Synthesis, Antitumor and Antioxidant Evaluation of Some New Antipyrine Based Azo Dyes Incorporating...

Copyright © 2012 by Modern Scientific Press Company, Florida, USA

International Journal of Modern Organic Chemistry, 2012, 1(3): 213-225

International Journal of Modern Organic Chemistry

Journal homepage: www.ModernScientificPress.com/Journals/IJOrgChem.aspx

ISSN: 2166-0174

Florida, USA

Article

Synthesis, Antitumor and Antioxidant Evaluation of Some New

Antipyrine Based Azo Dyes Incorporating Pyrazolone Moiety

M. A. Metwally 1, Yaser. A. Suleiman 3, M. A. Gouda 1, 2, Ammar N. Harmal 1, 3, *, A. M. Khalil 1

1 Department of Chemistry, Faculty of Science, Mansoura University, El-Gomhoria Street, Mansoura,

35516, Egypt 2 Department of Chemistry, Faculty of Science and Arts, Ulla, Taibah University, KSA 3 Department of Chemistry, Faculty of Education, Amran University, Yemen

* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.:

+2018-7619869, Fax: +2050-2246781.

Article history: Received 7 November 2012, Accepted 10 December 2012, Published 12 December

2012.

Abstract: Diazodization of 3-aminopyrazole 5 with NaNO2 in HCl/AcOH afforded the

diazonium salt 6, which coupled with 7, 8 or 11 in pyridine to give arylazo derivatives 9,

10, and 12, respectively. Heating of 12 in acetic acid gave the hydrazone 14. Condensation

of compound 14 with 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 15 gave

the pyrazolopyrimidine 16. Multicomponent reaction of 4-methoxy(chloro)benzaldehyde

17 or 18 and 2-mercaptoacetic acid with 5 in a mixture of pyridine/DMF afforded the

thiazolidinones 20 and 21, respectively. Treatment of 5 with one or two equivalent of 2-

chloro-1-phenylethanone 22 in a mixture of pyridine/DMF afforded the ethanone and

bisethanone derivative 23. Cyclization of 23 under influence of phosphrousoxy chloride

afforded the imidazole derivative 25. Newly synthesized compounds were screened for

their antitumor and antioxidant activities. The obtained results showed clearly that most of

compounds exhibited good antitumor activities and moderate antioxidant activities, while

compounds 4 and 20 exhibited broad spectrum of antitumor and antioxidant activities.

Keywords: antipyrine; pyrazole; pyrazolopyrmidine; mercaptoacetic acid; antitumor;

antioxidant activity.

1. Introduction

It has been known for many years that the azo compounds are the most widely used class of

dyes due to their versatile application in various fields such as the dyeing of textile fibers, the coloring

Int. J. Modern Org. Chem. 2012, 1(3): 213-226


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of different materials, in biological–medical studies and advanced applications in organic synthesis [1-

6].

Antipyrine or phenazone derivatives are well known compounds used mainly as analgesic and

antipyreticdrugs [7]. One of the best known antipyrine derivatives is 4-aminoantipyrine which is used

for the protection against oxidative stress as well as prophylactic of some diseases including cancer

and these are important directions in medicine and biochemistry [8, 9]. Reactive oxygen species

(ROS), including free radicals led to a reactive species that damage the living cell. Oxidative stress

may arise in a biological system after an increased exposure to oxidants, so the antioxidants play a

major role in protecting biological systems against such threats. Different types of antioxidants

(vitamins C and E, glutathione, lipoic acid and butylated phenols, etc.) have been widely used in

different fields of industry and medicine as compounds that interrupt radical chain oxidation processes,

causing thus a high scientific interest [10,11]. In addition, antipyrine derivatives are strong inhibitors

of cycloxygenase isoenzymes, platelet tromboxane and prostanoids synthesis [7,12].

The biological activity of these compounds has also been attributed to its scavenging activity

against reactive oxygen and nitrogen species, as well as to the inhibition of neutrophil’s oxidative

burst. However, besides its well recognized benefits, antipyrine derivatives have been associated with

potential adverse effects characterized by leukopenia, most commonly of neutrophils, causing

neutropenia in the circulating blood (agranulocytosis). It is worth to mention that there are studies

demonstrating that this adverse effect might be exaggerated [13,14]. Moreover, several pyrazole ring

systems are associated with diverse biological activities [15-17].

Therefore, it was thought of interest to combine the above mentioned boilable rings together in

a molecular framework to investigate the additive effect of these rings towards antioxidant and

antitumor activities.

2. Experimental

2.1. Instruments

All melting points are in degree centigrade (uncorrected) and were determined on Gallenkamp

electric melting point apparatus. The IR spectra were recorded (KBr) on a Mattson 5000 FTIR

Spectrophotometer at the Microanalytical Unit at Faculty of Science, Mansoura University. The 1H-

NMR spectra were carried out on a Varian Spectrophotometer at 300 MHz, using TMS as an internal

reference and DMSO-d6 as solvent at the Microanalytical Center, Cairo University. The 1H-NMR

spectra were measured on a 500 MHz (JEOL). Chemical shifts are given in δ (ppm) relative to TMS as

internal standard material at National Research Center, Dokki, Cairo. The mass spectra were recorded

on Shimadzu Qp-2010 plus at the Microanalytical Center, Cairo University. Elemental analyses (C, H



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and N) were carried out at the Microanalytical Center of Cairo University, Egypt, and the results were

found to be in good agreement with the calculated values. Biological activity was carried at

Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Egypt.

2.2. Synthesis

Synthesis of N'-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-iminopropane-

hydrazonoyl cyanide (4(

A well stirred solution of 4-aminoantipyrine 1 (1.02 g, 5 mmol) in conc. HCl (3 mL) and 2 mL

H2O was cooled in ice-bath and diazotized with the solution of NaNO2 (0.35 g, 5.1 mmol in 5 mL

H2O). The cold diazonium solution was added slowly to a well stirred solution of 3 (0.41 g, 5 mmol) in

ethanol (20 mL) containing sodium acetate (1.64 g, 20 mmol). The reaction mixture was stirred for

another 2 h. The crude product was filtered off, dried well and recrystallized from ethanol/benzene

mixture to give 4. Yield (78%); Orange crystals; m.p. 218-220 oC; IR (KBr): ν/cm-1 = 3299, 3203

(2NH), 2190 (CN), 1646 (CO), 1612 (C=N), 1490 (N=N); 1H-NMR(DMSO-d6) δ (ppm): 2.23 (s, 3H,

CH3, pyrazole), 2.29 (s, 3H, CH3-C=N), 3.22 (s, 3H, CH3-N), 7.32-7.54 (m, 5H, Ar-H), 7.64 (br, 1H,

C=NH), 7.97 (br, 1H, NH, hydrazo); MS (EI, 70 ev) m/z (%) = 298 (M++2, 2.14), 297 (M++1, 8.91),

296 (M+, 1.1), 254 (5.08), 226 (0.53), 214 (0.55), 202 (9.17), 188 (1.02), 173 (0.78), 158 (1.02), 145

(0.76), 130 (0.69), 119 (5.19), 109 (1.66), 91 (10.75), 83 (43.0), 77 (10.26), 56 (100.0), 55 (8.14).

Anal. Calcd. for C15H16N6O (296.33): C, 60.80; H, 5.44; N, 28.36%. Found: C, 60.86; H, 5.49; N,

28.46%.

Synthesis of 4-((3-amino-5-methyl-1H-pyrazol-4-yl) diazenyl)-1, 5-dimethyl-2-phenyl-1H-pyrazol-

3(2H)-one (5)

To a solution of 4 (1.48 g, 5 mmol) in dioxane (20 mL) and hydrazine hydrate (0.25 g; 98%, 5

mmol) was added. After refluxing for 4 h, the reaction mixture was cooled then the formed precipitate

was filtered off, dried and recrystallized from DMF/EtOH to give 5. Yield (90%); orange powder; m.p.

288-290 oC; IR (KBr): ν/cm-1 = 3451, 3262, 3201 (NH2, NH), 1641 (CO), 1610 (C=N), 1484 (N=N);

1H-NMR(DMSO-d6) δ (ppm): 2.29 (s, 3H, CH3-antipyrine), 2.33 (s, 3H, CH3-pyrazole), 3.19 (s, 3H,

CH3-antipyrine), 6.08 (br, 2H, NH2), 7.35-7.56 (m, 6H, Ar-H, NH-pyrazole); MS (EI, 70 ev) m/z (%) =

312 (M++1, 7.07), 311 (M+, 37), 296 (2.05), 282 (0.17), 268 (0.3), 251 (0.13), 227 (0.17), 215 (1), 202

(1.1), 191 (1.75), 177 (0.99), 158 (0.74), 137 (0.7), 119 (5.47), 77 (9.15), 56 (100). Anal. Calcd. for

C15H17N7O (311.34): C, 57.87; H, 5.50; N, 31.49%. Found: C, 57.96; H, 5.57; N, 31.53%.

Synthesis of 4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yllazo)-5-methyl-pyrazol-3-

yl-hydrazones (8, 10, 12 and 14)



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General procedure: To a well stirred cooled solution of 5 (1.56 g, 5 mmol) in a mixture of

acetic acid (10 mL) and conc. HCl (3 mL), a solution of NaNO2 (0.4 g, 5.1 mmol in 5 mL H2O) was

added dropwise. The above cooled diazonium solution was added slowly to a well stirred solution of

active methylene compounds namely; 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 7 (0.87 g, 5 mmol) or

1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-5(4H)-one 9 (1.32 g, 5 mmol), 2-cyanoacetohydrazide 11

(0.495 g, 5 mmol), 3-amino-1H-pyrazol-5(4H)-one 13 (0.495 g, 5 mmol), in pyridine (15 mL). The

reaction mixture was stirred for 2 h. The crude product was filtered off, dried well and crystallized

from the appropriate solvent to give compounds 8, 10, 12 and 14, respectively.

4-(4-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)diazenyl)-5-methyl-1H-pyrazol-3-

yl) 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (8)

Yield (85.0%); yellow crystals; m.p. 253-255 oC; IR (KBr): ν/cm-1 = 3419,

3380(2NH),1658(br, 2CO),1494(N=N); 1H-NMR(DMSO-d6) δ (ppm): 2.39(s, 3H, CH3-pyrazolone),

2.56(s, 3H, CH3-anti-pyrine), 2.79(s, 3H, CH3, pyrazole), 3.32(s, 3H, CH3-N), 7.20-7.93(m, 10H,

ArH), 13.00(br, 1H, NH-pyrazole), 13.68 (br, 1H, NH, hydrazon); MS (EI, 70 ev) m/z (%) = 497 (M+,

19.42), 483 (22.47), 468 (18.30), 439 (19.90), (20.39), 379 (5.94), 372 (23.27), 361 (20.22), 341

(21.99), 328 (18.30), 311 (13.16), 309 (20.71), 296 (24.88), 282 (69.50), 266 (5.62), 251 (13.32), 231

(18.62), 214 (24.88), 197 (12.68), 187 (26.32), 177 (7.87), 161 (8.67), 140 (21.67), 132 (17.82), 119

(35.47), 105 (19.26), 91 (81.38), 77 (100.0), 56 (54.25). Anal. Calcd. for C25H24N10O2 (496.52): C,

60.47; H, 4.87; N, 28.21%. Found: C, 60.55; H, 4.90; N, 28.25 %.


yl) 1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-5(4H)-one (10)

Yield (83.0%); yellow crystals; m.p. 226-228 oC; IR (KBr): ν/cm-1 = 3313(br, 2NH), 1658(br,

2CO), 1513(2N=N); 1H-NMR(DMSO-d6) δ (ppm): 2.29(s, 3H, CH3-pyrazolone), 2.47(s, 3H, CH3-

antipyrine), 2.49(s, 3H, CH3-pyrazole), 3.2(s, 3H, CH3-N), 7.38-7.58(m, 10H, ArH, NH-pyrazole, NH-

hydrazone). MS (EI, 70 ev) m/z (%) = 585(M+-1, 65.48), 361 (70.24), 331 (75), 309 (23.81), 296

(100), 268 (64.29), 248 (66.07), 236 (15.48), 217 (72.02), 201 (76.79), 184 (78.57), 171 (9.52), 155

(62.50), 140 (66.07), 134 (30.36), 112 (7.14), 99 (20.83), 95 (33.33), 83 (86.90), 56 (81.55), 53

(23.81). Anal. Calcd. for C25H22N12O6 (486.52): C, 51.19; H, 3.78; N, 28.66%. Found: C, 51.31; H,

7.44; N, 28.89 %.


yl) 2-cyanoacetohydrazide (12)



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Yield (86.0%); brown crystals; m.p. 226-228 oC; IR (KBr): ν/cm-1 = 3411, 3313, 3250, 3197(

NH2, 2NH), 2220, 1658, 1641(2CO), 1488(2N=N); 1H-NMR(DMSO-d6) δ (ppm): 2.55(s, 3H, CH3-

antipyrine), 2.65(s, 3H, CH3-pyrazole), 3.37(s, 3H, CH3-N), 3.95 (br, 2H, NH2,), 7.38-7.59 (m, 5H,

ArH), 8.79 (br, 1H, NH, pyrazole), 10.35(br, 1H, NHCO), 10.75(br, 1H, NH, hydrazone). MS (EI, 70

ev) m/z (%) = 421 (M+, 6.71), 403 (1.58), 365 (7.11), 296 (1.45), 280 (7.11), 269 (2.63), 255 (7.83),

214 (31.58), 206 (7.30), 188 (45.66), 175 (17.24), 165 (7.30), 151 (7.24), 130 (5.46), 119 (19.08), 105

(25.46), 97 (100.00), 96(98.36), 77 (78.42), 68 (36.18), 53 (23.62). Anal. Calcd. for C18H19N11O2

(421.42): C, 51.30; H, 4.54; N,36.56%. Found: C, 51.44; H, 4.63; N, 36.89.

Synthesis of 6-((4-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)diazenyl)-5-methyl-

1H-pyrazol-3-yl)diazenyl)-3-methyl-1-phenyl-8,9a-dihydro-1H-dipyrazolo[1,5-a:4',3'-e]pyrimidin-

7(6H)-one (16)

A solution of compound 14 (1.05 g, 2.5 mmol) and 5-chloro-3-methyl-1-phenyl-1H-pyrazole-

4-carbaldehyde 15 (0.55 g, 2.5 mmol) in pyridine (20 mL) and DMF (10 mL) was refluxed for 24 h.

The reaction mixture was left to stand at room temperature overnight, and the separated was filtered

and crystallized from a mixture of DMF/ethanol to give compound 16. Yield, 66%, yellow powder,

mp: UP 300 oC; IR (KBr): νmax, cm-1: 3382 (NH), 1635 (br, C=O), 1444 (2N=N); 1H NMR (DMSO-d6)

δ (ppm): 2.46 (s, 3H, CH3, antipyrine), 2.62 (s, 3H, C8-CH3), 2.69 (s, 3H, CH3-pyrazole), 3.13 (s, 3H,

CH3-N, antipyrine), 6.9-7.4 (m, 10H, Ar-H), 7.94 (br, 1H, NH-pyrazole), 8.1 (s, 1H, CH=N), 8.2 (br,

1H, NH-pyrazolone), 8.87 (br, NH-hydrazone); MS (EI, 70 ev) (m/z, %): 372 (M+ -(azoantipyrine),

9.1), 358 (53.9), 341 (67.81), 332 (2.1), 311 (4.0), 298 (0.6), 282 (1.5), 279 (5.1), 259 (1.7), 244 (129),

236 (2.1), 221 (1.5), 210 (2. 9), 181 (6.9), 159 (2.5), 150 (3.4), 130 (6.5), 121 (18.8), 109 (6.0), 91

(24.8), 77 (100.00), 56 (2.3), 53 (1.8). Anal. Calcd. for C29H27N13O2 (589.61): C, 59.07; H, 4.62; N,

30.88%. Found: C, 59.11; H, 4.66; N, 30.91%

Synthesis of 4-((3-(2-(4-methoxy(chloro)phenyl)-4-oxothiazolidin-3-yl)-5-methyl-1H-pyrazol-4-yl)

diazenyl)-2,3-dimethyl-1-phenyl-1,2-dihydropyrazol-5-one (20 and 21)

A solution of compound 5 (1.5 g, 5 mmol) and 4-methoxybenzaldehyde 17 (0.68 g, 5 mmol), 4-

chlorobenzaldehyde 18 (0.7 g, 5 mmol) and 2-mercaptoacetic acid 19 (0.45 g, 5 mmol) in a mixture of

DMF (10 mL) and pyridine (2 mL) was refluxed for 24 h. The reaction mixture was left to stand

overnight at room temperature. The separated solid product was filtered off, dried and recrystallized

from a mixture of ethanol/benzene to give 20 and 21, respectively.

4-((3-(2-(4-methoxyphenyl)-4-oxothiazolidin-3-yl)-5-methyl-1H-pyrazol-4-yl)diazenyl)-2,3-dimethyl-

1-phenyl-1,2-dihydropyrazol-5-one (20)



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Yield, 73%, yellow crystals, mp: 124-126 oC; IR (KBr): νmax, cm-1: 3197 (NH), 1644 (2CO),

1506 (N=N).; 1H NMR (DMSO-d6) δ (ppm): 2.39 (s, 3H, CH3-antipyrine), 2.53 (s, 3H, CH3-pyrazole),

3.11 (s, 3H, CH3-N), ), 3.29 (br, 2H, CH2) 3.78-3.81 (m, 4H, CH,OCH3), 6.97-7.73 ( m, 9H, ArH), 9.4

(S, 1H, NH, pyrazole). MS (EI, 70 ev) (m/z, %): 503 (M+, 11.24), 455 (8.82), 443 (19.01), 429 (50.22),

415 (18.24), 401 (8.75), 361 (1.75), 337 (17.71), 335 (10.81), 311 (100.0), 295 (8.39), 292 (11.61), 269

(24.80), 253 (5.99), 251 (0.41), 228 (10.62), 213 (8.79), 203 (45.68), 190 (2.14), 98 (6.90), 84 (23.32),

58 (1.01). Anal. Calcd. for C25H25N7O3S (503.58): C, 59.63; H, 5.00; N, 19.47%. Found: C, 59.75; H,

5.09; N, 19.89%

4-((3-(2-(4-chlorophenyl)-4-oxothiazolidin-3-yl)-5-methyl-1h-pyrazol-4-yl)diazenyl)-2,3-dimethyl-1-

phenyl-1,2-dihydropyrazol-5-one (21)

Yield, 75%, Brown crystals, mp: 246-7oC; IR (KBr): νmax, cm-1: 3261 (NH), 1646 (br, 2CO),

1486 (N=N).; 1H NMR (DMSO-d6) δ (ppm): 2.41 (s, 3H, CH3-antipyrine), 2.62 (s, 3H, CH3-pyrazole),

3.15 (s, 3H, CH3-N), 3.37 (br, 2H, CH2) 3.78-3.81 (br, 1H, CH), 7.32-7.79 (m, 9H, ArH), 9.5 (s, 1H,

NH-pyrazole). MS (EI, 70 ev) (m/z, %) = 510 (M++2, 8.5) 508 (M+, 24.63), 508 (M+, 32.03), 379

(23.98), 368 (100.0), 351 (23.33), 338 (25.28), 322 (8.87), 313 (31.08), 311 (6.23), 297 (4.72), 296

(18.74), 288 (27.79), 263 (14.29), 249 (34.72), 229 (26.88), 213 (36.02),192 (22.11), 182 (14.89), 172

(44.11), 158 (13.38), 148 (20.82), 132 (39.74), 116 (4.03), 98 (77.84), 96 (26.23), 81 (43.64), 60

(36.93), 53 (17.49). Anal. Calcd. for C24H22ClN7O2S (508): C, 56.74; H, 4.37; N, 19.30%. Found: C,

56.91; H, 4.40; N, 19.41%.

1,5-dimethyl-4-((5-methyl-3-((2-oxo-2-phenylethyl)amino)-1H-pyrazol-4-yl)diazenyl)-2-phenyl-1H-

pyrazol-3(2H)-one (23)

A solution of 5 (1.56 g, 5 mmol) and 2-chloro-1-phenylethanone 22 (2.14 g, 5 mmol) in a

mixture of pyridine (20 mL) and DMF (10 mL) was refluxed for 3 h. The reaction mixture was left to

stand at room temperature overnight. The separated solid product was filtered off, dried and

crystallized from a mixture of ethanol/benzene to give 23.

Yield, 80%, Brown crystals, mp: 255-257 oC; IR (KBr): νmax, cm-1: 3350, 3203 (2NH), 1660

(br, 2CO), 1490 (N=N).; 1H NMR (DMSO-d6) δ (ppm): 2.46 (s, 3H, CH3-antipyrine), 2.62 (S, 3H,

CH3-pyrazole), 2.68 (s, 3H, CH2-C=O), 3.24 (S, 3H, CH3-N, antipyrine), 7.33-8.11 (m, 10H, ArH),

8.19 (br, 1H, NH), 9.12 (br, 1H, NH-pyrazole). MS (EI, 70 ev) (m/z, %): 430 (M++1, 12.65), 429 (M+,

36.65), 415 (2.15), 400 (1.42), 374 (0.60), 352 (13.31), 336 (1.34), 328 (9.03), 313 (4.68), 311 (0.63),

297 (1.10), 296 (2.14), 287 (3.66), 274 (9.59), 260 (33.16), 246 (2.67), 231 (4.56), 223 (2.06), 204

(1.54), 190 (4.34), 180 (1.07), 155 (2.39), 140 (1.68), 128 (12.4), 115 (2.58), 105 (94.30), 91 (2.79), 77



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(100.00), 56 (10.33). Anal. Calcd. for C23H23N7O2 (429.47): C, 64.32; H, 5.40; N, 22.83%. Found: C,

64.91; H, 5.44; N, 22.89%.

2,3-dimethyl-4-((6-methyl-3-phenyl-5,7a-dihydro-1H-imidazo[1,2-b]pyrazol-7-yl)diazenyl)-1-phenyl-

1,2-dihydropyrazol-5-one (25)

A solution o f 23 (0.86 g, 2 mmol) and phosphrousoxychloride (0.76 g, 5 mmol) in DMF (10

mL) was refluxed for 6 h .The reaction mixture was left to stand at room temperature overnight. The

separated solid product was filtered off, dried and recrystallized from a mixture of ethanol/benzene to

give 25.

Yield, 75%, yellow crystals, mp: 245-247 oC; IR (KBr): νmax, cm-1: 3220 (NH), 1648 (CO),

1620 ( C=N), 1497 (N=N ). 1H NMR (DMSO-d6) δ (ppm): 2.40 (s, 3H, CH3-antipyrine), 2.63 (s, 3H,

CH3-pyrazole), 3.24 (s, 3H, CH3-N), 3.77 (br, 1H, NH), 7.33-8.20 (m, 11H, ArH.). MS (EI, 70 ev)

(m/z, %): 411 (M+, 0.17), 367 (0.25), 341 (0.50), 299 (0.20), 284 (0.32), 279 (0.43), 256 (0.16), 242

(0.66), 227 (1.32), 213 (3.71), 199 (2.41), 185 (4.61), 171 (3.11), 157 (3.02), 149 (8.62), 129 (11.56),

121 (8.17), 98 (9.01), 95 (17.59), 81 (44.65), 69 (100.0), 55 (67.63), 50 (1.87). Anal. Calcd. for

C23H21N7O (411.46): C, 67.14; H, 5.14; N, 23.83%. Found: C, 67.91; H, 5.44; N, 23.89%.

Synthesis of (E)-1,5-dimethyl-4-((3-methyl-1-(2-oxo-2-phenylethyl)-5-((2-oxo-2-phenylethyl)amino)-

1H-pyrazol-4-yl)diazenyl)-2-phenyl-1H-pyrazol-3(2H)-one (24)

An equimolecular amounts of 5 (1.56 g, 5 mmol) and 2-chloro-1-phenylethanone 22 (1.54 g, 10

mmol) in DMF (30 mL) containing a catalytic amount of pyridine (1 mL) was refluxed for 5 h. The

reaction mixture was poured onto ice cold water (250 mL), filtered off, washed with petroleum ether

(60-80) and recrystallized from a mixture of ethanol/benzene to give compounds 24.

Yield, 75%, Brown crystals, mp: 160-162 oC; 1H-NMR (DMSO-d6) δ (ppm): 2.26 (s, 3H,

CH3-anti), 2.33 (s, 3H, CH3, pyrazole), 2.64 (2, 2H, CH2-NH), 3.16 (S, 3H, CH3-N) ), 3.28 (S, 2H,

CH2-N=), 7.04-8.14 (m, 15H, ArH), 8.22 (s, 1H, NH). MS (EI, 70 ev) (m/z, %): 549 (M++2, 62.20),

548 (M++1, 10.24), 547 (M+, 70.87), 542 (58.27), 521 (66.14), 511 (68.50), 494 (51.18), 480 (49.61),

461 (40.94), 445 (66.14), 418 (64.57), 394 (44.88), 390 (57.48), 377 (64.57), 362 (42.52), 338 (61.42),

334 (51.97), 311 (61.42), 307 (61.42), 301 (3.15), 290 (43.31), 282 (68.50), 273 (51.97), 253 (51.97),

245 (68.50), 213 (32.28), 206 (55.91), 192 (22.05), 176 (48.82), 156 (61.42), 151 (55.12), 138 (61.42),

121 (44.88), 100 (100.0), 88 (53.54), 72 (26.77), 65 (21.26), 54 (12.60). Anal. Calcd. for C31H29N7O3

(547.62): C, 67.99; H, 5.34; N, 17.90%. Found: C, 68.01; H, 5.54; N, 18.1%.

2.3. Antitumor Activity Assay



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Different concentrations of the tested compounds were prepared (ED100, ED50 and ED25 μg/

DMSO). The amount of DMSO was adjusted to give a final concentration of 0.1%. Ascites fluid was

obtained from the peritoneal cavity of the donor animal from National Cancer Institute, Cairo, Egypt,

which contain Ehrlich cell was as aseptically aspirated. The cells were grown partially floating and

attach in a suspension culture (RPMI 1660 medium, Sigma Chemical Co., St. Louis, USA),

supplemented with 10% foetal bovine serum (GIBCO, UK). They were maintained at 37 oC in

humidified atmosphere with 5% CO2 for 2 h. The viability of the cell used in control experiments

(DMSO only without drug) exceeded 95% as determined by microscopically examination using a

hemocytometer and trypan blue stain (stain only the dead cells).

2.4. Antioxidant Activity Assay

Antioxidant activity determinations were evaluated from the bleaching of ABTS derived

radical cations. The radical cation was derived from ABTS [2,2'-azino-bis (3-ethyl benzothiazoline-6-

sulfonic acid)] was prepared by reaction of ABTS (60 μL) with MnO2 (3 mL, 25 mg/mL) in phosphate

buffer solution (10 μM, pH 7, 5 mL) After shaking the solution for a few minutes, it was centrifuged

and filtered. The absorbance (A control) of the resulting green-blue solution (ABTS radical solution)

was recorded at λmax 734 nm. The absorbance (A test) was measured upon the addition of (20 μL of 1

mg/mL) solution of the tested sample in spectroscopic grade MeOH/buffer (1:1, v/v) to the ABTS

solution. The decrease in the absorbance is expressed as % inhibition which calculated from this

equation: % Inhibition= [A (control) – A (test)/A (control)] 100.

Ascorbic acid (20 μL, 2 mM) solution was used as standard antioxidant (positive control).

Blank sample was run using solvent without ABTS.

3. Results and Discussion

3.1. Synthesis

The synthetic strategies adopted to obtain the target compounds are depicted in Schemes 1-4. 4-

((3-Amino-5-methyl-1H-pyrazol-4-yl)diazenyl)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one 5 was

obtained according reported method [18] via coupling of 1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-

1H-pyrazole-4-diazonium chloride 2 with 3-iminobutanenitrile 3 in ethanol containing sodium acetate

followed by reaction of the formed N'-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-

imino-propanehydrazonoyl cyanide 4 with hydrazine hydrate in 1,4-dioxane. Diazotization of

aminopyrazole 5 with sodium nitrite in a mixture of acetic acid and hydrochloric acid afforded the

corresponding diaznium salt 6 which coupled with 3-methyl-1-phenyl-1H-pyrazol-5(4H)-one 7 [19] or



221

1-(2,4-dinitrophenyl)-3-methyl-1H-pyrazol-5(4H)-one 8 in pyridine afford the corresponding

hydrazones 9, 10 (Scheme 1).

Furthermore, coupling of 6 with 2-cyanoacetohydrazide 11, in pyridine gave the corresponding

hydrazone 12. Reaction of compound 12 under reflux in acetic acid gave the corresponding pyrazone

derivative 14. In another rout compound 14 was obtained via coupling of 6 with 3-amino-1H-pyrazol-

5(4H)-one 13 [18].

Reaction of compound 14 with 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 15

[20] gave the corresponding pyrazolopyrimidine derivative 16 (Scheme 2).

Scheme 1



222

Scheme 2

It was found that multicomponent reactions (MCRs) have attracted the attention of synthetic

organic chemists for building highly functionalized organic molecules and pharmacologically

important heterocyclic compounds [8], thus multicomponent reactionof 4-methoxybenzaldehyde 17 or

4-chlorobenzaldehyde 18 and 2-mercaptoacetic acid 19 with 5 in a mixture of pyridine/DMF afforded

4-((3-(2-(4-substituted)-4-oxothiazolidin-3-yl)-5-methyl-1H-pyrazol-4-yl)diazenyl)-2,3-dimethyl-1-

phenyl-1,2-dihydropyrazol-5-one 20 and 21, respectively (Scheme 3) [21].

Scheme 3

Treatment of 5 with one equivalent of 2-chloro-1-phenylethanone 5 in a mixture of

pyridine/DMF afforded the ethanone derivative 23, whereas with 22 two equivalent of 22 afforded the

bis ethanone derivative 24, two equivalent of cyclization of 23 under influence of phosphrousoxy

chloride afforded the imidazole derivative 25 (Scheme 4).



223

Scheme 4

3.2. Pharmacology

3.2.1. Antitumor Activity

Antipyrine derivatives were tested for cytotoxicity against a well known established model

EAC in vitro [22,23]. Results for the ED100, ED50, ED25 and IC50 values of the active compounds are

summarized in Table 1. The data showed clearly that most of compounds have good activities.

Compounds 12 and 16 have moderate activities, whereas compounds 9, 21, 23 and 25 have weak

activities. Thus, it would appear that introducing of pyrazole, hyrdrazo and pyrazolotriazine moieties

decrease the anti-tumor properties of N'-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-

2-iminopropanehydrazonoyl cyanide 4. By comparing the results obtained of the investigated

compounds to their structures the following structure activity relationships (SAR's) were postulated: (i)

antipyrine derivative 4 is more potent than 5-florouracil (5-Fu) which may be attributed to the

replacement of pyrimidine moiety with the antipyrine moiety, (ii) compound 5 is less potent than

compound 4 which may be due to conversion of the iminobutanenitrile moiety into pyrazole, (iii) most

of the hydrazo derivatives are more potent than the pyrazolotriazine which may be attributable to

presence of hydrazone moiety, and (iv) compound 20 is more potent than 21 which may be due to

presence of methoxy moiety.



224

3.2.2. Antioxidant Activity

The antioxidant activities of pyrazole derivatives were evaluated same as reported by Lissi et

al. [23]. The data showed clearly that compound 20 has good activity, while compounds 4 and 21

exhibited moderate activities. On the other hand, the other compounds showed weak activities (Table

2). Thus, it would appear that introducing of aminopyrazole moiety enhances the antioxidant properties

of aminoantipyrine ring system.

Table 1. Ehrlich ascites carcinoma cells (EAC) in vitro assay for the investigated compounds

Where, IC50 is the inhibitive concentration and ED100, ED50, and ED25 are the effective doses at 25, 50, and 100 μl,

respectively, of the compounds used. The dead % refers to the % of the dead tumor cells and 5-Fu is 5-fluorouracil as a

well known cytotoxic agent.

Table 2. Antioxidant activity assay for the investigated compounds

Compound No. ABTS

Absorbance of samples % Inhibition

Control of ABTS 0.507 0.0

Ascorbic acid 0.039 92.30

4 0.090 80.93

5 0.338 28.38

8 0.395 16.31

10 0.266 43.64

12 0.350 23.94

14 0.301 36.22

16 0.396 22.88

20 0.037 92.70

21 0.116 77.12

23 0.288 43.19

24 -- --

25 0.259 48.91

Compound No. IC50 μg ED100 µg ED50 µg ED25 µg

5-Fu 32.26 98.2 70.1 40

4 0.46 94.9 68.7 49.8

5 1.71 98.8 80 63.3

9 25.56 93 64.9 49.2

10 1.05 98.2 70.1 40

12 16.49 95.6 70 53.7

14 1.51 97.3 79.2 58.3

16 15.91 95 69.3 50

20 0.3 100 98.4 93

21 26.9 99 86 68.2

23 34.62 94 64 41.1

24 -- -- -- --

25 25.67 94 68 48.3



225

4. Conclusions

The objective of the present study was to synthesize and evaluate the antitumor and

antioxidant activities of some novel pyrazole direvatives with the hope of discovering new

structure leads serving as antitumor and antioxidant gents. The data showed clearly that most of

compounds have good antitumor activities and weak antioxidant activities, while compounds 4

and 20 exhibited broad spectrum of antitumor and antioxidant activities.

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