alexa Synthesis and Antimicrobial Activity of Some New 3,5-Disubstituted Pyrazoles and Isoxazoles

ISSN: 2161-0444

Medicinal Chemistry

  • Research Article   
  • Med chem (Los Angeles) 2017, Vol 7(12): 371
  • DOI: 10.4172/2161-0444.1000483

Synthesis and Antimicrobial Activity of Some New 3,5-Disubstituted Pyrazoles and Isoxazoles

Simha Pulla Reddy, Gudi Yamini, Donthamsetty V Sowmya, Venkatapuram Padmavathi and Adivireddy Padmaja*
Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
*Corresponding Author: Adivireddy Padmaja, Department of Chemistry, Sri Venkateswara University, Tirupati-517 502, Andhra Pradesh, India, Tel: +919440350194, Email: [email protected]

Received Date: Dec 01, 2017 / Accepted Date: Dec 04, 2017 / Published Date: Dec 11, 2017

Abstract

A new class of 3,5-disubstituted pyrazoles and isoxazoles were prepared from the Michael acceptors 1-furanyl / thiophenyl / pyridinyl-3-indole-prop-2-en-1-ones under ultrasonication and evaluated for antimicrobial activity. Amongst all the tested compounds fluoro substituted thiophene linked compounds 12b and 18b displayed promising antibacterial activity particularly against Bacillus subtilis and antifungal activity against Aspergillus niger. Furthermore, compounds with more number of electron withdrawing groups showed higher antimicrobial activity. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.

Keywords: Pyrazoles, Isoxazoles; Antimicrobial activity

Introduction

The five membered heterocyclic compounds with two heteroatoms particularly pyrazoles and isoxazoles have considerable interest in various fields because of their wide range of pharmacological and physical applications. A number of pyrazole and isoxazole derivatives possess antimicrobial [1-6], anti-inflammatory [7,8], anticancer [9,10], analgesic [11,12], anticonvulsant [13,14], anthelmintic and antioxidant [15,16] activities. Besides, pyrazole containing drugs celecoxib demonstrates anti-inflammation effect and inhibits COX- 2 [17] rimonabant functions as cannabinoid receptor and is utilized in obesity treatment [18] fomepizole inhibits alcohol dehydrogenase and sildenafil inhibits phosphodiesterase [19]. Isoxazole motif is ubiquitous in many natural products such as ibotenic acid, muscimol, isoxazole-4-carboxylic acid and drugs like valdecoxib, leflunomide, cloxacillin, oxacillin [20-24], dicloxacillin [25], isocarboxazide [26] and sulfisoxazole [27]. Among the various methods for the synthesis of pyrazoles, 1,3-dipolar cycloaddition and [2+3] cyclocondensation reactions are the prominent ones [28]. Among the different methods of isoxazole synthesis, [2+3] cycloaddition of 1,3-dipoles to alkynes and the reaction of hydroxylamine with 1,3-diketone or an α,β-unsaturated ketones have gained importance [29]. Moreover, the activated olefins are valuable intermediates in a variety of synthetic transformations and useful as building blocks in the synthesis of carbocyclic and heterocyclic compounds. In fact, we have exploited various activated olefins to develop pyrazoles and isoxazoles and studied their biological properties [30-33]. Recently, ultrasound method has been successfully employed as non-conventional method, to promote 1,3-dipolar cycloadditions [34]. Hence, development of pharmacologically active heterocycles adopting simple and efficient methodologies is one of the major challenges for organic chemists. With this background and in continuation of our studies in this direction, the present work synthesis and antimicrobial activity of 3,5-disubstituted pyrazoles and isoxazoles under ultrasonication has been taken up.

Experimental Protocols

All the chemicals were purchased from commercial sources and used without further purification. Melting points were determined in open capillaries on a Mel-Temp apparatus and are uncorrected. The homogeneity of the compounds was checked by TLC (silica gel H, BDH, hexane/ethyl acetate, 3:1). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were given in cm-1. The 1H NMR spectra were recorded in CDCl3/DMSO-d6 on a Jeol JNM λ-400 MHz spectrometer. The 13C NMR spectra were recorded in CDCl3/DMSO-d6 on a Jeol JNM spectrometer operating at λ-100 MHz. High-resolution mass spectra were recorded on Micromass Q-TOF micromass spectrometer using electrospray ionization. All chemical shifts were reported in δ (ppm) using TMS as an internal standard. The microanalyses were performed on a Perkin- Elmer 240C elemental analyzer. The temperature was measured by flexible probe throughout the reaction. Ultrasonication was performed in a Bandelin Sonorex RK 102H ultrasonic bath operating at frequency of 35 KHz.

General procedure for the synthesis of (E)-1-(furan-2-yl)-3-(1Hindol- 3-yl)prop-2-en-1-one (5a, b) / (E)-1-(5-bromothiophen-2-yl)- 3-(1H-indol-3-yl)prop-2-en-1-one (6a, b) / (E)-3-(1H-indol-3-yl)-1- (pyridin-4-yl)prop-2-en-1-one (7a, b)

To a solution of substituted indole-3-carboxaldehyde (1) (1.0 mmol) in anhydrous methanol (4 mL), 2-acetyl furan (2) / 2-acetyl- 5-bromothiophene (3)/4-acetyl pyridine (4) (1.0 mmol) were added followed by diisopropylethylamine (DIPEA) (0.33 mmol) and subjected to ultrasonication at a frequency of 35 KHz at room temperature for 60- 80 min. After completion of reaction (monitored by TLC), the contents of the flask were allowed to cool and poured into ice water. It was neutralized with 10% acetic acid. The separated solid was filtered, dried and recrystallized from 2-propanol.

(E)-1-(Furan-2-yl)-3-(1H-indol-3-yl)prop-2-en-1-one (5a): M.p. 190-191°C, yield 87%; IR (KBr) (cm-1): 3329 (NH), 1683 (C=O), 1632 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 6.93 (d, 1H, HB, J=15.4 Hz), 7.54-7.90 (m, 8H, Ar-H) 8.02 (d, 1H, HA, J=15.4 Hz), 11.74 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 133.9 (C-HB), 137.1 (C-HA), 176.4 (C=O), 111.3, 112.9, 113.1, 113.8, 115.3, 118.6, 120.8, 125.2, 125.6, 134.4, 147.8, 152.4 ppm (aromatic carbons). HRMS (m/z): 260.0687 [M+Na]; Anal. calcd. for C15H11NO2: C, 75.94; H, 4.67; N, 5.90%. Found: C, 76.05; H, 4.70; N, 6.02%.

(E)-3-(5-Fluoro-1H-indol-3-yl)-1-(furan-2-yl)prop-2-en-1-one (5b): M.p. 163-165°C, yield 89%; IR (KBr) (cm-1): 3322 (NH), 1675 (C=O), 1634 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 6.76 (d, 1H, HB, J=15.2 Hz), 7.63-7.65 (m, 7H, Ar-H) 8.13 (d, 1H, HA, J=15.2 Hz), 11.98 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 134.0 (CHB), 137.5 (C-HA), 176.9 (C=O), 110.6, 112.7, 113.5, 113.4, 115.4, 117.7, 125.3, 125.4, 134.6, 147.3, 153.3, 157.0 ppm (aromatic carbons). HRMS (m/z): 278.0593 [M+Na]; Anal. calcd. for C15H10FNO2: C, 70.58; H, 3.95; N, 5.49%. Found: C, 70.66; H, 3.97; N, 5.64%.

(E)-1-(5-Bromothiophen-2-yl)-3-(1H-indol-3-yl)prop-2-en- 1-one (6a): M.p. 202-204°C, yield 90%; IR (KBr) (cm-1): 3326 (NH), 1672 (C=O), 1628 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 7.21 (d, 1H, HB, J=15.6 Hz), 7.65-7.88 (m, 7H, Ar-H) 8.15 (d, 1H, HA, J=15.6 Hz), 11.94 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 125.0 (C-HB), 138.8 (C-HA), 180.4 (C=O), 112.4, 112.6, 113.8, 120.6, 121.2, 122.6, 122.7, 132.2, 132.6, 133.8, 137.5, 148.1 ppm (aromatic carbons). HRMS (m/z): 353.9569 [M+Na]; Anal. calcd. for C15H10BrNOS: C, 54.23; H, 3.03; N, 4.22%. Found: C, 54.33; H, 3.08; N, 4.39%.

(E)-1-(5-Bromothiophen-2-yl)-3-(5-fluoro-1H-indol-3-yl)prop- 2-en-1-one (6b): M.p. 177-179°C, yield 92%; IR (KBr) (cm-1): 3318 (NH), 1676 (C=O), 1631 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 7.04 (d, 1H, HB, J=15.8 Hz), 7.52-7.83 (m, 6H, Ar-H) 8.02 (d, 1H, HA, J=15.8 Hz), 12.02 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 126.3 (C-HB), 138.4 (C-HA), 180.7 (C=O), 112.7, 113.3, 114.0, 121.8, 122.3, 122.9, 124.1, 132.7, 137.3, 140.1, 148.3, 157.6 ppm (aromatic carbons). HRMS (m/z): 371.9470 [M+Na]; Anal. calcd. for C15H9BrFNOS: C, 51.45; H, 2.59; N, 4.00%. Found: C, 51.57; H, 2.63; N, 4.16%.

(E)-3-(1H-Indol-3-yl)-1-(pyridin-4-yl)prop-2-en-1-one (7a): M.p. 209-211°C, yield 88%; IR (KBr) (cm-1): 3336 (NH), 1688 (C=O), 1638 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 7.23 (d, 1H, HB, J=15.9 Hz), 7.61-7.92 (m, 9H, Ar-H) 8.17 (d, 1H, HA, J=15.9 Hz), 12.01 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 137.6 (C-HB), 144.2 (CHA), 188.2 (C=O), 110.2, 112.5, 112.8, 114.6, 120.6, 121.4, 122.9, 125.0, 134.5, 140.9, 151.6 ppm (aromatic carbons). HRMS (m/z): 271.0847 [M+Na]; Anal. calcd. for C16H12N2O: C, 77.40; H, 4.87; N, 11.28%. Found: C, 77.49; H, 4.89; N, 11.39%.

(E)-3-(5-Fluoro-1H-indol-3-yl)-1-(pyridin-4-yl)prop-2-en- 1-one (7b): M.p. 186-188°C, yield 91%; IR (KBr) (cm-1): 3330 (NH), 1686 (C=O), 1633 (C=C); 1H NMR (400 MHz, DMSO-d6): δ 7.08 (d, 1H, HB, J=15.7 Hz), 7.59-7.86 (m, 8H, Ar-H) 8.23 (d, 1H, HA, J=15.7 Hz), 12.09 (s, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 134.1 (C-HB), 144.5 (C-HA), 188.4 (C=O), 110.8, 112.9, 114.5, 114.8, 121.5, 125.4, 125.5, 135.8, 140.3, 150.6, 157.2 ppm (aromatic carbons). HRMS (m/z): 289.0753 [M+Na]; Anal. calcd. for C16H11FN2O: C, 72.17; H, 4.16; N, 10.52%. Found: C, 72.30; H, 4.20; N, 10.66%.

General procedure for the synthesis of 3-(furan-2-yl)-5-(1Hindol- 3-yl)-2-pyrazoline (8a, b) / 3-(5-bromothiophen-2-yl)- 5-(1H-indol-3-yl)-2-pyrazoline (9a, b) / 3-(pyridin-2-yl)-5- (1H-indol-3-yl)-2-pyrazoline (10a, b)

A mixture of 5/6/7 (1 mmol), hydrazine hydrate (1.5 mmol) and ethanol (6 mL) was subjected to ultrasonication at a frequency of 35 KHz at room temperature for 70-90 min. After completion of the reaction (monitored by TLC), the contents of the flask were poured onto crushed ice. The separated residue was extracted with dichloromethane. The organic layer was washed with water, brine and dried (an. Na2SO4). The solvent was removed under vacuum. The resultant residue was purified by column chromatography (silica gel, 60-120 mesh) using hexane/ethyl acetate (4:1) as eluent.

3-(Furan-2-yl)-5-(1H-indol-3-yl)-2-pyrazoline (8a): M.p. 208- 210°C, yield 80%; IR (KBr) (cm-1): 3338 (NH), 1640 (C=C), 1578 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.24 (dd, 1H, HX, JAX=6.5 Hz, JMX=10.7 Hz), 4.28 (dd, 1H, HM, JAM=12.5 Hz, JMX=10.9 Hz), 4.43 (dd, 1H, HA, JAM=12.5 Hz, JAX=6.5 Hz), 6.69-7.73 (m, 8H, Ar- H), 7.85 (bs, 1H, NH-pyrazoline), 10.21 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.6 (C-4ꞌ), 44.9 (C-5ꞌ), 143.6 (C-3ꞌ), 110.2, 110.6, 113.1, 118.3, 120.5, 122.8, 126.4, 129.6, 131.0, 136.2, 142.4, 150.7 (aromatic carbons). HRMS (m/z): 274.0956 [M+Na]; Anal. calcd. for C15H13N3O: C, 71.70; H, 5.21; N, 16.72%. Found: C, 71.83; H, 5.24; N, 16.92%.

3-(Furan-2-yl)-5-(5-fluoro-5-1H-indol-3-yl)-2-pyrazoline (8b): M.p. 178-180°C, yield 82%; IR (KBr) (cm-1): 3320 (NH), 1636 (C=C), 1577 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.32 (dd, 1H, HX, JAX=6.8 Hz, JMX=10.9 Hz), 4.13 (dd, 1H, HM, JAM=12.8 Hz, JMX=10.9 Hz), 4.46 (dd, 1H, HA, JAM=12.8 Hz, JAX=6.8 Hz), 6.73- 7.78 (m, 7H, Ar-H), 7.88 (bs, 1H, NH-pyrazoline), 10.32 (bs, 1H, NHindole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.4 (C-4ꞌ), 44.6 (C- 5ꞌ), 143.9 (C-3ꞌ), 113.2, 113.8, 117.4, 119.3, 123.6, 127.1, 129.2, 131.6, 136.8, 143.4, 150.6, 151.3 (aromatic carbons); HRMS (m/z): 292.0862 [M+Na]; Anal. calcd. for C15H12FN3O: C66.91; H, 4.49; N, 15.60%. Found: C, 67.02; H, 4.50; N, 15.78%.

3-(5-Bromothiophen-2-yl)-5-(1H-indol-3-yl)-2-pyrazoline (9a): M.p. 212-214°C, yield 84%; IR (KBr) (cm-1): 3328 (NH), 1639 (C=C), 1584 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.34 (dd, 1H, HX, JAX=6.3 Hz, JMX=10.2 Hz), 4.19 (dd, 1H, HM, JAM=12.4 Hz, JMX=10.2 Hz), 4.66 (dd, 1H, HA, JAM=12.4 Hz, JAX=6.3 Hz), 6.92- 7.68 (m, 7H, Ar-H), 7.82 (bs, 1H, NH-pyrazoline), 10.56 (bs, 1H, NHindole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.7 (C-4ꞌ), 45.1 (C- 5ꞌ), 139.4 (C-3ꞌ), 111.3, 112.5, 118.4, 118.7, 120.7, 121.9, 125.3, 127.2, 128.7, 132.3, 134.1, 151.8 (aromatic carbons); HRMS (m/z): 367.9842 [M+Na]; Anal. calcd. for C15H12BrN3S: C, 52.03; H, 3.49; N, 12.14%. Found: C, 52.13; H, 3.50; N, 12.36%.

3-(5-Bromothiophen-2-yl)-5-(5-fluoro-1H-indol-3-yl)-2- pyrazoline (9b): M.p. 181-183°C, yield 82%; IR (KBr) (cm-1): 3324 (NH), 1642 (C=C), 1577 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.38 (dd, 1H, HX, JAX=6.5 Hz, JMX=10.3 Hz), 4.23 (dd, 1H, HM, JAM=12.3 Hz, JMX=10.3 Hz), 4.68 (dd, 1H, HA, JAM=12.3 Hz, JAX=6.5 Hz), 7.01-7.82 (m, 6H, Ar-H), 7.89 (bs, 1H, NH-pyrazoline), 10.62 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.1 (C-4ꞌ), 45.4 (C-5ꞌ), 138.9 (C-3ꞌ), 117.6, 120.4, 111.8, 113.2, 125.4, 126.7, 128.3, 132.1, 136.4, 140.8, 151.2, 152.4 (aromatic carbons); HRMS (m/z): 385.9739 [M+Na]; Anal. calcd. for C15H11BrFN3S: C, 49.46; H, 3.04; N, 11.54%. Found: C, 49.58; H, 3.06; N, 11.79%.

3-(Pyridin-2-yl)-5-(1H-indol-3-yl)-2-pyrazoline (10a): M.p. 227-229°C, yield 83%; IR (KBr) (cm-1): 3327 (NH), 1646 (C=C), 1589 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.43 (dd, 1H, HX, JAX=5.5 Hz, JMX=10.7 Hz), 4.10 (dd, 1H, HM, JAM=11.8 Hz, JMX=10.7 Hz), 4.64 (dd, 1H, HA, JAM=11.8 Hz, JAX=5.5 Hz), 7.14-7.88 (m, 9H, Ar- H), 7.91 (bs, 1H, NH-pyrazoline), 11.63 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.6 (C-4ꞌ), 45.6 (C-5ꞌ), 142.9 (C-3ꞌ), 121.2, 137.5, 113.1, 117.3, 123.5, 124.7, 125.1, 127.3, 128.6, 130.4, 151.6 (aromatic carbons); HRMS (m/z): 285.1116 [M+Na]; Anal. calcd. for C16H14N4: C, 73.26; H, 5.38; N, 21.36%. Found: C, 73.20; H, 5.36; N, 21.28%.

3-(Pyridin-2-yl)-5-(5-fluoro-1H-indol-3-yl)-2-pyrazoline (10b): M.p. 201-203°C, yield 79%; IR (KBr) (cm-1): 3341 (NH), 1643 (C=C), 1596 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.45 (dd, 1H, HX, JAX=5.7 Hz, JMX=10.2 Hz), 4.18 (dd, 1H, HM, JAM=11.6 Hz, JMX=10.2 Hz), 4.67 (dd, 1H, HA, JAM=11.6 Hz, JAX=5.7 Hz), 7.23- 7.90 (m, 8H, Ar-H), 7.94 (bs, 1H, NH-pyrazoline), 11.76 (bs, 1H, NHindole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.5 (C-4ꞌ), 45.2 (C-5ꞌ), 142.3 (C-3ꞌ), 121.4, 138.7, 113.4, 117.8, 121.4, 123.7, 127.5, 128.2, 129.9, 151.3, 152.7 (aromatic carbons); HRMS (m/z): 303.1022 [M+Na]; Anal. calcd. for C16H13FN4: C, 68.56; H, 4.67; N, 19.99%. Found: C, 68.66; H, 4.70; N, 20.18%.

General procedure for the synthesis of 3-(furan-2-yl)-5-(1Hindol- 3-yl)-2-isoxazoline (14a, b) / 3-(5-bromothiophen-2- yl)-5-(1H-indol-3-yl)-2-isoxazoline (15a, b) / 3-(pyridin-2- yl)-5-(1H-indol-3-yl)-2-isoxazoline (16a, b)

A solution of 5/6/7 (1 mmol), hydroxylamine hydrochloride (1.1 mmol) in ethanol (6 mL) was kept under ultrasonication at room temperature for 60-70 min. The progress of the reaction was monitored by TLC. After completion of the reaction, the contents of the flask were poured onto crushed ice. It was extracted with dichloromethane. The organic layer was washed with water, brine and dried (an. Na2SO4). The solvent was removed under reduced pressure. The resultant residue was purified by column chromatography (silica gel, 60-120 mesh) using hexane/ethyl acetate (4:1) as eluent.

3-(Furan-2-yl)-5-(1H-indol-3-yl)-2-isoxazoline (14a): M.p. 168- 170°C, Yield 79%; IR (KBr) (cm-1): 3335 (NH), 1644 (C=C), 1588 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.26 (dd, 1H, HX, JAX=6.6 Hz, JMX=10.5 Hz), 4.52 (dd, 1H, HM, JAM=12.6 Hz, JMX=10.5 Hz), 4.98 (dd, 1H, HA, JAM=12.6 Hz, JAX=6.8 Hz), 6.88-7.87 (m, 8H, Ar- H), 10.48 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.5 (C-4ꞌ), 45.8 (C-5ꞌ), 141.2 (C-3ꞌ), 113.8, 117.2, 119.7, 120.8, 122.8, 127.9, 129.6, 134.5, 138.1, 140.3, 151.6, 156.4 (aromatic carbons); HRMS (m/z): 275.0796 [M+Na]; Anal. calcd. for C15H12N2O2: C, 71.42; H, 4.79; N, 11.10%. Found: C, 71.51; H, 4.78; N, 11.26%.

3-(Furan-2-yl)-5-(5-fluoro-1H-indol-3-yl)-2-isoxazoline (14b): M.p. 182-184°C, yield 81%; IR (KBr) (cm-1): 3343 (NH), 1635 (C=C), 1579 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.30 (dd, 1H, HX, JAX=6.7 Hz, JMX=10.8 Hz), 4.56 (dd, 1H, HM, JAM=12.7 Hz, JMX=10.8 Hz), 5.08 (dd, 1H, HA, JAM=12.7 Hz, JAX=6.7 Hz), 6.91-7.89 (m, 7H, Ar-H), 10.53 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 43.2 (C-4ꞌ), 45.3 (C-5ꞌ), 141.6 (C-3ꞌ), 113.9, 114.2, 119.4, 127.7, 129.3, 134.5, 138.4, 138.8, 151.6, 140.6, 151.5, 158.1 (aromatic carbons); HRMS (m/z): 293.0708 [M+Na]; Anal. calcd. for C15H11FN2O: C, 66.66; H, 4.10; N, 10.37%. Found: C, 66.61; H, 4.12; N, 10.40%.

3-(5-Bromothiophen-2-yl)-5-(1H-indol-3-yl)-2-isoxazoline (15a): M.p. 217-218°C, yield 78%; IR (KBr) (cm-1): 3332 (NH), 1637 (C=C), 1587 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.41 (dd, 1H, HX, JAX=5.8 Hz, JMX=10.6 Hz), 4.57 (dd, 1H, HM, JAM=12.4 Hz, JMX=10.6 Hz), 5.12 (dd, 1H, HA, JAM=12.4 Hz, JAX=5.8 Hz), 7.12-8.09 (m, 7H, Ar-H), 10.71 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.8 (C-4ꞌ), 45.7 (C-5ꞌ), 140.2 (C-3ꞌ), 114.2, 114.6, 117.3, 120.2, 121.8, 123.5, 125.7, 127.0, 128.5, 130.6, 139.3, 151.8 (aromatic carbons); HRMS (m/z): 368.9681 [M+Na]; Anal. calcd. for C15H11BrN2OS: C, 51.89; H, 3.19; N, 8.07%. Found: C, 51.97; H, 3.18; N, 8.25%.

3-(5-Bromothiophen-2-yl)-5-(5-fluoro-1H-indol-3-yl)-2- isoxazoline (15b): M.p. 187-189°C, yield 80%; IR (KBr) (cm-1): 3336 (NH), 1632 (C=C), 1584 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.47 (dd, 1H, HX, JAX=5.8 Hz, JMX=10.6 Hz), 4.57 (dd, 1H, HM, JAM=12.4 Hz, JMX=10.6 Hz), 5.12 (dd, 1H, HA, JAM=12.4 Hz, JAX=5.8 Hz), 7.12-8.09 (m, 6H, Ar-H), 10.78 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 41.4 (C-4ꞌ), 45.9 (C-5ꞌ), 140.7 (C- 3ꞌ), 117.6, 117.9, 117.8, 118.3, 120.9, 123.8, 128.0, 129.1, 136.6, 139.5, 152.0, 152.3 (aromatic carbons); HRMS (m/z): 386.9579 [M+Na]; Anal. calcd. for C15H10BrFN2OS: C, 49.33; H, 2.76; N, 7.67%. Found: C, 49.44; H, 2.79; N, 7.90%.

3-(Pyridin-2-yl)-5-(1H-indol-3-yl)-2-isoxazoline (16a): M.p. 216-218°C, yield 86%; IR (KBr) (cm-1): 3338 (NH), 1631 (C=C), 1585 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.52 (dd, 1H, HX, JAX=6.2 Hz, JMX=10.8 Hz), 4.62 (dd, 1H, HM, JAM=12.2 Hz, JMX=10.8 Hz), 5.20 (dd, 1H, HA, JAM=12.2 Hz, JAX=6.2 Hz), 7.26-8.24 (m, 9H, Ar- H), 11.96 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.4 (C-4ꞌ), 45.8 (C-5ꞌ), 140.1 (C-3ꞌ), 123.3, 136.9, 113.7, 117.1, 121.5, 124.9, 126.4, 127.7, 129.0, 140.3, 151.5 (aromatic carbons); HRMS (m/z): 286.0956 [M+Na]; Anal. calcd. for C16H13N3O: C, 72.99; H, 4.98; N, 15.96%. Found: C, 73.11; H, 4.96; N, 16.22%.

3-(Pyridin-2-yl)-5-(5-fluoro-1H-indol-3-yl)-2-isoxazoline (16b): M.p. 186-188°C, yield 82%; IR (KBr) (cm-1): 3335 (NH), 1633 (C=C), 1582 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.56 (dd, 1H, HX, JAX=6.4 Hz, JMX=10.5 Hz), 4.65 (dd, 1H, HM, JAM=12.2 Hz, JMX=10.5 Hz), 5.23 (dd, 1H, HA, JAM=12.2 Hz, JAX=6.4 Hz), 7.31- 8.27 (m, 8H, Ar-H), 11.98 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 42.7 (C-4ꞌ), 45.5 (C-5ꞌ), 141.6 (C-3ꞌ), 123.9, 138.5, 117.7, 121.0, 124.7, 126.8, 127.6, 128.8, 129.2, 151.9, 152.3 (aromatic carbons); HRMS (m/z): 304.0862 [M+Na]; Anal. calcd. for C16H12FN3O: C, 68.32; H, 4.30; N, 14.94%. Found: C, 68.41; H, 4.32; N, 15.14%.

General procedure for the synthesis of 3,5-diaryl pyrazoles (11 a, b / 12 a, b / 13 a, b) and 3,5-diaryl isoxazoles (17 a, b / 18 a, b / 19 a, b)

A solution of 8 / 9 / 10 / 14 / 15/ 16 (1 mmol) in xylene (7 mL) and chloranil (1.2 mmol) were subjected to ultrasonication at reflux temperature for 1-2 hrs. Then, it was treated with 5% NaOH solution. The organic layer was separated and repeatedly washed with water and dried (an. Na2SO4). The solvent was removed in vaccuo. The solid obtained was purified by recrystallization from 2-propanol.

3-(Furan-2-yl)-5-(1H-indol-3-yl)pyrazole (11a): M.p. 212-214°C, yield 73%; IR (KBr) (cm-1): 3342 (NH), 1638 (C=C), 1575 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 6.83-7.72 (m, 9H, Ar-H), 7.63 (bs, 1H, NH-pyrazole), 10.46 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.3 (C-4ꞌ), 131.0 (C-5ꞌ), 132.5 (C-3ꞌ), 109.8, 110.6, 111.4, 112.8, 113.2, 118.7, 120.9, 125.3, 125.7, 134.6, 141.1, 152.5 (aromatic carbons); HRMS (m/z): 272.0800 [M+Na]; Anal. calcd. for C15H11N3O: C, 72.28; H, 4.45; N, 16.86%. Found: C, 72.22; H, 4.48; N, 17.02%.

3-(Furan-2-yl)-5-(5-fluoro-1H-indol-3-yl)pyrazole (11b): M.p. 183-185°C, yield 75%; IR (KBr) (cm-1): 3337 (NH), 1648 (C=C), 1598 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 6.94-7.67 (m, 8H, Ar-H), 7.68 (bs, 1H, NH-pyrazole), 10.53 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.1 (C-4ꞌ), 131.3 (C-5ꞌ), 132.7 (C-3ꞌ), 110.4, 110.7, 110.9, 113.3, 113.6, 117.8, 124.3, 125.5, 134.8, 141.6, 153.1, 157.4 (aromatic carbons); HRMS (m/z): 290.0706 [M+Na]; Anal. calcd. for C15H10FN3O: C, 67.41; H, 3.77; N, 15.72%. Found: C, 67.49; H, 3.78; N, 15.91%.

3-(5-Bromothiophen-2-yl)-5-(1H-indol-3-yl)pyrazole (12a): M.p. 217-219°C, yield 73%; IR (KBr) (cm-1): 3342 (NH), 1647 (C=C), 1594 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.32-7.71 (m, 8H, Ar- H), 7.84 (bs, 1H, NH-pyrazole), 10.84 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.1 (C-4ꞌ), 130.6 (C-5ꞌ), 133.5 (C-3ꞌ), 111.0, 112.2, 112.8, 114.2, 120.5, 121.9, 122.5, 124.3, 132.4, 136.9, 142.4, 153.4 (aromatic carbons); HRMS (m/z): 365.9681 [M+Na]; Anal. calcd. for C15H10BrN3S: C, 52.34; H, 2.93; N, 12.21%. Found: C, 52.44; H, 2.92; N, 12.45%.

3-(5-Bromothiophen-2-yl)-5-(5-fluoro-1H-indol-3-yl)pyrazole (12b): M.p. 184-186°C, yield 70%; IR (KBr) (cm-1): 3346 (NH), 1640 (C=C), 1583 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.41-7.78 (m, 7H, Ar-H), 7.79 (bs, 1H, NH-pyrazole), 10.86 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.6 (C-4ꞌ), 131.8 (C-5ꞌ), 133.3 (C- 3ꞌ), 110.2, 111.7, 112.4, 113.8, 114.6, 122.2, 123.1, 124.5, 135.3, 142.7, 153.8, 157.6 (aromatic carbons); HRMS (m/z): 383.9582 [M+Na]; Anal. calcd. for C15H9BrFN3S: C, 49.74; H, 2.50; N, 11.60%. Found: C, 49.82; H, 2.52; N, 11.79%.

3-(Pyridin-2-yl)-5-(1H-indol-3-yl)pyrazole (13a): M.p. 220- 222°C, yield 76%; IR (KBr) (cm-1): 3344 (NH), 1649 (C=C), 1596 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.20-8.03 (m, 10H, Ar-H), 7.84 (bs, 1H, NH-pyrazole), 11.21 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.2 (C-4ꞌ), 131.4 (C-5ꞌ), 133.2 (C-3ꞌ), 110.3, 112.7, 111.6, 114.3, 120.1, 121.4, 122.2, 125.1, 134.6, 139.1, 148.4 (aromatic carbons); HRMS (m/z): 283.0960 [M+Na]; Anal. calcd. for C16H12N4: C, 73.83; H, 4.65; N, 21.52%. Found: C, 73.92; H, 4.68; N, 21.73%.

3-(Pyridin-2-yl)-5-(5-fluoro-1H-indol-3-yl)pyrazole (13b): M.p. 208-210°C; yield 73%; IR (KBr) (cm-1): 3348 (NH), 1656 (C=C), 1591 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.25-8.09 (m, 9H, Ar-H), 7.87 (bs, 1H, NH-pyrazole), 11.28 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.5 (C-4ꞌ), 131.9 (C-5ꞌ), 133.5 (C-3ꞌ), 110.9, 112.4, 114.6, 120.6, 122.7, 125.3, 125.5, 135.7, 139.4, 148.9, 157.3 (aromatic carbons); HRMS (m/z): 301.0865 [M+Na]; Anal. calcd. for C16H11FN4: C, 69.06; H, 3.98; N, 20.13%. Found: C, 69.17; H, 4.01; N, 20.36%.

3-(Furan-2-yl)-5-(1H-indol-3-yl)isoxazole (17a): M.p. 174- 176°C, yield 79%; IR (KBr) (cm-1): 3334 (NH), 1644 (C=C), 1588 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 6.91-7.69 (m, 9H, Ar-H), 10.72 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.5 (C-4ꞌ), 131.6 (C-5ꞌ), 132.3 (C-3ꞌ), 110.2, 111.4, 112.5, 112.7, 113.9, 120.5, 120.3, 121.1, 132.4, 133.9, 141.2, 152.7, (aromatic carbons); HRMS (m/z): 273.0649 [M+Na]; Anal. calcd. for C15H10N2O2: C, 71.99; H, 4.03; N, 11.19%. Found: C, 72.12; H, 4.08; N, 11.47%.

3-(Furan-2-yl)-5-(5-fluoro-1H-indol-3-yl)isoxazole (17b): M.p. 187-188°C, yield 77%; IR (KBr) (cm-1): 3338 (NH), 1639 (C=C), 1585 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.01-7.72 (m, 8H, Ar-H), 10.78 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.9 (C-4ꞌ), 132.0 (C-5ꞌ), 132.9 (C-3ꞌ), 109.8, 110.5, 111.7, 113.4, 113.9, 118.1, 125.6, 126.4, 135.2, 141.9, 153.5, 157.2 (aromatic carbons); HRMS (m/z): 291.0546 [M+Na]; Anal. calcd. for C15H9FN2O2: C, 67.16; H, 3.38; N, 10.44%. Found: C, 67.27; H, 3.41; N, 10.66%.

3-(5-Bromothiophen-2-yl)-5-(1H-indol-3-yl)isoxazole (18a): M.p. 221-223°C, yield 74%; IR (KBr) (cm-1): 3341 (NH), 1655 (C=C), 1592 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.21-7.85 (m, 8H, Ar- H), 10.89 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 108.7 (C-4ꞌ), 132.1 (C-5ꞌ), 133.4 (C-3ꞌ), 110.5, 111.9, 112.3, 113.8, 118.4, 121.1, 122.8, 126.3, 132.4, 136.0, 143.2, 154.0 (aromatic carbons); HRMS (m/z): 366.9517 [M+Na]; Anal. calcd. for C15H9BrN2OS: C, 52.19; H, 2.63; N, 8.11%. Found: C, 52.31; H, 2.67; N, 8.36%.

3-(5-Bromothiophen-2-yl)-5-(5-fluoro-1H-indol-3-yl)isoxazole (18b): M.p. 204-206°C, yield 78%; IR (KBr) (cm-1): 3339 (NH), 1651 (C=C), 1589 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.29-7.87 (m, 7H, Ar-H), 10.94 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.2 (C-4ꞌ), 132.7 (C-5ꞌ), 133.8 (C-3ꞌ), 111.2, 112.0, 112.7, 114.2, 118.7, 121.8, 123.1, 127.3, 136.3, 143.6, 154.5, 157.9 (aromatic carbons); HRMS (m/z): 384.9422 [M+Na]; Anal. calcd. for C15H8BrFN2OS: C, 49.60; H, 2.22; N, 7.71%. Found: C, 49.53; H, 2.23; N, 7.86%.

3-(Pyridin-2-yl)-5-(1H-indol-3-yl)isoxazole (19a): M.p. 224- 226°C; yield 71%; IR (KBr) (cm-1): 3343 (NH), 1657 (C=C), 1595 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.32-8.14 (m, 10H, Ar-H), 11.34 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.6 (C-4ꞌ), 132.3 (C-5ꞌ), 133.6 (C-3ꞌ), 110.4, 111.6, 111.9, 114.5, 120.7, 121.8, 122.5, 125.3, 134.8, 139.2, 149.4 (aromatic carbons); HRMS (m/z): 284.0800 [M+Na]; Anal. calcd. for C16H11N3O: C, 73.55; H, 4.24; N, 16.08%. Found: C, 73.64; H, 4.26; N, 16.26%.

3-(Pyridin-2-yl)-5-(5-fluoro-1H-indol-3-yl)isoxazole (19b): M.p. 198-200°C, yield 69%; IR (KBr) (cm-1): 3345 (NH), 1653 (C=C), 1598 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 7.36-8.17 (m, 9H, Ar-H), 11.37 (bs, 1H, NH-indole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 109.8 (C-4ꞌ), 132.5 (C-5ꞌ), 133.9 (C-3ꞌ), 111.2, 113.1, 114.8, 121.4, 122.9, 125.8, 126.0, 136.3, 139.8, 149.2, 157.6 (aromatic carbons); HRMS (m/z): 302.0706 [M+Na]; Anal. calcd. for C16H10FN3O: C, 68.81; H, 3.61; N, 15.05%. Found: C, 68.74; H, 3.60; N, 15.21%.

Antimicrobial Testing

The compounds 8-19 were dissolved in DMSO at different concentrations of 50 and 100 μg/well.

Cells

Bacterial strains S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae and fungi A. niger, P. chrysogenum were obtained from Department of Applied Microbiology, Sri Padmavathi Mahila Visvavidyalayam, Tirupati.

Antibacterial and antifungal assays

The in vitro antimicrobial studies were carried out by agar well diffusion method against test organisms [35,36]. Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100 μl) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petriplate. Various concentrations of DMSO dissolved compounds (50, 100 μg/ well) were added into the wells by using sterile pipettes. Simultaneously the standard antibiotics, Chloramphenicol for antibacterial activity and Ketoconazole for antifungal activity (as positive control) were tested against the pathogens. The samples were dissolved in DMSO which showed no zone of inhibition acts as negative control. The plates were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. After appropriate incubation, the diameter of zone of inhibition of each well was measured. Duplicates were maintained and the average values were calculated for eventual antibacterial activity.

Broth dilution test was used to determine minimum inhibitory concentration (MIC) of the above mentioned samples [37,38]. Freshly prepared nutrient broth was used as diluents. The 24 h old culture of the test bacteria S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae and fungi A. niger, P. chrysogenum were diluted 100 folds in nutrient broth (100 μl bacterial cultures in 10 ml NB). Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 20, 40 μl of stock solution contains 6.25, 12.5, 25, 50, 100, 200 μg/well of the compounds) were added to the test tubes containing the bacterial and fungal cultures. All the tubes were incubated at 37°C for 24 h for bacteria and at 28°C for 48 h for fungi. The tubes were examined for visible turbidity using NB as control. Control without test samples and with solvent was assayed simultaneously. The lowest concentration that inhibited visible growth of the tested organisms was recorded as MIC. To determine the minimum bactericidal concentration (MBC) [39] and minimum fungicidal concentration (MFC) [40] for each set of test tubes in the MIC determination, a loopful of broth was collected from those tubes which did not show any growth and inoculated on sterile nutrient broth (for bacteria) and PDA (for fungi) by streaking. Plates inoculated with bacteria and fungi were incubated at 37°C for 24 h and at 28°C for 48 h, respectively. After incubation, the lowest concentration was noted as MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed.

Results and Discussion

Chemistry

The Michael acceptors (E)-1-(furan-2-yl)-3-(1H-indol-3-yl) prop-2-en-1-one (5), (E)-1-(5-bromothiophen-2-yl)-3-(1H-indol-3- yl)prop-2-en-1-one (6) and (E)-3-(1H-indol-3-yl)-1-(pyridin-4-yl) prop-2-en-1-one (7) were utilized as synthons to synthesize a new class of 3,5-disubstituted pyrazoles and isoxazoles. The synthetic intermediates 5, 6 and 7 were in turn obtained by the Claisen-Schimdt reaction of indole-3-carboxaldehyde (1) with 2-acetylfuran (2) / 2-acetyl-5-bromothiophene (3) / 4-acetylpyridine (4) in the presence of diisopropylethylamine (DIPEA) in methanol under ultrasonication (Scheme 1). It was observed that these compounds were obtained in shorter reaction times with high yield in ultrasonication method when compared with the conventional method. The 1H NMR spectra of compounds 5a, 6a and 7a exhibited two doublets were observed at 6.93, 7.21, 7.23 and 8.02, 8.15, 8.17 which were accounted to olefin protons HA and HB. The coupling constant values JAB=15.4, 15.6 and 15.9 Hz revealed that they possess trans geometry. In addition to these, a broad singlet was observed at 11.74, 11.94 and 12.01 ppm was assigned to NH which disappeared on deuteration. The enone moiety present in 5 / 6 / 7 was exploited to develop pyrazoline and isoxazoline rings adopting [2+3] cyclocondensation. Thus, the cyclocondensation reaction of compounds 5, 6 and 7 with hydrazine hydrate under ultrasonication furnished 3-(furan-2-yl)-5-(1H-indol- 3-yl)-2-pyrazoline (8), 3-(5-bromothiophen-2-yl)-5-(1H-indol-3-yl)- 2-pyrazoline (9) and 3-(pyridin-2-yl)-5-(1H-indol-3-yl)-2-pyrazoline (10) respectively (Scheme 2). The 1H NMR spectra of compounds 8a, 9a and 10a displayed an AMX splitting pattern due to methine and methylene protons of pyrazoline ring. Thus the three double doublets present at δ 4.43, 4.28, 3.24 in 8a; at 4.66, 4.19, 3.34 in 9a and at 4.64, 4.10, 3.43 ppm in 10a were accounted to HA, HM and HX, respectively. Moreover, two broad singlets appeared at δ 7.85, 7.82 and 7.91 and at 10.21, 10.56 and 11.63 ppm were assigned to NH of pyrazoline and indole rings in addition to signals due to aromatic protons. The signals due to NH disappeared on deuteration. The oxidation of compounds 8, 9 and 10 with chloranil in xylene provided 3-(furan-2-yl)-5-(1H-indol-3-yl) pyrazole (11), 3-(5-bromothiophen-2-yl)-5-(1H-indol-3-yl)pyrazole (12) and 3-(pyridin-2-yl)-5-(1H-indol-3-yl)pyrazole (13), respectively. The absence of an AMX splitting pattern due to pyrazoline ring protons in the 1H NMR spectra of compounds 11a, 12a and 13a indicated that aromatization occurred. The singlets corresponding to C4ꞌ-H of pyrazole appeared at downfield region and merged with aromatic protons. Apart from this, two broad singlets present at δ 7.63, 7.84, 7.96 and 10.46, 10.84, 11.21 ppm were assigned to NH of pyrazole and indole moieties. The signals due to NH disappeared when D2O was added.

medicinal-chemistry-synthesis

Scheme 1: Synthesis of (E)-1-(aryl)-3-(1H-indol-3-yl)prop-2-en-1-ones.

medicinal-chemistry-pyrazolines

Scheme 2: Synthesis of 3-(aryl)-5-(1H-indol-3-yl)-2-pyrazolines and Pyrazoles.

Similarly, the cyclocondensation of 5, 6 and 7 with hydroxylamine hydrochloride in ethanol under ultrasonication yielded 3-(furan-2-yl)-5- (1H-indol-3-yl)-2-isoxazoline (14), 3-(5-bromothiophen-2-yl)-5-(1Hindol- 3-yl)-2-isoxazoline (15) and 3-(pyridin-2-yl)-5-(1H-indol-3-yl)- 2-isoxazoline (16), respectively. The 1H NMR spectra of compounds 14a, 15a and 16a displayed an AMX splitting pattern due to isoxazoline ring protons. The three double doublets present at δ 4.98, 4.52, 3.26 (14a); at 5.12, 4.57, 3.41 (15a) and 5.20, 4.62, 3.52 (16a) ppm were assigned to HA, HM and HX, respectively. Besides, the broad singlet observed at δ 10.48 (14a), 10.71 (15a) and 11.96 ppm (16a) was attributed to NH of indole which disappeared on deuteration. Furthermore, the aromatized products 3-(furan-2-yl)-5-(1H-indol-3-yl)isoxazole (17), 3-(5-bromothiophen-2-yl)-5-(1H-indol-3-yl)isoxazole (18) and 3-(pyridin-2-yl)-5-(1H-indol-3-yl)isoxazole (19) were obtained by the dehydrogenation of compounds 14, 15 and 16 with chloranil in xylene under ultrasonication (Scheme 3). The 1H NMR spectra of compounds 17a, 18a and 19a presented a singlet due to equation at much downfield region and merged with aromatic protons. Moreover, a broad singlet observed at δ 10.72, 10.89 and 11.34 ppm was assigned to NH of indole ring which disappeared when D2O was added. In fact, the target compounds were obtained in shorter reaction times with high yield under ultrasonication when compared with conventional method. The structures of all the synthesized compounds were further ascertained by IR, 13C NMR, HRMS and microanalyses.

medicinal-chemistry-isoxazolines

Scheme 3: Synthesis of 3-(aryl)-5-(1H-indol-3-yl)-2-isoxazolines and Isoxazoles.

Biological evaluation

Antimicrobial activity: The compounds 8-19 were evaluated for antimicrobial activity at two concentrations (50 and 100 μg/well) by agar well diffusion and broth dilution methods. The results regarding the antibacterial activity presented in Table 1 and Figure 1 indicated that Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) were more susceptible against the tested compounds than Gramnegative bacteria (Pseudomonas aureginosa and Klebsiella pneumoniae). The compounds 8b, 9a, 10a and 14b displayed low activity whereas the compounds 8a and 14a showed no activity. Amongst all the compounds 12b (39 μg/well) and 18b (41 μg/well) displayed higher antibacterial activity than the standard Chloramphenicol (38 μg/well) particularly against B. subtilis. However, the remaining compounds showed moderate to good activity. The compounds having pyrazole (11-13) and isoxazole units (17-19) displayed higher antibacterial activity than pyrazoline (8-10) and isoxazoline rings (14-16). The presence of electron withdrawing groups on the aromatic ring enhanced the activity.

medicinal-chemistry-antibacterial

Figure 1: The in vitro antibacterial activity of compounds 8-19.

Zone of inhibition (mm)
Compound Gram-positive bacteria Gram-negative bacteria
S. aureus B. subtilis P. aeruginosa K. pneumoniae
50 µg/well 100 µg/well 50 µg/well 100 µg/well 50 µg/well 100 µg/well 50 µg/well 100 µg/well
8a - - - - - - - -
8b - 7 ± 1 - 8 ± 3 - - - 7 ± 1
9a 11 ± 2 13 ± 1 12 ± 3 14 ± 3 8 ± 2 10 ± 1 11 ± 2 14 ± 1
9b 17 ± 1 19 ± 3 20 ± 1 22 ± 2 16 ± 3 18 ± 1 20 ± 3 21 ± 1
10a 10 ± 1 12 ± 3 11 ± 2 14 ± 1 8 ± 1 11 ± 3 7 ± 2 9 ± 1
10b 15 ± 2 17 ± 1 16 ± 3 19 ± 2 12 ± 3 15 ± 1 13 ± 1 16 ± 2
11a 13 ± 1 15 ± 2 22 ± 1 25 ± 3 9 ± 2 11 ± 1 16 ± 3 18 ± 2
11b 23 ± 3 25 ± 3 27 ± 2 30 ± 2 16 ± 1 19 ± 3 25 ± 2 27 ± 1
12a 19 ± 2 21 ± 1 25 ± 2 28 ± 3 15 ± 3 17 ± 1 23 ± 2 25 ± 3
12b 25 ± 2 28 ± 2 35 ± 1 39 ± 1 21 ± 1 24 ± 3 29 ± 2 32 ± 1
13a 17 ± 3 19 ± 1 25 ± 1 28 ± 1 14 ± 3 16 ± 1 21 ± 2 24 ± 3
13b 24 ± 1 27 ± 3 29 ± 3 32 ± 1 19 ± 1 21 ± 3 25 ± 2 28 ± 1
14a - - - - - - - -
14b - 8 ± 2 - 10 ± 1 - - - 9 ± 3
15a 15 ± 1 17 ± 2 19 ± 3 21 ± 1 14 ± 3 17 ± 2 18 ± 1 20 ± 3
15b 22 ± 3 25 ± 1 27 ± 2 28 ± 1 20 ± 1 23 ± 3 24 ± 1 26 ± 2
16a 13 ± 1 15 ± 2 17 ± 2 19 ± 1 13 ± 2 15 ± 2 16 ± 1 19 ± 3
16b 19 ± 3 21 ± 3 25 ± 3 27 ± 3 17 ± 1 19 ± 3 22 ± 3 25 ± 2
17a 16 ± 3 18 ± 1 23 ± 1 27 ± 3 11 ± 2 13 ± 1 18 ± 3 20 ± 2
17b 27 ± 2 29 ± 1 29 ± 3 32 ± 1 19 ± 3 22 ± 2 29 ± 1 33 ± 3
18a 22 ± 1 25 ± 1 28 ± 3 30 ± 2 17 ± 2 19 ± 1 24 ± 3 27 ± 2
18b 28 ± 3 31 ± 3 37 ± 1 41 ± 1 23 ± 1 26 ± 2 32 ± 3 35 ± 1
19a 18 ± 3 20 ± 1 26 ± 2 29 ± 3 12 ± 2 15 ± 3 26 ± 1 29 ± 2
19b 25 ± 2 31 ± 1 32 ± 1 34 ± 2 20 ± 3 23 ± 2 27 ± 1 30 ± 3
Chloramphenicol 33 ± 1 35 ± 3 34 ± 2 38 ± 3 27 ± 1 30 ± 3 40 ± 2 42 ± 1
Control (DMSO) - - - - - - - -

- No activity; ± Standard deviation

Table 1: The in vitro antibacterial activity of compounds 8-19.

All the tested compounds 8-19 inhibited the spore germination against the fungi Aspergillus niger and Penicillium chrysogenum except 8a and 14a (Table 2 and Figure 2). In general, all the compounds showed comparatively higher antifungal activity against A. niger than P. chrysogenum. Amongst all the tested compounds 12b (37 μg/well) and 18b (39 μg/well) displayed higher antifungal activity than the standard drug (36 μg/well). It was observed that thiophene linked compounds (9, 12, 15 and 18) inhibited the spore germination against the tested fungi when compared with the other compounds. The MIC, MBC and MFC values of the tested compounds are shown in Table 3. The compounds 12b and 18b exhibited low MIC values. The MBC value of compounds 12b and 18b is 2 × MIC in case of B. subtilis and MFC value of 12b and 18b is 2 × MIC in case of A. niger.

medicinal-chemistry-compounds

Figure 2: The in vitro antifungal activity of compounds 8-19.

Zone of inhibition (mm)
Compound Gram-positive bacteria Gram-negative bacteria
S. aureus B. subtilis P. aeruginosa K. pneumoniae
50 µg/well 100 µg/well 50 µg/well 100 µg/well 50 µg/well 100 µg/well 50 µg/well 100 µg/well
8a - - - - - - - -
8b - 7 ± 1 - 8 ± 3 - - - 7 ± 1
9a 11 ± 2 13 ± 1 12 ± 3 14 ± 3 8 ± 2 10 ± 1 11 ± 2 14 ± 1
9b 17 ± 1 19 ± 3 20 ± 1 22 ± 2 16 ± 3 18 ± 1 20 ± 3 21 ± 1
10a 10 ± 1 12 ± 3 11 ± 2 14 ± 1 8 ± 1 11 ± 3 7 ± 2 9 ± 1
10b 15 ± 2 17 ± 1 16 ± 3 19 ± 2 12 ± 3 15 ± 1 13 ± 1 16 ± 2
11a 13 ± 1 15 ± 2 22 ± 1 25 ± 3 9 ± 2 11 ± 1 16 ± 3 18 ± 2
11b 23 ± 3 25 ± 3 27 ± 2 30 ± 2 16 ± 1 19 ± 3 25 ± 2 27 ± 1
12a 19 ± 2 21 ± 1 25 ± 2 28 ± 3 15 ± 3 17 ± 1 23 ± 2 25 ± 3
12b 25 ± 2 28 ± 2 35 ± 1 39 ± 1 21 ± 1 24 ± 3 29 ± 2 32 ± 1
13a 17 ± 3 19 ± 1 25 ± 1 28 ± 1 14 ± 3 16 ± 1 21 ± 2 24 ± 3
13b 24 ± 1 27 ± 3 29 ± 3 32 ± 1 19 ± 1 21 ± 3 25 ± 2 28 ± 1
14a - - - - - - - -
14b - 8 ± 2 - 10 ± 1 - - - 9 ± 3
15a 15 ± 1 17 ± 2 19 ± 3 21 ± 1 14 ± 3 17 ± 2 18 ± 1 20 ± 3
15b 22 ± 3 25 ± 1 27 ± 2 28 ± 1 20 ± 1 23 ± 3 24 ± 1 26 ± 2
16a 13 ± 1 15 ± 2 17 ± 2 19 ± 1 13 ± 2 15 ± 2 16 ± 1 19 ± 3
16b 19 ± 3 21 ± 3 25 ± 3 27 ± 3 17 ± 1 19 ± 3 22 ± 3 25 ± 2
17a 16 ± 3 18 ± 1 23 ± 1 27 ± 3 11 ± 2 13 ± 1 18 ± 3 20 ± 2
17b 27 ± 2 29 ± 1 29 ± 3 32 ± 1 19 ± 3 22 ± 2 29 ± 1 33 ± 3
18a 22 ± 1 25 ± 1 28 ± 3 30 ± 2 17 ± 2 19 ± 1 24 ± 3 27 ± 2
18b 28 ± 3 31 ± 3 37 ± 1 41 ± 1 23 ± 1 26 ± 2 32 ± 3 35 ± 1
19a 18 ± 3 20 ± 1 26 ± 2 29 ± 3 12 ± 2 15 ± 3 26 ± 1 29 ± 2
19b 25 ± 2 31 ± 1 32 ± 1 34 ± 2 20 ± 3 23 ± 2 27 ± 1 30 ± 3
Chloramphenicol 33 ± 1 35 ± 3 34 ± 2 38 ± 3 27 ± 1 30 ± 3 40 ± 2 42 ± 1
Control (DMSO) - - - - - - - -

- No activity; ± Standard deviation

Table 2: The in vitro antifungal activity of compounds 8-19.

Compound Minimum inhibitory concentration MIC (MBC/MFC) mg/well
S. aureus B. subtilis P. aeruginosa K. pneumoniae A. niger P. chrysogenum
12b 50(200) 6.25(12.5) 50(>200) 100(>200) 12.5(25) 25(100)
18b 25(100) 6.25(12.5) 50(200) 100(>200) 12.5(25) 25(100)
Chloramphenicol 12.5 6.25 6.25 6.25 - -
Ketoconazole - - - - 6.25 12.5

- No activity; ± Standard deviation

Table 3: MIC, MBC and MFC of compounds 12b and 18b.

The structure-activity relationship of the tested compounds revealed that thiophene linked compounds showed greater activity than furan and pyridine moieties. Further, it was observed that aromatized heterocycles 11-13 and 17-19 showed greater antimicrobial activity than non-aromatized compounds 8-10 and 14-16. Moreover aromatized heterocyclic compounds having isoxazole unit displayed slightly higher activity than pyrazole unit. This is may be due to the presence of electron withdrawing oxygen atom. It was also noticed that compounds having electron withdrawing fluoro substituent on aromatic ring enhanced the activity when compared with the unsubstituted ones. Besides, the compounds having more number of electron withdrawing groups showed increased antimicrobial activity. Amongst all the compounds 12b and 18b were found to be potential antimicrobial agents particularly against Bacillus subtilis and Aspergillus niger. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.

Conclusion

A new class of 3,5-disubstituted pyrazoles and isoxazoles were prepared from the Michael acceptors 1-furanyl / thiophenyl / pyridinyl- 3-indole-prop-2-en-1-ones under ultrasonication and evaluated for antimicrobial activity. In fact, the target compounds were obtained in shorter reaction times with high yield under ultrasonication when compared with conventional method. Amongst all the tested compounds 12b and 18b displayed promising antimicrobial activity particularly against Bacillus subtilis and Aspergillus niger. The presence of electron withdrawing fluoro substituent on the aromatic ring enhanced the activity than the unsubstituted ones. Furthermore, compounds with more number of electron withdrawing groups showed higher antimicrobial activity. This result indicates that compounds 12b and 18b can be used as lead compounds in the future studies.

Acknowledgements

One of the authors, G. Yamini is thankful to University Grants Commission (UGC), New Delhi for the sanction of UGC-BSR fellowship. The authors are also thankful to Prof. P. Uma Maheswari Devi, Department of applied microbiology, Sri Padmavathi Mahila Visvavidyalayam, Tirupati for providing necessary facilities to carry out the antimicrobial activity.

References

Citation: Reddy SP, Yamini G, Sowmya DV, Padmavathi V, Padmaja A (2017) Synthesis and Antimicrobial Activity of Some New 3,5-Disubstituted Pyrazoles and Isoxazoles. Med Chem (Los Angeles) 7: 371-380. Doi: 10.4172/2161-0444.1000483

Copyright: © 2017 Reddy SP, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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