Tetrahedron Letters 58 (2017) 4166–4168
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Synthesis, characterization and biological evaluation of purine nucleoside analogues Shankaraiah Malthum a, Naveen Polkam a, Tejeswara Rao Allaka a, Kalyani Chepuri b, Jaya Shree Anireddy a,⇑ a b
Centre for Chemical Sciences and Technology, IST, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085, Telangana State, India Centre for Biotechnology, IST, Jawaharlal Nehru Technological University Hyderabad, Kukatpally, Hyderabad 500085, Telangana State, India
a r t i c l e
i n f o
Article history: Received 26 April 2017 Revised 13 September 2017 Accepted 15 September 2017 Available online 18 September 2017 Keywords: Cytotoxicity Doxorubicin Purine nucleosides Sonogashira coupling
a b s t r a c t We present a convenient route for the synthesis of C6-amino-C50 -N-cyclopropyl carboxamido-C2-alkynylated purine nucleoside analogues 11a–g via Sonogashira coupling reaction. The nine step synthesis is easy to perform, employing commercially available reagents. Compound 9 is used as key intermediate for the synthesis of analogues 11a–g. Synthetic intermediates and final products are appropriately characterized by IR, 1H NMR, 13C NMR and Mass. The modified nucleoside analogues 11a–g is evaluated for in vitro anticancer activity against MDA-MB-231 and Caco-2 cell lines. Screening data reveals that compounds 11b and 11e displayed potent IC50 value of 7.9, 6.8 mg/mL respectively against MDA-MB-231 and of 7.5, 8.3 mg/mL respectively against Caco-2 than the standard drug doxorubicin, thus establishing the potential anti-cancer properties of these newer derivatives. Ó 2017 Elsevier Ltd. All rights reserved.
Introduction Nucleoside analogues are a versatile family of drugs for several life threatening diseases due to their multiple mechanisms of action and potential indications.1 Modified nucleosides have been a focus of several recent studies,2,3 including modification of both the sugar and the hetero cyclic base. Many chemical modifications of the nucleobase and/or sugar have been developed for the 2, 6 and 50 positions. The chemical diversity of nucleoside analogues is achieved by halogenation, azotation, N-conjugation, methylation, saturation, ring opening, protection by polar groups, alkynylation and other modifications. From the literature findings, it can be inferred that many nucleosides encompass diverse C-substituents at 6th position of purine base possessing broad spectrum of biological activities. Extensive structure activity relationship studies have shown that the nucleosides bearing C-substituents at 50 position of sugar moiety exhibited potential anticancer activity.4 In addition, earlier studies established that the introduction of an alkynyl group in C2 position of purine base enhances the recognition effect at both A2a and A3aRs.5 Recently, some reports on the modified purine nucleosides active against different bacterial strains and cancer cells have been published.6,7 Nevertheless, significant improvements continue to appear. Ribavirin, AZT, Fludarabine, Cladribine are the examples of heterocyclic base and/or sugar mod-
⇑ Corresponding author. E-mail address:
[email protected] (J.S. Anireddy). https://doi.org/10.1016/j.tetlet.2017.09.041 0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.
ified drugs against antiviral and anticancer diseases.8,9 SGI-110, CMX157 etc., are under phase I/II preclinical trials. Several nucleoside and nucleotide analogues have also undergone drug repositioning; PMEG was initially developed as an antiviral agent but it was also shown to exhibit anticancer activities.10 Similarly certain cytotoxic nucleoside analogues were found to possess antiviral or antibacterial properties. Azidothymidine and gemcitabine possess antibiotic properties and have synergistic actions against highly resistant s. aureus bacteria.11,12 Besides their classical use in cancer and virology, some nucleoside and nucleotide analogues, and related compounds have been used in various other indications like hyperuricaemia, immunosuppression, neuroprotection and cardioprotection.13 Recently, Rieger Jayson et al. have utilised the strategy of incorporating alkynes into the adenosines and elicited the obtained derivatives as efficient A2a receptor agonist whereas Beauglehole Anthony et al. have reported on alkoxy-carbonyl-aminoalkynyl-adenosines for treating A2a receptor agonist diseases. Based on multiple mechanisms of action and drug repositioning of nucleoside analogues, we presumed that modified nucleoside analogues would be efficacious against anticancer activities. Preparation of C2, C6 and C50 modified nucleoside derivatives was successfully accomplished from commercially available guanosine as outlined in Scheme 1. Acetyl group was selectively introduced at C20 , C30 and C50 hydroxyl groups of guanosine using acetic anhydride in a one-pot reaction (step-i). It is interesting to find that due to low nucleophilicity of amino group, it doesn’t involve in the course of the reaction and acetylation step proceeds as per
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S. Malthum et al. / Tetrahedron Letters 58 (2017) 4166–4168 Table 1 Anti-cancer activity of compounds 11a–g. Compound
11a 11b 11c 11d 11e 11f 11g Doxorubicin
IC50 (mg/mL)a MDA-MB-231
Caco-2
18.8 ± 0.62 7.9 ± 0.54 12.1 ± 2.01 11.7 ± 0.8 6.8 ± 0.53 21.1 ± 2.83 23.4 ± 0.48 8.9 ± 0.9
20.9 ± 1.04 7.5 ± 0.17 19.4 ± 1.23 12.6 ± 0.16 8.3 ± 0.81 19.2 ± 1.81 13.2 ± 0.94 9.1 ± 1.0
a The IC50 value was expressed as the mean ± standard deviation of 50% inhibitory concentrations of triplicate experiments.
Scheme 1. Reagents and conditions: (i) Ac2O, dry DMF, dry pyridine, 75 °C, 4.15 h, 70%; (ii) POCl3, N,N-Dimethylaniline, Tetra methyl ammonium chloride, Acetonitrile, 0 °C to rt, 16 h, 64.1%; (iii) t-Butyl nitrite, CH2I2, Acetonitrile, 80 °C, 4.50 h, 44%; (iv) Liquid NH3, 78 °C to rt, 18 h, 80%; (v) PTSA, 2,2-Dimethoxypropane, Acetone, rt, 4 h, 70%; (vi) KMnO4, KOH, H2O, rt, 72 h, 78.6%; (vii) EDCl, HOBT, DMAP, Cyclopropylamine, DMF, TEA, rt, Overnight, 70.3%; (viii) 50% Formic acid, 80 °C, 1.5 h, 73.4%; (ix) Pd(PPh3)2Cl2, Cuprous iodide, dry DMF, dry acetonitrile, dry triethylamine, rt, 24 h.
expectation. The acetylated purine nucleoside 2 was purified by reverse-phase HPLC (gradient: acetonitrile and ammonium acetate 0–50%). In step-ii the carbonyl group of nucleobase was converted to chloro derivative 3 by reaction with POCl3 and N,N-dimethyl aniline. The C2 amino group was directly converted to iodo derivative 4 by reacting with t-butyl nitrite and diiodomethane (step-iii). To our great delight, the envisaged nucleophilic aromatic substitution with NH3 at C6 position of purine in compound 4 took place easily and this was combined with cleavage of acetate protecting groups in compound 5 (step-iv). Regio selective protection of the C20 and C30 diol moiety of compound 5 (step-v) with 2,2dimethoxy propane in PTSA (p-Toluene sulfonic acid) yielded compound 6. The C-50 hydroxyl group in sugar moiety of 6 was subsequently oxidized (step-vi) with KMnO4 (Potassium permanganate) to carboxylic acid derivative 7. In step-vii, commercially available HOBt (1-Hydroxybenzotriazole) was used for the conversion of C50 carboxylic group to amide derivative 8. Deprotection (step-viii) of C20 and C30 diol moiety was carried out by treatment with formic acid to afford the compound 9. On a general note, Pd catalyzed cross-coupling reactions14,15 have proven for the efficient alkynylations of many iodo or bromo substituted nucleoside derivatives with terminal acetylenes. Regardful of this, the synthesis (step-ix) of different C2 alkynylated nucleoside analogues 11a–g was performed using Sonogashira coupling16 on the corresponding C2 iodo derivative 9 with terminal alkyne derivatives 10a–g. The resulting compound was purified by silica gel column chromatography and/or reverse-phase HPLC. Further, the C2 alkynyl functional group was interpreted as a synthon and be exploited with click chemistry to furnish diverse heterocyclic derivatives17 which are amenable for further modifications.
In this work, we report a facile straight forward nine step synthesis of C50 -N-cyclopropyl carboxamido-C6-amino-C2-alkynylated purine nucleoside analogues as well as their anticancer activities.18,19 The structures of the products were confirmed by 1H NMR, 13C NMR, IR and Mass spectral data. In the 1H NMR spectra of compounds 2, 3 and 4 signals for the acetyl protons in sugar moiety were observed at d 2.16–2.25 ppm. The protons of C2 NH2 of compounds 3 and 4 appeared as singlets at d 6.52 and 5.13 ppm. Broad singlets of OH groups in sugar moiety were occurred in compound 5. The signals of geminal CH3 protons of compounds 6, 7 and 8 were appeared as singlets at d 1.4–1.6 ppm. C-50 carboxylic proton of compound 7 was observed at 11.0 ppm as singlet. The amide protons of compounds 8 and 9 were observed at d 7.2–8.2 ppm as singlets. The 13C NMR spectra further confirmed the structures of reaction intermediates 2–9 and title analogues 11a–g. The IR spectra of compounds 2–9 and 11a–g were in good agreement with the proposed structures: the absorption bands were in the regions 1615.73 (COOMe str), 3163.09 (N@CH str), 873.86 (CACl str), 789.72 (C-I str), 3426.78 (OH str), 3433.03 (COOH str), 1600 (CONH str) and 1203.51 (CAN str) cm1. The synthesized/modified purine analogues 11a–g are evaluated for in vitro anti-proliferative activity against human breast cancer cell line MDA-MBA-231 and human colon cancer cell line Caco-2 using MTT assay taking doxorubicin (DXN) a known anticancer drug as a reference compound. Table 1 contains the activity of modified purine analogues expressed as IC50 values. It is interesting to find that compounds that 11b (IC50 = 7.5 mg/mL) and 11e (IC50 = 6.8 mg/ mL) displayed superior activity than DXN against Caco-2 and MDA-MB-231 cell lines respectively indicating their selectiveness towards these cell lines. Compounds 11c and 11d have displayed a moderate inhibitory effect against MDA-MB-231 cell lines and 11d and 11g exhibited a similar effect against Caco-2 cell lines. To conclude, we have described a convenient and efficient multi-step protocol for the synthesis of target compounds 11 by utilizing Sonogashira reaction. The final derivatives were tested for anti-cancer efficacy against MDA-MB-231, Caco-2 cell lines. Among the screened, products 11b and 11e exhibited promising cytotoxic activity than doxorubicin (standard anticancer agent). Acknowledgments One of the authors (MSR) is thankful to University Grants Commission (UGC), New Delhi, Govt. of India for financial assistance in the form of a senior research fellowship. Authors MSR, JSA are thankful to TEQIP-II for aiding improved laboratory facilities. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tetlet.2017.09. 041.
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References 1. Lars PJ, David D, Fabien Z, Charles D. Nat Rev Drug Discov. 2013;12:447–464. 2. Wei-chu X, Wright EG, Brown CN, et al. Bioorg Med Chem Lett. 2011;21:4197–4202. 3. Shelton RJ, Burt RS, Peterson AM. Bioorg Med Chem Lett. 2011;21:1484–1487. 4. Martina ET, Zhe N, Stephen M, et al. Bioorg Med Chem Lett. 2004;14:3165–3168. 5. Dilip KT, Moshe C, Andrei AI, Athena MK, Zhan-Guo G, Kenneth AJ. J Med Chem. 2009;52:7580–7592. 6. Abdalla EAH, Reham AIAE, William BP, Paula WA, John AS. Bioorg Chem. 2016;65:9–16. 7. Jadd R, Scott RB, Matt AP. Bioorg Med Chem Lett. 2011;21:1484–1487. 8. Paeshuyse J, Dallmeier K, Neyts J. Curr Opin Virol. 2011;1:590–598.
9. Kolesar MJ, Morris KA, Kuhn GJ. J Oncol Pharm Pract. 1996;2:160–181. 10. Holy A, Votruba I, Metra A, et al. Antiviral Res. 1990;30:295–311. 11. Michael MPB, Clausen AR, Stephen LWO, Frank MA, Birgutte MP, Jure P. J Antimicrob Chemother. 2007;60:510–520. 12. Elwell LP, Ferone R, Freeman GA, et al. Antimicrob Agents Chemother. 1987;31:274–280. 13. Sharma NK, Mahadevan N, Balakumar P. Cardiovasc Taxicol. 2013;13:22–31. 14. Fairlamb IJS. Tetrahedron. 2005;61:9661–9662. 15. Hocek M. Eur J Org Chem. 2003;2003:245–254. 16. Chinchilla R, Najera C. Chem Rev. 2007;107:874–922. 17. Kolb CH, Sharpless BK. Drug Discov Today. 2003;8:1128–1137. 18. Bonev B, Hooper J, Parisot J. J Antimicrob Chemother. 2008;61:1295–1301. 19. Mosmann T. J Immunol Methods. 1983;65:55–63.
