Synthesis and cytotoxicity of 4′-C- and 5′-C-substituted toyocamycins

Article abstract of DOI:10.1016/S0968-0896(00)00221-2

Toyocamycin and some analogues have shown potent antitumor activities; however, none of them could be used clinically primarily owing to their cytotoxicity to normal human cells. In order to overcome the weakness of these nucleoside analogues, substitution of a variety of modified sugars for the ribofuranose was explored in our laboratories with expectation that certain sugar-modified toyocamycin analogues may be selectively cytotoxic to cancer cells. In this article, we report synthesis and cytotoxicity of 4′-C- and 5′-C-substituted toyocamycins, which were prepared via the condensations of 4-C- and 5-C-substituted ribofuranose derivatives 11, 12, 13, 20, 21, and 26 with the silylated form of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (27) and subsequent debromination and debenzoylation. When compared to the parent toyocamycin, all these analogues showed much lower cytotoxicity to human prostate cancer cells (HTB-81), mouse melanoma cancer cells (B16) as well as normal human fibroblasts. Compound 1e showed a significant cytotoxicity to the prostate cancer cells and a moderate selectivity. The results suggested that sugar modifications, especially those that may affect phosphorylation of nucleosides, could alter cytotoxicity profile significantly.

Full text of DOI:10.1016/S0968-0896(00)00221-2

Bioorganic & Medicinal Chemistry 9 (2001) 163±170
Synthesis and Cytotoxicity of 40-C- and 50-C-Substituted
Toyocamycins
Esmir Gunic,a Jean-Luc Girardet,a Zbigniew Pietrzkowski,b Cathey Eslerb
and Guangyi Wanga,*
aChemistry Laboratories, ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa Mesa, California 92626, USA
bCancer Biology Laboratories, ICN Pharmaceuticals, Inc., 3300 Hyland Avenue, Costa Mesa, California 92626, USA
Received 21 June 2000; accepted 15 August 2000
AbstractÐToyocamycin and some analogues have shown potent antitumor activities; however, none of them could be used clini-
cally primarily owing to their cytotoxicity to normal human cells. In order to overcome the weakness of these nucleoside analogues,
substitution of a variety of modi®ed sugars for the ribofuranose was explored in our laboratories with expectation that certain
sugar-modi®ed toyocamycin analogues may be selectively cytotoxic to cancer cells. In this article, we report synthesis and cyto-
toxicity of 40-C- and 50-C-substituted toyocamycins, which were prepared via the condensations of 4-C- and 5-C-substituted ribo-
furanose derivatives 11, 12, 13, 20, 21, and 26 with the silylated form of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (27) and
subsequent debromination and debenzoylation. When compared to the parent toyocamycin, all these analogues showed much
lower cytotoxicity to human prostate cancer cells (HTB-81), mouse melanoma cancer cells (B16) as well as normal human ®bro-
blasts. Compound 1e showed a signi®cant cytotoxicity to the prostate cancer cells and a moderate selectivity. The results suggested
that sugar modi®cations, especially those that may a€ect phosphorylation of nucleosides, could alter cytotoxicity pro®le sig-
ni®cantly. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
be selectively cytotoxic to cancer cells. If a compound
can be selectively phosphorylated in cancer cells, a selec-
tive inhibition of cancer cells might be achieved.14,15 In
order to search for such a nucleoside analogue, we syn-
thesized a series of sugar-modi®ed toyocamycin analo-
gues having a lower alkyl, alkenyl, or alkynyl substituent
on the ribose moiety of toyocamycin. In this article we
report the synthesis and in vitro cytotoxicity of 40-C- and
50-C-substituted toyocamycins (1b±g, 2a±d, and 3a±c), as
shown in Chart 1.
Many toyocamycin analogues have been synthesized and
evaluated for antitumor and antiviral activities since
toyocamycin was isolated four decades ago.1 9 Although
some toyocamycin analogues are potent inhibitors of
many cancer cells, they are also toxic to normal human
cells. Many e€orts have been made to improve their
selectivity pro®les. Unfortunately, none of the toyoca-
mycin analogues are used clinically. It was reported that
the toxicity pro®le of neplanocin A, another antibiotic
nucleoside, was improved when a methyl group was
introduced at its 60-carbon.10 50-C-Methyl toyocamycin
and sangivamycin were also reported,11 but no detailed
Chemistry
biological studies were given.
A
few nonphos-
The 40-C- and 50-C-substituted toyocamycins were syn-
thesized via the condensations of 4-C- and 5-C-substituted
ribofuranose derivatives with the silylated form of 4-
phorylatable, 50-modi®ed analogues of toyocamycin and
sangivamycin designed as protein kinase inhibitors were
reported.12,13 Apparently, 50-C-substituted toyocamycin
analogues as anticancer and antiviral agents have not
been intensively explored. In other words, it was not
known whether C-branched nucleoside analogues can
amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine by
a
similar procedure as those used for preparation of toyo-
camycin.16,17 Synthesis of the modi®ed ribofuranoses is
shown in Scheme 1. The 5(R)-allylribofuranose deriva-
tive 5 was prepared according to a previously published
procedure.18 Reaction of 419 with ethynylmagnesium
bromide a€orded 6 in good yield, which was a mixture of
the 5(R)- and 5(S)-isomers (1:1). A controlled catalytic
*Corresponding author. Tel.: +714-545-0100 ext. 4165; fax: +714-
668-3141; e-mail: gwang@icnpharm.com
0968-0896/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00221-2

164
E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
O-5; removal of 2,3-O-isopropylidene; benzoylation at
O-2 and O-3; and acetylation at O-1.
Treatment of 4 with formaldehyde in the presence of
sodium hydroxide yielded 14, which was selectively
protected with 4,40-dimethoxytrityl (DMT) at the 4a-
hydroxymethyl to give 15. The benzoylation of 15 and
the subsequent removal of DMT gave 16, which was
converted to the 4-C-methyl derivative 17 by a Barton
type deoxygenation.22,23 Compounds 17 and the 4,5-O-
dibenzoylated form of 14 (not shown) were converted to
18 and 19, respectively, by treatment with TFA at low
temperature and the subsequent benzoylation. Com-
pounds 18 and 19 were acetylated in the presence of
sulfuric acid to give the 1-O-acetylribofuranoses 20 and
21, respectively. Compound 14 was selectively protected
with t-butyldimethylsilyl (TBS) at the 4a-hydroxy-
methyl to give 22 in good yield. Tritylation of 22 at the
5-hydroxyl and the subsequent removal of the TBS
group a€orded 23. Compound 23 was converted to an
aldehyde, which was subjected to a Wittig reaction to
give 24 in very good yield. After hydrogenation of 24
over palladium, the resulting product was subjected to
the same sequence of reactions as 5±7 to give the 4-C-
ethyl derivative 26.
Chart 1.
The Vorbruggen condensations (Scheme 2)16,17,24 of the 1-
O-acetylated ribofuranose derivatives 11, 12, 13, 20, 21,
and 26 with the fully silylated form of 4-amino-6-bromo-5-
cyanopyrrolo[2,3-d]pyrimidine (27)25 in the presence of
trimethylsilyl tri¯ate yielded compounds 28±35, respec-
tively. The removal of bromine in compounds 28±32 was
accomplished by treatment with zinc in acetic acid,26
hydrogenation of 6 over nickel boride20 a€orded 7 (R/S
ratio, 1:1). The 5(R)-isomer (7a) of 7 was also prepared
following a published procedure.21 Compounds 5±7 were
converted to 11±13 in moderate to good yields, respec-
tively, through a sequence of reactions: benzoylation at
Scheme 1. (a) HCꢀCMgBr, THF, 40 to 0 ꢁC, 1.50 h, 77% for 6; (b) H2, Ni2B, (CH2NH2)2, EtOH, rt, 10 days, 63%; (c) BzCl, pyridine, rt, over-
night; (d) TFA/H2O, 0 ꢁC, 1.5 h; (e) same as c; (f) Ac2O, AcOH, H2SO4, rt, overnight, 37% for 11, 55% for 12, 63% for 13 (4 steps); (g) CH2O,
NaOH, dioxane/H2O, rt, overnight, 85%; (h) DMT-Cl, pyridine, rt, overnight; (i) same as c; (j) 80% AcOH, rt, 2 h; (k) PHOC(S)Cl, DMAP,
CH3CN, rt, 2 h; (l) (TMS)3SiH, ACCN, toluene, 100 ꢁC, 15 h; (m±o) same as d±f, 28% for 20 (8 steps), 73% for 21 (4 steps); (p) TBDMS-Cl,
pyridine, rt, 24 h; (q) same as h; (r) TBAF, THF, 3 days, 65% (3 steps); (s) DMSO, DCC, TFA, pyridine, rt, 8 h, 89%; (t) Ph3PˆCH2, ether, rt, 6 h,
99% (2 steps); (u) H2, Pd/C, rt, 6 h; (v) same as c; (x±z) same as d±f, 58% (5 steps).

E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
165
followed by debenzoylation, to a€ord compounds 1c,
1d, 1f, 2b and 2c, respectively, in moderate to good
yields, with 1c and 2b as an isomeric mixture and 1d and
2c as another isomeric mixture. Compounds 1d and 2c
could be separated by chromatography, and compounds
1c and 2b were only partially separated. Compounds
33±35 were subjected to catalytic hydrogenolysis over
Pd/C, followed by debenzoylation, to give 3a±c, respec-
tively, in good yields. The mixture of 1c and 2b was
hydrogenated over palladium to give a mixture of 1e
and 2d, which was separated by chromatography.
Compound 1g was obtained from the catalytic hydro-
genation of 28 over palladium and the subsequent
debenzoylation. Compounds 1b and 2a were reported
previously11 and synthesized recently in our laboratory
by a di€erent synthetic route.24
Scheme 2. (a) 1. HMDS, (NH4)2SO4, re¯ux, overnight; 2. TMSOTf,
ClCH2CH2Cl, re¯ux, 3 days; (b) Zn, AcOH, rt, overnight (for 28±32);
(c) H2, Pd/C, rt, 6 h (for 33±35); (d) 1. NH3/MeOH, rt, overnight; 2.
NaOAc/DMF, 120 ꢁC, 5 h, 51% for 1f, 62% for 1c and 2b, 29% for 1d
and 2c, 69% for 3a, 62% for 3b and 54% for 3c (3 steps).
Assignments of the selective protection site of 14
From previous publications,27,28 we could anticipate
that the 4a-hydroxymethyl of 14 would be selectively
protected. As can be seen from Scheme 1, compounds
15 and 22 were prepared in good yields from such
selective protections of 14. In order to make certain that
the selective protection took place at the 4a-hydroxy-
methyl rather than at the O-5 position of 14, we have
investigated the stereochemical assignments. The major
product (77%) and the minor product (8%) from the
tritylation of 14 with DMT-Cl were methylated, respec-
tively. The methylated products were separately sub-
jected to treatment with TFA at 0 ꢁC to remove DMT
and isopropylidene protecting groups. The reactions of
the deprotected products with p-tosyl chloride gave the
tosyl derivatives 36 (from the major tritylation product)
and 37 (from the minor tritylation product), respec-
tively. The tosylates 36 and 37 were treated separately
with sodium hydride in THF at 50 ꢁC. One reaction was
complete within 2 h with formation of two products
while the other reaction did not proceed at all even after
24 h. The two products formed from the ®rst reaction
were tentatively assigned as 3-O,4-C-methylene and 2-
O,4-C-methylene ribofuranose derivatives 38 and 39,
respectively (Scheme 3).29 As can be seen from a ball±
stick model, the ring formation could only occur to
compound 36 since its tosyl group is at the 4a-hydro-
xymethyl and adjacent to the 2- and 3-hydroxyls, which
allowed us to determine, without ambiguity, that the
major tritylation product which led to the formation of
36 is compound 15 as shown in Scheme 1 and the minor
tritylation product is compound 23 (Scheme 1). By
comparison of the two NMR spectra (identical) of 23
from the two routes, it is evident that the selective sily-
lation also took place at the 4a-C-hydroxymethyl of 14.
Scheme 3. (a) MeI, NaH, THF, rt; (b) TFA/H2O, 0 ꢁC; (c) TsCl, pyr-
idine, rt; (d) NaH, THF, 50 ꢁC.
nucleosides simply through the assignments of the 5-C-
substituted ribofuranose intermediates. The R con®g-
uration at the C-5 of 5 was assigned in a previous pub-
lication.18 The 5(R)-isomer (7a) of 7 prepared according
to a published procedure21 was converted to 1d. Com-
parison of the proton NMR spectrum of 1d with those
of the two separated products from 7 (ꢂ1:1 mixture)
could clearly distinguish 1d and 2c. 50(R)-C-Ethyltoyo-
camycin (1e), the fully hydrogenated product of 1d, has
identical proton NMR spectrum to that of one of the
hydrogenated products of 1c and 2b mixture.
Assignments of con®gurations of the C-50 positions
In vitro cytotoxicity
In the 50-C-substituted toyocamycins, the C-50 can have
either R or S con®guration, which depends on the
orientation of the substituents at the C-50. Since the
condensations of the 1-O-acetylated ribofuranose deri-
vatives with the silylated base and the subsequent reac-
tions did not change the con®gurations at the C-5, we
can determine the con®gurations at the C-50 of the
Compounds 1c±g, 2b±d and 3a±c as well as known
compounds 1a,16 1b and 2a11,24 were evaluated for their
cytotoxicity to human prostate cancer cells, mouse mel-
anoma cancer cells, and normal human ®broblasts. The
results (Table 1) indicated that all the compounds tested
showed a dramatic reduction in cytotoxicity to both

166
E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
Table 1. Cytotoxicity (EC50a mM) of 40-C- and 50-C-substituted toyoca-
mycins
A usual work-up procedure was used for most of the
reactions in the Experimental section: the mixture was
diluted with ethyl acetate (or methylene chloride),
washed sequentially with water (or brine), dilute sodium
bicarbonate, and water (or brine), dried (Na2SO4), ®l-
tered, and concentrated to dryness at reduced pressure.
The crude product was puri®ed by ¯ash chromato-
graphy on silica gel.
Compound
HTB-81b
B-16c
NHFd
0.004
40.5
59.5
64.6
68.1
>100
>100
41.7
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
3a
3b
3c
0.012
17.3
17.2
19.1
10.3
0.005
45.3
>100
85.6
98.5
>100
>100
19.6
>100
52.1
44.4
5(R,S)-C-Ethynyl-2,3-O-isopropylidene-1-O-methyl-ꢀ-D-
ribofuranoses (6). A solution of 419 (4.0 g, 19.8 mmol) in
anhydrous THF (20 mL) was added to a stirred solution
of ethynylmagnesium bromide (0.5 M in THF, 80 mL)
at 40 ꢁC under argon. The mixture was warmed up to
0 ꢁC during 1.5 h, poured into ice/water, and neutralized
with acetic acid. After the usual work-up, the residue was
chromatographed (ethyl acetate:hexanes, 1:3) to give
3.48g (77%) of 6 as a colorless solid (ratio of the two iso-
mers, 1:1); 1H NMR (CDCl3) d 1.31, 1.33 (2 s, 3H, Me),
1.48, 1.49 (2 s, 3H, Me), 2.51 (dd, J=2.4, 0.6 Hz, 0.5 H,
=CH), 2.54 (dd, J=2.1, 0.6 Hz, 0.5H, =CH), 3.45, 3.50
(2 s, 3H, OMe), 3.90±4.02 (m, 1H, OH), 4.32±4.48 (m,
2H), 4.59 (d, J=6.0 Hz, 1H), 4.81 (d, J=5.7 Hz, 0.5H),
5.01 (d, J=5.7 Hz, 1H), 5.02 (d, J=6.3 Hz, 0.5H).
5.8
9.9
11.7
22.1
23.6
23.7
15.2
82.5
11.5
14.9
19.8
53.6
95.7
30.8
>100
44.4
>100
>100
aEC50 is the concentration of compound that caused 50% reduction in
absorbance at 490 nm relative to untreated cells using MTS assay.
bHTB-81, human prostate cancer cells.
cB-16, mouse melanoma cells.
dNHF, normal human ®broblasts.
cancer and normal cells when compared with toyoca-
mycin (1a). Compounds 1b, 1c, 1d, 1e, 2b, 2c, and 2d still
retain moderate to signi®cant cytotoxicity to the prostate
cancer cells (HTB-81), but less cytotoxicity to normal
cells (NHF). It was noted that three 50(R)-substituted
compounds (1c, 1d, 1e) have less cytotoxicity to NHF
cells than their 50(S)-isomers (2b, 2c, 2d) although both
types of isomers have comparable cytotoxicity to HTB-
81 cells. These results indicated a moderate selectivity in
cytotoxicity for the 50(R)-isomers, which implicated that
there might be a possibility to ®nd more selectively
cytotoxic compounds through sugar modi®cations.
2,3-O-Isopropylidene-1-O-methyl-5(R,S)-C-vinyl-ꢀ-D-
ribofuranoses (7). A mixture of 6 (two isomers, 6.1 g,
26.73 mmol) and nickel boride (3.5 g) in ethanol (200 ml)
containing ethylenediamine (5.8 mL) was shaken in a
hydrogenation apparatus (10 psi hydrogen) for 10 days.
The catalysts were ®ltered and washed with ethanol, and
the ®ltrate was concentrated to dryness. Chromato-
graphy (ethyl acetate:hexanes, 1:2) gave 4.2 g (63%) of 7
(two isomers, 1:1) as a syrup and 1.1 g of recovered 5.
1H NMR (CDCl3) d 1.30, 1.32 (2 s, 3H, Me), 1.48 (s,
3H, Me), 3.40, 3.46 (2 s, 3H, OMe), 3.94, 4.10 (2 br s,
1H, OH), 4.25±4.42 (m, 2H), 4.56±4.61 (m, 1H), 4.79±
4.87 (m, 1H), 4.95, 4.98 (2 s, 1H), 5.22±5.48 (m, 2H,
vinyl), 5.80±5.96 (m, 1H, vinyl).
In summary, we have reported synthesis and in vitro
cytotoxicity evaluation of a number of new toyocamy-
cin analogues containing 40-C- and 50-C-substituted
ribofuranoses. The cytotoxicity of these compounds to
human prostate cancer (HTB-81) and mouse melanoma
cancer cells (B-16) as well as normal human ®broblasts
was dramatically reduced compared to that of toyoca-
mycin. Several compounds (1c, 1d, 1e, 2b, 2c, 2d)
retained moderate to signi®cant cytotoxicity to the can-
cer cells, with compound 1e demonstrating a moderate
selectivity. Further evaluation of these compounds for
cytotoxicity to other types of cancer cells and their
antiviral activity are under way.
2,3-O-Isopropylidene-1-O-methyl-5(R)-C-vinyl-ꢀ-D-ribo-
furanose (7a). A solution of 419 (1.0 g, 5.0 mmol) in
anhydrous THF (25 mL) was added to a stirred solution
of vinylmagnesium bromide (1.0 M in THF, 14.8 ml) in
THF (115 mL) at 20 ꢁC. The reaction mixture was
stirred at 10 ꢁC for 2 h. Similar work-up as described
for 5 and subsequent chromatography (1% methanol in
dichloromethane) gave 0.41 g (36%) of 7a as a syrup; 1H
NMR (CDCl3) d 1.30 (s, 3H, Me), 1.47 (s, 3H, Me), 3.46
(s, 3H, OMe), 3.94 (s, 1H, OH), 4.27±4.34 (m, 2 H), 4.58
(d, J=6.0 Hz, 1H), 4.80 (d, J=6.0 Hz, 1H), 5.00 (s, 1H,
1-H), 5.26±5.31 (m, 1H, -CˆCH2), 5.41±5.48 (m, 1H,
-CˆCH2), 5.81±5.92 (m, 1H, -CHˆC).
Experimental
1H NMR spectra were recorded on a Varian 300 spec-
trometer and tetramethylsilane was used as the internal
standard. Elemental analysis was conducted by NuMega
Resonance, Inc., San Diego, CA. Melting points were
measured on a capillary Melting Point Measurement
Apparatus and are uncorrected. Anhydrous solvents
were purchased from Aldrich or Fluka without further
treatment unless noted. Thin layer chromatography
plates and silica gel for column chromatography were
supplied by ICN Biomedicals. Solvent ratios are based
on volume in cases where a solvent mixture was used.
1-O-Acetyl-5(R)-C-allyl-2,3,5-tri-O-benzoyl-ꢀ-D-ribofur-
anose (11). A solution of 518 (4.49 g, 18.38 mmol) and
benzoyl chloride (2.7 mL, 23.2 mmol) in anhydrous pyr-
idine (40ml) was stirred at room temperature overnight
and quenched with water. After the usual work-up, the
residue was puri®ed by chromatography (15% ethyl ace-
tate in hexanes) to give 6.26g of the benzoylated product,
which was dissolved in cold TFA:water (9:1, 60mL) and
stood at 0 ꢁC for 1.5 h. Solvent was evaporated at 0 ꢁC,

E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
167
1
and the residue was dissolved in methanol/toluene and
concentrated to dryness. The crude was subjected to
benzoylation as described above to give 5.30 g of 8,
which was dissolved in a mixture of acetic acid (18 ml)
and acetic anhydride (2.3 mL). Under cooling with ice,
sulfuric acid (96%, 250 mL) in acetic acid (5 mL) was
added, and the resulting mixture was stirred at room
temperature overnight. After the usual work-up, the crude
was puri®ed by chromatography (ethyl acetate:hexanes,
1:4) to give 2.82g (37%, 4 steps) of 11 as a colorless solid;
1H NMR (CDCl3) d 2.14 and 2.35 (2 s, 3H, Ac), 2.50±2.70
(m, 2H), 4.68±4.75 (m, 1H), 5.06±5.21 (m, 2H), 5.48±5.92
(2 m, 3H), 6.02±6.12 (m, 1H), 6.38 (d, J=0.9 Hz, 2/3H),
6.60 (d, J=4.5 Hz, 1/3H), 7.10±8.18 (m, 15H).
used directly in the next reaction. H NMR (CDCl3) d
1.34 (s, 3H, Me), 1.53 (s, 3H, Me), 3.34 (s, 3H, OMe), 3.87
(m, 2H), 4.41±4.54 (m, 2H), 4.74 (m, 2H), 5.04 (s, 1H),
7.43±7.62 (m, 3H, Bz), 8.07 (m, 2H, Bz).
1-O-Acetyl-2,3,5-tri-O-benzoyl-4-C-methyl-ꢀ-D-ribofura-
nose (20). A solution of 16 (the whole product obtained
above), DMAP (4.3 g, 35 mmol), and phenoxy-
thiocarbonyl chloride (2.4 mL, 17 mmol) in acetonitrile
(174 ml) was stirred at room temperature for 2 h, con-
centrated to dryness, dissolved in methylene chloride,
and washed with 0.5 M hydrochloric acid. After the
usual work-up, the residue was dried and dissolved in
toluene (93 mL), and tris(trimethylsilyl)silane (9.0 ml,
29 mmol) and 1,10-azobis(cyclohexanecarbonitrile) (0.71 g,
2.9 mmol) were added. The reaction mixture was stirred
at 100 ꢁC for 15 h, cooled, and concentrated. The resi-
due was puri®ed by chromatography (1±2% methanol in
dichloromethane) to give 17 as a syrup, which was con-
verted to 20 (1.70 g, 28%, 8 steps) by the same procedure
as described for 11. Recrystallization from ethyl acetate/
hexanes gave a colorless, crystalline solid (b-isomer); 1H
NMR (CDCl3) d 1.61 (s, 3H, Me), 1.96 (s, 3H, Ac), 4.49
(dd, J=58.5 Hz, 11.4 Hz, 2H), 5.84 (d, J=5.4 Hz, 1H),
5.98 (d, J=5.1 Hz, 1H), 6.43 (s, 1H), 7.26±7.64 (m, 9H,
Bz), 7.81±8.14 (m, 6H, Bz).
1-O-Acetyl-2,3,5-tri-O-benzoyl-5(R,S)-C-ethynyl-ꢀ-D-
ribofuranoses (12). A colorless syrup (R:S, 1:1; a:b, 1:2)
was prepared in 55% yield from 6 by the same proce-
dure as described for 11. 1H NMR (CDCl3) d 1.75, 2.13,
2.14 and 2.19 (4 s, 3H), 2.48, 2.54, 2.63, and 2.64 (4 d,
J=2.1 Hz, 1H), 4.79±4.87 (m, 1H), 5.69±5.82 (m, 1H),
5.93-6.19 (m, 2H), 6.39 (s, 1/3H), 6.43 (s, 1/3H), 6.68 (d,
J=4.2 Hz, 1/6H), 6.78 (d, J=4.8 Hz, 1/6H), 7.28±8.16
(m, 15H).
1-O-Acetyl-2,3,5-tri-O-benzoyl-5(R,S)-C-vinyl-ꢀ-D-ribo-
furanoses (13). A colorless syrup (R:S, 1:1; a:b, 1:2)
was prepared in 63% yield from 7 by the same proce-
dure as described for 11. 1H NMR (CDCl3) d 1.77, 2.12,
2.13 and 2.19 (4 s, 3H), 4.71±4.78 (m, 1H), 5.30±5.61 (m,
3H), 5.73±6.03 (m, 3H), 6.38 (s, 1/3H), 6.42 (s, 1/3H),
6.63 (d, J=4.5 Hz, 1/6H), 6.76 (d, J=4.5 Hz, 1/6H),
7.28±8.15 (m, 15H).
1-O-Acetyl-4-C-benzoyloxymethyl-2,3,5-tri-O-benzoyl-
ꢀ-D-ribofuranose (21). Prepared from 14 (3.0 g,
12.8 mmol) by the same procedure as described for 11.
1
Total yield: 5.82 g (73%) as a syrup (a:b ratio, 1:2). H
NMR (CDCl3) d 2.05 and 2.18 (2 s, 3H, Ac), 4.69±4.92
(m, 3H), 5.83±5.88 (m, H), 6.18 (d, J=5.4 Hz, 2/3H),
6.24 (d, J=6.3 Hz, 1/3H), 6.51 (s, 2/3H), 6.74 (d, J=4.5
Hz, 1/3H), 7.20±8.20 (m, 20H, Bz).
4-C-Hydroxymethyl-2,3-O-isopropylidene-1-O-methyl-ꢀ-
D-ribofuranose (14). To a stirred solution of 419 (20.22
g, 0.1 mol) and formaldehyde (37% aq., 76 mL) in 1,4-
dioxane (380 mL) at 0 ꢁC was added aqueous NaOH
(2.0 M, 188 mL). The reaction mixture was stirred at
room temperature overnight, neutralized with 10%
AcOH, and concentrated to half the volume. After the
usual work-up, the residue was puri®ed by chromato-
graphy (4% methanol in chloroform) to give 20.2 g
5-O-(4,40-Dimethoxytrityl)-4-C-hydroxymethyl-2,3-O-iso-
propylidene-1-O-methyl-ꢀ-D-ribofuranose (23). A solu-
tion of 14 (4.5 g, 19 mmol) and tert-butyldimethylsilyl
chloride (3.4 g, 23 mmol) in anhydrous pyridine (96 mL)
was stirred at room temperature for 24 h, quenched
with water (5 mL), and concentrated. After the usual
work-up, the residue (22) was dried and dissolved in
anhydrous pyridine, and 4,40-dimethoxytrityl chloride
(8.4 g, 25 mmol) was added. The reaction mixture was
stirred at room temperature overnight, quenched with
methanol, and concentrated. After the usual work-up,
the residue was dried and dissolved in THF (57 mL),
and TBAF (1.0 M in THF, 23 mL) was added. After
24 h at room temperature, more TBAF (3.8 mL) was
added, and the mixture was stirred for an additional
36 h. The solvent was evaporated and the residue was
chromatographed (ethyl acetate:hexanes, 1:1) to give
1
(85%) of 14 as a colorless solid; H NMR (CDCl3) d
1.33 (s, 3H, Me), 1.51 (s, 3H, Me), 2.37 (t, 1H, OH), 3.4
(m, 1H), 3.44 (s, 3H), 3.60±3.82 (m, 4H), 4.67 (d, J=6.0
Hz, 1H), 4.86 (d, J=6.3 Hz, 1H), 5.00 (s, 1H).
5-O-Benzoyl-4-C-hydroxymethyl-2,3-O-isopropylidene-1-
O-methyl-ꢀ-D-ribofuranose (16). A solution of 14 (3.5 g,
15 mmol) and 4,40-dimethoxytrityl chloride (6.0 g,
18 mmol) in anhydrous pyridine (78 mL) was stirred at
room temperature overnight, quenched with methanol
(6 mL) at 0 ꢁC, and concentrated. After the usual work-
up, the residue was puri®ed by chromatography (20±
25% ethyl acetate in hexanes) to give 6.2 g (77%) of 15
as a colorless foam, which was subjected to benzoyl-
ation as described before. The crude product was dis-
solved in toluene and concentrated to dryness. The
residue was dissolved in 80% acetic acid (174 mL), and
the mixture stood at room temperature for 2 h and con-
centrated to dryness. Chromatography (1±2% methanol
in dichloromethane) gave 16 as a white foam, which was
1
6.6 g (65%, 3 steps) of 23 as a colorless foam; H NMR
(CDCl3) d 1.27 (s, 3H, Me), 1.49 (s, 3H, Me), 2.15 (m,
1H, OH), 3.17 (s, 3H, OMe), 3.27 (m, 2H), 3.79 (s, 6H,
OMe), 3.86±4.01 (m, 2H), 4.45 (m, 2H), 4.91 (s, 1H),
6.84 (m, 4H, DMT), 7.20±7.46 (m, 9H, DMT).
5-O-(4,40 -Dimethoxytrityl)-2,3-O-isopropylidene-1-O-
methyl-4-C-vinyl-ꢀ-D-ribofuranose (24). A solution of
TFA (0.49 mL, 6.4 mmol) and pyridine (1.6 mL, 19mmol)
in DMSO (11 mL) was added to a stirred solution of 23

168
E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
(6.9 g, 13 mmol) and DCC (6.6 g, 32 mmol) in a mixture
of toluene (26 mL) and DMSO (66 mL) at 5 ꢁC. The
reaction mixture was stirred at ambient temperature for
8 h and cooled to 0 ꢁC. Ethyl acetate (80 mL) and a
solution of oxalic acid (1.8 g, 19 mmol) in methanol
(10 mL) were added, and the mixture was stirred at
room temperature overnight. The precipitate was ®ltered
and washed with a 1:1 mixture of hexanes and ethyl
acetate. After the usual work-up, the residue was puri®ed
by chromatography (ethyl acetate:hexanes, 1:3) to give
6.1 g (89%) of the 4-C-formyl product as a colorless foam.
under argon for 3 days, poured into ice-water (50 mL)
containing sodium bicarbonate (3 g), extracted with
chloroform, and the combined organic layer was
washed with water, dried over sodium sulfate, and con-
centrated. Chromatography on silica gel (ethyl acetate:
hexanes, 2:3) gave 1.8 g (66%) of 28 as a colorless solid.
A mixture of 28 (750 mg 1.02 mmol) and zinc dust
(0.53 g, 8.16 mmol) in acetic acid (25 mL) was stirred at
room temperature for 4 h, then more zinc dust (0.53 g,
8.16 mmol) was added, and the mixture was stirred for
another 7 h. The solid was ®ltered, and the ®ltrate was
concentrated to dryness. After the usual work-up, the
residue was dissolved in ammonia-saturated methanol
(40 mL), and the resulting solution stood at ambient
temperature overnight. Solvent was evaporated, and the
resulting residue and sodium acetate (20 mg) in anhy-
drous DMF (20 mL) was stirred at 120ꢁC for 5 h. After
evaporation, the residue was puri®ed by chromato-
graphy (4% methanol in ethyl acetate) to give 145 mg
(77%, last two steps) of 1f as a colorless solid: mp 165±
167 ꢁC; 1H NMR (DMSO-d6) d 2.05±2.27 (m, 2H),
3.67±3.74 (m, 1H), 3.79 (m, 1H), 4.16 (m, 1H), 4.42 (dd,
J=12.0 and 5.7 Hz, 1H), 4.97±5.09 (m, 2H), 5.17 (d,
J=4.5 Hz, 1H, OH), 5.39 (d, J=6.6 Hz, 1H, OH), 5.46
(d, J=4.8 Hz, 1H, OH), 5.76±5.92 (m, 1H), 5.97 (d, 1H,
J=6.9 Hz), 6.92 (br s, 2 H, NH2), 8.19 (s, 1H), 8.42 (s,
1H). Anal. calcd for C15H17N5O4: C, 54.38; H, 5.17; N,
21.14. Found: C, 54.37; H, 5.04; N, 21.08.
A solution of sodium pentoxide (2.5 g, 22 mmol) in
benzene (34 mL) was added to a stirred suspension of
methylphosphonium bromide (8.8 g, 25 mmol) in ether
(250 mL) under argon. The mixture was stirred at room
temperature for 6 h, and a solution of the 4-C-formyl
product (6.0 g, 11 mmol) in ether (30 mL) was added.
The resulting mixture was stirred at room temperature
overnight, quenched with brine at 0 ꢁC. After the usual
work-up, the residue was chromatographed (ethyl acetate:
hexanes, 1:3) to give 5.9 g (99%) of 24 as a colorless
foam; 1H NMR (CDCl3) d 1.26 (s, 3H, Me), 1.42 (s, 3H,
Me), 3.20 (m, 5H), 3.79 (s, 6H, OMe), 4.38 (d, J=6.0
Hz, 1H), 4.50 (d, J=6.0 Hz, 1H), 4.90 (s, 1H), 5.30 (dd,
J=11.1 Hz, 1.8 Hz), 5.45 (dd, J=17.4 Hz, 1.8 Hz), 6.20
(dd, J=17.4 Hz, 11.1 Hz), 6.82 (m, 4H, DMT), 7.20±
7.46 (m, 9H, DMT).
1-O-Acetyl-2,3,5-tri-O-benzoyl-4-C-ethyl-ꢀ-D-ribofura-
nose (25). A suspension of 10% Pd/C (50% water, 508
mg) and 24 (5.0 g, 9.4 mmol) in methanol (254 mL) was
shaken in a hydrogenation apparatus (5 psi hydrogen)
at room temperature for 6 h. The catalyst was ®ltered
and washed with methanol. The solvent was evapo-
rated, and the residue was dried by evaporation with
anhydrous pyridine and dissolved in pyridine (75 mL).
Benzoyl chloride (1.2 ml, 10 mmol) was added, the
reaction mixture was stirred at room temperature for
15 h, then cooled to 0 ꢁC. Methanol (5 mL) was added,
and the solvents were evaporated under reduced pres-
sure. Ethyl acetate, hexane and brine were added, and
the organic extract was washed with brine, dried over
sodium sulfate, ®ltered and evaporated to dryness. The
crude product 25 was converted to 26 by the same pro-
cedure as described for 11. Total yield (ratio of a:b, 1:2):
4-Amino-5-cyano-7-(5(R)-C-ethynyl-ꢀ-D-ribofuranosyl)-
pyrrolo[2,3-d]pyrimidine (1c) and 4-amino-5-cyano-7-
(5(S)-C-ethynyl-ꢀ-D-ribofuranosyl)pyrrolo[2,3-d]pyrimi-
dine (2b). Prepared as a mixture (1:1) from 12 by the
same procedure as described for 1f. Yield: 62% (3
steps). The two isomers were partially separated by a
¯ash chromatography on silica (4% methanol in ethyl
acetate). 1c (50(R)-isomer): H NMR (DMSO-d6) d 3.42
1
(s, 1H), 3.91 (d, J=4.2 Hz, 1H), 4.16 (t, J=4.5 Hz, 1H)
4.40±4.45 (m, 2H), 5.36 (d, J=4.2 Hz, 1H), 5.45 (d,
J=6.6 Hz, 1H), 6.07 (d, J=7.5 Hz, 1H), 6.24 (d, J=5.4
Hz, 1H), 6.95 (br s, 2H), 8.21 (s, 1H), 8.34 (s, 1H). 2b
(50(S)-isomer): 1H NMR (DMSO-d6) ꢀ 3.39 (s, 1H), 3.90
(m, 1H), 4.10 (m, 1H) 4.39±4.45 (m, 2H), 5.34 (d, J=4.8
Hz, 1H), 5.50 (d, J=6.6 Hz, 1H), 6.09 (m, ), 6.95 (br, s,
2H), 8.22 (s, 1H), 8.38 (s, 1H). Anal. calcd for C14H13
N5O4: C, 53.33; H, 4.16; N, 22.21. Found: C, 53.04; H,
4.06; N, 22.15.
1
58% (5 steps) as a colorless syrup; H NMR (CDCl3) d
1.05 and 1.11 (2 t, J=7.6 Hz, 3H, CH3), 1.94 and 2.10
(2 m, 2H, CH2), 1.98 and 2.14 (2 s, 3H, Ac), 4.40±4.58
(m, 2H), 5.85 (m, 1H), 6.02 and 6.06 (2 d, J=6.3 Hz and
5.7 Hz, 1H), 6.45 and 6.66 (2 d, J=1.2 Hz and 4.8 Hz,
1H), 7.34±8.15 (m, 15H, Bz).
4-Amino-5-cyano-7-(5(R)-C-vinyl-ꢀ-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine (1d) and 4-amino-5-cyano-7-(5(S)-
C-vinyl-ꢀ-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (2c).
Prepared as a mixture (1:1) from 13 by the same proce-
dure as described for 1f. Chromatography (3% metha-
nol in ethyl acetate) gave 1d (17%, 3 steps) and 2c
(12%, 3 steps), both as a colorless solid. Compound 1d
was also prepared starting from 7a in 20% yield (3
steps). 1d (50(R)-isomer): mp 228±230 ꢁC (recrystallized
4-Amino-5-cyano-7-(5(R)-C-allyl-ꢀ-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine (1f). A mixture of 4-amino-6-bromo-5-
cyanopyrrolo[2,3-d]pyrimidine (1.05 g, 4.41 mmol), HMDS
(75 ml), anhydrous m-xylene (25 mL), and ammonium sulfate
(50 mg) was re¯uxed under argon overnight, concentrated to
dryness, and dried under vacuum for 1 h. The residue together
with 11 (2.0g, 3.67 mmol) was dissolved in anhydrous 1,2-
dichloroethane (80 mL), the resulting solution was cooled
to 10ꢁC, and TMSOTf (1.3 ml, 7.3 mmol) in 1,2-dichlor-
oethane (5 mL) was added. The solution was re¯uxed
1
from water); H NMR (DMSO-d6) d 3.90 (m, 1H), 4.04
(m, 1H), 4.23 (m, 1H), 4.46 (m, 1H), 5.15 (d, J=10.8
Hz, 1H), 5.20 (d, J=4.2 Hz, 1H, OH), 5.34 (d, J=17.1
Hz, 1H), 5.40 (d, J=6.6 Hz, 1H, OH), 5.83±5.94 (m,
1H,), 5.87 (d, J=3.3 Hz, 1H, OH), 6.01 (d, J=7.5 Hz,

E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
169
1H), 6.96 (br s, 2H, NH2), 8.21 (s, 1H), 8.43 (s, 1H).
Anal. calcd for C14H15N5O4: C, 52.99; H, 4.76; N,
22.07. Found: C, 53.02; H, 4.69; N, 21.88. 2c (50(S)-iso-
mer): mp 165±167 ꢁC (recrystallized from methanol and
1H), 4.58 (m, 2H, including 1 OH), 5.17 (d, J=5.1 Hz,
1H, OH), 5.21 (t, J=5.7 Hz, 1H, OH), 5.36 (d, 1H,
J=7.2 Hz, OH), 6.06 (d, 1H, J=7.2 Hz), 6.90 (br s, 2H,
NH2), 8.20 (s, 1H), 8.42 (s, 1H). Anal. calcd for
C13H15N5O5 H2O: C, 46.02; H, 5.05; N, 20.64. Found:
C, 46.25; H, 4.69; N 20.67.
1
.
water); H NMR (DMSO-d6) d 3.90 (t, J=3.0 Hz, 1H),
4.10 (dd, J=8.4 and 3.6 Hz, 1H), 4.18 (m, 1H), 4.34 (dd,
J=11.1 and 4.8 Hz, 1H), 5.08 (dt, J=9.0 and 1.5 Hz,
1H) 5.20 (d, J=4.8 Hz, 1H, OH), 5.27 (dt, J=15.3 and
1.8 Hz, 1H), 5.45 (d, J=6.0 Hz, 1H, OH), 5.62 (d,
J=6.3 Hz, 1H, OH), 5.85±5.98 (m, 1H,), 6.00 (d, J=6.0
Hz, 1H), 6.92 (br s, 2H, NH2), 8.21 (s, 1H), 8.44 (s, 1H).
Anal. calcd for C14H15N5O4: C, 52.99; H, 4.76; N,
22.07. Found: C, 53.08; H, 4.67; N, 21.91.
4-Amino-5-cyano-7-(5(R)-C-propyl-ꢀ-D-ribofuranosyl)-
pyrrolo[2,3-d]pyrimidine (1g). A colorless solid was pre-
pared from 1f by the same hydrogenation and
deprotection procedure as described for 3c. Yield: 23%
(3 steps): mp 184±186 ꢁC (recrystallized from methanol);
1H NMR (DMSO-d6) d 0.86 (t, J=6.3 Hz, 3H), 1.20±
1.50 (m, 4 H), 3.62 (m, 1H) 3.76 (m, 1H), 4.11 (br s,
1H), 4.41 (dd, J=11.4 and 5.4 Hz, 1H), 5.15 (d, J=4.2
Hz, 1H, OH), 5.32 (d, J=4.8 Hz, 1H, OH), 5.37 (d,
J=6.3 Hz, 1H, OH), 5.96 (d, J=6.9 Hz, 1H), 6.92 (br s,
2H, NH2), 8.19 (s, 1H), 8.42 (s, 1H).
4-Amino-5-cyano-7-(5(R)-C-ethyl-ꢀ-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine (1e) and 4-amino-5-cyano-7-(5(S)-
C-ethyl-ꢀ-D-ribofuranosyl)pyrrolo[2,3-d]pyrimidine (2d).
A mixture of 1c and 2b (1:1, 150 mg, 0.476 mmol) and
10% Pd/C (100 mg) in methanol (50 mL) was shaken in
a hydrogenation apparatus (20 psi hydrogen) for 3 h.
The catalysts were ®ltered and washed with methanol,
and the ®ltrate was concentrated to dryness. Chroma-
tography (4% methanol in ethyl acetate) gave 60 mg
(39%) of 1e and 70 mg (46%) of 2d, both as a colorless
solid. 1e (50(R)-isomer): mp 187±188 ꢁC (recrystallized
4-Amino-5-cyano-7-(4-C-methyl-ꢀ-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine (3a). A colorless solid was pre-
pared from 20 by the same procedure as described for
3c. Yield: 69% (3 steps); mp 179±180 ꢁC (recrystallized
1
from water); H NMR (DMSO-d6) d 1.28 (s, 3H, Me),
3.32±3.51 (m, 2H), 4.01 (t, J=4.8 Hz, 1H), 4.60 (dd,
J=12.9 and 6.3 Hz, 1H), 5.14 (d, J=4.8 Hz, 1H, OH),
5.32 (m, 2H, OH), 6.02 (d, J=6.9 Hz, 1H), 6.91 (br s,
2H, NH2), 8.20 (s, 1H), 8.44 (s, 1H). Anal. calcd for
C13H15N5O4: C, 51.15; H, 4.95; N, 22.94. Found: C,
50.92; H, 4.85; N, 22.85.
1
from methanol); H NMR (DMSO-d6) d 0.89 (t, J=7.2
Hz, 3H), 1.20±1.50 (m, 2H), 3.54 (m, 1H) 3.77 (dd,
J=4.2 and 2.4 Hz, 1H), 4.12 (m, 1H), 4.41 (m, 1H), 5.16
(d, J=4.2 Hz, 1H, OH), 5.33 (d, J=4.8 Hz, 1H, OH),
5.38 (d, J=5.7 Hz, 1H, OH), 5.97 (d, J=7.2 Hz, 1H,),
6.91 (bs, 2H, NH2), 8.19 (s, 1H), 8.43 (s, 1H). Anal.
calcd for C14H17N5O4: C, 52.66; H, 5.37; N, 21.93.
Found: C, 52.84; H, 5.38; N, 21.99. 2d (50(S)-isomer):
mp 163±165 ꢁC (methanol); 1H NMR (DMSO-d6) d 0.88
(t, J=7.8 Hz, 3H), 1.45 (m, 2H), 3.48 (m, 1H) 3.86 (t,
J=3.3 Hz, 1H), 4.08 (dd, J=8.7 and 3.9 Hz, 1H), 4.30
(dd, J=11.4 and 5.7 Hz, 1H), 5.12 (d, J=4.8 Hz, 1H,
OH), 5.20 (d, J=6.6 Hz, 1H, OH), 5.44 (d, J=6.0 Hz,
1H, OH), 6.02 (d, J=5.4 Hz, 1H), 6.91 (br s, 2H, NH2),
8.20 (s, 1H), 8.46 (s, 1H). Anal. calcd for C14H17N5O4:
C, 52.66; H, 5.37; N, 21.93. Found: C, 52.58; H, 5.35; N,
21.77.
4-Amino-5-cyano-7-(4-C-ethyl-ꢀ-D-ribofuranosyl)pyr-
rolo[2,3-d]pyrimidine (3b). Prepared from 26 by the
same procedure as described for 3c. Yield: 62% (3 steps)
as aꢁcolorless solid (recrystallized from water): mp 207±
1
209 C; H NMR (DMS-d6) d 0.83 (t, J=12.5 Hz, 3H,
CH3CH2), 1.63 (m, 2H, CH3CH2), 3.47 (m, 2H), 4.02 (t,
J=4.8 Hz, 1H), 4.66 (dd, J=12.9 and 7.2 Hz, 1H), 5.11
(d, J=4.8 Hz, 1H, OH), 5.27 (m, 2H, OH), 6.00 (d,
J=7.8 Hz, 1H), 6.91 (br s, 2H, NH2), 8.20 (s, 1H), 8.44
(s, 1H). Anal. calcd for C14H17N5O4: C, 52.66; H, 5.37;
N, 21.93. Found: C, 52.53; H, 5.29; N, 21.77.
4-Amino-5-cyano-7-(4-C-hydroxymethyl-ꢀ-D-ribofurano-
syl)pyrrolo[2,3-d]pyrimidine (3c). The same condensa-
tion procedure as described for 1f gave 3.70 g of 35 as a
colorless solid from 21 (4.32 g, 7.07 mmol).
Assignment of the tritylation and silylation site of
compound 14
The major tritylation product 15 was added to a stirred
solution of NaH (2 equiv) in THF, followed by addition
of MeI (1.2 equiv). The reaction mixture was stirred at
room temperature overnight, cooled, and quenched
with dilute acetic acid until pH 7 was reached. After the
usual work-up, the crude was puri®ed by a short chro-
matography (EtOAc/hexane). The resulting product
was dissolved in cold TFA:water (9:1) and stirred at
0 ꢁC for 3 h. Methanol was added, and the mixture was
concentrated at 0 ꢁC in vacuo. After a quick chromato-
graphy (0±5% methanol in methylene chloride), the
product was dissolved in pyridine, and p-tosyl chloride
(2 equiv) was added. The mixture was stirred at room
temperature for 24 h, cooled to 0 ꢁC, and quenched with
water. After the usual work-up and puri®cation by ¯ash
chromatography, the dried tosyl derivative (36) was
added to a suspension of NaH (3 equiv) in THF. The
A mixture of 35 (3.6g, 4.41mmol) and 10% Pd/C
(500mg) in anhydrous dioxane (150mL) containing tri-
ethylamine (1.5 mL) was shaken in a hydrogenation
apparatus (20 psi hydrogen) for 5 h. The catalysts were
®ltered and washed with chloroform, and the ®ltrate was
concentrated to dryness. After the usual work-up, the
residue was dissolved in ammonia-saturated methanol
(200 mL), and the resulting solution stood at room
temperature overnight. Solvent was evaporated, and the
residue and sodium acetate (50 mg) were heated in
DMF (50 mL) at 120 ꢁC for 5 h and then concentrated
to dryness. Chromatography (12% methanol in ethyl
acetate) gave 1.17ꢁg (54%, 3 steps) of 3c as a colorless
solid: mp 216±218 C (recrystallized from methanol); 1H
NMR (DMSO-d6) d 3.55 (m, 4H) 4.14 (t, J=4.8 Hz,

170
E. Gunic et al. / Bioorg. Med. Chem. 9 (2001) 163±170
resulting mixture was stirred at 50 ꢁC for 2 h. After the
usual work-up, chromatography (20% acetone in
methylene chloride) gave the 4-C,3-O-methylene ribo-
furanose derivative (38) and 4-C,2-O-methylene ribofur-
anose derivative (39) in a ratio of 1:2, both as colorless
solid; 1H NMR (CDCl3) of 39: d 2.54 (d, J=5.7 Hz, OH),
3.38 (s, 3H, OMe), 3.41 (s, 3 H, OMe), 3.73 (d, 1H, J=7.8
Hz, 40-H), 3.74 (s, 2H, 5-H), 3.98 (d, J=7.8 Hz, 1H, 40-H),
4.01 (s, 1H, 2-H), 4.23 (d, J=5.4 Hz, 1H, 3-H), 4.77 (s,
1H, 1-H). 1H NMR (CDCl3) of 38: d 3.39 (s, 3H, OMe),
3.44 (s, 3H, OMe), 3.48, 3.55 (AB, J=9.9 Hz, 2H, 5-H),
4.06 (dd, J=1.5, 5.7 Hz, 1H, 2-H), 4.37 (d, J=7.2 Hz, 1H,
40-H), 4.84 (d, J=7.2 Hz, 1H, 40-H), 5.05 (d, J=1.5 Hz,
1H, 1-H), 5.07 (d, J=5.7 Hz, 1H, 3-H).
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The minor tritylation product 23 was subjected to the
same reactions as 15 to give 37, which was intact after it
was treated with sodium hydride in THF at 50ꢁC for 24h.
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