Development of Anti-Cancer Agent

Development of Anti-Cancer Agent Design, ultrasound assisted synthesis and in-vitro anticancer activity of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor cell lines Sachin K. Bhosale, Shreenivas R. Deshpande  and Rajendra D. Wagh Design, ultrasound asisted synthesis and in-vitro anticancer activity of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor cell lines Abstract: In an attempt to develop an effective and safer anticancer agent, 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde 5 have been designed and synthesized under ultrasonication by formylation of 3-(4-chlorophenyl)-sydnone 4 and characterized by spectral studies. The ultrasonic method of synthesis was found to be simple, ecofriendly, reduces reaction time and gave good yield when compared with traditional methods of synthesis. Anticancer activity of the compounds were tested against 60 human tumor cell lines and compared with standard drug Vincristine sulphate. Cytotoxicity evaluation revealed that compound 5 is most potent against NCI-H23 (Non-Small cell lung cancer) with comparative activity of Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31 (Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma). In future, research and modification of compounds to different derivatives may lead to development of potent anticancer drug. Keywords: Anticancer, 1, 2, 3-oxadiazol-5-olate, Formylation, Ultrasonication INTRODUCTION Mesoionic sydnone compounds have shown a variety of biological activities including antitumor ((Kier et al., 1964, Dunkley et al., 2003, Satyanarayana et al., 1995, Kavali et al., 2000, Dunkley et al., 2003, Fregly et al., 1964, Stewart et al., 1965, Roche et al., 1965). It is thought that the ionic resonance structures of the heterocyclic ring promote significant interactions with biological molecules. In 1992 a series of 4-substituted-3-nitrophenylsydnones were synthesized and evaluated by Grynberg et al for anticancer activity and it was found that the 4-chloro and the 4-pyrrolidino compounds significantly enhanced the survival of Sarcoma 180 (S180), Ehrlich carcinoma (Ehrlich) and Fibrous histiocytoma (B10MCII) tumor bearing mice(Dunkley et al., 2003). It was also found that the larger hetero rings; p-piperidino and p-morpholino, were less potent (Kier et al., 1964, Dunkley et al., 2003). Herein we report the design, synthesis, and biological evaluation of two para-chloro substit uted analogues of sudnone molecules 4 and 5. These were tested for 60 human tumor cell lines at the National Cancer Institute for antitumor activity at a minimum of five concentrations at 10-fold dilutions.The synthesized chloro substituted sydnones (4 and 5) passed the primary assay by inhibiting the growth of at least one cell line to less than 32%, and both are proved to be moderate active and versatile against each cell line. MATERIALS AND METHODS All chemicals and reagents were purchased form from Sigma-Aldrich, Mumbai, India. Melting points of the intermediates and the final products were recorded using a Systolic melting point apparatus and are reported uncorrected. Thin layer chromatographies (TLC) were performed on E-Merck precoated 60 F254 plates and the spots were rendered visible by exposing to UV light. Infra red spectra were recorded in KBr discs using Jasco FTIR 1460 Plus spectrometer. NMR spectra were obtained on a BRUKER AVANCE II 400 NMR spectrometer at 500 MHz for 1H and 125 MHz for 13C, the chemical shifts are expressed in ÃŽ ´ (ppm) downfield from tetramethylsilane (TMS). Electron impact mass spectra were recorded on WATERS, Q-TOF MICROMASS (LC-MS) instrument. Elemental analyses (C, H, N) were in full agreement with the proposed structures within  ±0.4% of the theoretical values. The ultrasonic irradiation was performed by using a Biotechnics India TM ultrasonic cleaner bath, model 1510, AC input 115 V, outp ut 50 W, 1.9 liters with a mechanical timer (60 min with continuous hold) and heater switch, 47 KHz. Synthesis of Ethyl N-(4-chlorophenyl) glycinate (1) A mixture of Para-Chloroaniline (2.80 g, 0.02mol) and chloroethyl acetate (2.12 mL, 0.02mol) was added to the solution of ethanol (20 mL) and anhydrous sodium acetate (3.28 g, 0.04mol) under ultrasonication conditions and allowed to react for 120 min. The mixture was diluted with 20 mL of water and cooled in refrigerator for overnight and recrystallized in ethanol to yield 1 (83%), mp 114-116  ºC. IR (KBr): 3327, 2950, 2934, 2879, 1756, 1069; 1H NMR (400 MHz, DMSO-d6): ÃŽ ´ 1.21 (t, 3H, COOCH2CH3), 3.76 (s, 1H, NH), 4.29 (s, 2H, CH2), 4.54 (q, 2H, COOCH2CH3), 6.83-7.21 (m, 4H, Ar-H); 13C NMR (40 MHz, DMSO-d6): ÃŽ ´ 14.65, 44.73, 62.07, 115.12, 123.22, 129.01, 146.26, 173.13. Synthesis of N-(4-chlorophenyl) glycine (2) Ethyl N-(4-chlorophenyl) glycinate (4.26 g, 0.02mol) and sodium hydroxide (1.2 g, 0.030mol) in ethanol was heated under ultrasonication conditions at 65  °C for 15 min and allowed to cool and acidified with dil HCl. Yield 87 %, mp 146-148  ºC. IR (KBr): 3319, 3277, 2951, 2937, 2879, 1703, 1063; 1H NMR (400 MHz, DMSO-d6): ÃŽ ´ 4.29 (s, 2H, CH2), 6.39 (s, 1H, COOH), 6.55 (s, 1H, NH), 6.79-7.27 (m, 4H, Ar-H); 13C NMR (40 MHz, DMSO-d6): ÃŽ ´ 44.48, 115.02, 124.16, 130.02, 145.97, 171.98. Synthesis of N-nitroso (4-chlorophenyl) glycine (3) Ice cold solution of the N-(4-chlorophenyl) glycine (3.72 g, 0.02mol) was added in crushed ice water (40 mL). A solution of sodium nitrite (1.38 g, 0.02mol) in ice cold water (10 mL) was added dropwise under ultrasonication condition at 0  °C for 10 min. The reaction mixture was filtered and precipitated by adding concentrated hydrochloric acid to the filtrate. Precipitate was filtered and recrystallized in methanol to yield 3 (81%), mp 109-111  ºC. IR (KBr): 3255-2521, 2923, 2849, 1711, 1569, 1325, 1062; 1H NMR (400 MHz, DMSO-d6): ÃŽ ´ 5.02 (s, 2H, CH2), 6.93-7.45 (m, 4H, Ar-H), 11.55 (s, 1H, COOH); 13C NMR (40 MHz, DMSO-d6): ÃŽ ´ 49.42, 120.75, 128.29, 130.43, 138.78, and 169.25. Synthesis of 3-(4-chlorophenyl) sydnone (4) Acetic anhydride (30 mL) was added to 3 (5.40 g, 0.0252 mol) under ultrasonication condition at room temperature for 60 min. The reaction mixture was left overnightat room temperature. The solution was poured slowly into cold water which was very well stirred. The crude sydnone was filtered, dried and recrystallized in ethanol to yield 4 (93%), mp 139-141  ºC. IR (KBr): 3181, 1748, 1053. 1H NMR (400 MHz, DMSO-d6): ÃŽ ´ 7.23 (s, 1H, sydnone), 7.49-8.13 (m, 4H, Ar-H). 13C NMR (40 MHz, DMSO-d6): ÃŽ ´ 123.15, 126.49, 131.53, 136.13, 141.13, 170.07 Synthesis of 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde (5) N-Methylformanilide 2.84g (0.0210mol) and phosphoryl chloride (3.17g, 0.0205mol) were mixed under ultrasonication conditions for 10 min. After 0.5 h, 3.0 g (0.0186mol) of 4 was added portionwise with swirling and cooling under ultrasonication below 350C. Hydrogen chloride was evolved vigorously. After standing overnight, the viscous, dark-brown mixture was dissolved in 15 ml. of acetone and poured (stirring) into 75 ml. of ice water. The yellow-orange precipitate was filtered, washed (cold water), and dried. Yield 56%, mp 75-77 0C. MS (M+) (m/e) 223.99, 225.996, 225.002. IR (KBr): 1790 (Ñ ´C=O sydnone ring), 1640 (Ñ ´C=O aldehyde). 1H NMR (400 MHz, DMSO-d6): ÃŽ ´ 7.2, 7.2, 7.3, 7.3 (Cl-Ph-), 9.61 (Sydnone-4-CHO). 13C NMR (40 MHz, DMSO-d6): ÃŽ ´ 127, 129, 129, 130, 130, 134 (Cl-Ph-) 190 (CHO). Element analysis: C; 48.13, H; 2.24, N; 12.47, O; 21.37 Scheme 1-Synthesis of 3-(substituted aryl)-4-sydnonecarboxaldehyde (5) under ultrasonication conditions 1.3. Anticancer screening The synthesized compound was screened for preliminary anticancer assay by National Cancer Institute (NCI), Bethesda, USA in an in vitro 60 human tumor cell panel. The process utilized 60 different human tumor cell lines of the leukemia, Non-small cell lung, Colon, CNS, melanoma, ovarian, renal, Prostrate and breast cancers which was aimed in showing selective growth inhibition or cell killing of particular tumor cell lines by specific compound. The screening begins with the evaluation of selected compounds against these 60 cell lines at a single dose of 10-5 M. The output from the single dose screen is reported as a mean graph of the percent growth of treated cells. It allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0) (Roschke et al., 2003, Lorenzi et al., 2009, Mingyi et al., 2013, Al-Suwaidan et al., 2013, Senff-Ribeiro et al., 2004, Butkovic et al., 2011, Lorenzi et al., 2009, Mingyi et al., 2013, Al-Suwaidan et al., 2013, Sen ff-Ribeiro et al., 2004, Butkovic et al., 2011). RESULT Compund 5 showed highly cytotoxic activity against NCI-H23 cell line (Non-Small cell lung cancer) as compared to standard drug Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31(Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma). Details of % growth inhibition for comp. 4 (NSC 35759) 5 (NSC 774943/1) as shown in table 1. Table1 One dose mean graph for compound 4 (NSC: 35759) and compound 5 (NSC: 774943 / 1) Conc: 1.00E-5 Molar Table 2 Percent Growth inhibition of synthesized compounds against most effective cancer cell lines Figure1 Percentage growh inhibition for cytotoxic activity evaluation of synthesized ompounds Figure 2 Percentage growh inhibitions for cytotoxic activity evaluation of compound 4 Figure 3 Percentage growh inhibitions for cytotoxic activity evaluation of compound 5 Figure 4 Percentage growh inhibitions for cytotoxic activity evaluation of standard anticancer drug Vincristine sulphate. DISCUSSION Molecules synthesized under ultrasonication conditions. The method provides several advantages over current reaction methodologies, including a simple work up procedure. The ultrasonic method of synthesis was also found to be simple, ecofriendly, reduces reaction time and gave good yield when compared with traditional methods of synthesis. Cytotoxicity evaluation revealed that out of 60 human tumor cell lines, compound 5 is highly effective against SNB-75 (CNS cancer) and UO-31 (Renal cancer) human tumor cell lines.Compund 5 showed highly cytotoxic activity against NCI-H23 cell line (Non-Small cell lung cancer) as compared to standard drug Vincristine sulphate. Compound 5 also had shown moderate cytotoxicity against T-47D (Breast cancer), CAKI-1, UO-31(Renal cancer), HL-16TB (Leukemia), NCI-H23, NCI-H522 (Non-Small cell lung cancer), SNB-75 (CNS cancer), MALME-3 (Melanoma). CONCLUSION In the process of anticancer drug discovery, to find new potential anti-lung cancer agent, we designed and synthesized molecule 5. As compund 5 showed highly cytotoxicity against NCI-H23 cell line (Non-Small cell lung cancer) in comparision to standard drug Vincristine sulphate, in future, research and development with designing desired modifications of molecule 4 and 5 may develop in safer and effective potential anticancer molecules. ACKNOWLEDGEMENT Authors are thankful to BCUD, University of Pune, INDIA (Project-13PHM000018) for financial assistance, NCI, USA for carrying out anticancer activity, SAIF Punjab University for spectral study, JNTU Hyderabad and Dr. Dhake A. S., SMBT College of Pharmacy, Dhamangaon, Nasik (MS) India for providing necessary facilities to carry out the research work. COMPETING INTERESTS The authors declare that they have no competing interest. REFERENCES Kier LB, Dhawan D and Fregley MJ (1964) Synthesis of alkyl substituted sydnone carboxylic acids. J. Pharm. Sci., 53: 677. Dunkley CS and Thoman CJ (2003) Synthesis and biological evaluation of a novel phenyl substituted sydnone series as potential antitumor agents. Bioorg Med. Chem. Lett., 13(17): 2899-2901. Satyanarayana K and Rao MN (1995) Synthesis, antiinflammatory, analgesic and antipyretic testing of 4-[1-oxo-(3-substituted aryl)-2-propenyl]-3-phenylsydnones and of 3-[4-[3-(substituted aryl)-1-oxo-2-propenyl] phenyl] sydnone J. Pharm. Sci., 84(2):263-6. Kavali JR and Badami BV (2000) 1, 5-Benzodiazepine derivatives of 3-arylsydnones: synthesis and antimicrobial activity of 3-aryl-4-[2†²-aryl-2†², 4†², 6†², 7†²-tetrahydro-(1†²H)-1†², 5†²-benzodiazepine-4†²-yl] sydnones. IL Farmaco, 55: 406-409. Fregly MJ, Kier LB and Dhawan D (1964) Chloruretic, naturetic and depressor activities of some substituted sydnones. Tox Appl. Pharmacol., 6:529 Stewart TG and Kier LB (1965) Synthesis of several mesoionic 1, 3,4-thiadiazoles. J. Pharm. Sci., 54:731. Roche EB and Kier LB (1965) Synthesis of two 4,5- Dialkylsydnone J Pharm. Sci., 54: 1700. Dunkley CS and Thoman CJ (2003) Synthesis and biological evaluation of a novel phenyl substituted sydnone series (VII) as potential antitumor agents. ChemInform, 34:129. Garraway LA, Widlund HR, Rubin MA, Getz G, Berger AJ, Ramaswamy S, Beroukhim R, Milner DA, Granter SR, Du J, Lee C, Wagner SN, Li C, Golub TR, Rimm DL, Meyerson ML, Fisher DE and Sellers WR (2005). Integrative genomic analyses identify MITF as a lineage survival oncogene amplified in malignant melanoma. Nature, 436 (7047):117-122. Adams S, Robbins FM, Chen D, Wagage D, Holbeck SL, Morse HC 3rd, Stroncek D and Marincola FM (2005). HLA class I and II genotype of the NCI-60 cell lines. J. Transl. Med., 3(1):11. Roschke AV, Tonon G, Gehlhaus KS, McTyre N, Bussey KJ, Lababidi S, Scudiero DA, Weinstein JN and Kirsch IR (2003) Karyotypic complexity of the NCI-60 drug screening panel. Cancer Res, 63(24):8634-8647. Lorenzi PL, Reinhold WC, Varma S, Hutchinson AA, Pommier Y, Chanock SJ, Weinstein JN (2009) DNA Fingerprinting of the NCI 60 cell line panel. Mol. Cancer Ther, 8(4):713-24. Mingyi M, Longru S and LouMei Ji (2013) Synthesis and biological evaluation of Combretastatin A-4 derivatives containing a 3-O-substituted carbonic ether moiety as potential antitumor agents. Chemistry Central Journal, 7(1):179. Al-Suwaidan IA, Alanazi AM Abdel-Aziz AA, Mohamed MA and El-Azab AS (2013). Design, synthesis and biological evaluation of 2-mercapto-3-phenethylquinazoline bearing anilide fragments as potential antitumor agents: Molecular docking study. Bioorganic medicinal chemistry letters, 23:3935-3941. Senff-Ribeiro A, Echevarria A, Silva EF, Franco CRC, Veiga SS and Oliveira MBM (2004) Cytotoxic effect of a new 1, 3, 4-thiadiazolium mesoionic compound (MI-D) on cell lines of human melanoma. Br. J. Cancer; 91(2): 297–304. Butkovic K, Marinic Z and Sindler-Kulyka M (2011) Synthesis of 3-(o-stilbenyl) sydnone and 3-(o-stilbenyl)-4-substitutedsydnone derivatives and their antitumor evaluation. ARKIVOC, 1:15