Hydroxamic acid derivatives as potent peptide deformylase inhibitors and antibacterial agents

Article abstract of DOI:10.1021/jm000018k

Low-molecular-weight β-sulfonyl- and β-sulfinylhydroxamic acid derivatives have been synthesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most of the compounds synthesized and tested displayed antibacterial activities that cover several pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these compounds as antibacterial agents is discussed with respect to selectivity, intracellular concentrations in bacteria, and potential for resistance development.

Full text of DOI:10.1021/jm000018k

2324
J . Med. Chem. 2000, 43, 2324-2331
Hyd r oxa m ic Acid Der iva tives a s P oten t P ep tid e Defor m yla se In h ibitor s a n d
An tiba cter ia l Agen ts
Christian Apfel, David W. Banner, Daniel Bur, Michel Dietz, Takahiro Hirata, Christian Hubschwerlen,*
Hans Locher, Malcolm G. P. Page, Wolfgang Pirson, Ge´rard Rosse´,† and J ean-Luc Specklin
Preclinical Research, F. Hoffmann-La Roche Ltd., CH-4070 Basle, Switzerland
Received J anuary 18, 2000
Low-molecular-weight â-sulfonyl- and â-sulfinylhydroxamic acid derivatives have been syn-
thesized and found to be potent inhibitors of Escherichia coli peptide deformylase (PDF). Most
of the compounds synthesized and tested displayed antibacterial activities that cover several
pathogens found in respiratory tract infections, including Chlamydia pneumoniae, Mycoplasma
pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. The potential of these
compounds as antibacterial agents is discussed with respect to selectivity, intracellular
concentrations in bacteria, and potential for resistance development.
In tr od u ction
addition reaction of trimethylsilyl ketene to the ap-
propriate aldehyde, R2CHO.12,13 The key carboxylic acid
intermediates 4 were obtained by two different routes:
either by 1,4-conjugated addition of thiols on â-substi-
tuted acrylic acids 2 or by CsF-catalyzed ring opening
of â-lactones 3 with thiols (Scheme 1). The carboxylic
acids 4 were then converted into the corresponding
protected hydroxamate derivatives 5 either by reaction
with O-benzyl- or O-TBDMS-protected hydroxylamines
in the presence of DCC or, alternatively, by loading onto
hydroxylamine-Wang resin after activation with HATU
(O-(7-a za benzotria zol-1-yl)-N,N,N′,N′-tetra methyl-
uronium hexafluorophosphate). The hydroxamates 5
were oxidized either to the corresponding sulfones 6
with Oxone or mCPBA or to sulfoxides 7 with 3-phenyl-
2-(phenylsulfonyl)oxaziridine.14 Deprotection using stand-
ard methods (Scheme 1) afforded the target sulfones 8
and sulfoxides 9 (Table 1). Separation of the two
enantiomers of the O-benzyl hydroxamate 6d by pre-
parative HPLC on a chiral column gave the two enan-
tiomers (R)-8d and (S)-8d after subsequent deprotec-
tion.
Antibiotic resistance among Gram-positive bacteria
(staphylococci, enterococci, and streptococci) is becoming
a major health concern. Therefore, there is an urgent
need to discover antibiotics with new modes of action.
The protein synthesis machinery is targeted by many
successful drugs including chloramphenicol, tetracy-
clines, macrolides, and aminoglycosides. In this respect,
peptide deformylase (PDF), an enzyme that removes the
formyl group from the N-terminal methionine of nascent
polypeptides, represents an interesting and unexploited
target since this enzyme is conserved and believed to
be essential in eubacteria1-3 and is not required for
protein synthesis in eukaryotes.
Ra tion a le
Screening for inhibitors of E. coli PDF resulted in the
identification of actinonin (1), a natural hydroxamic acid
derivative earlier described as an aminopeptidase in-
hibitor4,5 and an antibacterial agent.6 PDF is a metallo-
enzyme which has recently been recognized to utilize
iron (Fe2+ 7-10
as the catalytic metal for N-formyl
)
The R-hydroxy-â-sulfinylhydroxamic derivatives 15a
and 15b were obtained starting from trans-2-hepten-1-
ol (Scheme 2). A sequence employing a modified Sharp-
less epoxidation using L- or D-diethyl tartrate15,16 fol-
lowed by RuO4 oxidation afforded the trans-epoxy acids
11a (2R,3S) and 11b (2S,3R), respectively. Further
treatment of 11a and 11b with thiophenol in the
presence of LiClO4 on alumina and subsequent reaction
with O-tetrahydropyranylhydroxylamine17 in the pres-
ence of carbonyldiimidazole gave the sulfide derivatives
13a and 13b. Oxidation with 3-phenyl-2-(phenylsulfon-
yl)oxaziridine and final deprotection with acidic Am-
berlyst IR15 afforded derivatives 15a (2S,3R) and 15b
(2R,3S) as mixture of diastereoisomers, due to the
nonstereoselective oxidation of the sulfide.
hydrolysis. The metal is coordinated to two histidine
residues, one cysteine residue, and a molecule of water,
which can donate a proton to Glu133 (Chart 1). The
X-ray structure of the E. coli PDF suggests a mechanism
for the deformylation of peptides.10,11 On the basis of
the structures of 1 and of the general substrates A, we
hypothesized that the complex formed with a hydrox-
amic acid mimics the transition state and therefore
predicted that smaller hydroxamic acid derivatives of
type B should be PDF inhibitors.
In this paper we describe the synthesis and the
evaluation of â-sulfonyl- and â-sulfinylhydroxamic acids
as PDF inhibitors and antibacterial agents.
Ch em istr y
The 3-substituted â-lactones 3 were prepared accord-
ing to literature precedent by an efficient [2+2] cyclo-
Resu lts a n d Discu ssion
1. In h ibition of th e Na tive E. coli P DF . The
â-sulfonylhydroxamic acid derivatives 8 were found to
be potent inhibitors of the native E. coli PDF. The
structure-activity relationship (SAR) is presented in
* To whom correspondence should be addressed. Tel: 41 61 688
5967. Fax: 41 61 688 6459. E-mail: christian.hubschwerlen@roche.com.
† Present address: Selectide Corp., A Subsidiary of Aventis Phar-
maceuticals, Tucson, AZ 85737.
10.1021/jm000018k CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/20/2000

Hydroxamic Acids as PDF Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2325
Ch a r t 1. Mechanism for the Deformylation of Peptides and Rational Design of PDF Inhibitors
Sch em e 1a,b
a
Reagents: methods A-D refer to general procedures described in the Experimental Section; (a) methods A & C: 2, DIPEA; methods
B & D: 3, CsF; (b) method A: NH2OBn, DDC; methods B & D: NH2O-Wang resin, HATU; method C: NH2OTBDMS, DCC; (c) method
A: Oxone; method B: mCPBA; methods C & D: 3-phenyl-2-(phenylsulfonyl)oxaziridine; (d) method A: H2, Pd/C; method B: TFA; (e)
b
method C: KF; method D: TFA. R1 and R2 are defined in Table 1.
Sch em e 2a,b
The activity of the inhibitors is also largely dependent
on the stereochemistry at the carbon in â-position to the
hydroxamic acid. This is illustrated with compound 8d ,
where one enantiomer turned out to be 100 times more
active than the other. An X-ray crystal structure of 8d
bound to PDF was determined (Figure 1). It is clearly
seen that the (R)-enantiomer is strongly bound in the
active site, which is consistent with modeling consid-
erations. The same configuration is found at the corre-
sponding C(â)-position in actinonin.
a
Reagents: (a) RuO4, NaIO4; (b) PhSH, LiClO4/Al2O3; (c)
The binding mode of (R)-8d (Figure 1) suggested that,
since only one sulfone oxygen is involved in a hydrogen-
bonding interaction and the other appears to make no
participating interaction with the protein, the corre-
sponding sulfoxide might bind just as well, or even
better. Compared to the corresponding â-(arylsulfonyl)-
hydroxamic acid template, the â-(arylsulfinyl)hydrox-
amic acid template gave slightly less potent inhibitors
(compare 9a and 8d or 9b and 8m ), but a similar trend
in the SAR was observed in both classes. The sulfoxide
functionality introduces an additional uncontrolled
chiral center leading to a mixture of epimers. Modeling
suggests that only one of the four isomers present in
the mixture can make a good hydrogen-bonding interac-
tion with the backbone NH of Ile44 (Figures 1 and 2).
The higher IC50 values observed for the sulfoxides 9 are
consistent with this suggestion.
NH2OTHP, CDI, HBT; (d) 3-phenyl-2-(phenylsulfonyl)oxaziridine;
(e) Amberlyst IR15. The enantiomeric series 10b-15b was
obtained using (-)-diethyl D-tartrate in the preparation of 10b.
b
Table 2. The activity of these inhibitors is strongly
influenced by the nature of the substituent R2, which
occupies the same position in the enzyme as the me-
thionine side chain of the natural substrates does.
Moreover, stronger inhibition was obtained with un-
substituted aromatic rings or linear aliphatic chains
(propyl to pentyl). Although the maximum activity was
observed with the n-butyl side chain (e.g. 8d ), E. coli
PDF can also accommodate bulkier groups (e.g. 8h ). For
the substituent R1, which could, in principle, mimic the
peptide backbone of the substrates, best activities were
obtained with aryl groups (e.g. 8l-p ). The nature and
the position of the substituent on the phenyl ring both
played an important role in determining the affinity
(compare 8m and 8n or 8m and 8p ). In particular, the
N-acetyl group in 8o induced the lowest IC50 value due
to further favorable contacts with the enzyme. Benzyl,
cycloalkyl, and long alkyl substituents led to less potent
inhibitors (e.g. 8i-k ).
Inspection of the complex of E. coli PDF with (R)-8d
(Figure 1) indicated that only small substituents are
tolerated in the R-position. As shown in Figure 2, an
R(S)-hydroxyl substituent was predicted to make favor-
able interactions through two additional donor and
acceptor H-bonds with residue Gly45, whereas an R(R)-

2326 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Ta ble 1. Preparation of â-Sulfonyl- and â-Sulfinylhydroxamic Acids
Apfel et al.
compd
R1
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
hexyl
cyclohexyl
benzyl
2-naphthyl
4-MeO-phenyl
3-MeO-phenyl
4-AcNH-phenyl
4-Br-phenyl
phenyl
4-MeO-phenyl
3-MeO-phenyl
2-naphthyl
4-Br-phenyl
phenyl
R2
R3
n
methoda
yieldb (%)
elemental anal.c
MS (m/z)
8a
8b
8c
8d
(R)-8d
(S)-8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
methyl
ethyl
propyl
butyl
butyl
butyl
H
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
1
1
1
1
A
A
A
A
Ag
Ag
A
A
A
A
A
A
A
A
A
B
A
B
C
C
D
C
D
E
45
67
74
82
97
98
63
67
62
51
90
76
21
95
79
2
88
10
22
11
5
45
13
63
34
NA
NA
C12H17NO4S
C13H19NO4S
NA
243d
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
OH
258.0e
272.0e
286.1e
284.1f
284.1f
300.3e
306.2e
348.2e
294.2e
292.3e
290.3f
299d
NA
pentyl
phenyl
C14H21NO4S
C15H15NO4S
C16H15NO6S
C13H13NO5S
C13H27NO4S
C13H25NO4S
C14H21NO4S
C17H21NO4S
C14H21NO5S
NA
C15H22N2O5S
NA
C13H19NO3S
NA
NA
NA
benzo[1,3]dioxol-5-yl
2-furanyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
butyl
334.2e
314.2e
314.2c
341.2e
362 364f
268.3f
298.4f
298.3f
318.2e
345 347f
284.1f
284.1f
9a
9b
9c
9d
9e
NA
NA
NA
15a h
15bi
phenyl
E
a
b
d
Methods A-E refer to general procedures described in the Experimental Section. For the final step. c C, H, N, S. EI (M). e ISP (M
+ H)+. f ISN (M - H)-. Compound obtained through HPLC separation on a chiral column. (2S,3R). (2R,3S).
g
h
i
Ta ble 2. Inhibition of Native PDF and in Vitro Antibacterial
Activity Against Selected Strains
MIC (µg/mL)
M. catarrhalis RA21
<0.06
PDF-Fea
IC50 (µM)
compd
E. coli DC2
1
8a
8b
8c
0.006
22.00
3.000
0.160
0.035
0.016
1.900
0.140
0.160
1.100
0.094
0.530
0.230
0.190
0.023
0.031
0.086
0.011
0.120
0.100
0.099
0.094
0.064
0.210
0.280
7.400
1
>32
>64
64
8
>32
>64
4
8d
<0.25
<0.25
16
<0.06
8
1
4
4
<0.25
16
0.5
0.5
2
1
2
<0.25
<0.25
0.25
<0.25
<0.125
<0.06
4
(R)-8d
(S)-8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
9a
9b
9c
9d
2
>128
2
32
16
32
64
8
128
8
F igu r e 1. X-ray crystal structure of (R)-8d bound to Ni-PDF.
The inhibitor is shown as ball-and-stick in white. The protein
CR trace is in blue, with selected active site residues in green.
The S1′ subsite of the protein is represented as a blue surface.
The Ni2+ ion is shown as a magenta ball, with red dashed
bonds to its three protein ligands and blue dashed bonds to
the two hydroxamate oxygens. The hydrogen bond from the
NH of Ile44 to a sulfone oxygen is also indicated.
16
16
16
16
1
2
4
2
1
9e
15a
15b
by these hydroxamic acids (e.g. compound 8d with IC50
0.096 and 0.34 µM, respectively, and compound 9a with
IC50 0.046 and 0.24 µM, respectively).
1
32
a
Inhibition of the isolated E. coli enzyme demonstrated to
contain mainly iron (IC50’s are mean values of at least 2 experi-
ments).
3. In h ibition of En d op r otea ses. The â-sulfonyl-
and â-sulfinylhydroxamic acid derivatives 8 and 9,
respectively, turned out to be highly selective for PDF
over representative endopeptidases found in the car-
diovascular system (Table 3). Less selectivity was
observed with respect to various matrix metallopro-
teases (MMP-1, -13, -7, -12).18-21 The relevance of this
latter finding for the use of this class of derivatives as
antibiotics remains to be evaluated, but it appears that
hydroxyl substituent would collide with the side chain
of Leu91 (Figure 1). This was confirmed by the greater
potency of 15a over 15b in the â-sulfinyl series.
2. In h ibition of P DF fr om Oth er Or ga n ism s. PDF
from B. subtilis and S. pneumoniae, used as purified
recombinant native enzymes, were similarly inhibited

Hydroxamic Acids as PDF Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2327
C. pneumoniae, M. pneumoniae, H. influenzae, and M.
catarrhalis.
5. Up t a k e of In h ib it or s 8d a n d 9a in E. coli.
Several lines of evidence indicate that antibacterial
activity is indeed related to inhibition of the peptide
deformylase.22 Nevertheless, the antibacterial activity
is generally lower than expected from the strong enzyme
inhibition. Therefore the intracellular uptake of radio-
labeled 8d and 9a was studied in E. coli (Figure 3). Both
compounds were rapidly taken up into the strain DC2,
which has a mutation in lipopolysaccharide (LPS),
allowing the outer membrane to become more perme-
able.23,24 An equilibrium concentration was established
within 3 min. The steady-state level reached was only
about one-fourth of that expected for equilibration.
Addition of the uncoupler CCCP (carbonyl cyanide
m-chlorophenyl hydrazone) at equilibrium clearly in-
creased the steady-state level of accumulation (Figure
3, open symbols). Using the parental strain, E. coli
UB1005, the accumulation was even lower, with a value
approximately 6-fold less than the equilibrium level
(Figure 3). The lower accumulation strongly indicates
that the outer membrane acts as a barrier for the
penetration of these compounds. Furthermore, the effect
of CCCP suggests the existence of an active efflux
system. We assume that both effects contribute to the
low intracellular concentration. Similar results were
obtained when the uptake under conditions close to the
ones used for in vitro antibacterial activity tests (pres-
ence of Iso-Sensitest broth (Oxoid); data not shown) was
measured.
F igu r e 2. Model predicting the binding mode of 15a . Color
scheme as in Figure 1. The predicted hydrogen-bonding
interaction with Gly45 is indicated.
Ta ble 3. Inhibition of Several Endopeptidases and MMPs by
Selected PDF Inhibitors
IC50 (µM)a
compd PDF-Fe ACE
ECE NEP COL-1 COL-3 MAT HME
>50 18 0.11 0.022 1.10 0.012
>100 2.50 0.150 0.079 2.10 0.042
>100 15 0.300 0.018 1.20 0.009
8d
0.035
81
34
(R)-8d 0.016
(S)-8d
8l
8j
1.9
0.023
0.230 >100
0.100 >100
>100
12.4 ∼450 0.50 0.070 0.009 1.50 0.013
78 1.65 0.054 0.100 0.29 0.014
>100 2.60 0.093 ndb ndb ndb
9a
a
ACE, angiotensin-converting enzyme; ECE, endothelin-con-
verting enzyme; NEP, neutral endopeptidase; COL-1, collagen-
ase-1 (MMP-1); COL-3, collagenase-3 (MMP-13); MAT, matrilysin
b
The sulfoxide 9a showed 8-16 times higher anti-
microbial activity than the sulfone 8d against E. coli
DC2. However, the intracellular concentration of both
compounds in E. coli DC2 was about 10 ng/mg dry
weight of cells at 100 µM of the external concentration.
Thus the higher antimicrobial activity of 9a compared
to 8d could not be explained by different uptake and
must be related to other factors.
(MMP-7); HME, human macrophage elastase (MMP-12). nd, not
determined.
this cross-reactivity would not be prohibitive for short-
term treatment. In addition, the opportunities for more
selective inhibitors are still not fully exploited. The
structural differences between the substrate recognition
sites of PDF and MMPs appear to allow the design and
synthesis of more selective inhibitors.
6. In Vitr o Resista n ce Develop m en t in E. coli.
The development of resistance was tested in vitro in E.
coli with 9a and found to occur in high frequency.22
Resistance development in S. aureus against actinonin
was also reported recently.25 Resistant mutants had a
slow growth phenotype as has been found in trans-
formylase/deformylase double knock-out mutants. This
indicates the presence of an escape mechanism, prob-
ably through formylation independent initiation, as is
the case in eukaryotes.3
4. An tiba cter ia l Activity in Vitr o. The PDF inhibi-
tors were routinely tested for antibacterial activity with
the permeable outer membrane mutant E. coli DC2 and
the pathogen M. catarrhalis RA21. Antibacterial activity
was observed for most compounds. This was especially
the case for the most potent enzyme inhibitors (Table
2). However, there was not a good correlation between
the IC50 values and the minimum inhibitory concentra-
tions (MIC). For example, compounds having long linear
R2 side chains (compare 8e and 8c) showed better
antibacterial activity than expected. We propose this
may be related to the greater lipophilicity of the
molecule, connected with better permeation of the
bacterial membrane. â-Sulfinylhydroxamic acid deriva-
tives were found to be consistently more potent against
bacteria than their corresponding â-sulfonyl derivatives
despite the stronger enzyme inhibition observed in the
latter series. The â-sulfinylhydroxamic acid derivative
9a and its corresponding â-sulfonyl derivative 8d were
further characterized as representative examples. The
antibacterial spectrum of these compounds is presented
in Table 4. Activity against the major pathogens S.
aureus, E. coli, and S. pneumoniae was observed albeit
at relatively high concentrations. In contrast, chemo-
therapeutically relevant activity was observed against
Con clu sion
Potent inhibition of bacterial PDF has been achieved
with low-molecular-weight hydroxamic acid derivatives
that were designed and synthesized based on the
discovery of actinonin in screening for inhibitors of E.
coli PDF. The â-(arylsulfonyl)- and â-(arylsulfinyl)-
hydroxamic acids 8 and 9 displayed useful antibacterial
activity. The sulfoxide 9a and the sulfone 8d were
further characterized as representative examples of
these classes, and their antibacterial spectra cover some
interesting pathogens found in respiratory tract infec-
tions such as C. pneumoniae, H. influenzae, M. pneu-
moniae, and M. catarrhalis. However, the development
of resistance toward these antibiotics in vitro22,25 as well

2328 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Apfel et al.
Ta ble 4. In Vitro Antibacterial Activity of Selected Inhibitors of PDF Against Different Species
MIC (µg/mL)
bacteria
erythromycin
1
8d
9a
Escherichia coli ATCC 25922
>64
4
64
2
128 (128)a
8 (2)
32
0.5
Escherichia coli DC2b
Pseudomonas aeruginosa ATCC 27853
Stenotrophomonas maltophilia 1AC739
Moraxella catarrhalis RA21
>64
>128
>128 (>128)
>128
<0.25
0.06
1
32
16
4
<0.125
2
<0.06
0.06
0.25
2
>64
<0.125
<0.125
>64
0.125
<0.25 (<0.25)
4 (2)
Haemophilus influenzae 11
2
>64
4
Mycoplasma pneumoniae ATCC 29342
Chlamydia pneumoniae AR-39
Staphylococcus aureus ATCC 25923
Enterococcus faecalis ATCC 29212
Enterococcus faecium vanA E25_1
Bacillus subtilis ATCC 585969
Streptococcus pneumoniae ATCC 49619
Streptococcus pneumoniae 1/1c
1 (0.5)
0.25
0.25
16
0.5 (0.5)
256 (128)
>128
32
>128
>128
8
32
128
>128
>128
0.25
32
>128
1 (1)
128 (128)
128
64
a
b
Values in parentheses refer to the (R)-8d enantiomer. Outer membrane permeable mutant. c Penicillin-resistant.
F igu r e 3. Uptake of hydroxamic acid derivatives in E. coli. Uptake of 100 µM each of (A) 3-[14C]-radiolabeled 9a or (B) 3-[14C]-
radiolabeled 8d , in E. coli DC2 (outer membrane permeable mutant) (b) or E. coli UB1005 (wild type) (9). Open symbols indicated
the uptake after 100 µM CCCP was added at 60 min, in DC2 (O) and UB1005 (0).
as a recent report26 stating that not all bacteria require
tal analyses are indicated by the symbol of the elements;
analytical results were within 0.4% of the theoretical values.
the formylation pathway poses some questions as to the
Abbr evia tion s: DIPEA, N-ethyldiisopropylamine; DCE,
dichloroethane; NMP, N-methylpyrrolidinone.
Meth od A: (RS)-3-(P h en ylsu lfon yl)h ep ta n oic Acid Hy-
d r oxya m id e (8d ). (a ) (RS)-3-(P h en ylsu lfa n yl)h ep ta n oic
validity of PDF as target for broad-spectrum antibacte-
rial chemotherapy.
The low intracellular inhibitor concentrations ob-
served in bacteria are certainly another disadvantage
for broad chemotherapeutic application of the described
hydroxamic acid derivatives. It remains to be seen if
PDF inhibition is a valid approach for the development
of antibacterials.
Acid Ben zyloxyam ide (5d; R1 ) P h ; R2 ) Bu ; R ) NHOBn ).
A solution of 2-heptenoic acid (650 mg; 5.0 mmol), PhSH (0.55
mL; 5.3 mmol) and DIPEA (0.96 mL; 5.5 mmol) in DMF (5
mL) was stirred at 60 °C for 48 h. The DMF was evaporated
under reduced pressure and the residue, dissolved in CH2Cl2
(20 mL), was reacted with BnONH2 (855 mg; 5.3 mmol),
1-hydroxybenzotriazole (766 mg, 5.0 mmol) and DCC (1.04 g;
5.0 mmol). After stirring overnight, the solution was diluted
with CH2Cl2 and washed with 1% NaHCO3 (10 mL), worked
up and chromatographed (AcOEt/n-hexane) affording 1.2 g of
Exp er im en ta l Section
Gen er a l. Solvents were dried by filtration through Al2O3
(neutral, Brockmann no. 1) when necessary and stored over a
bed of molecular sieves (3 Å). Hydroxylamine-Wang resin
(200-400 mesh, 1.04 mmol/g) was purchased from Bachem.
All organic solutions obtained after extraction were worked
up by washing with H2O and brine, dried over MgSO4 and
evaporated under reduced pressure with a H2O bath temper-
ature below 35 °C. Chromatography was performed using
Merck silica gel 60 (particle size 40-63 µm). Chiral separation
was performed on a 250-4 Chiralpak AD column (solvent
EtOH/n-hexane, 6:4). TLC was performed on Merck TLC plates
(silica gel 60 F254) and the plates were visualized with Cl2 and
a solution of o-toluidine. Proton NMR spectra were recorded
on a Bruker FT AC-250 (250 MHz) spectrophotometer in
DMSO-d6 solution (unless indicated otherwise). Chemical
shifts (δ) are reported in ppm relative to Me4Si as internal
standard. Coupling constants (J ) are given in Hz. IR spectra
were recorded on a Nicolet FT IR 20 SXB. Ion spray and EI
mass spectra were measured on a Finnigan MAT SSQ 7000
and Perkin-Elmer Siex API III recorder, respectively. Elemen-
1
a yellow oil (70%). H NMR: 0.83 (t, J ) 6, 3H); 1.2-1.6 (m,
6H); 2.23 (2dd, J ) 15 and 6, 2H); 3.50 (m, 1H); 4.78 (s, 2H);
7.38 (s, 10H); 11.1 (s, 1H).
(b) (RS)-3-(P h en ylsu lfon yl)h ep ta n oic Acid Ben zyloxy-
a m id e (6d ). A solution of 5d (1.2 g; 3.5 mmol) in acetone (75
mL) was treated dropwise with a solution of Oxone (3.8 g; 6.3
mmol) in H2O (25 mL) and stirred overnight at room temper-
ature. The acetone was evaporated under reduced pressure
and the aqueous layer was extracted twice with AcOEt. The
combined organic layers were worked up and chromatographed
(AcOEt/n-hexane, 2:1) affording 0.86 g of a colorless oil (65%).
IR (KBr): 1654, 1446, 1304, 1148 cm-1. 1H NMR: 0.77 (t, J )
6, 3H); 1.2-1.5 (m, 5H); 1.70 (m, 1H); 2.20 (dd, J ) 7.5 and
15, 1H); 2.50 (dd, J ) 6 and 15, 1H); 3.5 (m, 1H); 4.72 (s, 2H);
7.6-7.9 (m, 5H); 11.20 (s, 1H).
(c) (RS)-3-(P h en ylsu lfon yl)h ep ta n oic Acid Hyd r oxy-
a m id e (8d ). A solution of 6d (375 mg; 1 mmol) in MeOH (15
mL) was hydrogenated over 10% Pd/C (100 mg). After stirring

Hydroxamic Acids as PDF Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2329
for 2 h, the reaction mixture was diluted with CH2Cl2 and the
catalyst was filtered off. The filtrate was evaporated under
reduced pressure and the residue was crystallized from
CH2Cl2/hexane. The solid was purified by chromatography
(CH2Cl2/MeOH, 9:1) affording 234 mg (82%) of colorless
residue was purified by chromatography (AcOEt/n-hexane)
affording 1.8 g of light brown material (81%). MS (ISN): 366.2
(M - H)-. IR (film): 1659, 1529, 1256, 835 cm-1 1H NMR:
.
-0.06 and 0 (2s, 2 × 3H); 0.7-1.6 (m, 18H); 2.12 (d, J ) 6,
2H); 3.4 (m, 1H); 7.3 (m, 5H); 10.6 (s, 1H).
1
material. IR (film): 1666, 1447, 1303 cm-1. H NMR: 0.76 (t,
(b) Mixtu r e of (RS)- a n d (SR)-3-[(RS)-Ben zen esu lfin yl]-
h ep ta n oic Acid Hyd r oxya m id e (9a ). A solution of 5d ′ (1.8
g; 5 mmol) in CH2Cl2 (50 mL) was treated with 3-phenyl-2-
(phenylsulfonyl)oxaziridine (1.34 g; 51 mmol). The resulting
suspension was stirred for 2 h then evaporated to dryness
under reduced pressure. The residue was purified by chroma-
tography (AcOEt/hexane, 1:1) affording 290 mg of colorless
solid (22%). Mp: 138 °C. MS (ISN): 268.3 (M - H)-. IR
J ) 6, 3H); 1.2-1.5 (m, 5H); 1.75 (m, 1H); 2.2 (dd, J ) 8 and
15, 1H); 2.45 (dd, J ) 6 and 15, 1H); 3.57 (m, 1H); 7.8-7.95
(m, 5H); 8.95 (s, 1H); 10.61 (s, 1H).
Meth od B: (RS)-3-(4-Br om oben zen esu lfon yl)h ep ta n -
oic Acid Hyd r oxya m id e (8p ). (a ) 4-Bu tyloxeta n -2-on e (3;
R2 ) Bu ). A solution of trimethylsilyl ketene (6.4 g; 55 mmol)
in CH2Cl2 (20 mL) was added dropwise at -20 °C to a solution
of valeraldehyde (4.3 g; 50 mmol) and 0.5 M BF3‚Et2O solution
(1 mL) in CH2Cl2 (40 mL). After stirring at 0 °C for 1 h, 2
drops of H2O was added and the mixture was further stirred
at room temperature. The solvent was evaporated under
reduced pressure and the residue, dissolved in CH3CN (30 mL),
was further treated with KF hydrate (1 g). The mixture was
stirred for 2 h, the solid filtered off and the filtrate evaporated.
The residue was purified by chromatography (hexane/CH2Cl2/
AcOEt, 8:1:1) leaving 3.4 g (53%) of colorless oil. 1H NMR: 0.93
(t, J ) 6, 3H); 1.34-1.47 (m, 6H); 1.74-1.9 (m, 2H); 3.05 (dd,
J ) 5 and 18, 1H); 3.51 (dd, J ) 5 and 18, 1H); 4.46-4.55 (m,
1H).
1
(film): 1668, 1635, 11468, 1443, 1017 cm-1. H NMR: 0.87 (t,
J ) 7, 3H); 1.2-1.75 (m, 6H); 1.85 (dd, J ) 6 and 15; 1H); 2.1
(dd, J ) 8 and 15, 1H); 3.0 (m, 1H); 7.6 (m, 5H); 8.8 (s, 1H);
10.5 (br, 1H).
Meth od D: (RS)- a n d (SR)-3-[(RS)-4-Br om oben zen e-
su lfin yl]h ep ta n oic Acid Hyd r oxya m id e (9e). (a ) Oxid a -
tion w ith 2-(P h en ylsu lfon yl)-3-p h en yloxa zir id in e. The
resin (0.25 mmol) obtained in step 3 of method B was washed
with DCE (3 × 4 mL) and treated with a 0.25 M solution of
2-(phenylsulfonyl)-3-phenyloxaziridine (2.6 mL; 0.28 mmol) in
CH2Cl2. The reaction mixture was shaken for 10 h and filtered.
The resin was sequentially washed with DCE (3 × 4 mL),
isopropyl alcohol (3 × 3 mL) and DCE (3 × 4 mL). This was
used directly in the next step.
(b) 3-Bu tyl-3-(4-br om op h en ylsu lfa n yl)p r op ion ic Acid
(4p ). 4-Bromothiophenol (0.41 g; 2.2 mmol) was added to a
suspension of CsF (0.34 g; 2.2 mmol) in DMF (4 mL). The
suspension was stirred for 10 min, then treated with 3p (0.14
g; 1.1 mmol). The reaction was stirred overnight then filtered
and evaporated. The residue was purified by chromatography
(CH2Cl2/MeOH, 95:5) affording 0.29 g (83%) of pale yellow oil.
(b) (RS)- a n d (SR)-3-[(RS)-4-Br om oben zen esu lfin yl]-
h ep ta n oic Acid Hyd r oxya m id e (9e). The above-described
resin was treated as in the preparation of 8p (method B). The
crude product was purified by preparative HPLC to give 12
mg (13%) of 9e. MS (ISN): 345.2 and 347.2 (M - H)-. 1H
NMR: 10.4 (s, 1H, NH); 8.76 (s, 1H, OH); 7.76 (d, J ) 8, 2H);
7.54 (d, J ) 8, 2H); 2.99-3.10 (m, 1H); 1.98-2.18 (m, 2H);
1.12-1.48 (m, 6H); 0.84 (t, J ) 7, 3H).
1
MS (ISN): 315; 317 (M - H)-. H NMR: 0.83 (t, J ) 6, 3H);
1.18-1.7 (m, 6H); 2.38-2.62 (m, 2H); 3.36-3.50 (m, 1H); 7.36-
7.36 (d, J ) 8, 2H); 7.52-7.56 (d, J ) 8, 2H); 12.4 (b, 1H).
(c) Loadin g on a Hydr oxylam in e-Wan g Resin . Hydroxyl-
amine-Wang resin (250 mg; 0.25 mmol, 1% cross-linking, 38-
75 µΜ, 1 mmol/g) was swollen with DMA (5 mL). A solution
of 4p (120 mg; 0.38 mmol) in DMA (0.4 mL), was treated with
a 0.5 N solution of HATU (0.7 mL; 0.38 mmol) in NMP and
with a 1.5 N solution of DIPEA (0.7 mL; 1.13 mmol) in NMP.
After 5 min of shaking at room temperature, the solution was
added to the hydroxylamine-Wang resin. The reaction mixture
was shaken for 2 h, filtered off, and washed with DMA (3 × 4
mL) and with isopropyl alcohol (3 × 3 mL). This resin was
used directly in the next reaction step. ATR (attenuated total
Meth od E: 1:1 Mixtu r e of (2S,3R)-3-[(R)- a n d (S)-Ben -
zen esu lfin yl]-2-h yd r oxyh ep ta n oic Acid Hyd r oxya m id e
(15a ). (a ) (2S,3S)-(3-Bu tyloxir a n yl)m eth yl Alcoh ol (10a ).
A solution of tetraisopropyl orthotitanate (1.85 mL) in CH2Cl2
(300 mL) was cooled to -25 °C and treated sequentially with
6 g of powered, activated molecular sieves (4 Å) and 1.43 mL
of (+)-diethyl l-tartrate. The solution was further stirred for
15 min then treated with 45 mL of a 5 M solution of t-BuO2H
in CH2Cl2. The resulting solution was stirred for 30 min at
this temperature then treated dropwise with a solution of
trans-2-hepten-1-ol (12 g) in CH2Cl2 (40 mL). The reaction was
stirred at -20 °C overnight and quenched by addition of H2O
(60 mL). The mixture was further stirred at -24 °C for 30
min, then treated with a 30% NaOH solution (7 mL; saturated
with salt). The organic layer was worked up and evaporated
to leave a colorless oil. The excess of t-BuO2H was eliminated
by azeotropic distillation with toluene. The residue was
purified by chromatography (AcOEt/hexane, 3:7) and crystal-
lization from n-hexane at -20 °C. 1H NMR (CDCl3): 0.91 (t, J
) 6, 3H); 1.3-1.8 (m, 6H); 2.9 (m, 2H); 3.63 (m, 1H); 3.89 (m,
1H). [R]D -33° (MeOH; c 1). (2R,3R)-enantiomer 10b: [R]D
+32° (MeOH; c 1).
reflection) FTIR: 1670 cm-1
.
(d ) Oxid a tion w ith m CP BA. The above-described resin
(0.25 mmol) was washed with DCE (3 × 4 mL) and then
treated with a 0.25 M solution of mCPBA (4.0 mL; 1.0 mmol)
in CH2Cl2. The reaction mixture was shaken for 3 h, filtered
off, and washed with DCE (3 × 4 mL), isopropyl alcohol (3 ×
3 mL) and DCE (3 × 4 mL). This resin was used directly in
the next reaction step.
(e) (RS)-3-(4-Br om ob en zen esu lfon yl)h ep t a n oic Acid
Hyd r oxya m id e (8p ). The above-described resin was treated
with a solution of TFA/H2O/CH2Cl2 (4 mL; 70:2:28). The
reaction mixture was shaken for 3 h, filtered off and washed
with TFA/H2O/CH2Cl2 (2 mL; 70:2:28). The combined eluates
were evaporated. The residue was purified by preparative
HPLC (YMC Pack Pro C18 column, 5 µm, 120 Å, 50 × 20 mm;
H2O/CH3CN gradient) yielding 14 mg (10%) of solid.
Meth od C: Mixtu r e of (RS)- a n d (SR)-3-[(RS)-Ben zen e-
su lfin yl]h ep ta n oic Acid Hyd r oxya m id e (9a ). (a ) (RS)-3-
(P h en ylsu lfa n yl)h ep t a n oic Acid (ter t-Bu t yld im et h yl-
silyl)h yd r oxya m id e (5d ′; R1 ) P h , R2 ) Bu , R ) NHOT-
BDMS). A solution of trans-2-heptenoic acid (796 mg; 6 mmol),
PhSH (793 mg; 7 mmol) and DIPEA (1.54 mL; 9 mmol) in DMF
(10 mL) was stirred at 100 °C for 48 h. The DMF was
evaporated, and the residue, dissolved in CH2Cl2 (20 mL), was
treated with NH2O-TBDMS (1.06 g; 7.2 mmol) and DCC (1.56
g; 7 mmol). The solution was stirred for 4 h then treated with
Fuller’s earth (0.5 g) and the resulting suspension stirred for
30 min and filtered. The filtrate was concentrated and the
(b) (2R,3S)-3-Bu tyloxir a n e-2-ca r boxylic Acid (11a ). A
solution of 10a (9.5 g; 73 mmol) in a mixture of CH2Cl2/CH3CN/
H2O (450 mL; 1:1:2.5) was treated sequentially with RuO2 (122
mg; 0.073 mmol) and NaIO4 (63 g; 294 mmol) while the pH
was kept at 4.5 by addition of 2 N NaOH solution then 1 N
NaHCO3 solution by automatic titration. After stirring for 24
h, NaIO4 (31 g; 144 mmol) was added and the reaction was
further stirred. The pH of the reaction was brought to 8.5 by
addition of saturated Na2CO3 solution and the reaction mixture
was diluted with CH2Cl2 (400 mL). The organic layer was
separated and washed with H2O (100 mL). The combined
aqueous layers were acidified to pH 2 with 6 N H2SO4 at 0 °C
and extracted with CH2Cl2 (3 × 200 mL). The organic layer
was worked up to leave 3 g (28%) of colorless liquid. MS
1
(ISN): 143.2 (M - H)-. H NMR (CDCl3): 0.92 (t, J ) 6, 3H);
1.3-1.75 (m, 6H); 3.20 (dt, J ) 2 and 6, 1H); 3.26 (d, J ) 2,

2330 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12
Apfel et al.
1H); 10.0 (b, 1H). [R]D -19° (CHCl3; c 1). (2S,3R)-enantiomer
11b: [R]D +26° (CHCl3; c 1).
incubation at 37 °C for 18-24 h. Iso-Sensitest broth (Oxoid)
was used as the test medium. It was supplemented as
appropriate for testing fastidious bacteria. Susceptibility of M.
pneumoniae was tested in PPLO broth with phenol red.28
Activity against C. pneumoniae was tested with cell culture
of Hep-2 cells in 96-well microtiter plates.29
(c) (2S,3R)-2-Hydr oxy-3-(ph en ylsu lfan yl)h eptan oic Acid
(12a ). A suspension of LiClO4/Al2O3 (2 mmol/g; 6 g; 12 mmol)
in CH2Cl2 was treated with PhSH (1.32 g; 12 mmol) and stirred
at room temperature for 30 min. The resulting suspension was
treated with 11a (0.86 g; 6 mmol) and the resulting mixture
was further stirred overnight. The mixture was filtered and
evaporated. The residue was purified by chromatography
(CH2Cl2/MeOH, 9:1) affording 430 mg (28%) of colorless gum.
1H NMR: 0.82 (t, J ) 6, 3H); 1.2-1.7 (m, 6H); 3.4 (m, 1H);
4.02 (m, 1H); 7.40 (m, 5H).
Up ta k e of Ra d iola beled 8d a n d 9a in E. coli Cells. E.
coli DC2 is a LPS altered outer membrane hyperpermeable
mutant23,24 of parental strain UB1005. 3-[14C]-Radiolabeled 8d
and 9a (specific activity, 11.3 and 13.0 mCi/mmol, respectively)
were used for the study. Bacteria were grown to the late
exponential phase at 37 °C in medium A (0.7% K2HPO4, 0.3%
KH2PO4, 0.05% trisodium citrate, 0.01% MgSO4‚7H2O, 0.1%
(NH4)2SO4) supplemented with 0.2% glucose, 0.1% casamino
acids, 1 µg/mL thiamine chloride, and 10 µg/mL methionine.
Cells were harvested, washed twice with 50 mM of MOPS-
KOH buffer (pH 6.6) and suspended to OD600 ) 20 with the
same buffer containing 0.2% glucose and 100 µg/mL chlor-
amphenicol. Cells were vigorously shaken at 37 °C for 20 min.
Uptake was started by adding 100 µL of radiolabeled com-
pound to the same volume of cells (0.76 mg dry weight) and
incubated at 30 °C. At indicated times, 3 mL of wash buffer
(50 mM MOPS-KOH, pH 6.6, 5 mM citrate, and 1% DMSO)
was added and cells were immediately filtered through What-
man GF/F filters. Filters were washed, dried, and counted with
a liquid scintillation counter. We suggest that the washing
procedure causes some leakage of the compounds which can
explain the difference between the maximal equilibrium
measured under uncoupling conditions and the expected
calculated level. The intracellular concentration was calculated
with cell volume, determined by the method reported by
Rottenberg30 as 1.55 µL/mg dry weight of cells for DC2 strain.
Str u ctu r e Deter m in a tion . For crystallization and X-ray
structure determination, E. coli PDF containing nickel was
used.9 Crystals of space group C2 were obtained under similar
conditions to what these authors used and soaked with 1 mM
8d in 54% ammonium sulfate, 100 mM Tris, pH 8.5. Diffrac-
tion data were collected at 15 °C to 3.0 Å on an Enraf Nonius
FR571 generator equipped with a graphite monochromator
and a Siemens Hi-Star detector. Data were processed with the
program XDS31 and reduced with the CCP4 package.32 The
unit cell parameters were a ) 142.91 Å, b ) 63.90 Å, c ) 84.44
Å, â ) 123.1°. The inhibitor density, and in particular the
stereochemistry, was clear from a difference map computed
against a previously determined model of Ni-PDF. The inhibi-
tor complex was refined with program CNS33 to a final R-factor
of 22.4% (R-free 26.2%) with tight geometrical constraints (rms
bond errors 0.007 Å, rms angle errors 1.29°). Modeling and
figure generation were performed with the in-house program
Moloc.34
(d) (2S,3R)-2-Hydr oxy-3-(ph en ylsu lfan yl)h eptan oic Acid
(Tetr a h yd r op yr a n -2-yloxy)a m id e (13a ). A solution of 1,1′-
carbonyldiimidazole (331 mg; 2 mmol) and 1-hydroxybenzo-
triazole (276 mg; 2 mmol) in CH3CN (10 mL) was sequentially
treated with 12a (432 mg; 17 mmol) and after 30 min NH2-
O-THP (220 mg; 19 mmol). The mixture was then heated
under reflux overnight. The reaction mixture was evaporated
under reduced pressure and the residue was redissolved in
CH2Cl2. The organic layer was washed with NaHCO3, and
further worked up and chromatographed (n-hexane/AcOEt,
1
2:1) leaving 110 mg (18%) of colorless oil. H NMR: 0.85 (t, J
) 6, 3H); 0.75-1.75 (m, 12H); 3.45 and 4.05 (2m, 2 × 1H);
4.90 (m, 1H); 6.0 (d, J ) 6, 1H); 7.2-7.5 (m, 5H); 11.05 (2s,
1H).
(e) (2S,3R)-3-Ben zen esu lfin yl-2-h ydr oxyh eptan oic Acid
(Tetr a h yd r op yr a n -2-yloxy)a m id e (14a ). A solution of 13a
(110 mg; 0.31 mmol) dissolved in CH2Cl2 (10 mL) was reacted
with 3-phenyl-2-(phenylsulfonyl)oxaziridine (89 mg; 0.34 mmol)
overnight at room temperature. The organic layer was worked
up and chromatographed (n-hexane/AcOEt, 1:1) leaving 60 mg
(52%) of a colorless foam. MS (EI): 285 (M - THP - H)-. IR
1
(MIR): 1680, 1443, 1114, 1034, 1018 cm-1. H NMR: 0.57 (t,
J ) 6, 3H); 0.75-1.75 (m, 12H); 2.85 (m, 1H); 3.45 and 4.05
(2m, 2 × 1H); 4.60 (d, J ) 6, 1H); 4.90 (d, J ) 5, 1H); 6.15 (t,
J ) 6, 1H); 7.61 (m, 3H); 7.79 (m, 2H); 11.2 (2s, 1H).
(f) 1:1 Mixtu r e of (2S,3R)-3-[(R)- an d (S)-Ben zen esu lfin -
yl]-2-h yd r oxyh ep ta n oic Acid Hyd r oxya m id e (15a ). A
solution of 14a (35 mg, 0.1 mmol) dissolved in a mixture of
CH2Cl2/MeOH/H2O (3:2:1; 6 mL) was treated with Amberlyst
IR15 (10 mg) for 3 days. The mixture was filtered and washed
with MeOH. The solvents were evaporated under reduced
pressure and the residue was purified by chromatography
(AcOEt) leaving 18 mg (63%) of colorless powder. MS (ISN):
1
284.3 (M - H)-. H NMR: 0.58 (t, J ) 6, 3H); 0.75-1.75 (m,
6H); 2.85 (m, 1H); 4.55 (dd, J ) 2 and 5, 1H); 6.05 (d, J ) 6,
1H); 7.61 (m, 3H); 7.79 (m, 2H); 8.77 (s, 1H); 10.7 (s, 1H).
En zym e In h ibition . E. coli PDF was isolated, as recom-
binant enzyme from E. coli, in its native iron containing form
and purified according to the literature method.9 PDF activity
was measured at room temperature in a microtiter plate
format with 5 mM formyl-methionine-alanine-serine (f-MAS)
at pH 7.2 (100 mM MOPS-KOH, 250 mM KCl, 0.4% BSA, 100
µg/mL catalase) in a total volume of 50 µL. The reaction was
initiated by the addition of PDF, Fe-PDF with approximately
1100 U/mg (final concentrated: 5.7 nM), and stopped after 5
min. with 200 µL of fluorescamine. After 30 min at room
temperature, the optical density was measured at 380 nm and
the amount of hydrolyzed substrate determined against a
standard curve of MAS which was run in parallel. Inhibitors
were tested at a final concentration of 1% DMSO.
Ack n ow led gm en t. We are grateful to our col-
leagues from the Central Units for performing the
spectroscopic measurements and for the determination
of the microanalysis and our colleagues from NRRC for
running the enzyme inhibition screen. We also thank
Dr. Ph. Huguenin and R. Preiswerk for the synthesis
of the radiolabeled compounds 8d and 9a ; E. Bald, F.
Gerber, E. Philipp, B. Prud’hon, D. Zulauf, C. Lacoste,
B. Rutten, and O. Partouche for their excellent technical
assistance; and Dr. H. Matile for testing antichlamydial
activity. For enzyme preparation, we thank Dr. B.
Taka´cs, R. Remy, and B. Gsell. For crystal preparation,
we thank M. Stihle and A. D’Arcy. We thank also D.
Blum and Dr. B. Lo¨ffler for assays with ACE, ECE, and
NEP and R. T. MacKenzie for determination of metallo-
protease inhibition. We thank Dr. V. Wagner, Univer-
sity of Heidelberg, Germany, for valuable advice on
enzyme preparation and properties.
Inhibition of ACE, ECE, NEP and several matrix metallo-
proteases (MMP) was determined according to known litera-
ture procedures.18-21
In Vitr o An tiba cter ia l Activity. Bacterial strains used
in this study were from our own in-house strain collection.
They were obtained from the American Type Culture Collec-
tion (ATCC) and from various hospitals and kept at - 80 °C.
The minimal inhibitory concentrations (MICs) of the test
compounds were determined by microdilution following the
guidelines of the National Committee for Clinical Laboratory
Standards.27 The MIC of a compound was defined as the lowest
concentration that prevents visible growth of bacteria after
Su p p or t in g In for m a t ion Ava ila b le: Yield of prepara-
tion and MS and NMR data of intermediates 4-6. This

Hydroxamic Acids as PDF Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 12 2331
(18) Carmel, A.; Ehrlich-Rogozinsky, S.; Yaron, A. A fluorometric
assay for angiotensin-I converting enzyme in human serum.
Clin. Chim. Acta 1979, 93, 215-220.
material is available free of charge via the Internet at
http://pubs.acs.org.
(19) Carvalho, K. M.; Boileau, G.; Camargo, A. C. M.; J uliano, L. A
highly selective assay for neutral endopeptidase based on the
cleavage of a fluorogenic substrate related to Leu-enkephalin.
Anal. Biochem. 1996, 237, 167-173.
Refer en ces
(1) Meinnel, T.; Lazennec, C.; Villoing, S.; Blanquet, S. Structure-
function relationships within the peptide deformylase family.
Evidence for a conserved architecture of the active site involving
three conserved motifs and a metal ion. J . Mol. Biol. 1997, 267,
749-761.
(2) Mazel, D.; Coic, E.; Blanchard, S.; Saurin, W.; Marliere, P. A
survey of polypeptide deformylase function throughout the
eubacterial lineage. J . Mol. Biol. 1997, 266, 939-949.
(3) Mazel, D.; Pochet, S.; Marliere, P. Genetic characterization of
polypeptide deformylase a distinctive enzyme of eubacterial
translation. EMBO J . 1994, 13, 914-923.
(4) Lelievre, Y.; Bouboutou, R.; Cartwright, T. Inhibition of synovial
collagenase by actinonin. Structure-activity relationship. Pathol.
Biol. 1989, 37, 43-46.
(5) Umezawa, H.; Aoyagi, T.; Tanaka, T.; Suda, H.; Okuyama, A.;
Naganawa, H.; Hamada, M.; Takeuchi, T. Production of actino-
nin, an inhibitor of aminopeptidase M, by actinomycetes. J .
Antibiot. 1985, 38, 1629-1630.
(6) Broughton, B. J .; Chaplen, P.; Freeman, W. A.; Warren, P. J .;
Wooldridge, K.; Wright, D. E. Studies concerning the antibiotic
actinonin. VIII Structure-activity relations in the actinonin
series. J . Chem. Soc., Perkin Trans. 1 1975, 9, 857-860.
(7) Rajagopalan, P. T. R.; Yu, X. C.; Pei, D. Peptide deformylase, a
new type of mononuclear iron protein. J . Am. Chem. Soc. 1997,
119, 12418-12419.
(8) Becker, A.; Schlichting, I.; Kabsch, W.; Schultz, S.; Wagner, A.
F. V. Structure of peptide deformylase and identification of the
substrate binding site. J . Biol. Chem. 1998, 273, 11413-11416.
(9) Groche, D.; Becker, A.; Schlichting, I.; Kabsch, W.; Schultz, S.;
Wagner A. F. V. Isolation and crystallization of functionnally
competent Escherichia coli peptide deformylase forms containing
either iron or nickel in the active site. Biochem. Biophys. Res.
Commun. 1998, 246, 342-346.
(10) Becker, A.; Schlichting, I.; Kabsch, W.; Groche, D.; Schultz, S.;
Wagner A. F. V. Iron centre, substrate recognition and mecha-
nism of peptide deformylase. Nat. Struct. Biol. 1998, 5, 1053-
1058.
(20) Schweizer, A.; Loeffler, B. M.; Rohrer, J . Palmitoylation of the
three isoforms of human endothelin-converting enzyme-1. Bio-
chem. J . 1999, 340, 649-656.
(21) Knight, C. G.; Willenbrock, F.; Murphy, G. A novel coumarin-
labeled peptide for sensitive continuous assays of the matrix
metalloproteinases. FEBS Lett. 1992, 296, 263-266.
(22) Apfel, C. Manuscript in preparation.
(23) Richmond, M. H.; Clark, D. C.; Wotton, S. Indirect method for
assessing the penetration of â-lactamase-nonsusceptible penicil-
lins and cephalosporins in Escherichia coli strains. Antimicrob.
Agents Chemother. 1976, 10, 215-218.
(24) Rocque, W. J .; Fesik, S. W.; Haug, A.; McGroarty, E. J . Polycation
binding to isolated lipopolysaccharide from antibiotic-hypersus-
ceptible mutant strains of Escherichia coli. Antimicrob. Agents
Chemother. 1988, 32, 308-313.
(25) Margolis, P.; Hackbarth, C.; White, R.; Lopez, S.; Trias, J .
Resistance to deformylase inhibitor VRC483 is caused by muta-
tions in formylase transferase. 39th Annual ICAAC Meeting, San
Francisco, CA, 1999; Abstract No. 1795, Book of Abstracts, 334.
(26) Newton, D. T.; Creuzenet, C.; Mangroo D. Formylation is not
essential for initiation of protein synthesis in all eubacteria. J .
Biol. Chem. 1999, 274, 22143-22146.
(27) National Committee for Clinical Laboratory Standards. Methods
for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically, 3rd ed.; National Committee for Clinical
Laboratory Standards: Villanova, PA, 1993; Vol. 13, No. 25, M7-
A3.
(28) Kaku, M.; Ishida, K.; Irifune, K.; Mizukane, R.; Takemura, H.;
Yoshida, R.; Tanaka, H.; Usui, T.; Tomono, K.; Suyama, N.;
Koga, H.; Kohno, S.; Hara, K. In vitro and in vivo activities of
sparfloxacin against Mycoplasma pneumoniae. Antimicrob. Agents
Chemother. 1994, 38, 738-741.
(29) Roblin, P. M.; Montalban, G.; Hammerschlag, M. R. Susceptibili-
ties to clarithromycin and erythromycin of isolates of Chlamydia
pneumoniae from children with pneumonia. Antimicrob. Agents
Chemother. 1994, 38, 1588-1589.
(11) Hao, B.; Gong, W.; Rajagopalan, R.; Zhou, Y.; Pei, D.; Chan, M.
K. Structural basis for the design of antibiotics targeting peptide
deformylase. Biochemistry 1999, 38, 4712-4719.
(30) Rottenberg, H. Proton electrochemical potential gradient in
vesicles, organelles, and prokaryotic cells. Methods Enzymol.
1989, 172, 63-84.
(12) Ruden, R. A. Trimethylsilylketene. Acylation and Olefination
Reactions. J . Org. Chem. 1974, 39, 3607-3608.
(31) Kabsch, W. Evaluation of single-crystal X-ray diffraction data
from a position-sensitive detector. J . Appl. Crystallogr. 1988,
21, 916-924.
(13) Woon, H.; Romo, D. Studies of asymmetric [2+2] cycloaddition
of silylketenes and aldehydes employing Ti-TADDOL catalysts.
Tetrahedron Lett. 1998, 39, 2877-2880.
(32) Bailey, S. Collaborative Computational Project, Number 4. The
CCP4 suite: programs for protein crystallography. Acta Crys-
tallogr. 1994, D50, 760-763.
(14) Davis, A. F.; Stringer, O. D. Chemistry of oxaziridines. 2.
Improved synthesis of 2-sulfonyloxaziridines. J . Org. Chem.
1982, 47, 1774-1775.
(33) Bru¨nger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros,
P.; Grosse-Kunstleve, R. W.; J iang, J .-S.; Kuszewski, J .; Nilges,
M.; Pannu, N. S.; Read, R. J .; Rice, L. M.; Simonson, T.; Warren,
G. L. Crystallography and NMR system: a new software suite
for macromolecular structure determination. Acta Crystallogr.
1998, D54, 905-921.
(15) Wang, Z.; Zhou, W. Asymmetric epoxidation of allylic alcohol
by modified Sharpless reagent. Tetrahedron 1987, 43, 2935-
2944.
(16) Paddon-J ones, G.; Moore, C.; Brecknell, D.; Koenig, W.; Kitching
W. Synthesis and absolute stereochemistry of Hagen’s-Gland
lactones in some parasitic waspes (hymenoptera: braconidae).
Tetrahedron Lett. 1997, 38, 3479-3482.
(17) Oguri, T.; Shioiri, T.; Yamada, S. Amino acids and peptides. XV.
New synthesis of R-amino acids by amination of R-metalated
carboxylic acids. Chem. Pharm. Bull. 1975, 23, 167-172.
(34) Gerber, P. R.; Mu¨ller, K. MAB: A generally applicable molecular
force field for structure modelling in medicinal chemistry. J .
Comput.-Aided Mol. Des. 1995, 9, 251-268.
J M000018K