Structural and thermodynamic studies of proline-containing peptidomimetics

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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;46-48 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 acid46 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 chloroformate49 followed by selective hydrolysis of ester group with 1M NaOH to give the corresponding acid 4’ in 71% total yield (Scheme 2.2).

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).4,5 base N OH base S RCN + NH2OH.HCl R + NH2OH.HCl NH2 R NH 2.
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.
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 electron-withdrawing 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, R1, R2 = H). Treatment of amines with ammonia affords N-unsubstituted amidoximes respectively (Scheme 2.4). N OH N OH base R NCS R H Cl  C NO R1R2NH R N OH NR1R2.
Other syntheses of amidoximes, involve the amidoxime-derived starting materials, thus are less general, can be realized by reactions: a) reduction of nitrosolic acid RC(=NOH)-NO with H2S and nitrolic acid RC(=NOH)-NO2 by a reduction with Pt; reduction of hydroxyamidoximes like PhC(=NOH)-NHOH with SO2; b) action of hydroxylamine on amidine hydrochlorides RC(=NH)NH2.HCl, amidrazones RC(=NH)-NHNH2, iminoethers RC(=NH)-OR’ and on imidoyl chlorides RC(=NH)-Cl; c) action of ammonia and amines on oximinoethers RC(=NHOH)-OR’; d) a reductive ring opening of 1,2,4-oxadiazoles or 1,2,4-oxadiazolin-5-ones upon reduction with LiAlH4 and 1,2,5-oxadiazoles with H2 on a Pd/C catalyst.4

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-α-amino 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 7-imino-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.
To investigate the scope of this novel method, the coupling of 2-cyanonicotinic acid (4) with several commercially available methyl esters of L-α-amino acids (such as Ala, Phe, Pro, Gly, Val, Leu, Trp and CysTr) was examined (Scheme 2.5). As activating agent, N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in the presence of 1-hydroxybenzotriazole (HOBt) and triethylamine was used.
1H NMR analysis of the reaction products confirmed that, in most cases, the formation of open chain methyl esters of (2S)-N-(2-cyanopyridin-3-yl)carbonyl substituted amino acids 5 was followed by further intramolecular cyclization and giving a pyrrolidine ring and production of the tautomeric methyl esters of (2S)-2-(7-imino-5-oxo-5,6-dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)alkanoic acids 6 (Scheme 2.5). The reaction proceeds with nucleophilic attack by the amide nitrogen lone pair on the the sp carbon and further transfer of a proton.

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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-2-carboxamides with amidoxime groups at position 3. The synthesis was carried out in two steps utilizing the strategy elaborated before for the pyridine series61: coupling of 3-cyanopyrazine-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 protocol61 (Table 2.4). As an activating agent N-ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) in the presence of 1-hydroxybenzotriazole (HOBt) was used. As a solvent, tetrahydrofuran instead of dichloromethane was taken due to the low solubility of the starting material in dichloromethane.

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 1,2,4-oxadiazoles have been utilized in the design of mimetics, as peptidomimetic building blocks,69-73 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).

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 nОw 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, Boc-protected 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. 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.80

Table of contents :

Chapter 1: Introduction
1. Introduction
2. Pyridine and pyrazine heterocycles as scaffolds for peptidomimetics
3. Amidoximes and masked amidoximes as prodrugs of amidines, an arginine mimics .
4. Amidoximes as NO donors
Chapter 2: Synthesis of Pyridine and Pyrazine-based Peptidomimetics
1. Introduction
2. Synthetic plan
3. Synthesis of starting 2-cyanonicotinic and 3-cyanopyrazine-2-carboxylic acids
4. Synthesis of peptidomimetics bearing amidoxime function
4.1 Synthesis of amidoximes
4.2 Synthesis of pyridine based amidoximes
4.3 Synthesis of pyrazine based amidoximes
5. Studies towards the synthesis of 1,2,4-oxadiazoles via amidoxime ester units
5.1 Overview of synthetic approaches to 3,5-disubstituted 1,2,4-oxadiazoles via amidoxime esters
5.2 Acylation of amidoximes with further conversion into amino acid derived 1,2,4- oxadiazoles
6. Studies towards the synthesis of 1,2,4-triazoles via N-acylamidrazones
6.1 Overview of synthetic approaches via N-acylamidrazones
6.2 Synthesis of pyridine(pyrazine)-based N-acylamidrazones with further conversion into amino acid derived 1,2,4-triazoles
7. Synthesis of hydrazide modified turn mimics
8. Conclusions
Chapter 3: Structural Analysis
1. Introduction
2. Methods and techniques of conformational study
2.1 Infrared absorption spectroscopy (IR)
2.2 Nuclear Magnetic Resonance spectroscopy (NMR)
2.2.1 One dimensional NMR
2.2.2 Two dimensional NMR
2.3 Molecular modeling
3. Structural and thermodynamic studies of proline-containing peptidomimetics
3.1 Conformational preferences and thermodynamic studies of proline derivatives .
3.2 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
3.2.1 FT-IR and NMR investigations
3.2.2 Molecular modeling
3.2.3 cis-trans isomerization study
3.2.4 A tentative correlation between toxicity and structure
4. Stuctural analysis of esterified amidoximes and oxadiazoles 8, 8’ and 9, 9’
4.1 Conformational analysis of alanine and phenylalanine derivatives 8, 8’ (a,b) and 9, 9’ (a,b)
4.2 Conformational analysis of the proline derivatives 8c, 8’c and 9c, 9’c
5. Structural and thermodynamic studies of acylamidrazones 10, 10’
6. Stuctural analysis of hydrazide modified peptidomimetics 12, 12’
7. Conclusions
Chapter 4: Preliminary Biologycal Evaluation of Amidoximes as NO Donors
General conclusions and perspectives
Chapter 5: Experimental
1. General Methods
2. Experimental procedures
References 

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