Process for preparing imetelstat
The present invention relates to a process for preparing the telomerase inhibitor imetelstat using a 3 steps per cycle phase support bound process comprising the steps of deprotection of the 3′-amino group of the support-bound oligonucleotide, coupling with a 5′-phosphoramidite, and sulfurization with an acyl disulfide, characterized by the absence of an additional capping step in each cycle that is used to prevent unreacted 3′-amino oligonucleotide groups from reacting during subsequent cycles. Imetelstat has formula below.
1. A method of synthesizing the N3′→P5′ thiophosphoramidate oligonucleotide imetelstat of formula
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′-O-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer of formula (B′ n ) wherein n=2 to form an internucleoside N3′→P5′-phosphoramidite linkage;
d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3′→P5′ thiophosphoramidate;
e) repeating 11 times in successive order the deprotection step b), the coupling step c) with a protected 3′-aminonucleoside-5′-O-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B′ n ) wherein the protected nucleoside base B′ in monomer (B′ n ) is successively the protected nucleobase B 3 to B 13 in the respective 11 coupling steps, and the sulfurization step d);
f) removing the acid-labile protecting group PG; and
g) cleaving and deprotecting imetelstat from the solid-phase support;
characterized in that no additional capping step is performed in any of the reaction steps a) to e).
2. The method as claimed in claim 1 wherein imetelstat is further converted into its sodium salt.
3. The method as claimed in claim 1 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.
4. The method as claimed in claim 3 wherein the acyl disulfide is PADS.
5. 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.
6. The method as claimed in claim 5 wherein PADS is dissolved in a mixture of 2,6-lutidine with acetonitrile.
7. The method as claimed in claim 6 wherein the PADS solution is aged between 4 and 14 hours before use.
8. The method as claimed in claim 1 wherein the acid-labile protecting group PG is selected from triphenylmethyl, p-anisyldiphenylmethyl, and di-p-anisylphenylmethyl.
9. The method as claimed in claim 1 wherein the acid-labile protecting group PG is removed by treatment with an acidic solution.
10. The method as claimed in claim 1 wherein the protecting group on the adenine, cytosine and guanine base in the monomer of formula (B′ n ) is a base-labile protecting group selected from acetyl, benzoyl, isobutyryl, dimethylformamidinyl, and dibenzylformamidinyl.
11. The method as claimed in claim 1 wherein the coupling step c) is performed using an activator selected from tetrazole, 5-(ethylthio)-1H-tetrazole, 5-(4-nitro-phenyl)tetrazole, 5-(2-thienyl)-1H-tetrazole, triazole, and pyridinium chloride.
12. The method as claimed in claim 1 wherein step f) 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.