Inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase by lipophilic phosphonates: SAR, QSAR, and crystallographic studies

Article abstract of DOI:10.1021/jm200363d

1-Deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) is a novel target for developing new antibacterial (including antituberculosis) and antimalaria drugs. Forty-one lipophilic phosphonates, representing a new class of DXR inhibitors, were synthesized, among which 5-phenylpyridin-2-ylmethylphosphonic acid possesses the most activity against E. coli DXR (EcDXR) with a K _i of 420 nM. Structure-activity relationships (SAR) are discussed, which can be rationalized using our EcDXR:inhibitor structures, and a predictive quantitative SAR (QSAR) model is also developed. Since inhibition studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been performed well, 48 EcDXR inhibitors with a broad chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a MtDXR:inhibitor complex reveals the flexible loop containing the residues 198-208 has no strong interactions with the 3,4-dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide implications in the future design and development of potent DXR inhibitors.

Full text of DOI:10.1021/jm200363d

ARTICLE
pubs.acs.org/jmc
Inhibition of 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase by
Lipophilic Phosphonates: SAR, QSAR, and Crystallographic Studies
Lisheng Deng,†,§ Jiasheng Diao,†,§ Pinhong Chen,† Venugopal Pujari,z Yuan Yao,† Gang Cheng,†
Dean C. Crick,z B. V. Venkataram Prasad,‡ and Yongcheng Song*,†
†Department of Pharmacology and ‡Verna and Marrs McLean Department of Biochemistry and Molecular Biology,
Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States
zDepartment of Microbiology, Colorado State University, 1682 Campus Delivery, Ft. Collins, Colorado 80523, United States
S Supporting Information
b
ABSTRACT: 1-Deoxy-D-xylulose-5-phosphate reductoisome-
rase (DXR) is a novel target for developing new antibacterial
(including antituberculosis) and antimalaria drugs. Forty-one
lipophilic phosphonates, representing a new class of DXR
inhibitors, were synthesized, among which 5-phenylpyridin-2-
ylmethylphosphonic acid possesses the most activity against
E. coli DXR (EcDXR) with a Ki of 420 nM. Structureꢀactivity
relationships (SAR) are discussed, which can be rationalized
using our EcDXR:inhibitor structures, and a predictive quanti-
tative SAR (QSAR) model is also developed. Since inhibition
studies of DXR from Mycobacterium tuberculosis (MtDXR) have not been performed well, 48 EcDXR inhibitors with a broad
chemical diversity were found, however, to generally exhibit considerably reduced activity against MtDXR. The crystal structure of a
MtDXR:inhibitor complex reveals the flexible loop containing the residues 198ꢀ208 has no strong interactions with the 3,4-
dichlorophenyl group of the inhibitor, representing a structural basis for the reduced activity. Overall, these results provide
implications in the future design and development of potent DXR inhibitors.
’ INTRODUCTION
studies and recent clinical trials.5 However, since 1 is a highly
polar molecule with great water solubility, it has a short half-life
(∼1 h) in plasma as well as poor oral availability. In addition,
many pathogens, such as Mycobacterium tuberculosis, the causa-
tive agent of tuberculosis that kills ∼1.6 million people each year,
are completely resistant to 1 and sensitive bacteria can also easily
develop drug resistance. This is because 1 is actively transported
into cells via glycerol-3-phosphate transporter GlpT.6 Pathogens
without GlpT or with a mutant GlpT are therefore not affected
by 1 due to limited cellular uptake.2d,7 Great interest has there-
fore been generated to develop potent inhibitors of DXR.8ꢀ10
A potential solution is to develop lipophilic DXR inhibitors
that are able to passively permeate into bacterial cells. Medicinal
chemistry studies based on the structures of 1 and 2 have been
carried out for the past decade.8 Although most modifications
resulted in compounds with largely reduced activity, derivatives
of 1 and 2 having an R-substituent still possess the majority of the
inhibitory activity.8j The best in this series are compounds 3 and
4 with a 3,4-dichlorophenyl group, which have similar enzyme
activity as 1 but showed greatly increased activity against
P. falciparum (EC50 of 90 nM for 3 and 250 nM for 4 vs 1100 nM
for 1),8j presumably due to enhanced cellular uptake resulting
Isoprenoid biosynthesis, producing very important substances
such as isoprenyl (e.g., farnesyl and undecaprenyl) diphosphates,
vitamins, steroids, and terpenoids, is essential to the growth of all
organisms. Isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP) are two common precursors for isopre-
noid biosynthesis. In humans and animals, the mevalonate
pathway is used to synthesize IPP and DMAPP (Figure 1). On
the other hand, the vast majority of bacteria and apicomplexan
parasites such as Plasmodium falciparum, the causative agent of
malaria, use the non-mevalonate (or methylerythritol phosphate,
MEP) isoprene biosynthesis pathway discovered in the 1990s
(Figure 1).1 Since humans do not have the 7 enzymes in the
pathway, the non-mevalonate pathway, especially the second
enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase
(DXR), has attracted much interest for its potential to be a
new anti-infective drug target.2
DXR catalyzes the isomerization and reduction of 1-deoxy-D-
xylulose-5-phosphate (DXP) to 2-C-methyl-D-erythritol-4-phos-
phate (MEP) with a Mg2þ and NADPH being the enzyme
cofactors (Figure 1). Fosmidomycin (1, Chart 1) (as well as its
analogue FR900098 (2)) is a very potent DXR inhibitor3 and has
strong antibacterial activity against many Gram-negative bacteria
such as Escherichia coli and Pseudomonas aeruginosa.4 Particularly
remarkable is its profound antimalaria activity in preclinical
Received: March 28, 2011
Published: May 11, 2011
r
2011 American Chemical Society
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Figure 1. Non-mevalonate and mevalonate isoprene biosynthesis pathways.
Chart 1. Structures of Known DXR Inhibitors 1ꢀ10
from their lipophilicity. In addition, only a few DXR inhibitors
have been identified that are structurally distinct from 1 and 2.9
Two bisphosphonates (5 and 6, Chart 1) bearing a hydrophobic
pyridine and isoquinoline side chain are relatively weak inhibitors
(IC50: 4 and 7 μM).9a We recently used a coordination chemistry
based design to find a lipophilic, drug-like DXR inhibitor 7 (IC50:
1.4 μM) without a phosphonate group, which exhibited good
activity against a broad spectrum of pathogenic bacteria.9b More-
over, in order to probe the hydrophobic nature of the DXR active
site, we identified three new lipophilic phosphonate inhibitors
8ꢀ10 having a pyridine or quinoline group with IC50 values as
low as 0.84 μM.9c
Our X-ray crystallographic studies of E. coli DXR (EcDXR) in
complex with 8ꢀ10 reveal a new inhibitor binding mode as well
as an important role of Trp211 in inhibitor recognition.9c The
great plasticity of a flexible loop (residues 205ꢀ215) of EcDXR
allows the indole ring of Trp211 to form πꢀπ stacking and
charge transfer interactions with the electron-deficient pyridine/
quinoline group of these inhibitors. Previously reported crystal
structures of EcDXR in complex with 1,11 5, and 69a also show
favorable hydrophobic interactions between Trp211 and the
inhibitors. In addition, our structures show that the conforma-
tional change of the flexible loop opens up a mainly hydrophobic
pocket, where the pyridine/quinoline ring of 8ꢀ10 is located.
These structures have been used to model the binding modes of
other lipophilic DXR inhibitors, such as compounds 3 and 4.
Here, we report synthesis, inhibitory activity, structureꢀactivity
relationships (SAR) and quantitative SAR (QSAR) of 41 lipo-
philic phosphonate and related compounds against E. coli DXR.
In addition, since M. tuberculosis is a Gram-positive bacterium
(which behaves very differently from Gram-negative bacteria,
e.g., E. coli) and causes enormous human mortalities, we also
report the activity of 48 EcDXR inhibitors with a broad range
of chemical diversity against M. tuberculosis DXR (MtDXR). The
results show that these inhibitors generally have considerably
lowered affinity to MtDXR. In addition, to investigate the
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possible structural basis for the reduced activity, a crystal
structure of MtDXR:inhibitor complex is disclosed, showing
the flexible loop of MtDXR, especially the residue Trp203 (the
aligned counterpart of EcDXR Trp211) does not have strong
interactions with the hydrophobic group of the inhibitor.
inactive likely due to its electron-donating 5-benzyloxy group,
which is also sterically more demanding. Compounds 32 and 33
bearing a 6-bromo and -phenyl group, respectively, show
slightly weaker inhibitory activity (Ki = 3.6 and 3.5 μM) against
EcDXR than does 8. Since compound 9 possesses a greatly
improved potency, five derivatives 34ꢀ38 were made in an
effort to find more active inhibitors. However, neither electron-
withdrawing -F/-Cl (for 34 and 35, Ki = 1.1 and 1.6 μM)
nor electron-donating -Me/-OMe (for 36 and 37, Ki = 3.9 and
3.6 μM) on the 5-phenyl ring provide enhanced potency. 38
with a bulkier naphthyl substituent is also a weak inhibitor with
a Ki of 13.9 μM. These results show that introducing a 5-phenyl
group leads to the strongest EcDXR inhibitor 9 among this class
of compounds.
Finally, the phosphonate group in compounds 8 and 9 was
replaced by an isosteric carboxyl, hydroxamate, or sulfonate
group with a rationale that the replacement with a less polar,
negatively charged group could result in a higher lipophilicity,
thereby improving cellular uptake. However, compounds 39ꢀ44
that are analogues of 8 with either a carboxyl or a hydroxamate
group exhibit no inhibitory activity against EcDXR at 100 μM.
Similarly, compounds 45ꢀ48, which are analogues of 9 contain-
ing a carboxyl, hydroxamate, or sulfonate group, are also inactive.
These results show that the phosphonate group is essential to the
tight binding of this class of inhibitors.
’ RESULTS AND DISCUSSION
SARs of Lipophilic Phosphonates in EcDXR Inhibition. In
our previous study,9c pyridine/quinoline containing phospho-
nate compounds 8ꢀ10 were identified to be a new class of
EcDXR inhibitors. On the basis of the structure of 8, we
synthesized a total of 41 compounds to analyze the structureꢀ
activity relationships (SAR) of this class of compounds. These
compounds were tested to inhibit recombinant EcDXR, and their
inhibition constants (Ki) are shown in Table 1.
First, compounds 11ꢀ13 (Table 1) were synthesized to find
the optimal linker length between the pyridine ring and the
phosphonate group. Compound 11 without a linker has essentially
no activity on EcDXR at 100 μM, while 12 and 13 having two and
three methylenes are active with Ki values of 18.6 and 9.5 μM,
respectively. However, compared to 8 with a Ki of 2.3 μM, they
are ∼8ꢁ and 4ꢁ less potent, suggesting that one methylene
linker is best for DXR inhibition.
Second, we optimized the aromatic ring of 8 in order to find
more potent DXR inhibitors. Compounds 14 and 15 with a
pyridin-3-yl and pyridin-4-yl group were found to be active (Ki = 5.0
and 3.5 μM). In addition, other N-containing aromatic hetero-
cyclic compounds 10 (with quinoline), 16 (with pyrazine), and
17 (with pyrimidine) also exhibit inhibitory activity against
EcDXR, with Ki values ranging from 3.5 to 11.2 μM. However,
phenyl ring containing compounds 18ꢀ20 with a variety of
linker length (n = 0ꢀ2) are inactive. Since the 3,4-dichlorophenyl
group is present in potent EcDXR inhibitors 3 and 4 (Chart 1)
with antimalaria activity,8j compound 21 having a 3,4-dichlor-
ophenyl group was prepared, but it was found to have no activity.
Compounds 22 and 23 containing electron-donating hydroxyl
groups also possess no inhibitory activity. To further probe the
electronic effects of the aromatic ring, compounds 24 and 25
were synthesized, which have hydroxyl (or tautomeric carbonyl)
substituted pyridine rings that are more electron-rich. Compared
to unsubstituted parent compounds 14 and 12, these two
compounds are essentially inactive. These results indicate that
a highly electron-deficient aromatic ring, such as pyridine, quino-
line, and pyrimidine, is needed for DXR inhibition.
Third, compounds 26ꢀ38 were synthesized to see which
substituents on the pyridine ring are favored. Compared to the
parent compound 8, compound 26 with a 4-bromo substituent
has a slightly weakened inhibitory activity with a Ki of 3.5 μM. 27
with a 4-phenyl group exhibits better activity (Ki = 1.1 μM), while
compound 28 bearing a 4-(4-fluorophenyl) group possesses a
reduced activity (Ki = 3.5 μM). For compounds 9 and 29ꢀ31
that have a 5-substituent, compound 9 with a 5-phenyl group
shows a Ki of 0.42 μM against EcDXR, which is 5ꢁ as potent as 8,
while compound 29 with a 5-bromo group exhibits a weaker
inhibition (Ki = 6.7 μM). Compound 30 with a 5-hydroxyl group
has strong activity with a Ki of 0.7 μM, being ∼3ꢁ as potent as 8.
This is rather unexpected given that 5-hydroxylpyridine is more
electron-rich and, according to the SAR found above, should lead
to a reduced activity. However, the high activity of 30 could be
rationalized by molecular modeling using the crystal structure of
the EcDXR:8 complex, as discussed below. Compound 31 is
SAR Rationalization. With the availability of the crystal
structures of EcDXR:8ꢀ10 complexes, it is possible to rationalize
the structureꢀactivity relationships observed above. Our crystal-
lographic studies show that there are two common interactions
between EcDXR and these inhibitors.9c The first one is the πꢀπ
stacking and charge transfer interactions between the electron-
deficient pyridine/quinoline group of 8ꢀ10 and the indole ring
of the residue Trp211, with the two parallel rings having a
distance of ∼3.5 Å. To see if compounds 11ꢀ13 are potentially
able to form such interactions, we docked these compounds
into the crystal structure of DXR:8 complex, using the pro-
gram Glide12 in Schr€odinger,13 and the results are shown in
Figure 2aꢀc. The phosphonate group of 11 is predicted to be
located very close to the phosphonate binding site of 8. However,
its pyridine ring cannot form a πꢀπ interaction with the indole
ring of Trp211, which might be responsible for its non-activity.
The docking results of compounds 12 and 13 indicate their
pyridine rings can be parallel to the indole, thanks to their longer,
flexible linkers. The modeling studies are in line with the
experimental data showing 12 and 13 are active against EcDXR
(Table 1). In addition, the importance of Trp211 in inhibitor
recognition also determines that an electron-deficient aromatic
ring is needed for EcDXR inhibition, because of its stronger
electrostatic interaction with the electron-rich indole ring. More-
over, the crystal structure of DXR:9 complex reveals that the
conformation of Trp211 is different from that of DXR:8, with the
indole ring flipping ∼180° in order to have more hydrophobic
interactions with the 5-phenyl group of 9.9c This might account
for the improved activity of compound 9.
The second key interaction is the tight binding of the
phosphonate group in 8ꢀ10, with all of the three negatively
charged O atoms forming a network of electrostatic and H-bond
interactions with the residues Lys227, Asn226, Ser221, and
Ser185. However, a carboxylate, hydroxamate, or sulfonate group
possesses considerably reduced polarity as well as negative
charges at the physiological pH. Lack of activity of compounds
39ꢀ48 against EcDXR can therefore be rationalized.
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Table 1. Structures of Compounds 8ꢀ48 and Their Ki Values against EcDXR and MtDXR
a No inhibition observed at 100 μM.
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and 8ꢀ48) were constructed, minimized, and aligned with
compound 8 as a template, using the “flexible ligand alignment”
module in Maestro15 in Schr€odinger, which recognizes common
features within the molecules (e.g., similar partial charge, hydro-
phobicity, aromaticity, and H-bond donor/acceptor). The
aligned compounds were imported into Phase, and a partial
least-squares (PLS) method was used to correlate the EcDXR
activities of these compounds with the Phase field data calculated
from their 3-D structures. The QSAR model yielded r2 = 0.95, q2
(no. of factors) = 0.78 (5), F-test = 158.5, and a pKi error of 0.30,
as shown in Table 3 and Figure 3a. Five leave-5-out tranining/
test sets were applied to further validate the model, obtaining a
good predictivity with r2 g 0.94, q2 g 0.62, F-test g 123.5, as
shown in detail in Table 3. The compound alignment and the
3-D QSAR virtualization showing negative ionic, electron-with-
drawing, hydrophobic, H-bond donor Phase fields are shown
in Figure 3bꢀf. The results are in good agreement with
the qualitative SAR observations. For example, the negatively
charged phosphonate group (Figure 3c) and the electron-
deficient aromatic ring (Figure 3d) are important for the
activity. A hydrophobic group (e.g., phenyl) at the 5-position
of the pyridine ring of compound 9 is favored (Figure 3e). In
addition, the negative charge (due to chelating Mg2þ) and the
electron-withdrawing feature of the hydroxamate group as well
as the hydrophobic nature of the carbon skeleton of compounds
1ꢀ4 contribute together to their superior EcDXR inhibitory
activities (Figure 3cꢀe).
Figure 2. Lowest energy docking structures of compounds (A) 11, (B)
12, (C) 13, and (D) 30 in the EcDXR:8 structure, together with
superimposed structure of 8 (in yellow).
Table 2. Ki Values of 1ꢀ7 against EcDXR and MtDXR
Inhibition of MtDXR. Since the discovery of the non-meva-
lonate isoprene biosynthesis pathway, DXRs from many species,
including bacteria, plants, and parasites, have been cloned and
characterized.3,5a,7,16 However, inhibition studies of DXRs other
than E. coli DXR have not been well performed, except that 1 has
been tested and shown broad activity on these enzymes. To this
end, we are particularly interested in DXR from M. tuberculosis
(MtDXR). This bacterium is a major human pathogen, causing
deaths of ∼1.6 million people a year worldwide. In addition, it
has become more and more resistant to first line treatments such
as isoniazid and rifampicin during the past decade.17 Previous
studies demonstrated that M. tuberculosis exclusively uses the
non-mevalonate pathway and DXR is essential for its survival,2d,7
suggesting DXR is a target for developing new antituberculosis
drugs, but the bacterium is completely resistant to the treatment
of 1 due to lack of cellular uptake. Without GlpT (glycerol-3-
phosphate transporter), 1 cannot penetrate the M. tuberculosis
cell membrane and reach the target in cytosol. Nevertheless, 1
was found to be a strong inhibitor of MtDXR. However, no other
EcDXR inhibitors have been tested against MtDXR.
We examined the activity of 48 EcDXR inhibitors (compounds
1ꢀ48) on recombinant DXR from M. tuberculosis. It is also
noteworthy that these inhibitors represent a wide range of
chemical diversity. Compounds 1 and 2 are highly polar molecules
containing a hydroxamate and a negatively charged phosphonate
group, while 3 and 4 are their derivatives with a hydrophobic 3,
4-dichlorophenyl side chain.8j Compounds 5 and 6 are isoquino-
line/pyridine containing bisphosphonates,9a which are extremely
polar with 4 negative charges at the physiological pH. Distinct
from the above compounds, 7 is a lipophilic, drug-like DXR
inhibitor without a phosphonate.9b Moreover, all of compounds
8ꢀ48 consist of a lipophilic side chain and a polar headgroup
(phosphonate, carboxylate, hydroxamate, or sulfonate).
compound ID
EcDXR Ki (μM)
MtDXR Ki (μM)
1
2
3
4
5
6
7
0.027
0.019
0.058
0.036
6.3
0.14
0.13
1.2
1.5
32.7
NI a
21.8
15.3
0.70
a No inhibition observed at 100 μM.
Moreover, molecular modeling could be used to explain why
compound 30 with the electron-donating 5-hydroxy group
possesses unexpected, strong inhibition (Ki = 0.7 μM). As shown
in Figure 2d, the docking result of 30 in the DXR:8 structure
indicates that, in addition to the phosphonate binding and the
πꢀπ stacking with Trp211, the 5-hydroxy group of 30 forms a
hydrogen bond with the carbonyl of Asn210. This extra H-bond
could compensate the weakened πꢀπ/charge transfer interac-
tions between 30 and the protein.
QSAR Study in EcDXR Inhibition. The above qualitative SAR
analysis and rationalization demonstrate that a complex combi-
nation of electronic, steric, hydrophobic, and H-bonding features
of the substituents affect the activity of these compounds. In
order to understand these observations in a quantitative and
predictive manner, we next carried out a 3-D quantitative
structure activity relationship (QSAR) study, using the program
Phase14 in Schr€odinger. Since compounds 1ꢀ4 (Chart 1) are
more potent EcDXR inhibitors (Table 2, Ki: 19ꢀ58 nM) than
8ꢀ10 and known to occupy the same binding site, they were also
included in an effort to generate a broader and more predictive
QSAR model. In addition, for those inactive compounds in
Table 1 against EcDXR, their Ki values were arbitrarily assigned
to be 300 μM for QSAR calculation. The 45 compounds (1ꢀ4
Activities of compounds 1ꢀ7 against MtDXR as well as EcDXR
are shown in Table 2. In our study, the reference compound 1
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Table 3. QSAR Results for EcDXR Inhibition Using Phase
exhibited a Ki value of 140 nM against MtDXR, which is very
close to that of 150 nM found in a previous study2d (calculated
from the IC50 of 310 nM). It is noted that in both assays
physiologically relevant Mg2þ (4 mM) was used as the cofactor.
1 was also reported to have a lower IC50 value of 80 nM using
phase pKi predictions
pKi
(M)a
training
set
test
set 1b
test
test
test
test
compound
set 2
set 3
set 4
set 5
2.1 mM of Mn .
2þ 18 However, such a high concentration of Mn2þ
1
7.57
7.72
7.24
7.44
5.64
6.38
5.10
3.52
4.73
5.02
5.30
5.46
4.95
5.46
3.52
3.52
3.52
3.52
3.52
3.52
3.52
3.52
5.46
5.96
5.46
5.17
6.15
3.52
5.46
5.46
5.96
5.80
5.41
5.46
4.86
3.52
3.52
3.52
3.52
3.52
3.52
3.52
3.52
3.52
3.52
7.67
7.95
7.13
7.13
5.57
5.85
4.76
3.72
4.54
5.18
4.88
5.03
5.40
6.09
3.29
4.20
3.72
3.78
3.51
3.68
3.48
3.53
5.61
5.60
5.53
5.16
5.50
3.29
5.29
5.60
5.88
5.82
5.80
5.75
4.67
3.36
3.53
3.59
3.57
3.38
3.31
3.68
3.50
3.59
3.89
0.95
0.78
158.5
5
7.58
7.84
6.94
7.10
5.51
5.81
4.75
3.65
4.57
5.22
4.88
4.97
5.34
6.07
3.50
4.12
3.74
3.72
3.58
3.59
3.45
3.55
5.66
6.09
6.06
5.10
5.47
3.22
5.25
5.56
5.86
5.82
5.79
5.71
5.09
3.29
3.52
3.59
3.59
3.33
3.33
3.70
3.48
3.58
3.80
0.95
0.68
7.82
7.48
7.06
6.93
5.66
5.77
4.77
3.79
4.59
5.24
4.93
5.10
5.69
6.15
3.36
4.36
3.61
3.91
3.34
3.84
3.57
3.56
5.75
5.84
5.80
5.22
5.58
3.38
5.42
5.74
5.77
5.69
5.67
5.66
4.58
3.33
3.81
3.50
3.50
3.32
3.36
3.32
4.02
3.35
3.87
0.94
0.65
7.42
7.99
7.14
7.20
5.59
5.86
4.80
3.80
4.48
5.15
4.92
5.10
5.40
6.19
3.33
4.13
3.46
3.76
3.55
3.75
4.35
3.53
5.61
5.53
5.46
5.16
5.57
3.43
5.28
5.58
5.85
5.86
5.84
5.70
4.79
3.41
3.53
3.58
3.54
3.42
3.24
3.62
3.52
3.55
3.86
0.95
0.62
7.78
8.07
7.15
6.97
5.62
5.91
4.65
3.62
4.54
5.18
4.83
4.93
5.46
6.05
3.27
4.18
3.62
3.68
3.47
3.64
3.46
3.50
5.64
5.57
5.49
5.18
5.52
3.29
5.27
5.61
5.90
5.76
5.78
5.88
4.66
3.30
4.05
3.63
3.57
3.36
3.50
3.83
3.80
4.19
3.95
0.95
0.76
7.62
7.93
7.16
7.17
5.64
5.94
4.82
4.06
4.62
4.74
4.89
5.06
5.43
6.15
3.32
4.19
3.71
3.76
3.58
3.65
3.48
3.56
5.65
5.57
5.49
5.20
5.56
3.28
5.35
5.57
5.97
5.91
5.88
5.82
4.72
3.41
3.48
3.57
3.53
3.33
3.41
3.68
3.44
3.59
3.86
0.96
0.76
is not present in any organisms and the Km value for the substrate
DXP was not measured in the experiment. This result therefore
cannot be used to compare to our data. In addition, we
determined the Ki value of 1 for EcDXR to be 27 nM, which is
also similar to a literature value of 21 nM.19 Therefore, our study
shows that 1 binds to MtDXR ∼5ꢁ more weakly than EcDXR.
Likewise, compound 2, another extremely potent EcDXR
inhibitor,8j was found to possess a Ki of 19 nM against EcDXR
in our experiment, while the Ki of 2 for MtDXR is 130 nM,
indicating again the inhibitor has ∼6ꢁ less affinity to the M.
tuberculosis enzyme. Even more drastic are the differences
between the Ki values of compounds 3 and 4 for these two
DXR proteins. The two compounds bearing a bulky R-(3,
4-dichloro)phenyl group are potent inhibitors of EcDXR with
Ki values of 58 and 36 nM, approaching the activities of their
parent compounds 1 and 2. However, their inhibition constants
against MtDXR are 1.2 and 1.5 μM, being ∼10ꢁ less active than
1 and 2 (KiMtDXR = 0.14 and 0.13 μM). In addition, compared to
EcDXR, the binding of compounds 3 and 4 to MtDXR exhibits
enormous 21- and 42-fold affinity losses. These results show that
introducing an R-(3,4-dichloro)phenyl group is highly disfavored
with respect to targeting MtDXR. Similarly, bisphosphonates
(5 and 6) and compound 7 (Table 2) were also found to be
greater than 5ꢁ and 31ꢁ less active against the M. tuberculosis
enzyme, respectively.
The Ki values of compounds 8ꢀ48 against MtDXR are shown
in Table 1. Except for compounds 8 and 10, these compounds
possess considerably reduced binding affinities to MtDXR. The
best EcDXR inhibitor 9 (KiEcDXR = 0.42 μM) in this series has a
7-fold affinity loss to MtDXR (KiMtDXR = 3.2 μM). In addition, it
appears that the M. tuberculosis enzyme is very sensitive to any
additional substituents on 9. For example, compound 34 with
only a small 40-fluoro substituent, which is still a strong EcDXR
inhibitor (KiEcDXR = 1.1 μM), is much less active against MtDXR
with a KiMtDXR of 50.7 μM. Other derivatives of 9, compounds
35ꢀ37 with a 40-chloro, 40-methyl, and 30-methoxy substituent,
are essentially inactive against MtDXR. On the other hand,
compounds 8 and 10 possess slightly improved Ki values (1.6
and 6.9 μM, respectively) for MtDXR, compared to their KiEcDXR
values of 2.3 and 7.9 μM.
2
3
4
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
The inhibition study of M. tuberculosis DXR is of interest, since
it shows most inhibitors, including the reference compounds 1
and 2, have significantly reduced binding affinities to the enzyme
(KiEcDXR . KiMtDXR). More problematic is that the substrate
DXP, however, binds to MtDXR twice as tightly as EcDXR
MtDXR
EcDXR
(Km
= 47.1 μM2d vs Km
= 99 μM20). These two
factors render the inhibition of M. tuberculosis DXR in the
presence of DXP even more difficult and make it a great challenge
to design potent MtDXR inhibitors.
Crystallographic Studies of MtDXR. Due to the limited
number of active MtDXR inhibitors, our attempts for QSAR
studies in MtDXR inhibition did not yield a predictive model.
X-ray crystallography was used to investigate the possible
structural basis for the generally reduced inhibitor affinity to
the protein. DXR is highly conserved across the species using the
non-mevalonate pathway.9c MtDXR and EcDXR share an overall
r2
q2
F-test
N
131.5 128.3 123.5 165.8 151.7
5
4
5
4
5
n
45
40
40
40
40
40
a An arbitrary pKi value of 3.52 (Ki = 300 μM) is given to all inactive
compounds. b Bold values represent predicted activities of compounds
that were not included in the training set.
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Figure 3. (A) Correlation between the experimental EcDXR inhibitory activity and predicted activity by the phase QSAR model, showing r2 = 0.95. (B)
Flexible alignment of compounds (only heavy atoms and polar hydrogens displayed for clarity), with compound 9 shown in a ball and stick model and 1
in a tube model. (C) Phase negative ionic fields, pink favorable, yellow disfavored. (D) Electron-withdrawing fields, red favorable. (E) Hydrophobic
fields, cyan favorable, purple disfavored. (F) H-bond donor fields, orange disfavored.
∼40% sequence identity and 69% similarity (Supporting Infor-
mation Figure S1), with the active site showing even higher
homology (∼50% identity). In fact, despite the 5ꢁ affinity
difference, 1 binds to the two DXRs in a very similar fashion,
as shown in the aligned MtDXR:1 (PDB code: 2JCV)18 and
EcDXR:1 (PDB code: 2EGH)11d structures (Figure S2). Three
common interactions are (1) the electrostatic/H-bonds between
of 4 found on the surface of monomer B, likely due to nonspecific
binding. Each of the two Mn2þ ions is anchored to the protein via
coordination to three carboxylate groups of the residues Asp151,
Glu153, and Glu222. Moreover, the binding of both NADPH
molecules in the monomers A and B closely resemble those
found in the MtDXR:1 structure. Since monomer A mimics the
solution state of the MtDXR:4 complex better, the discussion
below is focused on monomer A. In addition, the racemic form of
4 was used in the crystallization experiment and it is not
unambiguous to assign the absolute configuration of the
R-carbon atom of the inhibitor. As shown in Figure S4, good
models for (R)- and (S)-4 can be generated, in which the
phosphonates, hydroxamates, and 3,4-dichlorophenyl groups
are located almost in the same sites, but the conformations of
the linker carbon skeletons differ from each other. We therefore
assigned 50% occupancy for each enantiomer in the final
structure. While (R)-4 is used for the following discussion on
inhibitorꢀprotein interactions, the (S)-4 molecule possesses
essentially the same properties.
Close-up views of the active site of the MtDXR:4 structure and
proteinꢀligand interactions are shown in Figure 4a and Support-
ing Information Figure S5, respectively. The phosphonate group
of 4 is located in the phosphonate binding site of 1, with all of the
three O atoms forming hydrogen bonds and/or electrostatic
interactions with the residues Lys219, Asn218, Ser213, and
Ser177. The hydroxamate group of 4 is found to chelate
Mn2þ, with the distances between the two O atoms and the
metal ion being 2.04 and 2.51 Å. These two features are common
to all known structures of DXR:1 complexes. The 3,4-dichloro-
phenyl group of 4 is located in a hydrophobic cavity, sur-
rounded by Pro265, Ser245, and Trp203. However, there is no
πꢀπ stacking interaction between the 3,4-dichlorophenyl and
the indole ring of Trp203. Moreover, it is remarkable that the
the phosphonate and the protein; (2) metal ion (Mg2þ or Mn2þ
)
chelation by the hydroxamate group; and (3) the hydrophobic
interaction of the inhibitor backbone with a conserved Trp residue
(Trp211 for EcDXR and Trp203 for MtDXR), which also shields
the active site from the solvent. From the two DXR:1 structures, it
is still unclear what factor determines the affinity difference.
We turned to compound 4 for its larger affinity difference
(KiEcDXR = 36 nM vs KiMtDXR = 1500 nM). In addition, 4 is also
10ꢁ less active than 1 against MtDXR, while these two com-
pounds have similar activity on EcDXR. We determined the
X-ray crystal structure of MtDXR in complex with 4, Mn2þ, and
NADPH, at 2.5 Å. Details of data processing and refinement are
shown in Table 4 and the overall structure of the MtDXR:4
complex illustrated in Figure S3a. Although the protein crystal-
lizes as a homodimer, only one monomer (monomer A) contains
inhibitor 4 as well as Mn2þ and NADPH in the active site. In
addition, a flexible loop consisting of the residues 198ꢀ208,
which acts as a “cap” opening and closing when the substrate or
an inhibitor enters the active site, adopts a closed conformation
(Figure S3b). The other monomer (monomer B) includes a
Mn2þ, SO42ꢀ, and NADPH in the active site, with the flexible
loop in a widely open conformation due to crystal packing
interactions (Figure S3c).18 No inhibitor is found in the active
2ꢀ
site of monomer B, while a SO4 ion is located in the
phosphonate binding site of 1. The above phenomena can also
be observed in the MtDXR:1 structure.18 There is one molecule
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electron density for the residues 198ꢀ203 in the flexible loop is
relatively weak with high B factor values, showing that this part of
the loop flips around and has no strong interactions with the
inhibitor 4. On the contrary, the loop in the MtDXR:1 structure is
bound well near the center of the active site, with the indole ring of
Trp203 covers on top of the relatively hydrophobic carbon
skeleton of 1 (Figure 4b). It can therefore be inferred that the
weak interactions between Trp203 and the 3,4-dichlorophenyl ring
could account for the reduced activity of compound 4 on MtDXR.
Next, we analyzed the binding differences of compound 4 in
MtDXR and EcDXR. Although the crystal structure of EcDXR in
complex with 4 has not been available, a model of how 4 binds to
the protein has been generated in our previous study9c by using
the crystal structure of the EcDXR:8 complex, which is also
supported by experimental data. The modeled EcDXR:4 struc-
ture was aligned with the MtDXR:4 structure, and as shown in
Figure 4c, the two molecules of 4 can almost be superimposed
onto each other and possess a similar binding pose. Nevertheless,
a notable difference exists with respect to the conformation of the
flexible loops of the two proteins. The EcDXR loop (in purple) is
located closer to the inhibitor, with the Trp211 indole ring
forming a πꢀπ stacking interaction with the 3,4-dichlorophenyl
group of 4, affording a tight binding mode. This is also in line with
the potent inhibitory activity of 4 (KiEcDXR = 36 nM). On the
other hand, the loosely bound loop of MtDXR might be again
responsible for the considerably lowered affinity of 4 to the
enzyme (KiMtDXR = 1500 nM).
The exact origin for the weak interaction between Trp203 in
MtDXR and compound 4 is unclear, but should be attributed to
factors from both the protein and the inhibitor. From the side of
the protein, the flexible loop of EcDXR has one conformationally
more rigid proline residue (Pro209), while that of MtDXR
contains two proline residues (Pro201 and Pro207), which could
restrict the conformational change of Trp203 from having a
favorable πꢀπ stacking interaction with the 3,4-dichlorophenyl
group of inhibitor 4. However, this might not be the only reason,
since Trp203 is still able to move closely to 1 (Figure 4b). From
the side of the inhibitor, the relatively weak electron deficiency on
the 3,4-dichlorophenyl ring could be another unfavorable factor.
For example, phosphonate compound 21 containing this group
(Table 1) has essentially no activity against both EcDXR and
MtDXR. The more electron-deficient pyridine analogue 8 is a
good DXR inhibitor and, interestingly, shows slightly improved
activity on MtDXR over EcDXR, suggesting a pyridine contain-
ing analogue of 4 could be particularly active against MtDXR.
Synthesis Aspect. Compounds 1ꢀ6 were prepared according
to published methods8j,9a and 39ꢀ42 are commercially available.
Compounds 7ꢀ10, 14, 15, 21, and 23 were from our previous
studies.9b,c Scheme 1 shows the general synthetic methods for
making the other compounds. 2-Bromopyridine or bromoben-
zene was converted to their corresponding phosphonate diethyl
ester, using either a palladium catalyzed reaction or a lithiumꢀ
halogen exchange followed by treatment with diethyl chloro-
phosphate. The esters thus obtained were hydrolyzed with
Table 4. Data Processing and Refinement Statistics
Data Processing
Wavelength (Å)
Space group
1.542
P21
Unit cell dimensions
a, b, c (Å)
67.25, 65.18, 85.41
90.0, 101.3, 90.0
100ꢀ2.56(2.6ꢀ2.56)
22365 (1144)
93.9 (94.5)
R, β, γ (°)
Resolution (Å)
Unique reflections
Completeness (%)
Redundancy
3.9 (3.8)
Rsym (%)
13.6 (51.8)
I/σ (I)
11.2 (2.2)
Refinement
Resolution (Å)
33.45ꢀ2.55 (2.71ꢀ2.55)
21222 (3505)
20.8/27.8
5866
Number of reflections used in working set
R/Rfreea (%)
Number of non-hydrogen atoms
Number of solvent waters
119
Mean B-factor from Wilson plot (Å2)
Mean B-factor, protein atoms (Å2)
Mean B-factor, solvent atoms (Å2)
Mean B-factor, inhibitors (Å2)
Mean B-factor, NADPH (Å2)
Mean B-factor, Mn2þ (Å2)
Mean B-factor, SO42- (Å2)
49.90
52.42
44.21
59.47
51.54
59.17
56.24
Root mean square deviations from ideality
Bond length (Å)
0.008
1.3
Bond Angle (°)
a A subset of the data (5%) was excluded from the refinement and used
to calculate Rfree
.
Figure 4. (A) Active site of the MtDXR:4 structure. The protein backbone is shown as green curved lines, 4 as a ball and stick model, and Mn2þ as a
brown sphere. (B) Crystal structure of MtDXR:4 (in green) superimposed with the structure of MtDXR:1 (in orange). For clarity, only the flexible loops
containing residues 198ꢀ208 of the two structures are shown as curved lines. (C) Crystal structure of MtDXR:4 (in green) superimposed with a
modeled structure of EcDXR:4 complex (in purple). Only the flexible loops (residues 198ꢀ208 for MtDXR and 205ꢀ215 for EcDXR) of the two
structures are shown as curved lines.
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Scheme 1a
after hydrolysis, compounds 45 and 46. Hydroxamates 43, 44,
and 47 were synthesized from their corresponding acids with an
amide forming reaction with O-benzylhydroxylamine, followed
by hydrogenation. Finally, compound 48 was made from sodium
sulfite and 2-chloromethyl-5-phenylpyridine.
’ CONCLUSION
Our SAR/QSAR and crystallographic studies of E. coli and
M. tuberculosis DXRs have resulted in several novel observations
that have implications in the design of potent inhibitors. First, a
total of 41 lipophilic phosphonate and related compounds were
synthesized and their inhibitory activity tested against E. coli
DXR, with the best compound 9 (5-phenylpyridin-2-ylmethyl-
phosphonic acid) possessing a KiEcDXR of 420 nM. Second, SAR
studies show that (1) an electron-deficient aromatic ring, espe-
cially a pyridine, and a phosphonate headgroup are essential for
the inhibition; (2) one methylene linker between these two
groups is optimal; and (3) certain substituents (e.g., 5-phenyl or
5-hydroxyl) on the aromatic ring provide enhanced activity. In
addition, these SARs can be rationalized using the crystal
structures of the EcDXR:8 and EcDXR:9 complexes. We also
developed a 3-D QSAR model for EcDXR inhibition that had
good predictivity. Third, the 41 compounds as well as 7 known
EcDXR inhibitors with a broad range of chemical diversity were
tested against M. tuberculosis DXR, in an effort to find new
antituberculosis agents. The vast majority of the inhibitors were
found to exhibit considerably weakened activity against MtDXR,
compared to that on EcDXR. In particular, fosmidomycin and its
derivatives 1ꢀ4 with low nM Ki values against EcDXR show
5ꢀ40ꢁ lower affinity to MtDXR (KiMtDXR: 130ꢀ1500 nM).
Fourth, the X-ray crystal structure of MtDXR in complex with 4 is
disclosed. The fosmidomycin moiety of 4 exhibits an expected
binding mode, with the phosphonate located in the favored site
and the hydroxamate group chelating the central metal ion.
However, the flexible loop of MtDXR shows relatively weak
electron density in the structure, in which Trp203 indole ring has
no πꢀπ stacking interaction with the 3,4-dichlorophenyl group
of 4. This could represent a structural basis for the considerably
lowered activity of 4 against MtDXR.
a Reagents and conditions: (i) PH(OEt)2, Pd(Ph3P)4, Ph3P, DIPEA,
toluene, reflux; (vi) n-BuLi, ClP(O)(OEt)2, THF, ꢀ78 °C; (iii) TMSBr,
CH3CN, rt, then MeOH/H2O; (iv) P(OEt)3, 120 °C, overnight; (v)
RB(OH)2, Pd(Ph3P)4, Na2CO3, toluene/H2O (v/v 3:1), 90 °C; (vi)
m-CPBA, CHCl3; (vii) Ac2O, 90 °C; (viii) KOH, MeOH; (ix) SOCl2,
CH2Cl2; (x) LDA, 2-bromoethylphosphonate, THF, ꢀ78 °C; (xi) NaH,
tetraethyl diphosphonate, THF, 0 °C; (xii) H2, 5% Pd/C, MeOH; (xiii)
NH2OBn, HOBT, EDC, TEA, CH2Cl2; (ixv) Na2SO3, H2O reflux.
bromotrimethylsilane to afford compounds 11 and 18. Com-
pounds 12, 16, 17, 19, 20, 22, and 24 with 1 or 2 methylene
linkers were synthesized from their corresponding chloride with
an Arbuzov reaction, followed by bromotrimethylsilane
mediated hydrolysis. For making pyridine containing phospho-
nates 26ꢀ38, a substituted 2-picoline, which was either com-
mercially available or prepared from a bromo-substituted
2-picoline using a Suzuki coupling, was oxidized with m-chloro-
perbenzoic acid (m-CPBA) to give a pyridine oxide. It was then
treated with Ac2O at 90 °C followed by hydrolysis (KOH) in
MeOH to afford a 2-hydroxymethyl pyridine, which was reacted
with thionyl chloride to give the corresponding chloride. It can
then be readily converted to 26ꢀ38, using an Arbuzov reaction
followed by hydrolysis. To make compound 13 with a three-
methylene linker, 2-picoline was treated with lithium diisopro-
pylamide (LDA) followed by alkylation with diethyl 2-bromo-
ethylphosphonate. The resulting diethyl ester was hydrolyzed
with bromotrimethylsilane to give 13. The reaction of the sodium
salt of tetraethyl diphosphonate with 2-methoxypyridine-5-car-
baldehyde produced an R,β-unsaturated phosphonate, which
subjected to hydrogenation (5% Pd/C) and hydrolysis to give
compound 25. Suzuki coupling of methyl 5-bromopyridine-2-
carboxylate or -2-acetate with benzeneboronic acid afforded,
’ EXPERIMENTAL SECTION
All reagents were purchased from Alfa Aesar (Ward Hill, MA) or
Aldrich (Milwaukee, WI). Compounds 1ꢀ6 were prepared according to
published methods8j,9a and 39ꢀ42 are commercially available. Com-
pounds 7ꢀ10, 14, 15, 21, and 23 were from our previous studies.9b,c All
compounds were characterized by 1H, 31P, and/or 19F NMR on a Varian
(Palo Alto, CA) 400-MR spectrometer. The purities were monitored by
a Shimadzu Prominence HPLC with a Zorbax C18 or C8 column (4.6 ꢁ
250 mm) or 1H (at 400 MHz) absolute spin-count quantitative NMR
analysis with imidazole as an internal standard. The purities of all
compounds were found to be >95%.
General Method A. A chloride (1 mmol) was heated with triethyl
phosphite (5 mmol) at 120 °C under N2 overnight. Upon removal of
excess triethyl phosphite under reduced pressure, the residue was
purified with flash column chromatography to give the corresponding
phosphonic acid diethyl ester.
General Method B. To a solution of a phosphonic acid diethyl
ester (1 mmol) in dry CH3CN (3 mL) was slowly added bromotri-
methylsilane (3 mmol) at 0 °C. After stirring overnight at room
temperature, the solution was evaporated to dryness and MeOH
(5 mL) was added to the residue. The solvent was removed under
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reduced pressure again and the residue redissolved in MeOH (5 mL).
Neutralization with 2 M NaOH to pH = 8 gave a disodium salt of a
phosphonic acid as a white powder, which may be recrystallized in H2O/
acetone if needed.
General Method C. A mixture of a bromide (1 mmol), a boronic
acid (1.5 mmol), Na2CO3 (424 mg, 4 mmol), and Pd(Ph3P)4 (58 mg,
5 mmol %) in toluene/H2O (v/v 3:1, 9 mL) was heated to 90 °C under
N2 overnight. The product was extracted with EtOAc (3 ꢁ 10 mL) and
purified with flash column chromatography.
was stirred for 1 h, followed by addition of 2-bromoethylphosphonate
(960 μL, 5 mmol). After 2 h, the reaction was warmed to room
temperature and stirred for additional 1 h. The reaction was quenched
with statured NH4Cl (10 mL) and extracted with EtOAc (3 ꢁ 25 mL).
The combined organic phases were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The residue was purified with
column chromatography (silica gel, EtOAc) to give the corresponding
diethyl phosphonate, which was hydrolyzed, following the general
method A, to afford compound 13 as a white powder (516 mg, 33.4%
yield). 1H NMR (400 MHz, D2O): δ 8.30 (d, J = 4.8 Hz, 1H), 7.77ꢀ7.71
(m, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.19 (m, 1H), 2.74 (t, J = 7.6 Hz, 2H),
1.82ꢀ1.75 (m, 2H), 1.41ꢀ1.32 (m, 2H). 31P NMR (162 MHz, D2O):
δ 21.8.
General Method D. To a solution of a 2-methylpyridine (25 mmol)
in CHCl3 (100 mL) was added m-CPBA (11.2 g with 50% purity,
32.5 mmol). After stirring for 5 h, the reaction was quenched by adding
saturated NaHCO3 (30 mL) and the aqueous layer extracted with
CH2Cl2 (15 mL). The combined organic phases were washed with 2 N
HCl (15 mL), dried over Na2SO4, filtered, and concentrated under
reduced pressure to give the corresponding pyridine oxide, which was
used without further purification. It was then treated with acetic
anhydride (50 mL) at 90 °C for 12 h and the resulting dark solution
concentrated under reduced pressure. The residue was dissolved in
MeOH (30 mL) and KOH (2.1 g, 15 mmol) added at 0 °C. After stirring
for 3 h at room temperature, the solvent was removed and the residue
treated with saturated NaHCO3 (10 mL) and extracted with EtOAc
(3 ꢁ 10 mL). The combined organic layers were dried, filtered,
concentrated, and purified with flash column chromatography (silica
gel, hexane/EtOAc: 1/1) to give a pyridin-2-ylmethyl alcohol. To a
solution of the alcohol (1 mmol) in CH2Cl2 (5 mL) was added thionyl
chloride (0.22 mL, 3 mmol) solution in CH2Cl2 (2 mL) at 0 °C.
The reaction was warmed to room temperature and stirred overnight.
Upon removal of the solvent, the residue was treated with saturated
Na2CO3 (5 mL) and extracted with CH2Cl2 (3 ꢁ 10 mL). The
combined organic layers were dried, filtered, and evaporated to give
the corresponding chloride, which was used without purification,
following the general methods A and B, to produce a phosphonic acid
disodium salt as a white powder.
Pyridin-2-ylphosphonic Acid Disodium Salt (11). To a
solution of 2-bromopyridine (790 mg, 5 mmol), diethyl phosphite
(967 μL, 7.5 mmol), and diisopropylethylamine (DIPEA, 5 mL, 25 mmol)
in toluene (40 mL) were successively added Ph3P (1.31 g, 5 mmol) and
Pd(Ph3P)4 (290 mg, 5 mmol %) under N2. The reaction mixture was
refluxed overnight. After removing solvent under reduced pressure, the
residue was purified with column chromatography (silica gel, EtOAc) to
give the corresponding phosphonate (350 mg), which was hydrolyzed,
following the general method A, to give compound 11 as a white powder
(320 mg, 31.5% yield). 1H NMR (400 MHz, D2O): δ 8.41 (d, J = 4.8 Hz,
1H), 7.69ꢀ7.67 (m, 2H), 7.23ꢀ7.21 (m, 1H). 31P NMR (162 MHz,
D2O): δ 6.0.
Pyrazin-2-ylmethylphosphonic Acid Disodium Salt (16).
To a solution of 2-methylpyrazine (1 mL, 22 mmol) in the CCl4
(80 mL) were added N-chlorosuccinimde (4.27 g, 31.5 mmol) and
benzoyl peroxide (0.26 g, 1.1 mmol). The reaction mixture was stirred
overnight. Upon removal of the solvent under reduced pressure, the
residue was purified with column chromatography (silica gel, hexane/
EtOAc: 6/1) to give the corresponding chloride, which was used
following the general methods A and B, to give compound 16 as an
1
off-white powder (1.46 g, 30.4% yield). H NMR (400 MHz, D2O):
δ 8.47 (d, J = 1.6, 1H), 8.38 (s, 1H), 8.31 (d, J = 1.6 Hz, 1H), 3.10
(d, J = 20.4 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.9.
Pyrimidin-4-ylmethylphosphonic Acid Disodium Salt
(17). Compound 17 was prepared from 4-methylpyrimidine, following
a similar procedure as 16, as an off-white powder (158 mg, 37% yield).
1H NMR (400 MHz, D2O): δ 8.84 (m, 1H), 8.46 (d, J = 4.8 Hz, 1H),
7.44 (s, 1H), 3.03 (d, J = 20.4 Hz, 2H). 31P NMR (162 MHz, D2O):
δ 13.9.
Phenylphosphonic Acid Disodium Salt (18). To a solution of
bromobenzene (468 mg, 3 mmol) in THF (9 mL) was added n-BuLi
(1.23 mL, 2.93 M, 3.6 mmol) at ꢀ78 °C. After stirring 1.5 h, diethyl
chlorophosphate (776 mg, 4.5 mmol) was added. The reaction mixture
was stirred for additional 3 h at the same temperature, quenched with
saturated NH4Cl (10 mL), and extracted with EtOAc (3 ꢁ 10 mL). The
combined organic phases were dried over Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified with flash
column chromatography (silica gel, hexane/EtOAc: 1/1) to give the
diethyl phosphonate, which was hydrolyzed, following the general
method B, to afford compound 18 as a white powder (340 mg, 56%
overall yield). 1H NMR (400 MHz, D2O): δ 7.30ꢀ7.21 (m, 5H). 31
NMR (162 MHz, D2O): δ 17.2.
P
Benzylphosphonic Acid Disodium Salt (19). Compound 19
was prepared from benzyl chloride (250 mg, 2 mmol), following the
general methods A and B, as a white powder (293 mg, 68.1% yield). 1H
NMR (400 MHz, D2O): δ 7.20ꢀ7.04 (m, 5H), 2.13 (d, J = 20.0 Hz,
2H). 31P NMR (162 MHz, D2O): δ 21.6.
2-Phenylethylphosphonic Acid Disodium Salt (20). Com-
pound 20 was prepared from phenylethylchloride (280 mg, 2 mmol),
following the general methods A and B, as a white powder (300 mg, 65%
yield). 1H NMR (400 MHz, D2O): δ 7.16ꢀ7.01 (m, 5H), 2.21 (m, 2H),
2.13 (m, 2H). 31P NMR (162 MHz, D2O): δ 19.5.
2-(2-Hydroxyphenyl)ethylphosphonic Acid Disodium Salt
(22). Compound 22 was prepared from 2-chloroethylphenol (160 mg,
1 mmol), following the general methods A and B, as a white powder
(94mg, 40.5%yield).1HNMR(400MHz, D2O): δ7.10 (d, J= 7.2 Hz, 2H),
6.99 (t, J = 7.2 Hz, 1H), 6.79 (t, J = 7.2 Hz, 1H), 6.73 (t, J = 7.2 Hz, 1H),
2.61 (m, 2H), 1.57 (m, 2H). 31P NMR (162 MHz, D2O): δ 22.5.
5-(1-Hydroxy-pyridin-2-one)methylphosphonic Acid Di-
sodium Salt (24). To a solution of 6-methoxypyridine-3-carbaldehyde
(274 mg, 2 mmol) in methanol (6 mL) was slowly addedNaBH4 (114 mg,
3 mmol) at 0 °C. After 1 h at room temperature, the solvent was re-
moved under reduced pressure and the reaction quenched with water
2-(Pyridin-2-yl)ethylphosphonic Acid Disodium Salt (12).
To a solution of 2-(pyridin-2-yl)ethanol (2.46 g, 20 mmol) was added
slowly thionyl chloride (5.74 mL, 80 mmol) at 0 °C. The reaction
mixture was heated to 80 °C for 3 h. Upon removal of excessive thionyl
chloride under reduced pressure, the residue was treated with saturated
NaHCO3 (30 mL) and extracted with CH2Cl2 (3 ꢁ 20 mL). The
combined organic layers were dried over anhydrous Na2SO4, filtered,
and evaporated to give the crude chloride, which was used for the next
step without purification. Compound 12 was obtained from the chloride
following the general methods A and B as a white powder (3.8 g, 82%
yield). 1H NMR (400 MHz, D2O): δ 8.29 (d, J = 4.8 Hz, 1H), 7.77ꢀ7.73
(m, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.21 (m, 1H), 2.93ꢀ2.68 (m, 2H),
1.80ꢀ1.72 (m, 2H). 31P NMR (162 MHz, D2O): δ 22.4.
3-(Pyridin-2-yl)propylphosphonic Acid Disodium Salt
(13). To a solution of diisopropylamine (1.51 mL, 10.8 mmol) in
THF (12 mL) was added dropwise n-BuLi (2.46 mL, 2.93 M, 7.2 mmol)
at ꢀ10 °C. After 30 min, the solution was cooled to ꢀ78 °C and
2-picoline (558 mg, 6 mmol) was added slowly. The reaction mixture
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and extracted with EtOAc (3 ꢁ 10 mL). The combined organic phases
were dried over Na2SO4, filtered, and concentrated under reduced
pressure. The resulting crude alcohol was dissolved in CH2Cl2 (8 mL)
and a solution of SOCl2 (0.43 mL, 6 mmol) in CH2Cl2 (5 mL) added
slowly at 0 °C. After 3 h, the solvent was removed under vacuum. The
residue was treated with saturated Na2CO3 (10 mL) and extracted with
CH2Cl2 (3 ꢁ 10 mL). The combined organic layers were dried, filtered,
and evaporated to give the corresponding chloride, which was used
following the general method A to give the diethyl phosphonate. It
(1 mmol) was dissolved in CHCl3 (5 mL) and m-CPBA (690 mg, 50%, 2
mmol) added. After 5 h, the reaction was quenched by adding saturated
NaHCO3 (10 mL) and the aqueous layer extracted with CH2Cl2
(15 mL). The combined organic phases were washed with 2 N HCl
(5 mL), dried over Na2SO4, filtered, and concentrated under reduced
pressure to give the crude product. It was refluxed in acetyl chloride
(5 mL) for 1 h. After excessive acetyl chloride was removed, the residue
was washed with diethyl ether and hydrolyzed, following the general
Method B, to give compound 24 as a white powder (152 mg, 61% overall
yield). 1H NMR (400 MHz, D2O): δ 7.98 (s, 1H), 7.42 (d, J = 8.8 Hz,
1H), 6.63 (d, J = 8.8 Hz, 1H), 2.72 (d, J = 18.8 Hz, 2H). 31P NMR
(162 MHz, D2O): δ 18.6.
a white powder in an overall 21% yield from 5-benzyloxy-2-methyl-
1
pyridine. H NMR (400 MHz, D2O): δ 8.01 (s, 1H), 7.39ꢀ7.28 (m,
7H), 5.08 (s, 2H), 2.90 (d, J = 19.6 Hz, 2 H). 31P NMR (162 MHz,
D2O): δ 16.3.
(6-Bromopyridin-2-yl)methylphosphonic Acid Disodium
Salt (32). It was prepared following the general method D as a white
powder in an overall 45% yield from 6-bromo-2-methylpyridine.
1H NMR (400 MHz, D2O): δ 7.52ꢀ7.46 (m, 1H), 7.32 (s, 1H), 7.30
(s, 1H), 2.93 (d, J = 19.6 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.3.
(6-Phenylpyridin-2-yl)methylphosphonic Acid Disodium
Salt (33). It was prepared following the general method D as a white
powder in an overall 40% yield from 6-bromo-2-methylpyridine.
1H NMR (400 MHz, D2O): δ 7.77ꢀ7.64 (m, 3H), 7.48ꢀ7.32 (m, 5H),
3.03 (d, J = 20 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.6;
5-(4-Fluorophenyl)pyridin-2-ylmethylphosphonic Acid
Disodium Salt (34). It was prepared following the general method D
as a white powder in an overall 32% yield from 5-bromo-2-methyl-
pyridine. 1H NMR (400 MHz, D2O): δ 8.48 (s, 1H), 7.85 (d, J = 8.0 Hz,
1H), 7.59ꢀ7.56 (m, 2H), 7.44 (d, J = 8.8 Hz, 1H), 7.13 (t, J = 8.8 Hz,
2H), 2.98 (d, J = 20 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.5.
19F NMR (376 MHz, D2O): δ ꢀ115.8.
2-(Pyridin-2-one-6-yl)ethylphosphonic Acid Disodium Salt
(25). To a solution of tetraethyl diphosphonate (1.1 mL, 5.5 mmol) in
THF (15 mL) was added NaH (242 mg, 6.1 mmol, 60% in oil) at 0 °C.
6-methoxypyridine-2-carbaldehyde (730 mg, 5 mmol) was added to the
resulting solution. After 2 h at room temperature, the reaction mixture was
quenched with saturated NH4Cl and extracted with EtOAc (3 ꢁ 15 mL).
The combined organic phases were dried over Na2SO4, filtered, and
concentrated under reduced pressure. The crude product was hydro-
genated with5% Pd/C in methanol(15 mL) for2 h to affordthe saturated
ester, which was hydrolyzed, following the general method B, to afford
5-(4-Chlorophenyl)pyridin-2-ylmethylphosphonic Acid Di-
sodium Salt (35). It was prepared following the general method D as a
white powder in an overall 36% yield from 5-bromo-2-methylpyridine.
1H NMR (400 MHz, D2O): δ 8.50 (s, 1H), 7.86 (d, J = 8.0 Hz, 1H),
7.56ꢀ7.40 (m, 5H), 2.98 (d, J = 20 Hz, 2H). 31P NMR (162 MHz,
D2O): δ 15.4.
5-(4-Methylphenyl)pyridin-2-ylmethylphosphonic Acid Di-
sodium Salt (36). It was prepared following the general method D as a
white powder in an overall 28% yield from 5-bromo-2-methylpyridine.
1H NMR (400 MHz, D2O): δ 8.50 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.51
(d, J = 8.0 Hz, 2H), 7.42 (d, J = 7.2 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 2.97
(d, J = 20 Hz, 2H), 2.24 (s, 3H). 31P NMR (162 MHz, D2O): δ 15.5.
5-(3-Methoxyphenyl)pyridin-2-ylmethylphosphonic Acid
Disodium Salt (37). It was prepared following the general method D
as a white powder in an overall 32% yield from 5-bromo-2-methyl-
pyridine. 1H NMR (400 MHz, D2O): δ 8.56 (s, 1H), 7.84 (d, J = 8.0 Hz,
1H), 7.38ꢀ7.35 (m, 2H), 7.20ꢀ7.17 (m, 2H), 6.91 (d, J = 8.0 Hz, 1H),
3.77 (s, 3H), 3.10 (d, J = 20 Hz, 2H). 31P NMR (162 MHz, D2O):
δ 16.2.
1
compound 25 as a white powder (816 mg, 66% yield). H NMR (400
MHz, D2O): δ 7.49ꢀ7.41 (m, 1H), 6.32ꢀ6.23 (m, 2H), 2.61 (m, 2H),
1.57 (m, 2H). 31P NMR (162 MHz, D2O): δ 19.3.
(4-Bromopyridin-2-yl)methylphosphonic Acid Disodium
Salt (26). It was prepared following the general method D as a white
powder in anoverall 41% yield from 4-bromo-2-methylpyridine. 1H NMR
(400 MHz, D2O): δ 8.07 (d, J = 7.2 Hz, 1H), 7.58 (s, 1H), 7.32 (d, J = 7.2
Hz, 1H), 2.89 (d, J = 19.6 Hz, 2H). 31P NMR (162 MHz, D2O): δ 14.8.
(4-Phenylpyridin-2-yl)methylphosphonic Acid Disodium
Salt (27). It was prepared following the general method D as a white
5-(Naphthalen-2-yl)pyridin-2-ylmethylphosphonic Acid Di-
sodium Salt (38). It was prepared following the general method D as a
white powder in an overall 27% yield from 5-bromo-2-methylpyridine.
1H NMR (400 MHz, D2O): δ 8.57 (s, 1H), 7.98ꢀ7.78 (m, 5H),
7.63ꢀ7.61 (m, 1H), 7.44ꢀ7.38 (m, 3H), 2.97 (d, J = 20 Hz, 2H). 31P
NMR (162 MHz, D2O): δ 15.5.
1
powder in an overall 51% yield from 4-bromo-2-methylpyridine. H
NMR (400 MHz, D2O): δ 8.30 (d, J = 4.4 Hz, 1H), 7.72 (s, 1H), 7.70
(s, 1H), 7.66 (s, 1H), 7.45ꢀ7.39 (m, 4H), 3.01 (d, J = 19.6 Hz, 2H). 31P
NMR (162 MHz, D2O): δ 15.6.
4-(4-Fluorophenyl)pyridin-2-ylmethylphosphonic Acid
Disodium Salt (28). It was prepared following the general method D
as a white powder in an overall 37% yield from 4-bromo-2-methylpyr-
idine. 1H NMR (400 MHz, D2O): δ 8.25 (d, J = 7.2 Hz, 1H), 7.74 (t, J =
8.8 Hz, 2H), 7.62 (s, 1H), 7.38 (m, 1H), 7.16 (t, J = 8.8 Hz, 2H), 2.97 (d,
J = 20 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.5. 19F NMR (376
MHz, D2O): δ ꢀ116.2.
N-Hydroxy-pyridine-2-carboxamide (43). To a solution of
pyridine-2-carboxylic acid (411 mg, 3 mmol), NH2OBn (525 μL, 4.5
mmol), and triethylamine (1.26 mL, 9 mmol) in the CH2Cl2 (20 mL)
were added HOBT (405 mg, 3 mmol) and N-ethyl-N0-(3-amino-
propyl)carbodiimide hydrochloride (864 mg, 4.5 mmol), respectively.
The reaction mixture was stirred overnight at room temperature. The
reaction was quenched with water (10 mL) and extracted with CH2Cl2
(3 ꢁ 10 mL). The combined organic phases were washed successively
with saturated Na2CO3 (10 mL) and brine, dried, filtered, and evapo-
rated. The resulting oil was purified with flash column chromatography
(silica gel, hexane/EtOAc: 2/1) to give the amide, which was hydro-
genated with 10% Pd/C in methanol (12 mL) for 3 h to afford com-
pound 43 as a pale yellow oil (201 mg, 48.9% overall yield). 1H NMR
(400 MHz, D2O): δ 8.46ꢀ8.44 (m, 1H), 7.90ꢀ7.86 (m, 1H),
7.82ꢀ7.80 (m, 1H), 7.50ꢀ7.47 (m, 1H).
(5-Bromopyridin-2-yl)methylphosphonic Acid Disodium
Salt (29). It was prepared following the general method D as a white
1
powder in an overall 46% yield from 5-bromo-2-methylpyridine. H
NMR (400 MHz, D2O): δ 7.52ꢀ7.46 (m, 1H), 7.32 (s, 1H), 7.30
(s, 1H), 2.93 (d, J = 19.6 Hz, 2H). 31P NMR (162 MHz, D2O): δ 15.3.
(5-Hydroxylpyridin-2-yl)methylphosphonic Acid Diso-
dium Salt (30). It was prepared from 31 by hydrogenation with 5%
Pd/C in 99% yield as a white powder. 1H NMR (400 MHz, D2O): δ 7.64
(m, 1H), 7.30ꢀ7.28 (m, 2H), 2.96 (d, J = 19.2 Hz, 2H); 31P NMR (162
MHz, D2O): δ 14.9.
N-Hydroxy-(pyridin-2-yl)acetamide (44). It was prepared
from pyridin-2-ylacetic acid following the similar procedure as 112 as
(5-Benzyloxypyridin-2-yl)methylphosphonic Acid Diso-
dium Salt (31). It was prepared following the general method D as
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a colorless oil (169 mg, 37% overall yield). 1H NMR (400 MHz, D2O):
δ 8.32ꢀ8.30 (m, 1H), 7.72ꢀ7.68 (m, 1H), 3.56 (s, 2H).
The inhibition assay for MtDXR was carried out similarly as the
EcDXR experiment. It was done in a 96-well microplate using the
purified MtDXR (50 nM), 4 mM MgCl2, 100 μM DXP, 50 μM NADPH
in 50 mM HEPES buffer (pH = 7.6) containing 25 μg/mL BSA.
Compounds were incubated with MtDXR for 10 min at 30 °C before
adding DXP. The initial velocities were calculated and the IC50 values for
inhibitors obtained by using Prism. The IC50s used for calculating Ki
values were from at least three independent experiments with standard
deviations of less than 20%. Ki values were calculated using the formula
Ki = IC50/(1 þ [S]/Km), where [S] is the concentration of DXP
(100 μM) and Km is the literature value of 47.1 μM.2d
Molecular Modeling. Docking studies were performed as pre-
viously reported9b,c using Schr€odinger suite 2010,13 which includes all of
the programs described below. Crystal structures of E. coli DXR were
prepared using the module “protein preparation wizard” in Maestro 9.115
with default protein parameters: water molecules were removed, hydro-
gens added, inhibitors extracted, and NADPH remained in the protein
structure for docking. H-bonds were then optimized and the protein was
energy-minimized using OPLS-2005 force field. A receptor grid, which is
large enough to contain the whole active site, was generated using
Glide12 without constraints. Compounds were built, minimized using
OPLS-2005 force field in Maestro, and then docked into the prepared
protein structure using Glide (docking parameters: standard-precision
and dock flexibly).
5-Phenylpyridine-2-carboxylic Acid (45). The methyl ester
of compound 45 was prepared from 5-bromopyridine-2-carboxylate
(324 mg, 1.5 mmol), following the general method C. It was dissolved in
MeOH (5 mL) and 3 equiv of KOH was added at 0 °C. After stirring 3 h
at room temperature, the solvent was removed under vacuum. The
residue was dissolved in H2O (1 mL), acidified with 2 M HCl, and
extracted with ethyl acetate (30 mL). The organic layer was concen-
trated and purified with column chromatography (silica gel, hexane/
EtOAc: 5/1) to give 45 as a white solid (214 mg, 72% yield). 1H NMR
(400 MHz, D2O): δ 8.68 (s, 1H), 8.03ꢀ8.02 (m, 1H), 7.82 (d, J = 8.4 Hz,
1H), 7.61 (s, 1H), 7.59 (s, 1H), 7.44ꢀ7.35 (m, 3H).
(5-Phenylpyridin-2-yl)acetic Acid (46). It was prepared from
5-bromopyridine-2-acetate (350 mg, 1.5 mmol) following the similar
procedure as 45, as a white solid (257 mg, 81% yield). 1H NMR (400
MHz, D2O): δ 8.82 (s, 1H), 8.60 (d, J = 7.2 Hz, 1H) 7.83 (d, J = 7.2 Hz,
1H), 7.62 (s, 1H), 7.61 (s, 1H), 7.47ꢀ7.44 (m, 3H), 4.01 (s, 2H).
N-Hydroxy-(5-phenylpyridin-2-yl)acetamide (47). It was
prepared from 46 (212 mg, 1 mmol) following the similar procedure
1
as 43, as a white solid (164 mg, 73% yield). H NMR (400 MHz, d6-
DMSO): δ 10.73 (br, 1H), 8.86 (s, 1H), 8.75 (s, 1H), 8.00 (d, J = 8.0 Hz,
1H), 7.70 (s, 1H), 7.68 (s, 1H), 7.50ꢀ7.46 (m, 2H), 7.42ꢀ7.40 (m, 2H),
4.01 (s, 2H).
QSAR studies were performed with default settings using Phase14
(version 3.2). All compounds were built and geometry- and energy-
minimized using the OPLS-2005 force field in Maestro. Compound
alignment was carried out using the “Flexible Ligand Alignment” module
in Maestro with compound 8 as a template. The aligned compounds
were imported into the Phase program. A partial least-squares (PLS)
method was used to correlate the activities of these compounds with the
3-D structural features calculated by Phase with default settings. The
QSAR model was then validated by performing five leave-5-out training/
test sets.
Crystallization and Structure Refinement. The crystallization
of MtDXR was performed according to a published procedure.18 In brief,
purified MtDXR (10 mg/mL) was mixed with an equal volume of the
crystallization buffer containing 2 mM MnCl2, 3 mM NADPH, 0.2 M
(NH4)2SO4, 25% poly(ethylene glycol) 3350, and 0.1 M Bis-Tris, pH
5.7. The protein crystals appeared in ∼1 week at room temperature,
which were soaked for 1 h in the crystallization buffer containing an
inhibitor (0.2 M). Data were collected to a resolution of 2.56 Å using a
Rigaku FR-Eþ SuperBright X-ray source at Baylor College of Medicine
and were then processed using the program HKL2000.21 The initial
structure was obtained by molecular replacement using the program
Phaser22 with the coordinates of 2JCZ as a target. The refinement was
carried out using the program CNS.23 Since the inhibitor 4 was
synthesized in a racemic form, both (R)- and (S)-4 were built into the
model with the occupancy of each enantiomer set to 0.5. The data
collection and refinement statistics are provided in Table 4.
5-Phenylpyridin-2-ylmethanesulfonic Acid Sodium Salt
(48). The mixture of 2-chloromethyl-5-phenylpyridine (203.5 mg, 1 mmol)
and sodium sulfite (126 mg, 1 mmol) in the water (3 mL) was heated under
reflux for 12 h. The solvent was concentrated under reduced pressure and
the solid obtained was filtered and washed with diethyl ether to give 48 as a
white powder (240 mg, 97%). 1H NMR (400 MHz, D2O): δ 8.76 (s, 1H),
8.10 (d, J = 8.4 Hz, 1H), 7.73ꢀ7.48 (m, 6H), 4.37 (s, 2H).
DXR Inhibition Assay. E. coli DXR expression, purification, and
activity assay were carried out as previously reported.9b,c In brief, the
inhibition assay was performed in a 96-well microplate using the purified
EcDXR (10 nM), 2 mM MgCl2, 100 μM DXP, 50 μM NADPH in
50 mM HEPES buffer (pH = 7.5) containing 25 μg/mL BSA (bovine
serum albumin). Inhibitors were preincubated with EcDXR for 10 min at
30 °C, before adding DXP to initiate the reaction. The process was
monitored at 340 nm with a Beckman DTX-880 microplate reader. The
initial velocities of wells containing increasing concentrations of an
inhibitor were calculated and input into Prism (version 3.0, GraphPad
Software, Inc., La Jolla, CA). The IC50 values were then obtained by
using standard doseꢀresponse curve fitting in the software. The IC50s
used for calculating Ki values were the mean values of at least three
experiments with standard deviations of less than 20%. Ki values were
calculated using the formula Ki = IC50/(1 þ [S]/Km), where [S] is
the concentration of DXP (100 μM) and Km is the literature value of
99 μM.20
M. tuberculosis DXR was expressed and purified as described in the
literature.18 BL21-DE3 strain (Lucigen) was transformed and cultured at
37 °C in LB medium containing ampicillin (50 μg/mL). Upon reaching
an optical density of ∼0.6 at 600 nm, DXR expression was induced for 3 h
by adding 0.42 mM isopropylthiogalactoside at 37 °C. Cells were then
harvested and resuspended in 50 mM NaH2PO4 (pH 8.0), 300 mM
NaCl (buffer A). After addition of 1 mM phenylmethylsulfonyl fluoride
and sonication at 0 °C, the lysate was centrifuged at 20 000 rpm for
20 min and the supernatant was collected and subjected to an affinity
column chromatography using the Talon cobalt resin (Clontech). The
resin was washed with 5 mM imidazole in buffer A, and then the protein
was eluted with 150 mM imidazole in buffer A. After desalting (using
HiTrap, GE Healthcare) to 20 mM Tris pH 7.5, 150 mM NaCl, 2%
glycerol, the protein was concentrated and stored in small aliquots
at ꢀ80 °C.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures S1ꢀS5 showing Seq-
b
uence alignment for EcDXR and MtDXR, superimposed structures
of EcDXR:1 and MtDXR:1, the overall structure of the MtDXR:4
complex, the electron density of inhibitor 4, and Ligplot interaction
diagram for (R)-4 in MtDXR. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
The coordinates for the Mycobacterium tuberculosis DXR:4 com-
plex have been deposited in Protein Data Bank as entry 3RAS.
4732
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Journal of Medicinal Chemistry
ARTICLE
’ AUTHOR INFORMATION
J. Infect. Dis. 2004, 189, 901–908. (d) Borrmann, S.; Lundgren, I.;
Oyakhirome, S.; Impouma, B.; Matsiegui, P. B.; Adegnika, A. A.; Issifou,
S.; Kun, J. F.; Hutchinson, D.; Wiesner, J.; Jomaa, H.; Kremsner, P. G.
Fosmidomycin plus clindamycin for treatment of pediatric patients aged
1 to 14 years with Plasmodium falciparum malaria. Antimicrob. Agents
Chemother. 2006, 50, 2713–2718. (e) Borrmann, S.; Adegnika, A. A.;
Moussavou, F.; Oyakhirome, S.; Esser, G.; Matsiegui, P. B.; Ramharter,
M.; Lundgren, I.; Kombila, M.; Issifou, S.; Hutchinson, D.; Wiesner, J.;
Jomaa, H.; Kremsner, P. G. Short-course regimens of artesunate-
fosmidomycin in treatment of uncomplicated Plasmodium falciparum
malaria. Antimicrob. Agents Chemother. 2005, 49, 3749–3754. (f)
Oyakhirome, S.; Issifou, S.; Pongratz, P.; Barondi, F.; Ramharter, M.;
Kun, J. F.; Missinou, M. A.; Lell, B.; Kremsner, P. G. Randomized
controlled trial of fosmidomycin-clindamycin versus sulfadoxine-pyri-
methamine in the treatment of Plasmodium falciparum malaria. Anti-
microb. Agents Chemother. 2007, 51, 1869–1871.
Corresponding Author
*Address: Department of Pharmacology, Baylor College of
Medicine, 1 Baylor Plaza, Houston, TX 77030. Tel: 713-798-7415.
E-mail: ysong@bcm.edu.
Author Contributions
§These authors contributed equally.
’ ACKNOWLEDGMENT
This work was supported by a grant (R21AI088123) from the
National Institute of Allergy and Infectious Diseases (NIAID) to
Y.S., grants AI049151 and AI065357 from NIAID to D.C.C., and
a grant (Q1279) from Robert Welch Foundation to B.V.V.P. The
recombinant M. tuberculosis DXR expression plasmid was kindly
provided by Dr. T. Alwyn Jones (Uppsala University, Sweden).
We thank the staff of the X-ray Crystallography Facility at Baylor
College of Medicine for assistance in data collection.
(6) Sakamoto, Y.; Furukawa, S.; Ogihara, H.; Yamasaki, M. Fosmido-
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tuberculosis and fosmidomycin resistance is due to a lack of uptake. BMC
Microbiol. 2008, 8, 78–86.
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’ ABBREVIATIONS
DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase;EcDXR,
DXR from E. coli; MtDXR, DXR from Mycobacterium tuberculosis;
DXP, 1-deoxy-D-xylulose-5-phosphate; IPP, isopentenyl dipho-
sphate; DMAPP, dimethylallyl diphosphate; MEP, 2-C-methyl-
D-erythritol-4-phosphate; SAR, structure activity relationship;
QSAR, quantitative structure activity relationship.
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