A new development of matrix metalloproteinase inhibitors: Twin hydroxamic acids as potent inhibitors of MMPs

Article abstract of DOI:10.1016/j.bmcl.2005.03.002

Starting from the observation that the CbzNH(CH₂)₂ side chain of the potent MMP-2/MMP-14 inhibitor, benzyl-(3R)-4-(hydroxyamino)-3- [isopropoxy(1,1′-biphenyl-4yl-sulfonyl)amino]-4-oxobutylcarbamate, (R)-1 lies in a hydrophobic region

Full text of DOI:10.1016/j.bmcl.2005.03.002

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2311–2314
A new development of matrix metalloproteinase inhibitors: twin
hydroxamic acids as potent inhibitors of MMPs
Armando Rossello,a,* Elisa Nuti,a Maria Pia Catalani,a Paolo Carelli,a
Elisabetta Orlandini,a Simona Rapposelli,a Tiziano Tuccinardi,a Susan J. Atkinson,b
Gillian Murphyb and Aldo Balsamoa
a
`
Dipartimento di Scienze Farmaceutiche, Universita degli Studi di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
bDepartment of Oncology, University of Cambridge, Hills Road, Cambridge CB2 2XY, UK
Received 25 November 2004; revised 24 February 2005; accepted 2 March 2005
Available online 30 March 2005
Abstract—Starting from the observation that the CbzNH(CH2)2 side chain of the potent MMP-2/MMP-14 inhibitor, benzyl-(3R)-4-
(hydroxyamino)-3-[isopropoxy(1,10-biphenyl-4yl-sulfonyl)amino]-4-oxobutylcarbamate, (R)-1 lies in a hydrophobic region (S1)
exposed to the solvent of the protease active site, we hypothesized that an aminoethylcarboxamido chain structurally related to that
of (R)-1 might be an useful tool to bind another linker stretching out from the protein. This would be able to interact either with a
enzyme region adjacent to the active site, or with other molecules of matrix metalloproteinases (MMPs), or other proteins of the
extracellular matrix (ECM) that may be involved in the enzyme activation. On these basis we describe new dimeric compounds
of type 2, twin hydroxamic acids, obtained by the joint of two drug entities of (R)-1 linked in P1 by extendable semirigid linkers.
Type 2 compounds are potentially able to undergo more complex inhibitor–enzyme interactions than those occurring with mono-
meric compounds of type 1, thus influencing positively the potency, selectivity and/or cytotoxicity of the new compounds.
Ó 2005 Elsevier Ltd. All rights reserved.
During tumour progression, an overexpression of ma-
trix metalloproteinases (MMPs) is often responsible
for the deregulation of extracellular matrix (ECM) func-
tions.1,2 Normally, the homeostasis of MMPs is main-
tained by tissue inhibitors (TIMPs), but in cancer
progression, control over MMPs activity is lost.3 Other
proteases, such as uPA, MMPs and furin-like serine pro-
teases, are responsible for the activation of specific
MMPs, such as MMP-2 (gelatinase A), MMP-9 (gelatin-
ase B) and MMP-14 (a membrane-associated MMP,
which is the principal activator of pro-MMP-2). These
three specific MMPs play a significant role in some neo-
plastic processes, and are directly involved in metastatic
tumour dispersion and angiogenesis.4–8 More recently,
MMP-2 has been shown to play other important roles
in tumours, for example, increasing the resistance to
apoptosis, activating EGF receptors and promoting cel-
lular proliferation.9–12 All these data, taken together,
indicate that MMP-2, MMP-9 and MMP-14 may repre-
sent important targets to develop new potential antican-
cer drugs.13–21 As
a matter of fact, the recent
development of some synthetic MMPi (matrix metallo-
proteinase inhibitors) possessing a good potency and
selectivity towards the two gelatinases A and B, together
with the discovery that some of these inhibitors that are
active on MMP-2 exhibit important pro-apoptotic ef-
fects on tumour cell cultures, has confirmed the possibil-
ity of their potential use as anti-tumour agents.22–29
Nowadays, new compounds possessing inhibitory activ-
ity and selectivity on the MMPs, which are over-ex-
pressed in some kinds of tumours, are viewed as useful
tools in particular with a view to the control of the
viability and invasiveness of cancer cells.2,15
In previous studies on new MMPi, we developed a class
of arylsulfonamido-based hydroxamic acid inhibitors of
type A, substituted on their sulfonamido nitrogen with
an oxyalkyl side chain, instead of the hydrogen atom
of type B compounds, or an alkyl side chain of type C
compounds. This simple structural modification allowed
us to obtain new potent and selective inhibitors of
MMP-2 and MMP-9, which are able to block in vitro
Keywords: MMP-inhibitors; MMP-2/MT1-MMP selective inhibitors;
Antiangiogenic agents; Twin hydroxamic acids; MMPi; Arylsulfon-
amido-based hydroxamate.
*
Corresponding author. Tel.: +39 050 2219562; fax: +39 050
2219605; e-mail: aros@farm.unipi.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.002

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A. Rossello et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2311–2314
tumour cell invasion.30 Among the two type
A
rigidity to the linker chain and potentially to interact
with appropriate parts of the enzyme (Fig. 2).32
inhibitors, the ones with the (R) configuration, which
bear on the carbon alpha to the hydroxamic group an
alkyl group (R) of increasing hindrance, showed better
biopharmacological properties with respect to the
unsubstituted analogues. In particular, the compound
of type A in which R = (CH2)2NHCbz R1 = i-Pr and
R2 = Ph ((R)-1) showed a very good MMP inhibitory
There are numerous examples, in the field of drug re-
search, of twin drugs containing two pharmacophoric
groups linked in a single molecule, some of the hydrox-
amic acid type, but none in the field of MMPi.33–36
profile, with
a
high potency towards MMP-2
The synthesis of the two twin inhibitors (R,R)-2a and
(R,R)-2b is outlined in Scheme 1. As a start, the Cbz
derivative (R)-4 was hydrogenated with H2 and Pd/
10% on charcoal in a 1:1 MeOH/AcOH mixture to yield
the deprotected salt (R)-5.31 Acylation of (R)-3 with the
appropriate acyl dichloride (6a) or (6b), in anhydrous
DMF in the presence of N-methylmorpholine (NMM),
gave the dimeric tert-butyl esters (R,R)-7a and (R,R)-
7b, respectively. Acidic cleavage of 7a and 7b to acids
(R,R)-8a and (R,R)-8b, followed by their reaction with
O-(tert-butyldimethylsilyl)hydroxylamine (TBDMSO–
NH2) in the presence of EDCI, gave the O-silylate bis-
hydroxamate (R,R)-9a and (R,R)-9b. Acid cleavage of
(R,R)-9a,b yielded the corresponding bishydroxamic
acids (R,R)-2a and (R,R)-2b. The acid dichloride 6b,
used for the synthesis of (R,R)-7b, was prepared by acyl-
ation of 4-aminobutyric acid 10 with isophthaloyl
dichloride 6a, to yield the symmetrical diacid 11b, which
was then converted to 6b with oxalyldichloride in a pyr-
idine/toluene mixture.
(IC50 = 0.41 nM), MMP-9 (IC50 = 16 nM) and MMP-
14 (IC50 = 7.7 nM), and was able to block angiogenesis
in the chemioinvasion model on HUVEC cells com-
pletely, at a submicromolar concentration.31 A docking
study carried out on the complex of (R)-1 with MMP-2
showed that its CbzNH(CH2)2 side chain lies in a hydro-
phobic region of the active site exposed to the solvent
(S1) (see green arrow in Fig. 1), without hindering the
fit of the inhibitor with the enzyme.32
This observation prompted us to hypothesize that an
aminoethylcarboxamido chain structurally related to
that of (R)-1 might be a useful tool to bind another lin-
ker stretching out from the protein, able to interact
either with an enzyme region adjacent to the active site,
or with other MMPs molecules or other proteins of the
ECM, which may be involved in the enzyme activation.
We believe that the possibility, for the dimeric com-
pounds of type 2, to give an inhibitor–enzyme interac-
tion more complex than that allowed for monomeric
compounds of type 1, may influence positively the po-
tency and/or selectivity of the new compounds, com-
pared with those of 1. As a result, we designed the
new twin inhibitors (R,R)-2a,b, formally obtained by
linking two of the fundamental frames (R)-331 of (R)-1
by a spacer like an isophthaloyl group [(R,R)-2a] or an
N,N0-bis(4-aminobutanoyl)-isophthalamide moiety [(R,R)-
2b], in order to study the effects of this type of structural
manipulation on the MMP-2/MMP-9/MMP-14 inhibi-
tory activity of compounds of type 1. As a core, in the
linker in this study, we used the isophthaloyl group,
either simple or symmetrically linked to two c-aminobu-
tyric portions, in view of its ability to confer a partial
The bishydroxamates (R,R)-2a and (R,R)-2b, the di-
meric acid (R,R)-8a, a precursor of (R,R)-2a, and the
monomeric reference inhibitors (R)-1, whose molecular
skeleton is duplicated in the twin inhibitor (R,R)-2a,b,
Figure 1. Docking of MMP-2 with monomeric inhibitor (R)-1.
Figure 2.

A. Rossello et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2311–2314
2313
Scheme 1. Reagents and conditions: (i): H2, Pd/C 10%, MeOH, AcOH; (ii) 6a or 6b, NMM, anhyd DMF; (iii) TFA, anhyd DCM; (iv)
TBDMSONH2, EDCI, anhyd DCM; (v) TFA, anhyd DCM; (vi) H2N(CH2)3CO2H, (10) NaOH, Et2O, H2O; (vii) C2O2Cl2, Py, toluene.
were tested on some MMPs (1, 2, 9 and 14) to evaluate
their inhibitory properties.30
Both (R,R)-2a and (R,R)-2b are less selective than (R)-
1 on the MMP-2 enzyme.
The results, expressed in terms of their IC50 and selecti-
vity for MMP-2, are reported in Table 1.
As expected for a compound lacking the hydroxamic
acid moiety, the carboxylic analogue of (R,R)-2a
((R,R)-8a) proved to be practically inactive towards
the two MMPs screened, MMP-2 and MMP-9.
For MMP-2, the two dimers (R,R)-2a and (R,R)-2b
show an IC50 in the nM range, which is 480- and 24-fold
more than that of the monomeric inhibitor (R)-1,
respectively. In the case of MMP-1, only (R,R)-2b shows
an appreciable activity, with a submicromolar IC50 va-
lue, which is 40-fold higher than that displayed towards
MMP-2, whilst (R,R)-2a and (R)-1 are practically
inactive.
The more active of the two twin inhibitors ((R,R)-2b)
was evaluated for cytotoxicity by means of the Trypan
Blue assay on HT1080 cells, in comparison with the
monomeric analogue (R)-1 (see Table 2).37
As can be seen, (R,R)-2b is well tolerated by the cells at a
dose lower than 1 lM; cytotoxic effects for (R,R)-2b
become significant at a dose of 10 lM, where this com-
pound shows 10% dead cells. At this same dose, the
monomeric analogue (R)-1 is about four times more
toxic, with 35% dead cells.
The two dimers (R,R)-2a and (R,R)-2b show an IC50 va-
lue in the submicromolar range against MMP-9, but in
this case dimer (R,R)-2b is twice as potent as (R)-1, while
(R,R)-2a proves to be less potent. Both (R,R)-2a and
(R,R)-2b show an MMP-9/MMP-2 selectivity lower
than (R)-1. As regards MMP-14, the twin inhibitor
(R,R)-2b shows a high inhibitory potency, albeit with
an IC50 value four times higher than that of the refer-
ence compound (R)-1; on the contrary, compound
(R,R)-2a shows an IC50 value in the micromolar range.
These preliminary results indicate that, even if the
dimerization of MMP inhibitors of type A, like (R)-1,
appears to be unable to enhance the selectivity of the
new compounds towards the MMP screened, neverthe-
less, at least in the case of (R,R)-2b, it appears to
Table 1. MMPi profile of (R,R)-2a,b, -8a dimers and monomeric inhibitor (R)-1
Compound
IC50 nM ( SD)a
MMP-1
MMP-2
197 42
MMP-9
MMP-14
(R,R)-2a
(R,R)-2b
(R,R)-8a
(R)-1
10,000 1333 (50)b
397 79 (40)b
n.t.
983 40 (4.9)b
8.3 1.4 (0.8)b
>30,000
1490 130 (7.6)b
34 14 (3.5)b
n.t.
9.8
>1000
2
>3000 (>7300)b
0.41 0.01
16 2 (39)b
7.7 2 (18.7)b
a MMPi assay.30
b Selectivity for MMP-2 over each of the other MMPs is expressed as the ratio of the IC50 value for MMPn over the value for MMP-2.

2314
A. Rossello et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2311–2314
Table 2. Cytotoxicitya with twin inhibitor (R,R)-2b and monomeric
inhibitor (R)-1
23. Wada, C. K.; Holms, J. H.; Curtin, M. L.; Dai, Y.;
FlorJancic, A. S.; Garland, R. B.; Guo, Y.; Heyman, H.
R.; Stacey, J. R.; Steinman, D. H.; Albert, D. H.; Bouska,
J. J.; Elmore, I. N.; Goodfellow, C. L.; Marcotte, P. A.;
Tapang, P.; Morgan, D. W.; Michaelides, M. R.; David-
sen, S. K. J. Med. Chem. 2002, 45, 219.
24. Supuran, C. T.; Casini, A.; Scozzafava, A. Med. Res. Rev.
2003, 23, 535.
25. Zook, S. E.; Dagnino, R., Jr.; Deason, M. E.; Bender, S.
L.; Melnick, M. J. WO Patent 97/20824; 1997, 127,
108945. Chem. Abstr. 1995, 123, 2870.
Compound
% Dead cellsb
1 lM
5 lM
10 lM
(R,R)-2b
(R)-1
0
4
5
20
10
35
a Trypan Blue test on HT1080 cells.
b Dead cells over total cells, living and dead, percentage at dose
screened.37
26. Supuran, C. T.; Scozzafava, A. In Proteinase and Pepti-
dase Inhibitors: Recent Potential Targets for Drug Devel-
opment; Smith, H. J., Simons, C., Eds.; Taylor & Francis:
London & New York, 2002; pp 35–61.
27. Bernardo, M. M.; Brown, S.; Li, Z.-H.; Fridman, R.;
Mobashery, S. J. Biol. Chem. 2002, 277, 11201.
28. Rabbani, S. A.; Harakidas, P.; Guo, Y.; Steinman, D.;
Davidsen, S. K.; Morgan, D. W. Int. J. Cancer 2000, 87,
276.
improve the activity on MMP-1 and MMP-9. Moreover,
dimerization of (R)-1 to (R,R)-2b seems to reduce the
cellular cytotoxicity of this new class of twin inhibitors
appreciably.
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37. HT1080 cells stably transfected to overexpress MT1 MMP
were seeded into 24-well culture dishes at 105 cells/well in
selection medium and incubated at 37 °C overnight. The
following day, cells were washed twice to remove serum
and then incubated in 300 lL/well DMEM with insulin,
transferrin and selenium supplements (Sigma, UK) added.
Test compounds ((R,R)-2b, (R)-1) at 0, 1, 5 and 10 lM
were added to duplicate wells. After a further 24 h, media
were removed for zymographic analysis and the cells were
washed in PBS. The cells from each well were harvested by
trypsinization, resuspended in PBS and mixed 1:1 with
trypan blue (0.4% w/v, Sigma, UK). After 5 min, a sample
of cell suspension from each well was removed and
introduced into a haemocytometer. The total number of
cells in the upper grid was counted and a separate count
was taken of the cells, which had taken up the blue dye,
that is, the dead cells. The process was repeated by
counting the cells in the lower grid. Percentage toxicity for
each compound at the different concentrations was calcu-
lated as a mean of the values for duplicate wells, after
subtracting the values for the wells without either
compound.
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