Synthesis and Antiproliferative Activity of Novel Neocryptolepine-Hydrazides Hybrids

Article history: Received on: 27/05/2017 Accepted on: 08/07/2017 Available online: 30/10/2017 By combining the structural features of neocryptolepine and hydrazides, novel hybrid compounds 8a-d and 9a-j were synthesized using a simple and convenient method. Evaluation of these compounds against human hepatocellular carcinoma (HepG2) cell line using MTT assay was performed. Compound 8c was identified as the most active anticancer agent as it showed effectiveness at low concentration with IC50 2.51 μM on the tested cell line when compared with reference drug 5-FU. It is worth to note that all tested compounds showed antiproliferative activity at very lower micro molar concentrated when compared with the reference drug except compounds 9a, 9e, and 9j which were less active than 5-FU. The anticancer screening results of the tested compounds provide an encouraging framework that could lead to the development of potent new anticancer agents based on neocryptolepine core structure.


INTRODUCTION
Nitrogen-containing polycyclic structures based on privileged templates have drawn significant interest owing to their ability to imitate the natural product chemical probes that play an important role for the drug discovery process (Butler, 2004).In this context, the tetracyclic heteroaromatic compounds, cryptolepine 1 and neocryptolepine 2 (cf.Fig. 1) which are naturally occurred as indoloquinoline alkaloids isolated from the shrub Cryptolepis sanguinolenta, that is growing in west Africa, used for treatment of endemic disease such as malaria fever and various disorder of the body are important due to their wide spectrum of biological properties (Cimanga et al., 1996;El Sayed et al., 2009;2011;2012;Jonckers et al., 2002;Lavrado et al., 2010;Lu et al., 2013;Prakash et al., 2011;Mei et al., 2013;Wang et al., 2013, Wang et al., 2012;Xu and Pieters 2013;Emam et al., 2015;Okada et al., 2016;Larghi et al., 2015;Peng et al., 2012) However, a direct comparison of the cytotoxicity of cryptolepine and neocryptolepine demonstrated that the latter was much less cytotoxic and high affinity towards DNA.(Bailly et al., 2000;Guittat et al., 2003).
In addition we aim to improve the biological activity of the parent compound 2 by substituting it with different side chains.As part of our ongoing program for developing anticancer agents based on a natural product scaffolds herein, we synthetized novel analogues of neocryptolepine compounds bearing sulfonyl-and carbonyl hydrazide moieties and evaluated their anticancer activity against the most common cancer in Egypt hepatocellular carcinoma (Ibrahim et al., 2014).

General methods
All 1 HNMR experiments (solvent DMSO-d 6 ) were carried out with a 400MHz varian and Bruker Avance at the main chemical warfare laboratories, Egypt.Chemical shifts are reported in part per million (ppm) relative to the respective solvent or tetramethylsilane (TMS).The mass spectroscopy experiments were recorded on thermos scientific trace 1310 gas chromatograph at Fungi National Centre, Al-Azhar University and IR spectroscopy & Melting points (m.p) were performed at Cairo University, Egypt.The biological activity analysis was carried out at Medical Research Division, National Research Center, Cairo, Egypt.All reactions were followed by thin layer chromatography (TLC) on kiesel gel F254 precoated plates (Merck).Starting materials sulfonyl and carbonyl hydrazides were synthesized and purified according to literature procedures (Goyal et al., 2016;Brandes, et al., 2005;Bader et al., 2008;Kumaraswamy et al., 2008;Surendiran et al., 2012;xie, et al., 2012).Moreover, the compounds 3 and 4 were commercially available and purchased from Sigma-Aldrich.The required intermediates 5, 6 and 7 were prepared by adopting the earlier reported procedures (Wang et al., 2014).

Evaluation of cell proliferation by MTT assay
The number of viable HepG 2 cells after treatment with different concentration of the compounds was evaluated by the MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay as reported previously with slight modification (Maurya et al., 2011) .In brief, after evaluation of cell count and viability by trypan blue dye, HepG 2 cells (1x10 4 cells/well) were seeded in a 96-well plate in triplicate and were allowed to adhere and spread for 24 h.The tested compounds were dissolved in 500µl Dimethyl sulfoxide (DMSO) to have stock solution of 100 mM, as the final concentration of DMSO in the culture medium never exceeded 0.2% (v/v) (Ranganathan et al., 2015) and then various concentrations of tested compounds were prepared by further diluting in complete medium to have final concentration of 0.01, 0.1, 1, 10, and 100µM.
In the next day the medium was replaced with fresh medium with the indicated concentrations of tested compounds and cells were allowed to grow for 72 h.Four hours before completion of incubation, 10μl of MTT (5 mg/mL in PBS w/o Ca, Mg, Lonza Verviers SPRL Belgium, cat#17-516F) was added in each well.After completing the incubation, 100μl of Dimethyl sulfoxide (DMSO) was added to each well, the 96 well plates were centrifuged for 5 minutes at 4000 rpm to precipitate the formazan crystals.Color developed after the reaction was measured at 490 nm using Bio-Tekmicro plate reader.The experiment was conducted in triplicate.
Data were calculated as percent of cell viability by the following formula: % cell viability = (Mean absorbance in test wells / Mean absorbance in control wells) 100.The effect of tested compounds on the morphology of treated hepatocellular carcinoma cells was investigated by the light microscope and then photographed by SONY CYBER-SHORT (El-Far et al., 2009).

Chemistry
The pathways to assemble the neocryptolepine core structure 2 have been developed starting from easily accessible intermediates as depicted in Scheme 1, this approach allowed us to synthesize new analogues with a varied substitution pattern at B ring.The 11-chloroneocryptolepines 7, the key intermediate for the diversification of the parent natural compound, neocryptolepine 2 was initially obtained according to the route depicted in Scheme 1.The synthetic methodology for 7 was achieved starting from 1H-methyl indole-3-carboxylate 3 and Nmethylaniline 4. The intermediates 2-(phenylamino)-1H-indole-3 -carboxylate 5, obtained via chlorination with Nchlorosuccinimide (NCS) in the presence of 1,4dimethylpiperazine followed by addition of aniline derivative 4 as trichloroacetate salt, was cyclized in boiling diphenyl ether to afford 5,6-dihydro-11H-indolo [2,3-b] quinolin-11-one 6 which, was dehydroxy chlorinated with POCl 3 to give 11-chloronorneocryptolepine 7 in good yield as shown in Scheme 1.
It should be notable that the yield and the reaction time for the cyclization of the anilino derivative 5 to afford the teteracyclic indoloquinolone 6 are very crucial to the amount of the solvent, diphenyl ether used.It was observed that performing the reaction in less amount of solvent makes the cyclization step cleaner, faster, and higher yield.With the key compound 7 in hand, the sulfonylhydrazido neocryptolepine derivatives 8 and carbonylhydrazido neocryptolepine have been synthesized through the incorporation of basic nitogen side-chains at the C-11 position of the B ring of neocryptolepine scaffold with different sulfonyl or carbonyl hydrazide substitutions.
The introduction of the sulfonamido side chain is expected to contribute to the DNA intercalation binding ability through hydrogen bonding motives.Furthermore, the incorporation of the ionisable amine functionality with different linker chain would promote the electrostatic interactions with the negatively charged phosphate groups of DNA backbone.In each case, the compounds were designed to incorporate structural features that might increase DNA binding affinity, cell viability, and cytotoxicity towards the cancer cells.The indoloquinoline scaffold was selected due to its welldocumented DNA binding capability and to its DNA binding interactions through intercalation (Bailly et al., 2000;Guittat et al., 2003;Peczynska−Czoch et al., 1994).Therefore, indoloquinolines may be considered promising scaffolds for the development of novel selective anticancer drugs.Therefore, the key intermediates 11-chloro neocryptolepine 7 was used further for the diversification of the neocryptolepine core at the C-11 position.Thus, the reaction of 7 with carbonylhydrazide derivatives in DMF under reflux and in presence of triethyl amine as a base catalyst yielded the corresponding 11-carbohydrazide neocryptolepine analogues 9 smoothly in very good yield according to the route depicted in Scheme 1.The NMR results for all products are in good agreement with the chemical structure of the compounds and well correspond with the bond formation of the terminal side-chain nitrogen to the sulfonyl or carbonyl group (cf.experimental part for details).The spectra of the known isolated products were in agreement with literature data.The plausible mechanism of this reaction involves the nucleophilic aromatic substitution (S NAr ) of the amino group at a chloride atom of 7 followed by elimination of chloride ion as HCl which reacts with triethyl amine to form triethylammonium chloride salt and the neocryptolepine conjugates 8 and 9 as shown in Scheme 2.

Anticancer Activity with structure-activity relationships (SARs)
The anticancer activity evaluation of the prepared compounds have been assessed against human liver cancer cell line (HepG 2 ) using the MTT assay.For comparison, 5-flurouracil (5-FU) was used as a reference anticancer drug.Dimethyl sulfoxide (DMSO) used as a control for the cancer cells.Cell viability was assessed using the MTT assay.The key results obtained for compounds 8a-d and 9a-j toward hepatocellular carcinoma (HepG2) cells are shown in Table 2, Figure 2.  Results from three separate experiments were recorded and the percentage of viable cells was calculated as percent of cell viability by the following formula % cell viability = (Mean absorbance in test wells / Mean absorbance in control wells) 100.The cell viability was observed following 72 h of exposure to all compounds at doses of 0.01, 0.1, 1, 10 and 100 μM of compounds.The results revealed that compounds 8c, 9b, 9f and 9g with diphenylazo, furyl, tetracarbazolyl and ciprofloxacinyl substituents at the hydrazide side chain of C-11 position of the neocryptolepine core exhibited the highest activity against HepG 2 cell line with IC 50 : 2.51, 4.17, 4.24 and 5.02 respectively, On the other hand, compounds 9h, 9d, 8a, 9c, 8b, and 8d with naphthofuryl, oxobutanoyl, tolyl, phenyl, 4-fluorophenyl, 2,4,6triisopropylphenyl moieties at the hydrazide side chain of C-11 exhibited strong activity against HepG 2 cell line with IC 50 : 10. 71, 11.50, 12.60, 15.10, 15.10, and 20 respectively, While compounds 9i, 9j, 9a and 9e with benzofuranyl, indoleyl, pyridinyl, (4isobutylphenyl)ethyl substituents at the hydrazide side chain of C-11 showed lower potency when compared by 5-FU with IC 50 : 28.11, 40.95, 52.50 and 73.95 respectively.In summary, neocryptolepine has been confirmed as a useful lead compound for the development of new anticancer compounds.Our initial goal to prepare synthetic derivatives bearing hydrazide side chain with a higher anticancer activity could be achieved resulting in several compounds with a higher potency than the reference drug 5-FU.

CONCLUSION
In conclusion, a series of novel neocryptolepinehydrazides hybrids bearing sulfonyl and carbonyl hydrazide moieties have been synthesized.The in vitro anticancer activity was evaluated on in vitro human liver cancer cell model (HepG2).The screening results suggested that the parent natural product neocryptolepine was remarkably influenced by various substituents on the B ring and at the N-terminal of the hydrazide moiety.Moreover, the data revealed that, among the tested compounds, four candidates (compounds 8c, 9b, 9f and 9g) showed high effectiveness on the HepG2, and could be considered as useful templates for further development to obtain more potent anticancer agent(s).Further studies to assess the effect of these compounds on other cancer cell biomarkers are currently underway in our lab.