SYNTHESIS AND BIOLOGICAL SCREENING OF SOME CINCHOPHENIC ACID DERIVATIVES

Transcription

1 113 P a g e International Standard Serial Number (ISSN): International Journal of Universal Pharmacy and Bio Sciences 3(3): May-June 2014 INTERNATINAL JURNAL F UNIVERSAL PHARMACY AND BI SCIENCES IMPACT FACTR 1.89*** Pharmaceutical Sciences ICV 5.13*** RESEARCH ARTICLE!!! SYNTHESIS AND BILGICAL SCREENING F SME CINCHPHENIC ACID DERIVATIVES Jyoti Chaudhary *, Khushbu Patel, Dr. C. N. Patel Department of Medicinal Chemistry, Shri Sarvajanik Pharmacy College, Shri Sarvajanik Vidya Sankul, Near Arvind Baug, Mehsana, Gujarat, India. KEYWRDS: Cinchophenic acid, Quinoline-4-carboxylic acid, Anti-bacterial activity, Anti-fungal activity. For Correspondence: Jyoti Chaudhary * Address: Department of Medicinal Chemistry, Shri Sarvajanik Pharmacy College, Shri Sarvajanik Vidya Sankul, Near Arvind Baug, Mehsana, Gujarat, India. jyotichaudhary_6391@ya hoo.com ABSTRACT The major drawback of current treatment of infectious diseases are challenging due to resistance to antimicrobial agents and their side effects. It is necessary to continue the search for new antibacterial agents with higher potency and lesser side effects. The increasing incidence of fungal infection also associated with unsatisfactory therapeutic treatment in immuno-compromized patients and the emergence of azole resistant fungal strains have stimulated the search for alternative antifungal drugs with higher potency and broader spectrum of activity along with a greater metabolic stability.cinchophenic acid derivatives are the heterocyclic compounds with considerable therapeutic and pharmacological properties. In this view, the series of cinchophenic acid derivatives were synthesized and evaluated for antibacterial and antifungal activities. Cinchophenic acid was synthesized by Dobner reaction between pyruvic acid,4-methoxy benzaldehyde and aniline followed by formation of acyl chloride as an intermediate by using oxalyl chloride. Then cinchophenic acid derivatives (6a-g) were synthesized by reaction of intermediate with different amines followed by schotten baumen reaction. The purity of the compounds was checked by TLC monitoring and all the synthesized compounds were characterized by UV, IR, Mass and 1 H- NMR spectroscopy. The antibacterial and antifungal activities of synthesized compounds were tested by cup plate method.

2 114 P a g e International Standard Serial Number (ISSN): INTRDUCTIN 1-14 : An infection is the invasion of the body s natural barriers by microscopic organisms like bacteria, fungi, virus etc. which multiply to create symptoms of infectious disease. Microbes are living organisms that multiply frequently and spread rapidly. They include bacteria (e.g., Staphylococcus aureus, which causes some staph infections), viruses (e.g., influenza, which causes the flu), fungi (e.g., Candida albicans, which causes some yeast infections), and parasites (e.g., Plasmodium falciparum, which causes malaria). Antibiotic resistance is a type of drug resistance where a micro-organism has developed the ability to survive exposure to an antibiotic. It is a one of the problem of global proportions affecting virtually all bacteria which commonly cause human illness. The emergence of antibiotic resistance is an evolutionary process that is based on selection for organisms that have enhanced ability to survive doses of antibiotics that would have previously been lethal. Antibiotics like Penicillin and Erythromycin, which used to be one-time miracle cures are now less effective because bacteria have become more resistant. Antibiotics themselves act as a selective pressure that allows the growth of resistant bacteria with in a population and inhibits susceptible bacteria. Antibiotic selection of pre-existing antibiotic resistant mutants with in bacterial populations was demonstrated in 1943 by the Luria Delbruck experiment. Survival of bacteria of ten results from an inheritable resistance. Any antibiotic resistance may impose a biological cost. Spread of antibioticresistant bacteria may be hampered by reduced fitness associated with the resistance, which is disadvantageous for survival of the bacteria when antibiotic is not present. Additional mutations, however, may compensate for this fitness cost and aids the survival of these bacteria.antibiotic resistant microorganisms, sometimes referred to as" super bugs", may contribute to there-emergence of diseases which are currently well- controlled. NDM-1 is a newly identified enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics. An antifungal drug is medication used to treat fungal infections such as athletes foot, ringworm, candidacies (thrush), serious systemic infections such as cryptococcal meningitis and others. Such drugs are usually obtained by doctor s prescription or purchased over-the-counter (TC). Future Antifungal Pipeline: Inhibitors of New Targets: Inositol phosphoryl ceramide synthase inhibitors Inositol phosphoryl ceramide synthase was identified as an attractive target for antifungals through the traditional method of natural product screening. Inositol phosphoryl ceramide synthase is specific to fungi and is essential for fungal growth through its involvement in sphingolipid biosynthesis. The

3 115 P a g e International Standard Serial Number (ISSN): prototypical natural product, Aureobasidin A (25), was active in a systemic mouse candidiasis model following both oral and intravenous administration. The aureobasidin class of natural products produced by Aureobasidium pullulans are active against many pathogenic fungi, including Candida albicans, Cryptococcus neoformans, and Histoplasma capsulatum, and are fungicidal against Candida albicans. N-myristoyl transferase inhibitors Myristoyl coenzyme A (CoA) protein N-myristoyl transferase (NMT) is one of the first antifungal targets discovered by a genetic based approach. NMTs catalyze the transfer of the rare cellular fatty acid, myristate, from myristoyl CoA to the Nterminal glycines of eukaryotic proteins. Genetic studies have shown that fungal pathogens require NMT to maintain their viability in vitro and in vivo. To date, there is no NMT inhibitor development candidates reported, possibly because of the difficulties in obtaining broad-spectrum antifungal agents with appropriate metabolic stability. Isoleucyl-tRNA synthetase inhibitors (ITRS) ITRS is the target of Icofungipen (26), which is a highly novel clinical development compound for the treatment of candida infections. Icofungipen is a synthetic derivative of the naturally β- amino acid Cispentacin (27) (derived from Bacillus cereus and Streptomyces setonii) that blocks isoleucyl-trna synthetase, resulting in the inhibition of protein synthesis and growth of fungal cells. Icofungipen, like Fluconazole has the advantage of renal clearance to drive highly predictive pharmacokinetics and avoid drug-drug interactions as a result of hepatic cytochrome P450-based clearance. Like Fluconazole, it is also a small, highly soluble and orally bioavailable molecule. Although active against a number of candida species, including Fluconazole resistant strains, it has only moderate antifungal potency in vitro (minimum inhibitory concentration (MIC) = 8-64 μg ml-1 with no fungicidal activity). 2. MATERIAL AND METHD Melting points were determined in open capillary tubes and by using Differential Scanning. Colorimetry(DSC). The IR spectra were recorded in FT-IR and bruker alpha ATR and 1H NMR spectra were recorded in CDCL 3 with TMS as internal standard on a Bruker spectrometer at 400 MHz respectively. Mass spectra were obtained using LCMS Shimadzu instrument. All physical and spectral characteristics are shown in Table 3.1 and 3.2.

4 116 P a g e International Standard Serial Number (ISSN): Synthesis of 2-(4-methoxyphenyl)quinoline-4-carboxylic acid (4) A mixture of aniline (9.13g,0.1 mol), 4-methoxy benzaldehyde (12.14g, 0.1 mol) and pyruvic acid (8g, 0.1 mol) placed in a ml of two necked flask was subjected to MW irradiation (400 W) for 9-10 minutes. After completion of the reaction (monitored by TLC), the reaction mixture was allowed to cool and putted it for 24 hours. The solid was came out after 24 hours which was collected by filtration, dried and recrystallized it from methanol. 2.2 Synthesis of 2-(4-methoxyphenyl)quinoline-4-carbonyl chloride (5) 2-(4-methoxyphenyl)quinoline-4-carboxylic acid, (2.79 g, 0.01 mol) was taken and dichloromethane (6 ml) was added to it, to form a suspension. To this suspension,oxalylchloride (1.3 ml 0.01 mol) in 10 ml dichloromethane was added to form a clear solution.the solution was concentrated to give yellow liquid of acid chloride of cinchophen, which was used for further reaction without purification. 2.3 Synthesis of 2-(4-methoxyphenyl)quinoline-4-substituted carboxamide (6a-g ) To 2-(4-methoxyphenyl)quinoline-4-carbonyl chloride, (2.97 ml, 0.01 mol) substituted anilines (0.01 mol) in dichloromethane (5 ml) was added and stirred for 5-6hrs. Aqueous NaH was added to the solution to neutralize excess of HCl.The resulting organic layer was separated and ether was added to it. Evaporate ether, yellow solid was obtained which was recrystalized from methanol.

5 117 P a g e International Standard Serial Number (ISSN): SCHEME F SYNTHESIS : NH 2 Aniline CH + + H 3 C CH3 4-methoxybenzaldehyde R H 2-oxopropanoic acid Dobner reaction Ethanol MW 245W 9-10 mint CH N 2-(4-methoxyphenyl)quinoline- 4-carboxylic acid xalyl chloride stirr for 4-5hrs Heat 4 CH 3 NH substituted amines Cl NaH N stirr for 5-6 hrs N 2-(4-methoxyphenyl)quinoline -4-substituted carboxamide CH 3 2-(4-methoxyphenyl)quinoline-4- carbonyl chloride CH 3 6a-g 5 R 4-F, 3-Cl-4-F, 5-Cl-2-CH 3, 4-Cl, 4-N Cl

6 118 P a g e International Standard Serial Number (ISSN): Biological Screening The microbiological assay is based upon a comparison of inhibition of growth of micro-organisms by measured concentrations of test compounds with that produced by known concentration of a standard antibiotic. Two methods generally employed are turbidometric (tube-dilution) method and cylinder plate (cup-plate) method Antibacterial Activity The synthesized compounds were tested for their antibacterial activity by cup-plate method. The agar solution spread over the disc and Ciprofloxacin was employed as the standard drug against the bacterial strains. The antibacterial activities of all the compounds were studied against grampositive bacteria (Staphylococcus aureus and Bacillus subtilis) and gram-negative bacteria (E.coli) The compounds were tested in various concentrations (200,, and ug/ml). The exhibited values of antibacterial activity and graphical plots of the compounds are shown in Table 3.3 and 3.4 and Figure 3.1 and 3.2 respectively Antifungal Activity The synthesized compounds were tested for their antifungal activity by cup-plate method. Fluconazole used as standard drug against the pathogenic strains of candida albicans in various concentrations. The compounds were tested in various concentrations (200,, and ug/ml). The observed values and graphical plots of anti fungal activities are given in Table 3.5 and 3.6 and Figure 3.3 and 3.4 respectively. 3. RESULT AND DISCUSSIN 3.1 Physical and Spectral characteristics All the compounds synthesized were pale yellow coloured solids. All compounds were soluble in methanol, chloroform and other solvents like ethanol, acetone, DCM. The melting points of compounds were in the range of C. None of compounds were freely soluble in water Spectral characteristics UV spectra UV spectra of all the compounds were studied in UV-1700 Shimadzu spectrophotometer. UV spectra of all the compounds were studied in methanol. All the compounds were found to have absorption λmax in range of 200 nm to 300nm.

7 119 P a g e International Standard Serial Number (ISSN): IR spectra IR spectra of all compounds were recorded on FT-IR 8400S Shimadzu spectrophotometer using KBR and bruker Alpha ATR. All the synthesized compounds have shown characteristic of NH stretching in the range of cm -1, C-Cl around cm -1 and C= around cm -1. Mass spectra Mass spectra were obtained using ESI LCMS Shimadzu instrument from Synzeal Laboratory. All compounds possess characteristics molecular ion peak M-1,M +, M+1, M+2,M+4 peaks. 1H NMR spectra The 1H NMR spectra of some of the compounds were studied in Bruker advanced-ii NMR-400Mhz instrument using CDCL 3 as solvent and TMS as an internal standard. All the compounds shows characteristic chemical shift from TMS in terms of δ, ppm. All the compounds showed multiplet in the range from ppm due to presence of aromatic rings and at around 10 due to presence of amine. Table Physical characteristics of 2-(4-methoxyphenyl)quinoline-4- substituted carboxamide R NH N CH 3 Compound R Moecular Mol Wt. mp Yield R f Code Formula (g/mol) ( º C) (%w/w) 4 - C 17 H 13 N % C 17 H 12 N 2 Cl % a 2-Cl C 23 H 17 N 2 2 Cl b 4-F C 23 H 17 N 2 2 F c 3-Cl-4-F C 23 H 16 N 2 2 ClF d 4-Cl C 23 H 17 N 2 2 Cl e 3,4-Cl C 23 H 16 N 2 2 Cl f 4-N 2 C 23 H 17 N g 5-Cl-2-CH 3 C 24 H 19 N 2 2 ClN

8 120 P a g e International Standard Serial Number (ISSN): Table: 3.2 Spectral characteristics of 2- ( 4- methoxy phenyl) quinoline- 4- substituted carboxamide Compound code UV λmax (nm) IR (ν, cm -1 ) str.(-h), str.(ar.c-h), 1678 str. (C=), 1649 str. (C=N) str.(ar-c-h), str.(c=), str.(c-cl) 6a str(-nh), 3061 str.(ar-c- H), 1688 str.(c=), 1632 str.(c=n), 696,644 str.(c-cl) 6b str(-nh), 3010 str.(ar-c- H), 1683 str.(c=), 1638 str.(c=n),1030 str.(c-f) 6c str(-nh), 3060 str.(ar-c- H), 1682 str.(c=), 1640 str.(c=n),1123,1031 str.(c- F),689 str.(c-cl) 6d str(-nh), 3010 str.(ar-c- H), 1680 str.(c=), 1632 str.(c=n),743,689 str.(c-cl) 6e str(-nh), 3030 str.(ar-c- H), 1683 str.(c=), 1640 str.(c=n),695,753 str.(c-cl) 6f str(-nh), 3070 str.(ar-c- H), str.(c=), 1640 str.(c=n),1274 str.(n=) 6g str(-nh), 3020 str.(ar-c- H), str.(c=), str.(c=n), 697 str.(c-cl) Mass 1 H NMR (δ, ppm) (m/z) CDCL (M + ), 299.7(M+2) 388.8(M + ), 390.7(M+2) 3.81(s, 3H, Ar-CH 3 ), 7.08(d,CH-Ar-CH 3 ), 7.24(t, CH-Ar-Cl), 7.41(t, CH-Ar-Cl), 7.57( d, CH-Ar-Cl), 7.73( d, CH-Ar-Cl), 7.76(t, CH-quinoline), 7.91&7.99(m, CHquinoline), (m,CH-ring), (s, 1H, amide) 372.4(M + ) 3.80(s, 3H, Ar-CH 3 ), 7.06(d,CH-Ar-CH 3 ), 7.15(d, CH-Ar-Cl), 7.60(d,CH-Ar-F), 7.73(d,CH-Ar-Cl), 7.76(t,CH-quinoline), 7.91&7.99(m,CHquinoline), (m,CH-ring), 10.22(s,1H, amide) 405.7(M-1), 406.9(M + ), 408.7(M+2) 388.9(M + ), 390.7(M+2) 423.8(M + ), 425.9(M+2), 427.4(M+4) 398.9(M-1), 399.7(M + ) 402.7(M + ), 404.6(M+2)

9 121 P a g e International Standard Serial Number (ISSN): Compound code Table: 3.3 Antibacterial activities of synthesized compounds Concentration (μg/ml) Zone of inhibition (mm) Gram +ve Gram -ve S.aureus B.subtilis E.coli 6a b c d e f g Ciprofloxacin

10 Zone of inhibition (mm) 122 P a g e International Standard Serial Number (ISSN): Figure 3.1 : Histogram of Antibacterial activity S.aureus B.subtilis E.coli 6a 6b 6c 6d 6e 6f 6g ciprofloxacin Concentration (μg/ml) Table: 3.4 MIC of Antibacterial Activity Minimum Inhibitory Concentrations (μg\ml) Compound code S.aureus B.subtilis E.coli 6a 200 6b 200 6c 150 6d 200 6e 6f 6g Ciprofloxacin

11 Concentration(µg/ml) 123 P a g e International Standard Serial Number (ISSN): Figure 3.2: Histogram of MIC (Antibacterial Activity) Minimum Inhibitory Concentration (µg/ml) 6a 6b 6c 6d 6e 6f 6g Ciprofloxacin Compound Code S.aureus B.subtilis E.coli 3.3 Discussion for antibacterial activity: All synthesized compounds were screened for antibacterial activity by cup plate method at different concentration ranges from to µ g/ml. Compounds 6a,6c and 6d have shown highest zone of inhibition against S. aureus having MIC 200,150 and µ g/ml respectively. Compounds 6b and 6d have shown highest inhibition against B. subtilis having MIC and 200µ g/ml respectively. Compounds 6b and 6c have shown highest zone of inhibition against E.coli. having MIC 200 and µ g/ml respectively. ur study revealed that electron withdrawing groups increase antibacterial activity. All synthesized compounds were found to be less potent compared to the standard drug ciprofloxacin.

12 124 P a g e International Standard Serial Number (ISSN): Antifungal activity Table: 3.5 Antifungal activities of synthesized compounds Compound code Concentration (μg/ml) Zone of inhibition (mm) C.albicans 6a b c d e f g fluconazole

13 Concentration(µg/ml) Zone of inhibition (mm) 125 P a g e International Standard Serial Number (ISSN): Antifungal activity C.albicans 0 6a 6b 6c 6d 6e 6f fluconazole Concentration (μg/ml) Figure 3.3: Histogram of Antifungal Activity Table: 3.6 MIC of Antifungal Activity Minimum Inhibitory Concentrations (μg\ml) Compound code C.albicans 6a 6b 200 6c 6d 150 6e 6f 6g Fluconazole 12.5 Figure 3.4: Histogram of MIC (Antifungal Activity) Minimum Inhibitory Concentrations (μg\ml) 0 C.albicans Compound code

14 126 P a g e International Standard Serial Number (ISSN): Discussion for antifungal activity : All synthesized compounds were screened for antifungal activity by cup plate method at different concentration ranges from to µ g/ml. Compounds 6b and 6d have shown highest zone of inhibition against C. albicans having MIC 200 and 150µ g/ml respectively. Compounds 6c and 6g have shown moderate activity while compounds 6a, 6e, and 6f have shown lowest activity. ur study revealed that electone withdrawing groups increase antifungal activity. All synthesized compounds were found to be less potent compared to the standard drug fluconazole. 4. CNCLUSIN All the synthesized compounds were characterized by UV, IR, Mass and some of them by 1 H-NMR spectroscopy & reports of them support the structures of compounds. 2-(4-methoxy quinoline) carboxylic acid was synthesized by using Micro Wave technique with better yield in less time compared to conventional route of synthesis. As per synthesis point of view all the synthesized final compounds have good yield between 50-70%. All the synthesized final compounds were screened for antibacterial and antifungal activities by Cup Plate Method against standard reference drug ciprofloxacin and fluconazole respectively. Evaluation of antibacterial and antifungal screening revealed that compounds 6b, 6c and 6d with electron withdrawing functional groups having good activity. REFERENCES: 1. Pelczar MJ., Chan ECS., Krieg NR. Microbiology; 5 th edition; Tata McGraw Hill Publishing Company Limited, New Delhi, (1993), 73-98, Fredrickson J, Zachara J and Balkwill D, Geomicrobiology of high level nuclear waste contaminated vadose sediments. Appl. Environ. Microbiol. (2004) 70, Jain NK. Pharmaceutical Microbology; 1 st edition; Vallabh Prakashan, Delhi, (2001), Goyal RK. Derasari and Gandhi s Elements of Pharmacology; 15 th edition; B.S.Shah Prakashan, Ahmedabad, (2005),

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