INTRODUCTION:

The organic compound containing pyrazole nucleus has wide applications in medicinal chemistry as well as considerable interest in the chemotherapeutic activity. Pyrazole and its synthetic analogues have been found to exhibit industrial, agricultural and some biological application ^1-5. The ring system plays an important role in many biological processes, and many therapeutic agents contain pyrazole moiety. For example some alkyl, aryl substituted pyrazoles have pronounced sedative action on the central nervous system⁶. Certain alkyl pyazoles also shown significant analgesic, antipyretic, bacteriostatic, bactericidal and fungicidal activities.^7-9Chalcones (1,3-diaryl-2-propene-1-ones) are of a high interest due to their wide spectrum of biologically activity and used in as starting materials in the synthesis of a series of heterocyclic compounds^10-13 like, isoxazoles, quinolinones, thiadiazines, benzofuranones, benzodiazepine, tetrahydro-2-chromens¹⁴, flavones etc. Moreover, these are important intermediates in many addition reactions of nucleophiles due to inductive polarization of carbonyl group at the β-position.

RESULTS AND DISCUSSION:

Our continuation work on chalcones as precursors in the synthesis of various heterocycles,^15-18 we have planned to synthesize some new chalcones according to Claisen-Schimidt condensation method¹⁹ by condensing substituted

acetophenones with 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde. The structures of the various synthesized compounds were assigned on the basis of spectral and elemental analysis. Furthermore, these pyrazole chalcones were screened for their in vitro antimicrobial activity.

The starting 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxyaldehyde was prepared by chloro formylation²⁰ of pyrazolone 1 under Vilsmeir-Haack reaction condition (Scheme-1). Initially, we carried the condensation of simple aromatic acetophenone 3 with 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxyaldehyde 2 in ethanol to give the corresponding product in good yield (Sheme-2). The reaction went to completed smoothly within 1 hr. As encouraged by this result, we turned to change the condensation of hetero acetophenones with same pyrazole carbaldehyde 2 to formed respective product in high yield (Sheme-3).

IR spectra of chalcone showed the characteristic band at 1640-1650 cm^-1due to carbonyl stretching vibration. ¹H NMR spectra of the compounds showed the characteristic doublets at δ 7.4-7.6 ppm due to α, β-unsaturated protons. However, these doublets coalesced with aromatic protons. The phenolic proton was as singlet δ11-12 ppm, while the other aromatic and aliphatic protons were appeared at expected region. The mass spectra of the compounds showed molecular ion peak were correlated with their molecular weight of that respected compound.

Scheme-1

Scheme-2

Scheme-3

Table-1 Physico-chemical data synthesized chalcone derivatives 4(a-h)

Entry	Product	R₁	R₂	R₃	Yield (%)	M.P. (°C)
1	4a	H	H	Cl	86	168
2	4b	I	H	Cl	88	174
3	4c	Br	H	Cl	89	152
4	4d	Cl	H	Cl	91	141
5	4e	H	CH₃	Cl	86	128
6	4f	I	CH₃	Cl	88	156
7	4g	Br	CH₃	Cl	90	185
8	4h	H	H	CH₃	85	132

Table-2 Physico-chemical data synthesized chalcones 4(i-j)

Entry	Product	Hetero	Yield (%)	M.P. (°C)
9	4i		90	148
10	4j		92	159

Table-3 Antimicrobial data of compounds derivatives (4a-j).

Compound	Bacteria		Fungi
Compound	E. coli	B. subtilis	A. niger	P. chrysogenum
4a	6	6	4	5
4b	6	7	2	--
4c	5	5	7	6
4d	8	8	5	5
4e	--	6	6	4
4f	5	4	5	5
4g	7	5	6	8
4h	--	6	--	4
4i	6	8	5	6
4j	2	6	2	5
Penicillin	10	14	11	12

Zone of inhibition was expressed in mm.

The results of the antimicrobial (antibacterial and antifungal) activity data are given in Table-3. Compounds 4a, 4b, 4c, 4d, 4f, 4i, and 4j were found to moderate active against all the bacterial strains. Compounds 4b, 4d and 4i were displayed good zone of inhibition against B. subtillis. Only the compound 4d was showed promising activity against both bacterial strains. The antifungal screening data revealed that, all the compounds showed moderated activity towards A. niger. Only the compounds 4c and 4g were showed good zone of inhibition for fungal strains.

Experimental:

Melting points were uncorrected determined in an open capillary tube. The purity of the products was checked by TLC on pre-coated sheets of silica gel of 0.25 mm thickness. IR spectra were recorded (KBr cm^-1) on FTIR shimadzu spectrometer. ¹H NMR spectra were recorded in DMSO-d₆ on Avance-300 MHz spectrometer using TMS as an internal standard. The mass spectra were recorded on EI-shimadzu GC- Mass spectrometer.

General procedure for synthesis of chalcone derivatives 4(a-h)

An equimolar mixture of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxyaldehyde 2 (1 mmol), substituted acetophenone 3 (1 mmol), KOH (2 mmol) were stirred in ethanol at 40 °C for 1 hr. After completion of the reaction (checked by TLC), the crude mixture was worked up in ice cold water (100 mL) and acidified with dil HCl. Solid get separated was filtered and dried. The crude product was crystallized from acetic acid. Similarly other analogues of the series were synthesized by the same procedure.

General procedure for synthesis of chalcone derivatives starting from hetero acetophenone 4(i-j)

An equimolar mixture of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carboxyaldehyde 2 (1 mmol), hetero substituted acetophenone (1 mmol), KOH (2 mmoL) were stirred in ethanol at 40 °C for 1 hr. After completion of the reaction (monitored by TLC), the crude reaction mixture was worked up in ice cold water (100 mL) and acidified with dil HCl. The solid get separated was filtered and dried. The crude product was crystallized from acetic acid to give the corresponding product. Similarly other analogues of the series were synthesized by the same procedure.

Spectroscopic data of selected compounds:

4a: IR (KBr): 3158 (-OH), 1650 (>C=O), 1595 (-C=N); ¹H NMR (DMSO-d₆): δ 2.32 (s, 3H, CH₃), δ 7.05-8.42 (m, 10H, Ar-H + CH=CH), δ 11.58 (s, 1H, OH) ppm; M.S. (m/z): 371[M⁺]; Anal. Calcd for C₁₉H₁₄O₂N₂Cl₂: C, 61.14; H, 3.78; N, 7.51%. Found: C, 61.20; H, 3.60; N, 7.56%.

4d: IR (KBr): 3165 (-OH), 1656 (>C=O), 1599 (-C=N); ¹H NMR (DMSO-d₆): δ 2.36 (s, 3H, CH₃), δ 7.12-8.35 (m, 9H, Ar-H + CH=CH), δ 11.76 (s, 1H, OH) ppm; M.S. (m/z): 407[M⁺]; Anal. Calcd for C₁₉H₁₃O₂N₂Cl₃: C, 55.98; H, 3.21; N, 6.87%. Found: C, 56.13; H, 3.11; N, 6.98%.

4g: IR (KBr): 3148 (-OH), 1652 (>C=O), 1602 (-C=N); ¹H NMR (DMSO-d₆): δ 2.26 (s, 3H, CH₃), δ 2.38 (s, 3H, CH₃), δ 7.16-8.48 (m, 8H, Ar-H + CH=CH), δ 11.98 (s, 1H, OH) ppm; M.S. (m/z): 466[M⁺]; Anal. Calcd for C₂₀H₁₅O₂N₂Cl₂Br: C, 51.53; H, 3.22; N, 6.01%. Found: C, 51.65; H, 3.38; N, 6.15%.

4j: IR (KBr): 1646 (>C=O), 1596 (-C=N); ¹H NMR (DMSO-d₆): δ 2.35 (s, 3H, CH₃), δ 7.16-8.48 (m, 15H, Ar-H + CH=CH), δ 11.56 (s, 1H, OH) ppm; M.S. (m/z): 485[M⁺]; Anal. Calcd for C₂₄H₁₈O₂N₂Cl₂S₂: C, 59.38; H, 3.74; N, 5.77%. Found: C, 59.52; H, 3.65; N, 5.89%.

Antimicrobial activity

The antimicrobial activities of the synthesized compounds 4(a-j) were determined by agar diffusion method.^21-22 The compounds were evaluated for antibacterial activity against Escherichia coli and Bacillus subtilis. The antifungal activity was studied against Aspergillus niger and Penicillium chrysogenum. The antibiotic Penicillin (10 µg/mL) was used as reference drug for both antibacterial and antifungal activity for comparison. Dimethyl sulphoxide (1%, DMSO) was used a control.

The culture strains of bacteria were maintained on nutrient agar slant at 37±0.5 °C for 24 h. The antibacterial activity was evaluated using nutrient agar plate seeded with 0.1 mL of respective bacterial culture strain suspension prepared in sterile saline (0.85%) of 10⁵ CFU/mL dilutions. The wells of 6 mm diameter were filled with 0.1 mL of compound solution at fixed concentration 10 µg/mL separately for each bacterial strain. All the plates were incubated at 37±0.5 °C for 24 h. Zone of inhibition of compounds in mm were noted.

For antifungal activity, all the culture strains of fungi maintained on potato dextrose agar (PDA) slant at 27±0.2 °C for 24-48 hrs, till sporulation. Spore of strains were transferred in to 5 mL of sterile distilled water containing 1% Tween-80 (to suspend the spore properly). The spores were counted by haemocytometer (10⁶CFU/mL). Sterile PDA plate was prepared containing 2% agar; 0.1 mL of each fungal spore suspension was spread on each plate and incubated at 27±0.2 °C for 12 hrs. After incubation well prepared using sterile cork borer and each agar well was filled with 0.1 mL of compound solution at fixed concentration 10 µg/mL. The plates were kept in refrigerator for 20 minutes for diffusion and then incubated at 27±0.2 °C for 24-28 hrs. After incubation, zone of inhibition of compounds were measured in mm, along with standard.

CONCLUSION:

In summary, we have synthesized a series of some new pyrazolo based chalcones by conventional Claisen-Schimidt condensation method. The newly synthesized compounds were screened for antimicrobial activity. It was also concluded that the compound 4d was showed promising antibacterial activity. Only the compounds 4c and 4g were showed good zone of inhibition against tested fungal strains. Now it is clear that these compounds possess antibacterial and antifungal properties.

ACKNOWLEDGMENT:

The authors are sincerely thankful to Principal, Yeshwant Mahavidyalaya, Nanded for providing laboratory facilities and also to the Director of IICT, Hyderabad for providing instrumental facilities.

REFERENCES

1. El-Kashef H, El-Emary T, Gasquet M, Timon-David P, Maldonado J and Vanelle P. Pharmazie. 2000; 55: 572.

2. Taha M, Moukha-chafiq O, Lazrek H, Vasseur J and Imbach J. Nucleosides Nucleotides Nucleic Acids. 2001; 20: 955.

3. Vicentini C, Forlani G, Manfrini M, Romagnoli C and Mares D. J. Agric Food Chem. 2002; 50: 4839.

4. Brzozonsiki Z and Saczawski F. Eur J Med Chem. 2002; 37: 709.

5. Hough L, Nalwalk J, Stadel R, Timmerman H, Leurs R, Paria B., Wang X and Dey S. J Pharmacol Exp Ther. 2002; 14: 303.

6. Vichlyaev VI, Batulin KS, Grandberg II, Kost AN. Farmakol. J. Toksikol. 1962; 25: 27.

7. Hernab E and Gabliks J. J. Cancer Chemtherapy Rept. 1961; 14: 85.

8. Rich S, Horsfall J.Phytopathology. 1952; 42: 457.

9. Potts KT, Comprehensive Heterocyclic Chemistry, Vol 5, Part 4A, 1986, Pergamon Press, Oxford.

10. Wang S, Yu G, Lu J, Xiao K, Hu Y and Hu, H. Synthesis. 2003; 487.

11. Prakash O, Kumar A, Sadana A, Prakash R, Singh PS, Claramunt MR, Sanz D, Alkoratie I, Elguero. J, Tetrahedron. 2005; 61: 6642-665.

12. Prasad RY, Rao LA, Prasoona L, Murali K, Kumar RP. Bio-Org. Med. Chem. Lett. 2005; 15: 5030-5034.

13. Raghavan S, Anuradha K. Tetrahedron Lett. 2002; 43: 5181-5183

14. Sarda SR, Puri VA, Rode AB, Dalawe TN, Jadhav WN, Pawar R.P, Arkivoc. 2007; xvi: 242.

15. Dawane BS, Vibhute YB, Konda SG, and Mali MR, Asian J. Chem. 2008; 20 (6): 4199-4204.

16. Khobragade CN, Bodade RG, Shine MS, Deepa RR, Bhosale RB and B. S. Dawane BS. J. Enzyme Inhibition and Med. Chem. 2008; 3: 341-346.

17. B. S. Dawane, S. G. Konda, R. G. Bodade, R. B. Bhosale, J .Het.Chem., 47(1), 2010, 237-241.

18. Dawane, B. S.; Konda, S. G.; Shaikh, B. M.; Bhosale, R. B, Acta Pharm., 59, 2009, 473-482.

19. Vogel’s text book of practical organic chemistry, 5^th edition Longman London, 1989.

20. Schwartz A, Bete G, Kovari Z, Boscky Z, farkas O, Matyus P. Theochem. 2000; 528:49-57

21. Fairbrother RW and Martyn G. J. Clin. Patho. 1951; 4: 374-377.

22. Gould JC and Bowie JH J. Edinb. Med., 1952; 59:178-199.

Received on 15.09.2009 Modified on 24.10.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 90-93