Structure-activity relationships of the peptide deformylase inhibitor BB-3497: Modification of the P2′ and P3′ side chains

Article abstract of DOI:10.1016/S0960-894X(03)00533-X

Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to both the P2′ and P3′ side chains. Enzyme inhibition and antibacterial activity data revealed that a variety of substituents are tolerated at the P2′ and P3′ positions of the inhibitor backbone. The data from this study highlights the potential for modification at the P2′ and P3′ positions to optimise the physicochemical properties.

Full text of DOI:10.1016/S0960-894X(03)00533-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2715–2718
Structure–Activity Relationships of the Peptide Deformylase
Inhibitor BB-3497: Modification of the P20 and P30 Side Chains
Stephen J. Davies,* Andrew P. Ayscough, R. Paul Beckett, John M. Clements,
Sheila Doel, Lisa M. Pratt, Zoe M. Spavold, S. Wayne Thomas and Mark Whittaker
British Biotech Pharmaceuticals Limited, Watlington Road, Oxford OX4 6LY, UK
Received 26 March 2003; revised 19 May 2003; accepted 22 May 2003
Abstract—Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial
SAR around this lead for modifications to both the P20 and P30 side chains. Enzyme inhibition and antibacterial activity data
revealed that a variety of substituents are tolerated at the P20 and P30 positions of the inhibitor backbone. The data fromthis study
highlights the potential for modification at the P20 and P30 positions to optimise the physicochemical properties.
# 2003 Elsevier Ltd. All rights reserved.
Chemistry
Bacterial peptide deformylase (PDF) belongs to a sub-
family of metalloproteases that catalyse the removal of
the N-terminal formyl group from newly synthesised
proteins.1 PDF is essential in prokaryotes and con-
served throughout the eubacteria. It is therefore con-
sidered an attractive target for developing new
antibacterial agents.2,3
The preceding paper11 described the approach used in
identifying the optimum distance between the metal
binding group and the P10 sidechain, together with the
preferred P10 side chains for PDF enzyme affinity and
antibacterial activity. The n-butyl substituent at the P10
position was optimal for enzyme activity and it was the
common intermediate 4, detailed in Scheme 2, that was
used in exploring the effect of sequential modification of
substituents at the P20 and P30 positions. We were able
to make use of a precursor previously reported in the
asymmetric route to BB–349712 to access 4.
We reported previously4 that the N-formyl hydroxyl-
amine BB-3497 (Fig. 1) is an effective inhibitor (IC50=7
.
nM) of the Escherichia coli PDF Ni enzyme, exhibiting
potent antibacterial activity both in vitro and in vivo. In
a subsequent communication,5 we have reported the
structure-based design around the metal binding group.
We now wish to report on the systematic modification
of the P20 and P30 side chains indicated in Figure 1,
describing the synthetic approach to this work and the
biological data that resulted fromthe screening process.
X-ray crystallographic data6ꢀ10 had revealed that these
regions of the PDF enzyme would tolerate a diverse
range of substitution and hence it was our intention to
explore the effect of introducing a variety of sterically
demanding and/or polar substituents at the P20 and P30
positions to direct the optimisation of this class of
inhibitor.
*Corresponding author. Tel.: +44-1235-441732; fax: +44-1235-
441509; e-mail: stephen.davies@evotecoai.com
Figure 1. BB-3497 structure–activity relationships.
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00533-X

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S. J. Davies et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2715–2718
Compounds with modified P20 substituents were pre-
pared by coupling the appropriate amino acid–NMe2
derivative 3 (Scheme 1) with 2-[(benzyloxy-formyl-
amino)-methyl]-hexanoic acid 4 as detailed in Scheme 2.
synthesis approach gave the amide compound 10. Cat-
alytic hydrogenation of the Cbz group provided the P30
amide analogue 11 without prior purification. Sub-
sequent peptide coupling conditions of 11 to carboxylic
acid 4 gave compound 7, as shown in Scheme 2.
Removal of the O-benzyl protecting group by Pd-cata-
lysed hydrogenation provided the N-formyl hydroxyl-
amines 8a–n, as detailed in Table 2. Purification of 8a–n
was achieved by preparative HPLC.
A series of benzyloxycarbonyl (Cbz) protected l-amino
acids (1) was coupled with dimethylamine, via a parallel
synthesis approach using standard peptide coupling
conditions (Scheme 1). The unpurified amides (2) were
then subjected to Pd-catalysed hydrogenation to give
the required l-amino acid–NMe2 (3) in quantitative
yields.
Standard peptide coupling conditions of 3 with inter-
mediate 4 followed by catalytic hydrogenation, afforded
the N-formyl hydroxylamine derivative 6 (Scheme 2). A
comprehensive list of amino acids investigated is shown
in Table 1.
Similarly, for modification of the P30 substituent
(Scheme 3), Cbz-l-tert-leucine 9 was treated with a
variety of secondary amines as detailed in Table 2.
Standard peptide coupling conditions in a parallel
Scheme 3. (i) Secondary amine, EDC, HOAt, DMF, 60–85%; (ii) H2,
Pd/C, MeOH, quant.
Results and Discussion
All compounds were screened for activity against the
PDF.Ni enzyme and selected Gram-positive and Gram-
negative bacterial strains.13
The majority of substituents investigated at the P20
position (6a–w) are reasonably well tolerated (Table 1).
This correlates with recent X-ray crystallographic data4
of BB-3497 suggesting that the P20 side chain is mostly
solvent exposed and makes little contact with the PDF
enzyme. Incorporation of certain polar groups, (6p–q,
6u) results in a 30-fold reduction in enzyme activity but
the arginine residue (6r) offers some potential for
enhancing the physicochemical properties of the inhibi-
tors through the introduction of appropriate polar resi-
dues. It is, however, noteworthy that the tert-leucine of
BB-3497 and the related S-methyl penicillamine (6h)
showed the best in vitro enzyme inhibition and anti-
bacterial profile.
Scheme 1. (i) Me2NH, EDC, HOAt, DMF, 68–95%; (ii) H2, Pd/C,
MeOH, quant.
A variety of sterically demanding groups are tolerated
at the P30 position, suggesting little interaction with the
enzyme (Table 2). Replacement of the dimethylamido
functionality of the P30 side chain (BB-3497) revealed
that the methyl groups can be linked to form a five- or
six-membered ring (8d–j) which can be further fused to
a second ring (8l–m). Replacement of the amide func-
tionality14 by an ester (8a) or methyl ketone (8c) was
tolerated but acidic or basic group incorporation caused
a modest reduction in PDF enzyme activity (8a vs 8b
and 8f vs 8g).
In this study, modifications to the P20 and P30 side
chains have not demonstrated an improved micro-
biological profile over BB-3497. Indeed, compounds
Scheme 2. (i) 3a–w, EDC, HOAt, DMF, 50–75%; (ii) 11a–n, EDC,
HOAt, DMF, 60–80%; (iii) H2, Pd/C, MeOH, then purification by
preparative HPLC.

S. J. Davies et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2715–2718
2717
Table 1. PDF enzyme inhibition and antibacterial activity of BB-
3497 and its P20 analogues
with similar PDF activity often had a markedly different
antibacterial profile (e.g., 6k vs 6n), highlighting the
significant activity profile seen for BB-3497. However,
the results suggest the potential for modification at the
P20 and P30 positions to optimise physicochemical
properties.3
Modifications adjacent to the metal binding group and
to the n-butyl substituent are limited due to the steric
requirements for binding the active site metal and size of
the hydrophobic P10 pocket.10,11 Substituents that
closely mimic the methionine of the substrate provide
optimal PDF enzyme inhibition. In contrast, a range of
substituents and functional groups are tolerated by
PDF at the P20 and P30 positions. This observation is in
line with crystallographic data that suggest these groups
are largely exposed to solvent. These structural studies
have also highlighted the importance of the backbone
amide bonds for enzyme inhibition through hydrogen
bonding interactions with the enzyme.
Compd
P20 amino acid
(NH-R2-CO)
E. coli
.
PDF Ni
IC50 (nM)
E. coli
MIC (mM) MIC (mM)
S. capitis
6a
6b
6c
6d
6e
6f
Gly
Ala
Val
Leu
Cha
Ile
100
40
20
20
30
20
7
50
25
25
50
100
25
12.5
100
25
200
100
>200
100
25
50
100
12.5
BB-3497 t-Leu (S–isomer)
25
>200
25
6g
6h
6I
6j
6k
6l
6m
6n
6p
6q
6r
6s
6t
6u
6v
6w
t-Leu (R-isomer)
Pen(SMe)
Cys(Bn)
Ser
Val(b-OH)
Val(b-OMe)
Asp(b-Bn)
Glu(b-Bn)
Lys
Lys(e-NMe2)
Arg
Phe
Phe(4-Cl)
Tyr
40
6
70
50
20
20
100
20
200
300
20
10
50
300
80
400
12.5
50
25
In addition to PDF inhibition, there are clearly a range of
factors to consider in identifying an agent with a suitable
antibacterial profile. Factors that affect drug distribution
such as bacterial membrane permeability, efflux and
metabolism are all clearly important in the identification
of a compound as a new class of antibiotic.
12.5
25
>200
>200
>200
>200
>200
100
>200
>200
>200
>200
100
25
200
>200
>200
50
50
In conclusion, a series of BB-3497 analogues with
modifications along the pseudopeptide backbone have
been synthesised. The results indicate a preference for
the tert-butyl group at P20 and for further developing
the P30 group where the P30 benzylpiperidinyl sub-
stituent (8j) enhances Gram-positive activity. This has
been a focus of our continuing programme and will be
reported in due course.
100
>200
>200
50
l-Tic
Pro
S. capitis, Staphylococcus capitis.
Acknowledgements
Table 2. PDF enzyme inhibition and antibacterial activity of BB-
3497 and its P30 analogues
The authors wish to thank Drs. Stephen East and Gilles
Pain for support and contributive discussions.
References and Notes
Compd
P30 (R3)
E. coli
.
E. coli
S. capitis
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PDF Ni MIC (mM) MIC (mM)
IC50 (nM)
BB-3497
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
NMe2
OMe
OH
7
30
80
20
10
20
50
10
10
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20
30
40
9
12.5
50
>200
25
200
>200
100
25
25
50
50
25
25
6.25
100
50
50
100
Me
25
25
25
25
Pyrrolidin-1-yl
Morpholin-4-yl
4-Me-piperazin-1-yl
4-Me-piperidin-1-yl
4-Ac-piperidin-1-yl
4-EtO2C-piperidin-1-yl
4-Bn-piperidin-1-yl
N(Me)c-Hexyl
100
100
>200
200
>200
>200
50
Decahydroquinoliny-1-yl
Tetrahydroquinolin-1-yl
N(Bn)CH2CH2Ph
90
>200

2718
S. J. Davies et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2715–2718
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R. A.; Clements, J. M.; Doel, S.; Grew, C.; Launchbury,
S. B.; Perkins, G. M.; Pratt, L. M.; Smith, H. K.; Spavold,
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13. PDF inhibition assays and MICs were performed as
described previously.4
14. The methyl ester analogue 8a was prepared fromester-
ification of Cbz-l-tert-leucine using diazomethane in di-
chloromethane. Saponification at the final stage of the
synthesis afforded the carboxylic acid 8b. The ketone com-
pound 8c was prepared by MeLi addition to the correspond-
ing Weinreb amide.