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Synthesis of 4’-Thio-Modified-Nucleosides and Nucleotides

1. INTRODUCTION

2. SYNTHESIS OF 4’-THIO-MODIFIED NUCLEOSIDES

Modified nucleotides are of great significance in the treatment of viral infections. Sofosbuvir, a sugar-modified nucleotide prodrug for the treatment of HCV and Tenofovir Disoproxil, an acyclic nucleotide pro-drug used for the treatment of HIV and chronic HBV, are excellent examples of this important class of compounds.

Thionucleosides have also been identified to have potential as novel treatments for HCV and other flavivirus infections.1 Nucleotide analogues still pose significant challenges in terms of both synthesis and purification. However, scientists at Concept Life Sciences in collaboration with Shire, have developed a robust and reliable synthesis of 4’-thio-modified-nucleosides and corresponding nucleotides.2Starting from the readily available D-Ribonic acid-1,4-lactone, 3 to 10 g of various nucleosides have been synthesised in 10 to 15 steps (the bases used were uracil, 2-thio-uracil, cytosine, 5-Me-cytosine, guanine and adenine). See section 2.The nucleosides were then converted to the corresponding nucleotides using either the method of Bogachev3 or Moffatt4 in 3 to 6 chemical steps which were purified by ion exchange column chromatography to give 150 to 500 mg of the targets. See sections 3 and 4.

4. PURIFICATION

5. CONCLUSION

aHuxley, A., aQueva S., aWainwright P., aZhang X., aGlen R., aHull J. and bDe Rosa F.a Concept Life Sciences Ltd., Chapel-en-le-Frith, Derbyshire, SK23 0PG, UK | b Shire, 300 Shire Way, Lexington, MA 02421, USA

All nucleotides were purified by ion exchange column chromatography (DEAE Sepharose fast flow) eluting with a gradient of triethylammonium bicarbonate solution from 0.01 M to 0.5 M. The advantages of ion exchange over reverse phase preparative chromatography are as followed:

• Very good recovery• Scalable (500 mg isolated in one run)• Good separation of mono-, di- and triphosphate• Triphosphate obtained as its Et3N salt which is more stable

than the common Na+ salt or the free acid

3. FROM NUCLEOSIDE TO NUCLEOTIDE

1. The Bogachev approachThe protected nucleosides 8a to 8c were converted to their corresponding nucleotides in three steps. Addition of di-tert-butyl diisopropylphosphoramidite and oxidation with H2O2 was followed by TFA mediated deprotection to give the mono-phosphate 11 in good yield. The triphosphates 12a, 12a’, 12b and 12c were then obtained (25 to 62% yield, 150 to 500 mg) in a one pot procedure developed by Bogachev3 using TFAA as an activating agent followed by treatment with a nucleophilic catalyst and inorganic pyrophosphate.

Pyrimidine based thio-nucleosides 8 were prepared using the chemistry shown in scheme 1. 330 g of compound 5 was obtained from 500 g of 1 using literature procedures5. Oxidation of 5 with m-CPBA gave two isomers which were separated, since only the isomer shown below was able to react in the Pummerer reaction. This afforded the fully protected pyrimidine nucleosides 7a, 7b and 7c (3 to 10 g). Further modification of the base followed by silyl deprotection gave nucleosides 8a, 8a’, 8b and 8c, which were used as the starting materials for the synthesis of the corresponding nucleotides. Purine based nucleosides were prepared as shown in scheme 2, where sulfoxide 9 was synthesised in order to functionalise the 2’ position which was part of another project.

REFERENCES

1. “1’,4’-Thionucleosides for the Treatment of HCV”; US2014/0364446 A1 2. WO 2014/152513 A1 3. Bogachev, V.S. Synthesis of deoxynucleoside 5’-triphosphates using trifluoroacetic anhydride as activation reagent. Russ. J. Bioorg. Chem., 1996, 22, 599-604 4. Moffatt, J.G.; Khorana, H.G. J. Am. Chem. Soc., 1961, 83, 649-658; Moffatt, J.G. Can. J. Chem., 1964, 42, 599-604 5. Jayakanthan, K.; Johnston, B.D.; Pinto, B.M. Carbohydrate Research, 2008, 343, 1790-1800

SCHEME 1

Conditions: i. Acetone, H2SO4, 93%; ii. MsCl, Et3N, DCM, quant.; iii. KOH, H2O, 73%; iv. TBDMSCl, imidazole, DCM, quant.; v. NaBH4, THF/MeOH, 86%; vi. MsCl, Et3N, DCM, 91%; vii. Na2S.9H2O, DMF, 62%; viii. m-CPBA, DCM, 35%; ix. Base, TMSOTf, Et3N, DCM/Toluene 40 to 63%; x. TBAF, THF, 86% of 8a, 95% of 8b; xi. a) POCl3, triazole, Et3N, CH3CN, b) NH4OH, dioxane, c) TBAF, THF, 74% overall yield of 8a’/ 89% overall yield of 8c

SCHEME 2

Conditions: xii. a)TFA, THF/H2O, 88%, b) Py., TIPDSCl2, 64%, c) Py., BzCl, 64%, d) m-CPBA, DCM, 82%; xiii. a) 6-chloropurine, 2,6-lutidine, TMSOTf, DCE/CH3CN, 42%, b) AcOH, TBAF, THF, 99%, c) NH3, MeOH, 71%, d) perchloric acid, acetone, 80%; xiv. a) 6-chloro-2-aminopurine, 2,6-lutidine, TMSOTf, DCE/CH3CN, 17%, b) AcOH, TBAF, THF, 99%, c) mercaptoethanol, NaOMe, MeOH, 30%, d) perchloric acid, acetone, 80%

2. The Moffatt approachThe approach described above was unsuccessful for nucleosides bearing a purine base. Therefore, the Moffatt procedure4 was investigated. The phosphorylation remains the same as described above. However, the monophosphate 14 was then converted to its piperidine adduct 15 which was reacted with tri-n-butylammonium pyrophosphate to give the nucleotides 12d and 12e in 50 to 75% yield (200 to 500 mg).

SCHEME 1

Conditions: i. Di-tert-butyl diisopropylphosphoramidite, imidazole, imidazole.HCl then H2O2, 60 to 85%; ii. TFA, DCM/H2O, quant.; iii. a) TFAA, Et3N, N,N-dimethylaniline, CH3CN, b) 1-methylimidazole, Et3N, CH3CN, c) tris(tetrabutylammonium)pyrophosphate, CH3CN, 25 to 62%.

SCHEME 2

Conditions: i. a) TFA, DCM/H2O, quant., b) DMFDMA, MeOH, 80%, c) Di-tert-butyl diisopropylphosphoramidite, imidazole, imidazole.HCl then H2O2, 40 to 60 %; ii. a) NH3, MeOH, 90 to 99%, b) TFA, DCM/H2O, quant.; iii. Piperidine, 2,2’-Dipyridyldisulfide, PPh3, DMSO then NaI in acetone, 80 to 95%; iv. 0.4M tri-n-butylammonium pyrophosphate in DMF, 40 to 70%.

During our collaboration with Shire, chemists from Concept Life Sciences have acquired and developed outstanding knowledge and skill towards the synthesis of 4’-thio-modified-nucleosides and nucleotides. Starting from 500 g of D-Ribonic acid-1,4-lactone, we have succeeded in isolating 150 to 500 mg of several 4’thio-nucleotides. We also established a reliable method to purify nucleotides on scale up to 1 g by ion exchange column chromatography (DEAE Sepharose fast flow) enabling the synthesis of these important modified nucleotides for a variety of applications including potential treatments to combat viral infections.