Bis-substituted malonic acid hydroxamate derivatives as inhibitors of human neutrophil collagenase (MMP8)

Article abstract of DOI:10.1021/jm980112p

Malonic acid hydroxamate derivatives bis-substituted at the methylene group were synthesized as potential nonpeptidic inhibitors of human neutrophil collagenase (MMP8). The presence of an aromatic residue both at the C2 malonic acid position and in the C-

Full text of DOI:10.1021/jm980112p

J . Med. Chem. 1998, 41, 3041-3047
3041
Bis-Su bstitu ted Ma lon ic Acid Hyd r oxa m a te Der iva tives a s In h ibitor s of Hu m a n
Neu tr op h il Colla gen a se (MMP 8)
Erich Graf von Roedern,†,∇ Hans Brandstetter,†,| Richard A. Engh,†,‡ Wolfram Bode,† Frank Grams,§ and
Luis Moroder*,†
Max-Planck-Institut fu¨r Biochemie, Am Klopferspitz 18A, D-82152 Martinsried, Germany, Department of Biotechnology,
Boehringer Mannheim GmbH, D-82372 Penzberg, Germany, and Department of Chemical Research,
Boehringer Mannheim GmbH, D-68305 Mannheim, Germany
Received February 19, 1998
Malonic acid hydroxamate derivatives bis-substituted at the methylene group were synthesized
as potential nonpeptidic inhibitors of human neutrophil collagenase (MMP8). The presence of
an aromatic residue both at the C2 malonic acid position and in the C-terminal tail for
hydrophobic interactions with the surface-exposed S1 binding site and the S1′ pocket of the
enzyme, respectively, was found to be sufficient for submicromolar inhibition potencies. For
optimal insertion of the aryl amide group into the hydrophobic S1′ pocket, spacing of the
C-terminal phenyl group by at least a 3C-chain was required. In view of these results the
achiral indan-2,2-dicarboxylic acid was used to mimic the 2-benzyl-2-methylmalonic acid residue,
and its derivatization to the 3-phenylpropyl amide hydroxamate produced a potent, achiral,
low-mass inhibitor of MMP8 (Ki ) 0.3 µM), the binding mode of which was unambiguously
determined by X-ray crystallographic analysis.
In tr od u ction
residue, as well as varying the methylene substituent
of the malonic acid residue as a pseudo-P1 residue.19
Thereby X-ray structural analyses of selected MMP8/
inhibitor complexes fully confirmed the non-substrate-
like binding mode of these malonic acid-based inhibi-
tors.20 By conversion of the peptidic to nonpeptidic
malonic acid derivatives, submicromolar inhibitory po-
tencies were obtained if the following two conditions
were met: (i) sufficiently large hydrophobic pseudo-P1′
residues to ensure strong interactions with the hydro-
phobic S1′ pocket of MMP8 and (ii) monosubstitution
at the methylene group of the malonic acid moiety with
alkyl or aryl groups for additional binding to the surface-
exposed S1 subsite.19
The X-ray analyses of complexes of matrix metallo-
proteinases (MMPs) with synthetic hydroxamate- or
thiol-type inhibitors revealed binding of the inhibitors
to the active-site cleft of the enzymes in extended
conformations and in a manner similar to the binding
mode of the substrates,1-15 except for the thermolysin
inhibitor (2R,S)-HONH-Mal(i-Bu)-Ala-Gly-NH2.16,17 This
malonic acid-based hydroxamate binds to human neu-
trophil collagenase (MMP8) with the hydroxamate act-
ing as a bidentated chelator of the active-site Zn2+ ion.15
The hydrophobic substituent at the malonic acid moiety
is directed toward the edge strand of the active-site cleft
in a hydrophobic interaction with the S1 subsite of the
enzyme. In contrast to the succinic acid hydroxamates
which represent the most potent MMPs inhibitors
known so far,18 the malonic acid-based inhibitor lacks
a spacer between the chelating group and the hydro-
phobic group. Correspondingly, the position of the side
chain of the subsequent amino acid residue, i.e., of Ala,
becomes similar but not identical to that of P2′ residues
in substrates, while the residual peptidic tail is inserted
into the S1′ pocket, thus leading to an overall â-turn-
like conformation of the inhibitor in the enzyme-bound
state.
In the present study a bis-substitution at the meth-
ylene group of the malonic acid hydroxamate was
analyzed as a possible approach to improve inhibition
of MMP8 by low-molecular-weight nonpeptidic hydrox-
amates.
Resu lts a n d Discu ssion
Ch em istr y. Monoalkylation of diethyl malonate at
the C2 position was performed by standard methods
with alkyl halides.19 In a subsequent alkylation step
the 2,2-bis-substituted diethyl malonates were obtained
following essentially known procedures as outlined in
Scheme 1.21-23 Conversely, the cyclic bis-substituted
malonic acid diethyl esters were prepared in one step
employing suitable dibromo derivatives.24,25 The result-
ing 2,2-bis-substituted malonic acid diethyl esters were
then hydrolyzed with 1 equiv of KOH to produce the
monoethyl esters which in turn were condensed with
(benzyloxy)amine using EDCI/HOBT as coupling re-
agent to avoid the guanidine adduct formation.19 After
an additional saponification step with excesses of KOH,
the resulting benzyl-protected malonic acid hydroxam-
ates were amidated with arylamines using again the
In a previous study we have analyzed the possibility
of improving the binding affinity of malonic acid-based
compounds for MMP8 by iterative changes in the
peptidic tail at the pseudo-P1′ residue and in the P2′
* Corresponding author. Tel: 49-89-8578-3905. Fax: 49-89-8578-
2847. E-mail: moroder@biochem.mpg.de.
† Max-Planck-Institut fu¨r Biochemie.
‡ Boehringer Mannheim GmbH, Penzberg.
§ Boehringer Mannheim GmbH, Mannheim.
∇ Current address: Hoechst Marion Roussel, HMR Forschung
Chemie, 65929 Frankfurt, Germany.
| Current address: Department of Chemistry, MIT, 77 Massachu-
setts Ave., Cambridge, MA 02139.
S0022-2623(98)00112-5 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/30/1998

3042 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Graf von Roedern et al.
Ta ble 1. Inhibition of MMP8 with Chiral and Achiral
Sch em e 1. General Synthetic Route Used for the
Synthesis of the Chiral and Achiral Malonic Acid-Based
MMP8 Inhibitors
Bis-Substituted Nonpeptidic Malonic Acid Hydroxamates
EDCI/HOBt procedure. Finally, hydrogenolytic cleav-
age of the benzyl group from the hydroxamate over Pd/C
catalyst led to the target hydroxamic acids.
MMP 8 In h ibition by Bis-Su bstitu ted Ma lon ic
Acid Hyd r oxa m a tes. In our previous study on mono-
substituted malonic acid-based MMP inhibitors, both
branched alkyl and aryl derivatives such as 2-isobutyl,
2-benzyl, or 2-(2-phenyl)ethyl compounds were found to
increase the inhibitory potency19 by hydrophobic inter-
actions with the S1 subsite as clearly revealed by X-ray
analysis.15,20 In an attempt to further exploit the S1
binding site, we have synthesized bis-substituted mal-
onic acid derivatives, and a comparison of the inhibitory
potency of 2-isobutyl-2-methylmalonic acid hydroxamate
ethyl ester (2) (Ki ) 41 µM) with that of the related
2-benzyl-2-methyl derivative 5 (Ki ) 11 µM) indicated
a clear advantage of an aromatic substituent (Table 1).
To answer the question of whether the bis-substituted
derivatives retain the binding mode of the monosubsti-
tuted malonic acid-based inhibitors, X-ray crystal-
lographic analysis of the MMP8/5 complex was per-
formed. Apparent disorder in the crystals of MMP8/5
prevented the unambiguous determination of a single
structure, possibly because both stereoisomers may be
bound in the crystals. An initial difference electron
density map supported the binding mode shown in
Figure 1 where the hydroxamate group acts as a
bidentated chelator of the active-site Zn2+ ion, the
benzyl group covers a hydrophobic patch on the surface
of the enzyme, and the following carbonyl group is
hydrogen-bonded to Leu160NH whereas the aliphatic
tail extends to the S1′ pocket. However, the resolution
of the electron density map does not rule out binding of
the alternate stereoisomer with the aromatic group
inserted into the S1′ pocket and the ethyl ester group
interacting at the exposed site. Indeed, the electron
density map of a second crystal seems to favor this
interpretation although again not unambiguously. Si-
multaneous binding of both stereoisomers would support
the hypothesis of similar binding free energies for both.
Since from volume considerations the aliphatic binding
in S1′ is likely weaker than that of the aromatic group,
the hydrophobic interactions of the exposed benzyl
group would have to compensate for this loss.
*For (standard errors of the Ki values, see the Experimental
Section.
MMP8 was analyzed. Taking into account the beneficial
effect of C-terminal aromatic amides observed for of
monosubstituted malonic acid hydroxamates,19 the
amides 7-11 were synthesized which differ in the
number of methylene groups as spacers of the phenyl
group from the amide. While the anilide 7 showed a
4-fold weaker inhibitory potency (Ki ) 44 µM) than the
ester 5, with the benzyl amide 8 (Ki ) 1.1 µM) a
remarkably more potent inhibitor with a 10-fold en-
hanced binding affinity was obtained. Interestingly, the
affinity for the enzyme was found to decrease again
remarkably with the homobenzyl amide 9 (Ki ) 9.6 µM),
while further extension of the C-terminus with three
(10) or four (11) methylene groups resulted in signifi-
cantly improved inhibitory potencies with Ki values in
the submicromolar range, i.e., 0.24 and 0.38 µM, re-
spectively. Modeling experiments performed with com-
pounds 10 and 11 using the binding mode of 5 as
template indicate better occupancy of the S1′ pocket and
thus improved hydrophobic interactions with this en-
zyme subsite than with 8. Attempts to similarly
improve inhibition of MMP8 with para-, meta-, or ortho-
chloro substitutions in the benzyl amide 8 failed (Ki
In view of these results, in a first series of compounds
the methyl/benzyl substitution pattern at the malonic
acid was retained and the effect of different C-terminal
derivatizations for interaction with the S1′ subsite of

Malonic Acid Hydroxamates as MMP8 Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3043
In crystals of the MMP8/21 complex the structure of
the achiral inhibitor could be unambiguously deter-
mined, although the electron density around the indan
C2 carbon is somewhat weaker (Figure 2). Minor
adjustments of the malonic acid moiety and of the
substituent, correlated with the indan pucker, might be
a reasonable explanation. A comparison of the position
of inhibitor 21 in the active-site cleft of the enzyme with
that of compound 5 (Figure 3) shows an overall transla-
tion of the common groups away from the S1′ pocket;
however, hydrogen bonding of the malonic acid carbonyl
to Leu160NH is retained and the indan group interacts
with the hydrophobic surface formed mainly by the side
chain of Ile159, whereas the C-terminal benzyl group
inserts into the S1′ pocket.
Similarly to what was observed for the 2-benzyl-2-
methyl-substituted derivatives, replacement of the C-
terminal benzyl residue with the 3-phenylpropyl group
(22) leads to improved interactions in the hydrophobic
S1′ binding site with a Ki value of 0.3 µM that is almost
identical with that of compound 10. An enhanced
hydrophobic interaction at the S1 site was attempted
with the 6,7-dihydro-5H-dibenzo[a,f]cycloheptene-6,6-
dicarboxylic acid as an achiral mimetic of a 2,2-diben-
zylmalonic acid moiety. Its conversion to the hydrox-
amate 3-phenylpropyl amide 24, however, led to inhibi-
tion of MMP8 with a Ki of 1.9 µM which is 15-fold higher
than that of the related indan derivative 22. Modeling
experiments were predicting for compound 24 the
binding of one aromatic group in a manner very similar
to that of the indan moiety and an additional hydro-
phobic interaction by the second benzene moiety with
the edge of the hydrophobic S1′ pocket. Therefore, the
experimentally observed lower inhibition potency of 24
might be attributed more to energetic than entropic
factors since the side chain of compound 24 should be
almost as rigid as in 22.
F igu r e 1. One of the two possible binding modes of (2R,S)-
HONH-Mal(Me/Bn)-OEt (5) to MMP8 as derived from X-ray
crystallographic analysis. In the binding mode represented in
this figure, the benzyl group interacts with the S1 subunit and
the ethyl ester is inserted into the S1′ pocket.
values in the range of 1.8-1.7 µM). Similarly, with
para-, meta-, or ortho-methyl derivatives of 8 the Ki
values remained in the range of 1.4-9 µM. Introduction
of a para-carboxyl group into the benzyl amide 8, as a
potential interaction partner for the Arg222 residue on
the bottom of the S1′ pocket of MMP8, led to a Ki value
of 41 µM. This result would suggest that in contrast to
that expected from the modeling experiments described
in the Experimental Section, a buried salt bridge
between the para-carboxyl group and the Arg side chain
does not occur and that interactions with the S1′ subsite
are significantly reduced. By constraining conforma-
tionally the C-terminal benzyl amide residue as an
R-naphthyl amide (13) weak inhibition was again
obtained. Similarly unsuccessful was the derivatization
of the bis-substituted malonic acid hydroxamate to the
1,2,9,10-tetrahydroisochinolide (14) as a rigid mimetic
of the benzyl or 2-phenylethyl amide; the inhibition
potency was found to be identical to that of the 2-phe-
nylethyl amide 9.
Attempts to improve the solubility of the inhibitors
as well as the hydrogen-bonding network with the edge
strand of the active-site cleft of MMP8 led us to replace
the 2-methyl group of compound 8 with an acetamido
(16) or hydroxy (19) group. Both hydroxamates showed
slightly reduced inhibitory potencies compared with the
parent compound 8 (Table 1).
MMP 8 In h ib it ion b y Ach ir a l Cyclic 2,2-Bis-
Su bstitu ted Ma lon ic Acid Hyd r oxa m a tes. The bis-
substituted malonic acid derivatives described above
were all obtained as racemates. To bypass this draw-
back achiral 2,2-cyclic derivatives of the malonic acid
hydroxamates were examined for their affinity for
MMP8. For this purpose the indan-2,2-dicarboxylic acid
was prepared as a mimetic of the 2-benzyl-2-methyl-
malonic acid and converted to the hydroxamate benzyl
amide 21. As expected from modeling experiments, it
showed an inhibitory potency that compared well to that
of compound 8 (Table 1).
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Solvents and reagents used in
the synthesis were of the highest quality commercially avail-
able. Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 was pur-
chased from Bachem (Heidelberg, Germany). TLC silica gel
60 plates were from Merck AG (Darmstadt, Germany), and
compounds were visualized with the chlorine/tolidine or per-
manganate reagent. FAB-MS spectra were recorded on a
Finnigan MAT 900 spectrometer and the NMR spectra on
AMX 400 and AMX 500 (Bruker) spectrometers.
En zym e Assa y. The catalytic domain of MMP8 (Phe79,-
Gly242-MMP8) was used for the inhibition experiments. En-
zyme assays were performed at 25 °C in 10 mM CaCl2, 100
mM NaCl, 50 mM Tris/HCl (pH 7.6) using 8 nM enzyme
concentration and the fluorogenic substrate Dnp-Pro-Leu-Gly-
Leu-Trp-Ala-D-Arg-NH2 (1 × 10-5 M). Enzyme kinetics was
performed essentially as described by Stack and Gray,26 and
the increase in fluorescence at 350 nm was monitored over a
period of 100 s with a luminescence spectrometer LS 50B
(Perkin-Elmer) to determine initial rates of hydrolysis. Evalu-
ation of the kinetic data was performed as reported by
Copeland et al.,27 and the IC50 values were determined with
the program Enzfitter (Version 1.05, 1987, Elsevier-Biosoft);
average standard errors for the IC50 values were (5-10%.
Assuming Michaelis-Menten kinetics, the Ki values were then
calculated from the IC50 values according to the equation: Ki
) IC50‚Km/(Km + [S]).
X-r a y Cr ysta llogr a p h y. Crystals of the MMP8 (Phe79,-
Gly242-MMP8) complexed with inhibitor 21 were grown with
sitting drop vapor diffusion using conditions similar to those

3044 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Graf von Roedern et al.
F igu r e 2. X-ray structure of HONH-Ind-NHBn (21) in the MMP8/inhibitor complex superimposed with the 2.0 Å 2Fo - Fc electron
density map.
F igu r e 3. Comparison of the binding mode of the inhibitors 5 and 21 to the active site of MMP8.
previously reported.15,20 Drops consisting of 2 µL of inhibitor
solution (50 mM in aqueous MeOH), 2 µL of enzyme solution
(4.5 mg/mL in MES/NaOH, pH 6), and 3 mL of precipitant
(0.1 M MES/NaOH, pH 6.0, 10% PEG 6000, 3% MPD) were
equilibrated at room temperature against a reservoir solution
of 0.8 M phosphate buffer at pH 6. Crystals of P212121
symmetry and cell dimensions a, b, c ) 33.0, 69.5, 72.7 Å grew
out of precipitated protein after several weeks. A total of 8075
nonredundant reflections were processed for overall complete-
ness of 71% in the resolution range 6-2 Å after application of
a 2σ threshold; the R-merge was 6% overall and 27% in the
highest resolution shell.
program package INSIGHT/DISCOVER (version 2.96, MSI
Technologies, San Diego, CA) with the implemented force field
CVFF and a dielectric constant of 1. By keeping the protein
structure fixed and the hydroxamate function as a ligand of
the active-site Zn2+ as determined in the MMP8/5 or MMP8/
21 complexes as a constraint, alternative orientations and
conformations of potential new inhibitors were generated and
energy-minimized to convergence. For inspection of the pos-
sible binding mode of newly designed ligands and to gain
information about hydrogen-bonding and hydrophobic interac-
tions, interaction sites have been generated with the program
LUDI.28
Two crystals of the MMP8/5 complex were obtained by
soaking MMP8 crystals in inhibitor solution for several hours.
Data were measured to a theoretical completeness of at least
90%, but poorer crystal quality compared to the MMP/21
crystals resulted in an overall completeness of 50% to 2.3 Å
after 2σ cutoff for crystal no. 1 (cell dimensions 33.1, 69.0, 72.7)
and 74% to 2.3 Å for crystal no 2 (cell dimensions 33.0, 69.1,
72.7). All data were collected using a Siemens ×1000 multi-
wire area detector using Siemens data collection and process-
ing software (FRAMBO/SADIE/ASTRO/SAINT).
The refinement was performed with XPLOR, and the
structures have R-factors of 23.5% for MMP/21 to 2.0 Å
(without solvent) and 21.2% for crystal no. 1 and 23.5% for
crystal no. 2 of MMP/5 to 2.3 Å (with 42 solvent molecules).
Com p u ta tion a l Meth od s. Modeling experiments were
performed on the crystal structure of MMP815 using the
Syn th esis. (2R,S)-Bn ONH-Ma l(Me/i-Bu )-OEt (1). Di-
ethyl 2-isobutyl-2-methylmalonate21 (8.98 g, 39.0 mmol) was
saponified in 100 mL of EtOH/water (4:1) with KOH (2.18 g,
39.0 mmol) to the monoethyl ester by refluxing the mixture
for 2 days. The solvent was evaporated, and (2R,S)-KO-Mal-
(Me/i-Bu)-OEt was precipitated from EtOH with ether: yield
5.41 g (58%). To (2R,S)-KO-Mal(Me/i-Bu)-OEt (3.60 g, 15.0
mmol) and (benzyloxy)amine hydrochloride (2.40 g, 15 mmol)
in 15 mL of THF were added HOBT (1.5 g, 10.0 mmol) and
EDCI (3.30 g, 17 mmol). The reaction mixture was stirred
overnight at room temperature, diluted with AcOEt, and
washed with 5% KHSO4, 5% NaHCO3, and water. The organic
layer was dried over MgSO4 and evaporated to dryness. The
crude product was purified by flash chromatography on silica
gel (100 g, AcOEt/n-hexane, 1:4): yield 2.20 g (48%) as a
colorless oil; TLC (AcOEt/n-hexane, 1:2) Rf 0.5; FAB-MS m/z

Malonic Acid Hydroxamates as MMP8 Inhibitors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 3045
1
308.2 [M + H+]; Mr ) 307.17 calcd for C17H25NO4; H NMR
(DMSO-d6) δ 11.05 (s, 1H, NHOBn), 7.30-7.45 (m, 5H, C6H5),
4.86 (s, 2H, CH2-C6H5), 4.05 (q, 2H, CH2CH3), 1.73 (dd, 2H,
CH2 (i-Bu)), 1.58 (m, 1H, CH (i-Bu)), 1.30 (s, 3H, CH3 (Mal)),
1.18 (m, 3H, CH2CH3), 0.8 (2d, 6H, 2 CH3 (i-Bu)).
[M + H+]; Mr ) 312.1 calcd for C18H20N2O3; 1H NMR (DMSO-
d6) δ 10.50 (s, 1H, NHOH), 8.85 (s, 1H, NHOH), 8.22 (dd, 1H,
NHBn), 7.05-7.35 (m, 10H, 2 C6H5), 4.30 (m, 2H, CH2
(NHBn)), 3.09 + 3.17 (2d, 2H, CH2 (Mal(Bn)), 1.17 (s, 3H, CH3
(Mal)).
(2R,S)-HONH-Ma l(Me/i-Bu )-OEt (2). Compound 1 (0.15
g, 0.50 mmol) was hydrogenated in MeOH over Pd/C catalyst
under a low stream of H2 for 1 h. The catalyst was filtered
off, and the filtrate was evaporated to dryness: yield 0.105 g
(93%); TLC (AcOEt/n-hexane, 1:1) Rf 0.5; FAB-MS m/z 218.1
(2R,S)-HONH-Mal(Me/Bn )-NH-CH2CH2-P h (9). The com-
pound 6 (155 mg, 0.5 mmol) was coupled with 2-phenylethy-
lamine (121 mg, 1.0 mmol), and the resulting O-benzylhydrox-
amate was hydrogenated as reported for 7: yield 120 mg (75%)
over the two steps; TLC (CHCl3/MeOH, 9:1) Rf 0.55; FAB-MS
m/z 327.2 [M + H+]; Mr ) 326.2 calcd for C19H22N2O3; 1H NMR
(DMSO-d6) δ 10.42 (s, 1H, NHOH), 8.78 (s, 1H, NHOH), 7.75
(dd, 1H, NH-(CH2)2-Ph), 7.05-7.35 (m, 10H, 2 C6H5), 3.28
+ 2.70 (m, 4H, (CH2)2-Ph), 3.05 + 3.17 (2d, 2H, CH2 (Mal-
(Bn)), 1.13 (s, 3H, CH3 (Mal)).
1
[M + H+]; Mr ) 217.1 calcd for C10H19NO4; H NMR (DMSO-
d6) δ 10.58 (s, 1H, NHOH), 8.69 (s, 1H, NHOH), 4.15 (q, 2H,
CH2CH3), 1.73 (dd, 2H, CH2 (i-Bu)), 1.58 (m, 1H, CH (i-Bu)),
1.30 (s, 3H, CH3 (Mal)), 1.18 (m, 3H, CH2CH3), 0.90 (d, 6H, 2
CH3 (i-Bu)).
(2R,S)-HO-Ma l(Me/Bn )-OEt (3). Diethyl 2-benzyl-2-me-
thylmalonate22 (4.0 g, 15.0 mmol) was saponified in EtOH/
water (4:1) with KOH (0.84 g, 15.0 mmol) at room temperature
for 2 days to the monoethyl ester. The bulk of the solvent was
evaporated, and the solution was diluted with water and
extracted with ether. The aqueous layer was acidified with 2
N HCl to pH 1.5 and extracted with ether; the combined
extracts were washed with water, dried over MgSO4, and
evaporated to dryness: yield 2.3 g (64%) of colorless crystals;
FAB-MS m/z 237.1 [M + H+]; Mr ) 236.1 calcd for C13H16O4;
1H NMR (DMSO-d6) δ 12.95 (s br, 1H, COOH), 7.1-7.3 (m,
5H, C6H5), 4.12 (m, 2H, CH2CH3), 3.1 (2d, 2H, CH2C6H5), 1.18
(m, 6H, CH3 (Mal), CH3 (Et)).
(2R,S)-Bn ONH-Ma l(Me/Bn )-OEt (4). To compound 3
(1.20 g, 5.0 mmol) and (benzyloxy)amine hydrochloride (0.96
g, 6.0 mmol) in THF was added NMM (1.01 g, 10 mmol)
followed by HOBT (0.65 g, 5.0 mmol) and EDCI (0.97 g, 5.1
mmol). The reaction mixture was stirred overnight at room
temperature, diluted with AcOEt, and washed with 5%
KHSO4, 5% NaHCO3, and water. The organic layer was dried
over MgSO4 and evaporated to dryness: yield 1.05 g (62%) as
a colorless oil; TLC (AcOEt/n-hexane, 1:2) Rf 0.3; FAB-MS m/z
342.2 [M + H+]; Mr ) 341.2 calcd for C20H23NO4; 1H NMR
(DMSO-d6) δ 11.30 (s, 1H, NH), 7.10-7.30 (m, 10H, 2 C6H5),
4.11 (m, 2H, CH2CH3), 3.1 (2d, 2H, CH2C6H5), 1.15-1.20 (m,
6H, CH3 (Mal), CH3 (Et)).
(2R,S)-HONH-Mal(Me/Bn )-NH-CH2CH2CH2-P h (10). The
title compound was obtained by amidation of 6 (155 mg, 0.5
mmol) with 3-phenylpropylamine (135 mg, 1.0 mmol) followed
by hydrogenation as described for 7: yield 132 mg (75%) over
the two steps; TLC (CHCl3/MeOH, 9:1) Rf 0.6; FAB-MS m/z
341.2 [M + H+]; Mr ) 340.1 calcd for C20H24N2O3; 1H NMR
(DMSO-d6) δ 10.41 (s, 1H, NHOH), 8.87 (s, 1H, NHOH), 7.73
(dd, 1H, NH-CH2CH2CH2-Ph), 7.10-7.30 (m, 10H, 2 C6H5),
3.04 (m, 4H, NH-CH2CH2CH2-Ph, CH2 (Bn)), 2.50 (dd, 2H,
NH-CH2CH2CH2-Ph), 1.71 (q, 2H, NH-CH2CH2CH2-Ph),
1.17 (s, 3H, CH3).
(2R,S)-HONH-Ma l(Me/Bn )-NH-CH2CH2CH2CH2-P h (11).
Compound 6 (155 mg, 0.5 mmol) was coupled with 4-phenyl-
butylamine (147 mg, 1.0 mmol), and the resulting O-benzyl-
hydroxamate was hydrogenated as described for 7: yield 123
mg (66%) over the two steps; TLC (AcOEt/n-hexane, 1:1) Rf
0.45; FAB-MS m/z 355.1 [M + H+]; Mr ) 354.2 calcd for
C
21H26N2O3; 1H NMR (DMSO-d6) δ 10.38 (s, 1H, NHOH), 8.85
(s, 1H, NHOH), 7.73 (dd, 1H, NH-CH2CH2CH2CH2-Ph),
7.10-7.35 (m, 10H, 2 C6H5), 3.04 (m, 4H, NH-CH2CH2CH2-
CH2-Ph, CH2 (Bn)), 2.50 (dd, 2H, NH-CH2CH2CH2CH2-Ph),
1.58-1.76 (m, 4H, NH-CH2CH2CH2CH2-Ph), 1.17 (s, 3H,
CH3).
(2R,S)-HONH-Ma l(Me/Bn )-NHBn -(p-COOH) (12). To 6
(155 mg, 0.5 mmol) in 2 mL of THF was added 4-aminometh-
ylbenzoic acid methyl ester hydrochloride (202 mg, 1.0 mmol)
followed by NMM (101 mg, 1.0 mmol), HOBT (75 mg, 0.5
mmol), and EDCI (100 mg, 0.6 mmol). The reaction mixture
was stirred overnight, diluted with AcOEt, and washed with
5% KHSO4, 5% NaHCO3, and water. The solution was dried
over MgSO4 and evaporated to dryness. The methyl benzoate
moiety was saponified in EtOH/water (4:1) with KOH (160 mg,
3.0 mmol) at room temperature for 4 h. The EtOH was
evaporated and the solution diluted with water and extracted
with ether. The aqueous layer was acidified with 2 N HCl to
pH 1.5 and extracted with ether; the combined extracts were
washed with water, dried over MgSO4, and evaporated to
dryness. The residue was hydrogenated in MeOH as described
for 2: yield 73 mg (41%) over the three steps; TLC (CHCl3/
MeOH, 4:1) Rf 0.3; FAB-MS m/z 357.2 [M + H+]; Mr ) 356.1
calcd for C19H20N2O5; 1H NMR (DMSO-d6) δ 13.3 (br, 1H,
COOH), 10.45 (s, 1H, NHOH), 8.73 (br, 1H, NHOH), 8.17 (dd,
1H, NHBn), 7.91 + 7.65 (2d, 4H, C6H4), 7.05-7.35 (m, 5H,
C6H5), 4.28 (m, 2H, CH2 (NHBn)), 3.09 + 3.17 (2d, 2H, CH2
(Mal(Bn)), 1.13 (s, 3H, CH3).
(2R,S)-HONH-Ma l(Me/Bn )-OEt (5). Compound 4 (0.16 g,
0.47 mmol) was hydrogenated as reported for compound 2:
yield 0.11 g (93%); TLC (AcOEt/n-hexane, 1:1) Rf 0.3; mp 89-
90 °C; FAB-MS m/z 252.2 [M + H+]; Mr ) 251.1 calcd for
C
13H17NO4; 1H NMR (DMSO-d6) δ 10.52 (s, 1H, NHOH), 8.84
(s, 1H, NHOH), 7.10-7.30 (m, 5H, C6H5), 4.11 (m, 2H, CH2-
CH3), 3.1 (2d, 2H, C6H5), 1.18 (m, 6H, CH3 (Mal), CH3 (Et)).
(2R,S)-Bn ONH-Ma l(Me/Bn )-OH (6). The monoethyl ester
4 (1.02 g, 3.0 mmol) was saponified in THF/water (3:1) with
KOH (0.68 g, 12.0 mmol) for 2 h at 80 °C and worked up as
described for 3. The product was isolated by precipitation from
ether with n-pentane: yield 0.78 g (84.5%); FAB-MS m/z 314.2
1
[M + H+]; Mr ) 313.1 calcd for C18H19NO4; H NMR (DMSO-
d6) δ 13.1 (br, 1H, COOH), 11.30 (s, 1H, NH), 7.10-7.30 (m,
10H, 2 C6H5), 4.82 (s, 2H, CH2 (OBn)), 3.1 (2d, 2H, CH2 (Bn)),
1.23 (s, 3H, CH3 (Mal)).
(2R,S)-HONH-Ma l(Me/Bn )-NHP h (7). To 6 (155 mg, 0.5
mmol) and aniline (95 mg, 1.0 mmol) in 2 mL of THF were
added HOBT (75 mg, 0.5 mmol) and EDCI (100 mg, 0.6 mmol).
After 12 h the reaction mixture was diluted with AcOEt and
washed with 5% KHSO4, 5% NaHCO3, and water. The organic
layer was dried over MgSO4 and evaporated to dryness. The
residue was hydrogenated in MeOH as described for 2: yield
80 mg (54%) over the two steps; TLC (AcOEt/n-hexane, 1:1)
Rf 0.2; FAB-MS m/z 299.1 [M + H+]; Mr ) 298.1 calcd for
(2R,S)-HONH-Ma l(Me/Bn )-r-n a p h th yla m id e (13). Cou-
pling of 6 (155 mg, 0.5 mmol) with R-naphthylamine (143 mg,
2.0 mmol) followed by hydrogenation of the O-benzylhydrox-
amate was performed as described for 7: yield 95 mg (55%)
over the two steps; TLC (CHCl3/MeOH, 9:1) Rf 0.5; mp 112-
114 °C; FAB-MS m/z 349.2 [M + H+]; Mr ) 348.1 calcd for
17H18N2O3;1H NMR (DMSO-d6) δ 10.63 (s, 1H, NHOH), 9.61
C
21H20N2O3; 1H NMR (DMSO-d6) δ 10.85 (s, 1H, NHOH), 10.38
C
(s, 1H, NH (naphthyl)), 9.10 (s, 1H, NHOH), 7.17-7.95 (m,
12H, R-naphthyl + C6H5), 3.20 + 3.30 (2d, 2H, CH2C6H5), 1.17
(s, 3H, CH3).
(s, 1H, NHPh), 8.90 (s, 1H, NHOH), 7.6 + 7.05-7.35 (m, 10H,
2 C6H5), 3.23 (s, 2H, CH2C6H5), 1.26 (s, 3H, CH3).
(2R,S)-HONH-Ma l(Me/Bn )-NHBn (8). Coupling of 6 (155
mg, 0.5 mmol) with benzylamine (107 mg, 1.0 mmol) followed
by hydrogenation of the O-benzylhydroxamate was performed
as described for 7: yield 71 mg (40%) over the two steps; TLC
(CHCl3/MeOH, 9:1) Rf 0.5; mp 112-114 °C; FAB-MS m/z 313.1
(2R,S)-HONH-Ma l(Me/Bn )-1,2,9,10-tetr a h yd r oisoch in o-
lid e (14). The title compound was prepared by coupling 6 (155
mg, 0.5 mmol) with 1,2,9,10-tetrahydroisochinoline (143 mg,
2.0 mmol) and subsequent hydrogenation of the O-benzylhy-

3046 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Graf von Roedern et al.
droxamate as reported for 7: yield 105 mg (62%) over the two
steps; TLC (CHCl3/MeOH, 9:1) Rf 0.5; mp 112-114 °C; FAB-
Bn ONH-In d -OH (20). Indan-2,2-dicarboxylic acid diethyl
ester24 (4.05 g, 15 mmol) was saponified to the monoethyl ester
as described for 3 and then reacted with (benzyloxy)amine
hydrochloride following the procedure for 4. The resulting
BnONH-Ind-OEt was again saponified with KOH as described
for 6: yield 1.98 g of soft crystals (43% over the three steps);
TLC (CHCl3/MeOH, 4:1) Rf 0.7; FAB-MS m/z 312.3 [M + H+];
1
MS m/z 339.2 [M + H+]; Mr ) 338.1 calcd for C20H22N2O3; H
NMR (DMSO-d6) δ 10.65 (s, 1H, NHOH), 8.83 (s, 1H, NHOH),
7.05-7.25 (m, 9H, arom (tetrahydroisochinolide) + C6H5), 4.60
+ 3.60 + 2.80 (3m, 6H, 3 CH2 (tetrahydroisochinolide)), 3.15
(s, 2H, CH2C6H5Mal(Bn)), 1.17 (s, 3H, CH3).
1
Mr ) 311.1 calcd for C18H17NO4; H NMR (DMSO-d6) δ 13.0
(2R,S)-Bn ONH -Ma l(NH Ac/Bn )-OE t (15). (2R,S)-EtO-
Mal(NHAc/Bn)-OH (mp 127-129 °C)28 (1.40 g, 5.0 mmol) was
condensed in THF/DMF (2:1) with (benzyloxy)amine hydro-
chloride (0.96 g, 6 mmol) by the HOBT/EDCI method in the
presence of NMM as auxiliary base and worked up as described
for 4. The product was precipitated from AcOEt with ether/
n-pentane: yield 0.57 g (30%); TLC (AcOEt/n-hexane, 1:1) Rf
0.2; FAB-MS m/z 385.2 [M + H+]; Mr ) 384.1 calcd for
(br, 1H, COOH), 11.26 (s, 1H, NH), 7.15-7.35 (m, 9H, C6H4 +
C6H5), 4.84 (s, 2H, CH2C6H5), 3.56 (m, 4H, CH2).
HONH-In d -NHBn (21). Compound 20 (155 mg, 0.5 mmol)
was coupled with benzylamine (107 mg, 1.0 mmol) and
deprotected by hydrogenation as described for 7: yield 66 mg
(43%) over the two steps; TLC (CHCl3/MeOH, 9:1) Rf 0.4; mp
158 °C dec; FAB-MS m/z 311.1 [M + H+]; Mr ) 310.1 calcd for
21H24N2O5; 1H NMR (DMSO-d6) δ 11.44 (s, 1H, NHOBn), 7.67
C
18H18N2O3; 1H NMR (DMSO-d6) δ 10.66 (s, 1H, NHOH), 8.83
C
(s, 1H, NHOH), 8.18 (dd, 1H, NHBn), 7.05-7.30 (m, 9H, C6H4
+ C6H5), 4.26 (d, 2H, CH2C6H5), 3.45 (s, 4H, 2 CH2).
(s, 1H, NHAc), 7.34-7.38 (m, 5H, C6H5 (OBn)), 7.21-7.28 (m,
3H, 2H-3′ + H-4′ (Bn)), 6.99 (d, 2H, 2H-2′ (Bn)), 4.70 + 4.76
(2d, 2H, CH2 (OBn)), 4.08 (q, 2H, CH2CH3), 3.43 + 3.52 (2d,
2H, CH2 (Bn)), 1.94 (s, 3H, CH3 (Ac)), 1.16 (t, 3H, CH2CH3).
HONH-In d -NH-CH2CH2CH2-P h (22). BnONH-Ind-OH
(20) (155 mg, 0.5 mmol) was coupled with 3-phenylpropylamine
(135 mg, 1.0 mmol) and then hydrogenated as reported for 7:
yield 134 mg (80%) over the two steps; TLC (CHCl3/MeOH,
9:1) Rf 0.5; mp 148 °C; FAB-MS m/z 339.2 [M + H+]; Mr )
339.1 calcd for C20H22N2O3; 1H NMR (DMSO-d6) δ 10.53 (s,
1H, NHOH), 8.79 (s, 1H, NHOH), 7.62 (dd, 1H, NH-CH2CH2-
CH2-Ph), 7.07-7.30 (m, 9H, C6H4 + C6H5), 3.42 (s, 4H, 2 CH2
(Ind)), 3.06 (quart, 2H, NH-CH2CH2CH2-Ph), 2.49 (dd, 2H,
NH-CH2CH2CH2-Ph), 1.67 (q, 2H, NH-CH2CH2CH2-Ph).
Bn ONH-Ma l(Dbch )-OH (23). 6,7-Dihydro-5H-dibenzo[a,f]-
cycloheptene-6,6-dicarboxylic acid diethyl ester25 (3.36 g, 10
mmol) was saponified to the monoethyl ester as described for
3 and then reacted with (benzyloxy)amine hydrochloride
following the procedure for 4. Finally, BnONH-Mal(Dbch)-
OEt was saponified as described for 6: yield 1.73 g (45%) of a
colorless powder over the three steps; TLC (CHCl3/MeOH, 4:1)
Rf 0.7; FAB-MS m/z 388.0 [M + H+]; Mr ) 387.1 calcd for
(2R,S)-HONH-Ma l(NHAc/Bn )-NHBn (16). Compound 15
(1.92 g, 5 mmol) was saponified with KOH (0.80 g, 15.0 mmol)
in THF/water (4:1) by refluxing for 10 h. The reaction mixture
was diluted with water, extracted with ether, and then
acidified to pH 1.5 with 2 N HCl. The product was extracted
with AcOEt; the extracts were washed with water, dried with
MgSO4, and evaporated to small volume. Upon precipitation
with n-pentane (2R,S)-BnONH-Mal(NHAc/Bn)-OH was ob-
tained as a homogeneous compound (TLC, CH3CN/H2O, 4:1;
Rf 0.65) in almost quantitative yield.
BnONH-Mal(NHAc/Bn)-OH (178 mg, 0.5 mmol) was con-
densed with benzylamine (112 mg, 1.0 mmol) by HOBT/EDCI
in THF, and the reaction mixture was worked up as described
for 7 with additional purification by silica gel chromatography
using AcOEt/n-hexane (1:2) as eluent. The resulting BnONH-
Mal(NHAc/Bn)-NHBn was hydrogenated in MeOH over Pd/C
catalyst as described for 2: yield 70 mg (42%) over the three
steps; TLC (CHCl3/MeOH, 9:1) Rf 0.2; dec > 100 °C; FAB-MS
m/z 356.2 [M + H+]; Mr ) 355.1 calcd for C19H21N3O4; 1H NMR
(DMSO-d6) δ 10.78 (s, 1H, NHOH), 8.92 (s, 1H, NHOH), 8.57
(dd, 1H, NHBn), 7.40 (s, 1H, NHAc), 6.90-7.30 (m, 10H, 2
C6H5), 4.26 (m, 2H, CH2 (NHBn)), 3.59 + 3.52 (2d, 2H, CH2
(Bn)), 1.90 (s, 3H, CH3).
C
24H21NO4; 1H NMR (DMSO-d6) δ 13.0 (br, 1H, COOH), 11.43
(s, 1H, NH), 7.15-7.45 (m, 13H, arom), 4.66 (s, 2H, CH2C6H5),
3.35 (m, 4H, 2 CH2 (Dbch)).
HONH-Ma l(Dbch )-NH-CH2CH2CH2-P h (24). Compound
23 (193 mg, 0.5 mmol) was coupled with 3-phenylpropylamine
(135 mg, 1.0 mmol) and then hydrogenated as described for 7:
yield 136 mg (66%) over the two steps; TLC (CHCl3/MeOH,
9:1) Rf 0.6; mp 199 °C; FAB-MS m/z 415.3 [M + H+]; Mr )
414.2 calcd for C26H26N2O3; 1H NMR (DMSO-d6) δ 10.52 (s,
1H, NHOH), 8.80 (s, 1H, NHOH), 7.62 (dd, 1H, NH-CH2CH2-
CH2-Ph), 7.10-7.40 (m, 13H, arom), 3.29 (s, 4H, 2 CH2
(Dbch)), 3.10 (ddd, 2H, NH-CH2CH2CH2-Ph), 2.50 (t, 2H,
NH-CH2CH2CH2-Ph), 1.70 (ddd, 2H, NH-CH2CH2CH2-Ph).
Abbr evia tion s: MMP, matrix metalloproteinase; MMP8,
human neutrophil collagenase; EDCI, 1-ethyl-3-(3-(dimethy-
lamino)propyl)carbodiimide hydrochloride; HOBT, 1-hydroxy-
benzotriazole; Mal(R,R′), 2,2-bis-substituted malonyl; i-Bu,
isobutyl; Me, methyl; Bn, benzyl, Ph, phenyl; Ind, indan-2,2-
dicarboxy; Dbch, 6,7-dihydro-5H-dibenzo[a,f]cycloheptene-6,6-
dicarboxy; NMM, N-methylmorpholine.
EtO-Ma l(OBn /Bn )-OEt (17). Diethyl benzyloxymalonate30
(5.33 g, 20 mmol) was added to a solution of sodium (0.51 g,
22 mmol) in EtOH and reacted with benzyl bromide (4.3 g, 25
mmol) under refluxing for 4 h. The solvent was evaporated
and the residue dissolved in ether. NaBr was filtered off, the
filtrate evaporated, and the product isolated by distillation (180
°C, 0.01 mmHg): yield 4.2 g (59%) of a yellow oil; FAB-MS
1
m/z 357.2 [M + H+]; Mr ) 356.2 calcd for C21H24O5; H NMR
(DMSO-d6) δ 7.18-7.40 (m, 10H, 2 C6H5), 6.31 (s, 2H, CH2
(OBn)), 4.17 (m, 4H, 2 CH2CH3), 3.46 (s, 2H, CH2 (Bn)), 1.16
(t, 6H, 2 CH2CH3).
(2R,S)-Bn ONH-Ma l(OBn /Bn )-OEt (18). Compound 17
(3.56 g, 10 mmol) was saponified to the monoethyl ester as
described for 3 and then reacted with (benzyloxy)amine
hydrochloride following the procedure for 4: yield 1.17 g of a
slightly yellow oil (27% over the two steps); TLC (AcOEt/n-
hexane, 1:1) Rf 0.7; FAB-MS m/z 434.2 [M + H+]; Mr ) 433.2
calcd for C26H27NO5; 1H NMR (DMSO-d6) δ 11.25 (s, 1H,
NHOBn), 7.18-7.40 (m, 15H, 3 C6H5), 6.31 (s, 2H, CH2
(NHOBn)), 4.65 (s, 2H, CH2 (OBn)), 4.23 (m, 2H, CH2CH3),
3.42 (s, 2H, CH2 (Bn)), 1.28 (t, 3H, CH2CH3).
Ack n ow led gm en t. The authors thank Prof. H.
Tschesche (University of Bielefeld, Germany) for provid-
ing the catalytic domain of MMP8, Ms. I. Buhrow for
the mass spectra, and Dr. I. Sonnenbichler for the NMR
spectra. This study was supported by Boehringer
Mannheim GmbH with a postdoctoral fellowship for
E.G.V.R.
(2R,S)-HONH-Ma l(OH/Bn )-NHBn (19). The ethyl ester
18 (1.17 g, 2.7 mmol) was saponified as described for 6 and
then condensed with benzylamine following the procedure
reported for 7. Deprotection was performed as described for
2: yield 280 mg (33%) over the three steps; TLC (CHCl3/
MeOH, 9:1) Rf 0.45; mp 138 °C dec; FAB-MS m/z 315.1 [M +
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