Asymmetric synthesis of BB-3497 - A potent peptide deformylase inhibitor

Article abstract of DOI:10.1016/S0960-894X(01)00509-1

By screening a library of metalloenzyme inhibitors, the N-formyl-hydroxylamine derivative BB-3497 was identified as a potent inhibitor of Escherichia coli peptide deformylase with antibacterial activity both in vitro and in vivo. The homochiral synthesis of BB-3497, involving a novel asymmetric Michael addition reaction is described.

Full text of DOI:10.1016/S0960-894X(01)00509-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2585–2588
Asymmetric Synthesis of BB-3497—A Potent Peptide
Deformylase Inhibitor
Lisa M. Pratt,* R. Paul Beckett, Stephen J. Davies, Steven B. Launchbury,
Andrew Miller,y Zoe M. Spavold, Richard S. Todd and Mark Whittaker
British Biotech Pharmaceuticals Limited, Watlington Road, Cowley, Oxford OX4 6LY, UK
Received 18 April 2001; revised 26 June 2001; accepted 18 July 2001
Abstract—By screening a library of metalloenzyme inhibitors, the N-formyl-hydroxylamine derivative BB-3497 was identified as a
potent inhibitor of Escherichia coli peptide deformylase with antibacterial activity both in vitro and in vivo. The homochiral
synthesis of BB-3497, involving a novel asymmetric Michael addition reaction is described. # 2001 Elsevier Science Ltd. All rights
reserved.
Introduction
There have been several reports describing inhibitors of
Escherichia coli peptide deformylase,4À11 but few of
All ribosome-mediated synthesis of proteins starts with
a methionine residue. In bacteria and the organelles of
eukaryotes, the amino group of the methionyl moiety
carried by the initiator tRNAfMet is N-formylated by
formyltransferase prior to its incorporation into a poly-
peptide.1 This is thought to be required for proper protein
synthesis initiation. Consequently, N-formylmethionine
is always present at the N-terminus of a nascent bacter-
ial polypeptide. However, in bacteria most mature pro-
teins donot retain the N-formyl group or the terminal
methionine residue. Following translation, the formyl
group is hydrolysed by the metalloenzyme peptide
deformylase (PDF, EC 3.5.1.31), which is necessary
before further processing at the N-terminus by methio-
nine aminopeptidase (EC 3.4.11.18) can take place.
Deformylation is therefore a crucial step in bacterial
protein biosynthesis and PDF is essential for bacterial
growth. The gene encoding PDF (def ) is present in all
sequenced pathogenic bacterial genomes, making it an
attractive target for antibacterial chemotherapy.2 It has
been generally accepted that eukaryotic organelles do
not have a deformylase activity. However, very recently
a human PDF homologue was identified which is pre-
sumed to be involved in deformylation in the mito-
chondrion.3 The function and sub-cellular location of
this putative gene product have yet to be confirmed.
these possess significant antibacterial activity. The
naturally occurring antibiotic actinonin, 1, for which the
molecular target was previously unknown, has also been
shown to be a potent inhibitor of PDF.12
By screening a proprietary library of potential metallo-
enzyme inhibitors we identified the N-formyl-hydroxyl-
amine derivative BB-3497, 2, as a potent and selective
PDF inhibitor with moderate antibacterial activity and
13
in vivoefficacy.
The structural relationship between
actinonin, BB-3497 and a typical PDF substrate is illu-
strated (Fig. 1). BB-3497 was originally prepared in a
non-stereoselective manner and the absolute stereo-
chemistry was tentatively assigned on the basis of
matrix metalloproteinase (MMP) inhibitory activity.
Here we describe the asymmetric synthesis of BB-3497
and proof of stereochemistry by small molecule X-ray
crystallography.
Chemistry and Enzyme Inhibitory Activity
BB-3497 was initially synthesised by the method of
Roques et al. (Scheme 1),14 whereby the b-(N-hydroxy)-
aminoacid fragment 5 was prepared by conjugate
addition of O-benzyl-hydroxylamine to 2-n-butyl acrylic
acid, 4. Peptide coupling to l-tert-leucine N,N-dimethyl-
amide, followed by formylation afforded a mixture of
protected N-formyl-hydroxylamine derivatives RS-6 and
SS-6, which were separated by column chromatography
*Corresponding author. Fax: +44-1865-780814; e-mail: ayscough@
britbio.co.uk
yPresent address: Vertex Pharmaceuticals (Europe) Limited, 88 Milton
Park, Abingdon, Oxford OX14 4RY, UK.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00509-1

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L. M. Pratt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2585–2588
Figure 1. Structural relationship between actinonin (1), BB-3497 (2) and a generic PDF substrate (3).
Scheme 1. Non-asymmetric synthesis of BB-3497 (see ref 14). Reagents and conditions: (a) piperidine, HCHO, EtOH, 80 ꢀC o/n; (b) H2NOBzl,
80 ꢀC, o/n; (c) HCOOH, Ac2O; (d) pentafluorophenol, EDC, CH2Cl2; (e) H-TleNMe2, DMF, 35 ꢀC; (f) separate diastereoisomers (flash chromato-
graphy); (g) H2, Pd/C, EtOH.
and individually deprotected by hydrogenolysis to pro-
vide BB-3497 (from less polar diastereoisomer of 6) and
BB-3554 (from more polar diastereoisomer of 6). The
absolute stereochemistry of these compounds was
unknown, but was inferred by studying their matrix
metalloproteinase inhibitory activity (Table 1). It was
clear that although BB-3497 had relatively weak activity
against the MMP enzyme family it was more potent
than BB-3554. Since it is well known that the preferred
P10 stereochemistry for MMP inhibition is R, corre-
number of possible approaches that we investigated was
the asymmetric counterpart of the conjugate addition
described above, in which the new asymmetric centre
was generated by a highly stereoselective protonation
step (Scheme 2).
The homochiral acrylate 7 was prepared under standard
conditions via the mixed anhydride. We found that
addition of O-benzyl-hydroxylamine to 7 at ambient
temperature proceeded in >90% d.e. and that crystal-
lisation as the tosyl salt afforded a single diastereo-
isomer of the Michael adduct, 8.17 The (S)-benzyl
SuperquatTM chiral auxiliary18 was preferred over the
conventional (4S)-benzyloxazolidin-2-one chiral aux-
iliary of Evans19 because of its lower propensity to
undergo oxazolidinone ring opening. After liberation of
the free base, removal of the SuperquatTM auxiliary was
performed under the less stringent conditions described
by Davies et al.18 We chose to formylate 5 before
coupling to the tert-leucine fragment because the inter-
mediate 9 could be used for synthesis of a wide variety
15
sponding to a natural l-aminoacid in the substrate,
we tentatively assigned BB-3497 as N-[2R-(N-formyl-N-
hydroxy-aminomethyl)-hexanoyl]-S-tert-leucine N,N-
dimethylamide. It is also noteworthy that while BB-
3497 is equipotent with actinonin against E. coli PDF, it
is significantly less potent against several of the MMP
enzymes, and hence more selective. This observation
appears tobe in general fro the
N-formyl-hydroxy-
lamine class and may offer some advantages in a clinical
setting.
0
In order to provide sufficient BB-3497 for detailed bio-
logical evaluation, and to facilitate an analogue syn-
thesis programme, we sought an asymmetric synthetic
route to the b-(N-hydroxy)amino acid derivative 5. At
the time we were unaware of any approaches to this
class of compound in the literature.16 Amongst a
o f P2 and P30 modified analogues as well as BB-3497
itself. The original assignment of stereochemistry of BB-
3497 was supported by a small molecule crystal struc-
ture of the benzyl precursor RS-6,20 (Fig. 2) and later by
the crystal structure of E. coli PDF/BB-3497 complex.13
The stereochemical outcome of the conjugate addition
Table 1. Metalloenzyme inhibition profile of BB-3497, BB-3554 and actinonin against E. coli PDF.Ni, matrix metalloproteinases (MMP), neutral
endopeptidase (NEP; enkephalinase) and angiotensin converting enzyme (ACE)
IC50 (nM)a
PDF.Ni
MMP-1
MMP-2
MMP-3
MMP-7
NEP
ACE
BB-3497
BB-3554
Actinonin
7
70
10
2000
>100,000
1100
15000
>100,000
3000
>100,000
>100,000
6000
>100,000
>100,000
60% Ib
50,000
NDc
6700
>100,000
NDc
>100,000
aThe IC50 represents the concentration of inhibitor (nM) required to decrease enzyme activity by 50%.
b% inhibition at 100 mM.
cNot determined.

L. M. Pratt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2585–2588
2587
Scheme 2. Asymmetric synthetic route to BB-3497. Reagents and conditions: (a) tBuCOCl, Et3N then 3-lithio-4-benzyl-5,5-dimethyl-oxazolidin-2-
one, THF, À78 ꢀC; (b) (i) H2NOBzl, rt, o/n; (ii) pTsOH, EtOAc; (c) (i) 1 M Na2CO3, EtOAc; (ii) LiOH, aq THF, 0 ꢀC; (d) HCOOAc, THF;
(e) H-TleNMe2, HOAt, EDC, DMF; (f) H2, Pd/C, EtOH.
Acknowledgements
We wish to thank Professor Bill Clegg and Dr Mark
Elsegood, University of Newcastle, for the small mol-
ecule crystal structure determination and Wayne
Thomas, British Biotech Pharmaceuticals Limited, for
the biological screening.
References and Notes
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Figure 2. X-ray crystal structure of RS-6.
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Figure 3. Chiral enolate in conjugate addition.
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We have identified a novel asymmetric conjugate addi-
tion reaction that provides a key intermediate required
in the synthesis of homochiral BB-3497. The full scope
of this reaction has yet to be explored. The absolute
stereochemistry of BB-3497 corresponds to that of a
natural formyl-methionyl peptide substrate of PDF.
Modification of the tert-leucine amide fragment of BB-
3497 to provide compounds with substantially improved
antibacterial activity will be reported elsewhere.
Lobell, M.; Palan, S.; Thomas, W.; Whittaker, M.; Baker,
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L. M. Pratt et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2585–2588
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vent removed in vacuo to obtain a yellow oil (59 mmol). This
was treated with 1 equiv p-toluene sulfonic acid and crystallised
from ethyl acetate to obtain the Michael adduct 8 as a white
crystalline solid.
18. Davies, S. G.; Sanganee, H. J. Tetrahedron: Asymmetry
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17. A mixture of acrylate
7
(63 mmol) and O-benzyl-
20. Crystallographic data for RS-6 is deposited with the
Cambridge Crystallographic Data Centre (Supplementary
Publication No: CCDC 160714) and can be obtained, free of
charge, on application to CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK; e-mail: deposit@ccdc.cam.ac.uk
hydroxylamine (126 mmol) was stirred at ambient temperature
o/n. The crude mixture was dissolved in ethyl acetate and
washed sequentially with 1 M HCl, 1 M Na2CO3 and brine.
The organics were dried over anhydrous MgSO4 and the sol-