key: cord-0813189-8cwmna06
authors: Penjarla, Srishylam; Kumar Sabui, Subir; Sudhakar Reddy, Dhande; Banerjee, Shyamapada; Yella Reddy, Paidi; Penta, Santhosh; Sanghvi, Yogesh S.
title: An Efficient Synthesis Tetrazole and Oxadiazole Analogues of Novel 2’-Deoxy-C-Nucleosides and their Antitumor Activity
date: 2020-10-21
journal: Bioorg Med Chem Lett
DOI: 10.1016/j.bmcl.2020.127612
sha: 046f28ae08011c766f45b43baa2cbb3aa0eaf5ea
doc_id: 813189
cord_uid: 8cwmna06

Various tetrazole and oxadiazole C-nucleoside analogues were synthesized starting from pure α- or β-glycosyl-cyanide. The synthesis of glycosyl-cyanide as key precursor was optimized on gram-scale to furnish crystalline starting material for the assembly of C-nucleosides. 1,2,4-Oxadizole C-nucleosides were synthesized via two independent routes. First, both anomers of glycosyl-cyanide were transformed into tetrazole nucleosides followed by acylative rearrangement to furnish 1,2,4-oxadiazoles in high yields. Second, the glycosyl-cyanide was converted into an amidoxime which upon ring closure offered an alternative pathway for the assembly of 1,2,4-oxadizoles in an efficient manner. These protocols offer an easy access to otherwise difficult to synthesize C-nucleosides in good yield and protecting group compatibility. These C-nucleosides were evaluated for their antitumor activity. This work paves a path for facile assembly of library of new chemical entities useful for drug discovery.

The presence of five-membered heterocyclic system is also a common feature in raltegravir, 11 an antiviral drug for treatment of HIV. Interestingly, the oxadiazole ring system is also present in ataluren 12 and zibotentan 13 used for the treatment of Duchenne muscular dystrophy (DMD) and prostate cancer, respectively (Figure -2) . These facts and other reports on the promising biological activity of various regioisomeric oxadiazoles inspired us to synthesise and evaluate the biological activity of novel C-glycosides assembled from 2'-deoxyribose and oxadiazoles. Figure-2 In particular, the oxadiazole ring 14 is an essential part of the pharmacophore favouring ligand binding, act as a flat aromatic linker to place substituents in the proper orientation and finally mimics as bioisoster of esters, amides, carbamates, and hydroxamic esters. The 1,2,3-oxadiazole ring is unstable. 1,2,4-Oxadiazole, 1,2,5-oxadiazole, and 1,3,4-oxadiazole are well known and appear in numerous marketed drugs. We have chosen 1,2,4-oxadiazole and 1,3,4oxadiazole moiety for C-nucleoside synthesis. We envisioned the synthesis of a common building block by C-C bond formation at C1 and further integration of the heterocycle to assemble these C-nucleosides. We utilized the glycosyl cyanide as the key starting material which is obtained as mixture of /-cyanide anomers from 1-chloro carbohydrate by reaction with trimethyl silyl cyanide in the presence of a Lewis acid as catalyst. These two anomers of glycosyl cyanide were transformed into novel C-nucleosides. The main advantage of this strategy resides in availability of stereochemically pure glycosyl cyanide that is transformed into C-nucleosides without anomerization at the C1 position. The C-nucleosides of both anomers of 2'-deoxyriboside of tetrazoles and oxadiazoles synthesised are reported for the first time and the methodologies developed are general, which can be applied to construct other structurally diverse anomerically pure C-nucleosides.

Herein, we describe a modular synthesis approach allowing rapid assembly of C-nucleoside library in an efficient manner. Synthesis of a common building block by C-C bond formation at C1 and further integration of the heterocycle was the key feature of our approach. We have divided our study in three parts: (i) synthesis and separation of glycosyl cyanine anomers on multigram scale, (ii) synthesis of 1,2,4-oxadiazole C-nucleoside via amidoxime intermediate and (iii) synthesis of 1,3,4-oxadozle via acylative rearrangement of tetrazole. (i) Synthesis and separation of glycosyl cyanide anomers on multigram scale: The glycosyl cyanide is one of the most important type of C-glycosyl intermediate, which is usually obtained as mixture of cyanide anomers from commercially available Hoffer's chloro sugar 15 by reaction with trimethyl silyl cyanide in the presence of a Lewis acid as catalyst (Scheme 1). Synthesis of 2'-deoxy glycosyl cyanine anomers (1a and 1b) have been reported 16 only on small-scale. Since 2'-deoxy glycosyl cyanine anomers (1a and 1b) are the key starting materials for our study, it was essential to optimize the yield and anomeric ratio with an ultimate objective of making it in hundred-gram quantity. Because chloro-sugar is devoid of neighbouring group participation, selective stereochemical outcome is challenging. Hence development of process which is robust and greener was undertaken. We screened various Lewis acids and solvents to improve yield and obtain better ratio of anomers in favour of selectivity. -Anomer 1b is desirable to produce C-nucleoside having resemblance to the naturally abundant 2'-deoxynucleosides. Upon screening of various Lewis acids, FeCl 3 afforded best : = 5.7:1 ratio in 70% yield (entry 7 Table 1 ). Next solvent screening using nitromethane, toluene, 1,2-dichloroethane, tetrahydrofuran, acetonitrile, 1,2dimethoxyethane, acetone and dimethyl formamide failed to improve the : ratio and yield compared to the reaction performed in dichloromethane. Considering desired : ratio, yield (93%) and scalability, SnCl 4 was chosen as preferred Lewis acid for the present study. It is important to note that low temperature is essential for -selectivity and high yield. Further optimization effort is underway in our laboratory to find a robust process with non-toxic Lewis acid.

The two anomers of glycosyl cyanides were easily separated by silica gel column chromatography and anomeric configuration was established by 1 H NMR experiments. The SnCl 4 protocol (entry 1 Table 1 ) was scaled-up to furnish 366g of the pure -anomer required for the transformation into various C-nucleosides containing five membered heterocycles. This route is the largest scale synthesis of glycosyl cyanide 1b reported to date in high yield.

(ii) Synthesis of 1,2,4-oxadiazole C-nucleoside via amidoxime intermediate:

Nitrile functional group has served as an excellent handle to install several heterocyclic rings. Separately, both anomers of glycosyl cyanide (1a and 1b) were converted into amidoxime (2a and 2b) following Tiemann protocol 17 using hydroxylamine hydrochloride under basic condition. Reaction was performed with NH 2 OH.HCl in presence of Hünigs base, instead of using NH 2 OH.HCl and Na 2 CO 3 was reported by Adelfinskaya et al. 18 Excellent yields were obtained for both anomers. These amidoxime derivatives were then converted into 1,2,4-oxadiazoles derivatives (5-6 and 7-11) following two distinct protocols (Scheme 2). First, sequential synthesis of O-acylated amidoximes using acetyl chloride followed by cyclization to 1,2,4-oxadiazole ring (5-6) using alkaline DMSO solution. Whereas the second protocol involve direct cyclization of amidoximes 19 to 1,2,4-oxadiazoles 20-27 using orthoformate or acid anhydride in presence of BF 3 .Et 2 O as Lewis acid. The later protocol is shorter and offered higher yields compared to the first route. Deprotection of p-tolyl group was accomplished using NaOMe in methanol at room temperature in excellent yield except for 7a and 7b. Multiple product formation was observed on TLC for 7a and 7b. This phenomenon of multiple product formation can be attributed to the deprotonation 28,29 of acidic C5-H of 1,2,4oxadiazole by NaOMe followed by ring opening and rearrangement. 3 and AlCl 3 in excellent yield. Our attempt to utilize same protocol starting with glycosyl cyanide 1a or 1b resulted in low yield of tetrazole derivative along with other unidentified products. Therefore, tetrazole derivatives [31] [32] [33] (18a and 18b) were successfully synthesized in good yield from both anomers of glycosyl cyanide (1a and 1b) using azide click reaction with copper and cupric sulphate in DMF at 120°C. Unprotected tetrazole nucleosides (19a and 19b) were obtained by cleaving tolyl protecting group using sodium methoxide in methanol at room temperature (Scheme 3). The conversion of tetrazole to 1,3,4-oxadiazole derivatives 34-38 was achieved either by reacting tetrazole derivatives with carboxylic acid anhydride in presence of hydroquinone under reflux or by reacting with carboxylic acid chloride in pyridine. [39] [40] [41] [42] The Deprotection of p-tolyl group was executed using NaOMe in methanol at room temperature in excellent yield. However, the deprotection protocol suffers from a drawback for C5-unsubstituted and C5-substitution with electron withdrawing groups. In both cases, multiple product formation was observed due to the ring opening of the oxadiazole ring. This decomposition can be explained by the nucleophilic addition of NaOMe to C5-carbon and ring opening. 43 The postulated mechanism 44 of this conversion is illustrated in Scheme-4. 5-Substituted tetrazole undergoes N2acylation upon treatment with acylation reagent due to steric bulk of 5-substitution. This unstable intermediate (INT-1) then ring opens via nitrogen extrusion and formation of N-acyl nitrilimine as putative intermediates (INT-2 and  INT-3) . These intermediates are then cyclized to form 1, 3, 4-oxadiazoles (20) (21) (22) (23) (24) (25) in good yield. Structural elucidation of the new compounds described in this study was based on NMR and mass spectral data. 

Various regio-isomeric five-membered oxadiazoles based 2'-deoxy-C-nucleosides were synthesized for the first time in good yield and high purity. All C-nucleosides were assembled from pure or -anomer of glycosyl cyanide. The synthesis of glycosyl cyanide as key starting material was established on large-scale and in excellent yield. The easy accessibility of glycosyl cyanide further allows its utility in design of therapeutic oligonucleotides. 47 The synthetic methodologies developed in this study are general and offer future scope to generate other nucleoside analogues for SAR study. Biological evaluation was carried out for synthesised compounds and shows reasonable cytotoxicity in five different tumor cell lines. Studies on antiviral activity of these compounds is in progress and it will be published elsewhere.

Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement: SS acknowledge the help and support of Director, NIT Raipur for allowing him to register and pursue his PhD. SS also thanks Mr. Madika Chandrakanth for his help in preparation of manuscript. 

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