Synthesis of unnatural 7-substituted-1-(2-deoxy-β-D-ribofuranosyl)isocarbostyrils: "Thymine replacement" analogs of deoxythymidine for evaluation as antiviral and anticancer agents

Article abstract of DOI:10.1081/NCN-100105246

A group of unnatural 1-(2-deoxy-β-D-ribofuranosyl)isocarbostyrils having a variety of C-7 substituents [H, 4,7-(NO₂)₂, I, CF₃, CN, (E)-CH=CH-I, -C≡CH, -C≡C-I, -C≡C-Br, -C≡C-Me], designed as nucleoside mimics, were synthesi

Full text of DOI:10.1081/NCN-100105246

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SYNTHESIS OF UNNATURAL 7-SUBSTITUTED-1-(2-
DEOXY-β-D-RIBOFURANOSYL)ISOCARBOSTYRILS†:
“THYMINE REPLACEMENT” ANALOGS OF
DEOXYTHYMIDINE FOR EVALUATION AS ANTIVIRAL AND
ANTICANCER AGENTS
Ebrahim Naimi a , Weili Duan a , Leonard I. Wiebe a & Edward E. Knaus b
a Faculty of Pharmacy and Pharmaceutical Sciences , University of Alberta , Edmonton,
Alberta, T6G 2N8, Canada
b Faculty of Pharmacy and Pharmaceutical Sciences , University of Alberta , Edmonton,
Alberta, T6G 2N8, Canada
Published online: 17 Aug 2006.
To cite this article: Ebrahim Naimi , Weili Duan , Leonard I. Wiebe & Edward E. Knaus (2001) SYNTHESIS OF UNNATURAL 7-
SUBSTITUTED-1-(2-DEOXY-β-D-RIBOFURANOSYL)ISOCARBOSTYRILS†: “THYMINE REPLACEMENT” ANALOGS OF DEOXYTHYMIDINE
FOR EVALUATION AS ANTIVIRAL AND ANTICANCER AGENTS, Nucleosides, Nucleotides and Nucleic Acids, 20:8, 1533-1553, DOI:
10.1081/NCN-100105246
To link to this article: http://dx.doi.org/10.1081/NCN-100105246
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(8), 1533À1553 (2001)
SYNTHESIS OF UNNATURAL
7-SUBSTITUTED-1-(2-DEOXY-b-D-
RIBOFURANOSYL)ISOCARBOSTYRILS{:
``THYMINE REPLACEMENT'' ANALOGS
OF DEOXYTHYMIDINE FOR EVALUATION AS
ANTIVIRAL AND ANTICANCER AGENTS
Ebrahim Naimi, Weili Duan, Leonard I. Wiebe, and
Edward E. Knaus*
Faculty of Pharmacy and Pharmaceutical Sciences, University of
Alberta, Edmonton, Alberta, Canada T6G 2N8
ABSTRACT
A
group of unnatural 1-(2-deoxy-ꢀ-D-ribofuranosyl)isocarbostyrils
having a variety of C-7 substituents [H, 4,7-(NO2)2, I, CF3, CN ,
(E)-CHˆCH-I, -CꢀCH, -CꢀC-I, -CꢀC-Br, -CꢀC-Me], designed as
nucleoside mimics, were synthesized for evaluation as anticancer and
antiviral agents. This class of compounds exhibited weak cytotoxicity in a
MTT assay (CC50 ˆ 10À 3 to 10À 5 M range) with the 4,7-dinitro deriva-
tive being the most cytotoxic, relative to thymidine (CC50 ˆ 10À 3 to
10À 5 M range), against a variety of cancer cell lines. The 4,7-dinitro, 7-I
and 7-CꢀCH compounds exhibited similar cytotoxicity against non-
‡
transfected (KBALB, 143B), and HSV-1 TK gene transfected (KBALB-
STK, 143B-LTK) cancer cell lines possessing the herpes simplex virus
‡
type 1 (HSV-1) thymidine kinase gene (TK ). This observation indicates
that these compounds are not substrates for HSV type-1 TK, and are
therefore unlikely to be useful in gene therapy based on the HSV gene
therapy paradigm.
{Nucleoside-like numbering is used by analogy to nucleoside nomenclature.
*Corresponding author. Fax: (780) 492-1217; E-mail: eknaus@pharmacy.ualberta.ca
1533
Copyright # 2001 by Marcel Dekker, Inc.
www.dekker.com

1534
NAIMI ET AL.
INTRODUCTION
The speci®c H-bonding patterns of the Watson-Crick A-T and G-C base
pairs serve an important role with respect to the storage and replication of
biological information1. Accordingly, the design of enzymatically replicable
``unnatural base pairs'' may constitute a method to expand the biological and
chemical genetic alphabet potential of DNA beyond the natural A-T and G-
C pairs. Romesberg et al.2 recently reported the unnatural hydrophobic
7-propynylisocarbostyril nucleoside mimic 1 that contains an isocarbostyril
moiety, in place of the natural uracil base, that can not form H-bonds. The
unnatural self-pair 1-1 is able to signi®cantly stabilize duplex DNA relative
to the natural A-T and G-C base pairs. In spite of the fact that the mimic 1
does not have shape complimentarity to any native base, the 50-triphosphate
of 1 is still incorporated opposite itself (1-1 pair) with reasonable eciency by
the Klenow fragment of E. coli DNA polymerase 1 (KF) such that the 1-1
base pair is the most ``orthogonal'' base pair relative to the natural bases,
reported to date. Although KF (DNA polymerase) inserts 1-triphosphate
opposite 1 with reasonable eciency, continued synthesis (chain elongation)
proceeds ineciently. This is attributed to the assumption that the unnatural
base pair 1-1 assumes a geometry that incorrectly positions the 30-OH of the
growing strand for nucleophilic reaction with the incoming nucleoside tri-
phosphate2. These results suggest that nucleoside mimics such as 1 should be
cytotoxic to rapidly multiplying cancer cells (inhibit tumor growth) and/or
act as antiviral agents due to their selective phosphorylation by virus-infected
cells3. Credence for this latter concept is provided by the discovery that the
unusual bicyclic 2,3-dihydrofuro[2,3-d]pyrimidin-2-one moiety present in
compounds 2b, which are derivatives of 2a described previously4,5, exhibit
potent and selective inhibition (300-fold greater potency than acyclovir with
no detectable in vitro cytotoxicity) of varicella-zoster virus (VZV)6. (see
Figure 1). This latter observation indicates that certain large bicyclic het-
erocyclic base moieties are tolerated with respect to retention of antiviral
ecacy. It was therefore anticipated that 7-substituted nucleoside mimic
derivatives of 1 may be useful as anticancer or antiviral agents, and as
radiopharmaceutical agents to image7. or chemotherapeutic agents to treat8,
‡
herpes simplex virus type-1 thymidine kinase positive (HSV-1 TK ) gene-
transfected tumors (gene therapy of cancer)9. We now report the synthesis,
and antiviral-anticancer activities for a group of 1-(2-deoxy-b-D-ribofur-
anosyl)-7-substituted-isocarbostyrils (1, 7, 9, 11, 13, 15, 18, 20, 23, 24)
designed as 5-substituted-20-deoxyuridine (thymidine) mimics.
CHEMISTRY
Nucleoside mimics are frequently synthesized by reaction of an
O-trimethylsilylated base mimic2, or an arylcadmium reagent10, with the

ANALOGS OF DEOXYTHYIMIDINE
1535
Figure 1. Structures of thymidine, 1-(2-deoxy-b-D-ribofuranosyl)-7-propynylisocarbostyril
(1), and some unusual 3-(2-deoxy-b-D-ribofuranosyl)-6-substituted-2,3-dihydrofuro[2,3-d]
pyrimido-2-one derivatives 2.
well known Ho€er a-chloro sugar synthon [3,5-bis-O-(p-toluoyl)-2-deoxy-
a-D-ribofuranosyl chloride]11. Accordingly, 3,5-bis-O-(p-chlorobenzoyl)-2-
deoxy-a-D-ribofuranosyl chloride (3) was used in place of the Ho€er a-
chloro sugar synthon since it is a more stable and readily accessible
crystalline reagent10. Coupling the a-chloro sugar 3 to the 2-O-tri-
methylsilyl derivative of isocarbostyril (4), which was generated from the
reaction of 4 with N,O-bis(trimethylsilyl)acetamide, was performed in
MeCNat 25 ꢁC (Scheme 1). As reported for a similar reaction using 3,5-
bis-O-(p-toluoyl)-2-deoxy-a-D-ribofuranosyl chloride2, this coupling reac-
tion yielded a mixture of the 3,5-bis-O-(p-chlorobenzoyl) b-D-5 (27%) and
a-D-6 (39%) anomers that were readily separated by ¯ash silica gel
column chromatography. Subsequent removal of the O-(p-chlorobenzoyl)
protecting groups by treatment with NaOMe a€orded b-D-7 (90%) and b-
D-8 (94%), respectively.
The con®guration at the C-10 carbon, and the conformation, of the
ꢀ-D-7 and a-D-8 anomers possessing an isocarbostyril moiety was ana-
lyzed by nuclear Overhauser enhancement (NOE) 1H NMR di€erence
spectroscopy (Figure 2). Selective irradiation of the H-40 signal (b-D-7)
resulted in an enhancement of the H-10 signal (6.8%), and irradiation of
the H-200 signal gave a 6.0% enhancement of the H-10 signal, which
support the assignment of the b-con®guration12. The C-8 hydrogen of the
isocarbostyril moiety is oriented in the direction of the sugar ring since
NOE enhancements of H-8 were observed upon selective irradiation of
H-20 (6.4%) and H-30 (3.6%). In contrast, irradiation of the H-10 signal of
a-D-8 provided enhancements of cis-H-20 (6.4%) and H-30 (1.9%).

1536
NAIMI ET AL.
Scheme 1. Reagents and conditions: (a) N,O-bis(trimethylsilyl)acetamide, MeCN, 25 ꢁC,
30 min, and then 3, SnCl4, 0 ꢁC, 30 min; (b) NaOMe, MeOH, 25 ꢁC, 30 min; (c) NH4NO3,
(CF3CO)2O, 25 ꢁC, 1 h; (d) ICl, CH2Cl2, re¯ux; (e) CuI, KF, DMF at 60 ꢁC, and then
ClCF2CO2Me, 120 ꢁC, 6 h; (f) Zn(CN)2, (PPh3)4Pd, DMF, 80À90 ꢁC, 6 h.
The failure to observe an enhancement of H-10 upon irradiation of H-40
indicates these two hydrogens are on opposite faces of the sugar moiety
which is indicative of the a-con®guration12.
Nitration13 of the 3,5-bis-O-(p-chlorobenzoyl) ester b-D-5 using
ammonium nitrate in tri¯uoroacetic anhydride a€orded the 4,7-dinitro
derivative, which on deprotection with NaOMe in MeOH at 25 ꢁC, gave 1-(2-
deoxy-b-D-ribofuranosyl)-4,7-dinitroisocarbostyril (9, 18% overall yield
from b-D-5). The 1H NMR spectrum for 9 indicated that nitration occurred
at the expected C-7 position since H-8 appeared as a singlet at d 9.57, and at

ANALOGS OF DEOXYTHYIMIDINE
1537
Figure 2. Some NOE studies to study the con®guration and conformation of 1-(2-deoxy-b-
D-ribofuranosyl)isocarbostyril (b-D-7), and 1-(2-deoxy-a-D-ribofuranosyl)isocarbostyril
(a-D-8) in MeOH-d4 at 22 ꢁC.
either C-4 or C-5 since a doublet (J ˆ 2.4 Hz), doublet (J ˆ 9.1 Hz) and a
doublet of doublets (J ˆ 9.1, 2.4 Hz) were present at d 8.93, 8.75 and 8.66,
respectively for three phenyl protons located on the isocarbostryril ring
system. A semiempirical PM3 calculation showed the electron densities in
isocarbostyril (4) are C-3 ( À 0.031), C-4 ( À 0.129), C-5 ( À 0.065), C-6
( À 0.117) and C-7 ( À 0.193). These data suggest electrophilic nitration
should occur at the more electron-rich C-4 position, relative to C-5, on the
phenyl moiety to give the 4,7-dinitro product.
Iodination of the 3,5-bis-O-(p-chlorobenzoyl) ester b-D-5 using ICl in
CH2Cl2 a€orded the 7-iodo derivative 10 (87%), which on deprotection with
NaOMe in MeOH gave 1-(2-deoxy-b-D-ribofuranosyl)-7-iodoisocarbostyril
(11, 82%). Synthesis of the 7-tri¯uoromethyl derivative 13 was carried out
using a reaction reported by Chen et al.14 Thus, the protected 7-iodo com-
pound 10 was treated with methyl chlorodi¯uoroacetate in the presence of
CuI and KF in DMF at 120 ꢁC to give the 3,5-bis-O-(p-chlorobenzoyl) ester
12 (71%), followed by deprotection with NaOMe in MeOH to a€ord 1-(2-
deoxy-b-D-ribofuranosyl)-7-tri¯uoromethylisocarbostyril (13, 97%). The 7-
cyano derivative 15 was also prepared from the protected 7-iodo derivative
10 by cyanation with zinc cyanide in the presence of (PPh3)4Pd(0)15, and then
deprotection using NaOMe in MeOH.
Synthesis of the (E)-7-(2-trimethylsilylvinyl) derivative 17 was per-
formed using a methodology similar to that reported previously16 for the

1538
NAIMI ET AL.
Scheme 2. Reagents and conditions: (a) (Ph3P)2PdCl2, MeCN, 60 ꢁC, 24 h; (b) ICl, MeCN,
0 ꢁC, 15 min.
synthesis of (E)-5-(2-iodovinyl)-20-¯uoro-20-deoxyuridine (IVFRU), as illu-
strated in Scheme 2. Accordingly, the coupling reaction of the unprotected 7-
iodo compound 11 with (E)-1-trimethylsilyl-2-tributylstannylethene (16)17
1
catalyzed by (Ph3P)2PdCl2 a€orded 17 (83%). The H NMR spectrum for
17 showed a JCHˆCH ˆ 18.9 Hz coupling constant that is indicative of the
(E)-7-(-CHˆCH-TMS) stereochemistry. Reaction of the (E)-7-(2-tri-
ꢁ
methylsilylvinyl) compound 17 with ICl in MeCNat 0 C yielded the corre-
sponding (E)-7-(2-iodovinyl) product 18 (70%). Proton NMR spectral
analysis of 18 indicated that only the (E)-isomer was produced (JCHˆCH
14.7 Hz).
ˆ
Reaction of 1-[3,5-bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofurano-
syl]-7-iodoisocarbostyril (10) with TMS-CꢀC-H in the presence of
(Ph3P)2PdCl2 and CuI in Et3N18 a€orded the corresponding 7-(trimethylsi-
lyl)ethynyl derivative 19 (78%), which upon treatment with NaOMe in
MeOH at 20 ꢁC resulted in simultaneous ester cleavage and removal of the
TMS group to give the 7-ethynyl product 20 (98%) as illustrated in Scheme 3.
The 7-iodoethynyl 23, or 7-bromoethynyl 24, compounds were prepared by
reaction of the 7-(trimethylsilyl)ethynyl compound 19 with either N-iodo-
succinimide, or N-bromosuccinimide, in the presence of AgNO3 catalyst in
DMF19, and then removal of the p-chlorobenzoyl protecting groups in the
sugar moiety using NaOMe in MeOH. The coupling reaction of 1-[3,5-bis-O-
( p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-iodoisocarbostyril (10) with
Me-CꢀC-H in the presence of (Ph3P)2PdCl2, CuI and dry Et3N, using a
procedure similar to that reported by Romesberg et al.2, and then ester
hydrolysis using NaOMe in MeOH, a€orded the target product 1-(2-deoxy-
b-D-ribofuranosyl)-7-propynylisocarbostyril (1, 61% overall yield from 10).

ANALOGS OF DEOXYTHYIMIDINE
1539
Scheme 3. Reagents and conditions: (a) H-CꢀC-SiMe3, (Ph3P)2PdCl2, CuI, Et3N , 50À60 ꢁC,
5 h; (b) NaOMe, MeOH, 25 ꢁC, 20 min; (c) N-iodosuccinimide, AgNO3, DMF, 0 ꢁC for 2 h, and
then 25 ꢁC for 1 h; (d) N-bromosuccinimide, AgNO3, DMF, 0 ꢁC for 2 h, and then 25 ꢁC for 4 h;
(e) NaOMe, MeOH, 25 ꢁC, 15 min; (f) (Ph3P)2PdCl2, CuI, Et3N, Me-CꢀC-H, 25 ꢁC, 5 h.
BIOLOGICAL RESULTS AND DISCUSSION
Nucleoside mimics, in which the natural thymine base in thymidine is
replaced by an unnatural isocarbostyril base, could induce new properties
which the natural nucleosides lack thereby providing a strategy to design a
new class of isocarbostyril nucleoside mimics. Isocarbostyril mimics possess
a number of potentially desirable features that include i) irreversible inhi-
bition of thymidylate synthase (TS), by the 50-monophosphate, upon reac-
tion of the nucleophilic -SH group of TS at the electron de®cient C-8
position of the isocarbostyril ring to exhibit a cytotoxic anticancer e€ect, ii)
utilization of the 50-monosphosphate as a substrate for the synthesis of
``false'' DNA that would stop chain elongatation resulting in a cytotoxic
antiviral=anticancer e€ect, and iii) increased lipophilicity which could
enhance their ability to penetrate the blood-brain-barrier (BBB) to improve
their ecacy for the treatment of brain viral infections and brain tumors.
A
group of 1-(2-deoxy-ꢀ-D-ribofuranosyl)-7-substituted-isocarbostyrils

1540
NAIMI ET AL.
having a variety C-7 substituents [H, NO2, I, CF3, CN , E()-CHˆCH-I,
-CHꢀCH, -CꢀC-I, -CꢀC-Br, -CꢀC-Me] were investigated to probe the
e€ect of size, electronic, hybridization [sp (-CꢀC-), sp2 (-CHˆCH-)] and
lipophilic parameters upon biological activity. Substituent selection could
also in¯uence oral bioavailability, metabolic stability and pharmacokinetic
properties. All of these factors are potential determinants of anti-
viral=anticancer ecacy.
A group of 7-substituted [H, 4,7-(NO2)2, I, CN , E( )-(-CHˆCH-I), -
CHꢀCH, -CꢀC-I, -CꢀC-Br, -CꢀC-Me] isocarbostyril compounds, and the
reference compounds 5-iodo-20-deoxyuridine (IUDR), 5-¯uoro-20-deoxyur-
idine (FUDR) and thymidine, were evaluated using the MTT cytotoxicity
assay20 (see Table 1). These 7-substituted compounds exhibited weak cyto-
toxicity (CC50 ˆ 10À 3 to 10À 5 M range), even when compared to thymidine
(CC50 ˆ 10À 3 to 10À 5 M range), against KBALB, KBALB-STK, 143B,
143B-LTK, EMT-6 and R-970-5 cancer cell lines. The 4,7-dinitro compound
9 was the most cytotoxic (CC50 ˆ 10À 5 M range) agent. A comparison of the
cytotoxicities against the KBALB cell line showed the cytotoxicity potency
order was 4,7-dinitro 97-CꢀC-I 23 > 7-CꢀC-Br 24 ꢂ 7-CꢀCH 20 ꢂ 7-I
11 ꢂ 7-(E)-CHˆCH-I 18 ꢂ 7-CN 15 ꢂ 7-CꢀC-Me 1 > 7-H 7. The I and Me
groups are potential isosteres since their van der Waal's radius are 2.15 and
2.0 A, respectively. This concept is supported by the observation that the
cytotoxicity for 7-CꢀC-I 23 is greater than 7-CꢀC-Me 1 toward KBALB
cells. The 4,7-dinitro 9, 7-I 11 and 7-CꢀCH 20 compounds exhibited similar
cytotoxicity against non-transfected (KBALB, 143B), and the corresponding
transfected (KBALB-STK, 143B-LTK) cancer cell lines possessing the herpes
‡
simplex virus type 1 (HSV-1) thymidine kinase gene (TK ). This comparison,
for these compounds, indicates that expression of the viral TK enzyme did
not provide a gene therapeutic e€ect.
The weak, or absence of, anticancer=antiviral ecacy for this novel
class of 1-(2-deoxy-ꢀ-D-ribofuranosyl)-7-substituted-isocarbostyrils could be
due to a number of factors. For example, it is possible that the sugar moiety
does not undergo phosphorylation by thymidine kinase (TK) to the 50-
monophosphate (50-MP). Support for this explanation is based on the
observations that there are generally negligible di€erences in anticancer/
antiviral activities between non-transfected (KBALB, 143B) and viral TK-
transfected (KBALB-STK, 143B-LTK) cell lines. In this respect, one would
anticipate that preferential phosphorylation to the 50-MP by transfected cells,
and subsequent transformation to the active 50-triphosphate (50-TP), would
have resulted in a greater cytotoxic e€ect since the 50-TP of 1 has been shown
to deter chain elongation2. Alternatively, the group of nucleoside mimics
evaluated may be devoid of anticancer=antiviral activity due to the fact that
they are not inhibitors of thymidylate synthase (TS). Credence for this pos-
sibility is based on the belief that the anticancer activity exhibited by 5-
¯uoro-20-deoxyuridine is primarily due to inhibition of DNA biosynthesis by

ANALOGS OF DEOXYTHYIMIDINE
1541
Table 1. In V|tro Cell Cytotoxicity of 1-(2-Deoxy-b-D-ribofuranosyl)-7-substituted-iso-
carbostyrils Determined Using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
Bromide (MTT) Assay
Cellular Toxicity (CC50, M) Toward Various Cell Linesa
Compd
KBALBb KBALB-STKc
143Bd
143B-LTKc
EMT-6e
R-970-5f
7
9
11
15
18
20
23
24
2.5610À 3
2.7610À 5
3.3610À 4
5.5610À 4
5.0610À 4
3.1610À 4
7.5610À 5
2.0610À 4
5.5610À 4
9.7610À 5
6.0610À 11
À
À
À
À
À
2.6610À 5
3.2610À 5 3.0610À 5 2.0610À 5 4.0610À 5
2.6610À 4 1.5610À 4 2.8610À 4 1.8610À 4
3.0610À 4
À
À
À
À
À
À
À
À
À
À
2.7610À 4
2.6610À 4 2.0610À 4 2.3610À 4 2.3610À 4
À
À
À
À
À
À
À
À
À
À
À
À
À
À
À
1
IUDRg
FUDRh
1.0610À 5
7.0610À 3 7.4610À 3 3.8610À 4 6.2610À 4
8.8610À 11 9.0610À 5 1.0610À 4 9.0610À 12 5.5610À 5
Thymidine 9.5610À 5
1.0610À 4
À
À
1.3610À 4 2.5610À 3
aThe molar concentration of the test compound that killed 50% of the cells (or 50% cell
survival) upon incubation for 3À5 days at 37 ꢁC in a humidi®ed atmosphere of 95% air and
5% CO2 (Mean value, n ˆ 6).
bTransformed ®broblast sarcoma cell line.
cThese cells were transfected by, and expressed, the herpes simplex virus type 1 thymidine
kinase (HSV-1 TK) gene.
dHuman osteosarcoma cell line.
eMouse mammary carcinoma cell line.
fHuman osteosarcoma cell line.
gIUdR ˆ 5-iodo-20-deoxyuridine.
hFUdR ˆ 5-¯uoro-20-deoxyuridine.
blocking TS, the enzyme which catalyzes the methylation of 20-deoxyuridine-
50-monophosphate to thymidine-50-monophosphate21,22. Furthermore, inhi-
bition of TS is the mechanism by which certain nucleosides such as (E)-5-(2-
iodovinyl)-20-deoxyuridine and 5-(1-azidovinyl)-20-deoxyuridine exhibit their
cytostatic e€ect23.
CONCLUSIONS
The novel class of 1-(2-deoxy-ꢀ-D-ribofuranosyl)-7-substituted-iso-
carbostyrils reported here lacks anticancer=antiviral activity. The results
obtained in this study suggest that these isocarbostyril mimics are not suitable
thymidine mimics. In the event that the failure of this class of compounds to
undergo phosphorylation to the 50-monophosphate is responsible for the
lackof anticancer=antiviral activities observed, it is possible that 50-cycloSal-
pronucleotide derivatives designed to adhere to the kinase-bypass concept24,25

1542
NAIMI ET AL.
could result in the transformation of these inactive unnatural nucleoside
mimics into a class of e€ective anticancer=antiviral agents.
EXPERIMENTAL SECTION
General Methods. Melting points were determined with a Thomas
1
Hoover capillary apparatus and are uncorrected. H NMR, 13C NMR, and
19F NMR spectra were measured on a Bruker AM-300 spectrometer in
CDCl3, MeOH-d4, or DMSO-d6. Proton chemical shifts (d) are given relative
to internal TMS (d 0). 13C NMR spectra were acquired using the J modu-
lated spin echo technique where methyl and methine carbon resonances
appear as positive peaks and methylene and quaternary carbons appear as
negative peaks, and carbon chemical shifts (d) are given relative to CDCl3 (d
77). Fluorine chemical shifts (d) are given relative to external C6F6 (d 0).
Infrared spectra were recorded on a Nicolet Magna 550 IR spectrometer
using air as reference. Elemental analyses were performed by the Micro-
Analysis Service Laboratory, Department of Chemistry, University of
Alberta. Silica gel 60A (Silicycle Co., 230À400 mesh) was used for all silica
gel column ¯ash chromatography separations. 3,5-Bis-O-(p-chlorobenzoyl)-
2-deoxy-a-D-ribofuranosyl chloride (3) was prepared according to the pre-
viously reported procedure10. (E)-1-Trimethylsilyl-2-tributylstannylethene
(16) was prepared according to the literature procedure17. All other reagents
were purchased from the Aldrich Chemical Co. The semiempirical PM3
calculation for isocarbostyril (4) was determined using the Alchemy 2000
simulation program, Version 2 (SciVision, Burlington, MA, U.S.A.).
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]isocarbosty-
ril (5) and 1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-a-D-ribofuranosyl]isoc-
arbostyril (6). Dry MeCN(220 mL) and bis(trimethylsilyl)acetamide (17 mL,
68 mmol) were added to isocarbostyril (4, 10.0 g, 68 mmol) with stirring at
25 ꢁC. Dissolution occurred within 10 min. Following a 30 min reaction time,
additional MeCN(240 mL) and 3 (24.0 g, 56 mmol) were added. The reaction
mixture was cooled to 0 ꢁC, SnCl4 (13.8 mL of a 1 M solution in CH2Cl2,
13.8 mmol) was added dropwise, and the reaction was allowed to proceed at
0 ꢁC for 30 min with stirring. EtOAc (1000 mL) was added, and the resulting
solution was consecutively extracted with saturated NaHCO3 (36200 mL)
and then brine (200 mL). The organic extracts were dried (Na2SO4), the
solvents were removed in vacuo, and the residue obtained was puri®ed by
¯ash silica gel column chromatography. Elution with CH2Cl2-EtOAc
(40 : 040 : 1, v=v) gave the b-anomer 5 (10.1 g, 27%) as white crystals (hexane-
CH2Cl2, mp 185À186 ꢁC), and the a-anomer 6 (14.3 g, 39%) as white crystals
(hexane-CH2Cl2, mp 172À173 ꢁC), respectively.
ꢀ-Anomer 5: 1H NMR (CDCl3) d 8.42 (d, J ˆ 7.6 Hz, 1H, H-8),
7.95À8.04 (m, 4H, ortho-benzoyl hydrogens), 7.38À7.69 (complex

ANALOGS OF DEOXYTHYIMIDINE
1543
multiplets, 8H, H-3, H-4, H-5, H-6, meta-benzoyl hydrogens), 6.83 (dd,
J ˆ 8.2, 5.8 Hz, 1H, H-10), 6.48 (d, J ˆ 7.6 Hz, 1H, H-7), 5.64 (br d,
J ˆ 6.7 Hz, 1H, H-30), 4.68À4.70 (m, 2H, H-50), 4.55À4.63 (m, 1H, H-40),
2.90 (ddd, J ˆ 14.3, 5.8, 1.6 Hz, 1H, H-20a), 2.34 (ddd, J ˆ 14.3, 8.2, 7.0 Hz,
1H, H-20b); 13C NMR (CDCl3) d 165.21, 165.09, 161.67, 140.15, 139.91,
136.59, 132.61, 131.13, 130.91, 128.90, 127.81, 127.59, 127.02, 125.90, 125.81,
125.14, 106.73, 85.51, 82.43, 75.47, 64.62, 38.54. Anal. calcd. for
C28H21Cl2NO6: C, 62.46; H, 3.93; N, 2.60. Found: C, 62.56; H, 3.87; N, 2.64.
a-Anomer 6: 1H NMR (CDCl3) d 8.43 (d, J ˆ 8.2 Hz, 1H, H-3),
7.20À8.20 (complex multiplets, 12H, H-4, H-5, H-6, H-8, benzoyl
hydrogens), 6.65 (dd, J ˆ 6.7, 1.5 Hz, 1H, H-10), 6.57 (d, J ˆ 7.6 Hz, 1H, H-
7), 5.63 (d, J ˆ 6.1 Hz, 1H, H-30), 4.93À4.99 (m, 1H, H-40), 4.64 (dd, J ˆ 11.9,
4.9 Hz, 1H, H-50a), 4.56 (dd, J ˆ 11.9, 5.2 Hz, 1H, H-50b), 3.03 (ddd, J ˆ 15.9,
6.7, 6.1 Hz, 1H, H-20b), 2.69 (d, J ˆ 15.9 Hz, H-20a); 13C NMR (CDCl3) d
165.14, 164.69, 161.63, 139.91, 139.85, 136.78, 132.45, 130.98, 130.89, 128.87,
128.61, 127.79, 127.49, 127.37, 126.80, 125.93, 125.71, 105.62, 88.03, 84.81,
75.23, 64.45, 38.89. Anal. calcd. for C28H21Cl2NO6: C, 62.46; H, 3.93; N,
2.60. Found C, 62.48; H, 3.89; N, 2.68.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)isocarbostyril (7). NaOMe (30 mg,
0.559 mmol) was added to a suspension of 5 (100 mg, 0.186 mmol) in MeOH
(4 mL), and the mixture was stirred at 25 ꢁC for 30 min. The reaction was
quenched via addition of NH4Cl (100 mg), and the solvent was removed in
vacuo to give a residue that was puri®ed via ¯ash silica gel column chro-
matography using MeOH-CH2Cl2 (1:9, v=v) as eluent to a€ord 7 (43 mg,
1
90%) directly as white crystals: mp 109À110 ꢁC; H NMR (CD3OD) d 8.29
(d, J ˆ 8.2 Hz, 1H, H-3), 7.77 (d, J ˆ 7.6 Hz, 1H, H-8), 7.70 (dt, J ˆ 7.9,
1.2 Hz, 1H, H-5), 7.61 (d, J ˆ 7.9 Hz, 1H, H-6), 7.51 (ddd, J ˆ 8.2, 7.9, 3.7 Hz,
1H, H-4), 6.72 (d, J ˆ 7.6 Hz, 1H, H-7), 6.67À6.72 (m, 1H, H-10), 4.38À4.46
(m, 1H, H-30), 3.99 (dd, J ˆ 7.3, 3.7 Hz, 1H, H-40), 3.82 (dd, J ˆ 11.9, 3.6 Hz,
1H, H-50a), 3.75 (dd, J ˆ 11.9, 3.9 Hz, 1-H, H-50b), 2.41 (ddd, J ˆ 13.4, 6.1,
3.4 Hz, 1H, H-20a), 2.18 (ddd, J ˆ 13.4, 7.0, 6.7 Hz, 1H, H-20b); 13C N MR
(CD3OD) d 163.4, 138.4, 133.8, 128.1, 127.9, 127.7, 127.1, 126.4, 107.9, 88.8,
86.4, 72.3, 63.0, 41.9. Anal. calcd. for C14H15NO4: C, 64.35; H, 5.78; N, 5.36.
Found: C, 64.28; H, 5.81; N, 5.42.
1-(2-Deoxy-a-D-ribofuranosyl)isocarbostyril (8). NaOMe (0.15 g,
2.79 mmol) was added to a suspension of 6 (0.50 g, 0.93 mmol) in MeOH
(20 mL), and the reaction was allowed to proceed at 25 ꢁC for 30 min with
stirring. The reaction was quenched via addition of NH4Cl (300 mg), and the
solvent was removed invacuo to give a residue that was puri®ed by ¯ash silica
gel column chromatography using MeOH-CH2Cl2 (1:9, v=v) as eluent to
1
a€ord 8 (0.23 g, 94%) as a white foam: mp 86À88 ꢁC; H NMR (CD3OD) d
8.25 (d, J ˆ 8.2 Hz, 1H, H-3), 7.69 (d, J ˆ 7.6 Hz, 1H, H-8), 7.67 (ddd, J ˆ 7.6,
7.0, 1.2 Hz, 1H, H-5), 7.59 (d, J ˆ 7.6 Hz, 1H, H-6), 7.47 (ddd, J ˆ 8.2, 7.0,

1544
NAIMI ET AL.
1.2 Hz, 1H, H-4), 6.70 (d, J ˆ 7.8 Hz, 1H, H-7), 6.53 (dd, J ˆ 7.3, 3.1 Hz, 1H,
H-10), 4.33À4.40 (m, 2H, H-30, H-40), 3.68 (dd, J ˆ 11.9, 4.3 Hz, 1H, H-50a),
3.61 (dd, J ˆ 11.9, 4.3 Hz, 1H, H-50b), 2.82 (ddd, J ˆ 14.3, 7.3, 7.1 Hz, 1H, H-
20b), 2.10 (ddd, J ˆ 14.3, 3.1, 3.0 Hz, 1H, H-20a); 13C NMR (CD3OD) d
163.4, 138.6, 133.7, 128.2, 127.9, 127.8, 127.7, 127.1, 126.4, 107.3, 90.6, 88.4,
72.5, 63.4, 42.5. Anal. calcd. for C14H15NO4: C, 64.35; H, 5.78; N, 5.36.
Found C, 64.26; H, 5.73; N, 5.30.
1-[3,5-Bis-O-(p-chlorobenzoyl)-b-D-ribofuranosyl]-4,7-dinitroisocarbo-
styril (9). Tri¯uoroacetic anhydride (10 mL) and ground NH4NO3 (0.45 g,
5.58 mmol) were added in aliquots to a solution of 5 (1.0 g, 1.86 mmol) in dry
CH2Cl2 (10 mL), and the reaction was allowed to proceed at 25 ꢁC for 1 h
with stirring. The resultant yellow solution was poured onto cold water
(100 mL), neutralized with NaHCO3, and extracted with CH2Cl2
(36100 mL). The combined organic extracts were washed consecutively with
water (26100 mL), and then brine (2650 mL), prior to drying (Na2SO4).
The solvent was removed invauco, and the residue was puri®ed via ¯ash silica
gel column chromatography using hexane-EtOAc (5:1, v=v) as eluent to give
the bis-3,5-O-ester (0.24 g, 21%) directly as pale yellow crystals: mp
1
170À171 ꢁC; H NMR (CDCl3) d 9.25 (d, J ˆ 2.4 Hz, 1H, H-3), 9.09 (s, 1H,
H-8), 8.85 (d, J ˆ 9.2 Hz, 1H, H-6), 8.61 (dd, J ˆ 9.2, 2.4 Hz, 1H, H-5),
7.88À8.04 (m, 4H, ortho-benzoyl hydrogens), 7.30À7.49 (m, 4H, meta-ben-
zoyl hydrogens), 6.59 (dd, J ˆ 7.6, 5.8 Hz, 1H, H-10), 5.69 (d, J ˆ 6.7 Hz, 1H,
H-30), 4.83À4.89 (m, 3H, H-40, H-50), 3.15 (ddd, J ˆ 13.4, 5.8, 1.2 Hz, 1H, H-
20a), 2.41 (ddd, J ˆ 13.4, 7.6, 7.0 Hz, 1H, H-20b); 13C NMR (CDCl3) d 165.04,
165.01, 159.6, 146.8, 140.4, 140.0, 134.4, 133.1, 131.1, 130.7, 129.0, 128.8,
128.2, 127.4, 127.2, 125.6, 124.7, 124.3, 87.6, 84.0, 75.1, 64.1, 39.6. NaOMe
(39 mg, 0.72 mmol) was added to a suspension of the bis-3,5-O-ester obtained
above (0.14 g, 0.24 mmol) in MeOH (5.5 mL), and the reaction was allowed
to proceed with stirring at 25 ꢁC for 30 min. The reaction was quenched via
addition of NH4Cl (0.15 g), the solvent was removed invacuo, and the residue
obtained was puri®ed via ¯ash silica gel column chromatography using
MeOH-CH2Cl2 (1:9, v=v) as eluent to a€ord 9 (70 mg, 89.5%) directly as
1
light yellow crystals: mp 175À176 ꢁC; H NMR (DMSO-d6) d 9.57 (s, 1H,
H-8), 8.93 (d, J ˆ 2.4 Hz, 1H, H-3), 8.75 (d, J ˆ 9.1 Hz, 1H, H-6), 8.66 (dd,
J ˆ 9.1, J ˆ 2.4 Hz, 1H, H-5), 6.37 (t, J ˆ 5.9 Hz, 1H, H-10), 5.36 (d,
J ˆ 4.3 Hz, 1H, 30-OH), 5.27 (t, J ˆ 4.5 Hz, 1H, 50-OH), 4.28À4.38 (m, 1H, H-
30), 3.96À4.03 (m, 1H, H-40), 3.60À3.72 (m, 2H, H-50a, H-50b), 2.40À2.50 (m,
1H, H-20a), 2.27 (ddd, J ˆ 12.0, 6.1, 5.9 Hz, 1H, H-20b); 13C NMR (DMSO-
d6) d 159.4, 145.8, 137.3, 133.0, 127.9, 127.7, 125.0, 124.0, 123.0, 88.3, 86.8,
69.5, 60.3, 41.0. Anal. calcd. for C14H13N3O8: C, 47.87; H, 3.73; N, 11.96.
Found: C, 47.84; H, 3.63; N, 11.82.
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-iodoisoc-
arbostyril (10). A solution of ICl (3.04 g, 18.4 mmol) in CH2Cl2 (30 mL) was
added dropwise to a solution of 5 (8.0 g, 14.88 mmol) in dry CH2Cl2 (96 mL)

ANALOGS OF DEOXYTHYIMIDINE
1545
with stirring, the reaction mixture was heated to re¯ux temperature, and then
cooled to 25 ꢁC. Saturated NaHCO3 (200 mL) was added followed by the
drop wise addition of saturated Na2S2O3 until a clear solution was main-
tained. Extraction with CH2Cl2 (500 mL), washing the aqueous fraction with
CH2Cl2 (26200 mL), drying the combined CH2Cl2 extracts (Na2SO4), and
removal of the solvent invacuo gave a residue that was puri®ed by ¯ash silica
gel column chromatography. Elution with hexane-EtOAc (7:1, v=v) as eluent
a€orded a pale yellow oil (10.8 g) which was recrystallized from CH2Cl2-
1
hexane to give 10 (8.64 g, 87%) as white crystals: mp 145À146 ꢁC; H N MR
(CDCl3) d 8.44 (dd, J ˆ 7.9, 1.2 Hz, 1H, H-3), 8.01À8.05 (m, 4H, ortho-
benzoyl hydrogens), 7.85 (s, 1H, H-8), 7.72À7.82 (m, 1H, H-5), 7.65 (dd,
J ˆ 7.6, 1.2 Hz, 1H, H-6), 7.57 (t, J ˆ 7.9 Hz, 1H, H-4), 7.40À7.49 (m, 4H,
meta-benzoyl hydrogens), 6.80 (dd, J ˆ 8.5, 5.5 Hz, 1H, H-10), 5.66 (d,
J ˆ 6.7 Hz, 1H, H-30), 4.74À4.84 (m, 2H, H-50), 4.60À4.66 (m, 1H, H-40), 2.93
(ddd, J ˆ 14.1, 5.5, 1.5 Hz, 1H, H-20a), 2.36 (ddd, J ˆ 14.1, 8.5, 6.7 Hz, 1H,
H-20b); 13C NMR (CDCl3) d 165.1, 165.0, 160.8, 140.1, 139.8, 136.6, 133.5,
131.6, 130.9, 128.9, 127.9, 127.6, 127.4, 126.0, 123.3, 85.5, 82.8, 75.4, 72.6,
64.4, 38.8. Anal. calcd. for C28H20Cl2INO6: C, 50.62; H, 3.03; N, 2.10.
Found: C, 50.42; H, 3.00; N, 2.14.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-iodoisocarbostyril (11). NaOMe
(488 mg, 9.04 mmol) was added to a suspension of 10 (2.0 g, 3.01 mmol) in
MeOH (68 mL), and reaction was allowed to proceed at 25 ꢁC for 30 min with
stirring. The reaction was quenched via addition of NH4Cl (1.5 g), the solvent
was removed in vacuo, and the residue obtained was puri®ed via ¯ash silica
gel column chromatography using MeOH-CH2Cl2 (1:9, v=v) as eluent to
1
yield 11 (0.95 g, 82%) directly as white crystals: mp 158À159 ꢁC; H N MR
(CD3OD) d 8.30 (s, 1H, H-8), 8.25 (d, J ˆ 7.9 Hz, 1H, H-3), 7.70 (dd, J ˆ 7.9,
7.0 Hz, 1H, H-5), 7.69 (d, J ˆ 7.9 Hz, 1H, H-6), 7.58 (dd, J ˆ 7.9, 7.0 Hz, 1H,
H-4), 6.62 (dd, J ˆ 6.7, 6.4 Hz, 1H, H-10), 4.40À4.48 (m, 1H, H-30), 3.98À4.05
(m, 1H, H-40), 3.85 (dd, J ˆ 11.9, 3.3 Hz, 1H, H-50a), 3.76 (dd, J ˆ 11.9,
3.7 Hz, 1H, H-50b), 2.40À2.50 (complex m, 1H, H-20a), 2.20 (ddd, J ˆ 13.7,
6.7, 6.4 Hz, 1H, H-20b); 13C NMR (CD3OD) d 162.6, 138.4, 134.7, 131.5,
128.9, 128.8, 128.7, 124.2, 89.0, 86.6, 72.8, 72.0, 62.6, 42.3. Anal. calcd. for
C14H14INO4: C, 43.43; H, 3.64; N, 3.61. Found: C, 43.32; H, 3.69; N, 3.72.
1-(3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-tri¯uoro-
methylisocarbostyril (12). A mixture of 10 (1.0 g, 1.51 mmol), CuI (0.43 g,
2.26 mmol), and anhydrous KF (0.13 g, 2.26 mmol) in dry DMF (5 mL) was
stirred under argon as the temperature was increased. When the reaction
temperature reached 60 ꢁC, ClCF2CO2Me (0.32 mL, 3.01 mmol) was added,
and the reaction was allowed to proceed at 120 ꢁC for 6 h under an argon
atmosphere. After cooling to 25 ꢁC, the reaction mixture was poured onto
ice-water (100 mL), the solution was ®ltered, and the residue was washed with
CH2Cl2 (3650 mL) and the aqueous layer was extracted with CH2Cl2
(36100 mL). The combined CH2Cl2 extracts were washed with water

1546
NAIMI ET AL.
(100 mL), the organic fraction was dried (Na2SO4), and the solvent was
removed in vacuo. The residue obtained was puri®ed via ¯ash silica gel
column chromatography using hexane-EtOAc (9:1, v=v) as eluent to give 12
1
(0.65 g, 71%) as a white foam: mp 85À86 ꢁC; H NMR (CDCl3) d 8.46 (d,
J ˆ 7.9 Hz, 1H, H-3), 7.90À8.04 (m, 4H, ortho-benzoyl hydrogens), 7.96 (s,
1H, H-8), 7.72À7.84 (m, 2H, H-5, H-6), 7.62 (ddd, J ˆ 8.2, 7.9, 4.0 Hz, 1H,
H-4), 7.36À7.50 (m, 4H, meta-benzoyl hydrogens), 6.71 (dd, J ˆ 8.2, 5.5 Hz,
1H, H-10), 5.63 (d, J ˆ 6.4 Hz, 1H, H-30), 4.73À7.85 (m, 2H, H-50), 4.64À4.70
(m, 1H, H-40), 2.99 (dd, J ˆ 14.6, 5.5 Hz, 1H, H-20a), 2.32 (ddd, J ˆ 14.6, 8.2,
6.7 Hz, 1H, H-20b); 19F NMR (CDCl3) d 100.79 (s, CF3).
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-tri¯uoromethylisocarbostyril
(13).
A solution of NaOMe in methanol (6 mL of 0.4 M) was added to 12 (0.60 g,
1 mmol), and the reaction was allowed to proceed with stirring at 25 ꢁC for
30 min. The reaction was quenched via addition of NH4Cl (0.5 g), the sol-
vent was removed in vacuo, and the residue was puri®ed via ¯ash silica gel
column chromatography using MeOH-CH2Cl2 (1:9, v=v) as eluent to give
13 (0.32 g, 97%) directly as white crystals: mp 163À164 ꢁC; 1H N MR
(CD3OD) d 8.53 (s, 1H, H-8), 8.35 (d, J ˆ 8.2 Hz, 1H, H-3), 7.74À7.83 (m,
2H, H-5, H-6), 7.59 (ddd, J ˆ 8.2, 7.9, 3.2 Hz, 1H, H-4), 6.61 (t, J ˆ 6.4 Hz,
1H, H-10), 4.42À4.50 (m, 1H, H-30), 4.01À4.08 (m, 1H, H-40), 3.89 (dd,
J ˆ 11.9, 3.0 Hz, 1H, H-50a), 3.77 (dd, J ˆ 11.9, 3.3 Hz, 1H, H-50b), 2.50
(ddd, J ˆ 13.1, 6.4, 4.0 Hz, 1H, H-20a), 2.25 (ddd, J ˆ 13.1, 6.7, 6.4 Hz, 1H,
H-20b); 13C NMR (CD3OD) d 162.8, 134.5, 132.8, 130.1 (q, J ˆ 5.5 Hz, C-8),
129.9, 129.8, 126.5, 125.6 (q, J ˆ 270.3 Hz, CF3), 124.2, 107.7 (q, J ˆ
31.8 Hz, C-7), 89.2, 87.1, 72.0, 62.4, 42.5; 19F NMR (CD3OD) d 103.59 (s,
CF3). Anal. calcd. for C15H14F3NO4: C, 54.71; H, 4.28; N, 4.25. Found: C,
54.74; H, 4.20; N, 4.14.
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-cyanoiso-
carbostyril (14). Compound 10 (1.0 g, 1.51 mmol) was added to Zn(CN)2
(107 mg, 0.91 mmol) and (Ph3P)4Pd (70 mg, 0.06 mmol) in dry DMF (2 mL),
and the yellow slurry was heated at 80À90 ꢁC under an argon atmosphere
with stirring for 6 h. The reaction mixture was cooled to 25 ꢁC, toluene
(100 mL) was added, the mixture was washed with 2NNH 4OH (2650 mL)
and then brine (50 mL), and the organic fraction was dried (Na2SO4).
Removal of the solvent in vacuo gave a residue that was puri®ed via ¯ash
silica gel column chromatography using hexane-EtOAc (6:1, v=v) as eluent to
give 14 (0.71 g, 84%) as white crystals (hexane-CH2Cl2): mp 185À186 ꢁC; IR
1
(KBr): 2221 (CN), 1726, 1669 (CˆO) cmÀ 1; H NMR (CDCl3) d 8.34 (d,
J ˆ 7.9 Hz, 1H, H-3), 8.04 (s, 1H, H-8), 7.87À7.95 (m, 4H, ortho-benzoyl
hydrogens), 7.68À7.78 (m, 2H, H-5, H-6), 7.55 (ddd, J ˆ 7.9, 5.5, 2.7 Hz, 1H,
H-4), 7.31À7.42 (m, 4H, meta-benzoyl hydrogens), 6.54 (dd, J ˆ 7.9, 5.5 Hz,
1H, H-10), 5.55 (dd, J ˆ 6.4, 1.5 Hz, 1H, H-30), 4.64À4.81 (m, 2H, H-50),
4.54À4.62 (m, 1H, H-40), 2.94 (ddd, J ˆ 13.1, 5.5, 1.5 Hz, 1H, H-20a), 2.17
(ddd, J ˆ 13.1, 7.9, 6.7 Hz, 1H, H-20b); 13C NMR (CDCl3) d 165.1, 164.9,

ANALOGS OF DEOXYTHYIMIDINE
1547
160.4, 140.2, 140.0, 134.6, 133.9, 132.9, 131.0, 130.8, 130.7, 129.0, 128.8,
128.6, 128.1, 127.4, 127.3, 124.8, 124.0, 123.9, 115.3, 91.8, 86.5, 83.3, 75.1,
64.0, 39.1.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-cyanoisocarbostyril (15). NaOMe
(75 mg, 1.38 mmol) was added to a suspension of 14 (0.26 g, 0.46 mmol) in
MeOH (8 mL), and the mixture was stirred at 25 ꢁC for 30 min. The reaction
was quenched via addition of NH4Cl (0.3 g) and the solvent was removed
in vacuo to give a residue that was puri®ed via ¯ash silica gel column chro-
matography using MeOH-CH2Cl2 (1:19, v=v) as eluent to a€ord 15 (0.13 g,
98%) directly as white crystals: mp 140À141 ꢁC; IR (KBr): 2232 (CN), 1722
1
(CˆO) cmÀ 1; H NMR (CD3OD) d 8.65 (s, 1H, H-8), 8.28 (dd, J ˆ 7.9,
1.2 Hz, 1H, H-3), 7.86 (ddd, J ˆ 8.2, 7.9, 1.5 Hz, 1H, H-5), 7.75 (dd, J ˆ 7.9,
1.2 Hz, 1H, H-6), 7.60 (ddd, J ˆ 8.2, 7.9, 1.2 Hz, 1H, H-4), 6.51 (dd, J ˆ 6.4,
6.1 Hz, 1H, H-10), 4.42À4.48 (m, 1H, H-30), 4.18À4.25 (m, 1H, H-40), 3.88
(dd, J ˆ 12.2, 3.0 Hz, 1H, H-50a), 3.77 (dd, J ˆ 12.2, 3.3 Hz, 1H, H-50b), 2.52
(ddd, J ˆ 13.7, 6.1, 3.7 Hz, 1H, H-20a), 2.20 (dt, J ˆ 13.7, 6.4 Hz, 1H, H-20b);
13C NMR (CD3OD) d 162.1, 138.2, 135.0, 134.6, 129.4, 128.7, 125.9, 124.5,
116.7, 91.8, 89.3, 87.4, 71.7, 62.3, 42.6. Anal. calcd. for C15H14N2O4: C,
62.93; H, 4.92; N, 9.78. Found: C, 62.87; H, 4.85; N, 9.53.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-(E)-(2-trimethylsilylvinyl)isocarbos-
tyril (17). (Ph3P)2PdCl2 (102 mg, 0.145 mmol) was added to a mixture of 11
(0.56 g, 1.45 mmol) and (E)-1-trimethylsilyl-2-tributylstannylethene (1.78 g,
3 mmol) in dry MeCN(22 mL) under argon, and the mixture was stirred
vigorously at 60 ꢁC for 24 h. Removal of the solvent invacuo gave a residue
that was puri®ed via ¯ash silica gel column chromatography using hexane-
EtOAc (1:3, v=v) as eluent to give 17 (0.43 g, 83%) as white crystals
1
(hexane-CH2Cl2): mp 132-133 ꢁC; H N MR (CDC3l) d 8.41 (d, J ˆ 8:2 Hz,
1H, H-3), 7.76 (d, J ˆ 7:6 Hz, 1H, H-6), 7.67 (dd, J ˆ 8:5; 6:7 Hz, 1H, H-5),
7.57, (s, 1H, H-8), 7.48 (dd, J ˆ 8:2; 6:7 Hz, 1H, H-4), 7.10 (d, J ˆ 18:9 Hz,
1H, CH ˆ CHTMS), 6.54 (dd, J ˆ 7:0; 6:4 Hz, 1H, H-10), 6.32 (d,
J ˆ 18:9 Hz, 1H, CH ˆ CHTMS), 4.65À4.72 (m, 1H, H-30), 4.16À4.22 (m,
1H, H-40), 4.02À4.10 and 3.65À3.74 (two m, 1H each, 30-OH, 50-OH),
3.88À4.00 (m, 2H, H-50), 2.47À2.58 (m, 2H, H-20), 0.18 (s, 9H, SiMe3); 13C
NMR (CDCl3) d 161.7, 136.9, 135.2, 132.6, 132.1, 128.2, 126.9, 125.2,
124.9, 122.5, 117.9, 87.9, 87.3, 71.7, 62.6, 40.4, À 1.1.
1-(2-Deoxy-b-D-ribofuranosyl)-7-(E)-(2-iodovinyl)isocarbostyril (18).
After dissolution of 17 (0.25 g, 0.7 mmol) in dry MeCN(12 mL), ICl (114 mg,
0.7 mmol) was added immediately, and the reaction was allowed to proceed
at 0 ꢁC for 15 min with stirring. Removal of the solvent in vauco gave a
residue that was puri®ed by ¯ash silica gel column chromatography using
hexane-EtOAc (1:3, v=v) as eluent to give a pale yellow oil which was
recrystallized from CH2Cl2-hexane to yield 18 (0.20 g, 70%) directly as white
1
crystals: mp 125À126 ꢁC; H NMR (CD3OD) d 8.35 (d, J ˆ 8:2 Hz, 1H,
H-3), 8.01 (s, 1H, H-8), 7.72À7.85 (m, 2H, H-5, H-6), 7.71 (d, J ˆ 14:7 Hz,

1548
NAIMI ET AL.
1H, CH ˆ CHI), 7.52À7.62 (m, 1H, H-4), 6.93 (d, J ˆ 14:7 Hz, 1H,
CH=CHI), 6.66 (dd, J ˆ 6:7; 6:4 Hz, 1H, H-10), 4.42À4.50 (m, 1H, H-30),
3.97À4.02 (m, 1H, H-40), 3.86 (dd, J ˆ 11:9; 3:0 Hz, 1H, H-5 a), 3.77 (dd,
J ˆ 11:9; 3:7 Hz, 1H, H-5 b), 2.44 (ddd, J ˆ 13:4; 6:7; 6:2 Hz, 1H, H-20a), 2.24
(ddd, J ˆ 13:4; 6:7; 6:2 Hz, 1H, H-20b); 13C NMR (CD3OD + DMSO-d6) d
162.5, 139.9, 135.7, 134.1, 128.8, 127.9, 126.1, 126.0, 124.1, 117.8, 89.1, 86.5,
78.7, 71.8, 63.2, 42.3. Anal. calcd. for C16H16INO4: C, 46.50; H, 3.90; N,
3.38. Found: C, 46.46; H, 3.75; N, 3.15.
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl)-7-(2-trimethyl-
silylethynyl)isocarbostyril (19). A mixture of 10 (2.0 g, 3.01 mmol), CuI
(90 mg), (Ph3P)2PdCl2 (110 mg), and (trimethylsilyl)acetylene (0.85 mL,
6.02 mmol) in dry Et3N(180 mL) was stirred at 50 À60 ꢁC for 5 h. The solvent
was removed in vacuo, the residue was dissolved in CH2Cl2, washed with
saturated aqueous ethylenediaminetetraacetic acid disodium salt and then
brine, the organic fraction was dried (Na2SO4), and the solvent was removed
in vacuo. The residue obtained was puri®ed via ¯ash silica gel column chro-
matography using hexane-EtOAc (7:1, v=v) as eluent to give 19 (1.5 g,78%)
as white crystals (hexane-CH2Cl2): mp 102À103 ꢁC; 1H NMR (CDCl3) d 8.44
(d, J ˆ 7:9 Hz, 1H, H-3), 8.00À8.05 (m, 4H, ortho-benzoyl hydrogens), 7.94
(d, J ˆ 7:6 Hz, 1H, H-6), 7.82 (s, 1H, H-8), 7.76 (dd, J ˆ 7:6; 7:0 Hz, 1H, H-
5), 7.60 (dd, J ˆ 7:9; 7:0 Hz, 1H, H-4), 7.41À7.49 (m, 4H, meta-benzoyl
hydrogens), 6.80 (dd, J ˆ 8:2; 5:5 Hz, 1H, H-10), 5.64 (br d, J ˆ 6:7 Hz, 1H,
H-30), 4.85 (dd, J ˆ 12:2; 3:7 Hz, 1H, H-50a), 4.72 (dd, J ˆ 12:2; 3:4 Hz, 1H,
H-50b), 4.60À4.65 (m, 1H, H-40), 2.94 (dd, J ˆ 14:4; 5:5 Hz, 1H, H-20a), 2.32
(ddd, J ˆ 14:4; 8:2; 7:9 Hz, 1H, H-20b), 0.26 (s, 9H, SiMe3); 13C N MR
(CDCl3) d 165.1, 164.9, 160.7, 140.4, 140.0, 139.7, 135.5, 133.0, 131.0, 130.8,
130.0, 128.86, 128.82, 127.7, 127.5, 124.9, 122.1, 102.1, 98.9, 98.3, 85.6, 82.7,
75.4, 64.5, 38.7, 0.01.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-ethynylisocarbostyril (20). A solution
of NaOMe in methanol (18.5 mL of 0.2 N) was added to 19 (0.50 g,
0.79 mmol), and the reaction was allowed to proceed with stirring at 25 ꢁC for
20 min. The reaction was quenched via addition of NH4Cl (0.5 g) and the
solvent was removed in vacuo. Puri®cation of the residue via ¯ash silica gel
column chromatography using MeOH-CH2Cl2 (1:19, v=v) as eluent a€orded
20 (0.22 g, 98%) directly as white crystals: mp 170À171 ꢁC; 1H N MR
(CD3OD) d 8.30 (d, J ˆ 8:2 Hz, 1H, H-3), 8.15 (s, 1H, H-8), 7.94 (d,
J ˆ 7:9 Hz, 1H, H-6), 7.76 (ddd, J ˆ 8:5; 7:9; 1:5 Hz, 1H, H-5), 7.54 (ddd,
J ˆ 8:5; 8:2; 0:9 Hz, 1H, H-4), 6.62 (dd, J ˆ 6:7; 6:4 Hz, 1H, H-10), 4.39À4.47
(m, 1H, H-30), 3.98À4.02 (m, 1H, H-40), 3.84 (dd, J ˆ 12:2; 3:4 Hz, 1H, H-
50a), 3.76 (dd, J ˆ 12:2; 3:7 Hz, 1H, H-50b), 3.73 (s, 1H, H-CꢀC), 2.41 (ddd,
J ˆ 13:7; 6:4; 3:7 Hz, 1H, H-20a), 2.21 (ddd, J ˆ 13:7; 6:7; 6:7 Hz, 1H, H-20b);
13C NMR (CD3OD) d 165.0, 139.7, 136.8, 135.9, 131.1, 131.0, 128.4, 128.3,
104.6, 91.6, 89.3, 84.9, 74.7, 65.3, 44.8. Anal. calcd. for C16H15NO4: C, 67.35;
H, 5.29; N, 4.90. Found: C, 67.30; H, 5.32; N, 4.91.

ANALOGS OF DEOXYTHYIMIDINE
1549
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-iodoethy-
nylisocarbostyril (21). N-Iodosuccinimide (214 mg, 0.95 mmol) and ground
AgNO3 (9 mg, 0.053 mmol) were added to a solution of 19 (0.50 g,
0.79 mmol) in dry DMF (5 mL) under argon at 0 ꢁC with stirring. The
reaction vessel was wrapped with aluminium foil to protect from light, and
the reaction mixture was stirred at 0 ꢁC for 2 h, and then at 25 ꢁC for 1 h. The
mixture was cooled to 0 ꢁC, mixed with cold water (100 mL), and extracted
with CH2Cl2 (36100 mL). The combined organic fractions were washed with
water and then brine, dried (Na2SO4), the solvent was removed invacuo, and
the residue was puri®ed via ¯ash silica gel column chromatography using
hexane-EtOAc (6:1, v=v) as eluent to give 21 (0.48 g, 88%) as white crystals
1
(hexane-CH2Cl2): mp 165-166 ꢁC; H NMR (CDCl3) d 8.40 (d, J ˆ 7.7 Hz,
1H, H-3), 7.94 (s, 1H, H-8), 7.85À7.95 (m, 4H, ortho-benzoyl hydrogens),
7.49À7.72 (m, 2H, H-5, H-6), 7.42À7.49 (m, 4H, meta-benzoyl hydrogens),
7.30À7.40 (m, 1H, H-4), 6.68À7.70 (m, 1H, H-10), 5.52À5.61 (m, 1H, H-30),
4.48À4.82 (m, 3H, H-50, H-40), 2.80À3.02 (m, 1H, H-20a), 2.20À2.35 (m, 1H,
H-20b).
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-iodoethynylisocarbostyril (23).
A
solution of NaOMe in MeOH (2.2 mL of 0.4 N) was added to 21 (0.25 g,
0.36 mmol) and the reaction was allowed to proceed at 25 ꢁC for 15 min. The
reaction was quenched via addition of NH4Cl (0.30 g), the solvent was
removed in vacuo, and the residue was puri®ed by ¯ash silica gel column
chromatography using MeOH-CH2Cl2 (1:19, v=v) as eluent to give 23 (0.14 g,
1
94%) directly as white crystals: mp 157À158 ꢁC; H NMR (CD3OD) d 8.29
(d, J ˆ 8.2 Hz, 1H, H-3), 8.07 (s, 1H, H-8), 7.84 (d, J ˆ 7.6 Hz, 1H, H-6), 7.73
(ddd, J ˆ 8.2, 7.9, 1.2 Hz, 1H, H-5), 7.54 (ddd, J ˆ 8.2, 7.9, 1.2 Hz, 1H, H-4),
6.60 (dd, J ˆ 6.7, 6.4 Hz, 1H, H-10), 4.35À4.46 (m, 1H, H-30), 3.98À4.01 (m,
1H, H-40), 3.84 (dd, J ˆ 12.2, 3.4 Hz, 1H, H-50a), 3.76 (dd, J ˆ 12.2, 3.9 Hz,
1H, H-50b), 2.45 (ddd, J ˆ 13.4, 6.4, 3.4 Hz, 1H, H-20a), 2.20 (ddd, J ˆ 13.4,
7.0, 6.7 Hz, 1H, H-20b); 13C NMR (CD3OD) d 162.3, 137.3, 134.3, 133.5,
128.6, 128.5, 125.7, 103.6, 89.0, 88.4, 86.7, 72.1, 62.7, 42.3, 14.1. Anal. calcd.
for C16H14INO4: C, 46.73; H, 3.43; N, 3.40. Found: C, 46.87; H, 3.39; N,
3.44.
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-(2-bro-
moethynyl)isocarbostyril (22). N-Bromosuccinimide (0.16 g, 0.9 mmol) and
ground AgNO3 (9 mg, 0.053 mmol) were added to a solution of 19 (0.45 g,
0.71 mmol) in dry DMF (5 mL) under argon at 0 ꢁC. The reaction vessel was
wrapped with aluminium foil to protect from light, and the reaction mixture
was stirred at 0 ꢁC for 2 h and then at 25 ꢁC for 4 h. The reaction mixture was
cooled to 0 ꢁC, mixed with cold-water (100 mL), and extracted with CH2Cl2
(36100 mL). The combined organic extracts were washed with water and
then brine, the organic fraction was dried (Na2SO4), and the solvent was
removed in vacuo. The residue was puri®ed by ¯ash silica gel column chro-
matography using hexane-EtOAc (6:1, v=v) as eluent to give 22 (0.34 g, 75%)

1550
NAIMI ET AL.
as white crystals (hexane-CH2Cl2): mp 151À152 ꢁC. Product 22 was hydro-
lzyed to 24 as indicted below.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-(2-bromoethynyl)isocarbostyril (24).
A solution of NaOMe in MeOH (1.6 mL of 0.4 N) was added to 22 (0.17 g,
0.265 mmol) with stirring and the reaction was allowed to proceed at 25 ꢁC
for 15 min. The reaction was quenched via addition of NH4Cl (0.15 g) and the
solvent was removed in vacuo. Puri®cation of the residue by ¯ash silica gel
column chromatography using MeOH-CH2Cl2 (1:19, v=v) as eluent yielded
24 (87 mg, 90%) directly as white crystals: mp 127À128 ꢁC; 1H N MR
(CD3OD) d 8.76 (s, 1H, H-8), 8.70 (d, J ˆ 8.2 Hz, 1H, H-3), 8.27 (dd, J ˆ 7.9,
1.2 Hz, 1H, H-6), 7.69 (ddd, J ˆ 7.9, 7.0, 1.5 Hz, 1H, H-5), 7.48 (ddd, J ˆ 8.2,
7.0, 0.9 Hz, 1H, H-4), 6.50 (dd, J ˆ 6.4, 6.1 Hz, 1H, H-10), 4.36À4.44 (m, 1H,
H-30), 3.97À4.05 (m, 1H, H-40), 3.74À3.90 (m, 2H, H-50), 2.47 (ddd, J ˆ 13.7,
6.4, 4.0 Hz, 1H, H-20a), 2.18 (ddd, J ˆ 13.7, 6.7, 6.1 Hz, 1H, H-20b); 13C
NMR (CD3OD) d 167.0, 136.4, 135.4, 134.3, 128.4, 128.1, 126.3, 126.2, 126.0,
108.1, 89.1, 87.2, 72.0, 62.7, 52.2, 42.4. Anal. calcd. for C16H14BrNO4: C,
52.76; H, 3.87; N, 3.84. Found: C, 52.82; H, 3.76; N, 3.69.
1-[3,5-Bis-O-(p-chlorobenzoyl)-2-deoxy-b-D-ribofuranosyl]-7-propyny-
lisocarbostyril (25). Dry Et3N(70 mL), (Ph P)2PdCl2 (53 mg, 0.075 mmol),
3
and CuI (0.057 g, 0.3 mmol) were added to 10 (1.0 g, 1.51 mmol), the mixture
was cooled to À 78 ꢁC, and propyne was added until a ®nal volume of 93 mL
was reached. The reaction vessel was sealed, and the reaction was allowed to
proceed at 25 ꢁC with stirring for 5 h. Excess propyne was allowed to escape
by venting the reaction vessel, and the solvent was removed in vacuo. The
residue was dissolved in CH2Cl2 (200 mL) and consecutively extracted with
5% EDTA (2650 mL), water (100 mL) and then brine (50 mL). The organic
fraction was dried (Na2SO4), the solvents were removed in vacuo, and the
residue was puri®ed by ¯ash silica gel column chromatography using hexane-
EtOAc (7:1, v=v) as eluent to give 25 (0.59 g, 68%) as a white foam: mp
1
125À126 ꢁC; H NMR (CDCl3) d 8.42 (d, J ˆ 7.9 Hz, 1H, H-3), 7.98À8.04
(m, 4H, ortho-benzoyl hydrogens), 7.92 (d, J ˆ 7.6 Hz, 1H, H-6), 7.73 (ddd,
J ˆ 8.2, 7.6, 1.5 Hz, 1H, H-5), 7.66 (s, 1H, H-8), 7.54 (dd, J ˆ 8.2, 7.9 Hz, 1H,
H-4), 7.38À7.47 (m, 4H, meta-benzoyl hydrogens), 6.84 (dd, J ˆ 8.2, 5.7 Hz,
1H, H-10), 5.62À5.66 (m, 1H, H-30), 4.79 (dd, J ˆ 12.2, 3.7 Hz, 1H, H-50a),
4.71 (dd, J ˆ 12.2, 3.4 Hz, 1H, H-50b), 4.56À4.62 (m, 1H, H-40), 2.86 (ddd,
J ˆ 14.3, 5.7, 1.5 Hz, 1H, H-20a), 2.34 (ddd, J ˆ 14.3, 8.2, 7.0 Hz, 1H, H-20b),
2.01 (s, 3H, CH3); 13C NMR (CDCl3) d 165.1, 164.9, 160.7, 140.0, 139.7,
136.0, 132.7, 131.0, 130.9, 128.8, 128.7, 127.7, 127.5, 127.3, 125.1, 125.0,
102.8, 89.0, 85.2, 82.5, 75.3, 64.5, 38.6, 4.2.
1-(2-Deoxy-ꢀ-D-ribofuranosyl)-7-propynylisocarbostyril (1). A solution
of NaOMe in MeOH (4.2 mL of 0.4 N) was added to 25 (0.4 g, 0.69 mmol) and
the reaction was allowed to proceed at 25 ꢁC for 15 min with stirring. The
reaction was quenched via addition of NH4Cl (0.3 g) and the solvent was
removed invacuo to give a residue that was puri®ed by ¯ash silica gel column

ANALOGS OF DEOXYTHYIMIDINE
1551
chromatography using MeOH-CH2Cl2 (1:19, v=v) as eluent to a€ord 1
1
(186 mg, 90%) as a white foam: mp 145À146 ꢁC; H NMR (CD3OD) d
8.22À8.32 (m, 1H, H-3), 7.95 (s, 1H, H-8), 7.84 (d, J ˆ 7.6 Hz, 1H, H-6),
7.65À7.76 (m, 1H, H-5), 7.52À7.60 (m, 1H, H-4), 6.63 (dd, J ˆ 7.5, 6.3 Hz, 1H,
H-10), 4.39À4.48 (m, 1H, H-30), 3.98À4.04 (m, 1H, H-40), 3.83 (dd, J ˆ 12.1,
3.5 Hz, 1H, H-50a), 3.76 (dd, J ˆ 12.1, 3.7 Hz, 1H, H-50b), 2.42 (ddd, J ˆ 13.7,
6.2, 3.5 Hz, 1H, H-20a), 2.17 (ddd, J ˆ 13.7, 7.4, 6.5 Hz, 1H, H-20b), 2.10 (s, 3H,
CH3); 13C NMR (CD3OD) d 162.4, 138.4, 137.6, 134.7, 131.5, 128.8, 128.2,
126.0, 124.2, 103.9, 90.0, 89.0, 86.7, 72.1, 62.7, 42.1, 3.9. Anal calcd. for
C17H17NO4: C, 68.21; H, 5.72; N,4.67. Found: C, 68.43; H, 5.61; N, 4.52.
In Vitro Cell Cytotoxicity (MTT assay). KBALB, KBALB-STK,
human 143B, human 143B-LTK and R-970-5 cells were cultured in complete
DMEM medium supplemented with 10% fetal bovine serum (FBS), and
EMT-6 cells were cultured in complete WAYMOUTH medium in 10% FBS.
Exponentially growing cells were trypsinized, centrifuged, resuspended in
growth medium, and the cell number was readjusted to 86103 cells=mL.
Cells were seeded into 96-well plates at 86102 cells=well, and incubated at
37 ꢁC in a humidi®ed 5% CO2 atmosphere for 24 h.
The test compound was dissolved in DMEM medium, and 100 mL of
this solution was added to cells in 96-well plates to produce the preselected
test compound concentration. DMEM medium (100 mL) was added to con-
trol wells. The plates were incubated for 3 days at 37 ꢁC in a humidi®ed
atmosphere consisting of 95% air and 5% CO2. At the end of the incubation,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT,
Sigma) was dissolved in phosphate-bu€ered saline (PBS) to produce a con-
centration of 5 mg/mL, ®ltered through a 0.45 mm membrane ®lter, and
diluted (1:5) with pre-warmed DMEM medium. A 50 mL aliquot of this
solution was added to each well, and the plates were incubated at 37 ꢁC for
4 h. The medium was removed from the wells, dimethyl sulfoxide (150 mL)
was added to each well, and the plates were placed on a shaker for 15 min to
dissolve the formazan crystals. The absorbance at 540 nm (A540) was mea-
sured immediately in each well using a scanning multi-well spectro-
photometer (ELISA reader). A540 values, corrected for the absorbance in
medium blanks, re¯ected the concentration of viable cells. The CC50 values
reported are the test drug concentration that reduced the A540 to 50% of the
control value (mean value, n ˆ 6). This assay20, which depends on the
metabolic reduction of MTT to colored formazan, measures cytostatic and
cytotoxic e€ects of the test drug.
ACKNOWLEDGMENTS
We are grateful to the Canadian Institutes of Health Research (Grant.
No. MOP-14480) for ®nancial support of this research, and to the Faculty of

1552
NAIMI ET AL.
Pharmacy & Pharmaceutical Sciences, University of Alberta, for a graduate
recruitment scholarship to one of us (E.N.).
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Received November 20, 2000
Accepted February 7, 2001