Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: A structure-activity study

Article abstract of DOI:10.1021/jm9900164

Several phosphinic pseudo-tripeptides of general formula R-XaaΨ(PO₂- CH₂)Xaa'-Yaa'-NH₂ were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P₁' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.

Full text of DOI:10.1021/jm9900164

2610
J . Med. Chem. 1999, 42, 2610-2620
P h osp h in ic P seu d o-Tr ip ep tid es a s P oten t In h ibitor s of Ma tr ix
Meta llop r otein a ses: A Str u ctu r e-Activity Stu d y†
Stamatia Vassiliou,§ Artur Mucha,∇ Philippe Cuniasse,‡ Dimitris Georgiadis,§ Karine Lucet-Levannier,‡
,[
Fabrice Beau,‡ Rama Kannan, Gillian Murphy,| Vera Kna¨uper,| Marie-Christine Rio, Paul Basset,
Athanasios Yiotakis,§ and Vincent Dive*,‡
CEA, De´partement d’Inge´nierie et d’Etudes des Prote´ines, 91191 Gif/ Yvette Cedex, France, Department of Chemistry,
Laboratory of Organic Chemistry, University of Athens, Panepistiomiopolis, Zografou, Athens, Greece,
School of Biological Sciences, University of East Anglia Norwich, Norfolk NR4 7TJ , U.K.,
Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire (IGBMC), CNRS/ INSERM/ Universite´ Louis Pasteur,
BP 163 67404 Illkirch Cedex, France, and Institut of Organic Chemistry, Biochemistry and Biotechnology,
Technical University of Wroclaw, Wybrzeze Wyspiankiego 27, 50-370 Wroclaw, Poland
Received J anuary 11, 1999
Several phosphinic pseudo-tripeptides of general formula R-XaaΨ(PO2-CH2)Xaa′-Yaa′-NH2 were
synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A
and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With
the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly
potent inhibitors of matrix metalloproteinases, provided they contain in P1′ position an unusual
long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence
of the R and Xaa′ substituents in this series may contribute to the design of inhibitors able to
block only a few members of the matrix metalloproteinase family.
In tr od u ction
act as a cancer cell survival factor and could be more
involved in tumor formation rather than in tumor
growth.17,18
Matrix metalloproteinases (MMPs), also known as
matrixins, form a group of structurally related extra-
cellular zinc endoproteinases collectively able to degrade
all components of the extracellular matrix.1 MMPs are
believed to be mediators of both normal and pathological
tissue-remodeling processes. Their overexpression has
been observed and linked with a variety of diseases,
including cancer, arthritis, multiple sclerosis, and
arteriosclerosis.2-8 Therefore, there has been a substan-
tial interest in developing MMP synthetic inhibitors for
a variety of therapeutic indications.9,10
Among the 15 members of the human MMP multi-
gene family,11 stromelysin-3 (MMP-11), which was first
described in fibroblastic cells of invasive breast carci-
noma,12 displays a number of unusual features: mature
forms of MMP-11 appear unable to degrade any major
extracellular matrix component;13,14 MMP-11 is secreted
in an active form,15,16 whereas most of the other secreted
MMPs must be activated extracellularly; and finally
while most MMPs possess a conserved leucine in their
S1′ pocket, this residue is replaced by glutamine in
MMP-11. These findings suggest that MMP-11 may
have a unique role in tissue-remodeling processes,
including those associated with tumor progression, and
may represent a particular target in the development
of MMP inhibitors. In this respect, it is worth noting
that recent studies on the role of MMP-11 in cancer
development led to the conclusion that MMP-11 may
The development of an inhibitor program directed
toward MMP-11 has been hampered by the lack of a
simple test for routine assay of MMP-11. Recently, we
described the first fluorogenic synthetic substrates that
are cleaved efficiently by MMP-11,19 allowing the de-
velopment of an inhibitor screening program. Most of
the MMP inhibitors developed over the last 10 years
possess a zinc-binding function attached to a peptide
framework, which binds to the primed regions of the
catalytic active site. The vast majority of inhibitors
described incorporate hydroxamate or carboxylate as
zinc-binding groups.20 Several studies suggest that the
hydroxamate function is the best zinc-chelating group
for development of potent inhibitors of MMPs. However,
concerns about the unfavorable pharmacokinetics of
hydroxamates, poor solubility, and potential for chronic
toxicities arising from metabolic activation of the hy-
droxamate group have stimulated the development of
MMPs inhibitors based on other zinc-chelating groups.
Pseudo-peptides, containing a phosphinic group as a
zinc-chelating function, may represent an interesting
inhibitor design alternative. Recent progress in the
solid-phase synthesis of phosphinic peptides makes it
possible to use either parallel or combinatorial chem-
istry for rapid development of potent inhibitors of zinc-
metalloproteases.21-23 Furthermore, in contrast to pep-
tidic inhibitors containing a thiol, a hydroxamate, or a
carboxylate function, phosphinic peptides, as good mim-
ics of the substrate in the transition state, interact with
both the primed and unprimed side of the active site
cleft.24 This property can be exploited to optimize
inhibitor selectivity.
† Abbreviations: MMP, matrix metalloproteinases; MMP-1, colla-
genase-1; MMP-2, gelatinase-A; MMP-7, matrilysin; MMP-8, collage-
nase-2; MMP-9, gelatinase-B; MMP-11, stromelysin-3; MMP-14, mem-
brane type-1 matrix metalloproteinase.
‡ De´partement d’Inge´nierie et d’Etudes des Prote´ines.
§ University of Athens.
| University of East Anglia Norwich.
CNRS/INSERM/Universite´ Louis Pasteur.
In the present report, several phosphinic peptides
were synthesized and evaluated as inhibitors of MMP-
∇ Technical University of Wroclaw.
[ Deceased April 28, 1999.
10.1021/jm9900164 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/17/1999

Phosphinic Pseudo-Tripeptides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2611
Ch a r t 1. Phosphinic Synthons
Sch em e 2a
Sch em e 1a
a
Reagents and conditions: (a)HMDS,110°C,H2CdC(R3)COOC2H5,
EtOH, 70 °C; (b) TMSCl, 0 °C, rt, H2CdC(R3)COOC2H5, rt, EtOH.
11. The potencies of these inhibitors were also deter-
mined toward other MMPs in order to decipher some
specificity rules, which could be used to develop com-
pounds able to block only a subset of the MMP family.
These inhibitors were designed according to some
important structural elements present in the best
substrates of MMP-11: an alanine or phenylalanine
residue in the P1 position and an unusual amino acid,
containing a long aryl-alkyl side chain, in the P1′
position.
Ch em istr y
The inhibitors of the present study were prepared by
conventional solid-phase peptide synthesis.21 This strat-
egy relies on the synthesis of two different types of
phosphinic synthon (Chart 1). These two types of
synthon were obtained from a common phosphinic
synthon precursor (compound 9). Compounds 9 were
prepared via a Michael addition of R-substituted-acryl-
ates to monosubstituted phosphinates 8 (Scheme 1).
Four different methods were used to obtain the required
R-substituted-acrylates (Scheme 2). In method a, ap-
propriate bromides were added to diethyl malonate
affording diethyl monosubstituted malonate derivatives
type 1, which gave the monoesters type 2 after saponi-
fication.25 Ethyl R-substituted-acrylates 3a -e were
obtained by treatment of ethyl monosubstituted mal-
onates 2 and paraformaldehyde, under Knoevenagel
reaction. Compound 3f was obtained by substitution of
the acetoxy group of acrylate 4, by nucleophilic attack
of the appropriate Grignard reagent, in the presence of
CuI (method b). To avoid simultaneous attack of the two
esters in acrylate 4, this reaction must take place at
low temperature (-78 °C).26
Several attempts to prepare compound 3g by method
a or b were unsuccessful. This compound was finally
prepared by method c,27 outlined in Scheme 2: Ethyl
R-bromomethylacrylate was converted to acrylate 5 by
treatment with benzenesulfinic sodium salt. The sulfinic
acrylate 5 was treated with tri-n-butyl-tin hydride, in
the presence of a catalytic amount of AIBN, to afford
acrylate 6. Compound 3g was obtained by refluxing
acrylate 6 with bromoethylnaphthalene, in the presence
of a catalytic amount of AIBN.
a
Reagents and conditions. Method a: (a) CH2(COOC2H5)2,
EtOH, Na; (b) KOH, EtOH, HCl; (c) paraformaldehyde, piperidine/
pyridine. Method b: (a) R(CH2)nMgBr, abs. Et2O, THF, cat. CuI,
-78 °C. Method c: (a) PhSO2Na, MeOH; (b) n-Bu3SnH, AlBN,
benzene; (c) R(CH2)nBr, AlBN, benzene. Method d: (a) Na, Et2O;
(b) MeO-Na+, MeOH; (c) Et2O, -15 °C, rt, 2 h.
2, method d). Conversion of the appropriate alcohols or
thiols to sodium salts gave white solids in high yields
(85-95%). Substitution of ethyl R-bromomethyl acrylate
by these sodium salts was easily achieved in dry ether.
Phosphinic synthons type 9 were prepared according
to methods previously described in the literature.21,28
From compounds 9, synthons 11 were prepared in two
steps21 (Scheme 3). In the first step, the silver salt of
compound 9 was reacted with 1-adamantyl bromide to
give the corresponding ester (compound 10). The second
step was the saponification of the ethyl ester which,
despite the adamantyl ester, was completely selective.
From synthons type 11, compounds 17-34 were pre-
pared by solid-phase synthesis, using a Rink amide
resin as a solid support.
Synthon 13 was obtained from compound 11 by a two-
step reaction, decarbobenzyloxylation of compound 11,
with ammonium formate as the hydrogen donor and
palladium/carbon as the catalyst in methanol, and then
introduction of the Fmoc protecting group. Using the
synthon 13, free amino phosphinic peptide compound
15 was prepared by classical Fmoc solid-phase peptide
synthesis. This intermediate, still linked to the resin,
was the common precursor to prepare compounds 35-
45.
Ethyl alkoxy or alkylthio methacrylates type 7 were
prepared by reaction of the corresponding alcohol or
thiol anions with ethyl R-bromomethyl acrylate (Scheme

2612 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Vassiliou et al.
Sch em e 3a
a
Reagents and conditions: (a) Ad-Br, Ag2O, CHCl3; (b) 0.6 NaOH, MeOH; (c) aq HCl; (d) H2N-Aaa-Rink (R), HBTU, DIEA, NMP; (e)
TFA/CH2Cl2/triisopropylsilane/H2O 5:4:0.5:0.5; (f) HCOO-NH4+, 10% Pd/C, MeOH; (g) Fmoc-Cl, 20% Na2CO3, dioxane; (h) 20% piperidine/
NMP; (i) R-OH, HBTU, DIEA, NMP; (j) R-Cl, DIEA, CH2Cl2; (k) BrCH2COBr, DIEA, CH2Cl2; (l) aniline derivatives, DIEA, DMSO.
Ta ble 1. Influence of the R3 Substituent (P1′ Position)
Ki (nM)
MMP-11
mST3
MMP-2
gel-A
MMP-9
gel-B
MMP-14
MT1-MMP
MMP-1
HFC
MMP-7
matrilysin
MMP-8
HNC
compd
R3
17
18
19
20
21
CH2-φ
CH2-CH2-φ
CH2-CH2-CH2-φ
CH2-O-CH2-φ
CH2-S-CH2-φ
350
51
100
175
36
250
80
31
250
14
280
60
23
44
6
2030
270
92
550
26
24% @ 2 µM
23% @ 2 µM
30% @ 2 µM
15% @ 2 µM
45% @ 2 µM
8% @2 µM
3% @2 µM
4% @2 µM
1% @2 µM
2% @2 µM
240
20
8
19
<0.5
Resu lts
from a benzyl to a phenylpropyl group (compounds 17-
19), led to a 30-fold increase in inhibitory potency. In
terms of selectivity, MMP-2, MMP-9, MMP-14, and
MMP-8 preferentially bound inhibitor containing a
phenylpropyl group, whereas MMP-11 accommodated
a phenylethyl group better in this position. Comparison
of compounds 19, 20, and 21 illustrated the influence
With the exception of MMP-7 and MMP-1, the results
reported in Table 1 demonstrate that phosphinic pseudo-
tripeptides can behave as highly potent inhibitors of
MMPs, provided they contain in their P1′ position an
unusual amino acid side chain. In the case of MMP-8,
the lengthening of the side chain in the P1′ position,

Phosphinic Pseudo-Tripeptides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2613
Ta ble 2. Influence of the R3 Substituent (P1′ Position)
Ki (nM)
MMP-11
mST3
MMP-2
Gel-A
MMP-9
Gel-B
MMP-14
MT1-MMP
MMP-1
HFC
MMP-7
matrilysin
MMP-8
HNC
compd
R3
22
23
24
25
26
27
28
29
30
31
CH3
CH2-CH-(CH3)2
CH2-CH2-φ
CH2-CH2-CH2-φ
CH2-CH2-CH2-CH2-φ
CH2-O-CH2-φ
CH2-S-CH2-φ-OMe
2-CH2-naphthyl
2-(CH2)2-naphthyl
(CH2)6-CH3
2670
22
8.8
5
33
16
2
74
12
34
0% @ 2 µM
0% @2 µM
0% @10 µM
192
660
105
580
545
22
1350
125
271
20% @2 µM
45% @ 2 µM
10% @ 2 µM
23% @ 2 µM
5% @ 2 µM
9% @ 2 µM
13% @ 2 µM
0% @ 5 µM
0% @ 2 µM
27% @ 2 µM
0% @2 µM
210
9% @1 µM
202
275
20
145
85
6
330
30
65
110
10
70
55
3
675
55
30
40
45
2.5
4.3
20
0.7
230
34
6
7% @2 µM
8% @2 µM
7% @2 µM
3% @2 µM
20% @2 µM
1800
2100
4% @2 µM
75
Ta ble 3. Influence of the Yaa′ Residue (P2′ Position)
Ki (nM)
MMP-11
mST3
MMP-2
Gel-A
MMP-9
Gel-B
MMP-14
MT1-MMP
MMP-1
HFC
MMP-7
matrilysin
MMP-8
HNC
compd
Yaa′
32
25
33
34
Ala
20% @ 1 µM
31% @2 µM
20
6% @2 µM
260
45% @2 µM
10
14% @2 µM
245
2960
105
10% @10 µM
1282
9% @ 2 µM
23% @ 2 µM
0% @2 µM
9% @ 2 µM
0% @2 µM
8% @2 µM
0% @2 µM
4% @2 µM
240
2.5
2% @1 µM
25
L-Trp
D-Trp
Dpa
5
1% @ 1 µM
27
of a heteroatom in the γ position of the P1′ side chain.
As compared to a carbon atom (compound 19), the
presence of an oxygen in this position decreased the
inhibitory potency (compound 20), while a sulfur sig-
nificantly increased the potency of the inhibitor (com-
pound 21). The magnitude of these substitutions on the
inhibitor affinity depended on the MMP.
thyl group, demonstrate that the S1′ pocket of some
MMPs is large enough to accept such a bulky group. In
terms of selectivity, MMP-11 preferred to bind a naph-
thylethyl rather than a linear heptyl group (compounds
30 and 31), whereas the reverse was true for MMP-8
and MMP-9. With the exception of MMP-1 and MMP-
7, the most potent inhibitor of MMPs in this series was
compound 28. The presence of a sulfur atom in the P1′
side chain of compound 28 accounts in part for its high
potency. As compared to the potency of compounds
containing either a methyl or an isobutyl side chain in
their P1′ position (compounds 22 and 23), the high
potency of compound 28 illustrates the critical role of
an unusual residue in the P1′ position in the affinity of
phosphinic peptides for MMPs.
Substitution of tryptophan in the P2′ position of the
inhibitor by alanine dramatically decreased potency
(Table 3). The same effect was observed with modifica-
tion of tryptophan chirality. However, the rather high
potency displayed by compound 34 suggests that other
aromatic side chains can be tolerated in this position,
depending on the MMP.
The potency of the phosphinic peptides could also be
increased by substituting the P1 side chain of the
inhibitor with a benzyl group (Table 2, compounds 24,
25, 27; as compared to compounds 18, 19, 20 in Table
1). In this series of compounds, the substitution of a
phenylethyl group in the P1′ position by a phenylpropyl
slightly increased the potency of the inhibitor for MMP-
11, while more substantial effects on potency were
observed for the other MMPs. This result is mainly due
to the fact that MMP-11 very well accommodates a
phenylethyl group, as compared to other MMPs. Ac-
cordingly, compound 24 is one of the most selective
MMP-11 inhibitors of this series. Comparison of the
potency of compounds 25 and 26 revealed that all MMPs
preferred a phenylpropyl group to a butylphenyl one in
the P1′ position. However, such a modification did not
affect the inhibitor potency to the same extent with
respect to the different MMPs. This explains the selec-
tivity of compound 26 for MMP-8. The rather high
potencies of compounds 29 and 30, containing a naph-
As phosphinic peptide chemistry makes it possible to
develop inhibitors which interact with the unprimed
enzyme subsites, several inhibitors containing a variety
of substituents in the P2 position were synthesized in
order to determine the influence of these modifications.

2614 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Ta ble 4. Influence of the R1 Substituent (P2 Position)
Vassiliou et al.
The presence of an acetyl group on the N-terminal side
of the P1 position of the inhibitor is sufficient to yield a
potent inhibitor of MMP-8 (compound 35, Table 4).
Overall, the substitution of the acetyl by different bulky
groups had a limited effect on the affinity of these
molecules for MMP-8. In contrast, in this series of
compounds, for MMP-11 the inhibitory potencies vary
from 20 nM (compound 35) to 0.9 nM (compound 41).
Compound 43, an example of a P2 modification influenc-
ing inhibitor selectivity, was less potent on MMP-2,
MMP-9, and MMP-14 than on MMP-11 and MMP-8.
Surprisingly, compounds 40 and 41 turned out to be the
only inhibitors in this study able to block MMP-1
activity potently and MMP-7 activity less so.
Discu ssion
MMPs are increasingly implicated in a wide range of
disease states and have therefore become the focus of
considerable efforts aimed at developing potent inhibi-
tors.10,20 However, in the light of MMP involvements in
the control of several important physiological functions,
it became apparent that selective as well as broad-
spectrum MMP inhibitors should be developed.
This goal turns out to be difficult to achieve, as the
amino acid content of the MMP binding sites is highly
conserved.11 The resolution of the 3D-structure of some
MMP catalytic domains has, nevertheless, provided
interesting information, which has been used to classify
the MMPs in terms of the size of their S1′ pocket.29-35

Phosphinic Pseudo-Tripeptides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2615
According to these structural studies, MMP-1 and
MMP-7 both possess a “small S1′ pocket”. The floor of
the S1′ pocket of MMP-1 and MMP-7 is characterized
by the presence, respectively, of the guanidinium group
of Arg-214 and the phenol group of Tyr-214. In contrast,
with the exception of MMP-11, most of the other MMPs
are characterized by the presence of a leucine, at the
position equivalent to Arg-214 in MMP-1 or Tyr-214 in
MMP-7.11 In these MMPs, the S1′ pocket corresponds
to a large cavity, forming a channel that extends
through the whole body of the enzyme catalytic domain.
Based on the size of their S1′ pocket, MMP-1 and MMP-7
should prefer to accommodate an isobutyl side chain in
the P1′ position of inhibitor, rather than a long aryl-
alkyl side chain. The higher potency of compound 23
for MMP-1 and MMP-7, as compared to inhibitors
containing a longer side chain (compounds 24-28), is
consistent with this proposal. However, the unexpect-
edly high potency of compound 41 toward MMP-1 and
MMP-7 demonstrates that in some cases, inhibitors
containing a long side chain in their P1′ position are able
to interact potently with MMP-1 and MMP-7. The
recent resolution of the crystal structure of a complex
between MMP-7 and a carboxylate peptide inhibitor,
containing in its P1′ position a biphenylpropyl side
chain, offers an explanation of how the binding of such
inhibitors to MMP-7 is possible.36 The analysis of this
complex reveals the occurrence of a dramatic confor-
mational shift in the S1′ pocket of the enzyme, creating
a large S1′ tunnel capable of accommodating the biphe-
nylpropyl side chain of the inhibitor. The same confor-
mational transition has also been observed in MMP-1,
explaining the ability of these MMPs to interact with
inhibitors bearing a long side chain in the P1′ position.36
The existence of such conformational flexibility in the
S1′ pocket of MMP-7 and MMP-1 makes it more difficult
to predict the behavior of inhibitors toward these two
MMPs and to develop selective inhibitors of MMPs.
Con clu sion
This work represents the first study aiming at explor-
ing the potential of phosphinic peptide derivatives as
inhibitors of MMP-11. This study has uncovered some
preferences of MMP-11 for particular substitutions in
the P1′ and P2 positions of the inhibitor, which should
be useful for the development of more selective MMP-
11 inhibitors. The positive contribution of a sulfur atom
in the γ position of the P1′ side chain of the inhibitor is
an interesting observation which should be more sys-
tematically exploited in the future. The structure-
activity relationships established from this work, to-
gether with the related contributions of other groups
to MMP inhibitors,37-39 should contribute to the devel-
opment of inhibitors with specificity for small sets of
MMPs. However, as discussed above, the presence of
some flexibility at the level of the S1′ pocket of MMPs,
in addition to the binding sites highly conserved be-
tween the different MMP members, will make this goal
particularly difficult to accomplish.
Exp er im en ta l Section
Gen er a l P r oced u r es. Commercially available reagents
and starting materials were from Aldrich, Sigma, Novabio-
chem, and Lancaster. Solid-phase synthesis of the phosphinic
peptides was performed in an Advanced Chemtech 357 mul-
tiple synthesizer on a Rink amide resin, using Fmoc strategy.
HPLC analyses and preparative purifications were carried
out on a VYDAC C18 analytical column and a VYDAC C18
preparative column, utilizing the following methods. Solvent
A ) 0.1% TFA and 10% CH3CN in water. Solvent B ) 0.1%
TFA and 90% CH3CN in water. The following gradients were
used. Gradient 1: t ) 0 min (0% B), t ) 10 min (30% B), t )
45 min (45% B). Gradient 2: t ) 0 min (0% B), t ) 10 min
(30% B), t ) 45 min (50% B). Gradient 3: t ) 0 min (0% B), t
) 10 min (35% B), t ) 45 min (50% B). Gradient 4: t ) 0 min
(0% B), t ) 10 min (35% B), t ) 45 min (55% B). Gradient 5:
t ) 0 min (0% B), t ) 10 min (35% B), t ) 45 min (60% B).
Gradient 6: t ) 0 min (0% B), t ) 10 min (40% B), t ) 40 min
(60% B). Gradient 7: t ) 0 min (0% B), t ) 10 min (45% B), t
) 40 min (65% B). Gradient 8: t ) 0 min (0% B), t ) 10 min
(45% B), t ) 45 min (70% B). These purifications were
performed on Gilson gradient system equipped with a variable
wavelength detector. Compounds were detected at 280 and 230
nm.
It was important to determine the behavior of MMP-
11, which is characterized by the presence of a glutamine
at the floor of its S1′ pocket and is thus predicted to
possess a “small S1′ pocket” like MMP-1 and MMP-7.
Our present data demonstrate that MMP-11 is able to
tightly bind inhibitors containing different types of
unusual long side chains. MMP-11 thus behaves more
like the MMPs harboring a leucine in their S1′ pocket
than MMP-1 and MMP-7. This apparent discrepancy
between a proposed model of MMP-11 and our observa-
tions could be resolved by suggesting that a conforma-
tional transition could also occur in MMP-11, allowing
the size of the S1′ pocket of this enzyme to change from
a small to a large tunnel cavity. However, the energetic
cost of this conformational transition should be lower
in MMP-11 than in MMP-1 and MMP-7. Consequently,
the interaction of inhibitors containing a long side chain
in their P1′ position is a more favorable process in MMP-
11 than in MMP-1 and MMP-7. This property favors
the development of potent MMP-11 inhibitors that do
not block MMP-1 and MMP-7 but makes it much more
difficult to develop potent MMP-11 inhibitors that do
not interact with the MMPs containing a leucine in their
S1′ pocket.
1H, 13C, and 31P NMR spectra were recorded on a Bruker
DPX 250 spectrometer. All NMR experiments were carried out
at 298 K in CDCl3. Chemical shifts of proton resonances and
13C resonances were measured relative to the residual chlo-
roform resonance at 7.26 ppm in proton spectra and 77.36 ppm
in 13C spectra. Chemical shift 31P resonances were measured
relative to the H3PO4 resonance at 0 ppm. 1H and 13C
resonance assignments were made using a combination of
homo- and heteronuclear experiments including COSY, TOC-
SY, HMQC, and HMBC experiments. For compounds of type
9, two 13C resonances corresponding to two diastereoisomers
were observed for the carbon atoms bound to the phosphorus
atom (indicated by * in the NMR characterization).
Phosphinic peptides purities were established by analytical
HPLC and mass spectroscopy. ES-MS spectra were recorded
on a Micromass Platform II instrument (Micromass, Altrin-
cham, U.K.).
Gen er a l P r oced u r es To P r ep a r e r-Alk yla cr yla t es.
Three different methods were utilized depending on the nature
of the R-substituent of the acrylate (Scheme 2; Table 5).
Meth od a (Sch em e 2). Acrylates 3 a -e were synthesized
according to a procedure described previously.25 Diethyl ma-
lonate (10 mmol) was added over a period of 10 min to a
solution of sodium ethoxide (10 mmol of sodium in 11 mL of
absolute ethanol). This solution was stirred at 50 °C for 1 h.
Bromide (10 mmol) was added dropwise to this reaction

2616 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Vassiliou et al.
Ta ble 5
Ta ble 6
compd 7
compd 3
R
n
method
bp (mmHg), °C
yield, %
R-X
yield, %
a
b
c
d
e
f
phenyl
phenyl
phenyl
phenyl
naphthyl
methyl
naphthyl
0
1
2
3
0
5
1
a
a
a
a
a
b
c
64-65 (0.01)40
90-95 (0.7)25
112-115 (0.6)40
120-125 (0.005)40
oil
80
76
87
78
25
40
13
a
b
c
4-MeO-C6H4-CH2-S
C6H4-CH2-S
C6H4-CH2-O
80
88
85
Ta ble 7
compd 9 R2
101-105 (8)
a
g
oil
R3
method yield, % Rf
a
b
c
d
e
f
g
h
i
H
H
H
H
H
CH2-Ph
CH2-CH2-Ph
CH2-CH2-CH2-Ph
CH2-O-CH2-Ph
CH2-S-CH2-Ph
a
a
a
a
a
a
a
a
a
a
a
b
b
80
95
83
61
60
85
79
75
81
64
40
60
30
0.41
0.47
0.57
0.69
0.48
0.77
0.80
0.82
0.68
0.61
0.63
0.66
0.65
mixture, which was stirred for 6 h, at 50 °C. Ethanol was
removed under vacuum and diethyl ether was added. This
solution was washed with water and brine, dried over Na2-
SO4, and concentrated to give an oily residue, which after
distillation gave compounds type 1, in yields ranging from 40
to 65%. A solution of KOH (10 mmol) in ethanol (8 mL) was
added to a solution of the diester 1 (10 mmol) in ethanol (8
mL). This mixture was stirred for 16 h. After evaporation of
the organic solvent, the residue was treated with water and
extracted with diethyl ether. The aqueous phase was acidified
with 6 N HCl and extracted twice with diethyl ether. The
organic phases were dried over Na2SO4 and evaporated to give
compounds type 2, in yields ranging from 65 to 90%. Paraform-
aldehyde was added (6 mmol) to a solution of monoester 2 in
pyridine (0.8 mL) and piperidine (0.05 mL). This mixture was
stirred and heated at 50-55 °C for 3 h. After addition of diethyl
ether, the organic phase was washed with water, 3 N HCl,
dried over Na2SO4, and concentrated to give compounds type
3, in yields ranging from 25 to 87%.
Meth od b, (Sch em e 2). The acrylate 3f was synthesized
according to a procedure described previously.26 Under argon
atmosphere, a solution of CH3(CH2)5Br (1.65 g, 10 mmol) in
absolute diethyl ether (15 mL) was added dropwise to a flask
containing Mg (0.22 g, 11 mmol) and I2 (catalyst), over a period
of 90 min. This reaction mixture was refluxed for 1 h. After
CuI (0.18 mmol) was added, the temperature of the mixture
was lowered to -78 °C. A solution of compound 426 (1.15 g,
6.7 mmol) in Et2O:THF (10 mL) was then slowly added to this
reaction mixture which was stirred for 30 min at room
temperature. After treatment of the mixture with 0.5 N HCl,
5% NaHCO3, and water, the organic layer was dried over Na2-
SO4. The solvent was removed under vacuum, and the result-
ing residue was purified by column chromatography, using
petroleum ether (40-60 °C)/ether (13:1) as eluent. The pure
acrylate, compound 3f, was obtained in a yield of 40%.
Meth od c (Sch em e 2). The acrylate 3g was synthesized
according to a procedure described previously.27 A mixture of
bromomethylacrylate40 (1.9 g, 10 mmol) and benzenesulfinic
sodium salt (3.3 g, 20 mmol) in methanol (40 mL) was refluxed
for 12 h. The solvent was removed, and diethyl ether was
added. The solution was washed with water and brine, dried
over Na2SO4, and concentrated to give 2 g of compound 5 (80%)
as an oil. Tri-n-butyltin hydride (4.7 g, 16.4 mmol) and 2.2′-
azobisisobutyronitrile (AIBN) (0.15 g, 0.96 mmol) were added
to a mixture of sulfinic acrylate 5 (2 g, 8 mmol) in dry benzene
(40 mL). This mixture was refluxed for 1.5 h. Water was added,
and the extracted organic layer was dried over Na2SO4 and
concentrated. The crude product was purified by column
chromatography, using petroleum ether (40-60 °C)/ether (9:
1) as eluent, to give 2.9 g of compound 6 (90%). The alkyl-
stannane 6 (1.45 g, 3.6 mmol) and 2-bromoethylnaphthalene
(0.39 g, 1.8 mmol) were dissolved in dry benzene (10 mL). After
addition of AIBN (0.064 g, 0.39 mmol), the reaction mixture
was refluxed for 2 h. After addition of water, the organic phase
was separated, washed with water, dried over Na2SO4, and
concentrated to dryness. Compound 3g (0.06 g, 13%) was
obtained after purification of the residue by column chroma-
tography, using petroleum ether (40-60 °C)/ether (9:1) as
eluent.
Ph CH2-CH2-Ph
Ph CH2-CH2-CH2-Ph
Ph CH2-CH2-CH2CH2-Ph
Ph CH2-(CH2)5-CH3
Ph CH2-O-CH2-Ph
Ph CH2-S-CH2-Ph-OMe
Ph 2-CH2-naphthyl
Ph 2-(CH2)2-naphthyl
j
k
l
m
a
CHCl3/MeOH/AcOH (7:0.5:0.5).
Syn th esis of Eth yl S-Alk yl Meth a cr yla te, 7a a n d 7b.
A solution of the appropriate thiol (10 mmol) in MeOH (15
mL) was added dropwise to a stirred ice-cold solution of sodium
(9 mmol) in MeOH (20 mL) over 30 min. After the thiol was
added, the mixture was concentrated to dryness and Et2O was
added. The precipitated salt was cooled in an ice-water bath.
The product was filtered, washed with cold Et2O, and dried
over P2O5 to give the sodium salt in 85-95% yield. To a
mixture of the sodium salt (10 mmol) suspended in dry Et2O
(40 mL) and cooled in an ice-water bath, a solution of
R-bromomethyl acrylate (9 mmol) in Et2O (20 mL) was added
dropwise over 45 min. This solution was stirred for 30 min at
0 °C and 1-2 h at room temperature. This reaction mixture
was diluted with water (20 mL), and the organic layer was
washed with water, dried over Na2SO4, and evaporated.
Compounds 7a and 7b were purified by column chromatog-
raphy, using petroleum ether (40-60 °C)/Et2O (8:2) as eluent.
Syn th esis of Eth yl O-Alk yl Meth a cr yla te, 7c. Small
pieces of sodium (10 mmol) were added to dry Et2O (20 mL)
in a well-dried flask. A solution of benzyl alcohol (10 mmol) in
dry Et2O (10 mL) was added to this reaction mixture over 2
h, under gentle reflux. The mixture was refluxed for an
additional 6 h. The white precipitate was cooled in an ice-
water bath, filtered, washed with cold dry Et2O, and dried over
P2O5 to give the sodium salt in 85-95% yield. The reaction of
this salt with R-bromomethyl acrylate and the purification of
7c were similar to those described above.
Gen er a l P r oced u r e for th e P r ep a r a tion of P h osp h in ic
Syn th on s Typ e 9 (Sch em e 1). These phosphinic synthons
were prepared according to two procedures described previ-
ously: method a21 or method b28 (Scheme 1, Table 7). Mono-
substituted phosphinate derivatives were synthesized accord-
ing to a procedure described previously.41
Syn th esis of P h osp h in ic Syn th on s 9a -9k , Meth od a .
A suspension of the N-benzyloxycarbonyl phosphinic acid 8 (1
mmol) and hexamethyldisilazane (5 mmol) was heated at 110
°C for 1 h, under nitrogen. The appropriate acrylate (1.3 mmol)
was then added dropwise over 15 min. This reaction mixture
was stirred for an additional 3 h at 110 °C, allowed to cool to
70 °C, and ethanol (3 mL) was added dropwise. After cooling
to room temperature, the reaction mixture was concentrated.
The residue was purified by column chromatography, using
chloroform/methanol/acetic acid (7:0.5:0.5) as eluent, to give
compounds 9 (yields reported in Table 7).
Syn th esis of P h osp h in ic Syn th on s 9l, 9m , Meth od b.
Diisopropylamine (2.1 mmol) and trimethylsilyl chloride (2.1
mmol) were added to an ice-cold solution of phosphinic acid 8
(1 mmol) in chloroform (2 mL), and the mixture was stirred
at room temperature for 3 h. After cooling to 0 °C, the
appropriate ethyl acrylate (1.4 mmol) was added, and the
Syn th esis of Eth yl r-Alk oxy or Alk ylth io Meth a cr yl-
a tes. Synthesis of ethyl R-alkoxy or alkylthio methacrylates
was achieved by the reaction of the corresponding alcohol or
thiol anions with ethyl R-bromomethyl acrylate (Scheme 2,
method d; Table 6).

Phosphinic Pseudo-Tripeptides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2617
reaction mixture was stirred at room temperature for 16 h.
After EtOH was added dropwise, the solvents were removed
under vacuum. Compounds 9l and 9m were obtained after a
chromatography step, as described above.
(COOCH2CH3), 53.3/51.75* (C6H5CH2CHP), 39.7 (C6H5CH2-
CH2CH2CH), 35.7 (C6H5CH2CH2CH2CH), 33.6 (C6H5CH2-
CH2CH2CH), 28.4 (C6H5CH2CH2CH2CH, 29.1/28.0* (PO2CH2),
13.9 (COOCH2CH3). 31P NMR: 53.1/52.2* (PO2CH2).
NMR Ch a r a cter iza tion of Typ e 9 P h osp h in ic Syn -
t h on s. 9a . 1H NMR: δ 7.04-7.30 (C6H5CH2O, C6H5CH2C),
5.32 (NH), 5.12 (C6H5CH2O), 4.11 (COOCH2CH3), 4.02 (CH3CH),
2.88 (C6H5CH2CH), 2.27/1.84 (PO2CH2), 1.91/1.79 (C6H5CH2-
CH), 1.24 (CH3CH), 1.23 (COOCH2CH3). 13C NMR: 175.3
(COOCH2CH3), 156.1 (C6H5CH2OCO), 126.2-140.6 (C6H5-
CH2O, C6H5CH2C), 45.8/46.2* (CH3CH), 67.5 (C6H5CH2O), 61.4
(COOCH2CH3), 40.05 (C6H5CH2CH), 35.7 (C6H5CH2CH)), 29.7/
28.9* (PO2CH2), 14.5 (CH3CH), 14.1 (COOCH2CH3). 31P
NMR: 54.1/54.32* (PO2CH2).
9b. 1H NMR: δ 7.05-7.33 (C6H5CH2O, C6H5CH2CH2C), 5.36
(NH), 5.12 (C6H5CH2O), 4.10 (COOCH2CH3), 4.05 (CH3CH),
2.85 (C6H5CH2CH2CH), 2.55 (C6H5CH2CH2CH), 2.25/1.84 (PO2-
CH2),1.95/1.83 (C6H5CH2CH2CH),1.28 (CH3CH),1.26 (COOCH2-
CH3). 13C NMR: 174.9 (COOCH2CH3), 156.2 (C6H5CH2OCO),
126.4-141.4 (C6H5CH2O, C6H5CH2CH2C), 45.3/46.05* (CH3CH),
67.6 (C6H5CH2O), 61.4 (COOCH2CH3), 39.2 (C6H5CH2CH2CH),
35.9 (C6H5CH2CH2CH), 33.4 (C6H5CH2CH2CH), 28.8/28.5*
(PO2CH2)), 14.6 (CH3CH), 14.5 (COOCH2CH3). 31P NMR:
54.45/54.23* (PO2CH2).
9h . 1H NMR: δ 7.0-7.26 (C6H5CH2O, C6H5CH2CHP, C6H5-
CH2CH2CH2CH2CH),5.40(NH),4.99(C6H5CH2O),4.10(COOCH2-
CH3), 4.25 (C6H5CH2CHP), 3.26/2.82 (C6H5CH2CHP), 2.80
(C6H5CH2CH2CH2CH2CH), 2.52 (C6H5CH2CH2CH2CH2CH),
2.23/1.75 (PO2-CH2), 1.62/1.53 (C6H5CH2CH2CH2CH2CH),
1.52 (C6H5CH2CH2CH2CH2CH), 1.25 (C6H5CH2CH2CH2CH2-
CH), 1.20 (COOCH2CH3). 13C NMR: 175.3 (COOCH2CH3),
156.5 (C6H5CH2OCO), 142.6-126.4 (C6H5CH2O, C6H5CH2CHP,
C6H5CH2CH2CH2CH2CH), 67.45 (C6H5CH2OCO), 61.4 (COOCH2-
CH3), 52.2/50.9* (C6H5CH2CHP), 39.7 (C6H5CH2CH2CH2-
CH2CH), 35.8 (C6H5CH2CH2CH2CH2CH), 34.0 (C6H5CH2CH2-
CH2CH2CH),31.2(C6H5CH2CH2CH2CH2CH),26.4(C6H5CH2CH2CH2CH2-
CH), 29.3/28.43* (PO2CH2), 14.2 (COOCH2CH3). 31P NMR:
53.1/52.3* (PO2CH2).
9i. 1H NMR: δ 7.16-7.27 (C6H5CH2O, C6H5CH2CHP), 5.46
(NH), 4.98 (C6H5CH2O), 4.20 (COOCH2CH3), 4.26 (C6H5-
CH2CHP), 3.28/2.85 (C6H5CH2CHP), 2.81 (CH3(CH2)5CH2CH),
2.23/1.75 (PO2CH2), 1.63/1.50 (CH3CH2CH2CH2CH2CH2CH2-
CH), 1.20 (CH3(CH2)5CH2CH), 1.23 (COOCH2CH3), 0.83
(CH3(CH2)5CH2CH). 13C NMR: 175.37 (COOCH2CH3), 156.4
(C6H5CH2OCO), 137.6-127.5 (C6H5CH2O, C6H5CH2CHP), 67.23
(C6H5CH2OCO), 61.2 (COOCH2CH3), 52.8/51.25* (C6H5CH2-
CHP), 39.31 (CH3(CH2)5CH2CH), 31.97/29.3/26.65/22.81 (CH3-
(CH2)5CH2CH), 14.7 (COOCH2CH3) 14.5 (CH3(CH2)5CH2CH),
29.73/28.45* (PO2CH2). 31P NMR: 54.4/53.9* (PO2CH2).
9j. 1H NMR: δ 7.36-7.16 (C6H5CH2O, C6H5CH2CHP, C6H5-
CH2OCH2CH), 5.48 (NH), 4.97 (C6H5CH2O), 4.15 (COOCH2-
CH3), 4.45 (C6H5CH2OCH2CH), 4.30 (C6H5CH2CHP), 3.65
(C6H5CH2OCH2CH), 3.28/2.85 (C6H5CH2CHP), 3.12 (C6H5CH2-
OCH2CH), 2.27/2.0 (PO2CH2), 1.22 (COOCH2CH3). 13C NMR:
173.2 (COOCH2CH3), 156.4 (C6H5CH2OCO), 138.1-127.2 (C6H5-
CH2O, C6H5CH2CHP, C6H5CH2OCH2CH), 77.42 (C6H5CH2-
OCH2CH), 73.44 (C6H5CH2OCH2CH), 67.47 (C6H5CH2OCO),
61.6 (COOCH2CH3), 52.65/50.9* (C6H5CH2CHP), 39.93 (C6H5-
9c. 1H NMR: δ 7.04-7.29 (C6H5CH2O, C6H5CH2CH2CH2C),
5.29 (NH), 5.1 (C6H5CH2O), 4.10 (COOCH2CH3), 4.03 (CH3CH),
2.92 (C6H5CH2CH2CH2CH), 2.56 (C6H5CH2CH2CH2CH), 2.27/
1.84 (PO2CH2), 1.95/1.81 (C6H5CH2CH2CH2CH), 1.52 (C6H5-
CH2CH2CH2CH), 1.25 (CH3CH), 1.20 (COOCH2CH3). 13C
NMR: 176 (COOCH2CH3), 156.4 (C6H5CH2OCO), 126.6-141.3
(C6H5CH2O, C6H5CH2CH2CH2C), 46.3/45.1* (CH3CH), 67.4
(C6H5CH2O), 61.7 (COOCH2CH3), 39.2 (C6H5CH2CH2CH2CH),
35.7 (C6H5CH2CH2CH2CH), 32.9 (C6H5CH2CH2CH2CH), 28.3
(C6H5CH2CH2CH2CH), 29.2/28.3* (PO2CH2), 14.4 (CH3CH),
14.1 (COOCH2CH3). 31P NMR: 53.2/52.17* (PO2CH2).
1
9d . H NMR: δ 7.26-7.32 (C6H5CH2OCO, C6H5CH2OCH2-
CH), 5.41 (NH), 5.09 (C6H5CH2OCO), 4.12 (COOCH2CH3), 4.48
(C6H5CH2OCH2CH), 4.05 (CH3CH), 3.65 (C6H5CH2OCH2CH),
3.09 (C6H5CH2OCH2CH), 2.25/2.02 (PO2CH2), 1.35 (CH3CH),
1.20 (COOCH2CH3). 13C NMR: 173.2 (COOCH2CH3), 156.3
(C6H5CH2OCO), 138.2-127.9 (C6H5CH2OCO, C6H5CH2OCH2-
CH), 77.4 (C6H5CH2OCH2CH), 73.5 C6H5CH2OCH2CH), 67.64
(C6H5CH2OCO), 61.77 (COOCH2CH3), 46.8/45.2 (CH3CH),
40.13 (C6H5CH2OCH2CH), 25.8/24.2* (PO2CH2), 14.4 (CH3CH),
14.1 (COOCH2CH3)). 31P NMR: 54.3 (PO2CH2).
CH2OCH2CH), 26.4/24.9* (PO2CH2), 13.94 (COOCH2CH3). 31
NMR: 53.7/53.59 (PO2CH2).
P
9k . 1H NMR: δ 7.36-7.18 (C6H5CH2O, C6H5CH2CHP, CH3-
OC6H5CH2SCH2CH),5.41(NH),4.98(C6H5CH2O),4.15(COOCH2-
CH3), 4.30 (C6H5CH2CHP), 3.65 (CH3OC6H5CH2SCH2CH),
2.75/2.50 (CH3OC6H5CH2SCH2CH), 3.0 (CH3OC6H5CH2SCH2-
CH), 2.25/2.02 (PO2CH2), 1.35 (CH3OC6H5CH2SCH2CH), 1.22
(COOCH2CH3). 13C NMR: 173.6 (COOCH2CH3), 159 (C6H5-
CH2OCO), 130.4-127.2 (C6H5CH2O, C6H5CH2CHP, CH3OC6H5-
CH2SCH2CH), 67.8 (C6H5CH2OCO), 61.7 (COOCH2CH3), 52.1/
50.8* (C6H5CH2CHP), 39.6 (CH3OC6H5CH2SCH2CH), 35.6
(CH3OC6H5CH2SCH2CH), 35.0 (CH3OC6H5CH2SCH2CH), 29.1/
27.9* (PO2CH2), 14.5 (COOCH2CH3). 31P NMR: 54.07/53.76*
(PO2CH2).
1
9e. H NMR: δ 7.26-7.33 (C6H5CH2O, C6H5CH2SCH2CH),
5.41 (NH), 5.12 (C6H5CH2O), 4.15 (COOCH2CH3), 4.08 (CH3CH),
3.65 (C6H5CH2SCH2CH), 2.75/2.50 (C6H5CH2SCH2CH), 3.0
(C6H5CH2SCH2CH), 2.25/2.02 (PO2CH2), 1.35 (CH3CH), 1.22
(COOCH2CH3). 13C NMR: 173.5 (COOCH2CH3), 156.2 (C6H5-
CH2OCO), 138.1-127.4 (C6H5CH2O, C6H5CH2SCH2CH), 67.8
(C6H5CH2OCO), 61.7 (COOCH2CH3), 46.7/44.9* (CH3CH), 39.2
(C6H5CH2SCH2CH), 36 (C6H5CH2SCH2CH), 34.56 (C6H5-
CH2OCH2CH), 28.2/26.8* (PO2CH2), 14.4 (CH3CH), 14.1
(COOCH2CH3). 31P NMR: 54.41/54.23* (PO2CH2).
9l. 1H NMR: δ 7.0-7.80 (C6H5CH2O, C6H5CH2CHP, C10H7),
5.28 (NH), 4.88 (C6H5CH2O), 4.02 (COOCH2CH3), 4.12 (C6H5-
CH2CHP), 3.20/2.74 (C6H5CH2CHP), 3.18 (C10H7CH2CH), 3.06/
2.99 (C10H7CH2CH), 2.18/1.85 (PO2CH2), 1.03 (COOCH2CH3).
13C NMR: 177.6 (COOCH2CH3), 158.0 (C6H5CH2OCO), 132.8-
126.1 (C6H5CH2O, C6H5CH2CHP, C10H7), 67.8 (C6H5CH2OCO),
62.1 (COOCH2CH3), 53.5/51.63* (C6H5CH2CHP), 41.6 (C10H7-
CH2CH), 40.34 (C10H7CH2CH), 34.45 (C6H5CH2CHP), 29.15/
28.22* (PO2CH2),14.13 (COOCH2CH3). 31P NMR: 54.40/53.88*
(P (PO2-CH2)).
9f. 1H NMR: δ 7.0-7.3 (C6H5CH2O, C6H5CH2CHP, C6H5-
CH2CH2CH), 5.61 (NH), 5.0 (C6H5CH2O), 4.15 (COOCH2CH3),
4.02 (C6H5CH2CHP), 3.17/2.83 (C6H5CH2CHP), 2.92 (C6H5-
CH2CH2CH), 2.55 (C6H5CH2CH2CH), 2.22/1.84 (PO2-CH2),
1.97/1.83 (C6H5CH2CH2CH), 1.23 (COOCH2CH3). 13C NMR:
177.6 (COOCH2CH3), 158.0 (C6H5CH2OCO), 141-127.1 (C6H5-
CH2O, C6H5CH2CHP, C6H5CH2CH2CH), 53.5/52.1* (C6H5-
CH2CHP), 68.5 (C6H5CH2OCO), 63.05 (COOCH2CH3), 39.8
(C6H5CH2CH2CH), 35.5 (C6H5CH2CH2CH), 33.1 (C6H5CH2CH2-
CH), 28.9/27.7* (PO2CH2),14.1 (COOCH2CH3). 31P NMR: 51.34
(PO2CH2).
9g. 1H NMR: δ 7.14-7.26 (C6H5CH2O, C6H5CH2CHP, C6H5-
CH2CH2CH2CH), 4.98 (NH), 4.94 (C6H5CH2O), 4.13 (COOCH2-
CH3), 4.20 (C6H5CH2CHP), 3.23/2.83 (C6H5CH2CHP), 2.87
(C6H5CH2CH2CH2CH), 1.51 (C6H5CH2CH2CH2CH), 2.52 (C6H5-
CH2CH2CH2CH), 2.21/1.75 (PO2CH2), 1.53/1.45 (C6H5CH2-
CH2CH2CH), 1.21 (COOCH2CH3). 13C NMR: 177.2 (COOCH2-
CH3), 157.7 (C6H5CH2OCO), 142.3-126.6 (C6H5CH2O, C6H5-
CH2CHP, C6H5CH2CH2CH2CH), 68.3 (C6H5CH2OCO), 62.3
9m . 1H NMR: δ 7.0-7.80 (C6H5CH2O, C6H5CH2CHP, C10H7),
5.60 (NH), 5.01 (C6H5CH2O), 4.02 (COOCH2CH3), 4.32 (C6H5-
CH2CHP), 3.27/2.92 (C6H5CH2CHP), 2.98 (C10H7CH2CH2CH),
2.85/2.73 (C10H7CH2CH2CH), 2.65 (C10H7CH2CH2CH), 2.18/
2.05 (PO2CH2), 1.28 (COOCH2CH3). 13C NMR: 177.6 (COOCH2-
CH3), 158.0 (C6H5CH2OCO), 132.8-126.1 (C6H5CH2O, C6H5-
CH2CHP, C10H7), 68.4 (COOCH2CH3), 62.6 (COOCH2CH3),
54.1/52.0* (C6H5CH2CHP), 39.98 (C10H7CH2CH2CH), 36.5
(C10H7CH2CH2CH), 37.2 (C10H7CH2CH2CH), 34.3 (C6H5CH2-
CHP), 29.1/28.3* (PO2CH2),14.5 (COOCH2CH3)). 31P NMR:
54.08 (PO2CH2).
Gen er a l P r oced u r e for th e P r ep a r a tion of P h osp h in ic

2618 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14
Vassiliou et al.
Ta ble 8
Ta ble 9
compds
10, 11 R2
yield, % yield, % Rf of Rf of
(for 10) (for 11) 10a 11b
compd
HPLC gradients
calcd mass
ES-MS (M + H)
R3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
1
1
3
2
1
1
2
3
5
4
4
7
6
6
6
2
4
6
2
3
2
4
2
3
7
4
8
4
5
590.23
604.25
618.26
620.24
636.22
590.23
632.28
680.28
694.29
708.31
696.27
742.26
716.28
730.29
674.32
579.25
694.29
760.26
602.27
708.31
693.31
727.27
743.26
703.29
771.21
715.29
800.19
765.33
807.38
590.5
604.2
618.7
620.6
636.5
590.5
632.6
680.7
694.8
708.9
696.7
742.8
716.8
730.9
674.8
579.6
694.7
761.7
602.7
709
693.8
728.2
744.2
703.9
772.7
715.8
801.8
765.8
808
a
b
c
d
e
f
g
h
i
H
H
H
H
H
CH2-Ph
CH2-CH2-Ph
CH2-CH2-CH2-Ph
CH2-O-CH2-Ph
CH2-S-CH2-Ph
95
97
97
93
72
89
75
95
96
91
93
91
95
80
81
85
90
92
95
97
95
78
80
94
92
86
0.70 0.53a
0.72 0.59a
0.73 0.21
0.77 0.42a
0.72 0.50a
0.61 0.42
0.66 0.42
0.63 0.44
0.78 0.56
0.71 0.26
0.66 0.46
0.53 0.38
0.57 0.35
Ph CH2-CH2-Ph
Ph CH2-CH2-CH2-Ph
Ph CH2-CH2-CH2-CH2-Ph
Ph CH2-(CH2)5-CH3
Ph CH2-O-CH2-Ph
Ph CH2-S-CH2-Ph-OMe
Ph 2-CH2-naphthyl
Ph 2-(CH2)2-naphthyl
j
k
l
m
a
b
Hex/AcOEt/AcOH (3:3:0.2). CHCl3/MeOH (9.5:0.5).
Syn th on s Typ e 10 (Sch em e 3). The type 9 synthon (1 mmol)
and 1-adamanthylbromide (1.2 mmol) were dissolved in chlo-
roform (10 mL). This reaction mixture was refluxed. Silver
oxide (2 mmol) was then added in five equal portions, over 50
min. This solution was refluxed for an additional 30 min. After
removal of solvents, the residue was treated with diethyl ether
and filtered through Celite. The filtrates were concentrated.
The residue was purified by column chromatography using
chloroform/2-propanol (9.8:0.2) as eluent. The compounds type
10 were obtained in yields reported in Table 8.
Gen er a l P r oced u r e for th e P r ep a r a tion of P h osp h in ic
Syn th on s Typ e 11 (Sch em e 3). One milliliter of 4 N NaOH
was added dropwise to a stirred solution of type 10 compounds
(1 mmol) in methanol (5.5 mL). The reaction mixture was
stirred for 18 h. The solvent was then removed, and the residue
was diluted with water and acidified with 0.5 N HCl in an
ice-water bath. The aqueous phase was extracted with ethyl
acetate (2 × 15 mL). The combined organic layers were dried
over Na2SO4 and concentrated to give compounds type 11 as
white solids in yields reported in Table 8.
equiv of the phosphinic synthon was used and that the reaction
was allowed to proceed for 60 min. Cleavage of the peptides
from the resin, together with the cleavage of the side chain
protection groups, was performed using a solution of TFA in
dichloromethane containing 5% triisopropylsilane and 5%
water.
P r oced u r e for th e P r ep a r a tion of P h osp h in ic Syn -
th on s Typ e 13. To a solution of methanol (7 mL), containing
compound 11g (0.64 g 1.0 mmol) and ammonium formate (0.19
g, 3.0 mmol), was added 10% Pd/C (0.15 g). After 12 min at
room temperature, the catalyst was removed by filtration
through Celite, and the filtrate was evaporated to dryness.
Methylene chloride was added to the residue, and the solution
was evaporated to dryness. This procedure was repeated twice.
The resulting residue was dissolved in 20% Na2CO3 (2 mL).
This reaction mixture was concentrated in vacuo until half of
the volume was removed, and then water (1 mL) and dioxane
(2 mL) were added. The mixture was ice-cooled, and a solution
of Fmoc-Cl (0.31 g, 1.20 mmol) in dioxane (2 mL) was added
dropwise over a period of 1 h. The solution was stirred for 2 h
at 4 °C and 4 h at room temperature, and the reaction mixture
was then diluted with water (15 mL), cooled in an ice-water
bath, and acidified to pH 2 with 2 N HCl. The solid product
which precipitated was quickly taken up in diethyl ether, and
the organic layer was rinsed with water, dried over Na2SO4,
and evaporated to dryness to give the crude product 13g, which
was purified by silica gel column chromatography using
chloroform/methanol (9.5:0.5) as eluent. The pure product 13g
was obtained in 52% (0.38 g) yield.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
35-45. These phosphinic peptides were prepared as described
above, using phosphinic synthons type 13. The Fmoc group of
compound type 14 was removed using 20% piperidine/NMP
solution. Compounds 40-43 were prepared by coupling indole-
2-carboxylic acid, 4,6-dichloro indole-2-carboxylic acid, 2-quino-
line carboxylic acid, and 6-8 dichloro-coumarine-3-carboxylic
acid to the free amino-phosphinic peptide compound type 15,
bound to the resin. These acids were incorporated by multiple
couplings, using 3 equiv of the acid to be coupled with 3 equiv
of HBTU and 10 equiv of DIEA. Compounds 35 and 36 were
prepared by coupling acetyl and benzyloxyacetyl chlorides to
the free amino-phosphinic peptide compound 15. Solutions (0.5
M) of chlorides and DIEA in dichloromethane were used, in
25-fold excess. Compounds 37-39 were prepared from com-
pound 15 by a two-step acylation-alkylation procedure using
bromoacetic bromide. The conditions used to incorporate the
bromide derivative were similar to those described for chlorides
coupling. Aniline derivatives (50 equiv) were alkylated using
a solution of DIEA in DMSO. Cleavage of the peptides from
the resin, together with the cleavage of the side chain protec-
tion groups, was performed as described above.
P u r ifica tion a n d Ch a r a cter iza tion of Com p ou n d s 17-
45. Purification of the diastereoisomeric forms of the phos-
phinic peptides was achieved by reverse-phase HPLC, using
different types of gradients (Table 9). Typically, four main
HPLC peaks were observed for all phosphinic peptides pre-
pared. Mass spectroscopy analysis of these four peaks showed
that they corresponded to the different diastereoisomeric forms
of these phosphinic peptides. Correctness of the phosphinic
peptide structure was checked by ES-MS. The data reported
in Tables 1-4 correspond to the most potent diastereoisomer
of the phosphinic compounds.
P r oced u r e for th e P r ep a r a tion of Com p ou n d Typ e 14.
This phosphinic peptide was obtained following the protocol
described for the synthesis of compounds type 12.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
17-34. The phosphinic pseudo-peptides were prepared by
conventional solid-phase peptide synthesis, on a Rink amide
resin, using a Fmoc strategy. Typically, 3 equiv of a Fmoc
amino acid, 3 equiv of HBTU, and 5 equiv of diisopropylethyl-
amine (DIEA) in N-methylpyrrolidone (NMP) were added to
the resin, and the coupling reaction was allowed to proceed
for 30 min. The Fmoc N-protection group was removed with a
30% solution of piperidine in NMP. The coupling of the
phosphinic synthons (compound type 11) was performed using
the coupling conditions described above, except that only 1.5
En zym es. Recombinant human full-length pro-MMP-1, pro-
MMP-2, pro-MMP-9, and pro-MMP-8 were expressed in NSO
myeloma cells and purified as described before.42,43 The pro-
enzymes were activated immediately prior to use. Pro-MMP-1

Phosphinic Pseudo-Tripeptides
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 14 2619
was treated with trypsin (10 µg/mL) for 25 min at 25 °C,
followed by bovine pancreatic trypsin inhibitor (100 µg/mL).
Pro-MMP-2, pro-MMP-9, and Pro-MMP-8 were activated with
4-aminophenylmercuric acetate.44 Catalytic domains of human
MMP-14 and MMP-7 and mouse MMP-11, expressed in
Escherichia coli BL21 DE3 pLysS cells, were obtained and
purified as described before.45
Nationale Franc¸aise contre le Cancer, the Comite´ du
Haut-Rhin, the Fondation de France, the BIOMED 2
(Contract BMH4CT96-0017) and BIOTECH 2 (Contract
ERBBIO4CT96-0464) Programs, and a grant to P.
Chambon from the Fondation J eantet.
En zym e Assa ys. Enzyme inhibition assays were carried
out in 50 mM Tris/HCl buffer, pH 6.8, 10 mM CaCl2 at 25 °C.
With the exception of MMP-11, all the assays were performed
in black 96-well plates. Progress curves were monitored by
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following the increase in fluorescence at 400 nm (λ ) 340
ex
nm), induced by the cleavage of the Mca-Pro-Leu-Gly-Leu-Dpa-
Ala-Arg-NH2 fluorogenic substrate by MMPs.46 The conditions
of a typical experiment were 200 µL of buffer, 0.2-5 nM MMP,
and 6 µM Mca-substrate. Inhibitors were dissolved at various
concentrations in buffer containing the MMP. The reaction was
then immediately initiated by addition of 1-2 µL of a stock
solution of substrate in DMSO. Under these conditions, steady-
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Data were collected for 30 min.
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Mca-Pro-Leu-Ala-Cys(OMeBn)-Trp-Ala-Arg-Dpa-NH2 substrate,
as described before.19
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evident from the progress curves. For those inhibitors where
time-dependence of inhibition was observed, inhibitor and
enzyme were preincubated for at least 1 h before addition of
the substrate.
For some highly hydrophobic compounds, binding of the
inhibitor to the 96-well plates was detected, biasing the
determination of the Ki value. In this case, these compounds
were studied in quartz cells.
Deter m in a tion of th e Ki Va lu es. For each MMP, initial
rate measurements in the absence of inhibitor were made for
eight different substrate concentrations. From these data, Km
values were determined using the graphical direct linear plot
method.47 A correction factor for each substrate concentration,
determined experimentally as described before,19 was used to
correct the fluorescence observed at high substrate concentra-
tions for the inner filter effect. The Km values determined for
MMP-1, MMP-2, MMP-7, MMP-8, MMP-9, MMP-11, and
MMP-14 were, respectively, 100, 49, 67, 61, 14, 0.2, and 21
µM.
For each inhibitor, percentages of inhibition were deter-
mined in triplicate experiments at five concentrations. Inhibi-
tor concentrations were selected in order to observe a 20-80%
range of inhibition. Ki values were determined using the
method proposed by Horovitz and Levitski.48 This approach
explicitly takes into account the effect of the substrate, enzyme,
and inhibitor concentrations and applies to the situation of
both standard and tight-binding inhibition.
Equ ip m en t a n d In str u m en ta tion . Assays were per-
formed in black flat-bottomed 96-well plates (nonbinding-
surface plates 999999, Corning Costar France.). With these
plates, normal progress curves were observed for all MMPs,
avoiding the requirement for prior BSA coating of the plates
to prevent abnormal kinetics. Fluorescence signals were
monitored using a Biolumin 960 photon-counter spectropho-
tometer (Molecular Dynamics), equipped with a temperature
device control and a plate shaker. For assays performed in
quartz cells, fluorescence measurements were performed with
a Perkin-Elmer LS 50 spectrophotometer.
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Ack n ow led gm en t. We thank I. Stoll for technical
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Commissariat a` l’Energie Atomique, the Institut Na-
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Centre National de la Recherche Scientifique, the Cen-
tre Hospitalier Universitaire Re´gional, the Bristol-
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Association pour la Recherche sur le Cancer, the Ligue

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