The development of β-selective glycosylation reactions with benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides: enabling practical multi-gram syntheses of 4'-Thio-2'-deoxycytidine (T-dCyd) and 5-aza-4’-thio-2’-deoxycytidine (aza-T-dCyd) to s

Article abstract of DOI:10.1080/15257770.2020.1832694

The lack of effective methods to perform direct β-selective glycosylation reactions with 2-deoxy-1,4-dithio-D-erythro-pentofuranosides has long been a significant stumbling block for the multi-gram synthesis of 4’-thio-2’-deoxy nucleosides. In addition, p

Full text of DOI:10.1080/15257770.2020.1832694

Nucleosides, Nucleotides & Nucleic Acids
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lncn20
The development of β-selective glycosylation
reactions with benzyl substituted 2-deoxy-1,4-
dithio-D-erythro-pentofuranosides: enabling
practical multi-gram syntheses of 4'-Thio-2'-
deoxycytidine (T-dCyd) and 5-aza-4’-thio-2’-
deoxycytidine (aza-T-dCyd) to support clinical
development
Donn G. Wishka , Omar D. Lopez , Vladimir F. Rudchenko , Guangfei Huang ,
Robert Bahde , Vineet Kumar , Sergiy M. Denysenko , Lianhao Zhang , Mianji
Zhang , Beverly A. Teicher & Joel Morris
To cite this article: Donn G. Wishka , Omar D. Lopez , Vladimir F. Rudchenko , Guangfei
Huang , Robert Bahde , Vineet Kumar , Sergiy M. Denysenko , Lianhao Zhang , Mianji Zhang ,
Beverly A. Teicher & Joel Morris (2020): The development of β-selective glycosylation reactions
with benzyl substituted 2-deoxy-1,4-dithio-D-erythro-pentofuranosides: enabling practical multi-
gram syntheses of 4'-Thio-2'-deoxycytidine (T-dCyd) and 5-aza-4’-thio-2’-deoxycytidine (aza-
T-dCyd) to support clinical development, Nucleosides, Nucleotides & Nucleic Acids, DOI:
10.1080/15257770.2020.1832694
To link to this article: https://doi.org/10.1080/15257770.2020.1832694
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NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
https://doi.org/10.1080/15257770.2020.1832694
The development of b-selective glycosylation reactions
with benzyl substituted 2-deoxy-1,4-dithio-D-erythro-
pentofuranosides: enabling practical multi-gram
syntheses of 4’-Thio-2’-deoxycytidine (T-dCyd) and 5-
aza-4’-thio-2’-deoxycytidine (aza-T-dCyd) to support
clinical development
Donn G. Wishkaa,c, Omar D. Lopeza,c, Vladimir F. Rudchenkob, Guangfei
Huangb, Robert Bahdea, Vineet Kumara, Sergiy M. Denysenkob, Lianhao
Zhangb, Mianji Zhangb, Beverly A. Teichera, and Joel Morrisa
aDevelopmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, The National
b
Cancer Institute, Rockville, MD, USA; Alchem Laboratories Corporation, Alachua, FL, USA;
cFrederick National Laboratory for Cancer Research, Frederick, MD, USA
ABSTRACT
ARTICLE HISTORY
Received 24 August 2020
Accepted 1 October 2020
The lack of effective methods to perform direct b-selective
glycosylation reactions with 2-deoxy-1,4-dithio-D-erythro-pen-
tofuranosides has long been a significant stumbling block for
the multi-gram synthesis of 4’-thio-2’-deoxy nucleosides. In
addition, previously reported methods for the preparation of
appropriately substituted 2-deoxy-1,4-dithio-D-erythro-pento-
furanosides have proven problematic for large scale synthesis.
To address these issues, herein we describe the modification
and optimization of previously reported methods to allow for
the convenient large scale synthesis of benzyl substituted 2-
deoxy-1,4-dithio-D-erythro-pentofuranosides. Furthermore, we
describe the development of reaction conditions for b-select-
ive glycosylation reactions of benzyl substituted 2-deoxy-1,4-
dithio-D-erythro-pentofuranosides with both N4-benzoylcyto-
sine and 5-aza-cytosine to enable the practical multi-gram
syntheses of the clinical candidates 4’-thio-2’-deoxycytidine (T-
dCyd) and 5-aza-4’-thio-2’-deoxycytidine (aza-T-dCyd). Taken
together, these new synthetic developments have made pos-
sible the preclinical and early clinical development of these
important anticancer agents at the National Cancer Institute.
KEYWORDS
4’-thio-2’-deoxycytidine;
glycosylation; large scale
synthesis; DNMT1; T-dCyd;
aza-T-dCyd
1. Introduction
Cytidine analogs remain an area of active drug discovery and development
having produced four FDA approved drugs for the treatment of cancer.
DNA Methyl Transferase I (DNMT1),[1] a maintenance methyltransferase
CONTACT Donn G. Wishka
donn.wishka@nih.gov
Frederick National Laboratory for Cancer Research,
Building 322, Room 104 Frederick, MD 21702, USA.
ß 2020 The Author(s). Published with license by Taylor & Francis Group, LLC
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives
License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction
in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

2
D. G. WISHKA ET AL.
Figure 1. Structures of cytidine analogs.
that contributes to the hypermethylation and silencing of tumor suppressor
genes, is a major molecular target of two of these drugs, azacytidine and
decitabine (Figure 1). The relatively low response rates and adverse effects
of these two DNMT1 inhibitors in clinical use has prompted the develop-
ment of new, modified nucleoside derivatives to address these issues.
Indeed, 40-thio-modified nucleosides such as 40-thio-20-deoxycytidine (T-
dCyd) 1 (NSC764276) and 5-aza-40-thio-20-deoxycytidine (aza-T-dCyd) 2
(NSC777586) potently deplete DNMT1 while inhibiting tumor growth in
both in vitro and in vivo models[2] with less observed toxicity than the cor-
responding 4-oxo-pentofuranose derived compounds aza-cytidine and deci-
tabine. As a result, both T-dCyd 1 and aza-T-dCyd 2 are currently in
Phase I clinical trials at The National Cancer Institute (NCT02423057 and
NCT03366116, respectively).
2. Results and discussion
The syntheses of both T-dCyd (1)[3] and aza-T-dCyd (2)[4] have been
previously reported on small scale. However, due to compounding gross
inefficiencies in both the synthesis of the intermediate benzyl 2-deoxy-1,4-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
3
Scheme 1. General approach for the preparation of benzyl 2-deoxy-1,4-dithio-D-erythro-
pentofuranoside.
dithio-D-erythro-pentofuranoside (5) and the subsequent glycosylation
chemistry, each of the reported approaches proved impractical for the reli-
able multi-gram production of these compounds required for preclinical
and early clinical development. Our objective was to develop practical
multi-gram syntheses for both T-dCyd (1) and aza-T-dCyd (2) that
addressed the inadequacies of the earlier methods in order to facilitate the
preclinical advancement of these compounds and to establish a robust sup-
ply chain to support clinical development.
Our initial efforts were directed at addressing the inefficiencies in the
synthesis of the intermediate benzyl 2-deoxy-1,4-dithio-D-erythro-pentofur-
anoside (5). The synthesis of benzyl 3,5-di-O-benzyl-2-deoxy-1,4-dithio-D-
erythro-pentofuranoside (4) was originally described by Walker et al.[5]
starting from 2-deoxy-D-erythro-ribose (3) (Scheme 1). A key step in the
syntheses of T-dCyd (1) and aza-T-dCyd (2) reported by Secrist et al.[2]
and Tirwari et al.[3] involved the debenzylation of 4 to provide intermediate
5. Attempted scale up of this deprotection (BCl3, DCM, ꢀ78 ꢁC) revealed
the capricious nature of this reaction as it was found to provide steeply
diminishing returns as the scale exceeded 90 grams of substrate. This limi-
tation necessitated serial batchwise processing for this step thereby creating
an untenable labor-intensive bottleneck for the synthesis of bulk amounts
of diol 5. In an effort to avoid this bottleneck we initially turned to chemis-
try first described by Uenishi et al.[6] for the asymmetric synthesis of ethyl
5-acetoxy-3-silyloxy-2-deoxy-4-thio-D-erythro-pentofuranoside (14). The
modifications to this chemistry and the adaptations required to enable the
bulk synthesis of the key intermediate (4-octyloxybenzyl) 2-deoxy-1,4-
dithio-D-erythro-pentofuranoside (16) are outlined in Schemes 2 and 3.
The desire to minimize the foul odors associated with the preparation of
low molecular weight thioethers led to the selection of the inoffensive 4-
octyloxybenzyl mercaptan (8)[7] as a key building block for the synthesis of
diol 16. The synthesis of mercaptan 8 is outlined in Scheme 2. Starting
with 4-hydroxy-benzaldehyde (6), the synthesis of mercaptan 8 was accom-
plished on kilogram scale in six linear steps in two reaction pots in 76%
overall yield after filtration of crude 8 through a silica pad. This was car-
ried out by alkylation of 4-hydroxy-benzaldehyde (6) with 1-bromooctane,
which was immediately followed by aldehyde reduction with sodium

4
D. G. WISHKA ET AL.
Scheme 2. Synthesis of 4-octyoxylbenzyl mercaptan (8).
Scheme 3. The asymmetric synthesis of (4-octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-pento-
furanoside (16).
borohydride in methanol to afford crude 4-octyloxybenzyl alcohol (7).
Conversion of benzyl alcohol 7 to the corresponding benzyl chloride was
achieved by treatment with aqueous 12 N hydrochloric acid and layer sep-
aration. Treatment of the crude benzyl chloride with thiourea in refluxing
acetonitrile was followed by aqueous base driven hydrolysis of the inter-
mediate thioguanidine in refluxing 13 N sodium hydroxide to give the thio-
carbonate sodium salt. Decomposition of this intermediate salt with
aqueous 12 N hydrochloric acid cleanly afforded 4-octyloxybenzyl mercap-
tan (8) in 76% overall yield after phase separation and filtration through a
silica pad.
The asymmetric synthesis of the key intermediate (4-octyloxybenzyl) 2-
deoxy-1,4-dithio-D-erythro-pentofuranoside (16) generally follows the route
described by Uenishi et al.[5] but with some modifications necessary to
ultimately enable scale up (Scheme 2). For instance, we greatly streamlined

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
5
the reported 6 step synthesis of unsaturated ester 10 to a two-step scaleable
protocol. In the event, in situ generation of allylmagnesium chloride by
treatment of allyl chloride with magnesium metal in the presence of triethyl
orthoformate afforded 4,4-diethoxybut-1-ene (9) in 86% yield. Catalytic
cross-metathesis of butene-acetal 9 with an excess of ethyl acrylate in the
presence of 2 mol % Grubbs (II) catalyst provided an 87% yield of unsatur-
ated ester 10. Reduction of ester 10 to the corresponding allylic alcohol
with diisobutylaluminum hydride, followed by Sharpless catalytic asymmet-
ric epoxidation gave ((2S,3S)-3-(2,2-diethoxyethyl)oxiran-2-yl)methanol
(11) in 73% overall yield. Subsequent reaction of alcohol 11 with carbon
disulfide in the presence of lithium hexamethyldisilazane, followed by pro-
tection of the intermediate secondary alcohol as the t-butyldiphenylsilyl
ether, afforded 1,3-oxathiolane-2-thione 12 in 79% overall yield. The
replacement of the originally reported TBS ether with the more robust
TBDPS ether at this point in the synthesis proved crucial in preserving the
yields on scale for the next three steps. The conversion of 12 to the corre-
sponding thiirane 13 was efficiently achieved in excellent yield through
treatment with potassium carbonate in methanol. Further treatment of 13
with a mixture of potassium acetate in glacial acetic acid and acetic anhyd-
ride yielded the intermediate ethyl 5-acetoxy-3-t-butyldiphenylsilyloxy-2-
deoxy-4-thio-D-erythro-pentofuranoside (14). Subsequent reaction of the
ethoxy-ether 14 with 4-octyloxybenzyl mercaptan (8) in the presence of
boron trifluoride diethyletherate resulted in conversion to the correspond-
ing (4-octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-pentofuranoside 15 in
75% yield. Hydrolysis of 15 with lithium hydroxide resulted in removal of
both the acetate and t-butyldiphenylsilyl protecting groups to afford diol 16
as a mixture of anomers in 78% yield. Notably, the final deprotection has
been performed on greater than 500-gram scale without the requirement
for batch-wise processing.
Concurrently we were able to modify and optimize the route originally
described by Walker et al.[4] starting from 2-deoxy-D-erythro-ribose (3) as
shown in Scheme 4. In order to eliminate the capricious benzyl deprotec-
tion of thioglycoside 4 we proposed to replace the 3,5-dibenzyl protecting
scheme from the reported chemistry with more labile benzoyl protecting
groups. We were cognizant that the more labile protecting scheme would
necessitate some modifications to the downstream chemistry, but we were
confident that the benefits would justify the effort. Further, in the interests
of odor control, the 4-octyloxybenzyl group was swapped for the benzyl
thioglycoside. In the event, 2-deoxy-D-erythro-ribose (3) was converted to
methyl glycoside 17 in the presence of dry HCl in methanol. Benzoyl pro-
tection of 17 at the 3 and 5 hydroxy positions was catalyzed by DMAP to
efficiently provide diester methyl glycoside 18. Treatment of 18 with 4-

6
D. G. WISHKA ET AL.
Scheme 4. Modification of the chemistry of Walker et al.[4] to enable the bulk synthesis of (4-
octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-pentofuranoside (16).
octyloxybenzyl mercaptan (8) under catalysis with boron trifluoride diethyl
etherate afforded dithioacetal 19 in 70% overall yield from 3. Mitsunobu
inversion of alcohol 19 afforded formate 20 in 82% yield. Cleavage of for-
mate 20 was accomplished with dry hydrochloric acid in methanol/THF to
afford a 95% yield of inverted alcohol 21. Formate cleavage under acidic
conditions was determined to be essential to prevent acyl migration.
Activation of alcohol 21 as the mesylate 22 was followed by cyclization of
22 in the presence of tetra n-butylammonium iodide and triethylamine to
provide 3,5-dibenzoyl protected 2-deoxy-1,4-dithio-D-erythro-pentofurano-
side 16a in 69% yield from alcohol 21. The deprotection of 16a was
uneventfully carried out by treatment with aqueous lithium hydroxide to
give (4-octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-penofuranoside 16 as
a mixture of anomers in 96% yield. As with the modified chemistry of
Uenishi et al.[5] described earlier, this revised route has been successfully
executed on large scale to produce bulk 16.
With robust chemistry in place to efficiently provide bulk quantities of
diol 16 we turned our attention to addressing the inefficiencies in the gly-
cosylation reactions of protected 16 with derivatives of cytosine such as
N4-benzoyl-cytosine (23a) and aza-cytosine (23b).
There is rich chemical literature[8] describing effective methods for the
conversion of 1-thioglycosides or 1-acetates of 2-deoxy-D-erythro-pentofur-
anose to the corresponding cytidine or aza-cytidine containing nucleosides
in a manner that reliably affords a preponderance of the desired C1-b

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
7
Scheme 5. General approach for the glycosylation of N4-benzoyl-cytosine (23a) and 5-aza-cyto-
sine (23 b).
anomer. Unfortunately, the same wealth of literature methods cannot be
found for the direct conversion of the thioglycosides or the corresponding
acetates of 2-deoxy-4-thio-D-erythro-pentofuranose to the corresponding
C1-b anomers of the nucleosides of immediate interest, T-dCyd (1) and 5-
aza-T-dCyd (2). Indeed, the reaction of thioglycosides of 2-deoxy-1,4-thio-
D-erythro-pentofuranoses with silylated nucleobases is generally reported
[2,3]
to favor formation of the undesired C1-a anomer or, at best, a 1:1
mixture of anomers. Furthermore, in the course of our studies it became
apparent that the lack of chemical equivalence between pentofuranosyl thi-
oglycosides and 4-thiopentofuranosyl thioglycosides meant that application
of known b-selective base addition methodology from the former system to
the latter system was an unlikely path forward. As a result, we embarked
upon an effort to identify conditions that would allow the successful
b-selective addition of a silylated-base to an appropriately functionalized
(4-octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-pentofuranoside.
The general approach utilized for reacting an appropriately functional-
ized 1,4-dithio-D-erythro-pentofuranoside with N4-benzoyl-cytosine (23a)
or 5-aza-cytosine (23b) after silylation is outlined in Scheme 5. The obser-
vations made upon the application of this approach toward the preparation
of T-dCyd (1) and 5-aza-T-dCyd (2) with regard to the interplay between
the nature of the diol protection scheme at R1 and R2, the leaving group at
C1 (R3) on the 4-thio-D-erythro-pentofuranosides (16a-g), the reaction
conditions, and the b/a ratio obtained for the protected nucleoside prod-
ucts (24a-f) are tabulated in Table 1.
The data in Table 1 points to some general trends that appear to favor
increased selectivity for the formation of the desired b anomer of protected
nucleosides 24a-f. For instance, it is clear from entries 16a and 16b, where
R1 and R2 are benzoyl, and R3 is either the 4-octyloxybenzylthio as in 16a
or acetate as in 16b that reaction of silylated N4-benzoyl-cytosine with the
thioglycoside 16a affords a more favorable b/a ratio (1:1) than reaction

8
D. G. WISHKA ET AL.
Table 1. Conditions for the glycosylation of N4-benzoyl-cytosine (23a) and 5-aza-cyto-
sine (23 b).
Compound
(24)/Observed Isolated Yield
R1, R2 or R1
Entry
þ R2
Bz-, Bz-
R3
Z
C
b Conditions
b/a Ratio
b Anomer
16a
NIS, MeCN
24a
1:1
22-25%
50-60oC
16b
16b’
16c
16d
16e
Bz-, Bz-
-OAc
-OAc
-OAc
C
N
C
N
N
TMSOTf, MeCN, 0oC
to rt
24a
1:2
24b
1:2
24c
2:3
24d
4:5
24d
5:6
25%
15%
32%
30%
27%
Bz-, Bz-
TMSOTf,
MeCN, 0oC to rt
TMSOTf,
Bn-, Bn-
MeCN, 0oC to rt
TMSOTf,
Ac-, -TBDPS
Ac-, -TBDPS
-OCHO/-OAc
MeCN, 0oC to rt
NBS, DCM
0oC to rt
16f
-(iPr)2SiOSi(iPr)2-
-(iPr)2SiOSi(iPr)2-
-OAc
N
C
TMSOTf,
24e
3:5
24f
5:1
25% (SFC)
54%
MeCN, 0oC to rt
NBS, DCM
0oC
16g
16g’
-(iPr)2SiOSi(iPr)2-
N
NBS, DCM
0oC
24e
4:1
44%
with the acetate 16b (1:2). The comparable amount of isolated b anomer 24a
despite the superior ratio afforded by thioglycoside 16a is the result of the
improved chemical conversion obtained upon reaction with the acetate 16b.
Likewise, entry 16b’ demonstrates that reaction of silylated 5-aza-cytosine
with the same acetyl pentofuranoside 16b provides a 1:2 b/a ratio of the 5-
aza-cytidine analog 24b. The lower isolated yield of b anomer 24b (15% vs
25% for 24a) is the result of the consistently poorer reactivity observed for
the reaction of silylated 5-aza-cytosine (23b) with acetyl 3,5-diacyl substituted
2-deoxy-4-thio-D-erythro-pentofuranosides. A further complication observed
while using 24b for the late stage synthesis of 5-aza-T-dCyd (2) involves the
well-documented[9] hydrolytic instability of the aza-cytosine ring in protic
solvents under basic conditions. This property proves to be a serious liability
during the final deprotection of 24b to give 5-aza-T-dCyd (2) and leads to
significant loss of material. One way we sought to minimize the hydrolytic
lability of the aza-cytosine ring during final deprotection was to adopt 3,5-
hydroxyl protection schemes for the 2-deoxy-4-thio-D-erythro-pentofurano-
side derivative that reduced the need to employ base in protic solvents above
room temperature during deprotection.
Along these lines, the introduction of a t-butyldiphenylsilyloxy group at
the 3 position and an acetate at the 5 position of formyl/acetyl 4-

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
9
thiopentofuranoside 16d[10] and (4-octyloxybenzyl) 1,4-dithio-pentofurano-
side 16e while using silylated 5-aza-cytosine (23b) as the nucleophile
afforded a nearly 1:1 ratio of b and a anomers and an improved isolation
of b anomer 24d for both substrates (27-30% respectively for 16d & 16e
versus 15% for bis-benzoate protected entry 16b’). In this case, deprotec-
tion of 24d by sequential treatment with tetra-n-butylammonium fluoride
and catalytic sodium methoxide (-5 to 0 ꢁC) cleanly precipitated 5-aza-T-
dCyd (2) from the reaction mixture in 75% yield.[11] These results sug-
gested that silyl protection of the 3 and 5 hydroxy groups might offer the
potential for both improved b/a ratios and enhanced reactivity with sily-
lated nucleobases, particularly the more sluggish 5-aza-cytosine, (23b), as
well as improved recovery on final deprotection of the b anomer to give
5-aza-T-dCyd (2).
Initially, as shown in entry 16f, dual protection the 3 and 5 hydroxy
groups of the acetyl 2-deoxy-4-thio-D-erythro-pentofuranoside with the tet-
raisopropyldisiloxane (TIPDS) protecting group afforded the 8-membered
silylcycle of the acetate 16f. Reaction of 16f with silylated 5-aza-cytosine
(23b) led to a slightly improved b/a ratio of 3:5 and an isolated yield of
25% for 24e after chromatography under SFC conditions. In this instance
anomer separation through SFC chromatography was required due to the
net a selectivity of this acetate coupling along with an unfavorable elution
order that made fractionation of the b and a anomers of 24e over silica gel
impractical.
In contrast to these results, dual protection the 3 and 5 hydroxyls of the
1,4-dithio-D-erythro-pentofuranoside with the tetraisopropyldisiloxane
(TIPDS) protecting group afforded the 8-membered silylcycle of the thio-
glycoside 16g. Gratifyingly, reaction of 16g with silylated N4-benzoyl-cyto-
sine (23a) gave the precursor to T-dCyd (1), 24f, in a b/a ratio of 5:1 and
an isolated yield of 54% of b anomer after silica gel column chromatog-
raphy (entry 16g). Similarly, reaction of 16g with silylated 5-aza-cytosine
(23b) gave the precursor to 5-aza-T-dCyd (2), 24e, in a b/a ratio of 4:1
and an isolated b anomer yield of 44% after column chromatography over
silica gel (entry 16g’).
On the basis of these results, our experience indicates that the three
essential elements required for the b-diastereoselective addition of nucleo-
bases 23a and 23b after silylation to derivatives of 2-deoxy-4-thio-D-
erythro-pentofuranose 16a-g are: one, dual protection of the 3 and 5
hydroxyl groups via the 8-membered silylcycle derived from 1,3-dichloro-
1,1,3,3-tetraisopropoxydisiloxane; two, a thioglycoside as donor; and three,
the use of N-bromo-succinimide as the activator[12] in dichloromethane.
The successful application of this methodology to the multi-gram synthe-
sis of T-dCyd (1) is outlined in Scheme 6. Protection of diol thioglycoside

10
D. G. WISHKA ET AL.
Scheme 6. The multi-gram synthesis of T-dCyd (1) (NSC764276).
Scheme 7. The multi-gram synthesis of 5-aza-T-dCyd (2) (NSC777856).
16 as the tetraisopropyldisiloxane gave silylcycle 16g in excellent yield.
Reaction of protected thioglycoside 16g with silylated N4-benzoyl-cytosine
23a in the presence of N-bromosuccinimide, followed by fractionation of
the b and a anomers provided protected b anomer 24f in 49% yield.
Treatment of bis silylether 24f with tetra-n-butylammonium fluoride gave a
93% yield of the corresponding diol. Finally, cleavage of the diol/benzamide
with methanolic ammonia (7 M, rt) afforded T-dCyd (1, NSC764276) in
94% isolated yield.
Similarly, the multi-gram synthesis of 5-aza-T-dCyd (2) is outlined in
Scheme 7. Starting with protected thioglycoside 16g, reaction with 5-aza-
cytosine 23b after silylation in the presence of N-bromosuccinimide fol-
lowed by fractionation of the b and a anomers provided protected b

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
11
anomer 24e in 44% yield. Finally, treatment of bis silylether 24e with
ammonium fluoride in refluxing MeOH[13] afforded 5-aza-T-dCyd (2,
NSC777586) in 73% yield.
Crystallization of T-dCyd (1) and aza-T-dCyd (2) after deprotection rou-
tinely provides API that is remarkably devoid of impurities with enantio-
meric excesses and diastereomeric excesses typically greater than 99% and
generally approaching the limits of detection. Comparison of the impurity
profiles for T-dCyd (1) and aza-T-dCyd (2) prepared from diol 16 derived
from both 2-deoxy-D-ribose (following the methods of Walker et al.[4])
and the modified stereoselective synthesis described by Uenishi et al.[5]
revealed no detectable points of difference.
3. Conclusions
Through the modification and optimization of existing chemistry[4,5] we
have identified two robust methods for the bulk synthesis of the key inter-
mediate (4-octyloxybenzyl) 2-deoxy-1,4-dithio-D-erythro-pentofuranoside
(16). Furthermore, we have identified conditions for the hitherto unre-
ported b-selective glycosylation reaction between the 3,5-tetraisopropyldisi-
loxane protected 2-deoxy-1,4-dithio-D-erythro-pentofuranoside (16g) and
the silylated bases of N4-benzoyl-cytosine (23a) and aza-cytosine (23b) to
conveniently afford multi-gram quantities of T-dCyd (1) and aza-T-dCyd
(2), respectively. Taken together, these developments represent a significant
improvement over reported methods for accessing multi-gram quantities of
these important compounds. In addition, the b-selective glycosylation
methodology enabled with thioglycoside 16g may find utility in the synthe-
sis of other 2-deoxy-4-thio-D-erythro-pentofuranose derived nucleosides
of interest.
Preliminary work to expand the scope of the glycosylation reaction utiliz-
ing 16g beyond N4-benzoyl-cytosine (23a) and aza-cytosine (23b) to other
nucleobases including uracil, thymine, and 5-fluoro-cytosine has been initi-
ated. Early efforts in this regard appear promising. The determination of
the practical value of this approach will be reported in due course.
4. Experimental
4.1. General methods
R
V
Melting points were measured using an Electrothermal Mel-Temp 3.0
capillary melting point apparatus that was used without correction.
Reactions were monitored with TLC using silica gel 60 F254 coated glass
plates from Merck KGAa. Purification on laboratory scale via column chro-
R
R
V
V
matography was carried utilizing Biotage or Combiflash automated

12
D. G. WISHKA ET AL.
chromatography systems and the commercially available columns specified.
Purification on preparative scale was carried out as stated utilizing slurry
1
packed silica gel for flash chromatography (230-400 mesh). H NMR and
13C NMR spectra were recorded in solution in appropriate deuterated sol-
vents on either a Varian 400 spectrometer at 400 and 100 MHz, respectively
or a Bruker 300 Advance spectrometer at 300 and 75 MHz respectively.
The chemical shifts are expressed in parts per million (ppm), using the
deuterated solvent as internal reference. The multiplicities of the signals are
indicated by the following abbreviations: s, singlet; d, doublet; t, triplet; q,
quadruplet; and m, multiplet, and coupling constants are expressed in
Hertz. Combustion analysis data was obtained from either Atantic
Microlab, Norcross, GA or Robertson Microlit Laboratories,
Ledgewood, NJ.
4.2. Chemical synthesis
4.2.1. Synthesis of 4-octyloxyphenylmethane thiol (8)
4.2.1.1. Preparation of 4-octyloxyphenylmethanol (7). A mixture of 4-hydroxy-
benzaldehyde (6) (430.0 g, 3.52 mol), 1-bromooctane (714.9 g, 3.70 mol) and
potassium carbonate (515.7 g, 3.73 mol) in acetonitrile (3.4 L) was refluxed
overnight and cooled to room temperature. The solid was filtered off, and
the filtrate was concentrated under reduced pressure to give 848.3 g (103%)
of crude 4-octyloxybenzaldehyde. The product was dissolved in methanol
(2.6 L) and sodium borohydride (44.4 g, 1.17 mol) was added portion-wise
to the formed solution, keeping the temperature below 15 ꢁC. The reaction
mixture was stirred at room temperature for 1 h. A solution of NaOH
(14.33 g, 0.358 mol) in water (200 mL) was added followed by ethyl acetate
(1.7 L) and brine (0.5 L). The organic solution was separated, dried over
sodium sulfate and evaporated under reduced pressure. Heptane (1 L) was
added to the residue and the formed mixture was cooled to 4 ꢁC. The solid
was filtered off, washed with ice-cooled heptane, and dried under vacuum
to give 765.4 g (92%) of crude compound 7, which was used in the follow-
ing step without further purification. 1H NMR spectrum (300 MHz,
CDCl3): d 7.26 (m, 2H), 6.87 (m, 2H), 4.59 (s, 2H), 3.95 (m, 2H), 1.78 m,
3H), 1.47-1.29 (m, 10 H), 0.89 (m, 3H).
4.2.1.2. Preparation of 4-octyloxyphenylmethanethiol (8). A mixture of 4-octy-
loxyphenylmethanol (7) (765.42 g, 3.24 mol), concentrated HCl (600 mL)
and acetonitrile (1.7 L) was stirred overnight at room temperature.
Thiourea (296.0 g, 3.89 mol) and acetonitrile (600 mL) were added. The
mixture was heated to reflux for 2 h, cooled to room temperature and kept
overnight. A solution of sodium hydroxide (518.7 g, 12.97 mol) in water

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
13
(1 L) was added. The mixture was heated to reflux temperature for 3 h and
cooled to 10 ꢁC. Concentrated HCl (600 mL) was added keeping the tem-
perature below 15 ꢁC. The mixture was extracted with MTBE (3 L) and the
extract was dried over magnesium sulfate and concentrated under reduced
pressure. Heptane (1 L) was added to the residue, and the mixture was
evaporated. Heptane (1.5 L) was added to the residue. The milky solution
was kept overnight and filtered through a silica gel pad (500 g). The filtrate
1
was evaporated to give 679.2 g (83%) of compound 8 as a colorless oil. H
NMR spectrum (300 MHz, CDCl3): d 7.23 (m, 2H), 6.84 (m, 2H), 3.95 (m,
2H), 3.66 (m, 2H), 1.77 (m, 3H), 1.21-1.59 (m, 10H), 0.88 (m, 3H).
4.2.2. The asymmetric synthesis of (4S,5R)-4-Hydroxy-5-hydroxymethyl-2-((4-
(octyloxy)benzyl)thio)-tetrahydrothiophene, (16)
4.2.2.1. Preparation of 4,4-Diethyoxybut-1-ene (9). The following procedure is
an adaptation of the method reported by Cloux, R.; Schlosser, M. Helv.
Chim. Acta. 1984, 67, 1470–1474. To a mixture of magnesium (grit,
ꢂ99.0% (KT) (Aldrich: 63040-250 G-F) (31.8 g, 1.31 mol) and triethoxyme-
thane (123 mL, 0.738 mol) was added 3-chloroprop-1-ene (53.2 mL,
0.653 mol) via addition funnel at a rate of 1 drop every 4 seconds, keeping
the temperature under 105 ꢁC. The reaction mixture was vigorously stirred
with an overhead stirrer. After addition was complete, the mixture was
allowed to cool to room temperature overnight (14 h). The reaction mixture
was then chilled to 0 ꢁC, treated with aqueous saturated NH4Cl solution
(200 mL), and stirred vigorously for 4 h. The aqueous layer was then
extracted with Et2O (3 ꢃ 150 mL). The organic layers were combined, dried
(MgSO4), and concentrated, resulting in a clear and colorless residue. To
the clear residue was added a solution of acetic acid (20 mL, 349 mmol),
sodium acetate (10 g, 122 mmol), and water (50 mL, 2.78 mol). The mixture
was stirred for 2.5 h. Sodium bicarbonate (32.4 g, 386 mmol) and water
(about 200 mL) was then slowly added to the mixture. When evolution of
CO2 stopped, the aqueous layer was extracted with Et2O (2 ꢃ 150 mL). The
organic layers were combined, dried (MgSO4), and concentrated to afford
1
compound 9 (80.6 g, 559 mmol, 86% yield) as clear and colorless liquid. H
NMR (400 MHz, CDCl3) d 5.86-5.75 (m, 1H); 5.14-5.06 (m, 1H); 4.54-4.51
(m, 1H); 3.66-3.62 (m, 2H); 3.55-3.49 (m, 2H); 1.23-1.19 (m, 6H).
4.2.2.2. Preparation of ethyl (E)-5,5-diethoxypent-2-enoate (10). To a solution
of 4,4-diethoxybut-1-ene 9 (60.2 g, 417 mmol) and ethyl acrylate (182 mL,
1.67 mol) in DCM (1.39 L) was added [1,3-bis-(2,4,6-trimethylphenyl)-2-
imidazolidinylidene]dichloro(phenylmethylene)(tricyclohexylphosphino)ru-
thenium (Grubbs II Catalyst) (3.54 g, 4.17 mmol) and the reaction mixture
was heated/refluxed at 45 ꢁC for 2 h. A 2nd portion of Grubbs II Catalyst

14
D. G. WISHKA ET AL.
(350 mg) was added. The reaction mixture was then heated to 45 ꢁC for
14 h. The reaction mixture was allowed to cool to room temperature and
the solvent was then removed to afford a black residue. The residue was
diluted with DCM and transferred to a 500 mL round-bottom flask. DCM
was removed by rotary evaporation. The 500 mL round bottom flask was
then attached to a short path distillation head and product was purified by
distillation. Ethyl acrylate and residual DCM was first removed by applying
heat and vacuum, starting at 60 ꢁC and 200 mbar and gradually increasing
to 80 ꢁC and 14 mbar vacuum. Product was then recovered at 98 ꢁC and
about 1 torr to afford compound 10 (73.3 g, 338 mmol, 81% yield) as clear
1
and colorless oil. H NMR (400 MHz, CDCl3) d 6.92 (m, 1H), 5.90 (d,
J ¼ 16 Hz, 1H), 4.58 (t, J ¼ 8 Hz, 1H), 4.19 (q, J ¼ 16, 8 Hz, 2H), 3.67 (m,
2H), 3.55 (m, 2H), 2.54 (t, J ¼ 8 Hz, 2H), 1.29 (t, J ¼ 8 Hz, 3H), 1.21 (t,
J ¼ 8 Hz, 6H).
4.2.2.3. Preparation of (E)-5,5-Diethoxypent-2-en-1-ol. Ethyl (E)-5,5-diethoxy-
pent-2-enoate (10) (63 g, 291 mmol) was dissolved in DCM (1.21 L) in a 5 L
three-neck round-bottom flask under nitrogen. The solution was cooled to
ꢀ78 ꢁC and treated dropwise via addition funnel with DIBAL-H in DCM
(699 mL, 699 mmol) over 2.5 h. The reaction was maintained at-78 ꢁC for
2 h. The reaction mixture was then quenched with ethyl acetate (28.5 mL,
291 mmol) and diluted with 1 L saturated sodium potassium tartrate. The
mixture was stirred overnight as it warmed to room temperature. The
layers were separated, and the aqueous layer was extracted once with DCM
(200 mL). The organic layers were combined, dried (MgSO4) and concen-
trated in vacuo to a colorless oil. The crude material was dissolved in a
minimum amount of hexane/DCM, loaded onto a 125 g RediSep Rf filter
column and was purified using a 330 g RediSep Rf Gold Silica Gel column,
eluting with a 0-30% EtOAc/hexane gradient. The appropriate fractions
were collected to afford (E)-5,5-diethoxypent-2-en-1-ol (41 g, 235 mmol,
1
81% yield) as a clear yellow oil. H NMR (400 MHz, CDCl3) d 5.70 (m,
2H), 4.50 (t, J ¼ 8 Hz, 1H), 4.11 (m, 2H), 3.65 (m, 2H), 3.52 (m, 2H), 2.38
(t, J ¼ 7 Hz, 2H), 1.19 (t, J ¼ 8 Hz, 6H).
4.2.2.4. Preparation of ((2S,3S)-3-(2,2-Diethoxyethyl)oxiran-2-yl)methanol (11).
A 50-L jacked reactor, equipped with overhead stirrer, thermometer, and
argon inlet, was charged with a solution of (þ)-diethyl L-tartrate (213.4 g,
1.04 mol) in anhydrous DCM (16 L) and activated molecular sieves (1 kg).
A solution of titanium tetraisopropoxide (245.17 g, 862.6 mmol) in anhyd-
rous DCM (0.5 L) was added dropwise at ꢀ30 ꢁC followed by addition of a
5.5 M solution of tert-butyl hydroperoxide (1.57 L, 8.63 mol) while keeping
the temperature under ꢀ25 ꢁC. The reaction mixture was stirred at this

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
15
temperature for 45 min, and a solution of (E)-5,5-diethoxypent-2-en-1-ol
(742.9 g, 4.31 mol) in anhydrous DCM (1 L) was added at ꢀ25 ꢁC. The
reaction mixture was stirred at this temperature for 7 h and warmed up to
0 ꢁC. A solution of citric acid (55.24 mol, 287.5 mmol) in water (250 mL)
was added. The reaction mixture was stirred at this temperature for 1 h
and a solution of triphenylphosphine (1.19 Kg, 4.53 mol) in anhydrous
DCM (2 L) was added dropwise while keeping the temperature under 5 ꢁC.
Anhydrous magnesium sulfate (500 g) and Celite (300 g) were added and
the mixture was stirred at 5 ꢁC for 5 h. The solids were filtered off through
a Celite pad and the filtrate was concentrated under reduced pressure. The
residue was dissolved in a mixture of ethyl acetate and heptanes (8 L, 1:4).
The formed solution was cooled to ꢀ9 ꢁC. Additional solids were filtered
off, washed with a mixture of ethyl acetate and heptanes (2 L, 1:4), and the
filtrate was evaporated. The residue was purified by column chromatog-
raphy (silica gel, 10 kg; ethyl acetate/heptanes, 1:4, then 2:3) to give 598.4 g
1
(73%) of compound 11 as a pale oil. H NMR (400 MHz, CDCl3) d 4.65
(m, 1H), 3.88 (m, 1H), 3.62 (m, 2H), 3.50 (m, 2H), 3.05 (m, 1H), 2.94 (m,
1H), 1.91 (m, 2H), 1.84 ꢀ 1.71 (m, 2H), 1.19 (m, 6H).
4.2.2.5. Preparation of (R)-4-((S)-3,3-diethoxy-1-hydroxypropyl)-1,3-oxathiolane-
2-thione. An oven-dried 250 mL two-neck round-bottom flask under nitro-
gen was charged with carbon disulfide (60.3 mL, 10 mol), dry THF
(475 mL) and ((2S,3S)-3-(2,2-diethoxyethyl)oxiran-2-yl)methanol (11)
(47.6 g, 250 mmol) and the solution was cooled to ꢀ78 ꢁC. The colorless
solution was treated dropwise with sodium bis(trimethylsilyl)amide in THF
(300 mL, 300 mmol) over 1 h. The deep orange solution was stirred at
ꢀ78 ꢁC for a total of 2 h and was quenched at ꢀ78 ꢁC with acetic acid
(17.9 mL, 313 mmol). The reaction then was stirred without the cooling
bath until it warmed to ꢀ30 ꢁC, was treated with 225 g silica gel (230-400
mesh), and the mixture was concentrated to dryness overnight. The crude
R
V
plug was placed in a CombiFlash dry load cartridge, eluted directly onto
a 340 g UltraSphereTM SNAP cartridge with a 0-60% EtOAc/hexane gradi-
ent into two fractions. First eluting fraction was combined and concen-
trated
to
afford
10.15 g
(12%)
of
(R)-4-((S)-3,3-diethoxy-1-
((trimethylsilyl)oxy)propyl)-1,3-oxathiolane-2-thione as a deep yellow oil.
Second eluting fraction was combined and concentrated to afford 53.1 g
(80%) of (R)-4-((S)-3,3-diethoxy-1-hydroxypropyl)-1,3-oxathiolane-2-thione
as an amber oil. The TMS ether (10.15 g, 30.0 mmol) was dissolved in
methanol (110 mL) in a 500 mL one-neck round-bottom flask. The solution
was treated with acetic acid (5.4 mL, 94 mmol) and the reaction was stirred
for 19 h at room temperature. The reaction was diluted with toluene
(200 mL) and concentrated to afford an additional 8.0 g (12%) of the title

16
D. G. WISHKA ET AL.
1
compound as a dark yellow oil. Alcohol: H NMR (400 MHz, CDCl3) d
5.16 ꢀ 5.08 (m, 1H), 4.87 ꢀ 4.78 (m, 1H), 4.69 (m, 1H), 4.04 ꢀ 3.87 (m,
3H), 3.69 (m, 2H), 3.58 ꢀ 3.43 (m, 2H), 1.90 (m, 1H), 1.88 ꢀ 1.73 (m, 1H),
1
1.20 (m, 6H). TMS ether: H NMR (400 MHz, CDCl3) d 4.93 (m, 1H), 4.76
(m, 1H), 4.61 (m, 1H), 4.10 (m, 1H), 3.97 (m, 1H), 3.68 ꢀ 3.52 (m, 2H),
3.51 ꢀ 3.38 (m, 2H), 1.90 (m, 1H), 1.79 (m, 1H), 1.26 (s, 1H), 1.25 ꢀ 1.13
(m, 6H), 0.15 (d, J ¼ 0.7 Hz, 9H).
4.2.2.6. Preparation: (R)-4-((S)-1-((tert-Butyldiphenylsilyl)oxy)-3,3-diethoxy-
propyl)-1,3-oxathiolane-2-thione (12). (R)-4-((S)-3,3-Diethoxy-1-hydroxy-
propyl)-1,3-oxathiolane-2-thione (11.78g, 44.2 mmol) was dissolved in DMF
(44mL) in a 500mL one-neck round-bottom flask under nitrogen. The solu-
tion was treated with imidazole (7.53g, 111 mmol) in a single portion fol-
lowed by tert-butylchlorodiphenylsilane (TBDPS-Cl) (23.00mL, 88 mmol)
and the reaction was stirred for 72h. The mixture was diluted with EtOAc
(200 mL), stirred vigorously with 50% saturated 1:1 sodium bicarbonate/
sodium chloride (100 mL) for 10 minutes, and the layers were separated. The
organic layer was washed with 50% saturated sodium chloride (3ꢃ 50mL),
dried (MgSO4) and concentrated in vacuo to give an amber oil. The crude
R
material was dissolved in hexane and was loaded onto a 50g UltraSilV SNAP
R
cartridge. The column was eluted over a 100g UltraSilV SNAP cartridge with
R
a 0-10% EtOAc/hexane gradient while collecting on a BiotageV system. The
appropriate fractions were combined and concentrated to afford 19.28 g
1
(86%) of compound 12 as a golden oil. H NMR (400 MHz, CDCl3) d
7.74ꢀ 7.60 (m, 4H), 7.50ꢀ 7.32 (m, 6H), 4.82 (m, 1H), 4.70 (m, 1H),
4.46m, 1H), 4.23 (m, 1H), 4.04 (m, 1H), 3.50ꢀ 3.22 (m, 3H), 3.12 (m, 1H),
1.90 (m, 1H), 1.72 (m, 1H), 1.11 (m, 3H), 1.05 (s, 9H), 1.10ꢀ 0.92 (m, 6H).
4.2.2.7. Preparation of tert-Butyl((S)-3,3-diethoxy-1-((S)-thiiran-2-yl)propoxy)di-
phenylsilane (13). (R)-4-((S)-1-((tert-Butyldiphenylsilyl)oxy)-3,3-diethoxy-
propyl)-1,3-oxathiolane-2-thione (12) (11.9 g, 23.57 mmol) was dissolved in
anhydrous methanol (140 mL) in a 250 mL one-neck round-bottom flask
under nitrogen and the mixture was cooled to 0 ꢁC. The yellow solution was
treated with powdered potassium carbonate 325 mesh (3.75 g, 27.1 mmol)
and the reaction was stirred 3 h at 0 ꢁC. The mixture was diluted with 1:1
diethyl ether/hexane (500 mL), the insoluble material was removed by filtra-
tion through anhydrous potassium carbonate, and the filtrate was concen-
trated in vacuo to a yellow oil. The crude material was dissolved in a
R
minimum amount of hexane, loaded onto a 50 g UltraSphereV SNAP cart-
R
ridge and was eluted with a 0-4% EtOAc/hexane gradient on a BiotageV sys-
tem. The appropriate fractions were combined and concentrated in vacuo to
1
afford 9.67 g (92%) of compound 13 as a colorless oil. H NMR (400 MHz,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
17
CDCl3) d 7.71 (m, 4H), 7.47 ꢀ 7.32 (m, 6H), 4.91 (m, 1H), 3.66 ꢀ 3.28 (m,
5H), 2.90 (m, 1H), 2.12 ꢀ 2.00 (m, 2H), 1.84 (m, 1H), 1.52 (m, 1H), 1.16 (m,
6H), 1.02 (s, 9H).
4.2.2.8. Preparation of ((2 R,3S)-3-((tert-Butyldiphenylsilyl)oxy)-5-ethoxytetrahy-
drothiophen-2-yl)methyl acetate (14). tert-Butyl((S)-3,3-diethoxy-1-((S)-
thiiran-2-yl)propoxy)diphenylsilane (13) (9.67 g, 21.74 mmol) was dissolved
in a mixture of acetic acid (25 mL, 437 mmol) and acetic anhydride (30 mL,
318 mmol) in a 250 mL one-neck round-bottom flask under nitrogen. The
solution was treated with potassium acetate (10.67 g, 109 mmol) and was
placed in an oil bath at 120 ꢁC for 4 h. The mixture was cooled to room tem-
perature, diluted with toluene (500 mL), the solid material was removed by
filtration, and the filtrate was concentrated to dryness. The residue was dis-
R
solved in a minimum amount of DCM, was loaded onto a 50 g UltraSphereV
R
SNAP cartridge, and was eluted over a 100 g UltraSilV SNAP cartridge with a
0-5% EtOAc/hexane gradient (0-25% of a 20% EtOAc/hexane stock solution)
R
on a CombiFlashV system. The appropriate fractions were combined and con-
centrated to afford 8.6 g (86%) of compound 14 as 2:3 b/a anomeric mixture
1
as a pale oil. H NMR (400 MHz, CDCl3) d 7.71 ꢀ 7.59 (m, 4H), 7.46 ꢀ 7.33
(m, 6H), 5.15 (m, 1H), 4.50 ꢀ 4.15 (m, 1H), 4.04 (m, 1H), 3.87 (m, 1H),
3.72 ꢀ 3.58 (m, 1H), 3.56 ꢀ 3.47 (m, 1H), 3.26 (m, 1H), 2.29 ꢀ 2.06 (m, 2H),
1.88 (m, 3H), 1.16 (m, 3H), 1.05 (s, 9H).
4.2.2.9. Preparation of ((2 R,3S)-3-((tert-Butyldiphenylsilyl)oxy)-5-((4-octyloxy-
benzyl)thio)- tetrahydrothiophen-2-yl)methyl acetate (15). Boron trifluoride
diethyl etherate (278.32 g, 1.96 mol) was added drop wise under argon to a
stirred solution of ((2 R,3S)-3-((tert-butyldiphenylsilyl)oxy)-5-ethoxytetrahy-
drothiophen-2-yl)methyl acetate (14) (455.5 g, 980.5 mmol) and 4-octyloxy-
phenylmethanethiol (8) (252.42 g, 1.0 mol) in anhydrous DCM (4.6 L) at 0 ꢁC.
The reaction mixture was stirred at this temperature for 2 h. Triethylamine
(238.12 g, 2.35 mol) was added dropwise while keeping the temperature under
5 ꢁC, followed by water (2 L). The reaction mixture was stirred for 1 h. The
organic solution was separated, dried (MgSO4) and evaporated. The residue
was purified by column chromatography (silica gel, ethyl acetate/heptanes,
1:15, then 1:9, and then 1:4) to give 486.9 g (75%) of compound 15 as a pale
oil. 1H NMR spectrum (300 MHz, CDCl3): d 7.79-7.59 (m, 4H), 7.45-7.35 (m,
6H), 7.2 (m, 2H), 6.82 (m, 2H), 4.57 (m, 0.7 H), 4.45 (m, 0.7H), 4.22-4.04 (m,
0.4H), 3.95-3.85 (m, 3H), 3.85-3.65 (m, 2.4H), 3.5 (m, 1H), 2.25-2.00 (m, 1H),
1.87 (m, 3H), 1.75 (m, 2H), 1.50-1.24 (m, 10H), 1.06 (m, 9H), 0.87 (m, 3H).
4.2.2.10. Preparation of (4S,5R)-4-Hydroxy-5-hydroxymethyl-2-((4-(octyloxy)ben-
zyl)thio)-tetrahydrothiophene, (16). A mixture of a solution of ((2 R,3S)-3-

18
D. G. WISHKA ET AL.
((tert-butyldiphenylsilyl)oxy)-5-((4-octyloxybenzyl)thio)-tetrahydrothio-
phen-2-yl)methyl acetate (15) (492.1 g, 739.9 mmol) in THF (5.7 L) and a
solution of lithium hydroxide monohydrate (310.5 g, 7.4 mol) in water
(1.9 L) was heated to reflux for 70 h. The mixture was cooled to room tem-
perature and methyl-t-butylether (MTBE) (2 L) was added. The organic
solution was separated and the aqueous layer was extracted with MTBE
(1 L). The combined organic layers were dried (Na2SO4) and evaporated to
give 485.5 g of crude product. The crude material was dissolved under heat-
ing in heptanes (3 L). The formed solution was cooled to room tempera-
ture, and then to 0 ꢁC overnight. The formed solid was filtered off, washed
with ice-cold heptanes (0.5 L) and dried under vacuum to give 212.0 g
(75%) of compound 16. The filtrate was evaporated. The residue was puri-
fied by column chromatography (silica gel, ethyl acetate/heptanes, 1:9, then
1:4, and then 7:3) to give an additional 10.2 g (3.6%) of compound 16. The
two crops of target compound were combined to afford 222.2 g (78%) of
1
compound 16. H NMR spectrum (300 MHz, CDCl3): d 7.21 (d, J ¼ 8.5 Hz,
2H), 6.84 (d, J ¼ 8.5 Hz, 2H), 4.57 (m, 1H), 4.36 (t, J ¼ 6.3 Hz, 1H), 3.93, (t,
J ¼ 6.6 Hz, 2H), 3.83-3.43 (m, 4H), 3.45 (m, 1H), 2.40-2.13 (m, 4H), 1.77
(m, 2H), 1.50-1.20 (m, 10H), 0.89 (m, 3H).
4.2.3. Synthesis of (4S,5R)-4-Hydroxy-5-hydroxymethyl-2-((4-(octyloxy)ben-
zyl)thio)-tetrahydrothiophene, (16) from D-erythro-2-deoxy-ribose
4.2.3.1. Preparation of (2 R,3S)-2-(hydroxymethyl)-5-methoxytetrahydrofuran-3-
ol (17). A 3.0 M solution of hydrogen chloride in methanol (15.0 mL,
45.1 mmol, 0.05 eq) was added drop-wise to a stirred suspension of 2-deoxy-
D-ribose (3) (120.90 g, 901 mmol) in anhydrous methanol (700 mL) at ambi-
ent temperature. The reaction mixture became a brown solution and was
stirred for 1.5 hours. Silver carbonate (7.77 g, 28.2 mmol) was added and the
reaction mixture was stirred for 1 h (pH at 8). The solids were filtered off
R
V
through a Celite pad and rinsed with methanol (300 mL). The filtrate was
evaporated under reduced pressure. THF (100 mL x 2) was added to the resi-
due and the resulting mixture was evaporated twice to remove traces of
methanol. The crude product was dried under high vacuum overnight to give
129.12 g (96.7%) of compound 17 as a brown oil. Product was used in the
next step without any further purification. 1H NMR (300 MHz, CDCl3) d 5.15
(m, 1H), 4.45 (m, 0.5H), 4.17 (m, 1H), 4.05 (m, 0.5H), 3.75-3.58 (m, 1H), 3.38
(s, 3H), 2.65 (bs, 2H), 2.30-1.90 (m, 2H).
4.2.3.2. Preparation of ((2 R,3S)-3-(benzoyloxy)-5-methoxytetrahydrofuran-2-
yl)methyl benzoate (18). A solution of benzoyl chloride (269.26 g,
191.55 mmol, 2.2 eq) in anhydrous DCM (60 mL) was added to a stirred
solution of (2 R,3S)-2-(Hydroxymethyl)-5-methoxytetrahydrofuran-3-ol (17)

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
19
(129.00 g, 870.68 mmol) containing 4-N,N-dimethylamino-pyridine (10.61 g,
87 mmole) and pyridine (166.74 g, 2108 mmol, 2.4 eq) in anhydrous DCM
(950 mL) cooled at ꢀ7 ꢁC through a dropping funnel during a period of
one hour while keeping the internal temperature below 0 ꢁC. After the add-
ition was complete, the reaction mixture was spontaneously warmed up to
ambient temperature and stirred overnight. After the reaction was com-
pleted, the reaction mixture was cooled in an ice bath and quenched with
water (600 mL). The cooling bath was removed, and the mixture was
stirred at ambient temperature for one hour. The layers were separated.
The organic layer was stirred with a solution of sodium bicarbonate
(60.00 g, 714 mmol) in water (600 mL) for two hours, followed by stirring
with a solution of citric acid monohydrate (36.64 g, 174.1 mmol, 0.2 eq) in
water (500 mL) for one hour. The organic layer was dried (MgSO4) and
evaporated under reduced pressure to give 325 g of the crude dibenzoyl
ester compound 18 as a brown oil. Product was used in the next step with-
1
out further purification. H NMR (300 MHz, CDCl3) d 8.18-8.10 (m, 4H),
7.62-7.41 (m, 6H), 5.70-5.42 (m, 1H), 5.27-5.18 (m, 1H), 4.69-4.42 (m, 3H),
3.44 (s, 2H), 3.37 (s, 1H), 2.62-2.19 (m, 3H).
4.2.3.3. Preparation of (2 R,3S)-2-Hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pen-
tane-1,3-diyldibenzoate (19). 4-Octyloxybenzyl-mercaptan (8) (201.01 g,
0.796 mol, 2.5 eq) was added to a stirred solution of ((2 R,3S)-3-(benzoy-
loxy)-5-methoxytetrahydrofuran-2-yl)methyl benzoate (18) (114.16 g,
0.320 mol) in anhydrous DCM (1 L) cooled in a dry-ice/water bath
(-1.9 ꢁC), followed by drop-wise addition of boron trifluoride etherate
(90.92 g, 0.641 mol, 2.0 eq) while keeping the internal temperature below
0 ꢁC. After the addition was complete, the reaction mixture was stirred in
an ice bath (1.0 ꢁC) for 3.5 hours and was added to a stirred mixture of
potassium carbonate (265.6 g, 1.92 mol, 6 eq) in water (1 L) and DCM
(300 mL) cooled in an ice bath (8-15 ꢁC). After the addition, the ice bath
was removed. The mixture was stirred for 50 minutes and then filtered
R
V
through a Celite 545 pad. The layers of the filtrate was separated. The
organic layer was dried (Na2SO4) and evaporated to give a yellowish oil
(307.9 g). The crude product was purified by silica gel column chromatog-
raphy (eluted with ethyl acetate/heptane, 1:9, and then 1:7.5) to give 186.8 g
1
(70%) of compound 19 as a colorless oil. H NMR (300 MHz, CDCl3) d
8.17-8.00 (d, J ¼ 7.9 Hz, 2H), 7.15-7.03 (d, J ¼ 7.9 Hz, 2H), 7.60-7.50 (d,
J ¼ 7.9 Hz, 2H), 7.55-7.39 (m, 4H), 7.17-7.29 (d, J ¼ 8.7 Hz, 2H), 7.15-7.03
(d, J ¼ 8.7 Hz, 2H), 6.72-6.69 (d, J ¼ 8.7 Hz, 2H), 6.70-6.58 (d, J ¼ 8.7 Hz,
2H), 5.42 (m, 1H), 4.60-4.27 (m, 2H), 4.05 (m, 1H), 3.87-3.59 (m, 7H),
2.79 (m, 1H), 2.45 (m, 1H), 2.15 (m, 1H), 1.80-1.65 (m, 4H), 1.50-1.20 (m,
18 H), 0.95-0.79 (m, 6H).

20
D. G. WISHKA ET AL.
4.2.3.4. Preparation of (2S,3S)-2-(formyloxy)-5,5-bis((4-(octyloxy)benzyl)thio)pen-
tane-1,3-diyl dibenzoate (20). Triphenylphosphine (117.64 g, 0.449 mol, 2 eq)
was added to a stirred solution of (2 R,3S)-2-hydroxy-5,5-bis((4-(octyloxy)-
benzyl)thio)pentane-1,3-diyl dibenzoate (19) (185.95 g, 0.224 mol) in anhyd-
rous THF (1.4 L). The resulting solution was cooled in an ice bath. Formic
acid (20.65 g, 0.448 mol, 2 eq) was added, followed by addition of DIAD
(90.7 g, 0.448 mol, 2 eq) dropwise while keeping the internal temperature
below 9 ꢁC. After the addition, the reaction mixture was left in the cooling
bath and spontaneously warmed up to ambient temperature with stirring
overnight. It was diluted with heptane (500 mL), followed by aq NaHCO3
(18.84 g, 0.224 mol in 500 mL of water). The mixture was stirred for
30 minutes. The layers were separated. The aqueous layer was extracted
with heptane (200 mL) again. The combined organic layers were dried
(MgSO4) and concentrated under reduced pressure. The residue (510 g)
was treated with ethyl acetate (500 mL) and heptane (1 L) and stirred for
20 minutes. Solids were filtered off, and the filtrate was concentrated under
reduced pressure to give a yellowish oil (316.8 g) which was purified by sil-
ica gel column chromatography (eluted with ethyl acetate/heptane 1:9) to
1
give compound 20 (155.96 g, 81.1% yield) as a yellowish oil. H NMR
(300 MHz, CDCl3) d 8.09 (s,1H), 7.95 (m, 4H), 7.59 (m, 2H), 7.45 (m, 4H),
7.09 (m, 4H), 6.65 (m, 4H), 5.68 (m, 1H), 5.42 (m, 1H), 4.45 (m, 1H), 4.29
(m, 1H), 3.82-3.63 (m, 8H), 3.55 (m, 1H), 2.31 (m, 1H), 2.05 (m, 1H), 1.75
(m, 4H), 1.50-1.20 (m, 18H), 0.84 (m, 6H).
4.2.3.5. Preparation of (2S,3S)-2-Hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pen-
tane-1,3-diyl dibenzoate (21). Hydrogen chloride solution (4.0 M in dioxane,
45.2 mL, 0.181 mol) was added to a stirred solution of (2S,3S)-2-(formyloxy)-
5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,3-diyl dibenzoate (20) (155.00 g,
0.181 mol) in THF (183 mL) and MeOH (1373 mL). The cloudy solution was
stirred at room temperature for 2 hours and concentrated under reduced pres-
sure (bath temperature at 31 ꢁC) and then under high vacuum. The residue
was dissolved in DCM (1.2 L). The resulting solution was washed with water
(1.2 L), dried (MgSO4), concentrated under reduced pressure and then under
high vacuum overnight to give compound 21 (143.6 g, 96%) as a yellowish
oil. This product was used in the next step without any further purification.
1H NMR (300 MHz, CDCl3) d 7.99 (d, J ¼ 7.9 Hz, 2H), 7.90 (d, J ¼ 7.9 Hz,
2H), 7.55 (m, 4H), 7.39 (m, 4H), 7.06 (m, 4H), 6.67 (m, 4H), 5.49 (m, 1H),
4.35 (m, 2H), 3.91-3.64 (m, 8H), 3.57 (m, 1H), 2.39 (m, 1H), 2.24 (m, 1H),
2.15 (m, 1H), 1.80-1.65 (m, 4H), 1.45-1.21 (m, 18 H), 0.84 (m, 6H).
4.2.3.6. Preparation of (2S,3S)-2-((methylsulfonyl)oxy)-5,5-bis((4-(octyloxy)ben-
zyl)thio)pentane-1,3-diyl dibenzoate (22). A solution of MsCl (23.9 g,

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
21
0.209 mol, 1.2 eq) in anhydrous DCM (30 mL) was added dropwise to a
stirred solution of (2S,3S)-2-hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pen-
tane-1,3-diyl dibenzoate (21) (144.36 g, 0.174 mol) in anhydrous DCM
(950 mL) cooled in an ice bath, followed by a solution of triethylamine
(21.1 g, 0.209 mol, 1.2 eq) in anhydrous DCM (20 mL) dropwise while keep-
ing the internal temperature below 5 ꢁC. After the addition, the reaction
mixture was left in the ice bath, and was spontaneously warmed up to
ambient temperature while stirring overnight. The reaction was quenched
with water (800 mL) and the resulting mixture was stirred at room tem-
perature for one hour. The layers were separated. The organic layer was
washed with a solution of sodium bicarbonate (17.5 g, 0.209 mol) in water
(800 mL) for 40 minutes, and then with a solution of citric acid monohy-
drate (17.5 g, 0.083 mol) in water (500 mL) for 30 minutes. The organic
layer was dried (MgSO4), and concentrated under reduced pressure at
27 ꢁC to give compound 22 (159.54 g, 101.0% yield) as a brown oil. The
crude product was used in the next step without any further purification.
1H NMR (300 MHz, CDCl3) d 8.15 (d, J ¼ 7.9 Hz, 2H), 7.91 (d, J ¼ 7.9 Hz,
2H), 7.77 (m, 2H), 7.47 (m, 4H), 7.15 (d, J ¼ 8.7 Hz, 2H), 7.05 (d,
J ¼ 8.7 Hz, 2H), 6.67 (m, 4H), 5.63 (m, 1H), 5.01 (m, 1H), 4.55 (m, 1H),
4.38 (m, 1H), 4.84-4.65 (m, 8H), 3.58 (m, 1H), 2.98 (s, 3H), 2.36 (m, 1H),
2.18 (m, 1H), 1.75 (m, 4H), 1.45-1.20 (m, 18 H), 0.84 (m, 6H).
4.2.3.7. Preparation of ((2 R,3S)-3-(benzoyloxy)-5-((4-(octyloxy)benzyl)thio)tetra-
hydrothiophen-2-yl)methyl benzoate (16a). A solution of (2S,3S)-2-((methyl-
sulfonyl)oxy)-5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,3-diyl dibenzoate
(22) (159.51 g, 175.81 mmol), triethylamine (35.58 g, 351.6 mmol, 2 eq) and
n-Bu4NI (129.88 g, 351.62 mmol, 2 eq) in acetonitrile (2.44 L) was heated at
75-77 ꢁC for 7 days. The reaction mixture was concentrated under reduced
pressure. The residue was mixed with ethyl acetate/heptane (1.28 L, 1:3) for
60 minutes. The solids were filtered off, and the filtrate was concentrated
under reduced pressure to give a sticky black oil. The residue (203.41 g)
was purified by silica gel column chromatography (ethyl acetate/heptane, 1:
12) to afford compound 16a (71.63 g, 69% yield, a mixture of two anomers)
1
as a white solid. H NMR (300 MHz, CDCl3) d 8.19-7.91 (m, 4H), 7.55 (m,
2H), 7.41 (m, 4H), 7.24 (d, J ¼ 8.7 Hz, 2H), 6.83 (d, J ¼ 8.7 Hz, 2H), 5.78
(m, 0.75H), 5.65 (m, 0.25H), 4.59-4.38 (m, 3), 4.11 (m, 0.25H), 3.96-3.76
(m, 4.75H), 2.75 (m, 0.25H), 2.59-2.29 (m, 1.75H), 1.76 (m, 2H), 1.45-1.20
(m, 9 H), 0.84 (m, 3H).
4.2.3.8. Preparation of (2 R,3S)-2-(hydroxymethyl)-5-((4-(octyloxy)benzyl)thio)te-
trahydrothiophen-3-ol (16). A mixture of ((2 R,3S)-3-(benzoyloxy)-5-((4-
(octyloxy)benzyl)thio)tetrahydrothiophen-2-yl)methyl
benzoate
(16a)

22
D. G. WISHKA ET AL.
(70.97 g, 119.7 mmol) with THF (710 mL) and lithium hydroxide monohy-
drate (20.09 g, 478.mmol, 4 eq.) in water (120 mL) was stirred at room tem-
perature for 48 hours. The reaction mixture was diluted with MTBE
(700 mL) and brine (700 mL) and stirred for 15 minutes. The layers were
separated. The aqueous layer was extracted with MTBE (500 mL) again.
The combined organic layers were dried over sodium sulfate, concentrated
under reduced pressure to give a light brown oil (53.52 g). The residue was
mixed with heptane (500 mL) and then concentrated under reduced pres-
sure to give a white solid (51.73 g). The crude product was stirred with
heptane (500 mL) for two hours and then kept at ꢀ23 ꢁC overnight. The
product was collected by filtration to give a white solid (46.05 g, 95.3% pur-
ity by HPLC). This solid was dissolved in DCM (500 mL) and was stirred
with a saturated aqueous solution of sodium bicarbonate (500 mL) for one
hour. The mixture was filtered through a F-fritted funnel and the layers
were separated. The aqueous layer was extracted with DCM (200 mL) again.
The combined organic layers were dried (Na2SO4), filtered through a
R
V
Celite 545 pad, concentrated under reduced pressure and then under high
vacuum for 72 hours to give compound 16 (44.2 g, 96.0% yield) as a white
1
solid. H NMR (300 MHz, CDCl3) d 7.21 (d, J ¼ 8.7 Hz, 2 H), 6.84 (d,
J ¼ 8.7 Hz, 2 H), 4.56 (m, 1 H), 4.35 (t, J ¼ 6.0 Hz, 1H), 3.93 (t, J ¼ 6.6 Hz,
2H), 3.65-3.79 (m, 4 H), 3.44 (m, 1 H), 2.34 (m, 2 H), 2.22 (m, 2 H), 1.77
(m, 2 H), 1.45 (m, 2 H), 1.29 (m, 8 H), 0.89 (t, J ¼ 6.9 Hz, 3H); m.p. 69.0-
70.5 ꢁC; Combustion Analysis for C20H32O3S2. Calculated: C, 62.46; H,
8.39; S,16.68, Found: C, 62.52; H, 8.42; S,16.41.
4.2.4. Synthesis of of 4-Amino-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetra-
hydrothiophen-2-yl)pyrimidin-2(1H)-one (1) (NSC764276, T-dCyd)
4.2.4.1. Preparation of (6aR,9aS)-2,2,4,4-tetraisopropyl-8-((4-(octyloxy)benzyl)th-
io)tetrahydro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocine
(16 g).
(2R,3S)-2-
(Hydroxymethyl)-5-((4-(octyloxy)benzyl)thio)tetrahydrothiophen-3-ol (16)
(598.8 g, 1.56 mol) was combined with imidazole (265 g, 3.89 mol, 2.5
equiv.) in dry DMF (3.1 L) in a 5 three-neck round-bottom flask under
argon. The solution was cooled to 2-5 ꢁC, and treated with 1,3-dichloro-
1,1,3,3-tetraisopropyldisiloxane (589.4 g, 1.87 mol, 1.2 equiv.) dropwise over
1.5 h while maintaining the reaction temperature below 5 ꢁC. The reaction
was stirred for 21 h as the cooling bath was allowed to expire. The reaction
mixture was then poured into cold water (8 L) in a 50 L reactor. The mix-
ture was extracted with EtOAc (3 ꢃ 5 L) and the combined organic layer
was washed with 50% saturated sodium chloride solution (3 ꢃ 3.5 L). The
organic layer was dried (MgSO4) and the volatiles were removed in vacuo
to provide a crude oil (1132.5 g). The crude material was purified by chro-
matography over 8 Kg silica gel (230-400 mesh) while eluting with 97%

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
23
EtOAc/heptane (57 L). The appropriate fractions were combined and con-
1
centrated to afford 927.5 g of compound 16g in 95% yield as a pale oil. H
NMR (400 MHz, CDCl3) d 7.20 (d, J ¼ 8.7 Hz, 2H), 6.81 (d, J ¼ 8.4 Hz, 2H),
4.59 (m, 0.2H), 4.22 (m, 2H), 4.05 (m, 1), 3.91 (m, 2H), 3.86 ꢀ 3.76 (m,
3H), 3.44 (m, 0.8H), 3.33 (m, 0.2), 2.50 (m, 0.8H), 2.40-2.18 (m, 0.4H),
2.00 (m, 0.8H), 1.75 (m, 2H), 1.45-1.20 (m, 10H), 0.92-1.19 (m, 28H), 0.89
(m, 3H).
4.2.4.2. Preparation of N-(2-oxo-1-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahy-
dro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-1,2-dihydropyrimidin-4-
yl)benzamide (24f). A 3 L three-neck round-bottom flask under argon was
charged with N4-benzoyl-cytosine (23a) (270.2 g, 1.26 mol), ammonium sul-
fate (4.115 g, 0.031 mol), and hexamethyldisilazane (HMDS) (1.3 L). The
suspension was heated to reflux for 18 h. The resulting solution (slight tur-
bidity) was cooled to room temperature and the excess HMDS was
removed in vacuo to afford the silylated base as a white solid. The solid
was dissolved in anhydrous DCM (2.25 L) and this solution was used dir-
ectly in the addition step.
(6aR,9aS)-2,2,4,4-Tetraisopropyl-8-((4-(octyloxy)benzyl)thio)tetrahydro-6H-
thieno[3,2-f][1,3,5,2,4]trioxadisilocine (16g) (525.2, 0.838 mol) was dissolved
in anhydrous DCM (2.26L) in a 50L reactor and the solution of silylated
N4-benzoyl-cytosine (23a) in DCM was added. The reaction mixture was
cooled to 0 ꢁC while stirring under argon. The solution was treated with N-
Bromosuccinimide (164g, 0.92 mol, 1.1 equiv.) in five portions over
30minutes. The reaction mixture was stirred at 0 ꢁC for 1.5 h. TLC (5%
EtOAc/heptane) was used to monitor the consumption of 16g. After 16g
was consumed completely (1.5h), the reaction was quenched with sodium
thiosulfate (305g in 2.5 L of water) and stirred for 0.5h at 0 ꢁC. The mixture
was filtered through a pad of Celite 209. The organic layer was separated,
and the aqueous layer was extracted with DCM (2ꢃ 2 L). The combined
organic layers were dried (MgSO4) and the filtrate was concentrated in
vacuo to give 788 g of crude product. Purification by flash chromatography
over 12Kg silica gel (230-400 mesh) eluting with a 20-50% EtOAc/heptane
gradient followed by chromatography of mixed fractions ultimately afforded
1
241.6 g (49%) of compound (24f) as an off-white solid. H-NMR (300 MHz,
CDCl3) d 8.82 (d, J ¼ 7.8Hz, 1H), 8.64 (br s, 1H),7.89 (d, J ¼ 7.8 Hz, 2H),
7.68-7.45 (m, 4H), 6.11 (d, J ¼ 6.9 Hz, 1H), 4.43-4.31 (m, 1H), 4.16 (dd,
J ¼ 12.9, 3.0 Hz, 1H), 4.00 (d, J ¼ 12.6 Hz, 1H), 3.41-3.35 (m, 1H), 2.63-2.50
(m, 1H), 2.37 (dd, J ¼ 13.2, 5.1 Hz, 1H), 1.20-0.80 (m, 28H).
4.2.4.3. Preparation of N-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydro-
thiophen-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide. A 3 L three-necked

24
D. G. WISHKA ET AL.
round-bottom flask under argon was charged with N-(2-oxo-1-((6aR,8R,9aS)-
2,2,4,4-tetraisopropyltetrahydro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-
1,2-dihydropyrimidin-4-yl)benzamide (24f) (236.4 g, 0.40 mol) and anhydrous
THF (1.3 L). The solution was cooled to 0 ꢁC, treated slowly dropwise with a
solution of tetra-n-butylammonium fluoride in THF (1 M, 1.21 L, 1.21 mol, 3
equiv.) over 1 h, and then it was stirred at that temperature for 2 h. Keeping
the bath temperature below 30 ꢁC, the reaction mixture was concentrated to
dryness. The residue was dissolved in DCM (2.5 L) and this solution was
slowly added to water (2.5 L) with vigorous stirring. After the addition was
complete the mixture was stirred an additional 0.5 h. The formed solid was
collected by filtration and the filter cake was washed successively with DCM
(0.5 L) and water (0.5 L). The solid was dried under vacuum at 40 ꢁC for 18 h
to afford 129.5 g (93%) of N-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)te-
trahydrothiophen-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide
as
a
1
white solid. H-NMR (300 MHz, CD3OD) d 8.78 (d, J ¼ 7.5 Hz, 1H), 7.97 (d,
J ¼ 7.8 Hz, 2H), 7.68-7.61 (m, 2H), 5.57-7.50 (m, 2H), 6.40 (t, J ¼ 6.4 Hz, 1H),
4.45 (q, J ¼ 4.5 Hz, 2H), 3.81 (d, J ¼ 5.4 Hz, 2H), 3.46 (q, J ¼ 5.4 Hz, 1H),
2.56-2.46 (m, 1H), 2.36-2.26 (m, 1H).
4.2.4.4. Preparation of 4-Amino-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)te-
trahydrothiophen-2-yl)pyrimidin-2(1H)-one (1) (NSC764276, T-dCyd). A sus-
pension
of
N-(1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)
tetrahydrothiophen-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl)benzamide
(129.5 g, 0.373 mol) in anhydrous methanol (4.7 L) was treated with a solu-
tion of ammonia in methanol (7 M, 470 mL, 3.29 mol). The suspension was
stirred 16 h at room temperature. The clear solution was evaporated to dry-
ness and the residue was stirred with acetonitrile (1 L) containing 15 mL
methanol overnight. The solid was collected by filtration, washed with
300 mL acetonitrile, and dried under high vacuum at 40 ꢁC overnight to
afford 85.5 g of compound 1 in 94% yield with 99.9% HPLC purity but
with 8 mol% methanol by proton NMR. The 85.5 g of solid 1 was dissolved
in water (2.5 L) and was dried by lyophilization to remove the residual
methanol. The lyophilized solid was dried under vacuum at 45 ꢁC afford
84.1 g (93%) of compound 1 (NSC764276, T-dCyd) as a fine white solid.
1H-NMR (300 MHz, DMSO-d6) d 7.93 (d, J ¼ 7.5 Hz, 1H), 7.17 (s, 1H),
7.15 (s, 1H), 6.35 (dd, J ¼ 8.1, 6.9 Hz, 1H), 5.77 (d, J ¼ 7.2 Hz, 1H), 5.20 (d,
J ¼ 3.9 Hz, 1H), 5.10 (t, J ¼ 5.4 Hz, 1H), 4.33 (t, J ¼ 3.3 Hz, 1H), 3.65-3.45
(m, 2H), 3.30-3.22 (m, 1H), 2.20-2.00 (m, 2H). 13C-NMR (75 MHZ,
DMSO-d6) d 165.3, 155.5, 142.2, 94.8, 73.5, 63.8, 60.6, 58.8, 41.7. Melting
Point: 215-216 ꢁC. Combustion analysis: Calculated for C9H13N3O3S: C,
44.43; H, 5.39; N, 17.27; S, 13.18. Found: C, 44.51; H, 5.25; N, 17.23;
S, 13.36.

NUCLEOSIDES, NUCLEOTIDES AND NUCLEIC ACIDS
25
4.2.5. Synthesis of 4-Amino-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahy-
drothiophen-2-yl)-1,3,5-triazin-2(1H)-one (2), (NSC777856, 5-Aza-T-dCyd)
4.2.5.1. Preparation of 4-Amino-1-((6aR,8R,9aS)-2,2,4,4-tetraisopropyltetrahy-
dro-6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-1,3,5-triazin-2(1H)-one (24e).
A suspension of 5-azacytosine (23b) (150.1 g, 1.34 mol) and ammonium
sulfate (4.4 g, 0.033 mol) in hexamethyldisilazane (HMDS, 980 mL) was
refluxed in 2-L round-bottomed flask while stirring under argon atmos-
phere for 21 h. The solution was cooled to room temperature and the
excess of HMDS was removed in a rotary evaporator to give a white solid.
The residue was dissolved in anhydrous DCM (1 L) and the solution was
used directly below.
(6aR,9aS)-2,2,4,4-Tetraisopropyl-8-((4-(octyloxy)benzyl)thio)tetrahydro-
6H-thieno[3,2-f][1,3,5,2,4]trioxadisilocine (16g) (280 g, 446.5 mmol) was
dissolved in anhydrous DCM (1.3 L) and the solution of the silylated azacy-
tosine was added. The resulting solution was cooled to 0 ꢁC while stirring
under argon atmosphere. N-Bromosuccinimide (87 g, 491.1 mmol, 1.1
equiv.) was added to the cooled mixture in 5 portions over 30 minutes and
the resulting mixture was stirred at 0 ꢁC for 1.5 h. Thin-layer chromatog-
raphy was used to monitor consumption of starting material 16g (5% Ethyl
acetate/heptane). After 16g was consumed completely, the reaction mixture
was quenched with sodium thiosulfate (160 g in 1.8 L of water) and stirred
for 0.5 h at 0 ꢁC. The mixture was filtered through a pad of Celite 209
(200 g). The organic layer was separated, and the aqueous layer was
extracted with DCM (1.5 L ꢃ 2). The combined organic layers were dried
(Na2SO4), filtered and concentrated in a 10-L rotary evaporator to give the
crude product as a 4:1 anomeric mixture (420 g). Fractionation of the crude
material was achieved by column chromatography over 3.5 Kg of silica gel
(230-400 mesh) while eluting with a 30-100% ethyl acetate/DCM gradient
followed by chromatography of mixed fractions to ultimately afford 90.6 g
1
(42%) of compound 24e as a white foam. H-NMR (300 MHz, CDCl3) d
8.99 (s, 1H), 6.22 br s, 1H), 5.95 (d, J ¼ 6.6 Hz, 1H), 5.60 (br s, 1H), 4.40-
4.26 (m, 1H), 4.13 (dd, J ¼ 12.8, 3.0 Hz, 1H), 3.97 (dd, J ¼ 7.0, 0.9 Hz, 1H),
3.38-3.30 (m, 1H), 2.57-2.44 (m, 1H), 2.33 (dd, J ¼ 13.4, 5.4 Hz, 1H), 1.18-
0.80 (m, 28 H).
4.2.5.2. Preparation of 4-Amino-1-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)te-
trahydrothiophen-2-yl)-1,3,5-triazin-2(1H)-one (2), (NSC777856, 5-Aza-T-dCyd).
A suspension of 4-amino-1-(6aR,8R,9aS)-2,2,4,4-tetraisopropyl-tetrahydro-
6H-thieno[3,2-f][1,3,5,2,4]
trioxadisilocin-8-yl)-1,3,5-triazin-2(1H)-one
(24e) (90 g, 184.9 mmol) and ammonium fluoride (34.3 g, 924.4 mmol) in
anhydrous methanol (1.3 L) was heated to 60-65 ꢁC for 2.5 h. The mixture
was cooled to 15 ꢁC and stirred for 1 h. The precipitated solid was collected

26
D. G. WISHKA ET AL.
in a filter funnel and washed with anhydrous methanol (2 ꢃ 30 mL) to give
28 grams of compound 2 (NSC-777856, 5-Aza-T-dCyd) as white solid after
drying. The mother liquor was mixed with Celite 209 (30 g), concentrated
to dryness and purified by chromatography on silica gel (150 g) eluting
with ethyl acetate/ethanol in a ratio 5:3 (4 L) and then with ethanol (4 L) to
give 7 g of crude product. The column isolate was stirred with methanol
(70 mL) for 30 min., followed by filtration to provide an additional 3.5 g of
2 as white solid after drying. The two lots of 2 were combined to afford a
1
total of 31.5 g (73%) of compound 2 as a fine white solid. H NMR
(400 MHz, DMSO- d6) d 8.67 (s, 1H), 7.50 (d, J ¼ 5.6 Hz, 2H), 6.06 (t,
J ¼ 7.0 Hz, 1H), 5.21 (d, J ¼ 4.0 Hz, 1H), 5.10 (t, J ¼ 5.4 Hz, 1H), 4.30 (q,
J ¼ 4.1 Hz, 1H), 3.60 (dt, J ¼ 11.7, 5.9 Hz, 1H), 3.53 (dt, J ¼ 11.2, 5.4 Hz,
1H), 3.25 (td, J ¼ 5.9, 3.5 Hz, 1H), 2.22 (dddd, J ¼ 19.7, 12.8, 9.7, 4.5 Hz,
2H). 13C NMR (101 MHz, DMSO- d6) d 165.95, 157.66, 153.80, 73.72,
63.45, 59.99, 59.17, 42.05. Melting Point: 207-209 ꢁC, d. Combustion
Analysis. Calculated for C8H12N4O3S: C, 39.34; H, 4.95; N, 22.94; S, 13.12.
Found: C, 39.28; H, 4.90; N, 22.79; S, 12.93.
Funding
This project was funded in whole or in part with Federal funds from the National Cancer
Institute, National Institutes of Health, under Contracts No. HHSN261200800001E,
HHSN261201600066C, HHSN261201700007I and NO2CM-2012-00020. DGW, ODL, RB,
SMD, LZ, and JM are co-inventors of the World Patent Application WO 2019152459 A1.
The content of this publication does not necessarily reflect the views or policies of the
Department of Health and Human Services, nor does mention of trade names, commercial
products, or organizations imply endorsement by the U.S. Government.
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