Selectivity of inhibition of matrix metalloproteases MMP-3 and MMP-2 by succinyl hydroxamates and their carboxylic acid analogues is dependent on P3′ group chirality

Article abstract of DOI:10.1016/S0960-894X(00)00719-8

Structure-activity relationships are described for a series of succinyl hydroxamic acids 1a-o and their carboxylic acid analogues 2a-o as inhibitors of matrix metalloproteases MMP-3 and MMP-2. For this series (P1′=(CH₂)₃Ph, P′2=t-Bu)

Full text of DOI:10.1016/S0960-894X(00)00719-8

Bioorganic & Medicinal Chemistry Letters 11 (2001) 567±570
Selectivity of Inhibition of Matrix Metalloproteases MMP-3 and
MMP-2 by Succinyl Hydroxamates and their Carboxylic Acid
Analogues is Dependent on P30 Group Chirality
M. Jonathan Fray,a,* M. Frank Burslemb and Roger P. Dickinsona
aDepartment of Discovery Chemistry, P®zer Global Research and Development, Sandwich, Kent CT139NJ, UK
bDepartment of Discovery Biology, P®zer Global Research and Development, Sandwich, Kent CT139NJ, UK
Received 9 June 2000; revised 27 November 2000; accepted 19 December 2000
AbstractÐStructure±activity relationships are described for a series of succinyl hydroxamic acids 1a±o and their carboxylic acid
analogues 2a±o as inhibitors of matrix metalloproteases MMP-3 and MMP-2. For this series (P10=(CH2)3Ph, P20=t-Bu)
selectivity for the inhibition of MMP-2 was found to be strongly dependent on P30. # 2001 Elsevier Science Ltd. All rights
reserved.
Matrix metalloproteases (MMPs) constitute a family of
structurally similar zinc-dependent proteases, which are
involved in the remodelling, repair and degradation of
extracellular matrix proteins, both as part of normal
physiological processes and in pathological conditions.1
For example, MMPs are thought to play a key role in
the post-partum remodelling of reproductive tissue,2
angiogenesis, and tissue repair and re-epithelialisation
of wounds by invading ®broblasts, keratinocytes and
endothelial cells.3 In these conditions, MMP function is
closely regulated due to their high destructive potential.
In contrast, abnormal levels of MMPs have been impli-
cated in conditions such as arthritis (MMP-3 and 13),4
tumour metastasis (MMP-2 and 9)5 and periodontal
disease (MMP-8).6 In order to help understand the role
of a particular MMP in pathological processes it would
be useful to have selective inhibitors available.
At the time we started this project, several structural
classes of MMP inhibitor with nanomolar potency were
known, for example, BB94 (batimastat; Fig 1).
Figure 1. Structure and enzyme inhibition pro®le of BB94 (batimastat)
(data generated in our laboratories).
Although it was known that selectivity over MMP-1
could be readily achieved by increasing the size of the
P10 substituent,7 there were few examples of MMP-3
inhibitors with appreciable selectivity over MMP-2,8
and several authors have reasoned that the diculty in
obtaining MMP-3 selective compounds is due to the
relatively open nature of the S30 binding site.9,10 How-
ever, this site should be smaller in MMP-2 because a
Tyr occupies the corresponding position of a Thr in
MMP-3.11 Thus, we prepared a series of hydroxamic
and carboxylic acids (1a±o and 2a±o) with fairly large and
rigid P30 substituents which we hoped would be accom-
modated into the S30 site of MMP-3 but not MMP-2.
This paper describes our initial work to optimise
potency and selectivity for MMP-3 inhibition over
MMP-2 and the discovery that certain P30 binding
groups can dramatically in¯uence activity against
MMP-2.
*Corresponding author. Tel.: +44-1304-640206; fax: +44-1304-640200;
e-mail: jonathan_fray@sandwich.p®zer.com
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00719-8

568
M. J. Fray et al. / Bioorg. Med. Chem. Lett. 11 (2001) 567±570
Chemistry
butyl because succinyl hydroxamates without bulky
P20 groups are less stable at high and low pH.18
The syntheses of 1a±o and 2a±o are shown in Scheme 1.
Aryl P30 groups were initially investigated because they
had been reported to increase potency and selectivity for
MMP-3.19 As can be seen, 1b and 1c were indeed more
potent but retained selectivity for MMP-2. The
cycloalkyl analogues 1d and 1e increased MMP-3 inhi-
bition slightly and the selectivity for MMP-2 was
diminished, and even a t-butyl group (1f) was tolerated
by both enzymes. Examination of the X-ray structure of
MMP-320 suggested introducing more bulky P30 groups
with a bend, such as benzyl, but since the S30 binding
site is solvent-exposed, we decided to use branched
groups to ensure that at least one of the groups would
protude into S30. For the a-methylbenzylamides 1g and
1h, potency and selectivity was found to be strongly
dependent on the chirality of the P30 group, with the R-
enantiomer retaining similar potency to 1a against
MMP-3, but causing a loss in potency of about 200-fold
against MMP-2. Levy et al. have reported P30 SARs in a
related series (7) and observed a loss of potency against
both MMP-2 and 3.10 The same stereochemical pref-
erence was observed for 1i±l. It was interesting that the
tertiary P30 groups in 1m and 1o did not increase the
MMP-3 selectivity, whereas the benzyhydryl analogue
1n was the most selective of all. Workers at British Bio-
tech have independently reported that the benzhydryl
group can increase selectivity for MMP-3: for example,
compound 8.8b
Two routes were used to access intermediates 3a±o. In
the ®rst, coupling of N-Boc-tert-leucine with various P30
amines (EDC, HOBt, Et3N or i-Pr2NEt, CH2Cl2) fol-
lowed by removal of the Boc protection a€orded amines
4. However, coupling of tertiary amines by this method
led to signi®cant racemisation, but this was readily
overcome by replacing HOBt with HOAt.12 The amines
4 were then coupled to acid 5.13 Alternatively, coupling
tert-leucine benzyl ester with 5 followed by hydro-
genolysis gave acid 6 which was coupled with amines
without epimerisation using Carpino's conditions.14
Deprotection of the t-butyl esters 3a±o a€orded the
carboxylic acids 2a±o which were converted in two steps
to the hydroxamic acids 1a±o by reaction with O-allyl-
hydroxylamine followed by palladium catalysed reductive
removal of the allyl group.15
Results and Discussion
Inhibition of MMP-3 and MMP-2 was measured by
the method of Knight et al.16 (with minor modi®ca-
tions) including using the ¯uorogenic peptide substrate
Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)
17
-NH2 which has approximately equal kcat/Km values
for each enzyme and results are shown in Table 1. The
selectivities shown are expressed as the ratios of the two
IC50s (MMP-2/MMP-3), and ratios for MMP-3 selec-
tive compounds are indicated in bold type. Our starting
point for exploration of SAR was the N-methyl analo-
gue 1a13 which is highly selective for MMP-2. The
phenpropyl P10 group was chosen because it
enhanced MMP-3 inhibition,6 and we kept P20=t-
Broadly similar potency and selectivity trends were
observed for the corresponding carboxylic acids 2a±o,
except that the potency against both enzymes was mark-
edly lower than the hydroxamates. Selectivity for MMP-2
was also higher for the carboxylates, with only the benz-
hydryl analogue 2n showing any selectivity for MMP-3.
Scheme 1. Reagents and conditions: (a) RNH2, EDC, HOBt, Et3N or i-Pr2NEt, CH2Cl2, 20 ꢀC, 18 h, 73±96%; (b) RNH2, EDC, HOAt, Et3N or
i-Pr2NEt, CH2Cl2, 38±96%; (c) HCl(g), CH2Cl2 or EtOAc, 0 ꢀC, 82±100%; (d) TFA, CH2Cl2, 0 ꢀC, 78% for 4o (only example); (e) EDC, HOBt,
i-Pr2NEt, CH2Cl2, 20 ꢀC, 18 h, 61±100%; (f) H2 (3 bar), Pd/C, EtOH/H2O, 20 ꢀC, 99%; (g) RNH2, PyAOP, collidine, CH2Cl2, 0±20 ꢀC, 3±18 h,
86±97%; (h) HCl(g), CH2Cl2 or EtOAc, 0±20 ꢀC, 81±100%; (i) TFA/CH2Cl2 (1:1), 20 ꢀC, 4 h,À67±100%; (j) O-allylhydroxylamine, EDC, HOBt
or HOAt, DMF, 20 ꢀC, 18 h or PyAOP, i-Pr2NEt, 20 ꢀC, 3±8 h, 22±95%; (k) NH+4 HCO2 , cat. Pd(OAc)2 2PPh3, EtOH/H2O (4:1), re¯ux,
.
30min±2 h, 35±90%. EDC= N-ethyl-N0-dimethylaminopropylcarbodiimide, HOBt=1-hydroxy-1H-1,2,3-benzotriazole, HOAt=7-aza-1-hydroxy-
1,2,3-benzotriazole, PyAOP=7-azabenzotriazol-1-yloxytris(pyrrolidino)phosphonium hexa¯uorophosphate.

M. J. Fray et al. / Bioorg. Med. Chem. Lett. 11 (2001) 567±570
569
Table 1. Inhibition of MMP-3 and MMP-2 by succinyl hydroxamates 1a±o and related acids 2a±o
1 X=NHOH
2 X=OH
MMP-2
IC50 (nM)
Cmpd
R
MMP-3
IC50 (nM)
MMP-2
IC50a (nM)
Selectivity ratio
MMP-2/MMP-3
MMP-3
IC50 (nM)
Selectivity
ratio
MMP-2/MMP-3
a
b
c
d
e
f
CH3
Ph
4-Pyridyl
c-C5H9
c-C6H11
t-Bu
48
13
4.4
6.8
19
21
0.34
1
0.2
1.6
1.3
5.8
1/141
1/13
1/22
1/4.2
1/15
1/3.6
410
475
275
11
1/37
301/17
6.4
54
1/46
1/63
3400
56038
10,000
1/15
84
50
1/15
1/56
g
h
i
9
0.38
1/24
1.5/1
1/5.5
1.7/1
1/1.5
4.4/1
2800
4061
18
53501580
1/3.4
330
3.3
5000
6100
8500
3200
1/15
1/1.2
1/1.4
1.1/1
j
4062
22
5100
6200
3500
k
l
15
25
110
m
n
o
C(CH3)2Ph
CHPh2
C(CH3)Ph2
73
48
570
3.4
414
26
1/21
8.6/1
1/22
>10,000
625
>10,000
280
2430
780
1/>35
3.9/1
1/>13
aIC50s are an average of two determinations.
of MMP-3 which show the S30 binding site to be open
and solvent exposed. However, potency of inhibition of
MMP-2 can be reduced by up to 1000-fold through a
suitable choice of an a-substituted benzylic group.
Where the substituent is not phenyl, the chirality of the
Conclusions
We have shown that varying the size of the P30 group
has relatively little impact on the potency of MMP-3
inhibition in accord with X-ray crystallographic structures

570
M. J. Fray et al. / Bioorg. Med. Chem. Lett. 11 (2001) 567±570
P30 group is critical. Whilst we do not have X-ray
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11 and the resulting steric clash reduces binding anity.
It is striking that the SARs at P30 are di€erent to 7,10
suggesting that the choice of the other substituents (P10,
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Acknowledgements
The authors wish to thank Mrs. G. B. Easter, Mrs. E. J.
Fairman and Mr. D. P. Winslow for measuring enzyme
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M. Sproates for preparing the compounds, Dr. A. Alex
for molecular modelling studies and sta€ of the Physical
Sciences Dept. for measuring spectroscopic data.
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