Synthesis and Biological Activities of Novel Arylazopyrazolones Substituted with Thiazolyhydrazone

Abstract

The 4-(1H)-benzotriazoyl methyl amino benzoate 3 was prepared by Mannich reaction of benzotriazole 1, ethyl-paminobenzoate 2 and formaldehyde. The prepared compound 3 then react with hydrazine hydrate results in the 4-(1H)-benzotriazoyl methyl amino benzoyl hydrazide 4. This compound on condensation with pre-prepared different ethyl 2-(2-(4-(4-substituted phenyl)thiazol-2-yl)hydrazono)-3-oxobutanoates 6a-d, furnished 1-(4-((1H-benzo[d] [1,2,3] triazol-1-yl)methyl amino) benzoyl)-4-(2-(4-(4-substituted phenyl)thiazol-2-yl) hydrazono)-3-methyl-1H-pyrazol-5(4H)-one 7a-d. All the compounds 7a-d was characterized by spectral studies. The compounds showed significant antimicrobial activity against various bacteria and fungi.

INTRODUCTION

Many pyrazolines and substituted pyrazolines derivatives are well known for their biological and pharmacological activities,1−5 which exhibit an anti-inflammatory,6 fungicidal,78 bactericidal, antipyretic,910 anti depressant,11−14 anticonvulsant1314 and protein kinase inhibitors.1516 These pyrazolone derivatives were investigated as thermal stabilizers for rigid PVC.1718 On the other hand, many azopyrazolone dyes have been utilized as chromogenic reagents forcolourimetric determinations1920 and as indicator for complexometric titrations.21 Also, there are some arylazopyrazolone dyes having potent antimicrobial activities. 22 Arylazopyrazoles are generally prepared by combination of aryl-azo-ethyl actoacetate derivatives and hydrazine derivatives.23−27 The benzotriazole is found as an important heterocyclic compound. They reveal valuable pharmacological properties and clinical applications.28−31 It is prime application is as corrosion inhibitors for copper or copper alloys.32 The area in which the merged molecule like aryl azo pyrazole-benzotriazole has not been developed in spite of good biological properties of both these compounds. Hence the present paper comprises the synthesis and characterization of aryl azo pyrazole-benzotriazole derivatives shown in Scheme 1.

 

EXPERIMENTAL

All chemicals used were of laboratory grade. Ethyl-4- amino benzoate, Benzotriazole and various substituted 2- amino-4-phenyl thiazole derivatives 5a-d were prepared by reported method.3334−36 Melting points were determined in open capillary tubes and were uncorrected. IR spectra were recorded in KBr pellets on a Nicolet 760D spectrometer. 1H NMR and 13C NMR spectra were recorded in DMSO with TMS as internal standard on a Bruker spectrometer at 400 MHz and 100 MHz, respectively. LCMS of selected samples taken on LC-MSD-Trap-SL_01046 instrument.

Scheme 1.Synthesis of arylazopyrazole derivatives.

Synthesis of 4-(1H)-benzotriazolyl methyl aminobenzoate 3

A mixture of 1H-Benzotriazole 1 (0.02 mole), formaldehyde (0.02mole) and ethyl-4-amino benzoate 2 (0.02mole) in ethanol (50 ml) was heated under reflux for 4 h. Subsequequently, ethanol was distilled off and the pasty mass obtained, which was triturated with petroleum ether (40− 60 ℃). The solid 4-(1H)-benzotriazolyl methylamino benzoate 3, which was isolated and dried. Yield 68%, m.p. 146−147℃. IR (ν, cm−1, KBr) 3034−3086 (C−H aromatic), 2965 (CH2), 1197−1255 (C−N), 725 (C=O of ester), 2890− 2910, 1456 (C−H). 1H NMR (400 MHz, δ, ppm, DMSOd6) 5.7(s, 2H, CH2), 3.2 (s, 1H, NH), 6.56−8.2 (m, 8H, Ar−H), 4.32 (q, 2H, −O−CH2), 1.32 (t, 3H, CH3). 13C NMR (100MHz, δ, ppm, DMSO) 114.3−149.1 (Ar−C), 75.7 (CH2), 13.9 (CH3), 62.1 (CH2), 170.4 (CO). LC-MS: m/z 305 (M+). Anal. Calcd for C16H16N4O2 (296) C, 64.85; H, 5.44; N, 18.91. Found: C, 64.8; H, 5.4; N, 18.9.

Synthesis of 4-(1H)-benzotriazolyl methyl amino benzoyl hydrazide 4

4-(1H)-benzotriazolylmethyl amino benzoate 3 (0.05mole) was refluxed with hydrazine hydrate (0.05 mole) in absolute ethanol for 8 to10 hours. It was cooled and kept overnight. The solid so obtained was filtered and recrystallized from ethanol. Yield 63%, m.p. 77−78 ℃. IR (ν, cm−1, KBr) 3034−3086 (C−H aromatic), 2965 (CH2), 1197−1255 (C−N), 1725 (C=O of ester), 3450, 1630 (NH2). 1H NMR (400 MHz, δ, ppm, DMSO-d6) 3.95 (s, 2H, NH2), 8.21−6.56 (m, 8H, Ar−H), 9.66 (s, 1H, CONH), 5.7 (s, 2H, CH2), 3.2 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 114.3− 149.1 (Ar−C), 75.7 (CH2), 170.4 (CO). LC-MS: m/z 291 (M+). Anal. Calcd for C14H14N6O (282): Calcd.: C, 59.56; H, 5.00; N, 29.77. Found: C, 59.5; H, 4.9; N, 29.7.

General Procedure for the Synthesis of Various Ethyl 3-oxo-2-(2-(substituted phenyl thiazol-2-yl) hydrazono) butanoate 6a−d

To a various substituted phenyl thiazole Amine 5a−d (0.01 mole) was dissolved in a mixture of HCl (8 ml) and water (6 ml) and cooled to 0 ℃ in ice bath. To it a cold aqueous solution of sodium nitrate (0.03 mole) was added. The diazonium salt solution was filtered into a cooled solution of ethyl actoacetate (0.01 mole) and sodium acetate (0.12 mole) in ethanol (50 ml). The resulting solid was washed with water and recrystallized from EtOH/MeOH.

Physical Properties and Spectral Data

Ethyl 2-(2-(4-(p-phenyl) thiazol-2-yl) hydrazono)-3 oxobutanoate 6a

Yield 78%; m.p. 145−147℃. IR (ν, cm−1, KBr) 3030− 3085 (C−H aromatic), 2920, 1465 (CH3, CH2), 1290 (C−N), 1725−1765 (C=O), 1148 (C−O), 3369 (N−H), 1580 (C=N), 1597, 1504 (Thiazole ring), 1550 (C−S−C). 1HNMR [400 MHz, δ, ppm, DMSO-d6) 1.34 (t, 3H, CH3), 4.29 (q, 2H, COCH2), 2.35 (s, 3H, COCH3), 7.48−7.94 (m, 6H, ArH), 7.42 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 107.8−172.6 (Ar−C), 134.6 (C=N), 14.4, 27.3 (CH3), 61.4 (CH2), 165.4, 195.4 (CO). LC-MS: m/z 329 (M+). Anal. Calcd for C15H15N3O3S (317): C, 56.77; H, 4.76; N, 13.24; S, 10.10. Found: C, 56.7; H, 4.7; N, 13.2; S, 10.0.

Ethyl 2-(2-(4-(4-bromophenyl)thiazol-2-yl) hydrazono)- 3-oxobutanoate 6b

Yield 74%; m.p. 142−143 ℃. IR (ν, cm−1, KBr) 3030− 3085 (C−H aromatic), 2920, 1465 (CH3, CH2), 1290 (C−N), 1725−1765 (C=O), 1148 (C−O), 3369 (N−H), 1580 (C=N), 1597, 1504 (Thiazole ring), 1550 (C−S−C), 550 (C−Br). 1H NMR (400 MHz, δ, ppm, DMSO-d6) 1.34 (t, 3H, CH3), 4.29 (q, 2H, C COCH2), 2.35 (s, 3H, COCH3), 7.42−7.90 (m, 5H, ArH), 7.42 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 114.8−143.6 (Ar−C), 123.8 (C−Br), 14.2, 26.9 (CH3), 61.4 (CH2), 165.4, 195.4 (CO). LC-MS: m/z 405 (M+). Anal. Calcd for C15H14N3O3BrS (394): C, 45.47; H, 3.56; N, 10.60; Br, 20.16; S, 8.09. Found C, 45.4; H, 3.5; N, 10.5; Br, 20.1; S, 8.0.

Ethyl-2-(2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)- 3-oxobutanoate 6c

Yield 75%; m.p. 141−143 ℃. IR (ν, cm−1, KBr) 3030− 3085 (C−H aromatic), 2920, 1465 (CH3, CH2), 1290 (C− N), 1725−1765 (C=O), 1148 (C−O), 3369 (N−H), 1580 (C=N), 1597, 1504 (Thiazole ring), 1550 (C−S−C), 1205 (R−O−Ar). 1H NMR (400 MHz, δ, ppm, DMSO-d6]: 3.65 (s, 3H, OCH3), 1.34 (t, 3H, CH3), 4.29 (q, 2H, COCH2), 2.35 (s, 3H, COCH3), 7.42−7.90 (m, 5H, ArH), 7.42 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 114.8− 160.6 (Ar−C), 56.2 (O−CH3), 14.2, 26.9 (CH3), 61.4 (CH2), 165.4, 195.4 (CO). LC-MS: m/z 356 (M+). Anal. Calcd for C16H17N3O4S (347): C, 55.32; H, 4.93; N, 12.10; S, 9.23. Found: C, 55.2; H, 4.9; N, 12.0; S, 9.0.

Ethyl 3-oxo-2-(2-(4-p-tolylthiazol-2-yl) hydrazono) butanoate 6d

Yield 78%; m.p. 147−149 ℃. IR (ν, cm−1, KBr) 3030− 3085 (C−H aromatic), 2920, 1465 CH3, CH2), 1290 (C−N), 1725−1765 (C=O), 1148 (C−O), 3369 (N−H), 1580 (C=N), 1597, 1504 (Thiazole ring), 1550 (C−S−C). 1H NMR (400 MHz, δ, ppm, DMSO-d6]: 2.42 (s, 3H, CH3), 1.34 (t, 3H, CH3), 4.29 (q, 2H, COCH2), 2.35 (s, 3H, COCH3), 7.32− 7.80 (m, 5H, ArH), 7.42 (s, 1H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 107.8−172.6 Ar−C), 134.6 (C=N), 14.4, 27.3 (CH3), 61.4 (CH2), 165.4, 195.4 (CO). 13C NMR (100 MHz, δ, ppm, DMSO) 108.8−156.6 (Ar−C), 14.2, 21.8, 26.9 (CH3), 61.4 (CH2), 165.4, 195.4 (CO). LC-MS: m/z 336 (M+). Anal. Calcd for C16H17N3O3S (331): C, 57.99; H, 5.17; N, 12.68; S, 9.68. Found: C, 57.9; H, 5.1; N, 12.6; S, 9.6.

General Procedure for Synthesis of 1-(4-((1H-Benzotriazol- 1-yl)methylamino)benzoyl)-3-methyl-4-(2-(substituted phenylthiazol-2-yl) hydrazono)-1H-pyrazol- 5(4H)-ones 7a−d

To a various ethyl 3-oxo-2-(2-(substituted phenylthiazol- 2-yl) hydrazono) butanoate 6a−d (0.002 mole) dissolved in glacial acetic acid (20 ml), a solution of 4-((1Hbenzotriazol- 1-yl) methyl amino) benzo hydrazide 4 (0.002 mole) in 25 ml of glacial acetic acid was added and the mixture was refluxed 10−12 h. It was then cooled and allowed to stand overnight. The resultant solid was filtered off, dried and crystallized from methanol to give desired product1-(4-((1H-benzotriazol-1-yl)methyl amino) benzoyl)- 3-methyl-4-(2-(substituted phenylthiazol-2-yl) hydrazono)- 1H-pyrazol-5(4H)-ones 7a−d.

Physical Properties and Spectral Data

1-(4-((1H-benzo[d][1,2,3]triazol-1-yl)methylamino) benzoyl)-3-methyl-4-(2-(4-phenyl thiazol-2-yl) hydrazono)- 1H-pyrazol-5(4H)-one 7a

Yield 72%; m.p. 206−208 ℃. IR (ν, cm−1, KBr) 3034− 3086 (C-Haromatic), 2920, 1465 (CH3, CH2), 1290 (C−N), 1725−1765 (C=O), 1695−1540 (C=N), 3369 (N−H), 1597, 1504 (Thiazole ring), 1550 (C−S−C). 1H NMR (400 MHz, δ, ppm, DMSO-d6) 2.35 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.80−7.82 (m, 14H, ArH), 11.62, 6.97 (s, 2H, NH).13C NMR (100 MHz, δ, ppm, DMSO) 110.8−151.6 (Ar−C), 11.6 (CH3), 59.8 (CH2), 164.3, 170.5 (CO), 172.1−129.3 (C=N). LC-MS: m/z 543 (M+). Anal. Calcd for C27H21N9O2S (535): C, 60.55; H, 3.95; N, 23.54; S, 5.99. Found: C, 60.5; H, 3.9; N, 23.5; S, 5.9.

1-(4-((1H-benzo[d] [1,2,3] triazol-1-yl) methylamino) benzoyl)-4-(2-(4-(4-bromophenyl)thiazol-2-yl) hydrazono)- 3-methyl-1H-pyrazol-5(4H)-one 7b

Yield 68%; m.p. 206−208 ℃. IR (ν, cm−1, KBr) 3034− 3086 (C−H aromatic), 2920, 1465 (CH3, CH2), 1290 (C−N), 1725−1765 (C=O), 1695−1540 (C=N), 3369 (N−H), 1597, 1504 (Thiazole ring), 1550 (C−S−C), 550 (C−Br). 1H NMR (400 MHz, δ, ppm, DMSO-d6) 2.35 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.80−7.82 (m, 14H, ArH), 11.62, 6.97 (s, 2H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 110.8−151.6 (Ar−C), 11.6 (CH3), 59.8 (CH2), 164.3, 170.5 (CO), 172.1− 129.3 (C=N), 123.8 (C−Br). LC-MS: m/z 628 (M+). Anal. Calcd for C27H20N9O2SBr (613): C, 52.77; H, 3.28; Br, 13.00; N, 20.52; S, 5.22. Found: C, 52.7; H, 3.2; Br, 12.9; N, 20.5; S, 5.1.

1-(4-((1H-benzo[d][1,2,3] triazol-1-yl) methylamino) benzoyl)-4-(2-(4-(4-methoxyphenyl)thiazol-2-yl) hydrazono)- 3-methyl-1H-pyrazol-5(4H)-one 7c

Yield 64%; m.p. 213−216 ℃. IR (ν, cm−1, KBr) 3034− 3086 (C−H aromatic), 2920, 1465 (CH3, CH2), 1290 (C− N), 1725−1765 (C=O), 1695−1540 (C=N), 3369 (N−H), 1597, 1504 (Thiazole ring), 1550 (C−S−C), 1148 (C−O), 1205 (R−O−Ar). 1H NMR (400 MHz, δ, ppm, DMSO-d6) 3.65 (s, 3H, OCH3), 2.35 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.80−7.82 (m, 13H, ArH), 11.62, 6.97 (s, 2H, NH). 13C NMR (100 MHz, δ, ppm, DMSO) 110.8−151.6 (Ar−C), 11.6 (CH3), 56.2 (O−CH3), 59.8 (CH2), 164.3, 170.5 CO), 172.1−129.3 (C=N). LC-MS: m/z 580 (M+). Anal. Calcd for C28H23N9O3S (565): C, 59.46; H, 4.10; N, 22.29; S, 5.67. Found: C, 59.4; H, 4.0; N, 22.2; S, 5.6.

1-(4-((1H-benzo[d][1,2,3] triazol-1-yl) methylamino)benzoyl)- 3-methyl-4-(2-(4-p-tolyl thiazol-2-yl) hydrazono)- 1H-pyrazol-5(4H)-one 7d

Yield 67%; m.p. 211−213 ℃. IR (ν, cm−1, KBr) 3034− 3086 (C−H aromatic), 1290 (C−N), 2920, 1465 (CH3, CH2), 1725−1765 (C=O), 1695−1540 (C=N), 3369 (N−H), 1597, 1504 (Thiazole ring), 1550 (C−S−C). 1H NMR (400MHz, δ, ppm, DMSO-d6) 2.42 (s, 3H, CH3), 2.35 (s, 3H, CH3), 5.64 (s, 2H, CH2), 6.80−7.82 (m, 13H, ArH), 11.62, 6.97 (s, 2H, NH). 13C NMR (100MHz, δ, ppm, DMSO) 110.8− 151.6 (Ar−C), 11.6, 21.7 (CH3), 59.8 (CH2), 164.3, 170.5 (CO), 172.1−129.3 (C=N). LC-MS: m/z 564 (M+). Anal. Calcd for C28H23N9O2S (549): C, 61.19; H, 4.22; N, 22.94; S, 5.83. Found: C, 61.1; H, 4.2; N, 22.9; S, 5.8.

Biological Screening

Antibacterial activities

Antibacterial activities of all the compounds were studied against Gram-positive Bacteria (Bacilus subtilis and Staphyllococcus aureus) and Gram-negative Bacteria (E. coil and Klebsiella promioe) at a concentration of 50 μg/ml by Agar cup plate method. Methanol system was used as control in this method. Under similar condition using sulphonamide as a standard for comparison carried out control experiment. The area of inhibition of zone measured in mm. Compound 7b and 7c found more active against the above microbes. Other compounds were found more active against the above microbes. The antibacterial activities all compounds are shown in Table 1.

Table 1.(Activity Index) std = Zone of Inhibition of the sample/ Zone of Inhibition of the standard.

Antifungal activity

The fungicidal activity of all the compounds was studied at 1000 ppm concentration in vitro. Plant pathogenic organisms listed in Table 2. The antifungal activities of all the samples were measured by cup plate method.36 Each of the plant pathogenic strains on potato dextrose agar (PDA) medium. Such a PDA medium contained potato 200 gms, dextrose 20 gms, agar 20 gms and water 1 liter. Five days old cultures were employed. The compounds to be tested were suspended (1000 ppm) in a PDA medium and autoclaved at 120 ℃ for 15 min, at 15 atm pressure. These medium were poured into sterile Petri plate and the organisms were inoculated after cooling the Petri plate. The percentage inhabitation for fungi was calculated after five days using the formula given below.

Percentage of inhibition = 100(X−Y)/X

Where, X: Area of colony in control plate

Y: Area of colony in test plate

The fungicidal activity all compounds are shown in Table 2.

The antifungal activity of all the compounds measured for various plant pathogens. Inspection of the result shown in Table 2 indicates that all compounds are good toxic for fungi. These compounds almost inhibit the fungi about 89%. Hence produced compounds can be employed as garden fungicides. Further work in the direction is in progress.

Table 2.Antifungal activity of the compounds 7a−d

 

RESULTS AND DISCUSSION

The compound 4 (hydrazide) has been synthesized successfully as the Mannich reaction reported previously.23−27 The synthesis of 5a−d has been performed based on the method reported.3435 From these compounds the novel compounds 6a−d have been synthesized. The compounds 6a−d reacted with 4 to give the corresponding compounds 7a−d. All the compounds were confirmed on the basis of the elemental analysis and spectroscopic investigation. IR spectrum of 4 revealed characteristic bands at 3450, 1630 (NH2) and confirmatory by 1H NMR δ3.95 (s, 2H, NH2). Further, IR spectroscopic investigation of 6a−d reveals bands at 1580 (C=N) and 1H NMR δ7.42 (s,1H, NH).

IR spectra of compounds 7a−d shows 1290 (C−N), 3369 (N−H), 1695−1540 (C=N) and 1H NMR 11.62, 6.97 (s, 2H, NH). The examination of these data reveals that the IR band and 1H NMR signals are appropriate to the correspond structure of compound.

The final structure of all compounds was confirmed by 13C NMR and LC-MS data, i.e. The compounds 7a shows the molecular ion peak m/z 543 give the molecular weight of 7a i.e. 535. All these facts confirm the structures 7a−d.

 

CONCLUSION

A number of aryl azo pyrazole derivatives have been synthesized and characterized by elemental as well as spectral analysis. All these compounds screened for antibacterial and antifungal activity. The compounds 7b and 7c exhibited more antibacterial activity comparable to the standard drug Sulphonamide. The antifungal activity of all the compounds measured for various plant pathogens. Inspection of the result indicates that all compounds are good toxic for fungi. Hence produced compounds can be employed as garden fungicides. Further work in the direction is in progress.