Acylprolinamides: A new class of peptide deformylase inhibitors with in vivo antibacterial activity

Article abstract of DOI:10.1016/j.bmcl.2012.04.086

A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.

Full text of DOI:10.1016/j.bmcl.2012.04.086

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4028–4032
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Acylprolinamides: A new class of peptide deformylase inhibitors with
in vivo antibacterial activity
Jeffrey M. Axten a, , Jesús R. Medina a, Charles W. Blackledge a, Céline Duquenne a, Seth W. Grant a,
⇑
Mark A. Bobko a, Tony Peng a, William H. Miller a, Theresa Pinckney a, Timothy F. Gallagher a, Swarupa
Kulkarni a, Thomas Lewandowski a, Glenn S. Van Aller a, Rimma Zonis a, Paris Ward b, Nino Campobasso b
a Infectious Diseases Center of Excellence for Drug Discovery, Antibacterial Discovery Performance Unit, GlaxoSmithKline, Collegeville, PA 19426, USA
b Computational and Structural Chemistry, GlaxoSmithKline, Collegeville, PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization
of blood stability and potency provided compounds with improved pharmacokinetics that were suitable
for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in
two respiratory tract infection models.
Received 6 March 2012
Revised 16 April 2012
Accepted 17 April 2012
Available online 25 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Peptide deformylase
PDF inhibitor
Antibacterial
Antibiotic
The emergence of drug resistant bacteria continues to hamper
the effectiveness of existing antibacterial agents. Demand for
new classes of antibiotics to fight resistant bacteria stimulated a
surge in research over the last 15 years to discover and develop
antibiotics with new modes of action to treat infections resistant
to common therapies. Drugs interfering with bacterial protein syn-
thesis are proven and effective in treating hospital and community
bacterial infections.1 Therefore, unexploited protein synthesis
machinery is attractive as a target for drug discovery and can offer
advantages in avoiding resistance in the clinic. Peptide deformyl-
ase (PDF) is an essential bacterial metalloenzyme that catalyzes
the hydrolytic removal of the N-formyl group from the terminal
methionine during protein maturation.2 Although bacterial PDF
was identified more than 40 years ago,3 it was not until the late
1990s that a stable preparation of the native enzyme was available,
which led to assay development, screening and subsequent reports
F
O
N+
-O
H
O
O
F
O
O
N
HN
N
O
O
O
O
H
N
N
N
O
N
H
N
H
N
H
N
H
N
N
H
N
OH
N
O
OH
OH
BB-83698
(Phase I)
LBM-415
(Phase I)
GSK1322322
(Phase II)
Figure 1. Structures of PDF inhibitors dosed in the clinic.
⇑
Corresponding author. Tel.: +1 610 917 7899.
E-mail address: jeffrey.m.axten@gsk.com (J.M. Axten).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.086

J. M. Axten et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4028–4032
4029
O
O
of a variety of inhibitor chemotypes and preclinical candidates.4
The PDF inhibitors BB-83698,5 LBM-415,6 and GSK13223227
(Fig. 1) have advanced to clinical trials.
O
R
N
H
HO
O
HN
OBn
O
O
In the present Letter, we describe our discovery and evaluation
of a series of PDF inhibitors based on an acylprolinamide template,
as exemplified by structures 3–5 (Scheme 1).
1
N
N
H
OH
b, f
a
The syntheses of compounds 3–5 is shown in Scheme 1.8 The
N-hydroxyformamide metal chelating functionality, and P10
cyclopentyl methyl groups were selected based on historical
SAR.5–7 The key intermediate prolinamide 2 was prepared by stan-
3
O
H2N
O
O
O
RO
O
N
N
H
dard coupling of the previously reported acid 19 with
L
-prolina-
c, f
HN
O
O
OBn
mide. The imides
3 and acylcarbamate derivatives 4 were
N
N
OH
H
2
prepared by acylation of the primary carboxamide with the corre-
sponding acid chloride or chloroformate. The acylation can be car-
ried out conveniently and efficiently by premixing the reactants in
THF, and slowly adding 2–2.2 equiv of LHMDS at low temperature.
Deprotection of the benzyl group afforded the N-hydroxyforma-
mide 3 or 4. The use of unreduced Pd/BaSO4 is critical in most cases
to minimize over-reduction to the formamide.10 The acylurea ana-
logs were prepared by several methods. Acylureas are accessible by
substituting carbamoyl chlorides for acid chlorides as described
above. When the isocyanate is available, heating with 2 neat or
in toluene provides the acylureas. Subsequent deprotection of the
benzyl group provided the N-hydroxyformamides 5.
d or e, f
g,h,i
4
O
O
RNH
PhO
O
O
HN
HN
O
O
O
O
j
N
N
OH
H
N
N
OH
H
5
6
Scheme 1. (a) L-prolinamide, EDCI, HOBt, NMM, DMF; (b) R(C@O)Cl, LHMDS, THF,
ꢀ78 °C; (c) RO(C@O)Cl, LHMDS, THF, ꢀ78 °C; (d) R1R2 N(C@O)Cl, LHMDS, THF,
ꢀ78 °C; (e) RNCO, toluene, 120 °C; (f) H2, Pd/BaSO4 (unreduced), MeOH; (g) H2, Pd/
C, MeOH; (h) TBDMSCl, imidazole, DMF; (i) PhO(C@O)Cl, LHMDS, THF, ꢀ78 °C, then
1 N HCl; (j) RNH2, MeOH.
A higher throughput synthesis of acylureas, eliminating the use
of isocyanates and the final hydrogenation step, was developed
from compound 6 which can be easily prepared on large scale
Table 1
aPDF enzyme activity and antibacterial activity of acylprolinamides
Entry
R1
PDF IC50 (nM)
MIC (lg/mL)
S. a.
S. p.
H. i.
S. a.
S. p.
H. i.
M. c.
E. f.
Oxford
1629
Q1
1502
7
3a
3b
3c
3d
3e
3f
3g
3h
3i
CH3–
12
8
5
4
10
5
4
1
1.5
0.8
1.3
0.7
1.2
0.43
0.5
16
13
10
7
18
9
1
4
2
1
2
4
4
2
1
2
0.25
0.125
0.5
0.5
2
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
1
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
60.06
8
16
4
2
4
8
2
1
1
1
4
2
4
1
1
1
0.5
2
>64
4
4
2
4
1
1
1
2
0.5
0.5
2
2
8
>64
4
8
CH3OCH2–
(CH3)2CH–
(CH3)2CHCH2–
(CH3)3C–
Cyclobutyl–
Cyclopropyl–
Cyclopentyl–
Cyclohexyl–
Phenyl–
CH3–
CH3CH2–
(CH3)2CH–
(CH3)3C–
Cyclobutyl–
Cyclopentyl–
Cyclohexyl–
Pyran-4-yl–
Piperidine-4-yl–
H
1
1
1
1
2.3
2.5
3.7
5.2
4.5
2.2
2
3.4
2.4
1.4
1.5
4.4
5.9
2.2
2.1
2
5.5
6.1
5.2
3.8
13
7.7
4.7
3.2
5.8
3.1
2.3
3.1
2.7
4.8
6.1
4.9
3
0.5
0.5
1
1
1
0.5
0.25
1
0.5
0.5
0.25
1
>64
2
0.5
0.5
1
0.125
0.25
0.5
0.25
0.25
0.25
1
0.5
0.25
0.25
0.25
0.125
0.125
0.25
8
3j
0.28
1.9
0.5
4a
4b
4c
4d
4e
4f
4g
4h
4i
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.125
0.25
0.25
0.25
0.5
1
1
0.5
16
0.54
0.48
0.52
0.51
0.26
0.3
0.66
1.4
0.91
0.46
0.44
0.31
0.22
0.3
2
1
1
1
0.125
CH3–
CH3CH2–
0.25
0.5
2
1
0.5
2
4
2
4
8
(CH3)2CH–
(CH3)3C–
Cyclobutyl
Cyclopentyl
Cyclohexyl
PhCH2–
1.6
1.2
0.9
2.4
1.8
2
1
0.125
0.125
0.125
0.125
0.125
60.06
0.25
1
1
1
0.5
1
0.125
0.5
0.5
0.5
60.06
0.5
0.5
2
0.52
0.1
0.2
0.93
1.7
0.86
1
4
0.19
0.12
0.19
2.1
0.35
0.37
0.35
0.41
6.5
3.6
4.1
20
4.4
4.7
5.9
6.1
5j
5k
5l
5m
5n
5o
5p
Ph(CH2)2–
Ph(CH2)3–
H2C@CHCH2–
4-pyridinyl-(CH2)2–
3-pyridinyl-(CH2)2–
2-pyridinyl-(CH2)2–
2-pyraziny-(CH2)2–
10
1
2
2
1
1.7
1.4
1.4
1.4
2
1
2
2
(continued on next page)

4030
J. M. Axten et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4028–4032
Table 1 (continued)
Entry
R1
PDF IC50 (nM)
MIC (lg/mL)
S. a.
S. p.
H. i.
S. a.
S. p.
H. i.
M. c.
E. f.
Oxford
1629
Q1
1502
7
5q
5r
5s
5t
HO(CH2)2–
HO(CH2)3–
H2N(CH2)2–
H2NC(O)(CH2)2–
1H-imidazole-4-yl-(CH2)2–
3.8
3.2
2.3
3.3
1.3
1.1
8.8
6.9
7
6.3
4.3
4
4
16
32
64
2
2
16
8
0.5
0.125
0.125
2
0.5
0.5
32
16
>64
>64
>64
0.77
0.38
0.72
0.15
1
>64
2
5u
8
16
bS. aureus, S. pneumoniae or H. influenzae PDF activity was measured as previously described.13 Enzyme activity was measured in a coupled assay system where formate
released from substrate is oxidized by formate dehydrogenase thereby reducing one molecule of NAD–NADH resulting in an increase in absorbance at 340 nM. Enzyme
reactions were initiated by adding PDF to 96 well plates containing all other assay components in a total volume of 50 lL. For S. aureus and H. influenzae PDF, final reaction
conditions were 50 mM potassium phosphate (pH 7.6), 5 units/ml FDH, 7 mM NAD, 5% DMSO, 2 nM enzyme, and Km concentrations of fMAS peptide. Conditions for S.
pneumoniae PDF were identical except that 2 mM NAD and 33 pM enzyme were used. Reaction velocities were measured for 20 min at 25 °C in a Molecular Devices
SpectraMax plate reader. Data reported is the average of at least 2 runs.
cWhole-cell antimicrobial activity was determined by broth microdilution using the National Committee for Clinical Laboratory Standards (NCCLS) recommended procedure
(Document M7-A4). The compound was tested in serial two-fold dilutions ranging from 0.06 to 64 mcg/ml. The minimum inhibitory concentration (MIC) was determined as
the lowest concentration of compound that inhibited visible growth. A mirror reader was used to assist in determining the MIC endpoint.
Figure 2. Crystal structure of 3j (shown as yellow stick model) bound to S. pneumoniae PDF: (a) View of active site highlighting hydrogen bonding of the acylprolinamide; (b)
view showing surface channel occupied by phenyl group of the imide.
without the need for chromatography (Scheme 1). The prolinamide
2 was converted to the TBDMS protected N-hydroxyformamide,
and acylated with phenyl chloroformate to provide 6 after acidic
aqueous workup. Since the acylureas can be easily prepared from
6 by mixing with an amine in suitable solvent, this was our pre-
ferred synthetic route for acylurea analog preparation.
accept a wide range of functionality of various sizes (see Table
1). Consequently, we directed our research to the optimization of
the physical properties and pharmacokinetics by modifying the
group that projects into this channel.
Imides 3a–j generally have poor oral availability and very high
clearance in rats, consistent with our finding that they are not
The imides prepared were found to be very potent PDF inhibi-
tors. Table 1 illustrates a range of substitution that is tolerated.
The acetyl imide 3a has the weakest potency, with IC50s for the
Staphylococcus aureus and Streptococcus pneumoniae PDF enzymes
of 12 and 4 nM, respectively. As the alkyl group of the acyl imide
is extended and branched the activity slightly improves, however
the tert-butyl analog 3e exhibits a slight loss in activity with a cor-
responding increase in MIC. The cycloalkyl and phenyl analogs 3f–j
generally are more potent enzyme inhibitors, and have favorable
antibacterial activity against S. aureus and S. pneumoniae.
An X-ray crystal structure of 3j bound to S. pneumoniae PDF was
solved at 1.8 Å resolution (Fig. 2).11 As shown in Figure 2a, the N-
hydroxyformamide is bound to the active site metal (Ni2+) and
the cyclopentylmethyl group fills the P10 pocket. The proline imide
carbonyl interacts with the protein through hydrogen bonding to a
conserved water and the OH of Y166, and the imide NH is engaged
in hydrogen bonding with the carbonyl of a backbone glycine. The
phenyl moiety occupies a large surface channel (Fig. 2b) that can
Table 2
aBlood stability data for acylprolinamide derivatives in rat and human blood
Entry
% recovery @ 2 h
Rat
Human
3e
3j
4c
4d
4e
4f
4g
4h
5c
5d
5g
5j
29.0 21.0
27.2 10.5
80.6 7.3
NT
0
49.6 14.4
52.0 10.4
31.7 15.3
57.5 10.0
45.8 6.4
23 16.9
110.9 15.3
99.7 1.5
103 11.3
92.2 9.4
27.6 31.6
0
0
0.4 39.1
9.5 18.6
58.6 19.4
NT
NT
NT
a
Samples (n = 3) were spiked into fresh human blood to yield target concen-
trations of 2000 ng/mL and incubated at 37 °C for 120 min. NT = not tested.

J. M. Axten et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4028–4032
4031
Table 3
Pharmacokinetic data (n = 3) for compound 5c
i.v.
p.o.
(ng.h/mL/mg/kg)
Dose (mg/kg)
Clp (ml/min/kg)
Dose (mg/kg)
DNAUCa
%F
(0–inf)
Rat
Dog
Monkey
2.5
4.5
3.0
119.9 14.8
36.3 4.1
29.4 3.3
27.1
6.5
5.1
43.3 0.6
629.9 46.9
111.4 42.1
ꢁ30b
100 ND
20
9
a
Clp = plasma clearance; DNAUC = dose normalized AUC; F = oral bioavailability.
Estimated from a non-crossover study.
b
10
antibacterial activity varies and is likely related to changes in lipo-
philicity, with more polar analogs exhibiting much higher MICs.
For example, primary alcohols (5q and 5r) have slightly diminished
antibacterial activity, whereas the more polar amine and imidazole
derived analogs (4i, 5s–u) are significantly less active in the whole
cell assay even though they maintain potent enzyme activity. This
might be due to decreased permeability or increased bacterial
efflux.
Compound 5c was selected for in vivo studies based on its blood
stability and robust activity in broader antibacterial profiling. Phar-
macokinetic data is shown in Table 3.12 In rats, Compound 5c has
high clearance and 30% bioavailability. The high clearance impacts
the exposure indicated by the relatively low DNAUC. In dogs and
monkeys, compound 5c has good exposure and exhibits moderate
to high clearance at low doses, with 100% and 20% bioavailability,
respectively.
(a)
9
8
7
6
5
4
3
2
1
0
Limit of
detection
Amox/Clav
350/50 mg/kg
75 mg/kg
Compound 5c
Controls
37.5 mg /kg
150 mg/kg
The efficacy of acylurea 5c was evaluated in rat respiratory tract
infection models.12 Oral b.i.d. dosing (4 days) resulted in a reduc-
tion of S. pneumoniae 1629 bacterial lung counts, with decreases
as high as 3.86log10 cfu/mL at the 75 and 150 mg/kg doses com-
pared to the non-treated control (Fig. 3a). Additionally, compound
5c was effective against H. influenzae H-128 when dosed b.i.d. for
2 days at a dose of 300 mg/kg, reducing bacterial counts by
4.21log10 cfu/mL (Fig. 3b).
In summary, we have discovered a new class of PDF inhibitors
containing an acylprolinamide functionality. Lead compounds
from this class have potent, broad spectrum antibacterial activity
against major pathogens prevalent in community acquired bacte-
rial infections. Adjusting the electrophilicity to enhance the blood
stability of the acylprolinamide functionality led to acylureas,
which have desirable physiochemical properties and pharmacoki-
netics. In vivo efficacy was demonstrated with compound 5c in
rat lung infection models of S. pneumoniae and H. influenzae, high-
lighting the utility of the acylprolinamide class of PDF inhibitors as
antibacterial agents.
9
8
7
6
5
4
3
2
1
0
(b)
Limit of
detection
Control
Levofloxacin Compound 5c
100 mg/kg 300 mg/kg
Figure 3. N = 6 for each group. (a) Efficacy of compound 5c (MIC = 0.25
amoxicillin/clavulinic acid (MIC = 0.03
ratory tract infection model. Compounds were dosed orally b.i.d. for 4 days starting
1 h post-infection; (b) efficay of compound 5c (MIC = 1 g/mL) and Levofloxacin
(MIC = 0.016 g/mL) in an H. influenzae H-128 rat respiratory tract infection model.
lg/mL) and
l
g/mL) in a S. pneumoniae 1629 rat respi-
l
l
Compounds were dosed orally b.i.d. for 2 days starting 1 h post-infection.
References and notes
stable in blood (Table 2). We suspected that the susceptibility of
imides to degradation by esterases and amidases was the primary
source of this instability. We reasoned that the blood stability
might be improved if the imide carbonyl was less electrophilic.
This led to the synthesis of the acylcarbamates 4 and acylureas 5.
Although there was little improvement in stability of the acylcar-
bamates 4, there was a dramatic improvement in blood stability
of the acylureas 5, particularly in human blood, where >89% of
compounds remained after a 2 h incubation.
The acylcarbamates and acylureas maintained good antibacte-
rial potency and spectrum. The acylcarbamates 4a–i have slightly
increased potency in the enzyme assay, but display remarkable
improvement in antibacterial activity, particularly against S. pneu-
moniae 1629 (see Table 1). The acyclcarbamates 4a–h and acylure-
as 5a–p also display very potent enzyme inhibition, generally
<10 nM, coupled with potent antibacterial activity against S. aur-
eus, S. pneumoniae, Haemophilus influenzae and Moraxella catarrhal-
is. Although target potency is similar amongst the compounds, the
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11. At 15.5 mg/ml, Streptococcus pneumoniae PDF was complexed with 1 mM 3j for
16 h at 4 °C, drops were applied using the mosquito and grown over a period of
2–8 days at 22 °C in a 400 nL hanging drop. The drop was composed of 200 nL
of the protein/inhibitor complex, 50 nL of previously grown crushed crystals
(seeds suspended in ꢁ3.3 M AmSO4, 3% Peg 400 and 0.1 M Hepes pH 7.5) from
apo Streptococcus pneumoniae PDF and 150 nL well composition 0.2 M
Potassium fluoride and 20% (w/v) PEG 3350. X-ray diffraction data were
collected at the Advanced Photon Source on sector 21f, the Life Sciences
Collaborative Access Team. The structure was solved by molecular replacement
and refined with Phenix. Refine (Adams, P. D.; Grosse-Kunstleve, R. W.; Hung,
L.-W.; Ioerger, T. R.; McCoy, A. J.; Moriarty, N. W.; Read, R. J.; Sacchettini, J. C.;
Sauter, N. K.; Terwilliger, T. C.. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2002,
58, 1948–1954). The model was built with COOT (‘Coot: model-building tools
for molecular graphics’ Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 2004, 60, 2126–2132 Part 12). Figures were made with Pymol
(DeLano, Warren L. The PyMOL Molecular Graphics System, 2009. DeLano
Scientific LLC, Palo Alto, California, USA. http://www.pymol.org). X-ray data
were submitted to the Protein Data Bank (PDB ID = 4EOX).
12. All studies were conducted after review by the GSK Institutional Animal Care
and Use Committee and in accordance with the GSK Policy on the Care, Welfare
and Treatment of Laboratory Animals.
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