Abstract
A novel class of 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazoles 25-32 were synthesized and evaluated for their in vitro antibacterial and antifungal activities. Four Compounds, which all possessed electron withdrawing functional groups (–Cl, –NO2, –Br) 27, 28, 30 and 32 were more potent against the tested bacterial/fungal strains than the standard bacterial and fungal drugs ciprofloxacin and fluconazole respectively.
Introduction
Various structurally diverse indazole nucleus have aroused great interest in the past and recent years due to their wide variety of biological properties such as antimicrobial activity [Citation1], inhibitors of protein kinase B/Akt [Citation2], antiprotozal agents [Citation3], antichagasic activity [Citation3], leishmanocidal activity [Citation3], trypanocidal activity [Citation3], inhibitors of S-adenosyl homocysteine/methylthio adenosine (SAH/MTA) nucleosides [Citation4], potent activator of the nitric oxide receptor [Citation5], inhibit platelet aggregation [Citation5].
In recent years there has been a great deal of interest in exploiting more than one proximal functional groups for designing novel structures capable of performing a variety of functions. The present study describes the use of 6-carbethoxy-3,5-diarylcyclohex-2-enone [Citation6], an intermediate with three versatile functional groups i.e., ketone, olefin and ester for the synthesis of fused indazole derivatives. In continuation of our earlier work on the synthesis of various bio active heterocyclic nucleus [Citation7–11], we wish to report the development of indazoles on 6-carbethoxy-3,5-diarylcyclohex-2-enone derivatives thus paving the way for a novel class of 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole.
Experimental
Microbiology
Materials
All the bacterial strains namely Staphylococcus aureus, β-Heamolytic streptococcus, Vibreo cholerae, Salmonella typhii, Shigella felxneri, Escherichia coli, Klebsiella pneumonia, Pseudomonas and fungal strains namely Aspergillus flavus, Mucor, Rhizopus and Microsporum gypsuem are clinical strains and are get hold of from Faculty of Medicine, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India.
In vitro antibacterial and antifungal activity (Disc Diffusion method)
The in vitro activities of the compounds were tested in Sabourauds dextrose broth (SDB) (Hi-media, Mumbai) for fungi and nutrient broth (NB) (Hi-media, Mumbai) for bacteria by the Disc Diffusion method [Citation12]. The respective hydrochlorides of the test compounds 25-32 were dissolved in water to obtain 1 mg mL− 1 stock solution and the different concentrations [100, 200, 500 ppm (μg/mL)] are prepared from the stock solution. Seeded broth (broth containing microbial spores) was prepared in NB from 24 h old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1°C while fungal spores from 1 to 7 days old Sabourauds agar (Hi-media, Mumbai) slant cultures were suspended in SDB. Sterile paper disc of 5 mm diameter was saturated with the three different concentrations and such discs were placed in each seeded agar plates. The petri plates were incubated in BOD incubator at 37°C for bacteria and at 28°C for fungi. The zone of inhibition was recorded by visual observations after 24 h of inhibition for bacteria and after 72-96 h of inhibition for fungi. Moreover, the zone of inhibition was measured by excluding the diameter of the paper disc. Ciprofloxacin was used as standards for bacteria and fluconazole as standard for fungi under analogous conditions.
In vitro antibacterial and antifungal activity (Minimum inhibitory concentration (MIC) method)
Minimum inhibitory concentration (MIC) in μg/mL values is carried out by two-fold serial dilution method [Citation13]. The respective hydrochlorides of the test compounds 23-27 were dissolved in water to obtain 1 mg mL− 1 stock solution. Seeded broth (broth containing microbial spores) was prepared in NB from 24 hrs old bacterial cultures on nutrient agar (Hi-media, Mumbai) at 37 ± 1°C while fungal spores from 1 to 7 days old Sabourauds agar (Hi-media, Mumbai) slant cultures were suspended in SDB. The colony forming units (cfu) of the seeded broth were determined by plating technique and adjusted in the range of 104-105 cfu/mL. The final inoculums size was 105cfu/mL for antibacterial assay and 1.1-1.5 X 102 cfu/mL for antifungal assay. Testing was performed at pH 7.4 ± 0.2 for bacteria (NB) and at a pH 5.6 for fungi (SDB). 0.4 mL of the solution of test compound was added to 1.6 mL of seeded broth to form the first dilution. One milliliter of this was diluted with a further 1 mL of seeded broth to give the second dilution and so on till six such dilutions were obtained. A set of assay tubes containing only seeded broth was kept as control. The tubes were incubated in BOD incubators at 37 ± 1°C for bacteria and 72-96 h for fungi. The minimum inhibitory concentrations (MICs) were recorded by visual observations after 24 h (for bacteria) and 72-96 h (for fungi) of incubation. Ciprofloxacin was used as standard for bacteria studies and Fluconazole was used as standards for fungal studies.
Chemistry
Performing TLC assessed the reactions and the purity of the products. All the reported melting points were taken in open capillaries and were uncorrected. IR spectra were recorded in KBr (pellet forms) on a Nicolet-Avatar–330 FT-IR spectrophotometer and note worthy absorption values (cm− 1) alone are listed. 1H, D2O exchanged 1H and 13C NMR spectra were recorded at 400 MHz and 100 MHz respectively on Bruker AMX 400 NMR spectrometer using DMSO-d as solvent. Two-dimensional HSQC spectra were recorded at 500 MHz and on Bruker DRX 500 NMR spectrometer using DMSO-d as solvent. The ESI + ve MS spectra were recorded on a Bruker Daltonics LC-MS spectrometer. Satisfactory microanalysis was obtained on Carlo Erba 1106 CHN analyzer.
By adopting the literature precedent, 1,3-diaryl-prop-2-en-1-ones 9-16 [Citation14] and 6-carbethoxy-3,5-diarylcyclohex-2-enone 17-24 [Citation6] were prepared.
Typical procedure for the synthesis of 4,6-diphenyl-4,5-dihydro-3-hydroxy-2[H]-indazole 25
A solution of 6-carbethoxy-3,5-diarylcyclohex-2-enone, 17 (0.1 mol) in methanol (40 mL) was treated with hydrazine hydrate (0.15 mol) and refluxed for 5 h. The reaction mixture was cooled and then poured over crushed ice. The crude product 25 was recrystallized twice using methanol as solvent. IR (KBr) (cm− 1): 3425, 3060, 2922, 2863, 1607, 1515, 1446, 1369, 758, 695; 1H NMR (δ ppm): 2.90, 3.1-3.2 (m,2H,H5), 4.19 (dd,1H,H4), 6.75 (d,1H,H7), 9.7 (s,1H,H2), 11.53 (s,1H,OH), 7.10-7.48 (m,10H,H-Arom.); 13C NMR (δ ppm): 34.2 C-4, 36.2 C-5, 98.3 C-9, 113.4 C-7; 136.2 C-8; 125.0-128.5 C-Arom.; 140.2, 145.3 ipso-C's; 157.2 C-3.
In the D2O exchanged 1H NMR spectrum, a broad peak at 11.53 ppm due to –OH proton at C-3 and a broad peak at 9.74 ppm due to –NH proton at C-2 disappeared.
In the HSQC spectrum, one bond correlation (34.2/4.19) between C-4 and H4a occurs. The 13C resonance at 36.2 ppm has correlations with methylene protons H5a (36.2/2.90; 36.2/3.20) and hence C-5 resonates at 36.2 ppm. The 13C resonance at 113.4 ppm correlations with doublet at 6.75 ppm. The doublet at 6.75 ppm is conveniently assigned to H7. The cross peak (113.4/6.75 ppm) confirms that the 13C resonance at 113.4 ppm is due to C-7. The 13C resonances at 98.3, 136.2, 157.2 ppm have no correlations with protons and hence it is due to quaternary carbons. Among the quaternary carbon resonances, the 13C resonance at 140.2, 143.2 ppm is assigned to ipso carbons. The 13C resonance at 136.2 and 157.2 ppm are due to the C-8 and C-3 carbons. The signal at 98.3 ppm is due to C-9 carbon and the C-6 carbon is merged with aromatic carbons.
The compounds 26-32 were synthesized similarly.
6-phenyl-4,5-dihydro-4-p-tolyl-3-hydroxy-2[H]-indazole 26
IR (KBr) (cm− 1): 3419, 3060, 3062, 2919, 2858, 1593, 1516, 757, 695; 1H NMR (δ ppm): 2.21 (s,3H,CH3 at phenyl ring); 2.87, 3.1-3.2 (m,2H,H5), 4.14 (dd,1H,H4), 6.75 (d,1H,H7), 8.30 (s,1H,H2), 10.98 (s,1H,OH), 7.02-7.47 (m,9H,H-Arom.); 13C NMR (δ ppm): 20.5 CH3 at phenyl ring; 33.8 C-4, 36.3 C-5, 98.5 C-9, 113.3 C-7; 136.3 C-8; 125.0-128.5 C-Arom.; 134.8,140.2,141.1,142.2 ipso-C's; 156.4 C-3.
4-(4-chlorophenyl)-4,5-dihydro-6-phenyl-3-hydroxy-2[H]-indazole 27
IR (KBr) (cm− 1): 3404, 3054, 2928, 1600, 1535, 1491, 757, 694; 1H NMR (δ ppm): 2.87, 3.1-3.2 (m,2H,H5), 4.20 (dd,1H,H4), 6.76 (d,1H,H7), 8.35 (s,1H,H2), 11.5 (s,1H,OH), 7.16-7.47 (m,9H,H-Arom.); 13C NMR (δ ppm): 33.7 C-4, 36.1 C-5, 97.8 C-9, 113.4 C-7; 136.2 C-8; 125.1-128.5 C-Arom.; 130.5, 140.1, 141.2, 144.2 ipso-C's; 156.3 C-3.
4-(4-nitrophenyl)-4,5-dihydro-6-phenyl-3-hydroxy-2[H]-indazole 28
IR (KBr) (cm− 1): 3422, 3076, 2924, 2847, 1599, 1515, 1437, 1345, 753, 695; 1H NMR (δ ppm): 2.86, 3.20-3.25 (m,2H,H5), 4.34 (dd,1H,H4), 6.76 (d,1H,H7), 9.70 (s,1H,H2), 11.70 (s,1H,OH), 7.25-8.11 (m,9H,H-Arom.); 13C NMR (δ ppm): 34.6 C-4, 36.0 C-5, 97.3 C-9, 113.5 C-7; 136.2 C-8; 123.4-128.8 C-Arom.; 140.1, 146.2 ipso-C's; 157.2 C-3.
4-(4-methoxyphenyl)-4,5-dihydro-6-phenyl-3-hydroxy-2[H]-indazole 29
IR (KBr) (cm− 1): 3417, 3065, 3052, 2930, 2836, 1608, 1511, 1443, 1373, 760, 696; 1H NMR (δ ppm): 2.85, 3.10-3.20 (m,2H,H5), 3.64 (s,3H,OCH3 at phenyl ring); 4.11 (dd,1H,H4), 6.73 (d,1H,H7), 8.40 (s,1H,H2), 10.70 (s,1H,OH), 7.03-7.45 (m,9H,H-Arom.); 13C NMR (δ ppm): 33.4 C-4, 36.4 C-5, 54.9 –OCH3 at phenyl ring; 98.7 C-9, 113.4 C-7; 136.2 C-8; 125.0-128.5 C-Arom.; 137.2, 140.3, 141.0, 157.5 ipso-C's; 156.3 C-3.
6-(4-bromophenyl)-4,5-dihydro-4-phenyl-3-hydroxy-2[H]-indazole 30
IR (KBr) (cm− 1): 3402, 3093, 3000, 2927, 2830, 1608, 1526, 1439, 1349, 807, 736; 1H NMR (δ ppm): 2.85, 3.10-3.20 (m,2H,H5), 4.16 (dd,1H,H4), 6.76 (d,1H,H7), 9.70 (s,1H,H2), 11.72 (s,1H,OH), 7.10-7.82 (m,9H,H-Arom.); 13C NMR (δ ppm): 34.4 C-4, 36.1 C-5, 97.8 C-9, 113.3 C-7; 135.2 C-8; 126.0-129.7 C-Arom.; 131.4, 131.9, 135.2, 139.2 ipso-C's; 157.5 C-3.
6-(4-methylphenyl)-4,5-dihydro-4-phenyl-3-hydroxy-2[H]-indazole 31
IR (KBr) (cm− 1): 3422, 3062, 3051, 2928, 2834, 1603, 1510, 1441, 1370, 764, 691; 1H NMR (δ ppm): 2.81, 3.13-3.18 (m,2H,H5), 2.24 (s,3H,CH3 at phenyl ring); 4.08 (dd,1H,H4), 6.70 (d,1H,H7), 8.38 (s,1H,H2), 11.21 (s,1H,OH), 7.08-7.38 (m,9H,H-Arom.); 13C NMR (δ ppm): 33.0 C-4, 36.7 C-5, 21.8 –CH3 at phenyl ring; 99.2 C-9, 112.6 C-7; 134.2 C-8; 124.7-127.9 C-Arom.; 137.0, 140.5, 141.5, 157.2 ipso-C's; 157.2 C-3.
4-(4-tolylphenyl)-4,5-dihydro-6-(3-nitrophenyl)-3-hydroxy-2[H]-indazole 32
IR (KBr) (cm− 1): 3428, 3022, 2925, 2852, 1609, 1525, 1489, 1448, 818, 701; 1H NMR (δ ppm): 2.85, 3.10-3.25 (m,2H,H5), 4.16 (dd,1H,H4), 6.77 (d,1H,H7), 9.82 (s,1H,H2), 10.90 (s,1H,OH), 6.95-8.24 (m,8H,H-Arom.); 13C NMR (δ ppm): 33.4 C-4, 36.3 C-5, 99.0 C-9, 113.5 C-7; 137.0 C-8; 116.0-131.4 C-Arom.; 137.0, 141.9, 148.2, 157.6 ipso-C's; 156.1 C-3.
Results and discussion
Chemistry
The synthetic strategy for the formation of 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole, a fused indazole derivative involves three steps which are described as follows: Condensation of appropriate acetophenone 1-4 and appropriate benzaldehyde 5-8 in the presence of sodium hydroxide yields the respective 1,3-diaryl-prop-2-en-1-ones 9-16. The respective α,β-unsaturated ketones 9-16 on treatment with ethyl acetoacetate in the presence of sodium ethoxide gives 6-carbethoxy-3,5-diarylcyclohex-2-enone 17-24. The formed ketones 17-24 on treatment with hydrazine hydrate in refluxing methanol gives 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole 25-32. The schematic representation and analytical data for the synthesized compounds 25-32 are furnished in Scheme and respectively. The structures of the compounds are elucidated by melting points, elemental analysis, MS, FT-IR, NMR (1H & 13C), D2O exchanged 1H-NMR, two-dimensional HSQC spectroscopic data.
Scheme 1. Synthetic pathway for the formation of 4,6-diaryl-4,5-diydro-3-hydroxy-2[H]-indazole
![Scheme 1. Synthetic pathway for the formation of 4,6-diaryl-4,5-diydro-3-hydroxy-2[H]-indazole](/cms/asset/5a7c010d-88fb-466e-aa1b-42ff85e6658c/ienz_a_281189_f0001_b.gif)
Table I. Analytical data of compounds 25-32.
Antibacterial activity
All the newly synthesized novel target molecule 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole 25-32 were tested for their antibacterial activity in vitro (Tables and ) against S. aureus, β-H. streptococcus, V. cholerae, S. typhii, S. felxneri, E. coli, K. pneumonia and Pseudomonas. Ciprofloxacin was used as standard drug; whose zone of inhibition (mm) values for S. aureus, β-H. streptococcus, V. cholerae, S. typhii, S. felxneri, E. coli, K. pneumonia and Pseudomonas was 25, 28, 23, 22, 23, 24, 26 and 23 mm respectively. Minimum inhibitory concentration (MIC) in μg/mL values is reproduced in Tables and . In general all the synthesized novel 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazoles 25-32 exerted a wide range of modest antibacterial activity in vitro against the tested organisms. All the compounds 25-32 were active against all the tested bacterial strains. Four Compounds, which all possessed electron withdrawing functional groups (–Cl, –NO2, –Br) 27, 28, 30 and 32 were more potent against the tested bacterial strains than the standard drug Ciprofloxacin.
Table II. In vitro profile of compounds 25-28 against test bacteria and fungi.
Table III. In vitro profile of compounds 29-32 against test bacteria and fungi.
Table IV. In vitro antibacterial activities (MIC) values for compounds 25-32.
Table V. In vitro antibacterial activities (MIC) values for compounds 25-32.
Antifungal activity
The in vitro antifungal activity () of the synthesized novel heterocyclic compounds, 25-32 was studied against the fungal strains viz., A. flavus, Mucor, Rhizopus and M. gypsuem. Fluconazole was used as a standard drug whose zone of inhibition (mm) values for A. flavus, Mucor, Rhizopus and M. gypsuem was 20 ± 0.5 zone of inhibition (mm) against all the tested fungi. Minimum inhibitory concentration (MIC) in μg/mL values is reproduced in . In general, all the synthesized compounds exerted a wide range of modest in vitro antifungal activity against all the tested organisms. Moreover, of all the compounds tested, compounds 27, 28, 30 and 32 are more effective against the tested fungal strains than the standard drug, Fluconazole.
Table VI. In vitro antifungal activities (MIC) values for compounds 25-32.
Conclusion
A close examination of the in vitro antibacterial and antifungal activity profile in differently substituted novel 4,6-diaryl-4,5-dihydro-3-hydroxy-2[H]-indazole 25-32 against the tested bacterial strains viz. S. aureus, β-H. streptococcus, V. cholerae, S. typhii, S. felxneri, E. coli, K. pneumonia and Pseudomonas and the fungal strains viz., A. flavus, Mucor, Rhizopus and M. gypsuem respectively, provides a better structure activity relationship correlate, which may be summarized as follows:
Results of this study show that the nature of substituent on the phenyl ring viz., chloro, nitro as well as the bromo functions at the meta and para positions of the aryl moieties are determinant for the nature and extent of the activity of the synthesized compounds, which might have influences on their inhibiting mechanism of actions. The method of action of these compounds is unknown. These observations may promote a further development of our research in this field. Further development of this group of compounds may lead to compounds with better pharmacological profile than standard drugs and serve as templates for the construction of better drugs to combat bacterial and fungal infection.
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