Synthesis of 4′-substituted 2′-deoxy-4′-thiocytidines and its evaluation for antineoplastic and antiviral activities

Article abstract of DOI:10.1016/j.tet.2019.06.044

4′-Azido- (7), 4′-C-fluoromethyl- (8) 4′-C-ethynyl- (9) and 4′-C-cyano- (10) 2′-deoxy-4′-thiocytidines have been synthesized. In this study, it was found that the isolated yield of 4′-thiouracil nucleoside 13 in a Lewis acid-promoted Vorbrüggen-type glycosidation utilizing 12 was better than that of the electrophilic glycosidation reaction between silylated uracil and 11. This improved result prompted us to perform the glycosidation utilizing 36 and 43 for the synthesis of 37 and 44. Introduction of the azido group was carried out by nucleophilic substitution in the 4′-benzoyloxy derivative 22a. On the other hand, 9 and 10 were synthesized by way of the chemical manipulation of the hydroxymethyl group at the 4′-position of 46. Evaluation of the antineoplastic activity of 2 and 7–10 against human B-cell (CCRF-SB) and T-cell leukemia (Molt-4) cell lines revealed that 4′-azido- (7) and 4′-C-fluoromethyl- (8) derivatives exhibited cytotoxic activity whereas no cytotoxicity was observed in the 4′-C-ethynyl- (9) and 4′-C-cyano- (10) derivatives as well as the parent compound 2. Compound 7 was also found to possess promising antiviral activity against VZV and HSV-1 without any cytotoxity against HEL host cells. It is noteworthy that 7 exhibited potent inhibitory activities against the thymidine kinase-deficient (TK^?) mutant of VZV and HSV-1.

Full text of DOI:10.1016/j.tet.2019.06.044

Accepted Manuscript
Synthesis of 4′-substituted 2′-deoxy-4′-thiocytidines and its evaluation for
antineoplastic and antiviral activities
Kazuhiro Haraguchi, Hiroki Kumamoto, Kiju Konno, Hideki Yagi, Yutaka Tatano, Yuki
Odanaka, Satoko Shimbara Matsubayashi, Robert Snoeck, Graciela Andrei
PII:
S0040-4020(19)30716-1
https://doi.org/10.1016/j.tet.2019.06.044
TET 30436
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 31 May 2019
Revised Date: 25 June 2019
Accepted Date: 27 June 2019
Please cite this article as: Haraguchi K, Kumamoto H, Konno K, Yagi H, Tatano Y, Odanaka Y,
Shimbara Matsubayashi S, Snoeck R, Andrei G, Synthesis of 4′-substituted 2′-deoxy-4′-thiocytidines and
its evaluation for antineoplastic and antiviral activities, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.06.044.
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ACCEPTED MANUSCRIPT
Synthesis of 4’-substituted 2’-deoxy-4’-thiocytidines and its Evaluation
for Antineoplastic and Antiviral Activities
1
)
1)
Kazuhiro Haraguchi, * Hiroki Kumamoto,
2
)
2)
2)
3)
Kiju Konno, Hideki Yagi, Yutaka Tatano, Yuki Odanaka,
3
)
4)
4)
Satoko. Shimbara Matsubayashi, Robert Snoeck, Graciela Andrei,
1
) Department of Pharmaceutical Sciences, Nihon Pharmaceutical University,
0281 Komuro, Inamachi, Kita-adachi-gun, Saitama 362-0806, Japan
) Department of Pharmaceutical Sciences, International University of Health and
Welfare, 2600-1 Kitakanemaru, Ohtawara City, Tochigi 324-8501
1
2
3
1
4
) School of Pharmacy, Showa University, Hatanodai 1-5-8, Shinagawa-ku, Tokyo,
42-8555, Japan
) Rega Institute for Medical Research, Katholieke Universiteit Leuven,
B-3000 Leuven, Belgium

ACCEPTED MANUSCRIPT
Phone +81-48-721-1155
Fax +81-48-721-6718
harakazu@nichiyaku..ac.jp
Keywords: nucleoside; thio-sugar; glycal; glycosidation; antineoplastic activity;
antiviral activity
2

ACCEPTED MANUSCRIPT
TOC GRAPHIC
Synthesis of 4’-substituted 2’-deoxy-4’-thiocytidines and its Evaluation for Antineoplastic and
Antiviral Activities
Kazuhiro Haraguchi, Hiroki Kumamoto, Kiju Konno, Hideki Yagi, Yutaka Tatano, Yuki Odanaka, Satoko.
Shimbara Matsubayashi, Robert Snoeck, Graciela Andrei
NH2
N
PgO
S
N
O
HO
X
S
R
PgO
HO
R = N3, CH F, C
Pg = protecting group
I: X = H
≡
CH, C N
≡
2
II: X = CH OPg
2
promising antineoplastic and
antiviral agents
3

ACCEPTED MANUSCRIPT
Abstract
’-Azido- (7), 4’-C-fluoromethyl- (8) 4’-C-ethynyl- (9) and 4’-C-cyano- (10)
’-deoxy-4’-thiocytidines have been synthesized. In this study, it was found that the
4
2
isolated yield of 4’-thiouracil nucleoside 13 in a Lewis acid-promoted Vorbrüggen-type
glycosidation utilizing 12 was better than that of the electrophilic glycosidation reaction
between silylated uracil and 11. This improved result prompted us to perform the
glycosidation utilizing 36 and 43 for the synthesis of 37 and 44. Introduction of the
azido group was carried out by nucleophilic substitution in the 4’-benzoyloxy derivative
2
2a. On the other hand, 9 and 10 were synthesized by way of the chemical manipulation
of the hydroxymethyl group at the 4’-position of 46.
Evaluation of the antineoplastic activity of 2 and 7-10 against human B-cell
(CCRF-SB) and T-cell leukemia (Molt-4) cell lines revealed that 4’-azido- (7) and
4
’-C-fluoromethyl- (8) derivatives exhibited cytotoxic activity whereas no cytotoxicity
was observed in the 4’-C-ethynyl- (9) and 4’-C-cyano- (10) derivatives as well as the
parent compound 2. Compound 7 was also found to possess promising antiviral
activity against VZV and HSV-1 without any cytotoxity against HEL host cells. It is
noteworthy that 7 exhibited potent inhibitory activities against the thymidine
-
kinase-deficient (TK ) mutant of VZV and HSV-1.
4

ACCEPTED MANUSCRIPT
1
. Introduction
Nucleoside analogues are recognized as an important class of biologically active
1
-21
compounds, especially as antiviral and antitumor agents.
Among their
sugar-modified analogues, 4’-thionucleosides, in which the oxygen atom in the furanose
ring is replaced with a sulfur atom, have attracted much attention since the discovery of
the antiviral and antitumor activities of 4’-thiothymidine (1) and
2
2-24
2
’-deoxy-4’-thiocytidine (2) (Figure 1).
O
NH2
N
Me
NH
HO
HO
N
O
N
O
S
S
HO
'-thiothymidne (
HO
2'-deoxy-4'-thiocytidine (
and
4
1
)
2)
Figure 1. Structures of compounds
1
2.
Recently, synthesis of 2’-substituted derivatives 3-6 of 2 and their evaluation for
25
antineoplastic activity has been reported (Figure 2). In this study, 4’-thioFAC 4 has
emerged as highly potent antineoplastic agent. Furthermore, some
’-deoxy-4’-thiocytidine derivatives exhibited antiviral activities.
a
2
5b
2
5

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NH2
N
HO
N
O
3
4
5
6
: X = N , Y = H
: X = F, Y = H
: X = Y = F
3
S
X
: X = Y = CH2
HO
Y
Figure 2. Structures of 2'-substituted 2'-deoxy-4'-thiocytidines 3-6.
To expand the structure-activity relationships of 2, we have been interested in the
antineoplastic and antiviral activities of 4’-substituted congeners 7-10 because
introduction of a substituent into the 4’-position of nucleoside has been found to lead to
2
6
biologically-active agents. Herein, we describe the results of the synthesis of 7-10 and
their evaluation for antineoplastic and antiviral activities.
NH2
N
N
O
7: R = N
3
HO
S
8
9
: R = CH2F
R
: R = C
≡
CH
N
HO
10: R = C
≡
Figure 3. Structures of the target molecules 7-10.
2
2
. Results and Discussion
.1. Chemistry
The synthetic plan is outlined in Scheme 1. 4’-Azido-2’-deoxy-4’-thiocytidine 7
would be obtained on the basis of the synthetic protocol of the respective thymidine
6

ACCEPTED MANUSCRIPT
2
7
nucleoside (Path A). Thus, 4-thiofuranoid glycal I is subjected to electrophilic
glycosidation and subsequent Pb(OBz) -mediated dibenzoyloxylation to give
4
2
8
4
’-benzoyloxy-2’-deoxy-4’-thiouridine derivative II.
Lewis-acid
promoted
nucleophilic substitution of II with TMSN and conversion of the uracil moiety to
3
cytosine base would provide the first target molecule 7. In the synthesis of the
4
’-branched 2’-deoxy-4’-thiocytidines 8-10, glycal III might be a suitable glycosyl
donor on the basis of the recently published procedure in which 9 has been already
2
9
synthesized utilizing IV as a glycosyl donor (Path B). The chemical transformation of
the substituent at the 4’-position of V would furnish 8-10.
O
NH
nucleophilic
substitution
Path A
X = H
N
O
BzO
BzO
S
7
β-face selective
electrophilic
glycosidation
TBDMSO
II
i-Pr
O
X
S
Si
O
i-Pr
O
N
Si O
NH
i-Pr
i-Pr
transformation of
CH OH
2
i-Pr
O
O
Path B
Pg = protecting group
S
Si
O
i-Pr
8
- 10
Y
I: X = H
X = CH OPg
2
III: X = CH OPg
2
Si
O
IV: X = C≡CSiEt3
i-Pr
i-Pr
V
Y = CH2F
CH OH
=
2
Scheme 1. Synthetic plan for the target molecules 7-10.
Initially, preparation of 4’-benzoyloxy-2’-deoxy-4’-thiouridine derivative II was
7

ACCEPTED MANUSCRIPT
carried out (Scheme 2). N-Iodosuccimide (NIS)-mediated electrophilic glycosidation
between 3,5-O-(tetraisopropyldisiloxane-1,3-diyl)-4-thiofuranoid glycal 11 and silylated
uracil gave the β-anomer of the protected 2’-deoxy-2’-iodo-4’-thiouridine 13 as a sole
3
0,31
product, albeit the isolated yield of 13 was moderate (57% yield).
To improve the
isolated yield of 13, Lewis-acid promoted Vorbrüggen-type glycosidation was examined.
Thus, the desired glycosyl donor 12 was prepared as single stereoisomer in 93%
isolated yield by reacting 11 with pivalic acid in the presence of NIS in CH CN-CH Cl .
3
2
2
When 12 was reacted with silylated uracil in the presence of TMSOTf in
CH CN/CH Cl 13 was isolated in 87% yield. The isolated yield in the two step
3
2
2,
sequence for the synthesis of 13 was superior to that of the electrophilic glycosidation;
1% (11→12→13) vs. 57% (11→13). Bu SnH-Mediated radical reduction of 13 using
8
3
Et B as initiator in toluene at −60 °C provided 14 in 98% yield. Compound 14 was
3
converted to di-O-acetate 15 in quantitative yield in two steps in one pot manner.
2
4
’-Deoxy-4’-thiourudine 16 was obtained from 15 by treating with methanolic ammonia.
’,5’-Unsaturated uracil nucleoside 19 was prepared in 47% yield in 4 steps from 15; 1)
reaction of 16 with I /Ph P/pyridine, 2) acetylation of 17, 3) elimination of HI of 18
2
3
with DBN. Compound 19 was converted to 20 and subsequent silylation of 20 gave 21
in 61% yield in two steps from 19. When 21 was treated with Pb(OBz) in PhMe at rt, a
4
8

ACCEPTED MANUSCRIPT
mixture
of
two
products
was
formed.
The
target
molecule
4
’-benzoyloxy-2’-deoxy-4’-thiouracil nucleoside 22a was obtained in 63% yield with
the α- -configuration as evidenced by HMBC correlation (H-5’/5’-OCOPh) and NOE
L
experiment (H-2’b (α-H)/CH -5’a, Si-Me/CH -5’a and Si-tert-Bu/CH -5’b). The other
2
2
2
product was the ring-expanded compounds 22b (28%), their thiopyranosyl structures
being evident from the observed HMBC correlations between C-1’/H-5’a and
C-1’/H-5’b. By comparing with the result obtained by the Pb(OAc) -mediated
4
2
7
di-acetoxylation, in which the respective 4’-acetoxynucleoside was obtained in 42%
yield along with the pyranosyl counterpart (34% yield), the use of Pb(OBz) gave
4
improved isolated yield of the desired 4’-benzoyloxy-4’-thionucleoside. With
4
’-benzoyloxy nucleoside 22a in hand, nucleophilic substitution was examined. Thus,
when 22a was reacted with Me SiN (5 equiv) in the presence of SnCl in CH Cl at
3
3
4
2
2
−30 °C, the desired 4’-α-azido derivative 23a was obtained in 61% isolated yield along
with the 4’-β-azido 23b (20% yield) (Scheme 3). The depicted stereochemistry of 23a
was assigned on the basis of NOE experiment of the respective 5’-O-acetyl derivative
2
5 (H-6/H-5’a and H-6/H-5’b) derived in two steps from 23a via 24. Compound 25 was
converted to the respective cytosine nucleoside 27 (Scheme 4) through ammonolysis of
the 4-O-(2,4,6-triisopropylbenzenefulfonyl) (TPS) ester 26. Compound 27 was
9

ACCEPTED MANUSCRIPT
transformed into the tri-acetate 28, which was subjected to treatment of NH in MeOH
3
to give the target molecule 7.
i
O
NH
O
i-Pr
O
Si
i-Pr
O
Si
O
I
i-Pr
O
Si
N
O
ii
iii
S
S
S
v
i-Pr
i-Pr
i-Pr
O
O
O
Si O
i-Pr
Si O
i-Pr
R1
Si O
i-Pr
i-Pr
i-Pr
i-Pr
1
1
1
2
3: R1 = I
4: R1 = H
1
1
iv
O
N
O
O
O
N
NH
NH
NH
NH
R3
N
S
O
N
O
O
O
ix
xii
BzO
S
S
S
BzO
BzO
BzO
2
R O
4
R O
TBDMSO
TBDMSO
15: R2 = Ac, R3 = OAc
19: R4 = Ac
22a
22b
vi
x
6: R2 = H, R3 = OH
7: R2 = H, R3 = I
20: R4 = H
1
1
xi
vii
21: R4 = TBDMS
18: R2 = Ac, R3 = I
viii
Scheme 2. Synthesis of 4'-benzoyloxy-2'-deoxy-4'-thiouridine derivative 22a. Reagents and Coditions: (i) 1) uracil, BSA, CH
3
CN,
2
) NIS, CH Cl2 (57%);(ii) NIS, benzoic acid, CH CN (93%) ;(iii) 1)uracil, BSA, CH CN, 2) TMSOTf, CH Cl (87%), (iv) Bu SnH, Et B,
2
3
3
2
2
3
3
toluene (98%); (v) 1) Bu NF then Ac O (100%), (vi) NH /MeOH, (vii) ! , Ph P, pyridine, dioxane, (viii) Ac O, i-Pr NEt, DMAP, CH CN;
4
2
3
2
3
2
2
3
(
ix) 1) DBN, CH
3
CN (47% in 4 stepsfrom 15), (x) NH
3
/MeOH, (xi) TBDMSCl, imidazole, DMF (61% in 2 steps from 19); (xii) Pb(OBz)
4
,
toluene, 22a (63%) and 22b (28%)
O
N
O
N
NH
NH
R1
O
RO
O
i
ii
S
S
22a
R2
N
N
N
TBDMSO
TBDMSO
1
2
24: R = H
2
2
3a: R = CH OBz, R = N
iii
2
3
25: R = Ac (83% from 23a)
1
2
3b: R = N , R = CH OBz
3
2
Scheme 3. Synthesis of 4'-azido-2'-deoxy-4'-thiouridine derivative 25. Reagents and Coditions:
i) TMSN , SnCl , CH Cl , 23a (61%) and 23b (20%); (ii) NaOMe, MeOH; (iii) Ac O, i-Pr NEt,
(
3
4
2
2
2
2
DMAP, CH CN, (83% from 23a)
3
10

ACCEPTED MANUSCRIPT
H
Ac
N
R
N
N
N
i
AcO
N
O
iii
AcO
O
iv
S
S
25
7
N
N
N
N
N
N
TBDMSO
AcO
2
2
6: R = OTPS
7: R = NH2
2
8
Scheme 4. Synthesis of 4'-azido-2'-deoxy-4'-thiocytidine 7. Reagents and Coditions: (i) TPSCl, K CO , CH CN;
2
3
3
(
ii) NH OH, THF; (iii) 1) Bu NF, THF, 2) Ac O, i-Pr NEt, DMAP, CH CN (75% from 25); (iv) NaOMe, MeOH (88%)
4 4 2 2 3
We then turned our attention to the synthesis of 4’-branched
’-deoxy-4’-thiocytidines 8-10. Initially, preparation of the glycosyl donor 36 for
’-C-fluoromethyl congener 8 was carried out (Scheme 5). The aldehyde 29 obtained
2
4
2
9
from -ribose according to the published procedure was converted into 31 through 30
D
in three steps. Compound 31 was subjected to β-elimination by reacting with tert-butyl
lithium to give the 4-thiofuranoid glycal 32 in 89% isolated yield. Protection of the
hydroxyl groups of 32 with 1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS) group
(85% yield) followed by TsOH-mediated methanolysis (98% yield) of the
dimethoxytrityl ether 33 gave the 4-C-α-hydroxymethyl glycal 34. Reaction of 34 with
DAST in the presence of Na CO at −10 °C gave 4-C-fluoromethyl glycal 35 in 85%
2
3
yield. The desired glycosyl donor 36 could be obtained in 97% yield as a single
stereoisomer under the identical conditions as for the synthesis of 12. Next,
TMSOTf-mediated glycosidation between 36 and silylated uracil gave
2
’-deoxy-4’-thiouracil nucleoside 37 in 72% isolated yield. Tin-radical mediated
11

ACCEPTED MANUSCRIPT
reduction of 37 gave 38 (99% yield). Cytosine nucleoside 40 could be obtained from 38
through 39 as for the synthesis of 27. Transformation of 40 to the acetate 41 (59% yield
in 3 steps from 38) followed by removal of the protecting group of 41 (78% yield)
gave the target 4’-C-fluoromethyl-2’-deoxy-4’-thiocytidine 8.
TBSO
O
O
i
1
HO
v
R O
S
iv
S
S
H
2
DMTrO
R O
O
O
O
O
HO
32
30: R1 = TBSOCH , R = H
2
29
2
ii, iii
1: R1 = H, R = DMTr
2
3
O
O
O
I
O
vii
O
S
viii
S
S
Si
Si
Si
3
O
F
R O
F
O
O
O
O
O
Si
Si
Si
36
35
3
3: R3 = DMTr
4: R3 = H
vi
3
O
N
R4
NH
N
O
xi
ix
N
O
O
5
S
R O
Si
S
O
F
F
R O
X
O
5
Si
4
5
3
4
4
9: R = OTPS, R = TIPDS
xii
xiii
xiv
3
7: X = I
x
4
5
0: R = NH , R = TIPDS
2
38: X = H
4
5
1: R = NHAc, R = Ac
4
, R5 = H
8
: R = NH
2
Scheme 5. Synthesis of 4'-C-fluoromethyl-2'-deoxy-4'-thiocytidine 8. Reagents and Coditions: (i) 1) NaBH , MeOH (98%),
4
(
ii) DMTrCl, Et N, CH Cl , (iii) Bu NF, THF (95% in 2 steps); (iv) 1) tert-BuLi,THF, 2) AcOH (89%); (v) 1) TIPDSCl, imidazole,
3 2 2 4
DMF (85%), (vi) 1% TsOH/MeOH, CHCl (98%); (vii) DAST, NaHCO , CH Cl (85%); (viii) pivalic acid, NIS, CH CN-CH Cl
2
3
3
2
2
3
2
(97%); (ix) 1) uracil, BSA, CH CN, 2) TMSOTf (72%), (x) O , Bu SnH, Et B,toluene (99%); (xi) 1) TPSCl, K CO , CH CN,
3 2 3 3 2 3 3
(
xii) NH OH, THF, (xiii) Bu NF thef Ac O (59% in 3 steps), (xiv) NaOMe, MeOH (78%)
4
4
2
Finally, synthesis of 4’-C-ethynyl- (9) and 4’-C-cyano- (10) 2’-deoxy-4’-thiocytidine
was carried out (Schemes 6 and 7). 4-C-Acetoxymethyl glycal 42, which was obtained
from 34 in 96% yield, was used as starting material. According to the synthetic
12

ACCEPTED MANUSCRIPT
sequence for 8, 42 was converted to glycosyl donor 43 (95% yield) and the subsequent
glycosidation utilizing 43 provided 44 in 80% yield. Radical reduction of 44 gave 45
(99% yield) and subsequent treatment of 45 with methanolic ammonia furnished
4
’-C-hydroxymethyl derivative 46 in 81% yield. Oxidation of the primary alcohol of 46
with Dess-Martin periodinane provided the aldehyde 47 in 94% yield. Reaction of 47
with Ph P=CHBr at −40 °C gave dibromoolefine 48 which was subsequently treated
3
32
with butyl lithium to give 4’-C-ethynyl nucleoside 49 in 38% yield in two steps.
Compound 49 was converted to the cytosine derivative 52 as for the synthesis of 27.
Compound 52 was transformed into 4’-C-ethynyl-2’-deoxy-4’-thiocytidine 9 by
treatment with methanolic ammonia. All spectral data of 9 were consisted with that of
2
9
the previously synthesized compound.
13

ACCEPTED MANUSCRIPT
O
N
NH
O
O
O
ii
O
O
I
iii
O
i
S
S
S
Si
Si
34
Si
AcO
Si
O
AcO
Si
O
AcO
Si
O
X
O
O
O
42
43
4
4
4: X = I (80%)
5: X = H (99%)
iv
R1
N
O
N
NH
N
O
2
O
v
O
viii
R O
S
S
Si
R
C
C
H
O
2
O
R O
Si
4
5
9: R1 = OH, R2 = TIPDS (38% from 47)
0: R1 = OTPS, R2 = TIPDS
ix
x
xi
51: R1 = NH
52: R1 = NHAc, R2 = Ac (68% from 49)
9: R1 = NH , R2 = H ( (97%)
, R2 = TIPDS
vi
46: R = CH
2
OH (81%)
7: R = CHO (94%)
8: R = CH CBr
2
4
4
vii
xii
=
2
2
Scheme 6. Synthesis of 4'-C-ethynyl-2'-deoxy-4'-thiocytidine 9. Reagents and Coditions: (i) 1) Ac O, i-Pr NEt, DMAP, CH CN (98%),
2
2
3
(
ii) DNIS, benzoic acid, CH
NH -MeOH (81%); (vi) Dess-Martin periodinane, CH
ix) 1) TPSCl, K CO , CH CN, (x) NH OH, THF, (xi) Bu
3
CN (95%); (iii) 1) uracil, BSA, CH
3
CN, 2) TMSOTf, CH
2
−
Cl
2
(80%); (iv) Bu
3
SnH, Et
3
B, toluene (99%); (v)
+
3
2
Cl
2
(94%); (vii) BrCH
NF thef Ac
2
PPh
3
Br , KOtert-Bu, THF; (viii) BuLi, THF (38% from 47);
-MeOH (97%)
(
2
3
3
4
4
2
O (68% from 49), (xii) NH
3
For the synthesis of 10, the aldehyde 47 was converted to the oxime 53 by reacting
H NOH in pyridine and subsequent treatment of 53 with MsCl/Et N in CH Cl gave the
2
3
2
2
4
’-C-cyano nucleoside 54 in 77% yield in two steps (Figure 7). Compound 54 was
converted to cytosine nucleoside 57 in 91% yield in 3 steps (54→55→56→57). Finally,
the target 4’-C-cyano-2’-deoxy-4’-thiocytidine 10 could be obtained from 57 in 90%
yield by treatment with methanolic ammonia.
14

ACCEPTED MANUSCRIPT
R2
N
O
N
NH
O
N
O
3
i
R O
O
iii
S
S
Si
4
7
R1
C
N
O
3
O
R O
Si
2
3
5
5
5
1
5: R = OTPS, R = TIPDS
iv
2
3
6: R = NH , R = TIPDS
1
v
2
5
3: R = CH=NOH
2
3
7: R = NHAc, R = Ac (91% from 54)
ii
vi
1
3
4
5
4: R = C
≡N (77% from 47)
0: R = NH , R = H (90%)
2
Scheme 7. Synthesis of 4'-C-cyano-2'-deoxy-4'-thiocytidine 10. Reagents and Coditions: (i) 1) H NOH,
2
pyridine;(ii) MsCl, Et N, CH Cl (77% from 47); (iii) 1) TPSCl, K CO , CH CN, (iv) NH OH, THF, (v)
3
2
2
2
3
3
4
Bu NF thef Ac O (91% from 54), (vi) NH -MeOH (90%)
4
2
3
2
.2. Biological evaluation
The synthesized 4’-substituted 2’-deoxy-4’-thiocytidines (7-10) were evaluated for
their cytotoxic activities in two human cancer cell lines: B-cell leukemia (CCRF-SB)
and T-cell leukemia (Molt-4) cell lines employing MTT assay. The resulting cytotoxic
activity data of 7-10 as well as the positive control doxorubicin were presented in Table
1
. Although 2 has been reported to show high cytotoxicity against human T-cell
leukemia CCRF-CEM (IC50 3.5 µM for a mixture of β and α-anomer of 2), no
cytotoxicity was observed against both CCRF-SB and Molt-4. Similarly, 4’-ethynyl- (9)
and 4’-cyano- (10) 2’-deoxy-4’-thiocytidine did not show cytotoxic activity against both
the above cell lines. On the other hand, 4’-azido-(7) exhibited cytotoxicity (IC 7.14
5
0
µM for CCRF-SB and 2.72 µM for Molt-4). Most potent cytotoxicity was seen in the
15

ACCEPTED MANUSCRIPT
case of the 4’-fluoromethyl derivative (8); (IC 3.19 µM for CCRF-SB and 2.24 µM
5
0
for Molt-4) although the potency of 7 and 8 was one tenth (for CCRF-SB) and one
hundredth (for Molt-4) of that of reference doxorubicin.
Table 1. Antineoplastic activities of 2 and 7-10
a
IC50 (µM)
compound
b
c
CCRF-SB
Molt-4
7
.14
.19
2.72
2.24
7
8
9
0
2
3
> 100
> 100
>
>
100
100
0
100
1
> 100
.28
0.060
doxorubicin
a MTT assay
b human B-cell leukemia
c human T-cell leukemia
Next, antiviral activities of 7 - 10 were also evaluated against the following viruses:
human cytomegalovirus strains AD-169 and Davis, varicella-zoster virus (VZV) strain
-
OKA, VZV/TK strain 07-1, human cytomegalovirus AD-164 and Davis, herpes
-
simplex virus type-1 (HSV-1) strain KOS, thymidine kinase-deficient (TK ) HSV-1
KOS strain resistance to ACV, HSV-2 strain G. These assays were based on the
inhibition of virus-induced cytopathicity or plaque formation (for HCMV and VZV) in
human embryonic lung fibroblasts (HEL).
The parent compound 2 was found to be highly toxic to host HEL cell. 4’-ethynyl- (9)
16

ACCEPTED MANUSCRIPT
and 4’-cyano- (10) 2’-deoxy-4’-thiocytidines have not possessed remarkable antiviral
activities. On the other hand, promising antiviral results could be obtained in the case of
4
’-azido- (7) and 4’-fluoromethyl- (8) 2’-deoxy-4’-thiocytidine. By comparing the
activities of 7 with that of 8, 7 was found to show more potent inhibitory activities
against most of the above cell lines and less cytotoxicity to HEL host cell than that of 8.
+
It was noteworthy that antiviral activity of 7 against VZV (TK , OKA) (EC of 0.49
50
µM), which was ten times more potent than that (EC of 5.31 µM) of Acyclovir. Its
5
0
-
antiviral activity against VZV (TK , 07-1) was also potent (0.76 µM of EC ) while the
5
0
inhibitory activity of Acyclovir was less potent (53.51 µM of EC ). Furthermore, the
5
0
-
r
growth inhibitory activity (EC of 0.56 µM) of 7 for HSV-1 (TK KOS ACV ) was
5
0
found not to diminish its activity, by comparing to that (EC of 0.75 µM) of HSV-1
5
0
(
KOS) and the activity was three times more potent than that of Ganciclovir (EC of
50
1
.8 µM).
17

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Table 2. Antiviral activities of 7-10
a
Antiviral activities EC50 (µM)b
c
Antiviral activities EC50 (µM)
CytotoxicityCC50 (µM)
HCMV
HCMV
VZV
VZV
(TK , 07-1)
HSV-1
(KOS)
HSV-1
(TK KOS ACV )
HSV-2
(G)
cell growth
compound
+
-
-
r
(
AD-169 strain) (Davis strain) (TK , OKA)
7
8
9
7.64
28.1
6.34
0.43
0.76
0.75
0.55
0.70
>100
10.64
2.38
20.0
1.65
38.07
41.5
0.39
8.28
21.5
1.13
6.47
0.71
2.98
0.57
26.75
ND
>100
>100
10
>20
<0.42
3.31
>100
0.44
5.89
20.0
28.5
13.4
2
<0.42
0.42
ND
0.4
0.6
ND
1.8
ND
<0.16
>350
Gancclovir
Acyclovir
0.032
0.9
>
250
5.31
53.51
>250
a: Effective concentration required to reduce virus plaque formation by 50%. Vurus input was 100 plaque forming units(PFU).
b: Required to reduce virus-induced cytopathogenicity by 50%.
c: Cytotoxic concentration required to reduce human enbrryomic lung cell (HEL) growth by 50%.
ND: not determined
3
. Conclusions
In this study, 4’-azido- (7), 4’-C-fluoromethyl- (8) 4’-C-ethynyl- (9) and 4’-C-cyano-
10) 2’-deoxy-4’-thiocytidine have been synthesized. It was found that isolated yield of
’-thiouracil nucleoside 13 in Lewis acid-promoted Vorbrüggen-type glycosidation
(
4
utilizing 12 was better than that of electrophilic addition reaction between silylated
uracil and 11. This improved result prompted us to perform the glycosidation utilizing
3
6 and 43 for the synthesis 4’-thionucleosides 37 and 44. Introduction of the azido
group was carried out by nucleophilic substitution of 4’-benzoyloxy derivative 22a
according to the published protocol for the synthesis of 4’-azido-4’-thiothymidine. In
the cases of 9 and 10, chemical manipulation of the hydroxymethyl group of 46 was
utilized because the susceptibility of the ethynyl group for electrophilic NIS and the
18

ACCEPTED MANUSCRIPT
inertness of the reactivity of 4-C-cyano-4-thiofuranoid glycal exerted by
electron-withdrawing character of the cyano group. Therefore, an alternative route to 9
was provided in this study.
Evaluation of the antineoplastic activity of 2 and 7-10 against human B-cell
(
CCRF-SB) and T-cell leukemia (Molt-4) cell lines revealed that 4’-azido- (7) and
’-C-fluoromethyl- (8) derivatives exhibited cytotoxic activity whereas no cytotoxicity
was observed in 4’-C-ethynyl- (9) and 4’-C-cyano- (10) as well as the parent compound
’-deoxy-4’-thiocytidine (2). Compound 7 was also found to possess promising
4
2
antiviral activity against VZV and HSV-1 without any cytotoxity against HEL host cell.
It is noteworthy that 7 exhibited potent inhibitory activities against thymidine
-
kinase-deficient (TK ) mutant of VZV and HSV-1.
4
4
. Methods
.1. General Methods
1
13
Melting points are uncorrected. H and C NMR spectra were recorded either at 400
MHz or at 500 MHz. Chemical shifts are reported relative to Me Si. Mass spectra
4
(MS) were taken in FAB or ESI modex. Column chromatography was carried out on
silica gel. Thin-layer chromatography (TLC) was performed on silica gel. When
19

ACCEPTED MANUSCRIPT
necessary, analytical samples were purified by high performance liquid
chromatography (HPLC). THF was distilled from benzophenone ketyl.
4
1
.2.
-O-Pivaloyl-2-deoxy-2-iodo-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio
-β-D-ribofuranose (12)
To a solution of 11 (428.3 mg, 1.14 mmol) in CH CN (4.0 mL)/CH Cl (3.0 mL)
3
2
2
was added pivalic acid (582.1 mg, 5.70 mmol) and NIS (513.0 mg, 2.28 mmol) at 0
C under Ar atmosphere and the mixture was stirred for 6 h. The reaction mixture
was partitioned between CHCl /saturated NaHCO -0.2M Na S O and silica gel
°
3
3
2
2
3
column chromatography（hexane/ethyl acetate = 40/1）of the organic layer gave 12
1
(
637.4 mg, 93%) as a syrup; H NMR (CDCl ) δ 1.04-1.09 (28H, m, Si-i-Pr), 1.18
3
(
9H, s, C(CH ) ), 3.50-3.52 (1H, m, H-4), 3.61 (1H, dd, J = 4.6 and J3, 4 = 9.2 Hz,
3
3
2, 3
H-3), 3.91 (1H, dd, J4,5a = 2.9 and J5a,5b = 12.9 Hz, H-5a), 4.06 (1H, dd, J4,5b = 2.9
and J5a,5b = 12.9 Hz, H-5b), 4.57 (1H, d, J2, 3 = 4.6 Hz, H-2), 6.03 (1H, s, H-1); NOE
’
13
experiment: H-1/H-4, H-2/H-5b ; C NMR (CDCl ) δ: 12.8, 12.8, 13.2, 13.3, 17.1,
3
1
7.2, 17.3, 17.3, 17.4, 17.4, 17.5, 26.8, 38.6, 38.8, 52.7, 59.4, 83.6, 176.6.
＋
+
FAB-MS(m/z): 1027(M ＋H); ESI-MS (m/z) 625 (M + Na); ESI-HRMS (m/z):
＋
calcd for C H O INaSSi : 625.13066, found: 625.13083 (M ＋Na).
2
2
43
5
2
20

ACCEPTED MANUSCRIPT
4
1
.3.
-[2-Deoxy-2-iodo-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-
D
-ribof
uranosyl]uracil (13)
To a suspension of uracil (64.4 mg, 0.57 mmol) in CH CN (2.5 mL) was added
3
BSA (0.28 mL, 1.14 mmol) at rt under Ar atmosphere and the mixture was stirred
for 1 h. To the clear solution was added a solution of 12 (229.0 mg, 0.38 mmol) in
CH CN (4.0 mL)/CH Cl (2.0 mL) and TMSOTf (0.27 mL, 1.52 mmol) at 0 °C
3
2
2
under Ar atmosphere and the mixture was stirred 7 h. The reaction mixture was
partitioned between CHCl /saturated NaHCO -0.2M Na S O and silica gel column
3
3
2
2
3
chromatograph y（ hexane/ethyl acetate = 2/1）of the organic layer gave 13 (202.9 mg,
1
8
7%) as a foam; H NMR (CDCl ) δ 0.87-0.97 and 1.06-1.14 (28H, each as m,
3
Si-i-Pr), 3.33 (1H, dd, J2’, 3’ = 4.6 and J3’, 4’ = 9.2 Hz, H-3’), 3.63-3.65 (1H, m, H-4’),
.02 (1H, d, J5’a,5’b = 12.9 Hz, H-5’a), 4.13 (1H, dd, J4’,5’b = 3.4 and J5’a,5’b = 12.9 Hz,
4
H-5’b), 4.44 (1H, d, J2’, 3’ = 4.6 Hz, H-2’), 5.70 (1H, dd, J5, NH = 2.3 and J5, 6 = 8.6 Hz,
H-5), 6.03 (1H, s, H-1’), 8.44 (1H, dd, J5, 6 = 8.6 Hz, H-6), 8.50 (1H, br, NH); NOE
1
3
experiment: H-6/H-3’, H-6/H-5’b, H-2’/H-5’a, H-2’/H-5’b; C NMR (CDCl ) δ:
3
1
1
2.5, 13.1, 13.3, 13.5, 16.9, 16.9, 17.1, 17.2, 17.4, 17.5, 38.2, 53.5, 58.0, 67.5, 71.0,
+
01.6, 141.2, 150.6, 163.5. ESI-MS (m/z) 635 (M + Na); ESI-HRMS (m/z): calcd
21

ACCEPTED MANUSCRIPT
＋
for C H O N INaSSi : 635.08986, found: 635.09042 (M ＋Na).
2
1
37
5
2
2
4
1
.4.
-[2-Deoxy-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-
D
-ribofuranos
yl]uracil (14)
To a solution of 13 (229.0 mg, 0.38 mmol) in toluene (8.0 mL) was added Bu SnH
3
(
0.25 mL, 0.92 mmol) and Et B (0.23 mL, 0.23 mmol) at −60 °C under Ar atmosphere
3
and the mixture was stirred under O atmosphere overnight. The reaction mixture was
2
purified by silica gel column chromatography（hexane/ethyl acetate = 2/1）on silica gel
1
to give 14 (219.9 mg, 98%) as a foam; H NMR (CDCl ) δ 0.92-1.14 (28H, each as m,
3
Si-i-Pr), 2.26 (1H, dd, J2’a, 3’ = 5.2 and J2’a, 2’b = 13.5 Hz, H-2’a), 2.46-2.58 (1H, ddd, J1’,
=
6.9, J2’b, 3’ = 14.6 and J2’a, 2’b = 13.5 Hz, H-2’b), 3.32-3.34 (1H, m, H-4’), 3.97 (1H, d,
J5’a,5’b = 12.6 Hz, H-5’a), 4.13 (1H, dd, J4’,5’b = 3.4 and J5’a,5’b = 12.6 Hz, H-5’b), 4.40
1H, ddd, J2’a, 3’ = 5.2, J2’b, 3’ = 14.6 and J3’, 4’ = 8.0 Hz, H-3’), 5.70 (1H, dd, J5, NH = 2.3
and J5, 6 = 8.0 Hz, H-5), 5.99 (1H, d, J1’,2’b = 6.9 Hz, H-1’), 8.31 (1H, d, J = 8.0 Hz,
2
’b
(
5,6
1
3
H-6), 8.57 (1H, br, NH); C NMR (CDCl ) δ: 12.4, 13.1, 13.2, 16.9, 16.96, 17.01, 17.3,
3
1
7.4, 17.5, 43.4, 54.2, 58.06, 58.11, 70.2, 101.6, 141.5, 150.8, 163.5. FAB-MS (m/z) 509
+
(
M + Na); ESI-HRMS (m/z): calcd for C H O N NaSSi : 509.19322, found:
2
1
38
5
2
2
＋
5
09.19326 (M ＋Na).
22

ACCEPTED MANUSCRIPT
4
.5. 1-[3,5-Di-O-acetyl-2-deoxy-4-thio-β-
D
-ribofuranosyl]uracil (15)
To a solution of 14 (287.1 mg, 0.59 mmol) in THF (5.0 mL) was added Bu NF (1M
4
THF solution) (1.30 mL, 1.30 mmol) at 0 °C under Ar atmosphere and the mixture was
stirred under Ar atmosphere 2 h. To the reaction mixture was added Ac O (0.22 mL,
2
2
.36 mL) at 0 °C under Ar atmosphere and the reaction mixture was stirred overnight.
The reaction mixture was partitioned between CHCl /saturated NaHCO and silica gel
3
3
column chromatograph y（ 3% MeOH in CH Cl ）of the organic layer gave 15 (201.5 mg,
2
2
1
1
00%) as foam; H NMR (CDCl ) δ 2.13 and 2.14 (6H, each as s, Ac), 2.21 (1H, ddd,
3
J1’, 2’a = 4.0, J2’a, 3’ = 9.2 and J2’a, 2’b = 10.3 Hz, H-2’a), 2.59 (1H, ddd, J1’, 2’b = 2.9, J2’b, 3’
=
=
6
1
.3 and J2’a, 2’b = 10.3 Hz, H-2’b), 3.72 (1H, ddd, J3’, 4’ = 2.3, J4’, 5’a = 6.3 and J4’, 5’b
0.3 Hz, H-4’), 4.21 (1H, dd, J4’, 5’a = 7.5 and J5’a,5’b = 11.7 Hz, H-5’a), 4.34 (1H, dd,
J4’,5’b = 5.8 and J5’a,5’b = 11.7 Hz, H-5’b), 5.38-5.40 (1H, m, H-3’), 5.87 (1H, d, J5, 6 = 8.0
Hz, H-5), 6.54 (1H, d, J1’,2’a = 4.0 and J1’,2’b = 2.9 Hz, H-1’), 7.81 (1H, d, J = 8.0 Hz,
5,6
1
3
H-6), 9.49 (1H, br, NH); C NMR (CDCl ) δ: 20.8, 21.0, 39.7, 52.5, 61.4, 64.9, 76.4,
3
+
1
03.7, 140.0, 150.7, 162.8, 170.1, 170.5; ESI-MS (m/z) 351 (M + Na); ESI-HRMS
＋
(
m/z): calcd for C H O N NaS: 351.06213, found: 351.06268 (M ＋Na).
13 16 6 2
4
.6. 1-[3-O-Acetyl-2,5-dideoxy-4-thio-β-D-glycero-4-eno-β-D-ribofuranosyl]uracil
(19)
23

ACCEPTED MANUSCRIPT
Compound 15 (198.5 mg, 0.60 mmol) was treated with methanolic ammonia (10 mL)
at rt overnight. The reaction mixture was evaporated to dryness and dried in vacuo
overnight. To a solution of 16 in dioxane (8.0 mL) was added pyridine (0.12 mL, 1.50
mmol), Ph P (314.7 mg, 1.20 mmol) and I (304.6 mg, 1.20 mmol) at rt under Ar
3
2
atmosphere and the reaction mixture was stirred for 4 h. The reaction mixture was
quenched with MeOH and silica gel column chromatography（3% MeOH in CH Cl ）
2
2
of the reaction mixture gave 17 (209.1 mg, 98%) as a solid. To a solution of 17 (209.1
mg, 0.61 mmol) in CH CN (5.0 mL) was added i-Pr NEt (0.53 mL, 3.05 mmol), Ac O
3
2
2
(0.17 mL, 1.83 mmol) and DMAP (37.9 mg, 0.31 mmol) at rt under Ar atmosphere and
the reaction mixture was stirred for 5 h. The reaction mixture was partitioned between
CHCl /saturated NaHCO and silica gel column chromatography（hexane/ethyl acetate
3
3
=
1/2）of the organic layer gave 18 (178.9 mg, 74%) as a foam. To a solution of 18 in
CH CN (4.0 mL) was added DBN (0.23 mL, 1.83 mmol) at rt under Ar atmosphere and
3
the reaction mixture was stirred overnight. The reaction mixture was neutralized with
AcOH, partitioned between CHCl /saturated NaHCO and silica gel column
3
3
chromatography（hexane/ethyl acetate = 1/2）of the organic layer gave 19 (101.0 mg,
1
6
5% from) as a foam; H NMR (CDCl ) δ 2.13 (3H, s, Ac), 2.21 (1H, ddd, J
= 8.4,
3
1’, 2’a
J2
’a, 3’
= 5.2 and J2’a, 2’b = 14.0 Hz, H-2’a), 2.59 (1H, ddd, J1’, 2’b = 6.3, J2’b, 3’= 3.4 and J2’a,
24

ACCEPTED MANUSCRIPT
=
’b
14.0 Hz, H-2’a), 5.26 (1H, s, H-5’a), 5.51 (1H, s, H-5’b), 5.84 (1H, dd, J2’a, 3’= 5.2
2
and J2’b, 3’ = 3.4 Hz, H-3’), 5.87 (1H, d, J5, 6 = 8.0 Hz, H-5), 6.73 (1H, dd, J1’,2’a = 8.4 and
1
3
J1’,2’b = 6.3 Hz, H-1’), 7.59 (1H, d, J = 8.0 Hz, H-6), 9.73 (1H, br, NH); C NMR
5,6
(
CDCl ) δ: 21.4, 41.4, 61.9, 77.1, 104.2, 110.3, 140.1, 144.6, 151.0, 163.5, 170.3.
3
＋
FAB-MS(m/z): 291 (M ＋Na); ESI-HRMS (m/z): calcd for C H O N NaS: 291.04100,
11
12
4
2
＋
found: 291.04126 (M ＋Na).
4
1
.7.
-[3-O-(tert-Butyldimethylsilyl)-2,5-dideoxy-4-thio-β-D-glycero-4-eno-β-D-ribofura
nosyl]uracil (21)
Compound 19 (259.3 mg, 1.00 mmol) was treated with methanolic ammonia (25 mL)
at rt for 4 h. The reaction mixture was evaporated to dryness and dried in vacuo
overnight. To a solution of 20 in DMF (15.0 mL) was added imidazole (340.4 mg, 5.0
mmol) and TBDMSCl (452.1 mg, 3.0 mmol) at rt under Ar atmosphere and the reaction
mixture was stirred overnight. The reaction mixture was partitioned between ethyl
acetate/H O and silica gel column chromatography（hexane/ethyl acetate = 2/1）of the
2
1
organic layer gave 21 (208.7 mg, 61%) as a foam.; H NMR (CDCl ) δ 0.11 and 0.12
3
(
6H, each as s, Si-Me), 0.91 (9H, s, Si-tert-Bu), 2.06-2.08 (1H, m, H-2’a), 2.39-2.44
1H, m, H-2’b), 4.74 (1H, t, J2’a, 3’ = J2’b, 3’ = 5.2 Hz, H-3’), 5.12 (1H, s, H-5’a), 5.32 (1H,
(
25

ACCEPTED MANUSCRIPT
s, H-5’b), 5.82 (1H, d, J5, 6 = 8.0 Hz, H-5), 6.57 (1H, dd, J1’,2’a = J1’,2’b = 6.9 Hz, H-1’),
13
7
.63 (1H, d, J = 8.0 Hz, H-6), 9.42 (1H, br, NH); C NMR (CDCl ) δ : −4.73, −4.65,
5,6 3
1
8.1, 25.7, 29.7, 44.4, 61.4, 76.3, 103.2, 106.4, 140.3, 148.7, 150.3, 162.5.
＋
+
FAB-MS(m/z): 1027(M ＋H); ESI-MS (m/z) 363 (M + Na); ESI-HRMS (m/z): calcd
＋
for C H O N NaSSi: 363.11691, found: 363.11719 (M ＋Na).
1
5
24
3
2
4
.8. Dibenzoyoxylation of 21 with Pb(OBz) : Formation of 22a and 22b
4
To a solution of 21 (146.8 mg, 0.43 mmol) in toluene (7.0 mL) was added Pb(OBz)4
(1.34 g, 1.94 mmol) at rt under Ar atmosphere and the mixture was stirred overnight.
The reaction mixture was filtered through a celite pad. The filtrate was neutralized with
Et N and partitioned between CHCl /saturated aq NaHCO . Column chromatography
3
3
3
(hexane/ethyl acetate = 2/1-1/1) of the organic layer gave 22a (158.3 mg, 63%, foam,)
and 22b (71.4 mg, 28%, foam).
1
4
.8.1. Physical data of 22a: H NMR (CDCl ) δ 0.06 and 0.18 (6H, each as s), 0.94 (9H,
3
s, Si-tert-Bu), 2.34 (1H, ddd, J1’, 2’a = 10.0, J2’a, 3’ = 3.4 and J2’a, 2’b = 12.9 Hz, H-2’a),
2
1
6
7
.57-2.63 (1H, m, H-2’b), 4.87 (1H, t, J2’a,3’ = J2’b,3’ = 3.4 Hz, H-3’), 4.90 (1H, d, J5’a,5’b =
2.0 Hz, H-5’a), 5.34 (1H, d, J5’a,5’b = 12.0 Hz, H-5’b), 5.66 (1H, d, J5,6 = 8.0 Hz, H-5),
.73 (1H, dd, J1’,2’a = 10.0 and J1’,2’b = 5.7 Hz, H-1’), 7.35-7.38, 7.47-7.52, 7.58-7.66,
.89-7.94 and 8.02-8.06 (11H, each as m, Ph and H-6), 9.18 (1H, br, NH); NOE
26

ACCEPTED MANUSCRIPT
experiment: : H-2’b (α-H)/CH -5’a, Si-Me/CH -5’a and Si-tert-Bu/CH -5’a; HMBC:
2
2
2
13
CH -5’/CH CO-5’ and CH -5’/CH CO-5’; C NMR (CDCl ) δ: −5.1, −4.5, 17.9, 42.2,
2
a
3
2b
3
3
6
1.6, 62.1, 101.1, 103.7, 128.4, 128.7, 129.5, 129.6, 133.2, 133.8, 140.1, 149.9, 150.6,
+
1
63.2, 164.5, 165.4; ESI-MS (m/z) 605 (M + Na); ESI-HRMS (m/z): calcd for
＋
C H O N NaSSi : 605.17482, found: 605.17505 (M ＋Na).
29
34
7
2
2
1
4
.8.2. Physical data of 22b: H NMR (CDCl ) δ −0.08 and 0.14 (6H, each as s, Si-Me),
3
0
.88 (9H, s, Si-tert-Bu), 2.30 (1H, ddd, J1’,2’a = 2.9, J2’a,3’ = 4.6 and J2’a, 2’b = 13.5 Hz,
H-2’a), 2.63 (1H, ddd, J1’,2’b = 12.3, J2’b,3’ = 1.7 and J2’a, 2’b = 13.5 Hz, H-2’b), 3.93 (1H,
d, J5’a,5’b = 13.7 Hz, H-5’a), 4.09 (1H, dd, J3’,5’b = 1.7 and J5’a,5’b = 13.7 Hz, H-5’b),
5
.38-5.39 (1H, m, H-3’), 5.74 (1H, d, J5,6 = 8.0 Hz, H-5), 6.33 (1H, dd, J1’,2’a = 2.9 and
J1’,2’b = 12.3 Hz, H-1’), 7.33 (1H, d, J5,6 = 8.0 Hz, H-6), 7.41-7.45, 7.49-7.51, 7.56-7.57,
1
3
7
.62-7.64, 8.00-8.01 and 8.10-8.11 (10H, each as m, Ph), 8.92 (1H, br, NH); C NMR
(
CDCl ) δ: −5.1, −4.4, 17.8, 25.7, 29.5, 39.1, 48.7, 68.5, 100.9, 103.4, 128.4, 128.7,
3
1
30.0, 133.6, 133.8, 140.3, 149.9, 163.2, 163.5, 164.1; HMBC: C-1’/H-5’a and
+
C-1’/H-5’b. FAB-MS (m/z) 605 (M + Na); ESI-HRMS (m/z): calcd for
＋
C H O N NaSSi: 605.17482, found: 605.17481 (M ＋Na).
29
34
7
2
4
5
.9. Reaction of 22a with azidotrimethylsilane: formation of 1-[4-Azido-
-O-benzoyl-3-O-(tert-butyldimethylsilyl)-2-deoxy-4-thio-β- -ribofuranosyl]uracil
D
27

ACCEPTED MANUSCRIPT
(23a)
and
1-[4-Azido-
5
-O-benzoyl-3-O-(tert-butyldimethylsilyl)-2-deoxy-4-thio-α-
L
-ribofuranosyl]uracil
(
23b)
To a solution of 22a (40.5 mg, 0.069 mmol) in dichloromethane (2.5 mL) was added
azidotrimethylsilane (46.5 µL, 0.35 mmol) and SnCl (1 M dichloromethane solution)
4
(0.21 mL, 0.21 mmol) at −50 °C under Ar atmosphere and the mixture was stirred at
−
30°C for overnight. The reaction mixture was partitioned between CHCl and saturated
3
NaHCO and preparative TLC (hexane/ethyl acetate = 2/1) of the organic layer gave
3
2
4
0
2
1
3a (21.3 mg, 61%, syrup) and 23b (10.0 mg, 20%, syrup).
-1
1
.9.1. Physical data for 23a: IR (neat) 2113 cm (N ); H NMR (CDCl ) δ 0.13 and
3
3
.16 (6H, each as s, Si-Me), 0.94 (9H, s, Si-tert-Bu), 2.35 (1H, ddd, J1’,2’a = 9.7, J2’a,3’
.9 and J2’a,2’b = 13.5 Hz, H-2’a), 2.51 (1H, ddd, J1’,2’b = 6.3, J2’b,3’ = 1.1 and J2’a,2’b
=
=
3.5 Hz, H-2’b), 4.27 (1H, br, H-3’), 4.67 (1H, d, J5’a,5’b = 11.5 Hz, H-5’a), 4.86 (1H, d,
J5’a,5’b = 11.5 Hz, H-5’b), 5.90 (1H, d, J5,6 = 8.6 Hz, H-5), 6.78 (1H, dd, J1’,2’a = 9.7 and
J1’,2’b = 6.3 Hz, H-1’), 7.46-7.49, 7.59-7.64 and 8.03-8.05 (5H, each as s, Ph) 7.63 (1H,
1
3
d, J5,6 = 8.6 Hz, H-6), 8.57 (1H, br, NH); C NMR (CDCl ) δ −4.9, −4.8,� 18.1, 25.7,
3
4
1
2.6, 59.8, 67.1, 77.4, 84.0, 103.5, 128.6, 129.0, 129.8, 133.8, 140.1, 150.3, 162.7,
+
65.9; FAB-MS (m/z) 526 (M + Na); ESI-HRMS (m/z): calcd for C H O N NaSSi:
2
2
29
5
5
28

ACCEPTED MANUSCRIPT
＋
5
26.15509, found: 526.15570 (M ＋Na).
-1
1
1
4
.9.2. Physical data for 23b: IR (neat) 2120 cm (N ); H NMR (CDCl ) δ H NMR
3 3
(
CDCl ) δ 0.15 and 0.19 (6H, each as s, Si-Me), 0.95 (9H, s, Si-tert-Bu), 2.27 (1H, ddd,
3
J1’,2’a = 7.4, J2’a,3’ = 4.0 and J2’a,2’b = 13.5 Hz, H-2’a), 2.59 (1H, ddd, J1’,2’b = 6.3, J2’b,3’
=
5
.2 and J2’a,2’b = 13.5 Hz, H-2’b), 4.52 (1H, t, J2’a,3’ = J2’b,3’ = 4.6 Hz, H-3’), 4.54 (1H, d,
J5’a,5’b = 11.5 Hz, H-5’a), 4.58 (1H, d, J5’a,5’b = 11.5 Hz, H-5’b), 5.68 (1H, d, J5,6 = 8.0
Hz, H-5), 6.60 (1H, dd, J1’,2’a = 7.4 and J1’,2’b = 6.3 Hz, H-1’), 7.48-7.52, 7.62-7.65 and
1
3
8
.05-8.07 (5H, each as s, Ph) 7.64 (1H, d, J5,6 = 8.0 Hz, H-6), 8.54 (1H, br, NH); C
NMR (CDCl ) δ −5.0, −4.5, 17.9, 25.6, 42.8, 61.9, 67.7, 78.6, 87.9, 104.2, 128.6, 129.1,
3
+
1
29.9, 133.6, 140.0, 150.3, 162.3, 165.7; FAB-MS (m/z) 526 (M + Na); ESI-HRMS
＋
(
m/z): calcd for C H O N NaSSi: 526.15509, found: 526.15588 (M ＋Na).
22 29 5 5
4
.10.
-O-(tert-butyldimethylsilyl)-2-deoxy-4-thio-β-
To a solution of 23a (60.1 mg, 0.12 mmol) in MeOH (3.5 mL) was added NaOMe
9.7 mg, 0.18 mmol) at 0 °C under Ar atmosphere and the mixture was stirred at rt
overnight. Silica gel column chromatography (2% MeOH in CH Cl ) of the reaction
1-[5-O-Acetyl-4-azido-
3
D
-ribofuranosyl]uracil (25)
(
2
2
mixture gave 24 (42.1 mg, 88%, syrup). To a solution of 24 (42.1 mg, 0.11 mmol) in
CH CN (3.0 mL) was added i-Pr NEt (63 µL, 0.36 mmol), Ac O (23 mL, 0.24 mmol)
3
2
2
29