Olga Ovdiichuk

IBMM Florine Cavelier Dr

Prof Claude Taillefumier

Prof Marc Le Borgne

Université Claude Bernard Lyon

Examinator Zoia Voitenko

Prof Taras Shevchenko

Prof Alain Durand

LCPM Marie-Christine Averlant-Petit Cr

Dr Olga V Hordiyenko

Prof Florine Cavelier

L E Marc

Borgne

Dr Jacques Bodiguel

Dr Regis Vanderesse

Dr Guillaume Pickaert

Dr Samir Acherar

Dr Olivier Fabre

Mme Mathilde

Dr Emmanuel Wenger

New Variety of Pyridine and Pyrazine-Based Arginine Mimics: Synthesis, Structural Study and Preliminary Biological Evaluation

ABSTRACT

Heterocycle-containing amino acids have become important precursors in the design of peptidomimetics. The pyridine and pyrazine rings can improve the binding affinity and bioavailability of potential drugs. Moreover, an enzymatic degradation resistance is known to be more pronounced in peptidomimetics with heterocyclic cores as non-peptidic fragments. In addition, an amidoxime group introduced into the structure represents potent pharmacophore due to the ability for its in vivo oxidation to amide with a subsequent release of NO (antiaggregatory activity) or their reduction to amidine, an excellent mimic of the arginine side chain. The use of amidoximes as amidine prodrugs allows to overcome unfavorable physicochemical and pharmacokinetic properties. This work hence describes the synthesis, structural study and preliminary biologycal evaluation of new variety of pyridine and pyrazine-based peptidomimetics. The first part of the thesis is devoted to the design and convenient synthesis of novel peptidomimetics bearing amidoxime function. Moreover, the introduction of an additional amino acid through different linkage like hydrazide, ester or heterocyclic unit (1,2,4-oxadiazole, 1,2,4-triazole) was of our great interest. 1,2,4-oxadiazole and 1,2,4-triazole rings are known as isosteric replacement of the amide and/or ester group due to its high resistance to metabolic degradation that has been widely used in peptide mimicry. Thus, several chemical functionalization of new scaffolds were studied. We have developed the acylation of amidoximes with further microwaveassisted condensation into amino acid derived 1,2,4-oxadiazoles. The synthesis of 1,2,4triazoles was performed via N-acylamidrazones. The latter were synthesized in mild conditions using a new approach from the pyrrolopyridines(pyrazines) precursors in good to excellent yields. On further developing constrained peptidomimetics, we also synthesized hydrazide modified turn mimics derived from amidoximes.

The second section describes structural analysis of the prepared compounds by NMR, IR spectroscopic studies and molecular modelling. Crystal structures of some compounds were analyzed by X-Ray diffraction study. Conformational preferences and thermodynamic studies of proline-containing peptidomimetics were investigated and both seriespyridine and pyrazine ones were compared. Examination of a new ProPhe pyrazine-based pseudotripeptide revealed the hydrogen bond formation between the proton of the OH and the carbonyl oxygen of the C-terminal phenylalanine and the hydrogen bond that adopts a sevenmembered -turn conformation. Therefore, a dramatic increase of the trans rotamer up to 98% was observed in weakly polar solvent, which is CHCl 3 . Hydrazide modified peptidomimetics adopt a turn structure in solution also stabilized by the hydrogen bond forming C 10pseudocycle. Conformational studies confirmed that these heterocyclic moieties can be used to increase rigidity and the pyrazine core could stronger effect on conformation stabilization.

The third part of the thesis is dedicated to the preliminary results of the NO release assay on amidoximes. All compounds revealed release NO in concentration sufficient for pharmaМologiМal ОffОМts (≥1 µM). A tОntativО МorrОlation bОtwООn struМturО anН aМtivity was performed. Chapter 1: Introduction

DEDICATION

Introduction

This work is a continuation of joint research on the synthesis of heterocycle-based peptidomimetics that mimic -turn, which had begun between Dr. Olga Hordiyenko (Taras Shevchenko National University of Kyiv) and Dr. Axelle Arrault (LCPM, ENSIC, Universite de Lorraine, Nancy). The previous study was focused on the synthesis of pseudopeptides where turn structure was determined by central 1H-isoindole unit that was connected at 1 and 3 positions with peptide chains. Several key molecules were synthesized from mono to pentapeptide, and it was shown that intramolecular hydrogen bonding could provide turn structures (Biitseva A. V., Rudenko I. V., Hordiyenko O. V., Jamart-Grégoire B., Arrault A., Eur. J. Org. Chem., 2012, 23, 4387).

In order to continue joint research on combining amino acids with heterocyclic core as a turn guiding factor, the introduction of pyridine and pyrazine 2,3-cyano acids into peptide chain was suggested to give promissing peptidomimetics. This study became the aim of the PhD theses in co-tutelle between the Taras Shevchenko National University of Kyiv and the LCPM, Universite de Lorraine. The bibliography search, editing of articles and some analyses (elemental analysis, LC-MS and IR for some products) were performed at the Taras Shevchenko National University of Kyiv while the synthesis and conformational analysis of peptidomimetics have been done in the Laboratoire de Chimie-Physique Macromoléculaire, Universite de Lorraine.

Pyridine and pyrazine heterocycles as scaffolds for peptidomimetics

The combination of aromatic or heterocyclic rings with a peptide motif represents one of the possible strategies towards peptidomimetics. Heterocycle-containing amino acids have become important precursors in the design of peptidomimetics. This approach is advanced for introduction of conformational restrictions which leads to a more stable and bioavailable product, hence favoring recognition and pharmacological properties of peptide-based drugs and decreasing toxicity. Moreover, a resistance to peptidase degradation is known to be more pronounced in peptidomimetics with heterocyclic cores as non-peptidic fragments. In this thesis we describe, the study of new pyridine and pyrazine-based peptidomimetics: synthesis, conformational behaviour and preliminary biological evaluation.

The pyridine ring, naturally occurring in the vitamins, nicotinic acid (vitamin B 3 /niacin) and pyridoxine (vitamin B 6 ), a number of alkaloids, such as nicotine, pyridine, is therefore notable for a wide range of biological activities (Figure 1.1). Moreover, niacin and nicotinamide, are precursors of the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) in vivo. 1 The pyrazine ring can improve the binding affinity and bioavailability of potential drugs. A number of compounds containing a pyrazine moiety are in late stage clinical trials (VE-821an ATR inhibitor) or have reached the market such as, bortezomib -the first therapeutic proteasome inhibitor, eszopiclone -a nonbenzodiazepine sedative hypnotic etc (Figure 1). Consequently, the six-member pyridine and pyrazine heterocycles are utilized in many pharmaceutical actives and thus they have been chosen as scaffolds of the corresponding peptidomimetics.

The benefits of the amidoxime group and modified amidoxime moieties (amidoxime esters and oxadiazoles) are further emphasized in Chapter 1 of the manuscript.

Amidoximes and masked amidoximes as prodrugs of amidines, an arginine mimics

Investigations in the chemistry as well as in the biological applications of amidoximes up to 2008 are summerized in the reviews [3-5]. However, a number of publications and patents appeared to date, in particular concerning the use of amidoximes and its derivatives as prodrugs of amidines in drug design and the way of their activation.

Amidoximes are compounds bearing both a hydroxyimino and an amino group at the same carbon atom which makes them structurally close to amidines, amides and hydroxamic acids. Amidines are often used as mimetics of the guanidine side chain of arginine which is responsible for pharmacological effects. Variety of drugs and drug candidates possesses the amidine moiety, such as thrombine inhibitors, factor Xa and VIIa inhibitors, glycoprotein IIb/IIIa receptor antagonists, and in other drugs. [6][7][8] However, due to their high hydrophilicity they are protonated at sp 2 nitrogen atom, forming a highly mesomerically stabilized cation under physiological conditions (Scheme 1.1). The cations formed are essential for interactions with the negatively charged carboxylates or targeted molecules, but prevent an absorption from the gastrointestinal tract. 9

NH H 2 N R N+ H 2 N R H H N H 2 N R H H +H + -H + Scheme 1.

Protonation of amidines

This problem can be solved by using prodrugs -bioreversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the pharmacologically active agent with the promoiety being non-toxic (Figure 1.2). 10,11 The development of prodrugs have become a tool for improving physicochemical, biopharmaceutical or pharmacokinetic properties of active parent drugs.

Figure 1.2 Prodrug concept

An amidoxime group introduced into the structure is not protonated under physiological conditions, thus it can be employed as an amidine replacement. The prodrug approach "AmiНoximОs instОaН of amiНinОs" has a growing intОrОst НuО to improved physicochemical properties and oral bioavailabilities. 5 Promoiety enzymatic and/or chemical transformation

Drug Promoiety

Drug

Recently it has been shown that the N-reductive enzyme system is responsible for the activation of amidoxime prodrugs in mitochondries. 12,13 This enzyme system is able to reduce a variety of N-hydroxylated derivatives (including amidoximes) in the presence of NADH. It This prodrug strategy has been successfully applied to a wide range of drug molecules.

Firstly it was developed for the aromatic diamidine pentamidine, an antiprotozoal drug (Scheme 1.2). 14,15 The replacement of amidine functionalities into amidoxime led to improved bioavailability and solubility. To optimize the pharmacokinetic profile, many studies have been devoted to new pentamidine analogues. Although, N,N-bis(acetoxy)pentamidine showed a clearly increased lipophilicity and oral bioavailability in animal studies it has very low water solubility. 16 Further prodrug principles were transferred to pentamidine (hydroxylation, conjugation with amino acids etc.) 17,18 , however N,N'-bis(succinyloxy)pentamidine exhibited clearly superior solubility and pharmacokinetic behavior as compared to the other prodrugs of pentamidine. 19 The activation of the prodrug proceeds via hydrolysis by esterases and the mARC enzyme system reduction and is hence the double prodrug strategy. Amidoximes have widely been studied as antitrombotic agents. A known example is ximelagatran (Exanta®, Astra Zeneca), which was the first approved direct thrombin inhibitor for oral application (Figure 1.4). 20,21 This pharmacologically inactive molecule is transformed in vivo in two steps into the active form melagatran. 22 Ximelagatran showed a significant improvement of oral bioavailability in human (about 20%), compare with melagatran with oral bioavailability of only 5%. Unfortunately, a hepatotoxicity that forced the market withdrawal in 2006 has been reported. Dabigatran etexilate (Pradaxa®)the only available oral thrombin inhibitor, is prodrug of the actual active substance dabigatran (Figure 1.5). 23 To overcome some disadvantages of Pradaxa, dabigatran amidoxime succinic acid ester has been patented as prodrug with excellent solubility, appropriate stability, and good oral bioavailability. 24 Recently, the amidoxime prodrug strategy was applied to another serine protease inhibitor upamostat (Mesupron®, WX-671), which has passed clinical phase II trials (Figure 1.6). 25,26 It is the orally available prodrug of an active form WX-UK1, the urokinase inhibitor.

The bioactivation by the mARC-containing N-reductive enzyme system has also been studied. The development of new antiviral agents can solve both major problems of known neuraminidase inhibitors (NAIs), a drug resistance and unfavorable pharmacokinetic (PK)

properties. Oseltamivir (Tamiflu®) -the first orally available NAI that was marketed in 2000 (Scheme 1.3). 27 Its amidoxime prodrug was shown to possess a better PK profile with comparable oral bioavailability (31% versus 36% for oseltamivir). 28 It includes hydroxylation the functional group at both nitrogen atoms of amidines to further reduce its basic character (the pK a from 11.6 in case of amidines and 4.8 for amidoximes to 3.8 for N,N-dihydroxybenzamidines) and increase lipophilicity. The additional reaction step necessary for in vivo reduction of an additional hydroxy group delays the degradation to amidines leading to extended bioavailability.

Amidoximes as NO donors

Nitric oxide (NO) is an endothelium-derived relaxing factor (EDRF) that plays a key role in many bioregulatory systems including cardiovascular through platelet aggregation inhibition and vascular relaxation, nervous system by neurotransmission and immune stimulation. 32,33 Owing to the properties highlighted, there is great interest in the molecules called nitric oxide donors (NO-donors).

NO is mainly biosynthesized endogenously from L-arginine by O The oxidative cleavage of the C=N(OH) bond of NOHA has been found to be also catalyzed by liver microsomal cytochromes P450 with formation of citrulline and nitrogen oxides (NO, NO 2 -...). 34 Similarly, NO is formed in vivo by rat liver microsomal oxidation of exogenous compounds containing an amidoxime or a related function. Firstly it was shown for p-hexyloxy-benzamidoxime which is transformed to the corresponding arylamide and nitrogen oxides by a cytochromes P450-dependent oxidation with NADPH and O 2 (Scheme 1.6). 35 C oxidative conversion to the respective amide derivative and nitrogen oxides. 36 The formation of NO was confirmed by the formation of the cytochromes P450 Fe(II) -NO complex detected in visible spectroscopy.

More detailed study of the oxidation of C=N(OH) bond was reported by Jousserandot et al. 37,38 Variety of aldoximes, ketoximes, amidoximes and hydroxyguanidines were studied for the formation of NO 2 -upon microsomal oxidation in the presence of the cofactors of cytochromes P450, NADPH and O 2 . It was found that the rate of the reaction depends on the number of nitrogen-containing electron-donating substituents on the C=N(OH) carbon atom in the order N,N-disubstituted N'-hydroxyguanidines>N-substituted N'-hydroxyguanidines>N,N-disubstituted amidoximes>amidoximes>ketoximes. A mechanism of the reaction was studied and oxidation appeared to be mainly due to O 2 .

-derived from the oxidase function of cytochrome P450, which was suggested from inhibitory effect of superoxide dismutase (SOD). On the contrary, the formation of the sideproducts nitriles was not inhibited by SOD thus was due to a cytochrome-P450-iron active species.

NO release and antithrombotic effects of several arylazoamidoximes were investigated Antithrombotic effects showed phenyl-, 4-chlorophenyl-and 1-naphthyl substituted azoamidoximes whereas compounds with the strongest electron donating (4methoxyphenylazo-methanamidoxime) and electron withdrawing (4-fluorophenylazomethanamidoxime) properties did not influence the thrombus formation in arterioles. In addition, the effect of SOD on the formation of NO was tested and revealed no influence in case of phenylazomethaneamidoxime indicating the oxidation by a P450-Fe(III)-OO . species.

In contrast the formation of NO from 4-methoxyphenylazo-methanamidoxime was significantly suppressed by the addition of superoxide dismutase and hence the release of O 2

.

is responsible for the oxidation.

A fast preliminary screening of potential NO precursors was carried out by Koikov et al. in order to reveal the unknown NO generating potential of the well-known oximes, amidoximes and hydroxamic acids (Figure 1.11). 40 R Other scientists exemined some p-substituted aromatic amidoximes on their ability to produce endothelium-independent vasorelaxation in the rat aorta (Figure 1.12). 41 R N The order of relaxant activity of amidoxime, ketoxime and N-hydroxyguanidine derivatives has been shown to be opposite to the order of reactivity of the same compounds for P450-dependent oxidative cleavage of the C=NOH bond. The substituted benzamidoximes (BZA) were all active, the electron-poor 4-chloro-and 4-nitro-being more potent than the unsubstituted BZA and substituted by the electron-rich groups N-cyclohexyl and 4-methoxy derivatives. The most active studied compound, 4-chlorobenzamidoxime has shown to cause endothelium-independent relaxation in the rat aorta through activation of guanylyl cyclase by NO. Failure of P450 and NO synthase inhibitors to blunt relaxation lend no support to the hypothesis of the involvement of a P450 or NO synthase in the relaxant effect of compounds with a C=NOH function in the rat aorta. Therefore, it was indicated that non α-amino acid-substituted benzamidoximes can act as efficient NO synthase-independent activators of the cyclic guanosine-γ′,5′-monophosphate (cGMP) pathway in the rat aorta.

Chalupsky et al. demonstrated that in the isolated rat aorta, formamidoxime and other oxime derivatives increased NO levels and induced the relaxant effect. 42 The NO-cyclic GMP pathway was shown to be involved in the relaxant properties of oxime derivatives. In the later study, these substances were evaluated on their vasodilator properties on rats, however, only 3 out of 5 derivatives active in vitro were also active in vivo. 43 Formamidoxime surpassed the effects of both other efficient substances and induced pronounced dose-dependent blood pressure reduction. Nitric oxide was shown to be responsible for a considerable part of blood pressure changes elicited by amidoxime compound.

Amidoximes with the imidazole scaffold were prepared and tested for their ability to donate NO and for their intraocular pressure (IOP) lowering via the activation of guanylate cyclase (Figure 1.13). 44 The ability of amidoximes and 1,2,4-oxadiazoles to release NO in the presence of a thiol co-factor in order to design new anti-inflammatory agents was investigated by Ispikoudi et al. 45 NO donation with simultaneous production of nitrile, was tested in vitro by acting of reducing agents like L-cysteine or thiophenol. Although the amidoximes tested did not release NO in the presence of a thiol factor, acetylated 5-amino-1,2,4-oxadiazole derivatives exhibited high NO release.

N

Chapter 2: Synthesis of Pyridine and Pyrazine-Based Peptidomimetics

Introduction

The purpose of this project is the synthesis of pyridine and pyrazine-based peptidomimetics possessing o-substitution in non-peptidic fragment which can promote turnlike structure thus providing additional conformational rigidity. In order to achieve this goal, compounds bearing amino acid substuent and amidoxime function as a replacement of amidine one have been investigated. Moreover, the introduction of an additional amino acid through different linkage like hydrazide, ester or heterocyclic unit (1,2,4-oxadiazole, 1,2,4triazole) is of our great interest.

Compounds of pyrine series were numbered like 1 -12 while the corresponding pyrazine analogues as 2' -12'. Difference in amino acid residues was indicated using letters a-h for pyridine derivatives and a-d in case of pyrazine counterparts.

Synthetic plan

A synthetic route to desired pyridine and pyrazine-based peptidomimetics is shown in

Synthesis of starting 2-cyanonicotinic and 3-cyanopyrazine-2-carboxylic acids

Preparation of the starting acid 4 was perfomed according to a known literature procedure; [46][47][48] the synthetic route is depicted in Scheme 2.2. Thus, commercialy available quinolinic anhydride 1 was first quickly hydrolyzed with aqueous ammonia (28%), similar to the method developed for phthalamic acid 46 and used later in the synthesis of the pyridine analogue by Spiessens and Anteunis, 48 to give 2-carbamoylnicotinic acid (2). The reaction proceeds selectively with formation of the product 2 and no isomeric 3-carbamoylpicolinic acid was obtained. It can be explained by the effect of the electronegative nitrogen atom that favors electrophilic character of the carbon of the closer carbonyl group thus promoting the attack on this carbon. We also suggested the hydrogen bonding between the proton of ammonia molecule and the pyridine nitrogen lone pair which can support the attack giving the acid 2 (Scheme 2.2). Then, ester 3 was obtained by treatment of acid 2 with methyl chloroformate, which was followed by hydrolysis with sodium hydroxide (1M) to give the corresponding 2-cyanonicotinic acid (4) in 28% overall yield. 47,48 Starting 3-cyanopyrazine-2-carboxylic acid 4' was synthesized from readily available 3-carbamoylpyrazine-2-carboxylic acid 2', which was treated with methyl chloroformate 49 followed by selective hydrolysis of ester group with 1M NaOH to give the corresponding acid 4' in 71% total yield (Scheme 2.2).

Cl

Synthesis of peptidomimetics bearing amidoxime function 4.1 Synthesis of amidoximes

The most commonly used method for the synthesis of amidoximes involve condensation of nitriles with hydroxylamine hydrochloride (Scheme 2.3).

Scheme 2.3 Synthesis of amidoximes

The experimental procedure consists of liberating the hydroxylamine from its hydrochloride by means of sodium carbonate, adding the appropriate nitrile and alcohol and finally keeping the mixture at 60-80 °C for several hours to obtain amidoximes in high yields.

Potassium or sodium hydroxide, sodium methoxide or ethoxide in place of sodium carbonate can lead to considerable reduced yields. Action of an excess of hydroxylamine may ameliorate the percentage in case of aromatic amidoximes. This reaction is also very useful, since nitriles are compounds readily available.

The development of new technologies and techniques enabled the attachement of amidoximes on a solid support. The reaction proceed by treating resin-bound nitriles with hydroxylamine, and the resulting resin bounded amidoximes can be further used for library generation and screening in drug discovery. 50,51 Moreover, amidoximes can be synthesized using ionic liquid-phase organic synthesis (IoLiPOS) methodology that has several advantages over solid support. 52 The reaction of hydroxylamine with thioamides can be used in case if they are more accessible that the corresponding nitriles or to afford amidoximes containing electronwithdrawing groups (Scheme 2.3).

The reaction of hydroxamoyl chlorides with ammonia leads to amidoximes via dehydrohalogenation step and formation of the nitrile oxide (Scheme 6, R 1 , R 2 = H).

Treatment of amines with ammonia affords N-unsubstituted amidoximes respectively

(Scheme 2.4). R N H OH NCS R N Cl OH R C N O R N NR 1 R 2 OH R 1 R 2 NH base

Synthesis of pyridine based amidoximes

In our attempts to develop a new variety of arginine peptidomimetics with an amidinoheteroaroyl motif in a peptide chain, we elaborated the synthesis of nicotinic acid based amino acid units bearing an amidoxime function on the pyridine ring as precursors of the corresponding amidine-containing pseudopeptides.

We envisioned that the coupling of 2-cyanonicotinic acid 4 with methyl esters of L-αamino acids with further conversion of the cyano residue into amidoxime group might be employed as a basic strategy for the synthesis of the target pyridine-based peptidomimetics.

The amination reaction in the series of α-cyanopyridinecarboxylic acids was studied earlier, 48,53 and it was shown that conversion of the methyl or ethyl esters of 2-cyanonicotinic acid 4 into the corresponding cyano amide was unsuccessful, and produced only cyclic 7imino-6,7-dihydro-5H-pyrrolo [3,4-b]pyridin-5-one upon reaction with ammonia. Similar bicyclic imines were obtained by treatment of cyano esters with primary amines. 53 To investigate the scope of this novel method, the coupling of 2-cyanonicotinic acid When the alanine, valine and leucine methyl esters were used, the formation of both products 5 and 6 in different ratios, with an increasing amount of the cyclic form 6, was observed while in the case of phenylalanine, glycine, tryptophan, the individual cyclic esters 6b,d,g were exclusively isolated. The total yield of the amino acid derivatives 5 and 6 was 60-80% after purification by column chromatography (Table 2.1). We also succeeded in separation of the open 5 and cyclic 6 forms of the alanine, valine and leucine derivatives by flash column chromatography, and in identification and characterization of the individual components by means of LC/MS and 1 H NMR, 13 C NMR and IR spectroscopy. The assignment of the synthesized compounds to open-chain or closed structure was possible on the basis of comparative analysis of their 1 H NMR spectra (Table 2.1) and X-ray crystallography data.

The only product formed from the reaction of methyl glycinate with acid 4 was analysed by single-crystal X-ray diffraction. It was shown that this product has the cyclic structure 6d (Figure 2.1). In the isolated pyrrolopyridines 6a-g the amino acid residue is attached to the endocyclic nitrogen atom, and not to the exocyclic imine nitrogen atom as was assumed for the structure of the major products in the similar reaction of ester 3 with ethyl or benzyl amines. 53 Two products were also isolated when ester 3 was reacted with benzylhydrazine, and their structures were assigned as isomeric pyrrolopyridines possessing a benzylamino substituent at either the imino or the pyrrole nitrogen atom. 55 Along with tryptophan derivative 6g, a very small amount of crystals of the unexpected substance A was isolated from the reaction mixture (Figure 2.2). 56 In contrast to the major cyclic product 6g, this 1 H, 13 C NMR spectra) yielded two peaks in variable ratios, which corresponded to the same molecular ions (Table 2.2). It was found that these major and minor peaks appear as a result of reversible intramolecular cyclization/recyclization of compounds 5 and 6 during analysis.

For each compound, the major peak value was around 70-100% and the minor peak was less than 30%.

(2S)-1-[(2-cyanopyridin-3-yl)carbonyl]pyrrolidine-2- carboxylate (5c)
Most amide bonds in peptides exist almost exclusively in the trans configuration. The cis form is energetically unfavorable, largely due to steric hindrance. The cyclic structure of proline lowers the steric hindrance, making both the cis and trans forms nearly equally energetically stable. 57,58 Due to this fact, two sets of signals were observed in the 1 H NMR spectrum of proline derivative 5c. In this context, structural and thermodynamic studies of proline-containing pseudopeptides were performed (see Chapter 3).

The open-chain methyl esters of (2S)-N-(2-cyanopyridin-3-yl)carbonyl-substituted amino acids 5a,c,e,f exhibited a characteristic cyano group IR absorption band at around 2237 cm -1 . The band intensity was weak, similar to the almost undetectable absorption of the reference compound, 2-cyanonicotinic acid (4). 48 The next step in the synthesis of amidine prodrugs consisted of transformation of the cyano group into an amidoxime function. Methyl esters of (2S)-N-[2-(N'hydroxycarbamimidoyl)pyridin-3-yl]carbonyl-substituted amino acids (7) were obtained as individual products in 67-79% yield from the corresponding methyl esters 5a,e,f,h and/or methyl esters 6a,b,e-g by treatment with hydroxylamine hydrochloride in methanol (Table 2.3). 4 The corresponding proline analogue 7c was synthesized similarly from the cyano derivative 5c in 70% yield (Scheme 2.7). The cyano transformation reaction proceeded upon stirring the mixture at room temperature overnight, and the crude products were purified by flash column chromatography. Both open-chain 5 and the corresponding cyclic 6 amino acid derivatives gave the same open-chain amidoximes 7 as the sole product. No cyclic amidoximes of type I, the formation of which could be expected according to a previously reported condensation of the benzene analogue 3-imino-1-oxoisoindoline with hydroxylamine that led to 3-(hydroxyimino)-1-oxoisoindole (II), 59 were found (Table 2.3, Figure 2.4).

NH O N OH II

Figure 2.4

The formation of pseudopeptides 7 from esters of type 6 proceeds via pyrrolidine ring opening by hydroxylamine to afford the same open-chain amidoximes as those obtained from the corresponding cyano esters 5 (Scheme 2.8). However, a formation of small amount of (7Z)-7-(hydroxyimino)-6,7-dihydro-5H-pyrrolo [3,4-b]pyridin-5-one (Scheme 2.8, III) as a side product was observed during the workup and/or further column purification of the corresponding amidoximes.

Synthesis of pyrazine based amidoximes

Recently, amino acid derived pyrazine carboxamides have been synthesized from pyrazine-2,3-dicarboxylic anhydride and amino acids in refluxing toluene, and the conversion was accompanied by decarboxylation along with pyrazine-2,3-dicarboximide formation in some cases. 60 Our studies began with the synthesis of amino acid derived pyrazine-2carboxamides with amidoxime groups at position 3. The synthesis was carried out in two steps utilizing the strategy elaborated before for the pyridine series 61 : coupling of 3cyanopyrazine-2-carboxylic acid (3) with several methyl esters of L-α-amino acids was followed by further reaction of the obtained nitriles with hydroxylamine hydrochloride.

Esters of (2S)-N-(3-cyanopyrazin-2-yl)carbonyl substituted amino acids 5' were prepared by coupling of 3-cyanopyrazine-2-carboxylic acid (4') with commercially available methyl esters of the L-alanine, L-phenylalanine and L-proline amino acids according to a previously developed protocol 61 (Table 2.4). As an activating agent N-ethyl-N'- (3dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in the presence of 1hydroxybenzotriazole (HOBt) was used. As a solvent, tetrahydrofuran instead of dichloromethane was taken due to the low solubility of the starting material in dichloromethane.

Table 2.4 Synthesis of Ala-, Phe-, Pro-containing pseudodipeptides 5'-7' 1). 61 Therefore, one could conclude by analogy with [61] that pyrrolopyrazines 6'a,b adopt the E configuration at the C=N exocyclic bond and syn orientation of the NH proton to the heterocyclic ring.

N OH O N N R Cl H 3 N CO 2 Me N N N O N CO 2 Me R H N X O N N CO 2 Me R NH 2 OH . HCl N N NH 2 OH X O N CO 2 Me R Et 3 N, MeOH rt
Amidoximes 7' could be prepared efficiently from single compounds 5' or 6' as well as from the mixture of 5' and 6' in a reaction with hydroxylamine hydrochloride (Table 2.4).

Thus, as in the case of the pyridine analogs, the products 7' were formed as a result of addition to nitriles 5' as well as pyrrolidine ring opening of pyrrolopyrazines 6'. As follows from the X-Ray crystallography data, compound IV adopts the Z configuration at the C=N bond. The molecule has a shot contact between H1-O1 2.43 Å (this is shorter than the sum of the van der Waals radii of hydrogen and oxygen atoms which is 2.72 Å).

In addition, all compounds 5', 6' and 7' and reaction conditions were compared with similar ones from the pyridine series that were described previously (5-7, Table 2.5). 61 The coupling reaction proceeded easily for pyridine analogs in contrast to pyrazine ones because of a low solubility of the latter that led to decreased yields (Table 2.5). The EDCI/HOBt protocol was applied in both cases with the difference in solvents, DCM was used for the coupling with 2-cyanonicotinic acid (4) and THF was found to be the best solvent for 3cyanopyrazine-2-carboxylic acid (4'). The ratio of isolated isomeric Ala-containing open form 5'a and cyclic pyrrolopyrazines 6'a vary as in the case of cyanonicotinic acid 4. But when Phe was coupled, we succeed in isolation of a small portion of open form 5'b along with a majoring pyrrolopyrazine 6'b unlike for 2-cyanonicotinic acid (4) where only the cyclic pyrrolopyridine analog 6b was obtained.

The comparison of NH proton signals in the 1 H NMR spectra of the pyridine and pyrazine derivatives 5, 5' revealed a low field shift above 1 ppm for the pyrazine compounds probably due to possiblО NH…N hyНrogОn bonНing or an NH... interaction 62,63 that was absent in the case of pyridine core. The general tendency for pyrrolidine ring opening of 6' by hydroxylamine to afford the open-chain amidoximes 7' was observed; the same structures were obtained from the corresponding cyano esters 5' similar to the formation of pyridine based amidoximes 7.

Additionally, we decided to extend this protocol in order to synthesize novel pseudotripeptide with the N-terminal pyrazine amidoxime motif in the backbone. Prolinephenylalanine dipeptide was chosen to discover the steric effects of the C-terminal substitution in proline on the amide isomer equilibrium of the model prolyl surrogate.

Likewise, o-substitution in non-peptidic fragment with dipeptide can provide additional conformational rigidity.

The target molecule was prepared utilizing the same strategy elaborated for nicotinic and pyrazinic acids based pseudodipeptides bearing an amidoxime residue. 61,64 A synthetic

Studies towards the synthesis of 1,2,4-oxadiazoles via amidoxime ester units

The 1,2,4-oxadiazole motif is an attractive heterocycle contained in many pharmaceuticals with widespread biological applications. 65,66 1,2,4-Oxadiazole ring have long been known as isosteric replacement of the amide and/or ester group due to its high resistance to metabolic degradation. 67,68 A number of nonproteinogenic α-amino acid-derived 1,2,4oxadiazoles have been utilized in the design of mimetics, as peptidomimetic building blocks, [69][70][71][72][73] dipeptidyl peptidase IV (DPP-4) inhibitors (A), 74 inhibitors of the Src SH2 domain (B), 75,76 sphingosine kinase inhibitors (C) 77,78 and immunomodulators (D) 79 (Figure 2.7). On the other hand, esterification and/or condensation of amidoximes presents a route towards the novel double prodrugs of amidines. 29,80,81 Hence, the synthesis and characterization of novel esterified amidoximes and 1,2,4-oxadiazoles are topics of our great interest. Additionally, we briefly describe an overview of synthetic approaches to 3,5disubstituted 1,2,4-oxadiazoles.

NH 2 N O F F F Y X N R HN H N O R 1 N N O R 2 O R 3 H 2 O 3 PO N O N nC 8 H 17 H 2 N R N N O R 1 NH 2 R 2 NHR 3 X = N; Y = O X = O; Y = N A B C D
5.1 Overview of synthetic approaches to 3,5-disubstituted 1,2,4oxadiazoles via amidoxime esters 1,2,4-Oxadiazoles can generally be formed by the reaction of an amidoxime with a carboxylic acid or acid chloride followed by an intramolecular cyclodehydration (Scheme 2.11). [START_REF] Jochims | Comprehensive Heterocyclic Chemistry II[END_REF]82,[START_REF] Hemming | Comprehensive Heterocyclic Chemistry III[END_REF] NOH

NH 2 R N NH 2 R O R' O N N O R R' R'COX Cyclodehydration Scheme 2.11 Classical route to 1,2,4-oxadiazoles
The synthesis can be performed with the isolation of acylated intermediate in two

steps or as a one-pot reaction without isolating the ester.

The two steps general route was followed for the preparation of 1,2,4-oxadiazole intermediates to inhibitor B (Figure 2.7). 75 Amidoximes were coupled to the appropriate amino acid derivatives, giving the intermediate O-acylamidoxime which underwent cyclization in refluxing pyridine followed by Boc deprotection (Scheme 2.12).

X O BocHN R 1 NH 2 N R 2 HO H 2 N R 1 N N O R 2 O O BocHN R 1 N R 2
NH 2 + DME, rt (X = OSu) or EDC.HCl, HOBt, DIEA, DCM, DMF, rt (X = OH) 1) Pyridine, reflux 2) TFA, DCM, rt Scheme 2.12 1,2,4-Oxadiazoles synthesis reported by Buchanan et al. [75] A library of twenty 1,2,4-oxadiazoles in a range of electron-rich and electron-poor benzamidoximes and both aromatic and aliphatic carboxylic acids was synthesized in a one-pot procedure (Scheme 2.13). [START_REF] Deegan | [END_REF] The authors used 1,1'-carbonyldiimidazole (CDI) as a reagent for both formation and cyclodehydration of O-acyl benzamidoximes. Another coupling method was used by Katritzky and co-workers using N-protected (αaminoacyl)benzotriazoles to achieve the synthesis of chiral 1,2,4-oxadiazoles (Scheme 2.17). 87 The O-acylation of amidoximes with benzotriazole derivatives occurred immediately after treatment with Et 3 N (1 eq.) in EtOH at room temperature. Subsequent refluxing of intermediates in EtOH in the presence of Et 3 N proceeded quickly with the cyclization into 1,2,4-oxadiazoles in 70-94% yield with good (>97%) retention of chirality. 70 N-Protected amino acyl fluorides were first coupled to amino acid-derived amidoximes in presence of NMM in ethanol. After 15 min, an equimolar quantity of NaOAc was added and the reaction mixture was refluxed for 3 h. The resulting 1,2,4-oxadiazolyl dipeptides were isolated after workup followed by purification in a yield exceeding 60%. The one-pot procedure was also used by the same laboratory for the synthesis of other peptidomimetics. 69 In some cases prolonged heating at high temperatures is the reason for the low yield of the desired products or their total absence. Consequently, approaches have been proposed for improvement of the cyclodehydration: the use of basic reagents, microwave irradiation or ultrasound. [88] The numerous acyl amidoximes were examined in different concentration of TBAF in THF (from 0.1 to 1.0 equivalent) affording the 1,2,4-oxadiazole derivatives in high yields.

RCO 2 H R 1 R 2 N NH 2 OH R 1 R 2 N NH 2 O R O R 1 R 2 N O N R 1)
O N N N PgHN R N R' HO H 2 N Et 3 N O N N R' R NHPg + EtOH, reflux R =
Synthesis of a new generation of chroman/catechol hybrids bearing 1,2,4-oxadiazole with using tetrabutylammonium fluoride as a mild and efficient catalyst was reported by Koufaki and co-workers (Scheme 2.22). 89 The alternative basic reagent has been suggested recently by Otaka (Scheme 2.25). 91 3,5-Disubstituted 1,2,4-oxadiazoles with a wider range of functionality have been obtained in good yields utilizing tetrabutylammonium hydroxide (TBAH). Reaction times were reduced compared with those of TBAF and this catalyst does not lead to corrosion of reaction vessels.

R 1 NH 2 N O O R 2 N N O R 1
R 2 TBAH (0.1 eq. 40% w/W aqueous solution) THF (0.4 M), rt, 10 min R 1 , R 2 = Alk, Ar Scheme 2.25 1,2,4-Oxadiazoles synthesis reported by Otaka et al. [91] An efficient method for DBU-catalyzed cyclization of acylamidoximes was developed by Lukin and Kishore to promote the formation of 1,2,4-oxadiazoles in 85-97% yield (Scheme 2.26). 92 The one-pot transformation was effected by CDI to form imidazolides of carboxylic acids followed by cyclodehydration in the presence of DBU at 60 °C for 1.5-2 hours.

O OH R N HO H 2 N R' 1) CDI, CH 3 CN, rt R O O N R' NH 2 DBU N O N R' R 60 °C, 1.5-2 h 2)
, 30 min Scheme 2. 26 94 The condensation of carboxylic acid esters and amidoximes in refluxing toluene in the presence of potassium carbonate was employed to synthesize a variety of oxadiazoles in moderate to excellent yields. The application of microwave energy can be employed to shorten reaction times and/or increase product purities and yields and to decrease formation of side products. 82,96 The one-pot microwave-assisted synthesis of variously disubstituted The starting acid was first treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and NMM in THF for 30 min at room temperature. The corresponding amidoxime and toluene were then added to the activated ester and the reaction mixture was irradiated for 5 min to afford 1,2,4-oxadiazoles in 62-95% yield.

O OMe R 1 N NH 2 R 2 OH R 3 OR 4 O O OR 4 N NH 2 R 2 OH R 3 O N N N N O R 2 R 2 N N O R 1 R 2 + K 2 CO 3 (
A microwave-assisted synthesis of 2-trifluoroacetylthiophene oxadiazoles as potent and selective class II human histone deacetylase (HDAC) inhibitors was achived by Muraglia et al. using 5-(trifluoroacetyl)thiophene-2-carboxylic acid activated with CDI and different amidoximes (Scheme 2.31). 98 A further equivalent of CDI and microwave irradiation at 140 °C for 2 min gave the oxadiazole inhibitors. [99] An ultrasound-promoted synthesis of 3-trichloromethyl-5-alkyl(aryl)-1,2,4oxadiazoles was achieved by Bretanha and co-workers using trichloroacetoamidoxime and acyl chlorides (Scheme 2.33). 100 The compounds were prepared in better yields and shorter reaction times compared to the conventional method.

S OH O F 3 C O S O O F 3 C O N NH 2 R S F 3 C O N N O R 1)
N NH 2 Cl 3 C OH O R Cl AcOEt N O N Cl 3 C R + )))

Acylation of amidoximes with further conversion into amino acid derived 1,2,4-oxadiazoles

We describe herein an easy microwave assisted synthetic route for the preparation of a Recently, it has been shown that conjugation of amidoximes with L-valine amino acid can enhance water solubility and bioavailability of the molecules, thus the latter was employed as one of the carboxylic residues. To the best of our knowledge, the synthesis of 1,2,4-oxadiazolylpyridines(pyrazines)

nОw
with amino acid substituted heteroaromatic and a chirally substituted 1,2,4-oxadiazole ring is unprecedented. The only examples of pyridines and pyrazines bearing 1,2,4-oxadiazole with proline 74,101-103 and phenylalanine 104 unit have been described recently.

L-phenylalanine amino acid was introduced into proline derivatives 7c and 7'c in order to investigate a propensity for cis amide bonds which is high when Pro is preceeded by an aromatic residue. 105 This is considered as a stabilizing Ar-Pro interaction in the cis conformation. Hence, the amidoximes 7c, 7'c were reacted with Boc-protected phenylalanine to produce Phe-amidoxime esters 8c and 8'c in quantitative yields (Table 2 Indeed, the population of the molecules 8c, 8'c in the cis conformation was slightly increased relative to that in 7c, 7'c (see Chapter 3).

Studies towards the synthesis of 1,2,4-triazoles via N-acylamidrazones

The 1,2,4-triazole scaffold displays a wide range of biological activities and can be used as amide bond replacement (isoster) in order to enhance stability towards proteolysis and introduction of rigidity that has been widely used in peptide mimicry. [106][107][108] This ring system can act as both hydrogen bond acceptor and donor which makes it useful in establishing intermolecular features in interactions between peptide ligands and receptors. Thus, various . N-acylamidrazones substrates could be used as common intermediates to synthesize 1,2,4-triazoles. 112 We proposed the approach including the pyrrolidine ring opening of cyclic precursors 6, 6', the same as for the amidoxime synthesis, with Boc-L-amino acid hydrazides in order to obtain N-acylamidrazones. This step would be followed by an intramolecular condensation of the latter and formation of 3,5-disubstituted-1,2,4-triazoles (Scheme 2.35). 35 Proposed route to amino acid derived 1,2,4-triazoles Herein, we describe our attempts at the synthesis of 1,2,4-triazole-derived pseudopeptides, including an overview of synthetic approaches to 3,5-disubstituted-1,2,4triazoles via N-acylamidrazones.

N H O H 2 N NH N N N R 1 R 2 NHCbz N N N Ph X NHR O N H R 2 NH 2 N N N Ar R 1 A B C
N X N O NH CO 2 Me R Boc-AA'-NHNH 2 N X N NH 2 N H O HN O NHBoc R' CO 2 Me R  N X O HN CO 2 Me R HN N N R' NHBoc X = CH, N Scheme 2.

Overview of synthetic approaches via N-acylamidrazones

Among the numerous methods leading to N-acylamidrazones, the condensation of amidrazones and carboxylic acids (or carbonyl chlorides) is one of the most exemplified in the literature. As has been reported, amidrazones are readily available precursors, and reaction of nitriles with hydrazine in refluxing ethanol was used for their preparation (Scheme 2.36). 113,114 This is then reacted with adamantancarbonyl chloride and tolylcarbonyl chloride They were used as precursors to 1,2,4-triazole-based tridentate ligands for Pt(II) complexes. 37 1,2,4-Triazoles synthesis from amidrazones reported by Mydlak et al. and Sanning et al. [116, 117] With the purpose of building triazole heterocycles for catalytic application (Pd(II) complexes), the amidrazones were prepared by reacting the nitriles of the isoxazole and isothiazole series with hydrazine hydrate (Scheme 2.38). 118 The reactions proceeded in methanol or ethanol solution at 50 °C for 2h. The target amidrazones were obtained in 80-95% yields. The latter were further acylated with acetyl chloride followed by thermal cyclization to the desired triazolylisoxazoles(isothiazoles) in glacial acetic acid in 85-98% yields. 49 1,2,4-Triazoles synthesis from amidines reported by Balo et al. [128] The condensation of acyl and phenyl hydrazides with amidines to afford Nacylamidrazones, followed by thermal cyclization, was discovered by Francis et al. for preparing 3,5-disubstituted-1,2,4-triazoles in high yields (Scheme 2.50). 129 50 1,2,4-Triazoles synthesis from amidines reported by Francis et al. [129] This approach was also applied by Sanning and co-workers for the synthesis of 1,2,4triazole-based ligands for Pt(II) complexes. 117 Treatment of trifluoroacetic acid ethyl ester with hydrazine hydrate generated in situ 2,2,2-trifluoroacetic hydrazide which was refluxed with the corresponding diamidine hydrochlorides and NaOH to give the 1,2,4-triazole derivatives (Scheme 2.51). With the same purpose, a suspension of dihydrazide in n-BuOH reacted with t-butyl and benzamidine hydrochloride after treatment with NaOMe in MeOH.

N N N 2 H 4 x H 2 O N NH HN NH 2 Cl R O EtOH, rt N NH HN N H O R Na 2 CO 3 DMF 0 ° -rt R = Ada, Tol, t-Bu Ethylene glycol 180 °C, 2 h N HN N N R N HN NH H 2 N HN NH N H R O N N NH R Scheme 2.
N X CN R 2 N 2 H 4 . H 2 O EtOH (X=O) or MeOH (X=S) N X R 2 H 2 N NNH 2 N X R 2 H 2 N N HN Ac AcOH N X R 2 N NH N 50 °C, 2h (X=O) or rt, 10 min (X=S) AcCl Et 3 N, Et 2 O 3h 4h R 1 X = O; R 1 = H, R 2 = Ar X = S; R 1 = R 2 = Cl R 1 R 1 R 1 Scheme 2.
N N N N H N H R Pg R = H, CH 3 , CH 2 Ph, CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , methylene-3-indole Pg = Cbz,
N N N N R 1 OMe HN N N N N R 1 NNHCOR 2 H 2 N NH 2 NHCOR 2 DMSO, H + (cat.), r.t. R 1 = Ar R 2 = Alk,
NHNH 2 O NH 2 R 1 R 2 NH.HCl NH 2 R 3 N H O NH 2 R 1 R 2 N R 3 NH 2 NH 2 R 1 R 2 N H N N R 3 NaOCH 3 , EtOH Chlorobenzene reflux R 1 , R 2 = H, Cl, -CH=CH-CH=CH- R 3 = 2-C 4 H 3 O, 2-C 4 H 3 S, C 6 H 5 , 3,4-C 6 H 4 (OCH 3 ) 2 Scheme 2.
NH NH 2 NH NH 2 O NHNH 2 R O NHNH 2 R EtOH NH N H H N O R 2 NaOMe EtOH (EtOH/Et 2 O; EtOH/chlorobenzene) reflux HN N N R HN N N R  Xylene + R = Ar + Scheme 2.
N HN NH 2 NH NH 2 R N OCH 3 O NH O HN H 2 N NH 2 NH NH 2 R 1 N N N NH HN N N R 1 R 1 OMe N N N NH HN N N F 3 C CF 3 R *2HCl CF 3 COOEt, N 2 H 4 xH 2 O, NaOH THF, reflux, 24 h *HCl , NaOMe n-BuOH, reflux, 2h R = H (28%), OCH 3 (44%) R 1 = t-Bu (37%), Ph (53%) Scheme 2.

Synthesis of pyridine(pyrazine)-based N-acylamidrazones with further conversion into amino acid derived 1,2,4-triazoles

The application of pyrrolopyridines(pyrazines) 6, 6' (a,b) as precursors was chosen for the synthesis of N-acylamidrazones and further construction of amino acid derived 1,2,4triazoles. First, the hydrazide of Boc-protected phenylalanine was synthesized from phenylalanine methyl ester starting with NH 2 protection using di-tert-butyl dicarbonate followed by treatment with hydrazine monohydrate giving the corresponding precursor in quantitative yield (Table 2.9). Second, the preparation of N-acylamidrazones was performed by the reaction of 6, 6' (a,b) with BocPheNHNH 2 in MeOH at room temperature (Table 2.9).

No cyclic pyrrolopyridines(pyrazines) derivatives 10'', the formation of which could be expected according to a previously reported condensation of the 1-imino-1H-isoindol-3-amine and its pyrazine analogue with amino acid hydrazides that afforded 1H-isoindole-and 5Hpyrrolo [3,4-b]pyrazine-based peptidomimetics, were found. 130,131 Table 2.9 Synthesis of the N-acylamidrazone derivatives 10, 10' (a,b) The formation of pyrrolopyridines 6a,b was suggested to be a result of termal decomposition followed by further intramolecular cyclization giving a pyrrolidine ring.

N X N O R CO 2 Me NH Boc-Phe-NHNH 2 MeOH N X O HN NH 2 N CO 2 Me R N H O NHBoc N O R CO 2 Me N NH O NHBoc N X X = CH, N 6, 6' (a

Synthesis of hydrazide modified turn mimics

On further developing constrained peptidomimetics, we are interested in the synthesis and conformational study of hydrazide modified turn mimics derived from amidoximes 7,7'.

The synthesized peptidomimetics might be considered as the hybride structures of azapeptides 132 with substitution of a nitrogen for the α-carbon center and aminoxy peptides 133,134 which are backbone-modified peptidomimetics with oxygen atom between the nitrogen atom and one of the backbone carbon atom (Figure 2.9). 2.11). The phenylalanine products 12b and 12'b were obtained in 60 and 69% yields after purification by HPLC preparative. In the case of alanine derivatives 12a and 12'a, the reaction yields did not exceed 44% due to lower reactivity of the corresponding amidoxime with triphosgene which led to an increased amount of side product derived from the condensation of phenylalanine hydrazide with triphosgene. Attempts to improve the yield by changing times and temperatures were unsuccessful and need further improvement.

NH 2 N O HN CO 2 Me R N X O O HN NH O N H Ph Boc H N N H O N R 2 H N O R 3 O R 1 H N O O R Azapeptide R 1 , R 2 , R 3 = side chains n R = side chain, n = 1,

Conclusions

The synthesis of various novel 2,3-substituted pyridine(pyrazine) non-peptidic turn structures possessing amidoxime, esterified with amino acid and hydrazide modified amidoxime, amino acid derived N-acylamidrazone, chiral 1,2,4-oxadiazole and and 1,2,4triazole residues has been developed (Scheme 2.52). This readily available variety of nicotinic and pyrazine acid based pseudopeptides with an amidoxime function on the heteroaromatic ring might be considered as potential amidine prodrugs, an arginine peptidomimetics and/or as nitric oxide donors for treatment of cardiovascular diseases.

Furthermore, the synthesis of new 3,5-disubstituted 1,2,4-oxadiazoles has been presented. It was shown that the use of optimized microwave-assisted conditions with a reduced reaction time afforded products in good to high yields (58-94%).

A new straightforward and mild synthesis of amino acid derived N-acylamidrazones via pyrrolidine ring opening by the hydrazide of amino acid has been reported. The pyrazinebased N-acylamidrazones appeared the effective precursors for cyclodehydration into the corresponding chiral α-amino acid-derived 1,2,4-triazoles. Moreover, the use of catalytic amount of HOAc along with microwave irradiation reduced reaction time and increased product yields up to 87%. Unfortunately, we were not able to obtain pyridine-linked 1,2,4triazoles due to high thermo and solvent sensibility. This represented the major limitation of the developed protocol.

Finally, the first condensation of the amidoximes with Boc-protected hydrazide of amino acid in order to obtain hybride turn mimics with amidoxime-carbonyl-hydrazide linkage was performed. Products were obtained in moderate to good yields (40-69% yield).

Introduction

The second chapter describes structural analysis of the compounds including hydrogen bond investigation, cis-trans conformational preferences and thermodynamic studies of the proline derivatives and Z/E isomerism study of the acylamidrazone compounds.

Methods and techniques of conformational study

Physico-chemical tools that were used to collect structural informations and identify interactions responsible of the structuration are presented below. The implemented strategy has combined spectroscopic methods, such as Nuclear Magnetic Resonance (NMR) and

Infrared spectroscopy (IR), and molecular dynamics.

Infrared absorption spectroscopy (IR)

This analytical method was used to determine the involvement of NH and CO functions in hydrogen bond formation.

In the IR spectra of peptides the most interesting bands are found in the C=O bond stretching vibrations region (amide I bands in the region 1750-1580 cm -1 ) and the N-H stretching vibrations region (3520-3200 cm -1 ). Hydrogen bond of type C=O…H-N shifts the absorpsion bands to lower wavelengths and an absolute value of the shift reflect the relative strength of the interactions. It is commonly accepted in the literature that amidic NH which is bonded has IR absorpsion band below 3400 cm -1 . 133,[135][136][137] Unfortunately, the application field of this spectroscopic method is often limited by the transparency of solvents in the explored frequency range, by the solubility of molecules, by self-assembling in these environments and by the multiplicity and the overlapping of NH or CO bands that make difficult the assignment. To overcome this problem in some cases the deconvolution of bands is necessary. This has been realized in the spectroscopic software OPUS (Bruker) using the Levenberg-Maquardt mathematic algorithms.

Nuclear magnetic resonance spectroscopy (NMR)

Nuclear magnetic resonance is a crucial experimental technique in investigating the molecular properties in solution.

One dimensional NMR (1D)

As a labile proton, NH proton is really sensitive to modifications of its environment, as for example, the formation or break of a hydrogen bond. Therefore, four experiments can be used:

-NH chemical shift study: if the NH proton is involved in hydrogen bond, the nucleus will be deshielded that will led to the deplacement of chemical shift to low field.

-The effect of solvents: it consists of following the evolution of NH chemical shift while adding a polar solvent (DMSO) in a nonpolar solvent (CDCl 3 ). The NH proton which is not involved in an intramolecular hydrogen bond will be rapidly shifted to low fiОlН rОgion (high Δδ). In contrary, if it is involved in the intramolecular bonding, the chemical shift will be little affected by adding the polar solvОnt. ThО Δδ of these chemical shifts will be very low in case of strong intramolecular interaction.

-The temperature dependence: in the solvent -hydrogen bond acceptor, the temperature rise leads to the breaking of intermolecular hydrogen bonds and the evolution of the chemical shift; vice versa, the chemical shift of NH proton is independent on the temperature if it is involved in intramolecular hydrogen bond, as its not sufficient for breaking this type of interaction. This technique allows also to study cis/trans conformational equilibrium for proline-containing componds and to calculate thermodynamic parameters.

1D NMR study allows also measuring the population of the amide AA-Pro isomers in equilibrium mixture of cis/trans rotamers in solution. This method was efficiently used by teams of Dorman 138,139 and Beausoleil 140 in order to make the assignment of the isomer geometry of proline or pseudoproline derivatives based on the chemical shift values for the signals of the αand δ-carbons. In the 13 C NMR spectra, the α-carbon signal of the transisomer appears upfield to that of the cis-isomer. The δ-carbon signal of the trans-isomer appears downfield from that of the cis-isomer. Furthermore, the -carbon of the cis-isomer appears upfield to that of the trans-isomer and the -carbon of the cis-isomer appears downfield from that of the trans-isomer.

Two dimensional NMR (2D)

Unambiguous assignment of signals in pseudopeptides is obtained by using different two dimensional NMR experiments:

-Homonuclear through-bond correlations: COSY (Correlated SpectroscopY), TOCSY (Totally Correlated SpectroscopY) that show the correlations between coupled spins or all spins in a spin system.

-Through-space correlations: ROESY (Rotating frame Overhauser Effect SpectroscopY) and NOESY (Nuclear Overhauser Effect SpectroscopY) establishing correlations between nuclei which are physically close to each other (within about 5 Å) regardless of whether there is a bond between them.

-Heteronuclear through-bond correlations: Heteronuclear single-quantum correlation spectroscopy (HSQC) and Heteronuclear multiple-bond correlation spectroscopy (HMBC) that detect correlations between nuclei of two different types which are separated by one bond (HSQC) or of about 2-4 bonds (HMBC).

Molecular modelling

Molecular modelling calculations allow to visualize 3D structures of molecules for predicting, analyzing the properties and the behavior of the molecules with the equations of quantum (ab initio methods) and classical (molecular mechanic and molecular dynamic) physics.

Structural and thermodynamic studies of proline-containing peptidomimetics

Conformational preferences of proline derivatives

The singular behavior of proline in peptides and protein folding represents another way to introduce different turn motives into the amino acid backbone due to its N-terminal extremity that is involved in a cycle. However, unlike the majority of natural amino acids that adopt the trans configuration of peptidic bond, increased cis content along the Xaa-Pro bond is significant since both the cis and trans isomers are energetically similar. 141 In this context, we studied the effect of substitution with proline on the conformational behavior of the products. Information concerning the conformation adopted by compounds 5c, 3.1). The population of the amide isomers were measured in the 1 H NMR spectra by integration of all well-resolved proton signals. The major contributions into cis-trans equilibrium mixture have been clearly determined by NMR spectroscopy to be the trans conformers (Table 3.3). Interestingly, in most cases the content of the trans isomer was about 63-77% while cis isomer was 23-37%. However, when 5'c was studied, a decreased trans/cis ratio to 54/46 was observed. We assumed the presence of a noncovalent intramolecular n  * interaction between the N lone pair of the pyrazine ring and the antibonding orbital of the C=O of the pyrrolidine ring for all Pro-based derivatives (Figure 3.2, structure I). However, there is an increased amount of the cis conformation for cyano compound 5'c, as the trans product is stabilized by a relatevely weaker interaction between the more electronegative oxygen and the carbonyl (Figure 3.2, structure II). Evidence for this nonbonding interaction can be downfield shift for H α of the Pro residue in the cis-5'c only (Table 3.2). This assumption has been verified using the corresponding distances from molecular dynamics simulation of the proline derivatives 7'c and 8'c (Table 3.4). 3.5). Values of ΔH° anН ΔS° were calculated from linear least-squares fits of the data in the plots to eq. 1.

lnK t/c = (-ΔH°/R)(1/T) + ΔS°/R (1)
All derivatives have a slight positive slope demonstrating a temperature-dependence.

For the Pro derivative 5'c and 7c, a reduction in the magnitude of K t/c was observed, demonstrating a smaller enthalpic preference to the trans conformation.

Although differences in entropy favor the cis isomer, the trans isomer in all cases is favored by enthalpy. The equivalent constants decrease with increasing temperature in the order 8'c 9'c 9c>7'c 5c 8c 7c 5'c (Table 3.5). The energy barrier (ΔG ‡ ) for amide isomerization in 5c was determined by NMR spectroscopy. The equilibrium between cis and trans rotamers of 5c shown in Figure 3.1, is related to rate constant k, which can be calculated using the Eyring equation (2).

k = (k B *T/h)*e (-ΔG ‡/RT) (2)
In this equation, k B is Boltzmann's Мonstant, h is PlanМk's Мonstant, R is thО gas Мonstant anН ΔG ‡ is the Gibbs free energy of activation. Hence, when the exchange rate between cis and trans of 5c is slow, two sets of signals are observed in the NMR spectrum, one for each rotamer. In contrast, with a fast rate of interconversion, only the average signal is present. Between these two limits a broad and flat peak should appear. The temperature when this happens is called the coalescence temperature, T c . At T c , the rate constant is given by equation (3).

k = (π*Δ )/√β = β.ββ*Δ (3)
where Δ is the difference in Hertz of the same proton frequencies of two isomers at a slow rate of interconversion. At this point, ΔG ‡ can be calculated using equation (4).

ΔG ‡ = RT c [22,96+ln(T c /Δ )] (4)
Knowing ΔG ‡ , it is possible to calculate the rate constant k at different temperatures using the Eyring equation 2, assuming that there is no changing in entropy on changing the temperature.

Finally, the half-life for rotation is given by equation (5).

t 1/2 = ln2/k (5)
Thus, a series of 1 H NMR spectra were recorded in DMSO-d 6 at increasing temperatures until the resonances for the two isomer populations were observed to coalesce at 90 °C (Figure 3.4).

Stuctural and thermodynamic analysis of pseudotripeptide methyl (2S)-2-({[(2S)-1-({3-[(Z)-(hydroxyamino)(imino)methyl]pyrazin-2-yl}carbonyl)pyrrolidin-2-yl]carbonyl}amino)-3-phenylpropanoate 7'd

Structural properties of the pyrazine amidoxime motif incorporated at N-terminus of the pseudotripeptide chain were investigated in order to determine its use in future applications. Information concerning the conformation adopted in solution by experiments with enough structural informations to calculate 3D structures of our molecules, neither good quality mono crystals could be obtained. The mobility of the peptidic part is probably a reason for these factual situations in both cases. Especially several conformations have been observed in NMR spectra. The strategy, repeatedly used successfully by our group and others, is to highlight the presence of hydrogen bonds. 63,64,136,137 For that purpose two spectroscopic methods were used combined to molecular dynamic calculations.

FT-IR and NMR investigations

First, FT-IR spectroscopy in solution was applied in order to observe the NH and CO stretching vibrations at around 3500-3200 cm -1 and 1600-1800 cm -1 region, respectively.

Second, 1D 1H-NMR spectra in CDCl 3 /DMSO-d 6 varying ratio highlights the labile protons not influenced by the environment changes because they are involved in hydrogen bonds.

FT-IR experiment of 7'd at 25 °C shows a large peak at 3319 cm -1 which means that at least one NH and/or OH proton is involved in the hydrogen bond. In the carbonyl region of the spectrum, six bands are observed at 1743 cm -1 , 1722 cm -1 , 1679 cm -1 , 1670 cm -1 , 1656 cm - 1 and 1653 cm -1 (Figure 3.5, Table 3.6). The number of bands indicates that at least two of the three carbonyl groups of the molecule are involved in hydrogen bonds. According to their wavelengths, the two bands at 1743 cm -1 and 1722 cm -1 were assigned to the ester C=O free and involved in the hydrogen bond, respectively. The next four bands were assigned using the absorbance spectra of the shorter peptides with one amino acid and the pyrazine amidoxime motif 7'b and 7'c (Table 3.6, Figure 3.6). Therefore, the amide stretching vibration at 1679 cm -1 corresponds to C=O of the peptide bond between proline and phenylalanine as it is close to the value obtained for AOPzPhe (7'b). The bands at 1670 and 1656 cm -1 were attributed to the amide bond of the pyrazine ring and proline-phenylalanine dipeptide in both free and bonded forms, respectively.

The C=N stretching vibration of the amidoxime group was then assigned to the band at 1653 cm -1 .

Results issued from IR spectroscopy experiments were complemented by 1 This observation means that in solution, the structure of 7'd is in equilibrium between at least two conformations. These results are consistent with the presence of the cis/trans isomers about the X-Pro peptide bond. Moreover, in both sets the amide proton of phenylalanine reveals no significant shift (Δδ = +0.22 and +0.42 ppm), which means that for both conformations the NH proton is involved in an intramolecular hydrogen bond. This result is consistent with the large band below 3400 cm -1 in the IR spectrum, which probably is the envelope of several bands.

Hence, the NH amide group of phenylalanine is involved in the hydrogen bond with the carbonyl group of pyrazine close to it (band at 1656 cm -1 ) and adopt a seven-mОmbОrОНturn conformation (Table 17).

Therefore, to confirm our observations, molecular modeling calculations in explicit solvent and without restraints have been undertaken. With regards to the proline residue, two conformers were observed in NMR spectra of the proline derivatives and we decided to investigate further forward the cis/trans isomerization of 7'd. In particular, we decided to consider the thermodynamics for trans and cis isomers of the prolyl peptide bond.

Molecular modeling

cis-trans isomerization study

Hydrogen between AOPzPro (7'c) and AOPzProPhe (7'd) may be due in part to interaction of the NH group with either the imide nitrogen lone pair or the imide carbonyl oxygen 140,146 or due to the stabilizing effect of the n  * interaction between the pyrazine nitrogen and C co of the pyrrolidine ring.

The thermodynamic parameters of the cis/trans isomerization for prolyl amide bond of 7'd were calculated from linear least-squares fits of the data in these plots to eq. 1 (Table 3.8).

Although all proline-containing molecules demonstrate a temperature-dependence with a slight positive slope (Figure 3.

A tentative correlation between toxicity and structure

The cellular toxicity of compounds was tested in order to verify a possibility of their use as therapeutics (Figure 3.14). The vibrations in the C=O region also did not show extra bands that could correspond to hydrogen bonded carbonyls (Table 3.9). and 3395 cm -1 , respectively (Figure 3 3.10). We have suggested the mechanism different to the route proposed for the amidoxime decomposition which involves nucleophilic attack of the NH 2 on the carbon of amide carbonyl, as in this case the formation of 6 does not occur (Scheme 2.8). A proposed mechanism includes nucleophilic attack of the NH on the carbon of C=N acylamidrazone followed by cyclyzation depicted in Scheme 3.2.

Stuctural analysis of hydrazide modified peptidomimetics 12, 12'

The assignment of all NH protons of the pyrazine-based hydrazide products was made by means of COSY and NOESY NMR spectra at first. As for the other pyridine and pyrazine derivatives, the comparison of the NH-amide protons revealed a downfield shift above 1 ppm ongoing from pyridine derivatives 12a,b to pyrazine analoges 12'a,b (Table 3.17). To highlight the presence of hydrogen bonds two spectroscopic methods were used.

First, FT-IR spectroscopy in solution in order to observe the NH and CO stretching vibrations at around 3500-3200 cm -1 and 1600-1800 cm -1 regions, respectively. Second, 1 H-NMR spectra in CDCl 3 /DMSO varying ratio hightlight the labile protons not influenced by the environment changes because they are involved in hydrogen bonds.

FT-IR experiment of pyridine derivatives 12a,b at 25°C showed a broad peak below 3400 cm -1 which consists of at least two NH protons of the hydrazide fragment.

Unfortunately, it was not possible to distinguish the different NH bands under this peak (Figure 3.26). In the carbonyl region of the spectrum, seven bands were observed (Figure 3.26, Table 3.18). Number of bands indicates that one of the carbonyl in the molecule is involved in hydrogen bond. According to their wavelengths, two bands at 1693 cm -1 and 1668 cm -1 were assigned to the amide carbonyl free and involved in a hydrogen bond, respectively. In case of 12b, similar tendencies were observed in the NH and C=O regions (Figure 3.27, Table 3.18). Therefore, two bands at 1699 cm -1 and 1669 cm -1 were attributed to the free and bonded amidic carbonyl. The FT-IR spectra of pyrazine-based analogues in the NH region also revealed a broad shoulder below 3400 cm -1 , however we did not succeed to assign the stretching vibrations in the C=O region (Figure 3. This kind of NOE pattern of compound 12b supported the view that it adopts a turn structure. Simulation in DMSO showed that this hydrogen bond is present in DMSO as well.

Likewise, the pyrazine amidoxime coupled with proline-phenylalanine dipeptide induces the hydrogen bond that adopts -turn conformation. Therefore, a dramatic increase of the trans rotamer up 98% was observed in weakly polar solvent, such as CHCl 3 . Experimental data provide evidence for the retention of the NH into hydrogen bonding in DMSO, which is in agreement with the low sensitivity of the NH proton resonance to solvation. This interaction and/or the n  * interaction between the pyrazine nitrogen and C co of the pyrrolidine ring significantly augments the cis isomer population in polar solvents. Thus, 7'd appears to be interesting as a model pseudotripeptide for conformational study of the cis/trans isomerization for prolyl amide bond.

Conformational analysis of esterified amidoximes 8, 8' and oxadiazoles 9, 9' did not reveal hydrogen bonding exept for the oxadiazole 9'c with the weak interraction between the NHBoc proton and the imide CO oxygen.

The Z/E isomerism in acylamidrazones was found to be dependent on the heterocycle and was studied by means of NMR spectroscopy and quantum chemical calculations. The

Griess method

One means to investigate nitric oxide formation is to measure nitrite (NO 2 -), which is one of two primary, stable and nonvolatile breakdown products of NO. This assay relies on a diazotization reaction that was originally described by Griess in 1879. 149 We pre-incubate microtube during 1 minute at 37°C. Then, 50 L of NADPH (1mM) in each tube is added. The reaction is initiated and the mixture incubates at 37°C for 10 minutes. 100 L of the working solution is taken from the medium and heated for 3min at 100°C. Microtube is centrifugated at 10000rpm during 15min at 4°C. We take this 100 L to achieve the determination of nitrite ions by Griess.

Two controls were prepared: control 1 without microsomes for each amidoxime and control 2 without amidoxime. Absorbance at 570nm

Concentration of nitrite (µM)

Concentration of nitrite was calculated from fits of the absorbance data to equation:

A = a[NO 2 - ] + b
The preliminary results are shown in Table 4.1. between the values for the pyridine and pyrazine analogs (7, 7' (a) -7, 7' (c)) showed better effect of the pyridine ring for phenylalanine and proline derivatives, however in the case of alanine-derived amidoximes, the pyrazine product appeared to be more efficiant than its pyridine counterpart. The correlation between the amino acid side chains and the nitrite concentration of the pyridine derivatives let us to put them in the order Phe Val Cys(Tr) Pro LОu Trp Ala. In the case of pyrazine compounds the opposite ability to NO release is observed: Ala Pro Phe.

In conclusion, amidoxime compounds were tested for nitric oxide release and all derivatives showed NO release in concentration sufficient for pharmacological effects (≥1 µM). A tentative correlation between the structures and the activity has been done.

General Conclusions and Perspectives

The aim of this project was the synthesis, structural study and preliminary biological evaluation of pyridine and pyrazine-based arginine mimics. This goal was accomplished with the synthesis of six-membered heterocyclic carboxylic acids with neighbor amidoxime moiety and their further modification. Target structures were functionalized to give novel esterified with amino acid and hydrazide modified amidoximes, amino acid derived N-acylamidrazones, chiral 1,2,4-oxadiazole and 1,2,4-triazole derivatives, bearing amidine motif incorporated into peptide chain. Hence, the synthesized molecules may be considered as arginine turn mimics possessing azine (pyridine or pyrazine) linkage.

A new convenient and mild synthesis of amidoximes and N-acylamidrazones via pyrrolidine ring opening by hydroxylamine and hydrazide of amino acid has been reported.

Structures of products were studied by NMR, IR spectroscopic studies and molecular modelling. Crystal structures of some compounds were analyzed by X-Ray diffraction study.

Some compounds revealed intramolecular hydrogen bonding that promote turn-like structure.

It was shown that pyridine and the pyrazine moiety in some cases can be used as a structural motif to stabilize the cis conformation of Pro-peptidomimetics. The nitrogen of the pyrazine ring can also interect with the NH-amide proton which prevents nucleophilic attack of the NH nitrogen on the carbon of C=N, allowing the 1,2,4-triazole condensation and giving the higher yields of the 1,2,4-oxadiazole cyclization. Amidoxime compounds were tested for nitric oxide release demonstrating NO release in concentrations 0.83 -2.06 µM. Some tentative structure-activity relations have been presented. The most promising compounds will be subjected to further tests in vivo.

In perspective, amidoximes could be tested for there ability to be reduced in vitro/in vivo into pharmacologically active amidines, an arginine mimics.

General Methods

The chemicals were purchased as the highest purity commercially available and were used without further purification. Dry THF was obtained by distillation over sodium and benzophenone. Methanol, acetonitrile and DMF were purchased in anhydrous form.

Microwave-assisted reactions were performed using CEM Focused Microwave acids parameters (ff99SB). Ptraj was employed for the analysis of 25000 structures in order to assess the possibility of a hydrogen bond formation.

Cytocompatibility with vascular smooth muscle cell

The cytocompatibility of pyrazine amidoxime derivatives AOPzPro (7'c), AOPzPhe (7'b), AOPzProPhe (7'd) were evaluated on rat smooth muscle cells (SMC) line (A-10) ATCC ® CRL-1476™. SMC wОrО grown in МomplОtО mОНium maНО of DulbОММo's MoНifiОН EaglО's

Medium DMEM supplemented with 10% (v/v) FBS, 2% (v/v) glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin mixture, and 4 mM sodium pyruvate. Cells were cultivated at 37 °C under 10% (v/v) of CO 2 in a humidified incubator. SMC were seeded in 96-wells plates at 60,000 cells/well 48 h before exposure to pyrazine amidoxime derivatives for 24 h (complete medium was used as control). Pyrazine amidoxime derivatives were firstly resuspended in 100% methanol at a concentration of 10 -1 , 10 -2 , 10 -3 and 10 -4 M. Finally, pyrazine amidoxime derivatives were diluted in complete medium for final concentrations of 10 -6 , 10 -5 , 10 -4 , 10 -3 M. Cytocompatibility through metabolic activity was checked with MTT assay. Briefly, 0.5 mg/mL MTT was incubated with the cells for 4 h. Then, 250 µL DMSO was added under stirring for 10 min to extract the formazan crystals. The absorbance was read at 570 nm with a reference at 630 nm using EL 800 microplate reader (Bio-TEK Instrument, INC ® , France). Metabolic activity in the presence of pyrazine amidoxime derivatives was compared to the control condition (culture medium alone).

Statistical analysis

Results

Synthesis of Compounds 5, 6 (a-f); General Procedure

To a solution of 2-cyanonicotinic acid (4; 740 mg, 5 mmol) in CH 2 Cl 2 (30 mL) were added Et 3 N (1.39 mL, 10 mmol), the corresponding methyl ester of an amino acid hydrochloride (5 mmol) anН HOBt (0.675 mg, 5 mmol). ThО mixturО was stirrОН at 0 °С anН EDCI (0.967 g, 5.05 mmol) was added. Then, the mixture was stirred at r.t. overnight. The mixture was diluted in CH 2 Cl 2 (50mL); then, the solution was washed with 0,1M HCl (3×15 mL) and brine (20 mL), dried over MgSO 4 and concentrated in vacuo.

Methyl (2S)-2-{[(2-cyanopyridin-3-yl)carbonyl]amino}propanoate (5a)

Methyl (2S)-2-[(7E)-7-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-3-(1Hindol-3-yl)propanoate (6g)

Synthesis of Compounds 7a-h; General Procedure

To a solution of the methyl ester of a (2S)-N-

Methyl (2S)-2-({[2-(N'-hydroxycarbamimidoyl)pyridin-3-yl]carbonyl}amino)-3-(1Hindol-3-yl)propanoate (7g)

Methyl (2S)-2-[({2-[amino(hydroxyimino)methyl]pyridin-3-yl}carbonyl)amino]-3-(tritylsulfanyl)propanoate (7h)

Synthesis of Compounds 5', 6' (a-c); General Procedure:

To a solution of 3-cyanopyrazine-2-carboxylic acid (3) (0.740 g, 5 mmol) in THF (30 mL) were added: Et 3 N (1.39 mL, 10 mmol), the corresponding methyl ester of an amino acid hyНroМhloriНО (5 mmol) anН HOBt (0.675 g, 5 mmol). ThО mixturО was stirrОН at 0 °С anН EDCI (0.967 g, 5.05 mmol) was added. Then, the mixture was stirred at r.t. overnight. The precipitate was filtered and the filtrate was evaporated under vacuo. The residue was diluted with CH 2 Cl 2 (50mL), washed with a solution of 0,1M HCl (3×15 mL), brine (20 mL), then dried with MgSO4 and concentrated in vacuo. Purification by flash column chromatography (AcOEt/petroleum ether, 7:3) gave 6'b as a yellow oil (1.007 g, 65%).

Methyl (2S)-2-{[(3-cyanopyrazin-2-yl)carbonyl]amino}propanoate (5'a)

IRμ max =

Boc-Pro-Phe-OMe

Compound is commercially available, but was prepared according to the general procedure for the synthesis of compounds 5,6(a-f). It was prepared by coupling of Boc-Pro-OH hydrochloride (1.08 g, 5 mmol) and H-Phe-OMe (1.08 g, 5 mmol) using EDC (0.97 g, 5.05 mmol), HOBt (0.77 g, 5 mmol) and triethylamine (1.4 mL, 10 mmol) in CH 2 Cl 2 (30 mL). The mixture was stirred at room temperature overnight and then washed with a solution of 0.1M HCl (3×15 mL), brine (20 mL), dried with MgSO 4 and then the solvent was removed in vacuo. The product was purified by flash chromatography, eluting with AcOEt/Petroleum ether, 2/1; yield: 92% (1.73 g).

H-Pro-Phe-OMe TFA salt

Compound is commertially available, but was synthesized from Boc-Pro-Phe-OMe (0.752 g, 2 mmol) by treatment with 30% trifluoroacetic aciddichlorometham (10 mL) during one hour. The solvent was then evaporated in high vacuo. A beige solid (0.772 g, 99%) was obtained.

Methyl (2S)-2-[({(2S)-1-[(3-cyanopyrazin-2-yl)carbonyl]pyrrolidin-2-yl}carbonyl)amino]-3-phenylpropanoate (5'd)

N N CN O N H N O Ph CO 2 Me
To a solution of dipeptide Boc-Pro-Phe-OMe (0.390 g, 1 mmol), acide 4' (0.149 g, 1 mmol)

and HOBt (0.168 g, 1.1 mmol) in THF (5 mL) at 0° C, HATU (0.380 g, 1 mmol) and DIEA (0.4 mL, 2.1 mmol) were added. After 15 min at 0° C and 10 hours at room tempereture, the solvent was evaporated and the crude product was purified by flash column chromatography (AcOEt/CH 2 Cl 2 , 7:3) to give 5'd as a light-yellow oil (0.830 g, 68%).

Synthesis of

Methyl (2S)-2-({[(2S)-1-({3-[(Z)-(hydroxyamino)(imino)methyl

Methyl (2S)-1-({2-[(Z,5S

Methyl (2S)-2-[({3-[(Z,5S

)-1-amino-5-isopropyl-

Synthesis of Compounds 9, 9' (a-c); General Procedure:

A 5 mL CEM microwave process vessel was charged with 8, 8' (a-c; 0.2 mmol) in CH 2 Cl 2 (5 mL) and the vessel was capped. The mixture was stirred and heated under microwave conditions (300 W) at 150 °C for 25 min. The solution was then concentrated under vacuum and chromatographed on silica gel to give 1,2,4-oxadiazoles 9, 9' (a-c).

Methyl (2S)-2-({[2-(5-{(1S)-1-[(tert-butoxycarbonyl)amino]-2-methylpropyl}-1,2,4oxadiazol-3-yl)pyridin-3-yl]carbonyl}amino)propanoate (9a)

, J = 7.1 Hz, 3H, CH IR: max = 3424, 3311,

Methyl (2S)-2-({[2-(5-{(1S)-1-[(tert-butoxycarbonyl)amino]-2-methylpropyl}-1,2,4oxadiazol-3-yl)pyridin-3-yl]carbonyl}amino)-3-phenylpropanoate (9b)

Methyl (2S)-1-{[2-(5-{(1S)-1-[(tert-butoxycarbonyl

Methyl

IR (CHCl 3 ): max = 3442, 3395, 1742, 1715, 1689 cm -1 .

Methyl (2S)-1-{[3-(5-{(1S)-1-[(tert-butoxycarbonyl)amino]-2-phenylethyl}

Boc-(S)Phe-OMe

To a solution of H-Phe-OMe hydrochloride (3.23 g, 15 mmol) in saturated aqueous NaHCO 3 (150 mL), Di-tert-butyl dicarbonate (3.43 g, 15.75 mmol) was added. The reaction mixture was stirred for two days. The organic phase was extracted with EtOAc (3×50 mL) and washed with HCl 10% (2×50 mL), NaHCO 3 10% (50 mL) and finally with brine (50 mL). The resulting solution was dried with MgSO 4 and concentrated under vacuum. The colorless solid was obtained in quantitative yield (4.2 g); mp 46 °C.

Boc-(S)Phe-NHNH 2

To a solution of Boc-Phe-OMe (4.03 g, 14.4 mmol) in MeOH (50 mL), hydrazine monohydrate (2.79 mL, 57.6 mmol) was added. The resulting solution was vigorously stirred until completion (monitored by TLC) and co-evaporated several times with CH 2 Cl 2 at reduced pressure until complete elimination of the hydrazine monohydrate. The colorless solid was obtained in quantitative yield (4.0 g); mp 128 °C. White solid; yield: 0.420 g (82%); mp 188-189 °C. The corresponding amidoxime 7, 7' (a,b; 0.8 mmol, 1 eq.) was added to triphosgene (0.4 mmol, 0.5 eq.) in CH 2 Cl 2 (10 mL) at 0 °C and the mixture was warmed to r.t. and stirred for 2.5 h. To the above mixture a solution of Boc-(S)Phe-NHNH 2 (1.08 mmol, 1.5 eq.) and DIEA

IRμ max =

Synthesis of

(2.16 mmol, 3 eq.) in CH 2 Cl 2 (10 mL) at 0 °C was added. The reaction was allowed to warm to r.t. and stirred overnight. The reaction mixture was washed with water (2×15 mL), brine (20 mL) and concentrated to give a crude which was purified by preparative NP-HPLC, using

X-Ray Diffraction Study of Compounds 6d and 7b

Intensities of reflections were measured on an automatic «Xcalibur 3» diffractometer (graphite monochromated Mo Kα radiation, CCD-НОtОМtor ω sМanning). All struМturОs wОrО solved by direct method using SHELX97 package. 151 Positions of the hydrogen atoms were loМatОН from ОlОМtron НОnsity НiffОrОnМО maps anН rОfinОН by "riНing" moНОl with Uiso = nUeq of carrier non-hydrogen atom (n = 1.5 for methyl group and n = 1.2 for other hydrogen atoms). Full-matrix least-squares refinement against F

Synthèse des amidoximes

Lors de nos tentatives de développement de nouvelles variétés de peptidomimétiques 1).

En ОffОt, quО МО soit р partir Н'un Нérivé Н'aМiНО aminé НО struМture ouverte 5 ou de sa forme correspondante cyclique 6 un unique produit de type amidoxime 7 est obtenu.

AuМunО amiНoximО МyМliquО n'a été rОportéО (sa formation aurait pu êtrО attОnНuО selon la condensation précédemment rapportée de l'analogue de benzène 3-imino-1oxoisoindoline avec l'hydroxylamine).

Le seul produit formé à partir de la réaction du glycinate de méthyle avec l'acide 4 a été analysé par diffraction des rayons X du monocristal. Les résultats montrent que ce produit adopte la structure 6d (Figure 1). Les spectres RMN de ces composés couplés à la proline ont révélé deux jeux de signaux dans le CDCl 3 à température ambiante: ils correspondent aux rotamères des liaisons amide cis et trans qui sont énergiquement proches dans le cas de la proline à la différence de la majorité des acides aminés naturels pour lesquels adoptent préférentiellement une configuration trans de la liaison peptidique. A partir des spectres RMN 1 H, RMN 13 C et NOESY des dérivés 5c, 5'c, 7c, 7'c, il a été possiblО Н'attribuОr Оt Н'estimer le rapport approximatif de ces rotamères. Les pics de corrélation observés dans les spectres NOESY de 5c correspondent à l'interaction spatiale entre le proton en position C4 du cycle pyridine et les protons en position C5 du résidu pyrrolidine et, de ce fait, indiquent que le produit majoritaire est l'isomère trans (en conséquence, le produit minoritaire est l'isomère cis) (Figure 6).

Produit

, cm -1 (C=O ester)

, cm -1 (C=O amide)

, cm 11).

Conclusion

Les différentes études menées dans ce travail de thèse ont permis la synthèse de nouvelles familles de peptidomimétiques ciblés, avec pour objectif de promouvoir des structures coudées stables afin de donner de la rigidité par rapport à leurs équivalents Initially, we have developed a convenient synthesis of new peptidomimetics with amidoxime function as a replacement of amidine one. The latter can imitate arginine residue in biological structures. Chemical functionalization of these scaffolds led to novel 2,3-disubstituted pyridine(pyrazine) turn structures bearing amidoximes esterified with amino acid or modified with amino acid hydrazides, N-acylamidrazones, 1,2,4-oxadiazole and 1,2,4-triazole residues. Additionally, all structures were analyzed by NMR, IR, molecular modelling and XRD. Conformational studies confirmed that pyridine and pyrazine heterocycles can be used to increase rigidity and the pyrazine core has stronger effect on conformation stabilization. Examination of a new ProPhe pyrazine-based pseudotripeptide revealed the hydrogen bond formation between the proton of the OH and the carbonyl oxygen of the C-terminal phenylalanine. This hydrogen bond adopts a seven-membered γturn conformation. Therefore, a dramatic increase of the trans rotamer up to 98% was observed in weakly polar solvent, which is CHCl3.

Finally, preliminary results of the NO release assay on amidoximes demonstrated sufficient values for pharmacological effect.

NOUVEAUX MIMES D'ARGININE A MOTIF PYRIDINIQUE OU PYRAZINIQUE: SYNTHESE, ETUDE STRUCTURALE ET EVALUATION BIOLOGIQUE PRELIMINAIRE

Acides aminés, mimes, synthèse, étude structurale, donneurs de NO Ce travail décrit la synthèse, l'étude structurale et une première évaluation des propriétés biologiques d'une nouvelle famille de mimes de l'arginine issus de motifs pyridine et pyrazine.

Pour cela, nous avons mis au point la synthèse de nouveaux peptidomimétiques dans lequels la fonction amidine est remplacée par une amidoxime mime potentiel du résidu arginine. La fonctionnalisation chimique de ces motifs a permis à partir de la pyridine (ou pyrazine) 2,3disubstituée l'obtention de nouveaux coudes non-peptidiques possédant des motifs amidoximes estérifiés par des acides aminés ou modifiés par des hydrazides d'acides aminés, des Nacylamidrazones et des résidus de 1,2,4-oxadiazole et de 1,2,4-triazole. L'analyse structurale réalisée par RMN, IR, modélisation moléculaire et par XRD a confirmé les propriétés des hétérocycles pyridine et pyrazine, en particulier dans la stabilisation des conformations et le noyau pyrazine qui semble jouer un rôle essentiel sur la stabilisation de la conformation. L'étude d'un nouveau pseudotripeptide à base de pyrazine-ProlylPhenylalanine a révélé la formation d'une liaison hydrogène entre le proton du groupe OH et l'oxygène du carbonyle de la phénylalanine C-terminale. La liaison hydrogène, ferme un pseudocycle à 7 atomes (C7). De plus, une augmentation considérable du rotamère trans (jusqu'à 98%) dans un solvant faiblement polaire (CHCl3) a été observée.

Enfin, les premières amidoximes testées ont permis de révéler la libération de NO en concentration suffisante pour produire un effet pharmacologique.

2

 2

Figure 1 . 1

 11 Figure 1.1 Selected examples of biologically active pyridine and pyrazine compounds

Figure 1 . 3 A

 13 Figure 1.3 A simplified illustration of N-reductive enzyme system

5 Scheme 1 . 2

 512 Scheme 1.2 The activation of the pentamidine-prodrugs

Figure 1 . 4

 14 Figure 1.4 Antitrombotic amidoxime prodrug and its active drug

Figure 1 . 5

 15 Figure 1.5 Prodrugs of dabigatran thrombin inhibitor

Figure 1 . 6

 16 Figure 1.6 Prodrug of the urokinase inhibitor

Scheme 1 . 3

 13 Scheme 1.3 Development of oseltamivir derivatives

Scheme 1 . 4 Figure 1 Figure 1 .7 1 , 2 , 4 -Figure 1 . 8 Figure 1 Figure 1 . 9

 141112418119 Scheme 1.4 Metabolic reduction of N,N-dihydroxybenzamidines as double prodrugs

Scheme 1 . 5

 15 Scheme 1.5 Biosynthesis of NO

2 + 2 P450Scheme 1 . 6

 2216 Scheme 1.6 Reaction of amidoxime with liver microsomal cytochromes P450

byFigure 1 . 10

 110 Figure 1.10 Azoamidoximes testet for NO release

Figure 1 . 11

 111 Figure 1.11 Oximes, amidoximes and hydroxamic acids tested as nitric oxide donors

2 OHFigure 1 . 12

 2112 Figure 1.12 Amidoximes tested for relaxant activity in the rat aorta

Figure 1 . 13

 113 Figure 1.13 Tested imidazole amidoximes

Scheme 2 . 1 .X 2 . 1

 2121 Scheme 2.1. We decided to employ 2-cyanonicotinic and 3-cyanopyrazine-2-carboxylic acids as starting products. The corresponding amidoximes can be synthesized in two steps to give different pyridine and pyrazine-bazed pseudodipeptides. Additionally, the same intermediates can be utilized for the reaction with amino acid hydrazides in order to obtain Nacylamidrazones that can be used to produce 1,2,4-triazole-containing scaffolds. Esterification of amidoximes either with amino acid or with hydrazide of amino acid displays a way towards pseudotripeptides or double prodrugs of amidines. The cyclization of the latter can give pharmaceutically important 1,2,4-oxadiazole derivatives.

Scheme 2 . 2

 22 Scheme 2.2 Synthesis of 2-cyanonicotinic and 3-cyanopyrazine-2-carboxylic acids

Scheme 2 . 4

 24 Scheme 2.4 Synthesis of amidoximes from hydroxamoyl chlorides

( 4 )Scheme 2 . 5

 425 Scheme 2.5 Synthesis of methyl esters of (2S)-(2-cyanopyridin-3-yl)carbonyl substituted amino acids 5a,e,f,h and methyl esters of (2S)-2-(7-imino-5-oxo-5,6-dihydro-6H-pyrrolo[3,4b] pyridin-6-yl) alkanoic acids 6a,b,d-g

Figure 2 . 1

 21 Figure 2.1 Molecular structure of compound 6d according to results of the X-ray diffraction study, with the atom numbering used in the crystallographic analysis; thermal ellipsoids are shown at 50% probability level

  unexpected material has an open structure that is regioisomeric to the nonisolated open form of 6g. Even though the starting acid 4 was obtained as individual compound of 100% purity (according to LC/MS data), the presence of the isomeric ester A could result from an undetected 3-cyanopicolinic acid impurity in the 2-cyanonicotinic acid (4).

Figure 2 . 2

 22 Figure 2.2 Structures of A and 6g

Scheme 2 . 7

 27 Scheme 2.7 Synthesis of methyl (2S)-1-{[2-(N'-hydroxycarbamimidoyl)pyridine-3yl]carbonyl}pyrrolidine-2-carboxylate 7c

Figure 2 . 3

 23 Figure 2.3 Molecular structure of compound 7b according to results of the X-ray diffraction study; thermal ellipsoids are shown at 50% probability level

2 OHScheme 2 . 8

 228 Scheme 2.8 Plausible mechanism of amidoxime 7 and oxime III formation via pyrrolidine ring opening by hydroxylamine

aand 6 '

 6 Isolated yields. Along with the desired methyl esters of amino acid 5'a,b, a tautomeric pyrrolopyrazine products 6'a,b were produced. Separation and purification of derivatives 5' was achieved by column chromatography and allowed us to isolate the open 5'a,b and cyclic 6'a,b products in different ratios (Table 2.4). When alanine was used, the content of both products 5'a and 6'a was approximately the same while in the case of phenylalanine the cyclic form 6'b largly predominated. The coupling with proline methyl ester necessarily afforded only the open-chain pyrazine derivative 5'c in 49% yield. The IR spectra of 5'a-c showed a characteristic cyano group absorption band at 2240, 2237 and 2239 cm -1 , respectively. By analogy to the pyridine counterparts, the 1 H NMR spectra of the coupling products 5', 6' revealed NH proton resonances at δ = 8.26 and 8.20 ppm for the open-chain compounds 5'a,b, and a downfield shift of δ = 9.51 and 9.46 ppm for the tautomeric cyclic derivatives 6'a,b. The values are very close to the values of those for the pyridine series 9.34 (Ala derivative 6a) and 9.29 (Phe derivative 6b) ppm (see Table

The 1 H

 1 NMR spectra of the amidoximes 7' revealed a chemical shift of δ = 5.45 ppm for the NH 2 protons of the alanine derivative and δ = 5.82 ppm for the phenylalanine one. X-Ray diffraction analysis of the amidoxime 7'b (Figure 2.5) revealed Z configuration at C=N bond of the amidoxime group similarly to the pyridine analog 7b. However, unlike for the pyridine derivative 7b, the NH group of the pyrazine compound 7'b is projected more inward (NH…N distance is 3.14 Å, NH…N anglО 78°).

Figure 2 . 5 Figure 2 . 6

 2526 Figure 2.5 Molecular structure of methyl (2S)-2-({[3-(N'-hydroxycarbamimidoyl)pyrazin-2yl]carbonyl}amino)-3-phenylpropanoate 7'b according to results of the X-ray diffraction study; thermal ellipsoids are shown at 50% probability level The formation of (7Z)-7-(hydroxyimino)-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-one as a side product was observed only after long storage of the pyrazine amidoximes 7'. Moreover, in our attempts to grow a crystal of 7'a we were able to obtain the crystal of the corresponding pyrrolo[3,4-b]pyrazin-5-one oxime IV as a by-product (Figure 2.6).

Table 2 . 5

 25 Comparison of compounds 5, 5' (a,b) and 6, 6' (a,b)

Scheme 2 . 9

 29 Scheme 2.9 Synthesis of pseudotripeptide 7'd

Figure 2 . 7 Scheme 2 . 10

 27210 Figure 2.7 Representative bioactive compounds containing α-amino acid-derived 1,2,4oxadiazoles

Pg 1 =Scheme 2 .20 1 , 2 , 4 -

 12124 Scheme 2.19 1,2,4-Oxadiazoles synthesis reported by Sureshbabu et al.[70]

  variОty of Мhiral α-amino acid derived 1,2,4-oxadiazoles. The following two-step reaction led to the desired 1,2,4-oxadiazoles 9, 9' which were submitted to the ring formation under microwave irradiation of the intermediate acylated amidoximes 8, 8' (Table 2.6). Firstly, Bocprotected L-valine was esterified with the corresponding amidoximes 7, 7' (a,b) using DCC/DMAP in anhydrous acetonitrile to obtain O-acyl amidoximes 8, 8' (a,b) in good yields.

1, 2 , 4 -

 24 triazoles containing chiral α-amino acids have been designed and synthesized. L-Tryptophane bearing 1,2,4-triazoles A have been reported as ghrelin receptor (GHS-R1a) ligands 109 , lysine derivatives B exhibit histone deacetylase (HDAC) inhibitor activity 110 with high metabolic stability and dipeptido-1,2,4-triazole derivatives C posess a high level of central nervous system (CNS) activity 111 (Figure 2.8)

Figure 2 . 8

 28 Figure 2.8 Selected biologically active 1,2,4-triazoles containing chiral α-amino acids

1 , 2 , 4 -

 124 Scheme 2.36 1,2,4-Triazoles synthesis from amidrazones reported byDumur et al. [115]

38 1, 2 , 4 -

 24 Scheme 2.38 1,2,4-Triazoles synthesis from amidrazones reported byBumagin et al. [118]

Scheme 2 .47 1 , 2 , 4 - 1 , 2 , 4 -

 2124124 Scheme 2.47 1,2,4-Triazoles synthesis from thioamides reported byBhuniya et al. [127]

82 The formation of acylamidrazones 10 ,Table 2 . 10

 8210210 10', similarly to the reaction with hydroxylamine, proceeded via pyrrolidine ring opening by hydrazide of amino acid and gave products 10a,b and 10'b in very good yields as analytically pure powders. However, we did not succeed to increase the yield of acylamidrazone 10'a above 50%. Comparison of the reactivity of our starting material and known previously described acylamidrazone precursors towards the corresponding hydrazide nucleophiles allowed us to compare them with highly reactive epoxy imidates, the precursors of epoxy acylamidrazones.126 Therefore, we have found a new approach towards a straightforward and mild synthesis of N-acylamidrazones.The presence of two sets of chemical shifts in the 1 H and13 C NMR spectra of the products in DMSO-d 6 and duplication of signals in IR spectra demonstrated the existence of (Z)/(E) amide isomerism (see Chapter 3). Following literature analogies, the cyclization of N-acylamidrazones 10, 10' (a,b) into the corresponding 1,2,4-triazoles was attempted in different solvents (toluene, CH 3 CN, THF, 1,4-dioxane, MeOH) at elevated temperatures (60 -150 °C). However, we were able to convert only the pyrazine-based acylamidrazones 10'a,b while in case of the pyridine counterparts the reaction lead to the pyrrolopyridines 6a,b and conversion into the target 1,2,4-triazoles did not exceed 10% under any of the tested conditions (Table 2.10). The conventional cyclization of acylamidrazones 10'a,b in toluene at 110 °C for 18 h furnished triazoles 11'a,b in 45 and 64% yield respectively. Alternatively, microwave assisted reaction of 10'a,b and cat. HOAc in CH 3 CN offered several advantages: reduced reaction time up to 20 min and increased yields up to 87%. Synthesis of the target amino acid-derived 1,2,4-triazoles

Figure 2 . 9

 29 Figure 2.9 Comparison of 12, 12' with a azapeptide and a aminoxy peptide

Scheme 2 . 52

 252 Scheme 2.52 Summary of all target compounds obtained

5' c ,Figure 3 . 1

 c31 Figure 3.1 Correlation in pyridine derivatives 5c, 7c-9c

  Additionally, the comparison of the α proton chemical shifts revealed unclear at first sight, downfield shift of the H  signal in cis 5'c ( = 4.80 ppm) compare to trans isomer H  ( = 4.64 ppm) that is probably a result of a deshielding effect by the ring current of neighboring pyrazine cycle (Table3.2). The upfield shift for 5c, 7c-9c, 7'c-9'c can be a result of shielded H  due to the syn position to the carbonyl oxygen of the ester group or due to the shielding effect by the pyrazine ring (Figure3.2, structure I).

Figure 3 . 2

 32 Figure 3.2 Trans/cis conformational states of proline-containing derivatives 5'c, 7'c, 8'c and 9'c

  anglОs (N…C=O) arО lОss of this valuО that НОМrОasОs the probability of the n  * interraction between the nitrogen lone pair and the amide carbonyl, whereas thО mОan θ 2 angles (O…C=O) allow the attack.The trans conformation of 9'c is stabilized by the plausible H-bonding of Phe NHBoc and proline carbonyl, although an n  * interaction may also contribute to its stabilization. Furthermore we investigated the impact of temperature (in the range 25 -90° C) on the trans/cis equilibrium constant (K t/c ). The K t/c values were measured by determining the relative concentrations of each isomer by integrating of all well-resolved proton signals in 1 H NMR spectrum in DMSO-d 6 at each temperature. The results arО illustratОН in Van't Hoff analysis (Figure 3.3, Table

Figure 3 . 3

 33 Figure 3.3 Van't Hoff plots for 5c, 5'c, 7c-9c, 7'c-9'c in DMSO-d 6

Figure 3 . 4

 34 Figure 3.4 Temperature dependence of 1 H NMR chemical shifts of 5c in DMSO-d 6

pseudotripeptide 7 '

 7 d was obtained by combining 1 H NMR and FT-IR absorption Unfortunately, it has been difficult to perform NOESY or ROESY

Figure 3 . 5

 35 Figure 3.5 FT-IR spectra of pseudotripeptide 7'd in CHCl 3 (10 mM)

Figure 3 . 6

 36 Figure 3.6 IR spectrum (C=O deconvoluted bands) of AOPzPhe (7'b) and AOPzPro (7'c) recorded in CHCl 3 (10 mM)

  H NMR experiments in solvent mixtures, which showed that two sets of signals are present at every solvent mixture ratio (Figure 3.7).

Figure 3 . 7

 37 Figure 3.7 Effect of varying ratios of solvent in the mixture of CDCl 3 and DMSO-d 6 on the chemical shifts of the NH proton of 7'd (3 mM)

  Molecular dynamics calculations on the molecule 7'd and on the simplified pseudodipeptide analogues AOPzPro (7'c) and AOPzPhe (7'b) were run in CHCl 3 and DMSO.

  DMSO in CDCl3)The superimposition of the three structures issued from simulation in CHCl 3 shows that the amidoxime-pyrazine moiety introduces rigidity of the molecules. (Figure3.8). The superimpositions of the structures two by two show the good conservation of the structure of the motif since the rmsd on the position of amidoxime-pyrazine atoms are below 0.5 Å (Figures 4 -6 in Appendix).

Figure 3 . 8 Table 3 . 7 Figure 3 . 9

 383739 Figure 3.8 Superposition of 7'd (blue) and its pseudodipeptide analogues AOPzPhe (7'b, green) and AOPzPro (7'c, pink); graphic representations were done in the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC

Figure 3 . 10 Figure 3 . 11

 310311 Figure 3.10 Solution conformations of pseudotripeptide 7'd; hydrogen bonds (orange dots) in in solution (CHCl 3 ) as obtained from molecular dynamics simulation

  bonds in CHCl 3 solution of 7'd augment extremely the trans rotamer population up to 98%. Therefore, variable temperature 1 H NMR studies of the cis/trans conformational equilibrium were performed on the synthesized tripeptide 7'd. The effects of temperature (in the range 25 -90 °C) on the values of K t/c are illustrated by Van't Hoff plots in Figure 3.12. The corresponding results were compared to those obtained for the simple Procontaining amidoxime 7'c.

Figure 3 .

 3 Figure 3.12 Van't Hoff plots for 7'd in D 2 O and DMSO-d 6

  3), the tripeptide 7'd shows negative slope in both D 2 O and DMSO-d 6 (Figure 3.12).

  , 3.12). Hence, C-terminal proline substitution with the phenylalanine amino acid has significant influence on the cis/trans equilibrium and thermodynamics of 7'd.The free energy of activation (ΔG ‡ ) was determined to be 75.3 kJ/mol at the coalescence temperature 70°C in D 2 O (Table3.8, Figure3.13). The rate of interconversion at T c = 70 °C was found to be 24.4 s -1 (the separation of the OMe group signals, at δ 3.78 ( = 1135 Hz) and 3.74 ppm ( = 1124 Hz), Δ = 11 Hz) and the half-life, t 1/2 = 63×10 -3 s.

Figure 3 . 12

 312 Figure 3.12 Temperature dependence of 1 H NMR chemical shifts of 7'd in D 2 O

Figure 3 . 14 4 . Stuctural analysis of esterified amidoximes 8 , 8 ' and oxadiazoles 9, 9 ' 4 . 1 Figure 3 .

 3144889413 Figure 3.14 In vitro cytocompatibility of the three different pyrazine amidoxime derivatives (AOPzPhe (7'b), AOPzPro (7'c) and AOPzProPhe (7'd)) on smooth muscle cell line (A-10) compared to control cells (culture medium). A-10 cells were treated with the indicated concentrations of pyrazine amidoxime derivatives for 24 h at 37 °C. Viability was estimated

Figure 3 .Figure 3 . 17

 3317 Figure 3.16 NH stretching vibrations of oxadiazole derivatives 9, 9' (a,b) in CHCl 3 (10 mM)It may be due to involvement of the NH amide protons and the pyrazine moiety in an NH…π intОraМtion (Figure3.17).62,63

Figure 3 . 18

 318 Figure 3.18 Effects of varying ratios of solvent in the mixture of CDCl 3 /DMSO-d 6 on the chemical shifts of the NH protons of 9'a,b (3 mM)

4. 2 Figure 3 .

 23 Figure 3.19 NH stretching vibrations of proline derivatives 8c, 8'c and 9c, 9'c in CHCl 3 (10 mM)The investigation of the CO-stretching bands in CHCl 3 did not give us to separate the Hbonded CO band presumably due to the low intensity and/or overlapping of some bands (Figure3.20). However, we supposed a weak interaction of the NH with amide carbonyl as CO ester should be oriented outwarН to form C=O…C=O an n  * interaction similarly to 7'c and 8'c (Table3.4).

Figure 3 .Figure 3 . 21 ). Table 3 .Figure 3 .Figure 3 . 22 5 . 10 '

 332133322510 Figure 3.20 IR spectrum (C=O deconvoluted bands) of 9'c

Scheme 3 . 1 Figure 3 .Figure 3 . 24

 313324 Scheme 3.1 Possible isomerism in N-acylamidrazones 10 (X = CH) and 10' (X = N) Two sets of signals were observed in the 1 H NMR spectra for most of the protons of compounds 10, 10' indicating the presence of only two forms. We abandoned plausible tautomeric forms A and B, as two singlets due to NH 2 signals were observed in the 1 H NMR spectra corresponding to each isomer. A COSY and NOESY experiments were performed for assignment of two populations. A NOE peak between the =N-NHand the NH 2 protons of compound 10b was observed for each conformation which indicated the presence of only Z isomers around C=N double bond and supported the occurrence of Z and E rotamers due to amide-type isomerism (Figure 3.23).

6 Scheme 3 . 2 Figure 3 . 25

 632325 Scheme 3.2 Suggested mechanism of intramolecular cyclization in non hydrogen bonding solvents for pyridine-based compounds 10a,b

Figure 3 .

 3 Figure 3.26 FT-IR spectra of 12a in CHCl 3 (10 mM)

Figure 3 .

 3 Figure 3.27 FT-IR spectra of 12b in CHCl 3(10 mM)

Figure 3 .

 3 Figure 3.28 FT-IR spectra of 12'a, 12'b in CHCl 3 (10 mM)

Figure 3 . 29

 329 Figure 3.29 Effects of varying ratios of solvent in the mixture of CDCl 3 /DMSO-d 6 on the chemical shifts of the NH protons of 12a,b and 12'a,b (3 mM)

Figure 3 .Figure 3 . 31

 3331 Figure 3.30 NOESY spectrum of 12b in CHCl 3

  latter showed amide-type isomerizm in pyridine derivatives 10a,b and Z/E isomerization around C=N bond in pyrazine acylamidrazones 10'a,b. Furthermore, testing the effect of solvent polarity on the E/Z isomeric ratio, a partial decomposition of the pyridine derivatives 10a,b after adding CDCl 3 was observed. The instant formation of the pyrrolopyridines 6a,b was detected by LCMS analysis and NMR spectroscopy. This was explained by stabilized effect of the hydrogen bond between the NH-amide proton and the nitrogen of the pyrazine ring in non hydrogen bonding solvents. was found that the hydrazide-modified peptidomimetics 12,12' adopt the turn structure stabilized by the hydrogen bond between the C=O amide and the NH-hydrazide forming C 10 -pseudocycle.

Scheme 4 . 1

 41 Scheme 4.1 Chemical reactions involved in the measurement of NO 2 -using the Griess Reagent System

Figure 4 . 1

 41 Figure 4.1 Nitrite Standard reference curve

[ 2 - 2 OH 13 С

 2213 cyanopyridine-3-yl]carbonyl substituted amino acid 5 and/or the methyl ester of a (2S)-2-(7-imino-5-oxo-5,6-dihydro-6H-pyrrolo[3,4b]pyridine-6-yl)alkanoic acid 6 (2 mmol) in MeOH (15 mL) was added Et 3 N (0.56 mL, 4 mmol) followed by hydroxylamine hydrochloride (0.278 mg, 4 mmol), and the reaction was stirred at r.t. overnight. The mixture was then concentrated to dryness. The residue was dissolved in EtOAc (100 mL) and the solution was washed with H 2 O (20 mL). The organic layer was dried with MgSO 4 and the solvent was removed in vacuo. The residue was purified by column chromatography. Purification by flash column chromatography (CH 2 Cl 2 /EtOH, 91:9) gave 7a as a white solid; yield: 0.362 g (68%); mp 74-75 °С. IR (KBr): 3470, 3322, 3067, 2918, 2849, 1739, 1731, 1641, 1582, 1565, 1547, 1538, 1454, 1216 cm -1 . 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.60 (d, J = 4.5 Hz, 1H, H Ar ), 7.92 (d, J = 7.8 Hz, 1H, H Ar ), 7.46 (d, J = 7.2 Hz, 1H, NH), 7.36 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 5.47 (s, 2H, NH 2 ), 4.75 (m, 1H, CH), 3.75 (s, 3H, OCH 3 ), 1.48 (d, J = 7.2 Hz, 3H, CH 3 ). NMR (75 MHz, CDCl 3 , 25 °C): δ = 174.1, 168.

2 OH 13 С

 213 Ester 7b was obtained as a white solid of analytical purity without further purification; yield: 0.527 g (77%); mp 17β °С. IR (KBr): 3359, 3201, 3020, 2886, 1723, 1672, 1644, 1586, 1575, 1537, 1276, 934 cm -1 . 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.60 (d, J = 4.8 Hz, 1H, H Ar ), 7.88 (d, J = 7.8 Hz, 1H, H Ar ), 7.63 (d, J = 7.5 Hz, 1H, NH), 7.37 -7.19 (m, 6H, 6×H Ar ), 5.49 (br s, 2H, NH 2 ), 5.11 (dd, J = 13.5, 6.0 Hz, 1H, CH), 3.72 (s, 3H, OCH 3 ), 3.24 -3.21 (m, 2H, CH 2 ). NMR (100 MHz, DMSO-d 6 +CCl 4 , 1:1, 25 °C): δ = 171.9, 167.

  (300 MHz, DMSO): δ = 11.63 (s, 1H, OH), 11.36 (s, 1H, NH), 8.84 (d, J = 4.9 Hz, 1H, H Ar ), 8.19 (dd, J = 7.8, 1.1 Hz, 1H, H Ar ), 7.61 (dd, J = 7.7, 4.9 Hz, 1H, H Ar ).

13 C

 13 NMR (75 MHz, DMSO): δ = 165.39, 154.16, 153.16, 143.98, 131.65, 125.52, 125.06.

Compounds 7'a- d ; 2 OH

 d2 General Procedure: To a solution of (2S)-N-(3-cyanopyrazin-2-yl)carbonyl-substituted amino acids 5' and/or methyl esters of methyl (2S)-2-[(5E)-5-imino-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6yl]alkanoic acids 6' (2 mmol) in MeOH (15 mL) was added Et 3 N (0.56 mL, 4 mmol) followed by hydroxylamine hydrochloride (0.278 g, 4 mmol) and the reaction was stirred at r.t. overnight. The mixture was then evaporated to dryness. The residue was dissolved in ethyl acetate (100 mL) and washed with H 2 O (20 mL). The organic layer was dried with MgSO 4 and the solvent was removed in vacuo. The residue was purified by column chromatography. Purification by flash column chromatography (CH 2 Cl 2 /MeOH, 95:5) gave 7'a as a paleyellow oil (0.449 g, 84%). IRμ max = 3462, 3328, 3066, 2995, 2954, 2847, 1733, 1646, 1537, 1454, 1435, 1340, 1215, 1161 cm -1 . 1 H NMR (300 MHz, CDCl 3 )μ δ = 8.57 (Н, J = 2.3 Hz, 1H, H Ar ), 8.49 (d, J = 2.3 Hz, 1H, H Ar ), 7.71 (d, J = 7.5 Hz, 1H, NH), 5.45 (br s, 2H, NH 2 ), 4.70 (pseudo-p, J = 7.2 Hz, 1H, CH), 3.70 (s, 3H, OCH 3 ), 1.45 (d, J = 7.2 Hz, 3H, CH 3 ).

13 C 2 OH 13 C 2 OH

 132132 NMR (75 MHz, CDCl 3 )μ δ = 17γ.8, 165.5, 150.4, 146.7, 145.0, 145.0, 14γ.5, 5γ.β, 49.β, 18.5. ESI calculated for C 10 H 13 KN 5 O 4 [M+K] + m/z = 306.0603, found 306.0599. Purification by flash column chromatography (CH 2 Cl 2 /MeOH, 93:7) gave 7'b as a lightyellow solid (0.432 g, 63%); mp 154-157 °C. IRμ max = 3474, 3350, 3060, 2926, 1717, 1675, 1645, 1547, 1435, 1161, 971 cm -1 .1 H NMR (300 MHz, DMSO-d 6 )μ δ = 9.97 (s, 1H, OH), 8.79 (Н, J = 7.5 Hz, 1H, NH), 8.71 (d, J = 2.3 Hz, 1H, H Ar ), 8.64 (d, J = 2.3 Hz, 1H, H Ar ), 7.32-7.20 (m, 5H, 5H Ar ), 5.82 (s, 2H, NH 2 ), 4.70 (dt, J = 7.2, 6.8 Hz, 1H, CH), 3.57 (s, 3H, OCH 3 ), 3.19 -2.99 (m, 2H, CH 2 ). NMR (75 MHz, DMSO-d 6 )μ δ = 171.γ, 165.5, 148.6, 147.5, 14γ.9, 14γ.5, 14β.6, 1γ6.9, 129.1, 128.2, 126.5, 53.8, 51.7, 37.1. ESI calculated for C 16 H 18 N 5 O 4 [M+H] + m/z = 344.1353, found 344.1353. Purification by flash column chromatography (CH 2 Cl 2 /MeOH, 93:7) gave 7'c as a white solid (0.545 g, 93%); mp 135-138 °C.

  Purification by flash column chromatography (CH 2 Cl 2 /MeOH, 95:5) gave 7'd as a lightyellow solid (0.669 g, 76%); mp 59-62 °C. IR (ATR): max = 3517, 3402, 3319 (NH, OH), 1743, 1722, 1679, 1670, 1656, 1653 (C=O, C=N) cm -1 . IRμ max = 3483, 3311, 3064, 2960, 2924, 2853, 1739, 1642, 1528, 1454, 1409, 1259, 1066, 798, 700 cm -1 . 1 H NMR (300 MHz, CDCl 3 , 25 °C, K t/c = 1γ.γ0)μ δ = 9.4β (s, 1H, OH), 8.55 anН 8.49 (Н, J = 2.4 Hz, 1H, H trans/cis Ar ), 8.52 and 8.41 (d, J = 2.4 Hz, 1H, H trans/cis Ar ), 7.78 (d, J = 9.2 Hz, 1H, NH), 7.27 -6.97 (m, 5H, 5H trans/cis Ph ), 5.68 and 5.42 (2×s, 2H, NH 2 trans , NH 2 cis ), 5.10 and 4.69 (td, m, J = 9.4, 6.5 Hz, 1H, CH

Synthesis of Compounds 8 , 8 'N

 88 (a-c); General Procedure: Amidoxime 7, 7' (a-c; 1 mmol) was dissolved in anhydrous acetonitrile (20 mL). After addition of N-t-butoxycarbonyl-L-valine(phenylalanine) (1.3 mmol), 4-DMAP (0.011 g, 0.09 mmol) and DCC (0.268 g, 1.3 mmol) the solution was stirred for 24h at r.t.. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography.Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 3:7) gave 8a as a white solid (0.298 g, 64%); mp 139 -141 °C; IR (CHCl 3 ): max = 3438, 3408, 1767, 1741, 1712, 1670, 1651 cm -1 . 1 H NMR (300 MHz, CDCl 3 ): δ = 8.64 (dd, J = 4.7, 1.5 Hz, 1H, H Ar ), 8.00 (dd, J = 7.8, 1.5 Hz, 1H, H Ar ), 7.58 (d, J = 6.8 Hz, 1H, NH), 7.44 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 5.85 (s, 2H, NH 2 ), 5.08 (d, J = 8.6 Hz, 1H, NHBoc), 4.79 (p, J = 7.1 Hz, 1H, CHCH 3 ), 4.26 (dd, J = 8.3, 6.1 Hz, 1H, CHCH(CH 3 ) 2 ), 3.74 (s, 3H, OCH 3 ), 2.14 (dq, J = 13.4, 6.7 Hz, 1H, CH(CH 3 ) 2 ), 1.52 (d, J = 7.2 Hz, 3H, CH 3 ), 1.44 (s, 9H, (CH 3 ) 3 ), 0.99 (dd, J = 11.5, 6.8 Hz, 6H, (CH 3 ) 2 ).

  column chromatography (CH 2 Cl 2 /AcOEt, 3:7) gave 8b as a light-yellow solid (0.492 g, 91%); mp 149 -151 °C; IR (CHCl 3 )μ max = 3435, 3408, 1766, 1743, 1711, 1670, 1648 cm -1 .

  column chromatography (CH 2 Cl 2 /MeOH, 95:5) gave 8c as a light-yellow solid (0.523 g, 97%); mp 84 -87 °C; IR (CHCl 3 )μ max = 3437, 3396, 1768, 1743, 1708, 1650, 1634 cm -1 .

13 C

 13 column chromatography (CH 2 Cl 2 /AcOEt, 3:7) gave 8'b as a white solid (0.363 g, 67%); mp 86-89 °C. IR: max = 3326, 2969, 2877, 2856, 1738, 1688, 1681, 1643, 1514, 1367, 1171, 898, 670 cm -1 . IR (CHCl 3 ): max = 3438, 3408, 1766, 1745, 1712, 1686, 1645 cm -1 . 1 H NMR (300 MHz, CDCl 3 ): δ = 8.70 (d, J = 2.0 Hz, 1H, H Ar ), 8.61 (d, J = 2.0 Hz, 1H, H Ar ), 7.72 (d, J = 7.4 Hz, 1H, NH), 7.30 -7.18 (m, 5H, 5H Ar ), 5.70 (s, 2H, NH 2 ), 5.15 (d, J = 8.7 Hz, 1H, NH), 5.05 (m, 1H, CH), 4.38 -4.25 (m, 1H, CH), 3.73 (s, 3H, OCH 3 ), 3.26 (d, J = 5.6 Hz, 2H, CH 2 ), 2.16 (m, 1H, CH), 1.45 (s, 9H, (CH 3 ) 3 ), 1.01 and 0.97 (2×d, J = 6.8 Hz, 6H, (CH 3 ) 2 ). NMR (75 MHz, CDCl 3 ): δ = 172.0, 170.1, 163.9, 156.4, 155.7, 146.9, 145.6, 145.0, 144.5, 136.4, 130.1, 129.2, 127.7, 80.7, 58.6, 54.5, 53.0, 38.5, 32.0, 29.0, 19.7, 18.5. ESI calculated for C 26 H 34 KN 6 O 7 [M+K] + m/z = 581.2117, found 581.2121. Methyl (2S)-1-({3-[(Z,5S)-1-amino-5-benzyl-9,9-dimethyl-4,7-dioxo-3,8-dioxa-2,6diazadec-1-en-1-yl]pyrazin-2-yl}carbonyl)pyrrolidine-2-carboxylate (8'c) column chromatography (CH 2 Cl 2 /AcOEt, 3:7) gave 8'c as a light-yellow solid (0.

  column chromatography (CH 2 Cl 2 /AcOEt, 1:1) gave 9c as light-yellow oil (0.060 g, 58%). IR (CHCl 3 )μ max = 3436, 3358, 1743, 1714, 1640 cm -1 .

N

  Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 4:6) gave 9'b as a colorless oil (0.100 g, 95%).

1 H 13 C

 113 NMR (300 MHz, CDCl 3 )μ δ = 7.5β (s, 1H, NHNH 2 ), 7.32 -7.16 (m, 5H, H Ph ), 5.19 (d, 1H, J = 8.1 Hz, NHBoc), 4.32 (dd, 1H, J = 15.0, 7.3 Hz, CH), 3.65 (br, 2H, NH 2 ), 3.10 -2.97 (m, 2H, CH 2 ), 1.34 (s, 9H, (CH 3 ) 3 ). NMR (75 MHz, CDCl 3 )μ δ = 17β.6, 156.8, 1γ7.1, 1β9.9, 1β9.4, 1β7.8, 81.β, 55.4, γ9.β, 29.0. Synthesis of Compounds 10, 10' (a,b); General Procedure: Methanolic (25 mL) suspension of the methyl ester of a (2S)-2-(imino-oxo-dihydro-6Hpyrrolo[3,4-b] (hetero)aryl)alkanoic acid 6, 6' (a,b; 1 mmol) and Boc-(S)Phe-NHNH 2 (1.1 mmol) was stirred overnight at r.t. Then, the precipitate was filtered off, washed with cold methanol (10 mL) and dried in vacuo.

  Compounds 11'a,b; General Procedure: A 5 mL CEM microwave process vessel was charged with 10'a,b (0.2 mmol) in CH 3 CN (5 mL) with addition of cat.HOAc (2%) and the vessel was capped. The mixture was stirred and heated under microwave conditions (150 W) at 110 °C for 20 min. The solution was then concentrated under vacuum and purified by NP-HPLC with a gradient of MeOH (1-5% v/v) in CHCl 3 to yield 1,2,4-triazoles 11'a,b. solid; yield: 0.084 g (85%); mp 177-180 °C.

CHCl 3 and

 3 MeOH as eluents. NP-HPLC (CHCl 3 /MeOH, 95:5) gave 12a as a white solid (0.183 g, 44%); mp 71-74 °C.

  NP-HPLC (CHCl 3 /MeOH, 95:5) gave 12b as a white solid (0.311 g, 60%); mp 102-105 °C.

  NP-HPLC (CHCl 3 /MeOH, 95:5) gave 12'b as a colorless solid (0.358 g, 69%); mp 107-110 °C.

AppendixFigure 1 :Figure 2 :Figure 3 :Figure 4 :

 1234 Figure 1: NOESY NMR spectrum of 5c

Figure 5 :

 5 Figure 5: Superposition of AOPzProPhe (in blue) and AOPzPro (in pink) on backbone atoms from H of amidoxime to N of proline residue next to the pyrazine ring. The rmsd on these 7 atoms is 0.411 Å.

Figure 6 :

 6 Figure 6: Superposition of AOPzPhe (in green) and AOPzPro (in pink) on backbone atoms from H of amidoxime to C of carbonyl next to the pyrazine ring. The rmsd on these 6 atoms is 0.209 Å.

Figure 7 :

 7 Figure 7: Structure of AOPzPro obtained from molecular dynamics simulationin explicit solvent boxes CHCl 3 (a) and DMSO (b)

2 P450Schéma 1 XSchéma 2 Schéma 3

 2123 OxyНation Н'amiНoximО en présence de cytochromes P450 de microsomes de foie Résultats Plan de synthèse La voie de synthèse envisagée pour accéder aux différents peptidomimétiques en série pyridine et pyrazine est décrite dans le Schéma 2. = C, N; R, R' = résidus d'acides aminés (AA) Stratégie de synthèse des peptidomimetiques ciblés Suivant la série étudiée, cette stratégie utilise les acides 2-cyanonicotinique et 3cyanopyrazine-2-carboxylique comme produits de départ. La substitution en positions 2,3 de МОs МyМlОs azotОs présОntО l'avantagО НО fournir une rigidité conformationnelle supplémОntairО, НonМ Н'inНuirО unО struМturО préférОntiОllО. Plus partiМulièrОmОnt, la présence de la fonction amidoxime en position ortho permet de mimer un -turn rigide se retrouvant habituellement dans la structure de l'arginine, précurseur physiologique de NO. Les amidoximes correspondantes sont alors synthétisées en deux étapes pour générer ensuite les différents pseudodipeptides dans les deux séries. Synthèse des acides 2-cyanonicotinique et 3-cyanopyrazine-2-carboxylique ConМОrnant la sériО pyrazinО, lО Мomposé НО Нépart, р savoir l'aМiНО 3-cyanopyrazine-2-carboxylique 4' a été synthétisé р partir НО l'aМiНО γ-carbamoylpyrazine-2-carboxylique 2' commercial. Après traitement par du chloroformiate de méthyle et hydrolyse sélective de la fonМtion ОstОr par unО solution aquОusО НО souНО 1M, l'aМiНО Нésiré 4' est généré avec un bon rendement global (71%).

Schéma 4

 4 d'arginine possédant un motif amidinohétéroaroyle dans une chaîne peptidique, nous avons élaboré une voie de synthèse de molécules pseudopeptidiques (acide nicotinique couplé à un acide aminé) et où la fonction amidoxime est portée par le cycle pyridinique et ceci en tant que précurseurs des pseudopeptides correspondants contenant de l'amidine. Cette stratégie permet Н'aММéНОr aux pОptiНomimétiquОs Оn sériО pyridinique et a été envisagée de la façon suivante: en premier lieu, par couplage de l'acide 2-cyanonicotinique 4 avec des esters méthyliques d'acides L-α-aminés puis, par conversion du résidu cyano en groupement amidoxime. Pour étudier la portée de cette nouvelle stratégie, le couplage de l'acide 2-cyanonicotinique 4 a été testé avec plusieurs esters méthyliques d'acides L-α-aminés (tels que Ala, Phe, Pro, Gly, Val, Leu, Trp et CysTr) commerciaux (Schéma 4). Le chlorhydrate de N-éthyl-N'-(3-diméthylaminopropyl)carbodiimide (EDCI) a été utilisé comme agОnt Н'aМtivation Оn présОnМО НО 1-hydroxybenzotriazole (HOBt) et de triéthylamine. Synthèse des esters méthyliques des (2S)-N-[2-(N'-hydroxycarbamimidoyl)pyridin-3-yl]carbonyles substitués par des aminoacides 7a-

Figure 1 Figure 2

 12 Figure 1 Structure moléculaire du compose 6d (radiocristallographie)

Figure 3 4 'Schéma 5 5 'et 6 'Tableau 2

 345562 Figure 3 Structures moléculaires des composés 7b (étude par diffraction des rayons X)

Figure 4 Schéma 6

 46 Figure 4 Structure moléculaire du (2S)-2-({[3-(N'-hydroxycarbamimidoyl)pyrazin-2yl]carbonyl}amino)-3-phénylpropanoate de méthyle 7'b (étude par diffraction des rayons X)

Figure 5

 5 Figure 5 Structure moléculaire de la (7Z)-7-(hydroxyimino)-6,7-dihydro-5H-pyrrolo[3,4b]pyrazin-5-oneII (en accord avec les résultats par diffraction des rayons X)

Schéma 7 Tableau 4

 74 Synthèse des pseudotripeptides 7'd L'analysО des propriétés structurales du motif pyridine amidoxime ajouté en Nterminal du pseudo peptide a été faite afin de déterminer son utilisation pour de futures applications. La spectroscopie infrarouge (FT-IR) combinée à la RMN du proton ont permis Н'apportОr des réponses quant à la conformation adoptée par 7'd en solution. Les expériences FT-IR de 7'd à 25°C montrent une bande large à 3319 cm -1 synonyme qu'au moins proton NH et/ou OH est impliqué dans une liaison hydrogène. Dans la région de carbonyle six bandes sont présentes (Tableau4). Région Н'adsorption des C=O de AOPzPhe (7'b), AOPzPro (7'c) et AOPzProPhe (7'd) dans le chloroforme à température ambiante (10 mM)

Figure 7

 7 Figure 7 Variation des déplacements chimiques du proton NH de 7'd en fonction du ratio CDCl 3 /DMSO-d 6 (3 mM)

Figure 8 Tableau 5

 85 Figure 8 Conformation de pseudopeptide 7'd issue de calculs de dynamique moléculaire en solvant explicite; les liaisons hydrogène sont représentées en orange

Figure 9

 9 Figure 9 Diagramme de Ramachandran du résidupseudotripeptide 7'd Enfin, une étude concernant la toxicité cellulaire des composés a été réalisée afin de vérifier leur potentielle utilisation comme agents thérapeutiques (Figure 10).

Figure 10 Tableau 6

 106 Figure 10 Etude de cytocompatibilité des trois dérivés pyrazineamidoxime (AOPzPhe (7'b), AOPzPro (7'c) et AOPzProPhe (7'd)) réalisée in vitro sur la lignée cellulaire de muscle lisse (A-10) par rapport aux cellules témoins (culture moyen). Les cellules A-10 ont été traitées avec les concentrations indiquées de dérivés 7 pendant 24 h à 37° C. La viabilité a été estimée par le test MTT.

Figure 11 6 , 6 'Schéma 8 Schéma 9

 116689 Figure 11 Interaction intramoléculaire supposée dans le dérivé oxadiazole 9'c

Figure 13

 13 Figure 13 Effets NOE observés pour les différents isomères 10b

6 Schéma 10

 610 Mécanisme suggéré de cyclisation intramoléculaire des composés en série pyridine 10a, b dans des solvants non donneurs de liaison hydrogène Cette cyclisation intramoléculaire est observée dans moins de 5% des analogues pyrazine 10' et l'oММurrОnМО du conformère Z diminue lorsque la polarité du solvant augmente. L'analysО НО МОs résultats montre clairement que dans les solvants polaires tels que le DMSO, tous les composés présentent une structure stabilisée par une liaison hydrogène. Ceci suggèrО qu'unО liaison hyНrogènО ОntrО lО NH amiНО Оt l'atomО Н'azotО Нu noyau pyrazinО pourrait stabiliser les dérivés pyrazine et peut exister également dans des solvants non favorablОs р l'établissОmОnt НОs liaisons hyНrogènО, favorisant la МyМlisation intramolОМulairО en précurseur 6'. Si l'on МonsiНèrО lОs conformations de plus basses énergies pour les composés acylamidrazones 10', calculées à partir de la fonction densité en chimie quantique, on remarque que dans 10'a et 10'b, unО liaison hyНrogènО NH…N Оst possiblО avОМ НОs distances interatomiques de 2,41 Å Оt β,γ9 Å Оt НОs anglОs NH…N НО 99° Оt 100° respectivement (Figure 14).

Figure 14

 14 Figure 14 Formation de la liaison hydrogène dans les conformations de plus basses énergies 10'a,b

Figure 15 Schéma 12 Tableau 11

 151211 Figure 15 Structure des peptidomimétiques 12 et 12' La synthèse de ces molécules a été réalisée par condensation de la fonction amidoxime avec l'hydrazide de la phénylalaninО par ajout Н'unО liaison МarbonylО supplémentaire. Ce couplage a été réalisé à partir des amidoximes 7, 7' (a,b) en série pyridine ou pyrazine traitées par du triphosgène et l'hydrazide de la phénylalanine préalablement obtenu en présence de diisopropyléthylamine dans le dichlorométhane (Schéma 12). Les dérivés de la phéylalanine 12b et 12'b ont été isolés après purification par CLHP préparative avec des rendements de 60 et 69% de rendements. Dans le cas des dérivés d'alanine 12a et 12'a, les rendements de réaction n'ont pas НépassОr 44%: la réactivité plus faible de l'amidoxime correspondante visà-vis du triphosgène en est la cause et a conduit à une quantité accrue de produit secondaire dérivé de la condensation de l'hydrazide de la phénylalanine avec le triphosgène.

Figure 16

 16 Figure 16 Représentation des interactions NOE observées pour le composé 12b dans CDCl 3 Par conséquent, les mimes peptidiques modifiés avec un motif hydrazide 12,12' adoptent un coude stabilisé par une liaison hydrogène entre le C=O amide et le NH hydrazide formant ainsi un pseudocycle-C10. Un autre argument en faveur Н'unО liaison hydrogène faible NH…N est la faible accessibilité au solvant décrite dans les expériences de mélange de solvant à différentes concentration (10% DMSO-d 6 ) (Figure 17).

Figure 17 12 'Schéma 13

 171213 Figure 17 Structures des mimes peptidiques -hydrazides 12, 12'

  peptidiques. Différentes amidoximes à motif pyridinique (ou pyrazinique) disubstitué ont, dans un premier temps, été synthétisées. Dans un second temps, cette fonction amidoxime qui a été estérifiée par des acides aminés et des hydrazides a permis ensuite diverses transformations: en N-acylamidrazones, 1,2,4-oxadiazoles et 1,2,4-triazoles (liens pseudopeptidiques). L'intérêt НО МО travail Оst МlairОmОnt l'obtОntion Н'un largО évОntail НО nouvОllОs struМturОs pОptiНomimétiquОs р partir Н'un mêmО préМursОur. Il a été mis en évidence que l'acide 2(3)-cyanohétéroaromatique réagit avec des esters méthyliques de différents acides aminés naturels pour donner deux types d'esters d'acides aminés, un sous sa forme ouverte (dérivé cyano) et un sous sa forme fermée, pyrrolo-pyridine (ou pyrazine) qui génèrent des amidoximes à chaîne ouverte par action de l'hydroxylamine. De plus, on a trouvé que ces mêmes précurseurs cycliques présentaient un intérêt majeur dans la synthèse directe et douce de N-acylamidrazones via l'ouverture du cycle pyrrolidine par l'hydrazide d'un acide aminé. La fermeture en un cycle 1,2,4-oxadiazole ou 1,2,4-triazole a été réalisée sous irradiation par micro-ondes avec des conditions optimisées et des temps de réaction réduits. Une première condensation d'amidoximes avec de l'hydrazide d'un acide aminé a permis d'obtenir des composés coudés hybrides avec une liaison de type amidoximecarbonyle-hyНraziНО. L'étuНО struМturalО a prouvé quО МОs pОptidomimétiques modifiés par cet hyНraziНО aНoptaiОnt la struМturО Н'un МouНО stabilisé par unО liaison hyНrogènО forméО ОntrО lО C=O НО l'amiНО Оt lО NH НО l'hyНraziНО formant ainsi un psОuНoМyМlО en C 10 . L'analysО conformationnelle réalisée sur les dérivés contenant la proline a démontré la tОnНanМО généralО qu'a МОt aМiНО aminé partiМuliОr р augmОntОr la proportion en isomère trans. L'amidoxime obtenue en série pyrazine et couplée au dipeptide Pro-Phe semble attrayant afin НО l'utilisОr МommО psОuНotripОptide modèle pour l'étude conformationnelle de l'isomérisation cis/trans de la liaison prolylamide. De plus, les études conformationnelles des composés synthétisés ont confirmé que les motifs hétérocycliques pyridine et pyrazine présОntОnt l'intérêt Н'augmОnter la rigidité et pus particulièrement le noyau pyrazine qui pourrait avoir un effet plus fort sur la stabilisation de la conformation. Les amidoximes synthétisées ont ensuite été testés afin de mesurer leur capacité de libération d'oxyde nitrique démontrant ainsi la libération de NO en concentration suffisante pour НОs ОffОts pharmaМologiquОs (≥ 1 M). En Мomparaison avОМ la moléМulО НО référОnМО, la 4-chlorobenzamidoxime, certains composés présentent une capacité de relargage de NO assez intéressante. Cependant, aucune rationalisation satisfaisante en terme de relation structure-aМtivité n'a pu êtrО ОnМorО établiО avОМ МОs prОmiОrs résultats. En perspective, les composés les plus prometteurs seront soumis à d'autres tests in vivo. Les amidoximes pourraient également être testées pour leur capacité à être réduites in vitro/in vivo en amidines pharmaМologiquОmОnt aМtivОs, mîmОs НО l'argininО.NEW VARIETY OF PYRIDINE AND PYRAZINE-BASED ARGININE MIMICS: SYNTHESIS, STRUCTURAL STUDY AND PRELIMINARY BIOLOGICAL EVALUATIONAmino acids, mimetics, synthesis, structural study, NO donorsThis work describes the synthesis, structural study and preliminary biological evaluation of new variety of pyridine and pyrazine-based arginine mimics.

Table 2 .

 2

	Ester	R	H 2 , d (J=4.8 Hz)	, ppm (J, Hz) H 3 , dd (J, Hz) H 4 , d (J, Hz)	NH	Yield, (%) a
	5a 6a	Me (Ala)	8.79 8.86	7.61 (8.1, 4.8) 7.57 (8.1, 4.8)	8.15 (8.1) 8.14 (8.1)	7.15 b 9.34	15 58
	6b	Bn (Phe)	8.81	7.51 (7.8, 4.8)	8.06 (7.8)	9.29	76
	5c	Side chain of Pro-				-	87
		residue
	6d	H (Gly)	8.87	7.58 (7.8, 4.8)	8.17 (7.8)	9.33	58
	5e 6e	i-Pr (Val)	8.81 8.87	7.62 (9.0, 4.8) 7.58 (9.0, 4.8)	8.18 (9.0) 8.15 (9.0)	6.97 9.33	37 34
	5f 6f	i-Bu (Leu)	8.78 8.81	7.61 (8.1, 4.8) 7.54	8.15 (8.1) 8.09 (6.6)	7.06 c 9.37	9 76
	6g	N H (Trp)	8.69	7.36 (7.8, 4.8)	7.91 (7.8)	9.33	83
	5h	CH 2 STr (CysTr)	8.77	7.46 (7.8, 4.8)	8.04 (7.8)	7.36 -7.02	60

1 Yield and 1H NMR data for compounds 5a,e,f,h and 6a,b,d-g a Isolated yeald. b d, j = 5.7 Hz. c d, j = 6.9 Hz.

Table 2 .

 2

	Entry	[М+1]	Major peak (%)	Minor peak (%)
	5a	234	68 -96	4 -32
	6a	234	74 -100	0 -26
	6b	310	87	13
	6d	220	78 -94	6 -22

2 LC/MS data for esters 5a,e,f and 6a,b,d,e,g a a The values were determined on the basis of three LC/MS analyses for each of the compounds 5a,e and 6a,d,e,g, and one LC/MS analysis each for 5f, 6b. 87% Scheme 2.6 Synthesis of methyl

Table 2 . 3

 23 Synthesis of the methyl esters of (2S)-N-[2-(N'-hydroxycarbamimidoyl)pyridin-3-

	yl]carbonyl-substituted amino acids 7a,b,e-h
		O	R			O	R
	N N	NH CN 6 (a,b,e-h) CO 2 Me NH CO 2 Me N O R 5 (a,e,f)	NH 2 OH . HCl Et 3 N, MeOH, r.t. 67-79%	N N	NH N OH CO 2 Me CO 2 Me N N OH NH 2 7 (a,b,e-h) I O R
	5, 6		R	7	Isolated yield (%)
	5a, 6a		Me	7a		68
	6b		Bn	7b		77
	5e, 6e		i-Pr	7e		79
	5f, 6f		i-Bu	7f		70
	6g		N	7g		67
			H
	6h		CH 2 STr	7h		94

  Isolated yield. b d, J = 7.4 Hz. c d, J = 7.9 Hz. d d, J = 5.7 Hz. e Not isolated.

	N N	H	N O N 6'b CO 2 Me Ph	-	9,46	68	N	H	6b O N N CO 2 Me Ph	-	9,29	76
						2240	8,26 b	23		HN	O	CO 2 Me	2240	7,15 d	15
			5'a				N	CN 5a
					Ph								Ph
	N H	N	O	CO 2 Me	2237	8,20 c	3		HN	O	CO 2 Me	-e	-e	-e
	N	CN 5'b				N	CN 5b
	N N	H	O N N 6'a CO 2 Me	-	9,51	28	N	H	O 6a N N CO 2 Me	-	9,34	58

a

Scheme 2.21 1

  ,2,4-Oxadiazoles synthesis reported byGangloff et al.

	Gangloff et al. found that in the presence of tetrabutylammonium fluoride (TBAF) at
	room temperature alkanoyl-and aroyloxyamidines were converted to the corresponding 3,5-
	disubstituted-1,2,4-oxadiazoles (Scheme 2.21). 88
	R HO	N	NH 2	acylator, DIPEA CH 2 Cl 2 , 0 °C	R'	O	R O	N	NH 2	THF, rt Bu 4 NF	R'	N	R N O

  1,2,4-Oxadiazoles synthesis reported by Lukin and Kishore[92]

	Another base, caesium carbonate, was used by Lloyd and co-workers to promote the
	formation of 1,2,4-oxadiazole-based precursor to pyrazolodihydropyrimidines as potent and
	selective KV1.5 blockers (Scheme 2.27). 93
	Boc	N	CO 2 H	+	N	NH 2 OH	1) EDC, HOBt, DCM 2) Cs 2 CO 3 , THF 89%	Boc	N	O	N N
	Scheme 2.27 1,2,4-Oxadiazoles synthesis reported by Lloyd et al. [93]
	Mono-and bis-1,2,4-oxadiazoles were prepared in one-pot by the method reported by
	Amarasinghe et al. (Scheme 2.28).

Scheme 2.30 Microwave

  -assisted synthesis of 1,2,4-oxadiazoles reported byPorcheddu et al.

									1,2,4-oxadiazoles
	including amino acid-derived 1,2,4-oxadiazoles with 100% enantiomeric purity was
	discovered by Porcheddu et al. (Scheme 2.30). 97
							N	OH
	R 1 COOH	CDMT, NMM, THF 30 min, rt	R 1	O O	N N	N OMe OMe	NH 2 toluene, MW, R 2 160 °C, 5 min	R 2	N N O	R 1
				[97]

Scheme 2.32 Microwave

  -assisted synthesis of 1,2,4-oxadiazoles reported byWang et al.

						Method A:
						HBTU, PS-BEMP, CH 3 CN
						MW, 160 °C, 15 min
	R	O OH	+	R'	N	NH 2 OH	R'	N N O	R
						Method B:
						i) PS-PPh 3 , CCl 3 CN
						MW, 100 °C, 5 min
						ii) DIEA, THF
						MW, 150 °C, 15 min
						CDI, DMF, rt, 30 min
						2) R-C(NOH)NH 2 , rt, 12 h
				CDI, DMF
		MW, 140 °C, 5 min
	Scheme 2.31 Microwave-assisted synthesis of 1,2,4-oxadiazoles reported by Muraglia et al.
						[98]
	Wang et al. combined the use of commercially available polymer-supported reagents
	with microwave heating to prepare libraries of 1,2,4-oxadiazoles (Scheme 2.32). 99

Scheme 2.34 Ionic

  liquid-phase strategy toward1,2,4-oxadiazoles reported byDuchet et al.

								HO	CN				CN
	N [HOC 2 mim][PF 6 ] N OH	PF 6			OH Ionic Liquid Phase IL	O DCC, DMAP 2% CH 3 CN, rt, 24 h 92%	IL	O	O
							NH 2						NH 2
	NH 2 OH . HCl, KOH EtOH, 0 °C, 30 min then nitrile, reflux, 18 h	IL	O	O	N OH	RCO 2 H DCC, DMAP 6-8% CH 3 CN, rt, 48 h	IL	O	O	N O	O	R
	82%
							R
						N	O						R
	H 2 O reflux, 24 h	IL	O			N	MeONa MeOH, reflux, 24 h	IL	OH	+		N	N	O
	66-98%		O									O
												O
							[52]
								15 min
								84-98 %
	Scheme 2.33 Ultrasound-promoted synthesis of 1,2,4-oxadiazoles reported by Bretanha et al.
							[100]
	A novel ionic liquid-phase strategy toward 3,5-disubstituted 1,2,4-oxadiazoles was
	developed by Duchet et al. for application in solution-phase combinatorial synthesis (Scheme
	2.34). 52 The arylnitrile bound to the ionic liquid moiety (1-(2-hydroxyethyl)-3-methyl
	imidazolium hexafluorophosphate ([HOC 2 mim][PF 6 ])) was chosen as task-specific ionic
	liquid (TSIL). The starting ionic liquid-phase (ILP) was produced from 1-methylimidazole
	and 2-chloroethanol. The esterification of 4-cyanobenzoic acid activated with DCC was
	followed by the reaction with hydroxylamine to obtain the arylamidoxime functionalized ILP.
	Acylation of amidoximes with a series of aliphatic, aromatic and heteroaromatic carboxylic
	acids in the presence of DCC and DMAP provided the target ILP bound O-acyl amidoximes.
	The cyclization of the latter to 1,2,4-oxadiazoles grafted on the ionic liquid phase, was carried
	out in deionized water for 24 h under reflux. Complete cleavage of the compounds was
	achieved by treatment with MeONa in refluxed methanol.

  80

Table 2 .

 2

						R						R
		O	R			HN	CO 2 Me				HN	CO 2 Me
	N X	NH N NH 2	OH CO 2 Me	Boc-Val-OH DCC, DMAP, CH 3 CN, rt	X N	O N NH 2	O	O	NHBoc	MW 150 °C 25 min DCM	X N	O N O N	NHBoc

6 Synthesis of 1,2,4-oxadiazoles pseudopeptides 9, 9' (a,b) 7,7' (a,b) 8,8' (a,b) 9,9' (a,b) a Isolated yield. b Conversion is determined by crude LC/MS. Heating esters 8, 8' (a,b) under microwave irradiation afforded the 1,2,4-oxadiazoles 9, 9' (a,b) in high yields (Table 2.6). Different solvents, temperatures and prolonged heating were explored in order to provide the best conversion. Phenylalanine derivative 8'b was selected as a model compound. The results are summarized in Table 2.7. The reaction in dichloromethane at 150 °C for 25 min under microwave heating afforded the highest conversion in the shortest time as judged by LC/MS analysis (entry 3). Table 2.7 Optimization of microwave assisted condensation conditions for 8'b as a model compound a Conversion is determined by crude LC/MS.

Table 2 . 8

 28 Synthesis of 1,2,4-oxadiazoles pseudopeptides with proline residue

		N		CO 2 Me			N	CO 2 Me				N	CO 2 Me
	N X	O N NH 2	OH	Boc-Phe-OH DCC, 4-DMAP CH 3 CN	X N	O N NH 2	O	O	NHBoc Ph	MW 150 °C 25 min DCM	X N	O N N O	NHBoc Ph
	7,7' c					8,8' c					9,9' c
	Entry			X		Yield, %			Conversion, %
	1			CH	8c	quantitative		-
	2			CH	9c	58				65
	3			N	8'c	quantitative		-
	4			N	9'c	76				80

.8). Further condensation under MW heating of the solution of 8c, 8'c in DCM (closed vessel, 300W, 150 °C, 25 min) delivered the 1,2,4-oxadiazole derivatives 9c and 9'c in 58% and 76% yields respectively.

Table 2 .

 2 11 Synthesis of hydrazide modified peptidomimetics

									CO 2 Me		Ph
	X	O	NH	R	CO 2 Me	BocPheNHNH 2 Cl 3 CO OCCl 3 O	X	HN	O	HN R O	NH	NHBoc
	N		N	OH	DIPEA, DCM, rt	N		N	O	O
		NH 2					NH 2
		7, 7' (a,b)					12, 12' (a,b)
	Entry			X	R				Isolated yield, %
	1				CH	Me (Ala)	12a			40
	2				CH	Bn (Phe)	12b			60
	3				N	Me (Ala)	12'a			44
	4				N	Bn (Phe)	12'b			69

The conformations of peptidomimetics 12 and 12' in solution wОrО stuНiОН (see Chapter 3).

Table 3 . 1

 31 Selected carbon chemical shifts in pyrrolidine ring of proline derivatives 5'c, 7'c-9'c in CDCl 3The δ resonances of the trans conformers vary from 48.3 to 49.5 ppm and lie within the range 47.2 -48.4 ppm for the cis, respectively. Furthermore, the -carbon signal of the trans isomer appears upfield from the cis isomer and the -carbon of the trans rotamer is downshifted relative to that of cis rotamer.

	Entry Product C α		C		C		C δ
			cis	trans	cis	trans	cis	trans	cis	trans
	1	5'c	61.2	60.2	31.8	29.4	22.6	25.6	48.4	49.5
	2	7'c	61.1	59.3	31.5	30.2	23.5	25.2	47.3	48.3
	3	8'c	61.4	59.4	31.5	30.2	23.5	25.4	47.2	48.7
	4	9'c	61.2	59.5	31.7	30.1	23.3	25.5	47.4	48.8

Table 3 . 2

 32 Chemical shifts of the α proton derived from 1 H NMR (CDCl 3 , 300K)

		5c	5'c	7c, 7'c	8c	8'c	9c	9'c
	δH α (trans)	4.71	4.64	4.74	4.80	4.77	4.66	4.60
	δH α (cis)	4.28	4.80	4.11	4.29	4.28	4.01	4.26
	Δδ α	-0.43	+0.16	-0.63	-0.51	-0.49	-0.65	-0.34

Table 3 .

 3

	Pyridine	cis-	trans-	Pyrazine	cis-	trans-
	derivative	rotamer, %	rotamer, %	derivative	rotamer, %	rotamer, %
	5c	23	77	5'c	46	54
	7c	32	68	7'c	26	74
	8c	37	63	8'c	29	71
	9c	27	73	9'c	24	76

3

The population of amide isomers 5c, 5'c, 7c-9c, 7'c-9'c measured from the 1 H NMR spectra in CDCl 3

Table 3 .

 3

4 Dimensions with standard deviations of the possible n  * interactions in the proline derivatives 7'c and 8

'c 7'c 8'c Mean distance d 1 (N…C) Mean distance d 2 (O…C) Mean distance d 1 (N…C) Mean distance d 2 (O…C

	58.6±2.6°	95.2±14.5°	70±4.6°	110.9±15.9°

) 2.99±0.087 Å 3.07±0.175 Å 3.10±0.17 Å 2.95±0.084 Å Mean angle θ 1 (N…C=O) Mean angle θ 2 (O…C=O) Mean angle θ 1 (N…C=O) Mean angle θ 2 (O…C=O) Indeed, all mean distances found (d 1 and d 2 ) arО ≤ γ.β Å, where the Van der Waals surfaces of the carbonyl oxygen and ester carbon (d 2 ) and the nitrogen lone pair and amide carbon (d 1 ) interpenetrate. Interestingly, the n  * interaction of the two carbonyl groups in 8'c seems to be stronger, according to the measured distance d 2 of 2.95 Å, which is less than for 7'c, where d 2 is equal to 3.07 Å (Table

3

.4). The allowed angles are reminiscent of the Bürgi-Dunitz trajectory for nucleophilic attack on a carbonyl carbon (~107°). ThО mОan θ 1

Table 3 .

 3

	Proline	K t/c	ΔH 0	ΔS 0	ΔG 0	300K
	derivative	DMSO-d6	(kJ mol -1 )	(J mol -1 K -1 )	(kJ mol -1 )
		(CDCl 3 )
	5c	2.33	-3.05	-2.96	-2.16

5 Thermodynamic parameters for compounds 5c, 7c-9c, 5'c, 7'c-9'c. Additionaly the trans/cis equilibrium constant at 300K is given

Table 3 .

 3 6 FT-IR adsorption in the C=O stretching regions for 10 mM concentration samples

	of AOPzPhe (7'b), AOPzPro (7'c) and AOPzProPhe (7'd) in chloroform at room temperature
										O
	N N	O	NH N OH CO 2 Me	N N	N	CO 2 Me N OH O	N N	N	O N C7	OH H N	CO 2 Me Ph
		NH 2		NH 2		NH 2
		AOPzPhe (7'b)	AOPzPro (7'c)	AOPzProPhe (7'd)
	Product	, cm -1			, cm -1					, cm -1
		(C=O ester)			(C=O amide)				(C=N)
	AOPzPhe	1743/1691			1680					1659
	AOPzPro	1743			1655					1642
	AOPzProPhe	1743/1722			1679
						1670/1656

Table 3 . 8

 38 Thermodynamic parameters for compound AOPzProPhe (7'd). Additionally the trans/cis equilibrium constant at 300K is given As we can see the K t/c values of 7'd are dependent on temperature such that the trans isomer becomes favored as the temperature increase, in other words, it is favored by enthropy. The cis rotamer is favored by enthalpy showing opposite behavior to the other proline derivatives (Tables 3.5, 3.8, Figures 3.3

		K t/c	ΔH 0	ΔS 0	ΔG 0	300K	ΔG ‡
		at 300K	(kJ mol -1 )	(J mol -1 K -1 )	(kJ mol -1 )	(kJ mol -1 )
	7'd -DMSO-d 6	1.12	1.82	6.96	-0.27		-
	7'd -D 2 O	1.37	3.75	14.43	-0.58		75.3

Table 3 .

 3

			O		O
			HN		R
			X	O	O	H
			N	N	O	N	O
			NH 2		O
	X, R		C=O		C=O		, cm -1 C=O	C=O	C=N
	CH, Me	8a	1767		1741		1712	1670	1651
	CH, Bn	8b	1766		1743		1711	1670	1648
	N, Me	8'a	1764		1743		1711	1688	1645
	N, Bn	8'b	1766		1745		1712	1686	1645

9

Stretching vibrations in the C=O region of compounds 8, 8' (a,b) in CHCl 3

(10 mM)

Table 3 .

 3 10 Сhemical shifts and solvent sensitivity of the NH protons in 8'a,b and 9

'a,b δNH (ppm) Δδ (ppm) δNH (ppm) Δδ

								 (ppm) =
								 (DMSO-d 6 ) -
							(ppm)	 (CDCl 3 )
		8'a	9'a		8'b	9'b		9'a	9'b
	NH-amide 7.82 8.05	+0.23	7.72 7.95	+0.23	1.17	1.28
	NHBoc	5.17 5.20	+0.03	5.15 5.19	+0.04	2.59	2.58
	It is also notable that this interaction is little affected by solvation (up to 20% of
	DMSO-d

6 ) (Δδ NHamidic = +0.43 and +0.39 ppm; Figure 3.18).

Table 3 .

 3 11 Сhemical shifts of the NH protons of 8a,

b and 9a,b δNH (ppm) Δδ (ppm) δNH (ppm) Δδ

		8a	9a		8b	9b	(ppm)
	NH-amide	7.58	6.78	-0.8	7.34	6.81	-0.53
	NHBoc	5.08	5.39	+0.31	5.12	5.44	+0.32

Table 3 .

 3 13 Absolute and relative energies (in atomic units and kcal/mol, respectively) for isomers of N-acylamidrazones calculated at B3LYP level of theory using 6-31G(d,p) basis set : Energies include both electronic and thermal energies. Geometries were obtained by energy minimization at thО statОН lОvОl of thОory. ΔE was МalМulatОН as a НiffОrОnМО bОtwООn ОnОrgiОs of thО most stablО Мonfigurations (underligned values).The energy barriers (ΔG ‡ ) and other parameters for amide isomerization in 10a,b and Z/E isomerization around C=N bond in 10'a,b were determined by NMR spectroscopy using equations 3-5 (Table3.14). 1 H NMR spectra were recorded at increasing temperatures in DMSO-d 6 until the coalescence of the two isomer populations was observed. The barrier for isomerization in 10a was calculated to be 74.73 kJ/mol at 65 °C while in the case of 10b -75.69 kJ/mol at 80 °C. This difference is probably due to the influence of the more bulky

	X, R	E I [a.u.]	E II [a.u.]	E III [a.u.]	E IV [a.u.]	ΔE [kcal/mol]
	CH, Me (10a) -1751.286540 -1751.294366 -1751.280429 -1751.277908	4.91
	CH, Bn (10b) -1982.258785 -1982.265883 -1982.256207 -1982.252817	4.45
	N, Me (10'a) -1767.328071 -1767.335808 -1767.331841 -1767.326273	2.49
	N, Bn (10'b) -1998.299683 -1998.306963 -1998.300874 -1998.298101	3.82

Notephenylalanine residue on the amide isomerization. The barriers in pyrazine derivatives 10'a and 10'b were calculated to be 75.55 kJ/mol at 80 °C. For the Z/E isomerization around C=N bond in 10'a,b the difference in C-terminal amino acid residue does not influence on the rotational barrier values.

Table 3.14 Rotational barriers for the Z/E isomerization Compound T

c , K Δν, Hz k, s -1 t 1/2 , s ΔG ‡ at T c , kJ/mol

  Δthe separation of the OMe groups signals in DMSO-d 6 A resonance assignment of the NH chemical shifts was performed for 10, 10' which exhibited two sets of signals, the downfield NH resonances being associated with the major E amide isomers of 10a,b and with the Z isomers around C=N bond for 10'a,b (Table3.15). However, it was not possible to distinguish the NH-amide protons for 10' due to overlapping of signals with the CH protons of the pyrazine ring.

	10a	338	9	20.0	0.03	74.73
	10b	353	21	46.6	0.01	75.69
	10'a	353	22	48.8	0.01	75.55
	10'b	353	22	48.8	0.01	75.55

Note:

Table 3 .

 3 15 Comparison of the chemical shifts of the NH protons for two ismers in DMSO-d 6We have also determined isomeric ratio of compounds in DMSO-d 6 (Table3.16). The population of the Z conformer slightly decreases with increasing size of the R group in amino acid residue which is presumably due to some steric hindrance. We next wanted to test the effect of solvent polarity on the isomeric ratio. Surprisingly, a partial decomposition of the pyridine derivatives 10a,b occurred after adding CDCl 3 (Table3.16, Scheme 3.2). The proton

		δNH-N=		Δδ	δNH-amide	Δδ	δNHBoc		Δδ
		E	Z		E	Z		E	Z
	10a	9.86	9.86	0	8.77	8.54	0.23	7.01	6.21	0.80
	10b	9.91	9.87	0.04	9.03	8.64	0.39	7.00	6.11	0.89
		Z	E	Δδ	Z	E	Δδ	Z	E	Δδ
	10'a	9.91	9.91	0	-	-	-	7.03	6.38	0.65
	10'b	9.93	9.92	0.01	-	-	-	7.02	6.32	0.70

signals of the pyrrolopyridines 6a,b appear in a mixture DMSO-d 6 /CDCl 3 and become predominant with increasing the ratio up to 1:3. The instant formation of 6a,b was also detected by LCMS analysis.

Table 3 .

 3

		E/Z ratio
	DMSO-d 6 /CDCl 3	1:0	1:1	1:3
	10a	1/0.63	-a	-b
	10b	1/0.56	-c	-d
		Z/E ratio
	10'a	1/0.70	1/0.66	1/0.50
	10'b	1/0.67	1/0.69	1/0.55

16

Influence of solvent polarity on the E/Z isomeric ratio of compounds 10, 10' a Mixture containing 10a and 6a (~45% of 6a); b Mixture containing 10a and 6a (~65% of 6a); c Mixture containing 10b and 6b (~35% of 6a); d Mixture containing 10b and 6b (~55% of 6a).

Table 3 .

 3 17 NH chemical shifts of peptidomimetics 12, 12' in CDCl 3(3 mM)

			CO 2 Me	HN	Boc
			HN	R	O	Ph
		X	O	HN	NH
		N	N	O		O
			NH 2
	X, R					 (ppm)
		NHBoc	NH-amide	NH-hydrazide	NH-hydrazide
	CH, Me (12a)	5.02	6.37	7.39	8.22
	CH, Bn (12b)	5.03	6.44	7.74	8.36
	N, Me (12'a)	5.01		7.4	7.86	8.3
	N, Bn (12'b)	5.13	7.27	7.84	8.34

Table 3 .

 3 18 Carbonyl stretching vibrations of pyridine-based peptidomimetics 12a,b

							O
			O		O		HN	O
			HN		R	O		Ph
				O	HN	NH
			N	N	O		O
			NH 2
							ν, cm -1
		C=O	C=O	C=O	C=O	C=O free	C=O bonded	C=N
	12a (R = Me)	1755	1742	1708	1703	1693	1668	1661
	12b (R = Bn)	1754	1745	1713	1705	1699	1669	1657

Table 3 .

 3 19 Solvent sensitivity of the NH protons in 12a

,b and 12'a,b Product  (ppm) =  (DMSO-d 6 ) - (CDCl 3 )

		NHBoc	NH-amidic	NH-hydrazide	NH-hydrazide
	12a	2.09	2.6	2.82	0.35
	12b	1.92	2.47	2.3	0.18
	12'a	2.12	1.76	2.33	0.4
	12'b	1.98	1.88	2.35	0.48
	More information on the conformation in solution was obtained from NOESY
	experiment recorded in CDCl 3 . Two correlations of NH-hydrazide protons were found in
	NOESY spectrum of 12b showing the spatial proximity of NH-hydrazide with CH of the N-

terminal Phe moiety and NH-hydrazide with CH of the C-terminal Phe residue (Figure

3

.30).

Table 4 . 1

 41 Preliminary results of the NO release assay on amidoximes

	Entry	Compound	[NO 2 µM -] 1	[NO 2 µM -] 2	[NO 2 µM -] 3	Mean value (µM)	Standard deviation
		N			CO 2 Me
	1		O			1.34	2.62	1.22	1.73	0.776
		N	N		OH
		NH 2		7c
		N		CO 2 Me
	2	N	O			1.95	0.98	0.53	1.15	0.726
		N	N	OH
		NH 2		7'c
		O			STr
	3	N	NH N	OH CO 2 Me	3.81	0.99	1.15	1.98	1,584
		NH 2
			7h
		O
	4		NH	CO 2 Me	1.50	2.62	-	2.06	0.792
		N	N		OH
		NH 2
			7b

  are shown as mean ± standard error of the mean (sem) values. Statistical comparisons were performed using the two-way ANOVA woth Bonferroni posttest. p < 0.05 was considered significantly different. Statistical analyses were performed using the GraphPad NMR (300 MHz, DMSO-d 6 , 25 °C): δ = 8.94 (d, J = 4.8 Hz, 1H, H Ar ), 8.48 (d, J = 8.1 Hz,1H, H Ar ), 7.88 (dd, J = 8.1, 4.8 Hz, 1H, H Ar ), 3.95 (s, 3H, OMe). .05 mL, 143 mmol) and the mixture was allowed to reach r.t. and stirred for 2 h. Water and NaHCO 3 (sat.) ~ 1:1 were added and the mixture was extracted with CH 2 Cl 2 .The organic phase was washed with H 2 O, dried over MgSO 4 , and concentrated to give the

	Experimental procedures
	2-Сarbamoylnicotinic acid (2) 46
	Prism software (GraphPad Software version 5.0, San Diego, USA) O 13 С NMR (75 MHz, DMSO-d 6 , 25 °C): δ = 163.0, 153.9, 138.8, 132.4, 129.4, 127.5, 116.2, title compound 2. 53.1. OH NH 2 Yellow solid; yield: 9.22 g (87%); mp 70-7β ˚C. 1 NMR (γ00 MHz, DMSO-d 6 , 25 °C): δ = N O 8.89 (s, 2H, 2H Ar ), 4.09 (s, 3H, OMe). 13 С NMR (75 MHz, DMSO-d 6 , 25 °C): δ = 162.8,
	Acid 2 was prepared from quinolinic anhydride (1) and aqueous ammonia according to a 2-Cyanonicotinic acid (4) 48 147.7, 146.8, 146.6, 131.4, 115.0, 54.5.
	literature procedure. 46 A suspension of anhydride 1 (7.45 g, 50 mmol) in 28% NH 4 OH (37 O
	mL) was stirred at 70 °C for 10 min, whereupon the reactant had dissolved. After the mixture was cooled to 0 °C, a precipitation was performed by adding 12M Сl (dropwise) while N OH 3-cyanopyrazine-2-carboxylic acid (4')
	cooling. The precipitate was collected by filtration and dried. O
	N	OH
	N
	Methyl 2-Cyanonicotinate (3) 47,48
	O
		O
	N	N
	To a stirred suspension of 2-carbamoylnicotinic acid (2; 7.47 g, 45 mmol) in CH 2 Cl 2 (50 mL)
	at 0 °С were added Et 3 N (13.08 mL, 94 mmol) and methyl chloroformate (7.65 mL, 99 Methyl 3-cyanopyrazine-2-carboxylate (3') 49
	mmol). After the reaction mixture was stirred at r.t. for 8 h, it was diluted with CHCl 3 (50 mL)
	with hexane and dried. and washed with H 2 O, dried over MgSO 4 and concentrated in vacuo. The product was washed O N O
	N

White solid; yield: 4.65 g (56%); mp 176 ˚C (Lit.

48

175 ˚C). 1 NMR (300 MHz, DMSO-d 6 , 25 °C): δ = 13.26 (s, 1H, COOH), 8.67 (dd, J = 4.8, 1.4 Hz, 1H, H Ar ), 8.04 (dd, J = 7.8, 1.3 Hz, 2H, 2×H Ar ), 7.72 -7.44 (m, 2H, NH 2 ). 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 168.1, 167.1, 150.4, 149.6, 136.7, 129.2, 125.2.

Pale yellow solid; yield: 5.76 g (79%); mp 80-8γ ˚C (Lit.

48

89 °C). IR (KBr): 3090, 2959, 2241, 1726, 1578, 1287 cm -1 . 1 N A mixture of ester 3 (3.16 g, 19.5 mmol), MeOH (60 mL) and 1M NaOH (22 mL) was stirred at r.t. for 8 h, and then concentrated in vacuo. The residue was diluted with water (20 mL) and CH 2 Cl 2 (50 mL), anН thОn thО solution was aМiНifiОН with 1 Сl unНОr Мooling. ThО resulting precipitate was collected, washed with H 2 O and hexane, and dried in vacuo. Pale-yОllow soliНν yiОlНμ 1.84 g (64%)ν mp 186 ˚C [Lit. 48 β14 ˚C, 185 ˚C (sublimation)]. 1 NMR (γ00 MHz, DMSO-d 6 , 25 °C): δ = 14.25 (s, 1H, COOH), 8.92 (dd, J = 4.8, 1.5 Hz, 1H, H Ar ), 8.47 (dd, J = 8.1, 1.5 Hz, 1H, H Ar ), 7.86 (dd, J = 8.1, 4.8 Hz, 1H, H Ar ). 13 С NMR (100 MHz, DMSO-d 6 + CCl 4 , 1:1, 25 °C): δ = 164.2, 153.5, 139.1, 133.1, 130.9, 127.2, 116.4. Anal. Calcd for C 7 H 4 N 2 O 2 : C, 56.76; H, 2.72; N, 18.91. Found: C, 56.74; H, 2.73; N 18.88. N To a stirred suspension of pyrazine-2,3-НiМarboxyliМ aМiН monoamiНО β' (10.86 g, 65 mmol) in CH 2 Cl 2 (100 mL) at 0 °С wОrО aННОН Et 3 N (19.00 mL, 136.5 mmol) and methyl chloroformate (11N A mixturО of γ' (0.978 g, 6.0 mmol), MОOH (50 mL) anН 1M NaOH (1β mL) was stirrОН at r.t. for 3 h and then concentrated in vacuo. The residue was diluted with water and CH 2 Cl 2 , anН thОn aМiНifiОН with 1 Сl unНОr Мooling. ThО rОsulting prОМipitatО was Мollected, washed with H 2 O and hexane, and dried in vacuo. Pale-yellow solid; yield: 0.742 g (82%); mp 157-158 ˚C. 1 NMR (γ00 MHz, DMSO-d6, 25 °C)μ δ = 9.00 (Н, J = β.γ Hz, 1H, H Ar ), 8.96 (d, J = 2.3 Hz, 1H, H Ar ). 13 С NMR (75 MHz, DMSO-Н6, β5 °C)μ δ = 164.5, 155.1, 146.6, 144.6, 128.1, 116.7.

-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6- yl]propanoate (6a)

  NMR (75 MHz, CDCl 3 , 25 °C): δ = 171.1, 165.9, 159.6, 154.8, 150.4, 132.1, 127.1, 125.5, 53.3, 48.6, 15.7. Anal. Calcd for C 11 H 11 N 3 O 3 : C, 56.65; H, 4.75; N, 18.02. Found: C, 56.45; H, 4.77; N, 17.96. NMR (75 MHz, CDCl 3 , 25 °C): δ = 168.5, 166.0, 159.8, 154.8, 150.4, 132.1, 127.0, 125.6, 53.1, 40.0. Anal. Calcd for C 10 H 9 N 3 O 3 : C, 54.79; H, 4.14; N, 19.17. Found: C, 54.57; H, 4.15; N, 19.10.

	O 1H, CHNH), 3.76 (s, 3H, OCH 3 ), 1.85 -1.66 (m, 3H, CH+CH 2 ), 0.99 -0.95 (2d, 6H, O
	NH CN N N O NH С NMR (75 MHz, CDCl 3 , 25 °C): δ = 173.5, 163.9, 153.0, 137.5, 135.49, 131.8, 127.3, O CO 2 Me N (CH 3 ) 2 ). O N CO 2 Me 13
	N Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 5a as a white solid; CN LCMS: MH + , 234. Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 5c as a pale-yellow oil; yield: 1.127 g (87%). Purification by flash column chromatography (AcOEt/petroleum ether, 1:1) gave 6e as a 116.5, 53.2, 52.4, 42.0, 25.5, 23.4, 22.5. LCMS: MH + , 220. white solid; yield: 0.438 g (34%); mp 141 °С. Anal. Calcd for C 14 H 17 N 3 O 3 : C, 61.08; H, 6.22; N, 15.26. Found: C, 60.89; H, 6.23; N, 15.22. yield: 0.177 g (15%); mp 135-1γ7 °С. IR (KBr): 3273, 3080, 2997, 2941, 2237 (CN), 1739, 1648, 1581, 1548, 1226 cm -1 . 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.79 (d, J = 4.8 Hz, 1H, H Ar ), 8.15 (d, J = 8.1 Hz, 1H, H Ar ), 7.61 (dd, J = 8.1, 4.8 Hz, 1H, H Ar ), 7.15 (d, J = 5.7 Hz, 1H, NH), 4.80 (m, 1H, CHNH), 3.78 (s, 3H, OMe), 1.56 (d, J = 7.2 Hz, 3H, CH 3 ). Methyl (2S)-2-[(7E)-7-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-3-phenylpropanoate (6b) N N O CO 2 Me IR (KBr): 2952, 2237 (CN), 1736, 1634, 1428, 1407, 1175 cm -1 . 1 IR (KBr): 3271, 2968, 1734, 1668, 1406, 1212, 1096 cm -1 . LCMS: MH + , 276. Methyl (2S)-2-{[(2-cyanopyridin-3-yl)carbonyl]amino}-3-methylbutanoate (5e) 1 NMR (γ00 MHz, CDCl 3 , 25 °C): δ = 9.33 (br s, 1H, NH), 8.87 (d, J = 4.8 Hz, 1H, H Ar ), NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.74 and 8.70 (2×d, J = 4.5 Hz, 1H, H Ar ), 7.90 and O 8.15 (d, J = 9.0 Hz, 1H, H Ar ), 7.58 (dd, J = 9.0, 4.8 Hz, 1H, H Ar ), 4.80 (d, J = 9.0 Hz, 1H, 7.79 (2×d, J = 7.8 Hz, 1H, H Ar ), 7.62 -7.51 (m, 1H, H Ar ), 4.71 and 4.28 (2m, 1H, CH), 3.77 and 3.55 (2s, 3H, OCH 3 ), 3.51 -3.38 (m, 2H, CH 2 N), 2.42 -2.31 (m, 1H, CH 2 ), 2.21 -1.77 (m, 3H, CH 2 CH 2 ). N N CHNH), 3.68 (s, 3H, OMe), 2.92 -2.80 (m, 1H, CH(CH 3 ) 2 ), 1.20 (d, J = 6.0 Hz, 3H, CH 3 ), Methyl (2S)-2-[(7E)-7-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-4-CO 2 Me H CN 0.88 (d, J = 6.0 Hz, 3H, CH 3 ). methylpentanoate (6f)
	NH Purification by flash column chromatography (AcOEt/petroleum ether, 1:1) gave 5e as a 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 170.3, 166.2, 160.2, 154.9, 150.1, 132.2, 127.2, O
	white solid; yield: 0.488 g (37%); mp 142-143 °С. 125.3, 58.7, 52.9, 28.8, 21.8, 20.1. N
	N	CO 2 Me
	NH
	IR (KBr): 3275, 3252, 3201, 3062, 3030, 2955, 2923, 2853, 1741, 1731, 1666, 1408, 1256 Purification by flash column chromatography (AcOEt/petroleum ether, 1:1) gave 6f as a light-
	cm -1 . H Ar ), 7.62 (dd, J = 9.0, 4.8 Hz, 1H, H Ar ), 6.97 (br m, 1H, NH), 4.79 (m, 1H, CHNH), 3.78 (s, yellow oil; yield: 1.046 g (76%).
	3H, OCH 3 ), 2.39 -2.28 (m, 1H, CH(CH 3 ) 2 ), 1.07 (d, J = 6.0 Hz, 3H, CH 3 ), 1.03 (d, J = 6.0 IR (KBr): 3276, 2956, 1734, 1665, 1593, 1405, 1257, 1209 cm -1 .
	Methyl Hz, 3H, CH 3 ). 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 172.5, 164.0, 153.1, 137.9, 135.8, 131.6, 127.4, O 2, 166.0, 159.7, 154.7, 149.9, 137.6, 132.0, N O 116.7, 58.9, 53.2, 32.2, 19.7, 18.5. O 2.36 (m, 1H, CH 2 ), 1.95 -1.90 (m, 1H, CH 2 ), 1.44 (m, 1H, CH(CH 3 ) 2 ), 0.90 and 0.85 (2d, J (2S)-2-[(7E)-7N N NH 129.5, 129.0, 127.3, 127.0, 125.1, 54.2, 53.4, 35.1. N NH CO 2 Me N H CO 2 Me = 5.7 Hz, 6H, (CH 3 ) 2 ). Anal. Calcd for C 13 H 15 N 3 O 3 : C, 59.76; H, 5.79; N, 16.08. Found: C, 59.55; H, 5.81; N, 16.12. N CN CO 2 Me Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 6a as a white solid; Anal. Calcd for C 17 H 15 N 3 O 3 : C, 66.01; H, 4.89; N, 13.58. Found: C, 65.81; H, 4.91; N, 13.53. LCMS: MH + , 310. Acetate 6d was obtained as a white solid of analytical purity without further purification; yield: 0.63 g (58%); mp 160 °С. LCMS: MH + , 262. 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 171.0, 166.1, 159.8, 154.7, 150.06, 132.0, 127.0, Purification by flash column chromatography (AcOEt/petroleum ether, 1:1) gave 5f as a white solid; yield: 0.117 g (9%); mp 117-118 °С. 125.3, 53.0, 51.6, 37.7, 25.6, 23.7, 21.7.
	yield: 0.671 g (58%); mp 90 °С. IR (KBr): 3294, 3238, 1743, 1731, 1665, 1403, 1410, 1258 cm -1 . Methyl (2S)-1-[(2-cyanopyridin-3-yl)carbonyl]pyrrolidine-2-carboxylate (5c) IR (KBr): γ198, γ057, β9γ7, 17γ6, 1669, 159γ, 1440, 1β81, 1β67, 1β48 м -1 . 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 9.33 (s, 1H, NH), 8.87 (dd, J = 4.8, 1.5 Hz, 1H, H Ar ), Methyl methylbutanoate (6e) 1 . (2S)-2-[(7E)-7-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl]-3-IR (KBr): 3280, 3080, 2956, 2871, 2237 (CN), 1740, 1666, 1645, 1584, 1545, 1407, 1205 cm -Anal. Calcd for C 14 H 17 N 3 O 3 : C, 61.08; H, 6.22; N, 15.26. Found: C, 60.85; H, 6.24; N, 15.21.
	1 8.17 (dd, J = 7.8, 1.5 Hz, 1H, H Ar ), 7.58 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 4.66 (s, 2H, CH 2 ), 3.77 NMR (300 MHz, CDCl 3 , 25 °C): δ = 9.34 (br s, 1H, NH), 8.86 (d, J = 4.8 Hz, 1H, H Ar ),
	(s, 3H, OCH 3 ).

13

С NMR (75 MHz, CDCl 3 , 25 °C): δ = 173.

4

, 163.6, 153.0, 137.4, 135.53, 132.0, 127.3, 116.5, 53.4, 49.7, 18.7. Anal. Calcd for C 11 H 11 N 3 O 3 : C, 56.65; H, 4.75; N, 18.02. Found: C, 56.48; H, 4.76; N, 17.95. LCMS: MH + , 234. 8.14 (d, J = 8.1 Hz, 1H, H Ar ), 7.57 (dd, J = 8.1, 4.8 Hz, 1H, H Ar ), 5.24 (q, J = 7.2 Hz, 1H, CH), 3.73 (s, 3H, OCH 3 ), 1.74 (d, J = 7.2 Hz, 3H, CH 3 ).

13 С Purification by flash column chromatography (AcOEt/petroleum ether, 7:3) gave 6b as a light-yellow solid; yield: 0.953 g (76%); mp 72-7γ °С. 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 9.29 (s, 1H, NH), 8.81 (d, J = 4.8 Hz, 1H, H Ar ), 8.06 (d, J = 7.8 Hz, 1H, H Ar ), 7.51 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 7.20 -7.14 (m, 5H, 5×H Ar ), 5.47 (dd, J = 10.8, 5.4 Hz, 1H, CH), 3.78 (s, 3H, OCH 3 ), 3.72 -3.59 (m, 2H, CH 2 ). 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 170.13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 172.4, 165.

2, 164.8, 152.1, 151.9, 138.2, 137.9, 136.4, 136.2, 131.3, 127.4, 127.2, 116.0, 61.4, 59.7, 53.3, 53.1, 49.6, 47.5, 31.8, 29.9, 25.5, 23.3. Anal. Calcd for C 13 H 13 N 3 O 3 : C, 60.22; H, 5.05; N, 16.21. Found: C, 59.98; H, 5.07; N, 16.15. LCMS: MH + , 260.

Methyl [(7E)-7-imino-5-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl]acetate (6d) 13 С IR (KBr): 3270, 3081, 2972, 2238 (CN), 1730, 1645, 1549, 1205 cm -1 . 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.81 (d, J = 4.8 Hz, 1H, H Ar ), 8.18 (d, 9.0 Hz, 1H, Anal. Calcd for C 13 H 15 N 3 O 3 : C, 59.76; H, 5.79; N, 16.08. Found: C, 59.57; H, 5.80; N, 16.04. LCMS: MH + , 262. Methyl (2S)-2-{[(2-cyanopyridin-3-yl)carbonyl]amino}-4-methylpentanoate (5f) 1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.78 (d, J = 4.8 Hz, 1H, H Ar ), 8.15 (d, J = 8.1 Hz, 1H, H Ar ), 7.61 (dd, J = 8.1, 4.8 Hz, 1H, H Ar ), 7.06 (d, J = 6.9 Hz, 1H, NH), 4.87 -4.79 (m, 1 NMR (γ00 MHz, CDCl 3 , 25 °C): δ = 9.37 (br s, 1H, NH), 8.81 (s, 1H, H Ar ), 8.09 (d, J = 6.6 Hz, 1H, H Ar ), 7.54 (m, 1H, H Ar ), 5.23 -5.16 (m, 1H, CHNH), 3.64 (s, 3H, OCH 3 ), 2.44 -

Methyl (2S)-2-{[(2-cyanopyridin-3-yl)carbonyl]amino}-3-(tritylsulfanyl)propanoate (5h)

  NMR (75 MHz, CDCl 3 ): δ = 169.32, 165.85, 159.41, 154.88, 150.19, 145.06, 132.25, 130.32, 128.94, 127.41, 127.11, 125.34, 67.98, 53.39, 52.64, 31.38.

		O
		N	NH
	N		CO 2 Me
		NH
	Purification by flash column chromatography (CH 2 Cl 2 /CH 3 CN, 8:2) gave 6g as a yellow
	solid; yield: 1.436 g (83%); mp 119-1β1 °С.
	IR (KBr): 3396, 3266, 3067, 1732, 1664, 1429, 1407, 1260 cm -1 .
	1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 9.33 (s, 1H, NH), 8.69 (d, J = 4.8 Hz, 1H, H Ar ), 8.29
	(s, 1H, NH Ar ), 7.91 (d, J = 7.8 Hz, 1H, H Ar ), 7.58 (d, J = 7.5 Hz, 1H, H Ar ), 7.36 (dd, J = 7.8,
	4.8 Hz, 1H, H Ar ), 7.18 (d, J = 7.8 Hz, 1H, H Ar ), 7.09 -6.98 (m, 3H, 3×H Ar ), 5.58 (dd, J =
	10.2, 4.8 Hz, 1H, CH), 3.95 -3.72 (m, 2H, CH 2 ), 3.77 (s, 3H, OCH 3 ).
		O	STr
		N H	CO 2 Me
	N	CN
	Purification by flash column chromatography (AcOEt/petroleum ether, 7:3) gave 5h as a
	light-yellow solid; yield: 1.521 g (60%); mp 78-81 °С.
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.77 (d, J = 4.8, 1H, H Ar ), 8.04 (d, J = 7.8 Hz, 1H, H Ar ),
	7.46 (dd, J = 7.8, 4.8 Hz, 1H), 7.36 -7.02 (m, 16H, NH, 15×H Ph ), 4.76 (dd, J = 11.1, 4.5 Hz,

13

С NMR (75 MHz, CDCl 3 , 25 °C): δ = 170.6, 166.1, 159.8, 154.5, 149.8, 136.6, 131.8, 127.8, 126.9, 125.0, 123.3, 122.4, 119.8, 119.0, 111.7, 111.6, 53.6, 53.3, 25.1. Anal. Calcd for C 19 H 16 N 4 O 3 : C, 65.51; H, 4.63; N, 16.08. Found: C, 65.29, H, 4.64; N, 16.02. LCMS: MH + , 349. 1H, CH), 3.52 (s, 3H, OCH 3 ), 3.46 -3.34 (m, 1H, CHH), 3.00 (dd, J = 13.3, 4.5 Hz, 1H, CHH). 13 C

  3, 151.2, 150.0, 147.3, 137.9, 131.7, 124.3, 53.0, 49.3, 18.2. Anal. Calcd for C 11 H 14 N 4 O 4 : C, 49.62; H, 5.30; N, 21.04. Found: C, 49.48; H, 5.32; N, 20.96.

	LCMS: MH + , 267.

-2-({[2-(N'-hydroxycarbamimidoyl)pyridin-3-yl]carbonyl}amino)-3- methylbutanoate (7e)

  J = 6.5 Hz, 2H, CH 2 ), 2.38 -2.20 and 1.96 -1.86 (2×m, 4H, CH 2 -CH 2 ). NMR (γ00 MHz, CDCl 3 , 25 °C): δ = 8.57 (d, J = 4.8 Hz, 1H, H Ar ), 7.92 (d, J = 7.8 Hz, 1H, H Ar ), 7.50 (d, J = 8.1 Hz, 1H, NH), 7.33 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 5.46 (s, 2H, NH 2 ), 4.62 (dd, J = 8.1, 5.1 Hz, 1H, CHNH), 3.69 (s, 3H, OCH 3 ), 2.22 -2.12 (m, 1H, CH), 0.94 (d, J = 6.9 Hz, 3H, CH 3 ), 0.91 (d, J = 6.9 Hz, 3H, CH 3 ). NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.62 (d, J = 4.8 Hz, 1H, H Ar ), 7.95 (d, J = 7.8 Hz, 1H, H Ar ), 7.41 -7.35 (m, 2H, NH+H Ar ), 5.46 (br s, 2H, NH 2 ), 4.84 -4.76 (m, 1H, CH), 3.76 (s, 3H, OCH 3 ), 1.78 -1.64 (m, 3H, CH 2 +CH), 0.98 (d, J = 6.3 Hz, 3H, CH 3 ), 0.96 (d, J = 6.3 Hz, 3H, CH 3 ). Anal. Calcd for C 14 H 20 N 4 O 4 : C, 54.54; H, 6.54; N, 18.17. Found: C, 54.36; H, 6.55; N, 18.10.

			O
	N
			OMe
		O
	N	N	OH
	NH 2
	Purification by flash column chromatography (CH 2 Cl 2 / 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 172.9, 168.3, 151.4, 150.1, 147.2, 138.4, 131.9, , 94μ6) gave 7c as a white solid; yield: 0.409 g (70%); mp 135-1γ6 °С. 124.5, 58.8, 52.8, 31.9, 19.4, 18.7.
	IR (KBr): 3521, 3159, 3117, 3080, 2886, 2867, 2835, 1732, 1651, 1608, 1537, 1471, 1433, Anal. Calcd for C 13 H 18 N 4 O 4 : C, 53.05; H, 6.16; N, 19.04. Found: C, 52.92; H, 6.18; N, 18.98.
	1368, 1199, 1177 cm -1 . LCMS: MH + , 295.
	1 NMR (300 MHz, CDCl 3 , 25 °C): δ = 8.57 (m, 1H, H Ar ), 8.08 (br s, 1H, OH), 7.69 and 7.61
	(2×dd, J = 7.8, 1.5 Hz, 1H, H Ar ), 7.39 -7.26 (m, 1H, H Ar ), 5.50 (br s, 2H, NH 2 ), 4.74 and 4.11
	(2dd, J = 8.6, 4.3 Hz, J = 6.1 Hz 1H, CH), 3.79 and 3.48 (2×s, 3H, OCH 3 ), 3.75 (m) and 3.25 Methyl (2S)-2-({[2-(N'-hydroxycarbamimidoyl)pyridin-3-yl]carbonyl}amino)-4-
	5, 149.9, 148.7, 147.4, Anal. Calcd for C 17 H 18 N 4 O 4 : C, 59.64; H, 5.30; N, 16.37. Found: C, 59.51; H, 5.32; N, 16.31. 137.4, 136.8, 131.7, 129.5, 128.4, 126.6, 123.3, 54.2, 51.9, 37.5. LCMS: MH + , 343. methylpentanoate (7f) N NH O CO 2 Me N NH 2 OH Purification by flash column chromatography (CH 2 Cl 2 /EtOH, 9:1) gave 7f as a white solid; yield: 0.431 g (70%); mp 114-115 °С. (t, Methyl NH O CO 2 Me N OH (2S)N IR (KBr): 3479, 3377, 3236, 3076, 2955, 1746, 1638, 1581, 1547, 1248 cm -1 .
	NH 2
	(2S)-1-{[2-(N'-hydroxycarbamimidoyl)pyridin-3-yl]carbonyl}pyrrolidine-2-carboxylate Purification by flash column chromatography (CH 2 Cl 2 /EtOH, 95:5) gave 7e as a white solid; 13 С NMR (75 MHz, CDCl 3 , 25 °C): δ = 174.1, 168.3, 151.4, 150.1, 147.0, 138.4, 131.9, (7c) yield: 0.465 g (79%); mp 120-121 °С. 124.5, 53.0, 52.2, 42.1, 25.5, 23.4, 22.7.
	IR (KBr): 3297, 1739, 1646, 1534, 1198, 1149 cm -1 .

13

С NMR (75 MHz, CDCl 3 , 25 °C): δ = 173.6, 173.3, 169.5, 169.2, 150.6, 150.3, 149.2, 145.7, 145.2, 137.2, 136.5, 131.5, 131.1, 124.4, 124.0, 61.2, 59.3, 52.9, 52.7, 48.9, 47.4, 31.5, 30.3, 25.2, 23.8. Anal. Calcd for C 13 H 16 N 4 O 4 : C, 53.42; H, 5.52; N, 19.17. Found: C, 53.25; H, 5.54; N, 19.10. LCMS: MH + , 293. 1 1 LCMS: MH + , 309.

  Ar ), 7.43 (d, J = 7.4 Hz, 1H, NH), 7.35 -7.00 (m, 16H, 16×H Ar ), 5.36 (s, 2H, NH 2 ), 4.67 (dd, J = 12.4, 5.4 Hz, 1H, CH), 3.58 (s, 3H, OCH 3 ), 2.69 (ddd, J = 27.4, 12.3, 5.4 Hz, 2H, CH 2 ).

	13 C NMR (75 MHz, CDCl 3 ): δ = 171.32, 168.09, 150.86, 149.90, 146.69, 144.85, 138.15,
	131.41, 130.00, 128.53, 127.41, 124.24, 67.37, 53.18, 52.72, 34.16.
	O		STr
		NH	CO 2 Me
	N	N	OH
	NH 2
	Purification by flash column chromatography (CH 2 Cl 2 /MeOH, 94:6) gave 7h as a light-
	yellow solid; yield: 1.015 g (94%); mp 76-80 °С.

H

-imino-7-oxo-5,7-dihydro-6H-pyrrolo[3,4-b]pyrazin-6- yl]propanoate (6'a)

  ESI calculated for C 10 H 11 N 4 O 3 [M+H] + m/z = 235.0838, found 235.0826.

		O
	N N Methyl (2S)-2-{[(3-cyanopyrazin-2-yl)carbonyl]amino}-3-phenylpropanoate (5'b) NH CN CO 2 Me
	Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 5'a as a pale-yellow
	solid (0.161 g, 23%); mp 71-72 °C.	O
	N IR: max = 3367, 3047, 2994, 2954, 2240 (CN), 1736, 1678, 1517, 1203, 1150 cm -1 . CN N NH CO 2 Me
	1 Purification by flash column chromatography (AcOEt/petroleum ether, 7:3) gave 5'b as a NMR (γ00 MHz, CDCl 3 , β5 °C)μ δ = 8.89 (Н, J = 2.3 Hz, 1H, H Ar ), 8.79 (d, J = 2.3 Hz,
	1H, H Ar ), 8.26 (d, J = 7.4 Hz, 1H, NH), 4.71 (pseudo-p, J = 7.3 Hz, 1H, CH), 3.72 (s, 3H, light-yellow solid (0.047 g, 3%); mp 97-99 °C.
	OCH 3 ), 1.49 (d, J = 7.2 Hz, 3H, CH 3 ). IRμ max = 3402, 3389, 3059, 3031, 2954, 2853, 2237 (CN), 1736, 1686, 1508, 1374, 1261cm -
	1 .
	Methyl 13 C NMR (75 MHz, CDCl 3 )μ δ = 171.9, 160.7, 147.8, 146.9, 145.4, 1γ6.1, 1β9.8, 1β9.4, N N CO 2 Me 128.0, 115.5, 54.2, 53.2, 38.6. (2S)-2-[(5E)-5N O ESI calculated for C 16 H 14 N 4 NaO 3 [M+Na] + m/z = 333.0954, found 333.0958.
		NH
	Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 6'a as a yellow oil
	(0.197 g, 28%).
	IRμ max = 3272, 3001, 2953, 1733, 1666, 1518, 1372, 1220, 1157 cm -1 .
	1 H NMR (300 MHz, CDCl 3 )μ δ = 9.51 (s, 1H, NH), 8.91 (Н, J = 2.6 Hz, 1H, H Ar ), 8.84 (d, J =
	2.6 Hz, 1H, H Ar ), 5.36 -5.29 (m, 1H, CH), 3.74 (s, 3H, OCH 3 ), 1.77 (d, J = 7.3 Hz, 3H, CH 3 ).

13

С NMR (75 MHz, CDCl 3 , β5 °C)μ δ = 17γ.0, 160.5, 147.9, 146.7, 145.6, 129.5, 115.4, 53.2, 48.8, 18.6. ESI calculated for C 10 H 10 N 4 NaO 3 [M+Na] + m/z = 257.0654, found 257.0645. 13 C NMR (75 MHz, CDCl 3 )μ δ = 170.6, 16γ.6, 157.1, 149.β, 148.7, 144.7, 144.7, 5γ.4, 48.8, 15.6.

1 H NMR (300 MHz, CDCl 3 )μ δ = 8.89 (Н, J = 2.3 Hz, 1H, H Ar ), 8.74 (d, J = 2.3 Hz, 1H, H Ar ), 8.20 (d, J = 7.9 Hz, 1H, NH), 7.29 -7.13 (m, 5H, 5H Ar ), 5.11 (dt, J = 8.0, 6.0 Hz, 1H, CH), 3.77 (s, 3H, OCH 3 ), 3.28 (2×dd, J = 8.1, 5.8 Hz, 2H, CH 2 ).

  3279, 3062, 3029, 2953, 2847, 1737, 1666, 1413, 1374, 1244, 1123 cm -1 .2.6 Hz, 1H, H Ar ), 7.19 -7.07 (m, 5H, 5H Ar ), 5.55 (dd, J = 10.9, 5.6 Hz, 1H, CH), 3.77 (s, 3H, OCH 3 ), 3.74 -3.61 (m, 2H, CH 2 ).

	13 C NMR (75 MHz, CDCl 3 )μ δ = 169.7, 16γ.7, 157.β, 149.1, 148.7, 144.γ, 144.β, 1γ7.β,
	129.4, 129.0, 127.3, 54.4, 53.4, 35.0.
	ESI calculated for C 16 H 14 N 4 NaO 3 [M+Na] + m/z = 333.0961, found 333.0958.
	Methyl (2S)-1-[1-(3-cyanopyrazin-2-yl)ethenyl]pyrrolidine-2-carboxylate (5'c)
		O
		N
	N	O
	N	CN
	IRμ max = 2955, 2887, 2239 (CN), 1738, 1637, 1455, 1373, 1171 cm -1 .
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.74 (s, 1H, H Ar ), 8.71 and 8.61 (2d, J = 2.3 Hz, 1H, H Ar ),
	4.80 and 4.64 (2dd, J = 8.4, 3.2 Hz, 1H, CH), 3.76 -3.64 (m, 2H, CH 2 N), 3.68 and 3.53
	(2s, 3H, OCH 3 ), 2.30 -1.90 (m, 4H, CH 2 CH 2 ).
	13 C NMR (75 MHz, CDCl 3 )μ δ = 17β.0 (17β.6), 16β.γ (16β.β), 15β.β (151.7), 146.1 (145.9),
	145.6 (144.6), 130.0 (130.8), 114.9 (115.2), 60.2 (61.2), 52.9 (52.8), 49.5 (48.4), 29.4 (31.8),
	25.6 (22.6).
	ESI calculated for C 12 H 12 KN 4 O 3 [M+K] + m/z = 299.0551, found 299.0541.

O

Purification by flash column chromatography (AcOEt/CH 2 Cl 2 , 1:1) gave 5'c as a light-yellow oil (0.382 g, 49%).

  H NMR (300 MHz, CDCl 3 , 25 °C, K t/c = 1.73): δ = 8.77 (s, 4H, H trans Ar ), 8.62 (d, J = 2.3 Hz, 1H, H cis Ar ), 8.35 (d, J = 2.2 Hz, 1H, H cis Ar ), 7.31 -7.05 (m, 18H, H trans/cis Ph ), 6.58 (d, J = 7.8 Hz, 1H, NH), 4.87 -4.74 and 4.68 (m, ddd, J = 13.8, 7.4, 4.8 Hz, 6H, CH trans/cis Phe, CH-

	α trans/cis Pro), 3.71, 3.69 and 3.85 -3.51 (s, s, m, 16H, OCH 3	trans , OCH 3	cis , CH 2 -δ trans/cis Pro),
	3.23 -3.05 and 2.96 -2.88 (2×m, 6H, CH 2	trans , CH 2
	(151.5), 146.2 (145.9), 145.6 (144.6), 136.6, 130.4 (131.1), 130.0 (129.5), 129.0 (129.3),
	127.4 (127.8), 115.2 (115.5), 61.5 (63.1), 54.2 (53.8), 52.9 (53.0), 49.9 (48.6), 38.3 (37.9),
	28.3 (32.3), 25.8 (22.4).
	ESI calculated for C 21 H 21 KN 5 O 4 [M+K]

1 cis Phe), 2.32 -1.78 and 1.67 -1.50 (2m, 16H, CH 2 CH 2 -, trans/cis Pro).

13 C NMR (75 MHz, CDCl 3 , 25 °C): δ = 172.3 (172.2), 170.6 (171.8), 163.4 (163.1), 152.5 + m/z = 446.1232, found 446.1225.

  NMR (300 MHz, CDCl 3 ): δ = 8.64 (d, J = 4.0 Hz, 1H, H Ar ), 7.88 (d, J = 7.0 Hz, 1H, H Ar ), 7.43 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 7.34 (d, J = 6.3 Hz, 1H, NH), 7.30 -7.18 (m, 5H, C 6 H 5 ), 5.83 (s, 2H, NH 2 ), 5.13 -5.07 (m, 2H, NHBoc + CHCH 2 ), 4.29 (dd, J = 8.4, 5.9 Hz, 1H, CHCH(CH 3 ) 2 ), 3.71 (s, 3H, OCH 3 ), 3.39 -3.22 (m, 2H, CH 2 ), 2.14 (dq, J = 13.4, 6.8 Hz, 1H, CH(CH 3 ) 2 ), 1.46 (s, 9H, (CH 3 ) 3 ), 0.98 (dd, J = 13.2, 6.8 Hz, 6H, (CH 3 ) 2 ).

	3,
	137.2, 133.0, 130.1, 129.1, 127.5, 125.6, 80.7, 58.6, 55.1, 52.8, 38.2, 31.9, 29.0, 19.7, 18.5.
	ESI calculated for C 27 H 35 N 5 NaO 7 [M+Na] + m/z = 564.2434, found 564.2429.

1 H 13 C NMR (75 MHz, CDCl 3 ): δ = 172.5, 170.1, 167.5, 156.4, 155.6, 150.2, 145.6, 138.

  H NMR (300 MHz, CDCl 3 ): δ = 8.58 -8.56 (m, 1H, H Ar ), 7.78 and 7.65 (2×dd, J = 7.8, 1.4 Hz, 1H, H Ar ), 7.45 and 7.34 (dd, J = 7.7, 4.8 Hz, 1H, H Ar ), 7.40 -7.11 (m, 5H, C 6 H 5 ), 5.50 (d, J = 25.0 Hz, 2H, NH 2 ), 5.11 (d, J = 6.1 Hz, 1H,NHBoc), 4.80 and 4.29 (dd, J = 8.2, 3.8 Hz, 1H, CH-α of Pro), 4.7γ -4.47 (m, 1H, CH of Phe), 3.80 and 3.54 (2×s, 3H, OCH 3 ), 3.96 -ESI calculated for C 27 H 33 N 5 NaO 7 [M+Na] + m/z = 562.2275, found 562.2272.

	3.66 and 3.60 -3.20 (2×m, 2H, CH 2 -δ of Pro), 3.33 -2.99 (m, 2H, CH 2 of Phe), 2.57 -2.30
	and 2.15 -1.60 (m, 4H, CH2CH2-, of Pro), 1.45 anН 1.44 (β×s, 9H, (CH 3 ) 3 ).
	13 C NMR (75 MHz, CDCl 3 ): δ = 174.0, 169.3, 169.2, 168.5, 168.1, 155.9, 154.7, 154.32,
	149.3, 149.2, 143.9, 143.6, 137.3, 137.0, 136.8, 136.7, 133.5, 133.1, 130.1, 130.0, 129.4,
	127.7, 125.9, 125.4, 80.8, 78.1, 77.7, 77.3, 61.5, 59.3, 54.6, 52.8, 52.7, 49.3, 48.8, 47.3, 39.5,
	39.3, 31.5, 30.3, 29.0, 26.3, 25.6, 25.4, 23.8.

1

  NMR (75 MHz, CDCl 3 ): δ = 173.6, 170.2, 164.0, 156.4, 155.7, 147.0, 145.5, 144.9, 144.4, 80.6, 58.6, 53.2, 49.2, 31.9, 28.9, 19.7, 18.7, 18.5. ESI calculated for C 20 H 31 N 6 O 7 [M+H] + m/z = 467.2244, found 467.2249.

	HN		CO 2 Me
	N	O	O
	N	N	O	NHBoc
	NH 2
	Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 3:7) gave 8'a as a white solid
	(0.391 g, 84%); mp 91-94 °C.
	IR: max = 3424, 3311, 3193, 2970, 2934, 2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670
	cm -1 .
	IR (CHCl 3 ): max = 3436, 3410, 1764, 1743, 1711, 1688, 1645 cm -1 .
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.68 (s, 1H, H Ar ), 8.61 (s, 1H, H Ar ), 7.82 (d, J = 6.7 Hz, 1H,
	NH), 5.78 (br s, 2H, NH 2 ), 5.17 (d, J = 8.5 Hz, 1H, NH), 4.83 -4.61 (m, 1H, CH), 4.36 -4.15

-1-yl]pyrazin-2-yl}carbonyl)amino]propanoate (8'a) (m, 1H, CH), 3.77 (s, 3H, OCH 3 ), 2.23 -2.08 (m, 1H, CH), 1.53 (d, J = 6.9 Hz, 3H, CH 3 ), 1.43 (s, 9H, (CH 3 ) 3 ), 1.01 (3H, d, J 7.1 Hz, (CH 3 ) 2 ), 0.97 (3H, d, J 7.0 Hz, (CH 3 ) 2 ).

13 C

  H NMR (300 MHz, CDCl 3 )μ δ = 8.67 (Н, J = 2.3 Hz, 1H, H Ar ), 8.61 -8.49 (m, 1H, H Ar ), 7.43 -7.11 (m, 5H, 5H Ar ), 5.58 (br s, 2H, NH 2 ), 5.15 (d, J = 7.8 Hz, 1H, NH), 4.84 -4.72 and 4.30 -4.27 (2m, 1H, CH), 4.65 (m, 1H, CH), 3.79 and 3.62 (2s, 3H, OCH 3 ), 3.46 (m, 2H, CH 2 N), 3.11 (m, 2H, CH 2 ), 2.40, 2.02 and 1.82 -1.52 (3m, 4H, CH 2 CH 2 ), 1.44 (m, 9H, (CH 3 ) 3 ).

	540 g, 100%); mp 96-98 °C.
	IR: max = 3444, 3330, 2977, 2935, 2878, 1742, 1711, 1644, 1498, 1455, 1365, 1163, 701 cm -
	1 .
	IR (CHCl 3 ): max = 3437, 3404, 1771, 1745, 1707, 1686, 1649 cm -1 .
	13 C NMR (75 MHz, CDCl 3 )μ δ = 17γ.4, 169.β, 166.γ, 155.9, 15γ.1, 150.6, 145.6 (145.1),
	143.4 (143.5), 141.1, 136.7 (136.9), 130.0, 129.4, 127.7, 80.8, 59.4 (61.4), 54.5, 52.9 (52.8),
	48.7 (47.2), 39.2, 30.2 (31.5), 28.9, 25.4 (23.5).
	ESI calculated for C 26 H 32 KN 6 O 7 [M+K] + m/z = 579.1984, found 579.1964.

1

  , J = 6.7 Hz, 6H, (CH 3 ) 2 ).

	13 C NMR (75 MHz, CDCl 3 ): δ = 180.4, 173.8, 167.7, 166.8, 155.8, 151.7, 143.6, 137.5,
	133.9, 125.6, 81.0, 54.2, 53.3, 49.5, 33.7, 28.9, 19.4, 18.8, 18.5.
	ESI calculated for C 21 H 30 N 5 O 6 [M+H] + m/z = 448.2190, found 448.2191.
	LC/MS: m/z = 470 [M+Na] + .

  , 133.6, 129.9, 129.2, 127.7, 125.5, 80.8, 54.4, 54.0, 53.0, 38.3, 33.5, 28.8, 19.3, 18.4. ESI calculated for C 27 H 33 N 5 NaO 6 [M+Na] + m/z = 546.2323, found 546.2323.

	Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 6:4) gave 9b as light-brown oil
	(0.085 g, 63%).
	IR (CHCl 3 )μ max = 3438, 3413, 1741, 1714, 1674 cm -1 .
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.78 (dd, J = 4.5, 1.1 Hz, 1H, H Ar ), 7.81 (dd, J = 7.7, 1.1 Hz,
	1H, H Ar ), 7.43 (dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 7.29 -7.09 (m, 5H, C 6 H 5 ), 6.81 (d, J = 7.6 Hz,
	1H, NH), 5.44 (d, J = 9.1 Hz, 1H, NHBoc), 4.98 -5.07 (m, 2H, 2×CH), 3.72 (s, 3H, OCH 3 ),
	3.31 -3.13 (m, 2H, CH 2 ), 2.24 (dd, J = 12.6, 6.3 Hz, 1H, CH(CH 3 ) 2 ), 1.40 (s, 9H, (CH 3 ) 3 ),
	0.95 (d, J = 6.7 Hz, 6H, (CH 3 ) 2 ).
	13 C NMR (75 MHz, CDCl 3 ): δ = 180.2, 172.2, 167.5, 166.8, 155.8, 151.6, 143.7, 137.1,
	136.3
	HN		CO 2 Me
		O
	N	N	NHBoc
	N O

LC/MS: m/z = 524 [M+H] + .

  2H, CH 2 -δ of Pro), 3.37 -3.05 (m, 2H, CH 2 of Phe), 2.36 -1.79 (m, 4H, CH2CH2-, of Pro), 1.39 (s, 9H, (CH 3 ) 3 ). , 81.0, 54.2, 53.3, 48.9, 33.8, 28.9, 19.2, 19.1, 18.5. ESI calculated for C 20 H 28 KN 6 O 6 [M+K] + m/z = 487.1705, found 487.1702. LC/MS: m/z = 471 [M+Na] + .

	13 C NMR (75 MHz, CDCl 3 ): δ = 180.4, 173.0, 167.7, 167.3, 155.4, 151.2, 142.2, 141.8,
	137.0, 136.6, 135.9, 135.5, 134.2, 134.1, 129.9, 129.4, 129.3, 128.0, 127.8, 126.0, 125.6, 81.1,
	81.0, 61.4, 59.3, 53.0, 52.9, 50.1, 49.3, 47.2, 40.8, 40.7, 31.6, 30.3, 30.2, 28.8, 25.4, 23.5.
	ESI calculated for C 27 H 31 N 5 NaO 6 [M+Na] + m/z = 544.2186, found 544.2167.
	LC/MS: m/z = 544 [M+Na] + .
	Methyl	(2S)-2-({[3-(5-{(1S)-1-[(tert-butoxycarbonyl)amino]-2-methylpropyl}-1,2,4-
	oxadiazol-3-yl)pyrazin-2-yl]carbonyl}amino)propanoate (9'a)
		HN		CO 2 Me
		N	O
		N	N	NHBoc
		N O
	Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 4:6) gave 9'a as a colorless oil
	(0.068 g, 75%).
	IR: max = 3424, 3311, 3193, 2970, 2934, 2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670
	cm -1 .
	IR (CHCl 3 ): max = 3442, 3397, 1740, 1716, 1688 cm -1 .
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.79 (s, 1H, H Ar ), 8.66 (s, 1H, H Ar ), 8.05 (d, J = 7.3 Hz, 1H,
	NH), 5.20 (d, J = 9.0 Hz, 1H, NH), 5.10 -4.90 (m, 1H, CH), 4.64 (pseudo-p, J = 7.3 Hz, 1H,
	CH), 3.71 (s, 3H, OCH 3 ), 2.32 -2.15 (m, 1H, CH), 1.44 (d, J = 7.1 Hz, 3H, CH 3 ), 1.38 (s, 9H,
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.88 -8.77 (m, 1H, H Ar ), 7.84 and 7.75 (2×dd, J = 7.7, 1.1 (CH 3 ) 3 ), 0.96 (d, J = 6.8 Hz, 6H, (CH 3 ) 2 ).
	Hz, 1H, H Ar ), 7.51 and 7.44 (2×dd, J = 7.8, 4.8 Hz, 1H, H Ar ), 7.31 -7.04 (m, 5H, C 6 H 5 ), 5.62 13 C NMR (75 MHz, CDCl 3 ): δ = 179.7, 173.5, 167.1, 162.4, 155.8, 146.6, 145.8, 144.5,
	143.3

-5.47 and 5.32 -5.22 (2×m, 1H, NH), 5.47 -5.32 (m, 1H, CH of Phe), 4.66 and 4.06 -3.95 (dd, m, J = 8.5, 4.4 Hz, 1H, CH-α of Pro), γ.79 anН γ.5γ (β×s, γH, OCH 3 ), 3.39 -3.75, 3.37 -3.13 (2×m,

-1,2,4- oxadiazol-3-yl)pyrazin-2-yl]carbonyl}pyrrolidine-2-carboxylate (9'c)

	N		CO 2 Me
	N	O
	N	N	NHBoc
	N O	Ph
	Purification by flash column chromatography (CH 2 Cl 2 /AcOEt, 4:6) gave 9'c as a light-yellow
	oil (0.079 g, 76%).
	IR: max = 3424, 3311, 3193, 2970, 2934, 2878, 1738, 1681, 1644, 1514, 1366, 1172, 893, 670
	cm -1 .
	IR (CHCl 3 ): max = 3438, 3362, 1746, 1714, 1650, 1615 cm -1
			71 and 3.50
	(2s, 3H, OCH 3 ), 3.40 -3.05 (2m, 4H, 2CH 2 ), 2.34 -1.79 (m, 4H, CH 2 CH 2 ), 1.24 (m, 9H,
	(CH 3 ) 3 ).
	13 C NMR (75 MHz, CDCl 3 )μ δ = 180.8 (180.6), 17β.8 (17β.7), 166.4, 165.4, 155.4, 150.8,
	145.7, 145.3 (145.1), 139.9, 135.9 (135.5), 130.0, 129.4, 127.9, 81.0, 59.5 (61.2), 53.1 (52.9),
	49.9, 48.8 (47.4), 40.9, 30.1 (31.7), 28.9, 25.5 (23.3).
	ESI calculated for C 26 H 30 KN 6 O 6 [M+K] + m/z = 561.1864, found 561.1858. LC/MS: m/z =
	545 [M+Na] + .

1

H NMR (300 MHz, CDCl 3 )μ δ = 8.7β (Н, J = 2.2 Hz, 1H, H Ar ), 8.65 and 8.56 (2d, J = 2.2 Hz, J = 2.3 Hz, 1H, H Ar ), 7.26 -6.97 (m, 5H, 5H Ar ), 5.62 and 5.17 (2d, J = 8.1 Hz, 1H, NH), 5.35 -5.37 (m, 1H, CH), 4.60 and 4.26 (2dd, J = 8.5, 4.0 Hz, 1H, CH), 3.

  H NMR (300 MHz, CDCl 3 ): δ = 7.27 -7.00 (m, 5H, H Ph ), 4.90 (d, J = 6.3 Hz, 1H, NH), 4.58

	-4.42 (m, 1H, CH), 3.64 (s, 3H, OCH 3 ), 3.11 -2.90 (m, 2H, CH 2 ), 1.34 (s, 9H, (CH 3 ) 3 ).
	13 C NMR (75 MHz, CDCl 3 )μ δ = 174.6, 156.1, 1γ7.γ, 1γ0.0, 1β9.5, 1β7.7, 80.7, 55.4, 5β.9,
	36.8, 29.0.

1

Synthesis of Compounds 12, 12' (a,b); General Procedure:

	Methyl (2S)-2-({[3-(5-{(1S)-1-[(tert-butoxycarbonyl)amino]-2-phenylethyl}-1H-1,2,4-
	triazol-3-yl)pyrazin-2-yl]carbonyl}amino)-3-phenylpropanoate (11'b)
	O
	N	NH	CO 2 Me
	N	N	NHBoc
	HN N	Ph
	Yellow solid; yield: 0.099 g (87%); mp 152-154 °C.
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.96 (d, J = 1.4 Hz, 1H, H Ar ), 8.71 -8.49 (m, 2H, NH amide
	and H Ar ), 7.33 -7.04 (m, 10H, 10×H Ar ), 5.49 (d, J = 8.5 Hz, 1H, NHBoc), 5.40 -5.19 (m,
	1H, CHNHBoc), 5.06 (dd, J = 13.9, 6.2 Hz, 1H, CHCOOCH 3 ), 3.77 (s, 3H, OCH 3 ), 3.41 -
	3.14 (m, 4H, 2×CH 2 ), 1.37 (s, 9H, (CH 3 ) 3 ).
	13 C NMR (75 MHz, CDCl 3 ): δ = 171.79, 164.82, 155.80, 153.55, 147.54, 142.89, 142.66,
	142.21, 137.71, 136.05, 130.19, 129.83, 129.30, 128.82, 127.96, 127.05, 80.04, 54.69, 53.22,
	50.79, 41.71, 38.54, 28.91.
	LC/MS: m/z = 572 [M+H] + .
	1 H NMR (300 MHz, CDCl 3 ): δ = 9.04 (s, 1H, H Ar ), 8.76 (d, J = 6.3 Hz, 1H, NH amide ), 8.69 (s,
	1H, H Ar ), 7.25 -7.09 (m, 5H, 5×H Ar ), 5.66 (d, J = 6.4 Hz, 1H, NHBoc), 5.42 -5.27 (m, 1H,
	CHNHBoc), 4.79 (pseudo-p, J = 7.2 Hz, 1H, CHCH 3 ), 3.82 (s, 3H, OCH 3 ), 3.45 -3.23 (m,
	2H, ), 1.59 (d, J = 7.1 Hz, 3H, CH 3 ), 1.37 (s, 9H, (CH 3 ) 3 ).
	13 C NMR (75 MHz, CDCl 3 ): δ = 173.20, 164.67, 164.44, 155.91, 152.90, 147.87, 143.22,
	142.55, 141.52, 137.61, 130.26, 128.95, 127.20, 80.24, 53.50, 50.78, 49.59, 41.66, 28.98,
	18.74.
	LC/MS: m/z = 496 [M+H] + .

  IntroductionUn composé peptidomimétique est une molécule dont la structure de base est non peptidique, mais dont les motifs structuraux miment les propriétés tridimensionnelles et fonМtionnОllОs НОs struМturОspОptiНiquОs. CОs Мomposés ont l'intérêt НО МonsОrvОr lОs caractéristiques avantageuses reliées aux peptides tout Оn s'affranМhissant НО МОrtains inconvénients. Effectivement, elles sont plus lentement dégradées par les protéases, augmentant ainsi considérablement leur stabilité et leur biodisponibilité.Pourquoi y intégrer une fonction amidoxime? Les amidines sont souvent utilisées comme mimes de la chaîne latérale guanidine de l'arginine, responsable des effets pharmacologiques. Une variété de médicaments et de candidats médicaments possèdent la fonction amidine, comme les inhibiteurs de thrombine, les inhibiteurs de facteur Xa et VIIa, les antagonistes des récepteurs aux glycoprotéines IIb / IIIa, ОtМ…. CОpОndant, en raison de leur grande hydrophilie, ils sont aisément protonés au niveau de l'atome d'azote sp 2 , formant un cation fortement stabilisé dans des conditions physiologiques qui empêche une absorption du tractus gastro-intestinal. Ce problème peut justement être contourné en utilisant des amidoximes comme prodrogues qui seront alors administrées par voie orale. Un groupe amidoxime introduit dans la structure ne sera pas protoné dans ces mêmes conditions physiologiques et peut donc être utilisé pour remplacer une amidine. Cette approche Н'utilisation НОs amiНoximОs comme prodrogues des amidines présente un intérêt croissant en raison НО l'amélioration notable des propriétés physico-chimiques et de biodisponibilité orale.

	Récemment, il a été montré que le système enzymatique N-réducteur est responsable de
	l'activation des prodrogues amidoxime dans les mitochondries. Une autre stratégie pour
	imaginer de nouvellespro drogues est l'utilisation d'amidoximes estérifiées et de 1,2,4-
	oxadiazol-5-ones ou 1,2,4-oxadiazoles avec la fonctionnalité amidine masquée afin de réduire
	le caractère basique et d'améliorer la biodisponibilité.
	UnО НОs stratégiОs possiblОs pour l'élaboration НО pОptiНomimétiques Оst Н'assoМiОr La seconde raison qui a orienté le choix de cette fonction amidoxime est sa
	des noyaux aromatiques ou hétérocycliques avec un motif peptidique. Les acides aminés transformation par diverses enzymes en fonction amide acМompagnéО Н'unО libération
	combinés à des hétérocycles sont devenus des précurseurs importants dans la conception de ultériОurО Н'unО moléМulО НО NO.
	peptidomimétiques. Dans cette thèse, nous décrivons l'étude des nouveaux peptidomimétiques L'oxyde nitrique (NO) est un médiateur endogène gazeux et radicalaire impliqué dans possédant un motif de type pyridine ou pyrazine. Les noyaux pyridiniques et pyraziniques se de nombreux processus physiologiques et joue un rôle clé dans de nombreux systèmes de bio-МaraМtérisОnt par un largО évОntail Н'aМtivités biologiquОs. A titrО Н'ОxОmplОs, lО motif régulation du tonus cardiovasculaire: relaxation vasculaire, inhibition de l'agrégation pyridine est naturellement présent dans les vitamines, l'acide nicotinique (vitamine B3 / plaquОttairО Оt égalОmОnt Нans Н'autrОs fonМtions biologiquОs ОssОntiОllОs μ nОurotransmission, niacine) et la pyridoxine (vitamine B6) et un certain nombre d'alcaloïdes comme la nicotine. De plus, la niacine et le nicotinamide sont des précurseurs des coenzymes nicotinamide réponsО immunitairО, proМОssus inflammatoirОs, lО МhoМ sОptiquО Оt Оnfin l'érОМtion.
	adénine dinucléotide (NAD) et nicotinamide adénine dinucléotide phosphate (NADP) in vivo. En raison de ces nombreuses propriétés, il y a un grand intérêt à élaborer de nouveaux
	composés -donneurs d'oxyde nitrique (donneurs de NO). La première oxydation des Un certain nombre de composés contenant un fragment pyrazine sont en phase finale amidoximes a été décrite par Andronik-Lion et al.en 1992 (Schéma 1) sur la p-hexyloxy-Н'Оssais МliniquОs (VE-821 -un inhibiteur de l'ATR) ou sont déjà commercialisés (bortezomib benzamidoximО qui Оst transforméО Оn présОnМО Н'oxygènО Оn arylamiНО correspondante a -le premier inhibiteur protéasome thérapeutique, eszopiclone -un sédatif hypnotique non libéré de l'oxyНО nitriquО Оn présОnМО НО МytoМhromОs P450 Оt НО NADPH (formО réНuit Нu bОnzoНiazépinО, ОtМ…). nicotinamide adénine dinucléotide phosphate).

, 6 et7 R 5 (rendement %) 6 (rendement %) 7 (rendement %)

  Dans lО Мas Н'utilisation НОs ОstОrs méthyliquОs НО l'alaninО, НО la valinО Оt НО la

	CH 2 STr (CysTr)	h	60	-	94
	La séparation des composés sous forme ouverte 5 et cyclique 6 a été réalisée par
	colonne chromatographique flash (cas des dérivés de la valine et de la leucine). La
	caractérisation des composes de structures ouvertes ou fermées ont été possible par
	comparaison entre les valeurs des déplacements chimiques des protons (RMN) et les données
	obtenues par radiocristallographie.
	L'étapО suivantО pour la synthèsО НО proНroguОs Н'amiНinОs МonsistО Оn la
	c, e-h transformation du groupement cyano en fonction amidoxime. Les amidoximes 7 sont alors
	L'analysО НОs proНuits НО réaМtion ОffОМtuéО par spОМtrosМopiО RMN 1 H confirme, obtenues à partir des esters méthyliques5a,e,f,h et/ou des esters méthyliques 6a,b,e-g avec de
	dans la majorité des cas, que la formation des esters méthyliques 5 des (2S)-N-(2-bons rendements (67-79%) par simplО traitОmОnt avОМ lО МhlorhyНratО НО l'hyНroxylaminО
	cyanopyridin-3-yl)carbonyles substitués a été suivie par une réaction de cyclisation dans le méthanol (Schéma 4, Table
	intramoléМulairО р l'originО Н'un МyМlО pyrrolidinique, générant les pyrrolopyridines
	tautomères 6.
	Me (Ala)	a	15	58	68
	Bn(Phe)	b	-	76	76
	Pro-résidu	c	87	-	70
	H(Gly)	d	-	58	-
	i-Pr (Val)	e	37	34	79
	i-Bu (Leu)	f	9	76	70
	Trp-résidu	g	-	83	67

leucine, la formation des deux produits 5 et6 est observée avec des ratios différents, alors qu'au МontrairО Нans lО Мas НО la phénylalaninО, НО la glyМinО Оt Нu tryptophanО, lОs ОstОrs cycliques 6b,d,g sont isolés exclusivement (Tableau 1). LО rОnНОmОnt global Н'obtОntion НОs dérivés 5 et 6 Оst НО l'orНrО НО 60-80% après purification par colonne chromatographique. Tableau 1 Rendements de la réaction de formation des composés 5

  Nous avons également décidé d'étendre ce protocole afin de synthétiser de nouveaux pseudotripeptides avec le motif «pyrazineamidoxime» en position N-terminale dans le squelette. Le dipeptide proline-phénylalanine a été sélectionné afin de pouvoir étudier les effets stériques de la substitution C-terminale de la proline sur l'équilibre d'isomère amide du substitut simplement substitué par la proline (modèle). De même, la substitution ortho sur un

	fragmОnt non pОptiНiquО par un НipОptiНО Нoit pОrmОttrО Н'augmОntОr la rigidité
	O Le couplage peptidique de l'acide 4'avec le dipeptide H-Pro-Phe-OMe a été réalisé en N H 5 NOE conformationnelle. CO 2 Me H H 4
	N présence de HATU/HOBt Оt a pОrmis Н'isolОr le nitrile correspondant 5'd avec un rendement N de 68%. Le traitement de ce dernier avec du chlorhydrate d'hydroxylamine en présence de
		Figure6 Corrélation observée par effet NOE dans le dérivépyridinique5c
	Tableau 3 Population cis/trans du lien amide des isomères 5c, 5'c, 7c-9c, 7'c-9'c mesurée à
	partir du spectre RMN 1 H effectué dans le CDCl 3
	X		Ratio pour 5 (trans/cis %)	Ratio pour 7 (trans/cis %)
	CH	c	77/23	68/32
	N	'М	54/46	74/26

Les attributions des dérivés de pyrazine couplés à la proline 5'c, 7'c ont été réalisés après étude des déplacements chimiques des spectres RMN

13

C (effectués dans le CDCl 3 ) des atomes de carbones α et δ. En effet dans les dérivés de la proline, le signal correspondant au carbone α du rotamère trans est blindé par rapport à celui du rotamère cis en raison de la position syn par rapport р l'atomО Н'oxygènО Нu МarbonylО НОs amides. Le signal du carbone δ, devrait être cependant plus blindé dans l'isomère cis. En effet, dans les conformères trans de 5'c, 7'c, les déplacements chimiques du carbone α se situent entre 59,3 et 60,2 ppm tandis que dans la forme cis, ils se retrouvent de 61,1 à 61,4 ppm. Les populations des deux isomères cis et trans de la liaison amide ont été mesurées grâce au spectre RMN 1 H par intégration des signaux bien résolus. Les contributions majoritaires de ce mélange ont été clairement déterminées par spectroscopie RMN comme étant les conformères trans (Tableau 3). triéthylamine a permis d'obtenir la molécule cible 7'd avec un bon rendement (Schéma 7).

  Les résultats obtenus à partir des analyses infrarouge ont ensuite été corroborés par des expériences 1D RMN dans des mélanges de solvants qui montrent que dans les conditions de la RMN à chaque instant deux jeux de signaux sont présents pour chaque point.

								-1
								(C=N)
	AOPzPhe	1743/1691			1680		1659
	AOPzPro	1743			1655		1642
	AOPzProPhe	1743/1722			1679
						1670/1656
	δ (ppm)	8.1 8.3
	Chemical Shift	7.5 7.7 7.9	0	20	40	60	80	100
				%DMSO (gradient DMSO in CDCl3)

1653 Le nombre de bandes dans les spectres infrarouge indique qu'au moins deux ou trois groupes carbonyle sont impliqués dans des liaisons hydrogène: ils ont été attribués en fonction de leur longueur Н'onНО: les deux bandes à 1743 cm -1 et 1722 cm -1 correspondent au C=O du groupe ester, respectivement libre et impliqué dans une liaison hydrogène. Les quatre bandes suivantes ont été attribuée à partir des spectres infrarouge des peptides plus petits 7'b et 7'c, comprenant un acide aminé et un motif pyrazineamidoxime (Tableau 4). Donc la vibration Н'élongation Н'un CO amide à 1679 cm -1 correspond au groupe carbonyle de la proline et est proche des valeurs obtenues pour AOPzPhe (7'b). Les bandes à 1670 cm -1 et 1656 cm -1 ont été attribuées au carbonyle amide entre le groupe pyrazine et le dipeptide proline-phenylalanine sous les formes liée et libre.

  (a,b) avec des rendements de 64-91%. La cyclodéshydratation assistée par micro-ondes, a quant à elle, donné les 1,2,4-oxadiazoles 9, 9' avec des rendements de 63-94%. Les rendements sont meilleurs en ce qui concerne les 1,2,4-oxadiazoles en série pyrazine et ceci peut être expliqué par l'effet stabilisant d'une interaction NH...N seulement envisageable sur ces dérivés de la pyrazine. En série pyridine, une réduction du rendement de l'orНrО НО 10% a été obsОrvéО suitО р la formation Оt НétОМtion par analysО LC/MS Н'un sous produit non désiré de réaction (pyrrolopyridine 6, Tableau 6).Ensuite, la L-phénylalanine a été introduite sur des amidoximes de proline 7c et 7'c afin d'étudier la propension de la liaison amide à être en conformation cis (qui est élevée dans le cas où la proline est précédée d'un résidu aromatique). Par conséquent, les amidoximes 7c,

		Les spectres infrarouge des composés amidoxime estérifiés 8, 8' montrent que dans le
	chloroforme, à une concentration de 10 mM seulement des NH non investis dans des liaisons
	hydrogène sont visibles.
		Les spectres infrarouge des dérivés pyrazine Ala-et Phe-9a,b, montrent que dans les
	mêmОs МonНitions НОux banНОs МorrОsponНant р l'élongation НОs liaisons NH ont НОs valОurs
	au-dessus de 3400 cm -1 . Alors que pour les dérivés pyrazine 1,2,4-oxadiazoles les fréquences
	de vibrations observées sont 3398 cm -1 et 3395 cm -1 correspondant à des NH impliqués dans
	des liaisons hydrogène.
		N		CO 2 Me					N	CO 2 Me			N	CO 2 Me
	N X	O N NH 2	OH	Boc-Phe-OH DCC, 4-DMAP CH 3 CN	X N	O N NH 2	O	O	NHBoc Ph	 150 °C 25 min DCM	X N	O N N O	NHBoc Ph
	CH 7,7' c		Me (Ala)	a			8,8' c	64			63 (66) 9,9' c
	CH			Bn (Phe)	b					91			81 (83)
	N X CH			Me (Ala) 8c	a	Rendement, % quantitatif	84		Conversion, % 75 (77) -
	N a Rendement après purification. Bn (Phe) CH 9c N 8'c N 9'c b Conversion déterminée par analyse LC/MS. b quantitatif 58 76 Le rapport entre les différents rotamères a été défini à partir des spectres RMN 1D 1 H 67 94 (96) 65 -80
	Les esters 8, 8' ont été chauffés sous irradiation par micro-ondes pour donner les et 13 C ainsi que des spectres 2D-NOESY. Dans les spectres NOESY de 8c et 9c, des pics de
	1,2,4-oxadiazoles 9, 9'. Différents solvants, températures et temps de réaction ont été explorés Мorrélation inНiquОnt quО l'isomèrО majoritairО Оst l'isomèrО cis. En ce qui concerne les

afin de trouver les conditions permettant la meilleure conversion. Par cette technologie, la réaction effectuée dans le dichlorométhane à 150 °C pОnНant β5 minutОs a pОrmis Н'obsОrvОr la meilleurО МonvОrsion (НétОrminéО par analysО par LC/MS) Оn un tОmps optimal Оt Н'obtОnir les O-acylamidoximes 8, 8' 7'c ont été mises en contact avec la phénylalanine N-protégée pour former les esters attendus 8c et 8'c avec des rendements quantitatifs (Tableau 6). Enfin, la condensation effectuée sous irradiation micro-ondes de la solution de 8c, 8'c dans du dichlorométhane (récipient fermé, 300W, 150 °C, 25 min) a fourni les dérivés de type 1,2,4-oxadiazole 9c et 9'c avec des rendements respectifs de 58% et 76%. Tableau 7 Synthèse de pseudopeptides possédant le cycle 1,2,4-oxadiazole (résidu proline) dérivés proline pyrazine 8'c et 9'c l'attribution des déplacements chimiques des carbones α et δ des spectres enregistrés dans le CDCl 3 ont été définis par rapport à celles des dérivés 5'c et 7'c précédemment réalisées. Dans les spectres infrarouge des dérivés pyrazine oxadiazole 9'c on observe des bandes à 3438 cm -1 et 3363 cm -1 correspondant aux NH libres et liés dans une liaison hydrogène respectivement. Ces bandes sont présentes à 10 mM ainsi qu'р НОs МonМОntrations НiluéОs Мonfirmant l'absОntО Н'intОraМtion intermoléculaires. L'analyse de la région des vibrations d'élongation НОs CO, Нans lО CHCl 3 n'a pas pОrmis l'attribution des bandes correspondant aux CO liés, probablement à cause de leur faiblО intОnsité Оt Нu rОМouvrОmОnt avОМ Н'autrОs banНОs. Nous avons égalОmОnt supposé qu'il pouvait exister une interaction faible entre le NH, lorsquО lО CO НО l'ОstОr Оst oriОnté vОrs l'ОxtériОur pour formОr unО intОraМtion C=O...C=O n * (Figure

Tableau 8

 8 Energies absolues et relatives (unité atomiques et kcal/mol, respectivement) pour les isomères des N-acylamidrazones calculées (B3LYP , 6-31G(d,p))

	X, R	E I [a.u.]	E II [a.u.]	E III [a.u.]	E IV [a.u.]	ΔE [kcal/mol]
	CH, Me (10a) -1751.286540 -1751.294366 -1751.280429 -1751.277908	4.91
	CH, Bn (10b) -1982.258785 -1982.265883 -1982.256207 -1982.252817	4.45

  Pour НОs quОstions Н'optimisation НО la quantité НО МytoМhromОs P450 issus НО microsomes de foie mis à notre disposition, les composés présentant la plus forte libération de NO ont été testés trois fois (7c, 7'c, 7h), ceux avec des valeurs plus modérées ont été testés deux fois (7b, 7'a, 7e, 7f) et les produits possédant la capacité de libération de NO la plus faible seulement qu'une seule fois (7'b, 7a, 7g). La comparaison НО l'ОffОt attОnНu a pu être effectuée avec la molécule modèle décrite dans la littérature (4-chlorobenzamidoxime), connue pour sa bonne capacité de libération de NO.Une corrélation provisoire entre les valeurs des analogues en série pyridine et pyrazine (7,7'(a)-7, 7'(c)) permet de conclure que le meilleur effet de libération de NO est observé pour les composés en série pyridine (résidus Phe et Pro). Par contre, concernant les amidoximes avec un résidu Ala, le produit au noyau pyrazinique semble être plus efficace que son homologue pyridinique. La corrélation entre les chaînes latérales d'acides aminés et la concentration en nitrite des dérivés pyridine permet un classement: PhО Val Cys(Tr) Pro LОu Trp Ala.Il est cependant à noter que dans le cas des composés en série pyrazine, il est observé une capacité opposéО р la libération НО NOμ Ala Pro Phe.

	N chlorobenzamidoxime CO 2 Me
	N		O N	OH			1.34	2.62	1.22	1.73	0.776
			NH 2				7c
		N				CO 2 Me
	N N	O NH 2 N			OH	7'c	1.95	0.98	0.53	1.15	0.726
		O				STr
	N		NH N	OH CO 2 Me	3.81	0.99	1.15	1.98	1,584
		NH 2						7h
		O
			NH			CO 2 Me	1.50	2.62	-	2.06	0.792
	N		N		OH
		NH 2						7b
		O
	N	NH				CO 2 Me	0.98	-	-	0.98	-
	N	N NH 2	OH		7'b
		O
	N		NH N	OH CO 2 Me	0.83	-	-	0.83	-
		NH 2						7a
		O
	N N	NH N NH 2	OH CO 2 Me	7'a	1.95	1.81	-	1.88	0.099
		O
	N		NH N	OH CO 2 Me	1.60	2.42	-	2.01	0.580
		NH 2						7e
		O
	N		NH N	OH CO 2 Me	1.45	1.40	-	1.43	0.035
		NH 2					7f
							NH
		O	NH			CO 2 Me	1.10	-	-	1.10	-
	N		N	OH
		NH 2						7g
			4-			2.40	3.39	1.26	2.35	1.066

H NMR (300 MHz, CDCl 3 ): δ = 8.46 (d, J = 3.6 Hz, 1H, H Ar ), 7.76 (dd, J = 7.7, 1.2 Hz, 1H,

H NMR (300 MHz, CDCl 3 )μ δ = 9.46 (s, 1H, NH), 8.8β (Н, J =

2.6 Hz, 1H, H Ar ), 8.75 (d, J =

ACKNOWLEDGMENTS

Lorraine for the collaboration on the NO-donors project and the biological testing of my compounds.

Chapter 3: Structural Analysis Chapter 4: Preliminary Biologycal Evaluation of Amidoximes as NO Donors

Chapter 5: Experimental part g/sm -3 , (MoKα) = 0.111 mm -1 , F(000) = 456. 10110 rОflОМtions mОasurОН up to βθ max = 64.4°, 3209 unique (R int = 0.0218) which were used in all calculations. Refinement was converged at wR 2 =0.1334 (all data), R 1 =0.047γ (βγγβ rОflОМtions with I>βσ(I)), GoF=1.0γ. unique (R int = 0.0129) which were used in all calculations. Refinement was converged at wR 2 =0.0957 (all data), R 1 =0.0γ54 (4β79 rОflОМtions with I>βσ(I)), GoF=1.0β.

X-ray Diffraction Study of 7'b

The structure was solved by direct method using OLEX2 software. Visualization and analysis of crystal structure was done in Mercury 3.8. 152 Positions of the hydrogen atoms were located from ОlОМtron НОnsity НiffОrОnМО maps anН rОfinОН by "riНing" moНОl with Uiso = nUОq of thО carrier atom (n = 1.5 for methyl and OH groups, n = 1.2 for other hydrogen atoms). Fullmatrix least-squares refinement of the structure against F 2 in anisotropic approximation for non-hydrogen atoms was converged to wR 2 = 0.104 (R 1 = 0.0390 for 3341 reflections with The structure was solved by direct method using OLEX2 software. Positions of the hydrogen atoms wОrО loМatОН from ОlОМtron НОnsity НiffОrОnМО maps anН rОfinОН by "riНing" moНОl with Uiso = nUeq of the carrier atom (n = 1.5 for methyl and OH groups, n = 1.2 for other hydrogen atoms). Full-matrix least-squares refinement of the structure against F 2 in anisotropic approximation for non-hydrogen atoms was converged to wR 2 = 0.1181 (R 1 = 0.045β for 681 rОflОМtions with I>βσ(I), S = 1.0918).