Peptide deformylase inhibitors with activity against respiratory tract pathogens

Article abstract of DOI:10.1016/j.bmcl.2003.10.013

A series of analogues of the peptide deformylase (PDF) inhibitor BB-3497 where the P3′ amide bond was replaced with a ketone functionality is described. The in vitro antibacterial profiling of these compounds revealed that they demonstrate activity agains

Full text of DOI:10.1016/j.bmcl.2003.10.013

Bioorganic & Medicinal Chemistry Letters 14 (2004) 59–62
Peptide deformylase inhibitors with activity against respiratory
tract pathogens
Stephen P. East,* R. Paul Beckett, Daniel C. Brookings, John M. Clements, Sheila Doel,
Kenneth Keavey, Gilles Pain, Helen K. Smith, Wayne Thomas, Alison J. Thompson,
Richard S. Todd and Mark Whittaker
British Biotech Pharmaceuticals Limited, Watlington Road, Oxford, OX4 6LY, UK
Received 13 August 2003; revised 8 October 2003; accepted 10 October 2003
Abstract—A series of analogues of the peptide deformylase (PDF) inhibitor BB-3497 where the P30 amide bond was replaced with a
ketone functionality is described. The in vitro antibacterial profiling of these compounds revealed that they demonstrate activity
against pathogens associated with respiratory tract infections.
# 2003 Elsevier Ltd. All rights reserved.
The emergence of multi-drug resistant bacterial isolates
from clinical settings has made the search for new clas-
ses of antibacterial agents with novel modes of action of
paramount importance. One of the new targets cur-
rently receiving widespread interest from both academic
and industrial research groups is peptide deformylase
(PDF).1 PDF is an iron-containing metalloenzyme
responsible for the removal of the N-formyl group from
the terminal methionine residue following protein
synthesis in bacteria.2 As deformylation is necessary
Figure 1. PDF enzyme inhibition and antibacterial activity of BB-3497
and the P30 methyl ketone 1.
before further processing of the newly synthesised pro-
tein can occur, PDF is essential for bacterial growth.
This, coupled with the observation that the gene encod-
ing PDF (def) is present in all sequenced pathogenic
bacterial genomes, has made PDF an attractive target
for antibacterial chemotherapy.
amide bond. At the outset, we considered that the
reduced peptidic nature of 1 compared to BB-3497 was an
attractive feature that may offer significant advantages
over more peptidic compounds in terms of metabolic sta-
bility and development potential. In this communication,
we present a focused SAR study on PDF inhibitors con-
taining a ketone group in the P30 position and report
compounds with excellent in vitro antibacterial activity.
Recently, we described a structure–activity relationship
(SAR) analysis of the pseudopeptide PDF inhibitor,
BB-3497 (Fig. 1).3,4 Following this preliminary SAR
evaluation, the P30 methyl ketone analogue (1) of BB-
3497 was selected as a lead compound for a new series
of PDF inhibitors as it showed good inhibitory activity
against the isolated PDF enzyme and retained some
antibacterial activity despite the removal of the P20-P30
From our earlier studies, we had established that the N-
formyl hydroxylamine was the optimum metal-chelating
group on the pseudopeptidic backbone of BB-3497.3
Additionally, we had found that substituents in the P10
position that mimicked the methionine residue in the
natural substrate closely, such as n-butyl and cyclo-
pentylmethyl, provided potent PDF inhibitors that dis-
played promising antibacterial activity.4a Consequently,
Keywords: Peptide deformylase; Inhibitors; Antibacterial.
* Corresponding author at present address: Evotec OAI, 111 Milton
Park, Abingdon, Oxon, OX14 4SD. Tel.: +44-1235-441416; fax:
+44-1235-441351; e-mail: stephen.east@evotecoai.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.013

60
S. P. East et al. / Bioorg. Med. Chem. Lett. 14 (2004) 59–62
we elected to fix these optimised parameters, together
with the t-butyl substituent in the P20 position4b (which
we anticipated might offer some protection to both the
amide and the ketone), in the design of our P30 ketone
series.
Following this study, we were particularly intrigued by
the encouraging microbiological data for the P30 aryl
ketones 7o (4-morpholinophenyl) and 7q (4-fluoro-
phenyl). We recognised that the combination of a fluoro
and an amino substituent on an aromatic ring had been
used to develop SAR in other classes of antibacterials
(notably the oxazolidinones10 and the quinolones11),
with the effect of improving antibacterial activity and/or
reducing metabolic liability. With this in mind, we were
eager to investigate their combined potential as anti-
bacterial fragments in our PDF inhibitors.
To access compounds in this P30 ketone series we
developed convergent routes from either the Weinreb
amide (2) or the pentafluorophenol (Pfp) ester (3) of N-
Boc-tert-Leu (Scheme 1). Thus, Grignard or alkyl/aryl
lithium addition to 2 or 35 provided ketone 4. Removal
of the N-Boc group with HCl/dioxane gave amine 5,
which, in our preferred synthetic route, was coupled to
carboxylic acid 66 (P10=n-butyl or cyclopentylmethyl)
activated using EDC and HOBt. The product from this
reaction (not shown) was then subjected to palladium-
catalysed hydrogenation to reveal the N-formyl hydro-
xylamine 7.7
A series of P30 4-aminoaryl ketones containing 0 (gen-
eral formula 11) and 1 (general formula 12) fluorine
substituent in the aromatic ring were synthesised in
parallel from 8 and 9 respectively using SNAr chemistry
(Scheme 2). Although the route that was used to access
compounds 12 allowed diversification at the final step,
the two-step route used for compounds 11 was preferred
as it suffered from fewer side processes. A series of P30
2-aminoaryl ketones (general formula 13) using the
same set of amines was also prepared via SNAr dis-
placements on the corresponding trifluoroaryl ketone
10.12 All of the compounds prepared by these routes
were purified by preparative HPLC at the final step.
Scheme 1. (i) RLi or RMgBr, Et2O or THF, ꢀ78 ꢁC to rt, 40–85%; (ii)
HCl, 1,4-dioxane, rt, 90–95%; (iii) 6, EDC, HOBt, DMF, 0 ꢁC to rt,
50–75%; (iv) H2, 10% Pd/C, MeOH, rt, 90–95%.
Compounds were tested in an Escherichia coli PDF.Ni
enzyme8 assay and in primary microbiology screens
against in-house strains of E. coli and Staphylococcus
capitis (S. capitis).9 Data from these assays on selected
compounds is recorded in Table 1.
Scheme 2. (i) HNR2, DMSO, 60 ꢁC, 16 h; (ii) 10% Pd/C, cyclohexene,
MeOH, reflux, 4 h; (iii) H2, 10% Pd/C, MeOH, rt; (iv) HNR2, DMSO,
40 ꢁC, 16 h.
An immediate improvement in PDF inhibition and
antibacterial activity was observed when the methyl
group in 1 was replaced with an aromatic or heteroaro-
matic group. Compounds 7a–s show excellent potency
against the isolated PDF enzyme with nearly all exam-
ples inhibiting at levels below 10 nM, irrespective of the
aromatic substituent in the P30 position. Interestingly,
both electron-donating (e.g., 7i) and electron-with-
drawing (e.g., 7r) substituents were tolerated on the aryl
ring without conferring a significant difference in anti-
bacterial activity. Additionally, we concluded that the
cyclopentylmethyl group in the P10 position provided
compounds that demonstrate at least a 2-fold improve-
ment in S. capitis MIC’s compared with the corres-
ponding n-butyl analogues.
Concomitant to this medicinal chemistry programme,
experiments to explore the mechanisms of resistance
against PDF inhibitors were undertaken in our research
group1d and by Margolis et al.13 These studies suggested
that the fmt gene that encodes the enzyme responsible
for N-formylation of methionine prior to protein
synthesis is essential in Streptococcus pneumoniae and
Haemophilus influenzae. Consequently, deformylation is
a necessary process for maturation of proteins in these
pathogens. As S. pneumoniae and H. influenzae are two
of the major pathogens associated with respiratory tract
infections (RTI’s), we concluded that the PDF inhibi-
tors were best suited to combat these types of infection.
Our in vitro screening panel of bacteria was altered to
reflect these observations and the P30 aminoaryl ketones

S. P. East et al. / Bioorg. Med. Chem. Lett. 14 (2004) 59–62
61
were tested against strains of S. pneumoniae, H. influen-
zae, and Moraxella catarrhalis, which is also responsible
for some RTI’s (Table 2).14
slight drop in potency against Gram -ve strains. The
presence of fluorine substituents in the aromatic ring
does not appear to have a detrimental effect on the in
vitro antibacterial profile of these PDF inhibitors.
From these microbiological results, we observe that the
incorporation of a focused series of amines in the 2- or
4-position of the aromatic ketones is well tolerated by
the enzyme. These compounds exert an excellent anti-
bacterial effect against S. pneumoniae, H. influenzae and
M. catarrhalis, although additional basic residues (11c–
13c and 11e–13e) in the amino substituent may cause a
Further to this study, 7o and 13a were progressed for
additional in vitro profiling15 against a panel of 104
organisms (Table 3). These compounds displayed an
improved spectrum of activity compared with BB-3497
with 13a having an antibacterial profile comparable to
amoxicillin-clavulanate (AMC).
Table 1. PDF and in vitro susceptibility profile for P30 ketones
Compds
P10
R
E. coli PDF.Ni IC50 (nM)
E. coli MICa (mM)
S. capitis MICa (mM)
1
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
Me
Ph
2-Pyridyl
2-Furyl
2-Benzofuryl
2-Benzofuryl
4-(OH)Ph
20
3
3
5
4
4
8
2
1
3
8
8
20
4
5
6
3
3
25
100
12.5
50
12.5
1.5
0.7
12
3.1
6.25
1.6
12
3.1
6.25
3.1
3.1
1.5
25
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
7s
12.5
12.5
12.5
6.25
6.25
6.25
6.25
12.5
3.1
6.25
12
6.25
50
6.25
12.5
12.5
3.1
6.25
25
Cyclopentylmethyl
n-Butyl
Cyclopentylmethyl
n-Butyl
Cyclopentylmethyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
n-Butyl
Cyclopentylmethyl
n-Butyl
Cyclopentylmethyl
n-Butyl
Cyclopentylmethyl
4-(OH)Ph
4-(MeO)Ph
4-(MeO)Ph
4-(NH2)Ph
4-(NHCOMe)Ph
4-(NHCOCF3)Ph
4-(NHSO2Me)Ph
4-(morpholino)Ph
4-(morpholino)Ph
4-(F)Ph
4-(F)Ph
4-(CN)Ph
4-(SO2Me)Ph
3.1
3.1
6.25
7
10
a MIC=minimum inhibitory concentration.
Table 2. PDF and in vitro susceptibility profile for P30 aminoaryl ketones
Compds
HNR2
E. coli
PDF.Ni IC50 (nM)
S. pneumoniae (6)
MIC (mg/mL)
H. influenzae (4)
MIC (mg/mL)
M. catarrhalis (3)
MIC (mg/mL)
7o
12a
13a
2
0.6
0.5
0.25–0.5
0.25
0.25–0.5
0.5–1
0.5–4
0.25–2
0.125
<0.125
<0.125
11b
12b
13b
2
2
1
0.25
0.25
0.25–0.5
0.25–2
0.125–2
0.25–2
<0.125
<0.125
<0.125
11c
12c
13c
2
6
2
0.25–0.5
0.25–0.5
0.25–1
0.5–2
0.125–4
2–4
<0.125
0.125
0.125–0.25
11d
12d
13d
7
2
2
<0.125–0.25
0.25
0.25
0.25–2
0.125–2
0.5–2
<0.125
<0.125
<0.125
11e
12e
13e
3
9
2
0.25–0.5
0.25–0.5
0.25–0.5
0.5–2
0.5–8
0.5–4
0.125–0.25
0.125–0.25
0.125–0.25
11f
12f
13f
2
1
2
1–2
0.5–1
0.5–1
0.25–2
0.125–2
0.25–2
<0.125
<0.125
<0.125
Number of strains tested against in parentheses

62
S. P. East et al. / Bioorg. Med. Chem. Lett. 14 (2004) 59–62
Table 3. Extended in vitro susceptibility profile for 7o and 13a
Compds
S. pneumoniae MIC (mg/mL) (40)
H. influenzae MIC (mg/mL) (35)
M. catarrhalis MIC (mg/mL) (29)
90%
Range
90%
Range
90%
Range
BB-3497
7o
13a
Penicillin
Ciprofloxacin
AMC
ꢃ16
2–>8
2
4
2
0.06–8
0.06–ꢃ16
0.5–4
ND
0.004–1
0.12–16
0.12
0.12
0.12
ND
0.06
0.12
0.015–0.12
0.015–0.12
0.03–0.12
ND
0.03–0.06
0.008–0.12
2
1
1
8
1
0.25–2
0.06–2
0.008–2
0.5–128
0.008–1
ND
0.015
1
Number of strains tested against in parentheses.
ND=not determined.
was usually detected as a side product (typically ꢂ10%)
In conclusion we have identified a new series of potent
PDF inhibitors that display in vitro antibacterial activ-
ity against pathogens commonly associated with
respiratory tract infections. Efforts to translate this in
vitro activity to in vivo efficacy are ongoing in our
research laboratories.
in this reaction.
Acknowledgements
6. Pratt, L. M.; Beckett, R. P.; Davies, S. J.; Launchbury,
S. B.; Miller, A.; Spavold, Z. M.; Todd, R. S.; Whittaker,
M. Bioorg. Med. Chem. Lett. 2001, 11, 2585.
7. Full experimental details on individual compounds can be
found in the patent literature. International publication
number: WO 00/61134.
We would like to thank Dr. Ryan Bragg of British Bio-
tech and Dr. Kirk Johnson of Genesoft Inc. for useful
discussions. We also thank Dr. Matthew Reid of Evotec
OAI for help with the interpretation of NMR spectra.
8. The natural ferrous-containing PDF enzyme oxidises
readily to the inactive ferric form. Consequently, for ease
of experimentation, we routinely test against the stable
nickel-containing enzyme that retains full catalytic activity.
9. For details of the PDF inhibition and microbiological
assays see: Clements, J. M.; Beckett, R. P.; Brown, A.;
Catlin, G.; Lobell, M.; Palan, S.; Thomas, W.; Whittaker,
M.; Wood, S.; Salama, S.; Baker, P. J.; Rodgers, H. F.;
Barynin, V.; Rice, D. W.; Hunter, M. G. Antimicrob.
Agents Chemother. 2001, 45, 563.
References and notes
1. (a) Pei, D. Emerging Ther. Targets 2001, 5, 23. (b)
Giglione, C.; Meinnel, T. Emerging Ther. Targets 2001, 5,
41. (c) Yuan, Z.; Trias, J.; White, R. J. Drug Discov.
Today 2001, 6, 955. (d) Clements, J. M.; Ayscough, A.;
Keavey, K.; East, S. P. Curr. Med. Chem.—Anti-Infective
Agents 2002, 1, 239.
10. Barbachyn, M. R.; Ford, C. W. Angew. Chem., Int. Ed.
2003, 42, 2010.
11. Koga, H.; Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T.
J. Med. Chem. 1980, 23, 1258.
12. The SNAr displacement was selective for ortho vs para
substitution to the ketone as verified by 19F NMR
experiments. Only the 2-aminoaryl ketones were isolated
following purification.
13. Margolis, P.; Hackbath, C.; Lopez, S.; Maniar, M.;
Wang, W.; Yuan, Z.; White, R.; Trias, J. Antimicrob.
Agents Chemother. 2001, 45, 2432.
14. All compounds were tested against an fmt mutant strain
of S. aureus and shown to have an MIC >32 mg/mL.
From this result we infer that PDF inhibition is the
mechanism of action.
15. Wise, R.; Andrews, J. M.; Ashby, J. Antimicrob. Agents
Chemother. 2002, 46, 1117.
2. (a) Adams, J. M.; Capecchi, M. Proc. Natl. Acad. Sci.
U.S.A. 1966, 55, 147. (b) Adams, J. M. J. Mol. Biol. 1968,
33, 571.
3. Smith, H. K.; Beckett, R. P.; Clements, J. M.; Doel, S.;
East, S. P.; Launchbury, S. B.; Pratt, L. M.; Spavold,
Z. M.; Thomas, W.; Todd, R. S.; Whittaker, M. Bioorg.
Med. Chem. Lett. 2002, 12, 3595.
4. (a) Davies, S. J.; Ayscough, A. P.; Beckett, R. P.; Bragg,
R. A.; Clements, J. M.; Doel, S.; Grew, C.; Launchbury,
S. B.; Perkins, G. M.; Pratt, L. M.; Smith, H. K.; Spavold,
Z. M.; Thomas, S. W.; Todd, R. S.; Whittaker, M.
Bioorg. Med. Chem. Lett. 2003, 13, 2709. (b) Davies, S. J.;
Ayscough, A. P.; Beckett, R. P.; Clements, J. M.; Doel,
S.; Pratt, L. M.; Spavold, Z. M.; Thomas, S. W.; Whit-
taker, M. Bioorg. Med. Chem. Lett. 2003, 13, 2715.
5. The tertiary alcohol, general formula 14, resulting from
double addition of the metallated species to Pfp ester 3