Benzodiazepine inhibitors of the MMPs and TACE

Article abstract of DOI:10.1016/S0960-894X(02)00633-9

A series of benzodiazepine inhibitors of the MMPs and TACE has been developed. These compounds display an interesting selectivity profile and should be useful tools for exploring the biological relevance of such selectivity.

Full text of DOI:10.1016/S0960-894X(02)00633-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2867–2870
Benzodiazepine Inhibitors of the MMPs and TACE
Frances C. Nelson,a,* Efren Delos Santos,a Jeremy I. Levin,a James M. Chen,a
Jerauld S. Skotnicki,a John F. DiJoseph,b Michele A. Sharr,b Amy Sung,b
Loran M. Killar,b Rebecca Cowling,a Guixian Jin,a
Catherine E. Rothb and J. Donald Albrighta
aWyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth Research, PO Box CN8000, Princeton, NJ 08543, USA
Received 27 March 2002; accepted 17 July 2002
Abstract—A series of benzodiazepine inhibitors of the MMPs and TACE has been developed. These compounds display an inter-
esting selectivity profile and should be useful tools for exploring the biological relevance of such selectivity.
# 2002 Elsevier Science Ltd. All rights reserved.
The matrix metalloproteinases (MMPs) are a family of
over 20 different enzymes that are involved in a variety
of biological processes including degradation of the
extracellular matrix, embryonic development, angio-
genesis, wound healing, apoptosis and normal tissue
remodeling. Under normal circumstances, the activity of
the MMPs is tightly controlled, as the disregulation of
these enzymes can lead to various disease states,
including osteoarthritis (OA),1 rheumatoid arthritis
(RA)1 and cancer.2 The ADAM class of enzymes is
structurally related to the MMPs and includes TNF-a
converting enzyme (TACE), the enzyme responsible for
cleaving membrane-bound TNF, which is believed to
have a major role in the pathology of rheumatoid
One problem that has been broached frequently during
the course of developing MMP inhibitors for clinical
trials is how to achieve selective inhibition of individual
members of the MMP and ADAM family of enzymes,
and whether such selectivity would be advantageous.
The current goal is to exploit known differences in the
binding domains of the various enzymes in order to
produce small molecule inhibitors with different selec-
tivity profiles and evaluate their efficacy and tox-
icological profiles.6 It is hoped that by doing so, we can
begin to address what type of selectivity is necessary in
order to minimize detrimental side effects of MMP and
TACE inhibitors while retaining their therapeutic
potential.
arthritis, inflammation and other disease states.3
A
variety of inhibitors of MMPs have been shown to be
potent inhibitors of TACE.4
Numerous examples of both peptide and nonpeptide
small molecule MMP inhibitors are known.6 In the area
of nonpeptide based inhibitors, CGS-27023A is a classic
example (Fig. 1A).7 We now wish to disclose our work
in this area using a benzodiazepine template as a frame-
work for designing potent MMP inhibitors (Fig. 1B).8
Given their role in the above diseases, it is expected that
small molecule inhibitors of MMPs and TACE would
have utility for treating many disorders. Thus, several
MMP inhibitors have already entered clinical trials for
the treatment of cancer and OA. Furthermore, there are
two clinically effective RA therapies available that block
the action of TNF-a, Enbrel1 and Remicade1, giving
credence to the idea that small molecule inhibitors of
TACE may be effective in treating RA.5
*Corresponding author. Tel.: +81-845-602-3746; fax:+1-845-602-
5561; e-mail: nelson@wyeth.com
Figure 1. (A) CGS-27023A; (B) general structure of benzodiazepine
inhibitors.
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00633-9

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F. C. Nelson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2867–2870
Scheme 1. Reagents and conditions: (a) Et3N, CH2Cl2; (b) 2-nitrobenzyl bromide, NaH, DMF; (c) ammonium formate, 10% Pd/C; (d) Et3N,
CH2Cl2, R1Cl; (e) sodium bicarbonate, CH3OH; (f) LiOH, THF, CH3OH, H2O; (g) (i) oxalyl chloride, DMF, CH2Cl2, 0 ꢀC; (ii) hydroxylamine
hydrochloride, Et3N, DMF.
These compounds exhibit potent inhibitory activity as
well as interesting selectivity profiles against MMPs and
TACE and as such may be useful in treating many of
the conditions stated above.
the activity for MMP-9 and MMP-13 remains largely
unaffected. Two notable compounds are 8e and 8l.
Compound 8e displays high potency against all four
enzymes while 8l shows a dramatic decrease in potency
for all four enzymes. This reduction in potency must
have an electronic component and is clearly not due just
to sterics. The biphenyl analogue 8i and the phenyl
derivative 8c are the most potent in the series with sub-
nanomolar IC50’s for both MMP-9 and MMP-13; 8i is
also the most selective member of the series with MMP-
1/MMP-13=132 and TACE/MMP-13=498. MMP-1 is
also sensitive to the size of the R1 substituent, however
the tolerance is somewhat different than TACE. Com-
pounds with an sp3 center a to the carbonyl (8j, MMP-
1=465 nM) are typically less active than those with an
sp2 center in the same position (8c, MMP-1=16 nM).
Chemistry
Synthesis of the benzodiazepines commences with the
sulfonylation of d,l-serine methyl ester with p-methoxy-
benzene sulfonyl chloride to give a good yield (79–84%)
of sulfonamide 3 (Scheme 1).9 Alternative sulfonyl
chlorides could be used to provide analogues with dif-
ferent R2 substituents. The sulfonamide is then alky-
lated with 2-nitrobenzyl bromide to give the nitro
intermediate in 54–69% yield. The moderate yield of
this reaction is due to alkylation of the primary alcohol
followed by elimination to give the a,b-unsaturated
ester as a by-product. The nitro group is then reduced
with ammonium formate and 10% palladium on carbon
in ethanol to give 65–74% of amine 4, after recrystalli-
zation from ethyl acetate. The benzodiazepine ring is
constructed via acylation of the aniline with con-
comitant elimination of the hydroxyl moiety to provide
5, which undergoes a Michael addition into the resulting
a,b-unsaturated ester to give 6 in 80–96% yield (two
steps). A variety of acid chlorides were used in this acy-
lation/elimination/cyclization sequence. Hydrolysis of
methyl ester 6, followed by subsequent transformation
to the hydroxamic acid gives the final products, 8 and 9.
Compounds 9a–d, where R2 is a butynyl ether were
prepared via the cleavage of the methyl ether in com-
pound 6 with BBr3, followed by alkylation of the
resulting phenol using the Mitsunobu protocol (DEAD,
PPh3, alcohol, THF, toluene). The butynyl analogues
were then converted to the hydroxamic acids as before.
In order to provide compounds with improved selectiv-
ity for MMP-13, compounds 8m–8p were prepared. In
other series, it is known that P10 substituents of this
nature have conferred increased selectivity for MMP-
13.6 Interestingly, this effect is not seen to any great
extent for 8m–8o. However, 8n does exhibit a decrease
in potency for TACE, although this is not accompanied
by a significant improvement in selectivity relative to 8c.
Example 8p, with the 4-pyridyl ether, displays the most
pronounced effects in this series with 381-fold selectivity
for MMP-13 over MMP-1 and 78-fold selectivity for
MMP-13 over TACE.
Select compounds were tested in two advanced models
(Table 2). In both models, CGS-27023A (Fig. 1A) was
used as a standard. This compound was the first of the
sulfonamide based MMP inhibitors and has been used
in clinical trials for the treatment of cancer.7 Initially,
compounds were screened in a dialysis tubing implant
assay to measure in vivo bioactivity versus MMP-13.11
In this assay a section of dialysis tubing containing
enzyme is implanted subcutaneously in the back of a rat
and inhibitor is administered ipor orally. After a period
of time the tubing is recovered and enzyme activity is
analyzed.
Discussion
The compounds synthesized as shown above were eval-
uated as inhibitors of MMP-1, MMP-9, MMP-13, and
TNF-a converting enzyme (TACE).10 All of the com-
pounds in Table 1 show selectivity for MMP-9 and
MMP-13 versus MMP-1 and TACE. As the size of R1
increases, the potency against TACE decreases, while
Many of the compounds performed quite well and were
equipotent or superior to CGS-27023A. Two com-
pounds, 8a and 8d, were tested by two routes of

F. C. Nelson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2867–2870
2869
Table 1. In vitro data for benzodiazepines 8a–8p Table 2. Dialysis and cartilage explant data for select compounds
Compd
R1
R2
MMP-1a MMP-9a MMP-13a TACEa
Compd
Dialysis activitya
(route, time)
Cartilage explant, activityc
(at 1 mM)
8a
OCH3
18
1.4
1
103
CGS-27023A
1
1
NT
8a
8a
8c
8d
8d
8e
8f
8g
8j
8m
8p
1.7 (2 h, ip)
0.90 (1 h, po)
1.2 (2 h, ip)
0.80 (1 h, ip)
0.49 (1 h, po)
1.1 (2 h, ip)
1.0 (1 h, po)
0.22 (2 h, ip)
0.47 (1 h, po)
IA
0.73
NT
8b
8c
8d
8e
8f
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
157
16
8
0.6
4
3
0.4
3
104
95
1.2
2.5
NT
NT
NT
NT
0.32 (1 h, po)b
171
20
69
aCompounds were dosed at 50 mpk and the data are expressed relative to
activity of CGS-27023A (such that control=1) versus MMP-13 in the same
experiment.
bCompound was dosed at 25 mpk.
cControl was CGS-27023A. Data are expressed as in the dialysis assay.
IA=inactive; NT=not tested.
1
1
13
34
2
2
95
Table 3. In vitro data for compounds 9a–9d
8g
8h
8i
523
55
18
4
26
2
207
271
199
318
157
808
157
528
254
157
Compd
R1
MMP-1a
MMP-9a
MMP-13a
TACEa
16
THP-1b
(%)
9a
835
228
77
60
53
0.7
13
2
0.4
7
9b
9c
260
165
841
95.5
35.8
33
34.6
9.5
29
20
59
10
44
25
59
8j
465
24
8k
8l
2
9d
aIC50, nM.
b% Inhibition @ 3 mM.
4982
61
187
2
317
2
cartilage explant model.12 All four compounds that
were tested provided a level of inhibition of cartilage
degradation that was comparable or superior to CGS-
27023A at a dose of 1 mM. Compound 8c, for example,
displayed 46.3% inhibition of cartilage degradation
while CGS-27023A gave 63% inhibition, again in the
same assay.
8m
8n
8o
59
2
2
35
2
4
Replacement of the methoxy group with but-2-ynyloxy
(Table 3) changes the selectivity dramatically for TACE.
Compounds 9a–9d all display a 1.6- to 6.9-fold increase
in potency for TACE relative to their methoxy sub-
stituted counterparts (8a–8d). Interestingly, the potency
for the other enzymes decreased with the addition of
this but-2-ynyloxy substituent. Notably, the selectivity
for TACE over MMP-1 is drastically improved, with
compound 9d showing an 84-fold preference for TACE.
These compounds were also tested for their ability to
inhibit TNF-a in a cellular assay using THP-1 cells.13 All
compounds displayed a reasonable level of inhibition at 3
mM. Upon dosing down, only 9a maintained a good level
of inhibition (44%) at 1 mM. Within the butynyl series, we
have therefore succeeded in providing compounds that are
selective for TACE over MMP-1 and have demonstrated
the ability to inhibit TNF-a in a cellular system.
8p
763
2
2
aIC50 (nM).
administration. Both showed a significant decrease in
activity when going from ipto po administration. The
poorest performer in this assay was 8g, which displayed
only 22% of the activity of the control at the 2 h time
point when dosed ip. Thus, while CGS-27023A gave
35.3% inhibition of MMP-13, 8g displayed a modest
7.8% inhibition when dosed in the same assay. Com-
pounds 8d, 8j and 8p had less than 50% of the activity
of the control when dosed orally. A small subset of the
compounds tested in dialysis went on to the bovine

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F. C. Nelson et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2867–2870
Chem. 2001, 8, 425. (b) Skotnicki, J. S.; Levin, J. I.; Killar, L.
Conclusion
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Carroll, B. J.; Goldstein, R.; Justice, M. R.; Zhu, L.; Hu, S.;
Melton, R. A.; Fryer, L.; Goldberg, R. L.; Doughty, J. R.;
Spirito, S.; Blancuzzi, V.; Wilson, D.; O’Byrne, E. M.; Ganu,
V.; Parker, D. T. J. Med. Chem. 1997, 40, 2525.
In conclusion, we have succeeded in synthesizing a
novel series of benzodiazepine inhibitors of the MMPs
and TACE. These compounds demonstrate an interest-
ing selectivity profile and several have also shown high
levels of activity in two advanced models. By appro-
priate manipulation of the R2 position, we have suc-
cessfully
transformed
the
methoxy-substituted
compounds into a series that now displays a vastly
improved selectivity profile for TACE. Given this data,
these compounds might be useful in the treatment of the
various disease states mediated by these enzymes and
serve as probes for the importance of selectivity in the
disease process.
8. (a) Albright, J. D; Delos Santos, E. G.; Du, X. U.S. Patent
6,071,903, 2000; Chem. Abstr. 2000, 133, 17487. (b) Delos
Santos, E. G.; Albright, J. D.; Nelson, F. C.; Du, X.; Levin, J.
I.; Chen, J.; Skotnicki, J. S.; Killar, L. M.; DiJoseph, J. F.;
Sung, A.; Sharr, M. A.; Roth, C. E.; Walter, T.; Cowling, R.;
Jin, G. Abstracts of Papers, 222nd National Meeting of the
American Chemical Society, Chicago, IL, Aug 26–30, 2001;
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9. All new compounds gave satisfactory 1H NMR, IR and
MS in accord with their assigned structure.
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