Inhibition of staphyloxanthin virulence factor biosynthesis in Staphylococcus aureus: In vitro, in vivo, and crystallographic results

Article abstract of DOI:10.1021/jm9001764

The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis inhibitors that inhibit the first co

Full text of DOI:10.1021/jm9001764

J. Med. Chem. 2009, 52, 3869–3880
3869
Inhibition of Staphyloxanthin Virulence Factor Biosynthesis in Staphylococcus aureus: In Vitro,
in Vivo, and Crystallographic Results†
Yongcheng Song,‡ Chia-I Liu,§,¶,⊥ Fu-Yang Lin,[ Joo Hwan No,[ Mary Hensler,# Yi-Liang Liu,[ Wen-Yih Jeng,§,¶,⊥
Jennifer Low,| George Y. Liu,| Victor Nizet,# Andrew H.-J. Wang,§,¶,⊥ and Eric Oldfield*,‡,[
Department of Chemistry and Center for Biophysics and Computational Biology, UniVersity of Illinois at UrbanasChampaign, 600 S. Mathews
AVenue, Urbana, Illinois 61801, Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei 11529, Taiwan, National Core Facility of
High-Throughput Protein Crystallography, Academia Sinica, Nankang, Taipei 11529, Taiwan, Institute of Biochemical Sciences, College of Life
Science, National Taiwan UniVersity, Taipei 10098, Taiwan, Department of Pediatrics and Skaggs School of Pharmacy and Pharmaceutical
Sciences, UniVersity of California, San Diego, 9500 Gilman DriVe, La Jolla, CA, and DiVision of Pediatric Infectious Diseases and
Immunobiology Research Institute, Cedars-Sinai Medical Center, Los Angeles, California 90048
ReceiVed February 11, 2009
The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor
for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis
inhibitors that inhibit the first committed step in its biosynthesis, condensation of two farnesyl diphosphate
(FPP) molecules to dehydrosqualene, catalyzed by the enzyme dehydrosqualene synthase (CrtM). The most
active compounds are phosphonoacetamides that have low nanomolar Ki values for CrtM inhibition and are
active in whole bacterial cells and in mice, where they inhibit S. aureus disease progression. We also report
the X-ray crystallographic structure of the most active compound, N-3-(3-phenoxyphenyl)propylphospho-
noacetamide (IC50 ) 8 nM, in cells), bound to CrtM. The structure exhibits a complex network of hydrogen
bonds between the polar headgroup and the protein, while the 3-phenoxyphenyl side chain is located in a
hydrophobic pocket previously reported to bind farnesyl thiodiphosphate (FsPP), as well as biphenyl
phosphonosulfonate inhibitors. Given the good enzymatic, whole cell, and in vivo pharmacologic activities,
these results should help guide the further development of novel antivirulence factor-based therapies for S.
aureus infections.
Introduction
colorless bacteria.9,10 In later work,6 it was shown that loss of
the STX pigment made S. aureus susceptible to killing by
reactive oxygen species (ROS, such as H2O2, ClO-, OH)
produced by neutrophils, blocking infectivity. Consequently,
inhibition of staphyloxanthin biosynthesis is a potential novel
target of anti-infective therapy against pigmented S. aureus
strains.11-13
Infections caused by Staphylococcus aureus are a growing
cause of concern1,2 because of the widespread development of
antibiotic resistance and the shortfall in the introduction of new
types of anti-infective agents. An alternative strategy that is now
gaining interest is targeting of bacterial virulence factors,3-5
molecules that are essential for bacterial growth and/or inva-
siveness in vivo. Since these factors are by definition not
essential for survival in vitro, screening for virulence factor
inhibitors can be challenging. However, at least in the case of
S. aureus,6,7 one important virulence factor is a brightly colored
carotenoid pigment, staphyloxanthin (STXa), whose biosynthesis
can be readily monitored spectrophotometrically. The carotenoid
is produced by the condensation of the C15 isoprenoid farnesyl
diphosphate (FPP) to form presqualene diphosphate and then
dehydrosqualene, followed by a series of oxidations and
glycosylations, in a series of reactions catalyzed by the enzymes
CrtM, N, O, P,...,8 and inhibition of these enzymes (e.g., CrtN
by diphenylamine) has been known for many years to result in
The first committed step in staphyloxanthin biosynthesis
(Figure 1) involves the condensation of two FPP molecules to
form dehydrosqualene. This reaction is thought to occur via a
presqualene diphosphate intermediate and is very similar (or
identical) to that catalyzed by squalene synthase (SQS) in plants,
animals, and some protozoa, where the squalene so produced
is then converted into sterols such as cholesterol, ergosterol,
and the plant sterols. It was thus of interest to see if any of the
many known SQS inhibitors previously developed as cholesterol
lowering drug leads might also have activity in blocking
staphyloxanthin biosynthesis and hence S. aureus virulence. We
recently reported that one class of inhibitor, phosphonosul-
fonates, did exhibit such activity.14 The phosphonosulfonates
(and related bisphosphonates) were developed earlier by Magnin
et al.15,16 using FPP as a “template”. The bisphosphonate
analogues of FPP tended to bind to bone and also caused
elevation of liver enzyme function, and the farnesyl side chain
was metabolically reactive. However, the phosphonosulfonates
did not have these drawbacks, and replacement of the farnesyl
side chain by a diphenyl ether removed the metabolic instability.
Using 1, we found good CrtM inhibition restored ROS (H2O2,
neutrophil) sensitivity and, importantly, observed a major
decrease in bacterial burden following S. aureus challenge in
mice.14 There are, however, many other conceivable backbones
(as well as side chains) that might also have good or even better
†
Crystal structure coordinates have been deposited in the Protein Data
Bank and will be released upon publication (2ZY1).
* To whom correspondence should be addressed. Phone: 217-333-3374.
Fax: 217-244-0997. E-mail: eo@chad.scs.uiuc.edu.
‡
Department of Chemistry, University of Illinois at UrbanasChampaign.
Institute of Biological Chemistry.
National Core Facility of High-Throughput Protein Crystallography.
National Taiwan University.
§
¶
⊥
[
Center for Biophysics and Computational Biology, University of
Illinois at UrbanasChampaign.
#
University of California, San Diego.
Cedars-Sinai Medical Center.
|
a Abbreviations: CrtM, dehydrosqualene synthase; FPP, farnesyl diphos-
phate; QSAR, quantitative structure-activity relationship; SQS, squalene
synthase; STX, staphyloxanthin.
10.1021/jm9001764 CCC: $40.75  2009 American Chemical Society
Published on Web 05/20/2009

3870 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
activity. To explore some of these possibilities, we were
particularly interested to see if it might be possible to reduce
backbone charge/acidity/polarity while still retaining CrtM
activity, since this might improve inhibitor uptake into bacterial
and host cells, as well as further reducing bone affinity. The
possibility that less polar analogues might still have good activity
is supported by the observation that, unlike bisphosphonate
inhibitors of farnesyl diphosphate synthase (FPPS), our pub-
lished CrtM results indicated that Mg2+ binding (which usually
involves binding to two anionic groups) is not essential for
potent phosphonosulfonate inhibition of CrtM.14 For example,
while 1 binds to CrtM with two Mg2+ (PDB file 2ZCQ), 2 binds
with only one Mg2+ (PDB file 2ZCR), and 3 has no Mg2+ at all
in its X-ray crystallographic structure (PDB file 2ZCS). So,
unlike the situation found with FPPS, it seemed likely that a
broad range of backbone structures having fewer anionic groups
might be developed, since the number of metal ions involved
in binding to CrtM is quite variable.
assay,17 as described in the Experimental Section, and are also
reported in Table 1. As can be seen in Table 1, the most potent
inhibitors (4-6) were all diphenyl ether phosphonoacetamides
(motif a, Figure 2) containing a (CH2)3 spacer between the
aromatic and amide moiety and had Ki values in the range
30-70 nM, slightly greater than the 20 nM found for the lead
phosphonsulfonate 1 in the same assay. The activities of these
three compounds in the inhibition of STX biosynthesis in S.
aureus are also shown in Table 1, from which we see that 5 is
the most active compound in this bacterial cell-based assay, with
an IC50 of 8 nM, much less than the IC50 of ∼100 nM reported
for 1, due presumably to improved cellular uptake. Substitution
of one and two Cl atoms on the diphenyl ether side chain had
relatively little effect on CrtM inhibition but decreased bacterial
STX production by ∼3-5× (with respect to 5) (Table 1).
Shortening the (CH2)n spacer by one CH2 group (14) decreased
activity in both assays (CrtM ∼40×; STX ∼400×), while
lengthening the spacer by one CH2 group (16) had an even larger
effect (CrtM IC50 ∼130×, STX ∼400×, again versus 5). We
also found that replacing the diphenyl ether side chain by a
biphenyl group (10) reduced both CrtM inhibition (by ∼20×)
and STX biosynthesis (∼600×) (Table 1). So the diphenyl ethers
containing a (CH2)3 spacer had the most activity, both in the
enzyme and in the whole bacterial cell assays.
We next investigated the effects of changing the PO3H2 group
to a SO3H group (Figure 2, motif b) or a -CO2H group (Figure
2, motif c). The sulfonoacetamide (11) had weak activity in
both assays: a Ki ) 0.81 µM in CrtM inhibition (∼20× higher
than 5) and an IC50 ) 15 µM in STX biosynthesis inhibition
(∼600× higher than 5) (Table 1). The results obtained with
the carboxylic acid analogue (21) in which the -PO3H group
was replaced with a -CO2H group were even worse, with Ki >
7 µM and an IC50 (STX) of 200 µM, ∼200× and ∼25000×
worse than with the phosphonoacetamide 5 (Table 1). So the
In this work, we describe the synthesis of, and inhibition by,
18 compounds encompassing the 11 basic structural motifs
(a-k) shown in Figure 2 in which Ar is an aromatic fragment.
Since some compounds were prepared as salts while others were
free acids, these motifs are shown for simplicity in their
protonated or free acid forms, a point we discuss later in the
text. These motifs were designed on the basis of the following
2-
-
ordering of activity is -PO3H2/-PO3 > -SO3H/-SO3
.
-CO2H/-CO2- in both CrtM and STX biosynthesis inhibition,
suggesting the likely importance of multiple electrostatic
interactions (and/or H-bonding) between CrtM and the inhibi-
tor’s anionic group. Also of interest is the observation that, at
least for the compounds where Ki values are accurately known
(i.e., they are not limit values, Table 1), the cell-based activity
values cover a larger range than do the CrtM enzyme inhibition
results, suggesting the importance of variations in bacterial cell
uptake between the different inhibitors.
ideas: In a, we reduced the (potentially) -3 formal side chain
2-
charge found in the phosphonosulfonates (-PO3H2/-PO3
,
-SO3H/-SO3-) to -2 but added a H-bond donor/acceptor
amide site, a feature suggested at least in part because of
synthetic accessibility. In all cases, the number of methylene
groups n in the “spacer” (Figure 2) was in the range n ) 2-4,
typically n ) 3, and the Ar groups were diphenyl ethers or
biphenyls. In b and c we reduced the side chain formal charge
to -1, but of course the sulfonic and carboxylic acids are
expected to have very different pKa values. In d and e we
investigated whether methyl substitutions might affect activity,
while in f we investigated the effect of modifying H-bond donor
ability. In g-i we further investigated the role of H-bonding,
while in j, we attempted to design a novel motif that might
facilitate metal binding. Finally, compound k (a phosphino-
methylphosphonate) was included as a nonhydrolyzable diphos-
phate analogue (replacing two -O- linkages with two -CH2-).
We also report the X-ray crystallographic structure of one of
the most potent CrtM/STX biosynthesis inhibitors (containing
motif a) bound to CrtM, which gives interesting new insights
into how this compound binds to its CrtM target.
On the basis of these observations, we next investigated the
effects of methyl substitutions on activity. In the case of the d
motif, dimethyl substitution on the acetamide CH2 (13) would
increase hydrophobicity, but this compound had worse CrtM
activity (J20×) and much worse (J62500× versus 5) activity
in STX biosynthesis, where it was essentially inactive (IC50
≈
0.5 mM, Table 1). In the case of the N-Me substituent (Figure
2, motif e), the single methyl group on nitrogen (12) reduced
activity by a factor of ∼20× versus 5 (to Ki ) 0.91 µM),
essentially the same as that seen with the dimethyl analogue
13, but in contrast to 13, there was still measurable STX
inhibition activity (IC50 ) 8.6 µM, ∼1000× worse than with
5). That is, the N-Me (12) and C(Me)2 (13) analogues have
essentially the same activity in CrtM inhibition (0.91 and 0.96
µM, respectively), while STX biosynthesis inhibition is very
different (8.6 and >500 µM for 12 and 13, respectively),
supporting again the idea that the -COCH2PO3H2 group is
important for cell-based activity. These results also suggested
to us that the amide NH group might be important in hydrogen
Results and Discussion
We synthesized a total of 18 compounds (4-21) based on
the 11 motifs shown in Figure 2, and the structures of these
compounds are shown in Table 1, rank-ordered in terms of
decreasing activity in CrtM inhibition. The Ki values were
determined by using a coupled diphosphate/phosphate release

Inhibition of Staphyloxanthin Biosynthesis
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3871
Figure 1. Schematic flowchart for staphyloxanthin and cholesterol biosynthesis from farnesyl diphosphate. The first committed step in both pathways
involves the head-to-head condensation of two molecules of farnesyl diphosphate to form presqualene diphosphate, catalyzed by the CrtM enzyme
in S. aureus, or squalene synthase in humans. In S. aureus, dehydrosqualene is then formed via ring-opening and elimination of diphosphate; in
humans, ring-opening is accompanied by an NADPH reduction step, resulting in squalene.
Given the apparent importance of this region for activity, it
was of interest to see if activity might be improved by
conversion of the NHCO amide moiety to a hydroxamate
(NOH·CO, Figure 2, motif f), which would provide alternative
possibilities for H-bond formation. We made three hydroxa-
mates, 8, 9, and 20, the first two species containing diphenyl
ether side chains, the third, a biphenyl. Interestingly, both the
dichloro and unsubstituted diphenyl ether phosphonohydrox-
amates had good activity in both CrtM and STX biosynthesis
inhibition (Table 1), although they were both less active than
the three most potent phosphonoacetamides. On average, the
hydroxamates were ∼6× less active against CrtM and ∼2×
less active in the STX biosynthesis assay (Table 1). Not
unexpectedly, the biphenyl hydroxamate (20) was even less
active, consistent with the weaker activity of the biphenyl
phosphonoacetamide, 10. So for activity, these results indicate
the importance of a phosphonoacetyl group, located in either
an acetamide or a hydroxamate group, suggesting the importance
of both electrostatic (H-bond) interactions between the phos-
phonate and the protein and, most likely, hydrogen bonding
between the amide (or hydroxamate) and the protein. To test
these ideas further, we next investigated motifs g-i, Figure 2,
Figure 2. Structural motifs present in the different inhibitors investigated.
using compounds 19, 17, and 18. Surprisingly, the sulfonamide
19 had no activity in either the CrtM or STX biosynthesis assay
(Table 1), suggesting a critical role for the amide/hydroxamate
CO group as an H-bond acceptor. Consistent with this, neither
bonding to the protein, since the NH to NMe substitution
reduced CrtM activity by ∼20× (Table 1).

3872 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
Table 1. Enzyme (CrtM, Ki), Pigment (STX, S. aureus, IC50), and Cell Growth (IC50) Inhibition Results for 4-21
a The value given are the Ki values for CrtM inhibition, in µM. b The values given are the IC50 values for STX (staphyloxanthin) virulence factor inhibition
in S. aureus and are in µM. c The values given are the Ki values for human squalene synthase inhibition (in vitro) and are in µM. d The values given are the
IC50 values for MCF-7 cell growth inhibition, in µM. e The values given are the IC50 values for NCI-H460 cell growth inhibition, in µM. f The values given
are the IC50 values for SF-268 cell growth inhibition, in µM.

Inhibition of Staphyloxanthin Biosynthesis
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3873
Table 2. Data Collection and Refinement Statistics for CrtM-5
Complex
the ester (17) nor the ketone (18) had significant activity in either
assay (Table 1).
CrtM-5
We then investigated two additional motifs, j and k in Figure
2. The hydroxamate (j, 15) had modest activity in both assays
(Ki, IC50 ∼4 µM) but was ∼100× (CrtM) to ∼500× (STX)
less active than was 5. The phosphinomethylphosphonate 7
(motif k) was a potent CrtM inhibitor (Ki ) 220 nM), but again
we believe, due to its increased polarity, it was ∼50× less
effective in STX biosynthesis inhibition in whole cells than was
the most potent phosphonoacetamide (5).
Finally, we investigated the effects of all 18 compounds on
human SQS inhibition, together with their activity in growth
inhibition of three human tumor cell lines (MCF-7, NCI-460,
and SF-268) (Table 1). Although inhibition of human SQS may
not be a particularly important toxicologic consideration, given
a choice, it would seem to be preferable to have a STX
biosynthesis inhibitor with poor activity against SQS, as opposed
to one with potent SQS activity, and as can be seen in Table 1,
several potent CrtM inhibitors do have relatively little activity
in the SQS assay. More important, in essentially all cases we
find no inhibition of human cell growth (in three human cell
lines, IC50 > 300 µM), supporting the idea that these compounds
will have low toxicity. In fact, only the ester 17 had significant
activity (IC50 ≈ 70-90 µM) on human cell growth, but since
this compound is inactive in STX biosynthesis inhibition, it is
not likely to be of interest.
Crystal Data Collection
radiation source
wavelength (Å)
space group
unit cell dimensions
a (Å)
NSRRC BL13B1
1.00000
P3221
80.59
b (Å)
c (Å)
80.59
90.02
resolution (Å)a
no. of reflections
completeness (%)
redundancy
Rmerge (%)
30-1.78 (1.84-1.78)
32804 (3224)
99.7 (100)
6.3 (6.2)
2.6 (33.1)
51.7 (4.6)
I/s(I)
Refinement
no. of reflections
Rwork (%)
Rfree (%)
31532 (2840)
18.2 (22.9)
21.9 (23.7)
Geometry deviations
bond lengths (Å)
bond angles (deg)
0.015
1.6
mean B-values (Å2)/no.
protein atoms
29.5/2353
29.2/24
53.1/507
compound atoms
water molecules
Ramachandran plot (%)
most favored
93.9
6.1
additionally allowed
Structure and Structure-Function Aspects. The results
described above are of interest, since they represent the
development of several new, chemically diverse virulence
inhibitors for S. aureus. However, the relationships between
structure and activity need to be explored in more detail in
order to obtain a better understanding of how, in particular,
the most active compounds act on their CrtM target. What
is interesting about the most active compounds, the phospho-
noacetamides, is that while they contain the same 3-(3-
phenoxyphenyl)propyl (PhOC6H4(CH2)3-) side chain as
found in the most active phosphonosulfonates,18 the “back-
bone” is clearly about two bond lengths greater in the new
compounds. So we next examined the effects of this common
feature on binding.
a Values in the parentheses are for the highest resolution shells.
3C. Interestingly, 5 binds to CrtM in a completely different
manner to that observed with FsPP or any of the three
phosphonosulfonates reported previously.14 Its polar phos-
phonate headgroup is located in the FsPP site 1, where it
makes a salt bridge with Arg45 and H-bond contacts with
Gln165 and Asn168, together with a complex H-bond
network with five H2O molecules (Figure 4). However, unlike
the situation found with 1, the diphenyl ether side chain
occupies the FPP site 2. In addition, the amide CO forms
H-bonds with Arg45 and two H2O molecules, and the amide
NH acts as an H-bond donor to Gln165 (Figure 4B).
Essentially, the increased length of 5 (over that present in
the phosphonosulfonates) is accommodated in the protein by
5 “bridging” both the FPP-1 and FPP-2 binding sites (Figure
3B,C).
In the CrtM enzyme, there are two binding sites for FPP,14
and in previous work we found that phosphonosulfonates
could bind to either one of these sites.14 The diphenyl ether
phosphonosulfonate 1 bound to site 1 and had interactions
with 2 Mg2+, while two biphenyl phosphonosulfonates (2,
3) bound to site 2 with 1 and 0 Mg2+, respectively. This
observation complicates any QSAR analysis, since there could
be 0, 1, or 2 Mg2+ present, and two different sites might be
occupied. Plus, with the phosphonoacetamides, the presence
of a longer backbone complicates the situation further, since,
for example, if the diphenyl ether is located in the same
region as found with 1, the headgroup will not fit. We
therefore next obtained the structure of a CrtM-5 complex
using X-ray crystallography to determine how 5 does in fact
bind to CrtM, in order to provide a better, albeit qualitative,
structure-based interpretation of the activity of one of the
most potent inhibitors. Data collection and refinement
statistics are given in Table 2, and full details are given in
the Experimental Section.
Given this new structure (PDB 2ZY1), it is now possible
to rationalize several of the SAR observations noted above.
Specifically, conversion of the -PO32- group in 5 to -SO3- or
-
-CO2 can be expected to result in the loss of many of the
H-bond/electrostatic interactions with Arg45, Gln165, and
Asn168 and the five H2O molecules. Second, conversion of the
amide NH to NMe will prevent H-bond formation with Gln165,
as will formation of an ester (17) or a ketone (18) in this region.
Likewise, conversion of the amide to the hydroxamate (9) results
in disruption of this H-bond interaction which may, however,
be partially compensated for by other conformational changes,
since 9 still retains quite good (Ki ) 320 nM) CrtM activity.
But how can we correlate our enzyme inhibition results with
cell based inhibition of STX biosynthesis, something that is
clearly desirable because we are interested in good inhibition
of virulence factor formation in cells and not just good CrtM
inhibitors (which might not actually get into cells)?
CrtM crystallizes in the P3221 space group, and there is
one molecule per asymmetric unit. The phosphonoacetamide
5 yielded well-resolved 2Fobs - Fcalc densities (Figure 3A),
and the refined structure of 5 (red) is shown superimposed
on FsPP (bound to CrtM) in Figure 3B and on 1-3 in Figure
In Vitro and in Vivo Results. When considering all
compounds investigated, we find that there is quite a good
correlation between the CrtM enzyme pKi () -log10 Ki, M)

3874 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
Figure 3. X-ray crystallographic results: (A) electron density for 5 in CrtM; (B) superposition of 5 (red) in the CrtM active site (2ZY1) with that
of two molecules (green, yellow) of S-thiolofarnesyl diphosphate (2ZCP); (C) superposition of 5 (red) with 1 (blue), 2 (yellow), 3 (cyan) in the
CrtM active site (2ZCQ, 2ZCR, 2ZCS).
Figure 5. Figure showing correlations between CrtM inhibition and
Figure 4. Interactions between 5 and different residues in the CrtM
STX biosynthesis inhibition: (A) data for the 14 compounds reported
in this work; (B) combination of 36 phosphonosulfonate inhibitor results
(ref 18) with the 14 compounds reported here; (C) combinatorial
descriptor search result for all 50 compounds tested (here and in ref
18) in CrtM and STX biosynthesis inhibition. The lower R2 value in
part B is likely due the high diversity of the large data set; the R2
improves to 0.68 by using the combinatorial descriptor approach.19
active site: (A) Pymol26 view; (B) Ligplot27 interactions.
and cell STX biosynthesis inhibition pIC50 () -log10 IC50,
M) values, as can be seen in Figure 5A where the R2 value
is 0.73 (for the 14 compounds having nonlimit Ki/IC50 values).
However, when we add results for the 36 compounds reported

Inhibition of Staphyloxanthin Biosynthesis
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3875
the presence of the coupling reagent N-ethyl-N′-(3-dimethyl-
aminopropyl)carbodiimide (EDC) to give, after hydrogenation
to remove the benzyl groups, a phosphonoacetamide (e.g.,
5). N-Hydroxyphosphonoacetamide compounds, such as 9,
were prepared from substituted hydroxylamine 29 and
diethylphosphonoacetyl chloride 23, after hydrolysis with
TMSBr (to remove ethyl phosphono-esters) and hydrogen-
ation (to remove O-benzyl protecting group), also shown in
Scheme 1. Compounds 13, 11, 17, 21, 14, and 16 were made
similarly, with a carbodiimide mediated amide/ester formation
reaction as the main step, as shown in Scheme 2.
Other compounds were made as outlined in Scheme 3.
Compound 15 is an analogue of 5 but has a hydroxamate
group in its side chain. Alkylation of ethyl dibenzylphospho-
noacetate with iodide 28 gave compound 30, as shown in
Scheme 3. The carboxylate ethyl ester was then selectively
hydrolyzed (1 M KOH) and the corresponding acid coupled
with O-benzylhydroxylamine under standard carbodiimide
conditions, followed by hydrogenation to remove three benzyl
protecting groups, to give 15. Compound 18 is a ꢀ-keto-
phosphonate analogue of 5, and its synthesis began with a
Suzuki coupling reaction of an ethyl 4-pentenoate derived
boron compound and 4-bromodiphenyl ether (Scheme 3),
affording carboxylate ester 31. Compound 31 was then
reacted with 2 equiv of lithium diethyl methylphosphonate
at -78 °C to give, after TMSBr mediated hydrolysis, 18.
The reaction of precursor amine 26 with methanesulfonyl
chloride produced methylsulfamide 32 (Scheme 3). This was
then treated with 2 equiv of butyllithium to give a dianion,
which was reacted with diethyl chlorophosphate and hydro-
lyzed with TMSBr, resulting in the phosphonosulfamide 19.
Compound 7 was made by an alkylation reaction of the
dianion of triethyl methylphosphinomethylphosphonate with
iodide 28, followed by hydrolysis.
Figure 6. In vivo results showing ∼96% reduction in S. aureus colony
formation in mice receiving treatment with 5.
previously to the correlation, the n ) 50 compound data set
exhibits a much worse correlation (Figure 5B) with an R2 )
0.42. This is similar to the results we reported previously
where we found for 10 different enzyme/cell assays that on
average the R2 value for the pKi/pIC50 correlation was only
0.32 (ref 19), making any predictions of cell-based activity,
based on enzyme activity, in some cases, impossible. The
large discrepancies found were, we proposed, likely to be
due to the neglect of factors that affect inhibitor uptake into
cells, and we described a general method in which this aspect
might be taken into consideration, by using a “combinatorial
descriptor approach”.19 That is, we described cell activity
by using the following equation:
pIC50(cell) ) apKi(enzyme) + bB + cC + d
Conclusions
where a-d are linear regression coefficients and where B and
C are mathematical descriptors (such as SlogP) chosen in a
combinatorial manner from a large series of potential descriptors
(such as the 230 descriptors in the program MOE20). Applying
this same method to the combined data set (50 compounds),
we now obtain (Figure 5C) R2 ) 0.68, a significant improvement.
To investigate in vivo activity, we selected the most potent
in vitro STX biosynthesis inhibitor (5) and carried out an
intraperitoneal challenge experiment with S. aureus in exactly
the same manner as reported previously for 1.14 We treated one
group of mice (n ) 9) with 0.5 mg of 5 twice per day (days
-1, 0, 1, and 2) and a second group (n ) 9) with equivalent
volume injections of PBS control. Upon sacrificing the mice at
72 h, S. aureus bacterial counts in the kidneys of the mice treated
with 5 were significantly lower than those of the control group
(p < 0.001). The median number of colony forming units (cfu)
in the untreated animals was 22 500 cfu/mL compared with 850
cfu/mL for the treated animals (Figure 6), about a 96% reduction
in surviving bacteria in the treatment group.
Synthetic Aspects. We outline here the synthetic schemes
used to prepare 4-21; full protocols for each compound are
given in the Experimental Section. A general synthetic route
to the most active phosphonoacetamide and N-hydroxyac-
etamide (hydroxamate) compounds is shown in Scheme 1.
The reaction of aldehyde 24 and sodium diethyl cyanometh-
ylphosphonate in THF gave, after hydrogenation, compound
25 in almost quantitative yield, which after reduction with 2
equiv of LiAlH4 and AlCl3 afforded amine 26. Amine 26
was then reacted with dibenzylphosphonoacetic acid 22 in
The results we have described here are of interest for a
number of reasons. First, we have synthesized a broad variety
of inhibitors of the dehydrosqualene synthase enzyme, CrtM,
exhibiting novel structural motifs. Second, we have determined
their activity in CrtM inhibition and in staphyloxanthin biosyn-
thesis in S. aureus. Third, we show that STX biosynthesis
activity for a broad range of inhibitors can be predicted from
enzyme inhibition results with quite good R2 values by using a
combinatorial descriptor approach. Fourth, we have demon-
strated that the most potent STX biosynthesis inhibitor in vitro
also has activity in vivo in preventing infection. Fifth, we show
that these compounds have no activity against three human cell
lines. Sixth, we have obtained the X-ray crystallographic
structure of the most potent STX biosynthesis inhibitor inves-
tigated here (5) bound to CrtM. The structure is unusual in that
the inhibitor actually bridges the FPP-1 and FPP-2 sites observed
previously,14 whereas the phosphonosulfonates reported previ-
ously bind to one or other of these sites but not to both. Many
of the major changes in CrtM activity between the different
motifs investigated can be interpreted in terms of the crystal-
lographic results, opening up the way to the further development
of this class of compound as novel anti-infective agents targeting
inhibition of the biosynthesis of the staphyloxanthin virulence
factor in S. aureus.
Experimental Section
General Synthesis Methods. General Method A. Triethyl
phosphonoacetate, or diethyl cyanomethylphosphonate (3.3

3876 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
Scheme 1. General Synthetic Routes to Phosphonoacetamides and N-Hydroxyphosphonoacetamidesa
a Reagents and conditions: (i) BuLi, then CO2, -78 °C, 63%; (ii) oxalyl chloride (2 equiv), 100%; (iii) NaH, diethyl cyanomethylphosphonate; (iv) H2,
Pd/C (5%); (v) LiAlH4 (2 equiv), AlCl3 (2 equiv); (vi) NaH, triethyl phosphonoacetate; (vii) LiAlH4 (2 equiv); (viii) MsCl, NEt3, then NaI (5 equiv); (ix)
O-benzylhydroxylamine (2 equiv), diisopropylethylamine, DMF, 80 °C, 50% overall from 24; (x) EDC, HOBt; (xi) NEt3; (xii) TMSBr (2 equiv), then
MeOH.
Scheme 2a
a Reagents and conditions: (i) NEt3; (ii) TMSBr (2 equiv), then MeOH, 48% for two steps; (iii) EDC, HOBt; (iv) DOWEX ion-exchange resin, H+ form,
85% for two steps; (v) H2, Pd/C (5%); (vi) KOH, MeOH/H2O, 66% for two steps; (vii) NaCN, DMF; (viii) LiAlH4 (2 equiv), AlCl3 (2 equiv).
mmol), was added dropwise to NaH (145 mg, 60% in oil, 3.6
mmol) suspended in dry THF (7 mL) at 0 °C. To the resulting
clear solution was added a benzaldehyde (3 mmol), and after
being stirred at room temperature for 0.5 h, the reaction mixture
was partitioned between diethyl ether (50 mL) and water (50
mL). The organic layer was dried and evaporated. The oily
residue was then hydrogenated in MeOH (15 mL) in the presence
of 5% Pd/C (50 mg). The catalyst was filtered and the filtrate
concentrated and dried in vacuo.
General Method B. The nitrile (or ester) obtained using general
method A was added slowly to 2 equiv of LiAlH4/AlCl3, or LiAlH4,
in dry THF at 0 °C. After the mixture was stirred at room temperature
for 2 h, the reaction was carefully quenched by adding a few drops of
water and the reaction mixture filtered and evaporated.

Inhibition of Staphyloxanthin Biosynthesis
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3877
Scheme 3a
a Reagents and conditions: (i) NaH, ethyl dibenzylphosphonoacetate, 68%; (ii) KOH, MeOH/H2O; (iii) O-benzylhydroxylamine, EDC, HOBt; (iv) H2,
Pd/C (5%), 31% from 30; (v) 9-BBN, then Pd(PPh3)4, K3PO4, 80 °C, 48%; (vi) diethyl methylphosphonate (2 equiv), BuLi, -78 C, 65%; (vii) TMSBr, then
MeOH; (viii) MsCl, NEt3; (ix) BuLi (2 equiv), -78 °C, then diethyl chlorophosphate, 76%; (x) BuLi (2 equiv), -78 °C, then 28, 58%.
General Method C. To a solution of a carboxylic acid (1 mmol)
and an amine (1 mmol) in CH2Cl2 (5 mL) were added N-ethyl-N′-(3-
dimethylaminopropyl)carbodiimide (EDC) (1.5 mmol) and 1-hydroxy-
benzotriazole (1 mmol). After the mixture was stirred for 2 h at room
temperature, 50 mL of ethyl acetate was added and the reaction mixture
washed successively with 1 N HCl (5 mL), water (5 mL), and saturated
NaHCO3 (5 mL), dried, and evaporated. The amide was purified using
flash chromatography (silica gel, ethyl acetate).
General Method D. A DMF solution (3 mL) containing a halide
(3 mmol), O-benzylhydroxylamine (6 mmol) and diisopropylethyl-
amine (3 mmol) was heated at 90 °C for 24 h. After the mixture
was cooled, diethyl ether (50 mL) was added and the mixture was
washed with H2O (20 mL), dried, and evaporated. The alkylated
hydroxylamine, such as 29, was purified by using column chro-
matography (silica gel; hexane/ethyl acetate, 6/1).
General Method E. To a diethyl phosphonate (1 mmol) in dry
CH3CN (3 mL) was added TMSBr (2 mmol) at room temperature.
After 6 h, the solution was evaporated and methanol (5 mL) added.
Neutralization with 1 N KOH to pH 8, followed by evaporation to
dryness and triturating with acetone, gave a white powder.
All reagents used were purchased from Aldrich (Milwaukee, WI).
The purities of all compounds were routinely monitored by using
1H and 31P NMR spectroscopy at 400 or 500 MHz on Varian (Palo
Alto, CA) Unity spectrometers. All compounds were of g95%
purity, as determined by combustion analysis. The details of these
syntheses are as follows.
Diethylphosphonoacetyl Chloride (23a). Compound 23a was
prepared by mixing diethylphosphonoacetic acid (1.5 mmol) with
oxalyl chloride (3 mmol) in benzene (5 mL) in the presence of one
drop of DMF for 1 h, followed by evaporation. The oily residue
was used immediately for the next reaction.
hyde (1 mmol), using general method A, and was then coupled
with dibenzylphosphonoacetic acid according to general method
C to give the dibenzyl ester of 4. The benzyl groups were
removed by catalytic hydrogenation (5% Pd/C in methanol for 1 h)
followed by neutralization with KOH to give compound 4 as a white
powder (245 mg, 48% overall yield). Anal. (C17H16Cl2-
K2NO5P·0.5CH3OH) C, H, N. 1H NMR (400 MHz, D2O): δ
1.60-1.70 (m, 2H, CH2); 2.35 (d, J ) 20 Hz, 2H, CH2P); 2.46 (t,
J ) 7.6 Hz, 2H, PhCH2); 2.98 (t, J ) 7.2 Hz, 2H, CH2N);
6.70-7.30 (m, 7H, aromatic). 31P NMR (D2O): δ 13.6.
N-[3-(3-Phenoxyphenyl)propyl]phosphonoacetamide Dipotassi-
um Salt (5). Amine 26 was prepared from 3-phenoxybenzaldehyde
(1 mmol) using general method A and was then coupled with
dibenzylphosphonoacetic acid according to general method C to give
the dibenzyl ester of 5. The benzyl groups were removed by
hydrogenation for 1 h, catalyzed with 5% Pd/C in methanol, followed
by neutralization with KOH to give compound 5 as a white powder
(307 mg, 62% overall yield). Anal. (C17H18K2NO5P·1.5H2O) C, H,
N. 1H NMR (400 MHz, D2O): δ 1.60-1.70 (m, 2H, CH2); 2.35 (d, J
) 20 Hz, 2H, CH2P); 2.46 (t, J ) 7.6 Hz, 2H, PhCH2); 2.98 (t, J )
7.2 Hz, 2H, CH2N); 6.70-7.30 (m, 9H, aromatic). 31P NMR (D2O):
δ 13.7.
N-[3-(3-(4-Chlorophenoxy)phenyl)propyl]phosphonoacetamide
Dipotassium Salt (6). 6 was prepared in the same way as 5, but
using 3-(4-chlorophenoxy)benzaldehyde (1 mmol) as starting ma-
terial, as a white powder (267 mg, 58% overall yield). Anal.
(C17H17ClK2NO5P·0.3KCl·1.5H2O) C, H, N. 1H NMR (400 MHz,
D2O): δ 1.60-1.70 (m, 2H, CH2); 2.37 (d, J ) 20 Hz, 2H, CH2P);
2.49 (t, J ) 7.6 Hz, 2H, PhCH2); 3.01 (t, J ) 7.2 Hz, 2H, CH2N);
6.70-7.30 (m, 8H, aromatic). 31P NMR (D2O): δ 13.5.
3-(3-Phenoxyphenyl)propylphosphinylmethylphosphonic Acid
Tripotassium Salt (7). Triethyl methylphosphinylmethylphospho-
nate (1 mmol) was treated with BuLi (2.2 mmol) in THF at -78
°C for 1 h, followed by addition of iodide 28 (1.1 mmol). The
reaction mixture was allowed to warm to room temperature over
3 h and was then quenched with saturated NH4Cl. The product
was purified with column chromatography (silica gel; ethyl acetate/
methanol, 20/1) and deprotected using general method E to give 7
3-(3-Phenoxyphenyl)propyl Iodide (28). Alcohol 27, obtained
from 3-phenoxybenzaldehyde (3 mmol) following general methods
A and B, in CH2Cl2 (10 mL) containing NEt3 (0.5 mL, 3.6 mmol)
was reacted with methanesulfonyl chloride (230 µL, 3 mmol) at 0
°C. After 1 h, diethyl ether (50 mL) and water (50 mL) were added
and the organic layer was collected, washed with 1 N HCl and
saturated NaHCO3, dried, and evaporated to dryness. The oily
residue was treated with NaI (1.35 g, 9 mmol) in acetone (7 mL)
at 60 °C for 1 h. The reaction mixture was then partitioned between
diethyl ether (50 mL) and water (50 mL) and the organic layer
washed with 5% Na2S2O3, dried, and evaporated to dryness to give
as
a white powder (320 mg, 62% overall yield). Anal.
(C17H19K3O6P2 ·H2O) C, H. 1H NMR (400 MHz, D2O): δ 1.30-1.60
(m, 6H, 3CH2); 1.75-1.85 (m, 2H, CH2P); 2.43 (t, J ) 7.6 Hz,
2H, PhCH2); 6.70-7.30 (m, 9H, aromatic). 31P NMR (D2O): δ 16.3
(s, 1P); 39.9 (s, 1P).
1
iodide 28. The iodide thus obtained is quite pure, according to H
and 13C NMR spectra, and may be used in the next step without
further purification.
N-Hydroxy-N-[3-(3-(3,4-dichlorophenoxy)phenyl)propyl]phos-
phonoacetamide Dipotassium Salt (8). 8 was prepared in the same
way as 9, but using 3-(3,4-dichlorophenoxy)benzaldehyde (3 mmol)
as starting material, as a white powder (428 mg, 28% overall yield).
N-[3-(3-(3,4-Dichlorophenoxy)phenyl)propyl]phosphonoacetam-
ide Dipotassium Salt (4). 3-(3-(3,4-Dichlorophenoxy)phenyl)-
propylamine was prepared from 3-(3,4-dichlorophenoxy)benzalde-

3878 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
Anal. (C17H16Cl2K2NO6P·KBr) C, H, N. 1H NMR (400 MHz, D2O):
δ 1.75-1.85 (m, 2H, CH2); 2.48 (t, J ) 7.6 Hz, 2H, PhCH2); 2.63
(d, J ) 20 Hz, 2H, CH2P); 3.46 (t, J ) 7.2 Hz, 2H, CH2N);
6.70-7.35 (m, 7H, aromatic). 31P NMR (D2O): δ 15.6.
N-[2-(3-Phenoxyphenyl)ethyl]phosphonoacetamide Dipotassium
Salt (14). 3-Phenoxybenzyl chloride (2 mmol) and NaCN (2.2
mmol) were stirred in DMF (2 mL) overnight at 60 °C. After the
mixture was cooled, diethyl ether (50 mL) was added and the
mixture was washed with water and the organic layer dried and
evaporated. 2-(3-Phenoxyphenyl)ethylamine was prepared from the
nitrile so obtained, using general method B, and was then coupled
with dibenzylphosphonoacetic acid according to general method
C to give the dibenzyl ester of 14. The benzyl groups were re-
moved by catalytic hydrogenation (5% Pd/C in methanol for 1 h)
followed by neutralization with KOH to give compound 14 as a
white powder (387 mg, 45% overall yield). Anal. (C16H16-
K2NO5P·H2O) C, H, N. 1H NMR (400 MHz, D2O): δ 2.39 (d, J )
20 Hz, 2H, CH2P); 2.58 (t, J ) 7.6 Hz, 2H, PhCH2); 3.05 (t, J )
7.2 Hz, 2H, CH2N); 6.70-7.30 (m, 9H, aromatic). 31P NMR (D2O):
δ 13.8.
N-Hydroxy-N-[3-(3-phenoxyphenyl)propyl]phosphonoacetam-
ide Dipotassium Salt (9). General method D with iodide 28 gave
substituted hydroxylamine 29 (1 mmol), which was reacted with
the acid chloride in the presence of NEt3 (2 mmol) in CH2Cl2 (5
mL) at 0 °C. After the mixture was stirred for 1 h, the coupling
product was purified by using column chromatography (silica gel,
ethyl acetate) and was then deprotected following general method
E. Hydrogenation (5% Pd/C, MeOH) gave compound 9 as a white
1
powder (312 mg, 56% overall yield). H NMR (400 MHz, D2O):
δ 1.65-1.75 (m, 2H, CH2); 2.42 (t, J ) 7.6 Hz, 2H, PhCH2); 2.66
(d, J ) 20 Hz, 2H, CH2P); 3.39 (t, J ) 7.2 Hz, 2H, CH2N);
6.70-7.20 (m, 9H, aromatic). 31P NMR (D2O): δ 15.8.
N-Hydroxy-2-phosphono-5-(3-phenoxyphenyl)pentamide Dipo-
tassium Salt (15). Iodide 28 was added to a cold DMF solution
containing ethyl dibenzylphosphonoacetate (1 equiv) and NaH (1.1
equiv). After the mixture was stirred for 3 h at room temperature,
the product 30 was purified by using column chromatography (silica
gel; hexane/ethyl acetate, 1/1). 30 was then treated with 3 N KOH
in EtOH/H2O (3:1) for 24 h and the resulting solution was reduced
in volume and then acidified with 3 N HCl, to give the correspond-
ing carboxylic acid. The acid so obtained was reacted with
O-benzylhydroxylamine, according to general method C, to give
protected 15, which was then hydrogenated in the presence of 5%
Pd/C in MeOH for 1 h to afford, after neutralization with KOH,
15 as a white powder (293 mg, 21% overall yield). Anal.
(C17H18K2NO6P·0.5C2H5OH) C, H, N. 1H NMR (400 MHz, D2O):
δ 1.25-1.75 (m, 4H, CH2); 2.20-2.50 (m, 3H, CH + PhCH2);
6.70-7.25 (m, 9H, aromatic). 31P NMR (D2O): δ 17.5.
N-[4-(3-Phenoxyphenyl)butyl]phosphonoacetamide Dipotassium
Salt (16). Iodide 28 (2 mmol) and NaCN (2.2 mmol) were stirred
in DMF (2 mL) overnight at 60 °C. After the mixture was cooled,
diethyl ether (50 mL) was added and the mixture washed with water
and the organic layer evaporated. 4-(3-Phenoxyphenyl)butylamine
was prepared from the nitrile so obtained, using general method
B, and was then coupled with dibenzylphosphonoacetic acid,
according to general method C, to give the dibenzyl ester of 16.
The benzyl groups were removed by catalytic hydrogenation (5%
Pd/C in methanol for 1 h) followed by neutralization with KOH to
give compound 16 as a white powder (475 mg, 51% overall yield).
Anal. (C18H20K2NO5P·1.5H2O) C, H, N. 1H NMR (400 MHz, D2O):
δ 1.55-1.70 (m, 4H, CH2); 2.39 (d, J ) 20 Hz, 2H, CH2P); 2.48
(t, J ) 7.6 Hz, 2H, PhCH2); 3.01 (t, J ) 7.2 Hz, 2H, CH2N);
6.70-7.30 (m, 9H, aromatic). 31P NMR (D2O): δ 13.5.
3-(3-Phenoxyphenyl)propyl Phosphonoacetate Dipotassium Salt
(17). Alcohol 27 was coupled with dibenzylphosphonoacetic acid
according to general method C to give dibenzyl ester of 17. The
benzyl groups were removed by catalytic hydrogenation (5% Pd/C
in methanol for 1 h) followed by neutralization with KOH to give
compound 17 as a white powder (180 mg, 42% overall yield). Anal.
(C17H17K2O6P) C, H. 1H NMR (400 MHz, D2O): δ 1.60-1.70 (m,
2H, CH2); 2.38 (d, J ) 20 Hz, 2H, CH2P); 2.44 (t, J ) 7.6 Hz, 2H,
PhCH2); 3.47 (t, J ) 7.2 Hz, 2H, CH2O); 6.70-7.30 (m, 9H,
aromatic). 31P NMR (D2O): δ 13.9.
2-Oxo-6-(4-phenoxyphenyl)hexylphosphonic Acid Dipotassium
Salt (18). 9-BBN (0.5 M in THF, 9 mL) was added to ethyl
4-pentenoate (3 mmol) at 0 °C and the reaction mixture stirred at
room temperature for 2 h. 4-Bromodiphenylether (3 mmol),
Pd(PPh3)4 (0.15 mmol), K3PO4 (6 mmol), and H2O (2 mL) were
then added, and the reaction mixture was refluxed overnight. The
organic layer was evaporated and purified by using column
chromatography (silica gel; hexane/ether, 6/1) to afford ester 31,
which was then reacted with 2 equiv of the lithium salt of diethyl
methylphosphonate at -78 °C, for 2 h. The reaction was quenched
with saturated NH4Cl, diethyl ether added to extract the product,
and the organic solvent removed. The oily residue was purified by
using column chromatography (silica gel, ethyl acetate) and
deprotected according to general method E to give compound 18
N-[3-(4-Biphenyl)propyl]phosphonoacetamide (10). 3-(4-Biphe-
nyl)propylamine was prepared from 4-phenylbenzaldehyde (1
mmol), using general method A, and was then coupled with
dibenzylphosphonoacetic acid according to general method C to
give the dibenzyl ester of 10. The benzyl groups were removed by
hydrogenation (catalyzed with 5% Pd/C in methanol) for 1 h,
followed by neutralization with KOH, to give compound 10 as a
white powder (222 mg, 65% overall yield). Anal.
(C17H20K2NO4P·0.25CH3OH) C, H, N. 1H NMR (400 MHz, D2O):
δ 1.65-1.75 (m, 2H, CH2); 2.31 (d, J ) 20 Hz, 2H, CH2P); 2.55
(t, J ) 7.6 Hz, 2H, PhCH2); 3.03 (t, J ) 7.2 Hz, 2H, CH2N);
7.20-7.55 (m, 9H, aromatic). 31P NMR (D2O): δ 12.8.
N-[3-(3-Phenoxyphenyl)propyl]sulfoacetamide (11). Amine 26
(1 mmol) was coupled with sulfoacetic acid (1 mmol) according
to general method C (without addition of 1-hydroxybenzotriazole)
to give 11. The product was purified by using column chromatog-
raphy (DOWEX ion-exchange resin, H+ form, methanol as eluent)
as an off-white powder (315 mg, 85% overall yield). Anal.
1
(C17H19NO5S) C, H, N. H NMR (400 MHz, D2O): δ 1.60-1.70
(m, 2H, CH2); 2.44 (m, 2H, PhCH2); 3.02 (m, 2H, CH2N); 3.59 (s,
2H, CH2S); 6.70-7.30 (m, 9H, aromatic).
N-Methyl-N-[3-(3-phenoxyphenyl)propyl]phosphonoacetamide
Dipotassium Salt (12). Amine 26 (1 mmol) was reacted with benzyl
chloroformate (ZCl, 1 mmol) in the presence of NEt3 to give
Z-protected amine 26 which was then methylated in THF with MeI
(1.5 equiv) and NaH (1.2 equiv) overnight. After hydrogenation
(5% Pd/C in MeOH) to remove the Z-protecting group, the
N-methylated amine 5 was coupled with dibenzylphosphonoacetic
acid, according to general method B, to give the dibenzyl ester of
12. The benzyl groups were removed by hydrogenation (5% Pd/C
in methanol) for 1 h, followed by neutralization with KOH to give
compound 12 as a white powder (220 mg, 50% overall yield). Anal.
(C18H20K2NO5P) C, H, N. The NMR spectrum of 12 showed that
two rotamers (with respect to the amide bond) exist with ratio of
1
∼45:55. H NMR (400 MHz, D2O): δ 1.60-1.80 (m, 2H, CH2);
2.35-2.45 (m, 2H, CH2P); 2.45-2.95 (m, 5H, Me and PhCH2);
3.10-3.40 (m, 2H, CH2N); 6.80-7.30 (m, 9H, aromatic). 31P NMR
(D2O): δ 13.6.
N-[3-(3-Phenoxyphenyl)propyl]phosphonodimethylacetamide Di-
potassium Salt (13). Diethyl phosphonodimethylacetate (3 mmol)
was treated with 3 N KOH (5 mL) in ethanol (8 mL) for 24 h,
followed by acidifiction with HCl to give the corresponding
carboxylic acid. As with compound 23a, the acid was then
converted to the acid chloride 23b, which was reacted with 1 equiv
of amine 26 in the presence of NEt3 in CH2Cl2 (5 mL) at 0 °C.
After the mixture was stirred for 1 h, the coupling product was
purified by using column chromatography (silica gel, ethyl acetate)
and was then deprotected following general method E to give 13
as
a white powder (335 mg, 21% overall yield). Anal.
(C19H22K2NO5P·0.5KBr·H2O) C, H, N. 1H NMR (400 MHz, D2O):
δ 1.09 (d, J ) 13.6 Hz, 6H, 2CH3); 1.60-1.70 (m, 2H, CH2); 2.46
(m, 2H, PhCH2); 2.99 (m, 2H, CH2N); 6.85-7.25 (m, 9H, aromatic).
31P NMR (D2O): δ 22.9.

Inhibition of Staphyloxanthin Biosynthesis
as
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 3879
a
white powder (312 mg, 20% overall yield). Anal.
Elmer MBA 2000 (Norwalk, CT) spectrophotometer. The IC50
values were obtained by fitting the OD data to a normal
dose-response curve, using GraphPad PRISM.
(C18H19K2O5P·0.5KBr·2H2O) C, H. 1H NMR (400 MHz, D2O): δ
1.30-1.50 (m, 4H, CH2); 2.44 (t, J ) 7.6 Hz, 2H, PhCH2); 2.54 (t,
J ) 7.6 Hz, 2H, CH2CO); 2.70 (d, J ) 20 Hz, 2H, CH2P);
6.80-7.25 (m, 9H, aromatic). 31P NMR (D2O): δ 11.0.
Human SQS Enzyme Expression, Purification, and Inhibition
Assay. E. coli expressing a human SQS construct were cultured in
Luria-Bertani medium supplemented with kanamycin (30 µg/mL)
and chloramphenicol (34 µg/mL) at 37 °C, until the cells reached
an OD of 0.4 at 600 nm, and were then induced at 37 °C for 4 h
by incubation with 1 mM isopropyl-1-thio-ꢀ-D-galactopyranoside.
Cells were harvested by centrifugation (10 min, 4000 rpm) and
resuspended in 10 mL of lysis/elution buffer (20 mM NaH2PO3,
pH 7.4, 10 mM CHAPS, 2 mM MgCl2, 10% glycerol, 10 mM
mercaptoethanol, 500 mM NaCl, 10 mM imidazole, and a protease
inhibitor cocktail), disrupted by sonication, and centrifuged at
16 000 rpm for 30 min. The supernatant (40 mL) was then applied
to a HiTrap nickel-chelating HP column (Amersham Biosciences).
Enzyme purification was performed according to the manufacturer’s
instructions using a Pharmacia FPLC system. Unbound protein was
washed off with 50 mM imidazole, and then the His6-HsSQS was
eluted with 1 M imidazole. Purity was confirmed by SDS-PAGE
electrophoresis. Fractions containing the enzyme were pooled and
dialyzed against buffer A (25 mM sodium phosphate, pH 7.4, 20
mM NaCl, 2 mM dithiothreitol, 1 mM EDTA, 10% glycerol, 10%
methanol), concentrated, then stored at -80 °C.
N-[3-(3-Phenoxyphenyl)propyl]phosphonomethylsufamide Di-
potassium Salt (19). Amine 26 prepared from 3-phenoxybenzal-
dehyde (3 mmol) using general method A was reacted with 1 equiv
of methanesulfonyl chloride in CH2Cl2 in the presence of 1.2 equiv
of NEt3 at 0 °C. After 1 h, 50 mL of ethyl acetate was added and
the reaction mixture was washed successively with 1 N HCl, water,
NaHCO3, then dried and evaporated. The oily residue was treated
with 2.2 equiv of BuLi at -78 °C for 1 h followed by addition of
0.6 equiv of diethyl chlorophosphate. The reaction mixture was
warmed to 0 °C over 1 h and then quenched with saturated NH4Cl.
Column chromatography (silica gel, ethyl acetate) followed by
hydrolysis using general method E gave 19 as a white powder (366
1
mg, 42% overall yield). Anal. (C16H18K2NO6P·KBr) C, H, N. H
NMR (400 MHz, D2O): δ 1.60-1.75 (m, 2H, CH2); 2.50 (t, J )
7.6 Hz, 2H, PhCH2); 2.87 (t, J ) 7.2 Hz, 2H, CH2N); 1.18 (d, J )
20 Hz, 2H, CH2P); 6.70-7.30 (m, 9H, aromatic). 31P NMR (D2O):
δ 4.4.
N-Hydroxy-N-[3-(4-methylbiphenyl)propyl]phosphonoacetam-
ide (20). Compound 20 was prepared in the same manner as 9, using
4-methylphenylbenzaldehyde as starting material, as a white powder
(175 mg, 48% overall yield). Anal. (C18H22NO5P·0.2HBr) C, H, N.
1H NMR (400 MHz, D2O): δ 1.70-1.80 (m, 2H, CH2); 2.21 (s, 3H,
Me), 2.46 (t, J ) 7.6 Hz, 2H, PhCH2); 2.66 (d, J ) 20 Hz, 2H, CH2P);
3.42 (t, J ) 7.2 Hz, 2H, CH2N); 7.0-7.30 (m, 8H, aromatic). 31P NMR
(D2O): δ 15.9.
N-[3-(3-Phenoxyphenyl)propyl]phosphonomalonamide Potassi-
um Salt (21). Amine 26 (1 mmol) was coupled with malonic acid
monoethyl ester according to general method C to give the ethyl
ester of 21, which was then hydrolyzed with 3 equiv of KOH in
MeOH/H2O for 1 h. The reaction mixture was acidified and
extracted with ethyl acetate, and the organic layer was evaporated.
The oily residue was dissolved in methanol, neutralized with KOH,
and evaporated to give 21 as a white powder (250 mg mg, 66%
overall yield). Anal. (C18H18KNO4 ·0.25KCl·0.5H2O) C, H, N. 1H
NMR (400 MHz, D2O): δ 1.80-1.90 (m, 2H, CH2); 2.62 (t, J )
7.6 Hz, 2H, PhCH2); 3.32 (s, 2H, CH2COO); 3.33 (m, 2H, CH2N);
6.70-7.40 (m, 9H, aromatic).
SQS activity was based on measuring the conversion of [3H]FPP
to [3H]squalene. Final assay concentrations were 50 mM MOPS
(pH 7.4), 20 mM MgCl2, 5 mM CHAPS, 1% Tween-80, 10 mM
DTT, 0.025 mg/mL BSA, 0.25 mM NADPH, and 7.5 ng of purified
recombinant human SQS. The reaction was started with the addition
of substrate (3HFPP, 0.1 nmol, 2.22 × 106 dpm), and the final
volume of the reaction was 200 µL. After incubation at 37 °C for
5 min, an amount of 40 µL of 10 M NaOH was added to stop the
reaction, followed by 10 µL of a (100:1) mixture of 98% EtOH
and squalene. The resulting mixtures were mixed vigorously by
vortexing. Then 10 µL aliquots were applied to 2.5 cm × 10 cm
channels of a silica gel thin layer chromatogram, and newly formed
squalene was separated from unreacted substrate by chromatography
in toluene-EtOAc (9:1). The region of the squalene band was
scraped and immersed in Hydrofluor liquid scintillation fluid and
assayed for radioactivity. IC50 values were calculated from the
hyperbolic plot of percent of inhibition versus inhibitor concentra-
tion, using GraphPad PRISM.
Enzyme and Biological Assays. CrtM Expression and Purifica-
tion. CrtM with a histidine tag was overexpressed in E. coli
BL21(DE3) cells and CrtM protein purified as described previ-
ously.14 A 50 mL overnight culture was transferred into 1 L LB
medium supplemented with 100 µg/mL ampicillin. Induction was
carried out with 1 mM IPTG for 4 h at 37 °C, when the cell culture
reached an OD of 0.6 at 600 nm. The cell extract was loaded onto
a Ni-NTA column and CrtM eluted by using a 100 mL linear
gradient of 0-0.5 M imidazole in 50 mM Tris-HCl buffer, pH 7.4.
CrtM Inhibition Assay. The condensation of farnesyl diphos-
phate was monitored by a continuous spectrophotometric assay for
phosphate releasing enzymes.1 The reaction buffer contained 50
mM Tris-HCl, 1 mM MgCl2, 450 µM FPP, pH 7.4. The compounds
investigated were preincubated with 2 µg of CrtM for 30 min at
20 °C. Reactions were carried out by using 96-well plates with
200 µL of reaction mixture in each well. The IC50 values were
obtained by fitting the inhibition data to a normal dose-response
curve in Origin 6.1 (OriginLab Corporation, Northampton, MA).
The Ki values reported are calculated on the basis of the substrate
concentrations used, the IC50 values found, and the kinetic constant
for CrtM, as described previously.14
Human Cell Growth Inhibition Assay. Three human cell lines
MCF-7, NCI-H460, and SF-268 were obtained from the National
Cancer Institute. Cells were cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum and 2 mM L-glutamine
at 37 °C in a 5% CO2 atmosphere with 100% humidity. A broth
microdilution method was used to calculate IC50 values for growth
inhibition by each compound. Cells were inoculated at a density
of 5000 cells/well into 96-well flat-bottom culture plates containing
10 µL of the test compound, previously half-log serial diluted (from
0.316 mM to 0.1 pM) for a final volume of 100 µL. Plates were
then incubated for 4 days at 37 °C in a 5% CO2 atmosphere at
100% humidity, after which an MTT ((3-(4,5-dimethylthiazole-2-
yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay (ATCC,
Manassas, VA) was used to quantify cell viability. The IC50 values
were obtained by fitting the OD data to a normal dose-response
curve, using GraphPad PRISM.
Murine Model of Kidney Infection. The 10-12 week old CD1
male mice (Charles River Laboratory) were randomized into two
groups at the start of the experiment and administered either 0.5
mg of BPH-652 or PBS control, ip, twice a day, starting on day 1
to day 2 (a total of eight doses). All mice were injected intraperi-
toneally (ip) with 108 early stationary phase S. aureus on day 0.
After 3 days, animals were euthanized, kidneys homogenized in
PBS, and plated on THA for quantitative bacterial culture.
Statistics. The significance of experimental differences in the
mouse in ViVo challenge studies were evaluated by use of the two-
tailed Student’s t test.
Staphyloxanthin Biosynthesis Inhibition Assay. The S. aureus
strain used was the WT clinical isolate (Pig1). S. aureus was
propagated in Todd-Hewitt broth (THB) or on THB agar (TBA;
Difco, Detroit, MI). For in vitro pigment inhibition studies, S. aureus
was cultured in THB (1 mL) in the presence of inhibitor compounds
for 72 h, in duplicate. Prior to assay, the bacteria were centrifuged
and washed twice in PBS. Staphyloxanthin was extracted with
MeOH, and the OD was determined at 450 nm using a Perkin-
X-ray Crystallography. Native CrtM was eluted from Ni-NTA
beads by incubation with factor Xa (Novagen) to cleave it from

3880 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
Song et al.
(9) Hammond, R. K.; White, D. C. Inhibition of vitamin K2 and carotenoid
synthesis in Staphylococcus aureus by diphenylamine. J. Bacteriol.
1970, 103 (3), 611–615.
(10) Hammond, R. K.; White, D. C. Inhibition of carotenoid hydroxylation
in Staphylococcus aureus by mixed-function oxidase inhibitors. J.
Bacteriol. 1970, 103 (3), 607–610.
(11) Daum, R. S. Removing the golden coat of Staphylococcus aureus.
N. Engl. J. Med. 2008, 359 (1), 85–87.
(12) Walsh, C. T.; Fischbach, M. A. Inhibitors of sterol biosynthesis as
Staphylococcus aureus antibiotics. Angew. Chem., Int. Ed. Engl. 2008,
47 (31), 5700–5702.
(13) Haebich, D.; von Nussbaum, F. “Superbugs bunny” outsmarts our
immune defense. ChemMedChem 2008, 3 (8), 1173–1177.
(14) Liu, C. I.; Liu, G. Y.; Song, Y.; Yin, F.; Hensler, M. E.; Jeng, W. Y.;
Nizet, V.; Wang, A. H.; Oldfield, E. A cholesterol biosynthesis
inhibitor blocks Staphylococcus aureus virulence. Science 2008, 319
(5868), 1391–1394.
(15) Magnin, D. R.; Biller, S. A.; Dickson, J. K., Jr.; Logan, J. V.;
Lawrence, R. M.; Chen, Y.; Sulsky, R. B.; Ciosek, C. P., Jr.; Harrity,
T. W.; Jolibois, K. G.; et al. 1,1-Bisphosphonate squalene synthase
inhibitors: interplay between the isoprenoid subunit and the diphosphate
surrogate. J. Med. Chem. 1995, 38 (14), 2596–2605.
(16) Magnin, D. R.; Biller, S. A.; Chen, Y.; Dickson, J. K., Jr.; Fryszman,
O. M.; Lawrence, R. M.; Logan, J. V.; Sieber-McMaster, E. S.; Sulsky,
R. B.; Traeger, S. C.; Hsieh, D. C.; Lan, S. J.; Rinehart, J. K.; Harrity,
T. W.; Jolibois, K. G.; Kunselman, L. K.; Rich, L. C.; Slusarchyk,
D. A.; Ciosek, C. P., Jr. R-Phosphonosulfonic acids: potent and
selective inhibitors of squalene synthase. J. Med. Chem. 1996, 39 (3),
657–660.
(17) Rieger, C. E.; Lee, J.; Turnbull, J. L. A continuous spectrophotometric
assay for aspartate transcarbamylase and ATPases. Anal. Biochem.
1997, 246 (1), 86–95.
the polyhistidine-containing N-terminal thioredoxin fusion tag. The
cleaved product was equilibrated with buffer containing 150 mM
NaCl, 5 mM DTT, 1 mM ꢀ-mercaptoethanol, 5% glycerol, and 20
mM Tris, pH 7.5 and then concentrated to 15 mg/mL. Native CrtM
crystals (space group P3221) were grown using the hanging-drop
method by mixing equal amounts of reservoir with 0.12-0.58 M
potassium sodium tartrate at room temperature. BPH-830 was
incorporated by soaking crystals with a solution of 5 (10 mM in
DMSO) for 3 h at room temperature. X-ray diffraction data were
collected at SPXF beamline BL13B1 at the National Synchrotron
Radiation Research Center (NSRRC), Hsinchu, Taiwan. All dif-
fraction images were recorded using an ADSD Q210 CCD detector,
and the data were indexed, integrated, and scaled by using the
HKL2000 package.21 The structure of the CrtM-5 complex was
determined by molecular replacement using CNS22 using the refined
native CrtM (PDB 2ZCO) as a search model. Iterative cycles of
model building with Xtalview23 and computational refinement with
CNS were performed, in which 5% reflections were set aside for
Rfree calculation.24 The stereochemical quality was assessed with
the program PROCHECK.25 Figures were obtained by using
Pymol.26
Acknowledgment. This work was supported by the United
States Public Health Service (Grant AI074832 to G.Y.L., Grant
HD051796 to V.N., and Grants AI074233, GM073216, and
GM65307 to E.O.). Y.S. was supported by a Leukemia and
Lymphoma Society Special Fellowship. Diffraction data were
obtained at the National Synchrotron Radiation Research Center
of Taiwan and was supported by grants from Academia Sinica
and the National Core Facility of High-Throughput Protein
Crystallography (Grant SC95-3112-B-001-015-Y to A.H.-J.W.).
(18) Song, Y.; Lin, F.-Y.; Yin, F.; Hensler, M.; Poveda, C. A. R.;
Mukkamala, D.; Cao, R.; Wang, H.; Morita, C. T.; Pacanowska, D. G.;
Nizet, V.; Oldfield, E. Phosphonosulfonate are potent, selective
inhibitors of dehydrosqualene synthase and staphyloxanthin biosyn-
thesis in Staphylococcus aureus. J. Med. Chem. 2009, 52 (4), 976–
988.
(19) Mukkamala, D.; No, J. H.; Cass, L. M.; Chang, T. K.; Oldfield, E.
Bisphosphonate inhibition of a Plasmodium farnesyl diphosphate
synthase and a general method for predicting cell-based activity from
enzyme data. J. Med. Chem. 2008, 51 (24), 7827–7833.
(20) Molecular Operating EnVironment (MOE); Chemical Computing
Group, Inc.: Montreal, Quebec, 2006.
(21) Otwinowski, Z.; Minor, W. ; Processing of X-ray diffraction data
collected in oscillation mode. Macromol. Crystallogr. 1997, 276, 307–
326.
(22) Brunger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.;
Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L.
Crystallography & NMR system: a new software suite for macromo-
lecular structure determination. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 1998, 54 (Part 5), 905–921.
(23) McRee, D. E. XtalView Xfit. A versatile program for manipulating
atomic coordinates and electron density. J. Struct. Biol. 1999, 125
(2V3), 156–165.
References
(1) Bancroft, E. A. Antimicrobial resistance: it’s not just for hospitals.
JAMA, J. Am. Med. Assoc. 2007, 298 (15), 1803–1804.
(2) Klevens, R. M.; Morrison, M. A.; Nadle, J.; Petit, S.; Gershman, K.;
Ray, S.; Harrison, L. H.; Lynfield, R.; Dumyati, G.; Townes, J. M.;
Craig, A. S.; Zell, E. R.; Fosheim, G. E.; McDougal, L. K.; Carey,
R. B.; Fridkin, S. K. Invasive methicillin-resistant Staphylococcus
aureus infections in the United States. JAMA, J. Am. Med. Assoc. 2007,
298 (15), 1763–1771.
(3) Treating Infectious Diseases in a Microbial World: Report of Two
Workshops on NoVel Antibacterial Therapeutics; National Research
Council: Washington, DC, 2006; pp 21-22.
(4) Wang, R.; Braughton, K. R.; Kretschmer, D.; Bach, T. H.; Queck,
S. Y.; Li, M.; Kennedy, A. D.; Dorward, D. W.; Klebanoff, S. J.;
Peschel, A.; DeLeo, F. R.; Otto, M. Identification of novel cytolytic
peptides as key virulence determinants for community-associated
MRSA. Nat. Med. 2007, 13 (12), 1510–1514.
(5) Escaich, S. Antivirulence as a new antibacterial approach for
chemotherapy. Curr. Opin. Chem. Biol. 2008, 12 (4), 400–408.
(6) Liu, G. Y.; Essex, A.; Buchanan, J. T.; Datta, V.; Hoffman, H. M.;
Bastian, J. F.; Fierer, J.; Nizet, V. Staphylococcus aureus golden
pigment impairs neutrophil killing and promotes virulence through
its antioxidant activity. J. Exp. Med. 2005, 202 (2), 209–215.
(7) Clauditz, A.; Resch, A.; Wieland, K. P.; Peschel, A.; Gotz, F.
Staphyloxanthin plays a role in the fitness of Staphylococcus aureus
and its ability to cope with oxidative stress. Infect. Immun. 2006, 74
(8), 4950–4953.
(24) Brunger, A. T. Assessment of phase accuracy by cross validation: the
free R value. Methods and applications. Acta Crystallogr. D 1993, 49
(1), 24–36.
(25) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M.
PROCHECK: a program to check the stereochemical quality of protein
structures. J. Appl. Crystallogr. 1993, 26, 283–291.
(26) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano
Scientific: Palo Alto, CA, 2008; http://www.pymol.org.
(27) Wallace, A. C.; Laskowski, R. A.; Thornton, J. M. Ligplot: a program
to generate schematic diagrams of protein-ligand interactions. Protein
Eng. 1995, 8, 127–134.
(8) Pelz, A.; Wieland, K. P.; Putzbach, K.; Hentschel, P.; Albert, K.; Gotz,
F. Structure and biosynthesis of staphyloxanthin from Staphylococcus
aureus. J. Biol. Chem. 2005, 280 (37), 32493–32498.
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