Design and studies of novel 5-substituted alkynylpyrimidine nucleosides as potent inhibitors of mycobacteria

Article abstract of DOI:10.1021/jm058167w

We herein report a new category of 5-substituted pyrimidine nucleosides as potent inhibitors of mycobacteria. A series of 5-alkynyl derivatives of 2′-deoxyuridine (1-8), 2′-deoxycytidine (9-14), uridine (15-17), and 2′-O-methyluridine (18, 19) were synthe

Full text of DOI:10.1021/jm058167w

7012
J. Med. Chem. 2005, 48, 7012-7017
Design and Studies of Novel 5-Substituted Alkynylpyrimidine Nucleosides as
Potent Inhibitors of Mycobacteria
Dinesh Rai,† Monika Johar,† Tracey Manning,† B. Agrawal,‡ Dennis Y. Kunimoto,§ and Rakesh Kumar*,†
Department of Laboratory Medicine and Pathology, Department of Surgery, and Department of Medicine,
Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, T6G 2H7, Canada
Received January 11, 2005
We herein report a new category of 5-substituted pyrimidine nucleosides as potent inhibitors
of mycobacteria. A series of 5-alkynyl derivatives of 2′-deoxyuridine (1-8), 2′-deoxycytidine
(9-14), uridine (15-17), and 2′-O-methyluridine (18, 19) were synthesized and evaluated for
their antimycobacterial activity in vitro. 5-Decynyl, 5-dodecynyl, and 5-tetradecynyl derivatives
showed the highest antimycobacterial potency against M. bovis and M. avium, with the 2′-
deoxyribose derivatives being more effective than the ribose analogues. Nucleosides bearing
short alkynyl side chains 5-ethynyl, 5-propynyl, 5-pentynyl, and 5-heptynyl were mostly not
inhibitory. Incorporation of a phenylethynyl function at the 5-position diminished the
antimicrobial effect. Furthermore, related bicyclic analogues (20-24) were devoid of antimy-
cobacterial activity, indicating that an acyclic side chain at the C-5 position of the pyrimidine
ring is essential for potent activity. Compounds 1-17 were synthesized by the Pd-catalyzed
coupling reactions of respective alkynes with 5-iodo derivatives of 2′-deoxyuridine, 2′-
deoxycytidine, and uridine. Intramolecular cyclization of 1 and 3-6 in the presence of Cu
afforded the corresponding bicyclic compounds 20-24. The investigated nucleosides are
recognized here for the first time to be potent inhibitors of mycobacteria. This class of compounds
could be of interest for lead optimization as antimycobacterial agents.
Introduction
M. tuberculosis and therefore is closely related to M.
tuberculosis. M. tuberculosis is most likely an evolved
form of M. bovis. M. bovis infections in humans have
been reported from 4000 to 5000 B.C. Interestingly, M.
bovis infections are back and causing TB in humans,
particularly those who are HIV-positive. In addition,
MDR strains of M. bovis have been isolated.8 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 in 19 patients.8 There is an ever-increasing
threat of drug-resistant TB appearing as an epidemic
in many developing as well as developed countries,
particularly because no new classes of drugs have been
especially developed for the treatment of tuberculosis
since 1967.3
Mycobacterium avium complex (MAC) infections, in
particular M. avium infections, are one of the most
serious complications among patients with AIDS.9,10
MAC infections are disseminated rather 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.9,10 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.11,12
TB is spread through the air when someone with
infectious TB disease coughs or sneezes. The control and
management of mycobacterial infections are highly
important not only for the survival of AIDS patients but
also for the people who are or will be infected with multi-
drug-resistant bacteria, people who develop resistance
after previous drug treatment, immunocompetent or
immunocompromised people in proximity to HIV pa-
tients, and patients with MAC infection.
Tuberculosis (TB) is an ancient enemy of humans. The
World Health Organization (WHO) estimates that one-
third of the world’s population is infected by Mycobac-
terium tuberculosis. TB infects eight million people and
causes 2-3 million deaths annually around the world.1-3
TB was declared a global health emergency by the WHO
in 1993. A number of factors have contributed to the
sharp increase in TB infections. The biggest factor is
that TB and human immunodeficiency virus (HIV) have
formed a new and deadly combination. In immunocom-
promised people, tuberculosis is much more likely to
cause infection, reactivate latent TB, and create a
greater number of active TB cases, leading to more
people who spread the disease. The increased mobility
of people from the developing world to developed
countries makes a ready pool for TB dissemination. For
both TB and HIV, misdiagnosis and noncompliance with
treatment regimens further compound the problem.
New TB strains are emerging and spreading that are
not susceptible to a number of available drugs, i.e.,
multidrug-resistant TB (MDR-TB).1,4,5 Although TB is
considered a single disease, it can be caused by several
microorganisms. Two groups of mycobacteria, M. tuber-
culosis and M. avium, pose a significant challenge to
the clinical management of tuberculosis in HIV-infected
patients and are often responsible for their death.6
Bacillus Calmette-Guerin (BCG)7 is an attenuated
strain of M. bovis that is more than 98% homologous to
* To whom correspondence should be addressed. Phone: (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.
10.1021/jm058167w CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/01/2005

Alkynylpyrimidine Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7013
Scheme 1a
The development of drug-resistant clinical isolates of
mycobacteria makes the investigation of new classes of
anti-TB agents a high priority, since new agents that
work by mechanisms different from those of current
drugs and are not cross-resistant with them are likely
the best long-term prospect to augment current therapy,
address the resistance crisis, and meet the global health
emergency.
Recently, we reported the in vitro antimycobacterial
activity of various 5-substituted pyrimidine nucleosides.
5-(1-Substitiuted alkyl)-2′-deoxyuridines exhibited po-
tent and selective in vitro antimycobacterial activity
against M. avium. Our studies have shown that novel
5-substituted alkyl side chains at the C-5 position of
pyrimidine nucleosides play a crucial role in their
antimycobacterial properties.13 Pyrimidine nucleosides
containing a C-5 alkynyl group have been shown to
possess significant antiviral and/or anticancer proper-
ties.14 The 5-alkynyl-2′-deoxyuridines with longer chain
alkynyl group at the C-5 position expressed appreciable
antiviral activity in contrast to the corresponding alkyl
derivatives that showed decreasing antiviral activity
with increasing C-5 side chain length.14 In an effort to
further explore the structure-activity relationships, in
the present investigation we have designed, synthesized,
and evaluated a new class of 5-substituted alkynyl
derived 2′-deoxyribose and ribose pyrimidine nucleo-
sides (1-19) for their antimycobacterial activity against
M. bovis and M. avium. It was postulated that un-
natural pyrimidine nucleosides can specifically target
the mycobacterial enzymes involved in their nucleic acid
synthesis by acting as their substrates and/or inhibitors
and inhibit the mycobacterial DNA and/or RNA syn-
thesis. We now report that 5-decynyl, 5-dodecynyl, and
5-tridecynyl analogues of 2′-deoxyuridine (5-7) and 2′-
deoxycytidine (12,13) exhibit marked inhibitory activity
against M. bovis and M. avium in vitro. In contrast,
nucleosides possessing shorter alkynyl side chains at
the 5-position [5-ethynyl, 5-propynyl, 5-pentynyl, 5-hep-
tynyl, and 5-(2-phenylethynyl)] and related analogues
in which the 5-substituent had undergone intramolecu-
lar cyclization to provide bicyclic analogues (20-24)
were devoid of antimycobacterial activity.
a Reagents: (i) H-CtC-R, Pd(PPh3)4, CuI, (i-Pr)2EtN, DMF,
room temp.
Scheme 2a
a Reagents: (i) H-CtC-R, Pd(PPh3)4, CuI, (i-Pr)2EtN, DMF,
room temp.
Scheme 3
excellent yields (Scheme 1, 2). Bicyclic compounds 20-
24 were prepared by the treatment of corresponding
5-alkynyl-2′-deoxyuridines (1, 3-6) with copper iodide
in methanol and triethylamine using reported proce-
dures16 (Scheme 3). The 1HNMR spectra of 20-24
provided conclusive evidence for the cyclization products
because the NH proton disappeared and a new olefinic
proton was introduced.
Chemistry
5-Ethynyl-2′-deoxyuridine (1) and 5-propynyl-2′-deox-
yuridine (2) were synthesized by the coupling reaction
of 3′,5′-di-O-p-toluyl-5-iodo-2′-deoxyuridine with trim-
ethylsilylacetylene and propyne, respectively, in tri-
ethylamine followed by deprotection with sodium meth-
oxide in methanol at room temperature using the
method of Robins et al.15 5-Pentynyl- (3), 5-heptynyl-
(4), 5-decynyl- (5), 5-dodecynyl- (6), 5-tetradecynyl- (7),
and 5-(2-phenylethynyl)- (8) 2′-deoxyuridines were pre-
pared by the treatment of 5-iodo-2′-deoxyuridine (25)
with respective terminal alkynes as described earlier.16
The synthetic route for the preparation of 5-alkynyl-2′-
deoxycytidines (10-14) and 5-alkynyluridines (15-17)
was adopted after slight modification over the method
used for 2′-deoxyuridine analogues.15,16 The Pd-cata-
lyzed coupling reaction of terminal alkynes and ary-
lacetylene with 5-iodo-2′-deoxycytidine (26) and 5-io-
douridine (27) proceeded readily to yield hitherto
unknown target compounds 10-17 in moderate to
Results and Discussion
5-Alkynylpyrimidine nucleosides (1-19) and the bi-
cyclic compounds 20-24 were evaluated in culture to
determine their antimicrobial effect against the multi-
plication of two mycobacteria (M. bovis, M. avium) using
the microplate alamar blue assay (MABA)17 at 1-100
µg/mL concentrations. Rifampicin and clarithromycin
were used as reference standards. Antimycobacterial
activity for this new class of compounds 1-24 is
summarized in Table 1. Among the different series of
5-alkynyl substituted 2′-deoxyuridines (1-8), 2′-deoxy-
cytidines (9-14), uridines (15-17), and 2′-O-meth-
yuridines (18, 19), a clear structure-activity relation-
ship (SAR) can be delineated. The nucleosides containing
a larger carbon chain at the C-2 carbon of the 5-sub-
stituent are potent inhibitors of M. bovis and M. avium
in these assays, demonstrating activity depending on

7014 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Rai et al.
Table 1. In Vitro Antimycobacterial Activity of Test Compounds against M. bovis and M. avium
antimycobacterial activity
M. bovis
M. avium
d
% inhibition
MIC90
% inhibition
(concentration, µg/mL)
MIC90
(µg/mL)
compd
R
R1
(concentration, µg/mL)a
(µg/mL)
4
O
(CH2)4CH3
(CH2)7CH3
(CH2)9CH3
(CH2)11CH3
(CH2)7CH3
(CH2)9CH3
60 (100, 50)
25 (100)
5
O
90 (100), 50 (50), 0 (10)
90 (100, 50), 70 (10), 25 (1)
90 (100, 50,10), 33 (1)
90 (100, 50), 10 (10)
90 (100, 50, 10), 25 (1)
100 (0.5-1)
100
50
30 (100)
6
7
O
O
NH2
NH2
75 (100), 0 (50)
75 (100), 30 (50)
75 (100), 50 (50), 20 (10),
100 (100), 60 (50), 10 (10)
90 (2)
10
12
50
10
13
100
2
2
Std1b
Std2b
0.5-1
NDc
NDc
95 (2)
a Antimycobacterial activity was determined at concentrations 100, 50, 10, and 1 µg/mL. b Positive control drugs. Std1 ) rifampicin,
Std2 ) clarithromycin. c ND ) not determined. d Concentration of compounds exhibiting 90% inhibition in mycobacterial growth.
the length of the alkynyl side chain. The short chain
containing 5-ethynyl (1), 5-propynyl (2, 9, 18, 19),
5-pentynyl (3, 10, 15), and heptynyl (11,16) derivatives
were devoid of any antimycobacterial activity, with the
exception of 5-heptynyl-2′-deoxyuridine (4), which was
moderately active for M. bovis (60% at 100 and 50 µg/
mL) and M. avium (25% at 100 µg/mL) (Table 1).
5-Decynyl, 5-dodecynyl, and 5-tetradecynyl side chains
appear to provide optimal activity against both myco-
bacteria. It was observed that increasing the C10 (de-
cyne) side chain to C12 (dodecyne) and further to C14
(tetradecyne) provided significantly enhanced biological
activities (Table 1). The inhibition in multiplication of
M. bovis by the two most potent 2′-deoxyuridine ana-
logues 5-dodecynyl-2′-deoxyuridine (6) and 5-tetrade-
cynyl-2′-deoxyuridine (7) was 90% (100, 50 µg/mL) and
70% (10 µg/mL) for 6 and 90% (100, 50, 10 µg/mL) for
7. The 5-decynyl (5) and 5-dodecynyl (6) analogues of
2′-deoxyuridine displayed moderate activity, while the
corresponding 2′-deoxycytidine derivatives (12, 13) ex-
hibited higher activity against both mycobacteria. In-
terestingly, inhibition of M. bovis by 5-dodecynyl-2′-
deoxycytidine (13) was similar to 5-tetradecynyl-2′-
deoxyuridine (7), and activity against M. avium was
superior. These results suggest that a longer carbon
chain at the C-2 of the 5-position is determinant of the
antimycobacterial activity. In addition, an amino group
at C-4 appears to be preferred for improved activity.
Interestingly, inclusion of the phenyl ring at the C-2
carbon atom of the 5-substituent in the present com-
pounds was detrimental to antimycobacterial activity,
as exemplified by 5-(2-phenylethynyl) analogues (8, 14,
17) that were inactive. Modification of the 5-alkynyl
substituent in the novel pyrimidine nucleoside (1, 3-6)
to the corresponding cyclic analogues 3-(2′-deoxy-â-D-
ribofuranosyl)-6-octynyl-2,3-dihydrofuro[2,3-d]pyimidin-
2-one (20-24) provided compounds that were devoid of
antimycobacterial activity, suggesting that an acyclic
substituent at the C-5 position of the pyrimidine ring
or intact pyrimidine motif [NHC(O)] is essential for
potent antimycobacterial activity. It thus appears that
in order to confer a substantial antimycobacterial effect,
the 5-alkynyl side chain should be linear and exceed
seven carbons. Further, the respective 5-alkynyluracils
were devoid of antimycobacterial activity (data not
shown), suggesting that antimycobacterial activity also
depends on the glycosyl part of these molecules.
Comparison of the anti M. bovis activity with anti M.
avium activity revealed that 5-decynyl- (5, 12), 5-dode-
cynyl- (6, 13), or 5-tetradecynyl- (7) 2′-deoxy nucleosides
possessed higher inhibitory activity against M. bovis
than M. avium.
To examine whether the most promising compounds
6 and 7 are acting as promiscuous inhibitors18 or specific
inhibitors of mycobacteria, their activity was determined
in the presence of various detergents (Figure 1). Initially
the effect of three detergents (Tween-20, Triton X-100,
and SDS) on BCG viability and growth was determined
(data not shown) in the presence of concentrations of
detergents up to their critical micellar concentrations
(cmc).19 The concentrations of detergents that had no
effect on BCG viability and growth were then used in
the assay to determine the activity of 6 and 7 (Figure
1). It was interesting to note that the inhibition profiles
of the two selected compounds did not change in the
presence of various detergents, suggesting that they are
not acting as promiscuous inhibitors but rather have
specific antimycobacterial activity. Compound 6 showed
increased inhibition (90%) at 10 µg/mL in the presence
of all detergents used, compared to that in the absence
of detergents (70%). In fact, at the lowest concentration
of compounds 6 and 7 (i.e., 1.0 µg/mL) in the presence
of detergent Triton X-100, the antimycobacterial activity
was also slightly increased compared to that of inhibi-
tion in the absence of detergents (Figure 1B,C vs Figure
1A) probably because of the detergent causing a reduc-
tion in nonspecific binding of compounds to plates. These
observations are consistent with previous studies18
where inhibition of â-lactamase activity by inhibitors
was increased in the presence of detergents. These
results clearly indicated that compounds 6 and 7 are
not promiscuous inhibitors whose activity is dependent
on their aggregation in biological medium but are
specific inhibitors of mycobacterial growth and could be

Alkynylpyrimidine Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7015
Figure 1. Effect of detergents on antimycobacterial activity of compounds 6 and 7. Panel A shows the dose response of compounds
6 and 7 in the absence of detergent. The control bacterial well gave an average reading of 90 991. Panel B shows the activity of
compound 6, and panel C shows the activity of compound 7 in the presence of three different detergents. Both compounds were
tested at four different concentrations (depicted by individual lines), as shown in each graph.
considered as a starting point for the design of thera-
peutic agents designed to treat mycobacterial infections.
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 deciphered.20 It encodes many of
the enzymes required for DNA and RNA synthesis and
for pyrimidine and purine nucleoside biosynthesis. It
is possible that active nucleoside analogues after their
metabolic conversion to phosphorylated forms by my-
cobacterial kinases may be selectively inhibiting its
DNA and/or RNA synthesis by acting as substrates and/
or inhibitors of metabolic enzymes of DNA/RNA syn-
thesis.
The promising compounds (4-13) were tested in vitro
for their toxicity against monkey kidney (Vero cells),
HepG2 cells, and human foreskin fibroblast (HFF cells)
cell lines up to 100 µg/mL concentration, where they did
not display toxicity up to the highest concentrations
tested (CC50 g 100 µg/mL).
of agents merits further studies to explore them as
potential chemotherapeutic agents for tuberculosis.
Additional in vitro and in vivo tests as well as studies
of structure-activity relationships and mechanism of
action on this class of compounds are ongoing in our
laboratories. Identification of new lead compounds
working by different mechanisms of action is required
for the treatment of tuberculosis because of the emerg-
ing global health threat.
Experimental Section
Melting points were determined with a Buchi capillary
1
apparatus and are uncorrected. H NMR spectra were deter-
mined for solutions in Me2SO-d6 on a Bruker AM 300 spec-
trometer using Me4Si 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 Whatman
MK6F silica gel microslides (25 µM thickness). 5-Iodo-2′-
deoxyuridine (25), 5-iodo-2′-deoxycytidine (26), and 5-iodou-
ridine (27) were purchased from Sigma-Aldrich Chemical Co.
5-Propynyl-2′-deoxycytidine (9), 5-propynyl-2′-O-methyluridine
(18), and 5-propynyl-2′-O-methylcytidine (19) were purchased
from Berry and Associates.
Summary
In conclusion, we present design, synthesis, and in
vitro biological evaluation of 5-alkynylpyrimidine nu-
cleosides that emerge as potent antimycobacterial agents.
2′-Deoxyuridine and 2′-deoxycytidine analogues pos-
sessing 5-decynyl, 5-dodecynyl, and 5-tetradecynyl sub-
stituents were found to be strong inhibitors of M. bovis
multiplication in vitro. It is noteworthy that this series
of agents is also active against M. avium. This new class
Preparation of 5-Alkynylpyrimidine Nucleosides. A
full procedure is provided for 5-pentynyl-2′-deoxycytidine (10)
and 5-pentynyluridine (15). For other analogues, only brief
spectroscopic data are presented.
5-Pentynyl-2′-deoxycytidine (10). Tetrakis(triphenylphos-
phine)palladium(0) (98 mg, 0.084 mmol), copper(I) iodide (32

7016 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22
Rai et al.
mg, 0.169 mmol), diisopropylethylamine (0.30 mL, 1.69 mmol),
and 1-pentyne (0.35 mL, 3.54 mmol) were added to a solution
of 5-iodo-2′-deoxycytidine (26) (300 mg, 0.85 mmol) in anhy-
drous dimethylformamide (25 mL). The orange reaction mix-
ture was stirred at room temperature for 4 h in a nitrogen
atmosphere; the progress of the reaction was monitored by
TLC in MeOH/CHCl3 (1:9, v/v). After the mixture was stirred
for 4 h, 15 drops of 5% of disodium salt of EDTA/H2O were
added to the reaction mixture and the contents were concen-
trated in vacuo. The resulting residue was purified on silica
gel column using CHCl3/MeOH (9:1, v/v) as eluent to yield 10
the progress of the reaction was monitored by TLC in MeOH/
CHCl3 (1:9, v/v). After the mixture was stirred overnight, 15
drops of 5% of disodium salt of EDTA/H2O was added to the
reaction mixture, and then the mixture was concentrated in
vacuo. The residue obtained was purified on silica gel column
using CHCl3/MeOH (9:1, v/v) as eluent to yield 15 (120 mg,
48%) as a syrup. 1H NMR (DMSO-d6) δ 11.60 (s, 1H, NH), 8.20
(s, 1H, H-6), 5.78 (d, J ) 5.2 Hz, 1H, H-1′), 5.40, 5.20 and 5.10
(m, 1H each, 5′-OH, 3′-OH, 2′-OH), 4.10-3.84 (m, 3H, H-2′,
H-3′, H-4′), 3.70-3.50 (m, 2H, H-5′), 2.35 (t, J ) 7.0 Hz, 2H,
R-CH2), 1.52 (m, 2H, â-CH2), 0.98 (t, J ) 7.3 Hz, 3H, CH3); 13
C
1
(185 mg, 74%) as a syrup. H NMR (DMSO-d6) δ 8.07 (s, 1H,
NMR (DMSO-d6) δ 13.27 (CH3), 20.71 (CH2), 21.59 (R-CH2),
60.39 (C-5′), 69.49 (C-2′), 72.82 (C-â), 73.68 (C-3′), 84.78 (C-
4′), 87.99 (C-1′), 93.03 (C-R), 99.03 (C-5), 142.66 (C-6), 149.61
(C-2), 161.55 (C-4). Anal. (C14H18N2O6) C, H, N.
H-6), 7.67 (s, 1H, NH), 6.72 (s, 1H, NH), 6.12 (t, J ) 6.5 Hz,
1H, H-1′), 5.22 (d, J ) 4.3 Hz, 1H, 3′-OH), 5.08 (t, J ) 5.0 Hz,
1H, 5′-OH), 4.20 (m, 1H, H-3′), 3.78 (m, 1H, H-4′), 3.50-3.68
(m, 2H, H-5′), 2.37 (t, J ) 7.0 Hz, 2H, R-CH2), 2.13 and 1.98
(2m, 2H, H-2′), 1.54 (m, 2H, â-CH2), 0.95 (t, J ) 7.3 Hz, 3H,
CH3); 13C NMR (DMSO-d6) δ 13.41 (CH3), 20.96 (CH2), 21.47
(R-CH2), 40.69 (C-2′), 60.97 (C-5′), 70.06 (C-3′), 72.08 (C-â),
85.17 (C-4′), 87.31 (C-1′), 90.32 (C-R), 95.40 (C-5), 143.35 (C-
6), 153.36 (C-2), 164.23 (C-4). Anal. (C14H19N3O4) C, H, N.
5-Heptynyl-2′-deoxycytidine (11). Yield 96%; 1H NMR
(DMSO-d6) δ 8.05 (s, 1H, H-6), 7.65 (s, 1H, NH), 6.68 (s, 1H,
NH), 6.11 (t, J ) 6.6 Hz, 1H, H-1′), 5.20 (d, J ) 4.0 Hz, 1H,
3′-OH), 5.04 (t, J ) 5.0 Hz, 1H, 5′-OH), 4.20 (m, 1H, H-3′),
3.72 (m, 1H, H-4′), 3.68-3.50 (m, 2H, H-5′), 2.40 (t, J ) 7.1
Hz, 2H, R-CH2), 2.15 and 1.97 (2m, 2H, H-2′), 1.54 (m, 2H,
â-CH2), 1.34 (m, 4H, 2 × CH2), 0.87 (t, J ) 7.0 Hz, 3H, CH3);
13C NMR (DMSO-d6) δ 13.82 (CH3), 19.0, 21.62, 27.73 (3 ×
CH2), 30.60 (R-CH2), 40.69 (C-2′), 61.01 (C-5′), 70.11 (C-3′),
71.90 (C-â), 85.20 (C-4′), 87.34 (C-1′), 90.37 (C-R), 95.62 (C-5),
143.32 (C-6), 153.38 (C-2), 164.25 (C-4). Anal. (C16H23N3O4) C,
H, N.
5-Decynyl-2′-deoxycytidine (12). Yield 82%; 1H NMR
(DMSO-d6) δ 8.05 (s, 1H, H-6), 7.68 (s, 1H, NH), 6.68 (s, 1H,
NH), 6.10 (t, J ) 6.6 Hz, 1H, H-1′), 5.20 (d, J ) 4.4 Hz, 1H,
3′-OH), 5.04 (t, J ) 4.9 Hz, 1H, 5′-OH), 4.20 (m, 1H, H-3′),
3.78 (m, 1H, H-4′), 3.65-3.50 (m, 2H, H-5′), 2.38 (t, J ) 7.2
Hz, 2H, R-CH2), 2.12 and 1.96 (2m, 2H, H-2′), 1.54 (m, 2H,
â-CH2), 1.30 (m, 10H, 5 × CH2), 0.85 (m, 3H, CH3); 13C NMR
(DMSO-d6) δ 13.89 (CH3), 19.01, 22.02, 28.04, 28.40, 28.47,
28.53 (6 × CH2), 31.20 (R-CH2), 40.69 (C-2′), 61.01 (C-5′), 70.11
(C-3′), 71.90 (C-â), 85.18 (C-4′), 87.34 (C-1′), 90.35 (C-R), 95.61
(C-5), 143.26 (C-6), 153.35 (C-2), 164.23 (C-4). Anal. (C19H29N3O4)
C, H, N.
5-Dodecynyl-2′-deoxycytidine (13). Yield 80.5%; 1H NMR
(DMSO-d6) δ 8.05 (s, 1H, H-6), 7.67 (s, 1H, NH), 6.70 (s, 1H,
NH), 6.12 (t, J ) 6.6 Hz, 1H, H-1′), 5.22 (d, J ) 4.0 Hz, 1H,
3′-OH), 5.05 (t, J ) 5.0 Hz, 1H, 5′-OH), 4.20 (m, 1H, H-3′),
3.79 (m, 1H, H-4′), 3.68-3.52 (m, 2H, H-5′), 2.38 (t, J ) 7.1
Hz, 2H, R-CH2), 2.12 and 1.98 (2m, 2H, H-2′), 1.54 (m, 2H,
â-CH2), 1.28 (m, 14H, 7 × CH2), 0.86 (m, 3H, CH3); 13C NMR
(DMSO-d6) δ 13.89 (CH3), 19.0, 22.02, 28.01, 28.36, 28.47,
28.61, 28.85, 28.90 (8 × CH2), 31.21 (R-CH2), 40.66 (C-2′), 61.00
(C-5′), 70.10 (C-3′), 71.89 (C-â), 85.17 (C-4′), 87.31 (C-1′), 90.30
(C-R), 95.61 (C-5), 143.26 (C-6), 153.29 (C-2), 164.21 (C-4).
Anal. (C21H33N3O4) C, H, N.
1
5-Heptynyluridine (16). Yield 47%; H NMR (DMSO-d6)
δ 11.58 (s, 1H, NH), 8.18 (s, 1H, H-6), 5.76 (d, J ) 4.9 Hz, 1H,
H-1′), 5.40, 5.17, 5.05 (m, 1H each, 5′-OH, 3′-OH, 2′-OH), 4.20-
3.80 (m, 3H, H-2′, H-3′, H-4′), 3.70-3.50 (m, 2H, H-5′), 2.35
(t, J ) 6.9 Hz, 2H, R-CH2), 1.52 (m, 2H, â-CH2), 1.48 (m, 4H,
2 × CH2), 0.9 (t, J ) 6.9 Hz, 3H, CH3); 13C NMR (DMSO-d6) δ
13.77 (CH3), 18.72, 21.56, 27.80 (3 × CH2), 30.40 (R-CH2), 60.43
(C-5′), 69.50 (C-2′), 72.62 (C-â), 73.65 (C-3′), 84.79 (C-4′), 87.99
(C-1′), 93.19 (C-R), 99.04 (C-5), 142.60 (C-6), 149.59 (C-2),
161.52 (C-4). Anal. (C16H22N2O6) C, H, N.
1
5-(2-Phenylethynyl)uridine (17). Yield 36.5%; H NMR
(DMSO-d6) δ 11.72 (s, 1H, NH), 8.50 (s, 1H, H-6), 7.60-7.38
(m, 5H, aromatic), 5.76 (d, J ) 4.6 Hz, 1H, H-1′), 5.48, 5.30,
5.12 (m, 1H each, 5′-OH, 3′-OH, 2′-OH), 4.12-3.98 (m, 3H,
H-2′, H-3′, H-4′), 3.80-3.58 (m, 2H, H-5′); 13C NMR (DMSO-
d6) δ 60.11 (C-5′), 69.17 (C-2′), 73.86 (C-3′), 82.29 (C-â), 84.63
(C-4′), 88.40 (C-1′), 91.65 (C-R), 98.08 (C-5), 122.27, 128.38,
128.48, 128.57, 131.00 (C-phenyl), 143.90 (C-6), 149.51 (C-2),
161.18 (C-4) Anal. (C17H16N2O6) C, H, N.
In Vitro Antimycobacterial Activity Assay (M. bovis,
M. avium). M. bovis (BCG) and M. avium (ATCC 25291) were
obtained from the American Type Culture Collection, Rock-
ville, MD. The antimycobacterial activity was determined
using the microplate alamar blue assay (MABA).17 Test
compounds were dissolved in DMSO at 100× of 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, 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 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 10× 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
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 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 spectro-
photometer. Visual MIC was defined as the lowest concentra-
tion 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 both M. bovis BCG and M. avium.
Rifampicin and clarithromycin were used as positive controls.
As negative controls, DMSO was added to the B well at a
concentration 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 of 60000-100000.
5-(2-Phenylethynyl)-2′-deoxycytidine (14). Yield 86%;
1H NMR (DMSO-d6) δ 8.32 (s, 1H, H-6), 7.80 (s, 1H, NH), 7.38-
7.62 (m, 5H, aromatic), 7.05 (s, 1H, NH), 6.12 (t, J ) 6.4 Hz,
1H, H-1′), 5.24 (d, J ) 4.3 Hz, 1H, 3′-OH), 5.14 (t, J ) 4.9 Hz,
1H, 5′-OH), 4.22 (m, 1H, H-3′), 3.80 (m, 1H, H-4′), 3.72-3.53
(m, 2H, H-5′), 2.20 and 2.05 (2m, 2H, H-2′); 13C NMR (DMSO-
d6) δ 40.81 (C-2′), 60.85 (C-5′), 69.87 (C-3′), 81.56 (C-â), 85.37
(C-4′), 87.37 (C-1′), 89.43 (C-R), 93.64 (C-5), 122.38, 128.31,
131.13 (C-phenyl), 144.76 (C-6), 153.26 (C-2), 164.64 (C-4).
Anal. (C17H17N3O4) C, H, N.
5-Pentynyluridine (15). To a solution of 5-iodoridine (27)
(300 mg, 0.810 mmol) in anhydrous dimethylformamide (25
mL) were added tetrakis(triphenylphosphine)palladium(0) (94
mg, 0.081 mmol), copper(I) iodide (31 mg, 0.162 mmol),
diisopropylethylamine (0.3 mL, 1.69 mmol), and 1-pentyne
(0.24 mL, 2.43 mmol). The reaction mixture was stirred at
room temperature for overnight under nitrogen atmosphere;

Alkynylpyrimidine Nucleosides
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 22 7017
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The antimycobacterial activity of compounds 6 and 7 was
also determined against BCG in the presence of various
detergents using MABA assay. For these experiments titrating
amounts of detergents, i.e., Tween-20, Triton X-100, and SDS
(Fisher Scientific, critical micellar concentrations (cmc): 0.05,
0.3, and 8.2 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 µg/mL (final concentration) for 15 min
before adding to the BCG wells in triplicate. The data are
shown as the average of the triplicates, and the standard
deviation was within 5%. Control wells included BCG cultured
with various amounts of all three detergents without com-
pounds. In the initial experiment, we determined the effect of
various concentrations of the three detergents on BCG growth.
Tween-20 had no effect on BCG growth at all four concentra-
tions, whereas Triton X-100 was toxic at 1 cmc, and SDS was
toxic to BCG at 1 and 0.1 cmc. Therefore, the compounds were
tested in the presence of nontoxic doses of detergents.
Cell Cytotoxicity Assay. Cell viability was measured
using the cell proliferation kit 1 (MTT; Boehringer Mannheim),
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
media was replaced with media containing drugs at concentra-
tions of 100, 50, 25, 12.5, 6.3, and 1.5 µg/mL. DMSO was also
included as control. Plates were incubated for 3 days at 37
°C. The color reaction involved adding 10 µL of 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 (absorption at 560-650 nm) following an overnight
incubation at 37 °C.
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Technology MIC Determination with Clinical Mycobacterium
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460 System for High-Throughput Screening of Compounds
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Acknowledgment. We are grateful to the Canadian
Institutes of Health Research (CIHR) for an Operating
Grant MOP-49415 (R.K. and D.Y.K.) for the financial
support of this research. B.A. is grateful to the Alberta
Heritage Foundation for Medical Research (AHFMR) for
a Medical Scholar Award.
Supporting Information Available: Results from el-
emental analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
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