Dual inhibitors of matrix metalloproteinases and carbonic anhydrases: Iminodiacetyl-based hydroxamate-benzenesulfonamide conjugates

Article abstract of DOI:10.1021/jm800964f

Matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) are two classes of zinc enzymes with different roles and catalytic targets, such as the degradation of most of the extracellular matrix (ECM) proteins and the regulation of the CO₂/

Full text of DOI:10.1021/jm800964f

7968
J. Med. Chem. 2008, 51, 7968–7979
Dual Inhibitors of Matrix Metalloproteinases and Carbonic Anhydrases: Iminodiacetyl-Based
Hydroxamate-Benzenesulfonamide Conjugates
Se´rgio M. Marques,† Elisa Nuti,‡ Armando Rossello,‡ Claudiu T. Supuran,§ Tiziano Tuccinardi,‡ Adriano Martinelli,‡ and
M. Ame´lia Santos*,†
Centro de Qu´ımica Estrutural, Instituto Superior Te´cnico, AV. RoVisco Pais 1, 1049-001 Lisboa, Portugal, Dipartimento di Scienze
Farmaceutiche, UniVersita` degli Studi di Pisa, Via Bonanno 6, 56126 Pisa, Italy, and Laboratorio di Chimica Bioinorganica, UniVersita` degli
Studi di Firenze, Room 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino (Firenze), Italy
ReceiVed July 30, 2008
Matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) are two classes of zinc enzymes with
different roles and catalytic targets, such as the degradation of most of the extracellular matrix (ECM)
-
proteins and the regulation of the CO2/HCO3 equilibrium in the cells, respectively. Both families have
isoforms which were proved to be involved in several stages of carcinogenic processes, and so the selective
inhibition of these enzymes might be of interest in cancer therapy. We report herein the design, synthesis,
and in Vitro evaluation of a series of compounds possessing the iminodiacetic acid as the main backbone
and two functional groups attached, namely, the hydroxamic acid and the arylsulfonamide (ArSO2NH2)
moieties, to enable the inhibition of MMPs and CAs, respectively. These compounds were demonstrated to
strongly inhibit both MMPs and CAs, some of them from the nanomolar to subnanomolar range. Furthermore,
a docking study for MMPs was reported for the most promising compound in order to investigate its binding
interactions with the different MMPs.
Introduction
that the use of broad-spectrum inhibitors could cause severe
side effects with dose-limiting consequences.8,9 Altogether, these
facts lead to the need of developing a new generation of MMP
inhibitors with high selectivity for the well-identified and
validated targets in each disease, namely, inhibiting the target
MMPs and sparing the antitarget proteins.10 To date, Overall
and Kleifeld9 identified MMP-1, -2, and -7 as anticancer drug
target MMPs and MMP-3, -8, -9, and -12 as antitargets.
However, considering the musculoskeletal problems that have
been associated with the inhibition of MMP-1,11 in spite of some
controversy,12 that enzyme is herein considered as an antitarget.
Therefore, in a cancer therapy, a candidate drug for targeting
the MMPs should be able to inhibit preferably MMP-2 and -7
and to spare some others, more specifically the antitargets MMP-
1, -3, -8, -9, and -12, in a ratio higher than 1000-fold IC50 or KI
units; nowadays, this is the major challenge in the design of
third generation MMP inhibitors.4,10,12
Matrix metalloproteinases (MMPs),a also designated as
matrixins, are a class of zinc-dependent endopeptidases able to
degrade all components of the extracellular matrix (ECM), such
as glycoproteins (e.g., collagen), proteoglycans, and fibrins, but
also other proteins such as growth-factor-binding proteins, cell-
surface receptors, cell-adhesion molecules, or even the precur-
sors of other proteinases. These enzymes are involved in
physiological processes related to the degradation of the
connective tissue, including tissue remodeling and repair,
embryonic development, and angiogenesis, but also other very
complex processes such as homeostatic regulation of the
extracellular environment and control of the innate immunity.1-3
However, some pathological situations are associated with
imbalance between MMPs and their endogenous regulators
(namely, the TIMPs, tissue inhibitors of MMPs), leading to an
overexpression of these enzymes, which may contribute to the
occurrence of several degenerative and inflammatory diseases.4,5
It is also well-known that MMPs are involved in several steps
of cancer progression, including tumor growth, angiogenesis
and apoptosis, as well as tissue invasion and formation of
metastasis, and their overexpression was detected in most types
of cancers.2,6 For this reason, MMPs have been considered for
long as promising targets in cancer therapy. However, due to
their complex roles, namely, with their signaling functions,
MMPs may also have protective roles against tumor progres-
sion.7 On the other hand, phase III clinical trials have shown
The R-carbonic anhydrases (CAs, EC 4.2.1.1) are a class of
widespread zinc-containing enzymes which catalyze the CO2/
-
HCO3 interconversion. They play a vital role for most living
organisms, participating in several physiological processes
related with the respiration and transport of those species, pH
homeostasis, several carbonate-dependent biosynthetic reactions,
bone resorption, etc. Among the 16 isoforms known in
vertebrates, considerable differences are associated with their
activity and subcellular localization. They can be cytosolic,
membrane-bound, mitochondrial, etc. and can be found in
various tissues, such as the gastrointestinal tract, nervous system,
kidneys, skin, and eyes.13 It is also known the involvement of
these enzymes in several pathologies (such as glaucoma,
epilepsy, hypertension, gastric and duodenal ulcers, tumors),
and so CA inhibitors have been largely investigated, and some
of them are in clinical use for the treatment of these diseases.
They belong mostly to the class of primary sulfonamides
(RSO2NH2), sulfamates (ROSO2NH2), and sulfamides
(RNHSO2NH2); one example is acetazolamide (AAZ,14 see
Figure 1), in clinical use for more than 50 years as a
* To whom correspondence should be addressed. Phone: 00351-
218419273. Fax: 00351-218464455. E-mail: masantos@ist.utl.pt.
†
Instituto Superior Te´cnico.
Universita` degli Studi di Pisa.
Universita` degli Studi di Firenze.
‡
§
a Abbreviations: MMP, matrix metalloproteinase; CA, carbonic anhy-
drase; ECM, extracellular matrix; TIMP, tissue inhibitor of MMPs; HIFR,
hypoxia inducible factor-R; ZBG, zinc-binding group; IDA, iminodiacetic
acid; SPE, 4-sulfamoylphenylethyl group; TACE, TNF-R converting
enzyme; SAR, structure-activity relationship; SMC, scaffold match
constraint.
10.1021/jm800964f CCC: $40.75
2008 American Chemical Society
Published on Web 11/19/2008

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7969
2),27 we report herein two series of related compounds, which
were designed mainly for dual inhibition of MMPs and CAs
(see general formula in Chart 1). A first series of compounds
(3a-c) was developed to evaluate the effect of introducing an
(R)-isopropyl group in the R-position to the hydroxamic moiety
of some of the previously reported inhibitors. Then, a second
series of compounds was synthesized, containing a hydroxamic
acid (X ) NHOH moiety) and a benzenesulfonamide group
(R2 ) NH-SPE, SPE ) 4-sulfamoylphenylethyl) (compounds
4, 6a-c) in order to further confer activity against CAs. Finally,
compound 7, conjugating both features of the two series was
developed, aiming at the double target inhibition.
Figure 1. The broad-spectrum MMP inhibitor CGS 27023A20 and
the reference CA inhibitors acetazolamide (AAZ)14 and N-(4-sulfa-
moylphenylethyl)-4-sulfamoylbenzamide (SPESB).21
Chemistry
All of the compounds reported herein possess the iminodi-
acetic acid (IDA) as the main scaffold, although they were
obtained from either IDA or D-valine as starting materials.
Except 4, all possess an N-sulfonylaryl moiety, which provides
an aromatic moiety and hydrogen bond acceptor atoms (from
the SO2 group), for hydrophobic interactions with the S1′ pocket
of the enzymes and H-bonds with the amino acid residues at
the entrance of this cavity, respectively.
For compounds 3a-c, possessing an alkyl group ((R)-isopropyl)
R-positioned relative to the hydroxamic acid, the starting material
was D-valine (see Scheme 1). This reagent was first N-substituted
by reaction with the appropriate arylsulfonyl chloride (aryl )
PhOCH3, PhPh, PhOPh), resulting in the corresponding secondary
sulfonamide. Considering the subsequent reaction steps, a protection
of the carboxylic acid was necessary. The benzyl group was
revealed to be the most suitable for this purpose, and the
esterification was performed with benzyl bromide in the presence
of triethylamine (TEA). Afterward, the coupling of one acetic
moiety with the sulfonamide was performed using tert-butyl
bromoacetate in dry DMF and excess of K2CO3, leading to
compounds 10a-c. The next step was the catalytic hydrogenolysis
of the benzyl ester using Pd/C, affording the corresponding
carboxylates 11a-c. The carboxylic groups of these intermediates
were condensed with the O-benzylhydroxylamine, upon previous
activation with ethylchloroformate (ECF) in the presence of
N-methylmorpholine (NMM), to afford the O-benzyl-protected
hydroxamic acids, 12a-c. Further tert-butyl and benzyl deprotec-
tion with TFA and catalytic hydrogenolysis, respectively, afforded
the final compounds 3a-c.
Compound 4 (Scheme 2) was prepared from N-benzylimino-
diacetic acid (BIDA), which was converted to the corresponding
anhydride, 14, upon treatment with oxalyl chloride, and subse-
quently underwent nucleophilic attack with (4-sulfamoylphenyl)-
ethylamine (SPEA) to yield 15. Further coupling of the carboxylic
group with hydroxylamine, using the ECF/NMM method described
above, afforded the final hydroxamic analogue, 4, possessing the
4-sulfamoylphenylethyl (SPE) moiety.
For the synthesis of 6a-c (Scheme 3), the corresponding
N-substituted dicarboxylic acids 16a-c were used as starting
materials, which were obtained upon derivatization of IDA, as
previously reported.27 In these cases, instead of the anhydride
intermediate route used for compound 4, the first step consisted
of peptide coupling of SPEA with one of the carboxylic groups
of 16a-c, involving activation with ECF/NMM, to obtain the
compounds 5a-c. This step was followed by a second
condensation of the remaining carboxylic group with hydroxy-
lamine using the same method, thus affording the final hydrox-
amic analogues 6a-c.
Figure 2. Catalytic coordination site of the Zn(II) in MMP-8 and
human carbonic anhydrase II (hCA II). In MMPs, the water molecule
is H-bonded to the carboxylate group of the glutamic acid, whereas in
CAs it forms a H-bond with the hydroxyl of a threonine residue, which
is H-bonded with a glutamic acid.22
antiglaucoma and antiepileptic drug.15 In spite of the long use
of CA inhibitors, only recently the roles of some specific
isoforms have been more clearly understood, namely, the
transmembrane CA IX, which is present only in very few normal
tissues but has its expression strongly increased in many types
of hypoxic tumors, namely, due to hypoxia inducible factor-R
(HIFR).16 CA IX was proved to be involved in acidification of
the extratumoral medium, contributing both to the acquisition
of metastatic phenotypes and to chemoresistance to weakly basic
anticancer drugs, and probably it also plays a role in providing
the bicarbonate necessary for the cell growth.17,18 These factors
point out the high interest of finding selective CA IX inhibitors,
which can spare the ubiquitous CA I and II, either to be used
in cancer therapy as antitumor drugs against hypoxic tumors or
to develop imaging fluorescent agents to target this very specific
hypoxic-related CA.19
Since both MMP and CA families have members that are related
to some carcinogenic and tumor progression processes, we thought
that it might be of great interest to exploit possible synergic effects
or “cross-reactivity” of dual inhibitors of these enzymes. To date,
only a few reports are known about compounds able to inhibit
both classes of metalloenzymes, with moderate cross-activity,
namely, compounds of hydroxysulfonamide,22 sulfonylated hy-
droxamate,22,23 and hydroxyurea24,25 types.
Although both MMPs and CAs possess a catalytic zinc ion
coordinated to three histidine residues and one water molecule/
hydroxide ion, the major differences between their catalytic sites
make it difficult for a zinc-binding group (ZBG) to be good for
both classes of enzymes (see Figure 2).22,26 However, following a
bifunctional approach, it is possible to conjugate in one molecule
two of the preferred ZBGs for MMPs and CAs, namely, a
hydroxamic acid and a primary arylsulfonamide, respectively.
Following our previous work reporting a series of iminodi-
acetylmonohydroxamate derivatives, which proved to act as
MMP inhibitors, some of them presenting high activity and
selectivity for MMP-2 vs MMP-1 (Chart 1, compounds 1 and
For the synthesis of compound 7, the starting material was
the intermediate 10c, prepared as discussed above, whose tert-

7970 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Marques et al.
Chart 1
Scheme 1a
a Reagents and conditions: (i) ArSO2Cl, TEA, 1:1 dioxane/H2O, room temperature; (ii) BnBr, TEA, MeCN, reflux; (iii) K2CO3, KI catalyst, DMF, room
temperature; (iv) H2, Pd/C, MeOH, room temperature; (v) ECF, NMM, THF, 0 °C; (vi) NH2OBn, MeOH, 0 °C; (vii) TFA, DCM, room temperature.
Scheme 2a
O-benzylhydroxylamine was coupled to the carboxylic acid of
19, forming the corresponding O-benzylhydroxamic acid inter-
mediate, 20. The use of O-protected hydroxylamine instead of
the nonprotected one (as in the synthesis of 6a-c) was preferred
herein, in order to avoid side reactions with the hydroxyl group
and guarantee a better overall yield. Catalytic deprotection of
20 with Pd/C afforded the final compound 7.
Results and Discussion
MMPs Inhibition. Most of the reported compounds were
tested against MMP-1, -2, -8, -9, -14, and -16, and the most
active ones were also tested on MMP-13 and TACE (TNF-R
converting enzyme), another zinc-dependent metalloproteinase
whose active site domain is homologous to that of MMPs. The
inhibition results obtained herein are presented in Table 1, which
also includes the values for CGS 27023A20 (see Figure 1), a
reference broad-spectrum sulfonamide-type inhibitor, as well
as two previously reported IDA derivatives (1 and 2).27
From a brief SAR analysis of the inhibitory profile of these
compounds, the type of the N-substituent group (R1 of Chart
a Reagents and conditions: (i) C2O2Cl2, DCM, reflux; (ii) SPEA, TEA,
MeCN, 0 °C; (iii) ECF, NMM, THF, 0 °C; (iv) NH2OH, MeOH, 0 °C.
butyl ester hydrolysis with TFA afforded 17 (see Scheme 4).
The SPE moiety was then introduced in this compound, by
coupling its carboxylic group with SPEA, as described before
for 5a-c. After catalytic hydrogenolysis of the benzyl ester,

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7971
Scheme 3a
a Reagents and conditions: (i) ECF, NMM, THF, 0 °C; (ii) SPEA, THF, 0 °C; (iii) NH2OH, MeOH, 0 °C.
Scheme 4a
a Reagents and conditions: (i) TFA, DCM, room temperature; (ii) ECF, NMM, THF, 0 °C; (iii) SPEA, THF, 0 °C; (iv) H2, Pd/C, MeOH, room temperature;
(v) NH2OBn, MeOH, 0 °C.
1) was shown to be the most determinant factor for the activity
and selectivity of the inhibitors. In fact, compound 4, possessing
a benzyl group as the R1 substituent, presented lower inhibitory
activity (high micromolar range) than its analogous compounds
with an arylsulfonyl group, 6a-c, which showed activities from
nanomolar to subnanomolar range. This fact is in agreement
with our previous work, where it was shown that inhibitors
possessing an arylalkyl group attached to the amine of the
iminodiacetylhydroxamate derivatives (usually acting as a P1′
substituent, which interacts with the S1′ subsite) presented much
weaker binding interaction with the catalytic site of the tested
enzymes than their analogues containing an arylsulfonyl group.
That difference is attributed to the ability of this group to get a
more favorable orientation of the aromatic group for interaction
within the S1′ pocket, as well as to provide extra O-donor atoms
to establish H-bonding interactions with residues Ala192 and
Glu40427,29 (numeration according to the human MMP-2
structure, entry P08253 of UniProtKB/Swiss-Prot database).30
Furthermore, among the arylsulfonyl derivatives, those pos-
sessing a phenoxybenzenesulfonyl group resulted to be more
active than the corresponding biphenylsulfonyl and methoxy-
benzenesulfonyl analogues, on all tested enzymes.
With regard to the introduction of an isopropyl group in the
R-position to the hydroxamate (as in compounds 3a-c), it was
mainly aimed at increasing the potency of the inhibitors and,
eventually, their bioavailability. In fact, it is known that P1
substituents often lead to enhancement of the inhibitory
activity,31,32 and on the other hand, an R-substituent may shield
the adjacent amide bond and reduce the metabolization of the
hydroxamic acid moiety.33 The (R)-isomer was chosen because
it is also known that this stereoisomer interacts more favorably
with the enzymes32 and, since most natural amino acids are (S)
isomers, (R) compounds are expected to be less likely to undergo
in ViVo metabolization.
Compounds 3a-c (see Table 1) revealed high inhibitory
activities against all of the tested MMPs (nanomolar or
subnanomolar), with 1.3-35-fold potency increase when com-
pared to their homologues without the R-(R)-isopropyl group
(i.e., 1, 2). The highest inhibition enhancements were observed
on MMP-1 and -8, with a concomitant reduction of the
selectivity for MMP-2 relative to these two enzymes. Since for
many inhibitors possessing an R-substituent, this group tends
to bind into the S1 cavity of the enzymes, identical behavior
was expected for these compounds. The S1 pocket does not
change much between the different MMPs; therefore, modifica-
tions in the P1 substituent of the inhibitors usually do not lead
to selectivity enhancement, while lipophilic groups in this
position have been reported to give broad-spectrum character-
istics to the inhibitors.34
The introduction of a 4-sulfamoylphenylethyl (SPE) moiety
as the R2 group (compounds 6a-c) also led to an increase of
activity toward all of the tested MMPs, as compared with their
homologues containing a carboxylic acid (e.g., 1, 2), thus
indicating that the SPE group must interact favorably with these
enzymes. However, this increase of activity was associated with
some decrease of selectivity for the cancer-related enzymes
(namely, MMP-2), particularly evident for compound 6c vs 2.
The carboxylic analogues (compounds 5b and 5c) of hydrox-
amic inhibitors (6b and 6c) were also tested on some MMPs in
order to compare the effect of these functional groups on the
activities. The results indicated that these compounds possess
moderate activity against the tested enzymes (from 2.2 to 8.2

7972 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Marques et al.
µM range). The phenoxybenzenesulfonyl-containing compound
(5c) was more active than the analogue with a biphenylsulfonyl
group, 5b, as happened with their hydroxamic homologues.
The best results were obtained with compound 7, which was
designed to conjugate the positive effects on MMP inhibition
resulting from the insertion of the R-(R)-isopropyl group, as in
3c, with those of the SPE substituent, as in 6c. Among the series
of tested MMPs inhibitors, 7 appears as the most active
compound, displaying high inhibitory activity (subnanomolar
toward all MMPs, except MMP-1).
The inhibitory activity toward TACE was also evaluated, but
only for the most promising compounds, 3c and 7. The results
showed moderate to high activity (2.3 µM for 3c and 55 nM
for 7) but still displaying selectivity for the MMPs (e.g.,
selectivity MMP-2/TACE of 4580 and 157, respectively). This
fact may be due to the main differences in the primary and
secondary structures of MMPs and TACE, namely, on the S1′
and S3′ sites, where the inhibitors are expected to bind with
different affinities.35,36
Globally, within these series of new compounds, some potent
MMP inhibitors were obtained, with activities in nanomolar to
subnanomolar range. Although with reduced selectivity for
MMP-2 over some antitargets in cancer therapy (e.g., MMP-8
and -9), in general they presented selectivity for MMP-2 over
MMP-1 and TACE.
CA Inhibition. The inhibitory activities of the SPE-containing
compounds were evaluated toward a set of physiologically
relevant CAs (CA I, II, and IX), and the results are shown in
Table 1, together with some others previously reported.37 As
can be seen from this table, all of the tested inhibitors presented
high activity against CA II and IX (nanomolar range), and the
carboxylic analogues (e.g., 5b, c) were shown to be more active
than the corresponding hydroxamates (e.g., 6b, c). In general,
all of the compounds demonstrated lower activity toward CA I
(high nanomolar to micromolar range) than AAZ and the
structurally related inhibitor N-(4-sulfamoylphenylethyl)-4-
sulfamoylbenzamide, SPESB21 (see Figure 1), and they pre-
sented similar potency against the CA II isoform.
Concerning the membrane-bound tumor-associated isozyme
CA IX, these compounds presented very high inhibitory activity,
in most cases higher than AZZ and SPESB (compound 7
displaying the best result, with a KI of 3.3 nM). They also
demonstrated a selectivity profile that favors the inhibition of
CA IX over the ubiquitous isoforms CA I and II (selectivities
CA IX/I ranging from 7.9 to 536 and CA IX/II from 1.3 to
5.4), which is a remarkable feature when developing potential
antitumor drugs.
Considering the overall inhibition profiles of the compounds
reported herein over MMPs and CAs, our studies revealed for
some of them high inhibitory potency against both classes of
enzymes. In particular, the conjugated introduction of an
isopropyl group in the R-position to the hydroxamate and the
SPE substituent on the IDA scaffold endowed compound 7 with
a subnanomolar activity on most MMPs and a high activity and
selectivity toward CA IX and CA II vs CA I. As previously
mentioned, based on the multifactorial nature of many disease
processes, the capacity of a drug to simultaneously target two
pathways associated to the same disease may represent a
favorable feature in therapy. Altogether, these results suggest
that these bifunctional compounds hold great potential as dual
drugs and may provide a more efficient approach to overcome
the complex processes of cancer. Further studies will be
developed to improve the activity and selectivity of this type

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7973
Figure 3. rmsd evaluation of the binding disposition of the ligands (in Å) docked into the respective MMPs, for the entire molecule and for the
ZBG, using different procedures (neutral hydroxamate of the ligand and anionic Glu- in the protein).
of compound toward those of MMPs and CAs really implicated
in cancer, such as MMP-2 and CA IX.
the docking with simple methods (without any constraint), ASP
predicted the binding with an average rmsd of 0.78 and 1.65 Å
for the ZBG and the whole ligand, respectively. ChemScore
also gave acceptable results but with slightly worse average rmsd
(0.78 Å for the ZBG and 1.73 Å for the ligands).
Molecular Modeling. In order to understand the interactions
of this type of inhibitor with the MMP active site, modeling
studies were carried out by docking compound 7 into all of the
assayed MMP structures. The best approach to be used in this
modeling study was based on a first evaluation of several
inhibitor-enzyme systems reported in the literature.
When the SMC was applied to the hydroxamate scaffold, the
prediction ability was improved for all of the scoring functions,
both for the ZBG and for the whole molecule. As displayed in
Figure 3, the ASP function still demonstrated the best ability
on predicting both the position of the ZBG (average rmsd of
0.31 Å) and the entire ligand molecule (rmsd of 1.26 Å).
Docking of Compound 7 with MMPs. In order to test the
reliability of our docking calculations, we first tested the ability
of GOLD software38 for that purpose by doing the docking of
30 MMP inhibitors into their crystal structures. These structures
represented all of the available crystal structures39 of MMP
complexed with inhibitors characterized by the presence of the
hydroxamic acid as ZBG. In the complex model, the ligand and
the enzyme were worked up (see Experimental Section), and
then the respective ligands were submitted to docking calcula-
tions into their corresponding protein using the program Gold
3.0. The three scoring functions available with that software
(i.e., GoldScore, ChemScore, and ASP), either without any
constraint or with a scaffold match constraint (SMC), were used.
In the SMC method, the hydroxamic moiety of the original
ligand was used as a scaffold to match the tested ligands. The
SMC was applied because it has recently shown good results
for the docking of MMP inhibitors.40 The first-ranked structures
obtained from these calculations were then compared with the
original complex, and the root-mean-square deviations (rmsd)
were determined for the heavy atoms of the ZBG (the molecular
segment CRCONO) and for the whole ligand molecules (see
Figure 3).
From this analysis we concluded that the most reliable method
to perform docking studies involving MMPs/hydroxamic inhibi-
tors with the Gold program is the ASP fitness function with a
hydroxamate SMC.
Compound 7 was then docked into all of the analyzed MMPs
by means of Gold software, applying the SMC-ASP procedure
discussed above. Figure 4 displays the results for MMP-2. The
hydroxamate group coordinates the zinc ion through the two
oxygen atoms and also establishes two H-bonds with the
enzyme, through the carboxylate of Glu404 and the carbonyl
of Ala192 (numeration according to UniProtKB/Swiss-Prot
database, entry P08253).30 The sulfonyl group of the SO2PhOPh
moiety forms H-bonds with the backbone of Leu191 and
Ala192, and the phenoxybenzene aromatic rings are accom-
modated in the S1′ cavity, forming van der Waals contact with
its hydrophobic residues, in particular with Leu399, Val400,
and Leu420. Regarding the isopropyl group, as expected from
the previous SAR analysis,32 it is placed in the S1 zone of the
enzyme, which is mostly hydrophobic, and allows lipophilic
interactions with Leu190. This may account for the higher
potency evidenced by this inhibitor (7) and others, relative to
the compounds without this R-substituent group (compounds 1
and 2).
The results show that, from the three scoring functions
supplied by Gold, in general ASP gives the best average
prediction of the corresponding binding conformation of the
inhibitors, in terms of both its coordination to the zinc and the
overall accommodation of the ligand in the catalytic site. For

7974 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Marques et al.
Figure 5. Compound 7 docked into the MMP-1 catalytic site.
mide group is too bulky to fit in the S1′ cavity, thus explaining
the decreased activity for this MMP subtype relative to the others
studied.
Conclusions
A set of new hydroxamic compounds based on the imino-
diacetyl scaffold with a variety of substituent groups was
developed and evaluated, aimed at obtaining potent and selective
inhibitors for some target matrix metalloproteinases (MMPs)
involved in cancer, but also providing inhibitory ability against
carbonic anhydrase isozymes (CAs), which are also involved
in this pathology. Among the different types of P1′ substituents,
the N-sulfonylaryl groups appeared to be the most relevant ones
to achieve the best activity and selectivity results on MMP
inhibition. Modifications at the R-position to the hydroxamate
by insertion of an isopropyl group, to improve the bioavailability
of inhibitors, in most cases led to significant enhancement of
inhibitory activity toward MMPs, although with some selectivity
reduction. A further substitution was performed at the remaining
OH group of the IDA, with the 4-sulfamoylphenylethyl group
(SPE), known to display inhibitory activity toward CAs. This
feature led to enhancement of the inhibitory activities both on
MMPs and on CAs (from nanomolar to subnanomolar range),
thus suggesting that these types of bifunctional compounds can
be potential double targeting drugs for use in the therapy of
some malignant tumors.
A docking study of the most interesting compound allowed
the understanding of its interaction with the various MMPs, thus
suggesting the basis of its activity profile.
In conclusion, the present paper demonstrates that the
introduction of the 4-sulfamoylphenylethyl group in the IDA
scaffold maintains the inhibitory potency on MMPs and also
provides a good activity against some CAs. This finding
encourages further studies toward the identification of new
selective dual inhibitors for those enzymes implicated in cancer,
such as MMP-2 and CA IX.
Figure 4. (a) Compound 7 docked into the MMP-2 catalytic site with
H-bonds displayed (slashed lines). (b) Surface of the binding site and
its subsites, green representing conserved and yellow nonconserved
regions, among MMP-2, -8, -9, -13, -14, and -16.
Concerning the SPE moiety, it seems to be well accom-
modated in the catalytic site and is able to establish several
interesting binding interactions with the enzyme. The NH group
of the amide connecting the SPE moiety to the IDA backbone
is able to form a H-bond with the backbone carbonyl group of
Gly189. The benzene ring of the SPE extends toward the S2′-S3′
region, over the phenol rings of Tyr395 and Tyr425, establishing
a close lipophilic interaction with these groups, and finally the
nitrogen of the sulfonamide group forms a H-bond with the
backbone carbonyl of Tyr425.
Compound 7 shows a high inhibitory activity with almost
all of the MMPs tested. The docking studies of this compound
into MMP-8, -9, -13,-14, and -16 suggested that the binding
interactions shown for the MMP-2 are also maintained in these
MMPs. As shown in the Supporting Information (Figures
S1-S5), compound 7 presented all of the main interactions
reported for MMP-2 also in MMP-8, -9, and -13, in particular
the binding of the SPE moiety in the S2′-S3′ region and
displaying the two H-bonds with the backbone of Gly189 and
Tyr425 (MMP-2 sequence number). In MMP-14 and -16, the
ligand showed the same type of accommodation observed for
the other MMPs; however, instead of the H-bond with the
backbone of Tyr425, the sulfonamido group forms a H-bond
with an asparagine residue that is not conserved and is
substituted by a tyrosine in the other analyzed MMPs (Tyr395
in MMP-2).
Experimental Section
(A) Chemistry. General Methods and Materials. Analytical
grade reagents were purchased from Aldrich, Sigma, and Fluka and
were used as supplied. Solvents were dried according to standard
methods.41 The chemical reactions were monitored by TLC using
alumina plates coated with silica gel 60 F254 (Merck). Column flash
chromatography separations were performed on silica gel Merck
230-400 mesh ASTM. Melting points were measured with a Leica
Galen III hot stage apparatus and are uncorrected. IR spectra were
recorded on a Bio-Rad Merlin, FTS 3000 MX. The 1H NMR spectra
were recorded on a Varian Unity 300 spectrometer at 25 °C.
Chemical shifts (δ) are reported in parts per million (ppm) from
standard internal references, namely, tetramethylsilane (TMS) for
Analysis of the primary sequence of the binding site of the
analyzed MMPs reveals that, as shown in Figure 4b, the region
in which inhibitor 7 interacts is highly conserved, thus explaining
the very close inhibition activities shown by this compound with
all of these MMPs.
The activity of 7 against MMP-1 is almost 30-fold worse
than for the other MMPs. As shown in Figure 5, MMP-1 is
characterized by a short S1′ pocket, and the ligand is not able
to display favorable binding interactions since the arylsulfona-

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7975
organic solvents and sodium 3-(trimethylsilyl)-[2,2,3,3-
d4]propionate (DSS) for D2O solutions. The following abbreviations
are used: s ) singlet, d ) doublet, t ) triplet, q ) quadruplet, m
) multiplet, dd ) double doublet, 2d ) two overlapping doublets,
and bs ) broad singlet. 13C NMR spectra were measured for some
representative compounds and were recorded with a Bruker Avance
II 400 spectrometer at 25 °C, and they are presented in Supporting
Information. Mass spectra (FAB) were performed in a VG TRIO-
2000 GC/MS instrument and ESI spectra on a Quattro LC mass
spectrometer (Micromass, Manchester, U.K.). The high-resolution
mass spectra (HRMS) were obtained with a high-resolution Fourier
transform ion cyclotron resonance (FTICR) instrument, Finnigan
FT/MS 2001-DT, equipped with a 3.0 T superconducting magnet,
by electron impact (EI), typically with 15 eV electron beam
energies, 5-µm emission currents, and 150 °C sample temperatures.
Elemental analyses were performed on a Fisons EA1108 CHNF/O
instrument.
General Method for Preparation of N-(Arylsulfonyl)-D-
valine Compounds (8a-c). To a solution of D-valine (0.50 g, 4.27
mmol) and triethylamine, TEA (0.89 mL, 6.40 mmol), in 1:1 water/
dioxane (100 mL) was added the appropriate arylsulfonyl chloride
(4.70 mmol), and the mixture was stirred for 12 h. The resulting
solution was concentrated, and the residue was dissolved in 5%
NaOH (50 mL) and washed with CH2Cl2 (4 × 50 mL); the aqueous
phase was acidified to pH 1-2 with concentrated HCl, and this
solution was extracted with CH2Cl2 (3 × 50 mL). After the organic
phase was dried over Na2SO4 and solvent evaporated under vacuum,
the pure products were obtained as white solids.
solution was extracted with water (2 × 40 mL), 5% NaOH (40
mL), and brine (40 mL). After drying over anhydrous Na2SO4 and
evaporation of the solvent, the residue was dissolved in methanol
(25 mL) and refluxed with active charcoal (0.5 g) for 3 h; filtration
and evaporation of the solvent gave a yellow oil, which had no
further purification.
N-(4-Phenoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
O-benzyl-D-valine (10c). 1H NMR (CDCl3) δ 7.83 (d, J ) 8.4
Hz, 2H, ArH), 7.40 (t, J ) 7.8 Hz, 2H, ArH), 7.35-7.19 (m, 6H,
ArH), 7.02 (d, J ) 8.1 Hz, 2H, ArH), 6.90 (d, J ) 8.7 Hz, 2H,
ArH), 5.00, 4.85 (dd, J ) 12.3, 12.0 Hz, 2H, CH2Ph), 4.24, 3.91
(dd, J ) 18.3, 18.6 Hz, 2H, CH2CO2tBu), 4.02 (d, J ) 10.5, 1H,
NCH(iPr)), 2.08-2.01 (m, 1H, CH(CH3)2), 1.44 (s, 9H, tBuH),
1.00 (d, J ) 6.6 Hz, 3H, CHCH3), 0.87 (d, J ) 6.6 Hz, 3H,
CHCH3); m/z (FAB) 498 (M - tBu + 2H)+, 576 (M + Na)+, 520
(M - tBu + H + Na)+.
N-(4-Methoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
O-benzyl-D-valine (10a) and N-(4-Biphenylsulfonyl)-N-tert-bu-
toxycarbonylmethyl-O-benzyl-D-valine (10b). See characterization
in Supporting Information.
Method for Preparation of N-(Arylsulfonyl)-N-tert-butoxy-
carbonylmethyl-D-valine Compounds (11a-c). The general method
for benzyl deprotection by catalytic hydrogenolysis was followed.
To a solution of the O-benzyl derivative 10a, 10b, or 10c (1.00 g)
in methanol (15 mL) was added 10% Pd/C (0.30 g), and the
suspension was stirred under H2 (4 bar) at room temperature for
3 h. After filtration and evaporation of the solvent, the residue was
taken into 2 M NaOH (50 mL) and washed with n-hexane (2 × 50
mL), and then the aqueous solution was extracted with ethyl acetate
(3 × 50 mL). The organic phase was dried over anhydrous Na2SO4,
and evaporation of the solvent afforded the pure product.
N-(4-Phenoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
D-valine (11c). Pale yellow solid; 79% overall yield for 10c and
11c; mp 97-99 °C; 1H NMR (CDCl3) δ 8.01 (d, J ) 8.7 Hz, 2H,
ArH), 7.36 (t, J ) 7.8 Hz, 2H, ArH), 7.17 (t, J ) 7.4 Hz, 1H,
ArH), 6.96 (d, J ) 7.8 Hz, 2H, ArH), 6.79 (d, J ) 8.4 Hz, 2H,
ArH), 4.24, 4.10 (dd, J ) 18.0, 18.3 Hz, 2H, CH2CO2tBu), 3.62
(d, J ) 10.2, 1H, NCH(iPr)), 1.98-1.91 (m, 1H, CH(CH3)2), 1.43
(s, 9H, tBuH), 0.86 (d, J ) 6.0 Hz, 3H, CHCH3), 0.25 (d, J ) 5.7
Hz, 3H, CHCH3); m/z (FAB) 452 (M - tBu + 2Na)+, 430 (M -
tBu + H + Na)+, 486 (M + Na)+.
N-(4-Phenoxybenzenesulfonyl)-D-valine (8c). The 4-phenoxy-
benzenesulfonyl chloride used herein was prepared according to
the literature.27 Final recrystallization with n-hexane afforded the
product as a white solid: yield 67%; mp 97-99 °C; 1H NMR
(CDCl3) δ 7.78 (d, J ) 9.3 Hz, 2H, ArH), 7.41 (t, J ) 8.0 Hz, 2H,
ArH), 7.22 (t, J ) 7.6 Hz, 1H, ArH), 7.06-6.99 (2d, 4H, ArH),
5.07 (d, J ) 9.6 Hz, 1H, NH), 3.84-3.79 (2d, 1H, NCH(iPr)),
2.16-2.09 (m, 1H, CH(CH3)2), 0.99 (d, J ) 6.6 Hz, 3H, CHCH3),
0.89 (d, J ) 6.6 Hz, 3H, CHCH3); m/z (FAB) 350 (M + H)+, 372
(M + Na)+.
N-(4-Methoxybenzenesulfonyl)-D-valine (8a) and N-(4-Biphe-
nylsulfonyl)-D-valine (8b). See characterization in Supporting
Information.
General Method for Preparation of N-(Arylsulfonyl)-O-
benzyl-D-valine Compounds (9a-c). To a solution of the respec-
tive intermediate 8a, 8b, or 8c (2.60 mmol) and TEA (0.54 mL,
3.90 mmol) in acetonitrile (30 mL) was added benzyl bromide (0.34
mL, 2.86 mmol), and the mixture was stirred overnight under reflux.
The solvent was evaporated, and the residue was dissolved in ethyl
acetate (30 mL) and then washed with 0.1 M HCl (3 × 30 mL);
after addition of n-hexane to the organic phase (10 mL), the solution
was washed with 5% NaOH (3 × 40 mL), the organic phase was
dried over anhydrous Na2SO4, and after evaporation of the solvent
and drying of the solid residue in vacuum, the pure products were
obtained as solids.
N-(4-Phenoxybenzenesulfonyl)-O-benzyl-D-valine (9c). Yellow
solid; 81% yield; mp 57-59 °C; 1H NMR (CDCl3) δ 7.75 (d, J )
9.0 Hz, 2H, ArH), 7.40 (t, J ) 8.1 Hz, 2H, ArH), 7.38-7.20 (m,
6H, ArH), 7.03 (d, J ) 7.8 Hz, 2H, ArH), 6.96 (d, J ) 8.7 Hz,
2H, ArH), 5.09 (d, J ) 9.6 Hz, 1H, NH), 4.92 (s, 2H, CH2Ph),
3.81-3.76 (2d, 1H, NCH(iPr)), 2.12-2.05 (m, 1H, CH(CH3)2),
0.96 (d, J ) 6.9 Hz, 3H, CHCH3), 0.84 (d, J ) 6.9 Hz, 3H,
CHCH3); m/z (FAB) 462 (M + Na)+, 440 (M + H)+.
N-(4-Methoxybenzenesulfonyl)-O-benzyl-D-valine (9a) and
N-(4-Biphenylsulfonyl)-O-benzyl-D-valine (9b). See characteriza-
tion in Supporting Information.
General Method for Preparation of N-(Arylsulfonyl)-N-tert-
butoxycarbonylmethyl-O-benzyl-D-valine Compounds (10a-c).
To a suspension of the derivative 9a, 9b, or 9c (2.07 mmol), K2CO3
(2.86 g, 20.7 mmol), and KI (0.069 g, 0.41 mmol) in dry DMF (20
mL) was added tert-butyl bromoacetate (0.61 mL, 4.14 mmol), and
the mixture was stirred at room temperature for 3 days. The mixture
was then taken into 1:1 n-hexane/ethyl acetate (40 mL), and that
N-(4-Methoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
D-valine (11a) and N-(4-Biphenylsulfonyl)-N-tert-butoxycarbo-
nylmethyl-D-valine (11b). See characterization in Supporting
Information.
Method for Preparation of N-(Arylsulfonyl)-N-tert-butoxy-
carbonylmethyl-O-benzyl-D-valinehydroxamic Acids (12a-c).
The general method for carboxylic acid/hydroxylamine coupling
was followed. To an ice-cooled solution of the intermediate 11a,
11b, or 11c (2.06 mmol) in dry THF (25 mL) was added
ethylchloroformate (ECF, 0.23 mL, 2.37 mmol) and N-methylmor-
pholine (NMM, 0.26 mL, 2.37 mmol); the mixture was stirred at 0
°C for 45 min, and the solids were subsequently filtered off.
Meanwhile, a solution of O-benzylhydroxylamine hydrochloride
(0.411 g, 2.58 mmol) in dry methanol was neutralized with KOH
(0.144 g, 2.58 mmol), stirring at 0 °C for 30 min, followed with
filtration. The first solution was then dropwise added to the
hydroxylamine solution, and the mixture was stirred at 0 °C for
4 h under N2. The reaction mixture was concentrated under vacuum
and the residue taken into ethyl ether (30 mL), which was then
washed with 1 M HCl (2 × 30 mL); the two layers were separated,
and to the organic phase was added petroleum ether (30 mL), and
the mixture was further washed with 5% NaOH (3 × 50 mL). After
drying the final organic phase over Na2SO4 and solvent removal,
the pure products were obtained.
N-(4-Phenoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
O-benzyl-D-valinehydroxamic Acid (12c). Pale yellow solid; 77%
yield; mp 45-48 °C; 1H NMR (CDCl3) δ 8.97 (s, 1H, CONHOBn),
7.84 (d, J ) 9.3 Hz, 2H, ArH), 7.46-7.36 (m, 7H, ArH), 7.22 (t,
J ) 7.3 Hz, 1H, ArH), 7.05-7.01 (2d, 4H, ArH), 4.92 (s, 2H,
CH2Ph), 4.14, 3.90 (dd, J ) 17.7, 18.3 Hz, 2H, CH2CO2tBu), 3.30

7976 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Marques et al.
(d, J ) 10.5, 1H, NCH(iPr)), 2.15-2.07 (m, 1H, CH(CH3)2), 1.47
(s, 9H, tBuH), 0.79 (d, J ) 6.9 Hz, 3H, CHCH3), 0.49 (d, J ) 6.3
Hz, 3H, CHCH3); m/z (FAB) 513 (M - tBu + 2H)+, 591 (M +
Na)+, 569 (M + H)+.
aminoacetic Acid (15). A suspension of N-benzyliminodiacetic
anhydride hydrochloride, 14 (1 g, 4.14 mmol), prepared as
previously reported,27 in dry acetonitrile (50 mL) was neutralized
with dry pyridine (0.34 mL, 4.14 mL) and left stirring for 30 min.
The resulting solution was added dropwise to an ice-cooled solution
of (4-sulfamoylphenyl)ethylamine (SPEA, 0.691 g, 3.45 mmol) in
dry acetonitrile (50 mL); the mixture reacted for 4 h, and it was
subsequently filtered and evaporated. The crude material was
washed with hot ethanol, affording the pure product as a white
N-(4-Methoxybenzenesulfonyl)-N-tert-butoxycarbonylmethyl-
O-benzyl-D-valinehydroxamic Acid (12a) and N-(4-Biphenyl-
sulfonyl)-N-tert-butoxycarbonylmethyl-O-benzyl-D-valinehy-
droxamic Acid (12b). See characterization in Supporting Information.
General Method for Preparation of N-(Arylsulfonyl)-N-
hydroxycarbonylmethyl-O-benzyl-D-valinehydroxamic Acids
(13a-c). To a solution of compound 12a, 12b, or 12c (1.40 mmol)
in dichloromethane (5 mL) was added trifluoroacetic acid (TFA,
3.3 mL), and the mixture was stirred at room temperature for 2 h.
The solution was concentrated in vacuum, the residue was taken
into 5% NaOH (40 mL), and this solution was washed with ethyl
ether (2 × 40 mL); the pH of the aqueous phase was lowered to 6
with 2 M HCl, and it was extracted with CH2Cl2 (3 × 20 mL).
The total organic phase was dried over Na2SO4, and the solvent
was evaporated in vacuum, affording the pure products as white
solids.
1
solid (0.529 g, 38% yield): mp 181-182 °C; H NMR (D2O, pD
ca. 3) δ 7.73 (d, J ) 8.7 Hz, 2H, ArH), 7.42-7.28 (m, 7H, ArH),
4.28 (s, 2H, CH2Ph), 4.04 (s, 2H, CH2CONHCH2), 3.94 (s, 2H,
CH2CO2H), 3.36 (t, J ) 6.4 Hz, 2H, NHCH2CH2PhSO2NH2), 2.75
(t, J ) 6.4 Hz, 2H, NHCH2CH2PhSO2NH2); m/z (FAB) 406 (M +
H)+.
N-Benzyl-N-{[2-(4-sulfamoylphenyl)ethylcarbamoyl]methyl}-
aminoacetohydroxamic Acid (4). The general method for car-
boxylic acid/hydroxylamine coupling, as described for compounds
12a-c, was followed with 15, but using hydroxylamine hydro-
chloride instead. After filtration and evaporation of the reaction
mixture, the residue was dissolved in water (20 mL), and extractions
were performed with ethyl acetate (6 × 20 mL); the organic phase
was then dried over anhydrous Na2SO4 and evaporated. Further
recrystallization of the crude with CH2Cl2/ethyl ether afforded the
pure product as a white solid (0.319 g, 77% yield): mp 70-71 °C;
1H NMR (D2O, pD ca. 3) δ 7.78 (d, J ) 7.2 Hz, 2H, ArH),
7.44-7.32 (m, 7H, ArH), 4.29 (s, 2H, CH2Ph), 3.98 (s, 2H,
CH2CONHCH2), 3.90 (s, 2H, CH2CONHOH), 3.47 (t, J ) 6.0 Hz,
N-(4-Phenoxybenzenesulfonyl)-N-hydroxycarbonylmethyl-O-
benzyl-D-valinehydroxamic Acid (13c). Herein, the extractions at
pH 6 were carried out with ethyl ether instead of CH2Cl2, after
which the organic phase was washed several times with 1%
1
Na2HPO4 solution. Yield 62%; mp 69-70 °C; H NMR (CDCl3)
δ 8.80 (s, 1H, CONHOBn), 7.80 (d, J ) 8.7 Hz, 2H, ArH),
7.43-7.38 (m, 7H, ArH), 7.23 (t, J ) 7.6 Hz, 1H, ArH), 7.05-7.01
(2d, 4H, ArH), 4.89 (s, 2H, CH2Ph), 4.29, 4.01 (dd, J ) 18.3, Hz,
2H, CH2CO2H), 3.36 (d, J ) 10.5, 1H, NCH(iPr)), 2.17-2.06 (m,
1H, CH(CH3)2), 0.79 (d, J ) 6.6 Hz, 3H, CHCH3), 0.47 (d, J )
6.0 Hz, 3H, CHCH3); m/z (FAB) 535 (M + Na)+, 513 (M + H)+.
N-(4-Methoxybenzenesulfonyl)-N-hydroxycarbonylmethyl-O-
benzyl-D-valinehydroxamic Acid (13a) and N-(4-Biphenylsul-
fonyl)-N-hydroxycarbonylmethyl-O-benzyl-D-valinehydroxam-
ic Acid (13b). See characterization in Supporting Information.
General Method for Preparation of N-(Arylsulfonyl)-N-
hydroxycarbonylmethyl-D-valinehydroxamic Acids (3a-c). Cata-
lytic hydrogenolysis of intermediate 13a, 13b, or 13c was performed
as described for 11a-c. Final recrystallization afforded the pure
products as white solids.
2H, NHCH2CH2PhSO2NH2), 2.84 (t,
J ) 6.0 Hz, 2H,
NHCH2CH2PhSO2NH2); m/z (FAB) 421 (M + H)+. Anal.
(C19H24N4O5S) C, H, N. S: calcd, 7.46; found, 6.98.
GeneralMethodforPreparationofN-(Arylsulfonyl)-N-{[2-(4-sulfa-
moylphenyl)ethylcarbamoyl]methyl}aminoacetic Acids (5a-c).
To an ice-cooled solution of the appropriate N-(arylsulfonyl)imi-
nodiacetic acid, 16a, 16b, or 16c, prepared according to the
literature27 (0.73 mmol), in dry THF (10 mL) were simultaneously
and slowly added two THF solutions (10 mL each) of NMM (0.085
mL, 0.77 mmol) and ECF (0.073 mL, 0.77 mmol). This mixture
was stirred for 45 min, and then a solution of SPEA (0.146 g, 0.73
mmol) in dry THF (10 mL) was added; the final solution was left
stirring at 0 °C for 4 h. The solids were filtered off, the solution
was concentrated under vacuum, and the residue was dissolved in
a 5% NaHCO3 solution (25 mL), which was then washed with
CH2Cl2 (3 × 25 mL). The resulting aqueous phase was acidified
(pH ca. 1-2) with 2 M HCl and then extracted with ethyl acetate
(3 × 25 mL), and the combined organic phases were dried over
anhydrous Na2SO4 and evaporated. After evaporation of the solvent,
recrystallization of the remaining residue with ethanol afforded the
pure products as white solids.
N-(4-Methoxybenzenesulfonyl)-N-hydroxycarbonylmethyl-D-
valinehydroxamic Acid (3a). Recrystallized from CH2Cl2/n-
1
hexane; 98% yield; mp 80-82 °C; H NMR (D2O, pD ca. 8) δ
7.83 (d, J ) 8.4 Hz, 2H, ArH), 7.11 (d, J ) 9.34 Hz, 2H, ArH),
4.28, 3.81 (dd, J ) 18.0, 18.3 Hz, 2H, CH2CO2tBu), 3.89 (s, 3H,
OCH3), 3.44 (d, J ) 10.5 Hz, 1H, NCH(iPr)), 1.97-1.89 (m, 1H,
CH(CH3)2), 0.90 (d, J ) 6.0 Hz, 3H, CHCH3), 0.76 (d, J ) 6.6
Hz, 3H, CHCH3); m/z (FAB) 361 (M + H)+, 383 (M + Na)+.
Anal. (C14H20N2O7S) C, N, S. H: calcd, 5.70; found, 5.11.
N-(4-Biphenylsulfonyl)-N-hydroxycarbonylmethyl-D-valine-
hydroxamic Acid (3b). Recrystallized from acetonitrile/ethyl ether;
N-(4-Biphenylsulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarbamoyl]-
1
methyl}aminoacetic Acid (5b). Yield 34%; mp 127-129 °C; H
NMR (D2O, pD ca. 8) δ 7.94-7.87 (2d, 4H, ArH), 7.76-7.69 (2d,
4H, ArH), 7.61-7.49 (m, 3H, ArH), 7.29 (d, J ) 7.8 Hz, 2H, ArH),
3.91 (s, 2H, CH2CONHCH2), 3.87 (s, 2H, CH2CO2H), 3.39 (t, J
) 6.6 Hz, 2H, NHCH2CH2PhSO2NH2), 2.79 (s, J ) 6.6 Hz, 2H,
NHCH2CH2PhSO2NH2); m/z (FAB) 532 (M + H)+, 554 (M +
Na)+.
1
96% yield; mp 100-102 °C; H NMR (CD3OD) δ 8.00 (d, J )
8.4 Hz, 2H, ArH), 7.81 (d, J ) 8.4 Hz, 2H, ArH), 7.69 (d, J ) 7.8
Hz, 2H, ArH), 7.51-7.41 (m, 3H, ArH), 4.49, 4.02 (dd, J ) 18.3
Hz, 2H, CH2CO2H), 3.64 (d, J ) 10.5, 1H, NCH(iPr)), 2.09-1.98
(m, 1H, CH(CH3)2), 0.88 (d, J ) 6.6 Hz, 3H, CHCH3), 0.83 (d, J
) 6.6 Hz, 3H, CHCH3); m/z (FAB) 429 (M + Na)+, 407 (M +
H)+. Anal. (C19H22N2O6S·2CH2Cl2) C, H, N. S: calcd, 5.57; found,
5.08.
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarba-
moyl]methyl}aminoacetic Acid (5c). Yield 46%; mp 119-121 °C;
1H NMR (D2O, pD ca. 9) δ 7.78 (d, J ) 7.8 Hz, 2H, ArH), 7.72
(d, J ) 7.8 Hz, 2H, ArH), 7.45 (t, 2H, J ) 8.0 Hz, ArH), 7.34-7.27
(m, 3H, ArH), 7.12 (d, J ) 7.8 Hz, 4H, ArH), 3.83 (s, 2H,
CH2CONHCH2), 3.80 (s, 2H, CH2CO2H), 3.40 (t, J ) 6.6 Hz, 2H,
N-(4-Phenoxybenzenesulfonyl)-N-hydroxycarbonylmethyl-D-valine-
hydroxamic Acid (3c). Recrystallized from ethyl ether/petroleum
1
ether; 97% yield; mp 80-82 °C; H NMR (CDCl3) δ 9.24 (bs,
1H, CONHOH), 7.78 (d, J ) 8.7 Hz, 2H, ArH), 7.42 (t, J ) 7.6
Hz, 2H, ArH), 7.23 (t, J ) 7.2 Hz, 1H, ArH), 7.07-7.02 (2d, 4H,
ArH), 4.37, 3.83 (dd, J ) 18.9 Hz, 2H, CH2CO2H), 3.55 (d, J )
10.2, 1H, NCH(iPr)), 2.25-2.16 (m, 1H, CH(CH3)2), 0.82 (d, J )
6.0 Hz, 3H, CHCH3), 0.37 (d, J ) 6.9 Hz, 3H, CHCH3); m/z (FAB)
445 (M + Na)+, 423 (M + H)+. Anal. (C19H22N2O7S·0.3Et2O) C,
N. H: calcd, 5.65; found, 5.21. S: calcd, 7.23; found, 6.70.
N-Benzyl-N-{[2-(4-sulfamoylphenyl)ethylcarbamoyl]methyl}-
NHCH2CH2PhSO2NH2), 3.40 (t,
J
)
6.8 Hz, 2H,
NHCH2CH2PhSO2NH2); m/z (FAB) 570 (M + Na)+, 548 (M +
H)+.
N-(4-Methoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarba-
moyl]methyl}aminoacetic Acid (5a). See characterization in
Supporting Information.
GeneralMethodforPreparationofN-(Arylsulfonyl)-N-{[2-(4-sulfa-
moylphenyl)ethylcarbamoyl]methyl}aminoacetohydroxamic Ac-

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7977
ids (6a-c). The general method for carboxylic acid/hydroxylamine
coupling, as for the preparation of compounds 12a-c, was followed
with intermediate 5a, 5b, or 5c and using hydroxylamine hydro-
chloride. The reaction mixture was filtered, and the solvent was
evaporated; recrystallization of the residue afforded the pure
products as solids.
N-(4-Methoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarba-
moyl]methyl}aminoacetohydroxamic Acid (6a). Recrystallized
from acetonitrile; white solid; 82% yield; mp 170-172 °C; 1H
NMR (D2O, pD ca. 9) δ 7.77-7.72 (2d, 4H, ArH), 7.35 (d, J )
8.4 Hz, 2H, ArH), 7.12 (d, J ) 9.0 Hz, 2H ArH), 3.88 (s, 3H,
OCH3), 3.79 (s, 2H, CH2CONHCH2), 3.71 (s, 2H, CH2CONHOH),
3.40 (t, J ) 6.8 Hz, 2H, NHCH2CH2PhSO2NH2), 2.84 (t, J ) 6.8
Hz, 2H, NHCH2CH2PhSO2NH2); m/z (FAB) 501 (M + H)+, 535
(M + K)+. Anal. (C19H24N4O8S2) C, H, S. N: calcd, 11.19; found,
11.68.
CH(CH3)2), 0.87-0.83 (2d, 6H, CH(CH3)2); m/z (FAB) 680 (M +
H)+, 702 (M + Na)+.
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)eth-
ylcarbamoyl]methyl}-D-valine (19). The title compound was
prepared following the general method for catalytic hydrogenolysis,
starting from 18 and using 1.5 bar of H2. Recrystallization with
ethyl ether afforded the pure product as a pale solid: yield 92%;
mp 97-99 °C; 1H NMR (CDCl3) δ 7.87 (d, J ) 8.4 Hz, 2H, ArH),
7.75 (d, J ) 9.0 Hz, 2H, ArH), 7.44-7.39 (m, 4H, ArH), 7.22 (t,
J ) 7.5 Hz, 1H, ArH), 7.06-7.01 (2d, 4H, ArH), 6.92 (bs, 1H,
CONHCH2), 4.95 (s, 2H, SO2NH2), 3.98, 3.80 (dd, J ) 17.4, 18.6
Hz, 2H, CH2CONHCH2), 3.84 (d, J ) 9.6 Hz, 1H, NCH(iPr)),
3.68-3.50 (2m, 2H, NHCH2CH2PhSO2NH2), 2.93 (q, J ) 5.7 Hz,
2H, NHCH2CH2PhSO2NH2), 2.03-1.90 (m, 1H, CH(CH3)2), 0.94
(d, J ) 6.3 Hz, 3H, CHCH3), 0.72 (d, J ) 6.3 Hz, 3H, CHCH3);
m/z (FAB) 612 (M + Na)+, 590 (M + H)+.
N-(4-Biphenylsulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarbamoyl]-
methyl}aminoacetohydroxamic Acid (6b). Recrystallized from
CH2Cl2; white solid; 85% yield; mp 165-166 °C; 1H NMR (D2O,
pD ca. 9) δ 7.98 (d, J ) 8.7 Hz, 2H, ArH), 7.89 (d, J ) 7.8 Hz,
2H, ArH), 7.79 (d, J ) 8.1 Hz, 2H, ArH), 7.71 (d, J ) 7.2 Hz,
2H, ArH), 7.54-7.45 (m, 3H, ArH), 7.34 (d, J ) 7.8 Hz, 2H, ArH),
3.83 (s, 2H, CH2CONHCH2), 3.78 (s, 2H, CH2CONHOH), 3.42
(t, J ) 7.1 Hz, 2H, NHCH2CH2PhSO2NH2), 2.85 (s, J ) 7.4 Hz,
2H, NHCH2CH2PhSO2NH2); m/z (HRMS) for (M + Na)+, calcd
569.11351, found 569.11466; for (M + H)+, calcd 547.13157,
found 547.13270. Anal. (C24H26N4O7S2 ·0.4CH2Cl2) C, N. H: calcd,
4.65; found, 4.24. S: calcd, 11.01; found, 11.44.
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)ethylcarba-
moyl]methyl}aminoacetohydroxamic Acid (6c). Recrystallized
from acetonitrile; pale solid; 31% yield; mp 151-153 °C; 1H NMR
(D2O, pD ca. 9) δ 7.78 (d, J ) 9.0 Hz, 2H, ArH), 7.74 (d, J ) 8.1
Hz, 2H, ArH), 7.48 (t, J ) 8.1 Hz, 2H, ArH), 7.36 (d, J ) 8.4 Hz,
2H, ArH), 7.32 (t, 1H, J ) 7.2 Hz, ArH), 7.17-7.12 (2d, 4H, ArH),
3.85 (s, 2H, CH2CONHCH2), 3.79 (s, 2H, CH2CONHOH), 3.41
(t, J ) 6.8 Hz, 2H, NHCH2CH2PhSO2NH2), 2.84 (t, J ) 6.8 Hz,
2H, NHCH2CH2PhSO2NH2); m/z (HRMS) for (M + Na)+, calcd
585.10843, found 585.10743; for (M + H)+, calcd 563.12648,
found 563.12580. Anal. (C24H26N4O8S2 ·0.4CH2Cl2) C, H, N, S.
N-(4-Phenoxybenzenesulfonyl)-N-hydroxycarbonylmethyl-O-
benzyl-D-valine (17). The synthesis of the title compound involved
the preparation of 10c, starting from 9c as previously described,
and subsequent tert-butyl deprotection was performed as for
compounds 13a-c. After evaporation of the reaction mixture, the
residue was washed with ethyl ether, the solid was dissolved in
ethyl acetate, and the resulting solution was washed with 1% NaOH;
after the organic phase was dried over anhydrous Na2SO4 and the
solvent was evaporated, the pure product was obtained as a pale
yellow solid: overall yield of 38% for 10c-17; mp 73-75 °C; 1H
NMR (CDCl3) δ 7.76 (d, J ) 8.4 Hz, 2H, ArH), 7.33 (t, J ) 7.8
Hz, 2H, ArH), 7.22-7.13 (m, 6H, ArH), 6.94 (d, J ) 7.8 Hz, 2H,
ArH), 6.79 (d, J ) 8.1 Hz, 2H, ArH), 4.97, 4.91 (dd, J ) 12.3,
12.0 Hz, 2H, CH2Ph), 4.19 (d, J ) 18.0, Hz, 1H, CH2CO2H),
3.90-3.83 (m, 3H, CH2CO2H, NCH(iPr)), 2.08-1.97 (m, 1H,
CH(CH3)2), 0.71-0.65 (2d, 6H, CH(CH3)2); m/z (FAB) 520 (M +
Na)+.
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)eth-
ylcarbamoyl]methyl}-O-benzyl-D-valinehydroxamic Acid (20).
Compound 20 was synthesized by a procedure identical to that used
for 12a-c, starting from 19. After concentration in vacuum of the
reaction mixture, the residue was dissolved in ethyl acetate, and
this solution was washed with 1 M HCl and water; the organic
phase was then dried over anhydrous Na2SO4 and the solvent
evaporated under vacuum. Flash column chromatography of that
residue was then performed with a 12:1 CH2Cl2/MeOH eluent (Rf
0.48); evaporation of the solvent under vacuum afforded the pure
product as a yellow hygroscopic solid (64% yield): 1H NMR
(CDCl3) δ 9.19 (s, 1H, CONHOBn), 7.83 (d, J ) 8.4 Hz, 2H, ArH),
7.77 (d, J ) 8.4 Hz, 2H, ArH), 7.39-7.31 (m, 9H, ArH), 7.21 (t,
J ) 6.9 Hz, 1H, ArH), 7.04-7.00 (m, 4H, ArH), 6.60 (t, J ) 6.0
Hz, 1H, CONHCH2), 4.90-4.79 (m, 4H, SO2NH2, CH2Ph), 3.99,
3.76 (dd, J ) 17.1 Hz, 2H, CH2CONHCH2), 3.59-3.43 (m, 2H,
NHCH2CH2PhSO2NH2), 3.42 (d, J ) 10.8 Hz, 1H, NCH(iPr)), 2.91
(t, J ) 6.0 Hz, 2H, NHCH2CH2PhSO2NH2), 2.04-1.99 (m, 1H,
CH(CH3)2), 0.81 (d, J ) 6.3 Hz, 3H, CHCH3), 0.50 (d, J ) 6.0
Hz, 3H, CHCH3); m/z (FAB) 695 (M + H)+, 717 (M + Na)+.
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)eth-
ylcarbamoyl]methyl}-D-valinehydroxamic Acid (7). Catalytic
hydrogenolysis of 20 was performed, using the above-described
general method, with 1.5 bar of H2. Final recrystallization from
ethyl acetate/ethyl ether afforded the pure product as a pale yellow
solid; yield 95%; mp 85-87 °C;1H NMR (D2O, pD ca. 9) δ
7.76-7.71 (2d, 4H, ArH), 7.44 (t, J ) 7.0 Hz, 2H, ArH), 7.36 (d,
2H, J ) 7.8 Hz, ArH), 7.28 (t, J ) 6.3 Hz, 1H, ArH), 7.10 (d, J
) 6.9 Hz, 4H, ArH), 4.20, 3.94 (dd, J ) 17.1, 18.0 Hz, 2H,
CH2CONHCH2), 3.63 (d, J ) 10.2 Hz, 1H, NCH(iPr)), 3.38-3.32
(m, 2H, NHCH2CH2PhSO2NH2), 2.80 (t, J ) 7.0 Hz, 2H,
NHCH2CH2PhSO2NH2), 1.84-1.97 (m, 1H, CH(CH3)2), 0.80 (d,
J ) 6.3 Hz, 3H, CHCH3), 0.73 (d, J ) 5.7 Hz, 3H, CHCH3); m/z
(FAB) 605 (M + H)+, 627 (M + Na)+. Anal. (C27H32N4O8S2 ·
0.4EtOAc) C, H, N, S.
(B) MMP Inhibition Assays.42,43 Recombinant human proge-
latinases A (pro-MMP-2) and B (pro-MMP-9) from transfected
mouse myeloma cells and MMP-16 and MMP-14 catalytic domains
were supplied by Prof. Gillian Murphy (Department of Oncology,
University of Cambridge, U.K.). Pro-MMP-1, pro-MMP-8, pro-
MMP-13, and TACE (ADAM-17) were purchased from Calbio-
chem. Proenzymes were activated immediately prior to use with
p-aminophenylmercuric acetate (APMA, 2 mM, for 1 h at 37 °C
for MMP-2, MMP-1, and MMP-8, 1 mM for 1 h at 37 °C for
MMP-9 and MMP-13).
For assay measurements, the inhibitor stock solutions (DMSO,
100 mM) were further diluted, at seven different concentrations
(0.01 nM-300 µM) for each MMP in the fluorometric assay buffer
(FAB: 50 mM Tris, pH ) 7.5, 150 mM NaCl, 10 mM CaCl2, 0.05%
Brij 35, and 1% DMSO). Activated enzyme (final concentration
2.9 nM for MMP-2, 2.7 nM for MMP-9, 1.5 nM for MMP-8, 0.3
nM for MMP-13, 1 nM for MMP-14 cd, 15 nM for MMP-16 cd,
2.0 nM for MMP-1, and 7.5 nM for TACE) and inhibitor solutions
were incubated in the assay buffer for 4 h at 25 °C. After addition
of 200 µM solution of the fluorogenic substrate Mca-Arg-Pro-Lys-
N-(4-Phenoxybenzenesulfonyl)-N-{[2-(4-sulfamoylphenyl)eth-
ylcarbamoyl]methyl}-O-benzyl-D-valine (18). A procedure similar
to that used for the preparation of 5a-c was followed, starting from
17. After the extractions were performed as mentioned in that case,
the organic extract was concentrated, and the residue was purified
by flash chromatography using 12:1 CH2Cl2/MeOH as eluent (Rf
0.60). Evaporation of the solvent under vacuum afforded the pure
product as a yellow solid: yield 72%; mp 60-62 °C; 1H NMR
(CDCl3) δ 7.88 (d, J ) 8.1 Hz, 2H, ArH), 7.68 (d, J ) 8.7 Hz,
2H, ArH), 7.44-7.22 (m, 10H, ArH), 7.04 (d, J ) 7.2 Hz, 2H,
ArH), 6.93 (d, J ) 8.7 Hz, 2H, ArH), 4.92, 4.80 (dd, J ) 12.3,
12.0 Hz, 2H CH2Ph), 4.73 (s, 2H, SO2NH2), 4.12, 3.85 (dd, J )
18.3, 17.7 Hz, 2H, CH2CONHCH2), 4.04 (d, J ) 9.9 Hz, 1H,
NCH(iPr)), 3.55 (q, J ) 6.8 Hz, 2H, NHCH2CH2PhSO2NH2), 2.93
(t, J ) 7.2 Hz, 2H, NHCH2CH2PhSO2NH2), 2.07-1.95 (m, 1H,

7978 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Marques et al.
Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2 (Sigma) for MMP-3 and
Mca-Lys-Pro-Leu-Gly-Leu-Dap(Dnp)-Ala-Arg-NH2 (Bachem)44 for
all of the other enzymes in DMSO (final concentration 2 µM), the
hydrolysis was monitored every 15 s for 20 min by recording the
increase in fluorescence (λex ) 325 nm, λem ) 395 nm) using a
Molecular Device SpectraMax Gemini XS plate reader. The assays
were performed in triplicate in a total volume of 200 µL per well
in 96-well microtiter plates (Corning, black, NBS). Control wells
lack inhibitor. The MMP inhibition activity was expressed in relative
fluorescent units (RFU). Percent of inhibition was calculated from
control reactions without the inhibitor. IC50 was determined using
the formula Vi/V0 ) 1/(1 + [I]/ IC50), where Vi is the initial velocity
of substrate cleavage in the presence of the inhibitor at concentration
[I] and V0 is the initial velocity in the absence of the inhibitor.
Results were analyzed using SoftMax Pro45 software and GraFit46
software.
(C) CA Inhibition Assays. Recombinant human CA isoforms
I, II, and IX were prepared as previously reported by our group,47,48
and their activity was assayed by a stopped-flow CO2 hydration
assay26 with an Applied Photophysics (Oxford, U.K.) stopped-flow
instrument. Phenol red (at a concentration of 0.2 mM) was used as
indicator, working at the absorbance maximum of 557 nm, with
10 mM Hepes (pH 7.5) as buffer, 0.1 M Na2SO4 (for maintaining
constant the ionic strength), following the CA-catalyzed CO2
hydration reaction for a period of 10-100 s. The CO2 concentrations
ranged from 1.7 to 17 mM for the determination of the kinetic
parameters and inhibition constants. For each inhibitor, at least six
records of the initial 5-10% of the reaction were used for
determining the initial velocity. The uncatalyzed rates were
determined in the same manner and subtracted from the total
observed rates. Stock solutions of inhibitor (1 mM) were prepared
in distilled deionized water with 10-20% (v/v) DMSO (which is
not inhibitory at these concentrations), and dilutions up to 0.1 nM
were done thereafter with distilled deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min at room
temperature prior to assay in order to allow for the formation of
the E-I complex. The inhibition constants were obtained by
nonlinearleast-squaresmethodsusingPRISM3,fromLineweaver-Burk
plots, as reported earlier, and represent the average from at least
three different determinations.47,48
(rmsd) between the positions of the heavy atoms was calculated,
this parameter being considered as a measure of the docking
accuracy.
Each scoring function was also tested with a scaffold match
constraint, where the hydroxamic moiety of the original ligand in
each crystal structure was used as the scaffold to be matched; the
scaffold match constraint weight, a parameter defining how closely
the ligand atoms should fit onto the scaffold, was set to 10.0.
Docking of Compound 7. The ligand was built using Maestro
7.5 and underwent CS and structure optimization with Macromodel,
using the same procedures described above. The structures of the
proteins were extracted from the RCSB Protein Data Bank and
subsequently treated in the same way as described before: for
MMP-2 (entry 1QIB, catalytic domain), MMP-1 (entry 1HFC),
MMP-8 (entry 1MNC), MMP-9 (entry 1GKC), MMP-13 (entry
830C), MMP-14 (entry 1BQQ), and MMP-16 (entry 1RM8). Since
the MMP-2 structure did not possess any ligand, it was aligned
with the MMP-9 complex (using Chimera software),51 and this
ligand was further used to define the zone of interest in MMP-2
for the docking calculations with Gold. To perform the docking
calculations on MMPs, we used the same conditions and parameters
that were previously used in the validation analysis, which revealed
the best prediction of the binding mode of the hydroxamic inhibitors
into the MMPs. These conditions involved, namely, the ASP scoring
function applying the SMC function.
Acknowledgment. The authors are grateful for financial
support given by the Portuguese Fundac¸a˜o para a Cieˆncia e
Tecnologia (postdoc grant SFRH/BPD/29874/2006 for S.M.M.
and project PDCT/QUI/56985/2004) and by the Fondazione
Monte dei Paschi di Siena (“Strumenti terapeutici innovativi
per la modulazione dell’attivita` di metalloproteasi della matrice
implicate nelle patologie tumorali cerebrali”).
Supporting Information Available: Docking results of inhibitor
7 with MMP-8, -9, -13, -14, and -16 and synthesis and characteriza-
tion of all compounds, as well as chromatographic and purity data
for target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(D) Docking of Ligands. MMP-Inhibitor Complex Struc-
tures and Docking. The X-ray structures of MMP-inhibitor
complexes with different hydroxamate-based inhibitors, available
to date, were taken from the Protein Data Bank39 and were used
for this analysis. Solvent molecules and counterions were removed
from the original structure whenever they were present, as well as
the structural Ca2+ ions in the proteins. Hydrogen atoms were added
by means of Maestro 7.5.49 The ligands were extracted from the
complexes and were subjected to a conformational search (CS) of
1000 steps, using a water environment model (generalized-Born/
surface-area model) by means of Macromodel.50 The algorithm used
was based on the Monte Carlo method with the MMFFs force field
and a distance-dependent dielectric constant of 1.0. The ligands
were then minimized using the conjugated gradient method until a
convergence value of 0.05 kcal/(Å ·mol) was reached, using the
same force field and parameters used for the CS. The minimized
ligands were docked in their respective proteins with the Gold
program,38 version 3.0.1. The region of interest used by Gold was
defined in order to contain the residues within 15 Å from the original
position of the ligand in the X-ray structures; the zinc ion was set
as possessing a trigonal-bipyramidal coordination. The “allow early
termination” option was deactivated while the possibility for the
ligand to flip ring corners was activated. The remaining Gold default
parameters were used, and the ligands were submitted to 100 genetic
algorithm runs applying the three fitness functions available with
Gold (GoldScore, ChemScore, and ASP), without any other
constraints. The best docked conformation for each scoring function
was then compared with the experimental conformation of the
ligand in the crystal structure and the root-mean-square deviation
(1) Sternlicht, M. D.; Werb, Z. How matrix metalloproteinases regulate
cell behavior. Annu. ReV. Cell DeV. Biol. 2001, 17, 463–516.
(2) Egeblad, M.; Werb, Z. New functions for the matrix metalloproteinases
in cancer progression. Nat. ReV. Cancer 2002, 2, 161–174.
(3) Parks, W. C.; Wilson, C. L.; Lo´pez-Boado, Y. S. Matrix metallopro-
teinases as modulators of inflammation and innate immunity. Nat. ReV.
Immunol. 2004, 4, 617–629.
(4) Vartak, D. G.; Gemeinhart, R. A. Matrix metalloproteases: underuti-
lized targets for drug delivery. J. Drug Target. 2007, 15, 1–20.
(5) Raffetto, J. D.; Khalil, R. Matrix metalloproteinases and their inhibitors
in vascular remodeling and vascular disease. A. Biochem. Pharmacol.
2008, 75, 346–359.
(6) Overall, C. M.; Lo´pez-Ot´ın, C. Strategies for MMP inhibition in cancer:
innovations for the post-trial era. Nat. ReV. Cancer 2002, 2, 657–672.
(7) Martin, M. D.; Matrisian, L. M. The other side of MMPs: protective
roles in tumor progression. Cancer Metastasis ReV. 2007, 26, 717–
724.
(8) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Matrix metallopro-
teinase inhibitors and cancer: trials and tribulations. Science 2002, 295,
2387–2392.
(9) Overall, C. M.; Kleifeld, O. Tumour microenvironmentsOpinion:
validating matrix metalloproteinases as drug targets and anti-targets
for cancer therapy. Nat. ReV. Cancer 2006, 6, 227–239.
(10) Overall, C. M.; Kleifeld, O. Towards third generation matrix metal-
loproteinase inhibitors for cancer therapy. Br. J. Cancer 2006, 94,
941–946.
(11) Reiter, L. A.; Robinson, R. P.; McClure, K. F.; Jones, C. S.; Reese,
M. R.; Mitchell, P. G.; Otterness, I. G.; Bliven, M. L.; Liras, J.; Cortina,
S. R.; Donahue, K. M.; Eskra, J. D.; Griffiths, R. J.; Lame, M. E.;
Lopez-Anaya, A.; Martinelli, G. J.; McGahee, S. M.; Yocum, S. A.;
Lopresti-Morrow, L. L.; Tobiassen, L. M.; Vaughn-Bowser, M. L.
Pyran-containing sulfonamide hydroxamic acids: potent MMP inhibi-
tors that spare MMP-1. Bioorg. Med. Chem. Lett. 2004, 14, 3389–
3395.

Metalloproteinase and Carbonic Anhydrase Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7979
(12) Fingleton, B. MMPs as therapeutic targetssStill a viable option. Semin.
Cell DeV. Biol. 2008, 19, 61–68.
molecular modelling study, synthesis and MMP-1/MMP-2 inhibition profile
of (R)- and (S)-N-hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-3-
methylbutanamides. Bioorg. Med. Chem. 2006, 14, 4260–4276.
(33) Peng, S. X.; Strojnowski, M. J.; Joanna K, Hu, B. J. S.; Eichhold,
T. H.; Wehmeyer, K. R.; Pikul, S.; Almstead, N. G. Gas chromato-
graphic-mass spectrometric analysis of hydroxylamine for monitoring
the metabolic hydrolysis of metalloprotease inhibitors in rat and human
liver microsomes. J. Chromatogr. B 1999, 724, 181–187.
(34) Terp, G. E.; Cruciani, G.; Christensen, I. T.; Jørgensen, F. S. Structural
differences of matrix metalloproteinases with potential implications
for inhibitor selectivity examined by the GRID/CPCA approach.
J. Med. Chem. 2002, 45, 2675–2684.
(35) Levin, J. I.; Chen, J. M.; Cheung, K.; Cole, D.; Crago, C.; Santos, E. D.; Du,
X.; Khafizova, G.; MacEwan, G.; Niu, C.; Salaski, E. J.; Zask, A.; Cummons,
T.; Sung, A.; J. Xu, Y. Z.; Xu, W.; Ayral-Kaloustian, S.; Jin, G.; Cowling,
R.; Barone, D.; Mohler, K. M.; Black, R. A.; Skotnicki, J. S. Acetylenic TACE
inhibitors. Part 1. SAR of the acyclic sulfonamide hydroxamates. Bioorg.
Med. Chem. Lett. 2003, 13, 2799–2803.
(36) Rao, B. G.; Bandarage, U. K.; Wang, T.; Come, J. H.; Perola, E.;
Wei, Y.; Tian, S.-K.; Saunders, J. O. Novel thiol-based TACE
inhibitors: Rational design, synthesis, and SAR of thiol-containing aryl
sulfonamides. Bioorg. Med. Chem. Lett. 2007, 17, 2250–2253.
(37) Santos, M. A.; Marques, S.; Vullo, D.; Innocenti, A.; Scozzafava, A.;
Supuran, C. T. Carbonic anhydrase inhibitors. Inhibition of cytosolic/
tumor-associated isoforms I, II and IX with iminodiacetic carboxylates/
hydroxamates also incorporating benzenesulfonamide moieties. Bioorg.
Med. Chem. Lett. 2007, 17, 1538–1543.
(13) Supuran, C. T. Carbonic anhydrases: catalytic and inhibition mech-
anisms, distribution and physiological roles. In Carbonic Anhydrase.
Its Inhibitors and ActiVators; Supuran, C. T.; Scozzafava, A.; Conway,
J., Eds. CRC Press: Boca Raton, London, and New York, 2004; pp
1-23.
(14) Maren, T. H. Carbonic anhydrase: chemistry, physiology, and inhibi-
tion. Physiol. ReV. 1967, 47, 595–781.
(15) Supuran, C. T.; Scozzafava, A.; Casini, A. Carbonic anhydrase
inhibitors. Med. Res. ReV. 2003, 23, 146–189.
(16) Scozzafava, A.; Mastrolorenzo, A.; Supuran, C. T. Carbonic anhydrase
inhibitors and activators and their use in therapy. Expert Opin. Ther.
Pat. 2006, 16, 1627–1664.
(17) Thiry, A.; Dogne´, J.-M.; Masereel, B.; Supuran, C. T. Targeting tumor-
associated carbonic anhydrase IX in cancer therapy. Trends Pharmacol.
Sci. 2006, 27, 566–573.
(18) Supuran, C. T. Carbonic anhydrases: novel therapeutic applications for
inhibitors and activators. Nat. ReV. Drug DiscoVery 2008, 7, 168–181.
(19) Alterio, V.; Vitale, R. M.; Monti, S. M.; Pedone, C.; Scozzafava, A.;
Cecchi, A.; Simone, G. D.; Supuran, C. T. Carbonic anhydrase
inhibitors: X-ray and molecular modeling study for the interaction of
a fluorescent antitumor sulfonamide with isozyme II and IX. J. Am.
Chem. Soc. 2006, 128, 8329–8335.
(20) MacPherson, L. J.; Bayburt, E. K.; Capparelli, M. P.; Carroll, B. J.; Goldstein,
R.; Justice, M. R.; Zhu, L.; Hu, S.-i.; 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. Discovery of CGS 27023A, a non-peptidic, potent,
and orally active stromelysin inhibitor that blocks cartilage degradation in
rabbits. J. Med. Chem. 1997, 40, 2525–2532.
(38) Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R.
Development and validation of a genetic algorithm for flexible docking.
J. Mol. Biol. 1997, 267, 727–748.
(21) Mincione, F.; Starnotti, M.; Menabuoni, L.; Scozzafava, A.; Casini,
A.; Supuran, C. T. Carbonic anhydrase inhibitors: 4-sulfamoyl-
benzenecarboxamides and 4-chloro-3-sulfamoyl-benzenecarboxamides
with strong topical antiglaucoma properties. Bioorg. Med. Chem. Lett.
2001, 11, 1787–1791.
(22) Scozzafava, A.; Supuran, C. T. Carbonic anhydrase and matrix
metalloproteinase inhibitors: sulfonylated amino acid hydroxamates
with MMP inhibitory properties act as efficient inhibitors of CA
isozymes I, II, and IV, and N-hydroxysulfonamides inhibit both these
zinc enzymes. J. Med. Chem. 2000, 43, 3677–3687.
(23) Nuti, E.; Orlandini, E.; Nencetti, S.; Rossello, A.; Innocenti, A.;
Scozzafava, A.; Supuran, C. T. Carbonic anhydrase and matrix
metalloproteinase inhibitors. Inhibition of human tumor-associated
isozymes IX and cytosolic isozyme I and II with sulfonylated
hydroxamates. Bioorg. Med. Chem. 2007, 15, 2298–2311.
(24) Campestre, C.; Tortorella, P.; Agamennone, M.; Preziuso, S.; Biasone,
A.; Nuti, E.; Rossello, A.; Gallina, C. Peptidyl 3-substituted 1-hy-
droxyureas as isosteric analogues of succinylhydroxamate MMP
inhibitors. J. Med. Chem. 2008, 43, 1008–1014.
(25) Temperini, C.; Innocenti, A.; Scozzafava, A.; Supuran, C. T. N-hydroxyureasa
versatile zinc binding function in the design of metalloenzyme inhibitors.
Bioorg. Med. Chem. Lett. 2006, 16, 4316–4320.
(26) Gupta, S. P.; Maheswaran, V.; Pande, V.; Kumar, D. A comparative
QSAR study on carbonic anhydrase and matrix metalloproteinase
inhibition by sulfonylated amino acid hydroxamates. J. Enzyme Inhib.
Med. Chem. 2003, 18, 7–13.
(27) Santos, M. A.; Marques, S. M.; Tuccinardi, T.; Carelli, P.; Panelli,
L.; Rossello, A. Design, synthesis and molecular modeling studies of
iminodiacetyl monohydroxamic acid derivatives as MMP inhibitors.
Bioorg. Med. Chem. 2006, 14, 7539–7550.
(28) Khalifah, R. G. The carbon dioxide hydration activity of carbonic
anhydrase. J. Biol. Chem. 1971, 246, 2561–2573.
(39) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.;
Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank.
Nucleic Acids Res. 2000, 28, 235–242.
(40) Tuccinardi, T.; Nuti, E.; Ortore, G.; Rossello, A.; Avramova, S. I.;
Martinelli, A. Development of a receptor-based 3D-QSAR study for
the analysis of MMP2, MMP3, and MMP9 inhibitors. Bioorg. Med.
Chem. 2008, 16, 7749–7758.
(41) Armarego, W. L. F.; Perrin, D. D. Purification of Laboratory
Chemicals, 4th ed.; Butterworth-Heinemann: Oxford, 1999.
(42) Knight, C. G.; Willenbrock, F.; Murphy, G. A novel coumarin-labelled
peptide for sensitive continuous assays of the matrix metalloprotein-
ases. FEBS Lett. 1992, 296, 263–266.
(43) Murphy, G.; Crabbe, T. Gelatinases A and B. Methods Enzymol. 1995,
248, 470–484.
(44) Neumann, U.; Kubota, H.; Frei, K.; Ganu, V.; Leppert, D. Charac-
terization of Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, a fluoro-
genic substrate with increased specificity constants for collagenases
and tumor necrosis factor converting enzyme. Anal. Biochem. 2004,
328, 166–173.
(45) SoftMax Pro 4.7.1 by Molecular Device.
(46) GraFit version 4 by Erithecus Software.
(47) (a) Cecchi, A.; Hulikova, A.; Pastorek, J.; Pastorekova, S.; Scozzafava,
A.; Winum, J.-Y.; Montero, J.-L.; Supuran, C. T. Carbonic anhydrase
inhibitors. Design of fluorescent sulfonamides as probes of tumor-
associated carbonic anhydrase IX that inhibit isozyme IX-mediated
acidification of hypoxic tumors. J. Med. Chem. 2005, 48, 4834–4841.
(b) Casey, J. R.; Morgan, P. E.; Vullo, D.; Scozzafava, A.; Mas-
trolorenzo, A.; Supuran, C. T. Carbonic anhydrase inhibitors. Design
of selective, membrane-impermeant inhibitors targeting the human
tumor-associated isozyme IX. J. Med. Chem. 2004, 47, 2337–2347.
(48) (a) Pastorekova, S.; Casini, A.; Scozzafava, A.; Vullo, D.; Pastorek, J.;
Supuran, C. T. Carbonic anhydrase inhibitors: the first selective, membrane-
impermeant inhibitors targeting the tumor-associated isozyme IX. Bioorg.
Med. Chem. Lett. 2004, 14, 869–873. (b) Vullo, D.; Innocenti, A.;
Nishimori, I.; Pastorek, J.; Scozzafava, A.; Pastorekova, S.; Supuran, C. T.
Carbonic anhydrase inhibitors. Inhibition of the transmembrane isozyme XII
with sulfonamidessa new target for the design of antitumor and antiglaucoma
drugs? Bioorg. Med. Chem. Lett. 2005, 15, 963–969.
(29) Marques, S. M.; Chaves, S.; Rossello, A.; Tuccinardi, T.; Santos, M. A.
Iminodiacetyl-monohydroxamate derivatives as potent and selective
MMP inhibitors. Met. Ions Biol. Med. 2006, 9, 117–121.
(30) Bairoch, A.; Apweiler, R. The SWISS-PROT protein sequence
database and its supplement TrEMBL in 2000. Nucleic Acids Res.
2000, 28, 45–48.
(31) Yoshiizumi, K.; Yamamoto, M.; Miyasaka, T.; Ito, Y.; Kumihara, H.;
Sawa, M.; Kiyoi, T.; Yamamoto, T.; Nakajima, F.; Hirayama, R.;
Kondo, H.; Ishibushi, E.; Ohmoto, H.; Inoue, Y.; Yoshino, K. Synthesis
and structure-activity relationships of 5,6,7,8-tetrahydropyrido(3,4-
b)pyrazine-based hydroxamic acids as HB-EGF shedding inhibitors.
Bioorg. Med. Chem. 2003, 11, 433–450.
(49) Maestro, version 7.5; Schro¨dinger Inc.: Portland, OR, 2005.
(50) Macromodel, version 8.5; Schro¨dinger Inc.: Portland, OR, 1999.
(51) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF ChimerasA
visualization system for exploratory research and analysis. J. Comput.
Chem. 2004, 25, 1605–1612.
(32) Tuccinardi, T.; Martinelli, A.; Nuti, E.; Carelli, P.; Balsano, F.; Uccello-
Barretta, G.; Murphy, G.; Rossello, A. Amber force field implementation,
JM800964F