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CA: Candida albicans, AF: Aspergillus fumigatus, TM: Trichophy- ton mentagrophytes, PM: Penicillium marneffei. Table 4 MIC and MFC of compounds 3 and 5.
Journal of Saudi Chemical Society (2013) 17, 237–243

King Saud University

Journal of Saudi Chemical Society www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis, characterization and antimicrobial evaluation of novel halopyrazole derivatives Zeba N. Siddiqui Asad U. Khan b a b

a,*

, Farheen Farooq a, T.N. Mohammed Musthafa aAnis Ahmad b,

Department of Chemistry, Aligarh Muslim University, Aligarh 202 002, India Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202 002, India

Received 9 February 2011; accepted 29 March 2011 Available online 3 April 2011

KEYWORDS 5-Chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde; Pyrazoles; Antibacterial activity; Antifungal activity

Abstract Two novel halopyrazole derivatives (3, 5) were synthesized from 5-chloro-3-methyl-1phenylpyrazole-4-carboxaldehyde (1) using appropriate synthetic routes. Newly synthesized compounds were characterized using elemental analysis, spectral data (IR, 1H NMR, 13C NMR and mass spectrometry) and were evaluated for their in vitro antimicrobial activity. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) were determined for the test compounds as well as for reference standards. The investigation of antimicrobial screening revealed that compounds (3, 5) showed good antibacterial and antifungal activities, respectively. ª 2011 King Saud University. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license.

1. Introduction In recent years, the number of life-threatening infectious diseases caused by multi-drug resistant Gram-positive and Gram-negative pathogen bacteria has reached an alarming level in many countries around the world (Berber et al., 2003; * Corresponding author. Tel.: +91 09412653054. E-mail address: [email protected] (Z.N. Siddiqui). 1319-6103 ª 2011 King Saud University. Production and hosting by Elsevier B.V. Open access under CC BY-NC-ND license. Peer review under responsibility of King Saud University. doi:10.1016/j.jscs.2011.03.016

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Mitscher et al., 1999). Morbidity and mortality because of enteric bacterial infection are the major health problems in some areas like the Indian subcontinent, portions of South America and tropical fraction of Africa (Qadri et al., 2005; Devasia et al., 2006). Every year millions of people are being killed by some or the other Gram-positive and Gram-negative strains of bacteria. These bacteria mostly lead to food poisoning, rheumatic, salmonellosis and diarrhea (Khan et al., 2008). Thus, antibiotics provide the main basis for the therapy of microbial (bacterial and fungal) infections. However, overuse of antibiotics has become the major factor for the emergence and dissemination of multi-drug resistant strains of several groups of microorganisms (Harbottle et al., 2006). Furthermore, the pharmacological drugs available are either too expensive or have undesirable side effects or contraindications (Berger, 1985). Thus, in light of the evidence of rapid global spread of resistant clinical isolates, the need to find new antimicrobial agents is of paramount importance.

238 Pyrazole derivatives, important heterocyclic analogs, are promising candidates in this context as they possess wide spectrum of biological activities. They are extensively studied and used as antimicrobial agents (Menozzi et al., 2004; Tandon et al., 2005; Agarwal et al., 2006; Akbas and Berber, 2005). Several pharmaceutical drugs including celecoxib (Penning et al., 1997) and rimonabant (Deng and Mani, 2008) utilize the pyrazole as their core molecular entity (Katritzky et al., 2001; Deng and Mani, 2006). Many pyrazole derivatives are reported to have the broad spectrum of biological activities, such as anti-inflammatory (Bekhit et al., 2008), antifungal (Prakash et al., 2008), herbicidal (Ohno et al., 2004), cytotoxic (Vera-Divaio et al., 2009) and A3 adenosine receptor antagonists (Baraldi et al., 2003). In particular, 5-chloropyrazole derivatives are reported to show potential antimicrobial (Kumar et al., 2009), analgesic and anti-inflammatory activities (Girisha et al., 2010). Numerous synthetic pyrazole derivatives are also found used in various pharmaceuticals, agrochemicals, photographic and other applications. Such examples of important pyrazole derivatives are natural products (S)-pyrazolylalanine, pyrazomycin and synthetic compounds sildenaffil, ionazolac, difenamizole, mepirizole etc. (Riyadh et al., 2010). In search of more potent antibiotics, herein we report the one pot, efficient and simple methodology for the synthesis of novel halopyrazole derivatives in good yields by using 5chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde as starting material. The synthesized compounds were screened for their in vitro antibacterial and antifungal activities.

2. Experimental 2.1. General Melting points were taken in Reichert Thermover instrument and are uncorrected. The IR spectra were recorded on Perkin Elmer RXI spectrometer in KBr. 1H NMR spectra were recorded on Bruker DRX-300 and Bruker Avance II-400 spectrometer using tetra methyl silane (TMS) as an internal standard. 13C NMR spectra were recorded on a Bruker DRX 400 Spectrometer (100 MHz) with DMSO. Mass spectra were recorded on JEOL-Accu TOF JMS-T100LC DART-MS spectrometer. Microanalytical data were collected using Carlo Erba analyzer model 1108. The purity of all compounds was checked by TLC on glass plates (20 · 5 cm) coated with silica gel (E-Merck G254, 0.5 mm thickness). The plates were run in chloroform–methanol (4:1) mixture and were visualized by iodine vapors. The compounds 1 (Pawar and Patil, 1994) and 2 (Jursic and Neumann, 2001) were synthesized from 3methyl-1-phenylpyrazole-5-one, 1,3-dimethylbarbituric acid, respectively by reported procedures. 2.2. Synthesis of compound 3 To a well stirred solution of 5-acetyl-1,3-dimethylbarbituric acid (4.52 mmol) in ethanol (12 ml) containing pyridine (0.5 ml), 5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde (4.52 mmol) was added in the portion. The reaction mixture was then stirred at room temperature for 1 h. Bromine (4.52 mmol) was then added dropwise to the vigorously stirred solution over a period of 20 min. After complete addition of Br2 the reaction mixture was further stirred for another 3 h.

Z.N. Siddiqui et al. The monobromoderivative 3 was precipitated, filtered off, and washed with 15 ml of ether to remove the excess of bromine. Further purification was made by recrystallization from the chloroform–methanol (4:1v/v) mixture. 2.2.1. (Z)-2-Bromo-3-(5-chloro-3-methyl-1-phenylpyrazol-4yl)-1-(1,3-dimethyl-2,4,6-pyrimidinetrione-5-yl)prop-2-ene-1one (3) Pale yellow crystals; m.p. 180–182 C, isolated yield 87%, IR (KBr, cm 1): 1730 (C‚O), 1689 (C‚O),1623 (C‚C); 1H NMR (400 MHz, CDCl3): dH 2.61 (s, 3H, CH3), 3.32 (s, 3H, N–CH3), 3.48 (s, 3H, N–CH3), 7.46–7.62 (m, 5H, Ar-H), 7.81 (s, 1H, Ha). 13C NMR (100 MHz, DMSO): dc 14.27, 27.73, 29.28, 38.94, 108.52, 124.53, 124.99, 128.40, 128.67, 128.79, 137.13, 148.57, 157.13, 165.12, 174.42. ESI–MS m/z: 480.6 (M+). Anal. Calcd. For C19H16N4O4BrCl: C, 47.57; H, 3.35; N, 11.67. Found: C, 47.42; H, 3.49; N, 11.56. 2.3. Synthesis of compound 5 A mixture of 5-chloro-3-methyl-1-phenylpyrazole-4-carboxaldehyde (4.52 mmol), 2,4-dinitrotoluene (4.52 mmol) and pyridine (0.3 ml) in ethanol (12 ml) was refluxed in a heating mantle for about 2.5 h. On completion of reaction (as checked by TLC), the reaction mixture was poured into 25 ml ice cold water, acidified with conc-HCl. The solid, thus, obtained was filtered washed with water, methanol, dried and purified by recrystallization from chloroform–methanol (3:2 v/v) mixture. 2.3.1. 5-Chloro-4-[2-(2,4-dinitrophenyl)-vinyl]-3-methyl-1phenyl-1H-pyrazole (5) Yellow crystals; m.p. 122–125 C, isolated yield 84%, IR (KBr, cm 1): 1597 (C‚C), 1535 (C‚N), 1345 (C–N). 1H NMR (300 MHz, CDCl3): dH 2.54 (s, 3H, CH3), 7.20 (d, 1H, J = 16.5 Hz, Ha), 7.35–7.57 (m, 5H, Ar–H), 7.66 (d, 1H, J = 16.5 Hz, Hb), 8.00 (d, 1H, J = 8.7 Hz, Hc), 8.45 (d, 1H, J = 8.7 Hz, Hd), 8.85 (s, 1H, He). 13C NMR (100 MHz, DMSO): dc 14.65, 115.11, 117.54, 120.84, 120.99, 124.98, 125.28, 128.72, 129.28, 129.39, 139.37, 147.12, 149.05, 151.85. ESI–MS m/z: 384.12 (M+). Anal. Calcd. For C18H13N4O4Cl: C, 56.19; H, 3.40; N, 14.56. Found: C, 56.06; H, 3.53; N, 14.51. 2.4. Antibacterial studies The newly synthesized compounds were screened for their antibacterial activity against Streptococcus pyogenes (clinical isolate), Methicillin resistant Staphylococcus aureus (MRSA +ve), Pseudomonas aeruginosa (ATCC-27853), Klebsiella pneumoniae (clinical isolate) and Escherichia coli (ATCC25922) bacterial strains by disk diffusion method (Cruickshank et al., 1975; Collins, 1976). A standard inoculums (1– 2 · 107 c.f.u./ml 0.5 McFarland standards) was introduced onto the surface of sterile agar plates and a sterile glass spreader was used for even distribution of the inoculums. The disks measuring 6 mm in diameter were prepared from Whatman no. 1 filter paper and sterilized by dry heat at 140 C for 1 h. The sterile disks previously soaked in a known concentration of the test compounds were placed in nutrient agar medium. Solvent and growth controls were kept. Ciprofloxacin (30 lg) was used as positive control while the disk poured in DMSO was used as negative control. The plates were inverted and incubated for 24 h at 37 C. The susceptibility was assessed

Synthesis, characterization and antimicrobial evaluation of novel halopyrazole derivatives Table 1

239

Antibacterial activity of compounds 3, 5 and positive control ciprofloxacin.

Compounds

Diameter of zone of inhibition (mm) Gram-positive bacteria

3 5 Standard DMSO

Gram-negative bacteria a

S. pyogenes

MRSA

P. aeruginosa

K. pneumoniae

E. coli

19.4 ± 0.4 19.2 ± 0.2 22.5 ± 0.4 –

19.4 ± 0.4 18.2 ± 0.2 21.5 ± 0.4 –

26.3 ± 0.4 25.2 ± 0.2 31.0 ± 0.2 –

14.6 ± 0.2 14.1 ± 0.4 19.0 ± 0.2 –

18.7 ± 0.4 17.5 ± 0.2 27.0 ± 0.4 –

Positive control (Standard); ciprofloxacin and negative control (DMSO) measured by the Halo Zone Test (unit, mm). a Methicillin resistant Staphylococcus aureus (MRSA +ve).

Table 2

MIC and MBC results of compounds 3, 5 and positive control ciprofloxacin.

Compounds

Gram-positive bacteria S. pyogenes

3 5 Standard

Gram-negative bacteria P. aeruginosa

K. pneumoniae

E. coli

MIC

MBC

MRSA MIC

MBC

MIC

MBC

MIC

MBC

MIC

MBC

12.5 12.5 6.25

50 100 12.5

25 25 6.25

50 50 12.5

25 50 12.5

100 100 25

25 50 6.25

100 100 25

50 25 6.25

100 100 25

MIC (lg/ml), minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (lg/ml), minimum bactericidal concentration, i.e., the lowest concentration of the compound for killing the bacteria completely.

Table 3 Antifungal activity of compounds 3 and 5. Positive control (Griseofulvin) and negative control (DMSO) measured by the Halo Zone Test (Unit, mm). Diameter of zone of inhibition (mm). Compounds

CA

AF

TM

PM

3 5 Standard DMSO

21.4 ± 0.2 21.2 ± 0.4 30.5 ± 0.2 –

19.9 ± 0.3 18.4 ± 0.2 26.5 ± 0.2 –

16.2 ± 0.2 15.3 ± 0.4 23.5 ± 0.3 –

13.5 ± 0.4 12.2 ± 0.2 21.5 ± 0.5 –

CA: Candida albicans, AF: Aspergillus fumigatus, TM: Trichophyton mentagrophytes, PM: Penicillium marneffei.

on the basis of diameter of zone of inhibition against Grampositive and Gram-negative strains of bacteria. Inhibition zones were measured and compared with the controls. The bacterial zones of inhibition values are given in Table 1. Minimum inhibitory concentrations (MICs) were determined by broth dilution technique. The nutrient broth, which

Table 4

contained logarithmic serially two fold diluted amount of test compound and controls was inoculated with approximately 5 · 105 c.f.u./ml of actively dividing bacteria cells. The cultures were incubated for 24 h at 37 C and the growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). To obtain the minimum bactericidal concentration (MBC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 18– 24 h of incubation at 35 C. MBC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The minimum inhibitory concentration and minimum bactericidal concentration are given in Table 2. 2.5. Antifungal studies Antifungal activity was also done by disk diffusion method. For assaying antifungal activity Candida albicans, Aspergillus fumigatus, Trichophyton mentagrophytes and Penicillium marneffei were recultured in DMSO by agar diffusion method

MIC and MFC of compounds 3 and 5. Positive control griseofulvin.

Comp

CA

AF

TM

PM

MIC

MFC

MIC

MFC

MIC

MFC

MIC

MFC

3 5 Standard

25 50 6.25

50 100 25

25 50 12.5

100 100 25

12.5 25 6.25

50 100 25

50 50 12.5

100 100 25

CA: Candida albicans, AF: Aspergillus fumigatus, TM: Trichophyton mentagrophytes, PM: Penicillium marneffei. MIC (lg/ml), minimum inhibitory concentration, i.e., the lowest concentration of the compound to inhibit the growth of fungi completely; MFC (lg/ml), minimum fungicidal concentration, i.e., the lowest concentration of the compound for killing the fungi completely.

240 Table 5

Z.N. Siddiqui et al. PBE and fungicidal/fungistatic activity (MFC/MIC) of compounds 3, 5.

Compounds

3 5 Ciprofloxacine Griseofulvin

PBE = 100/MIC

MFC/MIC

Bacteria tested

Fungi tested

SP

MRSA

PA

KP

EC

CA

AF

TM

PM

8 8 16 –

4 4 16 –

4 2 8 –

4 2 16 –

2 4 16 –

2 2 – 4

4 2 – 2

4 4 – 4

2 2 – 2

SP: S. pyogenes, MRSA: Methicillin resistant Staphylococcus aureus, PA: P. aeruginosa, KP: K. pneumoniae, EC: E. coli, CA: C albicans, AF: A. fumigatus, TM: T. mentagrophytes, PM: P. marneffei.

(Khan, 1997; Varma, 1998). Sabourauds agar media was prepared by dissolving peptone (1 g), D-glucose (4 g) and agar (2 g) in distilled water (100 ml) and adjusting pH to 5.7. Normal saline was used to make a suspension of spore of fungal

strain for lawning. A loopful of particular fungal strain was transferred to 3 ml saline to get a suspension of corresponding species. 20 ml of agar media was poured into each Petri dish. Excess of suspension was decanted and the plates were dried

Hd H Ha c

NO2

H3C EtOH-Br2

He N N

Cl

Hb

RT, stirring

NO2

No reaction

C6H5 5

EtOH-Pyridine, Reflux or (i) EtOH-Pyridine (ii) Br2, RT. Stirring

CH3 NO2

NO2 4 +

O O

H3C H N

H3C +

CH3

N O

Cl

N

Ha

O

N

O

(i) EtOH-Pyridine (ii) Br2, RT. Stirring

Br

N N

1

C6H5

2

70 °C DMF / POCl3

120 °C AcOH / POCl3 O

H3C

N N N C6H5

O

O

N

CH3 O

CH3

Scheme 1

O N

Cl

CH3

C6H5

O

H3C

Synthetic route of compounds 3 and 5.

O

N CH3

3

CH3 O

Synthesis, characterization and antimicrobial evaluation of novel halopyrazole derivatives by placing in an incubator at 37 C for 1 h. Using an agar punch, wells were made and each well was labeled. A control was also prepared in triplicate and maintained at 37 C for 3–4 days. The fungal activity of each compound was compared with griseofulvin as standard drug. Inhibition zones were measured and compared with the controls. The fungal zones of inhibition values are given in Table 3. The nutrient broth, which contained logarithmic serially two fold diluted amount of test compound and controls was inoculated with approximately 1.6 · 104–6 · 104 c.f.u./ml. The cultures were incubated for 48 h at 35 C and the growth was monitored. The lowest concentration (highest dilution) required to arrest the growth of fungi was regarded as minimum inhibitory concentration (MIC). To obtain the minimum fungicidal concentration (MFC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 48 h of incubation at 35 C. MFC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The minimum inhibitory concentration and minimum fungicidal concentration are given in Table 4. The PBE (percentual bacteriostatic efficiency, %) was obtained as, PBE = 100/MIC and fungicidal/fungistatic activity

(MFC/MIC) as obtained are presented in Table 5. The ratio MFC/MIC was calculated in order to determine if the compound had a fungistatic (MFC/MIC P 4) or fungicidal (MFC/MIC 6 4) activity and the results have been summarized in Table 5. 3. Results and discussion 3.1. Chemistry Due to the exceptional reactivity of formyl group in 5-chloro-3methyl-1-phenylpyrazole-4-carboxaldehyde, 1 was taken as synthon for the generation of novel bioactive molecular frameworks. Initially attempts were made to synthesize monobromo derivative 3 starting from 1 and 5-acetyl-1,3-dimethylbarbituric acid 2 in ethanol under basic medium followed by the subsequent addition of bromine with vigorous stirring at room temperature (Scheme 1). The reaction sequence has been outlined in Scheme 2. The reaction of heteroaldehyde 1 with heteroaryl active methyl compound 2 in ethanol under basic medium might have generated a,b-unsaturated system A. Dropwise

OH

O O

H3C H N N

241

O

H2C +

N N

1

CH3

N H

O

N

O

N

O

Cl CH3

CH3

C6H5

O

CH3 N

O

Cl

O

H3C

C6H5

2

-H2O

H

O

O

H3C N H

N N

O

N

CH3

O

Cl CH3

A C6H5

Br2

O

Br O

H3C N Br

N N C6H5

N

Cl 3

Scheme 2

CH3

O

CH3

N

CH3

H Br

N O

O H

H3C

O

-HBr

N C6H5

O

N

Cl B

Reaction sequence for the formation of product 3.

CH3

O

242 addition of bromine into the reaction mixture probably resulted in the formation of dibromodihydro derivative B, which on subsequent dehydrohalogenation afforded the required monobromo derivative of chalcone 3. In another reaction, efforts were made to synthesize styryl pyrazole 5, by the reaction of 1 with 2,4-dinitrotoluene 4 in refluxing ethanol with catalytic amount of pyridine. The reaction as visualized afforded the product 5 in quantitative yield in short span of time (2.5 h). The same reaction was carried under ethanol-pyridine, Br2 reaction condition did not afford the expected monobromo derivative, instead the reaction stopped at the styryl pyrazole product stage, 5 probably because of the steric hindrance from the bulky aromatic or heteroaromatic rings. This fact was further proved by the incompleteness of the bromination reaction of isolated styryl pyrazole 5 (Scheme 1). The structures of the compounds isolated were characterized by elemental and spectral analysis (IR, 1H NMR, 13C NMR and Mass spectrometry). The infrared (IR) spectrum of 3 showed the carbonyl absorption band of barbituric moiety at 1730 cm 1. The absorption bands for carbonyl group and carbon–carbon double bond of the a,b-unsaturated system appeared at 1689 and 1623 cm 1, respectively. The 1H NMR spectrum showed Ha proton as sharp upfield singlet at d 7.81. The aromatic protons of the N-phenyl-pyrazole-moiety were present in the form of multiplet at d 7.46–7.62. Six protons of N–CH3 groups were discernible as two sharp singlets at d 3.32 and 3.48 whereas protons of CH3 group of pyrazole unit were present in the form of another sharp singlet at d 2.61. The 13C NMR spectrum was in accordance with the structure. Further confirmation of the structure was done by mass spectrum, which showed M+ at 480.6 as base peak. The 1H NMR spectrum of 5 showed Ha and Hb protons as doublets at d 7.20 (J = 16.5 Hz), 7.66 (J = 16.5 Hz) respectively. The other peaks were observed at their normal position and are given in the experimental section. 3.2. Antimicrobial activity The investigation of antibacterial screening data (Tables 1 and 2) revealed that all the tested compounds showed moderate to good bacterial inhibition. All the compounds showed good inhibition against S. pyogenes, Methicillin resistant Staphylococcus aureus (MRSA +ve) P. aeruginosa, K. pneumoniae and E. coli species. MIC of compounds was in the range of 12.5–50 lg/ml. The MBC of compounds was found to be two or four folds higher than the corresponding MIC results. The antifungal screening data (Tables 3 and 4) showed moderate to good activity. The compounds, 3 and 5 showed good fungicidal activity against C. albicans, A. fumigates, T. mentagrophytes and P. marneffei fungal strains. MIC of compounds was in the range of 12.5–50 lg/ml. The MBC of the compounds was found to be two or four folds higher than the corresponding MIC results. Most of the compounds showed good fungicidal activity against various fungal strains (Table 5). 4. Conclusion In summary, a clean and convenient synthesis of novel halopyrazole derivatives has been developed. The procedure offers several advantages including mild reaction conditions as well as simple experimental and product isolation procedures, thus,

Z.N. Siddiqui et al. making the current protocol as a useful and interesting methodology for the synthesis of series of novel heterocycles in good yields from cheap and readily available starting materials. The antibacterial, antifungal screening data revealed that newly generated compounds are potential antimicrobial agents. The importance of such kind of work lies in the possibility that the new compounds might be more effective against microbes for which a thorough study regarding the structure– activity relationship, toxicity and their biological effects would be helpful in designing more potent antimicrobial agents. The other biological evaluations may furnish some other important applications.

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