Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides

Article abstract of DOI:10.1021/jm960465t

Carboxyalkyl peptides containing a biphenylylethyl group at the P₁' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination

Full text of DOI:10.1021/jm960465t

1026
J . Med. Chem. 1997, 40, 1026-1040
In h ibition of Str om elysin -1 (MMP -3) by P 1′-Bip h en ylyleth yl Ca r boxya lk yl
Dip ep tid es
Craig K. Esser,* Robert L. Bugianesi, Charles G. Caldwell, Kevin T. Chapman, Philippe L. Durette,
Narindar N. Girotra, Ihor E. Kopka, Thomas J . Lanza, Dorothy A. Levorse, Malcolm MacCoss, Karen A. Owens,
Mitree M. Ponpipom, J oseph P. Simeone, Richard K. Harrison,† Lisa Niedzwiecki,† J oseph W. Becker,∇
Alice I. Marcy,∇ Melinda G. Axel,∇ Amy J . Christen,‡ J oseph McDonnell,‡ Vernon L. Moore,‡ J ulie M. Olszewski,‡
Cheryl Saphos,‡ Denise M. Visco,‡ Frank Shen,| Adria Colletti,§ Philip A. Krieter,§ and William K. Hagmann
Departments of Medicinal Chemistry, Enzymology, Pharmacology, Biophysical Chemistry, Biometrics, and Drug Metabolism,
Merck Research Laboratories, P.O. Box 2000, Rahway, New J ersey 07065-0900
Received J une 28, 1996X
Carboxyalkyl peptides containing a biphenylylethyl group at the P1′ position were found to be
potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50
nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at
P1′, a tert-butyl group at P2′, and a methyl group at P3′ produced orally bioavailable inhibitors
as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse
pleural cavity. The X-ray structure of a complex of a P1′-biphenyl inhibitor and the catalytic
domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues
at P1′ proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4′-
fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentanedioic acid 1-(R(S)-tert-butylglycine methy-
lamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg
po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and
IL-1â injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-
induced arthritis) models of cartilage destruction but showed activity only in the MMP-3
injection model (ED50 ) 6 mg/kg iv).
In tr od u ction
biphenylylethyl-containing carboxylate inhibitors of
MMP-3 with enhanced enzyme potency and oral activity
and their evaluation in animal models of cartilage
degradation.
The matrix metalloproteinases are a family of zinc-
containing, calcium-dependent endopeptidases that are
capable of hydrolyzing the extracellular matrix of con-
nective tissues and basement membranes.1-7 Although
their roles in the normal and pathological turnover of
these tissues are not completely understood,8 the el-
evated levels of these enzymes, particularly stromel-
ysin-1 (MMP-3) and collagenase-1 (MMP-1), found in
the synovium and cartilage of osteoarthritis and rheu-
matoid arthritis patients have suggested a role for these
enzymes in these disease processes.9-15 Gelatinase A
(MMP-2) is particularly proficient at degrading base-
ment membranes and is thought to play a role in tumor
metastasis.16 Therefore, there has been substantial
interest in developing MMP inhibitors for a variety of
therapeutic indications.17-27
Ch em istr y
A synthesis of the carboxyalkyl peptide inhibitors is
shown in Scheme 1. Starting from commercially avail-
able (4-iodophenyl)butyric acid (1), the mixed anhydride,
obtained with trimethylacetyl chloride, was reacted with
the lithium anion of (S)-4-benzyl-2-oxazolidinone to
afford 2. Following procedures described by Evans,38
enantioselective Michael addition of the titanium eno-
late of the chiral oxazolidinone 2 to tert-butyl acrylate
provided 3 having the carboxylate functionality with a
suitable protecting group. Hydrolysis of the chiral
auxiliary with lithium hydroxide and hydrogen peroxide
yielded the carboxylic acid 4. Alkylation of the lithium
dianion of 4 with iodobutane produced 5 as a mixture
of diastereomers (∼3:1) in favor of the desired 4S,2R
isomer. Separation of the desired diastereomer of acid
5 was not efficient, so the mixture was converted to its
(trimethylsilyl)ethyl ester using (trimethylsilyl)ethanol
and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hy-
drochloride (EDC) and then purified by silica gel chro-
matography to separate the diester 6 as the pure 4S,2R
diastereomer. This diester contains the P1 and P1′
portion of our inhibitors along with the iodo functional-
ity at the 4-position of the phenyl ring and served as a
key intermediate in the modification of the P1′ portion
of our inhibitors.
We have focused on the discovery of potent, selective
inhibitors of MMP-3.28-30 By structure-activity rela-
tionship (SAR) studies and structural analysis, we and
others have discovered that the S1′ binding pockets of
MMP-3 and MMP-1 differ considerably. MMP-3 has a
deep, hydrophobic S1′ hole that extends through the
enzyme, whereas the S1′ pocket of MMP-1 is much
shallower.29,31-37 We also discovered that substituted
carboxyalkyl dipeptides have significant oral activity in
an animal model of MMP-3-induced macromolecular
degradation.30 Herein, we report the discovery of P1′-
* To whom correspondence and reprint requests should be sent.
† Department of Enzymology.
‡ Department of Pharmacology.
The 4-iodophenethyl intermediate was modified by
various carbon-carbon bond-forming methodologies
reported in the literature. Palladium-catalyzed boronic
acid coupling methodology described by Suzuki and
§ Department of Drug Metabolism.
∇ Department of Biophysical Chemistry.
| Department of Biometrics.
X Abstract published in Advance ACS Abstracts, February 15, 1997.
S0022-2623(96)00465-7 CCC: $14.00 © 1997 American Chemical Society

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1027
Sch em e 1
Sch em e 2
others39-43 introduced the biphenyl moiety into our
inhibitors as illustrated in Scheme 1. Reacting inter-
mediate 6 with substituted arylboronic acids in the
presence of palladium(0) and a base generated biphenyls
of the general structure 7 in satisfactory yields (50-
80%). The TMS-ethyl ester in 7 was removed with
tetrabutylammonium fluoride, and the resulting acid 8
was coupled with (S)-tert-butylglycine methylamide
hydrochloride in the presence of N-methylmorpholine
(NMM), N-hydroxybenzotriazole (HOBt), and EDC.
Cleavage of the tert-butyl ester with trifluoroacetic acid
(TFA) afforded the carboxyalkyl peptides 58 as listed
in Tables 1 and 2.
tion using Suzuki conditions was also performed later
in the synthetic scheme on the larger dipeptide 11
followed by deprotection with TFA to yield the desired
carboxylate inhibitors. However, the Suzuki coupling
proceeded in better yield on the diester 6 compared to
the dipeptide 11 (75% vs 55% yields).
A less convergent but more directed method for
modifying the P1′ portion of our inhibitors is shown in
Scheme 3. The desired P1′ moiety (13, for example) was
acylated with succinic anhydride under Friedel-Crafts
conditions to yield 4-keto-4-biphenylylbutyric acid 14.
The ketone was then reduced with hydrogen in the
presence of Pearlman’s catalyst to afford the substituted
Alternatively, as shown in Scheme 2, biphenyl forma-

1028 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
Sch em e 3
Sch em e 4
butyric acid 15 that was further modified in the same
manner as shown in Scheme 1.
with N-Boc-2-iodoindole in the presence of tris(diben-
zylideneacetone)dipalladium(0), triphenylarsene, and
zinc chloride (1.0 M in THF) to afford the desired
2-substituted indole 23.
Scheme 5 demonstrates the use of an aliphatic iodo
intermediate in carbon-carbon bond formation using
zinc/copper couple and palladium(II). Starting from the
diester 18 derived from L-homoserine, the hydroxy
functionality was converted to iodide 19 and then
reacted with an aryl halide in the presence of zinc/
copper couple and palladium(II)54 to afford the desired
biphenyl compound 20.
Other examples of carbon-carbon bond-forming meth-
ods used to modify the P1′ position of the diester
intermediate 6 are illustrated in Scheme 4. Reaction
of 6 with phenylacetylene in the presence of palladium-
(II) and copper(I)44-46 allowed the introduction of a two-
carbon spacer between the two phenyl groups of the
biphenyl inhibitors, as in 17. In the presence of palla-
dium(II) and a base, cycloheptene reacted with the aryl
halide 6 to give the cycloheptenyl intermediate 21.47 The
last equation in Scheme 4 illustrates the application of
Stille-type carbon-carbon bond-forming conditions.48-52
The iodo diester 6 was converted to its trimethyltin
derivative 22 using hexamethylditin in the presence of
palladium(0) and lithium chloride53 and then reacted
Biologica l Eva lu a tion
Compounds were evaluated as inhibitors of recombi-
nant human fibroblast MMP-3 and MMP-2 by methods

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1029
Sch em e 5
Ta ble 1. Inhibition of Human MMP-3 and MMP-2 by Carboxyalkyl Peptides
Ki, nMa
compd no.c
R1 (P1′)
R3 (P3′)
MMP-3
MMP-2
PLCAVb % inh @ 60 mpk, po
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
4-F
4-F
4-F
3-NH2
4-CF3
4-pyridyl
Ph
CH3
Ph
2.0 ( 0.2
4 ( 1
25 ( 2
72 ( 10
61 ( 4
93 ( 2
4 ( 6
78 ( 7
17 ( 9
84 ( 3
0
62 ( 3
22 ( 12
61 ( 10
90 ( 2
72 ( 6
87 ( 2
80 ( 17
84 ( 5
37 ( 11
21 ( 11
NDe
150 ( 10
17 ( 1
10 ( 1
27 ( 3
37 ( 3
280 ( 30
39 ( 4
3600 ( 500
25 ( 2
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3-CF3
4-Cl
1100 ( 100
13 ( 2
3,5-diCl
2,4-diCl
4-Ph
3-Cl,4-F
4-Br
3-F
2-F
4-CN
4-SCH3
4-SO2CH3
4-SOCH3
2,6-diCH3
4-CHO
4-(2-imidazolyl)
4-COOH
4-(5-tetrazolyl)
4-OCH3
2-SO2NH-t-C4H9
2-SO2NH2
4-CO2CH3
4-CONH2
4-CONHCH3
4-CON(CH3)2
1500 ( 100
12 ( 1
1600 ( 100
100 ( 10
84 ( 9
39 ( 4
19 ( 2
80 ( 8
11 ( 1
17 ( 2
21 ( 1
48 ( 5
11 ( 1
61 ( 7
9 ( 1
14 ( 2
5 ( 1
13 ( 1
21 ( 1
19 ( 2
52 ( 5
30 ( 2
1200 ( 100
15 ( 1
1300 ( 100
16 ( 1
7 ( 12
17 ( 7
13 ( 5
ND
90 ( 3
ND
25 ( 2
94 ( 8
690 ( 30
350 ( 40
13 ( 1
580 ( 50
1800 ( 100
21 ( 2
10% @10d
9400 ( 1000
31 ( 3
17% @10d
0% @ 10
32 ( 2
ND
ND
24 ( 1
38 ( 4
3 ( 4
49 ( 4
34 ( 4
7( 9
40 ( 3
50 ( 4
40 ( 20
a
All compounds listed were evaluated as inhibitors of human stromelysin-1 (MMP-3) and gelatinase A (MMP-2) by the methods previously
described ((SE).28 Percent inhibition ((SE) of MMP-3-mediated degradation of radiolabeled transferrin in the mouse pleural cavity
b
(PLCAV) after 60 mpk oral dose of inhibitor. c Compounds were purified to homogeneity and satisfactory elemental analyses or exact
d
masses from high-resolution mass spectrometry were obtained ((0.4% of calculated values). Indicates percent inhibition at 10 µM.
e Not determined.
previously described,28 and the results are reported in
Tables 1 and 2. It should be noted that, in addition to
Ki determinations against MMP-3 and MMP-2, several
compounds (24-26, 33, 65, 66) were evaluated as
inhibitors of recombinant human fibroblast MMP-1.
Each of these compounds proved to be an extremely
weak inhibitor of MMP-1 (Ki > 10 µM). Generally,
structures containing the biphenyl moiety at P1′ were
poor MMP-1 inhibitors, so evaluation of these com-
pounds against MMP-1 was not continued.
The methodology used to evaluate inhibition of MMP-
3-mediated degradation of a macromolecular substrate
by these compounds in vivo is similar to that described
for the rat pleural cavity.30,55 The inhibition of the
degradation of radiolabeled transferrin by human MMP-3
in the mouse pleural cavity was measured. Mice were
predosed orally with compound, after which a solution
of [3H]transferrin was injected intrapleurally. This was
followed immediately by the injection of trypsin-
activated human MMP-3. After 30 min, the animals

1030 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
Ta ble 2. Inhibition of Human MMP-3 and MMP-2 by Carboxyalkyl Peptides
Ki, nMa
compd no.c
R1 (P1′)
n-C3H7
CH2Ph
n-C3H7
R3 (P3′)
MMP-3
11 ( 1
1400 ( 200
140 ( 20
1500 ( 100
15 ( 1
2000 ( 200
65 ( 6
49 ( 5
12 ( 1
200 ( 10
120 ( 10
95 ( 10
3800 ( 200
1400 ( 100
74 ( 6
MMP-2
PLCAVb % inh @ 60 mpk, po
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
4-pyridyl
Ph
22 ( 2
89 ( 4
15 ( 9
NDe
16% @ 10d
47 ( 3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3-Ph
2400 ( 200
21 ( 1
6900 ( 700
760 ( 80
380 ( 30
49 ( 4
3000 ( 300
800 ( 100
1900 ( 200
1500 ( 100
810 ( 80
210 ( 20
ND
3-thienyl
1-naphthyl
CHdCHPh
CH2CH2Ph
2-indolyl
OPh
2-naphthyl
4-cycloheptyl
H
70 ( 9
19 ( 4
ND
ND
34 ( 20
39 ( 11
65 ( 8
48 ( 10
ND
2-THF
OCH2Ph
ND
ND
a
All compounds listed were evaluated as inhibitors of human stromelysin-1 (MMP-3) and gelatinase A (MMP-2) by the methods previously
described ((SE).28 Percent inhibition ((SE) of MMP-3-mediated degradation of radiolabeled transferrin in the mouse pleural cavity
b
(PLCAV) after 60 mpk oral dose of inhibitor. c Compounds were purified to homogeneity and satisfactory elemental analyses or exact
d
masses from high-resolution mass spectrometry were obtained ((0.4% of calculated values). Indicates percent inhibition at 10 µM.
e Not determined.
Ta ble 3. Pharmacokinetic Properties of 26a
mouseb
ratb
rabbitc
plasma t1/2 (h)
2.7
4.4
0.9
clearance (mL/min/kg)
Vdss (L/kg)
1.1
0.20
78%
1.3
0.32
60%
7.6
0.20
oral bioavailabilityd
NDe
a
Compound levels were determined by RP-HPLC relative to a
standard curve (see the Experimental Section). 20 mg/kg iv. c 12
b
mg/kg iv. 20 mg/kg po. e ND ) not determined.
d
and inflammatory arthritis. Inhibitor 26 was admin-
istered to rabbits intravenously 15 min prior to the
injection of activated human recombinant fibroblast
MMP-3 into the stifle joint according to the published
procedures.56,57 The contralateral joint served as the
control knee. In the IL-1â injection model,58 compound
26 was administered (12 mg/kg iv) six times every 2 h
from the time of the IL-1â injection. In both models,
synovial lavage fluid was analyzed for released pro-
teoglycan by an alcian blue assay.56,57
In the rat adjuvant-induced arthritis (AIA) model,60,61
inhibitor 26 was administered at 50 mg/kg po b.i.d.
throughout the 21 days of the experiment. Indomethacin
(1 mg/kg po b.i.d.) served as the positive control. Body
weights, foot volumes, and thymus and spleen weights
were used to measure efficacy. In the mouse collagen-
induced arthritis (CIA) model,62 26 was also adminis-
tered at 50 mg/kg po b.i.d. throughout the course of the
experiment. Indomethacin (1 mg/kg po s.i.d.), dexam-
ethasone (1 mg/kg po s.i.d.), and cyclophosphamide (5
mg/kg po s.i.d.) served as positive controls. Severity
scores and percent incidence were used to evaluate
efficacy. Levels of total drug concentration in plasma
were determined at the time of euthanasia.
F igu r e 1. Structure of an N-carboxyalkyl inhibitor of MMP-
3-mediated cartilage degradation in the rabbit.56-59
were sacrificed, and the pleural cavity was lavaged.
After centrifugation, the amount of trichloroacetic acid-
soluble and -precipitable counts were measured and
compared to controls to give a percent inhibition of
MMP-3 degradation of the transferrin. Results are
reported in Tables 1 and 2 under the heading PLCAV.
Previously, we had shown that an N-carboxyalkyl
dipeptide MMP inhibitor (L-696,418) was capable of
inhibiting proteoglycan release from cartilage in a rabbit
knee joint treated intraarticularly with human MMP-3
(Figure 1).56,57 That same inhibitor was also efficacious
(∼50%) in limiting the amount of proteoglycan release
from a rabbit knee joint treated intraarticularly with
human IL-1â.58,59 These two injection models represent
acute cartilage degradation initiated by exogenous
enzyme or cytokine. L-696,418 did not have sufficient
potency, selectivity, or duration of action to be consid-
ered for more chronic models of cartilage degradation.
On the basis of a more satisfactory pharmacokinetic
profile (Table 3), compound 26 (L-758,354) was chosen
to be evaluated in several models of cartilage destruction

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1031
F igu r e 3. Comparison of P3′-aryl vs -methyl substitution on
MMP-3 activity of P1′-4-n-propylphenethyl inhibitors.
F igu r e 2. Comparison of C- vs N-carboxyalkyl peptide
inhibitors of MMP-3 in the mouse pleural cavity assay.30
Resu lts a n d Discu ssion
The starting point for this study was the carboxyalkyl
dipeptide inhibitor 2(R)-[2-(4-n-propylphenyl)ethyl]-
4(S)-methylpentanedioic acid 1-((S)-leucine phenyla-
mide) amide (71), MMP-3 Ki ) 68 nM (Figure 2).30 More
importantly, this compound showed a significant in-
crease in oral activity compared to its N-carboxyalkyl
analog 72 as measured in the pleural cavity assay.
Figure 2 reports these results as the dose in mg/kg
required to inhibit 50% of the human MMP-3 degrada-
tion of transferrin in the mouse pleural cavity compared
to the control group (ED50). This result, combined with
the increased potency and selectivity for MMP-3 inhibi-
tion found for the 4-propylphenethyl group at the P1′
position,64 suggested that additional gains in potency
and/or oral activity might be achievable by further
modification of the P1′-phenethyl side chain in this class
of carboxyalkyl peptides.
In addition to modifying the P1′ position, we sought
to optimize other subsites of this class to achieve the
desired combination of potency, selectivity, and phar-
macokinetic properties for an MMP-3 inhibitor. An
increase in potency has been observed with cyclic
imido30,65-70 and lipophilic66,68 groups at the P1 position.
We found that the n-butyl group provided for enhanced
potency versus MMP-3 as well as improved oral activ-
ity,69 and thus, it became the substituent of choice for
the P1 position in this study. The tert-butyl group at
P2′ had been found to improve plasma stability and oral
absorption in a series of MMP-1 inhibitors.23,25,70 The
tert-butyl group sterically hinders hydrolysis of the P2′-
P3′ amide bond and was also held constant in this study.
Inhibition of MMP-3 had been shown to be enhanced
by a P3′-aryl group.28,71,72 However, metabolic cleavage
of the P2′-P3′ amide bond would produce an aromatic
amine as a potentially toxic byproduct (e.g., aniline).
Many groups have incorporated a methyl group at P3′
in their inhibitor designs,65,66,68,70,73,74 but this substitu-
tion led to a decrease in activity against MMP-3 in our
earlier designs.75 For example, compound 54 has a Ki
) 11 nM against MMP-3, but when the 4-pyridyl group
is replaced by methyl, the MMP-3 activity diminishes
to a Ki of 140 nM for compound 56 (Figure 3).
F igu r e 4. Effect of 4′-fluoro substitution in the P1′-bipheny-
lylethyl group on MMP-3 activity.
to both block this metabolism and possibly enhance
potency, P1′-4-biphenyl analogs were prepared. Our
major focus was on substituted biphenyl derivatives, as
the unsubstituted parent in a related series was a
somewhat less active MMP-3 inhibitor (Figure 4). Ad-
ditionally, it was assumed that substituents, particu-
larly electron-withdrawing ones, would decrease poten-
tial oxidative metabolism of the biaryl system, and this
protection may also extend to the connecting benzylic
position. Thus, compounds 24-26 provide the basis for
comparison with the inhibitors described herein.
The combination of biphenyl substitution at P1′ with
methyl substitution at P3′ enhanced the oral activity of
these inhibitors as measured in the mouse pleural cavity
assay. Compound 26, which combines a (4-fluorobiphe-
nylyl)ethyl side chain at P1′ and a methyl group at P3′,
shows almost a 3-fold improvement in oral activity in
the pleural cavity assay compared to the corresponding
analog containing a phenyl group at P3′ (25).
As seen in Table 1, halogen substitution on the second
(or outer) phenyl ring is generally well tolerated by both
MMP-3 and MMP-2 with a few exceptions. The poor
activity of the 3′,5′-dichloro analog 31 suggests a lack
of steric symmetry in the P1′ site, whereas a 3′-chloro
substituent in 34 is tolerated. Fluorine is acceptable
in any position (26, 36, 37). The 4′-trifluoromethyl
group in 28 is active but not at the 3′-position in 29.
Possibly, the high dipolar nature of this substituent
imparts unfavorable interactions in this region.
In compounds related to 54 where the P2′-P3′ amide
bond was stabilized by a P2′-tert-butyl group, it was
found that the n-propyl group was metabolized by
isolated rat liver microsomes and also in vivo in rats.76
Several metabolites were isolated and determined to be
oxidized derivatives of the n-propyl group. In an effort
The 4′-position will tolerate a wide variety of substi-
tution. In particular, the terphenyl analog 33 (three
phenyl rings in a linear arrangement) illustrates the
nature of the S1′ pocket of these two enzymes. Struc-
tural studies have shown that the S1′ site in MMP-3 is

1032 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
P2′, 0.95 Å. As a consequence of this movement, there
is a change in the hydrogen-bonding pattern (Support-
ing Information, Table 2). The shift in binding geometry
correlates with changing the atom at the P1′ N position
from a nitrogen in the L-702,842 complex to a carbon
in the L-764,004 complex. A nitrogen atom in this
position can form hydrogen bonds with Ala165O and
Glu202Oꢀ2, while a carbon atom must move away from
these polar atoms. A consequence of this shift is the
strengthening of the polar interaction between the P2′
N and Pro221O atoms in the complex with L-764,004:
these atoms are 3.36 Å apart in this complex as
compared with a separation of 3.82 Å in the complex
with L-702,842. Another significant difference is the
conformation of the P1′ side chain: in the complex with
L-702,842, the P1′-phenylethyl group adopts a ttg-
conformation, while the corresponding arrangement in
the complex with L-764,004 is g+tg- (Supporting Infor-
mation, Table 3). Despite this difference, the compa-
rable aromatic rings occupy roughly the same volume
in the two complexes. The interactions of the first ring
of the biphenyl moiety of L-764,004 with specific protein
groups are similar to those seen in the complex with
L-702,842: the ring is parallel with the side chain of
His201, the carbonyl oxygen atoms of Leu218 and
Tyr220 point toward the ring, and the side chain of
Tyr223 is directed toward the center of the ring. The
dihedral angle between the two rings is 49°, and the
second ring interacts with both sides of the S1′ subsite,
making van der Waals contacts with Ala217 and Leu218
on one side and with Leu191, Tyr223, His224, and
Leu226 members of the flexible loops on the outside of
the site.
F igu r e 5. Comparison of P3′-aryl vs -methyl substitution of
P1′-(4′-fluorobiphenylyl)ethyl inhibitors on MMP-3 and mouse
pleural cavity activities.
a hole that extends completely through the enzyme.33,34
Modeling studies with 33 show that the third phenyl
ring extends out the other side of this hole. It is
intriguing that the enzymes are able to accommodate
such a large, rigid structure into their deep, albeit tight
fitting, S1′ sites.
Compound 26 is highly protein bound (97.6% in rabbit
plasma and 99.8% in the presence of human serum
albumin).76 As such, its bioavailability might be ex-
pected to be reduced, and thus, a more polar inhibitor
was sought. It was found that polar or charged sub-
stituents at the P2′ or P3′ positions had little effect on
protein binding or measured log P values and that the
biphenylylethyl group was the dominating feature in
determining protein binding.
X-ray crystallographic studies of MMP-3 had shown
that the S1′ pocket contains several carbonyl oxygen
atoms that could serve as hydrogen bond acceptors,34
but a more definitive structure of a P1′-biphenyl inhibi-
tor complexed to the catalytic site of MMP-3 was needed
(Figure 6 and Supporting Information, Table 1). The
overall structure of the SLN-247 (residues 83-247)
complex with L-764,004 is quite similar to that of the
SLN-255 (residues 83-255) complex with L-702,842
when the structures are aligned on all R-carbon atoms,
the average deviation being only 0.38 Å (rms 0.48 Å),
despite the differences in construct, inhibitor, and
crystal lattice (Figure 7).33,34 The only R-carbon atoms
that are separated by more than 1.0 Å in the aligned
structures are at residues 190, 225, and 228, positions
in apparently mobile regions as judged by their higher
than normal temperature factors. The alkyl chain on
the unprimed side of the inhibitor lies in an extension
of the binding site groove, and the benzolactam lies in
a shallow depression in the enzyme surface defined by
Ala167 and the side chains of Tyr155, His166, and
Tyr168. In addition, this group makes a lattice-specific
stacking interaction with the side chain of Phe210 from
another residue. The two rings are parallel to each
other and approximately 3.8 Å apart.
As a result of modeling studies with this X-ray
structure, polar (40, 41, 43, 48-53) or charged (44-
46) substituents on the biphenyl group were introduced
in search of potential hydrogen-bonding interactions
within the S1′ pocket that would reduce overall protein
binding and possibly enhance potency. Unfortunately,
these structures were less potent than the halogenated
analogs. In addition, oral activity in the pleural cavity
model was significantly reduced. For example, com-
pound 51 had reasonably good activity against MMP-3
(Ki ) 24 nM) but was inactive in the pleural cavity assay
(3% inhibition at 60 mg/kg po).
Efforts to uncover a different scaffold that bound into
the S1′ pocket of MMP-3 more efficiently than the P1′-
4-biphenylylethyl side chain of 26 were also not suc-
cessful. For example, 57 (3-biphenylylethyl group at P1′)
and 59 (1-naphthylphenethyl group at P1′) lost 2 orders
of magnitude in potency against MMP-3, further sup-
porting the observation that the S1′ pocket is deep but
relatively narrow. Some of the compounds in Table 2
showed similar activity to 26 against MMP-3 in vitro
but did not exhibit comparable oral activity in the
pleural cavity assay. For example, compound 62 has a
Ki ) 12 nM but has low activity in the pleural cavity
assay (34% inhibition at 60 mg/kg). Overall, a number
of different hydrophobic scaffolds were explored that led
to comparable potencies against MMP-3, but none
exhibited any advantages over 26.
In contrast with this general similarity, there are
several significant differences in how the inhibitors
interact with the enzyme. First, when the structures
are aligned on the R-carbon atoms of the enzyme, there
is a shift of the bound inhibitor toward the primed side
of the binding site: the P1′ R-carbons are 0.85 Å apart;
Figure 8 summarizes the structural changes to com-
pound 72 that were investigated and the resulting
effects these changes had on the potency and pharma-
cokinetic properties of these inhibitors. Alkyl groups

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1033
F igu r e 6. S1′ site. The inhibitor L-764,004 (see Figure 7) in gray carbon atoms is shown together with all groups of the catalytic
core that surround the P1′-(4′-fluoro-4-biphenylyl)ethyl group (light green carbon atoms). The catalytic zinc is represented as a
violet sphere, and the zinc ligand His201 is shown at the rear of the figure.
F igu r e 8. Summary of the structure-activity relationship for
P1′-biphenylylethyl carboxylate inhibitors of MMP-3.
Although inhibitor 26 was a potent inhibitor of
MMP-3 and MMP-2 with excellent selectivity against
MMP-1, with reasonable pharmacokinetic properties in
rodents (Table 3), and with good oral activity in the
mouse MMP-3 pleural cavity assay, its poor activity in
subsequent models of cartilage destruction raised sig-
nificant questions regarding both the physical properties
of an efficacious inhibitor as well as the purported role
of MMP-3 in the articular cartilage degradation ob-
served in these models. Initially, inhibitor 26 was
evaluated in a model of MMP-3 injection into the rabbit
knee synovial cavity. The compound was administered
intravenously to circumvent any oral absorption barri-
ers in the rabbit as well as the poorer pharmacokinetic
profile seen in this species (Table 3) and was found to
have an ED50 of 6 mg/kg iv in this assay, thus providing
evidence that 26 was able to inhibit MMP-3 activity in
the knee joint.
With respect to articular cartilage degradation, the
MMP-3 injection model described above utilizes the
appropriate substrate (endogenous cartilage) in a tar-
geted tissue (the joint space) but employs exogenous
enzyme instilled into the synovial cavity, not enzyme
generated and released within the cartilage itself. The
injection of the cytokine IL-1â into the synovial cavity
has been shown to cause the concomitant synthesis of
F igu r e 7. Comparison of the hydrogen-bonding interactions
of two inhibitors complexed with the catalytic domain of
human MMP-3 as determined by X-ray crystallography.
at P1 and the biphenylylethyl group at P1′ increased the
intrinsic activity against MMP-3 and improved activity
in the in vivo pleural cavity model. Changing the
secondary amine to a methylene had a positive effect
on oral bioavailability, as did incorporating a tert-butyl
group at P2′ and a methyl group at P3′. The combination
of all these changes into compound 26 produced a potent
inhibitor of MMP-3 that was well absorbed and showed
good duration of action.

1034 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
MMPs and cartilage destruction.58,77 Thus, cartilage
proteoglycan release in the IL-1â injection model rep-
resents the effects of endogenous enzyme on endogenous
substrate. Inhibitor 26 is a potent inhibitor of rabbit
MMP-3 (Ki ) 3.2 nM) and was administered intrave-
nously at 2 times the ED50 obtained in the MMP-3
injection model (12 mg/kg iv) every 2 h to insure
adequate plasma concentrations. This compound had
no effect on proteoglycan release in this assay.
identified as having several inhibitors.86-88 Compound
26 does not inhibit the release of TNF-R from monocytes
nor the processing of proTNF-R,89 and thus, this com-
pound would not be expected to have an effect on the
involvement of TNF-R in the models described above.
As seen in Table 3, 26 had sufficient oral bioavail-
ability, plasma half-life, and clearance rates in rodents
to provide adequate coverage in these species following
oral dosing. Indeed, the plasma levels obtained at the
end of the AIA and CIA models suggest that more than
adequate plasma levels of 26 were available to inhibit
MMP-3. The high protein binding of 26 may have, in
part, contributed to its lack of activity in the rat AIA
and mouse CIA models, but even 99.9% protein binding
would still have provided 50-65 nM concentrations of
free drug available to inhibit active enzyme. The
activity of 26 in the mouse pleural cavity and rabbit
knee IL-1â injection models suggests that 26 is still able
to inhibit MMP-3-mediated processes in extravascular
spaces such as the pleural cavity and knee joint. The
pharmacokinetic parameters and protein binding of 26
are comparable to several efficacious nonsteroidal an-
tiinflammatory drugs (NSAIDs), such as indomethacin,
naproxen, and ibuprofen.90 However, the high plasma
levels of 26 do not guarantee sufficient cartilage pen-
etration to reach the site of MMP-3 proteinase action.
Despite a low molecular weight (MW ) 512), the
negative charge of 26 may limit its facile transport into
the highly sulfated environment of articular cartilage
resulting, perhaps, in relatively low cartilage concentra-
tions of inhibitor. On the other hand, at least at a very
high concentration (100 µM), 26 was able to prevent IL-
1â- or retinoic acid-induced degradation of bovine
articular cartilage.91 Neutral MMP inhibitors have been
demonstrated to be active in preventing proteoglycan
loss, albeit in different species and models of cartilage
destruction: L-696,418, an N-carboxyalkyl dipeptide
MMP-3 inhibitor, was efficacious (50%) in preventing
the loss of proteoglycan in the IL-1â injection model in
the rabbit,59 and CGS 27023A, a nonpeptide hydroxamic
acid, prevented the loss of cartilage proteoglycan in a
partial meniscectomy model in rabbits.92 In summary,
the lack of activity of 26 in the rat AIA and mouse CIA
models may be attributed to one or more contributing
factors: noninvolvement of MMP-3 and MMP-2 in these
models, the lack of inhibition of MMP-1 and/or TNF-R
processing, high protein binding, poor cartilage penetra-
tion and insufficient potency, and free drug levels.
Whereas the two rabbit models of MMP-mediated
joint destruction described above represent models of
acute cartilage degradation, the articular cartilage
degradation seen in arthritic diseases occurs over many
years.78,79 Inhibitor 26 was evaluated in two rodent
models of more chronic joint destruction: rat adjuvant-
induced arthritis (AIA) and mouse collagen-induced
arthritis (CIA).60-62 It had a more favorable pharma-
cokinetic profile in rodents than in the rabbit and one
which supported long term oral administration (Table
3). In the rat AIA model, compound 26 (50 mg/kg po
b.i.d.) was administered from day 0 and continued
throughout the 21 days of the experiment. Total drug
plasma levels of 26 were determined 5 h after the last
dose on day 21 and found to be 49.8 ( 13.5 µM.
Compound 26 failed to demonstrate efficacy in any of
the parameters examined in rat AIA (body weight, paw
swelling, and thymus and spleen weights). In the
mouse CIA model, 26 was administered at the same
dose (50 mg/kg po b.i.d.) throughout the 29 days of the
experiment. Total drug plasma levels of 26 were
obtained at the end of the experiment (65.0 ( 44.5 µM).
Again, 26 failed to have a significant effect on the degree
of incidence and severity of joint destruction evaluated
in this model. A similar result in the CIA model was
found with mice of the appropriate background lacking
the MMP-3 gene (MMP-3 knock-out mice).80
Several explanations for the lack of efficacy of a
compound such as 26 in protecting cartilage from MMP-
mediated destruction may be offered. Compound 26
was a potent inhibitor of mouse MMP-3 (mouse MMP-3
Ki ) 23 nM), so species differences can be discounted.
MMP-3 (and perhaps MMP-2) may not be involved in
the models of cartilage degradation in which 26 was
evaluated. The mouse CIA model was chosen because
immunolocalization studies identified MMP-3 and MMP-
3-generated cartilage cleavage products (VDIPEN) in
areas of cartilage degradation in this model.81,82 How-
ever, the lack of efficacy seen with 26 and with the
MMP-3 knock-out mice in the CIA model suggests that
this enzyme is not important in this model. A potent
MMP-1 and MMP-2 inhibitor was effective in limiting
cartilage and bone destruction in the rat AIA model,
suggesting that at least one of these two enzymes is
operative in this model.83 Rat enzyme data are not
available for 26, so the role of MMP-2 and MMP-3 in
rat AIA cannot be convincingly assessed with this
compound. Also, an inhibitor of MMP-1 and MMP-3
was effective in vivo in limiting collagen and proteogly-
can loss from implanted femoral head cartilage in rats,
suggesting that the MMP-1 component may be essential
in protecting cartilage from degradation.84
A definitive role for MMP involvement in cartilage
degradation still remains elusive. Progress is being
made to identify specific cleavage products by these
metalloproteinases, and these are being correlated with
both animal model results and clinical readouts. New
metalloproteinases including membrane-type MMPs93,94
and cytokine processing86-88 with overlapping substrate
specificities and inhibition profiles will make the process
even more arduous. In order to help provide a more
definitive role for MMP involvement in articular carti-
lage degradation, MMP inhibitors will not only need to
be potent, perhaps selective, and orally bioavailable with
appropriate pharmacokinetic parameters but may also
require physical properties necessary for effective pen-
etration of cartilage to inhibit their respective targets
at the site of action.
Tumor necrosis factor-R (TNF-R) is a cytokine that
has been shown to be involved in many aspects of
inflammation and tissue degradation.85 A metallopro-
teinase that cleaves the precursor of TNF-R has been

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1035
ethyl acetate/hexane to afford 4.18 g (75%) of the title
compound as a faint yellow oil: 1H-NMR (400 MHz, CDCl3) δ
7.58 (d, 2H), 6.91 (d, 2H), 2.59 (m, 2H), 2.40 (m, 1H), 2.25 (m,
2H), 2.02-1.81 (m, 3H), 1.73 (m, 1H), 1.41 (s, 9H).
Exp er im en ta l Section
Ch em istr y. Gen er a l P r oced u r es. Proton NMR spectra
were recorded on either a Varian XL-400 or Unity-500 NMR
spectrometer. Chemical shifts are given on the δ scale.
Spectra were measured at 20 °C for solutions in chloroform-d
or methanol-d4. Infrared spectra were obtained as thin films
on sodium plates on Perkin-Elmer 295 or 1310 spectrometers.
Mass spectra were determined on a LKB 9000 mass spectrom-
eter. Analytical results for compounds followed by elemental
symbols were +0.4% of calculated values unless otherwise
indicated and were determined by Robertson-Microlit Labo-
ratories, Inc., Madison, NJ . Thin layer chromatography was
performed on precoated silica gel GHLF254 plates (Analtech)
or Kieselgel-60, and visualization was effected with UV light,
iodine, or ceric sulfate (1%)-sulfuric acid (10% spray). Pre-
parative flash and medium pressure column chromatographies
were performed on silica gel 60 (EM Science; 40-63 µm).
4(S)-Ben zyl-3-[4-(4-iod op h en yl)b u t yr yl]oxa zolid in -2-
on e (2). To a solution of (p-iodophenyl)butyric acid (5.0 g, 17.2
mmol) in 50 mL of dry THF at 0 °C was added triethylamine
(3.12 mL, 22.4 mmol) followed by trimethylacetyl chloride (2.34
mL, 19.0 mmol), and the mixture was stirred for 1 h. In a
separate flask, n-butyllithium (8.34 mL, 20.9 mmol, 2.5 M in
hexanes) was added to a solution of (S)-(-)-4-benzyl-2-oxazo-
lidinone (3.66 g, 20.7 mmol) in 25 mL of dry THF at -78 °C.
This was stirred for 1 h and then added to the mixed anhydride
via canula. This mixture was allowed to warm from 0 °C to
room temperature with stirring overnight, then diluted with
1.0 N KHCO3, and extracted with ethyl acetate. The combined
organic layers were washed with water and brine, dried over
magnesium sulfate, and concentrated in vacuo. The crude
product was purified by medium pressure liquid chromatog-
raphy (MPLC) on a 40 × 350 mm silica gel column eluting
with 15% ethyl acetate/hexane to afford 7.21 g (97%) of the
title compound as a white solid: 1H-NMR (400 MHz, CDCl3)
δ 7.59 (d, 2H), 7.33-7.15 (m, 5H), 6.95 (d, 2H), 4.62 (m, 1H),
4.13 (d, 2H), 3.26 (dd, 1H), 2.93 (m, 2H), 2.72 (dd, 1H), 2.63 (t,
2H), 1.97 (m, 2H).
4(R)-[(4(S)-Ben zyl-2-oxooxa zolid in -3-yl)ca r bon yl]-6-(4-
iod op h en yl)h exa n oic Acid ter t-Bu tyl Ester (3). To a
solution of titanium tetrachloride (1.46 mL, 13.3 mmol) in 25
mL of dry (distilled over calcium hydride) methylene chloride
at 0 °C was added titanium isopropoxide (1.24 mL, 4.17 mmol).
This mixture was stirred for 5 min at 0 °C; then diisopropy-
lethylamine (3.05 mL, 17.5 mmol) was added. This was stirred
for 15 min; then a solution of 2 (7.21 g, 16.7 mmol) in 25 mL
of dry methylene chloride was added. This was stirred for 90
min at 0 °C; then tert-butyl acrylate (3.67 mL, 25.0 mmol) was
added. The reaction mixture was stirred overnight at 0 °C,
then allowed to warm to room temperature, and diluted with
saturated ammonium chloride. The aqueous layer was ex-
tracted with methylene chloride; then the combined organic
layers were washed with 2 N HCl, water, and brine, dried over
sodium sulfate, and concentrated in vacuo. The crude product
was purified by MPLC on a 40 × 350 mm silica gel column
eluting with 10% ethyl acetate/hexane to afford 7.52 g (80%)
of the title compound as a faint yellow oil: 1H-NMR (400 MHz,
CDCl3) δ 7.55 (d, 2H), 7.32 (t, 2H), 7.27 (t, 1H), 7.18 (d, 2H),
6.91 (d, 2H), 4.52 (m, 1H), 4.11 (d, 2H), 3.82 (m, 1H), 3.29 (dd,
1H), 2.68 (dd, 1H), 2.56 (m, 2H), 2.24 (m, 2H), 2.02 (m, 2H),
1.89 (m, 1H), 1.73 (m, 1H), 1.42 (s, 9H).
2(R)-[2-(4-Iod op h en yl)eth yl]p en ta n ed ioic Acid 5-ter t-
Bu tyl Ester (4). 3 (7.52 g, 13.4 mmol) was dissolved in a
mixture of 3:1 THF/water (200 mL) and cooled to 0 °C. To
this solution were added 30% hydrogen peroxide (6.09 mL, 53.7
mmol) and lithium hydroxide monohydrate (1.13 g, 26.8 mmol).
This mixture was stirred at 0 °C for 3 h; then a solution of
sodium sulfite (7.44 g, 59.1 mmol) in 40 mL of water was added
along with 100 mL of 0.5 N sodium bicarbonate. This mixture
was stirred for 2 h; then the THF was evaporated off on a
Rotovap. This aqueous mixture was diluted with 2 N HCl to
pH ) 2 and then extracted with ethyl acetate. The combined
organic layers were dried over sodium sulfate and concentrated
in vacuo. The crude product was purified by MPLC on a 40 ×
350 mm silica gel column eluting with 0.5% acetic acid in 20%
2(R)-[2-(4-Iod op h en yl)eth yl]-4-bu tylp en ta n ed ioic Acid
5-ter t-Bu tyl Ester (5). To a cooled solution of diisopropy-
lamine (3.50 mL, 24.98 mmol) in 20 mL of dry THF at -78 °C
was added n-butyllithium (10.39 mL, 2.5 M in hexanes). To
this was added a solution of 4 (4.18 g, 9.99 mmol) in a mixture
of 15 mL of dry THF and 10 mL of 1,3-dimethyl-3,4,5,6-
tetrahydro-2(1H)-pyrimidinone (DMPU). The reaction mixture
was stirred at -78 °C for 1 h and then warmed to 0 °C, and
iodobutane (2.27 mL, 19.99 mmol) was added in one portion.
This mixture was stirred overnight at 0 °C, warmed to room
temperature, and then diluted with 2 N HCl. The aqueous
layer was extracted with ethyl acetate. The combined organic
layers were washed with 2 N HCl, water, and brine, dried over
magnesium sulfate, and concentrated in vacuo. The resulting
oil was purified by MPLC on a 40 × 350 mm silica gel column
eluting with a gradient from 0 to 5% (5% acetic acid/MeOH)/
methylene chloride to afford 4.08 g (86%) of the title compound
as a mixture of diastereomers (ca. 2:1 S,R:R,R): 1H-NMR (400
MHz, CDCl3) δ 7.58 (d, 2H), 6.91 (d, 2H), 2.61 (m, 1H), 2.53
(m, 1H), 2.37 (m, 1H), 2.26 (m, 1H), 2.02-1.83 (m, 2H), 1.82-
1.68 (m, 2H), 1.54 (m, 2H), 1.39 (s, 9H), 1.23 (m, 4H), 0.84 (t,
3H).
2(R)-[2-(4-Iod op h en yl)et h yl]-4(S)-b u t ylp en t a n ed ioic
Acid 5-ter t-Bu tyl Ester 1-[2-(Tr im eth ylsila n yl)eth yl] Es-
ter (6). To a solution of 5 (1.48 g, 3.12 mmol) in 5 mL of THF
were added (trimethylsilyl)ethanol (0.537 mL, 3.74 mmol),
(dimethylamino)pyridine (76 mg, 0.62 mmol), and 1-[3-(dim-
ethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC)
(1.20 g, 6.24 mmol). This mixture was stirred at room
temperature overnight, then diluted with ethyl acetate, washed
with saturated sodium bicarbonate and brine, dried over
magnesium sulfate, and concentrated in vacuo. The resulting
oil was purified by MPLC on a 40 × 350 mm silica gel column
eluting with a gradient from 0 to 5% ethyl acetate/hexane. The
fractions containing the lower Rf spot were separated and
combined to afford 300 mg of the title compound as the pure
S,R diastereomer, and the mixed fractions (725 mg) were
combined and saved for further purification: 1H-NMR (400
MHz, CDCl3) δ 7.58 (d, 2H), 6.91 (d, 2H), 4.13 (t, 2H), 2.60-
2.42 (m, 2H), 2.33 (m, 1H), 2.22 (m, 1H), 1.90 (m, 2H), 1.76
(m, 1H), 1.54 (m, 2H), 1.40 (s, 9H), 1.23 (m, 4H), 0.98 (t, 2H),
0.84 (t, 3H), 0.03 (s, 9H).
2(R )-[2-[4-(3-Th ie n yl)p h e n yl]e t h yl]-4(S )-b u t ylp e n -
ta n ed ioic Acid 5-ter t-Bu tyl Ester 1-[2-(Tr im eth ylsila n yl)-
eth yl] Ester (7). To a solution of 6 (300 mg, 0.52 mmol) in a
mixture of 2 mL of toluene and 1 mL of ethanol were added
thiophene-3-boronic acid (74 mg, 0.57 mmol), 2.0 M aqueous
sodium carbonate (0.653 mL, 1.3 mmol), potassium bromide
(68 mg, 0.57 mmol), and tetrakis(triphenylphosphine)pal-
ladium (30 mg, 0.03 mmol). This mixture was stirred at 95
°C with protection from light for 6 h and then allowed to cool
to room temperature. The mixture was diluted with ethyl
acetate, washed with 2 N NaOH and brine, dried over
magnesium sulfate, and concentrated in vacuo. The resulting
oil was purified by MPLC on a 21 × 300 mm silica gel column
eluting with 4% ethyl acetate/hexane to afford 240 mg of the
title compound as a brown oil: 1H-NMR (400 MHz, CDCl3) δ
7.49 (d, J ) 8 Hz, 2H), 7.39 (d, J ) 2 Hz, 1H), 7.35 (d, J ) 2
Hz, 2H), 7.18 (d, J ) 8 Hz, 2H), 4.16 (t, J ) 7 Hz, 2H), 2.68-
2.50 (m, 2H), 2.40 (m, 1H), 2.24 (m, 1H), 1.92 (m, 2H), 1.81
(m, 1H), 1.71 (m, 1H), 1.55 (m, 2H), 1.40 (s, 9H), 1.23 (m, 4H),
0.98 (t, 2H), 0.85 (t, J ) 7 Hz, 3H), 0.03 (s, 9H).
2(R )-[2-[4-(3-Th ie n yl)p h e n yl]e t h yl]-4(S )-b u t ylp e n -
ta n ed ioic Acid 5-ter t-Bu tyl Ester (8). To a solution of 7
(240 mg, 0.45 mmol) in 1 mL of DMF was added 1.0 M
tetrabutylammonium fluoride (0.68 mL, 0.68 mmol) in THF.
This mixture was stirred at room temperature for 4 h, then
diluted with ethyl acetate, washed with 2 N HCl and brine,
dried over magnesium sulfate, and concentrated in vacuo. The
resulting oil was purified by MPLC on a 21 × 130 mm silica
gel column eluting with a gradient from 0 to 5% (5% acetic
acid/methanol)/methylene chloride to afford 190 mg of the title

1036 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
compound as a brown oil: 1H-NMR (400 MHz, CDCl3) δ 7.49
(d, J ) 8 Hz, 2H), 7.39 (d, J ) 2 Hz, 1H), 7.35 (d, J ) 2 Hz,
2H), 7.18 (d, J ) 8 Hz, 2H), 2.68-2.50 (m, 2H), 2.40 (m, 1H),
2.24 (m, 1H), 1.92 (m, 2H), 1.81 (m, 1H), 1.71 (m, 1H), 1.55
(m, 2H), 1.40 (s, 9H), 1.23 (m, 4H), 0.85 (t, J ) 7 Hz, 3H).
2(R )-[2-[4-(3-Th ie n yl)p h e n yl]e t h yl]-4(S )-b u t ylp e n -
ta n ed ioic Acid 1-((S)-ter t-Bu tylglycin e m eth yla m id e)
5-ter t-Bu tyl Ester Am id e (9). To a solution of 8 (190 mg,
0.45 mmol) in 2 mL of THF were added (S)-tert-butylglycine
methylamide hydrochloride (90 mg, 0.5 mmol), N-methylmor-
pholine (0.060 mL, 0.54 mmol), 1-hydroxy-7-azabenzotriazole
(74 mg, 0.54 mmol), and EDC (173 mg, 0.9 mmol). This
mixture was stirred at room temperature overnight, then
diluted with ethyl acetate, washed with saturated aqueous
sodium bicarbonate and brine, dried over magnesium sulfate,
and concentrated in vacuo. The crude product was purified
by MPLC on a 21 × 300 mm silica gel column eluting with
20% ethyl acetate/methylene chloride to afford 135 mg of the
title compound as a white solid: 1H-NMR (400 MHz, CDCl3)
δ 7.49 (d, J ) 8 Hz, 2H), 7.37 (d, J ) 2 Hz, 1H), 7.34 (d, J )
2 Hz, 2H), 7.17 (d, J ) 8 Hz, 2H), 6.23 (d, J ) 9 Hz, 1H), 5.97
(br, 1H), 4.26 (d, J ) 8 Hz, 1H), 2.80 (d, J ) 5 Hz, 3H), 2.60
(m, 1H), 2.46 (m, 1H), 2.28 (m, 1H), 2.11 (m, 1H), 1.92 (m,
2H), 1.76 (m, 1H), 1.55 (m, 2H), 1.37 (s, 9H), 1.23 (m, 4H),
1.00 (s, 9H), 0.84 (t, J ) 7 Hz, 3H).
2(R )-[2-[4-(3-Th ie n yl)p h e n yl]e t h yl]-4(S )-b u t ylp e n -
ta n ed ioic Acid 1-((S)-ter t-Bu tylglycin e m eth yla m id e)
Am id e (58). To a solution of 9 (135 mg) in 2 mL of methylene
chloride at 0 °C were added 0.5 mL of trifluoroacetic acid (TFA)
and 0.3 mL of anisole. This mixture was allowed to warm to
room temperature with stirring overnight and then concen-
trated in vacuo. The resulting oil was purified by MPLC on a
21 × 130 mm silica gel column eluting with a gradient from 0
to 5% (5% acetic acid/methanol)/methylene chloride to afford
60 mg of the title compound as a white solid: m/ z+ ) 501;
1H-NMR (500 MHz, CDCl3) δ 7.51 (br, 1H), 7.43 (d, J ) 8 Hz,
2H), 7.36 (d, J ) 2 Hz, 1H), 7.32 (m, 2H), 7.11 (d, J ) 8 Hz,
2H), 6.52 (br, 1H), 4.44 (d, J ) 8 Hz, 1H), 2.83 (d, J ) 4 Hz,
3H), 2.61-2.52 (m, 2H), 2.49 (m, 2H), 2.08 (m, 1H), 1.92 (m,
1H), 1.79 (m, 2H), 1.52 (m, 2H), 1.30 (m, 4H), 1.05 (s, 9H),
0.88 (t, J ) 6 Hz, 3H).
2(R)-[2-[4-(4-F lu or op h en yl)p h en yl]et h yl]-4(S)-b u t yl-
p en ta n ed ioic Acid 1-((S)-ter t-Bu tylglycin e 4-p yr id yla -
m id e) 5-ter t-Bu tyl Ester Am id e (12). To a solution of 11
(150 mg, 0.23 mmol) in a mixture of 2 mL of toluene and 1
mL of ethanol were added 4-fluorophenylboronic acid (35 mg,
0.25 mmol), 2.0 M aqueous sodium carbonate (0.283 mL, 0.57
mmol), and tetrakis(triphenylphosphine)palladium (13 mg,
0.01 mmol). This mixture was stirred at 95 °C with protection
from light for 6 h and then allowed to cool to room tempera-
ture. The mixture was diluted with ethyl acetate, washed with
2 N NaOH and brine, dried over magnesium sulfate, and
concentrated in vacuo. The crude product was purified by
MPLC on a 21 × 300 mm silica gel column eluting with a
gradient from 0 to 5% (10% ammonium hydroxide/methanol)/
methylene chloride to afford 80 mg of the title compound as a
faint yellow solid: 1H-NMR (400 MHz, CDCl3) δ 8.92 (br, 1H),
8.37 (d, J ) 6 Hz, 2H), 7.48 (dd, J ) 5 Hz, 2H), 7.45 (d, J ) 6
Hz, 2H), 7.42 (d, J ) 8 Hz, 2H), 7.19 (d, J ) 8 Hz, 2H), 7.08 (t,
J ) 8 Hz, 2H), 6.35 (br d, 1H), 4.42 (d, J ) 8 Hz, 1H), 2.68 (m,
1H), 2.51 (m, 1H), 2.31 (m, 1H), 2.15 (m, 1H), 2.03-1.86 (m,
2H), 1.80 (m, 1H), 1.70 (m, 2H), 1.57 (m, 1H), 1.40 (m, 1H),
1.36 (s, 9H), 1.23 (m, 2H), 1.10 (s, 9H), 0.84 (t, J ) 6 Hz, 3H).
2(R)-[2-[4-(4-F lu or op h en yl)p h en yl]et h yl]-4(S)-b u t yl-
p en ta n ed ioic Acid 1-((S)-ter t-Bu tylglycin e 4-p yr id yla -
m id e) Am id e (24). To a solution of 12 (80 mg) in 1 mL of
methylene chloride at 0 °C was added 0.250 mL of trifluoro-
acetic acid (TFA). This mixture was allowed to warm to room
temperature with stirring overnight and then concentrated in
vacuo. The resulting oil was purified by MPLC on a 21 × 130
mm silica gel column eluting with a gradient from 0 to 15%
(5% acetic acid/methanol)/methylene chloride to afford 29 mg
of the title compound as a white solid: m/ z+ ) 575; 1H-NMR
(400 MHz, CD3OD) δ 8.38 (d, J ) 6 Hz, 2H), 8.02 (d, J ) 8 Hz,
1H), 7.69 (d, J ) 7 Hz, 2H), 7.53 (dd, J ) 7 Hz, 2H), 7.40 (d,
J ) 8 Hz, 2H), 7.19 (d, J ) 8 Hz, 2H), 7.11 (t, J ) 9 Hz, 2H),
4.44 (d, J ) 8 Hz, 1H), 2.59 (t, J ) 8 Hz, 2H), 2.37 (m, 1H),
1.98 (s, acetate), 1.93 (m, 1H), 1.83 (m, 1H), 1.58 (m, 3H), 1.31
(m, 4H), 1.08 (s, 9H), 0.90 (t, J ) 7 Hz, 3H).
4-(4′-F lu or obip h en ylyl)-4-oxobu tyr ic Acid (14). A 500
mL three-neck, round-bottom flask equipped with a mechan-
ical stirrer, thermometer, and reflux condenser was charged
with methylene chloride (125 mL). 4-Fluorobiphenyl (20.0 g,
0.116 mol) was added followed by succinic anhydride (11.6 g,
0.116 mol). The mixture was cooled in an ice bath, and
aluminum chloride (30.9 g, 0.232 mol) was added in six equal
portions at 10-15 min intervals. The ice bath was applied as
needed to maintain the internal temperature near 15 °C. After
the last addition, the mixture was stirred at ambient temper-
ature for 90 min and then at gentle reflux (37-39 °C) for
another 90 min. The reaction mixture was then cooled in an
ice bath and poured into a stirred mixture of ice (300 g) and
concentrated hydrochloric acid (40 mL). Additional water (500
mL) was added, and the resulting solid was filtered, washed
with water, and dried by suction. The solid was recrystallized
from hot acetic acid to afford 19.6 g (62%) of the title compound
as a white crystalline solid: 1H-NMR (400 MHz, CDCl3) δ 8.02
(d, 1H), 7.62 (d, 1H), 7.58 (dd, 1H), 7.15 (t, 1H), 3.33 (t, 2H),
2.83 (t, 2H).
4-(4′-F lu or obip h en ylyl)bu tyr ic Acid (15). A mixture of
14 (19.6 g, 0.072 mol) in acetic acid (185 mL) and methanol
(75 mL) was stirred overnight at 60 °C in the presence of 20%
palladium hydroxide-on-carbon (1.2 g) under an atmosphere
of hydrogen gas. The catalyst was removed by filtration
through Celite and the filter washed with acetic acid. The
combined filtrate and washings were evaporated and coevapo-
rated several times with toluene. The solid was taken up in
warm acetic acid and the solution resubjected to the above
hydrogenolysis conditions at room temperature for 24 h. The
catalyst was removed as above, and the filtrate was evaporated
and coevaporated several times with toluene to afford 18.3 g
(98%) of the title compound as a white crystalline solid: 1H-
NMR (400 MHz, CDCl3) δ 7.51 (dd, 1H), 7.44 (d, 1H), 7.23 (d,
1H), 7.09 (t, 1H), 2.70 (t, 2H), 2.49 (t, 2H), 1.99 (m, 2H).
4(S)-Ben zyl-3-[4-(4′-flu or obip h en ylyl)b u t yr yl]oxa zol-
id in -2-on e (16). To a solution of 15 (18.3 g, 0.071 mol) in
methylene chloride (220 mL) was added N,N-dimethylforma-
mide (362 µL). The solution was cooled in an ice bath, and
oxalyl chloride (6.85 mL, 0.79 mol) was added dropwise with
stirring over 10 min. The mixture was stirred at 0 °C for 30
min and then at room temperature for 2 h. After evaporation,
the resulting acid chloride was dried for 1 h in vacuo. To a
solution of 4(S)-benzyl-2-oxazolidinone (11.4 g, 64.3 mmol) in
dry THF (185 mL) at -78 °C was added n-butyllithium (43.4
mL, 1.6 M in hexanes, 69.4 mmol) dropwise over 10 min while
maintaining the temperature at or below -60 °C. After 15
min, a solution of the acid chloride in THF (35 mL) was added
rapidly, and the mixture was stirred for 30 min at -78 °C.
The cooling bath was then removed, and stirring was contin-
ued for 1 h. Saturated ammonium chloride (55 mL) was then
added, and the reaction mixture was partitioned between ethyl
acetate and water. The organic layer was washed with
saturated brine solution, dried (Na2SO4), and evaporated. The
product was purified by flash silica gel chromatography eluting
with 15% ethyl acetate in hexane. The title compound was
1
obtained as a white crystalline solid: yield 22.5 g (76%); H-
NMR (400 MHz, CDCl3) δ 7.51 (dd, 1H), 7.47 (d, 1H), 7.18-
7.33 (m, 6H), 7.09 (t, 1H), 4.63 (m, 1H), 4.16 (m, 2H), 3.28 (dd,
1H), 3.00 (m, 3H), 2.70-2.79 (m, 2H), 2.06 (m, 2H).
2(R)-[2-[4-(P h en yleth yn yl)ph en yl]eth yl]-4(S)-bu tylpen -
ta n ed ioic Acid 5-ter t-Bu tyl Ester 1-[2-(Tr im eth ylsila n yl)-
eth yl] Ester (17). To a solution of 6 (600 mg, 1.04 mmol) in
a mixture of 2 mL of DMF and 2 mL of diisopropylamine were
added phenylacetylene (160 mg, 1.57 mmol), copper(I) iodide
(10 mg, 0.05 mmol), and bis(triphenylphosphine)palladium
chloride (37 mg, 0.05 mmol). This mixture was stirred at room
temperature overnight, then diluted with ethyl acetate, washed
with 2 N NaOH and brine, dried over magnesium sulfate, and
concentrated in vacuo. The crude product was purified by
MPLC on a 21 × 300 mm silica gel column eluting with 2%
ethyl acetate/hexanes to afford 550 mg of the title compound
as a reddish oil: 1H-NMR (400 MHz, CDCl3) δ 7.50 (d, J ) 8

Carboxyalkyl Dipeptides as Inhibitors of MMP-3
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6 1037
Hz, 2H), 7.42 (d, J ) 8 Hz, 2H), 7.32 (m, 3H), 7.13 (d, J ) 8
Hz, 2H), 4.14 (t, 2H), 2.66-2.50 (m, 2H), 2.36 (m, 1H), 2.22
(m, 1H), 1.91 (m, 2H), 1.80 (m, 1H), 1.54 (m, 2H), 1.40 (s, 9H),
1.23 (m, 4H), 0.98 (t, J ) 7 Hz, 2H), 0.85 (t, J ) 7 Hz, 3H),
0.03 (s, 9H).
(m, 2H), 1.39 (s, 9H), 1.23 (m, 4H), 0.98 (t, J ) 7 Hz, 2H), 0.83
(t, J ) 7 Hz, 3H), 0.03 (s, 9H).
4(R)-[2-[4-[1-(ter t-Bu toxyca r bon yl)-1H-in d ol-2-yl]p h e-
n yl]eth yl]-2(S)-bu tylp en ta n ed ioic Acid 5-ter t-Bu tyl Ester
1-[2-(Tr im eth ylsila n yl)eth yl] Ester (23). To a mixture of
22 (300 mg, 0.49 mmol) in 2 mL of N-methyl-2-pyrrolidinone
were added triphenylarsene (15 mg, 0.05 mmol), zinc chloride
(1.47 mL, 1.47 mmol, 1.0 M diethyl ether solution), N-(tert-
butyloxycarbonyl)-2-iodoindole (235 mg, 0.74 mmol), and tris-
(dibenzylideneacetone)dipalladium(0) (22 mg, 0.025 mmol).
The mixture was stirred at 60 °C for 2 h, cooled to room
temperature, diluted with ethyl acetate, washed with sodium
bicarbonate and brine, dried over sodium sulfate, and concen-
trated in vacuo. The crude product was purified by silica gel
chromatography eluting with 2.5% diethyl ether/hexanes to
afford 116 mg of the title compound: 1H-NMR (400 MHz,
CDCl3) δ 8.14 (d, 2H), 7.54 (d, 2H), 7.34-7.24 (m, 5H), 4.23 (t,
2H), 2.73-2.55 (m, 2H), 2.44 (m, 1H), 2.26 (m, 1H), 1.94 (m,
2H), 1.83 (m, 1H), 1.59 (m, 2H), 1.43 (s, 9H), 1.27 (m, 13H),
1.04 (t, 2H), 0.90 (t, 3H), 0.08 (s, 9H).
4(R)-(2-Iod oeth yl)-2(S)-bu tylp en ta n ed ioic Acid 5-ter t-
Bu tyl Ester 1-[2-(Tr im eth ylsila n yl)eth yl] Ester (19). To
a solution of toluene (25 mL) in a 50 mL round-bottom flask
fitted with a stir bar and rubber septum were added imidazole
(530 mg, 7.7 mmol), triphenylphosphene (2.9 g, 8.0 mmol), and
iodine (1.63 g, 6.7 mmol). To this was added 4(R)-(2-hydroxy-
ethyl)-2(S)-butylpentanedioic acid 5-tert-butyl ester 1-[2-(tri-
methylsilanyl)ethyl] ester (2.0 g, 5.15 mmol). The reaction
mixture was stirred between 40 and 50 °C under nitrogen until
starting material was gone and then cooled to 0 °C, and
saturated sodium bicarbonate (10 mL) was added with vigor-
ous stirring. Enough iodine was added until the yellow color
just persists; 10 mL of water was added, and the solution was
extracted with 3 × 10 mL of methylene chloride. The organic
layers were dried over MgSO4, filtered, and concentrated under
reduced pressure. Purification on silica gel of the crude
product (2.5 g) (hexane/ethyl acetate, 98:2) gave two products,
the more polar of which is the desired product (1.5 g, 58%):
1H-NMR (400 MHz, CDCl3) δ 4.17 (m, 2H), 3.19 (m, 2H), 3.08
(m, 2H), 2.5 (m, 1H), 2.25 (m, 1H), 2.15 (m, 1H), 2.04 (m, 1H),
1.95 (m, 1H), 1.55 (m, 2H), 1.44 (s, 9H), 1.25 (m, 4H), 1.00 (m,
2H), 0.89 (t, 2H, J ) 9 Hz), 0.03 (s, 9H).
X-r a y Cr ysta llogr a p h y. Recombinant human proSLN-247
was expressed in Escherichia coli, purified and activated to
the mature form (residues 83-247) essentially as described
for proSLN-255.95 The expression level of proSLN-247 was
similar to that of proSLN-255; however, upon lysis of the cells,
more proSLN-247 was prone to degradation thus lowering the
final yields of protein by 30-50%.
4(R)-(2-Biph en yl-3-yleth yl)-2(S)-bu tylpen tan edioic Acid
5-ter t-Bu t yl E st er 1-[2-(Tr im et h ylsila n yl)et h yl] E st er
(20). To a solution of 19 (373 mg, 0.75 mmol) in a 5 mL round-
bottom flask fitted with a Teflon stir bar and rubber septum
were added 75 mg of zinc/copper couple (1.15 mmol), 1 mL of
benzene, and 100 µL of dry dimethylformamide. The solution
was stirred under nitrogen for 4 h at 70 °C and then cooled to
25 °C. To this was added 3-bromobiphenyl (116 mg), and the
reaction mixture was sonicated with an ultrasonicator at 25-
55 °C until the starting material was consumed (∼2 h). The
solution was filtered to remove the solids, and the solvent was
removed under reduced pressure. Flash chromatography
(97.5:2.5 hexane/ethyl acetate) gave 80 mg of product (30%
yield): 1H-NMR (400 MHz, CDCl3) δ 7.57 (d, 2H, J ) 8 Hz),
7.4 (m, 4H), 7.34 (m, 2H), 7.15 (d, 1H, J ) 8 Hz), 4.17 (m, 4H),
2.65 (m, 1H), 2.60 (m, 1H), 2.45 (m, 1H), 2.25 (m, 1H), 1.95
(m, 1H), 1.85 (m, 1H), 1.55 (m, 2H), 1.38 (s, 9H), 1.25 (m, 4H),
1.00 (m, 2H), 0.84 (t, 2H, J ) 9 Hz), 0.02 (s, 9H).
4(R)-[2-(4-Cycloh ep t-2,3-en ylp h en yl)eth yl]-2(S)-bu tyl-
p en ta n ed ioic Acid 5-ter t-Bu tyl Ester 1-[2-(Tr im eth ylsi-
la n yl)eth yl] Ester (21). To a solution of 6 (250 mg, 0.435
mmol) in N,N-dimethylformamide (1 mL) were added tetrabu-
tylammonium chloride (121 mg, 0.435 mmol), potassium
acetate (128 mg, 1.30 mmol), palladium(II) acetate (2.5 mg,
0.011 mmol), and cycloheptene (254 µL, 2.18 mmol). The
reaction mixture was stirred overnight at room temperature
under a nitrogen atmosphere. It was then diluted with ethyl
acetate, washed with water, dried (Na2SO4), and evaporated.
The product was obtained pure by flash silica gel chromatog-
raphy eluting with 5% diethyl ether in hexane: yield 150 mg
(64%); 1H-NMR (400 MHz, CD3OD) δ 6.94-7.61 (m, 4H), 5.69-
5.89 (m, 2H), 4.18 (m, 2H), 1.40 (s, 9H), 1.89 (t, 3H), 0.08 (s,
9H).
4(R)-[2-[4-(Tr im eth ylsta n n a n yl)p h en yl]eth yl]-2(S)-bu -
tylp en ta n ed ioic Acid 5-ter t-Bu tyl Ester 1-[2-(Tr im eth -
ylsila n yl)eth yl] Ester (22). To a solution of 6 (2.0 g, 3.48
mmol) in 20 mL of dioxane was added triphenylphosphine (18
mg, 0.07 mmol), lithium chloride (162 mg, 3.83 mmol), hex-
amethylditin (0.872 mL, 4.53 mmol), and tetrakis(triph-
enylphosphine)palladium (201 mg, 0.17 mmol). This mixture
was stirred at 95 °C for 90 min, cooled to room temperature,
then diluted with ethyl acetate, washed with 2 N NaOH and
brine, dried over magnesium sulfate, and concentrated in
vacuo. The crude product was purified by MPLC on a 40 ×
350 mm silica gel column eluting with 2.5% ethyl acetate/
hexanes to afford 1.42 g (67%) of the title compound as a
colorless oil: 1H-NMR (200 MHz, CDCl3) δ 7.38 (d, J ) 8 Hz,
2H), 7.13 (d, J ) 8 Hz, 2H), 4.14 (t, 2H), 2.62-2.58 (m, 2H),
2.36 (m, 1H), 2.22 (m, 1H), 1.91 (m, 2H), 1.80 (m, 1H), 1.54
The inhibited complex was crystallized by hanging drop
vapor diffusion. Protein solution contained 16.5 mg/mL SLN-
255, 1.2 M excess L-764,004, 5.0 mM CaCl2, 0.02% NaN3, and
20 mM MES, pH ) 6.5; the reservoir contained 10% PEG-
6000, 5% saturated NH4Cl, 0.02% NaN3, and 0.1 M cacodylate,
pH ) 7.0. The crystals belong to the orthorhombic space group
C2221, with a ) 84.13, b ) 85.35, and c ) 55.25 Å, and
diffraction data extend to a resolution of 1.7 Å. Three-
dimensional diffraction data were collected using an R-Axis
IIC area detector and Cu KR radiation from a Rigaku RU-200
rotating anode X-ray generator. Data were processed using
the RAXIS package.96
The structure was solved by difference Fourier methods
using the protein portion of an isomorphous stromelysin
complex with a different inhibitor, which had been solved by
heavy-atom methods.34 Electron density corresponding to the
inhibitor was clearly visible in initial maps. Complete models
were constructed by interactive model building97 and refine-
ment using X-PLOR including one cycle of simulated anneal-
ing.98 A bulk solvent mask was included in the model, and in
the later stages of refinement, the model was confirmed by
10% simulated-annealing omit maps.99 The final model of the
inhibited complex comprised residues 88-247, two Zn2+ ions,
three Ca2+ ions, the bound inhibitor, and 143 ordered water
molecules. Coordinates have been deposited in the Protein
Data Bank,100 with access code 1HFS, and crystallographic
parameters are provided in the Supporting Information.
Biology. Mou se P leu r a l Ca vity Assa y. Mice (C57B1/
6J ; n ) 6-10)101 were predosed orally with compound in a
solution of 2% DMSO, 2% Cremophor, and 96% aqueous
solution of 0.25% carboxymethylcellulose. After the dosing
time, 0.2 mL of a 2.5 mg/mL solution of [3H]transferrin63 was
injected intrapleurally followed immediately by the injection
of 0.3 mL of a 100 µg/mL buffered solution (20 mM Tris, pH )
7.5, 0.15 M NaCl, 10 mM CaCl2, 0.02% NaN3, 0.05% Brij) of
trypsin-activated human MMP-3. After 30 min, the animals
were sacrificed by carbon dioxide asphyxiation, and the pleural
cavity was lavaged with 1 mL of cold buffer containing 20 mM
phenanthroline to prevent continued transferrin degradation
in vitro. The fluid was centrifuged (10 min at 3000 rpm); 50
µL of a 15% solution of trichloroacetic acid (TCA) or buffer
was added to a 200 µL aliquot of the supernatant. TCA
preparations were precipitated on ice for 30 min and then
centrifuged (5 min at 10 000 rpm). The supernatant (125 µL)
was then counted. A percent inhibition relative to controls
was calculated according to the equation below, and the results
are reported in Tables 1 and 2 under the heading PLCAV.

1038 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 6
Esser et al.
dioxide inhalation on day 29, and blood was collected to
determine plasma levels of 26. A statistical analysis was
conducted daily from day 16 to monitor the progress of the
study. Incidence between treatment groups was compared
using the Fisher’s exact test. Total severity scores in the
treatment group were compared to vehicle controls using a
nonparametric Wilcoxon test. Statistical significance was
determined by using a significance level of 0.05 throughout
the analysis. Inhibitor 26 had no statistically significant effect
on either the incidence or severity of collagen-induced arthritis
in this study. Total compound plasma level was determined
to be 33.3 ( 22.8 µg/mL at the end of the experiment.
Deter m in a tion of Com p ou n d 26 Levels in Blood . At
the time of euthanasia, blood was collected by venapuncture
of the caudal vena cava. The blood was placed in heparinized
tubes and spun and the resulting plasma used to determine
blood levels. Compound 24 was used as an internal standard.
A solution of 24 (1.5 µL in 50 µL of 1% BSA) was added to
0.15 mL of plasma; 2 mL of acetonitrile/TFA (100:0.01, v/v)
was added while the samples were vortexed. After centrifuga-
tion, the supernatant was diluted with 10 mL of water
containing 0.1% TFA. The samples were passed through a
Waters C-18 Sep-Pak cartridge and washed with 5 mL of 0.1%
TFA in water. The cartridge was then eluted with 4 mL of
methanol containing 0.1% TFA, which was then dried under
a stream of nitrogen. The extract was resuspended in 0.2 mL
of HPLC mobile phase (vide infra) and put into autosample
vials after sonication and centrifugation. The samples were
injected onto a Zorbax Rx-C8 column (0.46 × 25 cm) equili-
brated with 10 mM 1-octanesulfate/acetonitrile/methanol/TFA
(29:24:47:0.1, v/v) at a rate of 1.0 mL/min. The column eluate
was monitored at 268 nm. Concentrations were determined
by comparing the ratio of areas of 26 and 24 to a standard
curve.
TCA-soluble counts
% degradation )
total counts
% inhibition ) [(% degradation w/compd soln) -
(% degradation in buffer)]/[(% degradation w/o
MMP-3 added) - (% degradation in buffer)]
Ra bbit MMP -3 In jection Mod el. MMP-3-induced pro-
teoglycan release was induced by injecting one stifle joint of
female New Zealand white rabbits (Hazelton Farms, Denver,
PA; ∼3 months of age, n ) 4) with a phosphate-buffered saline
(PBS) solution (0.5 mL) containing 100 µg of trypsin-activated
human recombinant MMP-3 and the other with PBS;58 1 h
after MMP-3 injection, the rabbits were sacrificed by lethal
injection (Socumb), and the joints were lavaged with PBS (2
× 0.5 mL) and analyzed for sulfated proteoglycans by the
described alcian blue method.56,57 Inhibitor 26 (in 2% DMSO,
2% Cremophor, 96% PBS) was administered intravenously 15
min prior to the injection of MMP-3, and a dose that effectively
inhibited 50% of the proteoglycan release was obtained (ED50
) 6 mg/kg iv).
Ra bbit IL-1â In jection Mod el. IL-1â-induced proteogly-
can release was induced by injecting one stifle joint of female
New Zealand white rabbits (Hazelton Farms, Denver, PA; ∼
3 months of age, n ) 6) with 0.5 mL of a PBS solution
containing 327 ng of human recombinant IL-1â and the other
with the PBS;58 12 h later, the rabbits were sacrificed by lethal
injection, and the joints were lavaged with 0.5 mL of PBS.
Synovial lavage fluid was analyzed for sulfated proteoglycans
by the alcian blue assay. Inhibitor 26 (12 mg/kg iv in 2%
DMSO, 2% Cremophor, 96% PBS) was administered every 2
h from the time of the IL-1â injection through the end of the
experiment but failed to inhibit proteoglycan release compared
to vehicle controls.
R a t Ad ju va n t -In d u ced Ar t h r it is Mod el. Adjuvant-
induced arthritis (AIA) was induced in female Lewis rats
(∼150 g, n ) 10) by an intradermal injection of 0.5 mg of
Mycobacterium butyricum in light mineral oil in the left hind
foot pad. Body weights, radiographs, and foot volumes of the
contralateral paw were determined on days 0, 14, and 21.
Compound 26 (50 mg/kg po b.i.d.) and appropriate vehicles
were started on day 0 and continued throughout the experi-
ment. Blood (1 mL) was withdrawn by cardiac puncture under
methoxyflurane anesthesia 5 h after the last dose to determine
plasma levels of 26. Rats were sacrificed by carbon dioxide
inhalation on day 21. The thymus and spleen of all rats were
removed and weighed. General necropsy noted that abdomi-
nal, peritoneal, and thoracic cavities were normal. Indometha-
cin (1 mg/kg/day po s.i.d.) was used as a positive control.
Compound 26 failed to demonstrate efficacy in any of the
parameters examined in rat AIA (body weight, paw swelling,
and thymus and spleen weights). The compound level in
plasma collected 5 h after the final dose was 25.5 ( 6.9 µg/
mL. Indomethacin inhibited the paw swelling by 80% in this
experiment.
Mou se Colla gen -In d u ced Ar th r itis Mod el. Collagen-
induced arthritis was induced in 8-10 week old male B10.RIII
mice (J ackson Laboratories) by an intradermal injection of a
solution (100 µL) of lyophilized porcine type II collagen in
complete Freunds adjuvant (CFA; H37 Ra. Mycobacterium
tuberculosis; Difco) in the base of the tail. The mice were
boosted by injecting human recombinant IL-1â (327 ng in 100
µL of PBS) subcutaneously on days 13, 14, and 15 following
type II collagen injection. Each paw was scored starting on
day 16 using the following system: 0 ) normal, 1 ) one or
two swollen toes, 2 ) three or more toes swollen and/or
extending into the carpus or tarsus, 3 ) deformity of paw, 4
) ankylosis. Severity score was calculated by adding all of
the scores of each paw and obtaining a mean for that group.
Compound 26 (50 mg/kg po b.i.d., n ) 28) was administered
in a solution of 4% DMSO, 4% Cremophor, and 92% PBS, and
indomethacin (1 mg/kg po s.i.d., n ) 10), dexamethasone (1
mg/kg po s.i.d.), and cyclophosphamide (5 mg/kg po s.i.d., n )
10) were administered in an aqueous solution throughout the
course of the experiment. Animals were sacrificed by carbon
Su p p or tin g In for m a tion Ava ila ble: Tables of crystal-
lographic parameters (1 page). Ordering information can be
found on any current masthead page.
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