Synthesis, structure, and SAR of tetrahydropyran-based LpxC inhibitors

Article abstract of DOI:10.1021/ml500210x

In the search for novel Gram-negative agents, we performed a comprehensive search of the AstraZeneca collection and identified a tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indicated the tetrahydropyran engaged in the same hydrogen bonds and van der Waals interactions as other known inhibitors. Systematic optimization of three locales on the scaffold provided compounds with improved Gram-negative activity. However, the optimization of LpxC activity was not accompanied by reduced inhibition of MMPs. Comparison of the crystal structure of the native product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of a tetrahydropyran-based inhibitor indicates pathways for future optimization.

Full text of DOI:10.1021/ml500210x

Letter
pubs.acs.org/acsmedchemlett
Synthesis, Structure, and SAR of Tetrahydropyran-Based LpxC
Inhibitors
Kerry E. Murphy-Benenato,*,†,§ Nelson Olivier,‡ Allison Choy,† Philip L. Ross,‡ Matthew D. Miller,†,∞
Jason Thresher,† Ning Gao,‡ and Michael R. Hale†
‡
†Department of Chemistry, Infection Innovative Medicines, and Discovery Sciences, AstraZeneca R&D, Boston,
35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: In the search for novel Gram-negative agents, we performed
a comprehensive search of the AstraZeneca collection and identified a
tetrahydropyran-based matrix metalloprotease (MMP) inhibitor that
demonstrated nanomolar inhibition of UDP-3-O-(acyl)-N-acetylglucosamine
deacetylase (LpxC). Crystallographic studies in Aquifex aeolicus LpxC indi-
cated the tetrahydropyran engaged in the same hydrogen bonds and van der
Waals interactions as other known inhibitors. Systematic optimization of
three locales on the scaffold provided compounds with improved Gram-negative
activity. However, the optimization of LpxC activity was not accompanied by
reduced inhibition of MMPs. Comparison of the crystal structure of the native
product, UDP-3-O-(acyl)-glucosamine, in Aquifex aeolicus to the structure of
a tetrahydropyran-based inhibitor indicates pathways for future optimization.
KEYWORDS: Antibacterial, LpxC, Gram-negative bacteria, MMP, hydrophobe, Pseudomonas aeruginosa, Aquifex aeolicus
he need for new antibacterials active against Gram-negative
Tbacteria is becoming critical. Current therapies are be-
coming ineffective due to increasing resistance, leaving health-
care practicioners with limited treatment options for serious
bacterial infections.1−3 To compound the issue, the presence of
an additional cell membrane, incorporating lipopolysaccharide
(LPS) in the outer leaflet, confers an intrinsic degree of resistance
to Gram-negative pathogens.4−6 The presence of the LPS layer,
in addition to promiscuous efflux pumps, prevents agents
effective at killing Gram-positive bacteria from penetrating the
Gram-negative cellular envelope.
UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC)
has become the focus of a number of programs aimed at the
development of novel Gram-negative agents.7 LpxC is a cytosolic
zinc metallo-enzyme that catalyzes the deacetylation of UDP-3-
O-(acyl)-N-acetylglucosamine, the first nonreversible step in the
biosynthesis of lipid A, the main component and the substrate to
which LPS is attached in the outer membrane of most Gram-
negative bacteria.8,9 Because the biosynthesis of lipid A is essen-
tial for maintenance of the outer membrane, LpxC is an attractive
target for the development of new agents.
A number of inhibitors of LpxC have been reported in the
literature.10−15 One of the most well documented inhibitors is
CHIR-090 (1, Figure 1).16−18 Key structural features of CHIR-
090, which are consistent in other reported inhibitors, are a zinc-
binding group (hydroxamate) and a hydrophobic tail (hydro-
phobe), which sits in a narrow hydrophobic tunnel, usually
occupied by the fatty acid tail of the natural substrate. The major
difference between the various classes of inhibitors is the core
Figure 1. CHIR-090 (1) and tetrahydropyran-based MMP inhibitor (2).
that links the hydroxamate to the hydrophobe. CHIR-090
contains a threonine-based core, which has been shown to
engage in a key hydrogen bond with Lys238 in Pseudomonas
aeruginosa (P. aeruginosa) LpxC. Pfizer has reported on a series of
methylsulfone-based inhibitors, which also take advantage of this
hydrogen bonding interaction.12,13
In our efforts to identify novel inhibitors of LpxC, we per-
formed a screen of all hydroxamates present in the AstraZeneca
compound collection. Through this effort we identified
compound 2, a tetrahydropyran-based hydroxamate, which is a
derivative of a known inhibitor (RS-130830) of matrix metal-
loprotease (MMP)-2, -8, -9, and -13.19 Tetrahydropyran-based
compound 2 demonstrated potent inhibition of P. aeruginosa
LpxC (7.4 nM, Table 1), moderate cellular activity against
P. aeruginosa PAO1 (MIC = 50 μM), but poor activity against
wild-type Escherichia coli (E. coli ARC523 MIC >200 μM). We
were attracted to the tetrahydropyran since this structural motif
Received: May 22, 2014
Accepted: September 23, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Table 1. Antibacterial Activity of Tetrahydropyran-Based LpxC Inhibitors
MIC (μM)
P. aeruginosa LpxC IC50
P. aeruginosa ARC546 (ΔMexABCDXY-
P. aeruginosa ARC545
E. coli ARC524 (ΔTolC-
E. coli ARC523
(W3110)
a
(nM)
PAO1)
(PAO1)
W3110)
1
0.31 0.15 (n = 4)
7.4 2.6
<0.2
6.25
1.56
6.25
6.25
25
1.56
50
<0.2
50
<0.2
>200
12.5
>200
>200
>200
200
200
25
2
9
4.4 1.0
100
100
200
>200
>200
>200
50
1.56
>200
200
>200
50
11a
11b
11c
11d
11e
18a
18b
18c
18d
18e
18f
18g
18h
18i
23
8.7 2.8
14 2.1
92 43
33 6.5
>200
25
139 21
6.25
0.39
12.5
1.56
0.39
6.25
12.5
3.13
0.78
25
3.0 (n = 1)
7.5 2.4
0.78
3.13
0.78
3.13
3.13
6.25
1.56
0.78
12.5
0.39
<0.2
25
100
50
100
25
2.7 1.1
2.6 0.9
>200
100
100
100
50
12.5
200
50
5.8 1.2
10 2.8
6.1 3.2
50
2.0 0.7
6.25
>200
3.13
1.56
>200
100
1.7 (n = 2)
0.20 0.1
3.1 2.9 (n = 4)
82.5 (n = 1)
8.6 2.8
>200
25
<0.2
<0.2
25
25
25
27a
27b
>200
>200
3.13
6.25
a
All errors are reported as 1 standard deviation. n = 3 unless otherwise noted.
had not been employed in other LpxC inhibitors, yet it has
similarity when compared to the natural substrate, UDP-3-O-
(acyl)-N-acetylglucosamine. The oxygen of the tetrahydropyran
has the potential to take advantage of the key Lys238 hydrogen
bond in P. aeruginosa. In addition, the MMP literature has shown
a cyclic core inhibits metabolism of the hydroxamate.19−21 With
this initial hit, we looked to maintain the tetrahydropyran core
and optimize the hydrophobe. Our initial focus was improve-
ment of Gram-negative activity and minimization of any poten-
tial off-target activity, particularly, against MMPs.
Scheme 1. Synthesis of Tetrahydropyran-Based LpxC
Inhibitor 9
a
On the basis of the wealth of structural information and SAR
available for LpxC, we proposed that a more linear hydrophobe
would optimize substrate binding in the pocket.22,23 The phenyl-
acetylenephenyl hydrophobe, similar to CHIR-090, was intro-
duced to the tetrahydropyran core to see if we could improve the
activity against LpxC as compared to the initial hit 2. For the
initial studies, an ether linkage of the phenylacetylenephenyl to
the tetrahydropyran core was chosen as the starting point, but we
planned on investigating alternative linkers as well.
a
Reagents and conditions: (a) LDA, CH2I2, THF, −40 °C to rt
The synthesis of our first target is highlighted in Scheme 1. The
tetrahydropyran core building block (4) was derived from alkyl-
ation of the lithium enolate of 3 with diiodomethane. We syn-
thesized phenol 7 in four steps, employing Sonogashira coupling
and reductive amination. Alkylation of 7 with alkyl iodide 4 fol-
lowed by hydroxamate formation afforded the target inhibitor 9.
We were pleased to find that compound 9 demonstrated ex-
cellent biochemical potency against P. aeruginosa LpxC (4.4 nM,
Table 1). In addition, compound 9 exhibited similar cellular acti-
vity against P. aeruginosa as compound 2 and improved activity
against E. coli. On the basis of their excellent activity, we obtained
crystal structures of compounds 2 and 9 in Aquifex aeolicus
(A. aeolicus) LpxC in order to confirm binding modes and to
generate a tool to aid in further design and optimization.
As expected, the hydroxamates of each compound super-
imposed and interacted with the zinc atom of the LpxC enzyme
(80%); (b) 3,4-dihydro-2H-pyran, PTSA, K2CO3, Et2O, 0 °C (80%);
(c) 4-ethynylbenzaldehyde, 10 mol % Pd(PPh3)2Cl2, 5 mol % CuI,
Et3N, MeCN, 60 °C (79%); (d) morpholine, NaBH(OAc)3, AcOH,
DCE, 0 °C to rt; (e) HCl, MeOH; (f) 4, K2CO3, DMF, 120 °C (51%
over three steps); (g) LiOH, MeOH/THF/H2O (1:1:1), 60 °C
(95%); (h) NH2OTHP, diethylcyanophosphonate, Et3N, DCM; (i)
HCl, MeOH (88% over two steps).
(Figure 2A,B). Examination of the core section of the molecules
indicated a hydrogen bond interaction between the tetrahy-
dropyran oxygen and Lys227 in A. aeolicus (equivalent to Lys238
in P. aeruginosa), similar to the interaction observed with other
LpxC inhibitors.22−25 The carbon backbone of the tetrahy-
dropyran engaged in van der Waals interactions with the hydro-
phobic phenylalanine present in the UDP pocket. Examination of
the hydrophobe portion of the molecules indicated some
differences in binding. Whereas compound 2 had a kink in its
B
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
a
Scheme 2. Synthesis of Hydrophobe Analogues
Figure 2. Superimposed cocrystal structures of A. aeolicus LpxC.
Compound 2 in green (PDB 4U3B); compound 9 in purple (PDB
4U3D); zinc ion is the gray sphere; compound 9 A. aeolicus LpxC
protein in pink. (A) Protein α-carbon superposition of compound 2
versus compound 9 cocrystal structures; protein model removed for
clarity. (B) Compound 9 cocrystal structure with A. aeolicus Lys227
interaction (3.0 Å).
a
Reagents and conditions: (a) 4, K2CO3, DMF, 120 °C; (b) LiOH,
MeOH/THF/H2O (1:1:1), 60 °C; (c) NH2OTHP, diethylcyano-
phosphonate, Et3N, DCM; (d) HCl, MeOH; (e) AcCl, Et3N, DCM
0 °C (74%); (f) CBr4, PPh3, DCM, 0 °C to rt (28%); (g) C6H5CCH,
4 mol % Pd2(dba)3, (4-MeOPh)3P, Et3N, DMF, 85 °C (60%); (h)
LiOH, MeOH/THF/H2O (80%); (i) 3,4-dihydro-2H-pyran, PTSA,
K2CO3, Et2O (80%); (j) Me3SiCCH, 5 mol % Pd(PPh3)2Cl2, 5 mol %
CuI, Et3N, MeCN (67%); (k) K2CO3, MeOH (73%); (l) 2-methylbut-
3-yn-2-ol, 5 mol % NiCl2·6H2O, 5 mol % CuI, TMEDA, THF (28%);
(m) PPTS, EtOH, reflux (quant.).
hydrophobe, compound 9 was linear and extended further into
the hydrophobic tunnel. This difference explains the contrast in
E. coli activity between 2 and 9. It has been shown that com-
pounds that bind in this area of the pocket, like compound 9, are
more potent inhibitors of E. coli.26
introduce disruptive interactions in the terminus of the hydro-
phobe region since the MMPs and LpxC vary in their electro-
statics in this region.30−32 The chemistry to access these modified
inhibitors 18a−18i can be found in Scheme 3.
With the binding mode of the tetrahydropyran core con-
firmed, we examined whether the diphenylacetylene-based
hydrophobe was optimal. The synthesis of the inhibitors with
modified hydrophobes can be found in Scheme 2. The target
compounds (11a−11e) were synthesized by alkylation of the
desired hydrophobe-phenol with alkyl iodide 4, followed by
three steps to access the hydroxamates. The phenols 10a, 10b,
and 10c were accessed from commercial sources. Phenol 10d was
synthesized by acylation of 4-hydroxybenzaldehyde (12) fol-
lowed by olefination to provide dibromo-olefin 13. Subsequent
Sonogashira reaction and deprotection of the phenol provided
10d. Diacetylene substituted phenol 10e was synthesized via
Ni-catalyzed coupling with 2-methylbut-3-yn-2-ol.
The results from the hydrophobe screen can be found in
Table 1. Of the nonacetylene-based hydrophobes, biphenyl 11a
and ether 11b were the most active with LpxC IC50s <20 nM and
good MICs against a P. aeruginosa MexAB efflux pump knockout
mutant. Diacetylene compounds 11d and 11e, which presumably
fill the hydrophobic tunnel, each had activity against an efflux-
deficient strain of E. coli in which TolC, E. coli’s main efflux pump,
has been knocked out.27 However, both compounds had poor
activity against P. aeruginosa with efflux mutant MICs >25 μM.
On the basis of this data, we decided to continue our studies
with the phenyl acetylene phenyl-based hydrophobe utilized in
compound 9.
All of the new inhibitors exhibited potent P. aeruginosa IC50s,
ranging from 1.7 to 10 nM (Table 1). The compounds exhibited
a range of cellular activity with substituted piperazines 18a, 18c,
and 18h showing the lowest P. aeruginosa and E. coli MICs.
Compound 18h demonstrated the best overall profile.
We sought to improve the Gram-negative activity further by
modifying the ether linkage. On the basis of the promising
activity of the original MMP inhibitor hit 2, we decided to focus
on thio-ethers. Additionally, we reasoned that thio-ethers would
provide the opportunity to improve overall properties by varying
the oxidation state of the sulfur linkage.25,26,28,29
Targets 23 and 25 were accessed starting from 4-iodo-
benzenesulfonyl chloride (19, Scheme 4) in a similar manner to
the other targets. Racemic sulfoxide 25 was accessed via peroxide
oxidation of methyl ester 22.
As can be seen in Table 1, the S-linked compounds exhibited
excellent biochemical potency and improved P. aeruginosa and
E. coli cellular activity compared to the ethers, with racemic
sulfoxide 25 (Table 1) having the best cellular activity of any
analogue to date. Modifying the linkage resulted in improved
penetration of the cell membranes as evidenced by an equivalent
P. aeruginosa ARC546 MIC to CHIR-090 (1). However, efflux
was still an issue.
We then looked to optimize the activity of compound 9
through modifications of the hydrophobe terminus. In order
to optimize the Gram-negative activity we planned to intro-
duce more basic substitutents since the outer membrane of
P. aeruginosa has been shown to be penetrated by basic com-
pounds.28,29 To minimize off-target MMP activity, we planned to
With the identification of lead structures for LpxC inhibition,
we turned our attention to off-target activity since this would be
critical for the program to move forward. Because of the fact that
MMPs and LpxC are both zinc-dependent metalloenzymes and
our initial hit was derived from a series designed to inhibit
C
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 3. Synthesis of Modified Phenylacetylenephenyl
Hydrophobes
Table 2. MMP Activity of Tetrahydropyran-Based Inhibitors
a
% inhibition (10 μM)
compd
MMP-2
MMP-13
a
a
1
12
30
b
2
NT
94
92
93
97
99 0.4
9
82
75
80
96
18d
18h
25
a
b
% inhibition at 30 μM. Inhibition at 15 μM; n = 3; error is reported
as 1 standard deviation.
significant inhibition of MMP-2 and MMP-13, as opposed to
CHIR-090 (1), which showed low inhibition. Modifications of
the hydrophobe and linker did nothing to help reduce the MMP
inhibition as compared to the initial hit 2. This data indicates the
selectivity against MMPs is due to the identity of the core. The
lack of MMP selectivity by our compounds with an elongated
hydrophobe suggests that the MMP binding pocket retains
inherent plasticity to accommodate ligands of variable size and
rigidity.33
To understand these results we analyzed the catalytic domain
of human collagenase-1 and -3 (MMP-1 and -13, respectively)
cocrystal structures where each MMP was in complex with a
tetrahydropyran biphenylether compound.33 We also obtained a
crystal structure of the native product, UDP-3-O-(acyl)-glucos-
amine, complexed with A. aeolicus LpxC and compared it to the
structures of compounds 2 and 9 and the MMP complexes
(Figure 3A).34 Having established that the selectivity of 1 did not
stem from the hydrophobe, we turned our attention to the
remaining parts of the molecule in order to increase selectivity
over MMP. Since modifications to the hydroxamate were
expected to be detrimental to potency, we focused on modi-
fication of the tetrahydropyran ring. Analysis of the product-
bound cocrystal structure of A. aeolicus LpxC indicated that
the product pyrophosphate group covalently linked to the
glucosamine directly interacted with Lys227 and a protein loop
between β6′ and α2′ of Domain II (Figure 3A). We hypothesized
that similar interactions could be targeted by vectors extending
target engagement while conferring selectivity against MMPs.
With this information in hand we examined modifications to
the tetrahydropyran core (Scheme 5). Each modification main-
tained the acceptor functionality of the tetrahydropyran oxygen
in order to keep the interaction with Lys238 in P. aeruginosa as
suggested in the docking model (Figure 3B).
a
Reagents and conditions: (a) 4, K2CO3, DMF, 120 °C; (b) amine,
Na(OAc)3BH, AcOH, DCE, 0 °C to rt; (c) LiOH, MeOH/THF/H2O
(1:1:1), 60 °C; (d) NH2OTHP, 2-chloro-1-methyl-pyridinium iodide,
DIPEA, DMAP, DCM, 0 °C; (e) 4 N HCl, MeOH.
a
Scheme 4. Synthesis of Modified Linkers
As can be seen in Table 1, compounds 27a and 27b were
less potent than the optimal tetrahydropyran-based compounds
(9, 18a, 18c, 18h, and 25). Alcohol 27a exhibited a significant
loss in enzyme potency as well as cellular activity. Methyl ether
27b exhibited excellent biochemical potency (7 nM IC50) but
poor antibacterial activity against wild-type P. aeruginosa and
E. coli, presumably due to high efflux activity. On the basis of their
poor LpxC inhibition, we did not follow-up with MMP testing.
In summary, we identified a new class of tetrahydropyran-
based LpxC inhibitors. We were able to improve upon the acti-
vity of the original tetrahydropyran-based hydroxamate 2
through optimization of the hydrophobe and linker. We iden-
tified compounds 23 and 25, which exhibited nanomolar po-
tencies against P. aeruginosa LpxC enzyme, 25 μM MIC against
wild-type P. aeruginosa (PAO1), and excellent activity against
E. coli (MIC < 3.13 μM). Unfortunately we were not able to
dial out the potent MMP inhibition. Attempts to modify the
a
Reagents and conditions: (a) (i) Zn, Me2SiCl2, DCE, DMA, 75 °C;
(ii) AcCl, 50 °C (65%); (b) 4-ethynylbenzaldehyde, 10 mol %
Pd(PPh3)2Cl2, 5 mol % CuI, Et3N, MeCN, 60 °C (74%); (c)
morpholine, NaB(OAc)3H, AcOH, DCE, 0 °C to rt (quant.); (d) 4,
K2CO3, DMF, 120 °C (57%); (e) LiOH, MeOH/THF/H2O (1:1:1),
60 °C; (f) NH2OTHP, diethylcyanophosphonate, Et3N, DCM; (g)
HCl, MeOH; (h) H2O2, AcOH, 0 °C (53%).
MMPs, we examined the potential for off-target activity of our
LpxC inhibitors against representative MMPs. As can be seen in
Table 2, the tetrahydropyran-based LpxC inhibitors exhibited
D
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Figure 3. Cocrystal structure of A. aeolicus LpxC and P. aeruginosa docking model. UDP-3-O-(acyl)-glucosamine in orange; product bound form of A.
aeolicus LpxC protein in yellow; zinc ion is the gray sphere; P. aeruginosa docking model in cyan; modeled compound 27b in P. aeruginosa in blue. (A)
UDP-3-O-(acyl)-glucosamine bound cocrystal structure in A. aeolicus LpxC (4OZE). (B) Compound 27b modeled with P. aeruginosa Lys238
interaction.
a
Scheme 5. Synthesis of Modified Tetrahydropyran Cores
AUTHOR INFORMATION
Corresponding Author
*E-mail: kerry.benenato@modernatx.com.
■
Present Addresses
§Moderna Therapeutics, Inc., 200 Technology Square, Cambridge,
MA 02139, United States.
∞Semichem, Inc., Shawnee, Kansas 66216, United States.
Author Contributions
All authors have given approval to this manuscript.
Notes
a
Reagents and conditions: (a) LDA, CH2I2, THF, −40 °C to rt
(86%); (b) 7, K2CO3, DMF, 120 °C (56%); (c) LiOH, MeOH/THF/
H2O (1:1:1), 60 °C (>98%); (d) NH2OTHP, 2-chloro-1-methyl-
pyridinium iodide, DIPEA, DMAP, DCM, 0 °C; (e) 4 N HCl, MeOH
(14% over two steps).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are thankful for Kumar Srinivas and colleagues at Syngene
International, Ltd., Bangalore, India for compound synthesis,
Nikunj Agrawal, Barbara Arsenault, Jesse Brock, Michele
Johnstone, and Linda Otterson for MICs, Eurofins Panlabs
Taiwan, Ltd., for MMP data, Camil Joubran and Sharon
Tentarelli for generating characterization data.
tetrahydropyran core provided compounds (27a and 27b,
Scheme 5) with diminished potency. In general these com-
pounds demonstrate good penetration of the P. aeruginosa cell
membranes, as evidenced by their MICs against the efflux knock-
out. However, they are still not as potent as other analogues like
CHIR-090 (1), and penetration needs improvement. Future
designs will focus on alternative substitutions off the tetrahy-
dropyran core. It has been observed with other series that
building into the UDP pocket can result in higher efflux ratios;
however, this may be a result of poor influx.11 We plan on
building into the UDP pocket, trying to imitate the natural
substrate, with the goals of increasing LpxC affinity, improving
cellular activity, and reducing activity against MMPs.
ABBREVIATIONS
■
MMP, matrix metalloprotease; UDP, uridine diphosphate; MIC,
minimum inhibitory concentration; THP, tetrahydropyran;
LDA, lithium diisopropylamide; THF, tetrahydrofuran; MeCN,
acetonitrile; DCE, 1,2-dichloroethane; DMF, N,N-dimethylfor-
mamide; DCM, dichloromethane; PTSA, para-toluenesulfonic
acid; TMEDA, N,N,N′,N′-tetramethylethylenediamine; DMAP,
4-(dimethylamino)pyridine; DIPEA, N,N-diisopropylethyl-
amine; DMA, N,N-dimethylacetamide; NT, not tested
ASSOCIATED CONTENT
■
S
* Supporting Information
REFERENCES
Biochemical P. aeruginosa LpxC assay; cloning, expression, and
purification of LpxC enzyme; crystallization, X-ray data
collection, and structure determination; and compound syn-
thesis. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
(1) Giamarellou, H.; Poulakou, G. Multidrug-resistant Gram-negative
infections: what are the treatment options? Drugs 2009, 69, 1879−1901.
(2) Alekshun, M. N.; Levy, S. B. Molecular mechanisms of antibacterial
multidrug resistance. Cell 2007, 128, 1037−1050.
(3) Payne, D. J.; Gwynn, M. N.; Holmes, D. J.; Pompliano, D. L. Drugs
for bad bugs: confronting the challenges of antibacterial discovery. Nat.
Rev. Drug Discovery 2007, 6, 29−40.
(4) Silver, L. L. Challenges of antibacterial discovery. Clin. Microbiol.
Rev. 2011, 24, 71−109.
Accession Codes
Protein data bank codes are as follows; compound 2, 4U3B;
compound 9, 4U3D; UDP-3-O-(acyl)-glucosamine bound
cocrystal structure in A. aeolicus LpxC, 4OZE.
E
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
(5) Silhavy, T. J.; Kahne, D.; Walker, S. The bacterial cell envelope.
Cold Spring Harb Perspect. Biol. 2010, 2, a000414.
(6) Silver, L. L. Multi-targeting by monotherapeutic antibacterials. Nat.
containing sulfonamide hydroxamic acids: potent MMP inhibitors that
spare MMP-1. Bioorg. Med. Chem. Lett. 2004, 14, 3389−3395.
(21) Weisburger, J. H.; Weisburger, E. K. Biochemical formation and
pharmacological, toxicological, and pathological properties of hydroxyl-
amines and hydroxamic acids. Pharmacol. Rev. 1973, 25, 1−66.
(22) Gennadios, H. A.; Whittington, D. A.; Li, X.; Fierke, C. A.;
Christianson, D. W. Mechanistic inferences from the binding of ligands
to LpxC, a metal-dependent deacetylase. Biochemistry 2006, 45, 7940−
7948.
(23) Hernick, M.; Gennadios, H. A.; Whittington, D. A.; Rusche, K. M.;
Christianson, D. W.; Fierke, C. A. UDP-3-O-((R)-3-hydroxymyristoyl)-
N-acetylglucosamine deacetylase functions through a general acid-base
catalyst pair mechanism. J. Biol. Chem. 2005, 280, 16969−16978.
(24) Liang, X.; Lee, C. J.; Chen, X.; Chung, H. S.; Zeng, D.; Raetz, C.
R.; Li, Y.; Zhou, P.; Toone, E. J. Syntheses, structures and antibiotic
activities of LpxC inhibitors based on the diacetylene scaffold. Bioorg.
Med. Chem. 2011, 19, 852−860.
(25) Lee, C. J.; Liang, X.; Chen, X.; Zeng, D.; Joo, S. H.; Chung, H. S.;
Barb, A. W.; Swanson, S. M.; Nicholas, R. A.; Li, Y.; Toone, E. J.; Raetz,
C. R.; Zhou, P. Species-specific and inhibitor-dependent conformations
of LpxC: implications for antibiotic design. Chem. Biol. 2011, 18, 38−47.
(26) Lee, C. J.; Liang, X.; Gopalaswamy, R.; Najeeb, J.; Ark, E. D.;
Toone, E. J.; Zhou, P. Structural basis of the promiscuous inhibitor
susceptibility of Escherichia coli LpxC. ACS Chem. Biol. 2014, 9, 237−
246.
(27) Blair, J. M.; Piddock, L. J. Structure, function and inhibition of
RND efflux pumps in Gram-negative bacteria: an update. Curr. Opin.
Microbiol. 2009, 12, 512−519.
(28) O’Shea, R.; Moser, H. E. Physicochemical properties of
antibacterial compounds: implications for drug discovery. J. Med.
Chem. 2008, 51, 2871−2878.
(29) Nikaido, H.; Vaara, M. Molecular basis of bacterial outer
membrane permeability. Microbiol. Rev. 1985, 49, 1−32.
(30) Tuccinardi, T.; Nuti, E.; Ortore, G.; Rossello, A.; Avramova, S. I.;
Martinelli, A. Development of a receptor-based 3D-QSAR study for the
analysis of MMP2, MMP3, and MMP9 inhibitors. Bioorg. Med. Chem.
2008, 16, 7749−7758.
(31) Pirard, B.; Matter, H. Matrix metalloproteinase target family
landscape: a chemometrical approach to ligand selectivity based on
protein binding site analysis. J. Med. Chem. 2006, 49, 51−69.
(32) Lukacova, V.; Zhang, Y.; Kroll, D. M.; Raha, S.; Comez, D.; Balaz,
S. A comparison of the binding sites of matrix metalloproteinases and
tumor necrosis factor-alpha converting enzyme: implications for
selectivity. J. Med. Chem. 2005, 48, 2361−2370.
Rev. Drug Discovery 2007, 6, 41−55.
(7) Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F.
M.; Chen, M. H.; Patchett, A. A.; Galloway, S. M.; Hyland, S. A.;
Anderson, M. S.; Raetz, C. R. Antibacterial agents that inhibit lipid A
biosynthesis. Science 1996, 274, 980−982.
(8) Barb, A. W.; Zhou, P. Mechanism and inhibition of LpxC: an
essential zinc-dependent deacetylase of bacterial lipid A synthesis. Curr.
Pharm. Biotechnol. 2008, 9, 9−15.
(9) Raetz, C. R.; Guan, Z.; Ingram, B. O.; Six, D. A.; Song, F.; Wang, X.;
Zhao, J. Discovery of new biosynthetic pathways: the lipid A story. J.
Lipid Res. 2009, 50 (Suppl), S103−8.
(10) Szermerski, M.; Melesina, J.; Wichapong, K.; Loppenberg, M.;
Jose, J.; Sippl, W.; Holl, R. Synthesis, biological evaluation and molecular
docking studies of benzyloxyacetohydroxamic acids as LpxC inhibitors.
Bioorg. Med. Chem. 2014, 22, 1016−1028.
(11) Hale, M. R.; Hill, P.; Lahiri, S.; Miller, M. D.; Ross, P.; Alm, R.;
Gao, N.; Kutschke, A.; Johnstone, M.; Prince, B.; Thresher, J.; Yang, W.
Exploring the UDP pocket of LpxC through amino acid analogs. Bioorg.
Med. Chem. Lett. 2013, 23, 2362−2367.
(12) Montgomery, J. I.; Brown, M. F.; Reilly, U.; Price, L. M.;
Abramite, J. A.; Arcari, J.; Barham, R.; Che, Y.; Chen, J. M.; Chung, S.
W.; Collantes, E. M.; Desbonnet, C.; Doroski, M.; Doty, J.; Engtrakul, J.
J.; Harris, T. M.; Huband, M.; Knafels, J. D.; Leach, K. L.; Liu, S.; Marfat,
A.; McAllister, L.; McElroy, E.; Menard, C. A.; Mitton-Fry, M.; Mullins,
L.; Noe, M. C.; O’Donnell, J.; Oliver, R.; Penzien, J.; Plummer, M.;
Shanmugasundaram, V.; Thoma, C.; Tomaras, A. P.; Uccello, D. P.; Vaz,
A.; Wishka, D. G. Pyridone methylsulfone hydroxamate LpxC inhibitors
for the treatment of serious gram-negative infections. J. Med. Chem.
2012, 55, 1662−1670.
(13) Brown, M. F.; Reilly, U.; Abramite, J. A.; Arcari, J. T.; Oliver, R.;
Barham, R. A.; Che, Y.; Chen, J. M.; Collantes, E. M.; Chung, S. W.;
Desbonnet, C.; Doty, J.; Doroski, M.; Engtrakul, J. J.; Harris, T. M.;
Huband, M.; Knafels, J. D.; Leach, K. L.; Liu, S.; Marfat, A.; Marra, A.;
McElroy, E.; Melnick, M.; Menard, C. A.; Montgomery, J. I.; Mullins, L.;
Noe, M. C.; O’Donnell, J.; Penzien, J.; Plummer, M. S.; Price, L. M.;
Shanmugasundaram, V.; Thoma, C.; Uccello, D. P.; Warmus, J. S.;
Wishka, D. G. Potent inhibitors of LpxC for the treatment of Gram-
negative infections. J. Med. Chem. 2012, 55, 914−923.
(14) Mansoor, U. F.; Vitharana, D.; Reddy, P. A.; Daubaras, D. L.;
McNicholas, P.; Orth, P.; Black, T.; Siddiqui, M. A. Design and synthesis
of potent Gram-negative specific LpxC inhibitors. Bioorg. Med. Chem.
Lett. 2011, 21, 1155−1161.
(15) Liang, X.; Lee, C. J.; Zhao, J.; Toone, E. J.; Zhou, P. Synthesis,
structure, and antibiotic activity of aryl-substituted LpxC inhibitors. J.
Med. Chem. 2013, 56, 6954−6966.
(16) McClerren, A. L.; Endsley, S.; Bowman, J. L.; Andersen, N. H.;
Guan, Z.; Rudolph, J.; Raetz, C. R. A slow, tight-binding inhibitor of the
zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic
activity comparable to ciprofloxacin. Biochemistry 2005, 44, 16574−
16583.
(33) Lovejoy, B.; Welch, A. R.; Carr, S.; Luong, C.; Broka, C.;
Hendricks, R. T.; Campbell, J. A.; Walker, K. A.; Martin, R.; Van Wart,
H.; Browner, M. F. Crystal structures of MMP-1 and -13 reveal the
structural basis for selectivity of collagenase inhibitors. Nat. Struct. Biol.
1999, 6, 217−221.
(34) Olivier, N.; Miller, M. D. PDB 4OZE. www.rcsb.org.
(17) Barb, A. W.; McClerren, A. L.; Snehelatha, K.; Reynolds, C. M.;
Zhou, P.; Raetz, C. R. Inhibition of lipid A biosynthesis as the primary
mechanism of CHIR-090 antibiotic activity in Escherichia coli.
Biochemistry 2007, 46, 3793−3802.
(18) Barb, A. W.; Jiang, L.; Raetz, C. R.; Zhou, P. Structure of the
deacetylase LpxC bound to the antibiotic CHIR-090: Time-dependent
inhibition and specificity in ligand binding. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 18433−18438.
(19) Fisher, J. F.; Mobashery, S. Recent advances in MMP inhibitor
design. Cancer Metastasis Rev. 2006, 25, 115−136.
(20) Reiter, L. A.; Robinson, R. P.; McClure, K. F.; Jones, C. S.; Reese,
M. R.; Mitchell, P. G.; Otterness, I. G.; Bliven, M. L.; Liras, J.; Cortina, S.
R.; Donahue, K. M.; Eskra, J. D.; Griffiths, R. J.; Lame, M. E.; Lopez-
Anaya, A.; Martinelli, G. J.; McGahee, S. M.; Yocum, S. A.; Lopresti-
Morrow, L. L.; Tobiassen, L. M.; Vaughn-Bowser, M. L. Pyran-
F
dx.doi.org/10.1021/ml500210x | ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX