Journal Information
Journal ID (publisher-id): chemical
Title: Journal of the Korean Chemical Society
Translated Title (ko): 대한화학회지
ISSN (print): 1017-2548
ISSN (electronic): 2234-8530
Publisher: Korean Chemical Society대한화학회
INTRODUCTION▼
Fused pyran derivatives represent promising class of fused heterocycles which constitute structural unit of many therapeutic agents1 and natural products.2−4 Pyrano[2, 3-c]pyrazole derivatives are such compounds gained major attention due to their potent biological activities such as antimicrobial,5 anti-inflammatory,6 analgesic,7 molluscicidal8 and anticancer9 activity. Also, they are well known as biodegradable agrochemicals10 and pharmaceutical ingredients.11
The synthesis of pyrano[2, 3-c]pyrazole has been first reported by Junek and co-workers by the reaction between 3-methyl-1-phenylpyrazolin-5-one and tetracyano ethylene in the presence of triethylamine in ethanol.12 Thereafter, several synthetic strategies have been introduced for the synthesis of this nucleus by employing various catalysts which includes acidic13,14 or basic15−18 catalyst, organocatalyst,19 biocatalyst,20 heterogenous catalyst21 and phase transfer catalyst.22 Also, catalyst free synthesis of pyrano[2, 3-c]pyrazoles is reported by Mandha and his coworkers.23 In spite of reporting many synthetic methods for the synthesis of dihydropyrano[2, 3-c]pyrazole, there is a need for clean and efficient protocols to develop wide variety of such nucleus.
Fused pyrimidines attracted the major interest of synthetic chemist due to their resemblance with the nitrogen bases. Hence, the annulated pyrimidines have emerged as an important pharmacophore for many life-threatening diseases such as cancer, tuberculosis etc.24 Pyrimidine fused pyrano[2, 3-c]pyrazoles have gained considerable interest among such derivatives. Many researchers have reported the synthesis of pyrimidine fused pyrano[2, 3-c]pyrazoles which found to exhibit significant biological activities such as antimicrobial, anti-inflammatory and anticancer agents.25,26
The use of greener and safe reaction media in place of volatile solvents have been considered as an essential part of organic synthesis. To address the problems caused by volatile organic solvents liquid polymers or low melting polymers are developed as an alternate medium for the organic synthesis.27,28 In this direction, PEG-400 is emerged as an important reaction medium due to its thermal stability, non-toxicity, recyclability, and water solubility.29-31
Herewith, we are reporting facile synthesis of dihydropyrano[2, 3-c]pyrazole under catalyst free condition using PEG-400 as a medium. Also, further derivatization was done on the dihydropyrano[2, 3-c]pyrazole using formic acid to obtain pyrimidine fused pyrano-pyrazoles and were screened for their activity as antimicrobial agents against M. smegmatis, E. coli and S. aureus.
EXPERIMENTAL▼
General
Chemicals are procured commercially and are used as such without any purification. Progress of the reaction is monitored using thin layer chromatographic technique (TLC) using pre-coated aluminum sheets alichrosep silica gel60/UV254 with I2 and UV light as visualizing agents. Melting point is determined using Thiele’s tube in open capillary tube and is uncorrected. IR spectra are recorded on Shimadzu Infrared spectrometer-8400s using KBr as background. 1H and 13C NMR analysis is done with Bruker avance NMR-400 MHz and 100 MHz respectively using TMS as an internal standard. Elemental analysis is performed by using Perkin Elmer 2400 CHN elemental analyzer.
General Procedure for the Synthesis of 1, 4-dihydropyrano[2, 3-c]pyrazole Derivatives 4(a-h)
Mixture of malononitrile (1 mmol) and appropriate aromatic aldehyde (1 mmol) in PEG-400 (5 mL) was stirred at room temperature for 30 min. To this mixture 3-methyl-1-phenyl-2-pyrazolin-5-one (1 mmol) was added and refluxed at 100 °C for 40-45 minute to get the titled product. The reactions were monitored by TLC. The reaction mixture was then cooled to room temperature and added to crushed ice. It was stirred for about 30 min. and solid thus formed was filtered and washed with 70% ethanol. The crude products were recrystallised from ethyl acetate. The spectral data of the synthesized molecules are given below.
6-Amino-3-methyl-1, 4-diphenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4a): yield, 302 mg (92%); m.p. 168−170 °C, (Lit. 171−173 °C32a); FTIR (γmax, cm−1): 3450 (N-H), 2195 (CN), 1589 (C=N), 1256 (C-O-C). 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.79 (s, 3H, CH3), 4.92 (s, 1H, 4-H), 7.65 (s, 2H, NH2), 7.26−7.81 (m, 10H, Ar-H). 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm): 162.4, 145.7, 144.9, 140.5, 138.1, 133.8, 133.1, 132.3, 130.3, 129.8, 128.7, 127.6, 120.3, 120.1, 97.9, 56.9, 12.5. Anal. Calcd. for C20H16N4O: C, 73.15; H, 4.91; N, 17.06. Found: C, 73.17; H, 4.97; N, 17.09.
6-Amino-4-(4-chlorophenyl)-3-methyl-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5 carbonitrile (4b): yield, 304 mg (94%); m.p. 170−172 °C, (Lit. 173−174 °C32a); FTIR (KBr): νmax = 3456 (N-H), 3195 (Ar-C-H), 2202 (CN), 1595 (C=N), 752 (C-Cl); 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.79 (s, 3H, CH3), 4.73 (s, 1H, 4-H), 7.28−7.38 (m, 3H, Ar-H), 7.42 (d, 2H, J=8.0 Hz, Ar-H), 7.48−7.52 (m, 2H, Ar-H), 7.51 (s, 2H, NH2), 7.77−7.79 (d, 2H, J=8.0 Hz, Ar-H) ppm. 13C NMR (100 MHz, DMSO-d6): δ 159.9, 145.6, 144.4, 143.1, 137.9, 132.0, 130.1, 129.1, 126.7, 120.5, 120.3, 98.6, 58.2, 36.5, 13.0 ppm. Anal. Calcd. for C20H15ClN4O: C, 66.21; H, 4.17; N, 15.44. Found: C, 66.24; H 4.21; N, 15.45.
6-Amino-4-(2, 4-dichlorophenyl)-3-methyl-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4c): yield, 368 mg (93%); m.p. 184−186 °C, (Lit. 185−187 °C32a); FTIR (γmax, cm−1): 3458 (N-H), 2198 (CN), 1593 (C=N), 756 (C-Cl). 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.77 (s, 3H, CH3), 5.15 (s, 1H, 4-H), 7.31−7.47 (m, 5H, ArH) 7.50−7.52 (d, J=8 Hz, ArH) 7.63 (s, 2H, NH2), 7.77−7.79 (d, 2H, J=8 Hz, ArH) ppm. 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm):160.4, 145.3, 144.7, 139.7, 137.6, 133.5, 133, 132.9, 129.8, 129.8, 128.5, 126.7, 120.4, 120, 97.7, 56.6, 12.8. Anal. Calcd. for C20H14Cl2N4O: C, 60.47; H, 3.55; N, 14.10. Found: C, 60.50; H, 3.57; N, 14.13.
6-Amino-3-methyl-4-(4-methylphenyl)-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4d): yield, 312 mg (91%); m.p. 176−178 °C, (Lit. 177−179 °C31b); FTIR (γmax, cm−1): 3451 (N-H), 2201(CN), 1591 (C=N). 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.80 (s, 3H, CH3), 2.28 (s, 3H, CH3), 5.21 (s, 1H, 4-H), 7.28−7.44 (m, 5H, Ar-H), 7.46−7.48 (d, 2H, J=8.4 Hz, Ar-H), 7.52 (s, 2H, NH2), 7.58−7.60 (d, 2H, J=8.0 Hz, Ar-H) ppm. 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm): 161.3, 145.7, 144.5, 140.4, 136.9, 133.9, 133.3, 132.7, 130.2, 129.7, 128.4, 126.3, 120.9, 120.4, 96.9, 57.2, 15.6, 13.2 ppm. Anal. Calcd. for C21H18N4O: C, 73.67; H, 5.30; N, 16.36. Found: C, 73.70; H, 5.35; N, 16.39.
6-Amino-4-(4-methoxyphenyl)-3-methyl-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4e): yield, 331 mg (95%); m.p. 172−174 °C, (Lit. 172−173 °C32b); FTIR (γmax, cm−1): 3395 (N-H), 2192 (CN), 1595 (C=N). 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.78 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 4.88 (s, 1H, 4-H), 6.82 (d, 2H, J=8.0 Hz, Ar-H), 6.96 (s, 2H, NH2), 7.04 (d, 2H, J=8.0 Hz, Ar-H), 7.20−7.24 (m, 1H, Ar-H), 7.40 (d, 2H, J=8.0 Hz, Ar-H), 7.58 (d, 2H, J=8.0 Hz, Ar-H). 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm): 163.2, 145.1, 144.4, 139.2, 137.9, 133.9, 133.2, 132.4, 130.2, 129.5, 128.9, 126.8, 120.9, 120.5, 98.2, 72.4, 56.9, 13.1. Anal. Calcd. for C21H18N4O2: C, 70.38; H, 5.06; N, 15.63. Found: C, 70.40; H, 5.10; N, 15.66.
6-Amino-4-(3, 4-dimethoxyphenyl)-5-cyano-3-methyl-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole (4f): yield, 337 mg (89%); m.p. 192−194 °C, (Lit. 193−195 °C32b); FTIR (γmax, cm−1): 3386, 3323, 2198, 1658, 1633, 1595, 1515, 1388, 1257, 1022 cm−1. 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.82 (s, 3H, CH3), 3.72 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 4.63 (s, 1H, 4-H), 7.18−7.38 (m, 5H, ArH), 7.47 (s, 2H, NH2), 7.49 (d, 2H, J=8.0 Hz, ArH), 7.78 (d, 2H, J=8.0 Hz, ArH). 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm):159.7, 149.0, 148.2, 145.8, 144.2, 138.0, 136.5, 129.8, 128.5, 120.5, 120.3, 112.2, 112.0, 99.1, 58.8, 56.3, 55.5, 34.8, 13.2. Anal. Calcd. for C20H15N4O: C, 66.21; H, 4.17; N, 15.44. Found: C, 66.25; H, 4.21; N, 15.50.
6-Amino-3-methyl-4-(3-nitrophenyl)-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4g): yield, 345 mg (91%); m.p. 190−192 °C, (Lit. 188−190 °C32b); FTIR (γmax, cm−1): 3429 (N-H), 2117 (CN), 1599 (C=N). 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 1.79 (s, 3H, CH3), 5.02 (s, 1H, 4-H), 7.25 (s, 2H, NH2), 7.30−7.36 (m, 1H, Ar-H), 7.44−7.46 (m, 2H, Ar-H), 7.58 (d, 2H, J=8.4 Hz, Ar-H), 7.78 (d, 2H, J=8.4 Hz, Ar-H), 8.15 (d, 2H, Ar-H). 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm):159.7, 144.8, 143.2, 139.4, 138.6, 133.4, 132.9, 132.3, 129.8, 129.1, 128.6, 126.5, 120.9, 120.4, 98.4, 57.5, 13.7. Anal. Calcd. for C20H15N5O3: C, 64.34; H, 4.05; N, 18.76. Found: C, 64.31; H, 4.03; N, 18.77.
6-Amino-3-methyl-4-(4-nitrophenyl)-1-phenyl-1, 4-dihydropyrano[2, 3-c]pyrazole-5-carbonitrile (4h): yield, 349 mg (90%); m.p. 194−196 °C, (Lit. 194−196 °C32c); FTIR (γmax, cm−1): 3430, 3340, 2116, 1665, 1596, 1354, 1124, 832, 754 cm−1. 1H NMR (400 MHz, DMSO-d6): δ 1.80 (s, 3H, CH3), 4.96 (s, 1H, 4-H), 6.98 (s, 2H, NH2), 7.32−7.36 (m, 1H, ArH), 7.48−7.52 (m, 2H, Ar-H), 7.58 (d, 2H, J=8.4 Hz, Ar-H), 7.80 (d, 2H, J=8.4 Hz, Ar-H), 8.24 (d, 2H, Ar-H) ppm. 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm): 162.4, 146.1, 145.2, 140.1, 138, 133.9, 133.2, 132.7, 129.9, 129.5, 128.2, 126.3, 120.8, 120.1, 98.2, 57.1, 13.4. Anal. Calcd. for C20H15N5O3: C, 64.34; H, 4.05; N, 18.76. Found: C, 64.40; H, 4.08; N, 18.80.
General Procedure for the Synthesis of Pyrano[2, 3-c] pyrazolopyrimidinone Derivatives
Pyrano[2, 3-c]pyrazole derivatives and formic acid (3 ml) were refluxed at 90 °C for 8 hours. On completion (confirmed by TLC) of the reaction, reaction mixture was cooled at room temperature. Further, the mixture was poured in the beaker and crushed ice was added and stirred it at room temperature, until separation of solid takes place. Then, the crude precipitate was filtered using buchner funnel. Crude solid was recrystallized using ethanol:DMF (1:10).
4-(4-Chlorophenyl)-3-methyl-1-phenyl-4,6-dihydropyrazolo[4',3':5,6]pyrano[2, 3-d] pyrimidin-5(1H)-one (5b): yield, 348 mg (89%); m.p. 196−198 °C; FTIR (KBr): νmax= 3464 (NH), 1681 and 1724 (C=O of amide), 1600 (C=N), 756 (C-Cl) cm−1. 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm):2.16(s,3H, CH3 proton), 4.23(s, 1H, CH proton), 7.17 (s, 1H, imine CH), 7.31−7.33 (d, 2H, J= 8 Hz), 7.39−7.46 (m, 5H, Ar-H), 7.68-7.70 (d, 2H, J=8 Hz, Ar-H), 10.90 (s, 1H, NH) ppm. 13C NMR (100 MHz) - spectrum in DMSO-d6 (δ, ppm): 162.1, 161.4, 153.7, 150.4, 140.4, 137.5, 130.6, 130.2, 129.3, 129.1, 126.6, 125.8, 125.4, 120.7, 119.1, 107.2, 35.7, 13.4. Elemental Anal. Clacd. for C21H15ClN4O2: C, 64.54; H, 3.87; N, 14.34; Found: C, 64.57; H, 3.92; N, 14.29.
4-(2,4-Dichlorophenyl)-3-methyl-1-phenyl-4,6-dihydropyrazolo[4',3':5,6]pyrano[2, 3-d]pyrimidin-5(1H)-one (5c): yield, 358 mg (84%); m.p. 208−210 °C; FTIR (KBr): νmax=3456 (NH), 1684 and 1729 (C=O of amide), 1596 (C=N), 762 (C-Cl) cm−1. 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 2.13 (s,3H, CH3 proton), 4.25 (s, 1H, CH proton), 7.19 (s, 1H, imine CH), 7.29−7.31 (d, 1H, J=8 Hz), 7.35−7.42 (m, 5H, Ar-H), 7.62−7.64 (d, 1H, J=8 Hz, Ar-H), 7.78 (s, 1H, Ar-H), 11.10 (s, 1H, NH) ppm. 13C NMR (100 MHz, DMSO-d6): 162.6, 161.3, 153.9, 150.7, 140.5, 137.6, 131.5, 130.9, 129.5, 129.3, 127.1, 126.2, 125.8, 121.3, 119.5, 107.6, 36.2, 13.6. Elemental Anal. Calcd. for C21H14Cl2N4O2: C, 59.31; H, 3.32; N, 13.17; Found: C, 59.27; H, 3.30; N, 13.20.
3-Methyl-4-(3-nitrophenyl)-1-phenyl-4,6-dihydropyrazolo[4',3':5,6]pyrano[2,3-d]pyrimidin-5(1H)-one (5g): yield, 325 mg (81%); m.p. 218−220 °C; FTIR (KBr): νmax =3452 (NH), 1679 and 1731 (C=O of amide), 1604 (C=N), 1589 and 1378 (N-O str.) cm−1. 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 2.09 (s, 3H, CH3 proton), 4.27 (s, 1H, CH proton), 7.17 (s, 1H, imine CH), 7.24−7.26 (d, 1H, J=8 Hz), 7.31−7.40 (m, 5H, Ar-H), 7.59−7.61 (d, 1H, J=8 Hz, Ar-H), 7.83 (s, 1H, Ar-H), 11.17 (s, 1H, NH) ppm. 13C NMR (100 MHz, DMSO-d6): 162.9, 161.6, 154.2, 150.9, 140.7, 137.8, 131.6, 131.4, 129.7, 129.5, 127.5, 126.4, 121.0, 119.9, 119.3, 107.3, 36.6, 13.9. Elemental Anal. Calcd. for C21H15N5O4: C, 62.84; H, 3.77; N, 17.45; Found: C, 62.81; H, 3.81; N, 17.49.
3-Methyl-4-(3-nitrophenyl)-1-phenyl-4,6-dihydropyrazolo[4',3':5,6]pyrano[2, 3-d]pyrimidin-5(1H)-one (5h): yield, 341 mg (85%); m.p. 164−166 °C; FTIR (KBr): νmax =3449 (NH), 1679 and 1731 (C=O of amide), 1604 (C=N), 1589 and 1378 (N-O str.) cm−1. 1H NMR (400 MHz) - spectrum in DMSO-d6 (δ, ppm): 2.09 (s, 3H, CH3 proton), 4.27 (s, 1H, CH proton), 7.17 (s, 1H, imine CH), 7.24−7.26 (d, 1H, J=8 Hz), 7.31−7.40 (m, 5H, Ar-H), 7.59−7.61 (d, 1H, J=8 Hz, Ar-H), 7.83 (s, 1H, Ar-H), 11.17 (s, 1H, NH) ppm. 13C NMR (100 MHz, DMSO-d6): 162.9, 161.6, 154.2, 150.9, 140.7, 137.8, 131.6, 131.4, 129.7, 129.5, 127.5, 126.4, 121.0, 119.9, 119.3, 107.3, 36.6, 13.9. Elemental Anal. Calcd. for C21H15N5O4: C, 62.84; H, 3.77; N, 17.45; Found: C, 62.81; H, 3.81; N, 17.49.
RESULTS AND DISCUSSION▼
Initially, focus was to develop an efficient protocol for the reaction using dimethyl amino pyridine (DMAP) catalyst in the presence of suitable solvent. The solvents were selected based on their feasibility, environmental safety, and ease of handling. Based on that ethanol, water, PEG-400 and glycerol were selected for the reaction (Table 1). Hence, the reaction conditions were optimized by taking model reaction between equimolar mixture of 4-chloro benzaldehyde, malononitrile and 3-methyl-1-phenyl-2-pyrazolin-5-one in the presence of DMAP (10 mol %) catalyst in different testing solvents under reflux condition. The reaction gave the product in good yield in all the cases and it was found to be highest in the presence of PEG-400 as a solvent (92% yield in 35 min.). Further investigation was done on the optimization of catalyst concentration in PEG-400 (5 mL) by varying the catalyst concentration from 0 to 25 mol % (Table 2). Surprisingly, the same reaction carried out in the absence of catalyst with PEG-400 as a solvent gave highest yield of 94%, in 45 min. and was better than that with 20 mol % of DMAP in 40 min.
Table1.
Selection of solvent for the synthesis of dihydropyrano[2, 3-c]pyrazole
| Entry | Solvent (5 mL) | Temperature | Time (min.) | Yield (%)* |
|---|---|---|---|---|
| 1 | Ethanol | 80 °C | 90 | 90 |
| 2 | Water | 100 °C | 180 | 72 |
| 3 | PEG-400 | 100 °C | 35 | 92 |
| 4 | Glycerol | 80 °C | 60 | 88 |
Reaction condition: Malononitrile (1 mmol), 4-Chlorobenzaldehyde (1 mmol) and 3-Methyl-1-phenyl-2-pyrazolin-5-one (1 mmol) with DMAP catalyst (10 mol %).
*Isolated yield.
Table2.
Optimization of catalyst concentration for the synthesis of dihydropyrano[2, 3-c]pyrazole
| Entry | DMAP Conc. (mol %) | Time (min.) | Yield (%)* |
|---|---|---|---|
| 1 | 0 | 45 | 94 |
| 2 | 10 | 45 | 92 |
| 3 | 20 | 50 | 93 |
| 4 | 25 | 35 | 91 |
Reaction condition: Malononitrile (1 mmol), 4-Chlorobenzaldehyde (1 mmol) and 3-Methyl-1-phenyl-2-pyrazolin-5-one (1 mmol) with PEG-400 as solvent (5 mL) at 100 °C.
*Isolated yield.
Encouraged with this fascinating result, further investigation was done on optimizing the temperature by varying it from 90 to 110 °C considering five different temperatures (Table 3) in the presence of PEG-400 (5 mL) as a solvent. The highest yield was observed, when reactions were carried out at 100 °C for 45 min. Considering the reaction time and yield both catalysed and uncatalysed reaction gave the similar results. Therefore, the reactions carried out without a catalyst with the temperature of 100 °C found to be the best condition for the reaction in the presence of PEG-400 as reaction medium. Finally, PEG-400 mediated multicomponent synthesis of dihydro pyrano[2, 3-c] pyrazole derivatives considered to be an efficient green method under catalyst free condition.
Table3.
Optimization of reaction temperature for the synthesis of dihydropyrano[2, 3-c]pyrazole
| Entry | Temperature | Time (min.) | Yield (%)* |
|---|---|---|---|
| 1 | 90 °C | 60 | 87 |
| 2 | 95 °C | 50 | 89 |
| 3 | 100 °C | 45 | 94 |
| 4 | 105 °C | 30 | 90 |
| 5 | 110 °C | 30 | 82 |
Reaction condition: Malononitrile (1 mmol), 4-Chlorobenzaldehyde (1 mmol) and 3-Methyl-1-phenyl-2-pyrazolin-5-one (1 mmol) with PEG-400 as solvent (5 mL).
*Isolated yield.
Formation of products are confirmed by FTIR, 1H-NMR and 13C-NMR spectroscopy. FTIR spectrum showed the characteristic peaks in the region of 2116 and 3460 cm−1 for −CN and −NH2 respectively. It is further confirmed by the presence of characteristic peaks around δ 5.17 and 7.5 ppm corresponds to C-H proton at 4th position of pyran ring and NH2 proton respectively. All the spectral data are in good agreement with the reported values.32
The formed pyrano[2, 3-c]pyrazole derivatives were further derivatised by reacting with formic acid to form pyrimidine fused pyrano[2, 3-c]pyrazoles. Product formation was confirmed by FTIR and proton NMR analysis. The FT-IR spectra of final products shown the characteristic peaks at 3464, 1681 and 1724, 1600, 756 cm−1 for NH str, C=O of amide, C=N and C-Cl respectively. It was further confirmed by NMR spectroscopy, which shows the characteristic peaks at δ 10.90, 7.17, and 4.23 ppm which corresponds to NH proton, imine CH proton and CH proton respectively.
Antimicrobial Activity
Compounds were screened for their activity as antimicrobial agents against non-virulent M. smegmatis, E. coli and S. aureus. In-vitro preliminary screening was done using agar diffusion method for all the molecules (5a-h, 10 mM concentration in DMSO). Inhibition zone was recorded in millimeter and readings were taken in triplicates (Table 4). Penicillin (for antituberrcular activity) and ciprofloxacin (for antibacterial activity) was used as standard drug for comparison.
Table4.
Inhibitory zone (diameter) in mm of synthesized compounds against tested microbial strains by agar diffusion method
| Compound | E. col | S. aureus | M. smegmatis |
|---|---|---|---|
| 5a | 25 | 23 | 9 |
| 5b | 30 | 28 | 12 |
| 5c | 20 | 22 | 8 |
| 5d | 19 | 26 | 10 |
| 5f | 31 | 27 | 9 |
| 5g | 21 | 23 | 8 |
| 5h | 28 | 32 | 13 |
| Ciprofloxacin | 41 | 44 | - |
| Penicillin | 24 |
The preliminary in vitro antimicrobial screening results shows that the novel pyrimidine annulated dihydropyrano[2, 3-c]pyrazole derivatives reported here have the less potential to act as antitubercular, agents against M. smegmatis but have shown moderate to good activity against both S. aureaus and E. Coli. Compound 5b and 5f have shown good activity against E. coli (gram negative) and moderate activity against S. aureus (gram positive). Compound 5h having −NO2 substitution was best among all the molecules against S. aureaus and M. smegmatis. Minimum inhibitory concentration was not determined as there no potent activity on comparing with standards.
The possible improvements in the antitubercular activity can be further achieved by slight modifications in the substituents on the basic dihydropyrano[2, 3-c]pyrazole nucleus. In view of the above findings, and to identify new pharmacophores that may be of value in designing new, selective, less toxic antimicrobial agents which might serve as new templates.
CONCLUSION▼
It has been found that, PEG-400 mediated protocol is an efficient and green method for the synthesis of the precursor, dihydropyrano[2, 3-c]pyrazole derivative owing to their non-toxic and eco-friendly nature. These molecules were further reacted with formic acid to get pyrimidine fused dihydropyrano[2, 3-c]pyrazole. The antitubercular activity studies of the pyrimidine annulated dihydropyrano[2, 3-c]pyrazole derivatives have shown moderate activity. Further structural modification might improve the activity against Mycobacterium smegmatis. Most of the molecules have shown better antibacterial activity against E. coli and S. aureus.