Antimycobacterial activities of 5-alkyl (or halo)-3′-substituted pyrimidine nucleoside analogs

Article abstract of DOI:10.1016/j.bmcl.2011.11.114

Several 5-alkyl (or halo)-3′-azido (amino or halo) analogs of pyrimidine nucleosides have been synthesized and evaluated against Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium. Among these compounds, 3′-azido-5-ethyl-2′,3′-dideoxyuridine (3) was found to have significant antimycobacterial activities against M. bovis (MIC ₅₀ = 1 μg/mL), M. tuberculosis (MIC₅₀ = 10 μg/mL) and M. avium (MIC₅₀ = 10 μg/mL).

Full text of DOI:10.1016/j.bmcl.2011.11.114

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1091–1094
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimycobacterial activities of 5-alkyl (or halo)-30-substituted pyrimidine
nucleoside analogs
Naveen C. Srivastav a, Neeraj Shakya a, Sudha Bhavanam a, Amogh Agrawal a, Chris Tse a,
Nancy Desroches a, Dennis Y. Kunimoto b, Rakesh Kumar a,
⇑
a Department of Laboratory Medicine and Pathology, 1-71 Medical Sciences Building, Edmonton, AB, Canada T6G 2H7
b Department of Medicine, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2H7
a r t i c l e i n f o
a b s t r a c t
Several 5-alkyl (or halo)-30-azido (amino or halo) analogs of pyrimidine nucleosides have been synthe-
sized and evaluated against Mycobacterium bovis, Mycobacterium tuberculosis and Mycobacterium avium.
Among these compounds, 30-azido-5-ethyl-20,30-dideoxyuridine (3) was found to have significant antimy-
Article history:
Received 12 October 2011
Revised 22 November 2011
Accepted 28 November 2011
Available online 4 December 2011
cobacterial activities against M. bovis (MIC50 = 1
(MIC50 = 10 g/mL).
lg/mL), M. tuberculosis (MIC50 = 10
lg/mL) and M. avium
l
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Tuberculosis
Pyrimidine nucleosides
Tuberculosis (TB), a contagious disease that spreads through
aerosols, has reached to pandemic proportions, and is the second
leading cause of infectious deaths globally. Approx. 9 million peo-
ple developed TB and 1.7 millions died from this disease in the year
2009 alone.1 Although TB is considered as a single disease, it can be
caused by several mycobacterial species.2 Mycobacterium bovis and
Mycobacterium tuberculosis (Mtb) pose a significant challenge to the
clinical management of TB in immunocompetent as well as immu-
nocompromised people such as HIV infected patients.3 Infection
with HIV suppresses the host’s immune system, rendering them
more susceptible to TB infection, and/or allowing the reactivation
of latent TB infection.4 Co-infection of TB and HIV allows faster
progress and worsening of both diseases.5 Although HIV is consid-
ered to be the single most mortality factor for TB, anyone can get
infected with TB and some people are at higher risk such as aborig-
inals, the elderly, the homeless, the health care professionals, and
people living in unhygienic and crowded places.6
Bacillus Calmette Guerin (BCG) is an attenuated strain of
M. bovis that is more than >93% homologous to Mtb.7 In humans,
M. bovis infections have been reported from 4000–5000 BC. Unfor-
tunately, M. bovis infections have re-emerged and are causing TB in
humans particularly in HIV positive people.8 In addition, multi-
drug resistant (MDR) strains of M. bovis that are resistant to up
to 11 drugs have been isolated.9 M. avium infections are also one
of the most serious complications among patients with AIDS.10,11
Infections caused by M. avium are disseminated rather than
restricted to lungs. The first-line anti-TB drugs alone are ineffective
against this mycobacteria. The American Thoracic Society (ATS)
recommends
a three-drug regimen including clarithromycin
(CAM) or azithromycin (AZM), rifampin (RFP) or rifabutin (RBT),
and ethambutol (EB) for pulmonary MAC disease.12 Resistance to
the macrolides occur at such a rate that single drugs are inade-
quate.13 Growing resistance to available TB drugs has emerged as
a new threat to the world with new untreatable strains of M. tuber-
culosis, XDR-TB (Extensively drug-resistant TB).14 Drug resistance
in TB arises from spontaneous mutations in mycobacterial DNA,
and reversal to drug-sensitivity is not observed.15 Clinical drug-
resistance usually occurs due to insufficient treatment and poor
patient adherence of the otherwise treatable TB. The MDR/XDR
TB is caused by sequential accumulation of mutations in different
genes involved in individual drug-resistance.15 Subsequent trans-
mission of drug-resistant TB to other people exponentially aggra-
vates the problem. Therefore, new drug development is of
paramount importance to respond to this medical emergency,
especially the one’s targeted to distinct bacterial pathways.
In earlier studies, we noted that a 20-fluoro derivative of
thymidine displays appreciable inhibition of M. bovis (MIC50 = 50
l
g/mL).16 In our next studies, we observed that a 30-bromo analog
of thymidine, 30-bromo-20,30-dideoxythymidine was a better inhib-
itor of Mtb (H37Ra) (MIC50 = 5–10
l
g/mL).17 During our recent
work, we found that an ethyl group at the C-5 position of the uracil
base also contribute to the antimycobacterial activity where a
30-bromo derivative of 5-ethyl pyrimidine nucleoside provided
very good activity against M. bovis and Mtb (MIC50 = 5
However, none of these compounds were inhibitory against
lg/mL).
⇑
Corresponding author. Tel.: +1 780 492 7545; fax: +1 780 492 7521.
E-mail address: rakesh.kumar@ualberta.ca (R. Kumar).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.114

1092
N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1091–1094
M. avium.18 In contrast, we observed that 5-ethyl-20-deoxyuridine
was devoid of antimycobacterial activity.16
In this article, we have explored various 5-methyl (ethyl or halo)-
substituted pyrimidine nucleosides with a variety of 30-substituents
to determine their effect on antimycobacterial activity. Among the
compounds investigated, 5-ethyl analog (3) of 30-azido-20,30-dide-
O
O
N
R
HN
HN
(i)
O
O
N
O
O
HO
HO
5
6
, R = Cl
, R = Br
oxyuridine displayed significant inhibition of M. bovis (MIC50
=
N3
N3
4
1
l
g/mL), Mtb (MIC50 = 10 g/mL) and M. avium (MIC50 = 10 g/
l
l
mL) which was equiactive to cycloserine against M. bovis and Mtb,
and more active against M. avium than cycloserine.
Scheme 2. Reagents and conditions: (i) N-chlorosuccinimide, NaN3, DME–water,
room temperature to 45 °C, 70% (for 5); N-bromosuccinimide, dry DMF, room
temperature, 76% (for 6).
The nucleoside, 30-azido-5-ethyl-20,30-dideoxyuridine (3) was
synthesized using a one step process as compared to multi-step
synthesis reported by Herdewijn et al.19 Thus, the reaction of 5-
ethyl-20-deoxyuridine (1) with triphenylphosphine and diisopropyl
azodicarboxylate in dry CH3CN, followed by treatment of the ob-
tained 2,30-anhydro derivative 2 with NaN3 in dry DMF at 125–
130 °C gave the compound 3 in 40% yield (Scheme 1). Chlorination
of 30-azido-20,30-dideoxyuridine (4) using an efficient procedure
with N-chlorosuccinimide and NaN3 in DME-water provided 30-azi-
do-5-chloro-20,30-dideoxyuridine (5) in 70% yield in contrast to
only 45% yield reported by Aerschot et al.20 30-Azido-5-bromo-
20,30-dideoxyuridine (6) was also obtained in a superior yield
(76% Scheme 2) using one step method by N-bromosuccinimide
in dry DMF as compared to the procedure reported earlier.21 Lin
et al22 reported the synthesis of 30-amino-20,30-dideoxy-5-methy-
luridine (8) by hydrogenation of 30-azido-30-deoxythymidine (7)
followed by purification of the product using anion-exchange col-
umn. We have synthesized 8 by a modified method by the reaction
of 7 with triphenylphosphine in dry pyridine followed by the addi-
tion of NH4OH at room temperature. Trituration of the obtained
product with ethyl acetate gave pure 8 in 58% yield (Scheme 3).
The target nucleosides 30-iodo-20,30-dideoxy-5-ethyluridine
O
O
N
CH3
CH3
HN
HN
(i)
O
O
N
O
O
HO
HO
7
8
H2N
N3
Scheme 3. Reagents and conditions: (i) (a) Triphenylphosphine, dry pyridine, room
temperature; (b) NH4OH, room temperature, 58% (for 8).
17 and 18, after detritylation with 80% aqueous AcOH provided
30-fluoro-20,30-dideoxypyrimidine nucleosides 1924 and 20 in 46%
and 56% yields, respectively. 30-azido-5-fluoro-20,30-dideoxyuridine
(21) was synthesized using a reported method25
The compounds 3, 5, 6, 8, 12, 13, 16, 19–21, along with refer-
ence drugs, cycloserine, rifampicin and clarithromycin were tested
against three mycobacteria M. bovis (BCG), M. tuberculosis (H37Ra)
and M. avium using microplate alamar blue assay (MABA).26 The
results are summarized in Table 1. Of the compounds tested,
30-azido-5-ethyl-20,30-dideoxyuridine (3) was the most active
agent against BCG (MIC50 = 1
against BCG was found to be similar to that of cycloserine
(MIC50 = 1 g/mL), a second-line anti-TB drug. Encouragingly, Mtb
strain H37Ra was also inhibited by 3 (MIC50 = 10 g/mL) at 10-
times higher concentration than M. bovis. Similar pattern of activ-
ity against BCG (50% inhibition at 1 g/mL, MIC50 = 1 g/mL) and
Mtb H37Ra (60% inhibition at 10 g/mL, MIC50 = 5 g/mL) was also
exhibited by cycloserine. Further, it was interesting to note that 3
possessed activity against M. avium (MIC50 = 10 g/mL) also.
(12), 20,30-dideoxy-5-ethyluridine (13), 1-(2-deoxy-b-
D-lyxofur-
anosyl)-5-ethyluracil (16), 30-fluoro-20,30-dideoxy-5-trifluorome-
thyluridine (19) and 30-fluoro-20,30-dideoxy-5-ethyluridine (20)
were synthesized as described in Scheme 4. 50-O-Tritylation
followed by 30-O-mesylation of the trifluorothymidine (9) and
5-ethyl-20-deoxyuridine (1) yielded respective 50-O-trityl-30-O-me-
syl derivatives (10 and 11) in 79% and 85% yields, respectively.
Treatment of 11 with NaI in dry DME followed by detritylation pro-
vided 12 in 65% yield. Subsequent hydrogenation of 12 gave deio-
dinated product 13 in 85% yield. The compounds 12 and 13 were
synthesized using reported methods, however, during our reac-
tions, we obtained improved yields of 12 and 13 than reported ear-
lier.23 Basic hydrolysis of compounds 10 and 11 with NaOH in 90%
lg/mL). The MIC50 exhibited by 3
l
l
l
l
l
l
l
Although 3 was significantly less active than the reference drug
clarithromycin against M. avium, it was more potent than cycloser-
aqueous ethanol under reflux afforded 1-(5-O-trityl-2-deoxy-b-
D
-
ine (15% inhibition at 10 l
g/mL). It is noteworthy that the 30-azido
lyxofuranosyl)pyrimidines 14 and 15 in 19% and 97% yields,
respectively. Detritylation of 15 with 80% aqueous AcOH at 90 °C
yielded 16 in 72% yield. In order to prepare 30-fluoro derivatives
19 and 20, the compounds 14 and 15 were reacted with (diethyl-
amino)sulfur trifluoride (DAST) in dry THF/dry benzene at room
temperature to give 50-O-trityl-30-fluoro nucleoside derivatives
17 and 18 in 70% and 80% yields, respectively. The compounds
derivative of thymidine (7) did not provide inhibition of any myco-
bacteria in our earlier studies,16 suggesting that ethyl moiety at the
C-5 position in compound 3 contributes to the antimycobacterial
activity. Replacement of an azido group at the 30-position in com-
pound 7 by an amino substituent (8) was found to contribute to
modest activity against M. bovis. In contrast to 3, the other 30-azido
nucleosides, 30-azido-5-chloro-20,30-dideoxyuridine (5) and 30-azi-
do-5-bromo-20,30-dideoxyuridine (6) demonstrated lower inhibi-
tion of M. bovis (50% at 100
inhibition, respectively at 100
l
l
g/mL) and Mtb (50% and 30%
g/mL). Whereas, 30-azido-5-flu-
O
O
N
O
oro-20,30-dideoxyuridine (21) was inactive against all of the myco-
bacteria tested. These results further indicate that 5-ethyl
substituent plays an important role for antimycobacterial activity
in this series of compounds. Among other 5-ethyl analogs investi-
gated, only 30-fluoro derivative (20) showed some inhibition of M.
C2H5
(i)
C2H5
(ii)
C2H5
N
O
HN
HN
O
N
O
O
N
O
O
HO
HO
HO
bovis only at higher concentration (40% inhibition at 100 lg/mL). In
1
2
3
HO
N3
contrast, 30-iodo derivative viz. 30-iodo-20,30-dideoxy-5-ethyluri-
dine (12) was devoid of activity which was not surprising since
30-iodo-20,30-dideoxythymidine had not shown activity in our ear-
lier studies.17 There was no antimycobacterial activity of 30-deoxy
Scheme 1. Reagents and conditions: (i) triphenylphosphine, diisopropyl azodicar-
boxylate, dry CH3CN, ꢀ15 °C to 0 °C, 4 h, 45% (for 2); (ii) NaN3, dry DMF, 125–
130 °C, 5 h, 40% (for 3).

N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1091–1094
1093
O
O
N
O
N
O
C2H5
C2H5
R
R
HN
HN
HN
HN
(i)
O
N
O
O
O
N
O
O
O
O
HO
HO
HO
TrO
12
13
10, R = CF3
11, R = C2H5
9, R = CF3
1, R = C2H5
I
HO
MsO
(ii)
O
O
O
N
O
R
R
R
F
HN
HN
HN
HN
(iii)
(iv)
O
N
O
N
O
O
N
O
OH
O
O
O
R1
TrO
HO
HO
F
14, R = CF3, R1 = OTr
F
N3
17, R = CF3
18, R = C2H5
19, R = CF3
21
15, R = C2H5, R1 = OTr
16, R = C2H5, R1 = OH
(iv)
20, R = C2H5
Scheme 4. Reagents and conditions: (i) Trityl chloride, 4-(dimethylamino)pyridine, dry pyridine, 80 °C, 5–8 h; mesyl chloride, 0 °C, overnight, 79% (for 10), 85% (for 11); (ii)
NaOH, 90% aq. EtOH, reflux, 1.5–2 h, 19% (for 14), 97% (for 15); (iii) DAST, dry benzene, dry THF, room temperature, 2–3 h, 70% (for 17), 80% (for 18) (iv) 80% aqueous AcOH,
90 °C, 0.5 h, 65% (for 12), 72% (for 16), 46% (for 19), 56% (for 20).
Table 1
In vitro antimycobacterial activity of 5-substituted pyrimidine nucleosides against M. bovis, M. tuberculosis and M. avium
O
H3C
CH3
X
N
OCH3
H3C
N
HO
CH3
CH3
OH
H C
CH3
R
O
N
H3C
HO
H
HN
HO
O3
O
C2H5
C2H5
O
O
OH
O
HN
OH OH
O
O
O
NH
O
H2N
O
O
O
OCH3
CH3
OH
O
HO
N
CH3
O
O
N
NH
O
O
OH
O
N
HO
OH
R1
O
CH3
3, 5, 6, 8,
12,13,19-21
16
Cycloserine
Rifampicin
Clarithromycin
Compd
3
X
O
R
R1
Antimycobacterial activitya,% inhibition (concentration
l
g/mL)
M. avium (ATCC 25291)
100% @ 100 g/mL, 60% @ 50
g/mL
M. bovis (BCG)
M. tuberculosis (H37Ra)
C2H5 N3
95% @ 100 and 50
l
g/mL, 85% @ 10
lg/ 60% @ 100 and 50
lg/mL, 50% @ 10
l
g/mL
l
lg/
mL, 50% @ 1
lg/mL
mL, 50% @ 10 l
5
6
8
12
13
16
19
20
O
O
O
O
O
—
O
O
O
—
Cl
Br
N3
N3
50% @ 100
50% @ 100
l
l
l
g/mL
g/mL
g/mL
50% @ 100
30% @ 100
0
0
0
0
0
0
l
l
g/mL
g/mL
0
0
0
0
0
0
CH3
C2H5
C2H5
—
CF3
C2H5
F
NH2 50% @ 100
I
H
—
F
F
N3
—
0
0
0
0
25% @ 100 lg/mL
0
0
100% @ 100 and 50
10 g/mL
90% @ 2
100% @ 2
40% @ 100
0
lg/mL
21
0
Cycloserine
—
100% @ 100, 50 and 10
g/mL
lg/mL, 50% @
100% @ 50
lg/mL, 80% @ 15–20
lg/mL, 60% @
lg/mL, 15% @
1
l
10
lg/mL, 50% @ 5
l
g/mL
l
Rifampicin
Clarithromycin
—
—
—
—
—
—
100% @ 0.5
l
g/mL
100% @ 0.5–1
ND
lg/mL
l
g/mL
NDb
lg/mL
a
Antimycobacterial activity was determined at concentrations 100, 50, 25, 10, 5, 1 and 0.5
ND = not determined.
lg/mL.
b
derivative, 20,30-dideoxy-5-ethyluridine (13), and the 30-lyxo-hy-
droxy analog 1-(2-deoxy-b- -lyxofuranosyl)-5-ethyluracil (16) of
The XTT and 3H-thymidine incorporation assays were per-
formed to evaluate the toxicity of investigated compounds (3, 5,
6, 8, 12, 13, 16, 19–21) in vitro against a human hepatoma cell line
(Huh-7). These compounds were not toxic up to the highest con-
D
3. These results suggest that both ethyl and azido substituents at
the C-5 position of the uracil base and at the 30-position of the su-
gar moiety, respectively, are important determinants of anti-TB
activity. The 5-trifluoromethyl analog (19) of 30-fluorothymidine
showed no antimycobacterial activity in contrast to its 5-ethyl ana-
log 20, further supporting the role of 5-ethyl group in the antimy-
cobacterial activity.
centration tested, 100 lg/mL (CC50 >100 lg/mL).
In conclusion, our structure activity relationship studies indi-
cate that both 5-ethyl and 30-azido substituents together influence
the antimycobacterial activity of pyrimidine nucleosides. The 30-
azido-5-ethyl-20,30-dideoxyuridine (3) that emerged as the most

1094
N. C. Srivastav et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1091–1094
2. Addo, K. K.; Owusu-Darko, K.; Yeboah-Manu, D.; Caulley, P.; Minamikawa, M.;
Bonsu, F.; Leinhardt, C.; Akpedonu, P.; Ofori-Adjei, D. Ghana Med. J. 2007, 41, 52.
3. (a) Wells, C. D. Curr. Infect. Dis. Rep. 2010, 12, 192; (b) Ruíz, P. R.; Esteban, J.;
Guerrero, M. L. F. Clin. Infect. Dis. 2002, 35, 212.
4. Rosas-Taraco, A. G.; Arce-Mendoza, A. Y.; Caballero-Olín, G.; Salinas-Carmona,
M. C. AIDS Res. Human Retroviruses 2006, 22, 45.
effective and selective inhibitor, was equiactive to cycloserine
against M. bovis and Mtb H37Ra, and was more potent than cyclo-
serine against M. avium. Although compound 3 was significantly
less active than a first-line anti-TB drug rifampicin, it could serve
as a useful lead compound to improve our understanding of TB
drug-design, and develop and optimize therapeutic regimens for
TB infections. It is postulated that 3 after metabolic conversion to
phosphorylated forms by mycobacterial kinases may be selectively
inhibiting Mtb DNA and/or RNA synthesis by acting as substrate
and/or inhibitor of metabolic enzymes of DNA/RNA synthesis.
In vitro antimycobacterial activity assay: M. bovis (BCG), M. tuber-
culosis (H37Ra), and M. avium (ATCC 25291) were obtained from
the American Type Culture Collection, Rockville, MD. The antimy-
cobacterial activity was determined using the Microplate alamar
blue assay (MABA).17,26 Test compounds were dissolved in DMSO
at 10 mg/mL and subsequent dilutions were performed in 7H9GC
(Difco Laboratories, Detroit, Michigan) medium in 96 well plates.
For these experiments, each compound was tested at 100, 50, 25,
5. Mariani, F.; Goletti, D.; Ciaramella, A.; Martino, A.; Colizzi, V.; Fraziano, M. Curr.
Mol. Med. 2001, 1, 209.
6. Tuberculosis Infection Latent Tuberculosis Infection, Fact Sheet 2008,
Communicable Disease Control Department, Haliburton, Kawartha, Pine
Ridge District Health Unit, Ontario, Canada. Accessed on 23rd September
2011. Available via website www.hkpr.on.ca/uploadedFiles/TBdisease.pdf
7. Imaeda, T. Int. J. Syst. Bacteriol. 1985, 35, 147.
8. Koehler, C. S. W. Modern Drug Dis. 2002, 5, 47.
9. Guerrero, A.; Cobo, J.; Fortün, J.; Navas, E.; Quereda, C.; Asensio, A.; Cañón, J.;
Blazquez, J.; Gómez-Mampaso, E. Lancet 1997, 350, 1738.
10. Inderlied, C. B.; Kemper, C. A.; Bermudez, L. E. Clin. Microbiol. Rev. 1993, 6, 266.
11. (a) Falkinham, J. O., III Clin. Microbiol. Rev. 1996, 9, 177; (b) Mdluli, K.; Swanson,
J.; Fischer, E.; Lee, R. E.; Barry, C. E., III Mol. Microbiol. 2002, 27, 1223.
12. Griffith, D. E.; Aksamit, T.; Brown-Elliott, B. A.; Catanzaro, A.; Daley, C.; Gordin,
F.; Holland, S. M.; Horsburgh, R.; Huitt, G.; Iademarco, M. F.; Iseman, M.;
Olivier, K.; Ruoss, S.; von Reyn, C. F.; Wallace, R. J., Jr.; Winthrop, K. Am. J. Respir.
Crit. Care Med. 2007, 175, 367.
13. Heifets, L.; Mor, N.; Vanderkolk, J. Antimicrob. Agents Chemother. 1993, 37, 2364.
14. (a) XDR-TB-a Global Threat. Editorial. Lancet 2006, 368, 964.; (b) Wright, A.
Morbidity and Mortality Weekly Report (MMWR) 2006, 55, 301.
15. Zhang, Y.; Yew, W. W. Int. J. Tubercul. Lung Dis. 2009, 13, 1320.
16. Johar, M.; Manning, T.; Kunimoto, D. Y.; Kumar, R. Bioorg. Med. Chem. 2005, 13,
6663.
10, 5, 1, and 0.5 lg/mL concentrations in triplicates. The experi-
ments were repeated two times and the mean percent inhibition
is reported in the Table 1. The standard deviations were within
10% of the mean value.
17. Shakya, N.; Srivastav, N. C.; Desroches, N.; Agrawal, B.; Kunimoto, D. Y.; Kumar,
R. J. Med. Chem. 2010, 53, 4130.
18. Srivastav, N. C.; Shakya, N.; Desroches, N.; Agrawal, B.; Kunimoto, D. Y.; Kumar,
R., Unpublished results.
19. Herdewijn, P.; Balzarini, J.; De Clercq, E.; Pauwels, R.; Baba, M.; Broder, S.;
Vanderhaeghe, H. J. Med. Chem. 1987, 30, 1270.
20. Aerschot, A. V.; Everaert, D.; Balzarini, J.; Augustyns, K.; Jie, L.; Janssen, G.;
Peeters, O.; Blaton, N.; De Ranter, C.; De Clercq, E.; Herdewijn, P. J. Med. Chem.
1990, 33, 1833.
21. Lin, T.-S.; Chen, M. S.; McLaren, C.; Gao, Y.-S.; Ghazzouli, I.; Prusoff, W. H. J. Med.
Chem. 1987, 30, 440.
22. Lin, T.-S.; Prusoff, W. H. J. Med. Chem. 1978, 21, 109.
23. Chu, C. K.; Schinazi, R. F.; Ahn, M. K.; Ullas, G. V.; Gu, Z. P. J. Med. Chem. 1989, 32,
612.
In Vitro Cytotoxicity Assay. Human hepatoma cell line (Huh-7)
was used to determine the effect of test compounds 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.
Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) for an operating grant (MOP-49415) for the financial sup-
port of this research.
24. Experimental synthesis of 3’-Fluoro-2’,3’-dideoxy-5-trifluoromethyluridine (19).
The compound 17 (0.07 g, 0.13 mmol) was dissolved in 80% aqueous acetic acid
(5 mL) and heated at 90 °C for 0.5 h. Solvent was removed in vacuo and the
crude product thus obtained was purified on silica gel column using MeOH/
Supplementary data
CHCl3 (5:95, v/v) as eluent to give 19 (0.018 g, 46%) as solid; mp 198–200 °C. 1
H
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.114.
NMR (DMSO-d6) d 2.22–2.63 (m, 2H, H-20), 3.58–3.69 (m, 2H, H-50), 4.28 (dm,
J4’,F = 26.86 Hz, 1H, H-40), 5.22 (m, 1H, 5’-OH), 5.30 (dm, J3’,F = 53.10 Hz, 1H, H-
30), 6.14–6.19 (m, 1H, H-10), 8.76 (s, 1H, H-6), 11.59 (s, 1H, NH). Anal. Calcd for
C
10H10F4N2O4: C, 40.28; H, 3.38; N, 9.39. Found: C, 40.45; H, 3.47; N, 9.17.
References and notes
25. Lin, T.-S.; Guo, J.-Y.; Schinazi, R. F.; Chu, C. K.; Xiang, J.-N.; Prusoff, W. H. J. Med.
Chem. 1988, 31, 336.
1. World Health Organization. Global Tuberculosis Control: Surveillance,
Planning, and Financing. WHO Report 2010 WHO Press: Geneva, Switzerland,
2010. Also see the website: http://www.who.int/tb/publications/global_report/
2010/en/index.html
26. 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. J. Clin. Microbiol. 1998, 36, 362.