Dr Gilbert Kirsch

Prof. Dr Mohamed Attia Shaaban

Prof. Dr Fatma A Ragab

Véronique Mrs

SRSMC Vaillant

Dr Stéphanie Hesse

Dr Cristina Cebrian Avilà

Dr Philippe Pierrat

Dr Sandrine

Structure et Réactivité des Systèmes

Université de Lorraine, without her, this work could not be achieved, and who did me the NMR and elemental analysis

Introduction générale

Le thiophène est un élément de construction important pour des agents thérapeutiques et des applications industrielles (optique non-linéaire…).

Notre laboratoire a acquis au long des années une grande expérience dans la synthèse du noyau thiophénique en utilisant différentes approches. Dans ce travail nous décrivons une nouvelle synthèse de thiophène ; les 3-acétyl-2-aminothiophènes et l'étude de la réactivité de ces dérivés afin qu'ils soient utilisables par la suite pour la formation de molécules plus complexes.

Le premier but de notre travail était la synthèse des thiophènes fonctionnalisés. Dans le chapitre 1, nous décrivons la préparation des 3-acétyl-2-aminothiophènes par l'utilisation de la réaction de Gewald aux dépens de la cyanoacétone. Une autre synthèse de thiophènes est aussi décrite dans ce chapitre permettant la formation du noyau à partir du cétènes dithioacétal préparé à partir de la cyanoacétone.

La proximité des 2 groupements fonctionnels (amino et acétyle) sur le thiophène permet d'envisager le composé comme point de départ pour de nombreux autres dérivés en utilisant diverses réactions. Dans le chapitre 2, nous explorons cette réactivité par action du réactif de Vilsmeier-Haack en ciblant la synthèse de thiénopyridines. Nous comparons cette réactivité à celle des o-aminoacétophénones.

Afin de mieux comprendre le mécanisme de la cyclisation avec le réactif de Vilsmeier-Haack, nous proposons de protéger l'amine par alcoylation pour voir si l'accès à des thiénopyridines différentes est possible. Ceci est décrit dans le chapitre 3.

Dans le chapitre 4, la possibilité supplémentaire de condensation vers des polycycles, la réaction de Friedländer, est utilisée. Des conditions douces y seront recherchés pour celle-ci.

Les chlorothiénopyridines attendues précédemment seront étudiées dans le cas de l'amination par substitution nucléophile aromatique dans différentes conditions (directe, Ullmann et Buchwald-Hartwig). Tous ces couplages seront présentés dans le chapitre 5.

General introduction

Thiophene is very important building block in many chemical products used as pharmaceutical agents or industrial applications (non-linear optics…).

In our laboratory, we have long experience in synthesizing thiophene containing compounds using diversity of synthetic approach. In current work, we report a novel synthesis of thiophene derivatives; 3-acetyl-2aminothiophenes and also the study of their reactivity in order to use them as a launch point of many final products via different synthetic pathways.

The first task of our project was the synthesis of functionalized thiophenes. In chapter one we described how to prepare 3-acetyl-2aminothiophene using the simple classical Gewald's reaction starting from cyanoacetone. Another synthesis of thiophenes is described starting from cyanoacetone dithioacetals.

Neighbourhood of two functional group (amino and acetyl) on the thiophene ring qualified them to be the base of synthesis many novel derivatives applying different organic reactions. In chapter 2 we explore the reactivity of 3-acetyl-2aminothiophenes in reaction with Vilsmeier-Haack reagent looking for access to thienopyridines. We made a comparison to the reactivity of o-aminoacetophenones.

In a trial for more comprehension of the mechanism of cyclization with Vilsmeier-Haack reagent, we decided to protect the amino group to see whether the reaction is behaving differently and the access to functionalized thienopyridines is possible. This is described in chapter 3.

In chapter 4, another utilization of the reactivity of our starting material through application of Friedländer reaction is studied. Smooth conditions for the reaction are searched for. As chlorothienopyridines will be available from the former studies, we are looking at the reactivity of chlorine atom to nucleophilic amination using different reactions (Ullmann coupling, Buchwald-Hartwig coupling…). All these coupling are described in chapter 5. Introduction Thiophene is a very important building block of many heterocycles which possesses pharmacological characteristics. Wide varieties of drugs are containing thiophene in their structure, e.g. antiplatelet aggregation tetrahydrothieno [3,2-c]pyridine: ticlopidine® and clopidogrel®, 1 estrogen receptor modulator Benzo [b]thiophenes: Raloxifene® 2 and antipsychotic thieno [2,3-b] [1,5]benzodiazepine: olanzapine®. 3 Other nucleus like thieno [2,3-b]pyridine has provoked a great interest as a possible treatment of anxiety and depression, 4 bacterial infection, 5 inflammation, 6 leishmaniasis, 7 malaria 8 and autoimmune diseases. 9 Tacrine like thiophene was reported as a possible alternative of Tacrine in treatment of Alzheimer disease. 10 1.1. 1.3 The Hinsberg Synthesis Condensation between 1,2-diketones 7 and thiodiglycolate 6 led to synthesizing of thiophene carboxylic acids 8, sodium or potassium ethoxides served as catalyst (Scheme 3). Ethyl thioglycolate for synthesizing 2-ethyl carboxylate derivatives of thiophene 11 15 , compound 10 were obtained by Arnold; who synthesized it in a reaction between α methylene containing ketone 10 and Vilsmeier-Haack reagent (Scheme 4). 16 9 10 11 Scheme 4 General synthesis of 2-functionalized thiophene. 14 O. Hinsberg, "Synthetische Versuche Mit Thiodiglykolsäureester," Berichte Der Deutschen Chemischen Gesellschaft 43, no. 1 (January 1, 1910): 901-6, doi:10.1002/cber.191004301153. 15 S. Hauptmann et al., "Eine Neue Synthese Substituierter Thiophene Und Pyrrole," Tetrahedron Letters 9, no. 11 (January 1, 1968): 1317-19, doi:10.1016/S0040-4039(01)98945-2. 16 Z. Arnold and J. Žemlička, "Synthetische Reaktionen von Dimethylformamid IV. Darstellung von β-Chlorvinylaldehyden Aus Carbonylverbindungen," Collection of Czechoslovak Chemical Communications 24, no. 7 (1959): 2385-92, doi:10.1135/cccc19592385. This reaction has been extended to a three component reaction in one pot by our group, Na2S.9H2O was the sulphur source. It reacted with β-chloroacrolein derivatives 10 in DMF to produce an intermediate that cyclized in situ with an activated halide in basic medium producing the thiophene 12 as the final output. 17 Similarly, ketene dithioacetal is a key intermediate in synthesis of many aromatic heterocycles, especially thiophenes. 18 It was prepared for the first time by Freund 19 , our laboratory has long tradition in preparation of such kind of products using DMF as a solvent, and potassium carbonate as catalysing base. Methyl iodide or dimethylsulphate are the reagents in use as methylating agent (Scheme 6).

14

Y, Z are electron withdrawing groups Scheme 6 General synthesis of ketene dithioacetal, To cyclize compound 14, two pathways were reported: one by combination between sodium sulfide and activated halide, second synthetic pathway by reaction of ketene 17 Paul Cagniant and Gilbert Kirsch, "Méthode Générale de Synthèse de Composés Thiophénique Simples Ou Complexes Alcoylés, Arylés Ou Fonctionnalisés en Position-2," C. R. Acad. Sc. Paris, C, 281 (1975): 35-38. 18 Geoffroy Sommen, Alain Comel, and Gilbert Kirsch, "A Convenient Synthesis of 2,3,4,5-Functionalized Thieno [2,3-b]thiophenes.," ChemInform 34, no. 28 (July 15, 2003), doi:10.1002/chin.200328120. 19 Erich Freund, "Über Die Einwirkung von Schwefelkohlenstoff Auf Nitro-Methan," Berichte Der Deutschen Chemischen Gesellschaft (A and B Series) 52, no. 3 (March 8, 1919): 542-44, doi:10.1002/cber.19190520307. dithioacetal with thioglycolate. Both synthetic pathways are promoted by basic catalysis.

Sodium sulfide or thioglycolate are serving as the source of Sulphur in the furnished thiophene (Scheme 7). 20 aminothiophene by applying one-pot reaction in which a carbonyl compound containing a methylene group in the α-position reacts with elemental sulphur and activated nitriles to produce 2-aminothiophene. 22 It was considered so brilliant to enclose the inert sulphur within a heterocyclic nucleus and force it to react. 23 Gewald three component reaction has been accomplished using different kinds of carbonyl product which containing free methylene group in position α, the most common activated nitriles were malononitrile, cyanoacetic ester and primary cyanoacetamide. 24 This reaction has been widely applied since it was discovered to result in many derivatives including different electron-withdrawing groups in the 3-position (e.g., CN, COOR, CONH2 and COPh) (Scheme 10). 25 As it illustrated in Scheme 11, the reaction consists of Knoevenagel condensation between the carboyl containing compound and the activated nitrile which is followed by ring closure accomplished by sulphur atom attacking the cyano carbon. 27 22 Karl Gewald, Elfriede Schinke, and Horst Böttcher, "Heterocyclen Aus CH-Aciden Nitrilen, VIII. 2-Amino-Thiophene Aus Methylenaktiven Nitrilen, Carbonylverbindungen Und Schwefel," Chemische Berichte 99, no. 1 (January 1, 1966): 94-100, doi:10.1002/cber.19660990116. 23 Yijun Huang and Alexander Dömling, "The Gewald Multicomponent Reaction," Molecular Diversity 15, no. 1 (February 2011): 3-33, doi:10.1007/s11030-010-9229-6. 24 Roland Mayer and K. Gewald, "The Action of Carbon Disulfide and Sulfur on Enamines, Ketimines, and CH Acids," Angewandte Chemie International Edition in English 6, no. 4 (April 1, 1967): 294-306, doi:10.1002/anie.196702941; Huang and Dömling, "The Gewald Multicomponent Reaction." 25 M. S. Manhas et al., "Synthesis of Thieno-and Furo-Pyrimidinethiones," Journal of the Chemical Society C: Organic, no. 14 (January 1, 1969): 1937-39, doi:10.1039/J39690001937; K.-J Hwang and N.-K Choi, "A Facile Synthesis of 2-Aminothiophene Derivatives," Bull. Korean Chem. Soc. 12 (1991): 121; J D Ramanathan et al., "Biological-Activity Of Some 2-Amino 4, 5, 6, 7-tetrahydrobenzo [b]thiophenes and Their Derivatives," Journal of the Indian Chemical Society 55, no. 8 (1978): 822-825; P. Richter, D. Oertel, and F. Oertel, "Synthese von 3-Alkyl-5phenyl-2-thioxo-3H-1,2-dihydro-cycloalka [4,5]thieno [2,3-e] [1,2,4]triazepinen," Pharmazie 43, no. 11 (1988): 753-755. 26 Huang and Dömling, "The Gewald Multicomponent Reaction." 27 Scheme 12 Synthesis of tellurophene and selenophene; 28 Jirí Šibor and Pavel Pazdera, "Syntheses of Some New Five-Membered Heterocycles Containing Selenium and Tellurium," Molecules 1, no. 10 (February 4, 1997): 157-62, doi:10.1007/s007830050031. X= electron withdrawing group Scheme 14 Microwave assisted two component Gewald synthesis 29 Hülya Özbek, Ivana Veljkovic, and Hans-Ulrich Reissig, "Gewald Synthesis of Aminothiophene Carboxylic Acids Providing New Dipeptide Analogues," Synlett 2008, no. 20 (December 2008): 3145-48, doi:10.1055/s-0028-1087243. 30 Stéphanie Hesse, Enrico Perspicace, and Gilbert Kirsch, "Microwave-Assisted Synthesis of 2-Aminothiophene-3-Carboxylic Acid Derivatives, 3H-thieno [2,3-d]pyrimidin-4-One and 4-chlorothieno [2,3-d] Among various pathways of synthesizing thiophene, Gewald synthesis is very convenient synthetic procedure for production of 2-aminothiophene. Classic Gewald reaction was applied to produce different electron withdrawing group in position 3 (e.g., CN, COOR, CONH2 and COPh). Some Modified techniques has been processed in order to improve yield (e.g., Microwave) or to obtain certain products. First Synthesis of 3acetyl-2-aminothiophenes was approached by Eller et al. in 2006. 32 1.1.4 Synthesis of 3-aminothiophene 1.1.4.1 Starting from β-chloroacrolein β-chloroacrolein 10 could be involved in multicomponent reaction with hydroxyl amine hydrochloride and acetic anhydride to give acrylonitrile 34 which consequently reacted with sodium sulphide and activated halide to give 3-aminothiophene 35 in basic condition (Scheme 40). 33 31 Gernot A. Eller and Wolfgang Holzer, "First Synthesis of 3-Acetyl-2-Aminothiophenes Using the Gewald Reaction," Molecules (Basel, Switzerland) 11, no. 5 (2006): 371-76. 32 Ibid. 33 H. M. Sampath Kumar et al., "Efficient One-Pot Preparation of Nitriles from Aldehydes Using N-Methyl-Pyrrolidone," Synthesis 1999, no. 04 (April 1999): 586-87, doi:10.1055/s-1999-3450; Zhijun Wang, Richard Neidlein, and Claus Krieger, "A New Approach to the Synthesis of Heteroannulated 3,1-Oxazin-4-Ones from β-Enamino Esters and Phosgeneiminium Salts," Synthesis 2000, no. 02 (2000): 255-58, doi:10.1055/s-2000-6249; Sommen, Comel, and Kirsch, "A Convenient Synthesis of 2,3,4,5-Functionalized Thieno [2,3-b]

Synthesis of Cyanoacetone

Cyanoacetone 32 is colorless unstable oily liquid. It has to be freshly prepared in every time of performing the reaction and very shortly before launching it. 34 Sommen, Comel, and Kirsch, "A Convenient Synthesis of 2,3,4,5-Functionalized Thieno [2,3-b]thiophenes."

32

Scheme 18 Synthesis of cyanoacetone Cyanoacetone 32 was prepared from (E,Z)-3-aminocrotonitrile 39 by stirring in 6N

HCl solution for 3 h (Scheme 18). 35 Cyanoacetone 32 was serving as the activated nitrile as it contains cyano and acetyl group on the two sides of activated methylene group.

Selection of suitable catalyzing amine

By surveying the literature, many organic amines has been used as basic catalysis; morpholine, piperidine, trimethylamine… etc. From our experiments, we can arrange the bases in decreasing order according to their efficiency as the followinwing: piperidine > triethylamine > morpholine.

Selection of the solvent

Ethanol, methanol and DMF were the most reported solvent in use. Both ethanol and methanol was performing well during our reaction. We did not notice any effect on yield upon changing of the solvent.

Reactivity of ketones

This type of reaction were applied for wide ranges of ketones, nearly 40 different kinds of carbonyl derivatives were tried. Few examples gave products in yield varying from 8-86% depending on kind of ketones (Table 1). The time of the reaction was adjusted to be 24 hours which was found optimum, the temperature was between 55-65 °C.

Increasing the time of the reaction or temperature beyond the fore mentioned values were found to decrease the yield or led to produce an oily solution upon treatment with ice and water (Scheme 19). The best results were obtained from alicyclic ketones. The site of substitution on the ketone governed the output. 2-substitution as in case of 2-methylcyclohexanone, 2chlorocyclohexanone and 2-acetylcyclohexanone gave no reaction in contrarily to 3methyl, 4-methyl and 4-tert-butyl-cyclohexanone (Table 1). Also multi substitution in more than one site (3,3,5-timethylcyclohexanone) and Aromatic substitution (in case of alpha and beta tetralone) did not give any results. It appeared that, the liberty of alpha methylene on both sides of the ketone is very crucial in order to complete the reaction.

Increasing the number of the member of the ring beyond seven caused failure of achievement of the reaction, cyclooctanone and cyclododecanone gave no products.

In investigation of 1 H NMR results of the products, it was found that the common features between all spectra were those indicating the two functional group; the sharp singlet of methyl group of acetyl moiety and broad singlet which interpreted for NH2 group.

Compounds 49, 51 and 53, showed interesting mode of H-H coupling. Due to the presence of asymmetric carbon in position 5 for 49 and in position 6 for the last two. 3). 1). Branching of the ketone skeleton or presence of substituent on the chain resulted in abolishment of the reactivity of the ketone. This could be attributed to the free rotation of the long chain which may hamper the ring closure during formation of the thiophene ring.

Heterocyclic ketones

The presence of heteroatoms helped in augmenting the yields (66 and 72 % of the products of the reaction with 1-methylpiperidin-4-one 64 and tetrahydro-4H-thiopyran-4-one 62 respectively) (Table 1). When the member of the ring decreased than six, unidentified products was separated. Substitution on any site of the ring produced in no products.

Diketones

No products were obtained from reaction of cyanoacetone, S8 with the diketones, neither normal aliphatic nor alicyclic ketones reacted to give the desired products.

Aldehydes

In reaction of heptanal with other reactants under Gewald's conditions, no product was identified.

Synthesis trial of 3-acetyl-2-aminoselenophene by application of three component Gewald synthesis

To extend our varities of the products, a trial was done to replace elemental sulphur with selenium to produce selenophene rather than thiophene. 36 the key question of our synthesis.

It was prepared just a while before the 3-components reaction started by the way described before in Scheme 18.

Afterward, it was dissolved in DMF with potassium carbonate and stirred for two hours in room temperature. Carbon disulfide (3 equivalents) was added and the reaction temperature was retained in room temperature for additional two hours. Finally, one equivalent of dimethyl sulfate was added and the reaction mixture was agitated for four hours, after treatment with ice and water, a product was obtained.

When this product was investigated by 1

67 69

Scheme 24

No traces of compound 69 were identified, replacement sodium sulfide and activated halide with thioglycolate did not lead to the other cyclization (Scheme 25), also changing of the catalytic base to DBU did not result in formation of compound 69 (Scheme 24).

67 71

Scheme 25

The mode of cyclization of 2-(bis(methylthio)methylene)-3-oxobutanenitrile 67 was further explored by hydrazine hydrate to investigate the direction of the cyclization whether it is changing or not (Scheme 26).

67 73

Scheme 26 67 73

Scheme 27

Chapter 1 -Synthesis of 3-acetyl-2-aminothiophene

27

Compound 67 was mixed with one equivalent of hydrazine hydrate in dichloromethane, at room temperature. The reaction mixture was stirred and followed by TLC. After the accomplishment of the reaction (ca 2 hours) the pyrazole 73 obtained had the same mode of ring closure as the only obtained product (Scheme 26) (Table 2).

The mechanism could be described by the nucleophilic attack of one nitrogen atom of hydrazine on the carbonyl carbon followed by another nitrogen attack on the carbon which restrained between two sulphur atoms for ring closure. This accompanied by delocalization of the double bond and followed by loss of water and methanethiol molecule to provide the final product 73 (Scheme 27).

Conclusion

Gewald's procedure has not described before to produce 3-acetyl containing thiophene by three components method and the first synthesis of 3-acetyl-2aminothiophene by using such way was introduced. Many trials were applied to produce varieties of this nucleus but only few examples of ketones were reactive. The sensitivity of cyanoacetone and rapid decomposition of it, may be one of description of the low reactivity of such kind of reaction. Yield of alicyclic ketones and their reactivity were observed to be higher than normal aliphatic one, which could be attributed to the free rotation of unchained ends of the aliphatic ketone may halt the ring closure.

Diketones and substituted or branched aliphatic ketones did not respond to the reaction. Assisted microwave synthesis was helpful only in decreasing the reaction time, but did not help in increasing the yield. Replacement elemental Sulphur with selenium and application of the reported procedure did not produce selenophene.

Ketene dithioacetal (2-(bis(methylthio)methylene)-3-oxobutanenitrile 67) prepared from cyanoacetone was another option for synthesizing thiophene nucleus. The mode of cyclization of it was observed to be on the direction of carbonyl carbon and cyanothiophene was obtained, using hydrazine hydrate to produce pyrazole from same starting compound did not lead to any change in the pattern of cyclization. The necleophilic attack on carbonyl carbon end to formation of cyanopyrazole.

Chapter 1 -Synthesis of 3-acetyl-2-aminothiophene 29

Experimental part

All solvents and reagents were purchased from commercial sources unless otherwise noted. Melting points were determined with a Büchi 530 digital melting point apparatus and are uncorrected. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, using Me4Si as the internal standard. Hydrogen coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). Highresolution mass spectra were measured with a Micro-Tof-Q98 instrument in ESI mode.

Column chromatography was performed using silica gel (60M, 0.04-0.063 mm). Thinlayer chromatography (TLC) was performed using silica gel plates (POLYGRAM SIL G/UV254, 0.20 mm), which were visualised under UV light.

Cyanoacetone (32)

(E,Z)-3-Aminocrotonitrile (10 g, 0.12 mol) was dissolved in 6N HCl (28.5 mL), and the solution was stirred and heated to 80 °C for 3 hours. The reaction mixture was cooled, extracted several times with CH2Cl2, dried over anhydrous Na2SO4 and concentrated under vacuo.

Yield: 5 g (53%); colourless liquid.

1H NMR (400 MHz, CDCl3): δ = 3.56 (s, 2 H), 2.28 (s, 3 H).

3-Acetyl-2-aminothiophene Derivatives; General Procedure

Carbonyl compound (0.025 mol) was added to freshly prepared cyanoacetone (0.03 mol) in either MeOH or EtOH (40 ml). Sublimed sulphur S8 (0.03 mol) and piperidine (0.03 mol)

were added and the mixture was stirred and heated to 55-65 °C for 24 h. Ice was then added and the formed precipitate was filtered under vacuum and washed with water. The obtained solid was crystallised from a suitable solvent, with the exception of 57 and 63, which were collected directly without further purification.

1-(2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-yl)ethanone (47)

Yield: 61%; brown solid (toluene); mp 230 °C

2-(bis(methylthio)methylene)-3-oxobutanenitrile (67)

Freshly prepared cyanoacetone (3.5 g, 0.042 mol) was dissolved with K2CO3 (5.8 g, 0.042 mol) in 40 ml DMF, the solution was stirred for two hours in room temperature. Carbon disulfide (9.5 g, 0.13 mol) was added while the reaction mixture was stirred in room temperature for another two hours. Afterward, dimethyl sulfate (15.9 g, 0.13 mol) was added and the stirring continued till 4 hours, after treatment with ice and water, a product was obtained as precipitate, filtered under vacuo and crystallized from cyclohexane (5.3 g, yield 72%).

Yield: 72%; brown solid (cyclohexane); mp 60 °C. 39 Eller and Holzer, "First Synthesis of 3-Acetyl-2-Aminothiophenes Using the Gewald Reaction." 40 Haiyu Li, Red-light blue azo disperse dye with high color development strength and preparation method therefor, CN105238094 (A), issued January 13, 2016. By working in the reported conditions of 60 °C for five hours, [START_REF]PDB code:1C25[END_REF] an unknown product was separated as a major component. Increasing the time of the reaction did not help.

When the temperature was elevated to 100 °C for five hours, a mixture of the desired compound and an unknown by-product were obtained.

Time of the reaction

After adjustment of the temperature to be 100 °C, five hours of reaction working did not help to get the desired compound exclusively. Increasing the time made no difference in the result.

Number of the Vilsmeier-Haack reagent of equivalents

The number of equivalents of the VH reagent was found to have great influence upon the completion of the reaction. Using large excess of the reagent, caused the treatment of the reaction with ice and water to be very violent and exothermic due to presence of great extent of unreacted POCl3. Decreasing the number of equivalents down certain limit gave uncomplete transformation.

Mode of reagent addition

The mode of addition was also found to govern both the type of product formed and the yield. Addition of the reagent over seven hours found to be the best solution to [START_REF]PDB code:1C25[END_REF] In conclusion, addition of the reagent over seven hours at 100 °C then stirring the reaction mixture overnight at the same temperature appeared to be the best reaction conditions.. The reaction followed by TLC, and mostly the starting material was consumed between 20 to 24 hours. The reaction mixture was treated with ice and water, pH of the reaction adjusted to neutral by ammonium acetate. The reaction mixture was extracted with EtOAc several times and the residue was adsorbed on silica and the product was obtained in pure form after running of flash silica gel column chromatography (EtOAccyclohexane, 1:9). Recrystallization of all products was done by mixture of (MeOH-H2O, 1:1). In contrast, two doublet signals was identified and the coupling constant of them referred to that they are coupling with each other. This pattern of NMR spectrum proved formation of formyl free product (Figure 4).

The replacement of the benzene ring with a thiophene ring is likely to have an impact on the final products, and may prevent further attack of the Vilsmeier-Haack reactant (Scheme 43).

Mechanism

Mechanism of the cyclization is postulated to be happened by one or two attacks of VH reagent:

Scheme 46

All the reactants gave yields from low to moderate, with the exception of 63 and 65, which gave undefined mixtures on TLC, without any major component. Attempts to separate pure compounds from these mixtures by using flash chromatography failed (Table 3).

Conclusion

Thiophene compounds exhibited a different mode of cyclisation in the reaction with Vilsmeier-Haack reagent than that reported for the reaction with o-aminoacetophenone, which could be ascribed to the influence of the thiophene nucleus.

4-Chloro-3-methyl-2-propylthieno[2,3-b]pyridine (121)

Yield: 141 mg (25%); white solid; mp 36-38 °C (MeOH-H2O); R f = 0.48 (EtOAccyclohexane, 9:1). Noteworthy that the formyl free products were obtained as traces (0 -14% yield) in mixture with major products 131 .the temperature of the reaction was 90 °C (Scheme 49). 60

Results and discussion

In synthesis of 4-chlorothieno [2,3-b]pyridine, formyl analogue was not attainable by any mean. 61 Formylation was not obtained when compound 115 was subjected to VH reagent at different heating condition (65-100 ⁰C) which is expected from pyridine as an electron deficient nucleus (Scheme 50).

132 Scheme 50

We assumed that the thiophene ring stabilize the pyridine moiety by its inductive effect which prevented it from attack another iminium ion of VH reagent, to test our assumption and to make approach toward comprehension of reaction mechanism, we 60 Ibid.

61 Ahmed Abdelwahab, Atef Hanna, and Gilbert Kirsch, "Synthesis of Novel 3-Acetyl-2-Aminothiophenes and Investigation of Their Behaviour in the Reaction with Vilsmeier-Haack Reagent," Synthesis 48, no. 17 (June 10, 2016): 2881-88, doi:10.1055/s-0035-1561459.

Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 65 decided to construct N-protected starting materials. This protection was intended to be done by electron withdrawing group, like acetyl or pivaloyl groups.

N-acetylated analogue was simply prepared by well-known procedure 62 of refluxing gently with acetic anhydride. Compounds 59 and 61 required certain modification of the procedure. For these last two, thiophene was concurrently dissolved with one equivalent of acetyl chloride in excess of acetic anhydride and stirred at 35 °C as maximum temperature, after 30 minutes the starting material was completely transformed to product which was treated with water and extracted with ethyl acetate. It was subsequently exposed to flash column purification (EtOAc-cyclohexane, 1:3) for maximum purity.

The previous procedure was not suitable for compound 63 to obtain complete reaction, mixture of starting material and product were obtained regardless the reaction time. Increasing the reaction temperature led to produce a lot of by-products. When the compound was dissolved in dichloromethane, excess of acetic anhydride, 3 equivalent of acetyl chloride and 3 equivalent of triethyl amine were added then the reaction mixture was gently refluxed for 30 hours, the complete reaction was achieved. 4).

Room temperature

Chloroformylthienopyridine 112 was synthesized as major product, chlorothienoderivatives 113 also obtained in lesser yield, the reaction time in this case need to be beyond 48-72 hours in order to obtain maximum productivity and the number of equivalent of VH reagent was same to which was used in Scheme 44.

At 65 °C

When same previously used reagent equivalent was applied, chlorothienopyridine 113 was the major product in low yield, while formylated product 112 was traces. When this amount was doubled and added at once, yield of formylated product 112 raised to be around 94% -32%) while unformylated compounds 113 were 25% to 0% in 4-5 hours.

Dropping the reagent over long period of time resulted in excessive decrease in the yield of both products, the reagent amount made no difference (Table 4).

At 100 °C

The reagent was dropped over long period of time (5-7 hours) and the reaction left under stirring for 20-24 hours. 6 Maximum yield of chlorothienopyridines 113 was obtained. When the same amount of the reagent was added at once, yields of both folmylated 112 and non-formylated compounds 113 were dramatically decreased, but the formylated product 112 was in greater amount.

Pivaloyl derivative 134 was exposed to the same reaction condition of synthesis of chlrothienopyridine 6 and formylated product 132 was the only output of this reaction

67

(yield, 42%), the same protected analogue gave no reaction at room temperature and 19% yield only at 65 °C using doubled amount of reagent (Scheme 52).

132 Scheme 52

Working with the procedure we described 63 and the same percentage of reactants with the new starting material gave us a considerable improvement in the yield of chlorothienopyridine 113 in addition to cleaner reaction, while addition of double amount of the reagent at one time to the reactant led to formation of formylated analogue 112 as major product and exclusively in some cases, the reaction time in this case was decreased to be 4-5 hours (Table 4).

Differently from reported before, 64 acetyl group was not involved in cyclization. It is higher temperature (100 °C) gives chance to departure of the acetyl group in the acidic media. This made the amino group free at the early stage of the reaction and pyridine seems like not susceptible to the excessive iminium ion.

By the way, pivaloyl group is more stable than acetyl group. Working under higher temperature (100 ⁰C), and with the normal reagent quantity gave only the formylated product, that may emphasize our suggestion.

Mechanism

Scheme 53

The mechanism of reaction is postulated to occur by two possible routes. First mode of cyclization in which the acetyl group leaves just prior to the chlorination (intermediate All the products structure were verified by 1 H NMR and 13 C NMR; in 1 H NMR spectra; two sharp singlets appeared in region around 10.5 and 8.7 ppm which are characteristic for the formyl proton and the single proton of the pyridine ring respectively (Figure 5), while the formyl carbon appeared in region of 189 -188 ppm. To explore the reactivity of the obtained products we applied the method described by Hauptmann et al. 66 for compound 132, it exhibited high reactivity and the reaction was accomplished within 30 minutes at 70°C (Scheme 54).

135

Scheme 54

Conclusion

We reported here new synthetic pathway of both 4-chlorothieno [ N-protection provides us more knowledge about the mechanisms of cyclization and chlorination of 3-acetyl-2-aminothiophene. These two processes more reasonable to happen by two attacks of iminium ion rather than one. In case of our previous proposal;

that cyclization and chlorination take places by only one nucleophilic attack, 67 Nacetylation is supposed to produce merely chlrothienopyridine rather than 3-formyl derivatives.

N-(3-acetyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2-yl)acetamide (136)

Yield: 1.03 g (84%); green solid; mp 117-119 °C 1

N-(3-acetyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (138)

Yield: 1.17 g (96%); brown solid; mp 100-103 °C; (cyclohexane). 1

N-(3-acetyl-5-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (139)

Yield: 1 g (83%); brown solid; mp 148 °C (cyclohexane).

N-(3-acetyl-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)acetamide (143)

Yield: 1.17 (82%); beige-brown solid; mp 141-143 °C (cyclohexane).

N-(3-acetyl-4,5-dimethylthiophen-2-yl)acetamide (147)

Yield: 1 g (81%); brown solid; mp 115-119 °C (EtOH 96%).

N-(3-acetyl-4-methyl-5-propylthiophen-2-yl)acetamide (149)

Yield: 1 g (82%); brown solid; mp 58-60 °C; R f = 0.54 (EtOAc-cyclohexane, 1:3).

N-(3-acetyl-4-methyl-5-pentylthiophen-2-yl)acetamide (151)

Yield; 1.32 g (97%); brown solid; mp 40 °C; R f = 0.53 (EtOAc-cyclohexane, 1:3).

4-chloro-3-formylthieno[2,3-b]pyridine, general procedure

Vilsmeier-Haack reagent was prepared by previously mentioned method, in this case the amount of the reagent was: 5 ml of POCl3, and 20 mL of DMF for 2.5 mmol of the starting material. The reagent was added to the thiophene at one time, and stirred at 65 °C for 4-5 hours. At the end of the reaction, the solution was decomposed by mixture of ice and water and neutralized by sodium acetate, in case of formation of precipitate, filtration under vacuum was applied, the obtained solid was dissolved in ethyl acetate and washed 2-3 times with water, the residue was purified by silica gel column chromatography (EtOAc-cyclohexane, 1:9), otherwise the solution was directly extracted by ethyl acetate, and purified by the same way.

For preparation of 4-chlorothieno [2,3-b]pyridine, same previously mentioned procedure and reactant amount were employed (Chapter 2). using Lewis acids [START_REF] Mogilaiah | Lithium Chloride as an Efficient Catalyst for Friedlander Synthesis of 1,8-Naphthyridines via the Use of Microwave Irradiation and Pestle/Mortar[END_REF] , inorganic salts [START_REF] Desai | A Highly Efficient Synthesis of Trisubstituted Quinolines Using Sodium Hydrogensulfate on Silica Gel as a Reusable Catalyst[END_REF] , ionic liquids [START_REF] Abdollahi | Nanosized MCM-41 Supported Protic Ionic Liquid as an Efficient Novel Catalytic System for Friedlander Synthesis of Quinolines[END_REF] , branched catalysts [START_REF] Fang | Homogeneous Catalysis, Heterogeneous Recycling: A New Family of Branched Molecules with Hydrocarbon or Fluorocarbon Chains for the Friedländer Synthesis of Quinoline under Solvent-Free Conditions[END_REF] have been reported for this reaction. reaction was followed by TLC and the product was purified by column chromatography (Scheme 60). 88

Results and discussion

Friedländer condensation introduced us an additional pathway for synthesis of thieno [2,3-b]pyridine. This perspective is allowed by 3-acetyl-2-aminothiophene due to adjacency of both amino and acetyl group. Wide varieties of derivatives could be obtainable, by different α methylene containing carbonyl compound.

Despite the different behaviour of thiophene ring in certain reaction from aryl compound, it still behaves similarly in other cases. Friedländer condensation was reported as an applicable synthesis on thiophene and thiazole rings in presence of AlCl3. 89 In previously described examples of Friedländer condensation including thiophene, cyano group was the functional group on which the reaction took place. In our example, the acetyl group replaced the cyanide moiety and cerium ammonium nitrate (CAN), 90 was the catalyst of choice.

168 170

Scheme 61

The procedure, reported by V. Sridharan et al was mainly followed. The time of the reaction was determined by continuous following of the reaction using TLC. The reactants were mixed in same equivalency and refluxed in absolute ethanol. It was noticed that, whatever the time of reaction, thiophenes were not consumed completely. The catalyst Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction 96 effect appeared to be the same at all molar amounts, increasing the molar percentage from reported amount (15 mol%) to 30% did not make any effect on accomplishment of the reaction (Scheme 61) (Table 5).

The examples which tried through the practical work gave yield from low to moderated, compounds 63, 65 were not reactive, which gave another hint about influence of the heteroatom in the fused cycle upon the reactivity of the thiophene. It is worth to be mentioned that when the Friedländer reaction was tried between the 3-acetyl-2aminothiophe 45 and diketone 171, unknown products were obtained (Scheme 62). To explore the reactivity of chlorine atom, thienopyridines were exposed to nucleophilic substitution using different amines. This substitution was tried in different conditions, simple nucleophilic substitution, of Ullmann coupling conditions and Buchwald-Hartwig cross coupling.

Nucleophilic substitution

Reaction between amines and aromatic halide need more drastic condition than that described for alkyl halide. The substitution to be accomplished needs at least one of activating group to facilitate the replacement of the halogen e.g. nitro group (Scheme 64). 91

Role of ligand

The Ullmann reaction showed a lot of disadvantages; high temperature and need of high boiling polar solvents (Scheme 65) could be overcome by using of copper-ligand complexes. 100 Development of ligands for Ullmann type copper catalysed coupling reactions has been done to improve reaction conditions with wider substrate scope. Therefore, extensive research efforts have been devoted to promote alternative ligands to run over the limitations of copper catalysed reactions in Ullmann type couplings in order to reduce the catalyst loadings and increase the yields using moderate reaction conditions. 101 In 2001, in an effort for increase the copper (I) salts solubility in the organic solvent and hence increasing their catalytic capacity, Cu(phen)(PPh3)Br 206 and Cu(neocup)(PPh3)Br 207 were prepared and performed will in C-N coupling (Scheme 70 The reaction mechanism for this reaction has been demonstrated to proceed through steps includes oxidative addition of the aryl halide to a Pd(0) species; addition of the amine to the oxidative addition complex, deprotonation followed by reductive elimination (Scheme 74). 108

Oxidative addition step

Oxidative addition products [PdArXLn] exhibit anticipated catalytic activity 109 therefore; it has been received substantial attention in the literature in a trail to maximize the rate of the oxidative addition. The rate of oxidative addition depends on the electronic and steric properties of the catalyst and substrate; the more electron-rich and sterically unhindered the catalyst, the higher the rate of the oxidative addition. 110

Amine coordination step

The rate of amine coordination depends on the catalyst and the substrate properties.

When the amine substrate is more basic and/or is sterically unhindered, the rate of amine binding is higher. The rate of deprotonation of the bound amine is determined by the acidity of the amine, which increases on coordination to the metal. 111 still being used. The palladium catalyst must be in the (0) oxidation state before the catalytic cycle initiates, and therefore the palladium (II) in Pd(OAc)2 must be reduced prior to catalysis initiation 120

Results and discussion

Nucleophilic substitution

To explore the reactivity of the chlorine atom, thienopyridines were exposed to nucleophilic substitution using aniline as a nucleophile. The chlorine atom could not been replaced.

194 241

Scheme 80

The reaction was tried under different conditions, either in excess of aniline or in the presence of base like K2CO3, and in different temperatures (from 120 °C to reflux in nitrobenzene), different reaction vehicles were used (aniline, toluene, DMF or nitrobenzene). From all these trials no results were obtained. Noteworthy that, ligand in trial five (Cu(phen)(PPh3)Br) was prepared by reported procedure 132 through reacting of PPh3 with CuBr2 and 1,10-Phenanthroline in two steps.

Buchwald-Hartwig coupling

The coupled products were only accessible through Buchwald-Hartwig crosscoupling, which was mediated by palladium salt and suitable ligand in the presence of base and an inert gas atmosphere. 133 After several hours of heating in toluene, the starting material was consumed to different extents according to nature of both reactants.

The products were separated by using flash column chromatography. The yields were variable according to the substituents on the benzene ring of aniline derivatives, and the scaffold of thiophene partner.

The reported condition by Queiroz et al 134 appeared to be the most suitable one, the yield for reaction between compound 115 and aniline 194 was obtainable in quantitative amount after 5 hours. The results from other substrates were quite variable from good yield to null. As which could be anticipated, presence of electron withdrawing group in the aniline derivative resulted in decreasing the nucleophilicity of the amine, hence diminishing the reactivity and the yield (Table 7).

132 Gujadhur, Bates, and Venkataraman, "Formation of Aryl-Nitrogen, Aryl-Oxygen, and Aryl-Carbon Bonds Using Well-Defined Copper(I)-Based Catalysts."

133 Queiroz et al., "Palladium-Catalysed Amination of Electron-Deficient or Relatively Electron-Rich Benzo [b]thienyl Bromides -Preliminary Studies of Antimicrobial Activity and SARs." 134 Ibid.

The reaction was catalyzed by palladium acetate and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) as ligand in the presence of cesium carbonate and an inert gas atmosphere (Scheme 82).

242 243

Scheme 82

Some trials were done to find alternative combination of catalyst, ligand and solvent but they were not profitable. Using Pd(PPh₃)₄ instead of BINAP, replacement toluene by dioxane or changing the base to t-BuoK were not useful. For these substrates which gave incomplete reaction, elongation of the reaction time more than 5 hours did not help in consumption of the residual amount of the reactants.

N-phenylthieno[2,3-b]pyridin-4-amine, general procedure

In a three-necked round-bottom flask fitted with a condenser, septum, and thermometer, 4-chlorothieno [2,3-b]pyridine derivatives (0.54 mmol) was dissolved in anhydrous toluene (5 mL), and Cs2CO3 (1.2 mmol), Pd(OAc)2 (0.016 mmol) and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (0.022 mmol) were added. Argon gas was applied and the reaction mixture was heated to 70-80 °C. Aniline derivative (0.54 mmol) was dissolved in anhydrous toluene (3 mL) and added dropwise to the reaction mixture by using a syringe through the septum under argon. Upon complete addition, the temperature was raised to 110 °C and the progress of the reaction was followed by TLC until consumption of the reactants was complete (ca. 5 h). The reaction mixture was poured over ice and water, and extracted with EtOAc. The final pure product was obtained after silica gel column chromatography (cyclohexane -EtOAc, 7:3) for compound 241 and (cyclohexane -EtOAc, 9:1) for the rest and recrystallization from suitable solvent if it is needed.

N-Phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (241)

Yield: 149 mg (99%); yellow solid; mp 102-105 °C (cyclohexane); Rf = 0.

N-(4-chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (245)

Yield: 57%; yellow solid; mp 152-154 °C.

N-(4-fluorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (248)

Yield: 60%; yellow solid; mp 138-140 °C

N-(p-tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (250)

Yield: 83%; yellow solid; mp 106-108 °C

N-(o-tolyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (253)

Yield: 54%; yellow solid; mp 185 °C

N-(2-methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (255)

Yield: 54%; brown solid; mp 136-138 °C

N-(4-chloro-2-methylphenyl)-6-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3b]pyridin-4-amine (258)

Yield: 28%; yellow solid; mp 138 °C Significant interest in the thieno [2,3-b]pyridine nucleus has arisen during the last three decades. Many researchers have reported the use of compounds based on this scaffold as a possible treatment of anxiety and depression, 1 bacterial infection, 2 inflammation, 3 leishmaniasis, 4 malaria 5 and autoimmune diseases. 6 Vilsmeier-Haack reaction is very practical method for versatile synthetic purposes. 7 The Vilsmeier-Haack reagent (POCl 3 /DMF) has been widely used in formylation, chlorination and cyclisation processes. 8 The preparation of 3formylquinoline through such reactions in one step starting from o-aminoacetophenone, inspired by the synthesis of 3formylchromone from o-hydroxyacetophenone, was reported. 9 The impact of replacement of a benzene ring with thiophene in the aforementioned cyclisation has not been investigated before. To study this reaction, we decided to construct 3-acetyl-2-amino thiophene, which could be used as a starting material for this purpose.

N-(4-chlorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-b]pyridin

N R 2 R 1 O R 2 R 1 N Cl O Ac S 8 A Ac NH 2 R 2 R 1 8-86% DMF/POCl 3 B R 2
In our research group, we have long experience of synthesising the thiophene nucleus by diverse synthetic pathways including Gewald's reaction. 10 Gewald's reaction is a one-pot reaction in which a carbonyl compound containing a methylene group in the α-position reacts with elemental sulfur and activated nitriles to produce 2-aminothiophene. 11 This reaction has been widely applied since it was discovered to result in many derivatives including different electron-withdrawing groups in the 3-position (e.g., CN, COOR, CONH 2 and COPh). 12 In 2006, Eller et al. introduced the first derivative having an acetyl group in the 3-position by following a two-component pathway involving cyanoacetone and 1,4-dithiane (the source of sulfur at the same time). 13 Herein, a typical three-component Gewald reaction was applied in which the cyanoacetone, carbonyl compound, and elemental sulfur reacted to produce 3-acetyl-2-aminothiophene derivatives (Scheme 1). Different amines (morpholine, triethyl amine and piperidine) were tried as basic catalyst in either ethanol or methanol. From our experiments, we can arrange the bases in decreasing order according to their efficiency: piperidine > triethylamine > morpholine.

It was found that alicyclic ketones gave better results than straight-chain aliphatic ketones; the reactivity in this case depended on the type and the place of substituents in relation to the carbonyl group and the α-methylene. The yields appeared to be low to moderate, with 4-tert-butyl cyclohexanone and saturated heterocyclic ketones giving increased yield (4e, 4j and 4k gave 86, 72 and 65% respectively) (Table 1).

The Vilsmeier-Haack-Arnold reaction was applied for the second step to achieve the cyclisation. The influence of the thiophene nucleus on the reaction is clear. Several trials were applied to achieve the cyclisation. By working in the reported conditions of 60 °C for five hours, 9c an unknown product was separated as a major component. Increasing the time of the reaction did not help. When the temperature was elevated to 100 °C for five hours, a mixture of the desired compound and an unknown by-product were ob-SYNTHESIS0 0 3 9 -7 8 8 1 1 4 3 7 -2 1 0 X © Georg Thieme Verlag Stuttgart • New York 2016, 48, 2881-2888 paper

Paper Syn thesis

tained; increasing the time of the reaction did not lead to the production of the desired compounds exclusively. The number of equivalents of the Vilsmeier-Haack reagent was found to have great influence upon the completion of the reaction. The mode of addition was also found to govern both the type of product formed and the yield. Addition of the reagent over seven hours then stirring the reaction mixture overnight appeared to be the best reaction conditions. In contrast to previous reports on the synthesis of 3formylquinoline and 3-formylchromone, the product was free from a formyl group (Scheme 1). 9 The replacement of the benzene ring with a thiophene ring is likely to have an impact on the final products, and may prevent further attack of the Vilsmeier-Haack reactant.

All the reactants gave yields from low to moderate, with the exception of 4j and 4k, which gave undefined mixtures on TLC, without any major component. Attempts to separate pure compounds from these mixtures by using flash chromatography failed (Table 1).

Table 1 Gewald and Vilsmeier-Haack Reactions

Scheme 1 General method for the synthesis of 3-acetyl-2-amino-thiophene and 4-chlorothieno [2,3-b]pyridine

O N + R 2 R 1 O S 8 piperidine R 2

Syn thesis

The mechanism of cyclisation is postulated to involve attack of the Vilsmeier-Haack reagent. In this mode, the enol form (I) is attacked by the reagent to form intermediate (II), which gives, after losing water, the iminium salt (intermediate IV). The latter intermediate is cyclised to the final product 5 (Scheme 2).

Increasing the number of equivalents of reagent, the time of reaction, or the temperature did not result in the production of formylated derivatives. The inductive effect of the thiophene ring may stabilise the electron within the system, which could decrease the nucleophilicity of the pyridine ring.

To explore the reactivity of the chlorine atom, thienopyridines were exposed to nucleophilic substitution using aniline as a nucleophile either in excess or in the presence of base. The chlorine atom could not been replaced, and application of Ullmann coupling conditions 14 did not help in performing the reaction. The product 6a was only accessible through Buchwald-Hartwig cross-coupling, 15 which was mediated by palladium acetate and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP) as ligand 16 in the presence of caesium carbonate and an inert gas atmosphere. After five hours, the starting material was completely consumed and the product was separated by using flash column chromatography. The product was obtained in very high yield (99%; Scheme 3). In conclusion, we report here the first synthesis of 3acetyl-2-aminothiophene by using the three-component Gewald reaction. The compounds exhibit a different mode of cyclisation in the reaction with Vilsmeier-Haack reagent than that reported for the reaction with o-aminoacetophenone, 9c which could be ascribed to the influence of the thiophene nucleus. This simple, two-step reaction allows the construction of some novel 4-chlorothieno [2,3-b]pyridine derivatives from very simple building units. Compound 5b reacted further with aniline by palladium-catalysed C-N cross-coupling to give the coupled product in high yield.

All solvents and reagents were purchased from commercial sources unless otherwise noted. Melting points were determined with a Büchi 530 digital melting point apparatus and are uncorrected. 1 H and 13 C NMR spectra were recorded at 400 and 100 MHz, respectively, using Me 4 Si as the internal standard. Hydrogen coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). High-resolution mass spectra were measured with a Micro-Tof-Q98 instrument in ESI mode. Column chromatography was performed using silica gel (60M, 0.04-0.063 mm). Thin-layer chromatography (TLC) was performed using silica gel plates (POLYGRAM SIL G/UV254, 0.20 mm), which were visualised under UV light.

Cyanoacetone (1)

(E,Z)-3-Aminocrotonitrile (10 g, 0.12 mol) was dissolved in 6N HCl (28.5 mL), and the solution was stirred and heated to 80 °C for 3 h. The reaction mixture was cooled, extracted several times with CH 2 Cl 2 , dried over anhydrous Na 2 SO 4 and concentrated under vacuo.

Yield: 5 g (53%); colourless liquid.

3-Acetyl-2-aminothiophene Derivatives; General Procedure

Carbonyl compound (0.025 mol) was added to freshly prepared cyanoacetone (0.03 mol) in either MeOH or EtOH (40 mL). Sublimed sulfur S 8 (0.03 mol) and piperidine (0.03 mol) were added and the mixture was stirred and heated to 55-65 °C for 24 h. Ice was then added and the formed precipitate was filtered under vacuum and washed with water. The obtained solid was crystallised from a suitable solvent, with the exception of 4g and 4j, which were collected directly without further purification.

1-(2-Amino-5,6-dihydro-4H-cyclopenta[b]thiophen-3-yl)ethanone (4a)

Yield: 2.7 g (61%); brown solid; mp 230 °C (toluene).

S O CH 3 NH 2 R 1 R 2 HN(CH 3 ) 2 S Cl N R 1 R 2 HOPOCl 2 S HO CH 2 NH 2 R 1 R 2 S HO C NH 2 R 1 R 2 Cl N S Cl H C NH 2 R 1 R 2 N S Cl N R 1 R 2 H H N I II IV X - X - X - H 2 O 4 V 5 X -= PO 2 Cl 2 N H Cl X - H H S HO C NH 2 R 1 R 2

1-(2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)ethanone (4b)

Yield: 2.8 g (58%); brown solid; mp 114-117 °C (toluene). 1

1-(2-Amino-5-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-3yl)ethanone (4c)

Yield: 1.5 g (28%); greenish solid; mp 180-182 °C (abs. EtOH). 1 H NMR (400 MHz, CDCl 3 ): δ = 6.97 (s, 2 H), 2.83 (dd, J = 15.6, 4.5 Hz, 1 H), 2.60-2.53 (m, 2 H), 2.42 (s, 3 H), 2.30-2.15 (m, 1 H), 2.01-1.76 (m, 2 H), 1.51-1.36 (m, 1 H), 1.10 (d, J = 6.6 Hz, 3 H).

1-{2-Amino-6-(tert-butyl)-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl}ethanone (4e)

Yield: 5.4 g (86%); beige-brown solid; mp 155-158 °C (cyclohexane). 1 H NMR (400 MHz, CDCl 3 ): δ = 6.99 (s, 1 H), 2.88 (ddt, J = 15.9, 4.7, 1.8 Hz, 1 H), 2.66-2.49 (m, 2 H), 2.40 (s, 3 H), 2.37-2.28 (m, 1 H), 2.07-2.00 (m, 1 H), 1.56-1.46 (m, 1 H), 1.40-1.28 (m, 1 H), 0.94 (s, 9 H).

1-(2-Amino-4-methyl-5-propylthiophen-3-yl)ethanone (4h)

Yield: 4.9 g (30%); brown solid; mp 113-115 °C (cyclohexane).

1-(2-Amino-4-methyl-5-pentylthiophen-3-yl)ethanone (4i)

Yield: 0.45 g (8%); greenish solid; mp 112-113 °C (cyclohexane).

4-Chlorothieno[2,3-b]pyridine Derivatives; General Procedure

For preparation of the Vilsmeier reagent, POCl 3 (2.5 mL) was dropped into DMF (10 mL) at 0 °C over 15 min with stirring. The starting materials (2.5 mmol) was dissolved in a minimum amount of DMF and the Vilsmeier reagent was added dropwise over 7 hours to the stirred solution at 100 °C. The reaction mixture was stirred at 100 °C. The progress of the reaction was monitored by TLC until the consumption of the starting material was complete (ca. 20-24 h), then the reaction mixture was treated with ice and water, neutralised with ammonium acetate and extracted with EtOAc several times. The product was obtained in pure form after purification by silica gel column chromatography (EtOAc-cyclohexane, 1:9) followed by recrystallisation (MeOH-H 2 O, 1:1).

4-Chloro

N-Phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4amine (6a)

In a three-necked round-bottom flask fitted with a condenser, septum, and thermometer, 5b (120 mg, 0.54 mmol) was dissolved in anhydrous toluene (5 mL), and Cs 2 CO 3 (400 mg, 0.12 mmol), Pd(OAc) 2 (4 mg, 0.054 mmol) and 2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (13 mg, 0.022 mmol) were added. Argon gas was applied and the reaction mixture was heated to 70-80 °C. Aniline (50 mg, 0.54 mmol) was dissolved in anhydrous toluene (3 mL) and added dropwise to the reaction mixture by using a syringe through the septum under argon. Upon complete addition, the temperature was raised to 110 °C and the progress of the reaction was followed by TLC until consumption of the reactants was complete (ca. 5 h). The reaction mixture was poured over ice and water, and extracted with EtOAc. The final pure product was obtained after silica gel column chromatography (cyclohexane-EtOAc, 7:3) and recrystallisation (cyclohexane).

N-Phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4amine (6a)

Yield: 149 mg (99%); yellow solid; mp 102-105 °C (cyclohexane); R f = 0.50 (EtOAc-cyclohexane, 3:7).

Introduction

CDC25 (Cell Division Cycle

) is dual specificity phosphatase that play critical role in the division of eukaryotic cells. Up to date, three CDC25 isoforms were identified: A, B and C [1][2][3][4] . They dephosphorylate CDK/cyclin protein complexes which are key regulator of cell division. Noteworthy,CDC25B is needed for checkpoint recovery to repair DNA before mitosis 3,5 . CDC25A and CDC25B are overexpressed in many different human cancers: (e.g.: breast cancer: 70% and 57%, colorectal cancer: 47-53% esophageal cancer: 46-66% and 48-79% respectively) 1 . Consequently, they appear as potential target in cancer therapy. Wide range of different scaffolds has been reported as CDC25 phosphatase inhibitors including quinoids, dysidiolide and large collection of miscellaneous structures 6,7 . Chalcones have numerous biological activities containing antihypertensive, antifungal, anticonvulsant, antiviral, antioxidant, anti-inflammatory, antimalarial, anti-HIV, antiprotozoal, antimicrobial, antifilarial, 8,9 they also proved their antitumor activities through their action against many molecular targets 10 . The substituted chalcones in position 4 of ring (B) was even reported as CDC25 inhibitors with considerable activity (Figure 1) 11 .Coumarines are well known as anticancer 12 active compounds with many different mechanisms like kinase inhibitors, apoptosis induction, angiogenetic inhibition, HSP90 inhibitors, telomerase inhibitor and antimitotic. 13 Some coumarines have anticoagulant and cardiovascular properties (dicoumarol and warfarin) others have antibiotic activities as novobiocin and clorobiocin which are naturally occurring. 14 Geiparvarin is another example of coumarine with dual activities: MAO-B inhibitory and antitumor. 15 Coumarine has been combined with different pharmacophores, and showed antioxidant, anti-inflammatory, anticancer, and antimicrobial activities. 16 Some of them, hybridized with quinones 17,18 or dimerized thanks to a disulfide bond of various lengths (Figure 1) 19 are particularly active.

Figure 1. Examples of substituted and coumarines based chalcones inhibitors of CDC25s

Synergistically, coumarine and chalcones combined structure appeared as promising anticancer molecules. They showed their abilities against number of human cancer cell lines, 20 therefore, series of novel chalcones derived from coumarine were synthesized by aldol condensation. The obtained compounds were investigated by docking simulation and then evaluated in vitro on CDC25A and B enzymes.

Results and Discussion

Chemistry

All compounds were synthesized by well-known base-catalyzed aldol condensation 21 between 3acetyl coumarines and cinnamaldehyde. Previously, in our laboratory, substituted 3-acetyl coumarines were prepared starting from salicylaldehyde 22 and ethylacetoacetate 23,24 . Commercially available cinnamaldehyde and other synthesized derivatives were introduced as condensed partner. In addition cinnamaldehydes compounds were synthesized with two different methods: Method A consisted of the reduction of cinnamic acid using LiAlH(OtBu) 4 25 Method B was done by Wittig reaction 26 between a benzaldehyde and an acetal 27 . All prepared compounds were purified by column chromatography (cyclohexane: ethyl acetate 9:1).

Biological evaluation

Fluorimetric analysis Compounds 1 to 8 were evaluated in-vitro against recombinant human CDC25 phosphatases by fluorimitric method. (Table 1 The electrophilic substitution: Cl and Br on position 6 of the coumarine part led to inhibit CDC25A in higher extent than methoxy substituted analogue. The order of activity of these compounds was 4>3>2. This order was inversed in the case of CDC25B. Introduction of methoxy group on the phenyl moiety increased the activity of compound 4 toward CDC25B but it diminished with CDC25A (from 35.95% to 7.34%). Furthermore, comparing the activities of compounds 1, 6 and 7; it showed that the methoxy group substitution on the phenyl ring increased the activity against both isoforms. The substitution on position 7 of coumarine with N, N-diethylamino group (compound 5) increased the inhibitory activity on CDC25A: 40.64±0.92% and CDC25B: 54.94±1.13%). This compound was finally the most potent candidate among all tested structures (Table 1).

MALDI-TOFMS Matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOFMS) was applied to check if the synthesized structures may be a candidate to CDC25 inhibition. In case of inhibition, the applied procedure is also able to inform about its reversibility or irreversibility. 28 Compounds with the highest values in biological analysis as inhibitor (3, 4 and 5) gave a positive results in MALDI-TOFMS Their protonated molecular ion directly appeared in the mass spectrum meaning that they behaves as reversible inhibitors as described by Sibille et al. 29 and Bana et al. 17

Molecular modelling

The molecular surface scanning of CDC25B reveals that its 3D structure exhibits a large groove just close to active cysteine 30 . Thus, this site called swimming pool also appears as an interesting target for inhibiting CDC25 by the binding of small molecules. 31 Hence, the binding inside this catalytic site cannot only be taken into account as a dependable mode of inhibition. We suggested designing of chalcones derived from coumarine and investigating the designed products by docking simulation to test the mode of orientation of each moiety in relation to active and inhibitory sites. Possibility of formyl pyranone ring to surrogate the phosphate group of the native substrate was presumed 6 .

The surface structure of the CDC25B catalytic domain (1QB0) 32 , indicated that the distance between the shallow active site and the large groove is ~ 7.3 Å. This distance must correspond to the linker length between the position 3 of the coumarine and the position 1 of the ring (B) of the chalcone-like moiety for inhibitor in 3D model. Using CDC25B as key model, we found that conjugating ketone-diene chain (n=2) provided a better linker length than a single double bond (n=1) for our approach. Coumarine was one of the counterparts of choice while the other was substituted aromatic ring, and they were intended to be hybridized. The docking study was achieved using AUTODOCK Vina 33 . We assumed that the coumarine compound is able to bind the cysteine of the catalytic site of CDC25B. Our docking study showed that, for the half of the structures tested, the coumarine part rather fitted inside the swimming pool of CDC25B, while the chalcone part entered the active site. This is observed for compounds 1, 4, 5 (Figure 3) and 6 blocked whereas it is the contrary for compounds 2, 3, 7 and 8.

The docking with CDC25A was more challenging as the catalytic cysteine is located inside very shallow groove, with a very small cavity appearing unsuitable for fitting neither coumarine nor phenyl moiety of the compounds series. The docking results showed that the most of molecular poses are oriented in a way that the coumarine was toward the catalytic cysteine (cys430) which complying with phosphates group simulation assumption (1, 2, 3 (Figure 4), 4, 5, 6). (Table 1).

This orientations similar to phosphate cradling model of the native substrate 34 . Compounds 7 and 8 had their binding mode in the opposite direction. Docking could not give the explanation of the inhibitory activity 29,[35][36][37][38][39][40][41][42][43][44][45][46][47] , so it was necessary to synthesize these molecules and evaluated their biological activities with CDC25A and B then verification of their reversibility.

The CDC25s have two interesting sites: the active site and the swimming pool which can play an important role, so it would be efficient if both of them are tested in same time.

It has been suggested that the hybrid structure may be more efficient at inhibiting CDC25s. Indeed, the first moiety of the inhibitor could fit the binding pocket (i.e. the swimming pool) while the other part could bind to the active cysteine 48 .

Conclusion

The antitumor characteristics of both coumarine and chalcones inspired our approach to design hybrid structures could block both grooves of the protein Mediterr.J.Chem., 2015, 5(1), D. Dridi et al. 327 (active and inhibitory) highlighting the lack of toxicity in the same time. Several chalcones derived from coumarine were synthesized, the binding modes of them were tested by molecular modelling simulation, the probability of phosphate surrogating by coumarine ring has been shown by certain derivatives. Biological evaluation revealed some activity of the compounds. N, N-diethylamino substitution on position 7 of coumarine (compound 5) increasing the inhibitory activity on both isoforms of CDC25. The derivatives proved to perform reversible inhibition by MALDI-TOF analysis (compounds 3, 4 and 5). The designed scaffold could be considered as a basis of further lead optimization in order to obtain better inhibition.

Experimental Section

Chemistry

All solvents were used as purchased unless otherwise noted. Melting points were determined on a Buchi 530 digital melting point apparatus. 1 H and 13 C NMR spectra were recorded at 400 MHz and 100 MHz, respectively, using Me 4 Si as the internal standard. Hydrogen coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m). High resolution mass spectra were measured using a MicroTof-Q98 instrument in ESI mode. Column chromatography was performed using silica gel (60M, 0.04-0.063 mm). Thin layer chromatography (TLC) was performed using silica gel plates (POLYGRAM SIL G/UV 254 , 0,20mm). For the visualization, TLC plates were placed under UV light.

General procedure for synthesis of compounds 1 to 8

In round bottom flask, 1.1 mmole of cinnamaldehydes and 1 mmole of substituted 3-acetyl coumarine were mixed in ethanol, and stirred under reflux till dissolving of coumarine derivatives. Afterward, 5% mole of piperidine was added and the reaction retained under reflux for 24 h. When the reaction completed, the solution allowed to be cooled, and the product started to precipitate. The obtained solid was filtered and recrystallized using ethanol.

3-(1-Oxo-5-phenyl-2Z, 4Z-pentadien-1-yl)-2H-1benzopyran-2-one (1)

Yellow powder; Yield: 52%; Mp: 184°C. 49

6-Chloro-3-[1-oxo-5-(2,4-diméthoxyphenyl)-2E,4E-pentadien-1-yl]-2H-1-benzopyran-2-one (8)

Colorless solid, Yield: 20%; Mp: 80°C

Biological evaluation

Recombinant human CDC25 phosphatases were prepared by previously described method 19 . Assay took place in 96-well plates; buffer solution was adjusted in the following quantities: 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, and 0.1% Bovine Serum Albumine, pH 8.1. Incubation period of substrate 3-O-methylfluorescein phosphate with enzyme was 2 h in 30⁰C. Enzyme was exposed to the inhibitors for 20 min and the residual activity of the enzyme has been determined by fluorimetric method.

MALDI/TOFMS

All compounds were dissolved in ethanol at a concentration of 10 -2 M. The solution were then further diluted to 2 • 10 -3 M with ultrapure water. Phosphatases CDC25A was solubilized in Tris A buffer (50 mM Tris, 50 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol [DTT], pH 8.0) at a concentration of 8 • 10 -5 M. One hundred and fifty µl of CDC25A was incubated with 40 µl of compounds for 1 h. Solution was ultracentrifugated on Microcon filter unit with a mass cutoff of 30 kDa (Millipore). The unbound material was eliminated by centrifugation during 10 min at 12,000 rpm and washed three times by 100 µL of ultrapure water. The retentate was then dissolved in 20 µL of ultrapure water. Two µl of the retentate was spotted on the MALDI plate (2 spot for sample in two line) , evaporated to dryness at room temperature. Then one µl of saturated solution of DHB (dihydroxybenzoic acid) was deposited for one of spot and HCCA (αhydroxycyanocinnamic acid) was deposited for the second spot of each sample on the plate to evaluate the response of each compound with both of matrix, and conserved the spectrum which had the intense response.

The presence of the protonated molecular ion in the MS spectrum is corresponding to a reversible inhibitor activity.

Introduction

Inflammation is a general and essential response to any aggression and may be shown by a localized response or by a more generalized one. Inflammation is thought to be at the start of different diseases including obesity, cancer and neurodegenerative illness. The biosynthetic cascade of arachidonic acid leading to the formation of prostaglandins (PG), leukotrienes (LT), hydroxyeicosatetraenoic acids (HETE) have been shown to be involved in the inflammation as well as other processes. Blocking the cascade is a possible way to fight inflammation. Inflammation is the process by which leucocytes and material derived from the serum are directed to the site of tissue injury. Coumarins are widespread natural chemicals [1]. Many of these compounds have a pharmacological use [2]. In this review we will investigate coumarins and derivatives with anti-inflammatory activity. We will present this study in terms of the chemical structure: simple coumarins, furo-and pyrano-coumarins.

In the first part, naturally occurring coumarins will be presented. The second part will be devoted to synthetic coumarins.

Naturally Occurring Coumarins

Simple Coumarins

Figure 1 shows the plant derived coumarins described in the literature with anti-inflammatory activities. Coumarin 1 itself was used for the treatment of edema [3]. A general study on 2, 3, 5, 6, 7, has been run on TNBS (trinitrobenzenesulfonic acid) induced colitis [4]. The study showed that these coumarins are potential agents for treating the inflammation. The effect seems directly related to the Coumarin 1 itself was used for the treatment of edema [3]. A general study on 2, 3, 5, 6, 7, 8 has been run on TNBS (trinitrobenzenesulfonic acid) induced colitis [4]. The study showed that these coumarins are potential agents for treating the inflammation. The effect seems directly related to the antioxidant properties of the coumarins, in particular for daphnetin 6, esculin 5 and scoparone 3. Food containing coumarins could be a possible strategy to prevent Inflammation Bowel Diseases (IBD). Natural products like esculetin 4, fraxetin 7, daphnetin 6 and other related coumarin derivatives are recognized as inhibitors not only of the lipoxygenase and cycloxygenase enzymic systems, but also of the neutrophil-dependent superoxide anion generation [5].The study on the ethanolic extract of Artemisia capillaris Thunberg (Compositaea) used in traditional Chinese medicine for amelioration of skin inflammation showed inhibition activity on 5-lipooxygenase [6]. In particular, esculetin 4 had the property along with other compounds [6]. Experiments run on inflammation in animal models (xylene induced ear edema, carrageanan induced paw edema and carrageanan induced mouse pleuresy) [7] have shown that esculin 5, a glycoside of esculetin, attenuated the phenomena. In-vitro pro-inflammatory cytokines levels of TNF-α and IL-6 were reduced by esculin. It was also found that esculin significantly inhibited LPS (lipopolysaccharide) induced activation of MAPK pathway in peritoneal macrophages. Esculin could be a promising agent for treating inflammatory diseases in humans. Another study on rat peritoneal leukocytes using 16 different coumarins [8] has pointed out the effect on LTB 4 and TXB 2 generated by calimycin (A23187). Fraxetin 7, esculetin 4, daphnetin 6, 4-methylesculetin 9 were the most active on LTB 4 and herniarin 10, 4-methyl-5,7 dihydroxycoumarin 11 and daphnetin 6 on TXB 2 with IC 50 ranging from 1-75 µM. Daphnetin 6 appears often as an effective compound. The compound extracted from Daphnea odora was tested in collagen induced arthritis in rats [9]. The therapeutic effect was determined by the balance of Tregs and Th-17. The levels of these lymphocytes were evaluated by ELISA and those related receptors. by immunochemistry. Administration of daphnetin for 21 days highly alleviated the severity of arthritis by modulating the balance. In continuation looking at the power of daphnetin, some finding suggest that it might have a neuroprotective effect in stressed mice [10]. The role of daphnetin in microglial inflammatory response was explored. The study showed that all the pro-inflammatory mediators (IL-1B, TNFα induced by LPS) were strongly depressed in a dose dependent manner in BV 2 microglia and it inhibited the LPS-induced iNOS and COX-2, even NO formation by microglia. In general daphnetin inhibits microglial activation and proinflammatory responses by modulating a series of intracellular pathways (IKK/κB,MAPKs and Pt.3K/Akt).

Umbelliferone 12 has not been found to have anti-inflammatory properties but some of its derivatives did. Among these, aurapten 13 and umbelliprenin 14 were the most studied. These compounds are found especially in the Ferula species. Aurapten 13 from Ferula szowitsiana showed cancer chemopreventive properties suggested by anti-inflammatory activity of the compound [11]. A mini-review on the biological activity of umbelliprenin from Artemisia species has appeared [12] linking the activity to iNOS inhibition. A paper [13] describing an extract from Ferula szowitsiana describes the activity of 6 terpenoïd coumarins isolated (methyl galbanate, galbanic acid, farnesiferol A, badrakemone, umbelliprenin and aurapten). In this case methylgalbanate 15 was the best inhibitor of nitric oxide production; it was better than umbelliprenin isolated from the same species.

Three naturally occurring O-prenyl coumarins; 4-isopentenyloxy-5-methyl-coumarin 16, 6-isopentenyloxy, -7-methoxy-coumarin 17, 8-isopentenyloxy-7-methoxy-coumarin 18; from respectively Gerbera crocera and Gerbera serrata (Asteraceae), Haplophyllum pedicellatum (Rutaceae) and divers Artemisia species were prepared by synthesis [14]. They have been studied as dual anti-bacterial and anti-inflammatory compounds for controlling peritoneal diseases. U937-3xκB-LUC cell line was used to evaluate the ability to inhibit the activation of NF-κB signaling pathway induced by LPS. Compounds 17 and 18 were effective in the inhibition but not 16. In fact, these two compounds also had anti-bacterial activity and were examples of dual action.

Osthole 19, 8-prenyl-7-methoxy-coumarinapplied in clinical practice in Traditional Chinese Medicine is found in different plants (Angelica, Archangelica, Citrus, Clausena) and in high content in the mature fruit of Cnidium monnieri. The anti-inflammatory properties are described in a recent review ( [15] and ref cited therein). Osthole has also been described as decreasing ocular inflammation [16].

Murracarpin 20, structurally close to osthole and isolated from Murraya exotica showed strong anti-inflammatory and analgesic effect [17].

Ethylacetate extract of Canadian marple syrup contains many phenolics related compounds including coumarins. Fifteen pure compounds available were tested in LPS stimulated RAW264.7 murine macrophage and the decrease of nitric oxide and PGE 2 measured. Unfortunately, the coumarinic isofraxidin 21 was not active [18]. All the biological results of simple coumarins are summarized in (Table 1). 1 Carageenin paw edema in rat; 2 Arachidonic acid induced ear edema in mice; 3 LPS-stimulated mouse peritoneal macrophage; 4 LPS-stimulated BV2 microglia; 5 Aβ-stimulated BV2 microglia; 6 LPS-stimulated macrophage RAW264.7; 7 LPS-stimulated U937-3xxKB-LUC human monocytic cell line; kg is referring to the mice weight; % is referring to percentage of inhibition. * p < 0.05 versus negative control. ** p < 0.01 versus negative control.)

Condensed Coumarins

The most condensed coumarins isolated from natural sources are furo-and pyranocoumarins. A review in 2006 [19] reports the biological activities of prenyloxy-furocoumarins and the in-vivo remarkable anti-inflammatory effect. We will review the publications appearing since then.

Furocoumarins

Among furocoumarins (Figure 2), imperatorin 22 has been the most investigated. The substance is found in Angelica dahurica, Glhenia littoralis, Cnidium monnieri and Peucedanum praeruptorum as well as in other Apiaceae and Umbellifereae species. These plants are used in traditional Chinese and Korean Medicine. as antipyretic and analgesic. Most assays that investigated the behavior against inflammation were run on LPS activated RAW264.7 cells [20-26] and carrageanan induced mouse paw edema [21]. In general, inhibition of NOS as well as COX-2 were found to be the cause. Imperatorin has also proved to be efficient on allergic rhinitis [27], acute lung injury induced by LPS in mouse [28], on sebum production in human sebocytes in vitro [29] and it has had an anti-allergic effect on Th-2 mediated allergic asthma [30]. It has been stated that phellopterin 23 presents the same features as imperatorin concerning this activity [20]. Dimeric furocoumarins from the roots of Angelica dahurica had the same activity [31]. An isomer of imperatorin, isoimperatorin 24, from the roots of Angelica dahurica [32] or Cimicifugae rhizome [33] was found to inhibit COX-dependent phases or TNF-α induced expression indicating its anti-inflammatory properties.

Various furocoumarins extracted from Chinese herbs were tested for iNOS 5 inhibition [34,35]. Among the different furocoumarins: imperatorin 22, deltoin 25 and sphondin 26 gave the best results. As sphondin is also available in different Heracleum species it may provide a basis for the use of plant extracts against inflammation. For example, sphondin extracted from Heracleum laciniatum inhibits IL-1β induced PGE2 release in A549 cells [36]. This inhibition is mediated by suppressing of COX-2 expression and maybe at least in part through suppression of NF-κB activity. It is worth noting that the extract of fruits from Heracleum crenatifolium contains different furocoumarins and has anticonvulsivant activity in male albino mice [37]. A review on Heracleum persicum details the pharmacology of the furocoumarins in it [38].The biological results of furocoumarins are summarized in (Table 2).

Pyranocoumarins

Pyranocoumarins (Figure 3) have less been tested for this activity. The first compound of this class extracted was seselin 27 from Sigmathantus trifoliatus able to inhibit inflammatory hyperalgesia and having also antinociceptive effect [39]. Obtained from Seseli resinosum, calipteryxin 28 and a dihydroseselin derivative were associated in the inhibition of inflammatory enzymes (iNOS and COX-2) and cytokines (TNF-α and IL-6) via NF-κB, MAPK and Akt pathways [40]. (+)-Praeruptorin 29 from the roots of Peucedanum praerupotorum reduced NO production and release of IL-1β, IL-6 and TNF-α induced by LPS-stimulated macrophage [41,42]. Other derivatives anomalin 30 and corymbocoumarin 31 were obtained respectively from Saposhnikovia divaricata and Seseli gummniferum have been used in LPS-stimulated RAW264.7 macrophages [43]. Anomalin dose-dependently inhibited iNOS, COX-2 mRNA and protein expression. Concerning corymbocoumarin, it exerts its effect by suppressing NF-κB activation and inducing HO-1 expression [44]. The biological results of this kind of coumarins are summarized below in (Table 3). An isomer of imperatorin, isoimperatorin 24, from the roots of Angelica dahurica [32] or Cimicifugae rhizome [33] was found to inhibit COX-dependent phases or TNF-α induced expression indicating its anti-inflammatory properties.

Various furocoumarins extracted from Chinese herbs were tested for iNOS 5 inhibition [34,35]. Among the different furocoumarins: imperatorin 22, deltoin 25 and sphondin 26 gave the best results. As sphondin is also available in different Heracleum species it may provide a basis for the use of plant extracts against inflammation. For example, sphondin extracted from Heracleum laciniatum inhibits IL-1β induced PGE 2 release in A549 cells [36]. This inhibition is mediated by suppressing of COX-2 expression and maybe at least in part through suppression of NF-κB activity. It is worth noting that the extract of fruits from Heracleum crenatifolium contains different furocoumarins and has anticonvulsivant activity in male albino mice [37]. A review on Heracleum persicum details the pharmacology of the furocoumarins in it [38].The biological results of furocoumarins are summarized in (Table 2).

Pyranocoumarins

Pyranocoumarins (Figure 3) have less been tested for this activity. The first compound of this class extracted was seselin 27 from Sigmathantus trifoliatus able to inhibit inflammatory hyperalgesia and having also antinociceptive effect [39]. Obtained from Seseli resinosum, calipteryxin 28 and a dihydroseselin derivative were associated in the inhibition of inflammatory enzymes (iNOS and COX-2) and cytokines (TNF-α and IL-6) via NF-κB, MAPK and Akt pathways [40]. (+)-Praeruptorin 29 from the roots of Peucedanum praerupotorum reduced NO production and release of IL-1β, IL-6 and TNF-α induced by LPS-stimulated macrophage [41,42]. Other derivatives anomalin 30 and corymbocoumarin 31 were obtained respectively from Saposhnikovia divaricata and Seseli gummniferum have been used in LPS-stimulated RAW264.7 macrophages [43]. Anomalin dose-dependently inhibited iNOS, COX-2 mRNA and protein expression. Concerning corymbocoumarin, it exerts its effect by suppressing NF-κB activation and inducing HO-1 expression [44]. The biological results of this kind of coumarins are summarized below in (Table 3).

Synthetic Coumarins and Pyranocoumarins

As functionalized coumarins can be easily prepared through synthesis, different compounds not naturally found have been prepared and tested (Figure 5). Starting from 6-chloro and 6-methoxy 4-bromomethyl-coumarin, paracetamol ethers 32, 33, 34 have been prepared (Figure 4).

Synthetic Coumarins and Pyranocoumarins

As functionalized coumarins can be easily prepared through synthesis, different compounds not naturally found have been prepared and tested (Figure 4). Starting from 6-chloro and 6-methoxy 4-bromomethyl-coumarin, paracetamol ethers 32, 33, 34 have been prepared (Figure 5). Screening the activity using carrageenan induced edema in rats gave the best results for compounds 32 where R = Cl and OCH3 was in position 6 [45]. A series of esters prepared from coumarin-3-carboxylic acids 35 obtained from the corresponding salicaldehydes, were tested in LPS-induced lung inflammation and elastase induced lung injury [46]. The data obtained showed that all coumarins had anti-inflammatory properties and that anti-elastase activity is essential to reducing lung inflammation in-vivo. For looking at 15-LPO inhibitors, a series of O-prenylated coumarins were synthesized (isopentyloxy, farnesyloxy, geranyloxy in 4, 5, 6, 7, and 8 position) [47].

Synthetic Coumarins and Pyranocoumarins

As functionalized coumarins can be easily prepared through synthesis, different compounds not naturally found have been prepared and tested (Figure 5). Starting from 6-chloro and 6-methoxy 4-bromomethyl-coumarin, paracetamol ethers 32, 33, 34 have been prepared (Figure 4). Screening the activity using carrageenan induced edema in rats gave the best results for compounds 32 where R = Cl and OCH 3 was in position 6 [45]. A series of esters prepared from coumarin-3-carboxylic acids 35 obtained from the corresponding salicaldehydes, were tested in LPS-induced lung inflammation and elastase induced lung injury [46]. The data obtained showed that all coumarins had anti-inflammatory properties and that anti-elastase activity is essential to reducing lung inflammation in-vivo. For looking at 15-LPO inhibitors, a series of O-prenylated coumarins were synthesized (isopentyloxy, farnesyloxy, geranyloxy in 4, 5, 6, 7, and 8 position) [47]. 5-Farnesyloxycoumarin 36 showed the most potent activity against soybean lipoxygenase and 6-farnesyloxycoumarin 37 against human lipoxygenase. A great number of 7(2-oxoalkoxy)coumarins 38 have been prepared by condensation of hydroxycoumarins with α-chloroacetone [48] and the effects on expression of iNOS and COX-2 measured in comparison with 7-hydroxycoumarin. Result was that the substitution increased the activity compared to the unsubstituted one. Most active compounds were 38a and 38b. As it has been demonstrated, gold nanoparticles can reduce the expression of pro-inflammatory molecules [49], the idea to conjugate scopoletin 2 to gold nanoparticules emerged [50]. Scopoletin from Artemisia roxburghiana was conjugated in situ to gold nanoparticules prepared by reduction of tetrachloroauric acid. The NO inhibitory activity of scopoletin was unaffected by the conjugation (NOS was induced by LPS on J.774.2cell lines). However, the conjugation increased the prevention of oxidative burst of ROS in the whole blood phagocytes and isolated neutrophiles.

A methodology to prepare coumarins from ortho-benzoquinones has been used for the synthesis of benzopyranocoumarins 39 [51-53] analogs to benzo [1]khellactone. All tested compounds [52], highly inhibited soybean lipoxygenase, competing with DMSO for OH • and some induced protection against carrageenan induced rat paw edema. Another series [53] showed similar results for competition with DMSO for OH • , for O 2 -scavenging and lipoxygenase inhibition. The biological results of synthetic coumarins are summarized in (Table 4). Vilsmeier-Haack reaction is very applicable method for versatile synthetic purposes. 1 Vilsmeier-Haack reagent (POCl3/DMF) has been widely used in formylation, chloroformylation of ketones and cyclization. 2 Preparation of 3formylchromone from o-hydroxyacetophenone via such reaction through one step inspired synthesis of 3formylquinoline from o-aminoacetophenone. 3 Also, 2-acetamido-5 substituted thiophene 1 are reported to react with VH reagent under classical conditions (65 °C) to give mixture of compounds 2 and 3 (Scheme 1). 4 Adjusting the reaction condition allowed the authors to prepared 2 or 3. 6 Noteworthy that the reaction condition in case of 3 was harsh (reflux in VH reagent for 1.5 -4 hours). In a previous work we have already described the reaction of 3-acetyl-2-aminothiophenes 10 with VH reagent. Only 4chlorothieno [2,3-b]pyridine 12 was obtained in low to moderate yield when reaction run at 100 °C for 24 hours (at 65 °C mixture of unknown compound as major and 12 as minor was obtained). 6 Formylation was not obtained when compound 12 was subjected to VH reagent at different heating condition (65 -100 ⁰C) which is expected from pyridine as an electron deficient nucleus.

To make approach toward comprehension of reaction mechanism, we decided to construct N-protected starting materials. This protection was intended to be done by electron withdrawing group, like acetyl or pivaloyl groups.

All thiophenes were synthesized by three components classical Gewald's synthesis which we reported. 6 N-acetylated aminothiophenes were simply prepared by a known procedure for most compounds. 7 This procedure was required addition of acetyl chloride to a dissolved thiophene in acetic anhydride (11h and 11i) or by using trimethylamine as another additive to catalyse the reaction (compound 11j).

These N-acetylated products were treated by Vilsmeier-Haack reagent at 65 ⁰C and 100 ⁰C. At each temperature we used two equivalents of reagent (same equivalent of preparation of chlorothienopyridine 6 or double amount). Addition of the reagent was done at once or dropwise over nearly 7 hours. It was found that every alteration of VH reagent amount and mode of addition at different temperatures led to different yields and products.

Scheme 3 General synthesis of formylated chlorothieno [2,3-b]pyridine and formyl-free chlorothieno [2,3-b]pyridine from N-acetyl-3-amino-2-acetylthiophenes At 65 °C; when same previously used reagent equivalent was applied, chlorothienopyridine 12 was the major product in low yield, while formylated product 13 was traces. When this amount was doubled and added at once, yield of formylated product 13 raised to be around 94% -32%) while formyl-free compounds 12 were 25% to 0% in 4-5 hours. Dropping the reagent over long period of time resulted in excessive decrease in the yield of both products, the reagent amount made no difference.

At 100 °C the reagent was dropped over long period of time (5-7 hours) and the reaction left under stirring for 20-24 hours. 6 Maximum yield of chlorothienopyridines was obtained. When the same amount of the reagent was added at once, yields of both folmylated and formyl-free compounds were dramatically decreased, but the formylated product was in greater amount.

Noteworthy that performing the reaction at room temperature resulted in synthesizing chloroformylthienopyridine 12 as major product. The reaction time needed to be beyond 72 hours in order to obtain maximum productivity, using the same reagent equivalent which we described. 8 Pivaloyl derivative 14 was exposed to the same reaction condition of synthesis of chlrothienopyridine 6 and formylated product 12b was the only output of this reaction (yield, 42%), the same protected analogue gave no reaction at room temperature and 19% yield only at 65 °C using doubled amount of reagent (Scheme 4).

Scheme 4 Exclusive synthesis of formylated cholorothienopyridine under the condition of synthesis formul-free chlorothienopyridine Differently from reported before, 4 acetyl group was not involved in cyclization. It is suggested to play role by its electron withdrawing capability, which may result in stabilizing the double bond in intermediate IV (Scheme 5) making it more prone to another iminum ion attack. In contrary to the reaction of acetanilide, 5 in which N-acetylation allowed synthesizing the 4-chloroquinoline 5 in low yield or had no influence.

Using higher amount of the reagent, may result in rapid reaction and cyclization so acetyl group leaves the reactant after accomplishment of another nucleophilic attack (intermediate VII) (Scheme 5). Additionally, acetyl group protection is stable toward deacetylation at 65 °C.

In contrast, lower quantity of the reagent added dropwise and higher temperature (100 °C) gives chance to departure of the acetyl group in the acidic media. This made the amino group free at the early stage of the reaction and pyridine seems like not susceptible to the excessive iminium ion.

By the way, pivaloyl group is more stable than acetyl group. Working under higher temperature (100 ⁰C), and with the normal reagent quantity, 6 gave only the formylated product, that may emphasize our suggestion.

N-protection provides us more knowledge about the mechanisms of cyclization and chlorination of 3-acetyl-2aminothiophene. These two processes more reasonable to happen by two attacks of iminium ion rather than one. In case of our previous proposal; that cyclization and chlorination take places by only one nucleophilic attack, 6 N-acetylation is supposed to produce merely chlrothienopyridine rather than 3formyl derivatives.

The mechanism of reaction is postulated to occur by two possible routes. First mode of cyclization in which the acetyl group leaves just prior to the chlorination (intermediate IV). One HCl molecule help in liberation of acetyl group as acetyl chloride. The second pathway is assumed to happen at lower temperature, in which the acetyl group departure is retarded to allow the non-resonating double bond of pyridine ring Directing the reaction toward one of the two products could be considered as controllable by modification the conditions of the reaction; temperature, mode of addition (which was found very crucial) and amount of the reagent. Differently from what reported before for the synthesis of chloroquinoline, our product appeared to behave completely in opposite way. Chloroquinoline is formed as traces when acetamido acetophenone was used while chlorothieno [2,3-b]pyridin was formed by using of 3acetyl-2-aminothiophene. 6 Protection of the amino group of the last, made the production of formylated product reachable which is the normal result of this procedure with o-aminoacetopenone and o-hydroxyacetophenone.

14

 14

6 7 8 Scheme 3

 83 Scheme 3 Hinsberg thiophene Synthesis 1.1.2 Synthesis of thiophene using multicomponents reaction 1.1.2.1 General synthesis of 2-functionalized thiophene A synthetic pathway was established by reacting β-chloroacroline derivatives 10 with

10 12 Z= electron withdrawing group Scheme 5

 10125 Scheme 5 Synthesis of 2-functionalized thiophene.

Scheme 7 2 -

 72 Scheme 7 Cyclization of ketene dithioacetal into thiopheneThe reaction mechanisms are depicted in Scheme 8 and Scheme 9; in which 1,4Michael addition of the negative charge of sodium sulfide or the thioglycolate leads to formation of S-alkylated intermediate upon elimination of the methylsulfanyl group. These addition-elimination take place in two steps in case of thioglycolate or three steps with sodium sulphide. The last intermediate goes toward thiophene through cyclization according to Claisen, Thorpe-Ziegler or Dieckmann reaction to form the thiophene ring.21

19 20 21 X=

 21 Scheme 10 General scheme of Gewald synthesis 1.1.3.1.1 Mechanism of Gewald reaction 26

  Scheme 11 Mechanism of Gewald reaction 1.1.3.1.2 Modified Gewald reaction 1.1.3.1.2.1 Synthesis of tellurophene and selenopheneTellurophene and selenophene 24 were accessible via Gewald procedure by replacement elemental sulphur by tellurium or selenium (Scheme 12).28

28

 28

22 23 24 X=

 24 Se or Te while Y = COOEt, CONH2 or CN.

32 33 Scheme 15

 323315 Scheme 15Two component Gewald synthesis of 3-acetyl-2-aminothiophenes.

1 . 2 . 1

 121 Scheme 17

32 40 41 Scheme 19

 4119 Scheme 19 General method of 3-acetyl-2-amino-thiophene 1.2.1.4.1 Alicyclic ketones

Figure 1

 1 Figure 1 2D type of NMR (HSQC) of compound 49 Each aliphatic proton surrounding the centre of asymmetry resonating solely and quite distinctive from the other carried on same carbon. Correlating the peaks to every proton done using 2D type of NMR. HSQC and HMBC were employed to identify C-H direct and indirect coupling. For compound 49; proton with chemical shift δ = 1.51 -1.36ppm was found to be carried on carbon which had δ = 31.0 ppm while the other proton appeared more deshielded in δ = 2.01 -1.93 ppm. Carbon with chemical shift of 36.6 ppm had its protons separated (δ = 2.30 -2.15 and 2.83 ppm). Sole hydrogen signals was found at δ = 1.92 -1.76 ppm (Figure1). Same techniques were applied for 51 and 53 and in case of 51 it revealed that the single proton on carbon of δ = 27.8 ppm had chemical shift of 1.47 -1.35 ppm and carbon with δ = 29.2 ppm had protons viewed at region of δ = 2.72 -2.61, 2.85 -2.76 ppm while protons of carbon at δ = 32.9 ppm were in the area of δ = 2.21 -2.12, 2.61 -2.54 ppm (Figure2).

Figure 2

 2 Figure 2 2D type of NMR (HSQC) of compound 51 53 exhibited some difference from the previously investigated examples; protons on the aliphatic region had different chemical shifts even for those which are not adjacent to the asymmetric centre (δ = 26.3 ppm). Protons which are attached to it were resonating

Chapter 1 - 20 Figure 3

 1203 Figure 3 2D type of NMR (HSQC) of compound 53 1.2.1.4.2 Aliphatic ketones Among several normal aliphatic ketones which were tried, few examples only succeeded in giving moderate to low yield, increasing the length of the chain had influence on decreasing the reactivity of the reactant. The yields were inversely proportional with the length of the chain (36, 30 and 8 % for reaction with butanone 56, 2-hexanone 58 and 2-octanone 60 respectively) (Table1). Branching of the ketone skeleton or presence of

4 -methylcyclohexanone 42 ,

 442 selenium and cyanoacetone 32 were the three components of the reaction in presence of excess of triethylamine as catalyst in ethanol. The reaction was refluxed for two hours. When the reaction was treated by ice and water, selenium was separated and no formation of product was detected (Scheme 20).

32 42 43 Scheme 20

 4320 Scheme 20 Synthesis trial of 3-acetyl-2-aminoselenophene 1.2.3 Application of microwave assisted Gewald synthesis Microwave assisted synthesis was tried to investigate the effect of this technique on the yield and to shorten the reaction time. Cyclohexanone 44 (1 mmol), activated nitrile

  Scheme 22

  Nieto et al., "An Experimental and Theoretical NMR Study of NH-Benzimidazoles in Solution and in the Solid State." Chapter 2 -Reactivity of 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 50 maximize the yield of the required product and to decrease the undefined product to the minimum.

Figure 4 1 H

 1 Figure 4 1 H NMR spectrum of 4-chlorothieno[2,3-b]pyridine derivatives. The output of the reaction were surprisingly different from what we expected, the analysis of 1 H NMR results showed absence of formyl proton and no indication of presence of singlet signals characterized for the proton of quinolone ring which adjacent to the nitrogen atom (Figure 4).

4 -

 4 Chloro-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-b]pyridine (119) Yield: 313 mg (53%); white solid; mp 60-63 °C (MeOH-H2O); R f = 0.48 (EtOAccyclohexane, 9:1). 1 H NMR (400 MHz, CDCl3) δ = 8.30 (d, J = 5.0 Hz, 1 H), 7.25 (d, J = 5.0 Hz, 1 H), 3.45 -3.38 (m, 2 H), 3.00 -2.94 (m, 2 H), 1.99 -1.91 (m, 4 H).

Chapter 2 - 4 -

 24 Reactivity of 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 60 Chloro-3-methyl-2-pentylthieno[2,3-b]pyridine (122) Yield: 228 mg (36%); white solid; mp 36-38 °C (MeOH-H2O); R f = 0.48 (EtOAccyclohexane, 9:1).

Chapter 3 -Chapter 3 -Résumé chapitre 3 : 48 When

 33348 Scheme 44).3.1.1 Reaction of 2-acetamido-5 substituted thiophenes 2-Acetamido-5 substituted thiophene 123, reported to react with VH reagent under normal conditions to give mixture of products, when the conditions of the reaction adjusted to certain parameters, either 124 or 125 were formed by application of harsh condition (Scheme 47).54

Chapter 3 -

 3 Scheme 49

51 62Chapter 3 -

 513 Scheme 51

Chapter 3 -

 3 Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent

  suggested to play role by its electron withdrawing capability, which may result in stabilizing the double bond in intermediate IV (Scheme 53) making it more prone to another iminum ion attack. In contrary to the reaction of acetanilides 129, 65 in which Nacetylation allowed synthesizing the 4-chloroquinoline 130 in low yield or had no influence. Using higher amount of the reagent, may result in rapid reaction and cyclization so acetyl group leaves the reactant after accomplishment of another nucleophilic attack (intermediate VII) (Scheme 53). Additionally, acetyl group protection is stable toward deacetylation at 65 °C. In contrast, lower quantity of the reagent added dropwise and 63 Abdelwahab, Hanna, and Kirsch, "Synthesis of Novel 3-Acetyl-2-Aminothiophenes and Investigation of Their Behaviour in the Reaction with Vilsmeier-Haack Reagent." 64 Meth-Cohn and Narine, "A Versatile New Synthesis of Quinolines, Thienopyridines and Related Fused Pyridines." 65 Amaresh and Perumal, "A Novel One-Pot Synthesis of 4-Chloro-3-Quinolinecarboxaldehydes, 4-Chloroquinolines and 4-Chloro-3-Ethylquinolines Using Vilsmeier Reagent."

Chapter 3 -

 3 Scheme 53).

Figure 5 1 H

 51 Figure 5 1 H NMR spectrum of 4-chloro-3-formylthieno[2,3-b]pyridine derivatives. HRMS (ESI) was also used for more structure confirmation, it was noticed that all analysed compounds showed molecular ion: [M + H + CH3OH] + as base peak

  2,3-b]pyridine and 4-chloro-3-formylthieno[2,3-b]pyridine by application of N-protection. This acylation helped increasing the yield of unformylated products and introduction of mild synthesis of novel 4-chloro-3-formylthieno[2,3-b]pyridine. Directing the reaction toward one of the two products could be considered as controllable by modification the conditions of the reaction; temperature, mode of addition (which was found very crucial) and amount of the reagent. Differently from what reported before for synthesis of chloroquinoline, our product appeared to behave completely in opposite way. Chloroquinoline is formed as traces when acetamido acetophenone was used while chlorothieno[2,3-b]pyridin was formed by using of 3-acetyl-2-aminothiophene. Protection of the amino group of the last, made the production of formylated product reachable which is the normal result of this procedure with o-aminoacetopenone and o-hydroxyacetophenone.

Chapter 3 -

 3 Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 74 N-(3-acetylthiophen-2-yl)acetamide derivatives (136 -147), general procedure 1 g of 3-acetyl-2-aminothiophene derivatives was dissolved in 3 ml of acetic anhydride, the mixture was refluxed gently for 10-15 minutes, and quenched with water/ice, and then the solution was boiled gently, to breakdown the leftover acetic anhydride, the formed solid was filtered under vacuum and washed with water, dried and recrystallized in appropriate solvent if it needed.

Chapter 3 -

 3 Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 78 N-(3-acetylthiophen-2-yl)acetamide derivatives (149, 151), general procedure Compounds 59 or 61 (5.1 mmol) and acetyl chloride (0.4 g, 5.1 mmol) was dissolved and stirred in excess of acetic anhydride at 35 °C. After 30 minutes the solution was treated with water and ice and heated to decompose the acetic anhydride and then extracted with ethyl acetate. It was subsequently exposed to flash column purification (EtOAccyclohexane, 1:3) for maximum purity.

Chapter 3 - 4 -

 34 Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 81 Chloro-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-b]pyridine-3-carbaldehyde (137) Yield: 560 mg (94%); White solid; mp 160 -163 °C (cyclohexane); R f = 0.36 (EtOAccyclohexane, 1:9).

Chapter 3 - 4

 34 Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 86 mg (43%); yellow solid; mp 180 °C (cyclohexane); R f = 0.29 (EtOAccyclohexane, 1:9).

4 -Résumé chapitre 4 :

 44 Scheme 55

4. 1 . 1 Chapter 4 -86 165 166 7 -Chapter 4 -

 11474 Scheme 57). In the first mechanism 2-amino substituted carbonyl compound I and reactive active methylene compound II reacts in a rate-limiting to aldol adduct III. This intermediate loses water in an elimination reaction to unsaturated carbonyl compound and then loses water again in imine formation to quinoline derivative V. In the second mechanism the first step is Schiff base VI formation followed by aldol reaction to VII and elimination to quinoline derivative V.[START_REF] Joseph | Concerning the Mechanism of the Friedländer Quinoline Synthesis[END_REF]

1 2, 3 , 4 -

 134 Scheme 62

Chapter 5 - 108 Résumé chapitre 5 :

 51085 C-N coupling between 4-chlorothieno[2,3b]pyridine derivatives and aromatic amines Chapter 5-C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines (French summary) Couplage C-N entre les 4-chlorothiéno[2,3-b]pyridines et les anilines En possession d'analogues quinoléiniques chlorés, il était intéressant d'étudier leur réactivité dans le cadre de réaction de substitution nucléophile aromatique. Une brève introduction sur ce type de réaction ainsi que celles catalysées par les métaux de transition est présentée. Divers essais ont été faits, de manière directe, dans les conditions d'Ullmann et de Buchwald-Hartwig. Si les deux premières n'ont pas donné de résultats, la dernière méthode a permis le couplage avec des anilines. Synthesis of 4-chlorothieno[2,3-b]pyridine derivatives have introduced another perspective in their chemistry. Presence of chlorine atom, was motivation of more lead structure development and alteration. The liability of these atom would pave a new path toward synthesizing more derivatives, hopefully some of them will have certain biological activity.

  Scheme 64

Chapter 5 -Scheme

 5 Scheme 68).98

3

 3 Scheme 73

128

  Scheme 76). The target compound could act as DNA-binding agent which may be used as biological or medical relevant probes or drugs.127

Chapter 5 -Chapter 5 -Chapter 5 -Chapter 5 -

 5555 C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines 130

a

  Durant ces travaux, presque 80 nouveaux composés à noyau thiophénique ont été préparés. Les produits de départ -3-acétyl-2-aminothiophènes-ont été synthétisés par une classique réaction de Gewald utilisant la cyanoacétone, réaction non décrite précédemment. Lorsque ces composés sont étudiés dans les conditions de Vilsmeier-Haack-Arnold, ils présentent des comportements différents dans la cyclisation en thiénopyridines apparemment dues au remplacement du noyau benzénique d'oaminoacétophénones par le thiophène. Alors que la protection de l'amine par acétylation fait que le thiophène se comporte comme l'o-amino acétophénone, permettant l'accès au composé chloro-formylé, en contrôlant les conditions de réaction on peut également accéder au dérivé uniquement chloré. D'autre part l'autre voie pour construire les thiéno[2,3-b]pyridines, la réaction de Friedländer, a été étudiée et a permis de préparer d'autres thiénopyridinres. Le couplage C-N utilisant le palladium comme catalyseur (Buchwald-Hartwig) a été appliquée aux 4-chloro-thiéno[2,3-b]pyridines, produits de la réaction de Vilsmeier-Haack-Arnold, a conduit à la formation des N-phenylthiéno[2,3-b]pyridin-4-amines. Ce travail a donc été utilisé pour permettre la préparation de nouveaux dérivés thiophéniques ainsi que de dérivés condensés. Différents réactifs ont été utilisés qui ont permis l'accès à des composés divers à partir de la cyanoacétone de départ. Plusieurs médicaments ont le noyau thiophénique dans leur structure, par exemple la Ticlopidine® et le Clopidogre®l contre l'agrégation plaquettaire par inhibition du récepteur P2Y12, le Raloxifène®, modulateur du récepteur oestrogénique et l'Olanzapine® comme anti-psychotique. Nombreuses autres activités biologiques sont décrites dans la littérature : traitement de l'anxiété et de la dépression, bactéricide, anti-leishmania et antipaludique, contre les maladies auto-immunes. Pour cela, une étude biologique des produits préparés pourrait être considérée. Une seconde option serait d'utiliser les produits préparés pendant la Conclusion and Perspective (French) 139 thèse comme structure de base et par modifications structurales tenter de préparer des composés biologiquement actifs. Conclusion and Perspective 140 Conclusion and Perspective During this PhD nearly 80 novel thiophene based structure have been delivered. The starting material (3-acetyl-2-amino-thiophenes) in this work was prepared using simple classical Gewald's synthesis starting from cyanocacetone which was not reported before. When the new prepared thiophenes were studied under Vilsmeier-Haack-Arnold condition, they displayed different cyclization behaviour for the formation of the thienopyridines as a consequence of replacement of benzene ring from oaminoacetophenones by the thiophene ring. While the protection of amino group by acetylation made the thiophene behave like o-aminoacetophenone allowing access to interesting chloro-formyl derivatives, the formyl-free products still could be producible by controlling the reaction conditions. On other hand, another way for building thieno[2,3-b]pyridine, the Friedländer reaction, has been achieved and allows to prepare different thienopyridines . C-N coupling using palladium as catalyst (Buchwald-Hartwig) was also applicable on 4-chlorothieno[2,3-b]pyridines, the products of Vilsmeier-Haack-Arnold reaction. Nphenylthieno[2,3-b]pyridin-4-amines were produced in this way. This project has be used as a guide for planning synthesis of more thiophene derivatives and thiophene condensed scaffolds. Wide variety of different reagents have been involved in the different synthetic pathway from the starting cyanoacetone. Many drugs have thiophene in their core structure, e.g. Ticlopidine®, Clopidogrel® which are a group of drugs acting as antiplatelet aggregation by inhibition of P2Y12 receptor, additionally Raloxifene® (estrogen receptor modulator) and Olanzapine® as antipsychotic drug. Many other biological activities are reported in the literatures like treatment of anxiety, depression, bacterial infection, inflammation, leishmania, malaria and autoimmune diseases. Therefore the biological study of the synthetized products should to be considered. Lorraine University, SRSMC, 1 Boulevard Arago, 57070 Metz, France gilbert.kirsch@univ-lorraine.fr b Chemistry of Natural Compounds Department, National Research Centre, El-Behoos St. 33, Dokki-Cairo12622, Egypt

7 R 1 ,

 71 Py, MeOH or EtOH, 55-65 °C B: DMF, POCl 3 , 100 °C, 20-24 h C: PhNH 2 , Pd(OAc) 2 , BINAP, Cs 2 CO 3 , toluene, 110 °C, R 2 = -(CH 2 ) 4 -Received: 11.03.2016 Accepted after revision: 26.04.2016 Published online: 10.06.2016 DOI: 10.1055/s-0035-1561459; Art ID: ss-2016-z0173-op Abstract Through an investigation into the behaviour of 3-acetyl-2aminothiophene in the reaction with Vilsmeier-Haack reagent, an efficient method for the synthesis of 4-chlorothieno[2,3-b]pyridine derivatives has been established. Gewald's classical synthesis starting from cyanoacetone, ketones and elemental sulphur has been applied to produce the starting materials. Key words Vilsmeier-Haack, Gewald's reaction, thieno[2,3-b]pyridine, Buchwald-Hartwig, cross coupling

Scheme 3

 3 Scheme 3 Buchwald-Hartwig synthesis of N-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (6b)

17

 17

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 3.56 (s, 2 H), 2.28 (s, 3 H).

Scheme 2

 2 Scheme 2 Possible mechanism of cyclisation of 3-acetyl-2aminothiophene through Vilsmeier-Haack-Arnold procedures

  (400 MHz, CDCl 3 ): δ = 6.82 (s, 2 H), 2.92-2.87 (m, 2 H), 2.78-2.72 (m, 2 H), 2.44-2.38 (m, 2 H), 2.38 (s, 3 H). 13 C NMR (100 MHz, CDCl 3 ): δ = 193.7, 168.6, 141.0, 121.3, 112.2, 31.74, 29.1, 28.7, 27.3. HRMS (ESI): m/z calcd for [C 9 H 11 NOS + Na] + : 204.0454; found: 204.0464. Anal. Calcd for C 9 H 11 NOS: C, 59.64; H, 6.12; N, 7.73; S, 17.69. Found: C, 60.14; H, 6.10, N, 7.48; S, 17.25.

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 194.0, 164.3, 130.6, 117.3, 115.7, 36.6, 31.0, 30.6, 29.3, 24.5, 22.1. HRMS (ESI): m/z calcd for [C 11 H 15 NOS -H + Na] + : 231.0694; found: 231.0699. Anal. Calcd for C 11 H 15 NOS: C, 63.12; H, 7.22; N, 6.69; S, 15.32. Found: C, 62.84; H, 7.12; N, 6.53; S, 15.12. 1-(2-Amino-6-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-3 yl)ethanone (4d) Yield: 1.5 g (28%); greenish solid; mp 127 °C (cyclohexane).

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 6.79 (s, 2 H), 2.85-2.76 (m, 1 H), 2.72-2.61 (m, 1 H), 2.61-2.54 (m, 1 H), 2.41 (s, 3 H), 2.21-2.12 (m, 1 H), 1.95-1.86 (m, 2 H), 1.47-1.35 (m, 1 H), 1.07 (d, J = 6.5 Hz, 3 H).

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 194.0, 164.2, 130.2, 117.2, 115.6, 32.9, 31.3, 30.6, 29.2, 27.8, 21.4. HRMS (ESI): m/z calcd for [C 9 H 16 OS -H + Na] + : 232.0767; found: 232.0801. Anal. Calcd for C 11 H 15 NOS: C, 63.12; H, 7.22; N, 6.69; S, 15.32. Found: C, 62.61; H, 7.19; N, 6.88; S, 15.75.

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 194.0, 164.2, 130.6, 118.3, 115.5, 32.4, 30.5, 29.2, 27.2, 26.3, 24.7 HRMS (ESI): m/z calcd for [C 14 H 21 NOS + Na] + : 274.1207; found: 274.1236. Anal. Calcd for C 14 H 21 NOS: C, 66.89; H, 8.42; N, 5.57; S, 12.76. Found: C, 66.41; H, 8.42; N, 5.55; S, 13.03.1-(2-Amino-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-3 yl)ethanone (4f) Yield: 1.7 g (32%); brown solid; mp 129-130 °C (toluene).

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 2.56 (t, J = 7.6 MHz, 2 H), 2.47 (s, 3 H), 2.24 (s, 3 H), 1.57 (m, 2 H), 0.96 (t, J = 7.2, 3 H).

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 194.4, 163.3, 128.0, 120.0, 13.6, 117.0, 30.6, 29.2, 24.4, 15.86. HRMS (ESI): m/z calcd for [C 10 H 15 NOS + Na] + : 220.0767; found: 220.0781. Anal. Calcd for C 10 H 15 NOS: C, 60.88; H, 7.66; N, 7.10; S, 16.25. Found: C, 60.55; H, 7.58; N, 7.20; S, 16.70.

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 2.59-2.53 (m, 2 H), 2.46 (s, 3 H), 2.24 (s, 3 H), 1.58-1.51 (m, 2 H), 1.39-1.28 (m, 4 H), 0.92 (t, J = 6.9 Hz, 3 H).

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 194.4, 163.4, 127.9, 120.

Paper Syn thesis 13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 193.9, 163.1, 130.5, 116.2, 113.6, 31.0, 29.8, 26.1, 25.4. HRMS (ESI): m/z calcd for [C 9 H 11 NOS 2 + H] + : 214.0355; found: 214.0353. Anal. Calcd for C 9 H 11 NOS 2 : C, 50.67; H, 5.20; N, 6.57; S, 30.06. Found: C, 50.35; H, 5.16; N, 6.75; S, 29.44. 5 g (66%); black solid; mp 149-151 °C (CH 2 Cl 2 -MeOH).

  -6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-b]pyridine (5a) Yield: 156 mg (30%); white solid; mp 123 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1). 1 H NMR (400 MHz, CDCl 3 ): δ = 8.22 (d, J = 5.1 Hz, 1 H), 7.17 (d, J = 5.1 Hz, 1 H), 3.17-3.07 (m, 2 H), 3.03-2.92 (m, 2 H), 2.47-2.37 (m, 2 H).

13 C

 13 NMR (101 MHz, CDCl 3 ): δ = 167.2, 145.0, 144.0, 137.1, 136.7, 128.2, 120.0, 30.1, 29.9, 27.3. HRMS (ESI): m/z calcd for [C 10 H 8 ClNS + H] + : 210.0144; found: 210.0142. Anal. Calcd for C 10 H 8 ClNS•H 2 O: C, 55.55; H, 4.06; N, 6.47; S′ 14.88. Found: C, 55.20; H, 4.03; N, 6.28; H, 14.66. mg (43%); white solid; mp 67 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1).

  mg (36%); white solid; mp 112-115 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1).

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.23 (d, J = 5.1 Hz, 1 H), 3.40 (dd, J = 17.4, 5.

4 - 1 H

 41 Chloro-7-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (5d) Yield: 213 mg (36%); white solid; mp 54-56 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1). NMR (400 MHz, CDCl 3 ): δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.22 (d, J = 5.1 Hz, 1 H), 3.38-3.29 (m, 1 H), 3.08-2.90 (m, 2 H), 2.56-2.46 (m, 1 H), 2.07-1.96 (m, 2 H), 1.57-1.47 (m, 1 H), 1.14 (d, J = 6.5 Hz, 3 H).

13 C 1 H

 131 NMR (100 MHz, CDCl 3 ): δ = 162.5, 145.2, 138.5, 137.6, 130.2, 127.4, 120.7, 35.1, 30.7, 28.8, 26.5, 21.3. HRMS (ESI): m/z calcd for [C 12 H 12 ClNS + H] + : 238.0452; found: 238.0463. Anal. Calcd for C 12 H 12 ClNS: C, 60.62; H, 5.09; N, 5.89; S, 13.49. Found: C, 60.89; H, 5.13; N, 6.02; S, 13.07. 7-(tert-Butyl)-4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3b]pyridine (5e) Yield: 558 mg (80%); white solid; mp 75-77 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1). NMR (400 MHz, CDCl 3 ): δ = 8.30 (d, J = 4.8 Hz, 1 H), 7.23 (d, J = 4.8 Hz, 1 H), 3.50-3.40 (m, 1 H), 2.97-2.89 (m, 2 H), 2.70-2.60 (m, 1 H), 2.19-2.12 (m, 1 H), 1.66-1.60 (m, 1 H), 1.49-1.34 (m, 1 H), 1.00 (s, 9 H).

13 C 1 H

 131 NMR (100 MHz, CDCl 3 ): δ = 162.5, 145.2, 140.0, 137.5, 130.2, 127.8, 120.7, 44.5, 32.4, 27.8, 27.7, 27.2, 24.2. HRMS (ESI): m/z calcd for [C 15 H 18 ClNS + H] + : 280.0921; found: 280.0929. Anal. Calcd for C 15 H 18 ClNS: C, 64.38; H, 6.48; N, 5.01; S, 11.46. Found: C, 63.93; H, 6.55; N, 5.06; S, 10.96. mg (53%); white solid; mp 60-63 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1). NMR (400 MHz, CDCl 3 ): δ = 8.30 (d, J = 5.0 Hz, 1 H), 7.25 (d, J = 5.0 Hz, 1 H), 3.45-3.38 (m, 2 H), 3.00-2.94 (m, 2 H), 1.99-1.91 (m, 2 H), 1.82-1.72 (m, 4 H).

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 161.6, 144.9, 143.4, 137.4, 133.1, 130.3, 121.4, 32.0, 30.1, 28.3, 27.1, 26.60. HRMS (ESI): m/z calcd for [C 12 H 12 ClNS + H] + : 238.0452; found: 238.0458. Paper Syn thesis Anal. Calcd for C 12 H 12 ClNS: C, 60.62; H, 5.09; N, 5.89; S, 13.49. Found: C, 61.01; H, 5.38; N, 6.06; S, 13.48.4-Chloro-2,3-dimethylthieno[2,3-b]pyridine (5g) Yield: 49 mg (10%); white solid; mp 69-72 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1).

1 H

 1 NMR (400 MHz, DMSO-d 6 ): δ = 8.37 (d, J = 5.1 Hz, 1 H), 7.48 (d, J = 5.1 Hz, 1 H), 3.33 (s, 3 H), 2.53 (s, 3 H).

4 -

 4 Chloro-3-methyl-2-pentylthieno[2,3-b]pyridine (5i) Yield: 228 mg (36%); white solid; mp 36-38 °C (MeOH-H 2 O); R f = 0.48 (EtOAc-cyclohexane, 9:1).1 H NMR (400 MHz, DMSO-d 6 ): δ = 8.37 (d, J = 5.1 Hz, 1 H), 7.47 (d, J = 5.1 Hz, 1 H), 2.87 (t, J = 7.6 Hz, 2 H), 2.53 (s, 3 H), 1.63 (t, J = 8 Hz, 2 H), 1.37-1.30 (m, 4 H), 0.90-0.85 (m, 3 H). 13 C NMR (100 MHz, CDCl 3 ): δ = 162.0, 145.2, 141.7, 137.8, 130.9, 125.1, 121.0, 31.4, 30.6, 28.4, 22.4, 14.7, 14.0. HRMS (ESI): m/z calcd for [C 13 H 16 ClNS + H] + : 254.0765; found: 254.0770. Anal. Calcd for C 13 H 16 ClNS: C, 61.52; H, 6.35; N, 5.52; S, 12.63. Found: C, 61.82; H, 6.45; N, 5.60; S, 12.18.

1 H

 1 NMR (400 MHz, CDCl 3 ): δ = 8.02 (d, J = 5.5 Hz, 1 H), 7.34-7.29 (m, 2 H), 7.15 (d, J = 8.4, 2 H), 7.08 (t, J = 8.4, 1 H), 6.77 (d, J = 5.5 Hz, 1 H), 6.55 (s, 1 H), 3.05-3.02 (m, 2 H), 2.79-2.77 (m, 2 H), 2.00-1.66 (m, 4 H).

13 C

 13 NMR (100 MHz, CDCl 3 ): δ = 162.0, 147.

  .

Figure 2 .

 2 Figure 2. MALDI-TOF mass spectra (reflectron mode) of compounds 3, 4 and 5 from up chart to down (DHB matrix for compound 3 and HCCA matrix for compounds 4 and 5, C = 2 10 -4 M) and CDC25A incubated with them. The ion m/z=381.01, m/z=337.2 and m/z=374.2 corresponding to [M+H + ] of 3, 4 and 5 respectively.

Figure 3 . 4 .

 34 Figure 3. Compound 5 docking orientation with Figure 4. Docking of compound 3 with CDC25A CDC25B interacting by two hydrogen bonds with Tyr428 and Arg544 forming two Hydrogen bonds with Arg436

Figure 1 .

 1 Figure 1. Natural coumarins.

Figure 1 .

 1 Figure 1. Natural coumarins.

Figure 2 .

 2 Figure 2. Naturally occurring furocoumarins.

Figure 2 .

 2 Figure 2. Naturally occurring furocoumarins.

Figure 3 .

 3 Figure 3. Naturally occurring pyranocoumarins.

Figure 4 .

 4 Figure 4. Synthesis of paracetamol modified coumarins.

Figure 3 .

 3 Figure 3. Naturally occurring pyranocoumarins.

Figure 5 .

 5 Figure 5. Synthetic coumarins and derivatives.

5 -

 5 Farnesyloxycoumarin 36 showed the most potent activity against soybean lipoxygenase and 6oxoalkoxy)coumarins 38 have been prepared by condensation of hydroxycoumarins with α-chloroacetone[48] and the effects on expression of iNOS and COX-2 measured in comparison with

Figure 4 .

 4 Figure 4. Synthetic coumarins and derivatives.

Figure 4 .

 4 Figure 4. Synthesis of paracetamol modified coumarins.Figure 5. Synthesis of paracetamol modified coumarins.

Figure 5 .

 5 Figure 4. Synthesis of paracetamol modified coumarins.Figure 5. Synthesis of paracetamol modified coumarins.

4

 4

Scheme 1 derivatives 4 . 5 Scheme 2

 1452 Scheme 1 Synthesis of 2-chlorothieno[2,3-b]pyridine and 2-chloro-3formylthieno[2,3-b]pyridine.

of 8 (

 8 (intermediate VII) to attack another iminium ion. The chlorination occurs afterward by third molecule of VH reagent Synthesis Paper / PSP / Special Topic Template for SYNTHESIS © Thieme Stuttgart • New York 2017-01-30 page 3 intermediate VIII, IX). This last intermediate transformed to the final product 13 by water treatment at the end of the reaction (Scheme 6). To explore the reactivity of the obtained products we applied the method described by Hauptmann, s. et al.8 for compound 13b. It exhibited high reactivity and the reaction was complete within 30 minutes at 70°C (Scheme 5).

  we reported here new synthetic pathway of both 4-chlorothieno[2,3-b]pyridine and 4-chloro-3formylthieno[2,3-b]pyridine by application of N-protection.This acylation helped increasing the yield of unformylated products and introduction of mild synthesis of novel 4-chloro-3formylthieno[2,3-b]pyridine.

Scheme 5

 5 Scheme 5 Proposed mechanism for synthesis of formylated chlorothieno[2,3-b]pyridine and formyl-free chlorothieno[2,3-b]pyridine from N-acetyl-3-amino-2acetylthiophenes

  1.1.3.1.2.2 Synthesis of 2-amnothiophene derivatives by employing methyl 2siloxycyclopropanecarboxylates Methyl 2-siloxycyclopropanecarboxylates 25 replaced the carbonyl compound in reaction with the activated nitrile 26 (Scheme 13).29

	25	26	27
		Scheme 13

1.1.3.1.2.3 Microwave assisted two component Gewald synthesis Dithiane (thioacetaldehyde dimer) 28 reacted with activated nitrile compound 29 under microwave assisted condition to give the thiophene product 30 in very short time (2 minutes) and in good yield in comparison with classical synthesis (Scheme 14). 30 28 29 30

  thiophenes."

	10	34	35
		Scheme 16
	1.1.4.2 From malononitrile and cyanoacetate
	Another multicomponent example is synthesis of 3-aminothiophene 38 starting from
	cyano group containing compound (malononitrile or cyanoacetate) 36 in two step reaction
	(Scheme 17). 34

Table 1

 1 Products and yield of Gewald's reaction.Cyanoacetone 32 was an attractive subject as starting material to be involved in the multicomponent synthesis of functionalized thiophene in two step reactions. It contains active methylene group confined between cyano group and acetyl group, the direction of cyclization in the second step toward one of these two electron withdrawing groups was

	Ketone	Thiophenes	Yield
			61%
	46
		47
			58%
	44	45
			30%
	48	49

37

Germain Revelant et al., "Microwave-Assisted Synthesis of 5-Substituted 2-Aminothiophenes Starting from Arylacetaldehydes," Synthesis 2011, no. 18 (September 2011): 2935-40, doi:10.1055/s-0030-1261032.

Table 2

 2 Products

	Reagent	Product	Yield
			10%
	74	75
			36%
	76	77
			78%
	78
	80	79	46%
			44%
	81
		82

of the reaction between 2-(bis(methylthio)methylene)-3oxobutanenitrile 67 and activated halide, thioglycolate and hydrazine hydrate

-Amino-5-methyl-4,5,6,7-tetrahydrobenzo[b]thiophen-3-yl)ethanone (49)

	1-(2Yield: 28%; greenish solid (Abs. ethanol); mp 180-182°C
	Anal Calcd for C10H13NOS: C, 61.50; H, 6.71; N, 7.17; O, 8.19; S, 16.42. Found: C, 61.85;
	H, 6.65, N, 6.86; S, 16.18.

H NMR (400 MHz, CDCl3) δ = 6.94 (s, 2 H), 2.74 -2.67 (m, 2 H), 2.58 -2.52 (m, 2 H), 2.42 (s, 3 H), 1.86 -1.79 (m, 4 H). 13 C NMR (100 MHz, CDCl3) δ = 194.1, 163.9, 130.6, 117.6, 115.8, 30.7, 28.1, 24.7, 23.1, 22.9. HRMS (ESI): m/z calcd for [C10H13NOS + Na] + : 218.0610; found: 218.0602.

1-(2-Amino-5,7-dihydro-4H-thieno[2,3-c]thiopyran-3-yl)ethanone (63)

	1-
	Yield: 8%; greenish solid (cyclohexane); mp 112-113 °C
	1 H NMR (400 MHz, CDCl3) δ = 2.59 -2.53 (m, 2 H), 2.46 (s, 3 H), 2.24 (s, 3 H), 1.58 -
	Yield: 36%; brown solid; mp 154-157 °C 1.51 (m, 2 H), 1.39 -1.28 (m, 4 H), 0.92 (t, J = 6.9 Hz, 3 H).
	1 H NMR (400 MHz, CDCl3) δ = 6.87 (s, 2 H), 2.46 (s, 3 H), 2.23 (s, 3 H), 2.18 (s, 3 H). 13 C NMR (100 MHz, CDCl3) δ = 194.4, 163.4, 127.9, 120.3, 117.04, 31.3, 30.9, 27.3, 22.5,
	13 C NMR (100 MHz, CDCl3) δ = 194.4, 163.1, 128.4, 117.0, 113.8, 30.6, 15.8, 12.6. 15.8, 14.02.
	HRMS (ESI): m/z calcd for [C12H19NOS-H + Na] + : 247.0993; found: 247.1001.
	Anal Calcd for C12H19NOS: C, 63.96; H, 8.50; N, 6.22; S, 14.23. Found: C, 64.01; H, 8.34;
	N, 6.17; S, 14.35.
	Yield: 72% brownish solid; mp 167-170 °C
	1 H NMR (400 MHz, CDCl3) δ = 6.96 (s, 2 H), 3.62 (t, J = 1.6 Hz, 2 H), 3.02 (ddd, J = 5.8,
	4.6, 1.2 Hz, 2 H), 2.93 (t, J = 5.5 Hz, 2 H), 2.42 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 193.9, 163.1, 130.5, 116.2, 113.6, 31.0, 29.8, 26.1, 25.4.
	13C NMR (100 MHz, CDCl3) δ = 194.4, 163.3, 128.0, 120.0, 13.6, 117.0, 30.6, 29.2, 24.4, HRMS (ESI): m/z calcd for [C9H11NOS2 + H] + : 214.0355; found: 214.0353.
	13 C NMR (100 MHz, CDCl3) δ = 194.6, 161.4, 136.1, 121.1, 118.5, 31.7, 31.0, 29.8, 28.4, 15.86. Anal Calcd for C9H11NOS2: C, 50.67; H, 5.20; N, 6.57; S, 30.06. Found: C, 50.35; H, 5.16;
	27.6, 26.6. HRMS (ESI): m/z calcd for [C10H15NOS + Na] + : 220.0767; found: 220.0781. N, 6.75; S, 29.44.
	HRMS (ESI): m/z calcd for [C11H15NOS + Na] + : 232.0767; found: 232.0778. Anal Calcd for C10H15NOS: C, 60.88; H, 7.66; N, 7.10; S, 16.25. Found: C, 60.55; H, 7.58;

H NMR (400 MHz, CDCl3) δ = 6.62 (s, 2 H), 2.87 -2.79 (m, 2 H), 2.61 -2.59 (m, 2 H), 2.43 (s, 1 H), 1.89 -1.83 (m, 2 H), 1.75 -1.64 (m, 4 H). Anal Calcd for C11H15NOS: C, 63.12; H, 7.22; N, 6.69; O, 7.64; S, 15.32. Found: C, 62.99; H, 7.13; N, 7.11; S, 15.08. 1-(2-Amino-4,5-dimethylthiophen-3-yl)ethanone (57) HRMS (ESI): m/z calcd for [C8H11NOS + H] + : 170.634; found: 170.0633; calcd for [C8H11NOS+Na]+: 192.0454; found: 192.0456. Anal Calcd for C8H11NOS: C, 56.77; H, 6.55; N, 8.28; S, 18.95. Found: C, 56.65; H, 6.41; N, 9.07; S, 19.07. 1-(2-Amino-4-methyl-5-propylthiophen-3-yl)ethanone (59) Yield: 30%; brown solid (cyclohexane); mp 113-115 °C 1H NMR (400 MHz, CDCl3) δ = 6.89 (brs, 2 H), 2.56 (t, J = 7.6 MHz, 2 H), 2.47 (s, 3 H), 2.24(s, 3 H), 1.57 (m, 2 H), 0.96 (t, J = 7.2, 3 H). N, 7.20; S, 16.70.

1-(2-Amino-4-methyl-5-pentylthiophen-3-yl)ethanone (61)

(2-Amino-6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-3-yl)ethanone (65)

	Yield: 66% black solid (dichloromethane/methanol); mp 149-151 °C
	1 H NMR (400 MHz, DMSO-d 6 ) δ = 8.19 (s, 2 H), 3.24 (s, 2 H), 2.73 (t, J = 5.7 Hz, 2 H),
	2.57 (t, J = 5.7 Hz, 2 H), 2.31 (s, 3 H), 2.29 (s, 3 H).
	13C NMR (100 MHz, DMSO-d 6 ): δ = 192.2, 164.6, 113.3, 128.5, 113.21, 53.0, 52.0, 45.1,
	30.1, 27.7.
	HRMS (ESI): m/z calcd for [C10H14N2OS + H] + : 211.0900; found: 211.0891.
	Anal Calcd for C10H14N2OS.H2O: C, 52.54; H, 7.06; N, 12.25; S, 14.02. Found: C, 52.48;
	H, 7.01; N, 12.62; S, 14.04.

Methyl-2-(methylthio)thiophene-3-carbonitrile derivatives, general procedure Method A

	Chapter 1 -Synthesis of 3-acetyl-2-aminothiophene
	HRMS (ESI): m/z calcd for [C7H9NOS2 + H] + : 188.0198; found: 188.0226. For [C7H9NOS2
	+ Na] + : 210.0018; found: 210.0069.
	Anal Calcd for C7H9NOS2: C, 44.89; H, 4.84; N, 7.48; S, 34.24. Found: C, 45.03; H, 4.86;
	N, 7.40; S, 34.23.
	4-Compound 67 (20 mmol) and Na2S.9H2O (20 mmol) was dissolved in 40 ml DMF and the
	solution was stirred at 70 °C for one hour. Activated halide (40 mmol, 2 equivalent) was
	added and after stirring for 30 minutes at 70 °C, potassium carbonate (20 mmol) was
	introduced and the stirring was left for additional one hour at 70 °C. When the solution
	quenched with ice and water, solid precipitate of thiophene was appeared and collected
	by filtration through vacuum.
	67	68
	Scheme 28
	Method B
	Compound 67 (20 mmol) was dissolved in 40 ml DMF, ethyl thioglycolate 80 (20 mmol)
	and potassium carbonate (20 mmol) were added to the solution which stirred at 70 °C for
	three hour. Precipitate of the product was formed by decomposition of the reaction
	solution in ice and water, it was filtered under vacuum, washed by water and dried to give
	compound 79; yield, 46%
	36

67 79 Scheme 29 5-Acetyl-4-methyl-2-(methylthio)thiophene-3-carbonitrile (75)

	Yield: 10%
	1 H NMR (400 MHz, CDCl3) δ = 2.71 (s, 3 H), 2.64 (s, 3 H), 2.53 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 188.9, 157.5, 145.7, 136.1, 113.3, 112.3, 29.6, 18.4, 15.8.

Methyl 4-cyano-3-methyl-5-(methylthio)thiophene-2-carboxylate (77)

	Yield: 36%; beige-brown solid; mp 122 °C
	1 H NMR (400 MHz, CDCl3) δ = 3.90 (s, 3 H), 2.70 (s, 3 H), 2.63 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 188.9, 155.7, 146.5, 125.1, 113.1, 112.8, 62.4, 18.9, 14.9.
	HRMS (ESI): m/z calcd for [C9H9NO2S2 + H] + : 228.0147; found: 228.0174. For
	[C9H9NO2S2 + Na] + : 249.9967; found: 249.9992.
	Anal Calcd for C9H9NO2S2.3%H2O: C, 46.13; H, 4.20; N, 5.97; S, 27.36. Found: C, 47.56;
	H, 3.97; N, 6.18; S, 26.09.

Ethyl 4-cyano-3-methyl-5-(methylthio)thiophene-2-carboxylate (79)

	Yield: 78% (method A), 46% (method B); yellow solid; mp 114 °C.
	1 H NMR (400 MHz, CDCl3) δ = 4.36 (q, J = 7.1 Hz, 2 H), 2.70 (s, 3 H), 2.63 (s, 3 H), 1.39
	(t, J = 7.1 Hz, 3 H).
	13 C NMR (100 MHz, CDCl3) δ 160.9, 157.7, 147.5, 126.1, 113.4, 111.8, 61.4, 18.5, 14.9,
	14.3.
	HRMS (ESI): m/z calcd for [C10H11NO2S2 + H] + : 242.0304; found: 242.0344. For
	[C10H11NO2S2 + Na] + : 264.0123; found: 264.0156.
	Anal Calcd for C10H11NO2S2.2%H2O: C, 48.77; H, 4.72; N, 5.68; S, 26.04. Found: C,
	48.88; H, 4.56; N, 5.75; S, 25.51.

3-Methyl-5-(methylthio)thiophene-2,4-dicarbonitrile (82)

	Yield: 44%; brown solid; mp 122 °C.
	1 H NMR (400 MHz, CDCl3) δ = 2.63 (s, 3 H), 2.43 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 158.4, 151.4, 112.6, 112.1, 110.5, 104.6, 18.9, 15.1.
	HRMS (ESI): m/z calcd for [C8H6N2S2 + H] + : 195.0045; found: 195.0075.
	5

-Formyl-4-methyl-2-(methylthio)thiophene-3-carbonitrile (84)

	Introduction
	The neighbourhood of the acetyl and amino group on the skeleton of the annulated
	thiophenes was anticipated to make them reactive. The main motivation was to
	investigate the impact of thiophene ring on the behaviour and reactivity of the compounds
	toward certain reagents involving both functional group.
	2.1.1 Chemistry of 3-acetyl-2-aminothiophenes derivative
	There are few examples of 3-acetyl-2-aminothiophenes reactions were reported in
	the literatures, some of them were carried out on the amino group or on both acetyl and
	amino group for quninoline or thienopyrimidine synthesis by cyclization.
	2.1.1.1	Reaction of amino group
	2.1.1.1.1	Reaction with acetyl halogenides
	Some acetamide were prepared by reaction with different types of acetyl halides
	(Scheme 30). The starting compounds 86 in this reaction were prepared by the method
	mentioned in
	Yield: traces; solid
	1 H NMR (400 MHz, CDCl3) δ = 9.81 (s, 1 H), 2.64 (s, 3 H), 2.56 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 179.7, 163.0, 162.5, 148.9, 135.9, 112.7, 18.3, 13.3.

Scheme 15. 38 85 86 87 Scheme 30

	2.1.1.1.2 Acetylation
	Amino group reacted with acetic anhydride by the well-known procedure, to form
	acetylated analogue 89 (Scheme 31). 39
	86	88	89
		Scheme 31
	2.1.1.1.3 Azo coupling
	Some red-light blue azo disperse dyes were prepared by azo coupling between 2-
	amino-3-acetyl-5-nitrothiophene and compounds 91 (Scheme 32). 40
	38 Gernot A. Eller and Wolfgang Holzer, "Synthesis and Detailed Spectroscopic Characterization of Two Novel
	N-(3-acetyl-2-thienyl)acetamides," Molbank 2006, no. 6 (December 1, 2006): M520, doi:10.3390/M520.

90 91 92 Scheme 32

Scheme 43 2.2 Results and discussion

	3-Acetyl-2-aminothiophenes 86 underwent cyclization when reacted with β-keto ester 2.1.2.2 Chlorination (Scheme 42) through such reactions in one step starting, was reported. 51
	97 to produce the qunioline analogues 98 (Scheme 34). 42 Oxygen of carbonyl compound could be replaced by chlorine atom via electrophilic
	2.1.1.1.4.3 Friedländer cyclization attack of VH reagent (Scheme 39). 49
	Propylphosphonic anhydride (T3P ® ) was used as catalytic agent in condensation 102 103
	between 1-(2-amino-5-(tert-butyl)thiophen-3-yl)ethan-1-one 99 and Boc protected piperidin-4-one 100, by Friedländer reaction and quinolone 101 was obtained (Scheme 35). 43 Scheme 36 108 112 41 113 109 It used also for formylation of the aromatic heterocycles, as in the next example Scheme 44 Scheme 41 (Scheme 37), in which 3-methylthiophene 104 gave 3-methylthiophene-2-carbaldehyde Scheme 39 105 as major product (Scheme 37). 47 2.2.1 Reaction temperature
	2.1.2.3 Chloroformylation
	Ketones could react with VH reagent to yield β-chloroacrolein 50 (Scheme 40)
	99 (another example of this sort of reaction was described within chapter one in 100 101 Scheme 35 2.1.2 104 110 111 105 thiophene synthesis introduction in Scheme 4. Scheme 42 Scheme 37 2.1.2.1.1 Mechanism of formylation 48 Vilsmeier-Haack-Arnold reaction was applied to achieve the cyclisation of 3-acetyl-2.1.2.4.1 Mechanism of cyclization 2-aminothiophenes which prepared by Gewald's reaction. This step was inspired from Vilsmeier-Haack reaction Two attacks of VH reagent are assumed to happen. Firstly, the enol form acts as cyclization of o-hydroxyacetophenone 108 and o-aminoacetophenone 110, which were Vilsmeier-Haack reaction is very practical method for versatile synthetic purposes. 44 nucleophilic and linked to the carbon of the iminium ion, one HCl moiety left and after previously mentioned in Scheme 41 and Scheme 42. The impact of replacement of a The Vilsmeier-Haack reagent (POCl3/DMF) has been widely used in formylation, 106 107 intramolecular delocalization of the double bond, the lone pair of electrons of the hydroxyl benzene ring with thiophene in aforementioned cyclisation has not been investigated chlorination and cyclisation processes. 45 Scheme 40 group neutralize the positive charge of the carbon atom which results in ring closure. One before. To study this reaction, we decided to use the 3-acetyl-2-amino thiophene, as a
	2.1.2.1 Formylation Vilsmeier-Haack reaction was initially established for formylation purposes of molecule of phosphorodichloridic acid and dimethyl amine leaves and neutral molecule starting material. The influence of the thiophene nucleus on the reaction is clear ( 2.1.2.4 Cyclisation The preparation of 3-formylchromone 109 from o-hydroxyacetophenone 108 is obtained. The double bond of the last intermediate spontaneously attacks another Scheme 44). molecule of the iminium salt, after losing HCl and dimethyl amine respectively, the product activated aromatic compounds (Scheme 36). 46 (Scheme 41) and 3-formylquinoline 111 from o-aminoacetophenone 110 is hydrolyzed by water molecule and phosphorodichloridic acid separated (Scheme 43). 52 Several trials were applied to achieve the cyclisation, optimization of reaction
	conditions; temperature, time of the reaction, number of Vilsmeier-Haack reagent Scheme 38 equivalents, mode of addition and the treatment of the reaction mixture at the end of the
	49 Annamaria Lilienkampf, Mikael P. Johansson, and Kristiina Wähälä, "(Z)-1-Aryl-1-Haloalkenes as 51 Horst Harnisch, "Chromon-3-Carbaldehyde," Justus Liebigs Annalen Der Chemie 765, no. 1 (January 3, 1973): 8-14, doi:10.1002/jlac.19727650103; Akira Nohara, Tomonobu Umetani, and Yasushi Sanno, "A Facile reaction was very critical and seen to be effective on the occurrence of the reaction, sort
	Intermediates in the Vilsmeier Haloformylation of Aryl Ketones," Organic Letters 5, no. 19 (September 18, 2003): 3387-90, doi:10.1021/ol034914c; Su et al., "Recent Progress in the Use of Vilsmeier-Type Reagents"; A. F. Mironov Synthesis of Chromone-3-Carboxaldehyde, Chromone-3-Carboxylic Acid and 3-Hydroxymethylchromone," Tetrahedron Letters 14, no. 22 (January 1973): 1995-98, doi:10.1016/S0040-4039(01)96102-7; Carla I Nieto et al., of the products and the yield.
	et al., "A New, Convenient Synthesis of Pyrrolylacetylenes," Synthesis 1979, no. 07 (1979): 533-35, doi:10.1055/s-"An Experimental and Theoretical NMR Study of NH-Benzimidazoles in Solution and in the Solid State: Proton
	1979-28749. Transfer and Tautomerism," Beilstein Journal of Organic Chemistry 10 (July 16, 2014): 1620-29,
	47 Otto Meth-Cohn and Mark Ashton, "Regioselective Electrophilic formylation-3-Substituted Thiophenes as a 50 Arnold and Žemlička, "Synthetische Reaktionen von Dimethylformamid IV. Darstellung von β-doi:10.3762/bjoc.10.168.
	Case Study," Tetrahedron Letters 41, no. 15 (April 8, 2000): 2749-52, doi:10.1016/S0040-4039(00)00254-9. Chlorvinylaldehyden Aus Carbonylverbindungen"; G. Jagath Reddy et al., "Synthesis of Pyrazolo[1,5-a]pyrimido[4,3-52 K. C Rajanna et al., "Kinetics and Mechanism of Vilsmeier-Haack Synthesis of 3-Formyl Chromones Derived
	48 Gurnos Jones and Stephen P. Stanforth, "The Vilsmeier Reaction of Non-Aromatic Compounds," in Organic d] Benzopyrans and 2-Pyrazolo[1,5-a]pyrimidinylphenols from the Reaction of 5(3)-Amino Pyrazoles," Heterocyclic from O-Hydroxy Aryl Alkyl Ketones: A Structure Reactivity Study," Tetrahedron 52, no. 10 (March 4, 1996): 3669-82,
	Reactions (John Wiley & Sons, Inc., 2004). Communications 9, no. 5 (2011): 453-456, doi:10.1515/HC.2003.9.5.453. doi:10.1016/0040-4020(96)00043-9.

Table 3

 3

	Product of reaction between 3-acetyl-3-aminothiophene and VH reagent
	Thiophenes	Thieno[2,3-b]

pyridine Yield 47 114 4-Chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (115)

	Yield: 240 mg (43%); white solid; mp 67 °C (MeOH-H2O); R f = 0.48 (EtOAc-
	cyclohexane, 9:1).
	1 H NMR (400 MHz, CDCl3) δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.23 (d, J = 5.1 Hz, 1 H), 3.16
	(dd, J = 5.8, 2.7 Hz, 2 H), 2.89 (t, J = 4.2 Hz, 2 H), 1.98 -1.85 (m, 4 H).
	13 C NMR (101 MHz, CDCl3) δ = 162.0, 144.8, 139.1, 137.9, 130.6, 127.8, 120.8, 26.7,
	26.1, 22.5.
	HRMS (ESI): m/z calcd for [C11H10ClNS + Na] + : 246.0115; found: 246.0133.

Anal Calcd for C11H10ClNS: C, 59.05; H, 4.51; N, 6.26; S, 14.33. Found: C, 59.70; H, 4.58; N, 6.48; S, 14.08. 4-Chloro-6-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (116) Yield: 213 mg (36%); white solid; mp 112-115 °C (MeOH-H2O); R f = 0.48 (EtOAccyclohexane, 9:1).

1 H NMR (400 MHz, CDCl3) δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.23 (d, J = 5.1 Hz, 1 H), 3.40 (dd, J = 17.4, 5.6 Hz, 1 H), 2.97 -2.89 (m, 2 H), 2.64 -2.56 (m, 1 H), 2.03 -1.92 (m, 2 H), 1.60 -1.48 (m, 1 H), 1.17 (d, J = 6.6 Hz, 3 H). 13 C NMR (100 MHz, CDCl3) δ = 162.6, 145.1, 138.6, 137.6, 130.3, 127.8, 120.7, 35.0, 30.6, 28.9, 25.9, 21.7. Chapter 2 -Reactivity of 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 57 HRMS (ESI): m/z calcd for [C12H13ClNS] + : 238.0452; found: 238.0456. Anal Calcd for C12H12ClNS: C, 60.62; H, 5.09; N, 5.89; S, 13.49. Found: C, 60.44; H, 5.11; N, 5.82; S, 13.03.

4-Chloro-7-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (117) Yield: 213 mg (36%); white solid; mp 54-56 °C (MeOH-H2O); R f = 0.48 (EtOAccyclohexane, 9:1). 1 H NMR (400 MHz, CDCl3) δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.22 (d, J = 5.1 Hz, 1 H), 3.38 -3.29 (m, 1 H), 3.08 -2.90 (m, 2 H), 2.56 -2.46 (m, 1 H), 2.07 -1.96 (m, 2 H), 1.57 -1.47 (m, 1 H), 1.14 (d, J = 6.5 Hz, 3 H).

13 C NMR (100 MHz, CDCl3) δ = 162.5, 145.2, 138.5, 137.6, 130.2, 127.4, 120.7, 35.1, 30.7, 28.8, 26.5, 21.3. HRMS (ESI): m/z calcd for [C12H12ClNS + H] + : 238.0452; found: 238.0463. Anal Calcd for C12H12ClNS: C, 60.62; H, 5.09; N, 5.89; S, 13.49. Found: C, 60.89; H, 5.13; N, 6.02; S, 13.07.

7-(

tert-butyl)-4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (118)

	13 C NMR (100 MHz, CDCl3) δ = 162.5, 145.2, 140.0, 137.5, 130.2, 127.8, 120.7, 44.5,
	32.4, 27.8, 27.7, 27.2, 24.2.
	HRMS (ESI): m/z calcd for [C15H18ClNS + H] + : 280.0921; found: 280.0929.
	Anal Calcd for C15H18ClNS: C, 64.38; H, 6.48; N, 5.01; S, 11.46. Found: C, 63.93; H, 6.55;
	N, 5.06; S, 10.96.
	Yield: 558 mg (80%); white solid; mp 75-77 °C (MeOH-H2O); R f = 0.48 (EtOAc-
	cyclohexane, 9:1).

Chapter 2 -Reactivity of 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 58 1 H NMR (400 MHz, CDCl3) δ = 8.30 (d, J = 4.8 Hz, 1 H), 7.23 (d, J = 4.8 Hz, 1 H), 3.50 -3.40 (m, 1 H), 2.97 -2.89 (m, 2 H), 2.70 -2.60 (m, 1 H), 2.19 -2.12 (m, 1 H), 1.66 -1.60 (m, 1 H), 1.49 -1.34 (m, 1 H), 1.00 (s, 9 H).

Table 4

 4 Products and yields of VH reaction with N-protected 3-acetyl-2-

	aminothiophenes at 65 °C and 100 °C
	N-protected 3-acetyl-2-aminothiophenes	Thieno[2,3-b]pyridine	65 °C	Yield 100 °C	4-Chloro-3-formylthieno[2,3-b]pyridine	65 °C	Yield 100 °C
			0%	21%		94%	9%
	136	114				137
			25%	58%		60%	0%
	138	115				132
			0%	48%		86%	30%
	139	116				140

was provoked by addition of ice and water. The solution was left for 30 minutes for allowing maximum precipitation, the solid was collected by filtration under vacuum and washed by water, the solid was of sufficient purity to be used without further purification (yield: 1.16 g; 81%) Yield: 81%; grey solid; mp 112-116 °C

1

H NMR (400 MHz, CDCl3) δ = 12.79 (s, 1 H), 2.85 -2.70 (m, 2 H), 2.71 -2.66 (m, 2 H), 2.54 (s, 3 H), 1.89 -1.80 (m, 4 H), 1.35 (s, 9 H).

13

C NMR (100 MHz, CDCl3) δ 196.

8, 176.8, 149.8, 129.4, 127.1, 120.9, 39.3, 31.3, 27.5,

3-acetyl-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophen-2-yl)acetamide (145)

	Yield: 1 g (83%); beige-brown solid; mp 75-78 °C (EtOH 96%).
	1 H NMR (400 MHz, CDCl3) δ = 11.90 (s, 1 H), 2.93 -2.90 (m, 2 H), 2.76 -2.73 (m, 2 H),
	2.53 (s, 3 H), 2.25 (s, 3 H), 1.92 -1.86 (m, 2 H), 1.75 -1.64 (m, 4 H).
	Anal Calcd for C, 65.49; H, 7.90; N, 4.77; S, 10.93. Found: C, 65.51; H, 7.92; N, 5.26; S,
	10.95.

1

H NMR (400 MHz, CDCl3) δ = 12.26 (s,

1 H)

, 2.96 -2.83 (m, 1 H), 2.72 -2.49 (m, 2 H), 2.43 (s, 3 H), 2.39 -2.30 (m, 1 H), 2.19 (s, 3 H), 2.08 -1.88 (m, 1 H), 1.53 -1.32 (m, 1 H), 1.32 -1.03 (m, 1 H), 0.88 (s, 9 H). 13 C NMR (100 MHz, CDCl3) δ 196.3, 167.8, 149.5, 127.5, 124.1, 120.1, 53.2, 52.3, 45.23, 31.1, 29.7, 27.7, 23.7. HRMS (ESI): m/z calcd for [C16H23NO2S + Na] + : 316.1342; found: 316.1338. Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 77 N-(13 C NMR (100 MHz, CDCl3) δ = 197.5, 167.6, 145.7, 135.0, 131.4, 123.1, 31.9, 31.6, 29.3, 28.4, 27.6, 26.7, 23.71. HRMS (ESI): m/z calcd for [C13H17NO2S + H] + : 252.1058; found: 252.1049. Anal Calcd for C13H17NO2S: C, 62.12; H, 6.82; N, 5.57; S, 12.76. Found: C, 62.56; H, 6.70; N, 5.45; S, 12.65.

  Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent

	1 H NMR (400 MHz, CDCl3) δ = 12.31 (s, 1 H), 2.66 (t, J = 7.7 Hz, 2 H), 2.55 (s, 3 H), 2.33
	(s, 3 H), 2.26 (s, 3 H), 1.66 -1.54 (m, 2 H), 1.38 -1.29 (m, 4 H), 0.96 -0.85 (m, 3 H). Compounds 154 was prepared from 153 (0.3 g, 1.2 mmol) by same procedure reported
	13 C NMR (100 MHz, CDCl3) δ = 197.1, 167.7, 148.2, 130.0, 126.8, 122.0, 31.4, 31.3, 30.9, before.
	27.3, 23.8, 22.4, 15.3, 14.0.
	HRMS (ESI): m/z calcd for [C14H21NO2S + H] + : 268.1366; found: 268.1369.
	Anal Calcd for C14H21NO2S: C, 62.89; H, 7.92; N, 5.24; S, 11.99. Found: C, 62.75; H,
	7.89; N, 5.41; S, 12.08.
	Yield; 1 g (84%); brown solid; mp 132-135 °C (EtOH 96%).
	1 H NMR (400 MHz, CDCl3) δ = 2.34 (s, 1 H), 3.76 (s, 2 H), 3.10 (t, J = 5.8 Hz, 2 H), 2.97
	(t, J = 5.8 Hz, 2 H), 2.53 (s, 3 H), 2.29 (s, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 196.5, 167.9, 148.5, 129.1, 123.2, 121.3, 31.7, 29.2, 26.3,
	25.4, 23.7.
	HRMS (ESI): m/z calcd for [C11H13NO2S2 + H] + : 256.0460; found: 256.0460.
	79

N-(3-acetyl-4,7-dihydro-5H-thieno[2,3-c]thiopyran-2-yl)acetamide (153)

Compound 63 (1g, 4.6 mmol) dissolved in 10 ml of dichloromethane, excess of acetic anhydride, acetyl chloride (1.08 g, 13.8 mmol) and triethyl amine (1.4 g, 13.8 mmol) were added and the reaction mixture was gently refluxed for 30 hours, the solvent was evaporated and residue dissolved in acetone and treated by ice and water. The reaction mixture was boiled for decomposing the acetic anhydride and the formed precipitate was filtered under vacuum and washed with water and crystallized from EtOH-H2O.

Anal Calcd for C11H13NO2S2: C, 51.74; H, 5.13; N, 5.49; S, 25.11. Found: C, 51.94; H, 5.67; N, 5.17; S, 25.56. Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 80 4-chloro-5,8-dihydro-6H-thiopyrano[4',3':4,5]thieno[2,3-b]pyridine (154) Yield: 31 mg (11%); colorless solid; mp 154 °C R f = 0.29 (EtOAc-cyclohexane, 1:9). 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 5.0 Hz, 1 H), 7.26 (d, J = 5.0 Hz, 1 H), 3.93 (s, 2 H), 3.51 -3.43 (m, 2 H), 3.03 (t, J = 5.2 Hz, 2 H). 13C NMR (100 MHz, CDCl3) δ = 161.1, 145.9, 138.1, 134.7, 130.4, 127.5, 121.2, 28.7, 26.6, 25.9. HRMS (ESI): m/z calcd for [C10H8ClNS2 + H] + : 241.9859; found: 241.9864. Anal Calcd for C10H8ClNS2; C, 49.68; H, 3.34; N, 5.79; S, 26.52. Found: C, 49.99; H, 3.60; N, 5.52; S, 26.85

4-Chloro-7-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine-3- carbaldehyde (142)

	Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent
	Anal Calcd for C12H10ClNOS: C, 57.26; H, 4.00; N, 5.56; S, 12.74. Found: C, 57.89; H, 1 H NMR (400 MHz, CDCl3) δ = 10.55 (s, 1 H), 8.80 (s, 1 H), 3.34 -3.30(m, 1 H), 3.08 -4-Chloro-3-methyl-2-propylthieno[2,3-b]pyridine-5-carbaldehyde (150)
	4.28; N, 5.30; S, 12.22. 2.91 (m, 1 H), 2.92 -2.86 (m, 1 H), 2.55 -2.34 (m, 1 H), 2.01 -1.86 (m, 2 H), 1.51 -1.41
	4-Chloro-6-methyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine-3-(m, 2 H), 1.07 (d, J = 6.5 Hz, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 189.1, 166.6, 145.7, 141.2, 140.3, 129.9, 128.5, 124.4, carbaldehyde (140) 34.2, 30.6, 28.6, 26.7, 21.2. Yield: 350 mg (53%); yellow solid; mp 120-122 °C (cyclohexane); ; R f = 0.38 (EtOAc-
	cyclohexane, 1:9). Yield: 487 mg (77%); White solid; mp 102-105 °C; ; R f = 0.38 (EtOAc-cyclohexane, 1:9). HRMS (ESI): m/z calcd for [C13H12ClNOS + H] + : 266.0401; found: 266.04201. 1 H NMR (400 MHz, CDCl3) δ = 10.58 (s, 1 H), 8.75 (s, 1 H), 3.41 -3.33 (m, 2 H), 2.94 -1 H NMR (400 MHz, CDCl3) δ = 10.67 (s, 1 H), 8.88 (s, 1 H), 2.89 (t, J = 7.6 Hz, 2 H), 2.67
	Anal Calcd for C13H12ClNOS: C, 58.75; H, 4.55; N, 5.27; S, 12.06. Found: C, 58.59; H, 2.86 (m, 2 H), 1.95 -1.80 (m, 2 H), 1.76 -1.67 (m, 4 H). (s, 3 H), 1.85 -1.69 (m, 4 H), 1.06 (t, J = 7.3 Hz, 3 H).
	4.54; N, 5.12; S, 11.92.
	13 C NMR (100 MHz, CDCl3) δ = 189.2, 166.1, 145.7, 143.2, 141.6, 130.5, 126.4, 124.4,
	30.5 24.2, 15.0, 13.7.
	13 C NMR (100 MHz, CDCl3) δ = 189.0, 166.6, 145.6, 141.4, 141.1, 129.8, 128.8, 124.3,
	Yield: 346 mg (60%); White solid; mp 162-165 °C (cyclohexane); ; R f = 0.36 (EtOAc-44.4, 32.4, 28.0, 27.9, 27.2, 24.1. 1 H NMR (400 MHz, CDCl3) δ = 10.56 (s, 1 H), 8.77 (s, 1 H), 2.55 (s, 3 H), 2.44 (s, 3 H). 1 H NMR (400 MHz, CDCl3) δ = 10.65 (s, 1 H), 8.87 (s, 1 H), 2.89 (t, J = 7.6 Hz, 2 H), 2.64
	cyclohexane, 1:9). HRMS (ESI): m/z calcd for [C16H18ClNOS + H + CH4O] + : 340.1133; found: 340.11691. 13 C NMR (100 MHz, CDCl3) δ = 189.0, 165.8, 145.7, 141.4, 137.3, 126.8, 14.8, 14.1. (s, 3 H), 1.80 -1.66 (m, 2 H), 1.45 -1.35 (m, 4 H), 0.97 -0.88 (m, 3 H).
	1 H NMR (400 MHz, CDCl3) δ = 10.54 (s, 1 H), 8.77 (s, 1 H), 3.15 -3.12 (m, 2 H), 2.84 -2.80 (m, 2 H), 1.95 -1.78 (m, 4 H). 13 C NMR (100 MHz, CDCl3) δ = 189.1, 166.4, 145.7, 141.2, 140.7, 130.0, 128.8, 124.3, 26.9, 26.3, 22.4, 22.3. HRMS (ESI): m/z calcd for [C10H8ClNOS -CHO + H] + : 198.0139; found: 198.0165. 13 C NMR (101 MHz, CDCl3) δ = 189.2, 166.1, 145.7, 143.5, 141.5, 130.5, 126.2, 124.5, Anal Calcd for C16H18ClNOS: C, 62.43; H, 5.89; N, 4.55; S, 10.41. Found: C, 62.43; H, 5.90; N, 4.50; S, 10.35. 31.4, 30.5, 28.5, 22.4, 15.0, 13.92. Anal Calcd for C10H8ClNOS: C, 53.22; H, 3.57; N, 6.21; S, 14.21. Found: C, 53.25; H, Yield: 429 mg (65%); White solid; mp 148 -152 °C (cyclohexane) ; R f = 0.36 (EtOAc-cyclohexane, 1:9). 3.60; N, 6.04; S, 14.17. HRMS (ESI): m/z calcd for [C14H16ClNOS + H] + : 282.0719; found: 282.0732
	82 83 84

1

H NMR (400 MHz, CDCl3) δ 10.51 = (s, 1 H), 8.76 (s, 1 H), 3.21 -3.13 (m, 2 H), 3.04 -2.96 (m, 2 H), 2.50 -2.43 (m, 2 H).

13

C NMR (100 MHz, CDCl3) δ = 188.7, 171.0, 146.0, 145.3, 140.4, 137.4, 128.1, 124.2, 30.1, 27.4. HRMS (ESI): m/z calcd for [C11H8ClNOS + H] + : 238.0088; found: 238.0122. Anal Calcd for C11H8ClNOS: C, 55.58; H, 3.39; N, 5.89; S, 13.49. Found: C, 56.35; H, 3.44; N, 5.82; S, 13.10. [4,5]thieno[2,3-b]pyridine-3-carbaldehyde (132) HRMS (ESI): m/z calcd for [C12H10ClNOS + H] + : 252.0244; found: 252.0266. Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent Yield: 571 mg (86%); White solid; mp 148-152 °C (cyclohexane); ; R f = 0.40 (EtOAccyclohexane, 1:9). 1 H NMR (400 MHz, CDCl3) δ = 10.54 (s, 1 H), 8.76 (s, 1 H), 3.41 -3.31 (m, 1 H), 2.90 -2.81 (m, 2 H), 2.61 -2.54 (m, 1 H), 1.99 -1.83 (m, 2 H), 1.53 -1.43(m, 2 H), 1.10 (d, J = 6.5 Hz, 2 H). 13 C NMR (100 MHz, CDCl3) δ = 189.0, 166.3, 145.4, 141.4, 140.6, 130.2, 128.9, 124.4, 35.2, 30.3, 28.9, 26.1, 21.6. HRMS (ESI): m/z calcd for [C13H12ClNOS + H] + : 266.0401; found: 266.0397. Anal Calcd for C13H12ClNOS: C, 58.75; H, 4.55; N, 5.27; S, 12.06. Found: C, 58.82; H, 4.56; N, 5.11; S, 12.00.

7-(tert-butyl)-4-chloro-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine-3carbaldehyde (144) Yield: 246 mg (32%); White solid; mp 190-192 °C (cyclohexane); ; R f = 0.40 (EtOAccyclohexane, 1:9).

1 H NMR (400 MHz, CDCl3) δ = 10.55 (s, 1 H), 8.77 (s, 1 H), 3.50 -3.35 (m, 1 H), 2.96 -2.80 (m, 2 H), 2.63 -2.55 (m, 1 H), 2.18 -2.01 (m, 1 H), 1.58 -1.50 (m, 2 H), 1.41 -1.31 (m, 1 H), 0.92 (s, 9 H).

4-Chloro-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-b]pyridine-3carbaldehyde (146)

13 C NMR (100 MHz, CDCl3) δ = 189.3, 165.4, 145.3, 145.1, 141.3, 134.3, 129.9, 124.7, 31.7, 30.1, 28.4, 26.9, 26.4. HRMS (ESI): m/z calcd for [C13H12ClNOS + H] + : 266.0401; found: 266.0429. Anal Calcd for C13H12ClNOS: 58.75; H, 4.55; N, 5.27; S, 12.06. Found: 58.62; H, 4.50; N, 5.15; S, 12.22.

4-Chloro-2,3-dimethylthieno[2,3-b]pyridine-5-carbaldehyde (148) Yield: 354 mg (63%); yellow solid; mp 216-218 °C (cyclohexane); ; R f = 0.29 (EtOAccyclohexane, 1:9). HRMS (ESI): m/z calcd for [C12H12ClNOS + H] + : 254.0401; found: 254.0419. Anal Calcd for C12H12ClNOS: C, 56.80; H, 4.77; N, 5.52; S, 12.63. Found: C, 56.71; H, 4.79; N, 5.35; S, 12.48. 4-Chloro-3-methyl-2-pentylthieno[2,3-b]pyridine-5-carbaldehyde (152) Yield: 436 mg (62%); White solid; mp 65-67 °C (EtOH-H2O); ; R f = 0.42 (EtOAccyclohexane, 1:9). Anal Calcd for C14H16ClNOS: C, 59.67; H, 5.72; Cl, 12.58; N, 4.97; S, 11.38. Found: C, 59.97; H, 5.23; N, 5.25; S, 11.60.

  The solution was stirred for 30 minutes at 70°C. The reaction mixture was treated by ice and water. If precipitate was formed; it was filtered under vacuum, washed and dried, otherwise the solution was extracted by ethyl acetate and dried by Na2SO4 anhyd and concentrated under vacuum. The precipitate or the residue was recrystallized from ethanol.

	Chapter 3 -Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent
	Yield: 69 mg (55%); white solid; mp 168 °C (ethanol 96%).
	87

1

H NMR (400 MHz, CDCl3) δ = 10.54 (s, 1 H), 8.81 (s, 1 H), 3.86 (s, 2 H), 3.46 (t, J = 5.9 Hz, 2 H), 2.97 (t, J = 5.9 Hz, 2 H).

13

C NMR (101 MHz, CDCl3) δ 188.8, 165.35, 146.6, 141.6, 136.5, 130.0, 128.6, 124.6, 28.9, 26.7, 25.9. HRMS (ESI): m/z calcd for [C11H8ClNOS2 + H] + : 269.9814; found: 269.9821. Anal Calcd for C11H8ClNOS2: C, 48.98; H, 2.99; Cl, 13.14; N, 5.19; O, 5.93; S, 23.77. Found: C, 49.00; H, 3.14; N, 5.92; S, 23.47.

Ethyl 7,8,9,10-tetrahydrobenzo[4,5]thieno[2,3-b]thieno[2,3-d]pyridine-2carboxylate (156)

Compound 132 (0.1 g, 0.4 mmol) was dissolved with ethyl thioglycolate (0.05 g, 0.4 mmol) in DMF (3 ml) and potassium carbonate (0.06 g, 0.4 mmol) was introduced.

1

H NMR (400 MHz, CDCl3) δ = 8.97 (s, 1 H), 8.24 (s, 1 H), 4.46 (q, J = 7.1 Hz, 2 H), 3.07 -3.03 (m, 2 H), 2.98 -2.95 (m, 2 H), 2.08 -1.95 (m, 4 H), 1.46 (t, J = 7.1 Hz, 3 H). 13 C NMR (101 MHz, CDCl3) δ = 162.3, 155.3, 142.8, 142.6, 138.5, 133.4, 132.6, 129.3, 127.6, 127.3, 61.9, 25.7, 25.3, 23.0, 22.4, 14.3. HRMS (ESI): m/z calcd for [C16H15NO2S2 + H] + : 318.0617; found: 318.0626. Anal Calcd for C16H15NO2S2: C, 60.54; H, 4.76; N, 4.41; S, 20.20. Found: C, 60.72; H, 4.72; N, 4.80; S, 20.13.

  88 Vellaisamy Sridharan et al., "Cerium(IV) Ammonium Nitrate Is an Excellent, General Catalyst for the Friedländer and Friedländer-Borsche Quinoline Syntheses: Very Efficient Access to the Antitumor Alkaloid Luotonin A," The Journal of Organic Chemistry 74, no. 15 (August 7, 2009): 5715-18, doi:10.1021/jo900965f.89 Seck et al., "Synthesis of New Selenophene and Thiazole Analogues of the Tacrine Series," 2012; Seck, Thomae, and Kirsch, "Synthesis of Substituted Amino-Cycloalkyl[b]thieno-[3,2-e]pyridines"; Thomae, Kirsch, and Seck, "Synthesis of Selenophene Analogues of the Tacrine Series," May 2008.

90

Sridharan et al., "Cerium(IV) Ammonium Nitrate Is an Excellent, General Catalyst for the Friedländer and Friedländer-Borsche Quinoline Syntheses."

11-Methyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3-b]quinolone (177)

  Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction 101 13 C NMR (100 MHz, CDCl3) δ 163.4, 153.3, 140.3, 139.3, 136.5, 127.8, 126.8, 33.4, 30.9, 29.8, 27.4, 26.0, 23.2, 22.9, 15.0. : 258.1304; found: 258.1311. Anal. Calcd for C16H19NS: C, 74.66; H, 7.44; N, 5.44; S, 12.46. Found: C, 74.05; H, 7.44; Calcd for C17H21NS: C, 75.23; H, 7.80; N, 5.16; S, 11.81. Found: C, 75.03; H, 7.69; N, 5.46; S, 12.19.

	HRMS (ESI): m/z calcd for [C15H17NS + H] + : 244.1154; found: 244.1143. N, 5.70; S, 12.38. 2,12-Dimethyl-2,3,4,7,8,9,10,11-octahydro-1H-benzo[4,5]thieno[2,3-
	Anal. Calcd for C15H17NS: C, 74.03; H, 7.04; N, 5.76; S, 13.17. Found: C, 74.04; H, 7.44; 2,3,4,6-Tetramethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (178) b]cyclohepta[e]pyridine (180)
	N, 5.79; S, 12.76.
	11-Methyl-1,2,3,6,7,8,9,10-octahydrocyclohepta[b]cyclopenta[4,5]thieno[3,2-
	e]pyridine (176)
	Yield: 47%; orange solid; mp 100-102 °C. Yield: 33%; white solid; mp 120-122 °C.
	10-Methyl-2,3,6,7,8,9-hexahydro-1H-cyclopenta[4,5]thieno[2,3-b]quinolone (175) 1 H NMR (400 MHz, CDCl3) δ = 3.13 -2.98 (m, 1 H), 2.97 -2.75 (m, 2 H), 2.53 (s, 3H), Yield: 39%; yellow solid; mp 88 °C. 2.51 (s, 3 H), 2.43 -2.37 (m, 1 H) 2.20 (s, 3 H), 2.05 -1.86 (m, 2 H), 1.46 -1.36 (m, 1
	1 H NMR (400 MHz, CDCl3) δ = 3.25 -3.09 (m, 1 H), 3.04 -2.99 (s, 2 H), 2.91 -2.87 (m, H), 1.03 (d, J = 6.5 Hz, 3 H).
	Yield: 26%; orange solid; mp 114-116 °C. Yield: 38%; yellow solid; mp 116-118 °C. 2 H), 2.79 -2.72 (m, 2 H), 2.73 -2.59 (m, 1 H), 2.56 (s, 3 H), 2.01 -1.71 (m, 6 H), 1.60 13C NMR (100 MHz, CDCl3) δ = 157.0, 152.5, 140.2, 135.2,130.8, 127.5, 126.2, 34.3, -1.43 (m, 1 H), 1.15 (d, J = 6.5 Hz, 3 H). 31.3, 28.8, 27.8, 23.5, 21.3, 16.4, 14.9. 1 H NMR (400 MHz, CDCl3) δ = 3.02-3.00 (m, 4 H), 2.88-2.84 (m, 2 H), 2.76-2.73 (m, 2 H), 2.55 (s, 3H), 1.94 -1.84 (m, 8H). 13 C NMR (100 MHz, CDCl3) δ = 157.90, 152.9, 140.9, 135.2, 130.8, 127.7, 126.6, 36.6, HRMS (ESI): m/z calcd for [C15H20NS + H] + : 246.1311; found: 246.1311.

1

H NMR (400 MHz, CDCl3) δ = 3.19 -3.07 (m, 2 H), 3.02 (t, J = 7.4 Hz, 2 H), 2.61 (s, 3 H), 2.56 (s, 3 H), 2.53 -2.45 (m, 2 H), 2.29 (s,

3 H)

.

13

C NMR (100 MHz, CDCl3) δ = 162.1, 152.

4

, 140.7, 139.6, 136.7, 128.2, 126.2, 30.9, 29.7, 27.5, 23.2, 16.1, 14.7. HRMS (ESI): m/z calcd for [C13H15NS + H] + : 218.0998; found: 218.0997. Anal. Calcd for C13H15NS: C, 71.85; H, 6.96; N, 6.45; S, 14.75. Found: C, 71.35; H, 7.20; N, 6.99; S, 14.00. 1 H NMR (400 MHz, CDCl3) δ 3.17 -3.06 (m, 2 H), 3.06 -2.94 (m, 2 H), 2.78 -2.74 (m, 2 H), 2.56 -2.39 (m, 5 H), 2.03 -1.69 (m, 4 H). Yield: 46%; white solid; mp 148-150 °C. 1 H NMR (400 MHz, CDCl3) δ = 3.18 -3.08 (m, 4 H), 3.04 -3.00 (m, 2 H), 2.94 -2.85 (m, 2 H), 2.59 (s, 3 H), 2.55 -2.29 (m, 2 H), 1.92 -1.86 (m, 2 H), 1.78 -1.70 (m, 2 H), 1.68 -1.63 (m, 2 H). 13 C NMR (100 MHz, CDCl3) δ = 162.3, 159.4, 140.5, 137.9, 136.9, 132.7, 128.1, 38.9, 32.1, 31.1, 29.8, 28.1, 27.5, 27.4, 26.8, 15.8. HRMS (ESI): m/z calcd for [C16H19NS + H] + : 258.1311; found: 258.1312. 13 C NMR (100 MHz, CDCl3) δ = 158.1, 153.3, 140.2, 135.2, 130.6, 127.8, 126.8, 33.4, 28.1, 26.3, 26.2, 23.4, 23.2, 22.8, 22.6, 15.4. Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction 102 HRMS (ESI): m/z calcd for [C16H19NS + H] + 1 H NMR (400 MHz, CDCl3) δ = 3.19 -3.14 (m, 1 H), 2.91 -2.88 (m, 2 H), 2.61 (s, 3 H), 2.60 (s, 3 H), 2.58 -2.46 (m, 1 H), 2.28 (s, 3 H), 2.02 -1.84 (m, 2 H), 1.57 -1.47 (m, 1 H), 1.15 (d, J = 6.5 Hz, 1 H). 13 C NMR (101 MHz, CDCl3) δ = 157.1, 152.5, 140.4, 135.1, 130.8, 127.5, 126.2, 34.3, 31.3, 28.8, 27.8, 23.4, 21.3, 16.4, 14.9. HRMS (ESI): m/z calcd for [C15H19NS + H] + : 246.1311; found: 246.1339. 2,11-Dimethyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3-b]quinolone (179) 30.7, 29.4, 26.3, 26.0, 23.3, 22.7, 22.3, 21.8, 15.6. HRMS (ESI): m/z calcd for [C17H21NS + H] + : 272.1467; found: 272.1469. Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction 103 Anal. 1 H NMR (400 MHz, CDCl3) δ = 3.15 -3.02 (m, 3 H), 2.88 -2.72 (m, 4 H), 2.57 (s, 3 H), 2.53 -2.35 (m, 1 H), 1.93 -1.71 (m, 3 H), 1.69 -1.59 (m, 2 H), 1.59 -1.53 (m, 2 H), 1.49 -1.34 (m, 2 H), 1.07 (d, J = 6.5 Hz, 1 H).

13 C NMR (100 MHz, CDCl3) δ = 159.0, 150.3, 139.1, 135.4, 133.0, 128.0, 127.4, 36.7, 31.9, 30.7, 29.7, 29.4, 28.0, 27.2, 26.7, 26.0, 21.8, 16.4. 2,3,4,7-Tetramethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine (181) Yield: 9%; orange solid; mp 158-160 °C. Anal. Calcd for C15H19NS.5%H2O: C, 69.75; H, 7.97; N, 5.42; S, 12.41. Found: C, 69.72; H, 7.69; N, 5.84; S, 12.44.

Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction 104 3,

11-Dimethyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3-b]quinolone (182)

	Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction
	Anal. Calcd for C18H23NS: C, 75.74; H, 8.12; N, 4.91; S, 11.23. Found: C, 76.02; H,
	8.08; N, 5.60; S, 11.62.
	7-(tert-Butyl)-2,3,4-trimethyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridine
	(184)
	Yield: 39%; white solid; mp 120-122 °C.
	1 H NMR (400 MHz, CDCl3) δ = 3.18 -3.08 (m, 1 H), 3.03 -2.99 (m, 2 H), 2.96 -2.85
	(m, 1 H), 2.77 -2.73 (m, 2 H), 2.55 (s, 1 H), 2.52 -2.44 (m, 1 H), 2.02 -1.94 (m, 2 H),
	1.94 -1.83 (m, 5 H), 1.56 -1.45 (m, 1 H), 1.12 (d, J = 6.5 Hz, 3 H). Yield: 26%; orange solid; mp 134-136 °C.
	13 C NMR (100 MHz, CDCl3) δ = 158.1, 153.3, 140.2, 134.90, 130.4, 127.4, 126.8, 34.3,
	33.4, 31.3, 28.8, 27.8, 26.3, 23.4, 22.8, 21.3, 15.4.
	Yield: 21%; white solid; mp 126-128 °C.
	1 H NMR (400 MHz, CDCl3) δ = 3.23 -3.02 (m, 3 H), 3.05 -2.78 (m, 4 H), 2.65 (s, 3 H),
	2.57 -2.40 (m, 1 H), 2.02 -1.97 (m, 2 H), 1.93 -1.79 (m, 2 H), 1.75 -1.62 (m, 4 H), 1.55
	-1.45 (m, 1H), 1.13 (d, J = 6.5 Hz, 3 H).
	13 C NMR (100 MHz, CDCl3) δ = 159.4, 157.1, 138.8, 135.2, 132.9, 130.6, 127.7, 38.8,
	34.4, 32.0, 31.4, 28.8, 28.0, 27.9, 27.3, 26.8, 21.3, 16.2.
	105

HRMS (ESI): m/z calcd for [C17H21NS + H] + : 272.1467; found: 272.1474. Anal. Calcd for C17H21NS.2%H2O: C, 73.72; H, 7.86; N, 5.05; S, 11.57. Found: C, 73.21; H, 7.60; N, 5.62; S, 11.58. 12-Methyl-2,3,4,7,8,9,10,11-octahydro-1H-benzo[4,5]thieno[2,3b]cyclohepta[e]pyridine (183) HRMS (ESI): m/z calcd for [C18H23NS + H] + : 286.1624; found: 286.1644. 1 H NMR (400 MHz, CDCl3) δ = 3.24 -3.19 (m, 1 H), 2.94 -2.87 (m, 2 H), 2.63 (m, 1 H), 2.64 (s, 3 H), 2.61 (s, 3 H), 2.29 (s, 3 H), 2.15 -2.10 (m, 1 H), 1.60 (m, 1 H), 1.49 -1.33 (m, 1 H), 0.98 (s, 9 H). 13 C NMR (100 MHz, CDCl3) δ = 157.4, 152.6, 140.1, 136.1, 130.6, 127.8, 126.1, 44.5, 32.4, 31.9, 29.7, 27.8, 27.4, 24.8, 23.6, 22.7, 16.2, 14.9. HRMS (ESI): m/z calcd for [C18H25NS+ H] + : 288.1780; found: 288.1771. Anal. Calcd for C18H25NS: C, 75.21; H, 8.77; N, 4.87; S, 11.15. Found: C, 74.11; H, 8.74; N, 5.35; S, 10.94.

3-(

tert-Butyl)-11-methyl-1,2,3,4,7,8,9,10-octahydrobenzo[4,5]thieno[2,3- b]quinolone (185)

	Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction
	13 C NMR (100 MHz, CDCl3) δ = 158.1, 152.3, 140.7, 135.9, 131.0, 127.2, 126.4, 44.5,
	32.4, 31.9, 29.7, 28.5, 27.8, 27.4, 24.8, 23.6, 22.7, 22.1, 20.8, 16.2.
	HRMS (ESI): m/z calcd for [C20H27NS + H] + : 314.1937; found: 314.1977.
	Anal. Calcd for C20H27NS.2%H2O: C, 75.09; H, 8.73; N, 4.37; S, 10.02. Found: C, 75.30;
	H, 8.58; N, 4.91; S, 9.99.
	2,3,4-Trimethyl-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-b]pyridine (186)
	Yield: 34%; orange solid; mp 84-86 °C.
	13 C NMR (100 MHz, CDCl3) δ = 156.5, 152.6, 139.6, 139.5, 133.3, 131.5, 126.2, 31.7,
	29.7, 29.5, 27.2, 26.6, 23.7, 17.1, 15.1.
	Yield: 29%; orange solid; mp 86-88 °C.
	106

1

H NMR (400 MHz, CDCl3) δ = 3.26 -3.14 (m, 1 H), 3.07 -2.96 (m, 2 H), 2.96 -2.80 (m, 2 H), 2.78 -2.69 (m, 2 H), 2.70 -2.56 (m, 1 H), 2.53 (s, 3 H), 2.17 -2.04 (m, 1 H), 1.97 -1.79 (m, 4 H), 1.65 -1.53 (m, 1 H), 1.48 -1.34 (m, 1 H), 0.98 (s, 9 H). 1 H NMR (400 MHz, CDCl3) δ = 3.18-316 (m, 2 H), 2.93-2.91 (m, 2 H), 2.64 (s, 3 H), 2.61 (s, 3 H), 2.31 (s, 3 H), 1.96-1.90 (m, 2 H), 1.84 -1.69 (m, 4 H). HRMS (ESI): m/z calcd for [C15H19NS + H] + : 246.1311; found: 246.1306.

12-Methyl-2,3,4,7,8,9,10,11-octahydro-1H-cyclohepta[4,5]thieno[2,3-b]quinolone (187)

	Chapter 4 -Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction
	13 C NMR (100 MHz, CDCl3) δ = 159.0, 152.7, 140.4, 133.9, 131.8, 127.1, 126.9, 33.0,
	32.0, 29.5, 29.4, 27.3, 26.5, 23.3, 22.6, 22.4, 16.1.
	HRMS (ESI): m/z calcd for [C17H21NS + H] + : 272.1467; found: 272.1472.
	Anal. Calcd for C17H21NS.2%H2O: C, 73.72; H, 7.86; N, 5.05; S, 11.57. Found: C, 75.38;
	H, 7.61; N, 5.91; S, 11.92.
	13-Methyl-1,2,3,4,5,8,9,10,11,12-decahydrocyclohepta[4,5]thieno[2,3-
	b]cyclohepta[e]pyridine (188)
	13 C NMR (100 MHz, CDCl3) δ = 159.09, 156.5, 140.1, 138.7, 133.6, 133.1, 131.8, 38.5,
	31.9, 31.7, 29.8, 29.6, 28.2, 27.3, 26.8, 26.6, 17.1.
	Yield: 21%; orange solid; mp 77-78 °C.
	1 H NMR (400 MHz, CDCl3) δ = 3.24 -3.10 (m, 2 H), 3.05 -2.92 (m, 2 H), 2.92 (dd, J =
	12.0, 6.5 Hz, 2 H), 2.84 -2.72 (m, 2 H), 2.58 (s, 3 H), 1.95 -1.83 (m, 6 H), 1.83 -1.65
	(m, 2 H).
	107

Yield: 22%; brown solid; mp 164-166 °C.

1

H NMR (400 MHz, CDCl3) δ = 3.19 -3.13 (m, 2 H), 3.14 -3.08 (m, 2 H), 2.97 -2.89 (m, 4 H), 2.66 (s, 3 H), 1.95 -1.84 (m, 4 H), 1.83 -1.70 (m, 6 H), 1.68 -1.63 (m, 4 H). HRMS (ESI): m/z calcd for [C18H23NS + Na] + : 308.1443; found: 308.1439.

2,3,4-Trimethyl-5,6,7,8-tetrahydrothieno[2,3-b]quinolone (189)

	Yield: 20%; white solid; mp 97-100 °C.
	1 H NMR (400 MHz, CDCl3) δ = 3.06 -2.91 (m, 2 H), 2.75 -2.65 (m, 2 H), 2.55 (s, 3 H),
	2.41 (s, 3 H), 2.38 (s, 3 H), 1.90 -1.70 (m, 4 H).

13

C NMR (100 MHz, CDCl3) δ = 161.5, 150.9, 138.7, 133.0, 132.3, 126.0, 125.6, 30.7, 29.7, 26.0, 22.7, 20.8, 15.4, 14.1. HRMS (ESI): m/z calcd for [C14H17NS + H] + : 232.1154; found: 232.1136.

  . Pd(OAc)2 interacts in situ with BINAP to generate BINAP(O) and Consequences on the Mechanism of Their Oxidative Addition with Phenyl Iodide and PhPd(OAc)(PPh3)2 as Intermediate in the Heck Reaction," Organometallics 14, no. 12 (December 1, 1995): 5605-14, doi:10.1021/om00012a029; Christian Amatore, Anny Jutand, and Fouad Khalil, "Neutral palladium(0) Complexes from Pd(OAc)2 and Tri-2-Furylphosphine and Their Reactivity in Oxidative Addition of Phenyl Iodide," Arkivoc 2006, no. 4 (March 2, 2006): 38, doi:10.3998/ark.5550190.0007.405; Christian Amatore, Anny Jutand, and Audrey Thuilliez, "Formation of Palladium(0) Complexes from Pd(OAc)2 and a Bidentate Phosphine Ligand (Dppp) and Their Reactivity in Oxidative Addition," Organometallics 20, no. 15 (July 1, 2001): 3241-49, doi:10.1021/om0101137.

	Pd(0) (Scheme 75). 121
	Scheme 75
	5.1.3.4 Bases
	121 William J. Marshall and Vladimir V. Grushin, "Palladium(II) and Palladium(0) Complexes of BINAP(O) (2-
	(Diphenylphosphino)-2'-(Diphenylphosphinyl)-1,1'-Binaphthyl)," Organometallics 22, no. 3 (February 1, 2003): 555-
	62, doi:10.1021/om020838q.
	122 Guram, Rennels, and Buchwald, "A Simple Catalytic Method for the Conversion of Aryl Bromides to
	Arylamines."
	123 Johanne Renaud et al., "Selective Estrogen Receptor Modulators with Conformationally Restricted Side
	Chains. Synthesis and Structure-Activity Relationship of ERalpha-Selective Tetrahydroisoquinoline Ligands," Journal
	of Medicinal Chemistry 48, no. 2 (January 27, 2005): 364-79, doi:10.1021/jm040858p.

Choosing a suitable, sufficiently strong base is another key factor in palladiumcatalyzed amination reactions. tBuONa was the first base used

122

and it has been extensively used with dialkyl biaryl phosphine ligand systems by Buchwald and coworkers. tBuOK exhibits the same efficiency in some of these reactions 123 but both of these bases have some limitations because the functional group tolerance for substrates is limited.

124

Hydroxide bases KOH, NaOH have also been used recently because they are inexpensive, but they generally give lower reaction rates than alkoxides do.

125

Weak 120 Christian Amatore et al., "Evidence for the Ligation of Palladium(0) Complexes by Acetate Ions:

124

Surry and Buchwald, "Dialkylbiaryl Phosphines in Pd-Catalyzed Amination."

125

Huang et al., "Expanding Pd-Catalyzed C-N Bond-Forming Processes"; Debabrata Maiti et al., "Palladium-Catalyzed Coupling of Functionalized Primary and Secondary Amines with Aryl and Heteroaryl Halides: Two Ligands Suffice in Most Cases," Chemical Science (Royal Society of Chemistry: 2010) 2, no. 1 (January 1, 2011): 57-68, doi:10.1039/C0SC00330A.

Table 6

 6 Ullmann synthesis in presence of ligandVarious reported combination 131 of ligands, copper salts, solvents, bases and temperature was matched in order to identify the optimum reaction conditions. All the trial are summarized in the following table, and as it is shown below, all of them were utterly failure (Table6). Ullmann coupling trials Kwong, Klapars, and Buchwald, "Copper-Catalyzed Coupling of Alkylamines and Aryl Iodides"; Ma, Cai, and Zhang, "Mild Method for Ullmann Coupling Reaction of Amines and Aryl Halides"; Kelkar, Patil, and Chaudhari, "Copper-Catalyzed Amination of Aryl Halides"; Kong et al., "Copper-Catalyzed Synthesis of Substituted Quinolines via C-N Coupling/Condensation from Ortho -Acylanilines and Alkenyl Iodides"; Kunz, Scholz, and Ganzer, "Renaissance of Ullmann and Goldberg Reactions -Progress in Copper Catalyzed C-N-, C-O-and C-S-Coupling."

	5.2.2 Ullmmann reaction
	5.2.2.1 Ligand free trials
	Classical Ullmann synthesis 130 was tried by using copper powder or copper oxide (II)
	and K2CO3 in presence of toluene, DMF or nitrobenzene as solvent. Open air or inert gas
	environment was applied and no product was noticed.
	5.2.2.2

Table 7

 7 Products

of coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines.

7-(tert-butyl)-N-(4-chlorophenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin- 4-amine (260)

	Chapter 5-C-N coupling between 4-chlorothieno[2,3-b]pyridine derivatives and aromatic amines
	13 C NMR (100 MHz, CDCl3): δ = 161.4, 148.8, 146.1, 138.9, 135.5, 130.1, 127.6, 126.0,
	123.9, 121.4, 103.0, 35.3, 29.7, 29.1, 25.5, 21.6. 19.3 (d, J = 8.7 Hz, 2 H), 6.81 (d, J = 4.5 Hz, 1 H), 6.59 (s, 1 H), 3.28 -3.23 (m, 1 H), 3.06 -
	HRMS (ESI): m/z calcd for [C19H19ClN2S + H] + : 343.1030; found: 343.1013. 2.94 (m, 1 H), 2.92 -2.86 (m, 1 H), 2.72 -2.55 (m, 2H), 2.17 -2.13 (m, 1 H), 1.65 -1.54
	7-(tert-butyl)-N-phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-b]pyridin-4-amine (m, 1 H), 1.54 -1.46 (m, 1 H), 0.99 (s, 9 H).
	(259) 13 C NMR (101 MHz, CDCl3) δ = 162.2, 146.5, 146.1, 138.6, 135.5, 129.7, 129.2, 125.7,
	123.2, 121.2, 103.9, 44.4, 32.4, 28.1, 27.4, 27.2, 24.4.
	HRMS (ESI): m/z calcd for [C21H23ClN2S + H] + : 371.1343; found: 371.1361.
	N-phenyl-6,7,8,9-tetrahydro-5H-cyclohepta[4,5]thieno[2,3-b]pyridin-4-amine (261)
	Yield: 66%; yellow solid; mp 92 °C
	Yield: 56%; yellow solid; mp 160 °C
	1 H NMR (400 MHz, CDCl3) δ = 8.14 (d, J = 5.0 Hz, 1 H), 7.39 (t, J = 7.8 Hz, 2 H), 7.22 -
	13 C NMR (100 MHz, CDCl3) δ = 161.6, 147.1, 145.7, 139.7, 135.2, 129.7, 125.8, 124.5, 7.10 (m, 3 H), 6.92 (d, J = 5.3 Hz, 1 H), 6.41 (s, 1 H), 3.24 -3.15 (m, 2 H), 2.97 -2.87
	122.2, 121.0, 103.6, 44.4, 32.4, 29.7, 27.4, 27.2, 24.5. (m, 2 H), 1.97 -1.74 (m, 6 H).
	13 C NMR (100 MHz, CDCl3) δ = 161.1, 146.7, 145.8, 140.7, 138.9, 131.3, 129.7, 123.7,
	123.4, 121.3, 105.8, 30.9, 29.9, 29.4, 27.1, 26.6.
	1 H NMR (400 MHz, CDCl3) δ = 7.97 (s, 1 H), 7.25 (s, 1 H), 7.17 (s, 2 H), 6.46 (s, 1 H),
	Yield: 75%; yellow solid; mp 80-83 °C
	135

6.29 (s, 1 H), 3.18 -3.11 (m, 1 H), 2.86 -2.82 (m, 2 H), 2.60 -2.54 (m, 1 H), 2.18 (s, 3 H), 1.98 -1.90 (m, 2 H), 1.54 -1.45 (m, 1 H), 1.10 (d, J = 6.6 Hz, 3 H). 1 H NMR (400 MHz, CDCl3) δ = 8.10 (d, J = 5.3 Hz, 1 H), 7.42 (t, J = 7.9 Hz, 2 H), 7.26 (d, J = 7.6 Hz, 2 H), 7.18 (dd, J = 14.0, 6.5 Hz, 1 H), 6.86 (d, J = 5.3 Hz, 1 H), 6.70 (s, 1 H), 3.32 -3.28 (m, 1 H), 3.08 -3.00 (m, 1 H), 2.97 -2.83 (m, 1 H), 2.71 -2.57 (m, 1 H), 2.24 -2.10 (m, 1 H), 1.67 -1.56 (m, 1 H), 1.55 -1.48 (m, 1 H), 1.00 (s, 9 H). HRMS (ESI): m/z calcd for [C21H24N2S + H] + : 337.1733; found: 337.1766. HRMS (ESI): m/z calcd for [C18H18N2S + H] + : 295.1263; found: 295.12911.

Table 1 (

 1 continued)

  H NMR (400 MHz, CDCl 3 ): δ = 6.94 (s, 2 H), 2.74-2.67 (m, 2 H), 2.58-2.52 (m, 2 H), 2.42 (s, 3 H), 1.86-1.79 (m, 4 H). NMR (100 MHz, CDCl 3 ): δ = 194.1, 163.9, 130.6, 117.6, 115.8, 30.7, 28.1, 24.7, 23.1, 22.9. HRMS (ESI): m/z calcd for [C 10 H 13 NOS + Na] + : 218.0610; found: 218.0602. Anal. Calcd for C 10 H 13 NOS: C, 61.50; H, 6.71; N, 7.17; O, 8.19; S, 16.42. Found: C, 61.85; H, 6.65, N, 6.86; S, 16.18.

13

C

2-Amino-4,5-dimethylthiophen-3-yl)ethanone (4g)

  CDCl 3 ): δ = 194.6, 161.4, 136.1, 121.1, 118.5, 31.7, 31.0, 29.8, 28.4, 27.6, 26.6. HRMS (ESI): m/z calcd for [C 11 H 15 NOS + Na] + : 232.0767; found: 232.0778. Anal. Calcd for C 11 H 15 NOS: C, 63.12; H, 7.22; N, 6.69; O, 7.64; S, 15.32. Found: C, 62.99; H, 7.13; N, 7.11; S, 15.08. NMR (100 MHz, CDCl 3 ): δ = 194.4, 163.1, 128.4, 117.0, 113.8, 30.6, 15.8, 12.6. HRMS (ESI): m/z calcd for [C 8 H 11 NOS + H] + : 170.634; found: 170.0633; calcd for [C 8 H 11 NOS + Na] + : 192.0454; found: 192.0456. Anal. Calcd for C 8 H 11 NOS: C, 56.77; H, 6.55; N, 8.28; S, 18.95. Found: C, 56.65; H, 6.41; N, 9.07; S, 19.07.

	1 H NMR (400 MHz, CDCl 3 ): δ = 6.62 (s, 2 H), 2.87-2.79 (m, 2 H), 2.61-
	2.59 (m, 2 H), 2.43 (s, 1 H), 1.89-1.83 (m, 2 H), 1.75-1.64 (m, 4 H).
	13 C NMR (100 MHz, 1-(Yield: 1.5 g (36%); brown solid; mp 154-157 °C.
	1 H NMR (400 MHz, CDCl 3 ): δ = 6.87 (s, 2 H), 2.46 (s, 3 H), 2.23 (s, 3 H),
	2.18 (s, 3 H).

13 C

2-Amino-5,7-dihydro-4H-thieno[2,3-c]thiopyran-3-yl)etha- none (4j)

  3, 117.04, 31.3, 30.9, 27.3, 22.5, 15.8, 14.02. NMR (400 MHz, CDCl 3 ): δ = 6.96 (s, 2 H), 3.62 (t, J = 1.6 Hz, 2 H), 3.02 (ddd, J = 5.8, 4.6, 1.2 Hz, 2 H), 2.93 (t, J = 5.5 Hz, 2 H), 2.42 (s,3 H).

	HRMS (ESI): m/z calcd for [C 12 H 19 NOS -H + Na] + : 247.0993; found:
	247.1001.
	Anal. Calcd for C 12 H 19 NOS: C, 63.96; H, 8.50; N, 6.22; S, 14.23. Found:
	C, 64.01; H, 8.34; N, 6.17; S, 14.35.
	1-(Yield: 3.8 g (72%); brownish solid; mp 167-170 °C.

1 H

  Calcd for C 10 H 14 N 2 OS•H 2 O: C, 52.54; H, 7.06; N, 12.25; S, 14.02. Found: C, 52.48; H, 7.01; N, 12.62; S, 14.04.

	1 H NMR (400 MHz, DMSO-d 6 ): δ = 8.19 (s, 2 H), 3.24 (s, 2 H), 2.73 (t,
	J = 5.7 Hz, 2 H), 2.57 (t, J = 5.7 Hz, 2 H), 2.31 (s, 3 H), 2.29 (s, 3 H).
	13 C NMR (100 MHz, DMSO-d 6 ): δ = 192.2, 164.6, 113.3, 128.5, 113.3,
	113.2, 53.0, 52.0, 45.1, 30.1, 27.7.
	HRMS (ESI): m/z calcd for [C 10 H 14 N 2 OS + H] + : 211.0900; found:
	211.0891.
	Anal.

  Anal. Calcd for C 11 H 10 ClNS: C, 59.05; H, 4.51; N, 6.26; S, 14.33. Found: C, 59.70; H, 4.58; N, 6.48; S, 14.08.

	1 H NMR (400 MHz, CDCl 3 ): δ = 8.30 (d, J = 5.1 Hz, 1 H), 7.23 (d, J =
	5.1 Hz, 1 H), 3.16 (dd, J = 5.8, 2.7 Hz, 2 H), 2.89 (t, J = 4.2 Hz, 2 H),
	1.98-1.85 (m, 4 H).
	13 C NMR (101 MHz, CDCl 3 ): δ = 162.0, 144.8, 139.1, 137.9, 130.6,
	127.8, 120.8, 26.7, 26.1, 22.5.
	HRMS (ESI): m/z calcd for [C 11 H 10 ClNS + Na] + : 246.0115; found:
	246.0133.

  calcd for [C12 H 13 ClNS] + : 238.0452; found: 238.0456. Anal. Calcd for C 12 H 12 ClNS: C, 60.62; H, 5.09; N, 5.89; S, 13.49. Found: C, 60.44; H, 5.11; N, 5.82; S, 13.03.

	6 Hz, 1 H), 2.97-2.89 (m, 2 H), 2.64-
	2.56 (m, 1 H), 2.03-1.92 (m, 2 H), 1.60-1.48 (m, 1 H), 1.17 (d, J =
	6.6 Hz, 3 H).
	13 C NMR (100 MHz, CDCl 3 ): δ = 162.6, 145.1, 138.6, 137.6, 130.3,
	127.8, 120.7, 35.0, 30.6, 28.9, 25.9, 21.7.
	HRMS (ESI): m/z

Chloro-3-methyl-2-propylthieno[2,3-b]pyridine (5h)

  H NMR (400 MHz, CDCl 3 ): δ = 8.31 (d, J = 5.1 Hz, 1 H), 7.24 (d, J = 5.1 Hz, 1 H), 2.86 (t, J = 7.6 Hz, 2 H), 2.60 (s, 3 H), 1.80-1.76 (m, 2 H), 1.04 (t, J = 7.4 Hz, 3 H).

	13 C NMR (100 MHz, DMSO-d 6 ): δ = 160.8, 145.8, 136.7, 135.6, 129.8,
	125.3, 121.2, 14.3, 13.7.
	HRMS (ESI): m/z calcd for [C 9 H 8 ClNS + H] + : 198.0139; found:
	198.0141.
	Anal. Calcd for C 9 H 8 ClNS•H 2 O: C, 53.04; H, 4.20; N, 6.87; S, 15.70.
	Found: C, 53.34; H, 3.94; N, 7.00; S, 15.31.
	4-Yield: 141 mg (25%); white solid; mp 36-38 °C (MeOH-H 2 O); R f =
	0.48 (EtOAc-cyclohexane, 9:1).
	13 C NMR (100 MHz, CDCl 3 ): δ = 162.0, 145.2, 141.4, 137.9, 130.9,
	125.3, 121.0, 30.4, 24.2, 14.7, 13.8.
	HRMS (ESI): m/z calcd for [C 11 H 12 ClNS + H] + : 226.0452; found:
	226.0456.
	Anal. Calcd for C 11 H 12 ClNS: C, 58.53; H, 5.36; N, 6.20; S, 14.20. Found:
	C, 58.70; H, 5.48; N, 6.38; S, 13.87.

1

Table 1 .

 1 ) Inhibitory activity of product 1-8 with CDC25A and B (concentration of compounds 20 µM)

	compounds	CDC25A	CDC25B
	DMSO	(%) 0 3.81	(%) 0 1 .25
	1	24.02 3.98	30.42 2.66
	2	16.59 5.59	28.32 3.95
	3	28.65 3.23	18.39 1.29
	4	35.95 1.29	16.42 1.13
	5	40.64 0.92	54.94 1.13
	6	32.72 3.97	34.63 2.31
	7	28.56 2.38	32.89 1.09
	8	7.34 1.77	28.94 3.43
	Naphtoquinone	99.68 4.52	99.41 3.12

Table 2 .

 2 Synthesized derivatives and their molecular modeling interaction scores with CDC25 phosphatase A and B indicating hydrogen bonds (number and length) and energy.

	Compounds R1	R2	CDC25A			CDC25B
				Energy	HB		Energy	HB
				(Kcal/mol)	no° Length	(Kcal/mol)	no° Length
						(A°)			(A°)
	1	H	H	-4.5	3	2.4; 2.6	-6.9	1	2.8
	2	6-OMe	H	-5,6	1	2	-7.3	1	2.6
	3	6-Br	H	-5,4	2	2; 2.5	-6.5	1	2.7
	4	6-Cl	H	-5,6	2	2.5; 2.6	-7.5	1	2.7
	5	7-N(C 2 H 5 ) 2 H	-3.9	2	2.7	-7.7	2	2.5; 2.6
	6	H	2,4-OMe -6.2	1	2.9	-7.6	2	2; 2.7
	7	H	2,5-OMe -6.1	2	2.6; 2.9	-7.8	2	2.3; 2.7
	8	6-Cl	2,4-OMe -5,9	2	2.7; 2.9	-7.4	1	2.2

Table 1 .

 1 Some anti-inflammatory activities of Simple coumarins.

	Cpd	CPE% 1	IL-1β	IL-6	5-LOX	NF-κB	NO	TNF-α	Ref.
	4				IC 50 : 6.6 2				[5]
	5			<200 ng/mL (20 mg/kg) 3, **				<400 ng/mL (20 mg/kg) 3, **	[7]
			<3000 pg/mL					<500 pg/mL
	6		(160 µg/mL, 24 h) 4, *; <1000 pg/mL			Activity < 1 (160 µM) 4, *	<20 µM (160 µM) 4, **	(160 µg/mL, 24 h) 4, *; <400 pg/mL	[10]
			(160 µg/mL) 5, **					(160 µg/mL) 5, **
	14	39% (0.01 mmol/kg)			IC 50 : 72.5 nm				[12]
	15						<10 µM (10 µM) 6, *		[13]
	16					>80% (50 µg/mL) 1			[14]
	17					<10% (50 µg/mL) 1			[14]
	18					<5% (50 µg/mL) 7			[14]
	21					0% 6	0% 6	0% 6	[18]

Table 2 .

 2 Some anti-inflammatory activities of furocoumarins.

	Cpd	COX-2	IL-1β	IL-6	IL-10	NF-κB	NO	iNO	PGE 2	TNF-α	Ref.
			<20 pg/mL
	22	<40% (10 µg/mL) 3, ***	(10 µg/mL) 1, *; <300 pg/mL	<600 pg/mL (30 mg/kg) 2, *	<600 pg/mL (15 mg/kg) 2, **
			(30 mg/kg) 2, *

Table 3 .

 3 Some anti-inflammatory activities of pyranocoumarins.

	Cpd COX-2	IL-1β	IL-6	NF-κB	NO	iNO	PGE 2	TNF-α	Ref.
	28	<20% (30 µM) 1, ***	<40% (30 µM) 1, ***			<20 µM (30 µM) 1, ***; <40 µM (30 µM) 2, **	<20% (30 µM) 1, ***		<40% (30 µM) 1, ***	[40]
	29		<100 pg/mL (25 µg/mL) 1, **	<100 pg/mL (100 µM) 1, **		<15 µM (25 µg/mL) 1, **			<10000 pg/mL (25 µg/mL) 1, *	[41,42]
					<4000 RFU (50 µM)
	30	<60% (50 µM) 1, ***			for NF-κB-dependent alkaline phosphate		<60% (50 µM) 1, ***		<60% (50 µM) 1, ***	[43]
					(SEAP ) 1, ***
	31				<8000 RFU (60 µg/mL) 1, **	<20 µM (60 µM) 1, ***		<500 pg/mL (60 µM) 1, ***		[44]

Table 4 .

 4 Some anti-inflammatory activities of synthetic coumarins.

	Cpd	COX-2	CPE% 3	IL-6	LOX	NO	iNO	TNF-α	Ref.
	32, R = 6-Cl		69 (3 h); 60 (6 h) *						[45]
	35, R1 = 5-chloropyridin-3-yl; R2 = CH 3 COOCH 2			<600 pg/mL (1 µM/kg) 1, *				<1200 pg/mL (1 µM/kg) 1, *	[46]
	38a	-8 ± 17.4 (10 µM) 2 ; 37 ± 15.2 (100 µM) 2

Table 1

 1 products and yields of VH reaction with N-protected 3-acetyl-2-aminothiophenes at 65 °C and 100 °C

	N-protected 3-acetyl-2-aminothiophenes	Thieno[2,3-b]pyridine	65 °C	Yield 100 °C	4-Chloro-3-formylthieno[2,3-b]pyridine	65 °C	Yield 100 °C
			-	21%		94% 9%
	11a	12a			13a
			25% 58%		60% -
	11b	12b			13b
			-	48%		86% 30%
	11c	12c			13c
			11% 54%		65% 9%
	11d	12d			13d
			23% 30%		32%	22%
	11e	12e			13e
			14% 70%		53% traces
	11f	12f			13f
			-	55%		63% traces
	11g	12g			13g
			10% 85%		77% -
	11h	12h			13h

Seck et al., "Synthesis of New Selenophene and Thiazole Analogues of the Tacrine Series," 2012.

H NMR (400 MHz, CDCl3) δ =

2.80 (s, 3 H), 2.60 (s, 3 H), 2.49 (s, 3 H).13 C NMR (100 MHz, CDCl3) δ = 191.1, 181.5, 118.3, 106.2, 30.9, 21.1, 18.9.

Reactivity of N-protected 3-acetyl-2-aminothiophenes in reaction with Vilsmeier-Haack reagent 3.1

Reactivity of 3-acetyl-2-aminothiophenes in Friedländer condensation reaction. 4.1

H NMR (400 MHz, CDCl3) δ = 8.12 (d, J = 4.5 Hz, 1 H), 7.35 (d, J = 8.7 Hz,

H), 7.16

© Georg Thieme Verlag Stuttgart • New York -Synthesis 2016, 48, 2881-2888 A. B. Abdelwahab et al.

Molecules 2016, 21, 1322; doi:10.3390/molecules21101322 www.mdpi.com/journal/molecules

PMACI induced of HMC-1 cells;

LPS-induced of BALF (broncoalveolar lavage fluid);

LPS-stimulated macrophage RAW264.7';

Carageenin paw edema in rat;

In bone marrow-derived mast cell (BMMC);

IL-1β induced of A549 cells; kg is referring to the mice weight; % is referring to percentage of inhibition; * p < 0.05 versus negative control; ** p < 0.01 versus negative control; *** p < 0.001 versus negative control; **** p < 0.005 versus negative control.

LPS-stimulated macrophage RAW264.7;

SNP induced macrophage RAW264.7; % is referring to percentage of inhibition; * p < 0.05 versus negative control; ** p < 0.01 versus negative control; *** p < 0.001 versus negative control.

LPS induced alveolar macrophage;

J774 macrophage stimulated by LPS;

Carageenin paw edema in rat; kg is referring to the mice weight; % is referring to percentage of inhibition. * p < 0.05 versus negative control.

Scheme 6 Synthesis of compound 15 as an example proved the reactivity of novel formylated chlorothienopyridines.

C NMR (100 MHz, CDCl3) δ = 197.1, 167.7,

147.9, 127.3, 123.8, 121.9, 31.3, 23.8, 15.3, 12.5 (s).

H NMR (400 MHz, CDCl3) δ = 10.55 (s, 1 H), 8.80 (s, 1 H), 3.34 -3.30(m, 1 H), 3.08 -2.91 (m, 1 H),

2.92 -2.86 (m, 1 H), 2.55 -2.34 (m, 1 H), 2.01 -1.86 (m, 2 H), 1.51 -1.41 (m, 2 H), 1.07 (d, J = 6.5 Hz, 3 H).

Acknowledgements

Acknowledgment

Ministry of Higher Education (mission division), Cairo, Egypt is gratefully acknowledged for funding the PhD program of A.B.A.

Conclusions

Coumarins and derivatives, even those that have been known for a long time, are still a source of interesting biological active compounds. As the synthesis of these compounds is quite easy, access to a greater amount of derivatives is possible. Since the compounds are sometimes present in edible foods, these may serve as anti-inflammatory supply and as fiber containing plants are considered prebiotics this may help even more to fight inflammation.

Acknowledgment

Ministry of Higher Education (mission division), Cairo, Egypt and Plant Advanced Technologies (PAT) Company, Nancy, France are gratefully acknowledged for funding the PhD program of A.B.A.

from the first time I entered the field of scientific research, his support, enthusiasm, great kindness and his approval which allowed me to follow my PhD in University of Lorraine, and for his visit which he made from Egypt to France I am fully grateful to Plant Advanced Technology (PAT) Company, Nancy, France for giving And my very special grateful to my trainees whom I followed during my PhD period; Loik MANGIN and Benjamin HIRTZ who helped me in synthesizing some starting materials. This PhD project was supported by Ministry of Higher Education (mission division), Cairo, Egypt to whom I am very grateful for funding the PhD program and to the cultural office in the Egyptian embassy, Paris, France especially Mrs. Hanan ELSARKAWY for

Using the compounds which were produced during the PhD as lead structure for producing biologically active materials is also another option.

Annex

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All solvents and reagents were purchased from commercial sources unless otherwise noted. Melting points were determined with a Büchi 530 digital melting point apparatus and are uncorrected. 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, using Me4Si as the internal standard. Hydrogen coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q) and multiplet (m). High-resolution mass spectra were measured with a Micro-Tof-Q98 instrument in ESI mode. Column chromatography was performed using silica gel (60M, 0.04-0.063 mm). Thin-layer chromatography (TLC) was performed using silica gel plates (POLYGRAM SILG/UV254, 0.20 mm), which were visualised under UV light.

All thiophenes were prepared by the Gewald's synthesis which we reported, the procedure, experimental data and supporting information of compounds 12a-12i are available on the published article. 6

N-(3-acetyl-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)pivalamide (14)

Pivaloyl chloride (0.92 g, 7.7 mmol) was added to 10 ml of dichloromethane in which 1-(2-Amino-4,5,6,7tetrahydrobenzo [b]thiophen-3-yl)ethanone (1g, 5.1 mmol) was previously dissolved, triethyl amine (0.78 g, 7.7 mmol) was added and the solution was stirred at room temperature for 3 hours, afterward the solution was dried under vacuum to evaporate the solvent and the residue was dissolved in acetone and the precipitation was provoked by addition of ice and water. The solution was left for 30 minutes for allowing maximum precipitation, the solid was collected by filtration under vacuum and washed by water, the solid was of sufficient purity to be used without further purification (yield: 1.16 g; 81%) Yield: 1.16 g (81%); grey solid; mp 112-116 °C 1

N-(3-acetylthiophen-2-yl)acetamide derivatives (11a-11g

), general procedure 9 1 g of 3-acetyl-2-aminothiophene derivatives was dissolved in 3 ml of acetic anhydride, the mixture was refluxed gently for 10-15 minutes, and quenched with water/ice, and then the solution was boiled gently, to breakdown the leftover acetic anhydride, the formed solid was filtered under vacuum and washed with water, dried and recrystallized in appropriate solvent if it needed.

Synthesis

Paper / PSP / Special Topic

N-(3-acetylthiophen-2-yl)acetamide derivatives (11h,11i), general procedure

Corresponding thiophene (5.1 mmol) and acetyl chloride (0.4 g, 5.1 mmol) was dissolved and stirred in excess of acetic anhydride at 35 °C. After 30 minutes the solution was treated with water and ice and heated to decompose the acetic anhydride and then extracted with ethyl acetate. It was subsequently exposed to flash column purification (EtOAccyclohexane, 1:3) for maximum purity.

N-(3-acetyl-4-methyl-5-propylthiophen-2-yl)acetamide (11h)

Yield: 1 g (82%); brown solid; mp 58-60 °C; Rf = 0.54 (EtOAc-cyclohexane, 1:3).

N-(3-acetyl-4-methyl-5-pentylthiophen-2-yl)acetamide (11i)

Yield; 1.32 g (97%); brown solid; mp 40 °C; Rf = 0.53 (EtOAc-cyclohexane, 1:3).

N-(3-acetyl-4,7-dihydro-5H-thieno[2,3-c]thiopyran-2-yl)acetamide (11j)

1-(2-Amino-5,7-dihydro-4H-thieno[2,3-c]thiopyran-3-yl)ethanone (1g, 4.6 mmol) dissolved in 10 ml of dichloromethane, 3 ml of acetic anhydride, acetyl chloride (1.08 g, 13.8 mmol) and triethyl amine (1.4 g, 13.8 mmol) were added and the reaction mixture was gently refluxed for 30 hours, the solvent was evaporated and residue dissolved in acetone and treated by ice and water. The reaction mixture was boiled for decomposing the acetic anhydride and the formed precipitate was filtered under vacuum and washed with water and crystallized from EtOH 96%.

Yield; 1 g (84%); brown solid; mp 132-135 °C (EtOH 96%).

4-Chloro-3-formyl-thieno[2,3-b]pyridine, general procedure

Vilsmeier-Haack reagent was prepared by previously mentioned method, in this case the amount of the reagent was: 5 ml of POCl3, and 20 mL of DMF for 2.5 mmol of the starting material. The reagent was added to the thiophene at one time, and stirred at 65 °C for 4-5 hours. At the end of the reaction, the solution was decomposed by mixture of ice and water and neutralized by sodium acetate. In case of formation of precipitate, filtration under vacuum was applied, the obtained solid was dissolved in ethyl acetate and washed 2-3 times with water, the residue was purified by silica gel column chromatography (EtOAc-cyclohexane, 1:9), and otherwise the solution was directly extracted by ethyl acetate, and purified by the same way.

Supporting Information

YES (this text will be updated with links prior to publication)

Primary Data

NO (this text will be deleted prior to publication)

Synthesis

Paper / PSP / Special Topic