Design, Synthesis, and Properties of a Potent Inhibitor of Pseudomonas aeruginosa Deacetylase LpxC

Article abstract of DOI:10.1021/acs.jmedchem.7b00377

Over the past several decades, the frequency of antibacterial resistance in hospitals, including multidrug resistance (MDR) and its association with serious infectious diseases, has increased at alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphospho-3-O-(R-hydroxydecanoyl)-N-Acetylglucosamine by the enzyme LpxC. We approached this through the design, synthesis, and biological evaluation of novel hydroxamic acid LpxC inhibitors, exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and plasma-protein binding were improved while retaining potent anti-pseudomonal activity in vitro and in vivo.

Full text of DOI:10.1021/acs.jmedchem.7b00377

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Article
Design, Synthesis and Properties of a Potent Inhibitor
of Pseudomonas aeruginosa Deacetylase LpxC
Grazia Piizzi, David T. Parker, Yunshan Peng, Markus Rolf Dobler, Anup Patnaik, Som
Wattanasin, Eugene Liu, Francois Lenoir, Jill Nunez, John Kerrigan, David McKenney,
Colin S. Osborne, Donghui Yu, Leanne Lanieri, Jade Bojkovic, JoAnne Dzink-Fox, Maria-
Dawn Lilly, Elizabeth R. Sprague, Yipin Lu, Hongming Wang, Srijan Ranjitkar, Bing Wang,
Meir Glick, Lawrence G. Hamann, Ruben Tommasi, Xia Yang, and Charles R. Dean
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017
Downloaded from http://pubs.acs.org on May 26, 2017
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Discovery Chemistry
Ranjitkar, Srijan; Novartis Institutes for BioMedical Research Emeryville,
Infectious Diseases
Wang, Bing; Novartis Institutes for BioMedical Research, Global Discovery
Chemistry
Glick, Meir; Novartis Institutes for BioMedical Research, Global Discovery
Chemistry
Hamann, Lawrence; Novartis Institutes for BioMedical Research, Global
Discovery Chemistry
Tommasi, Ruben; Novartis Institutes for BioMedical Research, Global
Discovery Chemistry
Yang, Xia; Novartis Institutes for BioMedical Research Emeryville,
Infectious Diseases
Dean, Charles R. ; Novartis Institutes for BioMedical Research Emeryville,
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Design, Synthesis and Properties of a Potent Inhibitor of Pseudomonas aeruginosa
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Deacetylase LpxC
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Grazia Piizzi*1, David T. Parker1, Yunshan Peng1, Markus Dobler1, Anup Patnaik1, Som Wattanasin1,
Eugene Liu1, Francois Lenoir1, Jill Nunez1, John Kerrigan1, David McKenney2, Colin Osborne2, Donghui
Yu2,Leanne Lanieri2, Jade Bojkovic2, JoAnn Dzink-Fox2, Maria-Dawn Lilly2, Elizabeth R. Sprague3, Yipin
Lu1, Hongming Wang1, Srijan Ranjitkar2, Bing Wang1, Meir Glick4, Lawrence G. Hamann1, Ruben
Tommasi1, Xia Yang2 and Charles R. Dean*2.
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Global Discovery Chemistry1, Infectious Diseases Area2, Chemical Biology and Therapeutics3,
Lead discovery Informatics4, Novartis Institutes for BioMedical Research, Cambridge MA,
021391,3,4 and Emeryville CA, 946082
ABSTRACT
Over the past several decades the frequency of antibacterial resistance in hospitals, including
multiꢀdrug resistance (MDR) and its association with serious infectious diseases, has increased at
alarming rates. Pseudomonas aeruginosa is a leading cause of nosocomial infections, and
resistance to virtually all approved antibacterial agents is emerging in this pathogen. To address
the need for new agents to treat MDR P. aeruginosa, we focused on inhibiting the first
committed step in the biosynthesis of lipid A, the deacetylation of uridyldiphosphoꢀ3ꢀOꢀ(Rꢀ
hydroxydecanoyl)ꢀNꢀacetylglucosamine by the enzyme LpxC. We approached this through the
design, synthesis and biological evaluation of novel hydroxamic acid LpxC inhibitors,
exemplified by 1, where cytotoxicity against mammalian cell lines was reduced, solubility and
plasmaꢀprotein binding (PPB) was improved while retaining potent antiꢀpseudomonal activity in
vitro and in vivo.
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INTRODUCTION
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Antibiotic resistance has become very widespread, threatening our continued ability to treat
serious life threatening infections1. This issue is exacerbated in Gramꢀnegative pathogens, as
these bacteria are defined by the presence of an additional protective outer membrane (OM)2.
The OM is an asymmetrical bilayer with a phospholipid inner leaflet and an outer leaflet
comprised primarily of crossꢀlinked lipopolysaccharide (LPS)3. The unique OM architecture
provides an extra permeability barrier that protects the cells from toxic compounds and is
important for bacterial survival during infection. The OM can limit compound influx, and this
together with active efflux can prevent many antibacterial compounds from accumulating
sufficiently within the cell to fully inhibit their targets4, 5. Many clinically used antibiotics that
are effective against Gramꢀpositive bacteria (which lack an OM), are ineffective against Gramꢀ
negative pathogens. The combination of the OM and efflux exacerbates the development of
resistance to the limited armamentarium of Gramꢀnegative agents. The OM also impacts efforts
to discover and optimize new antibacterial scaffolds to achieve potent cellular activity against
Gramꢀnegative pathogens. Along with its role in forming the OM, LPS is essential for growth in
many important Gramꢀnegative pathogens, including Escherichia coli, Klebsiella pneumoniae
and P. aeruginosa. LPS is not essential for in vitro growth in certain Gramꢀnegative bacteria
such as some A. baumannii6, however, inhibition of lipid A biosynthesis may still be efficacious
in treating infections by these organisms7, and would be expected to increase susceptibility to
other antibiotics by disrupting the OM permeability barrier8. For these reasons, lipid A
biosynthesis9, 10 and OM assembly11 have been a major focus in antibacterial drug discovery.
LPS consists of a lipid anchor, lipidꢀA, which forms the outer leaflet of the OM, to which is
attached a core oligosaccharide and a repeating carbohydrate Oꢀantigen extending out from the
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cell surface3. The first committed step of lipid A biosynthesis is mediated by the zinc metallo
enzyme LpxC (UDPꢀ3ꢀOꢀ(acyl)ꢀNꢀacetylglucosamine deacylase (Figure 1).
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Oꢀantigen
B
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core
lipid A
phospholipid
Kdo2ꢀlipid
Figure 1. LpxC catalyzes the first irreversible step of lipid A biosynthesis in Gramꢀnegative
bacteria (A). Multiple downstream steps complete the biosynthesis of lipid A which is then
linked to a core oligosaccharide and flipped across the inner membrane where it is then linked to
Oꢀantigen oligosaccharide repeating units and transported out to form the outer leaflet of the
asymmetrical outer membrane (B).
LpxC is essential for growth of many Gramꢀnegative pathogens, including P. aeruginosa12 and
several groups designed potent inhibitors of LpxC enzymes13ꢀ29. However, despite extensive
medchem campaigns over the last decade (Figure 2), few inhibitors have reached the clinic and
no LpxC inhibitor has reached the market, underscoring the challenges to find a safe and
efficacious treatment for P.a. infections. One of the main challenges is to balance desirable
physicochemical properties, such as low PPB and high solubility, with potent antibacterial
activity and low cytotoxicity16.
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OH
O
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HN
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OH
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HN OH
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OH
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Merck
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Lꢀ573,655
1996
Chiron
CHIRꢀ090
2004
AstraꢀZeneca
Example 297
2010
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N
NH2
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ACHNꢀ975
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Example 513
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MeO
Pfizer
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Figure 2. Representative LpxC inhibitors resulting from drug discovery efforts over the last two
decades. Compound 31 (CHIRꢀ090) is described in Anderson et al31, Lꢀ573,655 is described in
Onishi et al10 and ACHNꢀ975 is described in Kasar et al30.
RESULTS
Our efforts towards the discovery of potent and safe LpxC inhibitors began with the selection of
a promising lead from Chiron’s efforts in the early 2000’s, hydroxamate 231. Similarly to 3126, 31,
32 (Figure 2), compound 2 has a zinc binding hydroxamate warhead with a hydrophobic tail that
occupies the hydrophobic tunnel in the LpxC enzyme where the natural substrate fatty acid
resides (Figure 3). While endowed with strong LpxC and antibacterial activity, compound 2
presented low aqueous solubility and undesireable in vitro cytotoxicity (Table 1). Thus, we
initiated a structureꢀbased design to identify modifications aimed at tackling both of these
liabilities. A docking model of the binding of hydroxamate 2 revealed the proximity of the βꢀ
amino group to the UDP pocket of LpxC active site (Figure 3). Given that this large, solventꢀ
exposed pocket is mostly unoccupied and possesses hydrophilic residues (e.g. Asp 196, Lys238
and Lys261) as well as backbone polar atoms, we targeted this region for further ligand
interaction with the addition of waterꢀsolubilizing groups onto hydroxamate 2.
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Figure 3. Docking model of 2 bound to P.a. LpxC. LpxC is represented as a surface with polar
residues colored (nitrogen is blue and oxygen is red). Compound 2 is shown in green sticks. The
catalytic zinc is represented by a green sphere with dashes showing coordination by protein and
2. Residues in the UDP pocket are labelled.
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We started by screening several waterꢀsolubilizing groups that could occupy the UDP pocket.
Our initial effort lead to analog 3 which retained excellent enzymatic potency, displayed greater
aqueous solubility and lacked in vitro cytotoxicity (Table 1). Unfortunately, antimicrobial
activity against wildꢀtype P.a. (PAO1) was 32 fold weaker than analog 1 (MIC = 32 µg/mL vs 1
µg/mL, Table 1). However, P.a. defective for efflux (indicated as MIC Pump in Table 1), or
having mutations leading to increased OM permeability (indicated as MIC Perm in Table 1) were
susceptible to both analogs 2 and 3, reflecting the retention of potent activity against the LpxC
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target. These results suggested that analog 3 did not accumulate sufficiently in wild type cells to
exert potent antibacterial activity.
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Table 1. Initial attempt to add hydrophilic group on 2 expanding into UDP pocket*.
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b
aCompound 2: HTꢀSol pH 6.8 = 0.009 µM, Cytotoxicity K562 EC50 = 29.5 µM; Compound 3:
HTꢀSol pH 6.8 = 0.392 µM, Cytotoxicity K562 EC50 > 100 µM. WT is wild type P. aeruginosa
PAO1 strain K76733, the efflux deficient (Pump) strain is a K767 derivative deleted for mexAB-
oprM (K1119)34. The drug hypersusceptible (Perm) strain is strain Z6135.
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Encouraged by these preliminary results on solubility and cytotoxicity, we decided to prepare
additional analogs with substitution on the βꢀamino group in order to improve compound
accumulation in wild type P.a.. We decided to replace the biphenyl acetylene tail of compound 2
and 3 with the biphenylethyl due to concerns over chemical and metabolic stability of the
conjugated alkyne. Compounds 4ꢀ7 (Table 1) summarize the in vitro activity of the UDP series.
Although excellent enzymatic potency similar to compound 3 was retained, we did not observe
the expected improvement in antimicrobial activity. Indeed, the added polar groups had a
negative impact on efflux properties and/or passive cell permeability (compounds 5ꢀ7 versus 4).
These compounds were prepared according to Scheme 1 starting with βꢀamino ester 831.
Acylation or reductive amination of 8 allowed for the installment of the desired functionalities
onto the βꢀamino group and furnished compounds 9aꢀ9d. Conversion of the resulting esters 9aꢀ
9d to the corresponding hydroxamic acids 3, 5ꢀ7 was carried out by standard conditions.
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Scheme 1. Synthesis of key compounds for UDP pocket exploration.a
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a
Reagents and conditions: (a) Alkyl chloride, DMF, 50 °C, 36 h (Compounds 3, 5, 6); (b) 3ꢀpyridine
carboxaldehyde, Sodium triacetoxyborohydride, DCE:DCM (2:1), rt, 16 h (Compound 7); (c) NH2OH(aq) (50%
by wt), rt, 2 days.
We then turned our attention to the group occupying the hydrophobic tunnel of the enzyme
active site. We questioned whether the extended linear and conjugated biphenylethyl tail could
be shortened to reduce its hydrophobicity and improve physicochemical properties.
Representative examples from this exploration are summarized in Table 336. Interestingly,
compound 10 with a replacement of the aromatic ring by a substituted cyclohexyl group retained
good target potency but had no appreciable antimicrobial activity in wt P.a.. Conformationally
constrained substituents on one aromatic ring (compounds 11-13) yielded weak activity, mostly
due to compromised target potency. Extending the aromatic ring with a flexible hydrocarbon tail
restored enzymatic potency (compound 14) but a propargyl tail, as in compound 15, proved to be
the best fit, as of geometry and polarity, for the hydrophobic tunnel.
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Table 2. Exploration of novel tails for the hydrophobic tunnel.
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Based on this initial propargyl tail result, we then explored whether the size or electronic
properties of the substituent in β relative to the primary amino group could provide additional
gains in potency. Compounds in Table 3 were prepared according to the procedures described36.
A scalable synthesis of racemic and enantioselective 1 (+/-) was disclosed in a recent publication
37. Enantioselective synthesis of compound 20 is noteworthy and described in Scheme 2.
Condensation of trifluoro acetal with (S)-(ꢀ)ꢀtertꢀbutanesulfinamide followed by
diastereoselective addition of ethylꢀ2ꢀ ((diphenylmethylene)amino)acetate provided the desired
chiral intermediate 17 in relatively low yield owing to instability of imine 16. Cleavage of the
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chiral auxiliary and protective group provided the diamino ester 18. Selective acylation and
treatment with hydroxylamine yielded the final compound 20.
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Scheme 2. Synthesis of Compound 20a
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a
Reagents and conditions: (a) Ti(OEt)4, (S)ꢀ2ꢀmethylpropaneꢀ2ꢀsulfinamide, Molecular sieves 4 A0, DCM,
Reflux, 8 h; (b) LHMDS, Ethylꢀ2ꢀ ((diphenylmethylene)amino)acetate, THF, ꢀ78 0C to rt, 36 h, 17% (2 steps); (c)
Anhydrous HCl in EtOH (d) 2M HCl(aq), 100% (2 steps); (e) Intermediate (A), NaHCO3(aq), 33%; (f)
NH2OH(aq), MeOH:MeCN, 28%.
Addition of an extra methyl group in the β position of compound 15 was well tolerated even
when considering the activity of racemic compound 1 (Table 3). However, replacement of the
methyl group with larger and/or electron withdrawing substituents lead to weaker target and
cellular potency (20-22).
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Table 3. SAR of the central core.
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To complete our SAR assessment of the propargyl ether series, we looked at the effect of the
stereochemistry at the β position (monomethyl series) and that of the α position (gemꢀdimethyl
series). Compound 15, and single enantiomers 1-(-) and 1-(+) in Table 4 were prepared
according to the procedure referenced above36. Single stereoisomer 30 was synthesized as
described in Scheme 3. The synthesis began with trityl protection of methyl Lꢀallothreoninate
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followed by conversion of the alcohol 23 to azide 24 with inversion of stereochemistry under
Mitsunobu conditions. Trityl group of 24 was deprotected under acidic condition to furnish the
amine 25, which was subsequently coupled to propargyl benzoic acid 26 using standard
amidation protocol to afford the amide 28 in good yield. Unlike the previously reported azide
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reduction with PPh3 that proved to be challenging in terms of purification, a smooth one pot
azide reduction of 27 with SnCl2 followed by Bocꢀprotection to 28 was accomplished. After
purification, 28 was deprotected to the corresponding amine 29 and the desired hydroxamic acid
30 was obtained by treatment of this ester with 50% NH2OH(aq) at room temperature.
Scheme 3. Synthesis of Compound 30 a.
aReagents and conditions: (a) TrCl, TEA, DCM, 0 C – rt, quantitative yield; (b) DPPA, DIAD, PPh3, THF, rt, 63%
o
(c) HCl, THF, rt, 85%; (d) HATU, DIPEA, DCM, rt, 51%; (e) SnCl2, MeOH, rt; (f) (Boc)2O, NaHCO3, DCM, rt;
83% (2 steps); (g) TFA, DCM, rt; (h) NH2OH (aq. 50%), MeOH, rt, 82 (2 steps).
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The activity of the isolated stereoisomers is summarized in Table 4. While the β position was not
sensitive to changes in stereochemistry (15 and 30), the α position of the gemꢀdimethyl series
showed a clear preference for target inhibition and resulted in greater cellular potency for 1-(-).
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Table 4. Effect of stereochemistry on enzymatic and antibacterial potency.
To gain further insight into the SAR of our inhibitors, we solved the coꢀcrystal structure of P.a.
LpxC with stereoisomer 1(-). We confirmed that the gemꢀdimethyl group in the β position was
well accommodated adjacent to the UDP pocket. Likewise, the propargyl ether tail fits nicely in
the hydrophobic tunnel, and the hydroxymate warhead retains optimal zinc coordination (Figure
4). Substitution of the phenyl acetylene with the propargyl ether reduced the mass and surface
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area of the compound yet maintains in vitro potency (similar target potency for compounds 15
and 1 versus compound 2). Comparing the P.a. LpxC complex crystal structures with
compounds having different tunnel binding groups (e.g. 31 vs compound 1) suggests that the
tunnel adapts to various tail groups via movement of the “gatekeeper” helix and neighboring
residues (aa 192ꢀ205). Specifically, the movement of this helix repositions Ile197 in the upper
part of tunnel to contribute a key nonpolar interaction with the propargyl tail (Figure 4).
Additionally, the position of the primary amine allows for strong water network linking the
NH3+ group of the ligand to the protein via optimum hydrogen bonding with waters.
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Figure 4. Coꢀcrystal structure of P.a. LpxC with stereoisomer 1(-). View of the hydrophobic
tunnel enclosing the inhibitor. The protein surface is shown within 5Å of the compound and
sidechains lining the tunnel are shown as sticks and labelled. Compound 1 is shown in green
sticks. The catalytic zinc is represented by a green sphere.
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With this promising lead (compound 1(-)) we next evaluated in vitro cytotoxicity and increased
aqueous solubility. These were the two key liabilities of our starting point (compound 2) we
aimed to improve on (Table 1), and for the propargyl ether tail analogs (15 and 1) both liabilities
had been addressed (Table 5).
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Table 5. Cytotoxicity and solubility of selected analogs.
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Due to the presence of a strong zincꢀbinding warhead, we determined whether our potential lead
candidate (compound 1 (-)) was devoid of offꢀtarget activity including known metalloproteases.
We confirmed strong selectivity of binding to LpxC (hHDAC 1ꢀ13 IC50 > 30 uM, hMMPs IC50 >
30 uM). Finally, the mouse, rat and human plasma protein binding (PPB) for compound 1(ꢀ)
determined using standard assays was 22.8, 22.1 and 80.7 % which was improved over that of
the starting compound 2 (91.4, 92.8 and 91.9 %). Based on potency, solubility and clean in vitro
cytotoxicity and selectivity profile, we selected 1 (-) for further in vitro microbiology and in vivo
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characterization. MIC values were determined for a small test panel of Gramꢀnegative pathogens
and this indicated that potent antibacterial activity of 1 (-) is limited primarily to P. aeruginosa
(Table 6).
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Table 6. Susceptibility of various Gramꢀnegative bacteria to compound 1(-).
MIC (ꢀg/mL)
Name
Strain #
1 (ꢁ)
1
Ciprofloxacin
0.5
Pseudomonas aeruginosa
Pseudomonas aeruginosa
Acinetobacter baumannii
Alcaligenes xylosus
Burkholderia cepacia
NB52001
NB52096
NB48015
NB56002
NB49016
2
2
>128
16
0.5
2
32
0.5
Stenotrophomonas maltophilia NB55011
32
0.5
Enterobacter aerogenes
Escherichia coli
NB24004
NB27047
NB29018
NB30007
NB32022
>128
64
> 16
≤ 0.03
≤ 0.03
≤ 0.03
≤ 0.03
Klebsiella pneumoniae
Klebsiella oxytoca
Proteus mirabilis
16
16
64
We then measured the antimicrobial activity of 1 (-) and comparator antibiotics against a broad
panel of 180 P. aeruginosa clinical isolates from the Novartis strain collection and this is
summarized in Table 7. This panel included 96 isolates that were resistant to multiple standard
antibiotics according to CLSI breakpoints. The MIC50 and MIC90 values against this panel were
1 µg/mL and 2 µg/mL, respectively, with a range of MIC values of ≤ 0.06 – 4 µg/mL. These
values were lower than what was found for the comparator imipenem. Of the antibiotics tested
here, only colistin exhibited a similar potency to 1 (-). This suggests that our novel LpxC
inhibitor would provide a broad coverage of P. aeruginosa clinical isolates.
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Table 7. Summary of MIC ranges, MIC50 and MIC90 of 1 (-) against 180 Pseudomonas
aeruginosa strains (MIC in µg/mL).
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Compound
1 (ꢁ)
Range
MIC50
MIC90
≤ 0.06 – 4
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2
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Amikacin
≤ 0.25 ꢀ >1024
≤ 0.125 ꢀ >128
≤ 0.25 ꢀ >1024
≤ 0.25 ꢀ >128
0.06 ꢀ > 64
0.5 ꢀ >128
0.5 ꢀ >128
≤ 0.125 ꢀ >64
≤ 0.03 ꢀ >64
≤ 0.03 ꢀ >64
≤ 0.25 ꢀ 4
16
2
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4
>128
>128
>128
>128
16
>128
>128
64
Tobramycin
Azithromycin
Ceftazidime
Imipenem
Piperacillin
Ticarcillin
Ciprofloxacin
Doripenem
Meropenem
Colistin
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128
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2
0.5
Next, a standard in vivo rodent i.v. pharmacokinetics PK study on 1(-) revealed good
blood/plasma exposure and halfꢀlife, and this information was used to select the doses and routes
of administration for assessment of in vivo efficacy. The efficacy of 1 (-) was evaluated in the
neutropenic mouse model of pneumonia, infecting with a clinical isolate of P. aeruginosa (MIC
= 1 µg/mL) and results are summarized in Figure 5. Mice were dosed twice a day (BID) with 1
(-), 2 and 8 hours post infection, and the bacterial burden of the lungs was evaluated at 24 hours
post infection. Bacterial stasis (static dose) was achieved at 7.1 mg/kg/day with 1 (-) and 20
mg/kg/day gave reductions in bacterial burden of the the infected mice of approximately 2ꢀlog
below stasis. At 20 mg/kg/day both 1 (-) and ciprofloxacin gave similar reductions in bacterial
lung burden at 25 hours post infection compared to noninfected animals.
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Figure 5. P.a. mouse lung efficacy, BID dosing.
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Static dose (mg/kg/day)
Cipro i.v. = 1.8
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1 (ꢁ) i.v = 7.1
Efficacy against P. aeruginosa in the mouse thigh model is shown in Figure 6. The bacterial load
(cfu/thigh) at the start of therapy was 4.99 log10 and increased to 8.95 log10 24 hours later in
vehicle treated mice. Treatment with 1(-) at a single dose of 320 and 160 mg/kg was sufficient to
significantly reduce the bacterial burden in thighs from vehicle treated animals to levels before
those at the start of treatment.
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Figure 6. Efficacy of 1 (-) against P. aeruginosa in the mouse thigh infection model.
Neutropenic mice were infected with P. aeruginosa and treated subcutaneously 2 hours later
with 1 (-) or vehicle * P<0.05, *** P<0.001 versus vehicle, KruskalꢀWallis ANOVA followed
by a Dunn's multiple comparison test.
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DISCUSSION AND CONCLUSIONS
Here we describe the design of small molecule hydroxamate inhibitors of LpxC as potential new
antibacterial agents for the treatment of serious P. aeruginosa infections. Hydroxamate 2 was
employed as a starting point for a comprehensive structure based medicinal chemistry effort to
established detailed SAR relationships, allowing for the elimination of cytotoxicity of
compound 2 and retention of potent antiꢀpseudomonal activity, exemplified by compound 1(-).
The antibacterial spectrum of 1(-) is largely confined to P. aeruginosa, however it has potent
and broad activity across a range of P. aeruginosa clinical isolates including those that are
resistant to currently used antibiotics. The specificity of compound 1(ꢀ) for P. aeruginosa is
consistent with several reports highlighting structural variation in the conformation/flexibility of
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the substrateꢀbinding tunnel of the LpxC enzyme among Gramꢀnegative bacteria that can in part
modulate susceptibility to LpxC inhibitors12, 21, 38ꢀ40. Recently, inhibitors having a diacetylene
moiety in the tail region were shown to overcome some of this intrinsic target variability21. We
hypothesize that the lack of a diacetylene in 1(ꢀ), along with its relatively small propargyl ether
substitution that is both less bulky and nonꢀlinear favors specific inhibition of P. aeruginosa
LpxC. There is, however, measureable antibacterial activity of compound 1(ꢀ) against other
Gramꢀnegatives (Table 7), and the MIC can vary among different strains or mutants, reaching
values as low as 1ꢀ2 µg/mL against efflux deficient or membrane permeable mutants of E. coli
(data not shown). These data suggest that the LpxC target is being inhibited in some Gramꢀ
negative bacteria other than P. aeruginosa, but there may be insufficient cellular accumulation of
the compound in wild type cells to mediate potent antibacterial activity. Indeed, the
physicochemical properties of novel inhibitors such as logD may differentially impact whole cell
activity against different Gramꢀnegative pathogens as was recently reported for a series of novel
topisomerase inhibitors41. This, combined with the potential differences in susceptibility of the
targets to inhibition by compound 1(ꢀ) described above, likely determines the clinically useful
spectrum of compound 1(ꢀ) antibacterial activity. Nonetheless, our data here show that inhibitors
such as 1(-) can be designed that may be expected to have good clinical coverage of the highly
adaptable and particularly recalcitrant pathogen P. aeruginosa. Interest in more pathogenꢀ
specific antibacterials has been increasing, proportional to the apparent difficulties in finding
broadꢀspectrum antibiotics, and given the notion that a broad spectrum may contribute to
antibacterial crossꢀresistance and also unintentionally disturb the human microbiome. Consistent
with the potent in vitro activity of 1(-), acceptable efficacy was observed in the neutropenic
mouse pneumonia and thigh models of infection using i.v. administration. These results would
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warrant further in vivo investigations to fully understand PK/PD parameters and subsequent
human dose predictions. As well, the translation of the excellent in vitro antibacterial activity of
1(-), to animal models of infection shown here further supports LpxC as an attractive target
against which new inhibitors with in vivo efficacy can be designed.
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EXPERIMENTAL SECTION
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Compound synthesis and characterization.
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Compound purity was assessed by HPLC and all tested compounds were > 95% except compound 6
(Table 2), which was 93%. All solvents employed were commercially available anhydrous grade,
and reagents were used as received unless otherwise noted. Flash column chromatography was
performed on either an Analogix Intelliflash 280 using Si 50 columns (32ꢀ63 ꢁm, 230ꢀ400 mesh,
60Å) or on a Biotage SP1 system (32ꢀ63 ꢁm particle size, KPꢀSil, 60 Å pore size).
NMR: proton spectra are recorded on a Bruker 400 MHz ultrashield spectrometer. Chemical
shifts are reported relative to dimethyl sulfoxide (δ 2.50) or tetramethylsilane (δ 0.0). Chemical
shifts (δ) are reported in parts per million (ppm), and signals are reported as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), or br s (broad singlet).
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LC/MS: Analytical LCꢀMS was conducted using an Agilent 1100 series with UV detection at
214 nm and 254 nm, and an electrospray mode (ESI) coupled with a Waters ZQ single quad
mass detector.
HPLC: Preparative high pressure liquid chromatography was performed using a Waters 2525
pump with 2487 dual wavelength detector and 2767 sample manager. purification utilizes a C8
or C18 column (30 x 100mm, 5 µm, brand: Sunfire or XTerra) and is performed with 3% nꢀ
propanol in 5%ꢀ95% ACN in H2O.
Synthesis of compound 20.
(S,E)-2-Methyl-N-(2,2,2-trifluoroethylidene)propane-2-sulfinamide (16). Synthesis of
sulfinamide 16 was performed according to previously described procedures42.
Ethyl(2S,3R)-3-(((S)-tert-butyl(hydroxy)-l4-sulfanyl)amino)-2-((diphenylmethylene)-
amino)-4,4,4-trifluorobutanoate (17). To a solution of Nꢀ(diphenylmethyleneglycine) ethyl
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ester (11.68 g, 43.68 mmol) in anhydrous THF (400 mL) at ꢀ78 C was added 1N LiHMDS in
THF (43.68 mL, 43.68 mmol) dropwise. The solution was stirred for 1 h and then the reaction
mixture of 16 in toluene (27.3 mmol, 53 mL) was added slowly. The reaction mixture was
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o
stirred at ꢀ78 C for 30 minutes, then quenched by addition of sat. aq. NH4Cl (150 mL), and
warmed to room temperature. The aqueous phase was extracted with EtOAc. The combined
organic phases were washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residue was chromatographed on silica gel (gradient: 10% to 40% EtOAc/heptane)
to afford compound 17 (2.15 g) as the major isomer. Found m/z ES+ = 468. Assignment of its
absolute stereochemistry is based on similar previously described reactions43.
Ethyl (2S,3R)-2,3-diamino-4,4,4-trifluorobutanoate (18). To a solution of compound 17
(200 mg, 0.427 mmol) in anhydrous ethanol (1.15 mL) was added 4N HCl in 1,4ꢀdioxane (0.32
mL, 1.28 mmol). The reaction mixture was stirred at room temperature for 1 h. The volatiles
were evaporated under reduced pressure. To the residue was added in sequence THF (2 mL) and
aq. 2M HCl (0.43 mL). The reaction was stirred at room temperature for 2 hrs. The reaction
mixture was then diluted with aq. 1M HCl (15 mL). The aqueous phase was washed with diethyl
ether. The aqueous layer was freeze dried to afford 18 (90 mg; ES+ m/z = 146).
4-(But-2-yn-1-yloxy)benzoyl chloride (intermediate A) was prepared by refluxing the
carboxylic acid 26 (2.0 g, 10.53 mmol; the synthesis of compound 26 can be found in supporting
information), thionyl chloride (10.2 mL) and catalytic amount of DMF in dichloromethane (50
mL) overnight. The volatiles are removed under reduced pressure to afford intermediate A which
was used directly in the next step in preparing compound 19.
Ethyl (2S,3R)-3-amino-2-(4-(but-2-yn-1-yloxy)benzamido)-4,4,4-trifluorobutanoate (19).
A mixture of 18 (40 mg, 0.147 mmol) in 1,4ꢀdioxane (1.1 mL), intermediate A (30.65 mg, 0.147
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mmol), NaHCO3 (49.4 mg, 0.588 mmol) and water (1.1 mL) was stirred at room temperature for
36 hs. Then the reaction mixture was diluted with water and extracted with dichloromethane. The
organic layer was dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude
residue was then purified with HPLC (3% nꢀpropanol/Acetonitrile/H2O) to give compound 19
(18 mg). Found ES+ m/z = 373.
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N-((2S,3R)-3-Amino-4,4,4-trifluoro-1-(hydroxyamino)-1-oxobutan-2-yl)-4-(but-2-yn-1-
yloxy)benzamide (20). To a solution of 19 (18 mg, 0.05 mmol) in 2:1 acetontrile/methanol
(2.25 mL) was added 50% aq. hydroxylamine (2 mL) and the reaction mixture was stirred at
room temperature overnight. The reaction mixture was then purified with preparative HPLC to
give compound 20 (5 mg). Found ES+ m/z = 360 [M+H]+. 1H NMR (400 MHz, MeOD) δ 7.84 (d,
2H), 7.03 (d, 2H), 5.00 (d, 1H), 4.73 (s, 2H), 3.91 (m, 1H), 1.81 (s, 3H).
Synthesis of compound 30.
(2S,3R)-Methyl 3-hydroxy-2-(tritylamino)butanoate (23). To a solution of Lꢀthreonine
methyl ester (3.85 g, 22.7 mmol) in anhydrous DCM (30 mL) was added anhydrous TEA at 0 oC.
Then a solution of Trityl chloride (6.59 g, 23.15 mmol) in DCM (30 mL) was added dropwise in
5 min. The reaction mixture was stirred at room temperature overnight. The solid was filtered out
and the filtrate was washed with aq. sat. NaHCO3 solution and the aq. layer was extracted with
DCM twice. All organic layers were combined, dried over Na2SO4 and filtered. Volatiles were
1
removed under reduced pressure to afford quantitive yield of the title compound. H NMR (400
MHz, Chloroformꢀd) δ 7.54 – 7.46 (m, 6H), 7.31 (dtd, J = 8.3, 4.4, 3.6, 2.6 Hz, 6H), 7.24 (qt, J =
6.1, 0.9 Hz, 3H), 3.86 (td, J = 8.1, 7.0, 5.2 Hz, 1H), 3.43 (d, J = 7.5 Hz, 1H), 3.19 (s, 3H), 1.23
(d, J = 6.2 Hz, 3H).
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(2S,3S)-Methyl 3-azido-2-(tritylamino)butanoate (24). To a mixture of (2S,3R)ꢀmethyl 3ꢀ
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hydroxyꢀ2ꢀ(tritylamino)butanoate (2.35 g, 6.26 mmol) and PPh3 (2.46 g, 9.39 mmol) in THF(30
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mL) was added a solution of DIAD (2.03, 9.51 mmol) in THF (15 mL) dropwise at 0 C. Then a
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solution of DPPA (2.84 g, 10.01 mmol) in THF (20 mL) was added dropwise over 5 min. Then
the reaction mixture was stirred at rt overnight. Volatiles are removed under reduced pressure
and the residue was purified with silicaꢀgel chromatography (10ꢀ15% EtOAc/heptane) and
another silicaꢀgel chromatography (30% DCM/heptane) to afford the title compound (1.59 g,
1
63%). H NMR (400 MHz, Chloroformꢀd) δ 7.59 – 7.50 (m, 6H), 7.40 – 7.26 (m, 6H), 7.26 –
7.18 (m, 3H), 3.92 – 3.80 (m, 1H), 3.50 (d, J = 7.0 Hz, 1H), 3.23 (s, 3H), 3.02 (d, J = 19.4 Hz,
1H), 1.26 (d, J = 6.9 Hz, 3H).
Methyl (2S,3S)-2-azido-3-azidobutanoate (25). To a solution of (2S,3S)ꢀmethyl 3ꢀazidoꢀ2ꢀ
(tritylamino)butanoate (1.1 g, 2.74 mmol) in THF (20 mL) was added a HCl solution (2M in
o
Et2O, 13.7 mL, 27.4 mmol) at 0 C. Then the reaction mixture was stirred at rt overnight.
Volatiles were removed under reduced pressure and to the residue was added anhydrous Et2O.
White solid can be seen formed on the bottom of the flask. Rinse with anhydrous Et2O twice and
keep the white solid which was then dried under vacuum to afford crude HCl salt of the title
1
compound (0.50 g) which was used directly in the next step without further purification. H
NMR (400 MHz, DMSOꢀd6) δ 8.80 (s, 4H), 4.25 (d, J = 3.6 Hz, 2H), 3.78 (s, 3H), 1.39 (d, J =
6.6 Hz, 3H).
(2S,3S)-methyl 3-azido-2-(4-(but-2-ynyloxy)benzamido)butanoate (27). A mixture of HCl
salt of methyl (2S,3S)ꢀ2ꢀazidoꢀ3ꢀazidobutanoate (307 mg, 1.58 mmol) from previous step, 4ꢀ
(butꢀ2ꢀynyloxy)benzoic acid (26) (455mg, 2.39 mmol; its synthesis can be found in supporting
information), HATU (955 mg, 2.51 mmol), DIPEA (928 mg, 7.18 mmol) in DCM (10 mL) was
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stirred at rt for 4 h. Volatiles were removed under reduced pressure and the residue was purified
with silicaꢀgel chromatography (10ꢀ20% EtOAc/Heptane) three times to afford the title
compound (403 mg, 51%). ES+ m/z = 331.1 [M+H]+; 1H NMR (400 MHz, Chloroformꢀd) δ 7.84
– 7.77 (m, 2H), 7.09 – 7.00 (m, 2H), 6.88 (d, J = 7.7 Hz, 1H), 4.90 (dd, J = 7.8, 3.6 Hz, 1H), 4.73
(q, J = 2.3 Hz, 2H), 4.04 (qd, J = 6.9, 3.7 Hz, 1H), 3.86 (s, 3H), 1.89 (t, J = 2.3 Hz, 3H), 1.48 (d,
J = 6.9 Hz, 3H).
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(2S,3S)-methyl2-(4-(but-2-ynyloxy)benzamido)-3-(tert-butoxycarbonylamino)butanoate
(28). To a solution of (2S,3S)ꢀmethyl 3ꢀazidoꢀ2ꢀ(4ꢀ(butꢀ2ꢀynyloxy)benzamido)butanoate (400
mg, 1.21 mmol) in MeOH (5 mL) was added SnCl2 (2.30 g, 12.11 mmol). The reaction mixture
was stirred at rt for 3 h. Volatiles were removed under reduced pressure. Then to the residue was
added DCM (10 mL) and aq. sat. NaHCO3 (20 mL) followed by Boc2O (794, 3.64 mmol). The
reaction mixture was stirred at rt for 2 h followed by extracting with DCM (5x). All DCM layers
were combined and dried over anhydrous Na2SO4. The solid was filtered out and the filtrate was
concentrated followed by purification with silicaꢀgel chromatography (10ꢀ25% EtOAc/heptane)
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to afford white solid as the title compound (409 mg, 83%). ES+ m/z = 405.2 [M+H]+; H NMR
(400 MHz, Chloroformꢀd) δ 7.84 (d, J = 8.8 Hz, 2H), 7.64 (d, J = 5.8 Hz, 1H), 7.08 – 6.97 (m,
2H), 5.10 (d, J = 7.8 Hz, 1H), 4.77 (d, J = 4.4 Hz, 1H), 4.72 (q, J = 2.3 Hz, 2H), 4.23 (m, 1H),
3.81 (s, 3H), 1.89 (t, J = 2.3 Hz, 3H), 1.49 (s, 9H), 1.29 (d, J = 6.9 Hz, 3H).
(2S,3S)-methyl 3-amino-2-(4-(but-2-ynyloxy)benzamido)butanoate (29). To a solution of
(2S,3S)ꢀmethyl 2ꢀ(4ꢀ(butꢀ2ꢀynyloxy)benzamido)ꢀ3ꢀ(tertꢀbutoxycarbonylamino)butanoate (480
mg, 1.19 mmol) in DCM (10 mL) was added TFA (1.83 mL, 2.71 g, 23.74 mmol). The reaction
mixture was stirred at rt for 1 h. Volatiles were removed under reduced pressure to afford the
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Journal of Medicinal Chemistry
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residue as the TFA salt of the title compound which was used directly in the next step. ES+ m/z
= 305.2 [M+H]+.
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N-((2S,3S)-3-amino-1-(hydroxyamino)-1-oxobutan-2-yl)-4-(but-2-ynyloxy)benzamide
(30). To the residue obtained from previous step was added MeOH (3 mL) and NH2OH (aq.
50%, 2 mL) and the solution was stirred at rt overnight. White solid can be seen precipitated out.
The solid was filtered, rinsed with water multiple times, then with MeOH, dried under vacuum to
afford the title compound (297 mg, 82%). HRMS: C15H19N3O4 [M+H]+, calc. 306.1448, found
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306.1454; H NMR (400 MHz, DMSOꢀd6) δ 8.14 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.8 Hz, 2H),
7.01 (d, J = 8.8 Hz, 2H), 4.80 (q, J = 2.3 Hz, 2H), 4.60 (bs, 2H), 4.16 (t, J = 7.5 Hz, 1H), 3.13 (q,
J = 6.8 Hz, 1H), 1.83 (t, J = 2.3 Hz, 3H), 1.02 (d, J = 6.5 Hz, 3H).
The synthesis of other compounds described in this manuscript can be found in supporting
information.
Determination of inhibition of LpxC activity (IC50 determination)
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The assay was carried out using the LCꢀMS/MS method as described previously . The LpxC
substrate, UDPꢀ3ꢀOꢀRꢀ3ꢀhydroxydecanoyl)ꢀNꢀacetylglucosamine, was synthesized by the
Alberta Research Council (Alberta, Canada). Briefly, assays were performed in a 96ꢀwell plate
containing PaLpxC (1nM) and LpxC inhibitor (various concentrations initially preꢀincubated for
30 minutes at room temperature prior to the addition of substrate at 2µM). Reactions were
conducted for 20 minutes at room temperature before being quenched with the stop solution
(20% acetic acid). Product formation was quantified using the LCꢀMS/MS method. The IC50
(concentration of inhibitor resulting in half maximal enzyme activity) was determined in XLFit
by fitting the data with the following equations and using curve fit dose response model 205:
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