Peptidomimetic inhibitors of bacterial peptide deformylase

Article abstract of DOI:10.1016/j.bmcl.2011.04.132

A series of N-formyl hydroxylamine peptide deformylase inhibitors containing a cyclic azaamino acid moiety between the P1′ and P3′ substituents are presented. Selected compounds display antibacterial activity against pathogens associated with respiratory

Full text of DOI:10.1016/j.bmcl.2011.04.132

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4032–4035
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Peptidomimetic inhibitors of bacterial peptide deformylase
⇑
Stephen P. East , Andrew Ayscough, Ian Toogood-Johnson, Steven Taylor, Wayne Thomas
Vernalis (R&D) Ltd., Granta Park, Great Abington, Cambridge CB21 6GB, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of N-formyl hydroxylamine peptide deformylase inhibitors containing a cyclic azaamino acid
moiety between the P10 and P30 substituents are presented. Selected compounds display antibacterial
activity against pathogens associated with respiratory tract infections with representative compounds
showing excellent MICs against Haemophilus influenzae.
Received 29 March 2011
Revised 26 April 2011
Accepted 27 April 2011
Available online 11 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Peptide deformylase
Peptidomimetic
Azapeptide
Antibacterial
Metalloprotease
Peptide deformylase (PDF) is an iron-containing metalloenzyme
involved in post-translational modification of nascent polypeptides
in bacterial cells.1 Specifically PDF cleaves the N-formyl group from
the terminal methionine residue following ribosomal protein syn-
thesis. Deformylation is a critical step before further processing of
the newly synthesized protein can occur and the def gene, which
encodes PDF, is present in all sequenced bacteria. Gram-positive
bacteria may have additional functional genes that encode other
PDFs and eukaryotic homologues of PDF have also been identified.
This has raised concerns over possible specificity issues but re-
cently Meinnel and co-workers have demonstrated that selective
inhibitors for bacterial PDF can be identified through exploiting
differences in the S0 binding pocket.2 As such, PDF remains an
interesting molecular target for the development of new antibacte-
rial agents and also antitubercular drugs.3
that links the P10 and P30 positions of our PDF inhibitors. This strat-
egy and indeed attempts to identify non-peptidic PDF inhibitors
has been adopted by others.14–16 For example, in our previous pub-
lication we replaced the P20-P30 amide bond with a ketone and we
found that we could retain good antibacterial activities against
bacterial strains associated with respiratory tract infections
(RTIs).17 This communication describes alternative modifications
to the peptidic backbone of our N-formyl hydroxylamine PDF
inhibitors where the P20 amino acid is replaced with a cyclic azaa-
mino acid, generic formula I. The compounds presented here have
been evaluated for their inhibitory activity against bacterial PDF
isolated from Escherichia coli.
Nature has already provided examples of antibiotics that inhibit
PDF including the peptidic hydroxamic acid actinonin (1).4–6 Addi-
tionally, several groups have reported synthetic hydroxamic acids
or N-formyl hydroxylamines related to the structure of 1 that dis-
play potent antibacterial activity via inhibition of PDF.1,7–9 To date,
these research efforts have culminated in the progression of two
PDF inhibitors into Phase I clinical trials, LBM-415 (2)10,11 and a
compound from our laboratories, BB-83698 (3)12,13 (Fig. 1).
As part of our research effort to identify a back-up to 3 we
sought to mimic the peptidic structure by replacing the amino acid
OH
N
OH
N
O
F
O
O
O
H
N
HO
N+
O
O
H
N
N
H
N
H
O
1
2
P1'
O
n
OH
N
P3'
O
OH
N
H
N
O
O
N
O
N
N
O
N
H
O
H
R
O
⇑
Corresponding author at present address: Evotec (UK) Ltd, 114 Milton Park,
Abingdon, Oxon, OX14 4SA, United Kingdom. Tel.: +44 1235 441735.
P2'
3
I (n=1 or 2)
E-mail address: stephen.east@evotec.com (S.P. East).
This work was performed at British Biotech Pharmaceuticals Limited.
Figure 1. PDF inhibitors
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.132

S. P. East et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4032–4035
4033
n
i, ii
iii
iv
ZHN
n
n
OBn
N
OH
N
NHBoc
HN
NBoc
N
N
O
O
NBoc
NBoc
4
5a (n=1)
5b (n=2)
O
O
H
H
7a (n=1)
8a (n=1)
7b (n=2)
8b (n=2)
v
OBn
N
(n=1)
OH
O
O
H
n
6
OH
N
vi
vii or viii
then ix
n
OBn
N
HN
N
O
O
N
R
N
N
O
.2HCl
N
H
H
H
O
O
10
H
11a-25a (n=1)
11b-25b (n=2)
9a (n=1)
9b (n=2)
Scheme 1. Reagents and conditions: (i) 1,3-dibromopropane, NaH, DMF, rt, 89% (n = 1) or 1,4-dibromobutane, NaH, DMF, rt, 94% (n = 2); (ii) H2, 10% Pd/C, MeOH, rt, 78–100%;
(iii) 6, HATU, DIPEA, DMF 0 °C to rt, 52% (n = 1) or 6, HATU, DIPEA, DMF 0–60 °C, 7% (n = 2); (iv) H2, 10% Pd/C, MeOH, rt, 90%; (v) BF3ꢁEt2O, CH2Cl2, AcOH, rt, 95%; (vi) 6, HATU,
DIPEA, DMF 0 °C to rt, 65%; (vii) acid chloride, DIPEA, CH2Cl2, 0 °C to rt; (viii) isocyanate, EtOAc, rt; (ix) H2, 10% Pd/C, MeOH, rt, yields typically >75% (2 steps).
Bioisosteric replacement of amino acids with azaamino acids
is a peptidomimetic strategy18 which has been applied previ-
ously to a number of protease inhibitor programmes.19 Changing
the asymmetric carbon centre for a nitrogen atom usually cause
minimal disruption to the conformation of the molecule and so
binding affinities of compounds containing an azaamino acid
are expected to be comparable to the corresponding amino acid
containing analogues. Additionally, azaamino acids may be ex-
pected to be more stable, particularly to hydrolytic cleavage by
proteolytic enzymes. We,20 and others,21 reported N-formyl
hydroxylamine and hydroxamic acid PDF inhibitors containing
cyclic amino acids in the P20 position (e.g., 2). As an extension
of this work, we have investigated N-formyl hydroxylamines
containing azaproline (azPro) and azapipcolinic acid (azPip).
From our previous SAR analysis,20 together with available crys-
tallographic information,22 the major point of diversity for this
investigation was the P30 position.
against Gram-positive (Streptococcus pneumoniae) and Gram-nega-
tive bacteria (Haemophilus influenzae and Moraxella catarrhalis)
responsible for RTIs (Table 1).
Most of the compounds examined are potent (sub
lM) PDF
inhibitors indicating that the presence of the azPro or azPip were
tolerated by the enzyme. In general, the IC50’s for 5- or 6-mem-
bered ring P20 matched pairs are broadly similar (typically 1–5ꢀ
difference) suggesting that the enzyme does not have a preference
for either ring size. In line with our previous observations20, the en-
zyme accepts a variety of changes in the P30 position. Compounds
with a 6-membered aryl/heteroaryl ring directly attached to the
0
azPro/Pip carbonyl generally have higher IC50 s (13–17) with the
3-pyridyl substituent (17) being the least-favoured of the modifi-
cations investigated, particularly on the azPip scaffold. In contrast
the 5-membered furan containing compounds (18 and 19) pro-
vided some of the most potent PDF inhibitors in this study. The
0
N-linked analogues (21–25) also show low IC50 s against the
AzPro analogues were synthesized according to the route illus-
trated in Scheme 1. Orthogonally protected hydrazine 4 (prepared
from commercially available N-Boc hydrazine in quantitative yield)
was cycloalkylated using 1,3-dibromopentane and sodium hy-
dride. The Z-protecting group was then selectively removed via
standard hydrogenation conditions and the resulting N-Boc pro-
tected pyrazolidine 5 was coupled to carboxylic acid 623 using
HATU to give intermediate 7a. Deprotection of the O-benzyl group
in 7a provided N-formyl hydroxylamine 8a. Alternatively, the Boc
group in 7a could be removed using boron trifluoride etherate
and the resultant analogue 9a was capped with acid chlorides, to
give compounds 11a–20a or isocyanates to afford compounds
enzyme.
Aliphatic (8, 11, 12, 20) and aryl/heteroaryl (13–19) compounds
demonstrate good antibacterial activity against Gram-positive and
Gram-negative organisms. The compounds containing the azPip
P20 are consistently more active when compared with the corre-
0
sponding azPro compounds, even when the IC50 s are worse, for
example, 13b versus 13a. The explanation for this is not clear but
we can speculate that they are possibly less susceptible to efflux
mechanisms. The most interesting compound in terms of a good
balance between Gram-positive and Gram-negative activity is the
2-furyl derivative 18b.
Compounds 20–25 containing the P20P30 NH display good
21a–25a following O-benzyl deprotection by
a
palladium-
antibacterial activity against S. pneumoniae (MICs 2–16 lg/ml).
catalyzed hydrogenation reaction.
They also show excellent activity against M. catarrhalis and H.
influenzae. Some examples 21, 23 and 24 showed MICs that are
amongst the best PDF inhibitors that we have reported against
H. influenzae.17 The data for the azPro and azPip compounds
was similar.
We also examined the antibacterial activity of two related
hydroxamic acids containing the azPip P20 group with two of the
more interesting P30 substituents. These compounds, 29 and 30,
were prepared according to the synthetic route illustrated in
Scheme 2. Advanced intermediate 27, which was prepared from
26,25 was coupled to 10 using EDAC and HOAt to provide com-
pound 28. Subsequent capping with phenyl isocyanate or 2-furoyl
chloride gave 29 and 30 respectively after acid-promoted removal
of the hydroxamic acid protecting group.
A similar route was investigated for the azPip analogues
however the coupling of 5b with 6 gave compound 7b in an
unacceptably poor yield of <10%. This route did lead to ana-
logue 8b following deprotection of the O-benzyl group but it
did not represent a viable synthesis to investigate the capping
step. Fortunately the unprotected cyclic hydrazine 10, prepared
according to literature procedures,24 underwent coupling with 6
using HATU to provide adduct 9b ready for treatment with acid
chlorides and isocyanates. These reactions were followed by
hydrogenation of the O-benzyl group to give compounds 11b–
25b.
All of the compounds were tested in a PDF inhibition assay and
their minimum inhibitory concentrations (MICs) were determined

4034
S. P. East et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4032–4035
Table 1
PDF IC500s and MICs for compounds 8, 11–25
n
OH
N
N
O
N
O
H
R
O
I
MICb
(lg/ml)
a
Compd
R
n
PDF Ni E. coli IC50 (nM)
S. pneumoniae (4)
H. influenzae (3)
M. catarrhalis (1)
8a
8b
OtBu
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
1
2
30
9
60
200
40
200
70
500
50
100
20
60
60
300
300
40%@1
30
5
10
6
30
60
10
20
7
10
10
20
10
10
30
9
8-16
4–16
8
1–8
8
0.5–2
16
0.5–2
4–16
0.5–4
16
0.5–2
1–4
1–8
0.25–8
4–16
4–8
2–16
1–4
16–32
1–>32
4–8
0.5–2
2–>32
2–8
<0.125
<0.125
0.125
0.25
0.06
0.06
0.06
0.125
0.06
0.5
0.125
0.5
0.125
0.25
0.125
nd
<0.125
<0.125
0.06
<0.03
0.06
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
18a
18b
19a
19b
20a
20b
21a
21b
22a
22b
23a
23b
24a
24b
25a
25b
CH2OMe
Cyclohexyl
Ph
4-MeOPh
4-FPh
0.5–4
16–32
2–16
8
4-Pyridyl
3-Pyridyl
2-Furyl
2–>32
nd
lM
nd
4–8
0.5–2
4
<0.125–1
<0.125–0.25
1–>32
0.125–8
2–16
0.25–2
<0.125–2
<0.125–4
1–2
3-Furyl
0.5–2
4–8
0.5–4
4–16
2–4
8–16
4–8
4–8
2–4
8–16
4–8
2–8
4
Bn
0.25
NHPh
<0.125
<0.125
0.25
<0.03
<0.125
<0.125
<0.125
<0.125
0.06
NH(2-MeOPh)
NH(3-MeOPh)
NH(4-FPh)
NH(4-CF3Ph)
0.5–2
<0.125–2
<0.125–4
<0.125–2
<0.125–4
2–8
0.25–16
<0.125
a
The natural ferrous-containing PDF enzyme oxidises readily to the inactive ferric form. For ease of experimentation, enzyme inhibition was tested routinely using the
stable nickel-containing enzyme, which retains full catalytic activity.
b
Minimum number of strains tested against in parentheses; nd: no data.
Table 2
PDF IC50’s and MICs for hydroxamic acids
ref 25
O
O
Compd PDF Ni E. coli
MICb
S. pneumoniae (4) H. influenzae (3) M. catarrhalis (1)
(lg/ml)
OBn
OH
O
O
a
IC50 (nM)
HO
N
H
O
O
27
29
21b
30
20
20
8
4–8
2–4
2–4
1–4
nd
<0.125
0.06
26
<0.125–4
0.06–0.125
<0.125–0.25
i
18b
5
0.5–2
<0.125
a
The natural ferrous-containing PDF enzyme oxidises readily to the inactive
ferric form. For ease of experimentation, enzyme inhibition was tested routinely
using the stable nickel-containing enzyme, which retains full catalytic activity.
O
O
ii or iii
b
Minimum number of strains tested against in parentheses; nd: no data.
N
N
HO
N
O
O
N
H
N
H
then iv
N
H
O
O
R
O
29 R= NHPh
30 R = 2-furyl
28
The PDF inhibitory activity and antibacterial activity for 29 and
30 are shown in Table 2. Results from these assays suggest that
both the hydroxamic acid and N-formyl hydroxylamine metal
binding group provide similarly potent PDF inhibitors with
comparable antibacterial activity against Gram-positive and
Scheme 2. Reagents and conditions: (i) 10, EDAC, HOAt, DIPEA, CH2Cl2, 0 °C to rt,
50%; (ii) phenyl isocyanate, EtOAc, rt, 100%; (iii) 2-furoyl chloride, DIPEA, CH2Cl2, rt,
91%; (iv) HCl, MeOH, rt, 75%.

S. P. East et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4032–4035
4035
10. Fritsche, T. R.; Sader, H. S.; Cleeland, R.; Jones, R. N. Antimicrob. Agents
Chemother. 2005, 49, 1468.
11. Osborne, C. S.; Neckermann, G.; Fischer, E.; Pecanka, R.; Yu, D.; Manni, K.;
Goldovitz, J.; Amaral, K.; Dzink-Fox, J.; Ryder, N. S. Antimicrob. Agents
Chemother. 2009, 53, 3777.
12. Lofland, D.; Difuntorum, S.; Waller, A.; Clements, J. M.; Weaver, M. K.;
Karlowsky, J. A.; Johnson, K. J. Antimicrob. Chemother. 2004, 53, 664.
13. Ramanathan-Girish, S.; McColm, J.; Clements, J. M.; Taupin, P.; Barrowcliffe, S.;
Hevizi, J.; Safrin, S.; Moore, C.; Patou, G.; Moser, H.; Gadd, A.; Hoch, U.; Jiang, V.;
Lofland, D.; Johnson, K. W. Antimicrob. Agents Chemother. 2004, 48, 4835.
14. Aubart, K.; Zalacain, M. Prog. Med. Chem. 2006, 44, 109.
15. Lee, S. K.; Choi, K. H.; Lee, S. J.; Lee, J. S.; Park, J. Y.; Kim, B. M.; Lee, B. J. Bioorg.
Med. Chem. Lett. 2011, 21, 133.
Gram-negative organisms. This observation is consistent with our
previous findings.26
In conclusion, we have presented a new series of N-formyl
hydroxylamines containing an azPro or azPip residue. Compounds
such as 18b are less peptidic than our previously disclosed PDF
inhibitors and significantly, they retain good enzymatic potency
and the potential for broad-spectrum activity against pathogens
associated with RTIs. The antibacterial activity against Gram-
negative organisms, in particular H. influenzae, is amongst the most
promising data we have reported.
16. Yu, L.; Zhou, W.; Wang, Z. Bioorg. Med. Chem. Lett. 2011, 21, 1541.
17. East, S. P.; Beckett, R. P.; Brookings, D. C.; Clements, J. M.; Doel, S.; Keavey, K.;
Pain, G.; Smith, H. K.; Thomas, W.; Thompson, A. J.; Todd, R. S.; Whittaker, M.
Bioorg. Med. Chem. Lett. 2004, 14, 59.
18. Gante, J. Angew. Chem., Int. Ed. Engl. 1994, 33, 1699.
19. Zega, A. Curr. Med. Chem. 2005, 12, 589.
20. Davies, S. J.; Ayscough, A. P.; Beckett, R. P.; Clements, J. M.; Doel, S.; Pratt, L. M.;
Spavold, Z. M.; Thomas, S. W.; Whittaker, M. Bioorg. Med. Chem. Lett. 2003, 13,
2715.
21. Jain, R.; Sundram, A.; Lopez, S.; Neckermann, G.; Wu, C.; Hackbarth, C.; Chen,
D.; Wang, W.; Ryder, N. S.; Weidmann, B.; Patel, D.; Trias, J.; White, R.; Yuan, Z.
Bioorg. Med. Chem. Lett. 2003, 13, 4223.
22. 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.
23. Hunter, M. G.; Beckett, R. P.; Clements, J. M.; Whittaker, M.; Davies, S. J.;
Pratt, L. M.; Spavaold, Z. M.; Launchbury, S. B. Int. Pat. Appl. WO01010835,
2001.
24. Stetter, H.; Spangenberger, H. Chem. Ber. 1958, 91, 1982.
25. Pratt, L. M.; Keavey, K. N.; Pain, G. D.; Mounier, L. F. Int. Pat. Appl. WO01010834,
2001.
26. 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.
References and notes
1. (a) Pei, D. Emerging Ther. Targets 2001, 5, 23; (b) Clements, J. M.; Ayscough, A.;
Keavey, K.; East, S. P. Curr. Med. Chem. Anti Infect. Agents 2002, 1, 239; (c)
Johnson, K. W.; Lofland, D.; Moser, H. E. Curr. Drug Targets Infect. Disord. 2005, 5,
39; (d) Jain, R.; Chen, D.; White, R. J.; Patel, D. V.; Yuan, Z. Curr. Med. Chem. 2005,
12, 1607; (e) Leeds, J. A.; Dean, C. R. Curr. Opin. Pharmacol. 2006, 6, 445.
2. Boularot, A.; Giglione, C.; Petit, S.; Duroc, Y.; Alves de Sousa, R.; Larue, V.;
Cresteil, T.; Dardel, F.; Artaud, I.; Meinnel, T. J. Med. Chem. 2007, 50, 10.
3. Sharma, A.; Khuller, G. K.; Sharma, S. Expert Opin. Ther. Targets 2009, 13, 753.
4. Chen, D. Z.; Patel, D. V.; Hackbarth, C. J.; Wang, W.; Dreyer, G.; Young, D. C.;
Margolis, P. S.; Wu, C.; Ni, Z. J.; Trias, J.; White, R. J.; Yuan, Z. Y. Biochemistry
2000, 39, 1256.
5. Chu, M.; Mierzwa, R.; He, L.; Xu, L.; Gentile, F.; Terracciano, J.; Patel, M.; Miesel,
L.; Bohanon, S.; Kravec, C.; Cramer, C.; Fischman, T. O.; Hruza, A.; Ramanathan,
L.; Shipkova, P.; Chan, T. M. Tetrahedron Lett. 2001, 42, 3549.
6. Yoo, J. S.; Zheng, C. J.; Lee, S.; Kwak, J. H.; Kim, W. G. Bioorg. Med. Chem. Lett.
2006, 15, 4889.
7. Shi, W.; Duan, Y.; Qian, Y.; Li, M.; Yang, L.; Hu, W. Bioorg. Med. Chem. Lett. 2010,
20, 3592.
8. Lee, S. K.; Choi, K. H.; Lee, S. J.; Suh, S. W.; Kim, B. M.; Lee, B. J. Bioorg. Med. Chem.
Lett. 2010, 20, 4317.
9. Shi, W.; Ma, H.; Duan, Y.; Aubart, K.; Fang, Y.; Zonis, R.; Yang, L.; Hu, W. Bioorg.
Med. Chem. Lett. 2011, 21, 1060.