Efficient large-scale synthesis of 5′-O-dimethoxytrityl-N 4-benzoyl-5-methyl-2′-deoxycytidine

Article abstract of DOI:10.1080/15257770600726059

An efficient process to synthesize 5′- O -dimethoxytrityl-N 4 -benzoyl-5-methyl-2 ′-deoxycytidine in high yield and quality is described. Final benzoylation was improved by developing a method to selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-kilogram quantities for routine use in oligonucleotide therapeutics. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770600726059

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Efficient Large-Scale Synthesis of 5′-O-
Dimethoxytrityl-N 4-Benzoyl-5-Methyl-2′-
Deoxycytidine
Bruce S. Ross a , Mingming Han a & Vasulinga T. Ravikumar a
a Isis Pharmaceuticals, Inc., Carlsbad, California, USA
Version of record first published: 24 Feb 2007.
To cite this article: Bruce S. Ross, Mingming Han & Vasulinga T. Ravikumar (2006): Efficient Large-
Scale Synthesis of 5′-O-Dimethoxytrityl-N 4-Benzoyl-5-Methyl-2′-Deoxycytidine, Nucleosides,
Nucleotides and Nucleic Acids, 25:7, 765-770
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Nucleosides, Nucleotides, and Nucleic Acids, 25:765–770, 2006
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770600726059
EFFICIENT LARGE-SCALE SYNTHESIS OF
5ꢀ-O-DIMETHOXYTRITYL-N4-BENZOYL-5-METHYL-2ꢀ-DEOXYCYTIDINE
Bruce S. Ross, Mingming Han, and Vasulinga T. Ravikumar
Isis
2
Pharmaceuticals, Inc., Carlsbad, California, USA
An efficient process to synthesize 5ꢁ-O-dimethoxytrityl-N4-benzoyl-5-methyl-2 ꢁ-deoxycytidine in
2
high yield and quality is described. Final benzoylation was improved by developing a method to
selectively hydrolyze benzoyl ester impurities. This inexpensive approach was scaled up to multi-
kilogram quantities for routine use in oligonucleotide therapeutics.
Keywords Oligonucleotides; Antisense; 5ꢁ-O-DMT-N4-Benzoyl-5-Methyl-2ꢁ-Deoxycyti-
dine; Deoxynucleoside
INTRODUCTION
Phosphorothioate-linked nucleic acid analogs have found widespread
application in therapeutic drug development and molecular biology.[1–3]
Increased resistance to nuclease digestion displayed by phosphorothioate
diester DNA and RNA analogs has prompted use of these molecules for
current and investigational antisense treatment of a variety of diseases.[4,5]
Several antisense phosphorothioate oligodeoxyribonucleotides (ODN) are
currently undergoing clinical evaluation and an antisense drug for treat-
ment of CMV retinitis (Vitravene) has reached the market. Although
phosphorothioate ODN are showing excellent promise as safe and effective
therapeutic agents, their profiles are not yet ideal. Reported high-dose
effects of phosphorothioate ODN include immune cell stimulation, comple-
ment activation, and introduction of blood clotting abnormalities. Although
these effects are seen only at doses of oligonucleotide above those required
for pharmacological activity, investigations of chemical modifications to
enhance therapeutic index and facilitate delivery have been pursued.
Chemical modification of antisense oligonucleotides can confer additional
Received 31 October 2005; accepted 1 February 2006.
Address correspondence to Vasulinga T. Ravikumar, Isis Pharmaceuticals, Inc., 2282 Faraday Ave.,
Carlsbad, CA 92008. E-mail: vravikumar@isisph.com
765

766
B. S. Ross et al.
resistance to nucleases, longer serum half-life, and reduced toxicity. Mod-
ifications can also increase affinity of an antisense oligonucleotide for its
complementary target RNA, resulting in enhanced potency and specificity.
One among various modifications to be largely explored is the use of
5-methyl cytosine and its deoxy analog. 5-Methyl-2ꢁ-deoxycytidine has been
shown to possess interesting biological properties that have been reviewed
extensively.[6–8]
Although 5-methyl-2ꢁ-deoxycytidine can be isolated from salmon sperm
DNA, the quantity is limited due to low percentage content. The previously
reported[9–13] routes involve drastic conditions such as reflux at 140◦C for
72 h or the use of sodium hydride, which is not desirable in large-scale
synthesis. Recently an efficient amination method was reported by Komatsu
et al.[14] A much simpler alternative method involving amination of 5ꢁ-O-
DMT thymidine is described here. In addition, use of DMT thymidine as
starting material (current procedure) instead of thymidine (literature pro-
cedure) for the amination step affords easy organic extraction of product.
Synthesis of the title compound is shown in Scheme 1.
EXPERIMENTAL
General Methods
Melting points (uncorrected) were recorded using a Mel-Temp ap-
paratus. TLC was performed on Silica Gel 60 precut plates (240–400;
Merck). Reagent grade trimethylsilyl chloride, phosphorous oxychloride,
1,2,4-triazole, 4,4ꢁ-dimethoxytrityl chloride, pyridine, sodium hydrogen car-
bonate, ammonium hydroxide, dichloromethane, triethylamine, ethyl ac-
etate, hexanes, methanol, and anhydrous N ,N -dimethylformamide were
purchased from Aldrich Chemical Company, Inc., and used without further
purification. Thymidine was purchased from Samchully Pharmaceuticals,
SCHEME 1 Synthesis of 5ꢁ-O-DMT-N 4-benzoyl-2ꢁ-deoxycytidine.

Large-Scale Synthesis of 5ꢁ-O-Dimethoxytrityl-N4-Benzoyl-5-Methyl-2ꢁ-Deoxycytidine 767
South Korea, and benzoic anhydride was purchased from Chem Impex
Inc. Low water acetonitrile (<50 ppm) was purchased from Burdicken and
Jackson and used without further purification. Note; Trimethylsilyl chloride
and phosphorous oxychloride are corrosive and the reaction should be
conducted in a well-ventilated hood.
5ꢁ-O-Dimethoxytritylthymidine (2). A 50-L glass reactor equipped with air
stirrer and argon gas line was charged with thymidine (1.0 kg, 4.13 mol)
and anhydrous pyridine (6 L) at ambient temperature. 4,4ꢁ-Dimethoxytrityl
chloride (1.47 kg, 4.34 mol) was then added as a solid in four portions
over 1 h. After an additional 30 min, TLC indicated about 95% product,
2% thymidine, 5% DMT chloride and by-products, and 2% 3ꢁ,5ꢁ-bis-O-
dimethoxytritylthymidine (Rf in ethyl acetate 0.45, 0.05, 0.98, and 0.95,
respectively.). Saturated aqueous sodium bicarbonate solution (4 L) and
dichloromethane were added with stirring. An additional 18 L of water was
added. After stirring and allowing the phases to separate, the organic layer
was transferred to a second 50-L vessel. The aqueous layer was extracted
with additional dichloromethane (2 × 2 L). The combined organic layer
was washed with water (10 L) and then concentrated in a rotary evaporator
to a foamy material. This foam was redissolved in dichloromethane (3.5
L), and added to the reactor followed by water (6 L) and hexanes (13
L). The mixture was vigorously stirred and seeded to give a fine white
suspended solid at the interface. After stirring for 1 h, the suspension was
removed by suction through a 1/2-inch-diameter Teflon tube into a 20-L
suction flask. From the suction flask the suspension was poured onto a
25-cm Coors Buchner funnel, washed with water (2 × 3 L) and a mixture of
hexanes-dichloromethane (4:1, 2 × 3 L), and allowed to air dry overnight
in stainless steel pans (1-inch deep). The product was further dried in a
vacuum oven (75◦C/0.1 mm) for 48 h to a constant weight (2.07 kg; 93%)
of a white solid; mp 122–124◦C. TLC indicated trace contamination by
3ꢁ,5ꢁ-bis-O-dimethoxytritylthymidine.
5ꢁ-O-Dimethoxytrityl-2ꢁ-deoxy-5-methylcytidine (3). A 50-L Schott glass-
lined steel reactor equipped with an mechanical stirrer, reagent ad-
dition pump (connected to an addition funnel), heating/cooling sys-
tem, internal thermometer, and argon gas line was charged with 5ꢁ-O-
dimethoxytritylthymidine (3.00 kg, 5.51 mol), anhydrous acetonitrile (25
L), and triethylamine (12.3 L, 88.4 mol). The mixture was chilled with
stirring to −10◦C. Trimethylsilyl chloride (2.1 L, 16.5 mol) was added over
30 min while maintaining the temperature below −5◦C, followed by a wash
of anhydrous acetonitrile (1 L). The reaction was mildly exothermic and
copious hydrochloric acid fumes were liberated over the course of addition.
The reaction was allowed to warm to 0◦C and the progress confirmed by
TLC (Rf of starting material to trimethylsilyl product was 0.43 to 0.84 in
ethyl acetate–hexanes 4:1). Upon completion, 1,2,4-triazole (3.05 kg, 44
mol) was added to reaction mixture that was cooled to −20◦C. Phosphorous

768
B. S. Ross et al.
oxychloride (1035 mL, 11.1 mol) was added over 1 h to maintain the
temperature between −20◦C and −10◦C, followed by a wash of anhydrous
acetonitrile (1 L). The reaction was warmed to 0◦C and allowed to stir for
1 h. TLC indicated a complete conversion to the 1,2,4-triazole product (Rf
0.83 to 0.34 with the product spot glowing in long UV light). The reaction
was cooled to −15◦C and water (5 L) was slowly added at a rate to maintain
the temperature below 10◦C (caution—initially very exothermic) in order
to quench the reaction and to form a homogenous solution. Approximately
one half of reaction volume (22 L) was transferred by air pump to a first
work-up vessel. Ethyl acetate (12 L) and water (8 L) were added and the
mixture was stirred and the water layer transferred to a second work-up
vessel. The organic layer was washed with additional water (8 L). The
combined water layer was back-extracted with ethyl acetate (6 L). The water
layer was discarded and the organic layers were combined and concentrated
in a 20-L rotary evaporator to a foam. The foam was co-evaporated once with
anhydrous acetonitrile (4 L) to remove ethyl acetate.[15] The second half of
the reaction was treated in the same way and concentrated in a separate
flask. Each residue was dissolved in 1,4-dioxane (3 L) and concentrated
aqueous ammonium hydroxide (750 mL) was added. The solution became
homogenous in a few minutes and the reaction was allowed to stand
overnight.[16] TLC indicated the reaction to be complete (product Rf 0.35
in ethyl acetate-methanol 4:1). The reaction mixture was concentrated on
a rotary evaporator to a dense foam, redissolved in ethyl acetate (4 L),
transferred to a 50-L glass reactor vessel, and extracted with water (2 × 4
L) to remove 1,2,4-triazole. The water was back-extracted with ethyl acetate
(2 L). The organic layers were combined, concentrated to about 8 kg total
weight, and cooled to 0◦C. After overnight standing, the first crop was
collected on a 25-cm Coors Buchner funnel and washed repeatedly with
ethyl acetate until a white free-flowing powder remained (3 × 3 L) and then
with ethyl ether (2 × 3 L). The solid was transferred to stainless steel pans
(1-inch deep) and allowed to dry overnight. The filtrate was concentrated to
an oily product, redissolved in ethyl acetate (2 L), cooled, and seeded with
a sample of pure compound. The second crop was collected and washed
as before (with solvents proportional to those used for the first crop). The
filtrate was extracted and seeded as before one more time to yield a third
crop. After air drying, the three crops were separately dried in a vacuum
oven (50◦C, 0.1 mm, 24 h) to a constant weight of 1750, 600, and 200 g,
1
respectively. TLC and H NMR of the three crops were comparable and
were combined for a total of 2.55 Kg (85%) as fine white crystals.[17] The
mother liquor still contained product with minor impurities. If desired, the
mother liquor may be purified by silica gel chromatography using a gradient
of methanol (0–25%) in ethyl acetate to increase the yield.
Preparation of 5ꢁ-O-Dimethoxytrityl-2ꢁ-deoxy-N4-benzoyl-5-methyl-2ꢁ-deoxycytid-
ine (4). 5ꢁ-O-Dimethoxytrityl-5-methyl-2ꢁ-deoxycytidine (1500 g, 2.76 mol)

Large-Scale Synthesis of 5ꢁ-O-Dimethoxytrityl-N4-Benzoyl-5-Methyl-2ꢁ-Deoxycytidine 769
was dissolved in anhydrous DMF (4.5 kg) at ambient temperature in a
50-L glass reactor vessel equipped with air stirrer and argon line. Benzoic
anhydride (749 g, 3.31 mol) was added and the reaction was stirred at
ambient temperature for 22 h. TLC (0.25 Rf, CH2Cl2-ethyl acetate 4:1)
indicated about 98% complete reaction. The reaction mixture was diluted
with toluene (9 L) and then triethylamine (722 mL, 5.52 mol) was added.
After stirring for 10 min, water (10 L) was added. The mixture was stirred,
allowed to separate, and the aqueous layer (14 L) was removed. The organic
layer was washed two more times (2 × 10 L) in the same manner. Finally,
the organic layer (ca. 11 L) was dried with anhydrous sodium sulfate (500
g for 0.5 h) and concentrated in a rotary evaporator (20 L) until about 7 L
solvent was removed. The residue was transferred to a 12-L three-necked
reaction flask equipped with a mechanical stirrer and diluted with 6 L
toluene. Potassium tert-butoxide (500 g, 4.45 mol) was added over a period
of 10 min. The reaction mixture was stirred for 15 h at ambient temperature
under an argon atmosphere. A pink color developed during this period of
time and the solution became viscous and difficult to stir. Another portion of
potassium tert-butoxide (140 g, 1.25 mol) was added over a period of 5 min
and the reaction mixture was stirred for an additional 24 h. The reaction
mixture was washed with 10% citric acid solution (2 × 9 L), 5% aqueous
sodium bicarbonate solution (8 L), and water (2 × 10 L). The organic layer
was concentrated in a rotary evaporator flask (20 L) to remove about half
of the solvent. The partially concentrated solution was run through a short
silica gel column (2.5 kg of silica gel in a 6-L sintered glass funnel), washing
with a mixture of ethyl acetate-hexane (1:1) until the eluate became nearly
light and no polar impurity had come through. The eluate was concentrated
until the formation of foam. The white foam (1460 g, 82% yield, 99.5%
purity by HPLC) was obtained as the final product after drying (0.1 mmHg,
25◦C) for 24 h.
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16. The reaction with ammonium hydroxide is usually complete in 1 h.
17. The product had a melting point between 215 and 217◦C.