Commercial-scale synthesis of protected 2′-deoxycytidine and cytidine nucleosides

Article abstract of DOI:10.1081/NCN-120022956

Transformation of 2′-deoxyuridine and uridine analogs to protected 2′-deoxycytidine and cytidine analogs has been investigated by two different methods. First, traditional triazolation protocol and second p-nitrophenoxylation method. Our studies conclude

Full text of DOI:10.1081/NCN-120022956

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Commercial-Scale Synthesis of Protected 2′-
Deoxycytidine and Cytidine Nucleosides
Kikkeri J. Divakar a c , Chitra M. Sawant a , Y. A. Mulla a , Deepak V. Zemse a , Sakina M.
Sitabkhan a , Bruce S. Ross b & Yogesh S. Sanghvi b
a Research and Development, Innovasynth Technologies (India) Limited, (Formerly a Division
of Indian Organic Chemicals Limited), Khopoli District, Raigad, Maharashtra, India
b Isis Pharmaceuticals Inc., Carlsbad, California, USA
c R&D Division, Indofil Chemicals Company, S. V. Road, Azad Nagar, Sandoz Baug P.O., Thane,
400 607, Maharashtra, India
Available online: 31 Aug 2006
To cite this article: Kikkeri J. Divakar, Chitra M. Sawant, Y. A. Mulla, Deepak V. Zemse, Sakina M. Sitabkhan, Bruce S. Ross &
Yogesh S. Sanghvi (2003): Commercial-Scale Synthesis of Protected 2′-Deoxycytidine and Cytidine Nucleosides, Nucleosides,
Nucleotides and Nucleic Acids, 22:5-8, 1321-1325
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1321–1325, 2003
Commercial-Scale Synthesis of Protected 20-Deoxycytidine
and Cytidine Nucleosides
1
1
Kikkeri J. Divakar,1, Chitra M. Sawant, Y. A. Mulla,
Deepak V. Zemse,1 Sakina M. Sitabkhan,1
Bruce S. Ross,2 and Yogesh S. Sanghvi2
*
1Research and Development, Innovasynth Technologies (India) Limited,
(Formerly a Division of Indian Organic Chemicals Limited),
Khopoli District, Raigad,
Maharashtra, India
2Isis Pharmaceuticals Inc., Carlsbad, California, USA
ABSTRACT
Transformation of 20-deoxyuridine and uridine analogs to protected 20-deoxy-
cytidine and cytidine analogs has been investigated by two different methods.
First, traditional triazolation protocol and second p-nitrophenoxylation method.
Our studies conclude that the triazolation method is better and suitable for com-
mercial scale–up.
Key Words: Cytidine; Deoxycytidine; Triazolation; 4-Nitrophenoxylation.
*Correspondence: Kikkeri J. Divakar, R&D Division, Indofil Chemicals Company, S. V.
Road, Azad Nagar, Sandoz Baug P.O., Thane 400 607, Maharashtra, India; Fax: +91 22
2589 8369; E-mail: divakj@yahoo.com.
1321
DOI: 10.1081/NCN-120022956
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Divakar et al.
INTRODUCTION
With the progress of several antisense oligonucleotide drugs through the clinical
trials and consequent requirement of large quantities of these products, an increased
demand for larger quantity of protected nucleoside building blocks was inevi-
table.[1,2] Protected 20-deoxycytidine and cytidine analogs, which are also required
in larger quantities, are generally prepared from: (a) 20-deoxycytidine (dC) or cyti-
dine (C); (b) 20-deoxyuridine (dU) or uridine (U); (c) thymidine.
The second option is more attractive for reasons of economy and easy access of
starting materials. Several methods have been reported describing conversion of
dU=U derivatives to the corresponding dC=C derivatives. We have investigated
two approaches, first through triazolation and secondly via 4-nitrophenoxylation
of dU=U derivatives, with special emphasis on their usefulness for commercial scale
production.
CHEMISTRY AND DISCUSSION
Protected dC and C building blocks, which are required for the synthesis of
deoxyribo- and ribo-oligonucleotides respectively are synthesized either from dC
or C[3] or alternatively, from dU or U. The latter option is more attractive for rea-
sons of cost and accessibility of raw materials. For example, we estimate that the cost
of protected dC 8 synthesized from dU is lower by 15–20% when compared to that
synthesized from dC.
Therefore, we investigated the synthesis of dC and C building blocks, starting
from dU and U. Many methods are available for the conversion of 4-oxopyrimidine
nucleosides to the corresponding 4-aminopyrimidine nucleosides.[4] Among these
methods, the 4-triazol-1-yl-pyrimidine nucleoside mediated procedure had been
successfully employed[5] for the conversion of 50-O-DMT protected thymidine to the
corresponding 4-amino analog. As a result, we opted for this method for the pro-
posed conversion of other 4-oxopyrimidine nucleosides 1 (See Sch. 1) to the 4-
aminopyrimidine nucleosides 8.
Several variants of this method are available in the literature for the synthesis
of 4-triazol-1-yl pyrimidine nucleosides 3. Literature method describes addition of
the suitably protected 4-oxo-pyrimidine nucleoside to a solution of phosphoryl-tris-
triazolide [formed in situ by the reaction of phosphorous oxychloride and 1,2,4-
(1H)-triazole in the presence of triethylamine] in acetonitrile. The product is isolated
after quenching (triethylamine þ water mixture) and partitioning between an organic
solvent and saturated sodium bicarbonate solution.[6] We designate this as Method A.
In another variant, designated as Method B, the protected 4-oxopyrimidine
nucleoside to be triazolated is mixed with 1,2,4-triazole and triethylamine and to this
cooled mixture is added phosphorous oxychloride. The 4-(1,2,4-(1H) -triazolyl pro-
duct 4 or 6 is isolated as above.[7] We decided to try both variants for the synthesis of
the required 4-amino-pyrimidine nucleosides 8.
Thus, 50-O-(4,40-Dimethoxytrityl) -deoxyuridine 1a was treated with acetic an-
hydride in pyridine to yield 2a or trimethyl chlorosilane in acetonitrile containing
triethylamine to provide 5a. All attempts to triazolate 2a by either method A or

Commercial-Scale Synthesis of Cytidine Nucleosides
1323
Scheme 1.
method B failed in our hands. Furthermore, treatment of the product of this reaction
after workup with 1,4-dioxane and aqueous ammonia did not yield 50-(4,40-
dimethoxytrityl)-deoxycytidine 8a. We rationalized this outcome as due possibly
to the instability of the 4-(1,2,4-(1H)-triazolyl derivative 3a or 6a. Interestingly,
examples of triazolation mediated conversion of protected dU to the corresponding
dC derivative were unavailable in the literature. Recently, such instability of the tri-
azolyl derivative has also been observed by another group.[8]
As an alternative approach, we attempted the conversion by the procedure
described by Miah et al.[7] While Miah et al. used the unprotected nucleoside sub-
strates for their transformations, we used either 2a or 5a as the starting materials.
This change required the work up of the product differently. Treatment of a mixture
of 2a and N-methylpyrrolidine or triethylamine in acetonitrile with phosphorous
oxychloride and 4-nitrophenol followed by work up of the concentrated reaction
mixture by partitioning between ethyl acetate and sodium bicarbonate solution gave

1324
Divakar et al.
the 4-nitrophenoxy derivative 4a. Similarly, treatment of 5a with phosphorous
oxychloride and 4-nitrophenol in the presence of N-methyl pyrrolidine (or N-
methyl-morpholine) followed by work up of the concentrated reaction mixture by
partitioning between ethyl acetate and sodium bicarbonate solution afforded the 4-
nitrophenoxy derivative 7a. Compound 4a or 7a was not purified but treated with
a mixture of aqueous ammonia and 1,4-dioxane to obtain 8a in 57% yield. This pro-
duct was contaminated with 4-nitrophenol and required purification by silica gel
chromatography. On small-scale (<10 g), this procedure looked promising. How-
ever, further scale-up to pilot–plant level (5 Kg) and removal of traces of 4-nitrophe-
nol by chromatography proved time consuming and expensive. The overall yield of
8a from 1a was only 30–40% on pilot scale. Furthermore, ammonolysis of 7a
required 60 h for completion resulting in higher plant occupation cost.
Reverting to triazolation, we focused our attention on the following aspects that
could improve the yield of 4-triaolyl derivative 3a: (a) use of anhydrous solvents and
reagents and inert conditions during reaction; (b) ensuring efficient stirring of the
reaction mixture and temperature control; (c) ensuring efficient heat dissipation dur-
ing the addition of phosphorous oxychloride. Measures (b) and (c) were proved
essential by the reaction calorimetry study of the triazolation step which indicated
that the addition of phosphorous oxychloride to the protected nucleoside 5 was
prone to form ‘‘hot spots’’.
After many attempts, successful triazolation of 1a was accomplished employing
method B. The key to success was avoiding the isolation of triazolyl intermediate 3a.
The crude product was subjected to ammonolysis under usual conditions to provide,
after work up, 8a which was purified by column chromatography. An overall yield of
70% was obtained for the crystalline 8a on a 10 kg scale.
Similarly, 1b was protected on 30-end as acetate 2b and the product triazolated
by method A in good yields (70%). Unlike in case of the dU derivatives, here, tri-
azolation proceeded without any problems. For the conversion of 2b to 8b via
method B on a larger scale, ‘‘transient’’ silyl protection offered the advantage of
reducing two process steps. After ammonolysis and chromatographic purification,
a 86% yield of 50-O-(4,40-dimethoxytrityl)-20-O-methoxyethyl-5-methylcytidine 8b
was achieved. This transformation is now routinely performed on 10–20 kg scales.
We have also utilized the triazolation process to convert 50-O-(4,40-dimethoxytri-
tyl)-20-O-methyl-uridine 1c to 50-O-(4,40-dimethoxytrityl)-20-O-methyl cytidine 8c in
overall yields of 66% on kilo scale.
In summary, we have shown that while both the 4-(1,2,4-1H-triazolyl)- and
4-(4-nitrophenoxy)-derivatives are useful intermediates for conversion of 4-oxopyri-
midine nucleosides to the corresponding 4-aminopyrimidine nucleosides on smaller
scales, on commercial scale conversions, the former process offered cleaner product
in higher yields and at lower cost.
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