Phosphinic acid-based MMP-13 inhibitors that spare MMP-1 and MMP-3

Article abstract of DOI:10.1016/S0960-894X(03)00413-X

Phosphinic acid-based inhibitors of MMP-13 have been investigated with the aim of identifying potent inhibitors with high selectivity versus MMP-1. Independent variation of the substituents on a P₁′ phenethyl group and a P₂ benzyl gr

Full text of DOI:10.1016/S0960-894X(03)00413-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2331–2336
Phosphinic Acid-Based MMP-13 Inhibitors That Spare
MMP-1 and MMP-3
Lawrence A. Reiter,* Peter G. Mitchell, Gary J. Martinelli, Lori L. Lopresti-Morrow,
Sue A. Yocum and James D. Eskra
Pfizer Inc, Global Reseach & Development, Groton Laboratories, Eastern Point Rd., Groton, CT 06340, USA
Received 19 February 2003; revised 17 April 2003; accepted 22 April 2003
Abstract—Phosphinic acid-based inhibitors of MMP-13 have been investigated 0with the aim of identifying potent inhibitors with
high selectivity versus MMP-1. Independent variation of the substituents on a P1 phenethyl g0roup and a P2 benzyl group improved
potencies in both cases around 3-fold over the unsubstituted parent. Combining improved P1 and P2 groups into a single molecule
gave an inhibitor with a 4.5 nM IC50 against MMP-13 and which is 270-fold selective over MMP-1.
# 2003 Elsevier Science Ltd. All rights reserved.
The quest to identify therapeutically useful inhibitors of
MMP’s has entered its third decade1, and this quest
remains to be fulfilled. The early studies, which were
initiated in the pre-genomics era, aimed at inhibiting
MMP-1 and focused on identifying molecules which
were potent and which had favorable pharmacokinetic
(PK) properties. These early endeavors have been
replaced with more fundamental work aimed at identi-
fying the many MMP’s present in humans, assessing
their relative abundance and distribution, and most cri-
tically, determining their relevance to different disease
states.2 The search for therapeutically useful molecules
has likewise evolved. Early studies focused on peptidic
structures some of which displayed potent MMP-1
inhibition but which had unknown (and un-appreciated
lack of) specificity and poor PK properties. While pro-
gress has been made in identifying compounds with
improved PK in both peptidic and non-peptidic motifs,
clinical experience with broad spectrum inhibitors has
revealed a common, intolerable side effect, musculo-
skeletal syndrome (MSS).3 Avoiding MSS is likely to
require identification of the MMP(s) responsible for the
side effect and the discovery and development of
appropriately selective inhibitors which in an ideal case,
would inhibit only the target MMP(s).
That MMP-1 plays a role in the development of MSS
has been postulated4 and efforts to identify MMP
inhibitors that spare MMP-1 have been reported.4,5 The
target MMPs for these studies, though, have varied
depending on which disease is being studied. In the case
of osteoarthritis, the presence of MMP-13 in OA carti-
lage tissue in humans,6,7 its ability to turn over type II
collagen,6,8 its co-localization with cleaved type II col-
lagen in OA cartilage tissue,9 and it degradative prop-
erties in cartilage,10 all point to its likely role in the
pathology of the disease.11 Additional evidence that
MMP-13 is a key driver of cartilage destruction (vs
MMP-1) was provided by the study of a relatively broad
spectrum inhibitor, CGS 27023A,12 versus 1, a MMP-1-
sparing MMP-13 inhibitor, in the bovine nasal cartilage
(BNC) model of cartilage degradation (Fig. 1).13 In this
model CGS 27023A displayed an IC50 for inhibition of
hydroxyproline release of ꢀ50 nM which is about 10-
fold higher than its IC50 against recombinant MMP-13
(3 nM) and 3-fold higher than its MMP-1 IC50 (18 nM).
The phosphinate 1 displayed an IC50 in the BNC model
of ꢀ500 nM, about 10-fold higher than its MMP-13
IC50 (70 nM), consistent with the result with CGS
27023A. Furthermore, the IC50 of 1 in the BNC model
is about 40-fold less than its MMP-1 IC50 (22,000 nM)
clearly demonstrating that MMP-1 does not play sig-
nificant role in this model.
We recently reported a series of phosphinate-based
inhibitors, including 2, the t-butylglycine-containing
analogue of 1 (MMP-13 IC50 30 nM, MMP-1 IC50
*Corresponding author. Tel.:+1-8604-413-681; fax:+1-8606-861-176;
e-mail: lawrence_a_reiter@groton.pfizer.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00413-X

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L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2331–2336
methylacrylate.15 The use of an excess of cuprate drove
the reaction to completion leading to a crude product
containing the desired acrylate and the substituted tol-
uene derived from the quenched cuprate. This latter,
neutral impurity was easily removed after the next syn-
thetic step in which the mono-substituted phosphinic
acid 4, prepared through an Arbuzov reaction of the
corresponding benzyl bromide with bis(trimethyl-
silyl)phosphonite,16 was reacted with each a-substituted
acrylate.17 The crude di-substituted phosphinic acid was
purified by absorption onto MP-carbonate (Argonaut
Technologies) resin, washing to remove non-acidic
components and release of product with TFA in
CH2Cl2. The phosphinates thus prepared were pure by
NMR and LC-MS. Subsequent saponification and
selective coupling of the carboxylic acid to S tert-leucine
N-methylamide using PyBOP18 gave the crude desired
compounds as a mixture of diastereomers. Preparative
RP (reverse phase) chromatography yielded the pure
compounds 5–13.
The synthesis of the P2 analogues made use of inter-
mediate 14 in order to facilitate the rapid preparation of
the target compounds (Scheme 2). This was prepared
from benzyl a-phenethylacrylate and 4-iodobenzyl-
bromide as previously described.14 The intermediate
mono-substituted phosphinic acid adduct of bis(tri-
methylsilyl)phosphonite and benzyl a-phenethylacrylate
was partially resolved (ꢀ3/1 S/R) by one crystallization
of its S a-methylbenzylamine salt.19 Negishi coupling of
14 with benzyl zinc reagents20 gave the intermediates
containing the various P2 substituents. Conversion into
the target phosphinic acids 15–27 was achieved as pre-
viously described.14 Owing to the partial resolution of
the earlier intermediate, the diastereomeric mixtures
were all biased toward the more potent isomer. Never-
theless, if the diastereomeric mixture was sufficiently
potent, the pure isomer was isolated by preparative RP
chromatography (e.g., 15, 18, 21, and 26).
Figure 1. Inhibition of IL-1 induced degradation of bovine nasal car-
tilage (BNC) explants.
>30,000 nM), that take advantage of binding into
pockets on both sides of the scissile bond.14 In parti-
0
cular, key interactions occur in the S2 and S1 pockets
and with the catalytic zinc atom. The premise of this
work was that, through the interaction with more sub-
sites of the enzyme, the identification of selective com-
pounds may be possible. Since our group is focused on
potential treatments for osteoarthritis, we wished to
continue to explore this premise in the context of MMP-
13 inhibition. We especially sought compounds with
>100-fold selectivity versus MMP-1. The most potent
MMP-13 inhibitor identified in our previous work was
3, having an MMP-13 IC50 of 21 nM and 37-fold selec-
tivity over MMP-1 (Table 1). Although 2 was more
selective versus MMP-1, the somewhat greater potency
of 3 and the0 synthetic accessibility and lower molecular
weight of P1 phenethyl derivatives led us to explore the
latter sub-series. Herein we report the results of our stud-
0
stituents, 28, was prepared from 4-(2-methoxy)benzyl-
The analogue combining the better P1 and P2 sub-
benzyl bromide and ethyl a-(4-chlorophenyl)ethylacrylate
as described above for the other P1 analogues.
0
0
Substitution on the terminus of the phenethyl P1 group
modulated MMP-13 potency and/or selectivity versus
MMP-1. In all cases but one, substitution either had a
slight effect (e.g., 6) or modestly increased MMP-13
potency from 2- to 4-fold. The exception, the 2-methyl
derivative 8, was substantially less active. Although
increases in potency were only modest at best, these,
coupled with modest reductions in MMP-1 potency, led
to selectivities of greater than 100-fold for some of the
4-substituted derivatives. The loss of MMP-1 potency
for compounds with additional substitution at C-4 of
0
ies in which we have varied the P2 and P1 substituents
seeking potent and selective MMP-13 inhibitors.
0
the phenylethyl group is consistent with the MMP-1 S1
pocket being shallow.
0
0
The synthesis of P1 analogues was accomplished in 4
In contrast to the considerable published SAR for P1
synthetic steps (Scheme 1). Appropriately substituted
ethyl a-phenethylacrylates were prepared through reac-
tion of various benzyl cuprates with ethyl a-bromo-
substitution of MMP-13 inhibitors,2 little is known
about the S2 pocket of MMP’s.21 As a result, we chose
to initially survey the effect of fluoro, methyl and

L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2331–2336
Table 1. Variation of P1 and P2 residues. MMP-13 and MMP-1 activity
2333
0
CPDa
Y
X
MMP-1b
IC50 (nM)d
MMP-13c
IC50 (nM)d
MMP-1/ MMP-13
IC50 ratio
Ratio of S/R isomers
0
at P1 side chain
3
5
6
7
8
9
H–
H–
H–
H–
H–
H–
H–
H–
H–
H–
2-F–
3-F–
H–
2-F–
3-F–
4-F–
2-Me–
3-Me–
4-Me–
4-Cl–
4-Br–
4-MeO–
H–
H–
H–
H–
H–
H–
H–
H–
H–
770ꢁ300
280ꢁ40
21ꢁ9
10ꢁ3
37
28
45
41
<40
28
82
130
150
210
38
28
22
46
62
55
110
70
96
25
27
49
21
270
100/0
99/1
860ꢁ190
530ꢁ110
12,000ꢁ900
120ꢁ10
19ꢁ6
100/0
100/0
100/0
100/0
100/0
100/0
100/0
100/0
96/04
75/25
74/26
96/04
80/20
79/21
99/01
78/22
79/21
86/14
86/14
98/02
73/23
100/0
13ꢁ3
>300 (3)
4.3ꢁ0.8
7.7ꢁ2.4
8.3ꢁ2.8
9.4ꢁ1.2
7.7ꢁ2.0
18ꢁ7.8
58ꢁ41
44ꢁ31
24ꢁ21
37ꢁ13
29ꢁ9
10
11
12
13
15
16
17
18
19
20
21
22
23
24
25
26
27
28
630ꢁ30
1100ꢁ200
1400ꢁ100
1600ꢁ200
690ꢁ320
1600ꢁ300
970ꢁ50
4-F–
2-Me–
3-Me–
4-Me–
2-MeO–
3-MeO–
4-MeO–
2-Cl–
2-Et–
2-EtO–
2-CF3–
2-MeO–
1100ꢁ500
2300ꢁ1000
1600ꢁ300
770ꢁ290
3000ꢁ800
5200ꢁ500
430ꢁ190
350ꢁ40
7.0ꢁ3.8
43ꢁ18
54ꢁ23
17ꢁ7
H–
H–
H–
H–
13ꢁ7
540ꢁ190
650ꢁ370
1200ꢁ10 (2)
11ꢁ4
31ꢁ23
4-Cl–
4.5ꢁ2.2
1
0
aAll compounds were characterized by H NMR, MS and HPLC. The latter assured purity and allowed the diastereomeric ratio, if any, of the P1
side chain to be determined.
bDetermined using full-length MMP-1 by the method of Bickett (ref 22).
cDetermined using full-length MMP-13 by the method of Bickett (ref 22).
dValues are the meanꢁS.D. of 3 determination unless otherwise noted (n).
methoxy groups at the 2-, 3- and 4-positions of the
terminal P2 benzyl group. In contrast to the findings
with P1 substituents, most such substitutions led to
Having achieved our key goal of identifying compounds
with >100-fold selectivity over MMP-1, we next exam-
ined the selectivity of this series of compounds versus
other MMP’s. All of the compounds in Table 1 were
tested against MMP-2 and MMP-3; however, only data
for selected compounds, 3, 11, 21 and 28, are presented
in Table 2; these data are representative of what was
found for the entire series. The compounds were in
general very selective for MMP-13 versus MMP-3 with
virtually all analogues being 100-fold or more selective.
On the other hand, selectivity versus MMP-2 was
absent. In fact, most of these compounds were more
potent against MMP-2 than against MMP-13. In our
earlier report,14 we argued that the difference in poten-
cies of 3 and 29, a P2 ‘debenzyl’ analogue, indicated that
the P2 benzyl group is interacting with the S2 pocket of
MMP-13. Similarly, the MMP-2 potencies of 3 and 29
vary 11-fold suggesting that, as with MMP-13, the P2
benzyl group is interacting with the S2 pocket (or
another adjacent region) of MMP-2. To broaden the
profile of the series, selected compounds were also
screened against MMP-8 and MMP-12, two other deep
pocket MMPs. As with MMP-2, selectivity for MMP-13
inhibition over either MMP-8 or MMP-12 was not
observed (Table 2). Also, like MMP-2, the ꢀ10-fold
0
decreases in activity. However, within each substituent
set, the 2-substituted derivatives were the most potent
with the 2-methoxy analogue 21 being both more potent
and more selective than 3. The observed increase in
selectivity of 21 derives from its increased MMP-13
potency (3-fold) as MMP-1 potency remained the same.
This suggested that a 2-substituent may be interacting in
a favorable way with the S2 pocket of MMP-13 while
enjoying little additional positive interaction with the S2
pocket of MMP-1. In order to possibly capitalize on
this, we prepared additional 2-substituted derivatives,
24–27. However, none of these were more potent or
selective than 21.
Having defined improved substituents for both the P2
benzyl group and the P1 phenethyl group, we then pre-
0
pared 28 which combines these substituents—a 2-meth-
oxybenzyl group at P2 and a 4-chlorophenethyl group at
0
P1 —in a single molecule. As we had hoped, the sub-
stituents in this compound did display modest syner-
gism; excellent potency (MMP-13 IC50 4.5 nM) and
high selectivity versus MMP-1 (270-fold) was observed.

2334
L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2331–2336
Scheme 1.
Scheme 2.
shift in potency between the ‘de-benzyl‘ analogue 29 and
3 suggests that the inh0ibitors are interacting in the S2
pocket as well as the S1 pocket.
against other MMPs was not observed. However,
comparisons of MMP-2, -8, -12 and -13 potencies for
the ‘de-benzyl’ analogue 29 versus 3 (ꢀ10-fold differ-
ence in all cases) suggests that key interactions are
0
Through systematic variation of substituents we have
improved the potency of phosphinate-based MMP-13
inhibitors over those reported earlier. Furthermore,
high selectivity between MMP-13 and MMP-1 has been
demonstrated. With the exception of MMP-3, selectivity
occurring with both the P2 and P1 groups. Therefore,
further manipulation of these groups may lead to com-
pounds selective towards these other enzymes. Selectiv-
ity against these particular MMP’s may or may not
ultimately be sought depending on whether selectivity

L. A. Reiter et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2331–2336
2335
Table 2. MMP-13, MMP-3, MMP-2, MMP-8 and MMP-12 activity of selected phosphinic acids
MMP-13a
(IC50 nM)f
MMP-3b
(IC50 nM)f
MMP-2c
(IC50 nM)f
MMP-8d
(Ki nM)f
MMP-12e
(IC50 nM)f
3
11
21
28
29g
21ꢁ9
1600ꢁ500
900ꢁ100
800ꢁ200
1600ꢁ400
2700 (1)
10ꢁ4
0.77ꢁ0.44
0.56ꢁ0.10
2.1ꢁ0.1
2.4ꢁ0.4
12ꢁ2
8.2ꢁ2.5
1.7ꢁ0.7
4.0ꢁ0.8
5.0ꢁ4.2
93ꢁ12
8.3ꢁ2.8
7.0ꢁ3.8
4.5ꢁ2.2
210ꢁ80
2.6ꢁ0.6
6.6ꢁ2.4
6.6ꢁ0.5
110 (1)
Ratio of IC50’s 29 versus 3
10
—
11
1611
aDetermined using full-length MMP-13 by the method of Bickett (ref 22).
bDetermined using full-length MMP-3 by the method of Nagase (ref 23).
cDetermined using full-length MMP-2 by the method of Knight (ref 24).
dDetermined using the catalytic domain of MMP-8 by the method of Bickett (ref 22).
eDetermined using the catalytic domain of MMP-12 by the method of Bickett (ref 22).
fValues are the meanꢁSD of 3 determinations unless otherwise noted (n).
0
gCompound 29, reported in our earlier work (ref 14), is a 62/38 mixture of S/R P1 isomers.
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versus MMP-1 is sufficient to avoid MSS or on what
other MMPs are eventually identified as being key
contributors to this undesired side effect.
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