INTRODUCTION:

Thiourea derivatives exhibit diverse biological and pharmacological activities. Derivatives of N-aryl- or N-heteroarylthioureas are known as potential inhibitors of HIV-1,¹ antihyperthyroid² acaricidal³ wide spectrum of anthelmintic⁴ anti-inflammatory and analgetic agents.⁵ Various symmetrical and unsymmetrical thioureas, N, N’-disubstituted thioureas have shown remarkable anti-inflammatory activitiy. Derivatives of N-aroyl- or N-heteroaryl thioureas are known for their better anti-inflammatory activity and low toxicity.

Disubstituted thioureas possessing N-substituent like pyrimidine, quinazoline, acridinyl thiophene etc have displayed appreciable anti-inflammatory activity. Literature survey reveals N-substituted thioueras 1-(para-nitrobenzoyl)-3-(2, 3-dimethyl-oxo-1-phenylpyrazolin-4-yl)-thiourea,⁶ N-(4-alkoxyphenyl)-N’-(2-alkylthio-6-methyl-4- pyrimidinyl) thiocarbamides,⁷ 1- (2-phenyl quinazolin-3-yl-4(3H)-one)-3-substituted thioureas,⁸ have displayed considerable anti-inflammatory activities. Literature survey revealed that 2-amino 4-substitued thiazoles⁹ and their various derivatives such as 2-(2, 4-disubstituted-thiazole-5-yl)-3-aryl-3Hquinazoline- 4-ones,¹⁰ 3-[4'(p-chlorophenyl) thiazol-2'-yl]-2-[(substituted azetidinone/thiazolidinone)-aminomethy]-6-bromoquinazolin-4-ones,¹¹ 4-oxothiazolidine and its 5-arylidenes,¹²(Z)-4-((2,4-dioxothiazolidin-5-ylidene) methyl)-N-(4-substituted phenylthiazol-2-yl) benzene sulfonamides and 2-substituted-N-(4-Substituted-phenylthiazol-2yl) acetamides,¹³ thiazolyl-N-Ph piperazines,¹⁴ 2-(4-arylthiazol-2-yl-amino)-n-aryl acetamides¹⁵ have displayed considerable anti-inflammatory activity. When methane sulphonamido moiety was incorporated in the heteocycles the modified products are found to have appreciable anti-inflammatory activity with COX-2 selectivity.¹⁶ Literature revealed that there is no attention on the synthesis of thioureas having 2-sulphonyl amino thiazol-4-yl methyl moiety. Considering the pharmacological importance of thioureas and disbstituted thiazoles, here we decided to synthesize new thioureas like 1- [(2- (methylsulphonyl amino) thiazole-4-yl) methyl]-3- arylthioureas so as to obtain the compounds/ leads with better anti-inflammatory activity with reduced side effects.

MATERIAL AND METHODS:

Chemicals and reagents:

Reactions were monitored by thin layer chromatography. TLC was performed with Merck precoated TLC plates, silica gel 60F254 with thickness of 0.25mm and spots were visualized by irradiation with ultraviolet light (254 nm). Melting points were determined by open capillary method and are uncorrected. IR spectra were recorded on Bruker alpha ATR spectrophotometer. ¹H NMR was carried out on Bruker apparatus at DRX-300 MHz, using TMS as internal reference and DMSO-d6 as medium. Chemical shifts (δ values) are expressed in parts per million (ppm). Mass spectra have been scanned on DART-MS (ESI+) and on JMS 100LC, AccuTOF spectrometers. Elemental analysis was performed on Perkin–Elmer 2400 CHNS Elemental analyzer at SAIF CDRI Lucknow, India.

Scheme 1. Synthesis of 1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3- aryl thioureas.

Synthesis:

Synthesis of 2-amino-4-(isothiocynatomethyl) thiazole (2)

A mixture of 2-amino-4(chloromethyl) thiazole hydrochloride (1) (22 mmol), potassium thiocynate (26 mmol), and potassium iodide (12 mmol) in methanol (40 mL) was heated at reflux for 2 h. The solvent was concentrated under reduced pressure, and to the resulting residue 10 % aqueous NH₃ (80 mL) was added.Then the mass was added and was extracted with DCM (2×60 mL).The organic layer was dried using anhydrous K₂CO₃. The solvent was removed from the dried organic solution and the crude product obtained was crystallized from benzene, yield: 76% (2). m.p:105-106^oC (Lit.105-106^oC).¹⁶

Synthesis of 2-methylsuphonyl amino-4-(isothiocynatomethyl) thiazole (3)

To a stirred solution of 2-amino-4-(isothiocynatomethyl) thiazole (2) (15 mmol) in DCM (20 ml) triethyl amine (16.5 mmol) was added and the reaction mixture was cooled to 0-5 0C.Then methylsulphonyl chloride (16.5 mmol) was added in portions. After complete addition of the methylsulphonyl chloride the reaction mixture was further stirred at rt. for 24 h. The Progress of the reaction was monitored by TLC .The reaction mixture was then poured on water to remove un-reacted methylsulphonyl chloride and triethyl amine hydrochloride .Organic layer was separated, dried on sodium sulphate and concentrated to give crude solid which on crystallization with ethanol:DMF yielded the compound (3) with 81% yield. m.p:185-187 °C.

2-methylsuphonyl amino-4-(isothiocynatomethyl) thiazole (3):

IR (cm^-1): 3310 (N-H stretch), 3020 (C-H stretch, aromatic), 2960 (C-H stretch, aliphatic symme.), 2811 (C-H stretch, ansym.), 2163 (N=C=S stretch), 1620 (C=N), 1558 (C=C), 1280 (S=O assym.) and 1132 (S=O symm.).¹H NMR (DMSO-d6, 300 MHz) δ (ppm) : 2.92 (s, 2H, CH₂), 4.21 (s, 3H, -SO₂CH₃), 6.85 (s,1H,thiazolyl), 12.61(s, 1H, exchangable with D₂O).MS (ESI+ mode): m/z (% intensity) : 250 (M,+), .Elemental Analysis : M.F-C₆H₇N₃O₂S₃: Found % ( Calculated %): C, 28.82 (28.90); H, 2.80 (2.83); N, 16.82 (16.85); S, 38.50 (38.58).

Synthesis of 1-[(2-(methylsulphonyl amino) thiazole-4-yl) methyl]-3- arylthioureas (4a-f):

A mixture 2-methylsuphonyl amino-4-(isothiocynatomethyl) thiazole (10 mmol) and aryl amine (10 mmol) was dissolved in pyridine (10 mL) and the reaction solution was refluxed for 30 min and cooled to r.t. It was then poured on ice water. The crude solid obtained was filtered vacuum dried and crystallized from ethanol. Similarly other compounds of the series were prepared and their characteristic physical data is recorded.

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3- phenyl thiourea (4a)

Yield-82%, mp- 70-73°C IR (cm^-1): 3362 (N-H bend.), 3231 (N-H bend.), 3213 (N-H bend.), 3028 (Ar-H strech), 2928 (C-H stretch), 2828 (C-H stech aliphatic), 1710 (C=N), 1520 (N-H bend.), 1371 (S=O) and 1177 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.23(s, 2H,-CH₂), 4.45(s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 6.88-7.3 (m, 5H, Ar-H) 9.8(s,-NH, exchangeable with D₂O), 10.6 (-NH, exchangeable with D₂O), 12.20 (-NH, exchangeable with D₂O). MS (ESI+ mode): 343 (M,+), Elemental Analysis: M.F- C₁₂H₁₄N₄O₂S₃: Found% (Calculated %): C, 42.01 (42.09); H, 4.10 (4.12); N, 16.33 (16.36); S, 28.01 (28.09).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(4-Chloro) phenyl thiourea (4b)

Yield-79%, mp- 107-109°C IR (cm^-1): 3393 (N-H bend.), 3254 (N-H bend.), 3233(N-H bend.), 3049 (Ar-H strech), 2948 (C-H stretch), 2853 (C-H stretch aliphatic), 1733 (C=N), 1539 (N-H bend.), 1387 (S=O) and 1192 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.48(s, 2H,-CH₂), 4.18(s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 6.90-7.62 (m, 4H, Ar-H), 9.92(s,-NH, exchangeable with D₂O), 10.28 (-NH, exchangeable with D₂O), 12.60 (-NH, exchangeable with D₂O). MS (ESI+ mode): 361.01 (M, +), 363.01 (M,+2)Elemental Analysis : M.F- C₁₂H₁₃ClN₄O₃S₂: Found% (Calculated %): C, 39.92 (39.94); H,3.64 ( 3.63); Cl, 9.81 (9.83); N, 15.51 (15.53); O,13.31( 13.30); S,17.78 ( 17.77).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(4-Fluro) phenyl thiourea (4c)

Yield-77%, mp- 118-121°C IR (cm^-1): 3380(N-H bend.), 3239 (N-H bend.), 3222 (N-H bend.), 3037(Ar-H strech), 2936 (C-H stretch), 2839 (C-H strech aliphatic), 1720 (C=N), 1528 (N-H bend.), 1376 (S=O) and 1181 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.32 (s, 2H,-CH₂), 3.98 (s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 7.34-7.66 (m, 4H, Ar-H), 9.78 (s,-NH, exchangeable with D₂O), 10.12 (-NH, exchangeable with D₂O), 12.30 (-NH, exchangeable with D₂O). MS (ESI+ mode): 345.04 (M,+). Elemental Analysis: M.F-C₁₂H₁₃FN₄O₃S₂: Found% (Calculated %): C, 41.83 (41.85); H, 3.79 (3.80); F, 5.53 (5.52); N, 16.25(16.27); O, 13.94 (13.94); S, 18.6 (18.62).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(4-Methyl) phenyl thiourea (4d)

Yield-72%, mp- 147-149°C IR (cm^-1): 3358 (N-H bend.), 3227 (N-H bend.), 3209 (N-H bend.), 3025 (Ar-H strech), 2924 (C-H stretch), 2823 (C-H stech aliphatic), 1702 (C=N), 1510 (N-H bend.), 1366 (S=O) and 1171 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.20 (s, 2H,-CH₂), 3.92(s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 6.82-7.32 (m, 4H, Ar-H) , 9.7(s,-NH, exchangeable with D₂O), 10.2 (-NH, exchangeable with D₂O), 12.12 (-NH, exchangeable with D₂O). MS (ESI+ mode): 341.07 (M, +), Elemental Analysis: M.F-C₁₃H₁₆N₄O₃S₂: Found% (Calculated %): C, 45.86 (45.87); H, 4.74 (4.74); N, 16.45 (16.46); O, 14.08 (14.10); S, 18.81 (18.84).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(3-Chloro) phenyl thiourea (4e)

Yield-67%, mp- 92-95°C IR (cm ^-1): 3390 (N-H bend.), 3250 (N-H bend.), 3231 (N-H bend.), 3046 (Ar-H strech), 2944 (C-H stretch), 2848 (C-H stech aliphatic), 1728 (C=N), 1536 (N-H bend.), 1384 (S=O) and 1190 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.43 (s, 2H,-CH₂), 4.10 (s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 6.90-7.60 (m, 5H, Ar-H), 9.88 (s,-NH, exchangeable with D₂O), 10.26 (-NH, exchangeable with D₂O), 12.52 (-NH, exchangeable with D₂O). MS (ESI+ mode): 361.01 (M, +), 363.01 (M, + 2) Elemental Analysis: M.F- C₁₂H₁₃ClN₄O₃S₂: Found% (Calculated %): C, 39.93 (39.94); H, 3.65 (3.63); Cl, 9.80 (9.83); N, 15.52 (15.53); O, 13.30 (13.30); S, 17.76 (17.77).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(4-Methoxy) phenyl thiourea (4f)

Yield-80%, mp- 160-162°C IR (cm^-1): 3384 (N-H bend.), 3244 (N-H bend.), 3226 (N-H bend.), 3041 (Ar-H strech), 2940 (C-H stretch), 2844 (C-H stech aliphatic), 1723 (C=N), 1531 (N-H bend.), 1379 (S=O) and 1184 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.38(s, 2H,-CH₂), 4.02(s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 6.82-7.32 (m, 4H, Ar-H), 9.84 (s,-NH, exchangeable with D₂O), 10.20 (-NH, exchangeable with D₂O), 12.40 (-NH, exchangeable with D₂O). MS (ESI+ mode): 357.06 (M, +). Elemental Analysis: M.F- C₁₃H₁₆N₄O₄S₂: Found% (Calculated %): C, 43.80 (43.81); H, 4.50 (4.52); N, 15.70 (15.72); O, 17.94 (17.96); S, 17.97 (17.99).

1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3-(4-Bromo) phenyl thiourea (4g)

Yield-77%, mp- 120-121°C IR (cm^-1): 3377 (N-H bend.), 3235 (N-H bend.), 3217(N-H bend.), 3032 (Ar-H strech), 2933 (C-H stretch), 2835 (C-H stech aliphatic), 1716 (C=N), 1523 (N-H bend.), 1372 (S=O) and 1177 (S=O).¹H NMR (DMSO-d6, 400MHz) δ (ppm) : 1.28 (s, 2H,-CH₂), 3.98 (s, 3H,-SO₂CH₃), 6.85 (s,1H,thiazolyl), 7.09-8.10 (m, 4H, Ar-H), 9.74 (s,-NH, exchangeable with D₂O), 10.08 (-NH, exchangeable with D₂O), 12.30 (-NH, exchangeable with D₂O). MS (ESI+ mode): 404.96 (M, +), 404.96 M, +2). Elemental Analysis: M.F- C₁₂H₁₃BrN₄O₃S₂: Found% (Calculated %): C, 35.55 (35.56); H, 3.22 (3.23); Br, 19.70 (19.72); N, 13.81 (13.82); O, 11.83 (11.84); S, 15.80 (15.82).

In vitro anti-inflammatory activity by HRBC membrane stabilization method:

Fresh whole human blood was collected and it was mixed with equal volumes of sterilized Alsever’s solution (Dextrose 2%, Sodium citrate 0.8%, Citric acid 0.05%, Sodium chloride 0.42%, and Distilled water 100mL). This blood solution was centrifuged at 3000 rpm for 10 min and was washed three times with equal volume of normal saline. The volume of the blood is measured and reconstituted as 10% v/v suspension with normal saline. The reaction mixture consists of 1.0mL of test sample of different concentrations in normal saline and 0.5mL of 10% HRBC suspension, 1mL of 0.2M phosphate buffer, 1ml hypo saline were incubated at 37°C for 30 min and centrifuged at 3,000 rpm for 30 minutes. The hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560nm wavelength. Each experiment was performed in triplicate. Dichlofenac sodium was used as standard and distilled water as control in this study.¹⁷ Where the blood control represents 100% lysis or zero percent stability, the percentage of HRBC hemolysis calculated by formula,

% Hemolysis = (O.D of Test Sample / O.D of Control) × 100.

The concentration of a compound, where 50% of its maximal effect is observed (EC50) using graph pad prism was measured.¹⁸

RESULTS AND DISCUSSION:

Chemistry:

The synthetic sequence is outlined in Scheme 1.Synthesis of the target molecules was carried using starting material 2-amino-4-chloromethyl thiazole hydrochloride (1) which was freshly synthesized by cyclocondensing 1, 3-dichloroacetone and thiourea by following Hantzsch method. 2-Amino-4-(chloromethyl)thiazole hydrochloride (1) was then condensed in methanol with ammonium thiocynate in the presence of potassium iodide in methanol and obtained 2-amino-4-(isothiocynatomethyl)-thiazole (2). The 2-amino derivative (2) when mesylated with methyl sulphonyl chloride in DCM using triethylamine as catalyst at 0^oC gave desired 2-methylsuphonamido-4-(isothiocynatomethyl) thiazole (3). To synthesize thioureas from alkyl isothiocynate is relatively difficult as compared with aryl or aroyl isothiocynates as alkyl isothiocynates are less reactive.We have tried the additionof aryl amines and 2-methylsuphonyl amino-4-(isothiocynatomethyl) thiazole (3) in acetone/DCM, in presence of catalyst but couldn’t succeed to get the desired thioureas with moderate.to good yields.When the same reaction was carried in refluxed pyridine we got desired 1-((2- (methylsulphonyl amino) thiazole-4-yl) methyl)-3-arylthioureas (4a-f) in excellent yield. Here pyridine acted as medium as well as base.

The structures of intermediate and all new 1- [(2- (Methylsulphonyl amino) thiazole-4-yl) methyl]-3- aryl thioureas (4a-f) have been elucidated by elemental analyses, I.R., ¹H NMR, and Mass spectroscopic measurements. IR spectra of intermediate(3)diagnostic peak such as 2163 (N=C=S stretch), 1620 (C=N), 1558 (C=C), 1280 (S=O assym.) and 1132 (S=O symm.) are observed.¹H NMR spectra of intermediate (3) display peak at 2.92 (s, 2H, CH₂), 4.21 (s, 3H, -SO₂CH₃), 6.85 (s,1H,thiazolyl), 12.61(s, 1H, exchangable with D₂O, -NHSO₂CH₃) and MS (ESI⁺mode): 250 (M, +) which suppotes the stucture 2-methylsuphonyl amino-4-(isothiocynatomethyl) thiazole . In the IRspectra of compounds (3a-g) along with other diagnostic peak N, N diubstituted thioureas secondary amine N-H bending vibrations are observed (3227-3254cm^-1) and (3209-3233) is recorded. ¹H NMR spectra all final compounds display all significant and distinctive broad D₂O exchangeable secondary amino peaks in (9.70-9.92) and (12.12-12.60) range prove disubstituted thiourea strucures of final compounds. Mass spectra of titled compounds are in good agreement with molecular weight calculated and structures proposed for (4a-f).

Anti-inflammatory activity:

Anti-inflammatory agents inhibit the cyclooxygenase enzymes which are responsible for the conversion of arachidonic acid to prostaglandins. Because human red blood cell (HRBC) membranes are similar to these lysosomal membrane components, the prevention of hypotonicity induced HRBC membrane lysis was taken as a measure in estimating anti-inflammatory activity.¹⁷ Anti-inflammatory activity done by Human Red Blood Cell (HRBC), DFS stabilizes the membrane, thereby reducing the hemolysis. Thus with the increase in the component are prevented from leaking, thus as the concentration of DFS increases, the O.D decreases thereby decreasing the effect of the tonicity caused by hypo saline. Thus, HRBC membrane stabilization method¹⁹ was used to estimate anti-inflammatory activity. The result of the in vitro membrane stabilization activity of synthesized thiazolyl thioureas (4a-f) is presented in Table 1, Fig.1 and Fig.2. According to these results all the compounds showed dose dependent inhibition of hemolysis. Compound 6b(EC₅₀ = 6.60 ± 0.08), 6c (EC₅₀ = 8.93 ± 0.11) and 6d (EC₅₀ = 4.76 ± 0.04) displayed very good activity among the series as compared to standard Diclofenac sodium (EC₅₀ = 13.24). Other compound 6f (EC₅₀ = 14.10 ± 0.05) showed moderate activity and 4a (22.32 ± 0.17), 4e (21.28 ± 0.15), 4f (20.57 ± 0.10) had exhibited lower anti-inflammatory activity as compared to standard DCS.

CONCLUSION:

A new series of N-aryl, N’-thiazolyl thioueras with methylsulphonylamino pharmacophore has been synthesized by convenient synthesis and characterized by different spectral and elemental analyses. The newly synthesized thioureas (4a-g) exhibited good in vitro anti-inflammatory activity compared with Standard Diclofenac sodium. The compounds 4b, 4c and 4d exhibited most promising anti-inflammatory activity.

ACKNOWLEDGEMENT:

Author is thankful to Professor R. A. Mane for valuable discussions. Author is thankful to CDRI Lucknow for spectral analysis and Haffkine Institute, Mumbai for evaluating biological activity.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 08.06.2017 Modified on 10.07.2017

Asian J. Research Chem. 2017; 10(4):477-482.

DOI:10.5958/0974-4150.2017.00077.3

Compound	% hemolysis at different concentrations 1ug/ml 5ug/ml 25ug/ml 50ug/ml				(EC₅₀ ± SD)
4a	59.50	65.03	74.84	79.75	22.32 ± 0.17
4b	65.64	79.14	88.34	95.70	6.60 ± 0.08
4c	66.87	80.36	90.79	98.15	8.93 ± 0.11
4d	73.71	80.98	85.27	91.41	4.76 ± 0.04
4e	56.44	62.57	72.39	80.36	21.28 ± 0.15
4f	63.41	70.55	73.61	80.36	20.57 ± 0.10
4g	55.21	63.80	79.14	77.30	14.10 ± 0.05
DCS	61.34	63.19	65.64	70.55	13.24 ± 0.09