Process development for the synthesis of 5′-O-(4,4′ -dimethoxytrityl)-N2-isobutyryl-2′-O-(2-methoxyethyl) -guanosine - A potential precursor for the second generation antisense oligonucleotides: An improved process for the preparation of 2′-O-a

Article abstract of DOI:10.1081/NCN-120022957

An efficient four step process for the preparation of 5′ -O-(4,4′-dimethoxytrityl)-N2-isobutyryl-2′-O-(2-methoxyethyl) -guanosine 1 was developed. Direct 2′-O-alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive and commercially ava

Full text of DOI:10.1081/NCN-120022957

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1327–1330, 2003
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Process Development for the Synthesis of 5 -O-(4,4 -
2
0
Dimethoxytrityl)-N -isobutyryl-2 -O-(2-methoxy-
ethyl)-guanosine—A Potential Precursor for the
Second Generation Antisense Oligonucleotides:
An Improved Process for the Preparation
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of 2 -O-Alkyl-2,6-diaminopurine Riboside
1
1
1
Shabbir Ali S. Taj, P. Gurumurthy, R. Suresh, S. Narayanan,
1
1
S. Suman Meenakshi, and Yogesh S. Sanghvi
2,
*
1
Shasun Chemicals and Drugs Limited, Chennai, India
2
Isis Pharmaceuticals Inc., Carlsbad, California, USA
ABSTRACT
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An efficient four step process for the preparation of 5 -O-(4,4 -dimethoxytrityl)-
2
0
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N -isobutyryl-2 -O-(2-methoxyethyl)-guanosine 1 was developed. Direct 2 -O-
alkylation of 2,6-diaminopurine riboside 2 was accomplished via inexpensive
and commercially available reagents such as KOH, DMSO and alkyl halides at
room temperature in 4–6 hrs. Pure 2 -O-(2-methoxyethyl)-DAPR 3 was isolated
by crystallization from methanol. Enzymatic deamination of 3 followed by selec-
tive N -isobutyrylation and 5 -O-dimethoxytritylation furnished desired 1 in high
yield and purity. Fully optimized four step synthetic process has been scaled up to
the pilot plant level.
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2
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*Correspondence: Yogesh S. Sanghvi, ISIS Pharmaceuticals, 2292 Faraday Avenue, Carlsbad,
92008 CA, USA; Fax: þ1 760 603 4655; E-mail: ysanghvi@isisph.com.
1
327
DOI: 10.1081/NCN-120022957
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

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Taj et al.
INTRODUCTION
Antisense technology is a novel drug discovery method. Understanding the
genetic basis of complex disease process using antisense may open the doors to creat-
ing safer and more efficient drugs. In addition to the first generation phosphoro-
0
thioate oligonucleotides, the 2 -O-alkylribonucleotides have been recently developed
as second-generation oligonucleotide analogues with improved antisense proper-
[1]
ties. Therefore, it is increasingly important to develop an efficient, cost effective,
non-hazardous, industrially feasible and eco-friendly process for the synthesis of
various modified nucleosides required to construct these oligonucleotides. Among
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0
2
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these 5 -O-(4,4 -dimethoxytrityl)-N -isobutyryl-2 -O-(2-methoxyethyl)-guanosine (5 -O-
2
DMT-N -Ibu-2 -MOE-G) 1 serves as one of the key building blocks required for the
synthesis of second generation oligonucleotides.
0
[
2]
RESULTS AND DISCUSSION
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In order to synthesize nucleoside 1, regioselective alkylation at the 2 -OH posi-
tion of guanosine is essential. Literature survey revealed variety of methods available
but none of them appear to be suitable from industrial perspective and commercial
0
scale-up. For example, 2 -O-alkylguanosine can be synthesized via glycosylation of
[3]
D-ribose. This method involves too many steps and formation of undesired
0
a-anomer resulted in low overall yield. Selective alkylation at 2 -OH group of guano-
sine can also be effected by simultaneous protection of 3 - and 5 -OH groups using
expensive protecting group such as TIPDS-Cl followed by alkylation using non-
0
0
[4]
[5]
practical bases such as NaH or BEMP. Direct alkylation of 2,6-diaminopurine
riboside using NaH=DMF followed by expensive column chromatographic purifica-
[6]
tion is reported but not feasible for large-scale production (Sch. 1).
Scheme 1. Direct alkylation of DAPR, literature protocols.

0
2
-O-Alkyl-2,6-diaminopurine Riboside
1329
Scheme 2. i) KOH (2.6 equiv), DMSO (25 volumes), 2-methoxyethyl bromide (2.5 equiv),
r.t., 7h; ii) adenosine deaminase, sodium phosphate buffer, pH 7.2; iii) acetonitrile=TMS-Cl
(6.0 equiv)=r.t.=3h, Ibu-Cl (1.5 equiv)=r.t.=3h, aq. ammonia (pH 6.4), acetonitrile=aq. acetone;
ꢀ
iv) acetonitrile, 2,6-lutidine (3.3 equiv), DMT-Cl (1.01 equiv)=45 C=3h, EtOAc.
In the wake of these shortcomings, we have developed a suitable process
0
applicable on industrial scale for the synthesis of 1 (Sch. 2). Direct 2 -O-alkylation
of 2,6-diaminopurineriboside 2 was efficiently performed using inexpensive reagents
such as KOH, DMSO and 2-methoxyethyl bromide at room temperature in
0
4
–6 hrs. Changing various reaction parameters, high 2 -regioselectivity was obtained
0
furnishing 62–67% of the desired product 3 in the crude reaction mixture. Pure 2 -
O-(2-methoxyethyl)-2,6-DAPR 3 could be isolated from a complex mixture of other
0
by-products by simple crystallization from methanol in 40% yield. Structures of 2 -
0
and 3 -regioisomers were established by X-ray diffraction analysis. Further
improvements in the yield of 1 were realized when crude reaction mass after alkyla-
0
tion was directly subjected to deamination using ADA to furnish 2 -MOE-G 4 in
45% overall yield and 99.8% product purity. Silylation of 4 using TMS-Cl and iso-
butyryl chloride followed by de-protection provided crude product 5. Crystalliza-
tion of crude 5 from acetonitrile and aqueous acetone furnished 99.8% product
0
purity in 95% yield. Treatment of 5 with freshly crystallized 4,4 -dimethoxytrityl
chloride in acetonitrile and 2,6-lutidine afforded 1 in 85% yield and 99.8% product
purity. Suitable HPLC conditions were also developed which enabled us to separate
and evaluate all intermediates, final product and impurities during synthesis of 1.
The improved protocol has been successfully scaled-up on the pilot plant scale
(
>1Kg).

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Taj et al.
SUMMARY
An efficient process for the preparation of 1 in high purity and yield has been
developed using simple reaction conditions and inexpensive reagents. Purification
of intermediates and final product has been achieved by crystallization avoiding
column chromatography. Improved process has been successfully implemented in
0
0
pilot plant with reproducible results. Structures of 2 and 3 -MOE-DAPR has been
confirmed by single crystal X-ray diffraction method. Suitable HPLC methods have
been developed to monitor the reactions and estimate the product purities.
ACKNOWLEDGMENTS
We are thankful to managements of Shasun Chemicals, Isis Pharmaceuticals and
Austin Chemicals for encouragement and support, Prof. K.K. Balasubramanian for
useful discussions and QC (Shasun) for analytical support.
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