Step 1. General procedure for the synthesis of 6-(chloromethyl)-N²-(4-substituted aryl) - 1, 3, 5-triazine-2, 4-diamine 2(A-G):

Methanol (100 ml) was placed in a 250 ml two necked flasks fitted with a mechanical stirrer and reflux condenser. Taken 3 gm of Sodium in small pieces is dropped down the condenser into the stirred methanol. This resultant solution was cooled to room temperature and 0.05 Mol of aryl biguanide 1(A-G) was added. The mixture was stirred at room temperature for 20 min. The sodium chloride that precipitates was separated by filtration on a Buchner funnel and washed with 10 ml of methanol.

The filtrates, which contain aryl biguanide was placed in a 250 ml three-necked flask assembled with a mechanical stirrer, a drying tube containing calcium chloride and a dropping funnel. Later on, 0.1 Mol of ethyl chloroacetate was added at room temperature with stirring. The mixture was stirred at room temperature for 14 hrs, during which time 6-(chloromethyl)-N²-(4-substituted aryl) - 1, 3, 5-triazine-2, 4-diamine 2(A-G) precipitates as a white solid which was separated by filtration and air-dried, weighed the triazine. The methanol filtrate was added to 150 ml of cold water. The mixture was cooled in an ice bath with stirring for 2 hrs and filtered to remove an additional triazine. The triazine was recrystallized from 250 ml of dioxane, using 2 gm of decolourizing carbon and filtering hot, then dried and weighed.

Step 2. b.i. General Procedure for the synthesis of 1-(4-Substituted phenyl)-2-(1-phenylethylidene) hydrazine 5(a-d):

Dissolved 0.25 Mol of acetophenone in 100 ml of ethanol into 500 ml round bottom flask. To the above flask added 0.25 Mol of substituted phenyl hydrazine with 2 ml of glacial acetic acid and refluxed the reaction mixture for 5-6 hrs. The progress of the reaction was analyzed by the TLC method using silica gel G as the stationary phase and toluene: ethyl acetate (7:3) as a mobile phase. After the completion of the reaction, the reaction mixture was cooled to room temperature and solid crystals were obtained. The product was separated by filtration, washed with ethanol and dried to give substituted acetone phenylhydrazine 5(a-d).

Step 2. b.ii. General Procedure for the synthesis of 1-(4-Substituted phenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 6(a-d):

Taken 50 ml of dry DMF into 500 ml flat bottom flask and then 6 ml of phosphorus oxychloride (POCl₃) added dropwise under stirring at 0-5⁰C. After the complete addition of phosphorus oxychloride (POCl₃), the reaction mixture was stirred at this temperature for 15- 20 min. In the above mixture 0.05 Mol of freshly prepared 1-(4-Substituted phenyl)-2-(1-phenylethylidene) hydrazine 5(a-d) was added and heated on the water bath for 5-6 hrs. The succession of the reaction was monitored by the TLC technique using silica gel G as the stationary phase and toluene: ethyl acetate (7:3) as a mobile phase. After completion of the reaction, the reaction mixture was cool to room temperature and then poured on crushed ice. The solid crystals were separated and collected by filtration. The product was washed with cold water to remove acidic impurity. It was dried and recrystallised from DMF- Methanol to obtain pure substituted pyrazole aldehyde 6(a-d).

Step 3. General procedure for synthesis of 6-(chloromethyl)-N²-(1-(4-substituted phenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-aryl-1, 3, 5-triazine-2, 4-diamine 7(Aa-Gd):

To a solution of required 6-(chloromethyl)-N²-(4-substituted aryl) - 1, 3, 5-triazine-2, 4-diamine 2(A-G) 0.01 Mol in water, 1-2 ml of glacial acetic acid was added and maintained the pH between 5-6. To this solution, an equimolar quantity of 1-(4-Substituted phenyl)-3-phenyl-1H-pyrazole-4-carbaldehyde 6(a-d) in alcohol was added as shown in scheme 1. The reaction mixture was refluxed for 8-10 hrs. The resulting products 7(Aa-Gd) obtained after cooling, filtered the product and recrystallized from ethanol.

Physical and Spectral data of synthesized compounds:

6-(chloromethyl)-N²-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-phenyl-1, 3, 5-triazine-2, 4-diamine (7Aa):

C₂₆H₁₉Cl₂ N₇(500.38), mp (⁰C) 154-156; % Yield 72; IR ν-max (cm−1): 1652 (C=N), 3249 (N-H, stretching), 3392 (pyrazole N-N), 1660 (pyrazole C=N), 2869 (C-H, aliphatic ), 1472 (C=N, aliphatic ), 2917 (Ar, C-H), 1732 (Ar C=C), 765 (C-Cl), 840 (Ar-Cl); ¹H NMR (400 MHz,DMSO-d₆) (δ, ppm): 9.42 (Ar, C-NH), 7.9 (1-pyrazole, CH), 7.49-7.56 (4-Cl-benzene),7.41-7.79 (benzene), 6.81-7.63 (N-benzene), 9.13 (CH methine), 4.28 (CH₂ methylene); ₁₃C-NMR (20989.8 Hz, CDCl₃): δ 149.21, 134.12, 109 (pyrazole), 132.89, 138.12, 133.02, 137.63, 139.52, 127.53, 128.64, 129.32, 119.42, 117.12 (benzene), 162.34, 165.21, 177.08 (triazine), 44.6 (aliphatic), 161.38 (imine); Mass spectra: M⁺ (499.11), M+2 (501.10)

6-(chloromethyl)-N²-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-phenyl-1,3,5-triazine-2,4-diamine (7Ab):

C₂₆H₁₉ClF N₇(483.93), mp (⁰C) 173-175; % Yield 78; IR ν-max (cm−1): 1658 (C=N), 3254 (N-H, stretching), 3380 (pyrazole N-N), 1667 (pyrazole C=N), 2861 (C-H, aliphatic ), 1469 (C=N, aliphatic ), 2921 (Ar, C-H), 1738 (Ar C=C); 770 (C-Cl), 1236 (Ar-F); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.44 (Ar, C-NH), 7.8 (1-pyrazole, CH), 7.24-7.60 (4-F-benzene),7.43-7.76 (benzene), 6.83-7.66 (N-benzene), 9.11 (CH methine), 4.25 (CH₂ methylene)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-phenyl-1,3,5-triazine-2,4-diamine (7Ac):

C₂₆H₁₉ClBr N₇(544.83), mp (⁰C) 180-182; % Yield 83; IR ν-max (cm−1): 1648 (C=N), 3260 (N-H, stretching), 3375 (pyrazole N-N), 1662 (pyrazole C=N), 2859 (C-H, aliphatic ), 1466 (C=N, aliphatic ), 2914 (Ar, C-H), 1733 (Ar C=C); 774 (C-Cl), 756 (Ar-Br);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.41 (Ar, C-NH), 7.5 (1-pyrazole, CH), 7.51-7.60 (4-Br-benzene),7.44-7.77 (benzene), 6.82-7.64 (N-benzene), 9.11 (CH methine), 4.27 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-phenyl-1,3,5-triazine-2,4-diamine(7Ad):

C₂₆H₁₉ClN₈O₂ (510.93), mp (⁰C) 193-195; % Yield 80; IR ν-max (cm−1): 1650 (C=N), 3265 (N-H, stretching), 3354 (pyrazole N-N), 1667 (pyrazole C=N), 2862 (C-H, aliphatic), 1470 (C=N, aliphatic ), 2924 (Ar, C-H), 1739 (Ar C=C); 765 (C-Cl), 1326 (Ar-NO₂);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.41 (Ar, C-NH), 7.4 (1-pyrazole, CH), 8.11-8.43 (4-NO₂-benzene),7.43-7.75 (benzene), 6.84-7.67 (N-benzene), 9.12 (CH methine), 4.29 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(pyridin-4-yl)-1,3,5-triazine-2,4-diamine (7Ba):

C₂₅H₁₈Cl₂ N₈(501.37), mp (⁰C) 177-179; % Yield 75; IR ν-max (cm−1): 1662 (C=N), 3243 (N-H, stretching), 3381 (pyrazole N-N), 1665 (pyrazole C=N), 2849 (C-H, aliphatic ), 1478 (C=N, aliphatic ), 2930 (Ar, C-H), 1728 (Ar C=C); 768 (C-Cl), 846 (Ar-Cl), 1266 (C=N, Pyridine);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.9 (1-pyrazole, CH), 7.47-7.54 (4-Cl-benzene),7.43-7.80 (benzene), 6.99-8.46 (Pyridine), 9.14 (CH methine), 4.24 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(pyridin-4-yl)-1,3,5-triazine-2,4-diamine (7Bb):

C₂₅H₁₈ClF N₈(484.92), mp (⁰C) 185-187; % Yield 77; IR ν-max (cm−1): 1664 (C=N), 3257 (N-H, stretching), 3379 (pyrazole N-N), 1671 (pyrazole C=N), 2864 (C-H, aliphatic ), 1473 (C=N, aliphatic ), 2923 (Ar, C-H), 1742 (Ar C=C); 774 (C-Cl), 1233 (Ar-F), 1263 (C=N, Pyridine);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.45 (Ar, C-NH), 7.81 (1-pyrazole, CH), 7.27-7.62 (4-F-benzene), 7.42-7.78 (benzene), 6.97-8.48 (Pyridine), 9.14 (CH methine), 4.25 (CH₂ methylene)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-(pyridin-4-yl)-1,3,5-triazine-2,4-diamine (7Bc):

C₂₅H₁₈ClBr N₈ (545.82), mp (⁰C) 190-192; % Yield 81; IR ν-max (cm−1): 1636 (C=N), 3248 (N-H, stretching), 3358 (pyrazole N-N), 1652 (pyrazole C=N), 2864 (C-H, aliphatic ), 1456 (C=N, aliphatic), 2918 (Ar, C-H), 1738 (Ar C=C); 778 (C-Cl), 759 (Ar-Br), 1265 (C=N, Pyridine); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.46 (Ar, C-NH), 7.83 (1-pyrazole, CH), 7.54-7.62 (4-Br-benzene), 7.43-7.82 (benzene), 6.94-8.45 (Pyridine), 9.16 (CH methine), 4.3 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(pyridin-4-yl)-1,3,5-triazine-2,4-diamine (7Bd):

C₂₅H₁₈ClN₉O₂ (511.92), mp (⁰C) 202-204; % Yield 69; IR ν-max (cm−1): 1650 (C=N), 3268 (N-H, stretching), 3363 (pyrazole N-N), 1671 (pyrazole C=N), 2868 (C-H, aliphatic ), 1473 (C=N, aliphatic ), 2930 (Ar, C-H), 1742 (Ar C=C); 769 (C-Cl), 1329 (Ar-NO₂), 1255 (C=N, Pyridine); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.42 (Ar, C-NH), 7.84 (1-pyrazole, CH), 8.13-8.46 (4-NO₂-benzene), 7.44-7.74 (benzene), 6.91-8.47 (Pyridine), 9.11 (CH methine), 4.24 (CH₂ methylene);₁₃C-NMR (20989.8 Hz, CDCl₃): δ 151.23, 133.34, 105.86 (pyrazole), 145.9, 134.21, 133.02, 126.36, 128.26, 127.73, 118.53 (benzene), 154.9, 108.7 (4-pyridine), 163.42, 166.26, 178.14 (triazine), 43.4 (aliphatic), 160.89 (imine); Mass spectra: M⁺ (511.23), M+2 (513.12)

6-(chloromethyl)-N²-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (7Ca):

C₂₆H₁₈Cl₂N₈O₂(545.38), mp (⁰C) 187-189; % Yield 67; IR ν-max (cm−1): 1676 (C=N), 3252 (N-H, stretching), 3360 (pyrazole N-N), 1663 (pyrazole C=N), 2870 (C-H, aliphatic ), 1478 (C=N, aliphatic ), 2940 (Ar, C-H), 1736 (Ar C=C); 772 (C-Cl), 856 (Ar-Cl), 1332 (Ar-NO₂); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.79 (1-pyrazole, CH), 7.45-7.58 (4-Cl-benzene), 7.44-7.81 (benzene), 6.86-8.03 (4-NO₂-benzene), 9.15 (CH methine), 4.32 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (7Cb):

C₂₆H₁₈ClFN₈O₂(528.92), mp (⁰C) 205-207; % Yield 72; IR ν-max (cm−1): 1652 (C=N), 3263 (N-H, stretching), 3368 (pyrazole N-N), 1665 (pyrazole C=N), 2869 (C-H, aliphatic), 1471 (C=N, aliphatic), 2925 (Ar, C-H), 1740 (Ar C=C); 769 (C-Cl), 1239 (Ar-F), 1338 (Ar-NO₂);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.43 (Ar, C-NH), 7.84 (1-pyrazole, CH), 7.26-7.68 (4-F-benzene), 7.43-7.77 (benzene), 6.89-8.12 (4-NO₂-benzene), 9.12 (CH methine), 4.23 (CH₂ methylene)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (7Cc):

C₂₆H₁₈ClBrN₈O₂ (589.83), mp (⁰C) 191-193; % Yield 80; IR ν-max (cm−1): 1640 (C=N), 3242 (N-H, stretching), 3360 (pyrazole N-N), 1659 (pyrazole C=N), 2876 (C-H, aliphatic ), 1462 (C=N, aliphatic ), 2928 (Ar, C-H), 1735 (Ar C=C); 781 (C-Cl), 760 (Ar-Br), 1345 (Ar-NO₂); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.84 (1-pyrazole, CH), 7.53-7.68 (4-Br-benzene), 7.45-7.76 (benzene), 6.88-8.07 (4-NO₂-benzene), 9.16 (CH methine), 4.35 (CH₂ methylene)

6-(chloromethyl)-N²-(4-nitrophenyl)-N⁴-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-1,3,5-triazine-2,4-diamine (7Cd):

C₂₆H₁₈ClN₉O₄(555.93), mp (⁰C) 212-214; % Yield 73; IR ν-max (cm−1): 1668 (C=N), 3254 (N-H, stretching), 3366 (pyrazole N-N), 1680 (pyrazole C=N), 2872 (C-H, aliphatic ), 1465 (C=N, aliphatic), 2926 (Ar, C-H), 1746(Ar C=C); 774 (C-Cl), 1336 (Ar-NO₂), 1342 (Ar-NO₂);¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.46 (Ar, C-NH), 7.88 (1-pyrazole, CH), 8.15-8.46 (4-NO₂-benzene), 7.43-7.84 (benzene), 6.86-8.21 (4-NO₂-benzene), 9.21 (CH methine), 4.25 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(4-methoxyphenyl)-1, 3, 5-triazine-2,4-diamine (7Da):

C₂₇H₂₁Cl₂N₇O(530.41), mp (⁰C) 167-169; % Yield 76; 1656 (C=N), 3252 (N-H, stretching), 3380 (pyrazole N-N), 1667 (pyrazole C=N), 2878(C-H, aliphatic), 1473 (C=N, aliphatic ), 2930 (Ar, C-H), 1736 (Ar C=C), 772 (C-Cl), 838 (Ar-Cl), 1260 (C-O), 2780 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.46 (Ar, C-NH), 7.81 (1-pyrazole, CH), 7.51-7.58 (4-Cl-benzene),7.48-7.81 (benzene), 7.05-7.56 (N-benzene), 3.84 (CH, methoxy), 9.12 (CH methine), 4.22 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (7Db):

C₂₇H₂₁ClFN₇O(513.95), mp (⁰C) 186-188; % Yield 82; 1660 (C=N), 3263 (N-H, stretching), 3382 (pyrazole N-N), 1671 (pyrazole C=N), 2874(C-H, aliphatic), 1478 (C=N, aliphatic), 2934 (Ar, C-H), 1728 (Ar C=C), 765 (C-Cl), 1238 (Ar-F), 1272 (C-O), 2787 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.41 (Ar, C-NH), 7.79 (1-pyrazole, CH), 7.26-7.62 (4-F-benzene), 7.44-7.73 (benzene), 7.12-7.58 (N-benzene), 3.82 (CH, methoxy), 9.17 (CH methine), 4.3 (CH₂ methylene); ₁₃C-NMR (20989.8 Hz, CDCl₃): δ 150.32, 133.46, 107.8 (pyrazole), 133.71, 137.82, 133.59, 138.68, 140.12, 126.63, 128.04, 129.48, 118.72, 116.29 (benzene), 163.45, 164.93, 178.2 (triazine), 56.36 (CH methoxy), 43.8 (aliphatic), 162.49 (imine); Mass spectra: M⁺ (513.13), M+2 (515.15)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-(4-methoxyphenyl)-1, 3, 5-triazine-2, 4-diamine (7Dc):

C₂₇H₂₁ClBrN₇O(574.86), mp (⁰C) 195-197; % Yield 68; 1668 (C=N), 3246 (N-H, stretching), 3373 (pyrazole N-N), 1686 (pyrazole C=N), 2855 (C-H, aliphatic), 1473 (C=N, aliphatic), 2940 (Ar, C-H), 1750 (Ar C=C), 779 (C-Cl), 757 (Ar-Br), 1262 (C-O), 2774 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.51 (Ar, C-NH), 7.82 (1-pyrazole, CH), 7.52-7.62 (4-Br-benzene), 7.45-7.71 (benzene), 7.07-7.56 (N-benzene), 3.85 (CH, methoxy), 9.2 (CH methine), 4.27 (CH₂ methylene)

6-(chloromethyl)-N²-(4-methoxyphenyl)-N⁴-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-1, 3, 5-triazine-2, 4-diamine (7Dd):

C₂₇H₂₁ClN₈O₃ (540.96), mp (⁰C) 209-211; % Yield 71; 1649 (C=N), 3252 (N-H, stretching), 3347 (pyrazole N-N), 1680 (pyrazole C=N), 2857 (C-H, aliphatic ), 1476 (C=N, aliphatic ), 2945 (Ar, C-H), 1748 (Ar C=C), 784 (C-Cl), 1342 (Ar-NO₂), 1250 (C-O), 2790 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.41 (Ar, C-NH), 7.83 (1-pyrazole, CH), 8.15-8.46 (4-NO₂-benzene), 7.44-7.8 (benzene), 7.23-7.45 (N-benzene), 3.82 (CH, methoxy), 9.14 (CH methine), 4.26 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-p-tolyl-1, 3, 5-triazine-2,4-diamine (7Ea):

C₂₇H₂₁Cl₂N₇(514.41), mp (⁰C) 172-174; % Yield 74; 1670 (C=N), 3243 (N-H, stretching), 3374 (pyrazole N-N), 1652 (pyrazole C=N), 2865(C-H, aliphatic ), 1469 (C=N, aliphatic ), 2938 (Ar, C-H), 1745 (Ar C=C), 778 (C-Cl), 850 (Ar-Cl), 2810 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.8 (1-pyrazole, CH), 7.48-7.54 (4Cl-benzene),7.42-7.8 (benzene), 7.01-7.25 (N-benzene), 2.36 (CH, methyl), 9.18 (CH methine), 4.23 (CH₂ methylene)

6-(chloromethyl)-N2-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N4-p-tolyl-1, 3, 5-triazine-2, 4-diamine (7Eb):

C₂₇H₂₁ClFN₇(497.95), mp (⁰C) 188-190; % Yield 78; 1662 (C=N), 3268 (N-H, stretching), 3376 (pyrazole N-N), 1675 (pyrazole C=N), 2825(C-H, aliphatic ), 1449 (C=N, aliphatic ), 2946 (Ar, C-H), 1735 (Ar C=C), 771 (C-Cl), 1230 (Ar-F), 2807(C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.42 (Ar, C-NH), 7.81 (1-pyrazole, CH), 7.25-7.61 (4-F-benzene), 7.44-7.76 (benzene), 7.14-7.35 (N-benzene), 2.36 (CH, methyl), 9.16 (CH methine), 4.34 (CH₂ methylene)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-p-tolyl-1, 3, 5-triazine-2,4-diamine (7Ec):

C₂₇H₂₁ClBrN₇(558.86), mp (⁰C) 201-203; % Yield 85; 1677 (C=N), 3243 (N-H, stretching), 3365 (pyrazole N-N), 1684 (pyrazole C=N), 2858 (C-H, aliphatic), 1437 (C=N, aliphatic), 2944 (Ar, C-H), 1756 (Ar C=C), 779 (C-Cl), 762 (Ar-Br), 2800 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.84 (1-pyrazole, CH), 7.53-7.63 (4-Br-benzene), 7.42-7.73 (benzene), 7.06-7.25 (N-benzene), 2.35 (CH, methyl), 4.31 (CH₂ methylene)

6-(chloromethyl)-N²-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-N⁴-p-tolyl-1, 3, 5-triazine-2,4-diamine (7Ed):

C₂₇H₂₁ClN₈O₂(524.96), mp (⁰C) 214-216; % Yield 83; 1644 (C=N), 3257 (N-H, stretching), 3343 (pyrazole N-N), 1672 (pyrazole C=N), 2847 (C-H, aliphatic ), 1472 (C=N, aliphatic ), 2960 (Ar, C-H), 1752 (Ar C=C), 780 (C-Cl), 1348 (Ar-NO₂), 2794 (C-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.48 (Ar, C-NH), 7.87 (1-pyrazole, CH), 8.18-8.47 (4-NO₂-benzene), 7.46-7.78 (benzene), 7.07-7.28 (N-benzene), 2.33 ( CH, methyl), 9.13 (CH methine), 4.24 (CH₂ methylene)

6-(chloromethyl)-N²-(4-chlorophenyl)-N⁴-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-1, 3, 5-triazine-2, 4-diamine (7Fa):

C₂₆H₁₈Cl₃N₇(534.83), mp (⁰C) 177-179; % Yield 68; 1678 (C=N), 3250 (N-H, stretching), 3364 (pyrazole N-N), 1656 (pyrazole C=N), 2860 (C-H, aliphatic ), 1470 (C=N, aliphatic ), 2940 (Ar, C-H), 1759 (Ar C=C), 776 (C-Cl), 853 (Ar-Cl), 857 (Ar-Cl); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.52 (Ar, C-NH), 7.78 (1-pyrazole, CH), 7.47-7.59 (4-Cl-benzene),7.49-7.86 (benzene), 7.28-7.69 (4-Cl-benzene), 9.17 (CH methine), 4.37 (CH₂ methylene)

6-(chloromethyl)-N²-(4-chlorophenyl)-N⁴-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-1, 3, 5-triazine-2, 4-diamine (7Fb):

C₂₆H₁₈Cl₂FN₇(518.37), mp (⁰C) 184-186; % Yield 78; 1672 (C=N), 3266 (N-H, stretching), 3371 (pyrazole N-N), 1667 (pyrazole C=N), 2834 (C-H, aliphatic ), 1454 (C=N, aliphatic ), 2948 (Ar, C-H), 1740 (Ar C=C), 779 (C-Cl), 1236 (Ar-F), 838 (Ar-Cl) ; ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.51 (Ar, C-NH), 7.84 (1-pyrazole, CH), 7.34-7.67 (4-F-benzene), 7.43-7.8 (benzene), 7.28-7.61 (4-Cl-benzene), 9.15 (CH methine), 4.29 (CH₂ methylene)

N²-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-6-(chloromethyl)-N⁴-(4-chlorophenyl)-1, 3, 5-triazine-2, 4-diamine (7Fc):

C₂₆H₁₈Cl₂BrN₇(579.28), mp (⁰C) 191-193; % Yield 81; 1648 (C=N), 3246 (N-H, stretching), 3361 (pyrazole N-N), 1680 (pyrazole C=N), 2868 (C-H, aliphatic ), 1439 (C=N, aliphatic ), 2946 (Ar, C-H), 1764 (Ar C=C), 782 (C-Cl), 766 (Ar-Br), 845 (Ar-Cl); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.46 (Ar, C-NH), 7.82 (1-pyrazole, CH), 7.53-7.64 (4-Br-benzene), 7.45-7.73 (benzene), 7.28-7.65 (4-Cl-benzene), 9.18 (CH methine), 4.38 (CH₂ methylene)

6-(chloromethyl)-N²-(4-chlorophenyl)-N⁴-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methylene)-1, 3, 5-triazine-2, 4-diamine (7Fd):

C₂₆H₁₈Cl₂N₈O₂ (545.38), mp (⁰C) 206-208; % Yield 87; 1653 (C=N), 3256 (N-H, stretching), 3348 (pyrazole N-N), 1676 (pyrazole C=N), 2837 (C-H, aliphatic ), 1478 (C=N, aliphatic ), 2969 (Ar, C-H), 1758 (Ar C=C), 785 (C-Cl), 1356 (Ar-NO₂), 848 (Ar-Cl); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.82 (1-pyrazole, CH), 8.15-8.47 (4-NO₂-benzene), 7.42-7.89 (benzene), 7.32-7.65 (4-Cl-benzene), 9.15 (CH methine), 4.34 (CH₂ methylene)

4-(4-(chloromethyl)-6-((1-(4-chlorophenyl)-3-phenyl-1H-pyrazol-4-yl) methyleneamino)-1, 3, 5-triazin-2-ylamino) benzenesulfonamide (7Ga):

C₂₆H₂₀Cl₂N₈O₂S(579.46), mp (⁰C) 180-182; % Yield 67; 1672 (C=N), 3254 (N-H, stretching), 3366 (pyrazole N-N), 1661 (pyrazole C=N), 2865 (C-H, aliphatic ), 1473 (C=N, aliphatic ), 2942 (Ar, C-H), 1762 (Ar C=C), 777 (C-Cl), 854 (Ar-Cl), 1337 (S=O), 923 (S-N), 3337(N-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.42 (Ar, C-NH), 7.81 (1-pyrazole, CH), 7.48-7.57 (4-Cl-benzene),7.43-7.84 (benzene), 7.27-7.68 (N-benzene), 7.39 (Sulphonamide, NH), 9.2 (CH methine), 4.36 (CH₂ methylene)

4-(4-(chloromethyl)-6-((1-(4-fluorophenyl)-3-phenyl-1H-pyrazol-4-yl) methyleneamino)-1, 3, 5-triazin-2-ylamino) benzenesulfonamide (7GB):

C₂₆H₂₀ClFN₈O₂S(563.01), mp (⁰C) 189-191; % Yield 76; 1661 (C=N), 3237 (N-H, stretching), 3346 (pyrazole N-N), 1655 (pyrazole C=N), 2843 (C-H, aliphatic ), 1427 (C=N, aliphatic ), 2935 (Ar, C-H), 1725 (Ar C=C), 773 (C-Cl), 1233 (Ar-F), 1325 (S=O), 928 (S-N), 3342 (N-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.48 (Ar, C-NH), 7.88 (1-pyrazole, CH), 7.34-7.67 (4-F-benzene), 7.46-7.81 (benzene), 7.36-7.67 (N-benzene), 7.41 (Sulphonamide, NH), 9.23 (CH methine), 4.32 (CH₂ methylene)

4-(4-((1-(4-bromophenyl)-3-phenyl-1H-pyrazol-4-yl) methyleneamino)-6-(chloromethyl)-1, 3, 5-triazin-2-ylamino) benzenesulfonamide (7Gc):

C₂₆H₂₀ClBrN₈O₂S(623.91), mp (⁰C) 206-208; % Yield 80; 1629 (C=N), 3247 (N-H, stretching), 3352 (pyrazole N-N), 1661 (pyrazole C=N), 2857 (C-H, aliphatic ), 1430 (C=N, aliphatic ), 2936 (Ar, C-H), 1739 (Ar C=C), 757 (C-Cl), 770 (Ar-Br), 1333 (S=O), 927 (S-N), 3328 (N-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.47 (Ar, C-NH), 7.86 (1-pyrazole, CH), 7.54-7.67 (4-Br-benzene), 7.47-7.85 (benzene), 7.27-7.68 (N-benzene), 7.42 (Sulphonamide, NH), 9.22 (CH methine), 4.27 (CH₂ methylene)

4-(4-(chloromethyl)-6-((1-(4-nitrophenyl)-3-phenyl-1H-pyrazol-4-yl) methyleneamino)-1, 3, 5-triazin-2-ylamino) benzenesulfonamide (7Gd):

C₂₆H₂₀ClN₉O₄S(590.01), mp (⁰C) 218-220; % Yield 84; 1656 (C=N), 3238 (N-H, stretching), 3347 (pyrazole N-N), 1667 (pyrazole C=N), 2873 (C-H, aliphatic ), 1481 (C=N, aliphatic ), 2958 (Ar, C-H), 1746 (Ar C=C), 785 (C-Cl), 1323 (Ar-NO₂), 1346 (S=O), 932 (S-N), 3328 (N-H); ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 9.42 (Ar, C-NH), 7.81 (1-pyrazole, CH), 8.18-8.47 (4-NO₂-benzene), 7.46-7.82 (benzene), 7.36-7.66 (N-benzene), 7.37 (Sulphonamide, NH), 9.24 (CH methine), 4.29 (CH₂ methylene)

RESULT AND DISCUSSION:

Antimicrobial Activity:

All the synthesized compounds 7(Aa-Gd) were screened for their in vitro antimicrobial activity using Gram-positive, Gram-negative bacteria and fungi. Four different cultures, two for Gram-positive (Staphylococcus aureus, Streptococcus aureus) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) were tested with the synthesized compounds. Three strains were used for antifungal activity i.e. Candida albicans, Aspergillus fumigate and Aspergillus flavus.

A. Antibacterial Activity:

The antibacterial activity was performed by the cup and plate method (diffusion technique). The fresh culture of bacteria was obtained by inoculating bacteria in nutrient broth media and incubated at 37 ±2⁰C for 18-24 hrs. This fresh culture was mixed with nutrient agar media and poured into sterile Petri plates by pour plate method, by following aseptic technique. After solidification of the media, six bores were made at equal distance by using a sterile steel cork borer (8mm diameter). Into these cups, 100 µg/ml concentration solutions of standard drug and synthesized compounds were introduced. Dimethylformamide was used as a control. After the introduction of standard drugs and synthesized compounds, the plates were placed for proper diffusion of the drug into media for about 2 hrs. After 2 hrs, the plates were incubated in a BOD incubator and maintained at 37 ± 0.5⁰C for 18-24 hrs. After the incubation period, the plates were observed for the zone of inhibition by using a Hi-antibiotic zone reader. Results were evaluated by comparing the zone of inhibition shown by the synthesized compounds with the standard drug. The results were the average value of the zone of inhibition measured in millimetre of three sets. The results were shown in Table 1. The standard drug was dissolved in distilled water and the synthesized compounds were dissolved in a minimum quantity of DMF and diluted with water to get desired concentrations. The benzyl penicillin was used as a standard drug against Staphylococcus aureus and Streptococcus aureus and Streptomycin was used against Escherichia coli and Pseudomonas aeruginosa.

Table 1: In vitro antibacterial activity of compounds 7(Aa-Gd)

Compounds	Zone of inhibition in mm (100 µg/ml)
Compounds	*Staphyl. aureus*	*Strepto. aureus*	*E. coli*	*P. aerug.*
6Aa	12	11	13	10
6 Ab	11	10	12	11
6Ac	14	12	13	12
6Ad	13	14	15	12
6Ba	13	12	10	12
6Bb	12	11	10	13
6Bc	14	13	11	14
6Bd	15	13	14	12
6Ca	13	12	12	14
6Cb	14	11	12	13
6Cc	13	10	13	12
6Cd	12	11	13	13
6Da	15	14	14	13
6Db	14	14	13	11
6Dc	15	13	12	11
6Dd	16	15	13	12
6Ea	14	12	11	12
6Eb	13	12	12	10
6Ec	14	13	12	12
6Ed	15	13	13	11
6Fa	13	12	13	11
6Fb	14	12	11	12
6Fc	14	13	12	12
6Fd	14	12	11	11
6Ga	15	13	14	12
6Gb	15	12	13	11
6Gc	14	13	13	13
6Gd	14	14	12	12
Negative Control (DMF)	5	NA	NA	NA
Benzyl Penicillin (Positive Control)	20	20	NA	NA
Streptomycin (Positive Control)	NA	NA	23	21

Staphyl. Aureus = Staphylococcus aureus, Strepto. Aureus = Streptococcus aureus, E. coli = Escherichia coli, P. aerog = Pseudomonas aeroginosa

B. Antifungal activity:

The synthesized compounds were screened against two selected fungal strain Candida albicans and Aspergillus fumigates by using the diffusion method. The 48 hrs old fungal culture inoculated into nutrient broth by following aseptic techniques and incubated for 48 hrs at 37 ±2⁰C in a BOD incubator. This culture was mixed with well sterilized and cooled potato- dextrose agar media and poured into Petri plates by pour plate method. After solidification, six bores were made equal distance by using a sterile steel cork borer (8mm in diameter). Into these cups, 100 µg/ml concentration of standard drug and synthesized compounds along with control N, N'- dimethylformamide were introduced. These plates were placed for 2 hrs for proper diffusion. After 2 hrs, the Petri plates were transferred to BOD incubator and maintained at 37 ±2⁰C for 24-36 hrs. After the incubation period, the plates were taken to measure the zone of inhibition by using Hi- antibiotic zone reader. Results were evaluated by comparing the zone of inhibition measured in millimetre of three sets. The results were tabulated in Table 2. The standard drug was dissolved in distilled water and the synthesized compounds were dissolved in a minimum quantity of DMF and diluted to get the required concentration. Ketoconazole was used as a standard drug.

Table 2: In vitro antifungal activity of synthesized compounds 7(Aa-Gd)

Compounds	Zone of inhibition in millimetre (100 µg/ml)
Compounds	*Candida albicans*	*Aspergillus fumigates*	Aspergillus flavus
6Aa	15	13	14
6Ab	14	12	13
6Ac	15	12	13
6Ad	13	11	12
6Ba	14	13	13
6Bb	12	10	12
6Bc	15	12	14
6Bd	16	12	14
6Ca	15	11	13
6Cb	16	13	13
6Cc	16	14	14
6Cd	17	13	12
6Da	14	12	13
6Db	16	13	12
6Dc	17	14	15
6Dd	18	14	15
6Ea	15	12	13
6Eb	15	11	12
6Ec	16	12	12
6Ed	17	13	13
6Fa	16	14	13
6Fb	17	13	14
6Fc	17	12	14
6Fd	18	13	15
6Ga	17	12	15
6Gb	18	13	15
6Gc	18	14	14
6Gd	19	14	16
Negative Control (DMF)	NA	NA	NA
Ketoconazole (Positive Control)	23	20	22

CONCLUSION:

In conclusion, we accomplished the synthesis of the library of nitrogen-containing heterocyclics i.e. in conjugation with 1, 3, 5-triazine and pyrazole have been synthesized and evaluated against Gram-positive and Gram-negative bacterial strains as well as fungal strains. All the synthesized compounds showed significant antibacterial activity of the compound Da, Dc, Dd, Ed, Ga and Gb exhibited the highest and the rest of the compounds displayed moderate antibacterial activity against mentioned strains of bacteria. Further, all the compounds have been evaluated against three fungal strains i.e. Candida albicans, Aspergillus fumigates and Aspergillus flavus. Compounds Cd, Dc, Ed, Fb, Fc, Fd, Ga, Gb, Gc and Gd displayed the highest antifungal activity as compared to Ketoconazole. Hence, this study provides a road map to the design and synthesis of N-containing heterocycles as novel antimicrobial agents.

ACKNOWLEDGEMENT:

The authors are very grateful to the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Bhupal Nobles’ University, Udaipur, Rajasthan, India for proper support and guidance.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.11.2020 Modified on 19.01.2021

Research J. Pharm. and Tech 2022; 15(1):236-244.

DOI: 10.52711/0974-360X.2022.00039