Anticancer, Antibacterial, Antifungal Activities for Hybrid Probes of Aromatic Amine and Barbituric Acid

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1 /s GSTF International Journal of Chemical Sciences (JChem) Vol.1 No.2, May 2014 Anticancer, Antibacterial, Antifungal Activities for Hybrid Probes of Aromatic Amine and Barbituric Acid Bhaveshkumar D. Dhorajiya, Bharatkumar Z. Dholakiya*, Nimisha Singh Department of Applied Chemistry, S.V. National Institute of Technology, Surat , Gujarat, India. E- mail: Tel: , Fax No: * Corresponding Author Abstract- Cancer and microbes today remains one of the most deadly diseases in the world. In search of novel anticancer and antimicrobial probes, a series of newly hybrid molecules synthesized by combining the structural features of aromatic amines and barbituric acid, using the concept of green chemistry. This approach was accomplished efficiently using the aqueous medium to give the corresponding products in a high yield. The newly synthesized compounds were characterized by spectral analysis FT-IR, 1 H NMR, 13 C-NMR, HMBC, MASS and Elemental Analysis. Evaluations of these molecules have been carried out using four human cancer cell lines against Doxorubicin as a standard drug. Moreover all the synthesized barbiturates probes were evaluated for their antimicrobial activity against five bacterial strains and four fungal strains. Taken together, this study has led to the development of a convenient, a highly yielded and an environmentally friendly synthetic method of highly promising leads for anticancer and antimicrobial compounds. Graphical Abstract- Keyword Green Chemistry, N-Formylation, Knoevenagel Condensation, Anticancer, Antimicrobial activies. 1. INTRODUCTION Cancer and resistant to microbes continuing to be a major health problem in both developed and developing countries. Surpassing heart disease, it is taking the position of number one killer due to various worldwide factors. Several anticancer agents including taxol, vinblastine, vincristine, etoposide, camptothecin and its derivatives (topotecan and irinotecan), mitoxantrone, [1] 5-fluorouracil, indomethacin etc. are in clinical use all over the world. However, these drugs suffer from various side effects like low blood pressure, bone marrow suppression, gastrointestinal toxicity, constipation and hair loss. Therefore, the lack of effective chemotherapy of cancer is continuously inciting the scientific community to explore new chemical entities for the effective and safe cure of cancer. The pivotal role of small chemical entities (like amino acids, glucose, nucleobase) and the acceptability of their synthetic analogues in the biological systems may be the reason for the design and development of about 80% of the drugs based on small organic molecules. [2] Due to the biological significance of barbiturates as anti-convulsant, [3] anti-hypnotic, [4] anticancer, [5] and anti-inflammatory agents [6], we have chosen these molecular entities to suitably combine DOI: / _

2 them through carbon nitrogen-carbon bond formation for creating new hybrid molecules. Investigations of new molecules for their tumor growth inhibitory activities over four human cancer cell lines have been reported in this work. All the novel derivatives were screened for their antimicrobial activity against five bacterial strains and four fungal strains and their results are discussed. In continuation of our interest in the development of newly synthetic anticancer and antimicrobial agents, we further reported herein a convenient, an environmentally friendly and novel approach for the synthesis of aromatic amines based barbiturates in aqueous medium with a very good yield using the concept of green chemistry. The significance of this protocol is to avoid the use of organic solvents, catalyst as well as use of simple reaction conditions. II. EXPERIMENTAL This work reports synthesis of barbituric acid by Hantzch reaction according to reported procedure [7]. In this study, a series of 5-subtituted barbiturates B(1-19)(Table 1) has been synthesized using one pot three component systems by condensation reaction of various aromatic amines A(1-19), Formic acid 2 and Barbituric acid 3 in aqueous medium with moderate to good yields (63-78 %) (Scheme 1) in accordance with the mechanism suggested in literature [8]. Initially different aromatic amines (1 mmol) and formic acid (4 mmol) were charged in a three neck flat bottom flask equipped with condenser, thermometer and stirrer. Deionized water was charged as a green solvent in the flask. Reaction mass was continuously stirred for 2-3 hrs and temperature was maintained at 60 O C. Progress of reaction was monitored by TLC using hexane-ethylacetate (7:3) as the elluent system. After completion of the reaction, reaction mass was cooled to room temperature and in situ barbituric acid (1 mmol) was charged. Reaction mass was further stirred at reflux temperature for 3-4 hrs. After completion of the reaction, mass was filtered, washed with water/methanol and dried. Scheme 1. Synthesis for hybrid probes of aromatic amine and barbituric acid. TABLE I. 5-SUBSTITUTED BARBITURATES B (1-19) Sr. No Amine (Z)-5- arylaminomethylene pyrimidine 2, 4, 6-trione derivatives Yield (%) B-1 73 B-2 65 B-3 67 B

3 B-5 59 B B-6 74 B B-7 78 B B-8 69 TABLE II. 5-SUBSTITUTED BARBITURATES 5FN (1-10). B-9 76 Sr. No Amine Barbituric acid Based Schiff-Base Yield (%) FN-1 73 B FN-2 54 B FN-3 28 B FN-4 64 B FN-5 59 B FN-6 35 B FN-7 97 B FN

4 FN-9 97 FN-9 76 FN FN TABLE III. 5-SUBSTITUTED THIOBARBITURATES 5FN (1-10 ). Amine Thiobarbituric acid Based Yield Sr. No Schiff-Base (%) FN-1' FN-2 FN-3 88 FN-4 58 FN-5 36 FN-6 55 FN-7 78 FN-8 69 III. RESULT AND DISCUSSION A. Spectral Analysis The structures of all the new synthesized compounds were confirmed by FT-IR, 1 H NMR, 13 C-NMR, HMBC, Elemental Analysis and molecular weight confirmed by mass spectrometry. The important infrared spectral bands and their tentative assignments for barbiturates of N- formylated aromatic amines were recorded on a Perkin Elmer Spectrum RX-FTIR using KBr disks. The characteristic peak of FTIR spectra of synthesized compounds were observed at cm -1, cm -1 and cm -1 due to presence of C=C group of aromatic ring, C=O group and N-H group of pyrimidine ring respectively. The IR spectra of compounds B(1 19) revealed a characteristic bands at cm -1 confirming the presence of >C=CH-NH- group. The 1 H NMR data of compounds revealed signals between 7.27 and 8.05 δ ppm for aromatic protons of substituted phenyl ring. 1 H NMR data of compounds revealed signals at 8.62 δ ppm confirms sp hybridization of carbon of >C=CH-NH- and J value around Hz confirms trans-geometry i.e. (Z)- configuration. 13 C NMR spectra of compounds showed characteristic peak around 78.71, 94.21, , , , , , , , and δ ppm. ; ESIMS: m/z (M + 2). All synthesized analogues B (1-19) showed the strong absorption bands (λ max) in the range of nm owing to the π-π* transition as well as presence of exocyclic CH of the barbituric acid in the UV spectra [9]. B. Anticancer Activity 27

5 Anticancer activity was performed by growth inhibition test according to procedure adopted by the National Cancer Institute (NCI, USA). [10-11]. In this test, the cells were inoculated onto standard 96- well microtiter plates for one hour. The cell concentrations were adjusted according to the cell population doubling time (PDT): ml 1 for MCF-7 2 and NCI-H460 (PDT = h), ml 1 for SF-268 cell lines (PDT = 33 h) Test agents were then added in five, 10-fold dilutions ( mol l 1 ) and incubated for further 72 hours. Working dilutions were freshly prepared on the day of testing. The effect of the vehicle solvent (DMSO) on the growth of these cell lines were also evaluated in all the experiments by exposing untreated control cells by adjusting its concentration to be the same as in working concentrations. All synthesized compounds were tested for anticancer activity using four human cancer cell lines: MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), SF-268 (CNS cancer) and WI 38 (fibroblast cells) using Doxorubicin as a standard drug. The results are summarized in Table IV. TABLE IV. ANTICANCER ACTIVITY OF SYNTHESIZED COMPOUNDS B (1-19) Anticancer Activity (Effect on the growth Inhibition of human cancer cell lines) GI50 (μmol l 1 ) MCF-7 NCI-H460 SF-268 WI 38 B1 2.6 ± ± ± ± 10.2 B ± ± ± ± 6.3 B ± ± ± ± 22.6 B4 4.6 ± ± ± ± 0.1 B ± ± ± ± 1.8 B ± ± ± 1.6 > 100 B ± ± ± ± 12.9 B ± ± ± ± 4.6 B ± ± ± ± 8.2 B ± ± ± ± 12.7 B ± ± ± ± 8.0 B ± ± ± ± 2.8 B ± ± ± ± 12.7 B ± ± ± ± 10.6 B ± ± ± ± 22.3 B ± ± ± ± 8.3 B ± ± ± > 100 B ± ± ± >100 B ± ± ± ± 4.7 Doxorub icin 0.04 ± ± ±0.007 > 100 GI50 mean value ± standard error of mean of 3 independent experiments performed in duplicate. MCF-7- breast adenocarcinoma, NCI-H460- non-small cell lung cancer, SF-268 (CNS cancer), WI 38- fibroblast cells. C. Antibacterial and Antifungal Activity Antibacterial and Antifungal activity was done by broth microdilution method [12]. Antibacterial activities was performed against Streptococcus pyogenes, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia as the bacterial strains (Table 3) whereas antifungal activity was performed against Candida albicans, Aspergillus clavatus, Trichophyton rubrum and Penicillium wild strain as fungal strain (Table V-X). TABLE V. ANTIBALCTRIAL ACTIVITY B (1-19) Minimum Inhibitory Concentration (MIC) expressed in μg/ml Gram (+Ve) Bacteria Gram (-Ve) Bacteria Sp., Sa., Ec., Pa., Kp., B B B B B B B B B B B B B B B B B B B Ciprofloxacin

6 Sp., Streptococcus pyogenes, Sa., Staphylococcus aureus, Ec., Escherichia coli, Pa., Pseudomonas aeruginosa, Kp., Klebsiella pneumonia. TABLE VI. ANTIFUNGAL ACTIVITY B (1-19) Fungal Strains Ca., Ac., Tr., PWS B >1000 > B B >1000 B > >1000 B5 >1000 >1000 > B6 >1000 > B B >1000 B9 500 > B >1000 > B >1000 >1000 >1000 B >1000 >1000 B B B > B16 > >1000 B > >1000 B >1000 > B >1000 > Griseofulvin Ca., Candida albicans, Ac., Aspergillus clavatus, Tr., Trichophyton rubrum, PWS., Penicillum Wild Strain. TABLE VII. ANTIBALCTRIAL ACTIVITY 5FN (1-10) Minimum Inhibitory Concentration (MIC) expressed in μg/ml Gram (+Ve) Bacteria Gram (-Ve) Bacteria Sp., Sa., Ec., Pa., Kp., 5FN FN FN FN FN FN FN FN TABLE VIII. ANTIFUNGAL ACTIVITY 5FN (1-10) Compounds Fungal Strains Ca., Ac., Tr., PWS 5FN > FN FN >1000 5FN >1000 5FN FN FN > FN-8 >1000 >1000 >1000 >1000 5FN-9 >1000 > >1000 5FN-10 > Griseofulvin Ca., Candida albicans, Ac., Aspergillus clavatus, Tr., Trichophyton rubrum, PWS., Penicillum Wild Strain. TABLE IX. ANTIBALCTRIAL ACTIVITY 5FN (1-10 ) MIC (Minimun Inhibitory Concentration) (μg/ml) Gram (+Ve) bacteria Gram (-Ve) bacteria Sa. Sp. Ec. Pa. Kp. 5FN FN FN FN FN FN FN FN FN FN Ciprofloxacin FN FN Ciprofloxacin Sp., Streptococcus pyogenes, Sa., Staphylococcus aureus, Ec., Escherichia coli, Pa., Sp., Streptococcus pyogenes, Sa., Staphylococcus aureus, Ec., Escherichia coli, Pa., Pseudomonas aeruginosa, Kp., Klebsiella pneumonia. Pseudomonas aeruginosa, Kp., Klebsiella pneumonia. 29

7 TABLE X. ANTIFUNGAL ACTIVITY 5FN (1-10 ) Fungal Strains Ca., Ac., Tr., PWS 5FN > FN > FN FN FN FN >1000 5FN-7 > FN >1000 >1000 5FN >1000 >1000 >1000 5FN > Griseofulvin Ca., Candida albicans, Ac., Aspergillus clavatus, Tr., Trichophyton rubrum, PWS., Penicillum Wild Strain. IV. CONCLUSION In summary, we have successfully synthesized hybrid molecules of aromatic amines and barbituric acid through green route synthetic protocol. All synthesized compounds were evaluated for their anticancer and antimicrobial activities. Out of set of 19 molecules 3 molecules shows significant anticancer activities against MCF-7, NCI-H460 and SF-268 whereas 16 molecules exhibit potent antitumor activity against WI-38 cell lines. From anti microbial activity three molecules exhibits significant activity against both the grampositive as well as gram negative bacteria. On the other hand, two molecules having electron donating methyl functionality as substituent on phenyl ring also showed highly potent activity against T. Rubrum as the fungal strain where as three molecules were found to exhibit equipotent against T. rubrum as a fungal strain using griseofulvin as standard drug. V. ACKNOWLEDGEMENT We are very much thankful to Gujarat Council on Science and Technology for financial support (Project Grant No. GUJCOST/MRP/ /12-13/04/1319). REFERENCES [1] P. Singh, M. Kaur, P. Verma P Design, synthesis and anticancer activities of hybrids of indole and barbituric acids--identification of highly promising leads, Bioorg. Med. Chem. Lett. Vol. 19, pp [2] B. Meunier, Hybrid molecules with a dual mode of action: dream or reality? Acc. Chem. Res. Vol. 41, pp , [3] R. A. Nicoll, J. M. Wojtowicz, The effects of pentobarbital and related compounds on frog motoneurons Brain Res. Vol. 191, pp , [4] L. Y. M. Huang, J. L. Barker, Pentobarbital stereospecific actions of (+) and (-) isomers revealed on cultured mammalian neurons. Science. Vol. 207, pp , [5] B. D. Dhorajiya, A. S. Ibrahim, F. A. Badria, B. Z. Dholakiya, Design and synthesis of novel nucleobase based barbiturate derivatives as potential anticancer agents. Med Chem Res, DOI: /s [6] L. S. Goodman, A. Gilman In The Pharmacological Basis of Therapeutics Mc Graw-Hill: New Delhi, pp [7] Jacobson, U. S. pat. 2,090, 594, [C. A. 31, 7068] [8] B. D. Dhorajiya, B. Z., Dholakiya Green chemistry multi-component approach for N- formylation and Knoevenagel condensation for synthesis of thiobarbiturates in aqueous system, Res Chem Intermed, DOI /s [9] R. M. Silverstein, F. X. Webster Spectrometric identification of organic compounds, 6 th edn. Wiley, New York, pp [10] M. R. Boyd, K.D. Paull, Some practical considerations and applications of the National Cancer Institute in vitro anticancer drug discovery screen. Drug Dev Res. Vol. 34, pp [11] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren, H. Bokesch, S. Kenney and M.R. Boyd, J. Nat Can Inst, vol. 82, pp [12] National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial susceptibility testing: twelfth informational supplement. ISBN , M100-S12 (M7)

8 Dr. Bharatkumar Z. Dholakiya received his M.Sc. (Gold Medal) and Ph.D. (Gold Medal) degree in Industrial Chemistry and Chemistry from Sardar Patel University, Vallabh Vidhyanagar, Gujarat, India, in 1996 and 2006 respectively. Also,he has completed his post doctoral study on ultra-high efficiency biodiesel manufacturing from Institute of Environmental and Industrial Science (IEIS), Texas State University, San Marcos, Texas, USA in He is having 15 yrs. of teaching and research experience. Also, guided 10 M.Sc. and 6 Ph.D. thesis. He is now working as an Assistant Professor at Applied chemistry Department, Sardar Vallabhbhai National Institute of Technology (SVNIT), Surat, Gujarat, India. His research interest focuses on organic synthesis, polymer for specialty applications and the design of novel heterogeneous solid acid catalysts and their applications towards biodiesel production. Nimisha Singh completed her M.Sc., Chemistry from S. V. National Institute of Technology. Her current research interest is synthesis, characterization and bioassay of heterocyclic scaffold. Bhaveshkumar D. Dhorajiya completed his Master degree in organic chemistry from Sardar Patel University, Gujarat. Currently he is doing his Ph.D. under the guidance of Dr. Bharatkumar Z. Dholakiya at S.V. National Institute of Technology, Surat. His research interest is synthesis, characterization and bioassay of barbiturates and thiobarbiturates based heterocyclic scaffold. 31