MMPs inhibitors: New succinylhydroxamates with selective inhibition of MMP-2 over MMP-3

Article abstract of DOI:10.1016/S0960-894X(03)00590-0

Some ilomastat analogues featuring an isobutylidene group or a 2-substituted indole nucleus were synthesized to evaluate their inhibitory activities against gelatinase A and stromelysin-1. Potent MMP-2 inhibition and good selectivity for that enzyme have

Full text of DOI:10.1016/S0960-894X(03)00590-0

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2843–2846
MMPs Inhibitors: New Succinylhydroxamates with Selective
Inhibition of MMP-2 over MMP-3
Valerie Marcq,a Catherine Mirand,a,* Martine Decarme,b
Herve Emonardb and William Hornebeckb
a
´
IFR 53, UMR/CNRS 6013, Faculte de Pharmacie, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
b
´ ´
IFR 53 FRE/CNRS 2534, Faculte de Medecine, 51 rue Cognacq-Jay, 51096 Reims Cedex, France
Received 11 December 2002; revised 30 April 2003; accepted 11 June 2003
Abstract—Some ilomastat analogues featuring an isobutylidene group or a 2-substituted indole nucleus were synthesized to evalu-
ate their inhibitory activities against gelatinase A and stromelysin-1. Potent MMP-2 inhibition and good selectivity for that enzyme
have been observed for compounds 1a, 2 and 22.
# 2003 Elsevier Ltd. All rights reserved.
In humans, the MMP family comprises at least 22
memberswhich can hydrolyze virtually any component
of the extracellular matrix.1 Their involvement in all
stages of cancer progression is now well documented
and intensive efforts have been made to design MMP
inhibitorsascancer therapeutical agents. However, data
from clinical trials have revealed undesirable side effects
which can be partly attributed to the broad spectrum
MMP inhibitory activity of those drugs.2
We therefore prepared ilomastat (galardin1) analogues
featuring an isobutylidene group at S10 (1) and some
0
modificationsat S2 and S30 subsites (2 and 3) in order
to increase their overall hydrophobicity.
Gelatinase A (MMP-2) plays an active role in both
tumor growth by favoring tumor angiogenesis and
matrix invasion of cancer cells as melanoma cells. Its
activation iscatalyzed mainly by MT and MT2 MMP
1
at the cell plasma membrane; on the contrary, other
MMPs are processed through proteolytic cascades
where stromelysin-1 (MMP-3) occupies a central posi-
tion. In previous investigations,3 we observed that long
chain unsaturated fatty acids, but not their saturated
counterparts, displayed MMP inhibitory activity.
Besides, those compounds were found to exhibit higher
inhibitory activity towardsMMP-2 ascompared to
MMP-34 although both MMPswere described to con-
tain deep S10 hydrophobic pockets. We thus anticipated
that introduction of an unsaturation at that site could
bring specificity to MMP inhibitors.
Chemistry
The access to the targeted molecules required the pre-
paration of alkylidenesuccinic monoesters (Scheme 1).
Our first attempts, using as the most direct approach,
the Stobbe condensation between diethylsuccinate and
isobutyraldehyde led to the expected half-ester with low
5
yield, asfrequently observed. So we turned to the Wit-
tig reaction, utilizing the process developed by Mc
Combie to prepare arylidenesuccinates.6 The phosphor-
ane generated in situ by reaction between methyl mal-
eate 4 and tri-n-butyl phosphine was reacted with
isobutyraldehyde to yield the dimethyl alkylidene succi-
nate 5 asa 9:1 E/Z mixture.7 We also applied the
*Corresponding author. Tel.: +33-326-913587; fax: +33-326-918029;
e-mail: catherine.mirand@univ-reims.fr
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00590-0

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V. Marcqet al. / Bioorg. Med. Chem. Lett. 13 (2003) 2843–2846
Emmons–Wadsworth–Horner reaction,8 following a
procedure described by Dhokte et al.9 The phosphonate
6 was condensed with isobutyraldehyde to yield the
diethyl alkylidene succinate 7 asa 1:2 E/Z mixture.
Both diesters 5 and 7 were then hydrolyzed and the
resulting diacids were dehydrated, thus leading to the
anhydrides 8a (single E-isomer) and 8b (E:Z mixture),
respectively. Finally, refluxing 8a in benzyl alcohol pro-
vided the monoester 9a asthe single E-isomer while 8b
treated by methanol or benzyl alcohol gave 9ba and 9bb
as E/Z mixtures.
geometry and (iii) the importance of an amide function
at the C-terminal part of the molecule.
Substituted indole dehydro derivatives such as 3 were
our next target. To thisend we applied a method
reported in a preceding paper:14 the pseudopeptide
backbone wasgenerated through the reaction of a suc-
cinyl aminomalonate with a quaternarized gramine15
and the hydroxamate wasintroduced at the beginning
of the reaction sequence (Scheme 3).16 Thus, the 2-(suc-
cinylamino)malonate 16 wasprepared by reaction of
9bb with diethyl aminomalonate. Unfortunately,
hydrogenolysis of the benzylic ester was accompanied
by hydrogenation of the double bond and led to the
racemic carboxylic acid 17. However, the synthesis was
continued, at aim to obtain substituted indole deriva-
The dehydro analoguesof ilomatsat
1a and 1b were
then prepared from succinates 9a and 9ba and (l)-tryp-
tophan methylamide 1010 via a classical pathway11
(Scheme 2). Coupling of 10 with monoester 9a mediated
by DCC gave the succinyltryptophanamide 12a. After
deprotection of the benzyl ester, the free acid 13a was
converted in two steps12 into the pure E-hydroxamic
acid 1a.13
tivesof ilomatsat. Thusthe benzyl hydroxamate
18
derived from 17 wascondenesd with Nb-methyl-tetra-
hydro-b-carboline methyliodide 19, affording the indole
derivative 20. Hydrolysis-decarboxylation of 20 fol-
lowed by amidation gave the methyl amide 21 which
wasconverted into the hydroxamic acid 22.17 This
compound, even asa mixture of four diastereomerswas
biologically assayed.
Alternatively, tryptophanamide 12b obtained from 10
and methyl monoester 9ba led to the E/Z 1:1 mixture 1b.
A similar sequence of reactions gave access to the E-
ethyl carboxylate 2 from ethyl tryptophanate 11 and 9bb
via 14.
Biological Evaluation
With the compounds 1a, 1b, 2 in hand we were able to
evaluate the influence of three parameters: (i) the pres-
ence of a double bond at P10, (ii) the effect of its
Compounds 1a, 1b, 2 and 22 were tested in vitro for
inhibition of MMP-2 and MMP-3 and compared to
ilomastat.18 The results are shown in Table 1.
Scheme 1. Reagents and conditions: (a) isobutyraldehyde, (n-Bu)3P, THF, 25 ꢀC, 96 h, 56%; (b) isobutyraldehyde, NaH, Et2O, 25 ꢀC, 3 h, 100%;
(c) NaOH, H2O, 25 ꢀC, 72 h, 75%; (d) AcCl, reflux, 3 h, 100%; (e) ROH, reflux, 55–61%.
ꢀ
ꢀ
.
Scheme 2. Reagentsand conditions: (a) DCC, CH 2Cl2, 25 C, 16 h, 49–61%; (b) H2, Pd/C, EtOH, 25 C, 3–4 h, 100%; (c) LiOH H2O, THF/H2O
ꢀ
ꢀ
ꢀ
.
(3:1), 25 C, 3 h, 96%; (d) NH2OBn HCl, Et3N, DCC, HOBt, CH2Cl2, 25 C, 16 h, 77–100%; (e) H2, Pd/C, EtOH, 25 C, 5.5 h, 90–100%.

V. Marcqet al. / Bioorg. Med. Chem. Lett. 13 (2003) 2843–2846
2845
Scheme 3. Reagentsand condition:s (a) diethyl aminomalonate, DCC, CH 2Cl2, 25 ꢀC, 2 h, 57%; (b) H2, Pd/C, EtOH, 25 ꢀC, 4.5 h, 79%; (c)
ꢀ
ꢀ
.
.
NH2OBn HCl, Et3N, DCC, HOBt, CH2Cl2, 25 C, 20 h, 84ꢀ%; (d) NaH, THF, reflux, 16 h, 83%; (e) LiOH H2O, THF/H2O (3:1), 25 C, 72 h, then
ꢀ
.
HCl!pH 4, reflux, 16 h, 69%; (f) MeNH2 H2O, EtOH, 25 C, 24 h, 59%; (g) H2, Pd/C, EtOH, 25 C, 4.5 h, 98%.
sistent with the work of Martin and colleagues20 on
other constrained inhibitors such as the cyclopropane
derivatives 24a and 24b.
Table 1. Inhibition data
Compd
MMP-2a
MMP-3b
Selectivityc
Ilomastatb
1a
1b
2
12b
22
0.104
1.3
19.6
5.97
NId
5.73
0.191
179
910
2340
NId
665
2
138
46
392
—
Replacement of the methylamide residue at P30 by an
ethoxycarbonyl group in compound 3 caused about a 5-
fold decrease in potency against MMP-2 but a sig-
nificant enhancement of selectivity.
116
Similarly, Castelhano and coworkers21 have reported
that replacement of the P20–P30 amide bond of ilomastat
by imidazole maintained potency against MMPs and
induced selectivity for MMP-1 and MMP-8 over
MMP-3.
aIC50 (nm): valuesare meansof three experiments.
bData generated in our laboratories.
cSelectivity: MMP-3; IC50; MMP-2 IC5O
dNI: no inhibition.
.
On the other hand, a selectivity for MMP-3 over MMP-
2 may be obtained by replacement of the methyl group
of methylamide by a benzhydryl22 or a-methylbenzyl23
group.
The dehydroderivative 1a waslespotent ascompared to
ilomastat against MMP-2 (10-fold higher IC50) and MMP-
3 (1000-fold higher IC50). A similar important decrease of
potency wasreported for the ‘dehydroilomatsat’
(ꢁ300-fold higher MMP-1 Ki).19
23
The substituted indole derivative 22 showed lower inhi-
bitory activities, but a selectivity for MMP-2 was also
observed. This result suggests that selectivity against
MMP-2 over MMP-3 can also be modulated by modi-
fication of the P20 group. Work isin progressto prepare
the single isomer with the ‘right’ configuration at the
two stereogenic centers.
To exclude the possibility that binding of compounds to
exosites distinct from the MMP active site might be
responsible for the observed effect, compound 12b
bearing a methoxy group in the hydroxamate moiety
4
However, introduction of an insaturation at S10 subsite
provided about a 100-fold greater selectivity for MMP-2
inhibition over MMP-3, that isa 70-fold increaes as
compared to ilomastat.
wasevaluated for itsMMP inhibitory capacity.
proved to be uneffective on both MMP-2 and MMP-3.
It
Although 1a, 2 and 22 are not aseffective asilomastat,
their selectivity for MMP-2 makes these derivatives
interesting for more detailed SAR studies.
Potency and selectivity depend on the geometry of the
double bond: the equimolar E/Z mixture 1b displayed
substancially lower inhibitory potency than the pure
E-isomer.
Acknowledgements
Compounds 1a,b can be considered as conformationaly
constrained analogues of ilomastat. The double bond
confersto the E-isomer 1a a conformation close to that
of the biological active conformation of ilomastat while
the Z-isobutylidene group cannot be easily accom-
modated in the S10 pocket. Thisobesrvation iscon-
We thank the ‘Ministere de l’Education Nationale, de la
Recherche et de la Technologie’ for financial support,
the ‘ville de Reims’ for a PhD fellowship (V.M.), P.
Sigaut (MS), and C. Petermann (NMR) for spectro-
scopic recordings. Fruitful discussions with Professor J.
Levy have been appreciated.

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V. Marcqet al. / Bioorg. Med. Chem. Lett. 13 (2003) 2843–2846
4.55–4.40 (m, 1H), 3.30–2.92 (m, 4H), 2.70–2.50 (m, 1H), 2.61
References and Notes
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1
13. 1a: H NMR (DMSO-d6) d 10.82 (s, 1H), 10.57 (brs, 1H),
8.87 (s, 1H), 8.14 (d, 1H, J=8 Hz), 7.92 (m, 1H), 7.62 (d, 1H,
J=7.7 Hz), 7.34 (d, 1H, J=7.7 Hz), 7.15 (s, 1H), 7.07 (t, 1H,
J=7.7 Hz), 6.99 (t, 1H, J=7.7 Hz), 6.04 (d, 1H, J=9.8 Hz),