Growth inhibition of Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium in vitro: Effect of 1-β-D-2′-arabinofuranosyl and 1-(2′-deoxy-2′-fluoro-β-D-2′-ribofuranosyl) pyrimidine nucleoside analogs

Article abstract of DOI:10.1021/jm0703901

The resurgence of tuberculosis and the emergence of multiple-drug-resistant strains of Mycobacteria necessitate the search for new classes of antimycobacterial agents. We synthesized a series of 1-β-D-2′- arabinofuranosyl and 1-(2′-deoxy-2′-fluoro-β-D-rib

Full text of DOI:10.1021/jm0703901

3696
J. Med. Chem. 2007, 50, 3696-3705
Growth Inhibition of Mycobacterium boWis, Mycobacterium tuberculosis and Mycobacterium
aWium In Vitro: Effect of 1-â-D-2′-Arabinofuranosyl and
1-(2′-Deoxy-2′-fluoro-â-D-2′-ribofuranosyl) Pyrimidine Nucleoside Analogs
Monika Johar,† Tracey Manning,† Christopher Tse,† Nancy Desroches,† B. Agrawal,‡ Dennis Y. Kunimoto,§ and
Rakesh Kumar*,†
Department of Laboratory Medicine and Pathology, 1-71 Medical Sciences Building, Department of Medicine, Department of Surgery,
Faculty of Medicine and Dentistry, UniVersity of Alberta, Edmonton, AB, Canada T6G 2H7
ReceiVed April 3, 2007
The resurgence of tuberculosis and the emergence of multiple-drug-resistant strains of Mycobacteria
necessitate the search for new classes of antimycobacterial agents. We synthesized a series of 1-â-D-2′-
arabinofuranosyl and 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl) pyrimidine nucleosides possessing diverse
sets of alkynyl, alkenyl, alkyl, and halo substituents at the C-5 position of the uracil and investigated their
effect on activity against M. tuberculosis, M. boVis, and M. aVium. Among these molecules, 5-alkynyl-
substituted derivatives emerged as potent inhibitors of M. boVis, M. tuberculosis, and M. aVium. Nucleosides
1-â-D-2′-arabinofuranosyl-5-dodecynyluracil (5), 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-dodecynyluracil
(24), and 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tetradecynyluracil (25) showed the highest antimyco-
bacterial potency against M. boVis and M. tuberculosis. The MIC90 exhibited by compounds 5, 24, and 25
was similar or close to that of the reference drug rifampicin. The most active compounds 5, 24, and 25 were
also found to retain sensitivity against a rifampicin-resistant strain of M. tuberculosis H37Rv at similar
concentrations. Some of these analogs also revealed in vitro antimicrobial effect against several other gram-
positive pathogens.
Introduction
the clinical management of tuberculosis in HIV-infected patients
and are often responsible for their death.8 For both TB and HIV,
misdiagnosis and noncompliance with treatment regimens
further compound the problem and facilitate the development
of drug resistance.7,9
Mycobacterium tuberculosis (M. tuberculosisa), Mycobacte-
rium aVium (M. aVium), and Mycobacterium boVis (M. boVis)
are clinically significant species of the genus Mycobacterium
that are causative agents of tuberculosis (TB), killing over 2-3
million people annually worldwide.1 TB bacilli are highly
contagious, airborne, slow-growing, gram-positive, aerobic, acid-
fast mycobacteria. The World Health Organization (WHO)
estimates that between 1 and 2 billion people are latently
infected with TB bacilli.2-4 Approximately 8 million people
develop active disease each year.5 TB is the world’s second
most common cause of death from infectious disease, after
acquired immuno deficiency syndrome (AIDS).6
TB and human immunodeficiency virus (HIV) have formed
a new and deadly combination. There is a resurgence in the
incidence of TB in developed and developing countries with
high rates of HIV-TB coinfection. The increase in TB incidence
is strongly associated with the prevalence of HIV infection.7
M. tuberculosis and M. aVium pose a significant challenge to
M. aVium complex (MAC) infections, in particular, M. aVium
infections, are one of the most serious complications among
patients with AIDS.10,11 MAC infections are disseminated rather
than being restricted to the lungs. Clinical management of MAC
infections is very difficult because many of the first-line anti-
TB drugs are ineffective against it.10,11 New macrolides, such
as clarithromycin and azithromycin, are used for the treatment
of MAC, however, resistance occurs at such a rate that single
drugs are inadequate for therapy.12,13
The problem of tuberculosis is further complicated by the
emergence of new TB strains, which are not susceptible to a
number of available drugs, that is, multidrug-resistant TB
(MDR-TB).2,14,15 Around 50 million people have been infected
with MDR-TB. MDR-TB is defined as resistant to at least
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.16,17 A 100%
correlation has been observed in XDR-TB with HIV coinfection
and mortality.16
Bacillus Calmette Guerin (BCG)18 is an attenuated strain of
M. boVis that is more than 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. In addition,
MDR strains of M. boVis have been isolated.19 In Europe,
primary MDR-TB caused by M. boVis has been found to be
resistant to 11 anti-TB drugs with a mean survival of 44 days
* To whom correspondence should be addressed. Tel.: (780) 492-7545.
Fax: (780) 492-7521. E-mail: rakesh.kumar@ualberta.ca.
† Department of Laboratory Medicine and Pathology.
‡ Department of Surgery.
§ Department of Medicine.
a Abbreviations: TB, tuberculosis; M. boVis, Mycobacterium boVis; M.
tuberculosis, Mycobacterium tuberculosis; M. aVium, Mycobacterium aVium;
MAC, Mycobacterium aVium complex; MDR-TB, multidrug resistant
tuberculosis; XDR-TB, extensively drug resistant tuberculosis; BCG,
Bacillus Calmette Guerin; MABA, microplate alamar blue assay; HFF,
human foreskin fibroblast; CMC, critical micellar concentrations; CFU,
colony forming unit; GI, growth index; S. aureus, Staphylococcus aureus;
S. epidermis, Staphylococcus epidermis; E. faecalis, Enterococcus faecalis;
B. subtilis, Bacillus subtilis; S. pneumoniae, Streptococcus pneumoniae; S.
pyogenes, Streptococcus pyogenes; S. typhimurium, Salmonella typhimurium;
E. coli, Escherichia coli; P. Vulgaris, Proteus Vulgaris; P. aeruginosa,
Pseudomonas aeruginosa; L. monocytogenes, Lysteria monocytogenes; E.
aerogenes, Enterobacter aerogenes.
10.1021/jm0703901 CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/29/2007

Growth Inhibition of Mycobacterium In Vitro
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3697
in 19 patients.19 There is an ever-increasing threat of drug-
resistant TB appearing as an epidemic in many developing
countries as well as developed countries, particularly because
no new drugs have been introduced to treat TB for over 40
years.4
luracil (25) display very promising activity against M. boVis
and M. tuberculosis in vitro. Encouragingly, these compounds
were also found to retain sensitivity against drug-resistant M.
tuberculosis. These studies extend our previous observations
and suggest that this class of compounds has potential to serve
as new antituberculosis agents for drug-sensitive and drug-
resistant mycobacteria.
Because of the global health problems associated with TB,
the increasing rate of multiple drug-resistant TB and the high
rate of a co-infection with HIV, the discovery and development
of potent new anti-TB agents without cross-resistance with
current antimycobacterial agents are urgently needed.
The M. tuberculosis genome has been fully sequenced, which
has greatly facilitated the identification of potential new and
unique targets for the design of new classes of selective anti-
TB drugs.20 Pyrimidine biosynthesis is an essential step in the
progression of TB. Significant differences exist in the details
and the regulatory mechanisms of the pyrimidine biosynthesis
pathway in bacteria and mammals, suggesting that novel
nucleoside derivatives could target new biochemical mecha-
nisms, potentially allowing the design and development of novel
antituberculosis drugs.20 It is postulated that unnatural pyrimi-
dine nucleosides can specifically target the mycobacterial
enzymes involved in their nucleic acid synthesis by acting as
their substrates or inhibitors and inhibit the mycobacterial DNA
and RNA synthesis.
A number of pyrimidine nucleoside derivatives in which the
2′-hydroxyl group has the opposite configuration (1-â-D-2′-
arabinofuranosyl) to that of a ribonucleoside, exhibit potent
antiviral, and anticancer properties.21 Among them, 1-â-D-2′-
arabinofuranosyl-5-(1-propynyl)uracil (1a, netivudine) is one of
the most effective and selective analogs for the treatment of
varicella zoster virus infection, possessing very good pharma-
cokinetic properties with long plasma half-life.22 In addition,
incorporation of a fluorine atom at the 2′-position of 2′-
deoxyuridine derivatives with a variety of 5-substituents has
provided compounds that in many instances are excellent
substrates for phosphorylation by kinases. In earlier studies, it
has been observed that the presence of an arabino hydroxyl
group and the ribofluoro group at the C-2′ position greatly
enhances resistance to phosphorolysis of the glycosyl bond in
the deoxyribose derivatives while retaining biological activities
of the parent nucleosides.23
In ongoing efforts to develop new and effective therapies for
mycobacterial infections, our previous investigations of 5-sub-
stituted pyrimidine nucleosides led to the identification of
5-(alkyn-1-yl)uracil 2′-deoxy nucleosides with good inhibitory
activity against M. boVis (MIC90 ) 10-50 µg/mL) and M.
aVium (MIC75 ) 100 µg/mL) in cell-based assays.24,25 In these
studies, we observed an important role of the carbohydrate
portion because the corresponding uracil derivatives were
inactive.24 We reasoned that replacement of the 2′-deoxyribose
by a sugar moiety resistant to glycosidic bond cleavage might
produce compounds with analogous structural features and
enhanced biological profile. In the present studies, we now report
the synthesis and antituberculosis activity of 1-â-D-2′-arabino-
furanosyl (3-8, 9-16, 19, 20) and 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl) (22-26, 27-33, 36, 37) pyrimidine nucleoside
analogs with diverse 5-alkynyl, 5-alkenyl, 5-alkyl, and 5-ha-
loalkyl substituents against M. boVis, M. tuberculosis, and M.
aVium in vitro. Nucleosides possessing alkenyl, alkyl, and
halogen groups at the 5-position were moderately active or
devoid of antimycobacterial activity. However, our investiga-
tions revealed that 1-â-D-2′-arabinofuranosyl-5-dodecynyluracil
(5), 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-dodecynyluracil
(24), and 1-(2′-deoxy-2′fluoro-â-D-ribofuranosyl)-5-tridecyny-
Chemistry
The target 1-â-D-arabinofuranosyl-5-alkynyluracils (3-8)
were synthesized in one step by the Pd-catalyzed coupling of a
series of terminal alkynes with 1-â-D-arabinofuranosyl-5-
iodouracil (2)26 in DMF in 40-54% yields (Scheme 1).
Structures of 3-8 were characterized by H NMR, 13C NMR,
1
and microanalysis. Reaction of 2 with ethylacrylate, bis-
(triphenyl)palladium(II) chloride, and triethylamine, using a
procedure reported by us,26 yielded the (E)-5-(2-ethoxycarbo-
nylvinyl) analog (9). Alkaline hydrolysis of 9 afforded the (E)-
5-(2-carboxyvinyl) derivative (10).27
The nucleoside 1-â-D-arabinofuranosyl-5-(1-hydroxy-2-io-
doethyl)uracil (11)28 was synthesized by the reaction of 1-â-
D-arabinofuranosyl-5-vinyluracil (1b) with iodine in the presence
of the oxidizing agent, iodic acid. Treatment of (11) with
methanolic sulfuric acid yielded 12.28 The compounds 5-(2,2-
dibromo-1-hydroxyethyl)-(13) and 5-(2,2-dichloro-1-hydroxy-
ethyl)-(14) were prepared by the reaction of (E)-5-(2-bromovinyl)-

3698 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Johar et al.
Scheme 1a
Scheme 3a
a Reagents and conditions: (i) H-C t C-R, Pd(PPh3)4, CuI, (i-Pr)2EtN,
DMF, RT; (ii) CH2dCHCOOEt, (Ph3P)2PdCl2, Et3N (9); (iii) 0.5 N KOH
(10).
Scheme 2a
a Reagents and conditions: (i) H-CtC-R, Pd(PPh3)4, CuI, (i-Pr)2EtN,
DMF, RT; (ii) CH2dCHCOOEt, (Ph3P)2PdCl2, Et3N (27); (iii) 0.5 N KOH
(28); (iv) N-chlorosuccinimide, H2O, 25 °C (29); N-bromosuccinimide, H2O,
25 °C, (30); I2, KIO3, H2O, H2SO4 (31).
synthesized by the electrophilic addition reactions of 1-(2′-
deoxy-2′-fluoro-â-D-ribofuranosyl)uracil (34) with dilute solu-
tions of the appropriate halogen in acetic acid followed by
treatment with aqueous ammonia.23 The preparation of hitherto
unknown compounds 36 and 37 proceeded from 1-(2′-deoxy-
2′-fluoro-â-D-ribofuranosyl)uracil (34) and 1-(2′-deoxy-2′-fluoro-
â-D-ribofuranosyl)-5-methyluracil (35) and utilized essentially
the same methodology developed for the synthesis of corre-
sponding arabinosyl analogs (19, 20; Scheme 4).
a Reagents and conditions: (i) acetic anhydride, dimethylaminopyridine,
25 °C; Lawesson’s reagent, 1,4-dioxane, reflux; NH3, MeOH, 0-25 °C.
(1c) and (E)-5-(2-chlorovinyl)-(1d) 1-â-D-arabinofuranosyluracils,
with N-bromosuccinimide or N-chloro succinimide in 1,4-
dioxane, respectively.26 A mild and efficient reaction of 1-â-
D-arabinofuranosyluracil (17) with N-bromosuccinimide and
sodium azide in 1,2-dimethoxyethane (DME) at 25 °C yielded
the 5-bromo compound (15) in quantitative yield.29 A similar
chlorination reaction of 17 using N-chlorosuccinimide and
sodium azide in DME at 45 °C gave the 5-chloro product (16).29
The 4-thio arabinosyl nucleosides (19, 20) were prepared starting
from commercially available compounds 17 and 18 in a single
step without isolating protected nucleoside intermediates. Thus,
acetylation of 17 or 18 using acetic anhydride and dimethy-
laminopyridine at 25 °C, followed by thiolation with Lawesson’s
reagent in 1,4-dioxane and subsequent deacetylation of the crude
products, gave 4-thione products (19, 20) in 38 and 34% yields,
respectively (Scheme 2).
For the synthesis of 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
5-alkynyluracils (22-26), similar methodologies were used that
we had adopted for the preparation of compounds 3-8,
involving the Pd-catalyzed coupling reaction of terminal
acetylenes with 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-io-
douracil (21; Scheme 3). A coupling reaction of 21 with
ethylacrylate followed by alkaline hydrolysis of the obtained
ester (27)30 afforded compound 28,30 as described above for
arabino analogs (9, 10). The halohydrin derivatives, 29-31,
were readily prepared by the regiospecific addition of HO-X
(X ) Cl, Br, I) to the vinyl substituent of (E)-5-(2-ethoxycar-
bonylvinyl)-27 and (E)-5-(2-carboxyvinyl)-28, 1-(2′-deoxy-2′-
fluoro-â-D-ribofuranosyl)uracils according to the procedure
reported earlier30 (Scheme 3). Compounds 32 and 33 were
Results and Discussion
All of the test compounds were evaluated in vitro against M.
boVis, M. tuberculosis, and M. aVium by the microplate Alamar
blue assay (MABA)31 at 1-100 µg/mL concentrations. Rifampi-
cin and clarithromycin were used as reference standards.
Antimycobacterial activity for the compounds 3-16, 19, 20,
22-33, 36, and 37 is summarized in Table 1. The 5-substituted
pyrimidine nucleoside derivatives, modified in the carbohydrate
and base moiety, investigated here for their antimycobacterial
effect, can mainly be divided into four different structural
classes: (i) 5-alkynyl analogs (3-8, 22-26), (ii) 5-alkenyl
analogs (9, 10, 27, 28), (iii) 5-alkyl analogs (11-14, 20, 29-
31, 37), and (iv) 5-halo analogs (15, 16, 32, 33). Among the
nucleosides possessing 5-alkynyl substituents (3-8, 22-26), a
clear structure-activity relationship (SAR) can be delineated.
The compounds containing a longer carbon chain at the C-2
carbon of the 5-substituent, namely, 1-â-D-arabinofuranosyl-5-
decynyluracil (4), 1-â-D-arabinofuranosyl-5-dodecynyluracil (5),
1-â-D-arabinofuranosyl-5-tridecynyluracil (6), 1-â-D-arabino-
furanosyl-5-tetradecynyluracil (7), 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-5-decynyluracil (23), 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-5-dodecynyluracil (24), 1-(2′-deoxy-2′-fluoro-â-
D-ribofuranosyl)-5-tridecynyluracil (25), and 1-(2′-deoxy-2′-
fluoro-â-D-ribofuranosyl)-5-tetradecynyluracil (26), are potent
inhibitors of M. boVis and M. tuberculosis (MIC90 )1-100 µg/
mL range), with the activity depending on the length of the

Growth Inhibition of Mycobacterium In Vitro
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3699
Scheme 4a
a Reagents and conditions: (i) bromine (32) or chlorine (33), CH3COOH, 25 °C, NH4OH; (ii) acetic anhydride, dry pyridine, 0-25 °C; Lawesson’s
reagent, 1,4-dioxane, reflux; NH3, MeOH, 0-25 °C.
Table 1. In Vitro Antimycobacterial Activity of 1-â-D-2′-Arabinofuranosyl and 1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl) Pyrimidine Nucleoside Analogs
antimycobacterial activity
M. boWis (BCG)
% inhibitiona
(concn µg/mL)
25 (100)
M. tuberculosis (H37Ra)
M. aWium (ATCC 25291)
d
d
d
MIC90
% inhibitiona
MIC90
% inhibitiona
MIC90
cmpd
R
(µg/mL)
(concn µg/mL)
(µg/mL)
(concn µg/mL)
(µg/mL)
3
4
C5H11
C8H17
25 (100)
0 (100)
50 (100)
100 (100), 50 (50),
30 (10)
100
100 (100), 50 (50),
10 (10)
100
5
C10H21
100 (100, 50, 10),
75 (1)
1-5
100 (100, 50, 10),
75 (1)
1-5
100 (100), 55 (50)
50-100
6
7
8
C11H23
C12H25
100 (100, 50), 50 (10)
100 (100, 50), 50 (10)
100 (100), 50 (50)
10-50
10-50
100
100 (100, 50), 50 (10)
100 (100, 50), 50 (10)
100 (100), 50 (50)
10-50
10-50
100
25 (100)
0 (100)
25 (100)
9
CHdCHCOOEt
CHdCHCOOH
CH(OH)CH2I
CH(OMe)CH2I
CH(OH)CHBr2
CH(OH)CHCl2
Br
Cl
H
CH3
C5H11
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
50 (100)
100 (100), 75 (50),
50 (10), 30 (1)
100 (100, 50, 10),
70 (1)
100 (100, 50, 10),
90 (1)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
50 (100)
10
11
12
13
14
15
16
19
20
22
23
0 (100)
0 (100)
50 (100)
100 (100), 100 (50),
50 (10), 25 (1)
100 (100, 50, 10),
70 (1)
C8H17
50-100
1-5
50-100
1-5
24
25
C10H21
C11H23
100 (100), 50 (50)
100 (100), 50 (50)
50-100
50-100
1
100 (100, 50, 10),
90 (1)
1
26
27
28
29
30
31
32
33
C12H25
CHdCHCOOEt
CHdCHCOOH
CH(OH)CH(Cl)COOEt
CH(OH)CH(Br)COOEt
CH(OH)CH(I)COOH
Br
Cl
H
100 (100, 50), 50 (10)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
50 (100, 50)
25 (100)
25 (100)
10-50
100 (100, 50), 50 (10)
0 (100)
0 (100)
0 (100)
0 (100)
10-50
20 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
0 (100)
90 (2)
0 (100)
0 (100)
20 (100)
25 (100)
25 (100)
100 (0.5-1)
ND
36
37
CH3
std1b
std2b
100 (0.5-1)
0.5-1
ND
0.5-1
ND
2
2
NDc
95 (2)
a Antimycobacterial activity was determined at concentrations 100, 50, 10, and 1 µg/mL. b Positive control drugs; std 1 ) rifampicin, std2 ) clarithromycin.
c ND ) not determined. d Concentration of compounds exhibiting 90% inhibition in mycobacterial growth.
alkynyl side chain. The short chain containing 1-â-D-arabino-
furanosyl-5-heptynyluracil (3) and 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-5-heptynyluracil (22) derivatives were moderately
active for M. boVis and M. tuberculosis (25-50% inhibition at
100 µg/mL; Table 1). 5-Dodecynyl, 5-tridecynyl, and 5-tet-
radecynyl side chains appear to provide optimal activity against

3700 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Johar et al.
both mycobacterial species. The maximum inhibition of bacterial
growth in the case of 2′-arabinofuranosyl analogs (3-7) was
obtained by the dodecynyl moiety, whereas in 2′-fluororibo-
furanosyl derivatives (22-26), maximum activity was provided
by dodecynyl and tetradecynyl chain-bearing derivatives. To
delineate the structural requirements for optimal potency, beyond
the necessity of a longer alkyl chain at the C-2 of the 5-position,
we sought to replace the flexible alkyl chain by a rigid ring
system in compounds 3-7, allowing us to examine the effect
of a para-alkyl-substituted phenyl group at the terminal carbon
of the alkynyl chain. We note that inclusion of the N-
propylphenyl ring did not improve antimycobacterial activity,
as 1-â-D-arabinofuranosyl-5-(4-n-propylphenylethynyl)uracil (8)
was only moderately active (MIC90 )100 µg/mL). The most
potent compounds of the 5-alkynyl series, 5, 24 (MIC90 )1-5
µg/mL), and 25 (MIC90 )1.0 µg/mL), inhibited the growth of
M. boVis and M. tuberculosis at concentrations similar or
approaching that of the reference drug, rifampicin (MIC90
)0.5-1.0 µg/mL). The 5-tridecynyl- (25) analog of 1-(2′-deoxy-
2′-fluoro-â-D-ribofuranosyl)uracil displayed superior activity
compared to that of the corresponding 1-â-D-arabinofuranosy-
luracil derivative (6) against both mycobacteria. Further, inhibi-
tion of M. boVis and M. tuberculosis by 5-heptynyl- (22, 50%
inhibition at 100 µg/mL) and 5-decynyl- (23, 50% inhibition at
10 µg/mL) analogs of 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
uracil was improved over 1-â-D-arabinofuranosyl-5-heptynylu-
racil (3, 25% inhibition at 100 µg/mL) and 1-â-D-arabinofura-
nosyl-5-decynyluracil (4, 50% inhibition at 100 µg/mL). These
studies suggest that a fluoro substituent at the 2′-position of
the sugar moiety is preferred for improved activity.
deoxyribose analogs (75% inhibition at 100 µg/mL), suggesting
that variations in the glycosyl part of the pyrimidine nucleosides
investigated not only modulate the antimycobacterial activity,
but also influence the activity spectrum. Comparison of the
activity against M. aVium with M. boVis or M. tuberculosis
revealed that 5-heptynyl (3, 22), 5-decynyl (4, 23), 5-dodecynyl
(5, 24), 5-tridecynyl (6, 25), or 5-tetradecynyl (7, 26) nucleosides
possessed much higher inhibitory activity against M. boVis and
M. tuberculosis than M. aVium.
To extend our understanding of the structural requirements
for potent activity, we also examined the effect of alkenyl, alkyl
and haloalkyl substituents at the C-5 position of the pyrimidine
ring in the 1-â-D-arabinofuranosyl and 1-(2′-deoxy-2′-fluoro-
â-D-ribofuranosyl) nucleosides. Introduction of alkenyl groups
led to a dramatic reduction in antimycobacterial efficacy in both
1-â-D-arabinofuranosyluracil (9, 10) as well as 1-(2′-deoxy-2′-
fluoro-â-D-ribofuranosyl)uracil (27, 28) derivatives. Similarly,
nucleosides with shorter chain alkyl groups at the 5-position of
the uracil ring, namely, 1-â-D-arabinofuranosyl-5-(1-hydroxy-
(or methoxy)-2-iodoethyl)uracil (11, 12), 1-â-D-arabinofurano-
syl-5-(1-hydroxy-2,2-dihaloethyl)uracil (13, 14), 1-â-D-arabino-
furanosyl-4-thiothymine (20), and 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-4-thiothymine (37) or longer chain alkyl groups,
namely, 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-[1-hydroxy-
2-chloro-2-(ethoxy carbonyl)ethyl]uracil (29), 1-(2′-deoxy-2′-
fluoro-â-D-ribofuranosyl)-5-[1-hydroxy-2-bromo-2-(ethoxycar-
bonyl)ethyl]uracil(30),and1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
5-[1-hydroxy-2-iodo-carboxyethyl]uracil (31) were inactive up
to a concentration of 100 µg/mL against M. boVis, M. tuber-
culosis, and M. aVium. These results suggest that alkynyl
functionalities contribute to antituberculosis activity in com-
pounds 3-8 and 22-26.
To further establish the structural requirements at the 5-posi-
tion for antimycobacterial activity, we sought to determine the
potential of 5-halo analogs of 1-â-D-arabinofuranosyluracil (15,
16) and 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)uracil (32, 33).
Within these, none of the compounds 15, 16, or 32 showed
inhibitory effects against M. boVis, M. tuberculosis, and M.
aVium, except moderate activity of 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-5-chlorouracil (33) against M. boVis (50% inhibi-
tion at 50 µg/mL). The thio-compounds, 1-â-D-arabinofuranosyl-
4-thiouracil (19) and 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
4-thiouracil (36) did not exert any antimycobacterial activity
similar to other thio derivatives 20 and 37.
Interestingly, the activity of 5-dodecynyl analogs of 1-â-D-
arabinofuranosyluracil (5) and 2′-deoxy-2′-fluoro-â-D-ribofura-
nosyluracil (24) was significantly increased, but it was reduced
for the 5-tetradecynyl derivatives (7, 26), as compared to their
corresponding 2′-deoxyuridines [90% inhibition at 50-100 µg/
mL (5-dodecynyl) and 10 µg/mL (5-tetradecynyl), respectively]
against M. boVis.24 The activity of compounds 5 and 24 against
M. tuberculosis (H37Ra) was also significantly improved as
compared to their corresponding 2′-deoxyuridine congeners re-
ported previously24 (MIC90 ) 50-100 µg/mL, unpublished
data). These studies suggest that potent antimycobacterial activ-
ities are not solely dependent on the 5-alkynyl side chain, but
are also modulated by the carbohydrate portion of the nucleo-
sides. In our studies, we also synthesized 5-alkynyl (decynyl,
dodecynyl, and tetradecynyl) uracils that were found to be
inactive against M. boVis and M. tuberculosis (0% inhibition at
100 µg/mL, unpublished results). These results clearly demon-
strate that the glycosyl part is crucial and plays an important
role in the antimycobacterial activities of these molecules.
To examine whether the most promising compounds are
acting as promiscuous inhibitors whose activity is dependent
upon their aggregation in biological medium or specific inhibi-
tors of mycobacteria, we determined the activity of selected
nucleosides 1-â-D-arabinofuranosyl-5-dodecynyluracil (5) and
1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tridecynyluracil (25)
in the presence of detergents (Figure 1). Initially, the effect of
two detergents (Tween-20 and Triton X-100) on M. boVis
viability and growth was determined (data not shown) in the
presence of concentrations of detergents up to their critical
micellar concentrations (CMC).32,33 The concentrations of
detergents that had no effect on M. boVis viability and growth
were then used in the assay to determine the activity of 5 and
25 (Figure 1). It was interesting to note that the inhibition
profiles of the two selected compounds did not change in the
presence of both detergents, suggesting that they are not acting
as promiscuous inhibitors, but rather have specific antimyco-
bacterial activity.
With regard to antimycobacterial activity against M. aVium,
observations similar to those described for activities against M.
boVis and M. tuberculosis were made where 1-â-D-arabinofura-
nosyl-5-dodecynyluracil (5), 1-(2′-deoxy-2′-fluoro-â-D-ribofura-
nosyl)-5-dodecynyl uracil (24), and 1-(2′-deoxy-2′-fluoro-â-D-
ribofuranosyl)-5-tridecynyluracil (25) exhibited the most potent
activity (MIC90 ) 50-100 µg/mL; Table 1). The influence of
the 2′-ribofluoro substituent was again noted on the antituber-
culosis activity as compound 25 provided significantly enhanced
inhibition of M. aVium (MIC90 ) 50-100 µg/mL) in comparison
to the respective 2′-arabinohydroxyl analog 6 (25% inhibition
at 100 µg/mL). We note that the activity of nucleosides 5 and
24 was improved (50% inhibition at 50 µg/mL) but diminished
for the compounds 7 and 26 (0-20% inhibition at 100 µg/mL)
against M. aVium when compared with their respective 2′-
Finally, the most active inhibitors 5, 24, and 25 were also
evaluated for their activity toward the rifampicin-resistant strain

Growth Inhibition of Mycobacterium In Vitro
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3701
Figure 1. The effect of detergents on the antimycobacterial activity of compounds 5 and 25. Panel A shows the dose response of compounds 5
and 25 in the absence of detergent. The control bacterial well gave an average reading of 89 897. Panel B shows the activity of compound 5, and
panel C shows the activity of compound 25 in the presence of two different detergents. Both compounds were tested at four different concentrations
(depicted by individual lines), as shown in each graph.
Table 2. In Vitro Antibacterial Activities of Pyrimidine Nucleosides against Gram-Positive and Gram-Negative Bacteria
mean inhibitory concentrationa (MIC100) µg/mL (% inhibition)
cmpd
Ecc
Eac
Pvc
Pac
Stc
Sec
Sac
Bsc
Lmc
Spyc
Spnc
100
(100)
100
(100)
100
(100)
>100
Efc
23
>100
>100
>100
>100
>100
100
(100)
100
(100)
100
(100)
100
100
(100)
100
(100)
100
(100)
100
>100
100
(100)
100
(100)
100
(100)
>100
100
(100)
100
(100)
100
(100)
>100
100
(100)
100
(100)
100
(100)
>100
24
25
26
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(100)
>100
100, 10, 1
(100)
(50)
(100)
>100
100, 10, 1
(100)
(100)
ref1b
ref2b
>100
100
(100)
>100
>100
100
(100)
>100
>100
>100
>100
100
(100)
>100
>100
100
(100)
(50)
>100
100
(100%)
(50)
>100
>100
>100
>100
100, 10, 1
(100)
100, 10, 1
(100)
100, 10, 1
ref3b
>100
(100)
(100)
(100)
(100)
a Antibacterial activity against Gram-positive and Gram-negative bacteria was determined at concentrations 100, 50, 10, and 1 µg/mL using microtiter
broth dilution technique. Concentration of compounds exhibiting 100% inhibition in bacterial growth. b Positive control drugs; ref 1 ) isoniazid, ref 2 )
rifampicin, ref 3 ) vancomycin at 1 µg/mL. c Ec ) E. coli; Ea ) E. aerogenes; Pv ) P. Vulgaris; Pa ) P. aeruginosa; St ) S. typhimurium; Se ) S.
epidermidis; Sa ) S. aureus; Bs ) B. subtilis; Lm ) L. monocytogenes; Spy ) S. pyogenes; Spn ) S. pneumoniae; Ef ) E. faecalis.
of M. tuberculosis, H37Rv by BACTEC assay.34 Intriguingly,
all of the compounds exerted MIC90s against drug-resistant
mycobacteria at concentrations ranging from 1 to 10 µg/mL,
where rifampicin showed no activity at 2 µg/mL and isoniazid
provided 100% inhibition at 1 µg/mL.
An in vitro phosphorolysis study of selected pyrimidine
nucleosides 5 and 25, in the presence of E. coli thymidine
phosphorylase at 37 °C for 30 min was carried out to determine
their glycosidic bond stability.35 Up to the maximum time
investigated, cleavage products of 5 and 25 were not obtained
(<10%) in contrast to control thymidine (>70% cleavage in
30 min), suggesting that they are resistant to glycosidic bond
cleavage.
The precise mechanism of action of the compounds inhibiting
mycobacterial multiplication in this study is not clear yet. The
complete genome sequence of M. tuberculosis has been

3702 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Johar et al.
deciphered.20 It encodes many of the enzymes required for DNA
and RNA synthesis and pyrimidine and purine nucleoside
biosynthesis. It is possible that our active nucleoside analogs,
after their metabolic conversion to phosphorylated forms by
mycobacterial kinases, may be selectively inhibiting its DNA
and RNA synthesis, by acting as substrates and inhibitors of
metabolic enzymes of DNA/RNA synthesis.
The compounds 3-16, 19, 20, 22-33, 36, and 37 were also
tested for MICs for a variety of gram-positive (S. aureus, S.
epidermis, E. faecalis, B. subtilis, S. pneumonaie, S. pyogenes,
L. monocytogenes) and gram-negative (S. typhimurium, E. coli,
P. Vulgaris, P. aeruginosa) bacteria. In these species, compounds
23-26 showed a specific spectrum of action, being active
mainly toward gram-positive pathogens. Compounds 1-(2′-
deoxy-2′-fluoro-â-D-ribofuranosyl)-5-decynyluracil (23), 1-(2′-
deoxy-2′-fluoro-â-D-ribofuranosyl)-5-dodecynyluracil (24), and
1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tridecynyluracil (25)
exhibited activity against S. aureus, S. epidermis, E. faecalis,
S. pneumonaie, S. pyogenes, and L. monocytogenes (MIC100
)100 µg/mL; Table 2). Compound 1-(2′ -deoxy-2′-fluoro-â-
D-ribofuranosyl)-5-tetradecynyluracil (26) possessed activity
only against S. aureus and S. epidermis (MIC100 )100 µg/mL).
MICs obtained at higher concentrations against these bacteria
suggested that they possess higher specificity for mycobacterial
species.
Chemical shifts are given in ppm relative to TMS as an internal
standard. The assignment of all exchangeable protons (OH, NH)
was confirmed by the addition of the D2O. Microanalyses were
within (0.4% of theoretical values for all elements listed, unless
otherwise indicated. 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). 1-â-D-
arabinofuranosyl-5-iodouracil (2), 1-(2′-deoxy-2′-fluoro-â-D-ribo-
furanosyl)-5-iodouracil (21), 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
uracil (34), and 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)thymine
(35) were synthesized using procedures as described earlier.23,29
Preparation of 5-Alkynyl Pyrimidine Nucleosides. A full
procedure is provided for 1-â-D-arabinofuranosyl-5-heptynyluracil
(3). For other 5-alkynyl analogs, only brief spectroscopic data are
presented.
1-â-D-Arabinofuranosyl-5-heptynyluracil (3). Tetrakis(triph-
enylphosphine) palladium(0) (94 mg, 0.081 mmol), copper(I) iodide
(31 mg, 0.162 mmol), diisopropylethylamine (0.28 mL, 1.62 mmol),
and 1-heptyne (0.32 mL, 2.43 mmol) were added to a solution of
1-â-D-arabinofuranosyl-5-iodouracil (2; 300 mg, 0.81 mmol) in
anhydrous dimethylformamide (30 mL). The orange-colored reac-
tion mixture was stirred at room temperature for 19 h in a nitrogen
atmosphere (the progress of the reaction was monitored by TLC in
MeOH/CHCl3 (12:88; v/v). After 19 h of stirring, 15 drops of 5%
disodium salt of EDTA/H2O were added to the reaction mixture
and the contents were concentrated in vacuo. The resulting residue
was purified on a silica gel column using CHCl3/MeOH (95:5, v/v)
as eluent to yield 3 (120 mg, 44%) as a solid; mp 175-180 °C
(dec); 1H NMR (DMSO-d6) δ 0.87 (t, J ) 7.0 Hz, 3H, CH3), 1.23-
1.39 (m, 4H, 2 × CH2), 1.48 (m, 2H, â-CH2), 2.34 (t, J ) 7.0 Hz,
2H, R-CH2), 3.50-3.65 (m, 2H, H-5′), 3.72 (m, 1H, H-4′), 3.89
(m, 1H, H-3′), 3.98 (m,1H, H-2′), 5.11 (t, J ) 5.2 Hz, 1H, 5′-OH),
5.48 and 5.60 (m, 1H each, 2′-OH, 3′-OH), 5.95 (d, J ) 4.3 Hz,
1H, H-1′), 7.79 (s, 1H, H-6), 11.57 (s, 1H, NH); 13C NMR (CD3-
OD) δ 14.33 (CH3), 20.17, 23.28, 29.49 (3 × CH2), 32.24 (R-CH2),
62.38 (C-5′), 72.60 (C-â), 76.96, 77.46 (C-2′ and C-3′), 86.44 (C-
4′), 87.58 (C-1′), 94.84 (C-R), 99.84 (C-5), 145.77 (C-6), 151.19
(C-2), 164.77 (C-4). Anal. (C16H22N2O6) C, H, N.
The MTT test was performed to evaluate the toxicity of
promising compounds (4-8, 22-26) in vitro against Vero cells
and human foreskin fibroblast (HFF cells). No significant
toxicity was observed up to a concentration of 100 µg/mL (CC50
>100 µg/mL).
Summary
Identification of new lead antimycobacterial compounds that
work by different mechanisms of action is necessary for the
treatment of tuberculosis due to re-emergence of infection
globally and resistance to existing drugs. In this work, among
a series of 5-substituted 1-â-D-2′-arabinofuranosyl and 1-(2′-
deoxy-2′-fluoro-â-D-ribofuranosyl) pyrimidine nucleosides, 1-â-
D-2′-arabinofuranosyl-5-dodecynyluracil (5), 1-(2′-deoxy-2′-
fluoro-â-D-ribofuranosyl)-5-dodecynyluracil (24), and 1-(2′-
deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tridecynyluracil (25) emerged
with potent antimycobacterial activity at concentrations similar
or close to the reference drug. Importantly, the most efficacious
analogs are also active against drug-resistant M. tuberculosis.
The salient feature of the identified molecules, that is, potent
broad spectrum activity against drug-sensitive and drug-resistant
strains of mycobacteria as well as several other gram-positive
bacterial organisms, is an important step for the identification
of new antituberculosis agents with different resistance patterns
or mechanisms of action. It is envisioned that compounds 5,
24, and 25 would have better ADME/T properties as compared
to their 2′-deoxyuridine derivatives.22,23 Further biochemical
studies to elucidate the mechanism of action of these compounds
and in vivo tests are warranted. The new series of inhibitors
identified in this report hold promise for the development of a
new class of agents for TB infections.
1-â-D-Arabinofuranosyl-5-decynyluracil (4). Yield 49%; mp
1
185-189 °C (dec); H NMR (DMSO-d6) δ 0.85 (t, J ) 7.0 Hz,
3H, CH3), 1.22-1.38 (m, 10H, 5 × CH2), 1.46 (m, 2H, â-CH2),
2.34 (t, J ) 7.0 Hz, 2H, R-CH2), 3.54-3.63 (m, 2H, H-5′), 3.73
(m, 1H, H-4′), 3.89 (m, 1H, H-3′), 3.98 (m, 1H, H-2′), 5.10 (t, J )
5.3 Hz, 1H, 5′-OH), 5.47 and 5.59 (m, 1H each, 2′-OH, 3′-OH),
5.95 (d, J ) 4.3 Hz, 1H, H-1′), 7.78 (s, 1H, H-6), 11.56 (s, 1H,
NH); 13C NMR (CD3OD) δ 14.46 (CH3), 20.21, 23.72, 29.79, 30.04,
30.26, 30.34 (6 × CH2), 33.02 (R-CH2), 62.39 (C-5′), 72.60 (C-â),
76.96, 77.47 (C-2′ and C-3′), 86.46 (C-4′), 87.59 (C-1′), 94.84 (C-
R), 99.84 (C-5), 145.61 (C-6), 151.17 (C-2), 164.77 (C-4). Anal.
(C19H28N2O6) C, H, N.
1-â-D-Arabinofuranosyl-5-dodecynyluracil (5). Yield 48%; mp
1
182-187 °C (dec); H NMR (DMSO-d6) δ 0.85 (t, J ) 7.0 Hz,
3H, CH3), 1.23-1.37 (m, 14H, 7 × CH2), 1.47 (m, 2H, â-CH2),
2.34 (t, J ) 7.0 Hz, 2H, R-CH2), 3.53-3.62 (m, 2H, H-5′), 3.73
(m, 1H, H-4′), 3.89 (m, 1H, H-3′), 3.98 (m, 1H, H-2′), 5.11 (t, J )
5.2 Hz, 1H, 5′-OH), 5.48 and 5.60 (m, 1H each, 2′-OH, 3′-OH),
5.96 (d, J ) 4.3 Hz, 1H, H-1′), 7.78 (s, 1H, H-6), 11.57 (s, 1H,
NH); 13C NMR (CD3OD) δ 14.45 (CH3), 20.21, 23.72, 29.79, 30.04,
30.29, 30.46, 30.67, 30.72 (8 × CH2), 33.07 (R-CH2), 62.39 (C-
5′), 72.60 (C-â), 76.96, 77.47 (C-2′ and C-3′), 86.48 (C-4′), 87.59
(C-1′), 94.85 (C-R), 99.85 (C-5), 145.62 (C-6), 151.18 (C-2), 164.78
(C-4). Anal. (C21H32N2O6) C, H, N.
Experimental Section
1-â-D-Arabinofuranosyl-5-tridecynyluracil (6). Yield 41%; mp
1
Melting points were determined with a Buchi capillary apparatus
180-186 °C (dec); H NMR (DMSO-d6) δ 0.85 (t, J ) 7.0 Hz,
1
and are uncorrected. H NMR, 19F NMR, and 13C NMR spectra
3H, CH3), 1.18-1.38 (m, 16H, 8 × CH2), 1.46 (m, 2H, â-CH2),
2.34 (t, J ) 7.0 Hz, 2H, R-CH2), 3.55-3.63 (m, 2H, H-5′), 3.73
(m, 1H, H-4′), 3.89 (m, 1H, H-3′), 3.97 (m, 1H, H-2′), 5.10, 5.47
and 5.59 (m, 1H each, 2′-OH, 3′-OH, 5′-OH), 5.95 (d, J ) 4.27
Hz, 1H, H-1′), 7.79 (s, 1H, H-6), 11.56 (s, 1H, NH); 13C NMR
(CD3OD) δ 14.45 (CH3), 20.21, 23.75, 29.79, 30.04, 30.29, 30.48,
were determined for solutions in Me2SO-d6 or CD3OD on a Bruker
AM 300 spectrometer. 13C NMR (J modulated spin echo) spectra
were determined for selected compounds, where methyl and
methyne carbon resonances appear as positive peaks and methylene
and quaternary carbon resonances appear as negative peaks.

Growth Inhibition of Mycobacterium In Vitro
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3703
30.67, 30.74, 30.76 (9 × CH2), 33.09 (R-CH2), 62.39 (C-5′), 72.60
(C-â), 76.97, 77.48 (C-2′ and C-3′), 86.47 (C-4′), 87.59 (C-1′), 94.84
(C-R), 99.84 (C-5), 145.63 (C-6), 151.18 (C-2), 164.77 (C-4). Anal.
(C22H34N2O6) C, H, N.
C-2′, J ) 185.7 Hz), 95.28 (C-R), 101.31 (C-5), 144.05 (C-6),
151.00 (C-2), 164.57 (C-4). Anal. (C16H21FN2O5) C, H, N.
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-5-decynyluracil (23).
Compound 23 was prepared from 21 by using the procedure
1
described for the synthesis of 3 in 86% yield as a foam. H NMR
1-â-D-Arabinofuranosyl-5-tetradecynyluracil (7). Yield 40%;
1
(DMSO-d6) δ 0.85 (t, J ) 6.7 Hz, 3H, CH3), 1.21-1.39 (m, 10H,
5 × CH2), 1.47 (m, 2H, â-CH2), 2.33 (t, J ) 6.7 Hz, 2H, R-CH2),
3.55-3.81 (m, 2H, H-5′), 3.86 (m, 1H, H-4′), 4.08-4.22 (m, 1H,
H-3′), 5.02 (dm, J2′,F ) 52.8 Hz, 1H, H-2′), 5.31 (t, J5′,OH ) 4.9
Hz, 1H, 5′-OH), 5.60 (d, J3′,OH ) 6.7 Hz, 1H, 3′-OH), 5.85 (d, J1′,F
) 16.2 Hz, 1H, H-1′), 8.23 (s, 1H, H-6), 11.63 (s, 1H, NH); 13C
NMR (CD3OD) δ 14.45 (CH3), 20.20, 23.72, 29.75, 30.02, 30.26,
30.33 (6 × CH2), 33.03 (R-CH2), 60.69 (C-5′), 69.29 and 69.07
(d, C-3′, J ) 16.5 Hz),72.40 (C-â), 84.78 (C-4′), 90.05 and 89.60
(d, C-1′, J ) 34.1 Hz), 96.24 and 93.77 (C-2′, J ) 186.8 Hz),
95.28 (C-R), 101.31 (C-5), 144.07 (C-6), 151.02 (C-2), 164.58 (C-
4); 19F NMR (DMSO-d6) (C6F6 external standard): δ -34.9 (ddd,
J2′,F ) 53.7, J1′,F ) 17.1, J3′,F ) 21.9 Hz, F-2′). Anal. (C19H27-
FN2O5) C, H, N.
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-5-dodecynyluracil (24).
Compound 24 was prepared from 21 by using the procedure
described for the synthesis of 3 in 75% yield as a syrup. 1H NMR
(DMSO-d6) δ 0.85 (t, J ) 6.7 Hz, 3H, CH3), 1.21-1.39 (m, 14H,
7 × CH2), 1.47 (m, 2H, â-CH2), 2.33 (t, J ) 6.7 Hz, 2H, R-CH2),
3.54-3.80 (m, 2H, H-5′), 3.86 (m, 1H, H-4′), 4.10-4.20 (m, 1H,
H-3′), 5.02 (dm, J2′,F ) 52.8 Hz, 1H, H-2′), 5.32 (t, J5′,OH ) 4.9
Hz, 1H, 5′-OH), 5.58 (d, J3′,OH ) 6.7 Hz, 1H, 3′-OH), 5.86 (d, J1′,F
) 16.2 Hz, 1H, H-1′), 8.23 (s, 1H, H-6), 11.64 (s, 1H, NH). Anal.
(C21H31FN2O5) C, H, N.
mp 180-185 °C (dec); H NMR (DMSO-d6) δ 0.85 (t, J ) 7.0
Hz, 3H, CH3), 1.24-1.37 (m, 18H, 9 × CH2), 1.47 (m, 2H, â-CH2),
2.34 (t, J ) 7.0 Hz, 2H, R-CH2), 3.54-3.65 (m, 2H, H-5′), 3.73
(m, 1H, H-4′), 3.89 (m, 1H, H-3′), 3.98 (m,1H, H-2′), 5.09 (t, J )
5.2 Hz, 1H, 5′-OH), 5.46 and 5.59 (m, 1H each, 2′-OH, 3′-OH),
5.95 (d, J ) 4.3 Hz, 1H, H-1′), 7.78 (s, 1H, H-6), 11.56 (s, 1H,
NH); 13C NMR (CD3OD) δ 14.47 (CH3), 20.22, 23.75, 29.79, 30.04,
30.29, 30.49, 30.67, 30.74, 30.77, 30.79 (10 × CH2), 33.09 (R-
CH2), 62.39 (C-5′), 72.60 (C-â), 76.97, 77.48 (C-2′ and C-3′), 86.48
(C-4′), 87.59 (C-1′), 94.85 (C-R), 99.84 (C-5), 145.63 (C-6), 151.17
(C-2), 164.78 (C-4). Anal. (C23H36N2O6) C, H, N.
1-â-D-Arabinofuranosyl-5-(4-n-propylphenylethynyl)uracil (8).
1
Yield 54%; mp 217-221 °C (dec); H NMR (DMSO-d6) δ 0.88
(t, J ) 7.0 Hz, 3H, CH3), 1.60 (m, 2H, CH2), 2.57 (m, 2H, CH2),
3.57-3.68 (m, 2H, H-5′), 3.76 (m, 1H, H-4′), 3.92 (m, 1H, H-3′),
4.03 (m, 1H, H-2′), 5.17, 5.49 and 5.65 (m, 1H each, 2′-OH, 3′-
OH, 5′-OH), 5.99 (d, J ) 4.6 Hz, 1H, H-1′), 7.20-7.45 (m, 4H,
aromatic), 8.05 (s, 1H, H-6), 11.72 (bs, 1H, NH); 13C NMR (CD3-
OD) δ 14.04 (CH3), 25.53, 38.91 (2 × CH2), 62.32 (C-5′), 77.03,
77.35 (C-2′ and C-3′), 81.12 (C-â), 86.50 (C-4′), 87.74 (C-1′), 93.82
(C-R), 99.39 (C-5), 121.56, 129.53, 132.35, 144.51 (C-phenyl),
146.18 (C-6), 151.10 (C-2), 164.38 (C-4). Anal. (C20H22N2O6)
C, H, N.
1-â-D-Arabinofuranosyl-4-thiouracil (19). To a stirred suspen-
sion of 1-â-D-arabinofuranosyluracil (17; 1 g, 4.09 mmol), in acetic
anhydride (25 mL), 4-dimethylamino pyridine (50 mg, 0.41 mmol)
was added. The reaction mixture was stirred at room temperature
for 5 h. Acetic anhydride was removed in vacuo, and the syrup
was coevaporated with ethanol (2 × 25 mL). The residue obtained
was dried on high vacuum. To this residue, Lawesson’s reagent
(2.0 g, 4.94 mmol) and dry 1,4-dioxane (50 mL) were added. The
mixture was refluxed for 3 h. After the reaction mixture had cooled,
the solvent was removed in vacuo. The crude product thus obtained
was treated with a saturated solution of ammonia in methanol (20
mL) and stirred at room temp for 3 h. The reaction mixture was
concentrated in vacuo, and the residue was purified on a silica gel
column using chloroform/methanol (9:1, v/v) as the eluent to afford
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tridecynyluracil (25).
Compound 25 was prepared from 21 by using the procedure
described for the synthesis of 3 in 88% yield as a syrup. 1H NMR
(DMSO-d6) δ 0.85 (m, 3H, CH3), 1.22-1.37 (m, 16H, 8 × CH2),
1.46 (m, 2H, â-CH2), 2.33 (t, J ) 6.7 Hz, 2H, R-CH2), 3.55-3.82
(m, 2H, H-5′), 3.87 (m, 1H, H-4′), 4.23-4.09 (m, 1H, H-3′), 5.02
(dm, J2′,F ) 52.5 Hz, 1H, H-2′), 5.30 (t, J5′,OH ) 4.9 Hz, 1H, 5′-
OH), 5.59 (d, J3′,OH ) 6.4 Hz, 1H, 3′-OH), 5.86 (d, J1′,F ) 17.4
Hz, 1H, H-1′), 8.22 (s, 1H, H-6), 11.63 (s, 1H, NH); 13C NMR
(CD3OD) δ 14.46 (CH3), 20.20, 23.75, 29.75, 30.01, 30.29, 30.49,
30.65, 30.75, 30.77 (9 × CH2), 33.08 (R-CH2), 60.69 (C-5′), 69.29
and 69.07 (d, C-3′, J ) 16.5 Hz),72.41 (C-â), 84.80 (C-4′), 90.05
and 89.60 (d, C-1′, J ) 34.1 Hz), 96.24 and 93.77 (d, C-2′, J )
186.8 Hz), 95.29 (C-R), 101.32 (C-5), 144.05 (C-6), 151.01 (C-2),
164.58 (C-4); 19F NMR (DMSO-d6) (C6F6 external standard): δ
-35.0 (ddd, J2′,F ) 53.7, J1′,F ) 17.1, J3′,F ) 21.9 Hz, F-2′). Anal.
(C22H33FN2O5) C, H, N.
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-5-tetradecynylu-
racil (26). Compound 26 was prepared from 21 by using the
procedure described for the synthesis of 3 in 79% yield as a syrup.
1H NMR (DMSO-d6) δ 0.84 (t, J ) 6.7 Hz, 3H, CH3), 1.21-1.37
(m, 18H, 9 × CH2), 1.45 (m, 2H, â-CH2), 2.33 (t, J ) 6.7 Hz, 2H,
R-CH2), 3.55-3.79 (m, 2H, H-5′), 3.86 (m, 1H, H-4′), 4.06-4.23
(m, 1H, H-3′), 5.02 (dm, J2′,F ) 53.4 Hz, 1H, H-2′), 5.29 (t, J5′,OH
) 5.2 Hz, 1H, 5′-OH), 5.59 (d, J3′,OH ) 6.4 Hz, 1H, 3′-OH), 5.85
(d, J1′,F ) 17.1 Hz, 1H, H-1′), 8.22 (s, 1H, H-6), 11.62 (s, 1H,
NH); 13C NMR (CD3OD) δ 14.47 (CH3), 20.22, 23.75, 29.75, 30.00,
30.29, 30.49, 30.65, 30.77, 30.79, 30.93 (10 × CH2), 33.08 (R-
CH2), 60.69 (C-5′), 69.29 and 69.07 (d, C-3′, J ) 16.5 Hz), 72.41
(C-â), 84.80 (C-4′), 90.04 and 89.59 (d, C-1′, J ) 34.1 Hz), 96.24
and 93.77 (d, C-2′, J ) 186.8 Hz), 95.28 (C-R), 101.31 (C-5),
144.04 (C-6), 151.0 (C-2), 164.57 (C-4). Anal. (C23H35FN2O5)
C, H, N.
1
19 (400 mg, 38%) as a syrup. H NMR (DMSO-d6) δ 3.59 (m,
2H, H-5′), 3.76 (m, 1H, H-4′), 3.89 (m, 1H, H-3′), 4.02 (m, 1H,
H-2′), 5.05 (t, J ) 5.2 Hz, 1H, 5′-OH), 5.49 and 5.63 (m, 1H each,
2′-OH, 3′-OH), 5.95 (d, J ) 4.3 Hz, 1H, H-1′), 6.28 (d, J ) 7.3
Hz, 1H, H-5), 7.56 (d, J ) 7.6 Hz, 1H, H-6), 12.69 (s, 1H, NH);
13C NMR (DMSO-d6) δ 60.64 (C-5′), 74.94, 75.38 (C-2′ and C-3′),
85.17 (C-4′), 85.79 (C-1′), 111.19 (C-5), 137.38 (C-6), 147.55 (C-
2), 189.78 (C-4). Anal. (C9H12N2O5S) C, H, N.
1-â-D-Arabinofuranosyl-4-thio-5-methyluracil (20). This prod-
uct was synthesized by the procedure described for the synthesis
of 19 starting from 1-â-D-arabinofuranosyl-5-methyluracil (18) as
1
a syrup in 34% yield. H NMR (DMSO-d6) δ 1.96 (s, 3H, CH3),
3.62 (m, 2H, H-5′), 3.75 (m, 1H, H-4′), 3.91 (m, 1H, H-3′), 4.04
(m, 1H, H-2′), 5.13 (t, J ) 5.2 Hz, 1H, 5′-OH), 5.48 and 5.61 (m,
1H each, 2′-OH, 3′-OH), 5.95 (d, J ) 4.6 Hz, 1H, H-1′), 7.69 (s,
1H, H-6), 12.70 (s, 1H, NH). Anal. (C10H14N2O5S) C, H, N.
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-5-heptynyluracil (22).
Compound 22 was prepared from 21 by using the procedure
described for the synthesis of 3 in 77% yield as a syrup. 1H NMR
(DMSO-d6) δ 0.87 (t, 3H, CH3), 1.23-1.36 (m, 4H, 2 × CH2),
1.48 (m, 2H, â-CH2), 2.34 (t, 2H, R-CH2), 3.57-3.82 (m, 2H, H-5′),
3.87 (m, 1H, H-4′), 4.05-4.22 (m, 1H, H-3′), 5.03 (dm, J2′,F )
50.6 Hz, 1H, H-2′), 5.30 (t, J5′,OH ) 5.2 Hz, 1H, 5′-OH), 5.59 (d,
J3′,OH ) 6.7 Hz, 1H, 3′-OH), 5.86 (d, J1′,F ) 16.8 Hz, 1H, H-1′),
8.22 (s, 1H, H-6), 11.62 (s, 1H, NH); 13C NMR (CD3OD) δ 14.33
(CH3), 20.17, 23.28, 29.45 (3 × CH2), 32.21 (R-CH2), 60.69 (C-
5′), 69.09 and 69.29 (d, C-3′, J ) 17.6 Hz), 72.39 (C-â), 84.79
(C-4′), 90.05 and 89.59 (C-1′, J ) 35.2 Hz), 96.24 and 93.78 (d,
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-4-thiouracil (36). To
an ice-cold solution of 1-(2′-deoxy-2′-fluoro-â-D-ribofuranosyl)-
uracil (34; 600 mg, 2.44 mmol) in dry pyridine (40 mL) was added
acetic anhydride (0.58 mL, 6.1 mmol) dropwise with stirring. The
reaction mixture was stirred at 0 °C for 1 h and then at room
temperature for 24 h. Pyridine was removed in vacuo. The residues
thus obtained were taken in dry 1,4-dioxane (50 mL) and Lawes-
son’s reagent (1.18 g, 2.92 mmol) was added. The reaction mixture
was refluxed for 2 h. After the reaction mixture had cooled, solvent

3704 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15
Johar et al.
was removed in vacuo. The obtained crude product was treated
with a saturated solution of ammonia in methanol (20 mL) and
stirred at room temp for 2 h. The reaction mixture was concentrated
in vacuo and purified on silica gel column using chloroform/
methanol (95:5, v/v) as solvent to yield 36 (280 mg, 44%) as a
syrup. 1H NMR (DMSO-d6) δ 3.56-3.82 (m, 2H, H-5′), 3.89 (m,
1H, H-4′), 4.05-4.19 (m, 1H, H-3′), 5.06 (dm, J2′,F ) 53.0 Hz,
The data is shown as the average of the triplicates, and the standard
deviation was within 5%. Control wells included M. boVis cultured
with various amounts of both detergents without the test compounds.
In the initial experiment, we determined the effect of various
concentrations of the detergents on M. boVis growth. Tween-20
had no effect on M. boVis growth at all four concentrations, whereas
Triton X-100 was toxic at 1 CMC. Therefore, the compounds were
tested in the presence of nontoxic doses of detergents.
1H, H-2′), 5.24 (t, J5′,OH ) 5.2 Hz, 1H, 5′-OH), 5.62 (d, J3′,OH
)
6.4 Hz, 1H, 3′-OH), 5.84 (d, J1′,F ) 17.1 Hz, 1H, H-1′), 6.28 (d, J
) 7.3 Hz, 1H, H-5), 7.87 (d, J ) 7.6 Hz, 1H, H-6), 12.77 (s, 1H,
NH); 13C NMR (DMSO-d6) δ 58.91 (C-5′), 67.10 and 66.88 (d,
C-3′, J ) 16.5 Hz), 83.14 (C-4′), 87.89 and 87.44 (d, C-1′, J )
34.1 Hz), 94.66 and 92.20 (d, C-2′, J ) 185.7 Hz), 112.31 (C-5),
135.38 (C-6), 147.38 (C-2), 190.10 (C-4). Anal. (C9H11FN2O4S)
C, H, N.
Antimycobacterial Activity against a Drug-Resistant Strain
of M. tuberculosis: The activity of compounds 5, 24, and 25 was
determined against rifampicin-resistant M. tuberculosis H37Rv
(ATCC 35838, resistant to rifampicin at 2 µg/mL) using a
radiometric-BACTEC assay.34 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 containing radiometric 7H12 (BACTEC 12B) media
and incubated 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, media with
bacteria inoculum at 1:100, and media alone. As reference drugs,
rifampicin and isoniazid were used at their MIC90 concentration.
All 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 (4GI) was recorded for each compound. Percent
inhibition was defined as (GI of test sample/GI of control) × 100.
For the no drug control, the 4GI 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.
In Vitro Antibacterial Activity Assay. A total of 12 bacterial
strains Staphylococcus aureus (ATCC 25923), Staphylococcus
epidermis (ATCC 14990), Enterococcus faecalis (ATCC 29212),
Bacillus subtilis (ATCC 6633), Streptococcus pneumoniae (ATCC
49619), Streptococcus pyogenes (ATCC 19615), Salmonella typh-
imurium (Clinical isolate), Escherichia coli (ATCC 25922), Proteus
Vulgaris (ATCC 49132), Pseudomonas aeruginosa (ATCC 13048),
Lysteria monocytogenes (ATCC 15313), and Enterobacter aero-
genes were used for the determination of the in vitro antibacterial
activity of the studied compounds. The in vitro antibacterial activity
was studied by determining their minimum inhibitory concentrations
(MICs) by means of the broth microdilution 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.
1-(2′-Deoxy-2′-fluoro-â-D-ribofuranosyl)-4-thio-5-methylu-
racil (37). This product was synthesized according to the procedure
described for 36 starting from 1-(2′-deoxy-2′-fluoro-â-D-ribofura-
nosyl)-5-methyluracil (35) as a syrup in 55% yield. 1H NMR
(DMSO-d6) δ 1.94 (s, 3H, CH3), 3.57-3.85 (m, 2H, H-5′), 3.89
(m, 1H, H-4′), 4.07-4.24 (m, 1H, H-3′), 5.06 (dm, J2′,F ) 53.0
Hz, 1H, H-2′), 5.33 (t, J5′,OH ) 5.2 Hz, 1H, 5′-OH), 5.62 (d, J3′,OH
) 6.7 Hz, 1H, 3′-OH), 5.86 (d, J1′,F ) 16.5 Hz, 1H, H-1′), 8.02 (s,
1H, H-6), 12.78 (s, 1H, NH). Anal. (C10H13FN2O4S) C, H, N.
In Vitro Antimycobacterial Activity Assay (M. tuberculosis,
M. boWis, M. aWium): M. boVis (BCG), M. tuberculosis (H37Ra),
and M. aVium (ATCC 25291) were obtained from the American
Type Culture Collection, Rockville, MD. The antimycobacterial
activity was determined using the MABA assay.31 Test compounds
were dissolved in DMSO at 10 mg/mL, 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, and 1 micrograms/mL in triplicates. The
experiments were repeated two times and the mean percent
inhibition is reported in the table. The most promising compounds
were further repeated at 2-fold dilutions to obtain MIC90 values.
The standard deviations were within 10%. 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 8 with bacteria alone (B) and 8 with media alone (M).
Plates were incubated for 6 days and then 20 µL of 10× 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 to 48 h for visual
color change from blue to pink and read by spectrophotometer (at
excitation 530/525 and emission 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 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
calculated as (test well-M bkg/B well-M bkg) × 100. The lowest
drug concentration effecting an inhibition of ∼90% was considered
as the MIC90. Similar methodology was used for all M. boVis, M.
tuberculosis, and M. aVium. Rifampicin and clarithromycin were
used as positive controls. As negative controls, DMSO (2 µL, 1
µL, 0.2 µL, 0.02 µL) was added to the B well at concentrations
similar to that 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
60 000 and 100 000.
Cell Cytotoxicity Assay. Cell viability was measured using the
cell proliferation kit 1 (MTT; Boehringer Mannheim), as per
manufacturer’s instructions. Briefly, a 96-well plate was seeded
with Vero cells or HFF cells at a density of 2.5 × 105 cells per
well. Cells were allowed to attach for 6-8 h, and the medium was
replaced with medium containing drugs at concentrations of 100,
50, 25, 12.5, 6.3, and 1.5 µg/mL. DMSO was also included as a
control. Plates were incubated for 3 days at 37 °C. The color
reaction involved adding 10 µL MTT reagent per well, incubating
4 h at 37 °C and then adding 100 µL of solubilization reagent.
Plates were read on an ELISA plate reader (Abs 560-650 nm)
following an overnight incubation at 37 °C.
Acknowledgment. Financial support from the Canadian
Institutes of Health Research (CIHR) for this work (Operating
Grant MOP-49415) is gratefully acknowledged. B.A. is the
recipient of Alberta Heritage Foundation for Medical Research
(AHFMR) Senior Scholar Award.
The antimycobacterial activity of compounds 5 and 25 was also
determined against M. boVis in the presence of various detergents
using MABA assay. For these experiments, titrating amounts of
detergents, that is, Tween-20, and Triton X-100 (Fisher Scientific,
CMC: 0.05 mM and 0.3 mM, respectively) at 1.0, 0.1, 0.01, and
0.001 CMC (final concentration) were preincubated with test
compounds at 100, 50, 10, and 1 microgram/mL (final concentra-
tion) for 15 min before adding to the M. boVis wells in triplicates.
Supporting Information Available: Elemental analysis data.
This material is available free of charge via the Internet at http://
pubs.acs.org.

Growth Inhibition of Mycobacterium In Vitro
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 15 3705
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