Evaluation of P1'-Diversified Phosphinic Peptides Leads to the Development of Highly Selective Inhibitors of MMP-11

Article abstract of DOI:10.1021/jm0308491

Phosphinic peptides were previously reported to be potent inhibitors of several matrixins (MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast cancer, a series of phosphinic pseudopeptides bearing a variety of P

Full text of DOI:10.1021/jm0308491

J . Med. Chem. 2004, 47, 325-336
325
Eva lu a tion of P 1’-Diver sified P h osp h in ic P ep tid es Lea d s to th e Develop m en t of
High ly Selective In h ibitor s of MMP -11
Magdalini Matziari,† Fabrice Beau,‡ Philippe Cuniasse,‡ Vincent Dive,‡ and Athanasios Yiotakis*,†
Department of Chemistry, Laboratory of Organic Chemistry, University of Athens,
Panepistimiopolis Zografou 15771, Athens, Greece, and De´partement d’Inge´nierie et d’Etudes des Prote´ines,
CEA, 91191 Gif/ Yvette Cedex, France
Received March 25, 2003
Phosphinic peptides were previously reported to be potent inhibitors of several matrixins
(MMPs). To identify more selective inhibitors of MMP-11, a matrixin overexpressed in breast
cancer, a series of phosphinic pseudopeptides bearing a variety of P1’-side chains has been
synthesized, by parallel diversification of a phosphinic template. The potencies of these
compounds were evaluated against a set of seven MMPs (MMP-2, MMP-7, MMP-8, MMP-9,
MMP-11, MMP-13, and MMP-14). The chemical strategy applied led to the identification of
several phosphinic inhibitors displaying high selectivity toward MMP-11. One of the most
selective inhibitors of MMP-11 in this series, compound 22, exhibits a Ki value of 0.23 µM
toward MMP-11, while its potency toward the other MMPs tested is 2 orders of magnitude
lower. This remarkable selectivity may rely on interactions of the P1’-side chain atoms of these
inhibitors with residues located at the entrance of the S1’-cavity of MMP-11. The design of
inhibitors able to interact with residues located at the entrance of MMPs’ S1’-cavity might
represent an alternative strategy to identify selective inhibitors that will fully differentiate
one MMP among the others.
In tr od u ction
some conformational flexibility in the S1’-specificity loop,
the size and the shape of the MMPs’ S1’-specificity
pocket change upon inhibitor binding.8 Such flexibility
in the S1’-specificity pocket has been frequently proposed
to explain the lack of specificity of numerous inhibitors
toward MMPs.7,8 Among the members of the MMP
family, stromelysin-3 (MMP-11), which was first de-
scribed to be expressed by fibroblastic cells surrounding
invasive breast carcinoma,9 displays a number of un-
usual features: mature forms of MMP-11 are unable
to degrade any major extracellular matrix proteins, and
whereas most of the MMPs should be activated extra-
cellularly, MMP-11 is secreted in active form.10,11 Recent
in vivo studies demonstrated that MMP-11 exhibits a
new and unexpected role for an MMP during tumoro-
genesis, acting as a cancer cell survival factor, instead
of increasing neo-angiogenesis or cancer cell prolifera-
tion, suggesting that MMP-11 is more involved in tumor
formation than in tumor growth.12
Matrix metalloproteases (MMPs) have been consid-
ered to be attractive targets for cancer therapy on the
basis of their overexpression and activation in malig-
nant tissues and their unique ability to degrade all
components of the extracellular matrix.1 Unfortunately,
the results of clinical trials performed with synthetic
MMP inhibitors were disappointing. Current under-
standing of the wide range of biological activities
mediated by MMPsscell death, proliferation, differen-
tiation, tumor-associated angiogenesis, malignant
conversionsmay explain the lack of efficacy of MMP
inhibitors in anticancer therapy.2-4 Indeed, such studies
suggest the assignment of unique functions to individual
MMPs; however, the exact role of each MMP during
tumor progression is still poorly understood. Clearly,
there is a need for the development of highly selective
inhibitors for the different MMPs in order to determine
the specific role of each MMP along the different stages
of tumor progression. This knowledge may lead to the
reconsideration of the use of MMP inhibitors in cancer
clinical trials and could also be of great value for their
use in other pathologies associated with MMPs’ over-
expression.5,6
We previously reported the development of the first
potent inhibitors of MMP-11, based on phosphinic
peptide chemistry, e.g. RXP03, (Scheme 1).13 Whereas
the developed compounds displayed weak potencies
toward MMP-1 and MMP-7, they turned out also to be
very potent inhibitors of MMP-8, MMP-2, and MMP-9.
This lack of specificity precludes further use of these
inhibitors in animal models to evaluate the effect of
MMP-11-specific inhibition on tumor growth. To develop
a new series of phosphinic pseudopeptide inhibitors of
MMP-11, the core structure of RXP03 was retained,
since this compound has proven to be active in vivo.14
Furthermore, like other phosphinic peptides,15,16 RXP03
was observed to be metabolically stable in vivo.17 In
addition, previous structure-activity studies have dem-
onstrated the positive contribution to the inhibitory
The development of highly selective inhibitors of
MMPs, able to differentiate between the 25 members
of this protease family, is a particularly difficult task,
since the topology and the nature of the residues in the
enzyme active site are highly conserved among the
different MMPs.7 Moreover, due to the occurrence of
* To whom correspondence should be addressed. Tel/Fax: 0030-210-
7274498. E-mail: yiotakis@chem.uoa.gr.
† University of Athens.
‡ CEA.
10.1021/jm0308491 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/17/2003

326 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Matziari et al.
Sch em e 1. Stucture of Phosphinic Peptide Inhibitors of
MMPs
ester I is subjected to Michael addition of Z-protected
silyl phosphonite, leading to dehydroalaninyl phosphinic
synthon 1, via a tandem Arbuzov addition/allylic rear-
rangement. Removal of the tert-butyl ester (compound
2) and coupling with L-tryptophanylamide affords the
dehydroalaninyl precursor 3, in 65% overall yield.
Compound 3 was used as a template for parallel
diversification of the P1’-position, with a wide variety
of side chains, as shown in Table 1, using commercially
available thiols, leading to the target pseudocysteine
phosphinic peptide inhibitors 4-26, in good to excellent
yields (Table 1).
On the basis of the common precursor 3, this strategy
allowed the synthesis of the inhibitors of the present
study in a parallel manner, expeditiously and cost-
effectively. Yield and reaction time for thiol additions
are given in Table 1.
Phosphinic inhibitor 27 was prepared from 25 by
Suzuki arylation, using phenylboronic acid, as shown
in Scheme 3, in 81% yield.
As an additional perspective of this strategy, it was
examined whether the synthetic procedure described
above can be applied in solid-phase synthesis (Scheme
4). Therefore, the possibility to directly diversify the
dehydroalaninyl phosphinic template 3 while anchored
to a solid support has been checked. Coupling of synthon
2 with the amino terminus of L-tryptophan, attached
to Rink amide resin, can be achieved by applying the
procedure used for the solution methodology. Interest-
ingly, a single coupling step with 1 equiv of the phos-
phinic block 2 was sufficient, as indicated by a negative
Kaiser test and proved by HPLC. Thiol addition can be
performed under identical conditions with those in
solution. The procedure was applied for the nucleophilic
addition of an aromatic thiol, 8, and an aliphatic
compound, 10, giving results identical with those cre-
ated using solution methodology. Conclusively, it has
been established that parallel diversification of phos-
phinic peptides can also take place on a solid support,
providing an interesting alternative with regard to
automatization and/or even for the preparation of longer
sequences.
potency of a sulfur atom, when present in the P1’-side
chain of phosphinic peptide inhibitors of MMPs. Such
sulfur-containing compounds can be prepared by paral-
lel chemical diversification of a common dehydroalaninyl
precursor. Therefore, phosphinic peptides displaying the
general structure depicted in Scheme 1 were selected
for this study.
Resu lts
Ch em istr y. The classical approach to prepare a
series of P1’-diversified phosphinic peptides would be
rather inconvenient, since the preparation of the 24
inhibitors reported herein would demand four synthetic
steps, as well as three column purifications for each
inhibitor. To avoid using this low-yielded, cumbersome,
and time-consuming procedure, a new chemical strategy
has been applied. As shown in Scheme 2, according to
previously applied procedures,13 2-(bromomethyl)acrylic
ester I is converted to the pseudocysteine precursors of
type II. Phosphinic synthons of type III are usually
obtained by a Michael addition of the silyl ester18,19 of
Z-protected phenylalanine phosphinic analogue20 to
acrylate II.21 Cleavage of carboxylic ester III and
coupling of IV with L-tryptophanylamide could afford
the target inhibitors 4-26 in low to moderate yields. It
is worth mentioning that such couplings, where the
hydroxyphosphinyl function is unprotected, proceed in
moderate to good yields, but they are strongly influenced
by the size and nature of the P1’-side chain.22 This factor
introduces an additional disadvantage to this method,
given the diversity of the P1’-side chains of the target
phosphinic inhibitors. Protection of the hydroxyphos-
phinyl group would be a safer choice,21 but it would
render the procedure even lengthier and would probably
create additional restrictions in terms of orthogonal
protection of all functional groups present at the target
molecules.
In h ibitor y P oten cies. Screening of the first set of
compounds was performed with phosphinic peptides in
which the side chain in the P1’-position possesses both
S- and R-configuration. Indeed, we previously reported
that in this family of phosphinic peptides, both configu-
rations of the P1’-side chain provide potent inhibitors
of MMPs.14 As shown in Figure 1, compounds in which
the sulfur atom is connected with arylalkyl (compounds
4-7) are rather potent inhibitors of MMP-11 and MMP-
8. The lack of potency of the examined compounds for
MMP-7 is consistent with previous observations and is
in agreement with the presence of a small S1’-pocket in
the active site of this MMP.7 Connection of the sulfur
atom with either aliphatic side chains (compounds 11-
13) or aliphatic side chains bearing a carboxylic ester
functionality (compounds 14-17) provided inhibitors
exhibiting lower potencies. Surprisingly, the cyclic
aliphatic compound 10, bearing a cyclohexyl group,
displayed a remarkable selectivity for MMP-11. Con-
siderable selectivity for MMP-11 was also observed with
compound 9, bearing a phenyl group. Remarkably, the
presence of a naphthyl group in compound 8 led to an
To overcome these drawbacks, we developed a new
synthetic strategy, which allows fast and easy diversi-
fication of the P1’-position of phosphinic peptides.23 As
shown in Scheme 2 (steps e-h), 2-(bromomethyl)acrylic

Highly Selective Inhibitors of MMP-11
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 327
Sch em e 2. General Synthetic Proceduresa
a
Reagents, conditions, and yields: (a) RSH (1.1 equiv), MeONa/MeOH (1.1 equiv) in Et2O, -15 °C, to room temperature, 2-3 h; (b)
Z(R)PhePO2H2 (0.77 equiv), HMDS (4 equiv), 110 °C, 4 h, under argon, then EtOH; (c) cleavage of carboxylic ester, TFA/DCM 50% or
saponification; (d) L-TrpNH2 (1 equiv), EDC‚HCl (4 equiv), HOBt (1 equiv), DIPEA (2 equiv), in DCM, rt, 1 h; (e) same as step b, X ) But,
94%; (f) TFA/DCM 50%, rt, 1 h, quantitative; (g) same as step d, 69%; (h) RSH (4 equiv), R given in Table 1, EtONa/EtOH or MeONa/
MeOH for compounds 14 and 16 (2 equiv), in THF, rt, 3-40 h, 62-98%.
Sch em e 3. Synthesis of Compound 27a
of compound 9 for MMP-7, this MMP was removed from
the set of MMPs taken under consideration. All these
compounds were purified by HPLC, under conditions
allowing the separation of the two diastereomeric forms
of these compounds. Data that are reported in Figure 2
are those corresponding to the diastereomers eluting
first from the HPLC column. Indeed, evaluation of the
two HPLC fractions obtained for each compound re-
vealed that the first eluting fraction displayed higher
potency toward MMP-11 than the second one. Inspection
of Figure 2 demonstrates that most of the selected
phenyl ring modifications dramatically increase the
selectivity of the inhibitors toward MMP-11. However,
this gain seems to correlate with a loss of potency of
the compounds toward MMP-11 by 1 order of magni-
tude. The substitution on the phenyl ring in the ortho-
position seems to play a key role on the selectivity, since
meta-modification provided inhibitors with lower selec-
tivity, as indicated by the potencies of compounds 22
and 26. Finally, to improve the potency of the inhibitor,
compound 27, which contains a longer side chain and
retains the ortho-substitution for selectivity control, was
prepared. Indeed, as exemplified by compound 5, in-
a
Reagents and conditions: PhB(OH)2 (1.2 equiv), Pd[P(Ph3)]4
(0.05 equiv), 2 M Na2CO3 (2 equiv), reflux in toluene, under argon,
48 h, 81%.
inhibitor that did not differentiate between MMP-11 and
MMP-8. Compound 8 turned out also to be a potent
inhibitor of MMP-8, as compared to compound 9. The
rather high potency and selectivity displayed by com-
pound 9 toward MMP-11 led us to develop a series of
analogues in which the phenyl ring was modified by
different kinds of substituents. Given the lack of potency

328 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Ta ble 1. Yields and Reaction Time for Compounds 4-26
Matziari et al.
Ta ble 2. Ki Values and Selectivity Factors of Compounds 18,
22, and 24 toward MMPs
Mod elin g Stu d ies. In an attempt to find a structural
basis for the unusual selectivity of the compounds
reported in Table 2, models of interaction between the
compound 18 and the active site of three MMPs (MMP-
11, MMP-8, and MMP-14) were developed. The selected
inhibitor displayed a potency of 110 nM toward MMP-
11 and was 260 and 160 times less potent toward MMP-
14 and MMP-8, respectively (Table 2). For each model,
the interaction energy between the P1’-side chain and
the residues of the MMP catalytic domain was calcu-
lated. On the basis of these calculations, the residues
displaying an interaction energy lower than a threshold
value of 0.1 kcal/mol were identified (Figures 3 and 4).
As shown in Figure 3, the P1’-side chain of compound
18 interacts with a limited set of MMP residues,
belonging to the so-called S1’-specificity pocket. Inspec-
tion of Figure 4 reveals that the interaction energy
values between the P1’-side chain and these residues
no.
MMP-11 MMP-14 MMP-2 MMP-9 MMP-13 MMP-8
18 Ki (µM)
0.11
1
0.23
1
0.41
1
30.1
266
160
705
30
4.65
41
30
132
200
484
3.91
35
35.6
157
148
358
4.7
42
15.7
69
200
484
18.4
163
38.9
171
200
484
Sa
22 Ki (µM)
Sa
24 Ki (µM)
Sa
73
a
S ) Ki(MMP-X)/Ki(MMP-11).
creasing the size of the P1’-side chains provided com-
pounds with higher inhibitory potencies. Unfortunately,
this novel compound did not display higher potency
toward MMP-11. Ki values and selectivity factors of the
most discriminating compounds are reported in Table
2. Compound 22 displays a Ki value of 0.23 µM toward
MMP-11 and its potency is 2 orders of magnitude lower
against other MMPs.

Highly Selective Inhibitors of MMP-11
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 329
F igu r e 1. Graphic representation of inhibitory potencies of compounds 4-17 toward MMPs. The compounds were tested as a
mixture of diastereoisomers.
Sch em e 4. Solid Phase Diversificationa
three MMPs, positions 338, 340, 342 of the S1’-cavity
(residues squared in Figure 4). Thus, these models did
not offer an explanation for the different affinities
displayed by the compound 18 toward the selected
MMPs.
Discu ssion
The aim of this study was to develop compounds able
to selectively inhibit MMP-11 over other MMPs coex-
pressed with MMP-11 in cancer tissues.3-5,9 Resolution
of the crystal structure of the catalytic domain of MMP-
11, in interaction with the phosphinic peptide inhibitor
RXP03, has revealed that MMP-11 belongs to the group
of MMPs characterized by the presence of a very deep
S1’-cavity in their active sites.24 This observation cor-
roborates the striking ability of MMP-11 to cleave
efficiently synthetic substrates harboring an unusually
long side chain in their P1’-position25 and to interact
tightly with inhibitors displaying the same structural
features.13 The presence of such S1’-cavity in MMP-11
explains also why the development of selective inhibitors
of this MMP has been a tedious task.13 Indeed, with the
exception of MMP-1 and MMP-7, which possess a small
S1’-cavity, most of the MMPs display a S1’-cavity similar
to the one observed in MMP-11.7 In connection with this,
it should be remarked that many of the inhibitors of
MMPs that claim to be highly selective are actually
a
Reagents and conditions: (a) 2 (1 equiv), EDC‚HCl (4 equiv),
HOBt (1 equiv), DIPEA (2 equiv), in DCM, rt, 1.5 h; (b) cyclohexyl
or naphthyl thiol (4 equiv), 1 M EtONa/EtOH (2 equiv), in THF,
rt; (c) TFA/TIS/H2O 95:2.5:2.5.
are not significantly different in the three models. This
applies even for residues not strictly conserved in the

330 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Matziari et al.
F igu r e 2. Graphic representation of inhibitory potencies of compounds 18-27 toward MMPs.
F igu r e 3. Model of interaction of compound 18 and the S1’-subsite of MMP-11. Created with CHARMM.31
compounds that essentially discriminate MMPs accord-
ing to the size of their S1’-cavity. For example, AG-3340
or prinomastat, one of the most cited selective inhibitor
of MMPs, differentiates MMP-2 (Ki ) 50 pM) from
MMP-1 and MMP-7 with a selectivity factor of 166 and
1080, respectively.26 However, toward MMP-3 and
MMP-13, two MMPs with a deep S1’-cavity, the selectiv-
ity factor of prinomastat is only 6 and 0.6, respectively
(MMP-2 as reference).26 Thus, to our knowledge, com-
pounds 22 and 24 are the most selective inhibitors of

Highly Selective Inhibitors of MMP-11
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 331
information provided by experimental approaches, such
as NMR, designed to probe the mobility of proteins.27
Previous design of selective inhibitors of MMPs was
mainly based on the development of compounds bearing
very long, unusual P1’-side chains able to interact with
residues located at the bottom of the MMP S1’-sub-
site.7,28 This strategy provides potent inhibitors of
MMPs, but as exemplified by compounds 4, 5, and 6,
such inhibitors do not display high selectivity, for the
reasons discussed above. The most selective compounds
reported in this study are characterized by the presence
of groups, at the ortho-position of the phenyl ring, that
interact with residues of MMPs located at the entrance
of the S1’-cavity. These results suggest that the develop-
ment of compounds able to probe the entrance of the
S1’-cavity, rather than its bottom part, might represent
an alternative strategy to obtain more selective inhibi-
tors of MMPs. This proposal is supported by the
observation that the upper segment of the S1’-loop,
located at the entrance of the S1’-cavity, displays high
sequence variability. Furthermore, comparison of the
X-ray structure of the catalytic domains of different
MMPs shows that the conformation of the upper part
of the S1’-loop is highly conserved, suggesting that this
segment of the S1’-loop is less flexible than its lower
part.29,30 The selectivity of the reported compounds for
MMP-11 represents a significant progress. Further
increase of their potencies by developing inhibitors
bearing P1’-side chains able to probe the entrance of the
S1’-cavity and fill this cavity may represent attractive
future approaches. Compound 27, developed according
to this proposal, did not display the expected increase
in potency. This result clearly shows the limit of
classical structure-based design to discover highly selec-
tive inhibitors of MMPs. Better understanding of the
factors governing the potency and selectivity of inhibi-
tors toward MMPs is of critical importance in order to
realize further significant progress in this field.
F igu r e 4. Interaction energies in kcal/mol of the P1’-side chain
of compound 18 with different residues of the S1’-subsite of
MMP-11, MMP-14, and MMP-8, calculated using the molec-
ular models of interaction of compound 18 with the catalytic
domains of MMP-11, MMP-14, and MMP-8 as described in the
Experimental Section. The residue names correspond to those
of MMP-11.
an MMP reported to date, which is able to differentiate
MMP-11 between other examined MMPs that share a
deep S1’-cavity. As far as the bound form of the enzyme
(EI complex) is considered, modeling and computational
studies performed with compound 18 did not offer
explanations for the better potency displayed by this
inhibitor toward MMP-11. These simulations indicated
that the interaction of compound 18 with the active sites
of MMPs do not imply steric hindrance nor noticeable
conformational shift of the residues surrounding the P1’-
side chain of this compound, when the RXP03-MMP-
11 complex is taken as reference.24 These results were
in agreement with the observation that the inhibitors
harboring bulkier groups (compounds 19-25) than a Br
atom displayed potency and selectivity similar to com-
pound 18. A better agreement between experimental
and calculated data may require taking into account
other contributions to the binding free energy, such as
entropy or solvation energy. The role of water molecules
observed in the S1’-cavity of MMPs, in specific positions,
remains also to be determined.24 As previously dis-
cussed, the development of accurate models in the
specific case of MMPs suffers from the inherent flex-
ibility of the S1’-loop.7,8 It should be remarked that the
occurrence of conformational changes at the S1’-loop
level was mostly observed upon binding of inhibitors
bearing a very long P1’-side chain and were suggested
to take place in order to avoid steric clash.7,8 Our models
showed that the binding of compound 18 involves no
steric clash and the P1’-side chain of this inhibitor does
not enter deeply in the S1’-cavity. Thus, the inherent
flexibility involving residues located in the bottom part
of the S1’-loop (bottom of the S1’-cavity) is not expected
to play any important role during the binding of
compound 18. However, we could not exclude the
occurrence of conformational changes in other parts of
the enzyme active site upon inhibitor binding. Most of
the available X-ray structures of MMPs correspond to
forms of MMPs complexed to inhibitors. Little informa-
tion is available regarding the conformational states of
MMPs free of inhibitors. Therefore, development of
accurate models to describe these states represents a
difficult task. As recently illustrated, better understand-
ing of the molecular motions involved in the MMP-
inhibitor interactions will be greatly assisted by the
Con clu sion s
Implementation of a chemical strategy to produce
rapidly P1’-diversified phosphinic pseudopeptides led to
the identification of inhibitors able to discriminate
MMP-11 from other MMPs with a two-order magnitude
of selectivity. These results confirm the efficacy of
phosphinic peptide chemistry for the development of
highly selective inhibitors of zinc-metalloproteases.
Further development of the phosphinic peptide chem-
istry, conjugated to experimental approaches providing
information on mobility of MMP-inhibitor complexes,
should contribute to the discovery of the expected
selective MMP inhibitors.
Exp er im en ta l Section
Gen er a l P r oced u r es. All of the compounds, for which
analytical and spectroscopic data are quoted, were homoge-
neous by TLC. TLC analyses were performed using silica gel
plates (E. Merck silica gel 60 F-254) and components were
visualized by the following methods: ultraviolet light absor-
bance and charring after spraying with a solution of (NH4)-
HSO4 and ninhydrin spray. The solvent systems used for TLC
developments were (a) CHCl3-MeOH-AcOH (7:2:1), (b)
CHCl3-MeOH-AcOH (7:0.5:0.5), (c) 1-butanol-AcOH-H2O (4:
1:1), (d) petroleum ether (bp 40-60 °C) AcOEt-AcOH (3:3:
0.2). Column chromatography was carried out on silica gel (E.
Merck, 70-230 mesh), height 35 cm, diameter 1.2 cm. Melting

332 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Matziari et al.
points provided are those of the diastereoisomeric mixturess
where presentsand are not corrected.
temperature and then evaporated to dryness. The residue was
treated with DCM and a solid precipitated. Filtration afforded
the product quantitatively (0.4 g) as a white solid: mp 179-
181 °C; TLC Rf(a) 0.57, Rf(c) 0.74; HPLC (column 1, gradient
The purity of phosphinic peptides was established by
analytical HPLC and mass spectrometry; ESMS spectra were
recorded on a Micromass Platform II instrument using nega-
tive ionization mode by Atheris Laboratories, 314 CH-1233
Bernex, Geneva, Switzerland.
1
1) 25.46; H NMR (200 MHz, DMSO-d6) δ 2.45-2.86 (m, 3H,
PCH2, PhCHH), 3.03-3.18 (m, 1H, PhCHH), 3.85-4.04 (m,
1H, PCH), 4.82-5.01 (br s, 2H, OCH2Ph), 5.78 (s, 1H, Cd
CHH), 6.16 (s, 1H, CdCHH), 7.12-7.64 (m, 11H, aryl, NH);
13C NMR (50 MHz, DMSO-d6) δ 29.3 (d, 1J PC ) 85.2 Hz, PCH2),
32.9 (CH2Ph), 51.8 (d, 1J PC ) 105.1 Hz, PCH), 65.1 (OCH2Ph),
126.2, 127.1, 127.6, 128.2, 129.0, 132.8, 137.3, 138.3, 138.6
HPLC analyses and preparative purifications were carried
out on a MZ-Analytical Column 250 × 4 mm, Kromasil, 100,
C18, 5µm (column 1), at a flow rate of 0.5 mL/min, an
analytical Hypersil C18 (column 2), at a flow rate of 1 mL/
min, and a semipreparative VYDAC C18 (column 3), at a flow
rate of 3 mL/min. Solvent A: 10% CH3CN, 90% H2O, 0.1%
TFA. Solvent B: 90% CH3CN, 10% H2O, 0.09% TFA. The
following gradients were used: Gradient 1: t ) 0 min (0% B),
t ) 10 min (25% B), t ) 45 min (75% B). Gradient 2: t ) 0
min (0% B), t ) 15 min (45% B), t ) 45 min (60% B). Gradient
3: t ) 0 min (0% B), t ) 10 min (15% B), t ) 45 min (40% B).
Gradient 4: t ) 0 min (0% B), t ) 15 min (35% B), t ) 45 min
(60% B). Gradient 5: t ) 0 min (0% B), t ) 15 min (35% B), t
) 45 min (65% B). Gradient 6: t ) 0 min (0% B), t ) 15 min
(50% B), t ) 45 min (75% B). Gradient 7: t ) 0 min (0% B), t
) 15 min (30% B), t ) 45 min (65% B). Gradient 8: t ) 0 min
(0% B), t ) 15 min (35% B), t ) 45 min (70% B). Gradient 9:
t ) 0 min (0% B), t ) 10 min (35% B), t ) 45 min (55% B).
Eluted peaks were detected at 280, 254, and 230 nm. Given
times correspond to two diastereoisomeric forms and are
counted in minutes.
1H, 13C, and 31P NMR spectra were recorded on a 200 MHz
Mercury Varian spectrometer. All NMR experiments were
carried at 298 K. 13C and 31P NMR spectra are fully proton
decoupled. 31P chemical shifts are reported on δ scale, in ppm,
downfield from 85% H3PO4.
Abbr evia tion s: AcOH, acetic acid; AcOEt, ethyl acetate;
But, tert-butyl; DCM, dichloromethane; DIC, disopropylcarbo-
diimide; DIPEA, diisopropylethylamine; DMF, dimethylfor-
mamide; DMSO, dimethyl sulfoxide; EDC‚HCl, 1-ethyl-3-(3’-
dimethylaminopropyl)carbodiimide‚HCl; Et2O, diethyl ether;
EtOH, ethanol; HMDS, 1,1,1,3,3,3-hexamethyldisilazane; HOBt,
1-hydroxybenzotriazole; MeOH, methanol; PE (40-60), petro-
leum ether (bp 40-60 °C); PhB(OH)2, phenylboronic acid; Pd-
[P(Ph)3]4, tetrakis(triphenylphosphine)palladium; TFA, trif-
luoroacetic acid; THF, tetrahydrofuran; TIS, triisopropylsilane;
Z, benzyloxycarbonyl.
3
3
(aryl, vinyl), 156.0 (d, J PC ) 4.2 Hz, OCONH), 167.6 (d, J PC
) 4.1 Hz, COOH); 31P NMR (81 MHz, DMSO-d6) δ 42.38;
ESMS m/z calcd for C20H21NO6P (M - H)- 402.1, found 402.1.
[(R),(S)]-(1-Ben zyloxyca r bon yla m in o-2-p h en yleth yl)-
{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]a llyl}-
p h osp h in ic Acid (3). To a suspension of compound 2 (1 mmol,
0.40 g) in DCM (20 mL) were added DIPEA, (1 mmol, 0.13 g,
0.17 mL), L-tryptophanylamide (1 mmol, 0.20 g), HOBt (1
mmol, 0.13 g), EDC‚HCl, (4 mmol, 0.77 g), and finally another
1 mmol of DIPEA. The reaction mixture was stirred for 1 h at
room temperature. Then it was diluted with DCM (60 mL) and
washed with a solution of 1 N HCl (3 × 5 mL), a saturated
solution of NH4HCO3 (3 × 1 mL), 1 N HCl to pH 1 and brine
(10 mL). The organic layer was dried over Na2SO4 and
concentrated in vacuo. Purification by flash column chroma-
tography using CHCl3-MeOH-AcOH (7:1:0.4), as eluent af-
forded the product (0.41 g, 69%) as a white solid, mp 176-
180 °C. TLC Rf(a) 0.55, Rf(c) 0.73; HPLC (column 1, gradient
1
1) 30.59; H NMR (200 MHz, DMSO-d6) δ 2.20-3.42 (m, 6H,
indolylCH2, PCH2, PhCH2), 3.73-4.02 (m, 1H, PCH), 4.33-
4.56 (m, 1H, indolylCH2CH), 4.75-5.05 (br s, 2H, OCH2Ph),
5.17 (s, 1H, CdCHH), 5.44 (s, 1H, CdCHH), 6.92-7.68 (m,
1
17H, aryl, NH); 13C NMR (50 MHz, DMSO-d6) δ 27.9 (d, J PC
1
) 78.6 Hz, PCH2), 33.6 (CH2Ph), 50.5 (d, J PC ) 103.9 Hz,
PCH), 54.7 (indolylCH2CH), 64.9 (OCH2Ph), 111.1, 118.3,
121.1, 123.9, 126.6, 127.4, 128.1, 129.3, 136.1, 137.5, (aryl,
vinyl), 139.9, 155.9 (OCONH), 168.6 (CH2dCCONH), 174.6
(CONH2); 31P NMR (81 MHz, DMSO-d6) δ 29.37; ESMS m/z
calcd for C31H32N4O6P (M - H)- 587.2, found 587.4.
Gen er a l P r oced u r e for Th iol Ad d ition , in Solu tion . To
a solution of compound 3 (0.05 mmol, 0.029 g) in freshly
distilled THF (3 mL) were added the appropriate thiol (0.2
mmol) and a 1 M solution of EtONa/EtOH (0.1 mmol, 0.1 mL),
except in the case of compounds 14 and 16, where MeONa/
MeOH was used instead of EtONa/EtOH. The reaction mixture
was stirred at room temperature (for reaction time, see Table
1) and then evaporated to dryness. After the addition of some
drops of H2O in the residue, CHCl3 (5 mL) was added and the
resulting solution was washed with a solution of 1 N HCl to
pH 1. The organic layer was dried over Na2SO4 and concen-
trated in vacuo. Dissolution of the mixture in the minimum
volume of CHCl3, precipitation with PE (40-60), filtration, and
washings of the residue with PE (40-60) afforded the products
as white solids. Further purification of the products can be
achieved by column chromatography using CHCl3-MeOH-
AcOH (7:0.7:0.4) as eluent system. Yields are given in Table
1.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-p h en eth ylsu lfa n ylp r op yl}p h osp h in ic a cid (4): TLC Rf-
(a) 0.55/0.69, Rf(c) 0.74/0.79; HPLC (column 2, gradient 1)
29.08/31.53; ESMS m/z calcd for C39H42N4O6PS (M - H)-
725.3, found 725.9; relative intensity of the molecular ion, 98%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(4-ch lor oben zylsu lfa n yl)p r op yl}p h osp h in ic a cid (5):
TLC Rf(a) 0.60/0.71, Rf(c) 0.78/0.84; HPLC (column 2, gradient
1) 30.17/32.61; ESMS m/z calcd for C38H39ClN4O6PS (M - H)-
745.2, found 745.8; relative intensity of the molecular ion, 98%.
Ch em istr y. (R)-2-[(1-Ben zyloxyca r bon yla m in o-2-p h en -
yleth yl)h yd r oxyp h osp h in oylm eth yl]a cr ylic Acid ter t-
Bu tyl Ester (1). A suspension of (R)-(1-benzyloxycarbony-
lamino-2-phenylethyl)phosphinic acid (10 mmol, 3.19 g) and
HMDS (50 mmol, 4.8 g, 6.3 mL) was heated at 110 °C for 1 h
under argon atmosphere. Acrylate I (X ) But) (13 mmol, 2.87
g) was added at this temperature dropwise for 15 min and the
reaction mixture was stirred for 3 h. Then, absolute EtOH (30
mL) was added dropwise. After cooling at room temperature,
the mixture was evaporated in vacuo. The residue was
dissolved in AcOEt (200 mL) and the resulting suspension was
washed with HCl (1 N, 2 × 50 mL). Purification by column
chromatography, using CHCl3-MeOH-AcOH (7:0.4:0.4) as
eluent, afforded the product (4.3 g, 94%) as white solid: mp
1
141-145 °C; TLC Rf(b) 0.53, Rf(d) 0.37; H NMR (200 MHz,
CDCl3/TFA-d1 ) 99.5/0.5) δ 1.47 (s, 9H, C(CH3)3), 2.60-3.05
(m, 3H, PCH2, PhCHH), 3.15-3.37 (m, 1H, PhCHH), 4.16-
4.35 (m, 1H, PCH), 4.85-5.06 (br s, 2H, OCH2Ph), 5.70-5.88
(m, 2H, NH, CdCHH), 6.23 (s, 1H, CdCHH), 7.02-7.32 (m,
10H, aryl); 13C NMR (50 MHz, CDCl3/TFA-d1 ) 99.5/0.5) δ 27.8
1
(C(CH3)), 30.8 (d, J PC ) 94.6 Hz, PCH2), 34.3 (CH2Ph), 51.3
(d, 1J PC ) 107.5 Hz, PCH), 66.5 (OCH2Ph), 81.7 (C(CH3)), 126.1,
127.6, 128.2, 129.1, 133.5, 136.5, 137.8, 138.1 (aryl, vinyl),
156.8 (OCONH), 167.3 (COOBut); 31P NMR (81 MHz, CDCl3/
TFA-d1 ) 99.5/0.5) δ 49.53; ESMS m/z calcd for C24H29NO6P
(M - H)- 458.2, found 458.5.
(R)-2-[(1-Ben zyloxyca r bon yla m in o-2-p h en yleth yl)h y-
d r oxyp h osp h in oylm eth yl]a cr ylic Acid (2). To a solution
of compound 1 (1 mmol, 0.46 g) in DCM (5 mL) was added
TFA (5 mL). The reaction mixture was stirred for 1 h at room
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(4-m eth oxyben zylsu lfa n yl)p r op yl}p h osp h in ic a cid (6):
TLC Rf(a) 0.65/0.76, Rf(c) 0.80/0.85; HPLC (column 2, gradient

Highly Selective Inhibitors of MMP-11
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 333
1) 27.65/28.90; ESMS m/z calcd for C39H42N4O7PS (M - H)-
[(R),(R,S),(S)]-{3-[(1-Ben zyloxyca r bon yla m in o-2-p h e-
n yleth yl)h yd r oxyp h osp h in oyl]-2-[1-ca r ba m oyl-2-(1H-in -
dol-3-yl)eth ylcar bam oyl]pr opylsu lfan yl}acetic acid, m eth -
yl ester (14): TLC Rf(a) 0.64/0.71, Rf(c) 0.78/0.81; HPLC
(column 2, gradient 1) 20.72/23.21, (column 3, gradient 2)
39.06/41.57; ESMS m/z calcd for C34H39N4O8PS (M - H)-
693.2, found 694.0; relative intensity of the molecular ion, 97%.
741.3, found 741.9; relative intensity of the molecular ion, 98%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(fu r a n -2-ylm eth ylsu lfa n yl)p r op yl}p h osp h in ic a cid (7):
TLC Rf(a) 0.60/0.67, Rf(c) 0.73/0.75; HPLC (column 2, gradient
1) 25.17/27.30; ESMS m/z calcd for C36H38N4O7PS (M - H)-
701.2, found 702.0; relative intensity of the molecular ion, 95%.
[(R),(R,S),(S)]-{3-[(1-Ben zyloxyca r bon yla m in o-2-p h e-
n yleth yl)h yd r oxyp h osp h in oyl]-2-[1-ca r ba m oyl-2-(1H-in -
d ol-3-yl)eth ylca r ba m oyl]p r op ylsu lfa n yl}a cetic a cid , eth -
yl ester (15): TLC Rf(a) 0.65/0.73, Rf(c) 0.78/0.82; HPLC
(column 2, gradient 1) 24.78/26.49, (column 3, gradient 2)
42.60/46.15; ESMS m/z calcd for C35H40N4O8PS (M - H)-
707.2, found 708.0; relative intensity of the molecular ion, 97%.
[(R),(R,S),(S)]-3-{3-[(1-Ben zyloxyca r bon yla m in o-2-p h e-
n yleth yl)h yd r oxyp h osp h in oyl]-2-[1-ca r ba m oyl-2-(1H-in -
d ol-3-yl)eth ylca r ba m oyl]p r op ylsu lfa n yl}p r op ion ic a cid ,
m eth yl ester (16): TLC Rf(a) 0.65/0.73, Rf(c) 0.78/0.82; HPLC
(column 3, gradient 2) 38.82/44.39; ESMS m/z calcd for
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(n a p h th elen e-2-ylsu lfa n yl)p r op yl}p h osp h in ic a cid (8):
TLC Rf(a) 0.53/0.65, Rf(c) 0.73/0.77; HPLC (column 1, gradient
1) 39.23/43.47, (column 2, gradient 9) 29.76/32.81; 1H NMR
(200 MHz, DMSO-d6) δ 1.98-2.31 (m, 2H, PCH2), 2.61-3.39
(m, 7H, SCH2, PCH2CH, PhCH2, indolylCH2), 3.82-4.18 (m,
1H, PCH) 4.25-4.58 (m, 1H, indolylCH2CH), 4.84-5.01 (br s,
2H, OCH2Ph), 6.93-7.89 (m, 24H, aryl, NH); 13C NMR (50
1
MHz, DMSO-d6) δ 27.9 (d, J PC ) 78.3 Hz, PCH2), 32.9 (CH2-
1
Ph), 41.9 (PCH2CH), 50.1 (d, J PC ) 104.0 Hz, PCH), 53.9
C
35H40N4O8PS (M - H)- 707.2, found 708.1; relative intensity
(indolylCH2CH), 65.2 (OCH2Ph), 111.1, 112.8, 118.6, 121.0,
123.3, 123.4, 126.4, 127.4, 128.3, 129.3, 136.1, 137.3, 138.6,
(aryl), 156.1 (OCONH), 172.1 (CHCCONH), 173.8 (CONH2);
31P NMR (81 MHz, DMSO-d6) δ 42.60, 43.28; ESMS m/z calcd
for C41H40N4O6PS (M - H)- 747.2, found 747.9; relative
intensity of the molecular ion, 97%.
of the molecular ion, 97%.
[(R),(R,S),(S)]-3-{3-[(1-Ben zyloxyca r bon yla m in o-2-p h e-
n yleth yl)h yd r oxyp h osp h in oyl]-2-[1-ca r ba m oyl-2-(1H-in -
d ol-3-yl)eth ylca r ba m oyl]p r op ylsu lfa n yl}p r op ion ic a cid ,
eth yl ester (17): TLC Rf(a) 0.65/0.73, Rf(c) 0.78/0.82; HPLC
(column 2, gradient 1) 22.31/27.50; ESMS m/z calcd for
C
36H42N4O8PS (M - H)- 721.3, found 722.0; relative intensity
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-p h en ylsu lfa n ylp r op yl}p h osp h in ic a cid (9): TLC Rf(a)
0.60/0.67, Rf(c) 0.74/0.78; HPLC (column 2, gradient 1) 27.09/
29.19, (column 3, gradient 4) 25.42/28.28; 1H NMR (200 MHz,
DMSO-d6) δ 1.82-2.05 (m, 2H, PCH2), 2.68-3.58 (m, 7H,
SCH2, PCH2CH, PhCH2, indolylCH2), 3.73-4.07 (m, 1H, PCH)
4.35-4.45 (m, 1H, indolylCH2CH), 4.83-4.99 (br s, 2H, OCH2-
Ph), 6.92-7.68 (m, 22H, aryl, NH); ESMS m/z calcd for
of the molecular ion, 97%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{3-(2-br om op h en ylsu lfa n yl)-2-[1-ca r ba m oyl-2-(1H-
in d ol-3-yl)eth ylca r ba m oyl]p r op yl}p h osp h in ic a cid (18):
TLC Rf(a) 0.61/0.68, Rf(c) 0.74/0.77; HPLC (column 2, gradient
1) 28.40/31.05, (column 3, gradient 4) 34.40/38.51; 1H NMR
(200 MHz, DMSO-d6) δ 1.89-2.28 (m, 2H, PCH2), 2.58-3.40
(m, 7H, SCH2, PCH2CH, PhCH2, indolylCH2), 3.77-4.09 (m,
1H, PCH), 4.37-4.57 (m, 1H, indolylCH2CH), 4.85-4.99 (br
s, 2H, OCH2Ph), 6.94-7.78 (m, 21H, aryl, NH); 13C NMR (50
C
37H38N4O6PS (M - H)- 697.2, found 697.6; relative intensity
of the molecular ion, 99%.
1
MHz, DMSO-d6) δ 27.7 (d, J PC ) 77.1 Hz, PCH2), 32.8 (CH2-
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-cycloh exylsu lfa n ylp r op yl}p h osp h in ic a cid (10): TLC
Rf(a) 0.71/0.80, Rf(c) 0.82/0.85; HPLC (column 1, gradient 1)
40.70/44.24, (column 2, gradient 1) 27.66/30.37; 1H NMR (200
MHz, DMSO-d6) δ 0.78-2.08 (m, 10H, CH2 of cyclohexyl),
2.10-3.42 (m, 10H, CHSCH2, CHSCH2, PCH2CH, PCH2,
PhCH2, indolylCH2), 3.80-4.16 (m, 1H, PCH) 4.28-4.56 (m,
1H, indolylCH2CH), 4.85-5.05 (br s, 2H, OCH2Ph), 6.80-7.78
(m, 17H, aryl, NH); 13C NMR (50 MHz, DMSO-d6) δ 25.5 (CH2
1
Ph), 42.1 (PCH2CH), 51.0 (d, J PC ) 103.5 Hz, PCH), 54.3
(indolylCH2CH), 65.1 (OCH2Ph), 111.2, 118.2, 120.7, 126.2,
127.5, 128.1, 129.0, 136.1, 137.2, 138.6, (aryl), 156.0 (OCONH),
172.0 (CHCCONH), 173.7 (CONH2); 31P NMR (81 MHz,
DMSO-d6) δ 45.30; ESMS m/z calcd for C37H37BrN4O6PS (M
- H)- 775.1, found 775.5; relative intensity of the molecular
ion, 99%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(2-isopr opylph en ylsu lfan yl)pr opyl}ph osph in ic acid (19):
TLC Rf(a) 0.60/0.67, Rf(c) 0.74/0.78; HPLC (column 3, gradient
4) 41.02/45.15; 1H NMR (200 MHz, DMSO-d6) δ 1.06-1.18 (m,
7H, CH(CH)3, CH(CH)3) 1.81-2.06 (m, 2H, PCH2), 2.61-3.56
(m, 7H, SCH2, PCH2CH, PhCH2, indolylCH2), 3.68-4.08 (m,
1H, PCH), 4.39-4.52 (m, 1H, indolylCH2CH), 4.80-4.98 (br
s, 2H, OCH2Ph), 6.95-7.72 (m, 21H, aryl, NH); ESMS m/z
calcd for C40H44N4O6PS (M - H)- 739.3, found 739.6; relative
intensity of the molecular ion, 97%.
[(R),(R ,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(2-eth ylp h en ylsu lfa n yl)p r op yl}p h osp h in ic a cid (20):
TLC Rf(a) 0.60/0.67, Rf(c) 0.74/0.78; HPLC (column 3, gradient
5) 37.18/41.35.1H NMR (200 MHz, DMSO-d6) δ 1.01-1.13 (m,
5H, CH2CH3, CH2CH3), 1.85-2.10 (m, 2H, PCH2), 2.59-3.50
(m, 7H, SCH2, PCH2CH, PhCH2, indolylCH2), 3.71-4.09 (m,
1H, PCH), 4.37-4.58 (m, 1H, indolylCH2CH), 4.82-4.96 (br
s, 2H, OCH2Ph), 6.99-7.79 (m, 21H, aryl, NH); ESMS m/z
calcd for C39H42N4O6PS (M - H)- 725.3, found 725.6; relative
intensity of the molecular ion, 98%.
1
of cyclohexyl), 27.7 (d, J PC ) 77.9 Hz, PCH2), 33.1 (CH2Ph),
1
42.5 (PCH2CH), 51.3 (d, J PC ) 102.9 Hz, PCH), 53.8
(indolylCH2CH), 65.1 (OCH2Ph), 111.1, 118.1, 121.0, 123.3,
123.6, 126.6, 127.4, 128.2, 128.9, 136.2, 137.3, 139.9, (aryl),
156.1 (OCONH), 173.3 (CHCCONH), 174.3 (CONH2); 31P NMR
(81 MHz, DMSO-d6) δ 45.94, 46.85; ESMS m/z calcd for
C
37H44N4O6PS (M - H)- 703.3, found 704.0; relative intensity
of the molecular ion, 99%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-isobu tylsu lfa n ylp r op yl}p h osp h in ic a cid (11): TLC Rf(a)
0.70/0.77, Rf(c) 0.82/0.85; HPLC (column 2, gradient 1) 26.70/
29.29; ESMS m/z calcd for C35H42N4O6PS (M - H)- 677.3,
found 677.4; relative intensity of the molecular ion, 98%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(3-m eth ylbu tylsu lfa n yl)p r op yl}p h osp h in ic a cid (12):
TLC Rf(a) 0.70/0.79, Rf(c) 0.82/0.86; HPLC (column 2, gradient
1) 28.29/31.15; ESMS m/z calcd for C36H44N4O6PS (M - H)-
691.3, found 691.8; relative intensity of the molecular ion, 98%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-car bam oy l-2-(1H-in dol-3-yl)eth ylcar bam oyl]-
3-(2,4-di-ter t-bu tylph en ylsu lfan yl)pr opyl}ph osph in ic acid
(21): TLC Rf(a) 0.62/0.67, Rf(c) 0.75/0.78; HPLC (column 3,
gradient 6) 26.39/29.82; 1H NMR (200 MHz, DMSO-d6) δ 1.25-
1.37 (m, 18H, C(CH)3, CH(CH)3) 1.83-2.07 (m, 2H, PCH2),
2.64-3.58 (m, 7H, SCH2, PCH2CH, PhCH2, indolylCH2), 3.74-
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-h exylsu lfa n ylp r op yl}p h osp h in ic a cid (13): TLC Rf(a)
0.71/0.80, Rf(c) 0.84/0.87; HPLC (column 2, gradient 1) 31.27/
34.30; ESMS m/z calcd for C37H46N4O6PS (M - H)- 705.3,
found 705.5; relative intensity of the molecular ion, 99%.

334 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2
Matziari et al.
4.12 (m, 1H, PCH), 4.40-4.57 (m, 1H, indolylCH2CH), 4.82-
5.02 (br s, 2H, OCH2Ph), 6.91-7.68 (m, 21H, aryl, NH); ESMS
m/z calcd for C45H54N4O6PS (M - H)- 809.4, found 809.7;
relative intensity of the molecular ion, 97%.
mmol, 86 mg) and Pd[P(Ph3)]4 (0.005 mmol, 5.8 mg) in toluene
(5 mL) was added a solution of 2 M Na2CO3 (0.2 mmol, 0.1
mL) and PhB(OH)2 (0.12 mmol, 14.6 mg) under argon atmo-
sphere. The reaction mixture was refluxed under argon, for
48 h. After cooling to room temperature, 2 N HCl was added
(pH 1) and the reaction mixture was extracted with AcOEt (2
× 10 mL), washed with water and brine (5 mL each), dried
over Na2SO4, and evaporated. Purification by flash column
chromatography, using CHCl3-MeOH-AcOH (7:0.3:0.3) as
eluent afforded the product (76 mg, 81%) as a white solid: TLC
Rf(a) 0.61/0.65, Rf(c) 0.75/0.77; HPLC (column 1, gradient 1)
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(2-m eth oxyph en ylsu lfan yl)pr opyl}ph osph in ic acid (22):
TLC Rf(a) 0.61/0.68, Rf(c) 0.74/0.77; HPLC (column 3, gradient
7) 34.72/37.28.1H NMR (200 MHz, DMSO-d6) δ 1.91-2.07 (m,
2H, PCH2), 2.59-3.49 (m, 7H, SCH2, PCH2CH, PhCH2, in-
dolylCH2), 3.86-4.01 (m, 4H, OCH3, PCH), 4.41-4.58 (m, 1H,
indolylCH2CH), 4.88-5.00 (br s, 2H, OCH2Ph), 6.92-7.75 (m,
1
51.01/53.00.; H NMR (200 MHz, DMSO-d6) δ 1.72-2.11 (m,
1
21H, aryl, NH); 13C NMR (50 MHz, DMSO-d6) δ 27.4 (d, J PC
2H, PCH2), 2.59-3.67 (m, 7H, SCH2, PCH2CH, PhCH2, in-
dolylCH2), 3.74-4.12 (m, 1H, PCH), 4.32-4.49 (m, 1H,
indolylCH2CH), 4.84-5.01 (br s, 2H, OCH2Ph), 6.94-7.81 (m,
26H, aryl, NH); ESMS m/z calcd for C44H41F3N4O7PS (M - H)-
857.2, found 857.3; relative intensity of the molecular ion, 95%.
Gen er a l P r oced u r e for Th iol Ad d ition , on Solid P h a se.
Synthesis was carried out on Rink amide AM resin with a
loading capacity 0.63 mmol/g. The resin was swollen using
DMF and deprotected with 20% piperidine in DMF. Resin (50
mg) was swollen in 0.5 mL of DMF and a solution of 3 equiv
of FmocTrpOH, 3 equiv of DIC, and 3 equiv of HOBt in 0.5
mL of DMF was added. Coupling was repeated if necessary.
The resin was swollen using DMF, deprotected with 20%
piperidine in DMF, and washed several times with DMF,
DCM, and Et2O. A solution of 2 (0.03 mmol, 1 equiv, 12.7 mg)
DIPEA, (0.06 mmol, 10 µL), HOBt (0.03 mmol, 4 mg), and
EDC‚HCl (0.12 mmol, 23 mg) in DCM was added to the resin
and allowed to stand at room temperature for 1.5 h. One
coupling was sufficient (negative Kaiser test). The resin was
washed several times with DCM, DMF, 2-propanol/water (8:
2), 2-propanol, DCM, and Et2O. Then freshly distilled THF (1
mL), 2-thionaphthol (for compound 8), or cyclohexylthiol (for
compound 10) (0.12 mmol) and a 1 M solution of EtONa/EtOH
(0.06 mmol, 0.06 mL) were added. The resin was allowed to
stand at room temperature (40 and 1 h respectively) and was
then filtered and washed several times with 2-propanol/water
(8:2), 2-propanol, DCM, and Et2O. Cleavage from the resin was
achieved using TFA/TIS/H2O 95:2.5:2.5. The residue was
concentrated in vacuo and precipitated with Et2O-PE (40-
60) (1:1). Yields were 71% and 96%, respectively.
) 77.5 Hz, PCH2), 32.7 (CH2Ph), 42.3 (PCH2CH), 51.1 (d, 1J PC
) 102.8 Hz, PCH), 53.9 (OCH3), 54.3 (indolylCH2CH), 65.0
(OCH2Ph), 111.2, 118.2, 120.6, 126.2, 127.4, 128.1, 129.0, 136.1,
137.2, 138.6, (aryl), 150.1 (CH3OC), 156.1 (OCONH), 171.9
(CHCCONH), 173.7 (CONH2); 31P NMR (81 MHz, DMSO-d6)
δ 46.15; ESMS m/z calcd for C38H40N4O7PS (M - H)- 727.2,
found 727.6; relative intensity of the molecular ion, 99%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(2,5-d im eth oxyp h en ylsu lfa n yl)p r op yl}p h osp h in ic a cid
(23): TLC Rf(a) 0.62/0.67, Rf(c) 0.75/0.77; HPLC (column 3,
gradient 8) 30.96/33.34; 1H NMR (200 MHz, DMSO-d6) δ 1.89-
2.05 (m, 2H, PCH2), 2.59-3.39 (m, 7H, SCH2, PCH2CH,
PhCH2, indolylCH2), 3.44-4.05 (m, 7H, OCH3, PCH), 4.38-
4.56 (m, 1H, indolylCH2CH), 4.84-5.02 (br s, 2H, OCH2Ph),
6.84-7.73 (m, 20H, aryl, NH); ESMS m/z calcd for C39H42N4O8-
PS (M - H)- 757.3, found 757.6; relative intensity of the
molecular ion, 99%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-car bam oyl-2-(1H-in dol-3-yl)eth ylcar bam oyl]-3-
(2-t r i flu o r o m e t h o x y p h e n y ls u lfa n y l)p r o p y l}p h o s -
p h in ic a cid (24): TLC Rf(a) 0.60/0.65, Rf(c) 0.74/0.77;
HPLC (column 3, gradient 4) 38.94/42.99; 1H NMR (200 MHz,
DMSO-d6) δ 1.90-2.08 (m, 2H, PCH2), 2.61-3.47 (m, 7H,
SCH2, PCH2CH, PhCH2, indolylCH2), 3.87-4.00 (m, 1H, PCH),
4.40-4.58 (m, 1H, indolylCH2CH), 4.87-5.03 (br s, 2H, OCH2-
Ph), 6.90-7.76 (m, 21H, aryl, NH); 13C NMR (50 MHz, DMSO-
1
d6) δ 26.9 (d, J PC ) 79.1 Hz, PCH2), 32.7 (CH2Ph), 42.1
(PCH2CH), 52.8 (d, 1J PC ) 99.6 Hz, PCH), 54.3 (indolylCH2CH),
64.3 (OCH2Ph), 110.4, 111.2, 116.3, 118.2, 120.6, 122.5, 126.2,
127.4, 128.1, 129.0, 132.5, 136.1, 137.2, 138.6, (aryl, CF3), 155.8
(OCONH), 172.7 (CHCCONH), 173.5 (CONH2); 31P NMR (81
MHz, DMSO-d6) δ 45.83; ESMS m/z calcd for C38H37F3N4O7-
PS (M - H)- 781.2, found 781.5; relative intensity of the
molecular ion, 99%.
In h ibitor P oten cy. Enzyme assays and determination of
the Ki values were performed as previously described in
detail.13
Molecu la r Mod elin g. Molecular models of the interaction
of the phosphinic inhibitor 18 with the catalytic domains of
three different MMPs were achieved using a protocol based
on molecular dynamics with the program CHARMM (version
27).31 The CHARMM force field version 22 was used.32 The
3D structures of the catalytic domains of MMP-11, MMP-14,
and MMP-8 used in this work were taken from the PDB (code
1HV5, 1BQQ, and 1MMB, respectively).
Geometrical and nonbonded parameters for the phosphinic
inhibitor were derived from ab initio quantum calculations
with the program Gaussian98.33 These calculations were done
at the MP2 level of theory, using a 6-31+G(d, p) basis set.
The starting structure of each complex was defined using
the coordinates of the complex of MMP-11 with a phosphinic
inhibitor determined by X-ray diffraction (pdb code 1HV5).24
As the present modeling study was aimed at defining the
interaction of the P1’-side chain of the inhibitor with the
different MMPs, the Z-NH part of the phosphinic compound
was omitted.
To preserve the structure of the protein during the relax-
ation of the complexes, harmonic restraints were applied to
the atomic position of several sets of atoms. The harmonic
constants were set to 30, 10, and 5 kcal mol-1 A-2 for the
cations and the residues chelating the cations, the non-
hydrogen backbone atoms, and the non-hydrogen side chain
atoms, respectively. No harmonic restraints were applied to
the residues located at a distance below 5 Å from the inhibitor.
During the calculations, the nonbonded interactions were
modeled using a Lennard-J ones function and a Coulombic
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
et h yl)-{3-(4-b r om o-2-t r iflu or om et h oxyp h en ylsu lfa n yl)-
2-[1-car bam oyl-2-(1H-in dol-3-yl)eth ylcar bam oyl]-pr opyl}-
p h osp h in ic a cid (25): TLC Rf(a) 0.60/0.65, Rf(c) 0.74/0.77;
HPLC (column 1, gradient 1) 46.56/50.45, (column 3, gradient
2) 33.38/42.75; 1H NMR (200 MHz, DMSO-d6) δ 1.87-2.08 (m,
2H, PCH2), 2.58-3.48 (m, 7H, SCH2, PCH2CH, PhCH2, in-
dolylCH2), 3.86-4.02 (m, 1H, PCH), 4.38-4.58 (m, 1H,
indolylCH2CH), 4.86-5.03 (br s, 2H, OCH2Ph), 6.87-7.78 (m,
20H, aryl, NH); ESMS m/z calcd for C38H36BrF3N4O7PS (M -
H)- 859.1, found 859.4; relative intensity of the molecular ion,
97%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(3-m eth oxyph en ylsu lfan yl)pr opyl}ph osph in ic acid (26):
TLC Rf(a) 0.61/0.68, Rf(c) 0.74/0.77; HPLC (column 2, gradient
1) 26.50/28.53; 1H NMR (200 MHz, DMSO-d6) δ 1.90-2.07 (m,
2H, PCH2), 2.57-3.46 (m, 7H, SCH2, PCH2CH, PhCH2, in-
dolylCH2), 3.84-4.03 (m, 3H, OCH3, PCH), 4.41-4.57 (m, 1H,
indolylCH2CH), 4.82-5.03 (br s, 2H, OCH2Ph), 6.92-7.77 (m,
21H, aryl, NH); ESMS m/z calcd for C38H40N4O7PS (M - H)-
727.2, found 727.9; relative intensity of the molecular ion, 98%.
[(R),(R,S),(S)]-(1-Ben zyloxyca r b on yla m in o-2-p h en yl-
eth yl)-{2-[1-ca r ba m oyl-2-(1H-in d ol-3-yl)eth ylca r ba m oyl]-
3-(3-t r iflu or om e t h oxyb ip h e n yl-4-ylsu lfa n yl)p r op yl}-
p h osp h in ic Acid (27). To a solution of compound 25 (0.1

Highly Selective Inhibitors of MMP-11
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 2 335
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J iracek, J .; Vazeux, G.; Chauvet, M. T.; Cuniasse, P.; Corvol, P.
RXP 407, a phosphinic peptide, is a potent inhibitor of angio-
tensin I converting enzyme able to differentiate between its two
active sites. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 4330-5.
(16) J unot, C.; Gonzales, M. F.; Ezan, E.; Cotton, J .; Vazeux, G.;
Michaud, A.; Azizi, M.; Vassiliou, S.; Yiotakis, A.; Corvol, P.;
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electrostatic term with a nonbonded cutoff of 15 Å. The
dielectric constant was set to 4.
The first step of the relaxation protocol consists of an initial
2000 cycle of adopted basis Newton-Raphson energy mini-
mization. Then, 100 000 steps of molecular dynamics using
the Verlet algorithm were undertaken. The integration step
was set to 0.0005 ps. The temperature was gradually increased
by 25 K each 100 steps to reach 450 K. This molecular
dynamics was followed by 5000 cycles of energy minimization.
The resulting structure was then analyzed and the interaction
energy between the atoms of the P1’-side chain and each
residue of the MMP was calculated using the potential energy
function of CHARMM.
(17) Dive, V.; Lucet-Levannier, K.; Georgiadis, D.; Cotton, J .; Vas-
siliou, S.; Cuniasse, P.; Yiotakis, A. Phosphinic peptide inhibitors
as tools in the study of the function of zinc metallopeptidases.
Biochem. Soc. Trans. 2000, 28, 455-60.
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Ack n ow led gm en t. This work was supported by the
FP5 PCRD program (QLK3-CT02-02136). The authors
would also like to thank M. C. Rio, Institut de Genetique
et de Biologie Moleculaire et Cellulaire, CNRS/INSERM
U184/ULP, BP 163, 67404, Illkirch, Cedex, C. U. de
Strasbourg, France, for the supply of the enzymes MMP-
11 and MMP-14, and G. Murphy, Strangeways Research
Laboratory, Cambridge, United Kingdom, for the supply
of the enzymes MMP-2, MMP-7, MMP-8, MMP-9, and
MMP-13.
(21) Yiotakis, A.; Vassiliou, S.; J iracek, J .; Dive, V. Protection of the
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V.; Rio, M. C.; Basset, P.; Moras, D. Crystal structure of the
Su p p or tin g In for m a tion Ava ila ble: Mass spectra and
HPLC chromatograms of compounds 4, 6, 7, 10-13, and 18-
21. This material is available free of charge via the Internet
at http://pubs.acs.org.
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