Pyridone methylsulfone hydroxamate LpxC inhibitors for the treatment of serious Gram-negative infections

Article abstract of DOI:10.1021/jm2014875

The synthesis and biological activity of a new series of LpxC inhibitors represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series have improved solubility and free fraction when compared to compounds in the previously described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative antibacterial activity to comparator agents.

Full text of DOI:10.1021/jm2014875

Article
pubs.acs.org/jmc
Pyridone Methylsulfone Hydroxamate LpxC Inhibitors for the
Treatment of Serious Gram-Negative Infections
Justin I. Montgomery,*,† Matthew F. Brown,† Usa Reilly,† Loren M. Price,† Joseph A. Abramite,†
Joel Arcari,† Rose Barham,† Ye Che,‡ Jinshan Michael Chen,† Seung Won Chung,†
Elizabeth M. Collantes,‡ Charlene Desbonnet,§ Matthew Doroski,† Jonathan Doty,†
Juntyma J. Engtrakul,∥ Thomas M. Harris,† Michael Huband,§ John D. Knafels,⊥ Karen L. Leach,§
#
Shenping Liu,⊥ Anthony Marfat,† Laura McAllister,† Eric McElroy,† Carol A. Menard, Mark Mitton-Fry,†
Lisa Mullins,§ Mark C. Noe,† John O’Donnell,§ Robert Oliver,† Joseph Penzien,§ Mark Plummer,†
Veerabahu Shanmugasundaram,‡ Christy Thoma,§ Andrew P. Tomaras,§ Daniel P. Uccello,† Alfin Vaz,∥
and Donn G. Wishka†
†Worldwide Medicinal Chemistry, ‡Computational Chemistry, §Antibacterials Research Unit, ∥Drug Metabolism, ⊥Structural Biology,
#Primary Pharmacology, Pfizer Worldwide Research and Development, 445 Eastern Point Road, Groton, Connecticut 06340,
United States
S
* Supporting Information
ABSTRACT: The synthesis and biological activity of a new series of LpxC inhibitors
represented by pyridone methylsulfone hydroxamate 2a is presented. Members of this series
have improved solubility and free fraction when compared to compounds in the previously
described biphenyl methylsulfone hydroxamate series, and they maintain superior Gram-negative
antibacterial activity to comparator agents.
antibacterial target as there is no human homologue and it is
highly conserved in Gram-negative bacteria. As no inhibitors
of LpxC have been approved to date, antibiotics from this
new chemical class would likely overcome pre-existing class-
specific mechanisms of resistance such as extended-spectrum
β-lactamases (ESBLs),6 Klebsiella pneumonia carbapenemases
(KPCs),7 and the recently described New Delhi metallo-β-
lactamase (NDM-1).8
Various small-molecule LpxC inhibitors have been disclosed
in patents and journal articles over the last two decades,9 but to
our knowledge, none have reached the clinic. An earlier report
from our laboratories describes biphenyl methylsulfone
hydroxamate inhibitors with impressive activity and spectrum.10
Unfortunately, the series suffered from high clearance, high
protein binding, and low solubility, and had high human
efficacious dose projections. In a typical medicinal chemistry
program targeting oral delivery of drug, free fraction (fu) has
little to no effect on the free concentration of drug achieved at
the target site, as clearance generally goes up as free levels
increase;11 however, an exception exists in the case of high
clearance drugs delivered intravenously.12 When these con-
ditions exist, free AUC is directly proportional to fu and fu has a
dramatic effect on dose predictions. For these reasons, we set
out to both improve solubility and increase fu in our series to
consequently increase our ability to deliver more drug in an
acceptable volume for iv delivery and improve free exposure
INTRODUCTION
■
As the antibiotic pipeline continues to decline, considerable
concern has developed around growing resistance in bacterial
species responsible for hospital-acquired infections.1 The
so-called “ESKAPE” pathogens,2 Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species,
account for a large proportion of nosocomial infections, and
while substantial progress toward the treatment of the resistant
Gram-positive ESKAPE organisms has been achieved, few, or in
many cases no, drugs exist for the multidrug resistant Gram-
negatives (KAPE),3 creating a high medical need for new agents
that can overcome these “bad bugs”.4 LpxC (UDP-3-O-(R-3-
hydroxymyristoyl)-N-acetylglucosamine deacetylase) is a metallo-
enzyme that catalyzes the first committed step in the biosynthesis
of lipid A, an essential component of the outer membrane of
Gram-negative bacteria (Scheme 1).5 LpxC is an attractive
Scheme 1. Deacetylation of UDP-3-O-(R-3-
hydroxymyristoyl)-N-acetylglucosamine Catalyzed by LpxC,
the First Committed Step in the Biosynthesis of Lipid A
Received: November 4, 2011
Published: January 18, 2012
© 2012 American Chemical Society
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a
respectively, all while maintaining the excellent antibacterial
activity and spectrum of the previously described biphenyl
methyl sulfones. Herein, we describe efforts to replace the
central phenyl ring in biphenyl methylsulfone hydroxamate 1
(Figure 1) with more polar groups culminating in the discovery
Scheme 3. Synthesis of Compound 10
Figure 1. Methylsulfone hydroxamate LpxC inhibitors.
of pyridone 2a as well as the SAR and antibacterial spectrum of
the pyridone methylsulfone hydroxamate series.
a
Reagents and conditions: (a) mCPBA, DCM; (b) AcOH, Δ; (c)
LiOH, THF, MeOH, H2O; (d) amide coupling w/THPONH2, THP-
ONH2, HATU, DIPEA, DMF; (e) THP deprotection; HCl, DCM,
MeOH.
CHEMISTRY
■
The synthesis of the hydroxamic acids 7 (Scheme 2) began
with base promoted alkylation of ethyl esters (4) with the
ab
,
Scheme 2. Synthesis of Methylsulfone Hydroxamic Acids
Various pyridone analogues were synthesized to explore the
SAR around the core of the hydroxamic acids 2 (Scheme 4).
Scheme 4. Synthesis of Modified Pyridone Methylsulfone
a
Hydroxamic Acids
a
Racemic synthesis shown: separation of enantiomers can be per-
fomed at the hydroxamic acid (7) stage using chiral chromatography.
b
Reagents and condition: (a) NaH, DMF; (b) Cs2CO3, DMF 50 °C;
(c) MeI, Cs2CO3, DMF; (d) LiOH, THF, H2O; (e) method A, CDI,
DMF, THPONH2; method B, (1) CDMT, NMM, 2-MeTHF, (2)
THPONH2; method C, EDC, HOBt, THPONH2; (f) method A,
Silicat Pd-Dppf, K2CO3, EtOH:H2O (9:1), 70 °C; method B,
PhB(OH)2, Pd EnCat, K2CO3, EtOH:H2O (9:1), 70 °C; (g) HCl
(h) PhB(OH)2, Pd(dppf)Cl2, K3PO4, 1,4-dioxane.
a
Reagents and conditions: (a) Alkylation: method A, Cs2CO3, THF,
65 °C; method B, K2CO3, DMF, rt; method C, K2CO3, TBAB, ACN,
Δ. (b) Suzuki−Miyaura coupling, PhB(OH)2, Pd(II) EnCat, K2CO3,
EtOH:H2O (9:1). (c) Saponification: method A, LiOH, THF, MeOH,
H2O; method B, KOH. (d) Amide coupling w/THPONH2: method
A, (1) HOBt, DIPEA (TEA), DCM (DMF), (2) THPONH2, EDCI;
method B, (1) CDMT, NMM, 2-MeTHF, (2) THPONH2. (e) THP
deprotection: method A, PPTS, EtOH; method B, HCl.
appropriate alkyl iodides 3, which were readily synthesized
from the corresponding alcohols. When ethyl (methylsulfonyl)-
acetate was used in the initial alkylation, a second alkylation
using methyl iodide was performed to afford the corresponding
ethyl esters 5. The aryl bromides 5 (R1 = Br) were converted to
the corresponding hydroxamic acids 7 via saponification of the
ethyl ester, amidation of the free acid with O-(tetrahydro-2H-
pyran-2-yl)hydroxylamine (THPONH2) to afford the protected
hydroxamic acid, Suzuki−Miyaura cross coupling using phenyl-
boronic acid, and acid hydrolysis to reveal the free hydro-
xamate. Alternatively, the Suzuki−Miyaura cross coupling can
be performed directly on ethyl esters 5 to provide coupled prod-
ucts 6, followed by the same end-game steps leading to hydro-
xamic acids 7. When desired, the two enantiomers of methyl-
sulfone hydroxamates 7 can easily be separated at this stage
through the use of chiral chromatography.
Synthesis of these analogues required base mediated alkylation
of the corresponding free pyridones 11 with the R-enantiomer
of alkyl bromide 12 to afford ethyl ester 13. A Suzuki−Miyaura
cross coupling was performed, when necessary, to afford the
desired phenyl analogue, which was converted to the hydro-
xamic acids 2 via the previously described sequence involving
saponification, amide coupling, and acid deprotection.
Intermediate 16 was synthesized via two pathways (Scheme 5).
The ethyl ester 13a was readily elaborated through metal-
mediated coupling reactions (Suzuki−Miyaura or Negishi),
followed by standard protocols for the conversion of the ester
to the THP protected hydroxamic acid 16. Alternatively, inter-
mediate 15 was efficiently synthesized from the ethyl ester 13a
and was primed for the two-step analoguing sequence to the
desired hydroxamic acids 17. Metal-mediated coupling (Suzuki−
Miyaura, Negishi, Sonogashira, or Grignard) of 15 to the
Compound 10 (Scheme 3) was obtained via a mCPBA
oxidation of the pyridyl intermediate 6a to afford the N-oxide 8,
which rearranged in refluxing acetic acid to a hydroxy pyridine.
Saponification gave the free acid 9, which was then converted
to the hydroxamic acid 10 as previously described.
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concentrations (MICs) are presented. Strain PA3919 is a
Pae strain lacking major efflux pumps (Δ(mexAB−OprM)
Δ(mexXY)Δ(MexZ)), while PA01 is a fully efflux-competent
wild type Pae strain.
Scheme 5. Synthesis of Substituted Pyridone Methylsulfone
Hydroxamic Acids
a
As solubility and fu are generally correlated with polarity,13
we began by introducing heteroatom substitutions within the
central ring (Table 1) with the goal of increasing polarity
without sacrificing activity. Pyridine 7a was less active than
phenyl analogue 1 by about 9-fold in enzyme potency and 4−5
dilutions in whole cell activity. Moving the nitrogen atom over
one position as in pyridine 7b slightly lowered activity; how-
ever, introducing polarity on both sides of the central ring as in
pyrimidine 7c resulted in a larger reduction of both enzyme and
wild type Pae whole cell activity (PA01). C-Linked pyridone
10 was only moderately active, but N-linked pyridone 2a
maintained favorable potency (Pae IC50 = 3.6 nM) and MICs
(PA01 = 0.5 μg/mL). In addition, cLogD was reduced by
1.7 units (2.2 for 1, 0.5 for 2a) with the phenyl-to-pyridone
substitution. The three monomethyl analogues 2b, 2c, and
2d were investigated, and while all had measurable activity, the
3-methyl analogue 2b was superior and was similar in activity to
the parent pyridone 2a. Fluorine was also tolerated in the
3- (2e) and 5-positions (2f), and in these analogues, PA01
activity was similar to analogue 2a. Finally, substituting in a
second nitrogen atom in the 3- (2g) or 6-position (2i) led to a
substantial loss of activity, but pyrimidinone 2h maintained
high antibacterial activity, and cLogP in this analogue was
further reduced to −1.4.
The cocrystal structure of pyridone 2a in Pae LpxC enzyme
(Figure 2) was solved, revealing binding similar to that seen for
hydroxamate methylsulfone headgroup containing biphenyl
compounds.10 Data collection and refinement statistics for the
costructure are presented in Table 2. The pyridone oxygen
oriented toward methionine 62 in a relatively hydrophobic
pocket rather than binding in the opposite orientation to inter-
act with threonine 190, which modeling had suggested was a
possibility, but this alternate binding mode would have
disrupted the H-bond between the hydroxamate carbonyl and
threonine 190. Key additional interactions of 2a with the LpxC
enzyme include the hydroxamate zinc binding motif, the
sulfone−lysine 238 interaction, the positioning of the sulfone
methyl group in a hydrophobic pocket, and multiple hydro-
phobic contacts at the tail end of the molecule. As in the
biphenyl series, a good opportunity for optimizing potency and
properties was through modification of the pendant phenyl
group.
a
Reagents and conditions: (a) Suzuki−Miyaura coupling: RB(OC(CH3)2,
Pd(dppf)Cl2, K3PO4, 2-MeTHF. (b) Negishi coupling: RZnBr, Pd(PPh3)4,
THF. (c) Saponification: LiOH. (d) Amide coupling w/THPONH2: (1)
CDMT, NMM,2-MeTHF; (2) THPONH2. (e) THP deprotection:
method A, PPTS, EtOH; method B, HCl. (f) Suzuki−Miyaura coupling:
method A, RB(OR′)2 or (MeBO)3, Pd(PPh3), K2CO3, 1,4-dioxane, H2O,
80 °C; method B, RB(OR′)2, Pd Encat, K2CO3, EtOH, H2O, 70 °C;
method C, RB(OR′)2, SiliaCat DPP-Pd, K2CO3, EtOH, H2O, 70 °C;
RB(OR′)2, Pd(dppf)Cl2, K2CO3, 1,4-dioxane, H2O, 80 °C. (g) Sonogashira
coupling: PhCCH, Pd(PPh3)4, CuI, DIPEA, THF, rt. (h) Grignard
reaction: C6H11MgBr, CuBr-Me2S, THF, −78 °C to rt.
appropriate boronate, zincate, acetylene, or Grignard species
afforded the corresponding THP protected hydroxamic acids 16.
Acid-promoted THP deprotection then gave the desired hydro-
xamic acids 17.
As shown in Scheme 6, hydroxamic acid 19 was prepared via
alkylative-oxidation of t-butylpyridine and the alkyl bromide 12
Scheme 6. Additional Transformations Not Covered by
Previous Schemes
a
Having discovered a more polar, but equally active replace-
ment for the central phenyl ring in 1, we set out to study the
SAR around the terminal ring in pyridone 2a (Table 3).
Replacing the phenyl ring with various pyridines (17a−c) led
to substantial loss of activity, highlighting the importance of
maintaining hydrophobic contacts in this area of the enzyme.
4-Pyridine 17c, in which the polarity is placed toward the exit
of the hydrophobic tunnel, was the most potent pyridine at
39 nM, but the 2-pyridine analogue 17a had better wild type
Pae activity. Surprisingly, pyridine 17d with an effective
methoxy substitution para to the pyridone ring connection,
was quite active, with 6.4 nM potency and a 2 μg/mL wild type
Pae MIC. Unfortunately, its Gram-negative spectrum was
narrower than pyridone 2a (vide infra, Table 7). Removing the
terminal phenyl completely and replacing it with a methyl
group (17e) was detrimental to enzyme potency but did not
obliterate whole-cell activity completely. Increasing the size of
a
Reagents and conditions: (a) (1) 100 °C, 1 h; (2) 1 N aq NaOH,
K3Fe(CN)6, 100 °C to rt. (b) Amide coupling w/THPONH2: method A,
(1) CDMT, NMM, 2-MeTHF, (2) THPONH2; method B, THPONH2,
HATU, DIPEA, DMF. (c) THP deprotection: method A, TFA, DMSO;
method B, HCl, DCM, MeOH. (d) C6H8, 10% Pd/C, EtOH, rt.
in the presence of potassium ferricyanide (III) and 1 N aq
NaOH, followed by THPONH2 amidation and THP depro-
tection. Alkene 16h was converted to the alkane 20 via transfer
hydrogenation followed by acidic THP deprotection.
RESULTS AND DISCUSSION
■
In the following tables, Pseudomonas aeruginosa (Pae) enzyme
potency (Pae IC50) as well as two minimum inhibitory
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Table 1. Central Ring SAR
a
b
Biological data for racemic compound. n = 1 IC50 data.
the pyridone series (17h), but upon incubation of this analogue
with glutathione, we saw covalent modification of the alkyne,
and the series was deprioritized. Alkyne replacements such as
alkene 17i and alkane 20 were also investigated, but these
isosteres dropped off in activity, especially against tougher Pae
stains in our MIC90 panel (data not shown).
Although over 500 pyridone analogues with substituted
phenyl groups were synthesized and characterized, only a
selection of molecules that illustrate key SAR points and/or
represent the most active analogues are presented in Table 4.
Introducing a single fluorine on the phenyl ring in any position
(17j−l) led to slightly improved activity over phenyl pyridone
2a and difluoro analogues (17m−o) had activity very similar to
2a. However, introducing 2,6-dimethyl substitution (17p) led
to a substantial loss of activity, illustrating that the hydrophobic
tunnel cannot comfortably accommodate these larger groups.
3,5-Dimethyl analogue 17q’s activity was closer to the parent
compound 2a but still fell off slightly. In addition, midsized
groups such as methoxy in the ortho position (17r) led to loss
of activity relative to analogue 2a. Methoxy in the meta position
as in 17s was about equally active to phenyl pyridone 2a.
Excitingly, para-methoxy derivative 17t was significantly more
active than 2a with wild type Pae MIC = 0.125 μg/mL.
Combining methoxy and fluorine substituents led to a number
Figure 2. Co-crystal structure of pyridone 2a in complex with
Pseudomonas aeruginosa LpxC (RCSB PDB ID 3UHM).
the alkyl group to tert-butyl (19) restored enzyme activity to
double digit nM numbers, suggesting beneficial hydrophobic
interactions in this area. The cyclohexyl analogue 17f was less
active than phenyl pyridone 2a, but the introduction of a single
unsaturation in cyclohexenyl pyridone 17g completely restored
that activity. The phenyl alkyne substituent was quite active in
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5-membered heterocyclic analogues 17ff, 17gg, and 17hh, all of
which had wild type Pae MICs of 0.25 μg/mL.
Table 2. Data Collection and Refinement Statistics (RCSB
PDB ID 3UHM)
While searching for highly active LpxC inhibitors with more
polarity than biphenyl methylsulfone 1, we were continuously
measuring solubility and free fraction to make sure improve-
ments were also made in these properties (Table 5). Aqueous
thermodynamic solubility (from crystalline solid forms) was
measured at three different pH levels: (1) pH 3, where the
hydroxamate is fully protonated, providing a measure of
intrinsic solubility, (2) pH 7.4, physiological conditions, and
(3) pH 9, our upper limit for IV formulation/delivery and a
pH where the hydroxamate is partially ionized. For biphenyl
analogue 1, we found relatively low single digit μg/mL solu-
bilities at low and neutral pH, but a significant increase at pH
9 to 260 μg/mL. Pyridone 2a had dramatically higher solubility
at all measured pH’s and over 16 mg/mL solubility at pH 9,
which surpassed our target of 5 mg/mL for iv formulation.
The more active pyridone analogue 17v, with additional
polarity on the phenyl ring had intrinsic solubility of 82 μg/mL
and ≥14 mg/mL at pH 9, but some of these solubility gains
were lost in the three-ring analogues exemplified by 17hh,
where solubility was only ∼3 mg/mL at pH 9 (still ∼10×
greater than biphenyl analogue 1 at the same pH).
Free fraction in rat and human plasma was measured using
equilibrium dialysis. Compound 1 was only 2% free in human
plasma. Fortunately, the pyridones demonstrated much lower
protein binding and were generally between 10% and 35% free.
Parent pyridone 2a was 28% free in rat plasma but only 14%
free in human plasma, while three-ring triazole 17hh was more
free in human plasma. Analogue 17v was one of our best com-
pounds in terms of free fraction, with >30% free in both
species. Figure 3 is a plot of free fraction vs cLogD7.4 for
multiple compounds (of varying activity) in the pyridone
methylsulfone hydroxamate series (green stars) and biphenyl
methylsulfone hydroxamate10 series (yellow circles). The blue
shading shows the optimal cLogD7.4 range for both achiev-
ing good MICs (data not shown) and acceptable free fraction,
Data Collection
space group
cell dimensions
a, b, c (Å)
P21
34.14, 80.97, 49.78
90, 95.5, 90
α, β, γ (deg)
a
resolution (Å)
50.0−1.5 (1.53−1.5)
0.049 (0.059)
24.1 (22.7)
Rmerge
I/σ
completeness (%)
redundancy
Refinement
resolution (Å)
no. reflns
87.3(84.1)
3.4 (2.5)
50−1.5 (1.54−1.50)
37,590 (2,757)
R
work/Rfree
no. atoms
protein
0.175 (0.137)/0.208 (0.193)
2,351
34
ligand/ion
water
242
rms deviations
bond lengths (Å)
bond angles (deg)
0.010
1.07
a
Statistics in the highest resolution shell are shown in parentheses.
Crystal diffracted to 1.26A, but was limited to 1.5 Å for completeness.
of quite active analogues (17u, 17v, 17w). Chlorine in the para
position was also found to increase potency as illustrated by
analogue 17x. Trifluoro analogue 17y was slightly less active
than 17x, but switching to a para chloro substituent (17z)
resulted in one of our most active analogues with a 0.52 nM
Pae LpxC IC50. Napthyl analogue 17aa picked up a little
activity over 2a but at the expense of increased cLogP (∼1.2
units difference). A number of quite active analogues were
found by adding a third ring (a heteroaromatic) to the para
position of the pyridone phenyl ring (17bb−17hh). Of
particular note are methoxy pyrimidine 17ee and various
Table 3. Pyridone Terminal SAR (Non-phenyl)
a
Biological data for racemic compound.
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Table 4. Pyridone Terminal SAR (Phenyl)
b
n = 1 IC50 data.
Table 5. Properties, Solubility, And Free Fraction of Select Analogues
thermosol μg/mL
compd
MW
cLogP
cLogD7.4
pH 3
pH 7.4
pH 9
260
rat fu
human fu
1
347.4
364.4
412.4
431.5
1.93
−0.80
−0.57
−0.60
2.17
0.51
0.56
0.09
7.3
410
82
3.9
480
130
9.2
0.026
0.28
0.32
0.13
0.023
0.14
0.31
0.25
2a
>16000
>14000
2740
17v
17hh
3.3
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and one can readily see the inherent fu advantage of the
pyridone series in this polarity range.
Table 7. MIC90s of Select Analogues and Control
Compounds
a
species (n), MIC90s (μg/mL)
compd
Pae (22) Eco (11) Kpn (22) Asp (11) Eae (11) Cfr (11)
ciprofloxacin
64
64
4
>64
>64
2
64
>64
16
16
8
64
32
1
32
4
32
>64
8
aztreonam
1
32
2a
2
8
32
16
8
8
2f
4
4
32
8
2h
4
8
16
32
64
16
32
16
16
17d
17h
17v
17x
17y
17z
17aa
17hh
16
2
8
>64
>64
>64
64
0.06
0.5
0.25
0.125
1
0.25
0.125
0.5
0.5
1
0.5
0.5
1
0.5
0.5
1
2
>64
>64
>64
64
1
0.5
0.25
0.06
0.06
0.06
0.5
0.5
0.125
0.25
0.25
0.125
0.25
0.125
0.125
1
1
a
Pae, Pseudomonas aeruginosa; Eco, Escherichia coli; Kpn, Klebsiella
Figure 3. Plot of human free fraction (fu) vs cLogD7.4 for two series.
pneumoniae; Asp, Acinetobacter spp.; Eae, Enterobacter aerogenes; Cfr,
Citrobacter freundii.
Table 6 contains rat PK for various methylsulfone analogues.
High clearance was observed for all compounds with the
Acinetobacter spp. (Asp), which was not surprising given the
drop in LpxC enzyme potency we observe when comparing
Acinetobacter baumanii IC50s to Pae IC50s (data not shown). Like
Pae, Eco, and Kpn, Enterobacter aerogenes (Eae) and Citrobacter
freundii (Cfr) MIC90s were quite good (generally <1 μg/mL) for
the more active pyridone analogues, clearly differentiating these
new LpxC inhibitors from the marketed comparators.
Table 6. Rat PK for Select Compounds
dose
CI
Vdss
T1/2
AUC
free AUC
compd (mg/kg) (mL/min/kg) (L/kg) (h) (μg·h/mL) (μg·h/mL)
1
10
10
10
10
57
78
72
32
3.8
3.2
2.9
2.1
2.3
5.2
0.075
0.59
0.74
0.68
28
1.7
1.0
17v
17hh
1.7
1.4
0.92
0.94
CONCLUSION
■
Efforts to improve on the previously reported LpxC inhibitor 1
which suffered from low solubility and high protein binding
resulted in the identification of a pyridone series of LpxC
inhibitors with excellent Gram-negative antibacterial activity,
improved solubility, and increased free fraction. Many of these
novel compounds have MIC90s of ≤1 μg/mL for multiple
species and have met our targets for solubility and free fraction,
which will enable us to achieve the required exposure to
determine a relevant therapeutic index. The results of further in
vivo experiments will be reported in due course.
exception of triazole 17hh (32 mL/min/kg). Total AUC
was relatively consistent for the original biphenyl lead 1
(2.9 μg·h/mL) and pyridone comparators 2a, 17v, and 17hh
(2.1 to 5.2 μg·h/mL), but as the efficacy of these LpxC inhi-
bitors is related to free AUC, an order of magnitude advantage
exists for the pyridones over biphenyl analogue 1, which, as
noted earlier, directly translates into lower human dose pre-
dictions (unpublished results).
Table 7 presents MIC90 values for multiple species of Gram-
negative bacteria for various methylsulfone hydroxamates in
comparison to biphenyl methylsulfone hydroxamate 1 and
approved agents. For Pseudomonas aeruginosa (Pae), all of the
novel LpxC inhibitors are superior to the marketed agents,
ciprofloxacin and aztreonam. In comparing compounds with
modification at the central ring and a terminal phenyl ring,
pyridone 2a has a better Pae MIC90 than fluoropyridone 2f
and pyrimidinone 2h. Compound 17d with a terminal methoxy
substituted pyridine ring, drops off significantly with a Pae
MIC90 of 16 μg/mL. Alkyne 17h is equally active to pyridone
2a, but substituted aryl pryidones 17v, 17x, 17y, 17aa, and
17hh have Pae MIC90 = 1 μg/mL. The most active analogue
for Pae was trihalogen substituted pyridone 17z with an
impressive MIC90 of 0.25 μg/mL for the 22 strains tested. For
Escherichia coli (Eco), pyridone 2a had a relatively weak MIC90
of 8 μg/mL, but substitution of the terminal aromatic ring
resulted in multiple analogues with very low MIC90s of <0.5
μg/mL. Similarly, for Klebsiella pneumoniae (Kpn), aryl ring
substitution led to quite low MIC90s. Amazingly, three-ring
triazole analogue 17hh had a Kpn MIC90 of only 0.125 μg/mL.
None of the new analogues were particularly active for
EXPERIMENTAL SECTION
■
LpxC Enzyme Assay. The assay measures the enzymatic
deacetylation of the synthetic lipid substrate UDP-3-O-(R-hydroxy-
decanoyl)-N-acetylglucosamine by mass spectrometry using a
computer-controlled fluidic system combined with a triple quadrupole
mass spectrometer (BioTrove RapidFire) and is similar to that
reported by Langsdorf et al.14 The enzyme assay was conducted in
384-well polypropylene plates (Thermo no. 4312) in a total volume of
50 μL containing 100 pM purified Pseudomonas aeruginosa LpxC
enzyme. The enzyme was expressed in E. coli and purified from cell
lysates using a method based on a published protocol.15 Briefly, cell
paste was resuspended in buffer containing 10 mM sodium phosphate,
ph 7.0, 0.1 mM zinc chloride, and 10 mM magnesium chloride, in
addition to Benzonase and EDTA-free protease inhibitor tablets. Cells
were lysed by passing through a microfluidizer, then spun at 45000g
for 1 h. The resulting supernatant was purified on a DEAE-Sepharose
column, using a 0−150 mM NaCl gradient. Fractions containing the
LpxC protein were pooled, concentrated, and purified on a Superdex
200 gel filtration column. The purified LpxC protein was stored at
−80 °C until use. The substrate buffer contained 100 mM Na2PO4,
1 mg/mL bovine serum albumin, pH 7.0. The enzyme buffer contained
5 mM Na2PO4 and 1.5 mg/mL bovine serum albumin, pH 7.5.
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Compounds (5 μL in 100% DMSO: compound final concentration
range 100 μM to 1.0 pM) were preincubated with 20 μL of LpxC
enzyme for 30 min at room temperature. The substrate UDP-3-O-(R-
hydroxydecanoyl)-N-acetylglucosamine, which was obtained from
Alberta Research Council (Alberta, Canada), was added to initiate
the assay, 25 μL at 1.0 μM (final concentration 0.5 μM). Assay plates
were sealed, incubated at room temperature for 60 min, and the
reaction stopped by the addition of 10 μL of 1 N hydrochloric acid.
The plates were read on the BioTrove RapidFire with mass spec
detection, and the product and substrate peak areas were determined.
A percent substrate conversion was calculated in Microsoft Excel. The
calculated values were entered into an internally developed (within
Pfizer) data analysis program (Sights), and curves were drawn based
on the percent substrate conversion data points. An 11-point dose
response was plotted and IC50 determinations were made by that
software package. Unless otherwise noted, IC50 values are reported as
the average of 3 or more runs (n) the standard deviation.
Antimicrobial Susceptibility Testing. The minimum inhibitory
concentration (MIC) values were determined using the broth micro-
dilution protocol according to the methods of the Clinical and
Laboratory Standards Insitute (CLSI).
Inoculum Preparation and Direct Colony Suspension. Bacterial
strains were grown on Tryptic Soy Agar with 5% Sheep Blood for
24 h prior to testing. Colonies of strains were suspended into sterile
saline until a turbidity standard of 0.5 McFarland was achieved
(approximately 1 × 108 CFU/mL). This suspension was diluted 1:400
in Mueller−Hinton broth (2.5 × 105 CFU/mL).
Drug Dilution Tray Preparation. Compounds are diluted in
appropriate solvent to 2200 μg/mL. The microbroth dilution stock
plates were prepared in 2-fold dilution series, 64 to 0.06 μg drug/mL
(high dilution series) and 0.25 to 0.00025 μg drug/mL (low dilution
series). For tube 1 of the high dilution series, 200 μL of the 2220
μg/mL stock was added to duplicate wells of a 96-well microtiter plate.
For tube 1 of the low concentration series, 200 μL of an 8.33 μg/mL
stock was added to duplicate rows of a 96-well microtiter plate. Serial
2-fold decremental dilutions were made using a BioMek FX robot
(Beckman Coulter Inc., Fullerton, California) with 10 of the remaining
11 wells, each of which contained 100 μL of the appropriate solvent/
diluent. Row 12 contained solvent/diluent only and served as the
control. Daughter plates were spotted (3.0 μL/well) from the stock
plates listed above using the BioMek FX robot, yielding the final
concentration of drug as described above.
Tray Inoculation. Organisms were inoculated (100 μL volumes)
using the BioMek FX robot. The final number of bacterial cells per
well was approximately 2.5 × 104 CFU/mL. The inoculated trays were
placed in stacks of no more than four and covered with an empty tray.
The trays were incubated for 16−20 h at 35 °C in an ambient air
incubator.
MIC Test Results. After inoculation and incubation, the degree of
bacterial growth was estimated visually with the aid of a test reading
mirror (Dynex Technologies 220−16) in a darkened room with a
single light shining directly through the top of the microbroth tray.
The MIC was the lowest concentration of drug which prevented
macroscopically visible growth under the conditions of the test.
Determining the MIC90. After all the MICs for each subset of
strains were read, the values were sorted from highest to lowest and
the MIC value where 90% of strains were inhibited at that value or
below was considered the MIC90.
Crystallization Procedure. LpxC protein was expressed and
purified as previously described.15 Prior to crystallization, LpxC was
diluted to a final concentration of 10 mg/mL in protein buffer with
zinc chloride and ligand was added to 1 mM. The mixture was
incubated at room temperature for 1 h before crystallization to allow
for complex formation. Sitting drops composed of equal volumes of
the LpxC/inhibitor complex and well solution were equilibrated over
the well solution at 22 °C. The crystals were grown in 0.1 M bis-tris
pH 6.5, 0.1 M sodium citrate tribasic, 10 mM zinc chloride, 0.25 M
guanidine HCl, and 28% polyethylene glycol 3350. Crystals were
cryoprotected by quickly dipping them into well solution contain-
ing 16% ethylene glycol before flash-freezing them in liquid nitrogen.
X-ray diffraction data were collected at beamline 17-ID of the
Advanced Photon Source facility. Structure refinement was carried out
using the program BUSTER. The ligand was built into omit electron
density after refinements with protein only, using the program COOT.
General Experimental. Starting materials, reagents, and solvents
were purchased from commercial sources unless otherwise specified.
NMR spectra were recorded on a Varian Unity 400 (available from
Varian Inc., Palo Alto, CA) at room temperature and 400 MHz for
proton. Chemical shifts are expressed in parts per million (δ) relative
to residual solvent as an internal reference (acetonitrile-d3) δH = 1.94
ppm and δC = 1.32 0.02 ppm, (chloroform-d) δH = 7.26 ppm and
δC = 77.16 ppm, (dimethylsulfoxide-d6) δH = 2.50 ppm and δC =
39.52 0.06 ppm, (methanol-d) δH = 3.31 ppm and δC = 49.00 ppm.
The peak shapes are as denoted: s, singlet; d, doublet; dd, doublet of
doublets; t, triplet; q, quartet; m, multiplet; br s, broad singlet. High-
resolution mass spectrometry (HRMS) was collected on an Agilent
(Wilmington, DE) 6220 Accurate Mass time-of-flight LC/MS operat-
ing in the electrospray ionization mode. The chromatography system
consisted of an Agilent (Wilmington, DE) 1200 binary pumping
system with the addition of an extra isocratic system and a dynamic
splitter for dilution of samples as they passed between the UV detector
and the mass spectrometer. The binary pump operated at 1.1 mL/min
of A = 10 mM ammonium formate adjusted to pH 3.5 in water and
B = 50:50 acetonitrile/methanol. Sample injections were typically
0.5 μL. Separations were effected using an Agilent (Wilmington, DE)
Zorbax Eclipse Plus C-18 (3.0 mm × 50 mm, 1.8 μm) column
operating at 60 °C. As samples eluted the UV detector, they were
diluted in the dynamic splitter with an isocratic pump flowing at
0.5 mL/min and using a solvent of 50:50 methanol/water. Samples
were diluted by factors ranging from 33:1 to 100:1. Data was pro-
cessed using the MassHunter software that was provided with the
instrument. Normal phase chromatography was performed using
Biotage Isolera One purification systems or Combiflash companions
(Teledyne Isco, Inc.) and commercially available prepacked silica
columns from Biotage, LLC., Silicycle, Inc., and Varian, Inc. Reverse
phase chromatography was performed using a Biotage Isolera One
purification system and prepacked Varian Sepra C18 columns or via
preparatory HPLC using a Agilent 1100 preparatory HPLC instru-
ment. The tested compounds were determined to be >95% pure via
HPLC.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental methods and synthetic procedures for all
intermediates and final compounds. This material is available
free of charge via the Internet at http://pubs.acs.org. Com-
pound 17v will be available to purchase from Sigma-Aldrich,
Tocris, and Toronto Research Chemicals.
Accession Codes
The RCSB PDB code for the cocrystal structure of compound
2a with LpxC is 3UHM.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: (860) 686-9120. E-mail: justin.montgomery@pfizer.
com.
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