Structure based design of an in vivo active hydroxamic acid inhibitor of P. aeruginosa LpxC

Article abstract of DOI:10.1016/j.bmcl.2012.01.140

Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial growth and is unique to these bacteria. The firs

Full text of DOI:10.1016/j.bmcl.2012.01.140

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2536–2543
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure based design of an in vivo active hydroxamic acid inhibitor
of P. aeruginosa LpxC
Joseph S. Warmus a, , Cheryl L. Quinn b, Clarke Taylor a, Sean T. Murphy a, Timothy A. Johnson a,
⇑
Chris Limberakis a, Daniel Ortwine a, Joel Bronstein b, Paul Pagano b, John D. Knafels c, Sandra Lightle c,
Igor Mochalkin c, Roger Brideau b, Terry Podoll d
a Department of Chemistry, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
b Antibacterial Pharmacology, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
c Discovery Technologies Structural and Analytical Sciences, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
d Phamacodyamics and Drug Metabolism, Pfizer Global Research and Development, Ann Arbor, MI 48105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Lipid A is an essential component of the Gram negative outer membrane, which protects the bacterium
from attack of many antibiotics. The Lipid A biosynthesis pathway is essential for Gram negative bacterial
growth and is unique to these bacteria. The first committed step in Lipid A biosynthesis is catalysis by
LpxC, a zinc dependent deacetylase. We show the design of an LpxC inhibitor utilizing a robust model
which directed efficient design of picomolar inhibitors. Analysis of physiochemical properties drove
design to focus on an optimal lipophilicity profile. Further structure based design took advantage of a
conserved water network over the active site, and with the optimal lipophilicity profile, led to an
improved LpxC inhibitor with in vivo activity against wild type Pseudomonas aeruginosa.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 16 November 2011
Revised 30 January 2012
Accepted 31 January 2012
Available online 16 February 2012
Keywords:
LpxC
Antibacterial
Gram negative
Structure based drug design
Hydroxamic acid
Pseudomonas aeruginosa
LipE
Lipophilic efficiency
Computer aided drug design
Infections caused by Gram negative bacteria are causing in-
creased alarm due to associated high morbidity and mortality
rates.1 Commonly found in the natural environment, Gram nega-
tive bacteria are usually clinically relevant as nosocomial patho-
gens.2 Pseudomonas aeruginosa is one of the leading causes of
hospital-acquired pneumonia and urinary tract infections and is
the major cause of death in Cystic Fribrosis patients.3 Isolation of
antibiotic resistant P. aeruginosa is increasing, with some isolates
reportedly resistant to all available antibiotics.4 The Infectious Dis-
eases Society of America has cited P. auruginosa as one of the six
bacterial pathogens with the highest medical need for new thera-
peutic agents.1 New antibiotics are needed to combat P. aeruginosa
infections, with new targets and chemical classes desired to avoid
cross-resistance with established classes.
roles in the integrity of the bacterial cells and do not have
direct counterparts in humans. UDP-3-O-(R-3-hyrdoxymyristoyl)-
N-acetyl glucosamine (LpxC) is an essential enzyme in LPS biosyn-
thesis LpxC catalyzes the first step in biosynthesis of lipid A, a key
structural factor of the LPS matrix.
While LpxC inhibitors with good MICs against P. aeruginosa
have been reported, these compounds have hurdles for develop-
ment as systemic therapies. Pharmacokinetic properties of
hydroxamates, mainly rapid metabolism and poor oral availability,
are generally not suited to high exposures required to treat sys-
temic infections. However, hydroxamic acids have been used as or-
ally active agents after careful optimization of pharmacokinetic
properties.5
LpxC inhibitors have been widely reported (Fig. 1) with much
effort focused on the hydroxamate class of inhibitors. The known
amphipathic benzoic acid derivative (2) binds weakly to LpxC
and binds solely to the hydrophobic channel.6 Inclusion of a zinc
binding hydroxamate ‘warhead’ greatly increases binding to LpxC,
as in TU-514 (3).7 Initial reports from Merck revealed an oxazoline
4 with a hydroxamic acid ‘warhead’ as a potent inhibitor of
Escherichia coli LpxC (ecLpxC), but showed weaker in vitro
The biosynthetic pathways of the lipopolysaccharide coat of
P. aeruginosa and other Gram negative bacteria presents an array
of tempting targets for new antibiotics in that they play essential
⇑
Corresponding author. Address: Pfizer Global Research and Development,
Eastern Point Road 220/235A, Groton, CT 06340, USA.
E-mail address: Joseph.Warmus@Pfizer.com (J.S. Warmus).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.140

J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
2537
OH
OH
NH
O
O
O
HO
O
OH
N
O
O
O
S
O
O
O
H
N
O
HO
O
O
N
H
NH
HO
7
1
BB-78485
4
2
3
TU-514
E. coli Ki ~650 nM
E. coli Ki ~20 nM
E. coli Ki ~50 nM
A. aeolicus Kd 2uM
P.aeruginosa C53 MIC = 4
OH
NH
OH
OH
H
O
O
O
H
N
N
N
H
OH
N
H
OH
HN
O
O
NH2
N
O
O
N
N
7 (LPC-011)
E. coli MIC=0.05 ug/mL
MIC WT Pa 0.7 ug/mL
5
CHIR-090
6
E. coli Ki ~2 nM
E. coli IC50 =0.6 nM
MIC WT Pa 12.5 ug/mL
Figure 1. Reported LpxC inhibitors.
inhibition against P. aeruginosa LpxC (paLpxC).8 Chiron has used an
amino acid link between the hydroxamic acid and a hydrophobic
biphenyl acetylene giving a low nM inhibitor of paLpxC 5 that also
Our main concern was the known metabolism of hydroxamic
acids, namely, glucoronidation or hydrolysis releasing hydroxyl-
amine. Our first efforts to improve metabolism was to sterically
inhibited E. coli growth (MIC 0.2
l
g/mL).9 Merck has reported a
hinder hydrolysis of the hydroxamic acid. We prepared a a-Me ser-
series of urea linked hydroxamic acids 6 based off the Chiron-
090 framework which showed excellent activity against ecLpxC
and demonstrated weak MICs against wild type pseudomonas.10
Recently, Toone, Zhou and co-workers have systematically investi-
gated a series of diphenyl-diacetylenes from the patent literature,
ine derivative 9 which did show acceptable stability in both human
and rat hepatocytes in vitro. However, potency dropped consider-
ably. In order to understand the loss of potency, we turned to
modeling.
Detailed analysis of the binding site of ecLpxC with inhibitors is
known in the literature,9,13 as are reports of QSAR models for
ecLpxC.14 A crystal structure and detailed analysis of the binding
site for Pseudomonas aeruginosa complexed with the inhibitor BB-
78485 1 has been published.15 For the purposes of this publication,
a brief description of the binding site, as relevant to our design, is
presented (Fig. 3). The binding pocket is composed of a pentacoor-
dinate Zn atom, coordinated by 2 histidines and an aspartic acid. A
hydrophobic channel is present which binds the myristoyl unit of
the substrate.
and both CHIR-090 (5 MIC = 1.6
l
g/mL À PA01) and LPC-011 (7)
show low MIC’s against wild type Pseudomonas.9
We set out to prepare LpxC inhibitors with low MIC against wild
type Pa and showing in vivo efficacy in a rodent model. Our goal
was to improve solubility and metabolic stability of our series rel-
ative to the known inhibitors in the literature. Although the acety-
lene and diacetylene compounds from Chiron showed good
potency and MICs, we were concerned about toxicity due to the al-
kyne (or dialkyne) moiety.11 We therefore used a biphenyl as a
hydrophobic pocket binding element. Optimization of the biphenyl
will be described in other publications. Chiron reported in the pat-
ent literature12 biphenyl LpxC inhibitors 8 that showed good po-
tency, however clearance in rodents was poor (Fig. 2). We used
this as a starting point for our effort.
OH
OH
O
O
H
H
N
N
N
H
OH
N
H
OH
O
O
9 (rac)
8
Pa IC50 = 286 nM
Pa IC50 = 8 nM
Pa MIC = >64 ug/mL
HHep t1/2 = 230 min
RHep t1/2 = 220 min
Pa MIC = 4 ug/mL
HHep t1/2 = 230 min
RHep t1/2 = 45 min
Figure 2. Initial effort to minimize metabolic liability.
Figure 3. Binding schematic for hydroxamic acid inhibitors.

2538
J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
Figure 4. Binding sites in LpxC.
Figure 5. Steric clash in the amide series of inhibitors.
There is also a large 20 Å deep sugar binding region, which
keeping the a-methyl serine sidechain. As can be seen in Table 1,
binds the UDP portion of the substrate. This is a rather hydrophobic
area of the active site which leads to basic patch near the top of the
cleft (Fig. 4).
the two ethers showed differing potencies, with 11 being more po-
tent. The most potent side chain was the methylene linker. Both
the ether 11 and the methylene linker 12 were highly ligand effi-
cient and had high LipEs (lipophilic efficiency).16 However, the
more lipophilic methylene linker 12 had higher clearance than
the more polar ether linker 11. Due to the more favorable cLogP,
hHep clearance and solubility, we felt the ether linker was the
most promising for further optimization.
We built a homology model from aquiflex e. coli (aaLpxC)13b and
binding modes of inhibitors were later confirmed by in house X-ray
crystallography with paLpxC. A model of the binding mode for 9
indicated a steric clash between the
the carbonyl of the amide (Fig. 5).
a-methyl of the serine and
We therefore investigated alternative linkers to the amide
which would remove this clash and therefore lead to improved
potency. To this end, we prepared methylene and ether linkers,
We felt that the biphenyl group was adequate to use as we
explored other areas of SAR. Also, while in theory the hydroxamic
acid moiety could be replaced with a surrogate, we felt that
Table 1
O
X-Y =
9
HO
N
H
N
H
X
Y
10
OH
O
O
11
12
O
Compound
PaIC50 (nM)
MIC (
l
g/mL) PAO280a (pump KO)
MIC (l
g/mL) UI-18a (WT)
Kinetic solubility (
lM)
hHep t1/2 (min)
clogD
LE
LipE
9
286
390
22
32
—
4
>64
—
>64
16
200
26
200
89
230
86
360
72
1.5
2.6
2.2
2.9
0.39
0.40
0.47
0.52
5.0
3.8
5.5
5.6
10
11
12
3.3
0.5
a
Chuanchuen R, et al. Antimicrob. Agents Chemother. 2001, 45, 428–432.
Shapiro M. A., et al. J. Antimicrob. Chemother. 1997, 39, 273–276.
b

J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
2539
OH
H
H
N
HO
O
N
HO
O
O
O
13
11 (racemic)
IC50 = 311 nM
MIC (WT) = > 64 ug/mL
IC50 = 22 nM
MIC (WT) = > 64 ug/mL
Figure 6. Targeted Lys interaction.
keeping this key feature to retain potency was warranted. How-
ever, the SAR leading to the sugar pocket (the serine) was mini-
mally explored, and held features that were amenable to
optimizing potency. This publication will report our SAR in this
area.
bond forming reaction (Scheme 1). This allowed us variability in
substitution with excellent control over the absolute stereochem-
istry of the newly formed centers. The biphenyl ether 16 was
formed under standard conditions in good yield. The auxiliary
was installed via the acid chloride and aldols with alkyl, aryl and
heteroaryl aldehydes were carried out with high stereoselectivity
to provide 20. Removal of the auxiliary via displacement with
the magnesium salt of hydroxyl amine provided the desired
hydroxamic acids 21 in moderate to good yields.
We utilized a robust homology model derived from the aaLpxC
crystal structure13b to guide our design. Initial X-ray co-crystal
structures of inhibitors with paLpxC confirmed the ability of our
homology model to predict bound conformations. As will be
shown, this was key to the design of our LpxC inhibitors.
As was seen in CHIR-0909,12,13b 5 and a urea from Merck10 6, a
serine hydroxyl is a key feature for these inhibitors. We propose
this is due to a hydrogen bonding interaction of the free hydroxyl
with the mobile Lys238, (equivalent to Lys 227 in aaLpxC), or a
water bound to this Lys, as seen in our model. We used this as
an anchor point for initial design. Removal of this hydroxyl in
our ether series causes a loss in activity (compare 13 to 11, Fig. 6).
In order to prepare variably substituted
a-methyl hydroxamic
acids, we utilized Davies’ ‘Super Quat’ auxiliary 1817 as the key
NaH, DMF/THF,
-20°C 30 mins,
R.T. 30 mins,
50°C 1 hour
LiOH,
THF/water
Br
O
O
O
O
O
O
O
OH
OH
15
99%
85%
16
17
14
1) SOCl2
1) LDA, -78°C,
O
2) Ti(OiPr)3Cl, -40°C
O
2)BuLi,
H
O
O
N
O
N
N
3) RCHO
25-54%
R
O
O
OH
O
O
O
20
19
75%
18
HONH2 HCl
MeMgBr,
O
O
OH
N
H
R
MeOH, -20°C, 3 h
15-88%
OH
21
Scheme 1. Synthetic route to hydroxamic acid inhibitors.

2540
J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
We prepared a set of alkyl and heteroaryl analogs to probe the
interactions in this pocket. Introduction of heteroaryl groups was
more fruitful. While the hydrophobic phenyl (21h) was poorly tol-
erated, the 2-pyridyl and 3-pyridyl were more potent and only
fivefold less potent than the methyl substitution, yet were equiva-
lent LipE to the methyl substitution 21a and b. These also showed
good MICs versus the PAO280 pump deficient organism. This indi-
cated that inclusion of a more polar group could be advantageous.
More polar five-membered nitrogen containing heterocyles
showed little improvement (21k and 21l), until liberation of an
H-bond donor, as in 21m and 21n. This caused a dramatic increase
in binding over the pyridyls. There was also an improvement in
MICs, both for the pump knockout and wild type organisms. Mod-
sugar pocket (Table 2). Introduction of a methyl substituent to
compound 11 (21a and 21b) increased binding approximately sev-
enfold, indicating non-specific, hydrophobic interactions. The en-
zyme showed little preference for the chirality of the hydroxyl
center with a small methyl substituent, supporting the hypothesis
that there was non-specific binding of this small alkyl group.
Increasing the steric bulk of the alkyl substituent was detrimental
to binding and MICs for the pump knockout PAO280 and lowered
LipE of these analogs. Interestingly, an ether (21g) showed an im-
proved binding and lipophilic efficiency or LipE,16 relative to a
straight chain alkyl group (21f) indicating the possibility of polar
Table 2
HO
R
3R
H
N
O
OH
O
21
Compd #
R
IC50 (nM) Pa LpxC
MIC (l
g/mL) PAO280b (pump KO)
MIC (l
g/mL) UI-18c (WT)
clogD
LipE
11
H
22
3
4
—
—
32
64
>64
>64
>64
—
>64
>64
2.2
2.6
2.6
2.9
3.2
3.6
3.8
2.7
3.9
5.5
5.9
5.8
5.2
3.9
3.2
3.2
4.9
3.4
21a
21b
21c
21d
21e
21f
21g
21h
Me (3S)
Me (3R)
0.5a
0.75a
3
32
64
—
>64
8
Et
8
iPr
72
160
90
28
51
iBu
nBu
EtOCH2CH2–
Ph
N
N
21i
21j
18
15
0.5
32
32
2.6
2.6
5.1
5.2
0.25
N
N
N
21k
21l
26
25
0.8
2
>64
33a
8
2.2
2.2
2.3
5.4
5.4
6.8
N
N
0.19a
0.125
N
HN
21m
N
NH
21n
21o
21p
0.4
3
0.25
0.25
0.125
4
8
8
2.2
2.3
2.6
7.2
6.2
6.5
O
N
N
O
0.8
O
N
21q
21r
0.4
0.4
0.06
6a
2.6
2.7
6.8
6.7
O
N
0.015a
1.75a
a
b
c
Arithmetic mean—2–6 MIC determinations.
Chuanchuen R, et al. Antimicrob. Agents Chemother. 2001, 45, 428–432.
Shapiro M. A., et al. J. Antimicrob. Chemother. 1997, 39, 273–276.

J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
2541
Figure 9. Model of isoxazole 21r binding to LpxC.
Table 3
Figure 7. Model of H-bond interaction of imidazole inhibitor 21m.
O
N
HO
H
eling of 21m (Fig. 7) indicated the possibility of an H-bond interac-
tion to a backbone carbonyl, along with a possible p-stack interac-
tion. This binding mode was confirmed by an X-ray co-crystal
N
O
OH
O
structure.
Continuing the exploration of small heterocycles, we examined
oxazoles and isoxazoles (21o–21r) which showed good potency
21r
Data for compound 21r
PaLpxc IC50 (nM)
0.376
MIC (
WT
lg/mL) (pump KO)
0.015
1.75
HuHep t1/2 (min)
Kinetic solubility @ pH 6.5 (lM)
20
59
against the knockout PA and had high LipEs. In particular, the
1,2-oxazoles showed good potency and MIC against the knockout
PA, and had high LipEs. The methyl oxazole 21r, while having a
slightly lower LipE than the unsubstituted 21q, did show improved
MIC against the wild type PA (wtPA). We believed that these oxaz-
oles were also benefitting from the p-stack interaction; however a
backbone H-bond was not possible with these analogs. In order to
understand the possible binding interactions of these inhibitors,
we looked at a number of in house co-crystal structures. This com-
parison indicated that there was a conserved water lattice across
the top of the active site (Figs. 8a and 8b), which extended from
Lys 238 to Asp 196. It appeared from our models that imidazole
21m was replacing a weakly bound water molecule in this
structure.
Figure 8a. Conserved water lattice.
An alternative binding mode for the heterocyclic moiety can be
accessed by the isoxazoles 21o–r. Modeling suggests these also
displace a weakly bound water molecule, but they now make an
interaction with Lys238 (Fig. 9).
While compound 21r showed improved MICs against wtPA, a
number physical and PK properties were less than desirable (Table
3). This compound exhibited high clearance and low solubility. Ef-
flux was still an issue as evidenced by the MIC dilution difference.
Since we felt we had an understanding of the SAR of this series, and
knew our model was predictive, we turned to a study of the phys-
ical properties to address these issues.
Lipophilicity would have the greatest effect on these issues,16b
although it was not clear if there would be a direct effect on efflux
in Pa. We did an analysis of a larger set of LpxC inhibitors from
multiple classes looking at solubility vs cLogD along with clear-
Figure 8b. Model of imidazole inhibitor 21m interaction with conserved water
lattice.

2542
J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
Figure 10. PK analysis of hydroxamic acid LpxC inhibitors.
O
N
O
OH
N
HO
HO
H
N
H
N
O
OH
O
OH
O
O
21r
clogD = 2.7
22
clogD = 1.9
IC50 = 0.4 nM
IC50 = 0.6 nM
Figure 11. Compound design targeting water lattice interaction and improve PK properties.
ance (Fig. 10). As can be seen, compounds that had the desired cri-
teria (solubility P 100 M, t1/2 >100 min) tended to cluster at low-
l
er cLogD as expected. In fact, by keeping cLogD 6 2.2, the odds of
having both criteria met improved dramatically (48% vs 3%). This
analysis fine tuned our design principles.
We therefore looked to lower LogD to the optimal range (1–2.2)
and pick up additional interactions in the active site. The sugar
Figure 12b. Model of interaction with tightly bound water.
pocket is large and becomes hydrophobic further away from the
catalytic site. It would also be difficult to reach toward the basic
patch without an undesirable increase in molecular weight. Also,
the basic patch would be highly solvated, and we felt that desolv-
ation penalties would negate gains made by interaction in this
area. We therefore targeted interaction with the more tightly
Figure 12a. Targeting bound water.

J. S. Warmus et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2536–2543
2543
of picomolar inhibitors. Analysis of physical properties drove de-
sign to focus on an optimal lipophilicity profile. Further taking
advantage of structural information, and identification of a con-
served water network over the active site, along with the optimal
profile, led to an improved LpxC inhibitor with in vivo activity
against wild type P. aeruginosa. Further work by our group on this
class of inhibitors has been reported.20
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.140. These data
include MOL files and InChiKeys of the most important compounds
described in this article.
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18. All in vivo testing was done in accordance with approved animal use protocols.
19. Moderate challenge levels of bacteria were used in these studies (ꢀ 10 LD50’s)
administered via the IP route. In total, 88% of vehicle treated mice (challenged)
expired across six experiments.
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Chen, J. M.; Collantes, E.; Chung, W.; Desbonnet, C.; Doty, J.; Doroski, M.;
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Data for compound
21r
0.4
2.7
6.7
22
PaLpxc IC50 (nM)
clogD
LipE
MIC (lg/mL)
(pump KO)
0.6
1.9
7.3
0.015
1.75
20
59
—
0.015
1
140
130
37
WT
HuHep t1/2 (min)
Kinetic solubility @ pH 6.5 (
In vivo versus WT (iv) PD50 (mg/kg)
l
M)
bound waters (Fig. 12a). Addition of a hydroxyl group to compound
21r lowered cLogD to 1.9 from 2.7 (Fig. 11), and by our model, is
predicted to hydrogen bond to a tightly bound water molecule
(Fig. 12b) or an acid residue (Asp196 in Fig. 12c).
The result of this change is shown in Table 4. While potency
against the enzyme slightly decreased, most likely due to an in-
crease in desolvation energy, we did see an improved profile. Clear-
ance decreased and solubility increased. We also saw less efflux in
the wild type organism, as the MIC dropped almost 1 dilution. Due
to the improved in vitro clearance and solubility, this compound
was tested in vivo18 and showed activity against wild type (UI-
18) P. aeruginosa through iv administration (PD50 = 37 mpk).19 No
effect was seen when dosed SC. Dosing SC caused aggravation
and the compound was most likely not absorbed. This is the first
LpxC compound in the peer reviewed literature to show in vivo
activity against a wild type P. aeruginosa challenge.
In summary, we utilized structural information, both X-ray co-
crystals and a robust homology model, that guided efficient design