Molecular Hybridization and Preclinical Evaluation of Imines From ...

Send Orders for Reprints to [email protected] 60

Anti-Infective Agents, 2015, 13, 60-64

Molecular Hybridization and Preclinical Evaluation of Imines From Para-substituted 4-phenyl 2-amino Thiazole Incorporated with Isatin Analogues as Antitubercular Agents Nice Joy1 and Bijo Mathew2,* 1

Department of Pharmaceutical Chemistry, University College of Pharmacy, Cheruvandoor, Kottayam-686631, Kerala, India; 2Division of Drug Design and Medicinal, Chemistry Research Lab., Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Palakkad-678004, Kerala, India Abstract: A series of isatin imines incorporated with 4-sustituted phenyl 2-amino thiazole was synthesized on the basis of molecular hybridization drug design principle. The structures of the derivatives were confirmed by IR, 1HNMR and Mass analyses. The titled derivatives were screened against M.tuberculosis strain H37RVusing alamar blue susceptibility test. Compound 3-{[4-(4hydroxyphenyl)-1, 3-thiazol-2-yl] imino}-1, 3-dihydro-2H-indol-2-one (IIT2) was found to be most active with a MIC of 6.25µg/ml. Preclinical evaluation of the compounds was ascertained by in silico toxicity and ADME parameters. It has been concluded that the hydrogen contributing groups present in the phenyl system of the titled scaffold favours the activity ratio.

Keywords: Analogues, antitubercular agents, isatin, mycobacteria, molecular hybridization, thiazole. INTRODUCTION Tuberculosis (TB) is considered as the most devastating infectious disease caused by different species of mycobacteria. The primary source of TB infection is viable tubercular bacilli, expelled in the environment by coughing, sneezing, shouting, and singing of a patient with active TB and the air is contaminated with these bacilli. Inhaled bacilli in a person are inoculated into his respiratory bronchioles and alveoli, usually towards the apex of the lung. When the inhaled microorganisms multiply to a sufficient extent, an antigen–antibody interaction is evoked by the cell-mediated T-lymphocytes. Tubercles are then formed because of accumulation of macrophages at the site of infection [1-3]. A number of anti-TB candidate are ineffective against this disease because of development of resistance strains. Targets from cell wall biosynthesis, nucleic acid biosynthesis, and many other biosynthetic pathways are being nowadays utilized for drug development of promising anti-tubercular agents [4]. The most interesting application of isatins in organic synthesis is undoubtedly due to the highly reactive C-3 carbonyl group that is a prochiral center as well. The reactions of isatin such as nucleophilic addition and spriroannulation mainly due to the reactive C-3 carbonyl group of the structure [5]. A detailed investigation on this aspect, reactive C3 carbonyl group of isatins led to successful design and synthesis of various imines connected by various carbocyclic *Address correspondence to this author at the Division of Drug Design and Medicinal, Chemistry Research Lab, Department of Pharmaceutical Chemistry, Grace College of Pharmacy, Palakkad-678004, Kerala, India; Tel: +91 9946 700219; E-mail: [email protected] 2211-3533/15 $58.00+.00

and heterocyclic rings with interesting pharmacological profile such as anti-HIV, anticancer, antitubercular and antimicrobial activities [6-11]. Molecular hybridization is a modern concept of drug design principle based on the combination of different pharmacophore moieties of different bioactive substances to produce a new hybrid compound with improved affinity and efficacy, when compared with the parent drugs [12]. So with the aim of this and the continuous program of the early research work on the antitubercular discovery and the synthesis of imines structural core [13-17], we planned to synthesis a new series of imines linked between the isatin analogues and phenyl substituted thiazole derivatives. The objective of our study was to find out a new chemical entity (NCE) of antitubercular agents with the aim of the molecular hybridisation principle. EXPERIMENTAL Chemistry Melting points of all the synthesized derivatives were determined by open-capillary tube method and values were uncorrected. IR spectra were recorded on Shimadzu FT/IR spectrometer on KBr pellets. 1H NMR were recorded on a Bruker 400 mhz 1H-NMR spectrometer using CDCl3 as the solvent. Mass spectra were recorded on a JEOL GCmate mass spectrometer. Synthesis General Synthesis of 4-substituted phenyl-1, 3-thiazol-2amines (1) 4-substituted acetophenone (0.05 mol), thiourea (0.1 mol) and iodine (0.05 mol) were triturated well in a mortar and © 2015 Bentham Science Publishers

Molecular Hybridization and Preclinical Evaluation of Imines

pestle. Mixture was then transferred into a dried round bottom flask fitted with a water condenser and refluxed for 7-8 hrs. The semi-solid obtained was washed with diethyl ether to remove unreacted acetophenone followed by saturated solution of sodium thiosulphate to remove unreacted iodine. It was then filtered and the residue obtained was dissolved in hot water. Strong ammonia solution was added into the filtrate until the pH was 8-9 in order to precipitate 2 amino 4 (p-substituted) phenyl thiazole. Recrystallization of the product was done from ethanol: chloroform mixture. Synthesis of Imines A mixture of 1 (0.01) and isatin (0.01) in ethanol 25 ml was refluxed for 6h in the presence of few drops of acetic acid. The product formed is filtered, washed with water and recrystallized from absolute ethanol. 3-[(4-phenyl-1, 3-thiazol-2-yl) imino]-1, 3-dihydro-2Hindol-2-one FT-IR (KBr) vmax/cm-1:3342(NH), 3432(Ar-OH), 1693 (C=O), 1618(C=N), 710(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.48(s, 1H, C5-Thiazole), 6.63-7.12(m, 8H, Ar-H), 10.32(s, 1H, NH). MS: m/z (M+1) +305. 3-{[4-(4-hydroxyphenyl)-1, 3-thiazol-2-yl]imino}-1,3dihydro-2H-indol-2-one FT-IR (KBr) vmax/cm-1:3342(NH), 3439(Ar-OH), 1695(C=O), 1619(C=N), 707(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.44(s, 1H, C5-Thiazole), 6.67-7.16(m, 8H, Ar-H), 9.31(Ar-OH), 10.25(s, 1H, NH). MS: m/z (M+1) +321. 3-{[4-(4-methoxyphenyl)-1, 3-thiazol-2-yl]imino}-1,3dihydro-2H-indol-2-one FT-IR (KBr) vmax/cm-1:3437(NH), 1693(C=O), 1634(C=N), 715(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 3.83 (s, 3H, OCH3), 6.59(s, 1H, C5-Thiazole), 6.69-7.72(m, 8H, Ar-H), 10.34(s, 1H, NH). MS: m/z (M+1) +335. 3-{[4-(4-nitrophenyl)-1, 3-thiazol-2-yl]imino}-1,3-dihydro2H-indol-2-one FT-IR (KBr) vmax/cm-1:3342(NH), 1686(C=O), 1621(C=N), 712(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.76 (s, 1H, C5-Thiazole), 6.81-7.34(m, 8H, Ar-H), 10.22(s, 1H, NH). MS: m/z (M+2) +350. 5-chloro-3-[(4-phenyl-1, 3-thiazol-2-yl)imino]-1,3-dihydro2H-indol-2-one FT-IR (KBr) vmax/cm-1:3358(NH), 1682(C=O), 1609(C=N), 714(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.32 (s, 1H, C5-Thiazole), 6.65-7.13(m, 7H, Ar-H), 10.11(s, 1H, NH). MS: m/z (M+2) +339. 5-chloro-3-{[4-(4-hydroxyphenyl)-1, 3-thiazol-2-yl] imino}1, 3-dihydro-2H-indol-2-one FT-IR (KBr) vmax/cm-1:3449(NH), 3432(Ar-OH), 1698(C=O), 1620(C=N), 717(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.40(s, 1H, C5-Thiazole), 6.61-7.14(m, 8H, Ar-H), 9.37 (Ar-OH), 10.16(s, 1H, NH). MS: m/z (M+2) +355.

Anti-Infective Agents, 2015, Vol. 13, No. 1

61

5-chloro-3-{[4-(4-methoxyphenyl)-1, 3-thiazol-2-yl] imino}1, 3-dihydro-2H-indol-2-one FT-IR (KBr) vmax/cm-1:3342(NH), 1695(C=O), 1612(C=N), 702(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 3.68 (s, 3H, OCH3), 6.59(s, 1H, C5-Thiazole), 6.61-7.16(m, 8H, Ar-H), 10.40(s, 1H, NH). MS: m/z (M+1) +369. 5-chloro-3-{[4-(4-nitrophenyl)-1, 3-thiazol-2-yl] imino}-1, 3-dihydro-2H-indol-2-one FT-IR (KBr) vmax/cm-1:3456(NH), 1690(C=O), 1609(C=N), 717(C-S-C). 1H NMR (CDCl3, 400 MHz, δ ppm): 6.59 (s, 1H, C5-Thiazole), 6.76-7.19(m, 7H, Ar-H), 10.15(s, 1H, NH). MS: m/z (M+1) +369. In silico Studies ADMET Prediction The druglikeness properties of the compounds were evaluated by Lipinski’s rule of 5. The current study also calculated the toxicity risks parameters such as mutagenicity, tumorigenic, irritation and reproductive effects of the synthesized derivatives. Toxicity prediction of the newly designed scaffold was retrieved from a web-based application for Organic Chemistry Portal (http://www.organic-chemistry.org/ prog). Prediction results are valued and colour coded. Properties with high risks shown in red. Whereas a yellow and green colour indicates medium risk and drug-conform behaviour respectively [18, 19]. Antitubercular Activity The anti-mycobacterial activity of synthesized compounds (IIT1-IIT8) were screened against M. tuberculosis using microplate Alamar Blue assay. This methodology is non-toxic, uses a thermally stable reagent and shows good correlation with proportional and BACTEC radiometric method. Briefly, 200µl of sterile deionzed water was added to all outer perimeter wells of sterile 96 wells plate to minimized evaporation of medium in the test wells during incubation. The 96 wells plate received 100 µl of the Middlebrook 7H9 broth and serial dilution of compounds were made directly on plate. The final drug concentrations tested were 100 to 0.2 -100µg/ml. Plates were covered and sealed with parafilm and incubated at 37oC for five days. After this time, 25µl of freshly prepared 1:1 mixture of Alamar Blue reagent and 10% tween 80 was added to the plate and incubated for 24 h. A blue colour in the well was interpreted as no bacterial growth, and pink colour was scored as growth. The MIC was defined as lowest drug concentration which prevented the colour change from blue to pink [20]. RESULTS AND DISCUSSION Chemistry The synthesis of the titled derivatives (IIT1-IIT8) was accomplished in two steps and is outlined in (Fig. 1). Initially the substituted acetophenone reacted with thiourea in presence of iodine by oxidative cyclization method generating respective 2-amino, 4-phenyl thiazole [21]. This intermediate was further condensed with isatin under Schiff reaction in the presence of acetic acid, which yielded the corre-

62

Anti-Infective Agents, 2015, Vol. 13, No. 1

sponding imines [22-24]. The FT-IR spectrum of compound IIT3 showed three bands at 1693 cm-1, 1634 cm-1 and 715 cm-1 which is due to C=O, C=N and C-S-C- respectively. In addition the 1H-NMR spectrum of IIT3 displayed a sharp singlet at δ 10.34 ppm which is due to NH proton and also a sharp singlet at δ 3.83 ppm for OCH3 of the phenyl ring. The product formation was further substantiated by its mass spectra, where in a mass peak of 335(M+1) was observed which is in accordance with its molecular formula C18H13N3O2S. The mass fragmentation pattern of the IIT3 is shown in (Fig. 2).

Fig. (1). Synthetic route of the titled derivatives.

In silico Studies ADMET Assessment The “drug- likeness” nature of the newly synthesized thiazole-isatin hybrids was assessed by “Lipinski rule of Five”. Interestingly all the derivatives have zero violation, which indicated their probability of drug-likeness nature (Table 1). From the assessment it was found that the ana-

Fig. (2). Mass fragmentation pattern of IIT3.

Joy and Mathew

logues such as IIT4 and IIT8 have high risk of tumorigenic, mutagenic and medium risk of reproductive effects since they are found to have a very reactive –N=O group that may give rise to toxicity alerts (Table 2). Antitubercular Activity The discovery of the potent antitubercular activity of isatin based analogues led in the past decade to extensive synthesis of related compounds. The preliminary in vitro antitubercular activity of the synthesized compounds (IIT1IIT8) evaluated by alamar blue assay method against M. tuberculosis H37Rv and the observed minimum inhibitory concentrations (MIC) are tabulated in Table 3. All the compounds showed comparatively good activity with a MIC ranges from 6.25 to 50 µg/ml when compared to the standard pyrazinamide. Among the series, compound 3-{[4-(4hydroxyphenyl)-1, 3-thiazol-2-yl] imino}-1, 3-dihydro-2Hindol-2-one (IIT2) was found to be most active with a MIC of 6.25µg/ml. The presence of OH and OCH3 group in the para position of phenyl system of IIT3, IIT5 and IIT7 also showed good antitubercular activity with a MIC of 12.5 µg/ml. These results also suggested that the presence of electron donating group in the para position of phenyl system can contribute promising antitubercular results. However in a preliminary structure activity relationship assessment, most of the active compound contains the presence of strong hydrogen-bonding group like OH and OCH3 . Thus, it seems that the formation of hydrogen bonds may play a significant role in the drug action of the current scaffold. So the presence of these substituents in the aromatic ring of the titled scaffold may contribute significant activity. Another interesting factor observed that the combination of two electron withdrawing groups like NO2 and Cl in the phenyl and isatin nucleus cannot favours the activity ratio.

Molecular Hybridization and Preclinical Evaluation of Imines

Table 1.

Anti-Infective Agents, 2015, Vol. 13, No. 1

Lipinski rule evaluation by www.molinspiration.com

Compound code

logP

H-bond donors

H-bond acceptor

MW

No rotatable bonds

No of violations

IIT1

3.96

1

4

305

2

0

11T2

3.48

2

5

321

2

0

11T3

4.02

1

5

335

3

0

11T4

3.92

1

7

350

3

0

11T5

4.62

1

4

339

2

0

11T6

4.14

2

5

355

2

0

11T7

4.67

1

5

369

3

0

11T8

4.59

1

7

3842

3

0

Table 2.

Toxicity risk assessment osiris property explorer.

Compound code

Tumorigenic

Mutagenic

Irritant

Reproductive effects

IIT1

No

Medium

No

No

11T2

No

Medium

No

No

11T3

No

Medium

No

No

11T4

High

High

No

Medium

11T5

No

Medium

No

No

11T6

No

Medium

No

No

11T7

No

Medium

No

No

11T8

High

High

No

Medium

Table 3.

63

Physical constant and antimycobacterial activity of the synthesized compounds (IIT1-IIT8).

Compound code

R

R1

Yield (%)

Mp ( oC)

Rf *

MIC (µg/ml)

IIT1

H

H

42

165-167

0.53

25

11T2

OH

H

52

143-146

0.72

6.25

11T3

OCH3

H

65

132-134

0.64

12.5

11T4

NO2

H

32

161-163

0.57

25

11T5

H

Cl

34

215-217

0.77

12.5

11T6

OH

Cl

72

201-203

0.62

12.5

11T7

OCH3

Cl

69

195-197

0.56

12.5

11T8

NO2

Cl

30

222-225

0.74

50

Pyrazinamide

-

-

-

-

-

3.125

*

TLC (Chloroform: Ethanol) - 9:1.

CONCLUSION In conclusion, the present report described the synthesis of isatin−thiazole hybrids connected by imine linker on the basis of molecular hybridisation principle and evaluated their

anti-tubercular activity. The promising activities of these titled compounds could be attributed to the incorporation of para phenyl substituted thiazole to the isatin. We claimed that the presence of para substituted hydroxyl group in the

64

Anti-Infective Agents, 2015, Vol. 13, No. 1

Joy and Mathew

phenyl nucleus of the mentioned scaffold improves the solubility and a crucial factor for hydrogen bonding with receptor. So it can be concluded that a combination of para phenyl substituted thiazole with isatin showed promising results in anti-tubercular activity and further modification is needed to obtain more efficacious antituberculosis compounds. In addition we also hypothesised that the combination of more electron donating and hydrogen bonding participating group in the 4th and 6th position of phenyl and isatin system could generate more promising antitubercular candidates. CONFLICT OF INTEREST

[10] [11] [12] [13]

[14]

The author(s) confirm that this article content has no conflict of interest. [15]

ACKNOWLEDGEMENTS The authors are highly thankful to Maratha Mandal’s Nathajirao G. Halgekar Institute of Dental Sciences and Research Centre, Belgaum for antitubercular activity. Our sincere thanks goes to IIRBS, Mahatma Gandhi University, Kottayam and SAIF IIT, Chennai, for carrying out the spectral analysis.

[16]

[17]

REFERENCES [1] [2] [3]

[4] [5] [6]

[7] [8] [9]

Daniel, T.M.; Bates, J.H.; Downes, K.A. History of tuberculosis. In: Bloom BR, editor. Tuberculosis: Pathogenesis, protection and control. Washington, DC: ASM Press; 1994. pp 13-24. Nardell, E. Pathogenesis of tuberculosis. In: Reichman LB, Herschfield E, editor. Lung biology in health and disease. New York: Marcel Dekker, Inc.; 1993. pp 103-123. Thomas, K.D.; Adhikari, A.V.; Telkar, S.; Chowdhury, I.H.; Mahmood, R.; Pal, N.K. Design, synthesis and docking studies of new quinoline-3-carbohydrazide derivatives as antitubercular agents. Eur. J. Med. Chem., 2011, 46, 5283-5292. Tripathi, R.P.; Tewari, N.; Dwivedi, N.; Tiwari, V.K. Fighting tuberculosis: an old disease with new challenges. Med. Res. Rev., 2005, 25, 93-131. Sing, G.S.; Desta, Z.Y. Isatins as privileged molecules in design and synthesis of spiro- fused cyclic frameworks. Chem. Rev., 2012, 112, 6104-6155. Pandeya, S.N.; Sriram, D.; Nath, G.; De Clercq, E. Synthesis, antibacterial, antifungal and anti- HIV activity of Schiff and Mannich bases of isatin with N-[6-chlorobenzthiazole-2-yl]thiosemicarbazide. Indian J. Pharm. Sci., 1999, 61, 358-361. Bal, T.R.; Anand, B; Yogeeswari, P.; Sriram, D. Synthesis and evaluation of anti-HIV activity of isatin β -thiosemicarbazone derivatives. Bioorg. Med. Chem. Lett., 2005, 15, 4451-4455. Solomon, V.R.; Hu, C.; Lee, H. Hybrid pharmacophore design and synthesis of isatin–benzothiazole analogs for their anti-breast cancer activity. Bioorg. Med. Chem., 2009, 17, 7585-7492. Aboul-Fadl, T.; Bin-Jubair, F.A.; Aboul-Wafa, O. Schiff bases of indoline-2, 3-dione (isatin) derivatives and nalidixic acid carbohydrazide, synthesis, antitubercular activity and pharmacophoric model building. Eur. J. Med. Chem., 2010, 45, 4578-4586.

Received: February 24, 2014

Revised: September 05, 2014

Accepted: September 05, 2014

[18]

[19] [20]

[21]

[22]

[23] [24]

Sridhar, S.K.; Saravanan, M.; Ramesh, A. Synthesis and antibacterial screening of hydrazones, Schiff and Mannich bases of isatin derivatives. Eur. J. Med. Chem., 2001, 36, 615-625. Prakash, C.R.; Raja, S. Synthesis, characterization and in vitro antimicrobial activity of some novel 5-substituted schiff and mannich base of isatin derivatives. J. Saud Chem. Soc., 2013, 17, 337-344. Viegas-Junior, C.;Danuello, A.; da Silva Bolzani, V.; Barreiro, E.J.; Fraga, C.A. Molecular hybridization: a useful tool in the design of new drug prototypes. Curr. Med. Chem., 2007, 14, 1829-1852. Mathew, B.; Mathew, G.E.; Sonia, G.; Kumar, A.; Charles, N.P. Design of 1-(furan-2-yl)-N-(5-substituted phenyl-1, 3, 4-thiadiazol -2-yl) methanimine derivatives as Enoyl-ACP reductase inhibitors: Synthesis, molecular docking studies and anti-tubercular activity. Bangladesh J. Pharmacol., 2013, 8, 242-248. Mathew, B.; Suresh, A.J.; Anbazhagan, S. Synthesis and in silico design of some novel imines of 5- amino-1, 3, 4-thiadiazole-2-thiol linked to (1h-benzimidazole-2-yl) 3-substituted phenyl prop-2enes. Indian J. Heterocycl. Chem., 2013, 22, 337-340. Mathew, B.; Suresh. J.; Anbazhagan, S.; Chidambaranathan N. Discovery of some novel imines of 2-amino, 5-thio, 1, 3, 4thiadiazole as mucomembranous protector. Synthesis, anti-oxidant activity and in silico PASS approach. J. Saud Chem. Soc., 10.1016/j.jscs.2013.01.002. Mathew, B.; Suresh. J.; Anbazhagan, S.; Hypnotic profile of novel imines from benzimidazole chalcones: Mechanism of Synthesis, DFT studies and In silico preclinical evaluation. Cent. Nerv. Syst. Agents in Med. Chem., 2013, 13, 207-216. Mathew, B.; Suresh, J.; Mathew, G.E.; Sonia, G.; Krishnan, G.K.; Design, synthesis, toxicity estimation and molecular docking studies of N-(furan-2-yl)-1-(5-substituted) phenyl-1, 3, 4.oxadiazol-2yl) methanimine as antitubercular agents. Indian J. Pharm. Sci., 2014, 76, 401-406. Lipinski, C.; Lombardo, F.; Dominy, B.; Feeney, P. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Drug Deliv. Rev., 1997, 23, 3-25. Sander, T.; Freyss, J.; Korff, M.V.; Reich, J.R.; Rufener ,C. OSIRIS, an entirely in-house developed drug discovery informatics system. J. Chem. Inf. Model., 2009, 49, 232-246. Lourenco, M.C.S.; deSouza, M.N.; Pinheiro, A.C.; Ferreira, M.L.; Goncalves, R.B.; Nogneira, T.C.; Peralta, M.A. Evaluation of antitubercular activity of nicotinic and isoniazid analogues. Arkivoc., 2009, 15, 181-191. Pandeya, S.N.; Sriram, D.; Nath, G.; Declerch, E. Synthesis, antibacterial, antifungal and anti-HIV activities of Schiff and Mannich bases derived from isatin derivative and N-[4-(49-chlorophenyl) thiazol-2-yl]thiosemicarbazide. Eur. J. Pharm. Sci., 2000, 35, 249254. Sinha, R.; Sara, U.V.S.; Khosa, R.L.; Stables. J.; Jain. J. In silico validation and structure activity relationship study of a series of pyridine-3-carbohydrazide derivatives as potential anticonvulsants in generalized and partial seizures. Cent. Nerv. Syst. agents Med. Chem., 2013, 13, 132-140. Guzel, O.; Karali, N.; Salman, A. Synthesis and anituberculosis activity of 5-methyl/trifluromethoxy 1H-indole-2,3-dione thiosemicarbazone derivatives. Biorg. Med. Chem., 2008, 16, 8976-8987. Banerjee, D.; Yogeeswari, P.; Bhat, P.; Thomas, A.; Srividya, M.; Sriram, D. Novel isatinyl thiosemicarbazones derivatives as potential molecule to combat HIV-TB co-infection. Eur. J. Med. Chem., 2011, 46, 106-121.