Regioselective Synthesis of Triazolo[3,4-e] purine Derivatives and their Anti-Cancer Activity against NCI-60 Cell Lines

Introduction: Due to the vast medicinal importance of purine nucleoside, a hybrid molecule of triazole with purine ring might explode a lead molecule in the pharma sector and based on the last decade’s studies suggested that the nitrogen-rich molecules possess a wide range of medicinal importance. Aim: Due to the vast application of purine nucleoside itself in the field of cancer research, we synthesized triazolo[3,4-e]purines and screened them for their anti-cancer study against NCI-60 cell lines by the protocol used by NIH. Materials and methods: The targeted molecules, 4-chloro-5a,6-dihydro-8-substitutedphenyl-1H-[1,2,4]triazolo[3,4-e]purine derivatives (4a-4h) were synthesized in a two-step procedure by nucleophilic substitution (SN) at C-2 chlorine followed by formation of the triazole ring by acid-catalyzed reaction in the polar protic solvent. Results: It was observed that the regioselective approach followed in C-2 chlorine replacement instead of C-6 chlorine during SN reaction. One-dose response of selected three molecules (4a, 4b, and 4c) showed that 4b (K-562: 64.47 μM & SR: 63.38 μM; mean GI50: 99.09 μM) was found to be more potent than 4a and 4c. Conclusions: We have described in this study the general synthetic method for triazolo[3,4-e]purines as an innovative class of potential anticancer agents. The dose-response curve in the sense of mean GI50 for three compounds across all 60 cell lines, 4b can be served as lead after necessary modification.


INTRODUCTION
In the field of synthetic organic chemistry and medicinal chemistry, nitrogen-based heterocyclic compounds attract more attention of researchers due to their extensive biologically active properties. 1 Among the group of such molecules, purine nucleoside has been found to be more potent than any other nitrogen-rich scaffolds in the field of cancer therapy. 2 Derivatives of purines are used in current drug discovery system in the area of treatment of cancer in most cases. 3 In 1953, 6-mercaptopurine was found to be the first purine analogue having cancer activity and was approved by the FDA. A number of structures possessing the purine nucleus have been reported to show diverse biological acti-vities (purinethol and fludarabine, leukemia; aciclovir, herpes infection; abacavir, HIV/AIDS; allopurinol, decrease uric acid levels in the blood). 5 Purine derivatives directly affect tumor cell growth by acting as active components in the biochemical processes in cancer and important in host and tumor interaction process. [6][7][8] The drug used in cancer therapy generally disturb the synthesis of DNA or snoop its function by one or other ways to kill or inhibit the cancer cell growth. 9 After concerning the potency of such active nucleoside, it is not surprising that the researcher focused on to study chemistry, synthesis or biochemical processes of purines in human host. 10 Simultaneously, one more nitrogen annoying molecules, i.e. triazoles are an equally important type of heterocyclic compounds responsible for the supreme pharmacological effect. 11 Literature shows the diversity of both triazoles and purine compounds in the area of pharmacology due to their properties as anticancer 12 , antileukemic [13][14][15] , anti-HIV-1 16,17 , antiviral 18,19 , antitubercular 20,21 , antimicrobial agents [22][23][24] , acid base indicator 25 , and dyes 26 .
In addition, triazolo purine compounds act as a model for metal-ligand interaction observed in biological systems. [27][28][29][30] So, keeping the importance of these compounds in mind, we have taken up an extensive project to synthesize a new series of 1,2,4-triazoles of purine base analogues with the primary aim to evaluate their cytotoxic potency. In this work, we have reported the synthesis and anticancer screening of 4-chloro-5a,6-dihydro-8-phenyl-1H- [1,2,4] triazolo [3,4-e]purine derivatives.

MATERIALS AND METHODS
All the chemicals and reagents were received from sigma-Aldrich and Merck. Silica gel plate G60 F254 (Merck) was used in thin layer chromatography to monitor the completion of the reaction. Visualization was made under UV light (254 nm and 365 nm). Infrared spectra of the compounds were recorded on IR Affinity-1S spectrophotometer (Shimadzu). 1 H (400 MHz) and 13 C (101.1 MHz) NMR spectra were recorded on a Bruker AVANCE II spectrometer in DMSO-d 6 . Mass spectrometer GCMS-QP 2010 (Shimadzu) was used to resolute the mass spectra of compounds and rotary evaporator was used for drying the compounds.

Methodology for anti-cancer screening
The anticancer screening was performed at the National Cancer Institute (NCI), Bethesda, Maryland, USA. The compounds were evaluated in 60 cell lines in a one-dose primary anticancer assay subsequent to the NCI preclinical antitumor drug discovery screen. 31 Human tumor cell lines of the cancer screening panel were grown according to the specific protocol they follow and reported in literature. 31,33 For drug addition, an experimental drug was solubilized in dimethyl sulfoxide. The solubilized drug was applied on tumor cell lines (according to the standard procedure they followed). 32

Synthetic part
The novel series of triazolo [3,4-e]purine derivatives were obtained by the sequence of reactions shown in Fig. 1. The substituents for the synthesis of target molecule with their % of yield and substituents effect on its cancer activity are summarised in Fig. 2.
Nitrogen-rich purine hydrazide (2) was synthesized by coupling of 2,6-dichloropurine (1) with hydrazine hydrate (40%). Purine hydrazide intermediate (2) on reaction with substituted phenyl aldehyde in the presence of the catalytic amount of hydrochloric acid gave cyclized product 4a-4h at C-2 position having good yield. Optimizations carried out were by without acid catalyst and by varying solvents but no major progress of the reaction was obtained even by using heating conditions.

Spectroscopic data analysis
The structures of the synthesized compounds were confirmed by IR, 1 H NMR, 13 C NMR, and mass spectral data. 1 H NMR spectra revealed a singlet at δ= 12 ppm ( 1 H) assigned to -NH of triazole aromatic ring, in addition to that, the presence of two more singlets in upfield region 3.0-3.8 δppm (purine five-membered ring -NH proton), 2.8-2.3 δppm (methine proton of purine five-membered ring) indicating the existence of this compound in the hydrazide form rather than the azo form. In compound 4c, 4f and 4g, one broad hump was observed at 10-11 δppm and 4-5 δppm due to tautomeric -NH proton in the five membered purine ring (see supplementary material). IR spectra of the synthesized compounds show a very weak peak at 3300 -3150 cm -1 due to tautomerisation of -NH stretching frequency. IR spectra of all newly synthesized compounds show a strong and sharp peak in the region of 1280-1230 cm -1 (C-N stretching) which indicates the presence of aromatic amines.

Anti-cancer results
After the screening, out of seven compounds, three compounds (4a, 4b, and 4c) were selected and evaluated for in vitro anticancer activity against NCI-60 cell-lines in nine different panels consisting of the leukemia cell line, the non-small lung cancer cell line (NSCLC), the colon cancer cell lines, CNS cancer cell-lines, melanoma cancer cell lines, the ovarian cancer cell lines, the renal cancer cell lines, the prostate cancer cell lines and the breast cancer cell-line. For all these cell line activities they follow the method given in literature. 31,34 Each cell lines have more than three sub-panels. Compounds 4a and 4b showed promising activity as compared to that of 4c. Compound 4a reduced the growth NSCLC (NCI-H522) and renal cancer cell line (A498) to near about 73%. The cell line growth data of compounds 4a, 4b, and 4c are given in Table 1.
Compound 4b showed maximum activity and gave better results in leukemia cancer cell lines (leukemia K-562 sub panel and leukemia SR sub panel cell lines; GI 50 values: 64.47% and 63.38% respectively). In one response parameter, GI 50 value was calculated for each used NCI-60 cell lines which correspond to the compound concentration causing 50% decreases in net cell growth. 35,36 Compound 4a showed promising GI 50 value (75%) against non-small cell lung cancer cell-line (NCI-H522) and maximum active compound 4b also showed promising GI 50 value (75%) against NSCLC (NCI-H522). These compounds have shown growth inhibition against the cancer cell line men- tioned above probably due to the presence of electron-donating influence of its substituents. 35 On getting the final result of the activity, we were surprised for the compound 4c because it does not show any remarkable values but the mean GI 50 value (98.98%) was excellent than the 4a (100.58%) and 4b (99.09%). The GI 50 and full panel meangraph midpoint (MG-MID) values respectively, are given in Table 2.

Mechanism of action
Purine and pyrimidines are necessary components for the synthesis of DNA and among them about 14 antimetabolites were approved by FDA as anticancer precursors. Generally, purine derivatives act as an anticancer agent via membrane transporter. 36 After entering the cell, it is converted into genetic material nucleotides by enzymes. These nucleotides then inhibit one or more enzymes on the basis of wrong selection of nucleotides which are essential for the next step of DNA synthesis. 37 It is known that tumour cells have several mutations that might lead to defective apoptosis-inducing pathways, and this is probably at the basis of the initial expansion of the population of neoplastic cells. On the basis of the above discussion, it was realised that mismatching of DNA (genetic material) lead to mutagenesis in host cell which may result in the anticancer property of purine scaffolds. As per the literature survey, we haven't found any mutation rate of purine nucleoside and/or purine-triazole adducts. 38,39 We have synthesized fused triazolopurines derivatives which possess free -NH at the same position which may act in the same way as depicted above. The substitution present on the phenyl ring is also responsible for the incorporation of DNA and RNA functionalities. Methoxy group present at 4 th position has around to bind over there and is only possible via -NH of triazole which shows the highest activity for inhibition of DNA synthesis.

Conflict of Interest
Authors declare that there is no conflict of interest.