Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase

Article abstract of DOI:10.1016/j.bmc.2014.01.017

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.

Full text of DOI:10.1016/j.bmc.2014.01.017

Bioorganic & Medicinal Chemistry 22 (2014) 1726–1735
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Reaction intermediate analogues as bisubstrate inhibitors
of pantothenate synthetase
Zhixiang Xu a, Wei Yin a, Leonardo K. Martinelli b, Joanna Evans c, Jinglei Chen a, Yang Yu a,
Daniel J. Wilson b, Valerie Mizrahi c, Chunhua Qiao a, , Courtney C. Aldrich b,
⇑
⇑
a College of Pharmaceutical Science, Soochow University, Suzhou 215123, China
b Center for Drug Design, University of Minnesota, MN 55455, USA
c MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Institute of Infectious Disease and Molecular
Medicine and Department of Clinical Laboratory Sciences, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town 7925, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target
for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in
mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pan-
toic acid with b-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the
synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl
adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submi-
cromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular dock-
ing studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is
assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than
5% of wild-type levels.
Received 18 October 2013
Revised 25 December 2013
Accepted 14 January 2014
Available online 23 January 2014
Keywords:
Pantothenate synthetase
Tuberculosis
Bisubstrate inhibitor
Adenylation
Ó 2014 Elsevier Ltd. All rights reserved.
Coenzyme A
1. Introduction
carbonyl activating group.7,8 Bioinformatics analysis has identified
the de novo biosynthetic pathway to pantothenate as an attractive
Tuberculosis (TB) caused by members of the Mycobacterium
tuberculosis (Mtb) complex is an ancient scourge that remains the
leading source of morbidity and mortality today with an estimated
nine million new cases and 1.4 million deaths in 2011, primarily in
the developing world.1 The recommended therapy by the World
Health Organization (WHO) for drug sensitive TB is known as di-
rectly observed treatment, short course (DOTS) and requires
6 months of treatment with isoniazid, rifampin, ethambutol, and
pyrazinamide.2 This extremely long duration of treatment is likely
due to the ability of Mtb to switch its metabolism to a nonreplicat-
ing state,3 the heterogeneous nature of the bacterial subpopula-
tions residing in different lesions types,3 and the lack of drug
penetration into the site of infection.4 In order to combat this glo-
bal health threat, new drugs are needed to shorten the treatment
duration and for drug resistant strains including multidrug-resis-
tant (MDR) TB and extensively drug resistant (XDR) TB.5,6
target for the development of antimicrobial agents since this path-
way is absent in mammals, but essential in prokaryotes.9–11 Bio-
synthesis of pantothenate is accomplished by four enzymes
encoded by the genes panB, pancC, panD, and panE. PanB and PanE
are responsible for the synthesis of pantoic acid, while PanD, an
aspartate
a-decarboxylase, produces b-alanine. PanC (Rv3602c,
pantothenate synthetase) then catalyzes the condensation of pan-
toic acid with b-alanine through a two-step adenylation–ligation
reaction.12 In the adenylation half-reaction, ATP and pantoic acid
react to form a pantoyl-adenylate intermediate (Fig. 1A). Following
the release of pyrophosphate, b-alanine binds and PanC catalyzes
its ligation with the activated carbonyl of the pantoyl-adenylate
to afford pantothenate. The detailed kinetic characterization of
PanC from Mtb shows it uses a bi-uni-uni-bi ping pong kinetic
mechanism with sequential ordered binding of ATP followed by
pantoic acid and sequential ordered release of pantothenate fol-
lowed by AMP (Fig. 1B).12 The apparent KM values for pantoic acid,
ATP, and b-alanine are 0.13, 2.6, and 0.8 mM, respectively. PanC is a
33 kDa protein that exists as a homodimer in solution. The active
site is located in the N-terminal domain (residues 1–186) while
the C-terminal domain (residues 187–309) covers the active site.13
Substrates must diffuse through a gate comprising residues 75–88,
which are flexible and disordered, but becomes ordered upon
formation of the pantoyl adenylate. The structures PanC from
Pantothenate, also known as vitamin B5 is a precursor to coen-
zyme A (CoA), an essential cofactor required in central and inter-
mediary metabolism where it serves as an acyl group carrier and
⇑
Corresponding authors. Tel.: +86 512 65882092 (C. Qiao), +001 612 6257956
(C. Aldrich).
E-mail addresses: qiaochunhua@suda.edu.cn (C. Qiao), aldri015@umn.edu
(C. Aldrich).
0968-0896/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2014.01.017

Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
1727
A
B
C
D
Figure 1. Pantothenate synthetase catalyzed reactions.
Mtb in complex with substrates, intermediates, and products have
2. Results and discussion
2.1. Chemistry
been solved providing
a step-by-step view of the PanC
reaction.13,14
Inhibitors of PanC have been identified by high-throughput
screening,15–17 fragment-based approaches,18–20 dynamic combi-
natorial chemistry,21 and through the rationale design of analogues
of the pantoyl-adenylate intermediate.22,23 The pantoyl-adenylate
intermediate mimic 1, which is epimeric at the C-2 position of
the pantoyl fragment, reported by Ciulli and co-workers is the
Synthesis of diastereomerically pure 1a was achieved starting
from commercially available (R)-2-amino-3,3-dimethylbutanoic
acid 6 that was first converted to the corresponding
a
-hydroxy
-lac-
acid 7 with retention of configuration via an intermediate
a
tone (Scheme 1).24 Protection of the resultant hydroxyl group as
a TBS ether provided 8, which was coupled with N-hydroxy-
succinimide (NHS) using DCC to afford the common reaction inter-
mediate 9. The pantoyl-adenylate analogue 1a was then prepared
most potent inhibitor yet reported with a Ki of 0.22 lM with re-
spect to ATP as the varied substrate at saturating concentrations
of pantoic acid and b-alanine using a coupled biochemical assay
that measures formation of AMP.23 In this paper, we report the de-
sign, synthesis, and biochemical evaluation of five inhibitors with
the C-2 stereocenter retaining the same configuration (i.e., R) as
the reaction intermediate 1a (Fig. 2). To prevent the intramolecular
lactonization (Fig. 1C), the terminal C-4 hydroxyl of the pantoyl
moiety was removed in 1a–4 while the pantoyl carbonyl group
was deleted in 5. All compounds were also evaluated against
wild-type Mtb and a PanC depleted Mtb strain.
O
O
O
(i)
(ii)
(iii)
OH
O
OH
OH
NH2
OH
OTBS
8
7
6
NH2
N
O
N
O
O
O
(iv), (v)
N
S
N
O
N
N
H
O
O
NH2
N
O
OTBS
OH
N
Y
Me Me
O O
1a
9
OH OH
S
N
N
N
O
NH2
X
H
Z
OH
R1
N
N
O O
S
OH R2
N
N
H2N
O
O
, R1 = H, R2 = OH, X = Y = O, Z = H
, R1 = OH, R2 = H, X = Y = O, Z = H
1a
2
O
O
3, R1 = H, R2 = F, X = Y = O, Z = H
10
4, R1 = H, R2 = OH, X = CH2, Y = O, Z = H
5, R1 = H, R2 = OH, X = O, Y = H2, Z = OH
Scheme 1. Reagents and conditions: (i) H2SO4, NaNO2, H2O, 0 °C ? rt, 16 h, 81%; (ii)
TBSCl, imidazole, DMF, 16 h, 81%; (iii) NHS, DCC, DME, 16 h, 60%; (iv) 10, Cs2CO3,
DMF, 24 h; (v) 4:1 TFA–H2O, 8 °C, 48 h, 7% from 10.
Figure 2. Reaction intermediate analogues of pantoyl-adenylate.

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Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
NH2
N
NH2
N
NH2
N
NH2
N
N
N
N
N
N
N
N
O
O
O
O O
(i), (ii)
(i)
S
S
(iii)
N
N
N
N
N
H2N
O
N
O
HO
RO
O
O
HO
OH
OTBS
Et3NH
OH
11
O
O
HO
OH
OTBS
19
4
12
13
, R=TBS
, R=H
(ii)
Scheme 4. Reagents and conditions: (i) 9, Cs2CO3, DMF, 20 h; (ii) 4:1 TFA–H2O, 8 °C,
48 h, 7% from 19.
NH2
N
NH2
N
N
N
N
O
O
O
O
O
(iv), (v)
N
S
N
S
H2N
O
O
N
N
H
O
O
O
OTBS
HO
OH
(iii)
(i)
(ii)
OH
OH
OH
O
OTBS
O
O
OH OH
21
OH
14
2
Ph
20
22
Scheme 2. Reagents and conditions: (i) TBSCl (6.0 equiv), imidazole, DMF, 16 h,
70%; (ii) 2:1:1 H2O–THF–TFA, 2 h, 81%;(iii) NaH, H2NSO2Cl, dioxane, 24 h, 60%; (iv)
9, Cs2CO3, DMF, 24 h; (v) 4:1 TFA–H2O, 20 °C, 60 h, 3% from 14.
OTs
(v)
N3
(iv)
(vi)
NH2
O
O
O
O
O
O
Ph
Ph
23
24
25
Ph
NH2
N
NH2
N
NH2
N
N
N
NO2
N
N
O
O
N
S
(i)
O
O
(ii)
N
N
(vii) (viii)
N
HO
O
TBSO
S
N
O
H
N
O
O
O
N
N
H
O
OH
27
H
OH
HO
OH
5
Ph
OH
OTBS
26
OH OH
11
15
NH2
N
N
N
NH2
NH2
N
N
N
N
N
N
N
H2N
O
(iii)
(iv)
N
N
TBSO
HO
O
O
O
O
27
TBSO
16
F
TBSO
F
Scheme 5. Reagents and conditions: (i) LiAlH4, THF, 0 °C, 2 h, 58%; (ii) PhCH(OEt)2,
CSA, CH2Cl2, reflux, 24 h, 85%; (iii) TsCl, Et3N, CH2Cl2, 16 h, 83%; (iv) NaN3, DMF,
reflux, 16 h, 93%; (v) H2, Pd/C, MeOH, 2 h, 75%; (vi) 4-nitrophenyl chlorosulfate,
CH2Cl2, ꢀ78 °C, 2 h, 50%; (vii) 27, Et3N, CH2Cl2, 4 Å molecular sieves; (viii) 4:1 TFA–
H2O, ꢀ35 °C, 4 h, 4% from 26.
17
NH2
N
NH2
N
N
N
N
N
O
O
O
O
O
(v), (vi)
S
S
N
H2N
O
N
N
O
O
O
H
(Scheme 3). Fluorination of 15 with diethylaminosulfur trifluoride
(DAST) afforded 16.29 After selective removal of the 50-OTBS
employing TFA in 2:1 H2O–THF, intermediate 17 was converted
to sulfamate 18. The 1H NMR data of 17 and 18 agreed with re-
ported analytical data,30 indicating that the C-20 configuration of
16 was R. Acylation of 18 with 9 followed by deprotection of the
TBS with 80% aqueous TFA provided 3.
Carbocyclic analogue 4 was prepared from the reported ariste-
romycin nucleoside analouge 1931 by acylation with 9 followed
by deprotection with 80% aqueous TFA (Scheme 4).
OH
TBSO
F
OH
F
18
3
Scheme 3. Reagents and conditions: (i) TBSCl, Et3N, DMF, 16 h, 65%; (ii) DAST,
pyridine, CH2Cl2, 6 h, 31%; (iii) 2:1:1 H2O–THF–TFA, 20 min, 92%; (iv) NaH,
H2NSO2Cl, dioxane, 16 h, 60%; (v) 9, Cs2CO3, DMF, 20 h; (vi) 4:1 TFA–H2O, 20 °C,
72 h, 4% from 18.
by acylation of 20,30-isopropylidene-50-O-(sulfamoyl)adenosine
1025 with 9 employing Cs2CO3 in DMF followed by concomitant
deprotection of the TBS and acetonide groups using aqueous triflu-
oroacetic acid (TFA).26
The synthesis of sulfamide 5 began from commercially available
)-(ꢀ)-pantolactone 20 that was first reduced to triol 21 with
(D
LiAlH4 as described by Burkart et al., then protected as the 1,3-ben-
zylidene acetal 22 (Scheme 5).32 Functional group interconversion
of primary alcohol 22 to amine 25 was performed by sequential
tosylation, azide displacement, and catalytic reduction. Sulfamoy-
lation of 25 with 4-nitrophenyl chlorosulfate afforded 26, which
was coupled to nucleoside 2733 followed by global deprotection
of the acetonide and benzylidene acetal with 80% aqueous TFA to
furnish 5.34
Analogue 2 that is epimeric at the C-20 position was synthesized
in five steps from vidarabine 11 (Scheme 2). Persilylation with ex-
cess TBSCl afforded 12 and regioselective deprotection of the pri-
mary 50-OTBS was accomplished under carefully controlled
conditions with TFA in 2:1 H2O–THF to furnish 13. Sulfamoylation
with freshly prepared sulfamoyl chloride27 in dioxane yielded 14,
which was coupled to 9 and deprotected with 80% aqueous TFA
to provide the arabinosyl nucleoside derivative 2.
2.2. PanC inhibition studies
The 20-fluoroadenosine analogue 328 was also prepared starting
from vidarabine 11 through regioselective protection of the less
sterically encumbered 30 and 50-hydroxyl groups to yield 15
Pantothenate synthetase (PanC) from Mtb was subcloned from
BAC-Rv222 (kindly provided by the Institut Pasteur) into pET28b

Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
1729
2.3. Computational modeling
The key interactions between the intermediate analogue (Pan-
AMP, Fig. 1A) and PanC involve the secondary hydroxyl and the
carbonyl groups of the pantoyl fragment, which form hydrogen
bonds with protein residues Gln72 and Thr39, respectively. In
the adenylate moiety, the two hydroxyls of ribose form hydrogen
bonds with Asp161, Gly158 and Gln 164, while the two nitrogen
atoms at the adenine moiety interact with Val187 and Met195.
To understand the binding selectivity of our designed PanC inhib-
itors, we modeled 2–5 into PanC binding site using the reported
X-ray structure of the complex of 1 with PanC (Fig. 4A and B).17
The removal of the carbonyl group in 5 results in a loss of critical
hydrogen bond interaction with Gln72 and explains the nearly
13-fold loss of affinity relative to 1a. For inhibitor 3, the 20-fluorine
atom maintained hydrogen bond with Asp161. Replacement of the
oxygen atom by carbon in inhibitor 4 does not change the com-
pound overall binding mode with the protein and 4 displayed only
a slight loss in binding affinity (Ki = 287 nM) compared to 1a.
Figure 3. Inhibition of PanC with inhibitor 1a. Assays were performed at varying
concentrations of inhibitor 1a (0–0.8 M) and pantoic acid, fixed concentrations of
ATP (2.6 mM) and b-alanine (2.4 mM).
l
2.4. Evaluation against whole cell M. tuberculosis
Table 1
Inhibition constants for 1a–5
Pantoyl-adenylate analogues 1a–5 were evaluated against
whole cell Mtb H37RvMA in 7H9 liquid medium; however, none
of the compounds displayed any growth inhibition up to 250 lM.
Inhibitor
Ki (lM)
1a
2
3
4
5
0.27 0.04
1.73 0.24
0.99 0.30
0.87 0.12
3.47 0.48
Notably, no whole cell activity against wild-type Mtb has yet been
observed or disclosed for any previously described PanC inhibi-
tor.15–23 Mizrahi and co-workers recently reported on the prepara-
tion of a Mtb panC conditional mutant that expresses less than 5%
wild-type PanC levels.38 Depletion of PanC renders this mutant
hypersensitive to target-specific inhibitors. In order to provide
evidence that 1a–5 possess some target-based activity, the com-
pounds were screened against this PanC-depleted strain, but no
activity was observed with up to 2 mM 1a–5. Although discourag-
ing, it is important to note that the enzyme potency of these bisub-
strate inhibitors is relatively modest compared to related
bisubstrate inhibitors for other adenylating enzymes in Mtb.39
and expressed in Escherichia coli BL21 (DE3) as described in Sec-
tion 4 to provide an N-terminal His-tagged protein with kinetic
parameters commensurate with the native enzyme.12 Kinetic
studies to evaluate enzyme inhibition of each compound toward
PanC were performed under initial velocity conditions using a
continuous coupled assay that measures production of pyrophos-
phate (see Section 4).35,36
3. Conclusion
Since compounds 1a–5 are bisubstrate inhibitors, designed to
bind both the pantoic acid and ATP binding pockets, we evaluated
inhibition with respect to pantoic at fixed non-saturating concen-
trations of ATP and saturating concentrations of the third substrate
b-alanine. Representative inhibition data for compound 1a are
shown in Figure 3. The double-reciprocal plots of initial velocity
versus pantoic acid concentration at different inhibitor concentra-
tions of 1a display a pattern of intersecting lines that converge at
the y-axis, indicating that the molecule act as a competitive inhib-
We have reported the synthesis as well as the biochemical and
biological characterization of five PanC inhibitors as potential anti-
tubercular agents. These compounds structurally mimic the pan-
toyl-adenylate reaction intermediate by bioisosteric replacement
of the labile acyl–phosphate moiety with either acyl–sulfamate
or sulfamide functional groups. We demonstrated that structural
variation of the glycosyl domain of these inhibitors is well toler-
ated and the hydrogen bond interactions between the inhibitors
and the active site residues Gln72 and Gln164 are critical for high
binding affinity. Surprisingly, despite their close mimicry of the
pantoyl-adenylate, none of the compounds were exceptionally po-
itor towards pantoic acid, in which Km values are increased, but
37
there is no effect on Vmax
.
The data were fitted to Eq. 1 for com-
M.
petitive inhibition yielding a Ki value of 0.27 0.04
l
Inhibitors 2–5 were similarly evaluated and shown to possess
identical modalities of inhibition with respect to pantoic acid
(see Table 1 for inhibition constants). Inhibitor 1a containing an
acyl–sulfamate linkage and the natural ribosyl subunit is the most
tent inhibitors with Ki ranging from ꢁ0.3 to 3
lM. By contrast, re-
lated adenylate inhibitors of the enzymes MbtA and BirA in Mtb
possess picomolar dissociation constants.40,41 Ciulli reported the
binding of 1 (as a mixture of C-2 epimers) to PanC by isothermal
titration calorimetry is accompanied by a large unfavorable entro-
potent (Ki = 0.27
lM) in the series while inhibitor 5 containing a
pic term (ꢀT
D
S = +8.8 kcal/mol),23 which may reflect the energetic
penalty associated with the disorder-to-order transition of the gate
residues (amino acids 75–88) that occurs upon ligand binding.13
Thus, despite an impressive favorable enthalpy of binding PanC
sulfamide linkage is the least potent (Ki = 3.47
l
M), an approxi-
mately 13-fold difference in affinity, demonstrating that the linker
carbonyl is more important than the terminal hydroxyl group of
the pantoyl fragment. Replacement of the ribofuranosyl ring oxy-
gen with a methylene in carbocyclic analogue 3 and substitution
of the 20-hydroxyl with a fluoro group in analogue 4 are reasonably
well tolerated resulting in a modest 2–3 fold loss in affinities.
Inversion of the 20-alcohol in analogue 2 results in more pro-
nounced 4–6 fold loss in potency.
(D
H = ꢀ18.2 kcal/mol),23 the overall potency of 1a is limited by
the large unfavorable entropy that is likely to restrict the potency
of all active-site directed inhibitors of this enzyme. Since the
inhibitors have limited potency, we used an Mtb strain wherein
the levels of PanC are depleted relative to wid-type, thereby

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Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
A
B
S
Met 195
HN
O
Met 40
O
2.0
H
Gln 72
O
1.9
O
S
O
H
N
Val 187
N
NH
N
H
H
N
HN
H2N
H
1.8
2.0
O
1.8
N
O
O
N
H
O
S
N
O
H
H
2.1
1.9
HO
2.3
O
N
O
2.3
O
HO
2.6
O
H
2.3
HO
H
H
NH
Asp 161
HN
NH
O
HN
O
Thr 39
H
pro 38
Gly 158
NH2
O
Gln 164
Figure 4. Docking results of the inhibitors in the active site of the closed model of E. coli pantothenate synthetase. (A) The detailed binding interactions of inhibitor 1 in the
active site of the protein. The inhibitor is shown as sticks with grey carbons. (B) The detailed hydrogen bonds are indicated with dashed lines and H-bond distances are given.
hypersensitizing the organism to inhibitors of PanC, in order to
provide evidence of target specificity. However, none of the com-
pounds demonstrated any activity against this knock-down strain
suggesting the compounds failed to accumulate intracellularly.
This could be caused by poor cell penetration or active efflux. Alter-
natively, PanC may not be particularly vulnerable to inhibition and
thus may not represent an ideal target for development of new
antitubercular agents.
or 400 MHz NMR Spectrometers at 25 °C and referenced to TMS.
Chemical shifts are reported in ppm using the residual solvent line
as internal standard. Splitting patterns are designed as s, singlet; d,
doublet; t, triplet; m, multiplet. HRMS spectra were acquired on an
Agilent Technologies 6220 Accurate-Mass TOF LC/MS. Analytical
thin-layer chromatography (TLC) was performed on the glass-
backed silica gel sheets (silica gel 60 Å GF254). All compounds
were detected using UV light (254 or365 nm). Analytical HPLC
was conducted on SHIMADZU LC-20AD. Prior to Ki measurement,
all compounds were determined to be >95% pure by HPLC based
on the peak area percentage.
4. Materials and methods
4.1. General procedures
4.1.1. (R)-2-Hydroxy-3,3-dimethylbutanoic acid (7)
To a solution of 6 (1.0 g, 7.62 mmol, 1.0 equiv) in 0.5 M H2SO4 at
0 °C was added a solution of NaNO2 (3.15 g, 45.6 mmol, 6.0 equiv)
in H2O (10 mL) dropwise over 30 min. After addition was complete
All chemicals (reagent grade) used were purchased from Sig-
ma–Aldrich (USA) and Sinopharm Chemical Reagent Co. Ltd (Chi-
na). 1H NMR spectra were measured on Varian Unity Inova 300

Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
1731
the solution was warmed to 23 °C and stirred overnight (ꢁ16 h).
The reaction mixture was diluted with H2O (30 mL), extracted with
diethyl ether (3 ꢂ 30 mL) and the combined organic layers were
dried (Na2SO4), and concentrated to afford 7 (806 mg, 81%) as a col-
orless oil, which was used directly in next reaction. 1H NMR
(400 MHz, CDCl3) d 3.90 (s, 1H), 1.02 (s, 9H); MS (ESIꢀ) calcd for
C6H11O3 [MꢀH]ꢀ 131.1, found 131.4.
4.1.5. 20,30,50-O-Tri(tert-butyldimethylsilyl)vidarabine (12)
To a solution of vidarabine 11 (200 mg, 0.748 mmol, 1 equiv) in
DMF (1.5 mL) were added imidazole (306 mg, 4.49 mmol,
6.0 equiv) and TBSCl (677 mg, 4.49 mmol, 6.0 equiv) and the reac-
tion was stirred overnight at 23°C. The reaction mixture was parti-
tioned between EtOAc and H2O and the organic layer was dried
(NaSO4), and concentrated. Purification by flash chromatography
(80:1 CH2Cl2–MeOH) afforded the title compound (319 mg, 70%)
as a white solid: 1H NMR (400 MHz, CDCl3) d 8.36 (s, 1H), 8.03 (s,
1H), 6.48 (d, J = 3.6 Hz, 1H), 5.59 (s, 2H), 4.34–4.32 (m, 1H),
4.19–4.16 (m, 1H), 4.03–3.98 (m, 1H), 3.88–3.84 (m, 2H), 0.94
(s, 9H), 0.92 (s, 9H), 0.72 (s, 9H), 0.15 (s, 6H), 0.09 (s, 3H), 0.08
(s, 3H), -0.08 (s, 3H), ꢀ0.45 (s, 3H); 13C NMR (75 MHz, CDCl3)
d 155.5, 153.0, 149.6, 140.8, 119.3, 86.5, 85.9, 78.3, 77.6, 63.0,
26.1, 25.9, 25.7, 18.5, 18.1, 17.9, ꢀ4.4, ꢀ5.2, ꢀ5.5; MS (ESI+) calcd
for C28H56N5O4Si3 [M+H]+ 610.4, found 610.0.
4.1.2. (R)-2-(tert-Butyldimethylsilyl)oxy-3,3-dimethylbutanoic
acid (8)
To a solution of 7 (2.06 g, 15.6 mmol, 1.0 equiv) in DMF (15 mL)
were added imidazole (5.09 g, 74.8 mmol, 4.8 equiv) and tert-
butylchlorodimethylsilane (5.64 g, 37.4 mmol, 2.4 equiv). The mix-
ture was stirred overnight at 23 °C then extracted with 1:1 ethyl
acetate–petroleum ether (3 ꢂ 30 mL). The organic layer was
washed successively with a 10% aqueous citric acid solution,
H2O, saturated aqueous NaHCO3, and brine, then dried (Na2SO4),
and concentrated. The residue was dissolved in methanol
(100 mL), then an aqueous 0.8 M K2CO3 solution (50 mL) was
added. After 4 h, 10% citric acid aqueous solution was added to ad-
just pH to 4. The mixture was extracted with ethyl acetate
(3 ꢂ 30 mL) and the combined organic layers were dried (Na2SO4)
and concentrated. The residue was purified by flash chromatogra-
phy (10:1 petroleum ether–ethyl acetate) to afford the title com-
pound (3.1 g, 81%) as a colorless oil: 1H NMR (400 MHz, CDCl3) d
9.64 (br s, 1H), 3.83 (s, 1H), 0.97 (s, 9H), 0.93 (s, 9H), 0.08 (s, 3H),
0.06 (s, 3H); 13C NMR (100 MHz, CDCl3) d 176.6, 80.0, 35.4, 26.0,
25.8, 18.3, -5.2 (2C); MS (ESIꢀ) calcd for C12H25O3Si [MꢀH]ꢀ
245.2, found 245.2.
4.1.6. 20,30-O-Di(tert-butyldimethylsilyl)vidarabine (13)
To a solution of 12 (50 mg, 0.082 mmol) in THF (0.5 mL) was
added 35% aqueous TFA (2 mL). The reaction mixture was stirred
for 2 h at 23 °C, then quenched with saturated aqueous NaHCO3
and extracted with EtOAc. The combined organic layers were dried
(NaSO4) and concentrated. Purification by flash chromatography
(80:1 CH2Cl2–MeOH) afforded the title compound (33 mg, 81%)
as a white solid: 1H NMR (400 MHz, CDCl3) d 8.30 (s, 1H), 8.05 (s,
1H), 6.44 (d, J = 4.0 Hz, 1H), 5.83 (s, 2H), 4.38–4.34 (m, 1H), 4.31–
4.27 (m, 1H), 4.13 (s, 1H), 4.08–4.04 (m, 1H), 3.92–3.88 (m, 2H),
0.94 (s, 9H), 0.67 (s, 9H), 0.16 (s, 6H), -0.06 (s, 3H), -0.41 (s, 3H);
13C NMR (75 MHz, CD3OD) d 156.8, 153.6, 149.7, 141.9, 119.5,
88.0, 87.1, 79.1, 78.4, 62.6, 26.2, 26.0, 18.6, 18.3, ꢀ4.2, ꢀ4.4,
ꢀ4.8, ꢀ5.4; MS (ESI+) calcd for C22H42N5O4Si2 [M+H]+ 496.3, found
496.0.
4.1.3. (R)-2,5-Dioxopyrrolidin-1-yl-2-(tert-butyldimethylsilyl)
oxy-3,3-dimethylbutanoate (9)
To a solution of 8 (1.99 g, 8.1 mmol, 1.0 equiv) in DME (50 mL)
at 0 °C, N-hydroxysuccinimide (1.87 g, 16.2 mmol, 2.0 equiv) and
DCC (2.34 g, 11.3 mmol, 1.4 equiv) were added and the mixture
was stirred overnight at 23 °C. The mixture was filtered through
a short pad of Celite washing with ethyl acetate and the filtrate
was concentrated. The residue was purified by flash chromatogra-
phy (1:1 petroleum ether–CH2Cl2) to afford the title compound
(1.60 g, 60%) as a colorless oil: 1H NMR (400 MHz, CDCl3) d 4.09
(s, 1H), 2.81 (br s, 4H), 1.05 (s, 9H), 0.92 (s, 9H), 0.12 (s, 3H), 0.09
(s, 3H); 13C NMR (100 MHz, CDCl3) d 169.1, 168.1, 78.5, 36.0, 25.7
(3C), 18.2, ꢀ5.2, ꢀ5.5; MS (ESIꢀ) calcd for C16H29NO5Si [M]ꢀ
343.2, found 342.8.
4.1.7. 20,30-O-Di(tert-butyldimethylsilyl)-50-O-(sulfamoyl)vidar-
abine (14)
To a solution of 13 (100 mg, 0.2 mmol, 1.0 equiv) in 1,4-dioxane
(5 mL) was added NaH (60% w/w in mineral oil, 60 mg, 1.5 mmol,
7.5 equiv). The reaction mixture was stirred for 1 h at 23 °C. Next,
NH2SO2Cl27 (58 mg, 0.5 mmol, 2.5 equiv) was added and the reac-
tion was stirred for 24 h, then quenched by the slow addition of 5:1
CH2Cl2–MeOH (10 mL). The reaction mixture was filtered through
silica gel and the filtrate was concentrated. Purification by flash
chromatography (30:1 CH2Cl2–MeOH) afforded the title compound
(70 mg, 60%) as a white solid: 1H NMR (400 MHz, CDCl3) d 8.22 (s,
2H), 6.48 (d, J = 3.2 Hz, 1H), 4.48 (dd, J = 10.4, 7.2 Hz, 1H), 4.40–4.24
(m, 4H), 0.98 (s, 9H), 0.72 (s, 9H), 0.22 (s, 6H), ꢀ0.02 (s, 3H), ꢀ0.43
(s, 3H); 13C NMR (75 MHz, DMSO-d6) d 155.9, 152.7, 149.1, 139.8,
118.3, 84.2, 81.9, 77.3, 76.5, 68.1, 25.7, 25.4, 17.6, 17.3, ꢀ4.6,
ꢀ4.7, ꢀ5.4, ꢀ5.8; MS (ESI+) calcd for C22H43N6O6SSi2 [M+H]+
575.2, found 574.8.
4.1.4. 50-O-[N-((R)-2-Hydroxy-3,3-dimethylbutanoyl)sulfamoyl]
adenosine (1a)
To a solution of 1025 (100 mg, 0.26 mmol, 1.0 equiv) in DMF
(1.0 mL) was added 9 (179 mg,0.52 mmol, 2.0 equiv) and Cs2CO3
(169 mg, 0.52 mmol, 2.0 equiv) at 23 °C. After stirring 24 h at
23 °C, the solution was concentrated in vacuo. Purification by flash
chromatography (500:6:1.5 CH2Cl2–MeOH–Et3N) afforded 50-O-{N-
[(R)-2-(tert-butyldimethylsilyl)oxy-3,3-dimethylbutanoyl]sulfa-
moyl}-20,30-O-isopropylideneadenosine triethylammonium salt
(72 mg) as a yellow solid. This compound was dissolved in 80%
aqueous TFA (2 mL). After stirring 48 h at 8 °C, the solution was
concentrated in vacuo. Purification by flash chromatography
(10:1 CH2Cl2–MeOH) afforded the title compound (8 mg, 7% yield
from compound 10) as a white solid: 1H NMR (400 MHz, CD3OD)
d 8.57 (s, 1H), 8.33 (s, 1H), 6.38 (s, 1H), 5.05–5.00 (m, 1H), 4.79–
4.77 (m, 1H), 4.74–4.70 (m, 1H), 4.40–4.37 (m, 1H), 4.14–4.10
(m, 1H), 3.62 (s, 1H), 0.99 (s, 9H); 13C NMR (100 MHz, CD3OD) d
179.0, 158.7, 150.3, 140.9, 140.6, 121.2, 95.3, 85.2, 80.3, 77.2,
71.7, 59.7, 36.1, 26.5; HRMS (ESIꢀ) calcd for C16H23N6O8S [MꢀH]ꢀ
459.1304, found 459.1310.
4.1.8. 50-O-[N-((R)-2-Hydroxy-3,3-dimethylbutanoyl)sulfamoyl]
vidarabine (2)
To a solution of 14 (420 mg, 0.70 mmol, 1.0 equiv) in DMF
(10 mL) was added 9 (497 mg, 1.40 mmol, 2.0 equiv) and Cs2CO3
(476 mg, 1.40 mmol, 2.0 equiv). The reaction was stirred for 24 h
then concentrated in vacuo. The residue was purified by flash chro-
matography (500:6:1.5 CH2Cl2–MeOH–Et3N) to afford 20,30-O-di
(tert-butyldimethylsilyl)-50-O-{N-[(R)-2-(tert-butyldimethylsilyl-
oxy)-3,3-dimethylbutanoyl]sulfamoyl}vidarabine triethylammo-
nium salt (80 mg) as a yellow solid. This intermediate was
dissolved in 80% aqueous TFA (2 mL). After stirring 60 h at 23 °C,
the solution was concentrated in vacuo. Purification by flash chro-
matography (10:1 CH2Cl2–MeOH) afforded the title compound
(10 mg, 3% yield from compound 14) as a white solid: 1H NMR
(400 MHz, CDCl3) d 8.58 (s, 1H), 8.19 (s, 1H), 6.38 (d, J = 5.2 Hz,

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Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
1H), 5.01 (d, J = 13.6 Hz, 1H), 4.74 (d, J = 2.4 Hz, 1H), 4.68 (dd,
J = 16.0, 2.4 Hz, 1H), 4.52 (t, J = 5.2 Hz, 1H), 3.95 (t, J = 5.2 Hz, 1H),
3.64 (s, 1H), 1.00 (s, 9H); 13C NMR (100 MHz, CD3OD) d 179.6,
158.7, 150.1, 142.2, 141.5, 121.3, 91.8, 85.7, 81.3, 80.2, 76.9, 59.8,
36.2, 26.5;HRMS (ESIꢀ) calcd for C16H23N6O8S [MꢀH]ꢀ 459.1304,
found 459.1318.
tion of 5:1 CH2Cl2–MeOH (10 mL) then the mixture was filtered
through silica gel and the filtrate was concentrated. Purification
by flash chromatography (30:1 CH2Cl2–MeOH) afforded the title
compound (72 mg, 60%) as a white solid: 1H NMR (300 MHz, CD3-
OD) d 8.26 (s, 1H), 8.21 (s, 1H), 6.31 (dd, J = 17.1, 2.7 Hz, 1H), 5.53
(ddd, J = 52.5, 4.5, 2.7 Hz, 1H), 4.94–4.91 (m, 1H), 4.46–4.40 (m,
1H), 4.32–4.26 (m, 2H), 0.97 (s, 9H), 0.21 (s, 3H), 0.19 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 156.2, 152.8, 148.8, 139.7, 119.1, 92.2
(d, J = 187.5 Hz), 86.1 (d, J = 33.8 Hz), 80.3, 70.2 (d, J = 15.0 Hz),
67.7, 25.6, 17.8, -4.9, -5.1;MS (ESI+) calcd for C16H28FN6O5SSi
[M+H]+ 463.2, found 463.2.
4.1.9. 30,50-O-Di(tert-butyldimethylsilyl)vidarabine (15)
To a solution of vidarabine 11 (2.0 g, 7.5 mmol, 1.0 equiv) in
DMF (40 mL) were added triethylamine (5.2 mL, 37 mmol,
5.0 equiv) and TBSCl (2.82 g, 18.7 mmol, 2.5 equiv). The reaction
mixture was stirred for 16 hat 23 °C then partitioned between
EtOAc and H2O. The organic layer was dried (NaSO4), and concen-
trated. Purification by flash chromatography (80:1 CH2Cl2–MeOH)
afforded the title compound (2.4 g, 65%) as a white solid: 1H NMR
(400 MHz, CDCl3) d 8.35 (s, 1H), 8.27 (s, 1H), 6.33 (d, J = 2.4 Hz, 1H),
5.65 (s, 2H), 4.88 (d, J = 10.0 Hz, 1H), 4.37–4.34 (m, 1H), 4.14 (d,
J = 10.0 Hz, 1H), 4.07 (s, 1H), 3.96 (d, J = 11.2 Hz, 1H), 3.81 (d,
J = 11.2 Hz, 1H), 0.93 (s, 18H), 0.15 (s, 6H), 0.14 (s, 6H); MS (ESI+)
calcd for C22H42N5O4Si2 [M+H]+ 496.3, found 496.4.
4.1.13. 20-Deoxy-20-fluoro-50-O-[N-((R)-2-hydroxy-3,3-dimethy-
lbutanoyl)sulfamoyl]adenosine (3)
To a solution of 18 (140 mg, 0.30 mmol, 1.0 equiv) in DMF
(8 mL) was added 9 (196 mg, 0.60 mmol, 2.0 equiv) and Cs2CO3
(206 mg, 0.60 mmol, 2.0 equiv) at 23 °C. The reaction mixture
was stirred for 20 h at 23 °C, then concentrated in vacuo. The
residue was purified by flash chromatography (500:6:1.5
CH2Cl2–MeOH–Et3N) to afford 30-O-(tert-butyldimethylsilyl)-50-
O-{N-[(R)-2-(tert-butyldimethylsilyloxy)-3,3-dimethylbutanoyl]-
sulfamoyl}-20-deoxy-20-fluoroadenosine triethylammonium salt
(80 mg) as a yellow solid. The compound was dissolved in 80%
aqueous TFA (1 mL) and the solution was stirred at 20 °C for
72 h. The solvent was removed in vacuo. Purification by flash
chromatography (10:1 CH2Cl2–MeOH) afforded the title compound
(5.0 mg, 4% yield from 18) as a white solid: 1H NMR (400 MHz,
CD3OD) d 8.56 (s, 1H), 8.30 (s, 1H), 6.68 (d, J = 6.8 Hz, 1H), 4.97
(dd, J = 51.6, 4.4 Hz, 1H), 4.86–4.82 (m, 2H), 4.76 (dd, J = 14.0,
2.8 Hz, 1H), 4.50 (dt, J = 18.8, 4.4 Hz, 1H), 3.63 (s, 1H), 0.98 (s,
9H); 13C NMR (100 MHz, CD3OD) d 179.1, 158.7, 150.4, 141.0,
140.5, 121.3, 94.3 (d, J = 192.0 Hz), 92.4 (d, J = 31.0 Hz), 84.8, 80.2
(d, J = 7.0 Hz), 71.5 (d, J = 16.0 Hz), 59.5, 36.2, 26.5;HRMS (ESIꢀ)
calcd for C16H22FN6O7S [MꢀH]ꢀ 461.1260, found 461.1280.
4.1.10. 30,50-O-Di(tert-butyldimethylsilyl)-20-deoxy-20-fluoroad-
enosine (16)
To a solution of 15 (100 mg, 0.20 mmol, 1.0 equiv) and pyridine
(0.20 mL, 10 mmol, 10 equiv) in CH2Cl2 (3 mL) at 23 °C was added
diethylaminosulfur trifluoride (DAST) (50.1 lL, 1.0 mmol,
5.0 equiv). The reaction mixture was stirred for 6 h at 23 °C then
quenched with 5% aqueous NaHCO3 (15 mL) and extracted with
CH2Cl2 (3 ꢂ 30 mL). The combined organic layers were dried
(NaSO4) and concentrated. Purification by flash chromatography
(50:1 CH2Cl2–MeOH) afforded the title compound (20 mg, 31%)
as a white solid: 1H NMR (400 MHz, CDCl3) d 8.32 (s, 1H), 8.16 (s,
1H), 6.25 (dd, J = 15.6, 1.6 Hz, 1H), 6.05 (s, 2H), 5.32 (dd, J = 52.8,
1.6 Hz, 1H), 4.73–4.64 (m, 1H), 4.16–4.12 (m, 1H), 4.02 (dd,
J = 12.0, 2.4 Hz, 1H), 3.79 (dd, J = 12.0, 2.4 Hz, 1H), 0.93 (s, 9H),
0.89 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H), 0.08 (s, 3H), 0.06 (s, 3H);
13C NMR (75 MHz, CDCl3) d 155.8, 153.3, 149.4, 139.3, 120.1, 93.0
(d, J = 191.3 Hz), 87.0 (d, J = 33.0 Hz), 84.0, 69.4 (d, J = 15.8 Hz),
61.3, 26.0, 25.8, 18.5, 18.2, ꢀ4.6, ꢀ4.9, ꢀ5.3, ꢀ5.4; MS (ESI+) calcd
for C22H41FN5O3Si2 [M+H]+ 498.3, found 498.3.
4.1.14. 50-O-[N-((R)-2-Hydroxy-3,3-dimethylbutanoyl)sulfamoy-
l]aristeromycin (4)
To a solution of 1931 (89 mg, 0.19 mmol, 1.0 equiv) in DMF
(5 mL) was added 9 (128 mg, 0.38 mmol, 2.0 equiv) and Cs2CO3
(122 mg, 0.38 mmol, 2.0 equiv). The reaction mixture was stirred
for 20 h at 23 °C then concentrated in vacuo. The residue was puri-
fied by flash chromatography (500:8:1.5 CH2Cl2–MeOH–Et3N) to
afford 50-O-{N-[(R)-2-(tert-butyldimethylsilyl)oxy-3,3-dimethylb-
utanoyl]sulfamoyl}-20,30-O-isopropylidenearisteromycin triethyl-
ammonium salt (50 mg) as a yellow solid. The compound was
dissolved in 80% aqueous TFA (1.0 mL) and the solution was stirred
at 8 °C for 48 h. The solvent was removed in vacuo. Purification by
flash chromatography (10:1 CH2Cl2–MeOH) afforded the title com-
pound (6 mg, 7% yield from compound 19) as a white solid: 1H
NMR (400 MHz, CD3OD) d 8.56 (s, 1H), 8.27 (s, 1H), 5.04 (d,
J = 4.8 Hz, 1H), 4.93 (dd, J = 13.6, 3.6 Hz, 1H), 4.55 (dd, J = 13.6,
2.8 Hz, 1H), 4.11–4.07 (m, 1H), 4.02 (d, J = 4.8 Hz, 1H), 3.69 (s,
1H), 3.05–2.96 (m, 1H), 2.80 (d, J = 10.0 Hz, 1H), 2.04 (d,
J = 14.4 Hz, 1H), 1.00 (s, 9H); 13C NMR (100 MHz, CD3OD) d 179.1,
158.6, 150.5, 143.5, 141.2, 121.7, 80.3, 77.5, 74.4, 67.4, 60.6, 44.0,
36.1, 33.5, 26.5; HRMS (ESIꢀ) calcd forC17H25N6O7S [MꢀH]ꢀ
457.1511, found 457.1510.
4.1.11. 30-O-(tert-Butyldimethylsilyl)-20-deoxy-20-fluoroadeno-
sine (17)
To a solution of 16 (100 mg, 0.20 mmol) in THF (0.5 mL) was
added 35% aqueous TFA (2 mL) at 23 °C. The solution was stirred
for 20 min, then quenched with saturated aqueous NaHCO3 solu-
tion. The mixture was extracted with EtOAc (3 ꢂ 10 mL) and the
combined extracts were dried (NaSO4), and concentrated. Purifica-
tion by flash chromatography (50:1 CH2Cl2–MeOH) afforded the ti-
tle compound (71 mg, 92%) as a white solid: 1H NMR (400 MHz,
CD3OD) d 8.37 (s, 1H), 8.19 (s, 1H), 6.27 (dd, J = 15.2, 4.0 Hz, 1H),
5.51 (dt, J = 52.8, 4.0 Hz, 1H), 4.79–4.72 (m, 1H), 4.15 (d,
J = 2.0 Hz, 1H), 3.90 (d, J = 12.4 Hz, 1H), 3.71 (dd, J = 12.8, 2.4 Hz,
1H), 0.96 (s, 9H), 0.18 (s, 3H), 0.17 (s, 3H); 13C NMR (75 MHz,
DMSO-d6)
d
156.2, 152.6, 148.8, 139.6, 119.2, 92.3 (d,
J = 189.0 Hz), 85.7 (d, J = 32.3 Hz), 84.7, 70.0 (d, J = 15.0 Hz), 60.3,
25.6, 17.9, -4.9, -5.1; MS (ESI+) calcd for C16H27FN5O3Si [M+H]+
384.2, found 384.2.
4.1.15. (4R)-5,5-Dimethyl-4-hydroxymethyl-2-phenyl-1,3-diox-
ane (22)
4.1.12. 30-O-(tert-Butyldimethylsilyl)-20-deoxy-20-fluoro-50-O-
(sulfamoyl)adenosine (18)
To a solution of 17 (100 mg, 0.26 mmol, 1.0equiv) in 1,4-diox-
ane (5 mL) was added NaH (60 dispersion w/w in mineral oil,
32 mg, 0.78 mmol, 3.0 equiv) at 23 °C. After 1 h, NH2SO2Cl27
(75 mg, 0.65 mmol, 2.5 equiv) was added and the solution was stir-
red for 16 h at 23 °C. The reaction was quenched by the slow addi-
To a mixture of 2132 (371 mg, 2.77 mmol, 1.0 equiv) and benz-
aldehyde dimethyl acetal (548 mg, 3.04 mmol, 1.1 equiv) in CH2Cl2
(300 mL) was added camphorsulfonic acid (50 mg, 0.28 mmol,
0.1 equiv) at 23 °C. The reaction mixture was refluxed for 24 h,
then saturated NaHCO3 aqueous solution was added to adjust the
pH to 7. The mixture was extracted with EtOAc, and the combined

Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
1733
organic extracts were washed with saturated aqueous NaCl, dried
(NaSO4), and concentrated. Purification by flash chromatography
(6:1 petroleum ether–EtOAc) afforded the title compound
(500 mg, 85%) as a white solid: 1H NMR (400 MHz, CDCl3) d 7.52
(d, J = 6.8 Hz, 2H), 7.43–7.33 (m, 3H), 5.51 (s, 1H), 3.69–3.60 (m,
5H), 1.14 (s, 3H), 0.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d 138.4,
129.2, 128.4, 126.4, 102.2, 86.0, 79.0, 61.6, 31.7, 21.5, 19.3; MS
(ESI+) calcd for C13H19O3 [M+H]+ 223.1, found 223.0.
J = 9.2 Hz, 2H), 7.50–7.33 (m, 7H), 5.45 (s, 1H), 3.80–3.70 (m, 2H),
3.61 (d, J = 11.2 Hz, 1H), 3.57–3.49 (m, 1H), 3.32–3.21 (m, 1H),
1.20 (s, 3H), 0.89 (s, 3H); 13C NMR (100 MHz, CDCl3) d 154.3,
145.8, 137.7, 129.5, 128.4, 126.4, 125.4, 122.6, 102.1, 82.8, 78.3,
44.1, 31.9, 21.1, 18.7; MS (ESI+) calcd for
C19H22N2O7SNa
[M+Na]+ 445.1, found 444.8.
4.1.20. 50-Amino-50-deoxy-50-N-[N-((R)-2,2-dimethylbutane-1,3-
diol-4-yl)sulfamoyl]adenosine (5)
4.1.16. (4R)-5,5-Dimethyl-2-phenyl-4-[(trifluoromethanesulfo-
nyl)oxy]methyl-1,3-dioxane (23)
To a mixture of 26 (120 mg, 0.28 mmol, 1.0 equiv) and 2733
(167 mg, 0.56 mmol, 2.0 equiv) in CH2Cl2 (10 mL) at 23 °C was
added 4 Å molecular sieves (80 mg) and Et3N (0.40 mL, 2.80 mmol,
10 equiv). The reaction was stirred for 24 h, then filtered and the
filtrate concentrated. Purification by flash chromatography (40:1
CH2Cl2–MeOH) afforded 50-amino-50-deoxy-50-N-(N-{[(2R,4R)-5,5-
dimethyl-2-phenyl-1,3-dioxan-4-yl]methylamino}sulfamoyl)-
20,30-O-isopropylideneadenosine (100 mg) as a white solid. The
compound was dissolved in 80% aqueous TFA (2 mL) at ꢀ35 °C.
After stirring 4 h at ꢀ35 °C, the solution was concentrated in vacuo.
Purification by flash chromatography (10:1 CH2Cl2–MeOH) affor-
ded the title compound (5 mg, 4% yield from 26) as a white solid:
1H NMR (400 MHz, CD3OD) d 8.28 (s, 1H), 8.23 (s, 1H), 5.90 (d,
J = 6.8 Hz, 1H), 4.91–4.89 (m, 1H), 4.36 (dd, J = 5.2, 2.0 Hz, 1H),
4.32–4.28 (m, 1H), 3.64–3.55 (m, 2H), 3.40–3.34 (m, 3H), 3.16
(dd, J = 12.4, 2.0 Hz, 1H), 2.85 (dd, J = 12.8, 10.0 Hz, 1H), 0.81 (s,
3H), 0.78 (s, 3H); 13C NMR (100 MHz, CD3OD) d 157.6, 153.9,
150.0, 142.5, 121.2, 91.6, 85.9, 76.3, 74.3,73.0, 70.2, 46.1, 45.8,
39.6, 21.8, 20.1;HRMS (ESIꢀ) calcd for C16H26N7O7S [MꢀH]ꢀ
460.1620, found 460.1635.
To a solution of 22 (135 mg, 0.61 mmol, 1.0 equiv) and Et3N
(0.2 mL) in CH2Cl2 (5 mL) was added TsCl (173 mg, 0.91 mmol,
1.5 equiv) at 0 °C. The mixture was then warmed to 23 °C and stir-
red for 16 h. The solvent was removed in vacuo and the residue
was purified by flash chromatography (10:1 petroleum ether–
EtOAc) to afford the title compound (190 mg, 83%) as a white solid:
1H NMR (400 MHz, CDCl3) d 7.80 (d, J = 8.0 Hz, 2H), 7.48–7.34 (m,
5H), 7.27 (d, J = 8.0 Hz, 2H), 5.43 (s, 1H), 4.28 (dd, J = 10.8, 2.0 Hz,
1H), 4.11–4.03 (m, 1H), 3.91–3.85 (m, 1H), 3.65 (dd, J = 30.0,
11.2 Hz, 2H), 2.43 (s, 3H), 1.10 (s, 3H), 0.87 (s, 3H); 13C NMR
(100 MHz, CDCl3) d 144.8, 138.0, 132.9, 129.8, 129.0, 128.2,
128.0, 126.2, 101.6, 82.5, 78.5, 69.5, 31.8, 21.7, 21.4, 18.8; MS
(ESI+) calcd for C20H24O5SNa [M+Na]+ 399.1, found 399.0.
4.1.17. (4R)-4-Azidomethyl-5,5-dimethyl-2-phenyl-1,3-dioxane
(24)
To a solution of 23 (185 mg, 0.49 mmol, 1.0 equiv) in DMF
(3 mL) was added NaN3 (96 mg, 1.47 mmol, 3.0 equiv) and the
mixture was refluxed at 100 °C for 16 h. The mixture was extracted
with diethyl ether and the combined organic layers were dried
(NaSO4), and concentrated. Purification by flash chromatography
(10:1 petroleum ether–EtOAc) afforded the title compound
(113 mg, 93%) as a white solid: 1H NMR (400 MHz, CDCl3) d 7.55
(d, J = 6.8 Hz, 2H), 7.43–7.32 (m, 3H), 5.58 (s, 1H), 3.81 (d,
J = 9.2 Hz, 1H), 3.74 (d, J = 11.2 Hz, 1H), 3.65 (d, J = 11.2 Hz, 1H),
3.46 (dd, J = 12.8, 9.2 Hz, 1H), 3.20 (d, J = 13.2 Hz, 1H), 1.16 (s,
3H), 0.84 (s, 3H); 13C NMR (100 MHz, CDCl3) d 138.1, 128.9,
128.3, 126.1, 101.7, 84.7, 78.8, 50.7, 32.3, 21.5, 18.9; MS (ESI+)
calcd for C13H17N3O2Na [M+Na]+ 270.1, found 270.0.
4.2. Pantothenate synthetase assay
4.2.1. Materials
Pantoic acid was synthesized as described by Rychlik,42 7-
methyl-6-thioguanosine (MesG) was purchased from Berry and
Associates (Dexter, MI, USA), E. coli TOPO and BL21 (DE3) cells
are from Invitrogen (Carlsbad, CA, USA), restriction enzymes and
Taq polymerase are from New England Biolabs (Ipswich, MA,
USA), the vector pET28b is from EMD Biosciences (San Diego, CA,
USA), the primers for PCR are from Integrated DNA Technologies
(Coralville, IA, USA) and the PFU polymerase is from Agilent Tech-
nologies (Wilmington, DE, USA). All other chemicals, biological
buffers, and the coupling enzymes inorganic pyrophosphatase
(I1643) and purine nucleoside phosphorylase (N8264) were pur-
chased from Sigma–Aldrich (St. Louis, MO).
4.1.18. (4R)-4-Aminomethyl-5,5-dimethyl-2-phenyl-1,3-dioxane
(25)
To a solution of 24 (100 mg, 0.4 mmol, 1.0 equiv) in MeOH
(3 mL) under nitrogen was added 10% w/w Pd/C (20 mg). The nitro-
gen atmosphere was replaced with an atmosphere of H2 and the
mixture was stirred at 23 °C for 2 h. The reaction mixture was then
filtered through Celite washing with MeOH and the filtrate was
concentrated to afford the title compound (67 mg, 75%) as a white
solid: 1H NMR (300 MHz, CDCl3) d 7.56–7.28 (m, 5H), 5.47 (s, 1H),
3.89 (br s, 2H), 3.60–3.52 (m, 3H), 2.88–2.67 (m, 2H), 1.08 (s, 3H),
0.78 (s, 3H); 13C NMR (75 MHz, CDCl3) d 138.6, 128.8, 128.2, 126.1,
101.9, 87.5, 78.8, 41.4, 32.0, 21.4, 19.0; MS (ESI+) calcd for
4.2.2. Cloning, expression and purification of recombinant PanC
from Mycobacterium tuberculosis
PanC (Rv3602c) was amplified from the H37Rv BAC Rv222
using PFU turbo and the primers GCGAGCAACCACATCGTCAC and
CGACTTCAGCATCGTCCGTAAC. The PCR product was treated with
Taq using standard procedures to add 30 overhanging adenosine
residues and then cloned into pCR2.1-TOPO. The resulting plasmid
(pCDD22) was used as a template to amplify the expression con-
struct using the primers GGCATATGACGATTCCTGCGTTCCATCCC
C
13H20NO2 [M+H]+ 222.1, found 222.2.
4.1.19. 4-Nitrophenyl-[((4R)-5,5-dimethyl-2-phenyl-1,3-dioxan-
4-yl)methyl]sulfamate (26)
and
GCTCAAGCTTCAGTTTCTCCAATGTGATTCGAGGATTGCCCGG
containing the restriction sites NdeI and HindIII respectively
(underlined). The resulting PCR product was cloned into pCR2.1
giving plasmid pCDD23. The construct was digested using NdeI
and HindIII and ligated into similarly digested pET28b yielding
pCDD24. The recombinant plasmid pCDD24 was transformed into
E. coli BL21 (DE3) electrocompetent cells and streaked-out on a
Luria Bertani (LB) agar plate with 100
incubated overnight at 37 °C. A single colony was selected to inoc-
ulate 50 mL of LB with 100
starting culture.
To a solution of 25 (60 mg, 0.27 mmol, 1.0 equiv) and 4-nitro-
phenol (375 mg, 2.7 mmol, 10.0 equiv) in CH2Cl2 (3 mL) was added
4-nitrophenyl chlorosulfate (192 mg, 0.81 mmol, 3.0 equiv) drop-
wise in CH2Cl2 (3 mL) at ꢀ78 °C. The reaction was stirred 2 h at
ꢀ78 °C, then diluted with CH2Cl2 and warmed to 23 °C followed
by consecutive washes with 1 M NaH2PO4 and H2O. The organic
layer was dried (Na2SO4) and concentrated. Purification by flash
chromatography (50:1 CH2Cl2–MeOH) afforded the title compound
(60 mg, 50%) as a white solid: 1H NMR (400 MHz, CDCl3) d 8.09 (d,
l
g mLꢀ1 of kanamycin and
l
g mLꢀ1 of kanamycin to be used as a

1734
Z. Xu et al. / Bioorg. Med. Chem. 22 (2014) 1726–1735
Terrific Broth (TB) cultures (4 L) were supplemented with
100
g mLꢀ1 kanamycin, and 10 mL of an overnight starting cul-
ture added to the media. The cultures were grown at 37 °C until
gradient of 0.05 prior to docking. The crystal structures of panto-
l
thenate synthetase (M. tuberculosis. and E. coli) (PDB code:
3IVG.pdb) complex were retrieved from the RCSB Protein Data
Bank (http://www.rcsb.org/pdb/home/home.do). All bound waters
were eliminated from the protein and hydrogens were added to
the protein. Each ligand was docked into the active site of panto-
thenate synthetase using the Surflex-Dock suite of SYBYL 1.3.
an OD600 of 0.5 and then induced with 0.5 mM of isopropyl
b- -1-thiogalactopyranoside (IPTG) for 4 h at 30 °C. The cells were
D
centrifuged at 6000g for 30 min and the pellets were stored at
ꢀ80 °C. The frozen pellets were thawed in lysis buffer (50 mM
HEPES, 300 mM NaCl, 10 mM imidazole, pH 8.0) and the cells were
disrupted by sonication on a Branson Sonifier (5 ꢂ 2 min, 30% duty
cycle, power 8) and centrifuged for 30 min at 50,000g to remove
cell debris. To the supernatant was added 1 mL of 50% Ni-NTA
agarose resin in 30% ethanol (Qiagen) and the final solution was
incubated at 4 °C for 1 h. The sample was then loaded onto a grav-
ity column and the resin was washed with 15 mL of wash buffer
(50 mM HEPES, 300 NaCl, 20 mM imidazole, pH 8.0) and eluted
with 2.5 mL of elution buffer (50 mM HEPES, 300 NaCl, 250 mM
imidazole, pH 8.0). In order to remove the imidazole from the sam-
ple, a desalting column (PD-10, GE HealthCare, Piscataway NJ, USA)
was used with the storage buffer (10 mM HEPES [pH 8.0], 1 mM
EDTA, 5% glycerol). Protein concentrations were determined using
4.4. Mtb whole-cell assays
Whole-cell screening of 1–5 was carried out against wildtype
H37RvMA43 and the PanC-depleted strain as previously
described.38 Briefly, cells were grown to an OD600 of ꢁ0.2 in 7H9
medium prior to 500-fold dilution. In the case of the panC knock-
down, the medium was supplemented with Hygromycin B (Hyg;
50
l
g/mL), Kanamycin (Km; 25
lg/mL) and Gentamicin (Gm;
5
lg/mL) in order to facilitate maintenance of the regulatory vec-
tors. Using a starting concentration of 4 mM, 2-fold serial dilutions
of each inhibitor were performed in 96-well microplates contain-
ing 50
(12.5
presence and absence of an hydrotetracycline (ATc; 20 ng/mL)
alone or ATc (20 ng/mL) together with pantothenate (50 g/mL).
A volume of 50 L of the diluted cell suspension was added to each
well, yielding a final volume of 100 L per well. Plates were incu-
l
L 7H9 medium, supplemented with Hyg (25
lg/mL), Km
e
280 = 15,930 Mꢀ1 cmꢀ1 for the native PanC with a hexa-histidine
l
g/mL) and Gm (2.5 g/mL) where appropriate, in both the
l
tag and the final yield of this protocol provided approximately
45 mg per liter of cell culture.
l
l
4.2.3. In vitro inhibition studies
l
Kinetic studies to evaluate PanC inhibition of each compound
were performed under initial velocity conditions using the MesG
coupled assay (EnzChek pyrophosphatase assay, Invitrogen) with
400 nM PanC, 100 mM HEPES (pH 8.0), 2.4 mM b-alanine, 10 mM
MgCl2, 0.04 unit of pyrophosphatase, 0.1 unit of purine nucleoside
phosphorylase, 0.2 mM 7-methyl-6-thioguanosine (MesG) and
varying concentrations of inhibitor, pantoic acid, and ATP in a total
bated at 37 °C and growth was observed visually following 7, 10
and 14 days of incubation.
Acknowledgements
This research was supported by grants from the National Natu-
ral Science Foundation of China (Grant Nos. 81072514, 21002067
to C.Q.), the Ministry of Education Scholarship Fund (The Jiangsu
‘333’ Project to C.Q.), the National Institutes of Health (Grant No.
AI070219 to C.C.A.), and the Bill and Melinda Gates Foundation
(HIT-TB) Project; to V.M.) and the South African Medical Research
Council (to V.M.).
volume of 100 lL containing up to 5% DMSO. Reactions were initi-
ated by addition of PanC. In this coupled assay, pyrophosphate gen-
erated from the PanC reaction is converted to phosphate by
pyrophosphatase. Purine nucleoside phosphorylase then catalyzes
the phosphorolysis of the substrate 7-methyl-6-thioguanosine
(MesG) to the chromogenic product 7-methyl-6-thioguanine that
is measured by an increase in absorbance at 360 nm. Experiments
were performed in 96-well plates (UV Half Area plate with Trans-
parent Bottom-Corning) and formation of 7-methyl-6-thiogua-
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