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Synthesis and Antimicrobial Activity of Pyrazolyl Triazoles
ISSN: 2161-0444

Medicinal Chemistry
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Synthesis and Antimicrobial Activity of Pyrazolyl Triazoles

Gaddam Lakshmi Teja1, Donthamsetty Venkata Sowmya1, Adivireddy Padmaja1, Vasikarla Kamala Prasad2 and Venkatapuram Padmavathi1*
1Department of Chemistry, Sri Venkateswara University, Tirupati, Andhra Pradesh, India
2Denisco Chemistry Private Limited, Hyderabad, Telangana, India
*Corresponding Author: Venkatapuram Padmavathi, Department of Chemistry, Sri Venkateswara University, Tirupati-517 502, Andhra Pradesh, India, Tel: +918772249666303, Email: [email protected]

Received Date: Nov 21, 2017 / Accepted Date: Nov 25, 2017 / Published Date: Nov 30, 2017

Abstract

Pyrazolyl triazoles (5) were prepared by 1,3-dipolar cycloaddition of diazomethane to Schiff base - 3-ethynyl- N-(heteroarylmethylene)aniline (3), followed by oxidation with I2 in DMSO and studied their antimicrobial activity. The pyridine and dichloropyridine substituted compounds 3d, 5c and 5d are potential antibacterial agents against Bacillus subtilis and 3d is a potential antifungal agent against Penicillium chrysogenum.

Keywords: Schiff base; Pyrazole; Triazole; Antimicrobial activity

Introduction

The Schiff bases are the core motifs in medicinal and pharmaceutical fields and exhibit antimicrobial [1], antioxidant [2], anticancer [3] and pesticidal [4] properties. They have also find applications as synthetic intermediates to synthesize a variety of heterocycles [5]. Further the azole derivatives are prominent players in pharmaceutical research. Some pyrazole containing drugs viz., Ramifenazone, Antipyrine, Phenylbutazone, Celebrex, Pyrazofurin, Fipronil, Rimonabant, Novalgine are used as drugs (Figure 1). Moreover, 1,2,3-triazole containing entities such as Tazobactam and Cefatrizine are clinically used for the treatment of bacterial infections (Figure 1). Thus molecules with azole motif in different pharmacological agents have made it an indispensible anchor for the articulation of new drugs. The pyrazoles were prepared by the reaction of hydrazines with 1,3-dicarbonyl compounds [6,7] and ynones [8]. Moreover, 1,3-dipolar cycloaddition of nitrile imines and diazoalkanes to alkynes produced pyrazoles [9]. On the other hand, 1,3-dipolar cycloaddition of azides with alkynes under thermal conditions, in the presence of copper or ruthenium catalysts resulted in 1,2,3-triazoles [10-12]. Transition-metal-free synthesis of 1,2,3-triazoles was reported by the cyclocondensation of organic azides with terminal alkynes in the presence of a catalytic amount of tetramethylammonium hydroxide [13].

medicinal-chemistry-triazole-moieties

Figure 1: Drugs containing pyrazole and triazole moieties.

Metal-free triazole synthesis was also reported by the modification of Sakai reaction of an amine and α,α-dichlorotosylhydrazones [14]. On the basis of these observations and our continued interest to develop novel heterocycles [15,16] it is proposed to construct new molecules having pyrazole and 1,2,3-triazole motifs at 1,3-positions of the phenyl moiety and to study their antimicrobial activity.

Experimental Protocols

Melting points were recorded on a Mel-Temp apparatus in open capillaries and are uncorrected. The purity of the compounds was checked by TLC (silica gel H, BDH, ethyl acetate/hexane, 1:3). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as KBr pellets and the wave numbers were mentioned in cm-1. The 1H and 13C NMR spectra were recorded in DMSO-d6 on a Bruker-400 spectrometer operating at 400 and 100 MHz, respectively. The chemical shifts are reported in δ (ppm) using TMS as an internal standard. The high-resolution mass spectra were recorded on micromass Q-TOF micromass spectrometer using electrospray ionization. The microanalyses were performed on a Perkin-Elmer 240C elemental analyzer. The progress of the reaction was monitored by TLC using silica gel plates and components were visualized under UV light (254 and 365 nm). 3-Aminophenylacetylene (1) was purchased from Sigma- Aldrich, India.

General procedure for the synthesis of 3-ethynyl-N- (heteroarylmethylene)aniline (3a-e)

An equimolar (1 mmol) quantity of 3-aminophenylacetylene and heteroaldehyde in absolute ethanol (20 mL) was refluxed on an oil bath for 4-6 hrs in the presence of a catalytic amount of glacial acetic acid. After completion of reaction (monitored by TLC), the solution was cooled, diluted with water (20 mL) and extracted with ethyl acetate (3 × 10 mL). The organic phase was dried over anhydrous Na2SO4. The solvent was removed under vacuum and the resultant solid was purified by column chromatography (silica gel, 60-120 mesh) using hexaneethyl acetate (4:1) as eluent.

3-Ethynyl-N-(1H-pyrrol-2-ylmethylene)aniline (3a): Yield 82%; m.p. 125-127°C; IR (KBr, cm-1): 1597 (C=N); 1H NMR (400 MHz, DMSO-d6): 4.18 (s, 1H, ≡C-H), 6.24 (t, 1H, C4-H, J=6.4 Hz), 6.48 (d, 1H, C3-H, J=6.4 Hz), 6.96 (d, 1H, C5-H, J=6.4 Hz), 7.05-7.35 (m, 4H, Ar-H), 8.71 (bs, 1H, NH-pyrrole), 8.81 (s, 1H, N=CH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 81.9 (-C≡CH), 82.8 (-C≡CH), 111.8, 119.2, 122.2, 124.6, 124.9, 127.8, 129.8, 130.6, 131.2, 148.6 (C-2, C-3, C-4, C-5, aromatic carbons), 155.7 (N=CH) ppm; HRMS (m/z): 217.2257 [M+Na]+; Anal. Calcd. for C13H10N2: C, 80.39; H, 5.19; N, 14.42%; Found: C, 80.28; H, 5.23; N, 14.51%.

3-Ethynyl-N-(furan-3-ylmethylene)aniline (3b): Yield 83%; m.p. 130-132°C; IR (KBr, cm-1): 1599 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 3.99 (s, 1H, ≡CH), 6.72 (d, 1H, C4-H, J=5.5 Hz), 6.91-7.57 (m, 4H, Ar-H), 7.69 (d, 1H, C5-H, J=5.5 Hz), 7.92 (s, IH, C2-H), 9.13 (s, IH, N=CH) ppm; 13C NMR (100 MHz, DMSO-d6) δ : 80.9 (-C≡CH), 82.1 (-C≡CH), 113.8, 114.2, 121.2, 124.1, 126.6, 129.2, 130.9, 140.2, 139.8, 143.6 (C-2, C-3, C-4, C-5 aromatic carbons), 159.2 (N=CH) ppm; HRMS (m/z): 218.2099 [M+Na]+; Anal. Calcd. for C13H9NO: C, 79.98; H, 4.65; N, 7.17%; Found: C, 79.89; H, 4.62; N, 7.27%.

3-Ethynyl-N-(pyridin-3-ylmethylene)aniline (3c): Yield 80%; m.p. 128-130°C; IR (KBr, cm-1): 1595 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 4.09 (s, 1H, ≡C-H), 7.02-7.66 (m, 4H, Ar-H), 7.79 (t, 1H, C5-H, J=4.2 Hz), 8.42 (d, 1H, C4-H, J=4.2 Hz), 8.76 (d, 1H, C6-H, J=4.2 Hz), 8.98 (s, 1H, C2-H), 9.16 (s, 1H, N=CH) ppm; 13C NMR (100 MHz, DMSO-d6) δ: 81.4 (-C≡CH), 82.5 (-C≡CH), 122.8, 123.9, 124.7, 125.2, 127.6, 129.5, 130.1, 131.8, 133.7, 147.2, 149.1, 151.9 (C-2, C-3, C-4, C-5, C-6, aromatic carbons), 154.8 (N=CH) ppm; HRMS (m/z): 229.2370 [M+Na]+; Anal. Calcd. For C14H10N2: C, 81.53; H, 4.89; N, 13.58%; Found: C, 81.64; H, 4.94; N, 13.65%.

3-Ethynyl-N-(3,5-dichloropyridin-4-ylmethylene)aniline (3d): Yield 78%; m.p. 133-135°C; IR (KBr, cm-1): 1564 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 4.19 (s, 1H, ≡C-H), 7.01-7.81 (m, 4H, Ar-H), 9.07 (s, 2H, C2-H, C6-H), 9.53 (s, 1H, N=CH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 82.2 (-C≡CH), 83.1 (-C≡CH), 123.4, 124.2, 125.3, 128.9, 129.5, 130.1, 131.3, 137.1, 138.2, 147.8, 148.6, (C-2, C-3, C-4, C-5, C-6, aromatic carbons), 155.6 (N=CH) ppm; HRMS (m/z): 298.1209 [M+Na]+; Anal. Calcd. for C14H8Cl2N2: C, 61.12; H, 2.93; N, 25.77%; Found: C, 61.19; H, 2.96; N, 25.64%.

3-Ethynyl-N-(1H-indol-3-ylmethylene)aniline (3e): Yield 79%; m.p. 135-137oC; IR (KBr, cm-1): 1585 (C=N); 1H NMR (400 MHz, DMSO-d6): δ 4.03 (s, 1H, ≡C-H), 7.12-8.01 (m, 9H, Ar-H), 8.92 (s, 1H, N=CH), 11.39 (bs, 1H, NH) ppm; 13C NMR (100 MHz, DMSO-d6): δ 81.9 (-C≡CH), 82.8 (-C≡CH), 103.2, 111.3, 118.2, 120.9, 121.6, 122.1, 124.2, 126.7, 127.2, 129.7, 130.9, 131.1, 137.3, 143.7 (aromatic carbons), 160.2 (N=CH) ppm; HRMS (m/z): 267.2858 [M+Na]+; Anal. Calcd. For C17H12N2: C, 83.58; H, 4.95; N, 11.47%; Found: C, 83.49; H, 4.99; N, 11.39%.

General procedure for the synthesis of 1-(3-(1H-Pyrazol-4-yl) phenyl)-5-(heteroaryl)-4,5-dihydro-1H-1,2,3-triazole (4a-e)

An ice cold ethereal solution of diazomethane (15 mL, 0.4 mmol) and triethylamine (0.1 mL) was added to Schiff base (3) (0.1 mmol) in dichloromethane (7 mL). The reaction mixture was kept at -15C for 44-48 hrs. The solvent was removed on a rotary evaporator and the resultant solid was recrystallized from ethanol.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(1H-pyrrol-2-yl)-4,5-dihydro- 1H-1,2,3-triazole (4a): Yield 72%; m.p. 163-165°C; IR (KBr, cm-1): 1595 (C=N), 1622 (C=C), 3296 (NH); 1H NMR (400 MHz, DMSO-d6): δ 2.14 (dd,1H, C5ꞌꞌ-H, HX, JAX=7.1 Hz, JMX=11.3 Hz), 2.75 (dd, 1H, C5ꞌ ꞌ-H, HM, JAM=13.3 Hz, JMX=11.3 Hz), 3.95 (dd,1H, C4ꞌꞌ-H, HA, JAM=13.3 Hz, JAX=7.1 Hz), 5.73 (d, 1H, C3-H, J=6.8 Hz), 6.00 (t, 1H, C4-H, J=6.8 Hz), 6.70 (d, 1H, C5-H, J=6.8 Hz), 7.16-7.41 (m, 4H, Ar), 8.25 (s, 1H, C3ꞌ-H), 8.53 (s, 1H, C5ꞌ-H), 9.01 (bs, 1H, NH-pyrrole), 10.75 (bs, 1H, NH- pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 58.9 (C-5ꞌꞌ), 74.8 (C-4ꞌꞌ), 108.7, 110.0, 111.5, 115.9, 118.7, 119.9, 120.3, 122.7, 124.3, 125.3, 128.0, 129.9, 130.1 (C-2, C-3, C-4, C-5, C-3ꞌ, C-4ꞌ, C-5ꞌ, aromatic carbons) ppm; HRMS (m/z): 301.3077 [M+Na]+; Anal. Calcd. For C15H14N6: C, 64.73; H, 5.07; N, 30.20%; Found: C, 64.80; H, 5.01; N, 30.26%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(furan-3-yl)-4,5-dihydro-1H- 1,2,3-triazole (4b):Yield 70%; m.p. 170-172°C; IR (KBr, cm-1): 1568 (C=N), 1630 (C=C), 3140 (NH); 1H NMR (400 MHz, DMSO-d6): 2.30 (dd, 1H, C5ꞌꞌ, HX, JAX=7.0 Hz, JMX=11.1 Hz), 2.81 (dd, 1H, C5ꞌꞌ, HM, JAM=13.1 Hz, JMX=11.1 Hz), 3.83 (dd, 1H, C4ꞌꞌ, HA, JAM=3.1 Hz, JAX=7.0 Hz), 6.28 (d, 1H, C4-H, J=4.7 Hz), 7.15 (d, 1H, C5-H, J=4.7 Hz), 7.25 (s, 1H, C2-H), 7.35-7.54 (m, 4H, Ar), 8.32 (s, 1H, C3ꞌ-H), 8.59 (s, 1H, C5ꞌ-H), 10.89 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 58.6 (C-5ꞌꞌ), 74.2 (C-4ꞌꞌ), 105.6, 108.9, 110.5, 112.7, 116.3, 117.1, 120.1, 123.2, 125.3, 130.2, 135.2, 137.5, 141.1 (C-2, C-3, C-4, C-5, C-3ꞌ, C-4ꞌ, C-5ꞌ, aromatic carbons) ppm; HRMS (m/z): 302.1033 [M+Na]+; Anal. Calcd. for C15H13N5O: C, 64.51; H, 4.69; N, 25.07%; Found: C, 64.58; H, 4.62; N, 25.16%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(pyridin-3-yl)-4,5-dihydro- 1H-1,2,3-triazole (4c): Yield 68%; m.p. 168-170°C; IR (KBr, cm-1): 1585 (C=N), 1642 (C=C), 3138 (NH); 1H NMR (400 MHz, DMSO-d6): δ 1.90 (dd, 1H, C5ꞌꞌ, HX, JAX=6.9 Hz, JMX=10.9 Hz), 2.56 (dd, 1H, C5ꞌꞌ, HM, JAM=12.9 Hz, JMX=10.9 Hz), 3.60 (dd, 1H, C4ꞌꞌ, HA, JAM=12.9 Hz, JAX=6.9 Hz), 7.10-7.42 (m, 4H, Ar), 7.56 (t, 1H, C5-H, J=4.5 Hz), 7.86 (s, 1H, C4-H, J=4.5 Hz), 8.14 (s, 1H, C3ꞌ-H), 8.43 (d, 1H, C6-H, J=4.5 Hz), 8.52 (s, 1H, C5ꞌ-H), 8.62 (s, 1H, C2-H), 10.70 (bs, IH, 2H, pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 57.9 (C-5ꞌꞌ), 73.0 (C-4ꞌꞌ), 116.2, 117.2, 118.3, 120.2, 121.3, 122.2, 123.4, 124.9, 125.4, 129.3, 133.0, 133.5, 146.6, 148.2 (C-2, C-3, C-4, C-5, C-6, C-3ꞌ, C-4ꞌ, C-5ꞌ, aromatic carbons) ppm; HRMS (m/z): 313.3189 [M+Na]+; Anal. Calcd. for C16H14N6: C, 66.19; H, 4.86; N, 28.95%; Found: C, 66.27; H, 4.81; N, 29.04%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(3,5-dichloropyridine-3-yl)- 4,5-dihydro-1H-1,2,3-triazole (4d): Yield 66%; m.p. 174-176°C; IR (KBr, cm-1): 1604 (C=N), 1635 (C=C), 3199 (NH); 1H NMR (400 MHz, DMSO-d6): 2.11 (dd, 1H, C5ꞌꞌ, HX, JAX=7.2 Hz, JMX=11.2 Hz), 2.80 (dd, 1H, C5ꞌꞌ, HM, JAM=13.0 Hz, JMX=11.2 Hz), 4.01 (dd,1H, C4ꞌꞌ, HA, JAM=13.0 Hz, JAX=7.0 Hz), 7.30-7.53 (m, 4H, Ar), 8,43 (s, 1H, C3ꞌ-H), 8.92 (s, 2H, C2-H, C6-H), 8.95 (s, 1H, C5ꞌ-H), 11.10 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 57.9 (C-5ꞌꞌ), 73.1 (C-4ꞌꞌ), 117.2, 118.5, 119.5, 121.1, 122.5, 124.3, 125.6, 129.7, 131.9, 133.4, 142.9, 144.7, 146.4 (C-2, C-3, C-4, C-5, C-6, C-3ꞌ, C-4ꞌ, C-5ꞌ, aromatic carbons) ppm; HRMS (m/z): 382.2027 [M+Na]+; Anal. Calcd. For C16H12Cl2N6: C, 53.56; H, 3.37; N, 23.40%; Found: C, 53.50; H, 3.33; N, 23.32%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(1H-indole-3-yl)-4,5-dihydro- 1H-1,2,3-triazole (4e): Yield 66%; m.p. 174-176°C; IR (KBr, cm-1): 1583 (C=N), 1620 (C=C), 3209 (NH); 1H NMR (400 MHz, DMSO-d6): δ 2.01 (dd, 1H, C5ꞌꞌ, HX, JAX=7.1 Hz, JMX=11.1 Hz), 2.53 (dd, 1H, C5ꞌꞌ, HM, JAM=13.1Hz, JMX=11.1 Hz), 3.79 (dd, 1H, C4ꞌꞌ, HA, JAM=13.1 Hz, JAX=7.2 Hz), 7.09-8.03 (m, 9H, Ar), 8.38 (s, 1H, C3ꞌ-H), 8.85 (s, 1H, C5ꞌ-H), 10.15 (bs, 1H, NH, indole), 10.85 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 59.0 (C-5ꞌꞌ), 73.5 (C-4ꞌꞌ), 110.3, 111.9, 118.2, 119.9, 120.9,122.5, 125.1, 125.8, 126.1, 127.2, 128.1, 128.9, 129.3, 130.4, 131.2, 132.7, 135.5 (C-3ꞌ, C-4ꞌ, C-5ꞌ, aromatic carbons) ppm; HRMS (m/z): 351.3680 [M+Na]+; Anal. Calcd. for C19H16N6: C, 69.50; H, 4.91; N, 25.59%; Found: C, 69.59; H, 4.96; N, 25.66%.

General procedure for the Synthesis of 1-(3-(1H-pyrazol-4- yl)phenyl)-5-(heteroaryl)-1H-1,2,3-triazole (5a-e)

The compound 4 (0.3 mmol) and iodine (2 mg, 2.2 mol%) were dissolved in dimethyl sulfoxide (8 mL) and refluxed for 3-4 hrs. The solution was poured onto crushed ice and the resultant solid was filtered, dried and purified by column chromatography (silica gel, 60- 120 mesh) using hexane-ethyl acetate (3:1) as eluent.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(1H-pyrrol-2-yl)-1H-1,2,3- triazole (5a): Yield 66%; m.p. 174-176C; IR (KBr, cm-1): 1592 (C=N), 1631 (C=C), 3312 (NH); 1H NMR (400 MHz, DMSO-d6): δ 6.03 (d, IH, C3-H, J=7.2 Hz), 6.20 (t, 1H, C4-H, J=7.2 Hz), 6.98 (d, 1H, C5-H, J=7.2 Hz), 7.39-7.73 (m, 4H, Ar), 8.03 (s, 1H, C5ꞌꞌ-H), 8.30 (s, 1H, C3ꞌ-H), 8.73 (s, 1H, C5ꞌ-H), 9.12 (bs, 1H, NH-pyrrole), 10.81 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 106.8 (C-3), 111.5 (C-4), 116.1, 120.1, 125.2, 126.5, 127.1, 128.1, 128.9, 129.6, 130.2, 130.9, 132.8, 133.5, 134.0 (C-2, C-5, C-3ꞌ, C-4ꞌ, C-5ꞌ, C-4ꞌꞌ, C-5ꞌꞌ, aromatic carbons) ppm; HRMS (m/z): 299.2919 [M+Na]+; Anal. Calcd. for C15H12N6: C, 65.21; H, 4.38; N, 30.42%. Found: C, 65.14; H, 4.34; N, 30.34%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(furan-3-yl)-1H-1,2,3-triazole (5b): Yield 64%; m.p. 178-180C; IR (KBr, cm-1): 1594 (C=N), 1638 (C=C), 3186 (NH); 1H NMR (400 MHz, DMSO-d6): δ 6.31 (d, 1H, C4- H, J=5.9 Hz), 7.25 (d, 1H, C5-H, J=5.9 Hz), 7.41 (s, 1H, C2-H), 7.51-7.89 (m, 4H, Ar), 8.01 (s, 1H, C5ꞌꞌ-H), 8.24 (s, 1H, C3ꞌ-H), 8.71 (s, 1H, C-5ꞌ-H), 10.95 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 107.8, 108.9, 119.8, 122.7, 125.2, 126.7, 127.9, 128.7, 129.5, 130.8, 131.5, 132.7, 133.8, 138.5, 144.1 (C-2, C-3, C-4, C-5, C-3ꞌ, C-4ꞌ C-5ꞌ, C-4ꞌꞌ, C-5ꞌꞌ, aromatic carbons) ppm; HRMS (m/z): 300.0845 [M+Na]+; Anal. Calcd. for C15H11N5O: C, 64.97; H, 4.00; N, 25.26%; Found: C, 65.05; H, 4.05; N, 25.18%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(pyridin-3-yl)-1H-1,2,3- triazole (5c): Yield 61%; m.p. 175-177°C; IR (KBr, cm-1): 1590 (C=N), 1654 (C=C), 3299 (NH); 1H NMR (400 MHz, DMSO-d6): δ 7.43-7.75 (m, 4H, Ar), 7.87 (t, 1H, C5-H, J=4.8 Hz), 7.91 (s, 1H, C5ꞌꞌ-H), 8.42 (d, 1H, C4-H, J=4.8 Hz), 8.49 (s, 1H, C3ꞌ-H), 8.70 (d, 1H, C6-H, J=4.8 Hz), 8.83 (s, 1H, C5ꞌ -H), 9.24 (s, IH, C2-H), 10.74 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 119.7, 122.7, 124.3, 126.1, 126.9, 127.8, 128.1, 128.9, 129.1, 130.6, 131.7, 132.8, 133.7, 134.5, 147.9, 149.1 (C-2, C-3, C-4, C-5, C-6, C-3ꞌ, C-4ꞌ, C-5ꞌ, C-4ꞌꞌ, C-5ꞌꞌ, aromatic carbons) ppm; HRMS (m/z): 311.2954 [M+Na]+; Anal. Calcd. for C16H12N6: C, 66.66; H, 4.20; N, 29.15%; Found: C, 66.78; H, 4.16; N, 29.26%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5(3,5-dichloropyridin-4-yl)-1H- 1,2,3-triazole (5d): Yield 63%; m.p. 181-183°C; IR (KBr, cm-1): 1568 (C=N), 1645 (C=C), 3256 (NH); 1H NMR (400 MHz, DMSO-d6): δ 7.62-7.88 (m, 4H, Ar-H), 8.01 (s, 1H, C5ꞌꞌ-H), 8.53(s, 1H, C3ꞌ-H), 8.91 (s, 1H, C5ꞌ-H), 8.96 (s, 2H, C2-H, C6-H), 11.18 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): 112.4, 117.2, 120.2, 126.5, 127.1, 128.3, 129.0, 129.9, 130.8, 131.6, 132.8, 133.7, 135.2, 143.3, 147.1 (C- 2, C-3, C-4, C-5, C-6, C-3ꞌ, C-4ꞌ, C-5ꞌ, C-4ꞌꞌ, C-5ꞌꞌ, aromatic carbons) ppm; HRMS (m/z): 380.1857 [M+Na]+; Anal. Calcd. for C16H10Cl2N6: C, 53.80; H, 2.82; N, 23.53%; Found: C, 53.89; H, 2.85; N, 23.66%.

1-(3-(1H-Pyrazol-4-yl)phenyl)-5-(1H-indole-3-yl)-1H-1,2,3- triazole (5e): Yield 62%; m.p. 194-196°C; IR (KBr, cm-1): 1581 (C=N), 1630 (C=C), 3226 (NH); 1H NMR (400 MHz, DMSO-d6): δ 7.22-8.21 (m, 9H, Ar-H), 8.01 (s, 1H, C5-H), 8.62 (s, 1H, C3ꞌ-H), 9.01 (s, 1H, C5ꞌ-H), 10.39 (bs, 1H, NH-indole), 11.01 (bs, 1H, NH-pyrazole) ppm; 13C NMR (100 MHz, DMSO-d6): δ 111.2, 112.3, 117.3, 119.8, 120.5, 121.2, 123.4, 125.7, 126.4, 127.1, 127.8, 128.5, 129.1, 129.8, 130.1, 132.5, 133.7, 134.2, 136.7 (C-3ꞌ, C-4ꞌ, C-5ꞌ, C-4ꞌꞌ, C-5ꞌꞌ, aromatic carbons) ppm; HRMS (m/z): 349.3435 [M+Na]+; Anal. Calcd. for C19H14N6: C, 69.92; H, 4.32; N, 25.75%; Found: C, 70.01; H, 4.37; N, 25.64%.

In vitro antimicrobial assay

The in vitro antimicrobial studies were determined by agar well diffusion method against test organisms [17-20]. Nutrient broth (NB) plates were swabbed with 24 hrs old broth culture (100 μl) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petri plate. Different concentrations of DMSO dissolved compounds (50, 75 and 100 μg/well) were added into the wells using micropipette. The standard antibiotics- Chloramphenicol for antibacterial activity and Ketoconazole for antifungal activity (as positive control) were tested against the pathogens simultaneously. 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 hrs for bacteria and at 28°C for 48 hrs for fungi. After an appropriate incubation, the diameter of zone of inhibition of each well was measured. The experiment was repeated twice and the average values were calculated for eventual antibacterial activity.

Broth dilution test was used to determine MIC of the samples [21,22]. Freshly prepared NB was used as diluents. The 24 hrs old culture of the bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Klebsiella pneumoniae and the fungi Aspergillus niger and Penicillium chrysogenum were diluted 100-folds in NB (100 μl bacterial cultures in 10 ml NB). Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 20 and 40 μl of stock solution contains 6.25, 12.5, 25, 50, 100 and 200 μg 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 hrs for bacteria and at 28°C for 48 hrs 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) [23] and minimum fungicidal concentration (MFC) [24] 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 NB (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 hrs, respectively. After incubation, the lowest concentration that kills the tested organisms was noted as MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed.

Biological activity

The compounds 3-5 were evaluated for antibacterial and antifungal activities at three different concentrations 50, 75 and 100 μg/well. Bacterial strains Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Klebsiella pneumoniae and fungi Aspergillus niger, Penicillium chrysogenum were obtained from Department of Microbiology, SV University, Tirupati.

Results and Discussion

Chemistry

The synthetic pathway adopted to achieve 3-ethynyl-N- (heteroarylmethylene)aniline (3), 1-(3-(1H-pyrazol-4-yl)phenyl)-5- (heteroaryl)-4,5-dihydro-1H-1,2,3-triazole (4) and 1-(3-(1H-pyrazol- 4-yl)phenyl)-5-(heteroaryl)-1H-1,2,3-triazole (5) is shown in Scheme 1. The Schiff base (3) was prepared by the reaction of 3-aminophenylacetylene (Sigma-Aldrich) with heteroaldehydes in the presence of glacial acetic acid in ethanol. The 1H NMR spectrum of 3a displayed two singlets at δ 4.18, 8.81 due to -C≡CH and -CH=N, two doublets at 6.49, 6.96 due to C3-H and C5-H, a triplet at 6.24 due to C4-H and a broad singlet at 8.71 ppm due to NH in addition to the signals of aromatic protons. The 1,3-dipolar cycloaddition of dipolar reagents across the double bond is one of the facile methods for the synthesis of five membered heterocycles. Although there are wide reports of the addition of diazomethane to acetylenes to get pyrazoles, the reaction of imines with diazomethane to develop 1,2,3-triazoles is sparsely reported [17]. We have synthesized a vulnerable synthetic intermediate 3 having both acetylene and imine functionalities which paves the way for the development of pyrazole and 1,2,3-triazole units in one pot under more facile and mild reaction conditions. Thus the treatment of compound 3 with diazomethane in the presence of triethylamine in ether at 0-15°C resulted in 1-(3-(1H-pyrazol-4-yl)phenyl)-5-(heteroaryl)-4,5-dihydro- 1H-1,2,3-triazole (4). The 1H NMR spectrum of 4a showed two singlets at δ 8.21, 8.53 due to C3ꞌ-H and C5ꞌ-H of pyrazole. The methylene and methine protons of 1,2,3-triazoline displayed AMX splitting pattern. The double doublets observed at 2.14, 2.75 and 3.95 were assigned to HX, HM and HA, respectively. The coupling constants JAM=13.3, JAX=7.1, JMX=11.3 Hz indicated that HA, HM are cis, HA, HX are trans and HM, HX are geminal. The pyrrole ring protons C3-H, C4-H and C5-H exhibited a doublet at 5.72, a triplet at 5.99 and a doublet at 6.70, respectively. In addition two broad singlets were observed at 9.01 and 10.75 ppm due to NH of pyrrole and pyrazole which disappeared on deuteration. Aromatization of 1,2,3-triazoline with I2 in DMSO resulted in 1-(3-(1H-pyrazol-4-yl)phenyl)-5-(heteroaryl)-1H-1,2,3-triazole (5). The absence of AMX splitting pattern and the presence of a singlet at δ 8.03 due to C5"-H of triazole indicated that aromatization occurred. In addition to this signals corresponding to pyrrole ring protons appeared at downfield region, C3-H at 6.03, C4-H at 6.20 and C5-H at 6.98 ppm which confirmed the extended conjugation. All the new compounds are further characterized by IR, 13C NMR, mass and elemental analysis.

medicinal-chemistry-synthesis

Scheme 1: Synthesis of 3-ethynyl-N-(heteroarylmethylene)aniline/1-(3-(1Hpyrazol- 4-yl)phenyl)-5-(heteroaryl)-4,5-dihydro-1H-1,2,3-triazole/1-(3-(1Hpyrazol- 4-yl)phenyl)-5-(heteroaryl)-1H-1,2,3-triazole.

Biological evaluation

In vitro antimicrobial activity: The results of antibacterial activity presented in Table 1 and Figure 2 revealed that except 3b, 4a and 4b, the remaining compounds are susceptible towards the tested bacteria. The compounds displayed higher activity on B. subtilis amongst Grampositive bacteria and on P. aeruginosa amongst Gram-negative bacteria. The pyrazolyl triazoles (5) exhibited higher activity than pyrazolyl triazolines (4) and phenylacetylene Schiff bases (3). However the latter compounds displayed more activity than 4. Amongst the compounds 5a-5e those having pyridine 5c and 5d exhibited higher activity. In fact 5d showed slightly more activity than 5c which may be due to the presence of electron withdrawing chloro substituent. On the other hand, 5e with indole exhibited higher activity than those having pyrrole 5a and furan 5b. Amongst latter compounds 5a showed greater activity than 5b. Similar effect of substituents was observed in compounds 3a-d and 4a-d also. The antibacterial activity exhibited by 5c and 5d was greater than the standard drug Chloramphenicol on B. subtilis at all tested concentrations. However, 3d displayed equal activity to the standard drug on B. subtilis and 5d on P. aeruginosa.

medicinal-chemistry-antibacterial-activity

Figure 2: The in vitro antibacterial activity of compounds 3a-5e.

Diameter of zone of inhibition (mm)
Compound Gram- positive bacteria Gram-negative bacteria
S. aureus B. subtilis P. aeruginosa K. pneumoniae
50 (mg/well) 75 (mg/well) 100 (mg/well) 50 (mg/well) 75 (mg/well) 100 (mg/well) 50 (mg/well) 75 (mg/well) 100 (mg/well) 50 (mg/well) 75 (mg/well) 100 (mg/well)
3a - - - 17 ± 3.2 19 ± 1.1 20 ± 1.1 - 12 ± 1.5 15 ± 1.8 - - 9 ± 1.4
3b - - - - - - - - - - - -
3c 21 ± 1.2 24 ± 2.2 26 ± 1.8 27 ± 3.1 28 ± 1.9 31 ± 1.9 17 ± 2.5 19 ± 1.8 21 ± 1.1 16± 1.8 17 ± 2.3 20 ± 1.5
3d 27 ± 1.8 29 ± 3.3 30 ± 3.2 32 ± 2.2 34 ± 1.8 38 ± 2.5 20 ± 2.2 23 ± 2.2 25 ± 3.2 19 ± 2.6 22 ± 3.2 24 ± 2.2
3e 18 ± 1.8 22 ± 1.1 24 ± 2.3 25 ± 2.2 27 ± 2.1 28 ± 2.3 16 ± 1.7 18 ± 1.2 20 ± 1.9 14 ± 0.8 15 ± 1.1 17 ± 1.1
4a - - - - - - - - - - - -
4b - - - - - - - - - - - -
4c 12 ± 1.3 19 ± 2.1 22 ± 1.1 23 ± 2.3 25 ± 1.1 26 ± 1.8 14 ± 1.3 16 ± 2.2 18 ± 1.8 12 ± 2.2 13 ± 1.8 15 ± 1.8
4d 15 ± 2.2 21 ± 1.8 23 ± 2.1 24 ± 1.1 26 ± 1.2 27 ± 1.4 15 ± 1.5 17 ± 1.7 19 ± 2.1 14 ± 1.1 16 ± 1.2 17 ± 1.3
4e - - - 13 ± 1.4 15 ± 2.7 18 ± 2.1 - - 11 ± 1.5 - - -
5a 24 ± 1.3 27 ± 2.1 28 ± 3.2 29 ± 2.1 31 ± 2.0 33 ± 2.2 18 ± 1.9 20 ± 2.5 22 ± 1.8 17 ± 0.8 19 ± 1.9 21 ± 2.8
5b 10 ± 1.8 16 ± 1.8 20 ± 2.2 21 ± 1.8 22 ± 2.3 23 ± 0.9 11 ± 1.2 14 ± 1.9 17 ± 1.2 10 ± 1.9 11 ± 2.2 13 ± 1.8
5c 28 ± 1.3 31 ± 2.1 33 ± 3.2 33 ± 2.3 35 ± 3.1 40 ± 1.3 22 ± 2.1 24 ± 1.3 26 ± 1.1 21 ± 2.1 23 ± 2.1 26 ± 3.3
5d 29 ± 2.1 32 ± 1.3 34 ± 2.2 34 ± 1.3 37 ± 2.1 41 ± 2.1 25 ± 0.9 27 ± 1.8 30 ± 1.2 24 ± 2.8 26 ± 2.6 27 ± 1.7
5e 26 ± 2.2 28 ± 2.2 29 ± 3.4 30 ± 2.1 33 ± 2.2 36 ± 3.1 19 ± 1.3 21 ± 3.5 23 ± 1.2 18 ± 3.1 20 ± 2.3 22 ± 2.1
Chloramphenicol 30 ± 3.3 33 ± 1.1 35 ± 2.1 32 ± 3.2 34 ± 2.1 38 ± 1.2 25 ± 3.2 27 ± 2.1 30 ± 2.2 38 ± 1.1 40 ± 2.2 42 ± 3.1
Control (DMSO) - - - - - - - - - - - -

(-) No activity; (±) Standard deviation

Table 1: The in vitro antibacterial activity of compounds 3a- 5e by agar-well diffusion method.

All the compounds inhibited spore germination of the tested fungi. The Schiff bases (3) displayed higher activity than pyrazolyl triazolines (4) and pyrazolyl triazoles (5). Amongst the latter compounds 5 showed greater activity than 4. Pyridine and dichloropyridine substituted compounds exhibited more activity than pyrrole, furan and indole substituted ones. The compound 3d displayed higher activity on P. chrysogenum than the standard drug Ketoconazole at all tested concentrations (Table 2 and Figure 3).

medicinal-chemistry-compounds-activity

Figure 3: The in vitro antifungal activity of compounds 3a-5e.

Diameter of zone of inhibition (mm)
Compound Fungi
A. niger P. chrysogenum
50 (mg/well) 75 (mg/well) 100 (mg/well) 50 (mg/well) 75 (mg/well) 100 (mg/well)
3a 19 ± 3.1 21 ± 1.4 24 ± 2.1 20 ± 1.3 22 ± 0.8 25 ± 2.3
3b 14 ± 3.2 15 ± 1.9 17 ± 3.2 16 ± 1.9 18 ± 3.1 19 ± 2.4
3c 25 ± 2.2 27 ± 1.6 30 ± 1.7 26 ± 2.1 27 ± 2.1 29 ± 1.4
3d 30 ± 1.7 32 ± 2.6 34 ± 1.7 36 ± 1.1 38 ± 1.4 39 ± 2.2
3e 21 ± 1.8 23 ± 2.5 27 ± 0.7 24 ± 1.9 26 ± 1.1 28 ± 1.5
4a - 9 ± 2.9 10 ± 0.8 - - 11 ± 1.1
4b - - 9 ± 32 - 9 ± 1.2 10 ± 1.2
4c 15 ± 1.5 17 ± 2.1 19 ± 2.1 15 ± 1.8 19 ± 2.2 21 ± 1.8
4d 16 ± 2.6 18 ± 2.3 20 ± 1.7 18 ± 0.8 20 ± 1.5 22 ± 3.2
4e - 13 ± 1.8 15 ± 2.4 - - 16 ± 1.2
5a 12 ± 2.8 14 ± 2.8 16 ± 2.3 14 ± 0.9 16 ± 2.4 17 ± 1.8
5b - - 11 ± 1.8 - 10 ± 1.8 13 ± 1.1
5c 18 ± 1.9 20 ± 1.3 23 ± 3.2 22 ± 1.5 23 ± 2.1 26 ± 3.2
5d 27 ± 1.4 30 ± 3.4 32 ± 0.9 28 ± 0.9 32 ± 0.9 33 ± 0.8
5e 17 ± 2.2 19 ± 3.2 21 ± 2.7 19 ± 1.9 22 ± 1.8 24 ± 2.8
Ketoconazole 31 ± 2.2 33 ± 3.5 36 ± 2.8 35 ± 1.8 36 ± 2.2 38 ± 3.1
Control (DMSO) - - - - - -

(-) No activity; (±) Standard deviation

Table 2: The in vitro antifungal activity of compounds 3a- 5e by agar-well diffusion method.

MIC, MBC and MFC of the compounds 3d, 5c, 5d and 5e: The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of the compounds tested are listed in Table 3. MIC is the lowest concentration of an antimicrobial that inhibit the visible growth of a microorganism (But it is not sure that the microorganisms are completely killed). The MBC/MFC is the lowest concentration of antibiotic required to kill a particular bacterium/fungus. The MBC/ MFC is assayed by performing an additional set of steps once the MIC is determined. The antimicrobials are usually regarded as bactericidal/ fungicidal if the MBC/MFC is not greater than four times the MIC [18]. The compounds 3d, 5c and 5d exhibited low MIC and the MBC is 2 × MIC towards B. subtilis whereas 5d displayed MBC is 2 × MIC towards P. aeruginosa also. On the other hand, the compound 3d showed low MIC and MFC is 2 × MIC on P. chrysogenum. The structure- activity relationship of the compounds revealed that the aromatized compounds 5 displayed higher activity than non- aromatized heterocylcles 4. The compounds having pyridine showed more activity and the activity enhanced by the presence of electron withdrawing chloro substituent.

Compound Minimum inhibitory concentration MIC (MBC/MFC) mg/well
Staphylococcus aureus Bacillus subtilis Pseudomonas aeruginosa Klebsiella pneumoniae Aspergillus niger Penicillium chrysogenum
3d 25 (100) 6.25 (12.5) 12.5 (50) 50 (200) 50 (200) 6.25 (12.5)
5c 25 (100) 6.25 (12.5) 12.5 (50) 25 (100) 100 (>200) 12.5 (50)
5d 12.5 (25) 6.25 (12.5) 6.25 (12.5) 12.5 (50) 50 (200) 12.5 (50)
5e 50 (200) 12.5 (50) 25 (100) 25 (100) 100 (>200) 12.5 (50)
Chloramphenicol 12.5 6.25 6.25 6.25 - -
Ketoconazole - - - - 6.25 12.5

(-) No activity

Table 3: MIC, MBC and MFC of compounds 3d, 5c, 5d and 5e.

Conclusion

A variety of pyrazolyl triazoles (5) were prepared by exploitation of acetylene and azomethine moieties present in 3-ethynyl-N- (heteroarylmethylene)aniline (3) by 1,3-dipolar cycloaddition of diazomethane followed by oxidation with I2 in DMSO. Pyridine substituted compounds showed more activity than those with pyrrole, furan and indole moieties. In fact, pyridine substituted Schiff base and pyrazolyl triazoles 3d, 5c and 5d exhibited potential antibacterial activity against Bacillus subtilis and 5d also on Pseudomonas aeruginosa. Further 3d displayed potential antifungal activity on Penicillium chrysogenum.

Acknowledgements

One of the authors G. Lakshmi Teja is grateful to University Grants Commission (UGC), New Delhi for sanction of UGC-BSR fellowship.

References

Citation: Teja GL, Sowmya DV, Padmaja A, Prasad VK, Padmavathi V, et al. (2017) Synthesis and Antimicrobial Activity of Pyrazolyl Triazoles. Med Chem (Los Angeles) 7:361-367. DOI: 10.4172/2161-0444.1000481

Copyright: © 2017 Teja GL, 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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