SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME NEW4-(SUBSTITUTED\ARYL)-((1, 3–DIPHENYL-1H-PYRAZOLE-4-YL) METHYLENEAMINO)-1, 5-DIMETHYL-2-PHENYLPYRAZOLILDIN-3-ONE DERIVATIVES

SYNTHESIS AND ANTIMICROBIAL ACTIVITY OF SOME NEW4-(SUBSTITUTEDARYL)-((1, 3–DIPHENYL-1H-PYRAZOLE-4-YL) METHYLENEAMINO)-1, 5-DIMETHYL-2-PHENYLPYRAZOLILDIN-3-ONE DERIVATIVES

Jabali J. Vora ¹, Akash R.  Patel *¹, Dinesh R. Patel ² and Sandip dholakia ³

Department of Chemistry, Hemchandracharya North Gujarat University ¹, Patan, Gujarat, India

The Urban Science College ², Mehsana, Gujarat, India

Shankersinh vaghelabapu institute of Pharmacy ³, Gandhinagar, Gujarat, India

ABSTRACT: Schiff Base derivatives are important class of compounds. They possess different types of Biological activities like antibacterial, antiviral, anti HIV, antifungal etc. Schiff base derivatives are prepared by the condensation of aldehyde and amine and these compounds are characterized by chemical and instrumental methods. Their important biological properties have been investigated

INTRODUCTION:Hydrazones, possessing an azomethine –NHN=CH- proton, constitute an important class of compounds for new drug development. Therefore, many researchers have synthesized these compounds as target structures and evaluated their biological activities. Hydrazones are synthesized by heating the appropriate substituted hydrazine /hydrazides with aldehydes and ketones in solvents like ethanol, methanol, butanol, glacial acetic acid, ethanol-glacial acetic acid. These are well known intermediates for the preparation of oxadiazolines, azetidinones, thiazolidinones and many other derivatives.

Hydrazones exhibit a wide range of pharmacological activities like Anti-cancer ¹, Anti-malarial ², and Anti-tubercular ³ etc.

A large number of substituted pyrazole derivatives are prepared and tested for variety of biological activities like Anti HIV ⁴, Antiviral ⁵, Ant parasitic ⁶ etc. Schiff base of 4-amonoantipyrine have a variety of applications in biological, clinical, analytical and pharmacological areas. Studies of a new kind of chemotherapeutic Schiff bases are now attracting the attention of biochemists. Earlier work reported that some drugs showed increased activity.

Deoxyriboncleic acid (DNA) is the primary target molecule for most anticancer and antiviral therapies according to cell biologists.

MATERIALS AND METHODS: The compounds N-[(1, 3 –diphenyl-1H-pyrazol-4-yl)methylene- 4H-(1, 2, 4 triazol- 3-amine (I_a-m) were obtained by following preparation method(I-a)  (figure 1).

FIG. 1: SCHEME FOR SYNTHESIS OF PREDICTED COMPOUNDS

 Synthesis of n- phenylamino- α- methyl- phenyl azomethine: A mixture of phenyl hydrazine (1.08gm, 0.01M) and aceptophenone (1.20gm, 0.01M) in absolute ethanol was refluxed in water bath for 4 hrs in presence of 1ml glacial acetic acid. Product obtained after cooling was crystallized from absolute ethanol ⁷.

Yield, 1.8gm (90%), M.P.: 64°C. (C₁₄H₁₄N₂; Calculated: C, 80.00; H, 6.66; N, 13.37%; Found: C, 79.92; H, 6.64; N, 13.34%).

This typical experimental procedure was followed to prepare other analogs of this series.

Synthesis of 1, 3- diphenyl- 1h- pyrazole- 4- carbaldehyde: N-Phenylamino-α-methyl-phenyl azomethine (0.84gm, 0.004M) was added in a mixture of Vilsmeier – Haack reagent (prepared by drop wise addition of 1.2ml POCl₃ I ice cooled 10ml DMF) and refluxed for 6hrs. The reaction mixture was poured into crushed ice followed by neutralization using sodium bicarbonate. Crude product was isolated and crystallized from methanol ⁷.

Yield, 2.16gm (87%), M.P.: 120°C. (C₁₆H₁₂N₂O; Calculated: C, 77.42; H, 4.48; N, 11.29%; Found: C, 77.39; H, 4.80; N, 11.28%). Exactly similar experimental procedure was followed to prepare other analogs of this series.

4-((1, 3–diphenyl-1h-pyrazol-4-yl) methyleneamino)-1, 5-dimethyl-2-phenylpyrazolildin-3-one: A mixture of 1, 3-diphenyl-1H-pyrazole-4-carbaladehyde (2.48gm, 0.01M) and 4-Amino Antipyrine (2.03gm, 0.01M) was taken in absolute ethanol and few drops of glacial acetic acid was added. Then the mixture was refluxed for 6h on water bath. The separated solid was filtered, washed and recrystallized from ethanol ⁸.

M.P. 127°C, Yield 89%, and C₂₇H₂₅N₅O; Calculated: C, 74.39; H, 5.78 N, 16.08; Found: C, 74.30; 1H, 5.75; N, 16.05%).

The same experimental procedure was utilized to prepare other analogs of this serial (Ia-m). Their physical constant data are given in Table 1.

TABLE 1: PHYSICAL AND CHEMICAL CHARACTERISTICS OF 4-((1, 3-DIPHENYL-1H-PYRZOL-4YL) METHYLENEAMONO)-1, 5-DIMETHYL-2-PHENYLPYRAZOLIDIN-3-ONE

y=yellow, w=white

The same experimental procedure was utilized to prepare other analogs of this series (Ia-m). The purity of synthesized compounds was checked by thin layer chromatography (TLC) on silica gel plate using ethyl acetate: cyclohexane (70: 30). Melting points were determined by open capillary method and are uncorrected. IR spectra are recorded on FT-IR Perkin-Elmer spectrophotometer RXI using KBr disc. ¹H NMR spectra are recorded on in CDCL₃ ON a Bruker DRX-400 MHz using TMS as inter standard. The chemical shifts are reported as parts per million (ppm) and ESI MS were determined on Discovery Make Thermo Spectrometer.

The characterization data of compounds (Ia-m)aredescribed in Table 1.

RESULTS AND DISCUSSION: The synthesis of 4-((1,3–diphenyl- 1H- pyrazol- 4-yl) methyleneamino)-1, 5-dimethyl-2-henyl  pyrazolildin -3-one derivatives (Ia-m)involved the reaction between appropriate 1, 3 –diphenyl- 1H-pyrazole- 4- carbaldehyde (I_a-m) and 4-Amino Antipyrine as described in the general procedure.

IR spectra showed the C=O stretching vibration peak at 1672.05 cm^-1 and The Schiff base also confirmed by an intense band of  C=N around 1408.49cm^-1. The other peaks of IR spectra also prove the structure of hydrazones derivatives. The nuclear magnetic resonance spectra (¹H NMR) showed the amine proton (N=C-H) at 9.7896 ppm and (N-CH) at 8.4761 ppm and the mass spectrum of comp. (I-a) shows the [M] ⁺ molecular ion (m/z = 435) a base peak.

Many times, due to collision of secondary ion with sample molecular ion, [M]⁺ or [m+1]⁺ is formed and is sometimes prominent base peak, which undergoes less fragmentation. As per the nitrogen rule, it must have even molecular weight, which is 435.12 (isotopic mass). 436 peak is 31 % of 435 [M] ⁺ peak indicating the presence of 28 carbon atoms (confirmed by the rule of thirteen). Fragments showed peaks at m/z 204 [(base peak), 231] and m/z     etc.

Antimicrobial Activity (Table 2 & 3): Antimicrobial activity testing was carried out by using Agar cup method. Each purified compound was dissolved in dimethyl sulfoxide (DMSO), sterilized by filtration using sintered glass filter and stored at 4⁰C. All the synthesized compounds were screened for their antibacterial and antifungal activities against the E. coli, P. auregenosa, S. aures, S. pyogenus and the fungi C. albicans, A. niger, and C. albicans.

The compounds were tested at 250, 100, 50 and 25 concentration using nutrient agar tubes. The highest dilution showing at least 99 % inhibition is taken as MBC (minimal bactericidal concentration). Control experiments were carried out under similar condition by using gentamycin, ampicillin and chloramphenicol for antibacterial activity and nystatin and greseofulvin for antifungal activity as standard drugs.

• E. coli:   In compression the standard drug ampicillin compound v-a, v-h,     v-k, v-I, v-m   Shows equal or higher antibacterial activity and the compound v-a, v-e, v-k, v-m possess equal antibacterial activity also compared to chloramphenicol.
• P. aeruginosa:   Compounds no v-h, v-j and v-m exhibit equal or greater antibiotics activity compare to ampicillin at 250µg/ml concentrations. Compounds v-h and v-m shows equal antibacterial activity against chloramphenicol at 250 µg/ml concentrations.
• S. aureus:    The compounds v-a, v-d, v-l shows equal or higher antibacterial activity compared to ampicillin at 250µg/ml concentrations. Compound v-g is equal antibacterial activity against chloramphenicol.
• S. Pyrognes:  compounds v-b, v-g, and v-m possess higher or equal antibiotics activity compared to ampicillin at 250µg/ml concentrations. Compounds v-j and v-k exhibit equal antibacterial activity against chloramphenicol.

Antifungal activity (Table 4 & 5): The synthesized heterocyclic compound are do not exhibit antibacterial activity at law concentrations (5µg/ml) when antifungal studies was carried with A. niger (Aspergillus), it was observed that no compounds shows equal antifungal activity as the standard drug greseofulvin. Compound v-b shows equal antifungal activity compared to nystatin, when the antifungal study was carried out with C. albicans.

The compounds v-f, v-g, v-k and v-I shows equal or higher antifungal activity compared to greseofulvin. In compression the standard nystatin compound v-f and v-g shows equal antifungal activity.

Spectral study of 4-((1, 3–diphenyl-1H-pyrazol-4-yl) methyleneamino)-1, 5- dimethyl- 2- phenylpyrazolildin-3-one (i-a) [isotopic weight = 435.12 g].

IR (KBr) cm^-1:1408.49 (C=N Stretching of Schiff base), 2921.64 (C-H Str. Asym.), 1356.02    (C-H def. sym.), 3124.68 (Ar C-H Stretching), 1591.68 (C=N Str. Of pyrazole ring), 1672.05 (C=O Str antipyrine moiety) and 1299.54 (C-N Str antipyrine moiety).

H NMR (CDCI₃) δ (ppm): 9.7896(1 H, –CH=N-), 8.4824 (1H, pyrazol ring), 8.4761 and 7.1851(2H, antipyrine ring) 7.2144-7.7695 (15 H, Ar-H), 2.3693(3H, N-CH3), 1.1318-1.1790(3H, C-CH3).

Mass Spectra (m/z) = 435 (M) ⁺, 436(M+2), 204,231.

TABLE 2: ANTIBACTERIAL ACTIVITY TABLE

Zone of Inhibition in milli meter, 5, 25, 50,100,250 = various concentration

TABLE 3: ANTIBACTERIAL ACTIVITY TABLE

Zone of Inhibition in milli meter, 5, 25, 50,100,250 = various concentration

 TABLE 4: ANTIFUNGAL ACTIVITY TABLE

Zone of Inhibition in milli meter, 5, 25, 50,100,250 = various concentration

TABLE 5: ANTIFUNGAL ACTIVITY TABLE

Zone of Inhibition in milli meter, 5, 25, 50,100,250 = various concentration

CONCLUSION: Some of the compounds synthesized shows promising antimicrobial activity in particular, the compound v-h v-m shows promising antimicrobial activity. It is there for important to anticipate that appropriate molecular mini-pulsation of these compounds may result in the compounds with potent antimicrobial action.

However, certain structural alterations did not increase antimicrobial activity and working ahead in that direction may give quite promising results.

ACKNOWLEDGEMENT: The authors are thankful to the Director, SAIF, Punjab University, Chandigarh, for providing the IR, ¹H NMR, MASS spectral data and are also thankful to Mr.  Dhansukh Rajani (Microbiologist), Micro care Laboratory, Surat, for antimicrobial screening. We are also thankful to Trusty, Shri Shankarsinh Vaghela Bapu, Jayendrakumariba, Shri c. J. Josh and my principal Shri Milan Satia, Shankarsinh Vaghela Bapu Institute of Pharmacy, Gandhinagar, Gujarat, India.

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