Synthesis of 3′-β-carbamoylmethylcytidine (CAMC) and its derivatives as potential antitumor agents

Article abstract of DOI:10.1039/b517602f

3′-β-Carbamoylmethylcytidine (CAMC) and its derivatives were synthesized using an intramolecular Reformatsky-type reaction promoted by SmI₂ as the key step. In vitro tumor cell growth inhibitory activity was evaluated and CAMC was found to exhi

Full text of DOI:10.1039/b517602f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of 3ꢀ-b-carbamoylmethylcytidine (CAMC) and its derivatives as
potential antitumor agents†
Satoshi Ichikawa,*a Noriaki Minakawa,a Satoshi Shuto,a Motohiro Tanaka,b Takuma Sasakib and
Akira Matsuda*a
Received 12th December 2005, Accepted 25th January 2006
First published as an Advance Article on the web 20th February 2006
DOI: 10.1039/b517602f
3ꢀ-b-Carbamoylmethylcytidine (CAMC) and its derivatives were synthesized using an intramolecular
Reformatsky-type reaction promoted by SmI2 as the key step. In vitro tumor cell growth inhibitory
activity was evaluated and CAMC was found to exhibit potent cytotoxicity against various human
tumor cell lines. From a structure–activity relationship study it was postulated that the cytotoxic
mechanism of action of CAMC did not require phosphorylation at the 5ꢀ-hydroxyl group. This study
provides a novel strategy for the development of a new type of antitumor nucleoside.
Introduction
Considerable attention has been focused on branched-chain
sugar nucleosides because of their biological importance.2–16
We have developed stereoselective synthetic methods for
a variety of branched-chain sugar nucleosides. During the
course of this project,17–25 it was found that 2ꢀ-branched-chain
sugar nucleosides such as 1-(2-deoxy-2-methylene-b-D-erythro-
pentofuranosyl)cytosine (DMDC,26–30 1) and 1-(2-C-cyano-2-
deoxy-b-D-arabino-pentofuranosyl)cytosine (CNDAC,31–36 2) were
potent antitumor agents, and they are now under phase I clinical
studies (Fig. 1).37 Consequently, we thought it might be interesting
to investigate the biological activity of 3ꢀ-branched-chain ribonu-
cleosides, which may have efficient antitumor and/or antiviral ac-
tivity similar to that of 2ꢀ-branched-chain sugar nucleosides. Thus,
we synthesized 3ꢀ-b-branched-chain sugar ribonucleoside analogs
and found that 1-(3-C-ethynyl-b-D-ribo-pentofuranosyl)cytosine
(ECyd,38–50 3) had remarkably potent antitumor activity. It is also
under phase I study.37 However, despite the promising biological
activity of these compounds, few synthetic studies have been
reported51 because of the lack of efficient synthetic methods for the
preparation of 3ꢀ-b-branched-chain sugar ribonucleoside analogs.
ECyd was synthesized via the construction of the corresponding
3-ethynyl ribose derivative from D-xylose followed by the intro-
Fig. 1 Structures of branched-chain nucleosides exhibiting antitumor
activity.
duction of a nucleobase via glycosylation.38,39,41,47 Although this
glycosylation is suitable for preparing sugar-modified nucleosides
with a variety of nucleobases, rather lengthy synthetic routes are
required. Therefore, we have developed an efficient and general
synthetic method for 3ꢀ-b-branched-chain sugar pyrimidine nucle-
osides starting from uridine as shown in Scheme 1.21 Introduction
of a carbon substituent at the 3ꢀ-b-position of the nucleoside was
achieved by the intramolecular SmI252–57-promoted Reformatsky-
type52 reaction of 5ꢀ-O-bromoacetyl-3ꢀ-ketouridine derivative A
(Scheme 1), in which the precursor of the carbon substituent was
installed at the b-face of the sugar moiety. Ring opening of the
resulting lactone B with a nucleophile should provide the target
3ꢀ-b-branched-chain sugar uridines. This method allowed us to
prepare a range of 3ꢀ-b-branched-chain pyrimidine ribonucleoside
analogs quite efficiently. During this study, we found that 3ꢀ-b-
carbamoylmethylcytidine (CAMC) showed cell growth inhibitory
activity against mouse leukemic L1210 cells. This finding is
rather unexpected because, from our previous structure–activity
aGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-
12, Nishi 6, Kita-ku, Sapporo, 060-0812, Japan. E-mail: matuda@
pharm.hokudai.ac.jp; Fax: +81-11-706-4980; Tel: +81-11-706-3228
bSchool of Pharmacy, Aichi Gakuin University, 2-11 Semori-dori, Chikusa-
ku, Nagoya, 464-865, Japan
† This paper constitutes Part 238 of the series Nucleosides and
Nucleotides.1
Scheme 1 Synthetic strategy for 3ꢀ-b-branched-chain pyrimidine nucleosides.
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relationship studies of Ecyd,47 a less bulky carbon-substituent at
the 3ꢀ-b-position is very important for potent antitumor activity.
Namely, increasing the size of the 3ꢀ-b-substituent by introducing
the alkyl group at the terminal acetylene or changing the ethynyl
group to an ethenyl or an ethyl group resulted in decreased
cytotoxicity. Generally nucleoside antimetabolites have to be
metabolically activated by phosphorylation at the 5ꢀ-hydroxyl
group. This substituent–structure relationship would be strongly
related to the substrate specificity of the uridine–cytidine kinase
2 (UCK2).44,58–60 Therefore, the cytotoxic mechanism of action
of CAMC would be dissimilar to that of ECyd. With these
considerations in mind, we decided to elucidate the structure–
activity relationship of CAMC.
Fig. 2 Structures of target compounds.
a bromoacetyl group to give the 5ꢀ-O-bromoacetyl derivative 9,21
the precursor to the intramolecular Reformatsky-type reaction.
Treatment of 9 with 2 equiv of SmI2 in THF at −78 ◦C afforded
the desired lactone 10 in good yield while the Reformatsky reaction
with Zn failed to give 10 under standard conditions. This might
be due to the strong ability of the samarium enolate to chelate the
3ꢀ-carbonyl oxygen forming a six-membered transition state. Since
a THF solution of 9 was added dropwise to a solution of SmI2 in
THF, protection of the acidic NH group in a uracil moiety of 9
was unnecessary. Ammonolysis of the lactone 10 at −70 ◦C gave
the 3ꢀ-carbamoylmethyluridine derivative 11. When the reaction
was carried out at room temperature, a large amount of uracil
was obtained along with the desired 11. A retro-aldol reaction of
the hydroxylactone 10 followed by base-promoted b-elimination at
the 1ꢀ, 2ꢀ-position of the resulting 5ꢀ-O-acetyl-3ꢀ-ketouridine would
possibly explain the formation of uracil. The 5ꢀ-primary hydroxyl
group of 11 was protected with a TBS group to give 12, followed
by dehydration of the amide using p-TsCl in pyridine to provide
the 3ꢀ-cyanomethyl derivative 13. Compounds 12 and 13 both were
converted to the corresponding cytidine derivatives 14 and 15 by
way of their 4-(4-dimethylamino)pyridinium derivatives followed
by ammonolysis. Finally, treatment of 11, 13, 14 and 15 with NH4F
Chemistry
The structure–activity relationship study for ECyd explored only
alkyl, alkenyl, and alkynyl groups at the 3ꢀ-position and not
polar functional groups containing oxygen or nitrogen, which
would have the ability to form additional interactions with target
enzyme(s) by hydrogen bonding. Given the structure–activity
relationship associated with ECyd, it is important to introduce
a less-bulky carbon substituent at the 3ꢀ-b-position. It should be
noted that 1-(3-C-ethynyl-b-D-ribo-pentofuranosyl)uracil (EUrd),
a uracil counterpart of ECyd, also exhibited potent cytotoxicity.
Consequently, we decided to introduce carbamoylmethyl and
cyanomethyl groups, which could be easily prepared from the key
lactone B at the 3ꢀ-b-position of uridine and cytidine. The synthesis
of the 3ꢀ-b-branched-chain sugar pyrimidine nucleosides 4–7
(Fig. 2) is shown in Scheme 2. 2ꢀ-O-TBS-3ꢀ-ketouridine 8, which
was easily obtained from uridine in three steps,51 was acylated with
Scheme 2 Synthesis of compounds 4–7. Reagents and conditions: (a) BrCH2COBr, CH2Cl2, −78 ◦C (70%); (b) SmI2, THF, −78 ◦C (85%);
(c) NH3–MeOH, −70 ◦C (98%); (d) TBSCl, imidazole, DMF, room temperature (99%); (e) TsCl, pyridine, reflux (81%); (f) TPSCl, DMAP, Et3N,
MeCN, room temperature, then NH4OH (76% for 14, 99% for 15); (g) NH4F, MeOH (99% for 4, 99% for 5, 79% for 6, 99% for 7).
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in refluxing MeOH gave 3ꢀ-b-carbamoylmethyluridine (4), 3ꢀ-b-
cyanomethyluridine (5), 3ꢀ-b-carbamoylmethylcytidine (CAMC,
6) and 3ꢀ-b-cyanomethylcytidine (7), respectively.
methyl ester 30 using I2 and KOH in MeOH. Conversion of 30 to
the cytosine derivative 31, followed by ammonolysis of the methyl
ester and deprotection of the TBS groups at the 2ꢀ and 5ꢀ-hydroxyl
groups afforded the 3ꢀ-b-carbamoylcytidine 17. Wittig reaction
Next, we studied the structure–activity relationship of
CAMC. As will be described in the biological activity section,
CAMC (6) exhibited a tumor cell growth inhibitory activity
against L1210 and KB cell lines. 3ꢀ-a-Carbamoylmethylcytidine
16, 3ꢀ-b-carbamoylcytidine 17 and 3ꢀ-b-carbamoylethylcytidine
18, which correspond to the 3ꢀ-epimer, one-carbon-truncated
and one-carbon-extended analogs of CAMC, respectively, were
designed to explore the suitable position of the carbamoyl group
(Fig. 3). Compound 16 was synthesized as shown in Scheme 3.
The aldol reaction between 21 and LiCH2CO2Et at −78 ◦C gave
the xylo-adduct 22 highly stereoselectively, and the corresponding
ribo-adduct was not obtained at all. The complete a-selectivity
exhibited in this reaction is typical of addition reactions of
nucleophiles to 3ꢀ-ketonucleoside derivatives when the 5ꢀ-hydroxyl
group is protected. This is one of the drawbacks in preparing 3ꢀ-
b-branched-chain sugar nucleosides.61 Thus, our method for the
preparation of 3ꢀ-b-branched pyrimidine nucleosides using the in-
tramolecular Reformatsky-type reaction was found to be effective.
In a manner similar to the preparation of the cytidine derivatives
14 and 15, 22 was converted to 23. Ammonolysis of 23 followed
by deprotection of the TBS groups afforded the desired 16.
The synthesis of 17 and 18 is summarized in Scheme 4. The
methyl ester 24 was prepared by a two-step sequence including
methanolysis of the lactone 10 followed by TBS protection of the
liberated 5ꢀ-hydroxyl group. Reduction of the methyl ester 24 with
DIBAL-H followed by mesylation of the resulting hydroxyl group
and displacement of the mesylate 26 with phenylselenide ion gave
27. Oxidation of 27 with mCPBA furnished a diastereomeric mix-
ture of selenoxides, which was heated to promote syn-elimination
to provide the 3ꢀ-ethenyluridine 28. Dihydroxylation of the alkene
followed by oxidative cleavage of the resulting diol gave the
aldehyde intermediate 29, which was further oxidized to give the
=
of the aldehyde 29 with Ph3P CHCO2Et gave 32. Conversion to
the cytosine base and catalytic hydrogenation of the branched-
chain double bond gave 34. Finally, TBS deprotection followed by
ammonolysis provided the 3ꢀ-b-carbamoylethylcytidine 18.
A further series of CAMC analogs 19 and 20 shown in Fig. 3 was
also synthesized to elucidate the structure–activity relationship.
The N-alkyl amide analogs 19a–d were prepared in a manner
similar to the synthesis of CAMC, which is summarized in
Scheme 5. Ring opening of the key lactone 10 with N-methylamine,
N-ethylamine, N-benzylamine or N,Nꢀ-dimethylamine gave the
corresponding N-alkyl amide analogs 35a–d, in good yields with
less retro-aldol reaction observed in the treatment of 10 with
methanolic ammonia, possibly because of the inherent increased
nucleophilicity of alkylamines compared to that of ammonia.
Protection of the liberated 5ꢀ-hydroxyl group with a TBS group
provided compounds 36a–d, which were converted to the cytosine
derivatives 37a–d by the same procedure as for the synthesis of 14.
Finally, deprotection of the TBS groups afforded the desired com-
pounds 19a–d. The N4-methylcytosine analog 20a and the N4,N4ꢀ-
dimethylcytosine analog 20b were also prepared as shown in
Scheme 6. Conversion of 12 to the 4-(4-dimethylamino)pyridinium
derivative followed by substitution with N-methylamine or N,Nꢀ-
dimethylamine gave 38a,b. Subsequently, the TBS groups at the 2ꢀ
and 5ꢀ positions were removed to provide the desired compounds
20a,b.
Biological activity
The in vitro tumor cell growth inhibitory activities of the newly
synthesized 3ꢀ-b-branched-chain sugar nucleoside analogs against
L1210 and KB cells were evaluated using the MTT assay, and the
Fig. 3 Structures of CAMC analogs.
Scheme 3 Synthesis of a-CAMC. Reagents and conditions: (a) LiCH2CO2Et, THF, −78 ◦C (95%); (b) TPSCl, DMAP, Et3N, MeCN, room temperature,
then NH4OH (71%); (c) NH3–MeOH, reflux; (d) NH4F, MeOH, reflux (2 steps 90%).
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Scheme 4 Synthesis of 17 and 18. Reagents and conditions: (a) K2CO3, MeOH, −20 ◦C; (b) TBSCl, imidazole, DMF, room temperature (2 times, 2
steps 87%); (c) DIBAL-H, THF, 0 ◦C (39%); (d) MsCl, DMAP, Et3N, CH2Cl2, 0 ◦C (92%); (e) PhSeSePh, NaBH4, EtOH, room temperature (98%);
(f) mCPBA, CH2Cl2, 0 ◦C, then DMF, 80 ◦C (2 steps 49%); (g) OsO4, NMO, aq. acetone, room temperature, then NaIO4 (h) I2, KOH, MeOH, room
temperature (80% from 28); (i) TPSCl, DMAP, Et3N, MeCN, room temperature, then NH4OH (97% for 30, 98% for 32); (j) NH3–MeOH, reflux;
=
(k) NH4F, MeOH, reflux (2 steps 87% for 17, 87% for 18); (l) Ph3P CHCO2Et, CH2Cl2, room temperature (83%); (m) H2, Pd/C, MeOH, room
temperature (quant.).
Scheme 6 Synthesis of N4-substituted CAMCs. Reagents and conditions:
(a) TPSCl, DMAP, Et3N, MeCN, room temperature, then R1R2NH (94%
for 38a, 86% for 38b); (b) NH4F, MeOH, reflux (98%); (b) NH4F, MeOH,
reflux (80% for 20a, 76% for 20b).
a broad spectrum of cytotoxicity in a range of human tumor cell
lines although the activities were moderate.
The effect of CAMC on the synthesis of DNA (incorporation of
Scheme 5 Synthesis of amide-substituted CAMCs. Reagents and condi-
[3H]thymidine), RNA (incorporation of [3H]uridine), and protein
(incorporation of [3H]leucine) was also examined with L1210 cells
(Fig. 4). At 10 lg mL−1 concentrations of CAMC, DNA synthesis
was inhibited by 83%. In addition to DNA synthesis, RNA and
protein syntheses were also inhibited by 48 and 61%, respectively.
Although CAMC has a ribonucleoside structure, it inhibited not
only RNA synthesis, but also DNA and protein synthesis. It might
be suggested that the mode of action of CAMC in exhibiting tumor
cell growthinhibitory activities might not be the same as theknown
nucleoside antimetabolites.
tions: (a) R1R2NH–DMF, room temperature (99% for 35a, 99% for 35b,
99% for 35c, 85% for 35d); (b) TBSCl, imidazole, DMF, room temperature
(99% for 36a, 92% for 36b, 93% for 36c, 93% for 36d); (c) TPSCl, DMAP,
Et3N, MeCN, room temperature, then NH4OH (73% for 37a, 99% for 37b,
93% for 37c, 69% for 37d); (g) NH4F, MeOH (86% for 19a, 70% for 19b,
71% for 19c, 92% for 19d).
results are summarized in Table 1. Among 4–7, only CAMC (6)
was a potent inhibitor of tumor cell growth against L1210 and KB
cell lines, with IC50 values of 0.33 and 5.61 lM, respectively. We
next evaluated the inhibitory spectrum of CAMC on the growth of
various human tumor cell lines in vitro (Table 2). CAMC showed
Most of the nucleoside antitumor agents have to be phospho-
rylated at the 5ꢀ-hydroxyl group by a nucleoside kinase to show
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Table 1 Growth inhibitory effects of 3ꢀ-b-branched nucleosides against
L1210 and KB cellsa
IC50/lM
L1210
KB
4
>100
>100
0.33
74
43
>100
>100
73
>100
>100
5
6 (CAMC)
5.61
7
>100
>100
>100
>100
100
14
16
17
18
19a
19b
19c
19d
20a
20b
>100
74
>100
>100
67
>100
>100
>100
>100
>100
>100
Fig. 5 Competitive effects of common nucleosides on the cytotoxicity of
CAMC (6) against the growth of L1210 cells. L1210 cells (104 cells/mL)
were seeded in a 96-well microplate, and treated with graded concentrations
of 6 and each common nucleosides were simultaneously added in tripicate
to each well. The plate was incubated for 3 d at 37 ◦C in a humidified
atmosphere of 5% CO2. The cytotoxicity of 6 was evaluated by MTT
assay.
17
a IC50 (lM) was given as a concentration of 50% inhibition of cell growth.
Table 2 Inhibitory effects of CAMC on several tumor cells in vitro
Cell lines
Origin
IC50a/lM
kinases would be competitive with that of CAMC, the cytotoxicity
should be largely reduced if CAMC required phosphorylation
to show cytotoxicity. This suggests that CAMC possesses a
unique property to exhibit growth inhibitory activity without
phosphorylation of the 5ꢀ-hydroxyl group. This was also indicated
by the fact that the inhibitory activity against L1210 cells of the
2ꢀ,5ꢀ-di-O-TBS-protected CAMC 14 still remained (IC50 = 43 lM).
The cyanomethyl analog 7 had no activity and it was suggested
that the amide oxygen and/or amide hydrogen(s) are important
in exhibiting activity. In addition, the N4-amino functionality on
the nucleobase moiety is also a significant factor for the activity
as seen by comparison of 6 with the uridine analog 4.
MKN-28
MKN-45
KATO-III
NUGC-4
AGS
Human stomach adenocarcinoma
Human stomach adenocarcinoma
Human stomach adenocarcinoma
Human stomach adenocarcinoma
Human stomach adenocarcinoma
11
12
37
10
8.7
10
13
11
5.7
0.33
Colo320DM Human colon adenocarcinoma
HT-1080
K562
KB
L1210
Human fibrosarcoma
Human leukemia
Human oral epidermal adenocarcinoma
Murine leukemia
a IC50 (lM) was given as a concentration of 50% inhibiton of cell growth.
To determine the structure–activity relationship of the car-
bamoyl group, we synthesized several CAMC analogs and evalu-
ated their activities (Table 1). The N4-alkyl derivatives 20a and 20b
showed weak cytotoxicity whereas the N-alkylamide derivatives
19a–d exhibited no cytotoxicity. These results suggested that both
hydrogen atoms on the carbamoyl group, which are hydrogen
bond donors, are necessary to exhibit tumor cell growth inhibitory
activity. The 3ꢀ-epimer of CAMC 18, the carbamoyl analog 19
and the carbamoylethyl analog 20 showed no cytotoxicity, which
would indicate that the orientation of the carbamoyl group is also
an important factor.
Phosphorylation of 5ꢀ-hydroxyl groups, the first metabolic step,
is one of the rate-limiting steps in the metabolic pathway of nucle-
oside derivatives and its efficiency sometimes plays an important
role in exhibiting cytotoxic activity. Generally, chemically modified
nucleosides result in decreased efficiency of phosphorylation,
which was also seen in the structure–activity relationship study of
ECyd. Development of antitumor nucleosides as antimetabolites
should overcome this drawback. This study suggests that CAMC
exhibited cytotoxic activity without phosphorylation, which is
unusual for antimetabolic nucleoside derivatives. The nucleoside
analog TSAO62 is an anti-HIV agent with HIV reverse transcrip-
tase (RT) inhibition as the mode of action. TSAO possesses TBS
groups at the 2ꢀ and 5ꢀ positions and inhibited HIV RT without
Fig. 4 The effect of CAMC (6) on the synthesis of DNA, RNA, and
protein. After L1210 cells (2.5 × 105 cells/mL) were incubated for 24 h,
6 was added to the culture and incubated for 1 h. Before cell harvest,
cells were pulse-labelled for 30 min with [3H]thymidine, [3H]uridine or
[3H]leucin. The radioactivity of the acid-insoluble fractions was measured
by a liquid scintillation counter.
cytotoxic activity. In order to evaluate whether CAMC acts as an
antimetabolite in DNA and/or RNA synthesis, the competitive
inhibition assay against a range of natural nucleosides was
conducted. However, the tumor cell growth inhibitory activity
of CAMC was not reduced in the presence of the nucleosides,
including cytidine (Fig. 5). Since the phosphorylation of these
natural nucleosides by certain nucleoside and/or nucleotide
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phosphorylation of the 5ꢀ-hydroxyl group and is known as a
non-nucleoside RT inhibitor (NNRTI). However, an antitumor
nucleoside analog with the above-mentioned mechanism would
be very rare. Structurally, ECyd shares a similar structure to
CAMC as 3ꢀ-b-branched ribonucleoside analogs. However, it is
phosphorylated at 5ꢀ-hydroxyl group and exhibits more than one
order of magnitude more potent than CAMC. Different biological
activity would be due to the different mode of action between
these agents. Therefore, further study of the mechanism of action
of CAMC is needed to elucidate new targets of CAMC.
(SiO2, 20% Me◦OH–CHCl3) to give 14 (830 mg, 76%) as a white
1
solid: mp 160 C (crystallized from MeOH–CHCl3): H NMR
(CDCl3, 500 MHz) d 8.04 (d, 1H, J = 8.2 Hz), 7.23 (br s, 1H), 6.31
(d, 1H, J = 7.2 Hz), 5.95 (br s, 1H), 5.49 (br s, 1H), 4.24 (br s, 1H),
4.06 (d, 1H, J = 7.3 Hz), 4.01 (d, 1H, J = 11.8 Hz), 3.89 (dd, 1H,
J = 2.0, 12.2 Hz), 2.61 (d, 1H, J = 15.1 Hz), 2.57 (d, 1H, J = 15.1
Hz), 0.96 (s, 9H), 0.85 (s, 9H), 0.17 (s, 3H), 0.06 (s, 3H), 0.01 (s,
3H), −0.10 (s, 3H); 13C NMR (CDCl3, 125 MHz) d173.0, 164.1,
156.2, 141.6, 92.9, 87.9, 85.3, 79.6, 26.3, 26.0, −4.4, −4.6, −5.1,
−5.4; FAB-MS m/z 530 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(cyanomethyl)-b-D-
ribo-pentofuranosyl]cytosine (15). Compound 15 was prepared
from 13 (102 mg, 0.20 mmol) as described above for the synthesis
of 14. After purification by column chromatography (SiO2, 5%
Conclusion
A series of 3ꢀ-b-branched pyrimidine nucleosides was synthesized
using an intramolecular Reformatsky-type reaction promoted by
SmI2 as the key step. Their tumor cell growth inhibitory activity
was evaluated and 3ꢀ-b-carbamoylmethylcytidine (CAMC, 6) was
found to exhibit potent tumor cell growth inhibitory activity. A
structure–activity relationship was conducted and it was found
that CAMC showed activity without phosphorylation at the 5ꢀ-
hydroxyl group. This study provided a novel strategy for the
development of a new type of antitumor nucleoside.
1
MeOH–CHCl3) to give 15 (110 mg, 99%) as a white glass: H
NMR (CDCl3, 500 MHz) d 7.75 (d, 1H, J = 7.4 Hz), 6.19 (d, 1H,
J = 6.8 Hz), 5.83 (d, 1H, J = 7.4 Hz), 4.18 (br s, 1H), 4.08 (d, 1H,
J = 6.7 Hz), 3.97 (dd, 1H, J = 2.7, 12.4 Hz), 3.93 (dd, 1H, J =
1.9, 12.4 Hz), 3.58 (s, 1H), 2.84 (d, 1H, J = 16.5 Hz), 2.67 (d, 1H,
J = 16.5 Hz), 0.96 (s, 9H), 0.89 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H),
0.02 (s, 3H), −0.06 (s, 3H); 13C NMR (CDCl3, 125 MHz) d164.7,
157.2, 147.9, 140.4, 122.3, 116.4, 97.2, 86.2, 84.3, 78.3, 62.5, 26.3,
26.0, −4.7, −4.8, −4.8, −4.9; FAB-MS m/z 511 (MH+).
Experimental
1-[3-C -(Carbamoylmethyl)-b-D-ribo-pentofuranosyl]cytosine
(6). A mixture of 14 (105 mg, 0.20 mmol) and NH4F (148 mg,
4.01 mmol) in MeOH (10 mL) was heated under reflux for 2 h. The
mixture was evaporated under reduced pressure, and the residue
was partitioned between CHCl3 (20 mL) and H2O (20 mL). The
aqueous layer was washed CHCl3 (20 mL × 2) and evaporated
under reduced pressure. The residue was dissolved in H2O, and
the charcoal was added until the UV absorption of the solution
disappeared. The suspension was mounted on the column, and
the charcoal was washed with H2O (50 mL) and eluted with 50%
aqueous MeOH. The UV positive fractions were collected and
concentrated to give 6 (47.5 mg, 79%) as a white powder: mp
253 ◦C dec (H2O–MeOH): 1H NMR (DMSO-d6, 500 MHz) d 7.90
(d, 1H, J = 7.4 Hz), 7.47 (br s, 1H), 7.17 (br s, 2H), 7.05 (br s,
1H), 5.88 (d, 1H, J = 7.9 Hz), 5.75 (br s, 1H), 5.41 (d, 1H, J = 6.2
Hz), 5.22 (t, 1H, J = 4.5 Hz), 5.13 (s, 1H), 3.98 (dd, 1H, J = 7.1,
7.1 Hz), 3.93 (br s, 1H), 3.66 (ddd, 1H, J = 2.9, 4.5, 9.8 Hz), 3.57
(ddd, 1H, J = 2.4, 4.5, 9.8 Hz), 2.55 (d, 1H, J = 16.3 Hz), 2.49 (d,
1H, J = 16.3 Hz); 13C NMR (DMSO-d6, 100 MHz) d 173.1, 165.3,
156.0, 142.4, 94.5, 87.3, 86.7, 77.3, 77.2, 76.4, 61.0; FAB-MS m/z
253 (MH+); Anal. Calcd for C11H16N4O6: C, 44.00; H, 5.37; N,
18.66. Found: C, 43.79; H, 5.35; N, 18.37.
General methods
NMR spectra were obtained on a JEOL EX270, JEOL GX270,
JEOL AL400 or Bruker ARX-500 and were reported in parts
per million (d) relative to tetramethylsilane (0.00 ppm) as an
internal standard otherwise noted. Coupling constant (J) was
reported in Hertz (Hz). Abbreviations of multiplicity were as
follows; s: singlet, d; doublet, t: triplet, q: quatlet, m: multiplet, br:
broad. Data were presented as follows; chemical shift (multiplicity,
integration, coupling constant). FAB-MS were obtained on a
JEOL JMS-HX101 or JEOL JMS-700TZ. Analytical thin layer
chromatography (TLC) was performed on Merck silica gel 60F254
plates. Normal-phase column chromatography was performed on
Merck silica gel 5715 or Kanto Chemical silica gel 60 N (neutral).
Flash column chromatography was performed on Merck silica gel
60. Dichloromethane and acetonitorile were distilled from P2O5
and then CaH2. Methanol was distilled from sodium metal or
directly used HPLC grade solvent from Kanto Chemical Co., Inc.
Toluene was distilled from sodium metal–benzophenone ketyl.
N,N-Dimethylformamide and dimethylsulfoxide were distilled
from CaH2 under reduced pressure or purchased in dehydrated
solvent from Kishida Chemical Co., Ltd. Tetrahydrofuran was
purchased in dehydrated stabilizer free solvent from Kanto
Chemical Co., Inc.
1-[3-C-(Cyanomethyl)-b-D-ribo-pentofuranosyl]cytosine
(7).
Compound 7 was prepared from 15 (281 mg, 0.68 mmol) as
described above for the synthesis of 4. After purification by
desalting (charcoal, 50% aqueous MeOH) to give 7 (205 mg,
99%) as a white powder: 1H NMR (DMSO-d6, 500 MHz) d 7.83
(d, 1H, J = 7.5 Hz), 7.27 (br s, 2H), 5.89 (d, 1H, J = 7.8 Hz),
5.77 (d, 1H, J = 7.5 Hz), 5.63 (d, 1H, J = 6.0 Hz), 5.51 (t, 1H,
J = 4.4 Hz), 5.42 (s, 1H), 3.97 (dd, 1H, J = 6.0, 7.8 Hz), 3.89
(br s, 1H), 3.67 (ddd, 1H, J = 3.0, 4.4, 12.4 Hz), 3.62 (ddd, 1H,
J = 2.4, 4.4, 12.4 Hz), 2.86 (d, 1H, J = 17.0 Hz), 2.78 (d, 1H,
J = 17.0 Hz); 13C NMR (DMSO-d6, 125 MHz) d 165.5, 155.8,
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(carbamoylmethyl)-b-
D-ribo-pentofuranosyl]cytosine (14). A solution of 1221 (1.05 g,
2.00 mmol), DMAP (487 mg, 4.00 mmol), and Et3N (0.28 mL,
4.00 mmol) in MeCN (50 mL) was treated with 2,4,6-
triisopropylbenzenesulfonyl chloride (TPSCl, 1.21 g, 4.00 mmol),
and the mixture was stirred at room temperature for 24 h. Then,
28% NH4OH (20 mL) was added, and the mixture was stirred
for additional 2 h. The mixture was evaporated under reduced
pressure, and the residue was purified by column chromatography
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146.7, 118.2, 94.7, 87.2, 85.3, 76.0, 75.7, 60.4; FAB-MS m/z 283
(MH+); ESI-HRMS calcd for C11H14N4NaO5 305.0862, found
305.0852 (MNa+).
301 (MH+). FAB-HRMS calcd for C11H17N4O6 301.1070, found
301.1162 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C -(methoxycarbonyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (24). A solution of 10
(3.18 g, 8.00 mmol) in MeOH (50 mL) was treated with K2CO3
(1.10 g, 8.00 mmol) at −70 ◦C for 3 h. The remaining K2CO3
was filtered off, and the filtrate was evaporated under reduced
pressure. The residue in DMF (50 mL) was treated with imidazole
(3.26 g, 48.0 mmol) and TBSCl (3.62 g, 24.0 mmol) at room
temperature for 30 min. The mixture was partitioned between
EtOAc (150 mL) and H2O (150 mL), and the organic layer was
washed with H2O (150 mL) and brine (100 mL), dried (Na2SO4),
filtered, and evaporated under reduced pressure. The residue was
purified by column chromatography (SiO2, 25% EtOAc–hexane)
to give 24 (2.30 g, 84%) as a white foam: 1H NMR (CDCl3,
500 MHz) d 8.47 (br s, 1H), 7.98 (d, 1H, J = 8.1 Hz), 6.18 (d,
1H, J = 7.3 Hz), 5.72 (dd, 1H, J = 2.2, 8.1 Hz), 4.37 (s, 1H), 4.08
(d, 1H, J = 7.2 Hz), 4.06 (dd, 1H, J = 1.1, 11.8 Hz), 4.04 (d, 1H,
J = 7.3 Hz), 3.91 (dd, 1H, J = 2.1, 11.8 Hz), 3.73 (s, 3H), 3.08 (s,
1H), 2.81 (d, 1H, J = 15.6 Hz), 2.62 (d, 1H, J = 15.6 Hz), 0.95
(s, 9H), 0.90 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H), 0.03 (s, 3H), −0.08
(s, 3H); 13C NMR (CDCl3, 125 MHz) d 172.3, 163.2, 151.0, 140.5,
103.5, 86.1, 85.1, 78.8, 78.3, 63.3, 40.7, 26.2, 25.8, 18.5, 18.0, 11.7,
−4.4, −4.5, −5.1, −5.5; FAB-MS m/z 517 (MH+).
1-[2,5-Di-O -(tert-butyldimethylsilyl)-3-C -(ethoxycarbonyl-
methyl)-b-D-xylo-pentofuranosyl]uracil
(22). n-Butyllithium
(1.51 mL, 2.50 mmol, 1.66 M solution in hexane) was added
dropwise to a solution of HMDS (528 mg, 2.50 mmol) in THF
(5 mL) at −10 ◦C and the mixture was stirred for 20 min, and
then cooled to −78 ◦C. Ethyl acetate (0.25 mL, 2.5 mmol) in THF
(5 mL) was added, and the mixture was stirred for 20 min at the
same temperature. A solution of 2163 (470 mg, 1.00 mmol) in THF
(2 mL) was added and the mixture was stirred for 30 min before
the addition of saturated aqueous NH4Cl (2 mL). The mixture
was partitioned between EtOAc (100 mL) and H2O (100 mL),
and the organic layer was washed with H2O (50 mL) and brine
(50 mL), dried (Na2SO4), filtered, and evaporated under reduced
pressure. The residue was purified by column chromatography
(SiO2, 40% EtOAc–hexane) to give 22 (547 mg, 95%) as a white
foam: 1H NMR (CDCl3, 500 MHz) d 7.95 (br s, 1H), 7.88 (d, 1H,
J = 8.2 Hz), 5.73 (s, 1H), 5.64 (dd, 1H, J = 2.2, 8.2 Hz), 4.47 (s,
1H), 4.26 (s, 1H), 4.19–4.12 (m, 3H), 4.02 (dd, 1H, J = 3.8, 11.1
Hz), 3.96 (dd, 1H, J = 4.5, 11.1 Hz), 2.82 (d, 1H, J = 17.0 Hz),
2.70 (d, 1H, J = 17.0 Hz), 1.27 (t, 3H, J = 8.0 Hz), 0.92 (s, 9H),
0.89 (s, 9H), 0.21 (s, 3H), 0.13 (s, 3H), 0.12 (s, 3H), 0.08 (s, 3H);
13C NMR (CDCl3, 125 MHz) d 172.1, 163.6, 150.4, 141.2, 100.8,
91.5, 84.1, 82.2, 79.4, 61.0, 35.9, 25.9, 25.8, 18.0, −4.3, −4.4,
−4.4; FAB-MS m/z 559 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(2-hydroxyethyl)-b-D-
ribo-pentofuranosyl]uracil (25). To a solution of 24 (2.90 g,
5.33 mmol) in THF (50 mL), DIBAL-H (21.7 mL, 0.93 M
solution in hexane, 21.3 mmol) was added dropwise at −20 ◦C
and the mixture was stirred for 2 h. After the addition of saturated
aqueous NH4Cl (20 mL), the mixture was extracted with EtOAc
(100 mL × 2). The combined organic layers were washed with
H2O (100 mL) and brine (50 mL), dried (Na2SO4), filtered, and
evaporated under reduced pressure. The residue was purified by
column chromatography (SiO2, 60% EtOAc–hexane) to give 25
(2.57 g, 39%) as a white foam and 24 (recovered, 1.25 g): 1H NMR
(CDCl3, 500 MHz) d 8.17 (br s, 1H), 8.03 (d, 1H, J = 8.4 Hz), 6.14
(d, 1H, J = 7.2 Hz), 5.72 (dd, 1H, J = 2.3, 8.2 Hz), 4.20 (s, 1H),
4.08 (d, 1H, J = 7.2 Hz), 4.00–3.88 (m, 4H), 2.92 (s, 1H), 2.31
(br s, 1H), 2.03 (ddd, 1H, J = 5.8, 11.9, 14.7 Hz), 1.81 (ddd, 1H,
J = 5.8, 11.3, 14.7 Hz), 0.95 (s, 9H), 0.90 (s, 9H), 0.14 (s, 3H), 0.13
(s, 3H), 0.04 (s, 3H), −0.07 (s, 3H); 13C NMR (CDCl3, 125 MHz)
d 163.0, 150.6, 103.0, 86.2, 85.5, 80.0, 78.6, 63.4, 58.6, 34.6, 18.3,
17.7, −4.5, −4.6, −4.7, −4.8; FAB-MS m/z 489 (MH+).
1-[2,5-Di-O -(tert-butyldimethylsilyl)-3-C -(ethoxycarbonyl-
methyl)-b-D-xylo-pentofuranosyl]cytosine (23). Compound 23
was prepared from 22 (450 mg, 0.81 mmol) as described above for
the synthesis of 14. After purification by chromatography (SiO2,
4% MeOH–CHCl3) to give 23 (318 mg, 71%) as a yellow foam: 1H
NMR (CDCl3, 500 MHz) d 7.85 (d, 1H, J = 8.6 Hz), 5.64 (br s,
1H), 5.71 (s, 1H), 4.28 (s, 2H), 4.18–4.10 (m, 3H), 4.03–3.98 (m,
2H), 2.82 (d, 1H, J = 17.1 Hz), 2.69 (d, 1H, J = 17.1 Hz), 1.25
(t, 3H, J = 7.1 Hz), 0.92 (s, 9H), 0.89 (s, 9H), 0.28 (s, 3H), 0.13
(s, 3H), 0.12 (s, 3H), 0.10 (s, 3H); NOE, irradiate H-3ꢀꢀa (d 2.69),
observed at H-1ꢀ (d 5.64, 1.1%); 13C NMR (CDCl3, 125 MHz) d
172.6, 166.3, 156.4, 142.9, 94.1, 93.3, 84.5, 82.6, 79.9, 61.5, 36.4,
26.5, 26.1, 18.6, −3.6, −3.6, 3.7, −4.9; FAB-MS m/z 558 (MH+).
1-[3-C -(Carbamoylmethyl)-b-D-xylo-pentofuranosyl]cytosine
(16). Compound 23 (106 mg, 0.20 mmol) was treated with
methanolic ammonia (20 mL), and the mixture was stirred at room
temperature for 2 h. The mixture was evaporated under reduced
pressure. The residue was dissolved in MeOH (5 mL), and the
solution was heated with NH4F (148 mg, 4.00 mmol) under reflux
for 2 h. The mixture was evaporated under reduced pressure. The
residue was suspended in aqueous MeOH, and the resulting white
solid was collected to give 16 (55 mg, 90%) as a white powder:
1H NMR (DMSO-d6, 500 MHz) d 7.75 (d, 1H, J = 7.4 Hz), 7.66
(br s, 1H), 7.27 (br s, 1H), 7.17 (br d, 2H), 5.99 (d, 1H, J = 4.9
Hz), 5.76 (s, 1H), 5.69 (d, 1H, J = 7.4 Hz), 5.61 (s, 1H), 4.80 (t,
1H, J = 4.6 Hz), 3.89 (d, 1H, J = 4.9 Hz), 3.80–3.70 (m, 2H), 3.57
(ddd, 1H, J = 2.4, 4.6, 13.0 Hz), 2.48 (d, 1H, J = 15.3 Hz), 2.41
(d, 1H, J = 15.3 Hz); 13C NMR (DMSO-d6, 125 MHz) d 172.0,
165.9, 156.4, 143.0, 94.1, 92.8, 84.4, 82.6, 79.9, 63.0; FAB-MS m/z
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(2-mesyloxyethyl)-b-
D-ribo-pentofuranosyl]uracil (26). A solution of 25 (2.03 g,
4.45 mmol), Et3N (935 lL) and DMAP (814 mg, 6.68 mmol)
in CH2Cl2 (30 mL) was treated with methanesulfonic anhydride
(1.16 g, 6.68 mmol), and the mixture was stirred at room
temperature for 30 min. After the addition of H2O (20 mL), the
organic layer was washed with brine (50 mL), dried (Na2SO4),
filtered, and evaporated under reduced pressure. The residue was
purified by column chromatography (SiO2, 40% EtOAc–hexane)
to give 26 (2.42 g, 92%) as a white foam: 1H NMR (CDCl3,
500 MHz) d 8.15 (br s, 1H), 8.00 (d, 1H, J = 8.0 Hz), 6.15 (d,
1H, J = 7.2 Hz), 5.73 (dd, 1H, J = 2.2, 8.0 Hz), 4.49–4.43 (m,
2H), 4.13–4.08 (m, 2H), 3.96 (dd, 1H, J = 1.6, 12.4 Hz), 3.90 (dd,
1H, J = 2.0, 12.4 Hz), 2.79 (s, 1H), 2.28 (ddd, 1H, J = 7.8, 14.4,
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15.0 Hz), 1.92 (ddd, 1H, J = 5.3, 11.1, 14.4 Hz), 0.96 (s, 9H),
0.89 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H), 0.04 (s, 3H), −0.07 (s, 3H);
13C NMR (CDCl3, 125 MHz) d 162.5, 150.4, 140.4, 103.1, 103.0,
86.0, 85.1, 78.3, 66.0, 63.4, 37.3, 32.3, 18.2, 17.8, −4.5, −4.6, −4.7,
−4.7; FAB-MS m/z 595 (MH+).
added, and the mixture was stirred for additional 10 min. The
mixture was partitioned between EtOAc (200 mL) and H2O
(100 mL), and the organic layers were washed with brine (50 mL),
dried (Na2SO4), filtered, and evaporated under reduced pressure
to give the crude aldehyde 29, which was used in the next step
without further purification. A solution of the aldehyde 29 in
CH2Cl2 (10 mL) was treated with a solution of I2 (132 mg,
0.52 mmol) and KOH (◦58 mg, 1.04 mmol) in MeOH, and the
mixture was stirred at 0 C for 1 h. The mixture was neutralized
with 1 M aqueous HCl and partitioned between EtOAc (100 mL)
and H2O (100 mL), and the organic layers were washed with brine
(50 mL), dried (Na2SO4), filtered, and evaporated under reduced
pressure. The residue was purified by column chromatography
(SiO2, 25% EtOAc–hexane) to give 30 (161 mg, 80%) as a white
foam: 1H NMR (CDCl3, 500 MHz) d 8.20 (br s, 1H), 7.74 (d, 1H,
J = 8.2 Hz), 6.06 (d, 1H, J = 7.3 Hz), 5.77 (dd, 1H, J = 3.7, 11.3
Hz), 3.80 (s, 3H), 3.78 (dd, 1H, J = 3.7, 11.3 Hz), 3.57 (s, 1H),
0.91 (s, 9H), 0.85 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), 0.04 (s, 3H),
−0.01 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 170.5, 163.0, 150.6,
140.4, 103.4, 86.7, 86.0, 79.9, 62.7, 52.9, 26.1, 25.7, 18.6, 18.1,
−4.7, −4.7, −5.2, −5.6; FAB-MS m/z 531 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(2-phenylselenylethyl)-
b-D-ribo-pentofuranosyl]uracil (27). A solution of diphenyl-
diselenide (1.47 g, 4.93 mmol) in EtOH (30 mL) was treated with
NaBH4 (610 mg, 16.1 mmol), and the mixture was stirred at room
temperature for 15 min. A solution of 26 (2.25 g, 3.97 mmol) in
EtOH (30 mL) was added, and the whole was stirred at 60 ◦C
for 1 h, and evaporated under reduced pressure. The residue was
dissolve in EtOAc (200 mL), and the solution was washed with
H2O (100 mL × 3) and brine (50 mL), dried (Na2SO4), filtered,
and evaporated under reduced pressure. The residue was purified
by column chromatography (SiO2, 30% EtOAc–hexane) to give
27 (2.44 g, 98%) as a white foam: 1H NMR (CDCl3, 500 MHz) d
7.98 (d, 1H, J = 8.1 Hz), 7.96 (br s, 1H), 7.50 (m, 2H), 7.25 (m,
3H), 6.11 (d, 1H, J = 7.2 Hz), 5.69 (dd, 1H, J = 2.2, 8.1 Hz), 4.11
(br s, 1H), 4.00 (d, 1H, J = 7.2 Hz), 3.91 (dd, 1H, J = 2.3, 12.0
Hz), 3.76 (d, 1H, J = 12.0 Hz), 3.21 (dt, 1H, J = 5.0, 12.0 Hz),
2.74 (dt, 1H, J = 5.0, 12.0 Hz), 2.62 (s, 1H), 2.12 (dt, 1H, J =
5.0, 14.1 Hz), 1.90 (dt, 1H, J = 5.0, 14.1 Hz), 0.94 (s, 9H), 0.81
(s, 9H), 0.13 (s, 3H), 0.11 (s, 3H), −0.10 (s, 3H), −0.13 (s, 3H);
13C NMR (CDCl3, 125 MHz) d 162.9, 150.6, 140.6, 133.3, 133.2,
129.8, 129.7, 127.5, 102.9, 86.3, 84.8, 79.7, 78.4, 63.4, 32.1, 26.0,
25.6, 11.5, −4.6, −4.7, −5.5, −5.6; FAB-MS m/z 657 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(methoxycarbonyl)-b-
D-ribo-pentofuranosyl]cytosine (31). Compound 31 was prepared
from 28 (79.5 mg, 0.15 mmol) as described above for the synthesis
of 14. After purification by column chromatography (SiO2, 1%
1
MeOH–CHCl3) to give 31 (78 mg, 97%) as a white foam: H
NMR (CDCl3, 500 MHz) d 7.75 (d, 1H, J = 7.4 Hz), 6.17 (d, 1H,
J = 6.9 Hz), 5.80 (d, 1H, J = 7.4 Hz), 4.62 (d, 1H, J = 6.9 Hz),
4.12 (t, 1H, J = 3.7 Hz), 3.86 (dd, 1H, J = 3.9, 16.2 Hz), 3.79
(dd, 1H, J = 3.9, 16.2 Hz), 3.78 (s, 3H), 3.77 (s, 1H), 0.90 (s, 9H),
0.84 (s, 9H), 0.07 (s, 3H), 0.06 (s, 3H), 0.00 (s, 3H), −0.02 (s, 3H);
FAB-MS m/z 511 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-ethenyl-b-D-ribo-pento-
furanosyl]uracil (28). A solution of 27 (2.38 g, 3.63 mmol) in
CH2Cl2 (25 mL) was treated with mCPBA (751 mg, 4.36 mmol)
at 0 ◦C, and the mixture was stirred for 10 min before the
addition of saturated aqueous Na2S2O3 (2 mL). The mixture was
partitioned between EtOAc (200 mL) and H2O (100 mL), and the
organic layers were washed with brine (50 mL), dried (Na2SO4),
filtered, and evaporated under reduced pressure. The residue was
dissolved in DMF (50 mL), and the solution was heated at 60 ◦C
for 1 h. The mixture was partitioned between EtOAc (200 mL)
and H2O (200 mL), and the organic layers were washed with H2O
(200 mL) brine (50 mL), dried (Na2SO4), filtered, and evaporated
under reduced pressure. The residue was purified by column
chromatography (SiO2, 25% EtOAc–hexane) to give 28 (851 mg,
49%) as a white foam and 27 (recovered, 990 mg, 47%): 1H NMR
(CDCl3, 500 MHz) d 8.11 (br s, 1H), 8.03 (d, 1H, J = 8.0 Hz), 6.22
(d, 1H, J = 7.4 Hz), 6.02 (dd, 1H, J = 10.5, 17.1 Hz), 5.73 (dd,
1H, J = 2.1, 8.0 Hz), 5.65 (dd, 1H, J = 1.6, 17.1 Hz), 5.39 (dd,
1H, J = 1.6, 10.5 Hz), 4.14 (d, 1H, J = 7.4 Hz), 4.03 (br s, 1H),
3.86 (dd, 1H, J = 2.2, 11.5 Hz), 3.65 (d, 1H, J = 11.5 Hz), 2.82 (s,
1H), 0.94 (s, 9H), 0.83 (s, 9H), 0.15 (s, 3H), 0.14 (s, 3H), −0.05 (s,
3H), −0.09 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 163.1, 150.6,
140.5, 136.2, 118.6, 102.9, 86.7, 86.3, 80.5, 79.4, 63.4, 18.3, 17.7,
−4.4, −4.4, −4.9, −4.9; FAB-MS m/z 657 (MH+).
1-[3-C-(Carbamoyl)-b-D-ribo-pentofuranosyl]cytosine (17).
A
solution of 30 (20.5 mg, 0.04 mmol) was dissolved in methanolic
ammonia (10 mL, saturated at 0 ◦C) and heated at 80 ◦C in a
sealed tube for 24 h. The mixture was allowed to cool to room
temperature and evaporated under reduced pressure. The residue
in MeOH (10 mL) containing NH4F (20 mg, 0.80 mmol) was
heated under reflux for 2 h. The mixture was evaporated under
reduced pressure, and the residue was partitioned between CHCl3
(50 mL) and H2O (50 mL). The aqueous layer was washed CHCl3
(50 mL × 2) and evaporated under reduced pressure. The residue
was dissolved in H2O, and the charcoal was added until the
UV absorption of the solution disappeared. The suspension was
mounted on the column, and the charcoal was washed with H2O
(50 mL) and eluted with 50% aqueous MeOH. The UV positive
fractions were collected and concentrated to give 17 (10.8 mg, 2
steps 87%) as a white powder: 1H NMR (DMSO-d6, 500 MHz) d
7.87 (d, 1H, J = 7.3 Hz), 7.50 (br s, 1H), 7.39 (br s, 1H), 7.21 (br s,
1H), 7.14 (br s, 1H), 5.92 (d, 1H, J = 7.0 Hz), 5.77 (d, 1H, J = 7.4
Hz), 5.75 (d, 1H, J = 6.1 Hz), 5.60 (s, 1H), 4.80 (t, 1H, J = 5.3 Hz),
4.26 (dd, 1H, J = 6.1, 7.0 Hz), 3.76 (br s, 1H), 3.45 (m, 2H); 13
C
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(methoxycarbonyl)-b-
D-ribo-pentofuranosyl]uracil (30). A solution of 28 (200 mg,
0.40 mmol) and 4-methylmorphrine N-oxide (48 mg, 0.80 mmol)
in acetone–H2O (9 : 1, 10 mL) was treated with OsO4 (5 mg/1 mL
t-BuOH solution, 0.50 mL, 0.20 mL), and the mixture was stirred
at room temperature for 3 d. NaIO4 (87 mg, 0.40 mmol) was
NMR (DMSO-d6, 125 MHz) d 174.5, 171.9, 165.4, 155.5, 141.7,
94.7, 86.9, 86.4, 79.2, 76.2, 61.2; EI-MS m/z 286 (M+); ESI-HRMS
calcd for C10H14N4NaO6 309.0811, found 309.0822 (MNa+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(trans-ethoxycarbonyl-
ethenyl)-bD-ribo-pentofuranosyl]uracil (32). A solution of 28
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(200 mg, 0.40 mmol) and NMO (48 mg, 0.80 mmol) in acetone–
H2O (9 : 1, 10 mL) was treated with OsO4 (5 mg/1 mL t-BuOH
solution, 0.50 mL, 0.20 mL), and the mixture was stirred at
room temperature for 3 d. NaIO4 (87 mg, 0.40 mmol) was added,
and the mixture was stirred for additional 10 min. The mixture
was partitioned between EtOAc (200 mL) and H2O (100 mL),
and the organic layers were washed with brine (50 mL), dried
(Na2SO4), filtered, and evaporated under reduced pressure to
give crude aldehyde 29, which was used in the next step without
further purification. A solution of the aldehyde 29 in CH2Cl2
The mixture was heated at 80 ◦C in a sealed tube for 24 h and
allowed cool to room temperature. The mixture was evaporated
under reduced pressure. The residue was partitioned between
CHCl3 (10 mL) and H2O (50 mL). The aqueous layer was washed
CHCl3 (10 mL × 2) and evaporated under reduced pressure. The
residue was dissolved in H2O, and the charcoal was added until UV
absorption of the solution was disappeared. The suspension was
mounted on the column, and the charcoal was washed with H2O
(50 mL) and eluted with 50% aqueous MeOH. The UV positive
fractions were collected and concentrated to give 18 (8.2 mg, 2
steps 87%) as a white powder: 1H NMR (DMSO-d6, 500 MHz) d
7.92 (d, 1H, J = 7.5 Hz), 7.21 (br s, 1H), 7.14 (br s, 1H), 7.29 (br s,
1H), 6.71 (br s, 1H), 5.84 (d, 1H, J = 7.8 Hz), 5.74 (d, 1H, J = 7.4
Hz), 5.24 (d, 1H, J = 6.6 Hz), 5.14 (t, 1H, J = 3.8 Hz), 4.70 (s,
1H), 3.93 (dd, 1H, J = 6.6, 7.8 Hz), 3.76 (br s, 1H), 3.51 (m, 2H),
2.31 (ddd, 1H, J = 5.6, 10.7, 15.7 Hz), 2.17 (ddd, 1H, J = 5.1,
10.8, 15.7 Hz), 1.87 (ddd, 1H, J = 5.1, 5.6, 13.6 Hz), 1.76 (ddd,
1H, J = 10.7, 13.6, 10.8 Hz); 13C NMR (DMSO-d6, 125 MHz) d
175.4, 165.4, 155.7, 142.9, 94.6, 88.0, 87.0, 77.9, 77.0, 61.1, 29.6,
29.2; EI-MS m/z 316 (M+); ESI-HRMS calcd for C12H18N4NaO6
337.1124, found 337.1124 (MNa+).
=
(10 mL) was treated with a solution of Ph3P CHCO2Et (167 mg,
0.48 mmol), and the mixture was stirred at room temperature for
1 h. The mixture was evaporated under reduced pressure, and
the residue was purified by column chromatography (SiO2, 25%
1
EtOAc–hexane) to give 32 (194 mg, 85%) as a white foam: H
NMR (CDCl3, 500 MHz) d 8.04 (d, 1H, J = 8.1 Hz), 8.01 (br s,
1H), 7.10 (d, 1H, J = 15.5 Hz), 6.35 (d, 1H, J = 15.5 Hz), 6.23
(d, 1H, J = 7.3 Hz), 5.75 (dd, 1H, J = 2.1, 8.1 Hz), 4.26 (d, 1H,
J = 7.3 Hz), 4.26 (q, 2H, J = 7.2 Hz), 4.12 (br s, 1H), 3.89 (dd,
1H, J = 2.2, 11.8 Hz), 3.63 (d, 1H, J = 11.8 Hz), 3.03 (s, 1H),
1.30 (t, 3H, J = 7.2 Hz), 1.00 (s, 9H), 0.82 (s, 9H), 0.19 (s, 3H),
0.18 (s, 3H), −0.08 (s, 3H), −0.07 (s, 3H); 13C NMR (CDCl3, 125
MHz) d 165.7, 162.9, 150.6, 145.9, 140.3, 125.3, 103.4, 87.0, 86.8,
80.6, 79.6, 63.5, 60.8, 26.2, 25.6, 14.5, −4.4, −4.8, −5.2, −5.6;
FAB-MS m/z 572 (MH+).
1-[2-O-(tert-Butyldimethylsilyl)-3-C -(N -methylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (35a). A solution of 10
(398 mg, 1.00 mmol) containing methylamine hydrochloride
(333 mg, 5.00 mmol) and Et3N (0.7 mL, 5.00 mmol) in DMF
(5 mL) was stirred at room temperature for 7 h. The mixture was
partitioned between EtOAc (100 mL) and H2O (100 mL). The
organic layer was washed with H2O (100 mL) and brine (50 mL),
dried (Na2SO4), filtered, and evaporated under reduced pressure.
The residue was purified by column chromatography (SiO2, 4%
1-[2,5-Di-O -(tert-butyldimethylsilyl)-3-C -(ethoxycarbonyl-
ethenyl)-b-D-ribo-pentofuranosyl]cytosine (33). Compound 33
was prepared from 32 (80 mg, 0.14 mmol) as described above for
the synthesis of 14. After purification by column chromatography
(SiO2, 4% MeOH–CHCl3), 33 (78 mg, 98%) was obtained as a
white foam: 1H NMR (CDCl3, 500 MHz) d 7.92 (d, 1H, J = 7.5
Hz), 7.10 (d, 1H, J = 15.3 Hz), 7.02 (br s, 2H), 6.33 (d, 1H, J =
15.3 Hz), 6.32 (d, 1H, J = 7.5 Hz), 5.96 (d, 1H, J = 7.2 Hz), 4.18
(m, 3H), 4.06 (br s, 1H), 3.82 (br d, 1H, J = 11.3 Hz), 3.59 (br d,
1H, J = 11.3 Hz), 3.42 (s, 1H), 1.29 (t, 3H, J = 7.2 Hz), 0.96 (s,
9H), 0.74 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H), −0.01 (s, 3H), −0.17
(s, 3H); FAB-MS m/z 569 (MH+).
1
MeOH–CHCl3) to give 35a (456 mg, 99%) as a yellow glass: H
NMR (CDCl3, 500 MHz) d 8.15 (br s, 1H), 7.70 (d, 1H, J = 8.2
Hz), 6.60 (d, 1H, J = 4.7 Hz), 5.79 (dd, 1H, J = 2.2, 8.2 Hz), 6.17
(d, 1H, J = 7.3 Hz), 4.27 (br s, 1H), 3.88 (dd, 1H, J = 2.4, 12.6
Hz), 3.76 (dd, 1H, J = 1.2, 12.6 Hz), 3.54 (s, 1H), 2.83 (d, 3H,
J = 4.7 Hz), 2.80 (d, 1H, J = 15.7 Hz), 2.56 (d, 1H, J = 15.7 Hz),
0.89 (s, 9H), 0.63 (s, 3H), −0.01 (s, 3H); 13C NMR (DMSO-d6, 125
MHz) d 170.9, 162.6, 104.6, 85.2, 85.0, 77.1, 76.9, 59.7, 25.6, 24.8,
−4.5, −4.6; FAB-MS m/z 430 (MH+).
1-[2,5-Di-O -(tert-butyldimethylsilyl)-3-C -(ethoxycarbonyl-
ethyl)-b-D-ribo-pentofuranosyl]cytosine (34). A solution of 33
(57 mg, 0.10 mmol) and 10% Pd/C (10 mg) in MeOH (5 mL)
was vigorously stirred under H2 atmosphere at room temperature
for 30 min. The catalyst was filtered off, and the filtrate was
evaporated under reduced pressure. The residue was purified by
column chromatography (SiO2, 4% MeOH–CHCl3) to give 34
1 - [2 - O - (tert - Butyldimethylsilyl) - 3 - C - (N - ethylcarbamoyl -
methyl)-b-D-ribo-pentofuranosyl]uracil (35b). Compound 35b
was prepared from 10 (398 mg, 1.00 mmol), ethylamine (408 mg,
5.00 mmol), and Et3N (0.7 mL, 5.00 mmol) as described above for
the synthesis of 35a. After purification by column chromatography
(SiO2, 5% MeOH–CHCl3) to give 35b (462 mg, 99%) as a yellow
glass: 1H NMR (CDCl3, 500 MHz) d 9.28 (br s, 1H), 7.78 (d, 1H,
J = 8.1 Hz), 6.80 (d, 1H, J = 5.3 Hz), 5.79 (d, 1H, J = 7.3 Hz),
5.71 (dd, 1H, J = 2.2, 8.1 Hz), 4.40 (d, 1H, J = 7.3 Hz), 4.20 (br s,
1H), 4.17 (s, 1H), 4.01 (br s, 1H), 3.80 (dd, 1H, J = 1.0, 12.1 Hz),
3.73 (dd, 1H, J = 5.0, 12.1 Hz), 3.26 (dq, 2H, J = 5.3, 7.1 Hz),
2.73 (d, 1H, J = 15.7 Hz), 2.51 (d, 1H, J = 15.7 Hz), 1.12 (t, 3H,
J = 7.1 Hz), 0.89 (s, 9H), 0.63 (s, 3H), −0.01 (s, 3H); FAB-MS
m/z 444 (MH+).
1
(58 mg, quant.) as a white foam: H NMR (CDCl3, 500 MHz)
d 7.75 (d, 1H, J = 7.4 Hz), 6.18 (d, 1H, J = 7.6 Hz), 5.76 (d, 1H,
J = 7.4 Hz), 4.18 (q, 2H, J = 7.4 Hz), 4.21 (br s, 1H), 3.94 (dd,
1H, J = 2.9, 12.1 Hz), 3.74 (dd, 1H, J = 2.0, 11.3 Hz), 3.67 (s,
1H), 2.63 (ddd, 1H, J = 1.2, 3.0, 12.8 Hz), 2.31 (ddd, 1H, J = 2.8,
6.9, 12.8 Hz), 1.28 (t, 3H, J = 7.4 Hz), 0.96 (s, 9H), 0.84 (s, 9H),
0.14 (s, 3H), 0.13 (s, 3H), −0.02 (s, 3H), −0.18 (s, 3H); FAB-MS
m/z 571 (MH+).
1-[3-C-(Carbamoylethyl)-b-D-ribo-pentofuranosyl]cytosine (18).
A solution of 33 (20.5 mg, 0.04 mmol) and NH4F (20 mg,
0.80 mmol) in MeOH (10 mL) was heated under reflux for 2 h. The
mixture was evaporated under reduced pressure, and the residue
was dissolved in methanolic ammonia (10 mL, saturated at 0 ◦C).
1-[2-O -(tert -Butyldimethylsilyl)-3-C -(N -benzylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (35c). Compound 35c
was prepared from 10 (398 mg, 1.00 mmol), benzylamine (546 lM,
5.00 lmol), and Et3N (0.7 mL, 5.00 mmol) as described above for
1292 | Org. Biomol. Chem., 2006, 4, 1284–1296
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the synthesis of 35a. After purification by column chromatography
(SiO2, 5% MeOH–CHCl3) to give 35c (405 mg, 80%) as a colorless
glass: 1H NMR (CDCl3, 500 MHz) d 8.10 (br s, 1H), 8.05 (d, 1H,
J = 8.3 Hz), 7.35–7.33 (m, 5H), 7.15 (t, 1H, J = 5.6 Hz), 6.15 (d,
1H, J = 7.3 Hz), 5.72 (dd, 1H, J = 2.3, 8.1 Hz), 4.56 (d, 2H, J =
11.2 Hz), 4.19 (br s, 1H), 4.07 (d, 1H, J = 7.3 Hz), 3.87 (dd, 1H,
J = 1.2, 12.5 Hz), 3.82 (dd, 1H, J = 2.1, 12.5 Hz), 3.40 (br s, 1H),
2.68 (d, 1H, J = 15.5 Hz), 2.44 (d, 1H, J = 15.5 Hz), 0.89 (s, 9H),
0.18 (s, 3H), −0.07 (s, 3H); 13C NMR (DMSO-d6, 100 MHz) d
169.9, 162.7, 150.9, 140.6, 140.5, 102.5, 86.8, 86.7, 84.8, 77.7, 77.6,
77.4, 60.7, 37.1, 35.7, 34.8, 25.5, −4.5, −4.6, −5.0, −5.0; EI-MS
m/z 316 (M+).
was prepared from 35c (404 mg, 0.80 mmol) as described above for
the synthesis of 12. After purification by column chromatography
(SiO2, 40% EtOAc–hexane) to give 36c (460 mg, 93%) as a colorless
glass: 1H NMR (CDCl3, 500 MHz) d 8.06 (br s, 1H), 8.01 (d, 1H,
J = 8.3 Hz), 7.37–7.26 (m, 5H), 7.13 (t, 1H, J = 5.7 Hz), 6.15 (d,
1H, J = 7.3 Hz), 5.72 (dd, 1H, J = 2.3, 8.1 Hz), 4.46 (t, 2H, J =
11.6 Hz), 4.19 (br s, 1H), 4.07 (d, 1H, J = 7.3 Hz), 3.87 (dd, 1H,
J = 1.2, 12.5 Hz), 3.82 (dd, 1H, J = 2.1, 12.5 Hz), 3.40 (br s, 1H),
2.68 (d, 1H, J = 15.5 Hz), 2.44 (d, 1H, J = 15.5 Hz), 0.96 (s, 9H),
0.89 (s, 9H), 0. 18 (s, 3H), 0.17 (s, 3H), 0.03 (s, 3H), −0.07 (s, 3H);
13C NMR (CDCl3, 125 MHz) d 169.4, 150.8, 140.6, 138.5, 128.5,
128.0, 127.8, 103.4, 86.2, 85.2, 78.8, 78.6, 63.3, 43.7, 41.0, 26.3,
25.8, −4.3, −4.4, −4.5, −5.3; FAB-MS m/z 620 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N,Nꢀ-dimethylcarb-
amoylmethyl)-b-D-ribo-pentofuranosyl]uracil (36d). Compound
36d was prepared from 35d (192 mg, 0.44 mmol) as described
above for the synthesis of 12. After purification by column
chromatography (SiO2, 40% EtOAc–hexane) to give 36d (205 mg,
85%) as a yellow glass: 1H NMR (CDCl3, 500 MHz) d 7.97 (br s,
1H), 7.88 (d, 1H, J = 8.1 Hz), 6.23 (d, 1H, J = 7.5 Hz), 5.71 (dd,
1H, J = 2.3, 8.1 Hz), 4.46 (br s, 1H), 4.04 (d, 1H, J = 7.4 Hz),
3.88 (dd, 1H, J = 1.2, 12.0 Hz), 3.86 (dd, 1H, J = 2.4, 12.0 Hz),
3.51 (s, 3H), 2.80 (d, 1H, J = 16.3 Hz), 2.71 (d, 1H, J = 16.3 Hz),
0.95 (s, 9H), 0.90 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H), 0.04 (s, 3H),
−0.07 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 170.2, 162.8, 150.4,
140.5, 102.8, 102.7, 86.5, 85.8, 78.6, 78.6, 63.5, 37.3, 35.4, 25.9,
25.6, −4.5, −4.5, −4.6, −4.7; FAB-MS m/z 558 (MH+).
1-[2-O-(tert-Butyldimethylsilyl)-3-C-(N,Nꢀ-dimethylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (35d). Compound 35d
was prepared from 10 (398 mg, 1.00 mmol), dimethylamine
hydrochloride (408 mg, 5.00 mmol), and Et3N (0.7 mL,
5.00 mmol) as described above for the synthesis of 35a. After
purification by column chromatography (SiO2, 5% MeOH–
1
CHCl3) to give 35c (441 mg, 99%) as a yellow glass: H NMR
(CDCl3, 500 MHz) d 8.19 (br s, 1H), 7.32 (d, 1H, J = 8.0 Hz),
5.77 (dd, 1H, J = 2.2, 8.0 Hz), 5.41 (d, 1H, J = 7.7 Hz), 4.75 (d,
1H, J = 7.7 Hz), 4.39 (br s, 1H), 3.83 (dd, 1H, J = 2.3, 13.1 Hz),
3.57 (dd, 1H, J = 1.0, 13.1 Hz), 3.03 (s, 3H), 2.97 (s, 3H), 2.99 (d,
1H, J = 16.5 Hz), 2.75 (d, 1H, J = 16.5 Hz), 0.89 (s, 9H), 0.16
(s, 3H), −0.06 (s, 3H); 13C NMR (DMSO-d6, 125 MHz) d 169.9,
162.8, 151.1, 140.8, 102.5, 86.4, 85.0, 77.5, 60.7, 33.3, 25.5, 17.5,
14.5, −4.6, −4.6; FAB-MS m/z 444 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-methylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (36a). Compound 36a
was prepared from 35a (300 mg, 0.70 mmol) as described above for
the synthesis of 12. After purification by column chromatography
(SiO2, 40% EtOAc–hexane) to give 36a (398 mg, 99%) as a white
foam: 1H NMR (CDCl3, 500 MHz) d 8.06 (br s, 1H), 8.03 (d, 1H,
J = 8.2 Hz), 6.79 (d, 1H, J = 4.7 Hz), 6.16 (d, 1H, J = 7.2 Hz),
5.73 (dd, 1H, J = 2.2, 8.1 Hz), 4.22 (br s, 1H), 4.06 (d, 1H, J = 7.2
Hz), 3.88 (br s, 2H), 3.52 (s, 1H), 2.81 (d, 3H, J = 4.7 Hz), 2.63
(d, 1H, J = 15.5 Hz), 2.51 (d, 1H, J = 15.5 Hz), 0.96 (s, 9H), 0.89
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-methylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]cytosine (37a). Compound 37a
was prepared from 36a (217 mg, 0.40 mmol) as described above for
the synthesis of 14. After purification by column chromatography
(SiO2, 10% MeOH–CHCl3) to give 37a (157 mg, 73%) as a white
1
glass: H NMR (CDCl3, 500 MHz) d 8.06 (d, 1H, J = 8.2 Hz),
7.05 (d, 1H, J = 4.5 Hz), 6.23 (d, 1H, J = 6.7 Hz), 6.15 (br s, 1H),
4.19 (br s, 1H), 4.05 (d, 1H, J = 6.7 Hz), 3.99 (d, 1H, J = 11.5
Hz), 3.87 (d, 1H, J = 11.5 Hz), 2.80 (d, 3H, J = 4.5 Hz), 2.60 (d,
1H, J = 15.1 Hz), 2.53 (d, 1H, J = 15.1 Hz), 0.95 (s, 9H), 0.84 (s,
9H), 0.17 (s, 3H), 0.16 (s, 3H), −0.01 (s, 3H), −0.03 (s, 3H); 13C
NMR (CDCl3, 125 MHz) d 171.5, 162.5, 155.2, 141.3, 93.6, 86.2,
85.5, 78.8, 78.1, 63.5, 27.6, 25.9, 24.7, −4.6, −4.7, −5.2, −5.3;
FAB-MS m/z 543 (MH+).
(s, 9H), 0.18 (s, 3H), 0.10 (s, 3H), 0.03 (s, 3H), −0.06 (s, 3H); 13
C
NMR (CDCl3, 125 MHz) d 170.2, 163.2, 151.0, 140.7, 103.4, 86.3,
85.2, 78.8, 78.5, 63.4, 40.7, 26.2, 25.8, −4.4, −4.5, −5.0, −5.5;
FAB-MS m/z 544 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-ethylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (36b). Compound 36 was
prepared from 35b (222 mg, 0.50 mmol) as described above for
the synthesis of 12. After purification by column chromatography
(SiO2, 40% EtOAc–hexane) to give 36b (257 mg, 92%) as a white
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-ethylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]cytosine (37b). Compound 37b
was prepared from 36b (280 mg, 0.50 mmol) as described above for
the synthesis of 14. After purification by column chromatography
(SiO2, 40% EtOAc–hexane) to give 37b (280 mg, 99%) as a colorless
1
1
foam: H NMR (CDCl3, 500 MHz) d 8.01 (d, 1H, J = 8.1 Hz),
glass: H NMR (CDCl3, 500 MHz) d 8.02 (d, 1H, J = 7.5 Hz),
7.70 (br s, 1H), 6.81 (br s, 1H), 6.16 (d, 1H, J = 7.1 Hz), 5.73 (d,
1H, J = 8.2 Hz), 4.21 (br s, 1H), 4.05 (d, 1H, J = 7.2 Hz), 3.87
(br s, 2H), 3.28 (q, 2H, J = 7.3 Hz), 2.60 (d, 1H, J = 15.4 Hz), 2.50
(d, 1H, J = 15.4 Hz), 1.13 (t, 3H, J = 7.3 Hz), 0.94 (s, 9H), 0.88 (s,
9H), 0.16 (s, 6H), 0.08 (s, 3H), −0.08 (s, 3H); 13C NMR (CDCl3,
125 MHz) d 170.0, 162.5, 151.2, 102.5, 86.5, 85.0, 77.6, 77.4, 60.6,
33.3, 25.9, 25.0, 17.6, 14.7, −4.7, −4.8, −5.2, −5.6; FAB-MS m/z
544 (MH+).
7.10 (br s, 1H), 6.31 (d, 1H, J = 7.5 Hz), 6.31 (d, 1H, J = 7.1 Hz),
4.15 (br s, 1H), 4.05 (d, 1H, J = 7.1 Hz), 3.95 (d, 1H, J = 11.8
Hz), 3.85 (d, 1H, J = 11.8 Hz), 3.27 (dq, 2H, J = 7.2 Hz), 2.59 (d,
1H, J = 16.8 Hz), 2.52 (d, 1H, J = 16.8 Hz), 1.13 (t, 3H, J = 7.2
Hz), 0.95 (s, 9H), 0.86 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H), −0.04 (s,
3H), −0.10 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 172.1, 163.4,
151.1, 140.6, 103.5, 86.5, 85.2, 78.7, 77.9, 62.9, 33.7, 25.5, 24.7,
17.5, 14.6, −4.6, −4.6, −5.0, −5.6; FAB-MS m/z 557 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-benzylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]uracil (36c). Compound 36c
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N-benzylcarbamoyl-
methyl)-b-D-ribo-pentofuranosyl]cytosine (37c). Compound 37c
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was prepared from 36c (404 mg, 0.80 mmol) as described above for
the synthesis of 14. After purification by column chromatography
(SiO2, 40% EtOAc–hexane) to give 37c (460 mg, 93%) as a
colorless glass: H NMR (CDCl3, 500 MHz) d 8.03 (d, 1H, J =
0.20 mmol) as described above for the synthesis of 6. After
purification by desalting (charcoal, 50% aqueous MeOH) to give
19c (55 mg, 71%) as a white powder: mp 235 ◦C dec (crystallized
from aqueous MeOH): 1H NMR (DMSO-d6, 500 MHz) d 8.45 (br
t, 1H, J = 5.8 Hz), 7.91 (d, 1H, J = 7.4 Hz), 7.33–7.22 (m, 5H),
7.16 (br s, 2H), 5.99 (d, 1H, J = 7.9 Hz), 5.78 (br s, 1H), 5.42 (d,
1H, J = 6.1 Hz), 5.20 (t, 1H, J = 4.4 Hz), 5.02 (s, 1H), 4.30 (d, 2H,
J = 11.6), 4.02 (dd, 1H, J = 6.1, 7.9 Hz), 3.96 (br s, 1H), 3.70 (ddd,
1H, J = 1.2, 4.4, 11.9 Hz), 3.58 (ddd, 1H, J = 1.2, 4.4, 11.9 Hz),
2.65 (d, 1H, J = 15.1 Hz), 2.61 (d, 1H, J = 15.1 Hz); 13C NMR
(DMSO-d6, 125 MHz) d 170.5, 165.5, 155.9, 142.4, 139.2, 128.2,
127.3, 126.7, 94.4, 87.4, 86.7, 77.4, 76.4, 61.0, 42.1; FAB-MS m/z
391 (MH+); FAB-HRMS calcd for C18H23N4O6 390.1539, found
390.1612 (MH+).
1
7.4 Hz), 7.56 (br s, 1H), 7.34–7.24 (m, 5H), 6.31 (d, 1H, J = 7.3
Hz), 5.88 (br s, 1H), 4.44 (s, 2H), 4.15 (br s, 1H), 4.06 (d, 1H,
J = 7.3 Hz), 3.97 (d, 1H, J = 11.8 Hz), 3.82 (dd, 1H, J = 1.9,
11.8 Hz), 2.65 (d, 1H, J = 15.1 Hz), 2.59 (d, 1H, J = 15.1 Hz),
0.95 (s, 9H), 0.82 (s, 9H), 0.16 (s, 3H), 0.15 (s, 3H), −0.04 (s, 3H),
−0.06 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 169.7, 158.2, 141.8,
138.6, 128.6, 127.6, 127.3, 106.6, 88.8, 86.5, 84.8, 79.3, 78.5, 63.1,
43.3, 40.9, 26.1, 25.6, −4.7, −4.8, −5.3, −5.6; FAB-MS m/z 619
(MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(N,Nꢀ -dimethylcarb-
amoylmethyl)-b-D-ribo-pentofuranosyl]cytosine (37d). Compound
37d was prepared from 36d (167 mg, 0.3 mmol) as described
above for the synthesis of 14. After purification by column
chromatography (SiO2, 40% EtOAc–hexane) to give 37d (115 mg,
69%) as a white solid: 1H NMR (CDCl3, 500 MHz) d 8.15 (d, 1H,
J = 6.5 Hz), 6.30 (br s, 1H), 6.23 (d, 1H, J = 7.3 Hz), 4.43 (br s,
1H), 4.03 (d, 1H, J = 7.3 Hz), 3.87 (br s, 2H), 2.98 (s, 3H), 2.96
(s, 3H), 2.76 (d, 1H, J = 16.8 Hz), 2.74 (d, 1H, J = 16.8 Hz), 0.93
(s, 9H), 0.88 (s, 9H), 0.12 (s, 3H), 0.10 (s, 3H), 0.02 (s, 3H), −0.01
(s, 3H); 13C NMR (CDCl3, 125 MHz) d 172.0, 162.6, 154.6, 143.5,
91.6, 87.6, 84.2, 79.0, 78.6, 63.5, 37.5, 34.7, 25.8, 25.1, −4.2, −4.3,
−5.3, −5.4; FAB-MS m/z 619 (MH+).
1 - [3 - C - (N,Nꢀ - Dimethylcarbamoylmethyl) - b - D - ribo - pento-
furanosyl]cytosine (19d). Compound 19d was prepared from 37d
(111 mg, 0.20 mmol) as described above for the synthesis of 6. After
purification by desalting (charcoal, 50% aqueous MeOH) to give
19d (61 mg, 92%) as a white powder: mp 220 ◦C dec (crystallized
from aqueous MeOH): 1H NMR (DMSO-d6, 500 MHz) d 7.96 (d,
1H, J = 7.3 Hz), 7.26 (br d, 1H, J = 5.8 Hz), 5.91 (d, 1H, J = 8.1
Hz), 5.75 (d, 1H, J = 7.3 Hz), 5.27 (t, 1H, J = 3.9 Hz), 5.26 (d,
1H, J = 5.4 Hz), 5.17 (s, 1H), 4.03 (br s, 1H), 3.97 (dd, 1H, J =
5.4, 7.3 Hz), 3.58 (br s, 2H), 2.99 (s, 3H), 2.85 (s, 3H), 2.80 (d, 1H,
J = 15.3 Hz), 2.70 (d, 1H, J = 15.3 Hz); 13C NMR (DMSO-d6,
125 MHz) d 171.5, 166.3, 156.2, 143.6, 95.2, 87.6, 87.1, 78.1, 77.3,
60.5, 36.5, 34.6; FAB-MS m/z 329 (MH+).
1-[3-C-(N-Methylcarbamoylmethyl)-b-D-ribo-pentofuranosyl]-
cytosine (19a). Compound 19a was prepared from 37a (108 mg,
0.20 mmol) as described above for the synthesis of 6. After
purification by desalting (charcoal, 50% aqueous MeOH) to give
19a (54 mg, 86%) as a white powder: mp 262 ◦C dec (crystallized
from aqueous MeOH): 1H NMR (DMSO-d6, 500 MHz) d 8.05 (d,
1H, J = 8.1 Hz), 7.90 (q, 1H, J = 4.5 Hz), 7.28 (d, 2H), 5.91 (d,
1H, J = 8.0 Hz), 5.68 (d, 1H, J = 8.1 Hz), 5.58 (d, 1H, J = 6.0
Hz), 5.25 (t, 1H, J = 3.9 Hz), 5.10 (s, 1H), 3.96–3.93 (m, 2H), 3.75
(ddd, 1H, J = 1.5, 3.9, 12.1 Hz), 3.59 (ddd, 1H, J = 1.5, 3.5, 12.7
Hz), 2.55 (d, 1H, J = 16.2 Hz), 2.54 (d, 1H, J = 16.2 Hz); 13C
NMR (DMSO-d6, 125 MHz) d 171.6, 166.1, 156.5, 143.0, 95.0,
88.0, 87.3, 77.9, 76.9, 61.6, 26.1; FAB-MS m/z 315 (MH+); FAB-
HRMS calcd for C12H19N4O6 314.1226, found 315.1318 (MH+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(carbamoylmethyl)-b-
D-ribo-pentofuranosyl]-4-N-methylcytosine (38a).
A
solution
of 12 (265 mg, 0.50 mmol), DMAP (182 mg, 1.50 mmol), and
Et3N (105 lL, 1.50 mmol) in MeCN (20 mL) was treated with
2,4,6-triisopropylbenzenesulfonyl chloride (TPSCl, 450 mg,
1.50 mmol), and the mixture was stirred at room temperature
for 24 h. Then, 50% aqueous methylamine (5 mL) was added,
and the mixture was stirred for additional 2 h. The mixture was
evaporated under reduced pressure, and the residue was purified
by column chromatography (SiO2, 5% MeOH–CHCl3) to give
38a (256 mg, 94%) as a yellow glass: 1H NMR (CDCl3, 500 MHz)
d 8.13 (br s, 1H), 7.79 (d, 1H, J = 8.1 Hz), 7.05 (br s, 1H), 6.24 (d,
1H, J = 7.2 Hz), 5.71 (br s, 1H), 5.47 (br s, 1H), 4.21 (br s, 1H),
4.10 (d, 1H, J = 7.2 Hz), 3.86 (br s, 2H, J = 11.8 Hz), 2.81 (d,
3H), 2.60 (t, 2H, J = 15.8 Hz), 0.93 (s, 9H), 0.86 (s, 9H), 0.18 (s,
3H), 0.17 (s, 3H), −0.01 (s, 3H), −0.10 (s, 3H); 13C NMR (CDCl3,
125 MHz) d 170.3, 162.5, 152.1, 92.6, 87.6, 84.2, 79.0, 78.4, 63.2,
34.3, 29.4, 25.1, 24.1, −4.5, −4.6, −4.8, −4.9; FAB-MS m/z 543
(MH+).
1-[3-C -(N -Ethylcarbamoylmethyl)-b-D-ribo-pentofuranosyl]-
cytosine (19b). Compound 19b was prepared from 37b (124 mg,
0.20 mmol) as described above for the synthesis of 6. After
purification by desalting (charcoal, 50% aqueous MeOH) to give
19b (55 mg, 70%) as a white powder: mp 244 ◦C dec (crystallized
from aqueous MeOH): 1H NMR (DMSO-d6, 500 MHz) d 8.32 (d,
1H, J = 7.8 Hz), 8.03 (t, 1H, J = 5.4 Hz), 6.95 (br s, 2H), 6.08 (d,
1H, J = 7.8 Hz), 5.93 (d, 1H, J = 7.8 Hz), 5.76 (d, 1H, J = 6.5 Hz),
5.33 (t, 1H, J = 3.2 Hz), 5.21 (s, 1H), 4.56 (dq, 2H, 5.4, 7.3 Hz),
4.02 (br s, 1H), 3.96 (dd, 1H, J = 6.5, 7.8 Hz), 3.77 (ddd, 1H, J =
1.3, 3.2, 12.5 Hz), 3.61 (ddd, 1H, J = 3.2, 3.2, 12.7 Hz), 2.55 (d,
1H, J = 15.3 Hz), 2.54 (d, 1H, J = 15.3 Hz).; 13C NMR (DMSO-
d6, 125 MHz) d 172.6, 165.2, 155.6, 143.2, 95.1, 88.5, 87.7, 78.0,
76.6, 63.5, 33.3, 17.4, 14.2; FAB-MS m/z 329 (MH+); ESI-HRMS
calcd for C13H20N4NaO6 351.1281, found 351.1293 (MNa+).
1-[2,5-Di-O-(tert-butyldimethylsilyl)-3-C-(carbamoylmethyl)-b-
D-ribo-pentofuranosyl]-4-N,Nꢀ-dimethylcytosine
(38b). Com-
pound 38b was prepared from 12 (167 mg, 0.30 mmol) as
described above for the synthesis of 38a. After purification by
column chromatography (SiO2, 5% MeOH–CHCl3) to give 38b
1
(240 mg, 86%) as a yellow glass: H NMR (CDCl3, 500 MHz) d
7.79 (d, 1H, J = 7.9 Hz), 7.10 (br s, 1H), 6.27 (d, 1H, J = 6.7 Hz),
5.82 (d, 1H, J = 7.6 Hz), 5.39 (br s, 1H), 4.18 (br s, 1H), 4.08 (d,
1H, J = 6.7 Hz), 3.38 (dd, 1H, J = 2.9, 12.2 Hz), 3.28 (s, 3H),
3.12 (s, 3H), 2.62 (d, 1H, J = 15.7 Hz), 2.54 (d, 1H, J = 15.7 Hz),
0.97 (s, 9H), 0.86 (s, 9H), −0.17 (s, 3H), −0.17 (s, 3H), −0.01 (s,
1-[3-C -(N -Benzylcarbamoylmethyl)-b-D-ribo-pentofuranosyl]-
cytosine (19c). Compound 19c was prepared from 37c (124 mg,
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3H), −0.10 (s, 3H); 13C NMR (CDCl3, 125 MHz) d 172.4, 163.3,
155.9, 141.4, 92.2, 87.0, 84.6, 79.4, 78.3, 63.2, 40.8, 26.3, 25.9,
−4.4, −4.6, −5.1, −5.4; FAB-MS m/z 543 (MH+).
13 S. Becouarn, S. Szernecki and J.-M. Valery, Tetrahedron Lett., 1995,
36, 873–876.
14 P. M. J. Jung, A. Burger and J.-F. Biellmann, Tetrahedron Lett., 1995,
36, 1031–1034.
15 S. P. Auguste and D. W. Young, J. Chem. Soc., Perkin Trans. 1, 1995,
1-[3-C-(Carbamoylmethyl)-b-D-ribo-pentofuranosyl]-4-N-methyl-
cytosine (20a). Compound 20a was prepared from 38a (108 mg,
0.20 mmol) as described above for the synthesis of 6. After
purification by desalting (charcoal, 50% aqueous MeOH) to give
20a (50 mg, 80%) as a white powder: mp 223 ◦C dec (crystallized
395–404.
16 A. B. Eldrup, C. R. Allerson, C. F. Bennett, S. Bera, B. Bhat, N. Bhat,
M. R. Bosserman, J. Brooks, C. Burlein, S. S. Carroll, P. D. Cook, K. L.
Getty, M. MacCoss, D. R. McMasters, D. B. Olsen, T. P. Prakash, M.
Prhavc, Q. Song, J. E. Tomassini and J. Xia, J. Med. Chem., 2004, 47,
2283–2295.
1
17 T. Kodama, S. Shuto, S. Ichikawa and A. Matsuda, J. Org. Chem., 2002,
from aqueous MeOH): H NMR (DMSO-d6, 500 MHz) d 7.96
67, 7706–7715.
(d, 1H, J = 7.4 Hz), 7.28 (br d, 1H), 7.24 (d, 2H), 5.93 (d, 1H, J =
7.9 Hz), 5.78 (d, 1H, J = 7.4 Hz), 5.50 (d, 1H, J = 6.0 Hz), 5.32
(t, 1H, J = 4.2 Hz), 5.10 (s, 1H), 4.03 (t, 2H, J = 4.2, 7.4 Hz),
3.97 (br s, 1H), 3.74 (ddd, 1H, J = 3.0, 4.2, 11.9 Hz), 3.62 (ddd,
1H, J = 3.2, 4.2, 11.9 Hz), 2.63 (d, 3H, J = 4.5 Hz), 2.57 (s, 2H);
13C NMR (DMSO-d6, 125 MHz) d 171.6, 163.0, 155.8, 141.3,
91.9, 87.6, 84.7, 78.8, 78.3, 63.7, 26.5; FAB-MS m/z 315 (MH+);
FAB-HRMS calcd for C12H19N4O6 314.1226, found 315.1330
(MH+).
18 M. Sukeda, S. Shuto, I. Sugimoto, S. Ichikawa and A. Matsuda, J. Org.
Chem., 2000, 65, 8988–8996.
19 M. Sukeda, S. Ichikawa, A. Matsuda and S. Shuto, J. Org. Chem., 2003,
68, 3465–3475.
20 M. Sukeda, S. Ichikawa, A. Matsuda and S. Shuto, Angew. Chem., Int.
Ed., 2002, 41, 4748–4750.
21 S. Ichikawa, S. Shuto, N. Minakawa and A. Matsuda, J. Org. Chem.,
1997, 62, 1368–1375.
22 S. Shuto, M. Kanazaki, S. Ichikawa and A. Matsuda, J. Org. Chem.,
1997, 62, 5676–5677.
23 S. Shuto, M. Kanazaki, S. Ichikawa, N. Minakawa and A. Matsuda,
J. Org. Chem., 1998, 63, 746–754.
24 S. Shuto, M. Kanazaki, S. Ichikawa and A. Matsuda, J. Org. Chem.,
1997, 62, 5676–5677.
25 M. Nomura, S. Shuto, M. Tanaka, T. Sasaki, S. Mori, S. Shigeta and
A. Matsuda, J. Med. Chem., 1999, 42, 2901–2908.
26 A. Matsuda, H. Itoh, K. Takenuki, T. Sasaki and T. Ueda, Chem.
Pharm. Bull., 1988, 36, 945–953.
27 A. Matsuda, M. Satoh, H. Nakashima, N. Yamamoto and T. Ueda,
Heterocycles, 1988, 27, 2545–2548.
28 T. Ueda, A. Matsuda, Y. Yoshimura and K. Takenuki, Nucleosides
Nucleotides, 1989, 8, 743–752.
29 K. Takenuki, H. Itoh, A. Matsuda and T. Ueda, Chem. Pharm. Bull.,
1990, 38, 2947–2952.
30 A. Matsuda, K. Takenuki, T. Sasaki and T. Ueda, J. Med. Chem., 1991,
34, 234–239.
31 A. Matsuda, Y. Nakajima, A. Azuma, M. Tanaka and T. Sasaki, J. Med.
Chem., 1991, 34, 2917–2919.
1-[3-C -(Carbamoylmethyl)-b-D-ribo-pentofuranosyl]-4-N,Nꢀ-
dimethylcytosine (20b). Compound 20b was prepared from 38b
(112 mg, 0.20 mmol) as described above for the synthesis of 6.
After purification by desalting (charcoal, 50% aqueous MeOH) to
1
give 20b (48 mg, 76%) as a yellow glass: H NMR (DMSO-d6 +
D2O, 500 MHz) d 7.47 (d, 1H, J = 7.7 Hz), 6.27 (d, 1H, J = 7.7
Hz), 5.87 (d, 1H, J = 8.4 Hz), 4.43 (dd, 1H, J = 3.6, 12.7 Hz), 4.23
(dd, 1H, J = 3.6, 12.7 Hz), 4.12 (br s, 1H), 3.62 (m, 1H), 3.11 (s,
6H), 2.76 (s, 2H); 13C NMR (DMSO-d6, 125 MHz) d 172.0, 163.2,
153.8, 141.4, 91.4, 87.8, 84.6, 79.0, 78.3, 63.4, 35.6, 29.5; FAB-MS
m/z 329 (MH+); ESI-HRMS calcd for C13H20N4NaO6 351.1281,
found 351.1280 (MNa+).
32 A. Azuma, Y. Nakajima, N. Nishizono, N. Minakawa, M. Suzuki, K.
Hanaoka, T. Kobayashi, M. Tanaka, T. Sasaki and A. Matsuda, J. Med.
Chem., 1993, 36, 4183–4189.
Acknowledgements
33 M. Tanaka, A. Matsuda, T. Terao and T. Sasaki, Cancer Lett., 1992,
64, 67–74.
34 A. Azuma, K. Hanaoka, A. Kurihara, T. Kobayashi, S. Miyauchi, N.
Kamo, M. Tanaka, T. Sasaki and A. Matsuda, J. Med. Chem., 1995,
38, 3391–3397.
35 A. Matsuda and A. Azuma, Nucleosides Nucleotides, 1995, 14, 461–471.
36 X. Liu, Y. Guo, Y. Li, Y. Jiang, S. Chubb, A. Azuma, P. Huang, A.
Matsuda, W. Hittelman and W. Plunkett, Cancer Res., 2005, 65, 6874–
6881.
We thank Ms H. Matsumoto and Ms A. Maeda (Center for In-
strumental Analysis, Hokkaido University) for elemental analysis
and Ms S. Oka (Center for Instrumental Analysis, Hokkaido
University) for measurement of mass spectra.
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