Among infections and chronic communicable diseases,tuberculosis’s yearly death toll (TB) is second only to HIV/AIDS. In the last few decades,there has been an enormous increase in drug resistant pathogens and a rise in multidrug resistantMycobacterium tuberculosisstrains. However,potent antimycobacterial drugs with effective efficacy have not been produced in the last 40 years ^[1]. Different research groups are working toward the development of novel anti-tubercular drugs for the discovery of newer classes of compounds which are structurally different from known anti-tubercular drugs ^{[2, 3, 4, 5, 6]}. The antimycobacterial activity of 5-aryl-1-isonicotinoyl-3-(pyridin-2-yl)-4,5-dihydro-1H-pyrazole derivatives and N¹ -nicotinoyl-3-(4-hydroxy-3-methylphenyl)-5-[(sub)phenyl]-2-pyrazolines have been reported ^{[7, 8]}. Further, various pyrazoline derivatives have been found to have antitubercular ^[9] and antiamoebic activity ^[10] (Fig. 1).

Fig. 1

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Fig. 1. Key anti-tubercular agents

There are additional benefits of the substituted pyrazolines, such as anti-tubercular ^[11],anti-inflammatory ^{[12, 13, 14, 15]},antimicrobial ^[16],anticancer ^[17],MAO inhibition ^[18],and anticonvulsant ^[19] activities. Taking into account the previously stated significance of heterocyclic compounds,in the present study,a series of novel hexahydro-3-(phenylindazol-2-yl)(pyridin-4-yl)methanones derived from the anti-tubercular drug isoniazid is synthesized and assessed for in vitro anti-tubercular potential. Various conventions for the control of TB involving three key drugs are currently utilized as a part of the regimen. These drugs, isoniazid,pyrazinamide and rifampicin,are hepatotoxic and may prompt drug-induced hepatitis. Subsequently,an attempt has been made in our continued research interests ^{[20, 21, 22, 23, 24, 25, 26, 27, 28, 29]} in the present study to synthesize some indazol-2-yl(pyridin-4-yl)methanones. 2. Experimental

The chemicals and reagents were acquired from Sigma-Aldrich (India) and SD-Fine (India) and utilized without further purification. Infrared spectra were recorded on a Perkin-Elmer 1720 FT IR spectrometer,with samples analyzed as KBr plates. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AC 400 MHz, spectrometer using tetramethyl silane (TMS) as an internal standar δ in CDCl₃chemical shiftsdin ppm. The HRMS spectra were recorded on a JEOLSX 102/D. GC mass spectra were recorded on a Perkin Elmer GC clarus 680 and Mass Spectrometer clarus 600, with ionization induced by electron impact. 2.1. General procedures for the synthesis of 2,6-bis(benzylidene) cyclohexanones (3a-k)

A mixture of cyclohexanone 1 (5 mmol),aldehydes 2 (10 mmol),and 20 mol% solid sodium hydroxide were taken in a clean mortar and ground well with a pestle at room temperature for 5-10 min. A sufficient quantity of 2 mol/L HCl was added to the reaction mixture. The resulting yellow solid material was washed with sodium bicarbonate solution and water ^[30],collected by filtration,dried,and purified by recrystallization using ethanol: chloroform (9:1).

2,6-Bisbenzylidenecyclohexanone (3a): Yield 94%,yellow crystals,Mp 117-119℃; FT-IR (KBr,cm^-1 )(C=O) 1660; ¹H NMR (400 MHz,CDCl₃):δ1.72 (t,2H,J=6 Hz),2.86 (t,4H,J=12 Hz, CH),7.27 (t,2H,J=7.2 Hz,Ar-H),7.34 (t,4H,J=7.6 Hz,Ar-H) 7.40 (d,4H,J=7.6 Hz,Ar-H),7.73 (s,2H,C-H); ¹³C NMR (100 MHz, CDCl₃):δ23.16,28.60,128.53,128.74,130.52,136.11,136.32, 137.10,190.58. C₂₀H₁₈O m/z: 274.1358,found 274.3006.

2,6-Bis(4-chlorobenzylidene)cyclohexanone (3b): Yield 92%, yellow crystals,Mp 146-148℃; FT-IR (KBr,cm^-1 )(C=O) 1666; ¹H NMR (400 MHz,CDCl₃):δ1.77-1.83 (m,2H,J=6 Hz),2.90 (t,4H, J=5.6 Hz,CH),7.39 (t,8H,J=6.4 Hz,Ar-H),7.73 (s,2H,C-H); ¹³C NMR (100 MHz,CDCl₃):δ22.97,24.21,28.53,28.74,35.19,128.25, 128.82,128.84,130.82,131.65,131.73,134.46,134.77,135.18, 135.94,136.26,136.57,137.98,190.05. C₂₀H₁₆Cl₂Om/z: 343.2464, found 342.1759.

2,6-Bis(4-methyl benzylidene)cyclohexanone (3c): Yield 94%, yellow crystals,Mp 164-166℃; FT-IR (KBr,cm^-1 )(C=O) 1660; ¹H NMR (400 MHz,CDCl₃):δ1.78 (t,2H,J=6 Hz),2.38 (s,6H,CH3), 2.93 (t,4H,J=5.6 Hz,C-H),7.21 (d,4H,J=7.6 Hz,Ar-H),7.38 (d,4H,J=7.6 Hz,Ar-H),7.77 (s,2H,C-H); ¹³C NMR (100 MHz, CDCl₃):δ 21.55,23.16,28.67,129.27,130.62,133.34,135.63, 137.01,138.95,190.58. C₂₂H₂₂Om/z: 302.4095,found 302.3822. 2.2. General procedures for the synthesis of hexahydro 3-phenylindazol-2-yl)(pyridin-4-yl)methanones (4d-j)

To a mixture of the bisbenzylidenecycloalkanones 3 (1 mmol), isoniazid (2 mmol),absolute ethanol (5 mL),a catalytic amount of p-toluene sulphonic acid (PTSA) (50 mg) was included as catalyst and refluxed for 8-10 h. The progress of the reaction was monitored by TLC on silica gel (petroleum ether:EtOAc = 6:4). After completion of the reaction,the mixture was cooled to room temperature and added drop wise into a beaker containing crushed ice and allowed to stand for 15 min. The product was separated, filtered,washed several times with water,and purified by recrystallization using ethanol.

7-Benzylidene-3,3a,4,5,6,7-hexahydro-3-phenylindazol-2-yl)(pyridin-4-yl)methanone (4a): Yield 88%,colorless solid,Mp 201-205℃; FT-IR (KBr,cm^-1 )(C=O) 1624.06; ¹H NMR (400 MHz, CDCl₃):δ1.41-1.51 (m,1H),1.69 (t,3H,J=11.6 Hz),1.83 (d,1H, J=12.8 Hz),2.21 (t,1H,J=13.6 Hz),3.00 (d,1H,J=15.6 Hz),3.42- 3.48 (m,1H),5.84 (d,1H,J=10.8 Hz),7.16 (d,2H,J=6.8 Hz),7.25- 7.36 (m,8H),7.85 (d,2H,J=8.0 Hz),8.72 (s,2H); ¹³C NMR (100 MHz,CDCl₃):δ 23.95,25.78,28.47,49.41,65.13,123.82, 126.28,128.07,128.10,128.47,129.00,129.86,129.93,130.09, 135.85,136.61,142.01,149.86,161.18,163.85. HRMS [EI,M+]m/z calcd. for C₂₆H₂₃N₃O 393.1840,found 393.4800.

7-(4-Chlorobenzylidene)-3-(4-chlorophenyl)-3,3a,4,5,6,7-hexahydroindazol-2-yl)(pyridin-4-yl)methanone (4b): Yield 84%,light yellow color solid,Mp 222-224℃; FT-IR (KBr,cm^-1 )(C=O) 1618.28; ¹H NMR (400 MHz,CDCl₃):δ0.83-0.92 (m,1H),1.22- 1.25 (d,2H,J=10.4 Hz),1.42-1.52 (m,1H),1.72-1.75 (d,1H, J=11.6 Hz),2.15-2.22 (t,1H,J=13.6 Hz),2.9-2.96 (d,1H, J=15.6 Hz),3.41-3.47 (m,1H),5.79-5.82 (d,1H,J=10.8 Hz), 7.09-7.10 (d,2H,J=7.6 Hz),7.18 (s,1H),7.24-7.26 (d,1H, J=6.4 Hz) 7.33-7.34 (d,4H,J=7.6 Hz),7.813-7.823 (t,2H, J=2.4 Hz),8.72-8.73 (d,2H,J=4.0 Hz); ¹³C NMR (100 MHz, CDCl₃):δ 23.82,25.74,28.44,49.20,64.58,123.72,127.66, 128.74,128.78,129.30,130.46,131.17,133.96,134.04,134.17, 135.13,141.67,149.95,160.79,163.92. HRMS [EI,M+]m/zcalcd. for C₂₆H₂₁Cl₂N₃O 462.0680,found 462.3700.

7-(4-Methylbenzylidene)-3,3a,4,5,6,7-hexahydro-3-(4-methylphenyl)indazol-2-yl)(pyridin-4-yl)methanone (4c): Yield 78%,colorless solid,Mp 195-198℃; FT-IR (KBr,cm^-1 )(C=O) 1620.21; ¹H NMR (400 MHz,CDCl₃):δ0.85-0.94 (m,1H),1.40- 1.49 (m,1H),1.71 (d,1H,J=11.2 Hz),1.81 (s,3H),2.16 (s,6H),2.98 (d,1H,J=15.6 Hz),3.38-3.45 (m,1H),5.79 (d,1H,J=1.2 Hz),7.04 (d,2H,J=7.6 Hz),7.16 (t,4H,J=6.0 Hz),7.24 (t,2H,J=7.6 Hz),7.82 (d,2H,J=4.4 Hz),8.70 (d,2H,J=4 Hz); ¹³C NMR (100 MHz,CDCl₃):δ 21.26,21.44,23.90,25.70,28.48,29.81,31.05,49.34,64.98, 123.80,126.18,129.15,129.31,129.65,129.76,129.90,133.00, 133.61,137.67,138.12,142.08,149.79,161.36,163.74. HRMS [EI, M+]m/zcalcd. for C₂₈H₂₇N₃O 421.2160,found 421.5300.

Other compounds 3d-k and 4d-j spectral data are presented in the Supporting information. 2.3. Anti-tubercular evaluation procedures

The anti-tubercular activity of compounds4d-j was surveyed againstM. tuberculosisusing the microplate Alamar Blue assay (MABA). Sterile deionized water (200mL) was added to all external edge wells of a sterile 96 well plate to minimize the dissipation of medium in the test wells amid incubation. The 96 well plates received 100mL of the Middlebrook 7H9 broth,and serial dilutions of test compounds (4d-j) were made. The final drug concentrations tested ranged from 100 to 0.8μg/mL. Plates were secured and fixed with parafilm and incubated at 37℃ for 5 days. After this time, 25mL of freshly prepared 1:1 mixture of Alamar Blue reagent was added to the plate (10%-80% volume) and incubated for 24 h. A blue color in the well was interpreted as no bacterial development,while the pink color was scored as development ^[31]. Dimethyl sulphoxide was used as control. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of a drug which prevented the color change from blue to pink and the MIC values. 3. Results and discussion 3.1. Synthesis

The synthesis of 2,6-bisbenzylidenecyclohexanones 3 and hexahydro-3-phenyl indazol-2-yl(pyridin-4-yl)methanones 4 derived from the anti-tubercular drug isoniazid is delineated in Scheme 1.

Scheme 1

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Scheme 1.Synthesis of indazol-2-yl(pyridin-4-yl)methanones.

The 2,6-bisbenzylidenecyclohexanones 3a-k,were acquired in good yield by means of an effective Claisen-Schmidt condensation reaction (using 20 mol% solid sodium hydroxide,grinding techniques,and a simple work up procedure) ^[30]. All the incorporated compounds were purified by recrystallization using (9:1) absolute ethanol and chloroform and were characterized by FTIR, ¹H NMR, ¹³C NMR and mass spectroscopic techniques (Supporting information).

The hexahydro-3-phenyl indazol-2-yl(pyridin-4-yl)methanones4d-j (Table 1) were acquired by refluxing isoniazid and 2,6-bisbenzylidenecyclohexanones 3 for 8-10 h in the presence of a catalytic amount ofp-toluene sulphonic acid in ethanol. Product yields increased when the reaction was performed with a 1:2 mole ratio of 2,6-bisbenzylidenecyclohexanone and isoniazid. All the reactions were free from by-products.

Table 1

Table 1 Synthesis of 2,6-bisbenzylidenecyclohexanones 3a and hexahydro-3-phenyl indazol-2-yl(pyridin-4-yl)methanones 4b

Table 1 Synthesis of 2,6-bisbenzylidenecyclohexanones 3a and hexahydro-3-phenyl indazol-2-yl(pyridin-4-yl)methanones 4b

In the NMR spectra,two of the benzylidine protons (C-H) of the 2,6-bisbenzylidene cyclohexanones,3 appeared as a singlet betweend7.7-7.91 for all the compounds except 2,6-bis((furan-2-yl)methylene)cyclohexanone (3f),which appeared atd6.51. The base-catalyzed reactions were emulated by means of an enolate anion donor species,and the kinetically favored proton expulsion is from the less substituteda-carbon. Likewise,the chemoselectivity of the reaction is achieved by the formation of the E isomer (on either side of the carbonyl group). This is attributable to the transition state which undergone elimination reaction to a syn double bond,for instance,due to an unfavorable steric interaction between the ketone substituent and the phenyl group. However,such interaction was missing in the transition state for elimination to the most favorable anti double bond.

The arylidene protons in theEisomers lie in the region of deshielding by the carbonyl group and subsequently show up at a higher chemical shift (downfield area) in the NMR spectrum. The methyl and methoxy protons showed up as singlet atd2.38 and betweend3.84 and 3.92,respectively. All the aliphatic protons showed up betweend1.71 and 3.02,and the remaining aromatic protons appeared betweend6.66 andd8.42. The molecular weight of all the synthesized target molecules 3a-k(Table 1) are further confirmed by their mass spectra.

In the NMR spectrum of indazol-2-yl(pyridin-4-yl)methanones 4,one of the methine protons of indazole ring showed up as a doublet at δ2.7 while the other methine proton showed up as multiplet at δ3.37,confirming the fusion of the isoniazid and arylidienylcyclohexanone. The free benzylidine proton showed up as a doublet at δ5.77,and the naphthylidine proton appeared at δ 6.74 as a doublet. The methyl and methoxy group protons showed up as singlets at δ2.16 andd3.79 respectively. All the remaining aromatic protons appeared atd6.87. The HRMS and LCMS of all the synthesized target molecules 4d-j further confirmed their structures.

To study the effects of the catalyst and solvent,the reactions were completed between 3a and isoniazid to provide 4a(Table 2) under reflux. The results indicated that among the acidic catalysts tested (Table 2,entries 1,3-7),the p-TSA emerged out as a best catalyst with a yield of 88% in 9 h followed by citric acid with a yield of 72% in 13 h (Table 2,entries 7,8). Among the different solvents tested in the presence ofp-TSA,the ethanol was effective, followed by isobutanol (Table 2,entries 8-10).

Table 2

Table 2 SOptimization of catalyst and solvent in the synthesis of indazol-2-yl(pyridin-4-yl)methanones4b

Table 2 Optimization of catalyst and solvent in the synthesis of indazol-2-yl(pyridin-4-yl)methanones4b

The optimization of catalyst and isoniazid amount used were investigated in the synthesis of hexahydro-3-phenyl indazol-2-yl(pyridin-4-yl)methanones and the results are summarized respectively in Tables 3 and 4. The results indicated that 50 mg p-TSA is necessary for highest yield (Table 3,entry 6). An increase amount ofp-TSA resulted in the decrease of yield due to hydrogen bonding interactions. In addition,isoniazid in 2 mmol likewised showed the best result with a yield of 88% (Table 4, entry 3). The decrease or increase in the amount of isoniazid have resulted in reduced yield of the desired product 4a,which are attributable to the decreased reactivity of isoniazid due to their respective intramolecular and intermolecular hydrogen bonding.

Table 3

Table 3 Optimization of catalyst amount (p-TSA).a

Table 3 Optimization of catalyst amount (p-TSA).a

Table 4

Table 4 Optimization of amount of isoniazid in the synthesis of indazol-2-yl (pyridin-4-yl)methanonesa

Table 4 Optimization of amount of isoniazid in the synthesis of indazol-2-yl (pyridin-4-yl)methanonesa

Among the tested compounds 4a-k,4g-j showed good antitubercular activity with the MIC value of 12.5μg/mL,which is comparable to the standard drugs (Table 5). All the remaining compounds showed mild anti-tubercular activity with MIC values at higher concentration,for instance,60μg/mL. 4. Conclusion

A series of indazol-2-yl(pyridin-4-yl)methanones 4 were acquired from 2,6-bisbenzylidenecyclohexanones 3and isoniazid and their anti-tubercular effects were also screened. The structures of the synthesized products were concluded from their FTIR, ¹H NMR, ¹³C NMR,and HRMS/LCMS spectroscopic results. Among the tested compounds 4 screened againstM. tuberculosisH37 Ra cell line by the microplate Alamar Blue assay,the compounds 4g-j, showed moderate anti-tubercular activity with a MIC 12.5μg/mL, comparable to those for standard medications (streptomycin,MIC, 6.25μg/mL,pyrazinamide,isoniazid and ciprofloxacin with MICs of 3.125μg/mL).

Table 5

Table 5 Anti-tubercular activities of compounds (4a-j).a

Table 5 Anti-tubercular activities of compounds (4a-j).a

The authors thank VIT University for providing us with research funding and laboratory facilities. The DST-VIT-FIST for FT-NMR and SIF-VIT University,Vellore is acknowledged for providing the NMR and GCMS facilities. The authors acknowledge Maratha Mandal Dental College,Belgaum for biological screening support.

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.cclet.2015.01.008.