2.1. Chemistry
All the chemicals and reagents used for synthesis were of laboratory grade. The completion of the reaction and purity of all the synthesized compounds was supervised by using chromatography technique. Shimadzu FTIR 8400S was used to record IR spectra by using KBr and the NMR spectra were recorded in NMR Varian-Mercury 300 MHz spectrometer in CDCl3 and values are expressed in ppm.
2.2. General synthetic procedures
The general reaction sequence for different title compounds is outlined in Scheme 1.
2.2.1. General synthetic procedure for 6-substituted-2-aminobenzothiazoles (BT 1-5)
Method A: BT 1
To 25 ml (0.02mol) Aniline, 25 ml conc. hydrochloric acid was added in a 250 ml beaker and the solution was heated till it becomes warmed. A saturated solution of ammonium thiocyanate in water was prepared and added slowly to the above and boiled until the solution become turbid. The turbid solution was poured into cold water with continuous stirring. Phenylthiourea, separated as precipitate was filtered and recrystallized from aqueous ethanol (80%). To 250 ml round bottom flask containing 75 ml of chloroform, phenylthiourea (0.098mol) was added and stirred it. Bromine in chloroform (5%) was added dropwise to above suspension till an orange-yellow colour appeared, the temperature was maintained at 0-5℃. The reaction mixture was stirred overnight (20 h). The precipitate obtained was filtered and washed with chloroform until the colour disappeared. This precipitate, the hydrobromide salt, was dissolved in acidified water (5% HCl) and basified with concentrated ammonia solution. The precipitate obtained was filtered, dried and recrystallized using ethanol and water mixture [23].
Method B: BT 2-5
To a 250 ml round bottom flask, ammonium thiocyanate (11.93 gm, 0.156mol) and 100 ml acetic acid was added and it was stirred until ammonium thiocyanate dissolved completely. Aniline (10 gm, 0.078mol) was added to above solution. To this, Bromine solution (0.02mol) in acetic acid was added to the reaction mixture maintained at temperature 0-5℃ till an orange-yellow colour appeared. The slurry was kept overnight (20 h). Filtered the reaction mixture, precipitate obtained was dissolved in water (200 ml) and filtered to remove any undissolved matter and this filtrate was basified with concentrated ammonia solution. The precipitate obtained was filtered, dried and recrystallized using ethanol: water (80:20) mixture [24].
2-aminobenzothiazole/ 1,3-benzothiazol-2-amine BT-1: yield 62%; White coloured solid; mp 124-126℃;1H NMR (CDCl3, 500 MHz) δ: 6.92-7.79 (m, 4H, Ar-H), 4.22 (s, 2H, NH2); IR (KBr): 3396.7, 3132.5, 1683, 1640.2, 1278, 811 cm-1.
6-methyl-1,3-benzothiazol-2-amine BT-2: yield 70%; Buff coloured solid; mp 140-144℃;1H NMR (CDCl3, 500 MHz) δ: 7.21-7.83 ( m, 3H, Ar-H)3.89 ( s, 2H, NH2), 2.32 (s, 3H, CH3) ; IR (KBr): 3390, 3005, 2938, 1625, 1531, 1275, 814 cm-1.
6-methoxy-1,3-benzothiazol-2-amine BT-3: yield 72%; Shiny brown coloured solid; mp 158-160℃;1H NMR (CDCl3, 500 MHz) δ: 7.17-8.04 ( m, 3H, Ar-H), 3.97 ( s, 2H, NH2), 3.75 (s, 3H, OCH3); IR(KBr): 3340, 3010, 2890, 1629, 1545, 1224, 1150, 813 cm-1
6-chloro-1,3-benzothiazol-2-amine BT-4: yield 73%; White coloured solid; mp 198-200℃;1H NMR (CDCl3, 500 MHz) δ:7.11-7.69 (m, 3H, Ar-H), 4.09 (s, 2H, NH2); IR (KBr): 3420, 3020,1645, 1541, 1220, 809, 715 cm-1.
6-nitro-1,3-benzothiazol-2-amine BT-5: yield 75%; Bright yellow coloured solid; mp 245-247℃1H NMR (CDCl3, 500 MHz) δ: 7.04-7.75 (m, 3H, Ar-H), 4.16 (s, 2H, NH2); IR (KBr): 3470, 3012, 1616, 1555, 1535, 1265 cm-1.
2.2.2. General synthetic procedure for 2-Chloro-N-(6-substituted-1,3-benzothiazol-2-yl) acetamide BTC (1-5)
To 250 ml round bottom flask, a suspension of the substituted 2-amino benzothiazole BT (1-5) (0.005mol), anhydrous Potassium carbonate (2.5 gm) and 1ml of TEA in dry chloroform (65 ml) was taken. To above suspension, chloroacetylchloride (0.006mol) was added and the mixture was refluxed for 4-5 hrs. The solvent was then evaporated and the resulting solid was washed with cold water and recrystallized using ethanol: water (80:20) mixture affording N-chloroacetyl derivatives BTC (1-5) [25].
N-(1,3-benzothiazol-2-yl)-2-chloroacetamide BTC-1: yield 72%; Cream coloured solid; mp 165-167℃; 1H NMR (CDCl3, 500 MHz) δ:11.19 ( s, 1h, CONH), 7.38-8.11 (m, 4H, Ar-H), 4.32 (s, 2H,CH2); IR (KBr): 3012.5, 1670.4, 1612.8, 1555.2, 1460.3, 1354.7, 1275.8, 725.3 cm-1.
2-chloro-N-(6-methyl-1,3-benzothiazol-2-yl) acetamide BTC-2: yield 77%; Light cream coloured solid; mp 184-186℃;1H NMR (CDCl3, 500 MHz) δ:11.34 (s, 1H, NH), 7.26-7.82 (m, 3H, Ar-H), 4.35 (s, 2H, CH2), 2.17 (s, 3H, CH3); IR (KBr): 3042.6, 1681.9, 1550.8, 1465.9, 1334.78, 1273, 709.8 cm-1.
2-chloro-N-(6-methoxy-1,3-benzothiazol-2-yl) acetamide BTC-3: yield 75%; Light brown coloured solid; mp 172-174℃;1H NMR (CDCl3, 500 MHz) δ:12.84 (s,1H,NH), 7.28-7.81 (m, 3H, Ar-H), 4.2 (s, 2H, CH2), 3.7 (s, 3H, OCH3); IR (KBr): 3078.4, 1666.5, 1604.8, 1550.8, 1465.9, 1348.3, 1265.3, 721.2 cm-1.
2-chloro-N-(6-chloro-1,3-benzothiazol-2-yl) acetamide BTC-4: yield 70%; Cream coloured solid; mp 208-210℃;1H NMR (CDCl3, 500 MHz) δ:10.83 (s, 1H, CONH), 7.63-8.11 (m, 3H, Ar-H), 4.69 (s, 2H, CH2); IR (KBr): 3033.20, 1664.2, 1640.2, 1582.2, 1567.1, 1371.1, 1278.5, 730.1 cm-1.
2-chloro-N-(6-nitro-1,3-benzothiazol-2-yl) acetamideBTC-5: yield 64%; Light brown coloured solid; mp 180-182℃;1H NMR (CDCl3, 500 MHz) δ:11.24 (s, 1H, CONH), 7.20-7.66 (m, 3H, Ar-H), 4.14 (s, 2H, CH2); IR (KBr): 3041.2, 1675.2, 1638.1, 1534, 1525, 1521, 1350, 1268.3, 722.3 cm-1.
2.2.3. General synthetic procedure for 2-(substituted amino)-N-(6-substituted-1,3- benzothiazole-2-yl) acetamide BTC (a-t)
To a solution of 2-chloro-N-(6-substituted-1,3-benzothiazol-2yl) acetamide BTC (1-5) (0.005mol) and amino derivatives (0.005mol) in dry chloroform was taken in 250 ml round bottom flask and refluxed for 1-2 hrs. The solvent was evaporated and resulting solid was washed with cold water. The solid thus obtained was purified by recrystallization using appropriate solvent gave corresponding BTC (a-t) with 50-70% yield [25].
N-(1,3-benzothiazol-2-yl)-2-(pyrimidin-2-yl amino) acetamide BTC a: yield 60% ; cream coloured solid ; mp 208-210℃ ; 1H NMR (DMSO-d6, 500 MHz) δ: 11.5 (s, 1H, NH), 6.9-8.6(m, 7H, Ar-H), 6.5(s, 1H, NH), 4.02(s, 2H, CH2); IR(KBr): 3371.6, 3140.2, 1767.1, 1735.9, 1651.1, 1566.2, 1315.8cm-1.
N-(1,3-benzothiazol-2-yl)-2-(pyridin-2-yl amino) acetamide BTC b: yield 53%; Brown coloured solid; mp 186-188 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 11.31(s, 1H, NH), 6.5-8.6(m, 8H, Ar-H), 4.5(s, 1H, NH), 3.8(s, 2H, CH2); IR (KBr): 3201.9, 3001.3, 2970.4, 1666.5, 1627.9, 1589.4, 1538.3, 1290.7 cm-1.
N-[2-(1,3-benzothiazol-2-ylamino)-2-oxoethyl] pyridine-3-carboxamide BTC c: yield 67%; light cream coloured solid; mp 190-192 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.28(s, 1H, NH), 8.59(s, 1H, NH), 7.39-8.0(m, 8H, Ar-H), 3.81(s, H, CH2); IR(KBr): 3321.1, 3110, 1778, 1614.1, 1568.1, 1554.5, 1321.2 cm-1.
N-[2-(1,3-benzothiazol-2-ylamino)-2-oxoethyl] benzamide (BTC d): yield 55%; light cream coloured solid; mp 122-124 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.5(s, 1H, NH), 8.1(s, 1H, NH), 7.39-7.87(m, 9H, Ar-H),4.1 (s, 2H, CH2) ; IR (KBr): 3301.1, 3082.4,2905.1,1687.5, 1628.2, 1528.2, 1527.5, 1285.1 cm-1.
N-(6-methyl-1,3-benzothiazol-2-yl)-2-(pyrimidin-2-ylamino) acetamide BTC e: yield 52%; Brown coloured solid; mp 236-238℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.3 (s, 1H, NH), 7.39-8.3 (m, 6H, Ar-H), 6.5 (s,1H, NH), 3.86 (s, 2H, CH2), 2.33 ( s, 3H, CH3); IR (KBr): 3011.1, 2949, 1625.3, 1625, 1519.2, 1515.2, 1280.9 cm-1.
N-(6-methyl-1,3-benzothiazol-2-yl)-2-(pyridin-3-ylamino) acetamide BTC f: yield 64%; Cream coloured solid; mp 125-127℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.35 (s, 1H, NH), 7.13-7.87 (m,7H, Ar-H), 4.44 (s, 1H, NH), 4.0 (s, 2H, CH2), 2.6 (s, 3H, CH3) ; IR (KBr): 3011.2, 2958.4, 1649.8, 1547.3, 1525, 1520.2, 1270 cm-1.
N-{2-[(6-methyl-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} pyridine-3-carboxamide BTC g: yield 50 %; Pale yellow coloured solid; mp 220-222 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.4 (s, 1H, NH), 8.6 ( s, 1H, NH), 7.12-7.87 (m, 7H, Ar-H), 4.0 (s, 2H, CH2), 2.66 (s, 3H, CH3 ) ; IR (KBr): 3117, 2978.1, 1645.5, 1535.2, 1515.2, 1465, 1288.4 cm-1.
N-{2-[(6-methyl-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} benzamide BTC h: yield 65%; Pale yellow coloured solid; mp 203-205 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.45 (s, 1H, NH), 8.6 ( s, 1H, NH), 7.13-8.6 (m, 8H, Ar-H) 4.0 ( s, 2H, CH2 ), 2.8 ( s, 3H, CH3 ) ; IR (KBr): 3110.1, 3058.1, 1757.1, 1732.9, 1651.1, 1599.2, 1318.3 cm-1.
N-(6-methoxy-1,3-benzothiazol-2-yl)-2-(pyrimidin-2-ylamino) acetamide BTC i: yield 60%; Brown coloured solid; mp 260-264 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.49 s, 1H, NH), 7.0-8.9 ( m, 6H, Ar-H), 6.7 ( s, 1H, NH ), 4.5 ( s, 3H, OCH3 ), 3.7 (s, 2H, CH2 ) ; IR (KBr): 3183.2, 3029.1, 2990.2, 1798.6, 1665.1, 1628.1, 1515.6, 1260.3, 1027 cm-1.
N-(6-methoxy-1,3-benzothiazol-2-yl)-2-(pyridine-3-yl amino) acetamide BTC j: yield 50%; Light cream coloured solid; mp 189-192 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.72 ( s,1H, NH), 7.12-8.32 ( m, 7H, Ar-H), 6.5 ( s, 1H, NH), 4.5( s, 3H, OCH3 ), 4.0 ( s, 2H, CH2); IR (KBr): 3294.5, 3000, 2910.4, 1666.5, 1620.2, 1535.3, 1498.2, 1288.4, 1049.3 cm-1.
N-{2-[(6-methoxy-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} pyridine-3-carboxamide BTC k: yield 52%; Light cream coloured solid; mp 169-198 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.87 (s, 1H, NH), 9.5 (s, 1H, NH), 7.12-8.32 ( m, 7H, Ar-H), 4.44 (s, 3H, OCH3), 4.06 (s, 3H, CH2); IR (KBr): 3274, 3069, 2989.2,1655.7, 1620, 1598.6, 1515.6, 1267, 1019.5 cm-1.
N-{2-[(6-methoxy-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} benzamide BTC l: yield 70%; Light yellow coloured solid; mp 142-144 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.0 (s, 1H, NH), 8.32 ( s, 1H, NH), 7.12-8.0 ( m, 8H, Ar-H),4.06 ( s, 2H, CH2 ), 3.80 (s, 3H, OCH3); IR (KBr): 3317.3, 3066.2, 2975.2, 1645.9, 1620.2, 1519.3, 1448.2, 1270.4, 1039.1 cm-1.
N-(6-chloro-1,3-benzothiazol-2-yl)-2-(pyrimidin-2-ylamino) acetamide BTC m: yield 55%; Brown coloured solid; mp 17.-172 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 13.01, (s, 1H, NH), 6.7-8.9 ( m, 6H, Ar-H), 5.5 ( s, 1H, NH), 3.83 (s, 3H, CH2); IR (KBr): 3099.8, 2897.6, 1660.5, 1638.2, 1567.4, 1524.3, 1268.1 cm-1.
N-(6-chloro-1,3-benzothiazol-2-yl)-2-(pyridin-3-ylamino) acetamide BTC n: yield 65%; Light cream coloured solid; mp 123-126 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.5 (s, 1H, NH), 6.7-8.9 (m, 7H, Ar-H), 6.00 ( s, 1H, NH), 4.0 (s, 2H, CH2 ); IR (KBr): 3090.5, 2925.1, 1660.1, 1659.2, 1548, 1525.24, 1269.28 cm-1; MS m/z: 320 [M-Cl]+.
N-{2-[(6-chloro-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} pyridine-3-carboxamide BTC o: yield 60%; Light cream coloured solid; mp 238-240 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.59 (s, 1H,NH), 8.6 (s, 1H, NH), 7.39-7.99 ( m, 7H, Ar-H), 3.85 (s, 2H, CH2 ); IR (KBr): 3026.1, 2918.1, 1666.5, 1620.2, 1538.3, 1512.2, 1268.1 cm-1.
N-{2-[(6-chloro-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} benzamide BTC p: yield 70%; Light yellow coloured solid; mp 226-229 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:1228 (s, 1H, NH), 8.4 ( s, 1H, NH), 7.39-8.2 (m, 8H, Ar-H), 3.85 (s, 2H, CH2); IR (KBr): 3009, 2877.8, 1797.1, 1712.8, 1651.1, 1558.5,1303.9 cm-1.
N-(6-nitro-1,3-benzothiazol-2-yl)-2-(pyrimidin-2-yl amino) acetamide BTC q: yield 62%; Yellow coloured solid; mp 198-201 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.3 (s, 1H, NH), 8.59 (s, 1H, NH), 7.12-8.0 (m, 6H, Ar-H), 4.06 ( s, 2H, CH2); IR (KBr): 3294.5, 3000, 2911.8,1791.1,1712.8, 1651.1,1558.5,1465.9,1309.8 cm-1.
N-(6-nitro-1,3-benzothiazol-2-yl)-2-(pyridin-2-yl amino) acetamide BTC r: yield 60%; Cream coloured solid; mp 123-125 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.53 (s, 1H, NH), 7.12-7.87 (m, 7H, Ar-H), 4.44 (s, 1H, NH), 3.84 (s, 2H, CH2); IR (KBr): 3394.8, 3015.2,2918.1,1666.5, 1620.2,1553.3,1512.2,1435, 1268.1 cm-1.
N-{2-[(6-nitro-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} pyridine-3-carboxamide BTC s: yield 65%; Yellow coloured solid; mp 248-250 ℃; 1H NMR (DMSO-d6, 500 MHz) δ: 12.45 (s, 1H, NH), 8.32 (s, 1H, NH), 7.39-8.6 ( m, 7H, Ar-H), 4.06 ( s, 2H, CH2) ; IR (KBr): 3314.3, 3080.2, 2900.1,1660.5,1637.2,1555.3,1522.1,1428.2,1254.2 cm-1.
N-{2-[(6-nitro-1,3-benzothiazol-2-yl) amino]-2-oxoethyl} benzamide BTC t: yield 65%; Yellow coloured solid; mp 240-242 ℃; 1H NMR (DMSO-d6, 500 MHz) δ:12.6 ( s, 1H,NH), 8.06 ( s,1H,NH), 7.39-8.59 ( m, 8H, Ar-H), 4.5 (s, 1H, CH2); IR (KBr): 3300.1, 3074.1,2968.2,1630.6,1621, 1598, 1564.1,1445.3,1227.9 cm-1.
2.3. Biological Activity Evaluation
All target compounds, BTC(a-t) were screened for their antibacterial, antifungal activity. The synthesized compounds were evaluated for their antimicrobial activity against gram positive, gram negative bacteria and fungi by determining-
- Zone of inhibition
- Minimum Inhibitory Concentration (MIC)
The synthesized compounds were evaluated for their antibacterial activity against two-gram positive (Staphylococcus aureus, Bacillus subtillis) and two-gram negative bacteria (E. coli, Pseudomonas aeruginosa) by using ciprofloxacin as reference antibacterial agent. Antifungal activity was evaluated against Candida albicans and Aspergillus niger. fluconazole was used as reference drug. The microbial strains were collected from microbiology laboratory of Waghire college, saswad.
2.3.1. Zone of inhibition using cup plate method
The compounds were evaluated for the antibacterial activity by cup plate method. The results were recorded for each tested compound as average diameter of inhibition zone of bacterial and fungal growth around the cup in mm. The nutrient agar medium containing peptone (1%), beef extract (0.5%), sodium chloride (0.8%) and agar (2.5%) in distilled water. The solution was sterilized for 20 min in an autoclave at 15 psi pressure at 121oC. The basal medium 15-20 ml was poured in the sterile petri-dishes. After solidification of medium, the microorganisms were sub-cultured and then holes of 6mm diameter were bored. To these cups, concentration of 50, 100, 150 μg/ml of the test compounds were added by micropipette. Petri dishes were kept in refrigerator to facilitate the diffusion for about 2 h. These plates were then incubated at 37°C for 48 h. The extent of inhibition was determined by measuring the diameter of the inhibition zone in mm. For antibacterial and antifungal study, the ciprofloxacin 150 μg/ml and 150 μg/ml of fluconazole were used as reference drugs respectively.
2.3.2. Minimal inhibitory concentration using two folds dilution method (MIC)
The broth dilution was performed by using microtiter plates. The nutrient broth was prepared using peptone (1%), beef extract (0.5%), sodium chloride (0.8%). It was added in microtiter plates except first well. First well of microtiter plate was used to check the sterility of the medium as negative control in which inoculum was not added. Stock solution of test compounds was prepared in DMSO (200μg/ml) followed by twofold dilution at concentrations of (100, 50, 25….3.125 μg/ml). The inoculums were added to the other all wells containing test compounds ranging from 100, 50, 25….3.125 μg/ml. The micro titer plates were then incubated at 37°C for 24 h for bacteria and 48 h for fungi and minimal inhibitory concentration were measured for the growth in the form of turbidity. The ciprofloxacin (150μg/ml) was used as reference drug for antibacterial study while fluconazole (150μg/ml) was used for antifungal study [26].
2.4. Molecular Docking
To check the type of interactions exists between the enzyme and the ligand the docking study is a promising tool. The prediction of predominant binding modes of a ligand with a protein of known three-dimensional structure is one of the important goals of ligand-protein docking. In present study, V Life MDS 3.5 software was used for docking purpose [27-31]. The structure of DNA gyrase protein (PDB: 3G75) was obtained from Protein Data Bank (http://www.rcsb.org).