The triphosphate of β-d-4′-C-ethynyl-2′,3′-dideoxycytidine is the preferred enantiomer substrate for HIV reverse transcriptase

Article abstract of DOI:10.1016/j.bmc.2006.09.061

The enantioselective synthesis of the β-d (1) enantiomer of 4′-C-ethynyl-2′,3′-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between t

Full text of DOI:10.1016/j.bmc.2006.09.061

Bioorganic & Medicinal Chemistry 15 (2007) 283–287
The triphosphate of b-D-40-C-ethynyl-20,30-dideoxycytidine is the
preferred enantiomer substrate for HIV reverse transcriptase
Maqbool A. Siddiqui and Victor E. Marquez*
Laboratory of Medicinal Chemistry, Center for Cancer Research, NCI-Frederick, NIH, Frederick, MD 21702, USA
Received 28 August 2006; accepted 26 September 2006
Available online 29 September 2006
Abstract—The enantioselective synthesis of the b-D (1) enantiomer of 40-C-ethynyl-20,30-dideoxycytidine confirms an earlier stereo-
chemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate
between the D- and L-configuration of nucleoside 50-triphosphates.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
lute structural proof for the b-D (1) enantiomer by
chemical synthesis. Taking advantage of a recently
developed asymmetric synthesis of c-lactone 3a,2 which
was utilized for the synthesis of chiral (R)-diacylglycerol
lactones, we developed an asymmetric approach for the
synthesis of b-D (1) to confirm the earlier assignment
based strictly on the biological data.
The b-D (1) and b-L (2) enantiomers of 40-C-ethynyl-
20,30-dideoxycytidine were previously isolated by a li-
pase-catalyzed trans-esterification resolution performed
on the N4-benzoyl protected nucleoside racemate.1 Each
pure enantiomer was obtained with 98% and 91% ee,
respectively.
2. Results and discussion
The trityl group on the available chiral c-lactone (3a)
was not ideal to keep for an extended synthesis because
of its ease of cleavage under acidic conditions, including
during silica gel chromatography. Therefore, the more
acid robust t-butyldiphenylsilyl ether was put in place
leading to compound 3c after two steps in 86% overall
yield (Scheme 1). Reduction of the lactone with diisobu-
tylaluminum hydride (DIBAL-H) proceeded in 91%
yield and acetylation of the resulting lactol (4a) with ace-
tic anhydride in pyridine led to the corresponding ace-
tate 4b in 96% yield. During catalytic transfer
hydrogenation to remove the benzyl group in 4b, the
OAc group was exchanged to form the methyl glycoside
4c, and although such transformation was not intended,
the outcome was inconsequential for the ensuing elabo-
ration of the nucleoside (Scheme 2).
The assignment of the absolute stereochemistry of 1 and
2 was based on the ability of HIV reverse transcriptase
(RT) to discriminate between the corresponding 50-tri-
phosphates, which was further confirmed by assaying
against an RT mutant (M184V) with a marked prefer-
ence for incorporating nucleosides in the D-configura-
tion.1 Although the conclusions from these studies
seemed well-substantiated, we wished to provide abso-
The free OH group in 4c was protected as an acetate (4d,
Scheme 2) with the hope that, at least to some degree,
distant neighboring group participation by the acetate
would form an intermediate cation that would favor at-
tack from the b-face (Fig. 1). Thus, the methyl glycoside
Keywords: 40-C-ethynyldideoxynucleosides; Enantioselective synthesis;
HIV reverse transcriptase.
*
Corresponding author. Tel.: +1 301 846 5954; fax: +1 301 846
6033; e-mail: marquezv@dc37a.nci.nih.gov
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.061

284
M. A. Siddiqui, V. E. Marquez / Bioorg. Med. Chem. 15 (2007) 283–287
R O
1
R O
1
O
B
c
O
O
OR
3
R O
2
R O
2
TBDPSO
3a, R = Tr
4a, R = TBDPS
1
1
R = Bn
R = Bn
2
2
d
O
a
b
R = H
3
3b, R = H
1
4b, R = TBDPS
O
1
R = Bn
2
R = Bn
O
2
e
R = Ac
3
3c, R = TBDPS
1
4c, R = TBDPS
1
R = Bn
2
R = H
2
Figure 1. Proposed sugar carbocation intermediate formed through
R = CH
3
3
anchimeric assistance of the distant acetate.
Tr = CPh
3
Bn = CH Ph
2
the absence of any participating group (Scheme 2).1 This
type of distant neighboring anchimeric assistance has
been observed in the synthesis of nucleosides.3,4
TBDPS = (Me) CPh Si
3
2
Scheme 1. Reagents and conditions: (a) TFA, CH2Cl2; (b) TBDPS-Cl,
imidazole, CH2Cl2; (c) DIBAL-H, toluene, ꢀ78 ꢁC; (d) Ac2O,
pyridine; (e) HCO2H/MeOH, Pd-black.
Separation of both b- and a-anomers was accom-
plished by flash column chromatography and a com-
plete identification of the stereochemistry was
accomplished by examining the coupling pattern of
the anomeric protons. Typically, for the b-anomer
the anomeric proton appears as a pseudotriplet.5 This
assignment for the b-anomer had been confirmed pre-
viously by X-ray crystallography during the synthesis
of ( )-1.1 The remaining sequence of steps involved:
(1) hydrolysis of the acetate to give compound 7
and (2) oxidation of the free alcohol to form a formyl
4d reacted under standard Vorbruggen conditions with
¨
persilylated N4-benzoylcytosine and trimethylsilyl
triflate (TMSOTf) as a Lewis acid catalyst to give the
expected nucleoside products with a b-5/a-6 ratio of
1.14 (Scheme 2). Although we do not have any direct
evidence to support the distant participation of the
acetate, use of this strategy improved the b/a anomer
ratio significantly relative to the 0.51 ratio obtained in
Me Si
COPh
N
3
N
N
NHBz
N
TBDPSO
AcO
O
O
R O
1
OSiMe
3
O
N
OR
3
+
TBDPSO
AcO
N
N
O
R O
2
b
O
4c, R = TBDPS
NHBz
1
R = H
R = CH
2
3
5
6
a
3
4d, R = TBDPS
1
R = Ac
2
R = CH
3
3
NHBz
NHBz
N
NHBz
N
N
O
d
TBDPSO
OHC
N
TBDPSO
AcO
N
c
TBDPSO
HO
N
O
O
O
O
O
5
7
8
NHBz
N
NH
N
NH
N
2
2
g
e
f
TBDPSO
Cl
N
O
TBDPSO
N
HO
N
O
O
O
O
O
9
10
1
Scheme 2. Reagents and conditions: (a) Ac2O, pyridine; (b) TMSOTf, CH2ClCH2Cl; (c) guanidine, EtOH/CH2Cl2 (9:1); (d) DCC, DMSO,
Cl2CHCO2H; (e) ClCH2P(C6H5)3Cl, n-BuLi, THF, ꢀ78 ꢁC; (f) n-BuLi, THF, ꢀ78 ꢁC; (g) NH4FÆ0.5H2O, MeOH, 80 ꢁC.

M. A. Siddiqui, V. E. Marquez / Bioorg. Med. Chem. 15 (2007) 283–287
285
group. Without isolation, Wittig olefination was per-
formed on intermediate 8 with (chloromethyl)triphe-
nylphosphonium chloride to produce the chlorovinyl
intermediate 9. Because of some persistent contamina-
tion with DCC from the oxidation step, even after
flash column chromatography, the following conver-
sion was performed without further purification. The
chlorovinyl group was converted to the ethynyl group
after treatment with n-butyllithium in tetrahydrofuran
to give the penultimate intermediate 10, obtained in
56% overall yield from 7. Final fluoride deprotection
of the silyl ether afforded the desired, enantiomer
(+)-b-1, which was identical in all respects to the
sample that was resolved chemico-enzymatically.1
J = 12.1 Hz, CHHOBn), 3.58 (d, 1H, J = 12.1 Hz,
CHHOBn), 3.58 (d, 1H, J = 10.5 Hz, CHHOH), 3.50
(d, 1H, J = 10.5 Hz, CHHOH), 2.48–2.67 (m, 2H, H-
3a,b), 2.05 (m, 2H, H-4a,b); FAB MS (m/z, relative inten-
sity) 237.2 (MH+, 22). Anal. Calcd for C13H16O4: C,
66.09; H, 6.83. Found: C, 66.23; H, 6.85.
4.1.2. (R)-5-[(2,2-dimethyl-1,1-diphenyl-1-silaprop-
oxy)methyl]-5-[(phenylmethoxy)-methyl]-3,4,5-trihydrofu-
ran-2-one (3c). A solution of 3b (2.30 g, 9.73 mmol) in
anhydrous CH2Cl2 was treated with imidazole (4.22 g,
61.98 mmol) and tert-butyldiphenylsilyl chloride
(TBDPS-Cl, 5.30 mL, 20.63 mmol). After stirring at
room temperature for 1.5 h, the solvent was removed
under vacuum and the residue was taken up in EtOAc
(200 mL). The organic solution was washed with water
(2· 100 mL), dried (MgSO4), and concentrated under
vacuum. The crude product was purified by flash col-
umn chromatography using a step gradient of hex-
anes:EtOAc (from 10:1 to 4:1) to give 4.51 g (97.6%)
3. Conclusions
In conclusion, we have described an enantioselective
method for the synthesis of the b-D (1) enantiomer of
40-C-ethynyl-20,30-dideoxycytidine starting from a chiral
c-lactone (3a). The synthesis of 1 verifies our earlier
assignment, which was based on the ability of HIV RT
and its M184V mutant to discriminate between the D-
and L-configuration of nucleoside triphosphates.6,7 Our
results provide further support for the use of RT as an
effective enzyme to help assign the absolute configura-
tion of nucleoside 50-triphosphates.
23
1
of 3c as an oil; ½aꢁ +9.28 (c 0.79, CHCl3); H NMR
D
(CDCl3)d 7.19–7.60 (m, 15H, Ph), 4.46 (AB q, 2H,
J = 11.9 Hz, CH2Ph), 3.69 (d,
1 H, J = 10.9 Hz,
CHHOBn), 3.61 (d, 1H, J = 10.9 Hz, CHHOBn), 3.50
(AB q, 2 H, J = 10.3 Hz, CH2OSi), 2.55 (m, 2H, H-
3a,b), 2.11 (irregular t, 2H, H-4a,b), 1.00 (s, 9H,
SiC(CH3)3); FAB MS (m/z, relative intensity) 475.4
(MH+, 20). Anal. Calcd for C29H34O4Si: C, 73.38; H,
7.22. Found: C, 73.38; H, 7.19.
4. Experimental
4.1. General
4.1.3. 5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)meth-
yl]-5-[(phenylmethoxy)methyl]-oxolan-2-ol (4a). A solu-
tion of 3c (3.02 g, 6.36 mmol) in dry toluene (50 mL)
was cooled to ꢀ78 ꢁC and treated slowly with a solution
of diisobutylaluminum hydride (DIBAL, 13.4 mL, 1 M
in hexane). After 1 h stirring the reaction was quenched
with methanol (15 mL) and the resulting mixture was
warmed to room temperature. The solvent was removed
under vacuum and the residue was dissolved in EtOAc
(200 mL) and extracted with water (50 mL). The organic
layer was separated, dried (MgSO4), and concentrated.
The crude product was purified by flash column chro-
matography using a step gradient of hexanes:EtOAc
(from 10:1 to 4:1) to give 2.765 g (91.2%) of 4a as an oily
All chemical reagents were commercially available.
Flash column chromatography was performed on silica
gel 60, 230–240 mesh (E. Merck), and analytical TLC
was performed on Analtech Uniplates silica-gel GF.
1
Routine H NMR spectra were recorded at 400 MHz
on a Varian INOVA instrument. Chemical shifts are
reported in d (ppm) relative to internal tetramethylsi-
lane. Optical rotations were obtained using JASCO
model P-1010 polarimeter. Positive-ion fast-bombard-
ment mass spectra (FABMS) were recorded on a VG
7070E mass spectrometer at an accelerating voltage of
6 kV and a resolution of 2000. Glycerol was used as
the sample matrix and ionization was effected by a beam
of xenon atoms. Elemental analyses were performed by
Atlantic Microlab Inc., Norcross, GA.
1
mixture of anomers; H NMR (CDCl3)d 7.20–7.65 (m,
15H, Ph), 5.35 and 5.43 (m, 1H, CHOH), 4.50 (m, 2H,
CH2Ph), 3.32–3.57 (m, 4H, CH2OBn and CH2OSi),
1.50 (br s, 1 H, OH), 1.63–2.13 (m, 4H, H-3a,b and H-
4a,b), 1.00–1.06 (s, 9H, SiC(CH3)3); FAB MS (m/z, rela-
tive intensity) 469.2 (MH+–H2O, 10). Anal. Calcd for
C29H36O4Si: C, 73.07; H, 7.61. Found: C, 73.31; H, 7.67.
4.1.1. (R)-5-(hydroxymethyl)-5-[(phenylmethoxy)methyl]-
3,4,5-trihydrofuran-2-one (3b). A solution of 3a (5.26 g,
10.90 mmol) in anhydrous CH2Cl2 (50 mL) was treated
with an equal volume of 50% trifluoroacetic acid
(TFA) in CH2Cl2 and stirred at room temperature for
1 h. After the addition of CH2Cl2 (100 mL), the solu-
tion was washed with water (2· 100 mL), dried
(MgSO4), and concentrated under vacuum. The crude
product was purified by flash column chromatography
4.1.4. 5-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)meth-
yl]-5-[(phenylmethoxy)methyl]-oxolan-2-yl acetate (4b).
A solution of 4a (2.76 g, 5.79 mmol) in anhydrous
pyridine (50 mL) was treated with acetic anhydride
(4 mL, 42.31 mmol) and stirred at room temperature
for 24 h. The solvent was removed under vacuum and
the crude product was purified by flash column
chromatography using EtOAc:hexanes:Et3N (40:10:1)
to give 2.94 g (98%) of 4b as an oily mixture of anomers;
1H NMR (CDCl3)d 7.15–7.65 (m, 15H, Ph), 6.21 and
6.23 (doublet, 1 H, J = 4.68 and 4.69 Hz, CHOAc),
using a step gradient of hexanes/EtOAc (from 4:1 to
1:2) to give 2.31 g (88.9%) of 3b as a foam; ½aꢁ
23
D
1
+10.08 (c 0.74, CHCl3); H NMR (CDCl3)d 7.20–7.35
(m, 5H, Ph), 4.50 (s, 2H, CH2Ph), 3.70 (d, 1H,

286
M. A. Siddiqui, V. E. Marquez / Bioorg. Med. Chem. 15 (2007) 283–287
4.40–4.60 (m, 2 H, CH2Ph), 3.400–3.70 (m, 4H,
CH2OBn and CH2OSi), 1.80–2.20 (m, 4H, H-3a,b and
H-4a,b), 2.00 and 1.92 (s, 3H, OCOCH3), 1.00–1.06 (s,
9H, SiC(CH3)3); FAB MS (m/z, relative intensity)
459.2 (MH+–CH3CO2H, 12) and FAB MS (m/z, relative
intensity) 557.2 (M+K+, 30). Anal. Calcd for
C31H38O5Si: C, 71.78; H, 7.38. Found: C, 71.86; H, 7.37.
was cooled to 0 ꢁC and treated with a solution of 4d
(0.703 g, 1.58 mmol) in anhydrous 1,2-dichloroethane
(30 mL). Trimethylsilyl triflate (0.55 mL, 3.01 mmol)
was added, the ice bath was removed, and the solu-
tion was stirred for 2.5 h. The reaction was quenched
by the addition of an aqueous saturated solution of
NaHCO3 (10 mL) and after adding CH2Cl2 (50 mL)
the product was extracted into the organic phase.
The organic solution was dried (MgSO4) and evapo-
rated to dryness under vacuum. The crude product
obtained was purified by flash column chromatogra-
phy using a step gradient of hexanes/EtOAc (2:1 to
1:4) to give the less polar a-isomer 6 (0.464 g,
46.4%) followed by the desired, more polar b-isomer
5 (0.525 g, 53%).
4.1.5. {2-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)me-
thyl]-5-methoxyoxolan-2-yl}methan-1-ol (4c). A solution
of 4b (2.94 g, 5.66 mmol) in 4.4% HCO2H in methanol
(100 mL) maintained under a blanket of argon was
treated with palladium black (0.75 g) and stirred. After
1 h, an equal amount of catalyst was added and stirred
for 1.5 h. The mixture was filtered through a pad of Cel-
iteꢂ and the pad was washed with MeOH (50 mL). The
filtrate was evaporated to dryness under vacuum and the
residue was purified by flash column chromatography
using a step gradient of hexanes/EtOAc (8:1 to 2:1) to
give 0.809 g (92.9%) of 4c as a thick oil containing a mix-
ture of anomers; 1H NMR (CDCl3)d 7.25–7.65 (m, 10H,
Ph), 4.84 and 4.97 (m, 1H, J = 4.68 and 4.69 Hz,
CHOCH3), 3.40–3.74 (m, 4H, CH2OH and CH2OSi),
3.14 and 3.30 (s, 3H, OCH3), 1.75–2.00 (m, 4H, H-3a,b
and H-4a,b), 1.00–1.06 (s, 9H, SiC(CH3)3); FAB MS
(m/z, relative intensity) 369.1 (MH+–CH3OH, 40) and
FAB MS (m/z, relative intensity) 439.1 (M+K+, 55).
Anal. Calcd for C23H32O4SiÆ0.25H2O: C, 68.19; H,
8.09. Found: C, 68.08; H, 8.10.
23
D
1
Compound 5: ½aꢁ +35.93 (c 0.54, CHCl3); H NMR
(CDCl3)d 8.12 (d, 1H, J = 7.8 Hz, H-6), 7.30–7.90 (m,
16H, H-5, Ph), 7.25 (br s, 1H, NH), 6.10 (irregular t,
1H, J ꢂ 5.3 Hz, H-10), 4.04 (AB s, 2H, CH2OAc),
3.73 (AB q, 2H, J = 10.2 H, CH2OSi), 2.68 (m, 1H,
H ꢀ 30a), 1.80–2.10 (m, 3H, H ꢀ 30b, H ꢀ 20a;b), 1.98 (s,
3H, OCOCH3), 1.00 (s, 9H, SiC(CH3)3); FAB MS
(m/z, relative intensity) 626.4 (MH+, 12). Anal. Calcd
for C35H39N3O6SiÆ0.25H2O: C, 66.69; H, 6.32; N, 6.67.
Found: C, 66.71; H, 6.41; N, 6.52.
1
Compound 6: H NMR (CDCl3)d 8.70 (br s, 1H, NH),
8.20 (d, 1H, J = 7.4 Hz, H-6), 7.30–7.90 (m, 16H, H-5,
Ph), 5.92 (irregular dd, 1H, J ꢂ 6.2, 4.0 Hz, H-10), 4.44
(d, 1H, J = 12.1 Hz, CHHOAc), 4.22 (d, 1H,
J = 12.1 Hz, CHHOAc), 3.54 (AB q, 2H, J = 10.3 Hz,
4.1.6.
{2-[(2,2-Dimethyl-1,1-diphenyl-1-silapropoxy)
methyl]-5-methoxyoxolan-2-yl}methyl acetate (4d). A
solution of 4c (0.785 g, 1.95 mmol) in anhydrous pyri-
dine (25 mL) was treated with acetic anhydride
(0.58 mL, 6.13 mmol) and stirred at room temperature
for 24 h. The solvent was removed under vacuum and
the crude product was purified by flash column chroma-
tography using a step gradient of hexanes/EtOAc (15:1
to 8:1) to give 0.842 g (97.1%) of 4d as a thick oil
containing a 1:1 mixture of anomers; 1H NMR
(CDCl3)d 7.30–7.65 (m, 10H, Ph), 4.98 (br d, 0.5H,
J = 3.5 Hz, CHOCH3), 4.92 (m, 0.5H, CHOCH3), 4.26
(d, 0.5H, J = 11.1 Hz, CHHOAc), 4.15 (q, 1H,
J = 11.1 Hz, CH2OAc), 4.85 (d, 0.5 H, J = 11.1 Hz,
CHHOAc), 3.73 (d, 0.5H, J = 9.9 Hz, CHHOSi), 3.61
(q, 1H, J = 9.9 Hz, CH2OSi), 3.42 (d, 0.5H, J = 9.9 Hz,
CHHOSi), 3.25 and 3.18 (s, 3H, OCH3), 2.00 and 2.01
(s, 3H OCOCH3), 1.76–2.05 (m, 4H, H-2a,b and
H-3a,b), 1.00–1.06 (s, 9H, SiC(CH3)3); FAB MS (m/z,
relative intensity) 411.2 (MH+–CH3OH, 40) and FAB
MS (m/z, relative intensity) 481.3 (M+K+, 100). Anal.
Calcd for C25H34O5Si: C, 67.84; H, 7.74. Found: C,
67.87; H, 7.84.
CH2OSi), 2.55 (m, 1H, H ꢀ 30 ), 2.00 (m, 2H, H ꢀ 2a0 ;b),
a
1.80 (m, 1H, H ꢀ 30 ), 2.04 (s, 3H, OCOCH3), 1.00 (s,
b
9H, SiC(CH3)3); FAB MS (m/z, relative intensity)
626.4 (MH+, 9).
4.1.8. (2R,5R) ꢀ N-(1-{5-[(2,2-dimethyl-1,1-diphenyl-1-
silapropoxy)methyl]-5-(hydroxymethyl)oxolan-2-yl}-
2-oxohydropyrimidin-4-yl)benzamide (7). A solution of
nucleoside 5 (0.707 g, 1.11 mmol) in anhydrous CH2Cl2
(3 mL) at 0 ꢁC was treated with a solution of guanidine
in ethanol (27 mL) and allowed to reach room tempera-
ture after 30 min [The solution of guanidine was pre-
pared
from
guanidine
hydrochloride
(0.70 g,
7.32 mmol) dissolved in ethanol (7 mL) and treated with
sodium ethoxide (3.5 mL of 21 wt% in ethanol,
10.7 mmol) at room temperature for 20 min. The precip-
itate was removed and the filtrate containing free guani-
dine was used immediately]. After the 30 min treatment
with guanidine, the solvent was removed under vacuum
and the crude residue was purified by flash column
chromatography using a step gradient of hexanes/
EtOAc (1:1 to 100% EtOAc) followed by
4.1.7. (2R,5R)-{2-[(2,2-dimethyl-1,1-diphenyl-1- silaprop-
oxy)methyl]-5-[2-oxo-4-(phenylcarbonylamino)hydropyri-
midinyl]oxolan-2-yl}methyl acetate (5). A suspension of
N4-benzoylcytosine (0.70 g, 3.25 mmol) in anhydrous
CH3CN (10 mL) was treated with bis(trimethylsilyl)tri-
fluoroacetamide (BSTFA, 10 mL). After 30 min the
reaction became homogeneous, the solvent was
removed under vacuum, and the residual oil dissolved
in anhydrous 1,2-dichloroethane (30 mL). This solution
CH2Cl2:MeOH (20:1) to give 0.577 g (87.5%) of 7 as a
23
D
white foam; ½aꢁ +45.00 (c 0.86, CHCl3); 1H NMR
(CDCl3)d 8.13 (d, 1H, J = 7.4 Hz, H-6), 7.30–7.90 (m,
16H, H-5, Ph), 7.25 (br s, 1H, NH), 6.13 (irregular t,
1H, J ꢂ 5.5 Hz, H-10), 3.76 (AB q, 2H, J = 10.9 Hz,
CH2OSi), 3.53 (AB s, 2H, CH2OH), 2.66 (m, 1H,
H ꢀ 40a), 1.86–2.40 (m, 3H, H ꢀ 40b, H ꢀ 30a;b), 1.00 (s,
9H, SiC(CH3)3); FAB MS (m/z, relative intensity)
584.1 (MH+, 55). Anal. Calcd for C33H37N3O5SiÆH2O:

M. A. Siddiqui, V. E. Marquez / Bioorg. Med. Chem. 15 (2007) 283–287
287
C, 65.86; H, 6.53; N, 6.98. Found: C, 65.98; H, 6.39; N,
6.75.
2.40 (m, 1H, H ꢀ 40b), 2.38 (s, 1H, CCH), 1.90–2.60
(m, 2H, H ꢀ 30a;b), 1.00 (s, 9H, SiC(CH3)3); FAB MS
(m/z, relative intensity) 474.1 (MH+, 14). HRMS
(FAB) calcd for C27H31N3O3Si (MH+, m/z) 474.2213.
Found 474.2232.
4.1.9. (2R,5R)-4-amino-1-{5-[(2,2,-dimethyl-1,1- diphen-
yl-1-silapropoxy)methyl]-5-ethynyloxolan-2-yl}-1-hydro-
pyrimidin-2- one (10). A stirred solution of 7 (1.39 g,
2.38 mmol) and dicyclohexylcarbodiimide (DCC,
1.48 g, 7.17 mmol) in anhydrous DMSO (50 mL) was
cooled to 0 ꢁC and treated with dichloroacetic acid
(0.10 mL, 1.21 mmol). The ice bath was removed and
stirring continued for 2 h. The suspension was filtered
and the solid washed with EtOAc (50 mL). The filtrate
was extracted with water (2· 50 mL) and the organic
phase was dried (MgSO4) and concentrated under
vacuum. The crude product was purified by flash
column chromatography using a step gradient of
hexanes/EtOAc (1:1 to 100% EtOAc) to give 1.30 g
(94.2%) of 8 as a foam. Because this product showed
some contamination with DCC it was used directly in
the next step.
4.1.10. (2R,5R)-(4-amino-1-[5-ethynyl-5-(hydroxymeth-
yl)oxolan-2-yl]-1-hydropyrimidin-2-one [(+)-1-(20,30-dide-
oxy-4-C-ethynyl-ribo-pentofuranosyl)cytosine](+)-1.
Ammonium fluoride (2.39 g, 51.9 mmol) was added to
a stirred solution of 10 (0.824 g, 1.73 mmol) in metha-
nol (60 mL) and heated to reflux for 1.5 h. The solution
was allowed to reach room temperature, concentrated
under vacuum to a reduced volume, and directly load-
ed onto a silica-gel column to be flashed chromato-
graphed using a step gradient of CH2Cl2/MeOH (20:1
to 2:1). The collected fractions containing the product
were combined and rechromatographed using a re-
verse-phase C-18 column eluted with water, followed
by a step gradient of H2O/MeOH (10:1 to 5:1). The de-
sired fractions were collected, combined, and lyophi-
lized to give 0.339 g (82.9%) of 1 as a white foam,
identical in all respects to the previously made chiral
A suspension of (chloromethyl)trimethylphosphonium
chloride (2.10 g, 6.04 mmol) in dry THF (20 mL) was
cooled to ꢀ78 ꢁC and treated with n-BuLi (1.6 M in hex-
anes, 3.8 mL). After stirring for 1 h, a solution of alde-
hyde 8 in THF (50 mL) was added. The dry ice/
acetone cooling bath was replaced with a wet-ice bath
(0 ꢁC) and stirred for 3 h. The reaction was quenched
with a saturated aqueous solution of NH4Cl (5 mL)
and the resulting mixture was extracted with EtOAc
(2· 50 mL). The combined organic extract was washed
with water (50 mL), dried (MgSO4), and concentrated
under vacuum. The crude product was purified by flash
column chromatography using a step gradient of
EtOAc/hexanes (1:1 to 4:1) to give 1.85 g of 9 still
contaminated with DCC. This material was also used
directly in the following step.
material obtained by the lipase-catalyzed resolution;
23
D
½aꢁ +45.84 (c 0.59, H2O).
Acknowledgments
The authors thank Drs. James A. Kelley and Christo-
pher Lai of the Laboratory of Medicinal Chemistry,
NCI-Frederic for mass spectral analyses. This research
was supported in part by the Intramural Research Pro-
gram of the NIH, Center for Cancer Research, NCI-
Frederick.
References and notes
A solution of 9 in dry THF (20 mL) at ꢀ78 ꢁC was kept
under a blanket of argon and treated with n-BuLi (1.6 M
in hexanes, 30 mL). The solution was stirred under ar-
gon for 2 h and the reaction was quenched with a satu-
rated aqueous solution of NH4Cl (20 mL). The solution
was then allowed to reach room temperature and
extracted with EtOAc (2· 100 mL). The combined
organic extract was washed with water (75 mL), dried
(MgSO4), and concentrated under vacuum. The crude
product was purified by flash column chromatography
using a step gradient of EtOAc/hexanes (2:1 to 100%
1. Siddiqui, M. A.; Hughes, S. H.; Boyer, P. L.; Mitsuya, H.;
Van, Q. N.; George, C.; Sarafianos, S.; Marquez, V. E.
J. Med. Chem. 2004, 47, 5041.
2. Kang, J.-H.; Siddiqui, M. A.; Sigano, D. M.; Krajewski,
K.; Lewin, N. E.; Pu, Y.; Blumberg, P. M.; Lee, J.;
Marquez, V. E. Org. Lett. 2004, 6, 2413.
3. McDonald, F.; Gleason, M. M. J. Am. Chem. Soc. 1996,
118, 6648.
4. Choi, W. J.; Ahn, H. S.; Kim, H. O.; Kim, S.; Chun, M. W.;
Jeong, L. S. Tetrahedron Lett. 2002, 43, 6241.
5. Townsend, L. B. In Synthetic Procedures in Nucleic Acid
Chemistry; Zorbach, W. M., Tipson, R., Eds.; Wiley:
London, 1970; p 299.
6. Sarafianos, S. G.; Das, K.; Clark, A. D.; Ding, J. P.; Boyer,
P. L.; Hughes, S. H.; Arnold, E. Proc. Natl. Acad. Sci. USA
1999, 96, 10027.
EtOAc) and CH2Cl2/MeOH (20:1 to 10:1) to give
23
0.595 g of 10 in 52.6% after three steps; ½aꢁ +31.58 (c
D
0.52, CHCl3); 1H NMR (CDCl3)d 7.95 (d, 1H,
J = 7.4 Hz, H-6), 7.30–7.62 (m, 10H, Ph), 6.20 (irregular
dd, 1H, J ꢂ 6.9, 2.9 Hz, H-10), 5.20 (d, 1H, J = 7.4 Hz,
H-5), 4.30 (d, 1H, J = 11.3 Hz, CHHOSi), 3.75 (d, 1H,
J = 11.3 Hz, CHHOSi), 2.72 (m, 1H, H ꢀ 40a), 2.30–
7. Gao, H. Q.; Boyer, P. L.; Sarafianos, S. G.; Arnold, E.;
Hughes, S. H. J. Mol. Biol. 2000, 300, 403.