Nucleosides and nucleotides. 158. 1-(3-C-ethynyl-β-D-ribo- pentofuranosyl)-cytosine, 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)uracil, and their nucleobase analogues as new potential multifunctional antitumor nucleosides with a broad spectrum of activity

Article abstract of DOI:10.1021/jm960537g

We previously designed 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)uracil (EUrd) as a potential multifunctional antitumor nucleoside antimetabolite. It showed a potent and broad spectrum of antitumor activity against various human tumor cells in vitro and in vivo. To determine the structure-activity relationship, various nucleobase analogues of EUrd, such as 5-fluorouracil, thymine, cytosine, 5-fluorocytosine, adenine, and guanine derivatives, were synthesized by condensation of 1-O-acetyl-2,3,5-tri-O-benzoyl-3-C-ethynyl- α-β-D-ribo-pentofuranose (6) and the corresponding pertrimethylsilylated nucleobases in the presence of SnCl₄ or TMSOTf as a Lewis acid in CH₃CN followed by debenzoylation. The in vitro tumor cell growth inhibitory activity of these 3'-C-ethynyl nucleosides against mouse leukemia L1210 and human nasopharyngeal KB cells showed that 1-(3-C-ethynyl-β-D-ribo- pentofuranosyl)cytosine (ECyd) and EUrd were the most potent inhibitors in the series, with IC₅₀ values for L1210 cells of 0.016 and 0.13 μM and for KB cells of 0.028 and 0.029 μM, respectively. 5-Fluorocytosine, 5- fluorouracil, and adenine nucleosides showed much lower activity, with IC₅₀ values of 0.42.5 μM, while thymine and guanine nucleosides did not exhibit any activity up to 300 μM. We next evaluated the tumor cell growth inhibitory activity of ECyd and EUrd against 36 human tumor cell lines in vitro and found that they were highly effective against these cell lines with IC₅₀ values in the nanomolar to micromolar range. These nucleosides have a similar inhibitory spectrum. The in vivo antitumor activities of ECyd and EUrd were compared to that of 5-fluorouracil against 11 human tumor xenografts including three stomach, three colon, two pancreas, one renal, one breast, and one bile duct cancers. ECyd and EUrd showed a potent tumor inhibition ratio (73-92% inhibition relative to the control) in 9 of 11 and 8 of 11 human tumors, respectively, when administered intravenously for 10 consecutive days at doses of 0.25 and 2.0 mg/kg, respectively, while 5- fluorouracil showed potent inhibitory activity against only one tumor. Such excellent antitumor activity suggests that ECyd and EUrd are worth evaluating further for use in the treatment of human cancers.

Full text of DOI:10.1021/jm960537g

J . Med. Chem. 1996, 39, 5005-5011
5005
Nu cleosid es a n d Nu cleotid es. 158. 1-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)-
cytosin e, 1-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)u r a cil, a n d Th eir Nu cleoba se
An a logu es a s New P oten tia l Mu ltifu n ction a l An titu m or Nu cleosid es w ith a
Br oa d Sp ectr u m of Activity1
Hideshi Hattori,† Motohiro Tanaka,‡ Masakazu Fukushima,§ Takuma Sasaki,‡ and Akira Matsuda*,†
Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060, J apan, Cancer Research
Institute, Kanazawa University, Takara-machi, Kanazawa 920, J apan, and Cancer Research Laboratory-2, Taiho
Pharmaceutical Company Ltd., Hanno 357, J apan
Received J uly 25, 1996X
We previously designed 1-(3-C-ethynyl-â-D-ribo-pentofuranosyl)uracil (EUrd) as a potential
multifunctional antitumor nucleoside antimetabolite. It showed a potent and broad spectrum
of antitumor activity against various human tumor cells in vitro and in vivo. To determine
the structure-activity relationship, various nucleobase analogues of EUrd, such as 5-fluoro-
uracil, thymine, cytosine, 5-fluorocytosine, adenine, and guanine derivatives, were synthesized
by condensation of 1-O-acetyl-2,3,5-tri-O-benzoyl-3-C-ethynyl-R,â-D-ribo-pentofuranose (6) and
the corresponding pertrimethylsilylated nucleobases in the presence of SnCl4 or TMSOTf as a
Lewis acid in CH3CN followed by debenzoylation. The in vitro tumor cell growth inhibitory
activity of these 3′-C-ethynyl nucleosides against mouse leukemia L1210 and human naso-
pharyngeal KB cells showed that 1-(3-C-ethynyl-â-D-ribo-pentofuranosyl)cytosine (ECyd) and
EUrd were the most potent inhibitors in the series, with IC50 values for L1210 cells of 0.016
and 0.13 µM and for KB cells of 0.028 and 0.029 µM, respectively. 5-Fluorocytosine,
5-fluorouracil, and adenine nucleosides showed much lower activity, with IC50 values of 0.4-
2.5 µM, while thymine and guanine nucleosides did not exhibit any activity up to 300 µM. We
next evaluated the tumor cell growth inhibitory activity of ECyd and EUrd against 36 human
tumor cell lines in vitro and found that they were highly effective against these cell lines with
IC50 values in the nanomolar to micromolar range. These nucleosides have a similar inhibitory
spectrum. The in vivo antitumor activities of ECyd and EUrd were compared to that of
5-fluorouracil against 11 human tumor xenografts including three stomach, three colon, two
pancreas, one renal, one breast, and one bile duct cancers. ECyd and EUrd showed a potent
tumor inhibition ratio (73-92% inhibition relative to the control) in 9 of 11 and 8 of 11 human
tumors, respectively, when administered intravenously for 10 consecutive days at doses of 0.25
and 2.0 mg/kg, respectively, while 5-fluorouracil showed potent inhibitory activity against only
one tumor. Such excellent antitumor activity suggests that ECyd and EUrd are worth
evaluating further for use in the treatment of human cancers.
In tr od u ction
is believed to be due mainly to their inhibition of DNA
synthesis in tumor cells, in which they are metabolized
to their corresponding 5′-diphosphates and 5′-triphos-
phates and inhibit ribonucleoside diphosphate reductase
(RDPR) and/or DNA polymerases. However, many solid
tumor cells grow slowly, and a drug has few chances to
encounter S-phase, in which DNA synthesis occurs.
Therefore, it is not enough for nucleoside antimetabo-
lites to only inhibit DNA synthesis in tumor cells.
However, it has been reported that these 2′-deoxycyti-
dine analogues also inhibit RNA synthesis to some
extent.3g,4,5e Although their inhibition of RNA synthesis
would not be a major mechanism of tumor cell death, it
is conceivable that this inhibition may contribute to the
antitumor efficacy against slow-growing solid tumors.
Therefore, in our search for more potent inhibitors of
tumor cell growth, we designed a nucleoside antime-
tabolite which inhibited both DNA and RNA syntheses.
Inhibition of the nucleotide biosynthesis of tumor cells
by antimetabolites is a classic but still important
approach in cancer chemotherapy.2 Although a nucleo-
side antimetabolite has been recognized as one of the
most important agents in cancer chemotherapy, only
5-fluorouracil (5-FU) and its prodrugs are currently used
clinically in J apan against solid tumors, such as breast
carcinoma, colorectal carcinoma, gastric adenocarci-
noma, and pancreatic adenocarcinoma. 2′-Deoxycyti-
dine analogues, such as 1-(2-deoxy-2-methylene-â-D-
erythro-pentofuranosyl)cytosine (DMDC),3 2′-deoxy-2′,2′-
difluorocytidine (gemcitabine),4 and 1-(2-C-cyano-2-
deoxy-â-D-arabino-pentofuranosyl)cytosine (CNDAC),5
have been developed as potent antitumor agents which,
unlike 1-â-D-arabinofuranosylcytosine (araC), are effec-
tive not only against leukemias and lymphomas but also
against a wide variety of solid tumors in vitro as well
as in vivo. The antitumor activity of these nucleosides
To inhibit RNA synthesis, a nucleoside antimetabolite
should be recognized as a substrate and/or an inhibitor
of ribonucleoside- and ribonucleotide-metabolizing en-
zymes, such as nucleoside and nucleotide kinases, CTP
synthetase, and RNA polymerases, and therefore should
* Author to whom all correspondence and reprint requests should
be addressed.
† Hokkaido University.
‡ Kanazawa University.
§ Taiho Pharmaceutical Co. Ltd.
X Abstract published in Advance ACS Abstracts, November 1, 1996.
S0022-2623(96)00537-7 CCC: $12.00 © 1996 American Chemical Society

5006 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Hattori et al.
Sch em e 1a
have two hydroxyl groups at the 2′- and 3′-positions with
a ribo-configuration.
On the other hand, RDPR is one of the most impor-
tant target enzymes for the inhibition of DNA synthe-
sis.6 RDPR catalyzes the conversion of all of the four
main ribonucleoside 5′-diphosphates (rNDPs) to the
corresponding 2′-deoxyribonucleoside 5′-diphosphates
(dNDPs) in a de novo pathway. The resulting dNDPs
are further phosphorylated to the corresponding 2′-
deoxyribonucleoside 5′-triphosphates (dNTPs), which
are essential for DNA synthesis, by nucleoside diphos-
phate kinase. Escherichia coli RDPR, which is a
prototype of mammalian enzymes, is the best character-
ized. The enzyme consists of two subunits, R1 and R2,
each of which is a homodimer. The R2 subunit contains
a tyrosyl radical stabilized by a µ-oxo-bridged binuclear
iron(III) center.7 This radical is transferred to certain
amino acid residues in the active site of the R1 subunit
in the catalytic site, which initiates the enzyme-
catalyzed reduction of the 2′-hydroxyl group in rNDPs,
by abstraction of the 3′-hydrogen atom in the ribose
moiety of the rNDPs.8 The amino acid residue corre-
sponding to radical formation has been postulated to
be Cys439 based on the results of site-directed mu-
tagenesis of the R1 subunit.9 X-ray crystallographic
studies of the R1 subunit have also suggested that
Cys439 is near the 3′-hydrogen of rNDP.10 Therefore,
as a functional group to react effectively and directly
with the thiyl radical, we selected an acetylene group
to be introduced at the 3′-position, since acetylene
derivatives are well-known to react with thiyl radicals
to form alkylthio vinyl sulfides.11
On the basis of these considerations, we previously
designed 1-(3-C-ethynyl-â-D-ribo-pentofuranosyl)uracil
(EUrd) as a potential multifunctional antitumor nucleo-
side and found that it showed potent tumor cell growth
inhibitory activity against various human tumor cell
lines in vitro and strong antitumor activity against
murine leukemia P388 and a variety of human solid
tumor xenografts in vivo.12 In this paper, we describe
the synthesis of various nucleobase analogues of EUrd,
such as 5-fluorouracil, thymine, cytosine, 5-fluorocy-
tosine, adenine, and guanine derivatives, to determine
the structure-activity relationship of tumor cell growth
inhibitory activity in vitro. We also evaluated the
antitumor activity of EUrd and its cytosine analogue
against various human tumor xenografts in vivo.
a
Conditions: (a) TMSCtCH, BuLi, THF, -78 °C, 99%; (b) (i)
Bu4NF, THF, rt, (ii) BzCl, pyridine, rt, 93% in two steps; (c)
aqueous 20% HCl, MeOH, rt; (d) BzCl, DMAP, pyridine, 100 °C,
87% in two steps; (e) concentrated H2SO4, AcOH, Ac2O, rt, 93%.
in 72% yield along with methyl 5-O-benzoyl-3-C-ethynyl-
2,3-O-isopropylidene-â-D-ribo-pentofuranose in 24% yield.
Subsequent acetolysis of 5 using concentrated H2SO4
in a mixture of AcOH and Ac2O gave 1-O-acetyl-2,3,5-
tri-O-benzoyl-3-C-ethynyl-R,â-D-ribo-pentofuranose (6)
in 93% yield as a syrup.
We next investigated the condensation of 6 with
pertrimethylsilylated nucleobases under Vorbru¨ggen
conditions.16 When 1.2 equiv of bis(TMS)cytosine was
reacted with 6 in the presence of 2 equiv of SnCl4 in
CH3CN, the desired 1-(2,3,5-tri-O-benzoyl-3-C-ethynyl-
â-D-ribo-pentofuranosyl)cytosine (7a ) was obtained as
a major product in 59% yield along with 8 in 26% yield.
To improve the yield of 7a , increased amounts of bis-
(TMS)cytosine and SnCl4 to 4 and 5 equiv, respectively,
were used to give 7a in 81%. A further increase in the
above reagents gave only a complex mixture. In a
similar manner, 5-fluorocytosine, 5-fluorouracil, thy-
mine, N6-benzoyladenine, and N2-acetylguanine nucleo-
sides 7b, 9b,c, 11, and 12, respectively, were prepared
in good yields by the condensation of 6 and the corre-
sponding pertrimethylsilylated nucleobases. During the
condensation with N2-acetylguanine, the N9-riboside 12
was obtained in 99% yield without detection of its N7-
isomer, which is usually observed during glycosylation
reactions of guanine derivatives with appropriate sug-
ars.
Condensation of bis(TMS)uracil (4 equiv) with 6 in
CH3CN in the presence of SnCl4 (4 equiv) gave a
mixture of N1-â-nucleoside 9a and N3-â-nucleoside 10
in yields of 58% and 20%, respectively. The structures
of these nucleosides were identified by comparison of
their UV spectra after deblocking (see the Experimental
Section). However, when trimethylsilyl triflate (4 equiv;
TMSOTf)17 was used as a Lewis acid instead of SnCl4,
the yield of the desired 9a was improved to 95%.
For deblocking of these nucleosides, 7a ,b, 9a -c, and
10-12 were treated with NH3/MeOH at room temper-
ature to give the corresponding 3′-C-ethynyl nucleosides
13a ,b, 14a -c, and 15-17 in good yields as crystals. The
structures of these nucleosides were confirmed by H-
NMR, mass spectrometry, and IR and UV spectropho-
tometry, along with elemental analyses.
Recently, J ung et al. reported the stereoselective
addition of ethynylcerium reagents to 5′-hydroxy-3′-keto
nucleosides from the â-face to give 3′-C-ethynyl-â-D-ribo-
pentofuranosyl nucleosides, although the reaction of 5′-
O-TBDMS-3′-keto derivatives with the same reagents
Syn th esis
The synthesis of 3-C-ethynyl-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.13 Addition of LiCtCTMS to 1 gave
the desired â-adduct 2 in 99% yield with high stereo-
selectivity.14 Desilylation of 2 with tetrabutylammo-
nium fluoride (TBAF) followed by benzoylation gave 3
in 93% yield. Treatment of 3 with aqueous 20% HCl in
MeOH for 6 h at room temperature gave methyl 5-O-
benzoyl-3-C-ethynyl-R,â-D-ribo-pentofuranoside (4), which
was subsequently benzoylated in a mixture of BzCl and
DMAP in anhydrous pyridine at 100 °C to give 5 in 87%
yield. When this benzoylation was performed at room
temperature, benzoylation of the tertiary alcohol at the
3-position did not occur.15 Furthermore, when 3 was
treated with anhydrous HCl/MeOH at room tempera-
ture for 2 days, followed by benzoylation, 5 was obtained
1

Antitumor Nucleosides with a Broad Spectrum of Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 5007
Sch em e 2a
a
Conditions: (a) pertrimethylsilylated nucleobase, SnCl4, CH3CN; (b) pertrimethylsilylated nucleobase, TMSOTf, CH3CN; (c) NH3/
MeOH.
gave 3′-C-ethynyl-â-D-xylo-pentofuranosyl nucleosides.18
However, for preparing the various nucleobase ana-
logues of the 3′-C-ethynyl-â-D-ribo-pentofuranosyl nu-
cleosides in this study, our glycosylation method is
superior because once an appropriate sugar precursor
for the condensation is synthesized, condensation with
the appropriate nucleobases gives a variety of the
desired nucleosides.
Ta ble 1. Inhibitory Effects of Various
3′-C-Ethynyl-â-D-ribo-pentofuranosyl Nucleosides on the
Growth of L1210 and KB Cells in Vitroa
IC50 (µM)
compounds
L1210
0.016
0.53
0.13
2.5
>300
0.69
>300
KB
0.028
0.46
0.029
1.4
ECyd (13a )
EFCyd (13b)
EUrd (14a )
EFUrd (14b)
EMUrd (14c)
EAdo (16)
>300
0.83
>300
Biologica l Activity
In vitro tumor cell growth inhibitory activity of 3-C-
ethynyl-â-D-ribo-pentofuranose nucleosides ECyd (13a ),
EFCyd (13b), EUrd (14a ), EFUrd (14b), EMUrd (14c),
EAdo (16), and EGuo (17) against mouse leukemia
L1210 and human nasopharyngeal KB cell lines was
investigated first. The results are summarized in Table
1. ECyd was the most effective inhibitor of tumor cell
proliferation in this series of nucleosides, with IC50
values of 16 and 28 nM against L1210 and KB cells,
respectively. EUrd was 8 times less active against
L1210 cells than ECyd but was equally active against
KB cells. The introduction of a fluorine atom to the
5-position of ECyd and EUrd reduced the activity about
20-50-fold, and EMUrd had no activity against either
cell line. These data may reflect the substrate specific-
ity of the first activation enzymes of these nucleosides,
such as uridine/cytidine kinase, which would recognize
the bulky substituents at the 5-position. EAdo exhibited
activity similar to EFCyd, while EGuo did not have such
properties.
EGuo (17)
a
Tumor cell growth inhibitory activity assay in vitro was
performed following the method of Carmichael et al.21 Tumor cells
(2 × 103 cells/well) were incubated in the presence or absence of
the compounds for 72 h. MTT reagent was added to each well,
and the plate was incubated for an additional 4 h. 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) represents the concentration at
which cell growth was inhibited by 50%.
two osteo, one leiomyo, one fibro, two melanoma, and
one nasopharyngeal tumor cell lines in vitro. The
results are summarized in Table 2. ECyd was effective
against 22 cell lines with IC50 values in the nanomolar
to subnanomolar range and was ineffective against two
cell lines. EUrd had potent inhibitory activity against
12 cell lines with IC50 values in the subnanomolar range
and was ineffective against six cell lines. We previously
compared the in vitro tumor cell growth inhibitory
activity of EUrd against several human tumor cell lines
with those of 5-fluorouridine (FUrd), 2′-deoxy-5-fluo-
rouridine (FdUrd), and 5-fluorouracil (5-FU) and found
that EUrd was about 6-650 times more potent than
We next evaluated ECyd and EUrd against 36 human
solid tumor cell lines, including eight stomach, four
colon, ten lung, three breast, two pancreas, two bladder,

5008 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Hattori et al.
Ta ble 2. Inhibitory Effects of ECyd and EUrd on the Growth
of Human Tumor Cell Lines in Vitroa
potent antitumor effect against the human tumor xe-
nografts used in this study, only one-eighth as much
ECyd was required as EUrd. This reflects the in vitro
potency of both nucleosides. The doses of ECyd, EUrd,
and FU used in this study were each the respective
maximum nontoxic dose. Since multiple-dose schedules
were not tested, we may not have used these drugs
under optimal conditions.
Although the optimal therapeutic schedule of ECyd
and EUrd is not yet known, such excellent antitumor
activity suggests that ECyd and EUrd are worth further
evaluation for use in the treatment of human cancer.
Further studies are in progress to determine whether
the mechanism responsible for their antitumor proper-
ties is related to the inhibition of both DNA and RNA
syntheses. If ECyd and EUrd act as multifunctional
antitumor nucleosides, they would represent a new
approach in anticancer chemotherapy.
IC50 (µM)
cell lines
MKN-28
MKN-45
KATO-III
NUGC-4
ST-KM
KKLS
STSA-1
NAKAJ IMA
Colo320DM
HCT-15
SW-48
SW-480
PC-8
PC-9
PC-10
QG-56
QG-95
Lu-65
QG-90
origin
ECyd
EUrd
stomach adenocarcinoma
0.21
0.065
0.052
0.031
0.22
0.086
0.23
1.9
0.78
0.06
0.075
>1.9
0.34
0.33
0.19
>1.9
0.15
0.28
0.089
1.0
0.0088
0.031
0.024
0.047
0.021
0.15
0.17
0.02
0.0082
>1.9
0.26
0.090
0.11
0.16
0.045
0.21
0.032
0.13
colon adenocarcinoma
lung adenocarcinoma
lung squamous-cell carcinoma
lung large-cell carcinoma
lung small-cell carcinoma
Exp er im en ta l Section
QG-96
NCI-H-82
NCI-H-417
MDA-MB-321 breast adenocarcinoma
YMB-1-E
MCF-7
0.39
0.12
0.043
0.024 >1.9
0.16
0.069
0.38
0.23
0.11
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
>1.9
0.2
1
ionizing voltage of 70 eV. The H-NMR spectra were recorded
on a J eol J NM-GX 270 (270 MHz) spectrometer or Bruker ARX
500 (500 MHz) spectrometer with tetramethylsilane 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 recorded 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-[2′-(tr im eth ylsilyl)eth yn yl]-r-D-r ibo-p en tofu r a n ose (2).
A hexane solution of BuLi (1.62 M, 27.8 mL, 45 mmol) was
added dropwise over 30 min to a solution of (trimethysilyl)-
acetylene (6.3 mL, 45 mmol) in THF (60 mL) at -78 °C under
Ar atmosphere. After being stirred for a further 30 min, a
solution of 5-O-(tert-butyldimethylsilyl)-1,2-O-isopropylidene-
R-D-erythro-pentos-3-ulose (1; 4.5 g, 15 mmol)13 in THF (30 mL)
was added dropwise over 10 min to the acetylide solution at
the same temperature. The mixture was stirred for 3 h and
then the reaction quenched by the addition of aqueous NH4Cl
solution (1 M, 60 mL). The mixture was warmed to room
temperature and extracted by EtOAc (35 mL × 3), which was
further washed with brine (20 mL × 3). The separated organic
phase was dried (Na2SO4) and concentrated to dryness in
vacuo. The residue was purified on a silica gel column (7.5 ×
8 cm), which was eluted with 5% EtOAc in hexane to give 2
(5.92 g, 99% as a white powder, which was crystallized from
PANC-1
Mia-PaCa-2
T24
KK-47
HOS
MG-63
A431
HT-1080
A375
pancreas adenocarcinoma
bladder carcinomab
osteosarcoma
>1.9
>1.9
0.015
0.028
0.12
0.054
0.21
0.089
1.8
>1.9
0.29
1.0
0.15
leiomyosarcoma
fibrosarcoma
melanoma
0.033
0.073
0.026
0.047
0.028
0.16
0.021
0.086
0.029
SK-MEL-28
KB
pharyngeal carcinoma
a
Tumor cell growth inhibitory activity assay in vitro was done
b
as described for Table 1. Transitional cell.
5-FU and comparable to FUrd and FdUrd.12 Therefore,
the activity of ECyd was superior to that of EUrd and
5-FU derivatives in vitro. On the basis of the mean
graph analysis19 (data not shown), both ECyd and EUrd
have a similar inhibitory activity spectrum and there-
fore may have a similar mechanism of action for
antitumor activity.
We next examined the antitumor effects of ECyd and
EUrd20 against human tumors (three stomach, three
colon, two pancreas, one renal, one breast, and one bile
duct cancers) as xenografts in nude mice in comparison
with 5-FU. The results are shown in Table 3. In this
experiment, the tumor inhibition ratio (%) was used as
a parameter of antitumor activity, and day 15 was
chosen for the final evaluation of the effects of treatment
on tumor growth. ECyd had a potent antitumor effect
on 9 of 11 tumor xenografts and a moderate effect on
Hucc-T1 bile duct and BxPC-3 pancreas tumor xe-
nografts, while EUrd showed a similar potency to ECyd
for 8 of 11 tumors but was only moderately effective
against NUGC-3 stomach, Hucc-T1 bile duct, and BxPC
pancreas tumors, when given in a daily intravenous
dose of 0.25 and 2 mg/kg, respectively, for 10 consecutive
days. We next compared the antitumor activity of 5-FU,
which has been used clinically against solid tumors,
especially stomach, colon, and breast cancers. When
5-FU was administered intravenously 14 consecutive
days at a dose of 15 mg/kg, it had a potent antitumor
effect only on H-81 stomach tumor but showed moderate
antitumor effects on other human tumors. To show a
hexane): mp 84-86 °C; FAB-MS m/ z 401 (MH+), 383 (M+
-
1
OH); H-NMR (CDCl3) 5.85 (d, 1 H, H-1, J 1,2 ) 3.8 Hz), 4.56
(d, 1 H, H-2, J 2,1 ) 3.8 Hz), 4.01-3.94 (m, 3 H, H-4, H-5), 3.05
(s, 1 H, 3-OH), 1.56, 1.37 (s, each 3 H, i-Pr), 0.91 (s, 9 H, t-Bu),
0.18 (s, 9 H, Me × 3), 0.06, 0.03 (s, each 3 H, Me). Anal.
(C19H36O5Si2) C, H.
5-O-Ben zoyl-3-C-eth yn yl-1,2-O-isopr opyliden e-r-D-r ibo-
p en tofu r a n ose (3). A THF solution of TBAF (1 M, 5.4 mL,
5.4 mmol) was added to a solution of 2 (1.44 g, 3.6 mmol) in
THF (15 mL). The mixture was stirred for 10 min at room
temperature, and the solvent was removed in vacuo. The
residue was coevaporated with anhydrous pyridine (5 mL ×
3) and dissolved in anhydrous pyridine (30 mL). BzCl (0.92
mL, 7.9 mmol) was added to the above solution at 0 °C, and
the mixture was stirred for 2 h at room temperature. The
solvent was removed in vacuo, and the residue was coevapo-
rated with toluene (×3). The residue was taken up in EtOAc
(50 mL) which was washed with H2O (25 mL) and aqueous
saturated NaHCO3 (25 mL × 3). The separated organic phase

Antitumor Nucleosides with a Broad Spectrum of Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 5009
Ta ble 3. Antitumor Effects of ECyd, EUrd, and 5-FU on Human Tumor Xenografts in Nude Micea
tumor inhibition ratio (%)
ECydb
EUrdc
5-FUd
tumor
origin
0.25 mg/kg × 10
2.0 mg/kg × 10
15 mg/kg × 14
AZ-521
H-81
NUGC-3
CO-3
KM12C
DLD-1
J RC-11
H-31
Hucc-T1
PAN-12
BxPC-3
stomach
stomach
stomach
colon
colon
colon
renal
breast
bile duct
pancreas
pancreas
92
84
80
83
88
78
73
76
55
75
59
81
92
54
90
81
73
75
82
64
81
35
25
83
ND
49
59
50
69
68
1
40
50
a
Tumor mass (2 mm3) was subcuanteously transplanted into nude mice. The tumor inhibition ratio was evaluated at day 15 and
calculated as follows: inhibition ratio (%) ) (A - B)/A × 100, where A is the average tumor weight in the control group and B is that in
b
the treated group. Each group consisted of eight nude mice. ECyd was administered intravenously for 10 consecutive days when the
tumor reached 60-100 mm3. c EUrd was administered intravenously for 10 consecutive days when the tumor reached 60-100 mm3.
d
Some of the data were taken from ref 12. 5-FU was administered intravenously for 14 consecutive days when the tumor reached 60-
100 mm3.
was dried (Na2SO4) and concentrated to dryness. The residue
was purified on a silica gel column (4 × 17 cm), which was
eluted with 0-10% EtOAc in hexane to give 3 (1.07 g, 93% as
a yellowish oil): FAB-MS m/ z 319 (MH+), 303 (M+ - Me); 1H-
with CHCl3 (10 mL) and successively washed with H2O (0.4
mL), aqueous saturated NaHCO3 (1.2 mL × 2), and H2O (0.4
mL × 2). The separated organic phase was dried (Na2SO4)
and concentrated to dryness in vacuo. The residue was
purified on a silica gel column (2.5 × 6 cm), which was eluted
with CHCl3 to give 6 (259 mg, 93% as an oil): EI-MS m/ z 528
(M+), 485 (M+ - Ac); 1H-NMR (CDCl3) 8.17-7.32 (m, 15 H,
Bz × 3), 6.77 (d, 0.33 H, R-H-1, J 1,2 ) 4.6 Hz), 6.39 (d, 0.67 H,
â-H-1, J 1,2 ) 1.5 Hz), 6.19 (d, 0.67 H, â-H-2, J 2,1 ) 1.5 Hz),
6.07 (d, 0.33 H, R-H-2, J 2,1 ) 4.6 Hz), 5.07-4.79 (m, 3 H, R,â-
H-4, H-5), 2.89 (s, 0.67 H, â-3-CtCH), 2.81 (s, 0.33 H, R-3-
CtCH), 2.14 (s, 2 H, â-Ac), 2.00 (s, 1 H, R-Ac). The ratio of
R: â anomer was 1:2. Anal. (C30H24O9) C, H.
1-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)cytosin e (13a ).
Stannic chloride (0.29 mL, 2.5 mmol) was added to a solution
of bis(TMS)cytosine [prepared from cytosine (222 mg, 2 mmol)
and (NH4)2SO4 (7 mg) in HMDS (2 mL)] and 6 (264 mg, 0.5
mmol) in CH3CN (4 mL) at 0 °C. The reaction mixture was
stirred for 18 h at room temperature and then diluted with
CHCl3 (12 mL) and aqueous saturated NaHCO3 (5 mL) with
vigorous stirring for 30 min. The resulting precipitates were
removed by filtration through a Celite pad, which was washed
with CHCl3. The filtrate was further washed with H2O (5 mL
× 2) and aqueous saturated NaHCO3 (5 mL). The separated
organic phase was dried (Na2SO4) and concentrated to dryness
in vacuo. The residue was purified on a silica gel column (2
× 15 cm), which was eluted with 5% MeOH in CHCl3 to give
7a (235 mg, 81% as a foam): FAB-MS m/ z 580 (MH+); 1H-
NMR (CDCl3) 8.15-7.31 (m, 15 H, Bz × 3), 7.76 (d, 1 H, H-6,
J 6,5 ) 7.5 Hz), 6.60 (d, 1 H, H-1′, J 1′,2′ ) 5.2 Hz), 6.06 (d, 1 H,
H-2′, J 2′,1′ ) 5.2 Hz), 5.72 (d, 1 H, H-5, J 5,6 ) 7.5 Hz), 4.96-
4.89 (m, 3 H, H-4′, H-5′), 2.88 (s, 1 H, 3′-CtCH).
NMR (CDCl3) 8.12-7.42 (m, 5 H, Bz), 5.97 (d, 1 H, H-1, J 1,2
)
3.8 Hz), 4.76 (dd, 1 H, H-5a, J 5a,4 ) 3.8 Hz, J 5a,b ) 12.0 Hz),
4.61 (dd, 1 H, H-5b, J 5b,4 ) 7.4 Hz, J 5b,a ) 12.0 Hz), 4.60 (d, 1
H, H-2, J 2, 1 ) 3.8 Hz), 4.23 (dd, 1 H, H-4, J 4,5a ) 3.8 Hz, J 4,5b
) 7.4 Hz), 3.02 (s, 1 H, 3-OH), 2.63 (s, 1 H, 3-CtCH), 1.62,
1.41 (s, each 3 H, i-Pr). Anal. (C17H18O6) C, H.
Meth yl 2,3,5-Tr i-O-ben zoyl-3-C-eth yn yl-r,â-D-r ibo-pen to-
fu r a n osid e (5). (A) A solution containing 3 (637 mg, 2.0
mmol) and camphorsulfonic acid (1.16 g, 5.0 mmol) in MeOH
(27 mL) was heated under reflux for 3 days under Ar
atmosphere. The solvent was removed, and the residue was
taken up in EtOAc (30 mL), which was washed with H2O (15
mL) and aqueous saturated NaHCO3 (15 mL × 3). The
separated organic phase was dried (Na2SO4) and concentrated
to dryness to give a crude 4, which was coevaporated with
anhydrous pyridine (×3) and dissolved into anhydrous pyridine
(30 mL). BzCl (2.3 mL, 20 mmol) and DMAP (370 mg, 3.0
mmol) were added to the above mixture at 0 °C, and the whole
was heated for 24 h at 100 °C. The solvent was removed in
vacuo and coevaporated with toluene (×3). The residue was
taken into EtOAc (20 mL), which was washed with H2O (10
mL) and aqueous saturated NaHCO3 (10 mL × 3). The
separated organic phase was dried (Na2SO4) and concentrated
to dryness in vacuo. The residue was purified on a silica gel
column (4 × 11 cm), which was eluted with 0-10% EtOAc in
hexane to give 5 (825 mg, 83% as a yellowish oil): EI-MS m/ z
500 (M+); 1H-NMR (CDCl3) 8.12-7.30 (m, 15 H, Bz × 3), 6.03
(d, 0.67 H, â-H-1, J 1, 2 ) 1.2 Hz), 5.82 (d, 0.33 H, R-H-1, J 1,2
)
4.4 Hz), 5.47 (d, 0.33 H, R-H-2, J 2,1 ) 4.4 Hz), 5.17 (d, 0.67 H,
â-H-2, J 2,1 ) 1.2 Hz), 5.10-4.78 (m, 3 H, R,â-H-4, H-5), 3.53
(s, 2 H, â-OMe), 3.45 (s, 1 H, R-OMe), 2.86 (s, 0.67 H, â-3-
CtCH), 2.78 (s, 0.33 H, R-3-CtCH). The ratio of R: â anomer
was 1:2. Anal. (C29H24O8) C, H.
Compound 7a (200 mg, 0.35 mmol) was treated with
methanolic ammonia (saturated at 0 °C, 9 mL) for 2 days at
room temperature, and the solvent was removed in vacuo. The
residue was purified on a silica gel column (2 × 15 cm), which
was eluted with 5-20% MeOH in CHCl3 to give 13a (83 mg,
90% as a white powder, which was crystallized from aqueous
(B) Compound 3 (318 mg, 1 mmol) was treated with aqueous
methanolic HCl [prepared from a mixture of AcCl (1.94 mL,
27.3 mmol), H2O (3.2 mL), and MeOH (8.86 mL)] for 6 h at
room temperature. The mixture was neutralized with Et3N
(4.5 mL), and the solvent was removed in vacuo. The residue
was partitioned between EtOAc (15 mL) and aqueous satu-
rated NaHCO3 (7 mL × 3), and the separated organic phase
was dried (Na2SO4) and concentrated to dryness. The residue
was further treated with BzCl (1.16 mL, 10 mmol) and DMAP
(184 mg, 1.5 mmol) in pyridine (15 mL) under heating at 100
°C for 24 h. The similar workup and purification gave 5 (435
mg, 87% as a yellowish oil; the ratio of R: â anomer was 1:2).
1-O-Acet yl-2,3,5-t r i-O-b en zoyl-3-C-et h yn yl-r,â-D-r ibo-
p en tofu r a n ose (6). Concentrated H2SO4 (0.11 mL) was
added to a solution of 5 (264 mg, 0.53 mmol) in a mixture of
AcOH (1.75 mL) and Ac2O (0.22 mL) at 0 °C. The whole was
stirred for 4 h at room temperature. The mixture was diluted
MeOH): mp 233-235 °C; EI-MS m/ z 267 (M+), 250 (M+
-
OH); IR (Nujol) 2115 cm-1 (CtC); UV λmax (H2O) 270 nm (ꢀ
9 100), λmax (0.5 N HCl) 280 nm (ꢀ 13 400), λmax (0.5 N NaOH)
1
272 nm (ꢀ 9 300); H-NMR (DMSO-d6) 7.86 (d, 1 H, H-6, J 6,5
) 7.7 Hz), 7.25, 7.18 (br s, each 1 H, NH), 5.88 (d, 1 H, H-1′,
J 1′,2′ ) 6.6 Hz), 5.84 (s, 1 H, 3′-OH), 5.79 (d, 1 H, 2′-OH, J 2′-OH,2′
) 6.6 Hz), 5.78 (d, 1 H, H-5, J 5,6 ) 7.7 Hz), 5.06 (dd, 1 H, 5′-
OH, J 5′-OH,5′a ) 4.4 Hz, J 5′-OH,5′b ) 5.5 Hz), 4.16 (t, 1 H, H-2′,
J ) 6.6 Hz), 3.92-3.89 (m, 1 H, H-4′), 3.74-3.71 (m, 2 H, H-5′),
3.55 (s, 1 H, 3′-CtCH). Anal. (C11H13N3O5) C, H, N.
1-(3-C-Eth yn yl-â-D-r ibo-pen tofu r an osyl)u r acil (14a). (A)
Trimethylsilyl triflate (0.39 mL, 2 mmol) was added to a
solution of bis(TMS)uracil [prepared from uracil (225 mg, 2
mmol) and (NH4)2SO4 (7 mg) in HMDS (2 mL)] and 6 (264 mg,
0.5 mmol) in CH3CN (4 mL) at 0 °C. The mixture was stirred
for 5 h at room temperature and then diluted with CHCl3 (12

5010 J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25
Hattori et al.
mL), which was further stirred with aqueous saturated
NaHCO3 (5 mL) for 30 min. The whole was washed with H2O
(5 mL × 2) and aqueous saturated NaHCO3 (5 mL). The
separated organic phase was dried (Na2SO4) and concentrated
to dryness in vacuo. The residue was purified by a silica gel
column (2 × 9 cm), which was eluted with CHCl3 to give 9a
(274 mg, 95% as a foam): FAB-MS m/ z 581 (MH+); 1H-NMR
(CDCl3) 8.13-7.30 (m, 15 H, Bz × 3), 8.05 (br s, 1 H, NH),
7.71 (d, 1 H, H-6, J 6,5 ) 8.2 Hz), 6.38 (d, 1 H, H-1′, J 1′,2′ ) 5.0
Hz), 6.01 (d, 1 H, H-2′, J 2′,1′ ) 5.0 Hz), 5.74 (dd, 1 H, H-5, J 5,6
) 8.2 Hz, J 5,NH ) 2.0 Hz), 4.98-4.87 (m, 3 H, H-4′, H-5′), 2.92
(s, 1 H, 3′-CtCH).
Compound 9a (150 mg, 0.26 mmol) was treated with
methanolic ammonia (saturated at 0 °C, 10 mL) for 2 days at
room temperature. The solvent was removed in vacuo, and
the residue was purified on a silica gel column (2 × 10 cm),
which was eluted with 5-15% MeOH in CHCl3 to give 14a
(59.4 mg, 85% as a white powder, which was crystallized from
MeOH): mp 226-228 °C; EI-MS m/ z 268 (M+); IR (Nujol)
2110 cm-1 (CtC); UV λmax (H2O) 261 nm (ꢀ 9 900), λmax (0.5 N
HCl) 261 nm (ꢀ 10 200), λmax (0.5 N NaOH) 262 nm (ꢀ 7 500);
1H-NMR (DMSO-d6) 11.35 (br s, 1 H, NH), 7.99 (d, 1 H, H-6,
J 6,5 ) 8.2 Hz), 5.93 (s, 1 H, 3′-OH), 5.86 (d, 1 H, 2′-OH, J 2′-OH,2′
) 6.7 Hz), 5.83 (d, 1 H, H-1′, J 1′,2′ ) 7.3 Hz), 5.69 (d, 1 H, H-5,
J 5,6 ) 8.2 Hz), 5.13 (t, 1 H, 5′-OH, J ) 4.5 Hz), 4.18 (dd, 1 H,
H-2′, J 2′,1′ ) 7.3 Hz, J 2′,2′-OH ) 6.7 Hz), 3.90-3.88 (m, 1 H, H-4′),
3.74-3.60 (m, 2 H, H-5′), 3.55 (s, 1 H, 3′-CtCH). Anal.
(C11H12N2O6) C, H, N.
(B) Stannic chloride (0.23 mL, 2.0 mmol) was added to a
solution of bis(TMS)uracil [prepared from uracil (225 mg, 2
mmol) and (NH4)2SO4 (7 mg) in HMDS (2 mL)] and 6 (264 mg,
0.5 mmol) in CH3CN (4 mL) at 0 °C. The mixture was stirred
for 2 days at room temperature, and workup was done as
described in the method A. The residue was purified on a silica
gel column (2 × 9 cm), which was eluted with CHCl3 to give
9a (167 mg, 58% as a foam). Further elution of the column
with 5% MeOH in CHCl3 gave 10 (57 mg, 20% as a foam).
Physical data of 10: FAB-MS m/ z 581 (MH+); 1H-NMR
(CDCl3) 9.26 (br s, 1 H, NH), 8.14-7.29 (m, 16 H, Bz × 3, H-6),
6.92 (d, 1 H, H-1′, J 1′,2′ ) 6.8 Hz), 6.69 (d, 1 H, H-2′, J 2′,1′ ) 6.8
Hz), 5.82 (d, 1 H, H-5, J 5,6 ) 7.6 Hz), 5.04-4.88 (m, 3 H, H-4′,
H-5′), 2.82 (s, 1 H, 3′-CtCH).
methanolic ammonia (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, which was eluted
with 5-20% MeOH in CHCl3 to give the desired 3′-C-ethynyl
ribonucleosides.
1-(3-C-E t h yn yl-â-D-r ibo-p en t ofu r a n osyl)-5-flu or ocy-
tosin e (13b). Reaction of pertrimethylsilylated 5-fluorocy-
tosine (2 mmol) with 6 for 12 h at room temperature gave 7b
(224 mg, 75% as a foam): FAB-MS m/ z 598 (MH+); 1H-NMR
(CDCl3) 8.15-7.31 (m, 15 H, Bz × 3), 7.77 (d, 1 H, H-6, J 6,5-F
) 6.1 Hz), 6.54 (d, 1 H, H-1′, J 1′,2′ ) 5.1 Hz), 6.03 (d, 1 H, H-2′,
J 2′,1′ ) 5.1 Hz), 4.97-4.96 (m, 2 H, H-5′), 4.91-4.89 (m, 1 H,
H-4′), 2.90 (s, 1 H, 3′-CtCH).
From 7b (189 mg, 0.32 mmol), 13b (81 mg, 89% as a white
powder, which was crystallized from MeOH) was obtained.
13b: mp 242 °C dec; EI-MS m/ z 285 (M+); IR (Nujol) 2115
cm-1 (CtC); UV λmax (H2O) 280 nm (ꢀ 7 800), λmax (0.5 N HCl)
1
290 nm (ꢀ 11 400), λmax (0.5 N NaOH) 280 nm (ꢀ 8 400); H-
NMR (DMSO-d6) 8.11 (d, 1 H, H-6, J 6,5-F ) 7.2 Hz), 7.82, 7.57
(br s, each 1 H, NH), 5.84 (dd, 1 H, H-1′, J 1′,2′ ) 6.9 Hz, J 1′,5-F
) 1.9 Hz), 5.81 (s, 1 H, 3′-OH), 5.72 (d, 1 H, 2′-OH, J 2′-OH,2′
)
6.6 Hz), 5.14 (t, 1 H, 5′-OH, J ) 4.5 Hz), 4.13 (dd, 1 H, H-2′,
J 2′,1′ ) 6.9 Hz, J 2′,2′-OH ) 6.6 Hz), 3.88-3.87 (m, 1 H, H-4′),
3.75-3.60 (m, 2 H, H-5′), 3.53 (s, 1 H, 3′-CtCH). Anal.
(C11H12FN3O5) C, H, F, N.
1-(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)-5-flu o r o -
u r a cil (14b). Reaction of pertrimethylsilylated 5-fluorouracil
(2 mmol) with 6 for 6.5 h at room temperature gave 9b (283
mg, 95% as a foam): FAB-MS m/ z 599 (MH+); 1H-NMR
(CDCl3) 8.16 (br s, 1 H, NH), 8.14-7.30 (m, 15 H, Bz × 3),
7.83 (d, 1 H, H-6, J 6,5-F ) 5.8 Hz), 6.35 (dd, 1 H, H-1′, J 1′,2′
)
4.9 Hz, J 1′,5-F ) 1.7 Hz), 5.79 (d, 1 H, H-2′, J 2′,1′ ) 4.9 Hz),
4.96-4.95 (m, 2 H, H-5′), 4.89-4.87 (m, 1 H, H-4′), 2.95 (s, 1
H, 3′-CtCH).
From 9b (276 mg, 0.46 mmol), 14b (121 mg, 92% as a white
powder, which was crystallized from MeOH) was obtained.
14b: mp >210 °C dec; EI-MS m/ z 286 (M+), 251 (M+ - OH);
IR (Nujol) 2120 cm-1 (CtC); UV λmax (H2O) 268 nm (ꢀ 7 700),
λmax (0.5 N HCl) 269 nm (ꢀ 8 400), λmax (0.5 N NaOH) 269 nm
1
(ꢀ 6 900); H-NMR (DMSO-d6) 11.87 (br s, 1 H, NH), 8.33 (d,
1 H, H-6, J 6,5-F ) 7.2 Hz), 5.93 (s, 1 H, 3′-OH), 5.83 (dd, 1 H,
H-1′, J 1′,2′ ) 7.2 Hz, J 1′,5-F ) 1.8 Hz), 5.82 (d, 1 H, 2′-OH,
J 2′-OH,2′ ) 6.4 Hz), 5.27 (t, 1 H, 5′-OH, J ) 4.1 Hz), 4.18 (dd, 1
H, H-2′, J 2′,1′ ) 7.2 Hz, J 2′,2′-OH ) 6.4 Hz), 3.92-3.91 (m, 1 H,
H-4′), 3.77-3.64 (m, 2 H, H-5′), 3.56 (s, 1 H, 3′-CtCH). Anal.
(C11H11FN2O6‚1/3 MeOH), C, H, F, N.
1-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)th ym in e (14c).
Reaction of pertrimethylsilylated thymine (2.0 mmol) with 6
for 27 h at room temperature gave 9c (290 mg, 98% as a
foam): FAB-MS m/ z 595 (MH+); 1H-NMR (CDCl3) 8.17-7.34
(m, 17 H, Bz × 3, H-6, NH), 6.44 (d, 1 H, H-1′, J 1′,2′ ) 5.9 Hz),
6.06 (d, 1 H, H-2′, J 2′,1′ ) 5.9 Hz), 4.98-4.95 (m, 1 H, H-4′),
4.93-4.90 (m, 2 H, H-5′), 2.91 (s, 1 H, 3′-CtCH), 1.72 (s, 3 H,
5-Me).
3-(3-C-E t h yn yl-â-D-r ibo-p en t ofu r a n osyl)u r a cil (15).
Compound 10 (55 mg, 0.1 mmol) was treated with methanolic
ammonia (saturated at 0 °C, 1.5 mL) for 2 days at room
temperature. The solvent was removed in vacuo, and the
residue was purified on a silica gel column (2 × 6 cm), which
was eluted with 5-15% MeOH in CHCl3 to give 15 (17 mg,
67% as a white powder, which was crystallized from aqueous
MeOH): mp 255 °C dec; EI-MS m/ z 250 (M+ - OH), 237 (M+
- CH2OH); IR (Nujol) 2230 cm-1 (CtC); UV λmax (H2O) 264
nm (ꢀ 7 000), λmax (0.5 N HCl) 264 nm (ꢀ 7 200), λmax (0.5 N
1
NaOH) 293 nm (ꢀ 9 900); H-NMR (DMSO-d6) 11.20 (br s, 1
H, NH), 7.47 (d, 1 H, H-6, J 6, ) 7.6 Hz), 6.09 (d, 1 H, H-1′,
5
J 1′,2′ ) 8.3 Hz), 5.67 (s, 1 H, 3′-OH), 5.62 (d, 1 H, 2′-OH, J 2′-OH,2′
) 7.0 Hz), 5.60 (d, 1 H, H-5, J 5,6 ) 7.6 Hz), 5.06 (dd, 1 H, H-2′,
J 2′,1′ ) 8.3 Hz, J 2′,2′-OH ) 7.0 Hz), 4.48-4.46 (m, 1 H, 5′-OH),
3.86-3.84 (m, 1 H, H-4′), 3.68-3.61 (m, 2 H, H-5′), 3.37 (s, 1
H, 3′-CtCH). Anal. (C11H12N2O6‚1/2 H2O) C, H, N.
From 9c (290 mg, 0.49 mmol), 14c (115 mg, 83% as a white
powder, which was crystallized from MeOH) was obtained.
14c: mp 113-118 °C; EI-MS m/ z 282 (M+); IR (Nujol) 2115
cm-1 (CtC); UV: λmax (H2O) 266 nm (ꢀ 8 800), λmax (0.5 N HCl)
265 nm (ꢀ 8 600), λmax (0.5 N NaOH) 267 nm (ꢀ 7 000); 1H-
NMR (DMSO-d6) 11.30 (br s, 1 H, NH), 7.85 (d, 1 H, H-6,
J 6,5-Me ) 1.0 Hz), 5.90 (s, 1 H, 3′-OH), 5.83 (d, 1 H, H-1′, J 1′,2′
) 7.5 Hz), 5.81 (d, 1 H, 2′-OH, J 2′-OH,2′ ) 6.7 Hz), 5.13 (t, 1 H,
5′-OH, J ) 4.4 Hz), 4.19 (dd, 1 H, H-2′, J 2′,1′ ) 7.5 Hz, J 2′,2′-OH
) 6.7 Hz), 3.88-3.86 (m, 1 H, H-4′), 3.74-3.65 (m, 2 H, H-5′),
3.56 (s, 1 H, 3′-CtCH), 1.73 (d, 3 H, 5-Me, J 5-Me,6 ) 1.0 Hz).
Anal. (C12H14N2O6‚MeOH) C, H, N.
Gen er a l Met h od for t h e Syn t h esis of 3′-C-E t h yn yl
Ribon u cleosid es. Stannic chloride (3 mmol for N6-benzoyl-
adenine and N2-acetylguanine, 2.5 mmol for 5-fluorocytosine,
and 2 mmol for 5-fluorouracil and thymine) was added to a
solution of pertrimethylsilylated nucleobases [2 mmol, pre-
pared from the nucleobase (2 mmol) and (NH4)2SO4 (7 mg, for
the pyrimidine base) or pyridine (2 mL, for the purine base)
in HMDS (6 mL)] and 6 (264 mg, 0.5 mmol) in CH3CN (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 blocked nucleosides, which were then treated with
9-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)a d en in e (16).
Reaction of pertrimethylsilylated N6-benzoyladenine (2.0 mmol)
with 6 for 7 h at room temperature gave 11 (261 mg, 74% as
1
a foam): FAB-MS m/ z 708 (MH+); H-NMR (CDCl3) 8.99 (br
s, 1 H, NHBz), 8.75 (s, 1 H, H-8), 8.49 (s, 1 H, H-2), 8.16-7.31
(m, 15 H, Bz × 3), 6.58 (d, 1 H, H-1′, J 1′,2′ ) 4.8 Hz), 6.56 (d,
1 H, H-2′, J 2′,1′ ) 4.8 Hz), 5.07-5.03 (m, 2 H, H-5′), 4.98-4.94
(m, 1 H, H-4′), 2.95 (s, 1 H, 3′-CtCH).
From 11 (234 mg, 0.33 mmol), 16 (80 mg, 83% as a white
powder, which was crystallized from H2O) was obtained. 16:
mp 149-152 °C; EI-MS m/ z 291 (M+), 274 (M+ - OH); IR

Antitumor Nucleosides with a Broad Spectrum of Activity
J ournal of Medicinal Chemistry, 1996, Vol. 39, No. 25 5011
(Nujol) 2110 cm-1 (CtC); UV λmax (H2O) 256 nm (ꢀ 12 700),
(4) Plunkett, W.; Huang, P.; Xu, Y.-Z.; Heinemann, V.; Grunewald,
R.; Gandhi, V. Gemcitabine: Metabolism, mechanisms of action,
and self-potentiation. Semin. Oncol. 1995, 22 (Suppl. 11), 3-10
and references cited therein.
λmax (0.5 N HCl) 257 nm (ꢀ 12 500), λmax (0.5 N NaOH) 260 nm
1
(ꢀ 13 000); H-NMR (DMSO-d6) 8.35 (s, 1 H, H-8), 8.14 (s, 1
H, H-2), 7.36 (br s, 2 H, NH2), 5.99 (s, 1 H, 3′-OH), 5.88 (d, 1
H, 2′-OH, J 2′-OH,2′ ) 7.2 Hz), 5.85 (d, 1 H, H-1′, J 1′,2′ ) 7.8 Hz),
5.60 (dd, 1 H, 5′-OH, J 5′-OH,5′a ) 7.4 Hz, J 5′-OH,5′b ) 4.0 Hz),
4.81 (dd, 1 H, H-2′, J 2′,1′ ) 7.8 Hz, J 2′,2′-OH ) 7.2 Hz), 4.01-
3.80 (m, 1 H, H-4′), 3.79-3.69 (m, 2 H, H-5′), 3.54 (s, 1 H, 3′-
CtCH). Anal. (C12H13N5O4‚2/3 H2O) C, H, N.
(5) (a) Matsuda, A.; Nakajima, Y.; Azuma, A.; Tanaka, M.; Sasaki,
T. 2′-C-Cyano-2′-deoxy-1-â-D-arabinofuranosylcytosine (CNDAC):
Design of a potential mechanism-based DNA-strand breaking
antineoplastic nucleoside. J . Med. Chem. 1991, 34, 2917-2919.
(b) Azuma, A.; Nakajima, Y.; Nishizono, N.; Minakawa, N.;
Suzuki, M.; Hanaoka, K.; Kobayashi, T.; Tanaka, M.; Sasaki,
T.; Matsuda, A. 2′-C-Cyano-2′-deoxy-1-â-D-arabinofuranosylcy-
tosine and its derivatives: A new class of nucleoside with a broad
antitumor spectrum. J . Med. Chem. 1993, 36, 4183-4189. (c)
Tanaka, M.; Matsuda, A.; Terao, T.; Sasaki, T. Antitumor
activity of a novel nucleoside, 2′-C-cyano-2′-deoxy-1-â-D-arabino-
furanosylcytosine (CNDAC) against murine and human tumors.
Cancer Lett. 1992, 64, 67-74. (d) Azuma, A.; Hanaoka, K.;
Kurihara, A.; Kobayashi, T.; Miyauchi, S.; Kamo, N.; Tanaka,
M.; Sasaki, T.; Matsuda, A. Chemical stability of a new antitu-
mor nucleoside, 2′-C-cyano-2′-deoxy-1-â-D-arabino-pentofurano-
sylcytosine (CNDAC) in alkaline medium: Formation of 2′-C-
cyano-2′-deoxy-1-â-D-ribo-pentofuranosylcytosine (CNDC) and its
antitumor activity. J . Med. Chem. 1995, 38, 3391-3397. (e)
Matsuda, A.; Azuma, A. 2′-C-Cyano-2′-deoxy-1-â-D-arabinofura-
9-(3-C-Eth yn yl-â-D-r ibo-p en tofu r a n osyl)gu a n in e (17).
Reaction of pertrimethylsilylated N2-acetylguanine (2.0 mmol)
with 6 for 2 h at room temperature gave 12 (327 mg, 99% as
a foam): FAB-MS m/ z 662 (MH+); 1H-NMR (CDCl3) 12.26 (br
s, 1 H, 1-NH), 10.36 (br s, 1 H, 2-NHAc), 8.38 (s, 1 H, H-8),
8.15-7.28 (m, 15 H, Bz × 3), 6.85 (d, 1 H, H-1′, J 1′,2′ ) 3.9
Hz), 6.32 (d, 1 H, H-2′, J 2′,1′ ) 3.9 Hz), 5.09-5.05 (m, 1 H, H-4′),
5.04-4.86 (m, 2 H, H-5′), 2.97 (s, 1 H, 3′-CtCH), 2.36 (s, 3 H,
2-NHAc).
Compound 12 (320 mg, 0.49 mmol) was treated with NaOMe
(0.25 M, 0.9 mL) in MeOH (30 mL) for 2 h at room tempera-
ture. The mixture was neutralized with Amberlite IRC-50S
(H+), and the resin was filtered off. The filtrate was concen-
trated, and the residue was dissolved in H2O (150 mL), which
was extracted with CHCl3 (100 mL × 2). The separated
aqueous phase was concentrated to dryness in vacuo, and the
residue was crystallized from H2O to give 17 (95 mg, 63%):
mp 225 °C dec; EI-MS m/ z 307 (M+); UV λmax (H2O) 253 nm
(ꢀ 13 000), λmax (0.5 N HCl) 252 nm (ꢀ 12 100), λmax (0.5 N
nosylcytosine (CNDAC):
A mechanism-based DNA-strand-
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1
NaOH) 263 nm (ꢀ 11 000); H-NMR (DMSO-d6) 8.35 (s, 1 H,
H-8), 8.14 (s, 1 H, H-2), 7.36 (br s, 2 H, NH2), 5.99 (s, 1 H,
3′-OH), 5.88 (d, 1 H, 2′-OH, J 2′-OH,2′ ) 7.2 Hz), 5.85 (d, 1 H,
H-1′, J 1′,2′ ) 7.8 Hz), 5.60 (dd, 1 H, 5′-OH, J 5′-OH,5′a ) 7.4 Hz,
J 5′-OH,5′b ) 4.0 Hz), 4.81 (dd, 1 H, H-2′, J 2′,1′ ) 7.8 Hz, J 2′,2′-OH
) 7.2 Hz), 4.01-3.80 (m, 1 H, H-4′), 3.79-3.69 (m, 2 H, H-5′),
3.54 (s, 1 H, 3′-CtCH). Anal. (C12H13N5O5) C, H, N.
Ack n ow led gm en t . This investigation was sup-
ported in part by Grants-in-Aid for Cancer Research
from the Ministry of Education, Science, Sports, and
Culture of J apan and the 2nd Term Comprehensive
Ten-Year Strategy for Cancer Control from the Ministry
of Health and Welfare of J apan.
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J M960537G