidant have been developed and biological evaluated in vitro (Figure 72). Two compounds 44c and 34 showed promising results and 34 was selected to be part of in vivo studies (currently ongoing in Pr. Inestrosa's team). This low yield could be explained by the formation of the corresponding ketene as a side product of the reaction. Nevertheless, alkyne 49 was obtained in 4 steps with an overall yield of 18% on a 400 mg scale, our attempts to obtain higher amidation reaction yields failed.

Scheme 11: Synthesis of compound 49

Azide synthesis

For the following of the synthesis, azide 48 was obtained in 10 steps according to a synthesis developed in the laboratory by Doctor Killian Oukoloff 231 . In the interest of clarity, the synthesis will be divided and discussed in four main steps.

Synthesis of intermediate 60

Coupling of the partners by CuAAC

The main step of this synthetic pathway consisted in associating the two partners, azide 48 and alkyne 49, using a Copper(I) catalysed Huisgen cycloaddition to form a 1,2,3-triazole core (Scheme 16). We managed to obtain the protected compound 47 by performing the reaction with copper iodide in acetonitrile with an excellent yield of 71%.

Scheme 16: Synthesis of the MTDL 34

The deprotection step had to be optimized compared to what had been done previously by Dr. Monika Bouet. Indeed, using a catalytic amount of CSA, as it was previously reported, was not effective enough to deprotect the hydroxyl group of 47 even after an extended time of reaction (more than 5 days).

After several tests, we managed to deprotect 47 to obtain the final MTDL 34 with a good yield of 70% by using 10 equivalents of dried CSA.

Moreover, a final purification step by semi preparative RP-HPLC was required to obtain the pure MTDL (e.g. purity > 95%). The major issue was to understand the HPLC signals (Figure 73), we found out that signal (A) and (B) were given by the same compound 34 that seemed to take a protonated form in the TFA buffer we used for analytical HPLC.

We thus managed to synthesize approximatively 150 mg of pure compound 34, a new conjugated MTDL with a huprine pharmacophore and a capsaicin fragment, in 16 steps using a convergent pathway.

Biological evaluation

All 9 conjugated MTDLS (Figure 74) were tested for different biological activities:

-cholinesterases inhibition, -BACE-1 inhibition, -antioxidant properties, -BBB permeability.

BACE-1 inhibition

The percentage of BACE-1 inhibition was evaluated using a peptide mimicking APP sequence as substrate and fluorescence intensities with and without the inhibitor were compared to calculate the percentage of inhibition. Only 6 compounds showed inhibiting properties against BACE-1 with values from 3.6 for 44d (entry 7) for the least potent to 55.6 for 44a (entry 4) for the more active.

Unfortunately, the main compounds of interest 34 didn't express an inhibitor character against BACE-1 (entry 3).

DPPH inhibition

Radical scavenging activities of the huprine-capsaicin hybrids were determined by DPPH assay 269 , an assay based on electron-transfer. Indeed, if free radicals are scavenged by the tested compounds, the stable free radical DPPH will turn from purple to yellow (Scheme 17).

Scheme 17: Principle of DPPH scavenging capacity assay 270

All compounds retained antioxidant properties from the parent compound capsaicin, nevertheless compound 34 proved slightly less active, (higher IC50 against DPPH) bringing to light that adding a triazole ring was disadvantageous for the antioxidant properties of the hybrid (entry 3).

BBB penetration

Finally, to determine the hybrids brain penetration, the parallel artificial membrane permeation for BBB developed by Di et al. 271 was used and lipid extract of porcine brain membrane was employed to perform in vitro permeability assays. All compounds were found CNS+ which suggests that they would be able to cross the BBB and reach the chosen therapeutic targets. It was also observed that increasing the linker's length enhanced the values, suggesting that increasing lipophilicity improved BBB crossing.

This hypothesis was supported by the results obtained for compound 34 (entry 3), indeed, 34 was designed with a triazole core increasing the compound's hydrophilicity and seemed to be the least suited to cross the BBB.

6. Crystal structure with cholinesterases 34 crystallographic structures with TcAChE and hBChE were solved, data were collected from crystal soaked for 12h in mother liquor solution with a 10 mM solution of 34. Main interactions with the compound and cholinesterases are reported in Table 4. 75) revealed that the MTDL's huprine moiety binds at the bottom of the enzyme, in the active site in a similar manner as the parent compound Huprine Y. π-stacking was observed between the aromatic system and Trp84, while the chlorine atom showed its importance by fitting in a hydrophobic cavity brought by Phe330, Tyr334, Trp432, Met437, and Ile439. Moreover, the triazole nitrogen was involved in a perpendicular πstacking with Phe290 and a H-bond interaction with Phe288. Another H-bond interaction was displayed between the amide nitrogen atom of 34 and Ser286. Finally, the capsaicin moiety also interacted with the enzyme through the formation of a water bridge between its ether oxygen and Tyr121.

Entry

Crystal structure with hBChE

A crystal structure of 34 with hBChE was also determined (Figure 76) and the chlorine of 34 was found in the active pocket of BChE at 4 Å from Leu286 and 4.1 Å from Trp231, this position lead to a π-stacking with Phe329. Moreover, the free aniline moiety of 34 in position 12 was positioned between Ser198 and His448 which effectively disrupted the catalytic triad. While the cyclohexene of 34 π-stacked with Trp84, the triazole ring interacted in the same way with Tyr334 but also did H-bonds with Asp70.

Finally, the capsaicin moiety interacted through the formation of an H-bond with Asn68. In this second project, granted by the experience and expertise we gathered in the field of complex linker conjugated MTLDs synthesis, we decided to tackle the challenge of the development of new series of smaller combined MTDLs, more likely to reach all the required drugability parameters. In order to reach this goal, the strategy was based on the use of two known pharmacophores bearing a common moiety to obtain such combined MTDLs (Figure 77).

Figure 77: Structure of the MTDLs series

The main advantages of this type of combined MTDL ligands are:

-the small molecular weights respecting the Lipinski's rules of five 272 , -a good CNS MPO score 273,274 , -a lower structural complexity and an easier synthesis.

Indeed, one of our goals was to develop a shorter and high yielding synthetic pathway as compared to the synthetic efforts required for the access to linker conjugated MTDLs we previously designed in the team. The synthesis of this type of combined ligands was a new project I was assigned to tackle in Professor Renard's team. Considering Pr. Renard's team expertise in the development of AChE inhibitors, and the pivotal role of this enzyme inhibition to minimize AD impacts, we designed our new series with a pharmacophore offering this property. In collaboration with Pr. Routier's team in ICOA

Orléans, who has a long expertise in the development of nAChR agonists, considering their proven efficacy to counteract CNS related disorders, we selected an α7 nAChR agonist as second pharmacophore. The site used to bind ACh and nicotine is an orthosteric binding site found in the extracellular domain between the receptor subunits and is composed of identical α-type subunits 284 (Figure 79). It has been described as a series of "loops" bordering the binding site that could be divided in two groups: loops A, B and C from the principal component and loops D, E and F from the complementary component. Because α7 nAChRs are homopentamers, their binding sites are identical and made up of aromatic residues such as Tyr97, Tyr157, Tyr196, Tyr203, Trp155 and Trp57, bridged cysteines Cys198 and Cys199 and a disulphide bridge 286 . This last moiety gives to the binding site an electronegative character neutralizing the positive charges carried by most of the nicotinic ligands 287 . Moreover, a cation-π interaction between the ACh's ammonium centre and Trp155 was proven to be involved in ACh binding to the receptors 288 . Indeed, the ammonium is placed in the centre of an "aromatic box" composed of most of the aromatic residues ( Figure 80). modulation of the receptor functions, particularly when interacting with G-proteins and, hence, Gprotein-coupled receptors 296 (Figure 82).

Modulation of α7 nAChRs

When the receptor is in presence of high concentrations of neurotransmitters, it undergoes conformational transitions known as the gating isomerisation or active state, in this state, the α7 nAChR is highly permeable to Ca 2+ . After responding to their agonists, the receptors' actions are terminated either by the removal of the ligand leading to a resting state in which the ion gate is closed or by a fast desensitization 297 in which the orthosteric site is still occupied by the neurotransmitter, but the ion channel is closed. In order to be able to interact again with other neurotransmitter, the receptor must transit to a resting state in which the orthosteric binding site is empty (Figure 83). 296

nAChRs allosteric modulators

Various α7 nAChRs PAM have been designed in order to potentiate α7 nAChRs ion channel opening and thus current in presence of ACh and they can be divided in two types:

-type I PAM which mainly enhance agonist-evoked peak current without delaying desensitisation and without reactivating properties toward the receptor (Figure 84), -type II PAM able to delay desensitisation and reactivate desensitized receptors (Figure 85).

AVL-3288 is a type I α7 nAChRs PAM currently in phase I clinical trial for schizophrenia and schizoaffective disorder 299 . Studies in rodent models have shown that this promising compound was able to correct sensory deficits and improve cognition with or without the presence of an agonist 300 .

Figure 85: Examples of type II α7 nAChRs PAM

As a conclusion, α7 nAChRs agonists and PAMs appear to be a viable therapeutic strategy against CNSrelated pathologies, nevertheless, their mode of action, binding sites and overall structures are diverse, SAR studies failed to lead to a rationalization of the pharmacophores structural requirements, and the complexity of the nAChRs structure and their allosteric character thus significantly complicate the drug design. Indeed, the structure reorganization and the fact that no human nAChR crystal structure has

been determined yet must be considered during the development of drugs targeting nAChRs.

Chosen structures

α7 nAChRs agonists

Our first choice was to develop combined MTDL including an α7 nAChRs agonist scaffold. We designed our MTDLs with the help of our collaborators in the ICOA (Orléans). Indeed, Professor Routier's team has a large expertise in the development of α7 nAChRs especially compounds displaying a quinuclidine fragment and a triazole core. Moreover, they developed a library of compounds able to bind to α7 nAChRs bearing either a tropane, a quinazoline or a quinuclidine moiety 307 . They also put their efforts in the development of another α7 nAChRs agonists series with a 1,2,3-triazole core which is a wellknown amide bond bioisoster and they were able to synthesize 30 new compounds bearing this core and a quinuclidine fragment. SAR studies showed that the quinuclidine fragment was essential to obtain good biological in vitro results. They also showed that the (R)-configuration was more active 307 Thus, once we had all this information in hands, we decided to work on a small fragment, easy to synthesize and displaying good biological results as a monomer structure. The fragment (R)-69 we chose to work on has a 1,2,3-triazole core, bears a quinuclidine moiety, which is also found in a series of already developed α7 nAChR agonists, and a phenyl ring.

AChE inhibitor

Even though we now know that AD is a multifactorial disease, achieving an efficient AChE inhibition has been the main strategy used to develop treatments against AD for years. Indeed, inhibiting AChE could help restoring the ACh levels, a key neurotransmitter, in the brain. To be effective, we decided to base our inhibitor scaffold on one well-known AChE inhibitor (e.g. Tacrine, Rivastigmine, Galantamine, Donepezil). After observing the structures of these four main AChE inhibitors, we had to choose the one fulfilling all the criteria:

-small pharmacophore, -easy synthesis, -common moiety with the other chosen pharmacophore (α7 nAChRs agonist).

We thus turned our attention towards the Rivastigmine scaffold because all the criteria were fulfilled but also because it was a scaffold that attracted less attention for MTDLs, and that was not tackled in the group, and we were interested by the challenge. 308 The carbamate moiety has thus proved to be a promising fragment to use for the design of MTDLs against AD targeting AChE, but to this date, such Rivastigmine based pharmacophore remains underused in the development of new anti-AD drugs.

Hence, in this context, we decided to work on this particular field of research with these two promising pharmacophore scaffolds bearing a common moiety which will allow us to reduce the MTDL's molecular weight and develop combined MTDLs (Figure 88). -more than 5 H-bond donors, -more than 10 H-bond acceptors, -a molecular mass over 500 g/mol -an octanol-water partition coefficient log P higher than 5

Nevertheless, in the past years it has been shown that defining the druglikeness of a compound by only following these four rules was misleading. For example, the number of FDA-approved drugs with a MW over 500 g/mol has increased while the number of FDA-approved drugs with a MW below 500 g/mol has stayed relatively constant (Figure 90). All these evaluated properties are weighted equally, with a score ranging from 0.0 to 1.0 for each property and thus a CNS MPO score of a molecule can range from 0.0 to 6.0. Moreover, Wager et al.

evaluated their new algorithm on a set of drugs and candidates (Figure 91) and brought to light that 74% of drugs had MPO scores over 4, a higher score when compared to the candidates (only 60%). This study suggested that CNS MPO score might be a potent tool to increase the probability of CNS targeted drugs survival to clinical trials.

CADD -A potent tool in drug discovery

As it was mentioned in the general introduction, over the past years, the cost of drug design and development has significantly increased along with high rates of clinical trial failures. Hence, to tackle this issue, the in silico approach has been developed to evaluate small molecules interactions with multiple targets (enzymes, proteins, etc). As shown on Figure 93, computer aided drug design (CADD) can be used to various goals like pharmacophore screening to find novel small molecules interacting with the receptors or to build innovative MTDLs, structures optimisation by QSAR studies, discovery or validation to predict druggability. In our work, we used CADD to predict the designed ligands binding affinities to AChE and to visualise their position inside the pocket's enzyme. As already mentioned, we did not use CADD to predict the desired ligands binding ability towards α7 nAChR, due to the multiple potential modes of interaction. We also performed such calculation to predict their druggability.

Docking protocol

To choose the more promising structures to synthesize, we decided to perform in silico studies to evaluate ligand's binding to AChE (by introducing rigid or with flexible residues depending on the ligands we were evaluating). We have made the choice to work only on the AChE binding and not on the other targets, especially the α7 nAChR which structure and potential allosteric binding pockets as well as their potential effect on the receptors remain unknown.

Docking studies are divided in 5 steps:

- Once the choice of the enzyme structure in the protein data base to start with is made, thanks to a software (AutoDock tool), the target must be prepared. Indeed, the point is to remove the water molecules and the ligand of the co-crystal structure, and to determine which residue is protonated at physiological pH to add the corresponding polar hydrogen, and thus have an accurate vision of the charge of each residue.

Then, a box must be chosen, it means that the active site of the enzyme will be defined by 3 coordinates (X, Y, Z) defining a "box" where the docking will take place, and the ligand could be placed. If the box is too small, the docking will not be accurate and the possible conformations of the ligand inside the enzyme is limited, but if the box is too large, the calculation will be long and tedious.

Choice and preparation of the ligands

Once the target prepared, the ligands three-dimensional structures must be determined, and this happens in two steps. First, the ligands are designed with ChemBioDraw, the polar residues can be protonated or deprotonated if needed. This is achieved through the use of the Chemicalize.fr software, which enables to evaluate the protonation states of the ligands at pH 7.4. In a second time, the energy levels of the ligands are minimized with Chem3D which uses a Merck Molecular Force Field, MMFF94 318 .

Docking calculation

Once the target and the ligand are ready to be docked, thanks to AutoDock Vina 319 software, the docking calculations can be launched. AutoDock Vina is a suite of automated docking tools designed to predict energy levels of a small molecules such as drug candidates bound to a receptor of known 3D structure. The main programme is used to perform the docking of the ligands to a set of grids calculated by the software describing the target enzyme, in this case, the AChE. This software scoring function has been designed by combining knowledge-based potentials using the PDBbind database which gathers binding affinity data (KD, Ki and IC50) for 1359 protein-ligand complexes 320 with empirical scoring functions, more precisely by using the AMBER force field (assisted model building with energy refinement) 321 . Moreover, this type of hybrid scoring function has been widely inspired by X-score, an empirical scoring function for structure-based binding affinity predictions 322 . X-score includes in its scoring terms accounting for deformation penalty, Van de Waals interactions, hydrogen bonding and hydrophobic effects, this last parameter is calculated through three different algorithms leading to an improvement of the docking accuracy when compared to conventional force field computation.

Evaluation of the results

After the calculations, we will obtain evaluation of binding affinities of the chosen ligand in different conformation (usually 9) within the active site of the enzyme. These results are viewed as a text file with negative energy values in kcal/mol. The calculation considers intramolecular forces of the covalent binding (length of the link, angle of the link, dihedral angle) and non-covalent binding intermolecular forces (electrostatic, H-bond, dipolar interactions, hydrophobicity, Van der Waals).

Reasonably, the given results with the lowest values should be the more stable conformations displaying a higher number, and stronger interactions.

Visualisation of the results

To be able to visualize the different predicted conformations, different softwares can be used, like

Pymol which enables to see the position of the ligand inside the catalytic pocket or like Discovery studio which enables to visualize the interactions of the ligand with the enzymes' residues or within residues.

The visualization is an important step of the studies allowing us to rationalize the binding energy values previously obtained, to evaluate the positioning of the ligand inside the pocket and its different interactions with the residues of the target.

Design and synthesis of the first family of Stigmine derivatives

In silico evaluations

The carbamate moiety could be placed on any position relative to the triazole (-o, -m or -p). Due to the strong steric hindrance and blocked configuration of the whole molecule induced by the relative position of the carbamate moiety, the "ortho" family was not investigated. Nitrogen substituents R1 and R2 could be either methyl or ethyl group. The selected structure presents one stereocenter, thus in order to select the structures to synthesize, we performed docking studies on 12 derivatives (6 with a (R) stereocenter and their (S) enantiomers) of a Stigmine derivative linked with an α7 nAChRs agonist (Figure 94). The docking was made with mAChE (PDB 1Q84)323 structure containing flexible residues (Trp86, Tyr72, Tyr124, Tyr337, Tyr341). In silico investigations gave us essential pieces of information concerning the MTDLs we designed. The three main results that needed to be taken into account were:

-the binding affinity between the ligand and the enzyme, -the proximity of the AChE catalytic serine (Ser203) with the ligand's carbamate, -the interactions of the whole structure with the main residues of the enzyme.

. Binding affinity and proximity with Ser203

After following the protocol given previously, we obtained the following results for the 12 compounds compiled in Table 5. This first evaluation brought to light that varying the quinuclidine's configuration didn't affect the results in a significant manner.

The binding affinities of the desired MTDLs were similar compared to the Rivastigmine's binding affinities. Moreover, the two types of derivatives were well positioned inside the enzymatic pocket and the quinuclidine fragment could also interact with the enzyme's peripheral site (Figure 95).

Nevertheless, it could be notified that derivatives (R)-75c and (R)-76c (entries 4 and 7) displayed a significant stronger empirical binding energy when bound to AChE. On the other hand, the meta derivatives apart from (R)-76c (entry 7) would bring the carbamate closer to the catalytic serine because of their preferential conformation. Indeed, as shown on Figure 95, the ligand (R)-76a's angle enables the MTDLs with a carbamate in the meta position to better interact with the catalytic serine.

Table 5: Docking results of (R)-75a-c, (R)-76a-c, (S)-75a-c and (S)-76a-c

Interaction with the main AChE residues

Due to the striking similarity of results between the derivatives, only interactions between a representative of the meta and para substituted compounds (R)-75a and (R)-76a with AChE will be discussed but the results could be extended to all other derivatives (Table 6). Table 6: Main interactions predicted in silico for compounds (R)-75a-c and (R)-76a-c

Entry

Interactions predicted for the "para" derivatives family

Concerning the interactions between AChE main residues and the para derivatives (Figure 96), we could observe π -π interactions between Tyr341 and the phenyl ring of the ligand (entry 1). The phenyl also interacted with Tyr337 in a π-lone pair interaction type (entry 2). Moreover, the ligand's carbamate was predicted to interact with Trp86 through a π-σ interaction (entry 3).

Figure 96: Representation of predicted interactions between mAChE residues and (R)-75a

Main interactions predicted in silico for with the "meta" derivatives family

In the case of the meta derivatives (Figure 97), less interactions were found which could explain some of the lower binding energies as compared to these we obtained previously. Indeed, we only predicted a π -π interaction between the triazole core and Tyr341 and Tyr124 (entry 1), and a π -cation interaction between the Trp286 and the protonated amine of the quinuclidine (entry 4). Surprisingly, we couldn't observe neither the ligand's carbamate nor phenyl ring interactions with residues of the enzyme. This could be explained by the conformation of the ligand and the carbamate position which probably hinder the optimal placement of the ligand inside the catalytic site.

Figure 97: Representation of predicted interactions between mAChE residues and (R)-76a

To date, only two co-crystal structures of Rivastigmine with TcAChE have been disclosed in the literature, the first one was determined by Bar-On et al. 324 in 2002 (PDB : 1GQR) but displays the structure of Rivastigmine after the cleavage of the carbamyle leaving group (NAP) (Figure 98). The second one concerns analogue metabolites of Rivastigmine (PDB 6EUE) which were developed by Due to the lack of information concerning the interactions of Rivastigmine with AChE, it was decided to perform docking studies with the Rivastigmine structure itself (Figure 99) to be able to compare the results with the one obtained for the designed MTDLs. Apart from the position of the Rivastigmine which is close to the one observed for the various designed MTDLs, we observed several interactions with the main AChE residues found either in the MTDLs study or in the Rivastigmine study. Indeed, concerning Rivastigmine, the phenyl ring was predicted to perform a π-stacking interaction with Tyr341 and Tyr124 while the ethyl substituent of the carbamate displayed a π-sigma interaction with Trp86 which are interactions already predicted for the designed MTDLs and the enzyme. Moreover, a H-bond formation has also been predicted between Tyr124 and the Rivastigmine's oxygen.

CNS MPO scores

Besides the fact that all the derivatives are in accordance with the Lipinski's rules, CNS MPO scores have been determined for all the compounds of the series and showed the same results of 5.4/6 which are closed to the result obtained for Rivastigmine, the parent compound (Table 7). Hence, the 12

proposed MTDLs have promising pharmacologic profiles.

Entry

Percepta software CNS activity prediction

The last in silico study that had to performed was the evaluation of the compounds ability to penetrate the brain and be CNS active by using the Percepta software. It could be noted that the relative position of the carbamate on the molecule did not affect the predicted results (Table 8). It could also be noted that the calculated Percepta evaluated LogP and pKa are close to the ones found when evaluating the CNS MPO scores, which shows a logical link between the CNS MPO score and the results obtained with the Percepta software. Indeed, some of the physicochemical properties used to calculate CNS MPO scores are also used to predict the BBB penetrations of potential novel drugs by Percepta software. Result can be extended to the (S) enantiomers Table 8

Entry

: Physicochemical properties determined with Percepta of Rivastigmine, (R)-75a-c and (R)-76a-c

As it is shown on Figure 100, all the designed MTDLs appeared to be promising compounds able to passively cross the BBB according to the Percepta software prediction. Moreover, compound (R)-75b

(and compounds (R)-76b) (Figure 100 -(B)) displayed the best results concerning their predicited aptitude to be CNS active. This study showed us that even though all the MTDLs have shown interesting results, the carbamate's substitution can affect the brain penetration : a carbamate substituted by two ethyl groups seems to be the optimal structure for this type of ligands, according to the predictive softwares we used.

Figure 100: Results of Brain penetration for CNS activity for (A) (R)-75a (B) (R)-75b (C) (R)-75c (D) Rivastigmine (red dots represent CNS inactive compounds, blue dots represent CNS active compounds and the circled black dot represent the tested compound)

Hence, these computing evaluations have brought to light that the designed MTDLs were all promising compounds potentially able to interact with AChE, regardless of the substituent type or position.

Moreover, the CNS MPO scores and results obtained from the Percepta software showed interesting properties concerning the ability of all compounds to cross the BBB an act directly at the CNS. We thus decided to undertake the synthesis of all compounds.

Synthesis of Stigmine's derivatives (series 1)

Retrosynthetic route

In order to obtain the new series of MTDLs 75a-c and 76a-c and their enantiomers (R)-75a-c and (R)-76a-c and (S)-75a-c and (S)-76a-c bearing a carbamate function, we decided to develop a convergent synthetic pathway using the Copper(I) catalysed Huisgen cycloaddition as a key step of the synthesis (Scheme 18). As seen earlier, 1,2,3-triazole is one of the most common bioisosters of amide bonds, and, due to its easy synthetic access since Sharpless 326 and Meldal 327 have shown that the Huisgen cycloaddition between azides and alkynes yielding such scaffolds could be copper catalysed, with a perfect control of the isomer formed (1-4 vs 1-5 substituted triazole ring), its use in medicinal chemistry has increased. This abundance is also supported by practical structural features such as a good stability to basic and acid hydrolysis and reductive or oxidative conditions 328 . Moreover, this heterocycle displays a noticeable dipolar moment which could induce hydrogen bonds formation and is prompt to favour pi-stacking interactions with the target and thus should enhance the MTDLs activity 329 .

Once racemic and enantiopure azides 77, (R)-77, (S)-77 and the desired alkynes 78a-c and 79a-c are obtained, we should thus be able to link easily the two parts of the MTDLs by the desired 1-4 substituted triazole ring formation through catalysed regioselective Huisgen cycloaddition, in order to yield the targeted new series of compounds against AD.

Scheme 18: 75a-c and 76a-c retrosynthesis through a SNi pathway 335 (Scheme 20) but that the diazo (-N=N) moiety was transferred from the diazotransfer agent's nitrogen to the primary amine.

Scheme 20: Wrong Sni diazotransfer mechanism 335

They also confirmed the hypothesis given by Nyffeler et al. 332 that the attack of the amine group occurred on the terminal nitrogen of the transfer agent. And they thus hypothesised that, taking into account the copper(II) salts catalysis, the mechanism involves a transfer of the two terminal nitrogen atoms of the triflyl azide, through an intermediate 5 members metallocycle (Scheme 21).

Scheme 21: Plausible postulated diazotransfer mechanism 332

Enantiopure azidoquinuclidine synthesis

As it was shown previously by the Professor Routier's team, α7 nAChR agonists bearing a quinuclidine have better affinities when the (R)-enantiomer is used. For most of them, the binding affinity is in the nanomolar range, which is promising for the expected values of our MTDLs 308 .

However, because of the enantiopure starting material (R)-81 cost (90€/g), we decided to work on a diastereoselective synthetic pathway from the commercially available and cheap ketone. This synthesis consisted in a diastereoselective reduction of compounds 86 obtained from ketone 80 and a chiral auxiliary. 335 Pandiakumar, A. K.; Sarma, S. P.; Samuelson, A. G. Mechanistic Studies on the Diazo Transfer Reaction. Tetrahedron Lett. 2014, 55 (18), 2917-2920.

Our first idea was to synthesise compound 86 bearing a (S)-methylbenzylamine as a chiral auxiliary.

Starting by a condensation between the ketone 80 and (S)-phenethylamine, we managed to isolate imine 85 with a low 25 % yield which could be potentially explained by stability issues. The next step was a diastereoselective reduction of compound 85. We decided to perform it with L-selectride in order to use mild conditions (Scheme 22). Unfortunately, we were not able to obtain compound 86 but only degradation products too complex to be identified.

Scheme 22: First Synthetic pathway to (R)-81

Because of the obtained products' complexity, we decided to try another synthetic pathway. We changed the chiral auxiliary and used (R)-2-methylpropane-2-sulfinamide to induce a diastereoselective reaction 336 . However, we could not synthesise compound 87 despite our attempts to perform a condensation between ketone 80 and (R)-2-methylpropane-2-sulfinamide, even with using a strong Lewis acid such as tetraethoxytitane(IV) (Scheme 23). Here again, we only obtained a complex mixture of unidentified degradation products. 336 Ruano, J. L. G.; Alemán, J.; Parra, A.; Cid, M. B. Preparation of N-p-Tolylsulfonyl-(E)-1-Phenylethylideneimine. In Organic Syntheses; American Cancer Society, 2007; pp 129-138.

Scheme 24: Synthesis of carbamoyl chloride 91c

Our first attempt to synthesize the alkynes was to use a Sonogashira coupling reaction between trimethylsilylacetylene and the bromophenols bearing the desired carbamate moiety. Yet, our first attempts, and the experience of the laboratory on Sonogashira coupling reactions led us to realize that this carbamate moiety was interfering with the halogen residue during the Sonogashira cross coupling process. Thus, we changed our approach and decided to begin with the Pd-catalysed cross coupling reaction prior to the carbamoylation of the phenol (Scheme 25). The desired alkynes were synthesized in only 3 steps following the same synthetic pathway for all of them, with modest to good yields.

Scheme 25: Retrosynthetic pathway for alkynes 78a-c and 79a-c

After a Pd-catalyzed Sonogashira coupling 338 reaction between iodophenols 93a-c and trimethylsilylacetylene in present of a catalytic amount of a tetrakis(triphenylphosphine)palladium (0), we performed a deprotection of the obtained compounds, using tetrabutylammonium fluoride in order to obtain alkynes 92a-c 339 . The following step was a carbamoylation reaction of the phenol on the alkynes and the corresponding carbamoyl chloride with potassium carbonate 340 giving us a series of new 6 alkynes 78a-c and 79a-c bearing respectively the N,N-dimethyl carbamate 91a, the N,N-

diethyl

Scheme 26: Alkynes 78a-c and 79a-c synthesis

At the beginning of the project, alkynes bearing the carbamate in the ortho position were also

targeted. Yet, we realised that, even if, the accordingly substituted alkyne could be obtained, steric hindrance induced by the carbamate position will preclude performing the CuAAC, the last step of the synthetic scheme. Nevertheless, we managed to obtain the 6 other alkynes (meta and para substituted) in only 3 steps with overall yields ranging from 51 to 73%.

Final key step of the synthesis: The Copper catalysed Huisgen cycloaddition

With the racemic and enantiopure azidoquinuclidines and the different alkynes in hands, we could perform the Huisgen cycloaddition using catalytic amounts of Cu(II) 341

Scheme 27: Final step of MTDLs synthesis

Our efforts to rationalise the different global yields we obtained for the MTDLs series yet failed. Indeed, the unreproducible and variable yields we obtained for the azide intermediate precluded such a rationalization. This poor reproducibility could be directly assigned to the azide formation step and the high volatility and lack of stability of this key intermediate. Thus, these poor reproducibility issues had a direct impact on the global MTDLs yields. The following global yields were obtained for the different targeted MTDLs (Table 9).

Entry

(S)-76c

Table 9: Global yields obtained for the 18 targeted MTDLs

As a conclusion, we managed to obtain 6 new racemic combined MTDLs in 6 steps with global yields from 9% to 32% (entries 1 to 6) and their enantiopure analogues in 5 steps with overall yields from 13% to 36% for the (R) derivatives (entries 7 to 12) and global yields from 14% to 34% for the (S) analogues (entry 13 to 18).

Biological results

Binding affinity toward α-7 nAChR

The 12 enantiopure compounds (R)-75a-c and (R)-76a-c and their enantiomers (S)-75a-c and (S)-76ac were evaluated for their binding affinities toward α7 nAChR by the Pr. Chalon's team (CHU de Tours)

and reported in Table 10. Experiments were performed on male Wistar rats beheaded on the day of the assay and both frontal cortices of each animal were removed and kept on ice. Binding was determined by incubating proteins extracts from the frontal cortices (25 µg), radiolabelled [ 125 I]αbungarotoxin (2 nM) , a known competitive antagonist of nAChR in the presence or not of each tested compound (10 -6 to 10 -10 M). Ki values corresponding to the ability of each compound to compete for nAChR binding with bungarotoxin were calculated according to Cheng and Prusoff procedure 342 . Assays were repeated in triplicate.

A first thing to observe is that, as expected, all the (R) compounds (entry 2 to 7) have significant better affinities toward α7 nAChR than the (S) compounds (entry 8 to 13). Indeed, the best candidate of the (S) series has a binding affinity of 13 nM (entry 9) whereas the lowest value for the (R) series is 2.3 nM (entry 6). We can also note as an unexpected and gratifying result that our most promising compound (entry 2) shows a better binding affinity that the monomer (entry 1) we based our work on. Indeed, the values ranges from 8 nM for the known α7 nAChR agonist (entry 1) to 0.35 nM for compound (R)-75a (entry 2). One of the main risks when associating two pharmacophores when developing MTDLs is that the combined compounds displays a decrease or even total loss of one of the targeted biological activities. On the contrary in our case, it seems that that the carbamate moiety, initially introduced on the α7 nAChR ligand in order to add an AChE inhibitory activity, tends to increase the ligand affinity toward the receptor.

Table 10: Affinity of (R)-75a-c, (R)-76a-c, (S)-75a-c and (S)-76a-c toward a7 nAChRs

AChE inhibition

Cholinesterases inhibition were evaluated for the 6 (R) enantiomers by the Pr. Bartolini's team on human AChE and BChE and the preliminary data are reported in Table 11, unfortunately (S) enantiomers could not be evaluated due to a contamination of the series. The capacity to inhibit AChE and BChE activities were assessed using the Ellman's method343 . Initial rate assays were performed at

Figure 101: time-dependent inhibition profile of (R)-75b

We were particularly pleased to observe that for some of the synthesized MTDLs displayed a better AChE inhibition potency (up to one order of magnitude) that parent carbamate Rivastigmine. These results could be explained by the additional interactions which were predicted in the preliminary in silico studies between the quinuclidine fragment and peripherical site of the enzyme. Only crystal structures of the enzyme in interaction with the MTDLs could confirm these hypotheses. These studies are in progress in Martin Weik's team (IBS, Grenoble), a longtime collaborator of Pr. Renard's team for AChE and BChE structures determination.

The biological evaluations of the MTDLs affinities toward α7 nAChRs and inhibitory potency against AChE have shown global encouraging results, and two compounds have shown to be the most promising, (R)-76a and (R)-76b (Figure 102). Indeed, these results allowed us to obtain conjugates between an α7 nAChR agonist pharmacophore with an AChE inhibitor pharmacophore with increased properties compared to both the parent monomers. The IC50 values toward hAChE decreased from 50 µM to values around 5 µM. For the α7 nAChR, affinities decreased from 8 nM for the monomer parent to values around 1 nM for the MTDLs. These results have brought to light that a rational choice of each pharmacophore, besides its known activity toward its designated receptor, could assist the other pharmacophore in order to increase its biological properties.

Figure 102: Most promising MTDLs obtained for the series

Since the phenol is released once AChE or BChE is inhibited, we will also need to evaluate the biological properties of these parent compounds towards α7 nAChR. The preliminary results we obtained only display a binding affinity for the MTDL with α7 nAChR, for the carbamate and the phenols, the next step will be to undertake electrophysiological experiments in order to evaluate the effect of the MTDL (agonist / antagonist / allosteric modulator) on the receptor. These experiments will be undertaken by Dr. Romulo Araoz and Dr. Denis Servent's team at CEA Saclay. This team possesses the know-how on HiClamp experiments on xenopus ovocytes overexpressing the human α7 nAChR and has already evaluated the effect of some of the MTDLs synthesized in Pr. Renard's team.

The set-up of these assays required an additional amount of the most promising MTDLs. I, thus, following the synthetic pathway we optimized, performed an upscaled synthesis of the following compounds (Table 12):

Entry Compound Structure Isolated mass (mg) 1 (R)-76a 30
2 (R)-75b 80

Table 12: Upscale synthesis of (R)-76a and (R)-75b

If an agonist activity or an allosteric modulation is observed, the next step will be to evaluate the BBB crossing ability for the best compounds obtained (a collaboration with Pr. Marie-Pierre Dehouck, Université d'Artois has been set up to this end). If the in silico prediction are confirmed, in vivo studies will be undertaken, but finding an industrial partner able to further develop these molecules would be the best strategy.

Docking studies

Following the previously explained procedure, we prepared the target, and the different possible ligands by varying the relative position of the phenyl rings and the substituents of the carbamates ( 12possible combinations of each enantiomer) (Figure 104).

Figure 104: Structures of the MTDLs evaluated in silico

The three key points to evaluate during this study were:

-the binding energy value, -the interactions with the main useful residues of the enzyme, -the proximity of the carbamate with the catalytic serine (Ser203).

We used a semi-rigid model of the enzyme starting from the same crystal structure of mAChE (PDB 1Q84) 323 and we choose to give flexibility to the most important catalytic site residues of the target which could be involved in the ligands interaction (Trp86, Tyr72, Tyr124, Tyr337, Tyr341).

Binding affinity and proximity with Ser203

After completion of the 24 docking simulations, we obtained the following results listed in Table 13.

Once again, the quinuclidine's configuration didn't affect the values and only (R)

Table 13: Docking results for (R)-96a-c, (R)-97a-c, (R)-98a-c and (R)-99a-c

Adding a phenyl ring to the structure, regardless to its position, increased the predicted binding affinities by approximatively 5 kcal/mol as compared to Rivastigmine, and decreased the predicted distance between the MTDL's carbamate and AChE catalytic serine (Figure 105). Moreover, compared to the results obtained for the first series of carbamate-based derivatives, even though the carbamateserine distance remained unchanged, the predicted binding affinities with mAChE for this second series of MTDLs were also improved by approximatively 5 kcal/mol. This observation was in accordance with the idea that adding a phenyl ring to the structure could lead to an increased stabilisation of the ligand inside the enzymatic pocket through new interactions with the main AChE residues.

Figure 105: Visualisation of representative docking results for (A) (R)-96a and (B) (R)-97a (C) (R)-98a (D) (R)-99a inside AChE catalytic site with PyMol

purple). The phenyl ring directly linked to the triazole moiety is predicted to undergo π-π stacking interactions with Trp286 and Tyr124, Tyr337 whereas Tyr341 is predicted to interact with the phenyl ring bearing the carbamate (entry 1). Moreover, Trp86 is predicted to interact with the two methyl of the carbamate in a π-σ-type interaction (entry 2).

Concerning (R)-97a (and its analogues (R)-97b to (R)-97c), displaying the second phenyl ring at the meta position towards the triazole moiety, and the carbamate at the meta position of this second phenyl ring, we also observed two types of interactions (Figure 107). First, there is a H-bond formation (in green) between the carbamate's oxygen and the Tyr337 (entry 3). Then, we observed various π-π stacking (in pink) between the AChE residues and the triazole moiety and the two phenyl rings. Indeed, the triazole ring interacts with Trp286 and Tyr341. The phenyl directly linked to the triazole, for its part, is predicted to undergo π-π stacking interactions with Trp286 and Tyr124. Finally, the second phenyl ring bearing the carbamate interacts in the same way with Tyr341 and Tyr 337 (entry 1).

Figure 107: Representation of predicted interactions between mAChE and (R)-97a

Regarding compound (R)-98a (and its analogues (R)-98b and (R)-98c), displaying the second phenyl ring at the para position towards the triazole moiety, and the carbamate at the meta position of this second phenyl ring, the main predicted interactions are π-π type interactions (in pink) (Figure 108).

Indeed, the triazole moiety interacts with Trp286, the phenyl ring directly linked to the triazole is predicted to interact with Tyr341 and Tyr124 while the second phenyl ring bearing the carbamate is predicted to interact with Trp86 and Tyr337 (entry 1). Moreover, Tyr337 is also predicted to interact with the carbamate's oxygen via a H-bond (entry 3) (in green).

Figure 108: Representation of predicted interactions between between mAChE and (R)-98a

Eventually, for compound (R)-99a (and its analogues (R)-99b and (R)-99c), displaying the second phenyl ring at the para position towards the triazole moiety, and the carbamate at the para position of this second phenyl ring, besides a predicted π-σ interaction (in purple) between one of the carbamate's methyl and Trp86 (entry 2), all the other predicted interactions include the phenyl rings and π-π stacking type interactions (Figure 109). Indeed, the phenyl ring directly linked to the triazole moiety is predicted to interact with Trp286 and Tyr124, while the second phenyl ring bearing the carbamate is predicted to interact with Tyr341 and Tyr337 (entry 1).

Table 15: Physicochemical properties and CNS MPO scores computed for (R)-96a-c, (R)-97a-c, (R)-98a-c and (R)-99a-c

Percepta software CNS activity prediction

As it was done for the first series of MTDLs we developed, the predicted properties of the MTDLs designed in this second part were also calculated and are reported in Table 16. It is important to notice that adding a phenyl ring to the structures increased the values of LogP, as it was previously demonstrated for the CNS MPO scores calculations, inducing a better value of LogPS. The remaining physicochemical properties do not seem to differ from the results obtained for the first series. At this time of the project, we decided to study only the (R)-enantiomers. Indeed, it was already experimentally showed that these enantiomers had the best binding affinities toward α-7 nAChRs and the docking studies did not show any significant differences between the two series.

New series synthesis

The first step of our study was to synthesize the bromoalkynes 101a and 101b from the corresponding commercially available aldehydes 100a and 100b

Our first approach to obtain 101a-b was to perform a Seyfert-Gilbert homologation 344 using the Bestmann-Ohira reagent synthesized in the laboratory (Scheme 28). Unfortunately, we were not able to isolate the expected product and only obtained complex NMR signals, we were not able to confirm if we formed any expected alkyne.

Scheme 28: First attempt for the synthesis of alkynes 101a and 101b

We changed our approach and found another pathway described in the literature by Liu et al. 346 The strategy was to synthesize the 1,1-dibromoalkene from the aldehyde, followed by a Corey-Fuchs elimination to yield the targeted alkyne. We performed a Ramirez' olefination of commercially available bromoaldehydes 100a and 100b with tetrabromomethane and triphenylphosphine and obtained the corresponding 1,1-dibromo alkenes 102a and 102b with global yields of ca. 50% (Scheme 29). These modest yields could be explained by the difficulties we had to purify the product. Indeed, after completion of the reaction, it is challenging to be able to separate the desired products from the remaining tetrabromomethane, moreover, the quantity of triphenylphosphine oxide formed during the reaction proved to be an issue when up-scaling the reactions. Once we had the desired alkenes, we performed a Corey-Fuchs reaction following the strategy proposed by Liu et al. who developed Corey-Fuchs reaction conditions avoiding the use of a strong base and low or high temperatures, therefore, amenable for a wide range of substrates. Their method was appropriate in our case, since the use of a strong lithium based base such as n-BuLi could have led to a halogen-metal exchange and thus the loss of the bromine atom, essential for the rest of the synthesis. Their method using tetrabutylammonium fluoride in presence of water, and triphenyl phosphine allowed us to obtain the desired alkynes 101a and 101b with good yields without observing the bromoalkyne formation. of 6 carbamates, the lack of time did not allow us to synthesize the diethyl carbamate based compounds.

Scheme 35: Last step of the synthesis to (R)-98a/c and (R)-99a/c

We thus were able to synthesize 10 new combined MTDLs based on the diphenyl linker, these compounds were obtained in 6 steps with the corresponding total yields from (R)-81 displayed on Table 17.

Entry Compound Number of steps

Global yield (%) One step remained to be undertaken to obtain the two remaining compounds to achieve this second combined MTDL series. The in vitro biological evaluation for the 10 obtained compounds is in progress both for cholinesterase inhibition and α7 nAChR binding affinity.

1 (R)-96a 4
2 (R)-97a 6
3 (R)-96b 6

Partial conclusion and perspective on the project.

As a conclusion, after in silico evaluations, we managed to develop two short synthetic pathways able to yield two new combined MTDLs series against AD based on stigmine based AChE inhibitors and quinuclidine based α7 nAChR agonists.

-For the first series of Stigmine derivatives, we managed to obtain 18 new compounds ( 6racemic and their enantiopure analogues). We obtained the 6 racemic compounds in 6 steps with global yields ranging from 9 to 32% and the enantiopure ligands were synthesized in 5

steps with global yields ranging from 13 to 36%, in both pathways, CuAAC was used as a key synthetic step. Preliminary in vitro biological evaluation were performed on these new MTDLs (cholinesterase inhibition profile and α7 nAChR affinity), and we obtained promising results

with two lead compounds (R)-76a and (R)-75b showing interesting in vitro results (Figure 111), with improvement of the activity on the two targets as compared to the parent compounds they are derived from. Further experiments are currently in progress in order to evaluated in more details their biological activities, such as their ability to cross the BBB or their precise activity on α7 nAChRs (agonist, antagonist or allosteric modulation). -For the second series, 10 out of 12 targeted compounds were obtained, through a more complex synthetic access, but we developed a 6 steps synthesis with yields ranging from 0.8 to 10%. We have been able to show that the Suzuki-Myaura reaction can be used on different boronic acids in order to add a phenyl ring to the desired MTDLs. For the last two compounds, only one step remained to be achieved. For this second series of combined MTDL, biological assays need to be performed and activities will be compared to these obtained for the first series in order to evaluate the added value of the additional phenyl ring for the design of new MTDLs targeting α7 nAChRs and AChE.

However, even though the two synthesis we developed are short and relatively effective, one bottleneck has been identified: the formation of the mandatory azide 77 and its enantiomer analogues (R)-77 and (S)-77 required to link the two pharmacophores with the triazole moiety needs to be improved. According to the literature, it could be possible to form the unstable azide in situ to circumvent the volatility and instability issues. Different groups have studied the in situ formation of a triazole starting from an amine and an alkyne 347,348,349,350 . Beckmann et al. were the first to develop this kind of one step one-pot procedure with a variety of commercially available amines (Scheme 36).

Based on the simple reaction between benzylamine and phenylacetylene, they managed to optimize the reaction conditions and obtained good yields under microwave irradiations 347 .

Entry Amine We evaluated such a one-pot two steps strategy in order to increase the reaction yields and have a repeatable process. microwave irradiations. Using this process, we were able to obtain the MTDL 75a in only one step and with a good 78% yield (Scheme 37), to be compared to the 22% yield obtained in the two steps process.

Scheme 37: One-pot synthesis of 75a

Unfortunately, when we tried the same experiment with the commercially available enantiopure 3aminoquinuclidines, we could not obtain the desired MTDLs. When each enantiopure amine was put under these reaction conditions, they did not react at all. We did not have time to look for a rational explanation for this unexpected negative result (a difference in the protonation state of the amine could be suspected). Yet, based on our successful assay on the racemic mixture, and on the one-pot two steps procedures described in the literature, we should be able to find appropriate reaction conditions to perform the CuAAC. Once this step optimized, we will have a robust, rapid and convergent synthetic pathway for the two series of a new MTDLs against AD.

Another approach to circumvent the instability and volatility of the azido intermediate would be to base our synthesis on Maisonneuve and Xie work 349 . They managed to synthesize disubstituted 1,2,3triazoles directly from aldehyde and amines. They managed to develop an innovative one-pot, threestep synthesis from various aldehydes and amines. Their method has a large range of applications and can be used on aryl aldehydes but also on glycosyl aldehydes. For the azide precursors, aminoesters, dipeptides or C-glycosyl amines have been used. Starting from an aldehyde, they performed a Seyfert-Gilbert homologation with the Bestmann-Ohira reagent, followed by the diazotransfer with the imidazole-1-sulfonyl azide as a transfer agent. The last step was the copper catalysed cycloaddition with CuSO4 as a source of copper (Scheme 38).

Scheme 38: three-step one-pot synthesis of 115 by Maisonneuve et al. 349 We could imagine this procedure for our compounds, in order to obtain (R)-75a-c and (R)-76a-c from 3-aminoquinuclidine (R)-81 and the commercially available hydroxybenzaldehydes and then perform the carbamate addition to exemplify and obtain our first MTDLs series with better yields and a shorter synthetic pathway (Scheme 39).

Scheme 39: Proposed two steps synthetic access to (R)-75a-c and (R)-76a-c

Chapter III: Conception of combined MTDLs based on the Donepezil scaffold

Aim and context of the project

In this third project, we tackled a new challenge of designing and synthesising novel combined MTDLs based on another well-known AChEI, Donepezil. Given the encouraging results obtained in the previous chapter, it was decided to keep the quinuclidine moiety developed by our collaborator, Pr.

Routier, as an α7 nAChRs agonist and to combine it to the Donepezil scaffold by the same common moiety used in the previous series of combined MTDLs (Figure 112). As a conclusion, Donepezil moiety has been widely used in the past decade as a potent AChE inhibitor in the design of innovative MTDLs because of its potency and ability to bind with AChE both at the catalytic and peripherical sites, giving the molecule an additional protective activity against Aβ aggregation. In this context, using the active Donepezil fragment combined to a quinuclidine moiety able to interact with α-7 nAChRs to build a MTDLs appears to be a judicious choice.

Two different MTDL series have thus been designed (Figure 118), with the quinuclidine attached in place of one of the two dimethoxy substituents of Donepezil, since this moiety points towards AChE PAS. The first series has been designed with a flexible linker between the ketone (which key role in binding to AChE gorge residues has been proven in the various X-ray crystallographic data obtained with Donepezil bound to AChE), and the second one with the complete indanone moiety. For this second series, the synthesis will be led on the diastereomeric mixture with the mixture of the two absolute configurations at the asymmetric centre on the indanone moiety of Donepezil. Since it has been shown that at physiological pH, this indanone could easily undergo racemization, and although only one of the two enantiomers of Donepezil tightly bind to AChE binding site, this compound is administrated as a racemic mixture 365 .

Docking studies

The procedure explained in chapter II was used to evaluate the binding affinities and the position of the combined MTDLs inside the chosen enzyme, rhAChE. The starting point for the docking experiment was this time a crystal structure of this enzyme co-crystalized with Donepezil (PDB 4EY7) 354 . The predicted binding energies are reported in Table 18. Noteworthy, the binding mode and positioning predicted for Donepezil corresponds to the one observed in the crystal structure we started from. 19.

Entry

Entry

Table 19: Interactions predicted between rhAChE and (R)-128, (R)-129, (R)-130 and (R)-131

Concerning compound (R)-128 (Figure 121), the predominant interactions predicted with the enzyme were π-stacking interactions (entry 1). Indeed, it can be observed that the phenyl attached to the triazole was predicted to interact with Trp286 and Tyr341, and the second phenyl carried by the piperidine bounded with Trp86 which also interacts with the protonated hydrogen of the piperidine through π-cation interaction (entry 4) . Moreover, H-bonds interactions (entry 2) were predicted between Tyr124 and the ketone of (R)-128 and between one of the triazole ring's nitrogen and Arg296.

Figure 121: Representation of interactions between rhAChE and (R)-128

Compound (R)-129's triazole has been predicted to interact with Tyr72 through π-stacking interactions while the phenyl it carries has been predicted to π-stack with Trp286, moreover, the second phenyl carried by the piperazine appeared to π-stack with two residues: Trp86 and His447 (entry 1). Moreover, (R)-129's ketone was predicted to interact with Phe295 via H-bond interactions (entry 2) while the protonated nitrogen of the piperazine appeared to interact with Asp74 and Tyr337 through π-cation interactions (entry 4), finally, Tyr337 has been predicted to interact with the hydrogen of this protonated nitrogen through a carbon-hydrogen bond (entry 3) (Figure 122).

Figure 122: Representation of interactions between rhAChE and (R)-129

Compound (R)-130's phenyl carried by the triazole moiety has been predicted to π-stack with Trp286

while the second phenyl carried by the piperidine was predicted to interact with Trp86 and His447 in the same way (entry 1). Moreover, Trp337 and Trp86 have been predicted to interact with the protonated piperidine's nitrogen through π-cation interactions (entry 4). It must be noted that the quinuclidine appeared to do carbon-hydrogen bonds with Ser293 (entry 3). finally, a H-bond could be observed between the ketone of compound (R)-130 and the amide of Phe295 (entry 2) (Figure 123).

Figure 123: Representation of interactions between rhAChE and (R)-130

Regarding the last tested compound of this study, compound (R)-131, three main interactions have been predicted: π-stacking (entry 1)and H-bond formation (entry 2). Firstly, the phenyl carried by the triazole moiety π-stacked with Trp286 which also π-stacked with the triazole moiety, while the second phenyl of the molecule, carried by the piperazine, π-stacked with Trp86. Two H-bonds were predicted to be formed between Tyr124 and simultaneously the ketone and the non-protonated piperazine nitrogen. Finally, two other type of interactions have been predicted: a π-cation interaction between Asp74 (entry 4) and the piperazine's protonated nitrogen and a carbon-hydrogen bond between Ser293 and the quinuclidine moiety (entry 3) (Figure 124).

CNS MPO score computation

It has been verified that all the studied compounds respected the Lipinski's rules and their CNS MPO scores have been calculated from 6 of their physicochemical properties 366,367 (Table 20). Because of the high molecular weight and strong pKa values of the new series of combined MTDLs, the obtained results were lower than what was calculated in the previous project, nevertheless, the calculations displayed good CNS MPO scores for compound (R)-129 (entry 3) and compound (R)-131 (entry 5).

Concerning compound (R)-128 (entry 2) and compound (R)-130 (entry 4), the scores were shown to be slightly lower because of a high logP value added to the size of the molecule and the high pKa, yet still within an acceptable score. Moreover, Donepezil, the parent monomer (entry 1) displayed a score of 4.4, a value in accordance with the scores obtained for compounds (R)-129 and (R)-131, with also physico-chemical properties close to the one obtained for the all series.

Entry

Percepta software analysis of the compounds 368

To complete the in silico studies on compounds (R)-128, (R)-129, (R)-130 and (R)-131, calculations via the percepta software were performed and the results are reported in Table 21. The LogP and most basic pKa values obtained were closed to the one obtained for the calculation of CNS MPO scores, and the evaluation show that even if compound (R)-128 (entry 1) and (R)-130 (entry 3) displayed low CNS MPO scores, the calculated probability for them to cross the BBB remained sufficient. Moreover, the representations given in Figure 126 have shown that the presence of a Nbenzylpiperazine or a N-benzylpiperidine and their relative positions did not affect the chances of the four compounds to cross the BBB. Indeed, every compound was represented with the CNS active group of compounds (represented with blue dots).

Entry

Figure 126: Results of Brain penetration computed ability for CNS activity for (A) (R)-128 (B) (R)-129 (C) (R)-130 (D) (R)-131 (E) Donepezil (red dots represent CNS inactive compounds, blue dots represent

CNS active compounds and the circled black dot represent the tested compound)

Series 2

As part of the project of designing innovative Donepezil-based combined MTDLs, a second series carrying the whole indanone moiety was also evaluated in silico to predict the profile of compounds (R)-132 to (R)-135 (Figure 127). Since Donepezil is a chiral compound, but which stereocenter is prompted to racemization at physiological pH, this series was designed as a diastereomeric mixture, but for the docking experiments, the most active compound bearing the (R) absolute configuration at the second stereocenter was used. As for the first Donepezil based MTDL, the differences are in the attachment point for the triazole moiety and the N-benzylpiperazine or N-benzylpiperidine moiety.

Docking studies

Using the procedure described in chapter II, compounds (R)-132 to (R)-135 were docked with a rigid model of rhAChE (PDB 4EY7) 354 to determine their binding affinities toward the enzyme, the results are reported in Table 22.

Entry

Table 22: Docking results for compounds (R)-132, (R)-133, (R)-134 and (R)-135

It can be noticed that the binding affinities toward AChE predicted for all the newly designed MTDLs (entries 2 to 5) were slightly better than the binding affinity displayed by Donepezil (entry 1).

Moreover, compounds (R)-132 (Figure 128 Interactions between the newly designed MTDLs and the main residues of rhAChE were visualised with Discovery software and are reported in Table 23.

interacting with the phenyl fragment of the indanone (entry 1) and Gln291 interacting with the quinuclidine moiety through a carbon-hydrogen bond (entry 4). In addition, the compound's ketone has been predicted to interact with Tyr124 via a H-bond formation (entry 3) (Figure 130). Regarding compound (R)-135 (Figure 132), the phenyl ring part of the indanone moiety appeared to be proned to π-stack with Trp286 while the benzyl of the molecule, the one carried by the piperazine, to π-stack with Trp86 and His447 (entry 1) and the protonated nitrogen of the piperazine moiety seemed to bind with Asp74 through a H-bond interaction while the second nitrogen of the piperazine appeared to bind with Tyr124 in the same way (entry 3). Finally, the carbon between the indanone and the piperazine moiety has been predicted to interact with Tyr341 through π-sigma interactions (Figure 132).

Figure 132: Representation of interactions predicted between rhAChE and (R)-135

With these various results in hands, we were able to reach the same conclusion as for the first series, indeed, the predicted interactions of compounds (R)-132 to (R)-135 were similar to the one displayed for Donepezil structure (Figure 125 -p.12), meaning that adding a new pharmacophore to the Donepezil fragment did not seem to disrupt the interactions of this AChEI with the enzyme.

CNS MPO score computation

Besides the fact that compounds (R)-132 to (R)-135 respected the Lipinski's rules, their CNS MPO scores were calculated and reported in All physico-chemical properties were obtained from chemicalize.com

Table 24 along with their main physicochemical properties. The four tested compounds displayed promising ability concerning their aptitude to cross the BBB, indeed, they all obtained scores close to -3 (Figure 133) and were all positioned in the CNS active the second series were of pivotal importance to induce an appropriate binding of the compounds inside AChE catalytic pocket due to the numerous interactions in which they were predicted to be involved with the main residues of the enzyme. Moreover, the chosen size of the compounds (and thus the length of the linker between the two pharmacophores) appeared to be the right choice, indeed, interactions between the quinuclidine moiety and AChE PAS could be considered with these types of combined MTDLs and could possibly offer the molecules a higher affinity for the enzyme, more potential binding interactions and additional biological properties. Moreover, the studies concerning their physicochemical properties, either using CNS MPO score calculation or Percepta software representation, were encouraging for all the eight compounds. Nevertheless, as it was described by Sugimoto et al. 351 , the most optimal structure of Donepezil as a AChEI is the one carrying a Nbenzylpiperidine, which is thus why we first focused our synthetic efforts on compounds (R)-128, (R)-130, (R)-132 and (R)-134.

Synthesis of the Donepezil based MTDLs

For the sake of clarity, the synthesis of the two series of combined Donepezil-based MTDLs will be presented separately but the investigations were performed in parallel. azidoquinuclidine (R)-77 using the protocol described in chapter II 369,370 and the required alkynes 136 and 137, the two partners could be easily conjugated in the last synthetic step through a CuAAC 371 yielding the desired 1,2,3-triazole moiety (Figure 134).

.2. Synthesis of 136

In a first intend, we decided to focus on the development of a robust synthetic pathway for one compound. Based on the docking experiment and starting materials available, we have chosen to work on compound 136 where the alkyne is in the para position, to simplify the synthetic process (Figure 135). The retrosynthetic strategy (Figure 135) could be summarized as follow: reduction and deprotection of the alkyne 138 should offer target product. Alkyne moiety could be obtained through Sonogashira cross coupling reaction, and the corresponding alcohol could be formed by an aldolization reaction between aldehyde 140 (readily obtained from commercially available alcohol 142), and commercially available 4-bromoacetophenone 141. The aldolization/dehydration pathway was chosen based on the work of Nagel et al. 372 , moreover, our bibliographic researches on recent literature 373,374,375 lead to the conclusion that it was the predominant and more effective pathway to synthesise this type of donepezil moiety. Firstly, Swern oxidation of commercially available (1-benzylpiperidin-4-yl)methanol 142 using oxalyl chloride 143 and DMSO in presence of triethylamine in dichloromethane, offered the corresponding aldehyde 140 376 in gram-scale with an excellent yield of 95%. The next step was an aldolization reaction between aldehyde 140 and the aromatic ketone 141 using basic conditions in THF 372 . LiHMDS was chosen as a base, and compound 139 was obtained in a low 16% yield which could be explained by the high dependence of the reaction to the quality of the base. Indeed, it was observed that, although such reaction has been described with similar compounds, we observed that with LiHMDS we had in stock (which was an already used bottle of LiHMDS), the reaction mixture was mainly composed of side products (or degradation products) giving complex NMR signals not allowing us to elucidate their structures. Finally, based on our expertise in the development of efficient Sonogashira coupling conditions, we tried to perform the coupling between compound 139 and ethynyltrimethylsilane in presence of copper(I) iodide with palladium tetrakis triphenylphosphine as the catalytic source of Pd(0), in a mixture composed of dichloromethane and triethylamine 377 . Yet, despite our efforts, only the starting materials and the ethynyltrimethylsilane homocoupling product were recovered.

Scheme 40: First synthetic pathway to 138 4.1.1.3

. Screening Sonogashira cross coupling reaction conditions

Despite a longer time of reaction (from 2h to 12h) to obtain compound 138, the conversion rate as estimated through 1 H NMR spectra of quenched samples of crude reaction mixture, could not be increased higher than 10% under these reactions conditions. At this point of the project, we decided to find out optimized reaction conditions for the Sonogashira coupling step. The 1-(3bromophenyl)ethan-1-one 144 was used as a test compound to evaluate the best conditions to perform the reaction, and obtained results are reported in Commun. Camb. Engl. 2011, 47 (18), 5295-5297.

Sonogashira coupling is a sensitive reaction (due to the unstable character of Pd(0) complexes), thus must usually be run in mild, air-free and moisture-free conditions in presence of a base, acting as the solvent, in order to neutralize the hydrogen halide produced during the coupling. Based on our expertise, we first decided to keep the triethylamine as a base and performed the reactions in THF to be able to heat the mixture, and we varied the palladium source by using instead of Pd(PPh3)4, a Pd(dppf)Cl2 complex with dichloromethane (entry 2) and commercially available Buchwald precatalyst, XPhos Pd G3, an air, moisture, and thermally stable complex of bulky phosphine and Pd(II) (entry 3) 378 .

In the literature 379,380 , Pd(dppf)Cl2 complex with dichloromethane was proven to be an efficient precatalyst for this type of coupling and, in our study, it seemed relevant to evaluate the efficiency of such a bidentate ligand. Moreover, XPhos Pd G3 was also investigated because of its low sensitivity toward oxygen that could be found in the reaction mixture, which allowed us to minimize degradation (through oxidation) or even complexation of the precatalyst. Yet, neither heating with Pd(PPh3)4 nor use of these two Pd(II) sources significantly improved the conversion rate of 144. The second strategy we used to optimise the procedure was to use separate palladium (II) source and phosphine in presence of triethylamine (entry 4), with PdCl2 as Pd source and Davephos as the phosphine, we managed to obtain a 38% conversion of 144, a promising result but which still requires improvement to be implemented at this stage of the synthesis. Building on this improvement, we decided to try other sources of palladium (either Pd(0) or Pd(II) species) available in the laboratory coupled with the most promising phosphine, DavePhos, and triethylamine, as shown on entry 5 and 6, the conversion was once again improved, and we obtained either using a Pd(II) source, namely Pd(OAc)2 or a Pd(0) source, namely Pd2(dba)3, in both cases conversions around 45%. The last parameter we assayed was the replacement of the base, changing the triethylamine by cesium carbonate (entry 7 and 8),

unfortunately, this base modification ended up in a drastic drop in conversion (down to 0%). As a conclusion of this short study, we managed to obtain a Sonogashira cross coupling reaction procedure allowing to reach a conversion of 144 of 46% NMR (entry 5) but we estimated that such a modest result was insufficient and decided to change the synthetic strategy.

.2. Alkyne 148 and 149 synthesis

As mentioned in the retrosynthetic analysis, we decided to perform the Sonogashira cross coupling reaction using a triflate instead of the bromine derivative, thus, commercially available 4hydroxyacetophenone 152 and 3-hydroxyacetophenone 153 were put in presence of trifluoromethanesulfonic anhydride and triethylamine (4 equiv. ) in dichloromethane 381 to give respectively compounds 150 and 151 bearing a triflate function in good 71% and 78% yields. Then, a Sonogashira coupling, using the non-optimized but more affordable procedure (in presence of PdCl2(PPh3)2 in refluxing DMF) was performed between ethynyltrimethylsilane and compounds 150 and 151, followed by a deprotection of the terminal alkyne using potassium carbonate to provide alkynes 148 and 149 in 3 steps with acceptable 25% and 26% overall yields over the three steps in a 400 mg scale (Scheme 42). We first assayed the aldolization reaction on the alkynylated indanones with the previously obtained aldehyde 140, in order to evaluate the influence of the free alkyne on this reaction. The attempt was first performed with only alkyne 166 but did not show any expected result. Indeed, the first assayed conditions using sodium methanolate384 in methanol and THF to achieve the aldolizationcrotonization reaction, despite an extended reaction time of 12h instead of 3h , allowed us only to recover the starting materials. The second attempt using a stronger base, sodium hydride in THF was also unsuccessful, and we were not able to obtain the desired compound 172 but only a highly complex mixture we were not able to identify.(Scheme 49).

Partial conclusion and perspectives

As a conclusion on this third project, even though we didn't manage to obtain the expected novel As noted in the introduction of this chapter, taking into account the low pKa of the indanone moiety of Donepezil, it has been shown that the asymmetric centre epimerizes at physiological pH, thus we can assume that MTDLs (R)-132 and (R)-134 could be obtained as 1/1 diastereosisomers mixtures, a diastereoselective aldolization reaction is thus not required (but could be undertaken if need be). The same experiments in the same conditions were performed in parallel between compounds 145, 150, 151 and 178 and 1-benzypiperazine 174, sadly, the obtained results were similar to what was obtained previously (Table 29). When 1-benzylpiperazine was set in reaction with compounds 150 and 151, NMR spectra showed complex signals assuming that only degradation products were recovered.

On the contrary, when the reaction was performed with alkynes 145 and 178, no degradation was witnessed but the starting materials were recovered without any trace of the formation of the targeted intermediates. These reactions thus still need to be implemented in order to complete MTDL series, and other aldolization akin reactions need to be assayed. 385

Background and PhD objectives

Alzheimer disease is a complex and multifactorial neurodegenerative disease discovered in 1906 by Doctor Alois Alzheimer characterised by a loss of memory and cognition. Worldwide, AD is the first cause of dementia for patients over 75 years old, moreover, the number of people with AD is dramatically increasing and could reach 135.5 million in 2050. Even though AD is a highly complex disease, scientists have been able to determine the two main histopathologies involved. The first described histopathology is the formation of β-amyloid plaques through the fibrillation of Aβ peptide, a peptide issued of the cleavage of APP by β-secretases and γ-secretases in an amyloidogenic pathway.

In addition, studies have shown that there is a strong affinity between the Aβ peptide and the homomeric α7-nAChR facilitating peptide's aggregation and the inhibition of neuroprotection signals. druglikeness gave clues that this compound might have great chances to cross the BBB (Table 30). and predicted interactions with each of the enzymes were observed (Figure 139). These structural determinations brought to light that the aniline moiety in position 12 of MTDL 34 was able to fit in BChE acyl pocket, fitting between Ser198 and His448 and to disrupt the catalytic triad, which could explain the potency of 34 as an inhibitor of both AChE and BChE.

Entry

Perspectives

Approximatively 150 mg of MTDL 34 were synthesised for in vivo assays. indeed, with the exciting in vitro results obtained for the conjugated MTDL 34 related to its potent AChE inhibition and its antioxidant character, our collaborators in Pr. In this second project, we designed in collaboration with Pr. Routier's team (UMR CNRS 7311 ICOA, Orléans), new combined MTDLs combining an AChE inhibitor, the Rivastigmine fragment, and an α7 nAChR agonist, the quinuclidine fragment (Figure 140). The first pharmacophore targeting AChE was chosen for its long lasting AChE inhibition due to a slowly reversible covalent inhibition mechanism through carbamoylation of the catalytic serine, and the nAChR agonist moiety made up of a quinuclidine and a triazole fragment was chosen as the second pharmacophore because of its proven efficiency to counteract CNS-related pathologies. The strategy was to design combined MTDLs sharing a common moiety of each parent pharmacophore in order to decrease the molecular weight of the drug candidate and to improve its CNS activity profile. 76a-c in 5 steps with overall yields ranging from 9% to 36% (Figure 140). The limiting step is the formation of quinuclidine azide, which proved highly unstable, and different strategies in which the azide was formed in situ were considered to have acceptable and reproducible yields. One attempt was made and gave an encouraging result with an improved yield compared to the two steps process in the synthesis of 75a from 81 and 78a (Scheme 54).

Scheme 54: One-pot synthesis of 75a

The molecules of this series were evaluated in vitro for their binding to α7 nAChR by Pr. Chalon (UMR INSERM 1253, iBrain, Tours), and it appeared, as expected and already brought to light by Pr. Routier, that the (R) enantiomers had a better affinity toward α7 nAChRs. From that point, the 6 (R) enantiomers were also evaluated for their potency to inhibit cholinesterases by Pr. Bartolini's team (University of Bologna, Italy)(Table 31). The first thing to notice was that the affinities toward α7 nAChRs were increased compared to the parent compound for all the MTDLs of the family and all the new molecules displayed values at a nanomolar level and even subnanomolar for (R)-75a (entry 3).

Moreover, we saw that two compounds appeared more promising than the others, given the IC50 results. Indeed compound (R)-76a (entry 4) and (R)-75b (entry 5) also displayed the best results

concerning their ability to inhibit AChE (and BChE for (R)-76a In the same project, building on this first success, we attempted to optimise the structure of the new MTDLs by modulating the common moiety. Based on the expertise of our collaborator Pr. Routier, it was decided to add a phenyl ring to the common moiety while the active fragments remained unchanged. After an evaluation of the structure in silico, we managed to synthesise 10 new MTDLs in 6 steps with overall yields from 0.8% to 10% (Figure 142). Once again, the limiting step was the azide formation. In vitro evaluations of the activity of these drug candidates towards AChE, BChE and α7 nAChRs are currently in progress. In prevision of this work, preliminary studies have been performed and every potential compound exerted good in silico results with binding energies around 9.5 kcal/mol, a sufficient proximity of the π interactions could be noted between the phenyl ring and Tyr337 also involved in a H-bond (entry 5)

with the double bounded oxygen of the carbamate.

Then, compound (R)-181b is foreseen to display various interactions such as π-π interactions (entry 1)

between Tyr341 and the phenyl ring and the thiophene, Tyr337 and the phenyl ring and Tyr337 also could be involved in a H-bond (entry 5) with the double bounded oxygen of the carbamate. A novel interaction was also noted, indeed, Tyr124 seemed to interact with the sulphur atom of the thiophene in a sulphur-X interaction type (entry 6).

Regarding compound (R)-182a, we could note that Trp286 was predicted to interact in a π-π type with the triazole moiety and the thiophene, Tyr341 was predicted to be involved in π-π interactions with the thiophene and the phenyl ring also interacting with Tyr337 (entry 1). Finally, Trp86 was predicted to interact with the methyl groups of the carbamate in a π -σ interaction (entry 3).

Compound (R)-182b displayed predicted π-π interactions (entry 1) involving 3 AChE residues: Tyr337

with the phenyl ring, Tyr341 with the phenyl ring and the thiophene, Trp86 with the thiophene. A πdonor interaction between Tyr124 and the phenyl ring was also shown (entry4).

Based on our expertise in the design of combined MTDLs and Professor Routier's expertise in the design and synthesis of α-7 nAChRs agonists carrying a quinuclidine fragment, as an example, compound (Figure 146). This last family will be synthesized as a diastereoisomers mixture, since it has been shown that the indanone moiety was prompt to epimerize in physiological conditions.

Global perspectives

Granted by the expertise we gathered in the field of combined and combined MTDLs synthesis in the treatment of AD, we imagined other combinations of pharmacophores. Firstly, it could be possible to increase the potency of carbamate-based MTDLs against AChE by varying the substituents of the carbamate's nitrogen while keeping the same α7 nAChR agonist moiety (Figure 148).Indeed, Sterling

Experimental part

Optical rotations were recorder on a Perkin Emler 341 polarimeter using the sodium D line (589 nm)

and the concentration given in g.100 mL -1 and the values in unit deg.mL.cm -1 . g -1 . Reactions under microwave conditions were carried out in an Anton Paar Monowave 300 apparatus equipped with an infrared external temperature sensor and a stirrer. Melting points were measured with a Stuart SMP30 melting-point apparatus.

Biochemistry

AChE inhibition Assay

The inhibitory activity on human cholinesterases was evaluated spectrophotometrically by the method inhibitor were preincubated at 37 ºC for 20 min followed by the addition of substrate. Blank solutions containing all components except AChE or BChE were prepared in parallel to account for the nonenzymatic hydrolysis of the substrate. Initial rate assays were performed at 37 °C with a Jasco V-530 double beam Spectrophotometer: the rate of increase in the absorbance at 412 nm was followed for 210 s. The velocity in the presence and absence of inhibitor were compared and % inhibition was calculated. The results were plotted by placing the percentage of inhibition in function of the decimal log of the final inhibitor concentration. Linear regression and IC50 values were calculated using Microcal Origin 3.5 software (Microcal Software, Inc).

Evaluation of the time-dependent carbamoylation of hAChE

The stopped time assay was performed, in which AChE and the tested compound at a concentration of 6.0 µM, a value close to its IC50 value, were mixed in the assay buffer at pH 8.0. After 5, 10, 20, 30 and 40 minutes incubation at 37 °C, the determination of residual activity of the AChE-catalyzed hydrolysis of ATCh was carried out by Ellman's method. [1] A parallel control (i.e., no inhibitors in the mixture) allowed to adjust activities measured at the same incubation times.

BBB permeation Assay

The parallel artificial membrane permeation assay for blood-brain barrier described by

General procedure D: Olefination

To a solution of bromobenzaldehyde (1 equiv) in dry DCM (0.5 M), CBr4 (2 equiv) was added, the mixture was cooled at 0°C. Then, a solution of PPh3 (4 equiv) in dry DCM (0.5 M) was added dropwise.

The reacting mixture was stirred for 30 min at room temperature. After completion determined by TLC analysis, the solution was concentrated under reduced pressure, the crude product was purified by column chromatography (Cyclohexane 100) to yield the product.

General procedure E: Corey-Fuchs

To a solution of olefin (1 equiv) in a mixture THF/ H2O (20/1 v/v 0.2 M), TBAF.3 H2O (6 equiv) and PPh3

(1.5 equiv) were added. Then, the reacting mixture was stirred at 80°C in a sealed tube for 2h30. After completion determined by TLC analysis, the solution was diluted with H2O, extracted with EtOAc (x3).

Organics layers were combined, dried over Na2SO4, filtered, and concentrated under reduced pressure.

The crude oil was purified by column chromatography (Cyclohexane 100) to yield the product.

General procedure F: Suzuki-Myaura cross coupling

To a mixture of derivative 105a-b (1 equiv) in toluene and EtOH (7/3 v/v 0.1 M) were successively added the desired boronic acid (1.2 eq.), K2CO3 (2.0 eq.) and Pd(PPh3)4 (10 mol %). The reaction mixture was degassed with Ar and irradiated under microwave for 20 minutes at 150°C. After cooling, the volatiles were removed under reduced pressure and the crude material purified by flash chromatography (DCM/MeOH 80/20, v/v).

General procedure G: Triflate addition

To a solution of 3'-hydroxyacetophenone (1 equiv) in dry DCM (0.9 M), was added Et3N (4 equiv). The solution was cooled to 0°C. Trifluoromethanesulfonic anhydride (1.5 equiv) was added slowly. After 2h at 0°C, the reacting mixture was stirred at rt for 12h. After completion determined by TLC analysis, the solution was diluted with water, extracted with DCM (x3). Organics layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column chromatography (Cyclohexane/Et2O 90/10, v/v).

General procedure H: Sonogashira cross coupling with PdCl2(PPh3)2

A solution of triflate derivative (1 equiv) in dry DMF and Et3N (7/3 v/v 0.3 M) was degazed with argon for 10 min. Ethynyltrimethylsilane (2 equiv) and PdCl2(PPh3)2 (0.05 equiv) were added to the mixture.

The reaction mixture was stirred at reflux for 4h. After completion determined by TLC analysis, the solution was cooled to rt, diluted with water and extracted with EtOAc. The organic layers were combined, washed with NaCl sat., dried over MgSO4, filtered and concentrated under reduced pressure. The crude oil was purified by column chromatography (Cyclohexane/Et2O 80/20, v/v).

General procedure I: TMS deprotection with K2CO3

To a solution of protected alkyne (1 equiv) in dry MeOH (0.3 M), was added K2CO3 (2.5 equiv). The solution was stirred at rt for 1h. After completion determined by TLC analysis, the solution was concentrated under reduced pressure, diluted with H2O, extracted with DCM (x3). Organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column chromatography (Cyclohexane/Et2O 80/20, v/v) Mass spectrometry analysis are missing for several compounds due to a technical issue with the spectrophotometer of the institute, HRMS analysis are scheduled to be performed in another institute soon.

4-hydroxy-3-methoxybenzonitrile (51)

To a solution of vanillin 50 (3 g, 19.7 mmol)

4-((tert-butyldimethylsilyl)oxy)-3-methoxybenzonitrile (52)

A solution of 51 (2.74 g, 18.3 mmol) the mixture was stirred at rt for 12h, then, the reaction mixture was extracted with Et2O (6x40 mL).

The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. The obtained pink solid was diluted in DCM and concentrated under reduced pressure ( 4times) to give 3-butynoic acid 55 as a white solid (2 g, 83%). Then, to a solution of amine 53 (1.92 g, 7.16 mmol) 2, 144.7, 131.1, 121.0, 120.2, 111.9, 77.5, 74.4, 55.6, 43.9, 27.5, 25.8, 18.6, -4.5 A mixture of 1,1,3,3-tetramethoxypropane 56 (60 g, 365 mmol, 1 equiv.) and aq. 2 M HCl (182 mL) was stirred at rt for 1.5h. To this mixture cooled at 0 °C was added successively and carefully an aq. 5 N NaOH within 30 min (pH = 7.5) and MeOH (183 mL). At 0 °C, dimethyl-3-oxoglutarate 57 (127.3 g, 731 mmol, 2 equiv.) was added slowly, followed by addition of MeOH (127 mL). The reaction mixture was allowed to warm to rt and stirred for 3 days. The reaction mixture was acidified to pH = 2.5 with HCl 37%. Filtrating fraction wise, washing with H2O and drying at desiccator in presence of P2O5 to afford the desired tetra ester 59, as white-grey solid (82 g, 58%). 0, 121.0, 118.7, 107.9, 64.9, 63.3, 41.6, 38.1, 33.4, 30.2, 29.3, 28.3 7, 137.1, 125.1, 109.0, 79.8, 64.6, 62.9, 42.0, 39.1, 36.8, 35.1, 34.5, 31.3, 29.3, 28.2, 27.7, 23.1; MS (CI) (m/z) 322 (100) [M] + .

4-((1R,5R)-spiro[bicyclo[3.3.1]nonane-3,2'-[1,3]dioxolan]-6-en-7-yl)butan-1-ol (64)

To a cooled suspension of LiAlH4 (1.1 g, 28.9 mmol, 3 equiv) in dry Et2O (60 mL), was added dropwise a solution of 63 (2.8 g, 8.68 mmol, 1 equiv) in dry Et2O (10 mL). The reaction mixture was stirred at 0°C for 2h then quenched with H2O (3.6 mL), then an aqueous solution of NaOH (5 M, 3.6 mL) and H2O (3.6 mL). The mixture was dried with MgSO4. The filtrate was concentrated under reduced pressure to afford 64 as a yellow oil (2.18 g, 100%). 7, 124.2, 109.0, 64.5, 62.9, 62.7, 41.89, 39.0, 37.0, 34.6, 31.3, 29.2, 27.7, 23.6; HRMS (ESI + ) (m/z) calcd. for C15H25O3 + [M+H] + 253.1798 found 253.1794.

7-(4-Hydroxybutyl)bicyclo[3.3.1]non-6-en-3-one (65)

A solution of 64 (2.18 g, 8.6 mmol, 1 equiv.) in acetone (25 mL) was prepared to which paratoluenosulfonic acid (0.164 g, 0.86 mmol, 0.1 equiv.) was added. This solution was stirred at rt for 4 h. Then, the reaction mixture was extracted with EtOAc (3 × 30 mL) and the combined organic layer was washed with H2O and then dried with MgSO4. The solvent was removed under reduced pressure. 136.4, 124.9, 62.7, 49.2, 46.8, 36.7, 35.5, 32.04, 31.39, 30.5, 30.4, 23.6; MS (ESI -) (m/z) 207 ( 100

1-ol (67)

In the two-necked dry round-bottomed flask, a suspension of 2-amino-4-chlorobenzonitrile 66 (1.709 g, 11.23 mmol, 1.6 equiv.) in dry 1,2-DCE (70 mL) was prepared and AlCl3 (1.6 g, 11.94 mmol, 1.7 equiv.) was added in one portion. This mixture was stirred for 10 min then a solution of 65 (1.46 g, 7 mmol, 1 equiv.) in 1,2-DCE (14 mL) was added dropwise within 5 min, then the mixture was refluxed at 95 °C for 16 h. After cooling down to rt, THF was added, followed by H2O and aq. 5 M NaOH, this mixture was stirred for another 30 min and organic solvents were evaporated under reduced pressure.

Residues were taken up in EtOAc and extracted with EtOAc, then organic phases were washed with Hz, 2H), 6.90 (d, J = 7.9 Hz, 1H), 6.74 (d, J = 1.1 Hz, 2H), 5.58 (d, br.), 4.36 (d, J = 11.5 Hz, 2H), 4.21 (t, J = 6.9 Hz, 2H), 3.76 (s, 3H), 3.30-3.02 (m, 4H), 2.90-2.82 (m, 2H), 2.82 (m, 1H), 2.41-2.37 (m, 1H), 1.97-1.92 (m, 6H), 1.72-1.51 (m, 2H), 0.97 (s, 9H), 0.10 (s, 6H); 13 C NMR (CD3OD, 75 MHz) δ 171. 8, 155.4, 154.2, 152.3, 145.4, 141.1, 139.5, 137.8, 133.5, 127.2, 126.0, 125.9, 124.6, 124.4, 121.7, 121.0, 120.6, 115.4, 112.9, 99.4, 55.9, 53.2, 50.9, 44.2, 37.1, 36.8, 33.7, 30.0, 29.5, 28.4, 27.6, 26.2, 24.9, 19.3, -4.5; 24.8, 25.4, 27.5, 28.1, 29.3, 30.0, 33.6, 36.0, 37.0, 50.8, 56.4, 112.4, 115.4, 116.1 , 119.3, 121.3, 125.7, 126.3, 127.7, 131.1, 135.4, 137.9, 139.5 , 140.5, 140.6, 149.0, 153.0, 156.7, 171.4 ; HRMS (ESI + ) calcd for C32H35 [START_REF] Sadigh-Eteghad | Beta-Amyloid Exhibits Antagonistic Effects on Alpha 7 Nicotinic Acetylcholine Receptors in Orchestrated Manner[END_REF] ClN6O3 + [M] + 587.2532 found 587.2534.

MS (ESI+

Ethyl(methyl)carbamic chloride (91c)

To a solution of triphosgene 90 (500 mg, 8.4 mmol, 1 equiv) in DCM (8 mL) was added sodium bicarbonate (1.4g, 16.8 mmol, 2 equiv). The mixture was cooled at -10°C and a solution of ethylmethylamine 89 (1.6 g, 5.6 mmol, 0.6 equiv) in DCM (1.5 mL) was added dropwise. The resulting mixture was stirred at room temperature for 12h. After completion determined by TLC analysis, the solution was filtered. The filtrate was concentrated under reduced pressure to give 91c as a yellow oil (1.02 g, 100%). 2, 149.0, 47.9, 46.3, 37.8, 36.0, 12.9, 12.2 (each carbon gives 2 signals because of the 2 rotamers of the amide).

Spectral data are in accordance with those reported previously: Fuchs, M.; Koszelewski, D.; Tauber, K.; Kroutil, W.; Faber, K. Chemoenzymatic Asymmetric Total Synthesis of (S)-Rivastigmine Using ω-Transaminases. Chem. Commun. 2010, 46 (30), 5500-5502.

4-ethynylphenol (92a)

92a was obtained from 4-iodophenol 93a (200 mg, 1.04 mmol) following the general procedure A and isolated as a purple oil (111.7 mg, 91%). 4, 131.5, 115.3, 114.1, 83.8, 75.9.

Spectral data are in accordance with those reported previously:

3-ethynylphenyl dimethylcarbamate (78a)

78a was obtained from 92b (300 mg, 2.55 mmol) and 91a following the general procedure B and isolated as a yellow oil (385.9 mg, 80%). 6, 151.3, 129.1, 125.5, 123.3, 122.7, 82.9, 77.8, 36.8, 36.5; HRMS (ESI + ) (m/z) calcd. for C11H12NO2 + [M+H + ] 190.0790 found 190.0792.

3-ethynylphenyl diethylcarbamate (78b)

78b was obtained from 92b (300 mg, 2.55 mmol) and 91b following the general procedure B and isolated as a yellow oil (354 mg, 64%).

1

H NMR (300 MHz, CDCl3) δ 7. 26 -7.22 (m, 2H), 7.20 (t, J = 1.7 Hz, 1H), 7.12 -7.03 (m, 1H), 3.48 -3.24 (m, 4H), 3.00 (s, 1H), 1.28 -1.06 (m, 6H); 13 C NMR (75 MHz, CDCl3) δ 153.9, 151. 4, 129.2, 128.9, 125.5, 123.2, 122.8, 82.9, 77.7, 42.2, 12.9; HRMS (ESI + ) (m/z) calcd. for C13H16NO2 + [M+H + ] 218.1103 found 218.1174.

3-ethynylphenyl ethyl(methyl)carbamate (78c)

78c was obtained from 92b (200 mg, 1.76 mmol) and 91c following the general procedure B and isolated as a yellow oil (251 mg, 70%). 5, 138.2, 124.1, 88.9, 44.2, 34.3, 33.9, 13.3, 12.5 (three

4-ethynylphenyl dimethylcarbamate (79a)

79a was obtained from 92a (300 mg, 2.55 mmol) and 91a following the general procedure B and isolated as a white solid (381 mg, 79%). 5, 151.9, 133.3, 121.9, 119.1, 83.21, 77.1, 36.7; HRMS (ESI + ) (m/z) calcd. for C11H12NO2 + [M+H + ] 190.0790 found 190,0866.

4-ethynylphenyl diethylcarbamate (79b)

79b was obtained from 92a (300 mg, 2.55 mmol) and 91b following the general procedure B and isolated as a white solid (443 mg, 80%). 8, 152, 133.3, 121.9, 118.9, 83.3, 77.0, 42.4, 13.5; HRMS (ESI + ) (m/z) calcd. for C13H16NO2 + [M+H + ] 218.1103 found 218.1180.

4-ethynylphenyl ethyl(methyl)carbamate (79c)

79c was obtained from 92a (200 mg, 1.76 mmol) and 91c following the general procedure B and isolated as a yellow oil (251 mg, 70%). 3, 151.4, 129.1, 125.5, 123.3, 122.8, 82.92, 77.8, 44.2, 34.4, 33.9, 29.8 9, 151.6, 147.2, 127.8, 126.7, 122.3, 119.1, 58.2, 52.4, 46.9, 36.7, 28.2, 19.9 1, 151.5, 147.1, 127.7, 126.6, 122.2, 119.1, 58.2, 52.4, 47.1, 42.2, 28.2, 20.0, 13.9 (two 6, 147.3, 126.7, 122.3, 119.3, 57.7, 53.6, 52.2, 47.2, 46.2, 44.2, 38.3, 28.1, 25.4, 23.6, 19.7, 18.9 (two carbons are missing despite an extended acquisition time); HRMS (ESI + ) (m/z) calcd. for

1-bromo-4-ethynylbenzene (101b)

101b was obtained from 102b (1g, 2.9 mmol, 1 equiv) following the general procedure E and isolated as a colorless oil (308 mg, 59%). 148.9, 143.3, 132.1, 130.9, 130.5, 130.0, 127.9, 125.6, 125.2, 122.0, 119.3, 115.6, 114.9, 58.5, 52.0, 47.8, 47.5, 29.0, 25.1, 20. 6.91 -6.82 (m, 1H), 5.22 (s, 1H), 4.32 -4.11 (m, 1H), 3.88 (d, J = 18.8 Hz, 1H), 3.55 (dd, J = 13.1, 6.6 Hz, 1H), 3.43 -3.33 (m, 2H), 3.08 (dd, J = 5.2, 3.6 Hz, 1H), 2.63 -2.47 (m, 1H), 2.36 -2.23 (m, 1H) 5, 149.0, 142.4, 133.9, 133.15, 132.9, 130.1, 130.0, 129.0, 127.9, 123.8, 122.5, 116.7, 51.2, 47.5, 47.8, 33.0, 32.9, 30.7, 28.5, 18.9 9, 133.7, 133.2, 133.0, 130.1, 130.0, 129.9, 129.4, 128.4, 127.0, 126.8, 126.7, 58.5, 51.9, 47.8, 47.5, 29.0, 25.1, 24.9, 19. (0.13 mmol, 1.5 equiv) following general procedure B and isolated as a pale yellow oil (13.34 mg, 40%). following general procedure B and isolated as a yellow oil (9.18 mg, 71%). 4, 143.7, 138.5, 137.2, 127.4, 125.9, 125.7, 123.0, 121.0, 120.30, 120.1, 117.5, 117.0, 54.1, 47.4, 42.8, 42.5, 40.2, 25.7, 24.2, 20.9, 15.4 H NMR (300 MHz, MeOD) δ 8.55 (s, 1H), 8.12 (t, J = 1. 6 Hz, 1H), 7.87 -7.80 (m, 5H),7.25 -7.18 (m, 1H), 4.99 -4.90 (m, 1H), 3.86 (dd, J = 14.4, 4.6 Hz, 1H), 3.67 -3.51 (m, 1H), 3.23 (d, J = 12.5 Hz, 2H) 8, 142.4, 139.3, 133.9, 132.3, 130.6, 130.3, 129.0, 127.9, 126.7, 125.6, 123.3, 122.5, 58.9, 52.2, 47.8, 47.5, 36.8, 29.1, 25.6, 20.6, 14.5 51%).

1

H NMR (300 MHz, MeOD) δ 8.65 (s, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 8.6 Hz, 2H), 7.67 -7.61 (m, 1H), 7.51 (ddd, J = 21.7, 12.3, 6.6 Hz, 2H), 7.22 (d, J = 8.7 Hz, 1H), 5.33 (s, 1H), 4.31 (dd, J = 14.1, 4.3 Hz, 1H) following general procedure B and isolated as a colourless oil (6.2 mg, 48%). 6, 148.7, 142.4,138.7, 132.3, 130.6, 130.0, 129.0, 127.9, 126.7, 125.4, 123.3, 122.5, 58.9, 52.4, 47.8, 47.5, 45.2, 34.5, 30.7, 29.2, 25.9, 20.5 (two for another 30 min at -78°C. The mixture was allowed to cool down to rt and water was added, the mixture was extracted with EtOAc, the combined organic layers were washed with water and brine and dried over Na2SO4. The solvent was removed under reduce pressure to give compound 140 as a yellow oil used without further purification (831.7 mg, 95%).

3-(1-benzylpiperidin-4-yl)-1-(4-bromophenyl)-3-hydroxypropan-1-one (139)

To a solution of LiHMDS 1M in THF (2 mL, 2 mmol, 1 equiv) was added a solution of 4'bromoacetophenone 141 (398 mg, 2 mmol, 1 equiv) in dry THF (5 mL) at -78°C. The mixture was stirred at that temperature for 15 min followed by the addition of a solution of compound 140 (415.7 mg, 2 mmol, 1 equiv) in dry THF (5 mL). The resulting mixture was stirred at -78°C for another 45 min. After completion determined by TLC, water (20 mL) was added to the mixture which was, then, allowed to warm up at rt. The pH was adjusted at pH = 4 with an aq. solution of HCl and the reaction mixture was extracted with EtOAc. The combined organic layer's pH was adjusted at pH = 9.5 with an aq. solution of NaOH, dried over Na2SO4 and the solvent was evaporated under reduce pressure to give compound 139 as a colourless oil without further purification (128 mg, 16%).

4-acetylphenyl trifluoromethanesulfonate (150)

150 was obtained from 4-hydroxyaetophenone 152 (2 g, 14.7 mmol, 1 equiv) following the general procedure G and isolated as an orange solid (3.05 g, 78%). 1, 152.5, 136.0, 130.7, 121.7, 116.6, 26.6.

The NMR spectra matched those reported in the literature for 4-acethylphenyl trifluoromethanesulfonate [CAS: 109613-00-5].

3-acetylphenyl trifluoromethanesulfonate (151)

151 was obtained from 3-hydroxyacetophenone 153 (2 g, 14.7 mmol, 1 equiv) following the general procedure G and isolated as a brown oil (2.7 g, 71%). 9, 149.9, 139.4, 130.8, 128.3, 125.9, 121.1, 116.7, 26.8.

The NMR spectra matched those reported in the literature for 3-acethylphenyl trifluoromethanesulfonate [CAS: 138313-22-1].

1-(4-((trimethylsilyl)ethynyl)phenyl)ethenone (178)

178 was obtained from 150 (2 g, 7.46 mmol, 1 equiv) following the general procedure H and isolated as an orange solid (1.07 g, 73%). To a solution of 159 (490 mg, 2.3 mmol, 1 equiv) in dry DCM were successively added 5bromoindanone 160 (388.3 mg, 1.84 mmol, 0.8 equiv), tetra-n-butylammonium bromide (7.4 mg, 0.023 mmol, 0.01 equiv) and NaOH aq. 10% (3.2 mL). The resulting mixture was stirred at reflux for 5h

and allowed to cool down to rt before being evaporated under reduced pressure. The obtained crude oil was recrystallized in MeOH to give compound 161 as a brown solid (410.9 mg, 44%).

3-oxo-2,3-dihydro-1H-inden-5-yl trifluoromethanesulfonate (171)

171 was obtained from 6-hydroxyindanone 169 (500 mg, 3.37 mmol, 1 equiv) following the general procedure G and isolated as a brown oil (678.4 mg, 72%). 9, 154.7, 149.2, 139.1, 128.7, 127.6, 121.0, 116.6, 36.9, 25.7.

Résumé

La maladie d'Alzheimer (MA) est une maladie neurodégénérative complexe caractérisée par une perte progressive de la mémoire et de la cognition. C'est la première cause de démence chez le sujet âgé et affecte environs 4.6 millions de personnes par an, selon un rapport de l'association « Alzheimer's disease International », le nombre de patients pourrait s'élever à 135.5 millions en 2050. Du fait de sa complexité, la MA reste incurable et seuls 4 médicaments aux vertus palliatives dont 3 visant à inhiber l'acétylcholinestérase (AChE) ont reçu une autorisation de mise sur le marché à ce jour. L'approche multi-cible parait particulièrement adaptée du fait du grand nombre de cibles potentielles de la pathologie, et du caractère multifactoriel de la maladie. Cette approche consiste à associer sur une seule molécule, plusieurs pharmacophores afin qu'ils puissent agir simultanément sur différentes cibles impliquées dans le processus neurodégénératif. Dans ce contexte, en parallèle de la resynthèse d'un ligand multi-cible conjugué alliant un inhibiteur d'AChE (IAChE) et un antioxydant, deux nouvelles familles de ligands multi-cibles conjuguées, combinant un IAChE et un agoniste des récepteurs nicotiniques α7 (α7 nAChR) ont été conçues et leur synthèse abordée. Dans le cas de la première famille, le fragment ivastigmine a été choisi pour sa capacité à inhiber de manière pseudo-irréversible l'AChE et a été conjugué à un motif quinuclidine, un puissant agoniste des α7 nAChRs impliqués dans la MA. En combinant ces deux fragments, il a été observé que les propriétés biologiques in vitro de chaque pharmacophore étaient améliorées. La structure de la seconde famille est basée sur le Donepezil, un IAChE réversible de plus forte affinité, combiné au même motif quinuclidine que dans la série précédente. Si des intermédiaires avancés ont été obtenus, un ou deux étapes restent à finaliser pour finaliser la synthèse de cette troisième famille de MTDL.

Mots-clés : maladie d'Alzheimer, ligand multi-cibles, acétylcholinestérase, antioxydant, récepteur nicotinique α7, huprine, rivastigmine, donepezil.

Abstract

Alzheimer's disease (AD) is a complex neurodegenerative disease characterised by a progressive loss of memory and cognition. Nowadays, 4.6 million new patients are identified every year and according to the "Alzheimer's diseases International" association, the number of patients could reach 135.5 million in 2050. Due to its complexity, AD remains uncurable and only 4 palliative drugs, of which 3 are acetylcholinesterase (AChE) inhibitors (AChEI), have been approved by FDA to date. AD being a multifactorial illness, with many potential targets involved in the pathology, the MTDL approach seems promising. This strategy associates in one single molecule, different pharmacophores (at least) acting on different targets involved in this CNS-related disorder. In this context, in parallel with the upscaled synthesis of a conjugated MTDL combining an AChEI inhibitor and an antioxidant, two new families of conjugated MTDLs associating an AChEI and a α7 nicotinic receptor (α7 nAChR) agonist have been investigated. The structure of the first family was based on a Rivastigmine scaffold, known to be a pseudo-irreversible AChE inhibitor, and a quinuclidine fragment, a potent α7 nAChR agonist. By combining these two fragments, it was brought to light that the in vitro biological properties were improved on both targets. The second family was based on a donepezil fragment, a more potent AChEI, and the same quinuclidine fragment than in the first family. Advanced intermediates have been obtained, and two last steps remain to be achieved for the completion of this third MTDL series.

Keywords: Alzheimer's disease, multi-target directed ligand, acetylcholinesterase, antioxidant, α7 nicotinic receptor, huprine, rivastigmine, donepezil

  PhD consisted in preparing a heterodimer Huprine-Capsaicin targeting acetylcholinesterase and oxidative stress induced by AD, based on the previous work of Dr. Sovy Chao, Dr. Killian Oukoloff and Dr. Monika Bouet, former researchers and in collaboration with the Professor Diego Muñoz-Torrero, pioneer in the development of huprine compounds as anti-AD drugs.

*

  Values were standardized to the values of Western Europe

Figure 2 :

 2 Figure 2: Evolution of AD (adapted from Dr. Alain Buisson's presentation -University of Grenoble Alpes)

Figure 3 :

 3 Figure 3: MR images (brain-extracted)[START_REF] Ledig | Structural Brain Imaging in Alzheimer's Disease and Mild Cognitive Impairment: Biomarker Analysis and Shared Morphometry Database[END_REF]

Figure 4 :Figure 5 :

 45 Figure 4: PET scan of an AD patient's brain 14 (discussed below) and structures of 11 C-PIB and 11 C-PMP

Figure 6 :

 6 Figure 6: Structures of Tau pathology biomarkers

Figure 7 :

 7 Figure 7: Mechanism of the neurotransmission 23

Figure 8 :

 8 Figure 8: Results of Sims et al. on cholinergic dysfunction in patients with dementia 26

Figure 9 :

 9 Figure 9: Non-amyloidogenic pathway of the APP cleavage

Figure 10 :Figure 11 :

 1011 Figure 10: Amyloidogenic pathway of the APP cleavage

Figure 12 :

 12 Figure 12: Interactions between the Aβ and α7 nAChR according to the concentration of Aβ 35

  Drechsel, D. N.; Hyman, A. A.; Cobb, M. H.; Kirschner, M. W. Modulation of the Dynamic Instability of Tubulin Assembly by the Microtubule-Associated Protein Tau. Mol. Biol. Cell 1992, 3 (10), 1141-1154.

Figure 14 :

 14 Figure 14: Illustration of the Tau protein polymerization[START_REF] Verwilst | Shedding Light on Tau Protein Aggregation: The Progress in Developing Highly Selective Fluorophores[END_REF]

Figure 16 :

 16 Figure 16: Mechanism of ACh and Rivastigmine decarbamoylation 53

Figure 18 :

 18 Figure 18: Studies of adverse effects of Memogain (A) and performance recovery (B)[START_REF] Maelicke | Memogain Is a Galantamine Pro-Drug Having Dramatically Reduced Adverse Effects and Enhanced Efficacy[END_REF]

Figure 20 :

 20 Figure 20: Mechanism of action of a nAChR with ACh 59

Figure 22 :

 22 Figure22: examples of α7 nAChR agonists with a quinuclidine[START_REF] Yang | The Current Agonists and Positive Allosteric Modulators of α 7 NAChR for CNS Indications in Clinical Trials[END_REF]

Figure 24 :

 24 Figure 24: Example of M1 mAChR agonists

Figure 25 :

 25 Figure 25: Structure and mechanism of a GluN1/GluN2 NMDA receptor[START_REF] Hansen | Structure, Function, and Allosteric Modulation of NMDA Receptors[END_REF]

Figure 26 :

 26 Figure 26: Examples of NMDA receptors antagonists

Figure 28 :

 28 Figure 28: Examples of α-secretase activators

Figure 29 :

 29 Figure 29: Examples of BACE-1 inhibitors

Figure 30 :

 30 Figure 30: levels of Aβ in mice's neocortex exposed 100mg/kg of thalidomide for 3 months 102

Figure 31 :

 31 Figure 31: levels of Aβ 4h after Umibecestat administration 103

Figure 32 :

 32 Figure 32: Examples of γ-secretase inhibitors and modulators

Figure 33 :

 33 Figure 33: Brain concentrations of Aβ42 (A) and Aβtotal (B) measured 4h after administration of a 10 or 30 mg/kg dose of EVP-0015962 105

Figure 37 :

 37 Figure 37: Microtubules stabilizers

Figure 38 :

 38 Figure 38: Antioxidants in clinical trials

Figure 39 :

 39 Figure 39: The contrast of traditional drug development (A) and drug repositioning (B) 125

Figure 40 :

 40 Figure 40: Example of drugs repositioned for AD

Figure 41 :

 41 Figure 41: Three main clinical scenarios for multitarget therapy 131

Figure 43 :

 43 Figure 43: Example of various MTDLs' types 131 (NSAID: nonsteroidal anti-inflammatory drug)

Figure 48 :

 48 Figure 48: Example of MTDLs inhibiting AChE and MAOs

Figure 49 :

 49 Figure 49: Examples of MTDLs inhibiting AChE and kinases

7. 3 . 5 .

 35 Other types of MTDLs -Combined molecules 5-hydroxytryptamine receptors (5HT4R) is part of a family of serotonin receptors. It is a glycosylated transmembrane protein active in the CNS to modulate the release of neurotransmitters such as ACh.

  dual 5HT4R agonist (Ki = 8.5 nM) and AChEI (IC50 = 16 nM) also promoting the non-amyloidogenic pathway of the APP cleavage.Moreover, the same group designed compound 32 in 2018 166 by structurally modifying the already existing Donecopride 31 which demonstrated good AChE inhibition (IC50 = 28.8 nM) and promising affinities toward 5HT4R (Ki = 37.5 nM) and σ1 receptor (Ki = 5.1 nM) which is a transmembrane protein also involved in CNS-related pathologies. Several compounds acting on σ1 receptor167,168 have been designed and disclosed effective neuroprotection in transgenic mouse models of AD, probably because of the wide range of modulatory role of the receptor on neurotransmitter responses, ACh and glutamate systems and Ca 2+ mobilisation 169 .

Figure 50 :

 50 Figure 50: Examples of combined MTDLs against AD therapeutic targets

Figure 52 :

 52 Figure 52: Team expertise in the design of MTDLs

Figure 53 :

 53 Figure 53: Structure of a conjugated MTDL targeting AChE and α7 nAChR

  development of MTDLs targeting at least two therapeutic targets. One of the objectives of the ANR project "Multiclick" 2012-2017 involving our team, and the ones of Dr. Jacques-Philippe Colletier (IBS, Grenoble), Pr. Diego Muñoz-Torrero (University of Barcelona) and Pr. Jacques Rouen (University of Caen Normandy), consisted in synthesizing new MTDLs targeting the inhibition of AChE and targeting other landmarks of AD such as accumulation of reactive oxygen species, thus for instance bearing antioxidant properties (Figure54). Thus, based on the expertise of Pr. Muñoz-Torrero's team176,177 and ours in the synthesis of huprine derivatives, we planned to develop huprine-based MTDL with anti-AChE activity and antioxidant properties. This project was initiated by Dr. Monika Bouet, postdoctoral fellow in our group, who managed to design a new series of conjugated huprine-capsaicin MTDLs showing highly promising in vitro results 178 to be beneficial in the treatment of AD (see below).

Figure 54 :

 54 Figure 54: Conjugated MTDLs structure

Figure 55 :

 55 Figure 55: Structure of 34

Scheme 3 :

 3 Scheme 3: AChE catalysed ACh hydrolysis

  (hAChE) structure was obtained thanks to co-crystallisation of AChE with fasciculine-2 , a toxin acting as an inhibitor through interaction with the peripheral site, limiting the high flexibility of this part of the enzyme which prevented crystals formation 187 . Moreover, AChE peripheral site role in Aβ fibrils formation has been shed light 188 , understanding this role was limited by the presence of this PAS binding toxin in the crystal structures.More recently, crystals structures of the enzyme without the PAS binding toxin were obtained by Dr.M.C. Franklin 189 and Dr. Florian Nachon's (in collaboration with Pr. Renard) teams 190 , thus allowing a more accurate determination of the structure and flexibility of this peripheral site. These various studies led to a precise visualisation of the enzyme conformation and mode of action (Figure56).

Figure 56 :

 56 Figure 56: TcAChE (A) and hAChE (B) structures 187

Figure 57 :

 57 Figure 57: hAChE three-dimensional representation: in green, the peripherical site; in purple the active site; in red, the acylation site, in blue the π-cation site (figure extracted from Dr. Killian Oukoloff's PhD thesis)

3. 1 .

 1 Huprine -a potent AChE inhibitor 3.1.1. Design of huprine scaffold Pr. Camps and Pr. Muñoz-Torrero's team developed in 2000 an innovative and promising scaffold inorder to obtain a more potent AChEI than those already existing 178 . Huprine was born from the fusion of the scaffolds of two well-known AChEIs, namely tacrine and (-)-huperzine A (Figure60). The huprine structure combines the carbobicyclic moiety from the (-)-huperzine A and the 4-aminoquinoline moiety of tacrine.

Figure 60 :

 60 Figure 60: Design of huprine scaffold

Figure 61 :

 61 Figure 61: 3D structure of huprine W with hAChE 216

Figure 62 :

 62 Figure 62: Huprine X and huprine Y structure

Figure 64 :

 64 Figure 64: Examples of huprines functionalised in position 12

Figure 65 :

 65 Figure 65: Examples of huprines functionalized in position 9

  Scheme 4: Hupresin® formation

Figure 66 :Figure 67 :

 6667 Figure 66: Structure of HupNR1, HupNR2 and cyanine 5.0

Figure 68 :

 68 Figure 68: Structure and pharmacological properties of Capsaicin 245

Figure 72 :

 72 Figure 72: Huprine-Capsaicin hybrid conjugated MTDLs series

Scheme 7 :

 7 Scheme 7: Retrosynthetic pathway to 47

Scheme 8 :

 8 Scheme 8: Synthesis of compound 53

Diketone 60

 60 Scheme 15: Synthesis of azide 48

Figure 73 : 1 Figure 74 :

 73174 Figure 73: 34 chromatogram with TFA buffer (analytic HPLC (Thermo Hypersil GOLD C18 column, 5 μm, 2.1 × 100 mm) with MeCN and TFA (aq. 0.1%; pH 2.0) as eluents [100% aq. TFA (5 min), then linear gradient from 0% to 100% (45 min) MeCN] at a flow rate of 0.25 mL.min -1

Figure 75 :Figure 76 :

 7576 Figure 75: Crystal structure of 34-TcAChE

Figure 78 :

 78 Figure 78: (A) α7 nAChR chimera in complex with alpha bungarotoxin (PDB 4HQP) 282 and (B) α7AChBP in complex with Iobeline (PDB 5AFH)283

Figure 79 :

 79 Figure 79: Schematic representation of an α7 nAChR 285

Figure 80 :

 80 Figure 80: Schematic representation of ACh binding site of AChBP 286

Figure 81 :

 81 Figure 81: Structure of mecamylamine, a channel blocker

Figure 82 :

 82 Figure 82: Ligand-binding sites in nAChRs 289 (EC = extracellular domain; TM= transmembrane domain)

Figure 83 :

 83 Figure 83: Schematic representation of α7 nAChR modulation mechanism (adapted from Basak et al. 298 )

  and that a phenyl (R)-69, thiophene (R)-70 or a benzofuran (R)-71 could be attached to the triazole core and lead to good α7 nAChRs binding ability in in vitro assays 308 (Figure86).

Figure 86 :

 86 Figure86: α7 nAChR agonists developed byRoutier et al. 308

  Nevertheless, a few Rivastigmine-based or carbamate-based MTDLs have been designed and evaluated in the past years (Figure87).Weinstock et al. 309 developed compound 72 by combining the N-ethyl-N-methylcarbamate fragment of Rivastigmine with the N-propargyl-(1R)-aminoindanone fragment of the rasagiline, an anti-Parkinson drug. The resulting new MTDL has been evaluated and showed a good AChE inhibitory activity in addition to a MAO-A/B inhibitory activity, it could also be noted that compound 72 enhances cognition and reduces oxidative stress. Then, carbamate-based derivatives such as compounds 73 and 74 have been designed by Sterling et al. 310 , the structures of these MTDLs were also based on a combination of a carbamate with a rasagiline moiety and are close to the structure of compound 72. Both compound 73 and 74 displayed promising inhibitory activities toward AChE and MAO-A/B, with a slightly better IC50 on AChE for compound 74 .

Figure 87 :

 87 Figure 87: Examples of carbamate-based MTDLs

Figure 88 :

 88 Figure 88: Chosen structure for the α7 nAChRs agonist / AChE inhibitor MTDLs

Figure 89 :

 89 Figure 89: Representation of the BBB 313

313

  Abbott, N. J.; Patabendige, A. A. K.; Dolman, D. E. M.; Yusof, S. R.; Begley, D. J. Structure and Function of the Blood-Brain Barrier. Neurobiol. Dis. 2010, 37 (1), 13-25.

Figure 90 :

 90 Figure 90: Number of FDA-approved oral drugs by time period (drugs with MW less than 500 are in blue and those above 500 are in red) 314

Figure 91 :

 91 Figure 91: CNS MPO desirability score 274

Figure 92 :

 92 Figure 92: Scale of LogPS (adapted from ACDLab technical document)

Figure 93 :

 93 Figure 93: Overview of computer aided drug designing aims 317

  Choice and preparation of the target (protein, enzyme, etc) -Choice and preparation of the ligands -Docking calculation -Evaluation of the results -Visualisation of the docked ligands Choice and preparation of the target To provide a more realistic estimation of what could really happen, the starting point should be a cocrystal structure of the enzyme-ligand complexes where the known ligand must be structurally close to the MTDL which has to be evaluated. Such structures can be found in the protein data bank (PDB), a database gathering the X-ray crystallographic structures of proteins or nucleic acid obtained by biologists and biochemists on different biomolecules.

Figure 94 :

 94 Figure 94: MTDLs evaluated in silico

Figure 95 :

 95 Figure 95: Visualisation of most stabilized conformation for compounds (A) (R)-75a and (B) (R)-76a within the enzymatic pocket as predicted by AutodockVina and represented with PyMol

Figure 98 :

 98 Figure 98: Active site of TcAChE after inhibition with Rivastigmine (adapted from Bar-On et al. 324 )

Figure 99 :

 99 Figure 99: Docking prediction of Rivastigmine within mAChE (A) main interactions (B) snapshot of Rivastigmine position in the active site

  salts, through in situ reduction of the Cu(II) salts by sodium ascorbate as a reductive agent to obtain catalytically active Cu(I) derivatives. Thus, a new series of 18 MTDLs was obtained with yields ranging from 9 to 36% (6 new ligands were synthesized, either as racemic mixtures 75a-c and 76a-c or each one of the enantiopure form (R)-75a-c and (R)-76a-c and (S)-75a-c and (S)-76a-c) (Scheme 27

342a

  Yung-Chi, C.; Prusoff, W. H. Relationship between the Inhibition Constant (KI) and the Concentration of Inhibitor Which Causes 50 per Cent Inhibition (I50) of an Enzymatic Reaction. Biochem. Pharmacol. 1973, 22 (23), 3099-3108. Values are expressed as an average of 3 values from 3 independent experiments +/-SEM; b Binding determined by incubating proteins extracts from the frontal cortices (25µg), radiolabelled [125I]α-bungarotoxin (2 nM) and the tested compounds (10 -6 to 10 -10 M). Ki values were calculated according to Cheng and Prusoff procedure 342

  37 °C with a Jasco V-530 double beam spectrophotometer. Stock solution of the tested compounds(3 mM) were prepared in methanol and diluted in methanol. The assay solution consisted of a 0.1 M phosphate buffer, pH 8.0, with the addition of 340 µM 5,5'-dithiobis(2-nitrobenzoic acid), 0.02 unit/mL human recombinant AChE (Sigma Chemical) or human serum BChE (Sigma Chemical), and 550 µM substrate (acetylthiocholine iodide -ACTh or butyrylthiocholine iodide -BTCh, respectively). Assay solutions with and without inhibitor were preincubated at 37 °C for 40 min in the case of hAChE-based assay and of 20 min in the case of hBChE-based assay followed by the addition of substrate. Blank solutions containing all components except hAChE or hBChE were prepared in parallel to account for the non-enzymatic hydrolysis of the substrate.The results of these assays showed that the combined MTDL displays an equal or even higher inhibition potency towards cholinesterases: compound (R)-76a (entry 3) and (R)-75b (entry 4) display an almost 10 time more efficient inhibitory activity against hAChE as compared to Rivastigmine. Compound (R)-76a also exerted a high inhibitory activity toward hBChE compared to the known inhibitory activity of Rivastigmine against BChE, this added property could also have a positive effect against cholinergic deficit and lead us to the conclusion that compound (R)-76a seemed to be the best candidate of the series. Moreover, time-dependent inhibition profile of (R)-75b has been first determined in order to define the necessary incubation time to evaluate inhibitory potency toward hAChE of the other derivatives (Figure101). The Stigmine derivatives cholinesterase inhibition mechanism is a slow and reversible process as we already mentioned. Hence, the results obtained for compound (R)-75b which must be incubated for 40 minutes to reach 50% of inhibition are in accordance with what could be expected for carbamate-based AChEI. of 40 min; b incubation time of 20 min; c not determined Values are expressed as an average of 3 values from 3 independent experiments +/-SEM Stock solution of the tested compound (3 mM) was prepared in methanol and diluted in methanol. Assays solutions of a 0.1 M phosphate buffer, pH 8.0, with the addition of 340 µM 5,5'-dithiobis(2-nitrobenzoic acid), 0.02 unit/mL human recombinant AChE (Sigma Chemical) or human serum BChE (Sigma Chemical), and 550 µM substrate (acetylthiocholine iodide -ACTh or butyrylthiocholine iodide -BTCh, respectively). Assay solutions with and without inhibitor were preincubated at 37 °C followed by the addition of substrate.Table 11: Cholinesterases Inhibition potency of (R)-75a-c and (RStopped time assay performed, in which AChE and (R)-75b at a concentration of 6.0 µM, a value close to its IC50 value, were mixed in the assay buffer at pH 8.0. After 5, 10, 20, 30 and 40 minutes incubation at 37 °C, the determination of residual activity of the AChE-catalyzed hydrolysis of ATCh was carried out by Ellman's method 343 .

7 .

 7 Synthesis of Stigmine's derivatives with an optimised linker 7.1. Rational choice a new linker The promising binding affinities of the MTDLs (R)-75a-c and (R)-76a-c with α7 nAChRs determined by Pr Sylvie Chalon's team, lead us to go one step further in order to increase these values by combining Pr Routier's expertise on α7 nAChRs and the expertise we gained in the development of combined MTDLs using CuAAC. Our preliminary results have shown that combining two pharmacophores, a carbamate and a quinuclidine, could end up in an increase of both the molecules affinity toward α7 nAChRs, and AChE inhibition potency. Moreover, it has been recently shown by Pr. Routier's team that adding a phenyl ring substituted in the meta position as represented in compound (R)-94 could increase the binding affinity as compared to the monomer (R)-69 bearing only one phenyl ring or a substituent in the para position (Figure 103) 344 .

Figure 103 :

 103 Figure 103: Second generation α-7 nAChR agonists developed by Routier et al.

Figure 109 : 7 . 1 . 2 .

 109712 Figure 109: Representation of predicted interactions between mAChE and (R)-99a

Figure 110 :

 110 Figure 110: Results of Brain penetration prediction for CNS activity for (A) (R)-96a (B) (R)-96b (C) (R)-96c (D) (R)-98a (E) (R)-98b (F) (R)-98c (red dots represent CNS inactive compounds, blue dots represent CNS active compounds and the circled black dot represent the evaluated compound

Figure 111 :

 111 Figure 111: Lead compounds for the first combined MTDLs series.

Scheme 36 :

 36 Scheme 36: Optimized reaction conditions for the one-pot diazo-transfer and Huisgen cycloaddition

Figure 112 :

 112 Figure 112: Structure of new combined MTDLs

Figure 113 :

 113 Figure 113: Donepezil structure

Figure 114 :

 114 Figure 114: Structures of studies N-benzylpiperazine 119 and N-benzylpiperidine 120 fragments

Figure 115 :

 115 Figure 115: Structure of Donepezil with hAChE 354 (a) Donepezil is shown in stereo view with residues from TcAChE (solved at 2.5 Å) superimposed. Donepezil nitrogen and oxygen atoms are coloured blue and red, respectively, while carbon atoms are coloured pink in the human-bound conformer and light blue in the Torpedo-bound conformer. Residue numbering refers to human acetylcholinesterase, with equivalent numbers for TcAChE in parentheses (b) Active site gorge of the superimposed hAChE and TcAChE in complex with Donepezil (residues W286 and Y341 of the hAChE are shown as sticks and TcAChE residues are omitted for clarity

Figure 116 :

 116 Figure 116: Examples of Donepezil-based ChE inhibitors with additional Aβ self-aggregation inhibition potency

Figure 117 :

 117 Figure 117: Examples of Donepezil based MTDLs (MAOI = MAO inhibitor)

Figure

  Figure 118: Structures of the targeted combined MTDLs

Figure 119 :

 119 Figure 119: First series of Donepezil based MTDLs

  It can be noted that compounds (R)-128 (Figure120-A), (R)-129 (Figure 120-B), (R)-130 (Figure 120-C) and (R)-131 (Figure 120-D) have obtained encouraging results, both in terms of binding energy and positioning within the enzyme binding site, compared to the reported binding affinity of Donepezil (entry 1).

Figure 120 :

 120 Figure 120: Docking of rhAChE with (A) (R)-128 (B) (R)-129 (C) (R)-130 (D) (R)-131 (Trp286, Tyr124, Tyr341, Tyr337 and Trp86 are represented in sticks)

Figure 124 :

 124 Figure 124: Representation of interactions between rhAChE and (R)-131

Figure 125 :

 125 Figure 125: rhAChE active site with bound Donepezil (adapted from Cheung et al. 354 )

Figure 127 :

 127 Figure 127: Second series of Donepezil based MTDLs

  -A), (R)-133 (Figure 128-B), (R)-134 (Figure 128-C) and (R)-135 (Figure 128-D) all appeared to be well oriented inside the enzyme binding pocket and the quinuclidine fragment was found to be located at the PAS.

Figure 128 :

 128 Figure 128: Docking of rhAChE with (A) (R)-132 (B) (R)-133 (C) (R)-134 (D) (R)-135 (Trp286, Tyr124, Tyr341, Tyr337 and Trp86 are represented in sticks)

Figure 130 :

 130 Figure 130: Representation of predicted interactions between rhAChE and (R)-133

Figure 131 :

 131 Figure 131: Representation of predicted interactions between rhAChE and (R)-134

  The obtained results were similar to those previously obtained for the first series of Donepezil based MTDL, compounds (R)-132 (entry 2) and compound (R)-134 (entry 4) displayed lower scores because of a higher molecular weight coupled with a strong LogP value, nevertheless, compounds (R)-133 (entry 3) and compound (R)-135 (entry 5), despite their size, expressed better CNS MPO scores thanks to a lower LogP value and pKa. Once again, the scores and main physicochemical properties values of the four tested compounds were in accordance with the score and physicochemical properties displayed by the parent monomer Donepezil (entry 1).

  compounds group (represented with blue dots). Apparently, changing the N-benzylpiperidine of compounds (R)-132 and (R)-134 by a N-benzylpiperazine in compounds (R)-133 and (R)-135 did not affect the predicted properties of the ligands to cross the BBB, moreover, the relative position of the N-benzylpiperidine (or N-benzylpiperazine) fragment did not appear to induce a major change either.

Figure 133 :

 133 Figure 133: Results of Brain penetration prediction for CNS activity for (A) (R)-132 (B) (R)-133 (C) (R)-134 (D) (R)-135 (E) Donepezil (red dots represent CNS inactive compounds, blue dots represent CNSactive compounds and the circled black dot represent the tested compound)

  To obtain the first series of combined MTDLs (R)-128 and (R)-130 based on a Donepezil scaffold we designed and validate with in silico evaluations, the rational approach was to use our gained expertise in the development of Copper(I) catalysed Huisgen cycloaddition protocols. After obtaining the (R)-

Figure 134 :

 134 Figure 134: Retrosynthetic pathway to (R)-128 and (R)-130

372

  Nagel, A. A.; Liston, D. R.; Jung, S.; Mahar, M.; Vincent, L. A.; Chapin, D.; Chen, Y. L.; Hubbard, S.; Ives, J. L. Design and Synthesis of 1-Heteroaryl-3-(

Figure 135 :

 135 Figure 135: Proposed synthetic route to 136

4. 1 . 2 .

 12 New approach to (R)-128 and (R)-130 4.1.2.1. Retrosynthetic pathway After finding out that the first strategy we developed presented at least two bottlenecks (at the aldolization and Sonogashira cross coupling steps), to efficiently obtain combined MTDLs (R)-128 and (R)-130, we designed a new synthetic pathway where the Copper(I)-catalysed Huisgen's cycloaddition is not the final step but a pivotal point introduced earlier in the procedure (Figure 136). The newly developed retrosynthesis scheme could be summarized in the three following key steps: an aldolization of (R)-146 (or (R)-147) and aldehyde 140, previously obtained, followed by a reduction should lead to compound (R)-128 (or (R)-130). Ketone (R)-146 (or (R)-147) could be obtained from the corresponding alkyne 148 (or 149) and azide (R)-77 through a Copper(I)-catalysed Huisgen's cycloaddition following the procedure used in the previous chapter, and a Sonogashira cross-coupling between ketone 150 (or 151) ,bearing a triflate instead of a bromine, and ethynyltrimethylsilane should offer compound 148 (or 149).

Figure 136 :

 136 Figure 136: Proposed retrosynthetic access to (R)-128 and (R)-130

Scheme 42 :

 42 Scheme 42: Synthesis of alkynes 148 and 149

  In parallel to the development of combined MTDLs (R)-128 and (R)-130, we designed a strategy to access combined MTDLs (R)-132 and (R)-134 through the same pathway using Copper(I) catalysed Huisgen cycloaddition to link the two partners (R)-77 and 154 (or 155) via the formation of a 1,2,3triazole moiety (Figure137). As a first example of the series, MTDL (R)-134 was chosen as a first synthetic target in order to validate the synthetic pathway before applying it on the other substituents.

Figure 137 :

 137 Figure 137: Retrosynthetic pathway to (R)-132 and (R)-134

Scheme 49 :

 49 Scheme 49: Attempt synthesis of 172

  Donepezil based MTDLs due to a lack of time and unexpected synthetic bottlenecks, we developed promising synthetic pathways for both series. Concerning the first series of MTDLs (R)-128 and (R)-130, we obtained a key intermediate (R)-147 in 4 steps with an overall 6% yield and only two step remains to be completed to obtain MTDLs (R)-130 through an aldolization followed by a reduction of the ketol with TMSI. Moreover, another quicker synthetic strategy could be considered, indeed, after an activation of alcohol 142 into the mesylate 173, an alkylation of (R)-147 with LDA could lead directly to the desired MTDL (R)-130 (the same synthetic pathways could be used to obtain its regioisomer (R)-compound (R)-134 and by extension the regioisomer (R)-132, could also be obtained through an alkylation of compound (R)-164 (and (R)-165) with the activated mesylate 173 in presence of LDA.

Scheme 52 :

 52 Scheme 52: Further work to obtain (R)-132 and (R)-134

  The second lesion is characterised by the neurofibrillary degeneration induced by the protein Tau hyperphosphorylation which leads to a loss of affinity with the microtubules, weakening the cytoskeleton causing neuronal death. Despite a better understanding of the biochemical mechanism involved in this CNS-related pathology, only three AChE inhibitors are available in the market (Donepezil, Galantamine and Rivastigmine) and one NMDA antagonist (Memantine). Unfortunately, all these drugs have only a palliative role, and act only in the early stages of AD. Even though, numerous molecules have been developed in the past years (almost 100 molecules were involved in phase I, II, III and IV clinical trials), there hasn't been any new drug commercialised in the past decade due to toxicity, various adverse effects and the lack of activity of assayed molecules, and AD remains incurable. Nevertheless, to tackle this new challenge, researchers have begun to adopt a new approach, the multi target-directed ligand approach, indeed, due to the multifactorial character of the pathology of AD, designing new drugs composed of two pharmacophores able to act on various targets of the pathology appeared to be a promising way toward innovative treatment of such CNS-related disorders. My PhD project falls within this particular context. Three different approach were undertaken. By collaborating with Professor Muñoz-Torrero (University of Barcelona), a novel and promising Huprine-Capsaicin heterodimer has been designed and biologically evaluated. Moreover, arising from another collaboration with Professor Routier (ICOA of Orléans) and Professor Chalon (CHU of Tours), the design and development of two innovative combined MTDLs families targeting AChE and α7 nAChRs simultaneously was tackled.

2. Objectif 1 :

 1 Synthesis of an Huprine-Capsaicin heterodimer 2.1. Review of the resultsIn this first project of my PhD, I worked on the synthesis of a conjugated MTDL combining a Huprine fragment and a capsaicin fragment connected with a triazole based linker (Figure138). Based on the research of Doctor Chao, Doctor Oukoloff and Doctor Bouet, former researchers in the team, I performed the up-scaled synthesis of a heterodimer bearing a Huprine scaffold, known to be a potent inhibitor of AChE, and a capsaicin moiety proven to have high antioxidant properties. Compound 34 had already been synthesised in the team by Doctor Bouet, nevertheless, an upscale of the synthesis was required in order to obtain 34 in a larger scale in prevision of the in vivo assays required, and this up-scale proved challenging.

Figure 138 :

 138 Figure 138: Structure of MTDL 34

Figure 139 :

 139 Figure 139: Crystal structures TcAChE (A) and hBChE (B) in complex with

Figure 140 :

 140 Figure 140: New family of MTDLs

aFigure 141 :

 141 Figure 141: Structure of (R)-76a and (R)-75b

Figure 142 :

 142 Figure 142: Structures of MTDLs (R)-96a-c, (R)-97a-c, (R)-98a/c and (R)-99a/c

Figure 143 :

 143 Figure 143: α-7 nAChRs designed by Routier et al.

  R)-181a could be obtained through a similar route to the one designed for the other quinuclidine based MTDL : a Copper(I)-catalysed Huisgen cycloaddition between azide (R)-77 and alkyne 183 could allow formation of the triazole moiety, alkyne 183 could be obtained in two steps through a Suzuki Miyaura coupling between alkyne 184 and 4-hydroxyboronic acid 107 followed by an acylation reaction of 184 with the carbamoyl chloride 91a (Figure145).

Figure 145 : 4 . 1 .

 14541 Figure 145: Proposed retrosynthetic route to (R)-181a

Figure 146 :

 146 Figure 146: Structures of (R)-128, (R)-130, (R)-132 and (R)-134

Figure 147 :Scheme 58 :

 14758 Figure 147: Structure of (R)-147

  et al.387 designed in 2002 dual inhibitors of AChE and MAO 187 from the Rivastigmine scaffold, and during their investigations, they observed that the nature of the nitrogen substituents seemed to directly influence AChE inhibitory activity. They brought to light that an aromatic substituent lead to the best IC50 values while a substitution with methyl and ethyl groups lead to a loss of activity. Thus, carbamate-based compounds such as (R)-188 could be promising and have increased AChE inhibitory activities compared to the MTDLs that were designed during my PhD.

Figure 148 :

 148 Figure 148: Other possible structures of urea-based MTDLs

Figure 149 :

 149 Figure 149: Possible structures of Donepezil-based and carbamate-based MTDLs

Figure 150 :

 150 Figure 150: Combined MTDLs targeting MAO-B with α7 nAChR agonists properties

BACE- 1

 1 inhibition Assay β-Secretase (BACE-1, Sigma) inhibition studies were performed by employing a peptide mimicking APP sequence as substrate (Methoxycoumarin-Ser-Glu-Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys-dinitrophenyl, M-2420, Bachem, Germany). The following procedure was employed: 5 μL of test compound (or DMSO, if preparing a control well) were pre-incubated with 175 μL of enzyme (in 20 mM sodium acetate pH 4.5 containing CHAPS 0.1% w/v) for 1 h at rt. The substrate (3 μM, final concentration) was then added and left to react for 15 min. The fluorescence signal was read at λem = 405 nm (λexc = 320 nm). The DMSO concentration in the final mixture was maintained below 5% (v/v) to guarantee no significant loss of enzyme activity. The background signal was measured in control wells containing all the reagents, except BACE-1 and subtracted. The fluorescence intensities with and without inhibitor were compared and the percent inhibition due to the presence of test compounds was calculated by the following expression: 100 -(IFi/IFo  100) where IFi and IFo are the fluorescence intensities obtained for BACE-1 in the presence and in the absence of inhibitor, respectively. To demonstrate inhibition of BACE-1 activity a peptidomimetic inhibitor (β-secretase inhibitor IV, Calbiochem) was serially diluted into the reactions' wells (IC50 = 13.0 ± 0.1 nM). DPPH radical scavenging activity Assay Radical scavenging activity of the capsaicin-huprine hybrids was determined by DPPH assay. Four different concentrations of the compounds (3-100 µM) dissolved in DMSO were incubated with a methanolic solution of DPPH (100 μM). The final concentration of DMSO was kept below 1%. After 30 min of incubation at rt in the dark, the absorbance at 517 nm was measured by a spectrophotometer. The percent inhibition for each concentration was calculated by comparison with the absorbance of DPPH solution. Dose-response curves were plotted and IC50 values were calculated by the software CurveExpert version 1.34 for Windows. The average values represent the mean of 3 experiments.

1 H

 1 azide 48 (374.5 mg, 1.02 mmol) and alkyne49 (403.8 mg, 1.22 mmol, 1.2 equiv) was prepared in HPLC grade MeCN(34 mL).CuI (194.26 mg, 1.02 mmol, 1 equiv) was added to the solution and the reaction mixture was stirred at rt for 12h. The reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column on silica gel (DCM/MeOH, 98/2 to 80/20, v/v) to give 47 (507 mg, 71%) as a yellow solid. NMR (CD3OD, 300 MHz) δ 8.33 (d, J = 9.1 Hz, 1H), 7.69 (d, J = 2.1 Hz, 2H), 7.57 (dd, J = 2.0 Hz, J = 9.1

1 H

 1 To a solution of 47(50 mg, 0.07 mmol) in dry DCM(2.4 mL), CSA (165 mg, 0.7 mmol, 10 equiv) and MeOH (1 mL) were added. The reaction mixture was stirred at rt for 24h.Progress of the reaction was checked by HPLC. Reaction mixture was quenched with aq. sat. NaHCO3, extracted with DCM. Organic layers were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by semi-preparative with MeCN and aq. AcONH (10 mM) (0% MeCN followed by linear gradient from 40% to 45% of MeCN in 20 min with a flow rate of 1mL/min). Dual visible detection was achieved at 230 and 550 nm to give 34 (29 mg, 70%) as a beige solid. NMR (300 MHz, CD3OD) δ 8.33(d, J = 9.1 Hz, 1H), 7.67 (d, J = 1.9 Hz, 1H), , 7.63 (s, 1H) , 7.42 (dd, J = 9.1 Hz, J = 1.9 Hz, 1H) ,6.86 (s, 1H) , 6.71 (m, 2H), 5.59 (d, J = 5.7 Hz,1H) , 4.29 (s, 2H) , 4.17 (t, J = 6.9 Hz, 2H) ,3.81 (s, 3H) , 3.66 (s, 2H) , 3.30 (m, 1H) , 3.11 (dd, J = 18.0 Hz, J = 5.4 Hz, 1H) , 2.82 (d, J = 18.0 Hz, 1H), 2.74 (m, 1H) , 2.37 (dd, J = 17.0 Hz, J = 3.3 Hz, 1H), 2.10-1.87 (complex signal, 5H), 1.61 (m, 2H), 1.30 (m, 2H); 13 C NMR (75 MHz, CD3OD) δ

1 H

 1 NMR (300 MHz, CDCl3) δ 3.48 (dq, J = 21.3, 7.2 Hz, 2H), 3.07 (d, J = 26.1 Hz, 3H), 1.21 (dt, J = 14.6, 7.2 Hz, 3H); 13 C NMR (75 MHz, CDCl3) δ 149.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.32 -7.29 (m, 2H), 7.26 -7.24 (m, 1H), 7.12 (dt, J = 6.6, 2.5 Hz, 1H), 3.09 (s, 3H), 3.06 (s, 1H), 3.01 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 154.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.32 -7.28 (m, 2H), 7.25 (s, 1H), 7.15 -7.10 (m, 1H), 3.51 -3.35 (m, 2H), 3.09 -2.95 (m, 4H), 1.27 -1.14 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 151.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.51 -7.45 (m, 2H), 7.11 -7.05 (m, 2H), 3.05 (d, J = 25.1 Hz, 6H), 3.04 (s, 1H); 13 C NMR (75 MHz, CDCl3) δ 154.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.44 -7.38 (m, 2H), 7.05 -6.99 (m, 2H), 3.34 (dd, J = 12.4, 6.5 Hz, 4H), 2.97 (s, 1H), 1.20 -1.11 (m, 6H); 13 C NMR (75 MHz, CDCl3) δ 153.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.48 (d, J = 8.89 Hz, 2H), 7.09 (d, J = 7.2 Hz, 2H), 3.44 (dq, J = 14.1, 6.9 Hz, 2H), 3.03 (dd, J = 13.7, 7.5 Hz, 4H), 1.21 (dt, J = 13.9, 7.0 Hz, 3H); 13 C NMR (75 MHz, CDCl3) δ 154.

1 H

 1 from alkyne 79a (83.25 mg, 0.44 mmol, 1 equiv) and 3-aminoquinuclidine 81 (100 mg, 0.44 mmol, 1 equiv) following the general procedure C and isolated as a brown powder(45 mg, 30%). NMR (300 MHz, CDCl3) δ 7.83 (d, J = 9 Hz, 2H),7.78 (s, 1H), 7.19 (d, J = 9 Hz, 2H), 4.65 (s, 1H), 3.71 (m, 1H), 3.50 (m, 1H), 3.07 (d, J = 30 Hz, 6H), 2.95 (m, 4H), 2.29 (m, 1H), 1.80 (m, 4H); 13 C NMR (75 MHz, CDCl3) δ 154.

1 H

 1 carbons are missing despite an extended acquisition time); HRMS (ESI + ) (m/z)calcd. for C20H28N5O2 + [was obtained from alkyne 79b (95.5 mg, 0.44 mmol, 1 equiv) and 3-aminoquinuclidine (R)-81 (100mg, 0.44 mmol, 1 equiv) following the general procedure C and isolated as a yellow oil(79.38 mg, 49%).NMR and MS data were in accordance with those reported above for 75b [α]D = 19.25 (c = 0.05 in CHCl3 at 22 °C) was obtained from alkyne 79b (95.5 mg, 0.44 mmol, 1 equiv) and 3-aminoquinuclidine (S)-81 (100mg, 0.44 mmol, 1 equiv) following the general procedure C and isolated as a yellow oil(76.14 mg, 47%).NMR and MS data were in accordance with those reported above for 75b [α]D = -19.45 (c = 0.05 in CHCl3 at 22 °C) from alkyne 79c (83.25 mg, 0.44 mmol, 1 equiv) and 3-aminoquinuclidine 81 (100 mg, 0.44 mmol, 1 equiv) following the general procedure C and isolated as a brown powder (59.58 mg, 38%). NMR (300 MHz, CDCl3) δ 7.82(d, J = 9.21 Hz, 2H), 7.78 (s, 1H), 7.19 (d, J = 7.8 Hz, 2H), 4.65 (m, 1H), 3.72 (dd, J = 13.9, 4.1 Hz, 1H), 3.55 -3.35 (m, 3H), 3.08 -2.97 (m, 3H), 2.96 -2.87 (m, 2H), 2.28 (dt, J = 6.1, 3.0 Hz, 1H),1.87 -1.61 (m, 4H), 1.54 -1.41 (m, 1H), 1.31 -1.11 (m, 4H); 13 C NMR (75 MHz, CDCl3) δ 151.

81 (

 81 was obtained from alkyne 79c (83.25 mg, 0.44 mmol, 1 equiv) and 3-aminoquinuclidine (R)-100 mg, 0.44 mmol, 1 equiv) following the general procedure C and isolated as a brown powder(46.48 mg, 30%).NMR and MS data were in accordance with those reported above for 75c [α]D = 12.16 (c = 0.05 in CHCl3 at23 °C)

1 H

 1 NMR (300 MHz, CDCl3) δ 7.49 -7.43 (m, 2H), 7.38 -7.32 (m, 2H), 3.12 (s, 1H). Spectral data are in accordance with those reported previously: Kuang, C.; Yang, Q.; Senboku, H.; Tokuda, M. Synthesis of (Z)-1-Bromo-1-Alkenes and Terminal Alkynes from Anti-2,3-Dibromoalkanoic Acids by Microwave-Induced Reaction. Tetrahedron 2005, 61 (was obtained from 101a (150 mg, 0.66 mmol, 1 equiv) and (R)-3-aminoquinuclidine (R)-81 (119.5 mg, 0.66 mmol, 1 equiv) following general procedure C and isolated as a white solid (138.46 mg, 63%).

  was obtained from 101b (150 mg, 0.66 mmol, 1 equiv) and (R)-3-aminoquinuclidine (R)-81 (119.5 mg, 0.66 mmol, 1 equiv) following general procedure C and isolated as a white solid (31.1 mg, 14%).

1 H

 1 NMR (300 MHz, MeOD) δ8.47 (s, 1H), 7.76 (d, J = 8.5 Hz, 2H), 7.63 -7.54 (d, J = 8.6 Hz, 2H), 4.91 - 4.86 (m, 1H), 3.77 (dd, J = 13.9, 4.2 Hz, 1H),3.58 -3.45 (m, 1H), 3.15 (d, J = 14.7 Hz, 1H), 3.02 -2.89 (m, 3H), 2.31 (dd, J = 6.2, 3.1 Hz, 1H), 1.96 -1.85 (m, 2H), 1.68 -1.53 (m, 2H).

  was obtained from (R)-105a (50 mg, 0.15 mmol, 1 equiv) and 3-hydroxyphenylboronic acid 107(24.82 mg, 0.

  0; HRMS (ESI + ) (m/z) calcd. for C21H23N4O + [M+H + ] 347.1794 in progress; was obtained from (R)-105b (27 mg, 0.08 mmol, 1 equiv) and 4-hydroxyphenylboronic acid 106 (13.42 mg, 0.097 mmol, 1.2 equiv) following general procedure F and isolated as a yellow oil(16.60 mg, 60%).

1 H

 1 NMR (300 MHz, MeOD) δ 8.06 (dd, J = 8.3, 2.4 Hz, 1H), 7.88(d, J = 8.4 Hz, 1H), 7.78 -7.40 (m, 5H),

  , 2.15 (t, J = 19.6 Hz, 1H), 2.04 (d, J = 10.0 Hz, 2H), 1.86 (s, 2H); 13 C NMR (75 MHz, MeOD) δ 158.

  ; HRMS (ESI + ) (m/z) calcd. for C21H23N4O + [M+H + ] 347.1794 in progress; [α]D = -35.5 was obtained from (R)-105b (31.1 mg, 0.09 mmol, 1 equiv) and 3-hydroxyphenylboronic acid 107(15.24 mg, 0.

  9; HRMS (ESI + ) (m/z) calcd. for C21H23N4O + [M+H + ] 347.1794 in progress; [α]D = -28.92 (c = 0was obtained from (R)-109 (30 mg, 0.08 mmol, 1 equiv) and dimethylcarbamoyl chloride 91a

1 H

 1 NMR (300 MHz, MeOD) δ 8.54 (s, 1H), 8.12 (t, J = 1.5 Hz, 1H), 7.89 -7.81 (m, 1H), 7.66 -7.42 (m, 4H), 7.12 (ddd, J = 8.0, 2.3, 1.1 Hz, 1H), 4.93 -4.86 (m, 1H), 3.78 (dd, J = 14.2, 5.0 Hz, 1H), 3.52 (ddd, J was obtained from (R)-109 (10.00 mg, 0.03 mmol, 1 equiv) and 91c (0.04 mmol, 1.5 equiv)

1 H

 1 NMR (300 MHz, MeOD) δ8.56 (s, 1H), 8.13 (d, J = 1.6 Hz, 1H), 7.89 -7.83 (m, 1H), 7.68 -7.41 (m, 4H), 7.12 (dd, J = 8.0, 1.2 Hz, 1H), 4.96 -4.87 (m, 1H), 3.81 (dd, J = 14.3, 5.0 Hz, 1H), 3.61 -3.48 (m, 2H), 3.43 (dd, J = 14.3, 7.3 Hz, 1H), 3.23 -3.11 (m, 2H), 3.04 -2.92 (m, 4H), 2.35 (dd, J = 6.2, 3.1 Hz, 2H), 1.94 (dd, J = 9.3, 4.3 Hz, 3H), 1.76 -1.56 (m, 3H), 1.21 (t, J = 7.1 Hz, 2H); 13 C NMR (75 MHz, MeOD) δ 148.

32 (

 32 . (three carbon signals are missing despite an extended acquisition time); HRMS (ESI + ) (m/z) calcd. for C25H30N5O2 + [M+H + ] 432.2321 in progress; [α]D = 43.was obtained from (R)-108 (10.00 mg, 0.03 mmol, 1 equiv) and dimethylcarbamoyl chloride 91a (0.04 mmol, 1.5 equiv) following general procedure B and isolated as a pale yellow oil (4.5 mg, 36%).

1

 1

  . (one carbon signal is missing despite an extended acquisition time); HRMS (ESI + ) (m/z) calcd. for C24H28N5O2 + [M+H + ] 418.2165 in progress; was obtained from (R)-108 (10.00 mg, 0.023 mmol, 1 equiv) and diethylcarbamoyl chloride 91b (0.033 mmol, 1.5 equiv) following general procedure B and isolated as a colourless oil(5.22 mg,

  , 3.99 (t, J = 12.2 Hz, 1H), 3.70 -3.36 (m, 8H), 3.34 (d, J = 6.1 Hz, 5H), 2.63 (dd, J = 6.5, 3.2 Hz, 1H), 2.21 (dt, J = 18.6, 9.1 Hz, 2H), 2.00 -1.84 (m, 2H), 1.51 (d, J = 7.3 Hz, 2H); HRMS (ESI + ) (m/z) calcd. for C26H32N5O2 + [M+H + ] 446.2478 in progress; [α]D = -34.5 (c = 0.05 in CHCl3 at 23 °C).Compound (R)-97b was obtained in too low quantities to perform 13 C NMR experiment. was obtained from (R)-108 (10.00 mg, 0.03 mmol, 1 equiv) and 91c (0.045 mmol, 1.5 equiv)

1 H

 1 NMR (300 MHz, MeOD) δ8.55 (s, 1H), 8.12 (s, 1H), 7.88 -7.80 (m, 1H), 7.75 -7.68 (m, 2H), 7.67 - 7.39 (m, 3H), 7.22 (d, J = 8.6 Hz, 1H), 4.91 (s, 1H), 3.81 (dd, J = 14.2, 4.6 Hz, 1H), 3.62 -3.37 (m, 3H), 3.28 -3.17 (m, 1H), 3.11 (d, J = 14.1 Hz, 1H), 2.99 (d, J = 13.1 Hz, 4H), 2.34 (dt, J = 5.6, 2.9 Hz, 1H), 2.00 -1.85 (m, 2H), 1.79 -1.49 (m, 3H), 1.30 (d, J = 5.7 Hz, 3H); 13 C NMR (75 MHz, MeOD) δ 152.

1 H

 1 carbons are missing despite an extended acquisition time); HRMS (ESI + ) (m/z) calcd. for C25H30N5O2 + [M+H + ] 432.2321 in progress; [α]D = -44.5 (c = 0.05 in CHCl3 at 23 °C). was obtained from (R)-111(16.63 mg, 0.05 mmol, 1 equiv) and dimethylcarbamoyl chloride 91a (0.07 mmol, 1.5 equiv) following general procedure B and isolated as a white solid(7.5 mg, 36%). NMR (300 MHz, MeOD) δ 8.38(d, J = 5.2 Hz, 1H), 7.86 -7.79 (m, 2H), 7.65 -7.59 (m, 2H), 7.46 - 7.29 (m, 2H), 7.00 (ddd, J = 7.9, 2.3, 1.2 Hz, 1H), 4.84 -4.76 (m, 1H), 3.71 (dd, J = 14.2, 5.1 Hz, 1H), 3.44

1 H

 1 NMR (300 MHz, CDCl3) δ 8.08 -8.02 (m, 2H), 7.40 -7.36 (m, 2H), 2.62 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 196.

1 H

 1 NMR (300 MHz, CDCl3) δ 8.00 -7.95 (m, 1H), 7.87 -7.84 (m, 1H), 7.62 -7.56 (m, 1H), 7.49 (ddd, J = 8.3, 2.5, 1.0 Hz, 1H), 2.60 (s, 3H); 13 C NMR (75 MHz, CDCl3) δ 195.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.91 -7.86 (m, 2H), 7.56 -7.51 (m, 2H), 2.59 (s, 3H), 0.26 (s, 9H). the solvent was evaporated under reduce pressure to give the crude aldehyde which was used in the next step of the synthesis without further purification.

1 H

 1 NMR (300 MHz, CDCl3) δ 7.72 (d, J = 8.1 Hz, 1H), 7.67 (s, 1H), 7.55 (d, J = 8.1 Hz, 1H), 6.72 (d, J = 9.7 Hz, 1H), 4.12 (s, 2H), 3.70 (s, 2H), 2.82 (t, J = 12.2 Hz, 2H), 2.49 (d, J = 9.7 Hz, 1H), 1.70 (d, J = 10.5 Hz, 2H), 1.57 (s, 2H), 1.47 (s, 9H).

  Bowen, D. M.; Smith, C. B.; White, P.; Davison, A. N. NEUROTRANSMITTER-RELATED ENZYMES AND INDICES OF HYPOXIA IN SENILE DEMENTIA AND OTHER ABIOTROPHIES. Brain 1976,

  Piazzi et al. designed in 2008 a series of coumarin derivatives 147 able to inhibit both AChE and BACE-1.

	Aβ peptide fragments aggregation 138 and accelerating the peptide assembly into fibrils conducting to AChE. Concerning the Aβ aggregates, 10 displayed promising inhibitory results and a 92% protection 7.3.1.2. BACE-1 inhibition 146
	the senile plaques' formation. Moreover, it appears that the Aβ1-42 aggregates neurotoxicity depends of PC12 cells against Aβ-induced apoptosis 145 .
	on the amount of AChE linked to the complexes 139,140 . Building compounds with a dual activity toward
	AChE and Aβ aggregation could be an attractive therapeutic strategy against AD (Figure 44). The first Compound 11 was one of their best candidates with a balanced biological profile against the two
	example of MTDLs targeting AChE were Tacrine homodimer designed by Rodriguez-Franco et al. 141 targets. Six years later, the same group launched a screening of benzophenone derivatives to identify
	such as compound 6 capable of interacting with the AChE catalytic site and the PAS simultaneously. novel hybrids inhibiting AChE and BACE-1 but most of the compounds were proven to be inactive. Zha
	This characteristic gave compound 6 various neuroprotective activities like a high and selective AChE et al. synthesized and biologically evaluated a novel series of 26 Tacrine-benzofuran hybrids, amongst
	inhibition compared to Tacrine and BACE-1 inhibition properties associated with AChE-induced Aβ which compound 12 showed sub nanomolar inhibitory activity toward AChE and micromolar potency
	aggregation 142 . In 2008, Kwon et al. 143 designed piperidine derivatives based on the Donepezil scaffold against BACE-1 148 . At the same time, Constanzo et al. designed a Donepezil-based series of derivatives
	binding to the AChE catalytic site but also to the peripherical site. Compound 7 and 8 were the more and evaluated their selectivity toward AChE compared to BChE. Among all the derivatives, 13 had a
	promising MTDLs, indeed, they showed good inhibitory activity toward AChE and a promising promising inhibiting activity toward BACE-1 despite a less potent AChE inhibition potency as compared
	percentage of aggregation inhibition. Moreover, 7 displayed a high inhibitory activity toward AChE and to the parent compound 149 .
	a good selectivity of AChE relative to BChE (120 times more selective). Compound 9 was developed by Another class of MTDLs inhibiting AChE and BACE-1 was investigated by different groups, the lead
	Camps et al. in 2008 by assembling two known AChEIs: Tacrine and Donepezil. It is a sub nanomolar compounds not only modulated the two desired targets catalytic activity, but also acted on other
	AChEI and a potent BChE inhibitor. Like Donepezil, 9 can interact with the enzyme peripherical site to targets of CNS-dependant pathologies. Firstly, Camps et al. 150 discovered a series of quinoline-
	inhibit the peptide aggregation 144 . More recently, compound 10, a curcumin like compound with a chlorotacrine, the lead compound 14 proved to bind to both AChE catalytic and peripherical sites
	cyclohexanone moiety, was studied and showed a strong inhibitory activity and selectivity toward endowing the molecule with Aβ aggregation inhibition properties. 14 also displayed anti BACE-1
	7.3.1. MTDLs targeting AChE and amyloid-related targets properties (80% of BACE-1 activity blocked with a 2.5 μM concentration).
	7.3.1.1. Aβ aggregation inhibition Figure 45: Example of MTDLs inhibiting AChE and BACE-1
	Different studies have identified a link between AChE and Aβ aggregation. Indeed, it has been brought Then, Cavalli et al. designed compound 15 called Memoquin acting on numerous targets involved in
	to light that AChE peripheral site might contribute to amyloid deposits development by enhancing the AD. Indeed, besides its inhibiting activity on AChE and BACE-1, Memoquin also turned out to prevent
	the Aβ aggregates to assembly automatically (IC50 = 5.93 μM) or by AChE induction (IC50 = 28.3 μM).
	Moreover, Memoquin proved to possess antioxidant properties, lowering the free radicals' formation
	by 44% and helps reducing Tau hyperphosphorylation 151 . In vivo studies have shown that Memoquin
	Figure 44: Example of MTDLs inhibiting AChE and Aβ aggregation was able to restore cholinergic deficit and invert neuronal apoptosis in mice 152 . Finally, Huang et al.
	designed a quinoxaline derivatives series of 17 compounds expressing BACE-1 and AChE inhibition and
	some of them displayed H3 receptor antagonist activity. Among the 17 molecules, only one, compound
	151 Cavalli, A.; Bolognesi, M. L.; Capsoni, S.; Andrisano, V.; Bartolini, M.; Margotti, E.; Cattaneo, A.; Recanatini, M.;
	145 Zha, G.-F.; Zhang, C.-P.; Qin, H.-L.; Jantan, I.; Sher, M.; Amjad, M. W.; Hussain, M. A.; Hussain, Z.; Bukhari, S. Melchiorre, C. A Small Molecule Targeting the Multifactorial Nature of Alzheimer's Disease. Angew. Chem. Int.
	N. A. Biological Evaluation of Synthetic α,β-Unsaturated Carbonyl Based Cyclohexanone Derivatives as Ed Engl. 2007, 46 (20), 3689-3692.
	Neuroprotective Novel Inhibitors of Acetylcholinesterase, Butyrylcholinesterase and Amyloid-β Aggregation. 152 Bolognesi, M. L.; Cavalli, A.; Melchiorre, C. Memoquin: A Multi-Target-Directed Ligand as an Innovative
	Bioorg. Med. Chem. 2016, 24 (10), 2352-2359. Therapeutic Opportunity for Alzheimer's Disease. Neurotherapeutics 2009, 6 (1), 152-162.

  2.2. Butyrylcholinesterase 2.2.1. Role of the BChEBChE(EC 3.1.1.8)) is a non-specific cholinesterase enzyme, part of the serine hydrolase family, and able to hydrolyse various choline-based esters. It is synthesised in several part of the human body (liver, plasma, pancreas) but its physiologic role remains unclear 202 . Nevertheless, due to its poor selectivity, BChE is capable to take over the role of AChE to hydrolyse ACh. Indeed, transgenic mice, knocked-out for the AChE coding gene can survive without AChE, meaning that BChE is able to replace AChE and protect the mice of an ACh excess 203 . In the case of CNS-related pathologies such as AD, while the AChE levels are increasing, the BChE levels remain unchanged and in advanced stages of the disease, BChE can substitute AChE 204 . Increased BChE levels in blood serum has thus been identified as a significant biomarker of AD development but is also involved in some cases of diabetes205,206 . Very recently, some selective BChE inhibitors have shown promising results in AD treatment207,208 .

  . Moreover, other investigations have proven that huprines have a slow on-rate constant (kon), indeed, an incubation of 30 min between the enzyme and the inhibitor is mandatory during the inhibition assays 223 . Moreover, ex vivo studies on mice's brains have reported the huprine compounds have shown promising pharmacological profiles and a CNS activity 217 .Interestingly, this combination of two AChEIs has also displayed agonist activities toward mAChRs and nAChRs224,225 , antagonist activities toward NMDA receptors, giving a neuroprotective character 226 to these molecules. They have also proven their capacity to activate protein kinase C and mitogen activated protein kinase, proteins involved in the promotion of the non-amyloidogenic pathway for the APP cleavage 227 .Due to their high potency against AChE, huprine scaffold has been used in the past years in the development of MTDLs against AD. For example, Pr. Muñoz-Torrero's team designed new hybrids by functionalising the position 12176,177,228 (Figure64). In 2010, they have designed a family of dual binding site AChEIs by conjugating a huprine scaffold and a donepezil moiety, compound 35, the lead molecule of the series, proved to be a potent AChE inhibitor and turned out to moderately act on AChE-induced and self-induced Aβ aggregation and BACE-1228 . Five years later, this lead MTDL was evaluated in vivo and showed that compound 35 had pro-cognitive effects on mice improving both short and long-term

	Figure 63: Inhibitory activities of huprine X and huprine Y toward cholinesterases
	;
	224 Muñoz-Torrero, D.; Pera, M.; Relat, J.; Ratia, M.; Galdeano, C.; Viayna, E.; Sola, I.; Formosa, X.; Camps, P.; Camins, A.; Pallàs, M.; Bartolini, M.; Mancini, F.; Andrisano, V.; Arce, M. P.; Rodríguez-Franco, M. I.; Bidon-Chanal,
	Badia, A.; Clos, M. V. Expanding the Multipotent Profile of Huprine-Tacrine Heterodimers as Disease-Modifying A.; Luque, F. J.; Camps, P.; Muñoz-Torrero, D. Novel Huprine Derivatives with Inhibitory Activity toward β-Amyloid
	Anti-Alzheimer Agents. Neurodegener. Dis. 2012, 10 (1-4), 96-99. Aggregation and Formation as Disease-Modifying Anti-Alzheimer Drug Candidates. ChemMedChem 2010, 5 (11),
	225 Roman, S.; Badia, A.; Camps, P.; Clos, M. V. Potentiation Effects of (+/-)Huprine X, a New Acetylcholinesterase 1855-1870.
	Inhibitor, on Nicotinic Receptors in Rat Cortical Synaptosomes. Neuropharmacology 2004, 46 (1), 95-102. 229 Giménez-Llort, L.; Ratia, M.; Pérez, B.; Camps, P.; Muñoz-Torrero, D.; Badia, A.; Clos, M. V. AVCRI104P3, a
	Novel Multitarget Compound with Cognition-Enhancing and Anxiolytic Activities: Studies in Cognitively Poor

Ronco, C.; Sorin, G.; Nachon, F.; Foucault, R.

; Jean, L.; Romieu, A.; Renard, P. Y. Synthesis and Structure-Activity Relationship of Huprine Derivatives as Human Acetylcholinesterase Inhibitors. Bioorg.

Med. Chem. 2009, 17 (13)

, 4523-4536.

222

Ros, E.; Aleu, J.; Gómez de Aranda, I.; Muñoz-Torrero, D.; Camps, P.; Badia, A.; Marsal, J.; Solsona, C. The Pharmacology of Novel Acetylcholinesterase Inhibitors, (+/-)-Huprines Y and X, on the Torpedo Electric Organ. Eur. J.

Pharmacol. 2001, 421 (2)

, 77-84.

223

Camps, P.; Cusack, B.; Mallender, W. D.; Achab, R. E.; Morral, J.; Muñoz-Torrero, D.; Rosenberry, T. L. Huprine X Is a Novel High-Affinity Inhibitor of Acetylcholinesterase That Is of Interest for Treatment of Alzheimer's Disease. Mol. Pharmacol. 2000, 57 (2), 409-417.

2226 Canudas, A. M.; Pubill, D.; Sureda, F. X.; Verdaguer, E.; Camps, P.; Muñoz-Torrero, D.; Jiménez, A.; Camins, A.; Pallàs, M. Neuroprotective Effects of (±)-Huprine Y on in Vitro and in Vivo Models of Excitoxicity Damage. Exp. Neurol. 2003, 180 (2), 123-130. 227 Ratia, M.; Giménez-Llort, L.; Camps, P.; Muñoz-Torrero, D.; Clos, M.; Badia, A. Behavioural Effects and Regulation of PKCα and MAPK by Huprine X in Middle Aged Mice. Pharmacol. Biochem. Behav. 2010, 95 (4), 485-493.

3.1.3. State of the art

3.1.3.1. Huprine functionalized in position 12

memory. Moreover, compound 35 exhibited anxiolytic-like actions and did not exerted significant adverse effects

229

. The same group also worked on the design of another AChE dual inhibitor, a huprine-tacrine heterodimer linked in position 12 of the huprine moiety, compound 36 exerted promising in vitro results by acting on several targets of CNS-related pathologies. Indeed, compound 36, besides its potent inhibitory activity against AChE, also proved to be a good BACE-1 inhibitor and to reduce AChE-induced and self-induced Aβ aggregation 177 . Finally, compound 37 was designed by adding a Tau aggregation inhibiting moiety to the huprine moiety, the new hybrid gave promising in vitro results against AChE, BChE and BACE-1. Moreover, ex vivo studies in brain slices of mice revealed that compound 37 was efficient to protect against the Aβ self-aggregation and Tau protein aggregation, in vivo evaluations also showed an increase in the levels of mature APP and a central Aβ lowering effect 230 .

228

Viayna, E.; Gómez, T.; Galdeano, C.; Ramírez, L.; Ratia, M.; Badia, A.; Clos, M. V.; Verdaguer, E.; Junyent, F.Middle-Aged Mice. Behav. Brain Res. 2015, 286, 97-103.

230

Viayna, E.; Sola, I.; Bartolini, M.; De Simone, A.; Tapia-Rojas, C.; Serrano, F. G.; Sabaté, R.; Juárez-Jiménez, J.; Pérez, B.; Luque, F. J.; Andrisano, V.; Clos, M. V.; Inestrosa, N. C.; Muñoz-Torrero, D. Synthesis and Multitarget Biological Profiling of a Novel Family of Rhein Derivatives as Disease-Modifying Anti-Alzheimer Agents. J.

Med. Chem. 2014, 57 (6), 2549-2567.

Table 1 :

 1 Even though the use of antioxidants in the treatment of AD in clinical patients remains controversial242,243 , natural compounds and hybrids based on these molecules have been developed in the past years and have displayed interesting properties in the treatment of neurodegenerative diseases.

	1	HupNIR1	649	668	299 794	0.41	122 915
	2	HupNIR2	650	665	312 049	0.43	134 181

Fluorescence characterization of HupNR1 and HupNR2 3.2. Antioxidants against AD -Use of natural compounds 242 Feng, Y.; Wang, X. Antioxidant Therapies for Alzheimer's Disease. Oxid. Med. Cell. Longev. 2012, 2012. 243 Colizzi, C. The Protective Effects of Polyphenols on Alzheimer's Disease: A Systematic Review. Alzheimers Dement.

Transl. Res. Clin. Interv. 2019, 5, 184-196.

  Besides Capsaicin's antioxidant properties, it has also been proven byJiang et al. that this alkaloid was able to promote neuroprotection in rats248 .Flavonoids are part of the natural polyphenols family and have been widely investigated in the field of medicinal chemistry thanks to their proven health beneficial properties. Their antioxidant capacity has been used in the development of drugs against cancer 249 , inflammatory pathologies 250 or neurodegenerative diseases 251 . The antioxidant activity of flavonoids is due to their chemical structure and particularly to the hydroxyl groups present in the molecules 252 and the high antioxidant potency Thanks to their neuroprotective effects, flavonoids have been investigated as anti-AD drugs, the two main examples are represented inFigure 69. In 2008, Shimmyo et al. reported that myricetine, a flavonol, displayed inhibiting activity against BACE-1 inducing a decrease in the production of Aβ 253 . It must be added that myricetine also showed promising neuroprotective effects because of its ability to modulate NMDA receptors, and thus, to reduce Ca 2+ influx and inhibit the ROS production induced by the glutamate 254 . Another promising therapeutic agent against AD is (-)-epigallocatechin (EGCG), a flavonol found in green tea, indeed, in vitro studies have shown that the molecule had a protective activity against Aβ-induced cytotoxicity by acting as an AChE inhibitor 255 . Moreover, EGCG displayed interesting ROS scavenging properties inducing neuroprotective effects against Aβ-induced neuronal apoptosis 256 . Finally, further studies have shown that ECGC was involved in the inhibition of aggregates induced by the hyperphosphorylated Tau protein 257 . -tocopherol (Figure70) is the most studied and the most biologically active form of vitamin E, which includes different fat-soluble compounds, because it is the main form found in tissues and is an essential micronutrient for humans.

	Chapter I: Huprine-Capsaicin hybrid design and biological evaluations
	3.2.2.2. Vitamin E
	3.2.2. Other antioxidants
	3.2.2.1. Flavonoids
	Scheme 5: ROS scavenging by flavonoids 251
	Reveal Novel Pharmacophore
	Features. Biochim. Biophys. Acta BBA -Gen. Subj. 2008, 1780 (5), 819-825.
	254 Wang, J.; Yu, Z.; Yu, Q.; Kong, Q.; Zhang, N.; He, L.; Yi, Y. Study on Neuroprotective Capacity of Key Flavonoids
	of Ampelopsis Grossedentata. J. Food Nutr. Res. 2014, 2 (12), 880-889.
	255 Okello, E. J.; Leylabi, R.; McDougall, G. J. Inhibition of Acetylcholinesterase by Green and White Tea and Their
	Simulated Intestinal Metabolites. Food Funct. 2012, 3 (6), 651.
	123

Sharma, S. K.; Vij, A. S.; Sharma, M. Mechanisms and Clinical Uses of Capsaicin. Eur. J. Pharmacol. 2013, 720 (1), 55-62.

245

Adaszek, Ł.; Gadomska, D.; Mazurek, Ł.; Łyp, P.; Madany, J.; Winiarczyk, S. Properties of Capsaicin and Its Utility in Veterinary and Human Medicine. Res.

Vet. Sci. 2019, 123, 14-19. 246 Delanty, N.; Dichter, M. A. Antioxidant Therapy in Neurologic Disease. Arch. Neurol. 2000, 57 (9)

, 1265-1270.

247

Dairam, A.; Fogel, R.

; Daya, S.; Limson, J. L. Antioxidant and Iron-Binding Properties of Curcumin, Capsaicin, and S-Allylcysteine Reduce Oxidative Stress in Rat Brain Homogenate. J.

Agric. Food Chem. 2008, 56 (9), 3350- 3356.

of flavonoids is explained by the mechanisms in which they are involved. Indeed, they are able to scavenge free radicals (Scheme 5) through formation of less reactive phenol based radical, or even quinones, and inhibit enzymes known to be involved in ROS generation such as microsomal monooxygenase, glutathione-S transferase or NADH oxidase by chelating the metal ions. Figure 69: Structures of flavonoids as anti-AD agents 253 Shimmyo, Y.; Kihara, T.; Akaike, A.; Niidome, T.; Sugimoto, H. Flavonols and Flavones as BACE-1 Inhibitors: Structure-Activity Relationship in Cell-Free, Cell-Based and in Silico Studies 256 Choi, Y.-T.; Jung, C.-H.; Lee, S.-R.; Bae, J.-H.; Baek, W.-K.; Suh, M.-H.; Park, J.; Park, C.-W.; Suh, S.-I. The Green Tea Polyphenol (-)-Epigallocatechin Gallate Attenuates β-Amyloid-Induced Neurotoxicity in Cultured Hippocampal Neurons. Life Sci. 2001, 70 (5), 603-614. 257 Wobst, H. J.; Sharma, A.; Diamond, M. I.; Wanker, E. E.; Bieschke, J. The Green Tea Polyphenol (-)-Epigallocatechin Gallate Prevents the Aggregation of Tau Protein into Toxic Oligomers at Substoichiometric Ratios. FEBS Lett. 2015, 589 (1), 77-83. αFigure 70: Structure of α-tocopherol It has been shown that vitamin E has a pivotal role in the CNS because of its antioxidant properties, indeed, it is able to scavenge free radicals via a hydrogen atom transfer between the molecule and the free radicals resulting to a non-radical product and a less reactive vitamin E radical able to interact with a reducing agent such as vitamin C or ubiquinol to restore vitamin E (Scheme 6) 258 . Moreover, a low concentration of vitamin E in the CNS has been proven to cause cellular atrophy and cognitive deficit induced by a decrease of dendritic branching of Purkinje neurons 259 . Scheme 6: Vitamin E radical oxygen species scavenging mechanism 260 258 Niki, E. Role of Vitamin E as a Lipid-Soluble Peroxyl Radical Scavenger: In Vitro and in Vivo Evidence. Free Radic. Biol. Med. 2014, 66, 3-12. 259 Ulatowski, L.; Parker, R.; Warrier, G.; Sultana, R.; Butterfield, D. A.; Manor, D. Vitamin E Is Essential for Purkinje Neuron Integrity. Neuroscience 2014, 260, 120-129. 260 Valko, M.; Izakovic, M.; Mazur, M.; Rhodes, C. J.; Telser, J. Role of Oxygen Radicals in DNA Damage and Cancer Incidence. Mol. Cell. Biochem. 2004, 266 (1/2), 37-56.

Table 2 )

 2 . IC50 on human cholinesterases was evaluated using the method of Ellman et al.267 .

	As previously reported 268 , capsaicin didn't show any inhibitory activity against AChE or BChE while
	huprine Y was a potent inhibitor of both cholinesterases with a high selectivity ratio of 169.2 in favour
	of AChE. The entire series displayed good inhibitory activities, especially compound 34 (entry 3), the
	only huprine-based hybrid functionalized in position 9 of the series, and 44c (entry 6) functionalized in
	position 12. Moreover, compound 34 (entry 3) showed an interesting result, indeed this compound is

Table 2

 2 Other properties of the conjugated MTDLs, capsaicin and huprine Y were assessed and reported in

	: In vitro evaluation of 34, 44a-e, 45a-b and 46 cholinesterases inhibition

Table 3 .

 3

	Entry	Compound	% inhibition	IC50	Pe
			(hBACE-1)	(µM)	(10 -6 cm s -1 )
				(DPPH)
	1	Capsaicin	na a	50.3 +/-8.3	10.3 +/-1.2 (CNS+)
	2	Huprine Y	na a	nd b	23.8 +/-2.7 (CNS+)
	3	34	na a	92.0 +/-12.4	8.8 +/-1.3 (CNS+)
	4	44a	55.6	68.5 +/-8.2	11.5 +/-1.1 (CNS+)
	5	44b	34.3	62.6 +/-11.6	14.9 +/-1.2 (CNS+)
	6	44c	10.6	55.4 +/-2.3	17.2 +/-1.0 (CNS+)
	7	44d	3.6	70.8 +/-20.8	20.1 +/-1.1 (CNS+)
	8	44e	23.7	64.6 +/-8.6	21.8 +/-1.3 (CNS+)
	9	46	na	71.4 +/-15.5	14.3 +/-0.3 (CNS+)
	10	45a	na	31.7 +/-8.7	16.8 +/-0.1 (CNS+)
	11	45b	21.6	54.6 +/-12.5	22.3 +/-1.1 (CNS+)

a not active b not determined

Table 3 :

 3

Other biological properties of 34, 44a-e, 45a-b and 46

Table 4 :

 4 Interactions of Huprine and 34 with the cholinesterases main residues 6.1. Crystal structure with TcAChE Crystal structure of TcAChE in complex with 34 with (Figure

  2. Nicotinic receptor 2.1. Overall structure nAChR are integral allosteric membrane proteins of 290 kDa made up of five subunits (identical or homologous) arranged symmetrically around an ionic channel 275 , in the context of my PhD and for the sake of clarity, only α7 nAChR's structure will be discussed, since this family of nAChR was chosen as a target for the second pharmacophore. Early electron microscopy observation of fish electric organ nAChR showed that the nAChR pentamer is a cylinder of 8 nm diameter and 16 nm length 276 . Even though no human α7 nAChR structure has been solved to date, the X-ray crystallography of AChBP structure gave a viable template of human nAChR 277 (Figure78). Indeed, AChBP, a soluble protein discovered in the freshwater snail Lymnaea stagnalis 278 , displays a high degree of conservation of amino acids present at the binding site 279 , despites a lower overall sequence identity with nAChRs (around 30%)280 . AChBP crystal structure shows that AChBP subunits are folded as β-sandwiches constituted by a 6-strand inner sheet and a 4-strand outer sheet connected by a conserved loop that includes two bridged cysteines, also found in all animal nAChRs 281 . Moreover, every AChBP molecule possesses 5 nicotinic ligand binding sites located between the subunits. Presence of a Lattice Structure in Membrane Fragments Rich in Nicotinic Receptor Protein from the Electric Organ of Torpedo Marmorata. FEBSLett. 1973, 33 (1), 109-113.277 Dellisanti, C. D.; Yao, Y.; Stroud, J. C.; Wang, Z.-Z.; Chen, L. Crystal Structure of the Extracellular Domain of NAChR Alpha1 Bound to Alpha-Bungarotoxin at 1.94 A Resolution. Nat. Neurosci. 2007, 10 (8), 953-962.

	278 Smit, A. B.; Syed, N. I.; Schaap, D.; van Minnen, J.; Klumperman, J.; Kits, K. S.; Lodder, H.; van der Schors, R. C.;
	van Elk, R.; Sorgedrager, B.; Brejc, K.; Sixma, T. K.; Geraerts, W. P. A Glia-Derived Acetylcholine-Binding Protein
	That Modulates Synaptic Transmission. Nature 2001, 411 (6835), 261-268.
	279 Shahsavar, A.; Gajhede, M.; Kastrup, J. S.; Balle, T. Structural Studies of Nicotinic Acetylcholine Receptors:
	Using Acetylcholine-Binding Protein as a Structural Surrogate. Basic Clin. Pharmacol. Toxicol. 2016, 118 (6), 399-
	407.

275

Hucho, F.; Changeux, J. P. Molecular Weight and Quaternary Structure of the Cholinergic Receptor Protein Extracted by Detergents from Electrophorus Electricus Electric Tissue. FEBS

Lett. 1973, 38 (1), 11-15.

276 Cartaud, J.; Benedetti, E. L.; Cohen, J. B.; Meunier, J. C.; Changeux, J. P.

280

Hansen, S. B.; Talley, T. T.; Radic, Z.; Taylor, P. Structural and Ligand Recognition Characteristics of an Acetylcholine-Binding Protein from Aplysia Californica. J.

Biol. Chem. 2004, 279 (23)

, 24197-24202.

281

Tasneem, A.; Iyer, L. M.; Jakobsson, E.; Aravind, L. Identification of the Prokaryotic Ligand-Gated Ion Channels and Their Implications for the Mechanisms and Origins of Animal Cys-Loop Ion Channels. Genome Biol. 2005, 6 (1), R4.

  Nickell, J. R.; Grinevich, V. P.; Siripurapu, K. B.; Smith, A. M.; Dwoskin, L. P. Potential Therapeutic Uses of Mecamylamine and Its Stereoisomers. Pharmacol. Biochem. Behav. 2013, 108, 28-43. 292 Changeux, J.-P. The Nicotinic Acetylcholine Receptor: A Typical 'Allosteric Machine.' Philos. Trans. R. Soc. B Biol. Sci. 2018, 373 (1749), 20170174. 293 Monod, J.; Wyman, J.; Changeux, J.-P. On the Nature of Allosteric Transitions: A Plausible Model. J. Mol. Biol.

1965, 12 (1), 88-118. 294 Changeux, J.-P.; Taly, A. Nicotinic Receptors, Allosteric Proteins and Medicine. Trends Mol. Med. 2008, 14 (3), 93-102. 295 Mulle, C.; Léna, C.; Changeux, J.-P. Potentiation of Nicotinic Receptor Response by External Calcium in Rat Central Neurons. Neuron 1992, 8 (5), 937-945.

Table 7 :

 7

		Compound	CLogP CLogD	TPSA	MW	HBD	pKa CNS MPO
					(Å 2 )	g/mol			score
	1	Rivastigmine	2.41	1.00	32.78	250.34	0	8.8	5.2
	2	(R)-75a	2.15	0.38	63.49	341.41	0	9.17	5.4
		and (R)-76a
	3	(R)-75b	2.86	1.10	63.49	369.47	0	9.17	5.4
		and (R)-76b
	4	(R)-75c	2.51	0.74	63.49	355.44	0	9.17	5.4
		and (R)-76c
	Result can be extended to the (S) enantiomers
	Physico-chemical properties obtained using Chemicalize.com

Physicochemical properties and CNS MPO score of Rivastigmine, (R)-75a-c and (R)-76a-c

  Based on the work ofBerdini et al., 3-aminoquinuclidine 81 was obtained through a one steps process: reductive amination of commercially available 3-quinuclidinone 80, with a large excess of ammonium formate and imine reduction achieved by catalytic hydrogenation (Pd/C) in order to obtain the primary amine 81 in a quantitative yield330 . The second step consisted in a diazotransfer reaction to form the azide from the amine. Freshly prepared triflyl azide as a transfer agent was used, in presence of potassium carbonate through a Cu(II) salts331,332 catalysed process (Scheme 19). Triflyl azide was prepared according to the protocol described ofTitz et al. 333 who developed a safe method to synthesize this potentially explosive reagent. Indeed, by replacing the dichloromethane as solvent by

	5.3.2. Azide Synthesis
	5.3.2.1. Racemic azidoquinuclidine synthesis
	330 Berdini, V.; Cesta, M. C.; Curti, R.; D'Anniballe, G.; Bello, N. D.; Nano, G.; Nicolini, L.; Topai, A.; Allegretti, M. A
	Modified Palladium Catalysed Reductive Amination Procedure. Tetrahedron 2002, 58 (28), 5669-5674.
	331 Alper, P. B.; Hung, S.-C.; Wong, C.-H. Metal Catalyzed Diazo Transfer for the Synthesis of Azides from Amines.
	Tetrahedron Lett. 1996, 37 (34), 6029-6032.

toluene, the formation of hazardous side products such as azidochloromethane and diazidomethane could be avoided. We managed to obtain the racemic 3-azidoquinuclidine 77, in pure form, yet in a modest isolated yield, since even though most of the organic azides are stable in various conditions, compounds with multiple azide functions, or displaying low molecular weight like compound 77 tend to be volatile or even explosive as was proven by

Bräse et al. 334

Thus, in our hands, azide 77 was favourably directly used in the next steps without purification. Scheme 19: Synthesis of 77 The mechanism of the diazotransfer reaction converting amine 81 into azide 77 has been studied by different groups. Pandiakumar et al. showed that the mechanism was not a simple azide (-N3) transfer 332 Nyffeler, P. T.; Liang, C.-H.; Koeller, K. M.; Wong, C.-H. The Chemistry of Amine-Azide Interconversion: Catalytic Diazotransfer and Regioselective Azide Reduction. J. Am. Chem. Soc. 2002, 124 (36), 10773-10778. 333 Titz, A.; Radic, Z.; Schwardt, O.; Ernst, B. A Safe and Convenient Method for the Preparation of Triflyl Azide, and Its Use in Diazo Transfer Reactions to Primary Amines. Tetrahedron

Lett. 2006, 47 (14)

, 2383-2385.

334

Bräse, S.; Gil, C.; Knepper, K.; Zimmermann, V. Organic Azides: An Exploding Diversity of a Unique Class of Compounds. Angew.

Chem. Int. Ed Engl. 2005, 44 (33), 5188-5240.

  enantiomers values are reported in the table.

							Carbamate -
	Entry	Compound	Relative position of the	R1	R2	Ebind	Ser203
			carbamate			(kcal/mol)	distance (Å)
	1	Rivastigmine	-meta	Me	Et	-7.5	8.7
	2	(R)-96a	-meta	Me	Me	-12.7	5.0
	3	(R)-96b	-meta	Et	Et	-12.2	5.3
	4	(R)-96c	-meta	Me	Et	-12.3	5.0
	5	(R)-97a	-para	Me	Me	-12.4	6. 8
	6	(R)-97b	-para	Et	Et	-12.3	6.9
	7	(R)-97c	-para	Me	Et	-12.4	6.9
	8	(R)-98a	-meta	Me	Me	-13.2	5.0
	9	(R)-98b	-meta	Et	Et	-12.2	4.9
	10	(R)-98c	-meta	Me	Et	12.5	4.9
	11	(R)-99a	-para	Me	Me	-12.3	6.4
	12	(R)-99b	-para	Et	Et	-12.2	6.9
	13	(R)-99c	-para	Me	Et	-12.2	6.5

Table 15

 15

	Entry	Compound	CLogP	CLogD	TPSA	MW	HBD	pKa CNS MPO
					(Å 2 )	g/mol			score
	1	(R)-75a and (R)-76a	2.15	0.38	63.49	341.41	0	9.17	5.4
		(Reminder)
	2	(R)-96a and (R)-97a	3.80	2.03	63.49	417.51	0	9.17	4.6
	3	(R)-96b and (R)-97b	4.51	2.74	63.49	445.56	0	9.17	3.7
	4	(R)-96c and (R)-97c	4.15	2.39	63.49	431.54	0	9.17	4.1
	5	(R)-98a and (R)-99a	3.80	2.03	63.49	417.51	0	9.17	4.6
	6	(R)-98b and (R)-99b	4.51	2.74	63.49	445.56	0	9.17	3.7
	7	(R)-98c and (R)-99c	4.15	2.39	63.49	431.54	0	9.17

(results can be extended for the respective (S)-enantiomers). It can be observed that adding a phenyl ring to the structure lowers the score compared to the first series derivatives and four of the compounds (entry 3 and 6) obtained a low CNS MPO score under 4. This decrease could be explained by the LogP and LogD values drastic increase, indeed, a high cLogP (or cLogD) is an indicator of the lipophilic character's increase for the MTDL.

However, Waring has shown that the theoretical optimal range of lipophilicity (logD value) was situated between 1 and 3 345 , granting our new MTDLs derivative with good physicochemical profiles even if their CNS MPO scores are lower than expected.

Table 17 :

 17 Global yields obtained for the 10 out of 12 targeted MTDLs

  The racemic 3-aminoquinuclidine 81 and the desired alkyne 79a were set in

	presence of the diazo transfer agent (TfN3) with two catalytic sources of copper at two different
	oxidative states, Cu(II) and Cu(0), namely CuSO4 and Cu powder, during 30 min at 80°C, under
	347 Beckmann, H. S. G.; Wittmann, V. One-Pot Procedure for Diazo Transfer and Azide-Alkyne Cycloaddition:
	Triazole Linkages from Amines. Org. Lett. 2007, 9 (1), 1-4.
	348 Smith, N. M.; Greaves, M. J.; Jewell, R.; Perry, M. W. D.; Stocks, M. J.; Stonehouse, J. P. One-Pot, Three-
	Component Copper-Catalysed 'Click' Triazole Synthesis Utilising the Inexpensive, Shelf-Stable Diazotransfer

Reagent Imidazole-1-Sulfonyl Azide Hydrochloride. Synlett 2009, 2009 (9), 1391-1394. 349 Maisonneuve, S.; Xie, J. One-Pot Synthesis of 1,4-Disubstituted 1,2,3-Triazoles from Aldehydes and Amines.

Synlett 2009, 2009 (18)

, 2977-2981.

350

Johansson, J. R.; Lincoln, P.; Nordén, B.; Kann, N. Sequential One-Pot Ruthenium-Catalyzed Azide-Alkyne Cycloaddition from Primary Alkyl Halides and Sodium Azide. J. Org. Chem. 2011, 76

(7), 2355-2359.

Table 20 :

 20 All physico-chemical properties were obtained from chemicalize.com

		Compound CLogP CLogD	TPSA	MW	HBD	pKa	CNS MPO
					(Å 2 )	g/mol			score
	1	Donepezil	4.21	2.48	38.77	379.5	0	9.12	4.4
	2	(R)-128	4.83	1.23	54.26	483.66	0	9.5	3.5
	3	(R)-129	3.69	1.44	57.50	484.65	0	9.18	4.2
	4	(R)-130	4.83	1.70	54.26	483.66	0	9.34	3.5
	5	(R)-131	3.69	1.89	57.50	484.65	0	8.72	4.4

Physicochemical properties and CNS MPO scores evaluated for compounds (R)-128, (R)-129, (R)-130 and (R)-131

366

Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Central Nervous System Multiparameter Optimization Desirability: Application in Drug Discovery. ACS Chem. Neurosci. 2016, 7 (6), 767-775. 367 Wager, T. T.; Hou, X.; Verhoest, P. R.; Villalobos, A. Moving beyond Rules: The Development of a Central Nervous System Multiparameter Optimization (CNS MPO) Approach To Enable Alignment of Druglike Properties. ACS Chem. Neurosci. 2010, 1 (6), 435-449.

Table 21 :

 21

		Compound	LogP	pKa	fu	LogPS	LogBB	Log(PS*fu)	Brain
									penetration
									for CNS
									activity
	1	(R)-128	4.10	9.28 0.036	-1.6	0.20	-3.2	Sufficient
	2	(R)-129	2.85	9.28	0.16	-2.1	0.16	-3.1	Sufficient
	3	(R)-130	4.03	9.30 0.044	-1.6	0.25	-3.2	Sufficient
	4	(R)-131	2.78	9.30	0.17	-2.2	0.15	-3.1	Sufficient

Physicochemical properties determined with Percepta software for (R)-128, (R)-129, (R)-130 and (R)-131

Table 24 :

 24 All physico-chemical properties were obtained from chemicalize.com

	Entry	Compound CLogP CLogD	TPSA	MW	HBD	pKa	CNS MPO
					(Å 2 )	g/mol			score
	1	Donepezil	4.21	2.48	38.77	379.5	0	9.12	4.4
	2	(R)-132	4.98	1.49	54.26	495.67	0	9.45	3.3
	3	(R)-133	3.83	1.57	57.50	496.66	0	9.18	4.0
	4	(R)-134	4.98	1.96	54.26	495.67	0	9.26	3.4
	5	(R)-135	3.83	2.04	57.50	496.66	0	8.72	4.2

Physicochemical properties and CNS MPO scores of (R)-132, (R)-133, (R)-134 and (R)-135 3.2.3. Percepta software CNS activity prediction Passive BBB penetration ability for compounds (R)-132, (R)-133, (R)-134 and (R)-135 has been predicted based on calculated physicochemical properties reported in Table 25. It is important to note that calculated LogP and most basic pKa values are in accordance with the values used to previously evaluate the CNS MPO scores and despite a lower CNS MPO score, compounds (R)-132 (entry 1) and (R)-134 (entry 3) obtained more encouraging prediction in this study, enough to be considered CNS active.

Table 25 :

 25

Physicochemical properties determined with Percepta software for (R)-132, (R)-133, (R)-134 and (R)-135

  376 van Greunen, D. G.; Cordier, W.; Nell, M.; van der Westhuyzen, C.; Steenkamp, V.; Panayides, J.-L.; Riley, D. L. Targeting Alzheimer's Disease by Investigating Previously Unexplored Chemical Space Surrounding the Cholinesterase Inhibitor Donepezil. Eur. J.Med. Chem. 2017, 127, 671-690.

Table 26 .

 26 377 Mercey, G.; Verdelet, T.; Saint-André, G.; Gillon, E.; Wagner, A.; Baati, R.; Jean, L.; Nachon, F.; Renard, P.-Y. First Efficient Uncharged Reactivators for the Dephosphylation of Poisoned Human Acetylcholinesterase. Chem.

  378 Anderson, K. W.; Buchwald, S. L. General Catalysts for the Suzuki-Miyaura and Sonogashira Coupling Reactions of Aryl Chlorides and for the Coupling of Challenging Substrate Combinations in Water. Angew. Chem. Int. Ed. 2005, 44 (38), 6173-6177. 379 Chinchilla, R.; Nájera, C. The Sonogashira Reaction: A Booming Methodology in Synthetic Organic Chemistry † . Chem. Rev. 2007, 107 (3), 874-922. 380 Zou, G.; Zhu, J.; Tang, J. Cross-Coupling of Arylboronic Acids with Terminal Alkynes in Air. Tetrahedron Lett. 2003, 44 (48), 8709-8711.

  The synthesis of Donepezil based MTDLs began with the preparation of the Donepezil moiety 154. To achieve the formation of this moiety, the first step was to prepare the aldehyde 159 from commercially available ethyl isonipecoate based on a synthesis previously developed in the team by Doctor Renou during his PhD 383 (Scheme 44). Jean, L.; Nachon, F.; Renard, P.-Y. Synthesis and in Vitro Evaluation of Donepezil-Based Reactivators and Analogues for Nerve Agent-Inhibited Human Acetylcholinesterase. RSC Adv. 2016, 6 (22), 17929-17940.Alcohol 157 was obtained from ethyl isonipecoate 156 through the reduction of the ethyl ester using LiAlH4 in THF with an excellent 92% yield. Next, the secondary amine of compound 157 was protected as a Boc-carbamate to give compound 158 in 50% yield. This alcohol was 158 finally oxidized in aldehyde 159 through the use of 2-iodoxybenzoic acid giving the unstable aldehyde 159, which was used without further purification in the following steps of the synthesis. To obtain the Donepezil moiety 154 an aldolization-crotonization process was performed between aldehyde 159 and 5bromoindanone 160 in presence of an aqueous solution of sodium hydroxide as a base, the required water / DCM mixture needed in this process required the additional use of tetra-n-butylammonium bromide as a phase transfer catalyst. The aldolization-crotonization process gave compound 161 in 44% yield. With this Donepezil moiety 161 in hands, a Sonogashira cross coupling reaction was assayed, yet, as shown previously with the acyclic MTDL series, despite various attempts, compound 162 could not be obtained and only ethynyltrimethylsilane homocoupling product was observed along with the recovery of the starting material 161 (Scheme 45).As a model compound, 5-bromoindanone 160 was used in this study to evaluate the reaction conditions for the Sonogashira cross-coupling step required to access to alkyne 154 or 155. Main results are reported on Table27.obtained could be estimated to reach only around 40% which precluded this synthetic route and required the implementation of another synthetic strategy. PPh3)2 in a mixture of dimethylformamide and triethylamine, and deprotected in presence of potassium carbonate, giving the final alkynes 166 and 167 in decent yields. The two alkynes were prepared in 3 steps with overall yields of respectively 46% and 37% (Scheme 48).

	4.2.2. New approach to (R)-132 and (R)-134
	4.2.2.1. Second retrosynthetic strategy to access to (R)-132 and (R)-134
	As for the synthesis of MTDLs (R)-128 and (R)-130, a new strategy was designed to tackle the challenge
	of obtaining combined MTDLs (R)-132 and (R)-134 (Scheme 47). The Copper(I) catalysed Huisgen
	4.2.1.2. Synthesis of the Donepezil moiety 154 or 155 Scheme 44: Synthesis of 159 383 Renou, J.; Dias, J.; Mercey, G.; Verdelet, T.; Rousseau, C.; Gastellier, A.-J.; Arboléas, M.; Touvrey-Loiodice, M.; Baati, R.; Scheme 45: Attempt synthesis of 162 4.2.1.3. Screening Sonogashira cross coupling reaction conditions on 5-bromoindanone derivatives. cycloaddition will be used at an earlier key step of the synthetic pathway between azide (R)-77 and alkynes 166 and 167, followed by an aldol based coupling of the obtained compounds (R)-164 and (R)-165 bearing a triazole with aldehyde 140. Scheme 47: New retrosynthesis strategy to (R)-132 and (R)-134 4.2.2.2. Synthesis of alkynes 166 and 167 Phenols carried by commercially available 5-hydroxyindanone 168 and 6-hydroxyindanone 169 were activated as triflates in presence of trifluoromethanesulfonic anhydride in triethylamine and dichloromethane 381 to give compounds 170 and 171 in good yields. The newly obtained compounds were then coupled with ethynyltrimethylsilane via a conventional Sonogashira coupling catalysed by PdCl2(Scheme 48: Synthesis of 166 and 167 4.2.2.3. Attempted synthesis of a pivotal compound 172

Table 28 )

 28 .

	Entry	Compound	R	Relative position of	Result
				the R substituent
	1	170	OTf	Position 5	No desired
					product
	2	171	OTf	Position 6	No desired
					product
	3	163	Protected alkyne	Position 5	No desired
					product
	4	175	Protected alkyne	Position 6	No desired
					product

Table 28 :

 28 Assays to obtain piperazine based intermediates 176 and 177

  Learmonth, D. A.; Soares, D. S. P. M. Substituted Nitrated Catechols, Their Use in the Treatment of Some Central and Peripheral Nervous System Disorders and Pharmaceutical Compositions Containing Them. WO0198250 (A1),December 27, 2001.

Table 30 :

 30 In vitro evaluation of 34 In addition, crystal structures of TcAChE and hBChE in complex with compound 34 were obtained by our partner Dr. Jacques-Philippe Colletier, Dr. Nicolas Coquelle and Dr. Martin Weik at IBS (Grenoble),

		Compound	IC50	IC50	Selectivity	IC50	Pe
			(nM)	(nM)	ratio	(µM)	(10 -6 cm s -1 )
			(hAChE)	(hBChE)		(DPPH)
	1	Huprine Y 1.07 +/-0.05	181 +/-15	169.2	nd b	23.8 +/-2.7 (CNS+)
	2	Capsaicin	>200 000	na a	/	50.3 +/-8.3	10.3 +/-1.2 (CNS+)
	3	34	1.06 +/-0.26	7.3 +/-0.6	6.9	92.0 +/-12.4	8.8 +/-1.3 (CNS+)
	a not active; b not determined

  ). As MTDLs targeting AChE and α7 nAChRs simultaneously, these two molecules represented in Figure141in appeared to have the most promising dual action.

	Entry	Compound	Ki (nM)	IC50 hAChE (µM) a IC50 hBChE (µM) b
			α7 nAChRs
	1	(R)-69 (reminder)	8 ± 4	nd c	nd
	2	Rivastigmine	nd	48	54
	3	(R)-75a	0.35 ± 0.05	64.2 + 1.5	191 + 32
	4	(R)-76a	1.5 + 0.4	5.32 + 0.67	2.74 + 0.08
	5	(R)-75b	1 + 0.2	4.98 + 0.21	nd
	6	(R)-76b	2.1 + 1	177 + 34	nd
	7	(R)-75c	2.3 + 1	56.5 + 1.9	nd
	8	(R)-76c	1.8 + 0.2	nd	nd

  of Ellman et al 393 . AChE stock solution was prepared by dissolving human recombinant AChE (E.C.3.1.1.7) lyophilized powder (Sigma, Italy) in 0.1 M phosphate buffer (pH = 8.0) containing Triton X-100 0.1%. Stock solution of BChE (E.C. 3.1.1.8) from human serum (Sigma, Italy) was prepared by dissolving the lyophilized powder in an aqueous solution of gelatine 0.1%. Stock solutions of the tested

compounds (1 mM for compounds in chapter I and 3 nM for compounds in chapter II) were prepared in MeOH and diluted in MeOH. Five increasing concentrations of each inhibitor were used, able to give an inhibition of the enzymatic activity in the range of 20-80%. The assay solution consisted of a 0.1 M phosphate buffer pH 8.0, with the addition of 340 M 5,5'-dithio-bis(2-nitrobenzoic acid), 0.02 unit/mL of human recombinant AChE, or BChE from human serum and 550 M of substrate (acetylthiocholine iodide (ATCh) or butyrylthiocholine iodide (BTCh), respectively). Assay solutions with and without

  Di et al. 394 was used to assess the brain penetration of the HC hybrids. The in vitro permeability (Pe) of the synthesized and fourteen commercial drugs through lipid extract of porcine brain membrane was determined using a mixture of phosphate-buffered saline (PBS)/EtOH 70:30. Assay validation was made by comparison of the experimental and reported Pe values of the commercial drugs, which showed a good correlation: Pe (exp) = 1.4974 Pe (lit) -0.8434 (R 2 = 0. 9428). From this equation and taking into account the limits established byDi et al. for BBB permeation, the following ranges of The filters were washed twice with 3 mL of 4 °C buffer, and the residual radioactivity was measured in a gcounter (2480 Gamma counter Wizard, Perkin Elmer). The IC50 values were determined graphically for each compound and the Ki calculated according to Cheng & Prusoff 395 . to afford azidoquinuclidine 77. Then, to a solution of azidoquinuclidine 77 (1 equiv) in THF was added terminal alkyne (1 equiv) in t-BuOH followed by the addition of CuSO4.5H2O (0.05 equiv) in water and sodium ascorbate (0.01 equiv) (THF/Water/t-BuOH 1/1/1 v/v/v 0.1 M). The reaction mixture was stirred at room temperature for 12h. The crude product was poured into water, extracted with DCM

	permeability were established: compounds of high BBB permeation (CNS+): Pe (10 -6 cm s -1 ) > 5.1; compounds of low BBB permeation (CNS-): Pe (10 -6 cm s -1 ) < 2.1, and compounds of uncertain BBB permeation (CNS±): 5.1 > Pe (10 -6 cm s -1 ) > 2.1. nAChRs binding affinity Assay Animals were killed by decapitation on the day of the assay and both frontal cortices of each animal were removed on ice and weighed (2 rats were used for each experiment). The tissue was homogenized in 10 vol of a pH 7.4 HEPES 15 mM buffer containing 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2 and 1.8 mM CaCl2 using an Ultraturrax T25. After 45.000x g centrifugation at 4°C for 10 min (J2-pH 7.4 TriseHCl buffer (50 mM TriseHCl, 120 mM NaCl, 5 mM KCl, 1mM MgCl2 and 2.5mM CaCl2) for 3h at 22°C. After incubation, samples were diluted in 3 mL of TriseHCl buffer at 4°C and then rapidly filtered through Whatman GF/C fiber filters soaked with 0.05% polyethylenimine (Sigma, St Quentin-concentrated under vacuo. The residue obtained was purified by column chromatography Fallavier, France). (4x). The combined organic layers were washed with water and brine, dried over Na2SO4, and (DCM/MeOH 80/20, v/v).

394

Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. High Throughput Artificial Membrane Permeability Assay for Blood-Brain Barrier. Eur. J.

Med. Chem. 2003, 38 (3), 223-232.

hybrids 21M/E, Beckman), the supernatant was eliminated, and the pellet was suspended in 2 mL of the same buffer. The protein concentration was measured according to Bradford using bovine serum albumin as standard. For competition studies, [ 125 I]α-bungarotoxin (2 nM) was incubated in the presence or absence of each tested compound (10 -6 to 10 -10 M) with 25 mg protein in a total volume of 1 mL in a

  in acetic acid(15 mL) was added hydroxylamine hydrochloride(2.05 g, 29.55 mmol, 1.5 equiv). The reaction mixture was stirred at 120°C for 1h. The white solution became orange. After cooling down to room temperature, the solution was poured into Et2O. Then, it was washed with water and aq. 5% NaOH, and extracted with Et2O. The organic layers were dried over MgSO4 and concentrated under reduced pressure, after what, the crude product was recrystallized from toluene to give 51 (2.74 g, 93%) as a white solid.

	1 H NMR (300 MHz, CDCl3) δ 7.17 (dd, J = 1.2 Hz, 8.1 Hz, 1H), 7.02 (s, 1H), 6.91 (d, 8.4 Hz, 1H), 6.18 (br,
	OH), 3.86 (s, 3H).

NMR spectrum are in accordance with those reported previously: Thompson, R.; Doggrell, S.; Hoberg, J. O. Potassium Channel Activators Based on the Benzopyran Substructure: Synthesis and Activity of the C-8 Substituent. Bioorg.

Med. Chem. 2003, 11 (8), 1663- 1668.

13 C NMR (75 MHz, CDCl3) δ 150.

  To a solution of 61(2.82g, 14.41 mmol) in dry THF (58 mL), NaHMDS (0.6 M in toluene, 64.83 mL, 26.78 mmol, 2.5 equiv) was added dropwise at -78°C. The solution was stirred at -78°C for 1.5h. Then, the

	1 H NMR (300 MHz, CDCl3) δ 3.95-3.88 (m, 2H), 3.86-3.80 (m, 2H), 2.53-2.44 (m, 4H), 2.34 (dd, J = 18.4,
	6.6 Hz, 2H), 1.88-1.72 (m, 6H).
	Spectral data are in accordance with those reported previously:
	Tomizawa, M.; Shibuya, M.; Iwabuchi, Y. Highly Enantioselective Organocatalytic Oxidative Kinetic
	Resolution of Secondary Alcohols Using Chirally Modified AZADOs. Org. Lett. 2009, 11 (8), 1829-1831.
	Spiro[bicyclo[3.3.1]non[6]ene-3,2'-[1,3]dioxolan]-7-yl trifluoromethanesulfonate (62)
	1 H NMR (300 MHz, CDCl3) δ 12.34 (s, 2H), 3.84 (s, 6H), 3.77 (s, 6H), 3.30 (s, 2H), 3.23 (t, J = 3.3 Hz, 2H),
	1.94 (t, J = 3.3 Hz, 2H) ; 13 C NMR (75 MHz, CDCl3) δ 171.8, 170.8, 168.1, 102.0, 52.7, 52.2, 50.3, 30.1,
	22.5; GC-MS (m/z) 384 [M] + .

solution of N-phenylbis(trifluoromethane) sulfonimide

(6.5 g, 18.

2 mmol, 1.7 equiv.) was added and the reaction mixture was stirred at -78 °C for 5 h, quenched with a sat. aq. NaHCO3 and allowed warm up to rt and extracted with Et2O. The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under vacuo. The crude product was purified by flash chromatography on silica gel (PE/EtOAc, 95/5, v/v) to yield desired product 62 as a colourless oil (3.99 g, 85%).

1

H NMR (300 MHz, CDCl3) δ 5.80

(d, J = 10.2 Hz, 1H), 3.96 (m, 4H), 2.66 (m, 3H), 2.32 (d, J = 17.5 Hz, 1H), 1.72-1.84 (m, 5H), 1.62 (dt, J = 12.8, 2.7 Hz, 1H)

;

Butyl-4-(spiro[bicyclo[3.3.1]non[6]ene-3,2′-[1,3]dioxolane]-7-yl)butanoate (63)

  this solution was stirred at 0 °C for 30 min and at 80 °C for 2 h. Then, this solution was cooled down to rt and quenched with H2O (3 mL). The solution of 62(3.99 g, 12.1 mmol, 1 equiv.) in dry THF (4 mL) was added and the mixture was degassed and backfilled with Ar during 5 min, then catalyst PdCl2(dppf)•DCM (0.99 g, 0.121 mmol, 0.1 equiv.) and K3PO4(3.85 g, 18.15 mmol, 1.5 equiv.) were introduced. This mixture was heated at 80 °C overnight. After cooling down to rt, H2O and EtOAc were added to the reaction mixture, and residues were extracted with EtOAc and washed with H2O and brine, dried with MgSO4 and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel (PE/EtOAc, 90/10, v/v) to yield desired product 63 as a yellow oil(2.80 g, 72%).

	1 H NMR (300 MHz, CDCl3) δ 5.45 (d, J = 5.8 Hz, 1H), 3.95-3.65 (m, 4H), 2.46-2.07 (m, 5H), 1.92 (m, 3H),
	1.84-1.51 (m, 8H), 1.44 (s, 9H);

; MS(CI) (m/z) 329 (100) [M+H] +

tert-

In the two-necked dry round-bottomed flask under Ar atmosphere, 9-BBN (0.5 M in THF, 58.8 mL, 29 mmol, 2.4 equiv.) was added dropwise at 0 °C to the alkene derivative

(2.34 mL, 14.5 mmol, 1.2 equiv.)

and 13 C NMR (75 MHz, CDCl3) δ 173.

  The crude product was purified by flash column chromatography on silica gel (PE /EtOAc 60/40, v/v) to give the desired product 65 as a colourless oil(1.46 g, 81%).

	1 H NMR (300 MHz, CDCl3) δ 5.42 (d, J = 5.7 Hz, 1H), 3.69-3.51 (m, 2H), 2.67 (s, 1H), 2.58 (s, 1H), 2.47

(m, 2H), 2.

35-2.19 (m, 4H)

, 2.03-1.78 (m, 5H), 1.53-1.34 (m, 4H); 13 C NMR (75 MHz, CDCl3) δ 213.0,

9-(4-Azidobutyl)-3-chloro-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinolin-12- amine (48)

  To a solution of 68 (790 mg, 1.88 mmol, 1 equiv) in dry DMF(19 mL), NaN3 (366.73 mg, 5.64 mmol, 3 equiv) was added. The reaction mixture was stirred at 90°C overnight. After completion determined by TLC, the mixture was allowed to cool down to rt and quenched with H2O. Then, it was extracted with EtOAc. The organic layers were washed with H2O, brine and dried over MgSO4, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel 2H), 2.93 (d, J = 17.5 Hz, 1H), 2.77-2.73 (m, 1H), 2.45 (dd, J = 16.9, 4.5 Hz, 1H), 2.02-1.94 (m, 3H), 1.82 (t, J = 6.6 Hz, 2H),1.35-1.28 (m, 4H); HRMS (ESI + ) (m/z) calcd. for C20H23 35 ClN5 + [M+H] + 368.1636 found 368.1632. Spectral data are in accordance with those reported previously: Ronco, C.; Carletti, E.; Colletier, J. P.; Weik, M.; Nachon, F.; Jean, L.; Renard, P. Y. Huprine Derivatives as Sub-Nanomolar Human Acetylcholinesterase Inhibitors: From Rational Design to Validation by X-Ray Crystallography. ChemMedChem 2012, 7 (3), 400-405.

(PE/EtOAc, 60/40, v/v then EtOAc/MeOH 9/1 v/v) to give 48 (414 mg, 60%) as an orange solid. 1 H NMR (300 MHz, CDCl3) δ 7.84 (d, J = 2.1 Hz, 1H), 7.63 (d, J = 9.0 Hz, 1H), 7.27 (dd, J = 9.0, 2.1 Hz, 1H), 5.52

(d, J = 5.3 Hz, 1H), 4.82 (br.s), 3.21-3.19 (m, 1H), 3.11 (dd, J = 17.5, 5.6 Hz, 1H), 3.10-3.07 (m,

  carbons are missing despite an extended acquisition time); HRMS (ESI + ) : (m/z) calcd. for C12H14NO2 + [M+H + ] 204.1032 found 204.1032.

  . HRMS (ESI + ) (m/z) calcd. for C12H14NO2 + [M+H + ] 204.1032 found 204.1030.

  (two carbons are missing despite an extended acquisition time); HRMS (ESI + ) (m/z)calcd. for C18H24N5O2 + [M+H] + 342.1925 found 342.1924; mp = 170 °C CDCl3) δ 7.83 (d, J = 9 Hz, 2H), 7.78 (s, 1H), 7.19(d, J = 9 Hz, 2H), 4.65 (s, 1H), 3.71 (m, 1H), 3.45 (m, 6H), 2.92 (m, 4H), 2.20 (m, 2H), 1.66 (m, 3H), 1.24 (m, 5H); 13 C NMR(75 MHz, CDCl3)

	δ 154.

1 H NMR (300 MHz,

1-bromo-3-ethynylbenzene (101a) 101a was

  obtained from 102a (500mg, 1.45 mmol, 1 equiv) following the general procedure E and isolated as a colorless oil (131 mg, 50%). CDCl3) δ 7.64 (t, J = 1.7 Hz, 1H), 7.49 (ddd, J = 8.1, 1.9, 1.1 Hz, 1H), 7.45 -7.38 (m, 1H), 7.19 (t, J = 7.9 Hz, 1H),3.12 (s, 1H).Spectral data are in accordance with those reported previously: Malamas, M. S.; Barnes, K.; Johnson, M.; Hui, Y.; Zhou, P.; Turner, J.; Hu, Y.; Wagner, E.; Fan, K.; Chopra, R.; Olland, A.; Bard, J.; Pangalos, M.; Reinhart, P.; Robichaud, A. J. Di-Substituted Pyridinyl Aminohydantoins as Potent and Highly Selective Human β-Secretase (BACE1) Inhibitors. Bioorg. Med.

	1 H NMR (300 MHz,

Chem. 2010, 18 (2), 630-639.

  MeOD) δ8.45 (s, 1H), 7.98 (t, J = 1.7 Hz, 1H), 7.79 -7.72 (m, 1H), 7.43 (ddd, J = 8.0, 1.9, 1.0 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 4.82 (s, 1H),3.84 -3.68 (m, 1H), 3.59 -3.44 (m, 1H), 3.30 (s, 1H), 3.08 -2.87 (m, 3H), 2.25 (d, J = 2.6 Hz, 1H), 1.86 (s, 2H), 1.56 (s, 2H).

1 H NMR (300 MHz, Spectral data are in accordance with those reported previously: Ouach, A.; Vercouillie, J.; Bertrand, E.; Rodrigues, N.; Pin, F.; Serriere, S.; Boiaryna, L.; Chartier, A.; Percina, N.; Tangpong, P.; Gulhan, Z.; Mothes, C.; Deloye, J.-B.; Guilloteau, D.; Page, G.; Suzenet, F.; Buron, F.; Chalon, S.; Routier, S. Bis(Het)Aryl-1,2,3-Triazole Quinuclidines as Α7 Nicotinic Acetylcholine Receptor Ligands: Synthesis, Structure Affinity Relationships, Agonism Activity, [18F]-Radiolabeling and PET Study in Rats. Eur. J.

Med. Chem. 2019, 179, 449-469.

  18 mmol, 1.2 equiv) following general procedure F and isolated as a yellow oil(45.19 mg, .51 (s, 1H), 7.80 (dd, J = 13.3, 7.8 Hz, 1H), 7.56 (d, J = 7.0 Hz, 1H), 7.51 - 7.38 (m, 1H), 7.26 (t, J = 7.8 Hz, 1H), 7.17 -7.08 (m, 2H), 6.83 -6.77 (m, 1H), 5.01 -4.91 (m, 1H),3.88 (dd, J = 14.2, 4.7 Hz, 1H), 3.66 -3.53 (m, 1H), 3.35 (s, 1H), 3.29 -3.15 (m, 1H), 3.10 -3.01 (m, 3H), 2.42 -2.32 (m, 2H), 1.96 (dd, J = 8.5, 4.1 Hz, 2H), 1.76 -1.55 (m, 2H);

	87%).

1 H NMR (300 MHz, MeOD) δ 813 C NMR (75 MHz, MeOD) δ 159.0,

  11 mmol, 1.2 equiv) following general procedure F and isolated as a yellow oil(16.63 mg, 53%). .67 -8.42 (m, 1H), 8.05 (dd, J = 8.5, 2.7 Hz, 1H), 7.86 (ddd, J = 12.9, 8.2, 4.8 Hz, 1H), 7.80 -7.51 (m, 4H), 7.50 -7.41 (m, 1H), 5.06 -4.97 (m, 1H), 4.00 -3.85 (m, 1H), 3.67 (ddd, J = 12.9, 8.5, 2.6 Hz, 1H), 3.34 (d, J = 5.6 Hz, 1H), 3.11 (dd, J = 15.9, 7.0 Hz, 3H), 2.45 -2.37 (m, 1H), 2.07

1 H NMR (300 MHz, MeOD) δ 8-1.93 (m, 2H), 1.71 (d, J = 4.1 Hz, 2H), 1.26 (d, J = 2.8 Hz, 2H); 13 C NMR (75 MHz, MeOD) δ 133.

  , 3.15 (s, 2H), 3.09 -2.96 (m, 6H), 2.41 -2.30 (m, 1H), 2.01 -1.89 (m, 2H), 1.66 (d, J = 12.3 Hz, 3H); 13 C

NMR (75 MHz, MeOD) δ 148.

  CDCl3) δ 9.63 (s, 1H), 7.32 -7.27 (m, 5H), 3.49 (s, 2H), 2.85 -2.76 (m, 2H), 2.27 - 2.19 (m, 1H), 2.09 (td, J = 11.2, 2.5 Hz, 2H), 1.91 -1.82 (m, 2H), 1.74 -1.59 (m, 2H). Spectral data are in accordance with those reported previously: Woo Lee, H.; Leol Yoo, C.; Uk Lee, S.; Kwon Kang, S. Effective Procedure for the Oxidative Cleavage of Olefins by OsO4-NaIO4. HETEROCYCLES 2009, 78 (7), 1837.

1

H NMR (300 MHz,

Wong, G. T.; Manfra, D.; Poulet, F. M.; Zhang, Q.; Josien, H.; Bara, T.; Engstrom, L.; Pinzon-Ortiz, M.; Fine, J. S.; Lee, H.-J. J.; Zhang, L.; Higgins, G. A.; Parker, E. M. Chronic Treatment with the γ-Secretase Inhibitor LY-411,575 Inhibits β-Amyloid Peptide Production and Alters Lymphopoiesis and Intestinal Cell Differentiation. J.Biol. Chem. 2004, 279 (13), 12876-12882.

Rogers, K.; Felsenstein, K. M.; Hrdlicka, L.; Tu, Z.; Albayya, F.; Lee, W.; Hopp, S.; Miller, M.-J.; Spaulding, D.; Yang, Z.; Hodgdon, H.; Nolan, S.; Wen, M.; Costa, D.; Blain, J.-F.; Freeman, E.; De Strooper, B.; Vulsteke, V.; Scrocchi, L.; Zetterberg, H.; Portelius, E.; Hutter-Paier, B.; Havas, D.; Ahlijanian, M.; Flood, D.; Leventhal, L.; Shapiro, G.; Patzke, H.; Chesworth, R.; Koenig, G. Modulation of γ-Secretase by EVP-0015962 Reduces Amyloid Deposition and Behavioral Deficits in Tg2576 Mice. Mol. Neurodegener. 2012, 7, 61.

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Remerciements

Abbreviations

Synthesis of intermediate 65

The newly obtained diketone 60 was then, monoprotected as an acetal 61 in presence of boron trifluoride etherate and a large excess of ethylene glycol, the remaining ketone group was transformed into the corresponding vinyl triflate to obtain 62 in good yield (57% in two steps). To synthesise compound 61, we performed a Suzuki-Miyaura coupling reaction between 62 and the 9-BBN borate prepared in situ through an addition of 9-BBN on tert-butyl 3-butenoate, the coupling reaction was catalysed with a catalytic amount of Pd(dppf)Cl2 and performed in a mixture of THF and H2O. A reduction of the synthesized ester 63 with LiAlH4 in diethyl ether allowed us to obtain the corresponding alcohol 64, then, the cyclic acetal was deprotected with PTSA in acetone to give compound 65 in 5 steps with an overall yield of 33% (Scheme 13).

Scheme 13: Synthesis of 65 4.3.3. Synthesis of the huprine scaffold 67

Once we had compound 65 in hands, we were able to perform a Friedlander condensation with the 2amino-4-chlorobenzonitrile 66 in presence of AlCl3 in 1,2-dichloroethane to obtain the characteristic huprine scaffold 67 with a 40% yield.

Scheme 14: Synthesis of the huprine scaffold 67 4.3.4. Final steps to obtain the azide 48

Once the newly obtained huprine scaffold's hydroxyl function was activated as a mesylate 68 with methanesulfonyl chloride and triethylamine in THF, we could perform a nucleophile substitution with sodium azide in DMF leading to the final azide 48 (Scheme 15). The huprine azide was obtained in 10 steps with an overall yield of 2% in a 600 mg scale. Yields obtained were in agreement with the process on racemic mixture, and we observed the same issues with the enantiopure compounds that were already observed with the racemic 3azidoquinuclidine 77 concerning their instability and volatility. For this reason, we will assume that the conversions of amines 81, (S)-81 and (R)-81 into the azide 77, (S)-77 and (R)-77 were total in the following steps of the synthesis.

Alkynes 78a-c and 79a-c synthesis

Once we managed to synthesize azides 77, (S)-77 and (R)-77, we had to work on the an easily feasible and convergent synthetic pathway to obtain the alkynes bearing a carbamate group. First, we had to synthesize one of the carbamates precursors, the methyl(ethyl) carbamoyl chloride, which was not commercially available (Scheme 24). In one easy step developed by Fuchs et al. 337 , we obtained 91c from triphosgene 89 and N-methylethanamine 90 in presence of sodium bicarbonate in a quantitative yield and without any purification required.

337 Fuchs, M.; Koszelewski, D.; Tauber, K.; Kroutil, W.; Faber, K. Chemoenzymatic Asymmetric Total Synthesis of (S)-Rivastigmine Using ω-Transaminases. Chem. Commun. 2010, 46 (30), 5500-5502.

Interaction with the main AChE residues

Due to the high similarities of the designed MTDLs, we will only discuss the results obtained for (R)-96a, (R)-97a, (R)-98a and (R)-99a compiled in Table 14. These results can be extended to the rest of the series.

Entry

R)-96a-c, (R)-97a-c, (R)-98a-c and (R)-99a-c

The results obtained and presented in Figure 110 were, as it could be expected, closed to the results obtained for the first series. Indeed, these calculations showed that all the MTDLs have promising predicted properties to cross the BBB and it appears that compounds carrying a carbamate substituted by two ethyl groups (R)-96b (and (R)-97b) and (R)-98b (and (R)-99b) (B and E Figure 110) could be the most viable compounds to penetrate the BBB.

Scheme 29: Synthesis of alkynes 101a and 101b

Liu et al. proposed a mechanism for the Corey-Fuchs reaction when TBAF is used as a base and in presence of water, according to their studies, the HO -appeared to come from the hydrolysis of TBAF (Scheme 30). Indeed, after optimisation of the reaction conditions, they highlighted the importance of water in the solvent mixture, and Katsumura also reported that TBAF.3H2O promoted the generation of terminal alkyne 101a over bromoalkyne 103 346 .

Scheme 30: Proposed mechanism for the Corey-Fuchs reaction under Liu et al. 346

reaction conditions

Once we had compounds 101a-b in hand, we were able to perform the Copper-catalysed Huisgen's cycloaddition with the (R)-azidoquinuclidine (R)-77, using the same reaction conditions we previously presented (Scheme 31) 341 . We obtained compounds (R)-105a and (R)-105b with poor to good yields.

Here again, the yields difference we obtained for the formation of the two compounds could not be explained rationally, apart from the fact that the azide is an unstable and volatile compound. for (R)-109, using tetrakis(triphenylphosphine)palladium 344 catalysis under microwave irradiations (Scheme 32). This optimized synthetic strategy allowed us to obtain readily the two desired compounds in good yields without any step optimisation.

Scheme 32: Suzuki-Myaura coupling reaction allowing access to (R)-108 and (R)-1098

Building on this success, we decided to synthesize compound (R)-110 and (R)-111 from (R)-105b using the same synthetic pathway (Scheme 33). Once again, we readily obtained the two key intermediates in good yields.

Scheme 33: Suzuki-Myaura coupling reaction allowing access to (R)-110 and (R)-111

The last step of our synthesis of this new series of MTDLs was the carbamate addition on the second phenyl ring (Scheme 34). By using the same conditions, we already optimized for the first MTDL series, we were able to obtain 6 new MTDLs bearing a quinuclidine fragment and the different carbamates derivatives.

Scheme 34: Last step of the synthesis to (R)-96a-c and (R)-97a-c

Due to the lack of time, we had to make a choice concerning the MTDLs to synthesize. We focused on the first series bearing the second phenyl ring in position 3, because the structure was the closest to the α7 nAChRs agonist structure developed by Pr. Routier's team. However, we wanted to design

MTDLs with the second phenyl in position 4 (Scheme 35) to be able to evaluate whether this difference in substitution position had an influence for the MTDLs biological activities but we obtained only 4 out

Entry

Table 23: Interactions predicted between rhAChE and (R)-132, (R)-133, (R)-134 and (R)-135

Concerning the interactions between compound (R)-132 and rhAChE (Figure 129), a potential πstacking interaction could be observed between the phenyl ring of the indanone and simultaneously Tyr341 and Trp286 (entry 1) and Tyr124 has been predicted to interact with the ketone fragment through H-bond interactions (entry 3) (Figure 129).

Figure 129: Representation of predicted interactions between rhAChE and (R)-132

Compound (R)-133's predicted interactions with rhAChE could be summarized as followed (Figure 130). π-stacking was foreseen between the ligand and various residues such as Trp286 and Tyr341

Scheme 41: Test reaction for the Sonogashira coupling Pd2(dba) (2.5 mol%) DavePhos (10 mol%) 0

Scheme 43: Synthesis of (R)-147 and further work to obtain (R)-130

With this key intermediate in hands, the next step will be to proceed to the aldolization reaction it between ketone (R)-147 and aldehyde 140 using the previously displayed basic conditions, then proceed to a reduction of the ketol following the procedure described by Richard Roberts 382 (use an excess iodotrimethylsilane in DCM) to have access to the MTDL (R)-130. Regioisomer MTDL (R)-128 could be obtained following the same synthetic route. If these two last steps are successfully achieved, optimization of the whole synthetic process could be undertaken: for the aldolization reaction, the base used could be changed for a bulkier one such as LiTMP, or other aldolization processes (such as the use of enamines) preventing the use of basic aldolization conditions with an aldehyde able to form an enol, conditions known to end up with aldehyde auto condensation and polymerization issues. For the Sonogashira cross coupling reaction, yields could be improved using the optimized conditions described in paragraph 4.1.1.3 (Pd(OAc)2 or Pd2dba3 as the Pd source and Davephos as a ligand).

Scheme 46: Test reaction for the Sonogashira coupling

Based on our results on the synthesis of 145, the acyclic ketone analogue of 163, we already observed that triethylamine was the base giving the best results during the assays and it was not modified during the Sonogashira cross coupling reaction conditions optimization assays we performed. It can be noted that the reaction conditions where a pre-formed Pd-ligand complex was used, such as Pd(PPh3)4 (entry 1), Pd(dppf)Cl2 complex with dichloromethane (entry 2) or XPhos Pd, G3 (entry 3), no product was formed and the starting material was recovered as it stands, and without noticeable degradation. Yet, when distinct source of palladium such as PdCl2 and phosphine like DavePhos were used, the conversion of 160 significantly increased (entry 4). Unfortunately, the maximum conversion we

General conclusion

carbamate moiety with the catalytic serine (Ser203) of mAChE and promising CNS MPO scores (Figure 144). 32. aldolization related reaction can be undertaken, for instance by using a secondary amine to form then enamine of ketone (R)-147 , or a primary amine followed by a basic treatment to form the azaenolate.

Entry

Use of a Lewis acid in order to form the silylenol ether could be a back-up solution, but we have to deal with the tertiary amine. Another possibility would be to perform an enolate alkylation instead of the aldolization by replacing the aldehyde by a halogenated compound or an activated alcohol.

Scheme 57: Possible synthesis of (R)-130

For the second series of Donepezil based MTDL based on an indanone moiety, more efforts have to be undertaken, since one additional reaction step has to be secured, namely the CuAAC reaction which

Chemistry

Chemical and reagents

All reagents were supplied by Sigma-Aldrich, TCI, Alfa Aesar and Acros organic and used without further purification. CH3CN, THF, toluene, DCM and Et2O were dried over alumina cartridges using a solvent and a quadrupole analyzer. GC/FID analyses were done on a Shimadzu GC2014 or SCION GC436.

Quantitative analyses were obtained by usage of a linear alkane as internal standard (C12, C14 or C18).

General method for GC and GC/MS routine analysis. Realized on a 30 meters DB5-MS (95% PDMS, 5% diphenylsiloxane), with 0.25 mm ID and 0.25 μm film thickness. Helium carrier was set at 1.7 mL/min constant flow rate. Oven program : 150°C for 2 minutes, then 20 K/min up to 270°C, and a final isotherm for 17 more minutes. Total analysis time : 25 minutes. In case of MS analysis, detection ranged from m/z 50 to 500, with a 4.5 min solvent cut time.

Analytic HPLC (Thermo Hypersil GOLD C18 column, 5 μm, 2.1 × 100 mm) was performed with CH3CN and 0.1% aq. TFA as eluents [100% aq. TFA (5 min), then linear gradient from 0% to 80% (40 min) CH3CN] at a flow rate of 0.25 mL/min. UV/Vis detection was achieved with the "Max Plot" (i.e., chromatogram at absorbance maximum for each compound) mode (220-700 nm).

Semi-preparative RP-HPLC (Syncronis Thermoscientific C18 column, 5 μm, 21.2 x 250 mm) with MeCN and aq. 10 mM AcONH as eluents [40% MeCN (5min), followed by linear gradient from 40% to 100% (35 min) of MeCN] at a flow rate of 1 mL/min. Dual visible detection was achieved at 250 and 550 nm.

General procedures

General procedure A: Sonogashira cross coupling with Pd(PPh3)4

A solution of triethylamine and DCM (2/1 v/v, 0.2 M) was degassed with Argon for 15 min. Then iodophenol (1 equiv), CuI (0.1 equiv), Pd(triphenylphosphine) tetrakis palladium (0.02 equiv) were added. Trimethylsilylacetylene (2 equiv) was added and the mixture was stirred overnight at room temperature and kept away from light. After completion determined by TLC analysis, the solution was concentrated under reduced pressure, diluted with H2O, extracted with Et2O (x2). Organics layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column chromatography (PE/EtOAc 90/10, v/v) to yield the desired protected alkyne.

Then, a solution of protected alkyne (1 equiv) in THF (0.25 M) was cooled at 0 °C, tetrabutylammonium fluoride (1.2 equiv) was added dropwise and the solution was stirred 30 min at room temperature.

After completion determined by TLC analysis, the solution was diluted with NH4Cl sat, extracted with EtOAc (x2). Organics layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column chromatography (PE/EtOAc 90/10, v/v) to yield the desired product.

General procedure B: Carbamate formation

A solution of alkyne (1 equiv), carbamyl chloride 91a-c (1.5 equiv), K2CO3 (1.5 equiv) in MeCN (0.15 M) was put under reflux for 4 h. After completion determined by TLC analysis, the solution was concentrated under reduced pressure, diluted with H2O, extracted with Et2O (x2). Organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude oil was purified by column chromatography (PE/EtOAc 80/20, v/v) to yield the product.

General procedure C: Copper(II)-catalyzed alkyne-azide cycloaddition (Click chemistry)

To a solution of sodium azide (8 equiv) in water was added toluene (1/1 v/v 0.3 M). The mixture was cooled to 0 °C. Trifluoromethanesulfonic anhydride (1.6 equiv) was added dropwise and the resulting mixture was stirred at 0 °C for 2h. The reaction mixture was poured into water and extracted with toluene (2x) to yield TfN3 which was directly used in the following diazo transfer reaction. 3aminoquinuclidine dihydrochloride 81 (1 equiv), K2CO3 (1 equiv), CuSO4.5H2O (0.01 equiv) were dissolved in a minimum amount of water. Then, TfN3 was added, followed by the addition of methanol (0.13 M) to obtain a homogeneous system. The reaction mixture was stirred at room temperature for 12h. The reaction mixture was poured into water and extracted with DCM. The combined organic layers were washed with water and brine and dried over Na2SO4.

Bicyclo[3.3.1]nonane-3,7-dione (60)

A suspension of 59 (60 g, 130 mmol) in a HCl 37% (130 mL), H2O (72 mL) and glacial acetic acid (72mL) was heated at 100 °C for 6h. After cooling at rt., the reaction mixture was poured into crushed ice (200 g) and extracted with DCM (3 x 100 mL). The combined organic layer was washed with aq. sat. NaHCO3 until pH 7, then washed with H2O (2 x 100 mL) and brine. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure to give a brown solid. This crude product was dissolved in acetone (80 mL) and precipitated salts were filtered. The filtrate was concentrated and washed with acetone/Et2O (5/95, v/v) to afford the desired diketone 60 as a beige solid (10.6 g, 45%).

Spiro[bicyclo[3.3.1]nonane-3,2'-[1,3]dioxolan]-7-one (61)

To a solution of diketone 60 (4g, 26.28 mmol) in dry DCM (520 mL) with molecular sieves 4 Å at 0°C, was added BF3•Et2O (6.48 mL, 52.6 mmol, 2 equiv.) and ethylene glycol (7.5 mL, 131.44 mmol, 5 equiv.).

The reaction mixture was heated at 40°C for 15h. After completion determined by TLC analysis, the solution was cooled down and filtered to remove residues. The solution was washed with aq. sat.

NaHCO3, extracted with DCM (4x). The combined organic layers were washed with water and brine, dried over MgSO4, and concentrated under vacuo to obtain 61 as a white solid (3.46g, 67%).

1-bromo-3-(2,2-dibromovinyl)benzene (102a)

102a was obtained from 3-bromobenzaldehyde 100a (2g, 10.8 mmol, 1 equiv) following the general procedure D and isolated as a yellow oil (1.89 g, 51%). Despite an extended time of acquisition, [START_REF] Oukoloff | PET and SPECT Radiotracers for Alzheimer's Disease[END_REF] C NMR experiment could not be performed on compound (R)-108.

(ddd, J = 14.2, 9.9, 2.3 Hz, 1H), 3.25 (s, 1H) 4, 152.0, 147.0, 141.8, 139.9, 129.7, 129.4, 127.1, 125.8, 123.5, 120.9, 120.5, 119ij.9, 57.7, 50.9, 46.4, 46.1, 35.5, 35.3, 27.8, 24.4, 19. (0.02 mmol, 1.5 equiv) following general procedure B and isolated as a colourless oil (1.73 mg, 32%). Compound (R)-99c was obtained in too low quantities to perform 13 C NMR experiment.

1-benzylpiperidine-4-carbaldehyde (140)

A solution of oxalyl chloride 143 (0.45 mL, 5.2 mmol, 1.2 equiv) in dry DCM (6 mL) was cooled down to -78°C and a solution of DMSO (0.77 mL, 10.8 mmol, 2.5 equiv) in dry DCM (5 mL) was added dropwise.

The reaction mixture was stirred at -78°C for 30 min and a solution of compound 142 (883 mg, 4.3 mmol, 1 equiv) in dry DCM (5 mL) was added dropwise. The resulting mixture was stirred at -78°C for another 30 min, Et3N (3 mL, 21.7 mmol, 5 equiv) was added dropwise to the mixture which was stirred Spectral data are in accordance with those reported previously:

1-(4-ethynylphenyl)ethenone (148)

148 was obtained from 178 (1 g, 5.1 mmol, 1 equiv) following the general procedure I and isolated as a brown oil (380 mg, 60%).

5-((trimethylsilyl)ethynyl)-2,3-dihydro-1H-inden-1-one (163)

163 was obtained from 170 (1.2 g, 4.28 mmol, 1 equiv) following the general procedure H and isolated as a yellow oil (683 mg, 70%). 2, 154.9, 136.8, 131.3, 130.5, 129.5, 123.7, 104.4, 98.4, 36.4, 25.7, -

6-ethynyl-2,3-dihydro-1H-inden-1-one (167)

167 was obtained from 175 (150 mg, 0.66 mmol, 1 equiv) following the general procedure I and isolated as a yellow oil (87.51mg, 85%). 4, 155.0, 133.4, 132.6, 130.2, 128.0, 126.8, 84.5, 82.3, 33.7, 22.8; mp = 88.8 °C.