Carbocyclic analogues of the potent cytidine deaminase inhibitor 1-(β- D-ribofuranosyl)-1,2-dihydropyrimidin-2-one (zebularine)

Article abstract of DOI:10.1021/jm980111x

Three carbocylic analogues of the potent cytidine deaminase inhibitor (CDA) zebularine [1-(β-D-ribofuranosyl)-l,2-dihydropyrimidin-2-one, 1a] were synthesized. The selected pseudosugar templates correspond, respectively, to the cyclopentenyl moiety of nep

Full text of DOI:10.1021/jm980111x

2572
J . Med. Chem. 1998, 41, 2572-2578
Ca r bocyclic An a logu es of th e P oten t Cytid in e Dea m in a se In h ibitor
1-(â-D-Ribofu r a n osyl)-1,2-d ih yd r op yr im id in -2-on e (Zebu la r in e)
Lak Shin J eong,†,‡ Greg Buenger,†,§ J ohn J . McCormack,| David A. Cooney,† Zhang Hao,†, and
Victor E. Marquez*,†
Laboratory of Medicinal Chemistry, Division of Basic Sciences, National Cancer Institute, National Institutes of Health,
Bethesda, Maryland 20892, and Department of Pharmacology, College of Medicine, and Vermont Regional Cancer Center,
University of Vermont, Burlington, Vermont 05401
Received February 18, 1998
Three carbocylic analogues of the potent cytidine deaminase inhibitor (CDA) zebularine [1-(â-
D-ribofuranosyl)-1,2-dihydropyrimidin-2-one, 1a ] were synthesized. The selected pseudosugar
templates correspond, respectively, to the cyclopentenyl moiety of neplanocin A (compound 4),
the cyclopentyl moiety of aristeromycin (compound 5), and a newly designed, rigid bicyclo-
[3.1.0]hexane moiety (compound 6). These three carba-nucleoside versions of zebularine were
fashioned to overcome the inherent instability of the parent drug. Each target compound was
approached differently using either convergent or linear approaches. The immediate precursor
to the cyclopentenyl analogue 4 was obtained by a Mitsunobu coupling of pseudosugar 7 with
2-hydroxypyrimidine. The cyclopentyl analogue 5 was linearly constructed from carbocyclic
amine 17, and the final target 6 was similarly constructed from the carbobicyclic amine 27.
Of the three target compounds, only 5 showed a significant level of inhibition against human
CDA, but it was 16 times less potent than zebularine (Ki ) 38 µM vs Ki(apparent) ) 2.3 µM).
Although these carbocyclic analogues appeared to be more stable than zebularine, replacement
of the electronegative CO4′ oxygen for the less electronegative carbon in 4-6 presumably
reduces the capacity of the pyrimidin-2(1H)-one ring to form a covalent hydrate, a step
considered crucial for the compound to function as a transition-state inhibitor of the enzyme.
In tr od u ction
This result underscores the importance of the double
bond in increasing the acidity of the OH proton in
covalent hydrates 2a and 2b, both of which function as
potent and tight-binding transition-state inhibitors of
CDA.5
Cytidine deaminase (CDA) is a widely distributed
enzyme that catalyzes the hydrolytic deamination of
cytosine nucleosides to the corresponding uracil nucleo-
sides.1,2 Clinically, the deamination of antileukemic
nucleosides, such as cytosine arabinoside (ara-C) and
5-aza-2′-deoxycytidine (5-aza-CdR), results in loss of
antitumor activity. Therefore, a strategy to prevent the
inactivation of these drugs is to use them concurrently
with CDA inhibitors.2 Zebularine (1a )3 and 5-fluoro-
zebularine (1b)3 are potent inhibitors of CDA which
have been proposed as candidates for use in combination
chemotherapy with ara-C or 5-aza-CdR.4
The crystal structures of the E. coli CDA complexed
with zebularine (1a ) and 5-fluorozebularine (1b) showed
that these inhibitors bind at the active site as covalent
hydrates at C4.5 For zebularine, the Ki value of the
single inhibitory diastereoisomer of 2a was estimated
to be 1.2 × 10-12 M when the equilibrium constant for
the hydration of 1a to 2a was taken into account.6 This
value is roughly 8 orders of magnitude lower than the
Km value for cytidine.6 By contrast, the fully reduced
transition-state analogue tetrahydrouridine (3, THU) is
a substantially weaker ligand compared to the hydrated
forms of zebularine (2a ) and 5-fluorozebularine (2b).6,7
Unfortunately, the zebularines, and even THU, have
some disadvantages in terms of stability. THU isomer-
izes in neutral (slow) and acidic (fast) aqueous solutions
to a pyranoside form devoid of inhibitory activity.8
Zebularine and 5-fluorozebularine, on the other hand,
are extremely sensitive to basic conditions and they too
follow a complex decomposition pathway that destroys
biological activity.9
† National Institutes of Health.
‡ Current address: College of Pharmacy, Ewha Women’s University,
Seoul, Korea.
§ Current address: Flint AG, Bubendorf, Switzerland.
| University of Vermont.
Carbocyclic nucleosides (carba-nucleosides) are gener-
ally more stable than conventional nucleosides to acidic
Current address: Food and Drug Administration, Rockville, MD,
USA.
S0022-2623(98)00111-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/16/1998

Carbocyclic Analogues of Zebularine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2573
Sch em e 1
Sch em e 2
and basic conditions by virtue of a stable C-N bond.10,11
Therefore, three carbocyclic pseudosugar templates
present in some carbocyclic nucleosides were chosen to
construct stable analogues of zebularine for evaluation
as inhibitors of CDA. The cyclopentenyl moiety of
neplanocin A,12 the simpler cyclopentyl moiety of aris-
teromycin,13 and the recently developed rigid bicyclo-
[3.1.0]hexane template14 were selected to build three
distinct carba-nucleoside versions (4-6) of zebularine.
Syn th esis
The first target compound which contained the cyclo-
pentenyl moiety of neplanocin A (cyclopentenyl zebul-
arine, 4) was synthesized as a racemate from the readily
accessible carba-sugar precursor (()-3-[(benzyloxy)-
methyl]-4,5-O-isopropylidene-2-cyclopenten-1-ol (7).15 Mit-
sunobu coupling16 of 7 with 2-hydroxypyrimidine af-
forded the desired N-alkylated product 8 in a modest
yield (Scheme 1). Removal of the isopropylidene group
with trifluoroacetic acid produced diol 9, which was then
converted to the diacetate 10, prior to the removal of
the benzyl ether group. Treatment of 10 with BCl3,
followed by acetate ammonolysis, gave the target race-
mic CPE-Z (rac-4). The lack of CDA inhibitory activity
of racemic CPE-Z (vide infra) did not warrant synthesis
of the single enantiomer corresponding to the natural
configuration of neplanocin A.
In principle, the cyclopentyl zebularine target 5 is just
one reduction step away from 4. However, regiospecific
reduction of this double bond is not easily achievable,17
plus the 2-oxopyrimidine ring is readily reduced under
catalytic hydrogenation conditions.8 Thus, we decided
to prepare the chiral zebularine analogue 5 by con-
structing the pyrimidin-2(1H)-one ring from carbocyclic
uridine via the corresponding 4-hydrazino intermediate
(Scheme 2). This approach was developed earlier by
Cech and Holy for the syntheses of pyrimidin-2(1H)-one
nucleosides from uridine precursors.18 The carbocyclic
component of 5 was synthesized from the known (-)-
2,3-(cyclohexylidenedioxy)-4-cyclopentenone (11), a use-
ful chiral building block developed by Borchardt et al.19
for the synthesis of carbocyclic nucleosides. From 11,
the requisite hydroxymethyl arm was introduced photo-
chemically from the less hindered face of the molecule
to give 12 as a single diastereoisomer. The resulting
free hydroxyl group was then protected as benzoate
ester to give 13. Sodium borohydride reduction of 13,
again occurring from the less hindered side, produced
a single alcohol product (14). NMR spectral analyses
of these compounds, which differ from those reported
by Borchardt et al. only in terms of the nature of the
primary alcohol protecting group (i.e., benzoyl vs tert-
butyl), were consistent with their data and confirmed
the proposed structures.19c Alcohol 14 was converted
to its methanesulfonate ester (mesylate 15), which was
subsequently displaced with NaN3 to give the carbocy-
clic azide 16. Reduction of the azido group to amine 17
enabled the construction of the uracil ring after con-
densation with 3-ethoxypropenoyl isocyanate, followed
by base-catalyzed cyclization of the intermediate acry-
olyl urea to give compound 18. Our approach to the
4-hydrazino intermediate 20, which is required for the
conversion of the uracil moiety to the pyrimidin-2(1H)-
one ring, involved a slight variation from the original
method of Cech and Holy.18 In their approach, the
leaving group X in structure 19 (Scheme 2) was a
methylthio group. Instead, we decided to activate the
C4-position as the 2,4,6-triisopropylbenzenesulfonate
ester (compound 19), which upon treatment with hy-
drazine afforded the 4-hydrazino intermediate 20. Cleav-
age of the hydrazino group was accomplished with silver
oxide in dioxane to give the antepenultimate intermedi-
ate 21, accompanied by some uridine byproduct 18.

2574 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
J eong et al.
Sch em e 3
the corresponding O-alkylated products (data not shown).
For that reason, we were forced to use the longer linear
approaches for the construction of the pyrimidin-2(1H)-
one ring of targets 5 and 6 (Schemes 2 and 3). Chro-
nologically, we started with the method of Cech and
Holy,18 which, while successful, was found to be lengthy
and difficult. The alternate method involving the acid-
catalyzed condensation of an N-substituted urea (i.e.,
28) with 1,1′,3,3′-tetraethoxypropane proved to be a
more effective approach.20,21
Biologica l Resu lts a n d Discu ssion
Compounds 4 and 6 showed no inhibition of mouse
or human CDA at concentrations greater than 100 µM.
Compound 5, however, did show inhibitory activity
against human CDA, although it was 16-fold less potent
than that of zebularine (Ki ) 38 µM versus Ki(apparent)
)
2.3 µM). Although all the target compounds selected
(4-6) are formal nucleoside analogues of zebularine by
virtue of having a common pyrimidin-2(1H)-one ring,
they failed to mimic zebularine in inhibiting CDA. For
compound 4, it can be argued that the flat cyclopentene
ring does not allow the primary alcohol group, equiva-
lent to the 5′-OH of ribose, to form an effective hydrogen
bond with the enzyme.5 Similarly, one can hypothesize
that for 5 and 6 deviations from the ideal ribose
conformation found in the zebularine-CDA complex5
might be responsible for the lower potency of the former
and inactivity of the latter. However, the most impor-
tant lesson from this exercise is that just nucleoside
mimicry is not enough, particularly when the enzymatic
reaction involves a chemical transformation of the base,
such as hydration. The replacement of an electronega-
tive CO4′ oxygen for the less electronegative carbon
undoubtedly resulted in a diminished inductive effect
which changed the electronic environment of the pyri-
midine ring and reduced its capacity to form a covalent
hydrate. The transmision of information between the
base and the sugar moiety has been well documented
for the naturally occurring nucleosides, where the
energetics for the protonation-deprotonation equilib-
rium are transmitted through the anomeric effect to
drive the conformational North-South equilibrium of
the sugar moieties.23 In a case closer to the zebularines,
which further supports the existence of a “cross-talk”
between the ribose moiety and the heterocyclic base, we
reported earlier that in sharp contrast with the known
instability of 5-azacytidine in aqueous solutionswhich
is rapidly converted to N-(formylamidino)-N′-â-D-ribo-
furanosyl urea and 1-â-D-ribofuranosyl-3-guanylurea
after the formation of a covalent hydratesthe corre-
sponding carbocyclic analogue was stable.24 Therefore,
while we have succeeded in synthesizing more stable
analogues of zebularine (4-6), the corresponding bio-
logical activity was either reduced or lost because the
capacity of zebularine to function as an effective CDA
inhibitor is strictly associated with its ability to form a
covalent hydrate of its pyrimidin-2(1H)-one aglycon. It
is this requirement that is also responsible for the
chemical transformation that initiates decomposition of
the drug.
Tandem base and acid hydrolysis on 21 afforded the
optically active target, cyclopentyl zebularine analogue
(-)- 5.
Since cyclopentyl zebularine [(-)-5] showed inhibitory
activity against CDA (vide infra), we decided to syn-
thesize the third target 6 also in optically pure form
(Scheme 3). In agreement with previous reports, opti-
cally active cyclopentenol 23 underwent stereoselectve
Simmons-Smith cyclopropanation to give alcohol 24 in
excellent yield.14f At this stage, the synthetic approach
followed was similar to the one used for the synthesis
of (-)-5: (a) conversion of the secondary alcohol to the
methanesulfonate ester (mesylate 25), (b) nucleophilic
displacement with NaN3 to give the carbobicyclic azide
26, and (c) reduction of the azido group to the carbobi-
cyclic amine 27. However, for the completion of the
pyrimidin-2(1H)-one ring, we decided to follow a simpler
method than the one of Cech and Holy.18 This alterna-
tive method involved the condensation of a â-dialdehyde,
or its equivalent synthon, with the corresponding car-
bobicyclic ureido nucleoside. This approach has been
used successfully in the synthesis of pyrimidine-2(1H)-
thione and 1-methylpyrimidin-2(1H)-one.20,21 Thus,
reaction of amine 27 with trimethylsilylisocyanate af-
forded cleanly, and in quantitative yield, the ureido-
bicyclo[3.1.0]hexane intermediate 28. This intermediate
was cyclized in the presence of the complementary
3-carbon fragment, 1,1′,3,3′-tetraethoxypropane, to give
the desired pyrimidin-2(1H)-one carba-nucleoside, which
was converted to the corresponding diacetate 29 to aid
in the isolation and purification. Although the overall
conversion of 28 to 29 was only 40%, this approach to
the pyrimidin-2(1H)-one ring was more efficient when
compared to the previous method. Removal of the
O-benzyl ether with BCl3 and base-catalyzed hydrolysis
of the acetate groups afforded the desired optically
active target (+)-6.
The three different strategies used for the syntheses
of these compounds deserve comment. It is known that
Mitsunobu reactions with ambident anions, such as
those from 2-oxopyridines22 and 2-oxopyrimidines,14a,c
give mixtures of N- and O-alkylated products. With the
carbocyclic allylic alcohol 7, the reaction gave predomi-
nantly the desired N-alkylated product 8 (the direct
precursor of target 4, Scheme 1). However, when either
14 or 24 were used, the reactions afforded exclusively
Exp er im en ta l Section
All chemical reagents were commercially available. Melting
points were determined on a Fisher-J ohns melting point
apparatus and are uncorrected. Column chromatography was

Carbocyclic Analogues of Zebularine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2575
performed on silica gel 60, 230-400 mesh (E. Merk), and
analytical TLC was performed on Analtech Uniplates silica
gel GF. Infrared spectra were recorded on a Perkin-Elmer
1600 Series FTIR. Proton and 13C NMR spectra were recorded
74.1, 104.4, 127.7, 128.3, 129.2, 138.2, 142.8, 147.5, 155.5,
166.3, 169.6, 170.0. Anal. (C21H22N2O6) C, H, N.
r el-(1R,4R,5S)-1-[3-(Hyd r oxym eth yl)-4,5-d ih yd r oxy-2-
cyclop en ten -1-yl]p yr im id in -2(1H)-on e (4). A stirred solu-
tion of 10 (0.147 g, 0.37 mmol) in CH2Cl2 (-78 °C) was treated
with 1.85 mL of BCl3 (1 M in CH2Cl2) for 2 h at -78 °C.
Methanol (25 mL) was added and the reaction mixture was
stirred for 40 min while allowing it to reach room temperature.
The solvent was evaporated and the residue was purified by
silica gel column chromatography with 5-25% methanol in
CH2Cl2 to give 0.069 g (59%) of crude diacetate. This com-
pound was immediately dissolved in saturated methanolic
ammonia (25 mL) and stirred at 0 °C in a sealed vessel for 3
h. The solvent was evaporated to give a residue which was
purified by silica gel column chromatography to give 0.051 g
(100%) of 4: mp 183-184 °C (EtOH-H2O); 1H NMR (Me2SO-
d6) δ 3.95 (m, 1 H, H-5′), 4.09 (m, 2 H, CH2OH), 4.37 (m, 1 H,
H-4′), 4.89 (m, 2 H, OH), 5.08 (d, 1 H, J ) 6.0 Hz, OH), 5.44
(br s, 1 H, H-1′), 5.54 (m, 1 H, H-2′), 6.42 (dd, 1 H, J ) 6.6, 4.1
Hz, H-5), 7.87 (dd, 1 H, J ) 6.6, 2.8 Hz, H-4), 8.51 (dd, 1 H, J
) 4.0, 2.8 Hz, H-6); 1H NMR (D2O) δ 3.58 (d, 1 H, J ) 5.6 Hz,
H-5′), 3.67 (s, 2 H, CH2OH), 4.05 (d, 1 H, J ) 5.6 Hz, H-4′),
4.92 (br s, H-1′), 5.19 (s, 1 H, H-2′), 6.05 (m, 1 H, H-5), 7.35
(m, 1 H, H-4), 7.94 (m, 1 H, H-6); 13C NMR (Me2SO-d6) δ 58.6,
68.1, 72.6, 76.8, 104.1, 122.9, 146.2, 152.1, 155.9, 165.5. Anal.
(C10H12N2O4) C, H, N.
(2R ,3R ,4R )-4-H yd r oxym e t h yl-2,3-(cycloh e xylid e n e -
d ioxy)-1-cyclop en ta n on e (12). A solution of (-)-4,5-(cyclo-
hexylidenedioxy)-2-cyclopentenone (11, 0.38 g, 1.96 mmol)19
and benzophenone (0.14 g, 0.78 mmol) in MeOH (50 mL) was
purged with argon for 60 min and then placed in a Rayonet
Reactor RMR-400 equipped with one 350 nm lamp. The
reaction mixture was photolyzed for 2 h. The mixture was
concentrated under reduced pressure and the residue was
purified by silica gel column chromatography using hexane:
EtOAc (1:1) as eluant to give 12 (0.22 g, 50%) as a colorless
syrup: 1H NMR (CDCl3) δ 1.30-1.70 (m, 10 H, cyclohexyl),
2.15 (d, 1 H, J ) 18.1 Hz, H-5a), 2.55 (m, 1 H, H-4), 2.75 (dd,
1 H, J ) 18.1, 9.2 Hz, H-5b), 3.69 (dd, 1 H, J ) 10.0, 3.7 Hz,
CHHOH), 3.85 (dd, 1 H, J ) 10.0, 3.2 Hz, CHHOH), 4.27 (d,
1 H, J ) 5.4 Hz, H-3), 4.68 (d, 1 H, J ) 5.4 Hz, H-2). Anal.
(C12H18O4) C, H.
on
a Bruker AC-250 instrument at 250 and 62.9 MHz,
respectively. Spectra were referenced to the solvent in which
they were run (7.24 ppm for CDCl3). Following the norm for
reporting NMR data in nucleosides, the identity of protons and
carbons on the pseudosugar ring (carbocyclic moiety) are
indicated by numbers with primes. Specific rotations were
measured in a Perkin-Elmer Model 241 polarimeter. Positive-
ion fast-atom bombardment mass spectra (FABMS) were
obtained 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.
r el -(1R ,4R ,5S )-1-[3-(B e n zy lo x y )m e t h y l]-4,5-O -is o -
p r op ylid en e-2-cyclop en ten -1-yl]p yr im id in -2(1H)-on e (8).
A solution of 715 (6.41 g, 23.2 mmol), triphenylphosphine (6.08
g, 23.2 mmol), and 2-hydroxypyrimidine (2.23 g, 23.2 mmol)
in DMF (50 mL) was treated dropwise with diethyl azodicar-
boxylate (DEAD, 3.65 mL, 23.2 mmol). The resulting solution
was stirred for 10 days at room temperature. The solvent was
evaporated and the residue partitioned between water and
EtOAc. The organic layer was washed with brine, dried
(Na2SO4), and evaporated to dryness. The oily residue re-
vealed two major bands on TLC (silica gel, 5% MeOH in
CHCl3). The lower band (Rf ) 0.27) corresponded to the
desired product. The entire batch was purified by column
chromatography with silica gel using, sequentially, 40% EtOAc
in hexanes, CHCl3, and 5% MeOH in CHCl3 as eluants to give
3.94 g (48%) of 8: mp 111-112 °C; 1H NMR (CDCl3) δ 1.34 (s,
3 H, CH3), 1.44 (s, 3 H, CH3), 4.26 (s, 2 H, PhCH2OCH2), 4.61
(s, 2H, PhCH2OCH2), 4.64 (d, 1H, J ) 5.7 Hz, H-5′), 5.24 (d, 1
H, J ) 5.7 Hz, H-4′), 5.45 (s, 1 H, H-1′), 5.65 (s, 1 H, H-2′),
6.30 (dd, 1 H, J ) 6.6, 4.1 Hz, H-5), 7.35 (s, 5 H, Ph), 7.57 (dd,
1 H, J ) 6.6, 2.8 Hz, H-4), 8.56 (m, 1 H, H-6); 13C NMR (CDCl3)
δ 25.6, 27.1, 66.5, 70.4, 73.1, 83.5, 83.8, 103.9, 112.3, 121.8,
127.6, 128.3, 137.6, 145.1, 150.8, 155.7, 165.7. Anal.
(C20H22N2O4) C, H, N.
r el-(1R,4R,5S)-1-[3-(Ben zyloxy)m eth yl]-4,5-d ih yd r oxy-
2-cyclop en ten -1-yl]p yr im id in -2(1H)-on e (9). A solution of
8 (0.565 g, 1.59 mmol) in a 1:1 mixture of water and CF3COOH
(6 mL) was stirred for 2 h at room temperature. The reaction
mixture was diluted with 15 mL of water and extracted with
CH2Cl2 (3 × 50 mL). The organic extract was washed with
brine, dried (Na2SO4), and evaporated. The residue was
purified by column chromatography on silica gel using 5-10%
MeOH in CH2Cl2 as eluant to give 0.355 g (71%) of 9 as a clear
oil: 1H NMR (Me2SO-d6) δ 4.03 (t, 1 H, J ) 5.5 Hz, H-5′), 4.12
(m, 2 H, PhCH2OCH2), 4.40 (d, 1 H, J ) 5.5 Hz, H-4′), 4.55 (s,
2 H, PhCH2OCH2), 4.70-5.20 (br s, 2 H, OH), 5.44 (m, 1 H,
H-1′), 5.69 (d, 1 H, J ) 1.5 Hz, H-2′), 6.43 (dd, 1 H, J ) 6.5,
4.1 Hz, H-5), 7.30-7.50 (m, 5 H, Ph), 7.90 (dd, 1 H, J ) 6.5,
2.8 Hz, H-4), 8.52 (dd, 1 H, J ) 3.9, 2.8 Hz, H-6); 13C NMR
(Me2SO-d6) δ 66.7, 68.6, 71.8, 72.5, 76.4, 104.2, 125.6, 127.5,
128.3, 138.4, 146.7, 147.4, 155.8, 165.6. Anal. (C17H18N2O4‚
0.25H2O) C, H, N.
(2R ,3R ,4R )-4-[(B e n zo y lo x y )m e t h y l]-2,3-(c y c lo h e x -
ylid en ed ioxy)-1-cyclop en ta n on e (13). A solution of 12
(0.88 g, 3.89 mmol) in pyridine (20 mL) was treated with
benzoyl chloride (0.54 mL, 4.67 mmol) and stirred at room
temperature for 3 h. The reaction mixture was reduced to
dryness and the residue was dissolved in EtOAc (200 mL),
washed with brine (100 mL), and dried (MgSO4). The solvent
was evaporated and the residue obtained (1.28 g) was used in
the following reaction without further purification: 1H NMR
(CDCl3) δ 1.30-1.75 (m, 10 H, cyclohexyl), 2.25 (d, 1 H, J )
18.1 Hz, H-5a), 2.90 (m, 2 H, H-4, H-5b), 4.30 (m, 2 H, H-3,
CHHOCOPh), 4.52 (dd, 1 H, J ) 10.0, 2.9 Hz, CHHOCOPh),
4.73 (d, 1 H, J ) 5.3 Hz, H-2), 7.40-8.15 (m, 5 H, Ph).
(1S,2S,3R,4R)-4-[(Ben zoyloxy)m et h yl]-2,3-(cycloh ex-
ylid en ed ioxy)cyclop en ta n -1-ol (14). A solution of 13 (1.28
g, 3.88 mmol) in MeOH (20 mL) was treated with NaBH4 (0.15
g, 3.88 mmol) in the presence of CeCl3‚7H2O (1.45 g, 3.88
mmol) at 0 °C while being stirred for 5 min. The mixture was
neutralized with glacial AcOH, and all volatiles were removed
under reduced pressure. The residue was dissolved in EtOAc
(200 mL), and the organic solution was washed with brine (100
mL), dried (MgSO4), and evaporated to dryness. The residue
was purified by silica gel column chromatography with hexane:
EtOAc (4:1) as eluant to give 14 (1.00 g, 78% from 12) as a
colorless syrup: 1H NMR (CDCl3) δ 1.31-1.78 (m, 10 H,
cyclohexyl), 1.80-2.05 (m, 2 H, H-5a,b), 2.55 (m, 1 H, H-4),
4.12-4.40 (m, 3 H, H-1, H-2, H-3), 4.42-4.65 (m, 2 H, CH2-
OCOPh), 7.49-8.15 (m, 5 H, Ph). Anal. (C19H24O5) C, H.
r el-(1R,4R,5S)-1-[3-[(Ben zyloxy)m eth yl]-4,5-O-a cetyl-2-
cyclop en ten -1-yl]p yr im id in -2(1H)-on e (10). A solution of
9 (0.497 g, 1.58 mmol) in pyridine (7 mL) was treated with
acetic anhydride (0.50 mL, 5.30 mmol) and stirred at room
temperature for 1.5 h. Methanol (30 mL) was added, and the
solvents were evaporated. The residue was purified by column
chromatography over silica gel with CH2Cl2 and 5% MeOH in
CH2Cl2 as eluants to give 0.422 g (67%) of 10 as a clear oil:
1H NMR (CDCl3) δ 2.02 (s, 3 H, CH3), 2.04 (s, 3 H, CH3), 4.11
(m, 2 H, PhCH2OCH2), 4.55 (s, 2 H, PhCH2OCH2), 5.22 (t, 1
H, J ) 6.0 Hz, H-5′), 5.90 (m, 2 H, H-2′, H-4′), 6.05 (m, 1 H,
H-1′), 6.30 (dd, 1 H, J ) 6.6, 4.0 Hz, H-5), 7.20-7.40 (m, 5 H,
Ph), 7.50 (dd, 1 H, J ) 6.6, 2.8 Hz, H-4), 8.55 (distorted t, 1 H,
H-6); 13C NMR (Me2SO-d6) δ 20.4, 20.5, 65.9, 67.7, 71.7, 73.7,
(1S,2S,3R,4R)-4-[(Ben zoyloxy)m et h yl]-2,3-(cycloh ex-
ylid en ed ioxy)cyclop en ta n -1-ol Meth a n esu lfon a te (15). A
stirred solution of 14 (1.07 g, 3.32 mmol) in pyridine (10 mL)
was treated with methanesulfonyl chloride (0.39 mL, 4.98

2576 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
J eong et al.
mmol) at 0 °C and allowed to reach room temperature during
the course of 2 h. All volatiles were removed under reduced
pressure, and the residue was purified by silical gel column
chromatography using CH2Cl2:MeOH (70:1) as eluant to give
15 (1.33 g, 100%) as a colorless syrup which was used in the
following step without further purification: 1H NMR (CDCl3)
δ 1.31-1.79 (m, 10 H, cyclohexyl), 2.01 (m, 1 H, H-5a), 2.35
(m, 1 H, H-5b), 2.59 (m, 1 H, H-4), 3.09 (s, 3 H, CH3), 4.20 (dd,
1 H, J ) 11.4, 6.0 Hz, CHHOCOPh), 4.31 (dd, 1 H, J ) 11.4,
5.6 Hz, CHHOCOPh), 4.54 (d, 1 H, J ) 5.7 Hz, H-3), 4.70 (t,
1 H, J ) 5.5 Hz, H-2), 5.15 (m, 1 H, H-1), 7.40-8.02 (m, 5 H,
Ph).
amount of debenzoylated product was also isolated (0.06 g) and
this was rebenzoylated after treatment with benzoyl chloride
in pyridine to give additional 18.
1-[(1′R,2′S,3′R,4′R)-4-(h yd r oxym et h yl)-2,3-d ih yd r oxy-
cyclop en ta n -1-yl]p yr im id in -2(1H)-on e (5). A stirred solu-
tion of 18 (0.20 g, 0.47 mmol) in CH2Cl2 (20 mL) was treated
with Et3N (0.26 mL, 1.88 mmol) and cooled to 5 °C. After the
addition of 1,2,4-triisopropylbenzenesulfonyl chloride (0.14 g,
0.85 mmol) and DMAP (0.014 g, 0.12 mmol), the resulting
solution was stirred for 20 h at room temperature. All volatiles
were removed under reduced pressure, and the residue was
purified through a short silica gel column using hexane:EtOAc
(3:1) as eluant to give intermediate 19 as an amorphous solid:
UV (MeOH) λmax 281.0 nm; 1H NMR (CDCl3) δ 1.10-1.75 (m,
31 H, cyclohexyl, CHMe2), 2.20-2.54 (m, 2 H, H-5′a,b), 2.90 (m,
1 H, H-4′), 4.15-4.49 (m, 3 H, H-3′, CH2OCOPh), 4.67 (t, 1 H,
J ) 6.3 Hz, H-2′), 5.01 (dd, 1 H, J ) 6.9, 4.2 Hz, H-1′), 6.08 (d,
1 H, J ) 7.10 Hz, H-5), 7.19 (s, 2 H, Ph), 7.38-8.09 (m, 6 H,
H-6, Ph). This material (19, 0.26 g, 3.77 mmol) was dissolved
in dioxane (20 mL) and stirred with anhydrous hydrazine (0.11
g, 3.77 mmol) for 1 h at room temperature. The solution was
concentrated under reduced pressure to give the crude 4-hy-
drazino analogue 20 (0.20 g, 0.46 mmol), which was im-
mediately dissolved in aqueous dioxane (20 mL dioxane, 0.4
mL H2O) and treated with Ag2O (0.32 g, 1.38 mmol) in the
presence of Et3N (0.13 mL, 0.92 mmol) for 4 h at reflux. The
reaction mixture was filtered through a pad of Celite and
washed with successively with EtOH (10 mL) and CH2Cl2 (20
mL). The filtrate was collected, evaporated to dryness, and
purified by silica gel column chromatography using CH2Cl2:
MeOH (50:1) as eluant to give, according to 1H NMR analysis,
a 1:1 mixture of the desired product 21 (0.12 g) and a uridine
byproduct identical to 18. The combined mixture was dis-
solved in MeOH (20 mL) and pyridine (0.4 mL) and stirred in
the presence of NaOMe (0.5 M, 0.90 mL) for 20 h at room
temperature. After neutralization of the reaction mixture with
glacial AcOH, all volatiles were removed under reduced
pressure. The residue was purified by silica gel column
chromatography using CH2Cl2:MeOH (20:1) as eluant to give
first the less polar product 22 (0.03 g), followed by the more
polar uridine compound corresponding to debenzoylated 18
(0.05 g). Product 22 (0.03 g) was dissolved in dioxane (10 mL)
and stirred at room temperature in the presence of 1 N HCl
(1 mL) for 20 h. The solution was concentrated under reduced
pressure and the residue was loaded on a short column of 50W-
80 resin (H+ form). After washing with water, compound 5
was eluted with 1 N NH4OH. Reducing to dryness, redissolv-
ing in water, and freeze-drying yielded 0.02 g of 5 (19% from
18) as a pale yellowish hygroscopic solid: UV (H2O, pH 7) λmax
307.0 nm (ꢀ 2390); [R]25D -32.0 (c 1, MeOH); 1H NMR (CDCl3)
δ 1.58 (m, 1 H, H-5′a), 2.15 (m, 1 H, H-5′b), 2.32 (m, 1 H, H-4′),
3.65 (m, 2 H, CH2OH), 3.99 (dd, 1 H, J ) 5.5, 3.5 Hz, H-3′),
4.38 (dd, 1 H, J ) 8.7, 5.5 Hz, H-2′), 4.78 (m, 1 H, H-1′), 6.55
(dd, 1 H, J ) 6.6, 4.3 Hz, H-5), 8.24 (dd, 1 H, J ) 6.6, 2.6 Hz,
H-4), 8.56 (dd, 1 H, J ) 4.2, 2.6 Hz, H-6); 13C NMR (CD3OD)
δ 28.9, 46.7, 64.5, 67.3, 73.7, 75.0, 106.4, 149.5, 158.5, 166.9;
FAB MS m/z (relative intensity) 227 (MH+, 35). Anal.
(C10H14N2O4‚1.6H2O) C, H, N.
(1R,2S,3R,4R)-1-Azid o-4-[(ben zoyloxy)m eth yl]-2,3-(cy-
cloh exylid en ed ioxy)cyclop en ta n e (16). A stirred solution
of 15 (1.36 g, 3.32 mmol) in DMF (20 mL) was treated with
sodium azide (2.16 g, 33.2 mmol) at 0 °C. After the addition,
the temperature was raised to 120 °C and the reaction was
continued for 20 h. All volatiles were removed under reduced
pressure, and the residue was dissolved in EtOAc (300 mL).
The organic solution was washed with brine (100 mL), dried
(MgSO4) and evaporated. The residue was purified by silica
gel column chromatography using hexane:EtOAc (8:1) as
eluant to give 16 (0.93 g, 81%) as a colorless syrup: IR (neat)
1
2103 (N3), 1720 (CO) cm-1; H NMR (CDCl3) δ 1.32-1.71 (m,
10 H, cyclohexyl), 1.72 (m, 1 H, H-5a), 2.35 (m, 1 H, H-5b), 2.61
(m, 1 H, H-4), 4.11 (m, 1 H, H-1), 4.20-4.40 (m, 2 H, CH2-
OCOPh), 4.45 (br d, 1 H, J ) 5.4 Hz, H-3), 4.55 (br d, 1 H, J
) 5.9 Hz, H-2), 7.40-8.11 (m, 5 H, Ph). Anal. (C19H23N3O4)
C, H, N.
(1R,2S,3R,4R)-1-Am in o-4-[(ben zyloxy)m eth yl]-2,3-(cy-
cloh exylid en ed ioxy)cyclop en ta n e (17). A stirred solution
of 16 (1.93 g, 2.61 mmol) in MeOH (15 mL) was hydrogenated
under a balloon in the presence of 10% Pd/C (0.15 g) for 20 h
at room temperature. The mixture was filtered through a pad
of Celite, and the solid cake was washed with MeOH. The
filtrate was evaporated and the residue was dissolved in EtOAc
(300 mL). The organic solution was washed with brine (100
mL), dried (MgSO4), and evaporated. The residue was purified
by silica gel column chromatography using CH2Cl2:MeOH (20:
1) as eluant to give 17 (0.70 g, 81%) as a colorless syrup: IR
(neat) 3376 (NH2), 1719 (CO) cm-1; 1H NMR (CDCl3) δ 1.30-
1.70 (m, 11 H, H-5a, cyclohexyl), 2.25 (m, 1 H, H-5b), 2.47 (m,
1 H, H-4), 3.45 (m, 1 H, H-1), 4.21 (dd, 1H, J ) 6.5, 3.2 Hz,
H-3), 4.30-4.50 (m, 2 H, CH2OCOPh), 4.55 (dd, 1 H, J ) 6.5,
3.6 Hz, H-2), 7.35-8.09 (m, 5 H, Ph). Anal. (C19H25NO4) C,
H, N.
1-[(1′R,2′S,3′R,4′R)-4-[(ben zoyloxy)m et h yl]-2,3-(cyclo-
h exylid en ed ioxy)cyclop en ta n -1-yl]u r a cil (18). A solution
of 3-ethoxypropenoyl isocyanate in benzene (20 mL), prepared
according to the standard literature procedure25 from 3-ethoxy-
propenoyl chloride (0.30 g, 2.23 mmol) and silver cyanate (0.62
g, 4.13 mmol), was added to a solution of 17 (0.28 g, 0.85 mmol)
in benzene (20 mL), while the temperature was maintained
at 10 °C. The pale yellow solution formed was stirred for 30
min at the same temperature and after that time the solvent
was removed under reduced pressure. The residue was
purified through a short silica gel column using CH2Cl2:MeOH
(50:1) as eluant to give the corresponding acyclic pyrimidine
precursor (0.30 g) as a colorless syrup. This intermediate was
immediately dissolved in DMF (15 mL), treated with concen-
trated NH4OH (20 mL), and heated to 110 °C for a period of 4
h, while ammonia gas was continuously bubbled into the
reaction mixture. After cooling to room temperatue, water (20
mL) and CH2Cl2 (100 mL) were added, and the organic layer
was washed with brine (30 mL) and dried (MgSO4). The
solution was evaporated and the residue was purified by silica
gel column chromatography using CH2Cl2:MeOH (20:1) as
eluant to give 18 (0.22 g, 60%) as a colorless white foam: UV
(MeOH) λmax 265.40 nm; 1H NMR (CDCl3) δ 1.20-1.75 (m, 10
H, cyclohexyl), 2.10-2.40 (m, 2 H, H-5′a,b), 2.51 (m, 1 H, H-4′),
4.42 (m, 3 H, H-3′, CH2OCOPh), 4.61 (t, 1 H, J ) 6.4 Hz, H-2′),
4.83 (dd, 1 H, J ) 6.9, 4.8 Hz, H-1′), 5.70 (dd, 1 H, J ) 8.0, 2.0
Hz, H-5), 7.15 (d, 1 H, J ) 8.0 Hz, H-6), 7.37-8.09 (m, 5 H,
Ph), 9.25 (br s, 1 H, NH). Anal. (C23H26N2O6) C, H, N. A small
(1R,2R,3S,4S,5S)-1-[(Ben zyloxy)m eth yl]-2,3-O-isop r o-
p ylid en e-4-h yd r oxybicyclo[3.1.0]h exa n e (24). A stirred
solution of 2315 (6.0 g, 21.66 mmol) in CH2Cl2 at 0 °C was
treated with diethyl zinc (1 M hexane, 65 mL, 65 mmol) and
CH2I2 (5.2 mL, 64.98 mmol). After 15 min, the mixture was
allowed to reach room temperature and stirring continued for
a total of 15 h. Aqueous saturated NH4Cl was carefully added,
and the resulting mixture was extracted with ether (500 mL),
washed successively with saturated NaHCO3 solution (100 mL)
and brine (100 mL), dried (MgSO4), and evaporated. The
residue was purified by silica gel column chromatography
using hexane:EtOAc (2:1) as eluant to give 24 (5.0 g, 79%) as
a colorless syrup: 1H NMR (CDCl3) δ 0.61 (dd, 1 H, J ) 8.6,
5.5 Hz, H-6a), 1.17 (t, 1 H, J ) 5.1 Hz, H-6b), 1.29 (s, 3 H,
CH3), 1.52 (s, 3 H, CH3), 1.69 (m, 1 H, H-5), 1.99 (br s, 1 H,
OH), 3.17 (d, 1 H, J ) 10.4 Hz, CHHOBn), 3.68 (d, 1 H, J )

Carbocyclic Analogues of Zebularine
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2577
10.4 Hz, CHHOBn), 4.55 (m, 4 H, PhCH2O, H-3, H-4), 4.90
(br d, 1 H, J ) 5.9 Hz, H-2), 7.25-7.39 (m, 5 H, Ph). Anal.
(C17H22O4) C, H.
(d,1 H, J ) 10.1 Hz, CHHOBn), 4.01 (d, 1 H, J ) 10.1 Hz,
CHHOBn), 4.55 (AB q, 2 H, J ) 11 Hz, PhCH2O), 5.10 (d, 1
H, J ) 7.1 Hz, H-3′), 5.18 (s, 1 H, H-4′), 5.89 (m, 2 H, H-2′,
H-5), 7.30-7.39 (m, 5 H, Ph), 8.48 (m, 2 H, H-4, H-6). Anal.
(C22H24O6N2) C, H, N.
(1R,2R,3S,4R,5S)-1-[(Ben zyloxy)m eth yl]-2,3-O-isop r o-
p ylid en e-4-a zid obicyclo[3.1.0]h exa n e (26). A stirred solu-
tion of 24 (0.74 g, 2.54 mmol) in pyridine (10 mL) was treated
with methanesulfonyl chloride (0.30 mL, 3.81 mmol) at 0 °C
for 2 h. The solution was then reduced to dryness, and the
residue was dissolved in EtOAc (200 mL). The organic solution
was washed with brine (100 mL), dried (MgSO4), and evapo-
rated to dryness under reduced pressure to give mesylate 25
(0.78 g). The crude mesylate was dissolved in DMF (15 mL)
and reacted with NaN3 (0.83 g, 12.7 mmol) at 100 °C for 18 h.
After reaching room temperature, the reaction mixture was
diluted with EtOAc (200 mL) and extracted with water (50
mL). The organic layer was washed with brine, dried (MgSO4),
and reduced to dryness. The residue was purified by silica
gel column chromatography using hexane:EtOAc (5:1) as
eluant to give 26 (0.70 g, 88%) as a colorless syrup: IR (neat)
2097 cm-1; 1H NMR (CDCl3) δ 0.82 (ddd, 1 H, J ) 9.2, 5.5, 1.3
Hz, H-6a), 1.01 (t, 1 H, J ) 5.1 Hz, H-6b), 1.25 (s, 3 H, CH3),
1.49 (s, 3 H, CH3), 1.58 (m, 1 H, H-5), 3.35 (d, 1 H, J ) 10.5
Hz, CHHOBn), 3.63 (d, 1 H, J ) 10.5 Hz, CHHOBn), 3.83 (s,
1 H, H-4), 4.51 (dd, 1 H, J ) 7.2, 1.3 Hz, H-2), 4.55 (s, 2 H,
PhCH2O), 5.02 (dd, 1 H, J ) 7.2, 1.0 Hz, H-3), 7.25-7.39 (m,
5 H, Ph). Anal. (C17H21O3N3‚0.4H2O) C, H, N.
(1R,2R,3S,4R,5S)-1-(Hyd r oxym eth yl)-2,3-d ih yd r oxy-4-
(p yr im id in -2-(1H )-on -1-yl)b icyclo[3.1.0]h exa n e (6).
A
stirred solution of 29 (0.07 g, 0.17 mmol) in CH2Cl2 (10 mL)
was cooled to -78 °C and treated with a 1 M solution of BCl3
in CH2Cl2 (1.2 mL). After 20 min, the mixture was concen-
trated under reduced pressure and the residue was purified
by silica gel column chromatography using CH2Cl2:MeOH (30:
1) as eluant to give the intermediate diacetate (0.028 g), which
was immediately treated with concentrated methanolic am-
monia (10 mL) in a sealed vessel at 0 °C for 3 h. After removal
of all the volatiles under reduced pressure, the residue was
purified by silica gel column chromatography using the same
solvent system to give 6 (0.018 g, 44%): UV (H2O) λmax 307.0
nm; [R]25 ) +99.4° (c 0.17, MeOH); 1H NMR (CD3OD) δ 0.72
D
(m, 1 H, H-6′a), 1.48 (m, 2 H, H-6′b, H-5′), 3.27 (d, 1 H, J )
11.7 Hz, CHHOH), 3.82 (d, 1 H, J ) 10.8 Hz, H-2′), 4.25 (d, 1
H, J ) 11.7 Hz, CHHOH), 4.59 (d, 1 H, J ) 6.8 Hz, H-3′), 4.85
(s, 1 H, H-4′), 6.56 (dd, 1 H, J ) 6.6, 4.4 Hz, H-5), 8.55 (m, 1
H, H-4), 8.66 (dd, 1 H, J ) 6.6, 2.6 Hz, H-6); 13C NMR (CD3OD)
δ 11.96, 23.55, 38.42, 64.10, 67.23, 71.87, 77.19, 106.40, 148.39,
158.19, 166.94; FAB MS m/z (relative intensity) 239 (MH+, 40).
Anal. (C11H14O4N2) C, H, N.
(1R,2R,3S,4R,5S)-1-[(Ben zyloxy)m eth yl]-2,3-O-isop r o-
p ylid en e-4-a m in obicyclo[3.1.0]h exa n e (27). A stirred so-
lution of 26 (0.70 g, 2.22 mmol) in a mixture of MeOH (20 mL)
and EtOAc (20 mL) was hydrogenated in a balloon in the
presence of Lindlar’s catalyst (0.14 g). After 3 h, the mixure
was filtered and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column
chromatography using CHCl3:MeOH (10:1) as eluant to give
27 (0.67 g, 100%) as a colorless syrup: 1H NMR (CDCl3) δ 0.82
(ddd, 1 H, J ) 9.0, 5.3, 1.2 Hz, H-6a), 0.99 (t, 1 H, J ) 5.0 Hz,
H-6b), 1.25 (s, 3 H, CH3), 1.31 (ddd, 1 H, J ) 9.2, 4.7, 1.3 Hz,
H-5), 1.49 (s, 3 H, CH3), 1.75 (br s, 1 H, NH2), 3.26 (d, 1 H, J
) 10.3 Hz, CHHOBn), 3.32 (s, 1 H, H-4), 3.72 (d, 1 H, J )
10.3 Hz, CHHOBn), 4.33 (dd, 1 H, J ) 7.1, 1.33 Hz, H-2), 4.55
(AB d, 2 H, PhCH2O), 5.05 (d, 1 H, J ) 7.1, 0.8 Hz, H-3). Anal.
(C17H23O3N‚0.2H2O) C, H, N.
(1R,2R,3S,4R,5S)-1-[(Ben zyloxy)m eth yl]-2,3-O-isop r o-
p ylid en e-4-u r eid obicyclo[3.1.0]h exa n e (28). A stirred so-
lution of 27 (0.20 g, 0.69 mmol) in THF (10 mL) was treated
with trimethylsilyl isocyanate (0.24 mL, 1.52 mmol) at room
temperature for 15 h. Although TLC revealed no change, the
mixture was concentrated under reduced pressure and the
residue was purified by silica gel column chromatography
using CH2Cl2:MeOH (15:1) as eluant to give 28 (0.23 g, 100%)
as a colorless syrup: 1H NMR (CDCl3) δ 0.72 (dd, 1 H, J )
8.6, 5.9 Hz, H-6a), 1.01 (t, 1 H, J ) 5.1 Hz, H-6b), 1.25 (s, 3 H,
CH3), 1.35 (dd, 1 H, J ) 8.9, 4.0 Hz, H-5), 1.47 (s, 3 H, CH3),
3.15 (d, 1 H, J ) 10.3 Hz, CHHOBn), 3.81 (d, 1 H, J ) 10.3
Hz, CHHOBn), 3.92 (br s, 2 H, NH2), 4.43 (d, 1 H, J ) 6.9 Hz,
H-2), 4.44 (s, 1 H, H-4), 4.51 (AB q, 2 H J ) 11.9 Hz, PhCH2O),
5.01 (d, 1 H, J ) 6.9, H-3) 5.18 (br s, 1 H, NH), 7.25-7.39 (m,
5 H, Ph). Anal. (C18H24O4N2‚0.25H2O) C, H, N.
Cytid in e Dea m in a se Assa y. Cytidine deaminase was
measured either as already reported by following the decrease
in absorbance at 282 nm that characterizes the conversion of
cytidine to uridine1,3,24 or by HPLC. For the HPLC method,
the reaction mixtures (100 µL) contained 100 µM of inhibitor,
various concentrations of cytidine ranging from 25 to 400 µM,
and 2 µL of a human liver CDA preparation1,26 in 10 mM Tris-
HCl, pH 8.4. After a 10 min incubation at 37 °C, the reaction
was terminated by heating to 95 °C for 1 min. The reaction
mixtures were then centrifuged at 12 500 rpm for 2 min. The
supernatant (50 µL) was analyzed by HPLC using a C18
reverse column (TOSOHAAS Bioseparation Specialists, 10 µm,
7.5 mm i.d. × 30 cm) with an eluant consisting of a mixture of
1 mL of 88% formic acid in water (4 L). Under isocratic
conditions, at 1 mL/min, the retention time for cytidine was
4.66 min. Uridine, with a retention time of 5.80 min, coeluted
with the inhibitor 5. Cytidine deaminase was measured by
following the decrease of the cytidine peak. Under these
conditons, the calculated Km for cytidine was 47 µM and the
Ki for 5 was 38 µM.
Refer en ces
(1) Wentford, D. F.; Wolfenden, R. Cytidine deaminase (from
Escherichia coli and human liver) Methods Enzymol. 1978, 51,
401-408.
(2) For a review, see Marquez, V. E. In Developments in Cancer
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(3) McCormack, J . J .; Marquez, V. E.; Liu, P. S.; Vistica, D. T.;
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(4) Laliberte, J .; Marquez, V. E.; Momparler, R. L. Potent inhibitors
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(5) (a) Betts, L.; Xiang, S.; Short, S. A.; Wolfenden, R.; Carter, C.
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(6) Frick, L.; Yang, C.; Marquez, V. E.; Wolfenden, R. Binding of
pyrimidin-2-one ribonucleoside by cytidine deaminase as the
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tion of the 4-hydroxyl group to its binding affinity. Biochemistry
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(1′R,2′R,3′S,4′R,5′S)-1′-[(Ben zyloxy)m et h yl]-2′,3′-d i-O-
a cet yl-4′-(p yr im id in -2(1H )-on -1-yl)b icyclo[3.1.0]h exa n e
(29). A stirred solution of 28 (0.24 g, 0.72 mmol) in EtOH (20
mL) was treated with concentrated HCl (0.3 mL) and heated
at 45 °C for 20 min. After the addition of 0.72 mL of 1,1′,3,3′-
tetraethoxypropane (1 M, EtOH), stirring was continued for
15 h at 45 °C. After volatiles were removed under reduced
pressure, the residue was treated with acetic anhydride (0.5
mL) and pyridine (6 mL), and the resulting solution was stirred
at room temperature for 15 h. The solution was reduced to
dryness, dissolved in EtOAc (300 mL), washed with brine (100
mL), dried (MgSO4), and evaporated. The residue was purified
by silica gel column chromatography using CH2Cl2:MeOH (30:
1) as eluant to give 29 (0.12 g, 40%) as a colorless syrup: UV
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chia coli: Purification, properties, and inhibition by the potential
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Chem. 1971, 246, 7561-7565.
1
(H2O) λmax 307 nm; H NMR (CDCl3) δ 0.92 (m, 1 H, H-6′a),
1.47 (m, 2 H, H-6′b, H-5′), 2.01 and 2.08 (s, 3 H, CH3), 3.17

2578 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
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