Synthesis and Biological Evaluation of Indole Fused Triazine Derivatives
Surendra Nath Pandeya1, Rajeev Kumar1*, Gopal Nath2 and Ashish Kumar Pathak1
1Department of Pharmacy, Saroj Institute of Technology and Management, Sultanpur Road, Lucknow-226002, U.P., India
2Department of Microbiology, Institute of Medical Science, Banaras Hindu University, Varanasi, U.P., India
*Corresponding Author E-mail: rverma.rajeev@gmail.com
ABSTRACT:
A series of indole fused triazine derivatives have been synthesized and characterized by elemental and spectral analysis, further the compounds have been screened for antimicrobial activity against ten strains of Gram (+) and Gram (-) bacteria. Compound 5a was found to be most active in the series against E. faecalis ATCC29212 with MIC 12.5 µg/mL. From the antimicrobial screening result, it was observed that the newly synthesized compounds 5a, 5i and 5j showed good antimicrobial activity compared with standard sulphamethoxazole.
KEYWORDS: Triazine, antibacterial activity, MIC value, indole nucleus.
The quest for a more reliable and suitable drugs is always fascinating and challenging. A number of drugs containing simple heterocyclic or a combination of different moieties have been in use these days.1 Antibacterial disease is very common all over the world. Currently used antimicrobial agents are not effective due to the resistance developed by the microbes. And therefore, it is an ongoing effort to synthesize new antimicrobial agents.2
1,2,4-Triazines and their derivatives have been widely studied in terms of their synthetic methodologies and reactivity since some of these derivatives were reported to have promising biological activities like A1 Adenosine receptor antagonists3, Age-related macular degeneration4, analgesic5, anti-inflammatory activities5, Anticancer Activity6,7, anticonvulsant activity8, antimicrobial activity9,10, antinociceptive activity11, Antiproliferative activity12, anxioselective agent13, Kinase inhibitor activity14, Muscle Relaxant activity15.
An indole nucleus have arose great attention in recent years due to their wide variety of biological activities and pharmacological studies as cytotoxicity activity16, Kinase inhibitors17, anticancer17, antiangiogenic agents17, antimicrobial activity18-21, 23, Anti- HIV22, anti-inflammatory24, analgesic activity24, Anticonvulsant activity25,26, Sedative-Hypnotic Activities26.
Based on the above observations, herein are reported the synthesis of various indole fused Triazine derivatives and evaluation of their antibacterial activity.
EXPERIMENTAL WORK:
General Procedures. Melting points were determined using open capillary tubes on a Thiel’s melting point apparatus and are uncorrected. The purity of the synthesized compounds was checked by TLC (solvent -Ethyl acetate: n-hexane=5:5, Visualising agent- iodine vapour) and column chromatography. FT-IR was recorded on a Perkin Elmer FT-IR spectrophotometer, 1H NMR spectra were recorded at 300 MHz on a Bruker DRX-300 FT-NMR spectrophotometer (CDRI, Lucknow). The elemental analysis was obtained on a Elementar Vario EL III instrument (CDRI, Lucknow).
N-Methylindoline-2, 3-dione (N-Methyl isatin)
Isatin (5.00 g) and dimethyl sulphate (0.033 mL) were added in dil. sodium hydroxide (1N) in round bottom flack. This mixture was refluxed for approx. 50 minute on oil bath. After this the reaction mixture was poured in beaker and evaporated under reduced pressure. Then solid comp. was obtained.
M.P .=136 0 C, Rf =0.10, and yield=83.94%
General procedure for bromination of isatin and its derivatives
Bromine (1.00 mL) was added in acetic acid (4.00 mL) in 100 ml beaker in ice cold condition with stirring. Isatin derivatives (1.00 g) were dissolved in acetic acid in 100 mL beaker with stirring. Bromine solution (0.35 mL) was mixed in isatin derivatives solution in ice cold condition with stirring. Then mixture was placed in freezer for 24 hours. The solid was formed, dried it.
Characterization of 5-Bromoindoline-2,3-dione (5-Bromo isatin)
M.P. =1840 C, Rf =0.70 and yield=25%
Characterization of 5-Bromo-1-methylindoline-2,3-dione (5-Bromo N-methyl isatin)
M.P. = 46 0 C, Rf =0.87, and Percentage yield=27%
General procedure for nitration of isatin and its derivatives
Isatin derivatives (1.00 g) were dissolved in acetic acid in 100 mL beaker. Then sulphuric acid (1.8 mL) was added drop wise in ice cold condition with stirring. The nitric acid (0.6 mL) was added in mixture, stirred for some time. Then mixture was placed in freezer for 24 hours. The solid precipited was formed, dried it.
Characterization of 5-Nitroindoline-2, 3-dione (5-Nitro isatin)
M.P. =230 0 C, Rf =0.79 and Percentage yield=67%
Characterization of 1-Methyl-5-nitroindoline-2,3-dione (N-methyl 5-nitro isatin)
M.P. = 255 0 C, Rf =0.72, and yield=27%
General procedure for synthesis of thio or semicarbazones of isatin derivatives (3a-l)
Isatin derivatives (1mole) were dissolved in boiling acetic acid (50 mL) in 100 mL beaker. Thio or semicarbazide (1mole) was dissolved in distil water (10 mL) in another 100 mL beaker. Then this solution was added in boiling isatin mixture. The mixture was boiled for approx. 20 min. with stirring (3a, 3c, 3e, 3g, 3i, 3k) or refluxed for about 10 hours (3b, 3d, 3f, 3h, 3j, 3l). After cooling the solid formed was filtered off, washed with acetic acid followed with water. The dried product was recrystallized from acetic acid affording yellow crystals.
1-(2-Oxoindolin-3-ylidene) semicarbazide (3a)
M.P. =240 0 C, yield=55%, IR (KBr) in cm-1: 3406 (20NH), 3315 (10NH), 1725 (C=O), 1621 (amide C=O), 1660 (C=N). 1H-NMR (DMSO) δ ppm: 7.23- 8.01 (m, 4H, Ar-H), 7.21 (s, 1H, NH), 6.49 (s, 2H, NH2), 8.39 (s, 1H, indole-NH).
1-(2-Oxoindolin-3-ylidene) thiosemicarbazide (3b)
M.P. =220 0 C, yield=64%, IR (KBr) in cm-1: 3456 (2°NH), 3388 (10NH), 1678(C=O), 1624 (C=N), 1019(C=S). 1H-NMR (DMSO) δ ppm: 7.33,-7.81 (m, 4H, Ar-H), 7.36 (s, 1H, NH), 2.22 (s, 2H, NH2), 8.25 (s, 1H, indole-NH).
1-(1-Methyl-2-oxoindolin-3-ylidene) semicarbazide (3c)
M.P. =255 0 C, yield=51%, IR (KBr) in cm-1: 3425 (2°NH), 3318 (NH), 2922 (-CH3), 1655 (C=O), 1672 (amide C=O), 1690 (C=N). 1H-NMR (DMSO) δ ppm: 7.39-7.98 (m, 4H, Ar-H), 7.17 (s, 1H, NH), 6.55 (s, 2H, NH2), 3.19 (s, 3H, CH3).
1-(1-Methyl-2-oxoindolin-3-ylidene) thiosemicarbazide (3d)
M.P. =190 0 C, yield=57%, IR (KBr) in cm-1: 3443 (2°NH), 3344 (10NH), 2909(-CH3), 1674 (C=O), 1613 (C=N), 1023 (C=S). 1H-NMR (DMSO) δ ppm: 7.23-7.91(m, 4H, Ar-H), 7.38 (s, 1H, NH), 2.49 (s, 2H, NH2), 3.09 (s, 3H, CH3).
1-(5-Bromo-2-oxoindolin-3-ylidene) semicarbazide (3e)
M.P. = 175 0 C, yield=48%, IR (KBr) in cm-1: 3442 (20NH), 3223 (10NH), 1732 (C=O), 1665 (amide C=O), 1615 (C=N), 544 (C-Br). 1H-NMR (DMSO) δ ppm: 7.57-8.01 (m, 3H, Ar-H), 7.38 (s, 1H, NH), 6.28 (s, 2H, NH2), 8.25 (s, 1H, indole-NH).
1-(5-Bromo-2-oxoindolin-3-ylidene) thiosemicarbazide (3f)
M.P. = 145 0 C, yield=77%, IR (KBr) in cm-1: 3448 (2°NH), 3378 (10NH), 2935 (-CH3), 1683 (C=O) 1604 (C=N), 1090 (C=S), 534 (C-Br). 1H-NMR (DMSO) δ ppm: 7.41-7.96 (m, 3H, Ar-H), 7.38 (s, 1H, NH), 2.49 (s, 2H, NH2), 8.41 (s, 1H, indole-NH).
1-(5-Bromo-1-methyl-2-oxoindolin-3-ylidene) semicarbazide (3g)
M.P. = 195 0 C, yield=16%, IR (KBr) in cm-1: 3475 (2°NH), 3363 (NH), 2937(-CH3), 1645 (C=O), 1626 (amide C=O), 1679 (C=N), 546(C-Br). 1H-NMR (DMSO) δ ppm: 7.69- 8.07 (m, 3H, Ar-H), 2.99 (s, 3H, CH3), 7.53 (s, 1H, NH), 6.67 (s, 2H, NH2).
1-(5-Bromo-1-methyl-2-oxoindolin-3-ylidene) thiosemicarbazide (3h)
M.P. = 150 0 C, yield=29%, IR (KBr) in cm-1: 3465 (2°NH), 3381 (NH), 2960(-CH3), 1740 (C=O), 1617 (C=N), 1064 (C=S), 534 (C-Br). 1H-NMR (DMSO) δ ppm: 7.69-8.10 (m, 3H, Ar-H), 7.42 (s, 1H, NH), 2.49 (s, 2H, NH2), 3.39 (s, 3H, CH3).
1-(5-Nitro-2-oxoindolin-3-ylidene) semicarbazide (3i)
M.P. =265 0 C, yield=36%, IR (KBr) in cm-1: 3436 (2°NH), 3319 (NH), 1742 (C=O), 1643 (amide C=O), 1651 (C=N), 1342 (C-NO2). 1H-NMR (DMSO) δ ppm: 8.23- 8.67 (m, 3H, Ar-H), 7.47 (s, 1H, NH), 6.81 (s, 2H, NH2), 8.89 (s, 1H, indole-NH).
1-(5-Nitro-2-oxoindolin-3-ylidene) thiosemicarbazide (3j)
M.P. =220 0 C, yield=51%, IR (KBr) in cm-1: 3434 (2°NH), 3323 (10NH), 1734 (C=O), 1664 (amide C=O), 1688 (C=N), 1545, 1323 (C-NO2), 1087 (C=S). 1H-NMR (DMSO) δ ppm: 8.47-8.78 (m, 3H, Ar-H), 7.85 (s, 1H, NH), 2.69 (s, 2H, NH2), 8.37 (s, 1H, indole-NH).
Table 1: Structures and physicochemical data of compounds 3a-l.
Code No. |
R1 |
R2 |
X |
Mol. For. |
Mol. Wt |
% yield |
M.P. (0C) |
Rf |
Log p* |
Elemental analysis Calculated (found) (%) |
3a |
H |
H |
O |
C9H8N4O2 |
204.18 |
55 |
240 |
0.53 |
-0.3 |
C52.94(52.97), H3.95(3.90), N27.44(27.48) |
3b |
H |
H |
S |
C9H8N4OS |
220.25 |
64 |
220 |
0.39 |
0.26 |
C49.08(49.05), H3.66(3.69), N25.44(25.47) |
3c |
CH3 |
H |
O |
C10H10N4O2 |
218.21 |
51 |
255 |
0.84 |
-0.07 |
C55.04(55.09), H4.62(4.67), N25.68(25.73) |
3d |
CH3 |
H |
S |
C10H10N4OS |
234.27 |
57 |
190 |
0.91 |
0.49 |
C51.27(51.31), H4.30(4.26), N23.91(23.94) |
3e |
H |
Br |
O |
C9H7BrN4O2 |
283.08 |
48 |
175 |
0.62 |
0.53 |
C38.19(38.23), H2.49(2.52), N19.79(19.75) |
3f |
H |
Br |
S |
C9H7BrN4OS |
299.14 |
77 |
145 |
0.47 |
1.09 |
C36.13(36.17), H2.36(2.39), N18.73(18.75) |
3g |
CH3 |
Br |
O |
C10H9BrN4O2 |
297.10 |
16 |
195 |
0.83 |
0.76 |
C40.43(40.46), H3.05(3.01), N18.86(18.90) |
3h |
CH3 |
Br |
S |
C10H9BrN4OS |
313.17 |
29 |
150 |
0.87 |
1.32 |
C38.35(38.39), H2.90(2.88), N17.89(17.94) |
3i |
H |
NO2 |
O |
C9H7N5O4 |
249.18 |
36 |
265 |
0.36 |
-0.86 |
C43.38(43.41), H2.83(2.87), N28.11(28.15) |
3j |
H |
NO2 |
S |
C9H7N5O3S |
265.24 |
51 |
220 |
0.86 |
-0.36 |
C40.75(40.79), H2.66(2.69), N26.40(26.44) |
3k |
CH3 |
NO2 |
O |
C10H9N5O4 |
263.20 |
42 |
250 |
0.62 |
-1.14 |
C45.63(45.67), H3.45(3.48), N26.61(26.65) |
3l |
CH3 |
NO2 |
S |
C10H9N5O3S |
279.27 |
27 |
230 |
0.90 |
-0.64 |
C43.01(43.05), H3.25(3.27), N25.08%) |
*Log p value calculated by Chem office 2004 software. TLC solvent was ethyl acetate: n-hexane=5:5.
Table2:Structures and physicochemical data of compounds 4a-l.
Code No. |
R1 |
R2 |
X |
Mol. For. |
Mol. wt. |
% yield |
M.P. (0C) |
Rf |
Log p* |
Elemental analysis Calculated (found) (%) |
4a |
H |
H |
O |
C9H8O2N4 |
204.19 |
72 |
250 |
0.51 |
-0.07 |
C52.94(53.01), H3.95(3.91), N27.44(27.47) |
4b |
H |
H |
S |
C9H8ON4S |
220.25 |
22 |
260 |
0.27 |
0.48 |
C49.08(49.12), H3.66(3.69), N25.44(25.45) |
4c |
CH3 |
H |
O |
C10H10O2N4 |
218.21 |
43 |
270 |
0.51 |
0.23 |
C55.04(55.07), H4.62(4.58), N25.68(25.72) |
4d |
CH3 |
H |
S |
C10H10ON4S |
234.28 |
36 |
210 |
0.63 |
0.78 |
C51.27(51.31), H4.30(4.27), N23.91(23.95) |
4e |
H |
Br |
O |
C9H7BrN4O2 |
283.08 |
67 |
275 |
0.28 |
0.76 |
C38.19(38.23), H2.49(2.53), N19.79(19.76) |
4f |
H |
Br |
S |
C9H7BrN4OS |
299.15 |
82 |
220 |
0.78 |
1.31 |
C36.13(36.17), H2.36(2.40), N18.73(18.79) |
4g |
CH3 |
Br |
O |
C10H9BrN4O2 |
297.11 |
85 |
240 |
0.35 |
1.06 |
C40.43(40.40),H3.059(3.09), N18.86(18.89) |
4h |
CH3 |
Br |
S |
C10H9BrN4OS |
313.17 |
34 |
230 |
0.83 |
1.61 |
C38.35(38.38), H2.90(2.93), N17.89(17.92) |
4i |
H |
NO2 |
O |
C9H7N5O4 |
249.18 |
67 |
295 |
0.60 |
-0.61 |
C43.38(43.36), H2.83(2.85), N28.11(28.07) |
4j |
H |
NO2 |
S |
C9H7N5O3S |
265.25 |
85 |
290 |
0.70 |
-0.10 |
C40.75(40.77), H2.66(2.60), N26.40(26.37) |
4k |
CH3 |
NO2 |
O |
C10H7N5O3 |
263.21 |
67 |
280 |
0.55 |
0.12 |
C45.63(45.60), H3.45(3.48), N26.61(26.58) |
4l |
CH3 |
NO2 |
S |
C10H9N5O3S |
279.28 |
78 |
295 |
0.45 |
0.63 |
C43.01(43.05), H3.25(3.22), N25.08(25.11) |
*Log p value calculated by Chem Draw 2004 software, TLC solvent was ethyl acetate: n-hexane=5:5.
Table 3: Structures and physicochemical data of compounds 5a-l
Code No. |
R1 |
R2 |
X |
Mol. For. |
Mol. Wt |
% yield |
M.P. (0C) |
Rf |
Log p* |
Elemental analysis Calculated (found) (%) |
5a |
H |
H |
O |
C9H6ON4 |
186.17 |
94 |
275 |
0.64 |
1.13 |
C58.06(58.02), H3.25(3.28), N30.09 (30.06) |
5b |
H |
H |
S |
C9H6N4S |
202.24 |
93 |
280 |
0.57 |
1.67 |
C53.45(53.48), H2.99(2.93), N27.70(27.68) |
5c |
CH3 |
H |
O |
C10H8ON4 |
200.2 |
78 |
280 |
0.62 |
1.91 |
C59.99(59.94), H4.03(4.01), N27.99(27.95) |
5d |
CH3 |
H |
S |
C10H8N4S |
216.26 |
94 |
280 |
0.65 |
2.46 |
C55.54(55.51), H3.73(3.69), N25.91(25.88) |
5e |
H |
Br |
O |
C9H5BrN4O |
265.07 |
21 |
290 |
0.52 |
1.95 |
C40.78(40.75), H1.90(1.87), N21.14(21.16) |
5f |
H |
Br |
S |
C9H5BrN4S |
281.13 |
61 |
250 |
0.63 |
2.5 |
C38.45(38.41), H1.79(1.82), N19.93(19.91) |
5g |
CH3 |
Br |
O |
C10H7BrN4O |
279 |
67 |
295 |
0.58 |
2.74 |
C43.03(42.98), H2.53(2.55), N20.07(20.11) |
5h |
CH3 |
Br |
S |
C10H7BrN4S |
295.16 |
38 |
260 |
0.40 |
3.29 |
C40.69(40.72), H2.39(2.43), N18.98(19.01) |
5i |
H |
NO2 |
O |
C9H5N5O3 |
231.14 |
88 |
>300 |
0.67 |
0.06 |
C46.76(46.72), H2.18(2.20), N30.30(30.33) |
5j |
H |
NO2 |
S |
C9H5N5O2S |
247.23 |
80 |
275 |
0.55 |
0.57 |
C43.72(43.70), H2.04(2.07), N28.33(28.36) |
5k |
CH3 |
NO2 |
O |
C10H7N5O3 |
263.21 |
60 |
280 |
0.61 |
-0.22 |
C48.98(48.99), H2.88(2.85), N28.56(28.59) |
5l |
CH3 |
NO2 |
S |
C10H7N5O2S |
261.26 |
65 |
>300 |
0.52 |
0.29 |
C45.97(45.95), H2.70(2.73), N26.81(26.84) |
*Log p value calculated by Chem Draw 2004software, TLC solvent was ethyl acetate: n-hexane=5:5
Table 4: In vitro antibacterial activities of compounds 5a-l against selected strains (MICs in μg /mL)
S. No. |
Name of bacteria |
5a |
5f |
5i |
5j |
5l |
Sulph. |
|||||
1- |
Staphylococcus aureus |
500 |
400 |
200 |
25 |
400 |
5000 |
|||||
2- |
E.coli ATCC25922 |
50 |
150 |
100 |
50 |
50 |
1250 |
|||||
3- |
E.faecalis ATCC29212 |
12.5 |
--- |
200 |
50 |
500 |
5000 |
|||||
4- |
H. pylori |
100 |
50 |
200 |
100 |
100 |
2500 |
|||||
5- |
Klebsiella oxytoca |
500 |
--- |
200 |
50 |
--- |
5000 |
|||||
6- |
Morganella morganii |
150 |
--- |
100 |
50 |
200 |
2500 |
|||||
7- |
Pseudo.aeruginosa ATCC27853 |
500 |
--- |
200 |
250 |
--- |
5000 |
|||||
8- |
Shigella flexinerii |
100 |
--- |
200 |
50 |
50 |
2500 |
|||||
9- |
Salmonella typhi |
400 |
200 |
150 |
25 |
--- |
2500 |
|||||
10- |
Vibrio cholera |
500 |
--- |
150 |
400 |
300 |
5000 |
|||||
Sulph.= Sulphamethoxazole as standard drug, --- =Insensitive (inactive)
1-(1-Methyl-5-nitro-2-oxoindolin-3-ylidene) semicarbazide (3k)
M.P. =250 0 C, yield=42%, IR (KBr) in cm-1: 3447 (2°NH), 3335 (10NH), 2949 (-CH3), 1765 (C=O), 1634 (amide C=O), 1674 (C=N), 1335 (C-NO2). 1H-NMR (DMSO) δ ppm: 8.13-8.79, (m, 3H, Ar-H), 7.19 (s, 1H, NH), 6.23 (s, 2H, NH2), 2.99 (s, 3H, CH3).
1-(1-Methyl-5-nitro-2-oxoindolin-3-ylidene) thiosemicarbazide (3l)
M.P. =230 0 C, yield=27%, IR (KBr) in cm-1: 3431 (2°NH), 3321 (NH), 2930 (-CH3), 1740 (C=O), 1666 (amide C=O), 1650 (C=N), 1344 (C-NO2), 1023 (C=S). 1H-NMR (DMSO) δ ppm: 7.99-8.61(m, 3H, Ar-H), 7.46 (s, 1H, NH), 2.39 (s, 2H, NH2), 2.88 (s, 3H, CH3).
General procedure for synthesis of triazine derivatives in presence of NaOH (4a-l)
Thio or semicarbazone derivatives (2.00 g) were dissolved in a boiling solution of sodium hydroxide (1M, 100 mL) in 250 mL conical flask. This mixture was refluxed for 3.00 hours. After cooling, the mixture was acidified with acetic acid. The solid formed was immediately filtered off, washed with water and dried affording the yellow solid.
6-(2-Aminophenyl)-1,2,4-triazine-3,5(2H,4H)-dione (4a)
M.P. =245-260 0C, yield=72%, IR (KBr) in cm-1: 3489 (NHstr, ArNH2), 3435 (NHstr, cyclic CONH), 1660 (C=O, cyclic CONH), 1654 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.66-7.77 (m, 4H, Ar-H), 4.47 (s, 2H, NH2), 7.55 (s, 1H, cyclic NNHCO), 10.47 (s, 1H, cyclic CONH).
6-(2-Aminophenyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4b)
M.P. = 260 0C, yield=22%, IR (KBr) in cm-1: 3457 (NHstr, ArNH2), 3317 (NHstr, cyclic CONH), 1065 (C=Sstr), 1659 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.76-7.53 (m, 4H, Ar-H), 4.61(s, 2H, NH2), 7.44 (s, 1H, cyclic NNHCS), 8.43 (s, 1H, cyclic CONH).
6-(2-(Methylamino) phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (4c).
M.P. = 270 0C, yield= 43%, IR (KBr) in cm-1: 3436 (NHstr, ArNH), 3342 (NHstr, cyclic CONH), 2884 (CHstr, CH3), 1664 (C=O, cyclic CONH), 1428 (CHbend, CH3), 1623 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.63-7.78 (s, 4H, Ar-H), 2.92 (s, 3H, CH3), 4.30(s, 1H, NH), 7.1(s, 1H, cyclic NNHCO), 10.54 (s, 1H, cyclic CONH).
3,4-Dihydro-6-(2-(methylamino)phenyl)-3-thioxo-1,2,4-triazin-5(2H)-one (4d)
M.P. = 210 0C, yield= 36%, IR (KBr) in cm-1: 3451 (NHstr, ArNH), 3331 (NHstr, cyclic CONH), 2901 (CHstr, CH3), 1609 (C=O), 1426 (CHbend, CH3), 1081 (C=Sstr), 1648 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.73-7.68 (m, 4H, Ar-H), 2.87 (s, 3H, CH3), 4.58 (s, 1H, NH), 7.31(s, 1H, cyclic NNHCS), 10.3 (s, 1H, cyclic CONH).
6-(2-Amino-5-bromophenyl)-1,2,4-triazine-3,5(2H,4H)-dione (4e)
M.P. =275 0C, yield= 67%, IR(KBr) in cm-1: 3429 (NHstr, ArNH2), 3322 (NHstr, cyclic CONH), 1687 (C=O, cyclic CONH), 570 (C-Br), 1674 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.72-7.69 (m, 3H, Ar-H), 4.66 (s, 2H, NH2), 7.54 (s, 1H, cyclic NNHCO), 10.37 (s, 1H, cyclic CONH).
6-(2-Amino-5-bromophenyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4f)
M.P. = 220 0C, yield= 82%, IR (KBr) in cm-1: 3363 (NHstr, ArNH2), 3327 (NHstr, cyclic CONH), 1695 (C=O), 1075 (C=Sstr), 580 (C-Br), 1663 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.51-7.94 (m, 3H, Ar-H), 4.43 (s, 2H, NH2), 7.66(s, 1H, cyclic NNHCS), 8.37 (s, 1H, cyclic CONH).
6-(5-Bromo-2-(methylamino) phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (4g).
M.P. = 240 0C, yield=85%, IR (KBr) in cm-1: 3445 (NHstr, ArNH), 3336 (NHstr, cyclic CONH), 2869 (CHstr, CH3), 1630 (C=O, cyclic CONH), 1412 (CHbend, CH3), 1377, 1323 (NOstr, NO2), 770 (CSstr), 1683 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.38-7.99(m, 3H, Ar-H), 3.10 (s, 3H, CH3), 4.27 (s, 1H, NH), 7.74 (s, 1H, cyclic NNHCO), 10.49 (s, 1H, cyclic CONH).
6-(5-Bromo-2-(methylamino)phenyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4h)
M.P. = 230 0C, yield=34%, IR (KBr) in cm-1: 3463(NHstr, ArNH), 3318 (NHstr, cyclic CONH), 2855 (CHstr, CH3), 1432 (CHbend, CH3), 578 (C-Br), 1085 (C=Sstr), 1639 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.39-7.58 (m, 3H, Ar-H), 3.02 (s, 3H, CH3), 4.76 (s, 1H, NH), 7.47 (s, 1H, cyclic NNHCS), 8.37 (s, 1H, cyclic CONH).
6-(2-Amino-5-nitrophenyl)-1,2,4-triazine-3,5(2H,4H)-dione (4i)
M.P. = 295 0C, yield= 67%, IR (KBr) in cm-1: 3439 (NHstr, ArNH2), 3328 (NHstr, cyclic CONH), 1678 (C=O, cyclic CONH), 1384, 1317 (NOstr, NO2), 1681 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.98-8.79 (m, 3H, Ar-H), 4.46 (s, 2H, NH2), 7.41(s, 1H, cyclic NNHCO), 10.39 (s, 1H, cyclic CONH).
6-(2-Amino-5-nitrophenyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4j)
M.P. =290 0 C, yield=85%, IR (KBr) in cm-1: 3467 (NHstr, ArNH2), 3346 (NHstr, cyclic CONH), 1364, 1312 (NOstr, NO2), 1059 (C=Sstr), 1685 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.91-8.89 (m, 3H, Ar-H), 4.76 (s, 2H, NH2), 7.77 (s, 1H, cyclic NNHCS), 10.61 (s, 1H, cyclic CONH).
6-(2-(Methylamino)-5-nitrophenyl)-1, 2, 4-triazine-3, 5(2H, 4H)-dione (4k)
M.P. = 280 0C, yield=67%, IR (KBr) in cm-1: 3433 (NHstr, ArNH), 2854 (CHstr, CH3), 1605 (C=O, cyclic CONH), 1459 (CHbend, CH3), 1377, 1323 (NOstr, NO2), 1605 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 7.03-8.69 (m, 3H, Ar-H), 3.11 (s, 3H, CH3), 4.15 (s, 1H, NH), 7.61 (s, 1H, cyclic NNHCO), 10.26 (s, 1H, cyclic CONH).
3,4-Dihydro-6-(2-(methylamino)-5-nitrophenyl)-3-thioxo-1,2,4-triazin-5(2H)-one (4l)
M.P. = 295 0C, yield=78 %, IR (KBr) in cm-1: 3445 (NHstr, ArNH), 2580 (CHstr, CH3), 1412 (CHbend, CH3), 1102 (C=Sstr), 1630 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 7.04-8.89 (m, 3H, Ar-H), 3.06 (s, 3H, CH3), 4.56 (s, 1H, NH), 7.71(s, 1H, cyclic NNHCS), 8.47 (s, 1H, cyclic CONH).
General procedure for synthesis of novel triazine derivatives in presence of acetic acid (5a-l)
Triazine derivatives 4a-l (1.00 g) were dissolved in acetic acid (100 mL) in 250 mL conical flask. Then this mixture was refluxed for about 2.30 hours. The solid formed was filtered off, washed with water and dried yielding the yellow solid. TLC solvent was ethyl acetate: n-hexane=5:5.
2H-[1,2,4]Triazino[5,6-b]indol-3(5H)-one (5a)
M.P. =275 0C, yield=94%, IR (KBr) in cm-1: 3458 (NHstr, indole-NH), 3338 (NHstr, cyclic CONH), 1650 (C=Ostr, cyclic CONH), 1669 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.53-7.45 (m, 4H, Ar-H), 4.64 (s, 1H, indole-NH), 7.02 (s, 1H, cyclic CONH).
2H-[1,2,4]Triazino[5,6-b]indole-3(5H)-thione (5b)
M.P. =280 0C, yield= 93%, IR (KBr) in cm-1: 3479 (NHstr, indole-NH), 3321 (NHstr, cyclic CONH), 1045(C=S), 1639 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.76-7.51 (m, 4H, Ar-H), 4.74 (s, 1H, indole-NH), 7.35 (s, 1H, cyclic CSNH).
5-Methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5c)
M.P. = 280 0C, yield=78%, IR (KBr) in cm-1: 3473 (NHstr, indole-NH), 3310 (NHstr, cyclic CONH), 2838 (CHstr, CH3), 1621 (C=Ostr, trizine-ring), 1489 (CHbend, CH3), 1625 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.67-7.56 (m, 4H, Ar-H), 2.92 (s, 3H, CH3), 7.26 (s, 1H, cyclic CONH).
5-Methyl-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione (5d)
M.P. = 280 0C, yield=94%, IR (KBr) in cm-1: 3451 (NHstr, indole-NH), 2901 (CHstr, CH3), 1457 (CHbend, CH3), 1004 (C=S), 1609 (cyclic C=N). 1H- NMR (DMSO) δ ppm: 6.89-7.61 (m, 4H, Ar-H), 2.96 (s, 3H, CH3), 7.19 (s, 1H, cyclic CSNH).
8-Bromo-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5e)
M.P. = 290 0C, yield= 21%, IR (KBr): 3434 (NHstr, indole-NH), 3365 (NHstr, cyclic CONH), 1635 (C=Ostr, trizine-ring), 1443 (CHbend, CH3), 551 (C-Br), 1627 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.58-7.69 (m, 3H, Ar-H), 4.23 (s, 1H, indole-NH), 7.29 (s, 1H, cyclic CONH).
8-Bromo-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione (5f).
M.P. = 250 0C, yield= 61%, IR (KBr) in cm-1: 3410 (NHstr, indole-NH), 3365 (NHstr, cyclic CONH), 2810 (CHstr, CH3), 1443 (CHbend, CH3), 579 (C-Br), 1603 (cyclic C=N), 1075 (C=S). 1H-NMR (DMSO) δ ppm: 6.54-7.61 (m, 3H, Ar-H), 4.08 (s, 1H, indole-NH), 7.33 (s, 1H, cyclic CSNH).
8-Bromo-5-methyl-2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one (5g)
M.P. = 295 0C, yield = 67%, IR (KBr) in cm-1: 3434 (NHstr, indole-NH), 3325 (NHstr, cyclic CONH), 1635 (C=Ostr, trizine-ring), 2806 (CHstr, CH3), 1443 (CHbend, CH3), 575 (C-Br). 1H-NMR (DMSO) δ ppm: 6.79-.91 (m, 3H, Ar-H), 2.86 (s, 3H, CH3), 7.49 (s, 1H, cyclic CONH).
8-Bromo-5-methyl-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione (5h)
M.P. = 260 0C, yield=38%, IR (KBr) in cm-1: 3434 (NHstr, indole-NH), 3321 (NHstr, cyclic CONH), 2859 (CHstr, CH3), 1469 (CHbend, CH3), 560 (C-Br), 1621 (cyclic C=N), 1046 (C=S). 1H-NMR (DMSO) δ ppm: 6.51-7.88 (m, 3H, Ar-H), 2.71(s, 3H, CH3), 7.39 (s, 1H, cyclic CSNH).
8-Nitro-2H-[1, 2, 4] triazino [5, 6-b] indol-3(5H)-one (5i)
m.p. = > 300 0C, yield=88%, IR (KBr) in cm-1: 3402 (NHstr, indole-NH), 2969 (NHstr, cyclic CONH), 1786 (C=Ostr, trizine-ring), 1578 (C=Nstr, trizine-ring), 1493, 1460 (NOstr, NO2), 1H-NMR (DMSO) δ ppm: 6.91-8.89 (m, 3H, Ar-H), 4.59 (s, 1H, indole-NH), 7.28 (s, 1H, cyclic CONH).
8-Nitro-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione (5j)
M.P. = 275 0C, yield=80%, IR (KBr) in cm-1: 3470 (NHstr, indole-NH), 3038 (NHstr, Cyclic CONH), 1344, 1304 (NOstr, NO2), 1083 (C=Sstr), 1665 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.78-8.58 (m, 3H, Ar-H), 4.95 (s, 1H, indole-NH), 7.48 (s, 1H, cyclic CSNH).
5-Methyl-8-nitro-2H-[1, 2, 4] triazino [5, 6-b] indol-3(5H)-one (5k)
M.P. = 280 0C, yield=60%, IR (KBr) in cm-1: 3457 (NHstr, indole-NH), 3337 (NHstr, cyclic CONH), 2847 (CHstr, CH3), 1626 (C=Ostr, trizine-ring), 1423 (CHbend, CH3), 1364, 1435(NOstr, NO2), 1692 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.87-8.61 (m, 3H, Ar-H), 2.82 (s, 3H, CH3), 7.42 (s, 1H, cyclic CONH).
5-Methyl-8-nitro-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione (5l)
M.P. = >300 0C, yield= 65%, IR (KBr) in cm-1: 3478 (NHstr, indole-NH), 3208 (NHstr, cyclic CONH), 2859 (CHstr, CH3), 1450 (CHbend, CH3), 1367, 1312 (NOstr, NO2), 1053 (C=Sstr), 1670 (cyclic C=N). 1H-NMR (DMSO) δ ppm: 6.84-8.53 (m, 3H, Ar-H), 2.85 (s, 3H, CH3), 7.49 (s, 1H, cyclic CSNH).
Physico-chemical characterization:
The physical data of compounds were presented in table 1, 2 and 3. The compounds were identified by elemental analysis, FT-IR, NMR data.
Biological evaluation:
In vitro antibacterial activity:
Synthesized compounds were evaluated for their in-vitro antibacterial activity against pathogenic bacteria. The agar dilution method was performed using Muller-Hinton agar (Hi-Media) medium. Suspension of each microorganism was prepared and applied to plates with serially diluted compounds (DMF, solvent control) to be tested and incubated (approx. 20 hours) at 370C. The minimum inhibitory concentration (MIC) was considered to be the lowest concentration that was completely inhibited growth on agar plates.
SUMMARY AND CONCLUSION:
1, 2, 4-triazine heterocyclic entity are very interesting components in terms of their biological properties, such as antifungal, antibacterial and herbicidal were studied. Synthesized compounds 5a-l were tested against a panel of microorganisms including Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli ATCC-25922, Morganella morganii, Vibrio cholerae, E.faecalis ATCC-2921, Pseudo.aeruginosa ATCC-27853, Shigella flexneri, Klebsiella oxytoca, H. pylori, Salmonella typhi,) using conventional agar-dilution method. The MIC (minimum inhibitory concentration) values were determined by comparison to sulphamethoxazole as reference drug.
Sensitivity testing was performed on all compounds. Which showed, some synthetic compounds (5b, 5c, 5d, 5e, 5g, 5h, 5k) were not sensitive against all bacteria and rest compounds were sensitive against all bacteria?
Some compounds were not active against specific bacteria 5f (Morganella morganii), 5h and 5f (Vibrio cholerae), 5f (E.faecalis ATCC29212), 5f and (Pseudo.aeruginosa ATCC27853), 5f (Shigella flexinerii), 5f and 5l (Klebsiella oxytoca), 5f (Salmonella typhi). Compounds 5a, 5i and 5j were active against all bacteria. Its activity was found to be 2.5 to 400 times better than reference drug against all bacteria.
The MIC values of synthesized compounds tested against organism displayed a significant activity with wide degree of variation (Table 4). On gram positive bacteria, Compounds 5a-l was found to be 10 to 200 times more active than reference drug. On gram negative bacteria, Compounds 5a-l was found to be 10 to 400 times more active than reference drug.
Compound 5a exhibited significant activity against E.coli ATCC25922 with MIC value 50µg/mL. Among all synthesized compounds, compound 5a showed better activity against E.faecalis ATCC29212 with MIC value 12.5µg/mL and 400 times more activity than reference drug. Other then this bacteria, this compound exhibited moderate activity.
Compound 5f exhibited significant activity against H. pylori with MIC value 50µg/mL and 50 times more activity on both bacteria than reference drug. Other then these bacteria, this compound showed moderate activity. Compound 5j exhibited greater activity against Salmonella typhi, Staphylococcus aureus with MIC value 25µg/mL and showed significant activity against E.coli ATCC25922, Morganella morganii, Shigella flexinerii, Klebsiella oxytoca, with MIC value 50µg/mL. Other then these bacteria, this compound showed moderate activity.
Compound 5i showed significant activity against E.coli ATCC25922 and Shigella flexinerii with MIC value 50µg/mL. Other then these bacteria, this compound have shown moderate activity.
Thiol group containing compounds (C=S) (5f, 5i and 5l) were found to be more active than keto group containing compounds (C=O) (5a, 5i). Nitro group containing compounds (NO2) (5i, 5j) were found to be active against all bacteria than other group containing compounds (Br).
Antibacterial screening revealed that synthetic compounds exhibited moderate activity as compared to standard.
CONCLUSION:
Antimicrobial studies revealed that the most promising compounds are 5a (2H-[1,2,4]triazino[5,6-b]indol-3(5H)-one) with MIC value 12.5µg/ml against E. Faecalis ATCC29212, and 5j (8-nitro-2H-[1,2,4]triazino[5,6-b]indole-3(5H)-thione) with MIC value 25µg/ml against Salmonella typhi. Based on the above studies, the promising compounds can be evaluated for in vivo antimicrobial studies as a future perspective.
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Received on 24.02.2010 Modified on 15.03.2010
Accepted on 28.03.2010 © AJRC All right reserved
Asian J. Research Chem. 3(3): July- Sept. 2010; Page 646-652