Improved gelatinase a selectivity by novel zinc binding groups containing galardin derivatives

Article abstract of DOI:10.1016/S0960-894X(03)00214-2

The synthesis of several analogues of galardin, a MMP inhibitor, are presented with their in vitro inhibitory activity against MMP-1 and MMP-2. These compounds contain a distinct Zinc Binding Group (ZBG). Those having a 2-acylated-heterocycle as well as a 2-arylamide function do not exhibit a good inhibition/selectivity against the enzymes tested. On the contrary, those that are based on a hydrazide scaffold present potent selectivity for MMP-2 versus MMP-1.

Full text of DOI:10.1016/S0960-894X(03)00214-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1783–1786
Improved Gelatinase A Selectivity by Novel Zinc Binding Groups
Containing Galardin Derivatives
Franck Auge,a William Hornebeck,b Martine Decarmeb and Jean-Yves Laronzea,*
a
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UMR 6013 ‘Isolement, Structure, Transformation et Synthese de Produits Naturelles’, Faculte de Pharmacie, IFR53 Biomolecules,
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Universite de Reims Champagne-Ardenne, 51 Rue Cognacq Jay, 51096 Reims Cedex, France
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b
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FRE 2534 CNRS, Faculte de Medecine, IFR53 Biomolecules, Universite de Reims Champagne-Ardenne,
51 Rue Cognacq Jay, 51096 Reims Cedex, France
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Received 3 December 2002; revised 21 February 2003; accepted 21 February 2003
Abstract—The synthesis of several analogues of galardin, a MMP inhibitor, are presented with their in vitro inhibitory activity
against MMP-1 and MMP-2. These compounds contain a distinct Zinc Binding Group (ZBG). Those having a 2-acylated-hetero-
cycle as well as a 2-arylamide function do not exhibit a good inhibition/selectivity against the enzymes tested. On the contrary,
those that are based on a hydrazide scaffold present potent selectivity for MMP-2 versus MMP-1.
# 2003 Elsevier Science Ltd. All rights reserved.
Zn-metalloproteases have been, for a long time, tar-
gets for new drugs. The major example was the pio-
neering work of Ondetti1 on inhibitors of angiotensin
converting enzyme, which led to the first anti-
hypertensive drug of this family (Captopril1, Fig. 1).
Nearly all the zinc-containing metalloprotease inhibi-
tors are designed around, at least, two pharmaco-
phores, a Zinc Binding Group (ZBG) and a peptide-
like group. These compounds behave as competitive
inhibitors, providing that the latter group can fit into
one or more substrate recognizing subsites of the target
enzyme and that the ZBG binding mode can mimic one
of the transition states occurring during the substrate
hydrolysis.
Figure 1. Examples of Zn-metalloprotease inhibitors containing a
ZBG.
countries. As a consequence, the hydroxamic acid func-
tion is no longer present in any major drug.
To avoid the use of hydroxamic acid in matrix metallo-
protease (MMP) inhibitors, other ZBG containing
compounds as carboxylate, phosphonate, thiol . . . were
synthesized. However, associated with this structural
change, there is an important loss of activity compared
to the hydroxamate counterpart.3 In continuation of
our work on MMP4 and MMP inhibitors,5 we have
synthesized and evaluated the MMP inhibitory capacity
of galardin6 derivatives having novel ZBGs which theo-
retically might behave as potent chelating agents sug-
gested by our previous calculations. Our challenge was
to find a new organic group able to bind to the catalytic
zinc ion of MMP with, not only a good affinity for this
site, but also a good specificity, in the view to avoid
undesirable effect, inherent in this type of inhibitor.7
Hydroxamic acid was soon found to be one of the most
powerful ZBG. However the use of this ZBG in drugs
led to extreme undesirable effects, due to the poor
bioavailability of such function and especially to its high
transition-metal binding ability. For example, bufex-
amac2 (p.butoxybenzylhydroxamic acid, Parfenac1), a
non-steroidal anti-inflammatory drug, typically used in
mild skin disorders, sometimes exhibit undesirable
effects by initiating multiform exudative erythema and
was therefore withdrawn from the market in many
*Corresponding author. Tel.: +33-326913589; fax : +33-326918025;
e-mail: jy.laronze@univ-reims.fr
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00214-2

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F. Auge et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1783–1786
1784
Although clinical trials have proved MMP inhibitors to
fail in last-stage metastatic cancers,8 it must be kept in
mind that they could be useful in the first stage of tumor
progression, provided they display MMP selectivity, in
order to preclude extreme side effects like musculoske-
letal ones.
Compounds 2a10 and 2b have been synthesized follow-
ing the method of Hoffman et al.,11 starting from arylic
ketones 3a and 3b (Scheme 1). As known procedures12
to prepare 4 from 3 were ineffective, we adapted the
Krapcho’s method13 for our purpose. The coupling of
the known chiral triflate14 5 gave b-dicarbonyl com-
pounds 6, further hydrolyzed and decarboxylated into
acids 7 with acceptable yield, without any racemization
of the surviving chiral center. Derivatives 7 were cou-
pled with N-methyltryptophanamide already synthe-
sized in our laboratory.5b
In this letter, we described our preliminary results on
new non-hydroxamate MMP antagonists, with selectiv-
ity towards MMP-2 versus MMP-1. Three types of
potential ZBGs have been devised and are shown in
Figure 2: 1–4 ligands where X could be a hard or soft
heteroatom (family A), 1–5 ligands (family B), hydra-
zides (family C), compared to hydroxamate (compound
D). All these ligands are mimics of one transition state
encountered in the MMP hydrolysis mechanism path-
way, according to Lovejoy et al.9
The above method could not be used for the pyridine
derivative 2c, because the intermediate 6c gave only very
small amounts of targeted acid 7c due to the hydroxide
induced the cleavage of the b-dicarbonyl system in this
case. Finally, 7c was prepared following Scheme 2 via
tert-butylester
9c.
Amidation
by
N-methyl-
tryptophamide did not proceed with DCC-HOBt as well
as with other more or less classical coupling reagent
usual in peptide syntheses. Fortunately the reaction
worked conveniently with 4-(4,6-dimethoxy-1,3,5-tria-
zin-2-yl)-methylmorpholinium chloride (DMTMM)
developed by Kunishima et al.15
Chemistry
Potential A-type ligands: 2-acylated heteroaromatic
compounds (2) (Fig. 3).
Scheme 1. Synthetic route to prepare compounds 2a and 2b. (i) NaH,
CO(OMe)2; (ii) NaH, 5, THF; (iii) LiOH, H2O, 0 ꢀC then PhH reflux;
(iv) N-methyltryptophanamide, DCC-HOBt, CH2Cl2.
Figure 2. Possible binding mode of the different ZBG described in this
paper.
Scheme 2. Synthetic route to prepare the compound 2c. (i) COlm2,
LDA-THF ꢁ78 ꢀC, MeCO2tBu; (ii) NaH, THF, 5; (iii) 1M TFA in
CH2Cl2 then LiOH; (iv) N-methyltryptophanamide, DMTMM,
MeOH.
Figure 3. Compounds belonging to family A.

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F. Auge et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1783–1786
1785
The synthesis of pyrrole derivative 2d cannot be carried
out by the above methods, since it needs protection of the
pyrrole nitrogen by a benzyl group which we were unable
to remove at the last stage of the synthesis. So, in view of
that, pyrrole was introduced in the latter stages of the
synthesis, thus avoiding its protection (Scheme 3).
Evan’s Chiral oxazolidine 11d was alkylated to the tert-
butylester 12d with complete control of the chirality of
the new center. The cleavage of the tert-butyl group
gave the acid 13d, which was further transformed into
morpholinamide 14d. The Vilsmeier–Haack arylation
and oxazolidine removal was performed in the same
experiment, unfortunately in low yield. Finally 15d was
coupled to N-methyltryptophanamide with DMTMM.
Scheme 4. Synthetic route to prepare compounds 16a, 16b, 16c. (i)
LiOH, H2O2 in THF/H2O, 0 ꢀC; (ii) N-methyltryptophanamide,
DMTMM, THF; (iii) 1M TFA in CH2Cl2, reflux; (iv) DMTMM +
conditions (see text).
Potential B-type ligands: arylamides 16 (Fig. 4).
They were prepared along Scheme 4: Evan’s chiral oxa-
zolidine 12d was cleaved to the acid ester 17, which was
coupled with N-methyltryptophanamide in the presence
of DMTMM. The tert-butyl group was removed by
TFA, in refluxing methylene chloride, giving acid 18.
Arylamide 16c was smoothly obtained from 18 and
2-aminothiazine in THF which was not the case for
weekly basic aniline and 2-aminopyridine, which are not
able to deprotonate acid 18 into its carboxylate coun-
terpart. Adding N-methylmorpholine (NMM) overcame
this problem, giving 16a and 16b in acceptable yields.
Potential C-type ligands: hydrazides 19 (Fig. 5).
Hydrazides 19b and 19c were prepared in low yield from
acid 18, via DCC-HOBt coupling of methylhydrazine
and phenylhydrazine, respectively.
Hydrazine itself did not cleanly react with 18 in these
conditions, but 19a was obtained in 45% yield from 18,
using the DMTMM/NMM system in THF. The same
approach allowed smooth preparation of sulfonylhy-
drazide 19d in 67% yield (DMTMM/NMM in THF). For
the best of our knowledge, DMTMM-assisted synthesis of
hydrazides has, until now, not been documented.
Biology
Preliminary assay was performed by gelatin zymo-
graphy on compounds 2, 16, 19 and also on galardin 1
and its carboxylic analogue 18 as comparison models.
All compounds were incorporated into the incubation
buffer at concentration from 10ꢁ4 to 10ꢁ7 mol Lꢁ1. We
used conditioned medium from HT 1080 fibrosarcome
cells as a source of gelatinases (MMP-2 and MMP-9).
Compounds 2a, 2b, 16a, 16c, 19b, 19c proved to be non
inhibitory in such an assay and, thus, were not con-
sidered for the determination of IC50 using synthetic
Scheme 3. Synthetic route for the compound 2d. (i) ref 16: (ii)
BrCH2CO2tBu, HMDS ꢁ78 ꢀC; (iii) TFA 1M CH2Cl2; (iv) DCC-
HOBt, morpholine; (v) 1/POCl2, 2/pyrrole, 3/LiOH, H2O2; (vi)
N-methyltryptophanamide, DMTMM.
Figure 5. Compounds belonging to family C.
Figure 4. Compounds belonging to family B.

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F. Auge et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1783–1786
1786
Table 1. Analytical data and biological activities on the most active compounds, compared to our model 18 and galardin
21
½aꢂD (solvent)a
Compd
Mp ꢀC
UV (lmax, nm)
IC50 MMP-1 (nM)
IC50 MMP-2 (nM)
2c
2d
16b
19a
19d
18
79–80
109–110
87–88
187–188
192–193
ꢁ8.6 (CH2Cl2)
+ 20.9 (CH2Cl2)
ꢁ28.5 (MeOH)
+ 7.2 (DMSO)
ꢁ8.3 (MeOH)
222,270,290
202,246,287
205,223,278,291
205,222,275,283,291
204,222,280,292
730
860
60
180
540
50
550
30
50
>104
6
20
Galaradin
3
0.4
ac=10 mg.mLꢁ1
.
substrates. Inhibition of recMMP-1 and recMMP-2
(VWRCalbiochem) by galardin derivatives was deter-
mined using DNP-Pro-b-cyclohexylAla-Gly-Cys(Mes)-
His-Ala-Lys(N-methylaminobenzoyl)-NH2 (VWRCal-
bio-chem) and MCA-Pro-Leu-Gly-Leu-Dap(Dnp)-
Ala-Arg-NH2 (Bachem), respectively, according to pre-
vious published procedures.5a,5c
Acknowledgements
The authors thank the Association pour la Recherche
sur le Cancer (ARC) for its financial support No. 9488.
References and Notes
Experiments were performed at 37 ꢀC, in a 50 mM Tris/
HCl, 150 mM NaCl and 5 mM CaCl2, pH 7.5 buffer,
under steady state conditions, and IC50 values are
reported in Table 1.
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Discussion
Clearly A-type potential ZBG are not effective as MMP-
inhibitors with the exception of compound 2c. This is in
spite of our calculation predicting a high stabilization of
the complex Zn2+(His)3 by 2-acylpyridine 2d. This lack
of activity cannot be solely attributed to the bulkiness of
the pyridine ring compared to the hydroxamate group,
but rather to the greater conformational rigidity of these
aromatic derivatives versus the galardin that hampers
the access of inhibitor to its target (unpublished mole-
cular mechanic results).
In the B family of ligands, 16b showed increased activ-
ity, comparatively to its A counterpart, 2c. This could
be rationalized as being due to the extra hydrogen bond
made with the carboxylate of the Glu residue involved
in the hydrolysis mechanism (Fig. 2).
6. Galardy, R. E. Drugs Future 1993, 18, 1109.
7. Giavazzi, R.; Taraboletti, G. Crit. Rev. Oncol. Hematol.
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8. Mikala, E.; Zena, W. Nat. Rev. Cancer 2002, 2, 163.
9. Lovejoy, B.; Hassel, A. M.; Luther, M. A.; Weigl, D.; Jor-
dan, S. R. Biochemistry 1994, 33, 8207.
10. All the compounds have been analysed by IR, 1H and 13
NMR(including correlation spectra) and HREIMS. They
gave satisfactory microanalyses.
C
Much more interesting is the sulfonylhydrazide 19d
nearly 20 times more active than the non-sulfonylated
derivative 19a, with a high selectivity toward MMP-2
versus MMP-1. Two factors can contribute to this
enhancement: the first one is the increased acidity of the
N–H, close to the SO2 electron-withdrawing group
which enhances H-bonding to the Glu residue (Fig. 2).
Such substitution also increases the partial negative
charge on this nitrogen strengthening the bonding to the
Zn ion.
11. Hoffman, R. V.; Kim, H.-O. J. Org. Chem. 1995, 60, 5107.
12. For example see: (a) Homeyer, A. H.; Groves, W. US
patent 2,630,437, 1953. (b) Levine, H.; Hauser, C. J. Am.
Chem. Soc. 1944, 66, 1768.
13. Krapcho, A. P.; Diamanti, J.; Caten, C.; Bingham, R.
Org. Synth. 1973, 198.
14. Bitan, G.; Gilon, C. Tetrahedron 1995, 51, 10513.
15. (a) Kunishima, M.; Kawashi, C.; Iwasaki, I.; Terao, K.
Tetrahedron Lett. 1999, 40, 5327. (b) Kunishima, M.; Kawa-
shi, C.; Morita, J.; Terao, K.; Iwasaki, I.; Tani, S. Tetrahedron
1999, 55, 13159.
16. Levy, D. E.; Lapierre, F.; Liang, W.; Ye, W.; Lange, C. W.;
Li, X.; Grobelny, D.; Casabonne, M.; Tyrrell, D.; Holme, K.;
Nadzan, A.; Galardy, R. E. J. Med. Chem. 1998, 41, 199.
17. Nemes, C.; Jeannin, L.; Laronze, M.; Seghir, H.; Auge, F.;
Laronze, J. Y. Tetrahedron 2000, 56, 5479.
Work is in progress to assign a more precise role to the
arylsulfonylhydrazide group to help enhance its MMP-2
affinity and selectivity. A structure activity study is also
in progress to optimize this compound by modifying the
terminal N-methyltryptophanamide group by unnatural
tryptophane derivatives.17