Design and synthesis of cyclic inhibitors of matrix metalloproteinases and TNF-alpha production.

Full text of DOI:10.1021/jm970849z

J . Med. Chem. 1998, 41, 1745-1748
1745
Sch em e 1a
Design a n d Syn th esis of Cyclic In h ibitor s
of Ma tr ix Meta llop r otein a ses a n d TNF -r
P r od u ction
Chu-Biao Xue,* Xiaohua He, J ohn Roderick,
William F. DeGrado,2 Robert J . Cherney,
Karl D. Hardman, David J . Nelson,
Robert A. Copeland, Bruce D. J affee,1 and
Carl P. Decicco*,3
The DuPont Merck Pharmaceutical Company,
Experimental Station, Wilmington, Delaware 19880-0500
Received December 19, 1997
The matrix metalloproteinases (MMPs) are a family
of tightly regulated enzymes involved in the catabolic
aspect of remodeling and maintenance of normal tissue.4
In pathophysiological conditions in which matrix deg-
radation is linked to progression, incidence and severity
of disease have been associated with high levels of MMP
activity. Diseases such as arthritis, cancer, and perio-
dontal and ocular disease all include as part of their
progression MMP expression and activity significantly
above basal levels.5 The design and synthesis of inhibi-
tors of key enzymes of the MMP family has become an
area of vigorous investigation in drug discovery, in
essence, targeting the restoration of balance to these
pathodegradative processes. A collection of these agents
have now progressed to the stage of clinical evaluations
in cancer and arthritis in humans.6
We, and others, have shown that a subset of MMP
inhibitors is also capable of inhibiting the production
of TNF-R in cells and in animals.7 A possible explana-
tion for this activity has been ascribed to the inhibition
of the recently discovered TNF-R converting enzyme
(TACE),8 which has been shown to cleave cell-associated
26 kd proTNF to its soluble 17 kd form. TACE has
sequence homology to the reprolysins or snake venom
metalloproteinases which are included in the Metzincin
family of enzymes.9 The central role of TNF-R in
inflammatory disorders such as rheumatoid arthritis10
suggests inhibition of TNF production to be a very
important therapeutic target for small molecule drug
design. In this communication, we disclose a new series
of cyclic inhibitors of MMPs that also inhibit TNF-R
release from human cells, possibly through the inhibi-
tion of TACE or a related enzyme that possesses TNF
processing activity.
Succinate-based hydroxamic acids have been shown
by a number of groups to be potent inhibitors of MMPs.5
From a conformational analysis of these inhibitors using
molecular modeling, and the study of inhibitor-enzyme
complexes, we have observed that the preferential
binding mode is an extended conformation.
The crystal structure of BB-16/MMP-3 (Figure 1)
shows that the P1′ isobutyl and P1 methyl groups align
toward an antiperiplanar arrangement (dihedral angle
) 61°). The P2′ O-Me-phenyl group is directed away
from the active site into solvent. Indeed the P2′ group
of this inhibitor seems to be outside of the active site
and therefore not involved in a strict P2′-S2′ interac-
tion. This conformation clearly has P1 and P2′ directed
a
Conditions: (a) LDA (Xc ) (S)-4-phenylmethyloxazolidinone);
(b) n-BuO2CCHO, 36%; (c) LiOH, H2O2, 74% (d) BnBr, DBU, 79%;
(e) NaH, BrCH2CO2t-Bu, 71%; (f) H2, Pd-C, 99%; (g) N-Cbz-L-
lysine-NHMe, BOP, 75%; (h) H2, Pd-C, 4 N HCl/dioxane, 99%; (i)
BOP, 50%; (j) 1N LiOH, 73%; (k) BnONH2‚HCl, BOP, 21%; (l) H2,
Pd-C, 62%.
away from the active site into solvent. We chose to
exploit this observation by examining P1-P2′ linkages,
which were designed to hold the succinyl-peptide inhibi-
tor in a conformation determined by modeling, and
supported by crystallographic evidence, to be correct for
optimal enzyme-inhibitor interaction. Various cyclic
molecules were considered, and computer modeling of
the best cycles designed were determined to hold all of
the inhibitor-enzyme hydrogen bonds and van der
Waals contacts in analogous positions to the acyclic
molecules. We disclose here two novel classes of cyclic
MMP inhibitors, new synthetic methods for their prepa-
ration, and the first example of incorporating an anti-
succinate peptidomimetic moiety into a macrocycle.11,12
In vitro enzyme data for representatives of the collage-
nases, stromelysins, and gelatinases (MMP-1, -3, and
-9); TNF inhibition data determined from a cellular
assay, and X-ray crystal structures of these novel
inhibitors complexed with MMP-3 are also presented.
Syn th esis. Cyclic molecule SC903 containing an
ether-diamide linkage was prepared as depicted in
Scheme 1. 2-Hydroxy-3-isobutylsuccinate 2 was pre-
pared through chiral oxazolidinone-controlled aldol
condensation of 1 with butyl glyoxylate to give a 3:1
ratio of diastereomers of 2.13 The optimum sequence
S0022-2623(97)00849-2 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/01/1998

1746 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Communications to the Editor
F igu r e 1. (a, left) BB-16 in an extended conformation. (b, right) X-ray structure of BB-16 in the active site of MMP-3.
Sch em e 2a
for preparing 3b entailed hydrolysis of the chiral
auxiliary and benzyl esterification of the intermediate
carboxylate, followed by alkylation of the secondary
alcohol with tert-butyl bromoacetate to give 3a . Hydro-
genolysis of the benzyl ester gave the free carboxylic acid
3b which was then coupled to N-Cbz-Lys-NHMe to give
4. Sequential deprotection of the Cbz and tert-butyl
ester groups followed by intramolecular cyclization
using BOP gave the 13-membered lactam 5. Conversion
to the cyclic test molecule was completed by hydrolysis
of the butyl ester and conversion to the benzyl protected
hydroxamic acid, followed by hydrogenolysis in the final
step to provide the hydroxamic acid SC903.
Starting from anti-succinate 6,14 cyclophane SE205
was prepared as shown in Scheme 2. Benzyl ester
formation and 9-BBN oxidation of the olefin to the
primary alcohol, followed by bromination with CBr4/
Ph3P and subsequent hydrogenation of the benzyl ester,
gave chiral bromo acid 7. The benzyl ester protecting
group was used in the sequence to facilitate chromato-
graphic purification of the intermediate oxidation prod-
uct. Coupling of the carboxylic acid 7 with Tyr-NHMe
gave the phenoxy bromide cyclization precursor 8. Cs2-
CO3 in DMF was used to effect a high-yield cyclization
to give 9. Removal of the tert-butyl ester with TFA,
followed by coupling of the free carboxylic acid with
O-benzylhydroxylamine and subsequent purification
(again the benzyl group was used for ease of chromato-
graphic purification of the penultimate product), fol-
lowed by catalytic hydrogenation, provided cyclophane
test molecule SE205.
Biologica l Resu lts a n d Discu ssion . Macrocycles
a
Conditions: (a) BnBr, DBU, 65%; (b) 9-BBN, 64%; (c) CBr4,
SC903 and SE205 were tested and found to be potent
Ph3P, 65%; (d) H2, Pd-C, 99%; (e) Tyr-NHMe‚HCl, TBTU, 75%;
(f) Cs2CO3, 90%; (g) TFA, 95%; (h) BnONH2, HATU, 60%; (i) H2,
inhibitors of MMP-1, -3, and -9 (see Table 1).15 Crystal
structures for both SC903 and SE205 complexed with
MMP-3 were also solved (Figures 2 and 3, respec-
tively).16 The figures depict the active site region of the
enzyme extending toward the carboxy terminus (prime
side) from the catalytic Zn (orange sphere). Analysis
of these structures reveals a similar binding motif for
both cyclic inhibitors which is comparable to the ex-
Pd/BaSO4, 99%.
tended conformation of the linear inhibitor BB-16 shown
in Figure 1, confirming our design hypothesis. Study
of the overlap of the cyclophane SE205 with BB-16 (not
shown) indicates that the macrocycle is functioning to
maintain the succinyl-peptide backbone in an energeti-

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11 1747
Ta ble 1. Comparison of in Vitro Data for SE205, SC903, and
BB-16
The cyclics were also determined to inhibit TNF
release from LPS-stimulated human whole blood,17
possibly through inhibition of TNF-R converting enzyme
or related metalloproteinase TNF processing enzyme.18
These data indicate that the cyclic molecules SE205 and
SC903 represent new leads in the pursuit of metallo-
proteinase inhibitors that are antiinflammatory through
inhibition of the key inflammatory mediator TNF.
SE205 in particular was found to have excellent water
solubility (13 mg/mL) in comparison to BB-16 (0.3 mg/
mL), making it an attractive prototypical molecule for
further studies in vivo.
Ki, nM
IC50, nM
MMP-1
MMP-3
MMP-9
TNF-WBA
SE205
SC903
BB-16
1.2
2.8
0.87
32.7
24.1
1.93
1.8
2.6
0.13
1200
6500
1800
Con clu sion . We have described the design and
synthesis of two novel cyclic inhibitors of MMPs, SC903,
and SE205. These molecules were cocrystallized with
MMP-3, and the high-resolution structures were solved.
The structures clearly show the cyclic inhibitors bound
in the predicted orientation. These molecules were also
determined to inhibit the release of TNF from human
cells stimulated with LPS. The implication for further
iterations in design are numerous, and we are currently
studying several variations of these prototypes in our
labs. The physical properties, absorption, metabolism,
and pharmacokinetics, as well as comprehensive SAR
and potential therapeutic value for these series, are the
subject of subsequent disclosures from our labs.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the synthesis of SE205 (Scheme 2), spectroscopic data
on SC903, and X-ray crystallographic data (5 pages). Ordering
information is given on any current masthead page.
F igu r e 2. X-ray structure of SC903 in the active site of MMP-
3.
Refer en ces
(1) Department of Inflammatory Diseases Research, DuPont Merck.
(2) Current address: Department of Biochemistry and Biophysics,
The University of Pennsylvania School of Medicine, Philadel-
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(3) To whom correspondence should be addressed.
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inhibitors of these enzymes.

1748 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 11
Communications to the Editor
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were vortexed and incubated for 15 min, and then 15 µL of LPS
(Salmonella typhimurium, 5 mg/mL in AIM-V media) was added,
and the cultures were incubated for 5 h at 37 °C in a humidified
atmosphere of 5% CO2. AIM-V media (750 µL) was added to each
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J M970849Z