Identification of novel potent bicyclic peptide deformylase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.01.014

Screening of our compound collection using Staphylococcus aureus Ni-Peptide deformylase (PDF) afforded a very potent PDF inhibitor with an IC₅₀ in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural information obtained from Pseudomonas aeruginosa Ni-PDF complexed with the inhibitor suggested the synthesis of a variety of analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds synthesized proved to be excellent PDF-Ni inhibitors and some showed increased antibacterial activity in selected strains.

Full text of DOI:10.1016/j.bmcl.2004.01.014

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1477–1481
Identification of novel potent bicyclic peptide
deformylase inhibitors
Valentina Molteni,a,* Xiaohui He,a Juliet Nabakka,a Kunyong Yang,a Andreas Kreusch,a
Perry Gordon,a Badry Bursulaya,a Ian Warner,a Tanya Shin,a Tanya Biorac,a
Neil S. Ryder,b Ron Goldberg,c John Doughtyc and Yun Hea
aGenomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA
bNovartis Institutes for Biomedical Research, 100 Technology Square, Cambridge, MA 02139, USA
cNovartis Institute for Biomedical Research, One Health Plaza, East Hanover, NJ 07936, USA
Received 11 October 2003; revised 23 December 2003; accepted 10 January 2004
Abstract—Screening of our compound collection using Staphylococcus aureus Ni–Peptide deformylase (PDF) afforded a very potent
PDF inhibitor with an IC50 in the low nanomolar range but with poor antibacterial activity (MIC). Three-dimensional structural
information obtained from Pseudomonas aeruginosa Ni–PDF complexed with the inhibitor suggested the synthesis of a variety of
analogues that would maintain high binding affinity while attempting to improve antibacterial activity. Many of the compounds
synthesized proved to be excellent PDF–Ni inhibitors and some showed increased antibacterial activity in selected strains.
# 2004 Elsevier Ltd. All rights reserved.
Bacterial resistance to many of the existing antibiotics is
a growing health concern.1 Therefore there is an urgent
need to identify new antibiotics with unexploited modes
of action. Peptide Deformylase (PDF) is an essential
bacterial metalloenzyme responsible for the removal of
the N-terminal formyl group from methionine residues
following protein synthesis.2 PDF has been considered
an attractive target for antibacterial chemotherapy.3
Several classes of inhibitors have already been investi-
gated to date,3e,g,4ꢀ9 and some compounds with pseu-
dopeptidic backbone are now in development.7,8,10 With
concerns about possible low specificity for MMPs and
poor metabolic stability associated with pseudopeptidic
inhibitors,11,12 there is still a need for the identification
of novel and structurally diverse non-peptidic PDF
inhibitors.
representative mammalian metalloenzymes (MMP-2
and MMP-13) and also showed a desirable solubility
and metabolic stability, as measured in rat liver micro-
somes. Despite its potency against the PDF enzyme, this
compound lacked antibacterial activity (MIC >128 mg/
mL) against Escherichia coli, Klebsiella pneumoniae, S.
aureus, Enterococcus faecalis, and two efflux-deficient E.
coli mutants.13 The compound, however, displayed
some activity against two selected strains of Moraxella
catarrhalis and Haemophilus influenzae (Table 2).13 The
selected H. influenzae and M. catarrhalis strains were
specifically selected from our collection on the basis of
their hyper susceptibility to known proprietary PDF
inhibitors currently under investigation (N.S. Ryder,
unpublished data).
Compound 1 was co-crystallized with Pseudomonas
aeruginosa-Ni–PDF (Fig. 2). The X-ray structure
showed that compound 1 chelates to the nickel center
Screening of our compound collection for inhibitory
activity against Staphylococcus aureus Ni–PDF led to
the discovery of a very potent inhibitor 1 with an IC50
of less than 5 nM (Fig. 1). Compound 1, with a metal
chelating hydroxamic acid attached to a benzothiazi-
none core, showed good selectivity for PDF over
Keywords: Peptide deformylase inhibitors.
* Corresponding author. Tel.: +1-858-332-4736; fax: +1-858-812-
1648; e-mail: vmolteni@gnf.org
Figure 1. Initial hit 1 from HTS.
0960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.014

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V. Molteni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1477–1481
through the hydroxamic acid moiety. In particular, the
carbonyl and hydroxyl oxygen atom of the hydro-
xamate group act as a bidentate ligand for the Ni2+
while the nitrogen atom forms a strong H-bond with the
carboxylate group of Glu135. The benzothiazinone ring
occupies the hydrophobic S10 pocket, which usually
binds the methionine side chain of the substrate and the
carbonyl oxygen on the benzothiazinone ring forms a
favorable hydrogen bond to Ile46-N. This particular
H-bond is conserved in all available PDF inhibitor
structures.
Emmons condensation with formaldehyde in the
presence of sodium methoxide afforded the alkene 37.
Compound 37 underwent Michael addition with O-
benzyl hydroxylamine hydrochloride under refluxing
conditions in DMF to give 38. Compound 38 was N-
formylated or N-acetylated by treatment with formic
acid/acetic anhydride or acetic anhydride respectively.
Removal of the benzyl group by hydrogenolysis affor-
ded the reverse hydroxamates, N-formyl hydroxamine 6
and the N-acetyl hydroxylamine 7, respectively. Com-
pound 38 was also directly converted into compound 8
by hydrogenolysis.
The interactions observed in the crystal structure in
combination with molecular modeling suggested that
substitution at N-4 on the benzothiazinone ring and
small modifications of the heterocycle could be per-
formed to improve the antibacterial activity without
interfering with the key interactions responsible for the
binding. Here we describe various modifications at N-4
and on the heterocyclic core and their effects on anti-
bacterial activity. In addition, the replacement of the
hydroxamic acid for other chelating moieties is descri-
bed, since hydroxamic acids have been associated with
poor bacterial penetration.4
The N-substituted analogues of compound 1 and of 3,4-
dihydro-1H-quinolin-2-one 31 were synthesized accord-
ing to the sequence shown in Scheme 3. Esters 39 and 40
were N-alkylated by treatment with NaH in DMF fol-
lowed by the appropriate alkylating agent. The esters
were then hydrolyzed by LiOH in THF/water solution.
The acids were then converted to the final hydroxamic
acids, following the procedure of Reddy.15
Sulfone 30 was prepared by oxidation of acid 41 with
m-CPBA to give 42 that was transformed to the corres-
ponding hydroxamic acid under the same conditions
described above.15 Any attempt to directly oxidize
compound 1 gave an inseparable mixture (Scheme 4).
The synthesis of compounds 2–35 will be described,
followed by a discussion of their biological activity.
The bidentate analogues (3, 4 and 5) were synthesized
using standard alkylation conditions (Scheme 1). In the
case of compound 5, 2.0 equivalents of K2CO3 were
used to ensure alkylation of both NH and OH.
The carbon isosteres of 1, 31 and 34 were synthesized
according to Scheme 5. Protection of compounds 43
and 44 followed by a-alkylation with tert-butylbromo-
acetate afforded compounds 45 and 46, respectively.
Hydrolysis of the ester and deprotection with TFA
The reverse hydroxamate 6 was synthesized as shown in
Scheme 2. The 4H-benzo[1,4]thiazin-3-one 36 was easily
chlorinated to afford the 2-chloro derivative that was
converted to the corresponding phosphonate by treat-
ment with triethylphosphite (Scheme 2). Horner–
Scheme 3. (i) NaH, DMF, RX; (ii) LiOH 3 M, THF–H2O (1:1); (iii)
ethyl chloroformate, N-methylmorpholine, THF; (iv) NH2OH (1 M in
MeOH) (10–80% for 4 steps).
Scheme 1. (i) K2CO3, RX, DMF.
Scheme 4. (i) mCPBA, CH2Cl2 (27%); (ii) ethyl chloroformate, N-
methylmorpholine, THF; (iii) NH2OH (1M in MeOH) (38% for 2
steps).
Scheme 2. (i) SO2Cl2, CH2Cl2 (99%); (ii) P(OEt)3, 100 ꢁC (79%); (iii)
NaOMe, HCHO (72%); (iv) NH2OBn, HCl, DMF, reflux; (76%); (v)
Ac2O, HCOOH for 6 (65%) Ac2O for 7 (60%); (vi) 10% Pd/C, 1,4-
cyclohexadiene, EtOH.
Scheme 5. (i) NaH, DMF, pMBCl (90–95%); (ii) LDA, THF,
BrCH2COOt-Bu (30–53%); (iii) TFA, reflux (71–93%); (iv) ethyl
chloroformate, N-methylphoporline, THF; (iv) NH2OH (1 M in
MeOH) (30–90% for 2 steps).

V. Molteni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1477–1481
1479
followed by conversion to the hydroxamic acid gave the
target compounds.
again due to steric hindrance. The monodentate hydro-
xylamine 8 and acid 917 were inactive.
The 8-bromo substituted compound 35 was prepared as
shown in Scheme 6. Aromatic nucleophilic substitution
of 2,6-dinitrochlorobenzene 47 with 2-mercapto succinic
isopentylester followed by reduction of nitro groups
with Fe/HCl afforded directly the cyclized compound.
The second amino group was then converted into a
diazonium salt which was displaced in situ with CuBr2
to give 48. The ester 48 was then converted to
compound 35 with standard procedures.
The crystal structure of the P. aeruginosa-Ni–PDF-1
complex (Fig. 2) clearly indicated that substituents at
N-4 should not affect PDF activity as they are located
outside the active site. Under the hypothesis that lack of
antibacterial activity is due to poor penetration, sub-
stitution at the N-4 position could be used to improve
the pharmacological properties of the compounds,
which in turn should result in improved antibacterial
Table 1. PDF–Ni IC50 for analogues of 1
Table 1 gives the S. aureus Ni–PDF IC50 for compounds
1–35.
It has been proposed that potent hydroxamic acid con-
taining PDF inhibitors may be inadequate antibacterial
agents due to poor penetration and the action of efflux
pumps.4 As a consequence, one possible way to achieve
antibacterial activity for compounds of type 1, would be
through replacement of the hydroxamic acid function-
ality, assuming that enzyme inhibitory activity could be
retained. Several groups have previously described the
replacement of the hydroxamic acid functionality with
alternative chelating moieties.6,10,16 For the few struc-
tural classes described the hydroxamic acid and the
N-formyl hydroxylamine have proven to be the only
chelating functionalities which would guarantee PDF
enzyme inhibition.6,10 However, it was shown that the
hydroxamic acid motif alone is not sufficient for PDF
inhibition and the nature of the backbone contribute
greatly to the extent of inhibition.6 Examples of PDF
inhibitors that do not contain hydroxamic acid but
other chelating moieties such as thiols have been also
described.16c,d On this basis we decided that it was
worthwhile to evaluate a series of analogues in which the
hydroxamic acid in 1 was replaced with other related
mono- or bidentate chelating moieties (Table 1).
a,b
X
R1
R2
IC50
Actinonin
<5
1
2
3
4
5
6
7
8
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
H
H
H
H
H
H
H
H
H
CONHOH
CONHNH2
CONHOCH3
CONHOBn
CONCH3OCH3
N(CHO)OH
N(COCH3)OH
NHOH
<5
330
74
230
>2000
10
427
>2000
>2000
37
9
COOH
10
11
12
13
14
15
16
17
Methyl
Isopentyl
Pentyl
Isohexyl
Bn
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
<5
<5
<5
12
29
41
302
c-Propylmethyl
c-Butylmethyl
c-Hexylmethyl
18
S
CONHOH
18
19
20
S
S
2-c-Hexylethyl
Phenethyl
CONHOH
CONHOH
57
<5
21
S
CONHOH
<5
The bidentate hydrazide 217 and the methyl and benzyl
hydroxamate 3 and 4 were still PDF inhibitors but less
potent than 1. The decreased potency of 4 relative to 3 is
due to increased steric hindrance, as observed from
modeling. The N-methyl methyl hydroxamate 5 was
inactive maybe also because of steric reasons. It may be
also possible that the N–H is involved in a hydrogen
bond. The crystal structure of compound 1 (Fig. 2)
indeed showed a hydrogen bond interaction with the
side chain of Glu 135. The reverse hydroxamate, N-for-
myl hydroxamine 6, was equipotent to 1, while the N-
acetyl hydroxylamine 7, was 40-fold less potent, once
22
23
24
25
26
S
S
S
S
S
2-Methoxyethyl
CH2CN
CH2CotBu
CH2CONH2
CH(CH3)CONH2
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
<5
515
50
168
44
27
S
CONHOH
58
28
29
S
S
CONHOH
CONHOH
397
343
30
31
32
33
34
35c
SO2
CH2
CH2
CH2
C¼C
S
H
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
CONHOH
>2000
73
63
H
Isopentyl
c-Propylmethyl
175
169
74
H
H
a IC50 (nM).
b Ni–PDF assays with the S. aureus enzyme were performed in
duplicate according to Clements et al.3e using f-MAS as substrate
and detecting the free amino group of the PDF product MAS with
fluorescamine. The detection limit of the assay was 5 nM.
c 8-Bromo substituted analogue.
Scheme 6. (i) 2-Mercapto-succinic acid diisopentyl ester, TEA, DMF,
0 ꢁC (45%); (ii) Fe, HCl, Water/EtOH (80%); (iii) t-BuONO, CuBr2,
CH3CN (25%); (iv) NaOH, H2O/EtOH (99%); (v) ethyl chloro-
formate, N-methylphoporline, THF; (vi) NH2OH (1 M in MeOH)
(17% for 2 steps).

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V. Molteni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1477–1481
Table 2. In vitro antibacterial activity against selected strains
Compd
MIC mg/mL
M. catarrhalis
NB50012
MIC mg/mL
M. catarrhalis
NB01050
MIC mg/mL
H. influenzae
NB65007
1
6
12
15
31
35
8
8
2
8
4
1
64
128
8
64
32
8
128
128
32
128
128
32
Figure 2. X-ray crystal structure of (R)-1 bound to Ni–PDF from P.
aeruginosa. A: Polar interactions between 1 (light blue carbon atoms)
and PDF from P. aeruginosa (grey carbon atoms) are shown in green.
The Ni2+ ion is colored in magenta, nitrogen atoms in blue, oxygen
atoms in red, and sulfur atoms in yellow. B: The compound is shown
as yellow ball and stick model, the protein is shown as a surface with
the Ni2+ ion in green, carbon atoms in white, nitrogen atoms in blue,
oxygen atoms in red, and sulfur atoms in dark yellow. X-ray analysis
revealed that it is the (R)-enantiomer that strongly binds to the active
site. This stereochemistry, was not confirmed by enzyme assay because
the separated enantiomers were not available, but the crystallographic
observation is in line with the stereochemistry observed for previously
described PDF inhibitors.4,10,14
improvement in PDF activity. In our series the same
substitution afforded a decrease of activity of 15-fold
(IC50=74 nM).
Compounds 2–35 were all tested for antibacterial activity
on the standard panel of four bacterial strains (E. coli,
K. pneumonia, S. aureus and E. faecalis). No significant
improvement of antibacterial activity was obtained
against these strains. Selected compounds were also
tested against the three hyper susceptible strains of M.
catarrhalis and H. influenzae (Table 2).
activity. Several N-4 substituted derivatives of the
hydroxamic acid 1 and the related N-formyl hydroxyl-
amine 6 (data not shown) were synthesized and tested.
The nature of some of the substitutions performed were
suggested by published data5 and Novartis unpublished
data on existing PDF inhibitors. As expected the sub-
stitutions were generally tolerated (Table 1). Very
potent hydroxamic acid derivatives were obtained with
both long linear and branched alkyl chains (11–13) and
with phenethyl and 2-ethyl [1,3] dioxolane substituents
(20 and 21) whereas cycloalkyl and benzyl substituents
afforded compounds with lower activity (15–19). This
was also the case for benzyl analogues substituted on
the benzene ring (data not shown). These results are in
contrast to modeling expectations but are in line with
what has been observed by Apfel et al.5 on a class of
related PDF inhibitors. Activity was retained with more
polar substitution (23–26) and in substituted acetamides
(27–29) but the compounds showed a decrease in activity
with respect to the parent molecule 1.
The N-pentyl substituted compound 12 showed
improved activity with respect to 1. The tetrahydro-
quinolone 31, despite the fact that it is a much less
potent PDF inhibitor than 1 (73 nM versus <5 nM)
showed a similar MIC profile. Compound 35 showed
the best improvement of antibacterial activity against
these selected strains. In addition, it had a MIC of 64
mg/mL against E. coli, despite its lower PDF activity
relative to 1 (74 nM versus <5 nM). These results
reinforce the observation of Clements et al.,3e that
variations of antibacterial activity cannot necessarily be
attributed to a corresponding variation in enzyme
inhibition.
Many factors that affect drug distribution, such as bac-
terial membrane permeability and efflux pumps con-
tribute to antibacterial activity. In order to try to
explain the poor antibacterial activity for the described
inhibitors we decided to study the intracellular uptake
of 1 in E. coli and S. aureus.18 It was found that com-
pound 1 was unable to permeate the E. coli cell mem-
brane, explaining the lack of activity in this particular
strain. When the experiment was performed with S.
aureus, it appeared that the compound entered the cell
at a concentration that was expected to be sufficient for
PDF inhibition. It should be noted that compound
molecules bound to the outer cell membrane and sub-
sequently released during cell analysis could contribute
to the measured intracellular compound concentration.
It is likely, even if not proven, that this is what
happened in the case of S. aureus.
In order to address the possibility that the lack of anti-
microbial activity of 1 was due to intracellular meta-
bolic oxidation, we prepared the corresponding sulfone
30 which was inactive against PDF–Ni (Table 1). This
result prompted us to prepare a series of carbon iso-
steres. The carbon analogue 31 and the corresponding
seven member ring analogue 34 were both PDF inhibi-
tors, even if less potent than 1, suggesting that sulfur
oxidation is not the cause of lack of antibacterial activ-
ity for compound 1. Derivatives of the carbon analo-
gues were also prepared. They followed the same SAR
trend as the sulfur analogue but with lower potency (32
versus 11, 33 versus 15).
This letter describes preliminary efforts to convert
potent PDF inhibitors into antibacterial agents. We
demonstrated that the PDF activity of 1 might be
maintained and improved with appropriate structural
modifications. In all cases the PDF enzyme inhibitory
activity did not translate to antibacterial potency.
Compound 35 was synthesized to establish a compar-
ison with the bicyclic urea described by Apfel et al.5
For that core the 8-bromo substitution gave a 40-fold

V. Molteni et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1477–1481
1481
Nevertheless, two compounds showed a clear improved
antibacterial activity on selected strains with respect to
1. Further investigation of the reasons underlying
the poor antibacterial activity of these potent PDF
inhibitors may be the topic of future reports.
5. Apfel, C.; Banner, D. W.; Bur, D.; Dietz, M.; Hubsch-
werlen, C.; Locher, H.; Marlin, F.; Masciadri, R.; Pirson,
W.; Stadler, H. J. Med. Chem. 2001, 44, 1847.
6. Thorarensen, A.; Douglas, M. R.; Rohrer, D. C.; Vosters,
A. F.; Yem, A. W.; Marshall, V. D.; Lynn, J. C.; Boha-
non, M. J.; Tomich, P. K.; Zurenko, G. E.; Sweeney,
M. T.; Jensen, R. M.; Nielsen, J. W.; Seest, E. P.; Dolak,
L. A. Bioorg. Med. Chem. Lett. 2001, 11, 1355.
Acknowledgements
7. Wise, R.; Andrews, J. M.; Ashby, J. Antimicrob. Agents
Chemother. 2002, 46, 1117.
The authors would like to thank Tom Hollenbeck for
MS experiments, Doris Hafenbradl, Kathryn Bracken,
Scott Lesley, Don Karenewsky and Peter Schultz for
helpful discussions.
8. Clements, J. M.; Ayscough, A. P.; Keavey, K.; East, S. P.
Curr. Med. Chem.-Anti-Infective Agents 2002, 1, 239.
9. Jones, R. N.; Rhomberg, P. R. J. Antimicrob. Chemother.
2003, 51, 157.
10. 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.
Atomic coordinates have been deposited with the RCSB
Protein Data Bank with access code 1S17.
11. Hidalgo, M.; Eckhardt, S. G. J. Natl. Cancer Inst. 2001,
93, 178.
12. Hu, X.; Nguyen, K. T.; Verlinde, C. L. M. J.; Hol,
W. G. J.; Pei, D. J. Med. Chem. 2003, 46, 3771.
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17. Compound commercially available.
18. E. coli and S. aureus were grown in LB medium to
OD600=0.6. Compound 1 was added to the E. coli cells
to a final concentration of 25 mM, 50 mM, 75 mM, respec-
tively, and a final concentration of 100 mM and 300 mM
for S. aureus. Samples were taken after 30 min, 1.5 h, and
3.0 h, respectively, and the cell pellet washed several times
with LB media before stored at ꢀ30 ꢁC overnight.
Untreated E. coli and S. aureus samples served as con-
trols. Thawed E. coli cells were resuspended in 200 mL
lysis buffer (20 mM Tris pH 7.8, 150 mM NaCl), and
lysed by sonication. For S. aureus, the lysis buffer con-
tained an additional 0.5 mg/mL Lysostaphin. After cell
lysis and pelleting the cell debris, the supernatant was
filtered through a 3K centricon spin concentrator and 100
mL of the filtrate was analyzed by mass spectrometry.
4. 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.