Synthesis and biological evaluation of inhibitors of thymidine monophosphate kinase from Bacillus anthracis

Article abstract of DOI:10.1080/15257770701845238

Nineteen lipophilic thymidine phosphate-mimicking compounds were designed and synthesized as potential inhibitors of thymidine monophosphate kinase of Bacillus anthracis, a Gram-positive bacterium that causes anthrax. These thymidine analogues were substi

Full text of DOI:10.1080/15257770701845238

Nucleosides, Nucleotides, and Nucleic Acids, 27:244–260, 2008
Copyright ꢀC Taylor & Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701845238
SYNTHESIS AND BIOLOGICAL EVALUATION OF INHIBITORS
OF THYMIDINE MONOPHOSPHATE KINASE FROM
BACILLUS ANTHRACIS
Youngjoo Byun,1 Susan R. Vogel,2 Andrew J. Phipps,2 Cecilia Carnrot,3
Staffan Eriksson,3 Rohit Tiwari,4 and Werner Tjarks4
1Department of Radiology, Johns Hopkins Medical Institutions, Baltimore,
Maryland, USA
2Department of Veterinary Biosciences and Center for Microbial Interface Biology,
The Ohio State University, Columbus, Ohio, USA
3Department of Anatomy, Physiology and Biochemistry, BMC, Swedish University
of Agricultural Sciences, Uppsala, Sweden
4College of Pharmacy and Center for Microbial Interface Biology, The Ohio State University,
Columbus, Ohio, USA
Nineteen lipophilic thymidine phosphate-mimicking compounds were designed and synthesized
2
as potential inhibitors of thymidine monophosphate kinase of Bacillus anthracis, a Gram-positive
bacterium that causes anthrax. These thymidine analogues were substituted at the 5ꢁ-postion with
sulfonamide-, amide-, (thio)urea-, or triazole groups, which served as lipophilic surrogates for
phosphate. Three of the tested compounds produced inhibition of B. anthracis Sterne growth and/or
thymidine monophosphate activity. Additional studies will be necessary to elucidate the potential of
this type of B. anthracis thymidine monophosphate inhibitors as novel antibiotics in the treatment
of anthrax.
Keywords Bacillus anthracis thymidine monophosphate kinase (BaTMPK); 5ꢁ-Modified
thymidine-based BaTMPK kinase inhibitors
Received 10 July 2007; accepted 12 October 2007.
This work was supported by grants from The Ohio State University (OSU) College of Pharmacy
(to W.T.), the OSU College of Veterinary Medicine, Department of Veterinary Biosciences (salary
support to A.J.P.), NIH grant RR-017505 (stipend for S.R.V.), and the Swedish Research Council (to
S.E.). Y.B. thanks OSU for financial support in form of the Presidential Fellowship. The authors thank
Professor Serge Van Calenbergh for generously providing 1-[2,3-dideoxy-3-(hydroxymethyl)-β-D-erythro-
pentofuranosyl]thymine and 1-[3-(aminomethyl)-2,3-dideoxy-β-D-erythro-pentofuranosyl]thymine.
Address correspondence to Werner Tjarks, The Ohio State University, College of Pharmacy, 500 W.
12th Ave., Columbus OH 43210, USA. E-mail: tjarks.1@osu.edu
244

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245
INTRODUCTION
Anthrax is an infectious disease that is caused by the Gram-positive
bacterium Bacillus anthracis. The primary concern with this bacterium
in the 21st century is its use as a biological weapon.[1] Ciprofloxacin,
doxycycline, and penicillin G procaine are approved in the US for the
treatment of anthrax and a vaccine (Biothrax) is available.[1,2] However,
resistance could be developed towards these antibiotics and vaccination
of large populations is difficult. This calls for the discovery of novel
antibiotics for the treatment of unvaccinated individuals who may be-
come exposed to anthrax or infected with antibiotic-resistant strains of
B. anthracis.
Key enzymes involved in the salvage and de novo pathways of DNA
synthesis, such as DNA polymerase, dihydrofolate reductase (DHFR),
thymidylate synthetase (TS), ribonucleotide reductase (RR), and 2ꢁ-
deoxynucleoside kinases (dNKs), are classic molecular targets for
anticancer- and antiviral chemotherapy.[3–5] Prokaryotes rely on the same
pathways for DNA synthesis.[6–9] With the exception of DHFR inhibitors
such as trimethoprim,[10] antibiotics targeting other key enzymes of these
pathway have not yet entered the clinical stage. Novel inhibitors of bacterial
DHFR,[7] TS,[8] RR,[9] and thymidine monophosphate kinase (TMPK)[6]
are currently under investigation. With respect to inhibiting growth of
B. anthracis, inhibitors of the de novo pathway enzymes TS and RR have
proven to be effective in biological studies.[9,11]
Thymidine monophosphate kinase of B. anthracis (BaTMPK) could
be an attractive target for the treatment of anthrax because thymidine
monophosphate (dTMP), supplied by both salvage and de novo pathways
of DNA synthesis, is diphosphorylated only by BaTMPK. To our knowledge,
there is no report on transport of thymidine diphosphate (dTDP) into
bacteria and, therefore, it is likely that dTDP production in B. anthracis
relies solely on the action of BaTMPK. Unless B. anthracis possesses an
unknown alternative mechanism to generate or transport dTDP, BaTMPK
provides an excellent molecular target for the discovery of novel antibi-
otics for the treatment of anthrax infections. For similar reasons, the
inhibition of TMPK in Mycobacterium tuberculosis by bicyclic nuclesoside
analogues has been explored for the treatment of multidrug-resistant
tuberculosis.[6]
Based on the discussion in the aforementioned, our group has ini-
tiated studies on the design and synthesis of 5ꢁ-substituted thymidine
(dThd) analogues that have the potential to function as inhibitors of
BaTMPK. These studies are the subject of this article, which also in-
cludes results from the preliminary biological evaluation of the pre-
pared BaTMPK inhibitors in enzyme- and in vitro growth inhibition
assays.

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Y. Byun et al.
FIGURE 1 3ꢁ-modified dThd analogues: 1-[2,3-Dideoxy-3-(hydroxymethyl)-β-D-erythro-pentofur-
anosyl]thymine (1) and 1-[3-(aminomethyl)-2,3-dideoxy-β-D-erythro-pentofuranosyl]thymine (2);
5ꢁ-subsituted antimicrobial nucleoside analogues: Nucleocidin (3) and salicyl-AMS (4).
RESULTS AND DISCUSSION
Design Strategies
Initially, we tested the in vitro growth inhibition of B. anthracis
Sterne by the dThd analogues 1-[2,3-dideoxy-3-(hydroxymethyl)-β-D-
erythro-pentofuranosyl]thymine (1) and 1-[3-(aminomethyl)-2,3-dideoxy-
β-D-erythro-pentofuranosyl]thymine (2) (Figure 1).[12] Both compounds
proved to be effective inhibitors of M. tuberculosis TMPK (Ki’s: 41 and
57 µM).[12] Unfortunately, neither compound produced any growth inhi-
bition of B. anthracis Sterne (IC50’s: > 1000 µM). Therefore, we decided
to pursue a different strategy for the design of BaTMPK inhibitors, which
was based on the introduction of groups at the 5ꢁ-position that potentially
could act as lipophilic replacements of phosphate in dTMP. Such inhibitors
could compete with endogenous dTMP in the substrate-binding pocket of
BaTMPK and thereby decrease thymidine triphosphate (dTTP) produc-
tion. The resulting imbalanced dNTP pool could affect DNA synthesis in
B. anthracis, and thus, reduce its growth and spread.
Bisubstrate inhibitors, consisting of adenosine and dThd units linked
through 4-6 phosphates (Ade-P4,5,6-dThd), proved to be powerful in-
hibitors of TMPK.[13,14] However, these compounds have high molecular
weights (>900 Da) and strongly ionic characters. Presumably, they cannot
cross cell membranes, which renders them unsuitable as therapeutics.
Compounds containing lipophilic phosphate-mimicking groups have been
explored extensively.[15–26] These mimics included, but were not lim-
ited to, sulfonamide-, sulfonate-, fluoride-, hydroxyl-, urea-, amide-, ester-,
carboxylate-, thiomethyl-, pyrrolidinedione-, and thiazolidine groups. Aro-
matic moieties were frequently included in these structures to increase
lipophilicity. Figure 2 shows four different design strategies for BaTMPK
inhibitors. The dThd scaffold was kept intact because it provides the
element of selective affinity for BaTMPK. Sulfonamide-, (thio)urea-, amide-,
or triazole groups were attached to the 5ꢁ-position of dThd to generate

BaTMPK Inhibitors
247
FIGURE 2 Design of dThd-based BaTMPK inhibitors substituted with lipophilic phosphate-mimicking
groups at the 5ꢁ-position.
hydrogen-bond interactions with amino acid residues in the phosphate-loop
(P-loop) region of BaTMPK. To increase lipophilicity, aromatic moieties
were bound to these groups.
Among many known nucleoside analogues with phosphate-mimicking
groups at the 5ꢁ-postion, the natural product nucleocidin (3), an inhibitor of
protein synthesis with broad antibacterial spectrum,[27–29] and salicyl-AMS
(4), an inhibitor of the siderophore biosynthesis, are of special interest (Fig-
ure 1).[30,31] Both agents demonstrated antimicrobial activity. Therefore, it
is reasonable to assume that BaTMPK inhibitor containing stable lipophilic
phosphate-mimics at the 5ꢁ-position should also be able to enter the interior
of B. anthracis.
Chemistry
Compounds 5 (Schemes 1–3) and 21 (Scheme 4) were used as starting
materials for all synthesized BaTMPK inhibitors. Both compounds were syn-
thesized according to previously reported procedures.[16,32,33] Sulfonamide-
linked dThd inhibitors (6–9) were prepared in 65–80% yield by reacting 5
with the appropriate sulfonyl chlorides [4-(methylsulfonyl)benzenesulfonyl
chloride for 6, 3-(methylsulfonyl)benzenesulfonyl chloride for 7, 2-
(methylsulfonyl)benzenesulfonyl chloride for 8, 1-naphthalenesulfonyl
chloride for 9] in the presence of (Scheme 1) triethylamine.
The thiourea-linked inhibitor 10 was prepared by reacting compound 5
with 2-(methythio)phenylisothiocyanate and a catalytic amount of DMAP

248
Y. Byun et al.
SCHEME 1 Reagents and conditions: (a) Sulfonyl chloride, TEA, DMF, rt, 6 hours.
in pyridine in 76% yield (Scheme 2). The meta- (11) and para-isomers
(12) of 10 were prepared in 73% and 83% yield by using 3-(methylthio)-
and 4-(methylthio) phenylisothiocyanate, respectively. Identical reaction
conditions were used for the synthesis of the urea-linked dThd inhibitors 13
and 14. Treatment of 3-(methylthio)- and 4-(methylthio)phenylisocyanate
with 5 afforded 13 (68% yield) and 14 (73% yield), respectively.
Various benzene carboxylic acids (see experimental section) were
coupled with 5 using 1-hydroxybenzotriazole (HOBt) and 1,3-dicyclo-
hexylcarbodiimide (DCC) to afford the amide-linked inhibitors 15–19 in
yields ranging from 38% to 58% (Scheme 3). When methyl hydrogen
phthalate was reacted with 5, compound 20 was obtained in 28% yield.
The formation of 20 was presumably facilitated by the close proximity of
the CO2Me- and the NH group in an intermediate amide, resulting in the
formation of the phthalimide via intramolecular cyclization, as shown in
Scheme 3.
The triazole-linked inhibitors 22–25 were prepared by reacting 21 with
the appropriate alkynes in 8–15% (Scheme 4) yields. The formation of
1,4-substituted-1,2,3-triazoles from azides and alkynes has been described
as “click chemistry.”[34] No protective groups were required in this reaction.
SCHEME 2 Reagents and conditions: a) Phenylisothiocyanate or phenylisocyanate, DMAP, pyridine, rt,
12 hours.

BaTMPK Inhibitors
249
SCHEME 3 Reagents and conditions: a) Benzoic acid, DCC, HOBt, DMF, rt, 12 hours.
Copper (I) is the most powerful catalyst of this 1,3-dipolar cycloaddition
of azides and alkynes.[35] We used the ‘copper (II)/ascorbate system,’
which generates the Cu (I) catalyst in situ by reducing CuSO4 with sodium
ascorbate.[36] To our knowledge, this is the first report on 5ꢁ-triazole-
substituted dThds.
Biology
Initially, the in vitro growth inhibiting effects of all target compounds
(6–20 and 22–25) on B. anthracis Sterne were tested. The results of these
studies are shown in Table 1. Compounds with IC50 values <200 µM (11,
23) and several representatives of compounds with IC50 values >200 µM (6,
SCHEME 4 Reagents and conditions: Alkyne, sodium ascorbate, CuSO4, H2O/t-BuOH (1:1), vigorous
stirring, rt, 48 hours.

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Y. Byun et al.
TABLE 1 Growth inhibition of B. anthracis Sterne and inhibition of BaTMPK activity by compounds
6–20 and 22–25
Growth inhibition IC50
BaTMPK
Growth inhibition IC50
BaTMPK
Compd.
(µM)a
inhibitionc,d Compd.
(µM)a
inhibitionc,d
6
7
8
>200
>200
>200
>200
>200
94
91
2
—
3
16
17
18
19
20
22
23
24
25
>200
>200
>200
>200
>200
—
—
—
—
—
—
9
10
11
12
13
14
15
84 11
69
69
85.1 (76.4–95.1)b
>200
6
9
—
—
—
>200
112.8 (78.2–194.2)b
>200
87
8
>200
>200
>200
—
—
>200
aIC50 after 6 hours incubation. Data are based at least on three measurements.
bLower and upper limits of 95% confidence using EPA PROBIT analysis program. Compound
concentrations ranged from 1 µM to 200 µM.
cRemaining enzyme activity (in %) as compared to enzyme activity without addition of inhibitor (100
%).
dMean SD values are based on at least three separate determinations.
8, 10, 12) were evaluated in enzyme assays with BaTMPK at concentrations
of 100 µM.
Only compound 11, with a thiourea linker and a SCH3 group at the
meta-position of the phenyl ring, and 23, with a triazole linker and a
fluorine atom at the para-position of the phenyl ring, showed moderate
growth inhibition in vitro with IC50’s of 85 µM and 113 µM, respectively.
Compounds 11 and 23 were at least 10 times better inhibitors of B. anthracis
Sterne growth than the dThd analogues 1 and 2. Compounds 11 and 12
produced moderate (∼30%) inhibition of BaTMPK. Only compound 11
showed both inhibition of B. anthracis Sterne growth and BaTMPK activity.
The inhibitory capacities of compounds 6, 8, 10, 11, 12, 23 on B. anthracis
thymidine kinase (BaTK)[11] activity were also tested and compounds 6,
8, 10, and 11 produced >50% inhibition at a concentration of 100 µM
(Usova, E., Byun, Y., Eriksson, S. and Tjarks, W., unpublished results).
However, Gram-positive bacteria have low utilization levels for TKs, and
thus, low dependence on salvage pathways for their DNA biosynthesis.[37,38]
Therefore, it is unlikely that BaTK inhibition played a significant role in
the growth inhibitory effect observed for 11. It is conceivable that the
moderate growth inhibiting effect of 23 was caused by interaction with
other intracellular kinases or even RR or DNA polymerase. The lack of
growth inhibition seen with most of the tested compounds could have been
related to poor transport into B. anthracis Sterne. Additional studies will be
necessary to elucidate the general potential of these BaTMPK inhibitors as

BaTMPK Inhibitors
251
novel antibiotics in the treatment of anthrax. Such studies would include
eukaryotic systems.
EXPERIMENTAL PROCEDURES
General
1H and 13C NMR spectra were obtained on Bruker 250 or 400 MHz (1H
resonance frequency) FT-NMR instruments. Chemical shifts are reported
in parts per million (ppm). The coupling constants are reported in Hertz
(Hz). High-resolution electrospray ionization (HR-ESI) mass spectra were
recorded on a Micromass QTOF-Electrospray mass spectrometer and a
3-Tesla Finnigan FTMS-2000 Fourier Transform mass spectrometer at The
Ohio State University Campus Chemical Instrumentation Center. Com-
pound visualization on Silica Gel 60 F254 precoated TLC plates (0.25
mm layer thickness) (Merck, Darmstadt, Germany) was attained by UV
light and KMnO4 spray. Reagent grade solvents were used for column
chromatography using Silica gel 60, particle size 0.040–0.063 mm (Merck,
Whitehouse Station, NJ, USA). Reagent grade chemicals were obtained
from commercial vendors and used as such. Anhydrous benzene and THF
were obtained using distillation from sodium benzophenone ketyl prior to
use. All reactions were carried out under argon atmosphere.
General procedure for the synthesis of sulfonamide-linked BaTMPK
inhibitors (6–9)
To a solution of 5 (60 mg, 0.25 mmol) and the indicated sulfonylchloride
(0.28 mmol) [see below] in DMF (5 mL) was added triethylamine (0.5 mL).
The resulting solution was stirred for 6 hours at room temperature. The
solution was cooled to 4◦C, triethylamine hydrochloride was removed by
filtration, and the filtrate was concentrated. The residue was triturated
with methanol to give a crystalline solid, which was collected by filtration.
Washing the solid with hexanes/ethyl acetate (1:1) afforded the following
products.
5ꢁ-Deoxy-5ꢁ-[4-(methylsulfonyl)benzenesulfonamido]thymidine (6). Sulfonyl-
chloride: 4-(Methylsulfonyl)benzenesulfonyl chloride; yield: 92 mg (80%);
Rf 0.10 (dichloromethane/methanol, 10:1); 1H NMR (DMSO-d6) δ 1.78 (s,
3H, CH3), 2.00–2.15 (m, 2H, H-2ꢁ), 2.98 (dd, 1H, H-5ꢁ, J = 13.6, 6.7 Hz),
3.11 (dd, 1H, H-5ꢁ, J = 13.6, 4.1 Hz), 3.29 (s, 3H, SO2CH3), 3.68-3.72
(m, 1H, H-4ꢁ), 4.12–4.15 (m, 1H, H-3ꢁ), 5.33 (d, 1H, OH, J = 4.3 Hz),
6.11 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.49 (s, 1H, H-6), 8.04 (d, 2H, ArH, J =
8.4 Hz), 8.13 (d, 2H, ArH, J = 8.4 Hz), 8.21 (s, 1H, NH), 11.25 (s, 1H,
NH); 13C NMR (DMSO-d6) δ 12.10 (CH3), 38.19 (C-2ꢁ), 43.14 (C-5ꢁ), 44.71
(SO2CH3), 70.84 (C-3ꢁ), 83.86 (C-1ꢁ), 84.69 (C-4ꢁ), 109.70 (C-5), 127.48

252
Y. Byun et al.
(ArC), 128.12 (ArC), 136.28 (C-6), 144.00 (ArC), 145.15 (ArC), 150.44
(C-2), 163.72 (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na)+ calcd 482.0668,
found 482.0672.
5ꢁ-Deoxy-5ꢁ-[3-(methylsulfonyl)benzenesulfonamido]thymidine (7). Sulfonyl-
chloride: 3-(Methylsulfonyl)benzenesulfonyl chloride; yield: 89 mg (78%);
1
Rf 0.16 (dichloromethane/methanol, 10:1); H NMR (MeOH-d4) δ 1.89
(d, 3H, CH3, J = 1.1 Hz), 2.00–2.23 (m, 2H, H-2ꢁ), 3.10–3.25 (m, 2H,
H-5ꢁ), 3.18 (s, 1H, SO2CH3), 3.78–3.84 (m, 1H, H-4ꢁ), 4.27–4.33 (m, 1H,
H-3ꢁ), 6.14 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.52 (d, 1H, H-6, J = 1.1 Hz), 7.82
(t, 1H, ArH, J = 7.9 Hz), 8.17 (d, 1H, ArH, J = 7.9 Hz), 8.18 (d, 1H,
ArH, J = 7.9 Hz), 8.40 (t, 1H, ArH, J = 1.7 Hz); 13C NMR (MeOH-d4)
δ 12.38 (CH3), 40.18 (C-2ꢁ), 44.17 (C-5ꢁ), 45.77 (SO2CH3), 72.47 (C-3ꢁ),
86.23 (C-1ꢁ), 86.61 (C-4ꢁ), 111.88 (C-5), 126.86 (ArC), 131.79 (ArC), 132.22
(ArC), 132.83 (ArC), 138.30 (C-6), 143.46 (ArC), 143.88 (ArC), 152.28
(C-2), 166.39 (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na)+ calcd 482.0668,
found 482.0666.
5ꢁ-Deoxy-5ꢁ-[2-(methylsulfonyl)benzenesulfonamido]thymidine (8). Sulfonyl-
chloride: 2-(Methylsulfonyl)benzenesulfonyl chloride; yield: 83 mg (72%);
1
Rf 0.16 (dichloromethane/methanol, 10:1); H NMR (MeOH-d4) δ 1.88
(d, 3H, CH3, J = 1.1 Hz), 2.15–2.25 (m, 2H, H-2ꢁ), 3.17 (dd, 1H, H-5ꢁ,
J = 13.4, 6.1 Hz), 3.24 (dd, 1H, H-5ꢁ, J = 13.4, 3.9 Hz), 3.40 (s, 1H,
SO2CH3), 3.82–3.86 (m, 1H, H-4ꢁ), 4.26–4.30 (m, 1H, H-3ꢁ), 6.11 (t, 1H,
H-1ꢁ, J = 6.9 Hz), 7.42 (d, 1H, H-6, J = 1.1 Hz), 7.86–7.90 (m, 2H, ArH),
8.21–8.24 (m, 1H, ArH), 8.27–8.31 (m, 1H, ArH); 13C NMR (MeOH-d4)
δ 12.46 (CH3), 40.10 (C-2ꢁ), 44.54 (C-5ꢁ), 45.90 (SO2CH3), 72.45 (C-3ꢁ),
85.85 (C-1ꢁ), 86.41 (C-4ꢁ), 111.94 (C-5), 132.73 (ArC), 133.82 (ArC), 134.92
(ArC), 135.57 (ArC), 138.03 (C-6), 139.68 (ArC), 140.20 (ArC), 152.17
(C-2), 166.39 (C-4); MS (HR-ESI) C17H21N3O8S2 (M+Na)+ calcd 482.0668,
found 482.0673.
5ꢁ-Deoxy-5ꢁ-(1-naphthalenesulfonamido)thymidine
(9). Sulfonylchloride:
1-Naphthalenesulfonyl chloride; yield: 70 mg (65%); Rf 0.15
(dichloromethane/methanol, 10:1); 1H NMR (MeOH-d4) δ 1.85 (d,
3H, CH3, J = 1.1 Hz), 2.12–2.19 (m, 2H, H-2ꢁ), 3.08 (dd, 1H, H-5ꢁ, J =
14.0, 6.0 Hz), 3.24 (dd, 1H, H-5ꢁ, J = 14.0, 4.1 Hz), 3.75–3.81 (m, 1H, H-4ꢁ),
4.23–4.27 (m, 1H, H-3ꢁ), 6.05 (t, 1H, H-1ꢁ, J = 6.9 Hz), 7.46 (d, 1H, H-6, J
=1.1 Hz), 7.53–7.70 (m, 3H, ArH), 8.00 (dd, 1H, ArH, J = 7.8, 1.5 Hz), 8.12
(d, 1H, ArH, J = 7.8 Hz), 8.20 (dd, 1H, ArH, J = 7.8, 1.5 Hz), 8.69 (dd, 1H,
ArH, J = 7.8, 1.5 Hz); 13C NMR (MeOH-d4) δ 12.33 (CH3), 40.08 (C-2ꢁ),
45.73 (C-5ꢁ), 72.56 (C-3ꢁ), 86.30 (C-1ꢁ), 86.84 (C-4ꢁ), 111.70 (C-5), 125.25
(ArC), 125.87 (ArC), 127.95 (ArC), 129.01 (ArC), 129.47 (ArC), 130.07
(ArC), 130.14 (ArC), 135.19 (ArC), 135.81 (ArC), 136.71 (ArC), 138.45
(C-6), 152.21 (C-2), 166.39 (C-4); MS (HR-ESI) C20H21N3O6S (M+Na)+
calcd 454.1049, found 454.1031.

BaTMPK Inhibitors
253
General procedure for the synthesis of (thio)urea-linked BaTMPK
inhibitors (10–14)
To a solution of 5 (60 mg, 0.25 mmol) and the indicated phenyliso-
cyanate or phenylisothiocyanate (0.28 mmol) [see below] in pyridine
(10 mL) was added 4-(dimethylamino)pyridine (6 mg, 0.05 mmol). The
resulting solution was stirred for 12 h at room temperature. The solvent was
evaporated and the residue was purified by silica gel column chromatogra-
phy using ethyl acetate/methanol (25:1) as the eluent to give the following
products.
5ꢁ-Deoxy-5ꢁ-{[2-(methylthio)anilinothiocarbonyl]amino}thymidine(10). Phenyl-
isothiocyanate: 2-(Methylthio)phenyl isothiocyanate; yield: 80 mg (76%);
1
Rf 0.30 (ethyl acetate/methanol, 25:1); H NMR (MeOH-d4) δ 1.88 (s,
3H, CH3), 2.22–2.25 (m, 2H, H-2ꢁ), 2.40 (s, 3H, SCH3), 3.83–3.98 (m,
2H, H-5ꢁ), 4.02–4.06 (m, 1H, H-4ꢁ), 4.34–4.38 (m, 1H, H-3ꢁ), 6.19 (t,
1H, H-1ꢁ, J = 6.9 Hz), 7.18 (dd, 1H, ArH, J = 7.8, 7.4 Hz), 7.23
(dd, 1H, ArH, J = 7.8, 7.4 Hz), 7.34 (d, 2H, ArH, J = 7.8 Hz), 7.52
(s, 1H, H-6); 13C NMR (MeOH-d4) δ 12.47 (CH3), 15.42 (CH3), 39.96
(C-2ꢁ), 49.65 (C-5ꢁ), 73.09 (C-3ꢁ), 86.34 (C-1ꢁ), 87.00 (C-4ꢁ), 111.81 (C-
5), 126.74 (ArC), 128.07 (ArC), 129.09 (ArC), 129.45 (ArC), 126.25
(ArC), 138.12 (C-6), 141.03 (ArC), 152.28 (C-2), 166.43 (C-4), 182.27
(C=S); MS (HR-ESI) C18H22N4O4S2Na (M+Na)+ calcd 445.0980, found
445.0995.
5ꢁ-Deoxy-5ꢁ-{[3-(methylthio)anilinothiocarbonyl]amino}thymidine (11). Phenyl-
isothiocyanate: 3-(Methylthio)phenyl isothiocyanate; yield: 77 mg (73%);
Rf 0.31 (ethyl acetate/methanol, 25:1); 1H NMR (MeOH-d4) δ 1.86 (d, 3H,
CH3, J = 1.0 Hz), 2.22–2.30 (m, 2H, H-2ꢁ), 2.45 (s, 3H, SCH3), 3.77–3.98
(m, 2H, H-5ꢁ), 4.04–4.10 (m, 1H, H-4ꢁ), 4.32–4.38 (m, 1H, H-3ꢁ), 6.21 (t,
1H, H-1ꢁ, J = 6.9 Hz), 7.02–7.04 (m, 1H, ArH), 7.06 (t, 1H, ArH, J =
6.9 Hz) 7.23 (d, 1H, ArH, J = 7.9 Hz), 7.27–7.30 (m, 1H, ArH), 7.50 (d, 1H,
H-6, J = 1.0 Hz); 13C NMR (MeOH-d4) δ 12.40 (CH3), 15.43 (CH3), 38.85
(C-2ꢁ), 50.31 (C-5ꢁ), 73.12 (C-3ꢁ), 86.10 (C-1ꢁ), 87.11 (C-4ꢁ), 111.97 (C-5),
121.61 (ArC), 122.71 (ArC), 124.50 (ArC), 130.51 (ArC), 138.16 (C-6),
140.15 (ArC), 141.43 (ArC), 152.30 (C-2), 166.36 (C-4), 182.66 (C=S); MS
(HR-ESI) C18H22N4O4S2Na (M+Na)+ calcd 445.0980, found 445.0995.
5ꢁ-Deoxy-5ꢁ-{[4-(methylthio)anilinothiocarbonyl]amino}thymidine (12). Phenyl-
isothiocyanate: 4-(Methylthio)phenyl isothiocyanate; yield: 88 mg (83%);
Rf 0.26 (ethyl acetate/methanol, 25:1); 1H NMR (MeOH-d4) δ 1.87 (s, 3H,
CH3), 2.22–2.28 (m, 2H, H-2ꢁ), 2.45 (s, 3H, SCH3), 3.82–3.96 (m, 2H, H-5ꢁ),
4.04–4.07 (m, 1H, H-4ꢁ), 4.34–4.38 (m, 1H, H-3ꢁ), 6.20 (t, 1H, H-1ꢁ, J =
6.9 Hz), 7.22–7.27 (m, 4H, ArH), 7.52 (s, 1H, H-6); 13C NMR (MeOH-d4)
δ 12.41 (CH3), 16.00 (CH3), 39.84 (C-2ꢁ), 49.71 (C-5ꢁ), 73.08 (C-3ꢁ), 86.17
(C-1ꢁ), 87.08 (C-4ꢁ), 111.87 (C-5), 126.34 (ArC), 128.43 (ArC), 138.17 (C-6),
152.29 (C-2), 166.38 (C-4), 182.80 (C=S); MS (HR-ESI) C18H22N4O4S2Na
(M+Na)+ calcd 445.0980, found 445.1007.

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5ꢁ-Deoxy-5ꢁ-{[3-(methylthio)anilinocarbonyl]amino}thymidine
(13). Phenyl-
isocyanate: 3-(Methylthio)phenyl isocyanate; yield: 69 mg (68%); Rf 0.36
1
(ethyl acetate/methanol, 15:1); H NMR (DMSO-d6) δ 1.77 (s, 3H, CH3),
2.04–2.19 (m, 2H, H-2ꢁ), 2.42 (s, 3H, SCH3), 3.19–3.48 (m, 2H, H-5ꢁ),
3.76–3.79 (m, 1H, H-4ꢁ), 4.15–4.16 (m, 1H, H-3ꢁ), 5.33 (d, 1H, OH, J =
4.2 Hz), 6.18 (t, 1H, H-1ꢁ, J = 7.4 Hz), 6.32 (t, 1H, NH, J = 5.8 Hz), 6.78 (d,
1H, ArH, J = 7.8 Hz), 7.05 (d, 1H, ArH, J = 7.8 Hz), 7.15 (t, 1H, ArH, J =
7.8 Hz) 7.43 (s, 1H, ArH), 7.52 (s, 1H, H-6), 8.58 (s, 1H, NH), 11.32 (s, 1H,
NH); 13C NMR (DMSO-d6) δ 12.06 (CH3), 14.57 (CH3), 38.39 (C-2ꢁ), 41.43
(C-5ꢁ), 71.13 (C-3ꢁ), 83.74 (C-1ꢁ), 85.30 (C-4ꢁ), 109.77 (C-5), 114.18 (ArC),
114.60 (ArC), 118.46 (ArC), 129.14 (ArC), 136.07 (C-6), 138.38 (ArC),
140.91 (ArC), 150.48 (C-2), 155.12 (C=O), 163.70 (C-4); MS (HR-ESI)
C18H22N4O5S (M+Na)+ calcd 429.1209, found 429.1214.
5ꢁ-Deoxy-5ꢁ-{[4-(methylthio)anilinocarbonyl]amino}thymidine
(14). Phenyl-
isocyanate: 4-(Methylthio)phenyl isocyanate, yield: 74 mg (73%); Rf 0.34
1
(ethyl acetate/methanol, 15:1); H NMR (DMSO-d6) δ 1.77 (s, 3H, CH3),
2.04–2.17 (m, 2H, H-2ꢁ), 2.40 (s, 3H, SCH3), 3.17–3.43 (m, 2H, H-5ꢁ),
3.75–3.78 (m, 1H, H-4ꢁ), 4.17–4.21 (m, 1H, H-3ꢁ), 5.37 (d, 1H, OH, J =
4.2 Hz), 6.15 (t, 1H, H-1ꢁ, J = 7.4 Hz), 6.88 (t, 1H, NH, J = 5.8 Hz), 7.14 (d,
2H, ArH, J = 8.6 Hz), 7.37 (d, 2H, ArH, J = 8.6 Hz), 7.66 (s, 1H, H-6), 9.38
(s, 1H, NH), 11.28 (s, 1H, NH); 13C NMR (DMSO-d6) δ 11.90 (CH3), 16.26
(CH3), 38.50 (C-2ꢁ), 40.50 (C-5ꢁ), 70.82 (C-3ꢁ), 83.39 (C-1ꢁ), 85.21 (C-4ꢁ),
109.80 (C-5), 118.06 (ArC), 127.96 (ArC), 128.47 (ArC), 136.13 (C-6),
138.60 (ArC), 150.45 (C-2), 155.49 (C=O), 163.71 (C-4); MS (HR-ESI)
C18H22N4O5S (M+Na)+ calcd 429.1209, found 429.1212.
General procedure for the synthesis of amide-linked BaTMPK inhibitors
(15–20)
To a solution of 5 (60 mg, 0.25 mmol) and the indicated benzoic acid
(0.30 mmol) [See below] in DMF (8 mL) was added 1-hydroxybenzotriazole
(HOBt, 41 mg, 0.30 mmol). A solution of 1,3-dicyclohexylcarbodiimide
(DCC, 62 mg, 0.30 mmol) in DMF (2 mL) was added slowly and the resulting
reaction mixture was stirred for 12 hours at room temperature. The solvent
was removed under reduced pressure and the residue was purified by silica
gel column chromatography using ethyl acetate/methanol (20:1) as the
eluent to give the following products.
5ꢁ-Deoxy-5ꢁ-[4-(sulfamoyl)benzamido]thymidine (15). Benzoic acid: 4-(Sulf-
amoyl)benzoic acid; yield: 51 mg (48%); Rf 0.22 (ethyl acetate/methanol,
1
15:1); H NMR (DMSO-d6) δ 1.77 (s, 3H, CH3), 2.07–2.18 (m, 2H, H-2ꢁ),
3.52–3.55 (m, 2H, H-5ꢁ), 3.88–3.90 (m, 1H, H-4ꢁ), 4.24–4.26 (m, 1H, H-3ꢁ),
5.33 (d, 1H, OH, J = 4.4 Hz), 6.15 (t, 1H, H-1ꢁ, J = 6.7 Hz), 7.48 (s, 2H,
NH2), 7.52 (s, 1H, H-6), 7.90 (d, 2H, ArH, J = 8.3 Hz), 8.01 (d, 2H, ArH,
J = 8.3 Hz), 8.82 (t, 1H, NH, J = 5.6 Hz), 11.30 (s, 1H, NH); 13C NMR

BaTMPK Inhibitors
255
(DMSO-d6) δ 11.96 (CH3), 38.50 (C-2ꢁ), 41.67 (C-5ꢁ), 71.26 (C-3ꢁ), 84.03
(C-1ꢁ), 84.71 (C-4ꢁ), 109.62 (C-5), 125.60 (ArC), 127.88 (ArC), 136.17 (ArC),
137.11 (C-6), 146.30 (ArC), 150.41 (C-2), 163.68 (C-4), 165.47 (C=O); MS
(HR-ESI) C17H20N4O7S (M+Na)+ calcd 447.0950, found 447.0953.
5ꢁ-Deoxy-5ꢁ-[4-(methylsulfonyl)benzamido]thymidine (16). Benzoic acid: 4-
(Methylsulfonyl)benzoic acid; yield: 56 mg (53%); Rf 0.27 (ethyl ac-
etate/methanol, 15:1); 1H NMR (DMSO-d6) δ 1.76 (s, 3H, CH3), 2.07–2.17
(m, 2H, H-2ꢁ), 3.28 (s, 3H, SO2CH3), 3.54–3.58 (m, 2H, H-5ꢁ), 3.90–3.94 (m,
1H, H-4ꢁ), 4.25–4.29 (m, 1H, H-3ꢁ), 5.40 (d, 1H, OH, J = 4.3 Hz), 6.15 (t,
1H, H-1ꢁ, J = 6.8 Hz), 7.53 (s, 1H, H-6), 8.02–8.18 (m, 4H, ArH), 9.02 (s, 1H,
NH), 11.38 (s, 1H, NH); 13C NMR (DMSO-d6) δ 12.10 (CH3), 38.11 (C-2ꢁ),
42.14 (C-5ꢁ), 44.19 (SO2CH3), 70.89 (C-3ꢁ), 83.81 (C-1ꢁ), 84.56 (C-4ꢁ), 110.11
(C-5), 127.40 (ArC), 128.64 (ArC), 139.50 (C-6), 143.33 (ArC), 150.64 (C-2),
164.02 (C-4), 165.62 (ArC); MS (HR-ESI) C18H21N3O7S (M+Na)+ calcd
446.0998, found 446.1002.
5ꢁ-Deoxy-5ꢁ-[4-(methylthio)benzamido]thymidine (17). Benzoicacid:4-(Methyl-
thio)benzoic acid; yield: 57 mg (58%); Rf 0.37 (ethyl acetate/methanol,
1
15:1); H NMR (DMSO-d6) δ 1.76 (s, 3H, CH3), 2.07–2.12 (m, 2H, H-2ꢁ),
2.51 (s, 3H, SCH3), 3.49–3.52 (m, 2H, H-5ꢁ), 3.88–3.90 (m, 1H, H-4ꢁ),
4.22–4.26 (m, 1H, H-3ꢁ), 5.32 (d, 1H, OH, J = 4.3 Hz), 6.14 (t, 1H, H-1ꢁ,
J = 6.8 Hz), 7.32 (d, 2H, ArH, J = 8.4 Hz), 7.51 (s, 1H, H-6), 7.81 (d, 2H,
ArH, J = 8.4 Hz), 8.58 (t, 1H, NH, J = 5.6 Hz), 11.30 (s, 1H, NH); 13C
NMR (DMSO-d6) δ 12.45 (CH3), 14.59 (CH3), 41.90 (C-2ꢁ), 49.08 (C-5ꢁ),
71.76 (C-3ꢁ), 84.84 (C-1ꢁ), 85.21 (C-4ꢁ), 110.47 (C-5), 125.48 (ArC), 128.32
(ArC), 130.47 (ArC), 136.88 (C-6), 143.51 (ArC), 150.99 (C-2), 164.59
(C-4), 167.14 (C=O); MS (HR-ESI) C18H21N3O5S (M+Na)+ calcd 414.1100,
found 414.1104.
5ꢁ-Deoxy-5ꢁ-[4-(methoxycarbonyl)benzamido]thymidine (18). Benzoic acid:
mono-Methyl terephthalate; yield: 45 mg (43%); Rf 0.37 (ethyl ac-
etate/methanol, 15:1); 1H NMR (DMSO-d6) δ 1.76 (s, 3H, CH3), 2.08–2.22
(m, 2H, H-2ꢁ), 3.51–3.56 (m, 2H, H-5ꢁ), 3.88 (s, 3H, OCH3), 3.88–3.92 (m,
1H, H-4ꢁ), 4.24–4.26 (m, 1H, H-3ꢁ), 5.31 (d, 1H, OH, J = 4.4 Hz), 6.14 (t, 1H,
H-1ꢁ, J = 6.7 Hz), 7.51 (s, 1H, H-6), 7.97 (d, 2H, ArH, J = 7.3 Hz), 8.04 (d,
2H, ArH, J = 7.3 Hz), 8.80 (t, 1H, NH, J = 5.0 Hz), 11.28 (s, 1H, NH); 13C
NMR (MeOH-d4) δ 11.93 (CH3), 38.36 (C-2ꢁ), 41.67 (C-5ꢁ), 52.33 (OCH3),
71.28 (C-3ꢁ), 84.06 (C-1ꢁ), 87.73 (C-4ꢁ), 109.60 (C-5), 127.62 (ArC), 129.11
(ArC), 131.79 (ArC), 136.15 (ArC), 138.34 (C-6), 150.41 (C-2), 163.68 (C-4),
165.65 (C=O), 165.68 (C=O), 169.36 (C=O); MS (HR-ESI) C19H21N3O7
(M+Na)+ calcd 426.1277, found 426.1285.
5ꢁ-Deoxy-5ꢁ-[3-(methoxycarbonyl)benzamido]thymidine (19). Benzoic acid:
Methyl hydrogen isophthalate; yield: 38 mg (38%); Rf 0.36 (ethyl ac-
etate/methanol, 15:1); 1H NMR (MeOH-d4) δ 1.82 (s, 3H, CH3), 2.25–2.30
(m, 2H, H-2ꢁ), 3.63–3.78 (m, 2H, H-5ꢁ), 3.93 (s, 3H, OCH3), 4.02–4.06
(m, 1H, H-4ꢁ), 4.34–4.38 (m, 1H, H-3ꢁ), 6.22 (t, 1H, H-1ꢁ, J = 7.4 Hz),

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Y. Byun et al.
7.50 (s, 1H, H-6), 7.59 (t, 1H, ArH, J = 7.8 Hz), 8.09 (dt, 1H, ArH, J
= 7.8, 1.2 Hz), 8.17 (dd, 1H, ArH, J = 7.8, 1.2 Hz), 8.50 (t, 1H, ArH,
J = 1.2 Hz); 13C NMR (MeOH-d4) δ 12.29 (CH3), 40.11 (C-2ꢁ), 42.88
(C-5ꢁ), 52.86 (OCH3), 73.06 (C-3ꢁ), 86.43 (C-1ꢁ), 87.03 (C-4ꢁ), 111.75 (C-5),
129.42 (ArC), 130.02 (ArC), 131.88 (ArC), 132.87 (ArC), 133.45 (ArC),
136.04 (ArC), 138.20 (C-6), 152.32 (C-2), 163.36 (C-4), 167.73 (C=O),
169.36 (C=O); MS (HR-ESI) C19H21N3O7 (M+Na)+ calcd 426.1277, found
426.1289.
5ꢁ-Deoxy-5ꢁ-(phthalimido)thymidine (20). Benzoic acid: Methyl hydrogen
phthalate; yield: 26 mg (28%); Rf 0.57 (ethyl acetate/methanol, 15:1);
1H NMR (DMSO-d6) δ 1.82 (s, 3H, CH3), 2.07–2.26 (m, 2H, H-2ꢁ), 3.78
(dd, 1H, H-5ꢁ, J = 14.2, 7.7 Hz), 3.85 (dd, 1H, H-5ꢁ, J = 14.2, 5.6 Hz),
3.99–4.04 (m, 1H, H-4ꢁ), 4.22–4.26 (m, 1H, H-3ꢁ), 5.37 (d, 1H, OH, J =
4.1 Hz), 6.14 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.56 (s, 1H, H-6), 7.83–7.92 (m, 4H,
ArH), 11.25 (s, 1H, NH); 13C NMR (DMSO-d6) δ 12.86 (CH3), 38.84 (C-2ꢁ),
47.37 (C-5ꢁ), 72.44 (C-3ꢁ), 83.19 (C-1ꢁ), 84.18 (C-4ꢁ), 109.55 (C-5), 123.14
(ArC), 131.43 (ArC), 134.54 (ArC), 136.12(C-6), 150.36 (C-2), 163.65 (C-4),
167.86 (C=O); MS (HR-ESI) C18H17N3O6 (M+Na)+ calcd 394.1015, found
394.1024.
General procedure for the synthesis of triazole-linked BaTMPK inhibitors
(22–25)
Compound 21 (65 mg, 0.25 mmol) and the indicated alkyne
(0.25 mmol) [see below] were dissolved in a mixture of water and tert-BuOH
(6 mL, 1:1). Freshly prepared sodium ascorbate solution (0.05 mL, 1 M
solution in water) was added, followed by the addition of freshly prepared
copper (II) sulfate pentahydrate (0.01 mL, 0.5 M solution in water). During
vigorous stirring of the reaction mixture at room temperature for 48 hours,
a precipitate formed. The reaction mixture was diluted with 20 mL of water
and cooled to 0◦C. A precipitate was collected by filtration and dried in
vacuum to give the following products.
5ꢁ-Deoxy-5ꢁ-[4-phenyl-(1,2,3)triazol-1-yl]thymidine (22). Alkyne: Ethynyl-
benzene; yield: 14 mg (15%); Rf 0.18 (dichloromethane/methanol, 10:1);
1H NMR (DMSO-d6) δ 1.68 (s, 3H, CH3), 2.07–2.21 (m, 2H, H-2ꢁ), 3.78 (s,
3H, OCH3), 4.11–4.14 (m, 1H, H-4ꢁ), 4.29–4.33 (m, 1H, H-3ꢁ), 4.67 (dd, 1H,
H-5ꢁ, J = 14.3, 6.8 Hz), 4.76 (dd, 1H, H-5ꢁ, J = 14.3, 4.5 Hz), 5.53 (d, 1H,
OH, J = 4.4 Hz), 6.19 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.24 (s, 1H, H-6), 7.31-7.44
(m, 3H, ArH), 7.84 (d, 2H, ArH, J = 7.5 Hz), 8.57 (s, 1H, ArH), 11.31 (s,
1H, NH); 13C NMR (DMSO-d6) δ 11.96 (CH3), 37.85 (C-2ꢁ), 51.09 (C-5ꢁ),
71.38 (C-3ꢁ), 83.72 (C-1ꢁ), 84.65 (C-4ꢁ), 115.16 (C-5), 122.19 (ArC-triazole),
125.10 (ArC), 127.87 (ArC), 128.87 (ArC), 130.59 (ArC), 138.20 (C-6),
146.35 (ArC-triazole), 152.32 (C-2), 163.36 (C-4); MS (HR-ESI) C18H19N5O4
(M+Na)+ calcd 392.1335, found 392.1332.

BaTMPK Inhibitors
257
5ꢁ-Deoxy-5ꢁ-[4-(4-fluorophenyl)-(1,2,3)triazol-1-yl]thymidine
(23). Alkyne:
1-Ethynyl-4-fluorobenzene;
yield:
13
mg
(13%);
Rf
0.14
(dichloromethane/methanol, 10:1); 1H NMR (DMSO-d6) δ 1.69 (s,
3H, CH3), 2.11–2.21 (m, 2H, H-2ꢁ), 4.09–4.14 (m, 1H, H-4ꢁ), 4.28–4.32 (m,
1H, H-3ꢁ), 4.67 (dd, 1H, H-5ꢁ, J = 14.5, 6.3 Hz), 4.76 (dd, 1H, H-5ꢁ, J = 14.5,
4.7 Hz), 5.52 (d, 1H, OH, J = 4.4 Hz), 6.19 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.23 (s,
1H, H-6), 7.28 (t, 2H, ArH, J = 8.7 Hz), 7.88 (dd, 2H, ArH, J = 8.3, 5.7 Hz),
8.55 (s, 1H, ArH), 11.28 (s, 1H, NH); 13C NMR (DMSO-d6) δ 12.00 (CH3),
37.88 (C-2ꢁ), 51.12 (C-5ꢁ), 70.52 (C-3ꢁ), 83.72 (C-1ꢁ), 83.98 (C-4ꢁ), 109.82
(C-5), 115.71 (ArC), 115.93 (ArC), 122.12 (ArC-triazole), 127.11 (ArC),
127.19 (ArC), 135.95 (C-6), 145.51 (ArC-triazole), 150.44 (C-2), 160.55
(ArC), 162.98 (ArC), 163.57 (C-4); MS (HR-ESI) C18H18FN5O4 (M+Na)+
calcd 410.1241, found 410.1241.
5ꢁ-Deoxy-5ꢁ-[4-(4-methoxyphenyl)-(1,2,3)triazol-1-yl]thymidine (24). Alkyne:
4-Ethynylanisole; yield: 11 mg (10%); Rf 0.18 (dichloromethane/methanol,
1
10:1); H NMR (DMSO-d6) δ 1.69 (s, 3H, CH3), 2.07–2.21 (m, 2H, H-2ꢁ),
3.78 (s, 3H, OCH3), 4.08–4.12 (m, 1H, H-4ꢁ), 4.28–4.32 (m, 1H, H-3ꢁ), 4.65
(dd, 1H, H-5ꢁ, J = 14.2, 6.7 Hz), 4.73 (dd, 1H, H-5ꢁ, J = 14.2, 4.5 Hz), 5.53
(d, 1H, OH, J = 4.3 Hz), 6.19 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.00 (d, 2H, ArH, J =
8.7 Hz), 7.24 (s, 1H, H-6), 7.76 (d, 2H, ArH, J = 8.7 Hz), 8.45 (s, 1H, ArH),
11.31 (s, 1H, NH); 13C NMR (DMSO-d6) δ 12.04 (CH3), 38.75 (C-2ꢁ), 51.94
(C-5ꢁ), 56.00 (OCH3), 71.44 (C-3ꢁ), 83.34 (C-1ꢁ), 84.65 (C-4ꢁ), 109.78 (C-5),
115.16 (ArC), 124.11 (ArC-triazole), 127.36 (ArC), 134.93 (ArC), 135.91
(C-6), 147.18 (ArC-triazole), 152.32 (C-2), 159.88 (ArC), 163.53 (C-4); MS
(HR-ESI) C19H21N5O5 (M+Na)+ calcd 422.1440, found 422.1452.
5ꢁ-Deoxy-5ꢁ-[4-(pyridine-3-yl)-(1,2,3)triazol-1-yl]thymidine (25). Alkyne: 3-
Ethynylpyridine; yield: 7 mg (8%); Rf 0.06 (dichloromethane/methanol,
1
8:1); H NMR (DMSO-d6) δ 1.68 (s, 3H, CH3), 2.08–2.20 (m, 2H, H-2ꢁ),
4.09–4.15 (m, 1H, H-4ꢁ), 4.27–4.33 (m, 1H, H-3ꢁ), 4.67 (dd, 1H, H-5ꢁ, J =
14.3, 6.5 Hz), 4.79 (dd, 1H, H-5ꢁ, J = 14.3, 4.9 Hz), 5.46 (d, 1H, OH, J =
4.3 Hz), 6.19 (t, 1H, H-1ꢁ, J = 6.8 Hz), 7.24 (s, 1H, H-6), 7.39–7.66 (m,
2H, ArH), 8.23 (d, 1H, ArH, J = 7.4 Hz), 8.69 (s, 1H, ArH), 8.86–8.89 (m,
1H, ArH), 11.29 (s, 1H, NH); MS (HR-ESI) C17H18N6O4 (M+Na)+ calcd
393.1287, found 393.1310.
Biological Studies
Cloning and Expression of Ba TMPK
In order to clone and express BaTMPK, the B. anthracis Sterne strain
◦
(34F2) was grown on nutrient agar plates for 12–18 hours at 37 C. The
bacteria were pelleted and genomic DNA was extracted according to Sam-
brook & Champ; Russell (Molecular Cloning, A Laboratory Manual, 2001). The
B. anthracis Sterne strain (34F2) tmpk gene (BAS0029) was amplified with

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Y. Byun et al.
the PCR primers (forward) 5ꢁ-CCACCATATGAAGGGATTATTTGTAACA-3ꢁ
and (reverse) 5ꢁ-CGGATCCTTATAACAATTTATCTTCTATAACCTG-3ꢁ. The
primers contained an Nde I and a BamH I site, respectively, to facilitate sub-
cloning into the pET-14b expression vector (Novagen). The PCR product
was cloned into the pCRꢀR 4-TOPO vector (Invitrogen, Carlsbad, CA, USA)
and sequenced by the automated dye terminator cycle sequencing method
(3700 DNA Analyzer, Applied Biosystems, Foster City, CA). The tmpk gene
was subcloned into the pET-14b expression vector and transformed into
the chemical competent E. coli strain, BL21 (DE3) pLysS (Novagen). The
bacteria were cultured as previously described[11,39] at 37◦C to an OD600
of 0.6. Recombinant BaTMPK was expressed by induction for 6 h with
0.4 mM IPTG at 25◦C. Purification of recombinant BaTMPK was carried
out as previously described[11 39]
PAGE.[40]
,
and the enzyme was verified with SDS-
In Vitro Toxicity Studies
In vitro toxicity studies were carried out as described previously by us.[11]
B. anthracis Sterne was cultured in M9 minimal media with amino acids for
12–18 hours at 37◦C with constant shaking. An aliquot of the overnight
culture was diluted with fresh M9 minimal media with amino acids to an
optical density in the range of 0.050 to 0.100 at 600 nm. Compounds 6–20
and 22–25 were dissolved in DMSO to produce stock solutions with 500 µM
concentrations. Low concentrations (200, 100, 10, and 1 µM of all tested
compounds in the first experiment and 200, 100, 50, 25, 12.5, and 6.25 µM
of 11 and 23 for next two experiments) were prepared by diluting the initial
stock solutions with the M9 minimal media. Each stock solution was freshly
prepared before the experiment. The stock solution (50 µL) and the culture
media (5 mL) were mixed and incubated at 37◦C with constant shaking.
An aliquot (0.5 mL) of the culture was removed at 6 hours and the optical
density at 600 nm was determined. The percentage of growth inhibition
test(A600 nm
)
was determined using the formula %I =
× 100. The minimal
inhibitory concentration (IC50) was determcionnetrdol(bAy60l0innmear regression analysis
using Pharmacokinetic compartment model 107 (inhibitory effect sigmoid
Emax model) implemented in WinNonlin Professional (version 5.0.1, Cary,
NC, USA).
)
Enzymatic Studies
Inhibition of BaTMPK activity by compounds 6, 8, 10, 11, 12, and 23
was followed by measuring ADP production in a coupled enzyme system
with pyruvate kinase and lactate dehydrogenase.[41] The reaction mixture
contained 0.6 µg BaTMPK, 1 mM ATP, 10 µM dTMP, 50 mM Tris-HCl (pH
7.6), 2 mM MgCl2, 5 mM DTT, 1 mM phosphoenolpyruvate, 2 units/mL

BaTMPK Inhibitors
259
pyruvate kinase, 2 units/mL lactate dehydrogenase, 100 mM NADPH, and
100 µM inhibitor. The reaction was performed at 37◦C with a Cary 3
spectrophotometer (Varian Techtron, Mulgrave, Australia).
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