J. Chil. Chem. Soc, 52, Nº 2 (2007) págs.: 1145-1149

SYNTHESIS OF NOVEL PYRAZOLE, COUMARIN AND PYRIDAZINE DERIVATIVES EVALUATED AS POTENTIAL ANTIMICROBIAL AND ANTIFUNGAL AGENTS

WAGNAT W. WARDAKHAN^*A AND NADIA A. LOUCA^B

^aNational Organization for Drug Control & Research (NODCAR), P.O. 29, Cairo A. R. Egypt. ^bHormones Department, National Research Center, Dokki, A. R. Egypt

Dirección para correspondencia

ABSTRACT

The reaction of cyanoacetylhydrazine with ω-bromoacetophenone gave the N’-[2-bromo-1-phenylethylidene]-2-isocyanoacetohydrazide (3). The latter compound underwent ready cyclization when treated with potassium cyanide to give the 1-(isocyanoacetyl)-3-phenyl-1H-pyrazol-5-amine (6). Compound 6 underwent a series of heterocyclization to give either coumarin, pyridazine, 1,2,4-triazine or thiophene derivatives. The antimicrobial and antifungal activities of the newly synthesized products were measured, using two Gram-negative (Escherichia coli and Pseudomonas aeruginosa), two Gram-positive bacteria (Bacillus subtilis and Bacillus cereus) and the antifungal activity using Candida albicans.

Key words: Cyanoacetylhydrazine, Pyrazole, Coumarin, Pyridazine

INTRODUCTION

The pyrazole ring is a prominent structural motif found in numerous pharmaceutically active compounds. This is mainly due to the ease preparation and the important biological activity. Pyrazole framework plays an essential role in biologically active compounds and therefore represents an interesting template for combinatorial as well as medicinal chemistry^1-10 Indeed, pyrazole-based derivatives have shown several biological activities as seen as anxiolytics,¹¹ GABA receptor antagonists and insecticides,¹² a potential PET ligand for CB1 receptors,¹³ anti-inflammatory and antimicrobial agents¹⁴ and growth inhibition activity.¹⁵

RESULTS AND DISCUSSION

In this work we report a novel method for the synthesis of pyrazole derivatives starting with cyanoacetylhydrazine (1) and (0-bromoaceto-phenone (2) followed by heterocyclization to the pyrazole derivative. Thus, the reaction between the two reagents in 1,4-dioxan at room temperature gave a single product with molecular formula C₁₁H₁₀N₃OBr. Two possible isomeric structures were considered for the reaction product depending on the way of condensation reaction, either 3 or 4. The possibility of structure 4 was ruled out on the basis of IR spectrum which showed the absence of any NH₂ stretching, and the 1H NMR spectrum which revealed the presence of two singlets at δ4.48, 5.23 corresponding to two CH₂ groups, a multiplet at δ7.26-7.31 for the phenyl protons and a singlet (D₂O exchangeable) at δ8.31 for the NH group. Such data are consistent with the structure of the hydrazide-hydrazone derivative 3. Compound 3, reacts with potassium cyanide to give the 1-(cyanoacetyl)-3-phenyl-1//-pyrazol-5-amine 6 via the intermediate formation of the acyclic intermediate 5. The IR spectrum of the reaction product showed the presence of an NH₂ stretching at 1) 3466, 3380 corresponding to an NH₂ group and only one CN group stretching at δ2256. Moreover, the 1H NMR spectrum showed the presence of a singlet at δ4.67 corresponding to CH₂ group, a singlet at δ4.88 (D₂O exchangeable) corresponding to NH₂ group, a singlet at δ6.56 for pyrazole H-4 and a multiplet at δ7.30-7.36 for phenyl protons. The ¹³C NMR spectrum showed δ18.2 (CH₂), 93.4 (pyrazol C-4), 117.1 (CN), 127.8, 128.8, 129.1, 129.3, 130.2, 143.2, 150.4 (C₆H₅, pyrazole C-3, C-5) and 198.5 (C=O).

Compound 6 underwent many chemical transformations due to the presence of the 1-cyanoacetyl moiety and the 5-amino group. Thus, the reaction of benzenediazonium chloride 7 with compound 6 at 0-5 oC gave the phenylhydrazone derivative δ. On the other hand, with the aromatic aldehydes 9a-c gave the arylidene derivatives 10a-c, respectively. The analytical and spectral data of the reaction products are consistent with the proposed structures (see experimental section). On the other hand, the reaction of compound 6 with salicylaldehyde 11 gave the coumarin derivative 12. The IR and 1H NMR spectra agree with the proposed structures (Scheme 1). Formation of many coumarin derivatives by this method has been reported before.^16-18

The amino group present in compound 6 is ready for diazotization in a way similar with that reported work by Elnagdi and Ren et al.^19,20 Thus, a solution of compound 6 in acetic/hydrochloric acids reacts with sodium nitrite at 0-5 ^oC to give the non isolable diazonium chloride salt 13. The reaction of the intermediate diazonium salt 13 with malononitrile (14) gave the hydrazone derivative 15. Structure of the latter product was based on the analytical and spectral data. Thus, IR spectrum showed the presence of NH stretching at υ 3460-3327 cm^-1, three CN groups stretching at υ 2255, 2227-2220 cm^-1. Compound 15 underwent ready cyclization when heated in ethanolic sodium hydroxide solution to give the pyrazolo[1,5:1,2]pyrimidino[1,6-6]-1,2,4-triazine derivative 17. Formation of the latter product is expected via the intermediate formation of the pyrazolo[1,5-a]pyrimidine derivative 16. Structure of compound 17 was established on the basis of analytical and spectral data (see experimental section). Moreover, compound 15 reacted with either hydrazine hydrate (18a) or phenylhydrazine (18b) to give the pyrazolo-4-yl-5-azopyrazole derivatives 19a and 19b, respectively (Scheme 2).

The reaction of compound 15 with malononitrile (14) in ethanol containing a catalytic amount of triethylamine gave the 1-pyrazol-5-yl-pyridazine derivative 21, the reaction took place through the intermediate formation of the acyclic structure 20 followed by Michael cyclization. Formation of the latter product was based on the analytical and spectral data (see experimental section) beside its synthesis via another reaction route. Thus, the reaction of the diazonium salt 13 with P-amino-a,y-dicyanocrotononitrile (22) at 0-5 oC afforded the acyclic hydrazone derivative 20. Heating of the latter product in ethanolic/triethylamine solution gave the same product 21 (mixed m.p. and finger print IR spectra).

The reaction of compound 15 with phenylisothiocyanate (23) gave the 1-pyrazol-5-yl-1,2,4-triazine derivative 24 (Scheme 3). The analytical and spectral data of the latter product are consistent with the proposed structure (see experimental section).

Compound 15 coupled with the diazonium salts 7, 25a and 25b to give the hydrazone derivatives 26a-c, respectively. Analytical and spectral data of the latter products are agree with the proposed structures (see experimental section). On the other hand, compound 15 reacts with acetophenone (27) in the presence of ammonium acetate at 140 ^oC to give the condensed product 28. The latter product reacts with elemental sulfur in the presence of triethylamine to afford the thiophene derivative 29 (Scheme 4).

IN VITRO ANTIMICTOBIAL AND ANTIFUNGAL ACTIVITIES EVALUATION

An evaluation of the antibacterial activity using two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and two Gram-positive bacteria (Bacillus subtilis and Bacillus cereus) and the antifungal activity using Candida albicans as a representative species of fungi was assessed for compounds. The minimal inhibitory concentration (MIC in µg/mL) was determined using an adaptation of agar streak dilution method based on radial diffusion.^21,22 In the same conditions different concentrated solutions of ampicillin (antibacterial) and cycloheximide (antifungal) were used as standards. The MIC was considered to be the lowest concentration of the tested compound which inhibits growth of bacteria or fungi on the plate. The diameters of the inhibition zones corresponding to the MICs are presented in

Table 1. The compounds tested are not active against Pseudomonas aeruginosa starting from DMSO solutions of 1000 µg/mL of each compound.

From the analysis of Table 1 it is possible to establishsome SARs. The only active compounds against E. coli in the concentrations tested are 10a, 17 and 20 (MIC 12.5 µg/mL), the substituted pyrazole moiety being the responsible for such activity. However, the annulated derivative 17 showed the highest activity. Against Gram + bacteria the MICs for 10c are much higher than those for 10a. Comparing 3 with 10a and 10c (the pyridine derivative), compound 3 (α-haloketone) seemed to be more active against B. cereus (MIC 3.13 µg/mL) but less active against B. subtilis. Against C. albicans 10b (4-chlorophenyl) and 10c (4-methoxyphenyl) present the same MIC (25 µg/mL) which is higher than the MIC obtained for 3 (50 µg/mL). Compound 12 (with the coumarin group) showed the highest ativity against C. albicans.

Comparing compounds 26a, 26b and 26c, one can notice that 26a (with the phenylhydrazono group) showed highest MIC value against C. albicans whereas 26c showed the least value. For the in vitro antimicrobial activity, suspensions of the microorganism were prepared to contain approximately 108 cfu/mL and the plates were inoculated. A stock solution of the synthesized

compound (1000 |lg/mL) in DMSO was prepared and graded dilutions of the tested compounds were incorporated in a cavity (depth 3 mm, diameter 4 mm) made in the center of the petridish (nutrient agar for antibacterial activity and Sabouraud vs dextrose agar medium for antifungal activity). The plates were incubated at 37 oC (for bacteria) and at 30 oC (for fungi) for 24 h in duplicate. Positive control using only inoculation and negative control using only DMSO in the cavity were carried out.

CONCLUSION

The work described in this article showed a novel method for the synthesis of pyrazoles and their derivatives starting from a hydrazide-hydrazone derivative obtained through the reaction of ffl-bromoacetophenone and cyanoacetylhydrazine. Thus, Upon using a variety of a-haloketones as starting materials a new field is opened through which a new series of pyrazoles that can undergo heterocyclization into a large number of fused heterocycles. Most of the synthesized products showed antimicrobial and antifungal activities.

EXPERIMENTAL

Melting points are uncorrected. IR spectra were recorded (KBr) on a Pye Unicam SP-1000 spectrophotometer. 1H NMR spectra were obtained on a Varian Gemini 200 MHz spectrometer in DMSO-d6 as solvent and TMS as internal reference. Chemical shifts are expressed as δppm.

iV-[2-bromo-l-phenylethylidene]-2-isocyanoacetohydrazide (3)

To a solution of cyanoacetylhydrazine (1) (0.01 mol, 1.0 g) in 1,4-dioxan (50 mL), ffl-bromoacetophenone (0.01 mol, 2.0 g) was added. The reaction mixture was stirred at room temperature for 6 hrs. The formed solid product was collected by filtration. White crystals from 1,4-dioxan, yield 70 % (2.23 g), m.p. 160 oC. Calculated for C₁₁H₁₀BrN₃O (280.12): C, 47.16; H, 3.60; Br, 28.52; N, 15.00; Found: C, 46.89; H, 3.25; Br, 28.18; N, 14.74.IR 1): 3480-3325 (NH), 3055 (CH aromatic), 2875 (CH₂), 2260 (CN), 1687 (C=O), 1665 (C=N), 1632 (C=C). 1H NMR δ: 4.48, 5.23 (2s, 4H, 2 CH₂), 7.26-7.31 (m, 5H CH5), 8.31 (s, 1H, NH).

l-(isocyanoacetyl)-3-phenyl-lff-pyrazol-5-amine (6)

To a solution of compound 3 (2.80 g, 0.01 mol) in ethanol (40 mL), a solution of potassium cyanide (2.8 g, 0.05 mol) was added. The reaction mixture was heated in a warm water bath at 60 oC for 30 min. then left with stirring at room temperature for an additional one night. The solid product formed upon pouring onto ice/water containing hydrochloric acid (till pH 6) was collected by filtration. Pale yellow crystals from ethanol, yield 78 % (1.76 g), m.p. 220-22 oC. Calculated for C₁₂H₁₀N₄O (226.23): C, 63.71; H, 4.46; N, 24.77; Found: C, 64.08; H, 4.21; N, 25.09. IR 1): 3466-3380 (NH₂), 3050 (CH aromatic), 2256 (CN), 1690 (C=O), 1655 (C=N), 1634 (C=C). 1H NMR δ: 4.67 (s, 2H, CH₂), 4.88 (s, 2H, NH ₂), 6.56 (s, 1H, pyrazole H-4), 7.30-7.36 (m, 5H, CH₅). ¹³C NMR δ: 18.2 (CH₂), 93.4 (pyrazol C-4), 117.1 (CN), 127.8, 128.8, 129.1, 129.3, 130.2, 143.2, 150.4 (C6H5, pyrazole C-3, C-5), 198.5 (C=O).

2-(5-Amino-3-phenyl-lii-pyrazol-l-yl)-2-oxo-A^r"-phenylethane-hydrazonoyl isocyanide (8) :

To a cold solution (0-5 oC) of compound 6 (2.26 g, 0.01 mol) in ethanol (40 mL) containing sodium acetate (8.0 g), benzenediazonium chloride (0.01 mol) [prepared by adding sodium nitrite solution (0.7 g, 0.01 mol) to a cold suspension (0-5 oC) of aniline (0.94 g, 0.01 mol) in the appropriate quantity of hydrochloric acid with continuous stirring] was added. The reaction mixture was kept at room temperature for an additional 1h and the formed solid product was collected by filtration. Orange red crystals from 1,4-dioxan, yield 70 % (2.31 g), m.p. 185-7 oC Calculated for C₁₈H₁₄N₆O (330.34): C, 65.44; H, 4.27; N, 25.44; Found: C, 65.92; H, 4.31; N, 25.86. IR 1): 3450-3321 (NH₂, NH), 3052 (CH aromatic), 2255 (CN), 1683 (C=O), 1658 (C=N), 1638 (C=C). 1H NMR δ: 4.90 (s, 2H, NH₂), 6.59 (s, 1H, pyrazole H-4), 7.32-7.39 (m, 10H, 2C H₅), 8.33 (s, 1H, NH).

l-[2-Cyano-3-phenylprop-2-enoyl]-3-phenyl-lH-pyrazol-5-amine (10a), l-[2-cyano-3-(4-chlorophenyl)-prop-2-enoyl]-3-phenyl-lH-pyrazol-5-amine (10b), l-[2-Cyano-3-(4-methoxyphenyl)-prop-2-enoyl]-3-phenyl-lH-pyrazol-5-amlne (10c) and 3-[(5-amino-3-phenyl-lH-pyrazol-l-yl)carbonyl]-2H-chromen-2-one (12)

General procedure: Equimolar amounts of compound 6 (2.26 g, 0.01 mol) in 1,4-dioxane containing triethylamine (0.5 mL) and either benzaldehyde (1.06 g, 0.01 mol), 4-chlorobenzaldehyde (1.43 g, 0.01 mol), 4-methoxybenzaldehyde (1.39 g, 0.01 mol) or salicyladehyde (1.08 g, 0.01 mol) were heated under reflux for 2 hrs then left to cool. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid was collected by filtration.

Compound 10a: Pale yellow crystals from ethanol, yield 66 % (2.07 g), m.p. 122-24 ^oC. Calculated for C₁₉H₁₄N₄O (314.12): C, 72.60; H, 4.49; N, 17.82; Found: C, 72.32; H, 4.69; N, 18.20. IR υ: 3488, 3350 (NH₂), 3066 (CH aromatic), 2256 (CN), 1688 (C=O), 1655 (C=N), 1630 (C=C). ¹H NMR δ: 4.78 (s, 2H, NH₂), 6.53 (s, 1H, pyrazole H-4), 7.26-7.35 (m, 10H, 2C₆H₅), 8.21 (s, 1H, CH=C).

Compound 10b: Pale yellow crystals from ethanol, yield 82 % (2.85 g), m.p. 187-90 ^oC. Calculated for C₁₉H₁₃ClN₄O (348.79): Calcd: C, 65.43; H, 3.76; N, 16.06; Found: C, 65.09; H, 4.16; N, 15.88. IR υ: 3471, 3364 (NH₂), 3060 (CH aromatic), 2248 (CN), 1686 (C=O), 1650 (C=N), 1636 (C=C). ¹H NMR δ: 4.74 (s, 2H, NH₂), 6.58 (s, 1H, pyrazole H-4), 7.25-7.34 (m, 9H, C₆H₅, C₆H₄), 8.14 (s, 1H, CH=C).

Compound 10c: Yellow crystals from ethanol, yield 72 % (2.47 g), m.p. 165-8 ^oC. Calculated for C₂₀H₁₆N₄O₂ (344.37): C, 69.76; H, 4.68; N, 16.27; Found: C, 69.08; H, 4.25; N, 16.37. IR υ: 3469, 3325 (NH₂), 3051 (CH aromatic), 2896 (CH₃), 2250 (CN), 1688 (C=O), 1653 (C=N), 1640 (C=C). ¹H NMR δ: 2.89 (s, 3H, CH₃), 4.73 (s, 2H, NH₂), 6.61 (s, 1H, pyrazole H-4), 7.29-7.36 (m, 9H, C₆H₅, C₆H₄), 8.19 (s, 1H, CH=C).

Compound 12: Orange crystals from 1,4-dioxan yield 80 % (2.64 g), m.p. 230-4 ^oC. Calculated for C₁₉H₁₃N₃O₃ (331.32): C, 68.88; H, 3.95; N, 12.68; Found: C, 69.31; H,4.42; N, 13.09. IR υ: 3423, 3310 (NH₂), 3056 (CH aromatic), 2875 (CH₃), 1690, 1693 (2 C=O), 1650 (C=N), 1631 (C=C). ¹H NMR δ: 4.65 (s, 2H, NH₂), 6.60 (s, 1H, pyrazole H-4), 7.30-7.39 (m, 9H, C₆H₅, C₆H₄), 8.21 (s, 1H, coumarin H-4).

{[1-(cyanoacetyl)-3-phenyl-1H-pyrazol-5-yl]hydrazono}malononitrile (15) and 2-amino-3-{[1-(cyanoacetyl)-3-phenyl-1H-pyrazol-5 yl] hydrazono} prop-1-ene-1,1,3-tricarbonitrile (20)

A cold solution (0-5 ^oC) of the diazonium salt 13 (0.01 mol) [prepared by adding sodium nitrite solution (0.70 g, 0.01 mol) to a cold solution (0-5 ^oC) of compound 6 (2.26 g, 0.01 mol) in acetic acid (20 mL) and hydrochloric acid (5.0 mL) with continuous stirring] was added to a solution of either malononitrile (0.66 g, 0.01 mol) or compound 22 (1.32 g, 0.01 mol) in ethanol (40 mL) containing sodium acetate (6.0 g), with stirring. After complete addition of the diazonium salt 13, the reaction mixture was stirred for an additional 6 hrs at room temperature and the formed solid product was collected by filtration.

Compound 15: reddish brown crystals from ethanol yield 68 % (2.96 g), m.p. 158-61 ^oC. Calculated for C₁₅H₉N₇O (303.28): C, 59.40; H, 2.99; N, 32.33; Found: C, 59.11; H, 3.38; N, 32.68. IR υ: 3460-3327 (NH), 3051 (CH aromatic), 2870 (CH₂), 2255, 2227-2220 (3 CN), 1690 (C=O), 1662 (C=N). ¹H NMR δ: 4.29 (s, 2H, CH₂), 6.63 (s, 1H, pyrazole H-4), 7.33-7.36 (m, 5H, C₆H₅), 8.30 (s, 1H, NH).

Compound 20: reddish brown crystals from 1,4-dioxan yield 70 % (2.58 g), m.p. 110 ^oC. Calculated for C₁₈H₁₁N₉O (369.34): C, 58.53; H, 3.00; N, 34.13; Found: C, 57.88; H, 2.68; N, 33.79. IR (υ/cm^-1) = 3485, 3345 (NH₂, NH), 3058 (CH aromatic), 2877 (CH₂), 2248, 2229-2220 (4 CN), 1688 (C=O), 1660 (C=N), 1633 (C=C). ¹H NMR δ = 4.37 (s, 2H, NH₂), 4.66 (s, 2H, CH₂), 6.69 (s, 1H, pyrazole H-4), 7.32-7.36 (m, 5H, C₆H₅), 8.36 (s, 1H, NH).

3-amino-6-oxo-9-phenyl-6H-pyrazolo[5’,1’:2,3]pyrimido[1,6-b][1,2,4]triazine-2-carbonitrile (17)

A solution of compound 15 (3.03 g, 0.01 mol) in ethanolic sodium hydroxide (5 %) was heated under reflux for 4 hrs then poured onto ice/water containing hydrochloric acid (till pH 6). The formed solid product was collected by filtration. Pale yellow crystals from ethanol yield 56 % (1.69 g), m.p. >300 ^oC. Calculated for C₁₅H₉N₇O (303.28): C, 59.40; H, 2.99; N, 32.33; Found: C, 60.08; H, 3.32; N, 31.98. IR υ: 3452, 3346 (NH₂), 3056 (CH aromatic), 2225 (CN), 1686 (CO), 1660 (C=N), 1636 (C=C). ¹H NMR δ:4.72(s,2H,NH₂), 6.67, 6.83 (2s, 2H, pyrazole H-4, pyrimidine H-5), 7.30-7.33 (m, 5H, C₆H₅).

3-{5-[-(3,5-diamino-3H-pyrazol-4-yl)diazenyl]-3-phenyl-1H-pyrazol-1-yl}-3-oxopropanenitrile (19a) and 3-{5- [(3,5-diamino-1-phenyl-1H-pyrazol-4-yl)diazenyl]-3-phenyl-1H-pyrazol-1-yl}-3-oxopropanenitrile (19b)

General procedure: Equimolar amounts of compound 15 (3.03 g, 0.01 mol) and either hydrazine hydrate (0.5 g, 0.01 mol) or phenylhydrazine (1.08 g, 0.01 mol) was heated under reflux for 30 min. then left to cool. The reaction mixture was evaporated under vacuum and the remaining product was triturated with diethyl ether. The formed solid product was collected by filtration.

Compound 19a: White crystals from ethanol yield 62 % (2.07 g), m.p. 196-8 ^oC. Calculated for C₁₅H₁₃N₉O (335.32): C, 53.73; H, 3.91; N, 37.59; Found: C, 54.08; H, 4.41; N, 37.93. IR (υ/cm^-1) = 3466-3346 (2 NH₂), 3062 (CH aromatic), 2252 (CN), 1685 (CO), 1657 (C=N), 1636 (C=C). ¹H NMR δ: 4.23 (s, 2H, CH₂), 4.68, 5.31 (2s, 4H, 2 NH₂), 6.33 (s, 1H, pyrazole H-4), 7.31-7.35 (m, 5H, C₆H₅).

Compound 19b: Pale yellow crystals from ethanol yield 55 % (2.26 g), m.p. 130 ^oC. Calculated for C₂₁H₁₇N₉O (411.42): Calcd: C, 61.31; H, 4.16; N, 30.64; Found: C, 60.99; H, 4.53; N, 30.84. IR (υ/cm^-1) = 3480-3328 (NH₂), 3056 (CH aromatic), 2220 (CN), 1687 (CO), 1650 (C=N), 1639 (C=C). ¹H NMR δ: 4.26 (s, 2H, CH₂), 4.65, 5.34 (2s, 4H, 2 NH₂), 6.37 (s, 1H, pyrazole H-4), 7.27-7.38 (m, 10H, 2 C₆H₅).

4-amino-1-[1-(cyanoacetyl)-3-phenyl-1H-pyrazol-5-yl]-6-imino-1,6-dihydropyridazine-3,5-dicarbonitrile (21)

Method A: A solution of compound 15 (3.03 g, 0.01 mol) in absolute ethanol (50 mL) containing triethylamine (0.5 mL), malononitrile (0.66 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 4 hrs then left to cool and the precipitated product was collected by filtration.

Method (B); A solution of compound 20 (3.69 g, 0.01 mol) in absolute ethanol (40 mL) containing triethylamine (0.5 mL) was heated under reflux for 2 hrs then poured onto ice water. The formed solid product was collected by filtration. Yellow crystals from 1,4-dioxan yield 66 % (2.34 g), m.p. 260-2 ^oC. Calculated for C₁₈H₁₁N₉O (369.34): C, 58.53; H, 3.00; N, 34.13; Found: C, 58.33; H, 3.17; N, 34.49. IR υ: 3466, 3315 (NH₂, NH), 3047 (CH aromatic), 2890 (CH₂), 2240, 2227-2220 (3 CN), 1691 (C=O), 1662 (C=N), 1636 (C=C). ¹H NMR δ: 4.67 (s, 2H, CH₂), 5.21 (s, 2H, NH₂), 6.67 (s, 1H, pyrazole H-4), 7.30-7.35 (m, 5H, C₆H₅), 8.33 (s, 1H, NH).

2-[1-(cyanoacetyl)-3-phenyl-1H-pyrazol-5-yl]-5-imino-4-phenyl-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazine-6-carbonitrile (24)

To a solution of compound 15 (2.26 g, 0.01 mol) in 1,4-dioxan (30 mL) containing triethylamine (0.5 mL), phenylisothiocyanate (1.30 g, 0.01 mol) was added. The reaction mixture was heated under reflux for 12 hrs then evaporated under vacuum. The remaining product was triturated with diethyl ether and the solidified product was collected by filtration. Orange crystals from acetic acid yield 55 % (2.41 g), m.p. 180-3 ^oC. Calculated for C₂₂H₁₄N₈OS (438.47): C, 60.26; H, 3.22; N, 25.56; Found: C, 59.99; H, 2.89; N, 25.84. IR υ: 3480-3321 (NH), 3050 (CH aromatic), 2888 (CH₂), 2240, 2225 (2 CN), 1689 (C=O), 1668 (exocyclic C=N), 1642 (C=C). ¹H NMR δ: 4.80 (s, 2H, CH₂), 6.64 (s, 1H, pyrazole H-4), 7.32-7.39 (m, 5H, C₆H₅), 8.29 (s, 1H, NH).

({1-[2-cyano-2-(phenylhydrazono)acetyl]-3-phenyl-1H-pyrazol-5-yl}h ydrazono)malononitrile (26a), {[1-[2-cyano-2-(phenylhydrazono)acetyl]-3-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-1H-pyrazol-5-yl]hydrazon o}malononitrile (26b), and ethyl 2-{1-[2-cyano-2-(phenylhydrazono)acetyl]-5-[2-(dicyanomethylene)-hydrazino]-1H-pyrazol-3-yl}-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate (26c).

General procedure: To a cold solution of compound 15 (3.03 g, 0.01 mol) in ethanol (50 mL) containing sodium hydroxide (10 mL, 20 % g) either of benzenediazonium chloride (7) (0.01 mol), or any of the 2-diazo-4,5,6,7-tet rahydrobenzo[b]thiophene derivatives 25a or 25b was added with continuous stirring. The reaction mixture, in each case was kept at room temperature for an additional 2 hrs and the formed solid product, in each case, was collected by filtration.

Compound 26a: Orange-red crystals from 1,5-dioxan yield 63 % (2.56 g), m.p. 144 ^oC. Calculated for C₂₁H₁₃N₉O (407.39): C, 61.91; H, 3.22; N, 30.94; Found: C, 62.08; H, 3.64; N, 31.41. IR υ: 3466-3310 (2 NH), 3060 (CH aromatic), 2246, 2225-2220 (3 CN), 1687 (C=O), 1665 (exocyclic C=N), 1639 (C=C). ¹H NMR δ: 6.65 (s, 1H, pyrazole H-4), 7.31-7.36 (m, 10H, 2 C₆H₅), 8.20, 8.33 (2s, 2H, 2 NH).

Compound 26b: Orange-red crystals from acetic acid yield 70 % (3.44 g), m.p. 220-3 ^oC. Calculated for C₂₄H₁₆N₁₀OS (492.12): C, 58.53; H, 3.27; N, 28.44. Found: C, 59.03; H, 2.87; N, 28.82. IR υ: 3486-3318 (2 NH), 3054 (CH aromatic), 2242, 2227-2220 (4 CN), 1690 (C=O), 1657 (C=N), 1643 (C=C). ¹H NMR δ: 2.23-2.27 (m, 4H, 2 CH₂), 2.33-2.36 (m, 4H, 2CH₂), 6.66 (s, 1H, pyrazole H-4), 7.26-7.35 (m, 10H, 2 C₆H₅), 8.21,8.32(2s, 2H, NH₂).

Compound 26c: Orange-red crystals from acetic acid yield 70 % (3.44 g), m.p. 220-3 ^oC. Calculated for C₂₆H₂₁N₉O₃S (539.57): C, 57.88; H, 3.92; N, 23.36; S, 5.94. Found: C, 58.03; H, 3.55; N, 23.72; S, 6.33. IR υ: 3486-3318 (2 NH), 3054 (CH aromatic), 2242, 2227-2220 (4 CN), 1690 (C=O), 1657 (C=N), 1643 (C=C). [¹H NMR δ: 2.23-2.27 (m, 4H, 2 CH₂), 2.33-2.36 (m, 4H, 2CH₂), 6.66 (s, 1H, pyrazole H-4), 7.26-7.35 (m, 10H, 2 C₆H₅), 8.22, 8.35 (2s, 2H, 2 NH)].

({1-[2-cyano-3-phenylbut-2-enoyl]-3-phenyl-1H-pyrazol-5-yl}hydrazo no)malononitrile (28)

Equimolar amounts of dry solid of compound 15 (3.03 g, 0.01 mol), acetophenone (1.20 g, 0.01 mol) and ammonium acetate (2.0 g) were heated in an oil bath at 140ºC for 1 h then left to cool. The solidified product was triturated with dilute ethanol and the formed solid product was collected by filtration. Pale yellow crystals from ethanol yield 55 % (2.27 g), m.p. 277-80 ^oC. Calculated for C₂₃H₁₅N₇O (405.41): C, 68.14; H, 3.73; N, 24.18. Found: C, 67.77; H, 3.25; N, 23.69. IR υ: 3455-3323 (NH), 3058 (CH aromatic), 2240, 2229-2220 (3 CN), 1687 (C=O), 1650 (C=N), 1633 (C=C). ¹H NMR δ: 2.89(s,3H, CH₃), 6.67 (s, 1H, pyrazole H-4), 7.29-7.38 (m, 10H, 2 C₆H₅), 8.24 (s, 1H, NH).

({1-[(4-amino-2-phenyl-3-thienyl)carbonyl]-3-phenyl-1H-pyrazol-5-yl}hydrazono)malononitrile (29)

A mixture of compound 28 (4.05 g, 0.01 mol) and elemental sulfur (0.32 g, 0.01 mol) in 1,4-dioxan (40 mL) containing triethylamine (0.5 mL) was heated under reflux for 1 h then left to cool. The solid product formed upon pouring onto ice/water containing few drops of hydrochloric acid was collected by filtration. Yallow crystals from 1,4-dioxan yield 50 % (2.18 g), m.p. 160 ^oC. Calculated for C₂₃H₁₅N₇OS (437.48): C, 63.15; H, 3.46; N, 22.41; S, 7.33. Found: C, 63.08; H, 3.40; N, 22.88; S, 7.70.

IR υ: 3480-3330 (NH₂, NH), 3052 (CH aromatic), 2225, 2221 (2 CN), 1689 (C=O), 1656 (C=N), 1630 (C=C). ¹H NMR δ: 4.88 (s, 2H, NH₂), 6.21, 6.64 (2s,2H, thiophene H-5, pyrazole H-4), 7.26 - 7.37 (m, 10H, 2C₆H₅), 8.26 (s, 1H, NH).

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