# Synthesis of Novel 1,2,4-Triazole Derivatives as Antimicrobial Agents via the Japp-Klingemann Reaction: Investigation of Antimicrobial Activities

Authors: Tasneem Taj; Ravindra R. Kamble; Atukuri Dorababu; Gangadhar Y. Meti
Journal: Journal of Chemistry
 (2013)

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## Abstract

In the present investigation, 1,2,4-triazole appended to pyrazoline and pyrazole rings (4a–g) using N-arylsydnone as synthon was prepared. The title compounds were subjected to Osiris property explorer for the oral bioavailability to analyze their drug likeness and drug score. Further, the compounds were subjected to the antimicrobial activity and analyzed the IC 50 and MIC values.


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## Body

## 1. Introduction

3-Arylsydnone, a class of mesoionic compounds, has been used as synthon extensively for the synthesis of various pharmaceutically potent molecules like pyrazole, phenylindazole, carbazole, pyrazoline, tetrazine, and 1,3,4-oxadiazole by 1,3-dipolar cycloaddition and  addition elimination reactions [1–3]. On ring insertion with hydrazine hydrate, 1,3,4-oxadiazole yields 1,2,4-triazole derivative [4]. 1,2,4-Triazolinone derivatives such as azafenidin and sulfentrazone have been reported as herbicides [5–9]. Synthesis of 1,2,4-triazoles fused to another heterocyclic ring has attracted wide spread attention due to their diverse applications as antibacterial, antidepressant, antiviral, antitumor, anti-inflammatory agents, pesticides, herbicides, dyes, lubricant, and analytical reagents [10].There is a considerable interest in chemotherapeutic activity of pyrazole derivatives. They have been reported to exhibit broad spectrum of biological effects [11]. Pyrazoles also possess a broad spectrum of biological effectiveness such as antidepressant [12] and antibacterial activity [13]. Besides, great interest in the pyrazole molecule has been stimulated by some promising agrochemical applications such as herbicides and fungicides [14–16]. Pyrazole-1-carboxamidopyrazole and 1-thiocarbamoyl-pyrazole showed impressive in vivo antitumour activity in experimental animals. One of the series of azoxypyrazoles, that is,1,1′-bis(2-hydroxyethyl)-3,5,3′,5′-tetra methylazoxypyrazole, has been reported to possess activity against ascitic forms of the Ehrlich,Landschiitz,and Sarcoma 180 tumours and against the P.388 lymphatic leukaemia in mice [17]. In view of these observations, we herein report the synthesis of novel molecular scaffolds containing pyrazole, pyrazolines, and 1,2,4-triazole ring using N-arylsydnone as a synthon in the hope to get lead compounds as antimicrobial agents.


## 2. Experimental

Melting points were determined in open capillaries. The IR spectra were recorded on Nicolet Impact 5200 USA FT IR using KBr pellets.1H NMR spectra were recorded on Bruker Varian 300-MHz FT NMR spectrometer with TMS as internal standard. EI mass spectral analyses were recorded on Shimadzu Japan QP2010 S model spectrometer, and elemental analyses were carried out using Heraus CHN rapid analyzer. The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using hexane and ethyl acetate. The pharmacological evaluation was carried out at the Biogenics, Hubli, Karnataka, India. The c log P values have been calculated using the Osiris molecular property explorer software for the structural analogues of the synthesized compounds and are uncorrected. The Schiff bases 2-[4-(1-acetyl-5-phenyl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-4-alkyl/arylamino-5-methyl-3-oxo-2,4-dihydro-[1,2,4]-triazoles (1a–g) were prepared as per the reported procedure [4]. Characterization and Spectral data of synthesized compounds are tabulated in Tables 1 and 2, respectively.Table 1
Characterization data of synthesized compounds.
Comp.
Melting point (°C)
Solvent of crystallization
Yield (%)
Formula(Molecular mass)
Analysis calcd./found
C
H
N
2a
130-131
Methanol
60
C27H25N7O2  (479)
67.6367.62
5.255.23
20.4520.46
2b
140-141
Methanol
62
C27H24ClN7O2  (513)
63.0963.07
4.714.72
19.0819.05
2c
131-132
Methanol
63
C27H24ClN7O2  (513)
63.0963.08
4.714.73
19.0819.04
2d
123-124
Ethanol
60
C27H24ClN7O2  (513)
63.0963.04
4.714.72
19.0819.05
2e
127-128
Methanol
62
C28H27N7O2  (493)
68.1468.12
5.515.53
19.419.2
2f
130-131
Ethanol
66
C27H25N7O3  (495)
65.44
5.09
19.79
2g
119–121
Ethanol
70
C28H27N7O3  (509)
65.4366.03
5.065.35
19.7819.23
3a
141-142
Ethanol
71
C32H30N8O4  (590)
65.0765.08
5.125.13
18.9718.96
3b
118–120
Ethanol
66
C32H29ClN8O4  (624)
61.4961.50
4.684.69
17.9317.90
3c
134-135
Ethanol
60
C32H29ClN8O4  (624)
61.4961.51
4.684.68
17.9317.92
3d
125-126
Ethanol
60
C32H29ClN8O4  (624)
61.4961.49
4.684.67
17.9317.89
3e
112–114
Ethanol
65
C33H32N8O4  (604)
65.5565.53
5.335.35
18.5318.50
3f
128-129
Ethanol
69
C32H30N8O5  (606)
52.3652.33
4.44.1
18.4718.49
3g
115-116
Ethanol
71
C33H32N8O5  (620)
63.363.4
5.205.21
18.0518.04
4a
158–160
Ethanol
52
C32H32N10O2  (586)
65.2965.28
5.485.49
23.7923.78
4b
145-146
Ethanol
57
C32H31ClN10O2  (622)
61.6861.65
5.015.02
22.4822.47
4c
121-122
Ethanol
68
C32H31ClN10O2  (622)
61.6861.65
5.015.02
22.4822.47
4d
143-144
Ethanol
64
C32H31ClN10O2  (622)
61.6861.65
5.015.02
22.4822.47
4e
130–132
Ethanol
70
C33H34N10O2  (602)
65.7665.75
5.695.68
23.2423.27
4f
149–151
Ethanol
72
C32H32N10O3  (604)
63.5663.57
5.335.34
23.1623.14
4g
128-129
Ethanol
68
C33H34N10O3  (618)
64.0664.04
5.545.55
22.6422.65Table 2
Spectral data of synthesized compounds.
Comp.
IR cm−1
1H-NMR δ ppm
13C-NMR
MSm/e
2a
3449–3348 (NH), 1709 and 1657 (C=O), 1603 (C=N)
2.20 (s, 3H, acetyl CH3), 2.45 (s, 3H, CH3), 3.67 (dd, 1H, [HA] CH2, J = 10.12 Hz), 4.25 (dd, 1H, [HB] CH2, J = 12.25 Hz), 4.89 (m, 1H, [HX] CH, JXA = 4 Hz, JXB = 7 Hz), 5.93 (s, 2H, NH2), 6.61–8.64 (m, 13H, Ar-H), 9.99 (s, 1H, N=CH)
479
2b
3450–3348 (NH), 1710, 1654 (C=O), 1605 (C=N)
2.24 (s, 3H, acetyl CH3), 2.40 (s, 3H, CH3), 3.70 (dd, 1H, [HA] CH2, J = 13.05 Hz), 4.23 (dd, 1H, [HB] CH2, J = 12.05 Hz), 4.90 (m, 1H, [HX] CH, JXA = 4.80 Hz, JXB = 8.43 Hz), 7.21–8.67 (m, 12H, Ar-H), 9.60 (s, 2H, NH2, D2O exchangeable), 9.109 (s, 1H, N=CH)
513 (M+), 515 (M+2)
2c
3444–3339 (NH), 1705 and 1650 (C=O), 1600 (C=N)
2.20 (s, 3H, acetyl CH3), 2.43 (s, 3H, CH3), 3.68 (dd, 1H, [HA] CH2, J = 11.05 Hz), 4.20 (dd, 1H, [HB] CH2, J = 13.05 Hz), 4.2 (m, 1H, [HX] CH, JXA = 4.45 Hz, JXB = 7.75 Hz), 7.25–8.70 (m, 12H, Ar-H), 9.55 (s, 2H, NH2, D2O exchangeable), 9.68 (s, 1H, N=CH)
513 (M+), 515 (M+2)
2d
3434–3324 (NH), 1701, 1652 (C=O), 1604 (C=N)
2.22 (s, 3H, acetyl CH3), 2.45 (s, 3H, CH3), 3.62 (dd, 1H, [HA] CH2, J = 12.03 Hz), 4.15 (dd, 1H, [HB] CH2, J = 11.1 Hz), 4.89 (m, 1H, [HX] CH, JXA = 7 Hz, JXB = 4 Hz), 7.15–8.68 (m, 12H, Ar-H), 9.45 (s, 2H, NH2, D2O exchangeable), 9.70 (s, 1H, N=CH)
513 (M+), 515 (M+2)
2e
3455–3335 (NH), 1705, 1654 (C=O), 1600 (C=N)
2.25 (s, 3H, acetyl CH3), 2.30 (s, 3H, CH3), 2.50 (s, 3H, CH3), 3.60 (dd, 1H, [HA] CH2, J = 11.80 Hz), 4.20 (dd, 1H, [HB] CH2, J = 11.1 Hz), 4.89 (m, 1H, [HX] CH, JXA = 7 Hz,JXB = 4 Hz), 6.50–7.108 (m, 12H, Ar-H), 7.1 (s, 2H, NH2, D2O exchangeable), 8.11 (s, 1H, N=CH)
493
2f
3460 (OH), 3449–3348 (NH), 1712, 1657 (C=O), 1603 (C=N)
2.22 (s, 3H, acetyl CH3), 2.47 (s, 3H, CH3), 3.65 (dd, 1H, [HA] CH2, J = 12.10 Hz), 4.25 (dd, 1H, [HB] CH2, J = 12.06 Hz), 4.109 (m, 1H, [HX] CH, JXA = 3.32 Hz,JXB = 4.32 Hz), 5.15 (s, 1H, OH, D2O exchangeable), 7.25–8.65 (m, 12H, Ar-H), 9.50 (s,  2H, NH2, D2O exchangeable), 9.69 (s, 1H, N=CH)
495
2g
3439–3325 (NH), 1715, 1654 (C=O), 1605 (C=N)
2.30 (s, 3H, acetyl CH3), 3.72 (s, 3H, –OCH3), 2.47 (s, 3H, CH3), 3.65 (dd, 1H, [HA] CH2, J = 10.10 Hz), 4.26 (dd, 1H, [HB] CH2, J = 12.06 Hz), 4.79 (m, 1H, [HX] CH, JXA = 4 Hz, JXB = 7 Hz), 6.50-7.50 (m, 12H, Ar-H), 8.23 (s, 2H, NH2, D2O exchangeable), 8.55 (s, 1H, N=CH)
586
3a
1711, 1658, 1605 (C=O), 1519 (C=N)
1.97 (s, 3H, acetyl CH3), 2.37 (s, 6H, CH3), 2.46 (s, 3H, C5–CH3), 3.31 (dd, 1H, [HA] CH2, J = 11.05 Hz), 3.69 (dd, 1H, [HB] CH2, J = 12.06 Hz), 4.63 (m, 1H, [HX] CH, JXA = 4.55 Hz, JXB = 8.43 Hz), 5.53 (s, 1H, NH (D2O exchangeable)), 7.19–8.24 (m, 13H, Ar-H), 9.99 (s, 1H, N=CH)
590
3b
3455 (NH), 1710, 1658, 1604 (C=O), 1532 (C=N)
1.72 (s, 3H, acetyl CH3), 2.30 (s, 6H, CH3), 2.40 (s, 3H, C5–CH3), 3.20–3.25 (dd, 1H, [HA] CH2, J = 12.03 Hz), 3.71 (dd, 1H, [HB] CH2, J = 12.05 Hz), 4.79 (m, 1H, [HX] CH, JXA = 3.34 Hz, JXB =  10.11 Hz), 5.55 (s, 1H, NH (D2O exchangeable)), 7.25–8.30 (m, 12H, Ar-H), 8.64 (s, 1H, N=CH)
624 (M+), 626 (M+2)
3c
3445 (NH), 1715, 1656, 1600 (C=O), 1524 (C=N)
1.69 (s, 3H, acetyl CH3), 2.30 (s, 6H, CH3), 2.45 (s, 3H, C5–CH3), 3.24–3.27 (dd, 1H, [HA] CH2, J = 12.00 Hz), 3.71 (dd, 1H, [HB] CH2, J = 12.05 Hz), 4.79 (m, 1H, [HX] CH, JXA = 7 Hz, JXB =  4 Hz), 5.58 (s, 1H, NH (D2O exchangeable)), 6.50–7.108 (m, 13H, Ar-H), 8.00 (s, 1H, N=CH)
624 (M+), 626 (M+2)
3d
3455 (NH), 1708, 1654, 1604 (C=O), 1530 (C=N)
1.75 (s, 3H, acetyl CH3), 2.35 (s, 6H, CH3), 2.49 (s, 3H, C5–CH3), 3.27–3.29 (dd, 1H, [HA] CH2, J = 11.05 Hz), 3.75 (dd, 1H, [HB] CH2, J = 12.07 Hz), 4.80 (m, 1H, [HX] CH, JXA = 7.73 Hz, JXB =  4.47 Hz), 5.57 (s, 1H, NH (D2O exchangeable)), 6.55–7.75 (m, 13H, Ar-H), 8.13 (s, 1H, N=CH)
624 (M+), 626 (M+2)
3e
3455 (NH), 1710, 1658, 1604 (C=O), 1532 (C=N)
1.68 (s, 3H, acetyl CH3), 2.36 (s, 6H, CH3), 2.40 (s, 3H, C5–CH3), 2.54 (s, 3H, CH3), 3.25–3.28 (dd, 1H, [HA] CH2, J = 13.00 Hz), 3.107 (dd, 1H, [HB] CH2, J = 12.07 Hz), 4.89 (m, 1H, [HX] CH, JXA = 5.54 Hz,JXB =  8.44 Hz), 5.56 (s, 1H, NH (D2O exchangeable)), 6.50–7.109 (m, 13H, Ar-H), 8.15 (s, 1H, N=CH)
604
3f
3550 (OH), 3457 (NH), 1713, 1658, 1604 (C=O), 1530 (C=N)
1.72 (s, 3H, acetyl CH3), 2.37 (s, 6H, CH3), 2.45 (s, 3H, C5–CH3), 3.14–3.31 (dd, 1H, [HA] CH2, J = 10.05 Hz), 3.72 (dd, 1H, [HB] CH2, J = 13.06 Hz), 4.75 (m, 1H, [HX] CH, JXA = 4.43 Hz, JXB = 8.88 Hz), 5.53 (s, 1H, NH (D2O exchangeable)), 5.60 (s, 1H, OH) (D2O exchangeable), 7.20–8.25 (m, 13H, Ar-H), 9.00 (s, 1H, N=CH)
606
3g
3455 (NH), 1708, 1654, 1604 (C=O), 1532 (C=N)
1.75 (s, 3H, acetyl CH3), 2.35 (s, 6H, CH3), 2.49 (s, 3H, C5–CH3), 3.75 (s, 3H, –OCH3), 3.27–3.29 (dd, 1H, [HA] CH2, J = 12.05 Hz), 3.75 (dd, 1H, [HB] CH2, J = 12.07 Hz), 4.80 (m, 1H, [HX] CH, JXA = 4.80 Hz, JXB =  8.48 Hz), 5.55 (s, 1H, NH (D2O exchangeable)), 6.55–7.75 (m, 13H, Ar-H), 8.15 (s, 1H, N=CH)
620
4a
3426 (NH), 1704, 1617 (C=O), 1519 (C=N)
1.18 (s, 1H, NH), 1.58 (s, 3H, acetyl CH3), 2.29 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.45 (s, 3H, C5–CH3), 3.63 (dd, 1H, [HA] CH2, J = 11.03 Hz), 3.73 (dd, 1H, [HB] CH2, J = 12.00 Hz),  4.24 (s, 6H, CH3), 5.51 (m, 1H, [HX] CH, JXA = 4.40 Hz, JXB =  11.00 Hz), 7.19–7.96 (m, 13H, Ar-H), 7.99 (s, 1H, C=NH)
10.73, 12.63, 21.95, 44.51, 60.57, 107.83, 119.80, 126.39, 129.60, 129.06, 130.12, 130.82, 139.46, 142.53, 151.29, 154.32, 155.87, 169.21
586
4b
3430 (NH), 1707, 1620 (C=O), 1594 (C=N)
1.16 (s, 1H, NH), 2.00 (s, 3H, acetyl CH3), 2.31 (s, 3H, CH3), 2.40 (s, 3H, CH3), 2.50 (s, 3H, C5–CH3), 3.70 (dd, 1H, [HA] CH2, J = 12.03 Hz), 3.107 (dd, 1H, [HB] CH2, J = 11.00 Hz),  4.30 (s, 6H, CH3), 5.55 (m, 1H, [HX] CH, JXA = 4.80 Hz, JXB =  11.42 Hz), 7.25–8.00 (m, 12H, Ar-H), 8.05 (s, 1H, C=NH)
10.56, 11.72, 21.44, 45.81, 60.51, 107.69, 119.03, 127.73, 29.72, 130.01, 131.12, 131.82, 139.92, 142.35, 150.58, 154.43, 155.62, 169.39
622 (M+), 624 (M+2)
4c
3420 (NH), 1710, 1625 (C=O), 1593 (C=N)
1.19 (s, 1H, NH), 2.05 (s, 3H, acetyl CH3), 2.34 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.52 (s, 3H, C5–CH3), 3.71 (dd, 1H, [HA] CH2, J = 14.03 Hz), 3.107 (dd, 1H, [HB] CH2, J = 12.05 Hz),  4.30 (s, 6H, CH3), 5.30 (m, 1H, [HX] CH, JXA = 4.5 Hz, JXB =  11.00 Hz), 7.30–7.12 (m, 12H, Ar-H) 8.00 (s, 1H, C=NH)
9.13, 11.79, 21.69, 42.11, 59.48, 105.39, 117.51, 125.39, 127.16, 127.76, 128.23, 128.62, 137.03, 142.35, 150.48, 153.32, 154.69, 167.36
622 (M+), 624 (M+2)
4d
3435 (NH), 1710, 1627 (C=O), 1560 (C=N)
1.16 (s, 1H, NH), 2.07 (s, 3H, acetyl CH3), 2.32 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.51 (s, 3H, C5–CH3), 3.109 (dd, 1H, [HA] CH2, J = 11.03 Hz), 3.80 (dd, 1H, [HB] CH2, J = 12.00 Hz),  4.35 (s, 6H, CH3), 5.56 (m, 1H, [HX] CH, JXA = 3.33 Hz, JXB =  9.89 Hz), 7.24–8.05 (m, 12H, Ar-H) 8.10 (s, 1H, C=NH)
8.79, 11.63, 22.41, 41.14, 58.44, 106. 43, 118.51, 124.82, 126.82, 127.62, 128.88, 128.96, 142.71, 151.51, 153.53, 154.46, 167.91
622 (M+), 624 (M+2)
4e
3420 (NH), 1710, 1625 (C=O), 1593 (C=N)
1.19 (s, 1H, NH), 2.05 (s, 3H, acetyl CH3), 2.30 (s, 3H, CH3), 2.43 (s, 3H, CH3), 2.47 (s, 3H, C5–CH3), 3.71 (dd, 1H, [HA] CH2, J = 14.03 Hz), 3.107 (dd, 1H, [HB] CH2, J = 13.05 Hz),  4.30 (s, 6H, CH3), 5.30 (m, 1H, [HX] CH, JXA = 4.00 Hz, JXB =  9 Hz), 7.30–7.92 (m, 12H, Ar-H), 7.99 (s, 1H, C=NH)
9.12, 11.81, 22.59, 41.12, 58.91, 106.34, 118.51, 124.32, 126.93, 127.71, 128.42, 128.69, 143.01, 151.01, 153.35, 154.79, 167.66
602
4f
3510 (OH), 3420 (NH), 1715, 1623 (C=O), 152 (C=N)
1.71 (s, 1H, NH), 2.10 (s, 3H, acetyl CH3), 2.35 (s, 3H, CH3), 2.45 (s, 3H, CH3), 2.49 (s, 3H, C5–CH3), 3.71 (dd, 1H, [HA] CH2, J = 12.05 Hz), 3.108 (dd, 1H, [HB] CH2, J = 15.05 Hz),  4.35 (s, 6H, CH3), 5.32 (m, 1H, [HX] CH, JXA = 4.43 Hz, JXB =  8.50 Hz), 5.50 (s, 1H, OH), 7.35–7.1 (m, 12H, Ar-H), 8.11 (s, 1H, C=NH)
9.15, 11.83, 22.58,14.12, 59.96, 107.39, 118.75, 125.39, 126.83, 127.91, 128.42, 128.84, 144.61, 151.48, 153.84, 154.86, 168.69
604
4g
3437 (NH), 1708, 1627 (C=O), 1567 (C=N)
1.67 (s, 1H, NH), 2.08 (s, 3H, acetyl CH3), 2.35 (s, 3H, CH3), 2.47 (s, 3H, CH3), 2.52 (s, 3H, C5–CH3), 3.73 (s, 3H, –OCH3), 3.80 (dd, 1H, [HA] CH2, J = 14.03 Hz), 3.2 (dd, 1H, [HB] CH2, J = 10.00 Hz),  4.37 (s, 6H, CH3), 5.56 (m, 1H, [HX] CH, JXA = 4.00 Hz, JXB = 11.00 Hz), 7.25–8.07 (m, 12H, Ar-H) 8.10 (s, 1H, C=NH)
8.47, 12.38, 23.82, 42.85, 59.36, 106.28, 118.84, 126.92, 127.84, 128.47, 129.02, 129.96, 143.44, 151.86, 154.95, 155.79, 169.61
618


## 3.  Synthesis of 2-[4-(1-Acetyl-5-alkyl/aryl-4,5-dihydro-1H-pyrazol-3-yl)phenyl]-5-methyl-4{(4-amino phenyl) methylideneamino}-2,4-dihydro-3H-1,2,4-triazol-3-one (2a–g)

To a mixture of compound (1a–g, 0.50 g, 0.001 mol) and tin (0.0015 mol), concentrated HCl (3.0 mL) was added dropwise and refluxed on water bath until the solution becomes clear (approximately 30 min). The reaction mixture was then cooled and made alkaline by the addition of aqueous NaOH solution. The amino derivative thus formed was extracted with THF. The amorphous pale brown compound (2a–g) was obtained after evaporation of the solvent and purified using methanol/ethanol.


## 4. Synthesis of 3-[2-{4-(E-(-[(1-Acetyl-5-p-a nisyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl]-5-methyl-3-oxo-4H-1,2,4-triazol-yl)imino methyl}phenylhydrazinylidene]-pentane-2,4-dione (3a–g)

The amino derivative (2a–g, 0.001 mol) was diazotized in HCl (0.40 mL) with cold solution of sodium nitrite (0.20 g, 0.001 mol in 2.0 mL of water) during a period of 45 minutes at 0–5°C. The diazotized solution was treated with acetylacetone (0.001 mol) and sodium acetate (0.005 mol) in ethanol (10 mL) during 15 minutes and further stirred for one hour. The reaction mixture was then poured into water and the solid obtained was collected by filtration and crystallized from ethanol to get intermediate 3a–g.


## 5. Synthesis of 4-{[E-{4-[(E)-(3,5-Dimethyl-1H-pyrazol-4-yl)-diazenyl] phenyl}methylidene]amino}-5-methyl-2-(1-acetyl-5,4-hydroxylphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl-2,4-dihydro-3H-1,2,4-triazol-3-one (4a–g)

A mixture of compound3a–g (0.0001 mol) and hydrazine hydrate 99% (0.004 mol) in ethanol (15 mL) was refluxed on a water bath for about 8 h. The solid obtained after cooling was filtered and washed with hot ethanol to get compounds 4a–g. Recrystallization from ethanol afforded the pale brown crystals.


## 6. Molecular Osiris Property Explorer [18, 19]

In view of the biological properties of azopyrazolobenzylidene derivatives as described in the introduction, it was thought of subjecting the newly synthesized molecules to molecular Osiris property explorer. Prediction of toxicity, drug likeness, and drug score by the computer programme Osiris provides the basis to avoid the experimental study of potentially harmful substances. In Osiris, toxicity risk assessment predicts mutagenicity, tumorigenicity, and irritating and reproductive effects. The Osiris property explorer is an integral part of Actelion’s in-house substance registration system developed by Thomas Sander at Actelion Pharmaceuticals Limited, Switzerland. The prediction process relies on a precompiled set of structure fragment that gives rise to toxicity alerts in case they are encountered in the structure currently drawn. These fragment lists were created by rigorously shredding all compounds in the database known to be active in a certain toxicity class. During shredding, the molecule was cut at every rotatable bonds leading to a set of core fragments. These in turn were used to reconstruct all possible bigger fragments being a substructure of the original molecule. Afterwards, a substructure search process determines the occurrence frequency of any fragment within all compounds of that toxicity class.


## 7. Antimicrobial Assay [20, 21]

The protocol for antimicrobial activity assay was as follows.


### 7.1. Antibacterial Assay

Agar diffusion method was used to analyze the bacteriaS. typhi and B. pyogenes. The standard antibiotic used was gentamycin. The media used was peptone (10 g), NaCl (10 g), and yeast extract (5 g) and agar in 1000 mL of distilled water. Initially, the stock cultures of bacteria were revived by inoculating in broth media and grown at 37°C for 18 h. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 18 h old cultures (100 μL, 104 cfu) and spread evenly on the plate. After 20 min, the wells were filled with different concentrations of samples. The control wells were filled with gentamycin. The sample preparation was done as 20 mg sample dissolved in 1 mL of solvent (DMSO). The stock sample concentration was 20 mg/mL. The concentrations screened were 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg. All the plates were incubated at 37°C for 18 h, and the diameter of the inhibition zones was noted, and MIC values are presented in Table 3.Table 3
Molecular Osiris property of compounds4a–g.
Entry no.
c log P
Mol Wt
Drug likeliness
Drug score
4a
6.08
580
-
1.34
0.05
4b
6.69
620
-
1.65
0.04
4c
6.69
620
-
2.10
0.04
4d
6.69
620
-
0.44
0.04
4e
6.40
600
-
3.14
0.04
4f
5.109
602
-
1.108
0.04
4g
5.4
616
-
1.84
0.04


### 7.2. Antifungal Assay

The fungi analyzed wereA. niger and C. albicans. The standard antifungal used was amphotericin B. The media used was Czapek-Dox agar: composition (g/L) sucrose (30.0 g), sodium nitrate (2.0 g), K2HPO4 (1.0 g), MgSO4.7H2O (0.5 g), KCl (0.5 g), FeSO4 (0.01 g), and agar (20 g). Initially, the stock cultures of fungi were revived by inoculating in broth media and grown at 27°C for 48 h. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 48 h old cultures (100 μL, 104 cfu) and spread evenly on the plate. After 20 min, the wells were filled with different concentrations of samples. The control wells were filled with amphotericin. The sample preparation was done as 20 mg sample dissolved in 1 mL of solvent (DMSO). The stock sample concentration was 20 mg/mL. All the plates were incubated at 27°C for 48 h, and the diameter of the inhibition zones was noted, and MIC values are presented in Table 2.


## 7.1. Antibacterial Assay

Agar diffusion method was used to analyze the bacteriaS. typhi and B. pyogenes. The standard antibiotic used was gentamycin. The media used was peptone (10 g), NaCl (10 g), and yeast extract (5 g) and agar in 1000 mL of distilled water. Initially, the stock cultures of bacteria were revived by inoculating in broth media and grown at 37°C for 18 h. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 18 h old cultures (100 μL, 104 cfu) and spread evenly on the plate. After 20 min, the wells were filled with different concentrations of samples. The control wells were filled with gentamycin. The sample preparation was done as 20 mg sample dissolved in 1 mL of solvent (DMSO). The stock sample concentration was 20 mg/mL. The concentrations screened were 0.0625, 0.125, 0.25, 0.5, 1.0, and 2.0 mg. All the plates were incubated at 37°C for 18 h, and the diameter of the inhibition zones was noted, and MIC values are presented in Table 3.Table 3
Molecular Osiris property of compounds4a–g.
Entry no.
c log P
Mol Wt
Drug likeliness
Drug score
4a
6.08
580
-
1.34
0.05
4b
6.69
620
-
1.65
0.04
4c
6.69
620
-
2.10
0.04
4d
6.69
620
-
0.44
0.04
4e
6.40
600
-
3.14
0.04
4f
5.109
602
-
1.108
0.04
4g
5.4
616
-
1.84
0.04


## 7.2. Antifungal Assay

The fungi analyzed wereA. niger and C. albicans. The standard antifungal used was amphotericin B. The media used was Czapek-Dox agar: composition (g/L) sucrose (30.0 g), sodium nitrate (2.0 g), K2HPO4 (1.0 g), MgSO4.7H2O (0.5 g), KCl (0.5 g), FeSO4 (0.01 g), and agar (20 g). Initially, the stock cultures of fungi were revived by inoculating in broth media and grown at 27°C for 48 h. The agar plates of the above media were prepared and wells were made in the plate. Each plate was inoculated with 48 h old cultures (100 μL, 104 cfu) and spread evenly on the plate. After 20 min, the wells were filled with different concentrations of samples. The control wells were filled with amphotericin. The sample preparation was done as 20 mg sample dissolved in 1 mL of solvent (DMSO). The stock sample concentration was 20 mg/mL. All the plates were incubated at 27°C for 48 h, and the diameter of the inhibition zones was noted, and MIC values are presented in Table 2.


## 8. Results and Discussion

The facile synthesis of 2-[4-(1-acetyl-5-aryl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl-2,4-dihydro-[1,2,4]-triazol-3-one from 3-[4-(5-aryl-4,5-dihydro-1H-pyrazolin-3-yl)-phenyl] sydnone by two successive ring conversions has been reported from our laboratory [4]. We thought of exploiting the synthetic utility of potential amino group in these compounds. The amino group was unreactive towards reagents like α-halogen esters, KCNS, nitrous acid, and so forth, indicating its weak nucleophilic character, which is due to strong electron-withdrawing 1,2,4-triazolin-2-one ring. In an attempt to prepare the azopyrazolobenzylidene derivatives, we thought of using the 4-(4-nitrobenzylideneamino)-2-(4-(1-acetyl-4,5-dihydro-5-aryl-1H-pyrazol-3-yl)phenyl)-5-methyl-2H-1,2,4-triazol-3(4H)-one 1a–g as starting material. The compounds 1a–g on reduction with Sn/HCl gave the compound 2a–g, and further reaction with sodium nitrite, sodium acetate, and acetylacetone yielded the 2,4-dione 3a–g (Japp-Klingemann reaction). The reaction of compound 3a–g with hydrazine hydrate afforded azopyrazolobenzylidene derivatives 4a–g (Figure 1). The mechanism of conversion of compounds 1a–g to 4a–g is presented in Figure 2.Figure 1Figure 2The mechanism involves diazotization of compound2a–g to give diazonium salt which on coupling with enolate form of acetylacetone gave diazo compound. Hydrazine hydrate was made to attack two carbonyl groups of the diazo compound nucleophilically, and subsequent dehydration of the formed intermediate gave final compounds 4a–g.The Schiff base of 2-[4-(1-acetyl-5-nitrophenyl-4,5-dihydro-1H-pyrazol-3-yl)-phenyl]-4-amino-5-methyl-2,4-dihydro-[1,2,4]-triazol-3-one 1a–g has been utilized as useful precursor for the synthesis of pyrazole derivatives. Exploitation of structure activity relationship (SAR) studies of such combination of heterocycles could lead to the development of congener with more therapeutic index than those of parent compounds. There is a considerable interest in chemotherapeutic activity of triazole and pyrazole derivatives, and hence, interesting pharmacological properties have been claimed. The synthesis of pyrazole built on the para position of phenyl ring attached to sydnone is reported from this laboratory [4]. These observations and the ease with which the compounds having two pyrazole and one 1,2,4-triazole ring encouraged us to explore present work. Though many pyrazole derivatives have been reported, it is for the first time that a molecule having two pyrazoles spaced with 1,2,4-triazole were synthesized starting from N-arylsydnones as synthons.The IR spectra of intermediate amino compounds2a–g showed bands for the asymmetric and symmetric stretching vibrations of NH2 in the range of 3455–3434 cm−1 and 3348–3324 cm−1, respectively. The carbonyl group of the 1,2,4-triazolin-2-one ring appeared as a sharp band at 1715–1705 cm−1 and that of acetyl group appeared around 1657–1650 cm−1. The C=N group showed a band at 1600–1653 cm−1. Compound 2e showed a band at 3455 cm−1 for OH stretching. The IR spectra of diones 3a–g showed a broad band at 3455–3439 cm−1 due to NH vibrations. The carbonyl group of the 2,4-dione moiety appeared at 1658–1652 cm−1, while that of the 1,2,4-triazolin-2-one ring appeared at 1715–1710 cm−1. Also, carbonyl of acetyl group appeared around 1600–1604 cm−1. The C=N group showed a band around 1519–1532 cm−1. Compound 2e showed a band at 3550 cm−1 for OH group. The IR spectra of the compounds 4a–g showed a broadband at 3437–3426 cm−1 due to NH vibrations. The IR spectra showed C=O group of 1,2,4-triazolinone ring at 1715–1704 cm−1 and C=O of acetyl group appeared at 1627–1620 cm−1. The absence of the C=O at 1609 cm−1 evidences the formation of the pyrazole ring. The C=N group showed a band at 1594–1519 cm−1.The HA and HB protons appeared as doublet due to geminal and vicinal coupling. These HA and HB differ in coupling with the Hx, and hence, they are also anisogamous. The HA proton appears as doublet of doublet in the range δ 3.109–3.31 ppm. The HB also appeared as doublet of doublet at δ 3.72–4.25 ppm. where JBA is in the range 12.25–17.23 Hz. The Hx always appeared as four-line spectrum in the range δ 3.72–5.56 ppm. The 1H NMR spectra of these compounds 2a–g showed a singlet at δ 8.23–9.50 ppm. (D2O exchangeable) for the NH2 protons. The singlets at δ 2.30–2.40 ppm. and 2.45–2.47 ppm. were observed for methyl protons of triazolinone (C5–CH3) and that of acetyl group respectively. Another singlet was observed around δ 8.55–9.99 ppm. was due to the imine proton and the aromatic protons were resonated in the range δ 7.19–8.69 ppm. The 1H NMR spectra of the compounds 3a–g showed a singlet for acetyl CH3 at δ 2.30–2.37 ppm. and a singlet at δ 1.68–1.97 ppm. for the two CH3 groups of the 2,4-dione; the C5–CH3 appeared at δ 2.45–2.49 ppm., and the imine –CH appeared at δ 8.15–9.99 ppm. The NH proton was observed at δ 5.53–5.57 ppm. (D2O exchangeable). The 1H NMR spectra of compounds 4a–g showed a singlet for acetyl CH3 at δ 1.58–2.08 ppm. and the two CH3 protons of the pyrazole ring as two singlets at δ 2.29–2.35 ppm. and δ 2.36–2.47 ppm. The C5–CH3 also appeared at δ2.45–2.52 ppm. as singlet. The compounds 2e, 3e, and 4e showed a singlet for three protons of C5–CH3 in the range 2.29–2.37 ppm. The compounds 2f, 3f, and 4f showed a singlet for –OH at 5.15, 5.60, and 5.50 ppm., respectively. Also, singlets for –OCH3 at δ 3.72, 3.75, and 3.11 ppm. were observed in the compounds 2 g, 3 g, and 4 g.


## 9. Biological Activity

Further, in the present investigation, attention has also been diverted on structure activity relationship (SAR) by way of computational studies applying Osiris property explorer and analyzed for their drug score. The synthesized title compounds were also screened for antibacterial and antifungal activities and calculated the IC 50 values (graphs are provided in the Supplementary file) (See Supplementary Material available online athttp://dx.doi.org/10.1155/2013/909706).


### 9.1. Molecular Osiris Property Explorer

It was possible to predict the biological activity of all the synthesized compounds in terms of their toxicity by employing toxicity risk assessment through Osiris property explorer which calculates on-the-fly various drug-relevant properties whenever a structure is valid. Prediction results are valued and color coded [22, 23]. Properties with high risks of undesired effects like mutagenicity or a poor intestinal absorption are shown in red, whereas a green color indicates drug-conform behavior. In the latter study, all the compounds were evaluated for the presence of various toxicity parameters like mutagenicity, tumorigenicity, reproductive affective effects, and irritation. In the Osiris explorer tool, the drug score is calculated based on the combination of toxicity risks (mutagenicity, tumorigenicity, irritation, and reproduction), drug likeness, and some physicochemical parameters such as c log P, log S (solubility), and molecular weight in one handy value than may be used to judge the compound’s overall potential to qualify for a drug (based on the Lipinski rule of five). The title compounds do not violate the Lipinski rule and they fall well in the range. The target compounds showed moderate to good drug score (0.12–0.36) that revealed their potential as safe lead compounds. The drug likeness value ranged from −3.14 to −0.44, whereas the drug score ranged from 0.04 to 0.05. The chloro substituent in the compounds 4b, 4c, and 4d was shifted to different positions (ortho, para, and meta) to study the change on the positional effect of substituent. Interestingly, these compounds showed almost the same drug score which indicates that there is no positional effect on these compounds. The compound 4g which has p-anisyl group showed less drug likeness and also the drug score was less. The compounds 4e and 4f which have electron donating groups show good drug score as depicted in Table 3. The compound 4a has also shown diversified effects based on drug likeness and drug score, but c log P values are well within the range as mentioned by the rule.


### 9.2. Antimicrobial Activity

MIC values for thein vitroantibacterial studies of the compounds 4a–g and the standard are represented in Table 4 which fall at 09.00–21.00 mg/mL for antibacterial, and antifungal lies in the range 24.00–72.00 g/mL. The antibacterial activity of the compounds 4a, 4e, 4f, and 4g against S. typhi andB. pyogenes showed excellent potencies compared to standard drugs gentamycin. The compounds 4a and4g(phenyl, p-anisyl) have shown excellent potency against S. typhi. The compounds 4a and 4f (phenyl, p-hydroxy) have shown excellent potency against B. pyogenes.The compound 4e has shown good potency against both the bacterial strains tested. The antibacterial activity may be attributed towards the electron donating groups attached to the phenyl ring. The compounds 4b, 4c, and 4d have shown moderate potencies towards antibacterial activity. The compounds 4b, 4c, and 4d have good results towards antifungal activity. The compound 4d (p-chloro) has shown excellent potency against both the fungal strains A. niger and C. albicanscompared to standard drug amphotericin. The other two compounds 4b and 4c have shown good results against both strains tested. The antifungal activity may be due to the presence of halogen group on the phenyl ring. All other compounds have shown moderate activity against the fungal strains. From the results, it is apparent that among the title compounds, all of them, have shown excellent to moderate activity at 9.00–21.00 mg/mL and 24.00–72.00 mg/mL against all the screened bacteria and fungi. The results are in well agreement with the drug scores obtained from the Osiris property explorer. MIC values of all the triazole derivates 4a–g are excellent and promising because of the presence two pyrazole rings in a single moiety making them more potent towards the bacterial and fungal strains.Table 4
Antimicrobial activities of the compounds (MIC, mg/mL)4a–g.
Entry no.
S. typhii
B. pyogenes
A. niger
C. albicans
4a
18.0
21.0
40.0
24.0
4b
12.0
10.0
65.0
42.0
4c
09.00
13.0
60.0
40.0
4d
14.0
09.00
72.0
43.0
4e
15.0
18.0
44.0
27.0
4f
16.0
22.0
38.0
25.0
4g
21.0
19.0
43.0
33.0
Gentamycin
25
25
—
—
Amphotericin
—
—
100
50
Control
DMSO
DMSO
DMSO
DMSOThe IC 50 values of antimicrobial activity revealed that the compounds (4f and 4g) and (4a and 4b) have shown very good antifungal activity against A. niger and C. albicans, respectively, whereas the compounds (4b and 4g) and 4c are potent against S. typhi and B. pyogenes, respectively.


## 9.1. Molecular Osiris Property Explorer

It was possible to predict the biological activity of all the synthesized compounds in terms of their toxicity by employing toxicity risk assessment through Osiris property explorer which calculates on-the-fly various drug-relevant properties whenever a structure is valid. Prediction results are valued and color coded [22, 23]. Properties with high risks of undesired effects like mutagenicity or a poor intestinal absorption are shown in red, whereas a green color indicates drug-conform behavior. In the latter study, all the compounds were evaluated for the presence of various toxicity parameters like mutagenicity, tumorigenicity, reproductive affective effects, and irritation. In the Osiris explorer tool, the drug score is calculated based on the combination of toxicity risks (mutagenicity, tumorigenicity, irritation, and reproduction), drug likeness, and some physicochemical parameters such as c log P, log S (solubility), and molecular weight in one handy value than may be used to judge the compound’s overall potential to qualify for a drug (based on the Lipinski rule of five). The title compounds do not violate the Lipinski rule and they fall well in the range. The target compounds showed moderate to good drug score (0.12–0.36) that revealed their potential as safe lead compounds. The drug likeness value ranged from −3.14 to −0.44, whereas the drug score ranged from 0.04 to 0.05. The chloro substituent in the compounds 4b, 4c, and 4d was shifted to different positions (ortho, para, and meta) to study the change on the positional effect of substituent. Interestingly, these compounds showed almost the same drug score which indicates that there is no positional effect on these compounds. The compound 4g which has p-anisyl group showed less drug likeness and also the drug score was less. The compounds 4e and 4f which have electron donating groups show good drug score as depicted in Table 3. The compound 4a has also shown diversified effects based on drug likeness and drug score, but c log P values are well within the range as mentioned by the rule.


## 9.2. Antimicrobial Activity

MIC values for thein vitroantibacterial studies of the compounds 4a–g and the standard are represented in Table 4 which fall at 09.00–21.00 mg/mL for antibacterial, and antifungal lies in the range 24.00–72.00 g/mL. The antibacterial activity of the compounds 4a, 4e, 4f, and 4g against S. typhi andB. pyogenes showed excellent potencies compared to standard drugs gentamycin. The compounds 4a and4g(phenyl, p-anisyl) have shown excellent potency against S. typhi. The compounds 4a and 4f (phenyl, p-hydroxy) have shown excellent potency against B. pyogenes.The compound 4e has shown good potency against both the bacterial strains tested. The antibacterial activity may be attributed towards the electron donating groups attached to the phenyl ring. The compounds 4b, 4c, and 4d have shown moderate potencies towards antibacterial activity. The compounds 4b, 4c, and 4d have good results towards antifungal activity. The compound 4d (p-chloro) has shown excellent potency against both the fungal strains A. niger and C. albicanscompared to standard drug amphotericin. The other two compounds 4b and 4c have shown good results against both strains tested. The antifungal activity may be due to the presence of halogen group on the phenyl ring. All other compounds have shown moderate activity against the fungal strains. From the results, it is apparent that among the title compounds, all of them, have shown excellent to moderate activity at 9.00–21.00 mg/mL and 24.00–72.00 mg/mL against all the screened bacteria and fungi. The results are in well agreement with the drug scores obtained from the Osiris property explorer. MIC values of all the triazole derivates 4a–g are excellent and promising because of the presence two pyrazole rings in a single moiety making them more potent towards the bacterial and fungal strains.Table 4
Antimicrobial activities of the compounds (MIC, mg/mL)4a–g.
Entry no.
S. typhii
B. pyogenes
A. niger
C. albicans
4a
18.0
21.0
40.0
24.0
4b
12.0
10.0
65.0
42.0
4c
09.00
13.0
60.0
40.0
4d
14.0
09.00
72.0
43.0
4e
15.0
18.0
44.0
27.0
4f
16.0
22.0
38.0
25.0
4g
21.0
19.0
43.0
33.0
Gentamycin
25
25
—
—
Amphotericin
—
—
100
50
Control
DMSO
DMSO
DMSO
DMSOThe IC 50 values of antimicrobial activity revealed that the compounds (4f and 4g) and (4a and 4b) have shown very good antifungal activity against A. niger and C. albicans, respectively, whereas the compounds (4b and 4g) and 4c are potent against S. typhi and B. pyogenes, respectively.


## 10. Conclusion

In conclusion, we have developed a simple and efficient method for the synthesis of novel pyrazolyl triazole derivatives through reduction of nitro group and further reacting with acetyl acetone and hydrazine hydrate. We also believe that the procedural simplicity, the efficiency, and the easy accessibility of the reaction partners give access to an array of heterocyclic frameworks. The results of the antimicrobial activity (MIC and IC 50) revealed that all of the 7 compounds showed excellent to moderate inhibition against the bacteria and fungi screened.



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*Source: 909706-2013-09-18.xml*