3′-Bromo analogues of pyrimidine nucleosides as a new class of potent inhibitors of mycobacterium tuberculosis

Article abstract of DOI:10.1021/jm100165w

Tuberculosis (TB) is a major health problem worldwide. We herein report a new class of pyrimidine nucleosides as potent inhibitors of Mycobacterium tuberculosis (M. tuberculosis). Various 2′- or 3′-halogeno derivatives of pyrimidine nucleosides containing uracil, 5-fluorouracil, and thymine bases were synthesized and evaluated for antimycobacterial activities. Among the compounds tested, 3′-bromo-3′-deoxy- arabinofuranosylthymine (33) was the most effective antituberculosis agent in the in vitro assays against wild-type M. tuberculosis strain (H37Ra) (MIC ₅₀ = 1 μg/mL) as well as drug-resistant (H37Rv) (rifampicin-resistant and isoniazid-resistant) strains of M. tuberculosis (MIC₅₀ = 1-2 μg/mL). Compound 33 also inhibited intracellular M. tuberculosis in a human monocytic cell line infected with H37Ra, demonstrating higher activity against intramacrophagic mycobacteria (80% reduction at 10 μg/mL concentration) than extracellular mycobacteria (75% reduction at 10 μg/mL concentration). In contrast, pyrimidine nucleosides possessing 5-fluorouracil base were weak inhibitors of M. tuberculosis. No cytotoxicity was found up to the highest concentration of compounds tested (CC₅₀ > 100-200 μg/mL) against a human cell line. Overall, these encouraging results substantiate the potential of this new class of compounds as promising antituberculosis agents.

Full text of DOI:10.1021/jm100165w

4130 J. Med. Chem. 2010, 53, 4130–4140
DOI: 10.1021/jm100165w
30-Bromo Analogues of Pyrimidine Nucleosides as a New Class of Potent Inhibitors of
Mycobacterium tuberculosis
Neeraj Shakya,† Naveen C. Srivastav,† Nancy Desroches,† Babita Agrawal,‡ Dennis Y. Kunimoto,§ and Rakesh Kumar*,†
†Department of Laboratory Medicine and Pathology, 1-71 Medical Sciences Building, ‡Department of Surgery, and §Department of Medicine,
Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
Received February 5, 2010
Tuberculosis (TB) is a major health problem worldwide. We herein report a new class of pyrimidine
nucleosides as potent inhibitors of Mycobacterium tuberculosis (M. tuberculosis). Various 20- or 30-halo-
geno derivatives of pyrimidine nucleosides containing uracil, 5-fluorouracil, and thymine bases were
synthesized and evaluated for antimycobacterial activities. Among the compounds tested, 30-bromo-
30-deoxy-arabinofuranosylthymine (33) was the most effective antituberculosis agent in the in vitro assays
against wild-type M. tuberculosis strain (H37Ra) (MIC50=1 μg/mL) as well as drug-resistant (H37Rv)
(rifampicin-resistant and isoniazid-resistant) strains of M. tuberculosis (MIC50=1-2 μg/mL). Compound
33 also inhibited intracellular M. tuberculosis in a human monocytic cell line infected with H37Ra,
demonstrating higher activity against intramacrophagic mycobacteria (80% reduction at 10 μg/mL
concentration) than extracellular mycobacteria (75% reduction at 10 μg/mL concentration). In contrast,
pyrimidine nucleosides possessing 5-fluorouracil base were weak inhibitors of M. tuberculosis. No
cytotoxicity was found up to the highest concentration of compounds tested (CC50 > 100-200 μg/mL)
against a human cell line. Overall, these encouraging results substantiate the potential of this new class of
compounds as promising antituberculosis agents.
Introduction
at least two first-line drugs, isoniazid and rifampicin, and
requires the use of second-line drugs. An outbreak of recently
recognized “extensively drug-resistant tuberculosis” or XDR-
TB, threatens the TB control globally. XDR-TB is MDR-TB
that is also resistant to three or more of the six classes of
second-line drugs.8,9 A 100% correlation has been observed in
XDR-TB with HIV coinfection and mortality.8
Mycobacterium tuberculosis (M. tuberculosis),a the causa-
tive agent of tuberculosis (TB), is one of the most serious
infections among immunocompromised and/or immunocom-
petent individuals and is the second leading cause of mortality
worldwide. According to the World Health Organization
(WHO), M. tuberculosis has infected approximately 2 billion
people globally.1
The current course of therapy with first-line TB drugs is
more than 40 years old. Because of the global health problems
associated with TB, the increasing rate of MDR-TB, and the
high rate of coinfection with HIV, the discovery and develop-
ment of potent new anti-TB agents, without cross-resistance
with current antimycobacterial agents, are urgently needed.
After transmission into an individual, M. tuberculosis
lodges in pulmonary alveoli, where it is engulfed by macro-
phages, and multiplies and survives within these macro-
phages. One major challenge in TB drug development is the
difficulty in identifying new compounds with activity against
intracellular mycobacteria. Newly discovered molecules ac-
tive against M. tuberculosis should be able to inhibit both
extracellular and intracellular mycobacterial replication.
The complete genome sequence of M. tuberculosis has been
deciphered,10 facilitating the identification of novel targets
for developing inhibitors of the replication of this organism.
A number of enzymes involved in purine and pyrimidine
metabolism differ significantly between mammals and myco-
bacteria and could be used as targets for antituberculosis drug
design, especially by nucleoside and nucleotide analogues.11-13
M. tuberculosis-encoded thymidine monophosphate kinase
(TMPKmt) has been put forward as one of the mycobacterial
targets. It has high affinity for deoxythymidine and could also
TB is a contagious disease that spreads through aerosol. TB
and human immunodeficiency virus (HIV) have formed a new
and deadlycombination. Thereisa resurgence inthe incidence
ofTBindeveloped and developingcountries withhighrates of
HIV-TB coinfection. The increase in the incidence of TB is
strongly associated with the prevalence of HIV infection.2
M. tuberculosis poses a significant challenge to the clinical
management of HIV-infected patients and is often responsible
for their death.3 For both TB and HIV, misdiagnosis and
noncompliance with treatment regimens further compound the
problem and facilitate the development of drug resistance.2,4,5
The emergence of new TB strains that are not susceptible to
a number of available drugs, that is, multidrug-resistant
tuberculosis (MDR-TB), has further complicated the man-
agement of this disease.5-7 Around 50 million people have
been infected with MDR-TB, which is defined as resistant to
*To whom correspondence should be addressed. Tel: 780-492-7545.
Fax: 780-492-7521. E-mail: rakesh.kumar@ualberta.ca.
aAbbreviations: TB, tuberculosis; MDR-TB, multidrug-resistant
tuberculosis; XDR-TB, extensively drug-resistant tuberculosis; M. tuber-
culosis, Mycobacterium tuberculosis; MABA, microplate Alamar blue
assay; CFU, colony-forming unit; GI, growth index; MIC, minimum
inhibitory concentration; CC, cytotoxic concentration.
r
pubs.acs.org/jmc
Published on Web 04/26/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4131
phosphorylate thymidine to TMP for M. tuberculosis.12 There-
fore, the design of pyrimidine nucleosides capable of interfer-
ing with nucleic acid biosynthetic pathways of mycobacteria is
an excellent approach for the investigation of new classes of
antimycobacterial agents.
the target compound 19 or a simultaneously detritylated
product 20 under the reaction conditions (Scheme 3).
In our next attempt for the synthesis of target compound
20, compound 13 was treated with methyltriphenoxy-
phosphonium iodide in dry pyridine at room temperature
whereby the formation of intermediate 17 and removal of
phenoxyphosphonium ion led to the O2,30-cyclonucleoside 18
in 57% yield. Compound 18 was then treated with pyridine
hydrochloride in DMF at room temperature, where chloride
ion attacked from the less-hindered R-face, yielding 19 in 34%
yield with overall retention of configuration (Scheme 3).
Compound 19 was deblocked with 80% aqueous acetic acid
at 90 °C to afford the required compound 20 in 64% yield.
1-(3-Iodo-2,3-dideoxy-β-D-erythro-pentofuranosyl)thymine
(22), 1-(3-iodo-2,3-dideoxy-β-D-erythro-pentofuranosyl)-5-
fluorouracil (25), and 1-(3-iodo-2,3-dideoxy-β-D-threo-pento-
furanosyl)-5-fluorouracil (26) were synthesized by the reac-
tion of sodium iodide (NaI) with respective nucleosides 3 and
4 in dry 1,2-dimethoxyethane (DME) at reflux temperature
followed by detritylation of obtained compounds using 80%
acetic acid to provide 22, 25, and 26 in 37, 45, and 1% yield,
respectively (Scheme 4).
In our earlier studies, we investigated various series of 20-,
30-, and 5-substituted deoxy and dideoxy pyrimidine nucleo-
side analogues, where nucleosides containing C-5-alkynyl
moieties exhibited potent activity against replicating myco-
bacterial cultures.14-19 In our continuing efforts to investigate
new anti-TB agents, our group has discovered a new class of
30-bromo-substituted pyrimidine nucleosides. In these studies,
we note that 30-bromo-30-deoxy-arabinofuranosyl thymine
(33) in particular displays the most potent inhibition of wild-
type M. tuberculosis (H37Ra), drug-resistant M. tuberculosis
(H37Rv, rifampicin- or isoniazid-resistant strains), as well as
M. tuberculosis (H37Ra) harbored intracellularly in a human
monocyte cell line. The new class of compounds investigated
did not show cytotoxicity on the viability and proliferation of
a human cell line. These promising results offer an opportu-
nity for further exploration of this new class of analogues as
antimycobacterial agents.
The stereochemistry of the compounds 7-10, 12, 20, 22, 25,
and 26was assignedon the basis of NMR spectra, whichare in
agreement with well-established assignments,21 that is, (i) C-20
and C-200 protons in the compounds with the erythro confi-
guration have either very close chemical shifts or overlapping
signals, while the same protons with the threo configuration
have chemical shifts generally separated from each other by
0.5-1 ppm, and (ii) in the erythro configuration, the C-10
proton appears as triplet, while in threo-, this proton appears
as a quartet (or double doublets).21,22
To prepare the target compounds 32-34 and 35-37, the
free hydroxyl functions of uridine (27a), 5-methyluridine
(27b), and 5-fluorouridine (27c) were protected using trityl
chloride followed by reaction with mesyl chloride to yield
compounds 28a-c, which were converted to corresponding
20,30-lyxo-epoxidederivatives 29a-c, using1 N NaOHin50%
(v/v) aqueous acetone at room temperature. The compounds
29a-c were then treated with ammonium bromide (NH4Br)
in absolute ethanol to yield their respective bromohydrins
30a-c, in addition to the bromohydrins 31a-c (Scheme 5).
In these reactions, bromide ion attacked exclusively from the
R-face either at C-20 or at C-30 due to the hindered lyxo
configuration of the epoxides. The predominant formation
of the 30-deoxy-30-halo-arabino compounds 30a-c as major
products indicates thatthe attackof bromideion is more facile
at C-30 instead of C-20. This may be due to (i) the favorable
conformation of the sugar rings facilitating attack at C-30 of
halide ion predominantly and (ii) the closer proximity of C-20
tothe electron-withdrawinganomeric center, whichmakes the
epoxide opening facile at C-30, with an oxygen atom intact at
C-20.23 The compounds 30a-c and 31a-c were deprotected
using 80% aqueous (v/v) acetic acid to afford the target
compounds 32-34 and 35-37, respectively (Scheme 5).
Chemistry
50-O-Tritylation of thymidine (1) and 5-fluoro-20-deoxy-
uridine (2) followed by mesylation at the 30-position afforded
the respective 50-O-trityl-30-O-mesyl derivatives (3 and 4) in
90 and 87% yield, respectively. The compound 3 upon treat-
ment with lithium bromide (LiBr) or lithium chloride (LiCl)
in anhydrous acetonitrile at reflux temperature for 24 and
80 h, respectively, yielded 50-O-trityl-30-bromothymidine (5)
(51%) and 50-O-trityl-30-chlorothymidine (6) (50%), which
were then converted to the target deblocked counterparts,
7 (36%) and 8 (57%), respectively, using 80% aqueous acetic
acid. However, when compound 4 was reacted with LiBr
under similar reaction conditions, three detritylated products
9-11 (due to release of HBr in situ because of trapped traces
of moisture) were isolated in 24, 18, and 1% yields, respec-
tively (Scheme 1). The erythro 30-bromo compound 9 could
have been formed via double Walden inversion20 where
attack of bromide ion occurred from the R-face, whereas the
threo isomer 10 was formed by direct SN2 displacement of
30-methanesulfonyl group by bromide ion from the β-face of
the carbohydrate moiety (Scheme 1). In contrast, a similar
reaction of compound 4 with LiCl in acetonitrile under the
same conditions provided only a threo chloride compound 12
(Scheme 2). The sole formation of threo chloride 12 (apart
from detritylation of starting material) can be explained by
facile attack of the chloride ion from the β-face, due to the
smaller size in comparison to the bromide ion. However,
apart from the steric factors, some electronic effects of the
C5-fluorine atom alsoseemto playanimportant role, sincethe
results differ from the 5-CH3 analogue (3) under the same
conditions (Scheme 1).
In an attempt to synthesize the desired 5-fluoro-30-chloro-
20,30-dideoxyuridine (20) with a chloride in the erythro
configuration, the 50-O-tritylated 5-fluoro-20-deoxyuridine
(13) was allowed to react with triphenylphosphine and
carbon tetrachloride (CCl4) in anhydrous dimethylforma-
mide (DMF) at room temperature for 24 h. However, in this
reaction, we obtained a compound with chloride in the threo
configuration (15) along with the formation of O2,30-cyclo-
nucleoside 16 (Scheme 3). Compound 16 failed to convert to
Results and Discussion
Pyrimidine nucleoside analogues 7-10, 12, 20, 22, 25, 26,
and 32-37 were first evaluated for their activity against the
multiplication of wild-type M. tuberculosis strain H37Ra by
the microplate alamar blue assay (MABA)24 at 1-100 μg/mL
concentrations. Rifampicin, isoniazid, and cycloserine were
used as reference drugs. The compounds that exhibited pro-
mising inhibition against the wild-type strain were further

4132 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Shakya et al.
Scheme 1a
a Reagents and conditions: (i) Trityl chloride, 4-(dimethylamino)pyridine, anhydrous pyridine, 80 °C, 5 h. (ii) Methanesulfonyl chloride, 0-5 °C.
(iii) LiBr or LiCl (appropriately) 24 and 80 h days, respectively, anhydrous CH3CN, reflux. (iv) 80% AcOH, 90 °C, 30 min. (v) LiBr, anhydrous CH3CN,
reflux, 22 h.
evaluated against drug-resistant strains of M. tuberculosis
H37Rv by the BACTEC assay.25 Isoniazid-resistant and
rifampicin-resistant strains of M. tuberculosis were used in these
studies. The antimycobacterial effect of potent compounds was
also determined against intracellular mycobacteria in a human
monocytic cell-line (THP-1) infected with M. tuberculosis
strain H37Ra. Concentrations of 25, 10, and 2 μg/mL were
tested using the colony-forming units (CFU) assay.26 The
results obtained for anti-TB activity of the compounds 7-10,
12, 20, 22, 25, 26, and 32-37 along with reference first-line and
second-line anti-TB drugs are summarized in Table 1. The
antimycobacterial activity data for 30-fluoro-20,30-dideoxythy-
midine (38) and 30,5-difluoro-20,30-dideoxyuridine (39) pre-
viously reported by us14 are also included in Table 1 for
comparison. The compounds investigated here for their anti-
mycobacterial effects are divided into four different categories
based on the positions of the halogen substituent in the
carbohydrate moiety: (1) pyrimidine analogues possessing
a halogen atom in the 30-erythro configuration (7-9, 20, 22,
and 25), (2) pyrimidine analogues possessing a halogen atom

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4133
in the 30-threo configuration (10, 12, and 26), (3) 30-bromo-
30-deoxy-arabinofuranosyl analogues (32-34), and (4) 20-
bromo-20-deoxy-xylofuranosyl compounds (35-37). Among
the 30-halogeno-20,30-dideoxythymidine compounds (7, 8, 22,
and 38) described in this paper, only 30-bromo-20,30-dideox-
analogue 8 provided some inhibition of M. tuberculosis,
whereas the activity of the bromo derivative 7 was signifi-
cantly improved as compared to the corresponding 30-fluoro
(38) compound, which showed no activity in earlier studies.14
Thus, the relative antimycobacterial activity order in the
30-halogeno-20,30-dideoxythymidine series was found to be
Br > Cl > FdI. Replacement of the methyl group at the C-
5 position of compounds 7, 8, and 22 by a fluoro substituent
provided compounds 9, 20, and 25 with reduced anti-TB
activity; the 30-bromo analogue (9) showed 50% inhibition at
50 μg/mL, whereas 30-chloro (20) and 30-iodo (25) analogues
exhibited 40 and 32% inhibition at 100 μg/mL, respectively.
The 30-fluoro derivative (39) in this series of compounds also
showed no inhibition of M. tuberculosis, similar to the
20-30-dideoxythymidine series (compound 38).14 Therefore,
a similar pattern of relative activity order was observed viz.
Br > Cl > F < I among compounds 9, 20, 25, and 39 except
the 30-iodo derivative (25). These results indicate that the
bromo substituent contributed more to anti-TB activity than
the chloro, fluoro, and iodo groups at the C-30 position.
Interestingly, inversion of the halogen atoms in the “up” or
threo configuration at the C-30 carbon atom in the pyrimidine
nucleoside analogues 10, 12 (MIC50 =25-50 μg/mL), and 26
proved to be more effective in comparison to their 30-halogenated
ythymidine [7, minimum inhibitory concentration (MIC)50
=
5-10 μg/mL] and 30-chloro-20,30-dideoxythymidine (8, MIC50=
50 μg/mL) exhibited appreciable inhibition of M. tuberculosis
(H37Ra). However, it is interesting to note that the chloro
Scheme 2a
a Reagents and conditions: (i) LiCl, anhydrous CH3CN, reflux, 24 h.
Scheme 3a
a Reagents and conditions: (i) Trityl chloride, 4-(dimethylamino)pyridine, anhydrous pyridine, 80 °C, 5 h. (ii) Triphenylphosphine, CCl4, anhydrous
DMF, room temperature, 24 h. (iii) 80% aqueous AcOH, 90 °C, 30 min. (iv) Methyltriphenoxyphosphonium iodide, anhydrous pyridine, room
temperature, 1 h. (v) Pyridine hydrochloride, anhydrous DMF, room temperature, 8 days.

4134 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Shakya et al.
Scheme 4a
a Reagents and conditions: (i) NaI, anhydrous DME, reflux, 20 h. (ii) 80% aqueous AcOH, 90 °C, 30 min.
“down” or erythro isomers (9, 20, and25, respectively). However,
compounds 10 and 12 were less inhibitory as compared to the
30-bromo-20,30-dideoxythymidine (7). It would be worthwhile to
investigate antimycobacterial activity of threo analogues of 7 in
our next study.
(H37Rv) of M. tuberculosis (Table 1). Encouragingly, we
found that both of the drug-resistant strains were suscep-
tible to these compounds at concentrations similar to those
for drug-sensitive M. tuberculosis strain H37Ra, suggesting
that the identified pyrimidine nucleoside compounds are
not cross-resistant to the currently available first-line anti-
tuberculosis drugs. There was no inhibition of the drug-
resistant strains by the respective drugs rifampicin or isoniazid
at 2 μg/mL.
Among the 30-bromo-30-deoxy-arabinofuranosyl analogues
(32-34) tested, 30-bromo-30-deoxy-arabinofuranosylthymine
(33) (MIC50 =1 μg/mL) showed the best antimycobacterial
activity against H37Ra. Intriguingly, compound 33 had
activity significantly higher than the 30-bromo compound 7
(MIC50=5-10 μg/mL), as well as a second-line anti-TB drug
cycloserine (MIC50 = 5 μg/mL) (Table 1). Similarly, in this
series of compounds, the 5-fluoro derivative (34) also had
improved activity (MIC50=25 μg/mL) over compound 9
(MIC50 =50 μg/mL), suggesting that incorporation of a
hydroxyl substituent at the C-20 position in the arabino
configuration contributes to antimycobacterial activity.
Of the 20-bromo-20-deoxy-xylo compounds (35-37) tested,
only thymine analogue 36 exhibited modest activity against
H37Ra replication (MIC50=25 μg/mL), which is significantly
reduced as compared to the corresponding 30-bromo deriva-
tives 33 and 7, suggesting that bromine at the C-30 position is
an influential factor in the carbohydrate portion. Also, in the
20-bromo class of compounds, the uracil analogue (35) and
5-fluorouracil analogue (37) were found to be inactive, sug-
gesting that a methyl group at the C-5 position is an important
determinant for the anti-TB activity. Interestingly, the same
observation was made with other compounds (7, 8, and 33)
investigated in this report.
Compounds 7 and 33 were also assayed for their activity
toward intracellular M. tuberculosis. In the human monocytic
cell line (THP-1) infected with H37Ra, both compounds
significantly inhibited intracellular mycobacterial growth at
the two concentrations tested (10 and 25 μg/mL). Biological
results reported in Table 1 indicate that both 7 and 33
exhibited a concentration-dependent inhibition of intrama-
crophagic mycobacterial growth. In this assay, compound 7
provided 50% reduction in the CFU at 10 μg/mL, whereas
compound 33 showed 80% reduction in the CFU counts at
10 μg/mL. These data of 7 and 33 correlate well with their
respective MICs against extracellular mycobacteria. In addi-
tion, weweresurprisedto note thatthe intracellular antimyco-
bacterial activity exhibited by both compounds 7 and 33
was similar or improved with respect to their inhibition of
extracellular mycobacterial replication. The potent activity
shown by compounds 7 and 33 against intracellular myco-
bacteria suggests that they have the ability to inhibit myco-
bacteria harbored within macrophages in infected individuals.
M. tuberculosis is an intracellular pathogen, which resides,
propagates, and hides within macrophages. Most people
with latent M. tuberculosis infection, but without an active
disease, containthispathogenintracellularlyforyearsalbeitin
In our next studies, the most promising compounds
emerging from this work (7 and 33) were examined for their
potency against rifampicin- and isoniazid-resistant strains

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4135
Scheme 5a
a Reagents and conditions: (i) Trityl chloride, 4-(dimethylamino)pyridine, anhydrous pyridine 80 °C, 5 h. (ii) Methanesulfonyl chloride, anhydrous
pyridine, 0-5 °C, 24 h. (iii) NaOH (1 N) in acetone:water (1:1, v/v), room temperature, 24 h. (iv) Ammonium bromide, absolute ethanol, reflux, 20 h. (v)
80% aqueous AcOH, 90 °C, 30 min.
nonreplicating form. Although our experiments were per-
formed with human monocytes infected with actively replicat-
ing mycobacteria, the concentration-dependent inhibition of
intracellularM. tuberculosis by both compounds, in particular
33, is a very significant observation.
(33), was found to be more potent than cycloserine, a
second-line drug, and could serve as a useful lead compound
for the development of a much needed new antituberculosis
agent.
The XTT and 3H incorporation assays were performed to
evaluate the toxicity of investigated compounds (7-10, 12, 20,
22, 25, 26, and 32-37) in vitro against a human hepatoma cell
line (Huh7). No toxicity was observed up to the highest
concentration tested, 100-200 μg/mL [cytotoxic concentra-
tion (CC)50 >100-200 μg/mL].
The precise mechanism of action of the compounds inhibit-
ing mycobacterial multiplication in this study is not yet clear.
It is possible that the active nucleoside analogues, after their
metabolic conversion to phosphorylated forms by mycobac-
terial kinases, may be selectively inhibiting DNA and/or RNA
synthesis, by acting as substrates and/or inhibitors of meta-
bolic enzymes of DNA/RNA synthesis.
In conclusion, a new class of pyrimidine nucleosides with
promising antituberculosis activity and low or no cytotoxicity
was identified. Compounds 7 and 33 emerged as the most
active analogues and exhibited activity toward both drug-
sensitive and drug-resistant strains of M. tuberculosis and had
the ability to inhibit intracellular growth of M. tuberculosis,
providing further evidence that this class of compounds has
immense promise. The best antimycobacterial agent identified
in this study, 30-bromo-30-deoxy-arabinofuranosylthymine
Experimental Section
Melting points were determined with a Buchi capillary appa-
ratus and are uncorrected. 1H NMR and 13C NMR spectra were
determined for samples in Me2SO-d6, CD3OD, or CDCl3 on a
Bruker AM 300 spectrometer using Me4Si (TMS) as an internal
standard. 13C NMR (J modulated spin echo) spectra were deter-
mined for selected compounds where methyl and methyne carbon
resonances appear as positive peaks and where methylene and
quaternarycarbonresonances appearasnegativepeaks. Chemical
shifts are given in ppm relative to TMS. The assignment of all
exchangeableprotons(OH, NH) was confirmedbythe additionof
D2O. All of the final compounds had >95% purity, determined
by microanalysis. Microanalysis results were within (0.4% of
theoretical values for all elements listed, unless otherwise indi-
cated. Silica gel column chromatography was carried out using
Merck 7734 silica gel (100-200 μM particle size). Thin-layer
chromatography (TLC) was performed with Machery-Nagel
Alugram SiL G/uv silica gel slides (20 μM thickness). Thymidine
(1), 5-fluoro-20-deoxyuridine (2), uridine (27a), 5-methyluridine
(27b), and 5-fluorouridine (27c) were purchased from Aldrich.
1-(2-Deoxy-3-O-methanesulfonyl-5-O-trityl-β-D-ribopentofura-
nosyl)thymine (3).20 A mixture of thymidine 1 (1.0 g, 4.13 mmol),
trityl chloride (1.95 g, 7.0 mmol), and 4-(dimethylamino)-
pyridine (0.01 g, 0.80 mmol) in anhydrous pyridine was heated

4136 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Shakya et al.
Table 1. In Vitro Antimycobacterial Activity of Pyrimidine Nucleosides against Wild-Type and Rifampicin- and Isoniazid-Resistant Strains of
M. tuberculosis and Intracellular M. tuberculosis
antimycobacterial activity (M. tuberculosis)
rifampicin-
resistant
(H37Rv)
isoniazid-
resistant
(H37Rv)
intracellular
(H37Ra)
wild-type (H37Ra)
b
MIC50
(μg/mL)
% inhibition
(concn μg/mL)a
% inhibition
(conc. μg/mL) a
% reduction
(concn μg/mL)c
compd
R
X
% inhibition (concn μg/mL)a
7
8
9
CH3
CH3
F
Br
Cl
Br
Br
Cl
Cl
I
100 (100), 82 (50), 50 (5-10)
68 (100), 50 (50)
80 (100), 50 (50)
100 (100), 63 (50), 40 (25)
90 (100), 60 (50), 40 (25)
40 (100)
5-10
50
50
50 (5-10)
NDd
ND
75 (25), 50 (10)
ND
ND
90 (25), 50 (10)
ND
ND
ND
10
12
F
25-50
25-50
ND
ND
ND
ND
F
ND
ND
20
22
F
ND
ND
ND
ND
CH3
F
0 (100)
32 (100)
ND
ND
25
26
I
ND
ND
ND
ND
F
I
40 (100)
24 (100)
ND
ND
32
33
H
ND
ND
CH3
F
100 (100), 50 (100), 75 (10), 50 (1)
91 (100), 50 (25)
0 (100)
1
25
70 (10), 50 (1-2)
ND
ND
75 (10), 50 (2)
ND
ND
>90 (25), 80 (10)
ND
34
35
H
ND
ND
36
37
CH3
F
80 (100), 60 (50), 50 (25)
0 (100)
0 (100)
25
ND
ND
ND
ND
ND
ND
38e
CH3
F
F
F
ND
ND
ND
ND
39f
0 (100)
100 (1)
ND
isoniazid
rifampicin
cycloserine
<1
<0.5
5
100 (1)
0 (2)
ND
0 (2)
100 (0.5-1)
ND
>98 (2.5)
>95 (2.5)
ND
100 (0.5-1)
100 (50), 80 (25), 50 (5)
a The antimycobacterial activity was determined at concentrations of 100, 50, 25, 10, 5, and 1 μg/mL. b Concentration of compounds exhibiting 50%
inhibition in mycobacterial growth. c % reduction of the number of surviving bacteria in the human monocyte cell line (THP-1) with respect to the
untreated control. d ND = not determined. e 30-Fluoro-20,30-dideoxythymidine.14 f 30,5-Difluoro-20,30-dideoxyuridine.14
at 80 °C for 5 h. The reaction was cooled to 0 °C, methanesulfo-
nyl chloride (0.89 g, 7.8 mmol) was added dropwise, and the
reaction mixture was stirred at 0 °C for 1 h. Then, the reaction
mixture was kept in the refrigerator for 24 h. After the addition
of H2O (1 mL), the solvent was evaporated, and the resulting oil
was diluted with chloroform (100 mL), washed with H2O (2 ꢀ
25 mL), dried (Na2SO4), and evaporated in vacuo. The residue
was purified by silica gel column chromatography using CHCl3/
MeOH (97:3, v/v) as the eluent to yield 3 (2.1 g, 90%) as a white
solid; mp 120-122 °C. 1H NMR (CDCl3): δ 1.47 (s, 3H, CH3),
2.43-2.53 (m, 1H, H-20), 2.65-2.72 (m, 1H, H-200), 3.03 (s, 3H,
CH3SO2), 3.50 (m, 2H, H-50), 4.32 (m, 1H, H-40), 5.41 (m, 1H,
H-30), 6.43 (m, 1H, H-10), 7.29-7.41 (m, 15H 50-O-trityl),7.54
(s, 1H, H-6), 8.52 (br s, 1H, NH).
(0.20 g, 2.30 mmol) in anhydrous acetonitrile (20 mL) was
refluxed for 24 h. The solvent was removed in vacuo, and the
residue was purified on a silica gel column using EtOAc/hexane
(20:80; v/v) as the eluent to yield 5 (0.05 g, 51%) as a syrup. 1H
NMR (DMSO-d6): δ 1.57 (s, 3H, CH3), 2.59-2.85 (m, 2H,
H-20), 3.27 (m, 2H, H-50), 4.24 (m, 1H, H-40), 4.77 (dd, J =14.65
and 7.32 Hz, 1H, H-30), 6.22 Hz (dd, J=7.32 and 4.88 Hz, 1H,
H-10), 7.25-7.49 (m, 15H, 50-O-trityl), 7.55 (s, 1H, H-6), 11.42
(s, 1H, NH).
1-(3-Chloro-2,3-dideoxy-5-O-trityl-β-D-erythro-pentofurano-
syl)thymine (6). For the synthesis of this compound, a similar
procedure was followed as described for compound 5 using LiCl
1
instead of LiBr. It was obtained in 50% yield as a syrup. H
NMR (CDCl3): δ 1.46 (s, 3H, CH3), 2.66 (m, 2H, H-20), 3.47 (m,
2H, H-50), 4.24 (m, 1H, H-40), 4.61 (dd, J=9.76 and 4.88 Hz, 1H,
H-30), 6.38 (t, J=6.10 Hz, 1H, H-10), 7.23-7.50 (m, 15H, 50-O-
trityl), 7.61 (s, 1H, H-6), 8.19 (s, 1H, NH).
1-(2-Deoxy-3-O-methanesulfonyl-5-O-trityl-β-D-ribopentofura-
nosyl)-5-fluorouracil (4). This compound was synthesized in
87% yield as a solid using a published procedure20 and used
directly in the next reaction. 1H NMR (CDCl3): δ 2.35-2.47 (m,
1H, H-20) and 2.70-2.80 (m, 1H, H-200), 3.05 (s, 3H, CH3SO2),
3.52 (m, 2H, H-50), 4.36 (m, 1H, H-40), 5.36 (m, 1H, H-30), 6.32
1-(3-Bromo-2,3-dideoxy-β-D-erythro-pentofuranosyl)thymine
(7). A solution of 5 (0.05 g, 0.09 mmol) in 80% aqueous acetic
acid (10 mL; v/v) was heated at 90 °C for 30 min. The solvent was
evaporated in vacuo. The residue so obtained was purified on a
silica gel column using MeOH/CHCl3 (4:96; v/v) as the eluent to
yield 7 (0.010 g, 36%) as a white solid; mp 150-152 °C, dec. 1H
NMR (DMSO-d6): δ 1.77 (s, 3H, CH3), 2.60 (m, 2H, H-20), 3.65
(m, 1H, H-10), 7.21-7.49 (m, 15H, 50-O-trityl), 7.78 (d, J6,F
=
6.10 Hz, 1H, H-6), 8.71 (br s, 1H, NH).
1-(3-Bromo-2, 3-dideoxy-5-O-trityl-β-D-erythro-pentofurano-
syl)thymine (5). A mixture of 3 (0.10 g, 0.18 mmol) and LiBr

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4137
(m, 2H, H-50), 4.19 (m, 1H, H-40), 4.63 (dd, J=12.2 and 6.10 Hz,
1H, H-30), 5.26 (t, J=4.88 Hz, 1H, 50-OH), 6.23 (t, J=6.71 Hz,
1H, H-10), 7.70 (d, J = 1.22 Hz, 1H, H-6), 11.36 (s, 1H, NH).
Anal. (C10H13BrN2O4) C, H, N.
(m, 1H, H-30), 5.33 (d, J = 4.88 Hz, 1H, 30-OH), 6.13 (dd, J =
8.55 and 6.71 Hz, 1H, H-10), 7.21-7.47 (m, 15 H, 50-O-trityl),
7.87 (d, J6,F = 6.71 Hz, 1H, H-6), 11.86 (s, 1H, NH).
1-(3-Chloro-2,3-dideoxy-5-O-trityl-β-D-threo-pentofuranosyl)-
5-fluorouracil (15) and 50-O-Trityl-O2,30-cyclo-5-fluoro-20-deoxy-
uridinium Chloride (16). A mixture of 13 (2.7 g, 11.8 mmol),
triphenylphosphine (1.45 g, 5.53 mmol), and CCl4 (1.7 g, 11.05
mmol) in dry DMF (10 mL) was kept at room temperature for 24
h, diluted with methanol, and evaporated to dryness. The residue
was chromatographed on silica gel using EtOAc/CH2Cl2 (30:70,
v/v) as the eluent to yield 15 (1.6 g, 57%) as a syrup. This product
was used in the next step.
1-(3-Chloro-2,3-dideoxy-β-D-erythro-pentofuranosyl)thymine
(8). Detritylation of compound 6 using the same procedure as
described above provided the title compound. This was ob-
1
tained in 57% yield as a white solid. The H NMR spectrum
obtained for 8 was identical to that reported previously;21 mp
180-182 °C. 1H NMR (DMSO-d6): δ 1.77 (s, 3H, CH3), 2.60 (m,
2H, H-20), 3.64 (m, 2H, H-50), 4.05 (m, 1H, H-40), 4.64 (dd, J=
9.16 and 4.27 Hz, 1H, H-30), 5.26 (br s, 1H, 50-OH), 6.23 (t, J=
6.71 Hz, 1H, H-10), 7.69 (s, 1H, H-6), 11.36 (s, 1H, NH). Anal.
(C10H13ClN2O4) C, H, N.
Subsequent elution from the column using MeOH/CHCl3
(10:90, v/v) as the eluent yielded 16 (0.11 g, 4%) as a syrup.
1H NMR (DMSO-d6): δ 2.50 and 2.65 (2 m, 2H, H-20), 3.14
1-(3-Bromo-2,3-dideoxy-β-D-erythro-pentofuranosyl)-5-fluoro-
uracil (9), 1-(3-Bromo-2,3-dideoxy-β-D-threo-pentofuranosyl)-
5-fluorouracil (10), and 1-(3-O-Methanesulfonyl-2-deoxy-β-D-ery-
thro-pentofuranosyl)-5-fluorouracil (11). A mixture of 4 (1.05 g,
1.85 mmol) and LiBr (2.09 g, 24.10 mmol) in CH3CN was
refluxed for 22 h. The solvent was removed in vacuo. The residue
was purified on a silica gel column using MeOH/CHCl3 (6.5:93.5;
v/v) as the eluent to yield 9 (0.135 g, 24%) as a white solid; mp
0
0
(d, J4 ,5 =6.10 Hz, 2H, H-50), 4.46 (m, 1H, H-40), 5.38 (m, 1H, H-
30), 5.90 (d, J = 3.05 Hz, 1H, H-10), 7.22-7.44 (m, 15H, 50-O-
trityl), 8.17 (d, J6,F=4.27 Hz, 1H, H-6). Anal. (C28H24ClFN2O4)
C, H, N.
50-O-Trityl-O2,30-cyclo-5-fluoro-20-deoxyuridinium Iodide (18).
Methyltriphenoxyphosphonium iodide (0.185, 0.41 mmol) was
added to 13 (0.10 g, 0.20 mmol) in dry pyridine (5 mL), and the
reaction mixture was stirred at room temperature for 1 h. After
the pyridine was removed in vacuo, ethyl acetate was added,
washed with 5% (w/v) aqueous sodium thiosulfate solution
(10 mL) followed by water (2 ꢀ 10 mL), and dried over Na2SO4.
The organic phase was concentrated and triiturated with ether to
1
144-146 °C, dec. H NMR (DMSO-d6): δ 2.52-2.73 (m, 2H,
H-20), 3.67 (m, 2H, H-50), 4.20 (m, 1H, H-40), 4.59 (dd, J=13.42
and 6.71 Hz, 1H, H-30), 5.40 (t, J=4.88, 1H, 50-OH), 6.15 (t, J=
4.88 Hz, 1H, H-10), 8.24 (d, J6,F = 7.32 Hz, 1H, H-6), 11.88
(d, JNH,F=4.88 Hz, 1H, NH). Anal. (C9H10BrFN2O4) C, H, N.
Subsequent elution with MeOH/CHCl3 (7:93; v/v) afforded
11 as an impure substance, which was purified on preparative
TLC using MeOH/CH2Cl2 (7:93; v/v) to yield 11 (0.008 g, 1%)
afford 18 (0.070 g, 57%) as a syrup. 1H NMR (DMSO-d6): δ 2.52
0
and 2.65 (2 m, 2H, H-20), 3.10 (d, J4 ,5 =7.32 Hz, 2H, H-5 ), 4.46
(m, 1H, H-40), 5.37 (m, 1H, H-30), 5.89 (d, J = 3.05 Hz, 1H, H-10),
7.16-7.45 (m, 15H, 50-O-trityl), 8.15 (d, J6,F=5.49 Hz, 1H, H-6).
Anal. (C28H24FIN2O4) C, H, N.
0
0
1
as a syrup. H NMR (DMSO-d6): δ 2.39-2.55 (m, 2H, H-20),
3.29 (s, 3H, CH3SO2), 3.64 (m, 2H, H-50), 4.18 (m, 1H, H-40),
5.26 (m, 1H, H-30), 5.42 (br s, 1H, 50-OH), 6.16 (t, J=6.70 Hz,
1H, H-10), 8.17 (d, J6,F=7.32 Hz, 1H, H-6), 11.55 (br s, 1H, NH).
Anal. (C10H13FN2O7S) C, H, N.
1-(3-Chloro-2,3-dideoxy-5-O-trityl-β-D-erythro-pentofuranosyl)-
5-fluorouracil (19). Pyridine hydrochloride (2.44 g, 21.1 mmol) was
added to 18 (1.05 g, 1.75 mmol) in dry DMF (5 mL), and the
reaction mixture was stirred at room temperature for 8 days. DMF
was removed in vacuo. Ethyl acetate (50 mL) was added, and the
ethyl acetate layer was washed with water (5 mL), dried over
Na2SO4, and concentrated in vacuo. The resulting residue was
purified on a silica gel column using EtOAc/hexane (40:60, v/v) as
the eluent to yield 19 (0.30 g, 34%) as a syrup. 1H NMR (DMSO-
d6): δ 2.53 and 2.72 (2 m, 2H, H-20), 3.30 (m, 2H, H-50), 4.11
(m, 1H, H-40), 4.72 (dd, J=13.43 and 6.71 Hz, 1H, H-30), 6.17 (m,
1H, H-10), 7.20-7.45 (m, 15 H, 50-O-trityl), 7.97 (d, J6,F=6.71 Hz,
1H, H-6), 11.92 (d, JNH,F=3.66 Hz, 1H, NH).
1-(3-Chloro-2,3-dideoxy-β-D-erythro-pentofuranosyl)-5-fluoro-
uracil (20). The title compound was obtained in 64% yield as a
white solid after detritylation of 19 using the same procedure as
described for compound 7; mp 152-154 °C. 1H NMR (DMSO-
d6): δ 2.52 (m, 2H, H-20), 3.65(m, 2H, H-50), 4.05(dd, J=7.93and
3.05 Hz, 1H, H-40), 4.60 (dd, J=11.60 and 5.49 Hz, 1H, H-30),
5.37 (t, J=4.88 Hz, 1H, 50-OH), 6.17(td, J=6.10, Hzand 1.22Hz,
1H, H-10), 8.20 (d, J6,F=7.32 Hz, 1H, H-6), 11.88 (s, 1H, NH).
Anal. (C9H10ClFN2O4) C, H, N.
Further elution with MeOH/CHCl3 (7:93; v/v) provided im-
pure 10, which was purified on preparative TLC using MeOH/
CH2Cl2 (7:93; v/v) as the eluent to yield 10 (0.10 g, 18%) as a
1
syrup. H NMR (DMSO-d6): δ 2.45 and 3.11 (2 m, 2H, H-20),
3.70 (m, 2H, H-50), 4.02 (m, J=10.38 and 4.88 Hz, 1H, H-40), 4.79
(m, 1H, H-30), 5.11 (t, J=4.88 Hz, 1H, 50-OH), 6.01 (ddd, J=5.49
Hz, 4.88 and 1.83 Hz, 1H, H-10), 8.01 (d, J6,F=6.71 Hz, 1H, H-6),
11.92 (s, 1H, NH). Anal. (C9H10BrFN2O4) C, H, N.
1-(3-Chloro-2,3-dideoxy-β-D-threo-pentofuranosyl)-5-fluoro-
uracil (12). Compound 4 (1.0 g, 1.76 mmol) was reacted with
LiCl (1.50 g, 35.4 mmol) in anhydrous acetonitrile in a similar
fashion as described for 5 to yield 12. This was obtained in 11%
yield as a semisolid. 1H NMR (DMSO-d6): δ 2.31 (m, J=15.26
and 1.83 Hz, 1H, H-20), 2.94-3.05 (m, 1H, H-200), 3.73 (m, 2H,
H-50), 4.15 (m, 1H, H-40), 4.77 (m, 1H, H-30), 5.05 (t, J=5.49 Hz,
1H, 50-OH), 6.01 (ddd, J=7.93 Hz, 3.05 and 1.83 Hz, 1H, H-10),
7.91 (d, J6,F = 7.32 Hz, 1H, H-6), 11.91 (s, 1H, NH). Anal.
(C9H10ClFN2O4) C, H, N.
1-(3-Chloro-2,3-dideoxy-β-D-threo-pentofuranosyl)-5-fluoro-
uracil (12) from 15. Detritylation of 15 using 80% acetic acid at
90 °C for 30 min provided compound 12 in 79% yield as a white
solid. The physical data were identical in all respects with that of
12 obtained in the previous reaction.
1-(3-Iodo-2,3-dideoxy-β-D-erythro-pentofuranosyl)thymine (22).
This compound was synthesized according to the published
procedure,20 in 37% yield as a solid. The physical data for 22
were identical to those reported.20
1-(2-Deoxy-5-O-trityl-β-D-erythro-pentofuranosyl)-5-fluoro-
uracil (13). A mixture of 5-fluoro-20deoxyuridine (2, 0.50 g,
2.0 mmol), trityl chloride (0.91 g, 3.26 mmol), and 4-(dimethyl-
amino)pyridine (0.03 g, 0.24 mmol) in anhydrous pyridine was
heated at 80 °C for 5 h. Pyridine was removed in vacuo. The
residue was diluted with water (20 mL) and extracted with ethyl
acetate (2 ꢀ 50 mL), dried over Na2SO4, and concentrated in
vacuo. The residue so obtained was purified on a silica gel
column using MeOH/CHCl3 (3:97, v/v) as the eluent to yield 13
(0.80 g, 81%) as a syrup. 1H NMR (DMSO-d6): δ 2.08-2.29 (m,
2H, H-20), 3.11 (dd, J=10.99 and 3.05 Hz, 1H, H-50), 3.27 (dd,
J = 10.38 and 5.49 Hz, 1H, H-500), 3.87 (m, 1H, H-40), 4.27
1-(3-Iodo-2,3-dideoxy-β-D-erythro-pentofuranosyl)-5-fluoro-
uracil (25) and 1-(3-Iodo-2,3-dideoxy-β-D-threo-pentofuranosyl)-
5-fluorouracil (26). A dried mixture of 4 (2.3 g, 4.06 mmol) and
NaI (10.89 g, 72.65 mmol) in dry DME (50 mL) was refluxed for
20 h. The resulting brown mixture was cooled and filtered, and
the filtrate was evaporated to a solid. The solid was dissolved in
methylene chloride (50 mL) and washed with 5% sodium thio-
sulfatesolution (2 ꢀ 50 mL). The organic layerwas finally washed
with water (25 mL), dried over Na2SO4, and concentrated to give
the crude product. Aqueous 80% AcOH (50 mL; v/v) was added
to the crude product, and the reaction mixture was heated at
90 °C for 30 min. The solvent was removed in vacuo, and the

4138 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10
Shakya et al.
crude product thus obtained was purified on a silica gel column
using EtOAc/hexane (60:40; v/v) as the eluent to give 25 (0.65 g,
45%) as a semisolid. 1H NMR (DMSO-d6): δ 2.63-2.70 (m, 2H,
H-20), 3.72 (m, 2H, H-50), 4.15-4.21 (m, 1H, H-40), 4.31 (dd, J=
16.78 and 8.24 Hz, 1H, H-30), 5.41 (t, J=5.18 Hz, 1H, 50-OH),
5.99-6.03 (m, 1H, H-10), 8.30 (d, J6,F=7.63 Hz, 1H, H-6), 11.82
(d, JNH,F=3.97 Hz, 1H, NH). Anal. (C9H10FIN2O4) C, H, N.
Further elution with EtOAc/Hexane (90:10; v/v) provided
impure 26, which was purified on preparative TLC using
MeOH/CHCl3 (10:90; v/v) as the eluent to yield 26 (0.040 g,
1%) as a syrup. 1H NMR (DMSO-d6): δ 2.50-2.56 (m, 1H, H-
20), 3.00-3.14, (m, 1H, H-200), 3.56-3.74 (m, 3H, H-40, H-50),
4.58 (dd, J=10.99 and 4.88 Hz, 1H, H-30), 5.17 (br s, 1H, 50-OH),
5.95 (m, 1H, H-10), 8.16 (d, J6,F=7.32 Hz, 1H, H-6), 11.92 (d,
30 min afforded 32 in 70% yield as a white solid; mp 184-
185 °C. 1H NMR (DMSO-d6): δ 3.66 (m, 2H, H-50), 4.07 (m, 1H,
H-40), 4.15 (pseudo triplet, J=7.93 and 6.71 Hz, 1H, H-30), 4.53
(q, J = 6.10 Hz,1H, H-20), 5.27 (br s, 1H, 50-OH), 5.58 (dd,
J =8.54 and 1.83 Hz, 1H, H-5), 6.13 (d, J=6.10 Hz, 1H, H-10),
6.24 (d, J=5.49 Hz, 1H, 20-OH), 7.70 (d, J = 7.93 Hz, 1H, H-6),
11.31 (s, 1H, NH). Anal. (C9H11BrN2O5) C, H, N.
1-(3-Bromo-3-deoxy-β-D-arabinofuranosyl)thymine (33). Detri-
tylation of 30b using the procedure described above for 32
afforded compound 33 in 81% yield as a white solid; mp
1
206-210 °C, dec. H NMR (DMSO-d6): δ 1.76 (s, 3H, CH3),
3.69 (m, 2H, H-50), 4.05 (m, 1H, H-40), 4.19 (pseudo triplet, J =
8.54 and 7.32 Hz, 1H, H-30), 4.55 (q, J = 13.43 Hz, 6.71 Hz, 1H,
H-20), 5.32 (t, J = 4.88 Hz, 1H, 50-OH), 6.12 (d, J = 6.10 Hz, 1H,
H-10), 6.19 (d, J = 6.10 Hz, 1H, 20-OH), 7.61 (s, 1H, H-6), 11.30
(s, 1H, NH). Anal. (C10H13BrN2O5) C, H, N.
JNH,F=4.88 Hz, 1H, NH). Anal. (C9H10FIN2O4) C, H, N.
1-(2,3-Anhydro-5-O-trityl-β-D-lyxofuranosyl)uracil (29a). Com-
pound 28a27 (1.6 g, 2.5 mmol) was dissolved in 1 N NaOH in
acetone-water (1:1, 36 mL). The resulting yellow solution was
kept at room temperature for 18 h and then neutralized with 1 N
HCl. The resulting precipitate was filtered. The residue was diluted
with CHCl3 (50 mL), washed with water (2 ꢀ 10 mL), dried
(Na2SO4), and concentrated in vacuo to give 29a (1.0 g, 86%) as a
semisolid. 1H NMR (DMSO-d6): δ 3.20 (m, 2H, H-50), 4.11 (m,
2H, H-20, H-30), 4.26(m, 1H, H-40), 5.62 (d, J =7.93 Hz, 1H, H-5),
6.11 (s, 1H, H-10), 7.22-7.44 (m, 15H, 50-O-trityl), 7.51 (d, J =
7.93 Hz, 1H, H-6), 11.45 (s, 1H, NH).
1-(3-Bromo-3-deoxy-β-D-arabinofuranosyl)-5-fluorouracil (34).
Detritylation of 30c using the procedure described above for 32
provided compound 34 in 58% yield as a white solid; mp 190-
192 °C. 1H NMR (DMSO-d6): δ 3.69 (m, 2H, H-50), 4.01-4.05
(m, 1H, H-40), 4.15 (pseudo triplet, J = 8.50 and 7.30 Hz, 1H,
H-30), 4.56 (q, J=7.32 Hz, 1H, H-20), 5.49 (t, J = 5.44 Hz 1H,
50-OH), 6.08 (dd, J = 6.10 and 1.83 Hz, 1H, H-10), 6.25 (d, J =
6.10 Hz, 1H, 20-OH), 8.11 (d, J6,F = 7.32 Hz, 1H, H-6), 11.87 (d,
JNH,F=4.27 Hz, 1H, NH). Anal. (C9H10BrFN2O5) C, H, N.
1-(2-Bromo-2-deoxy-β-D-xylofuranosyl)uracil (35). This com-
pound was obtained in 67% yield as a syrup after detritylation of
31a using the same procedure described above. 1H NMR
(DMSO-d6): δ 3.65-3.77 (m, 2H, H-50), 4.26-4.39 (m, 3H,
H-20, H-30, H-40), 4.89 (t, J = 4.88 Hz, 1H, 50-OH), 5.64 (dd, J =
8.54 and 1.83 Hz, 1H, H-5), 6.04 (d, J = 3.05 Hz, 1H, H-10), 6.09
(d, J = 1.83 Hz, 1H, 30-OH), 7.72 (d, J = 7.93 Hz, 1H, H-6),
11.39 (s, 1H, NH). Anal. (C9H11BrN2O5) C, H, N.
1-(2,3-Anhydro-5-O-trityl-β-D-lyxofuranosyl)thymine (29b). This
compound was synthesized in 99% yield as a white solid from 28b28
using the same procedure as mentioned above from 29a; mp
1
130-132 °C. H NMR (DMSO-d6): δ 1.64 (s, 3H, CH3), 3.22
(m, 2H, H-50), 4.07 (m, 2H, H-20, H-30), 4.23 (t, J = 4.88 and 5.49
Hz, 1H, H-40), 6.10(s, 1H, H-10), 7.22-7.50 (m, 16H, H-6 and 50-O-
trityl), 11.45 (s, 1H, NH).
1-(2-Bromo-2-deoxy-β-D-xylofuranosyl)thymine (36). This
compound was obtained after detritylation of 31b using the
procedure described above for compound 32, in 75% yield, as a
1-(2,3-Anhydro-5-O-trityl-β-D-lyxofuranosyl)-5-fluorouracil (29c).
The title compound was obtained in 91% yield as a syrup from
28c29 using the method described for compound 29a. 1H NMR
(DMSO-d6): δ 3.15-3.27 (m, 2H, H-50), 4.10 (s, 2H, H-20, H-30),
4.26 (m, 1H, H-40), 6.12 (d, J = 1.83 Hz, 1H, H-10), 7.24-7.48
(m, 15H, 50-O-trityl), 7.61 (d, J6,5F = 6.7 Hz, 1H, H-6), 12.00
(s, 1H, NH).
1-(3-Bromo-3-deoxy-5-O-trityl-β-D-arabinofuranosyl)uracil (30a)
and 1-(2-Bromo-2-deoxy-5-O-trityl-β-D-xylofuranosyl)uracil (31a).
A mixture of 29a (0.50 g, 1.07 mmol) and NH4Br (0.62 g,
6.33 mmol) in absolute ethanol (20 mL) was refluxed for 20 h.
Ethanol was removed in vacuo, and the residue was purified by
silica gel column chromatography using MeOH/CHCl3 (2:98,
v/v) as the eluent to yield 31a (0.08 g, 14%) as a syrup. 1H NMR
(DMSO-d6): δ 3.20-3.30 (m, 1H, H-50), 3.35-3.97 (m, 1H,
H-500), 4.22-4.37 and 4.47-4.57 (2 m, 3H, H-20, H-30, H-40),
5.49 (dd, J = 7.84 Hz, 2.44 and 1.8 Hz, 1H, H-5), 6.03 (d, J =
3.57 Hz, 1H, H-10), 6.13 (d, J =1.8 Hz, 1H, 30-OH), 7.22-7.47
(m, 16H, H-6, 50-O-trityl), 11.41 (s, 1H, NH).
Subsequent elution using CHCl3/MeOH (98:2, v/v) yielded
30a (0.26 g, 44%) as a syrup. 1H NMR (DMSO-d6): δ 3.25-3.45
(m, 2H, H-50), 4.15-4.35 (2 m, 2H, H-30, H-40), 4.54 (d, J =6.10
Hz, 1H, H-20), 5.33 (dd, J = 8.54 Hz, 2.44 and 1.83 Hz, 1H,
H-5), 6.15 (d, J = 6.10 Hz, 1H, H-10), 6.27 (d, J = 6.10 Hz, 1H,
20-OH), 7.22-7.45 (m, 15H, 50-O-trityl), 7.60 (d, J = 8.5 Hz, 1H,
H-6), 11.35 (s, 1H, NH).
1-(3-Bromo-3-deoxy-5-O-trityl-β-D-arabinofuranosyl)thymine
(30b), 1-(3-Bromo-3-deoxy-5-O-trityl-β-D-arabinofuranosyl)-5-
fluorouracil (30c), 1-(2-Bromo-2-deoxy-5-O-trityl-β-D-xylofura-
nosyl)thymine (31b), and 1-(2-Bromo-2-deoxy-5-O-trityl-β-D-
xylofuranosyl)-5-fluorouracil (31c). For the synthesis of these
compounds, the same method was used as described above for
compounds 30a and 31a. Compounds 30b,c and 31b,c were
obtained as syrups in 40, 26, 12, and 7% yield, respectively, and
were used directly in the subsequent deprotection reactions.
1-(3-Bromo-3-deoxy-β-D-arabinofuranosyl)uracil (32). Detri-
tylation of 30a using 80% aqueous acetic acid (v/v) at 90 °C for
1
syrup. H NMR (DMSO-d6): δ 1.76 (s, 3H, 5-CH3), 3.70 (m,
2H, H-50), 4.26 and 4.35 (2 m, 3H, H-20, H-30, H-40), 4.89 (t, J =
4.88 Hz, 1H, 50-OH), 6.04 (d, J = 3.66 Hz, 1H, H-10), 6.12, (d,
J = 3.05 Hz, 1H, 30-OH), 7.60 (s, 1H, H-6), 11.42 (s, 1H, NH).
Anal. (C10H13BrN2O5) C, H, N.
1-(2-Bromo-2-deoxy-β-D-xylofuranosyl)-5-fluorouracil (37).
Detritylation of 31c by the process used above provided 37, in
1
58% yield, as a syrup. H NMR (DMSO-d6): δ 3.73 (m, 2H,
H-50), 4.30 and 4.40 (2 m, 3H, H-20, H-30 and H-40), 4.94 (t, J =
6.10 Hz, 1H, 50-OH), 6.09 (d, J = 1.83 Hz, 1H, H-10) 6.14 (d, J =
3.66 Hz, 1H, 30-OH), 7.96 (d, J6,5F=7.32 Hz, 1H, H-6), 12.00 (d,
JNH,F=4.28 Hz, 1H, NH). Anal. (C9H10BrFN2O5) C, H, N.
In Vitro Antimycobacterial Activity Assay. M. tuberculosis
(H37Ra) was obtained from the American Type Culture Collec-
tion (Rockville, MD). The antimycobacterial activity was de-
termined using the MABA.24 Test compounds were dissolved in
DMSO at 10 mg/mL, and subsequent dilutions were performed
in 7H9GC (Difco Laboratories, Detroit, MI) medium in 96-well
plates. For these experiments, each compound was tested at 100,
50, 25 10, 5, and 1 μg/mL in triplicate. The experiments were
repeated two times, and the mean percent inhibition is reported
in Table 1. The standard deviations were within 10% of the
mean value. Frozen mycobacterial inocula were diluted in
medium 7H9GC and added to each well at final concentration
of 2.5 ꢀ 105 CFU/mL. Sixteen control wells consisted of eight
with bacteria alone (B) and eight with medium alone (M). Plates
were incubated for 6 days, and then, 20 μL of 10ꢀ alamar blue
and 12.5 μL of 20% Tween 80 were added to one M and one B
well. Wells were observed for an additional 24-48 h for visual
color change from blue to pink and read by spectrophotometer
(at excitation 530 nm/525 nm and emission 590 nm/535 nm) to
determine OD values. If the B well became pink by 24 h
(indicating growth), reagent was added to the entire plate. If
the B well remained blue, additional M and B wells were tested

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 10 4139
daily until bacterial growth could be visualized by color change.
After the addition of the reagent to the plate, cultures were
incubated for 24 h, and plates were observed visually for color
change and also read by spectrophotometer. Visual MIC was
defined as the lowest concentration of a compound that pre-
vented a color change from blue to pink. The percent inhibition
was calculated as (test well-M bkg./B well-M bkg.) ꢀ 100. The
lowest drug concentration affecting an inhibition of ∼50% was
considered as the MIC50. Rifampicin, isoniazid, and cycloserine
were used as positive controls. As negative controls, DMSO
(2, 1, 0.2, and 0.02 μL) was added to the B well at concentrations
similar to those of compound wells; M wells served as negative
controls. In most of the experiments, the M wells gave an OD of
3000-4000, and the B wells had OD values ranging between
60000 and 100000.
The color reaction involved adding 50 μL XTT reagents per
well and incubating for 4 h at 37 °C. Plates were read
on an enzyme-linked immunosorbent assay plate reader (Abs
450-500 nm). For the 3H-Tdr incorporation assay, Huh-7 cells
were plated at 1 ꢀ 104 cells/well in 96-well flat bottom plates.
Mediumcontaining compoundsatconcentrationsof200, 100, 50,
10, and 1 μg/mL was added to the plates in triplicate. DMSO was
also included as the control. Plates were incubated for 2 days at
37 °C. The wells were pulsed with 0.5 μCi/well [3H]-thymidine
(Amersham) for 12-18 h. After this, the plates were harvested on
filter papers (Perkin-Elmer) using a 96-well plate harvester
(Tomtech MACH III M). The levels of [3H]-thymidine incorpo-
rated into the DNA of proliferating cells were counted in
a Microbeta Trilux liquid scintillation counter (Perkin-Elmer).
Acknowledgment. We are grateful to the Canadian Insti-
tutes of Health Research (CIHR) for an operating grant
(MOP-49415) for the financial support of this research. B.A.
is a recipient of a Senior Scholar Award from the Alberta
Heritage Foundation for Medical Research (AHFMR).
Antimycobacterial Activity against Drug-Resistant Strains of
M. tuberculosis. The activity of compounds 7 and 33 was deter-
mined against rifampicin-resistant M. tuberculosis H37Rv
(ATCC 35838, resistant to rifampicin at 2 μg/mL) and isoniazid-
resistant M. tuberculosis H37Rv (ATCC 35822, resistant to
isoniazid at 2 μg/mL) using a radiometric BACTEC assay.25
This assay detects the metabolism of 14C-labeled palmitic acid,
where evolving 14CO2 is captured and counted as a measure of
mycobacterial growth and metabolism. The growing inoculum
(2.5-5.0 ꢀ 105 CFU/vial) was diluted in a BACTEC vial con-
taining radiometric 7H12 (BACTEC 12B) medium and incu-
bated at 37 °C. Two-fold dilutions of test compounds were
delivered to the inoculum-containing BACTEC vials. Negative
control vials consisted of media with bacteria inoculum, med-
ium with bacteria inoculum at 1:100, and medium alone. As
reference drugs, rifampicin and isoniazid were used at their
MIC90 concentrations. All of the vials were incubated at 37 °C,
and the growth index (GI) was determined in a BACTEC 460
instrument until the GI of the 1:100 inoculum controls reached
30. Vials were read daily, and a change in GI (ΔGI) was recorded
for each compound. The percent inhibition was defined as (GI of
test sample/GI of control) ꢀ 100. For the no drug control, the
ΔGI continued to increase and was much higher than the 1:100
inoculum control. The BACTEC assay was preferred with the
resistant strain, because the method provides a safe, enclosed,
and biocontained method to monitor the kinetics of drug
inhibition.
Intracellular Antimycobacterial Activity in Human Monocytic
Cell Line Infected with M. tuberculosis (H37Ra). Human mono-
cytic cell line (THP-1) was cultured at 2 ꢀ 105 cells/well in a
24-well tissue culture plate with acid-washed, sterilized glass
coverslips. A 100 nM concentration of PMA (phorbol myristate
acetate) was added to each well, and the plates were incubated
for 3 days. At this time, ∼107 CFU of M. tuberculosis was added
to each well and allowed to infect the macrophages for 3 h.
Nonphagocytosed mycobacteria were removed with the super-
natant, and the coverslips were moved to new 24-well plates.
Test compounds were added in triplicate to the infected cell
culture in concentrations of 25, 10, 5, or 1 μg/mL along with
control wells of 2.5 μg/mL rifampicin or isoniazid and DMSO/
media and incubated for 4 days. After the contents of each well
were lysed, aliquots of lysates were transferred to 7H11 plates
in serial dilution. The growing colonies were counted at
2-3 weeks. The experiment was repeated twice, and the percent
reduction in CFUs was calculated.
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