Synthesis of some new indole derivatives containing pyrazoles with potential antitumor activity

A series of new [1-(4-methoxybenzyl)indol-3-yl](1 H -pyrazol-1-yl)methanones, 1-(1-(4-methoxybenzyl)-1 H -indole-3-carbonyl)-3-subsituted-1 H -pyrazol-5(4H)-ones have been developed using the 1-(4-methoxybenzyl)-1 H -indole-3-carbohydrazide 1 as a key intermediate. The target compounds were tested in-vitro for tumor cell-growth inhibition.


Introduction
3][4] Indole derivatives constitute an important class of therapeutic agents in medicinal chemistry including anticancer, 5 antioxidant, 6 antirheumatoidal and anti-HIV 7,8 and also play a vital role in the immune system. 9,10Many indole derivatives are considered as the most potent scavenger of free radicals. 11Artificial receptors for biologically active molecules have attracted attention from the view point of molecular recognition. 12n addition, it was reported that various 3-substituted indoles had been used as starting materials for the synthesis of a number of alkaloids, agrochemicals, pharmaceuticals and perfumes. 13n the other hand, over the past two decades, pyrazole-containing compounds have received considerable attention owing to their diverse chemotherapeutic potentials including versatile antineoplastic activities.4][25] We have recently reported synthesis and antitumor activity of some indole derivatives containing 1,3,4-oxadiazole and 1,2,4-triazole A, and discovered that some of the analogs are potent and selective against various cancer cell lines. 26Combination of the pyrazole moiety with the indole nucleus may enhance these activities.In view of these previous findings, intriguing cytotoxicity of various indolyl azoles and in continuation of our interest in the functionalization of indoles (A, B, C and D), 29,30 and in the development of new synthetic methods, [26][27][28][29][30][31] we report herein on the synthesis of some new indole derivatives containing pyrazoles with potential antitumor activity.
The structures of compounds 2, 3 and 4 were confirmed by their spectral data (IR, 1 H NMR, 13 C NMR and MS) together with elemental analyses.The IR spectrum of compounds 2, 3 and 4 reveals the absence of characteristic absorption bands due to carbohydrazide NHNH2 function and showed new characteristic bands atd C≡N an2 due to NH 1-3420, 3320, 3150 and 2220 cm respectively.The 1 H NMR spectrum of compound 2 expectedly shows characteristic signals near  4.31, 1.37 assignable to CH2 and CH3 respectively and at  10.75 assignable to OH.Further confirmation was achieved by the 13 C NMR spectrum which showed signals at 49.35, 55.09 and 116.67 due to OCH3, CH2 and C≡N respectively.Chemical confirmation for the amino ester 2 was achieved by boiling with formamide at 210 ºC, the pyrazolo[3,4-d]pyrimidinone 5 resulted.The formation of the pyrimidinone 5 is clearly evidenced by disappearance of the characteristic bands which belongs NH2 and ester groups and showed new characteristic absorption bands atdue to NH and C=O respectively. 1- Treatment of the acid hydrazide 1 with ethyl acetoactetate led to the formation of the corresponding methyl-1H-pyrazol-5(4H)-one 6 in good yield, whereas when it was allowed to react with ethyl benzoylacetate under the same conditions, the open structure was obtained.The later compound could be cyclized via its boiling with high boiling point solvent (propanol) to afford the corresponding 1H-pyrazol-5(4H)-one 8 in good yield (Scheme 2).Interestingly, when the acid hydrazide was allowed to react with acetyl acetone in refluxing ethanol for 2 hours afforded directly the cyclized 3,5-dimethyl pyrazole derivative 9 in 64% yield.Whereas, heating the same acid hydrazide with benzoyl acetone under the same reaction conditions led to the formation the open structure 11.The later compound was cyclized via its boiling in propanol to afford the corresponding (1-(4-Methoxybenzyl)-1H-indol-3-yl)(3-methyl-5-phenyl-1H-pyrazol-1-yl)methanone 12 in relatively good yield (Scheme 3).
It is worthy to note that, when the carbohydrazide 1 was allowed to react with acetyl acetone in boiling ethanol for prolonged time, an unexpected debenzylation occurred and the (3,5dimethyl-1H-pyrazol-1-yl)(1H-indol-3-yl)methanone 10 was obtained.The debenzylation and the formation of compound 10 was confirmed by 1 H NMR which revealed the disappearance of the methoxy, phenyl and CH2 signals and showed new signal at  10.56 due to NH.Further confirmation for the structure of compound 10 was obtained by MS which showed the [M] + ion at m/z 239 (2%).

Scheme 3
Finally, treatment of the acid hydrazide with benzolyl acetonitrile in boiling ethanol rise to the formation of the corresponding 5-amino-3-phenyl-1H-pyrazole 13 in 75% yield.The IR spectrum of compound 13 showed characteristic absorption bands at ν 3450, 3330 due to NH2.
The biological activity of all synthesized target compounds was tested in vitro for antitumor activity using the Alamar Blue assay 32 on a panel of five human tumor cell lines at Zentaris, Germany.The cytotoxicity was evaluated on five different cell lines, cervix cancer (KB/HELA), ovarian carcinoma (SK-OV-3), brain cancer (SF-268), nonsmall-cell lung cancer (NCl-H460), and adenocarcinoma colon cancer (RKOP-27).The first screening was carried out at a predefined concentration of 3.16 µg/ml.If the compound led to more than 50% inhibition at this concentration it was evaluated for EC50 mean values (lM) from at least two experiments on those five different cell lines.It turned out that the amino nitrile 3 showed significant cell-growth inhibitory activity (>50%) at a fixed concentration of 3.16 µg/mL.Subsequent determination of EC50 concentrations from dose-response curves gave valid values for four cell lines (in the case of NCI H-460, EC50 was above the highest test concentration).The results are summarized in Table 1.