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ORIGINAL RESEARCH ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 3  |  Page : 138-144

Synthesis, antimicrobial activity, and docking study of some N3, N6-diphenylpyridazine-3,6-diamine derivatives as dihydrofolate reductase inhibitors


1 Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Shoolini University, Solan, Himachal Pradesh, India
2 Department of Pharmaceutical Chemistry, Narayan Institute of Pharmacy, Gopal Narayan Singh University, Sasaram, Bihar, India

Date of Web Publication1-Feb-2019

Correspondence Address:
Dr. Afroze Alam
Narayan Institute of Pharmacy, Gopal Narayan Singh University, Jamuhar, Sasaram - 803 521, Bihar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jphi.JPHI_37_18

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  Abstract 


Objective: The present study focussed on the synthesis of pyridazine analogs to explore broad-spectrum antimicrobial study. Since pyridazine analogs are not conventionally found in nature, and hence, its analogs are studied later.
Materials and Methods: All the synthesized compounds were characterized by spectroscopic techniques, namely, UV, IR,1HNMR, and mass spectrometry. Antimicrobial activity was screened by serial dilution method and absorbance was recorded using ELISA reader, subsequently minimum inhibitory concentrations were determined. Docking study was done into the active site of dihydrofolate reductase using Auto Dock 4.2.
Results: The present investigation about synthesis, characterization, and biological studies of some new pyridazine analogs were carried out to obtain potent and pharmacologically active compounds. The free energy of binding was in the range of −5.12 to −8.97 kcal/mole. In silico study report was in good tune with laboratory experiments.
Conclusions: Most of the compounds were moderate-to-good toward the antimicrobial activity. Compound AJ27 was found to be most active. Results of anti-microbial activity establishes the importance of N3, N6-diphenylpyridazine-3,6-diamine as the basic skeleton required for the antimicrobial activity.

Keywords: Antimicrobial activity, biphenyl, dihydrofolate reductase inhibitors, docking study, pyridazine analogs


How to cite this article:
Javed A, Sapra S, Alam A. Synthesis, antimicrobial activity, and docking study of some N3, N6-diphenylpyridazine-3,6-diamine derivatives as dihydrofolate reductase inhibitors. Int J Pharma Investig 2018;8:138-44

How to cite this URL:
Javed A, Sapra S, Alam A. Synthesis, antimicrobial activity, and docking study of some N3, N6-diphenylpyridazine-3,6-diamine derivatives as dihydrofolate reductase inhibitors. Int J Pharma Investig [serial online] 2018 [cited 2019 Feb 20];8:138-44. Available from: http://www.jpionline.org/text.asp?2018/8/3/138/251368




  Introduction Top


Antimicrobials are the broad classes of substances acting against microorganisms.[1],[2] Resistance to antimicrobial agents has been encountered as a major hindrance in clinical therapy and cure of many diseases and deserves scientific intervention to bring about some control measures.[3],[4] Several drug discovery strategies have evolved, including incremental improvements to existing antibiotics by chemical manipulation and the search for novel drug targets based on genomic approaches. There is a need to explore new chemical moieties for developing new drugs.[3],[5] Pyridazine derivatives are new and important compound of heterocyclic nature, containing two nitrogen atoms at 1 and 2 positions in a six-membered ring. A substantial numbers of drug molecules used in medicine contain a phenyl ring. Replacing these phenyl rings with Pyridazine rings opens an access to several thousands of diaza analogs, presenting more interaction possibilities, lower Log P values, improved crystalline salts, and more hydrogen bonding possibilities.[6],[7]

Pyridazines derivatives are class of heterocyclic compound that is of substantial interest because of the diverse range of their biological activity.[8] Pyridazine derivatives possess a wide range of activities such as antimicrobial,[9] antifungal,[10] antiviral,[11] anti-protozoan,[12] anti-inflammatory[13] diuretic,[14] antimalarial,[15] antitubercular,[16] and many others.[17] The pyridazine structure is found within some herbicides such as credazine, pyridafol, pyridate, and maleic hydrazide. Pyridazine is also found within the structure of several drugs, for instance, cefozopran, cadralazine, minaprine, hydralazine, and cilazapril.[18],[19],[20],[21],[22],[23] Biphenyl is a neutral molecule, and it is neutral due to the lack of functional group. However, it is present in many natural and pharmacologically active products. The substituted biphenyl derivatives possess a wide range of activity such as anti-hypertensive, antimicrobial, diuretic, anti-diabetic, antipsychotic, anxiolytic, and anti-inflammatory.[24],[25],[26],[27]


  Materials and Methods Top


Synthesis of hydrazine dihydrochloride

Concentration hydrochloric acid (0.110 moles) was added dropwise to cold hydrazine monohydrate (0.123 moles) with constant stirring. The solution was heated to evaporate water. The solution was brought back to room temperature and concentration HCl (0.113 moles) was again added under stirring in cold and precipitate formed was filtered and washed with methanol.[28]

Synthesis of 3,6-pyridaze diol (BM1)

A volume of 50 ml of water was added to 0.005 moles of hydrazine dihydrochloride. Then, 0.005 moles of maleic anhydride was added. The solution was kept in the boiling conditions for 3 h.[21]

Synthesis of 3,6-dichloropyridazine (BM2)

0.0446 moles of 3,6-pyridazine diol was refluxed with the excess of phosphorous oxychloride (3.2 moles) for 5 h. Excess reagent was removed by distillation. Then, it was put into cold water. Filters were used for filtration. Finally, the product was separated by removing water to obtain the pure product.[29]

General procedure for the synthesis of N3, N6-BIS Pyridazine-3,6-diamine analogs (AJ11-AJ30).

1 × 10−3 mole of 3,6-dichlopyridazine was mixed with 2 × 10−3 mole of variously substituted anilines. The reaction was refluxed for 8 h in toluene. The reaction mixture was assessed using thin-layer chromatography (TLC) and toluene was removed under negative pressure [Table 1]. The final product was recrystallized in methanol [Figure 1].
Table 1: Various substitutions at benzene ring

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Figure 1: General synthetic scheme

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Antimicrobial activity

Microbial strains used in the present study

Gram-negative bacteria: Staphylococcus aureus, Micrococcus luteus,  Escherichia More Details coli. Gram-positive bacteria: Pseudomonas aeruginosa, Pseudomonas aurantiaca. Fungi: Trichophyton rubrum, Cryptococcus laurentii.

Test procedure

The nutrient broth was used for the anti-microbial activity. 96 well plates were used for microdilution assay to estimate the minimum inhibitory concentration. A volume of 100 μl double strength media was poured in the first row and in remaining well 100 μl single strength media was poured. In the first row, 100 μg/ml of concentrated solution was added and further serial dilutions were made till ninth well so the concentration is as follows: 1000 μg/ml, 500 μg/ml, 250 μg/ml, 125 μg/ml, 62.5 μg/ml, 31.25 μg/ml, 15.6 μg/ml, 7.8 μg/ml, and 3.9 μg/ml.

The experiment was performed in triplicates. Inoculums of wells were done with 50 μl of standard inoculums of the test organism. Positive and negative controls were prepared in triplicates in remaining columns.

The plates were incubated at 37°C for 24 h and plates were read in the ELISA plate reader at 595 nm. Standard drugs used were ciprofloxacin and ketoconazole. The optical density of growth in each well was observed and then MIC was calculated.

Preparation of inoculums

For the preparation of inoculums automated media, i.e., The nutrient broth was taken to laminar air flow bench, and microbiological test tubes were half filled with media and then standard microbial stains were inoculated in that media and incubated at 37°C for 24 h.

Docking study

A library of 20 N3, N6-diphenylpyridazine-3,6-diamine derivatives was prepared using ChemDraw ultra 8.0. The 3D crystal structure of the di hydrofolate reductase (DHFR) was obtained from Protein Data Bank (PDB codes: 7 DFR). The 3D structures of DHFR, (from 7DFR) were used for the virtual screening. The co-crystallized DHFR and water molecules were removed, and Auto Dock 4.2 was used for molecular docking studies. Docking parameters were set to default values on the basis of Lamarckian genetic algorithm principle. The 20 compounds with the highest estimated free energy of binding were docked into the active site of DHFR [Table 2]. Synthesized compounds were biologically evaluated for antimicrobial study, and attempt was made to establish the structure-activity relationship of the compounds to explore new lead compounds [Figure 2].
Table 2: List of estimated free energy of binding of synthesized compounds

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Figure 2: Stereo view of the complex formed by DHFR and the docked compound (AJ-23).The amino acids: GLY34, ASN39, CYS37, LYS 166, CYS36 AND CYS36 were involved in interaction with compounds

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  Results Top


All the compounds were synthesized as per the standard procedure of schemes. Log P value was determined using UV spectrophotometer, and the data are presented.

Spectral characterizations

Spectral data of synthesized compounds (AJ-11–AJ-30)

AJ11 IR (KBr, cm−1): 3365 (NH), 3059 (C-H Stretch Aromatic), 1644 (C = N). 1H NMR (400MHz, DMSO-d6) δ, 6.80 (d, J = 11.2 Hz, 1H), 6.97 (m, 4H), 7.25 (d, J = 11.2 Hz, 1H), 7.39 (d, J = 8.2 Hz, 4H), 7.74 (m, 2H), 7.92 (s, 1H, NH), 13.7 (s, 1H, NH). Analytical calculated value for C16H14N4: C, 73.26; H, 5.38; N, 21.36. Analytical Observed value for C16H14N4: C, 73.29; H, 5.45; N, 21.39. m/z: 262.12.

AJ12 IR (KBr, cm−1): 3413 (NH), 2921 (C-H Aromatic). 1H NMR (400MHz, DMSO-d6): δ 6.90 (d, J = 9.4 Hz, 1H), 7.38 (d, J = 9.4 Hz, 1H), 7.60 (s, 1H, NH), 7.88 (s, 4H), 13.14 (s, 1H, NH). Analytical Calculated value for C16H10N4 Br6: C, 26.12; H, 1.10; N, 7.62, Br, 65.17. Analytical Observed value forC16H10N4 Br6: C, 26.09; H, 1.06; N, 7.58, Br, 65.12. m/z: 730.

AJ13 IR (KBr, cm−1): 3316 (NH), 1596 (C = N), 1555 and 1298 (C-NO2). 1H NMR (400MHz, CDCl3):δ 6.87 (d, J = 8.1 Hz, 2H, 5'-H), 6.93 (d, J = 10.1 Hz, 1H), 7.27 (d, J = 10.1 Hz, 1H), 7.36 (m, 2H, 4'-H), 7.41 (s, 2H, 1'-H), 7.59 (d, J = 7.6 Hz, 2H, 3'-H), 8.3 (s, 1H, NH), 14.2 (s, 1H, NH). Analytical calculated value for C16H12O4N6: C, 54.55; H, 3.43; N, 23.85; O, 18.17. Analytical observed value for C16H12O4N6: C, 54.48; H, 3.39; N, 23.79; O, 18.22. m/z: 352.

AJ14 IR (KBr, cm−1): 3984 (NH), 1588 and 1298 (C-NO2). 1H NMR (400MHz, DMSO-d6): δ 6.62 (d, J = 7.2 Hz, 4H, 1'-H), 6.93(d, J = 9.1 Hz, 1H) 7.29 (d, J = 9.1 Hz, 1H), 7.94 (d, J = 8.2 Hz, 4H, 2'-H), 8.4 (s, 1H, 1H), 14.03 (s, 1H, 1H). Analytical calculated value for C16H12O4N6: C, 54.55;H, 3.43; N, 23.85; O, 18.17. Analytical observed value forC16H12O4N6: C, 54.49;H, 3.39; N, 23.89; O, 18.23 m/z: 352.

AJ15 IR (KBr, cm−1): 3119 (NH), 2988 (C-H Aromatic), 1659 (C = N), 784 (C-Cl). 1H NMR (400MHz, CDCl3): δ 6.43 (d, J = 7.6 Hz, 2H, 5'-H), 6.60 (m, 2H, 4'-H), 6.86 (d, J = 9.4 Hz, 1H), 6.91 (m, 2H, 3'-H), 7.09 (d, J = 7.2 Hz, 2H, 2'-H), 7.26 (d, J = 9.4 Hz, 1H), 8.6 (s, 1H, NH), 12.6 (s, 1H, NH). Analytical calculated value for C16H12N4 Cl2: C, 58.02; H, 3.65; N, 16.92; Cl, 21.41. Analytical observed value for C16H12N4 Cl2: C, 58.09; H, 3.68; N, 16.86; Cl, 21.35. m/z: 330, 334.

AJ16 IR (KBr, cm−1): 3109 (NH), 2994 (C-H), 1654 (C = N), 790 (C-Cl). 1H NMR (400MHz, CDCl3): δ 6.39 (d, J = 7.3 Hz, 2H, 5'-H), 6.52 (s, 2H, 1'-H), 6.72 (d, J = 7.9 Hz, 2H, 3'-H), 6.89 (d, J = 9.8 Hz, 1H), 7.04 (m, 2H, 4'-H), 7.27 (d, J = 9.8 Hz, 1H), 8.5 (s, 1H, NH), 11.4 (s, 1H, NH). Analytical calculated value for C16H12N4 Cl2: C, 58.02; H, 3.65; N, 16.92; Cl, 21.41. Analytical observed value forC16H12N4 Cl2:C, 58.01; H, 3.69; N, 16.86; Cl, 21.42. m/z: 330, 334.

AJ17 IR(KBr, cm−1): 3361 (NH), 3156 (C-H Aromatic), 1611 (C = N), 784 (C-Cl). 1H NMR (400MHz, DMSO-d6):δ 6.50 (d, J = 7.2 Hz, 4H, 1'-H), 6.89 (d, J = 9.8 Hz, 1H), 7.12 (d, J = 7.8 Hz, 4H, 2'-H), 7.27 (d, J = 9.8 Hz, 1H), 8.3 (s, 1H, NH), 11.5 (s, 1H, NH). Analytical calculated value for C16H12N4 Cl2: C, 58.02; H, 3.65; N, 16.92; Cl, 21.41. Analytical observed value for C16H12N4 Cl2: C, 58.05; H, 3.62; N, 16.87; Cl, 21.38. m/z: 330, 334.

AJ18 IR(KBr, cm−1): 3316 (NH), 2988 (C-H Aromatic), 1659 (C = N), 1115 (C-F). 1H NMR (400MHz, CDCl3): δ 6.41 (d, J = 7.3 Hz, 4H), 6.92 (d, J = 10.2 Hz, 1H), 7.31 (d, J = 10.2 Hz, 1H), 7.47 (d, J = 8.1 Hz, 4H), 8.1 (s, 1H, NH), 13.8 (s, 1H, NH). Analytical calculated value for C18H12N4F6: C, 54.28; H, 3.04; N, 14.07; F, 28.62. Analytical observed value for C18H12N4F6: C, 54.32; H, 3.07; N, 14.11; F, 28.56. m/z: 398.

AJ19 IR(KBr, cm-1): 3428 (NH), 3082 (C-H Aromatic), 1614 (C = N), 788 (C-Cl). 1H NMR (400MHz, CDCl3): δ=6.89 (s, 4H, 2',4'-H), 6.95 (d, J = 9.8 Hz, 1H), 7.25 (d, J = 9.8 Hz, 1H), 8.4 (s, 1H, NH), 12.8 (s, 1H, NH). Analytical calculated value for C16H8N4 Cl6: C, 40.98; H, 1.72; N, 11.95, Cl, 45.36. Analytical observed value for C16H8N4 Cl6: C, 40.92; H, 1.66; N, 11.98, Cl, 45.39. m/z: 466.

AJ20 IR(KBr, cm−1): 3355 (NH), 2996 (CH Aromatic), 1670 (C = N), 614 (C-Br). 1H NMR (400MHz, CDCl3): δ 6.39 (d, J = 7.5 Hz, 2H, 5'-H), 6.71 (m, 2H, 3'-H), 6.96 (d, J = 9.7 Hz, 1H), 7.03 (m, 2H, 4'-H), 7.25 (d, J = 2.5 Hz, 2H, 2'-H), 7.36 (d, J = 9.7 Hz, 1H), 7.9 (s, 1H, NH), 11.6 (s, 1H, NH). Analytical calculated value for C16H12N4 Br2: C, 45.74; H, 2.88; N, 13.34; Br, 38.04. Analytical observed value for C16H12N4 Br2: C, 45.68; H, 2.82; N, 13.29; Br, 38.10. m/z: 419.9.

AJ21 IR(KBr, cm-1): 3371 (NH), 2992 (C-H Aromatic), 1670 (C = N), 616 (C-Br). 1H NMR (400MHz, CDCl3):δ 6.52 (d, J = 7.6 Hz, 2H, 5'-H), 6.73 (s, 2H, 1'-H), 6.86 (m, J = 7.8 Hz, 2H, 4'-H), 6.93 (d, J = 9.3 Hz, 1H), 6.99 (d, J = 2.6 Hz, 2H, 3'-H), 7.32 (d, J = 9.3 Hz, 1H), 8.1 (s, 1H, NH), 12.9 (s, 1H, NH).

Analytical calculated value for C16H12N4 Br2: C, 45.74; H, 2.88; N, 13.34; Br, 38.04. Analytical observed value for C16H12N4 Br2. C, 45.68; H, 2.89; N, 13.39; Br, 38.11. m/z: 418.

AJ22 IR(KBr, cm−1): 3439 (NH), 2840 (C-H Aromatic), 1619 (C = N), 668 (C-Br). 1H NMR (400MHz, DMSO-d6): δ 6.45 (d, J = 7.4 Hz, 4H, 1'-H), 6.89 (d, J = 9.3 Hz, 1H), 7.25 (d, J = 2.7 Hz, 4H, 2'-H), 7.28 (d, J = 9.3 Hz, 1H), 8.5 (s, 1H, NH), 13.3 (s, 1H, NH). Analytical calculated value for C16H12N4 Br2: C, 45.74; H, 2.88; N, 13.34; Br, 38.04. Analytical observed value for C16H12N4 Br2: C, 45.78; H, 2.92; N, 13.37; Br, 38.08. m/z: 418.

AJ23 IR(KBr, cm−1): 3447 (NH), 3070 (C-H Aromatic), 1681 (C = N), 1510 and 1339 (C-NO2)1 H NMR (400MHz, DMSO-d6): δ 6.97 (d, J = 9.3 Hz, 1H), 7.36 (d, J = 9.3 Hz, 1H), 7.9 (s, 1H, NH), 8.55 (s, 2H, 4'-H), 8.85 (s, 2H, 2'-H), 12.4 (s, 1H, NH). Analytical calculated value for C16H8O8N8 Br2: C, 32.02; H, 1.34; N, 18.67; Br, 26.63; O, 21.33. Analytical observed value for C16H8O8N8 Br2: C, 32.08; H, 1.38; N, 18.73; Br, 26.69; O, 21.39 m/z: 598, 602.

AJ24 IR(KBr, cm−1): 3455(NH), 3072(C-H Aromatic), 1603(C = N), 810(C-Cl), 652(C-Br). 1H NMR (400MHz, CDCl3): δ 6.64 (d, J = 8.6 Hz, 2H, 5'-H), 6.93 (d, J = 9.4 Hz, 1H), 7.19 (d, J = 2.5 Hz, 2H, 4'-H), 7.33 (d, J = 9.4 Hz, 1H), 7.37 (s, 2H, 2'-H), 7.8 (s, 1H, NH), 11.6 (s, 1H, NH). Analytical calculated value for C16H10N4 Cl2 Br2: C, 39.3; H, 2.06; N, 11.46; Cl, 14.5; Br, 32.68. Analytical observed value for C16H10N4 Cl2 Br2: C, 39.1; H, 2.11; N, 11.51; Cl, 14.2; Br, 32.73. m/z: 486, 494.

AJ25 IR(KBr, cm−1): 3415 (NH), 2901 (C-H aromatic), 1619 (C = N), 418 (C-I). 1H NMR (400MHz, CDCl3): δ 6.41 (m, 2H, 3'-H), 6.59 (d, J = 8.0 Hz, 2H, 5'-H), 6.89 (d, J = 9.4 Hz, 1H), 7.15 (m, 2H, 4'-H), 7.28 (d, J = 9.4 Hz, 1H), 7.41 (d, J = 7.3 Hz, 2H, 2'-H), 8.0 (s, 1H, NH), 11.8 (s, 1H, NH). Analytical calculated value for C16H12N4I2: C, 37.38; H, 2.35; N, 10.9; I, 49.37. Analytical observed value for C16H12N4I2: C, 37.33; H, 2.38; N, 10.5; I, 49.34. m/z: 513.9.

AJ26 IR(KBr, cm−1): 3410 (NH), 3042 (C-H Aromatic), 1659 (C = N). 1H NMR (400MHz, DMSO-d6):δ 6.62 (d, J = 8.0 Hz, 4H), 6.97 (d, J = 9.6 Hz, 1H), 7.28 (d, J = 9.6 Hz, 1H), 7.38 (d, J = 8.6 Hz, 4H), 8.3 (s, 1H, NH), 11.9 (s, 1H, NH). Analytical calculated value for C16H12N4I2: C, 37.38; H, 2.35; N, 10.9; I, 49.37. Analytical observed value forC16H12N4I2: C, 37.34; H, 2.31; N, 10.5; I, 49.33. m/z: 513.9.

AJ27 IR(KBr, cm−1): 3400-3000 (OH Broad Peak), 1659 (C = O), 1581 (C = N). 1H NMR (400MHz, DMSO-d6): δ 5.4 (s, 2H, OH), 6.59 (d, J = 7.2 Hz, 2H, 5'-H), 6.88 (d, J = 7.7 Hz, 2H, 4'-H), 6.98 (d, J = 9.6 Hz, 1H), 7.32 (d, J = 9.6 Hz, 1H), 7.35 (s, 2H, 2'-H), 9.2 (s, 1H, NH), 10.45 (s, 2H, COOH), 14.4 (s, 1H, NH). Analytical calculated value for C18H14O6N4: C, 56.55; H, 3.69; N, 14.65; O, 25.11. Analytical observed value forC18H14O6N4: C, 56.51; H, 3.66; N, 14.68; O, 25.15. m/z: 382.3.

AJ28 IR(KBr, cm−1): 3458 (NH), 2954 (OH Aromatic), 1674 (C = O ester), 1626 (C = N), 1283 (C-O ester). 1H NMR (400MHz, CDCl3): δ 3.81 (s, 6H, OCH3),3.82 (s, 6H, OCH3), 3.83 (s, 6H, COOCH3), 6.13 (s, 2H, 5'-H) 7.91 (d, J = 9.8 Hz, 1H), 7.28 (s, 2H, 2'-H), 7.36 (d, J = 9.8 Hz, 1H), 8.9 (s, 1H, NH), 13.8 (s, 1H, NH). Analytical calculated value for C24H26O8N4: C, 57.83; H, 5.26; N, 11.24; O, 25.68. Analytical observed value forC24H26O8N4: C, 55.74; H, 5.56; N, 11.79; O, 26.92. m/z: 498.2.

AJ29 IR(KBr, cm−1): 3416 (NH), 2961 (C-H Aromatic), 1616 (C = N). 1H NMR (400MHz, CDCl3): δ 1.32 (m, 6H, *CH3), 2.35 (s, 6H, CH3), 2.61 (m, 4H, *CH2), 6.47 (m, 2H, 3'-H), 6.68 (d, J = 2.6 Hz, 2H, 2'-H), 6.93 (d, J = 9.8 Hz, 1H), 7.12 (d, J = 7.6 Hz, 2H, 4'-H), 7.30 (d, J = 9.8 Hz, 1H), 8.3 (s, 1H, NH), 13.8 (s, 1H, NH). Analytical calculated value for C22H26N4: C, 76.27; H, 7.56; N, 16.17. Analytical observed value forC22H26N4: C, 76.24; H, 7.59; N, 16.19. m/z: 346.4.

AJ30 IR(KBr, cm−1): 3325 (NH), 3033 (C-H Aromatic), 2799 (C-H). 1H NMR (400MHz, CDCl3): δ 2.33 (s, 3H, CH3), 3.72 (s, 6H, OCH3), 6.62 (d, J = 8.6 Hz, 2H, 5'-H), 6.67 (d, J = 2.6 Hz, 2H, 4'-H), 6.90 (d, J = 10.1 Hz, 1H), 7.27 (d, J = 10.1 Hz, 1H), 7.39 (s, 2H, 2'-H), 8.6 (s, 1H, NH), 13.3 (s, 1H, NH). Analytical calculated value for C20H22O2N4: C, 68.55; H, 6.33; N, 15.99; O, 9.13. Analytical observed value forC20H22O2N4: C, 68.53; H, 6.37; N, 15.96; O, 9.17. m/z: 350.1.

Docking study

All the synthesized compounds were docked into active site of DHFR and the results are presented as estimated free energy of binding in [Table 2].

Antimicrobial activity

All the synthesized compounds were subjected to antimicrobial activity using five bacterial and two fungal stains. The MIC of all the synthesized compounds were calculated and reported in tabular form [Table 3]. Ciprofloxacin was used as a standard against bacterial stains, and ketoconazole was used as a standard against fungal stains.
Table 3: Anti-microbial activity of all synthesized compounds

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  Discussion Top


Starting material for the synthesis was obtained by the reaction between 3,6-pyridazine diol with phosphorous oxychloride [Table 1]. Final compounds were designed by the reaction between 3,6-dichlopyridazine and various anilines. An attempt was made to synthesize diphenylpyridazine analogs. All the reactions were monitored throughout by TLC. All the structures of final compounds were confirmed by IR, NMR, and mass spectrometry. The synthesized pyridazine analogs were screened for their antibacterial activity using broth microdilution method against S. aureus, M. luteus, E. coli, P. aeruginosa and P. aurantiaca. Ciprofloxacin was used as standard drug for comparison. The title compounds were also evaluated for their antifungal activity against T. rubrum and C. laurentii using Ketoconazole standard drug. The results revealed that synthesized compounds showed excellent broad-spectrum antimicrobial activity against all bacterial as well as fungal stains and compound AJ23 was found to be most active compound. However, since Nitroaromatic compounds are actually toxic and mutagenic and many are suspected or established carcinogens and which can be removed at this point in time. However, compound bearing hydroxyl group and carboxyl group was found to be most active next most active compound, i.e., Compound AJ27. From the literature, it has been observed that the incorporation of electron withdrawing substituent on phenyl ring causes increase in activity and are important structural requirement to be a good antimicrobial agent.

A careful analysis of all the data for the antimicrobial activity of all pyridazine analogs demonstrated an interesting finding that incorporation of one or more NO2, Cl, Br groups in the skeleton causes increase in activity. Incorporation of fluorine in methyl group instead of the main skeleton does not yields good results. Compounds having–OCH3 are less active than other compounds of the series. Addition of–COOH group on the main skeleton, increases the activity. Similarly, compounds bearing 2-iodo compounds are less active than 4-iodo compounds. Same finding is continued for bromine as well as chlorine-containing compounds in the same series. In drug receptor interaction study ligands were ranked according to docking score/estimated free energy of binding. The free energy of binding of ligands was in the range between −5.12 to −8.97 Kcal/mole. Top-ranked compound (AJ-23) and (AJ-12) with −8.97 and −7.31 Kcal/mole free energy of binding, respectively, were in correlation with wet laboratory experiments. The protein-ligand analysis also has shown its strong interactions with target protein and had six hydrogen bond interactions in (AJ-23) and five hydrogen bond interactions in (AJ-12). The excellent interactions of DHFR with all top ranked compounds indicated a high degree of coherent relationship between in silico approach and in vitro studies. Large numbers of hydrogen bond interactions exist between different amino acids of the DHFR and NO2/hydroxyl/methoxyl group present in ring A, B, and heterocyclic ring C. High anti-microbial activities of the compounds demand further in vivo and clinical studies, and these compounds might find an important place in the new array of molecules targeting DHFR-dependent biological functions. Keeping in view, the biological and pharmacological importance of the pyridazine derivatives and biphenyl derivatives, it is our endeavor, to bring two important moieties into the single molecular frame by appropriate synthetic routes. This will stand not only as a source for new biologically active compounds but also as a model for molecular conjunction in the design of new drugs. The in silico results of the study were in good tune with the laboratory work experiments, and nearly 80% of docking results were same as that of in vitro experiments. Ligand-protein interactions were profoundly found in the present study and gave an insight for further evaluation of study up to molecular level.


  Conclusions Top


All the test compounds (AJ11-AJ30) were screened for antimicrobial activity against five bacterial and two fungal stains, namely, S. aureus, M. luteus, E. coli, P. aeruginosa, P. aurantiaca, T. rubrum, C. laurentii. Most of the compounds showed remarkably good broad-spectrum activity ranging from 7.8 to 500 ug/ml. The remaining compounds have shown moderate activity. N3, N6-diphenylpyridazine-3,6-diamine is established as structural skeleton required for the antimicrobial activity. Compound AJ27 is found to be the most active compound. Docking studies revealed that carbonyl oxygen, NO2, OH, and OCH3 of the selected derivatives were involved in hydrogen bonding with various amino acids of the receptor. This confirms our hypothesis that conjugation of two pharmacophores might improve the pharmacological profile of synthesized compounds

Acknowledgment

The authors are thankful to Vice-chancellor Shoolini University for providing laboratory facilities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Maartens MM, Swart CW, Carolina H. Pohl CH, Kock LJ. Antimicrobials, chemotherapeutics or antibiotics? Sci Res Essays 2011;6:3927-9.  Back to cited text no. 1
    
2.
Etebu E, Arikekpar I. Antibiotics: Classification and mechanism of action with emphasis on molecular perspectives. Int J Appl Microbiol Biotechnol Res 2016;4:90-101.  Back to cited text no. 2
    
3.
Chopra I, Hesse L, O'Neill AJ. Exploiting current understanding of antibiotic action for discovery of new drugs. J Appl Microbiol 2002;92 Suppl:4S-15S.  Back to cited text no. 3
    
4.
Liwa AC, Jaka H. Antimicrobial resistance: Mechanisms of action of antimicrobial agents. In: Vilas M, editor. The Battle against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs. Spain: Formatex Research Center; 2015. p. 876-85.  Back to cited text no. 4
    
5.
Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74:417-33.  Back to cited text no. 5
    
6.
Asif MA. Mini review on antimicrobial activities of different substituted pyridazinone derivatives. Eur J Biol Sci 2016;8:142-57.  Back to cited text no. 6
    
7.
Wermuth CG. Are pyridazines privileged structures. Med Chem Commun 2011;2:935-43.  Back to cited text no. 7
    
8.
Asif M. The pharmacological importance of some diazines containing drug molecules. SOP Trans Org Chem 2014;1:1-16.  Back to cited text no. 8
    
9.
Al-Kamali AS, Al-Hazmi AA, Alhousami MH, Al-Masany MA. Synthesis and antibacterial activity of some novel thieno [2, 3-c] pyridazines using 3-amino-5-phenyl-2-ethoxycarbonylthieno [2, 3-c] pyridazine as a starting material. Arab J Chem 2014 ;7:775-80.  Back to cited text no. 9
    
10.
Islam M, Siddique AA. Synthesis, antituberculosis, antifungal and antibacterial activity of 3-substituted phenyl-6-substituted phenyl-1,2,4-triazolo[4,3-b] pyridazines. Acta Pol Pharm Drug Res 2010;66:555-62.  Back to cited text no. 10
    
11.
Wang Z, Wang M, Yao X, Li Y, Tan J, Wang L, et al. Design, synthesis and antiviral activity of novel pyridazines. Eur J Med Chem 2012;54:33-41.  Back to cited text no. 11
    
12.
Bendjeddou LZ, Loaëc N, Villiers B, Prina E, Späth GF, Galons H, et al. Exploration of the imidazo[1,2-b] pyridazine scaffold as a protein kinase inhibitor. Eur J Med Chem 2017;125:696-709.  Back to cited text no. 12
    
13.
Asif M. General study of pyridazine compounds against cyclooxygenase enzyme and their relation with analgesic, anti-inflammatory and anti-arthritic activities. Chron Young Sci 2010;1:3-9.  Back to cited text no. 13
  [Full text]  
14.
Inada Y, Shibouta Y, Shimakawa H, Nishikawa K, Kikuchi S 1,4-dimorpholino-7-phenylpyrido[3,4-d] pyridazine (SD-511) as a new type of diuretic agent. Arzneimittelforschung 1978;28:1105-11.  Back to cited text no. 14
    
15.
Bhatt A, Singh RK, Kant R. Synthesis of novel Imidazo[1,2-b] pyridazine derivatives and study of their biomedicinal efficacy. Chem Biol Lett 2016;3:38-43.  Back to cited text no. 15
    
16.
Rathish IG, Javed K, Ahmad S, Bano S, Alam MS, Akhter M, et al. Synthesis and evaluation of anticancer activity of some novel 6-aryl-2-(p-sulfamylphenyl)-pyridazin-3(2H)-ones. Eur J Med Chem 2012;49:304-9.  Back to cited text no. 16
    
17.
Asif M. Some recent approaches of biologically active substituted pyridazine and phthalazine drugs. Curr Med Chem 2012;19:2984-91.  Back to cited text no. 17
    
18.
Bansal RK. Heterocyclic Chemistry. 4th ed. New Delhi: New Age International Publishers; 2007.  Back to cited text no. 18
    
19.
Horning RH, Amstutz ED. The preparation of some dialkyl pyridazines. J Org Chem 1955;20:707-13.  Back to cited text no. 19
    
20.
Asif M, Singh A. Exploring potential, synthetic methods and general chemistry of pyridazine and pyridazone: A brief Introduction. Int J Chem Tech Res 2010;2:1112-28.  Back to cited text no. 20
    
21.
Mizzoni RH, Spoerri PE. Synthesis in the pyridazine series. I. Pyridazine and 3, 6-dichloropyridazine. J Am Chem Soc 1951;73:1873-4.  Back to cited text no. 21
    
22.
Abed HB, Mammolithi O, Bande O, Lommen GV, Herdewijn P. Strategy for the synthesis of pyridazine heterocycles and their derivatives. J Org Chem 2013;78:7845-58.  Back to cited text no. 22
    
23.
Ibrahim HM, Behbehani H. Synthesis of a new class of pyridazin-3-one and 2-amino-5-arylazopyridine derivatives and their utility in the synthesis of fused azines. Molecules 2014;19:2637-54.  Back to cited text no. 23
    
24.
Jain ZJ, Gide PS, Kankate RS. Biphenyls and their derivatives as synthetically and pharmacologically important aromatic structural moieties. Arab J Chem 2017;10:2051-66.  Back to cited text no. 24
    
25.
Dravyakat BR, Kawade DP, Khedekar PB, Bhusari KP. Design and syntheses of some new diphenylaminoisoxazolines as potent anti-inflammatory agent. Ind J Chem 2008;47B:1559-67.  Back to cited text no. 25
    
26.
Subramanyam C, Ramana KV, Rasheed S, Adam S, Raju CN. Synthesis and biological activity of novel diphenyl N-substituted carbamimidoylphosphoramidate derivatives. Phosphorus Sulfur Silicon Relat Elem 2013;188:1228-35.  Back to cited text no. 26
    
27.
Said SB, Abdelrazek H, Elezaby RF. Synthesis and biological activity of some 2,3- diphenylindole derivatives. Res Rev 2015;4:2319-22.  Back to cited text no. 27
    
28.
Harshman RC, Nicolaisen BH. Inventor; Olin Mathieson, Assignee. Manufacture of Hydrazine Dihydrohalides. United States Patent US2978296A; 4 April, 1961.  Back to cited text no. 28
    
29.
Jayashree BS, Alam A, Nayak Y, Kumar DV. Synthesis of 3-methylflavones and their antioxidant and antibacterial activities. Med Chem Res 2012;21:1991-6.  Back to cited text no. 29
    


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