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Journal of Drug Delivery and Therapeutics

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Open Access Full Text Article                                                                                                Research Article

Structural Establishment and Biological Evaluation of Newly Synthesized Nitrogen Containing Hetrocyclic Compound of Pyridazinone and its Derivatives

Shailendra Kumar Verma¹*, Anshika Choudhary¹, Yashpal Singla²

1. BIU College of Pharmacy, Bareilly International University, Bareilly (U.P), India
2. SunRise University, Alwar (Rajasthan), India

Email id: sshail2006@gmail.com  

INTRODUCTION

Pyridazinones have been reported to possess a variety of biological activities like antinociceptive¹, anticancer², anticonvulsant³, antithrombotic⁴, and antiinflammatory^5-6 activity. In present work we reported synthesis of some pyridazinone derivatives and their anticonvulsant activity which is not done earlier and is quite significant. Reaction between benzimidazole and succinic anhydride in presence of hydrofluoric acid produced 4-(1-H-benzo[d] imidazole-5-yl)-4-oxo-butanoic acid (MA). The product obtained from step-I reactes with hydrazine hydrate to produce 6-(1-H-benzo[d] imidazole-5-yl)-2,3,4,5-tetrahydro Pyridazin-3-one (MB). The product obtained from step-II then react with different aromatic benzaldehydes in presence of acetic anhydride gives different derivatives (M-1 to M-6)                    

MATERIALS AND METHODS

Chemistry

Melting points were determined by open capillary tube method in liquid paraffin bath and were uncorrected. Purity of the compounds was checked on pre-coated silica- gel G plate using Toluene: Ethyl acetate: Formic acid (5:4:1) as solvent system. Infrared (IR) spectra (KBr disc) were recorded on FT-IR Perkin Elmer. ¹H- nuclear magnetic resonance (¹H-NMR) spectra were recorded in DMSO using Bruker Avance-II 400 MHz NMR spectrophotometer. The chemical shifts were expressed in terms of δ (ppm) downfield from TMS and the mass spectra were recorded on Jeol Sx-102  (FAB) Mass Spectrometer

Synthetic Study

Synthesis of 4-(1-H-benzo[d] imidazole-5-yl) -4oxo-butanoic acid (MA) A mixture of benzimidazole (0.10 mol) and hydrofluoric acid (40 ml) was taken in a three neck flask  and refluxed on a water bath under anhydrous condition followed by addition of succinic anhydride (0.10 mol) in small quantities with continuous stirring. Stirring and heating were continued for 4 hrs and the content after leaving overnight at room temperature were poured into ice cold hydrochloric acid (2.5% v/v) followed by steam distillation. The aqueous solution was then concentrated to a small volume by evaporating on a water bath to obtain the crude compound. It was purified by dissolving in 5% w/v sodium bicarbonate solution and filtered, filtrate on acidification with dilute hydrochloric acid yielded MA. It was recrystallized from aq. ethanol.                                                                             

Yield: 70% ; m.p. 95-96⁰C ; R_f  0.65. Spectroscopic analysis: IR (cm^-1): 3408 (Ar.NH), 1708 (C=N), 1640 (C=O); ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36(1H,s), 12.05(1H,s), 8.19(1H,s), 7.52-8.00(4H,m), 6.61(1H,s).

Synthesis of 6-(1-H-benzo[d] imidazole-5-yl)-2,3,4,5-tetrahydro pyridazine-3-one (MB) alcoholic solution of MA (0.10 mol) was refluxed for 12 hrs with hydrazine hydrate (1 ml) in ethanol (25 ml). The reaction mixture was concentrated and then poured into ice cold water to obtain compound MB,  which was crystallized from ethanol.

Yield: 60% ; m.p. 115-116⁰C ; R_f  0.55. Spectroscopic analysis: IR (cm^-1): 3416 (Ar.NH), 1667.3 (C=O), 1645.1 (C=N), 1245.7 (C-N); ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36(1H,s), 8.19(1H,s), 7.70-8.06(3H,m), 7.00(1H,s), 6.49(1H,s), 5.7(1H,s).

Synthesis of Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane derivatives (M-1 to M-6) Compound MB (0.02 mol) was refluxed with each six different substituted benzaldehydes (0.01 mol) in the presence of acetic anhydride (10 ml) for 26 hrs (monitored with the help of TLC) and then poured into ice cold water to get compound M-1 to M-6 which was also crystallized from ethanol. 

Synthesis of Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-1) Yield: 40% ; m.p. 88-89⁰C ; R_f   0.49. Spectroscopic analysis: IR (cm^-1): 3423.4 (NH, aromatic), 3079.9 ( C-H, aromatic), 2944.0 (C-H, aliphatic), 1669.4 (C=O), 1620.8 (C=N);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 8.19 (d, 2H,NH-CH), 7.67-8.06 (m, 6H, Ar-H), 7.23-7.26 (m, 5H, Ar-H), 6.91 (s, 1H, Al-H), 6.39 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H); MS (m/z): 510.0 (M⁺).

Synthesis of p-methoxy Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-2) Yield: 72% ; m.p. 140-141⁰C ; R_f  0.52. Spectroscopic analysis: IR (cm^-1): 3415.0 (NH, aromatic), 2979.0 (C-H), 1656.4 (C=H), 1625.8 (C=N), 1302.0 (C-N);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 8.19 (d, 2H,NH-CH), 7.67-8.06 (m, 6H, Ar-H), 7.26-7.43 (m, 5H, Ar-H), 6.91 (s, 1H, Al-H), 6.39 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H), 3.83 (s, 3H, OCH₃);  MS (m/z): 535.0 (M⁺).

Synthesis of m- chloro Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-3) Yield: 55% ; m.p. 157-158⁰C ; R_f  0.42. Spectroscopic analysis: IR (cm^-1): 3435.1 (NH, aromatic), 2963.9 (C-H), 1687.4 (C=O), 1633.9 (C=N), 1302.0 (C-N), 711.3 (C-Cl);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 8.19 (d, 2H,NH-CH), 7.67-8.06 (m, 6H, Ar-H), 7.26-7.43 (m, 5H, Ar-H), 6.91 (s, 1H, Al-H), 6.43 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H);  MS (m/z): 543.0 (M⁺).

Synthesis of m- nitro Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-4) Yield: 65% ; m.p. 132-133⁰C ; R_f  0.82. Spectroscopic analysis: IR (cm^-1): 3428.6 (NH, aromatic), 2988.5 (C-H), 1656.7 (C=O), 1631.8 (C=N), 1513.5 (NO_2, aromatic asymmetric), 1378.5 (NO_2, aromatic symmetric);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 8.19 (d, 2H,NH-CH), 7.54-8.49 (m, 4H, Ar-H), 7.27-7.42 (m, 5H, Ar-H), 6.91 (s, 1H, Al-H), 6.39 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H); MS (m/z): 556.0 (M⁺).

Synthesis of o- nitro Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-5) Yield: 62% ; m.p. 140-141⁰C ; R_f  0.56. Spectroscopic analysis: IR (cm^-1): 3428.6 (NH, aromatic), 2988.5 (C-H), 1656.7 (C=O), 1631.8 (C=N), 1513.5 (NO_2, aromatic asymmetric), 1378.5 (NO_2, aromatic symmetric);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 8.19 (d, 2H,NH-CH), 7.57-8.06 (m, 10H, Ar-H), 6.91 (s, 1H, Al-H), 6.39 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H); MS (m/z): 556.0 (M⁺).

Synthesis of o- hydroxy Phenyl di-[6-(1-H-benzo[d] imidazole-5-yl)- pyridazine-3-one-2yl] methane (M-6) Yield: 60% ; m.p. 122-123⁰C ; R_f  0.48. Spectroscopic analysis: IR (cm^-1): 3422.8 (NH, aromatic), 3369.0 (O-H ) 2932.0 (C-H), 1679.7 (C=O), 1620.9 (C=N), 1387.8 (C-N);  ¹H-NMR (DMSO, 400 MHz, δ, ppm): 12.36 (d, 2H, N-H), 9.68 (d, 1H, OH), 8.19 (d, 2H, NH-CH), 7.26-8.06 (m, 11H, Ar-H), 6.91 (s, 1H, Al-CH), 6.39 (d, 2H, Ar-H) 5.79 (d, 2H, Ar-H);  MS (m/z): 556.0 (M⁺).

Reaction Scheme

Anticonvulsant Activity

Animals: Male albino mice (Swiss strains) weighing between 25-35 g were used in the study. They were housed in standard laboratory condition for a week before the experiments. Animals were housed in polypropylene cage with dust free rice husk as bedding material under laboratory condition with controlled environment of temperature (25°C ± 2°C)  and humidity (60% ± 10%). The animals were fasted over night. The animals were transferred to the laboratory 1 hr before starting experiment. The institutional ethical committee approved the study. Experiments were conducted between 9-14 h. Each mouse was used only once.

Maximal Electro-Shock-Induced Convulsion (MES): The anticonvulsant activity was performed by using MES method as reported (Krall et al.) on male Swiss albino mice (25-35 g). Animals were divided into different groups (control, test and standard) containing 5 animals each. The test samples (100 mg/kg) and phenytoin sodium (25 mg/kg) as a standard anticonvulsant drug were administered orally. Control animal received distilled water one hour prior to induction of convulsion. The mice were subjected to supramaximal electroshock of current intensity 60 mA, 50 Hz for 0.2 sec duration by electro-convulsometer through ear electrodes. The duration of tonic hind limb extensor phase and percentage of animal protected from convulsion have been summarised in Table 1. A complete abolition of hind limb extension was considered as 100 % protection.

Statistical Analysis

The values are expressed in mean ± SEM. The results were analysed by using ANOVA followed by student‘t’ test. Statistical significance on comparison with control group are indicated by *mark, ***P<0.001 highly significant, **P<0.01 significant and *P<0.5 moderately significant.

RESULTS AND DISCUSSION

The pyridazinone derivatives MA, MB, M-1toM-6 were synthesized according to scheme 1 by friedel-crafts acylation of appropriate hydrocarbons with succinic anhydride in the presence of hydrofluoric acid chloride.   The desired derivatives (M-1 to M-6) were produced. From the anticonvulsant screening data it has been found that M-2 and M-6 possess excellent anticonvulsant activity on the basis of extensor phase. M-1 and M-2 have hydrogen bonding property and electron withdrawing group at ortho and para position respectively, while M-4  possess meta electron withdrawing group and has minimum activity in this group. Thus we can conclude that anticonvulsant activity is increasing by substitution with orto and para directing electron withdrawing group, while decreasing with meta directing electron withdrawing group.

Table 1: Anticonvulsant activity of Pyridazinone derivatives

Animals were divided into eight group (1- 8) for studying anticonvulsant effect

n = Number of animals                            SEM = Standard error mean                     Standard Drug = Phenytoin sodium (25mg/kg)

Control = Distilled water                       R /D = Recovery/Death

Table 2: Structure & Physiochemical data of derivatives

Table 3: Characterization of derivatives

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