Nucleosides and nucleotides, 175. Structural requirements of the sugar moiety for the antitumor activities of new nucleoside antimetabolites, 1-(3- C-ethynyl-β-d-ribo-pentofuranosyl)cytosine and -uracil

Article abstract of DOI:10.1021/jm9801814

We previously designed 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)uracil (EUrd) and its cytosine congener (ECyd) as potential multifunctional antitumor nucleoside antimetabolites. They showed potent and broad-spectrum antitumor activity against various human

Full text of DOI:10.1021/jm9801814

2892
J . Med. Chem. 1998, 41, 2892-2902
Nu cleosid es a n d Nu cleotid es. 175. Str u ctu r a l Requ ir em en ts of th e Su ga r
Moiety for th e An titu m or Activities of New Nu cleosid e An tim eta bolites,
1-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)cytosin e a n d -u r a cil1
Hideshi Hattori,† Eisuke Nozawa,† Tomoharu Iino,† Yuichi Yoshimura,† Satoshi Shuto,† Yuji Shimamoto,‡
Makoto Nomura,†,‡ Masakazu Fukushima,‡ Motohiro Tanaka,§ Takuma Sasaki,§ and Akira Matsuda*,†
Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, J apan,
Cancer Research Laboratory-2, Hanno Research Center, Taiho Pharmaceutical Company Ltd., Hanno 357-8527, J apan, and
Cancer Research Institute, Kanazawa University, Takara-machi, Kanazawa 920-0934, J apan
Received March 23, 1998
We previously designed 1-(3-C-ethynyl-â-D-ribo-pentofuranosyl)uracil (EUrd) and its cytosine
congener (ECyd) as potential multifunctional antitumor nucleoside antimetabolites. They
showed potent and broad-spectrum antitumor activity against various human and mouse tumor
cells in vitro and in vivo. To clarify the structure-activity relationship of the sugar moiety,
various 3′-C-carbon-substituted analogues, such as 1-propynyl, 1-butynyl, ethenyl, ethyl, and
cyclopropyl derivatives, of ECyd and EUrd were synthesized. We also prepared 3′-deoxy
analogues and 3′-homologues of ECyd and EUrd with different configurations to determine
the role of the 3′-hydroxyl group and the length between the 3′-carbon atom and the ethynyl
group and a 2′-ethynyl derivative of ECyd to determine the spatial requirements of the ethynyl
group. The in vitro tumor cell growth inhibitory activities of these nucleosides against mouse
leukemic L1210 and human KB cells showed that ECyd and EUrd were the most potent
inhibitors in the series, with IC50 values of 0.016 and 0.13 µM for L1210 cells and 0.028 and
0.029 µM for KB cells, respectively. Only 3′-C-1-propynyl and -ethenyl derivatives of ECyd
showed greatly reduced cytotoxicity. We found that the cytotoxic activity of these nucleosides
predominantly depended on their first phosphorylation by uridine/cytidine kinase.
In tr od u ction
12 h after treatment, ECyd induces apoptotic cell death
in human gastric cancer MKN 45 cells with the wild-
type p53 gene.4
The development of nucleoside antimetabolites is
important for progress in anticancer chemotherapy. We
recently designed 1-(3-C-ethynyl-â-D-ribo-pentofurano-
syl)uracil (EUrd) as a potential multifunctional antitu-
mor antimetabolite which was expected to inhibit the
synthesis of both DNA and RNA in tumor cells.2 EUrd
shows potent tumor cell growth inhibitory activity
against a variety of human tumor cell lines in vitro and
strong antitumor activity against not only murine
leukemia P388 but also a variety of human solid tumor
xenografts in vivo. To clarify the structure-activity
relationship of the nucleobase moiety, we synthesized
various analogues and found that 1-(3-C-ethynyl-â-D-
ribo-pentofuranosyl)cytosine (ECyd) showed more po-
tent activity against human tumor cell lines in vitro
than EUrd, and lower doses were required to produce
maximum antitumor effects against human tumor xe-
nografts.3,4 Since the in vitro cytotoxicity of ECyd is
significantly reduced in the presence of cytidine or
uridine, ECyd may have to be phosphorylated by uri-
dine/cytidine kinase (UCK) to show its cytotoxicity.4 The
substrate function of ECyd for human cytidine deami-
nase is extremely low.4 ECyd has been shown to
strongly inhibit RNA synthesis by inhibiting RNA
polymerases5 and to slightly inhibit DNA synthesis.4 At
To clarify further structural requirements for the
antitumor activity of ECyd and EUrd, we describe here
the synthesis and in vitro cytotoxicity of various 3′-C-
carbon-substituted analogues, such as 1-propynyl, 1-bu-
tynyl, ethenyl, ethyl, and cyclopropyl derivatives, of
ECyd and EUrd. We also prepared 3′-deoxy analogues
and 3′-homologues of ECyd and EUrd with different
configurations to determine the role of the 3′-hydroxyl
group and the required length between the 3′-carbon
atom and the ethynyl group. To determine the spatial
requirements of the ethynyl group, the 2′-ethynyl
derivative of ECyd was also synthesized. Moreover, the
phosphorylation of selected nucleosides by partially
purified uridine/cytidine kinase from mouse Sarcoma-
180 ascites cells and its relationship to cytotoxicity are
also described.
Ch em istr y
The synthesis of 3-C-propynyl- and -butynyl-â-D-ribo-
pentofuranose derivatives is illustrated in Scheme 1.
The 3-ulose derivative 1 was easily obtained from
D-xylose in four steps in good yield.6 Addition of
LiCtCCH3 (or LiCtCCH2CH3) to 1 gave the desired
â-adduct 2a (or 2b) with high stereoselectivity.3,7 Desi-
lylation of 2a ,b with tetrabutylammonium fluoride
(TBAF) followed by benzoylation of the hydroxyl group
at the 5-position yielded 3a ,b, which were treated with
20% HCl in aqueous MeOH at room temperature to give
* To whom all correspondence and reprint requests should be
addressed. Phone: +81-11-706-3228. Fax: +81-11-706-4980. E-mail:
matuda@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ Taiho Pharmaceutical Co. Ltd.
§ Kanazawa University.
S0022-2623(98)00181-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/18/1998

ECyd and EUrd Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2893
Sch em e 1a
Sch em e 3a
a
(a) LiCtCMe or LiCtCEt, THF; (b) TBAF, THF; (c) BzCl,
pyridine; (d) HCl/aq MeOH; (e) BzCl, DMAP, pyridine; (f) cH2SO4,
AcOH, Ac2O.
Sch em e 2a
a
(a) Persilylated cytosine, SnCl4, MeCN; (b) NH3/MeOH; (c)
persilylated uracil, TMSOTf, MeCN; (d) TPSCl, DMAP, then
NH4OH.
furanose (13c). Since the 3-tertiary alcohol was hardly
benzoylated, 10 equiv of BzCl and 100 °C were required
to complete the reaction. The 3-C-cyclopropyl derivative
13e was prepared in a similar manner.
Reaction of 1 with ethylmagnesium bromide, however,
gave the desired 3-C-ethyl derivative together with
considerable amounts of a reduced secondary alcohol
(data not shown).8 Therefore, the 3-C-ethyl derivative
was prepared from 10.3 Compound 10 was first desi-
lylated with TBAF to give 11, which was hydrogenated
and then benzoylated to give 12. Hydrolysis of the 1,2-
O-isopropylidene group, as described above, gave 9d ,
which was subsequently benzoylated to furnish 13d .
a
(a) H2CdCHMgBr or cyclopropyllithium, THF; (b) TBAF, THF;
(c) BzCl, pyridine; (d) aq HCl, THF; (e) BzCl, DMAP, pyridine; (f)
ref 3; (g) H2, Pd-C, MeOH.
methyl 5-O-benzoyl-3-C-propynyl(and butynyl)-R,â-ribo-
pentofuranosides (4a ,b). Compounds 4a ,b were subse-
quently benzoylated with a mixture of BzCl and DMAP
in anhydrous pyridine at 100 °C to give 5a ,b. Subse-
quent acetolysis of 5a ,b using concentrated H2SO4 in a
mixture of AcOH and Ac2O gave 1-O-acetyl-2,3,5-tri-O-
benzoyl-3-C-propynyl(and butynyl)-R,â-ribo-pentofura-
noses (6a ,b), which were used for condensation with
persilylated uracil and cytosine.
Although 3′-C-ethenyl and -ethyl derivatives can be
prepared from ECyd and EUrd by partial or complete
reduction of the ethynyl group by hydrogenolysis, the
products and the starting material could not be ad-
equately separated by any chromatography conditions
we tested, even using HPLC, since these compounds had
very similar retention times. Since ECyd and EUrd are
highly potent tumor cell growth inhibitors, contamina-
tion by these nucleosides should be avoided when 3′-C-
ethenyl and -ethyl derivatives are tested against tumor
cells. Therefore, 3-C-ethenyl and -ethyl derivatives were
synthesized using a condensation method similar to that
described above. Reaction of 1 with vinylmagnesium
bromide in THF at -78 °C gave the desired â-adduct
7c, whose 5-O-TBS group was then deprotected by
TBAF (Scheme 2). Subsequent benzoylation gave 8c.
The isopropylidene group of 8c was removed by hy-
drolysis using 1.5% HCl in aqueous THF at reflux
temperature to yield 9c. The remaining hydroxyl
groups of 9c were benzoylated with BzCl in pyridine to
give 1,2,3,5-tetra-O-benzoyl-3-C-ethenyl-R,â-D-ribo-pento-
Condensation of 6a ,b with persilylated cytosine was
carried out in the presence of SnCl4 as a Lewis acid in
MeCN at room temperature to give the corresponding
cytosine nucleosides 14a ,b in good yields (Scheme 3).
More time was needed for the condensation with bulkier
terminal substituents at the ethynyl group. For the
condensation of persilylated uracil with 6a ,b, trimeth-
ylsilyl triflate (TMSOTf) was used instead of SnCl4 as
a Lewis acid to avoid the formation of undesired N3-
isomer3 and gave predominantly the desired N1-nucleo-
sides 16a ,b in good yields. For the synthesis of the 3′-
C-ethenyl, -ethyl, and -cyclopropyl nucleosides, the
corresponding sugar units 13c-e were reacted with
persilylated uracil in the presence of TMSOTf in MeCN
to give 16c-e, which were then transformed into the
corresponding cytosine nucleosides 14c-e by the usual
procedure. Treatment of these protected nucleosides
14a -e and 16a -e with NH3/MeOH gave the desired
nucleosides 15a -e and 17a -e, respectively, in good
yields.
To further study the structure-activity relationship
of ECyd and EUrd, we synthesized 1-(3-C-ethynyl-â-D-
xylo-pentofuranosyl)cytosine (31) and -uracil (30) (Scheme
4), which are the 3′-epimers of ECyd and EUrd. Reac-
tion of 2′,5′-di-O-TBS-3′-ketouridine derivative 26 with
HCtCMgBr in THF at -78 °C gave xylo-adduct 28 with
high stereoselectivity.9-11 In a similar manner, N4-
acetyl-3′-ketocytidine derivative 27 was converted into

2894 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Hattori et al.
Sch em e 4a
Sch em e 6a
a
(a) HCtCMgBr, THF; (b) NH4F, MeOH; (c) HCl/MeOH.
Sch em e 5a
a
(a) (i) Bz2O, DMAP, MeCN, (ii) HCl/MeOH; (b) 1,1′-thiocar-
bonyldiimidazole, DMAP, MeCN, CH2Cl2; (c) Bu3SnH, AIBN,
toluene; (d) TBSCl, imidazole, DMF; (e) NaOMe/MeOH, then HCl/
MeOH; (f) (i) TPSCl, DMAP, Et3N, MeCN, (ii) NH4OH.
To determine whether the 3′-OH in ECyd and EUrd
is required for their cytotoxicity, we synthesized their
3′-deoxy analogues, 47, 48, and 50, as illustrated in
Scheme 6. The starting material 40 was synthesized
as described previously.10b Selective benzoylation of the
5′-hydroxyl group of 40 gave 41, and subsequent acid
hydrolysis of the 2′-O-TBS group of 41 gave 42. Treat-
ment of 42 with 1,1′-thiocarbonyldiimidazole in MeCN
gave 2′,3′-cyclic thiocarbonate 43, which, without puri-
fication, was treated with Bu3SnH in the presence of
AIBN in toluene under reflux conditions to give an
inseparable mixture of the corresponding 3′-deoxy de-
rivatives 44 in a ratio of about 4:1 in a yield of 92%.
This mixture was separable on a silica gel column after
tert-butyldimethylsilylation of the 2′-hydroxyl group of
44 to give 45 and 46, respectively. Since a propargyl
radical at the 3′-position is more stable than the
corresponding 2′-secondary radical, we could not detect
any 2′-deoxy derivatives by TLC. The structures of
these nucleosides were confirmed by NOE experiments.
Compound 46 was converted into the corresponding
cytosine nucleoside 49. These nucleosides were depro-
tected to give 47, 48, and 50.
a
(a) BuLi, BrCH2CtCTMS, THF; (b) NH4F, MeOH; (c) (i) Bz2O,
DMAP, MeCN, (ii) TPSCl, DMAP, MeCN, (iii) NH4OH, (iv)
NaOMe, MeOH, (v) NH4F, MeOH.
xylo-derivative 29. These nucleosides were deprotected
by the usual method to give the desired nucleosides 30
and 31.
Previously, J ung et al. reported that treatment of 2′-
O-TBS-3′-ketouridine derivative 32 with cerium (tri-
methylsilyl)acetylide in THF at -78 °C gave the ribo-
pentofuranosyl derivative in good yield.10 Therefore, we
tried to synthesize 3′-C-2-propynyl derivatives, 3′-ho-
mologues of ECyd and EUrd, using this method (Scheme
5). However, attempts to prepare a cerium reagent by
reacting 3-bromo-1-(trimethylsilyl)-1-propyne with BuLi
followed by treatment with dehydrated CeCl3 failed. On
the other hand, reaction of 32 with a lithium salt of
1-(trimethylsilyl)-1-propyne (prepared by the reaction
of 3-bromo-1-(trimethylsilyl)-1-propyne with BuLi) gave
1-[2-O-TBS-3-C-(3-(trimethylsilyl)-2-propynyl)-â-D-ribo-
pentofuranosyl]uracil (33) in 12% yield along with its
3′-epimer 34 in 29% yield. From this reaction mixture,
uracil (35) was obtained in 10% yield. In this reaction,
6 equiv of the reagent was required to consume all of
the starting material. Compound 33 was deprotected
by NH4F in MeOH to give the desired uridine derivative
36, which was converted into the cytosine derivative 37
using the usual method. Xylo-epimers 38 and 39 were
also prepared by the same method. The stereochemistry
at the 3′-position of each compound was confirmed using
the NOE technique. For example, when the 1′-position
of 36 was irradiated, NOE enhancement (5.8%) was
observed at the 3′-down-OH but not at the methylene
protons of the propynyl group, while 2.8% enhancement
at the 3′-up-OH was observed when the 6-proton of the
uracil base of 38 was irradiated.
We previously reported that the reaction of 1-[3,5-O-
[1,1,3,3-tetraisopropyldisiloxane-1,3-diyl (TIPDS)]-â-D-
erythro-2-pentulofuranosyl]uracil and LiCtCR gave
only R-adducts.12 However, the reaction of N4-benzoyl-
cytosine derivative 51 with LiCtCTMS in THF at -78
°C gave the desired â-adduct 52 in 8.5% yield together
with the usual R-adduct 53 in 68% yield (Scheme 7).
The stronger coordination effects of a lithium cation to
the 2-carbonyl oxygen of the cytosine base moiety than
to that of uracil should give the â-adduct 52 in this case,
since the â-addition of MeMgBr8,13,14 to the 2′-keto group
of 4-ethoxy-1-[3,5-O-(TIPDS)-â-D-erythro-2-pentulofura-
nosyl]-2(1H)-pyrimidinone was explained by chelation
of the metal between 2- and 2′-carbonyls at the base
and sugar moieties, which delivered the methyl carban-
ion from the sterically more hindered â-face. These
protected nucleosides were deblocked in the usual

ECyd and EUrd Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2895
Sch em e 7a
Ta ble 1. Inhibitory Effects of Various 2′- and 3′-Substituted
Uracil and Cytosine Nucleosides on the Growth of L1210 and
KB Cells in Vitroa
IC50 (µM)
compd
L1210
0.016
24.5
129
60.8
269
>350
>350
>350
>350
>350
>350
>350
KB
0.028
14.6
108
74.3
258
>350
>350
>350
>350
>350
>350
>350
0.029
70.8
226
51.8
>350
>350
>350
>350
>350
>350
>350
ECyd
15a
15b
15c
15d
15e
31
37
39
50
55
57
EUrd
17a
17b
17c
17d
17e
30
36
38
47
48
0.13
354
338
148
>350
>350
>350
>350
>350
>350
>350
a
(a) LiCtCTMS, THF; (b) TBAF, THF; (c) NH3/MeOH.
manner to give the corresponding free nucleosides 55
and 57, respectively.
The structure of these nucleosides was confirmed by
1H NMR, mass, IR, and UV spectrophotometries along
with elemental analyses. The purities of 15d , 17d , 48,
and 50 were confirmed by HPLC without contamination
by ECyd or EUrd.
a
Tumor cell growth inhibitory activity assay in vitro was done
following the literature method.15 Each tumor cell (2 × 103 cells/
well) was incubated in the presence or absence of compounds for
72 h. MTT reagent was added to each well, and the plate was
incubated for 4 h more. The resulting MTT-formazan was dissolved
in DMSO, and the OD (540 nm) was measured. Percent inhibition
was calculated as follows: % inhibition ) [1 - OD (540 nm) of
sample well/OD (540 nm) of control well] × 100. IC50 (µM) is given
as the concentration at 50% inhibition of cell growth.
Biologica l Activity
The in vitro tumor cell growth inhibitory activities of
the newly synthesized nucleosides against murine leu-
kemia L1210 and human epidermoid KB cells were
evaluated using MTT assay,15 and these activities were
compared to those of ECyd and EUrd. The results are
summarized in Table 1. Among the nucleosides, ECyd
was the most potent inhibitor of tumor cell growth
against L1210 cells, with an IC50 value of 0.016 µM.
Against KB cells, ECyd and EUrd were equally potent,
with IC50 values of 0.028 and 0.029 µM, respectively.
Compound 15a , which has a methyl group instead of
the terminal hydrogen atom of the ethynyl group in
ECyd, showed 1500- and 520-fold lower activity against
L1210 and KB cells, respectively. Compound 15b,
which has a longer ethyl group, had dramatically lower
activity against both of the cells. Although 15c, which
has a relatively bulky ethenyl group instead of an
ethynyl group, showed better activity than 15b, its
activity was negligible compared to that of ECyd. Both
the 3′-C-ethyl and -cyclopropyl derivatives, 15d ,e, did
not show any significant growth inhibitory activity.
Additionally, the 3′-epimer, 3′-homologue and its epimer,
and 3′-deoxy derivative of ECyd, 31, 37, 39, and 50,
respectively, did not show any activity up to 350 µM
against either of the cells. Moreover, the 2′-ethynyl
derivative and its epimer, 55 and 57, had no activity
against either of the cells. This association between the
3′-substituent and cytotoxicity in the cytidine deriva-
tives is similar to that in the uridine series (Table 1).
Since ECyd and EUrd were proposed to be phosphoryl-
ated to their 5′-monophosphates by UCK in a competi-
tion study with cytidine or uridine,4 the bulkiness of the
substituents would be important for recognition of these
substrates by the kinase. Although 30, 31, 47, 48, and
50 have an ethynyl group at their 3′-position, they do
Ta ble 2. Phosphorylation of ECyd, EUrd, and Their Related
Compounds by UCK from Mouse Sarcoma-180 Ascites Cells
and Their Cytotoxicity against Sarcoma-180 Cells in Vitro
phosphorylating
activitya
(pmol/min/assay)
relative
activity
(%)
cytotoxicityb
IC50
compd
(µM)
Cyd
Urd
ECyd
EUrd
15a
15b
15c
15d
17c
812
755
207
141
160
0
0
0
9.3
6.9
100
93
26
17
20
0
0
0
1.1
0.9
0.0034
0.013
13.9
>30
>30
>30
>30
50
>30
a
See the Experimental Section for the assay method. The values
b
are means of duplicate experiments. See Table 1 for the assay
method.
not have a cis-diol at the 2′- and 3′-positions and
therefore would not be substrates of UCK.
To further examine the structure-cytotoxicity rela-
tionship of the sugar moiety of ECyd analogues, we
compared the first phosphorylations of selected ana-
logues by partially purified UCK from mouse Sarcoma-
180 ascites cells. The results are summarized in Table
2 along with their in vitro tumor cell growth inhibitory
activity (IC50) against S-180 cells. For uridine, cytidine,
ECyd, and EUrd, radiolabeled nucleosides are available,
and the substrate activity was measured in terms of the
conversion of each nucleoside into the corresponding 5′-
monophosphate. However, for other ECyd analogues,
radiolabeled nucleosides are not available, and phos-

2896 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Hattori et al.
1 mmol) in THF (2.5 mL) was added dropwise over 10 min to
the above mixture at the same temperature with stirring. After
30 min, the reaction was quenched by addition of aqueous NH4-
Cl (1 M, 5 mL). The solution was extracted by EtOAc (30 mL
× 3), and the separated organic phase was washed with brine
(3 mL × 3), dried (Na2SO4), and concentrated to dryness in
vacuo. The residue was purified on a silica gel column (2 × 9
cm) with 5% EtOAc in hexane to give 2a (320 mg, 93% as a
syrup): LRMS (FAB) m/z 327 (M+ - Me); IR (neat) 2255 cm-1
(CtC). Anal. (C17H30O5Si) C, H.
5-O-(ter t-Bu t yld im et h ylsilyl)-1,2-O-isop r op ylid en e-3-
C-(1-bu tyn yl)-r-D-r ibo-p en tofu r a n ose (2b). As described
for the synthesis of 2a , 2b was similarly prepared from 1. From
1 (302 mg, 1 mmol) and 1-butyne (about 0.4 mL), 2b (259 mg,
73% as a syrup) was obtained. 2b: LRMS (FAB) m/z 341 (M+
- Me); IR (neat) 2245 cm-1 (CtC). Anal. (C18H32O5Si) C, H.
5-O-Ben zoyl-1,2-O-isop r op ylid en e-3-C-(1-p r op yn yl)-r-
D-r ibo-p en tofu r a n ose (3a ). A THF solution of TBAF (1 M,
10 mL, 10 mmol) was added to a solution of 2a (3.42 g, 10
mmol) in THF (30 mL). The mixture was stirred for 20 min
at room temperature, concentrated to dryness, and coevapo-
rated with pyridine (×3). BzCl (2.9 mL, 25 mmol) was added
to a solution of the above residue in pyridine (50 mL) at 0 °C.
The whole was stirred for 4 h at room temperature and
concentrated to dryness. The residue was partitioned between
EtOAc (100 mL) and H2O (50 mL). The separated organic
phase was further washed with aqueous saturated NaHCO3
(50 mL × 3), dried (Na2SO4), and concentrated to dryness in
vacuo. The residue was purified on a silica gel column with
5-15% EtOAc in hexane to give 3a (2.47 g, 74% as a white
powder, which was crystallized from hexanes-EtOAc): mp
120-122 °C; LRMS (FAB) m/z 333 (MH+). Anal. (C18H20O6)
C, H.
5-O-Ben zoyl-3-C-(1-bu tyn yl)-1,2-O-isop r op ylid en e-r-D-
r ibo-p en tofu r a n ose (3b). Compound 3b was obtained as
described for the synthesis of 3a . From 2b (3.56 g, 10 mmol),
3b (2.98 g, 86% as a white powder, which was crystallized from
hexanes-EtOAc) was obtained. 3b: mp 110-113 °C; LRMS
(FAB) m/z 347 (MH+); IR (neat) 2245 cm-1 (CtC). Anal.
(C19H22O6) C, H.
Meth yl 2,3,5-Tr i-O-ben zoyl-3-C-(1-p r op yn yl)-r,â-D-r ibo-
p en tofu r a n ose (5a ). Compound 3a (2.3 g, 6.9 mmol) was
treated with a mixture of AcCl (13.4 mL, 179 mmol), H2O (22.1
mL), and MeOH (74.9 mL) for 8 h at room temperature. The
mixture was neutralized with Et3N (30 mL), and the solvent
was removed in vacuo. The residue dissolved in EtOAc (80
mL) was washed with H2O (45 mL) and aqueous saturated
NaHCO3 (45 mL × 3). The separated organic phase was dried
(Na2SO4), concentrated, and coevaporated with pyridine (×3).
BzCl (8 mL, 69 mmol) was added to a mixture of the above
residue and DMAP (91.27 g, 10.4 mmol) in pyridine (110 mL)
at 0 °C. The mixture was heated for 24 h at 100 °C, and the
cooled mixture was concentrated and coevaporated with
toluene (×3) in vacuo. The residue dissolved in EtOAc (150
mL) was washed with H2O (50 mL) and aqueous saturated
NaHCO3 (50 mL × 3). The separated organic phase was dried
(Na2SO4) and concentrated to dryness in vacuo. The residue
was purified on a silica gel column with 0-10% EtOAc in
hexane to give 5a (2.8 g, 80% as a yellowish syrup): LRMS
(FAB) m/z 515 (MH+), 483 (M+ - OMe). Anal. (C30H26O8) C,
H.
Meth yl 2,3,5-Tr i-O-ben zoyl-3-C-(1-bu tyn yl)-r,â-D-r ibo-
p en tofu r a n ose (5b). Compound 5b was prepared as de-
scribed for the synthesis of 5a . From 3b (2.2 g, 6.4 mmol), 5b
(3.0 g, 88% as a yellowish syrup) was obtained. 5b: LRMS
(FAB) m/z 529 (MH+), 497 (M+ - OMe). Anal. (C31H28O8) C,
H.
1-O-Acet yl-2,3,5-t r i-O-b en zoyl-3-C-(1-p r op yn yl)-r,â-D-
r ibo-p en tofu r a n ose (6a ). Concentrated H2SO4 (1.04 mL)
was added to a solution of 5a (2.57 g, 5 mmol) in a mixture of
AcOH (16.6 mL) and Ac2O (2.1 mL) at 0 °C. The mixture was
stirred for 30 min at room temperature and diluted with CHCl3
(50 mL), which was successively washed with H2O (5 mL),
aqueous saturated NaHCO3 (15 mL × 3), and H2O (5 mL ×
phorylation by UCK was quantitated in terms of the
consumption of these nucleosides using HPLC. When
cytidine and uridine were used as substrates, the
phosphorylating activity of UCK was 812 and 755 pmol/
min, respectively. ECyd and EUrd were phosphorylated
by 26% and 17%, respectively, relative to the phospho-
rylation of cytidine. Therefore, the substrate specificity
of UCK is reflected in ECyd and EUrd, and these
analogues are relatively good substrates of UCK. Al-
though 15a was a substrate of the kinase (its relative
activity was 20%), the cytotoxicity (IC50 ) 13.9 µM) of
15a against S-180 cells was 1000-fold less than that of
EUrd. Again, 17c and 50 were slightly phosphorylated
by the kinase but were not cytotoxic. The phosphoryl-
ation of other analogues 15b-d was not detected by this
method, and these data correlate with their cytotoxicity.
Therefore, these data suggest that the first phosphoryl-
ation of nucleosides by UCK is important for expression
of their cytotoxicity. The substrate specificity of UCK
from mouse S-180 ascites cells reflects the in vitro
cytotoxicity of these compounds against L1210 and KB
cells. The target enzyme of ECyd and EUrd that is
responsible for their cytotoxicity against tumor cells has
been proposed to be RNA polymerases.5 Therefore, after
phosphorylation by UCK, these 5′-monophosphates
should be further converted into the corresponding 5′-
triphosphates. In the case of 15a , 17c, and 50, the
efficiency of further phosphorylations by nucleotide
kinases and/or their inhibitory activities against RNA
polymerases remain to be elucidated.
In conclusion, the substrate specificity of UCK re-
garding ECyd and EUrd analogues is closely related to
their cytotoxicities against the tumor cells used in this
study. Only an ethynyl group at the 3′â-position in
cytidine and uridine was sufficiently tolerated by UCK.
Other bulkier substituents, such as 1-butynyl, ethyl,
cyclopropyl, and even ethenyl groups, did not provide
good substrates for UCK. The presence of a 2′,3′-cis-
diol in ECyd and EUrd with the ribo-configuration was
essential for its cytotoxicity. A further study is needed
to elucidate the effect of these substituents on sugar
puckering and its relationship to the substrate specific-
ity of UCK.
Exp er im en ta l Section
Gen er a l Met h od s. Melting points were measured on a
Yanagimoto MP-3 micromelting point apparatus (Yanagimoto,
J apan) and are uncorrected. Fast atom bombardment mass
spectrometry (FAB-MS) was done on a J EOL J MS-HX110
instrument at an ionizing voltage of 70 eV. The 1H NMR
spectra were recorded on a J EOL J NM-GX 270 (270 MHz) or
Bruker ARX 500 (500 MHz) spectrometer with tetramethyl-
silane as an internal standard. Chemical shifts are reported
in parts per million (δ), and signals are expressed as s (singlet),
d (doublet), t (triplet), m (multiplet), or br (broad). All
exchangeable protons were detected by disappearance on the
addition of D2O. UV absorption spectra were recorded with a
Shimadzu UV-240 spectrophotometer. IR spectra were re-
corded with a J EOL A-102 spectrometer. TLC was done on
Merck Kieselgel F254 precoated plates (Merck, Germany). The
silica gel used for column chromatography was YMC gel 60A
(70-230 mesh) (YMC Co., Ltd., J apan).
5-O-(ter t-Bu t yld im et h ylsilyl)-1,2-O-isop r op ylid en e-3-
C-(1-p r op yn yl)-r-D-r ibo-p en tofu r a n ose (2a ). A hexane
solution of BuLi (1.63 M, 1.84 mL, 3 mmol) was added
dropwise over 30 min to a solution of propyne (about 0.5 mL)
in THF (5 mL) at -78 °C under Ar. A solution of 1 (302 mg,

ECyd and EUrd Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2897
2). The separated organic phase was dried (Na2SO4) and
concentrated to dryness in vacuo. The residue was purified
on a silica gel column with 10-20% EtOAc in hexane to give
6a (2.64 g, 98% as a syrup): LRMS (FAB) m/z 543 (MH+), 483
(M+ - OAc). Anal. (C31H26O9) C, H.
1-O-Acetyl-2,3,5-tr i-O-ben zoyl-3-C-(1-bu tyn yl)-r,â-D-r ibo-
p en tofu r a n ose (6b). Compound 6b was prepared as de-
scribed for the synthesis of 6a . From 5b (2.69 g, 5.1 mmol),
6b (2.37 g, 83% as a yellowish syrup) was obtained. 6b: LRMS
(FAB) m/z 557 (MH+), 513 (M+ - Ac), 493 (M+ - OAc). Anal.
(C32H28O9) C, H.
residue taken up with EtOAc (100 mL) was successively
washed with H2O (40 mL) and aqueous saturated NaHCO3
(40 mL × 3). The separated organic phase was dried (Na2-
SO4) and concentrated to dryness. The residue was purified
on a silica gel column with 5-20% EtOAc in hexane to give
12 (2.94 g, 91% as a white powder, which was crystallized from
hexanes-EtOAc): mp 83-86 °C (lit.16 mp 84-85 °C); LRMS
(FAB) m/z 323 (MH+), 307 (M+ - Me). Anal. (C17H22O6) C,
H.
1,2,3,5-Tet r a -O-b en zoyl-3-C-et h en yl-r,â-D-r ibo-p en t o-
fu r a n ose (13c). A solution of 8c (1.1 g, 3.4 mmol) in a mixture
of aqueous HCl (1 M, 25 mL) and THF (25 mL) was heated
under reflux for 8 h. The mixture was cooled to room
temperature and neutralized with Et3N. The solvent was
removed in vacuo, and the residue taken up with EtOAc (50
mL) was successively washed with H2O (30 mL) and aqueous
saturated NaHCO3 (25 mL × 3). The separated organic phase
was dried (Na2SO4) and concentrated to dryness. The residue
was coevaporated with pyridine (×3) and dissolved in pyridine
(60 mL). BzCl (4 mL, 34 mmol) and DMAP (629 mg, 5.1 mmol)
were added to the above solution at 0 °C. The mixture was
heated at 100 °C for 24 h. The solvent was removed in vacuo
and coevaporated with toluene (×3). The residue taken up
with EtOAc (100 mL) was washed with H2O (50 mL) and
aqueous saturated NaHCO3 (50 mL × 3). The separated
organic phase was dried (Na2SO4) and concentrated to dryness.
The residue was purified on a silica gel column with 0-10%
EtOAc in hexane to give 13c (1.6 g, 63% as a syrup): LRMS
(FAB) m/z 471 (M+ - OBz).
5-O-(ter t-Bu t yld im et h ylsilyl)-1,2-O-isop r op ylid en e-3-
C-eth en yl-r-D-r ibo-p en tofu r a n ose (7c). A solution of 1
(3.02 g, 10 mmol) in THF (40 mL) was added dropwise over
30 min to a THF solution of vinylmagnesium bromide (1 M,
30 mL, 30 mmol), at -15 °C under Ar. The mixture was
stirred for 2 h at the same temperature. The reaction was
quenched by addition of aqueous NH4Cl (1 M, 50 mL), which
was extracted by EtOAc (35 mL × 3). The separated organic
phase was washed with brine (30 mL × 3), dried (Na2SO4),
and concentrated to dryness in vacuo. The residue was
purified on a silica gel column with 5% EtOAc in hexane to
give 7c (2.11 g, 64% as a syrup): LRMS (FAB) m/z 315 (M+
Me), 273 (M+ - t-Bu). Anal. (C16H30O5Si) C, H.
-
5-O-(ter t-Bu t yld im et h ylsilyl)-3-C-cyclop r op yl-1,2-O-
isop r op ylid en e-r-D-r ibo-p en tofu r a n ose (7e). A hexane
solution of BuLi (1.7 M, 14.7 mL, 25 mmol) was added
dropwise over 30 min to a solution of cyclopropyl bromide (2.1
mL, 25 mmol) at -78 °C under Ar. The mixture was stirred
for 30 min at the same temperature. A solution of 1 (4.5 g, 15
mmol) in THF (25 mL) was added dropwise over 10 min to
the above solution. After being stirred for 3 h, the reaction
was quenched by EtOAc (30 mL × 3), and the separated
organic phase was washed with brine (15 mL × 3), dried (Na2-
SO4), and concentrated to dryness in vacuo. The residue was
purified on a silica gel column with 5% EtOAc in hexane to
give 7e (3.3 g, 65% as a white powder, which was crystallized
from hexane): mp 57-59 °C; LRMS (FAB) m/z 345 (MH+),
329 (M+ - Me). Anal. (C17H32O5Si) C, H.
1,2,3,5-Tetr a -O-ben zoyl-3-C-eth yl-r,â-D-r ibo-p en tofu r a -
n ose (13d ). Compound 13d was prepared as described for
the synthesis of 13c. From 12 (1.75 g, 5.43 mmol), 13d (2.3 g,
72% as a yellowish syrup) was obtained. 13d : LRMS (FAB)
m/z 595 (MH+), 473 (M+ - OBz).
1,2,3,5-Te t r a -O-b e n zoyl-3-C-cyclop r op yl-r,â-D-r ibo-
p en tofu r a n ose (13e). Compound 13e was prepared as
described for the synthesis of 13c. From 8e (2.0 g, 6 mmol),
13e (2.4 g, 67% as a yellowish syrup) was obtained. 13e:
LRMS (FAB) m/z 501 (M+ - Bz), 485 (M+ - OBz).
5-O-Ben zoyl-3-C-eth en yl-1,2-O-isopr opyliden e-r-D-r ibo-
p en tofu r a n ose (8c). After desilylation of 7c (1.67 g, 5.1
mmol) by TBAF in THF, BzCl (1.72 mL, 15 mmol) was added
to the residue in pyridine (35 mL) at 0 °C. The mixture was
stirred for 4 h at room temperature. Workup was done as
described for the synthesis of 3a to give 8c (1.46 g, 90% as a
white powder, which was crystallized from hexane/EtOAc).
Gen er a l Meth od for th e Syn th esis of 3′-C-Su bstitu ted
Ribon u cleosid es. Stannic chloride (2.5 mmol for cytosine)
or TMSOTf (2 mmol for uracil) was added to a solution of
persilylated pyrimidine [2 mmol, prepared from the pyrimidine
(2 mmol) and (NH4)2SO4 (7 mg) in HMDS (6 mL)] and the
sugar (6a ,b, or 13c-e; 0.5 mmol) in MeCN (4 mL) at 0 °C.
The whole was stirred for the indicated period at room
temperature. The mixture was diluted with CHCl3 (12 mL)
and aqueous saturated NaHCO3 (5 mL) with vigorous stirring
for 30 min. The precipitate was removed by filtration through
a Celite pad, which was washed well with CHCl3. The
combined filtrate and washings were washed with H2O (5 mL
× 2) and aqueous saturated NaHCO3 (5 mL). The separated
organic phase was dried (Na2SO4) and concentrated in vacuo.
The residue was purified on a silica gel column to give the
desired protected nucleoside, which was then treated with
NH3/MeOH (saturated at 0 °C) for 2 days at room temperature.
The solvent was removed in vacuo, and the residue was
purified on a silica gel column with 5-20% MeOH in CHCl3
to furnish the desired 3′-C-substituted ribonucleosides.
8c: mp 110-111 °C; LRMS (FAB) m/z 321 (MH+), 305 (M+
-
Me). Anal. (C17H20O6) C, H.
5-O-Ben zoyl-3-C-cyclop r op yl-1,2-O-isop r op ylid en e-r-
D-r ibo-p en tofu r a n ose (8e). Compound 8e was prepared as
described for the synthesis of 8c. From 7e (3.0 g, 8.7 mmol),
8e (2.49 g, 86% as a white powder, which was crystallized from
hexane/EtOAc) was obtained. 8e: mp 109-111 °C; LRMS
(FAB) m/z 355 (MH+), 319 (M+ - Me). Anal. (C18H22O6) C,
H.
3-C-Eth yn yl-1,2-O-isop r op ylid en e-r-D-r ibo-p en tofu r a -
n ose (11). Compound 103 (8.0 g, 20 mmol) in THF (60 mL)
was desilylated by TBAF (1 M, 21 mL, 21 mmol) for 20 min at
room temperature to give 11 (4.1 g, 96% as a white powder):
LRMS (FAB) m/z 215 (MH+).
5-O-Ben zoyl-3-C-et h yl-1,2-O-isop r op ylid en e-r-D-r ibo-
p en tofu r a n ose (12). A mixture of 11 (214 mg, 1 mmol) and
10% Pd-C (50 mg) in MeOH (18 mL) was stirred under
atmospheric pressure of H2 for 1 h at room temperature.
Insoluble materials were removed by filtration, and the filtrate
was concentrated in vacuo. The residue was purified on a
silica gel column with 5% MeOH in CHCl3 to give 3-C-ethyl-
1,2-O-isopropylidene-R-D-ribo-pentofuranose (209 mg, 96% as
a white powder, which was crystallized from EtOH): mp 97-
100 °C; LRMS (FAB) m/z 219 (MH+). Anal. (C10H18O5) C, H.
BzCl (1.74 mL, 15 mmol) was added to a solution of the above
residue (2.18 g, 10 mmol) in pyridine (50 mL) at 0 °C. The
mixture was stirred for 1.5 h at room temperature, and the
solvent was removed and coevaporated with toluene (×3). The
1-[3-C-(1-P r op yn yl)-â-D-r ibo-p en tofu r a n osyl]cytosin e
Hyd r och lor id e (15a ). The reaction of persilylated cytosine
(2 mmol) with 6a (0.5 mmol) in the presence of SnCl4 (0.29
mL, 2.5 mmol) for 2.5 h at room temperature gave 14a (214
1
mg, 72% as a foam): LRMS (FAB) m/z 594 (MH+); H NMR
(CDCl3) 8.15-7.31 (m, 15 H, Bz × 3), 7.82 (d, 1 H, H-6, J 6,5
)
7.5 Hz), 6.52 (d, 1 H, H-1′, J 1′,2′ ) 4.6 Hz), 6.03 (d, 1 H, H-2′,
J 2′,1′ ) 4.6 Hz), 5.74 (d, 1 H, H-5, J 5,6 ) 7.5 Hz), 4.95-4.86 (m,
3 H, H-4′, 5′), 1.86 (s, 3 H, 3′-CtCCH3). From 14a (150 mg,
0.25 mmol), 15a (63 mg, 89% as a pale-yellow solid, which was
crystallized as an HCl salt from Et2O/EtOH) was obtained.
15a : mp 162-165 °C; LRMS (EI) m/z 281 (M+); IR (Nujol) 2245
cm-1 (CtC); UV λmax (H2O) 272 nm (ꢀ 8600), λmax (0.5 M HCl)
280 nm (ꢀ 12 200), λmax (0.5 M NaOH) 273 nm (ꢀ 8900); 1H
NMR (DMSO-d6 + D2O) 8.26 (d, 1 H, H-6, J 6,5 ) 7.9 Hz), 6.15

2898 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Hattori et al.
(d, 1 H, H-5, J 5,6 ) 7.9 Hz), 5.78 (d, 1 H, H-1′, J 1′,2′ ) 5.7 Hz),
4.08 (d, 1 H, H-2′, J 2′,1′ ) 5.7 Hz), 3.95-3.94 (m, 1 H, H-4′),
3.75 (dd, 1 H, H-5′a, J 5′a,4′ ) 4.7 Hz, J gem ) 12.0 Hz), 3.67 (dd,
1 H, H-5′b, J 5′b,4′ ) 2.5 Hz, J gem ) 12.0 Hz), 1.81 (s, 3 H, 3′-
CtCCH3). Anal. (C12H15N3O5‚HCl‚0.2H2O) C, H, N.
at room temperature gave 16c (284 mg, 98% as a foam): LRMS
1
(FAB) m/z 583 (MH+); H NMR (CDCl3) 8.17-7.44 (m, 16 H,
Bz × 3, H-6), 8.05 (br s, 1 H, NH), 6.52 (d, 1 H, H-1′, J 1′,2′
)
)
7.7 Hz), 6.41 (dd, 1 H, 3′-CHcdCHaHb, J c,a ) 17.4 Hz, J c,b
11.1 Hz), 6.03 (d, 1 H, H-2′, J 2′,1′ ) 7.7 Hz), 5.54 (dd, 1 H, H-5,
J 5,6 ) 8.2 Hz, J 5,NH ) 2.2 Hz), 5.43-5.41 (m, 2 H, 3′-CHcd
CHa,b), 5.25 (dd, 1 H, H-4′, J 4′,5′a ) 3.2 Hz, J 4′,5′b ) 3.7 Hz),
4.83 (dd, 1 H, H-5′a, J 5′a,4′ ) 3.2 Hz, J gem ) 12.6 Hz), 4.71 (dd,
1 H, H-5′b, J 5′b,4′ ) 3.7 Hz, J gem ) 12.6 Hz). From 16c (279
mg, 0.48 mmol), 17c (121 mg, 93% as a white foam, which
was crystallized from aqueous MeOH) was obtained. 17c: mp
219-222 °C; LRMS (EI) m/z 271 (MH+); UV λmax (H2O) 261
nm (ꢀ 10 000), λmax (0.5 M HCl) 261 nm (ꢀ 10 400), λmax (0.5 M
NaOH) 263 nm (ꢀ 7700); 1H NMR (DMSO-d6) 11.32 (br s, 1 H,
NH), 8.08 (d, 1 H, H-6, J 6,5 ) 8.0 Hz), 6.05 (dd, 1 H, 3′-CHcd
CHaHb, J c,a ) 17.2 Hz, J c,b ) 10.7 Hz), 5.95 (d, 1 H, H-1′, J 1′,2′
) 8.0 Hz), 5.70 (dd, 1 H, H-5, J 5,6 ) 7.8 Hz, J 5,NH ) 2.1 Hz),
5.48 (dd, 1 H, 3′-CHcdCHaHb, J a,c ) 17.2 Hz, J a,b ) 1.9 Hz),
5.46 (d, 1 H, 2′-OH, J ) 7.0 Hz), 5.26 (dd, 1 H, 3′-CHcdCHaHb,
J b,c ) 10.7 Hz, J b,a ) 1.9 Hz), 5.24 (t, 1 H, 5′-OH, J ) 4.2 Hz),
4.90 (s, 1 H, 3′-OH), 4.11 (dd, 1 H, H-2′, J 2′,1′ ) 8.0 Hz, J 2′,OH
) 7.0 Hz), 3.78-3.76 (m, 1 H, H-4′), 3.57-3.41 (m, 2 H, H-5′).
Anal. (C11H14N2O6) C, H, N.
1-[3-C-(1-P r opyn yl)-â-D-r ibo-pen tofu r an osyl]u r acil (17a).
The reaction of persilylated uracil (2 mmol) with 6a (0.5 mmol)
in the presence of TMSOTf (0.39 mL, 2 mmol) for 8 h at room
temperature gave 16a (241 mg, 81% as a foam): LRMS (FAB)
1
m/z 595 (MH+); H NMR (CDCl3) 8.14-7.30 (m, 15 H, Bz ×
3), 7.97 (br s, 1 H, NH), 7.78 (d, 1 H, H-6, J 6,5 ) 8.0 Hz), 6.32
(d, 1 H, H-1′, J 1′,2′ ) 4.5 Hz), 5.96 (d, 1 H, H-2′, J 2′,1′ ) 4.5 Hz),
5.75 (dd, 1 H, H-5, J 5,6 ) 8.0 Hz, J 5,NH ) 2.0 Hz), 4.93-4.84
(m, 3 H, H-4′,5′), 1.91 (s, 3 H, 3′-CtCCH3). From 16a (236
mg, 0.4 mmol), 17a (87 mg, 77% as a white powder, which
was crystallized from aqueous MeOH) was obtained. 17a : mp
211-213 °C; LRMS (EI) m/z 283 (MH+); IR (Nujol) 2240 cm-1
(CtC); UV λmax (H2O) 262 nm (ꢀ 9700), λmax (0.5 M HCl) 262
nm (ꢀ 9600), λmax (0.5 M NaOH) 263 nm (ꢀ 7700); 1H NMR
(DMSO-d6) 11.33 (br s, 1 H, NH), 7.97 (d, 1 H, H-6, J 6,5 ) 8.2
Hz), 5.80 (d, 1 H, H-1′, J 1′,2′ ) 6.9 Hz), 5.75 (d, 1 H, 2′-OH, J
) 6.5 Hz), 5.70 (s, 1 H, 3′-OH), 5.68 (dd, 1 H, H-5, J 5,6 ) 8.2
Hz, J 5,NH ) 2.0 Hz), 5.04 (t, 1 H, 5′-OH, J ) 4.6 Hz), 4.10 (dd,
1 H, H-2′, J 2′,1′ ) 6.9 Hz, J 2′,OH ) 6.5 Hz), 3.87-3.85 (m, 1 H,
H-4′), 3.73-3.62 (m, 2 H, H-5′), 1.83 (s, 3 H, 3′-CtCCH3). Anal.
(C12H14N2O6‚0.2H2O) C, H, N.
1-[3-C-(1-C y c lo p r o p y l)-â-D -r i b o-p e n t o fu r a n o s y l]-
u r a cil (17e). The reaction of persilylated uracil (2 mmol) with
13e (0.5 mmol) in the presence of TMSOTf [0.39 mL, 2 mmol;
after 48 h, further amounts of TMSOTf (0.39 mL, 2.0 mmol)
were added] for 51 h at room temperature gave 16e (269 mg,
90% as a foam): LRMS (FAB) m/z 597 (MH+); 1H NMR (CDCl3)
8.18-7.45 (m, 15 H, Bz × 3), 7.93 (br s, 1 H, NH), 7.68 (d, 1
H, H-6, J 6,5 ) 8.2 Hz), 6.48 (d, 1 H, H-1′, J 1′,2′ ) 7.7 Hz), 5.80
(d, 1 H, H-2′, J 2′,1′ ) 7.7 Hz), 5.60 (dd, 1 H, H-5, J 5,6 ) 8.2 Hz,
J 5,NH ) 1.7 Hz), 5.46 (dd, 1 H, H-4′, J 4′,5′a ) 3.8 Hz, J 4′,5′b ) 3.1
Hz), 4.98 (dd, 1 H, H-5′a, J 5′a,4′ ) 3.8 Hz, J gem ) 12.7 Hz), 4.81
(dd, 1 H, H-5′b, J 5′b,4′ ) 3.1 Hz, J gem ) 12.7 Hz), 2.09-0.56 (m,
5 H, 3′-cyclopropyl). From 16e (265 mg, 0.44 mmol), 17e (106
mg, 85% as a white foam, which was crystallized from EtOH/
Et2O) was obtained. 17e: mp 179-181 °C; LRMS (EI) m/z
285 (MH+); UV λmax (H2O) 261 nm (ꢀ 8600), λmax (0.5 M HCl)
261 nm (ꢀ 8900), λmax (0.5 M NaOH) 263 nm (ꢀ 6900); 1H NMR
(DMSO-d6) 11.27 (br s, 1 H, NH), 8.11 (d, 1 H, H-6, J 6,5 ) 8.1
1-[3-C-(1-Bu tyn yl)-â-D-r ibo-pen tofu r an osyl]cytosin e Hy-
d r och lor id e (15b). The reaction of persilylated cytosine (4
mmol) with 6b (1 mmol) in the presence of SnCl4 (0.59 mL, 5
mmol) for 19 h at room temperature gave 14b (412 mg, 68%
as a foam): LRMS (FAB) m/z 608 (MH+); 1H NMR (CDCl3)
8.15-7.31 (m, 15 H, Bz × 3), 7.84 (d, 1 H, H-6, J 6,5 ) 7.4 Hz),
6.54 (d, 1 H, H-1′, J 1′,2′ ) 4.6 Hz), 6.04 (d, 1 H, H-2′, J 2′,1′ ) 4.6
Hz), 5.72 (d, 1 H, H-5, J 5,6 ) 7.4 Hz), 4.97-4.85 (m, 3 H,
H-4′,5′), 2.25-2.20 (m, 2 H, 3′-CtCCH2CH3), 1.08 (t, 3 H, 3′-
CtCCH2CH3, J ) 7.5 Hz). From 14b (336 mg, 0.55 mmol),
15b (154 mg, 95% as a pale-yellow foam, which was crystal-
lized as an HCl salt from i-PrOH) was obtained. 15b: mp
181-184 °C; LRMS (FAB) m/z 295 (M+); IR (Nujol) 2240 cm-1
(CtC); UV λmax (H2O) 271 nm (ꢀ 8600), λmax (0.5 M HCl) 280
1
nm (ꢀ 12 300), λmax (0.5 M NaOH) 273 nm (ꢀ 9900); H NMR
Hz), 5.88 (d, 1 H, H-1′, J 1′,2′ ) 7.8 Hz), 5.68 (d, 1 H, H-5, J 5,6
)
(DMSO-d6 + D2O) 8.27 (d, 1 H, H-6, J 6,5 ) 7.8 Hz), 6.18 (d, 1
H, H-5, J 5,6 ) 7.8 Hz), 5.77 (d, 1 H, H-1′, J 1′,2′ ) 5.5 Hz), 4.08
(d, 1 H, H-2′, J 2′,1′ ) 5.5 Hz), 3.96-3.95 (m, 1 H, H-4′), 3.77-
3.67 (m, 2 H, H-5′), 2.22-2.17 (m, 2 H, 3′-CtCCH2CH3), 1.05
(t, 3 H, 3′-CtCCH2CH3, J ) 7.5 Hz). Anal. (C13H17N3O5‚HCl)
C, H, N.
8.1 Hz), 5.42 (d, 1 H, 2′-OH, J ) 5.4 Hz), 5.16 (t, 1 H, 5′-OH),
4.16 (s, 1 H, 3′-OH), 4.03 (dd, 1 H, H-2′, J 2′,1′ ) 7.8 Hz, J 2′,OH
) 5.4 Hz), 3.75-3.63 (m, 3 H, H-4′,5′), 1.04-0.20 (m, 5 H, 3′-
cyclopropyl). Anal. (C12H16N2O6) C, H, N.
1-[3-C-(1-Eth yl)-â-D-r ibo-p en tofu r a n osyl]u r a cil (17d ).
The reaction of persilylated uracil (8 mmol) with 13d (2 mmol)
in the presence of TMSOTf [1.55 mL, 8 mmol; after 26 h,
further amounts of TMSOTf (0.78 mL, 4 mmol) were added]
for 52 h at room temperature gave 16d (963 mg, 83% as a
foam): LRMS (FAB) m/z 585 (MH+); 1H NMR (CDCl3) 8.35
(br s, 1 H, NH), 8.19-7.46 (m, 16 H, Bz × 3, H-6), 6.48 (d, 1
H, H-1′, J 1′,2′ ) 7.8 Hz), 5.86 (d, 1 H, H-2′, J 2′,1′ ) 7.8 Hz), 5.48
(dd, 1 H, H-5, J 5,6 ) 8.2 Hz, J 5,NH ) 1.9 Hz), 5.22 (dd, 1 H,
H-4′, J 4′,5′a ) 3.1 Hz, J 4′,5′b ) 3.5 Hz), 4.87 (dd, 1 H, H-5′a, J 5′a,4′
) 3.1 Hz, J gem ) 12.7 Hz), 4.69 (dd, 1 H, H-5′b, J 5′b,4′ ) 3.5 Hz,
J gem ) 12.7 Hz), 2.90-2.04 (m, 2 H, 3′-CH2CH3), 0.95 (t, 3 H,
3′-CH2CH3, J ) 7.4 Hz). From 16d (369 mg, 0.63 mmol), 17d
(160 mg, 93% as a white foam, which was crystallized from
EtOH/Et2O) was obtained. 17d : mp 220-222 °C (lit.16 mp
217-218 °C); LRMS (EI) m/z 273 (MH+); UV λmax (H2O) 262
nm (ꢀ 9300), λmax (0.5 M HCl) 261 nm (ꢀ 9500), λmax (0.5 M
NaOH) 263 nm (ꢀ 7300); 1H NMR (DMSO-d6) 11.26 (br s, 1 H,
NH), 8.08 (d, 1 H, H-6, J 6,5 ) 8.1 Hz), 5.89 (d, 1 H, H-1′, J 1′,2′
) 7.7 Hz), 5.66 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 5.31 (d, 1 H, 2′-
OH, J ) 6.4 Hz), 5.15 (t, 1 H, 5′-OH), 4.49 (s, 1 H, 3′-OH),
3.91 (dd, 1 H, H-2′, J 2′,1′ ) 7.7 Hz, J 2′,OH ) 6.4 Hz), 3.82-3.80
(m, 1 H, H-4′), 3.57-3.55 (m, 2 H, H-5′), 1.67-1.60 (m, 2 H,
3′-CH2CH3), 0.93 (t, 3 H, 3′-CH2CH3, J ) 7.3 Hz). Anal.
(C11H16N2O6‚0.2H2O) C, H, N.
1-[3-C-(1-Bu tyn yl)-â-D-r ibo-pen tofu r an osyl]u r acil (17b).
The reaction of persilylated uracil (2 mmol) with 6b (0.5 mmol)
in the presence of TMSOTf [0.39 mL, 2 mmol; after 48 h,
further amounts of TMSOTf (0.19 mL, 1.0 mmol) were added]
for 53 h at room temperature gave 16b (294 mg, 97% as a
1
foam): LRMS (FAB) m/z 609 (MH+); H NMR (CDCl3) 8.15-
7.29 (m, 15 H, Bz × 3), 8.02 (br s, 1 H, NH), 7.82 (d, 1 H, H-6,
J 6,5 ) 8.2 Hz), 6.34 (d, 1 H, H-1′, J 1′,2′ ) 4.4 Hz), 5.97 (d, 1 H,
H-2′, J 2′,1′ ) 4.4 Hz), 5.75 (dd, 1 H, H-5, J 5,6 ) 8.2 Hz, J 5,NH
)
2.3 Hz), 4.93-4.84 (m, 3 H, H-4′,5′), 2.30-2.25 (m, 2 H, 3′-Ct
CCH2CH3), 1.12 (s, 3 H, 3′-CtCCH2CH3, J ) 7.5 Hz). From
16b (286 mg, 0.47 mmol), 17b (129 mg, 93% as a white foam,
which was crystallized from EtOH/Et2O) was obtained. 17b:
mp 139-142 °C; LRMS (EI) m/z 297 (MH+); IR (Nujol) 2245
cm-1 (CtC); UV λmax (H2O) 263 nm (ꢀ 9,500), λmax (0.5 M HCl)
263 nm (ꢀ 10 600), λmax (0.5 M NaOH) 263 nm (ꢀ 7600); 1H
NMR (DMSO-d6) 11.31 (br s, 1 H, NH), 7.96 (d, 1 H, H-6, J 6,5
) 7.8 Hz), 5.79 (d, 1 H, H-1′, J 1′,2′ ) 6.7 Hz), 5.74 (d, 1 H, 2′-
OH, J ) 6.4 Hz), 5.67 (s, 1 H, 3′-OH), 5.66 (d, 1 H, H-5, J 5,6
)
7.8 Hz), 4.99 (t, 1 H, 5′-OH, J ) 4.6 Hz), 4.08 (dd, 1 H, H-2′,
J 2′,1′ ) 6.7 Hz, J 2′,OH ) 6.4 Hz), 3.87-3.86 (m, 1 H, H-4′), 3.74-
3.64 (m, 2 H, H-5′), 2.23-2.19 (m, 2 H, 3′-CtCCH2CH3), 1.07
(t, 3 H, 3′-CtCCH2CH3, J ) 7.5 Hz). Anal. (C13H16N2O6‚
0.6H2O) C, H, N.
1-[3-C-(1-Eth en yl)-â-D-r ibo-pen tofu r an osyl]u r acil (17c).
The reaction of persilylated uracil (2 mmol) with 13c (0.5
mmol) in the presence of TMSOTf (0.39 mL, 2 mmol) for 39 h
Gen er a l P r oced u r e for Con ver sion of Ur a cil Nu cleo-
sid es in to Cytosin e Nu cleosid es. Triethylamine (2 equiv)
was added to a mixture of 16c, 16d , or 16e, TPSCl (2 equiv),

ECyd and EUrd Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2899
and DMAP (2 equiv) in MeCN. The mixture was stirred for
indicated period at room temperature. Concentrated NH4OH
(28%, 20 mL) was added to the mixture, and the whole was
further stirred for 2.5 h at room temperature. The solvent
was removed in vacuo, and the residue was purified by silica
gel column chromatography with 0-5% MeOH in CHCl3 to
give the corresponding blocked cytosine derivatives 14c-e,
which were further treated with NH3/MeOH (saturated at 0
°C, 10 mL) for 2 days at room temperature. The solvent was
removed in vacuo, and the residue was purified by silica gel
column chromatography with 0-20% MeOH in CHCl3 to give
the corresponding cytosine derivatives 15c-e.
1-(3-C-Eth en yl-â-D-r ibo-p en tofu r a n osyl)cytosin e (15c).
From 16c (400 mg, 0.96 mmol), 14c (345 mg, 86% as a foam)
was obtained. 14c: LRMS (FAB) m/z 582 (MH+); 1H NMR
(CDCl3) 8.12-7.38 (m, 16 H, Bz × 3, H-6), 6.61 (d, 1 H, H-1′,
J 1′,2′ ) 7.6 Hz), 6.35 (dd, 1 H, 3′-CHcdCHaHb, J c,a ) 17.4 Hz,
J c,b ) 11.0 Hz), 6.03 (d, 1 H, H-2′, J 2′,1′ )7.6 Hz), 5.61 (d, 1 H,
H-5, J 5,6 ) 6.9 Hz), 5.40-5.33 (m, 2 H, 3′-CHcdCHaHb), 5.20-
5.12 (m, 1 H, H-4′), 4.84-4.65 (m, 2 H, H-5′). From 14c (323
mg, 0.55 mmol), 15c (156 mg, 96% as a white powder, which
was crystallized from Et2O/EtOH) was obtained. 15c: mp
194-197 °C; LRMS (El) m/z 270 (MH+); UV λmax (H2O) 270
nm (ꢀ 8100), λmax (0.5 N HCl) 280 nm (ꢀ 11 700), λmax (0.5 N
NaOH) 272 nm (ꢀ 7400); 1H NMR (DMSO-d6) 7.93 (d, 1 H, H-6,
J 6,5 ) 7.5 Hz), 7.20, 7.17 (each br s, each 1 H, NH2), 6.05 (dd,
1 H, 3′-CHcdCHaHb, J c,a ) 17.2 Hz, J c,b ) 10.6 Hz), 5.93 (d,
1 H, H-1′, J 1′,2′ ) 7.9 Hz), 5.75 (d, 1 H, H-5, J 5,6 ) 7.5 Hz), 5.46
(dd, 1 H, 3′-CHcdCHaHb, J a,c ) 17.2 Hz, J a,b ) 2.0 Hz), 5.31
(d, 1 H, 2′-OH, J ) 6.7 Hz), 5.23 (dd, 1 H, 3′-CHcdCHaHb,
J b,c ) 10.6 Hz, J b,a ) 2.0 Hz), 5.21 (t, 1 H, 5′-OH, J ) 4.5 Hz),
4.77 (s, 1 H, 3′-OH), 4.13 (dd, 1 H, H-2′, J 2′,1′ ) 7.9 Hz, J 2′,OH
) 6.7 Hz), 3.76-3.75 (m, 1 H, H-4′), 3.55-3.39 (m, 2 H, H-5′).
Anal. (C11H15N3O5) C, H, N.
1-(3-C-Eth yn yl-â-D-xylo-p en tofu r a n osyl)u r a cil (30). A
THF solution of HCtCMgBr (0.5 M, 12 mL, 6 mmol) was
added to a solution of 2610b (470 mg, 1 mmol) in THF (10 mL)
at room temperature. The mixture was stirred for 60 min,
and aqueous NH4Cl (1 M, 10 mL) was added. The mixture
was diluted with EtOAc (50 mL), washed with H2O (10 mL ×
3) and brine (25 mL), and dried (Na2SO4). The organic phase
was concentrated to dryness, and the residue was purified on
a silica gel column with 2% MeOH in CHCl3 to give 28 (450
mg, 91% as a white solid): LRMS (EI) m/z 439 (M+ - t-Bu);
1H NMR (CDCl3) 8.16 (br s, 1 H, NH), 7.93 (d, 1 H, H-6, J 5,6
)
8.1 Hz), 5.80 (s, 1 H, 3′-OH), 5.60 (d, 1 H, H-5, J 5,6 ) 8.1 Hz),
5.35 (br s, 1 H, H-1′), 4.37-4.30 (m, 2 H, H-5′), 4.09 (m, 2 H,
H-2′,4′), 2.60 (s, 1 H, 3′-CtCH), 0.90 (s, 18 H, t-Bu × 2), 0.22-
0.14 (each s, each 3 H, Me × 4). Anal. (C23H40N2O6Si2) C, H,
N. A mixture containing 28 (300 mg, 0.6 mmol) and NH4F
(450 mg, 12 mmol) in MeOH (12 mL) was heated under reflux
for 17 h. The mixture was allowed to cool to room temperature
and concentrated to dryness, and the residue was purified on
a silica gel column with 10-20% MeOH in CHCl3 to give 30
(132 mg, 82% as a yellowish solid, which was crystallized from
EtOH/hexane): mp 112 °C; LRMS (EI) m/z 268 (M+); 1H NMR
(DMSO-d6) 9.38 (br s, 1 H, NH), 7.73 (d, 1 H, H-6, J 6,5 ) 8.0
Hz), 6.26 (d, 1 H, 2′-OH, J ) 6.8 Hz), 6.13 (s, 1 H, 3′-OH), 5.67
(s, 1 H, H-1′), 5.62 (d, 1 H, H-5, J 5,6 ) 8.0 Hz), 4.90 (t, 1 H,
5′-OH), 4.02 (dd, 1 H, H-4′, J 4′,5′a ) 2.4 Hz, J 4′,5′b ) 7.3 Hz),
3.89 (d, 1 H, H-2′, J ) 6.0 Hz), 3.77 (ddd, 1 H, H-5′a, J 5′a,4′
)
2.4 Hz, J 5′a,OH ) 5.7 Hz, J gem ) 12.2 Hz), 3.70 (ddd, 1 H, H-5′b,
J 5′b,4′ ) 7.3 Hz, J 5′b,OH ) 5.7 Hz, J gem ) 12.2 Hz), 3.56 (s, 1 H,
3′-CtCH). Anal. (C11H12N2O6‚H2O) C, H, N.
N 4-Ace t yl-1-[2,5-d i-O-(t er t -b u t yld im e t h ylsilyl)-3-C-
eth yn yl-â-D-xylo-p en tofu r a n osyl]cytosin e (29). Oxidation
of N4-acetyl-1-[2,5-bis-O-(tert-butyldimethylsilyl)-â-D-ribofura-
nosyl]cytosine (1.03 g, 2 mmol) was done according to the
literature10b to give 27 (910 mg, 88% as a colorless foam). A
THF solution of HCtCMgBr (0.5 M, 12 mL, 6 mmol) was
added dropwise to a solution of 27 (512 mg, 1 mmol) in THF
(10 mL) at 0 °C. The mixture was stirred for 1 h at the same
temperature. The reaction was quenched with an aqueous
solution of NH4Cl (1 M, 10 mL). The mixture was diluted with
EtOAc (60 mL), washed with H2O (20 mL × 2) and brine (20
mL), and dried (Na2SO4). The organic phase was concentrated
to dryness, and the residue was purified on a silica gel column
with 0-2% MeOH in CHCl3 to give 29 (450 mg, 83% as a
yellow foam): LRMS (EI) m/z 480 (M+). Anal. (C25H43N3O6-
Si2) C, H, N.
1-(3-C-E t h yl-â-D-r ibo-p en t ofu r a n osyl)cyt osin e (15d ).
From 16d (500 mg, 0.86 mmol), 14d (390 mg, 78% as a foam)
was obtained. 14d : LRMS (FAB) m/z 584 (MH+); 1H NMR
(CDCl3) 8.17-7.43 (m, 16 H, Bz × 3, H-6), 6.66 (d, 1 H, H-1′,
J 1′,2′ ) 6.7 Hz), 5.85 (d, 1 H, H-5, J 5,6 ) 7.4 Hz), 5.50 (d, 1 H,
H-2′, J 2′,1′ ) 6.7 Hz), 5.17 (m, 1 H, H-4′), 4.90 (dd, 1 H, H-5′a,
J 5′a,4′ ) 3.1 Hz, J gem ) 12.4 Hz), 4.71 (dd, 1 H, H-5′b, J 5′b,4′
)
3.4 Hz, J gem ) 12.4 Hz), 2.87-2.03 (m, 2 H, 3′-CH2CH3), 0.93
(t, 3 H, 3′-CH2CH3, J ) 7.3 Hz). From 14d (386 mg, 0.66
mmol), 15d (159 mg, 89% as a white powder, which was
crystallized from Et2O/EtOH) was obtained. 15d : mp 220-
221 °C; LRMS (El) m/z 272 (MH+); UV λmax (H2O) 271 nm (ꢀ
8000), λmax (0.5 N HCl) 280 nm (ꢀ 12 300), λmax (0.5 N NaOH)
1-(3-C-Eth yn yl-â-D-xylo-p en tofu r a n osyl)cytosin e (31).
Acetyl chloride (0.5 mL) was added to MeOH (9.5 mL), and
the mixture was stirred for 30 min at room temperature. To
this mixture was added 29 (400 mg, 0.74 mmol), and the
mixture was stirred for 2 days at room temperature. The
mixture was neutralized with Et3N and concentrated to
dryness, and the residue was purified on a silica gel column
with 17-25% MeOH in CHCl3 to give a crude oil, which was
further purified by charcoal chromatography with H2O and
then 10-100% MeOH in H2O to give 31 (130 mg, 67% as a
colorless crystalline solid): mp 126 °C; LRMS (EI) m/z 267
(M+); 1H NMR (DMSO-d6) 7.69 (d, 1 H, H-6, J 6,5 ) 7.5 Hz),
7.15, 7.05 (each br s, each 1 H, NH2), 6.14 (d, 1 H, 2′-OH, J )
5.9 Hz), 6.03 (s, 1 H, 3′-OH), 5.58 (d, 1 H, H-5, J 5,6 ) 7.5 Hz),
5.66 (s, 1 H, H-1′), 4.86 (t, 1 H, 5′-OH, J ) 5.6 Hz), 4.00 (dd,
1 H, H-4′, J 4′,5′a ) 2.2 Hz, J 4′,5′b ) 7.1 Hz), 3.85 (d, 1 H, H-2′,
272 nm (ꢀ 8200); 1H NMR (DMSO-d6) 7.92 (d, 1 H, H-6, J 6,5
)
7.3 Hz), 7.22, 7.14 (each br s, each 1 H, NH2), 5.85 (d, 1 H,
H-1′, J 1′,2′ ) 7.6 Hz), 5.74 (d, 1 H, H-5, J 5,6 ) 7.3 Hz), 5.19 (d,
1 H, 2′-OH, J ) 6.4 Hz), 5.12 (t, 1 H, 5′-OH, J ) 3.9 Hz), 4.39
(s, 1 H, 3′-OH), 3.94 (dd, 1 H, H-2′, J 2′,1′ ) 7.6 Hz, J 2′,OH ) 6.4
Hz), 3.79 (m, 1 H, H-4′), 3.57-3.50 (m, 2 H, H-5′), 1.65-1.59
(m, 2 H, 3′-CH2CH3), 0.92 (t, 3 H, 3′-CH2CH3, J ) 7.3 Hz).
Anal. (C11H17N3O5‚H2O) C, H, N.
1-(3-C-Cyclop r op yl-â-D-r ibo-p en t ofu r a n osyl)cyt osin e
(15e). From 16e (440 mg, 0.74 mmol), 14e (315 mg, 72% as a
foam) was obtained. 14e: LRMS (FAB) m/z 596 (MH+); 1H
NMR (CDCl3) 8.18-7.40 (m, 16 H, Bz × 3, H-6), 6.65 (d, 1 H,
H-1′, J 1′,2′ ) 7.5 Hz), 5.77 (d, 1 H, H-2′, J 2′,1′ ) 7.5 Hz), 5.57 (d,
1 H, H-5, J 5,6 ) 6.9 Hz), 5.41-5.39 (m, 1 H, H-4′), 4.97-4.86
(m, 2 H, H-5′), 2.08-0.52 (m, 5 H, 3′-cyclopropyl). From 14e
(312 mg, 0.52 mmol), 15e (138 mg, 94% as a white powder,
which was crystallized from Et2O/EtOH) was obtained. 15e:
mp 205-208 °C; LRMS (EI) m/z 284 (MH+); UV λmax (H2O)
270 nm (ꢀ 8100), λmax (0.5 M HCl) 279 nm (ꢀ 11 400), λmax (0.5
J 2′,OH ) 5.9 Hz), 3.78 (ddd, 1 H, H-5′a, J 5′a,4′ ) 2.2 Hz, J 5′a,OH
)
5.9 Hz, J gem ) 12.0 Hz), 3.71 (ddd, 1 H, H-5′b, J 5′b,4′ ) 7.1 Hz,
J 5′b,OH ) 5.9 Hz, J gem ) 12.0 Hz), 3.50 (s, 1 H, 3′-CtCH); 13C
NMR (MeOH-d4) 167.78, 158.31, 143.46, 94.84, 94.53, 88.16,
83.68, 81.31, 77.52, 76.91, 61.38. Anal. (C11H13N3O5) C, H,
N.
1
M NaOH) 263 nm (ꢀ 7700); H NMR (DMSO-d6) 7.94 (d, 1 H,
H-6, J 6,5 ) 7.5 Hz), 7.17, 7.13 (each br s, each 1 H, NH2), 5.85
(d, 1 H, H-1′, J 1′,2′ ) 7.6 Hz), 5.74 (d, 1 H, H-5, J 5,6 ) 7.5 Hz),
5.31 (d, 1 H, 2′-OH, J ) 6.3 Hz), 5.13-5.11 (m, 1 H, 5′-OH),
4.06 (s, 1 H, 3′-OH), 4.04 (dd, 1 H, H-2′, J 2′,1′ ) 7.6 Hz, J 2′,OH
) 6.3 Hz), 3.71-3.62 (m, 3 H, H-4′,5′), 1.03-0.19 (m, 5 H, 3′-
cyclopropyl). Anal. (C12H17N3O5‚1/3H2O) C, H, N.
1-[2-O-(ter t-Bu tyld im eth ylsilyl)-3-C-[3-(tr im eth ylsilyl)-
2-p r op yn yl]-â-D-r ibo-p en tofu r a n osyl]u r a cil (33) a n d 1-[2-
O-(ter t-Bu tyld im eth ylsilyl)-3-C-[3-(tr im eth ylsilyl)-2-p r o-
p yn yl]-â-D-xylo-p en t ofu r a n osyl]u r a cil (34). A hexane
solution of BuLi (1.63 M, 27.9 mL, 45.5 mmol) was added
dropwise over 20 min to a mixture of 3210b (2.70 g, 7.57 mmol)

2900 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Hattori et al.
and 3-bromo-1-(trimethylsilyl)-1-propyne (6.4 mL, 45.2 mmol)
in THF (25 mL) at -78 °C under Ar. The mixture was stirred
at the same temperature for 25 min, and AcOH (3 mL) was
added to the mixture, which was warmed to room temperature
and concentrated in vacuo. The residue taken up with EtOAc
(100 mL) was washed with H2O (50 mL), aqueous saturated
NaHCO3 (50 mL) and brine (50 mL), and dried (Na2SO4). The
solvent was removed in vacuo, and the residue was purified
on a silica gel column with 40-50% EtOAc in hexane to give
33 (427 mg, 12% as a foam) and 34 (1.03 g, 29% as a foam).
From the aqueous phases, uracil (85 mg) was obtained. 33:
LRMS (FAB) m/z 469 (MH+); HRMS (FAB) calcd for
C21H37N2O6Si2 469.2190, found 469.2168. Anal. (C21H36N2O6-
Si2) C, H, N. 34: LRMS (FAB) m/z 469 (MH+); HRMS (FAB)
calcd for C21H37N2O6Si2 469.2190, found 469.2215. Anal.
(C21H36N2O6S12‚0.4H2O) C, H, N.
H, H-1′, J 1′,2′ ) 7.9 Hz), 5.74 (d, 1 H, H-5, J 5,6 ) 7.4 Hz), 3.90
(d, 1 H, H-2′, J 2′,1′ ) 7.9 Hz), 3.85 (br s, 1 H, H-4′), 3.72 (dd, 1
H, H-5′a, J gem ) 12.1 Hz), 3.61 (dd, 1 H, H-5′b, J gem ) 12.1
Hz), 2.77 (br s, 1 H, 3′-CH2CtCH), 2.53 (dd, 1 H, 3′-CH2Ct
CH, J gem ) 17.0 Hz); 13C NMR (MeOH-d4) 167.15, 158.70,
144.31, 96.50, 90.90, 88.26, 80.75, 78.94, 78.51, 71.97, 62.10,
25.10. Anal. (C12H15N3O5‚0.8H2O) C, H, N.
1-[3-C-(2-P r op yn yl)-â-D-xylo-p en t ofu r a n osyl]cyt osin e
(39). Compound 39 was prepared as described for the
synthesis of 37. From 34 (100 mg, 0.2 mmol), 39 (58 mg,
quantitative as a solid) was obtained. 39: mp 137 °C; LRMS
(FAB) m/z 281 (M+); HRMS (EI) calcd for C12H13N3O5 281.1010,
found 281.1017; 1H NMR (DMSO-d6 + D2O) 7.21 (d, 1 H, H-6,
J 6,5 ) 7.6 Hz), 5.82 (d, 1 H, H-5, J 5,6 ) 7.6 Hz), 5.53 (s, 1 H,
H-1′), 3.90-3.88 (m, 2 H, H-2′,4′), 3.83 (dd, 1 H, H-5′a, J gem
)
11.9 Hz), 3.69 (dd, 1 H, H-5′b, J gem ) 11.9 Hz), 2.75 (br s, 1 H,
3′-CH2CtCH), the proton signals for 3′-CH2CtCH were
overlapped with those for DMSO; 13C NMR (MeOH-d4) 166.70,
157.07, 144.49, 94.97, 94.61, 87.26, 83.20, 81.08, 80.88, 72.39,
61.87, 24.38. Anal. (C12H15N3O5‚0.5MeOH) C, H, N.
1-[3-C-(2-P r opyn yl)-â-D-r ibo-pen tofu r an osyl]u r acil (36).
A mixture of 33 (46 mg, 0.1 mmol) and NH4F (74 mg, 2 mmol)
in MeOH (2 mL) was heated under reflux for 29 h. The solvent
was removed in vacuo, and the residue was purified on a silica
gel column with 5% MeOH in CHCl3 to give 36 (14 mg, 50%
1-[5-O-Ben zoyl-3-C-[(tr im eth ylsilyl)eth yn yl]-â-D-r ibo-
p en tofu r a n osyl]u r a cil (42). Bz2O (220 mg, 0.97 mmol) was
added to a solution of 4010b (370 mg, 0.81 mmol) and DMAP
(119 mg, 0.97 mmol) in a mixture of MeCN and CH2Cl2 (1:1,
8 mL) at 0 °C. After being stirred for 45 min at the same
temperature, EtOH (1 mL) was added to the mixture. The
solvent was removed in vacuo, and the residue taken up with
EtOAc (10 mL) was washed with aqueous HCl (0.1 M), aqueous
saturated NaHCO3, H2O, and brine, dried (Na2SO4), and
evaporated to dryness to give a crude 41. The residue
containing 41 was further treated with HCl/MeOH (5%, 12
mL) at room temperature for 15 min, which was neutralized
with aqueous saturated NaHCO3. The solvent was removed
in vacuo, and the residue was purified on a silica gel column
with 3-5% MeOH in CHCl3 to give 42 (201 mg, 56% as a
solid): LRMS (FAB) m/z 445 (MH+). Anal. (C21H24N2O7Si)
C, H, N.
1-[5-O-Ben zoyl-3-d eoxy-3-C-[(tr im eth ylsilyl)eth yn yl]-
â-D-r ibo- a n d -xylo-p en tofu r a n osyl]u r a cil (44). A mixture
of 42 (440 mg, 1 mmol) and N,N′-thiocarbonyldiimidazole (310
mg, 1.6 mmol) in a mixture of MeCN and CH2Cl2 (1:1, 20 mL)
was stirred for 2.5 h at room temperature. Water (1 mL) was
added to the mixture, and the whole was diluted with CH2Cl2
(100 mL), which was washed with H2O (50 mL) and brine (50
mL). The organic phase was dried (Na2SO4), concentrated to
dryness, and coevaporated several times with toluene. The
residue was dissolved in toluene (70 mL) containing AIBN (16
mg) and Bu3SnH (810 µL, 3 mmol), which was heated under
reflux for 90 min. The solvent was removed in vacuo, and the
residue taken up with EtOAc (50 mL) was washed with
aqueous KF (1 M, 50 mL) and brine (20 mL × 2). The organic
phase was dried (Na2SO4) and concentrated to dryness. The
residue was purified on a silica gel column with 0-2% MeOH
in CHCl3 to give 44 (390 mg, 92% as a foam): LRMS (FAB)
m/z 429 (MH+); HRMS (FAB) calcd for C21H25N2O6Si 429.1482,
found 429.1495. Anal. (C21H24N2O6Si‚0.2H2O) C, H, N.
1-[5-O-Ben zoyl-2-O-(ter t-bu tyld im eth ylsilyl)-3-d eoxy-
3-C-[(t r im et h ylsilyl)et h yn yl]-â-D-xylo-p en t ofu r a n osyl]-
uracil (46) and 1-[5-O-Benzoyl-2-O-(tert-butyldimethylsilyl)-3-
d eoxy-3-C-[(t r im et h ylsilyl)et h yn yl]-â-D-r ibo-p en t ofu r a -
n osyl]u r a cil (45). A mixture of 44 (280 mg, 0.65 mmol), tert-
butyldimethylsilyl chloride (245 mg, 1.63 mmol), and imidazole
(133 mg, 1.95 mmol) in DMF (6.5 mL) was stirred for 20 h at
80 °C under Ar. The mixture was partitioned between EtOAc
(20 mL) and H2O (10 mL × 3). The organic phase was further
washed with aqueous HCl (1 M, 20 mL), aqueous saturated
NaHCO3 (10 mL), and H2O (10 mL) and dried (Na2SO4). The
solvent was removed in vacuo, and the residue was purified
on a silica gel column with 20% EtOAc in hexane to give 46
(255 mg, 72% as a foam) and 45 (71 mg, 20% as a foam). 46:
LRMS (FAB) m/z 543 (MH+); HRMS calcd for C27H39N2O6Si2
543.2346, found 543.2352. Anal. (C27H38N2O6Si2‚0.2H2O)
C, H, N. 45: LRMS (FAB) m/z 543 (MH+); HRMS calcd for
1
as a solid): mp 104 °C; LRMS (FAB) m/z 282 (M+); H NMR
(DMSO-d6) 11.07 (br s, 1 H, NH), 8.02 (d, 1 H, H-6, J 6,5 ) 8.1
Hz), 5.89 (d, 1 H, H-1′, J 1′,2′ ) 7.9 Hz), 5.69 (d, 1 H, H-5, J 5,6
)
8.1 Hz), 5.53 (br d, 1 H, 2′-OH), 5.31 (br s, 1 H, 5′-OH), 5.02
(s, 1 H, 3′-OH), 3.88 (br s, 2 H, H-2′,4′), 3.83-3.60 (br s, 2 H,
H-5′), 2.82 (t, 1 H, 3′-CH2CtCH), the proton signals for 3′-
CH2CtCH were overlapped with those for DMSO; 13C NMR
(MeOH-d4) 166.41, 153.22, 143.57, 103.38, 88.91, 88.50, 80.99,
79.13, 78.43, 72.34, 62.33, 25.35. Anal. (C12H14N2O6) C, H,
N.
1-[3-C-(2-P r opyn yl)-â-D-xylo-pen tofu r an osyl]u r acil (38).
Compound 34 was deprotected as described for the synthesis
of 36. From 34 (100 mg, 0.2 mmol), 38 (58 mg, 96% as a solid)
was obtained. 38: mp 218 °C; LRMS (FAB) m/z 282 (M+); 1H
NMR (DMSO-d6) 11.24 (br s, 1 H, NH), 7.78 (d, 1 H, H-6, J 6,5
) 8.1 Hz), 6.03 (d, 1 H, 2′-OH), 5.58 (m, 2 H, H-5,1′), 5.27 (s,
1 H, 3′-OH), 4.80 (t, 1 H, 5′-OH), 3.91 (dd, 1 H, H-2′, J 2′,1′
0.9 Hz, J 2′,OH ) 5.6 Hz), 3.87 (dd, 1 H, H-4′, J 4′,5′a ) 3.6 Hz,
J 4′,5′b ) 6.3 Hz), 3.83 (ddd, 1 H, H-5′a, J 5′a,4′ ) 3.6 Hz, J 5′a,OH
)
)
5.6 Hz, J gem ) 12.0 Hz), 3.69 (ddd, 1 H, H-5′b, J 5′b,4′ ) 6.3 Hz,
J 5′b,OH ) 5.6 Hz, J gem ) 12.0 Hz), 2.78 (t, 1 H, 3′-CH2CtCH),
2.48 (d, 2 H, 3′-CH2CtCH); 13C NMR (MeOH-d4) 167.03,
152.80, 143.63, 101.55, 93.71, 87.23, 83.29, 81.01, 72.58, 62.09,
24.55. Anal. (C12H14N2O6‚H2O) C, H, N.
1-[3-C-(2-P r op yn yl)-â-D-r ibo-p en tofu r a n osyl]cytosin e
(37). A mixture of 33 (187 mg, 0.4 mmol), Bz2O (272 mg, 1.2
mmol), and DMAP (147 mg, 1.2 mmol) in MeCN (4 mL) was
stirred for 1 h at room temperature. The mixture was diluted
with EtOAc (20 mL), which was washed successively with
aqueous HCl (0.1 M, 20 mL), aqueous saturated NaHCO3 (20
mL), and H2O (20 mL × 2), and dried (Na2SO4). The solvent
was removed in vacuo. A mixture of the residue, Et3N (112
µL, 0.8 mmol), 2,4,6-triisopropylbenzenesulfonyl chloride (TP-
SCl; 242 mg, 0.8 mmol), and DMAP (98 mg, 0.8 mmol) in
MeCN (4 mL) was stirred for 12 h at room temperature, and
then NH4OH (28%, 3 mL) was added to the mixture, which
was further stirred for 30 min at room temperature. The
solvent was removed in vacuo, and the residue taken up with
EtOAc (20 mL) was washed with H2O (20 mL × 6). The
organic phase was dried (Na2SO4), and the solvent was
removed in vacuo. The residue was purified on a silica gel
column with 0-3% MeOH in CHCl3 to give protected 37, which
was treated with MeOH (5 mL) containing NaOMe (5 M, 24
µL) at room temperature for 27 h. The mixture was neutral-
ized with aqueous HCl (1 M), and the solvent was removed in
vacuo. A mixture of the residue and NH4F (300 mg, 8 mmol)
in MeOH (5 mL) was heated under reflux for 1.5 h. The
solvent was removed, and the residue was extracted with
CHCl3. The aqueous phase was absorbed to an active charcoal
column which was washed well with H2O and then with 50%
aqueous MeOH to give 37 (57 mg, 51% as a solid): mp 210-
213 °C; LRMS (FAB) m/z 250 (M+ - CH2OH); 1H NMR
(DMSO-d6 + D2O) 7.87 (d, 1 H, H-6, J 6,5 ) 7.4 Hz), 5.85 (d, 1
C
27H39N2O6Si2 543.2346, found 543.2349. Anal. (C27H38N2O6-
Si2) C, H, N.

ECyd and EUrd Derivatives
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2901
1-[5-O-Ben zoyl-2-O-(ter t-bu tyld im eth ylsilyl)-3-d eoxy-
3-C-[(tr im eth ylsilyl)eth yn yl]-â-D-xylo-p en tofu r a n osyl]cy-
tosin e (49). A mixture of 46 (120 mg, 0.22 mmol), TPSCl (133
mg, 0.44 mmol), Et3N (60 µL, 0.44 mmol), and DMAP (54 mg,
0.44 mmol) in MeCN (2 mL) was stirred for 19 h at room
temperature, and then NH4OH (28%, 2 mL) was added to the
mixture, which was further stirred for 45 min at the same
temperature. The solvent was removed in vacuo, and the
residue was purified on a silica gel column with 2% MeOH in
CHCl3 to give 49 (115 mg, 96% as a foam): LRMS (FAB) m/z
542 (MH+); HRMS (FAB) calcd for C27H40N3O5Si2 542.2506,
found 542.2505. Anal. (C27H39N3O5Si2) C, H, N.
1-(3-D e o x y -3-C-e t h y n y l-â-D -xyl o-p e n t o fu r a n o s y l)-
u r a cil (48). Compound 46 (100 mg, 0.18 mmol) was stirred
in MeOH (2 mL) containing MaOMe (5 M, 40 µL) at room
temperature. After 3.5 h, HCl/MeOH (2%, 2 mL) was added
to the mixture, which was stirred further for 16 h at the same
temperature. The solvent was removed and coevaporated
several times with EtOH. The residue was purified on a silica
gel column with 10% MeOH in CHCl3 to give 48 (48 mg,
quantitative as a foam): LRMS (EI) m/z 252 (M+); HRMS
(FAB) calcd for C11H12N2O5 252.0745, found 252.0723; 1H NMR
(D2O) 7.92 (d, 1 H, H-6, J 6,5 ) 8.1 Hz), 5.88 (d, 1 H, H-5, J 5,6
) 8.1 Hz), 5.83 (d, 1 H, H-1′, J 1′,2′ ) 3.0 Hz), 4.58 (dd, 1 H,
H-2′, J 2′,1′ ) 3.0 Hz, J 2′,3′ ) 3.5 Hz), 4.53 (ddd, 1 H, H-4′, J 4′,3′
) 6.3 Hz, J 4′,5′a ) 6.3 Hz, J 4′,5′b ) 4.3 Hz), 4.04 (dd, 1 H, H-5′a,
in vacuo. The residue was purified on a silica gel column with
25% EtOAc in hexane to give 53 (2.49 g, 68% as a white foam).
The column was successively eluted with 35% EtOAc in hexane
and then 50% EtOAc in hexane. From the former elution, 51
(490 mg, as a foam) was recovered, and from the latter elution,
52 (310 mg, 8.5% as a white foam) was obtained. 53: LRMS
(EI) m/z 685 (M+). Anal. (C33H51N3O7Si3) C, H, N. 52: LRMS
(EI) m/z 685 (M+). Anal. (C33H51N3O7Si3) C, H, N.
1-(2-C-Eth yn yl-â-D-a r a bin o-pen tofu r an osyl)cytosin e Hy-
d r och lor id e (57). A THF solution of TBAF (1 M, 4.1 mL,
4.1 mmol) was added to a mixture of 53 (810 mg, 1.2 mmol) in
THF (30 mL) containing AcOH (0.24 mL, 4.1 mmol). The
mixture was stirred for 40 min at room temperature and
concentrated to dryness in vacuo. The residue was purified
on a silica gel column with 4% MeOH in CHCl3 to give N4-
benzoyl-1-(2-C-ethynyl-â-D-arabino-pentofuranosyl)cytosine (56;
379 mg, 86% as a white powder): LRMS (EI) m/z 371 (M+).
Anal. (C18H17N3O6‚0.8EtOH) C, H, N. Compound 56 (250 mg,
0.67 mmol) was treated with NH3/MeOH (saturated at 0 °C,
10 mL) for 17 h at room temperature. The solvent was
removed in vacuo, and the residue was purified on a silica gel
column with 30% MeOH in CHCl3 to give a white powder,
which was crystallized from HCl/EtOH to give 57 (156 mg,
1
77%): mp 239-241 °C; H NMR (DMSO-d6) 9.78, 8.73 (each
br s, each 1 H, NH2), 7.99 (d, 1 H, H-6, J 6,5 ) 7.8 Hz), 6.51 (br
s, 1 H, 2′-OH), 6.14 (d, 1 H, H-5, J 5,6 ) 7.8 Hz), 6.12 (s, 1 H,
H-1′), 5.90 (br s, 1 H, 3′-OH), 3.87 (d, 1 H, H-3′, J 3′,4′ ) 4.4
Hz), 3.83 (dd, 1 H, H-4′, J 4′,3′ ) J 4′,5′a ) 4.4 Hz, J 4′,5′b ) 4.9 Hz),
3.66 (dd, 1 H, H-5′a, J 5′a,4′ ) 4.4 Hz, J gem ) 11.7 Hz), 3.60 (dd,
1 H, H-5′b, J 5′b,4′ ) 4.9 Hz, J gem ) 11.7 Hz), 3.59 (s, 1 H, 2′-
CtCH). Anal. (C11H13N3O5‚HCl) C, H, N.
J 5′a,4′ ) 6.3 Hz, J gem ) 12.3 Hz), 3.97 (dd, 1 H, H-5′b, J 5′b,4′
)
4.3 Hz, J gem ) 12.3 Hz), 3.30 (ddd, 1 H, H-3′, J 3′,2′ ) 3.5 Hz,
J 3′,4′ ) 6.3 Hz, J 3′,CtCH ) 2.6 Hz), 2.70 (d, 1 H, 3′-CtCH, J Ct
) 2.6 Hz); 13C NMR (MeOH-d4) 166.23, 152.38, 142.46,
CH,3′
102.06, 92.77, 82.35, 81.29, 80.24, 76.01, 63.14, 40.75. Anal.
(C11H12N2O5‚0.3MeOH) C, H, N.
1-(3-D e o x y -3-C-e t h y n y l-â-D -r i b o-p e n t o fu r a n o s y l)-
u r a cil (47). Compound 47 was prepared as described for the
synthesis of 48. From 45 (50 mg, 0.09 mmol), 47 (23 mg,
quantitative as a foam) was obtained. 47: LRMS (EI) m/z 252
(M+); HRMS (EI) calcd for C11H12N2O5 252.0745, found 252.0770;
1H NMR (D2O) 7.94 (d, 1 H, H-6, J 6,5 ) 8.1 Hz), 5.87 (d, 1 H,
H-5, J 5,6 ) 8.1 Hz), 5.85 (s, 1 H, H-1′), 4.53 (d, 1 H, H-2′, J 2′,3′
) 4.6 Hz), 4.35 (m, 1 H, H-4′, J 4′,3′ ) 10.3 Hz), 4.08 (br d, 1 H,
H-5′a, J gem ) 12.2 Hz), 3.87 (br d, 1 H, H-5′b, J gem ) 12.2 Hz),
3.18 (dd, 1 H, H-3′, J 3′,2′ ) 4.6 Hz, J 3′,4′ ) 10.8 Hz), 2.24 (s, 1
H, 3′-CtCH); 13C NMR (MeOH-d4) 166.37, 152.01, 142.37,
101.79, 93.74, 86.01, 78.70, 77.79, 74.50, 60.77, 36.13. Anal.
(C11H12N2O5‚0.57MeOH) C, H, N.
1-(3-D e o x y -3-C-e t h y n y l-â-D -xyl o-p e n t o fu r a n o s y l)-
cytosin e (50). Compound 50 was prepared as described for
the synthesis of 47. From 49 (100 mg, 0.18 mmol), 50 (59 mg,
quantitative as a foam) was obtained. 50: LRMS (EI) m/z 251
(M+); HRMS (EI) calcd for C11H13N3O4 251.0905, found 251.0913;
1H NMR (DMSO-d6 + D2O) 7.69 (d, 1 H, H-6, J 6,5 ) 7.5 Hz),
5.74 (d, 1 H, H-5, J 5,6 ) 7.5 Hz), 5.65 (br s, 1 H, H-1′), 4.21
(dd, 1 H, H-4′, J 4′,5′a ) 5.7 Hz, J 4′,5′b ) 4.6 Hz), 4.18 (br s, 1 H,
H-2′), 3.70 (dd, 1 H, H-5′a, J 5′a,4′ ) 5.7 Hz, J gem ) 11.5 Hz),
3.67 (dd, 1 H, H-5′b, J 5′b,4′ ) 4.6 Hz, J gem ) 11.5 Hz), 3.11 (s,
1 H, 3′-CtCH), 3.02 (br s, 1 H, H-3′); 13C NMR (MeOH-d4)
166.59, 156.92, 143.39, 95.24, 94.43, 83.03, 81.88, 80.18, 76.21,
63.16, 40.75.
1-(2-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)cytosin e Hy-
d r och lor id e (55). Compound 52 (360 mg, 0.53 mmol) was
desilylated as described above to give N4-benzoyl-1-(2-C-
ethynyl-â-D-ribo-pentofuranosyl)cytosine (54), which was de-
benzoylated to give 55 (95 mg, 60% as an HCl salt): mp 212-
213 °C; 1H NMR (DMSO-d6) 9.77 (br s, 1 H, NH), 8.77 (br s, 1
H, NH), 8.63 (d, 1 H, H-6, J 6,5 ) 7.8 Hz), 6.37 (br s, 1 H, 2′-
OH), 6.16 (d, 1 H, H-5, J 5,6 ) 7.8 Hz), 5.87 (s, 1 H, H-1′), 4.00
(d, 1 H, H-3′, J 3′,4′ ) 8.8 Hz), 3.84 (dt, 1 H, H-4′, J 4′,5′a ) J 4′,5′b
) 2.4 Hz, J 4′,3′ ) 9.3 Hz), 3.80 (dd, 1 H, H-5′a, J 5′a,4′ ) 2.4 Hz,
J gem ) 12.7 Hz), 3.61 (dd, 1 H, H-5′b, J 5′b,4′ ) 2.4 Hz, J gem
)
12.7 Hz), 3.53 (s, 1 H, 2′-CtCH). Anal. (C11H13N3O5‚HCl) C,
H, N.
P u r ifica tion of Ur id in e/Cytid in e Kin a se (UCK). Uri-
dine/cytidine kinase (UCK, EC 2.7.1.48) was highly purified
from ascitic Sarcoma-180 cells growing in mice. The tumor
cells (63.5 g wet weight) were homogenized with 4 volumes of
potassium phosphate buffer (10 mM, pH 7.5) containing DTT
(5 mM), KCl (25 mM), and MgCl2 (5 mM) and centrifuged at
105000g for 60 min. The resulting supernatant was treated
with ammonium sulfate (30-50% saturation) and centrifuged
at 105000g for 30 min. The precipitates were dissolved in
potassium phosphate buffer (10 mM, pH 7.5) containing DTT
(5 mM) and 5% glycerol, dialyzed against the same buffer, and
applied to a Sephacryl S-200 column (3 × 105 cm) equilibrated
with the same buffer. The active eluates containing UCK were
subjected to a Bio-Gel HT hydroxyapatite column (4 × 16 cm)
equilibrated with the same buffer. UCK was obtained by the
linear gradient elution of 10-100 mM phosphate buffer. The
eluates were dialyzed against the starting buffer and then
purified by chromatography on a DEAE-Sepharose CI-6B
column (3 × 15 cm). The purified enzyme preparation was
found not to contain any other enzymes such as CMP kinase,
CDP kinase, Cyd deaminase, or pyrimidine nucleoside phos-
phorylase. UCK activity was stable for more than 1 year when
stored at -80 °C.
N4-Ben zoyl-1-[3,5-O-(1,1,3,3-tetr a isop r op yld isiloxa n e-
1,3-d iyl)-2-C-[(tr im eth ylsilyl)eth yn yl]-â-D-r ibo-p en tofu r a -
n osyl]cytosin e (52) a n d N4-Ben zoyl-1-[3,5-O-(1,1,3,3-tetr a -
is o p r o p y ld is ilo x a n e -1,3-d iy l)-2-C-[(t r im e t h y ls ily l)-
eth yn yl]-â-D-a r a bin o-p en tofu r a n osyl]cytosin e (53).
A
hexane solution of BuLi (1.56 M, 10 mL, 16.1 mmol) was added
dropwise over 15 min to a solution of (trimethylsilyl)acetylene
(2.27 mL, 16.1 mmol) in THF (10 mL) at -78 °C under Ar.
After being stirred for 30 min, a solution of N4-benzoyl-1-[3,5-
O-(TIPDS)-â-D-erythro-pentofuran-2-ulosyl]cytosine (51; 3.15
g, 5.4 mmol) in THF (10 mL) was added dropwise over 10 min
to the above solution. The whole was stirred for 2.5 h at the
same temperature and was quenched by addition of aqueous
NH4Cl (1 M, 25 mL). The mixture was extracted with EtOAc
(50 mL × 3). The separated organic phase was washed with
brine (100 mL), dried (Na2SO4), and concentrated to dryness
Assa y of UCK. Phosphorylation of the test compounds by
UCK was measured according to the method described by
Ikenaka et al.17

2902 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Hattori et al.
(6) Yoshimura, Y.; Sano, T.; Matsuda, A.; Ueda, T. Synthesis of 6,3′-
methanocytidine, 6,3′-methanouridine, and their 2′-deoxyribo-
nucleosides. Chem. Pharm. Bull. 1988, 36, 162-167.
(7) Bevierre, M.-O.; De Mesmaeker, A.; Wolf, R. M.; Freier, S. M.
Synthesis of 2′-O-methyl-6,3′-ethanouridine and its introduction
into antisense oligonucleotides. Bioorg. Med. Chem. Lett. 1994,
4, 237-240.
(8) (a) Matsuda, A.; Itoh, H.; Takenuki, K.; Sasaki, T.; Ueda, T. Alkyl
addition reaction of pyrimidine 2′-ketonucleosides: Synthesis of
2′-branched-chain sugar pyrimidine nucleosides. Chem. Pharm.
Bull. 1988, 36, 945-953. (b) Fischer, J . C.; Horton, D. Synthesis
of chiral tertiary alcohols by Grignard additions to glycosulose
derivatives. J . Carbohydr. Nucleosides Nucleotides 1979, 6, 101-
126. (c) Rosenthal, A.; Mikhailov, S. N. Branched-sugars.
Modifications in the reaction of 1,2:5,6-di-O-isopropylidene-R-
D-ribo-hexofuranos-3-ulose with Grignard and organolithium
reagents. J . Carbohydr. Nucleosides Nucleotides 1979, 6, 237-
245.
(9) Hayakawa, H.; Tanaka, H.; Itoh, N.; Nakajima, M.; Miyasaka,
T.; Yamaguchi, K.; Iitaka, Y. Reaction of organometallic reagents
with 2′- and 3′-ketouridine derivatives; synthesis of uracil
nucleosides branched at the 2′- and 3′-positions. Chem. Pharm.
Bull. 1987, 35, 2605-2608.
(10) (a) J ung, P. M. J .; Burger, A.; Biellmann, J .-F. Rapid and efficient
stereocontrolled synthesis of C-3′-ethynyl ribo and xylonucleo-
sides by organocerium addition to 3′-ketonucleosides. Tetrahe-
dron Lett. 1995, 36, 1031-1034. (b) J ung, P. M. J .; Burger, A.;
Biellmann, J .-F. Diastereofacial selective addition of ethynyl-
cerium reagent and Barton-McCombie reaction as the key steps
for the synthesis of C-3′-ethynylribonucleosides and of C-3′-
ethynyl-2′-deoxyribonucleosides. J . Org. Chem. 1997, 62, 8309-
8314.
(1) Ra d ioch em ica l Assa y. The substrate activity of
radiolabeled Cyd, Urd, ECyd, and EUrd by partially purified
UCK was assayed by a radiochemical method which quanti-
tated the formation of the corresponding radioactively labeled
5′-monophosphates. The reaction mixture, in a total volume
of 125 µL, consisted of 50 mM Tris-HCl buffer (pH 8.0), 10
mM ATP, 10 mM NaF, 5 mM MgCl2, 1 mg/mL BSA, 0.6 mM
tritiated nucleoside (75 nmol/tube, 20 µCi/tube), and 50 mL of
the enzyme solution. The mixture was incubated at 37 °C for
15 min (for Cyd and Urd) or 60 min (for ECyd and EUrd),
heated in a boiling water bath for 3 min, and centrifuged at
3000 rpm for 10 min; 10 µL of the supernatant was then
spotted onto a 2.5 × 10-cm PEI-cellulose TLC plate (Merck
TLC plates, PEI-cellulose F precoated) and developed with
water. Phosphorylated compound at the origin was scraped
into a vial and extracted with 0.5 mL of 1 M HCl; 10 µL of
scintillator was then added, and the radioactivity was mea-
sured.
(2) HP LC Assa y. Phosphorylation of Cyd, Urd, ECyd,
EUrd, 15a -d , 17c, and 50 was measured by HPLC which
quantitated the consumption of these nucleosides by partially
purified UCK. The reaction mixture was similar to that
described above except that tritium-labeled nucleoside was by
nonlabeled nucleoside. After centrifugation of the boiled
reaction mixture, 50 µL of the supernatant was diluted with
200 µL of water; 10 µL of the sample was then loaded onto a
Chemcosorb 300-5C18 column (4.6 × 250 cm; Chemco Co.,
Ltd.) under the following chromatographic conditions: moni-
toring wavelength, 270 nm; flow rate, 1 mL/min; mobile phase,
1.5% MeOH for Cyd (7.1 min) and Urd (7.5 min), 2.5% MeOH
for ECyd (7.1 min), EUrd (9.8 min), 15a (8.5 min), 15c (9.5
min), 15d (8.3 min), and 17c (10.4 min), or 7.5% MeOH for
15b (9.3 min) and 50 (7.3 min) containing 0.01% trifluoroacetic
acid. The numbers in parentheses indicate the retention time.
(11) Huss, S.; Camarasa, M. J . Synthesis of 3′-C-ethynylnucleosides
of thymine. Tetrahedron 1991, 47, 1727-1736.
(12) (a) Yoshimura, Y.; Iino, T.; Matsuda, A. Stereoselective radical
deoxygenation of tert-propargyl alcohols in sugar moiety of
pyrimidine nucleosides: Synthesis of 2′-C-alkynyl-2′-deoxy-1-â-
D-arabinofuranosyl-pyrimidines. Tetrahedron Lett. 1991, 32,
6003-6006. (b) Iino, T.; Yoshimura, Y.; Matsuda, A. Synthesis
of 2′-C-alkynyl-2′-deoxy-1-â-D-arabinofuranosylpyrimidines via
radical deoxygenation of tert-propargyl alcohols in the sugar
moiety. Tetrahedron 1994, 50, 10397-10406. (c) Iino, T.; Shuto,
S.; Matsuda, A. Stereoselective synthesis of (2′S)-2′-C-alkyl-2′-
deoxyuridines. Nucleosides Nucleotides 1996, 15, 169-181. (d)
Awano, H.; Shuto, S.; Miyashita, T.; Ashida, N.; Machida, H.;
Kira, T.; Shigeta, S.; Matsuda, A. Synthesis and antiviral activity
of 5-substituted (2′S)-2′-deoxy-2′-C-methylcytidines and -uri-
dines. Arch. Pharm. Pharm. Med. Chem. 1996, 329, 66-72.
(13) Takenuki, K.; Itoh, H.; Matsuda, A.; Ueda, T. On the stereose-
lectivity of alkyl addition reaction of pyrimidine 2′-ketonucleo-
sides. Chem. Pharm. Bull. 1990, 38, 2947-2952.
Ack n ow led gm en t . This investigation was sup-
ported in part by a Grant-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education,
Science, Sports, and Culture of J apan and by the
Second-Term Comprehensive Ten-year Strategy for
Cancer Control form the Ministry of Health and Welfare
of J apan.
Su p p or tin g In for m a tion Ava ila ble: NMR data for the
nontarget compounds (8 pages). Ordering information is given
on any current masthead page.
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