INTRODUCTION

Cyanoacetamides are highly reactive compounds. They are extensively utilized as reactants or reaction intermediates, since the carbonyl and cyano functions in these compounds are suitably arranged to enable reactions with common bidentate reagents to form a variety of heterocyclic compounds [1–8]. Moreover, the active hydrogen on C2 in these compounds can take part in a variety of condensation and substitution reactions. In addition, many cyanoacetamide derivatives have been reported to exhibit antibacterial [9], anticoagulant [10], antifungal [11], antihistaminic [12], antileishhumanial [13], antimicrobial [14], and herbicidal properties [15]. The literature covering the chemistry of cyanoacetamide derivatives is limited. In particular, the review of the chemistry and reactions of cyanothioacetamides (in Russian) was published as far back as 1999 [16]. The main objective of the present work was to provide a comprehensive account of the synthetic utility of N-aryl- and/or N-heterylcyanoacetamides in constructing various organic heterocycles and to highlight their potential in developing better chemotherapeutic agents.

RESULTS AND DISCUSSION

The cyclocondensation of N-benzyl-2-cyanoacetamide with carbon disulfide provides thiazinone 3. The reaction presumably involves the formation of a non-isolable aza adduct 1 and its intramolecular cyclization via addition of the mercapto function to cyano group followed by a [1,3]-sigmatropic shift (Scheme 1).

Scheme
scheme 1

1.

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The 1H NMR spectrum of compound 3 displays a downfield signal at 8.21 ppm from the NH2 protons, a multiplet of aromatic protons at 7.25–7.33 ppm, an ethylene proton singlet at 5.86 ppm, and a doublet of the CH2N protons at 4.33 and 4.55 ppm. The 13C NMR spectrum shows C=S and C=O carbon signals at 167.69 and 161.02 ppm and a CH2 carbon at 85 ppm. The IR spectrum contains a C=O stretching band at 1650 cm–1 (the frequency is decreased by the +M effects of S and NH2).

Due to the presence of a cyclic enamine system, compound 3 can undergo heterocyclization to form interesting condensed polycyclic compounds. Thus, the cyclocondensation of compound 3 with maleic anhydride affords furopyrrole thiazine derivative 7 through the formation Michael adduct 4 and its cyclization via the condensation of the enamine nitrogen with the carboxyl group (with elimination of a water molecule) and subsequent intramolecular cycloaddition (Scheme 2).

Scheme
scheme 2

2.

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Compound 3 undergoes [3+3]-cycloaddition reaction with ethyl benzylidenecyanoacetate followed by hydrolysis, decarboxylation, and aromatization to furnish pyridothiazine derivative 10 (Scheme 3). The 1H NMR spectrum of compound 10 displays aromatic proton signals signal at 7.25–7.30 ppm and a benzylic signal at 5.16–5.25 ppm. The IR spectrum of 10 shows bands at 3330 and 1663 cm–1 due to NH2 and C=O stretching vibrations, respectively.

Scheme
scheme 3

3.

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Amidopyrrolothiazine 11 was obtained as a result of the cycloaddition reaction between compound 3 and benzylidene cyanoacetamide (Scheme 3). The 1H NMR spectrum of compound 11 displays a downfield broad signal of the NH2 protons at 10.31 ppm, as well as amide and benzylic proton signals at 5.12 and 4.16–4.20 ppm, respectively. The IR spectrum contains bands at 3300, 1650, and 1250 cm–1 from the NH2, C=O, and C=S groups, respectively. As shown in Scheme 3, compound 12 forms as a result of the attack of the enamine carbon in 3 to the polarized double bond in benzylidene malonitrile, after which the exocyclic amino group adds to the cyano function. The spectral characteristics of compound 12 are consistent with the proposed structure. The 1H NMR spectrum of compound 12 displays a downfield broad singlet for NH2 at 11.6 ppm, a signal of PhCH2 methylene protons at 5.26–5.27 ppm, as well as PhCH and CHCN methine proton signals at 5.10–5.12 and 4.36–4.37 ppm, respectively. The IR spectrum displays bands at 3300, 2198, 1650, 1547, and 1220 cm–1 assignable to the NH2, CN, C=O, C=C, and C=S groups, respectively.

Compound 3 undergoes ring opening under the action of urea or thiourea followed by cyclization to form pyrimidine derivative 14, whose air oxidation of leads to oxadiazolopyrimidine 15 or thiadiazolopyrimidine 16, respectively (Scheme 4). The 1H NMR of compound 16 shows a downfield signal for NH at 10.54 ppm, as well as aromatic and CH2N methylene proton signals at 7.26–7.32 and 3.98 ppm, respectively. The IR spectrum displays bands at 3300 (OH), 3170 (NH), and 1225 (C=S) cm–1.

Scheme
scheme 4

4.

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Treatment of thiazinthione derivative 3 with a mixture of NaOH and H2O2 leads to ring transformation to form mercaptopyrimidine 17 presumably via Dimorth rearrangement. At the same time, no disulfide 18 was detected (Scheme 5). The 1H NMR spectrum of compound 17 displays downfield singlets at 8.22, 8.20, and 8.19 ppm from the OH, NH, and SH protons, respectively.

Scheme
scheme 5

5.

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The ring transformation in compound 3 under the action of Br2 in acetic acid under reflux followed by benzylic bromination produces derivative 19 and no expected desulfurized product 20 (Scheme 6). The 1H NMR spectrum of compound 19 shows downfield signals of the NH, SH, and benzylic PhCHBr protons at 10.72, 9.63, and 4.98 ppm, respectively.

Scheme
scheme 6

6.

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Antimicrobial and antifungal activity testing. Compounds 7, 15, 16, and 19 were tested for in vitro antimicrobial activity against Gram-positive (S. aureus, S. faecalis, and B. subtilis) and Gram-negative bacteria (E. coli, N. gonorrhoeae, and P. aeruginosa). The antifungal activity of the compounds was tested against two fungi C. albicans and A. flavus. According to the resulting data (Table 1), compounds 15 and 19 showed inhibitory activity against S. aureus, E. coli, S. faecalis, B. subtilis, N. gonorrhoeae, P. aeruginosa, A. flavus, and C. albicans strains.

Table 1. Antibacterial and antifungal activity testing of the synthesized compounds, control: DMSO
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The biological activity of the test compounds was determined by a modified Kirby–Bauer disc diffusion method [17]. Briefly, 100 μl of a 100 mL of a solution of the test bacteria/fungi were grown in 10 mL of fresh media until they reached a count of approximately 108 cells/mL for bacteria and 105 cells/mL for fungi [18].

Anticancer activity testing. Compounds 7, 15, 16, and 19 were tested against HepG-2 (human hepatocellular cancer cell line) and HCT-116 cells (human colon cancer cell line) obtained from VACSERA Tissue Culture Unit (Cairo, Egypt). The procedures of testing are described in detail in [19–21].

The results of the testing of the effect of compounds 7, 15, 16, and 19 on the in vitro growth of HepG-2 cells after continuous exposure for 48 h are presented in Table 2.

Table 2. Effect of the synthesized compounds on HepG-2 cell growth
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All the test compounds were able to inhibit the growth of the test HepG-2 cells in a dose-dependent manner. As seen from Table 2, compound 15 showed the highest growth inhibitory activity, while not as high as the reference Doxorubicin. Compounds 7 and 19 exhibited a moderate growth inhibitory effect against HepG-2 cells, and compound 16 was the least active.

The cytotoxic activity of compounds 7, 15, 16 and 19 against HepG-2 cells was evaluated using solutions of different concentrations (0, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 µg/mL). The resulting cell viabities (%) and IC50 values (mg/mL) are listed in Table 3.

As seen from Table 3, compounds 15 and 19 showed a high cytotoxic activity against HepG-2 cancer cell line, whereas compounds 7 and 16 were moderately active.

Table 3. Cytotoxicity testing of the synthesized compounds against the HepG-2 cell line
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The results of the testing of the effect of compounds 7, 15, 16, and 19 on the in vitro growth of HCT-116 cells after continuous exposure for 48 h are presented in Table 4.

Table 4. Effect of the synthesized compounds on HCT-116 cell growth
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All the test compounds were able to inhibit the growth of HCT-116 cells in a dose-dependent manner. As seen from Table 4, compound 15 showed a strong inhibitory effect against HCT-116 cells line, but not as strong as the reference drug Doxorubicin. Compound 16 proved the least active, while compounds 7, 19 exhibited a moderate growth inhibitory effect. For the cytotoxicity testing of compounds 7, 15, 16 and 19 against the HCT-116 cell line, solutions of the same concentrations as in the cytotoxicity testing against the HepG-2 cell line. The resulting cell viabities (%) and IC50 values (mg/mL) are listed in Table 5.

Table 5. Cytotoxicity testing of the synthesized compounds against the HCT-116 cell line
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As seen from Table 5, compounds 15 and 19 showed a high cytotoxic activity against the HCT-116 cancer cell line, and compounds 7 and 16 were moderately active.

EXPERIMENTAL

The melting points were measured using an Electro thermal IA 9100 apparatus in open capillary tubes and are uncorrected. All experiments were carried out using dry solvents. Thin-layer chromatography (TLC) was performed on Merck Silica Gel 60 F254 plates with detection in UV light. The products were purified by crystallization. The IR spectra were recorded on a Pye Unicam Sp-3-300 or a Shimadzu FTIR 8101 spectrophotometer in KBr discs. The 1H and 13C NMR spectra were recorded on a Varian Mercury VX-300 spectrometer at 300 and 75.4 MHz, respectively, in DMSO-d6. All chemical shifts were expressed in ppm on the δ scale using TMS as internal reference. The elemental analysis and in vitro antimicrobial activity testing were performed at the Microanalysis Center, Cairo University, Giza, Egypt.

6-Amino-3-benzyl-2-thioxo-2H-1,3-thiazin-4(3H)-one (3). A solution of carbon disulfide (0.01 mol), compound 1 (0.01 mol), and potassium hydroxide (0.01 mol) in absolute ethanol (20 mL) was refluxed for 6 h and then poured into glacial acetic acid. The precipitate that formed was filtered off, washed with water, dried, and crystallized from ethanol. Yield 90%, white crystals, mp 138–142°C. IR spectrum, ν, cm–1: 3212 (NH2), 1650 (C=O), 1492 (C=N), 1295 (C=S). 1H NMR spectrum, δ, ppm: 4.44 d (2H, CH2N, J 7.0 Hz), 5.86 s (1H, CH. olefinic), 7.25–7.33 m (5Harom), 8.21 s (2H, NH2). 13C NMR spectrum, δ, ppm: 43.37 (CH2), 85.87 (=CH), 126.98 (phenyl carbons), 127.93, 128.70, 137.70, 140.12, 153.13, 156.06 (=C–NH2), 161.02 (C=O), 167.69 (C=S). Found, %: C 52.50; H 3.95; N 11.00; O 6.20; S 26.40. C11H10N2OS2. Calculated, %: C 52.77; H 4.03; N 11.19; O 6.39; S 26.62. M 250.33.

3-Benzyl-4-hydroxy-6-hydroxyfuro[3',2':4,5]pyrrolo[3,2-e][1,3]thiazine-2(3H)-thione (7) A solution of maleic anhydride (0.01 mol), compound 3 (0.01 mol), and sodium acetate (0.01 mol) in acetic acid (20 mL) was refluxed for 6 h and then poured into ice water. The precipitate that formed was filtered off, washed with water, dried and crystallized from acetic acid. Yield 94%, yellow powder, mp 78–82°C. IR spectrum, ν, cm–1: 3250 (OH), 3174 (NH), 1650 (C=O), 1575 (C=C), 1326 (C=S). 1H NMR spectrum, δ, ppm: 4.55 d (2H, CH2Ph, J 5.5 Hz), 5.00 d.d (2H, CH2, J 31.0 Hz), 5.43 d (1H, CH, J 35.5 Hz), 7.26–7.32 m (5Harom), 10.45 s (1H, OH). 13C NMR spectrum, δ, ppm: 42.22 (CH2), 127.45 (phenyl carbons), 127.69, 127.76, 128.32, 128.53, 128.59, 139.16 (=C–OH), 164.48 (imine carbon), 184.31 (=C–OH), 201.63 (C=S). Found, %: C 54.33; H 2.99; N 8.60; O 14.33; S 19.39. C15H10N2O3S2. Calculated, %: C 54.53; H 3.05; N 8.84; O 14.53; S 19.41. M 330.01.

7-Amino-3-benzyl-5-phenyl-2-thioxo-2H-pyrido[3,2-e][1,3]thiazin-4(3H)-one (10). A solution compound 3 (0.01 mol), (Z)-2-benzylidene-3-oxopentanenitrile (0.01 mol), and 3 drops of trimethylamine in DMF (20 mL) was refluxed for 6 h and then pour in several portions into HCl diluted with ice water. The precipitate that formed was filtered off, washed in water, dried, and crystallized from DMF. Yield (93%, yellow powder, mp 148–152°C. IR spectrum, ν, cm–1: 3330 (NH2), 1663 (C=O), 1563 (C=C), 1220 (C=S). 1H NMR spectrum, δ, ppm: 5.21 d (2H, CH2Ph, J 34.4 Hz), 7.25–7.30 m (13H, Harom and NH2). Found, %: C 64.00; H 4.00; N 11.00; O 4.19; S 16.97. C20H15N3OS2. Calculated, %: C 63.63; H 4.00; N 11.13; O 4.24; S 17.00. M 377.48.

7-Amino-3-benzyl-4-oxo-5-phenyl-2-thioxo-3,4,5,6-tetrahydro-2H-pyrido[3,2-e][1,3]thiazine-6-carboxamide (11). A solution of (Z)-2-cyano-3-phenylacrylamide (0.01 mol), compound 3 (0.01 mol), and 3 drops of TEA in DMF (20 mL) was refluxed for 6 h the solution was poured into dilute HCl. The precipitate that formed was filtered off, washed in water, dried, and crystallized from DMF. Yield 95%, brown powder, mp 118–122°C. IR spectrum, ν, cm–1: 3300 (NH2), 1650 (C=O), 1575 (C=C), 1250 (C=S). 1H NMR spectrum, δ, ppm: 4.18 d (2H, CH2Ph, J 8.0 Hz), 4.46–4.70 m (2H, CH–CH), 5.12 s (2H, NH2C=O), 7.30–7.32 m (10Harom), 10.32 d (2H, NH2, J 5.0 Hz). Found, %: C 59.00; H 3.20; N 13.11; O 3.98; S 15.00. C21H18N4O2S2. Calculated, %: C 59.70; H 3.82; N 13.26; O 4.00; S 15.20. M 422.52.

7-Amino-3-benzyl-4-oxo-5-phenyl-2-thioxo-3,4,5,6-tetrahydro-2H-pyrido[3,2-e][1,3]thiazine-6-carbonitrile (12). A mixture of 2-benzylidenemalononitrile (0.01 mol), compound 3 (0.01 mol), and 3 drops of TEA was refluxed in DMF (20 mL) for 6 h and then poured in several portions into dilute HCl. The precipitate that formed was filtered off, washed with water, dried, and crystallized from DMF. Yield 96%, beige powder, mp 128–132°C. IR spectrum, ν, cm–1: 3330 (NH2), 2198 (CN), 1650 (C=O), 1547 (C=C), 1220 (C=S). 1H NMR spectrum, δ, ppm: 11.6 s (2H, NH2), 7.31–7.35 m (10Harom), 5.27 d (2H, CH2Ph, J 8.5 Hz), 5.11 d (1H, PhCH, J 9.0 Hz), 4.37 d (1H, NC–CH, J 6.5 Hz). Found, %: C 60.00; H 3.60; N 13.70; O 3.55; S 15.66. C21H16N4OS2. Calculated, %: C 62.35; H 3.99; N 13.85; O 3.96; S 15.86. M 404.51.

6-Benzyl-7-hydroxy-3-imino-3H-[1,2,4]oxadiazolo[4,3-c]pyrimidine-5(6H)-thione (15). A mixture of urea (0.01 mol), compound 3 (0.01 mol), and sodium acetate (0.01 mol) was refluxed for 6 h in acetic acid (20 mL) and then poured into KOH solution. Then precipitate that formed was filtered off and crystallized from acetic acid. Yield 88%, orange powder, mp 83–87°C. IR spectrum, ν, cm–1: 3167 (OH), 2922 (NH), 1538 (C=C), 1328 (C=S). 1H NMR spectrum, δ, ppm: 4.55 d (2H, CH2Ph, J 5.6 Hz), 5.33 s (1H, NH), 7.25–7.33 m (6Harom), 10.42 s (1H, OH). 13C NMR spectrum, δ, ppm: 42.22 (CH2), 128.32 (phenyl carbons), 128.53, 128.59, 128.74, 128.89, 128.92, 164.43 (imine carbons), 172.67, 184.31 (=C–OH), 201.63 (C=S). Found, %: C 52.00; H 3.60; N 20.33; O 11.60; S 11.66. C12H10N4O2S. Calculated, %: C 52.54; H 3.67; N 20.43; O 11.67; S 11.69. M 274.30.

6-Benzyl-7-hydroxy-3-imino-3H-[1,2,4]thiadiazolo[4,3-c]pyrimidine-5(6H)-thione (16). A solution of thiourea (0.01 mol), compound 3 (0.01 mol), and sodium acetate (0.01 mol) was refluxed in acetic acid (20 mL) for 6 h and then poured into KOH solution and stirred until a precipitate formed. The precipitate was filtered off and crystallized from acetic acid. Yield 95%, orange powder, mp 158–162°C. IR spectrum, ν, cm–1: 3300 (OH), 3170 (NH), 1650 (C=O), 1530 (C=C), 1225 (C=S). 1H NMR spectrum, δ, ppm: 3.98 d (2H, CH2N, J 18.0 Hz), 4.54 d (2H, CH2Ph, J 6.0 Hz), 7.26–7.32 m (6Harom), 10.54 s (1H, NH). Found, %: C 49.00; H 3.40; N 19.00; O 5.30; S 22.00. C12H10N4OS2. Calculated, %: C 49.63; H 3.47; N 19.03; O 5.51; S 22.09. M 290.36.

1-Benzyl-6-hydroxy-4-sulfanylpyrimidine-2(1H)-thione (17). A mixture of compound 3 (0.01 mol), sodium hydroxide (0.01 mol) and 20 mL of hydrogen peroxide was refluxed for 4 h then pour in to ice water, precipitate was filtered off, washed in water, dried, and crystallized. Yield 97%, beige powder, mp 133–137°C. IR spectrum, ν, cm–1: 3350 (OH), 3215 (NH), 3100 (SH), 1744 (C=O), 1493 (C=C), 1394 (C=S). 1H NMR spectrum, δ, ppm: 4.36 d (2H, CH2Ph, J 7.0 Hz), 6.95–7.31 m (6Harom), 8.19 s (1H, SH), 8.20 s (1H, NH), 8.22 s (1H, OH). Found, %: C 50.00; H 4.00; N 11.00; O 6.30; S 25.00. C11H9N2OS2. Calculated, %: C 52.78; H 4.03; N 11.19; O 6.39; S 25.2. M 249.02.

1-[Bromo(phenyl)methyl]-6-hydroxy-4-mercaptopyrimidine-2(1H)-thione (19). A solution of compound 3 (0.01 mol) and brome (0.01 mol) and in 20 mL acetic acid was refluxed for 4 h then pour in potassium hydroxide solution, obtainable precipitate was filtered off, washed in water, dried and crystallized form acetic acid. Yield (95%), beige powder, mp 220–222°C. IR spectrum, ν, cm–1: 3273 (OH), 3100 (NH), 3012 (SH), 1645 (C=C), 1594 (C=S). 1H NMR spectrum, δ, ppm: 4.98 s (1H, PhCHBr), 7.31–7.65 m (5Harom), 9.63 s (1H, SH), 10.72 s (1H, NH). Found, %: C 40.00; H 2.66; N 8.44; O 4.80; S 19.40. C11H8BrN2OS. Calculated, %: C 40.13; H 2.76; Br 24.27; N 8.51; O 4.86; S 19.48. M 296.17.