Development of Anti-Cancer Agent

Development of Anti- crabmeat AgentDesign, ultrasonography assisted tax write-off and in-vitro antitumour exercise of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor carrell linesSachin K. Bhosale, Shreenivas R. Deshpandeand Rajendra D. WaghDesign, ultrasound asisted synthesis and in-vitro antitumor action at law of 3-(4-chlorophenyl) sydnone and 3-(4-chlorophenyl) sydnonecarboxaldehyde against 60 Human Tumor cubicleular phone linesAbstractIn an attempt to develop an good and safer antitumour agent, 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde 5 stool been designed and synthesized nether unhearableation by formylation of 3-(4-chlorophenyl)-sydnone 4 and characterized by phantasmal studies. The ultrasonic method of synthesis was engraft to be primary, ecofriendly, reduces reaction date and gave good yield when comp bed with traditional methods of synthesis. Anticancer activity of the obscures were tested against 60 human t umor carrell lines and compared with type drug vincristine sulphate. Cytotoxicity paygrade revealed that change 5 is most male against NCI-H23 (Non-Small carrel lung cancer) with comparative activity of Vincristine sulphate. heterogeneous 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 prison 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, UltrasonicationINTRODUCTIONMesoionic sydnone compounds have shown a variety of biologic 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 noteworthy 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 hefty histiocytoma (B10MCII) tumor bearing mice(Dunkley et al., 2003). It was also found that the larger hetero rings p-piperidino and p-morpholino, were slight potent (Kier et al., 1964, Dunkley et al., 2003). Herein we report the design, synthesis, and biological military rating of dickens para-chloro substituted analogues of sudnone molecules 4 and 5. These were tested for 60 human tumor cell lines at the subject area Cancer build 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 ont ogenesis of at least one cell line to less than 32%, and twain are proved to be moderate active and versatile against to each one cell line.MATERIALS AND METHODSAll chemicals and reagents were purchased form from Sigma-Aldrich, Mumbai, India. Melting points of the intermediates and the final products were save victimization a Systolic melting point apparatus and are report 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 sum total spectrometer. nuclear magnetic resonance spectra were obtained on a BRUKER AVANCE II 400 proton magnetic resonance spectrometer at 500 MHz for 1H and 125 MHz for 13C, the chemical shifts are expressed in (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 fu ll agreement with the proposed structures within 0.4% of the metaphysical values. The ultrasonic irradiation was performed by using a Biotechnics India TM ultrasonic cleaner bath, model 1510, AC input 115 V, output 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 form of Para-Chloroaniline (2.80 g, 0.02mol) and chloroethyl acetate (2.12 mL, 0.02mol) was added to the solution of grain alcohol (20 mL) and anhydrous atomic number 11 acetate (3.28 g, 0.04mol) nether ultrasonication conditions and allowed to react for 120 min. The mixture was diluted with 20 mL of water and cooled in refrigerator for nightlong and recrystallized in ethanol to yield 1 (83%), mp 114-116 C. IR (KBr) 3327, 2950, 2934, 2879, 1756, 1069 1H NMR (400 MHz, DMSO-d6) 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) 14.65, 4 4.73, 62.07, 115.12, 123.22, 129.01, 146.26, 173.13.Synthesis of N-(4-chlorophenyl) genus 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 C. IR (KBr) 3319, 3277, 2951, 2937, 2879, 1703, 1063 1H NMR (400 MHz, DMSO-d6) 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) 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 intemperate hydrochloric acid to the filtrate. Precipitate was filtered and recrystallized in methanol to yield 3 (81%), mp 109-111 C. IR (KBr) 3255-2521, 2923, 2849, 1711, 1569, 1325, 1062 1H NMR (400 MHz, DMSO-d6) 5.02 (s, 2H, CH2), 6.93-7.45 (m, 4H, Ar-H), 11.55 (s, 1H, COOH) 13C NMR (40 MHz, DMSO-d6) 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 remaining overnightat room temperature. The solution was poured slowly into cold water which was very hygienic stirred. The crude sydnone was filtered, dried and recrystallized in ethanol to yield 4 (93%), mp 139-141 C. IR (KBr) 3181, 1748, 1053. 1H NMR (400 MHz, DMSO-d6) 7.23 (s, 1H, sydnone), 7.49-8.13 (m, 4H, Ar-H). 13C NMR (40 MHz, DMSO-d6) 123.15, 126.49, 131.53, 136.13, 141.13, 170.07Synthesis of 3-(4-chlorophenyl)-4-sydnonecarboxaldehyde (5)N-Methylformanilide 2.84g (0.0210mol) and phospho ryl 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 (C=O sydnone ring), 1640 (C=O aldehyde). 1H NMR (400 MHz, DMSO-d6) 7.2, 7.2, 7.3, 7.3 (Cl-Ph-), 9.61 (Sydnone-4-CHO). 13C NMR (40 MHz, DMSO-d6) 127, 129, 129, 130, 130, 134 (Cl-Ph-) 190 (CHO). Element analysis C 48.13, H 2.24, N 12.47, O 21.37Scheme 1-Synthesis of 3-(substituted aryl)-4-sydnonecarboxaldehyde (5) under ultrasonication conditions1.3. Anticancer classingThe synthesized compound was screened for preliminary anticancer assay by Nation al 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 harvesting forbiddance or cell killing of particular tumor cell lines by specific compound. The screening begins with the valuation of selected compounds against these 60 cell lines at a single venereal infection 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, Senff-Ribeiro et al., 2004, Butkovic et al., 2011).RESULTCom pund 5 showed super 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 hoagyTable 2 Percent Growth inhibition of synthesized compounds against most achievementive cancer cell linesFigure1 Percentage growh inhibition for cytotoxic activity military rank of synthesized ompoundsFigure 2 Percentage growh inhibitions for cytotoxic activity evaluation of compound 4Figure 3 Percentage growh inhibitions for cytotoxic activity evaluation of compound 5Figure 4 Percentage growh inhibitions for cytotoxic activity eval uation of standard anticancer drug Vincristine sulphate.DISCUSSIONMolecules 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 exceedingly 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).CONCLUSIONIn the process of anticancer drug discovery, to find freshly say-so 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.ACKNOWLEDGEMENTAuthors 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.REFERENCESKier LB, Dhawan D and Fregley MJ (1964) Synthesis of alkyl substituted sydnone carboxylic acids. J. Pharm. 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