2-(2-oxo-1,4-dihydro-2H-quinazolin-3-yl)- and 2-(2,2-dioxo-1,4-dihydro-2H-2λ6-benzo [1,2,6]thiadiazin-3-yl)-N-hydroxy-acetamides as potent and selective peptide deformylase inhibitors

Article abstract of DOI:10.1021/jm000352g

Potent, selective, and structurally new inhibitors of the Fe(II) enzyme Escherichia coli peptide deformylase (PDF) were obtained by rational optimization of the weakly binding screening hit (5-chloro-2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-acetic acid hydra

Full text of DOI:10.1021/jm000352g

J . Med. Chem. 2001, 44, 1847-1852
1847
Articles
2-(2-Oxo-1,4-d ih yd r o-2H-qu in a zolin -3-yl)- a n d
2-(2,2-Dioxo-1,4-d ih yd r o-2H-2λ6-ben zo[1,2,6]th ia d ia zin -3-yl)-N-h yd r oxy-a ceta m id es
a s P oten t a n d Selective P ep tid e Defor m yla se In h ibitor s
Christian Apfel, David W. Banner, Daniel Bur,† Michel Dietz, Christian Hubschwerlen,‡ Hans Locher,‡
Fre´de´ric Marlin, Raffaello Masciadri,* Wolfgang Pirson, and Henri Stalder
Discovery Chemistry, F. Hoffmann-La Roche Ltd., CH-4070 Basle, Switzerland
Received August 14, 2000
Potent, selective, and structurally new inhibitors of the Fe(II) enzyme Escherichia coli peptide
deformylase (PDF) were obtained by rational optimization of the weakly binding screening hit
(5-chloro-2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-acetic acid hydrazide (1). Three-dimensional
structural information, gathered from Ni-PDF complexed with 1, suggested the preparation of
two series of related hydroxamic acid analogues, 2-(2-oxo-1,4-dihydro-2H-quinazolin-3-yl)-N-
hydroxy-acetamides (A) and 2-(2,2-dioxo-1,4-dihydro-2H-2λ6-benzo[1,2,6]thiadiazin-3-yl)-N-
hydroxy-acetamides (B), among which potent PDF inhibitors (37, 42, and 48) were identified.
Moreover, two selected compounds, one from each series, 36 and 41, showed good selectivity
for PDF over several endoproteases including matrix metalloproteases. However, these
compounds showed only weak antibacterial activity.
In tr od u ction
Scr een in g, X-r a y, a n d Mod elin g Resu lts
High-throughput screening of our compound reposi-
tory in an Escherichia coli PDF based assay afforded
the weakly binding (IC50 ) 27µM) hydrazide 1 (Scheme
1), which was successfully cocrystallized with E.coli Ni-
PDF (Figure 1): The hydrazide group of 1 was observed
to chelate the Ni2+ cation bidentally. The 5-chloro
substituent of hydrazide 1 was localized in the hydro-
phobic P1′ pocket which accommodates the side chain
of methionine in the natural enzyme/substrate complex.
The urea carbonyl group of 1 did not undergo a
hydrogen-bonding interaction with the enzyme. Com-
pound 2 (Scheme 1), the R-methyl analogue of hydrazide
1, was devoid of PDF inhibitory activity, indicating an
unfavorable steric interaction with the protein in the
vicinity of the hydrazide function. From our X-ray
structure of PDF complexed with actinonin,18 we knew
that the backbone NH of Ile44 forms an H-bond with
the substrate-like inhibitor actinonin. We superimposed
the two structures of PDF complexed with actinonin and
hydrazide 1, respectively, and speculated that a tetra-
hedral SO2 group instead of the in-plane carbonyl group
of 1 might undergo a similar hydrogen-bonding inter-
action with the backbone (Figure 2).
Peptide deformylase (PDF, EC 3.5.1.27) is believed
to be an essential enzyme in both Gram-positive and
Gram-negative bacteria.1-3 In eukaryotes, gene se-
quences similar to def (PDF gene) have been identified,
but their functions have not yet been rigorously estab-
lished.4,5 PDF catalyzes the removal of a formyl group
attached to the N-terminus of the leading methionine
from nascent polypeptide chains. Initial studies with
PDF were hampered by the fact that PDF was isolated
accidentally as an almost inactive but thermodynami-
cally stable zinc complex.6 The native enzyme, however,
turned out to be the oxidatively very labile Fe(II)
complex.7,8 The mechanism of action of PDF has been
extensively studied.9,10 The search for selective inhibi-
tors of PDF has relied on modifying the thermolysin
inhibitor thiorphane,11-13 the calpain inhibitor calpep-
tin,14 the naturally occurring antibacterial hydroxamate
actinonin,15-18 the angiotensin II receptor antagonists
from the biphenyl tetrazole class,19 and the anticholes-
teremic thyropropic acid.20 Recently, the orally bioavail-
able N-formyl hydroxylamine BB-3497 has given mo-
mentum to the validity of PDF as a novel antibacterial
target.21
On the basis of our X-ray and modeling studies, we
proposed the general structures A and B (Scheme 1),
wherein the hydrazide group of 1 was replaced by a
hydroxamic acid function resulting in stronger metal
coordination. Substituent R1 should be a small lipophilic
substituent (F, Cl, Br, or CF3), filling the hydrophobic
methionine pocket optimally. Substituent R2, which
points toward the surface of the enzyme, would probably
allow broader structural variation.
* To whom correspondence should be addressed: Raffaello Mas-
ciadri, Ph.D., F. Hoffmann-La Roche Ltd., Pharmaceuticals Division,
Discovery Chemistry PRBC-L, Bldg. 092/3.76, CH-4070 Basel, Swit-
zerland. Phone: 0041 61 688 75 04. Fax: 0041 61 688 64 59. E-mail:
raffaello.masciadri@roche.com.
† Current address: Actelion, Gewerbestr. 16, CH-4123 Allschwil,
Switzerland.
‡ Current address: Morphochem AG, Schwarzwaldallee 215, CH-
4058 Basel, Switzerland.
10.1021/jm000352g CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/05/2001

1848 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Apfel et al.
ortho position (10, R1 ) CF3).23 The resulting benzyl
bromides 8-10 were then used to monoalkylate glycine
ethyl ester with NaH in DMF (method B). Bisalkylation
was not observed since the benzyl bromides 8-10 are
deactivated by the bulk and the electron-withdrawing
nature of the two ortho substituents (NO2 and R1 ) Cl,
Br, CF3). The nitro groups of 11 and 12 were then
reduced chemoselectively by catalytic hydrogenation
over Raney nickel which left the sensitive substituents
(R1 ) Cl, Br) untouched (method C-1).24 The nitro group
of 13 was reduced simply by catalytic hydrogenation
over Pd/C in THF (method C-2). Ring closure of 14-18
was achieved with either bis(trichloromethyl)carbonate
in dioxane (method E)25 or with excess sulfamide in
refluxing pyridine (method F).26 The resulting com-
pounds 19-28 were directly transformed into the cor-
responding hydroxamic acids 34-43 with an excess of
potassium salt of hydroxylamine in THF/water at 0 °C
(method H).27 This transformation had to be controlled
carefully because under the basic reaction conditions
partial hydrolysis to the corresponding carboxylic acid
can occur. The target compounds 34-43 (R2 ) H) were
quite insoluble and precipitated either directly from the
reaction medium or from the organic layer after extrac-
tion. The more lipophilic target compounds 44-48 (R2
* H) could be extracted and purified by silica gel
chromatography, thus removing traces of the corre-
sponding carboxylic acid. The 1H NMR spectra of
compounds 34-48 revealed that they exist in solution
as mixture of E/Z-rotamers leading to doubling of most
signals. A more laborious route to the hydroxamic acids
34-48 involved 1,1′-carbonyldiimidazole coupling of the
acids derived from esters 21-33 with O-protected
hydroxylamines (Bn, THP, MOM, TBDMS), followed by
deprotection. While the coupling was straightforward,
clean deprotection was difficult to reproduce, and we
therefore abandoned this route. The library of final
compounds was extended by introducing lipophilic sub-
stituents R2 (benzyl, n-butyl, 3-phenylpropyl, and 1-cy-
clopropylmethyl) into compound 26 or 27 via standard
alkylation with the corresponding alkyl bromides (method
G), affording the intermediates 29-33, which were
transfomed into the corresponding hydroxamic acids
44-48 by method H.
Sch em e 1
F igu r e 1. X-ray crystal structure of hydrazide 1 bound to Ni-
PDF. The inhibitor is shown as ball-and-stick in white. The
protein CR trace is in cyan, with selected active site residues
in red. The Ni2+ ion is shown as a magenta ball, with blue
dashed bonds to its three protein ligands and to the hydrazide
oxygen and nitrogen.
Biologica l Resu lts a n d Discu ssion
1. In h ibition of Na tive E. coli P DF . The proposed
structural modifications of the initial screening hit 1
(Scheme 1) led to the discovery of potent inhibitors of
the native E. coli PDF (Table 1). Compound 36, the
hydroxamate analogue of the original hydrazide 1, was
90 times more potent. Compound 41, the SO2 analogue
of 36, was another 3 times more active, less than one
would have expected for an additional strong H-bond
but still beneficial. The small lipophilic substituent R1
(F, Cl, Br, CF3) was crucial for activity. The bromo
substituent was optimum in size, as demonstrated with
the most potent inhibitor 37, which was 40 times more
potent than its nonsubstituted analogue 34. In series
B, this phenomenon was less pronounced than in series
A, where the three compounds 41 (R1 ) Cl), 42 (R1 )
Br), and 43 (R1 ) CF3) were almost equally potent.
Attachment of a lipophilic substituent R2 to compound
41 did not improve the potency as demonstrated with
derivatives 44-47 (R2 ) benzyl, n-butyl, 3-phenylpropyl,
F igu r e 2. Model predicting the binding mode of hydroxamate
41. Color scheme is as in Figure 1. The predicted hydrogen-
bonding interaction with Ile44 is indicated with a white dashed
line.
Ch em istr y
The choice of our synthetic routes (Scheme 2) was
guided by the availability of suitable starting materials
already carrying the substituent R1. The 2-amino-
benzylamines 3 (R1 ) H) and 4 (R1 ) F) were monoalky-
lated with ethyl bromoacetate in dioxane at reflux
(method D).22 The 2-nitrotoluenes 5-7 (R1 ) Cl, Br, CF3)
were readily photobrominated (method A) despite the
presence of strongly deactivating substituents in the

Inhibitors of Peptide Deformylase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1849
Sch em e 2a
a
Reagents: (a) NBS, CCl4, cat. BPO, light, reflux (method A); (b) ethyl glycinate, NaH, DMF, rt (method B); (c) RaNi, H2, TEA, THF
(method C-1); or Pd/C, H2, THF (method C-2); (d) BrCH2CO2Et, TEA/dioxane, reflux (method D); (e) bis(trichloromethyl)carbonate, dioxane,
rt (method E); (f) sulfamide, pyridine, reflux (method F); (g) R2Br, NaH, DMF (method G); (h) 5 equiv NH2OK, THF/H2O, rt (method H).
Ta ble 1. Preparation, Analytical Data, Inhibition of Native PDF, and in Vitro Antibacterial Activities against Selected Strains
MIC (µg/mL)
yieldb
(%)
mp
(°C)
MS
(m/z)
IC50 (µM)
compd
R1
Cl
H
F
Cl
Br
CF3
H
R2
X
methoda
nd
D, E, H
D, E, H
A-C, E, H
A-C, E, H
A-C, E, H
D, F, H
elem. analysisc
C10H11ClN4O2
PDF Fef
E.c.g
H.i.h
M.c.i
1
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
H
H
H
H
H
H
H
H
H
H
H
CO
CO
CO
CO
CO
CO
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
nd
15
56
58
62
45
63
55
52
50
35
55
37
13
80
60
>300
190
215
209
208
198
foam
144
foam
85
145
foam
143
125
145
foam
255d
220e
238e
254e
300e
288e
256e
274e
290e
336e
324e
382e
346e
408e
344e
388e
27
50
>64
>64
16
32
64
>64
>64
>64
32
>64
64
nd
>64
64
nd
>64
16
2
C
C
C
C
10H11N3O3
1.900
0.870
0.310
0.049
0.230
0.760
0.870
0.120
0.098
0.130
2.000
0.590
0.290
0.110
0.069
10H10FN3O3
10H10ClN3O3
10H10BrN3O3
8
16
64
4
8
C11H10F3N3O3
C9H11N3O4S
>64
>64
16
>64
64
8
F
D, F, H
C9H10FN3O4S
C9H10ClN3O4S
C9H10BrN3O4S
C10H10F3N3O4S
C16H16ClN3O4S
C13H18ClN3O4S
C18H20ClN3O4S
C13H16ClN3O4S
C13H16BrN3O4S
Cl
Br
CF3
Cl
Cl
Cl
Cl
Br
A-C, F, H
A-C, F, H
A-C, F, H
A-C, F-H
A-C, F-H
A-C, F-H
A-C, F-H
A-C, F-H
>64
32
32
64
32
64
64
4
8
8
2
8
4
1
benzyl-
n-butyl
Ph(CH2)3-
c-PrCH2-
c-PrCH2-
>64
>64
>64
64
a
b
d
Methods A-H refer to the general procedures described in the Experimental Section. For the final step. c C, H, N. ISP (M + H)+.
e ISN (M - H)-. f Inhibition of the isolated E. coli enzyme demonstrated to contain mainly iron. E. coli DC2. H. influenzae 11. M.
catarrhalis RA21.
g
h
i
1-cyclopropylmethyl). In contrast to our modeling ex-
pectations, the N-benzyl derivative 44 was almost 20
times less active, and this was the case for a whole
series of benzyl analogues substituted on the benzene
ring (data not shown). The best substituent R2 was
1-cyclopropylmethyl (compound 47), which brought the
activity back to the level of 41. A combination of the
best substituents R1 (Br) and R2 (1-cyclopropylmethyl)
afforded the second most potent compound 48 of the
whole series.
Ta ble 2. Inhibition of Several Endopeptidases and MMPs by
the PDF Inhibitors
IC50 (µM)a
compd
PDF-Fe
ACE
ECE
NEP
COL-3
HME
36
41
0.31
0.12
90
>100
>10
>10
>100
>100
0.46
>0.5
>100
>100
a
ACE, angiotensin-converting enzyme; ECE, endothelin-con-
verting enzyme; NEP, neutral endopeptidase; COL-3, collage-
nase-3 (MMP-13); HME, human macrophage elastase (MMP-12).
2. In h ibition of En d op r otea ses. The hydroxamic
acid derivatives 36 and 41 were selective for PDF over
representative endopeptidases found in the cardiovas-
cular system (Table 2). Moreover, these compounds
showed selectivity for PDF over human macrophage

1850 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12
Apfel et al.
were dissolved in THF (50 mL) and hydrogenated (1 bar H2)
at room temperature over fresh Raney nickel (ca. 2 g suspen-
sion in water) until 3 equiv of H2 was absorbed. The mixture
was filtered through Celite and evaporated. Chromatogra-
phy: hexane/EtOAc 2:1. Yellow oil: 3 g (68%). 1H NMR: δ
1.27 (t, 3H), 1.70 (br, 1H), 3.41 (s, 2H), 3.99 (s, 2H), 4.18 (q,
2H), 4.77 (br, 2H), 6.55 (d, 1H), 6.73 (d, 1H), 6.97 (t, 1H).
elastase (MMP-12) and/or collagenase-3 (MMP-13), a
goal we had missed by our first approach.18
3. An tiba cter ia l in Vitr o Activity. The PDF inhibi-
tors were routinely tested for Gram-negative antibacte-
rial activity with the permeable outer membrane mu-
tant E. coli DC2, Haemophilus influenzae, and Moraxella
catarrhalis RA21 (Table 1). However, despite potent
enzyme inhibition, the hydroxamic acids 36-38, 41-
43, and 46-48 were only weakly active in the first two
strains. The results of pertinent uptake studies will be
published elsewhere.28 In the strain M. catarrhalis,
substantial antibacterial activity was observed.
Meth od C-2: (2-Am in o-6-tr iflu or om eth yl-ben zylam in o)-
a cetic Acid Eth yl Ester (18). Compound 13 (2.0 g, 7 mmol)
was dissolved in THF (5 mL) and hydrogenated (1 bar H2) over
10% palladium on charcoal (0.5 g). The mixture was filtered
through a pad of dicalite (5 g). The yellow filtrate was
1
evaporated. A yellow oil was obtained: 1.5 g (83%). H NMR:
δ 1.29 (t, J ) 8, 3H), 1.6 (br, 1H), 3.43 (s, 2H), 3.88 (s, 2H),
4.22 (q, J ) 8, 2H), 5.00 (br, 2H), 6.83 (d, J ) 8, 1H), 7.00 (d,
J ) 8, 1H), 7.14 (t, J ) 8, 1H).
Con clu sion s
Meth od D: (2-Am in o-6-flu or o-ben zylam in o)-acetic Acid
Eth yl Ester (15). 2-Amino-6-fluoro-benzylamine (1.54 g, 10.1
mmol) and TEA (3.06 mL, 20.2 mmol) were refluxed in
dioxane. Then a solution of ethyl bromoacetate (1.12 mL, 10.1
mmol) in dioxane (5 mL) was added slowly over 1 h. A
precipitate formed. Stirring at reflux was continued for 2 h.
Extraction: 2 × AcOEt, 2 × H2O. Chromatography: CH2Cl2/
Starting from a weakly binding screening hit, whose
binding mode could be elucidated by X-ray analysis, we
have rationally designed and synthesized highly potent
inhibitors of PDF, some of which bound 500 times better
than the initial screening hit. Upon broader testing, the
inhibitors were found to exhibit selectivity for PDF over
endoproteases, including matrix metalloproteinases
(MMPs). The antibacterial activity was reasonable in
M. catarrhalis, but only marginal in other Gram-
negative strains such as H. influenzae and E. coli DC2.
Our approach demonstrates how new structural scaf-
folds can be identified and optimized by applying a
combination of screening, X-ray, and medicinal chem-
istry.
1
MeOH 40:1. Yellow oil: 1.7 g (74%). H NMR: δ 1.28 (t, 3 H),
1.65 (br, 1H), 3.40 (s, 2H), 3.84 (s, 2H), 4.23 (q, 2H), 4.75 (br,
2H), 6.42 (m, 2H), 7.00 (dd, J ) 6, 1H).
Meth od E: (2-Oxo-5-tr iflu or om eth yl-1,4-d ih yd r o-2H-
qu in a zolin -3-yl)-a cetic Acid Eth yl Ester (23). Compound
18 (0.50 g, 2 mmol) and bis(trichloromethyl)carbonate (0.54
g, 2 mmol) were dissolved in dioxane (5 mL) and stirred at
room temperature for 24 h. Extraction: AcOEt, H2O. Chro-
matography: CH2Cl2/t-BuOMe 10:1. A colorless oil, which
crystallized spontaneously, was obtained: 0.10 g (46%); mp
1
161 °C. H NMR: δ 1.30 (t, J ) 8, 3H), 4.19 (s, 2H), 4.25 (q, J
Exp er im en ta l Section
) 8, 2H), 4.68 (s, 2H), 6.90 (t, J ) 4, 1H), 7.26 (t, J ) 4, 2H),
8.11 (s, 1H).
Gen er a l P r oced u r es. The following reagents and inter-
mediates were commercially available or prepared by litera-
ture procedures: 2-chloro-6-nitrobenzyl bromide 8 (commer-
cial), 2-bromo-6-nitrobenzyl bromide 9 (method A),29 N-(2-
chloro-6-nitrobenzyl)glycine ethyl ester 11 (method B),24 (2-
bromo-6-nitro-benzylamino)-acetic acid ethyl ester 12 (method
B),24 and (2-amino-benzylamino)-acetic acid ethyl ester 14
(method D).30 The benzyl bromides 8-10 are strong lacrima-
tors and should be manipulated in a ventilated hood. All
organic extracts were dried over Na2SO4, filtered, and evapo-
rated under vacuum. All chromatographic separations were
performed on prepacked Flash cartridges of KP-Sil silica, 32-
63 µm, 6 nm (12, 40, or 90 g), unless otherwise stated. 1H NMR
spectra (400 MHz) were measured in CDCl3 unless otherwise
stated. Chemical shifts (δ) are reported in ppm relative to Me4-
Si as internal standard. Coupling constants (J ) are given in
hertz. Ion spray and EI mass spectra were measured on a
Finningan MAT SSQ 7000 and Perkin-Elmer Siex API III
recorder, respectively.
Met h od F : (5-Ch lor o-2,2-d ioxo-1,4-d ih yd r o-2H -2λ6-
ben zo[1,2,6]th ia d ia zin -3-yl)-a cetic Acid Eth yl Ester (26).
Compound 16 (2.6 g, 11 mmol) and sulfamide (3.1 g, 32 mmol)
were refluxed in pyridine (20 mL) for 24 h. Extraction: 2 × 3
N HCl, 2 × 50% saturated NaCl. Chromatography: CH2Cl2.
The product crystallized spontaneously: 2.6 g (79%); mp 108
°C. 1H NMR: δ 1.27 (t, J ) 7, 3H), 3.84 (s, 2H), 4.22 (q, J ) 7,
2H), 4.85 (s, 2H), 6.65 (s, 1H), 6.67 (s, 1H), 7.10 (d, J ) 8, 1H),
7.17 (t, J ) 8, 1H).
Meth od G: (1-Ben zyl-5-ch lor o-2,2-d ioxo-1,4-d ih yd r o-
2H-2λ6-ben zo[1,2,6]th ia d ia zin -3-yl)-a cetic Acid Eth yl Es-
ter (29). NaH (55% in oil, 78 mg, 1.8 mmol) was dissolved in
DMF (5 mL) under argon, then 26 (0.5 g, 1.64 mmol) was
added carefully, and stirring continued for 5 min. Benzylbro-
mide (0.21 mL, 1.8 mmol) and catalytic Bu4NI (61 mg, 0.18
mmol) were added, and the solution was heated at 80 °C for 2
h. Extraction: 2 × AcOEt, 2 × H2O. Chromatography: hexane/
CH2Cl2 1:1, then CH2Cl2. A white solid was obtained: 437 mg
Meth od A: 2-Nitr o-6-tr iflu or om eth yl-ben zyl Br om id e
(10). 2-Nitro-6-(trifluoromethyl)toluene (5.00 g, 24 mmol),
N-bromosuccinimide (4.34 g, 24 mmol), and benzoyl peroxide
(0.1 g, 0.4 mmol) were dissolved in CCl4 (50 mL), heated at
reflux, and irradiated with light (500 W bulb) for 16 h. The
reaction mixture was filtered and concentrated. Chromatog-
raphy: hexane/AcOEt 20:1. A yellow oil was obtained: 5 g
1
(67%); mp 63 °C. H NMR: δ 1.30 (t, J ) 7, 3H), 3.88 (s, 2H),
4.24 (q, J ) 7, 2H), 4.89 (s, 2H), 5.00 (s, 2H), 6.68 (d, 1H), 7.07
(m, 2H), 7.28 (m, 1H), 7.28 (m, 4H).
Meth od H: N-Hydr oxy-2-(2-oxo-1,4-dih ydr o-2H-qu in azo-
lin -3-yl)-a ceta m id e (34). Hydroxylamine hydrochloride (552
mg, 7.9 mmol) was dissolved in ice-cooled 4 N KOH (3.9 mL)
and stirred for 5 min. A solution of 19 (350 mg, 1.6 mmol) in
THF (4 mL; for compounds 36, 38, and 43: 40 mL because of
low solubility!) was added, and stirring continued for 2 h at 0
°C. The resulting emulsion was neutralized with 4 N HCl (1.95
mL) and stirred for 15 min. Extraction: AcOEt, H2O. The
organic layer was not dried, but it was concentrated until the
product precipitated. The product was filtered off and dried
at 80 °C under vacuum. White crystals were obtained: 51 mg,
15%; mp 190 °C dec. 1H NMR (DMSO-d6) (5:1 mixture of
rotamers): δ 3.85 and 4.16 (2s, 2H), 4.47 (s, 2H), 6.76 (d, J )
8, 1H), 6.86 (t, J ) 8, 1H), 7.05 (d, J ) 8, 1H), 7.12 (t, J ) 8,
1H), 8.82 and 9.16 (2s, 1H), 9.20 and 9.26 (2s, 1H), 10.1 and
10.6 (2s, 1H).
1
(72%). H NMR: δ 4.93 (s, 2H), 7.62 (t, J ) 8, 1H), 7.95 (d, J
) 8, 1H), 8.05 (d, J ) 8, 1H).
Meth od B: (2-Nitr o-6-tr iflu or om eth yl-ben zyla m in o)-
a cetic Acid Eth yl Ester (13). Glycine ethyl ester hydrochlo-
ride (2.0 g, 19 mmol) and sodium hydride (0.93 g, 39 mmol)
were stirred under argon in DMF (30 mL) for 0.5 h. Then 10
(5.0 g, 18 mmol) in DMF (20 mL) was added dropwise over 15
min (slightly exothermic). Stirring at room temperature was
continued for 5 h. Extraction: AcOEt, H2O. Chromatography:
hexane/AcOEt 5:1. A yellow oil was obtained: 3.20 g (60%).
1H NMR: δ 1.28 (t, J ) 8, 3H), 2.11 (s, 1H), 3.35 (s, 2H), 4.19
(q, J ) 7, 4H), 7.54 (t, J ) 8, 1H), 7.87 (t, J ) 8, 2H).
Meth od C-1: N-(2-Am in o-6-ch lor oben zyl)glycin e Eth yl
Ester (16). Compound 11 (4.7 g, 18 mmol) and TEA (2.5 mL)
En zym e In h ibition . E. coli PDF was isolated, as recom-

Inhibitors of Peptide Deformylase
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 12 1851
binant enzyme from E. coli, in its native iron-containing form
and purified according to a published procedure.8 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
started by the addition of PDF and Fe-PDF with approximately
1100 U/mg (final concentration: 5.7 nM), and it was stopped
after 5 min with 200 µL of a 1.54 mM solution of fluorescamine
in a 20:1 mixture of 2-methoxy-ethanol and 0.2 M sodium
borate buffer (pH 8.5). 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.
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Inhibition of ACE, ECE, NEP, and several MMPs was
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In Vitr o An tiba cter ia l Activity. Bacterial strains used
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Str u ctu r e Deter m in a tion . For crystallization and X-ray
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conditions similar to those used by these authors and were
soaked with a 1 mM solution of hydrazide 1 in 54% ammonium
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generation were performed with the in-house program Moloc.40
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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 from NRRC for running the
enzyme inhibition screen. We also thank Dr. T. Hartung
and P. Dreier for the synthesis of the radiolabeled
compounds 36 and 41, and E. Bald, N. Nock, E. Philipp,
B. Prud’hon, C. Lacoste, B. Rutten, and O. Partouche
for their excellent technical assistance. For enzyme
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and properties.
Su p p or tin g In for m a tion Ava ila ble: Additional experi-
mental data (1H NMR, MS, yields, etc.) of all compounds not
listed in the Experimental Section. This material is available
free of charge via the Internet at http://pubs.acs.org.

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