Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase

Article abstract of DOI:10.1002/cmdc.201600342

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED₅₀

Full text of DOI:10.1002/cmdc.201600342

DOI: 10.1002/cmdc.201600342
Full Papers
Bacterial Cell Growth Inhibitors Targeting Undecaprenyl
Diphosphate Synthase and Undecaprenyl Diphosphate
Phosphatase
Yang Wang+,[a] Janish Desai+,[b] Yonghui Zhang,[a] Satish R. Malwal,[a] Christopher J. Shin,[a]
Xinxin Feng,[a] Hong Sun,[c] Guizhi Liu,[c] Rey-Ting Guo,[c] and Eric Oldfield*[a, b]
We synthesized a series of benzoic acids and phenylphosphon-
ic acids and investigated their effects on the growth of Staphy-
lococcus aureus and Bacillus subtilis. One of the most active
compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7,
ED50 ~0.15 mgmLꢀ1) acted synergistically with seven antibiotics
known to target bacterial cell-wall biosynthesis (a fractional in-
hibitory concentration index (FICI) of ~0.35, on average) but
had indifferent effects in combinations with six non-cell-wall
biosynthesis inhibitors (average FICI~1.45). The most active
compounds were found to inhibit two enzymes involved in
isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl di-
phosphate synthase (UPPS) and undecaprenyl diphosphate
phosphatase (UPPP), but not farnesyl diphosphate synthase,
and there were good correlations between bacterial cell
growth inhibition, UPPS inhibition, and UPPP inhibition.
Introduction
There is, without doubt, a pressing need for the development
of novel antibiotics with new structures and new targets to
help combat the development of drug resistance.[1] Over the
past ~70 years, many antibiotics that target bacterial cell-wall
biosynthesis—compounds such as penicillin, methicillin and
bacitracin—have been discovered and developed, and so the
enzymes that are used in cell-wall biosynthesis are attractive
drug targets. A simplified version of bacterial cell-wall biosyn-
thesis is shown in Figure 1. Initial steps involve formation of
the (C5) isoprenoids dimethylallyl diphosphate (DMAPP, 1) and
isopentenyl diphosphate (IPP, 2), formed in either the mevalo-
nate pathway (in Staphylococcus aureus, for example), or the 2-
C-methyl-d-erythritol 4-phosphate (MEP) pathway (in E. coli, for
example), with fosmidomycin inhibiting the MEP pathway.[2]
DMAPP then condenses, sequentially, with two molecules of
IPP to form the (C15) species, farnesyl diphosphate (FPP, 3), in
a reaction catalyzed by farnesyl diphosphate synthase (FPPS).
The FPP so produced then condenses with eight additional IPP
molecules to form (C55) undecaprenyl diphosphate (UPP, 4) in
a reaction catalyzed by undecaprenyl diphosphate synthase
(UPPS). UPPS is an attractive drug target as it is not used by
humans, and several inhibitors (e.g., tetramic/tetronic acids, di-
amidines, and benzoic acids) have been reported.[3] UPP is
then converted by undecaprenyl diphosphate phosphatase
(UPPP) to undecaprenyl phosphate (UP, 5), which acts as
a membrane “anchor” for the formation of glycosylated prod-
ucts (Lipid I, Lipid II), which are then converted into peptido-
glycan cell wall products, as outlined in Figure 1. Antibiotics
such as bacitracin, vancomycin, and methicillin target these
later stages in cell-wall biosynthesis, again as shown in
Figure 1. In this work, we sought to find novel benzoic acid in-
hibitors of UPPS and potentially UPPP since in earlier work,[3b]
we had found that 2-(3-(decyloxy)benzamido)-5-nitrobenzoic
acid (6, Figure 1) inhibited UPPS, and we reasoned that similar
lipophilic, anionic species might also mimic the UPPP sub-
strate, UPP. We thus synthesized 30 analogues of 6 (7–36), pri-
marily benzoic acids, but also four phosphonic acid analogues,
and tested them for activity against S. aureus and B. subtilis, as
well as against UPPS and UPPP, and in several cases FPPS and
a human cell line. We also investigated their possible synergis-
tic activity with a range of known antibiotics that either target,
or do not target, bacterial cell-wall biosynthesis.
[a] Dr. Y. Wang,+ Y. Zhang, Dr. S. R. Malwal, C. J. Shin, Dr. X. Feng,
Prof. E. Oldfield
Department of Chemistry,
University of Illinois at Urbana–Champaign,
600 South Mathews Avenue, Urbana, IL 61801 (USA)
E-mail: eo@chad.scs.uiuc.edu
[b] J. Desai,+ Prof. E. Oldfield
Center for Biophysics and Quantitative Biology,
University of Illinois at Urbana–Champaign,
1110 West Green Street, Urbana, IL 61801 (USA)
[c] H. Sun, G. Liu, Prof. R.-T. Guo
Industrial Enzymes National Engineering Laboratory,
Tianjin Institute of Industrial Biotechnology,
Chinese Academy of Sciences, Tianjin, 300308 (China)
Results and Discussion
In previous work we found that the benzoic acid 6 (Figure 1)
was a promising inhibitor of both E. coli UPPS (EcUPPS, IC50 =
3.0 mm) and S. aureus UPPS (SaUPPS, IC50 =0.49 mm). In later
work we found that several related lipophilic benzoic acids
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201600342.
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Figure 1. Schematic outline of cell-wall biosynthesis (in most bacteria) delineating the role of isoprenoid biosynthesis in the early stages of peptidoglycan for-
mation, together with the reactions targeted by several compounds discussed in the text.
had activity against Gram-positive (but not Gram-negative)
bacteria, with 6 having an ED50 value of 1.3 mm against B. subti-
lis, but no activity against E. coli, leading us to synthesize the
30 compounds whose structures and activities are shown in
Table 1. Full synthesis and characterization details are provided
in the Supporting Information.
against S. aureus. The most active bacterial cell growth inhibi-
tors were all lipophilic benzoic acids with highly electron-with-
drawing (-NO2, -OCF3) ring substituents (Table 1). We screened
11 and three other compounds (7, 14, and 18) against
a human cell line (NCI-H460) to assess toxicity, finding low ac-
tivity (a selectivity index of ~1000 for 11, Figure S2). The pres-
ence of electron-donating groups (13 and 14) decreased anti-
bacterial activity, as did the presence of substituents that
might hydrogen bond with water (19, 22, 27, and 29). The
phosphonic acids 19, 31, and 36 had little or no antibacterial
activity, although the diethyl phosphonate 21 was active, con-
sistent with the observation that all compounds with addition-
al ionizable groups on the benzoic acid have poor activity.
The question then arises: what might the targets of these in-
hibitors be? The compounds were designed based on the
UPPS inhibition precedent with 6, but FPPS and UPPP inhibi-
tion also seemed possible (with some carboxylic acids being
The rationale for the synthesis of these compounds was first
to investigate a series of compounds (7–16, 19, and 21) in
which we varied the substituent meta to the carboxyl group,
covering a wide range of Hammett sm values (0 for -H, 0.71
for -NO2), which we reasoned would affect the acidity of the
carboxyl group, the more electron-withdrawing groups possi-
bly yielding a better analogue (carboxylate) of the diphosphate
group (in FPP and UPP). We also investigated whether phos-
phonic acids (18, 31, 36) might be better inhibitors than ben-
zoic acids; we also investigated a broad range of other benzoic
acid ring-substitution patterns, as well as different “side
chains” (corresponding to the octyloxy group in 7, for exam-
ple), in order to probe both enzyme as well as cell activity. We
then investigated the cell growth inhibitory activity of these
compounds against S. aureus and B. subtilis, and ED50 values (in
mgmLꢀ1) are listed in Table 1 (rank ordered by SaUPPS inhibi-
tion, as discussed below). We also screened for activity against
the yeast Saccharomyces cerevisiae and the Gram-negative
E. coli, but there was no activity with any compound (ED50
values >200 mm). Typical dose–response curves for three com-
pounds—benzoic acids with electron-withdrawing (7) or elec-
tron-donating (13) ring substituents, plus a phosphonic acid
analogue 18—are shown in Figure 2. Dose–response curves for
all compounds tested are shown in Supporting Information
Figure S1. There were several active compounds amongst the
30 investigated, with the most active being the meta-trifluoro-
methoxy analogue 11, with an ED50 value of 0.082 mgmLꢀ1
Figure 2. Representative dose–response curves for three inhibitors (7, 13
and 18) against B. subtilis and S. aureus. Data are the meanꢁSD for dupli-
cate experiments.
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known potent FPPS inhibitors,[4] binding to an allosteric site).
To narrow down the possibilities and open up potential routes
to synergistic combinations, we next investigated whether one
of the most active compounds, fluorobenzoic acid 7, exhibited
synergistic, additive, indifferent, or antagonistic activity with
a broad range of antibiotics that act either by inhibiting bacte-
rial cell-wall biosynthesis, or by other mechanisms. The antibi-
otics that act by targeting cell-wall biosynthesis were ampicil-
lin, bacitracin, fosmidomycin, carbenicillin, vancomycin, fosfo-
mycin, and cefotaxime. The compounds that do not target
bacterial cell-wall biosynthesis were kanamycin, tetracycline,
sulfamethoxazole, trimethoprim, spectinomycin, and chloram-
phenicol. We determined the fractional inhibitory concentra-
tion index (FICI) values for each combination using the FICI for-
mula:[5]
MIC values of the most effective combination of drug A or B in
the presence of drug B or A. Using this method, FICI values of
<0.5 indicate synergy, >0.5 and <1.0 indicate additivity, >1
and <2 indicate an indifferent effect, and ꢂ2 indicates antag-
onism.[6] In addition, we evaluated isobolograms using the
method of Berenbaum.[7] FICI values are listed in Table 2 and
representative isobolograms are shown in Figure 3. All isobolo-
grams are given in Supporting Information Figure S3.
As can be seen in Table 2, there are very distinct differences
between the FICI values obtained with 7 and the cell-wall bio-
synthesis inhibitors, and those obtained with 7 and the non-
cell-wall biosynthesis inhibitors. For S. aureus, the mean FICI
with a cell-wall biosynthesis inhibitor is 0.32, for B. subtilis, 0.37.
These values represent synergism (i.e., FICI<0.5) and suggest
that 7 acts in the cell-wall biosynthesis pathway. With the non-
cell-wall biosynthesis inhibitors, the mean FICI for S. aureus is
1.42, and for B. subtilis, 1.47, meaning indifferent effects, as ex-
pected. Clearly, for both S. aureus and B. subtilis, compound 7
targets primarily one or more enzymes involved in cell-wall
biosynthesis and likely candidates that use long lipophilic
anionic substrates are UPPS and UPPP, and perhaps FPPS. We
next tested all compounds against UPPS (from S. aureus), since
MICðABÞ MICðBAÞ
FICI ¼ FICA þ FICB ¼
þ
MICðAÞ
MICðBÞ
in which FICA, FICB are the fractional inhibitory concentrations
of drugs A and B, MIC(A) and MIC(B) are the MIC values of
drugs A and B acting alone, and MIC(AB) and MIC(BA) are the
Table 2. Combinations of 7 and antibiotics against B. subtilis and S. aureus.
Antibiotic
MIC antibiotic [mgmLꢀ1
]
FIC antibiotic[a]
B. subtilis
MIC 7 [mgmLꢀ1
]
FIC 7[a]
FICI
fosmidomycin
carbenicillin
cefotaxime
vancomycin
fosfomycin
ampicillin
5
5
0.039ꢁ0.0053
0.22ꢁ0.073
0.17ꢁ0.026
0.27ꢁ0.24
1
1
1
1
1
1
1
0.13ꢁ0.056
0.15ꢁ0.071
0.14ꢁ0.081
0.17ꢁ0.063
0.063ꢁ0.022
0.23ꢁ0.084
0.36ꢁ0.14
0.17ꢁ0.056
0.37ꢁ0.10
0.31ꢁ0.085
0.44ꢁ0.25
0.22ꢁ0.15
0.53ꢁ0.11
0.53ꢁ0.17
Cell Wall
Biosynthesis
Inhibitors
2.5
0.5
200
0.5
200
0.16ꢁ0.15
0.30ꢁ0.072
0.18ꢁ0.089
bacitracin
Protein
Biosynthesis
Inhibitors
chloramphenicol
kanamycin
tetracycline
0.5
1.5
5
0.75ꢁ0.18
0.64ꢁ0.14
0.48ꢁ0.047
1
1
1
0.97ꢁ0.42
0.71ꢁ0.30
0.57ꢁ0.50
1.72ꢁ0.46
1.36ꢁ0.33
1.04ꢁ0.50
Nucleic Acid
Inhibitors
sulfamethoxazole
trimethoprim
200
0.5
0.78ꢁ0.21
0.66ꢁ0.13
1
1
0.94ꢁ0.64
0.85ꢁ0.29
1.72ꢁ0.67
1.51ꢁ0.32
S. aureus
carbenicillin
cefotaxime
vancomycin
fosfomycin
ampicillin
15
2
1.5
200
0.5
200
0.23ꢁ0.048
0.18ꢁ0.055
0.14ꢁ0.034
0.20ꢁ0.11
0.41ꢁ0.17
0.16ꢁ0.11
1
1
1
1
1
1
0.077ꢁ0.026
0.084ꢁ0.031
0.15ꢁ0.062
0.091ꢁ0.034
0.15ꢁ0.071
0.044ꢁ0.019
0.30ꢁ0.055
0.27ꢁ0.063
0.29ꢁ0.071
0.29ꢁ0.36
0.56ꢁ0.18
0.20ꢁ0.11
Cell Wall
Biosynthesis
Inhibitors
bacitracin
spectinomycin
chloramphenicol
kanamycin
40
5
1.5
0.5
0.75ꢁ0.43
0.88ꢁ0.47
0.89ꢁ0.68
0.24ꢁ0.11
1
1
1
1
0.81ꢁ0.32
0.81ꢁ0.31
0.83ꢁ0.29
0.51ꢁ0.24
1.56ꢁ0.54
1.69ꢁ0.56
1.72ꢁ0.74
0.75ꢁ0.26
Protein
Biosynthesis
Inhibitors
tetracycline
Nucleic Acid
Inhibitors
sulfamethoxazole
trimethoprim
200
15
0.54ꢁ0.13
0.69ꢁ0.32
1
1
0.98ꢁ0.37
0.57ꢁ0.25
1.53ꢁ0.39
1.26ꢁ0.41
inhibitors targeting cell-wall biosynthesis
inhibitors targeting nucleic acids and protein biosynthesis
inhibitors targeting cell-wall biosynthesis
0.37ꢁ0.14
1.47ꢁ0.28
0.32ꢁ0.12
1.42ꢁ0.37
B. subtilis
S. aureus
Mean FICIs:
inhibitors targeting nucleic acids and protein biosynthesis
[a] FIC values are reported as the meanꢁSD for duplicate experiments.
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Figure 3. Representative isobolograms for 7 with antibiotics having known mechanisms of action. a) 7+fosmidomycin in B. subtilis showing synergy
(FICI=0.17) of 7 with a cell-wall biosynthesis inhibitor (that targets 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) in the non-mevalonate pathway);
b) 7+sulfamethoxazole in B. subtilis showing an indifferent effect (FICI=1.72) of 7 with a nucleic acid biosynthesis inhibitor (that targets dihydropteroate syn-
thase); c) 7+bacitracin in S. aureus showing synergy (FICI=0.20) of 7 with a cell-wall biosynthesis inhibitor (that targets UPPP); d) 7+kanamycin in S. aureus
showing an indifferent effect (FICI=1.72) of 7 with a protein biosynthesis inhibitor (that targets ribosome function).
in earlier work we had found 6 was a UPPS inhibitor; we also
tested all compounds against a UPP phosphatase. UPPP is
a membrane protein and we used the fusion hybrid of E. coli
UPPP with Haloarcula marismortui bacteriorhodopsin,[8] which
is active in detergent-based assays and is inhibited by bacitra-
cin. We also tested the most active compounds against an
EcFPPS, but there was no inhibition (up to 200 mm). Typical
dose–response curves for SaUPPS and EcUPPP are shown in
Figure 4 and include, for UPPP, results for the known inhibitor
bacitracin, which has an IC50 value of 32 mm, as also reported
by Chang et al.[9] Full dose–response curves for all compounds
are in Supporting Information Figure S4 and all numerical re-
sults are listed in Table 1.
EcUPPS (PDB ID: 4H2O), which suggests a possible explanation
for the results reported herein. Specifically, 6 has a decyloxy
side chain, and the two molecules that bind to UPPS have rela-
tively solvent-exposed nitrobenzoate side chains. This exposure
will have an associated energy penalty. The shorter C8 side
chain in 8 could improve enzyme inhibition by decreasing this
solvent exposure, but the effect is small (0.33 mm vs. 0.49 mm).
However, with another pair of ligands, fluorobenzoate 7 with
a C8 side chain and fluorobenzoate 23 with a C6 side chain, we
find that the shorter-chain species has an IC50 value of 4.9 mm,
whereas the longer-chain (C8) species has a much smaller IC50
value (0.32 mm). Taken together, these results indicate that in-
hibitors with a C8 side chain and small, electron-withdrawing
ring substituents have close to optimum activity. The bulkier
methyl sulfone and diethyl phosphonate groups then—while
being very electron withdrawing—likely contribute to a de-
crease in protein binding affinity, because the added ligand
volume is more solvent exposed than with the smaller sub-
stituents. This binding mode (as seen with 6 in PDB ID: 4H2O),
in which the alkyloxy groups are buried in the interior of UPPS
while the benzoates are at or close to the surface, could also
explain why adding extra carboxylates or phosphonates de-
creases activity: the added groups can hydrogen bond with
the solvent.
As can be seen in Table 1, the most active cell growth inhibi-
tors are also some of the most potent UPPS and UPPP inhibi-
tors, with IC50 values as low as 320 nm (for UPPS) and 1.3 mm
(for UPPP). For the top eight most potent SaUPPS inhibitors
(7–11 and 13–15) there is a good correlation with reported
Hammett sm values (R2 =0.79), but this breaks down with bulk-
ier ring substituents. For example, the electron-withdrawing
group (EtO)2PO (sm =0.42) has weak activity. We have not
been able to obtain X-ray structures of SaUPPS with bound in-
hibitors, but we have reported the structure of 6 bound to
Interestingly, as can be seen in Figure 5a, there is a very
good correlation between the pED50 (ꢀlog10 ED50 [mm]) values
for S. aureus and B. subtilis cell growth inhibition (a Pearson
r value correlation coefficient, r=0.90), strongly suggesting
that the same targets are involved in both systems. As shown
in Figure 5b, the correlation for S. aureus cell growth versus
SaUPPS inhibition is also high, with r=0.85. Figure 5c shows
a Pearson r value correlation coefficient matrix[10] for all cell
and enzyme activities as well as for logD (estimated using the
www.chemicalize.org web portal, www.chemaxon.com), from
which it can be seen that both UPPS inhibition and UPPP in-
hibition correlate with cell growth inhibition. There are weaker
correlations between cell growth inhibition and logD, while
the enzyme inhibition results are essentially uncorrelated with
logD. Therefore, as expected, permeability/transport plays
a role in overall activity in cell growth inhibition. The enzyme/
Figure 4. Dose–response curves of various benzoic acid and phenyl phos-
phonic acid derivatives against SaUPPS and EcUPPP. The benzoic acids are
up to ~40ꢁ more potent UPPP inhibitors than bacitracin, a known UPPP in-
hibitor used as a topical antibiotic. Data are the meanꢁSD for duplicate ex-
periments.
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Figure 5. Correlations between cell growth and enzyme inhibition results. a) Correlation between S. aureus and B. subtilis cell growth inhibition based on
pED50 (ꢀlog10 ED50 [mm]) results; b) correlation between S. aureus cell growth inhibition and SaUPPS inhibition; c) Pearson r value correlation matrix/heat map
for S. aureus cell growth inhibition, B. subtilis cell growth inhibition, SaUPPS and EcUPPP enzyme inhibition (all based on pED50 or pIC50 values), and logD. The
Pearson r values are indicated, and red/orange=high correlation, green=low correlation.
cell correlations are better than those we discussed in a previ-
ous study,[11] in which we found for 10 different cell/putative
enzyme target inhibition assays (three from our research
group, seven randomly chosen from the literature) that on
average the correlation between the enzyme and cell assay re-
sults was r~0.55.
cases FPPS, finding several promising UPPS as well as UPPP in-
hibitors. The most potent UPPP inhibitor was the trifluorome-
thoxy analogue 11 which was ~40ꢁ more active than the
known UPPP inhibitor, the antibiotic bacitracin. It also inhibited
UPPS and was active against S. aureus (ED50 ~82 ngmLꢀ1). Elec-
tron-withdrawing substituents were essential for enzyme/cell
inhibition by the benzoic acids, presumably because they
make the benzoates much stronger acids (that are fully dissoci-
ated), although the presence of multiple acid groups de-
creased both enzyme and cell activity. The phosphonic acids
were worse UPPS/UPPP inhibitors and were mostly inactive in
cells. There were good correlations between cell growth inhibi-
tion and UPPS inhibition, as well as between cell growth inhib-
ition and UPPP inhibition, but UPPS inhibition was more
potent. Overall, the results are of interest since we show for
the first time that lipophilic benzoic acids inhibit both UPPP
and UPPS and are active in cells, where they act synergistically
with known cell-wall biosynthesis inhibitors and as such, they
may represent new leads for developing bacterial cell-wall bio-
synthesis inhibitors.
Also apparent from the results shown in Table 1 is that the
IC50 values for UPPP inhibition are, in general, somewhat larger
than those for UPPS inhibition, suggesting that in cells, UPPS,
and not UPPP, is the primary target. However, this is likely to
be an oversimplification, as the enzyme assays were all deter-
gent-based; substrate concentrations in cells are not known;
UPPP is a membrane protein, and inhibitors may accumulate
in membranes; and we used an EcUPPP–bacteriorhodopsin
fusion in our assay together with FPP, and not UPP. Neverthe-
less, as can be seen in Table 1, several of the most potent cell
growth inhibitors do target both enzymes, and inhibition of
two consecutive enzymes in the bacterial cell-wall biosynthesis
pathway is expected to result in enhanced activity over single-
target inhibition. Moreover, we find good enzyme inhibition/
cell growth correlations; plus, as shown in Figure 3, addition of
any of the known cell-wall biosynthesis inhibitors in assays
with 7 results in synergistic cell growth inhibition, with FICI
values of ~0.35, on average.
Experimental Section
Chemical syntheses: All chemicals were reagent grade. 1H and
13C NMR spectra were obtained on Varian Unity spectrometers
(Palo Alto, CA, USA) at 400 and 500 MHz for 1H NMR. HPLC–MS
analyses were performed with an Agilent LC–MSD Trap XCT Plus
system (Agilent Technologies, Santa Clara, CA, USA) with an 1100
series HPLC system including a degasser, autosampler, binary
pump, and multiple wavelength detector. Purity was determined
by HPLC–MS, and structures were characterized by 1H NMR and
HRMS. We synthesized 26 benzoic acids and four phenylphospho-
nates using the general methods shown in Scheme 1. Full synthe-
sis and characterization details are provided in the Supporting In-
formation, and details of the synthesis of two representative com-
pounds are given below.
Conclusions
The results reported herein are of interest, since we find that
several lipophilic benzoic acids with electron-withdrawing ring
substituents have activity against S. aureus and B. subtilis cell
growth. One of the most potent compounds exhibited syner-
gistic activity with numerous known cell-wall biosynthesis in-
hibitors (FICIavg ~0.35, n=13 different cell/inhibitor combina-
tions), but an indifferent effect (FICIavg ~1.45, n=12 cell/inhibi-
tor combinations) with non-cell-wall biosynthesis antibiotics.
We tested all compounds against UPPS and UPPP, and in some
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Scheme 1. General synthesis methods for A) benzoic acids and B) phenylphosphonates.
5-Fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7): The proce-
dure used to synthesize 7 is shown in Scheme 1A. 1-Bromooctane
b (7.7 g, 40 mmol) was added to a solution of methyl 3-hydroxy-
benzoate a (3.0 g, 20 mmol) in 20 mL DMF followed by the addi-
tion of potassium carbonate (5.5 g, 20 mmol). The solution was
heated at 808C and stirred for 12 h. After cooling to room temper-
ature, the solution was washed with water (100 mL), then extracted
with ethyl acetate (3ꢁ50 mL). The organic layer was dried over
Na2SO4 and solvent removed under vacuum. The residue was puri-
fied by flash chromatography on silica gel (hexane/EtOAc=20:1)
to give methyl 3-(octyloxy)benzoate c as a colorless oil (4.8 g,
90%). Compound c (4.0 g, 15 mmol) was dissolved in 20 mL THF,
and aqueous LiOH (1.8 g in 10 mL water) was added. The reaction
mixture was stirred at room temperature for 12 h, then concentrat-
ed under vacuum to remove solvent. The aqueous solution was
acidified with HCl to pH 1, upon which a white solid precipitated.
The suspension was extracted with ethyl acetate (3ꢁ30 mL). The
organic layer was dried over Na2SO4 and solvent removed under
vacuum. The 3-(octyloxy)benzoic acid d was obtained as a white
solid (2.5 g, 95%). Compound d (2.5 g, 10 mmol) was dissolved in
20 mL CH2Cl2, and oxalyl chloride (2 mL) was added. Then, one
drop of DMF was added as a catalyst. The reaction mixture was
stirred at room temperature for 12 h, then concentrated under
vacuum. The residue 3-(octyloxy)benzoyl chloride e was obtained
as a yellow liquid (3.4 g, 90%). Compound e (2.7 g, 10 mmol) was
dissolved in 20 mL CH2Cl2, then 2-amino-5-fluorobenzoic acid f
(1.6 g, 10 mmol) was added. After stirring for 5 min, NEt3 (2 mL)
was added. The reaction mixture was stirred at room temperature
for 12 h then washed with 2m HCl (2ꢁ30 mL) and water (30 mL).
The residue was concentrated under vacuum to give the final
pound e (540 mg, 2.0 mmol) was dissolved in 20 mL CH2Cl2 and
then diethyl(2-aminophenyl)phosphonate h (460 mg, 2.0 mmol)
was added. After stirring for 5 min, NEt3 (2 mL) was added. The re-
action mixture was stirred at room temperature for 12 h then
washed with 2m HCl (2ꢁ30 mL) and water (30 mL). The residue
was concentrated under vacuum to give compound i as a white
solid (780 mg, 85%). Compound i (460 mg, 1 mmol) was dissolved
in dry CH2Cl2 (15 mL), cooled to 08C, and Me3SiBr (1.2 mL, 9 mmol)
was added dropwise over 30 min. The mixture was then stirred for
two days at room temperature. The solvent was evaporated, and
the residue dried under vacuum for ~1 h. Dry methanol (20 mL)
was then added, and the mixture was stirred for 20 min at room
temperature. The solvent was removed, and the residue was dried
overnight to give the final product j (31) as a white solid (390 mg,
1
95%). H NMR ([D6]DMSO, 500 MHz): d=12.14 (s, 1H), 8.64 (dd, J=
4.5, 8.5 Hz, 1H), 7.65 (dd, J=7.5, 14.5 Hz, 1H), 7.59 (d, J=7.5 Hz,
1H), 7.54 (m, 2H), 7.44 (t, J=8.0 Hz, 1H), 7.16 (m, 2H), 4.03 (t, J=
6.5 Hz, 2H), 1.73 (m, 2H), 1.42 (m, 2H), 1.26–1.22 (m, 12H),
0.85 ppm (t, J=7.0 Hz, 3H); ESI HRMS: m/z [MꢀH]ꢀ calculated for
C21H27NO5Pꢀ: 404.1627, found: 404.1622. Purity of the product de-
termined by HPLC (Phenomenex C6-Phenyl 110A, 100ꢁ2 mm,
3 mm, 250 nm, tR =7.3 min): 93.1%.
B. subtilis growth inhibition assay: ED50 values for B. subtilis cell
growth inhibition were determined using a microdilution method.
A stationary overnight starter culture of B. subtilis (Bacillus subtilis
subsp. subtilis (Ehrenberg) Cohn ATCC 6051) was diluted 1000-fold
and grown to an OD600 value of ~0.3. This log-phase culture was
again diluted 500-fold into fresh LB broth to generate the working
solution; 200 mL of this working solution was transferred into each
well of a 96-well culture plate (Corning 3370). Inhibitors were then
added at 1 mm and sequentially diluted 3ꢁ to 46 nm, keeping
volume and culture broth composition constant. Plates were incu-
bated for 12 h at 378C, shaking at 200 rpm, then absorbance at
600 nm was measured to assess bacterial cell growth. ED50 values
were determined using nonlinear regression, whereas minimum in-
hibitory concentration (MIC) values for each antibiotic and 7 in the
synergy assays were calculated by using a Gompertz function in
Prism 5 (GraphPad Software Inc., La Jolla, CA, USA).
product
g (7) as a
light-yellow solid (3.0 g, 80%). 1H NMR
([D6]DMSO, 500 MHz): d=8.66 (dd, J=5, 9.5 Hz, 1H), 7.75 (d, J=
9.5 Hz, 1H), 7.54 (t, J=9.5 Hz, 1H), 7.48 (m, 3H), 7.19 (d, J=8.0 Hz,
1H), 4.03 (t, J=6.5 Hz, 2H), 1.72 (m, 2H), 1.41 (m, 2H), 1.26–1.22
(m, 8H), 0.84 ppm (t, J=7.0 Hz, 3H); 13C NMR (125 MHz): d=168.8,
164.3, 158.9, 156.9 (d, J=240 Hz), 137.4, 135.7, 130.1, 122.1 (d, J=
7.6 Hz), 121.0 (d, J=22.0 Hz), 118.9, 118.7 (d, J=6.5 Hz), 118.3,
117.0 (d, J=23.9 Hz), 112.1, 67.6, 31.2, 28.7, 28.6, 28.5, 25.4, 22.0,
13.9 ppm; ESI HRMS: m/z [M+H]+ calculated for C22H27FNO4
:
+
388.1924, found: 388.1924. Purity of the product determined by
HPLC (Phenomenex C6-Phenyl 110A, 100ꢁ2 mm, 3 mm, 250 nm,
tR =8.1 min): 99.7%.
S. aureus growth inhibition assay: As with the B. subtilis growth
inhibition assay, an overnight starter culture of S. aureus (Newman
strain) was diluted 1000-fold to create a working solution. Working
solutions were transferred into flat-bottom 96-well plates, and in-
hibitors were added at 1 mm and sequentially diluted 3ꢁ to 46 nm.
2-(N-Methyl-3-(octyloxy)benzamido)phenylphosphonic acid (31):
The procedure used to synthesize 31 is shown in Scheme 1B. Com-
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Full Papers
Plates were incubated at 378C, shaking at 200 rpm for 24 h. The
OD600 value was then measured to determine bacterial growth in-
hibition.
Supporting information: Enzyme and cell growth inhibition tables
and graphs, isobolograms, compound synthesis and characteriza-
1
tion, HPLC purity results, H NMR spectra of inhibitor compounds.
H460 cell toxicity: A broth microdilution method was used to de-
termine the growth inhibition ED50 values. Briefly, ~104 NCI-H460
cells suspended in 100 mL DMEM supplemented with 10% fetal
bovine serum (FBS), 4.5 gLꢀ1 glucose and l-glutamine and pre-
served with 1% penicillin–streptomycin were seeded in 96-well
plates (Corning Inc., Corning, NY, USA) and incubated at 378C
under a 5% CO2 atmosphere. The cells were cultured for 24 h then
incubated with various concentrations of compounds for another
24 h. Then, an MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide] cell proliferation assay (ATCC, Manassas, VA, USA)
was performed. Data from four experiments for each inhibitor
were pooled and then fitted to single dose–response curves. The
reported values are thus ꢁSEM (n=4).
Acknowledgements
This work was supported by the United States Public Health Ser-
vice (National Institutes of Health (NIH) grants CA158191 and
GM065307), a Harriet A. Harlin Professorship, the University of Illi-
nois Foundation/Oldfield Research Fund, and the National Natu-
ral Science Foundation of China (grants 31200053, 31300615,
31400678, and 31470240).
Keywords: benzoic acids
· cell-wall biosynthesis · drug
Synergy/antagonism assays: To investigate possible synergistic in-
teractions between compound 7 and a range of antibiotics, we
carried out two-drug combination assays. Bacterial cells were incu-
bated with a 3ꢁ gradient of antibiotic typically ranging from
40 mgmLꢀ1 to 18 ngmLꢀ1 (200 mgmLꢀ1 to 90 ngmLꢀ1 for bacitracin,
fosfomycin, and sulfamethoxazole) in the presence half-MIC con-
centrations of 7, in addition to a 3ꢁ gradient of 7 ranging from
40 mgmLꢀ1 to 18 ngmLꢀ1 in the presence of half-MIC concentra-
tions of each antibiotic. New MIC values were calculated by using
a Gompertz function in Prism 5 (GraphPad Software Inc., La Jolla,
CA, USA).
discovery · membrane proteins · Staphylococcus aureus
[1] Antibiotic Resistance Threats in the United States, 2013, US Department
of Health and Human Services, Centers for Disease Control and Preven-
tion (CDC): http://www.cdc.gov/drugresistance/pdf/ar-threats-2013-
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Enzyme inhibition assays: SaUPPS and EcUPPP were expressed
and purified as described previously.[3b,9] UPPS assays were carried
out using a phosphate release assay.[3b] Benzoic acid and phos-
phonic acid derivatives were prepared as 10 mm stock solutions in
DMSO, and were then serially diluted from 1 mm to 1 nm. Inhibi-
tors were incubated with 25 ng SaUPPS at room temperature for
10 min in a pH 7.5 buffer (50 mm HEPES, 150 mm NaCl, 10 mm
MgCl2, and 0.02% n-dodecyl-b-d-maltopyranoside) before adding
reaction mixture containing 5 mm FPP, 50 mm IPP, 3 UmLꢀ1 purine
nucleoside phosphorylase, 1 UmLꢀ1 inorganic phosphatase, and
~600 mm 7-methyl-6-thioguanosine (MESG), again in the same
buffer. Reactions were monitored for 15 min with the rate of in-
crease in absorbance at 360 nm taken as the rate of UPP synthesis.
IC50 values were calculated by using Prism 5 (GraphPad Software
Inc., La Jolla, CA, USA). The UPPP inhibition assay was carried out
using a malachite-green reagent as described previously.[12] The
same 10 mm inhibitor stock solutions and assay buffer as for the
SaUPPS assays were used to test for UPPP inhibition. Inhibitors
were incubated with 20 nm EcUPPP at room temperature for
15 min before adding FPP to 35 mm. Reaction mixtures were incu-
bated at 378C for 20 min, then quenched by adding 30 mL mala-
chite-green reagent. In this assay, the phosphate released from FPP
reacts with ammonium molybdate to form phosphomolybdate
(yellow), which then forms a complex (lmax ~620 nm) with mala-
chite-green, used to assess phosphatase activity. Phosphate release
was measured at 620 nm and quantified based on a phosphate
standard curve, and the OD620 values used to construct dose–re-
sponse curves.
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[12] A. A. Baykov, O. A. Evtushenko, S. M. Avaeva, Anal. Biochem. 1988, 171,
266–270.
Received: July 5, 2016
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FULL PAPERS
Y. Wang, J. Desai, Y. Zhang, S. R. Malwal,
C. J. Shin, X. Feng, H. Sun, G. Liu,
R.-T. Guo, E. Oldfield*
&& – &&
Bacterial Cell Growth Inhibitors
Targeting Undecaprenyl Diphosphate
Synthase and Undecaprenyl
Diphosphate Phosphatase
UPP-ward antibiotic trends: We synthe-
sized 30 benzoic and phenylphosphonic
acids and discovered several that inhibit
Staphylococcus aureus and Bacillus subti-
lis growth. They act synergistically with
antibiotics that target cell-wall biosyn-
thesis but not with other antibiotics.
The most potent compounds were
found to target undecaprenyl diphos-
phate phosphatase (UPPP) and unde-
caprenyl diphosphate synthase (UPPS),
and there were good correlations be-
tween enzyme and cell growth inhibi-
tion.
&
ChemMedChem 2016, 11, 1 – 10
www.chemmedchem.org
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ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!