Enantioselective synthesis and biological evaluation of 5-o-carboranyl pyrimidine nucleosides

Article abstract of DOI:10.1016/S0968-0896(99)00222-9

Base-modified carborane-containing nucleosides such as 5-o-carboranyl-2'-deoxyuridine (CDU) when combined with neutrons have potential for the treatment of certain malignancies. Lack of toxicity in various cells, high accumulation in cancer cells and intracellular phosphorylation are desirable characteristics for modified nucleosides used in boron neutron capture therapy (BNCT) for brain tumors and other malignancies. The aim of this work was to synthesize the two β-enantiomers of several 5-o-carboranyl-containing nucleosides. These derivatives may possess favorable properties such as high lipophilicity, high transportability, the ability to be phosphorylated, and resistance to catabolism. β-Isomers of 2',3'-dihydroxynucleosides and analogues containing a heteroatom in the sugar moiety were also synthesized. Carboranyl pyrimidine nucleosides were prepared either from the parent β-D-nucleoside, β-L-nucleoside, or by a coupling reaction. The dioxolane derivative 7Scheme 1Reagents and conditions: (a) 1,1,1,3,3,3-hexamethydisilazane, ammonium sulfate, reflux, 6 h; (b) tin(IV) chloride, CH₂Cl₂, 0°C, 2h; (c) tetrabutylammonium fluoride, 1 M sol in THF, 0°C, 3h. was prepared by a coupling reaction between protected 5-o-carboranyluracil (8, CU) and the corresponding protected heterocycle. Specific catalysts were used during the N-glycosylation process to favor the formation of the β-isomer. Biological evaluation of these new chiral 5-o-carboranyl pyrimidine derivatives indicated that most of these compounds have low toxicity in a variety of normal and malignant cells and achieved high cellular levels in a lymphoblastoid cell line. Increasing the number of hydroxyl groups on the sugar moiety decreased the cellular accumulation and serum binding to different extents. Five compounds were identified for further biological evaluation as potential agents for BNCT. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00222-9

Bioorganic & Medicinal Chemistry 7 (1999) 2759±2766
Enantioselective Synthesis and Biological Evaluation of
5-o-Carboranyl Pyrimidine Nucleosides
Nicolas S. Mourier, a,b Alessandra Eleuteri, a,b Selwyn J. Hurwitz, a,b
Phillip M. Tharnish a,b and Raymond F. Schinazi a,b,
*
aDepartment of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
bVeterans A€airs Medical Center, 1670 Clairmont Road, Decatur, GA 30033, USA
Received 17 May 1999; accepted 7 July 1999
AbstractÐBase-modi®ed carborane-containing nucleosides such as 5-o-carboranyl-20-deoxyuridine (CDU) when combined with
neutrons have potential for the treatment of certain malignancies. Lack of toxicity in various cells, high accumulation in cancer cells
and intracellular phosphorylation are desirable characteristics for modi®ed nucleosides used in boron neutron capture therapy
(BNCT) for brain tumors and other malignancies. The aim of this work was to synthesize the two b-enantiomers of several 5-o-
carboranyl-containing nucleosides. These derivatives may possess favorable properties such as high lipophilicity, high transport-
ability, the ability to be phosphorylated, and resistance to catabolism. b-Isomers of 20,30-dihydroxynucleosides and analogues con-
taining a heteroatom in the sugar moiety were also synthesized. Carboranyl pyrimidine nucleosides were prepared either from the
parent b-d-nucleoside, b-l-nucleoside, or by a coupling reaction. The dioxolane derivative 7 was prepared by a coupling reaction
between protected 5-o-carboranyluracil (8, CU) and the corresponding protected heterocycle. Speci®c catalysts were used during the
N-glycosylation process to favor the formation of the b-isomer. Biological evaluation of these new chiral 5-o-carboranyl pyrimidine
derivatives indicated that most of these compounds have low toxicity in a variety of normal and malignant cells and achieved high
cellular levels in a lymphoblastoid cell line. Increasing the number of hydroxyl groups on the sugar moiety decreased the cellular
accumulation and serum binding to di€erent extents. Five compounds were identi®ed for further biological evaluation as potential
agents for BNCT. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
30 ppm.4,5 In general, carborane-cluster-containing
compounds should be more e€ective than organo-boro-
nic acids due to their high boron content and lipophilic
properties.
High boron content molecules are desirable for the
boron neutron capture therapy (BNCT) used for the
treatment of gliomas, melanomas and malignancies.1,2
This therapeutic modality combines the high accumula-
tion of boron-containing compounds targeted to the
tumor cells with neutron irradiation to produce micro-
nuclear reactions within tumors.3 The nuclear reaction
occurs by irradiating stable non-radioactive 10B with
low energy thermal neutrons, leading to cell destruction
by the high linear energy transfer radiation released in
the neutron capture reaction. To be successful, this
concept requires that a sucient amount of a non-toxic
10B carrier is delivered to the cancer cells to produce this
nuclear reaction. It has been calculated that the e€ective
dose of 10B in the tumor should be in the range of 5 to
Several carboranyl compounds have already been
synthesized such as 5-(o-carboran-1-yl)-20-deoxyuridine
(1, d-CDU, Fig. 1)6,7 and 5-o-carboranyl-1-(2-deoxy-2-
¯uoro-b-d-arabinofuranosyl)uracil (d-CFAU).8 Our
group demonstrated that d-CDU is taken up by human
lymphocytic cells (CEM) and primary human peripheral
blood mononuclear (PBM) cells and is phosphorylated
intracellularly.7 The ®nding that the 50-monophosphate
of d-CDU is formed intracellularly suggested that the 5-
o-carboranyl moiety is well tolerated and can mimic a
pyrimidine nucleoside analogue. The entrapment of this
lipophilic molecule in cells o€ers the possibility to
enhance the tumor-to-blood and tumor-to-normal tis-
sue ratios for BNCT since malignant cells divide faster
than normal tissues in compartments such as the brain
where gliomas develop. The encouraging results
obtained with d-CDU or d-CFAU prompted us to
develop the enantiomeric synthesis of new 5-o-caboranyl
Key words: l-Nucleosides; boron neutron capture therapy; antiviral
activity.
* Corresponding author at Veterans A€airs Medical Center, Medical
Research -151, 1670 Clairmont Road, Decatur, GA 30033, USA. Tel.:
+1-404-728-7711; fax: +1-404-728-7726; e-mail: rschina@emory.edu
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00222-9

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N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
with the protected dioxolane sugar (9)14 using tin(IV)
chloride (SnCl4) yielded the b-protected nucleoside 7 in
good yield (Scheme 1). The mechanism of this reaction
involves the formation of an oxonium ion at the a-face
of the sugar moiety which mainly allows attack of the
silylated base on the b-face.15±17 An equimolar mixture
of the a and b forms was obtained when trimethylsilyl
tri¯uoromethanesulfonate (TMSOTf) was used. Depro-
tection was performed with 1 M tetrabutylammonium
¯uoride solution in THF, leading to the desired 5-o-
carboranyl derivative 7 which was characterized by
NOE experiments.
Figure 1. Structures of several enantiomers of 5-o-carboranyl pyr-
imidine nucleosides.
In order to avoid a-analogue formation for the synth-
esis of other derivatives, the synthesis was initiated
directly from the b-parent nucleoside. Compound 13
(Scheme 2) was a common intermediate for the synth-
esis of several l-derivatives. 30,50-Di-O-benzoyl-2,20-
anhydro-b-l-uridine (13) was obtained from l-arabi-
nose in good overall yield according to the procedure
described by Holy.18 Opening of the 2,20-anhydro link-
age of compound 13 with anhydrous HCl in DMF pro-
vided the 20-chloro-20-deoxy derivative 14. Reduction
with tri-n-butyltin hydride in the presence of AIBN as a
catalyst a€orded 30,50-di-O-benzoyl-20-deoxy-b-l-uri-
dine (17) in quantitative yield. Iodination with N-iodo-
succinimide and catalytic amounts of n-butyl disul®de
a€orded the 5-iodo derivative which was coupled with
(trimethylsilyl)acetylene in the presence of bis(triphenyl-
phosphine)palladium(II) chloride, copper iodide and
triethylamine. Desilylation was performed before the
carboranylation step. Decaborane was heated under
re¯ux in toluene and propionitrile with the formed
ethynyl derivative, yielding the 1-[b-l-(2-deoxyribo-
furanosyl)]-5-o-carboranyluracil (l-CDU, 2) after
deprotection with a 0.1 M sodium methoxide solution.
1-(b-d-Arabinofuranosyl)-5-o-carboranyluracil (3) was
obtained following the same procedure as l-CDU from
2,20-anhydro-1-(b-d-arabinofuranosyl)uracil, which was
prepared by the method of Hampton and Nichol.19
20-Hydroxyl group inversion involved the use of di-
phenyl carbonate which is safe and readily available.
nucleoside derivatives with both the d- or l-con®gura-
tion. Certain l-nucleosides can be phosphorylated as
well as their corresponding d-enantiomers and their
nucleotides have increased stability to phosphodies-
terases.9 In certain cases, l-enantiomers were found to
be more potent and less toxic than their corresponding
d-counterparts.10,11 One of the most important exam-
ples is the ( )-enantiomer of b-20,30-dideoxy-30-thiacyti-
dine (3TC), which is at least 10-fold less toxic than the
(+)-enantiomer and has potent and selective antiviral
activity.10,12 Based on these ®ndings, several new b-l-
and d-5-o-carboranyl pyrimidine nucleosides were syn-
thesized with potentially improved physicochemical and
biological properties.
Results
5-o-Carboranyl derivatives were prepared either by gly-
cosylation (compounds 5 and 7) or directly from the
parent b-d- or b-l-nucleoside (compounds 1±4 and 6).
Using the previously described synthesis of compound
5,13 the dioxolane analogue (7) was obtained by a cou-
pling procedure. The key intermediate, 5-o-carbor-
anyluracil (8), was prepared from 5-iodouracil in six
steps.13 Coupling of the silylated 5-o-carboranyl base
Scheme 1. Reagents and conditions: (a) 1,1,1,3,3,3-hexamethydisilazane, ammonium sulfate, re¯ux, 6 h; (b) tin(IV) chloride, CH2Cl2, 0ꢀC, 2 h; (c)
tetrabutylammonium ¯uoride, 1 M sol in THF, 0ꢀC, 3 h.

N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
2761
Scheme 2. Reagents and conditions: (a) methyl propiolate, 50% aqueous EtOH, re¯ux, 5 h; (b) BzCl, pyridine, 5ꢀC, 16 h; (c) HCl, 4 M sol in dioxane,
DMF, 90ꢀC, 90 min; (d) H2SO4, 0.2 M sol in DMF:H2O (1:1 v/v), 100ꢀC, 3 h; (e) CH3SO2Cl, pyridine, 5ꢀC, 18 h, rt, 3 h; (f) [CH3(CH2)3]3SnH,
AIBN, benzene, re¯ux, 1.5 h; (g) NH3/MeOH, rt, 4 h; (h) sodium benzoate, HMPA, 120ꢀC, 7 h; (i) N-iodosuccinimide, n-butyl disul®de, DMSO, rt,
18 h; (j) (trimethylsilyl)acetylene, bis(triphenylphosphine)palladium(II) chloride, CuI, Et3N, THF, 40ꢀC, 4 h; (k) TBAF, 1 M sol in THF, rt, 20 min;
(l) I2, CHCl3, 1 N aqueous HNO3, re¯ux, 2 h; (m) Ac2O, pyridine, rt, 18 h; (n) 0.1 N methanolic NaOMe, 5ꢀC, 18 h; (o) decaborane, propionitrile,
toluene, re¯ux, 6 h.
The iodination step was performed with iodine in
re¯uxing 0.1 M HNO3 solution in CHCl3 as described
by Schinazi et al.20 Although this reaction produced
lower yields compared to the precedent method using
N-iodosuccinimide, the advantages were that no DMSO
was used and reaction times were shorter.
1-(b-l-Arabinofuranosyl)-5-o-carboranyluracil (4) and
5-o-carboranyl-b-l-uridine (6) were synthesized from
the common intermediate 1-(b-l-arabinofuranosyl-3,5-
di-O-benzoyl)uracil (15) obtained by hydrolysis of
compound 13 under acidic conditions. Carboranylation
steps were conducted as described for compound 3. The

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N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
benzoyl protecting groups had to be removed prior to
iodination to avoid incomplete transformation and the
formation of byproducts. In addition, synthesis of 5-o-
carboranyl-b-l-uridine required 20-hydroxyl group
inversion of compound 15. This transformation was
initiated from the mesyl protected derivative 16 with
sodium benzoate in HMPA at 120ꢀC. Under mild
deprotection conditions less than 7% of nido derivatives
were detected by HPLC.13,21 These conversions to nido
isomers occurred most likely during the deblocking of
acetylated or benzoylated hydroxy functions of carbor-
anyl analogues under basic conditions.21 All carboranyl
derivatives tested demonstrated low toxicity against the
various cell lines used (Table 1). Accumulation of the
compounds in CEM cells and their sensitivities to the
presence of 10% serum in the medium decreased in
the order: CU > CDU > ara-CU > ribo-CU (Fig. 2).
The ratio of compound accumulated in the presence
of 10% serum to that accumulated in the absence of
serum ranged from 0.1 to 0.7 for CU and l-ribo-CU,
respectively. The d-enantiomers of CDU and ara-CU
were less sensitive to the presence of serum than their
l-counterparts.
considered for BNCT. When evaluated in di€erent cells,
l-enantiomers exhibited the lowest toxicity in CEM
cells. The relative toxicity in CEM cells was: b-d-ara-
CU > b-d-ribo-CU > b-d-CDU ꢁ b-d-dioxolane-CU
> b-l-ribo-CU ꢁ b-l-CDU > b-l-ara-CU. In addi-
tion, l-enantiomers gave similar cytotoxicity pro®les in
PBM and rapidly dividing Vero cells. Most of the
compounds tested demonstrated low toxicity in mela-
noma, prostate and lung cancer cells. Accumulation
experiments of the carboranyl derivatives in CEM cells
(Fig. 2) demonstrated that the presence of a sugar moi-
ety and the number and orientation of its substituent
hydroxyl groups can alter cellular accumulation and
serum binding. It was observed that the presence of an
aglycon, and increasing the number of hydroxyl func-
tions on this sugar moiety from two to three, decreased
the cellular accumulation and serum binding to di€erent
extents. This was expected since both biological para-
meters are in¯uenced by changes in lipophilicity. Fur-
ther biological testing is warranted to determine how
these structural changes alter the in vivo pharmacoki-
netics and tissue selectivity of these carboranyl-containing
agents.
Discussion
Conclusions
Although compounds described were primarily devel-
oped for BNCT, they were also tested for cytotoxicity
and antiviral activity in various normal and cancer cell
systems and for anti-HIV activity in primary human
lymphocytes. b-d-Dioxolane-CU (7) was the most
potent anti-HIV-1 analogue (EC50=4.6 mM, Table 1),
but its therapeutic index was approximately 2 in PBM
cells. It was also found to be moderately toxic in CEM
and rapidly dividing Vero cells at higher concentrations.
Low toxicity is a highly desirable property for compounds
This paper contains the ®rst report on the synthesis of
l-enantiomers of boron nucleoside analogues for
potential use in BNCT. Based on the relatively low in
vitro cytotoxicity in primary human PBM cells and
other cell lines (Table 1), and low protein-binding
properties (Fig. 2), b-l-ribo-CU (6) and b-l-ara-CU (4)
have desirable properties for BNCT. However, d- or
l-CDU (1 and 2) and the nucleoside base CU (8) should
also be considered since they accumulate in CEM cells
to the greatest extent.
Table 1. In vitro antiviral activity and cytotoxicity of several 5-o-carboranyl pyrimidines in various normal and cancer cell lines
Compound
HIV-1, EC50 (mM)a
Cytotoxicity, IC50 (mM)a
PBMb
CEMc
Verod
SK-MEL-28e
SK-MES-1f
LNCaPg
MCF-7h
9Li
b-d-CDU (1)
66.4
>100
44.7
>100
49.6
77.9
>100
>100
39.5
>100
81.9
71.6
55.0
68.5
13.8
81.1
31.6
68.1
58.7
17.3
65.9
47.2
103.0
90.1
51.0
44.0
83.6
64.8
85.4
>100
>100
>100
>100
>100
>100
>100
>100
44.4
52.4
>100
>100
>100
>100
>100
51.8
83.3
81.2
>100
>100
81.4
>100
97.0
b-l-CDU (2)
b-d-ara-CU (3)
b-l-ara-CU (4)
b-d-ribo-CU (5)
b-l-ribo-CU (6)
b-d-dioxolane-CU (7)
>100
>100
>100
>100
NDj
112
>100
>100
4.6
9.6
101.0
Cycloheximide
5-Fluorouracil
AZT
Ð
Ð
0.002
2.8
0.5
>100
0.14
90.5
14.0
2.1
6.8
29.0
1.3
6.1
>100
2.6
13.1
>100
2.4
30.0
>100
1.5
43.7
>100
0.2
0.8
>100
a
EC50 and IC50 are the median e€ective (antiviral) and inhibitory (cytotoxic) concentrations, respectively.
b
c
d
e
f
PBM: human peripheral blood mononuclear cells, cell count endpoint on day 5; MTT dye uptake endpoint on day 5.
CEM: human T-cell lymphoma, cell count endpoint on day 5; MTT dye uptake endpoint on day 4.
Vero: African green monkey kidney, cell count endpoint on day 3; MTT dye uptake endpoint on day 3.
SK-MEL-28: human lung squamous carcinoma, MTT dye uptake endpoint on day 4.
SK-MES-1: human lung squamous carcinoma, MTT dye uptake endpoint on day 5.
LNCaP: human prostate adenocarcinoma, MTT dye uptake endpoint on day 5.
MCF-7: human breast carcinoma, MTT dye uptake endpoint on day 4.
g
h
i
9L: rat glioma, MTT dye uptake endpoint on day 5.
ND: not determined.
j

N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
2763
Figure 2. Accumulation of 5-o-carboranyl pyrimidines in CEM cells for 2.5 h. Numbers in parentheses represent the ratio of accumulation in 10%
serum to accumulation without serum.
Experimental
General procedure B: reaction with trimethylsilyacetyl-
ene. To a solution of the corresponding iodinated deri-
vative (1.0 equiv), bis(triphenylphosphine)palladium(II)
chloride (0.1 equiv) and copper iodide (0.1 equiv) in
THF were added triethylamine (2.0 equiv) and (tri-
methylsilyl)acetylene (1.4 equiv). The reaction mixture
was heated at 40ꢀC for 4 h. The solvent was removed
under reduced pressure and the residue puri®ed by ¯ash
chromatography on silica gel using a gradient of hex-
ane±ethyl acetate as eluent.
Chemistry
Melting points (mp) were measured on an Electro-
thermal melting point apparatus and are uncorrected.
1H and 13C NMR spectra were recorded on a Varian
Unity Plus 400 spectrometer at 400 and 100.6 MHz,
respectively. Tetramethylsilane was used as an internal
standard and J values are given in Hz. Microanalyses
were performed by Atlantic Microlab, Norcross, Geor-
gia. Thin layer chromatography was performed on
Whatman silica gel HP-KF glass-backed plates. Merck
silica gel (grade 10184) was used for ¯ash column chro-
matography. Anhydrous solvents were purchased from
the Aldrich Chemical Company, Milwaukee, WI, and
used without further puri®cation or drying. Triethyl-
amine and propionitrile were dried by heating at 70±
80ꢀC with calcium hydride, then distilled at atmospheric
pressure. HPLC analysis were performed on a Hewlett±
Packard 1050 instrument. Decaborane (purity > 99%)
was purchased from Boron Biologicals Inc. (Raleigh,
NC). o-Carboranyluracil (CU, 8) was obtained as
described previously.13
General procedure C: deprotection of silylated hydroxy
functions. The protected nucleoside (1.0 equiv) was dis-
solved in THF and tetrabutylammonium ¯uoride, 1 M
solution in THF (2.0 equiv), was added. The resulting
solution was stirred at room temperature for 1±2 h and
then concentrated under reduced pressure. A solution of
the residue in CH2Cl2 (50 mL) was washed with H2O
(2Â50 mL) and saturated brine (50 mL), dried (MgSO4),
®ltered and concentrated in vacuo. The residue was
puri®ed by ¯ash chromatography on silica gel. Combi-
nation and evaporation of the fractions eluted with
hexane±ethyl acetate gradient a€orded the corresponding
unprotected derivative.
General procedure A: synthesis of iodinated nucleosides.
The unprotected nucleoside (1.0 equiv) and iodine (1.8
equiv) were suspended in a mixture of CHCl3 and 1 M
aqueous HNO3 (1:4/v:v). The resulting biphasic reac-
tion mixture was re¯uxed for 2 h, then refrigerated
overnight. The iodinated derivative was collected as a
crystalline solid by ®ltration and repeated washes with
Et2O in order to remove the excess iodine.
General procedure D: carboranylation reactions. A solution
of the corresponding unprotected ethynyl derivative
(1.0 equiv), decaborane (2.0 equiv) and propionitrile
(20.0 equiv) in toluene (20 mL) was re¯uxed for 4±6 h.
After cooling to room temperature, the solvents were
removed under reduced pressure. The residue was pur-
i®ed by ¯ash chromatography on silica gel, and combi-
nation of the fractions eluted with hexane±ethyl acetate

2764
N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
gradient a€orded the carboranyl derivative as colorless
solid.
1-(ꢀ-D-1,3-Dioxolane-4-yl)-5-o-carboranyluracil (7). A
mixture of 10 (18.0 mg, 0.03 mmol) in THF (3 mL) and
1 M tetra-n-butylammonium ¯uoride solution in THF
(30 mL, 0.06 mmol) was stirred at room temperature for
2 h. After the mixture was concentrated, the residue was
puri®ed by PLC (CHCl3:MeOH, 95:5) to yield 7
(6.1 mg, 56%) as a white solid. Mp 219±220ꢀC; 1H
NMR (DMSO-d6) d 1.20±3.00 (br, 10H, B10H10), 3.65
(br, 2H, H-50), 4.08 (m, 1H, H-20), 4.27 (m, 1H, H-20),
4.95 (m, 1H, H-40), 5.25 (br, 1H, OH, ex.), 6.14 (br, 1H,
B10H10CH), 6.16 (m, 1H, H-10), 8.09 (s, 1H, H-6). Anal.
calcd for C10H20B10N2O5: C, 33.70; H, 5.65; N, 7.86.
Found: C, 33.87; H, 5.98; N, 7.54.
General procedure E: speci®c deprotections of acetyl or
benzoyl groups under mild conditions. Protected carbor-
anyl analogues (1.0 equiv) and 0.1 M sodium methoxide
(3.3 equiv) in MeOH were stirred at 5ꢀC for 18 h. The
ion exchange resin DOWEX 50WX8-100 H+ form was
added until the pH was 7. The solution was ®ltered and
the resin washed with MeOH. The residue was puri®ed
by ¯ash chromatography on silica gel. Concentration
and evaporation of the fractions eluted with CH2Cl2±
MeOH gradient a€orded the desired compound.
1-[ꢀ-L-(2-Deoxyribofuranosyl)]-5-o-carboranyluracil (2).
Use of 30,50-di-O-benzoyl-5-ethynyl-20-deoxy-l-uridine
(20) a€orded 2 by the application of general procedures
1-[2-[(tert-Butyldiphenylsilyloxy)methyl]-ꢀ-D-1,3-dioxolan-
4-yl]-5-o-carboranyluracil (10). A mixture of 5-o-carbor-
anyluracil (8) (0.1 g, 0.39 mmol) in hexamethyldisilazane
(15 mL) and catalytic amount of ammonium sulfate was
re¯uxed for 3 h. The resulting clear solution was con-
centrated in vacuo under anhydrous conditions to yield
silylated-5-o-carboranyluracil as colorless oil. To a
solution of silylated base in dry dichloromethane (5 mL)
were added a solution of 914 (0.157 g, 0.39 mmol) in dry
dichloromethane and SnCl4 (0.47 mL, 0.47 mmol) at
5ꢀC, and the reaction mixture was stirred at room tem-
perature for 2 h under argon atmosphere. The reaction
mixture was quenched by saturated aqueous NaHCO3
solution (10 mL) and stirred for an additional 30 min at
room temperature. The organic layer was separated and
the aqueous layer was extracted with methylene chloride
(3Â30 mL). The combined organic layer was washed
with saturated aqueous NaHCO3 solution and water
and dried (anhydrous MgSO4). After ®ltration, the ®l-
trate was concentrated and the residue was puri®ed by
silica gel column chromatography (CHCl3:MeOH,
D and E. Mp 223±224ꢀC; H NMR (DMSO-d6) d 1.6±
1
2.8 (br, 10H, B10H10), 2.09 (m, 1H, H-20), 2.2 (m, 1H,
H-20), 3.60 (m, 2H, H-50), 3.87 (m, 1H, H-40), 4.25 (m,
1H, H-30), 5.15 (br, 1H, OH, ex.), 5.29 (br, 1H, OH, ex.),
6.10 (s, 1H, B10H10-CH), 6.15 (m, 1H, H-10), 8.33 (s, 1H,
H-10), 11.83 (s, 1H, NH, ex.); 13C NMR (DMSO-d6) d
59.29, 60.99, 70.34, 71.45, 85.45, 87.94, 105.06, 142.75,
148.76, 160.66. Anal. calcd for C11H22B10N2O5: C, 35.67;
H, 5.99; N, 7.56. Found: C, 35.53; H, 5.90; N, 7.47.
1-(ꢀ-D-Arabinofuranosyl)-5-o-carboranyluracil (3). Use
of 2,20-anhydro-1-(b-d-arabinofuranosyl)uracil20 a€or-
ded 3 by the application of general procedures A, B, C,
D and E. Mp 241ꢀC; H NMR (DMSO-d6) d 1.2±3.0
1
(br, 10H, B10H10), 3.60 (m, 2H, H-50), 3.80 (m, 1H, H-
40), 3.98 (m, 2H, H-30, H-20), 5.15 (br, 1H, OH, ex.), 5.58
(br, 1H, OH, ex.), 5.73 (br, 1H, OH, ex.), 6.03 (m, 2H,
H-10, B10H10CH), 7.94 (s, 1H, H-6), 11.87 (s, 1H, NH,
ex.). Anal. calcd for C11H22B10N2O6: C, 34.19; H, 5.74;
N, 7.25. Found: C, 34.17; H, 5.77; N, 7.28.
1
100:1) to yield 10 (19.3 mg, 10%) as white foam. H
NMR (CDCl3) d 1.06 (s, 9H, tBu), 1.60±2.80 (br, 10H,
B10H10), 3.89 (m, 2H, H-50), 4.16±4.24 (m, 2H, H-20),
5.17 (t, 1H, J=4.6 Hz, H-40), 5.64 (br, 1H, B10H10CH),
6.20 (d, 1H, J=4.8 Hz, H-10), 7.37±7.74 (m, 11H, aro-
matic, H-6), 8.92 (s, 1H, NH, ex.).
1-(ꢀ-L-Arabinofuranosyl)-5-o-carboranyluracil (4). Use
of compound 21 a€orded 4 by the application of general
procedures D and E. Mp 260±261ꢀC; 1H NMR
(DMSO-d6) d 1.6±2.8 (br, 10H, B10H10), 3.55±3.67 (m,
2H, H-50), 3.80 (m, 1H, H-40), 3.99 (m, 2H, H-30, H-20),
5.15 (br, 1H, OH, ex.), 5.55 (br, 1H, OH, ex.), 5.73 (m,
1H, OH, ex.), 6.00 (s, 1H, B10H10CH), 6.04 (d, 1H,
J=4.4 Hz, H-10), 7.95 (s, 1H, H-6), 11.87 (s, 1H, NH,
ex.); 13C NMR (DMSO-d6) d 54.79, 59.11, 60.44, 71.55,
74.85, 75.37, 85.21, 85.57, 103.36, 144.51, 148.82,
160.66. Anal. calcd for C11H22B10N2O6: C, 34.19; H,
5.74; N, 7.25. Found: C, 34.22; H, 5.66; N, 7.18.
2-Amino-ꢀ-L-arabinofurano[10,20:4,5]oxazoline (11). The
title compound was synthesized according to the proce-
dure of Holy and isolated in similar yield.18 Mp 180ꢀC;
1H NMR (DMSO-d6+D2O) d 3.25 ( m, 2H, H-50), 3.63
(m, 1H, H-40), 4.01 (m, 1H, H-30), 4.54 (m, 1H, H-20),
5.65 (d, 1H, J=6.0 Hz, H-10); 13C NMR (DMSO-d6) d
61.47, 75.55, 84.48, 87.99, 99.77, 162.11.
2,20-Anhydro-L-uridine (12). The title compound was
synthesized according to the procedure of Holy and
isolated in similar yield.18 Mp 239ꢀC; 1H NMR
(DMSO-d6) d 3.18±3.26 (m, 2H, H-50), 4.07 (m, 1H, H-
40), 4.38 (m, 1H, H-30), 4.98 (t, 1H, J=5.2 Hz, 50OH,
ex.), 5.20 (m, 1H, H-20), 5.85 (d, 1H, J=7.6 Hz, H-5),
5.88 (d, 1H, J=4.4 Hz, 30OH, ex.), 6.30 (d, 1H,
J=6.0 Hz, H-10), 7.84 (d, 1H, J=7.6 Hz, H-6).
1-(ꢀ-L-Ribofuranosyl)-5-o-carboranyluracil (6). Use of
compound 22 a€orded 6 by the application of general
procedures D and E. Mp 250±251ꢀC; 1H NMR
(DMSO-d6) d 1.6±2.8 (br, 10H, B10H10), 3.63 (m, 2H,
H-50), 3.93 (m, 1H, H-40), 3.97 (m, 1H, H-30), 4.05 (m,
1H, H-20), 5.14 (br, 1H, OH, ex.), 5.26 (br, 1H, OH,
ex.), 5.45 (br, 1H, OH, ex.), 5.78 (m, 1H, H-10), 6.03 (s,
1H, B10H10CH), 8.39 (s, 1H, H-6), 11.86 (s, 1H, NH,
ex.); 13C NMR (DMSO-d6) d 59.29, 60.32, 69.79, 71.43,
74.46, 85.02, 88.61, 105.36, 142.87, 149.00, 160.59. Anal.
calcd for C11H22B10N2O6: C, 34.19; H, 5.74; N, 7.25.
Found: C, 34.23; H, 5.77; N, 7.20.
30,50-Di-O-benzoyl-2,20-anhydro-L-uridine (13). The title
compound was synthesized according to the procedure
of Holy and isolated in similar yield.18 1H NMR
(DMSO-d6) d 4.37 (m, 2H, H-50), 4.85 (m, 1H, H-40),

N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
2765
5.72 (m, 1H, H-30), 5.78 (m, 1H, H-20), 5.90 (d, 1H,
J=7.6 Hz, H-5), 6.50 (d, 1H, J=6.0 Hz, H-10), 7.85±
8.07 (m, 11H, aromatic, H-6); 13C NMR (DMSO-d6) d
62.99, 77.13, 82.11, 86.06, 89.76, 108.88, 128.61, 128.91,
133.34, 133.77, 136.38, 159.20, 164.78, 165.09, 170.43.
H-50), 3.73 (m, 1H, H-40), 3.89 (m, 1H, H-30), 3.95 (m,
1H, H-20), 5.04 (t, 1H, J=5.2 Hz, 50OH, ex.), 5.46 (d,
1H, J=4.8 Hz, OH, ex.), 5.59 (m, 2H, H-5, OH, ex.),
5.99 (d, 1H, J=4.4 Hz, H-10), 7.63 (d, 1H, J=8.4 Hz, H-
6), 11.23 (s, 1H, NH, ex.).
30,50-Di-O-benzoyl-20-chloro-20-deoxy-L-uridine (14). The
title compound was synthesized according to the proce-
dure of Holy and isolated in similar yield.18 Mp 1660±
1-(ꢀ-L-Ribofuranosyl-2,3,5-tri-O-benzoyl)uracil (19). 1-
(b-l-Arabinofuranosyl-2-O-methanesulfonyl-3,5-di-O-
benzoyl)uracil (16) (1 g, 1.88 mmol) and sodium benzo-
ate (2.71 g, 18.8 mmol) were heated at 120ꢀC in HMPA
(20 mL) for 7 h. The products were allowed to cool to
room temperature and ethyl acetate (200 mL) was
added. The solution was washed with saturated brine
(3Â100 mL) and H2O (2Â100 mL), dried (Na2SO4), ®l-
tered and concentrated in vacuo. The residue was pur-
i®ed by ¯ash chromatography on silica gel, combination
and evaporation of the fractions eluted with
CH2Cl2:MeOH (9:1 v/v) a€orded the title compound as
a colorless solid (0.65 g, 1.17 mmol, 62%). 1H NMR
(DMSO-d6) d 4.70±4.77 (m, 3H, H-50, H-40), 5.69 (d,
1H, J=7.6 Hz, H-5), 5.93 (m, 2H, H-30, H-20), 6.16 (d,
1H, J=3.6 Hz, H-10), 7.38±8.02 (m, 11 H, aromatic, H-
6), 11.54 (s, 1H, NH, ex.).
ꢀ
1
167 C; H NMR (DMSO-d6) d 4.60±4.68 (m, 3H, H-5 ,
H-40), 5.27 (m, 1H, H-30), 5.69 (d, 1H, J=8.0 Hz, H-5),
5.74 (m, 1H, H-20), 6.17 (d, 1H, J=8.0 Hz, H-10), 7.52-
8.07 (m, 11H, aromatic, H-6), 11.56 (s, 1H, NH, ex.).
1-(ꢀ-L-Arabinofuranosyl-3,5-di-O-benzoyl)uracil
(15).
The title compound was synthesized according to the
procedure of Holy and isolated in similar yield.18 1H
NMR (DMSO-d6) d 4.45 (m, 1H, H-40), 4.52 (m, 1H, H-
30), 4.68 (m, 2H, H-50), 5.39 (m, 1H, H-20), 5.58 (d, 1H,
J=8.4 Hz, H-5), 6.20 (d, 1H, J=4.4 Hz, H-10), 6.28 (br,
1H, OH, ex.), 7.53±8.06 (m, 11H, aromatic, H-6), 11.20
(br, 1H, NH, ex.).
1-(ꢀ-L-Arabinofuranosyl-2-O-methanesulfonyl-3,5-di-O-
benzoyl)uracil (16). To a cooled (0ꢀC; ice/water bath)
solution of 1-(b-l-arabinofuranosyl-3,5-di-O-benzoyl)-
uracil (15) (3.04 g, 6.72 mmol) in pyridine (35 mL)
methanesulfonyl chloride (0.78 mL, 10.08 mmol) was
added dropwise. The solution was stirred at 5ꢀC for 18 h
and a further 0.3 mL (3.36 mmol) of methanesulfonyl
chloride added. Stirring was continued for 3 h at room
temperature, water (5 mL) was added and the solvent
removed under reduced pressure. The residue was dis-
solved in CHCl3 (200 mL), washed with H2O (200 mL),
10% HCl in water (200 mL) and saturated aqueous
NaHCO3 (200 mL), dried (Na2SO4), ®ltered and con-
centrated in vacuo. The residue was triturated with
EtOH (100 mL) and kept at room temperature over-
night. The title compound, a colorless solid, homo-
geneous by TLC, was collected by ®ltration (3.27 g,
30,50-Di-O-benzoyl-5-ethynyl-20-deoxy-L-uridine (20). Use
of compound 17 a€orded 20 by the application of
1
general procedures A, B and C. H NMR (CDCl3) d
2.32 (m, 1H, H-20), 2.80 (m, 1H, H-20), 3.02 (s, 1H,
ethynyl), 4.59 (m, 1H, H-40), 4.71 (m, 1H, H-50), 4.80
(m, 1H, H-50), 5.63 (m, 1H, H-30), 6.38 (m, 1H, H-10),
7.44±8.07 (m, 11H, aromatic, H-6).
1-(ꢀ-L-Arabinofuranosyl-2,3,5-tri-O-acetyl)-5-ethynylur-
acil (21). Use of compound 18 a€orded 21 as a color-
less glass by the application of general procedures A, B
and C. 1H NMR (DMSO-d6) d 1.97 (s, 3H, acetyl), 2.09
(s, 6H, acetyl), 4.18 (s, 1H, ethynyl), 4.26 (m, 1H, H-40),
4.35 (m, 2H, H-50), 5.27 (m, 1H, H-30), 5.40 (m, 1H, H-
20), 6.23 (d, 1H, J=4.8 Hz, H-10), 7.94 (s, 1H, H-6),
11.81 (s, 1H, NH, ex.).
1
6.16 mmol, 98%). H NMR (DMSO-d6) d 3.28 (s, 3H,
SO2CH3), 4.60 (m, 1H, H-40), 4.68 (m, 2H, H-50), 5.62
(m, 3H, H-5, H-30), 5.77 (m, 1H, H-20), 6.36 (d, 1H,
J=5.2 Hz, H-10), 7.50±8.08 (m, 11H, aromatic, H-6),
11.49 (s, 1H, NH, ex.).
1-(ꢀ-L-Ribofuranosyl-2,3,5-tri-O-acetyl)-5-ethynyluracil
(22). Use of unprotected compound 19 a€orded 22 by
1
the application of general procedures A, B and C. H
NMR (DMSO-d6) d 2.06 (s, 6H, acetyl), 4.21 (s, 1H,
ethynyl), 4.20±4.36 (m, 3H, H-50, H-40), 5.34 (m, 1H, H-
30), 5.47 (m, 1H, H-20), 5.89 (d, 1H, J=4.8 Hz, H-10),
8.15 (s, 1H, H-6), 11.82 (s,1H, NH, ex.).
30,50-Di-O-benzoyl-20-deoxy-L-uridine (17). 30,50-Di-O-
benzoyl-20-chloro-20-deoxy-l-uridine (14) (6.09 g, 12.9
mmol), tri-n-butyltin hydride (14.28 mL, 53.3 mmol)
and AIBN (0.05 g, 0.3 mmol) were re¯uxed in benzene
(130 mL) for 1.5 h. The solution was allowed to cool to
room temperature and the title compound, a colorless
solid, was collected by ®ltration (5.36 g, 12.28 mmol,
95%). The compound was recrystallized from boiling
EtOH. Mp 218±219ꢀC; 1H NMR (DMSO-d6) d 2.62 (m,
2H, H-20), 4.52 (m, 1H, H-40), 4.61 (m, 2H, H-50), 5.62
(m, 2H, H-5, H-30), 6.29 (m, 1H, H-10), 7.51±8.04 (m,
11H, aromatic, H-6), 11.42 (s, 1H, NH, ex.).
Antiviral and cytotoxicity assays
Anti-HIV-1 activity of the compounds was determined
in human peripheral blood mononuclear (PBM) cells as
described previously.22 Brie¯y, the antiviral EC50 and
cytotoxicities were obtained from the concentration±
response curve using the median e€ective method.23,24
The anti-cancer activity of the compounds was deter-
mined in CEM (human lymphoblastoid), LNCaP
(human metastatic prostate adenocarcinoma), SK-
MES-1 (human lung carcinoma), SK-MEL-28 (human
skin melanoma), MCF-7 (human breast carcinoma) and
9L (rat glioma) cells. Appropriate numbers of cells were
cultured with the drug for a speci®c number of days in
1-ꢀ-L-Arabinofuranosyluracil (18). Use of 1-(b-l-arabino-
furanosyl-3,5-di-O-benzoyl)uracil (15) (2.2 g, 4.86 mmol)
by the application of general procedure E a€orded 18.
Mp 234±235ꢀC; H NMR (DMSO-d6) d 3.59 (m, 2H,
1

2766
N. S. Mourier et al. / Bioorg. Med. Chem. 7 (1999) 2759±2766
96-well plates (PBM, LnCaP and SK-MES-1: 5 days;
CEM, SK-MEL-28, MCF-7 and 9L: 4 days; Vero: 3
days). Positive cytotoxic controls included 5-¯uorouracil,
an inhibitor of thymidylate synthetase, cycloheximide, an
inhibitor of rRNA transfer, and the antiretroviral
nucleoside AZT. Untreated cells exposed to solvent
were included as negative controls. After incubation,
actively metabolizing cells were quanti®ed using the Cell
Titer 96 Cell Proliferation Assay (MTT, Promega,
Madison, WI), as described by the manufacturer.
Barth, R. F.; Codogni, I. M.; Wilson, J. G. Chem. Rev. 1998,
98, 1515.
3. Barth, R. F.; Soloway, A. H.; Fairchild, R. G.; Brugger, R.
M. Cancer 1992, 70, 2995.
4. Fairchild, R. G.; Bond, V. P. Int. J. Radiat. Oncol. Biol.
Phys. 1985, 21, 831.
5. Zamenhof, R. G.; Kalend, A. M.; Bloomer, W. D. J. Natl.
Cancer Inst. 1992, 84, 1290.
6. Yamamoto, Y.; Seko, T.; Nakamura, H.; Nemoto, H.;
Hojo, H.; Mukaoi, N.; Hashimoto, Y. J. Chem. Soc., Chem.
Commun. 1992, 157.
7. Schinazi, R. F.; Goudgaon, N. M.; Fulcrand, G.; El Kattan,
Y.; Lesnikowski, Z.; Ullas, G.; Moravek, J.; Liotta, D. C. Int.
J. Radiat. Oncol. Biol. Phys. 1994, 28, 1113.
Accumulation in CEM cells
8. Fulcrand-El Kattan, G.; Goudgaon, N. M.; Ilksoy, N.;
Huang, J. T.; Watanabe, K. A.; Sommadossi, J.-P.; Schinazi,
R. F. J. Med. Chem. 1994, 37, 2583.
9. Shewach, D. S.; Liotta, D. C.; Schinazi, R. F. Biochem.
Pharmacol. 1993, 45, 1540.
10. Schinazi, R. F.; Chu, C. K.; Peck, A.; McMillan, A.;
Mathis, R.; Cannon, D.; Jeong, L.-S.; Beach, J. W.; Choi,
W.-B.; Yeola, S.; Liotta, D. C. Antimicrob. Agents Chemother.
1992, 36, 672.
11. Schinazi, R. F.; McMillan, A.; Cannon, D.; Mathis, R.;
Lloyd, R. M.; Peck, A.; Sommadossi, J.-P.; St Clair, M.; Wil-
son, J.; Furman, P. A.; Painter, G.; Choi, W.-B.; Liotta, D. C.
Antimicrob. Agents Chemother. 1992, 36, 2423.
12. Sommadossi, J.-P.; Schinazi, R. F.; Chu, C. K.; Xie, M.-Y.
Biochem. Pharmacol. 1992, 44, 1921.
All radiolabeled nucleosides were custom synthesized by
Moravek Biochemicals Inc. (Brea, CA), and were
labeled on the acidic proton of the carboranyl group.7
The speci®c activities of CU, b-d-ara-CU, b-d-ribo-CU,
b-d-CDU, b-l-ara-CU, b-l-ribo-CU and b-l-CDU
were 59.2, 6.7, 1.2, 1.8, 1.0, 2.3 and 23.1 Ci/mmol,
respectively. CEM cells (106/mL) were incubated at
37ꢀC in the presence of 3H-radiolabeled compound
(1 mM) in RPMI 1640 medium containing 0 and 10%
FBS for 2.5 h. The accumulation of carboranyl deriva-
tives was then measured by centrifuging quadruplicate
samples of the cell suspensions (200 mL) through silicone
oil, lysing the cells using formic acid and scintillation
counting.25 Counts per minute (cpm) were converted to
pmol/106 cells using linear regressions of pmol com-
pound versus cpm.
13. Goudgaon, N. M.; El Kattan, Y.; Xia, X.; McAtee, J.;
Soria, J.; Wey, S.-J.; Liotta, D. C.; Schinazi, R. F. Nucleosides
and Nucleotides 1997, 16, 2133.
14. Kim, H. O.; Ahn, A. J.; Alves, S. K.; Beach, J. W.; Jeong,
L. S.; Choi, B. G.; Van Roey, P.; Schinazi, R. F.; Chu, C. K.
J. Med. Chem. 1992, 35, 1987.
Acknowledgements
15. Vorbruggen, H.; Ho¯e, G. Chem. Ber. 1981, 114, 1256.
16. Graciet, J.-C.; Shi, J.; Schinazi, R. F. Nucleosides and
Nucleotides 1998, 17, 711.
17. Shi, J.; McAtee, J. J.; Wirtz, S. S.; Tharnish, P. M.; Juo-
dawlkis, A.; Liotta, D. C.; Schinazi, R. F. J. Med. Chem. 1999,
42, 859.
We are grateful to Dr. C. K. Chu (University of Geor-
gia, Athens, USA) for generously providing the dioxo-
lane heterocycle for coupling. This work was supported
by the Department of Energy DE-FG02-96ER62156
(RFS), the Department of Veterans A€airs (RFS) and
la Fondation pour la Recherche Medicale SE000618-01
(NSM). The synthesis of radiolabeled compounds was
supported by the National Institute of Health grant
5R44-CA65434 (RFS). We thank Susan Schlueter Wirtz
for excellent technical assistance.
18. Holy, A. Tet. Lett. 1971, 2, 189.
19. Hampton, A.; Nichol, A. W. Biochemistry 1966, 5, 2076.
20. Schinazi, R. F.; Chen, M. S.; Pruso€, W. H. J. Med. Chem.
1979, 22, 1273.
21. Fulcrand-El Kattan, G.; Lesnikowski, Z. J.; Yao, S.;
Tanious, F.; Wilson, W. D.; Schinazi, R. F. J. Am. Chem. Soc.
1994, 116, 7494.
22. Schinazi, R. F.; Sommadossi, J.-P.; Saalman, V.; Cannon,
D. L.; Xie, M.-Y.; Hart, G. C.; Smith, G. A.; Hahn, E. F.
Antimicrob. Agents Chemother. 1990, 34, 1061.
23. Chou, T. C.; Talalay, P. Adv. Enzyme Regul. 1984, 22, 27.
24. Belen'kii, M. S.; Schinazi, R. F. Antiviral Res. 1994, 25,1.
25. Hurwitz, S. J.; Terashima, M.; Mizunuma, N.; Slapak, C.
A. Blood 1997, 89, 3745.
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