Scientific paper
Synthesis and Biological Evaluation of Some Novel 3,5-Disubstituted-1,2,4-triazole Incorporated 2-Mercaptobenzothiazoles
Mohammed Afzal Azam,* Bhojraj Suresh, Naga Srinivas, Sumit Sachdev
and Raman Rajeshkumar
Department of Pharmaceutical Chemistry, J. S. S. College of Pharmacy, Ootacamund-643001, Tamil Nadu, India * Corresponding author: E-mail: afzal9azam@hotmail.com Received: 14-11-2011
Abstract
Several 2-mercaptobenzothiazole derivatives 5a-i containing 1,2,4-triazole moiety incorporating two additional substi-tuents were synthesized. All the newly synthesized compounds were tested for in vitro activity against certain strains of bacteria such as Enterococcus faecalis, Bacillus coagulans, Pseudomonas aeruginosa, Escherichia coli and Candida albicans. Compound 5a showed significant activity against the Gram-negative bacteria Escherichia coli. Compounds 5a-i were also screened for their antifungal activity against Candida albicans and compounds 5a, 5b, 5d and 5g displayed significant activity against this fungus. Some of these compounds were evaluated for their in vivo antiinflammatory activity, acute toxicity and ulcerogenic actions. Tested compounds 5g and 5h showed significant anti-inflammatory activity and significant gastrointestinal protection compared to the standard drug diclofenac sodium. Molecular modeling studies of the synthesized compounds are presented.
Keywords: 2-Mercaptobenzothiazoles, 1,2,4-triazole, antimicrobial activity, anti-inflammatory activity, ulcerogenic effect.
1. Introduction
Nonsteroidal anti-inflammatory drugs (NSAIDs), one of the most commonly used class of medications to treat inflammation and pain, block two different cyclo-oxygenases (COX-1 and COX-2). COX-2, found in joint and muscle, contributes to the pain and inflammation.1 Long term use of NSAIDs causes gastrointestinal (GI) disorders and renal toxicity2'3 because they also block the COX-1 enzyme, which protects4 the lining of the stomach from its acidic content. The introduction of the cyclooxygenase-2 (COX-2)-specific NSAIDs5 in the late 1990s promised a revolution in NSAID therapy because of their much higher specificity for the COX-2 system, but unfortunately evidence of cardiovascular side effects including an increased risk of myocardial infarction began to emerge6 causing these COX-2-specific NSAIDs to be withdrawn from the world market. Thus, the development of drugs with an effective anti-inflammatory profi-
le, but with fewer side effects than NSAIDs, would be beneficial.
A literature survey revealed that compounds containing 1,2,4-triazole moiety possess a promising biological properties like antimicrobial7 8 and anti-inflammatory.910 In addition 2-mercaptobenzothiazoles are also known to posses antimicrobial1112 and anti-inflammatory activities.1314 Based on the above observations and results of our docking study it appeared of interest to link the ben-zothiazole nucleus at the second position to some 1,2,4-triazole ring system so as to bring them in the same framework; therefore attempting to investigate the influence of such hybridization and structure variation on the anticipated biological activities, hoping to add some synergistic effects that would increase the biological significance of the target molecules. In the present investigation we aimed to synthesize 2-{[3-(3,5-disubstituted-1H-1,2,4-tria-zol-1-yl)propyl]sulfanyl}-1,3-benzothiazoles 5a-i to evaluate their antimicrobial, anti-inflammatory and ulceroge-nic activities.
2. Results and Discussion
2. 1. Chemistry
Synthesis of the titled compounds 5a-i was carried out as presented in the Scheme 1. Aryloxyacetic acids were prepared by reactions of appropriate phenols with chlo-roacetic acid in basic medium.15 4-Amino-3,5-disubstitu-ted-1,2,4-triazoles16 2a-i were synthesized by heating a mixture of the appropriate acid and hydrazine hydrate (85%) in an oil bath according to the procedure described in the literature. Intermediate 2-[(3-chloropropyl)sul-fanyl]-1,3-benzothiazole (3) was synthesized17 by stirring a mixture of 2-mercaptobenzothiazole and 1-bromo-3-chloropropane in dry toluene at 60 °C in the presence of powdered potassium carbonate. One-pot synthesis of the title compounds 2-{[3-(3,5-disubstituted-1H-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-benzothiazoles 5a-i via intermediate 4 was carried out by reactions21 (described by Ast-leford et al. in 1989) of triazoles 2a-i with compound 3 in refluxing isopropyl alcohol and subsequent treatment of the reaction mixture with conc. HCl, saturated sodium nitrite solution and saturated potassium carbonate solution.
Structures of the newly synthesized compounds were confirmed by the analytical and spectroscopic data. The infrared (IR) spectra of the synthesized compounds 5a-i showed characteristic absorption bands in the range 1703-1637 cm-1 for C=N and 2935-2908 cm-1 for CH2 stretching. The formation of the triazole ring in 5h was supported by its proton magnetic resonance (1H NMR) spectrum which showed three multiplet signals at S 8.40-7.86, 7.57-7.41 and 7.27-6.98 ppm integrated for four, ten and four aromatic protons, respectively. Two triplet signals observed at S4.12 and 2.58 ppm were assigned to the OCH2 and SCH2 fragments, while a multiplet signal at S 1.98-1.89 ppm was due to the CH2 fragment of the propyl group linking the 2-mercaptobenzothiazole and 1,2,4-triazole rings. The OCH2 fragments of 2-naphthoxy-methyl groups present on the third and fifth position of the 1,2,4-triazole ring resonated as a singlet signal at S 4.85 ppm. 13C NMR spectra were recorded for compounds 5f and 5h. In the 13C NMR spectrum of compound 5h azo-methine carbon of the benzothiazole ring exhibited a signal at S 167.10 ppm, whereas 3-C and 5-C carbons of triazole nucleus were observed at S 153.74 ppm. Three signals
Reagents and conditions:
(i)	NH2NH2.HzO, 200-270 °C, 4-6 h
(ii)	Br(CH2)3CI. toluene, K2C03, 60 °C, stirring^r.t., 1h
(iii)	isopropyl alcohol, reflux, 4448 h, 0-5 °C, conc. HCl, aq. NaN02, aq. K2C03
Scheme 1. Synthetic route of 2-{[3-(3,5-disubstituted-1ff-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-benzothiazoles 5a-i.
at S 40.58, 31.28 and 21.82 ppm were assigned to the NCH2, SCH2 and CH2 (propyl group) fragments, respectively. The signal at S 66.98 ppm was assigned to the OCH2 fragment of the 2-naphthoxymethyl group. Remaining carbon signals were observed at the expected chemical shift values. The mass spectrum of compound 5h displayed [M++2] peak at m/z 590 which is in agreement with the molecular formula C34H28N4O2S2. A base peak was observed at m/z 94 due to the fragment [C6H6N]+. and an intense peak at m/z 144 was assigned to the 2-naphthol fragment [C10H7O]+., which is consistent with the structure of 5h.
2. 2. Antimicrobial Activity
In vitro antimicrobial screening by the cup plate method18 displayed moderate to weak inhibitory activity (inhibition zone 13-16 mm) of the tested compounds 5a-h against Gram-positive bacterium Enterococcus fae-calis whereas the rest of the compounds were found to be inactive against the same organism (Table 1). Tested compounds showed no activity against Bacillus coagulans. Compound 5a having methyl group at the third and fifth position of the triazole ring showed significant inhibitory activity (inhibition zone 37 mm) against Escherichia coli, whereas the rest of the compounds showed a weak activity against the tested strains of Gram-negative bacteria. Compounds 5a, 5b 5d and 5f-h exhibited significant inhibitory activity (inhibition zone 17-21 mm) against Candida albicans. In this regard compound 5a having methyl group at the third and fifth position of the triazole ring showed maximum activity (inhibition zone 21 mm). From these result it is evident that substitution of small alkyl group at the third and fifth position of the triazole ring is optimal for activity against E. coli and C. albicans.
Table 1. Antimicrobial activity of 3,5-disubstituted-1,2,4-triazole incorporating 2-mercaptobenzothiazoles 5.
2. 3. Anti-inflammatory Activity
The anti-inflammatory activity results determined using the carrageenan induced paw oedema method19 (described by Winter et al. in 1962) in rats are summarized in Table 2. From these results it is evident that the tested compounds showed moderate to weak activity (9.5-42.8% protection) at 0.5 and 1 h after carrageenan injection compared to the reference drug diclofenac sodium (29.9 and 49.3% at a dose of 20 mg/kg). At the second, third and fourth hour four compounds, namely 5f and 5g-i exhibited significant protection (51.1-67.4%) against carrageenan induced oedema. In this regard maximum activity (67.4% protection) was observed at the third hour for compound 5g having 1-naphthyloxymethyl group at the third and fifth position of the triazole ring. It was observed that substitution with 1- or 2-naphthyloxymethyl groups (5g and 5h) at the third and fifth position of the triazole ring enhances the activity, whereas the substitution with the methyl group resulted in a marked decrease in activity. Moreover, a marked decrease in anti-inflammatory activity was also observed when electron withdrawing NO2 moiety was introduced in the phenyl rings (5c) attached to the third and fifth position of the 1,2,4-triazole ring.
2. 4. Ulcerogenic Effects
Synthesized compounds 5f, 5g and 5h were tested for their ulcerogenic potential according to the method reported by Cioli et al.20 The tested compounds showed low severity index (2.3 ± 0.3 to 3.5 ± 0.6) compared to the standard drug diclofenac sodium (severity index 4.4 ± 0.6). The maximum reduction in the ulcerogenic activity was found for compound 5h (severity index 2.3 ± 0.3). The tested compounds 5f and 5g also exhibited better GI safety profile as compared to the standard drug diclofenac sodium (Table 3).
Compound		Zone of inhibition (mm)a'b			
	E. f.	B. c.	P. a.	E. c.	C. a.
5a	-	-	11	37	21
5b	-	-	11	-	19
5c	-	-	11	-	13
5d	-	-	12	-	18
5e	16	-	12	-	16
5f	13	-	12	-	17
5g	14	-	12	-	18
5h	14	-	15	-	17
Ciprofloxacin	33	30	34	41	-
Ketoconazole	-	-	-	-	21
DMSO	-	-	-	-	-
Test compounds, ciprofloxacin and ketoconazole were tested at 100, 10 and 20 ng/mL concentration, respectively. a Average of three independent determinations. b - indicates no activity. E. f.: Enterococcusfaecalis; B. a.: Bacillus coagulans; P. a.: Pseudomonas aeruginosa; E. c.: Escherichia coli; C.a.: Candida albi-cans.
3. Experimental
3. 1. Chemistry
Reagents were of commercial grade and used as supplied. Melting points were determined in open glass capillaries and are uncorrected. The reaction progress and purity of the compounds were checked by thin-layer chroma-tography (TLC) on silica gel F254 plates from Merck. The IR spectra were recorded on KBr disks, using a Shimadzu 8400S FT-IR spectrophotometer. The 1H NMR and 13C NMR spectra were recorded using Bruker AV-III 500 spectrometer (500 MHz for 1H and 125 MHz for 13C with DMSO-d6 as the solvent. Chemical shifts are reported as S ppm using the solvent as the internal standard. EI-MS were obtained on Jeol GC Mate II instrument. Elemental analyses (C, H, N) were carried out on a Flash EA 1112 series instrument and were within ±0.4% of calculated values.
Table 2. Anti-inflammatory activity of some selected 3,5-disubstituted-1,2,4-triazole incorporating 2-mercaptobenzothiazoles 5 by the carrageenan induced rat paw oedema method.
Percent protection
Compd.	30 min	1 h	2 h	3 h	4 h
	Mean %	Mean %	Mean %	Mean %	Mean %
	protection	protection	protection	protection	protection
	±SEM	±SEM	±SEM	±SEM	±SEM
5a	10.4±0.8ca	28.3±0.7b	35.5±0.6a	37.2±0.8a	22.5±0.6c
5c	15.5±0.9b	34.1±0.9b	44.0±0.8a	46.3±0.6b	42.9±0.7b
5f	22.5±1.2b	42.8±0.7b	53.0±0.6b	62.3±0.5a	58.6±0.7b
5g	17.2±0.8a	37.7±0.5a	56.2±0.8b	67.4±1.1b	62.8±0.9a
5h	9.5±0.8b	32.0±1.1a	51.5±0.6b	62.4±0.5b	64.9±0.9a
5i	21.9±0.6b	38.3±0.9b	51.1±0.5a	59.6±0.8b	57.7±0.6b
Diclofenac	29.9±1.2c	49.3±0.9a	61.1±1.2b	72.0±0.9c	71.1±1.2b
sodium					
Test compounds 5 and diclofenac sodium were tested at 100 mg/kg and 20 mg/kg body weight, respectively. Result are expressed in mean ± SEM (n = 6). Significance levels: a P < 0.05, b P < 0.01 and c P < 0.001 compared with the respective control.
Table 3. Ulcerogenic effects of some selected 3,5-disubstituted-1,2,4-triazole incorporating 2-mercaptobenzothiazoles 5 by the Cioli's method.
Compound	Control 1% CMC	5f	5g	5h	Diclofenac sodium
Severity Index	0.23±0.9b	3.5±0.6a	2.7±0.8c	2.3±0.3a	4.4±0.6
a Test compounds 5 and diclofenac sodium were tested at 200 and 20 mg/kg body weight, respectively. Results are expressed in mean ± SEM (n = 6). Significance levels: a P < 0.05, b P < 0.01 and c P < 0.001 compared with the respective control.
Aryloxyacetic acids,15 4-amino-3,5-disubstituted-1,2,4-triazoles16 and 2-[(3-chloropropyl)sulfanyl]-1,3-ben-zothiazole17 were prepared as described in the literature.
General procedure for the synthesis of 2-{[3-(3,5-di-substituted-1H-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-benzothiazoles 5a-i. A mixture of the appropriate 4-ami-no-3,5-disubstituted-1, 2, 4-triazole (10.5 mmol) and 3-chloroproplybenzothiazole (2.44 g, 10 mmol) in isopropyl alcohol (20 mL) was refluxed for 44-48 h. After completion of the reaction the excess solvent was removed under vacuum and 20 mL water was added to the reaction mixture. The content was cooled to 5 °C in an ice bath and conc. HCl (1.8 mL, 2.2 mmol) was added followed by a drop-wise addition of saturated aqueous sodium nitrite solution (3 mL, 11 mmol). The mixture was allowed to warm to the room temperature and then it was neutralized with saturated potassium carbonate solution. The separated solid was filtered, washed thoroughly with water, dried and recrystallized from an appropriate solvent to yield the title compounds 5a-i.
2-{[3-(3,5-Dimethyl-1H-1,2,4-triazol-1-yl)propyl]sul-fanyl}-1,3-benzothiazole (5a). Solvent of crystallization:
acetone. Yield 55%, mp 118-120 °C. IR (KBr) v: 3003, 2972, 2835, 1668, 756 cm-1. 1H NMR (DMSO-d6): 8 7.63-7.17 (m, 4H, ArH), 4.21 (t, 2H, NCH2), 2.58 (t, 2H, SCH2), 2.12-2.01 (m, 2H, CH2), 1.95 (s, 6H, 2xCH3). MS: m/z 306 (M++2), 274, 239, 225, 210, 190, 179, 166, 137, 121, 105, 91, 77. Anal. Calcd. for C14H16N4S2: C, 55.24; H, 5.30; N, 18.40. Found: C, 54.87; H, 5.277; N, 18.36.
2-{[3-(3,5-Diphenyl-1H-1,2,4-triazol-1-yl)propyl]sul-fanyl}-1,3-benzothiazole (5b): Solvent of crystallization: acetone. Yield 62%, mp 188-190 °C. IR (KBr) v: 3023, 2837, 1670, 1597, 742 cm-1. 1H NMR (DMSO-d6): 8 8.34-7.79 (m, 4H, ArH), 7.63-7.39 (m, 10H, ArH), 4.01 (s, 2H, NCH2), 2.75 (t, 2H, SCH2), 2.02-1.96 (m, 2H, CH2). MS: m/z 428 (M+). Anal. Calcd. for C24H20N4S2: C, 67.226; H, 4.70; N, 13.07. Found: C, 67.31; H, 4.62; N, 13.12.
2-{[3,5-Bis(3,5-dinitrophenyl-1H-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-benzothiazole (5c): Solvent of crystallization: acetone. Yield 50%, mp 130-132 °C. IR (KBr) v: 3025, 2935, 1656, 1597, 1515, 1320, 750 cm1. 1H NMR (DMSO-d6): 8 8.37-7.87 (m, 6H, ArH),
7.74-7.48 (m, 4H, ArH), 4.34 (t, 2H, CH2), 2.64 (t, 2H, SCH2), 2.05-1.93 (m, 2H, CH2). MS: m/z 609 (M++1). Anal. Calcd. for C24H16N8O8S2: C, 47.37; H, 2.65; N, 18.41. Found: C, 47.31; H, 2.58; N, 18.47.
2-({3-[3,5-Bis(4-chlorophenyl)-1H-1,2,4-triazol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5d): Solvent of crystallization: acetone. Yield 57%, mp 148-150 °C. IR (KBr) v: 3063, 2926, 1676, 1597, 1046, 738, 842 cm1.1H NMR (DMSO-d6): 8 8.23-7.85 (m, 4H, ArH), 7.68-6.98 (m, 8H, ArH), 36.98 (t, 2H, NCH2), 2.51 (t, 2H, SCH2), 1.99-1.91 (m, 2H, CH2). MS: m/z 497 (M+). Anal. Calcd. for C24H18Cl2N4S2: C, 57.95; H, 3.65; N, 11.26. Found: C, 57.90; H, 3.58; N, 11.31.
2-( {3-[3,5-Bis(2,4-dichlorophenyl)-1H-1,2,4-triazol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5e): Solvent of crystallization: acetone. Yield 65%, mp 154-156 °C. IR (KBr) v: 3063, 2935, 1663, 1597, 1034, 741 cm1. 1H NMR (DMSO-d6): 8 8.34-7.78 (m, 6H, ArH), 7.64-7.42 (m, 4H, ArH), 4.12 (t, 2H, NCH2), 2.63 (t, 2H, SCH2), 2.08-2.01 (m, 2H, CH2). MS: m2/z 568 (M++2). Ana2l. Calcd. for C24H16Cl4N4S2: C, 50.90; H, 2.85; N, 9.89. Found: C, 50.82; H, 2.78; N, 9.92.
2-{[3-(3,5-Diphenoxymethyl-1H-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-benzothiazole (5f): Solvent of crystallization: chloroform and methanol (1:1). Yield 52%, mp 158-160 °C. IR (KBr) v: 3059, 2906, 1660, 1247, 1039, 748, 692, 735 cm-1.1H NMR (DMSO-d6): 8 8.34-7.82 (m, 4H, ArH), 7.35-6.97 (m, 10H, ArH), 4.79 (s, 4H, 2xOCH2), 3.94 (t, 2H, NCH2), 2.54 (t, 2H, SCH2), 2.07-1.99 (m, 2H, CH2). 13C NMR (DMSO-d6): 8 1671, 158.4, 152.0, 136.4, 129.9, 129.9, 127.0, 126.3, 125.1, 121.7, 121.1, 115.2, 66.4, 59.2, 41.2, 32.2, 23.3. MS: m/z 489 (M++1). Anal. Calcd. for C26H24N4O2S2: C, 63.91; H, 4.95; N, 11.47. Found: C, 63.89; H, 4.96; N, 11.50.
2-( {3-[3,5-Bis(1-naphthoxymethyl)-1H-1,2,4-triazol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5g): Solvent of crystallization: chloroform and methanol (1:1). Yield 48%, mp 208 °C. IR (KBr) v: 3055, 2935, 1650, 1252, 1032, 736 cm-1. 1H NMR (DMSO-d6): 8 8.33-7.79 (m, 4H, ArH), 7.61-7.50 (m, 10H, ArH), 7.34-6.85 (m, 4H, ArH), 4.63 (s, 4H, 2xOCH2), 4.25 (t, 2H, NCH2), 2.57 (t, 2H, SCH2), 2.11-2.02 (m, 2H, CH2). MS: m/z 589 (M++1). Anal. Calcd. for C34H28N4O2S2: C, 69.36; H, 4.79; N, 9.52. Found: C, 69.33; H, 4.72; NN, 9.49.
2-( {3-[3,5-Bis(2-naphthoxymethyl)-1H-1,2,4-triazol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5h): Solvent of crystallization: chloroform and methanol (1:1). Yield 52%, mp 210 °C. IR (KBr) v: 3062, 2927, 1659, 1245, 1028, 738 cm-1.1H NMR (DMSO-d6): 8 8.40-7.86 (m, 4H, ArH), 7.57-7.41 (m, 10H, ArH), 7.27-6.98 (m, 4H, ArH), 4.85 (s, 4H, 2xOCH2), 4.12 (t, 2H, NCH2), 2.58 (t,
2H, SCH2), 1.98-1.89 (m, 2H, CH2). 13C NMR (DMSO-d6): 8 167.1, 157.9, 153.7, 134.5, 127.8, 127.0, 126.8, 126.4, 125.7, 125.2, 123.6, 122.8, 122.6, 121.3, 121.2, 109.7, 106.2, 66.9, 40.5, 31.2, 21.8. MS: m/z 590 (M++2), 456, 435, 407, 381, 280, 258, 231, 216, 198, 171, 157, 144, 129, 110, 94, 83, 77, 65. Anal. Calcd. for C34H28N4O2S2: C, 69.36; H, 4.79; N, 9.52. Found: C, 693.34; H, 4.79; N, 9.48.
2-({3-[3,5-Bis(4-chlorophenoxymethyl)-1H-1,2,4-tria-zol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5i): Solvent of crystallization: ethanol and acetone (1:1). Yield 51%, mp 160 °C. IR (KBr) v: 3064, 2908, 1660, 1234, 1006, 742, 821 cm-1.1H NMR (DMSO-d6): 8 8.37-7.78 (m, 4H, ArH), 7.52-6.76 (m, 8H, ArH), 46.67 (s, 4H, 2xOCH2), 4.05 (t, 2H, NCH2), 2.54 (t, 2H, SCH2), 2.07-1.98 (m, 2H, CH2). MS: m/2z 559 (M++2). 2Anal. Calcd. for C26H22Cl2N4O2S2: C, 56.01; H, 3.98; N, 10.05. Found: C, 552.97; H,3.98; NN, 10.11.
3. 2. Antimicrobial Activity
Antibacterial activity was evaluated on nutrient agar (Hi-media) plates (37 °C, 24 h) against Enterococcus fae-calis, Bacillus coagulans, Pseudomonas aeruginosa and Escherichia coli by the cup plate method.18 Test compounds were also evaluated18 for their antifungal potential on Sabouraud dextrose agar (Hi-media) plates (26 °C, 48-72 h) against Candida albicans. Solutions of the test compounds, ciprofloxacin and ketoconazole were prepared in dimethylsulfoxide (DMSO) at the concentrations of 100, 10 and 20 |ag/mL, respectively. The results (Table 1) were recorded as the average diameter of inhibition zones (three independent determinations) of bacterial or fungal growth around the disks and are given in mm.
3. 3. Pharmacological Activity
Animals were procured from the animal facility of the J. S. S. College of Pharmacy, Ootacamund, Tamil Nadu, India and were maintained in colony cages at 23±2 °C with relative humidity of 45-50% and under 12 h light and dark cycle. They were fed with the standard rat pellet diet (Hindustan Liver Ltd., Mumbai). Prior approval of the Local Animal Ethical Committee was obtained to carry out the experimental work on animals. The synthesized compounds 5a, 5c, 5f, 5g, 5h and 5i were evaluated for their acute toxicity and anti-inflammatory activities. Data were statistically analyzed by one-way analysis of variance (ANOVA) followed by Student's t-test to assess the statistical significance.
3. 3.1. Acute Toxicity
Acute oral toxicity was performed for the synthesized compounds 5a, 5c, 5f, 5g, 5h and 5i following the Or-
ganization of Economic Cooperation and Development (OECD-423) guidelines (acute toxic class method). Swiss albino mice (n = 3) of either sex selected by random sampling were used for the study. The animals were fasted for 3-4 h with water ad libitum, after which the test compounds (suspension in 1% CMC) were administered orally at the doses of 50, 100, 250, 500 and 1000 mg/kg and the mice were observed for three days. No behavioral changes in animals were observed during the experiment and at the end hematological parameters were estimated and there was no observable change. In the present study, mortality was not observed even at 1000 mg/kg indicating that the compounds are nontoxic to animals.
3. 3. 2. Anti-inflammatory Activity
The acute anti-inflammatory activity results of the synthesized compounds was determined following the carrageenan induced paw oedema method19 in Wistar albino rats (n = 6) of either sex (155-160 g). The animals were fasted for 24 h before the experiment with free access to water. The test compounds and diclofenac sodium were administered orally as suspension (1.0% w/v CMC solution). The control rats received appropriate volumes of CMC solution orally. Thirty minutes after administration of the test compounds, 0.1 mL carrageenan solution (1.0% w/v in sterile saline) was injected into the sub-plantar tissue of the right hind paw of each rat. The volume of the paw was measured at different time intervals of 0.5, 1, 2 and 3 h after the carrageenan injection by the means of plethysmometer (UGO Basile 7140, India). The percentage protection against inflammation was calculated by the following formula,
(Vc-Vt)/Vc x100	(1)
where Vc is the oedema volume in control group and Vt is the oedema volume in groups treated with the test compounds. The anti-inflammatory activity results are summarized in Table 2.
3. 3. 3. Ulcerogenic Effects
The test compounds 5g and 5i were evaluated for their acute ulcerogenic effects according to the method of Cioli et al.20 in Wistar albino rats (n = 6) of either sex. The test compounds and diclofenac sodium were administered orally as suspension in 1% carboxymethyl cellulose (CMC). Control group received appropriate volumes of 1% CMC. Food but not water was removed 24 h before administration of the test compounds. After compound treatment, the rats were fed with normal diet for 17 h and then sacrificed. Their stomachs were removed, cut out along the greater curvature and washed with distilled water and then gently cleaned by dipping in normal saline. The mucosal damage was examined by means of a magnifying glass. For each stomach the mucosal damage was assessed according to the following scoring systems: 0.5 redness; 1.0 spot ulcers; 1.5 haemorrhagic streaks; 2.0 ulcers >3 but <5; 3.0 ulcers >5. The mean score of each treated group minus the mean score of control group was regarded as the severity index (Table 3).
3. 3. 4. Docking studies of 2-{[3-(3,5-disubstitu-
ted-1.ff-1,2,4-triazol-1-yl)propyl]sulfanyl}-1,3-
benzothiazoles
The type IV, cyclic AMP-specific phosphodiesterases (PDE4B)22-24 is particularly abundant in immune and inflammatory cells, where an increase of cAMP leads to the inhibition of the synthesis and the release of pro-inflammatory mediators.25 Due to their role in regulation of cell function, PDEs have become good clinical targets for the treatment of inflammation.26 Benzothiazole nucleus linked to the small heterocyclic moiety27 and triazole nucleus fused with the other heterocyclic system28 is exploited as a PDE4 inhibitor. Combining some of the structural features of ben-zothiazole and triazole in a single molecule may lead to a new class of compounds which may be explored for the identification as novel PDE4B inhibitors. Prompted by this hypothesis we initially became interested in the evaluation of 2- {[3-(3,5-disubstituted-1 #-1,2,4-triazol-1-yl)propyl]
Table 4. Docking study results of the 2-{[3-(3,5-disubstituted-1ff-1,2,4-triazol-1-yl) propyl] sulfanyl}-1,3-benzothiazoles 5.
Comp.	Glide	Glide	E-1a	E-2b	E-3c	E-4d	RMSD
	score	energy	(kcal/mol)	(kcal/mol)	(kcal/mol)	(kcal/mol)	
5h	-10.73	-57.67	-1.90	-7.63	-1.19	0	0.024
5i	-9.71	-55.08	-1.47	-7.35	-0.18	0	0.011
5g	-9.48	-51.49	-1.92	-7.85	0.08	0.21	0.011
5d	-8.80	-46.18	-1.47	-5.80	-0.29	0.21	0.021
5f	-8.61	-49.04	-1.22	-6.55	-0.24	0	0.028
5b	-8.37	-43.57	-1.87	-5.52	-0.17	0	0.033
5e	-8.16	-41.90	-1.82	-6.53	0.19	0	0.011
5c	-7.68	-52.85	-1.20	-5.51	-0.12	0	0.033
5a	-7.62	-33.59	-1.47	-4.21	-0.34	0.34	0.004
a Hydrophobic enclosure reward; b Lipophilic EvdW; c Electrostatic reward; d Rotational penalty
sulfanyl}-1,3-benzothiazoles (5a-i) for their potential affinities with respect to PDE4B through docking studies using the enzyme PDE4B co-crystallised with piclamilast29 as the target. This complex was obtained from the RCSB protein data bank under the PDB code 1XM4.
The structures of the selected compounds 5a-i are initially optimized using the Schrodinger Maestro version 9.2 software. The theoretical binding profile of each molecule was evaluated using Glide, version 5.7, Schrodinger, LLC, New York, NY, 2011 and the parameters such as the GLIDE scores, hydrophobic endurance reward, hydrophi-lic reward, RMSD, and penalties were obtained after docking of these molecules with PDE4B protein. The results are summarized in Table 4. The data clearly suggests that these molecules bind well with PDE4B. H-bonding interaction was observed in the case of compounds 5a-d, 5f and 5i. In the case of the compound 5i hydrogen bond interaction was observed between oxygen function of the one of the p-chlorophenoxymethyl residues and the magnesium metal ion mediated by a water molecule (Figure 1) whereas in compounds 5a and 5d hydrogen bonding was observed between the centre of the benzothiazole ring N(1) and the -NH2 group of the Gln443 residue of the
PDE4B protein (Figures 2 and 3) that is essential for nuc-leotide recognition and selectivity.30 In compounds 5b (phenyl) and 5c (4-nitrophenyl) hydrogen bonding was observed between the thiol group present at the second position of the benzothiazole ring and the magnesium metal ion mediated by a water molecule (Figures 4 and 5). In both compounds a second hydrogen bond was also observed between the centre of the benzothiazole ring N(1) and the carbonyl group (C=O) of the Met347 residue, whereas in the compound 5f hydrogen bonding was observed between oxygen function of one of the phenoxymethyl groups and a water molecule (Figure 6).
It is evident from the docking results (Table 4) that in compounds 5g and 5h one of the naphthyl rings is sandwiched in the hydrophobic clamp (hydrophobic enclosure reward -1.92 and -1.90 kcal/mol, respectively) (Figures 7 and 8) and the other naphthyl group is directed into the more capacious Q2 site adjacent to the methionine (Met431) at the entrance to the catalytic pocket, whereas in compounds 5b and 5e, phenyl or 2,4-dichlorophenyl groups are sandwiched by the hydrophobic clamp (hydrophobic enclosure reward -1.87 and -1.82 kcal/mol, respectively) (Figures 9 and 10). The remaining parts of
Figure 1. Docking of 5i at the active site of PDE4B.
Figure 3. Docking of 5d at the active site of PDE4B.
Figure 2. Docking of 5a at the active site of PDE4B.
Figure 4. Docking of 5b at the active site of PDE4B.
Figure 5. Docking of 5c at the active site of PDE4B.
Figure 7. Docking of 5g at the active site of PDE4B.
Figure 9. Docking of 5b at the active site of PDE4B.
the molecules are shown to extend into the catalytic domain in close proximity to both the Zn2+ and Mg2+ cations.
Figure 6. Docking of 5f at the active site of PDE4B.
Figure 8. Docking of 5h at the active site of PDE4B.
Figure 10. Docking of 5e at the active site of PDE4B.
Such an orientation would block the approach of cAMP to the catalytic domain and forms the basis for inhibiting
tion of the 1,2,4-triazole ring, showed maximal Glide scores (-10.73 kcal/mol). This may be attributed to an improved fit of the ligand into the Q1 subpocket, as defined by Card et al.,25 adjacent to the purine-scanning glutamine in the interior of the catalytic pocket.
In the compounds 5a and 5d benzothiazole ring (Figures 11 and 12) and in compound 5i p-chlorophe-noxymethyl group are sandwiched into the hydrophobic clamp (hydrophobic enclosure reward -1.47 kcal/mol, Figure 13). The remaining parts of the molecules are shown to extend into the catalytic domain in close proximity to both the Zn2+ and Mg2+ cations.
4. Conclusion
Figure 11. Docking of 5a at the active site of PDE4B.
Figure 12. Docking of 5d at the active site of PDE4B.
Figure 13. Docking of 5i at the active site of PDE4B.
PDE4B. In the present investigation compound 5h, having 2-naphthoxymethyl group at the third and fifth posi-
In the present investigation all the synthesized compounds 5 were found to be either weakly active or inactive against the tested strains of bacteria. Compound 5a showed maximal inhibitory activity against Candida albicans. On the other hand, compounds 5g and 5h showed significant anti-inflammatory activity with significant reduction of gastrointestinal toxicity (severity index 2.7±0.8 and 2.3 ± 0.3, respectively) in an animal model and this correlates well with the docking study result (GLIDE scores -9.48 and -10.73 kcal/mol, respectively). Hence 2-({3- [3,5-bis(1/2-naphthoxymethyl)-1#-1,2,4-triazol-1-yl]propyl}sulfanyl)-1,3-benzothiazole (5g and 5h) would represent a fruitful framework for the development of newer anti-inflammatory agents.
5. Acknowledgements
The authors are most grateful to the JSS University, Mysore for providing facilities.
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Povzetek
Pripravili smo več 2-merkaptobenzotiazolnih derivatov 5a-i, ki vsebujejo 1,2,4-triazolno enoto z vezanima dvema dodatnima substituentoma. Vse nove spojine smo in vitro testirali za morebitno aktivnosti proti določenim vrstam bakterij, kot so Enterococcus faecalis, Bacillus coagulans, Pseudomonas aeruginosa, Escherichia coli in Candida albicans. Spojina 5a je pokazala opazno aktivnost proti Gram-negativni bakteriji Escherichia coli. Spojinam 5a-i smo tudi določili morebitno delovanje proti glivam na primeru Candida albicans, spojine 5a, 5b, 5d in 5g so pokazale opazno aktivnost proti tej glivi. Za nekatere spojine smo tudi določili in vivo protivnetno aktivnost, akutno toksičnost in ulceroge-no aktivnost. Testirani spojini 5g in 5h sta pokazali pomembno protivnetno aktivnost in močno gastrointestinalno pro-tekcijo v primerjavi s standardnim zdravilom diklofenak natrij. Predstavljene so tudi študije molekulskega modeliranja pripravljenih spojin.