SOJ Materials Science & Engineering

A convenient and efficient method of halogen substituted 1-acetyl-3,5-diphenyl pyrazole derivatives were synthesized by the following two steps. In a first step, three halogen substituted acetophenones condensed with 3-hydroxy benzaldehydes through claisen-schmidt condensation reaction and which can be achieved by two solvents like ethanol and PEG-600. In a second step, the halogen substituted chalcones were condensed with hydrazine hydrate with acetic acid in the presence of piperidine as catalyst. The six synthesized compound structures were identified and confirmed by UV-Visible, FT-IR and 1H NMR and Mass spectral analysis respectively. The anti-bacterial activities of compounds have also been tested using Minimum Inhibitory Concentration (MIC) method with two different microorganisms like Staphylococcus aureus (MTCC3381) and Escherichia coli (MTCC739). The results of the studies infer that the synthesised compounds found to have excellent antibacterial activity against the selected pathogenic organisms.

Keywords: Halogen Substituted Chalcones; PEG-600; Hydrazine Hydrate; Piperidine

Flavonoids comprise a large family of plant derived polyphenolic compounds classified as flavonols, chalcones, aurones, flavanones, isoflavones, flavans, flavanonols, flavanols, and flavones differencing from each other in their structural group arrangements [1]. α,β-Unsaturated ketones display a wide range of pharmacological properties, including cytotoxicity towards different cancer cell lines [2,3], antibiotic [4], antibacterial [5], antiviral [6], anti-inflammatory [7] and hepatoprotective activities [8]. They are well known intermediates for synthesizing various heterocyclic compounds like pyrazoline and pyrimidine derivatives. A survey of literature in the recent past reveals that some pyrazole derivatives possess antibacterial [9], antiinflammatory [10] and antifungal effects [11]. In the view of the above mentioned facts and our continued interest in the synthesis of heterocyclic compounds derived from chalcone precursors [12,13], it was thought of interest to synthesize some new heterocyclic compounds containing pyrazole rings [14,15] and examine their antimicrobial properties.

Based on the careful analysis of the literature, in our present work a series of chalcones and 1-acetyl substituted pyrazoles compounds were synthesized. The synthesized compounds were characterized on the basis of UV-Visible, FTIR, ¹H NMR and mass spectral data. All the compounds were screened for their in vitro antibacterial activity against Gram positive strains (Staphylococcus aureus) and Gram negative strains (Escherichia coli) respectively.

The chemicals 3-hydroxybenzaldehyde (1) 4-flouroacetophenone (2a), 4-chloroacetophenone (2b), 4-bromoacetophenone (2c), hydrazine hydrate (3), acetic acid, PEG-600, sodium hydroxide and piperidine were obtained from Sigma Aldrich and were used as such without further purification. Silica gel (TLC and Column grade) were purchased from Merck. The solvents were purified as per the standard procedure.

FTIR spectra (KBr pellets) were measured using Alpha Bruker FTIR instrument scanning with the entire region of 4000 - 400 cm-1 with typical resolution of 1.0 cm-1. UV-Visible spectra were also recorder using Alpha Bruker UV spectrophotometer. The NMR spectra of the compounds have been recorded on Bruker AV400 spectrometer operating at 400 MHz for recording ¹H NMR spectra in DMSO solvent using TMS as internal standard. Mass spectra have been recorded on SHIMADZU spectrometer using chemical ionization technique. Melting points of all synthesized compounds have been determined in open glass capillaries on Mettler FP51 melting point apparatus and are uncorrected.

The substituted chalcones were synthesised by two methods by using ethyl alcohol and PEG-600 as solvents.

Method – A: To a stirred solution of 1 (0.0409 mol) and 2a – 2c (0.0409 mol) in ethanol (50 mL) cooled to 0°C. To this sodium hydroxide solution (3.27g in 20 mL, 0.0818 mol) were added drop wise over a period of 20 minutes. The reaction mixture was stirred at same temperature for 4-5 hours. Then the reaction mixture was kept in a refrigerator for overnight. The reaction was monitored by TLC which shows completion of reaction. The chilled reaction mixture warmed to room temperature, neutralised by using HCl (pH = 2-3), thrown out solid was stirred for a while and filtered, dried under vacuum. The purity of the product was checked by TLC (7:3, Hexane: Ethyl acetate as developing solvent) the solid obtained was recrystallized in methanol to yielded 4a- 4c.

Method – B: To a stirred suspension of 1 (0.0409 mol), 2a – 2c (0.0409 mol) and sodium hydroxide solution (3.27g in 20mL, 0.0818 mol) in PEG-600 (20 mL) heated to 65°C for 1-2 h. The completion of the reaction was monitored by TLC which shows consumption of starting material. The reaction mixture was cooled to 0°C, neutralised using HCl (pH = 2-3), thrown out solid was filtered and dried under vacuum. The purity of the product was checked by TLC (7:3, Hexane: Ethyl acetate as developing solvent) the solid obtained was recrystallized in methanol, yielded 4a-4c. Filtrate was concentrated to remove water leaving behind PEG-600. The recovered PEG-600 has been reused for further synthesis of chalcones.

To a stirred mixture of 4a-4c (0.0133 mol) in acetic acid (10 V), hydrazine hydrate (1.3 mL, 0.025 mol) and piperidine (2-3 drops) were added. The resulting reaction mixture was refluxed for 2-3 hours. The completion of reaction was monitored by TLC. The reaction mixture was poured into ice-cold water. The thrown out solid was filtered and dried under vacuum. The solid obtained was recrystallized in ethyl acetate; afford (5a-5c).

Melting Point : 140-141°C
Yield & R_f value : 94 % & 0.4324
UV –Visible (λ_max: nm) : 226 (π → π* transition), 314 (n → π* transition)
FTIR (cm^-1) : 3322 (O-H), 2966 (Aromatic C-H str), 2839 (CH), 1647 (C=O), 1581 (C=C str), 1218 (C-F chloro aromatic), 826 (C-H out plane bending)
¹H NMR (δ ppm) : 6.88 (d, J=7.6Hz, 1H), 7.32(d, J=7.6Hz, 1H), 7.84(d, J=15.6Hz, 2H), (400 MHz, DMSO-d₆) 6.74(d, J=15.6Hz, 2H), 8.26(s, 1H), 8.26-8.22(m, 1H), 7.42(d, J=8Hz, 1H), 7.26(d, 1H, J=8Hz), 9.638(s, 1H, Ar-OH) Figure 1
Mass (m/ z) : Calculated M.W: 242.07, Observed M.W: 243.1 (M+1) Figure 2.

Melting Point : 169 -170 °C
Yield & R_f value : 92% & 0.1081
UV –Visible (λ_max: nm) : 276 (π → π* transition), 316 (n → π* transition)
FTIR (cm^-1) : 3284 (O-H), 2941 (Aromatic C-H str), 1712(C=O), 1517 (C=C str), 1342 (C-F chloro aromatic), 754(C-H out plane bending)
¹H NMR (δ ppm) : 9.39(s, 1H, Ar-OH), 6.55-7.87 (m, 8H, Ar- H), 5.42-5.48(t, 1H, methine proton), (400 MHz, DMSO-d₆) 3.08 – 3.87(t, 2H, methylene proton), 2.30(s, 3H, acetyl proton) Figure 3
Mass (m/ z) : Calculated M.W 298.11, Observed M.W 299.2 Figure 4

Melting Point : 109 – 110 °C
Yield & Rf value : 90 % & 0.7187
UV –Visible (λ_max: nm) : 230 (π → π* transition), 345 (n → π* transition)
FTIR (cm^-1) : 3222 (O-H), 3029 (Aromatic C-H str), 2899 (CH), 1677 (C=O), 1580
(C=C str), 1089 (C-Cl chloro aromatic), 815 (C-H out plane bending)
¹H NMR (δ ppm) : 5.2 – 5.7 (2d, 2H, -CH=CH-), (400 MHz, DMSO-d₆) 6.9 – 8.2 (m, 8H, Ar-H), 11.09 (s, 1H, Ar-OH)
Mass (m/ z) : Calculated M.W 258.0448.402, Observed M.W 259.6

Melting Point : 176-177 °C
Yield & Rf value : 89 % & 0.2813
UV –Visible (λ_max: nm) : 206 (π → π* transition), 316 (n → π* transition)
FTIR (cm-1) : 3284 (O-H), 2938 (Aromatic C-H str), 1775(C=O), 1591 (C=C str), 1091 (C-Cl chloro aromatic), 828 (N-H out plane bending)
¹H NMR (δ ppm) : 9.34 (s, 1H, Ar-OH), 6.66-6.77 (m, 8H, Ar- H), 5.4-5.46(t, 1H, methine proton), (400 MHz, DMSO-d₆) 3.02 – 3.84(t, 2H, methylene proton), 2.50(s, 3H, acetyl proton)
Mass (m/ z) : Calculated M.W 314.08, Observed M.W 315.2

Melting Point : 124-125 °C
Yield & R_f value : 92 % & 0.6216
UV –Visible (λ_max: nm) : 230 (π → π* transition), 345 (n → π* transition)
FTIR (cm^-1) : 3320 (O-H), 3050 (Aromatic C-H str), 2899 (CH), 1666 (C=O), 1592 (C=C str), 1491 (C-Brbromo aromatic), 821 (C-H out plane bending)
¹H NMR (δ ppm) : 5.2 – 5.7 (2d, 2H, -CH=CH-),(400 MHz, DMSO-d₆) 6.9 – 8.2 (m, 8H, Ar-H), 11.09 (s, 1H, Ar-OH)
Mass (m/ z) : Calculated M.W 303.5, Observed M.W 306.6 (M+2)

Melting Point : 202-203 °C
Yield & R_f value : 90 % & 0.2973
UV –Visible (λ_max: nm) : 227 (π → π* transition), 264 (n → π* transition)
FTIR (cm^-1) : 3391 (O-H), 3065 (Aromatic C-H str), 1650(C=O), 1586 (C=C str), 1048 (C-Br bromo aromatic), 825 (C-H out plane bending)
¹H NMR (δ ppm) : 9.98 (s, 1H, Ar-OH), 6.81-7.95 (m, 8H, Ar- H), 5.46-5.52(t, 1H, methine proton),(400 MHz, DMSO-d₆) 3.02 – 3.83(t, 2H, methylene proton), 2.26(s, 3H, acetyl proton)
Mass (m/ z) : Calculated M.W 357.5, Observed M.W 359.5

As mentioned in the first step, the compounds 4a – 4c have been synthesised using two different solvents such as ethanol and PEG – 600 successively. The results obtained were encouraged in the case PEG-600 than ethanol as solvent. The usage of alcohol solvent cause environmental hazards and as well as cannot able to reuse again. In the later case it wholes good and reusable, ecofriendly and the reaction ends with shorter duration. As second step we have been synthesised 5a – 5c substituted pyrazoles. The reaction takes much more time when compared to the absence of piperidine.

Figure (1,2) revealed the 1HNMR and Mass spectra of 1-(4-fluorophenyl)-3-(3-hydroxyphenyl)prop-2-en-1-one (4a) respectively using compound 1 and 2 with ethanol or PEG-600 as solvent in the presence of sodium hydroxide has been shown in the scheme. Figure (3-4) revealed the 1HNMR and Mass spectra of 1-(3-(4-fluorophenyl)-4,5-dihydro-5-(3-hydroxyphenyl) pyrazol-1-yl)ethanone(5a) respectively using compound 4 and 3 with acetic acid in the presence of piperidine as catalyst as also been presented in the Scheme - I.

UV absorption and FTIR spectra of compound 4a has been provided a preliminary idea for the formation of product. According to the UV spectrum, presence of peaks at 226 and 314 nm clearly showed that the compound 4a has -CH=CH- group and hetero atom respectively. According to the FTIR, the presence of peak at 1580 cm-1 has clearly noticed the utilization of starting materials transforms into the product. Further, the corresponding peaks at 3322, 2966,1647 and 1581 cm-1 have been related to – OH, C-H aromatic, C-C, C=O stretching and aliphatic C=C stretching respectively in the compound 4a. The concerned mass of the compound 4a is in good agreement with the observed (243.1 m/ z) and calculated value (242.07 m/ z) were shown in Figure (2). Similarly, proton NMR strongly empowered for the formation of the product by its δ value at 9.638, 7.23 – 8.26, and 6.87 – 6.89 ppm corresponding to the O-H, Ar-H and -CH=CH- protons of compound 4a were mentioned in Figure (1).

UV absorption and FTIR spectra of compound 5a has provided a preliminary idea in confirmation the formation of product. According to the UV spectrum of compound 5a, presence of peaks at 276 and 358nm has been related to aromatic double bond and hetero atom respectively. According to the FTIR, the absence of peak at 1647 cm^-1 clearly observed the complete utilization of starting materials transformed into the product. Further, the corresponding peaks at 3284, 2941, 1712, 1517 and 828 cm^-1 for –OH, C-H aromatic stretching, C=O, C=C stretching and N-H bending vibrations respectively in the compound 5a. All such stretching and bending peaks have also been supported for the formation of the product. The concern mass of compound 5a are in good agreement with the observed (299.2 m/ z) and calculated value (298.11 m/ z) were mentioned in Figure (4). Similarly, proton NMR strongly empowered for the formation of the product by its δ value at 9.39, 6.55 - 7.87, 5.42 - 5.48, 3.08 - 3.87 and 2.30 ppm corresponding to the O-H, Ar-H, C-H, CH₂ and CO-CH₃ protons of compound 5a were mentioned in Figure (3).

The Minimum Inhibitory Concentration (MIC), which is considered as the least concentration of the sample which inhibits the visible growth of a microbe was determined by the broth dilution method. All the synthesized compounds were evaluated for their antimicrobial activity using broth dilution method to find the MIC value were tested against one gram positive and

one gram negative bacterial strains namely staphylococcus aureus and Escherichia coli respectively. The MIC values of synthesized compounds were given in the following Table 2. From the result, compound 4b and 5b found to have better against the staphylococcus aureus and Escherichia Coli than that of other synthesised compounds. This might be due the presence of chloro group with two nitrogen moieties enhances the overall performance of the compound against the pathogenic microorganisms. Similarly, fluoro and bromo substituted compound has some activity against the bacteria. However, these activities were less pronounced than the chloro-substituted compounds. Hence, the order of the antibacterial activity has been given in the following order:

Chloro substituted > Fluoro substituted > Bromo substituted

In the present work, 1-acetyl-3,5-diphenyl substituted pyrazole derivatives have been synthesized successfully by

Claisen-Schmidt reaction. In a first step substituted chalcones were synthesised by using ethanol and PEG-600 as solvent in presence of NaOH as catalyst. This protocol found to be a new protocol and green synthesis which offers non-toxic, inexpensive, water soluble and recyclable. In a second step, the chalcones were condensed with hydrazine hydrate in the presence of catalytic amount of piperidine. Use of piperidine as catalyst found to have lesser reaction time (less than an hour) whereas the same the reaction proceeds more than eight hours to complete in the absence of catalyst.

The chemical structures of compounds 4a – 4c and 5a – 5c have been confirmed using various spectral techniques viz., FTIR,

UV-Visible, Mass and 1H-NMR spectra and were found to be in agreement with the chemical structures expected.

The microbial activities halogen substituted chalcones and 1-acetyl substituted pyrazole derivatives were checked against the two microbes Staphylococcus aureus and Escherichia coli. The report of antimicrobial activity clearly showed that, the synthesized compounds of 4b and 5b has excellent activities towards the tested bacterial strains of both gram positive and gram negative. This might be due to the presence of chlorine and two nitrogen moieties in the molecule.

1. Grotewold E. The Science of Flavonoids. Springer. New York. 2006.
2. Konieczny MT, Konieczny W, Sabisz M, Skladanowski A, Wakieć R, Augustynowicz-Kopeć E, et al. Acid-catalyzed synthesis of oxathiolone fused chalcones. Comparison of their activity toward various microorganisms and human cancer cells line. Eur J Med Chem. 2007;42(5):729-733.
3. Kumar D, Kumar NM, Akamatsu K, Kusaka E, Harada H, Ito T. Synthesis and biological evaluation of indolyl chalcones as antitumor agents. Bioorg Med Chem Lett.  2010;20(13):3916-3919. doi: 10.1016/j.bmcl.2010.05.016.
4. Ducki S, Forrest R, Hadfield JA, Kendall A, Lawrence NJ, McGown AT, et al. Potent antimitotic and cell growth inhibitory properties of substituted chalcones. Bioorg Med Chem Lett. 1998;8(9):1051-1056.
5. Pandeya SN, Sriram D, Nath G, DeClercq E. Synthesis, antibacterial, antifungal and anti-HIV activities of Schiff and Mannich bases derived from isatin derivatives and N-[4-(4'-chlorophenyl)thiazol-2-yl] thiosemicarbazide. Eur J Pharm Sci. 1999;9(1):25-31.
6. Biradar JS, Sasidhar BS, Parveen R. Synthesis, antioxidant and DNA cleavage activities of novel indole derivatives. European Journal of Medicinal Chemistry. 2010;45(9):4074-4078.
7. Ganapathi M, Jayaseelan D, Guhanathan S. Microwave assisted efficient synthesis of diphenyl substituted pyrazoles using PEG-600 as solvent - A green approach. Ecotoxicol Environ Saf. 2015;121:87-92. doi: 10.1016/j.ecoenv.2015.05.002.
8. Matthews L, R Lo A, Forgacs,Celius L, Macpherson L, Harper P, Zacharewski T. Dioxin-dependent recruitment of AHR to promoter regions in mouse liver. Toxicol Lett. 2010;196(3):213-217.
9. Kumar Y, Green R, Wise DS, Wotring LL, Townsend LB. Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antifilarial and antitumor agents. 2. 2-Arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole. J Med Chem. 1993;36(24):3849-3852.
10. Shoman ME, Abdel-Aziz M, Aly OM, Farag HH, Morsy MA. Synthesis and investigation of anti-inflammatory activity and gastric ulcerogenicity of novel nitric oxide-donating pyrazoline derivatives. Eur. J. Med. Chem. 2009;44:3068-3076.
11. Cherkupally SR, Dasari CR, Vookanti Y, Adki N. Synthesis and antimicrobial study of bis-[thiadiazol-2-yltetrahydro-2H-pyrazolo[3,4-d][1,3]thiazole]methanes. Org. Commun. 2010;3(3):57-69.
12. Jayaseelan D, Ganapathi M, Guhanathan S. Microwave assisted efficient synthesis of diphenyl substituted pyrazoles using PEG-600 as solvent-A green approach. Ecotoxicol Environ Saf. 2015;121:87-92. doi: 10.1016/j.ecoenv.2015.05.002.
13. Turan-Zitouni G, Chevallet P, Kiliç FS, Erol K. Synthesis of some thiazolyl-pyrazoline derivatives and preliminary investigation of their hypotensive activity. Eur J Med Chem. 2000;35(6):635-641.
14. Ganapathi M, Jayaseelan D, Guhanathan S. Synthesis, Characterization and antimicrobial activities of 1-(3-(4-chlorophenyl)-4,5-dihydro-5-(4-hydroxyphenyl)pyrazol-1-yl)ethanone. International Journal of Frontiers in Science and Technology.  2015;3(3):1-16.  
15. Asiri AM, Khan SA. Synthesis and anti-bacterial activities of a bis-chalcone derived from thiophene and its bis-cyclized products. Molecules. 2011;16(1):523-531. doi: 10.3390/molecules16010523.