SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL ACTIVITIES OF PYRIMIDINE DERIVATIVES CONTAINING 2-PYRAZOLIN-5-ONE

Received on 20 November, 2013; received in revised form, 20 February, 2014; accepted, 09 March, 2014; published 01 May, 2014

SYNTHESIS, CHARACTERIZATION AND PHARMACOLOGICAL ACTIVITIES OF PYRIMIDINE DERIVATIVES CONTAINING 2-PYRAZOLIN-5-ONE

Mohamed Salah K. Youssef* and Ahmed Abdou O. Abeed

Department of Chemistry, Faculty of Science, Assiut University, Assiut, 71516, Egypt

ABSTRACT: A new series of thiazolo [3',2';1,2] pyrimido [5,4,e] pyrimidines and thiazolo [3',2';1,2] [1,2,4] triazolo [3'',4''-c]pyrimido [5,4-e] pyrimidines linked to 2-pyrazolin-5-one moiety were prepared by derivatization of the hydrazine functionality of the heterocyclic hydrazine with various reagents. The newly synthesized compounds were characterized by elemental analyses and spectroscopic techniques. The antimicrobial, anti-inflammatory, and analgesic activities of the synthesized compounds were evaluated using agar diffusion method, carrageenan-induced paw edema, and writhing assays, respectively. The finding of the study showed that, compounds 1a, b, 3a, b, 7b and 15a, b have excellent antimicrobial potency. In the anti-inflammatory activity, compounds 2a and 2b showed remarkable activities (30% and 32%, respectively). For the analgesic activity, the same compounds 2a and 2b gave significant activities (27% and 29%, respectively).

2-Pyrazolin-5-one, Antimicrobial, Anti-inflammatory, Analgesic

INTRODUCTION: The chemistry of pyrazoles has received considerable attention in recent years owing to their synthetic and attractive medicinal importance. Heterocyclic compounds bearing pyrazole nucleus have been reported to have a broad spectrum of pharmacological activities including potential biological ^1-5, anti-inflammatory and analgesic ^6-9 activities. In addition to these important medicinal applications, pyrazoles are also of great utility in synthetic organic chemistry ^10-12. On the other hand, the thiazolopyrimidines are used as starting materials for the preparation of new heterocyclic systems ^13-15 as well as their biological activity ^{16, 17}.

In the design of new drugs, the development of hybrid molecules through the combination of different pharmacophore in a single molecule may lead to compounds with improved pharmacological profile. In this study, prompted by these observations and in combination of our research program on the synthesis of novel heteerocycles linked to 2-pyrazolin-5-one ^18-21, the synthesis and the biological evaluation of pyrazolinyl thiazolopyrimidopyrimidines, and pyrazolinyl thiazolotriazolo pyrimidopyrimidines as hybrid molecules including different pharmacophore are aimed at.

MATERIALS AND METHODS:

Chemistry: All melting points are uncorrected. Thin layer chromatography (TLC) was performed on Silica Gel F₂₅₄ plates with visualization by UV or iodine vapour. FT-IR spectra (KBr disk) were recorded on a Pye-Unicam SP3-100 spectrophotometer. ¹H- and ¹³C NMR spectra were determined on JNM-LA Series 400 spectrometers with TMS as internal standard and the chemical shifts are in δ ppm. Mass spectra were recorded at 70 eV with JOEL JMS 600 spectrometer.

6-Hydrazino-3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-aryl-8-methyl-5H-thiazolo [3', 2': 1, 2]pyrimido[5,4-e]pyrimidines 1a,b: An equimolar mixture (5 mmol) of the chloro derivatives ²² and hydrazine hydrate (0.15 mL, 5 mmol) in ethanol (20 mL) was heated under reflux for 3h, then left to cool. The reaction mixture was poured onto ice-water mixture. The solid formed was collected by filtration, washed with water, dried, and recrystallized from ethanol to afford 1a,b.

6-Hydrazino-3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo[3',2': 1, 2] pyrimido[5,4-e]pyrimidine (1a): Yield: 81%; white needles; mp: 280-281 °C; IR (KBr): v (cm^-1) 3350-3100 (NHNH₂), 1640 (CO pyrazolone);¹HNMR (400MHz, CDCl₃): δ ppm 8.80 (s, 1H, NH_, D₂O exchangeable), 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 5.20 (s, 2H, NH₂, D₂O exchangeable), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, CH₃ pyrazolone); ¹³C NMR (400MHz, CDCl₃): 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 168 (C-NH), 170 (CO pyrazolone); MS m/z(%): 482.29 (M⁺) (79.10); Anal. Calcd. for C₂₅H₂₂N₈OS: C, 62.22; H, 4.60; N, 23.22; S, 6.64. Found: C, 62.20; H, 4.51; N, 23.20; S, 6.59.

6-Hydrazino-3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-p-chlorophenyl-8-methyl-5H-thia zolo[3',2':1,2]pyrimido[5,4-e]pyrimidine (1b): Yield: 75%; yellow needles; mp: 291-293 °C; IR (KBr): v (cm^-1) 3350-3100 (NHNH₂), 1640 (CO pyrazolone), 1570 (C=N); ¹HNMR (90 MHz, CDCl₃): δ ppm 8.80 (s, 1H, NH, D₂O exchangeable), 7.30-7.70 (m, 9H, aromatic protons), 7.00 (s, 1H, thiazole-H), 6.70 (s, 1H, pyrimidine-H), 6.55 (s, 1H, pyrazolone-H), 5.25 (s, 2H, NH₂, D₂O exchangeable), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, CH₃ pyrazolone); ¹³CNMR (400 MHz, CDCl₃): 15 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 51 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 146 (C=N triazole), 153 (C thiazole), 156 (C=N pyrazolone), 158 and 104 (C=C pyrimidine), 162 (C=N pyrimidine), 167 (C=N pyrimidine), 175 (CO pyrazolone). MS m/z (%): 518.87 (M+2) (20.05), 516.92 (M⁺) (60.12); Anal. Calcd. for C₂₅H₂₁N₈OSCl: C, 58.08; H, 4.09; N, 21.67; S, 6.20; Cl, 6.86. Found: C, 58.01; H, 3.99; N, 21.61; S, 6.21; Cl, 6.82.

General procedure for the synthesis of  12-aryl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4- yl)-5-methyl-3-thione-2,12-dihydrothiazolo [3', 2': 1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e]pyrimidines 2a,b: A mixture of 1a,b (5 mmol) and carbon disulfide (0.58 mL, 10 mmol) in pyridine (20 mL) was refluxed for 3h. The reaction mixture was left to cool, and then added to ice-water mixture. The solid formed was filtered, dried and recrystallized from ethanol to afford 2a,b.

12-Phenyl-10-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-methyl-3-thione-2,12-dihydrothia zolo[3',2':1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5, 4-e]pyrimidine (2a): Yield: 77%; pale yellow crystals; mp: 301-303 °C; IR (KBr): v (cm^-1) 3250 (NH), 1640 (CO pyrazolone), 1570 (C=N).¹HNMR (400 MHz, CDCl₃): δ ppm 8.50 (s, 1H, NH, D₂O exchangeable), 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s,1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³CNMR (400MHz, CDCl₃): 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 145 (C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 170 (CO pyrazolone), 189 (C=S); MS m/z (%): 524.30 (M⁺) (68); Anal. Calcd. for C₂₆H₂₀N₈OS₂: C, 59.52; H, 3.84; N, 21.36; S, 12.22. Found: C, 59.46; H, 3.80; N, 21.39; S, 12.25.

12-p-Chlorophenyl-10-(3-methyl-5-oxo-1-phenyl -2-pyrazolin-4-yl)-5-methyl-3-thione-2,12-di hydrothiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c] pyrimido[5,4-e]pyrimidine (2b): Yield: 78%; yellow crystals; mp: 310-312 °C; IR (KBr): v (cm^-1) 3250 (NH), 1640 (CO pyrazolone), 1570 (C=N); ¹H NMR (90 MHz, CDCl₃): δ ppm 8.50 (s, 1H, NH, D₂O exchangeable), 7.30-7.70 (m, 9H, aromatic protons), 7.10 (s, 1H, thiazole-H), 6.90 (s, 1H, pyrimidine-H), 6.60 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, CH₃ pyrazolone); ¹³C NMR (400MHz, CDCl₃): 15 (CH₃ pyrimidine), 17 (CH₃ pyrazolone), 48 (CH pyrimidine), 52 (CH pyrazolone), 88 (CH thiazole), 143-122 (C and CH aromatic rings), 141 (C=N triazole), 152 (C thiazole), 154 (C=N pyrazolone), 158 and 102 (C=C pyrimidine), 164 (C=N pyrimidine), 167 (C=N pyrimidine), 174 (CO pyrazolone), 191 (C=S);MS m/z (%): 559.95 (M+2) (22), 557.83 (M⁺) (66); Anal. Calcd. for C₂₆H₁₉N₈OS₂Cl: C, 55.86; H, 3.43; N, 20.04; S, 11.47; Cl, 6.34. Found: C, 55.81; H, 3.50; N, 20.09; S, 11.39; Cl, 6.41.

General procedure for the synthesis of 2-formyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)- 5-aryl-8-methyl-5H-thiazolo[3',2':1,2]pyrimido [5,4-e]pyrimidine)hydrazines 3a,b: A mixture of 1a,b (5 mmol) and excess of formic acid (10 mL) was heated under reflux for 2 h. The reaction mixture was cooled, and added to ice-water mixture. The solid formed was filtered, dried and recrystallized from benzene to afford 3a,b.

2-Formyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo[3', 2': 1,2]pyrimido[5,4-e]pyrimidine)hydrazine (3a): Yield: 82%; orange powder; mp: 293-294 °C; IR (KBr): v (cm^-1) 3300-3250 (NHNH), 3010 (CH aliphatic), 1695 (CO formyl), 1640 (CO pyrazolone);¹HNMR (400 MHz, CDCl₃): δ ppm 11.10 (s, 1H, CHO), 9.80 (s, 1H, NH D₂O exchangeable), 8.40 (s, 1H, NH, D₂O exchangeable), 8.30-8.00 (m, 10H, aromatic protons), 7.60 (s, 1H, pyrimidine-H), 7.50 (s, 1H, thiazole-H), 7.40 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 510.53 (M⁺) (72); Anal. Calcd. for C₂₆H₂₂N₈O₂S: C, 61.16; H, 4.34; N, 21.95; S, 6.28. Found: C, 61.22; H, 4.39; N, 21.86; S, 6.33.

2-Formyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-p-chlorophenyl-8-methyl-5H-thia zolo[3',2':1,2]pyrimido[5,4-e]pyrimidine)hydra zine (3b): Yield: 81%; yellow crystals; mp: 301-303 °C; IR (KBr): v (cm^-1) 3300-3250 (NHNH), 3010 (CH aliphatic), 1695 (CO formyl), 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 11.10 (s, 1H, CHO), 9.80 (s, 1H, NH, D₂O exchangeable), 8.40 (s, 1H, NH, D₂O exchangeable), 8.30-8.00 (m, 9H, aromatic protons), 7.60 (s, 1H, pyrimidine-H), 7.50 (s, 1H, thiazole-H), 7.40 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 547.03 (M+2) (22), 545.33 (M⁺) (72); Anal. Calcd. for C₂₆H₂₁N₈O₂SCl: C, 57.30; H, 3.88; N, 20.56; S, 5.88; Cl, 6.50. Found: C, 57.38; H, 3.97; N, 20.72; S, 5.79; Cl, 6.60.

General procedure for the synthesis of 12-aryl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin- 4-yl)-5-methyl-12H-thiazolo [3',2':1,2] [1,2,4] triazolo [3'',4''-c]pyrimido[5,4-e]pyrimidines 4a,b:

Method A: A solution of 1a,b (5 mmol) and excess of formic acid (5 mL) in glycerol (20 mL) was refluxed for 3h. The reaction mixture was added to ice-water mixture and the solid formed was filtered, dried and recrystallized from ethanol to afford 4a,b.

Method B: Fusion of the formyl hydrazines 3a,b (5 mmol) for 15 min. The solids formed were separated, washed with pet.ether 40-60 ºC and recrystallized from ethanol. The products were identical in all aspects (IR, ¹HNMR, MS and elemental analyses) with this prepared in method A.

12-Phenyl-10-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-methyl-12H-thiazolo[3',2':1,2] [1, 2, 4]triazolo[3'',4''-c]pyrimido[5,4-e]pyrimi dine (4a): Yield: 83%; yellow crystals; mp: 310-312 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone), 1580 (C=N);¹HNMR (400 MHz, CDCl₃): δ ppm 8.00 (s, 1H, triazole), 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³CNMR (400MHz, CDCl₃): 14(CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45(CH pyrimidine), 50(CH pyrazolone), 89(CH thiazole), 142-124 (C and CH aromatic rings), 145 (2C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 170 (CO pyrazolone); MS m/z (%): 492.46 (M⁺) (69); Anal. Calcd. for C₂₆H₂₀N₈OS: C, 63.40; H, 4.09; N, 22.75; S, 6.51. Found: C, 63.32; H, 3.99; N, 22.78; S, 6.45.

12-p-Chlorophenyl-10-(3-methyl-5-oxo-1-phenyl -2-pyrazolin-4-yl)-5-methyl-12H-thiazolo [3', 2': 1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e] pyrimidine (4b): Yield: 78%; yellow powder; mp: 321-323 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone), 1580 (C=N); ¹HNMR (90 MHz, CDCl₃); δ ppm 8.10 (s, 1H, triazole), 7.35-7.80 (m, 9H, aromatic protons), 7.00 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.60 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.30 (s, 3H, pyrazolone-CH₃); MS m/z (%): 529.21 (M+2) (18), 527.13 (M⁺) (54); Anal. Calcd. for C₂₆H₁₉N₈OSCl: C, 59.26; H, 3.63; N, 21.26;  S, 6.08; Cl, 6.73. Found: C, 59.31; H, 3.69; N, 21.31; S, 5.99; Cl, 6.80.

General procedure for the synthesis of ethyl 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-6- hydrazino-5-aryl-8-methyl-5H-thiazolo [3', 2': 1,2]pyrimido[5,4-e]pyrimidine carbazates 5a,b: A suspension of 1a,b (5 mmol) and excess of ethyl chloroformate (10 mL) was refluxed for 3 h. The solid formed was filtered, dried and recrystallized from ethanol to afford 5a,b.

Ethyl 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-6-hydrazino-5-phenyl-8-methyl-5H-thiazolo [3',2':1,2]pyrimido[5,4-e]pyrimidine carbazate (5a): Yield: 74%; yellow powder; mp220-222°C; IR (KBr): v (cm^-1) 3300-3250 (NHNH), 1710 (CO ester), 1640 (CO pyrazolone); ¹HNMR (400 MHz, CDCl₃): δ ppm 10.20 (s, 1H, NH, D₂O exchangeable), 9.20 (s, 1H, NH, D₂O exchangeable), 8.20-7.40 (m, 10H, aromatic protons), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 3.98 (q, 2H, CH₂CH₃, J = 7.5 Hz), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃), 1.50 (t, 3H, CH₂CH₃, J = 7.5 Hz); ¹³CNMR (400MHz, CDCl₃): 10 (CH₂CH₃), 15 (CH₃ pyrimidine), 17 (CH₃ pyrazolone), 35 (CH₂CH₃), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 102 (C=C pyrimidine), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 168 (C-NH), 175 (CO pyrazolone);MS m/z (%): 554.52 (M⁺) (70); Anal. Calcd. for C₂₈H₂₆N₈O₃S: C, 60.64; H, 4.73; N, 20.20; S, 5.78. Found: C, 60.40; H, 4.94; N, 19.90; S, 5.51.

Ethyl 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-6-hydrazino-5-p-chlorophenyl-8-methyl- 5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine carbazate (5b): Yield: 72%; pale yellow powder; mp: 231-233 °C; IR (KBr): v (cm^-1) 3300-3250 (NHNH), 1710 (CO ester), 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 10.20 (s, 1H, NH, D₂O exchangeable), 9.20 (s, 1H, NH, D₂O exchangeable), 8.20-7.40 (m, 9H, aromatic protons), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 3.98 (q, 2H, CH₂CH₃, J = 7.4 Hz), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃), 1.50 (t, 3H, CH₂CH₃, J = 7.4 Hz); MS m/z (%): 591.23 (M+2) (8), 589.33 (M⁺) (24); Anal. Calcd. for C₂₈H₂₅N₈O₃SCl: C, 57.09; H, 4.28; N, 19.02; S, 5.44; Cl, 6.02. Found: C, 57.12; H, 4.23; N, 18.99; S, 6.10; Cl, 6.10.

General procedure for the synthesis of 12-aryl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)- 5-methyl-3-oxo-2,12-dihydrothiazolo[3',2':1,2][1, 2,4]triazolo[3'',4''-c]pyrimido[5,4-e] pyrimidines 6a, b:

Method A: To hydrazino derivatives 1a,b (5 mmol) in pyridine (20 mL) was added ethyl chloroformate (0.43 mL, 5 mmol). The reaction mixture was refluxed for 3 h. After cooling, the solid formed was filtered, dried and recrystallized from benzene to afford 6a,b.

Method B: Ethyl 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-6-hydrazino-5-aryl-8-methyl-5H thiazolo[3,2-a]pyrimido[4,5-d]pyrimidine carbazates 5a,b were fused for 15 mins. The solids formed were washed with pet.ether 40-60ºC, filtered and recrystallized from ethanol. The products were identical in all aspects (IR, ¹HNMR, MS and elemental analyses) with those prepared in method A.

12-Phenyl-10-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-methyl-3-oxo-2,12-dihydrothiazolo [3',2':1,2][1,2,4]triazolo[3'',4''-c] pyrimido[5,4-e] pyrimidine (6a): Yield: 75%; orange crystals; mp: 295-297 °C; IR (KBr): v (cm^-1) 3350 (NH), 1640 (CO pyrazolone), 1620 (CO triazole);¹HNMR (400 MHz, CDCl₃): δ ppm 8.70 (s, 1H, NH_, D₂O exchangeable), 7.70-7.50 (m, 4H, aromatic protons), 7.35-7.10 (m, 6H, aromatic protons) 7.00 (s, 1H, thiazole-H), 6.70 (s, 1H, pyrimidine-H), 6.55 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, CH₃ pyrazolone); ¹³CNMR (CDCl₃, 400MHz): 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 145 (C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 155 (CO triazole), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 170 (CO pyrazolone); MS m/z (%): 508.44 (M⁺) (74); Anal. Calcd. for C₂₆H₂₀N₈O₂S: C, 61.40; H, 3.96; N, 22.03; S, 6.31. Found: C, 61.43; H, 3.90; N, 22.10; S, 6.23.

12-p-Chlorophenyl-10-(3-methyl-5-oxo-1-phenyl -2-pyrazolin-4-yl)-5-methyl-3-oxo-2,12-dihydro thiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c] pyrimido[5,4-e]pyrimidine (6b): Yield: 71%; pale orange crystals; mp: 311-313 °C; IR (KBr): v (cm^-1) 3350 (NH), 1640 (CO pyrazolone), 1620 (CO triazole); ¹HNMR (90 MHz, CDCl₃): δ ppm 8.80 (s, 1H, NH, D₂O exchangeable), 7.70-7.30 (m, 9H, aromatic protons), 7.10 (s, 1H, thiazole-H), 6.90 (s, 1H, pyrimidine-H), 6.60 (s, 1H, pyrazolone-H), 3.25 (s, 3H, pyrimidine-CH₃), 2.30 (s, 3H, pyrazolone-CH₃); MS m/z (%): 545.20 (M+2) (21), 543.81 (M⁺) (63); Anal. Calcd. for C₂₆H₁₉N₈O₂SCl: C, 57.51; H, 3.53; N, 20.64; S, 5.91; Cl, 6.53. Found: C, 57.47; H, 3.47; N, 20.62; S, 5.95; Cl, 6.56.

Synthesis of ethyl acetoacetate, 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-aryl-8- methyl-5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine hydrazones 7a,b: A mixture of the key intermediates 1a,b (5 mmol) and ethyl acetoacetate (0.64 mL, 5 mmol) in ethanol (20 mL) was refluxed for 2 h. On cooling, the solid formed was filtered, dried and recrystallized from dioxane to afford 7a,b.

Ethyl acetoacetate, 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-phenyl-8-methyl-5H-thia zolo[3',2':1,2]pyrimido[5,4-e]pyrimidine hydra- zone (7a): Yield: 75%; white powder; mp: 253-255 °C; IR (KBr): v (cm^-1) 3290 (NH), 1695 (CO ester), 1640 (CO pyrazolone);¹HNMR (90 MHz, CDCl₃): δ ppm 9.20 (s, 1H, NH pyrimidine, D₂O exchangeable), 8.70 (s, 1H, NH, D₂O exchangeable), 8.40-7.70 (m, 10H, aromatic protons), 7.50 (s, 1H, pyrimidine-H), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 4.10 (s, 2H, CH₂), 3.75 (q, 2H, CH₂CH₃, J = 9 Hz), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃), 1.50 (t, 3H, CH₂CH₃, J = 9 Hz), 1.30 (s, 3H, CH₃); MS m/z (%): 596.53 (M⁺) (49); Anal. Calcd. for C₃₁H₃₂N₈O₃S: C, 62.40; H, 5.34; N, 18.73; S, 5.38. Found: C, 62.38; H, 5.32; N, 18.84; S, 5.41.

Ethyl acetoacetate, 3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chlorophenyl-8-methyl-5H -thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine hydrazone (7b): Yield: 75%; yellow crystals; mp: 261-263 °C; IR (KBr): v (cm^-1) 3290 (NH), 1695 (CO ester), 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 9.20 (s, 1H, NH, D₂O exchangeable), 8.70 (s, 1H, NH, D₂O exchangeable), 8.50-7.70 (m, 9H, aromatic protons), 7.50 (s, 1H, pyrimidine-H), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 4.10 (s, 2H, CH₂), 3.75 (q, 2H, CH₂CH₃, J = 9 Hz), 3.20 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃), 1.50 (t, 3H, CH₂CH₃, J = 9 Hz), 1.30 (s, 3H, CH₃); MS m/z (%): 631.70 (M+2) (20), 631.10 (M⁺) (51); Anal. Calcd. for C₃₁H₃₁N₈O₃SCl: C, 58.99; H, 4.95; N, 17.75; S, 5.08; Cl, 5.62. Found: C, 58.94; H, 4.92; N, 17.71; S, 5.04; Cl, 5.68.

Synthesis of 6-(3-methyl-5-oxo-2-pyrazolin-1-yl)-3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin- 4-yl)-5-aryl-8-methyl-5H-thiazolo [3', 2': 1, 2] pyrimido [5,4-e]pyrimidines 8a,b:

Method A: A mixture of 1a,b (5 mmol) and ethyl acetoacetate (0.64 mL, 5 mmol) in ethanol (20 mL) containing potassium hydroxide (0.5 g) was refluxed for 3 h. The reaction mixture was cooled, neutralized with acetic acid.

The solid formed was filtered, dried and recrystallized from toluene to afford 8a,b.

Method B: Compounds 7a,b were fused for 15 mins. The solids formed were washed with pet.ether 40-60ºC, recrystallized from toluene. The products were identical in all aspects (IR, ¹HNMR, MS and elemental analyses) with the products of method A.

6-(3-Methyl-5-oxo-2-pyrazolin-1-yl)-3-(3-methyl -5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-phenyl-8- methyl-5H-thiazolo[3',2':1,2]pyrimido[5,4-e] pyrimidine (8a): Yield: 80%; white crystals; mp: 275-277 °C; IR (KBr): v (cm^-1) 1670 and 1640 (CO pyrazolone);¹HNMR (400 MHz, CDCl₃): δ ppm 8.30-7.80 (m, 10H, aromatic protons), 7.60 (s, 1H, pyrimidine-H), 7.40 (s, 1H, pyrimidine-H), 7.30 (s, 1H, thiazole-H), 7.10 (s, 2H, pyrazolone-H), 3.30 (s, 3H, pyrimidine-CH₃), 2.70 (s, 3H, pyrazolone-CH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 548.23 (M⁺) (52); Anal. Calcd. for C₂₉H₂₄N₈O₂S: C, 63.49; H, 4.40; N, 20.31; S, 5.84. Found: C, 63.51; H, 4.36; N, 20.29; S, 5.90.

6-(3-Methyl-5-oxo-2-pyrazolin-1-yl)-3-(3-methyl -5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chloro phenyl-8-methyl-5H-thiazolo[3',2':1,2] pyrimido [5,4-e]pyrimidine (8b): Yield: 71%; white powder; mp: 291-293 °C; IR (KBr): v (cm^-1) 1670 and 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 8.30-7.80 (m, 9H, aromatic protons), 7.60 (s, 1H, pyrimidine-H), 7.40 (s, 1H, pyrimidine-H), 7.30  (s, 1H, thiazole-H), 7.10 (s, 2H, pyrazolone-H), 3.30 (s, 3H, pyrimidine-CH₃), 2.70 (s, 3H, pyrazolone-CH₃), 2.40 (s, 3H, pyrazolone-CH₃). MS (EI, 70eV): m/z (%), 584.11 (M+2, 19), 582.43 (M⁺, 59). Anal. Calcd. for C₂₉H₂₃N₈O₂SCl (583.06): C, 59.74; H, 3.98; N, 19.22; S, 5.50; Cl, 6.08. Found: C, 59.80; H, 3.93; N, 19.18; S, 5.52; Cl, 6.04.

Synthesis of 6-(3,5-dimethyl-2-pyrazolin-1-yl)-3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)- 5-aryl-8- methyl-5H-thiazolo [3', 2': 1, 2] pyrimido [5,4-e]pyrimidines 9a,b: A solution of 1a,b (5 mmol) in ethanol was heated with acetyl acetone (0.51 mL, 5 mmol) for 3 h. On cooling, the solid formed was filtered, dried and recrystallized from acetic acid to afford 9a,b.

6-(3,5-Dimethyl-2-pyrazolin-1-yl)-3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine (9a): Yield: 78%; white crystals; mp: 253-255 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 7.90-7.50 (m.10H, aromatic protons), 7.40 (s, 1H, pyrimidine-H), 7.30 (s, 1H, thiazole-H), 7.10 (s, 2H, pyrazoline-CH₂), 3.30 (s, 3H, pyrimidine-CH₃), 2.80 (s, 6H, pyrazoline-CH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 546.12 (M⁺) (58); Anal. Calcd. for C₃₀H₂₆N₈OS: C, 65.91; H, 4.79; N, 20.50; S, 5.87. Found: C, 65.86; H, 4.72; N, 20.57; S, 5.93.

6-(3,5-Dimethyl-2-pyrazolin-1-yl)-3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chloro phenyl-8-methyl-5H-thiazolo [3', 2': 1, 2] pyrimido[5,4-e]pyrimidine (9b): Yield: 71%; white crystals; mp: 269-271 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 7.90-7.50 (m, 9H, aromatic protons), 7.40 (s, 1H, pyrimidine-H), 7.30 (s, 1H, thiazole-H), 7.10 (s, 2H, pyrazoline-CH₂), 3.30 (s, 3H, pyrimidine-CH₃), 2.80 (s, 6H, pyrazoline-CH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 581.98 (M+2) (17), 580.23 (M⁺) (51); Anal. Calcd. for C₃₀H₂₅N₈OSCl: C, 62.01; H, 4.34; N, 19.28; S, 5.52; Cl, 6.10. Found: C, 62.06; H, 4.36; N, 19.19; S, 5.57; Cl, 6.14.

Synthesis of 1,2,2-triacetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-aryl-8-methyl- 5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazines 10a,b: Compounds 1a,b (5 mmol) and excess of acetic anhydride (15 mL) were refluxed for 3 h. On cooling, the solid formed was filtered, dried and recrystallized from ethanol to afford 10a,b.

1,2,2-Triacetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo [3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazine (10a): Yield: 76%; orange crystals; mp: 268-270 °C; IR (KBr): v (cm^-1) 1680 (CO acetyl), 1670 (2 CO acetyl), 1640 (CO pyrazolone); ¹HNMR (400 MHz, CDCl₃): δ ppm 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.15 (s, 3H, pyrimidine-CH₃), 3.00 (s, 3H, COCH₃), 2.80 (s, 6H, 2 COCH₃), 2.30 (s, 3H, pyrazolone-CH₃); ¹³CNMR (400MHz, CDCl₃): 10 (CH₃ acetyl), 12 (2 CH₃ acetyl), 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 158 (N=C-NN pyrimidine), 162 (C=N pyrimidine), 165 (C=N pyrimidine), 169 (CO acetyl), 172 (CO pyrazolone), 175 (2CO acetyl); MS m/z (%): 608.11 (M⁺) (61);Anal. Calcd. for C₃₁H₂₈N₈O₄S: C, 61.17; H, 4.64; N, 18.41; S, 5.27. Found: C, 61.07; H, 4.69; N, 18.46; S, 5.24.

1,2,2-Triacetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chlorophenyl-8-methyl- 5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazine (10b): Yield: 70%; yellow crystals; mp: 281-283 °C; IR (KBr): v (cm^-1) 1680 (CO acetyl), 1670 (2 CO acetyl), 1640 (CO pyrazolone); ¹HNMR (400 MHz, CDCl₃): δ ppm 7.30-7.70 (m, 9H, aromatic protons), 7.00 (s, 1H, thiazole-H), 6.70 (s, 1H, pyrimidine-H), 6.55 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 3.00 (s, 3H, COCH₃), 2.80 (s, 6H, 2 COCH₃), 2.40 (s, 3H, pyrazolone-CH₃); MS m/z (%): 644.12 (M+2) (20), 642.21 (M⁺) (64); Anal. Calcd. for C₃₁H₂₇N₈O₄SCl: C, 57.89; H, 4.23; N, 17.42; S, 4.99; Cl, 5.51. Found: C, 57.85; H, 4.19; N, 17.44; S, 5.96; Cl, 5.45.

Synthesis of 2-acetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-aryl-8-methyl-5H- thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazines 11a,b: A mixture of 1a,b (5 mmol) and excess of acetic acid (20 mL) was refluxed for 3 h. The cooled reaction mixture was added to ice/water mixture. The solid formed was filtered, dried and recrystallized from benzene to afford 11a,b.

2-Acetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyra zolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo [3', 2':1,2]pyrimido[5,4-e]pyrimidine)hydrazine (11a): Yield: 79%; red crystals; mp: 290-292 °C; IR (KBr): v (cm^-1) 3250-3150 (NHNH), 1690 (CO acetyl), 1640 (CO pyrzolone);¹H NMR (90 MHz, CDCl₃): δ ppm 10.20 (s, 1H, NHCO, D₂O exchangeable), 9.20 (s, 1H, NH, D₂O exchangeable), 8.20-7.40 (m, 10H, aromatic protons), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.40 (s, 3H, COCH₃), 2.20 (s, 3H, pyrazolone-CH₃); MS m/z (%): 524.01 (M⁺) (59); Anal. Calcd. for C₂₇H₂₄N₈O₂S: C, 61.82; H, 4.61; N, 21.36; S, 6.11. Found: C, 61.86; H, 4.64; N, 21.38; S, 6.03.

2-Acetyl-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chlorophenyl-8-methyl-5H- thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazine (11b): Yield: 72%; orange crystals; mp: 284-286 °C; IR (KBr): v (cm^-1) 3250-3150 (NHNH), 1690 (CO acetyl), 1640 (CO pyrazolone); ¹HNMR (90 MHz, CDCl₃): δ ppm 10.20 (s, 1H, NHCO, D₂O exchangeable), 9.20 (s, 1H, NH, D₂O exchangeable), 8.20-7.40 (m, 9H, aromatic protons), 7.30 (s.1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.40 (s, 3H, COCH₃), 2.20 (s, 3H, pyrazolone-CH₃); MS m/z (%): 561.20 (M+2) (18), 559.11 (M⁺) (53); Anal. Calcd. for C₂₇H₂₃N₈O₂SCl: C, 58.01; H, 4.15; N, 20.04; S, 5.74; Cl, 6.34. Found: C, 57.96; H, 4.11; N, 20.00; S, 5.69; Cl, 6.32.

Synthesis of 12-aryl-3,5-dimethyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo [3',2':1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e]pyrimidines 12a,b: A suspension of 11a,b (2 mmol) in xylene (20 mL) and phosphoryl chloride (0.186 mL, 2 mmol) was refluxed for 6 h. On cooling, the solid formed was filtered, dried and recrystallized from ethanol to afford 12a,b.

12-Phenyl-3,5-dimethyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo[3',2':1,2] [1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e] pyrimidine (12a): Yield: 76%; white crystals; mp: 274-275 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone), 1560 (C=N);¹HNMR (400 MHz, CDCl₃): δ ppm 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃),2.90 (s, 3H, triazole-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³CNMR (CDCl₃, 400MHz): 10 (CH₃ triazole), 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 145 (C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 157 (C=N triazole), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 170 (CO pyrazolone); MS m/z (%): 506.41 (M⁺) (60); Anal. Calcd. for C₂₇H₂₂N₈OS: C, 64.01; H, 4.38; N, 22.12; S, 6.33. Found: C, 64.06; H, 4.31; N, 22.14; S, 6.39.

12-p-Chlorophenyl-3,5-dimethyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo [3', 2':1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e] pyrimidine (12b): Yield: 71%; yellow crystals; mp: 250-252 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone), 1560 (C=N);¹H NMR (90 MHz, CDCl₃): δ ppm 7.50-8.00 (m, 9H, aromatic protons), 7.00 (s,1H, thiazole-H), 6.70 (s, 1H, pyrimidine-H), 6.55 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.90 (s, 3H, triazole-CH₃), 2.30 (s, 3H, pyrazolone-CH₃); MS m/z (%): 542.11 (M+2) (24), 540.31 (M⁺) (71); Anal. Calcd. for C₂₇H₂₁N₈OSCl: C, 59.94; H, 3.91; N, 20.71; S, 5.93; Cl, 6.55. Found: C, 59.90; H, 3.87; N, 20.67; S, 5.90; Cl, 6.56.

Synthesis of 2-benzylidene-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-aryl-8-methyl- 5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazines 13a,b: An equimolar amounts of 1a,b (5 mmol) and benzaldehyde (0.51 mL, 5 mmol) in ethanol (20 mL) was refluxed for 3 h. On cooling, the solid formed was filtered, dried and recrystallized from ethanol to afford 13a,b.

2-Benzylidene-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-phenyl-8-methyl-5H-thiazolo [3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazine (13a): Yield: 79%; yellow crystals; mp: 285-287 °C; IR (KBr): v (cm^-1) 3205 (NH), 1640 (CO pyrazolone), 1590 (C=N);¹H NMR (90 MHz, CDCl₃): δ ppm 9.60 (s, 1H, NH, D₂O exchangeable), 8.20-7.50 (m, 15H, aromatic protons), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 5.00 (s, 1H, N=CH), 3.00 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³C NMR (400MHz, CDCl₃): 16(CH₃ pyrimidine), 17(CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 161 (C=N pyrimidine), 164 (C=N pyrimidine), 168 (N=CHPh), 170 (CO pyrazolone). MS m/z (%): 569.01 (M⁺) (53); Anal. Calcd. for C₃₂H₂₆N₈OS: C, 67.35; H, 4.59; N, 19.64; S, 5.62. Found: C, 67.28; H, 4.52; N, 19.60; S, 5.67.

2-Benzylidene-1-(3-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-5-p-chlorophenyl-8-methyl- 5H-thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine) hydrazine (13b): Yield: 73%; yellow powder; mp: 294-296 °C; IR (KBr): v (cm^-1) 3205 (NH), 1640 (CO pyrazolone), 1590 (C=N); ¹H NMR (400 MHz, CDCl₃): δ ppm 9.60 (s, 1H, NH, D₂O exchangeable), 8.20-7.50 (m, 14H, aromatic protons), 7.30 (s, 1H, pyrimidine-H), 7.20 (s, 1H, thiazole-H), 7.10 (s, 1H, pyrazolone-H), 5.00 (s, 1H, N=CH), 3.00 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃). MS m/z (%): 606.70 (M+2) (17), 604.81 (M⁺) (52); Anal. Calcd. for C₃₂H₂₅N₈OSCl: C, 63.52; H, 4.16; N, 18.52; S, 5.30; Cl, 5.86. Found: C, 63.49; H, 4.01; N, 18.55; S, 5.32; Cl, 5.90.

Synthesis of 12-aryl-5-methyl-3-phenyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H- thiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c] pyrimido[5,4-e]pyrimidines 14a,b: A suspension of 13a,b (2 mmol) and excess of thionyl chloride (20 mL) was heated on a water bath for 3 h. On cooling, the excess of thionyl chloride was removed by distillation. The residue was triturated with pet. ether 60-80°C, filtered, dried and recrystallized from toluene to afford 14a,b.

3,12-Diphenyl-5-methyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo[3',2':1,2] [1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e] pyrimidine (14a): Yield: 82%; orange powder; mp: 300-302°C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone) cm^-1.¹H NMR (400 MHz, CDCl₃): δ ppm 8.10-7.60 (m, 6H, aromatic protons), 7.44-7.06 (m, 9H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³C NMR (400 MHz, CDCl₃): 14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 120-143 (C and CH aromatic rings), 145 (C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103 (C=C pyrimidine), 160-162 (C=N pyrimidine and C=N triazole), 164 (C=N pyrimidine), 170 (CO pyrazolone); MS m/z (%): 568.70 (M⁺) (63); Anal. Calcd. for C₃₂H₂₄N₈OS: C, 67.59; H, 4.25; N, 19.71; S, 5.64. Found: C, 55.48; H, 4.27; N, 19.68; S, 5.60.

3-p-Chlorophenyl-5-methyl-12-phenyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H- thiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c] pyrimido[5,4-e]pyrimidine (14b): Yield: 74%; orange crystals; mp: 290-292 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone) cm^-1. ¹HNMR (90 MHz, CDCl₃): δ ppm 8.10-7.40 (m, 14H, aromatic protons), 7.00 (s, 1H, thiazole-H), 6.85 (s, 1H, pyrimidine-H), 6.70 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃).MS m/z (%): 604.12 (M+2) (22), 602.61 (M⁺) (67);Anal. Calcd. for C₃₂H₂₃N₈OSCl: C, 63.73; H, 3.84; N, 18.58; S, 5.32; Cl, 5.88. Found: C, 63.70; H, 3.81; N, 18.56; S, 5.34; Cl, 5.90.

Synthesis of 12-aryl-5-methyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo [3', 2':1,2]tetrazolo[3'',4''-c]pyrimido[5,4-e] pyrimidines 15a,b: A solution of sodium nitrite in water (15 mL 5%, 5 mmol) was added to a solution of 1a,b ( 5 mmol) in concentrated hydrochloric acid (10 mL) at 0°C over a period of 10 mins with stirring. The solid formed was filtered, dried and recrystallized from benzene to afford 15a,b.

12-Phenyl-5-methyl-10-(3-methyl-5-oxo-1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo[3',2':1,2] tetrazolo[3'',4''-c]pyrimido[5,4-e]pyrimidine (15a): Yield: 76%; white powder; mp: 270-272 °C; IR (KBr): v (cm^-1) 1640 (CO pyrazolone);¹HNMR (400 MHz, CDCl₃): δ ppm 7.64-7.24 (m, 4H, aromatic protons), 7.14-7.06 (m, 6H, aromatic protons), 6.92 (s, 1H, thiazole-H), 6.80 (s, 1H, pyrimidine-H), 6.71 (s, 1H, pyrazolone-H), 3.10 (s, 3H, pyrimidine-CH₃), 2.20 (s, 3H, pyrazolone-CH₃); ¹³CNMR (400 MHz, CDCl₃):14 (CH₃ pyrimidine), 16 (CH₃ pyrazolone), 45 (CH pyrimidine), 50 (CH pyrazolone), 89 (CH thiazole), 142-124 (C and CH aromatic rings), 145(C=N triazole), 154 (C thiazole), 155 (C=N pyrazolone), 159 and 103(C=C pyrimidine), 161(C=N pyrimidine), 164(C=N pyrimidine), 170(CO pyrazolone); MS m/z (%): 493.15 (M⁺) (80); Anal. Calcd. for C₂₅H₁₉N₉OS: C, 60.84; H, 3.88; N, 25.54; S, 6.50. Found: C, 60.80; H, 3.90; N, 25.60; S, 6.52.

12-p-Chlorophenyl-5-methyl-10-(3-methyl-5-oxo -1-phenyl-2-pyrazolin-4-yl)-12H-thiazolo [3', 2': 1,2]tetrazolo[3'',4''-c]pyrimido[5,4-e] pyrimidine (15b): Yield: 74%; white powder; mp: 279-280° C. IR (KBr): v (cm^-1) 1640 (CO pyrazolone); ¹HNMR (400 MHz, CDCl₃): δ ppm 7.50-8.00 (m, 9H, aromatic protons), 7.00 (s, 1H, thiazole-H), 6.70 (s, 1H, pyrimidine-H), 6.55 (s, 1H, pyrazolone-H), 3.20 (s, 3H, pyrimidine-CH₃), 2.30 (s, 3H, pyrazolone-CH₃); MS m/z (%): 529.70 (M+2) (19), 527.80 (M⁺) (63); Anal. Calcd. for C₂₅H₁₈N₉OSCl: C, 56.87; H, 3.44; N, 23.88; S, 6.07; Cl, 6.71. Found: C, 56.85; H, 3.47; N, 23.84; S, 6.11; Cl, 6.79.

Biological screening: The screened compounds dissolved in DMSO to get a solution of 5 % concentration. Filter paper discs (Watman No. 3 and 5 mm diameter) saturated with this solution. The discs placed on the surface of solidified Nutrient agar dishes seeded by the tested bacteria or Czapek´s Dox agar dishes seeded by the tested fungi. The diameter of inhibition zones (mm) were measured at the end of the incubation period (24h-48h at 37ºC for bacteria and for 4-7 days at 28ºC for fungi)²³. Discs saturated with DMSO used as control. Chloramphenicol and Clotrimazole used as reference substances. The biologically active compounds diluted with DMSO to prepare a series of concentrations in order to determine the minimum inhibitory concentration (MIC) of each compound. MICs calculated as mg/mL. The data of antibacterial and antifungal activities are given in Tables 1 and 2.

Anti-inflammatory screening: Paw edema inhibition test was used on albino rats by winter method ²⁴. Groups of five animals of both sexes (body weight 120–160 g), excluding pregnant females, were given a dose of test compound. Thirty minutes later, 0.20 mL of 1% freshly prepared carrageenan suspension in 0.9% NaCl solution was injected subcutaneously into the planter aponeurosis of the hind paw and the volume was measured 1-3 h later.

The mean increase of paw volume at each interval was compared with that of control group (five rats treated with carrageenan but not with test compound) at the same intervals and percent inhibition value calculated by the formula is given below.

% Anti-inflammatory activity = [1 - D_t / D_c] × 100

D_t and D_c are paw volumes of oedema in tested and control groups, respectively.

Analgesic screening: Acetic acid writhing test was performed on mice by following Davis method ²⁵. Groups of five mice of both sexes (body weight 20–30 g), pregnant females excluded, were given a dose of test compound. Thirty minutes later, the animals were injected 0.25 mL/mice of 0.5% acetic acid solution and writhes were counted during the following 60 mins. The mean number of writhes of each experimental group and percent decrease compared with control group (five mice not treated with test compounds) were calculated.

RESULTS AND DISCUSSION:

Chemistry: Our efforts focused on the introduction of chemical diversity in the molecular framework in order to synthesize pharmacologically interesting compounds of different compositions. This motivated us to design and synthesize new thiazolopyrimidopyrimidines, thiazolotriazolo pyrimidopyrimidines, and pyrazolinyl thiazolo pyrimidopyrimidines linked to 2-pyrazolin-5-one. A facile method for the synthesis of key intermediates 1a,b was achieved by the hydrazinolysis of the chloro derivatives ²¹ with hydrazine hydrate (Scheme 1).

The ¹H NMR and ¹³C NMR spectra of 1a,b were the basis of their structures elucidation. The IR spectrum for compound 1a showed absorption bands at range of 3100-3350 cm^-1 due to NHNH₂ groups. Moreover, the ¹H NMR spectra indicated the presence of signals for NH and NH₂ protons at δ 8.80 and 5.20 ppm, respectively. As illustrated in Scheme 1, the compounds 1a,b were reacted with carbon disulfide in pyridine producing the thiazolotriazolo pyrimidopyrimidine thiones 2a,b. On the other hand, when 1a,b were fused with formic acid or refluxed with an equimolar amount of formic in glycerol, they afford the formyl hydrazine analogues 3a,b or thiazolotriazolo pyrimido pyrimidines 4a,b, respectively. The latter compounds 4a,b were also prepared by another method on fusion of 3a,b. The structures of the latter compounds were established based on analytical and spectrum data. The IR spectra of 3a,b showed the presence of absorption bands at range of 1690-1695 cm^-1 for C=O formyl groups. Moreover, the ¹H NMR spectra showed, besides the expected signals for the pyrazoline ring systems, presence of signals for CHO proton at δ 11.10 ppm.

Ethyl thiazolo[3',2':1,2]pyrimido[5,4-e]pyrimidine carbazates 5a,b were obtained from the reaction of 1a,b with ethyl chloroformate. On the other hand, using of pyridine as a solvent in the latter reaction afforded thiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e]pyrimidines 6a,b which were also obtained when 5a,b were fused for 15 mins (Scheme 1).

The ¹H NMR and ¹³C NMR spectra of 1a, b were the basis of their structures elucidation. The IR spectrum for compound 1a showed absorption bands at range of 3100-3350 cm^-1 due to NHNH₂ groups. Moreover, the ¹H NMR spectra indicated the presence of signals for NH and NH₂ protons at δ 8.80 and 5.20 ppm, respectively. As illustrated in Scheme 1, the compounds 1a, b was reacted with carbon disulfide in pyridine producing the thiazolotriazolo- pyrimidopyrimidine thiones 2a, b. On the other hand, when 1a,b were fused with formic acid or refluxed with an equimolar amount of formic in glycerol, they afford the formyl hydrazine analogues 3a,b or thiazolotriazolo- pyrimidopyrimidines 4a,b, respectively. The latter compounds 4a, b were also prepared by another method on fusion of 3a, b. The structures of the latter compounds were established based on analytical and spectrum data. The IR spectra of 3a, b showed the presence of absorption bands at range of 1690-1695 cm^-1 for C=O formyl groups. Moreover, the ¹H NMR spectra showed, besides the expected signals for the pyrazoline ring systems, presence of signals for CHO proton at δ 11.10 ppm.

Ethyl thiazolo[3',2':1,2] pyrimido[5,4-e ]pyrimidine carbazates 5a,b were obtained from the reaction of 1a,b with ethyl chloroformate. On the other hand, using of pyridine as a solvent in the latter reaction afforded thiazolo[3',2':1,2][1,2,4]triazolo[3'',4''-c]pyrimido[5,4-e]pyrimidines 6a,b which were also obtained when 5a,b were fused for 15 mins (Scheme 1).

SCHEME 1: SYNTHESIS OF COMPOUNDS 1a, b - 6a, b. Reagents i)NH₂NH₂.H₂O; ii) CS₂ / pyridine; iii) HCOOH; iv) fusion; v) HCOOH/glycerol; vi) ClCOOEt; vii) fusion; viii) ClCOOEt/pyridine.

Reacting 1a,b treated with ethyl acetoacetate in ethanol afforded the hydrazones 7a,b. When the latter reaction was proceeded in alkaline medium such as potassium hydroxide dissolved in ethanol, the pyrazolinyl thiazolopyrimidopyrimidins 8a,b were obtained. Compounds 8a,b were prepared by another route via intramolecular cyclization of 7a,b when fused for 15 mins. As a part of the hydrazine group reactivity, the hydrazine compounds 1a,b were reacted with acetyl acetone to afford dimethyl pyrazolinylthiazolopyrimidopyrimidines 9a,b.

The IR spectra ofthelatterproductsexhibited disappearance of absorption bands for NHNH₂ group. Their ¹H NMR spectra showed a singlet signal for each at δ 2.80 ppm attributed to CH₃ protons of the new pyrazoline unit. Acetylation of the hydrazine derivatives 1a,b with Ac₂O yielded the triacetylated compounds 10a,b. However, monoacetylation for the same compounds were produced viarefluxingthecompounds with an equimolar amount of acetic acid to give 11a,b which on refluxing with POCl₃ in xylene gave methyl thiazolotriazolopyrimidopyrimidines 12a,b via the intramolecular cyclization. The structures of the compounds 11a,b and 12a,b were characterized by spectral and elemental analyses. The IR spectra of compounds 12a,b revealed the absence of characteristic absorption bands to NH function.

The ¹H NMR spectra have shown the absence of signals of 2 NH protons and the presence of signal at δ 2.90 ppm due to CH₃ protons of the new triazole ring. The condensation of 1a,b with benzaldehyde gave the corresponding Schiff bases 13a,b which were cyclized by treatment with SOCl₂ to afford compounds polycyclic compounds 14a,b. The ¹H NMR spectra of 14a,b showed disappearance of NH and CH protons of Schiff bases. On the other hand, their ¹³C NMR spectra showed C=N signals at δ 145 and 162 ppm. The diazotization of the key intermediates 1a,b by treatment with nitrous acid to produce thiazolo tetrazolpyrimidopyrimidines 15a,b (Scheme 2).

SCHEME 2: Synthesis of compounds 7a, b - 15a, b. Reagents i)AcCH₂COOEt/ EtOH; ii) AcCH₂COOEt/KOH / EtOH; iii) CH₂ (Ac)₂; iv) AcO₂; v) AcOH vi) POCl₃/ xylene; vii) ArCHO; viii) SOCl₂; ix) NaNO₂ / HCl.

Pharmacological activities:

Biological evaluation:

Evaluation of antibacterial activity: All of the synthesized compounds were evaluated for antibacterial activity against five pathogenic strains of bacteria (Staphylococcus aureus AUMC B-54, Bacillus cereus AUMC B-52, Escherichia coli AUMC B-53, Psedomonas aeruginosa AUMC B-73 and Serratia marcescens B-55).

The MIC (minimum inhibitory concentration) values were compared with Chloramphenicol as control agent. As indicated in Table 1, all of the synthesized compounds showed potent antibacterial activity against all the bacterial strains tested except the compounds 7a, 8b, 11a, b and 14a, b. Among all the compounds tested, compounds 1b, 12b, and 15b showed excellent MIC values. The rest of compounds exhibited moderate antibacterial activities.

Evaluation of antifungal activity: For the antifungal activity, The MIC values were compared with Clotrimazole as control agent. The final concentration of antibacterial agents was between 1.1 - 100 µg/mL. The compounds were tested againstsix antinfungal species, Candida albicans AUMC 1299, Geotrichum candidum AUMC 226, Fusarium oxysporum AUMC 5119, Aspergillus flavus AUMC 1276, Trichophyton rubrum AUMC 1804, and Scopulariopsis brevicaulis AUMC 361. From the results depicted in Table 2, we find that the target compounds 1a, b showed excellent antifungal activities for all the tested pathogens with MIC that ranged between 10 and 2.5 µg/mL. Furthermore, the rest of compounds showed good antifungal activity. In comparison of the substituent in aromatic ring at position-5, we note that substitution of hydrogen atom with chlorine increased the antifungal and antifungal activities of the compounds.

TABLE 1: ANTIBACTERIAL ACTIVITY OF THE SYNTHESIZED COMPOUNDS 1a, b - 15a, bMICS (µg/ML)

Ref^a. Chloramphenicol used as antibacterial standard.  (-) Indicates no activity.

TABLE 2: ANTIFUNGAL ACTIVITY OF THE SYNTHESIZED COMPOUNDS 1a,b - 15a,bMICS (µg/ML):

Ref^a. Clotrimazole used as antifungal standard.  (-) No activity.

Anti-inflammatory and analgesic activities: The anti-inflammatory activity of compounds1a, b, 2a, b, 4a, b, 6a, b, 8a, b, 9a, b, 12a, band14a, bwasmeasured using carrageenan-induced rat paw edema assay. As indicated in Table 3, the key intermediates1aand1bat 50 mg/kg p.o. exhibited good activities (15% and 16%, respectively).

The thiazolo thiazolopyrimidopyrimidines 2a,b, 4a,b 6a,b, 12a,b, 14a,b exhibited 30%, 32%, 17%, 18%, 21%, 22%, 24%, 26%, 28% and 29%, respectively. On the other hand, pyrazolinyl thiazolopyrimido pyrimidines 8a, b and 9a, b exhibited 19%, 20%, 19% and 20%, respectively, whereas ibuprofen exhibited 25% anti-inflammatory activity at 50 mg/kg p.o. The analgesic activity evaluation was carried out using acetic acid writhing assay ²⁵. The results are summarized in Table 3, the key compounds 1a and 1b at 50 mg/kg p.o. exhibited 11% and 14%, respectively. Compounds 2a, b, 4a, b 6a,b, 12a,b and 14a, b exhibited 27%, 29%, 13%, 14%, 22%, 24%, 20%, 21%, 24% and 26%, respectively.

Furthermore, pyrazolinyl thiazolo pyrimido pyrimidines 8a, b and 9a, b exhibited 18%, 19%, 16% and 18%, respectively. It is worth mentioning that compounds 2a, 2b and 14b exhibited excellent anti-inflammatory and analgesic activities.

TABLE 3: ANTI-INFLAMMATORY AND ANALGESIC ACTIVITIE EVALUATION OF COMPOUNDS 1a, b, 2a, b, 4a, b, 6a, b, 8a, b, 9a, b, 12a, b and 14a, b.

Structure-activity relationship (SAR): From the results depicted in Tables 1-3, we found that the key intermediates 1a and 1b showed promising pharmacological activities. This encouraged derivatization of the hydrazine functionality of these intermediates hopping to improve the pharmacological effects. Here, the chemistry showed three heterocyclic systems linked to 2-pyrazolin-5-one, the first one constituted thiazolopyrimido- pyrimidines 1a, b, 3a, b, 5a, b, 7a, b, 10a, b, 11a, b and 13a, b.

The second class constituted thiazolotriazolo-pyrimido pyrimidines 2a, b, 6a, b, 12a, b and 14a, b, while the latter class constituted pyrazolinyl thiazolopyrimido pyrimidines 8a, b and 9a, b. The three classes were tested against antimicrobial, anti-inflammatory, and analgesic activities.

In the antimicrobial activity, replacing the –NHNH₂ group in compounds 1a,b with –NHNH CHO, -NHNHCOOEt, -N(Ac)N(Ac)₂, NHNHAc, and –NHN=CHPh groups in compounds 3a,b, 5a,b, 7a,b, 10a,b, 11a,b and 13a,b, respectively, affected positive impact on the activity.

As the same, incorporation of new five-membered nitrogen containing ring as shown in compounds 2a,b, 4a,b, 6a,b, 8a,b, 9a,b, 12a,b and 14a,b, respectively. From the anti-inflammatory and analgesic results of compounds 2a, b, it is worth mentioning that incorporation of pyrazole unit to the key intermediates 1a, b gave significant activity (30% and 32%) for the anti-inflammatory and (27%, 29%) for analgesic activities.

CONCLUSION: In summary, we reported herein a simple and convenient method for the syntheses of new pharmacophore. The new pharmacophore was built via the combination of thiazolo-ppyrimidopyrimidine skeleton and 2-pyrazolin-5-one unit. The newly synthesized compounds were screened for antimicrobial, analgesic, and anti-inflammatory activities. Most of the compounds showed remarkable results.

ACKNOWLEDGEMENTS: The authors are thankful to Dr. Gamal Mohamed Zayed, Department of Pharmaceutical Scinces, Faculty of Pharmacy, Al-Azhar University for his kind assistance in performing the anti-inflammatory and analgesic screening as well as they thanks to Assiut University of Mycological Center (AUMC).

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    Youssef MSK and Abeed AAO: Synthesis, characterization and pharmacological activities of pyrimidine derivatives containing 2-pyrazoline-5-one.Int J Pharm Sci Res 2014; 5(5): 1705-20.doi: 10.13040/IJPSR.0975-8232.5 (5).1705-20

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