Synthesis of Some Novel 6-Nitro-8-Pyridinyl Coumarin And 6-Nitro-8-Pyranyl Coumarin Derivatives and Evaluation of Their Anti-Bacterial Activity

Abstract

Two novel series of 4-methyl-6-nitro-8-(pyridin-2-yl)-2-oxo-2Hchromen- 7-yl benzoates VI-VIII and 4-methyl-6-nitro-8-(pyran-6-yl)-2-oxo-2Hchromen- 7-yl benzoates IX-XI were synthesized starting from the 8-acetyl- 4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate V. All synthesized compounds were evaluated for their antibacterial activity using paper disc diffusion technique and agar dilution technique. Among of the tested compounds, the 8-(6-Amino-4-(2-bromophenyl)-5-thio-carbamoyl-4Hpyran- 2-yl)-4-methyl-6-nitro-2oxo-2H-chromen-7-yl benzoate Xh displayed the most promising antibacterial activity with MIC of 64, 512 and 128 μg/ ml against B. subtilis, S. aureus and E. coli, respectively

Keywords

6-Nitro-8-pyridinyl-(8-pyranyl) -coumarins, Antibacterial activity.

Introduction

Coumarin derivatives are a class of compounds that have great importance and interest due to their wide distribution in nature and their broad and diverse biological activities including anticoagulant [1], vasorelaxant [2], lipid lowering [3-5], antioxidant [6], antidepressant [7], anticonvulsant [8], antihistaminic [9], anticancer [10-12], antivira l[13-15], antiprotozoal [16], antimicrobial [17-20], antiinflammatory [21,22], analgesic and antipyretic [23] activities.

Interestingly, the coumarin nucleus constitutes a basic moiety in the skeleton of several antibiotics such as Novobiocin and its derivative Clorobiocin which were discovered to possess excellent activity against gram positive pathogens [17,20]. Referring to the literature, many coumarin derivatives bearing different heterocyclic moieties at position 8 [24] were reported to display promising antibacterial activity. Also, it was reported that coumarin derivatives containing nitro group in their structures possessed significant antimicrobial activity [25].

Based on this background, we achieved the synthesis and the evaluation of the antibacterial activity of two new series of coumarin derivatives bearing a nitro group at position 6 together with a pyridine or a pyran moiety at position 8 namely; 8-(pyridin-2-yl)-4-methyl- 6-nitro-2-oxo-2H-chromen-7-yl benzoates VI-VIII and 8-(pyran-6-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates IX-XI, respectively through the reactive key intermediate; 8-acetyl-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate V. (Schemes 1 and 2).

Results and Discussion

Chemistry

Scheme 1

Pechmann reaction [26] was used for the preparation of the 7-hydroxy-4-methyl-2H-chromen-2-one I. Preparation of 4-methyl-2-oxo-2H-chromen-7-yl acetate II was achieved by the condensation of I with acetyl chloride according to Limaye's method [27]. Then, Fries rearrangement [28,29] was used to prepare 8-acetyl-7-hydroxy-4-methyl-2H-chromen-2-one III. The 8-acetyl-4-methyl-2- oxo-2H-chromen-7-yl benzoate IV was prepared by direct heating of III with benzoyl chloride [30]. The 6-nitrocoumarin derivative V was prepared using a mixture of concentrated sulfuric/nitric acids (Scheme 1).

Synthesis of the required 6-oxopyridine derivatives VIa-i was performed via the one pot reaction of the 8-acetyl coumarin V with ethyl cyanoacetate, the appropriate aromatic aldehyde and excess ammonium acetate in refluxing n-butanol (Scheme 1). Similarly, 6-thioxopyridine derivatives VIIa-i and 6-iminopyridine derivarives VIIIa-i were obtained by reaction with thiocyanoacetamide and malononitrile, respectively, (Scheme 1).

a) CH₃COCl, reflux, 1.5 h (95%)

b) AlCl₃, 160-170oC, 2h (75%)

c) Benzoyl chloride, 160-170°C, 1.5 h (67.5%)

d) HNO₃/H₂SO₄, rt, 1 h (72.5%), e) Ethyl cyanoacetate/Aromatic aldehyde/Ammonium acetate, n-butanol, reflux, 5 h

f) Thiocyanoacetamide/Aromatic aldehyde/Ammonium acetate, n-butanol, reflux, 5 h

g) Malononitrile/Aromatic aldehyde/Ammonium acetate, n-butanol, reflux, 4 h

Scheme 2

On the other hand, treatment of the 8-acetyl coumarin derivative V with the appropriate aromatic aldehyde and ethyl cyanoacetate in the presence of few drops of piperidine in refluxing n-butanol afforded the titled ethyl pyran-3-carboxylate derivatives IXa-i. In addition, 3-thiocarbamoyl pyran derivatives Xa-i and 3-cyanopyran derivatives XIa-i were obtained by a similar reaction using thiocyanoacetamide and malononitrile, respectively, (Scheme 2).

a) Ethyl cyanoacetate/Aromatic aldehyde/Piperidine, n-butanol, reflux, 6 h

b) Thiocyanoacetamide/Aromatic aldehyde/Piperidine, n-butanol, reflux, 6 h

c) Malononitrile/Aromatic aldehyde/Piperidine, n-butanol, reflux, 5 h

Antibacterial screening

A preliminary in-vitro antibacterial evaluation was carried out on fifty five of the newly synthesized compounds, namely, 8-acetyl-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate V, 8-(pyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates VI-VIII and 8-(pyran-6-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates IX-XI using paper disc diffusion technique against Bacillus subtilis ATCC 5256 and Staphylococcus aureus ATCC 6571 (Gram-positive bacteria) and against Escherichia coli 0157:H₇ and Pseudomonas aeruginosa ATCC 27852 (Gram-negative bacteria) using Mueller Hinton agar medium (Oxoid).

When the sensitivity test [31] was first performed for all the fifty five compounds in the concentration of 50 μg/disc, it was found that fifteen compounds V, VIf, VIIId, VIIIe, VIIIf, VIIIh, IXb, IXc, IXe, IXi, Xa, Xg, Xh, XIe and XIi possessed growth inhibitory activity against only the gram-positive bacteria B. subtilis, while they had no inhibitory activity against neither the gram-positive bacteria S. aureus nor the gram-negative bacteria E. coli and P. aeruginosa. In addition, the rest of the assessed compounds did not show growth inhibitory activity against the selected microorganisms (Table 1).

Consequently, when the minimum inhibitory concentration [32] of the aforementioned fifteen compounds V, VIf, VIIId, VIIIe, VIIIf, VIIIh, IXb, IXc, IXe, IXi, Xa, Xg, Xh, XIe and XIi was determined using agar dilution technique against the same microorganisms, it was concluded that all of them were most active against B. subtilis and that they possessed variable activities against both S. aureus and E. coli while they were inactive against P. aeruginosa (Table 2).

Compared to the other compounds, the 8-(4-(2-bromophenyl)-3-thiocarbamoyl-2-imino-3,4-dihydropyran-6-yl) coumarin derivative Xh could be considered as the most promising antibacterial agent with MIC of 64, 512 and 128 μg/ml against B. subtilis, S. aureus and E. coli, respectively.

Conclusion

Fifty five new coumarin derivatives were successfully synthesized and screened for their antibacterial activity. The results of the sensitivity test revealed that only fifteen compounds; V, VIf, VIIId, VIIIe, VIIIf, VIIIh, IXb, IXc, IXe, IXi, Xa, Xg, Xh, XIe and XIi possessed growth inhibitory activity against only the gram-positive bacteria B. subtilis while the results of the minimum inhibitory concentration of these fifteen compounds showed that all of them were most active against B. subtilis and that they possessed variable activities against both S. aureus and E. coli while they were inactive against P. aeruginosa. It was also concluded that compound Xh showed the most promising antibacterial activity against B. subtilis, S. aureus and E. coli.

Experimental

Chemistry

Melting points were measured using Electro-thermal IA 9100 apparatus and were uncorrected. Elemental microanalysis was carried out on Elmentar Vario EL apparatus at the Micro Analytical Center, Faculty of Science, Cairo University. Infrared (IR) spectra were recorded on Bruker Vector 22 FT-IR spectrometer as potassium bromide discs at the Micro Analytical Center, Faculty of Science, Cairo University. Proton nuclear magnetic resonance (¹HNMR) spectra were determined on Varian mercury 300 MHz spectrometer at the Faculty of Science, Cairo University and on JEOL NMR EX-270 MHz spectrometer at the Central Services Lab, the Central Unit for Analysis and Scientific Services, National Research Center using tetramethylsilane (TMS) as an internal standard and either DMSO-d₆ or CDCl₃ as solvents. Mass spectra (MS) were performed on Shimadzu QP-2010 plus spectrometer using 70 eV, EI mode at the Micro Analytical Center, Faculty of Science, Cairo University. The reactions were monitored by thin layer chromatography (TLC) plates (silica gel 60 F254, aluminium sheets, Merck) using hexane/ethyl acetate (1:1 v/v) as eluent.

7-Hydroxy-4-methyl-2H-chromen-2-one (I)

In a flat bottomed flask equipped with a dropping funnel, 250 ml sulfuric acid was placed and the temperature was kept below 10°C. A solution of resorcinol (33 g, 0.3 mol) in ethyl acetoacetate (38 ml, 0.3 mol) was added dropwise. After complete addition, the reaction mixture was stirred at room temperature for 12 hours, and then poured onto ice-water. The formed precipitate was filtered off, washed with cold water, dissolved in 5% aqueous sodium hydroxide and then re-precipitated with diluted sulfuric acid. The separated product was filtered off, washed with cold water and crystallized from absolute ethyl alcohol. Yield: 90%. m.p.: 185- 186°C (as reported) [26].

4-Methyl-2-oxo-2H-chromen-7-yl acetate (II)

A mixture of I (14.08 g, 0.08 mol) and acetyl chloride (36 ml, 0.5 mol) was refluxed for one and half hours. The reaction mixture was then poured onto ice-water and the separated product was filtered off, washed with cold water and crystallized from absolute ethyl alcohol. Yield: 95%. m.p.: 150- 151°C (as reported) [27,28].

8-Acetyl-7-hydroxy-4-methyl-2H-chromen-2-one (III)

A mixture of the 7-acetoxycoumarin derivative II (32.7 g, 0.15 mol) and anhydrous aluminium chloride (66.67 g, 0.5 mol) was heated in an oil bath at 160-170°C for 2 hours. After cooling down, the reaction mixture was placed in an ice bath and decomposed with diluted hydrochloric acid. The formed solid was then filtered off, washed with cold water, dissolved in 5% aqueous sodium hydroxide and then re-precipitated with diluted hydrochloric acid. The obtained precipitate was filtered off, washed with cold water and crystallized from absolute ethyl alcohol. Yield: 75%. m.p.: 162-163°C (as reported) [29].

8-Acetyl-4-methyl-2-oxo-2H-chromen-7-yl benzoate (IV)

A mixture of the 8-acetyl-7-hydroxycoumarin III (17.44 g, 0.08 mol) and benzoyl chloride (59 ml, 0.5 mol) was refluxed for one and half hours at 160-170°C. The reaction mixture was cooled then poured onto ice-water. The water layer was then removed by decantation and the obtained gray sticky mass was suspended in the least amount of ethyl alcohol, filtered off, washed with diethyl ether and crystallized from glacial acetic acid. Yield: 67.5%. m.p.: 188-189°C (as reported) [30].

8-Acetyl-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (V)

To a solution of the 8-acetyl-7-benzoyloxycoumarin derivative IV (32.2 g, 0.1 mol) in sulfuric acid (30 ml) placed in an ice-salt bath to keep the temperature below 5°C, a mixture of nitric acid (8 ml) and sulfuric acid (10 ml), kept at the same temperature, was added drop wise with stirring while keeping the temperature below 5°C. After complete addition, the reaction mixture was stirred at room temperature for 1 hour then poured onto ice-water. The obtained precipitate was then filtered off, washed with cold water and crystallized from absolute ethyl alcohol. Yield: 72.5%. m.p.: 98-100°C. Analysis for C₁₉H₁₃NO₇, M.wt.: 367.31. Calcd.: %C, 62.13; H, 3.57; N, 3.81. Found: %C, 61.92; H, 3.61; N, 3.73. IR (KBr, u, cm^-1): 3065 (CH, aromatic), 2928 (CH, aliphatic), 1735 (3 C=O), 1615 and 1482 (C=C), 1546 and 1374 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.36 (s, 3H, CH₃), 2.53 (s, 3H, COCH₃), 6.16 (s, 1H, C-3 proton), 7.46-8.57 (m, 6H, aromatic protons). MS; m/z (R.I. %): [M⁺] 367 (0.34), 80 (100).

8-(4-Aryl-5-cyano-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates (VIa-i)

A mixture of the 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), ethyl cyanoacetate (0.1 ml, 0.001 mol) and ammonium acetate (0.62 g, 0.008 mol) in n-butanol (5 ml) was refluxed for 5 hours. After cooling down, the obtained precipitate was filtered off, washed s with diethyl ether and crystallized from absolute ethyl alcohol.

8-(4-(4-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIa)

Yield: 60%. m.p.: 176-178°C. Analysis for C₂₉H₁₆ClN₃O₇, M.wt.: 553.91. Calcd.: %C, 62.88; H, 2.91; N, 7.59. Found: %C, 62.97; H, 2.75; N, 7.71. IR (KBr, u, cm^-1): 3413 (NH), 2211 (C≡N), 1725 (3 C=O), 1530 and 1377 (NO₂). MS; m/z (R.I. %): [M⁺] 553 (9.02), [M⁺+2] 555 (2.16), 80 (100).

8-(5-Cyano-4-(4-nitrophenyl)-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIb)

Yield: 60%. m.p.: 174-175°C. Analysis for C₂₉H₁₆N₄O₉, M.wt.: 564.46. Calcd.: %C, 61.71; H, 2.86; N, 9.93. Found: %C, 61.52; H, 2.99; N, 9.80. IR (KBr, u, cm^-1): 3375 (NH), 2208 (C≡N), 1723 (3 C=O), 1521 and 1389 (2 NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 5.90 (s, 1H, C-3 proton), 6.41 (s, 1H, oxopyridine proton), 7.42-8.38 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(5-Cyano-4-(4-hydroxyphenyl)-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIc)

Yield: 70%. m.p.: 166-168°C. Analysis for C₂₉H₁₇N₃O₈, M.wt.: 535.46. Calcd.: %C, 65.05; H, 3.20; N, 7.85. Found: %C, 65.24; H, 3.42; N, 7.68. IR (KBr, u, cm^-1): 3376 (NH), 3193 (OH), 2206 (C≡N), 1720 (3 C=O), 1530 and 1383 (NO₂).

8-(5-Cyano-6-oxo-4-p-tolyl-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VId)

Yield: 82%. m.p.: 188-190°C. Analysis for C₃₀H₁₉N₃O₇, M.wt.: 533.49. Calcd.: %C, 67.54; H, 3.59; N, 7.88. Found: %C, 67.58; H, 3.63; N, 7.71. IR (KBr, u, cm^-1): 3425 (NH), 2213 (C≡N), 1732 (3 C=O), 1522 and 1371 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 2.35 (s, 1H, p-CH₃), 5.96 (s, 1H, C-3 proton), 6.50 (s, 1H, oxopyridine proton), 7.05-8.75 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable). MS; m/z (R.I. %): [M⁺] 533 (62.31), 86 (100).

8-(4-(3-Bromophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIe)

Yield: 62%. m.p.: 159-160°C. Analysis for C₂₉H₁₆BrN₃O₇, M.wt.: 597.36. Calcd.: %C, 58.21; H, 2.70; N, 7.02. Found: %C, 58.02; H, 2.55; N, 7.22. IR (KBr, u, cm^-1): 3426 (NH), 2208 (C≡N), 1728 (3 C=O), 1526 and 1381 (NO₂). MS; m/z (R.I. %): [M⁺] 597 (22.14), [M⁺+2] 599 (10.84), 406 (100).

8-(4-(2-Chlorophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIf)

Yield: 52%. m.p.: 150-151°C. Analysis for C₂₉H₁₆ClN₃O₇, M.wt.: 553.91. Calcd.: %C, 62.88; H, 2.91; N, 7.59. Found: %C, 62.67; H, 3.05; N, 7.56. IR (KBr, u, cm^-1): 3430 (NH), 2210 (C≡N), 1731 (3 C=O), 1527 and 1390 (NO₂).

8-(5-Cyano-4-(2-nitrophenyl)-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIg)

Yield: 56%. m.p.: 173-175°C. Analysis for C₂₉H₁₆N₄O₉, M.wt.: 564.46. Calcd.: %C, 61.71; H, 2.86; N, 9.93. Found: %C, 61.56; H, 2.91; N, 9.80. IR (KBr, u, cm^-1): 3414 (NH), 2210 (C≡N), 1722 (3 C=O), 1523 and 1382 (2 NO₂).

8-(4-(2-Bromophenyl)-5-cyano-6-oxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIh)

Yield: 56%. m.p.: 170-172°C. Analysis for C₂₉H₁₆BrN₃O₇, M.wt.: 597.36. Calcd.: %C, 58.21; H, 2.70; N, 7.02. Found: %C, 58.04; H, 2.56; N, 7.16. IR (KBr, u, cm^-1): 3419 (NH), 2210 (C≡N), 1729 (3 C=O), 1527 and 1385 (NO₂).

8-(5-Cyano-6-oxo-4-phenyl-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIi)

Yield: 67%. m.p.: 190-192°C. Analysis for C₂₉H₁₇N₃O₇, M.wt.: 519.46. Calcd.: %C, 67.05; H, 3.30; N, 8.09. Found: %C, 66.95; H, 3.41; N, 8.28. IR (KBr, u, cm^-1): 3424 (NH), 2211 (C≡N), 1715 (3 C=O), 1511 and 1373 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.34 (s, 3H, CH₃), 5.90 (s, 1H, C-3 proton), 6.56 (s, 1H, oxopyridine proton), 7.45-8.19 (m, 11H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(4-Aryl-5-cyano-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates (VIIa-i)

A mixture of 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), thiocyanoacetamide (0.1 g, 0.001 mol) and ammonium acetate (0.62 g, 0.008 mol) in n-butanol (5 ml) was refluxed for 5 hours. The reaction mixture was cooled; the formed precipitate was filtered off, washed with diethyl ether and crystallized from absolute ethyl alcohol.

8-(4-(4-Chlorophenyl)-5-cyano-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIa)

Yield: 91%. m.p.: 198-199°C. Analysis for C₂₉H₁₆ClN₃O₆S, M.wt.: 569.97. Calcd.: %C, 61.11; H, 2.83; N, 7.37. Found: %C, 60.01; H, 2.85; N, 7.50. IR (KBr, u, cm^-1): 3425 (NH), 2210 (C≡N), 1726 (2 C=O), 1500 and 1316 (NO₂), 1269 (C=S). MS; m/z (R.I. %): [M⁺] 569 (46.61), [M⁺+2] 571 (11.18), 179 (100).

8-(5-Cyano-4-(4-nitrophenyl)-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIb)

Yield: 74 %. m.p.: 216-217°C. Analysis for C₂₉H₁₆N₄O₈S, M.wt.: 580.52. Calcd.: %C, 60.00; H, 2.78; N, 9.65. Found: %C, 61.13; H, 2.64; N, 9.62. IR (KBr, u, cm^-1): 3355 (NH), 2207 (C≡N), 1718 (2 C=O), 1519 and 1387 (2 NO₂), 1267 (C=S). MS; m/z (R.I. %): [M⁺] 580 (61.32), [M⁺+1] 581 (5.66), 265 (100).

8-(5-Cyano-4-(4-hydroxyphenyl)-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIc)

Yield: 61%. m.p.: 194-195°C. Analysis for C₂₉H₁₇N₃O₇S, M.wt.: 551.53. Calcd.: %C, 63.15; H, 3.11; N, 7.62. Found: %C, 63.20; H, 3.18; N, 7.55. IR (KBr, u, cm^-1): 3343 (NH), 3202 (OH), 2205 (C≡N), 1717 (2 C=O), 1517 and 1379 (NO₂), 1270 (C=S). MS; m/z (R.I. %): [M⁺] 551 (20.60), [M⁺+1] 552 (19.29), 162 (100).

8-(5-Cyano-6-thioxo-4-p-tolyl-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIId)

Yield: 51%. m.p.: 184-186°C. Analysis for C₃₀H₁₉N₃O₆S, M.wt.: 549.55. Calcd.: %C, 65.57; H, 3.48; N, 7.65. Found: %C, 65.52; H, 3.42; N, 7.55. IR (KBr, u, cm^-1): 3348 (NH), 2205 (C≡N), 1722 (2 C=O), 1530 and 1379 (NO₂), 1268 (C=S).

8-(4-(3-Bromophenyl)-5-cyano-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIe)

Yield: 51%. m.p.: 160-162°C. Analysis for C₂₉H₁₆BrN₃O₆S, M.wt.: 613.42. Calcd.: %C, 56.69; H, 2.62; N, 6.84. Found: %C, 56.71; H, 2.46; N, 6.77. IR (KBr, u, cm^-1): 3334 (NH), 2207 (C≡N), 1719 (2 C=O), 1541 and 1378 (NO₂), 1268 (C=S). ¹HNMR (DMSO-d⁶, δ, ppm): 2.46 (s, 3H, CH₃), 6.20 (s, 1H, C-3 proton), 6.46 (s, 1H, thioxopyridine proton), 7.00-8.45 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(4-(2-Chlorophenyl)-5-cyano-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIf)

Yield: 81%. m.p.: 202-204°C. Analysis for C₂₉H₁₆ClN₃O₆S, M.wt.: 569.97. Calcd.: %C, 61.11; H, 2.83; N, 7.37. Found: %C, 60.87; H, 2.73; N, 7.32. IR (KBr, u, cm^-1): 3328 (NH), 2210 (C≡N), 1718 (2 C=O), 1533 and 1380 (NO₂), 1271 (C=S).

8-(5-Cyano-4-(2-nitrophenyl)-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIg)

Yield: 83%. m.p.: 140-142°C. Analysis for C₂₉H₁₆N₄O₈S, M.wt.: 580.52. Calcd.: %C, 60.00; H, 2.78; N, 9.65. Found: %C, 60.12; H, 2.68; N, 9.67. IR (KBr, u, cm^-1): 3326 (NH), 2206 (C≡N), 1722 (2 C=O), 1527 and 1376 (2 NO₂), 1267 (C=S). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 6.19 (s, 1H, C-3 proton), 6.46 (s, 1H, thioxopyridine proton), 7.05-8.65 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(4-(2-Bromophenyl)-5-cyano-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIh)

Yield: 66%. m.p.: 144-146°C. Analysis for C₂₉H₁₆BrN₃O₆S, M.wt.: 613.42. Calcd.: %C, 56.69; H, 2.62; N, 6.84. Found: %C, 56.82; H, 2.66; N, 6.80. IR (KBr, u, cm^-1): 3345 (NH), 2209 (C≡N), 1724 (2 C=O), 1532 and 1378 (NO₂), 1273 (C=S). ¹HNMR (DMSO-d₆, δ, ppm): 2.40 (s, 3H, CH₃), 6.21 (s, 1H, C-3 proton), 6.50 (s, 1H, thioxopyridine proton), 7.07-8.40 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(5-Cyano-4-phenyl-6-thioxo-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIi)

Yield: 79%. m.p.: 183-185°C. Analysis for C₂₉H₁₇N₃O₆S, M.wt.: 535.53. Calcd.: %C, 65.04; H, 3.20; N, 7.85. Found: %C, 64.06; H, 3.29; N, 7.83. IR (KBr, u, cm^-1): 3319 (NH), 2207 (C≡N), 1717 (2 C=O), 1550 and 1387 (NO₂), 1270 (C=S). ₁HNMR (DMSO-d₆, δ, ppm): 2.34 (s, 3H, CH₃), 6.27 (s, 1H, C-3 proton), 6.42 (s, 1H, thioxopyridine proton), 7.18-8.27 (m, 11H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(4-Aryl-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates (VIIIa-i)

A mixture of 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), malononitrile (0.066 g, 0.001 mol) and ammonium acetate (0.62 g, 0.008 mol) in n-butanol (5 ml) was refluxed for 4 hours then cooled. The formed precipitate was filtered off, washed with diethyl ether and crystallized from absolute ethyl alcohol.

8-(4-(4-Chlorophenyl)-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIa)

Yield: 90%. m.p.: 223-224°C. Analysis for C₂₉H₁₇ClN₄O₆, M.wt.: 552.92. Calcd.: %C, 62.99; H, 3.10; N, 10.13. Found: %C, 62.86; H, 3.13; N, 10.21. IR (KBr, u, cm^-1): 3441 and 3341 (2 NH), 2199 (C≡N), 1707 (2 C=O), 1622 (C=N), 1571 and 1381 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.44 (s, 3H, CH₃), 6.25 (s, 1H, C-3 proton), 6.89 (s, 1H, iminopyridine proton), 7.23-8.87 (m, 10H, aromatic protons, 2H, 2NH, D₂O exchangeable). MS; m/z (R.I. %): [M⁺] 552 (1.39), [M⁺+2] 554 (0.33), 64 (100).

8-(5-Cyano-6-imino-4-(4-nitrophenyl)-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIb)

Yield: 91%. m.p.: 228-229°C. Analysis for C₂₉H₁₇N₅O₈, M.wt.: 563.47. Calcd.: %C, 61.81; H, 3.04; N, 12.43. Found: %C, 61.87; H, 3.11; N, 12.32. IR (KBr, u, cm^-1): 3418 and 3337 (2 NH), 2203 (C≡N), 1713 (2 C=O), 1642 (C=N), 1571 and 1387 (2 NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.41 (s, 3H, CH₃), 6.32 (s, 1H, C-3 proton), 6.94 (s, 1H, iminopyridine proton), 7.02-8.33 (m, 10H, aromatic protons, 2H, 2NH, D₂O exchangeable).

8-(5-Cyano-4-(4-hydroxyphenyl)-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIc)

Yield: 88%. m.p.: 242-244°C. Analysis for C₂₉H₁₈N₄O₇, M.wt.: 534.48. Calcd.: %C, 65.17; H, 3.39; N, 10.48. Found: %C, 65.02; H, 3.45; N, 10.55. IR (KBr, u, cm^-1): 3435 and 3339 (2 NH), 3212 (OH), 2194 (C≡N), 1706 (2 C=O), 1619 (C=N), 1552 and 1386 (NO₂). MS; m/z (R.I. %): [M⁺] 534 (77.78), 175 (100).

8-(5-Cyano-6-imino-4-p-tolyl-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIId)

Yield: 95%. m.p.: 170-172°C. Analysis for C₃₀H₂₀N₄O₆, M.wt.: 532.50. Calcd.: %C, 67.67; H, 3.79; N, 10.52. Found: %C, 67.74; H, 3.61; N, 10.57. IR (KBr, u, cm^-1): 3436 and 3342 (2 NH), 2197 (C≡N), 1706 (2 C=O), 1620 (C=N), 1553 and 1383 (NO₂). MS; m/z (R.I. %): [M⁺] 532 (20.00), 110 (100).

8-(4-(3-Bromophenyl)-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIe)

Yield: 89%. m.p.: 210-211°C. Analysis for C₂₉H₁₇BrN₄O₆, M.wt.: 596.37. Calcd.: %C, 58.31; H, 2.87; N, 9.38. Found: %C, 58.43; H, 2.82; N, 9.50. IR (KBr, u, cm^-1): 3437 and 3342 (2 NH), 2198 (C≡N), 1712 (2 C=O), 1627 (C=N), 1568 and 1379 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.33 (s, 3H, CH₃), 6.27 (s, 1H, C-3 proton), 6.94 (s, 1H, iminopyridine proton), 7.14-8.85 (m, 10H, aromatic protons, 2H, 2NH, D₂O exchangeable).

8-(4-(2-Chlorophenyl)-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIf)

Yield: 91%. m.p.: 180-182°C. Analysis for C₂₉H₁₇ClN₄O₆, M.wt.: 552.92. Calcd.: %C, 62.99; H, 3.10; N, 10.13. Found: %C, 63.11; H, 3.04; N, 10.23. IR (KBr, u, cm^-1): 3448 and 3345 (2 NH), 2198 (C≡N), 1714 (2 C=O), 1620 (C=N), 1570 and 1381 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.40 (s, 3H, CH₃), 6.19 (s, 1H, C-3 proton), 6.93 (s, 1H, iminopyridine proton), 7.06-8.16 (m, 10H, aromatic protons, 2H, 2NH, D₂O exchangeable).

8-(4-(2-Nitrophenyl)-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIg)

Yield: 89%. m.p.: 248-249°C. Analysis for C₂₉H₁₇N₅O₈, M.wt.: 563.47. Calcd.: %C, 61.81; H, 3.04; N, 12.43. Found: %C, 61.69; H, 3.15; N, 12.34. IR (KBr, u, cm^-1): 3438 and 3376 (2 NH), 2199 (C≡N), 1708 (2 C=O), 1624 (C=N), 1562 and 1382 (2 NO₂).

8-(4-(2-Bromophenyl)-5-cyano-6-imino-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIh)

Yield: 99%. m.p.: 200-202°C. Analysis for C₂₉H₁₇BrN₄O₆, M.wt.: 596.37. Calcd.: %C, 58.31; H, 2.87; N, 9.38. Found: %C, 58.22; H, 2.85; N, 9.47. IR (KBr, u, cm^-1): 3447 and 3341 (2 NH), 2200 (C≡N), 1705 (2 C=O), 1620 (C=N), 1561 and 1383 (NO₂).

8-(5-Cyano-6-imino-4-phenyl-1,6-dihydropyridin-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (VIIIi)

Yield: 98%. m.p.: 188-189°C. Analysis for C₂₉H₁₈N₄O₆, M.wt.: 518.48. Calcd.: %C, 67.18; H, 3.50; N, 10.81. Found: %C, 67.01; H, 3.55; N, 10.90. IR (KBr, u, cm^-1): 3447 and 3342 (2 NH), 2201 (C≡N), 1710 (2 C=O), 1617 (C=N), 1560 and 1384 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.43 (s, 3H, CH₃), 6.48 (s, 1H, C-3 proton), 6.89 (s, 1H, iminopyridine proton), 7.06-8.07 (m, 11H, aromatic protons, 2H, 2NH, D₂O exchangeable).

Ethyl 4-aryl-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-2-imino-3,4-dihydro-2H-pyran-3-carboxylates (IXa-i)°C

A mixture of 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), ethyl cyanoacetate (0.1 ml, 0.001 mol) and 5 drops piperidine in n-butanol (5 ml) was refluxed for 6 hours. After cooling down, the formed precipitate was filtered off, washed with diethyl ether and crystallized from absolute ethyl alcohol.

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(4-chlorophenyl)-4H-pyran-3-carboxylate (IXa)

Yield: 57%. m.p.: 90-92°C. Analysis for C₃₁H₂₃ClN₂O₉, M.wt.: 602.98. Calcd.: %C, 61.75; H, 3.84; N, 4.65. Found: %C, 61.82; H, 4.02; N, 4.54. IR (KBr, u, cm^-1): 3438 (NH), 1713 (3 C=O), 1611 (C=N), 1566 and 1391 (NO₂).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(4-nitrophenyl)-4H-pyran-3-carboxylate (IXb)

Yield: 62%. m.p.: 80-82°C. Analysis for C₃₁H₂₃N₃O₁₁, M.wt.: 613.53. Calcd.: %C, 60.69; H, 3.78; N, 6.85. Found: %C, 60.62; H, 3.86; N, 6.72. IR (KBr, u, cm^-1): 3439 (NH), 1726 (3 C=O), 1617 (C=N), 1521 and 1382 (2 NO₂).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(4-hydroxyphenyl)-4H-pyran-3-carboxylate (IXc)

Yield: 62 %. m.p.: 110-111°C. Analysis for C₃₁H₂₄N₂O₁₀, M.wt.: 584.53. Calcd.: %C, 63.70; H, 4.14; N, 4.79. Found: %C, 63.79; H, 4.20; N, 4.83. IR (KBr, u, cm^-1): 3418 (NH), 3260 (OH), 1727 (3 C=O), 1603 (C=N), 1519 and 1379 (NO₂). MS; m/z (R.I. %): [M⁺] 584 (36.93), [M⁺+1] 585 (52.27), 129 (100).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-p-tolyl-4H-pyran-3-carboxylate (IXd)

Yield: 73%. m.p.: 84-86°C. Analysis for C₃₂H₂₆N₂O₉, M.wt.: 582.56. Calcd.: %C, 65.98; H, 4.50; N, 4.81. Found: %C, 66.18; H, 4.58; N, 4.77. IR (KBr, u, cm^-1): 3437 (NH), 1726 (3 C=O), 1602 (C=N), 1528 and 1378 (NO₂). MS; m/z (R.I. %): [M⁺] 582 (8.16), 64 (100).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(3-bromophenyl)-4H-pyran-3-carboxylate (IXe)

Yield: 64%. m.p.: 98-100°C. Analysis for C₃₁H₂₃BrN₂O₉, M.wt.: 646.43. Calcd.: %C, 57.51; H, 3.58; N, 4.33. Found: %C, 57.57; H, 3.42; N, 4.43. IR (KBr, u, cm^-1): 3435 (NH), 1724 (3 C=O), 1598 (C=N), 1532 and 1380 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 1.58 (t, 3H, CH₃), 2.38 (s, 3H, CH₃), 2.98 (d, 1H, iminopyran C-3 proton), 3.99 (t, 1H, iminopyran C-4 proton), 4.12 (q, 2H, OCH₂), 6.20 (s, 1H, C-3 proton), 6.97 (d, 1H, iminopyran C-5 proton), 7.18-8.57 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(2-chlorophenyl)-4H-pyran-3-carboxylate (IXf)

Yield: 65%. m.p.: 100-102°C. Analysis for C₃₁H₂₃ClN₂O₉, M.wt.: 602.98. Calcd.: %C, 61.75; H, 3.84; N, 4.65. Found: %C, 61.65; H, 3.91; N, 4.48. IR (KBr, u, cm^-1): 3430 (NH), 1724 (3 C=O), 1603 (C=N), 1540 and 1382 (NO₂).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(2-nitrophenyl)-4H-pyran-3-carboxylate (IXg)

Yield: 57%. m.p.: 86-88°C. Analysis for C₃₁H₂₃N₃O₁₁, M.wt.: 613.53. Calcd.: %C, 60.69; H, 3.78; N, 6.85. Found: %C, 60.73; H, 3.85; N, 6.74. IR (KBr, u, cm^-1): 3433 (NH), 1730 (3 C=O), 1607 (C=N), 1543 and 1378 (2 NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 1.58 (t, 3H, CH₃), 2.40 (s, 3H, CH₃), 2.95 (d, 1H, iminopyran C-3 proton), 3.89 (t, 1H, iminopyran C-4 proton), 4.00 (q, 2H, OCH₂), 6.19 (s, 1H, C-3 proton), 6.96 (d, 1H, iminopyran C-5 proton), 7.19-8.57 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable). MS; m/z (R.I. %): [M⁺] 613 (24.94), 69 (100).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-(2-bromophenyl)-4H-pyran-3-carboxylate (IXh)

Yield: 55%. m.p.: 112-114°C. Analysis for C₃₁H₂₃BrN₂O₉, M.wt.: 646.43. Calcd.: %C, 57.51; H, 3.58; N, 4.33. Found: %C, 57.70; H, 3.52; N, 4.37. IR (KBr, u, cm^-1): 3398 (NH), 1727 (3 C=O), 1599 (C=N), 1550 and 1380 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 1.58 (t, 3H, CH₃), 2.33 (s, 3H, CH₃), 3.04 (d, 1H, iminopyran C-3 proton), 4.04 (t, 1H, iminopyran C-4 proton), 4.40 (q, 2H, OCH2), 6.16 (s, 1H, C-3 proton), 6.98 (d, 1H, iminopyran C-5 proton), 7.18-8.58 (m, 10H, aromatic protons, 1H, NH, D2O exchangeable).

Ethyl 2-amino-6-(7-(benzoyloxy)-4-methyl-6-nitro-2-oxo-2H-chromen-8-yl)-4-phenyl-4H-pyran-3-carboxylate (IXi)

Yield: 52%. m.p.: 88-90°C. Analysis for C₃₁H₂₄N₂O₉, M.wt.: 568.53. Calcd.: %C, 65.49; H, 4.25; N, 4.93. Found: %C, 65.61; H, 4.31; N, 4.88. IR (KBr, u, cm^-1): 3436 (NH), 1732 (3 C=O), 1606 (C=N), 1527 and 1379 (NO₂).

8-(4-Aryl-3-thiocarbamoyl-2-imino-3,4-dihydro-2H-pyran-6-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates (Xa-i)

A mixture of 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), thiocyanoacetamide (0.1 g, 0.001 mol) and 5 drops piperidine in n-butanol (5 ml) was refluxed for 6 hours. The reaction mixture was left to cool; the formed precipitate was filtered off, washed with diethyl ether and crystallized from absolute ethyl alcohol.

8-(6-Amino-5-thiocarbamoyl-4-(4-chlorophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xa)

Yield: 79%. m.p.: 120-122°C. Analysis for C₂₉H₂₀ClN₃O₇S, M.wt.: 589.00. Calcd.: %C, 59.04; H, 3.42; N, 7.12. Found: %C, 59.12; H, 3.47; N, 7.01. IR (KBr, u, cm^-1): 3441 and 3400 (NH₂), 3280 (NH), 1724 (2 C=O), 1592 (C=N), 1525 and 1375 (NO₂), 1257 (C=S). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 2.96 (d, 1H, iminopyran C-3 proton), 3.57 (t, 1H, iminopyran C-4 proton), 6.20 (s, 1H, C-3 proton), 6.98 (d, 1H, iminopyran C-5 proton), 7.23-8.57 (m, 10H, aromatic protons, 1H, exchangeable NH), 9.98 (s, 2H, NH₂, D₂O exchangeable).

8-(6-Amino-5-thiocarbamoyl-4-(4-nitrophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xb)

Yield: 83%. m.p.: 116-118°C. Analysis for C₂₉H₂₀N₄O₉S, M.wt.: 600.56. Calcd.: %C, 58.00; H, 3.36; N, 9.33. Found: %C, 58.14; H, 3.42; N, 9.53. IR (KBr, u, cm^-1): 3432 and 3372 (NH₂), 3292 (NH), 1724 (2 C=O), 1595 (C=N), 1519 and 1377 (2 NO₂), 1271 (C=S).

8-(6-Amino-5-thiocarbamoyl-4-(4-hydroxyphenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xc)

Yield: 78%. m.p.: 154-155°C. Analysis for C₂₉H₂₁N₃O₈S, M.wt.: 571.56. Calcd.: %C, 60.94; H, 3.70; N, 7.35. Found: %C, 61.06; H, 3.71; N, 7.44. IR (KBr, u, cm^-1): 3446 and 3401 (NH₂), 3302 (NH), 3197 (OH), 1717 (2 C=O), 1593 (C=N), 1514 and 1374 (NO₂), 1263 (C=S). MS; m/z (R.I. %): [M⁺] 571 (23.27), [M⁺+1] 572 (12.16), 69 (100).

48-(6-Amino-5-thiocarbamoyl-4-p-tolyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xd)

Yield: 84%. m.p.: 86-88°C. Analysis for C₃₀H₂₃N₃O₇S, M.wt.: 569.58. Calcd.: %C, 63.26; H, 4.07; N, 7.38. Found: %C, 63.33; H, 4.17; N, 7.30. IR (KBr, u, cm^-1): 3429 and 3384 (NH₂), 3298 (NH), 1726 (2 C=O), 1597 (C=N), 1520 and 1376 (NO₂).1262 (C=S). MS; m/z (R.I. %): [M⁺] 569 (54.41), 199 (100).

8-(6-Amino-4-(3-bromophenyl)-5-thiocarbamoyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xe)

Yield: 79%. m.p.: 116-118°C. Analysis for C₂₉H₂₀BrN₃O₇S, M.wt.: 633.45. Calcd.: %C, 54.90; H, 3.18; N, 6.62. Found: %C, 56.84; H, 3.23; N, 6.47. IR (KBr, u, cm^-1): 3431 and 3380 (NH₂), 3292 (NH), 1726 (2 C=O), 1594 (C=N), 1527 and 1375 (NO₂), 1255 (C=S).

8-(6-Amino-5-thiocarbamoyl-4-(2-chlorophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xf)

Yield: 87%. m.p.: 106-108°C. Analysis for C₂₉H₂₀ClN₃O₇S, M.wt.: 589.00. Calcd.: %C, 59.04; H, 3.42; N, 7.12. Found: %C, 59.24; H, 3.55; N, 7.15. IR (KBr, u, cm^-1): 3429 and 3372 (NH₂), 3287 (NH), 1727 (2 C=O), 1596 (C=N), 1530 and 1377 (NO₂), 1266 (C=S).

8-(6-Amino-5-thiocarbamoyl-4-(2-nitrophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xg)

Yield: 72%. m.p.: 130-132°C. Analysis for C₂₉H₂₀N₄O₉S, M.wt.: 600.56. Calcd.: %C, 58.00; H, 3.36; N, 9.33. Found: %C, 58.05; H, 3.41; N, 9.25. IR (KBr, u, cm^-1): 3430 and 3379 (NH₂), 3293 (NH), 1723 (2 C=O), 1601 (C=N), 1530 and 1378 (2 NO₂), 1263 (C=S).

8-(6-Amino-4-(2-bromophenyl)-5-thiocarbamoyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xh)

Yield: 90%. m.p.: 88-90°C. Analysis for C₂₉H₂₀BrN₃O₇S, M.wt.: 633.45. Calcd.: %C, 54.90; H, 3.18; N, 6.62. Found: %C, 55.02; H, 3.12; N, 6.54. IR (KBr, u, cm^-1): 3429 and 3369 (NH₂), 3287 (NH), 1727 (2 C=O), 1596 (C=N), 1531 and 1375 (NO₂), 1262 (C=S). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 2.95 (d, 1H, iminopyran C-3 proton), 3.85 (t, 1H, iminopyran C-4 proton), 6.32 (s, 1H, C-3 proton), 6.93 (d, 1H, iminopyran C-5 proton), 7.15-8.58 (m, 10H, aromatic protons, 1H, exchangeable NH), 10.19 (s, 2H, NH₂, D₂O exchangeable).

8-(6-Amino-5-thiocarbamoyl-4-phenyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (Xi)

Yield: 83%. m.p.: 92-94°C. Analysis for C₂₉H₂₁N₃O₇S, M.wt.: 555.56. Calcd.: %C, 62.70; H, 3.81; N, 7.56. Found: %C, 62.75; H, 3.91; N, 7.45. IR (KBr, u, cm^-1): 3432 and 3381 (NH₂), 3297 (NH), 1727 (2 C=O), 1597 (C=N), 1528 and 1376 (NO₂), 1266 (C=S). ¹HNMR (DMSO-d₆, δ, ppm): 2.32 (s, 3H, CH₃), 2.95 (d, 1H, iminopyran C-3 proton), 3.80 (t, 1H, iminopyran C-4 proton), 6.26 (s, 1H, C-3 proton), 6.96 (d, 1H, iminopyran C-5 proton), 7.19-8.40 (m, 11H, aromatic protons, 1H, exchangeable NH), 9.99 (s, 2H, NH₂, D₂O exchangeable). MS; m/z (R.I. %): [M⁺] 555 (0.80), 91 (100).

8-(4-Aryl-3-cyano-2-imino-3,4-dihydro-2H-pyran-6-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoates (XIa-i)

A mixture of 8-acetyl-7-benzoyloxy-6-nitrocoumarin V (0.36 g, 0.001 mol), the appropriate aromatic aldehyde (0.001 mol), malononitrile (0.066 g, 0.001 mol) and 5 drops piperidine in n-butanol (5 ml) was refluxed for 5 hours. After cooling, the formed precipitate was filtered off, washed with diethyl ether and crystallized from absolute ethyl alcohol

8-(6-Amino-4-(4-chlorophenyl)-5-cyano-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIa)

Yield: 92%. m.p.: 182-183°C. Analysis for C₂₉H₁₈ClN₃O₇, M.wt.: 555.92. Calcd.: %C, 62.65; H, 3.26; N, 7.56. Found: %C, 62.60; H, 3.33; N, 7.49. IR (KBr, u, cm^-1): 3334 (NH), 2195 (C≡N), 1709 (2 C=O), 1616 (C=N), 1560 and 1379 (NO₂).

8-(6-Amino-5-cyano-4-(4-nitrophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIb)

Yield: 84%. m.p.: 160-161°C. Analysis for C₂₉H₁₈N₄O₉, M.wt.: 566.47. Calcd.: %C, 61.49; H, 3.20; N, 9.89. Found: %C, 61.52; H, 3.27; N, 9.91. IR (KBr, u, cm^-1): 3338 (NH), 2196 (C≡N), 1725 (2 C=O), 1611 (C=N), 1563 and 1384 (2 NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.39 (s, 3H, CH₃), 2.97 (d, 1H, iminopyran C-3 proton), 3.56 (t, 1H, iminopyran C-4 proton), 6.17 (s, 1H, C-3 proton), 6.99 (d, 1H, iminopyran C-5 proton), 7.04-8.59 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(6-Amino-5-cyano-4-(4-hydroxyphenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIc)

Yield: 85%. m.p.: 210-212°C. Analysis for C₂₉H₁₉N₃O₈, M.wt.: 537.48. Calcd.: %C, 64.80; H, 3.56; N, 7.82. Found: %C, 64.87; H, 3.68; N, 7.64. IR (KBr, u, cm^-1): 3335 (NH), 3192 (OH), 2195 (C≡N), 1707 (2 C=O), 1614 (C=N), 1557 and 1375 (NO₂). MS; m/z (R.I. %): [M⁺] 537 (23.18), [M⁺+1] 538 (25.75), 56 (100).

8-(6-Amino-5-cyano-4-p-tolyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XId)

Yield: 92%. m.p.: 245-247°C. Analysis for C₃₀H₂₁N₃O₇, M.wt.: 535.50. Calcd.: %C, 67.29; H, 3.95; N, 7.85. Found: %C, 67.34; H, 3.88; N, 7.75. IR (KBr, u, cm^-1): 3336 (NH), 2196 (C≡N), 1720 (2 C=O), 1614 (C=N), 1563 and 1377 (NO₂). MS; m/z (R.I. %): [M⁺] 535 (7.23), 80 (100).

8-(6-Amino-4-(3-bromophenyl)-5-cyano-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIe)

Yield: 96%. m.p.: 202-204°C. Analysis for C₂₉H₁₈BrN₃O₇, M.wt.: 599.37. Calcd.: %C, 58.02; H, 3.02; N, 7.00. Found: %C, 58.13; H, 3.16; N, 6.92. IR (KBr, u, cm^-1): 3339 (NH), 2199 (C≡N), 1727 (2 C=O), 1608 (C=N), 1572 and 1377 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.41 (s, 3H, CH₃), 2.99 (d, 1H, iminopyran C-3 proton), 3.39 (t, 1H, iminopyran C-4 proton), 6.38 (s, 1H, C-3 proton), 6.82 (d, 1H, iminopyran C-5 proton), 7.15-8.59 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(6-Amino-4-(2-chlorophenyl)-5-cyano-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIf)

Yield: 88%. m.p.: 213-215°C. Analysis for C₂₉H₁₈ClN₃O₇, M.wt.: 555.92. Calcd.: %C, 62.65; H, 3.26; N, 7.56. Found: %C, 62.80; H, 3.32; N, 7.61. IR (KBr, u, cm^-1): 3334 (NH), 2196 (C≡N), 1724 (2 C=O), 1617 (C=N), 1562 and 1379 (NO₂). ¹HNMR (DMSO-d₆, δ, ppm): 2.45 (s, 3H, CH₃), 3.01 (d, 1H, iminopyran C-3 proton), 3.35 (t, 1H, iminopyran C-4 proton), 6.21 (s, 1H, C-3 proton), 6.97 (d, 1H, iminopyran C-5 proton), 7.25-8.64 (m, 10H, aromatic protons, 1H, NH, D₂O exchangeable).

8-(6-Amino-5-cyano-4-(2-nitrophenyl)-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIg)

Yield: 88%. m.p.: 250-252°C. Analysis for C₂₉H₁₈N₄O₉, M.wt.: 566.47. Calcd.: %C, 61.49; H, 3.20; N, 9.89. Found: %C, 61.44; H, 3.11; N, 9.74. IR (KBr, u, cm^-1): 3333 (NH), 2199 (C≡N), 1713 (2 C=O), 1618 (C=N), 1563 and 1381 (2 NO₂). MS; m/z (R.I. %): [M⁺] 566 (27.93), 80 (100).

8-(6-Amino-4-(2-bromophenyl)-5-cyano-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIh)

Yield: 98%. m.p.: 246-248°C. Analysis for C₂₉H₁₈BrN₃O₇, M.wt.: 599.37. Calcd.: %C, 58.02; H, 3.02; N, 7.00. Found: %C, 57.85; H, 2.95; N, 7.16. IR (KBr, u, cm^-1): 3334 (NH), 2201 (C≡N), 1724 (2 C=O), 1622 (C=N), 1563 and 1385 (NO₂).

8-(6-Amino-5-cyano-4-phenyl-4H-pyran-2-yl)-4-methyl-6-nitro-2-oxo-2H-chromen-7-yl benzoate (XIi)

Yield: 94%. m.p.: 242-244°C. Analysis for C₂₉H₁₉N₃O₇, M.wt.: 521.48. Calcd.: %C, 66.79; H, 3.67; N, 8.06. Found: %C, 66.71; H, 3.54; N, 8.01. IR (KBr, u, cm^-1): 3341 (NH), 2198 (C≡N), 1723 (2 C=O), 1614 (C=N), 1568 and 1384 (NO₂).

Antibacterial screening

The antibacterial screening was performed at and all the used microorganisms were obtained from the culture collection of the Department of Microbiology and Immunology, Faculty of Pharmacy, Helwan University.

Sensitivity test using paper disc diffusion technique

The medium was sterilized at 120°C for 30 minutes using the autoclave then each 100 ml of it was inoculated with 1 ml of each microorganism suspension (105 cfu/ml) at 40-50°C and poured into Petri dishes to give a depth of 3-4 mm. Non sterile powders of the fifty five tested compounds were dissolved in DMSO (dimethyl sulphoxide) giving a concentration of 10,000 μg/ ml. Sterile filter paper discs each of 6 mm diameter were impregnated with 5 μl (50 μg) of the above solutions and then placed over the surface of the nutrient agar containing standardized inoculums of the test microorganisms. Novobiocin was used as a reference standard in the form of standard discs each of 30 μg/disc concentration. The plates were incubated at 37°C for 24 hours. The observed zones of inhibition after incubation were measured and compared to those of Novobiocin (Table 1).

Minimum inhibitory concentration (MIC) using agar dilution method

MIC of each of the synthesized compounds was determined by agar dilution method. A stock solution of each compound was prepared in the concentration of 1024 μg/ml DMSO and was two folds serially diluted up to 1 μg/ml. The test compounds were incorporated in specified quantity of molten sterile nutrient agar for anti-bacterial activity. A specified quantity of the medium containing the compound was poured into a Petri dish to give a depth of 3-4 mm and allowed to solidify. The microorganisms were applied using the multipoint inoculator to the plates with serially diluted compounds in DMSO to be tested and incubated at 37°C for 24 hours. The MIC was considered to be the lowest concentration of the test substance exhibiting no visible growth of bacteria on the plate. The observed MIC is presented in (Table 2).

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