A convenient, highly selective and eco-friendly N-Boc protection of pyrimidines under microwave irradiation

Abstract

Protected pyrimidine nucleobases are of major importance as intermediates in the synthesis of nucleoside analogues and molecules with biological interests. We describe herein a novel practical microwave-assisted N-Boc protection of pyrimidine nucleobases under mild conditions using silica gel, avoiding treatment steps, and in increased yield.

---

Pyrimidine systems are found in many drugs which interact with the synthesis and functions of nucleic acids such as the antitumor drug fluorouridine¹ (1), the anti-HIV drug lamivudine² (2) or the anti-HBV drug telbivudine³ (3), or the antimalarial drug pyrimethamine⁴ (4), (Fig. 1), to only quote some of them.

Fig. 1 Chemotherapeutics containing a pyrimidine moiety.

As chemical modifications of nucleobases are of paramount importance in the development of new drugs,⁵ their syntheses have been largely described.⁶ Focusing on the base moiety, a key step is the choice of the appropriate protecting groups for the N1 and N3 atoms of pyrimidines which have to meet several requirements such as: a differential protection at N1 and N3, a selective deprotection, and a removal under mild reaction conditions. There is an abundant literature relating to the protection of amino groups, such as by 9-fluorenyl-methoxycarbonyl (FMOC),⁷ tert-butyl carbamates (Boc), benzyl carbamate, benzamide, acetamide,⁸ phtalimides,⁹ triphenyl methyl amide¹⁰ and tosylamide.¹¹

Among these groups, one of the most used is the Boc; it's stable towards most nucleophiles and bases¹² and it can be removed cleanly and selectively under neutral conditions.¹³ This is of particular interest when working with biolabile groups such as found in nucleoside prodrugs.¹⁴ Surprisingly, only two teams have reported the synthesis of N-Boc-protected pyrimidines under basic conditions and in two steps. Gothelf et al.¹⁵ have prepared the N3-Boc-protected thymine (6) through the formation of N1,N3-di-Boc protected derivative and subsequent selective deprotection of N1-Boc group with K₂CO₃ in dioxane, in only 31% overall yield (Fig. 2). Porcheddhu et al.¹⁶ have described the synthesis of N4,N4-di-Boc cytosine (7) in two steps in moderate 60% yield, via the fully Boc protected cytosine which was then treated with aq. NaHCO₃ in MeOH at reflux. However, in spite of their potential utility, these methods suffer from some drawbacks such (a) a long reaction time implied by the first step, (b) unsatisfactory yields of the deprotection step and (c) cumbersome product isolation procedures. Thus, we present herein, a greener practical protocol to selectively synthesize N1-free, N3-Boc protected pyrimidine nucleobases (5–7, 8a–c) under milder conditions, shorter reaction time and in moderate to quantitatively yields.

Fig. 2 Structure of monoprotected pyrimidic nucleobases.

The general strategy involves two steps: (1) the use of microwave irradiation to reach quantitatively the fully protected pyrimidines (10a, b) and (2) the subsequent selective N1-deprotection by treatment with SiO₂ (ref. 17) in diethoxymethane/ethanol (9 : 1) as an eco-friendly surrogate to CH₂Cl₂/MeOH, to desired compounds 5 and 6, respectively (Scheme 1). It is important to quote that microwave activation, which ensures shorter reaction time, cleaner reactions and which has been applied to nucleosides and their precursors,¹⁸ has never been reported for either the Boc-protection or deprotection of pyrimidines.

Scheme 1 General approach to N1 free, N3-Boc-pyrimidines.

Thus, starting with uracil 9a, we applied the microwave assisted, solvent-free and catalyst-free procedure reported by Dighe et al.¹⁹ Unfortunately, in our hands, probably due to the insolubility of uracil derivatives in Boc₂O, the expected compounds were not isolated. We moved then to a solution-phase preparation of N1,N3-di-Boc uracil (10a) under microwave irradiation in dry diethoxymethane (DEM) as a replacement of THF, with an excess of Boc₂O, in presence of 0.35 equivalents of 4-N,N-(dimethylamino)pyridine (DMAP) at 70 °C. It is interesting to quote that DEM is an attractive alternative to conventional solvents as it is an eco-responsible solvent, with unique properties,²⁰ which make it a green industrial solvent.

After evaporation of all volatiles, compound 9a was almost quantitatively converted to 10a (based on TLC) and was engaged in the next step without the need for further purification. Table 1 summarizes the different conditions used for the N1 regioselective deprotection of 10a to 5.

Table 1 Preparation of N3-tert-butylcarbonyluracil (5) by selective deprotection of 10a with SiO₂

Entry	Solvent	Temperature	Reaction time	Yield (%)
a Reaction performed with SiO2 (60% w/w).
1^a	DCM	RT	5 h	43
2^a	DCM/MeOH (9/1)	RT	1 h 30 min	93
3	DCM/MeOH (9/1)	RT	12 h	77
4^a	DCM/MeOH (9/1)	60 °C	1 h	96
5^a	DCM/MeOH (9/1)	MW, 70 °C	2 min	97

---

Based on the work of Zhang et al.,¹⁷ the N1,N3-di-(Boc) uracil (10a) was reacted with SiO₂ in dichloromethane (entry 1). To avoid the full deprotection, the reaction was performed at room temperature and monitored by TLC. After 5 hours, the reaction was stopped by simple evaporation of volatiles, affording a solid deposit for flash chromatography. Under this condition, 5 was obtained in 44% yield. When increasing the polarity to CH₂Cl₂/MeOH (9/1), the desired compound 5 was afforded in 90 minutes at room temperature, in 93% yield (entry 2). The benefits of SiO₂ was confirmed (entry 3) observing the dramatically decreased yield from 93% to 77% yield obtained after 12 hours whereas the activation of the reaction over classical heating increases the yield and reduces the reaction time (entry 4). With our interest in shorter reaction time, the previous conditions were optimized under microwave activation to afford quantitatively the desired compound (entry 5).

Following the same procedure, N1 protected thymine (6) was obtained in good overall yields from 9b. This straight method offers thus a direct solid deposit for flash chromatography after evaporation of all volatiles, avoiding any pre-treatment.

To investigate the final regioselectivity of the Boc derivatives (N1 versus N3), the N1-alkylation was performed and the selectivity fully determined by NMR. For instance, compound 6 was derivatized to 12 with ethyl bromoacetate in the presence of K₂CO₃) (Scheme 2).

Scheme 2 Derivation at N1 position for NMR study.

Structure of 12 was determined by HMBC experiment, confirming the regioselectivity the deprotection step (Fig. 3).

Fig. 3 HMBC NMR analysis of 12.

Then we turned our attention to C5-halopyrimidines which are of great interest as useful synthetic building blocks for further C5 modifications. Surprisingly when applying this two-steps procedure to the C5-halogenopyrimidines (13a–c), we observed the minor formation of the expected N3-Boc uracils (8a–c) concomitant with the major N3-alkylation with a tert-butyl group (14a–c, respectively) (Scheme 3).

Scheme 3 Deprotection of bis-Boc halogenated uracils.

To explain the formation of 14a–c, we hypothesized that: (1) under our conditions (e.g. slightly acidic), the σ-electron withdrawing effect of the C5-halogens (fluoro, chloro, and bromo) can help to release the Boc at N3, and (2) the more nucleophilic N3 obtained can react with the released isobutene under microwave heating in closed vials. When the deprotection step was performed under ultrasounds, due to their “degassing effect”, only the unprotected C5-halogenated uracils were isolated in quantitatively yields.

In order to be as eco-friendly as possible, we have extended the investigation of the DEM/EtOH solvent system, phasing out the dichloromethane and in a less extend, the methanol. Under our optimized conditions (e.g., slightly acidic SiO₂ under microwaves), DEM is an attractive replacement solvent for DCM. Compared to methanol, ethanol is produced by factory fermentation of food crops and it's more ecological than methanol.

Thus, the final optimized conditions were SiO₂ 60% w/w in DEM/EtOH (9/1) under microwaves irradiation. Applied to cytosine, the N4,N4-di-Boc cytosine 7 was reached through 15 in 2 minutes under microwaves at 70 °C with more than 97% yield (Scheme 4).

Scheme 4 Synthesis of derivative 7 under eco-friendly conditions.

In summary, we have accomplished a convenient, highly selective and eco-friendly N-Boc protection of some pyrimidine nucleobases (5–7, 8a–c) under microwave irradiation. The mild and heterogeneous conditions are provided by SiO₂, acid enough to support the deprotection of carbamates. The microwave irradiation has proved to be the most efficient synthesis route, giving the desired compounds with quantitative (for 5–7) or moderate (for 8a–c) yields, in short reaction times. The eco-friendly diethoxymethane and ethanol were chosen as a greener alternative to more toxic dichloromethane and methanol, respectively. These advantages make from these reactions some powerful ecological alternatives, optimizing the synthesis of Boc-thymine and bis-Boc-cytosine and creating a synthetic way for Boc-uracil. This environmentally friendly approach represents a promising way to synthesize these necessary building blocks for the synthesis of nucleosides, of utmost importance in medicinal chemistry.

Acknowledgements

We are grateful for financial support from the FEDER (project COSMI), the Region Centre, the University of Orleans and the LabEx SYNORG (ANR-11-LABX-0029).

Notes and references

1. D. K. Goette, J. Am. Acad. Dermatol., 1981, 4, 633–649.
2. W. Choi, R. Schinazi, L. J. Wilson, S. Yeola and D. C. Liotta, J. Am. Chem. Soc., 1991, 113, 9377–9379.
3. A. Holy, Collect. Czech. Chem. Commun., 1972, 37, 4072–4087.
4. H. M. McIntosh, from The Cochrane Library, Update Software Ltd, 2001, Issue 4.
5. K. I. Kimura and T. D. H. Bugg, Nat. Prod. Rep., 2003, 20, 252–273.
6. (a) D. J. Brown, The Pyrimidines, Wiley-Interscience, New York, 1970; (b) D. J. Brown, in Comprehensive Heterocyclic Chemistry, Pergamon Press, Oxford, 1984, vol. 3, p. 84; (c) I. M. Lagoja, Chem. Biodiversity, 2005, 2, 1–48.
7. L. A. Carpino and G. Y. Han, J. Org. Chem., 1972, 37, 3404–3409.
8. W. Green and P. G. M. Wuts, in Protective groups in Organic Synthesis, John Wiley & Sons, New York, 3rd edn, 1999.
9. P. Y. Reddy, S. Kondo, T. Toru and Y. Ueno, J. Org. Chem., 1997, 62, 2652–2654.
10. M. Soroka and J. Zygmunt, Synthesis, 1988, 5, 370–372.
11. B. Nyasse, L. Greyn, H. L. S. Maia, L. S. Monteiro and U. Ragnarsson, J. Org. Chem., 1999, 64, 7135–7139.
12. (a) A. Sarkar, S. R. Roy, N. Parikh and A. K. Chakraborti, J. Org. Chem., 2011, 76, 7132–7140; (b) E. A. Englund, H. N. Gopi and D. H. Appella, Org. Lett., 2004, 6, 213–215; (c) R. Moumne, S. Lavielle and P. Karoyan, J. Org. Chem., 2006, 71, 3332–3334; (d) D. M. Shendage, R. Froehlich and G. Haufe, Org. Lett., 2004, 6, 3675–3678.
13. J. R. Hwu, M. L. Jain, S.-C. Tsay and G. H. Hakimelahi, Tetrahedron Lett., 1996, 37, 2035–2038.
14. M. Hamada, V. Roy, T. R. McBrayer, T. Whitaker, C. Ubina-Blanco, S. P. Nolan, J. Balzarini, R. Snoeck, G. Andrei, R. F. Schinazi and L. A. Agrofoglio, Eur. J. Med. Chem., 2013, 67, 398–408.
15. M. F. Jacobsen, M. M. Knudsen and K. V. Gothelf, J. Org. Chem., 2006, 71, 9183–9190.
16. (a) A. Porcheddu, G. Giacomelli, I. Piredda, M. Carta and G. Nieddu, Eur. J. Org. Chem., 2008, 5786–5797; (b) T. S. Li, S. F. Lu, L. Xing, G.-C. Lin, Z. Ghuan and Z.-J. Yang, J. Chin. Pharm. Sci., 2010, 19, 436–442.
17. M.-J. Zhang, X.-H. Yuan, M. Li, J.-Y. Zhao and L.-X. Gao, Chem. Res. Chin. Univ., 2007, 12, 2330–2332.
18. V. Roy, U. Pradère and L. A. Agrofoglio, Future Med. Chem., 2010, 2, 177–192.
19. S. N. Dighe and H. R. Jadhav, Tetrahedron Lett., 2012, 53, 5803–5806.
20. N. W. Boaz and B. Venepalli, Org. Process Res. Dev., 2001, 5, 127–131.

---

Footnote

† Electronic supplementary information (ESI) available: General procedure and ¹H, ¹³C NMR spectra are provided. See DOI: 10.1039/c4ra13033b

---