Synthesis and Characterization of Ethyl 7-Acetyl-2-substituted 3-( substituted benzoyl ) indolizine-1-carboxylates for in vitro Anticancer Activity

Indolizines are heteroaromatic compounds containing condensed five and six membered rings with bridging nitrogen. They are isoelectronic with indole and represent a group of heterocyclic compounds structurally related to purines. Indolizine skeletons with different degrees of unsaturation are present in wide variety of natural and unnatural azacyclic compounds. Most of the naturally occurring indolizines have been isolated from species of genus dendrobates (poison-arrow frogs) [1,2]; monomorium (ants) [3]; dendrobium (orchids) [4]; tylophora [5] and the leguminosae family (plants). Indolizine alkaloids display broad spectrum of biological activities. Polyhydroxylated indolizine alkaloids are excellent inhibitors of biologically important pathways. These include the binding and processing of glycoproteins [6], potent glycosidase inhibitor activities [7,8], activity against AIDS [9,10] as well as against other important pathologies [11]. The 1-azabicyclo[4,3,0]nonane (indolizine) framework occupies a special place in heterocyclic systems due to the presence of this structural assembly in a number of natural products of biological importance such as tabersonine [12], (-)-strychnine [13], (+)-vinblastine [14], (-)-monomorine [15], (-)-gephyrotoxin Synthesis and Characterization of Ethyl 7-Acetyl-2-substituted 3-(substituted benzoyl)indolizine-1-carboxylates for in vitro Anticancer Activity

Because of unexceptional potential of these indolizines, noteworthy advances on their synthesis and biological evaluation have gone unreported.A careful look at the indolizine framework would logically suggest that one step or one-pot simultaneously tandem construction of the N-C bond and C-C bond on to six membered nitrogen heterocycles (piperidine/ pyridine), in an appropriately organized manner using a suitable reagent would lead to the formation of the desired azabicyclo-(4.3.0)nonaneframe work [28][29][30][31][32].

EXPERIMENTAL
Commercially available chemicals were procured from Sigma Aldrich.Hot-air dried glass wares were used to carry out reactions under nitrogen atmosphere using dry solvents.Monitoring of chemical reactions was done on analytical thin layer chromatography (TLC) with Merck 60 F-254 silica-gel plates.NMR spectra ( 1 H and 13 C) were recorded at ambient temperature using CDCl3, DMSO-d6 as a solvent on 400 MHz Bruker-spectrometer using tetramethylsilane (TMS) as internal standard.Chemical shifts were showed in ppm (δ) and were referenced with TMS.LC-MS analysis was performed on Agilent LC-1200 series coupled with 6140 single quad mass spectro-meter with ESI +ve and -ve mode, MS range from 100-2000.IR spectra were recorded using KBr pellets on Brucker alpha FT-IR spectrometer.Perkin Elmer CHNS analyser was used for elemental analyses.

General procedure for the synthesis of ethyl 7-acetyl-3-(4-substituted benzoyl)-2-substituted indolizine-1-carboxylate:
To a solution of 4-acetyl-1-[2-(4-substituted phenyl)-2-oxoethyl]pyridinium bromide (0.0156 mol), in dry dimethyl formamide, substituted ethyl propiolate (0.0156 mol), K2CO3 (0.0156 mol, 2.15 g) was added and the reaction mixture was stirred at room temperature for 0.5 h.Completion of the reaction was monitored on TLC.After completion of the reaction, thesolvent was evaporated under reduced pressure and diluted with ethyl acetate.Organic layer was washed with water, brine and dried with sodium sulphate.The crude compounds were purified by column chromatography using 30 % n-hexane in ethyl acetate to afford the title compound 2a-2r and physical constants are tabulated in Table-1.

RESULTS AND DISCUSSION
The general route to obtain the title compounds 2a-r is illustrated in Scheme-I.Compounds 1a-f as intermediates were prepared by stirring 4-acetylpyridine with substituted phenacyl bromides separately in the presence of acetone medium at room temperature as shown in Scheme-I.The completion of reaction was monitored on thin layer chromatography (TLC), the solid deposited was filtered, dried under vacuum and recrystallized using ethanol as solvent.The yields of 1a-f obtained were up to 98-99 % and characterization was achieved by proton NMR, LC-MS and elemental analysis.Substituted indolizine compounds 2a-r have been synthesized by the reaction between 4-acetyl-1-[2-(substituted phenyl)-2-oxoethyl]pyridin-1-ium bromide and substituted alkynes in presence of anhydrous potassium carbonate in dimethyl formamide medium as depicted in Scheme-I.The reaction completion was observed on TLC and all the products have been achieved within 0.5 h with constant stirring.Column chromatography was used to purify products using 60-120 mesh silica gel using 30 % n-hexane in ethyl acetate as a solvent and the yield was found to be 54-79 %.The title compounds have been characterized by IR, NMR, LC-MS and elemental analysis.cLogP of the compounds was calculated using ChemBioDraw Ultra 13.0v and found to be in the range of 1.5990-5.7262.IR (KBr) spectrum of the compounds 2a-r had broad carbonyl (C=O) in the range of 1735-1650 cm -1 .Compounds 2m-2o having aryl nitrile group (Ar-C≡N) exhibited absorbance at 2227-2231 cm -1 .The proton NMR spectrum exhibited quartet (-CH2-) and triplet (-CH3) in the range of 4.29-4.45and 1.27-1.75ppm, respectively for ethyl ester group (-COOC2H5) and singlet (-CH3) in the range of 2.63-2.74ppm for acetyl group (-COCH3). 13C NMR spectrum of compound 2a exhibited carbonyl carbon of acetyl group (CH3CO) at 195.82 ppm.[M+H] + peak for all the synthesized compounds is observed in the mass spectrum.Results of elemental analysis were in good agreement with the calculated values of the proposed title compounds 2a-r.
Anticancer activity: Five of the selected test compounds 2b, 2h, 2i, 2q and 2r have been screened for in vitro anticancer activity against human cervix cancer cell line SiHa at 10, 20, 40 and 80 µg/mL concentration using sulforhodamine B assay [47,48].The activity was carried out at Advanced Centre for Treatment, Research and Education in Cancer, (ACTREC) Mumbai using adriamycin (ADR) (doxorubicin) as positive control and the results are presented in Table-2.Three of the compounds such as 2b, 2q and 2r at 80 µg/mL exhibited remarkable lowest cell growth promotion against human cervix cancer cell line SiHa of -63.1, 22.4 and -54.8, respectively, when compared to standard adriamycin -74.1.cLogP of test compounds 2b, 2q and 2r was 2.1814, 4.83 and 3.8061, respectively.

Conclusion
The research work is focused on the efficient synthesis of substituted indolizines analogues and the reactions performed are eco-friendly.Yield of the products including intermediates were satisfactory.In addition, some of the selected test compounds are subjected for anticancer activity and compounds 2b, 2q and 2r were found to show dose dependent anticancer activity at 10, 20, 40 and 80 µg/mL.

TABLE -
All of the products were characterized by spectral and physical data; b Yields after purification by column chromatography; c cLogP was calculated using ChemBioDraw Ultra 13.0v.