Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 2′-'up' fluoro (or hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.05.004

Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mechanisms of action is urgently required for the treatment of mycobacterial infections due to the emerging global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy) methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 2′-'up' fluoro (or hydroxy) nucleosides (1, 2, 4-6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among 2′-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro- β-d-arabinofuranosyl)-5-ethyluracil (13) exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with isoniazid. The most active compound emerging from these studies, 1-(β-d-arabinofuranosyl)-4-thio-5-hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC₅₀ = 0.5 μg/mL) and M. bovis (MIC₅₀ = 0.5 μg/mL) at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine (MIC₅₀ = 5.0 μg/mL), a second line drug. It also showed an additive effect when combined with isoniazid. Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC₅₀ = 1 μg/mL) at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21 (MIC₅₀ = 10 μg/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested (CC₅₀ > 100 μg/mL). These observations offer promise for a new drug treatment regimen to augment and complement the current chemotherapy of TB.

Full text of DOI:10.1016/j.bmc.2012.05.004

Bioorganic & Medicinal Chemistry 20 (2012) 4088–4097
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel 5-(ethyl or hydroxymethyl) analogs of 20-‘up’ fluoro (or
hydroxyl) pyrimidine nucleosides as a new class of Mycobacterium tuberculosis,
Mycobacterium bovis and Mycobacterium avium inhibitors
Neeraj Shakya a, Naveen C. Srivastav a, Sudha Bhavanam a, Chris Tse a, Nancy Desroches a,
Babita Agrawal b, Dennis Y. Kunimoto c, Rakesh Kumar a,
⇑
a Department of Laboratory Medicine and Pathology, 7-28 Heritage Medical Research Centre, University of Alberta, Edmonton, AB, Canada T6G 2H7
b Department of Surgery, University of Alberta, Edmonton, AB, Canada T6G 2H7
c 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
Article history:
Discovery of novel antimycobacterial compounds that work on distinctive targets and by diverse mech-
anisms of action is urgently required for the treatment of mycobacterial infections due to the emerging
global health threat of tuberculosis. We have identified a new class of 5-ethyl or hydroxy (or methoxy)
methyl-substituted pyrimidine nucleosides as potent inhibitors of Mycobacterium bovis, Mycobacterium
tuberculosis (H37Ra, H37Rv) and Mycobacterium avium. A series of 20-‘up’ fluoro (or hydroxy) nucleosides
(1, 2,4–6, 9, 10, 13, 16, 18, 21, 24) was synthesized and evaluated for antimycobacterial activity. Among
Received 22 March 2012
Revised 27 April 2012
Accepted 2 May 2012
Available online 15 May 2012
Keywords:
Tuberculosis
Pyrimidine nucleosides
Mycobacterium
20-fluorinated compounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-b-
exhibited promising activity against M. bovis and Mtb alone, and showed synergism when combined with
isoniazid. The most active compound emerging from these studies, 1-(b- -arabinofuranosyl)-4-thio-5-
hydroxymethyluracil (21) inhibited Mtb (H37Ra) (MIC50 = 0.5 g/mL) and M. bovis (MIC50 = 0.5 g/mL)
at low concentrations, and was ten times more potent against Mtb (H37Ra) than cycloserine
(MIC50 = 5.0 g/mL), a second line drug. It also showed an additive effect when combined with isoniazid.
Compound 21 retained sensitivity against a rifampicin-resistant (H37Rv) strain of Mtb (MIC50 = 1 g/mL)
D-arabinofuranosyl)-5-ethyluracil (13)
D
l
l
l
l
at concentrations similar to that for a rifampicin-sensitive (H37Rv) strain, suggesting that it has no cross-
resistance to a first-line anti-TB drug. In addition, the replication of M. avium was also inhibited by 21
(MIC50 = 10
(CC50 > 100
l
g/mL). No cellular toxicity of 13 or 21 was observed up to the highest concentration tested
l
g/mL). These observations offer promise for a new drug treatment regimen to augment and
complement the current chemotherapy of TB.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
TB-causing mycobacteria have evolved a highly efficient means
of aerosol transmission, and its extent is reflected in the estimate
that one third of the global population is latently infected with
Mtb.7 Around one in ten individuals in this infected population
develop active TB. In 2009 an estimated 9.4 million new cases of
TB were reported and 1.7 million people died.8 The expanding
threats of TB drug resistance and concurrent human infection with
immunodeficiency virus (HIV) and TB, compound the already crip-
pling burden of TB. HIV infection facilitates reactivation of Mtb
and results in more rapid progression from infection to active dis-
ease. Thus, the HIV/AIDS epidemic has been the primary cause of
the surge in TB cases over the past few decades.9
An outbreak of recently recognized ‘extensively drug-resistant
TB’ (XDR-TB) threatens the control of TB globally by making it
almost incurable.9 Although, most of the XDR-TB cases have been
reported among HIV-infected patients, there have been reports of
XDR-TB among HIV-negative patients.10 The currently available
TB treatment is inadequate. The long duration of a complex
Tuberculosis (TB) is caused predominantly by Mycobacterium
tuberculosis (Mtb), an obligate aerobic bacillus. The vast majority
of TB infections are caused by Mtb, but other closely related myco-
bacteria, Mycobacterium bovis and Mycobacterium avium can also
cause severe disease.1–3 M. bovis, in attenuated form, has been used
as BCG vaccine.1 However, recently M. bovis infections have re-
emerged and are causing TB in humans, particularly those who
are immunosuppressed.2,3 Further, multidrug resistant (MDR)
strains of M. bovis have also been found that are resistant to a large
number of anti-TB drugs.2,3 Clinical management of M. avium infec-
tion is very difficult since many of the first-line anti-TB drugs are
ineffective against them.4–6
⇑
Corresponding author. Tel.: +1 780 492 7545; fax: +1 780 492 7521.
E-mail address: rakesh.kumar@ualberta.ca (R. Kumar).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.05.004

N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
4089
treatment regimen and associated side effects result in treatment
non-compliance. Insufficient treatment with the current drug regi-
men allows rapid emergence of resistance and continuous spread of
the drug-sensitive as well as drug-resistant mycobacterial strains.11
Clearly, there is an urgent need to discover new treatments for TB by
either modifying the application of existing agents or introducing
new drugs. New anti-TB drugs are required to permit shorter treat-
ment times, reduce toxic side effects, and treat MDR and XDR-TB.
The identification of novel targets for developing new anti-TB
drugs has been facilitated by the complete genome sequencing of
Mtb.12 Several enzymes involved in purine and pyrimidine metab-
olism, and DNA and RNA synthesis differ significantly between
humans and Mtb.13 Therefore, nucleoside and nucleotide analogs
may interfere these targets thereby disrupting nucleic acid biosyn-
thetic pathways of mycobacteria.
An important area of anti-TB drug design is the investigation of a
new class ofpyrimidinenucleosides thatpossess substitutionsat the
C-5 position of the pyrimidine base including novel glycosyl moie-
ties which exhibit potent and selective antimycobacterial activity.
In our recent studies14, we reported various 5-methyl pyrimidine
nucleosides substituted at 20 and/or 30 positions which exhibited sig-
nificant antimycobacterial activity against Mtb (H37Ra and H37Rv)
Meldrum et al20 reported that it had selective cytotoxic activity
on a variety of tumor cell lines. In contrast, 5-hydroxymethyl
analogs of AZT (30-azido-20,30-dideoxythymidine) and FLT (30-flu-
oro-20,30-dideoxythymidine), had lower anti-HIV activity than
AZT and FLT. However, 5-hydroxymethyl-30-azido-20,30-dideoxy-
uridine and 5-hydroxymethyl-30-fluoro-20,30-dideoxyuridine were
significantly less toxic than AZT and FLT and no toxicity to host
cells was found up to a concentration of 400 l
M21 In addition,
the 5-hydroxymethyl analogs of thymidine inhibited the replica-
tion of Escherichia coli (E. coli)22, and Bacimethrin, a hydroxymethyl
analog of thymine is a known antibacterial agent for Salmonella
typhimurium (S. typhimurium) and Salmonella enterica (S. enterica).
Bacimethrin has been shown to inhibit the bacterial thiamine
biosynthetic pathway.23
Herein we report the synthesis and antimycobacterial activity
of a new class of 5-ethyl and 5-(hydroxymethyl or methoxy-
methyl) substituted pyrimidine analogs with various 1-(2-deoxy-
2-fluoro-b-
(9), 1-(2-deoxy-2-fluoro-30-(un)substituted-b-
(10, 13, 16) and 1-b- -arabinofuranosyl (18, 21, 24) moieties
against M. bovis, Mtb and M. avium, in vitro. Among these com-
pounds, 1-(3-bromo-2,3-dideoxy-2-fluoro-b- -arabinofuranosyl)-
5-ethyluracil (13) provided promising in vitro activity against M.
bovis and Mtb alone and in combination with isoniazid. 1-(b- -ara-
binofuranosyl)-4-thio-5-hydroxymethyluracil (21) displayed the
most potent inhibition of Mtb H37Ra (MIC50 = 0.5 g/mL), H37Rv
(MIC50 = 1.0 g/mL) and M. bovis (MIC50 = 0.5 g/mL). This com-
pound also retained activity for a rifampicin-resistant Mtb strain
H37Rv at a similar concentration (MIC50 = 1.0 g/mL). In addition,
it was encouraging to note that compound 21 exhibited activity
D
-arabinofuranosyl) (1, 2, 4–6), 2,30-anhydro derivative
D
-arabinofuranosyl)
D
D
(MIC50 = 1–25 lg/mL range). In this article, we report the antimyco-
bacterial activities of a novel class of 5-ethyl and 5-hydroxymethyl
derivatives of related pyrimidine nucleosides and their potential
as potent and selective anti-TB agents.
D
l
5-Ethyl-20-deoxyuridine (EDU), a ‘natural’ thymidine analog
with the same base-pairing property as thymidine,15 has been re-
ported to be non-mutagenic in several organisms.16 In the carbo-
hydrate portion of EDU, replacement of hydrogen with fluorine
(which is strongly electronegative and has the same van der Waals
radius as H) has been used to obtain favorable biological proper-
ties. It was subsequently shown that the introduction of a 20-flu-
oroarabino substituent enhanced the chemotherapeutic potential
l
l
l
against M. avium (MIC50 = 10.0 lg/mL).
2. Chemistry
of EDU.17 The 1-(2-deoxy-2-fluoro-b-
D-arabinofuranosyl) deriva-
20-Fluoroarabinosyl nucleoside, 1-(2-deoxy-2-fluoro-b-
D-arabi-
tive of EDU was found to be highly active against several herpes
viruses with much lower toxicity towards host cells compared to
other fluorinated nucleosides, partly due to its non-inhibitory ef-
fect on cellular DNA synthesis. Importantly, this modification lead
to immediate and sustained inhibition of hepatitis B virus replica-
tion in a woodchuck model with no apparent toxic effects.17,18
nofuranosyl)-5-ethyluracil (1) was prepared by a coupling reaction
of silylated 5-ethyluracil and the appropriate furanosyl bromide.24
Acetylation of 1 with acetic anhydride in anhydrous pyridine at
room temperature yielded 1-(3,5-di-O-acetyl-2-deoxy-2-fluoro-b-
D-arabinofuranosyl)-5-ethyluracil (2) in 99% yield. The compound
2 on thiation with Lawesson’s reagent in anhydrous 1,4-dioxane
5-Hydroxymethyl-20-deoxyuridine has been reported as
a
under refluxing conditions gave the crude 4-thio 30-50-di-acety-
potent inhibitor of vaccinia virus and herpes simplex virus.19
lated product
3
which upon deacetylation with methanolic
O
O
S
OTMS
C2H5
(ii)
C2H5
(i)
C2H5
C2H5
HN
HN
O
HN
N
O
O
N
N
TMSO
BzO
N
N
+
O
AcO
O
HO
O
AcO
O
F
F
F
2
1
3
(iii)
F
AcO
HO
AcO
Br
BzO
S
S
C2H5
C2H5
HN
HN
+
O
O
N
N
AcO
O
HO
O
F
F
4
5
HO
HO
Scheme 1. Reagents and conditions: (i) Acetic anhydride, anhydrous pyridine, 0 °C to room temperature, 24 h, 99% yield; (ii) Lawesson0s reagent, anhydrous 1,4-dioxane,
reflux, 3 h; (iii) methanolic ammonia, room temperature, 4 h (4, 25%; 5, 43% yield).

4090
N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
O
N
NH2
NHAc
C2H5
C2H5
C2H5
HN
N
O
N
O
(ii)
(i)
O
O
O
N
N
AcO
O
HO
(iii)
AcO
F
F
F
2
6
6a
AcO
HO
AcO
Scheme 2. Reagents and conditions: (i) (a) 2,4,6-Triisopropylbenzenesulfonyl chloride, 4-(dimethylamino)pyridine, anhydrous Et3N, anhydrous CH3CN, room temperature,
60 h, (b) NH4OH, room temperature, 24 h; (ii) acetic anhydride, anhydrous pyridine, 0 °C to room temperature, 24 h; (iii) K2CO3, MeOH, 0 °C, 30 min, 32% yield.
O
N
O
N
O
N
O
N
F
H
O
C2H5
C2H5
C2H5
C2H5
N
O
HN
O
HN
O
N
O
O
O
(i)
(ii)
O
HO
TrO
TrO
TrO
F
F
F
F
O
S
HO
HO
7
1
8
F
F
NEt2
(iii)
O
N
O
C2H5
C2H5
HN
O
N
O
(iv)
O
N
O
HO
HO
F
F
N3
9
10
Scheme 3. Reagents and conditions: (i) TrCl, 4-(dimethylamino)pyridine, anhydrous pyridine, 80 °C, 8 h, 89% yield; (ii) DAST, anhydrous CH2Cl2, -12 °C to room temperature,
6 h, 78% yield; (iii) 80% AcOH, 90 °C, 30 min, 82% yield; (iv) NaN3, anhydrous DMF, 125–130 °C, 12 h, 16% yield.
ammonia at room temperature for 4 h provided 1-(5-O-acetyl-2-
deoxy-2-fluoro-b- -arabinofuranosyl)-4-thio-5-ethyluracil (4) and
1-(2-deoxy-2-fluoro-b- -arabinofuranosyl)-4-thio-5-ethyluracil
with NaN3 in dry DMF at 125–130 °C provided 10 in 16% yield
(Scheme 3).
D
D
1-(3-Bromo-2,3-dideoxy-2-fluoro-b-D-arabinofuranosyl)-5-eth-
(5) in 25% and 43% yields, respectively (Scheme 1). The removal
rate of acetyl group at 50-O-position is expected to be faster than
acetyl group at 30-O-position. The formation of 4 could have
been resulted due to an intramolecular migration of acetyl moi-
ety from 30-O-position to 50-O-position. 1-(2-Deoxy-2-fluoro-b-
yluracil (13) was synthesized from compound 7 (Scheme 4). Com-
pound 7 upon treatment with methanesulphonyl chloride (mesyl
chloride) in anhydrous pyridine at 0 °C afforded the 30-O-mesyl-50-
O-trityl derivative 11 in 97% yield. Reaction of 11 with LiBr in anhy-
drous CH3CN under reflux or in anhydrous DMF at 110 °C, despite
prolonged heating (48 h), did not provide the desired 30-bromo
product 12. However, our attempt using NaBr as a source of bromide
anion (Brꢀ) was successful. Thus, compound 11 upon reaction with
NaBr in anhydrous DMF at 110 °C for 24 h gave the 30-bromo deriv-
ative 12 in 34% yield which after detritylation with 80% AcOH at
90 °C yielded the target nucleoside 13 in 60% yield.
D
-arabinofuranosyl)-5-ethylcytosine (6) was prepared by the
reaction of 2 with 2,4,6-triisopropylbenzenesulphonyl chloride
in the presence of 4-(dimethylamino)pyridine (DMAP) and
Et3N followed by treatment with NH4OH at room temperature.
The obtained compound 6 was impure and it was difficult to re-
move the impurities by repeated column chromatography.
Therefore, it was converted to 30,50-N-triacetylated derivative
6a which, after purification, was deblocked with K2CO3 in meth-
anol at 0 °C. The desired pure product 6 was obtained in an over-
all yield of 32%, starting from 2 (Scheme 2).
Reaction of 1 with triphenylmethyl chloride in dry pyridine at
80 °C in the presence of DMAP followed by treatment of the obtained
50-O-trityl derivative (7) with (diethylamino)sulfur trifluoride
(DAST) in anhydrous CH2Cl2 gave 2,30-anhydro nucleoside 8 in 78%
yield. Compound 8, upon reaction with 80% AcOH at 90 °C, yielded
the deblocked 2,30-anhydro product 9 in 82% yield. Azidation of 9
The 30-deoxy derivative, 1-(2,3-dideoxy-2-fluoro-b-
D-threo-
pentofuranosyl)-5-ethyluracil (16) was synthesized as illustrated
in Scheme 5. Initially we planned to synthesize 16 via 30-iodo deriv-
atives (14 and 15), since 30-iodo derivatives have been reported to be
easily deiodinated to give the 30-deoxy compound.25 However, our
attempts to synthesize the 30-iodo derivative 14 failed under various
reaction conditions (NaI/anhydrous DME, reflux, 24 h or NaI/anhy-
drous DMF, 110 °C, 24 h). Subsequently, we attempted synthesis of
the 30-deoxy derivative 16 by the reductive debromination of
compound 13. Compound 13 upon treatment with Et3N, in the

N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
4091
12
O
N
O
N
(ii, iii)
O
N
O
N
X
C2H5
C2H5
C2H5
C2H5
HN
O
HN
O
HN
O
HN
O
(iv)
(i)
(v)
O
O
O
O
TrO
TrO
HO
TrO
F
F
F
F
12
11
7
13
HO
MsO
Br
Br
Scheme 4. Reagents and conditions: (i) Mesyl chloride, anhydrous pyridine, 0–5 °C, 48 h, 97%, yield; (ii) LiBr, anhydrous CH3CN, reflux, 48 h; (iii) LiBr, anhydrous DMF, 110 °C,
48 h; (iv) NaBr, anhydrous DMF, 110 °C, 24 h, 34% yield; (v) 80% AcOH, 90 °C, 30 min, 60% yield.
O
N
O
N
O
N
O
N
C2H5
C2H5
C2H5
C2H5
HN
O
HN
O
HN
O
HN
O
(i) or (ii)
O
O
O
(iii)
O
X
TrO
R
HO
HO
F
F
F
F
13
11
16
MsO
I
Br
14, R = OTr
15, R = OH
Scheme 5. Reagents and conditions: (i) NaI, anhydrous DME, reflux, 24 h; (ii) NaI, anhydrous DMF, 110 °C, 24 h; (iii) H2, 10% Pd-C, Et3N, MeOH, room temperature, 2 h, 45%
yield.
S
O
N
O
N
S
O
N
CH2OH
CH2 OH
(iii)
CH2 OAc
(iv)
CH2 OAc
(v)
HN
HN
HN
HN
HN
O
O
O
O
O
N
N
(i) or (ii)
HO
O
HO
AcO
O
HO
O
HO
HO
O
HO
AcO
O
AcO
AcO
HO
AcO
HO
HO
AcO
21
20
18
17
19
(vi)
X
Scheme 6. Reagents and conditions: (i) Paraformaldehyde, 0.5 N KOH, 65 °C, 52 h, 9% yield; (ii) 37% formaldehyde, 0.5 N KOH, 65 °C, 18 h, 30% yield; (iii) acetic anhydride,
anhydrous pyridine, 0 °C to room temperature, 42% yield; (iv) Lawesson’s reagent, anhydrous 1,4-dioxane, reflux, 3.5 h, 40% yield; (v) K2CO3, MeOH, 0 °C, 1% yield; (vi)
paraformaldehyde, 0.5 N HCl, reflux, 12 h.
presence of 10% Pd-C, in methanol under hydrogen atmosphere gave
the desired compound 16 in 45% yield.
Reaction of 19 with p-toluenesulfonyl chloride, Et3N and 1-
methylpiperidine in dry acetonitrile at 0 °C followed by treatment
with NH4OH at 0 °C provided 22, which upon treatment with
K2CO3 in methanol at 0 °C, did not yield the desired product 23
1-b- -Arabinofuranosyluracil (17) was heated at 65 °C with
D
paraformaldehyde in 0.5 N aqueous KOH for 52 h to obtain the 5-
hydroxymethyl derivative 18, but this method provided 18 in poor
yield (9%). In order to optimize the yield of 18, we reacted com-
pound 17 with 37% formaldehyde in base as well as with parafor-
maldehyde using acidic (0.5 N HCl) conditions. It was found that
the yield of 18 was improved to 30% when 37% formaldehyde in
basic conditions was used, whereas the starting material (17)
was decomposed when it was heated with paraformaldehyde un-
der acidic conditions. In order to synthesize the 4-thio analog 21,
compound 18 was acetylated using acetic anhydride in anhydrous
pyridine to give a tetra-O-acetyl derivative 19 which was then re-
acted with Lawesson’s reagent in dry 1,4-dioxane to yield the pro-
tected 4-thio derivative 20 in 40% yield. Compound 20 was
deprotected using K2CO3 in dry methanol to afford the target
compound 21. (Scheme 6)
but resulted in 1-(b-
sine (24) in 54% yield. The possible mechanism for the conversion
of 22 to 24 may include the formation of ,b-unsaturated imine
D-arabinofuranosyl)-5-methoxymethylcyto-
a
intermediate 22a under basic conditions followed by Michael type
addition of methanol at C-5 of 22a to give 24 (Scheme 7).
3. Results and discussion
The antimycobacterial activities for this new class of 5-ethyl (1,
2, 4–6, 9, 10, 13, 16), 5-hydroxymethyl (18, 21) and 5-methoxy-
methyl (24) pyrimidine analogs were determined against myco-
bacteria (M. bovis, Mtb H37Ra, M. avium) using the microplate
alamar blue assay (MABA)26 at 0.5–100
l
g/mL concentrations.
The results are summarized in Table 1. Cycloserine, rifampicin,

4092
N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
NH2
CH2OH
N
O
N
HO
O
NH2
N
HO
CH2 OAc
(iii) X
N
23
(i)
(ii)
HO
19
O
O
AcO
NH2
NH2
N
(iii)
-OAc
CH2
AcO
CH2OCH3
N
O
N
22
CH3O-
HO
AcO
O
O
N
HO
O
HO
HO
24
HO
HO
22a
Scheme 7. Reagents and conditions: (i) p-Toluenesulfonyl chloride, Et3N, 1-methylpiperidine, anhydrous CH3CN, 0 °C, 4 h; (ii) NH4OH, 0 °C, 15 min, 30% yield; (iii) K2CO3, dry
MeOH, 0 °C, 30 min, 54% yield.
Table 1
In vitro antimycobacterial activity of test compounds against M. bovis, Mtb and M. avium
O
N
X
N
NH2
N
NH2
N
X
N
C2H5
C2H5
C2H5
CH2OH
CH2OCH3
HN
N
O
N
O
HN
HN
O
O
O
O
O
R1
O
HO
HO
O
HO
O
HO
HO
HO
F
F
F
R
HO
HO
HO
18, 21
1, 2, 4, 5, 10, 13, 16
6
9
24
Compd
X
R
R1
Antimycobacterial activity % inhibitiona (concentration
lg/mL)
M. bovis (BCG)
Mtb (H37Ra)
M. avium (ATCC 25291)
1
2
4
5
6
9
10
13
16
18
21
24
O
O
S
OH
OCOCH3
OH
OH
—
—
N3
Br
H
—
—
—
OH
OCOCH3
OCOCH3
OH
—
0
0
0
75 (100), 55 (50)
68 (100), 55 (50)
73 (100), 60 (50), 50 (10)
60 (100), 50 (50)
50 (100)
25 (100, 50)
0
78 (100), 65 (50), 56 (10), 50 (5)
25 (100)
0
100 (100, 50), 90 (10), 70 (5), 50 (0.5)
80 (100), 60 (50), 50 (10)
100 (50), 80 (15–20), 60 (10), 50 (5)
100 (0.5)
35 (100)
50 (100), 25 (50)
35 (100), 25 (50)
0
50 (100), 25 (50)
0
0
25 (100)
0
73 (100), 58 (50), 50 (10)
20 (100)
ND
80 (100), 65 (50), 50 (10)
65 (100), 55 (50)
50 (100)
25 (100, 50)
0
80 (100), 70 (50), 60 (10), 50 (5)
25 (100)
0
S
—
—
O
O
O
O
S
—
—
—
—
—
OH
OH
OH
—
—
—
—
—
—
100 (100, 50), 90 (10), 75 (5), 50 (0.5)
80 (100), 60 (50), 50 (10)
Cycloserine
Rifampicin
Clarithromycin
—
—
—
NDb
100 (0.5)
ND
90 (2)
100 (2)
ND
a
Antimycobacterial activity was determined at concentrations 100, 50, 25, 10, 5, 2, 1 and 0.5
ND = not determined.
lg/mL.
b
and clarithromycin were used as reference drugs. The compounds
that exhibited promising inhibition against the wild-type strain of
M. bovis were further evaluated against wild-type and rifampicin-
resistant strains of Mtb (H37Rv).
derivatives (2, 4) of 1 and 5 showed increased anti-TB activity when
compared to 1 and 5. This enhanced activity could partly be attrib-
uted to their increased lipophilicity. From this series of compounds,
the 30-bromo analog, 1-(3-bromo-2,3-dideoxy-2-fluoro-b-
D
-arabi-
Among the 5-ethyl series of compounds described here 1-(2-deoxy-
nofuranosyl)-5-ethyluracil (13) displayed the best antimycobacteri-
2-fluoro-b-
mL) and 1-(2-deoxy-2-fluoro-b-
(6, MIC50 = 100 g/mL) exhibited moderate inhibition of Mtb
(H37Ra), in contrast to 1-(2-deoxy-2-fluoro-b- -arabinofuranosyl)-5-
ethyluracil (1). However, it is interesting to note that the acetate
D
-arabinofuranosyl)-4-thio-5-ethyluracil (5, MIC50 = 50
l
g/
al activity against both M. bovis and Mtb (MIC50 = 5
the second-line anti-TB drug cycloserine (MIC50 = 5 lg/mL, Table 1).
l
g/mL) ,equal to
D-arabinofuranosyl)-5-ethylcytosine
l
This observation was consistent with our previous studies where an
analogous compound, 1-(3-bromo-3-deoxy-b- -arabinofurano-
syl)thymine (25), also exhibited the most potent activity against
D
D

N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
4093
Mtb (MIC50 = 1
l
g/mL)14 and M. bovis (40% inhibition at 1
l
g/mL,
by itself whereas 13 and 21 exhibited 56% and 90%, and 50% and
70% inhibition at 10 and 5 g/mL, respectively. Synergy was defined
unpublished results). Although the activity of 13 was not improved
over earlier compound 25 as expected, it was encouraging to note
that the efficacy of 13 was not completely lost but was only 5-fold
less than compound 25 against both M. bovis and Mtb. This suggests
that substitution of a methyl group for an ethyl group on the uracil
ring, and replacement of the hydroxyl at the 20-position by fluorine
are still acceptable by both mycobacterial species. The other 5-ethyl
l
as x/y < 1/z, where x is the growth index of the combination of
drugs, y is the lowest growth index of a single drug of the combina-
tion and z is the number of drugs in combination. For a two drug
combination, a quotient <0.5 indicates synergistic action whereas
a quotient of 0.5–1 reflects an additive effect.28 The synergy quo-
tient for the combination of compound 13 and isoniazid is 0.33,
which is lower than 0.5, suggesting a synergistic interaction. For
the combination of 21 and isoniazid, the synergy quotient was
0.8, reflecting an additive effect. This study suggests that our novel
nucleosides are acting at a different target, not interacting antago-
nistically, and would be effective when combined with current
drugs.
derivatives, O-2,30-anhydro-1-(2-deoxy-2-fluoro-b-
syl)-5-ethyluracil (9), 1-(3-azido-2,3-dideoxy-2-fluoro-b-
nofuranosyl)-5-ethyluracil (10) and 1-(2,3-dideoxy-2-fluoro-b-
threo-pentofuranosyl)-5-ethyluracil (16) had almost no, or very
low, inhibitory action against any mycobacteria, suggesting that
30-bromo substituent plays an important role in antimycobacterial
activity against Mtb in pyrimidine nucleosides. These results are in
agreement with our previous observations made with thymidine
analogs.14
D
-lyxofurano-
-arabi-
D
D
-
The XTT and 3H-thymidine incorporation assays were per-
formed to evaluate the toxicity of investigated compounds ( 1, 2,
4–6, 9, 10, 13, 16, 18, 21, 24) in vitro against a human hepatoma
cell line (Huh-7). These compounds were not toxic up to the high-
X
est concentration tested, 100 lg/mL (CC50 > 100 lg/mL). These re-
CH3
HN
sults were significant as the modifications sought at the C-5
position of the base and at 20 and/or 30 positions of the sugar moi-
ety of pyrimidine nucleoside derivatives described in this manu-
script did not contribute to enhanced toxicity as compared to
compounds reported in our previous studies.14,27
O
N
HO
O
HO
R
Due to the re-emergence of TB infection globally, and the resis-
tance of mycobacteria to existing drugs, there is an urgent need to
identify novel antimycobacterial compounds that work by new
mechanisms of action. In this work, a new class of pyrimidine
nucleosides was identified which has promising anti-TB activity
against several mycobacterial species [Bacillus Calmette Guerin
(BCG), Mtb H37Ra, H37Rv, M. avium] and has no cytotoxicity. Com-
pounds 13 and 21 emerged as the most efficacious analogs. Com-
pound 13 was found to be as active as cycloserine, a second-line
TB drug and demonstrated a synergistic effect in combination with
a first-line drug, isoniazid. Compound 21 has activity against vari-
ous mycobacterial species and with different isolates of Mtb. It
exhibited activity towards both drug-susceptible and drug-resis-
tant strains and was 10 times more potent than the reference drug
cycloserine. Thus the new inhibitors identified in this report hold
promise to be used alone or in combination for the development
of a new class of agents for TB infections.
Our results with 5-ethyl- and 5-hydroxymethyl series of pyrim-
idine nucleosides indicate that not only C-5, but other modifica-
tions e.g., substituents on the carbohydrate moiety, and C-4
position of the base, also play an important role in the determina-
tion of antimycobacterial activity. These observations provide us
with an impetus for designing a new series in the near future
which would integrate features of compounds 13 and 21, and rel-
evant compounds from our previous work.
25, R=Br, X=O
26, R=OH, X=O,
27, R=OH, X=S
Of the 5-hydroxymethyl derivatives, 4-thio analog, 1-(b-
furanosyl)-4-thio-5-hydroxymethyluracil (21), and 4-amino analog,
1-(b- -arabinofuranosyl)-5-methoxymethylcytosine (24), showed
potent anti-TB activity whereas 4-oxo analog, 1-(b- -arabinofur-
D-arabino-
D
D
anosyl)-5-hydroxymethyluracil (18) was found to be inactive. Sim-
ilar correlations were also observed in the 5-ethyl series of
compounds 1, 5 and 6, although 21 and 24 possessed much higher
activity. Encouragingly, anti-TB activity displayed by 21
(MIC50 = 0.5 lg/mL) was superior to 13 (MIC50 = 5 lg/mL), the most
active agent of 5-ethyl series. Further, the MIC50 exhibited by com-
pound 21 was ten times lower than the reference drug cycloserine
(MIC50 = 5
inhibition of M. avium (MIC50 = 10
prising since in our earlier studies27 1-(b-
mine (26) and 1-(b- -arabinofuranosyl)-4-thio-thymine (27) were
l
g/mL). In addition, compound 21 also showed marked
g/mL). These findings were sur-
-arabinofuranosyl)thy-
l
D
D
found to be inactive, suggesting that the hydroxymethyl group at
the C-5 position has an influence on antimycobacterial activity.
However, inactivity of compound 18 indicates that 5-hydroxy-
methyl group together with a thio substituent at the C-4 position
contribute to the potent antimycobacterial activity against Mtb as
well as M. avium.
The most active inhibitor, 21, was also evaluated for its activity
towards drug-susceptible and rifampicin-resistant strains of Mtb,
H37Rv. Intriguingly, compound 21 demonstrated an MIC50 of
4. Experimental section
1.0
bacteria. In these assays, rifampicin showed no activity against the
drug-resistant strain at 2.0 g/mL whereas isoniazid provided
100% inhibition at 1.0 g/mL.
We wanted to understand potential and possible interactions of
our new class of nucleoside analogs with current antimycobacterial
drugs, so we selected compounds 13 and 21 to test in combination
with isoniazid to determine their effect on the growth of Mtb
(H37Ra) in vitro. Compounds 13 and 21 were used at 10 and
lg/mL against both drug-susceptible and drug-resistant myco-
Melting points were determined with an electrothermal melting
point apparatus and are uncorrected. 1H NMR spectra were deter-
mined for samples in Me2SO-d6, CDCl3 or CD3OD on a Bruker AM
300 spectrometer using TMS as an internal standard. Chemical
shifts are given in ppm relative to TMS and signals are described
as s (singlet), d (doublet), t (triplet), br (broad signal), q (quartet),
m (multiplet), dm (doublet of multiplet), and dd (doublet of dou-
blets). The assignment of all exchangeable protons (OH, NH) was
confirmed by the addition of D2O. All of the final compounds had
>95% purity except 6a (>95% as a water adduct), determined by
microanalysis. Microanalysis results were within 0.4% of the theo-
retical values for all elements listed unless otherwise indicated. Sil-
ica gel column chromatography was carried out using Merck 7734
l
l
5
l
g/mL, respectively, and isoniazid at 0.5, 0.1 and 0.05
was encouraging to note that 13 provided synergistic inhibition of
Mtb (70% inhibition at 10 g/mL) whereas 21 showed an additive
effect (82% inhibition at 5 g/mL) when combined with isoniazid
at 0.1 g/mL. Isoniazid showed only 10% inhibition at 0.1 g/mL
lg/mL. It
l
l
l
l
silica gel (100–200 lM particle size). Thin–layer chromatography

4094
N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
(TLC) was performed with Machery-Nagel Alugram SiL G/UV silica
gel slides (20 M thickness). 1-b- -arabinofuranosyluracil (17)
was purchased from Aldrich.
acetic anhydride (0.71 mL, 7.54 mmol) in anhydrous pyridine
(30 mL) at room temperature. The triacetylated derivative was puri-
fied on a silica gel column using MeOH/CHCl3 (3:97, v/v) as the
eluent to give 6a (0.6 g) as a syrup.
l
D
4.1. 1-(3,5-Di-O-acetyl-2-deoxy-2-fluoro-b-
5-ethyluracil (2)
D
-arabinofuranosyl)-
To an ice cooled solution of 6a (0.6 g, 1.50 mmol) in methanol
(30 mL) was added K2CO3 (1.04 g, 7.52 mmol) and the reaction
mixture was stirred at 0 °C for 30 min. The solvent was removed
in vacuo and the crude product thus obtained was purified on a sil-
ica gel column using MeOH/CHCl3 (20:80, v/v) as the eluent to give
To an ice-cooled solution of 1 (1.0 g, 3.65 mmol) in anhydrous
pyridine (25 mL) was added acetic anhydride (0.82 mL, 8.72 mmol)
and the reaction mixture was stirred at 0 °C for 1 h. The stirring
was continued at room temperature for 23 h. Solvent was removed
in vacuo and the crude product thus obtained was purified on a sil-
ica gel column using MeOH/CHCl3 (2:98, v/v) as the eluent to give 2
6 (0.32 g, 32%) as a solid; mp 140–142 °C; ½a D
ꢁ
+107.76 (c 0.28,
MeOH); 1H NMR (DMSO-d6): d 1.02 (t, J = 7.35 Hz, 3H, CH3), 2.24
(q, J = 7.36 Hz, 2H, CH2), 3.53–3.65 (m, 2H, H-50), 3.76 (m, 1H, H-
40), 4.18 (dm, J3 ,F = 19.41 Hz, 1H, H-30), 4.95 (dm, J2 ,F = 52.63 Hz,
0
0
(1.30 g, 99%) as a solid; mp 50–52 °C; ½a D
ꢁ
+41.08 (c 0.33, MeOH);
1H, H-20), 5.12 (br s, 1H, 50-OH), 5.85 (br s, 1H, 30-OH), 6.11 (dd,
1H NMR (CDCl3): d 1.15 (t, J = 7.37 Hz, 3H, CH3), 2.13 (s, 3H, CH3),
2.17 (s, 3H, CH3), 2.38 (q, J = 7.34 Hz, 2H, CH2), 4.23–4.52 (m, 3H,
J1 ,F = 17.27 Hz, 4.08 Hz, 1H, H-10), 6.92 (br s, 1H, NH), 7.35 (br s,
0
1H, NH), 7.43 (s, 1H, H-6). Anal. Calcd for C11H16FN3O4 +0.85
H2O: C 45.78, H 6.18. Found: C 46.08, H 5.96.
H-40, H-50), 5.10 (dd, J2 ,F = 49.9 Hz, 2.74 Hz, 1H, H-20), 5.24 (dd,
0
J3 ,F = 16.64 Hz, 2.70 Hz, 1H, H-30), 6.22 (dd, J1 ,F = 22.44 Hz,
2.82 Hz, 1H, H-10), 7.29 (m, 1H, H-6), 8.53 (br s, 1H, NH). ES-MS
(+ve mode) = 359.1 (M+H)+; Anal. Calcd for C15H19FN2O7: C 50.28,
H 5.34, N 7.82. Found: C 50.48, H 5.47, N 7.65.
0
0
4.4. 1-(2-Deoxy-2-fluoro-5-O-trityl-b-D-arabinofuranosyl)-
5-ethyluracil (7)
To a dried mixture of 1 (3.0 g, 10.94 mmol), trityl chloride
(4.58 g, 16.43 mmol) and 4-(dimethylamino)pyridine (0.20 g,
1.64 mmol) was added in anhydrous pyridine (70 mL) and the
reaction mixture was heated at 80 °C for 8 h. The solvent was re-
moved in vacuo and the crude product thus obtained was purified
on a silica gel column using MeOH/CHCl3 (5:95, v/v) as the eluent
to give 7 (5.0 g, 89%) as a solid; mp 212–214 °C; 1H NMR (DMSO-
d6): d 0.83 (t, J = 7.32 Hz, 3H, CH3), 2.03 (q, J = 7.32 Hz, 2H, CH2),
3.17–3.35 (m, 2H, H-50), 3.97 (m, 1H, H-40), 4.24–4.39 (m, 1H, H-
4.2. 1-(5-O-Acetyl-2-deoxy-2-fluoro-b-D-arabinofuranosyl)-4-
thio-5-ethyluracil (4) and 1-(2-deoxy-2-fluoro-b-D-arabinofur
anosyl)-4-thio-5-ethyluracil (5)
To a dried mixture of 2 (2.60 g, 7.26 mmol) and Lawesson’s re-
agent (4.40 g, 10.88 mmol) was added anhydrous 1,4-dioxane
(50 mL). The reaction mixture was refluxed for 3 h, cooled to rt
and concentrated in vacuo to give the crude reaction mixture. This
mixture was then stirred with methanolic ammonia (40 mL) at
room temperature for 4 h. The solvent was removed in vacuo and
the crude product thus obtained was purified on a silica gel column
using MeOH/CHCl3 (2:98, v/v) as the eluent to give 4 (0.61 g, 25%)
as a solid; mp 108–110 °C; 1H NMR (DMSO-d6): d 1.06 (t,
J = 7.32 Hz, 3H, CH3), 2.06 (s, 3H, CH3), 2.48 (q, J = 7.32 Hz, 2H,
30), 5.04 (dm, J2 ,F = 52.49 Hz, 1H, H-20), 5.95 (d, J = 4.88 Hz, 1H, 30-
0
OH), 6.17 (dd, J1 ,F = 17.70 Hz, 4.27 Hz, 1H, H-10), 7.25–7.45 (m,
0
16H, 50-O-trityl and H-6), 11.48 (s, 1H, NH); ES-MS (+ve
mode) = 539.2 (M+Na)+; ES-MS (ꢀve mode) = 515.2 (MꢀH)ꢀ.
4.5. 2,30-O-Anhydro-1-(5-O-trityl-2-deoxy-2-fluoro-b-
D-lyxofur
anosyl)-5-ethyluracil (8)
CH2), 4.03–4.36 (m, 4H, H-30, H-40, H-50), 5.08 (dt, J2 ,F = 52.19 Hz,
0
3.66 Hz, 1H, H-20), 6.08 (d, J = 4.58 Hz, 1H, 30-OH), 6.13 (dd,
J1 ,F = 17.39 Hz, 3.97 Hz, 1H, H-10), 7.41 (s, 1H, H-6), 12.88 (s, 1H,
To a solution of 7 (0.66 g, 1.28 mmol) in anhydrous dichloro-
methane (30 mL) at ꢀ12 °C was added DAST (1.35 mL, 10.24 mmol)
and the reaction mixture was stirred at ꢀ12 °C for 30 min, followed
by stirring at room temperature for 5.5 h. The reaction mixture was
poured into 5% NaHCO3 (50 mL) and the aqueous layer was ex-
tracted with EtOAc (3 ꢂ 25 mL). The combined organic layers were
dried over anhydrous Na2SO4 and concentrated to give the crude
product, which was purified on a silica gel column using EtOAc as
the eluent to give 8 (0.50 g, 78%) as a syrup; 1H NMR (DMSO-d6): d
1.08 (t, J = 7.32 Hz, 3H, CH3), 2.23 (q, J = 7.32 Hz, 2H, CH2), 3.13 (d,
J = 6.71 Hz, 2H, H-50), 4.62 (m, 1H, H-40), 5.43 (t, J = 3.05 Hz, 1H, H-
0
NH); ES-MS (+ve mode) = 333.1 (M+H)+; ES-MS (ꢀve
mode) = 331.1 (MꢀH)ꢀ; Anal. Calcd for C13H17FN2O5S: C 46.98, H
5.16, N 8.43. Found: C 46.77, H 5.28, N 8.31.
Further elution with MeOH/CHCl3 (3:97, v/v) yielded 5 (0.91 g,
43%) as a solid; mp 95–97 °C; ½a D
ꢁ
+150.39 (c 0.33, MeOH); 1H
NMR (DMSO-d6): 1.06 (t, J = 7.33 Hz, 3H, CH3), 2.48 (q,
d
J = 7.33 Hz, 2H, CH2), 3.64 (m, 2H, H-50), 3.81 (m, 1H, H-40), 4.26
(dm, J3 ,F = 20.14 Hz, 1H, H-30), 5.11 (dt, J2 ,F = 52.79 Hz, 4.27 Hz,
1H, H-20), 5.22 (br s, 1H, 50-OH), 5.93 (d, J = 4.88 Hz, 1H, 30-OH),
0
0
6.13 (dd, J1 ,F = 12.82 Hz, 4.88 Hz, 1H, H-10), 7.72 (s, 1H, H-6),
0
12.83 (s, 1H, NH); ES-MS (+ve mode) = 291.1 (M+H)+; ES-MS (ꢀve
mode) = 289.1 (MꢀH)ꢀ; Anal. Calcd for C11H15FN2O4S: C 45.51, H
5.21, N 9.65. Found: C 45.65, H 5.32, N 9.47.
30), 5.92 (dm, J2 ,F = 51.26 Hz, 1H, H-20), 6.02 (m, 1H, H-10), 7.23–
0
7.41 (m, 15H, 50-O-trityl), 7.63 (s, 1H, H-6); ES-MS (+ve
mode) = 499.2 (M+H)+.
4.3. 1-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)-5-ethylcytosine
4.6. O-2,30-Anhydro-1-(2-deoxy-2-fluoro-b-
Dlyxofuranosyl)-
(6)
5-ethyluracil (9)
To
a
dried mixture of
2
(1.30 g, 3.63 mmol), 2,4,6-
A solution of 8 (0.50 g, 1 mmol) in 80% aqueous AcOH (40 mL)
was heated at 90 °C for 30 min. The solvent was removed in vacuo
and the crude product thus obtained was purified on a silica gel col-
umn using MeOH/CHCl3 (15:85, v/v) as the eluent to give 9 (0.21 g,
triisopropylbenzenesulfonyl chloride (3.47 g, 11.46 mmol) and 4-
(dimethylamino)pyridine (0.70 g, 5.73 mmol) was added anhydrous
acetonitrile (50 mL) followed by anhydrous Et3N (2.66 mL,
19.07 mmol). The reaction mixture was stirred at room temperature
for 60 h. NH4OH (40 mL) was added and the stirring was continued
at room temperature for a further 24 h. The solvent was removed in
vacuo and the crude product thus obtained was purified on a silica
gel column using MeOH/CHCl3 (15:85, v/v) as the eluent to give 6
as an impure syrup (1.38 g). The syrup was dried, acetylated with
82%) as a solid; mp 202–204 °C (dec.); ½aꢁD ꢀ40.64 (c 0.31, MeOH);
1H NMR (DMSO-d6): d 1.04 (t, J = 7.32 Hz, 3H, CH3), 2.23 (q,
J = 7.32 Hz, 2H, CH2), 3.44–3.58 (m, 2H, H-50), 4.36 (m, 1H, H-40),
5.16 (t, J = 5.49 Hz, 1H, 50-OH), 5.31 (t, J = 3.05 Hz, 1H, H-30), 5.89
(dt, J2 ,F = 50.66 Hz, 3.66 Hz, 1H, H-20), 5.95 (m, 1H, H-10), 7.57 (s,
0
1H, H-6); ES-MS (+ve mode) = 257.1 (M+H)+; Anal. Calcd for

N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
4095
C11H13FN2O4: C 51.56, H 5.11, N 10.93. Found: C 51.41, H 5.18, N
10.87.
for C11H14BrFN2O4: C 40.02, H 4.76, N 7.78. Found: C 39.82, H
4.45, N 8.07.
4.7. 1-(3-Azido-2,3-dideoxy-2-fluoro-b-
D
-arabinofuranosyl)-
4.11. 1-(2,3-Dideoxy-2-fluoro-b-Dthreo-pentofuranosyl)-5-ethyl
5-ethyluracil (10)
uracil (16)
To a dried mixture of 9 (0.10 g, 0.39 mmol) and NaN3 (0.026 g,
0.4 mmol) was added anhydrous DMF (10 mL). The reaction mix-
ture was heated at 125–130 °C for 12 h. Solvent was removed in
vacuo and the crude product thus obtained was purified on a silica
gel column using EtOAc/Hexane (50:50, v/v) as the eluent to give
To a solution of 13 (0.05 g, 0.15 mmol) in MeOH (10 mL) was
added Et3N (0.1 mL) and 10% Pd-C (0.035 g) and the reaction mix-
ture was stirred at room temperature under H2 atmosphere for 2 h.
The reaction mixture was filtered through celite and the filtrate ob-
tained was concentrated in vacuo. The crude product was purified
on preparative TLC using MeOH/CHCl3 (5:95, v/v) as the eluent to
10 (0.019 g, 16%) as syrup; 1H NMR (DMSO-d6):
d 1.03 (t,
J = 7.32 Hz, 3H, CH3), 2.23 (q, J = 7.32 Hz, 2H, CH2), 3.69 (m, 2H,
give 16 (0.017 g, 45%) as a solid; mp 120–122 °C; ½a D
ꢁ
+75.34 (c
H-50), 3.83 (m, 1H, H-40), 4.52 (ddd, J3 ,F = 21.97 Hz, 7.93 Hz,
0.28, MeOH); 1H NMR (DMSO-d6): d 1.02 (t, J = 7.32 Hz, 3H, CH3),
2.01–2.20 (m, 2H, H-30), 2.23 (q, J = 7.32 Hz, 2H, CH2), 3.51–3.68
(m, 2H, H-50), 4.09 (m, 1H, H-40), 5.07 (t, J = 5.49 Hz, 1H, 50-OH),
0
5.49 Hz, 1H, H-30), 5.40 (dt, J2 ,F = 53.10 Hz, 5.49 Hz, 1H, H-20),
0
5.42 (br s, 1H, 50-OH), 6.17 (dd, J1 ,F = 9.76 Hz, 5.49 Hz, 1H, H-10),
0
5.32 (dm, J2 ,F = 54.93 Hz, 1H, H-20), 6.02 (dd, J1 ,F = 15.87 Hz,
3.66 Hz, 1H, H-10), 7.62 (s, 1H, H-6), 11.43 (s, 1H, NH); ES-MS
(+ve mode) = 259.1 (M+H)+; Anal. Calcd for C11H15FN2O4: C 51.16,
H 5.85, N 10.85. Found: C 51.33, H 5.94, N 10.72.
0
0
7.59 (s, 1H, H-6), 11.45 (br s, 1H, NH); Anal. Calcd for C11H14FN5O4:
C 44.15, H 4.72, N 23.40. Found: C 44.41, H 4.77, N 23.16.
4.8. 1-(2-Deoxy-2-fluoro-3-O-mesyl-5-O-trityl-b-D-arabinofur
anosyl)-5-ethyluracil (11)
4.12. 1-(b-D-Arabinofuranosyl)-5-hydroxylmethyluracil (18)
To an ice cooled solution of 7 (5.65 g, 10.94 mmol) in anhydrous
pyridine (60 mL) was added mesyl chloride (1.69 mL, 21.82 mmol)
dropwise with stirring. The reaction mixture was kept in the refrig-
erator for 48 h. After the addition of water (2 mL), the solvent was
evaporated and the resulting residue was dissolved in CHCl3
(100 mL), washed with water (2 ꢂ 30 mL) and dried over anhy-
drous Na2SO4. The solvent was removed in vacuo and the residue
thus obtained was purified on a silica gel column using MeOH/
CHCl3 (3:97, v/v) as the eluent to give 11 (6.30 g, 97%) as a solid;
mp 90–92 °C; 1H NMR (DMSO-d6): d 0.86 (t, J = 7.32 Hz, 3H, CH3),
2.04 (q, J = 7.32 Hz, 2H, CH2), 3.24 (s, 3H, CH3), 3.30–3.47 (m, 2H,
H-50), 4.25 (m, 1H, H-40), 5.41 (m, 1H, H-30), 5.55 (dm,
4.12.1. Method A
A mixture of 1-b-D-arabinofuranosyluracil 17 (0.25 g, 1.1 mmol),
paraformaldehyde (0.06 g) and 0.5 N KOH (1.25 mL) was heated at
65 °C for 52 h. The reaction mixture was cooled to room temperature
and neutralized with Dowex 50 W-X-8–H+resin. After stirring for
1 h, the resin was filtered off, and washed with water (50 mL). The
combined filtrate and washings were evaporated in vacuo at room
temperature to an oily residue. The residue was purified on a silica
gel column using EtOAc/MeOH (92:8, v/v) as the eluent to yield 18
(0.025 g, 9%) as a solid; mp 162–164 °C; 1H NMR (DMSO-d6): d
J2 ,F = 56.15 Hz, 1H, H-20), 6.26 (dd, J1 ,F = 16.48 Hz, 4.27 Hz, 1H, H-
10), 7.25–7.45 (m, 16H, 50-O-trityl and H-6), 11.55 (s, 1H, NH);
ES-MS (+ve mode) = 617.2 (M+Na)+; ES-MS (ꢀve mode) = 593.2
(MꢀH)ꢀ.
3.52–3.70 (m, 2H, H-50), 3.73 (dd, J = 8.79 Hz, 4.80 Hz, 1H, H-40),
0
0
0
0
3.90 (m, 1H, H-3 ), 3.99 (m, 1H, H-2 ), 4.08–4.15 (m, 2H, 5–CH2),
4.95 (t, J = 5.39 Hz, 1H, 5–CH2OH), 5.01 (t, J = 5.11 Hz, 1H, 50-OH),
5.45 (d, J = 4.35 Hz, 1H, 30-OH), 5.53 (d, J = 5.11 Hz, 1H, 20-OH), 6.00
(d, J = 4.34 Hz, 1H, H-10), 7.58 (s, 1H, H-6), 11.31 (s, 1H, NH); ES-MS
(+ve mode) = 297.1 (M+Na)+; ES-MS (ꢀve mode) = 273.1 (MꢀH)ꢀ;
Anal. Calcd for C10H14N2O7: C 43.80, H 5.15, N 10.22. Found: C
43.94, H 5.36, N 10.05.
4.9. 1-(3-Bromo-2,3-dideoxy-2-fluoro-5-O-trityl-b-D-arabinofur
anosyl)-5-ethyluracil (12)
To a dried mixture of 11 (0.60 g, 1.0 mmol) and NaBr (1.99 g,
19.34 mmol) was added anhydrous DMF (30 mL). The reaction
mixture was heated at 110 °C for 24 h. The solvent was removed
in vacuo and the crude product thus obtained was purified on a sil-
ica gel column using EtOAc/Hexane (30:70, v/v) as the eluent to
give 12 (0.20 g, 34%) as a syrup; 1H NMR (CDCl3): d 0.98 (t,
J = 7.32 Hz, 3H, CH3), 2.22 (q, J = 7.32 Hz, 2H, CH2), 3.47 (m, 2H,
4.12.2. Method B
A mixture of 17 (0.25 g, 1.1 mmol), 37% formaldehyde (1.25 mL)
and 0.5 N KOH (1.25 mL) was heated at 65 °C for 18 h. The reaction
mixture was cooled to room temperature and neutralized with
Dowex 50 W-X-8–H+ resin. After stirring for 1 h, the resin was fil-
tered off, and washed with water (50 mL). The combined filtrate
and washings were evaporated in vacuo at room temperature to
an oily residue. The residue was purified on a silica gel column
using EtOAc/MeOH (92:8, v/v) as the eluent to yield 18 (0.086 g,
30%) as a solid. Physical data were identical to the product ob-
tained by method A.
H-50), 4.33 (m, 1H, H-40), 4.50 (ddd, J3 ,F = 23.19 Hz, 5.49 Hz,
0
1.83 Hz, 1H, H-30), 5.31 (dm, J2 ,F = 53.10 Hz, 1H, H-20), 6.36 (dd,
0
J1 ,F = 18.31 Hz, 3.66 Hz, 1H, H-10), 7.28–7.50 (m, 16H, 50-O-trityl
0
and H-6), 8.65 (br s, 1H, NH); ES-MS (+ve mode) = 601.1
(M+Na)+; ES-MS (ꢀve mode) = 577.1 (MꢀH)ꢀ.
4.10. 1-(3-Bromo-2,3-dideoxy-2-fluoro-b-
D-arabinofuranosyl)-
4.12.3. Method C
5-ethyluracil (13)
A mixture of 17 (0.25 g, 1.1 mmol), paraformaldehyde (0.06 g)
and 0.5 N HCl (0.8 mL) was refluxed for 12 h. TLC showed decom-
position of the starting material.
Detritylation of 12 using the procedure as described for 9 pro-
vided 13. Yield 60%. Compound 13 was recrystallized from EtOH.
mp 62–64 °C; ½a D
ꢁ
+50.15 (c 0.31, MeOH); 1H NMR (DMSO-d6): d
4.13. 1-(2,3,5-Tri-O-acetyl-b-D-arabinofuranosyl)-5-acetoxymeth
yluracil (19)
1.02 (t, J = 7.32 Hz, 3H, CH3), 2.22 (q, J = 7.32 Hz, 2H, CH2), 3.72
(m, 2H, H-50), 4.17 (m, 1H, H-40), 4.55 (ddd, J3 ,F = 20.75 Hz,
0
7.93 Hz, 6.10 Hz, 1H, H-30), 5.44 (t, J = 5.49 Hz, 1H, 50-OH), 5.61
To an ice cooled (0 °C) solution of 18 (0.19 g, 0.42 mmol) in
anhydrous pyridine (10 mL) was added acetic anhydride (0.21 g,
2.1 mmol) dropwise. The reaction mixture was stirred at 0 °C for
(dt, J2 ,F = 53.10 Hz, 6.10 Hz, 1H, H-20), 6.29 (dd, J1 ,F = 8.54 Hz,
0
0
6.10 Hz, 1H, H-10), 7.63 (s, 1H, H-6), 11.48 (s, 1H, NH). Anal. Calcd

4096
N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
1 h and then at room temperature for 24 h. Pyridine was removed
in vacuo followed by co-evaporation with ethanol (2 ꢂ 25 mL). The
resulting residue was purified on a silica gel column using EtOAc/
Hexane (70:30 v/v) as the eluent to yield 19 (0.13 g, 42%) as syrup;
1H NMR (CDCl3): d 2.05, 2.07, 2.16 and 2.17 (4s, 12H, 4 ꢂ CH3), 4.23
and 4.46 (2 m, 3H, H-40, H-50), 4.89 (s, 2H, 5-CH2), 5.12 (m, 1H, H-
30), 5.41 (dd, J = 3.66 Hz, 1.22 Hz, 1H, H-20), 6.30 (d, J = 4.27 Hz, 1H,
H-10), 7.73 (s, 1H, H-6), 8.46 (br s, 1H, NH); ES-MS (+ve
mode) = 443.1 (M+H)+, 465.0 (M+Na)+; ES-MS (ꢀve mode) = 441.1
(MꢀH)ꢀ.
4.18. In vitro antimycobacterial activity assay (M. bovis, Mtb,
and M. avium)
M. bovis (BCG), Mtb (H37Ra), and M. avium (ATCC 25291) were
obtained from the American Type Culture Collection, Rockville,
MD. Antimycobacterial activity was determined using the micro-
plate alamar blue assay (MABA).26 Test compounds were dissolved
in DMSO at 10 mg/mL and subsequent dilutions were made in
7H9GC medium (Difco Laboratories, Detroit, Michigan) in 96 well
plates. For these experiments, each compound was tested at 100,
50, 25, 10, 5, 1, and 0.5 lg/mL in triplicate. The experiments were
4.14. 1-(2,3,5-Tri-O-acetyl-b-
xymethyluracil (20)
D
-arabinofuranosyl)-4-thio-5-aceto
repeated two times and the mean percent inhibition is reported in
Table 1. The standard deviations were within 10% of the mean va-
lue. Frozen mycobacterial inocula were diluted in 7H9GC medium
and added to each well at a final concentration of 2.5 ꢂ 105 CFU/
mL. Sixteen control wells consisted of eight with bacteria alone
(B) and eight with medium alone (M). Plates were incubated for
To a dried mixture of 19 (0.12 g, 0.27 mmol) and Lawesson0s re-
agent (0.20 g, 0.48 mmol) was added anhydrous 1,4–dioxane
(10 mL). The reaction mixture was refluxed for 3.5 h, cooled to rt
and concentrated in vacuo. The crude product thus obtained was
purified on a silica gel column using CHCl3/MeOH (97.5:2.5 v/v)
as the eluent to yield 20 (0.05 g, 40%) as a syrup. The product
was deblocked as stated in the next step.
six days and then 20
lL of 10ꢂ alamar blue and 12.5
lL of 20%
Tween 80 were added to one M and one B well. Wells were ob-
served for an additional 24–48 h for visual color change from blue
to pink and read by spectrophotometer (at excitation 530/525 nm
and emission 590/535 nm) to determine OD values. If the B well
became pink by 24 h (indicating growth), reagent was added to
the entire plate. If the B well remained blue, additional M and B
wells were tested daily until bacterial growth could be visualized
by color change. After the addition of the reagent to the plate, cul-
tures were incubated for 24 h and plates were observed visually for
color change and read by spectrophotometer. MIC was defined
visually 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.) ꢂ 100. The lowest drug concen-
tration affecting an inhibition of ꢃ50% was considered as the
MIC50. Similar methodology was used for all (three) mycobacteria
strains. Cycloserine, rifampicin and clarithromycin were used as
4.15. 1-(b-D-Arabinofuranosyl)-4-thio-5-hydroxymethyluracil
(21)
Deprotection of 20 using the procedure as described for 6a?6
yielded compound 21. Yield 1%; syrup; 1H NMR (CD3OD): d 3.77–
3.84 (m, 2H, H-50), 3.94 (m, 1H, H-40), 4.08 (m, 1H, H-30), 4.17 (m,
1H, H-20), 4.48 (s, 2H, 5–CH2), 6.13 (d, J = 4.27 Hz, 1H, H-10), 7.80
(s, 1H, H-6). Anal. Calcd for C10H14N2O6S: C 41.37, H 4.86, N 9.65.
Found: C 41.11, H 4.80, N 9.86.
4.16. 1-(2,3,5-Tri-O-acetyl-b-D-arabinofuranosyl)-5-acetoxy
methylcytosine (22)
positive controls. As negative controls, DMSO (2, 1, 0.2, 0.02 lL),
To a solution of 19 (0.20 g, 0.45 mmol), triethylamine (0.09 g,
0.89 mmol) and 1-methylpiperidine (0.05 g, 0.50 mmol) in dry ace-
tonitrile (10 mL) was added p-toluenesulfonyl chloride (0.17 g,
0.89 mmol) in three portions at 0 °C with stirring. The reaction
mixture was stirred at 0 °C for 4 h. To this was added NH4OH
(5 mL) at rt and the reaction mixture stirred at rt for 15 min. Ace-
tonitrile was removed in vacuo and the resulting residue was puri-
fied on a silica gel column using EtOAc/MeOH (94:6 v/v) as the
eluent to yield 22 (0.06 g, 30%) as a syrup; 1H NMR (CDCl3): d
2.09 and 2.14 (2s, 6H, 2 ꢂ CH3), 2.17 (s, 6H, 2 ꢂ CH3), 4.23–4.35
and 4.58–4.68 (2 m, 3H, H-40, H-50), 5.05 (m, 1H, H-30), 5.09 (s,
was added to the B well at concentrations similar to those of com-
pound 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 60,000–100,000.
4.19. Antimycobacterial activity against a drug-resistant strain
of Mtb
The activity of compound 21 was determined against rifampi-
cin-resistant Mtb H37Rv (ATCC 35838, resistant to rifampicin at
2 l
g/mL) using a radiometric-BACTEC assay.29 This assay detects
0
2H, 5-CH2), 5.42 (dd, J = 3.66 Hz, 1.22 Hz, 1H, H-2 ), 6.30 (d,
the metabolism of 14C-labelled palmitic acid, where evolving
14CO2 is captured and counted as a measure of mycobacterial
growth and metabolism. The growing inoculum (2.5–5.0 ꢂ 105
CFU/vial) was diluted in a BACTEC vial containing radiometric
7H12 (BACTEC 12B) medium and incubated at 37 °C. Two-fold dilu-
tions of test compounds were delivered to the inoculum-containing
BACTEC vials. Negative control vials consisted of medium with bac-
teria inoculum, medium with bacteria inoculum at 1:100, and med-
ium 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 instru-
ment until the GI of the 1:100 inoculum controls reached 30. Vials
were read daily and a change in GI (d GI) was recorded for each
compound. Percent inhibition was defined as (GI of test sample/
GI of control) ꢂ 100. For the no drug control, the d GI 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.
J = 3.66 Hz, 1H, H-10), 8.07 (s, 1H, H-6), 8.49 and 10.49 (2s, 2H,
2 ꢂ NH); ES-MS (+ve mode) = 442.1 (M+H)+.
4.17. 1-(b-D-Arabinofuranosyl)-5-methoxymethylcytosine (24)
Treatment of 22 with K2CO3 using the procedure as described
for 6a?6 provided a crude product, which was purified on HPLC
to yield pure 24 (0.02 g, 54%) as a syrup; [HPLC tR 7.0 min, column,
OmniSpher 5 C-18 (100 mm ꢂ 4.6 mm) (from Varian Inc.), mobile
phase, CH3OH-H2O (40:60), flow rate, 1.0 mL/min, UV wavelength,
254 nm, column temperature, 25 °C]; ½a D
ꢁ
+83.41 (c 0.14, MeOH);
1H NMR (DMSO-d6): d 3.20 (s, 3H, OCH3), 3.53–3.62 (m, 2H, H-
50), 3.67–3.76 (m, 1H, H-40), 3.85–3.89 and 3.90–3.96 (2 m, 2H,
H-20, H-30), 4.09 (dd, J = 18.3 Hz, 12.2 Hz, 2H, 5-CH2), 5.42 (br s,
4H, 4 ꢂ OH), 6.01 (d, J = 4.27 Hz, 1H, H-10), 6.58 (br s, 1H, NH),
7.29 (br s, 1H, NH), 7.60 (s, 1H, H-6); ES-MS (+ve mode) = 288.0
(M+H)+, 310.0 (M+Na)+; ES-MS (ꢀve mode) = 285.9 (MꢀH)ꢀ; Anal.
Calcd for C11H17N3O6: C 45.99, H 5.96, N 14.63. Found: C 46.23, H
5.84, N 14.56.

N. Shakya et al. / Bioorg. Med. Chem. 20 (2012) 4088–4097
4097
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Acknowledgments
We are grateful to the Canadian Institutes of Health Research
(CIHR) for an operating grant (MOP-49415) for the financial sup-
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from the Alberta Heritage Foundation for Medical Research
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