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1. (WO2019011829) IMPROVED PROCESS FOR PREPARING IMETELSTAT
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Claims

1. A method of synthesizing the N3' - P5' thiophosphoramidate oligonucleotide imetelstat of formula

imetelstat


the method comprises of

a) providing a first 3'-amino protected nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;


b) deprotecting the protected 3'-amino group to form a free 3'-amino group;


c) reacting the free 3'-amino group with a protected 3'-aminonucleoside-5'-0-cyanoethyl- Ν,Ν-diisopropylaminophosphoramidite monomer of formula (B n), wherein B n with n = 2 is protected A, to form an internucleoside N3'- P5'-phosphoramidite linkage;

mer


d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3'- P5' thiophosphoramidate;

e) repeating 1 1 times in successive order the deprotection step b), the coupling step c) with a protected 3'-aminonucleoside-5'-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B n) wherein the nucleoside base B' of monomer (B n) is protected B except when B is thymine, and wherein Bn is successively nucleobase B3 to B13 in the respective 1 1 coupling steps, and the sulfurization step d);

f) removing the acid-labile protecting group PG; and

g) deprotecting and cleaving imetelstat from the solid-phase support;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

The method as claimed in claim 1 wherein imetelstat is further converted into its sodium salt.

The method as claimed in any one of claim 1 or claim 2 wherein the acyl disulfide is selected from dibenzoyl disulphide, bis(phenylacetyl) disulfide (PADS), bis(4-methoxybenzoyl) disulphide, bis(4-methylbenzoyl) disulphide, bis(4-nitrobenzoyl) disulphide and bis(4-chlorobenzoyl) disulfide.

The method as claimed in claim 3 wherein the acyl disulfide is PADS.

The method as claimed in claim 4 wherein PADS is dissolved in a mixture of 3-picoline or 2,6-lutidine with a co-solvent selected from acetonitrile, toluene, 1-methylpyrrolidinone and tetrahydrofuran.

The method as claimed in claim 5 wherein PADS is dissolved in a mixture of 2,6-lutidine with acetonitrile.

The method as claimed in claim 6 wherein the PADS solution is aged between 4 and 14 hours before use.

The method as claimed in any one of the preceding claims wherein the acid-labile group PG is selected from triphenylmethyl, p-anisyldiphenylmethyl, and di-p-anisylphenylmethyl.

9. The method as claimed in any one of the preceding claims wherein the acid-labile protecting group PG is removed by treatment with an acidic solution.

10. The method as claimed in any one of the preceding claims wherein the base-labile protecting group on an adenine, cytosine and guanine base in the monomer of formula (B n) is selected from acetyl, benzoyl, isobutyryl, dimethylformamidinyl, and dibenzylformamidinyl.

1 1. The method as claimed in any of the preceding claims wherein the coupling step c) is

performed using an activator selected from tetrazole, 5-(ethylthio)-1 H-tetrazole, 5-(4-nitro- phenyl)tetrazole, 5-(2-thienyl)-1 H-tetrazole, triazole, and pyridinium chloride.

12. The method as claimed in any of the preceding claims wherein step g) is performed by treatment with a basic solution.

13. The method as claimed in claim 12 wherein the basic solution is diethylamine dissolved in acetonitrile or aqueous ammonia dissolved in an alcohol, or a combination of both.