Pseudopeptides based on nicotinic acid with 4-amidoxime unit

The carbodiimide-mediated coupling of 4-cyanonicotinic acid with L-amino acid methyl esters takes place with the predominant formation of bicyclic compounds – methyl 2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)alkanoates, which are monoimino derivatives of 4-azaphthalimide. Under the action of hydroxylamine they undergo pyrrolidine cycle opening with the formation of pseudopeptides – coupling products of nicotinic acid with amino acid esters bearing an amidoxime function at position 4.

Amidoximes serve as amidine precursors due to their ability to be reduced by mARC-containing enzyme system to the amidines,¹ which are effective mimetics of the guanidine moiety of arginine in the development of anticoagulant drugs. Substitution of amidine group with amidoxime group improves oral bioavailability and has allowed the development of an "amidoximes instead of amidines" prodrug principle.² Amidoximes can also be oxidized in vivo to serve as donors of nitric oxide (NO)^3,4 that plays a crucial role in numerous physiological and pathological processes.⁵ Peptidomimetics with a benzamidine, as well as a benzamidoxime moiety have recently been discovered as potent and selective noncovalent furin inhibitors for the treatment of SARS CoV-2 virus infection.⁶ Recently, luminescence properties were found in the solid state of mixed-ligand Zn(II) acetate complexes with pyridine-4-amidoxime ligands.⁷

In continuation of our research on heterocyclic peptidomimetics possessing nonpeptidic β-turn structures, we herein report an efficient synthesis of novel pseudopeptides based on nicotinic acid that are positional isomers of the previously studied pseudopeptides, containing an amidoxime group at position 4 instead of position 2 of the pyridine nucleus.⁸

The synthetic route to the target compounds can be similar to those previously elaborated.^8,9 The scheme consisted of sequential coupling of 2-cyanonicotinic acid with esters of α-amino acids and subsequent transformation, by reaction with hydroxylamine, into amidoximes as individual derivatives of intermediate methyl esters of (2S)-N-[(2-cyanopyridin-3-yl)carbonyl]amino acids or cyclic tautomeric methyl esters of (2S)-2-(7-imino-5-oxo-5,7- dihydro-6H-pyrrolo[3,4-b]pyridin-6-yl)alkanoic acids, and their mixtures. In this work, 4-cyanonicotinic acid was coupled with amino acid esters using 1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (EDCI) as the coupling reagent. Obtained pseudopeptides were then treated with hydroxylamine.

According to the proposed route, the first task was to synthesize the starting 4-cyanonicotinic acid (1) (Scheme 1). An attempt was made to synthesize it by the method used for the 2-cyano isomer in our previous work,⁸ starting from the cinchomeronic anhydride through 4-carbamoylnicotinic acid (17% yield), which was transformed by interaction with methyl chloroformate into methyl 4-cyanopyridine- 3-carboxylate (9% yield) and then into acid 1 by a saponification. However, our efforts to select and to modify the reaction conditions did not allow to improve the low yields.

Scheme 1

Instead, we succeeded in synthesizing 4-cyanonicotinic acid (1) by ortho-lithiation of isonicotinonitrile (2) using lithium 2,2,6,6-tetramethylpiperidine (LiTMP) followed by carboxylation with dry ice according to modified literature procedure¹⁰ (Scheme 1). Based on the experimental results, we found that only a slight excess of LiTMP (1.15 equiv) was necessary to improve the yield of acid 1 in contrast to 2 equiv used in the original procedure. Carboxylation was carried out by pouring the reaction mixture on dry ice under Ar with vigorous stirring, which was also found more effective than the reverse addition of the electrophile to the lithiated isonicotinonitrile (2). The modification of the synthetic procedure allowed to obtain 4-cyanonicotinic acid (1) with an improved yield of 61%, which is higher compared to the 41% of the original procedure.

Subsequent coupling of 4-cyanonicotinic acid (1) with methyl esters of a number of L-α-amino acids (Gly, Ala, Val, Leu, Phe, Trp, Asp, Glu, Pro) was carried out using EDCI as the coupling agent in the presence of 1-hydroxybenzotriazole hydrate (HOBt).^8,9 The cyclization of cyanonicotinamides 3a–h occurred in situ. The total yield of amino acid derivatives 4a–h after purification by column chromatography was 80–88% (Table 1).

Table 1. NH proton chemical shifts and yields of compounds 4a–h

It was interesting to investigate how fast the coupling of 4-cyanonicotinic acid (1) proceeds and whether the basicity of tertiary amine used affects its final result. For a comparative experiment, a weaker base N-methylmorpholine (NMM) (pK_a 7.38) was chosen instead of Et₃N (pK_a 10.75), in the presence of which the cyclization rate of compounds 3a–h should not be fast.11 Thus, in the reaction of acid 1 with the aspartic acid dimethyl ester using NMM, the primary product with m/z 292 [M+H]⁺ (LC/MS), which apparently corresponded to cyanonicotinamide 3g (t_R 2.756 min), transformed into pyrrolopyridine 4g (t_R 2.878 min coincided with that of the individual product) much more slowly in the first hours of coupling compared to the reaction when Et₃N was used (Table 2). At the same time, when using NMM, the conversion of acid 1 into cyanonicotinamide 3g occurred faster. As shown by LC/MC results in both cases, the cyclization of amide 3g to compound 4g was completed in about 3 days.

Table 2. The ratio of acid 1 to cyanocarboxamide 3g and pyrrolopyridine 4g in reaction mixtures^*

Along with the absorption bands of the two C=O groups in the regions 1752–1736 and 1732–1711 cm^–1, in IR spectra of compounds 4a–h, C=N stretching vibrations appear at the region 1667–1658 cm^–1, and a sharp strong NH band appears at 3408–3209 cm^–1. An absorption band of the cyano group at the 2200 cm^–1 region which might belong to open form 3 is not observed in contrast to the derivatives of 2-cyanonicotinic acid.⁸

In comparison to the previously studied amino acid derivatives of 2-cyanonicotinic acid,⁸ for which the bicyclic tautomeric form based on dihydro-6H-pyrrolo[3,4-b]pyridine has been dominant along with acyclic 2-cyano-substituted amides, coupling products 4a–h have predominant cyclic tautomeric form of methyl 2-(1-imino-3-oxo-1,3-dihydro- 2H-pyrrolo[3,4-c]pyridin-2-yl)alkanoates. Characteristic signal of the C=NH imino group proton at the region of 10 ppm in the ¹H NMR spectra supports this hypothesis (Table 1). As follows from ¹H NMR spectra of compounds 4a–h (see Supplementary information file), they adopt the E-configuration at imino C=N bond when NH proton is orientated toward the aromatic ring in a similar manner to what was observed previously for isomeric compounds.⁸

Thus, the structure of pyrrolopyridine 4d was confirmed using the following NMR experiments (DMSO-d₆): ¹H, ¹³C-APT, COSY, ¹H–¹³C HSQC, ¹H–¹³C HMBC, and NOESY experiments (see Supplementary information file). It should be noted that the NOE correlation between the NH proton and the aromatic H-5 Py proton (position 5 of the pyridine ring) at 8.17 ppm is relatively strong (3%), indicating significant spatial proximity of these protons in the molecule.

Furthermore, it is interesting to mention the differences observed in the 1H NMR spectra of compound 4d when measured in DMSO-d₆ and CDCl₃. Both NH and H-5 Py being in proximity exhibit significant broadening in CDCl₃ compared to DMSO-d₆. This phenomenon can be attributed to the poor solvation of NH group in CDCl₃ as well as the slight acidity of CDCl₃. These factors can induce slow (in the NMR time scale) exchange processes involving the NH group, leading to its broadening, and consequently, the broadening of the H-5 Py proton peak at 7.66 ppm due to its spatial proximity.

The analysis of the 1H NMR spectra of pyrrolopyridine 4a in DMSO-d6 shows doubling of all proton signals, and most importantly of the NH proton signal, due to the probable occurrence of a mixture of Z- and E-isomers in the ratio of approximately 15:85. Prior to conducting the NOESY experiment on substrate 4a mixture illustrated in Figure 1, we have performed a temperature-dependent 1H NMR at 80°C in DMSO-d₆ (see Supplementary information file). The results indicated that the doubling of all signals in the ¹H NMR spectrum occurs neither due to rotamery issues of the aliphatic substituent nor slow (on the NMR time scale) E/Z isomerization at C=N bond.

Figure 1.

Structures of the E- and Z-isomers of compound 4a, confirmed by NOESY experiment (DMSO-d₆). ¹H NMR chemical shifts are shown in ppm.

The NOESY experiment proved (see Supplementary information file) to be highly beneficial in distinguishing the E- and Z-isomers of molecule 4a, as all relevant signals were well resolved in the ¹H NMR spectrum (Fig. 1). For the E-isomer, a strong NOE was observed between the pyridine H-5 Py at 8.19 ppm and NH at 10.52 ppm. Conversely, for the Z-isomer, an NOE was observed between the aliphatic CH₂ group at 4.65 ppm and NH at 9.92 ppm. In the ¹H NMR spectrum of compound 4a, a high-field shift of the NH and H-5 Py signals of the Z-isomer was observed relative to the signals of the corresponding protons of the E-isomer. The signals of the aliphatic fragment, on the contrary, were shifted to the low field in Z-isomer.

In the ¹³C NMR spectrum (APT) of compound 4a, a noticeable difference between E- and Z-isomers was also observed in the position of the carbon atom signals of the amidine fragment N=C–N (157.5 ppm (E) vs. 153.4 ppm (Z)), as well as signals of C-4 Py atom (139.0 ppm (E) vs. 143.9 ppm (Z)) and C-5 Py (117.2 ppm (Z) vs. 117.9 ppm (E)).

Slightly noticeable signals of the corresponding minor Z-isomers occurred in the same region in the ¹H NMR spectra of other synthesized acylamidines 4b–h at a level not exceeding 3%. This is possibly due to greater steric hindrances in the formation of Z-isomers affecting the cyclization of the corresponding cyanocarboxamides 3b–h.

¹H NMR spectra of pyrrolopyridines 4a–h in CDCl₃ showed only one set of signals. The NH signal appeared as a broadened singlet at about 9 ppm, high-field shifted by more than 1.5 ppm compared to the corresponding signal recorded in DMSO-d₆. In addition, the H-5 signal had the shape of a broadened doublet or a broadened singlet of reduced intensity. In the ¹³C NMR spectra in CDCl₃, the signals of C-4 Py and C-5 Py atoms during prolonged accumulation of the spectrum have been detected as barely noticeable broadened singlets of low intensity. This may be due to the dynamic processes in the molecules of compounds 4a–h occurring in CDCl₃ solution.

Compounds 4a–h are the corresponding monoimino derivatives of pyrrolo[3,4-c]pyridine-1,3(2H)-dione that have not yet been reported. The most studied are the 4-aza analogs of phthalimide, and cinchomeronic acid anhydride interaction with various amines is used in the synthesis of N-substituted 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-diones.¹² A milder procedure is the amination of methyl 3-chlorocarbonylisonicotinate, that was prepared from cinchomeronic acid obtained by conversion of the starting anhydride.¹³ N-Benzyl-substituted pyrrolo[3,4-c]pyridine- 1,3(2H)-diones have raised a particular interest as a new class of antimycobacterial substances.^14,15

N-Substituted 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives have been obtained as byproducts of synthesis of the corresponding imines derived from the hydrolysis of the imino group. Thus, during the isolation of the target reaction products, in particular, acylamidine 4b, a standard washing of the reaction mixture with 0.1 M HCl was used.^8,9 However, even short-term acid exposure reduced the yield of the target compound 4b due to the hydrolysis of the exocyclic imino group with the formation of dioxopyrrolopyridine 5b (Scheme 2). Compound 5b was isolated by flash chromatography, and its structure was confirmed by LC/MS, HRMS, NMR, and IR spectroscopy.

Scheme 2

It should be noted that the formation of hydrolysis products similar to compound 5b was observed even during the LC/MS analysis of purified acylamidines 4a–h, which is probably due to the use of a mobile phase consisting of formic acid (0.1%, v/v) via gradient elution. Another reason for the decrease in the yield of compound 4b during CH₂Cl₂ extraction was its partial solubility in water. Therefore, brine was subsequently used to wash the reaction mixture.

The dihydropyrroline ring of methyl (E)-2-(1-imino- 3-oxo-1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)alkanoates 4a–h under the action of hydroxylamine undergoes ring opening to give methyl esters of N-{4-[amino(hydroxyimino) methyl]pyridin-3-yl}carbonyl-substituted α-amino acids 6a–h as observed for similar previously studied compounds (Scheme 3).^8,9 However, the yields of compounds 6a–h using the previously described method were quite low and did not exceed 50%. Their noticeable solubility in water along with other reasons contributed to the loss of the products during the isolation. The replacement of Et₃N with NaHCO₃ to generate free NH₂OH in situ and avoiding the use of water during the isolation of the target compounds made it possible to increase their yields to more than 80%.

Scheme 3

As in the case of similar isoindolones,⁹ the formation of an undesirable but expected side product with m/z 164 [M+H]⁺, which undoubtedly assigned as 1-(hydroxyimino)-1,2- dihydro-3H-pyrrolo[3,4-c]pyridin-3-one, was observed. The content of the latter increased with an extension of time of keeping the reaction mixtures without separation, as well as with even a slight heating, which led to the decrease of the yields of open-chain compounds 6a–h. Therefore, it was necessary to work up the reaction mixture no later than 16 h after the start of the reaction, and to carry out all solvent evaporation at a temperature not higher than 40°C.

In ¹H NMR spectra of compounds 6a–h, characteristic two-proton signals are observed approximately at 5.8 ppm and intense single-proton signals at ~9.9 ppm which correspond to NH₂ and OH protons, respectively, of N'-hydroxycarbamimidoyl group. In the ¹³C NMR spectra of compounds 6a–h, the carbon signal of the amidoxime group located approximately at 150.6 ppm and only in the case of proline derivative 6i is shifted to high field (149.6 ppm), i.e., in general, this signal is shifted to high field by about 7 ppm in comparison to cyclic acylamidines 4a–h. It is also worth to note the shift of the H-5 Py signal to the high field by approximately 0.6 ppm associated with the Z-configuration of the C=N–OH double bond. In the ¹H NMR spectrum of amidoxime 6d in CDCl₃, the OH signal is not observed, probably due to rapid proton exchange. The position of the CONH group signal at 7.86 ppm should also be noted, which is shifted by almost 1 ppm to high field compared to this signal in DMSO-d₆ solution.

Single crystals of valine-derived amidoxime 6c (R = i-Pr) suitable for X-ray diffraction analysis have been grown from saturated CHCl₃–MeOH, 9:1 solution (Fig. 2). Compound

Figure 2.

Molecular structure of methyl (Z)-[4-(N'-hydroxycarbamimidoyl) nicotinoyl]-L-valinate (6c) with the atom numbering scheme. Thermal ellipsoids are shown at 50% probability level.

6c crystallizes in the chiral space group P2₁ that confirms the existence of a pure enantiomer in the crystal phase. The absence of a heavy atom makes it impossible to determine the absolute configuration of the asymmetric center C(8).

Two substituents of the pyridine ring are located in a vicinal position, which causes a significant steric repulsion between them. This repulsion is compensated by twisting of the exocyclic C(6)–C(3)–C(4)–C(7) angle up to 10.3(6)° and significant rotation of both substituents with respect to the aromatic cycle: C(5)–C(4)–C(7)–O(5) and C(4)–C(3)–C(6)–N(2) torsion angles are 62.3(6)° and 45.1(6)°, respectively. The C(4)–C(7) and C(3)–C(6) bond lengths (1.491(6) and 1.486(6) Å) also confirm some disturbance of conjugation between the π-systems of the carbamide fragment or the amino(hydroxyimino) methyl group and the aromatic cycle. In the crystal phase molecules 6c form layers parallel to the (100) crystallographic plane due to the formation of the N–H···O, N–H···N, and O–H···O intermolecular hydrogen bonds (Table 3).

Table 3. Characteristics of intermolecular hydrogen bonds in the crystal of compound 6c

Unlike other amino acids, in the case of proline, the formation of only a noncyclic product is clearly possible (Scheme 4). The presence of a partially double peptide bond N–CO leads to the appearance of cyanonicotinamide 3i as a pair of cis- and trans-isomers, in a ratio of 7:3. Similarly to the previously studied proline derivatives of 2-cyanonicotinic acid,⁸ it can be assumed that the major product (≈71%) is the trans-isomer, and the minor product is the cis-isomer (≈29%). Under similar conditions, a Pro amidoxime 6i was synthesized from cyano derivative 3i. Its ¹H NMR spectrum reveals doubling of signals, the intensity of which suggests that the ratio of trans-/cis-isomers is ~65:35.

Scheme 4

In conclusion, the dominant pathway of 4-cyanonicotinic acid coupling with L-amino acid methyl esters is the formation of bicyclic compounds methyl 2-(1-imino-3-oxo- 1,3-dihydro-2H-pyrrolo[3,4-c]pyridin-2-yl)alkanoates. Under the action of hydroxylamine they undergo the pyrrolidine cycle opening with the formation of the nicotinic acid based pseudopeptides with amidoxime function at position 4. The obtained substances can serve as precursors in the synthesis of pseudopeptides imitating β-turn by further modification of both the amino acid residue and the amidoxime group. The obtained pyridine-based amidoximes could serve as ligands for the elaboration of the metal complexes with luminescence properties.

Experimental

IR spectra have been registered on a PerkinElmer Spectrum BX FT-IR spectrometer for samples prepared as KBr pellets. ¹H and ¹³C NMR spectra were recorded on Bruker Avance DRX-400 (400 and 100 MHz, respectively), Bruker Avance DRX-500 (500 and 125 MHz, respectively), and Bruker Avance DRX-600 (600 and 150 MHz, respectively) spectrometers. TMS was used as internal standard. HPLC/MS have been recorded on an Agilent Technologies 1260 Infinity II Quaternary system (electrospray ionization at atmospheric pressure) equipped with an Infinity Lab Poroshell 120EC-C18 column (4.6 × 100 mm, 2.7 μm), solvent MeOH, gradient elution with 0.1% aqueous HCOOH in H₂O with increasing admixture of 0.1% HCOOH in MeOH. High-resolution mass spectra were recorded on an Agilent 6224 TOF LC/MS. Melting points have been determined using an OptiMelt automated melting point system. Flash column chromatography was carried out using silica gel 60, 40–63 μm.

All chemicals have been purchased from Enamine Ltd.

4-Cyanonicotinic acid (1). n-BuLi (44 ml, 0.110 mol, 2.5 M) was added slowly at –30°C to a stirred solution of 2,2,6,6-tetramethylpiperidine (16.25 g, 0.115 mol) in THF (100 ml). The obtained mixture was stirred for 45 min at this temperature and then was cooled down to –80°C. Isonicotinonitrile (2) (10.5 g, 0.10 mol) in THF (50 ml) was added dropwise to the mixture. After 1 h at –80°C, the reaction mixture was poured onto dry ice under Ar and kept for 4 h with vigorous stirring. Then it was evaporated to dryness, and the residue was dissolved in H₂O (100 ml). The resulting solution was acidified to pH 6 using 1 M HCl (230 ml) and extracted with EtOAc (3×150 ml). After evaporation of the solvent, a white powder of 4-cyanonicotinic acid (1) was obtained. Yield 9.03 g (61%), white powder, mp 174–176°C. IR spectrum, ν, cm^–1: 3468, 2462, 2240 (CN), 1909, 1719 (CO), 1668, 1600 (C=N), 1325, 1281, 1161, 1059, 854. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 8.01 (1H, d, J = 4.9, H-5 Py); 9.00 (1H, d, J = 5.1, H-6 Py); 9.25 (1H, s, H-2 Py). ¹³C NMR spectrum (125 MHz, DMSO-d₆), δ, ppm: 116.8 (CN); 120.9 (C-5 Py); 127.8 (C-3 Py); 128.8 (C-4 Py); 152.5 (C-2 Py); 154.8 (C-6 Py); 165.1 (COO). Found, m/z: 149.0347 [M+H]⁺. C₇H₅N₂O₂. Calculated, m/z: 149.0346.

Synthesis of methyl 2-(1-imino-3-oxo-1,3-dihydro-2Hpyrrolo[ 3,4-c]pyridin-2-yl)alkanoates 4a–h and 4-cyanonicotinamide 3i (General method). To a suspension of 4-cyanonicotinic acid (1) (0.37 g, 2.5 mmol) in CH₂Cl₂ (5 ml), HOBt (0.371 g, 2.75 mmol, 1.1 equiv), an appropriate methyl ester of L-amino acid hydrochloride (2.5 mmol), EDCI (0.502 g, 2.62 mmol, 1.05 equiv), and Et₃N (0.518 g, 5.12 mmol, 2.05 equiv) were added at 0°C. The mixture then was warmed up to room temperature and stirred overnight. Then the resulting clear amber solution was diluted with CH₂Cl₂ (10 ml) and brine (5 ml). The organic layer was separated and washed with brine (5 ml). The combined aqueous layer was further extracted with CH₂Cl₂ (10 ml). The combined organic layer then was dried over Na₂SO₄ and evaporated under reduced pressure. The residue was purified by flash column chromatography (gradient elution of MTBE–i-PrOH, 0:100–20:80) to afford the desired products.

Methyl (Z/E)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo- [3,4-c]pyridin-2-yl)acetate (4a). Yield 0.44 g (80%), paleyellow powder, mp 144–146°C. IR spectrum, ν, cm^–1: 3457, 3289 (N–H), 2958, 1748 (CO), 1732 (CO), 1664 (C=N), 1612, 1434, 1391, 1377, 1333, 1242, 1222, 1134, 946. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.69–3.70 (3H, m, CH₃O (Z + E)); 4.53 (1.68H, s, CH₂ (E)); 4.65 (0.32H, s, CH₂ (Z)); 7.93 (0.16H, d, J = 4.9, H-5 Py (Z)); 8.19 (0.84H, d, J = 5.0, H-5 Py (E)); 9.03 (0.16H, d, J = 4.9, H-6 Py (Z)); 9.07 (0.84H, d, J = 5.0, H-6 Py (E)); 9.08–9.09 (1H, m, H-2 Py (Z + E)); 9.92 (0.16H, s, NH (Z)); 10.52 (0.84H, s, NH (E)). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 40.1 (2 signals, CH₂ (Z), CH₂ (E)); 53.2 (CH₃O (E)); 53.3 (CH₃O (Z)); 117.2 (C-5 Py (Z)); 117.9 (C-5 Py (E)); 125.6 (C-3 Py (E)); 126.2 (C-3 Py (Z)); 139.0 (C-4 Py (E)); 143.9 (C-4 Py (Z)); 145.2 (C-2 Py (E)); 145.3 (C-2 Py (Z)); 153.4 (N=C–N (Z)); 155.7 (C-6 Py (Z)); 155.8 (C-6 Py (E)); 157.5 (N=C–N (E)); 166.7 (O=C–N (Z)); 166.9 (O=C–N (E)); 169.1 (2 signals, COO (Z), COO (E)). Found, m/z: 220.0720 [M+H]⁺. C₁₀H₁₀N₃O₃. Calculated, m/z: 220.0717.

Methyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)propanoate (4b). Yield 0.47 g (81%), pale-yellow powder, mp 135–137°C. IR spectrum, ν, cm^–1: 3443, 3288 (N–H), 3002, 1736 (CO), 1719 (CO), 1660 (C=N), 1426, 1415, 1312, 1277, 1262, 1224, 1133, 1091, 872. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.56 (3H, d, J = 107.1, CHCH₃); 3.62 (3H, s, CH₃O); 5.16 (1H, q, J = 7.2, CHCH₃); 8.18 (1H, d, J = 4.9, H-5 Py); 9.07–9.08 (2H, m, H-2,6 Py); 10.67 (1H, s, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 15.6 (CHCH₃); 47.9 (CHCH₃); 53.2 (CH₃O); 117.8 (C-5 Py); 125.5 (C-3 Py); 138.9 (C-4 Py); 145.2 (C-2 Py); 155.7 (C-6 Py); 157.2 (N=C–N); 166.7 (O=C–N); 171.2 (COO). Found, m/z: 234.0878 [M+H]⁺. C₁₁H₁₂N₃O₃. Calculated, m/z: 234.0873.

Methyl 2-(1,3-dioxo-1,3-dihydro-2H-pyrrolo[3,4-c]-pyridin-2-yl)propanoate (5b) was chromatographically isolated from the mixture with compound 4b (yield 0.26 g (46%)) and other products obtained according to the general method, which in this case has included washing the reaction mixture with 0.1 M HCl. Yield 0.20 g (34%), light-beige powder, mp 70–72°C. IR spectrum, ν, cm^–1: 3478, 3008, 1783 (CO), 1746 (CO), 1720 (CO), 1611, 1453, 1418, 1383, 1354, 1302, 1255, 1152, 1077, 886. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.55 (3H, d, J = 7.2, CHCH₃); 3.65 (3H, s, CH₃O); 5.06 (1H, q, J = 7.2, CHCH₃); 7.94 (1H, d, J = 4.8, H-5 Py); 9.15 (1H, d, J = 4.8, H-6 Py); 9.17 (1H, s, H-2 Py). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 15.5 (CHCH₃); 48.0 (CHCH₃); 53.6 (CH₃O); 118.0 (C-5 Py); 126.2 (C-3 Py); 139.8 (C-4 Py); 145.2 (C-2 Py); 157.2 (C-6 Py); 166.8 (O=C–N); 167.2 (O=C–N); 170.7 (COO). Found, m/z: 235.0715 [M+H]⁺. C₁₁H₁₁N₂O₄. Calculated, m/z: 235.0713.

Methyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)-3-methylbutanoate (4c). Yield 0.52 g (80%), pale-yellow powder, mp 114–116°C. IR spectrum, ν, cm^–1: 3383, 3291 (N–H), 3106, 2964, 1740 (CO), 1663 (C=N), 1594, 1436, 1422, 1411, 1359, 1290, 1217, 1094, 896. ¹H NMR spectrum (400 MHz, DMSO-d₆), δ, ppm (J, Hz): 0.77 (3H, d, J = 6.8, CH₃); 1.11 (3H, d, J = 6.6, CH₃); 2.62–2.71 (1H, m, β-CH); 3.58 (3H, s, CH₃O); 4.71 (1H, d, J = 8.7, α-CH); 8.19 (1H, d, J = 4.8, H-5 Py); 9.08–9.10 (2H, m, H-2,6 Py); 10.73 (1H, s, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 20.0 (CH₃); 22.0 (CH₃); 28.7 (β-CH); 53.0 (CH₃O); 57.7 (α-CH); 118.0 (C-5 Py); 125.2 (C-3 Py); 138.6 (C-4 Py); 145.4 (C-2 Py); 155.8 (C-6 Py); 157.9 (N=C–N); 167.1 (O=C–N); 170.1 (COO). Found, m/z: 262.1188 [M+H]⁺. C₁₃H₁₆N₃O₃. Calculated, m/z: 262.1186.

Methyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)-4-methylpentanoate (4d). Yield 0.58 g (85%), pale-yellow powder, mp 89–91°C. IR spectrum, ν, cm^–1: 3395, 3209 (N–H), 2960, 2871, 1740 (CO), 1660 (C=N), 1617, 1597, 1422, 1255, 1216, 1165, 1080, 866. ¹H NMR spectrum (500 MHz, CDCl₃), δ, ppm (J, Hz): 0.92 (3H, d, J = 6.7, CH₃); 0.96 (3H, d, J = 6.5, CH₃); 1.44–1.50 (1H, m, γ-CH); 1.97–2.02 (1H, m) and 2.42 (1H, br. s, CH₂); 3.72 (3H, s, CH₃O); 5.18–5.21 (1H, m, α-CH); 7.64 (1H, br. s, H-5 Py); 8.95 (1H, br. s, NH); 9.02 (1H, d, J = 4.9, H-6 Py); 9.16 (1H, s, H-2 Py). ¹³C NMR spectrum (125 MHz, CDCl₃), δ, ppm: 21.6 (CH₃); 23.5 (CH₃); 25.4 (γ-CH); 37.5 (CH₂); 51.5 (α-CH); 53.0 (CH₃O); 115.4 (br. s, C-5 Py); 125.3 (C-3 Py); 138.6 (br. s, C-4 Py); 145.7 C-2 Py); 154.6 (C-6 Py); 158.9 (br. s, N=C–N); 166.7 (O=C–N); 170.7 (COO). ¹H NMR (600 MHz, DMSO-d₆), δ, ppm (J, Hz): 0.84 (3H, d, J = 6.7, CH₃); 0.89 (3H, d, J = 6.5, CH₃); 1.40– 1.43 (1H, m, γ-CH); 1.83–1.88 (1H, m) and 2.26–2.31 (1H, m, CH₂); 3.61 (3H, s, CH₃O); 5.09–5.12 (1H, m, α-CH); 8.19 (1H, d, J = 4.9, H-5 Py); 9.08 (1H, d, J = 4.6, H-6 Py) 9.09 (1H, s, H-2 Py); 10.70 (1H, s, NH). ¹³C NMR (150 MHz, DMSO-d₆), δ, ppm: 21.6 (CH₃); 23.5 (CH₃); 24.9 (γ-CH); 37.2 (CH₂); 50.5 (α-CH); 52.8 (CH₃O); 117.4 (C-5 Py); 124.8 (C-3 Py); 138.3 (C-4 Py); 144.8 (C-2 Py); 155.3 (C-6 Py); 157.2 (N=C–N); 166.6 (O=C–N); 170.5 (COO). Found, m/z: 276.1343 [M+H]⁺. C₁₄H₁₈N₃O₃. Calculated, m/z: 276.1343.

Methyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)-3-phenylpropanoate (4e). Yield 0.63 g (81%), cream-colored powder, mp 115–117°C. IR spectrum, ν, cm^–1: 3438, 3288 (N–H), 2946, 1752 (CO), 1726 (CO), 1658 (C=N), 1432, 1412, 1218, 1117, 859. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.46–3.49 (2H, m, CH₂); 3.66 (3H, s, CH₃O); 5.39–5.42 (1H, m, α-CH); 7.05– 7.17 (5H, m, H Ph); 8.11–8.12 (1H, m, H-5 Py); 9.01 (1H, s, H-2 Py); 9.04 (1H, d, J = 5.0, H-6 Py); 10.64 (1H, s, NH). ¹³C NMR spectrum (100 MHz, DMSO-d₆), δ, ppm: 34.6 (CH₂); 53.4 (α-CH); 53.7 (CH₃O); 117.8 (C-5 Py); 124.9 (C-3 Py); 127.4 (C-4 Ph); 129.1 (C-3,5 Ph); 129.8 (C-2,6 Ph); 138.1 (C-1 Ph); 138.5 (C-3 Py); 145.2 (C-2 Py); 155.9 (C-6 Py); 157.3 (N=C–N); 166.7 (O=C–N); 170.3 (COO). Found, m/z: 310.1175 [M+H]⁺. C₁₇H₁₆N₃O₃. Calculated, m/z: 310.1186.

Methyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)-3-(1H-indol-3-yl)propanoate (4f). Yield 0.71 g (82%), orange powder, mp 91–93°C. IR spectrum, ν, cm^–1: 3408 (N–H), 3200, 2926, 1736 (CO), 1660 (C=N), 1617, 1413, 1270, 1108, 745. ¹H NMR spectrum (500 MHz, CDCl₃), δ, ppm (J, Hz): 3.69–3.96 (5H, m, CH₂, CH₃O); 5.51 (1H, br. s, α-CH); 6.99 (1H, s, H-2 indole); 7.02 (1H, dd, ²J = 7.7, ¹J = 7.3, H-6 indole); 7.10 (1H, dd, ²J = 7.3, ¹J = 7.6, H-5 indole); 7.24 (1H, d, J = 8.1, H-4 indole); 7.48 (1H, br. s, H-5 Py); 7.58 (1H, d, J = 7.9, H-7 indole); 8.05 (1H, s, NH indole); 8.86 (1H, br. s, C=NH); 8.90 (1H, d, J = 5.0, H-6 Py); 9.03 (1H, s, H-2 Py). ¹³C NMR spectrum (125 MHz, CDCl₃), δ, ppm: 24.8 (CH₂); 53.1 (α-CH); 53.4 (CH₃O); 111.4 (C-3 indole); 111.5 (C-7 indole); 115.3 (C-5 Py); 118.7 (C-4 indole); 119.7 (C-5 indole); 122.3 (C-6 indole); 122.9 (C-2 indole); 125.1 (C-3 Py); 127.5 (C-4a indole); 136.3 (C-7a indole); 138.5 (C-4 Py); 145.5 (C-2 Py); 154.4 (C-6 Py); 158.8 (N=C–N); 166.5 (O=C–N); 170.2 (O–C=O). ¹H NMR (600 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.55–3.58 (1H, m, CH₂); 3.68 (3H, s, CH₃O); 3.69–3.73 (1H, m, CH₂); 5.39–5.41 (1H, m, α-CH); 6.87 (1H, dd, ¹J = 7.6, ²J = 7.5, H-5 indole); 6.98 (1H, dd, ¹J = 7.4, ²J = 7.3, H-6 indole); 7.02 (1H, d, J = 2.4, H-2 indole); 7.25 (1H, d, J = 8.1, H-7 indole); 7.48 (1H, d, J = 7.9, H-4 indole); 8.11 (1H, d, J = 4.2, H-5 Py); 8.99 (1H, s, H-2 Py); 9.03 (1H, d, J = 5.0, H-6 Py); 10.62 (1H, s, C=NH); 10.71 (1H, s, NH indole). ¹³C NMR (150 MHz, DMSO-d₆), δ, ppm: 24.3 (CH₂); 52.9 (α-CH); 53.0 (CH₃O); 110.1 (C-3 indole); 111.8 (C-7 indole); 117.3 (C-5 Py); 118.4 (C-4 indole); 118.7 (C-5 indole); 121.3 (C-6 indole); 123.9 (C-2 indole); 124.7 (C-3 Py); 127.6 (C-4a indole); 136.4 (C-7a indole); 138.2 (C-4 Py); 144.7 (C-2 Py); 155.3 (C-6 Py); 157.0 (N–C=N); 166.4 (N–C=O); 170.1 (O–C=O). Found, m/z: 349.1292 [M+H]⁺. C₁₉H₁₇N₄O₃. Calculated, m/z: 349.1295.

Dimethyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)succinate (4g). Yield 0.62 g (85%), cream-colored powder, mp 103–105°C. IR spectrum, ν, cm^–1: 3410, 3287 (N–H), 2957, 1738 (CO), 1711 (CO), 1667 (C=N), 1609, 1415, 1376, 1311, 1232, 1172, 1134, 873. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.03–3.08 (1H, m) and 3.25–3.28 (1H, m, CH₂); 3.59 (3H, s, CH₃O); 3.63 (3H, s, CH₃O); 5.47–5.50 (1H, m, α-CH); 8.18 (1H, d, J = 5.0, H-5 Py); 9.09 (1H, d, J = 5.0, H-6 Py); 9.11 (1H, s, H-2 Py); 10.72 (1H, s, NH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 34.3 (CH₂); 48.7 (α-CH); 52.7 (CH₃O); 53.7 (CH₃O); 117.9 (C-5 Py); 125.3 (C-3 Py); 138.8 (C-4 Py); 145.4 (C-2 Py); 155.9 (C-6 Py); 157.1 (N–C=N); 166.6 (N–C=N); 170.0 (α-COO); 171.3 (β-COO). Found, m/z: 292.0931 [M+H]⁺. C₁₃H₁₄N₃O₅. Calculated, m/z: 292.0928.

Dimethyl (S)-2-(1-imino-3-oxo-1,3-dihydro-2H-pyrrolo-[3,4-c]pyridin-2-yl)pentanedioate (4h). Yield 0.67 g (88%), cream-colored crystalline powder, mp 72–74°C. IR spectrum, ν, cm^–1: 3455, 3295 (N–H), 2957, 1743 (CO), 1664 (C=N), 1610, 1410, 1277, 1176, 1115, 850. ¹H NMR spectrum (400 MHz, DMSO-d₆), δ, ppm (J, Hz): 2.34–2.40 (4H, m, 2CH₂); 3.48 (3H, s, CH₃O); 3.62 (3H, s, CH₃O); 5.09–5.12 (1H, m, α-CH); 8.16–8.19 (1H, m, H-5 Py); 9.06–9.08 (2H, m, H-2,6 Py); 10.68 (1H, s, NH). ¹³C NMR spectrum (100 MHz, DMSO-d₆), δ, ppm: 24.5 (β-CH₂); 30.8 (γ-CH₂); 51.7 (α-CH); 52.2 (CH₃O); 53.4 (CH₃O); 117.9 (C-5 Py); 125.4 (C-3 Py); 138.9 (C-4 Py); 145.3 (C-2 Py); 155.7 (C-6 Py); 157.6 (N=C–N); 167.1 (O=C–N); 170.5 (α-COO); 173.6 (γ-COO). Found, m/z: 328.0907 [M+Na]⁺. C₁₄H₁₅N₃NaO₅. Calculated, m/z: 328.0904.

Methyl cis/trans-(4-cyanonicotinoyl)-L-prolinate (3i). Yield 0.54 g (84%), beige crystalline powder, mp 116– 118°C. IR spectrum, ν, cm^–1: 3455, 2955, 2238 (CN), 1736 (CO), 1629, 1582, 1431, 1402, 1271, 1168, 1086, 854. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.85–2.00 (2.71H, m, β-CH₂ (cis), γ-CH2 (cis, trans)); 2.02– 2.08 (0.29H, m, β-CH₂ (cis)); 2.29–2.36 (1.00H, m, β-CH₂ (cis, trans)); 3.45 (0.87H, s, CH₃O (cis)); 3.47–3.51 (1.42H, m, δ-CH₂ (trans)); 3.62–3.67 (0.58H, m, δ-CH2 (cis)); 3.68 (2.13H, s, CH₃O (trans)); 4.54–4.58 (1H, m, α-CH (cis, trans)); 7.98 (0.29H, d, J = 5.1, H-5 Py (cis)); 8.02 (0.71H, d, J = 5.1, H-5 Py (trans)); 8.88–8.90 (0.58H, m, H-2,6 Py (cis)); 8.92–8.93 (1.42H, m, H-6 Py (trans)). ¹³C NMR spectrum (125 MHz, DMSO-d₆), δ, ppm: 23.4 (γ-C (cis)); 25.6 (γ-C (trans)); 29.9 (β-C (trans)); 31.8 (β-C (cis)); 47.7 (δ-C (cis)); 49.9 (δ-C (trans)); 52.9 (CH₃O (trans)); 53.3 (CH₃O (cis)); 59.8 (α-CH (trans)); 61.3 (α-CH (cis)); 116.0 (C-5 Py); 118.6 (CN (trans)); 119.2 (CN (cis)); 127.6 (C-3 Py (cis)); 127.8 (C-3 Py (trans)); 133.9 (C-4 Py (trans)); 134.2 (C-4 Py (cis)); 148.3 (C-2 Py (cis)); 148.8 (C-2 Py (trans)); 152.4 (C-6 Py (cis)); 152.6 (C-6 Py (trans)); 164.1 (O=C–N (trans)); 164.7 (O=C–N (cis)); 172.5 (COO (trans)); 172.7 (COO (cis)). Found, m/z: 260.1031 [M+H]⁺. C₁₃H₁₄N₃O₃. Calculated, m/z: 260.1030.

Synthesis of methyl esters of N-{4-[amino(hydroxyimino) methyl]pyridin-3-yl}carbonyl-substituted α-amino acids 6a–i (General method). To a stirred suspension of NH₂OH·HCl (0.175 g, 2.5 mmol, 2.5 equiv) in MeOH (2.5 ml), NaHCO₃ (0.212 g, 2.5 mmol, 2.5 equiv) was added at room temperature. After 15 min, appropriate pyrrolo[3,4-c]pyridine 4a–h or 4-cyanonicotinamide 3i (1 mmol) was added. The obtained mixture was stirred until the TLC showed the absence of starting material. The silica (1 g) was added to the obtained solution, and the solvent then was evaporated under reduced pressure at ambient temperature. The residue was purified by flash column chromatography (gradient elution of MTBE–MeOH, 0:100–20:80) to afford the desired product as a solid.

Methyl [4-(N'-hydroxycarbamimidoyl)nicotinoyl]glycinate (6a). Yield 0.21 g (83%), pale-yellow foam. IR spectrum, ν, cm^–1: 3342, 2925, 1744, 1648, 1594, 1561, 1376, 1220, 937. ¹H NMR spectrum (400 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.68 (3H, s, CH₃O); 4.00 (2H, d, J = 5.8, CH₂); 5.82 (2H, s, NH₂); 7.54 (1H, d, J = 5.2, H-5 Py); 8.59 (1H, s, H-2 Py); 8.65 (1H, d, J = 5.1, H-6 Py); 8.90 (1H, t, J = 5.8, CONH); 9.90 (1H, s, OH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 42.0 (CH₂); 52.9 (CH₃O); 123.7 (C-5 Py); 131.8 (C-3 Py); 140.5 (C-4 Py); 149.4 (C-2 Py); 150.5 (N=C–N); 151.5 (C-6 Py); 168.3 (O=C–N); 171.1 (COO). Found, m/z: 253.0933 [M+H]⁺. C₁₀H₁₃N₄O₄. Calculated, m/z: 253.0931.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-alaninate (6b). Yield 0.21 g (80%), pale-yellow powder, mp 58–60°C. IR spectrum, ν, cm^–1: 3451, 3330, 2960, 1740, 1645, 1595, 1558, 1455, 1380, 1220, 940, 667. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.36 (3H, d, J = 7.3, CHCH₃); 3.67 (3H, s, CH₃O); 4.41–4.47 (1H, m, CHCH₃); 5.82 (2H, s, NH₂); 7.53 (1H, d, J = 5.1, H-5 Py); 8.58 (1H, s, H-2 Py); 8.65 (1H, d, J = 5.1, H-6 Py); 8.83 (1H, d, J = 7.0, CONH); 9.88 (1H, s, OH). ¹³C NMR spectrum (100 MHz, DMSO-d₆), δ, ppm: 17.8 (CHCH₃); 49.0 (CHCH₃); 53.0 (CH₃O); 123.7 (C-5 Py); 131.7 (C-3 Py); 140.5 (C-4 Py); 149.5 (C-2 Py); 150.5 (N=C–N); 151.4 (C-6 Py); 167.5 (O=C–N); 173.8 (COO). Found, m/z: 267.1092 [M+H]⁺. C₁₁H₁₅N₄O₄. Calculated, m/z: 267.1088.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-valinate (6c). Yield 0.25 g (84%), colorless crystals, mp 171–173°C. IR spectrum, ν, cm^–1: 3508, 3377, 3282, 3040, 2976, 1740, 1638, 1597, 1572, 1365, 1259, 1201, 924, 662. ¹H NMR spectrum (400 MHz, DMSO-d₆), δ, ppm (J, Hz): 0.94 (6H, d, J = 6.8, 2CH₃); 2.07–2.15 (1H, m, β-CH); 3.67 (3H, s, CH₃O); 4.31–4.34 (1H, m, α-CH); 5.87 (2H, s, NH₂); 7.50 (1H, d, J = 5.1, H-5 Py); 8.58 (1H, s, H-2 Py); 8.65 (1H, d, J = 5.0, H-6 Py); 8.69 (1H, d, J = 8.0, CONH); 9.85 (1H, s, OH). ¹³C NMR spectrum (100 MHz, DMSO-d₆), δ, ppm: 19.4 (CH₃); 20.0 (CH₃); 30.8 (β-CH); 52.8 (CH₃O); 59.1 (α-CH); 123.9 (C-5 Py); 131.6 (C-3 Py); 140.5 (C-4 Py); 149.8 (C-2 Py); 150.8 (N=C–N); 151.4 (C-6 Py); 167.7 (O=C–N); 172.7 (COO). Found, m/z: 295.1404 [M+H]⁺. C₁₃H₁₉N₄O₄. Calculated, m/z: 295.1401.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-leucinate (6d). Yield 0.27 g (87%), pale-yellow foam. IR spectrum, ν, cm^–1: 3460, 3223, 2959, 2872, 1742, 1647, 1594, 1560, 1370, 1169, 939, 667. ¹H NMR spectrum (400 MHz, CDCl₃), δ, ppm (J, Hz): 0.90 (3H, d, J = 7.2, CH₃); 0.91 (3H, d, J = 7.2, CH₃); 1.54–1.73 (3H, m, CH₂, γ-CH); 3.69 (3H, s, OCH₃); 4.65–4.71 (1H, m, α-CH); 5.34 (2H, s, NH₂); 7.22 (1H, d, J = 5.1, H-5 Py); 7.86 (1H, d, J = 8.2, CONH); 8.45 (1H, d, J = 5.1, H-6 Py); 8.61 (1H, s, H-2 Py). ¹³C NMR spectrum (100 MHz, CDCl₃), δ, ppm: 22.1 (CH₃); 23.1 (CH₃); 25.2 (γ-CH); 41.3 (CH); 51.8 (α-CH); 52.8 (CH₃O); 123.2 (C-5 Py); 130.7 (C-3 Py); 138.8 (C-4 Py); 149.2 (C-2 Py); 151.1 (N=C–N); 151.2 (C-6 Py); 167.2 (O=C–N); 173.7 (COO). ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 0.90 (3H, d, J = 6.5, CH₃); 0.92 (3H, d, J = 6.5, CH₃); 1.51–1.56 (1H, m) and 1.61–1.67 (1H, m, CH₂); 1.70–1.77 (1H, m, γ-CH); 3.67 (3H, s, OCH₃); 4.41–4.46 (1H, m, α-CH); 5.80 (2H, s, NH₂); 7.52 (1H, d, J = 5.1, H-5 Py); 8.57 (1H, s, H-2 Py); 8.65 (1H, d, J = 5.1, H-6 Py); 8.81 (1H, d, J = 7.6, CONH); 9.83 (1H, s, OH). ¹³C NMR spectrum (125 MHz, DMSO-d₆), δ, ppm: 22.3 (CH₃); 23.8 (CH₃); 25.1 (γ-CH); 40.6 (CH); 51.8 (α-CH); 52.9 (CH₃O); 123.8 (C-5 Py); 131.6 (C-3 Py); 140.5 (C-4 Py); 149.6 (C-2 Py); 150.6 (N=C–N); 151.4 (C-6 Py); 167.6 (O=C–N); 173.7 (COO). Found, m/z: 309.1558 [M+H]⁺. C₁₄H₂₁N₄O₄. Calculated, m/z: 309.1557.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-phenylalaninate (6e). Yield 0.32 g (93%), white powder, 176–178°C. IR spectrum, ν, cm–1: 3390, 3308, 3065, 2955, 1748, 1731, 1663, 1594, 1518, 1372, 1214, 924, 704. ¹H NMR spectrum (600 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.00–3.13 (2H, m, CH₂); 3.64 (3H, s, OCH₃); 4.65–4.68 (1H, m, α-CH); 5.75 (2H, s, NH₂); 7.24 (1H, t, J = 7.1, H-4 Ph); 7.27–7.33 (4H, m, H Ph); 7.51 (1H, d, J = 5.1, H-5 Py); 8.38 (1H, s, H-2 Py); 8.63 (1H, d, J = 5.1, H-6 Py); 8.97 (1H, d, J = 7.7, CONH); 9.89 (1H, s, OH). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 37.7 (CH₂); 52.9 (CH₃O); 55.0 (α-CH); 123.7 (C-5 Py); 127.5 (C Ph); 129.3 (2C Ph); 130.1 (2C Ph); 131.4 (C-3 Py); 138.1 (C Ph); 140.3 (C-4 Py); 149.5 (C-2 Py); 150.6 (N=C–N); 151.4 (C-6 Py); 167.5 (O=C–N); 172.5 (COO). Found, m/z: 343.1397 [M+H]⁺. C₁₇H₁₉N₄O₄. Calculated, m/z: 343.1401.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-tryptophanate (6f). Yield 0.35 g (91%), pale-orange powder, mp 176–178°C. IR spectrum, ν, cm^–1: 3332, 3044, 2952, 1754, 1620, 1597, 1562, 1347, 1215, 755, 669. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 3.16–3.27 (2H, m, CH₂); 3.62 (3H, s, OCH₃); 4.68–4.72 (1H, m, α-CH); 5.81 (2H, s, NH₂); 7.01 (1H, dd, 1J = 2J = 7.5, H-5 indole); 7.09 (1H, dd, ¹J = 7.8, ²J = 7.5, H-6 indole); 7.26 (1H, d, J = 2.4, H-2 indole); 7.37 (1H, d, J = 8.1, H-7 indole); 7.52 (1H, d, J = 5.1, H-5 Py); 7.54 (1H, d, J = 7.9, H-4 indole); 8.47 (1H, s, H-2 Py); 8.64 (1H, d, J = 5.1, H-6 Py); 8.93 (1H, d, J = 7.5, CONH); 9.91 (1H, s, OH); 10.88 (1H, s, NH indole). ¹³C NMR spectrum (150 MHz, DMSO-d₆), δ, ppm: 28.0 (CH₂); 52.9 (OCH₃); 54.6 (α-CH); 110.3 (C-3 indole); 112.4 (C-7 indole); 119.0 (C-4 indole); 119.4 (C-5 indole); 122.0 (C-6 indole); 123.8 (C-5 Py); 124.9 (C-2 indole); 128.1 (C-4a indole); 131.6 (C-3 Py); 137.1 (C-7a indole); 140.5 (C-4 Py); 149.6 (C-2 Py); 150.7 (N–C=N); 151.4 (C-6 Py); 167.6 (N–C=O); 173.0 (O–C=O). Found, m/z: 382.1507 [M+H]⁺. C₁₉H₂₀N₅O₄. Calculated, m/z: 382.1510.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-aspartate (6g). Yield 0.27 g (82%), pale-yellow foam. IR spectrum, ν, cm^–1: 3453, 3350, 3042, 2956, 2852, 1738, 1649, 1594, 1557, 1439, 1373, 1222, 849, 667. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 2.79–2.90 (2H, m, CH₂); 3.63 (3H, s, OCH₃); 3.67 (3H, s, OCH₃); 4.81–4.85 (1H, m, α-CH); 5.80 (2H, s, NH₂); 7.53 (1H, d, J = 5.1, H-5 Py); 8.53 (1H, s, H-2 Py); 8.65 (1H, d, J = 5.2, H-6 Py); 8.87 (1H, d, J = 8.1, CONH); 9.86 (1H, s, OH). ¹³C NMR spectrum (100 MHz, DMSO-d₆), δ, ppm: 36.4 (CH2); 49.8 (α-CH); 52.7 (CH₃O); 53.3 (CH₃O); 123.6 (C-5 Py); 131.6 (C-3 Py); 140.4 (C-4 Py); 149.3 (C-2 Py); 150.4 (N=C–N); 151.5 (C-6 Py); 167.5 (O=C–N); 171.5 (α-COO); 171.9 (β-COO). Found, m/z: 325.1143 [M+H]⁺. C₁₃H₁₇N₄O₆. Calculated, m/z: 325.1143.

Methyl (Z)-[4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-glutamate (6h). Yield 0.29 g (86%), colorless foam. IR spectrum, ν, cm^–1: 3451, 3351, 3058, 2956, 1737, 1648, 1595, 1560, 1439, 1377, 1268, 1215, 938, 667. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.89–1.96 (1H, m) and 2.03–2.10 (1H, m, β-CH₂); 2.46–2.49 (2H, m, γ-CH₂); 3.60 (3H, s, CH₃O); 3.67 (3H, s, CH₃O); 4.41–4.46 (1H, m, α-CH); 5.81 (2H, s, NH₂); 7.53 (1H, d, J = 5.1, H-5 Py); 8.57 (1H, s, H-2 Py); 8.66 (1H, d, J = 5.1, H-6 Py); 8.82 (1H, d, J = 7.5, CONH); 9.83 (1H, s, OH). ¹³C NMR spectrum (125 MHz, DMSO-d₆), δ, ppm: 26.9 (β-CH₂); 36.4 (γ-CH₂); 52.3 (α-CH); 52.7 (CH₃O); 53.0 (CH₃O); 123.6 (C-5 Py); 131.7 (C-3 Py); 140.4 (C-4 Py); 149.5 (C-2 Py); 150.5 (N=C–N); 151.4 (C-6 Py); 167.8 (O=C–N); 172.8 (α-COO); 173.7 (γ-COO). Found, m/z: 339.1301 [M+H]⁺. C₁₄H₁₉N₄O₆. Calculated, m/z: 339.1299.

Methyl [4-(N'-hydroxycarbamimidoyl)nicotinoyl]-L-prolinate (6i). Yield 0.24 g (81%), pale-yellow powder, mp 92–94°C. IR spectrum, ν, cm^–1: 3446, 3342, 2956, 2882, 1740, 1624, 1594, 1444, 1401, 1202, 1177, 943, 668. ¹H NMR spectrum (500 MHz, DMSO-d₆), δ, ppm (J, Hz): 1.81–1.95 (3.00H, m, β-CH₂ (trans, cis), γ-CH₂ (cis, trans)); 2.33–2.42 (1.00H, m, β-CH₂ (trans, cis)); 3.15–3.25 (1.30H, m, δ-CH₂ (trans)); 3.46 (1.05H, s, CH₃O (cis)); 3.50–3.60 (0.70H, m, δ-CH2 (cis)); 3.69 (1.95H, s, CH₃O (trans)); 4.11–4.14 (0.35H, m, α-CH (cis)); 4.40–4.43 (0.65H, m, α-CH (trans)); 5.92 (1.30H, s, NH₂ (trans)); 5.98 (0.70H, s, NH₂ (cis)); 7.58 (0.35H, d, J = 5.2, H-5 Py (cis)); 7.61 (0.65H, d, J = 5.2, H-5 Py (trans)); 8.22 (0.35H, s, H-2 Py (cis)); 8.38 (0.65H, s, H-2 Py (trans)); 8.62 (0.35H, d, J = 5.2, H-6 Py (cis)); 8.66 (0.65H, d, J = 5.2, H-6 Py (trans)); 8.96 (1H, s, OH). ¹³C NMR spectrum (125 MHz, DMSO-d₆), δ, ppm: 23.7 (γ-CH₂ (cis)); 25.4 (γ-CH₂ (trans)); 30.2 (β-CH₂ (trans)); 31.2 (β-CH₂ (cis)); 47.1 (δ-CH₂ (cis)); 49.2 (δ-CH₂ (trans)); 52.9 (CH₃O (trans, cis)); 59.3 (α-CH (trans)); 61.2 (α-CH (cis)); 122.3 (C-5 Py (cis)); 122.5 (C-5 Py (trans)); 131.3 (C-3 Py (cis)); 131.6 (C-3 Py (trans)); 138.3 (C-4 Py (cis)); 138.7 (C-4 Py (trans)); 148.1 (C-2 Py (trans)); 148.3 (C-2 Py (cis)); 149.6 (N=C–N (trans)); 149.7 (N=C–N (cis)); 150.9 (C-6 Py (cis)); 151.0 (C-6 Py (trans)); 167.8 (N–C=O (trans)); 168.1 (N–C=O (cis)); 173.3 (COO (trans)); 173.5) (COO (cis)). Found, m/z: 293.1242 [M+H]⁺. C₁₃H₁₇N₄O₄. Calculated, m/z: 293.1244.

X-ray structural study of compound 6c. The colorless crystals of compound 6c (C₁₃H₁₈N₄O₄) are monoclinic. At 173K, a 9.5932(14), b 7.3711(10), c 9.9957(14) Å; β 95.633(10)°; V 703.41(17) Ǻ³; M_r 294.31; Z 2; space group P2₁; d_calc 1.390 g/cm³; μ(MoKα) 0.105 mm^–1; F(000) 312. Intensities of 8285 reflections (2344 independent, R_int 0.061) have been collected on a Bruker APEX II diffractometer (graphite monochromated MoKα radiation, CCD detector, φ- and ω-scanning, 2Θ_max 50°). The structure was solved by a direct method using the SHELXT package¹⁶ and refined using SHELXL program.¹⁷ Positions of the hydrogen atoms have been located from electron density difference maps and refined using riding model with U_iso = nU_eq (n = 1.5 for methyl groups and n = 1.2 for other hydrogen atoms) of the carrier atom. Hydrogen atoms participated in the formation of N–H···O, N–H···N, and O–H···O hydrogen bonds have been refined using isotropic approximation. Full-matrix least-squares refinement against F² in anisotropic approximation for non-hydrogen atoms using 2344 reflections was converged to wR₂ 0.165 (R₁ 0.057 for 2156 reflections with F > 4σ(F), S 1.071). The final atomic coordinates and crystallographic data for molecule 6c have been deposited at the Cambridge Crystallographic Data Center (deposit CCDC 2280261).

Supplementary information file, containing ¹H and ¹³C NMR spectra of the synthesized compounds is available at the journal's website http://springerlink.bibliotecabuap.elogim.com/journal/10593.

We thank Enamine Ltd. for providing support to the experiment. We are also grateful to the European Chemistry School for Ukrainians (ecpsfu.org) for support of Ukrainian scientists.