Nucleosides and nucleotides. 180. Synthesis and antitumor activity of nucleosides that have a hydroxylamino group instead of a hydroxyl group at the 2'- or 3'-position of the sugar moiety

Article abstract of DOI:10.1021/jm980466g

The design and synthesis of potential antitumor antimetabolites 2'- deoxy-2'-(hydroxylamino)uridine (15), -cytidine (19, 2'-DHAC), and -adenosine (35), their regioisomers, 3'-deoxy-3'-(hydroxylamino)uridine (40) and - cytidine (45, 3'-DHAC), and their 2'-deoxy analogues, 2',3'-dideoxy-3'- (hydroxylamino)uridine (49) and -cytidine (52, 3'-dDHAC), are described. We measured the pK(a) values of the hydroxylamino group in 15 and 40 using ¹³C NMR spectroscopy as a function of pH to be 2.9 and 3.4, respectively. We also found that these nucleosides gradually decomposed in neutral solution but not in acidic solution. This decomposition may be related to the generation of aminoxy radicals at the sugar moiety. The in vitro cytotoxicity of these nucleosides was evaluated using L1210 and KB cells. 2'-DHAC (19) inhibited the growth of L1210 and KB cells, with IC₅₀ values of 1.58 and 1.99 μM, respectively. 3'-DHAC (45) and 3'-dDHAC (52) were also cytotoxic against L1210 cells, with IC₅₀ values of 4.03 and 1.84 μM, respectively, but not against KB cells. The cytotoxicity of 2'-DHAC (19) and 3'-DHAC (45) against L1210 cells in vitro was reversed by the addition of cytidine, while that of 3'-dDHAC (52) was reversed by 2'-deoxycytidine. 2'-DHAC (19) and 3'-dDHAC (52) mainly inhibited DNA synthesis in L1210 cells, while 3'-DHAC (45) inhibited RNA synthesis. We also evaluated the antitumor activities of 2'- DHAC (19) and 3'-DHAC (45) against murine Meth-A fibrosarcoma cells in vivo. 2'-DHAC (19) was more active than 3'-DHAC (45) when administered intravenously on days 1-10 consecutively at 10 mg/kg/day. 2'-DHAC (19) inhibited tumor growth at a rate of 66.9%.

Full text of DOI:10.1021/jm980466g

5094
J . Med. Chem. 1998, 41, 5094-5107
Nu cleosid es a n d Nu cleotid es. 180. Syn th esis a n d An titu m or Activity of
Nu cleosid es Th a t Ha ve a Hyd r oxyla m in o Gr ou p In stea d of a Hyd r oxyl Gr ou p a t
th e 2′- or 3′-P osition of th e Su ga r Moiety1
Akira Ogawa,† Motohiro Tanaka,‡ Takuma Sasaki,‡ and Akira Matsuda*,†
Laboratory of Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University,
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, J apan, and Cancer Research Institute, Kanazawa University,
Takara-machi, Kanazawa 920-0934, J apan
Received August 10, 1998
The design and synthesis of potential antitumor antimetabolites 2′-deoxy-2′-(hydroxylamino)-
uridine (15), -cytidine (19, 2′-DHAC), and -adenosine (35), their regioisomers, 3′-deoxy-3′-
(hydroxylamino)uridine (40) and -cytidine (45, 3′-DHAC), and their 2′-deoxy analogues, 2′,3′-
dideoxy-3′-(hydroxylamino)uridine (49) and -cytidine (52, 3′-dDHAC), are described. We
measured the pKa values of the hydroxylamino group in 15 and 40 using 13C NMR spectroscopy
as a function of pH to be 2.9 and 3.4, respectively. We also found that these nucleosides
gradually decomposed in neutral solution but not in acidic solution. This decomposition may
be related to the generation of aminoxy radicals at the sugar moiety. The in vitro cytotoxicity
of these nucleosides was evaluated using L1210 and KB cells. 2′-DHAC (19) inhibited the
growth of L1210 and KB cells, with IC50 values of 1.58 and 1.99 µM, respectively. 3′-DHAC
(45) and 3′-dDHAC (52) were also cytotoxic against L1210 cells, with IC50 values of 4.03 and
1.84 µM, respectively, but not against KB cells. The cytotoxicity of 2′-DHAC (19) and 3′-DHAC
(45) against L1210 cells in vitro was reversed by the addition of cytidine, while that of 3′-
dDHAC (52) was reversed by 2′-deoxycytidine. 2′-DHAC (19) and 3′-dDHAC (52) mainly
inhibited DNA synthesis in L1210 cells, while 3′-DHAC (45) inhibited RNA synthesis. We
also evaluated the antitumor activities of 2′-DHAC (19) and 3′-DHAC (45) against murine
Meth-A fibrosarcoma cells in vivo. 2′-DHAC (19) was more active than 3′-DHAC (45) when
administered intravenously on days 1-10 consecutively at 10 mg/kg/day. 2′-DHAC (19)
inhibited tumor growth at a rate of 66.9%.
In tr od u ction
which contains a hydroxylamino moiety, potently in-
hibits DNA synthesis through the inhibition of ribo-
nucleotide diphosphate reductase.7 Therefore, if such
a substituent can be introduced into the sugar moiety
of a nucleoside, the result may be a unique nucleoside
with a variety of biological activities.
Uridine/cytidine kinase (UCK)-dependent chemo-
therapy is highly promising because the activity of UCK
is much higher in various human tumor tissues than
in nonneoplastic tissues.2,3 We have been developing
1-(3-C-ethynyl-â-D-ribo-pentofuranosyl)cytosine (ECyd)
as a new type of antitumor agent, especially for solid
tumors.4 This nucleoside is a relatively good substrate
of UCK,4c,e and the resulting 5′-monophosphate (ECMP)
is further metabolized to its corresponding 5′-triphos-
phate (ECTP),4f which is a potent and rather pure
inhibitor of RNA polymerase.4d,f Since RNA synthesis
occurs in every cell cycle except the M phase, the
inhibition of RNA synthesis is one of the most important
targets of cancer chemotherapy, especially for slow-
growing solid tumors. Therefore, it is important to
design nucleoside antimetabolites which are good sub-
strates of UCK for progress in cancer chemotherapy.
Recently, we designed and synthesized 2′-deoxy-2′-
(hydroxylamino)cytidine (2′-DHAC, 19).8 This nucleo-
side showed potent cytotoxicity against mouse leukemic
L1210 and human epidermoid KB cells in vitro and
antileukemic activity against the mouse P388 model in
vivo. 2′-DHAC (19) was found to be phosphorylated by
UCK and to predominantly inhibit DNA synthesis. We
also detected 2′-NHO‚ radicals of 2′-DHAC (19) in
neutral solution at room temperature by ESR, from
which we anticipated that the 5′-diphosphate of 2′-
DHAC (19) would inhibit ribonucleotide diphosphate
reductase, which is a rate-determining enzyme of DNA
synthesis. As described above, we now have two potent
antitumor nucleoside antimetabolites that are both
phosphorylated by UCK: one inhibits RNA synthesis,
while the other inhibits DNA synthesis. In this paper,
we report the improved synthesis of 2′-DHAC (19) and
2′-DHAU (15) in detail and the synthesis of their
regioisomers, 3′-deoxy-3′-(hydroxylamino)cytidine (45,
3′-DHAC) and -uridine (40, 3′-DHAU), and their 2′-
deoxy analogues, 2′,3′-dideoxy-3′-(hydroxylamino)uri-
dine (49) and -cytidine (52, 3′-dDHAC). We also de-
Hydroxylamine derivatives have interesting chemical
properties. They can be readily reduced to amines and
readily oxidized to nitrons. Additionally, the oxidation
of hydroxylamine by ceric sulfate5 and OH radicals6
produces NH2O‚ radicals. Furthermore, hydroxyurea,
* To whom all correspondence and reprint requests should be
addressed. Phone: +81-11-706-3228. Fax: +81-11-706-4980. E-mail:
matuda@pharm.hokudai.ac.jp.
† Hokkaido University.
‡ Kanazawa University.
10.1021/jm980466g CCC: $15.00 © 1998 American Chemical Society
Published on Web 11/06/1998

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5095
Sch em e 1a
a
(a) See ref 9; (b) H2, Pd-C, EtOH, rt; (c) DHF, PPTS, THF, rt; (d) TBAF, THF, rt; (e) Cs2CO3, MeOH, rt; (f) TBSCl, imidazole, DMF,
rt; (g) HCl, ROH; (h) (i) TPSCl, DMAP, Et3N, MeCN, 0 °C, rt, (ii) 28% NH4OH, rt; (i) TsOH‚H2O, MeOH, rt; (j) (i) aq HCHO, MeOH, rt,
(ii) NaBH3CN, rt; (k) (i) TMSCl, Et3N, DMAP, MeCN, rt, (ii) TPSCl, (iii) 28% NH4OH, rt.
scribe the chemical and antitumor properties of these
nucleosides with some discussion of the underlying
mechanisms.
The 5′-O-silyl group of 4 was deprotected with tetrabu-
tylammonium fluoride (TBAF) to give 5, and the cyclic
carbamate moiety of 5 was cleaved with Cs2CO3 in
MeOH to give 11. Finally, the THF group was removed
with 10% HCl in EtOH to give the desired 2′-deoxy-2′-
(hydroxylamino)uridine (15, 2′-DHAU) as a hydrochlo-
ride. However, since this method for preparing 15 is
rather lengthy, we devised a new route to prepare 4.
(2-Tetrahydrofuranyl)oxyamine (THFONH2) was pre-
pared via the reaction of N-hydroxyphthalimide with
2,3-dihydrofuran in the presence of pyridinium p-
toluenesulfonate, followed by treatment with hydra-
zine.10 THFONH2 was used instead of BnONH2 for the
first reaction with 1a to give 4 in 87% yield.
Ch em istr y
Recently, Sebesta et al. reported a new synthetic
method for preparing 2′-(alkoxyamino)uridines via the
intramolecular nucleophilic substitution of 2,2′-O-an-
hydrouridine derivatives.9 This method is highly useful
for synthesizing 2′-deoxy-2′-hydroxylamino pyrimidine
nucleosides, such as 2′-DHAU (15) and 2′-DHAC (19)
(Scheme 1). 5′-O-[tert-Butyldiphenylsilyl (TBDPS)]-2,2′-
O-anhydrouridine (1a ) was subsequently treated with
N,N′-carbonyldiimidazole and then O-benzylhydroxy-
lamine. The resulting 3′-O-(benzyloxyamino)carbonyl
derivative, without purification, was converted into the
corresponding 2′-(benzyloxyamino)-2′-N,3′-O-carbonyl
derivative 2 in good yield.9 When 2 was treated with
Cs2CO3 in MeOH, the cyclic carbamate was effectively
cleaved to give 2′-O-(benzyloxyamino)-2′-deoxyuridine
derivative 8 in good yield. However, hydrogenation of
the benzyl group of 8 using 10% Pd-C catalyst under a
H2 atmosphere did not give the desired 2′-hydroxy-
lamino derivative 9. On the other hand, hydrogenation
of 2 under the same conditions gave N-hydroxyl deriva-
tive 3 in 79% yield. Since the cyclic carbamate moiety
of 3 was not effectively cleaved by Cs2CO3 in MeOH,
the N-hydroxyl group was again protected with a
tetrahydrofuranyl group to give 4, which would be
deprotected in the final stage of the reaction sequence.
The desired cytosine counterpart 19 was synthesized
from 10, which was obtained from 4 as described above.
Protection of the 3′-hydroxyl group of 10 with a tert-
butyldimethylsilyl group gave 12, which was converted
into cytosine nucleoside 16 in the usual manner, fol-
lowed by deprotection of 16 with 30% HCl in EtOH to
give 2′-deoxy-2′-(hydroxylamino)cytidine (19, 2′-DHAC)
as a dihydrochloride.
Recently, we reported that 2′-DHAC showed potent
cytotoxicity against murine leukemia L1210 and human
epidermoid KB cells in vitro and antileukemic activity
against murine leukemia P388 models.8 To further
investigate the importance of the hydroxylamino group
at the 2′-position, 2′-NHOMe, -NHOBn, and -NMeOH
derivatives, 23, 22, and 27, respectively, were also
synthesized as shown in Scheme 1. 5′-O-DMTr-2,2′-O-

5096 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
Sch em e 2a
Sch em e 4a
a
(a) 28% NH4OH, dioxane, rt; (b) HCl/EtOH, 0 °C.
Sch em e 3a
a
(a) NH2OH‚HCl, pyridine, rt; (b) aq TFA, rt; (c) NaBH4, AcOH,
0 °C; (d) TBAF, THF, rt; (e) TBDMSCl, imidazole, DMF, rt; (f) (i)
TPSCl, DMAP, Et3N, MeCN, 0 °C, rt, (ii) 28% NH4OH, rt; (g)
DMTrCl, Et3N, CH2Cl2, rt; (h) TBAF, AcOH, THF, rt; (i) concd
HCl, EtOH, 0 °C.
a
(a) (i) MsCl, Et3N, DMAP, CH2Cl2, 0 °C, (ii) TBAF, THF, rt;
(b) TBDPSCl, imidazole, DMA, rt; (c) (i) Im2CO, pyridine, rt, (ii)
NH2OTHF, rt, (iii) DBU, THF, rt; (d) Cs2CO3, MeOH, rt; (e) 4 M
HCl, MeOH, rt.
analogues. We synthesized regioisomers of 2′-DHAC
(19) and 2′-DHAU (15), such as 3′-DHAU (40) and 3′-
DHAC (45), and their 2′-deoxy analogues, such as 3′-
dDHAU (49) and 3′-dDHAC (52). Stereoselective re-
duction of 3′-keto nucleosides to ribonucleosides was
achieved with the assistance of the 5′-free hydroxyl
group.12 The stereoselective addition of some carban-
ions into the 3′-keto nucleosides from the â-face was also
recently accomplished.4e,13 Therefore, the desired 3′-
hydroxylamino derivatives with a ribo-configuration can
be obtained by hydride reduction of the corresponding
3′-oximes14 when the 5′-hydroxyl group is not protected.
As shown in Scheme 4, the desired oxime derivative 37
was readily obtained from 3′-ketouridine derivative 36
in quantitative yield. The 5′-protecting group of 37 was
removed by treatment with aqueous 80% trifluoroacetic
acid (TFA) to give 38. The oxime group was reduced
using NaBH4 in AcOH to give the desired 39, which was
deprotected to yield 3′-DHAU (40). The stereochemistry
of the sugar moiety was confirmed at 41, which was
obtained after further protection of the hydroxy groups
with a TBDPS group. When the 3′â-proton of 41 was
irradiated, NOE enhancements were observed at the
2′â-proton (9.3%) and at the 4′-proton (1.2%). The
cytosine counterpart 45 was prepared from 39 by the
usual method, after protection of the 5′-hydroxy group
with a TBDPS group. However, isolation of 45 after
deprotection of 42 was troublesome due to its highly
polar character. Therefore, to ease the isolation, the
4-NH2 group of 42 was dimethoxytritylated to yield 43,
the protecting groups of which at the sugar moiety were
removed by treatment with TBAF to give 44. Finally,
44 was treated with concentrated HCl in EtOH to give
3′-DHAC (45) as a dihydrochloride.
anhydrouridine (1b) was selected as a starting material.
In a reaction sequence similar to the synthesis of 2′-
DHAC (19), 1b was converted into 22 and 23. Com-
pound 27 was prepared from 10, the THF group of
which was first deprotected to 24, followed by reductive
methylation in the presence of NaBH3CN and aqueous
HCHO to give 2′-NMeOH derivative 25. After trimeth-
ylsilylation of the hydroxy groups of 25, the uracil
moiety was converted into a cytosine by the usual
method, followed by deprotection to furnish 27.
Since hydroxyurea is known to inhibit DNA synthesis
due to inhibition of ribonucleotide diphosphate reduc-
tase,7 hydroxyurea derivative 29 was also prepared as
shown in Scheme 2. Compound 10 was treated with
NH3/MeOH to give 28, which was deprotected to furnish
29.
Adenine analogue 35 (2′-DHAA) was synthesized as
shown in Scheme 3. For the synthesis of 35, 3′,5′-O-
TIPDS-arabinofuranosyladenine (30),11 readily prepared
from 3′,5′-O-TIPDS-adenosine in two steps, was selected
as a starting material. If the 2′-hydroxyl group of the
arabinofuranosyladenine can be activated, a similar
intramolecular nucleophilic substitution can be realized
from the 3′-hydroxyl group. The 2′-hydroxyl group of
30 was first mesylated and then treated with TBAF to
give the desired 2′-OMs derivative 31, the 5′-position
of which was protected by a TBDPS group to give 32.
As in the cleavage of the 2,2′-O-anhydro linkage of 1,
32 was treated first with N,N′-carbonyldiimidazole and
then with THPONH2, followed by DBU to give 33, which
was, without purification, further treated with Cs2CO3
in MeOH to give 34 with the desired ribo-configuration,
in 40% yield from 32. Deprotection of 34 gave 2′-DHAA
(35) as a dihydrochloride.
The synthesis of 3′-dDHAU (49) and 3′-dHAC (52) is
shown in Scheme 5. Treatment of 4615 with NH2OH‚
HCl in pyridine gave oxime derivative 47 in good yield.
It is important to better understand the structure-
activity relationship of 2′-DHAC (19) to find more potent

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5097
Sch em e 5a
a
(a) NH2OH‚HCl, pyridine, rt; (b) concd HCl, MeOH, 0 °C; (c)
NaBH4, AcOH, 0 °C; (d) TBDPSCl, imidazole, DMF, rt; (e) (i)
TPSCl, DMAP, Et3N, MeCN, 0 °C, rt, (ii) 28% NH4OH, rt.
However, deprotection of the trityl group of 47 with HCl/
MeOH gave 48 along with uracil in a ratio of ca. 1:1.
Therefore, the glycosyl linkage of the oxime derivative
was still unstable against acid treatment, while the 2′-
deoxy-3′-keto nucleoside 46 was more labile against acid
treatment.15 Further treatment of the crude mixture
of 48 with NaBH4 in AcOH gave 3′-dDHAU (49) in 51%
yield from 47. The hydroxyl groups of 49 were silylated
to give 50, which was converted into cytosine derivative
51 in the usual manner, and deprotection of 51 was
carried out under concentrated HCl in MeOH to give
3′-dDHAC (52) as a dihydrochloride. The stereochem-
istry of the sugar moiety of 52 was confirmed using the
NOE technique. For example, when the 2′â-proton of
52 was irradiated, NOE enhancement (15.3%) was
observed at the 3′â-proton, while 1.8% enhancement was
observed at the 3′â-proton when the 2′R-proton was
irradiated.
We recently reported that generation of 2′-NHO‚
radicals can be detected by their ESR spectrum at room
temperature when 2′-DHAC is dissolved in phosphate
buffer at pH 7.0.8 However, none of these radicals are
detected in the same buffer at pH 1.0 (data not shown).
Simic and Hayon studied the generation of aminoxy
radicals in hydroxylamine as a function of pH by
hydroxyl radicals and concluded that protonation to
hydroxylamine reduces the ability to produce aminoxy
radicals due to the decreased reactivity of electrophilic
hydroxy radicals.6 Therefore, we became interested in
the pKa values of our nucleosides. We selected 2′-DHAU
(15) and 3′-DHAU (40) as models and measured their
pKa values at the NHOH groups using 13C NMR
spectroscopy. The dependence of the chemical shifts of
these nucleosides as a function of pH was measured,
and the results are shown in Figure 1. For 2′-DHAU
(15), the chemical shift for the 2′-carbon changes as a
function of pH, and the pKa value was calculated to be
2.9, while the pKa value was calculated to be 3.4 from
the change in the chemical shift of the 4′-carbon of 3′-
DHAU as a function of pH. On the basis of these data,
the hydroxylamino groups of both compounds would not
be protonated in pH 7.0 buffer, and these results are
consistent with the conclusion of Simic and Hayon.6
However, the pKa value of N-methylhydroxylamine
has been reported to be 5.96.16 Therefore, the pKa
F igu r e 1. Measurement of the pKa of the hydroxylamino
group of 2′-DHAU (A) and 3′-DHAU (B) as a function of pH
by 13C NMR spectroscopy. See Experimental Section for the
method.
values of 2′-DHAU (15) and 3′-DHAU (40) are quite low.
We anticipate that the low pKa values of these hydroxy-
lamino groups are due in part to approximation of the
vicinal hydroxyl group such as in intramolecular hy-
drogen bonding. The pKa value of 2′-amino-2′-deoxyuri-
dine and its derivatives has been reported to be 6.2, and
this low pKa value is explained by the presence of the
vicinal hydroxyl group as well as either the oxygen or
the nitrogen of the molecule.17
While measuring the pKa values of these nucleosides,
we observed some changes as functions of time and pH.
We previously anticipated that the generation of ami-
noxy radicals from 2′-DHAC 5′-diphosphate would be
important for inhibiting ribonucleotide reductase, which
might inhibit DNA synthesis in tumor cells. However,
if the formation of the aminoxy radical from 2′-DHAC
(19) itself is related to decomposition of the nucleoside,
it would reduce the antitumor activity of 2′-DHAC (19).
Therefore, we studied stabilities of 2′-DHAC (19) and
3′-DHAC (45) as functions of time and pH, and the
results are shown in Figure 2. Incubation of 2′-DHAC
(19; 1 mM) in phosphate buffer (100 mM, pH 7.0) was
carried out at 37 °C, and the stability was checked by
HPLC. The peak that was attributed to the starting
material gradually disappeared, and several broad
peaks were detected. One of the products was assigned
to be cytosine, but other products could not be assigned
because each peak contained several compounds based
1
on their H NMR spectra. The t1/2 of 2′-DHAC (19) was
calculated to be 2.6 h. When the reaction mixture was
bubbled with argon, the degradation rate was greatly
reduced, while bubbling with oxygen accelerated deg-

5098 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
Ta ble 1. Inhibitory Effects of Various 2′- and
3′-Hydroxylamine-Substituted Uracil and Cytosine Nucleosides
on the Growth of L1210 and KB Cells in Vitroa
IC50 (µM)
compounds
L1210
1.58
34.5
27.5
>300
>300
37.7
>300
4.03
3.63
>300
1.84
KB
1.99
27.3
49.6
>300
>300
269
>300
254
>300
>300
>300
2′-DHAC (19)
2′-DHAU (15)
2′-DHAA (35)
22
23
27
29
3′-DHAC (45)
3′-DHAU (40)
3′-dDHAU (49)
3′-dDHAC (52)
a
The inhibition of tumor cell growth in vitro was assayed as
described elsewhere.18 Tumor cells (2 × 103 cells/well) were
incubated in the presence or absence of 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: percent inhibition (%) ) [1 - OD (540
nm) of sample well/OD (540 nm) of control well] × 100. IC50 (µg/
mL) is the concentration that inhibits cell growth by 50%.
was also inactive against both types of cells. As
discussed below, these nucleosides should be activated
by certain nucleoside kinases to exhibit the cytotoxicity.
As a result, 29 would not act as a hydroxyurea deriva-
tive to inactivate ribonucleotide reductase since it would
not be phosphorylated.
F igu r e 2. Degradation of 2′-DHAC (19) and 3′-DHAC (45).
See Experimental Section for the method.
radation with a t1/2 of 1.1 h. On the other hand, when
2′-DHAC was dissolved in HCl-KCl solution at pH 1.0,
degradation was not detected either with or without
oxygen bubbling. In addition, 3′-DHAC (45) was also
found to decompose at a much faster rate than 2′-DHAC
(19), with a t1/2 of 1.9 h at pH 7.0 buffer without oxygen
or argon bubbling. Therefore, the decomposition of
these nucleosides is closely related to the generation of
aminoxy radicals.
Next, we evaluated the in vitro cytotoxicities of 2′-
DHAC (19), 3′-DHAC (45), and 3′-dDHAC (52) against
10 human tumor cell lines, including three leukemias,
one colon, two pancreas, one fibro, one breast, one
melanoma, and one hepatoma tumor cell lines. The
results are summarized in Table 2. 2′-DHAC (19) was
effective against nine cell lines with IC50 values in the
micromolar range and was inactive against one pan-
creas adenocarcinoma cell line (PANC-1). However, 3′-
DHAC (45) was active only against MCF-7 mammary
adenocarcinoma cells, and 3′-dDHAC (52) was effective
against only two leukemic cell lines, HL-60 and CCRF-
CEM.
Biologica l Activity
The inhibitory activity of the hydroxylamino nucleo-
sides against the in vitro growth of murine leukemia
L1210 and human epidermoid KB cells was evaluated
using the MTT assay (Table 1).18 2′-DHAC (19) inhib-
ited the growth of L1210 and KB cells (IC50 values of
1.58 and 1.99 µM, respectively) more potently than 2′-
DHAU (15) and 2′-DHAA (35) (IC50 values of 34.5 and
27.3 µM, and 27.5 and 49.6 µM, respectively). To
examine the structure-cytotoxicity relationship, the in
vitro cytotoxicities of 22 and 23, which have O-substi-
tuted hydroxylamines, and 27, which has an NMe
hydroxylamine, were tested. Compounds 22 and 23 did
not show any cytotoxicity up to 300 µM, while 27 showed
weak cytotoxicity against L1210 cells but not against
KB cells. These data suggest that the OH group in the
hydroxylamino group of 2′-DHAC (19) is essential for
its cytotoxicity. Since this OH group is also essential
for generating the aminoxy radical, these data support
a relationship between radical formation and cytotox-
icity. For 3′-hydroxylamino derivatives, 3′-DHAC (45),
3′-DHAU (40), and 3′-dDHAC (52), but not 3′-dDHAU
(49), were almost equally active against the growth of
L1210 cells, with IC50 values of 4.03, 3.63, and 1.84 µM,
respectively. Interestingly, these 3′-hydroxylamino nu-
cleosides were not cytotoxic against KB cells, while 2′-
hydroxylamino nucleosides were almost equally active
against both types of cells. Hydroxyurea derivative 29
We also evaluated the in vivo antitumor activity of
2′-DHAC (19) against P388 mouse leukemia cells that
had been implanted intraperitoneally into female CDF1
mice. When 2′-DHAC (19) was administered intraperi-
toneally on days 1-5 consecutively at a dose of 20 mg/
kg/day, it showed antitumor activity with a T/C value
of 167%, while all of the tumor-bearing mice (6 mice
were used as a control) that were not administered the
drug died on day 10.8 Under the same schedule, when
50 mg/kg/day 3′-DHAC (45) was administered intrap-
eritoneally, it showed antitumor activity with a T/C
value of 144%. We also evaluated the antitumor poten-
cies of 2′-DHAC (19) and 3′-DHAC (45) against murine
Meth-A fibrosarcoma cells in vivo, and the results are
summarized in Table 3. When the drug was adminis-
tered intravenously on days 1-10 consecutively, these
nucleosides showed dose-dependent antitumor activities
to reduce the tumor size. 2′-DHAC (19) was more active
than 3′-DHAC (45), and at 10 mg/kg/day it inhibited
tumor growth at a rate of 66.9%.
To estimate the metabolic pathways of 2′-DHAC (19),
3′-DHAC (45), and 3′-dDHAC (52), the inhibitory effects

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5099
Ta ble 2. Inhibitory Effects of 2′-DHAC (19), 3′-DHAC (45), and 3′-dDHAC (52) on the Growth of Various Human Tumor Cells
in Vitroa
IC50 (µM)
cell line
K562
origin
2′-DHAC (19)
3′-DHAC (45)
3′-dDHAC (52)
chronic myelogenous leukemia
promyelocytic leukemia
acute lymphoblastic leukemia
colon adenocarcinoma
pancreas adenocarcinoma
pancreas adenocarcinoma
fibrosarcoma
2.77
5.76
1.73
8.93
4.00
69.1
2.53
1.30
0.89
1.47
14.9
213
170
27.1
4.90
6.05
HL-60
CCRF-CEM
Colo320DM
MiaPaCa-2
PANC-1
HT-1080
MCF-7
133
>300
>300
>300
>300
30.3
27.7
>300
60.5
>300
107
6.91
28.2
274
mammary adenocarcinoma
melanoma
A375
HuH7
hepatoma
a
See Table 1 for the assay method.
Ta ble 3. Antitumor Effects of 2′-DHAC (19) and 3′-DHAC (45)
5′-monophosphates, which would be further phospho-
rylated by certain nucleotide kinases to their 5′-di- and
5′-triphosphates. As described above, the pKa values
of the hydroxylamino groups were quite low, and
therefore these groups are not protonated at physiologi-
cal pH. This characteristic might be important for them
to be substrates for kinases. The relationship between
aminoxy radical formation and antitumor activity is not
fully understood. However, the generation of such
radicals is at least partly related to their antitumor
activities, since the structure-activity relationship of
2′-DHAC (19) shows that the NOH group is required
for cytotoxicity and also for the generation of radicals.
Considering pyrimidine nucleotide metabolism, we an-
ticipated that 2′-DHAC (19), after phosphorylation to
its 5′-diphosphate with the generation of aminoxy
radicals, might inhibit ribonucleoside diphosphate re-
ductase (RDR), which is responsible for the de novo
biosynthesis of deoxyribonucleotides required for DNA
synthesis, and 2′-DHACTP might be responsible for
inhibiting RNA polymerase. 3′-DHAC (45) or 3′-dDHAC
(52) might inhibit RNA polymerases or DNA poly-
merases at their 5′-triphosphates.
In conclusion, we designed and synthesized several
nucleosides with a hydroxylamino group at the sugar
moiety and expected some chemical reactivity of the
hydroxylamino group. These nucleosides were cytotoxic
against several tumor cell lines both in vitro and in vivo.
Relatively stable aminoxy radical formation was ob-
served in 2′-DHAC (19)8 and 3′-DHAC (45), which might
be responsible for their antitumor activities and may
also lead to their degradation. Since the activity of
uridine/cytidine kinase is much higher in various hu-
man tumor tissues than in nonneoplastic tissues,2,3 2′-
DHAC (19) and 3′-DHAC (45) should be expected to
exhibit antitumor activities in a tumor-specific manner,
if prodrugs could be devised to prevent their decomposi-
tion in serum fluids.
on Murine Meth-A Fibrosarcoma in Vivoa
dose
(mg/kg × 10)
tumor
size (mg)
tumor inhibition
rate (%)
drug
control
740.4 ( 222.6
309.3 ( 10.6
245.3 ( 95.4
500.4 ( 109.6
404.7 ( 192.4
2′-DHAC (19)
2
10
2
58.2b
66.9b
32.4
3′-DHAC (45)
10
45.3
a
Meth-A cells (5 × 105/mouse) were subcutaneously trans-
planted into BALB mice. Drugs were intravenously administered
the day after transplantation for 10 days. p < 0.05.
b
of these nucleosides on the growth of L1210 cells were
examined in the presence or absence of common nucleo-
sides, such as cytidine, 2′-deoxycytidine, thymidine, and
adenosine. L1210 cells (104 cells/mL) were treated with
graded concentrations of 19, 45, or 52 separately and
in combination with each common nucleoside (final
concentration; 100 µM) for 72 h (Figure 3). The inhibi-
tory effects of 2′-DHAC (19) and 3′-DHAC (45) on the
growth of L1210 cells were reduced by the addition of
cytidine (Figure 3A,B, respectively). Therefore, the
antitumor activity of these nucleosides may require
phosphorylation by UCK. On the other hand, the
cytotoxicity of 3′-dDHAC (52) was reduced by the
addition of 2′-deoxycytidine (Figure 3C). This experi-
ment suggested that 3′-dDHAC (52) may be phospho-
rylated by deoxycytidine kinase (dCK).
We next examined the effects of these nucleosides on
DNA and RNA synthesis in L1210 cells by measuring
the incorporation of [3H]thymidine and [3H]uridine as
precursors, respectively, in the presence or absence of
these nucleosides (Figure 4). As a result, 2′-DHAC (19;
20 µg/mL) predominantly inhibited DNA synthesis in
L1210 cells while slightly inhibiting RNA synthesis
(Figure 4A), while 3′-DHAC (45; 50 µg/mL) inhibited
RNA synthesis time-dependently (Figure 4B). On the
other hand, the inhibition of DNA synthesis was a major
mechanism of the cytotoxicity of 3′-dDHAC (52; 50 µg/
mL) (Figure 4C). However, the inhibition of DNA and/
or RNA synthesis by 3′-DHAC (45) and 3′-dDHAC (52)
seemed to require a time lag. Interestingly, changes
in the position of the hydroxylamino group in the sugar
moiety apparently alter the inhibitory effects on DNA
and RNA synthesis.
Exp er im en ta l Section
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
ionizing voltage of 70 eV. The H NMR spectra were recorded
on a J EOL J NM-GX 270 (270-MHz) 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
Since the cytotoxicities of these nucleosides were
reduced by the addition of certain common nucleosides,
such as cytidine or 2′-deoxycytidine, while the hydrox-
yurea derivative 29 was not effective, these nucleosides
may need to be phosphorylated by UCK or dCK to their

5100 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
11.2 Hz), 3.81 (dd, 1 H, H-5′b, J 5′b,4′ ) 4.6, J gem ) 11.3 Hz),
1.05 (s, 9 H, t-Bu).
5′-O-(t er t -Bu t yld ip h e n ylsilyl)-2′-N ,3′-O-ca r b on yl-2′-
d eoxy-2′-(h yd r oxyla m in o)u r id in e (3). A mixture of 2 (15.2
g, 24.8 mmol) and Pd-C (10%, 2.0 g) in EtOH (300 mL) was
stirred at room temperature under H2 atmosphere for 24 h.
The mixture was filtrated through a Celite pad, and the filtrate
was concentrated in vacuo. The residue was crystallized from
EtOH and Et2O to give 3 (10.3 g, 79% as a white crystalline
solid): LRMS (FAB) m/z 522 [(M - H)+, 100%]; 1H NMR
(DMSO-d6) 12.50 (br s, 1 H, 2′-NOH), 10.30 (br s, 1 H, 3-NH),
7.68-7.37 (m, 11 H, H-6, Ph), 5.99 (m, 1 H, H-1′), 5.54 (d, 1
H, H-5, J 5,6 ) 7.9 Hz), 5.17 (dd, 1 H, H-3′, J 3′,2′ ) 8.3, J 3′,4′
)
5.5 Hz), 4.71 (d, 1 H, H-2′, J 2′,3′ ) 8.3 Hz), 4.20 (m, 1 H, H-4′),
3.91-3.83 (m, 2 H, H-5′), 1.00 (s, 9 H, t-Bu).
5′-O-(t er t -Bu t yld ip h e n ylsilyl)-2′-N ,3′-O-ca r b on yl-2′-
d eoxy-2′-[(2-t et r a h yd r ofu r a n yl)oxya m in o]u r id in e (4).
Meth od A. A suspension of 3 (10.3 g, 19.7 mmol) in THF (200
mL) containing DHF (2.97 mL, 39.3 mmol) and PPTS (988 mg,
3.93 mmol) was stirred at room temperature for 11 h. The
solvent was removed in vacuo, and the residue was dissolved
in EtOAc (250 mL), washed with H2O (250 mL × 2) and brine
(200 mL), dried (Na2SO4), and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(CHCl3:MeOH ) 50:1) to give 4 (11.7 g, quantitative as a white
foam): LRMS (FAB) m/z 594 (MH+, 7%); 1H NMR (CDCl3)
8.90, 8.79 (each br s, each 1 H, 3-NH), 7.65-7.31 (m, 11 H,
H-6, Ph), 6.22, 6.03 (each d, each 1 H, H-1′, J 1′,2′ ) 3.4 Hz),
5.65-5.41 (m, 2 H, H-5, THF), 5.18, 5.16 (each dd, each 1 H,
H-3′, J 3′,2′ ) 8.1, 8.5 Hz, J 3′,4′ ) 4.4, 5.8 Hz), 4.27 (m, 1 H, H-4′),
4.08-3.83 (m, 4 H, H-5′, THF), 2.17-1.75 (m, 4 H, THF), 1.10,
1.06 (each s, each 9 H, t-Bu). Anal. (C30H35N3O8Si) C, H, N.
Meth od B. A mixture of 1a (232 mg, 0.5 mmol) and N,N-
carbonyldiimidazole (122 mg, 0.75 mmol) in pyridine (5 mL)
was stirred at room temperature for 24 h. A pyridine solution
(1 mL) of NH2OTHF (206 mg, 2 mmol) was added to the above
mixture, and the mixture was stirred at the same temperature
for a further 5 h. The solvent was removed in vacuo, and the
residue taken up with AcOEt (20 mL) was washed with H2O
(10 mL × 3) and brine (10 mL), dried (Na2SO4), and concen-
trated in vacuo. DBU (15 µL, 0.1 mmol) was added to a
solution of the above residue in THF (5 mL), and the mixture
was stirred at room temperature for 24 h. The solvent was
removed, and the residue was purified on a silica gel column
to give 4 (257 mg, 87% from 1a ).
(2-Tetr a h yd r ofu r a n yl)oxya m in e. DHF (15.2 mL, 200
mmol) and PPTS (5.00 g, 19.9 mmol) were added to a stirred
solution of N-hydroxyphthalimide (16.3 g, 100 mmol) in 1,4-
dioxane (100 mL) at room temperature. After the mixture was
stirred at room temperature for 1 h, the mixture was poured
into H2O (600 mL). The resulting precipitates were collected
by filtration to give 18.8 g (81%) of white crystals: mp 128
°C; LRMS (FAB) m/z 234 (MH+, 40%); HRMS (FAB) calcd for
C
12H12NO4 234.0766, found 234.0759; 1H NMR (CDCl3) 7.83-
7.72 (m, 4 H, Ph), 5.79 (dd, 1 H, H-1), 4.34, 4.01 (each m, each
1 H, H-4), 2.32-1.93 (m, 4 H, H-2, H-3); 13C NMR (100 MHz,
CDCl3) 163.47, 133.99, 128.82, 123.11, 108.56, 69.00, 30.80,
22.56.
F igu r e 3. Competitive effects of common nucleosides on the
cytotoxicities of 2′-DHAC (A), 3′-DHAC (B), and 3′-dDHAC (C)
against the growth of L1210 cells.
Hydrazine hydrate (3.9 mL, 80 mmol) was added to a stirred
solution of the above crystals (9.32 g, 40.0 mmol) in a mixture
of CHCl3 (50 mL) and MeOH (10 mL) at 0 °C. After the
mixture was stirred at room temperature for 24 h, the mixture
was concentrated in vacuo. The residue was dissolved with
Et2O (100 mL), washed with aqueous NaOH (6 M, 100 mL)
and brine (100 mL), dried (Na2SO4), and concentrated in vacuo
to give (2-tetrahydrofuranyl)oxyamine (2.00 g, 49% as a yellow
oil): LRMS (EI) m/z 103 (M+, 0.1%); HRMS (EI) calcd for C4H9-
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).
2′-(Ben zyloxya m in o)-5′-O-(ter t-bu tyld ip h en ylsilyl)-2′-
N,3′-O-ca r bon yl-2′-d eoxyu r id in e (2). This compound was
prepared according to the previous method.9 From 1a (2.30
g, 4.95 mmol), 2 (2.50 g, 82% as a white foam) was obtained:
1H NMR (CDCl3) 8.40 (br s, 1 H, 3-NH), 7.61-7.32 (m, 16 H,
H-6, Ph), 6.77 (d, 1 H, H-1′, J 1′,2′ ) 8.1 Hz), 5.16-5.04 (m, 3 H,
H-3′, benzylic), 4.24 (dd, 1 H, H-2′, J 2′,1′ ) 8.1, J 2′,3′ ) 2.1 Hz),
1
NO2 103.0633, found 103.0637; H NMR (DMSO-d6) 5.83 (br
s, 2 H, NH2), 5.09 (dd, 1 H, H-1), 3.72 (m, 2 H, H-4), 1.89-
1.66 (m, 4 H, H-2, H-3); 13C NMR (MeOH-d4) 106.79, 66.15,
30.11, 23.59. Anal. (C4H9NO2‚0.1AcOEt) C, H, N.
2′-N,3′-O-Ca r bon yl-2′-d eoxy-2′-[(2-tetr a h yd r ofu r a n yl)-
oxya m in o]u r id in e (5). A solution of 4 (2.37 g, 3.99 mmol)
in THF (35 mL) was treated with TBAF (1 M in THF, 4.4 mL,
4.18 (m, 1 H, H-4′), 3.91 (dd, 1 H, H-5′a, J 5′a,4′ ) 4.0, J gem
)

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5101
F igu r e 4. Effects of 2′-DHAC (A), 3′-DHAC (B), and 3′-dDHAC (C) on the incorporation of [3H]thymidine and [3H]uridine into
DNA and RNA fractions of L1210 cells.
4.4 mmol) at room temperature for 24 h. The solvent was
removed in vacuo. The residue taken up with EtOAc (250 mL)
was washed with H2O (50 mL × 2) and brine (50 mL), and
the organic phase was dried (Na2SO4) and concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (EtOAc:EtOH ) 50:1) to give 5 (1.12 g, 79% as a
white foam): LRMS (FAB) m/z 356 (MH+, 16%); 1H NMR
(DMSO-d6) 11.50 (br s, 1 H, 3-NH), 7.86, 7.68 (each d, 1 H,
dried (Na2SO4), and concentrated in vacuo. The residue was
purified by silica gel column chromatography (CHCl3:EtOH
) 20:1) to give 10 (924 mg, 81% as a white crystalline solid):
1
mp 147-148 °C; LRMS (FAB) m/z 568 (MH+, 41%); H NMR
(CDCl3) 8.39 (br s, 1 H, 3-NH), 7.89, 7.84 (each d, 1 H, H-6,
each J 5,6 ) 8.3 Hz), 7.68-7.40 (m, 10 H, Ph), 6.41, 6.37 (each
d, 1 H, 2′-NH, J 2′,NH ) 3.0, 3.8 Hz), 6.06, 5.95 (each d, 1 H,
H-1′, J 1′,2′ ) 4.7, 7.3 Hz), 5.53-5.39 (m, 2 H, H-5, THF), 4.43
(m, 1 H, H-3′), 4.29 (m, 1 H, H-4′), 4.04 (m, 2 H, THF), 3.90
(m, 2 H, H-5′), 3.70-3.63 (m, 1 H, H-2′), 1.99-1.81 (m, 4 H,
THF), 1.10, 1.06 (each s, 9 H, t-Bu). Anal. (C29H37N3O7Si) C,
H, N.
3′-O-(ter t-Bu tyld im eth ylsilyl)-5′-O-(ter t-bu tyld ip h en yl-
silyl)-2′-d eoxy-2′-[(2-t et r a h yd r ofu r a n yl)oxya m in o]u r i-
d in e (12). TBSCl (2.79 g, 18.5 mmol) and imidazole (1.51 g,
22.2 mmol) were added to a stirred solution of 10 (3.50 g, 6.17
mmol) in DMF (50 mL) at room temperature under argon
atmosphere. After being stirred at room temperature for 24
h, the mixture was diluted with EtOAc (250 mL), washed with
H2O (250 mL × 3) and brine (250 mL), dried (Na2SO4), and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane:EtOAc ) 3:2) to give 12 (4.20
g, quantitative as a white foam): LRMS m/z 682 (MH+, 45%);
1H NMR (CDCl3) 8.19 (br s, 1 H, 3-NH), 7.80, 7.77 (each d, 1
H, H-6, J 5,6 ) 8.0, 8.3 Hz), 7.68-7.37 (m, 10 H, Ph), 6.11, 6.09
(each d, 1 H, 2′-NH, J 2′,NH ) 10.0, 10.1 Hz), 6.03, 5.99 (each d,
1 H, H-1′, J 1′,2′ ) 7.9, 8.3 Hz), 5.45 (d, 1 H, H-5, J 5,6 ) 8.0 Hz),
5.32 (m, 1 H, THF), 4.43 (m, 1 H, H-3′), 3.92-3.68 (m, 5 H,
H-2′, H-4′, H-5′), 1.91-1.68 (m, 4 H, THF), 1.10, 0.88 (each s,
9 H, t-Bu), 0.09, 0.07, 0.02, 0.00 (each s, 3 H, Me). Anal.
(C35H51N3O7Si2) C, H, N.
3′-O-(ter t-Bu tyld im eth ylsilyl)-5′-O-(ter t-bu tyld ip h en yl-
silyl)-2′-d eoxy-2′-[(2-t et r a h yd r ofu r a n yl)oxya m in o]cyt i-
d in e (16). Triethylamine (205 µL, 1.47 mmol) was added
dropwise to a stirred mixture of 12 (340 mg, 0.489 mmol),
TPSCl (455 mg, 1.47 mmol), and DMAP (24 mg, 0.196 mmol)
in MeCN (5 mL) at 0 °C under argon atmosphere. After being
stirred at room temperature for 2 h, NH4OH (28%, 5 mL) was
added to the mixture. After 3 h, the resulting mixture was
extracted with EtOAc (50 mL). The organic phase was washed
with H2O (20 mL × 3) and brine (20 mL), dried (Na2SO4), and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (CHCl3:MeOH ) 10:1) to give 16 (302
mg, 91% as a white foam): LRMS (FAB) m/z 681 (MH+, 31%);
1H NMR (CDCl3) 7.91-7.87 (m, 1 H, H-6), 7.70-7.36 (m, 12
H, 4-NH2, Ph), 6.21-6.14 (m, 2 H, H-1′, 2′-NH), 5.41-5.34 (m,
2 H, H-5, THF-1′′), 4.02-3.64 (m, 5 H, H-2′, H-4′, H-5′, THF),
1.94-1.69 (m, 4 H, THF), 1.11, 0.89 (each s, 9 H, t-Bu), 0.09,
0.08, 0.03, 0.01 (each s, 3 H, Me). Anal. (C35H53N4O6Si2) C,
H, N.
H-6, each J 5,6 ) 8.0 Hz), 6.12, 6.08 (each d, 1 H, H-1′, J 1′,2′
)
1.8, 4.0 Hz), 5.75, 5.65 (each d, 1 H, H-5, each J 5,6 ) 8.0 Hz),
5.53, 5.51 (each m, 1 H, THF), 5.22, 5.11 (br t, 1 H, 5′-OH),
5.16-5.07 (m, 1 H, H-3′), 4.76-4.72 (m, 1 H, H-2′), 4.15-4.06
(m, 1 H, H-4′), 3.88-3.77 (m, 2 H, THF), 3.72-3.61 (m, 2 H,
H-5′), 2.07-1.74 (m, 4 H, THF). Anal. (C14H17N3O8‚0.2H2O)
C, H, N.
2′-Deoxy-2′-[(2-t et r a h yd r ofu r a n yl)oxya m in o]u r id in e
(11). A solution of 5 (300 mg, 0.844 mmol) and Cs2CO3 (824
mg, 2.53 mmol) in MeOH (10 mL) was stirred at room
temperature for 23 h. The solvent was removed in vacuo, and
the residue was purified by silica gel column chromatography
(CHCl3:MeOH ) 15:2) to give 11 (236 mg, 85% as a white
solid): mp 153-155 °C; LRMS (FAB) m/z 330 (MH+, 76%); 1H
NMR (DMSO-d6) 11.30 (br s, 1 H, 3-NH), 7.86, 7.85 (each d, 1
H, H-6, J 5,6 ) 8.0, 8.2 Hz), 6.51, 6.42 (each d, 1 H, 2′-NH, J 2′,NH
) 7.8, 8.8 Hz), 5.89, 5.88 (each d, 1 H, H-1′, J 1′,2′ ) 7.6, 7.7
Hz), 5.67-5.64 (m, 1 H, H-5), 5.54, 5.41 (each d, 1 H, 3′-OH,
J 3′,OH ) 4.5, 5.2 Hz), 5.20 (m, 1 H, THF), 5.09 (m, 1 H, 5′-OH),
4.18 (m, 1 H, H-3′), 3.86 (m, 1 H, H-4′), 3.72 (m, 1 H, H-2′),
3.68 (m, 2 H, THF), 3.55 (m, 2 H, H-5′), 1.87-1.56 (m, 4 H,
THF). Anal. (C13H19N3O7‚1/3H2O) C, H, N.
2′-Deoxy-2′-(h ydr oxylam in o)u r idin e Hydr och lor ide (15,
2′-DHAU). Compound 11 (408 mg, 1.24 mmol) was dissolved
in EtOH (4.5 mL) containing concentrated HCl (500 µL) at 0
°C under argon atmosphere. After the mixture was stirred at
the same temperature for 24 h, the solvent was removed in
vacuo. The residue was coevaporated several times with
EtOH, and the resulting crystals were collected by filtration
to give 15 (353 mg, 96%): mp 160-162 °C; LRMS (FAB) m/z
352 [(MH + glycerin)+, 9%]; 1H NMR (MeOH-d4) 7.97 (d, 1 H,
H-6, J 5,6 ) 8.1 Hz), 6.41 (d, 1 H, H-1′, J 1′,2′ ) 6.8 Hz), 5.77 (d,
1 H, H-5, J 5,6 ) 8.1 Hz), 4.64 (dd, 1 H, H-3′, J 2′,3′ ) 6.2, J 3′,4′
)
3.0 Hz), 4.20 (dd, 1 H, H-2′, J 1′,2′ ) 6.8, J 2′,3′ ) 6.2 Hz), 4.15
(ddd, 1 H, H-4′, J 3′,4′ ) 3.0, J 4′,5′a ) 2.6, J 4′,5′b ) 2.9 Hz), 3.83
(dd, 1 H, H-5′a, J 4′,5′a ) 2.6, J gem ) 12.2 Hz), 3.75 (dd, 1 H,
H-5′b, J 4′,5′b ) 2.9, J gem ) 12.2 Hz). Anal. (C9H13N3O4‚HCl‚
0.2H2O) C, H, N.
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2′-d eoxy-2′-[(2-t et r a h y-
d r ofu r a n yl)oxya m in o]u r id in e (10). A solution of 4 (1.20
g, 2.02 mmol) and Cs2CO3 (2.00 g, 6.14 mmol) in MeOH (15
mL) was stirred at room temperature for 24 h. The solvent
was removed in vacuo, and the residue was dissolved in EtOAc
(100 mL), washed with H2O (100 mL × 3) and brine (100 mL),
2′-Deoxy-2′-(h yd r oxyla m in o)cytid in e Dih yd r och lor id e
(19, 2′-DHAC). Compound 16 (696 mg, 1.02 mmol) was

5102 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
dissolved in a mixture of concentrated HCl (3 mL) and MeOH
(7 mL) at room temperature under argon atmosphere. After
the mixture was stirred at room temperature for 24 h, the
resulting crystals were collected by filtration to give 19 (306
mg, 91%): mp 170-172 °C; LRMS (FAB) m/z 259 (MH+, 42%);
1H NMR (MeOH-d4) 8.40 (d, 1 H, H-6, J 5,6 ) 8.0 Hz), 6.45 (d,
1 H, H-1′, J 1′,2′ ) 6.0 Hz), 6.23 (d, 1 H, H-5, J 5,6 ) 8.0 Hz), 4.68
(dd, 1 H, H-3′, J 2′,3′ ) 6.2, J 3′,4′ ) 3.4 Hz), 4.30 (dd, 1 H, H-2′,
J 1′,2′ ) 6.0, J 2′,3′ ) 6.2 Hz), 4.23 (ddd, 1 H, H-4′, J 3′,4′ ) 3.4,
J 4′,5′a ) 2.6, J 4′,5′b ) 2.8 Hz), 3.88 (dd, 1 H, H-5′a, J 4′,5′b ) 2.6,
J gem ) 12.3 Hz), 3.75 (dd, 1 H, H-5′b, J 4′,5′b ) 2.8, J gem ) 12.3
Hz). Anal. (C9H14N4O6‚2HCl) C, H, N.
2,2′-O-An h yd r o-1-[5′-O-(4,4′-d im et h oxyt r it yl)-â-D-a r a -
bin ofu r a n osyl]u r a cil (1b). A mixture of 2,2′-O-anhydrou-
ridine (11.3 g, 50.0 mmol) and DMTrCl (19.5 g, 57.6 mmol) in
pyridine (450 mL) was stirred at room temperature for 12 h
under argon atmosphere. The solvent was removed in vacuo,
and the residue was dissolved in AcOEt (500 mL), washed with
H2O (100 mL × 2) and brine (100 mL), dried (Na2SO4), and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (CHCl3:MeOH ) 8:1) to give 1b (25.1
g, 95% as a white foam): 1H NMR (CDCl3) 7.32-6.74 (m, 14
H, H-6, Ph), 6.14 (d, 1 H, H-1′, J 1′,2′ ) 5.8 Hz), 5.95 (d, 1 H,
J 5,6 ) 7.4 Hz), 5.27 (dd, 1 H, H-2′, J 1′,2′ ) 5.6, J 2′,3′ ) 5.6 Hz),
5.05 (d, 1 H, 3′-OH, J 3′,OH ) 4.0 Hz), 4.47 (m, 1 H, H-4′), 4.38
(m, 1 H, H-3′), 3.75 (s, 6 H, OMe), 3.08 (dd, 1 H, H-5′a, J 4′,5′a
) 5.1, J gem ) 10.4 Hz), 2.96 (dd, 1 H, H-5′b, J 4′,5′b ) 7.0, J gem
) 10.3 Hz).
(dd, 1 H, H-3, J 2′,3′ ) 5.4, J 3′,4′ ) 3.0 Hz), 3.95 (ddd, 1 H, H-4′,
J 3′,4′ ) 3.0, J 4′,5′a ) 2.7, J 4′,5′b ) 2.6 Hz), 3.70 (s, 6 H, OMe),
3.62 (ddd, 1 H, H-2′, J 1′,2′ ) 6.8, J 2′,NH ) 9.2, J 2′,3′ ) 5.4 Hz),
3.43 (s, 3 H, 2′-NOMe), 3.38 (dd, 1 H, H-5′a, J 4′,5′a ) 2.7, J gem
) 10.1 Hz), 3.26 (dd, 1 H, H-5′b, J 4′,5′b ) 2.6, J gem ) 10.1 Hz),
0.82 (s, 9 H, t-Bu), 0.00, -0.02 (each s, each 3 H, Me).
2′-(N-Ben zyloxya m in o)-3′-O-(ter t-bu tyld im eth ylsilyl)-
2′-deoxy-5′-O-(4,4′-dim eth oxytr ityl)cytidin e (17). This com-
pound was prepared as described for the synthesis of 16. From
13 (2.50 g, 3.26 mmol), 17 (2.19 g, 88% as a white foam) was
obtained: LRMS (FAB) m/z 765 (MH+, 1%); HRMS (FAB) calcd
for C43H53N4O7Si 765.3683, found 765.3686; 1H NMR (CDCl3)
7.86 (d, 1 H, H-6, J 5,6 ) 7.8 Hz), 7.39-6.81 (m, 18 H, Ph), 6.19-
6.15 (m, 2 H, H-1′, 2′-NH), 5.34 (d, 1 H, H-6, J 5,6 ) 7.8 Hz),
4.76, 4.68 (each d, each 1 H, benzylic, each J ) 11.7 Hz), 4.38
(dd, 1 H, H-3′, J 2′,3′ ) 4.9, J 3′,4′ ) 4.9 Hz), 4.07 (m, 1 H, H-2′),
3.79 (s, 6 H, OMe), 3.59-3.52 (m, 2 H, H-4′, H-5′a), 3.25 (dd,
1 H, H-5′b, J 4′,5′b ) 2.9, J gem ) 10.7 Hz), 0.80 (s, 9 H, t-Bu),
0.01, -0.09 (each s, each 3 H, Me).
3′-O -(t er t -B u t y ld im e t h y ls ily l)-2′-d e o x y -5′-O -(4,4′-
dim eth oxytr ityl)-2′-(N-m eth oxyam in o)cytidin e (18). This
compound was prepared as described for the synthesis of 16.
From 14 (740 mg, 1.07 mmol), 18 (567 mg, 77% as a white
foam) was obtained: LRMS (FAB) m/z 689 (MH+, 0.2%);
HRMS (FAB) calcd for C37H49N4O7Si 689.3370, found 689.3367;
1H NMR (CDCl3) 7.91 (d, 1 H, H-6, J 5,6 ) 7.0 Hz), 7.36-6.78
(m, 13 H, Ph), 6.10-6.08 (m, 2 H, H-1′, 2′-NH), 5.31 (d, 1 H,
H-5, J 5,6 ) 7.0 Hz), 4.37 (m, 1 H, H-3′), 4.03 (m, 1 H, H-2′),
3.75 (s, 6 H, OMe), 3.56 (m 1 H, H-4′), 3.51 (m, 1 H, H-5′),
3.49 (s, 3 H, 2′-NOMe), 3.24 (dd, 1 H, H-5′a, J 4′,5′a ) 2.8, J gem
) 12.3 Hz), 0.78 (s, 9 H, t-Bu), 0.00, -0.01 (each s, each 3 H,
Me).
2′-(N-Ben zyloxya m in o)-2′-d eoxy-5′-O-(4,4′-d im et h ox-
ytr ityl)cytid in e (20). A mixture of 17 (2.00 g, 2.62 mmol)
and TBAF (1 M in THF, 2.88 mL, 2.88 mmol) in THF (25 mL)
was stirred at room temperature for 3 h. The solvent was
removed in vacuo, and the residue was dissolved in AcOEt (150
mL), washed with H2O (50 mL × 5) and brine (50 mL), dried
(Na2SO4), and concentrated in vacuo. The residue was purified
by silica gel column chromatography (CHCl3:MeOH ) 15:1)
to give 20 (1.70 g, quantitative as a white foam): LRMS (FAB)
m/z 651 (MH+, 1%); HRMS (FAB) calcd for C37H39N4O7
651.2818, found 651.2817; 1H NMR (CDCl3) 7.81 (d, 1 H, H-6,
J 5,6 ) 7.8 Hz), 7.38-6.81 (m, 18 H, Ph), 7.05 (br s, 1 H, 2′-
NH), 5.93 (d, 1 H, H-1′, J 1′,2′ ) 5.9 Hz), 5.43 (d, 1 H, H-5, J 5,6
) 7.8 Hz), 4.75, 4.69 (each d, each 1 H, benzylic, each J )
11.7 Hz), 4.33 (m, 1 H, H-3′), 4.17 (m, 1 H, H-2′), 3.79 (s, 6 H,
OMe), 3.67 (br s, 1 H, 3′-OH), 3.48 (m, 1 H, H-4′), 3.41 (dd,
1H, H-5′a, J 4′,5′a ) 2.0, J gem ) 10.7 Hz), 3.28 (dd, 1 H, H-5′b,
J 4′,5′b ) 4.9, J gem ) 10.7 Hz).
2′-(N-Ben zyloxya m in o)-2′-N,3′-O-ca r bon yl-2′-d eoxy-5′-
O-(4,4′-d im eth oxytr ityl)u r id in e (6). Compound 6 was pre-
pared as described for the synthesis of 4 (method B). From
1b (5.29 g, 10.0 mmol), 6 (4.89 g, 72% as a white foam) was
obtained: 1H NMR (CDCl3) 9.03 (br s, 1 H, 3-NH), 7.44-6.78
(m, 19 H, H-6, Ph), 5.44 (d, 1 H, H-1′, J 1′,2′ ) 8.0 Hz), 5.08-
5.02 (m, 4 H, H-5, H-3′, benzylic), 4.31 (dd, 1 H, H-2′, J 1′,2′
)
8.1, J 2′,3′ ) 1.7 Hz), 4.18 (dd, 1 H, H-4′, J 3′,4′ ) 9.4, J 4′,5′ ) 4.7
Hz), 3.78 (s, 6 H, OMe), 3.42 (dd, 1 H, H-5′a, J 4′,5′a ) 5.2, J gem
) 10.5 Hz), 3.38 (dd, 1 H, H-5′b, J 4′,5′b ) 4.1, J gem ) 10.5 Hz).
2′-N,3′-O-Ca r bon yl-2′-d eoxy-5′-O-(4,4′-d im eth oxytr ityl)-
2′-(N-m eth oxya m in o)u r id in e (7). Compound 7 was pre-
pared as described for the synthesis of 4 (method B). From
1b (1.06 g, 2.01 mmol), 7 (924 mg, 77% as a white foam) was
obtained: 1H NMR (CDCl3) 8.76 (br s, 1 H, 3-NH), 7.52 (d, 1
H, H-6, J 5,6 ) 8.1 Hz), 7.37-6.82 (m, 13 H, Ph), 6.06 (d, 1 H,
H-1′, J 1′,2′ ) 1.3 Hz), 5.50 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 5.32 (dd,
1 H, H-3′, J 2′,3′ ) 8.3, J 3′,4′ ) 5.7 Hz), 4.53 (dd, 1 H, H-2′, J 1′,2′
) 1.3, J 2′,3′ ) 8.3 Hz), 4.30 (ddd, 1 H, H-4′, J 3′,4′ ) 5.7, J 4′,5′a
)
3.4, J 4′,5′b ) 4.4 Hz), 3.96 (s, 3 H, 2′-NOMe), 3.79 (s, 6 H, OMe),
3.55 (dd, 1 H, H-5′a, J 4′,5′a ) 3.4, J gem ) 10.7 Hz), 3.51 (dd, 1
H, H-5′b, J 4′,5′b ) 4.4, J gem ) 10.7 Hz).
2′-(N-Ben zyloxya m in o)-3′-O-(ter t-bu tyld im eth ylsilyl)-
2′-d eoxy-5′-O-(4,4′-d im et h oxyt r it yl)u r id in e (13). Com-
pound 13 was prepared as described for the synthesis of 10.
From 6 (2.71 g, 4.00 mmol), 13 (2.66 g, 87% as a white foam)
was obtained: LRMS (FAB) m/z 766 (MH+, 3%); HRMS (FAB)
calcd for C43H52N3O8Si 766.3523, found 766.3535; 1H NMR
(CDCl3) 8.30 (br s, 1 H, 3-NH), 7.60 (d, 1 H, H-6, J 5,6 ) 8.2
Hz), 7.33-6.78 (m, 18 H, Ph), 6.05 (d, 1 H, 2′-NH, J 2′,NH ) 9.4
Hz), 5.90 (d, 1 H, H-1′, J 1′,2′ ) 7.1 Hz), 5.31 (dd, 1 H, H-5, J )
2.0, 8.1 Hz), 4.64, 4.61 (each d, each 1 H, benzylic, each J )
11.7 Hz), 4.32 (dd, 1 H, H-3′, J 2′,3′ ) 5.3, J 3′,4′ ) 2.7 Hz), 3.97
(m, 1 H, H-4′), 3.75 (s, 6 H, OMe), 3.57 (m, 1 H, H-2′), 3.43
(dd, 1 H, H-5′a, J 4′,5′a ) 2.8, J gem ) 10.9 Hz), 3.27 (dd, 1 H,
H-5′b, J 4′,5′b ) 2.6, J gem ) 10.9 Hz), 0.79 (s, 9 H, t-Bu), -0.04,
-0.09 (each s, each 3 H, Me).
2′-Deoxy-5′-O-(4,4′-d im eth oxytr ityl)-2′-(N-m eth oxya m i-
n o)cytid in e (21). This compound was prepared as described
for the synthesis of 20. From 18 (527 mg, 0.764 mmol), 21
(419 mg, 95% as a white foam) was obtained: LRMS (FAB)
m/z 575 (MH+, 2%); HRMS (FAB) calcd for C31H35N4O7
575.2505, found 575.2510; 1H NMR (CDCl3) 7.85 (d, 1 H, H-6,
J 5,6 ) 7.1 Hz), 7.39-6.84 (m, 13 H, Ph), 7.01 (s, 1 H, 2′-NH),
5.93 (d, 1 H, H-1′, J 1′,2′ ) 5.8 Hz), 5.44 (d, 1 H, H-5, J 5,6 ) 7.1
Hz), 4.35 (m, 1 H, H-3′), 4.32 (m, 1 H, H-2′), 3.92 (br s, 1 H,
3′-OH), 3.80 (s, 6 H, OMe), 3.66 (m, 1 H, H-4′), 3.58 (s, 3 H,
2′-NOMe), 3.45 (dd, 1 H, H-5′a, J 4′,5′a ) 2.6, J gem ) 10.7 Hz),
3.35 (dd, 1 H, H-5′b, J 4′,5′b ) 4.2, J gem ) 10.7 Hz).
2′-(N-Ben zyloxya m in o)-2′-d eoxycytid in e Dih yd r och lo-
r id e (22). A mixture of 20 (1.60 g, 2.46 mmol) in concentrated
HCl (2 mL) and EtOH (18 mL) was stirred at room tempera-
ture for 10 min. The solvent was removed in vacuo, and the
residue was dissolved in H2O (100 mL), washed with CHCl3
(30 mL × 5), and concentrated in vacuo to give 22 (840 mg,
98% as a white crystalline solid): mp 210 °C dec; LRMS (FAB)
m/z 349 (MH+, 49%); HRMS (FAB) calcd for C16H21N4O5
3′-O -(t er t -B u t y ld im e t h y ls ily l)-2′-d e o x y -5′-O -(4,4′-
dim eth oxytr ityl)-2′-(N-m eth oxyam in o)u r idin e (14). Com-
pound 14 was prepared as described for the synthesis of 10.
From 7 (850 mg, 1.41 mmol), 14 (828 mg, 85% as a white foam)
was obtained: LRMS m/z 690 (MH+, 1%); HRMS calcd for
1
1
C
37H48N3O8 690.3210, found 690.3192; H NMR (CDCl3) 8.08
349.1510, found 349.1524; H NMR (MeOH-d4) 8.32 (d, 1 H,
(br s, 1 H, 3-NH), 7.69 (d, 1 H, H-6, J 5,6 ) 8.1 Hz), 7.29-6.73
(m, 13 H, Ph), 5.96 (d, 1 H, 2′-NH, J 2′,NH ) 9.2 Hz), 5.88 (d, 1
H, H-1′, J 1′,2′ ) 6.8 Hz), 5.27 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 4.35
H-6, J 5,6 ) 7.9 Hz), 7.40 (s, 5 H, Ph), 6.47 (d, 1 H, H-1′, J 1′,2′ )
7.0 Hz), 6.13 (d, 1 H, H-6, J 5,6 ) 7.9 Hz), 5.12, 5.07 (each d,
each 1 H, benzylic, each J ) 9.2 Hz), 4.64 (dd, 1 H, H-3′, J 2′,3′

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5103
) 5.9, J 3′,4′ ) 2.5 Hz), 4.30 (dd, 1 H, H-2′, J 1′,2′ ) 6.7, J 2′,3′
6.2 Hz), 4.20 (ddd, 1 H, H-4′, J 3′,4′ ) 2.5, J 4′,5′a ) 2.5, J 4′,5′b
2.5 Hz), 3.83 (dd, 1 H, H-5′a, J 4′,5′a ) 2.6, J gem ) 12.4 Hz), 3.77
(dd, 1 H, H-5′b, J 4′,5′b ) 2.7, J gem ) 12.4 Hz); 13C NMR (100
MHz, MeOH-d4) 160.58, 148.05, 145.70, 134.04, 130.44, 129.65,
)
)
H-5, J 5,6 ) 7.4 Hz), 4.48 (m, 1 H, H-3′), 3.94 (m, 1 H, H-4′),
3.90 (dd, 1 H, H-5′a, J 4′,5′a ) 2.9, J gem ) 11.1 Hz), 3.75 (dd, 1
H, H-5′b, J 4′,5′a ) 1.9, J gem ) 11.1 Hz), 3.30 (m, 1 H, H-2′), 2.82
(s, 3 H, 2′-NMe), 1.11 (s, 9 H, t-Bu), 0.12, 0.08 (each s, each 9
H, Me).
95.71, 89.55, 86.57, 77.70, 70.88, 66.42, 62.29. Anal. (C16H20
N4O5‚2HCl) C, H, N.
-
2′-Deoxy-2′-(N-h yd r oxy-N-m et h yla m in o)cyt id in e Di-
h yd r och lor id e (27). A mixture of 26 (115 mg, 0.176 mmol)
in concentrated HCl (200 µL) and EtOH (1.8 mL) was stirred
at room temperature for 5 h. The solvent was removed in
vacuo. The residue was coevaporated several times with
EtOH, dissolved in H2O (25 mL), washed with CHCl3 (25 mL
× 3), and concentrated in vacuo. The residue was coevapo-
rated several times with EtOH to give 27 (51 mg, 84% as a
white crystalline solid): mp 205 °C dec; LRMS (FAB) m/z 273
2′-Deoxy-2′-(N-m eth oxya m in o)cytid in e Dih yd r och lo-
r id e (23). This compound was prepared as described for the
synthesis of 22. From 21 (200 mg, 0.348 mmol), 23 (102 mg,
85% as a white powder) was obtained: mp 170 °C dec; LRMS
(FAB) m/z 273 (MH+); HRMS (FAB) calcd for C10H17N4O5
1
273.1198, found 273.1184; H NMR (MeOH-d4) 8.39 (d, 1 H,
H-6, J 5,6 ) 7.9 Hz), 6.44 (d, 1 H, H-1′, J 1′,2′ ) 6.4 Hz), 6.22 (d,
1 H, H-5, J 5,6 ) 7.9 Hz), 4.65 (m, 1 H, H-3′), 4.32 (m, 1 H,
H-2′), 4.22 (m, 1 H, H-4′), 3.94 (s, 3 H, 2′-NOMe), 3.89 (dd, 1
H, H-5′a, J 4′,5′a ) 2.7, J gem ) 12.1 Hz), 3.87 (dd, 1 H, H-5′b,
J 4′,5′b ) 2.5, J gem ) 12.1 Hz). Anal. (C10H16N4O5‚2HCl‚4/5H2O)
C, H, N.
(MH+, 28%); HRMS (FAB) calcd for C10H17N4O5 273.1198,
1
found 273.1210; H NMR (MeOH-d4) 8.53 (d, 1 H, H-6, J 5,6
)
7.9 Hz), 6.61 (d, 1 H, H-1′, J 1′,2′ ) 5.6 Hz), 6.22 (d, 1 H, H-5,
J 5,6 ) 7.9 Hz), 4.69 (dd, 1 H, H-3′, J 2′,3′ ) 5.9, J 3′,4′ ) 3.6 Hz),
4.46 (dd, 1 H, H-2′, J 1′,2′ ) 5.6, J 2′,3′ ) 5.9 Hz), 4.22 (m, 1 H,
H-4′), 3.87 (dd, 1 H, H-5′a, J 4′,5′a ) 2.4, J gem ) 12.3 Hz), 3.80
(dd, 1 H, H-5′b, J 4′,5′a ) 2.1, J gem ) 12.3 Hz), 3.36 (s, 3 H, 2′-
NMe); 13C NMR (100 MHz, MeOH-d4) 160.87, 148.12, 146.01,
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′-d eoxy-2′-(N-h yd r oxy-
la m in o)u r id in e (24). A mixture of 10 (114 mg, 0.201 mmol)
and TsOH‚H2O (16 mg, 0.084 mmol) in MeOH (2 mL) was
stirred at room temperature for 3 days, and the reaction
mixture was neutralized by addition of saturated aqueous
NaHCO3. The solvent was removed in vacuo, and the residue
was extracted with AcOEt (25 mL). The organic layer was
washed with H2O (25 mL) and brine (25 mL), dried (Na2SO4),
and concentrated in vacuo. The residue was purified by silica
gel column chromatography (CHCl3:MeOH ) 10:1) to give 24
(83.0 mg, 83% as a white foam): LRMS (FAB) m/z 498 (MH+,
21%); HRMS (FAB) calcd for C25H32N3O6Si 498.2058, found
95.76, 88.67, 86.02, 73.91, 61.91, 58.33, 46.91. Anal. (C10H16
-
N4O5‚2HCl‚3/5H2O‚1/3EtOH) C, H, N.
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2′-[N-ca r b a m oyl-N-[(2-
tetr a h yd r ofu r a n yl)oxy]a m in o]-2′-d eoxyu r id in e (28).
A
mixture of 10 (594 mg, 1.00 mmol) in 1,4-dioxane (5 mL) and
NH4OH (28%, 5 mL) was stirred at room temperature for 24
h. The solvent was removed in vacuo, and the residue was
coevaporated several times with MeOH. The resulting white
solid was collected by filtration, washed MeOH, and crystal-
lized from EtOH to give 28 (380 mg, 62%): mp 173-174 °C;
LRMS (FAB) m/z 611 (MH+, 11%); HRMS (FAB) calcd for
C30H39N4O8Si 611.2537, found 611.2524; 1H NMR (DMSO-d6)
11.40 (br t, 1 H, 3-NH), 7.69-7.37 (m, 11 H, H-6, Ph), 6.55 (br
s, 2 H, NH2), 6.35, 6.26 (each d, 1 H, H-1′, J ) 3.9, 4.9 Hz),
1
498.2072; H NMR (CDCl3) 7.73 (d, 1 H, H-6, J 5,6 ) 8.1 Hz),
7.67-7.36 (m, 10 H, Ph), 6.20 (d, 1 H, H-1′, J 1′,2′ ) 6.6 Hz),
5.42 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 4.55 (dd, 1 H, H-3′, J 2′,3′
)
6.3, J 3′,4′ ) 3.6 Hz), 4.13 (m, 1 H, H-4′), 4.01 (dd, 1 H, H-5′a,
J 4′,5′a ) 2.0, J gem ) 11.8 Hz), 3.82 (dd, 1 H, H-5′b, J 4′,5′a ) 1.9,
J gem ) 11.8 Hz), 3.67 (dd, 1 H, H-2′, J 1′,2′ ) 6.6, J 2′,3′ ) 6.3 Hz),
1.08 (s, 9 H, t-Bu). Anal. (C25H31N3O6Si‚4/5H2O) C, H, N.
5.58-5.29 (m, 2 H, H-5, THF-1′′), 4.69 (dd, 1 H, H-3′, J 2′,3′
)
7.8, J 3′,4′ ) 4.4 Hz), 4.43 (m, 1 H, H-2′), 4.32, 4.26 (each t, 2 H,
THF-4′′, each J ) 7.6 Hz), 3.98-3.74 (m, 3 H, H-4′, H-5′), 2.02-
1.78 (m, 4 H, THF-2′′, THF-3′′), 0.99 (s, 9 H, t-Bu); 13C NMR
(100 MHz, CDCl3) 163.79, 163.66, 162.84, 162.77, 150.30,
150.03, 040.04, 139.57, 135.22, 134.98, 132.61, 131.96, 131.86,
129.70, 129.61, 129.48, 127.58, 127.54, 127.42, 112.07, 109.82,
102.56, 102.13, 85.50, 85.20, 84.24, 68.91, 68.48, 68.37, 68.30,
66.80, 66.55, 62.68, 62.24, 31.25, 30.92, 30.63, 26.76, 26.68,
23.89, 23.82, 23.69, 19.25. Anal. (C30H38N4O8Si) C, H, N.
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2′-d eoxy-2-(N-h yd r oxy-
N-m eth yla m in o)u r id in e (25). A mixture of 24 (498 mg, 1.00
mmol) in MeOH (10 mL) containing aqueous HCHO (37%, 333
µL, 4.00 mmol) was stirred at room temperature for 3 h.
NaBH3CN (943 mg, 15.0 mmol) was added to the above
mixture at room temperature. After the mixture was stirred
for 15 h, the solvent was removed in vacuo. The residue was
dissolved in AcOEt (50 mL), washed with H2O (20 mL × 3)
and brine (20 mL), dried (Na2SO4), and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(CHCl3:MeOH ) 20:1) to give 25 (308 mg, 60% as a white
foam): LRMS (FAB) m/z 512 (MH+, 100%); HRMS (FAB) calcd
for C26H34N3O6Si 512.2214, found 512.2220; 1H NMR (CDCl3)
7.86 (d, 1 H, H-6, J 5,6 ) 8.1 Hz), 7.66-7.35 (m, 10 H, Ph), 6.62
(d, 1 H, H-1′, J 1′,2′ ) 7.9 Hz), 5.51 (d, 1 H, H-5, J 5,6 ) 8.1 Hz),
4.51 (m, 1 H, H-3′), 4.18 (m, 1 H, H-4′), 4.02 (dd, 1 H, H-5′a,
J 4′,5′a ) 1.8, J gem ) 11.9 Hz), 3.83 (dd, 1 H, H-5′b, J 4′,5′a ) 1.7,
J gem ) 11.9 Hz), 3.31 (dd, 1 H, H-2′, J 1′,2′ ) 7.9, J 2′,3′ ) 5.5 Hz),
2.83 (s, 3 H, 2′-NMe), 1.12 (s, 9 H, t-Bu).
5′-O-(ter t-Bu t yld ip h en ylsilyl)-3′-O-(t r im et h ylsilyl)-2′-
d eoxy-2′-[N-[(t r im et h ylsilyl)oxy]-N-m et h yla m in o]cyt i-
d in e (26). A mixture of 25 (250 mg, 0.489 mmol), TMSCl (311
mL, 2.45 mmol), Et3N (681 mL, 4.89 mmol), and DMAP (24
mg, 0.196 mmol) in MeCN (5 mL) was stirred at room
temperature under argon atmosphere for 2 h. TPSCl (443 mg,
2.30 mmol) was added to the mixture at 0 °C. After the
mixture was stirred at room temperature for a further 3 h,
NH4OH (28%, 5 mL) was added to the mixture. After 2 h, the
mixture was diluted with AcOEt (100 mL), washed with H2O
(30 mL × 3) and brine (30 mL), dried (Na2SO4), and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (CHCl3:MeOH ) 20:1) to give 26 (125 mg,
39% as a white foam): LRMS (FAB) m/z 655 (MH+, 23%);
HRMS (FAB) calcd for C32H51N4O5Si3 655.3164, found 655.3140;
1H NMR (CDCl3) 7.77 (d, 1 H, H-6, J 5,6 ) 7.4 Hz), 7.69-7.33
(m, 10 H, Ph), 6.61 (d, 1 H, H-1′, J 1′,2′ ) 6.4 Hz), 5.33 (d, 1 H,
2′-(N-Ca r b a m oyl-N-h yd r oxyla m in o)-2′-d eoxyu r id in e
(29). A mixture of 28 (100 mg, 0.164 mmol) in concentrated
HCl (400 µL) and MeOH (1.6 mL) was stirred at 0 °C for 24 h.
The solvent was removed in vacuo, and the residue was
purified by silica gel column chromatography (CHCl3:MeOH
) 1:1) to give 29 (40 mg, 80% as a white solid): mp 163-164
°C; LRMS (FAB) m/z 303 (MH+, 26%); HRMS (FAB) calcd for
C
10H15N4O7 303.0939, found 303.0941; 1H NMR (DMSO-d6)
11.40 (br s, 1 H, 3-NH), 9.26 (br s, 1 H, 2′-NOH), 7.77 (d, 1 H,
H-6, J 5,6 ) 8.1 Hz), 6.37 (br s, 2 H, NH2), 6.19 (d, 1 H, H-1′,
J 1′,2′ ) 4.4 Hz), 5.62 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 4.98 (t, 1 H,
5′-OH, J 5′,OH ) 5.1 Hz), 4.64 (dd, 1 H, H-3′, J 2′,3′ ) 7.6, J 3′,4′
)
4.6 Hz), 4.18 (dd, 1 H, H-2′, J 1′,2′ ) 7.1, J 2′,3′ ) 7.1 Hz), 3.87
(m, 1 H, H-4′), 3.64 (ddd, 1 H, H-5′a, J 4′,5′a ) 2.2, J gem ) 10.0,
J 5′,OH ) 4.9 Hz), 3.49 (m, 1 H, H-5′b).
2′-O-(Meth ylsu lfon yl)-â-D-ar abin ofu r an osyladen in e (31).
A mixture of 30 (550 mg, 1.19 mmol), Et3N (248 µL, 1.78
mmol), DMAP (145 mg, 1.19 mmol), and MsCl (138 µL, 1.78
mmol) in CH2Cl2 (10 mL) was stirred at 0 °C for 2 h under
argon atmosphere. The solvent was removed in vacuo, and
the residue was dissolved in AcOEt (50 mL), washed with H2O
(20 mL) and brine (20 mL), dried (Na2SO4), and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (CHCl3:MeOH ) 15:1) to give a white foam,
which was treated with TBAF (1 M in THF, 2.20 mL, 2.20
mmol) in THF (10 mL) at 0 °C for 2 h. The reaction was
quenched by addition of AcOH (126 µL), and the solvent was
removed in vacuo. The residue was coevaporated several times

5104 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
with MeOH and Et2O, and the resulting crystals were collected
by filtration to give 31 (280 mg, 0.811 mmol, 68%): mp 230
°C dec; LRMS (FAB) m/z 346 (MH+, 9%); HRMS (FAB) calcd
for C11H16N5O6S 346.0820, found 346.0821; 1H NMR (DMSO-
d6) 8.30, 8.20 (each s, each 1 H, H-2, H-8), 7.37 (br s, 2 H,
145.70, 144.08, 120.37, 89.50, 85.57, 70.71, 67.25, 62.49. Anal.
(C10
H14N6O4‚2HCl‚H2O) C, H, N.
1-[2,5-O-Bis(ter t-bu tyldim eth ylsilyl)-3-deoxy-3-(h ydr oxy-
im in o)-â-D-er yth r o-p en tofu r a n osyl]u r a cil (37). A solution
of 36 (4.70 g, 9.98 mmol) and NH2OH‚HCl (1.74 g, 25.0 mmol)
in pyridine (100 mL) was stirred at room temperature for 20
min. The solvent was removed in vacuo, and the residue was
dissolved in AcOEt (250 mL), washed with H2O (50 mL × 3)
and brine (50 mL), dried (Na2SO4), and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(hexane:AcOEt ) 3:1) to give 37 (4.87 g, 9.98 mmol, quantita-
tive as a white foam): LRMS (FAB) m/z 486 (MH+, 20%);
HRMS (FAB) calcd for C21H40N3O6Si2 486.2453, found 486.2429;
1H NMR (CDCl3) 8.67-8.31, 7.86 (each br s, each 2 H, 3-NH,
3′-NOH), 7.94 (d, 1 H, H-6, J 5,6 ) 8.2 Hz), 7.78 (d, 1 H, H-6,
J 5,6 ) 8.1 Hz), 6.12 (d, 1 H, H-1′, J 1′,2′ ) 7.2 Hz), 5.97 (d, 1 H,
H-1′, J 1′,2′ ) 2.9 Hz), 5.77, 5.71 (each dd, 1 H, H-5, J ) 2.2, 8.1
Hz), 5.03 (m, 1 H, H-4′), 4.90 (d, 1 H, H-2′, J 1′,2′ ) 2.9 Hz),
4.81 (m, 1 H, H-4′), 4.62 (dd, 1 H, H-2′, J ) 1.3, 7.2 Hz), 4.15
(dd, 1 H, H-5′a, J 4′,5′a ) 1.2, J gem ) 11.1 Hz), 4.10 (dd, 1 H,
H-5′a, J 4′,5′a ) 2.3, J gem ) 11.8 Hz), 3.96 (dd, 1 H, H-5′b, J 4′,5′b
) 3.2, J gem ) 11.8 Hz), 3.86 (dd, 1 H, H-5′b, J 4′,5′b ) 1.8, J gem
) 11.1 Hz), 0.92, 0.90, 0.88, 0.86 (each s, each 9 H, t-Bu), 0.12,
0.11, 0.10, 0.08, 0.07, 0.06, 0.05, 0.04 (each s, each 3 H, Me);
13C NMR (CDCl3) 163.45, 163.14, 157.47, 155.45, 150.29,
150.12, 140.44, 139.71, 103.21, 102.46, 91.09, 86.51, 79.55,
74.68, 71.81, 63.21, 62.27, 25.90, 25.86, 25.63, 25.47, 18.47,
18.34, 18.16, 18.08, -4.72, -5.02, -5.20, -5.30, -5.35, -5.42,
-5.45. Anal. (C21H39N3O6Si2) C, H, N.
NH2), 6.52 (d, 1 H, J 1′, ) 6.2 Hz), 6.07 (d, 1 H, 3′-OH, J 3′,OH
2′
) 5.6 Hz), 5.32 (dd, 1 H, H-2′, J 1′,2′ ) 6.2, J 2′,3′ ) 6.2 Hz), 5.18
(ddd, 1 H, H-3′, J 2′,3′ ) 6.2, J 3′,OH ) 5.6, J 3′,4′ ) 6.4 Hz), 3.90
(m, 1 H, H-4′), 3.75 (m, 2 H, H-5′), 3.12 (s, 3 H, Me). Anal.
(C11H15N5O6S‚0.5MeOH) C, H, N.
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′-O-(m eth ylsu lfon yl)-â-
D-a r a bin ofu r a n osyla d en in e (32). A mixture of 31 (250 mg,
0.724 mmol), TBDPSCl (283 µL, 1.09 mmol), and imidazole
(99.0 mg, 1.45 mmol) in DMF (7 mL) was stirred at room
temperature for 12 h. The solvent was removed in vacuo, and
the residue was dissolved in AcOEt (50 mL), washed with H2O
(50 mL) and brine (50 mL), dried (Na2SO4), and concentrated
in vacuo. The residue was coevaporated several times with
MeOH and Et2O, and the resulting crystals were collected by
filtration to give 32 (360 mg, 85%): mp 198-200 °C; LRMS
(FAB) m/z 584 (MH+, 100%); HRMS calcd for C27H34N5O6SSi
1
584.1996, found 584.2002; H NMR (CDCl3) 8.23, 8.05 (each
s, each 1 H, H-2, H-8), 7.70-7.35 (m, 10 H, Ph), 6.52 (d, 1 H,
H-1′, J 1′,2′ ) 5.5 Hz), 5.67 (br s, 2 H, NH2), 5.25 (dd, 1 H, H-2′,
J 1′,2′ ) 5.5, J 2′,3′ ) 5.0 Hz), 4.90 (m, 1 H, H-3′), 4.08-3.99 (m,
3 H, H-4′, H-5′), 3.52 (br s, 1H, 3′-OH), 2.69 (s, 3 H, Me), 1.11
(s, 9 H, t-Bu). Anal. (C27H33N5O6SSi) C, H, N.
5′-O-(ter t-Bu t yld ip h en ylsilyl)-2′-d eoxy-2′-[(2-t et r a h y-
d r ofu r a n yl)oxya m in o]a d en osin e (34). A mixture of 32
(1.40 g, 2.40 mmol) and N,N′-carbonyldiimidazole (779 mg,
4.80 mmol) in pyridine (20 mL) was stirred at room temper-
ature for 30 min. NH2OTHP (1.40 g, 12.0 mmol) was added
to the mixture at room temperature. After the mixture was
stirred for a further 4.5 h, the solvent was removed in vacuo.
The residue was coevaporated several times with toluene and
dissolved in THF (20 mL) containing DBU (1.40 mL, 9.60
mmol). The mixture was stirred at room temperature for 24
h, and the solvent was removed in vacuo. The residue was
purified by silica gel column chromatography (CHCl3:MeOH
) 40:1) to give 33 as white foam, which was treated with Cs2-
CO3 (3.00 g, 9.21 mmol) in MeOH (20 mL). After 24 h, the
reaction mixture was neutralized by addition of aqueous HCl
(1 M), and the mixture was extracted with AcOEt (150 mL).
The organic phase was washed with H2O (50 mL × 2) and
brine (50 mL), dried (Na2SO4), and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(CHCl3:MeOH ) 40:1) to give 34 (584 mg, 40% as a white
foam): LRMS (FAB) m/z 605 (MH+, 100%); HRMS (FAB) calcd
for C31H41N6O5Si 605.2905, found 605.2902; 1H NMR (CDCl3)
8.09, 8.28 (each s, 1 H, H-8), 8.11 (s, 1 H, H-2), 7.64-7.30 (m,
10 H, Ph), 7.23 (br s, 1 H, 2′-NH), 6.18, 6.06 (each d, 1 H, H-1′,
J 1′,2′ ) 6.6, 7.3 Hz), 5.79 (br s, 2 H, 4-NH2), 4.83, 4.81 (each m,
1 H, H-3′), 4.69 (br s, 1 H, 3′-OH), 4.54 (m, 1 H, H-2′), 4.38 (m,
1H, THP), 4.13 (ddd, 1 H, H-4′, J ) 4.0, 4.0, 7.9 Hz), 3.99-
3.91 (m, 2 H, THP), 3.83 (m, 1 H, H-5′a), 3.59 (m, 1 H, H-5′b),
1-[2-O-(ter t-Bu t yld im et h ylsilyl)-3-d eoxy-3-(h yd r oxy-
im in o)-â-D-er yth r o-p en tofu r a n osyl]u r a cil (38). A solution
of 37 (3.50 g, 7.20 mmol) in aqueous TFA (80%, 25 mL) was
stirred at 0 °C for 30 min, and the solvent was removed in
vacuo. The residue was purified by silica gel column chroma-
tography (hexane:AcOEt ) 1:1) to give 38 (2.64 g, 99% as a
white foam): LRMS (FAB) m/z 372 (MH+, 31%); HRMS (FAB)
calcd for C15H26N3O6Si 372.1589, found 372.1592; 1H NMR
(CDCl3) 8.91 (br s, 1 H, 3′-NOH), 8.41 (br s, 1 H, 3-NH), 7.55
(d, 1 H, H-6, J 5,6 ) 8.1 Hz), 5.83 (d, 1 H, H-5, J 5,6) 8.1 Hz),
5.62 (d, 1 H, H-1′, J 1′,2′ ) 6.6 Hz), 5.06 (dd, 1 H, H-2′, J 1′,2′
)
6.6 Hz), 5.04 (m, 1 H, H-4′), 4.18 (dd, 1 H, H-5′a, J 4′,5′a ) 1.5,
J gem ) 12.0 Hz), 3.99 (dd, 1 H, H-5′b, J 4′,5′b ) 2.0, J gem ) 12.0
Hz), 0.86 (s, 9 H, t-Bu), 0.35, 0.13 (each s, each 3 H, Me). Anal.
(C15H25N3O6Si‚0.5H2O) C, H; N: calcd, 11.04; found, 10.33.
2′-O-(ter t-Bu tyldim eth ylsilyl)-3′-deoxy-3-(h ydr oxylam i-
n o)u r id in e (39). A solution of 38 (400 mg, 1.09 mmol) in
AcOH (3 mL) was added dropwise to a solution of AcOH (8
mL) containing NaBH4 (82 mg, 2.18 mmol) at 0 °C. After the
mixture was stirred at the same temperature for 2 h, the
solvent was removed in vacuo. The residue was diluted with
AcOEt (50 mL), washed with H2O (20 mL × 3) and brine (20
mL), dried (Na2SO4), and concentrated in vacuo. The residue
was purified by silica gel column chromatography (AcOEt) to
give 39 (378 mg, 92% as a white foam): LRMS (FAB) m/z 374
(MH+, 8%); HRMS (FAB) calcd for C15H28N3O6Si 374.1746,
found 374.1718; 1H NMR (CDCl3) 10.20 (br s, 1 H, 3-NH), 8.00
(d, 1 H, H-6, J 5,6 ) 8.1 Hz), 6.82 (br s, 1 H, 3′-NHOH), 5.74 (d,
1 H, H-5, J 5,6 ) 8.1 Hz), 5.69 (d, 1 H, H-1′, J 1′,2′ ) 2.8 Hz), 5.64
1.97-1.27 (m, 6 H, THP), 1.02 (s, 9 H, t-Bu). Anal. (C31H40
N6O5Si‚0.5H2O) C, H, N.
-
2′-Deoxy-2-(h yd r oxyla m in o)a d en osin e Dih yd r och lo-
r id e (35). A solution of 34 (400 mg, 0.661 mmol) in MeOH
(2.5 mL) was treated with HCl (4 M in 1,4-dioxane, 2.5 mL)
at room temperature for 24 h. The solvent was removed in
vacuo, and the residue was dissolved in H2O (15 mL), washed
with CHCl3 (15 mL × 3), and concentrated in vacuo. The
residue was suspended with Et2O, and the resulting crystals
were collected by filtration to give 35 (233 mg, 99%): mp 125
°C dec; LRMS (FAB) m/z 283 (MH+, 75%); HRMS (FAB) calcd
for C10H15N6O4 283.1154, found 283.1145; 1H NMR (MeOH-
d4) 8.72, 8.47 (each s, each 1 H, H-2, H-8), 6.72 (d, 1 H, H-1′,
J 1′,2′ ) 6.3 Hz), 4.84 (dd, 1 H, H-3′, J 2′,3′ ) 6.3, J 3′,4′ ) 3.3 Hz),
4.75 (dd, 1 H, H-2′, J 1′,2′ ) 6.3, J 2′,3′ ) 6.3 Hz), 4.26 (ddd, 1 H,
H-4′, J 3′,4′ ) 3.2, J 4′,5′a ) 3.0, J 4′,5′b ) 3.2 Hz), 3.86 (dd, 1 H,
H-5′a, J 4′,5′a ) 2.9, J gem ) 12.3 Hz), 3.80 (dd, 1 H, H-5′b, J 4′,5′b
) 3.2, J gem ) 12.2 Hz); 13C NMR (MeOH-d4) 151.83, 149.87,
(br s, 1 H, 3′-NHOH), 4.65 (dd, 1 H, H-2′, J 1′,2′ ) 2.8, J 2′,3′
4.7 Hz), 4.10 (ddd, 1 H, H-4′, J 3′,4′ ) 6.9, J 4′,5′a ) 1.3, J 4′,5′b
)
)
1.3 Hz), 4.02 (dd, 1 H, H-5′a, J 4′,5′a ) 1.3, J gem ) 12.1 Hz), 3.85
(dd, 1 H, H-5′b, J 4′,5′b ) 1.3, J gem ) 12.1 Hz), 3.77 (br s, 1 H,
5′-OH), 3.66 (dd, 1 H, H-3′, J 2′,3′ ) 4.7, J 3′,4′ ) 6.9 Hz), 0.93 (s,
9 H, t-Bu), 0.16 (s, 6 H, Me); 13C NMR (67.8 MHz, CDCl3)
164.69, 150.78, 142.41, 101.60, 92.78, 81.92, 73.82, 61.74,
61.24, 25.72, 17.95, -4.81, -4.87. Anal. (C15H27N3O6Si‚1/
3H2O) C, H, N.
3′-Deoxy-3-(h yd r oxyla m in o)u r id in e (40, 3′-DHAU). A
mixture of 39 (371 mg, 1.00 mmol) and TBAF (1 M in THF,
1.20 mL, 1.20 mmol) in THF (10 mL) was stirred at room
temperature for 1 h. The solvent was removed in vacuo, the
residue was coevaporated several times with MeOH, and the
resulting precipitates were collected by filtration to give 40
(185 mg, 71% as a white solid): mp 205-206 °C; LRMS (FAB)

2′-Deoxy-2′-(hydroxylamino)cytidine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25 5105
m/z 352 ((MH + glycerin)+, 7%); 1H NMR (DMSO-d6) 11.30
(br s, 1 H, 3-NH), 7.96 (d, 1 H, H-6, J 5,6 ) 8.2 Hz), 7.46 (br s,
1 H, 3′-NHOH), 5.83 (d, 1 H, H-1′, J 1′,2′ ) 5.2 Hz), 5.71 (br s,
1 H, 3′-NHOH), 5.64 (d, 1 H, H-5, J 5,6 ) 8.2 Hz), 5.13 (br s, 1
H, 5′-OH), 4.17 (m, 1 H, H-2′), 3.98 (ddd, 1 H, H-4′, J 3′,4′ ) 4.8,
J 4′,5′a ) 2.0, J 4′,5′b ) 2.8 Hz), 3.67 (d, 1 H, H-5′a, J gem ) 11.8
Hz), 3.53 (d, 1 H, H-5′b, J gem ) 11.8 Hz), 3.36 (m, 1 H, H-3′);
13C NMR (67.8 MHz, DMSO-d6) 163.24, 150.80, 140.67, 101.67,
88.57, 81.93, 72.78, 62.77, 61.87. Anal. (C9H13N3O6‚0.2MeOH)
C, H, N.
calcd for C30H33N4O7 561.2349, found 561.2339; 1H NMR
(CDCl3) 7.70 (d, 1 H, H-6, J 5,6 ) 7.4 Hz), 7.31-6.81 (m, 13 H,
Ph), 6.96 (br s, 1 H, 4-NH), 5.64 (s, 1 H, H-1′), 5.13 (d, 1 H,
H-5, J 5,6 ) 7.4 Hz), 4.44 (m, 1 H, H-2′), 4.11 (m, 1 H, H-4′),
3.87 (d, 1 H, H-5′a, J gem ) 11.4 Hz), 3.84 (d, 1 H, H-5′b, J gem
11.4 Hz), 3.76 (s, 6 H, OMe), 3.59 (m, 1 H, H-3′).
)
3′-Deoxy-3′-(h yd r oxyla m in o)cytid in e Dih yd r och lor id e
(45, 3′-DHAC). A mixture of 44 (56.0 mg, 0.099 mmol) in
concentrated HCl (100 µL) and EtOH (900 µL) was stirred at
0 °C for 24 h. The solvent was removed in vacuo. The residue
was dissolved in H2O (25 mL), washed with CHCl3 (10 mL ×
3), and concentrated in vacuo. The residue was suspended
with i-PrOH and Et2O, and the resulting crystals were
collected by filtration to give 45 (32.0 mg, 98%): mp 241 °C
2′-O-(ter t-Bu tyld im eth ylsilyl)-5′-O-(ter t-bu tyld ip h en yl-
silyl)-3′-[N-(ter t-bu tyld ip h en ylsilyl)oxya m in o]-3′-d eoxy-
u r id in e (41). A mixture of 39 (1.00 g, 2.68 mmol), TBDPSCl
(2.10 mL, 8.25 mmol), and imidazole (618 mg, 9.08 mmol) in
DMF (30 mL) was stirred at room temperature for 24 h. The
solvent was removed in vacuo, and the residue was dissolved
in AcOEt (150 mL), washed with H2O (50 mL × 3) and brine
(50 mL), dried (Na2SO4), and concentrated in vacuo. The
residue was purified by silica gel column chromatography
(hexane:AcOEt ) 5:1) to give 41 (2.06 g, 90% as a white
foam): LRMS (FAB) m/z 850 (M+, 12%); HRMS (FAB) calcd
for C47H63N3O6Si3 850.4099, found 850.4119; 1H NMR (CDCl3)
8.28 (br s, 1 H, 3-NH), 7.64-7.25 (m, 21 H, H-6, Ph), 5.85 (d,
1 H, H-1′, J 1′,2′ ) 2.4 Hz), 5.79 (d, 1 H, 3′-NH, J 3′,NH ) 9.2 Hz),
1
dec; LRMS (FAB) m/z 259 (MH+, 33%); H NMR (MeOH-d4)
8.25 (d, 1 H, H-6, J 5,6 ) 7.8 Hz), 6.21 (d, 1 H, H-5, J 5,6 ) 7.8
Hz), 5.93 (d, 1 H, H-1′, J 1′,2′ ) 4.6 Hz), 4.49 (dd, 1 H, H-2′, J 1′,2′
) 4.6, J 2′,3′ ) 6.0 Hz), 4.45 (m, 1 H, H-4′), 3.93 (dd, 1 H, H-3′,
J 2′,3′ ) 6.0, J 3′,4′ ) 5.8 Hz), 3.76 (dd, 1 H, H-5′a, J 4′,5′b ) 2.5,
J gem ) 12.2 Hz), 3.65 (dd, 1 H, H-5′b, J 4′,5′b ) 2.7, J gem ) 12.2
Hz); 13C NMR (MeOH-d4) 160.95, 148.46, 146.05, 95.01, 92.98,
81.87, 74.10, 62.37, 61.76. Anal. (C9H14N4O5‚2HCl‚0.5EtOH)
C, H, N.
1-[2,3-Did eoxy-3-(h yd r oxyim in o)-5-O-tr ityl-â-D-er yth r o-
p en tofu r a n osyl]u r a cil (47). A mixture of 5′-O-trityl-2′-
deoxyuridine (2.82 g, 5.99 mmol) and Dess-Martin reagent
(5.09 g, 12.0 mmol) in CH2Cl2 (50 mL) was stirred at room
temperature for 24 h. The mixture was diluted with AcOEt
(200 mL), washed with aqueous NaHCO3 (50 mL), aqueous
Na2S2O3 (50 mL), and brine (50 mL), dried (Na2SO4) and
concentrated in vacuo. A mixture of crude 46 and NH2OH‚
HCl (2.00 g, 28.8 mmol) in pyridine (50 mL) was stirred at
room temperature for 24 h. The solvent was removed in vacuo,
and the residue was dissolved in AcOEt (200 mL), washed with
H2O (50 mL) and brine (50 mL), dried (Na2SO4), and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane:AcOEt ) 1:2) to give 47 (2.10 g, 72%
as a white foam): LRMS (FAB) m/z 484 (MH+, 10%); HRMS
(FAB) calcd for C28H26N3O5 484.1872, found 484.1875; 1H NMR
(CDCl3) 9.73, 9.70 (each br s, 1 H, 3′-NOH), 9.27, 9.20 (each
br s, 1 H, 3-NH), 7.94, 7.83 (each d, 1 H, H-6, J 5,6 ) 8.3 Hz),
5.16 (d, 1 H, H-5, J 5,6 ) 8.1 Hz), 4.31 (dd, 1 H, H-2′, J 1′,2′
)
2.4, J 2′,3′ ) 4.8 Hz), 3.86 (m, 2 H, H-5′), 3.51 (dd, 1 H, H-4′,
J 3′,4′ ) 8.9, J 4′,5′ ) 4.1 Hz), 3.31 (ddd, 1 H, H-3′, J 2′,3′ ) 4.8,
J 3′,NH ) 9.2, J 3′,4′ ) 8.9 Hz), 1.08, 1.04, 0.88 (each s, each 9 H,
t-Bu), 0.16, 0.08 (each s, each 3 H, Me); NOE irradiate H-3′â,
observe H-2′â (9.3%) and H-4′ (1.2%), no other NOE enhance-
ment was observed. Anal. (C47H63N3O6Si3) C, H, N.
2′-O-(ter t-Bu tyld im eth ylsilyl)-5′-O-(ter t-bu tyld ip h en yl-
silyl)-3′-[N-(ter t-bu tyld ip h en ylsilyl)oxya m in o]-3′-d eoxy-
cytid in e (42). Compound 41 (2.10 g, 2.47 mmol) was con-
verted into 42 (2.02 g, 96% as a white foam) as described for
the synthesis of 16: LRMS (FAB) m/z 849 (MH+, 8%); HRMS
1
(FAB) calcd for C47H65N4O5Si3 849.4259, found 849.4225; H
NMR (CDCl3) 7.71-7.16 (m, 23 H, H-6, 4-NH2, Ph), 5.80 (s, 1
H, H-1′), 5.75 (d, 1 H, 3′-NH, J 3′,NH ) 11.3 Hz), 5.03 (d, 1 H,
H-5, J 5,6 ) 7.4 Hz), 4.38 (d, 1 H, H-2′, J 2′,3′ ) 4.1 Hz), 3.91-
3.80 (m, 2 H, H-5′), 3.52 (dd, 1 H, H-4′, J 3′,4′ ) 11.6, J 4′,5′ ) 4.2
Hz), 3.24 (ddd, 1 H, H-3′, J 2′,3′ ) 4.1, J 3′,NH ) 11.3, J 3′,4′ ) 11.6
Hz), 1.06, 1.03, 0.88 (each s, each 9 H, t-Bu), 0.29, 0.16 (each
s, each 3 H, Me). Anal. (C47H64N4O5Si3‚2H2O) C, N; H: calcd,
7.74; found, 7.29.
7.38-7.20 (m, 15 H, Ph), 6.35, 6.34 (each dd, 1 H, H-1′, J 1′,2′
)
6.9, 8.3 Hz), 5.28, 5.25 (each d, 1 H, H-5, J 5,6 ) 8.3 Hz), 4.94,
4.71 (each m, 1 H, H-4′), 4.02-2.75 (m, 4 H, H-2′, H-5′).
2′,3′-Dideoxy-3′-(h ydr oxylam in o)u r idin e (49, 3′-dDHAU).
A mixture of 47 (2.00 g, 4.14 mmol) in concentrated HCl (1
mL) and MeOH (9 mL) was stirred at room temperature for
12 h, and the reaction mixture was neutralized by addition of
saturated aqueous NaHCO3. The solvent was removed in
vacuo, and the residue was purified by silica gel column
chromatography (CHCl3:MeOH ) 10:1) to give white solids
(646 mg) as a mixture of 48 and uracil (about 1:1): LRMS
(FAB) m/z 242 (MH+, 41%); HRMS (FAB) calcd for C9H12N3O5
N4-(4,4′-Dim eth oxytr ityl)-2′-O-(ter t-bu tyldim eth ylsilyl)-
5′-O-(t er t -b u t yld ip h e n ylsilyl)-3′-[(t er t -b u t yld ip h e n yl-
silyl)oxya m in o]-3′-d eoxycyt id in e (43). A mixture of 42
(849 mg, 1.00 mmol), Et3N (237 µL, 1.70 mmol), and DMTrCl
(508 mg, 1.50 mmol) in CH2Cl2 (10 mL) was stirred at room
temperature for 6 h. The solvent was removed in vacuo, and
the residue was dissolved in AcOEt (50 mL), washed with H2O
(10 mL × 3) and brine (10 mL), dried (Na2SO4), and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (hexane:AcOEt ) 3:2) to give 43 (1.12 g, 97%
as a white foam): LRMS (FAB) m/z 1151 (MH+, 5%); HRMS
(FAB) calcd for C68H83N4O7Si3 1151.5569, found 1151.5580; 1H
NMR (CDCl3) 7.58-6.79 (m, 34 H, H-6, Ph), 6.67 (br s, 1 H,
4-NH), 5.77 (s, 1 H, H-1′), 5.73 (d, 1 H, 3′-NH, J 3′,NH ) 9.0 Hz),
4.75 (d, 1 H, H-5, J 5,6 ) 7.6 Hz), 4.33 (d, 1 H, H-2′, J 2′,3′ ) 3.9
Hz), 3.76 (s, 6 H, OMe), 3.76-3.64 (m, 2 H, H-5′), 3.27 (m, 1
1
242.0777, found 242.0792; H NMR (DMSO-d6) 11.30, 11.00
(each br s, 1 H, 3′-NOH), 10.90, 10.80 (each br s, 1 H, 3′-NH),
8.01, 7.82 (each m, 1 H, H-6), 6.19 (m, 1H, H-1′), 5.69-5.64
(m, 1 H, H-5), 5.02, 4.08 (m, 1 H, 5′-OH), 4.71, 4.47 (each m,
1 H, H-4′), 3.70-3.60 (m, 2 H, H-5′), 3.16-2.49 (m, 2 H, H-2′).
A solution of the above solids (580 mg) in AcOH (10 mL)
was added dropwise to a solution of AcOH (10 mL) containing
NaBH4 (182 mg, 4.81 mmol) at 0 °C. After being stirred for 1
h, the solvent was removed in vacuo. The residue was
coevaporated several times with toluene and purified by silica
gel column chromatography (CHCl3:MeOH ) 3:1) to give 49
(456 mg, 1.87 mmol, 51% from 47 as a white foam): LRMS
(FAB) m/z 244 (MH+, 31%); HRMS (FAB) calcd for C9H13N3O5
H, H-4′), 3.00 (ddd, 1 H, H-3′, J 2′,3′ ) 3.9, J 3′,NH ) 9.0, J 3′,4′
)
11.4 Hz), 1.04, 0.97, 0.89 (each s, each 9 H, t-Bu), 0.28, 0.17
(each s, each 3 H, Me).
N4-(4,4′-Dim eth oxytr ityl)-3′-d eoxy-3′-(h yd r oxyla m in o)-
cytid in e (44). A solution of 43 (505 mg, 0.438 mmol) in THF
(4 mL) containing AcOH (94 µL, 1.58 mmol) was treated with
TBAF (1 M in THF, 1.58 mL, 1.58 mmol) at room temperature
for 3 h. The solvent was removed in vacuo, and the residue
was dissolved in AcOEt (50 mL), washed with H2O (10 mL ×
5) and brine (10 mL), dried (Na2SO4), and concentrated in
vacuo. The residue was purified by silica gel column chroma-
tography (CHCl3:MeOH ) 8:1) to give 44 (217 mg, 88% as a
white powder): LRMS (FAB) m/z 561 (MH+, 3%); HRMS (FAB)
1
244.0933, found 244.0929; H NMR (DMSO-d6) 11.80 (br s, 1
H, 3-NH), 8.55 (d, 1 H, H-6, J 5,6 ) 8.1 Hz), 6.72 (dd, 1 H, H-1′,
J 1′,2′a ) 6.8 Hz, J 1′,2′b ) 6.8 Hz), 6.25 (d, 1 H, H-5, J 5,6 ) 8.1
Hz), 4.51 (m, 1 H, H-4′), 4.26-4.13 (m, 3 H, H-3′, H-5′), 2.81
(m, 1 H, H-2′a), 2.57 (ddd, 1 H, H-2′b, J 1′,2′b ) 6.6, J gem ) 14.2,
J 2′b,3′ ) 6.6 Hz); 13C NMR (MeOH-d4) 162.91, 150.19, 140.44,
101.65, 84.50, 83.07, 62.30, 35.42, 21.73. Anal. (C9H13N3O5‚
1.5MeOH) C, H; N: calcd, 14.43; found, 13.87.

5106 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 25
Ogawa et al.
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′,3′-d id eoxy-3′-[(ter t-bu -
tyld ip h en ylsilyl)oxya m in o]u r id in e (50). A mixture of 49
(319 mg, 1.31 mmol), TBDPSCl (1.02 mL, 3.94 mmol), and
imidazole (446 mg, 6.55 mmol) in DMF (10 mL) was stirred
at room temperature for 24 h. The mixture was diluted with
AcOEt (200 mL), washed with H2O (50 mL) and brine (50 mL),
dried (Na2SO4), and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane:AcOEt
) 2:1) to give 50 (809 mg, 85% as a white foam): LRMS (FAB)
m/z 720 (MH+, 49%); HRMS (FAB) calcd for C41H50N3O5Si2
showed the appropriate retention time. Each peak area ratio
was calculated with cytidine as a reference.
Effects of Com m on Nu cleosid es on th e in Vitr o Cyto-
toxicities of 2′-DHAC (19), 3′-DHAC (45), a n d 3′-d DHAC
(52). L1210 cells (104 cells/mL) (180 µL) were seeded in a 96-
well microplate, and 10 µL of graded concentrations of 2′-
DHAC (19), 3′-DHAC (45), or 3′-dDHAC (52) (final concentra-
tion: 10, 2, 0.4, 0.08 µg/mL) and 10 µL of each common
nucleoside (final concentration: 50 µM) were simultaneously
added in triplicate to each well. The plate was incubated for
3 days at 37 °C in a humidified atmosphere of 5% CO2. The
cytotoxicities of 2′-DHAC, 3′-DHAC, and 3′-dDHAC were
evaluated by the MTT assay.
Effects of 2′-DHAC (19), 3′-DHAC (45), a n d 3′-d DHAC
(52) on DNA a n d RNA Syn th eses. After L1210 cells (2.5 ×
105 cells/mL) were incubated for 24 h, 2′-DHAC (19), 3′-DHAC
(45), or 3′-dDHAC (52) was added to the culture at 20 µg/mL
and incubated for 1, 2, and 4 h. Before cell harvest, cells were
pulse-labeled for 30 min with 1 µCi/mL of [3H]thymidine (83.2
Ci/mmol) or [3H]uridine (23.1 Ci/mmol) purchased from Am-
ersham Life Sciences, Buckinghamshire, England. The ra-
dioactivity of the acid-insoluble fractions was measured by a
Beckman LS 6500 liquid scintillation counter (Beckman
Instruments, Fullerton, CA) using Scintisol EX-H (Dojin
Chemicals, Kumamoto, J apan).
An titu m or Effects of 2′-DHAC (19) a n d 3′-DHAC (45)
on Mu r in e F ibr osa r com a Meth -A. Five-week-old female
BALB/cCrj mice weighing about 20 g were purchased from
Charles River J apan Inc., Atsugi, J apan. They were main-
tained on a standard diet and water throughout the experi-
mental period under specific pathogen-free conditions. Six
mice were used for each group in these experiments. Murine
fibrosarcoma Meth-A was maintained weekly by serial trans-
plantation by injection into the abdominal cavity of BALB/c
mice. One-week-old Meth-A cells (5 × 105 cells) were subcu-
taneously transplanted to the right inguinal region of BALB/c
mice. 2′-DHAC (19) and 3′-DHAC (45) were intravenously
administered for 10 consecutive days at doses of 2 and 10 mg/
kg from the day after transplantation. Three weeks after
transplantation, mice were sacrificed and solid tumors were
dissected out and weighed. The tumor inhibition ratio (%) was
calculated as follows: tumor inhibition ratio (%) ) (1 - T/C)
× 100, where C is the mean tumor weight in the control group
and T is that in the treated group. Effectiveness was statisti-
cally evaluated by Student’s t-test. The criterion for statistical
significance was p < 0.05.
1
720.3289, found 720.3306; H NMR (CDCl3) 8.95 (br s, 1 H,
3-NH), 7.74 (d, 1 H, H-6, J 5,6 ) 8.1 Hz), 7.68-7.32 (m, 20 H,
Ph), 6.20 (dd, 1 H, H-1′, J 1′,2′a ) 6.3, J 1′,2′b ) 7.6 Hz), 5.35 (dd,
1 H, H-5, J ) 2.0, 8.1 Hz), 5.22 (d, 1 H, 3′-NH, J 3′,NH ) 5.6
Hz), 3.95-3.82 (m, 3 H, H-3′, H-5′), 3.59 (m, 1 H, H-4′), 2.48
(ddd, 1 H, H-2′a, J 1′,2′a ) 6.3, J gem ) 14.4, J 2′a,3′ ) 4.2 Hz), 1.99
(ddd, 1 H, H-2′b, J 1′,2′b ) 7.6, J gem ) 14.4, J 2′b,3′ ) 7.6 Hz), 1.09,
1.05 (each s, each 9 H, t-Bu). Anal. (C41H49N3O5Si2‚0.9H2O)
C, H, N.
5′-O-(ter t-Bu tyld ip h en ylsilyl)-2′,3′-d id eoxy-3-[(ter t-bu -
tyld ip h en ylsilyl)oxya m in o]cytid in e (51). Compound 50
(735 mg, 1.02 mmol) was converted into 51 (635 mg, 87% as a
white foam) as described for the synthesis of 16: LRMS (FAB)
m/z 719 (MH+, 1%); HRMS (FAB) calcd for C41H51N4O4Si2
719.3449, found 719.3442; 1H NMR (CDCl3) 7.86 (d, 1 H, H-6,
J 5,6 ) 7.3 Hz), 7.65-7.28 (m, 20 H, Ph), 6.18 (dd, 1 H, H-1′,
J 1′,2′a ) 6.3, J 1′,2′b ) 6.3 Hz), 5.33 (d, 1 H, H-5, J 5,6 ) 7.3 Hz),
5.19 (d, 1 H, 3′-NH, J 3′,NH ) 6.8 Hz), 3.97 (ddd, 1 H, H-4′, J 3′,4′
) 4.9, J 4′,5′a ) 2.9, J 4′,5′b ) 2.9 Hz), 3.86 (dd, 1 H, H-5′a, J 4′,5′a
) 2.9, J gem ) 11.2 Hz), 3.77 (m, 1 H, H-3′), 3.56 (dd, 1 H, H-5′b,
J 4′,5′b ) 2.9, J gem ) 11.2 Hz), 2.62 (ddd, 1 H, H-2′a, J 1′,2′a ) 6.3,
J gem ) 14.2, J 2′a,3′ ) 6.3 Hz), 1.97 (ddd, 1 H, H-2′b, J 1′,2′b ) 6.3,
J gem ) 14.2, J 2′b,3′ ) 7.8 Hz), 1.07, 1.03 (each s, each 9 H, t-Bu);
13C NMR (CDCl3) 165.28, 155.52, 141.06, 135.63, 135.61,
135.40, 135.24, 132.96, 132.38, 129.79, 129.69, 127.71, 127.67,
127.48, 127.46, 93.61, 85.90, 82.57, 64.23, 61.75, 36.75, 27.45,
27.07, 19.43, 19.29. Anal. (C41H50N4O4Si2‚1.6H2O) C, H, N.
2′,3′-Dideoxy-3′-(h ydr oxylam in o)cytidin e Dih ydr och lo-
r id e (52, 3′-d DHAC). A mixture of 51 (72 mg, 0.100 mmol)
in concentrated HCl (300 µL) and MeOH (700 µL) was stirred
at room temperature for 4 h. The solvent was removed in
vacuo. The residue was coevaporated several times with EtOH
and suspended with EtOH and hexane, and the resulting
crystals were collected by filtration to give 52 (32.0 mg,
quantitative): mp 175 °C dec; LRMS (FAB) m/z 243 (MH+,
28%); HRMS calcd for C9H15N4O4 243.1093, found 243.1095;
1H NMR (MeOH-d4) 8.36 (d, 1 H, H-6, J 5,6 ) 7.9 Hz), 6.27 (dd,
Ack n ow led gm en t . This investigation was sup-
ported in part by a Grant-in-Aid for Scientific Research
on Priority Areas from the Ministry of Education,
Science, Sports, and Culture of J apan and by the
Second-Term Comprehensive Ten-Year Strategy for
Cancer Control from the Ministry of Health and Welfare
of J apan.
1 H, H-1′, J 1′,2′a ) 6.6, J 1′,2′b ) 6.6 Hz), 6.17 (d, 1 H, H-5, J 5,6
7.9 Hz), 4.54 (ddd, 1 H, H-4′, J 3′,4′ ) 2.6, J 4′,5′a ) 2.6, J 4′,5′b
3.3 Hz), 4.31 (ddd, 1 H, H-3′, J 2′a,3′ ) 2.6, J 2′b,3′ ) 8.6, J 3′,4′
)
)
)
2.6 Hz), 3.93 (dd, 1 H, H-5′a, J 4′,5′a ) 2.6, J gem ) 11.9 Hz), 3.82
(dd, 1 H, H-5′b, J 4′,5′b ) 3.3, J gem ) 11.9 Hz), 2.85 (ddd, 1 H,
H-2′a, J 1′,2′a ) 6.6, J gem ) 15.2, J 2′a,3′ ) 2.6 Hz), 2.55 (ddd, 1 H,
H-2′b, J 1′,2′b ) 6.6, J gem ) 15.2, J 2′b,3′ ) 8.6 Hz); NOE: irradiate
H-2′â, observe H-1′ (1.9%), H-6 (5.8%), and H-3′â (15.3%),
irradiate H-2′R, observe H-1′ (17.9%) and H-3′â (1.8%), no other
NOE enhancement was observed;13C NMR (MeOH-d4) 160.84,
148.00, 145.94, 94.61, 88.46, 82.73, 62.97, 62.56, 35.12. Anal.
(C9H14N4O4‚2HCl‚0.4H2O) C, H, N.
Deter m in a tion of th e p Ka ’s of 2′-DHAU (15) a n d 3′-
DHAU (40) by 13C NMR Sp ectr oscop y. The nucleosides (50
mM) in 200 mM HCl-KCl, phosphate, borate, or acetate buffer
(H2O) adjusted to the pH given in Figure 1 and containing
10% D2O were measured at 27 °C. Each data point is shown
as ∆δ (ppm) ) δ (ppm) at the pH given in Figure 1 - δ (ppm)
at pH 0.57 for 15 or pH 0.50 for 40.
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