Inhibition of mycobacterial replication by pyrimidines possessing various C-5 functionalities and related 2′-deoxynucleoside analogues using in vitro and in vivo models

Article abstract of DOI:10.1021/jm100568q

Tuberculosis (TB) has become an increasing problem since the emergence of human immunodeficiency virus and increasing appearance of drug-resistant strains. There is an urgent need to advance our knowledge and discover a new class of agents that are distin

Full text of DOI:10.1021/jm100568q

6180 J. Med. Chem. 2010, 53, 6180–6187
DOI: 10.1021/jm100568q
Inhibition of Mycobacterial Replication by Pyrimidines Possessing Various C-5 Functionalities and
Related 20-Deoxynucleoside Analogues Using in Vitro and in Vivo Models
Naveen C. Srivastav,† Dinesh Rai,† Christopher Tse,† B. 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 May 10, 2010
Tuberculosis (TB) has become an increasing problem since the emergence of human immunodeficiency
virus and increasing appearance of drug-resistant strains. There is an urgent need to advance our
knowledge and discover a new class of agents that are distinct than current therapies. Antimycobacterial
activities of several 5-alkyl, 5-alkynyl, furanopyrimidines and related 20-deoxynucleosides were in-
vestigated against Mycobacterium tuberculosis. Compounds with 5-arylalkynyl substituents (23-26, 33,
35) displayed potent in vitro antitubercular activity against Mycobacterium bovis and Mycobacterium
tuberculosis. The in vivo activity of 5-(2-pyridylethynyl)-uracil (26) and its 20-deoxycytidine analogue,
5-(2-pyridylethynyl)-20-deoxycytidine (35), was assessed in BALB/c mice infected with M. tuberculosis
(H37Ra). Both compounds 26 and 35 given at a dose of 50 mg/kg for 5 weeks showed promising in vivo
efficacy in a mouse model, with the 20-deoxycytidine derivative being more effective than the uracil
analogue and a reference drug D-cycloserine. These data indicated that there is a significant potential in
this class of compounds.
Introduction
Chemotherapy with antituberculosis drugs such as strepto-
mycin, rifampicin and several six-membered aromatic ring
derivatives such as isoniazid (1), pyrazinamide (2), p-amino-
salicylic acid (3), and ethionamide (4) revolutionized TB
therapy in the 1970s and resulted in a rapid decline in
tuberculosis in many developed countries. However, there is
now an ever-increasing threat of drug-resistant TB becoming
epidemic in many countries because no new classes of TB-
specific drugs have been developed since rifampicin.2,3,7-9
Tuberculosis (TBa) is an ancient infectious disease that still
remains a leading cause of morbidity and mortality world-
wide. At the present time, nearly two billion people worldwide
are infected with the tubercle bacillus, and the prevalence of
active TB is increasing.1-3 The latent infection in many of
these individuals may reactivate sometime later in life. Tuber-
cle bacilli can be contained in the presence of effective cellular
immunity but immunocompromised status such as human
immunodeficiency virus (HIV) infection, cancer chemotherapy,
or use of immunosuppressive drugs in transplantation provides
the single most significant factor in reactivation of the latent TB
leading to full clinical disease.4,5
TB and HIV have formed a new and deadly combina-
tion. There is a resurgence in the incidence of TB with high
rates of HIV-TB coinfections. Mycobacterium tuberculosis
(M. tuberculosis) poses a significant challenge to the clinical
management of TB in HIV-infected patients and is often res-
ponsible for their death.6 Bacillus Calmette Guerin (BCG)6 is
an attenuated strain of Mycobacterium bovis that is >98%
homologous to M. tuberculosis and, therefore, is closely
related to M. tuberculosis. Interestingly, M. bovis infections
have re-emerged and are causing TB in humans, particularly
those who are HIV positive.
*To whom correspondence should be addressed. Phone: (780) 492-
7545. Fax: (780) 492-7521. Email: rakesh.kumar@ualberta.ca. Address:
Department of Laboratory Medicine and Pathology, 1-71 Medical
Sciences Building Faculty of Medicine and Dentistry, University of
Alberta, Edmonton, AB, Canada T6G 2H7.
a Abbreviations: TB, tuberculosis; M. bovis, Mycobacterium bovis; M.
tuberculosis, Mycobacterium tuberculosis; M. avium, Mycobacterium avium;
BCG, Bacillus Calmette Guerin; MABA, microplate alamar blue assay;
CFU, colony forming unit; B. subtilis, Bacillus subtilis; S. aureus, Staphylo-
coccus aureus; S. pneumoniae, Streptococcus pneumoniae; S. pyogenes, Stre-
ptococcus pyogenes; SAR, structure-activity relationship; INH, Isoniazid.
Drug resistance has been observed for all of the first-line
antimycobacterial drugs as well as for several second-line
r
pubs.acs.org/jmc
Published on Web 07/22/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6181
antituberculosis drugs.10 Although drug-resistant TB was
reported before the emergence of HIV, the problem of drug-
resistant TB was not as severe as it is today.6,10,11 Thus,
there exists an urgent need for the design of new classes of
antimycobacterial agents which the bacteria have never
encountered.
In our earlier studies, we synthesized and evaluated various
nucleoside compounds for their anti-TB activity.12-17 Among
different substituents, werevealedthatpyrimidinenucleosides
with acetylenic side chains at the C5 position exert re-
markable in vitro inhibition of M. bovis, M. avium, and
M. tuberculosis.12,14,15 In these studies, we also noted that 5-(1-
azido (or halo)-2-haloethyl) substituents and 5-alkylalkynyl
moieties at the C-5 position of the substituted pyrimidines
contributed to significant antimycobacterial activities.13,16 As
part of our ongoing program to identify agents that display
potent anti-TB activity and to further explore the SARs, we
have now designed, synthesized, and evaluated a new series of
5-alkyl (7, 9-16) and 5-alkyl (or aryl)alkynyl pyrimidines
(17-26, 28-31) for their antimycobacterial properties. In
these studies, we discovered that pyrimidines with 5-arylalk-
ynyl pharmacophores (23-26) exhibit potent inhibition of
M. bovis and M. tuberculosis in comparison to their 5-alkyl (7,
9-16) and 5-alkylalkynyl (17-22) counterparts. Among the
5-arylalkynyl pyrimidine compounds, 5-(2-pyridylethynyl)-
uracil (26) demonstrated most potent activity against
M. tuberculosis (H37Ra) and was tested for its efficacy in a
murine model of H37Ra infection. In our previous investiga-
tions, we observed that in order to confer a substantial
antimycobacterial effect, a glycosyl moiety at the N-1 position
of substituted uracil plays a crucial role.12,14,15 Further, an
amino group at C-4 appears to be preferred for improved anti-
TB activity in the 5-alkynyl-20-deoxyuridine series of
compounds.12 Encouraged by considerable in vivo inhibition
of mycobacteria by compound 26, we also synthesized 20-
deoxyuridine (33) and 20-deoxycytidine (35) derivatives of
5-(2-pyridylethynyl)-uracil (26) and investigated their poten-
tial as anti-TB agents. Although both nucleosides 5-(2-
pyridylethynyl)-20-deoxyuridine (33) and 5-(2-pyridyl-
ethynyl)-20-deoxycytidine (35) exhibited potent in vitro activ-
ity against M. tuberculosis (H37Ra) that were comparable,
compound 35 demonstrated no cytotoxicity, therefore, it was
selected for in vivo activity studies. Intriguingly, compound35
substantially reduced the bacterial load in lung, liver, and
spleen tissues in three out of five mice as compared to its uracil
analogue (26). For the first time, we report the antimycobac-
terial potential of 5-alkynyl analogues of pyrimidine nucleo-
sides in vivo.
Scheme 1a
a Reagents and conditions: (i) CH2dCHCOOEt, (Ph3P)2PdCl2,
Et3N; (ii) 0.5 N KOH (6); (iii) I2, KIO3, H2O, H2SO4 (7); (iv) vinyl
acetate, (Ph3P)2PdCl2, Et3N, 80 °C (8); (v) N-iodosuccinimide, H2O, 25
°C (9), N-halosuccinimide, MeOH, 25 °C (10-12), N-iodosuccinimide,
EtOH, 25 °C (13), iodine mono chloride, BrCH2CH2OH (14), iodine
mono chloride, FCH2CH2OH (15), iodine mono chloride, CF3CH2OH
(16), 25 °C.
Scheme 2a
Chemistry
Synthesis of 5-alkyl uracils (9-16) (Scheme 1) was carried
out using reported procedures described by us earlier.18 5-
Alkynyl pyrimidines (17-22, 24, 25) were prepared by the Pd-
catalyzed coupling reaction of 5-iodoracil (5) with appropriate
alkylacetylenes, arylacetylenes, and para substituted arylace-
tylenes to yield the target 5-alkynyl pyrimidines 17-22, 24, 25
as described in Scheme 2. The 5-(2-phenylethynyl)-uracil (23)
was synthesized using a previously reported method.19 Our
initial attempts to synthesize related 5-(2-pyridylethynyl)-
uracil (26) were unsuccessful using similar reaction conditions
a Reagents and conditions: (i) Pd(PPh3)4, CuI, (i-Pr)2EtN, DMF, N2
atmosphere, room temperature, 1-pentyne (17), 1-heptyne (18), 1-de-
cyne (19), 1-dodecyne (20), 1-tridecyne (21), 1-tetradecyne (22), pheny-
lacetylene (23), 4-n-propylphenylacetylene (24), 1-ethynyl-4-pentyl-
benzene (25), 2-ethynylpyridine (26); (ii) CuI, Et3N, dry MeOH, reflux.
Scheme 3

6182 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Srivastav et al.
Scheme 4a
a Reagents and conditions: (i) Pd(PPh3)4, CuI, (i-Pr)2EtN, 2-ethynylpyridine, DMF, N2 atmosphere, room temperature.
where an undesired dimer of 2-ethynylpyridine was obtained
in 30% yield (Scheme 3). The formation of the dimer, 1,4-
bis-(2-pyridyl)-buta-1,3-diyne (27), can be explained based on
oxidative homocoupling reactions20 as depicted in Scheme 3.
The structure of 27 was confirmed with the help of 1H NMR,
13C NMR spectroscopic data and microanalysis studies. To
avoid unexpected dimerization mentioned above, we carried
out the synthesis of compound 26 by taking extra precautions
and completely eliminating moisture and oxygen from the
reaction medium. The reaction flask was oven-dried and
cooled in a nitrogen atmosphere and the reaction performed
under positive pressure of nitrogen, yielding the desired
compound 26 in 87% yield.
the C-5 position. Together, these results suggest that in addition
to the 5-substituent, N-1 glycosyl moieties also play a role in the
antimycobacterial activity.
Encouragingly, among the pyrimidines (17-26), 5-arylalk-
ynyl uracil derivatives 23-26 emerged as potent inhibitors of
M. bovis and M. tuberculosis. 5-(4-n-Propylphenylethynyl)-
uracil (24), 5-(4-n-pentylphenylethynyl)-uracil (25), and 5-(2-
pyridylethynyl)-uracil (26) provided significant inhibition of
both mycobacteria tested, M. bovis (MIC50 = 1-5 μg/mL)
and M. tuberculosis (MIC50 = 10, 10, and 5 μg/mL, respec-
tively), where compound 26 showed the best activity against
H37Ra (Table 1). Among compounds 23-26, it appears that
the alkyl substituent present at the 4-position of the phenyl-
alkynyl group (as in 24, 25) is influential for increased anti-
mycobacterial effect because the compound 23 showed
reduced inhibition of both M. bovis and M. tuberculosis.
Replacement of the phenyl ring by a pyridinyl moiety (as in
26) in the 5-alkynyl side chain led to significantly improved
activity against both mycobacteria. The increased activity of
26 could possibly be due to increased interaction (H-bonding)
of the heteroaryl ring with mycobacterial enzymes. In com-
paring the antimycobacterial activities of the arylalkynyl
pyrimidines (24, 25) with their corresponding inactive 5-alkyl-
alkynyl derivatives (17, 18), we note that inclusion of a
phenyl ring completely reversed the activity of these mole-
cules. These studies reflect that aromatic acetylene moieties at
the 5-position of pyrimidine are crucial to provide antimyco-
bacterial activities and conformational freedom of the alkyl
side chain is not tolerated in this series of compounds.
Alternatively, antimycobacterial action of compounds
23-26 could be due to the shape and size of the side chains
that may produce steric hindrance. In contrast, inclusion of
the aryl or p-alkylaryl ring in the 5-alkynyl side chain of the
deoxy, dideoxy, and arabinouridine compounds was detrimen-
tal to antimycobacterial activities.12,14,15 These studies suggest
that pyrimidines 23-26 are perhaps working by different
mechanism of action or targets than nucleoside compounds.
The antimycobacterial activity of the dimer 27 was also eval-
uated. Surprisingly, compound 27 showed remarkable inhibi-
tion of BCG replication (80% at 0.5-1 μg/mL) and H37Ra
replication (80% at 1 μg/mL). However, it was found to be
cytotoxic [in the XTT cell cytotoxicity assay against a human
hepatoma cell line (Huh-7 cells) CC50 = 10 μg/mL] in compar-
ison to compounds 23-26 (CC50 = >100 μg/mL).
Cyclization21 of selective 5-alkynyl uracils (17-20) in the
presence of copper iodide and triethylamine in dry methanol
at reflux temperature yielded the corresponding bicyclic furano-
pyrimidines (28-31) in 38-64% yields (Scheme 2).
Synthesis of 5-(2-pyridylethynyl)-20-deoxyuridine (33) has
been described by Pichat and co-workers22 by the coupling
reaction of a protected 30,50-trimethylsilylated 5-iodo-20-deoxy-
uridine with alkynylzinc reagent in 17% overall yield. For the
synthesis of nucleoside 33 and its 20-deoxycytidine derivative
(35), we used methodologies similar to that of compounds
17-25. The single step Pd-catalyzed coupling reaction of
2-ethynylpyridine with respective 5-iodo-20-deoxy nucleosides
(32, 34) provided target compounds 33 and 35 in excellent
yields of 97% and 81%, respectively (Scheme 4).
Results and Discussion
5-Substituted pyrimidines (7, 9-31) and 20-deoxy nucleo-
sides (33, 35) were evaluated in vitro against M. bovis and
M. tuberculosis (H37Ra) by the microplate alamar blue assay
(MABA) at 1-100 μg/mL concentrations. Rifampicin, iso-
niazid (INH), and D-cycloserine were used as positive control
drugs. The results are summarized in Table 1. The 5-(1-
hydroxy (or alkoxy)-2-haloethyl) pyrimidines (7, 9-16, data
not shown) did not prove to be inhibitory against both
mycobacteria tested. In earlier studies,16 we observed that in
addition to 5-alkyl side chains, 5-alkylalkynyl (5-decynyl and
5-dodecynyl) moieties at the C-5 position of the specific N-1
substituted pyrimidines also contribute to significant antimyco-
bacterial activities. Therefore, to further establish the structural
requirements at the 5-position of pyrimidines for antimycobac-
terial activity, we explored various 5-alkyl (or aryl)alkynyl
pyrimidines (17-26). Within these, 5-alkylalkynyl substituted
pyrimidines (17-22, Table 1) were found to be inactive as
antimycobacterial agents despite their varying chain lengths at
Modification of the 5-substituents in the pyrimidines
17-20 to the corresponding bicyclic analogues 28-31 yielded
compounds with almost no antimycobacterial activity with

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6183
Table 1. In Vitro Antimycobacterial Activity of 5-Substituted Pyrimidines against M. bovis and M. tuberculosisa
M. bovis (BCG) % inhibition
(concentration μg/mL)
M. tuberculosis (H37Ra) % inhibition
(concentration μg/mL)
MIC50
[μg/mL]
compd
R
MIC50 [μg/mL]
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
33
35
CtC-(CH2)2CH3
CtC-(CH2)4CH3
CtC-(CH2)7CH3
CtC-(CH2)9CH3
CtC-(CH2)10CH3
CtC-(CH2)11CH3
CtC-C6H5
0
0
0
0
0
0
0
0
0
0
0
0
90 (100, 50), 40 (10)
100 (100), 70 (50,10), 50 (1-5)
90 (100), 60 (50,10), 50 (1-5)
90 (100), 75 (50,10), 60 (5)
100 (100,50,10), 80 (0.5-1)
90 (100), 50 (50,10)
50 (100)
[>10]
[1-5]
[1-5]
[<5]
90 (100, 50), 40 (10)
90 (100), 55 (50,10), 0 (5)
[>10]
[<10]
[10]
CtC-C6H4-n-propyl
CtC-C6H4-n-pentyl
CtC-(2-pyridyl)
90 (100), 50 (50,10), 0 (5)
90 (100), 70 (50), 60 (10), 50 (5)
[5]
[<0.5]
[10-50]
[100]
100 (100,50,10), 80 (1)
50 (100), 0 (10)
[<1]
[100]
(CH2)2CH3
(CH2)4CH3
0
(CH2)7CH3
(CH2)9CH3
0
0
0
0
CtC-(2-pyridyl)
CtC-(2-pyridyl)
90 (100, 50), 80 (10), 50 (1-5)
90 (100, 50), 80 (10), 50 (1-5)
100 (1)
[1-5]
[1-5]
[<1]
[<1]
[5]
90 (100), 78 (50), 68 (10), 50 (2.5-5)
90 (100, 50), 70 (10), 50 (2.5-5)
100 (1)
[2.5-5]
[2.5-5]
[<1]
rifampicinb
INHb,c
100 (1)
71 (10), 50 (5)
100 (1)
67 (10), 50 (5)
[<1]
[5]
cycloserineb
a Antimycobacterial activity was determined at concentrations 100, 50, 10, 5, 2.5, and 1 μg/mL. b Positive control drug. c INH = isoniazid.
the exception of a significant shift in the antimycobacterial
activity of compound 28 (MIC50 = 10 μg/mL, BCG; 100 μg/
mL H37Ra) when compared to its acyclic analogue 17.
Subsequently, we tested compound 26 for its efficacy in
female BALB/c mice infected with H37Ra at a dose of 50 mg/kg
by the intraperitoneal route as described in detail in the Experi-
mental Section. The mice were infected with M. tuberculosis
strain H37Ra. Encouragingly, compound 26, caused consider-
able reduction of the CFU counts in the lungs, liver, and spleen
of the drug treated animals compared with those of the un-
treated controls (Figure 1A). Out of the five mice, compound 26
decreased the bacterial load in the lungs and liver of three mice
(lungs: 75% inhibition in one, 50% inhibition in two; liver: 50%
inhibition in three mice), but in the spleen only one mouse (70%
inhibition) showed reduction in bacterial growth. Reduction
in CFUs in lungs by compound 26 was statistically significant
(P= 0.03) compared to untreated mice. However, the reduction
in CFU counts in liver and spleen by compound 26 showed a
trend but did not meet significance (P > 0.05). Compound 26
was found to be much less active than isoniazid administered at
25 mg/kg dose. However, no toxicity was observed in any of the
mice treated with 26.
observations, we investigated the antimycobacterial activity
of nucleoside derivatives of the most effective compound, 26.
Interestingly, both of the 20-deoxy pyrimidine nucleoside
analogues (33, 35) showed potent inhibition of M. bovis and
M. tuberculosis (MIC50 = 1-5, 2.5-5 μg/mL, respectively).
Encouragingly, antimycobacterial activity of 33 and 35
against M. tuberculosis was also improved (68-70% inhibi-
tion at 10 μg/mL) as compared to their parent pyrimidine
compound (26, 70% inhibition at 50 μg/mL). From these
results, it is clear that 2-pyridylethynyl functionality at the C-5
position has the ability to retain the antimycobacterial activity
in case of both pyrimidine and pyrimidine nucleosides.
Although compounds 33 and 35 were found to exert compar-
able in vitro activity against H37Ra, the 20-deoxycytidine
3
analogue 35 showed no cytotoxicity in XTT and H incor-
poration assays in Huh-7 cells up to a concentration of >200
μg/mL, whereas compound 33 showed 25% inhibition in cell
viability at 200 μg/mL in the XTT assay. Therefore, com-
pound 35 was selected to test its potency in the mouse model
(H37Ra). Drug treatment was started five days post infection
and was given for 5 weeks (5 days a week). Mice were treated
with an intraperitoneal dose of compound 35 at 50 mg/kg
formulated in 10% DMSO-saline. The efficacy of 35 was
compared to that of D-cycloserine at 50 mg/kg and isoniazid
at 25 mg/kg, which were administered as parallel treat-
ment. The results are described in Figure 1B. Compound 35
showed significant activity in three out of five mice (85-90%
In our previous investigations, we observed that in order
to confer a substantial antimycobacterial effect, a glycosyl
moiety at the N-1 position of substituted uracil plays a crucial
role. Further, an amino group at C-4 appears to be preferred
for improved anti-TB activity.12,14,15 Upon the basis of these

6184 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Srivastav et al.
Figure 1. Activities of compound 26, compound 35, cycloserine, and INH in murine model of tuberculosis. In vivo efficacy of compound 26
(A), compound 35 (B), cycloserine, and isoniazid (INH) against M. tuberculosis. Mice (n = 5 or 3) were treated intraperitonially 5 days/week for
five weeks. CFUs obtained from individual mouse organs (2 lungs, 1 liver, 1 spleen) are shown in the figure. Each symbol represents an
individual mouse.
reduction of the CFU in the lungs) compared to the untreated
control group. Compound 35 reduced the bacterial load to
∼50% in four out of five mice in the liver tissues. In the
spleens, compound 35 was slightly less effective, providing
∼50% inhibition ofthe CFUsinonly three mice. Reduction in
the mycobacterial load in lungs and livers of mice treated with
compound 35 was statistically significant (P < 0.05) com-
pared to untreated mice. Mice administered compound 35 at
50 mg/kg for 5 weeks showed no adverse effects in terms of
weight loss, behavioral changes, or in the findings of gross
necroscopy after the mice were euthanized. It is notable that
compound 35 showed moderate MIC values in vitro, it
exhibited significant efficacy in vivo. Although compound
35 was less active than isoniazid both in vitro and in vivo, it
exhibited similar in vitro and superior in vivo inhibition of
mycobacterial replication in all organs (lungs, spleen, liver) as
compared to another reference drug cycloserine (Figure 1B).
It has been reported that 50-70% of cycloserine is excreted
unchanged within 12-24 h following administration.23 The
improved in vivo inhibition exerted by 35 compared to
cycloserine could be due to factors such as longer plasma
half-life, higher stability, better bioavailability, better cellular
delivery, and/or different mechanism of action.
100 μg/mL except 18, suggesting a preferential action against
mycobacteria of the most active compounds (24-26, 33, 35).
Compound 5-heptynyl-uracil (18) possessed activity only
against the Gram-positive bacteria S. epidermis (MIC90 =
10 μg/mL), and S. aureus, S. pyogenes, E. fecalis, and
L. monocytogenes (MIC90 = 50 μg/mL).
The precise mechanism of action of the active compounds
inhibiting mycobacterial multiplication in this study is not
clear. The complete genome sequence of M. tuberculosis has
been deciphered.24 It encodes many enzymes required for
DNA and RNA synthesis and pyrimidine and purine nu-
cleoside biosynthesis. It is postulated that active compounds
may be inhibiting mycobacterial DNA and/or RNA synth-
esis by acting as substrates and/or inhibitors of metabolic
enzymes of DNA and/or RNA synthesis. It is also possible
that the active pyrimidinediones and pyrimidine nucleosides
could be acting at two different mycobacterial targets and/
or processes by virtue of dissimilarity in their structural
features.
The promising compounds 23-27, 33, and 35 were tested in
vitro for their toxicity for Huh-7 cells in concentrations up to
200 μg/mL using XTT and 3H thymidine incorporation
assays. No toxicity was observed except for compounds 27
(50% inhibition at 10 μg/mL concentration) and 33 (25%
inhibition at 200 μg/mL by XTT assay). These studies confirm
that the anti-TB activity of 23-26, 33, and 35 does not arise
due to cytotoxicity of these compounds.
The compounds 6, 7, 9-31, 33, 35 were also evaluated for
their antibacterial activities against several Gram-positive and
Gram-negative bacteria (Staphylococcus aureus, Staphylococcus
epidermis, Enterococcus fecalis, Bacillus subtilis, Streptococcus
pneumoniae, Streptococcus pyogenes, Listeria monocytogenes,
Salmonella typhimurium, Escherichia coli, Proteus vulgaris,
Pseudomonas aeruginosa). However, none of these compounds
exhibited any antibacterial activity at concentrations up to
Conclusion
Tuberculosis is a significant global health problem and a
high priority for research. There is an immense need to

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6185
7.64 (s, 1H, H-6), 11.15 (br s, 1H, NH), 11.28 (s, 1H, NH). 13
discover novel agents for the treatment of tuberculosis, which
work on targets and by mechanisms different from current
therapies. A group of 5-alkyl, alkylalkynyl, and arylalkynyl
analogues of pyrimidine and 20-deoxy pyrimidine nucleosides
were investigated for their antimycobacterial action. Com-
pounds 23-26, 33, and 35 showed significant antimycobac-
terial activity in vitro. Compound 26 and 35 demonstrated
encouraging in vivo efficacy and did not show in vitro or in
vivo toxicity. These results suggest that there is an excellent
potential in this class of compounds, particularly 5-arylalk-
ynyl derivatives, to develop new antimycobacterial agents.
Combination chemotherapy has been the most successful
strategy for the treatment of tuberculosis. Therefore, new
classes of compounds discovered in these studies have great
potential to be used in combination with current TB drugs to
shorten the duration of treatment, provide better compliance,
reduce toxicity, and avoid or delay the emergence of resistance
because they possess entirely different molecular structures
and are expected to have different mechanisms of action.
C
NMR (DMSO-d6) δ 13.85 (CH3), 18.75, 21.64, 27.89, 30.44 (4 ꢀ
CH2), 72.89 (C-β), 92.68 (C-R), 97.56 (C-5), 144.38 (C-6), 150.34
(C-2), 162.74 (C-4). Anal. (C11H14N2O2) C, H, N.
5-Decynyl-uracil (19). This was obtained as a solid in 89%
yield; mp 228-230 °C (dec). 1H NMR (DMSO-d6) δ 0.85 (t, J =
6.72 Hz, 3H, CH3), 1.20-1.50 (m, 12H, 6 ꢀ CH2), 2.33 (t, J =
6.72 Hz, 2H, R-CH2), 7.63 (s, 1H, H-6), 11.12 (s, 1H, NH), 11.28
(s, 1H, NH). 13C NMR (DMSO-d6) δ 13.92 (CH3), 18.77, 22.04,
28.18, 28.23, 28.47, 28.56 (6 ꢀ CH2), 31.20 (R-CH2), 72.88 (C-β),
92.67 (C-R), 97.56 (C-5), 144.33 (C-6), 150.33 (C-2), 162.72
(C-4). Anal. (C14H20N2O2) C, H, N.
5-Dodecynyl-uracil (20). This was obtained as a solid in 80%
yield; mp 201-203 °C (dec). 1H NMR (DMSO-d6) δ 0.85 (t, J =
7.20 Hz, 3H, CH3), 1.24-1.50 (m, 16H, 8 ꢀ CH2), 2.33 (t, J =
7.20 Hz, 2H, R-CH2), 7.62 (s, 1H, H-6), 11.20 (br s, 2H, 2 ꢀ NH).
13C NMR (DMSO-d6) δ 13.92 (CH3), 18.78, 22.06, 28.19, 28.33,
28.49, 28.65, 28.73, 28.91 (8 ꢀ CH2), 31.25 (R-CH2), 72.91 (C-β),
92.63 (C-R), 97.53 (C-5), 144.46 (C-6), 150.38 (C-2), 162.72
(C-4). Anal. (C16H24N2O2) C, H, N.
5-Tridecynyl-uracil (21). This was obtained as a solid in 51%
yield; mp 222-224 °C. 1H NMR (DMSO-d6) δ 0.85 (t, J = 7.02
Hz, 3H, CH3), 1.24-1.49 (m, 18H, 9 ꢀ CH2), 2.33 (t, J = 7.02
Hz, 2H, R-CH2), 7.63 (s, 1H, H-6), 11.13 (br s, 1H, NH), 11.27 (s,
1H, NH). 13C NMR (DMSO-d6) δ 13.93 (CH3), 18.78, 22.07,
28.20, 28.23, 28.35, 28.52, 28.69, 28.93, 28.98 (9 ꢀ CH2), 31.27
(R-CH2), 72.89 (C-β), 92.67 (C-R), 97.58 (C-5), 144.36 (C-6),
150.34 (C-2), 162.73 (C-4). Anal. (C17H26N2O2) C, H, N.
5-Tetradecynyl-uracil (22). This was obtained as a solid in
72% yield; mp 232-234 °C (dec). 1H NMR (DMSO-d6) δ 0.85
(t, J = 7.02 Hz, 3H, CH3), 1.23-1.51 (m, 20H, 10 ꢀ CH2), 2.33
(t, J = 7.02 Hz, 2H, R-CH2), 7.63 (s, 1H, H-6), 11.12 (s, 1H,
NH), 11.28 (s, 1H, NH). 13C NMR (DMSO-d6) δ 13.89 (CH3),
18.75, 22.03, 28.17, 28.34, 28.47, 28.52, 28.65, 28.75, 28.88, 28.94
(10 ꢀ CH2), 31.23 (R-CH2), 72.87 (C-β), 92.64 (C-R), 97.58
(C-5), 144.28 (C-6), 150.29 (C-2), 162.67 (C-4). Anal. (C18H28-
N2O2) C, H, N.
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, CDCl3, or CD3OD on a
Bruker AM 300 spectrometer using Me4Si as an internal stan-
dard. 13C NMR (J modulated spin echo) spectra were determined
for selected compounds where methyl and methyne carbon
resonances appear as positive peaks and where methylene and
quaternary carbon resonances appear as negative peaks. Chemi-
cal shiftsare giveninppm relativetoTMSas aninternalstandard.
The assignment of all exchangeable protons (OH, NH) was
confirmed by the addition of D2O. All of the final compounds
had >95% purity, determined by microanalysis. Microanalysis
results were within (0.4% of theoretical values for all ele-
ments listed unless otherwise indicated. Silica gel column chro-
matography 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). 5-Iodoracil (5), 5-iodo-20-deoxyuridine
(32), and 5-iodo-20-deoxycytidine (34) were purchased from
Aldrich.
5-(2-Phenylethynyl)-uracil19 (23). This was obtained as a solid
1
in 45% yield; mp >250 °C (dec). H NMR (DMSO-d6) δ 7.
38-7.47 (m, 5H, aromatic), 7.90 (d, J = 5.8 Hz, 1H, H-6), 11.37
(d, J = 6.1 Hz, 1H, NH), 11.45 (s, 1H, NH). Anal. (C12H8N2O2)
C, H, N.
5-(4-n-Propylphenylethynyl)-uracil (24). This was obtained as
a solid in 42% yield; mp >250 °C (dec). 1H NMR (DMSO-d6) δ
0.88 (t, J = 7.33 Hz, 3H, CH3), 1.52-1.64 (m, 2H, CH2), 2.56 (t,
J = 7.33 Hz, 2H, CH2), 7.22 (d, J = 8.24 Hz, 2H, aromatic), 7.36
(d, J = 8.24 Hz, 2H, aromatic), 7.87 (s, 1H, H-6), 11.35 (s, 1H,
NH), 11.42 (s, 1H, NH). 13C NMR (DMSO-d6) δ 13.45 (CH3),
23.63 (CH2), 36.95 (CH2), 81.87 (C-β), 91.56 (C-R), 96.94 (C-5),
119.75, 128.50, 130.82, 142.58 (C-phenyl), 145.29 (C-6), 150.22
(C-2), 162.28 (C-4). Anal. (C15H14N2O2) C, H, N.
5-(4-n-Pentylphenylethynyl)-uracil (25). This was obtained as
a solid in 60% yield; mp >250 °C. 1H NMR (DMSO-d6) δ 0.85
(t, J = 7.02 Hz, 3H, CH3), 1.23-1.33 (m, 4H, 2 ꢀ CH2),
1.51-1.61 (m, 2H, CH2), 2.58 (t, J = 7.63 Hz, 2H, CH2), 7.21
(d, J = 7.94 Hz, 2H, aromatic), 7.35 (d, J = 7.94 Hz, 2H,
aromatic), 7.87 (s, 1H, H-6), 11.35 (br s, 1H, NH), 11.41 (s, 1H,
NH). 13C NMR (DMSO-d6) δ 13.77 (CH3), 21.79, 30.17, 30.72,
34.84 (4 ꢀ CH2), 81.85 (C-β), 91.56 (C-R), 96.92 (C-5), 119.71,
128.44, 130.83, 142.83 (C-phenyl), 145.29 (C-6), 150.22 (C-2),
162.27 (C-4). Anal. (C17H18N2O2) C, H, N.
5-(2-Pyridylethynyl)-uracil (26). This was obtained as a solid
in 87% yield; mp >250 °C. 1H NMR (DMSO-d6) δ 7.36 (m, 1H,
aromatic), 7.51 (d, J = 7.93 Hz, 1H, aromatic), 7.81 (m, 1H,
aromatic), 7.99 (s, 1H, H-6), 8.57 (d, J = 4.27 Hz, 1H, aromatic),
11.48 (br s, 2H, 2 ꢀ NH). Anal. (C11H7N3O2) C, H, N.
1,4-Bis-(2-pyridyl)-buta-1,3-diyne (27). This was obtained as a
solid in 30% yield; mp 118-120 °C (dec). 1H NMR (DMSO-d6)
δ 7.49-7.53 (m, 2H, aromatic), 7.75-7.79 (m, 2H, aromatic),
7.85-7.93 (m, 2H, aromatic), 8.65 (d, J = 4.88 Hz, 2H,
Preparation of 5-Alkynyl-uracils. A full procedure is provided
for 5-pentynyl-uracil (17). For other analogues, only brief
spectroscopic data are presented.
5-Pentynyl-uracil (17). To a stirred solution of 5-iodoracil (5,
300 mg, 1.26 mmol) in anhydrous dimethylformamide (20 mL)
at room temperature in a nitrogen atmosphere were added
tetrakis(triphenylphosphine)palladium (0) (146 mg, 0.13 mmol),
copper(I) iodide (48 mg, 0.25 mmol), diisopropylethylamine
(0.44 mL, 2.52 mmol), and 1-pentyne (0.37 mL, 3.78 mmol).
The reaction mixture was stirred at room temperature in a
nitrogen atmosphere; the progress of the reaction was moni-
tored by TLC in MeOH/CHCl3 (10:90, v/v). After 18 h, 15 drops
of 5% of the disodium salt of EDTA/H2O were added to the
reaction mixture, and the mixture was concentrated in vacuo.
The residue obtained was purified on a silica gel column using
MeOH/CHCl3 (6:94, v/v) as an eluent to give 17 (200 mg, 89%)
1
as a syrup. H NMR (DMSO-d6) δ 0.98 (t, J = 7.33 Hz, 3H,
CH3), 1.43-1.55 (m, 2H, β-CH2), 2.32 (t, J = 7.02 Hz, 2H,
R-CH2), 7.64 (s, 1H, H-6), 11.16 (br s, 1H, NH), 11.28 (s, 1H,
NH). 13C NMR (DMSO-d6) δ 13.31 (CH3), 20.73, 21.64 (2 ꢀ
CH2), 73.04 (C-β), 92.49 (C-R), 97.55 (C-5), 144.38 (C-6), 150.33
(C-2), 162.73 (C-4). Anal. (C9H10N2O2) C, H, N.
Compounds 18-26 were prepared using the procedure as
described for 17.
5-Heptynyl-uracil (18). This was obtained as a syrup in 86%
yield. 1H NMR (DMSO-d6) δ 0.87 (t, J = 7.02 Hz, 3H, CH3),
1.28-1.53 (m, 6H, 3 ꢀ CH2), 2.33 (t, J = 7.02 Hz, 2H, R-CH2),

6186 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16
Srivastav et al.
aromatic). 13C NMR (CD3OD) δ 73.61 (C-β), 81.57 (C-R),
125.83, 129.94, 138.53, 142.11, 151.13 (C-phenyl). Anal.
(C14H8N2) C, H, N.
inocula were diluted in medium 7H9GC and added to each well
at 2.5 ꢀ 105 CFU/mL final concentration. Sixteen control wells
consisted of eight with bacteria alone (B) and eight with media
alone (M). Plates were incubated for an initial 6 days, and starting
from 6 days of incubation, 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 24-48 h for visual color change from blue to pink
and read by spectrophotometer (at excitation 530/525 and emis-
sion 590/535) 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
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 prevented
a color change from blue to pink. Percent inhibition was calcu-
lated as (test well-M bkg/B well-M bkg) ꢀ 100. Similar methodo-
logy was used for M. bovis BCG and M. tuberculosis. Rifampicin
was used as a positive control. As negative controls, DMSO was
added to the B well at a concentration similar to that in test
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 of 60000-100000.
In Vitro Antibacterial Activity Assay. Different bacterial
strains were used for the determination of the in vitro antibac-
terial activity of the studied compounds. The in vitro antibac-
terial activity was studied by determining their minimum
inhibitory concentrations (MICs) by means of the broth micro-
dilution method. Briefly, exponentially growing bacteria were
diluted in a liquid sterile medium to obtain a final inoculum of
1 ꢀ 104 CFU/mL and subsequently cultured with varying
dilutions of compounds for 16-20 h. The MICs were defined
as the lowest concentration at which bacterial growth was no
longer evident.
6-Propyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (28). To a
stirred solution of 17 (160 mg, 0.89 mmol) in methanol/triethyl-
amine (7:3) (30 mL), was added copper(I) iodide (35 mg, 0.18
mmol) at room temperature in a nitrogen atmosphere. The
reaction mixture was then heated to reflux and stirred for 3 h.
The solvent was removed in vacuo. The solid thus obtained was
redissolved in MeOH and filtered to remove the inorganic
impurities. The filtrate was concentrated and purified on a silica
gel column using MeOH/CHCl3 (10:90, v/v) as an eluent to give
28 (80 mg, 50%) as a solid; mp >250 °C (dec). 1H NMR
(DMSO-d6) δ 0.93 (t, J = 7.32 Hz, 3H, CH3), 1.59-1.70 (m,
2H, β-CH2), 2.60 (t, J = 7.32 Hz, 2H, R-CH2), 6.37 (s, 1H, H-5),
8.14 (s, 1H, H-4), 11.98 (br s, 1H, NH). 13C NMR (DMSO-d6) δ
13.32 (CH3), 19.77, 29.25 (2 ꢀ CH2), 99.57 (C-5), 105.87 (C-4a),
138.74 (C-4), 155.68 (C-2), 157.42 (C-6), 171.93 (C-7a). Anal.
(C9H10N2O2) C, H, N.
Compounds 29-31 were prepared using the procedure as
described for 28.
6-Pentyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (29). This
1
was obtained as a solid in 64% yield; mp >250 °C (dec). H
NMR (DMSO-d6) δ 0.87 (s, 3H, CH3), 1.30 (br s, 4H, 2 ꢀ CH2),
1.56-1.60 (m, 2H, β-CH2), 2.59-2.62 (m, 2H, R-CH2), 6.36 (s,
1H, H-5), 8.14 (s, 1H, H-4), 11.97 (br s, 1H, NH). Anal.
(C11H14N2O2) C, H, N.
6-Octyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (30). This was
obtained as a solid in 44% yield; mp 248-250 °C (dec). 1H NMR
(DMSO-d6) δ 0.83-0.85 (m, 3H, CH3), 1.24-1.35 (m, 10H,
5 ꢀ CH2), 1.58-1.65 (m, 2H, β-CH2), 2.60-2.64 (m, 2H,
R-CH2), 6.36 (s, 1H, H-5), 8.14 (s, 1H, H-4). Anal. (C14H20-
N2O2) C, H, N.
6-Decyl-2,3-dihydrofuro-[2,3-d]pyrimidin-2-one (31). This was
obtained as a solid in 38% yield; mp >250 °C (dec). 1H NMR
(CDCl3 þ CD3OD) δ: 0.83 (t, J = 7.02 Hz, 3H, CH3), 1.18-1.36
(m, 14H, 7xCH2), 1.62-1.67 (m, 2H, β-CH2), 2.62 (t, J = 7.33
Hz, 2H, R-CH2), 6.18 (s, 1H, H-5), 7.84 (s, 1H, H-4). Anal.
(C16H24N2O2) C, H, N.
In Vitro Cytotoxicity Assay. Human hepatoma cell line (Huh-7)
was used to determine the effect of compounds 23-27, 33, and
35 on human cell cytotoxicity using XTT and 3H-thymidine
assays. Cell viability was measured using the cell proliferation
kit II (XTT; Roche), as per manufacturer’s instructions. Briefly, a
96 well plate was seeded with Huh-7 cells at a density of 1 ꢀ 105
cells per well. Cells were allowed to attach for 6-8 h when the
medium was replaced with medium containing compounds at
concentrations of 200, 100, 50, 10, and 1 μg/mL. DMSO was also
included as control. Plates were incubated for 2 days at
37 °C. The color reaction involved adding 50 μL XTT reagents
per well and incubating for 4 h at 37 °C. Plates were read on an
Compounds 33, 35 were prepared using the procedure as
described for 17.
5-(2-Pyridylethynyl)-20-deoxyuridine (33). This was obtained
as a solid in 97% yield; mp 193-195 °C (dec). 1H NMR
(DMSO-d6) δ 2.09-2.22 (m, 2H, H-20), 3.62 (m, 2H, H-50),
3.81 (m, 1H, H-40), 4.25 (m, 1H, H-30), 5.18 (t, J = 4.88 Hz, 1H,
50-OH), 5.27 (d, J = 4.27 Hz, 1H, 30-OH), 6.12 (t, J = 6.71 Hz,
1H, H-10), 7.36 - 7.42 (m, 1H, aromatic), 7.53 (d, J = 7.93 Hz,
1H, aromatic), 7.83 (m, 1H, aromatic), 8.44 (s, 1H, H-6), 8.57 (d,
J = 4.88 Hz, 1H, aromatic), 11.75 (s, 1H, NH). Anal.
(C16H15N3O5) C, H, N.
3
ELISA plate reader (Abs 450-500 nm). For the H-Tdr incor-
poration assay, Huh-7 cells were plated at 1 ꢀ 104 cells/well in 96-
well flat bottom plates. Medium containing compounds at con-
centrations of 200, 100, 50, 10, and 1 μg/mL was added to the plate
in triplicates. DMSO was also included as 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 incorporated into the DNA of proliferating cells were
counted in a Microbeta Trilux liquid scintillation counter (Perkin-
Elmer).
5-(2-Pyridylethynyl)-20-deoxycytidine (35). This was obtained
as a solid in 81% yield; mp 224-226 °C (dec). 1H NMR
(DMSO-d6) δ 1.98-2.22 (m, 2H, H-20), 3.61 (m, 2H, H-50),
3.82 (m, 1H, H-40), 4.22 (m, 1H, H-30), 5.13 (t, J = 4.88 Hz, 1H,
50-OH), 5.22 (d, J = 4.27 Hz, 1H, 30-OH), 6.13 (t, J = 6.71 Hz,
1H, H-10), 7.08 (br s, 1H, NH2), 7.37 (m, 1H, aromatic), 7.77-
7.87 (m, 3H, aromatic and NH2), 8.38 (s, 1H, H-6), 8.56 (d, J =
4.88 Hz, 1H, aromatic). Anal. (C16H16N4O4) C, H, N.
In Vitro Antimycobacterial Activity Assay (M. bovis, M.
tuberculosis). M. bovis (BCG) and M. tuberculosis (H37Ra) were
obtained from the American Type Culture Collection, Rockville,
MD. The antimycobacterial activity was determined using the
microplate Alamar Blue assay (MABA).25 Test compounds were
dissolved in DMSO at 100ꢀ the highest final concentration used,
and subsequent dilutions were performed in 7H9GC (Difco
Laboratories, Detroit, Michigan) media in 96-well plates. For
these experiments, each compound was tested at 100, 50, 10, 5, 2.5,
and 1 μg/mL in triplicate. The experiments were repeated three
times, and the mean percent inhibition is reported in the table. The
standard deviations were within 10%. Frozen mycobacterial
Animals and Infection. All animal experimental protocols
used in this study were approved by the University of Alberta
Animal Care and Use Committee for Health Sciences, and
conducted in accordance with the guidelines of the University
of Alberta, Edmonton, Canada, and the Canadian Council on
Animal Care. Five-six week old female BALB/c mice were
purchased from Charles River Laboratories and were allowed
to acclimate for 1 week. Mice were inoculated iv in tail vein
with 0.1 mL of bacterial suspension containing approximately
2.5 ꢀ 105 CFU of M. tuberculosis H37Ra in PBS. The infected
mice were divided into groups (5 animals per group).

Article
Administration of Drugs. The 5-(2-pyridylethynyl)-uracil (26)
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 16 6187
(10) Dye, C.; Espinal, M. A.; Watt, C. J.; Mbiaga, C.; Williams, B. G.
Worldwide Incidence of Multidrug-Resistant Tuberculosis. J. In-
fect. Dis. 2002, 185, 1197–1202.
(11) Johnson, R.; Streicher, E. M.; Louw, G. E.; Warren, R. M.;
Helden, P. D. V.; Victor, T. C. Drug Resistance in Mycobacterium
tuberculosis. Curr. Issues Mol. Biol. 2006, 8, 97–112.
(12) Rai, D.; Johar, M.; Manning, T.; Agrawal, B.; Kunimoto, D. Y.;
Kumar, R. Design and Studies of Novel 5-Substituted Alkynylpyr-
imidine Nucleosides as Potent Inhibitors of Mycobacteria. J. Med.
Chem. 2005, 48, 7012–7017.
(13) Johar, M.; Manning, T.; Kunimoto, D. Y.; Kumar, R. Synthesis
and in Vitro Anti-Mycobacterial Activity of 5-Substituted Pyrimi-
dine Nucleosides. Bioorg. Med. Chem. 2005, 13, 6663–6671.
(14) Johar, M.; Manning, T.; Tse, C.; Desroches, N.; Agrawal, B.;
Kunimoto, D. Y.; Kumar, R. Growth Inhibition of Mycobacterium
bovis, Mycobacterium tuberculosis and Mycobacterium avium In
Vitro: Effect of 1-β-D-20-Arabinofuranosyl and 1-(20-Deoxy-20-
fluoro-β-D-20-ribofuranosyl) Pyrimidine Nucleoside Analogs.
J. Med. Chem. 2007, 50, 3696–3705.
(15) Rai, D.; Johar, M.; Srivastav, N. C.; Manning, T.; Agrawal,
B.; Kunimoto, D. Y.; Kumar, R. Inhibition of Mycobacterium
tuberculosis, Mycobacterium bovis, and Mycobacterium avium by
Novel Dideoxy Nucleosides. J. Med. Chem. 2007, 50, 4766–4774.
(16) Srivastav, N. C.; Manning, T.; Kunimoto, D. Y.; Kumar, R.
Studies on Acyclic Pyrimidines as Inhibitors of Mycobacteria.
Bioorg. Med. Chem. 2007, 15, 2045–2053.
(17) Srivastav, N. C.; Manning, T.; Kunimoto, D. Y.; Kumar, R. In
Vitro Anti-Mycobacterial Activities of Various 20-Deoxyuridine,
20- Arabinouridine and 20-Arabinofluoro-20-deoxyuridine Analo-
gues: Synthesis and Biological Studies. Med. Chem. 2006, 2, 287–
293.
(18) Kumar, R.; Knaus, E. E.; Wiebe, L. I.; Allen, T. M.; Tempest,
M. L. Synthesis and Cytotoxic Activity of 5-(1-hydroxy-2-
haloethyl)-, 5-Oxiranyl- and (E)-5-(2-Iodovinyl)-2,4-dichloro (or
Dimethoxy) Pyrimidines. Eur. J. Med. Chem. 1991, 26, 557–562.
(19) Hudson, R. H. E.; Moszynski, J. M. A Facile Synthesis of
Fluorophores Based on 5-Phenyl-Ethynyluracils. Synlett 2006,
18, 2997–3000.
was dissolved in poly(ethylene glycol) methyl ether. The 5-(2-
pyridylethynyl)-20-deoxycytidine (35) and control drugs cyclo-
serine and isoniazid were dissolved in 10% dimethyl sulfoxide
and saline. The drugs were frozen at -20 °C and thawed each
morning prior to administration. All of the compounds studied
were administered intraperitonially. The compounds and do-
sages were: 5-(2-pyridylethynyl)-uracil (26), 50 mg/kg; 5-(2-
pyridylethynyl)-20-deoxycytidine (35), 50 mg/kg; cycloserine,
50 mg/kg; isoniazid, 25 mg/kg. Control animals received equiva-
lent volumes of diluents only. Drug treatment was initiated
5 days after bacterial challenge and continued until week 6
(total treatment days = 25). Three days after the last treatment,
mice were euthanized and lung, liver, and spleen were removed
aseptically and individually homogenized. CFU counts per
organ were determined on 7H11 selective agar plates pur-
chased from BD Biosciences. The plates were incubated at
37 °C in ambient air for up to 4 weeks prior to counting the
colonies.
Statistical Analyses. Standard randomization procedure was
used in assigning the groups to mice after infection. To avoid
bias, the identity of groups of animals was blinded from the
person performing euthanization of mice and colony counts.
Statistical analyses were done by independent sample t test using
SPSS 16.0 software.
Acknowledgment. This work was supported by the Canadian
Institutes of Health Research (CIHR) operating grant (MOP-
49415). B.A. is a recipient of a Senior Scholar Award from the
Alberta Innovates-Health Solutions (Formerly Alberta Heritage
Foundation for Medical Research, AHFMR).
(20) Siemsen, P.; Livingston, R. C.; Diederich, F. Acetylenic Coupling:
A Powerful Tool in Molecular Construction. Angew. Chem., Int.
Ed. 2000, 39, 2632–2657.
References
(1) World Health Organization Tuberculosis Programme; http://www.
who.int/tb/en/, 2009.
(2) Willcox, P. A. Drug-Resistant Tuberculosis. Curr. Opin. Pulm.
(21) Robins, M. J.; Barr, P. J. Nucleic Acid Related Compounds. 31.
Smooth and Efficient Palladium-Copper Catalyzed Coupling of
Terminal Alkynes with 5-Iodo uracil Nucleosides. Tetrahedron
Lett. 1981, 22, 421–424.
Med. 2000, 6, 198–202.
ꢀ
(3) Mendez, A. P.; Raviglione, M. C.; Laszlo, A.; Binkin, N.; Rieder,
H. L.; Bustreo, F.; Cohn, D. L.; van Weezenbeek, C. S. B. L.; Kim,
S. J.; Chaulet, P.; Nunn, P. Global Surveillance for Antituber-
culosis-Drug Resistance, 1994-1997. N. Engl. J. Med. 1998, 338,
1641–1649.
(22) Vincent, P.; Pichat, J. B. Alcynyl-5-disoxy-20-uridines par Cou-
plages D’Organozinc Iques Acetyleniques avec L’Iodo-5O-30,50-
Bis(trimethylsilyl) Desoxyuridine, Catalyses par Des Com-
plexes Organopalladies et de Nickel. Tetrahedron Lett. 1981, 22,
945–947.
(4) Williams, B. G.; Dye, C. Antiretroviral Drugs for Tuberculosis
Control in the Era of HIV/AIDS. Science 2003, 301, 1535–1537.
(5) Corbett, E. L.; Watt, C. J.; Walker, N.; Maher, D.; Williams, B. G.;
Raviglione, M. C.; Dye, C. The Growing Burden of Tuberculosis:
Global Trends and Interactions With the HIV Epidemic. Arch.
Intern. Med. 2003, 163, 1009–1021.
(6) (a) Pozniak, A. Mycobacterial Diseases and HIV. J. HIV Ther.
2002, 7, 13–16. (b) Colditz, G. A.; Brewer, T. F.; Berkey, C. S.; Wilson,
M. E.; Burdick, E.; Fineberg, H. V.; Mosteller, F. Efficacy of BCG
Vaccine in the Prevention of Tuberculosis. Meta-Analysis of the
Published Literature. JAMA, J. Am. Med. Assoc. 1994, 271, 698–702.
(7) Wang, J. Y.; Lee, L.-N.; Lai, H. C.; Wang, S. K.; Jan, I. S.; Yu,
C. J.; Hsueh, P. R.; Yang, P. C. Fluoroquinolone Resistance in
Mycobacterium tuberculosis Isolates: Associated Genetic Muta-
tions and Relationship to Antimicrobial Exposure. J. Antimicrob.
Chemother. 2007, 59, 860–865.
(8) Lounis, N.; Roscigno, G. In Vitro and In Vivo Activities of New
Rifamycin Derivatives Against Mycobacterial Infections. Curr.
Pharm. Des. 2004, 10, 3229–3238.
(9) World Health Organization. Antituberculosis Drug Resistance in
the World. Report No. 4. The WHO/IUATLD Global Project on
Antituberculosis Drug Resistance Surveillance 2000-2007; World
Health Organization: Geneva, Switzerland, 2008.
(23) Cycloserine. Tuberculosis 2008, 88, 100-101.
(24) Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.;
Harris, D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.; Barry, C. E., III;
Tekala, F.; Badcock, K.; Bashman, D.; Brown, D.; Chillingworth,
T. R.; Connor, R.; Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.;
Hamlin, N.; Holroyd, S.; Hornsby, T.; Jagels, K.; Krogh, A.;
Mclean, J.; Moule, S.; Murphy, L.; Oliver, K.; Osborne, J.; Quail,
M. A.; Rajandream, M.-A.; Rogers, J.; Rutter, S.; Seeger
K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.; Taylor,
K.; Whitehead, S.; Barrell, B. G. Deciphering the Biology of
Mycobacterium tuberculosis from the Complete Genome Sequence.
Nature 1998, 393, 537–544.
(25) (a) Franzblau, S. G.; Witzig, R. S.; McLaughlin, J. C.; Torres, P.;
Madico, G.; Hernandez, A.; Degnan, M. T.; Cook, M. B.; Quenzer,
V. K.; Ferguson, R. M.; Gilman, R. H. Rapid, Low-Technology
MIC Determination with Clinical Mycobacterium tuberculosis
Isolates by Using the Microplate Alamar Blue Assay. J. Clin.
Microbiol. 1998, 36, 362–366. (b) Collins, L.; Franzblau, S. G.
Microplate Alamar Blue Assay versus BACTEC 460 System for
High-Throughput Screening of Compounds against Mycobacterium
tuberculosis and Mycobacterium avium. Antimicrob. Agents Chemo-
ther. 1997, 41, 1004–1009.