Synthesis, Tautomerism Study, Antimicrobial Evaluation and Cytotoxicity of Some New Bis(Arylazo)-Terpyrazoles

 

Farag Altalbawy^1,2*, Mohammed Alfadi¹

¹Department of Biological Sciences, University College of Duba, Tabuk University, Duba, KSA.

²National Institute of Laser Enhanced Sciences (NILES), Cairo University, Giza, Egypt.

 

*Email:[email protected]

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ABSTRACT

The chemistry of α-(arylhydrazono)-β-ketoaldehydes has recently received considerable attention and they are frequently employed as synthons in organic synthesis and their structural features are part of biologically active compounds. Thus, the present studies research describe the novel series synthesis of 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-arylhydrazono-3-oxo-propanal) 3a-i via coupling reactions of sodium 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxoprop-1-en-1-olate) (2) with arenediazonium chloride. Condensation of the compounds 3a-i with hydrazine hydrate afforded a new series of the bis(arylazo)-terpyrazole derivatives 5a-i as the end compounds. The newly prepared structures compounds were characterized as established on their spectroscopic spectra and rudimentary evauation. In addition, the spectral electronic absorption of compounds 5 was measured in various buffer solutions. The acidity constants pK's were calculated for the prepared series and their correlations versus Hammet substituent constants were investigated. The outcomes demonstraed that the title compound 5 exist in the bis-hydrazo form 5A. Producst 5a-i were screened in vitro for antibacterial and antitumor activity toward the HepG2 cell line.

Key words: Pyrazoles, Enaminones, Hydrazone, Tautomerism, Antimicrobial, Anti-tumor activities

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INTRODUCTION

Pyrazole and their ter-heterocycles have been presented with a considerable observation because of their broad spectrum of antitumor agents, kinase inhibitor applied in tumor therapeutics and anti-proliferative activities against many cancer cell lines [1-4]. Additionally, several heterocyclic compounds incorporating pyrazole moiety have a wide range of biological activities toward NOS inhibitors, antibacterial, monoamine oxidase inhibitors, antiamoebic and potent antimicrobial activity [5-10]. In addition, arylazo and arylhydrazo heterocycles are depicted in significant groups of commonly observed organic dyes. They are utilized in several of broad industrial uses like inkjet inks, dispersed dyes, laser products, reprographic technology and laser printing [11-13]. Thanks to their photodynamic therapy and laser non-linear optical properties (NLO) appropriate for several purposes including optical switching and molecular photoprobe, other arylazo dispersion dyes have also gained considerable interest [14-17]. β-Enaminones have been used as an important precursors for synthesis of heterocyclic systems [18, 19]. In an attempt to establish new precursors, the preparation of which is less expensive than β-enaminones and used in such heterocyclic synthesis, it was considered interesting to examine the synthesis and responces of type β-hydroxyenones, R-CO-CH=CHOH. Given the above literatures and continued our preceding efforts within the bioactive heterocyclic synthesis, bis-heterocyclic compounds [20-25] and our previous work to synthesize and study spectrophotometrically arylazo and bis-arylazo derivatives of heterocyclic dyes [26, 27] to surve their utility as photoprobes, and for hydrogen ion concentration sensing [28-30]. Herein, novel sodium 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxo-propenolate) reported the synthesis (2) and its reactions with hetaryldiazonium salts to yield bis-hydrazonal for the synthesis of bis(arylazo)-terpyrazoles and utility of the physical constrains approach for determining its pK's to elucidate the more best tautomeric form via correlation the acid dissociation constant with Hammett substituent's constants using Hammett equation. The nine synthesized dyes were tested for their antimicrobial and anti-cancer activities toward HepG2 cell lines.

MATERIALS AND METHODS

The melting point was measured with a Griffin melting point device. The IR- spectrum was measured with a Pye Unicam SP 2000 infrared spectrophotometer using the KBr wafer technology. The EI-MS spectrum was determined using the AE1MS 902 mass spectrometer. The mass spectrum was recorded by the GCMS-Q1000-EX Shimadzu and the GCMS 5988-A HP spectrometers, and the ionization voltage was 70 eV. ¹H-NMR (300MHz) was measured on a Varian Gemini instrument using a chemical shifts (δ) in terms of downfield ppm from TMS as an internal standard for deuterated dimethylsulphoxide (DMSO-d₆). Electronic absorption spectra were recorded on Perkin-Elmer Lambda 40 spectrophotometer. Elemental evalutions were conducted by the Microanalytical Center of Cairo University, Giza, Egypt.

Synthesis of sodium 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxoprop-1-en-1-olate) (2).

A mixture of 3,4-diacetylpyrazole derivative 1 (2.42 g, 10 mmol) and ethyl formate (1.48 g, 20 mmol) was fallen above a stirred dry ether (30 mL) solution containing MeONa (1.08 g, 20 mmol). The pure salt solid product obtained was filtrated, withered and used directly for further reactions.

Synthesis of 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxo-2-(2- arylhydrazono) -propanal) derivatives (3a-i)

General procedure.

3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxoprop-1-en-1-olate) (2) (0.342 g, 1 mmol) in C₂H₅OH (30 mL) and sodium acetate trihydrate (0.276g, 2 mmol). The solution of sodium was cooled in ice bath at 0-5 °C with stirring Salt by adding a properportion of diazonium salt to the cold solution and diazotizing the appropriate aniline derivative (2 mmol) in hydrochloric acid (6M, 2 mL) with sodium nitrite (0.14 g, 2 mmol) in H₂O (4 mL) was prepared. After adding all the diazonium salt solution, the mixture was stirred for an additional 30 min. while cooling in a ice-water bath. The solution was then left in the refrigerator for 3days. The separated precipitate was filterated, washed with H₂O, dried and recrysallized from the relevants solvent to provide the corresponding products 3a-i. The prepared compounds 3a-i are shown benearth along the physical constants.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(4-methoxyphenyl)-hydrazono-3-oxopropanal) (3a).

Yield (78%), mp 164-166 °C (EtOH); IR (KBr) ν_max: 3415 (NH), 3027, 2918 (C-H), 1725, 1688 (2C=O), 1025 (C-O-C) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.38 (s, 3H, CH₃), 3.73 (s, 6H, 2OCH₃), 7.26-7.89 (m, 13H, ArH), 10.33 (s, 2H, 2CHO), 12.81 (s, 2H, 2NH); MS m/z (%): 566 (M⁺, 1), 340 (18) 312 (32), 297 (48), 257 (100), 240 (30), 148 (40), 144 (21), 132, (10), 122, (15),116 (18), 103 (25), 91 (67), 76 (55), 51 (46). Anal. Calcd for C₃₀H₂₆N₆O₆ (566.56): C, 63.60; H, 4.63; N, 14.83. Found: C, 63.72; H, 4.50; N, 14.70%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(4-methylphenyl)hydrazono-3-oxopropanal) (3b).

Yield (82%), mp 152-154 °C (EtOH); IR (KBr) ν_max: 3410 (NH), 3019, 2935 (C-H), 1731, 1678 (2C=O), 1022 (C-O-C) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.22 (s, 3H, CH₃), 2.45 (s, 6H, 2CH₃), 7.22–7.75 (m, 13H, ArH), 10.35 (s, 2H, 2CHO), 12.87 (s, 2H, 2NH); MS m/z (%): 534 (M⁺, 2), 414 (32), 296 (38), 279 (48), 227 (100), 241 (42), 149 (38), 145 (27), 122, (17), 103 (32) , 91 (77), 76 (65), 63 (44). Anal. Calcd for C₃₀H₂₆N₆O₄ (534.56): C, 67.40; H, 4.90; N, 15.72. Found: C, 67.51; H, 4.78; N, 15.57%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(3-methylphenyl)hydrazono-3-oxopropanal) (3c).

Yield (72%), mp 147-149 °C (EtOH); IR (KBr) ν_max: 3398 (NH), 3036, 2965 (C-H), 1711, 1668 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.29 (s, 3H, CH₃), 2.66 (s, 6H, 2CH₃), 6.88–7.80 (m, 13H, ArH), 10.39 (s, 2H, 2CHO), 12.79 (s, 2H, 2NH); MS m/z (%): 534 (M⁺, 4), 414 (21), 295 (34), 281 (42), 226 (100), 240 (52), 149 (68), 147 (41), 122 (55), 116 (12), 103 (63) , 91 (80), 76 (66). Anal. Calcd for C₃₀H₂₆N₆O₄ (534.56): C, 67.40; H, 4.90; N, 15.72. Found: C, 67.61; H, 4.76; N, 15.63%

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxo-2-(2-phenylhydrazono)propanal) (3d). Yield 81%; mp 123-125 °C (EtOH) (Lit. mp 123-125 °C °C [31]).

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(4-chlorophenyl)hydrazono-3-oxopropanal) (3e).

Yield (86%), mp 193-195 °C (EtOH); IR (KBr) ν_max: 3378 (NH), 3030, 2965 (C-H), 1710, 1688 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.23 (s, 3H, CH₃), 6.58–7.56 (m, 13H, ArH), 10.32 (s, 2H, 2CHO), 12.86 (s, 2H, 2NH); MS m/z (%): 577 (M⁺+2, 2), 575 (M⁺, 5), 440 (33), 315 (24), 295 (56), 240 (100), 149 (28), 121(53), 114 (31), 105 (73), 91 (80), 76 (85), 51 (66). Anal. Calcd for C₂₈H₂₀C_l2N₆O₄ (575.40): C, 58.45; H, 3.50; N, 14.61. Found: C, 58.28; H, 3.35; N, 14.54%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(3-chlorophenyl)hydrazono-3-oxopropanal) (3f).

Yield (71%), mp 173-175 °C (EtOH); IR (KBr) ν_max: 3348 (NH), 3048, 2955 (C-H), 1711, 1668 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.29 (s, 3H, CH₃), 6.78–7.76 (m, 13H, ArH), 10.12 (s, 2H, 2CHO), 12.95 (s, 2H, 2NH); MS m/z (%): 577 (M⁺+2, 3), 575 (M⁺, 10), 440 (23), 315 (34), 295 (66), 241 (100), 149 (48), 132 (43), 121 (63), 115 (54), 105 (87), 91 (89), 76 (65), 51 (82). Anal. Calcd for C₂₈H₂₀C_l2N₆O₄ (575.40): C, 58.45; H, 3.50; N, 14.61. Found: C, 58.36; H, 4.39; N, 14.58%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(4-bromophenyl)hydrazono-3-oxopropanal) (3g).

Yield (80%), mp 184-186 °C (EtOH); IR (KBr) ν_max: 3410 (NH), 3041, 2965 (C-H), 1717, 1648 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.26 (s, 3H, CH₃), 7.04–7.96 (m, 13H, ArH), 10.07 (s, 2H, 2CHO), 12.82 (s, 2H, 2NH); MS m/z (%): 666 (M⁺+2, 5) 664 (M⁺, 6), 481(28), 402 (24), 387 (44), 295 (36), 242 (100), 149 (28), 132 (35), 115 (33), 105 (58), 91 (80), 76 (55), 51 (52). Anal. Calcd for C₂₈H₂₀Br₂N₆O₄ (664.30): C, 50.62; H, 3.03; N, 12.65. Found: C, 50.46; H, 3.00; N, 12.53%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(2-nitrophenyl)hydrazono-3-oxopropanal) (3h). Yield (68%), mp 204-206 °C (dioxane); IR (KBr) ν_max: 3401 (NH), 3037, 2975 (C-H), 1718, 1668 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.24 (s, 3H, CH₃), 7.24–8.06 (m, 13H, ArH), 10.12 (s, 2H, 2CHO), 12.90 (s, 2H, 2NH); MS m/z (%): 596 (M⁺, 2), 446 (68), 402 (45), 298 (32), 241 (100), 150 (38), 122 (47), 114 (36), 105 (47), 76 (85), 51 (42). Anal. Calcd for C₂₈H₂₀N₈O₈ (596.50): C, 56.38; H, 3.38; N, 18.78. Found: C, 56.27; H, 3.22; N, 18.55%.

3,3'-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(2-(4-nitrophenyl)hydrazono-3-oxopropanal) (3i).

Yield (64%), mp 264-266 °C (dioxane); IR (KBr) ν_max: 3431 (NH), 3035, 2955 (C-H), 1721, 1678 (2C=O), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.31 (s, 3H, CH₃), 7.34–8.26 (m, 13H, ArH), 10.28 (s, 2H, 2CHO), 13.2 (s, 2H, 2NH); MS m/z (%): 596 (M⁺, 7), 445 (77), 401 (52), 301 (22), 243 (100), 152 (39), 122, (36), 116 (22), 105 (51), 91 (66), 76 (54), 51 (81). Anal. Calcd for C₂₈H₂₀N₈O₈ (596.50): C, 56.38; H, 3.38; N, 18.78. Found: C, 56.52; H, 3.32; N, 18.69%.

Alternative synthesis of 3d

Carry out the same above coupling reaction using 1,1'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-(dimethyl -amino)prop-2-en-1-one) (4)  (0.352 g, 1 mmol) instead of 2. The precipitated product was filtrated, washed with H2O and lastly recrystallized from ethanol to provide a product proved identical in all respects (mp, mixed mp and IR spectra) with compound 3d, with those bought from coupling of 2 with PhN₂Cl but in 70% yield.

General method for synthesis of 5'-methyl-1'-phenyl-4,4''-bis(aryldiazenyl)- 1H,1'H,1''H-3,3':4',3''-terpyrazole derivatives 5a-i:

A mixture of the appropriate bis(2-arylhydrazono-3-oxo-propanal) derivatives 3a-i (1mmol) and hydrazine hydrate (2 mL) in dioxane (10 ml) was refluxed for 10-15 h. The formed solid after cooling the solution was filtered off and crtstallized from the proper solvent to give the corresponding ter-pyrazoles 5a-i. The products obtained are described below along with their analytical data.

4,4''-Bis((4-methoxyphenyl)diazenyl)-5'-methyl-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5a).

Yield (72%), mp 292-294 °C (DMF); IR (KBr) ν_max: 3419 (NH), 3025, 2918 (C-H), 1695 (C=N), 1024 (C-O-C) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.75 (s, 3H, CH₃), 3.71 (s, 6H, 2OCH₃), 7.46-7.89 (m, 15H, ArH and pyrazole-H), 10.24 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 558 (M⁺, 2), 412 (32), 380 (15) 292 (41), 226 (22), 152 (48), 137 (24), 103 (100), 91 (65), 76 (75), 51 (90). Anal. Calcd for C₃₀H₂₆N₁₀O₂ (558.59): C, 64.51; H, 4.69; N, 25.08. Found: C, 64.37; H, 4.53; N, 25.00%.

5'-Methyl-1'-phenyl-4,4''-bis(p-tolyldiazenyl)-1H,1'H,1''H-3,3':4',3''-terpyrazole (5b).

Yield (68%), mp 280-282 °C (dioxane); IR (KBr) ν_max: 3415 (NH), 3028, 2948 (C-H), 1690 (C=N), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.35 (s, 3H, CH₃), 2.41 (s, 6H, 2CH₃), 7.32-7.72 (m, 15H, ArH and pyrazole-H), 10.17 (s, 2H, D₂O-exchangeable, 2NH). MS m/z (%): 526 (M⁺, 4), 397 (21), 382 (42), 291(35), 225 (18), 159 (47), 116 (18), 103 (100), 91 (48), 76(59), 63 (38), 51 (67). Anal. Calcd for C₃₀H₂₆N₁₀ (526.59): C, 68.42; H, 4.98; N, 26.60. Found: C, 68.59; H, 5.10; N, 26.87%.

5'-Methyl-1'-phenyl-4,4''-bis(m-tolyldiazenyl)-1H,1'H,1''H-3,3':4',3''-terpyrazole (5c).

Yield (62%), mp 275-277 °C (DMF); IR (KBr) ν_max: 3412 (NH), 3025, 2966 (C-H), 1687 (C=N), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.34 (s, 3H, CH3), 2.39 (s, 6H, 2CH₃), 6.88-7.59 (m, 15H, ArH and pyrazole-H), 10.02 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 526 (M⁺, 8), 395 (34), 380 (22), 290 (44), 224 (38), 158 (77), 115 (48), 101 (100), 91 (42), 76 (61), 51 (68). Anal. Calcd for C₃₀H₂₆N₁₀ (526.59): C, 68.42; H, 4.98; N, 26.60. Found: C, 68.37; H, 5.03; N, 26.46%.

5'-Methyl-1'-phenyl-4,4''-bis(phenyldiazenyl)-1H,1'H,1''H-3,3':4',3''-terpyrazole (5d).

Yield 76%;  mp > 300 °C (DMF) (Lit. mp > 300 °C °C [31]).

4,4''-Bis((4-chlorophenyl)diazenyl)-5'-methyl-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5e).

Yield (73%), mp >300 °C (DMF); IR (KBr) ν_max: 3426 (NH), 3028, 2958 (C-H), 1665 (C=N), cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.29 (s, 3H, CH₃), 6.92-7.78 (m, 15H, ArH and pyrazole-H), 10.11 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 569 (M⁺+2, 4), 567 (M⁺, 14), 416 (21), 292 (35), 224 (36), 158 (42), 142 (23), 124 (25), 116 (48), 103 (100) , 91 (66), 76 (57), 51 (33). Anal. Calcd for C₂₈H₂₀C_l2N₁₀ (567.43): C, 59.27; H, 3.55; N, 24.68. Found: C, 59.14; H, 3.42; N, 24.51%.

4,4''-Bis((3-chlorophenyl)diazenyl)-5'-methyl-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5f).

Yield (71%), mp > 300 °C (DMF); IR (KBr) ν_max: 3432 (NH), 3046, 2972 (C-H), 1685 (C=N) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.31 (s, 3H, CH₃), 6.96-7.73 (m, 15H, ArH and pyrazole-H), 10.18 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 569 (M⁺+2, 5), 567 (M⁺, 17), 415 (22), 290 (45), 224 (25), 159 (65), 141 (53), 131 (25), 124 (44), 116 (40), 101 (100), 91 (34), 77 (87), 51 (52). Anal. Calcd for C₂₈H₂₀C_l2N₁₀ (567.43): C, 59.27; H, 3.55; N, 24.68. Found: C, 59.39; H, 3.47; N, 24.55%.

4,4''-Bis((4-bromophenyl)diazenyl)-5'-methyl-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5g).

Yield (76%), mp > 300 °C (DMF); IR (KBr) ν_max: 3443 (NH), 3029, 2978 (C-H), 1680 (C=N) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.34 (s, 3H, CH₃), 6.86-7.82 (m, 15H, ArH and pyrazole-H), 10.31 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 658 (M⁺+2, 6), 656 (M⁺, 7), 464 (34), 294 (28), 226 (54), 160 (39), 141 (18), 133 (15), 126 (26), 114 (41), 105 (100), 91 (62), 76 (54), 51 (39). Anal. Calcd for C₂₈H₂₀Br₂N₁₀ (656.33): C, 51.24; H, 3.07; N, 21.34. Found: C, 51.32; H, 3.05; N, 21.31%.

5'-Methyl-4,4''-bis(2-nitrophenyl)diazenyl)-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5h).

Yield (70%), mp > 300 °C (DMF); IR (KBr) ν_max: 3448 (NH), 3031, 2974 (C-H), 1685 (C=N) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.33 (s, 3H, CH₃), 6.96-7.79 (m, 15H, ArH and pyrazole-H), 10.11 (s, 2H, D₂O-exchangeable, 2NH); MS m/z (%): 588 (M⁺, 5), 440 (45) 291 (34), 238 (31), 223 (45) 158 (41), 144 (22), 103 (100), 91 (77), 76 (52), 51 (57). Anal. Calcd for C₂₈H₂₀N₁₂O₄ (588.53): C, 57.14; H, 3.43; N, 28.56. Found: C, 57.09; H, 3.30; N, 28.51%.

5'-Methyl-4,4''-bis(4-nitrophenyl)diazenyl)-1'-phenyl-1H,1'H,1''H-3,3':4',3''-terpyrazole (5i).

Yield (77%), mp > 300 °C (DMF); IR (KBr) ν_max: 3452 (NH), 3032, 2978 (C-H), 1688 (C=N) cm⁻¹; ¹H NMR (DMSO-d₆): δ 2.36 (s, 3H, CH₃), 7.15-7.98 (m, 15H, ArH and pyrazole-H), 10.26 (s, 2H, D₂O-exchangeable, 2NH). MS m/z (%): 588 (M⁺, 10), 442 (48) 290 (65), 237 (38), 224 (49) 156 (48), 101 (100), 92 (76), 76 (22), 51 (71). Anal. Calcd for C₂₈H₂₀N₁₂O₄ (588.53): C, 57.14; H, 3.43; N, 28.56. Found: C, 57.22; H, 3.37; N, 28.47%.

Prepared pK's Determination of bis(arylazo)-terpyrazole derivatives 5a-i

The rial methods go along with the analysis of the dissociation constants pKa and their estimates are based on the electronic absorbance-pH data as reported earlier in the literature [32, 33]. 

Antimicrobial evaluation

In this analysis, the Gram-negative bacteria PA and Gram +ve bacteria SP and ECand BS, and the fungal AF, SR, GC and CA strains were used for examining the prepared compounds for their antimicrobial and fungicde activities. The selected species were obtained from the regional center for biotechnology and mycology of Al-Azhar University, Cairo, Egypt [34-37].

Control antibacterial and antifungal

Control antibacterial as Ampicillin and Gentamicin and antifungal as Amphotericin B were purchased from Sigma-Aldrich (St Louis, MO, USA) and used as a comparable references

Anti-Tumor evaluation

The human liver cancer (HepG-2) cell line was acquired from the American Type Culture Collection (ATCC, Rockville, Marylan). Cells were cultured in RPMI-1640 medium and added with 10% inactivated fetal bovin serum and 50µg/mL gentamicin. The cells were maintained at 37°C in a humidified atmosphere 0f 5% CO₂ and were sub-cultured 2 to 3 times a week.

In the anti-cacer assays, the tumor cell lines were suspended in the medium at a concentration of 5x10⁴ cell/well on corning 96-well corning plates (6 repeats) achieving 8 concentrations for every compound. Six vehicle controls using medium or 0.5% DMSO were performed on every 96 well plates as controls. After 24 hours of incubating, the amount live cells was measured by MTT assay. Concisely, the medium is removed from the 96 well plates and replaced with 100 µL fresh RPMI 1640 medium without phenol red, followed by 10 µL, 12 µL MTT stock (5 mg of MTT in 1 mL of PBS) in each well comprising unprocessed controls. The 96-well plates was then incubated at 37°c and 5% CO₂ for 4 hours. An 85µL of the medium was removed from the wells, and 50µL of DMSO was added to every well mixed thoroughly with a pipette, and incubated at 37°c for 10 minutes. Subsequently, amicroplate reader (Sunrise, TECAN, Inc, USA) was used to measure the optical density at 590nm, the live cells count was measured, and the viability was (1-(ODt/ODc)) x 100% calculated as ODt is the average optical density of untreated cells. Plot the relationship between drug concentration and viable cells to acquire the stable curve for every tumor cell line after treatment with the indicated compound. The 50% inhibitory concentration (IC₅₀), which necessary to produce a toxic effects in 50% of intact cells, is a dose-response curve plot for every concentration with Graphed Prism software (San Diego, CA. USA) was decided from [38-41].

RESULTS AND DISCUSSION

Chemistry

The starting sodium 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-oxoprop-1- en-1-olate) (2) was prepared via a reaction of 1-phenyl-3,4-diacetyl-5-methylpyrazole (1) [11] with two equivalents of ethyl formate in etheric CH₃NaO solution. The sodium reactivity (pyrazole-diyl)bis(3-oxopropenolate) derivative 2 was allowed to react with arenediazonium salts to synthesize new series of bis-arylhydrazone derivatives carrying pyrazole moiety. Thus, couplling compound 2 with two moles of each of arenediazonium chlorides in ethanolic sodium acetate trihydrate afforded the corresponding bis-arylhydrazone coupling products 3a-i in decent to perfect yields (64-86%) (Figure 1).

The compound 3a-i structure was established based on spectral data and micro-analytical analysis (Ms, IR and ¹H NMR). The Ms spectra of all compounds 3 revealed, in each case, a respective molecular ion peak at the correct formula molecular weight. The infrared absorption spectra of hydrazones 3a-i showed in each derivative one band at 3,348-3,431 cm^-1 for NH stretching absorptions and two strong absorption bands observed at 1,648-1668 and 1,710-1,731 cm^-1 for the two stretching vibrations C=O groups. Their spectrum of ¹H NMR revealed in each case the expected singlet and multiplieds signals of the methyl and aromatic protons at δ 2.22-2.38 and 6.58-8.26 ppm, respectively. In addition these bands also showed two singlet bands one of them in the regions δ 10.02-10.39 assignable to -CHO proton and the other singlet in the region δ 12.79-13.2 ppm, due to hydrazone NH group. These δ values agree with those of the Z-isomers of (Ar₁COC(CHO)=NNHAr₂) which were previously reported in the literature to show their aldehydic proton signals in the region δ 9.50-9.63 and hydrazone proton band in the region δ 11.85-12.78 ppm [33, 42]. The ¹H-NMR spectrum of the E-isomers, as outlined in Figure 2 of the 3-aryl-3-oxo-2-arylhydrazonopropanals revealed the two singlet signals at δ 9.96-10.17 and δ 13.9-14.35 ppm for their aldehydic and hydrazone protons, respectively [31, 42]. This indicated that product 3 is present only in the Z-isomers (Figure 2).

In addition, to provide conclusive evidence of the assigned structure 3a-i, prepared by the condensation reaction of bisenaminose 4 [43] (compound 1 and 2 equivalents of dimethylformamide dimethylacetal (DMF-DMA) with benzene diazonium chloride in ethanol) coupling reaction was performed. the sodium acetate trihydrate presence  yielded a product that was found to be comparable to 3a at all points (IR, MS, mp. and mixed mp.) (Figure 1).

 

Figure 1.  Synthesis of bis-arylhydrazonopropanal derivatives 3a-i

 

Figure 2. Isomers of bis-arylhydrazonopyrazolederivatives 3a-i

 

Next, refluxing of the bis-arylhydrazone derivatives 3a-i with hydrazine hydrate in dioxane yielded the corresponding single products, 5a-i (as confirmed by the TLC spectrum of the crude products), as depicted in Figure 3. The assigned corrected structure of 5'-methyl-1'-phenyl-4,4''-bis(aryldiazenyl)-1H,1'H,1''H-3,3':4',3''- terpyrazole derivatives 5a-i were elucidated by their elemental analyses, ¹HNMR, infrared absorptions and mass fragmentations spectral data. The IR absorption bands of hydrazone derivatives 5a-i (See Experimental section) exhibited an absorption peak in each derivative in the ranges ν_max 3412-3452 cm^-1 due to the stretching hydrazone NH group in addition to the two expected peaks of CH aliphatic and C=N stretching. The mass fragmentation spectra of all terpyrazole 5a-i showed the molecular ion peak (M⁺) of each derivative with the correct formula weight (M⁺) for derivative (See Experimental section). The resulting spectroscopic analysis persistent with the designated structurales formula 5a-i, but differentiates between the two probable tautomeric types is incapable, the bis-arylhydrazone and bis-arylazo types 5A an 5B, respectively. UV-V spectrum was measured at various pH solutions To confirm the exact tautomeric structure of product 5.

Figure 3.  Synthesis of bis-arylazo-terpyrazole derivatives 5a-i.

 

Determination of pKa's and actual tautomeric form of the products 5a-i

The UV-Vis absorption spectra of terpyrazole derivatives 5a-i in ethanol outlined, in every scenario, two traits peaks, one in the region of 414-395 nm and the second absorption within the 337-313 nm range (Table 1). These UV-Vis data are typical of those of arylhydrazone chromophore [22, 26]. So it can be concluded that the studied dyes 5 are present in solution as one tautomeric form, namely the bis-arylhydrazone tautomeric form 5A.

To dispense additional evidence 0f the assignment of arylhydrazone 5A to studied products 5a-i, the pKa constants of the prepared series were calculated spectrophotometrically. A typical UV-Vis absorption spectrum of 5d buffers of various pH values is indicatwd (Figure 4). From the absorbance-pH spectra, the value of the acid dissociation constants pKa was determined [44, 45] using equation (1):

pK=pHi+log(Ab-Ai )/(  Ai-Aa )

(1)

 

Where A_i is the absorbance of the test solution at pH_i, and A_b and A_a are the absorbance values of a strongly base and acidic solution [40]. The dissociation acidity constants pKa values calculated for the prepared products 5a-i are outlined in (Table 1). Equations 2 and 3 comparable to the straight lines acquired are:

pKa=9.90-1.4 σx ,r2 = 0.94, s = ± 0.09)

(2)

 

pKa=9.91-1.4 σx- , r2 = 0.98, s = ± 0.07)

(3)

Where r and s are the correlation coefficient and standard deviation, respectively. The pKa data of compounds 5a-i from the values of r and s seem to be preferable associated with the enhanced Hammett substituent constant s^- rather than the Hammett substituent constant s are shown in (Figure 5). Such excellent correlations with Hammett substituent constant indicate that the studied compounds 5a-i exists in the bis-hydrazone Tautomeric form 5A in solution. This is because if the prepared compounds 5 exist as equilibrium mixture of 5A and 5B, as outlined in (Figure 3), no linear relationship is observed between pKa and s^-x. In addition, the reaction rate  r = 1.09 appears to indulge the bis-hydrazone form 5A as it is in decentagreement with previously described values for comparable bis-hydrazones rather than  bis-arylazo derivatives [32, 46].

(a)	(b)
Figure 4. (a) Electronic Absorption Spectra of compound 5d, in solution of different pH values (1:4 (v/v) dioxane-water) at 27 0C and μ=0.10.; (b) Correlation of log(Ab - Ai)/(Ai - Aa) at two maximum electronic absorption wave lengths with pH values of compound 5d.

Table 1. UV/Vis Spectra and acid dissociation constants pK of bis-arylhydrazonopyrazole derivatives 5a-i.

Compound No.	λmax nm (EtOH) (log ε)	σX	σ-X	pK	λmaxa	λmaxb	Δν cm-1	pK*
4a	401(4.12), 321(4.22)	−0.27	−0.27	10.29	393	475	4392	1.06
4b	397(4.06), 325(4.15)	−0.17	−0.17	10.15	392	469	4188	1.35
4c	395(4.00), 323(4.25)	−0.07	−0.07	9.90	388	467	4359	0.78
4d	392(4.15),313(4.10)	0.00	0.00	9.85	390	468	4273	0.87
4e	400(4.42), 325(4.40)	0.23	0.23	9.75	396	479	4375	0.56
4f	406(4.10), 337(4.12)	0.37	0.37	9.49	392	470	4233	0.60
Dg	409(4.40),330(4.45)	0.71	0.71	9.03	400	480	4166	0.28
4h	402(4.41), 335(4.33)	0.5	0.84	9.04	400	483	4296	0.02
4i	414(3.39), 337(3.54)	0.78	1.28	8.56	400	485	4381	-0.64

Note. ^a In acid medium; ^b in alkaline medium; ± s = 0.05.

 

Next, the acidity constants in excited state pK*'s of the studied compounds 5a-i were determined using the Forester energy cycle [28, 29]. According to this energy cycle the equation 4:

pK*=pK+(Dn/  0.625/T )

(4)

Here, pK and pK* are the acid dissociation constants in the ground state and the excited states, respectively and Dn the frequency difference in cm^-1 units between the maximum l_max absorption values. Any compound in acidic and alkaline media. The computations outcomes of are shown in (Table 1). The interrelation between these pK* data and sx is shown in (Figure 5). The linear equations 5 and 6 equivalent to such interrelations are:

pK*= 0.87-1.40 σx ,r2= 0.81, s = ± 0.28

(5)

 

pK*= 0.90-1.10 σx-,  r2 = 0.93, s = ± 0.17

(6)

Such linear equations indicate that studied series 5a-i has also predominantly the bis-hydrazone tautomeric form in their excited states [47, 48].

a)	b)
Figure 5. Correlation of pKa bis-arylhydrazonopyrazole derivatives 5a-i (a) with Hammett substituent constant, sX. (b) with enhanced Hammett substituent constant, s-X.

Antimicrobial evaluation

In recent study, the title-synthesized terpyrazole derivatives 5a-i were assessed for in vitro antimicrobial activity against Gram -ve bacteria such as E coli (EC), Pseudomonas aeruginosa (PA) and Gram-positive bacteria eg. Bacillis subtilis (BS), Streptococcus pneumoniae (SP) and for their in vitro fungicid activity towards Syncephalastrum racemosum (SR), Aspergillus fumigatus (AF), Candida albicans (CA), and Geotricum candidum (GC) fungal strains. Under similar conditions, the bactericides Gentamicin and Ampicillin and the antifungal Amphotericin B wer utilized as guide lines to determine the effectiveness of the compounds evaluated. The diameter of the inhibition zone (IZD) was used as the criterion for the antibacterial activity using diffusion technology (Table 2).

 

Table 2. Antimicrobial activities of the synthesized bis(arylazo)-terpyrazoless (5a-i).

Comp.	Inhibition zone diameter (cm)
	Gram (+)		Gram (−)		Fungi
Standard drugs	(SP)	(BS)	(PA)	(EC)	(AF)	(SR)	(GC)	(CA)
	23.8 ± 0.2	32.4 ± 0.3	17.3 ± 0.1	19.9 ± 0.3	23.7 ± 0.2	19.7 ± 0.2	28.7 ± 0.2	25.4 ± 0.1
5a	14.3 ± 0.3	14.9 ± 0.3	NA	NA	15.2± 0.5	11.3 ± 0.3	11.7 ± 0.5	NA
5b	12.1 ± 0.4	16.3 ± 0.3	NA	NA	16.4± 0.3	11.6 ± 0.3	12.0 ± 0.4	NA
5c	12.4 ± 0.3	15.2 ± 0.4	NA	NA	15.7 ± 0.3	12.1 ± 0.2	12.3 ± 0.3	NA
5d	15.3 ± 0.3	17.6 ± 0.4	10.1 ± 0.3	8.2 ± 0.2	15.6 ± 0.6	12.7 ± 0.3	11.4 ± 0.4	13.6 ± 0.6
5e	19.1 ± 0.4	22.8 ± 0.3	13.1 ± 0.4	20.3 ± 0.1	20.2 ± 0.6	16.4 ± 0.6	22.4 ± 0.6	17.9 ± 0.4
5f	17.8 ± 0.5	20.4 ± 0.3	12.1 ± 0.3	19.1 ± 0.1	17.3 ± 0.4	13.2 ± 0.3	19.0 ± 0.6	17.3 ± 0.4
5g	18.7 ± 0.6	20.7 ± 0.4	14.1 ± 0.4	17.3 ± 0.1	18.9 ± 0.6	15.4 ± 0.3	20.7 ± 0.3	19.4 ± 0.3
5h	14.8 ± 0.6	15.2 ± 0.7	10.3 ± 0.4	14.6 ± 0.3	14.1 ± 0.5	14.0 ± 0.4	13.7 ± 0.5	13.7 ± 0.5
5i	16.9 ± 0.4	19.8 ± 0.6	11.1 ± 0.3	15.7 ± 0.3	15.3 ± 0.6	14.4 ± 0.4	19.5 ± 0.4	16.2 ± 0.3

Note. Data are expressed in the form of mean ± SD. Mean zone of inhibition in mm ± standard deviation beyond well diameter; (6 mm) produced on a range of environmental and clinically pathogenic microorganism using (5 mg/mL) concentration of tested sample (100 µL was tested).

 

The outcomes summarized in (Table 2) indicated: SP and BS are sensitive to all examined compounds 5a-i; furthermore, EC and PA are sensitive to compounds 5d-i. All tested compounds except compounds 5a-c exhibited antifungal activity against the four tested fungal strains SR, AF, CA and GC. Whereas, compound 5a-c showed no the antifungal activity against the one fungal species CA. The high activity of all compounds tested is due to the pharmacological active presence of ter-pyrazole moietist in all compounds 5a-i. The inactivity of the ter-pyrazole derivatives 5a-c towards the tested CA and Gram-negative bacteria is caused by the presence ofelectron-donor groups.

Anti-tumoractivity

The antitumor activity of the newly synthesized hydrazones compounds 5a-i was decided toward the liver cancer cell line HEPG2, using doxorubicin as a reference drug. The data created was used to plot a dose-response curve in which the concentration of the test compounds needed to kill 50% of the cell population (µM) was determined. The cytotoxic activity was presented as the mean IC₅₀ of three independent experiments (Table 3), and the outcomes indicated that:

Table 3. The in vitro inhibitory activity of tested compounds expressed as IC₅₀ values (μM) ± standard deviation from five replicates against HEPG-2 cell line.

Compound No.	Ar	IC50 (mM)	Compound No.	Ar	IC50 (mM)
5a	4-MeOC6H4	11.7± 0.19	5f	3-ClC6H4	6.32 ± 0.24
5b	4-MeC6H4	12.48± 0.26	5g	4-BrC6H4	2.15 ± 0.12
5c	3-MeC6H4	14.34± 0.21	5h	2-NO2C6H4	4.4 ± 0.15
5d	C6H5	14.45± 0.18	5i	4-NO2C6H4	2.6 ± 0.09
5e	4-ClC6H4	4.35 ± 0.16	Doxorubicin	-	± 0.18

All compounds tested showed concentration-dependent inhibitory activity to the cancer cell lines. The lower IC₅₀ values of the chosen compounds revealed that, higher concentrations can be used for stronger antitumor activity. The outcomes are shown in (Table 3) and indicate that: Shier et al. In vitro inhibitory activity of test [49]. Compound 5e and compounds 5g-i have strong in vitro inhibitory activity, 5f is moderate while other compounds 5a-d are weak against the (HepG2) cell lines, and have the following order: 5g > 5i >5e >5h> 5f > 5a > 5b > 5c > 5d. this indication of the substituents at the position phenyl group in arylhydrazo moiety affects the in vitro inhibitory activity. The electron-withdrawing introduction group (nitro group > bromine atom > chlorine atom) at the 4-position of the phenyl group. In contrast, the introduction of the electron-donating group (methoxy group > methyl group) reduces the anticancer activity

CONCLUSION

In summary, the new bis(arylazo)-terpyrazole derivatives 5a-i were synthesized via simple coupling reaction of sodium 3,3'-(5-methyl-1-phenyl-1H-pyrazole-3,4-diyl) bis(3-oxoprop-1-en-1-olate) (2) with arenediazonium salt, followed by condensation of the products 3a-i with hydrazine hydrate. The newly synthesized compounds structures were confirmed by tools spectrum and also from the calculation of the pKa to determine the best tautomeric form. The in-vitro anti-bacterial potential of the final synthesized compounds 5a-i was determined to achieve their antimicrobial potential.

ACKNOWLEDGMENTS : This work was supported by the Tabuk University Foundation of Saudi Arabia (Grant no. S-1440-0191).

CONFLICT OF INTEREST : None

FINANCIAL SUPPORT : None

ETHICS STATEMENT : None