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Simple, fast and efficient synthesis of β –keto esters from the esters of heteroaryl compounds, its antimicrobial study and cytotoxicity towards various cancer cell lines R. Venkat Ragavan, V. Vijayakumar, K. Rajesh, B. Palakshi Reddy, S. Karthikeyan, N. Suchetha Kumari PII: DOI: Reference: To appear in: Received Date: Revised Date: Accepted Date: S0960-894X(12)00447-7 10.1016/j.bmcl.2012.04.008 BMCL 18919 Bioorganic & Medicinal Chemistry Letters 27 October 2011 22 March 2012 3 April 2012

Please cite this article as: Venkat Ragavan, R., Vijayakumar, V., Rajesh, K., Palakshi Reddy, B., Karthikeyan, S., Suchetha Kumari, N., Simple, fast and efficient synthesis of β –keto esters from the esters of heteroaryl compounds, its antimicrobial study and cytotoxicity towards various cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2012), doi: 10.1016/j.bmcl.2012.04.008

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Suchetha Kumari c O Ar O O O Ar O O O O O O + LiHMDS THF [Cross condensed desired product] 1-21 [Self condensed product] . Palakshi Reddy a. B. ∗. S. V. Rajesh a. Venkat Ragavan a. Vijayakumar a. K. its antimicrobial study and cytotoxicity towards various cancer cell lines R. esters from the esters of heteroaryl compounds.Graphical Abstract Simple. fast and efficient synthesis of β–keto Leave this area blank for abstract info. Karthikeyan b and N.

K. β-keto methyl (or) ethyl esters are the most preferred esters for these transformations. Deralakatte 574 162. Many hetero cyclic compounds such as pyrazolones.11 and bistrimethylsilylmalonate12. The synthesis of β-keto esters from ketones like caboxylation of ketone enolates16. always suffers by the steric effect of tert-butyl group. VIT University. Palakshi Reddy a. India. O Ar O O O O Ar O O O O O + LiHMDS THF 1-21 [Cross condensed desired product] [Self condensed product] Scheme 1. Department of Biochemistry. 18 synthesized β-keto esters by treating the esters with the enolate of ethyl acetate (or) tert-butyl acetate in the refluxing conditions but these transformations also require the large quantity of ethyl acetate. There are methods to synthesize β-keto esters from esters1. India. ARTICLE INFO Article history: Received Revised Accepted Available online Keywords: Heteroaryl esters β-keto esters LiHMDS Ttrans-esterification Cytotoxic studies ABSTRACT A series of β-keto esters were synthesised from heteroaryl esters and ethyl acetate using LiHMDS as base at -50 to -30°C. The increase in yields of cross condensed product were observed and the percentage of self condensed product was reduced drastically by applying the suitable base (LiHMDS).com .. School of Bio Sciences & Technology. Ohta et al. inconsistent yield and use of excess reagent. were prepared traditionally from their corresponding β-keto esters. βketo esters of N-protected amino acids were efficiently prepared by treating the corresponding esters with the enolate of tert-butyl acetate (prepared using LDA in THF) at -50°C to -78°C19. pyrimidones. its antimicrobial study and cytotoxicity towards various cancer cell lines R. All the synthesized compounds were subjected to test for their cytotoxicity towards various cancer cell lines and also tested for their antimicrobial activity towards various bacterial and fungal strains and some of them were found to have promising activity. All rights reserved. however these methods suffer by inconsistent yield in case of aliphatic acylation. Efficient synthesis of β-keto esters of heteroaryl compounds 1-21. e-mail: kvpsvijayakumar@gmail. isooxazolones with hydrazines.13. oxazoles. 13C NMR and mass spectral data. All these β-keto esters were characterized using 1H NMR. aldehydes3.Bioorganic & Medicinal Chemistry Letters journal homepage: www. fast and efficient synthesis of β–keto esters from the esters of heteroaryl compounds. Vijayakumar a. The most general and simple method to synthesize β-keto esters in these days is the reaction of dimethyl or ethyl carbonate with ketone in the presence of strong base also suffers by long reaction time. Venkat Ragavan a. Reactions of these β-keto-tert-butyl esters in the preparation of pyrazolones. thiazoles. India. tert-butoxide. A number of methods such as acylation of enolates of malonates4. Industrial Biotechnology Division. K. ——— ∗ Corresponding author. mixed malonate esters10.S. Tel. S.17 using carbon dioxide and carbon monoxide sources in the presence of palladium or transition metal catalysts. Vellore 632 014. Vellore 632 014.elsevier. B. School of Advanced Sciences.15. solvent and the minimum amount of ethyl acetate. Hegde Medical Academy. VIT University. Rajesh a. ∗. excess of strong base (sodium ethoxide. often. acylation of meldrum’s acid6-9. Karthikeyan b and N.: +91-416-220-2535. NaH) and refluxing conditions with longer time under the usual reaction conditions.com Simple. etc.2. a chelating effect has been employed to lock the enolate anion of malonate using lithium and magnesium salts14. fax: +91-416-224-3092.5. but these methods have main limitation in varying the substituents. pyrimidines. Apart from that these transformations suffer by large quantity of by products (formed by the self condensation of ethyl acetate in particular) and require tedious work up procedures to remove the same. A plausible mechanism is also depicted to prove the formation of trans-esterified products. Suchetha Kumari c a b c Organic Chemistry Division. V. amidines and hydroxylamine respectively. isooxazoles. 2011 Elsevier Ltd.

5) 25(6. In case of 17 the electron donating nature of N-ethyl group increases the electron density in the molecule.25) 28(6. 902). In case of 13 and 20. Effect of solvent and base on the yield of 8 Base LiHMDS (1. fumigates 18(12. Cleavage of the Boc and Cbz groups were observed within 10 min.25) 19(12. MIC in mg/mL given in parenthesis. 8 and 14 are highly active against all the bacterial stains tested (Though all the compounds are subjected to the study. In these cases (13 and 20). Reaction with three equivalent of LDA and three equivalent of ethyl acetate gave the moderate yield.25) 21(6.125) Penicillium 17(12.25) The fungicidal activity of β-keto esters of heterocyclic compounds are very good. ethyl acetate and the results are summarized in Table 1.25) 23(6. isoquinoline.5) 15(12. Aspergillus fumigates (NCIM No.25) 25(12.) KOtBu (3 eq. Electrophilicity of the heterocyclic esters will change drastically by changing the substituents.5) 26(6. 13C NMR and LC-MS spectral data.5) 27(3.5) 17(12.5 equivalents of LiHMDS and 7 equivalent of ethyl acetate at -50 to -30°C produced the better yield. Pseudomonas aeruginosa (ATTC-27853).) LDA (1.) NaOMe (2 eq.25) 22(6. From the impressive results of the biological activity further conversion of these β-keto esters into the heterocyclic compounds are required.25) 20(6. By applying the suitable base.5) 18(12. Pyrimidine 2-β-keto ester is moderately active. produced the Boc-cleaved starting material and the desired product in 56% yield. indole) were converted in to the corresponding β-keto esters in good yield.25) P.25) 22(12. Compound 9 was moderately active against all the stains and the β-keto esters of heterocyclic compounds shown a good bacterial activity in particular β-keto esters of benzothiazole. yield got improved to 78%. Antifungal activities of the newly synthesized compounds. imidazole. Presence of two methoxy groups in 16 decreases the electrophilicity of the esters by donating the electrons. 7 eq.25) 23(6. We have investigated newly synthesized β-ketoesters for their antibacterial activity against Escherichia coli (ATTC-25922).125) A. pyrazine are equipotent or even higher than the standard used in some cases.5 eq. 75 eq. Among the tested compounds.25) 23(6.25) 25(6. solvents. Penicillium marneffei (Recultured) and Trichophyton mentagrophytes (Recultured) in DMSO by the serial plate dilution method34-35. In the case of 16 and 17 the unexpected and interesting trans-esterification was observed instead of desired β-keto esters.0M THF) (3 eq. Solvent THF THF Toluene THF THF THF THF Temp -78°C -78°C -78°C -78°C Reflux 25°C -50°C to-30°C Yield (%)a 18b 56c 78d 9e 0f 0g 94h Table 3.) Ethylacetate 3 eq. aeruginosa 22(6. 7.) NaH (2 eq. Among the tested compounds. standard = Ciprofloxacin) Compound 2 7 8 9 14 standard S. 7 and 14 were moderately active against all the stains. Reaction using NaH gave only 9% yield. Standard = Ciclopiroxolamine) Compd 2 7 8 9 14 standard Trichophyton 17(12.25) 32(6. Compounds 2. The importance of these esters. the same methodology has been applied to synthesis various β-keto esters (1-21) using different heterocyclic esters to prove the generality of the reaction (Table 2).25) A.5) 23(6.25) 20(6. compounds 2.25) 23(12.25) 23(6. The experimental results clearly reveal that heterocyclic esters with even lower to moderate electrophilicity undergoes the condensation smoothly without unwanted byproducts to the greater extent. Esters of different heterocyclic compounds (Quinoline. solvent and the minimum amount of ethyl acetate we have increased the yields of the cross condensed product and the percentage of self condensed product was reduced drastically.5) 22(6. pyridine.25) 19(6.25) E. flavus 19(12.5) 27(3. coli 22(6.0M THF) (1 eq.5) 24(6. isoquinoline. Reactivity of the esters mainly depends upon the electrophilicity. 50 eq. compounds 8 and 9 are highly active against all the stains tested. Antifungal activity was determined by measuring the inhibition zone and compared with the standard Ciclopiroxolamine (Though all the compounds are subjected to the study only the compounds with good inhibition results given in Table 4). Benzothiazole and 2-pyrimidine β-keto esters . This may be due to the weaker electrophilicity of the ester. 3 eq.) LiHMDS (3 eq. pyrazine.25) 22(12. only the compounds with good inhibition results given in Table 3).25) 22(6. All these β-keto esters (1-21) were characterized through 1H NMR. Table 1. thiazole. In most of the cases we did not get the product when we tried to synthesize β-keto esters of some heteroaryl esters by applying the existing methods. in particular β-keto esters of isoquinoline and pyrazine are equipotent or even higher than the standard used. This may be due to the labile nature of the protecting groups particularly in indole systems.5) 24(6. whereas in the case of 20 the Cbz group was completely cleaved and gave the Cbz-cleaved starting material.Unfortunately the existing methods do not produce the β-keto ester of heteroaryls from the heteroaryl esters in good yield. Then the yield of cross condensed product was optimized with reference to the compound 8 by changing the equivalence of base. 10 eq. Other bases NaOMe and tert-BuOK did not give the product at all.5) 23(6. After finding the optimized condition. 7 eq.) LiHMDS (1. and Klebsiella pneumonia (recultured) bacterial strains by the disc diffusion method31-33. causes the complete transesterification in this particular case.0M THF) (3. reagent.5) 18(12. 524).pneumonia 23(6.5) 26(6.25) 17(12.25) Our initial attempt with one equivalent of LiHMDS as base was disappointing and produced only 18% of product. Finally a reaction with 3. So it is not easy to avoid the self-condensed product. we observed unexpected deprotection of the Boc and Cbz groups. To overcome the difficulties present in the synthesis of β-keto esters of heteroaryl esters as the continuation of our interest towards the synthesis of β-keto esters and pyrazolones20-30.25) 24(6.25) 16(12.25) K.25) 18(12. Table 4.25) 27(6. Antibacterial activity of the newly synthesized compounds (Zone of inhibition in mm. led us to develop a simple method to synthesize β-keto ethyl esters of hetero aryls in good yield by changing the base.25) 23(6. When the reaction was carried out with hydrocarbon solvent like toluene and LiHMDS as base. aureus 21(6. benzo-thiazole.5) 22(6. In compound 13 the Boc group was partially deprotected and Similarly all these newly synthesized β-ketoesters were screened for their antifungal activity against Aspergillus flavus (NCIM No.5) 22(6. trans-esterified products were not observed even in trace amount. MIC in mg/mL given in parenthesis. we attempted the synthesis of 1-21 from their corresponding heteroaryl esters and ethyl acetate using Scheme 1. Staphylococcus aureus (ATTC-25923). (Zone of inhibition in mm. equivalence of base reagent and reaction conditions. pyrimidine.

were moderately active. Synthesis of β-ketoesters by cross-Claisen condensation Compd. Table 2. Because of the good fungicidal activity further conversion of these β-keto esters into the heterocyclic compounds are required. 1 N Ester COOC2H 5 Product COCH2COOC2H 5 N Yield (%) 94 2 COOC2H5 N Br S COCH2COOC2H 5 N Br S 97 3 Cl S N COOC 2H5 Cl S N N N COCH2COOC2H 5 91 4 N N COOC2H 5 COCH 2COOC 2H5 88 5 N N COOC2H5 N N COCH 2COOC2H5 0a 6 COOC2H5 COCH 2COOC2H5 0a 7 COOC2H 5 N COCH 2COOC 2H5 N 85 8 Cl N N COOC2H 5 Cl COOC2H 5 N N N COCH2COOC2H 5 83 9 COCH2COOC2H 5 N N 72 10 N N COOC2H5 66 COCH2COOC2H 5 N 11 N COOC 2H 5 N COCH 2COOC2H 5 92 12 COOC2H 5 COCH 2COOC2H5 87 N N 13 COOCH 3 N Boc COCH 2COOC2H 5 N Boc 56 14 N COOC2H5 N N COCH 2COOC2H5 N 85 .

Calu-1 (human lung carcinoma) and HCT-116 (human colon cancer). Logarithmically growing cells were plated at a density of 5 × 103 cells/well in a 96-well tissue culture grade micro-plate and . Panc-1 (human pancreatic adenocarcinoma). H460 (human non small cell lung carcinoma). ACHN (human renal cell carcinoma). A panel of five cancer cells representing multiple cancers of clinical relevance were obtained from ATCC (American Type Culture Collection). namely. Compd 1 2 3 4 5 7 8 9 10 11 12 13 14 15 18 19 21 Std 1 Std 2 Screening concentration in µM 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Flavopiridol (1000 nM) Gemcitabine (1000 nM) H460 (Non Small cell Lung cancer) 20 14 24 20 13 34 34 17 14 19 10 39 20 14 19 87 36 71 73 HCT116 (Colon Cancer) 28 39 48 30 20 26 40 29 11 30 39 33 23 28 24 90 21 78 74 ACHN (Renal cancer) 20 31 37 34 15 30 30 30 30 30 31 30 10 21 25 93 25 71 73 Calu1 (Lung Cancer) 23 19 32 16 12 30 31 21 30 30 12 20 17 24 20 94 22 88 71 Panc-1 (Pancreatic cancer) 26 27 29 27 35 35 14 23 35 35 10 23 37 26 30 97 27 74 79 All the synthesised compounds 1-21 have been subjected to WST-1 cytotoxicity assay. Cells were maintained in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% heat inactivated Fetal Bovine Serum and kept in humidified 5% CO2 incubator at 37 °C.15 N N CH2CH 2OCH 3 COOC2H5 N N CH2CH 2OCH 3 COCH 2COOC2H5 76 16 H3CO N COOCH 3 OCH 3 COCH2COOC2H 5 H 3CO N OCH 3 0a 17 COOCH 3 N COCH 2COOC2H 5 N 0a 18 COOC2H 5 N COCH(CH 3)COOC 2H 5 N 89 19 N N COOC2H 5 N N COCH(CH 3)COOC2H 5 84 20 COOCH 3 N Cbz COCH 2COOC2H 5 N Cbz 0a 21 N a 78 COOC2H 5 N COCH2COOC2H 5 percentage of products in crude LC-MS Table 5. Cytotoxic activity of the cross condensed β-keto esters (1-21).

S.. fast and an efficient method has been developed to synthesis β−keto esters from their corresponding heteroaryl esters in mild conditions using LiHMDS as a base. 7. Petersen.7 14. 4. Soc. B.0 100. 24.. 3021.. S. Venkat Ragavan.0 2. 35. 1985. J. K.. K. H. 1988. Marmion. 2087. o142. S.. Acta. Vijayakumar. o3019..3 1 3 10 30 IC 50 (µM) H460 3. Tetrahedron. M. E67.. J. R. Acta. A. K.. Sumino.. H. D. Effect of compound 19 on proliferation of different cell lines. R. Mansour.. 15. H. V. Panc-1 and MCF10A cancer cell lines respectively. Sarveswari. 26. Clin. 3469. Sucheta Kumari. R. O. H. O. Chem. M. V. 501. 770. 1925. J.0 Calu1 1. J. Con (µM) 0. Riva. S. 29. R.8 21. M. 1987. Vijayakumar. M... Oikawa. HCT116.. S. 127. 19. P.. 196. R. The compound 19 was found to be inhibitive against all these cell lines and hence its % cytotoxicity for various concentrations has been observed (Table 6). R.. Elofson. Tetrahedron.8 26.. V. Lee.3 99. Vijayakumar.. F.. Sugano.2 100. Chem. J. Sarveswari. 2003. 12. 66. E66. Shahani. Japan). T. 16.8. S. Perkin. Table 6.. Medicinal Microbiology. I. H. T. Am. 20. Acta. Chem. B. 2000. R. Adkins... Sarveswari. Carrie. S. 25. 12th ed.allowed to recover overnight.. Y.8 92. A. E66.. 3622. R. K.. F. Venkat Ragavan. Fun.. . 43. Narasimhamurthy. 2010. Vijayakumar. 2009. R..4 82. G. Acta. T. 50.0 39. Kalde. R. 2011. T.4 22. Maryland..9 MCF10A -15. R. J. Cytotoxic activity of the cross condensed β-keto ester 19 at varying concentrations and its IC 50 (µM). 34. Qisairi. Synthesis.9 29. A. D. C. 2221. Y. Pratt. Tubio. A. Cryst. Synthetic. M.. Heterocyclic. T. S. K. L. V. 23. 1039.8 6. Agr. Maibaum. B.. H. Robinson. Ltd. compounds 19 and 8 have been found to be the promising candidates for anti cancer activity and microbial agent respectively and hence further attention of these compounds are in need. 2000. H. Yeap. 11. 21. 53. Chem. Rathore. Venkat Ragavan.9 46. I. o1697. Kohsuke. Heterocycl. H. Sarveswari. Hauser. Chem.. S. M.. o1357.. Sarveswari. monosodium salt assay). 2. 33. L. R. A.. Yadav.1 100. Hayakawa. 10. Chu. Acknowledgments Authors are grateful to Syngene International Pvt. Reddy. Phillips....2 Figure 1. Products. Yonemitsu.. D. R. T. 323. Venkat Ragavan. Prep.9 and 21.2 67.7 9. Collins. Chantal. Jones..5 -6. 1988.. Vijayakumar. P.5 2. E66. H.. London). Maleczka. 18. Cryst. C. Robinson. 1985. D. Hudicky. M. Cryst. Taylor. J. Suzuki. Acta. 71... R. 3.. S. J.6 6. T. S. Org.. (WST1 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2.. Venkat Ragavan. 9. J.3 3. 1811. A. Chem. Barbieri. L. 196. 32. Venkatesh. 27. 1944. Satake.. G. G. 1978. US Patent 4447444.. 2010. Mellows. 28. V.4 -28. Cryst. Acta. 2010. 20. Venkat Ragavan. 13. 1286. V.. R. K.2%. By plotting these values (Fig. Morrison. 3. C. 184. Trabazo. 1173. E67. Y. GmbH (Gesellschaft mit beschränkter Haftung. Food.2 -14. Fun. Fun. Motley. 37. Cryst. 1987. The cells were challenged with varying concentration of compounds for 48 h. 36. 38. Duguid. 2010.. Cytotoxicity of all the compounds have been determined by measuring the absorbance on Tecan Sapphire multi-fluorescence micro-plate reader (Tecan.. Trans. 2. 24. Med.8 60. Subba Reddy. Rossow.. C. M. Authors are thankful to the VIT management for their generous support and facilities. 2010. 2nd ed. Stahler. T. ACHN. 44. Sucheta Kumari. 5µL/well CCK-8 reagent was added and plates were incubated for 2 h. M.2 µM for H460. Seonane.6 44.. Proced... R. R. Skaggs. V. B. E. 2. L.. A. Vijayakumar. Microbiol. Nat. M. Am. W. Kojima. T. Proced.. Venkat Ragavan. C. 1989. J. D. Eur.6 55. Soc.2 2. K. 41. Rich. Ed. D. Vijayakumar. R..1) the IC 50 value for cancer cell lines has been found as 3. cell toxicity was determined by CCK-8 (Cell Counting Kit-8) reagent (Dojindo Molecular Technologies. Butterworth. R. Okamoto.9 6.0 100. In accordance with the manufacturer’s instructions36. Calu-1. 10. N.2 38. 1987. Ohta. Banerji. 15. J. 71. H. J.3 10. B. Chem. Swain. 2011. Chem.. 22. Chem.9 32.. Molecular and Cellular Biology.1 0. 14. All these compounds have been subjected to WTS-1 cyctotoxicity assay and antimicrobial screening. J. N. Cryst. Shahani. Venkat Ragavan.0 13.. Izawa. After 48 h of incubation. 3852. 2010. Med. 1-Alkyl-3-alkoxymethyl-4-alkoxy5-dialkyl carbamethoxy pyrazoles and use as aphicides.. o403.3. E66. K. H. Synthetic. 43.03 0. Acta. S. In summary simple. Sarveswari. 1985.. C. The study reveals the promising activity of 19 towards the above mentioned cancer cell lines and further investigations are in need. L. Tetrahedron. Org. Fun.3 24. 3163. 2000.1 -9.. R. Radesca. Venkat Ragavan.8 85.. J. Baumgarten.8 Panc-1 8. M. 2011. Soc. D. Lett. London. M. 2.... Each independent experiment was performed thrice and tabulated in Table 5. T.2 51. Chemical and Pharmaceutical Bulletin. S.3 44. W.. M. Int. o2760.. Fun. 61. J. 20. Fun. Shahani. Loh. 48. B. Rathke. 1. Hauser.. G. 1975..4 18. 221. Patrick. 31. Hamed.. N. M. Y. Shahani. 166. A.. W. 1978. 44.0 1.. Eur. K. Breslow. Hidenori. J. Commun. 17. English: company with limited liability) at a wavelength of 450 nm corrected to 650 nm and normalized to controls.. J.. Microbiological Methods.6 15. T. 1949. 869. o151. 2254. 8. Evans. 646. 453. A. Org.8 92. 20. Vijayakumar. S. Control cells received standard media containing dimethylsulfoxide vehicle at a concentration of 0. 2010. Prep. Vijayakumar.. Soc.. Monica. Takashi. 45. Acta. V.. R. 875. V. V.. Bengaluru for providing spectral facilities.. Int. K. N.. H. Eeshwaraiah.9 19. E. Am.01 0.. Waltersdorfer. Chem.. Loh.. References and notes 1. K. Cryst. E66. S. Chem. E67. J.. E. E. Sim. o2129. Trans 1. C. Sarveswari.3 ACHN -23. Tetrahedron. Shimabayashi. R. W. P. Mills. V. W.. Soc.. Warnock. Germany.. Schmidt.. Mori. D. Fun. Venkat Ragavan. J. (Churchil Livingstone. 45. R. R. 30. E. H. 1976. A.. Cryst. 5. Inc.0 27. H. 773. Kart... Mills. Vijayakumar.1 82. 1949. 1976. Chem. S. Lett. Org. 6. Venkat Ragavan.. Umpierrez. S. V. Commun. Brown. Kohei. Vijayakumar. A.. D. Vaultier.0 3 HCT116 9. Honda.6 67. Cruickshank.0 100.. 2005. Nowak. J. 33. Turchi.4-disulfo phenyl)]-2H-tetrazolium. Katayama. Khoukhi.. G.. G. A. 1990. C.

.Supplementary Material The experimental data along the spectral evidence can be found with the supplementary material.

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