Novel method for the synthesis of dinucleoside-(N3′ → P5′)-phosphoramidothioates

Tetrahedron Letters 58 (2017) 2276–2279

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Novel method for the synthesis of dinucleoside-(N30 ? P50 )phosphoramidothioates Katarzyna Kulik a,⇑, Renata Kaczmarek a, Janina Baraniak b, Katarzyna S´lepokura c, Sergei Gryaznov d a

Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódz´, Poland Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz University, Armii Krajowej 13/15, 42-201 Cze˛stochowa, Poland c Faculty of Chemistry, Wrocław University, F. Joliot-Curie Street 14, 50-383 Wrocław, Poland d AliosBioPharma, 260 E. Grand Ave, S. San Francisco, CA 9408, USA b

a r t i c l e

i n f o

Article history: Received 28 February 2017 Revised 21 April 2017 Accepted 28 April 2017 Available online 29 April 2017 Keywords: Nucleoside H-thiophosphonates Dinucleoside-(N30 ? P50 )phosphoramidothioates P-chirality Absolute configuration X-ray diffraction analysis

a b s t r a c t A new approach for the synthesis of dinucleoside-(N30 ? P50 )-phosphoramidothioates based on the Atherton–Todd reaction has been developed using nucleoside H-thiophosphonates and 30 -amino-20 ,30 dideoxynucleosides with an unprotected 50 -hydroxyl group. A mixture of P-diastereomers of dinucleosides was separated by column chromatography into fast migrating and slow migrating isomers. Based on single crystal X-ray diffraction analysis, the absolute configuration at the phosphorus atom in the slow eluting diastereomer of the dinucleoside-(N30 ? P50 )-phosphoramidothioate GNPSGNHTr was assigned as Rp. Ó 2017 Elsevier Ltd. All rights reserved.

Introduction Synthetic oligonucleotides1 are powerful molecular biological and biochemical research tools, which permit fast and efficient regulation of gene expression and the study of gene function both in vitro and in vivo.2 However, several important issues remain to be resolved before oligonucleotides become widely used pharmaceutical agents. Among these are: (i) increased thermodynamic stability of the complexes formed by the interaction of oligomers with their targets, (ii) specificity of the interactions, (iii) resistance to enzymatic degradation by exo- and/or endo-nucleases, and (iv) suitable pharmacological and pharmacokinetic profiles. To improve/modulate these properties, oligonucleotide analogues containing numerous chemical modifications within the nucleobase, sugar unit and internucleotide linkage have been reported.3 Among them oligonucleoside-(N30 ? P50 )-phosphoramidothioates (NPS-oligos) have received significant attention.4 These oligonucleotides containing a 30 -amino group replacing the 30 -oxygen of deoxyribonucleosides and one of the non-bridging oxygen replaced by sulfur, were designed to combine the best characteristics from both oligonucleotide-(N30 ? P50 )-phosphoramidates (NPO-oligos)5 and phosphorothioates (PS-oligos).6 Therefore, like ⇑ Corresponding author. E-mail address: [email protected] (K. Kulik). http://dx.doi.org/10.1016/j.tetlet.2017.04.094 0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.

NPO-oligos, NPS-oligos form very stable duplexes with complementary DNA and RNA strands,7 and possess nuclease resistance like PS-oligos.8 However, replacement of one of the two non-bridging oxygens by a sulfur atom in an internucleotide linkage generates asymmetry at the phosphorus atom. Hence, the synthesized oligonucleoside-(N30 ? P50 )-phosphoramidothioates are a mixture of 2n diastereomers (where n is the number of internucleotide thiophosphoramidate groups). Only a few synthetic strategies concerning oligonucleotide(N30 ? P50 )-phosphoramidates have been described in the literature.9 Among them, an approach based on phosphoramidite transfer methodology, which utilizes the key 30 -aminonucleoside–50 -phosphoramidite building blocks originally developed by Fearon and co-workers10 for the synthesis of NPO-oligos, offers an efficient method for the synthesis of stereorandom NPSoligonucleotides,4a,9a but does not afford stereodefined NPS-oligos. One might consider that the oxathiaphospholane chemistry employed for the synthesis of dinucleoside-(N30 ? P50 )phosphoramidothioates9b could also be used for the synthesis of P-stereodefined NPS-oligos, however, unsuccessful attempts at elongation of the oligonucleotide chain indicate that this is not a suitable method for obtaining NPS-oligos. Recently, it was demonstrated that a lipid-modified oligonucleotide-(N30 ? P50 )-phosphoramidothioate (GRN163L) is the most

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active inhibitor of human telomerase,11,12 synthesizing de novo d(TTAGGG)n repeats at chromosomal DNA ends. Whereas the activity of this enzyme is observed in 85% of all human tumors, most normal somatic cells either lack telomerase activity, or express it only at low levels. Therefore, telomerase constitutes an attractive anticancer target for rational drug development. Taking into account that in many cases interaction of P-chiral analogues of DNA with enzymes depends on their chirality,6,13 it can be assumed that the principle is also true with regard to oligonucleotide-(N30 ? P50 )-phosphoramidates and telomerase. Therefore, there is a need to develop methods for the stereospecific synthesis of such compounds. Due to our long standing interest in the synthesis of modified nucleosides and oligonucleotides,9b,14 we decided to develop a procedure for the preparation of stereodefined NPS-building blocks to use in the solid-phase synthesis of oligonucleotide chains possessing in alternate positions phosphoramidate and stereodefined phosphoramidothioate internucleotide linkages.

Results and discussion Herein, we present a new approach towards the synthesis of diastereomerically pure dinucleoside-(N30 ? P50 )-phosphoramidothioates based on Atherton–Todd type reaction15 by reacting nucleoside 50 -H-thiophosphonate with 30 -aminonucleoside in the presence of CCl4. In this approach N2-isobutyryl-30 -(trityl)amino-20 ,30 -dideoxyguanosino-50 -O-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite (1), synthesized as previously described,16 was used as the starting material. Dry H2S was bubbled through an MeCN solution of 1 for 5 min, followed by the addition of 1-H-tetrazole (Scheme 1). After aqueous work-up to remove the tetrazolide salts, 50 -H-phospho-

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nothioate diester 2 was isolated without further purification17 in an almost quantitative yield as a mixture of Rp/Sp diastereomers which were characterized by 31P NMR [d (C6D6) 72.37 and 72.17 ppm] and FAB-MS [m/z 712 (M+1)]. Subsequently, diester 2 was converted into the corresponding chlorothiophosphate in the presence of DIPEA and CCl4 (a halogenating agent), which without isolation was coupled with 30 amino-20 ,30 -dideoxynucleosides 3 (GiBu) or 4 (ABz). The progress of the reaction was monitored by 31P NMR and after 30 min full conversion of 2 into the desired dinucleoside phosphoramidothioate GNPSGNHTr18 [5: d (CH3OD) 73.00 and 71.26 ppm] or dinucleoside phosphoramidothioate ANPSGNHTr19 [6: d (CH3OD) 72.83 and 71.29 ppm] was observed. The dinucleoside-(N30 ? P50 )-phosphoramidothioates 5–6 were isolated as a mixture of diastereoisomers by column chromatography on silica gel in 57–62% overall yield. Their structures were confirmed by 1H NMR (see ESI) and MALDI-MS [5 m/z 1046 (M+1); 6 m/z 1064 (M+1)]. The reaction between the chlorothiophosphate intermediate and 30 -amino-20 ,30 -dideoxynucleoside 3 was carried out in dry DMF, whereas for 4, higher coupling efficiency was obtained using a mixture of acetonitrile/pyridine (1:1) as the solvent. Next, the diastereoisomers of dinucleoside phosphoramidothioates 5–6 were separated by silica gel column chromatography into the individual fast and slow eluting P-epimers. The separation of 5 was performed on silica gel (230–400 mesh) using MeOH in CHCl3 (1 ? 13%) containing 0.2% pyridine as eluent. The fast eluting isomer 5a was obtained in 29% yield, whereas a slow eluting diastereomer 5b was isolated in 30% yield. To separate ANPSGNHTr (6), silica gel 60H and 2-propanol in CHCl3 (5 ? 15%) as eluent was used. The fast eluting isomer 6a was isolated in 26% yield whereas the slow eluting isomer 6b was only obtained in 7% yield. It is worth noting that 30 -amino-20 ,30 -dideoxynucleosides 3, 4 contain an unprotected 50 -hydroxyl group, which theoretically,

Scheme 1. Synthetic approach toward the dinucleoside-(N30 ? P50 )-phosphoramidothioates (5, 6) based on the Atherton–Todd type reaction.

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K. Kulik et al. / Tetrahedron Letters 58 (2017) 2276–2279

Fig. 1. 31P NMR spectrum of the crude reaction mixture from the reaction of 2 with 3.

might have reacted with the chlorophosphorothioates as a competitive nucleophile providing the corresponding 50 ,50 -dinucleoside phosphorothioates. Inspection of the crude reaction mixture obtained when 2 was reacted with 3 using 31P NMR (Fig. 1), showed that in addition to signals corresponding to 5, resonances at d = 52.53 and 52.19 ppm were present which could be attributed to the presence of 50 ,50 -dinucleoside phosphorothioates. This by-product was isolated from the reaction mixture and mass spectrometry analysis revealed its identity as N2-isobutyryl-30 -(trityl)amino-20 ,30 -dideoxyguanosine-50 -O-(2-cyanoethyl)phosphorothioate (7). Most likely this compound was formed as a result of competitive reactions between the in situ generated chlorothiophosphate of 30 -amino20 ,30 -dideoxynucleoside and trace amounts of water present in the reaction medium. This result indicates that the reaction between H-thiophosphonate and 30 -aminonucleoside is highly regioselective, and likely due to the higher nucleophilicity of the 30 -amino group compared to the 50 -hydroxyl group in 30 -amino20 ,30 -dideoxyribose 3 and 4. In order to use dinucleoside phosphoramidothioate 5, 6 as building blocks for the synthesis of P-stereodefined NPS-oligos, the absolute configuration at the phosphorus atom needed to be determined. Single crystal X-ray diffraction can be used for unambiguous assignment of the absolute configuration at a stereogenic center. Numerous attempts at the crystallization of individual

Fig. 2. Single crystal X-ray structure of Rp-GNPSGNHTr (5b). Present in the crystal as 5bCHCl3 showing the atom-numbering scheme for selected atoms and symmetry– independent hydrogen bonds (dashed lines), and CAH  p contacts (dotted lines). Disorder, CHCl3 molecule and C-bonded H atoms not involved in hydrogen bonding have been omitted for clarity.

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diastereoisomers of 5 and 6 were undertaken, unfortunately, only for the slow eluting diastereomer of dinucleoside-(N30 ? P50 )-phosphoramidothioate GNPSGNHTr (5b) were diffraction quality crystals obtained from CHCl3. The performed X-ray analysis20 (see ESI) proved the R-configuration at the phosphorus atom in 5b (Fig. 2) containing the lower field resonance [dP (121 MHz, CH3OD) 73.00 ppm] in 31P NMR. Conclusion In summary, a new route for the synthesis of dinucleoside(N30 ? P50 )-phosphoramidothioates based on the Atherton–Todd reaction has been developed using nucleoside H-thiophosphonates and 30 -amino-20 ,30 -dideoxynucleosides as synthons. The main advantage of the presented approach is its regioselectivity which offers the possibility to use 30 -amino-20 ,30 -dideoxynucleosides with unprotected 50 -hydroxyl groups. The reported synthesis of dinucleoside-(N30 ? P50 )-phosphoramidothioates GNPSGNHTr, together with the recently described21 synthesis and assignment of the absolute configuration at the phosphorus center of dithymidyl-(N30 ? P50 )-phosphoramidothioate may open the way for the synthesis of P-stereodefined NPSoligos. These compounds could be applied as dimeric building blocks in the solid-phase synthesis of oligonucleotide chains possessing in defined positions stereodefined GNPSG and TNPST internucleotide linkages. Acknowledgment This study was financially supported by the Statutory Funds of CMMS PAS. 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.2017.04. 094. References 1. (a) Singh R, Murat P, Defrancq E. Chem Soc Rev. 2010;39:2054–2070; (b) Juliano RL, Ming X, Nakagawa O. Acc Chem Res. 2012;45:1067–1076. 2. (a) Kurreck J. Eur J Biochem. 2003;270:1628–1644; (b) Kole R, Krainer AR, Altman S. Nat Rev Drug Discovery. 2012;11:125–140. 3. Crooke ST. Curr Mol Med. 2004;4:465–487. 4. (a) Gryaznov S, Pongracz K, Matray T, et al. Nucleosides Nucleotides Nucleic Acids. 2001;20:401–449; (b) Asai A, Oshima Y, Yamamoto Y, et al. Cancer Res. 2003;63:3931–3939; (c) Shea-Herbert B, Pongracz K, Shay JW, Gryaznov S. Oncogene. 2002;21:638–642.

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5. (a) Gryaznov S, Chen JK. J Am Chem Soc. 1994;116:3143–3144; (b) Chen JK, Schultz RG, Lloyd DH, Gryaznov S. Nucleic Acids Res. 1995;23:2661–2668. 6. Eckstein F. Antisense Nucleic Acid Drug Dev. 2000;10:117–121. 7. Egli M, Gryaznov S. Cell Mol Life Sci. 2000;57:1440–1456. 8. (a) Gryaznov S. Biochim Biophys Acta. 1999;1489:131–140; (b) Skorski T, Perrotti D, Nieborowska-Skorska M, Gryaznov S, Calabretta B. Proc Natl Acad Sci USA. 1997;94:3966–3971. 9. (a) Pongracz K, Gryaznov S. Tetrahedron Lett. 1999;40:7661–7664; (b) Baraniak J, Korczyn´ski D, Stec WJ. J Org Chem. 1999;64:4533–4536. 10. Nelson JS, Fearon KL, Nguyen MQ, et al. J Org Chem. 1997;62:7278–7287. 11. (a) Gryaznov S, Asai A, Oshima Y, et al. Nucleosides Nucleotides Nucleic Acids. 2003;22:577–581; (b) Dikmen G, Ozgurtas T, Gryaznov S, Herbert BS. Biochim. Biophys. Acta. 2009;1792:240–247. 12. (a) Blackburn EH. Mol Cancer Res. 2005;3:477–482; (b) Shay JW, Wright WE. Nat Rev Drug Discovery. 2006;5:577–584; (c) Wong JMY, Collins K. Lancet. 2003;362:983–988. 13. Guga P. Curr Top Med Chem. 2007;7:695–713. 14. (a) Baraniak J, Kaczmarek R, Wasilewska E. Tetrahedron Lett. 2004;45:671–674; (b) Kaczmarek R, Baraniak J, Stec WJ. Synlett. 2009;14:2269–2273; (c) Radzikowska E, Baraniak J. Org Biomol Chem. 2015;13:269–276. 15. (a) Atherton FR, Openshaw HT, Todd AR. J Chem Soc. 1945;660–663; (b) Le Corre SS, Berchel M, Couthon-Gourvès H, Haelters JP, Jaffrès PA. Beilstein J Org Chem. 2014;10:1166–1196. 16. Zielinska D, Pongracz K, Gryaznov SM. Tetrahedron Lett. 2006;47:4495–4498. 17. Experimental procedure for 2: H2S was bubbled through the MeCN (4 mL) solution of 1 (0.5 mmol, 390 mg) for 5 min. Then, a 0.5 M solution of tetrazole in MeCN (1 mL) was added and the reaction mixture stirred at room temperature for 30 min. The solvent was evaporated and the residue dissolved in CH2Cl2, washed with saturated NaHCO3, and dried over anhydrous MgSO4. After solvent removal, product 2 was obtained as a white solid in 96% yield (341 mg). 31P NMR (C6D6) d: 72.37; 72.17 ppm; 1H NMR (CD3CN) d: 7.55 (m, 7H), 7.31 (m, 9H), 6.03 (t, J = 7.03 Hz, 1H), 4.17 (m, 1H), 4.05 (m, 1H), 3.86 (m, 1H), 3.55 (dd, J = 6.22 Hz, J = 11.74 Hz, 2H), 3.41 (m, 1H), 2.65 (m, 2H), 2.51 (t, J = 6.18 Hz, 1H), 1.59 (m, 2H), 1.14 (d, J = 6.49 Hz, 6H); FAB-MS: (M+1) m/z 712. 18. Experimental procedure for 5: A mixture of crude 2 (0.5 mmol, 356 mg) and 3 (0.5 mmol, 168 mg) were dissolved in DMF (7 mL), then CCl4 (0.52 mmol, 80 mg) and DIPEA (0.52 mmol, 67 mg) were added dropwise. After 30 min, the desired compound 5 was isolated as a mixture of diastereomers by silica gel chromatography [silica gel 200–300 mesh, eluent: MeOH in CHCl3 (1 ? 13%)] in 62% yield (324 mg).31P NMR (CH3OD) d: 72.61; 71.00 ppm; 1H NMR (CD3CN) d: 7.90 (s, 2H), 7.69 (s, 2H), 7.53 (m, 12H), 7.28 (m, 12H), 7.17 (m, 6H), 5.99 (t, J = 6.76 Hz, 2H), 5.95 (m, 2H), 4.42 (t, J = 9.87 Hz, 2H), 4.27 (m, 2H), 4.22 (m, 2H), 4.07 (m, 2H), 3.99 (m, 4H), 3.69 (m, 2H), 3.53 (m, 2H), 3.38 (m, 4H), 2.78 (m, 2H), 2.73 (m, 4H), 2.66 (m, 2H), 2.53 (d, J = 9.86 Hz, 2H), 2.45 (m, 2H), 2.05 (m, 2H), 1.64 (m, 2H), 1.17 (d, J = 9.23 Hz, 6H), 1.16 (d, J = 9.23 Hz, 6H), 1.12 (d, J = 6.77 Hz, 6H), 1.11 (d, J = 6.77 Hz, 6H); MALDI-MS: (M+1) m/z 1046. 19. Experimental procedure for 6: A mixture of crude 2 (0.5 mmol, 356 mg) and 4 (0.5 mmol, 178 mg) were dissolved in the mixture of MeCN/pyridine (1:1) (7 mL), then CCl4 (0.52 mmol, 80 mg) and DIPEA (0.52 mmol, 67 mg) were added dropwise. After 30 min the product 6 was isolated as a mixture of diastereomers by silica gel chromatography [silica gel 200–300 mesh, eluent: MeOH in CHCl3 (1 ? 13%)] in 57% yield (303 mg). 31P NMR (CH3OD) d: 72.83; 71.29 ppm; 1H NMR (CD3CN) d: 8.57 (s, 2H), 8.36 (s, 2H), 7.94 (m, 4H), 7.60 (s, 2H), 7.44 (m, 18H), 7.18 (m, 18H), 6.34 (t, J = 5.55 Hz, 2H), 5.74 (m, 2H), 4.83 (m, 2H), 4.450 (m, 2H), 4.21 (m, 4H), 4.06 (m, 10H), 3.92 (m, 2H), 3.60 (m, 8H), 3.08 (m, 6H), 2.72 (m, 6H), 1.06 (m, 6H); MALDI-MS: (M+1) m/z 1064. 20. Single crystal X-ray data have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC – 1506736. 21. Kaczmarek R, Kaz´mierski S, Pawlak T, Radzikowska E, Baraniak J. Tetrahedron. 2016;72:803–809.