Identification of novel potent hydroxamic acid inhibitors of peptidyl deformylase and the importance of the hydroxamic acid functionality on inhibition

Article abstract of DOI:10.1016/S0960-894X(01)00242-6

Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of prokaryotic prepared polypeptides, an essential step in bacterial protein synthesis. Screening of our compound collection using Staphylococ

Full text of DOI:10.1016/S0960-894X(01)00242-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1355–1358
Identification of Novel Potent Hydroxamic Acid Inhibitors of
Peptidyl Deformylase and the Importance of the
Hydroxamic Acid Functionality on Inhibition
Atli Thorarensen,a,* Martin R. Douglas, Jr.,b Douglas C. Rohrer,c Anne F. Vosters,b
Anthony W. Yem,b Vincent D. Marshall,b Janet C. Lynn,d Michael J. Bohanon,d
Paul K. Tomich,d Gary E. Zurenko,e Michael T. Sweeney,e Randy M. Jensen,f
f
James W. Nielsen,f Eric P. Seestf andLester A. Dolak
aMedicinal Chemistry 7254-209-615, Pharmacia, Kalamazoo, MI 49001-0199, USA
bProtein Science, Pharmacia, Kalamazoo, MI 49001-0199, USA
cComputer Aided Drug Discovery Research, Pharmacia, Kalamazoo, MI 49001-0199, USA
dDiscovery Technologies, Pharmacia, Kalamazoo, MI 49001-0199, USA
eBiology I, Pharmacia, Kalamazoo, MI 49001-0199, USA
fStructural, Analytical & Medicinal Chemistry Research, Pharmacia, Kalamazoo, MI 49001-0199, USA
Received9 January 2001; accepted20 March 2001
Abstract—Peptidyl deformylase (PDF) is a metallo protease that catalyzes the removal of a formyl group from the N-termini of
prokaryotic preparedpolypeptides, an essential step in bacterial protein synthesis. Screening of our compoundcollection using
Staphylococcus aureus PDF afforded a very potent inhibitor with an IC50 in the low nanomolar range. Unfortunately, the com-
pound that contains a hydroxamic acid did not exhibit antibacterial activity (MIC). In order to address the lack of activity in the
MIC assay and to determine what portion of the molecule was responsible for binding to PDF, we prepared several analogues. This
paper describes our findings that the hydroxamic acid functionality found in 1 is mainly responsible for the high affinity to PDF. In
addition, we identified an alternative class of PDF inhibitors, the N-hydroxy urea 18, which has both PDF andantibacterial
activity. # 2001 Elsevier Science Ltd. All rights reserved.
Antibacterial resistance to a wide range of available
antibiotics is a growing problem in the treatment of
bacterial infections.1À3 Currently available antibiotics
inhibit several essential biological pathways in bacteria.
Historically, antibacterial discovery programs have
focusedon protein targets, which have little homology
or resemblance to mammalian counterparts. Recent
advances in biology and automation have resulted in the
discovery of new targets at an impressive rate. Anti-
bacterial discovery programs can rely on target or cell
protein synthesis is essential for bacterial proliferation.
The metal in the native enzyme is thought to be iron,
but due to the extreme sensitivity of this protein during
purification it has been isolatedwith a variety of
cations in its active site including cobalt, nickel, and
9,10
zinc.7 Several solution8 andsoli-dstate
structural
studies have recently been reported. Recent reports
describing PDF inhibitors with metal chelating func-
tional groups such as hydroxamic acids,11,12 N-for-
15
mylhydroxyl-amine,13 thiol,14 andbiaryl acsid
basedassays utilizing high throughput screening for
4À6
demonstrate a current interest in PDF as an anti-
bacterial target.
leadgeneration.
Peptidyl deformylase (PDF) is a
metallo protease, which catalyzes the removal of a for-
myl group from the N-termini of polypeptides bio-
synthesizedin prokaryotes. This step in bacterial
Screening of our compoundcollection against Staphyl-
ococcus aureus PDF identified a very potent inhibitor 1
with an IC50 in the low nanomolar range (Fig. 1).16 To
our surprise, compound 1 did not exhibit antibacterial
activity (MIC). The inhibitor contains a hydroxamic
acid, a feature common to recently reported inhibitors
*Corresponding author. Tel.:+1-616-833-3962; fax: +1-616-833-
2232; e-mail: atli.thorarensen@am.pnu.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00242-6

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A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1355–1358
of PDF, which demonstrate antibacterial activity.11,12
The lack of antibacterial activity for this compoundwas
noteworthy, considering its potency against the PDF
enzyme. We set out to address the lack of antibacterial
activity in this series of compounds and to find a potent
inhibitor of PDF with activity in our MIC assay. We
have testedandpreparedanalogues ardessing two
issues: (a) the importance of the hydroxamic acid
moiety to binding, and (b) the possibility of an alter-
native functional group existing for the hydroxamic acid
with affinity for PDF.
In order to evaluate the importance of the hydroxamic
acid, the corresponding carboxyl, amide, and hydrazide
surrogates were prepared(Scheme 1). These analogues
contain a constant attachment of the backbone of 1 to
the metal coordination group and are able to coordinate
the metal in a mono- or bidentate fashion.
The hydrazide 12 that achieves a bidentate chelate with
the metal inhibitedPDF with an IC of 4.1 mM (Table
50
2). This is a similar metal coordination to the hydro-
xamate 1 but is 100-foldless potent. The remainder of
the compounds 9–10, which are monodentate ligands,
were inactive. These results clearly demonstrate that the
hydroxamic acid functionality is essential for the high
affinity of 1 against PDF.
Similarity searches of our compoundcollection identi-
fiedseveral relatedcompounds shown in Table 1. These
compounds (which also contain a hydroxamic acid)
exhibit a wide range of inhibition against PDF, but were
all devoid of any antibacterial activity. The shape of the
molecule appears to contribute significantly to the tight
binding of 1 to PDF. It is not sufficient to only have the
hydroxamic acid functionality, since both 6 and 7 were
inactive as inhibitors for PDF.
Crystal structures of PDF10 andour computer modeling
efforts suggestedthat 1 binds to PDF in a C-shape (Fig.
2a). Based on this data, it was our desire to design a new
set of inhibitors that wouldhave the potential of
achieving a five-memberedbidentate chelate basedon
the importance of the hydroxamic acid functionality
foundin 1. In order to increase the basicity of the car-
bonyl andthereby enhance its coordination, we elected
to pursue amides. The data in Table 1 suggest that the
basic nitrogen foundin 1 was not essential for PDF
activity. Our approach was to prepare a-hetero-
substitutedamides that couldattain a C-shapedcon-
formation similar to 1 basedon molecular modeling
(Fig. 2b).
Figure 1. Inhibitor of PDF identified by screening.
Table 1. Antibacterial and PDF activity of selected hydroxamic acids
SAUR SEPI SPNE ECOL
6674
PDFa
IC50 (mM)
9218
12084
9912
2
>128
>128 >128
>128
0.059
3
4
5
6
7
>128
>128
>32
>128 >128
>128 >128
>128
>128
>32
0.46
0.69
>32
>32
0.2
>128
>128
>128 >128
>128 >128
>128
>128
>50
Scheme 1.
Table 2. Antibacterial andPDF activity of alternative chelators
>100
SAUR
9218
SEPI
12084
SPNE ECOL
9912
PDF
IC50 (mM)
6674
aPDF assays were performedwith the S. aureus enzyme utilizing the
assay relying on coupling the PDF peptide product to an aminopepti-
dase as described in ref 17a (data shown). In order to validate PDF
inhibition, the assay described in ref 17b, in which formate dehy-
drogenase utilizes the formic acid released to reduce NAD, was per-
1
10
9
NHOH
OH
NH2
>64
>128
64
>64
>128
64
64
128
16
>64
>128
>128
>128
0.046
>100
>100
4.1
formed (data complementary). In addition, in selected cases where the
aminopeptidase17a was inhibitedby a compound, the assay
17a
was
modified to an HPLC end point assay measuring PDF product for-
mation, verifying PDF inhibition.17
12
NHNH2
64
128
32

A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1355–1358
1357
The synthesis of the series of analogues is outlinedin
Scheme 2. 1-Naphthylamine was acylatedwith a,a,a-tri-
fluoro-m-tolylacetic acid affording amide 13. The amide
was subsequently reduced to afford 14 in excellent
overall yield. Amine 14 was then acylatedwith several
a-hetero substituted acid chlorides, which upon depro-
tection afforded thiol 15 andhydroxy compound 16.
The corresponding hydroxamate and the hydrazine were
preparedby acylation of 14 with a phosgene equivalent,
followed by the addition of excess hydrazine or hydroxyl-
amine to afford the desired compounds 17 and 18.
methylene group between the coordination groups were
inactive against PDF, but the hydroxy compound 16
was active as an antibacterial agents (Table 3).19 Replace-
ment of the carbon in 16 with a nitrogen, such as in the
hydrazine urea 17, also results in complete loss of anti-
bacterial activity. A more significant observation is that
the N-hydroxyurea 18 is active against PDF with an
IC50 around2.2 mM andhas antibacterial activity. The
ability of a hydroxamic urea to coordinate PDF has not
been previously reported. In compound 18, we have
overcome the lack of MIC foundin 1 despite a decrease
in PDF activity. This functionality appears not to have
the same disadvantage as the hydroxamic acid. One can
speculate that the inactivity in the MIC assay of 1 is due
to efflux andthat 18 is not a high affinity substrate for
the efflux pumps.12
The compounds exist as atropisomers and as a mixture
of E/Z amide isomers, which is solvent dependent. The
energy barrier for a few derivatives18 was measuredto
be approximately ÁG{ of 24.0 kcal/mol. This data
indicates that the compounds rapidly equilibrate at
room temperature andthe separation of each enantio-
mer is not justifiable.
This report describes an initial effort to design inhibitors
of PDF with in vitro antibacterial activity. It was
demonstrated that the high affinity of compound 1 is
mainly due to the hydroxamic acid functionality, but
the backbone is also important. The initial lead 1 and
the understanding of its inhibition of PDF was subse-
quently utilizedto design several a-hetero substituted
amides. The urea derivative 18 was foundto have PDF
activity with an IC50 of 2.2 mM in addition to anti-
bacterial activity. One can speculate that the lack of
MIC activity of 1 is due to efflux or poor membrane
permeability. The discovery of 18 circumvents that
Amine 14 was foundto be inactive in both the MIC and
the PDF screen. Compounds 15–16 containing a
Figure 2. Modeling of analogues in the active site of PDF: (a) 1, and
(b) a-thioacetamide 15.
Scheme 2.

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A. Thorarensen et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1355–1358
Table 3. Antibacterial andPDF activity of a-hetero substituted
acetoamides
4. (a) Setti, E. L.; Micetich, R. G. Curr. Med. Chem. 1998, 5,
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9218 MIC 12084 MIC 9912 MIC 6674 MIC IC50
SEPI
SPNE
ECOL
PDF
(mM)
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15 CH2SH
16 CH2OH
17 NHNH2
18 NHOH
>128
32
>128
32
>128
>128
>128
32
>128
32
>128
32
>128
>128
>128
>128
>100
>100
>100
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2.2
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problem andfulfills our initial goal of a PDF inhibitor
with antibacterial activity. The SAR of this general
template andthe optimization of its antibacterial activ-
ity along with the search of additional polar functional
groups will be the topic of future reports.
Acknowledgements
12. Apfel, C.; Banner, D. W.; Bur, D.; Dietz, M.; Hirata, T.;
Hubschwerlen, C.; Locher, H.; Page, M. G. P.; Pirson, W.;
Rosse, G.; Specklin, J.-L. J. Med. Chem. 2000, 43, 2324.
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taker, M.; Davies, S. J.; Pratt, L. M.; Spavold, Z. M.;
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59097.
We acknowledge the Structural, Analytical and Medic-
inal Chemistry Unit at Pharmacia for analytical ser-
vices. Thanks to DavidR. Graber andSteven P. Tanis
for information andhelp regarding the preparation of
compounds 8–11, andto Bruce N. Rogers for critical
reading of this manuscript.
14. Huntington, K. M.; Yi, T.; Wei, Y.; Pei, D. Biochemistry
2000, 39, 4543.
References and Notes
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16. Preliminary disclosure: Identification of Novel Potent
Hydroxamic Acid Inhibitors of Peptidyl Deformylase; Poster,
French–American Chemical Society FACS VIII, Sonoma
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18. Compounds not shown but are related to 16.
19. The antibacterial activity of 16 is due to an unknown
mechanism andappears to be unrelatedto PDF.