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

Article abstract of DOI:10.1021/jm000027t

The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physiological functions in these organisms. Unsubstituted sulfonamides act a

Full text of DOI:10.1021/jm000027t

J . Med. Chem. 2000, 43, 3677-3687
3677
Ca r bon ic An h yd r a se a n d Ma tr ix Meta llop r otein a se In h ibitor s: Su lfon yla ted
Am in o Acid Hyd r oxa m a tes w ith MMP In h ibitor y P r op er ties Act a s Efficien t
In h ibitor s of CA Isozym es I, II, a n d IV, a n d N-Hyd r oxysu lfon a m id es In h ibit
Both Th ese Zin c En zym es1
Andrea Scozzafava and Claudiu T. Supuran*
Laboratorio di Chimica Inorganica e Bioinorganica, Universita` degli Studi, Via Gino Capponi 7, I-50121 Florence, Italy
Received J anuary 21, 2000
The 14 different carbonic anhydrase (CA, EC 4.2.1.1) isozymes as well as the 23 different matrix
metalloproteinases (MMPs) isolated up to now in higher vertebrates play important physi-
ological functions in these organisms. Unsubstituted sulfonamides act as high-affinity inhibitors
for the first type of these enzymes, whereas hydroxamates strongly inhibit the latter ones.
Since the active site geometry around the zinc ion in these two types of metalloenzymes is
rather similar, we tested whether sulfonylated amino acid hydroxamates of the type RSO2-
NX-AA-CONHOH (X ) H, benzyl, substituted benzyl; AA ) amino acid moiety, such as Gly,
Ala, Val, Leu) with well-known inhibitory properties against MMPs and Clostridium histolyti-
cum collagenase (ChC, another zinc enzyme related to the MMPs) might also act as CA
inhibitors. We also investigated whether N-hydroxysulfonamides of the type RSO2NHOH (which
are effective CA inhibitors) inhibit MMPs and ChC. Here we report several potent sulfonylated
amino acid hydroxamate CA inhibitors (with inhibition constants in the range of 5-40 nM,
against the human isozymes hCA I and hCA II, and 10-50 nM, against the bovine isozyme
bCA IV), as well as preliminary SAR for this new class of non-sulfonamide CA inhibitors. Some
N-hydroxysulfonamides also showed inhibitory properties (in the micromolar range) against
MMP-1, MMP-2, MMP-8, MMP-9, and ChC. Thus, the SO2NHOH group is a new zinc-binding
function for the design of MMP inhibitors. Both CA as well as MMPs are involved, among
others, in carcinogenesis and tumor invasion processes. On the basis of these findings, we
suggest that the mechanism of antitumor action with some hydroxamate inhibitors might also
involve inhibition of some CA isozymes (such as CA IX, CA XII, and CA XIV) present only in
tumor cell membranes, in addition to collagenases/gelatinases of the MMP type. Our data also
suggest that it should be possible to develop dual enzyme inhibitors that would strongly inhibit
both these metalloenzymes, CAs and MMPs, based on the nature of the R, AA, and X moieties
in the above formula. Compact X (such as H) and AA (such as Gly) moieties favor CA over
MMP inhibition, whereas bulkier X (benzyl, substituted benzyl, etc.) and AA (such as Val,
Leu) moieties and substituted-aryl R groups are advantageous for obtaining potent MMP and
ChC inhibitors, which show lower affinity for CA.
In tr od u ction
VI is secreted in saliva, whereas several acatalytic forms
are also known (CA VIII, CA X, and CA XI).2-15
The ubiquitous enzyme carbonic anhydrase (CA, EC
4.2.1.1) is present in Archaea, prokaryotes, and eukary-
otes as three different families of enzymes:2,3 the R-CA
(mainly in vertebrates and in some green plants), â-CA
(mainly in bacteria, algae, and green plants) and γ-CA
(only in Archaea) families, respectively.2,3 In higher
vertebrates, including humans, 14 different CA isozymes
have been described up to now2 that are involved in
crucial physiological processes connected with respira-
tion and transport of CO2/bicarbonate between metabo-
lizing tissues and the lungs, pH homeostasis, electrolyte
secretion in a variety of tissues/organs, and biosynthetic
reactions, such as lipogenesis, gluconeogenesis, and
ureagenesis, among others.2-7 Some of these isozymes
are cytosolic (such as CA I, CA II, CA III, and CA VII);
others are membrane-bound (CA IV, CA IX, CA XII, and
CA XIV). CA V is present only in mitochondria, and CA
In addition to the physiological reaction, the reversible
hydration of carbon dioxide to bicarbonate, CAs also
catalyze a variety of other reactions,16-25 such as alde-
hyde hydration16 and hydrolysis of carboxylic acid
esters,17,18 whereas esters of sulfonic19 or phosphoric20
acids also seem to act as substrates of these enzymes.
Other hydrolytic reactions in which CAs may partici-
pate, but which have not thoroughly been studied from
the mechanistic point of view, include hydrolyses of
diverse halogeno derivatives such as 2,4-dinitrofluo-
robenzene,21 benzyloxycarbonyl chloride,22 or sulfonyl
chlorides.23 Recently, the cyanate and cyanamide hy-
dration reactions catalyzed by several CA isozymes,
leading to suicide inhibitors of this enzyme, have also
been investigated spectroscopically (using Co(II)-sub-
stituted enzyme), kinetically, and by X-ray crystal-
lography by our group.24,25 On the other hand, CAs do
not possess peptidase activity,26,27 which in turn is the
only reaction catalyzed by the proteases of the matrix
* To whom correspondence should be addressed. Tel: +39-055-
2757551. Fax: +39-055-2757555. E-mail: cts@bio.chim.unifi.it.
10.1021/jm000027t CCC: $19.00 © 2000 American Chemical Society
Published on Web 09/13/2000

3678 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Scozzafava and Supuran
metalloproteinase (MMP) family with very great
efficiency.26-32
CA inhibitors. Some N-hydroxysulfonamides investi-
gated previously as CA inhibitors also showed inhibitory
properties (in the micromolar range) against MMP-1,
MMP-2, MMP-8, MMP-9, and a bacterial collagenase
(ChC) isolated from Clostridium histolyticum.
Specific inhibitors of both these types of zinc enzymes
are well-known, and some of them were clinically used
for more than 45 years (the sulfonamide CA inhibitors).
Inhibition of CAs by aromatic/heterocyclic sulfonamides
such as 1-5 has been successfully used in the treatment
of a variety of diseases such as glaucoma,4,33 epilepsy,34
congestive heart failure,5 mountain sickness,35 and
gastric and duodenal ulcers36 or as diuretic agents.37
MMPs on the other hand became targets for drug design
only recently, but several drugs of this type might reach
clinics soon, as anticancer or antiarthritis agents among
others.27,30-32
Resu lts
Syn th esis. The majority of the N-hydroxysulfona-
mides42 and some of the sulfonylated amino acid hy-
droxamates43,44 investigated here have previously been
reported by this group. Some of the compounds inves-
tigated in this paper are new (such as 7-9, 13-15, 19-
21, 25-33, 69, 71-80). They were synthesized by the
same methods used for the preparation of amino acid
hydroxamate/N-hydroxysulfonamide derivatives re-
ported in previous contributions from our group.42-44
CA In h ibitor y Activity. Inhibition data against
three CA isozymes, hCA I, hCA II, and bCA IV, with
the sulfonylated amino acid hydroxamates 7-45 and the
N-hydroxysulfonamides 46-80 are shown in Tables 1
and 2. The esterase activity of CA isozymes against
4-nitrophenyl acetate as substrate has been used in this
assay45 (see Experimental Section for details).
MMP a n d Ch C In h ibitor y Activity. Inhibition data
against four MMPs (MMP-1, MMP-2, MMP-8, and
MMP-9) and type II ChC with the compounds investi-
gated in the present paper (7-80) are shown in Tables
1 and 2. The thioester (AcProLeuGly-S-LeuLeuGlyOEt)
spectrophotometric method of Powers and Kam,46a
modified by J ohnson et al.,46b was used for assessing
the catalytic activity and inhibition of the four MMPs
mentioned above, whereas the amidolytic spectropho-
tometric method with FALGPA as substrate was used
for the ChC inhibition assays.47
Sp ectr oscop ic Stu d ies on Co(II)-Su bstitu ted En -
zym es. Electronic spectroscopic data for Co(II)-substi-
tuted hCA II and ChC and their adducts with standard
and new inhibitors of the type reported here are shown
in Table 3.
Recently this38 and other groups also showed39 that
some new types of sulfonamide CA inhibitors act as
efficient tumor cell growth inhibitors in vitro and in
vivo, by a mechanism of action that might involve
acidification of the intratumoral environment ensued
after CA inhibition38,39 or due to a reduced provision of
-
bicarbonate for nucleotide synthesis (HCO3 is the
substrate of carbamoyl phosphate synthetase II) as a
consequence of CA inhibition.39 On the other hand, some
new CA isozymes, such as CA IX,9 CA XII,12,15 and CA
XIV,13 were predominantly found to be present only in
tumor cells. These and other more classical isozymes,
such as CA I, CA II, and CA IV,40,41 were also shown to
be present and actively involved in other types of
proliferative diseases/processes, such as von Hippel-
Lindau renal tumors,15 progressive polycystic kidney
disease,40 acinar-ductal carcinomas of the pancreas,41
autoimmune or idiopathic chronic pancreatitis,41 and
apoptosis in some human pancreatic cancer cells.41 Since
many hydroxamate MMP inhibitors were developed just
as possible antitumor/antimetastatic agents,27,30-33 and
considering the fact that all inhibitors mentioned above
(of CAs or of MMPs) bind to the metal ion within the
active site cavity, it appeared of interest to further
explore the connections between CA and MMP inhibi-
tors, with the eventual evidence of a “cross-reactivity”
between them. Here we report the observation that
several potent MMP inhibitors of the sulfonylated amino
acid hydroxamate type also act as efficient CA inhibitors
(with inhibition constants in the range of 7-45 nM,
against the human isozymes hCA I and hCA II, and 10-
50 nM, against the bovine isozyme bCA IV), as well as
preliminary SAR for this new class of non-sulfonamide
Discu ssion
Ch em istr y. CAs and MMPs possess very similar
metal coordination spheres within their catalytic sites,
consisting of a Zn(II) ion coordinated by three histidines,
with the fourth ligand being a water molecule/hydroxide
ion, which is the nucleophile intervening in the catalytic
cycle of both enzymes (Figure 1).4,26,27,48,49
The main structural difference between these two
types of enzymes regards the residues with which the
zinc-bound water molecule/hydroxide ion interacts: in
CAs, the non-protein zinc ligand forms a hydrogen bond
with the hydroxyl moiety of Thr 199 (hCA II number-
ing), which in turn is hydrogen-bonded to the carboxy-
late of Glu 106, leading thus to a dramatic enhance of
nucleophilicity of the water molecule/hydroxide ion.4,25-27
In the case of MMPs, the zinc-bound water molecule
interacts with the carboxylate moiety of a conserved
glutamate residue (Glu 198 in MMP-8), probably form-
ing two hydrogen bonds with it.27,48,49 Thus, a very
effective nucleophile is formed again, which will attack
the amide scissile bond of the peptide substrate. The
principal difference between the enzymatic mechanisms
of CAs and MMPs consists of the fact that the nucleo-
philic adduct formed after the attack of the zinc-bound

CA and MMP Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3679
Ta ble 1. Inhibition of MMPs, ChC, and CAs with the Hydroxamates 7-45
Kia (nM)
MMP-9b
no.
R1
R
X
MMP-1b
MMP-2b
MMP-8b
ChCc
hCA Id
hCA IId
bCA IVd
7
8
9
H
H
H
H
H
H
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
4-MeO-C6H4
n-C4F9
C6F5
H
H
H
>200
145
>200
30
7
60
>200
150
>200
26
7
58
>200
139
>200
21
7
43
>200
155
>200
16
75
44
110
3.9
1.5
18
69
40
87
3.2
0.9
15
66
41
130
125
155
5.3
1.1
31
118
116
125
4.9
1.1
19
111
104
126
4.2
1.0
13
108
101
123
3.3
0.6
9
5.5
1.0
27
2.4
0.1
21
5.1
1.1
20
2.3
0.3
18
125
100
143
5
80
54
120
13
6
27
79
45
130
12
6
20
78
40
103
10
5
17
69
38
95
8
18
7
30
105
90
15
8
32
85
36
120
16
8
35
92
38
120
15
11
39
16
10
29
100
42
145
17
10
30
107
43
136
20
13
38
124
48
185
30
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34e
35e
36e
37f
38f
39f
40g
41g
42g
43h
44h
45h
C6H5CH2
C6H5CH2
C6H5CH2
H
H
H
C6H5CH2
C6H5CH2
C6H5CH2
H
H
H
C6H5CH2
C6H5CH2
C6H5CH2
H
H
H
C6H5CH2
C6H5CH2
C6H5CH2
2-O2NC6H4CH2
2-O2NC6H4CH2
2-O2NC6H4CH2
4-O2NC6H4CH2
4-O2NC6H4CH2
4-O2NC6H4CH2
2-O2NC6H4CH2
2-O2NC6H4CH2
2-O2NC6H4CH2
4-O2NC6H4CH2
4-O2NC6H4CH2
4-O2NC6H4CH2
2-ClC6H4CH2
2-ClC6H4CH2
2-ClC6H4CH2
1.2
42
121
96
>200
Me
Me
Me
Me
Me
Me
iPr
iPr
iPr
iPr
iPr
iPr
iBu
iBu
iBu
iBu
iBu
iBu
H
21
7
137
32
121
84
195
29
8
33
140
88
4.3
1.4
35
120
89
82
126
2.4
0.8
11
62
39
4.3
1.2
27
36
122
78
108
45
>200
>200
36
10
50
18
11
19
39
84
5
116
56
>200
100
74
170
118
107
>200
>200
160
>200
>200
>200
>200
>200
>200
>200
1.9
0.8
10
3.7
1.4
15
1.5
0.7
18
2.9
0.8
13
1.4
0.7
15
3.7
1.5
12
4.0
180
100
>200
>200
127
>200
>200
150
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
139
75
190
120
49
6
44
25
6
54
62
3
28
24
7
39
60
4
25
37
10
1.1
1.3
4.6
1.3
39
2.0
0.6
31
4.4
1.0
24
1.5
0.6
5
13
13
6
24
12
5
20
10
5
19
11
5
H
H
H
H
140
136
130
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
4-MeO-C6H4
n-C4F9
C6F5
28
5.0
1.7
21
11
5
6.4
1.3
27
4-MeO-C6H4
53
31
22
a
Ki values were obtained from Easson-Stedman56 plots using a linear regression program, from at least three different assays. Standard
errors were of 5-10%. With the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.46 c With FALGPA as
b
substrate, spectrophotometrically.47 With 4-NPA as substrate, by the esterase method, spectrophotometrically.45 e MMP and ChC
d
inhibition (but not CA inhibition) data from ref 43c. f ChC inhibition (but not MMP and CA inhibition) data from ref 58. ChC inhibition
g
h
(but not MMP and CA inhibition) data from ref 43b. ChC inhibition (but not MMP and CA inhibition) data from ref 59.
nucleophile to the substrate is the reaction product in
the case of CAs (the bicarbonate ion), whereas the
nucleophilic adduct is only a reaction intermediate in
the case of the MMPs (and also ChC).26,27 This is also
of crucial importance for the interaction of these en-
zymes with their inhibitors.
Inhibition of both CAs as well as MMPs is correlated
with the coordination of the inhibitor molecule (in
neutral or ionized state) to the catalytic metal ion, with
or without substitution of the metal-bound water mol-
ecule.4,26,27,30 Thus, CA and MMP inhibitors (abbreviated
as CAIs and MMPIs, respectively) must contain a zinc-
binding function attached to a scaffold that will interact
with other binding regions of the enzymes.4,26,27,30 In the
case of CAIs, unsubstituted aromatic/heterocyclic sul-
fonamides4,5 as well as N-hydroxysulfonamides42 proved
to be very effective inhibitors, with affinities in the low
nanomolar range for isozymes such as CA I, CA II, CA
IV, etc. These derivatives bind monodentately, as anions
(RSO2NH-) to the Zn(II) ion within the CA active site,
interacting also with several other active site residues,
by means of hydrogen bonds or hydrophobic interac-
tions.50 Residues situated at the entrance of the active
site, such as the histidine cluster comprising residues
His 64, His 3, His 4, and His 10, among others, seem to
be critically important for the formation of strong
enzyme-inhibitor adducts.4,33,50 In fact, it has been
observed that CAIs possessing elongated molecules, able
to interact with amino acid residues situated at the edge
of the active site entrance (and obviously with the zinc
ion, as mentioned above), are among the most efficient
ones (such as, for example, aminobenzolamide 6), and
this has also been explained theoretically in several
QSAR studies of one of our groups.51

3680 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Scozzafava and Supuran
Ta ble 2. Inhibition of MMPs, ChC, and CAs with the Hydroxysulfonamides 46-80
Kia (µM)
MMP-9b
no.
R
MMP-1b
MMP-2b
MMP-8b
ChCc
hCA Id
hCA IId
bCA IVd
46
Me
CF3
CCl3
n-C4F9
n-C8F17
Me2N
PhCH2
Ph
p-F-C6H4
p-Cl-C6H4
p-Br-C6H4
p-I-C6H4
p-CH3-C6H4
p-O2N-C6H4
m-O2N-C6H4
o-O2N-C6H4
3-Cl-4-O2N-C6H3
p-AcNH-C6H4
p-H2N-C6H4
4-MeO-3-H2N-C6H3
p-CH3O-C6H4
2,4,6-(CH3)3-C6H2
C6F5
>100
>100
>100
74
70
>100
>100
>100
>100
19
14
>100
>100
>100
>100
25
23
>100
>100
>100
>100
21
21
>100
>100
>100
>100
62
55
>100
>100
20
23
1.8
1.6
12
19
18
15
27
38
15
51
57
17
15
17
40
62
41
68
72
30
33
25
4.7
0.03
0.15
0.015
0.009
0.040
0.19
56
0.016
0.21
0.027
0.021
0.29
47e
48
49
50
51e
52e
53
95
79
74
71
71
66
65
42
37
59
30
29
34
30
33
40
73
70
72
70
65
72
77
21
23
38
14
9
15
11
14
21
76
74
75
70
69
71
64
15
16
24
16
12
21
10
16
36
83
78
73
76
66
69
60
14
13
23
13
10
24
9
12
34
80
83
79
80
72
70
88
36
32
62
29
18
50
13
20
39
0.46
0.026
0.019
0.021
0.018
0.013
0.070
0.009
0.005
0.005
0.004
0.029
0.037
0.044
0.051
0.062
0.0008
0.0011
0.0009
0.074
0.090
0.120
0.039
0.045
0.127
0.070
0.024
0.013
0.012
0.040
0.055
0.136
0.110
0.156
0.013
0.016
0.012
54e
55e
56e
57e
58e
59e
60e
61e
62e
63e
64e
65
66e
67e
68e
69
0.4
0.9
0.08
0.9
0.12
0.09
1.0
1.5
0.12
1.3
1.7
0.10
0.5
0.8
7
3-CF3-C6H4
o-HOOC-C6H4
70e
a
Ki values were obtained from Easson-Stedman56 plots using a linear regression program, from at least three different assays. Standard
errors were of 5-10%. With the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.46 c With FALGPA as
b
substrate, spectrophotometrically.47 With 4-NPA as substrate, by the esterase method, spectrophotometrically.45 e CA inhibition (but
d
not MMP and ChC inhibition) data from ref 42.
Ta ble 3. Spectral Data (in the range 400-750 nm) for Adducts
of Co(II)-hCA II and Co(II)-ChC with Standard and New
Inhibitors Reported Herea
band position (nm) and
adduct
pH
molar absorptivity (M-1 cm-1
)
Co(II)-hCA II
8.0 520 (280); 550 (380); 616.5 (280);
640 (260)
+acetazolamide 1 8.0 518 (390); 549 (220); 574 (530);
595 sh (500)
8.1 465 (100); 529 sh (90); 571 (100);
F igu r e 1. Active site coordination of the Zn(II) ion in human
carbonic anhydrase isozyme II (hCA II) and human collagenase
2 (MMP-8). The non-protein zinc ligand of hCA II may be a
hydroxide ion (as shown above) or a water molecule, depending
on the pH.
+thiocyanate
+nitrate
+acetate
+benzoate
+8
689 (9)
7.5 470 (100); 515 (80); 555 (110);
709 (9)
7.5 470 (100); 515 sh (80); 555 (110);
709 (9)
7.5 480 sh (110); 507 sh (170); 555 (110);
709 (9)
7.5 475 (110); 515 sh (80); 555 (115);
710 (9)
7.5 470 (110); 515 sh (90); 555 (110);
710 (9)
8.0 518 (320); 550 (300); 574 (380);
600 sh (480)
thiols, the phosphorus-based ligands, or the sulfodi-
imines, among others.27,30 The strongest class of MMPIs
is constituted by the hydroxamates.27,30 The interaction
of the catalytic domain of several MMPs with some
inhibitors has been recently investigated by means of
X-ray crystallography (Figure 2).32,48,49,52
+19
+50
As seen in Figure 2, hydroxamates bind bidentately
to the catalytic Zn(II) ion of the enzyme, which acquires
a distorted trigonal-bipyramidal geometry in this
way.48,49,52 The hydroxamate anion forms a short and
strong hydrogen bond with the carboxylate moiety of
Glu 219, which is oriented toward the unprimed binding
regions, whereas the NH hydroxamate participates in
a hydrogen bond with the carbonyl oxygen of Ala 182.
Thus, several strong interactions are achieved at the
zinc site, without any significant unfavorable contacts.
Recently, Christianson’s group investigated two simple
hydroxamates (methyl and trifluoromethyl hydroxa-
mates) as possible CAIs and reported the X-ray struc-
ture of such an adduct (hCA II-CF3CONHOH).53 This
compound binds to hCA II with an affinity of 3.8 µM,
+60
8.0 519 (370); 550 sh (380); 575 (360);
597 sh (400)
Co(II)-ChC
+8
6.5 530 sh (135); 585 (180)
6.5 505 sh (85); 563 (120); 598 (15)
6.5 505 sh (85); 562 (115); 597 (13)
6.5 505 sh (85); 563 (117); 597 (15)
6.5 503 sh (80); 562 (112); 598 (15)
6.5 501 sh (78); 563 (105); 598 (11)
6.5 501 sh (79); 563 (109); 598 (11)
+11
+23
+35
+65
+69
a
Enzyme concentrations were in the range 0.1-0.4 nM, at the
pH values specified in each case. Inhibitor concentrations were in
the range of 0.1-2 mM.
Depending on the zinc-binding function contained in
their molecule, MMPIs belong to several chemical
classes, such as the carboxylates, the hydroxamates, the

CA and MMP Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3681
Sch em e 1
F igu r e 2. Schematic representation for the binding of a
succinate hydroxamate inhibitor to MMP-7, as determined by
X-ray crystallography.48 The Zn(II)-ligand and hydrogen bond
interactions in the enzyme-inhibitor adduct are evidenced
(adapted and modified from ref 48).
aliphatic and aromatic N-hydroxysulfonamides of the
type 46-53, as well as aryl/hetaryl mono-/di-/tri-/
pentasubstituted derivatives, possessing a large variety
of substituents at the aromatic/heterocyclic moiety, of
types 54-79 (Table 2).
Synthetic variants used for the preparation of the
previously reported sulfonylated amino acid hydroxa-
mates43,44 have been modified for obtaining the new
compounds reported here (Scheme 1).
Reaction of sulfonyl chlorides 81 with amino acids 82
led to the alkyl/arylsulfonyl amino acids 83,43,44 which
were then protected at the NH moiety by the tert-
butyloxycarbonyl (Boc) group (with Boc-ON, 2-tert-
butoxycarbonyloxyimino-2-phenylacetonitrile).57 In sev-
eral cases (for Gly, Ala, and Val) the reaction procedure
was the one described above, whereas in other cases
(Leu) better yields were obtained when the sulfonyl
chlorides were directly reacted with the Boc-protected
amino acid. This procedure could anyhow be used
successfully for the preparation of the Gly, Ala, and Val
derivatives too. The Boc-protected derivatives 84 were
converted to the corresponding hydroxamic acids in the
standard manner, with hydroxylamine (or O-Boc-hy-
droxylamine) and carbodiimides,43,44 leading to deriva-
tives 85, which were deprotected (eventually both at the
NH and OH moieties) with TFA. The use of the O-Boc-
hydroxylamine (t-Bu-OCO-ONH2) did not show any
significant advantage over the simple H2NOH for the
synthesis of these hydroxamic acids. Derivatives 7-9,
13-15, 19-21, and 25-27 were obtained in this way.
Reaction of sulfonyl halides with hydroxylamine af-
forded the new N-hydroxysulfonamides 69 and 71-80,
by the method previously reported by our group.42
CA, MMP , a n d Ch C In h ibition . The inhibition data
with compounds 7-80, presented in Tables 1 and 2, led
to the following observations: (i) Sulfonyl amino acyl
hydroxamates possessing moieties of the type RSO2NH-
amino acyl (such as 7-9, 13-15, 19-21, and 25-27)
generally act as efficient CA inhibitors and are relatively
weak MMP and ChC inhibitors. Thus, for CAs, these
inhibitors generally showed affinities in the range of
7-50 nM (hCA I);, 8-45 nM (hCA II), and 10-40 nM
(bCA IV), whereas for the different MMPs investigated
here and ChC, their affinities were in the range of 40-
>200 nM. For the three types of investigated deriva-
tives, the most active were the pentafluorophenylsul-
fonyl derivatives, followed by the corresponding perfluoro-
butyl ones, whereas the least active were the corre-
sponding 4-methoxyphenyl-substituted compounds. For
CA inhibition, best activity was observed for the Gly
derivatives, followed by the corresponding Ala deriva-
F igu r e 3. Schematic binding of trifluoromethyl hydroxamate
to hCA II (adapted from ref 53).
but its interaction with the Zn(II) ion of CA active site
is very different from that of the classical sulfonamide
inhibitors. Thus, the ionized nitrogen atom of the
hydroxamate moiety is directly coordinated to Zn(II),
whereas a fluorine atom of the trifluoromethyl moiety
also participates in the interaction with the metal ion
(Figure 3). In addition, hydrogen bonds between the
hydroxamate OH and the active site residue Thr 199
were also evidenced,53 which further stabilize the E-I
adduct (Figure 3).
On the basis of all these data presented above, we
decided to test sulfonylated amino acid hydroxamates
(recently reported to act as strong MMPIs)43,44,54 as
potential CAIs. The following types of compounds were
included in this study: (i) Sulfonylated amino acid
hydroxamates possessing an unsubstituted RSO2NH-
amino acyl moiety. The amino acid hydroxamates
included in the study were the Gly, Ala, Val, and Leu
derivatives (Table 1). (ii) Sulfonylated amino acid hy-
droxamates possessing a substituted RSO2NX-amino
acyl moiety, where X is generally a benzyl or 2- or
4-substituted-benzyl group (the same amino acid hy-
droxamates as above were included in the study, Table
1). For all these types of compounds, three different
examples have been used (R moieties) from the large
series of available aliphatic, aromatic, and heterocyclic
derivatives reported previously by our group.43,44 They
included the perfluorobutyl, pentafluorophenyl, and
4-methoxyphenylsulfonyl moieties and were chosen in
such a way as to include a very potent, a slightly
weaker, and an even weaker MMPI. Anyhow, all three
groups incorporated in amino acid hydroxamates gener-
ally led to potent MMPIs, with affinities (for the most
active derivatives) in the (low) nanomolar range (5-15
nM) for the different MMPs and ChC.43,44 (iii) Simple

3682 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Scozzafava and Supuran
tives, which in turn were more active than the corre-
sponding Val and Leu derivatives. J ust the opposite was
generally true for MMP and ChC inhibition, with the
bulkier Val and Leu derivatives generally more inhibi-
tory than the corresponding Ala and Gly derivatives
(Table 1). (ii) Sulfonyl amino acyl hydroxamates pos-
sessing RSO2N(benzyl/substituted-benzyl)-amino acyl
moieties (such as 10-12, 16-18, 22-241, and 28-45)
were weak or very weak CA inhibitors but showed
excellent MMP and ChC inhibitory properties. Thus,
these compounds were generally 4-8 times weaker CA
inhibitors as compared to the corresponding unsubsti-
tuted compounds mentioned above, whereas their af-
finities for MMPs were very much enhanced as com-
pared to those of the corresponding unsubstituted
compounds. It was in fact reported that the benzyl
moiety of this type of hydroxamate inhibitors fits well
within the S2′ site of the protease, contributing sub-
stantially to the formation of strong E-I adducts.30,32,49
Obviously the different MMPs possess quite diverse
affinities for these derivatives, with important differ-
ences between the deep pocket (MMP-2, MMP-8, and
MMP-9) and the short pocket enzymes (MMP-1).30,32
Thus, as already shown previously by us for some
structurally related derivatives,43c the deep pocket
enzymes MMP-2, MMP-8, and MMP-9 are much more
susceptible to be inhibited by this class of hydroxamates
(Ki’s in the range of 0.6-20 nM) than collagenase 1,
MMP-1 (Ki’s in the range of 7-60 nM). Again the
pentafluorophenylsulfonyl derivatives were the most
active inhibitors, followed by the corresponding perfluo-
robutyl ones, whereas the least active were the corre-
sponding 4-methoxyphenyl-substituted compounds. The
Leu derivatives were generally more active than the
corresponding Val derivatives, which in turn were more
inhibitory than the Ala and Gly derivatives. (iii) Further
substitution (with nitro or chloro moieties, in position
2 or 4) of the P2′ benzyl moiety, such as in compounds
31-45, led to a slight enhancement of the MMP inhibi-
tory properties, to an enhancement of the ChC inhibi-
tory effects, and to a drastic reduction of the CA
inhibitory properties of the corresponding compounds
(Table 1). (iv) N-Hydroxysulfonamides 46-80 generally
act as effective CAIs, with affinities in the low nano-
molar range (against isozyme II) for the perhalo-
genoalkyl/aryl (49, 50, 68, 73), the nitroaryl-substituted
(59-62), or the heterocyclic (79) compounds. Other
substitution patterns of the R moiety generally led to
less effective CAIs (affinities in the range of 13-50 µM
for hCA I, 40-150 nM for hCA II, and 100-300 nM for
bCA IV) (Table 2). (v) N-Hydroxysulfonamides 46-80
generally act as moderately weak MMP and ChC
inhibitors (affinities in the 5-95 µM) with several
important exceptions. Thus, N-hydroxyperfluorophe-
nylsulfonamide (68) or the 2-hydroxy-3,5-dichlorophe-
nyl-substituted derivative 74 showed an enhanced
affinity against all the investigated MMPs (with MMP-1
and ChC more susceptible than MMP-2, MMP-8, and
MMP-9), whereas the best MMPIs in the entire series
(compounds 70 and 73) arrived at affinities of around
50-120 nM against the diverse MMPs investigated
here. It should be noted that both these compounds
possess an o-carboxyl group in the neighborhood of the
F igu r e 4. Proposed binding of a sulfonylated amino acid
hydroxamate (as monoanion) to the metal ion within the active
site of CA (M ) Zn(II) for the native enzyme or Co(II) for the
cobalt-substituted one).
SO2NHOH one, which makes probable the participation
of both these functionalities in the interaction with the
catalytic zinc ion of the MMPs and ChC. On the other
hand, the m-carboxy- and p-carboxyphenyl-substituted
derivatives 71 and 72 showed at least a 100-fold
decreased affinity to the investigated MMPs, as com-
pared to the ortho-substituted isomer 70 (Table 2).
The data of the two tables prove that potent CAIs can
be obtained from the class of investigated sulfonylated
amino acid hydroxamates. Although it was noted that
in addition to MMPs, hydroxamates also inhibit other
metalloproteinases, such as leucine aminopeptidase,
neprylysin, thermolysin,60a and tumor necrosis factor-
R,60b among others, affinities as high (in the nanomolar
range) were not evidenced up to now. Thus, our results
are quite promising for the eventual design of novel
types of potent CAIs or of compounds with a dual
activity, as both CAIs and MMPIs. It should be also
noted that the N-hydroxysulfonamides were not opti-
mized as MMPIs, since in addition to the new zinc-
binding function reported here for the first time (SO2-
NHOH), modifications of moieties that bind in the S1′,
S2′, and/or eventually S3′ sites of the enzyme have not
been performed. It is thus envisageable that potent
MMPIs can be obtained incorporating this new zinc-
binding function and peptidomimetic scaffolds that
should fit well in the primed binding sites of these
enzymes. Work is in progress in our laboratory for the
design of such compounds. All these data also seem to
validate our hypothesis that the new class of CAIs
reported here binds to the Zn(II) ion of the enzyme as
shown in Figure 4.
Sp ectr oscop ic Stu d ies on Co(II)-Su bstitu ted En -
zym es. To monitor the possible interaction of the new
inhibitors reported here with the active site of these
enzymes, the electronic spectra of cobalt(II)-substituted
hCA II and ChC and of their adducts with standard and
hydroxamate/N-hydroxysulfonamide inhibitors were re-
corded. The electronic spectral data of Table 3 indicate
the folowing information. Sulfonamides, such acetazola-
mide 1, bind to the Co(II) ion within the CA active site
giving rise to a pseudotetrahedral geometry of the metal
ion.69 Such adducts are characterized by intense spectra
with molar absorbances above 300 M-1 cm-1 69,70
The
.
four absorption maxima in the spectrum of the pure
enzyme (at 520, 550, 616.5, and 640 nm, respectively)
undergo notable changes when inhibitor is coordinated
to the metal ion. Thus, especially the last two maxima
are changed dramatically after complexation, colapsing
into a unique, broad maximum centered at 574-575 nm
and a shoulder at 595-600 nm. The tetrahedral geom-
etry of such E-I adducts has been confirmed by X-ray
crystallographic data for some of these complexes (of the
native or metal-substituted enzymes).50 Co(II) is pen-

CA and MMP Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3683
tacoordinated in the adducts with thiocyanate,71a
nitrate,71b acetate, or benzoate, as shown in the classical
studies of Bertini’s group.70,72 These spectra are char-
acterized by molar absorbances under 150 M-1 cm-1 69,70
and a different pattern of the four absorption maxima:
thus, a maximum appears under 490 nm (generally at
465-480 nm), whereas the two strong maxima in the
spectrum of pure Co(II)-hCA II, at 550 and 616.5 nm,
appear as a weak band, with the absorption maximum
at 555-575 nm. Additionally, another maximum at
689-709 nm is seen in the electronic spectra of penta-
coordinated Co(II) of such E-I adducts. The pentaco-
ordination of the metal ion in some of these complexes
has been then confirmed by the report of the X-ray
structure for some of these adducts: hCA II thiocyanate
by Liljas’ group71a and hCA II nitrate by Mangani’s
group.71b As seen from the data of Table 3, the spectra
of the adducts of Co(II)-hCA II with some hydroxamates
7-45 (such as 8 and 19) reported here are clearly of
the pentacoordinated type mentioned above, with molar
absorbances under 150 M-1 cm-1 and with an absor-
bance maxima distribution pattern as described above.
The spectra of the adducts of the two hydroxamates are
quite similar to those of the carboxylate adducts (ace-
tate, benzoate) previously investigated.70,72 On the
contrary, the spectra of the adducts of Co(II)-hCA II
with N-hydroxysulfonamides, such as 50 or 60 are
typical for the tetrahedral geometry of the metal ion,
proving that such derivatives bind similarly to the
unsubstituted sulfonamides within the CA active
site.42,69a The above data prompt us to propose the
binding mode of hydroxamate inhibitors within the hCA
II active site described schematically in Figure 4. Since
the secondary sulfonamide moiety of such compounds
would also possess good metal-coordinating properties,55
we expect a bidentate binding, in which both the
sulfonyl and hydroxamate moieties participate in the
interaction with the metal ion (the inhibitor has been
formulated as a monoanion, since at the pH at which
the experiments have been performed (7.5-8.0) at least
the hydroxamic acid group will be ionized).
major changes of this spectrum were evidenced: three
absorption maxima instead the two mentioned above
appeared: at 501-505, 562-563, and 597-598 nm,
respectively. These spectra are of low intensity (molar
absorbances around 80-120 M-1 cm-1 for the first two
maxima and around 11-15 M-1 cm-1 for the last one),
being characteristic of Co(II) in pentacoordinated
geometry.70-75 With the available data presented above,
one can only state that similarly to the sulfonylated
amino acid hydroxamates, N-hydroxysulfonamides also
bind to the metal ion within the ChC active site and
presumably also within the MMP active site, but the
way in which they coordinate the metal ion is unknown
for the moment.
Con clu sion s
We report a new class of effective CAIs: the sulfony-
lated amino acyl hydroxamates. The SARs for this class
of CAIs are quite different from those of the structurally
related sulfonylated hydroxamates with MMP inhibitory
action. Best CA inhibitory activity was seen for com-
pounds unsubstituted at the RSO2NH-amino acyl moi-
ety (with affinities in the low nanomolar range for
isozymes CA I, CA II, and CA IV), with the less bulky
Gly and Ala derivatives more active than the corre-
sponding Val and Leu compounds. The perfluorophen-
ylsulfonyl derivatives were the best inhibitors, followed
by the n-perfluorobutylsulfonyl derivatives, which in
turn were more active than the 4-methoxyphenylsulfo-
nyl derivatives. Substitution at the secondary sulfona-
mide moiety with benzyl or substituted-benzyl moieties
led to decreased CA inhibitory properties but to a drastic
increase of the MMP inhibitory effects for the corre-
sponding derivatives (against MMP-1, MMP-2, MMP-
8, and MMP-9). The same is true regarding ChC
inhibition with these compounds. We proposed a biden-
tate binding mode of these hydroxamates to the metal
ion of the CA active site, similarly to that of CF3-
CONHOH reported previously. We also report here a
novel zinc-binding function (SO2NHOH) that may be
incorporated into the molecule of novel types of MMPIs
and ChC inhibitors. Although these derivatives were not
optimized, some of them bind with Ki’s of around 50 nM
against the different investigated metalloproteases
(MMPs/ChC). Electronic spectroscopic studies of the Co-
(II)-substituted CA and ChC showed undoubtedly that
these new classes of inhibitors bind to the metal ion
within the enzyme active site. Thus, we proved here that
it is possible to design dual enzyme inhibitors, which
interact both with the diverse MMPs and with the CAs.
In contrast to Co(II)-substituted CAs, metal substitu-
tion of MMPs or ChC has been much less investigated.
Van Wart’s group reported the preparation of Co(II)-
ChC which possesses catalytic activity similar to that
of the zinc enzyme,69b but no spectroscopic studies of
E-I complexes were mentioned in this valuable study.
Since ChC could not be crystallized for the moment, and
its tridimensional structure is thus not available, elec-
tronic spectroscopic studies of Co(II)-substituted ChC
might offer interesting information regarding the bind-
ing of inhibitors within the active site of this bacterial
protease. Here we report for the first time the electronic
spectroscopic studies of ChC and of its adducts with
hydroxamate and N-hydroxysulfonamide inhibitors
(Table 3). Co(II)-ChC possesses a pH-dependent elec-
tronic absorption spectrum, with a maximum centered
at 585 nm and a shoulder at 530 nm, this spectrum
being relatively similar to that of Co(II)-substituted
carboxypeptidase A or thermolysin,73-75 two enzymes
in which the Zn(II) ion is coordinated - such as in ChC
- by two histidines and a glutamate.76 In the presence
of hydroxamate (such as 8, 11, 23, and 35) or N-
hydroxysulfonamide (such as 65 and 69) inhibitors,
Exp er im en ta l Section
Gen er a l. Melting points: heating plate microscope (not
corrected); IR spectra: KBr pellets, 400-4000 cm-1, Perkin-
Elmer 16PC FTIR spectrometer; 1H NMR spectra: Varian
300CXP apparatus (chemical shifts are expressed as δ values
relative to Me4Si as standard); elemental analysis: Carlo Erba
Instrument CHNS elemental analyzer, model 1106. All reac-
tions were monitored by thin-layer chromatography (TLC)
using 0.25-mm precoated silica gel plates (E. Merck). Analyti-
cal and preparative HPLC was performed on a reversed-phase
C
18 Bondapack column, with a Beckman EM-1760 instrument.
Amino acids, sulfonyl halides, benzyl halides, EDCI, diisopro-
pylcarbodiimide, Boc-ON, hydroxylamine, TFA, triethylamine,
5,5′-dithiobis(2-nitrobenzoic acid), FALGPA, buffers, and other
reagents used in the syntheses were commercially available

3684 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Scozzafava and Supuran
compounds from Sigma-Aldrich or Acros (Milan, Italy). The
thioester MMP substrate, AcProLeuGly-S-LeuLeuGlyOEt, was
from Bachem. Acetonitrile, acetone, methylene chloride (from
E. Merck, Florence, Italy), or other solvents used in the
synthesis were doubly distilled and kept on molecular sieves
in order to maintain them in anhydrous conditions.
methods (elemental analysis, within (0.4% of the theoretical
values; IR; 1H and 13C NMR spectroscopy) that confirmed their
structure. Data for a representative compound of each series
are shown below.
N-P en ta flu or op h en ylsu lfon yl-glycin e, 83 (R1 ) H, R
) p en ta flu or op h en yl): white crystals, mp 178-9 °C; 1H
NMR (DMSO-d6) δ 3.62 (s, 2H, CH2 of Gly), 9.13 (br s, 1H,
SO2NH), 11.75 (br s, 1H, COOH); 13C NMR (DMSO-d6) δ 40.6
(s, CH2 of Gly), 130.6 (s, C-3 of C6F5), 135.8 (s, C-2 of C6F5),
145.5 (s, C-4 of C6F5), 152.3 (s, C-1 of C6F5), 178.5 (s, CO2H).
Anal. Found: C, 31.62; H, 1.54; N, 4.33. C8H4F5NO4S re-
quires: C, 31.49; H, 1.32; N, 4.59.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
83. An amount of 10 mmol of sulfonyl halide (chlorides for the
pentafluorophenyl and 4-methoxyphenylsulfonyl derivatives
and fluoride for the pentafluorobutyl derivatives) and the
stoichiometric amount of amino acid (Gly, L-Ala, l-Val or L-Leu)
were suspended in 100 mL of acetone and magnetically stirred
at 0-4 °C for 5 min. A solution obtained by dissolving 200 mg
K2CO3 (20 mmol) in 15 mL of water was then added to the
reaction mixture, and stirring was continued at room temper-
ature for 4-6 h (TLC control). pH was brought to 7 with 5%
aqueous HCl, the acetone was evaporated in vacuo, the
obtained syrup was either directly recrystallized from ethanol-
water (3/1-5/1, v/v) or poured into 50 mL of cold water and
the obtained precipitate was filtered, dried and recrystallized
as above. Yields were in the range of 80-95%.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
84. An amount of 5 mmol of sulfonyl amino acid 83, 105 µL
(7.5 mmol) of triethylamine and 135 mg (5.5 mmol) of Boc-
ON were suspended in 20 mL of acetonitrile-water (1:1, v/v).
The mixture became homogeneous in about 1 h, and stirring
was continued at room temperature for additional 2-3 h (TLC
control). The organic solvent was evaporated under reduced
pressure, the residue was retaken in 25 mL of water, and the
2-hydroxyimino-2-phenylacetonitrile formed in the reaction
was extracted with 2 × 10 mL of ethyl acetate. The aqueous
layer was separated and acidified with a 5% citric acid solution
till pH 4, and the Boc-protected compounds 84 were extracted
in 100-150 mL of ethyl acetate. After evaporation of the
organic solvent, they were recrystallized from ethanol. Yields
of the protection step were in the range of 69-83%.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
85 a n d 86. An amount of 3 mmol of Boc-protected sulfonylated
amino acid 84 was dissolved in 50 mL of anhydrous acetonitrile
and magnetically stirred for 10 min at 0 °C. An amount of 570
mg (3 mmol) of EDCI‚HCl was then added and the reaction
mixture was magnetically stirred at room temperature for 15
min, then 90 µL (6 mM) of triethylamine and 210 mg of
hydroxylamine‚HCl (3 mmol) dissolved in 10 mL of water were
added to the reaction mixture and stirring was continued for
8-10 h at 0-4 °C (TLC control). The solvent was evaporated
in vacuo and the residue consisting of crude 85 was taken in
50 mL of methylene chloride and treated with 2 mL of TFA
(magnetic stirring at room temperature for 30 min). The
solvent and excess TFA were evaporated in vacuo; the residue
was taken up in ethyl acetate (50 mL), dried over sodium
sulfate, filtered, and purified by means of preparative HPLC
(C18 reversed-phase µ-Bondapack or Dynamax-60A (25 × 250
mm) column; 80% acetonitrile/8% methanol/12% water, 30 mL/
min). Yields were in the range of 45-65%.
Gen er a l P r oced u r e for th e P r ep a r a tion of N-Hyd r oxy-
su lfon a m id es. The N-hydroxysulfonamides of type 46-80
were prepared as previously described by this group,42 by
reaction of arylsulfonyl halides or sulfonic acid anhydrides,
with hydroxylamine in alcoholic-aqueous medium. An amount
of 10 mmol of sulfonyl halide (chloride or fluoride) was
dissolved/suspended in 50 mL of methanol and the stoichio-
metric amount (10 mmol, 0.69 g) of hydroxylamine hydrochlo-
ride dissolved in 10 mL of water was added. The mixture was
magnetically stirred at 4 °C for 10 min, then the calculated
amount of solid NaHCO3 was added and stirring was continued
for 4-6 h (TLC control) at room temperature, till all the
sulfonyl halide was consumed in the reaction with the nucleo-
phile. The solvent was evaporated under reduced pressure and
the precipitated N-hydroxysulfonamides were recrystallized
from ethanol or ethanol water. Yields were generally high (75-
85%).
N-P en ta flu or op h en ylsu lfon yl-glycin e h yd r oxa m a te,
1
8: white crystals, mp 211-2 °C; H NMR (DMSO-d6) δ 3.70
(s, 2H, CH2 of Gly), 8.70 (br s, 1H, NHOH), 9.10 (br s, 1H,
SO2NH), 10.55 (br s, 1H, NHOH); 13C NMR (DMSO-d6) δ 40.4
(s, CH2 of Gly), 130.5 (s, C-3 of C6F5), 135.3 (s, C-2 of C6F5),
145.8 (s, C-4 of C6F5), 152.2 (s, C-1 of C6F5), 176.8 (s,
CONHOH). Anal. Found: C, 30.24; H, 1.60; N, 8.63. C8H5F5N2-
O4S requires: C, 30.01; H, 1.57; N, 8.75.
N-n-P er flu or obu tylsu lfon yl-a la n in e h yd r oxa m a te, 13:
1
white crystals, mp 143-4 °C; H NMR (DMSO-d6) δ 1.51 (d,
3J HH ) 6.1 Hz, 3H, CHCH3 of Ala), 3.90 (q, 1H, CH of Ala),
8.65 (br s, 1H, NHOH), 9.18 (br s, 1H, SO2NH), 10.50 (br s,
1H, NHOH); 13C NMR (DMSO-d6) δ 21.8 (s, CHCH3 of Ala),
35.8 (s, CHCH3 of Ala), 176.0 (s, CONHOH). The four pen-
tafluorobutyl carbons were not seen in the conditions of the
experiments, similarly to the case of some perfluorobutyl-
containing aromatic sulfonamides previously reported by
Whiteside’s group.68 Anal. Found: C, 21.56; H, 1.75; N, 7.12.
C7H7F9N2O4S requires: C, 21.77; H, 1.83; N, 7.25.
N-4-Meth oxyp h en ylsu lfon yl-N-ben zyl-L-va lin e h yd r ox-
1
a m a te, 24: white crystals, mp 222-4 °C; H NMR (DMSO-
3
d6) δ 1.12 (d, J HH ) 6.7 Hz, 6H, CH(CH3)2 of Val), 2.23-2.55
(m, 1H, CH(CH3)2 of Val), 3.45 (s, 3H, CH3OC6H4), 3.77 (d,
3J HH ) 4.4 Hz, 1H, NCHCO of Val), 3.82 (s, 2H, CH2 of benzyl),
7.13-7.60 (m, 7H, Hortho of CH3OC6H4 and Harom of Ph), 8.00
3
(d, J HH ) 8.1 Hz, 2H, Hmeta of CH3OC6H4), 8.71 (br s, 1H,
NHOH), 10.51 (br s, 1H, NHOH); 13C NMR (DMSO-d6) δ 17.3
(s, CH(CH3)2 of Val), 30.1 (s, CH(CH3)2 of Val), 33.4 (s, CH3-
OC6H4), 41.4 (s, CH2 of benzyl), 61.1 (s, NHCH of Val), 130.3
(s, Cmeta of CH3OC6H4), 131.8 (s, Cpara of Ph), 133.5 (s, Cmeta of
Ph), 134.78 (s, Cortho of Ph), 135.5 (s, Cortho of CH3OC6H4), 141.6
(s, Cipso of Ph), 145.0 (s, Cipso of CH3OC6H4), 146.3 (s, Cpara of
CH3OC6H4), 174.2 (s, CONHOH). Anal. Found: C, 47.81; H,
6.19; N, 9.08. C12H18N2O5S requires: C, 47.67; H, 6.00; N, 9.27.
N -P e n t a flu or op h e n ylsu lfon yl-N -4-n it r ob e n zyl-gly-
cin e h yd r oxa m a te, 35: white crystals, mp 165-7 °C; 1H
NMR (DMSO-d6) δ 3.71 (s, 2H, CH2 of Gly), 4.39 (s, 2H, CH2
of benzyl), 7.21-7.67 (m, 2, Hortho of O2NC6H4), 8.21 (d, 2H,
Hmeta of O2NC6H4), 8.70 (br s, 1H, NHOH), 10.58 (br s, 1H,
NHOH); 13C NMR (DMSO-d6) δ 40.6 (s, CH2 of Gly), 45.6 (s,
CH2 of benzyl), 123.8 (s, Cmeta of O2NC6H4), 129.4 (Cortho of O2-
NC6H4), 130.7 (s, Cmeta of C6F5), 135.9 (s, Cortho of C6F5), 144.3
(s, Cipso of O2NC6H4), 145.5 (s, Cipso of C6F5), 147.9 (s, Cpara of
O2NC6H4), 152.3 (s, Cpara of C6F5), 178.3 (s, CONHOH). Anal.
Found: C, 39.77; H, 2.39; N, 9.02. C15H10F5N3O6S requires:
C, 39.57; H, 2.21; N, 9.23.
N -4-Me t h oxyp h e n ylsu lfon yl-N -2-n it r ob e n zyl-L-a la -
n in e h yd r oxa m a te, 39: pale yellow crystals, mp 194-5 °C;
3
1H NMR (DMSO-d6) δ 1.56 (d, J HH ) 6.5 Hz, 3H, CHCH3 of
Ala), 3.45 (s, 3H, CH3OC6H4), 3.81 (s, 2H, CH2 of benzyl), 3.98
(q, 1H, CH of Ala), 7.14-7.68 (m, 6H, Hortho of CH3OC6H4 and
Harom of 2-O2N-C6H4), 8.07 (d, 3J HH ) 8.1 Hz, 2H, Hmeta of CH3-
OC6H4), 8.78 (br s, 1H, NHOH), 10.56 (br s, 1H, NHOH); 13C
NMR (DMSO-d6) δ 20.5 (s, CHCH3 of Ala), 33.1 (s, CH3OC6H4),
34.9 (s, CHCH3 of Ala), 44.4 (s, CH2 of benzyl), 127.0 (s, C-5 of
2-O2N-C6H4), 129.3 (C-4 of 2-O2N-C6H4), 129.9 (C-3 of 2-O2N-
C6H4), 130.4 (s, Cmeta of CH3OC6H4), 131.3 (C-6 of 2-O2N-C6H4),
134.5 (C-2 of 2-O2N-C6H4), 135.3 (C-1 of 2-O2N-C6H4), 135.7
(s, Cortho of CH3OC6H4), 146.8 (s, Cipso of CH3OC6H4), 148.4 (s,
Cpara of CH3OC6H4), 175.3 (s, CONHOH). Anal. Found: C,
50.03; H, 4.80; N, 10.11. C17H19N3O7S requires: C, 49.87; H,
4.68; N, 10.26.
All the new compounds reported here were extensively
characterized by means of standard chemical and physical

CA and MMP Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3685
CA P r ep a r a tion s a n d Assa y. Human CA I and CA II
cDNAs were expressed in Escherichia coli strain BL21 (DE3)
from the plasmids pACA/hCA I and pACA/hCA II described
by Lindskog et al.61 (the two plasmids were a gift from Prof.
Sven Lindskog, Umea University, Sweden). Cell growth condi-
tions were those described by this group,62 and enzymes were
purified by affinity chromatography according to the method
of Khalifah et al.63 Enzyme concentrations were determined
spectrophotometrically at 280 nm, utilizing a molar absorp-
tivity of 49 mM-1 cm-1 for CA I and 54 mM-1 cm-1 for CA II,
respectively, based on Mr ) 28.85 kDa for CA I and 29.30 kDa
for CA II, respectively.64,65 CA IV was isolated from bovine lung
microsomes as described by Maren et al., and its concentration
has been determined by titration with ethoxzolamide.66
absorbance at 324 nm using an extinction coefficient for
FALGPA ꢀ305 ) 24 700 M-1 cm-1 in the above-mentioned
reaction buffer.47 Measurements were made using a Cary 3
spectrophotometer interfaced with a PC. Initial velocities were
thus estimated using the direct linear plot-based procedure
reported by van Wart and Steinbrink.47 Ki’s were then deter-
mined according to Easson-Stedman56 plots and a linear
regression program.
Sp ectr oscop ic Stu d ies on Co(II)-Su bstitu ted En zym es.
Co(II)-hCA II was prepared as described in ref 69a, whereas
Co(II)-ChC by a modification of the method described in ref
69b by removing zinc from the native enzyme in the presence
of 50 mM pyridine-2,6-dicarboxylic acid, followed by dialysis
against metal-free 50 mM Tris-H2SO4 buffer and addition of
the stoichiometric amount of Co(II) chloride. Electronic spectra
were registered with a Cary 3 spectrophotometer, at 25 °C (in
the buffer mentioned above), in the range of 400-800 nm,
working at enzyme concentrations of 0.1-0.4 mM and the pH
values specified in each case (generally of 6.5-8 pH units).
The E-I adducts of the two cobalt enzymes mentioned above
were obtained by spectral titration of the pure enzyme with
inhibitors, until reaching an inhibitor concentration of 0.1-2
mM in the spectral cuvette. The absorbance values of these
spectra were converted to extinction coefficients after back-
ground subtraction and normalization based on absorbance
averages at 550-575 nm for Co(II)-hCA II and 560-585 nm
for Co(II)-ChC, respectively.
Initial rates of 4-nitrophenyl acetate hydrolysis catalyzed
by different CA isozymes were monitored spectrophotometri-
cally, at 400 nm, with a Cary 3 instrument interfaced with an
IBM-compatible PC.45 Solutions of substrate were prepared
in anhydrous acetonitrile; the substrate concentrations varied
between 2 × 10-2 and 1 × 10-6 M, working at 25 °C. A molar
absorption coefficient ꢀ of 18 400 M-1 cm-1 was used for the
4-nitrophenolate formed by hydrolysis, in the conditions of the
experiments (pH 7.40), as reported in the literature.45 Non-
enzymatic hydrolysis rates were always subtracted from the
observed rates. Duplicate experiments were done for each
inhibitor concentration, and the values reported throughout
the paper are the mean of such results. 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 0.01 nM were done
thereafter with distilled-deionized water. Inhibitor and en-
zyme solutions were preincubated together for 10 min at room
temperature prior to assay, to allow for the formation of the
E-I complex. The inhibition constant Ki was determined as
described by Pocker and Stone.45 Enzyme concentrations were
3.6 nM for hCA II, 8.8 nM for hCA I, and 27 nM for bCA IV
(this isozyme has a decreased esterase activity14 and higher
concentrations had to be used for the measurements).
Ack n ow led gm en t. This research was financed by
EU Grant ERB CIPDCT 940051. Special thanks are
addressed to Drs. Giovanna Mincione, Angela Casini,
and Marc A. Ilies for their assistance in different phases
of this project.
Refer en ces
(1) This paper is part 93 of the series. Preceding part: Scozzafava,
A.; Supuran, C. T. Carbonic anhydrase inhibitors. Synthesis of
N-Morpholyl-thiocarbonylsulfenylamino aromatic/ heterocyclic
sulfonamides and their interaction with isozymes I, II and IV.
Bioorg. Med. Chem. Lett. 2000, 10, 1117-1120.
(2) (a) Supuran, C. T.; Scozzafava, A. Carbonic anhydrase inhibitors
and their therapeutic potential. Exp. Opin. Ther. Patents 2000,
10, 575-600. (b) Supuran, C. T. Carbonic anhydrase inhibitors.
In Carbonic Anhydrase and Modulation of Physiologic and
Pathologic Processes in the Organism; Puscas, I., Ed.; Helicon:
Timisoara, Romania, 1994; pp 29-111.
(3) Hewett-Emmett, D.; Tashian, R. E. Functional diversity, con-
servation and convergence in the evolution of the R-, â- and
γ-carbonic anhydrase gene families. Mol. Phyl. Evol. 1996, 5,
50-77.
(4) Smith, K. S.; J akubzick, C.; Whittam, T. S.; Ferry, J . G. Carbonic
anhydrase is an ancient enzyme widespread in prokaryotes.
Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 15184-15189.
(5) Maren, T. H. The links among biochemistry, physiology and
pharmacology in carbonic anhydrase mediated systems. In
Carbonic Anhydrase - From Biochemistry and Genetics to
Physiology and Clinical Medicine; Botre´, F., Gros, G., Storey,
B. T., Eds.; VCH: Weinheim; pp 186-207.
MMP a n d Ch C P r ep a r a tion s. Human purified MMPs
(MMP-1, MMP-2, MMP-8 and MMP-9) were purchased from
Calbiochem (Inalco, Milano, Italy). They were activated67 in
the assay buffer by adding bovine trypsin (from Sigma, 50 µL,
0.6 mg/mL) to the proenzyme, followed by incubation at 37 °C
for 10 min. The trypsin was then inactivated with aprotinin
(50 µL, 1.2 mg/mL). Initial rates for the hydrolysis of the
thioester substrate AcProLeuGly-S-LeuLeuGlyOEt, coupled to
the reaction with 5,5′-dithiobis(2-nitrobenzoic acid) were used
for assessing the catalytic activity and inhibition of the four
MMPs mentioned above, by the spectrophotometric method
of Powers and Kam,46a and J ohnson et al.46b The change of
46a
absorbance (ꢀ ) 19 800 M-1 cm-1
)
at 405 nm was monitored
continuously at room temperature, using a Cary 3 spectro-
photometer interfaced with a PC. A typical 100-µL reaction
contained 50 mM MES, pH 6.0, 10 mM CaCl2, 100 µM
substrate, 1 mM 5,5′-dithiobis(2-nitrobenzoic acid) and 5 nM
MMP. For the Ki determinations, DMSO solutions of the
inhibitor were included in the assay, resulting in a final
concentration of 2% DMSO in the reaction mixture. In these
conditions, Ki values varied from 5-10% in replicate experi-
ments. Ki’s were then determined by using Easson-Stedman56
plots and a linear regression program.
(6) Chegwidden, W. R.; Dodgson, S. J .; Spencer, I. M. The role of
carbonic anhydrase in metabolism and cell growth in animals.
In Carbonic Anhydrase - New Horizons; Chegwidden, W. R.,
Edwards, Y., Carter, N., Eds.; Birkhauser: New York, 2000; in
press.
(7) Cabiscol, E.; Levine, R. L. Carbonic anhydrase III oxidative
modification in vivo and loss of phosphatase activity during
aging. J . Biol. Chem. 1995, 270, 14742-14747.
Clostridium histolyticum highly purified collagenase and its
substrate FALGPA (furanacryloyl-leucyl-glycyl-prolyl-alanine)
were purchased from Sigma Chemical Co. (Milano, Italy), and
their concentrations were determined from the absorbance at
280 nm and the extinction coefficients furnished by the
supplier. The activity of such preparations was in the range
of 10 NIH units/mg solid. The potency of standard and newly
obtained inhibitors was determined from the inhibition of the
enzymatic (amidolytic) activity of the collagenase, at 25 °C,
using FALGPA as substrate, by the method of van Wart and
Steinbrink.47 The substrate was reconstituted as 5 mM stock
in 50 mM Tricine buffer, 0.4 M NaCl, 10 mM CaCl2, pH 7.50.
The rate of hydrolysis was determined from the change in
(8) Parkkila, A. K.; Scarim, A. L.; Parkkila, S.; Waheed, A.; Corbett,
J . A.; Sly, W. S. Expression of carbonic anhydrase V in pancreatic
beta cells suggests role for mitochondrial carbonic anhydrase
in insulin secretion. J . Biol. Chem. 1998, 273, 24620-24623.
(9) (a) Pastorek, J .; Pastorekova, S.; Callebaut, I.; Mornon, J . P.;
Zelnik, V.; Opavsky, R.; Zatovicova, M.; Liao, S.; Portetelle, D.;
Stanbridge, E. J .; Zavada, J .; Burny, A.; Kettmann, R. Cloning
and characterization of MN, a human tumor-associated protein
with a domain homologous to carbonic anhydrase an a putative
helix-loop-helix DNA binding segment. Oncogene 1994, 9, 2877-
2888. (b) Pastorekova, S.; Parkkila, S.; Parkkila, A. K.; Opavsky,
R.; Zelnik, V.; Saarnio, J .; Pastorek, J . Carbonic anhydrase IX,
MN/CA IX: analysis of stomach complementary DNA sequence
and expression in human and rat alimentary tracts. Gastroen-
terology 1997, 112, 398-408.

3686 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20
Scozzafava and Supuran
(10) Lovejoy, D. A.; Hewett-Emmett, D.; Porter, C. A.; Cepoi, D.;
Sheffield, A.; Vale, W. W.; Tashian, R. E. Evolutionary conserved,
“acatalytic” carbonic anhydrase-related protein XI contains a
sequence motif present in the neuropeptide sauvagine: the
human CA-RP XI gene (CA11) is embedded between the secretor
gene cluster and the DBP gene at 19q13.3 Genomics 1998, 54,
484-493.
(11) Bergenhem, N. C. H.; Hallberg, M.; Wise´n, S. Molecular char-
acterization of the human carbonic anhydrase-related protein
(HCA-RP VIII). Biochim. Biophys. Acta 1998, 1384, 294-298.
(12) (a) Tureci, O.; Sahin, U.; Vollmar, E.; Siemer, S.; Gottert, E.;
Seitz, G.; Parkkila, A. K.; Shah, G. N.; Grubb, J . H.; Pfreunds-
chuh, M.; Sly, W. S. Human carbonic anhydrase XII: cDNA
cloning, expression, and chromosomal localization of a carbonic
anhydrase gene that is overexpressed in some renal cell cancers.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 7608-7613. (b) Karhu-
maa, P.; Parkkila, S.; Tureci, O.; Waheed, A.; Grubb, J . H.; Shah,
G.; Parkkila, A.; Kaunisto, K.; Tapanainen, J .; Sly, W. S.;
Rajaniemi, H. Identification of carbonic anhydrase XII as the
membrane isozyme expressed in the normal human endometrial
epithelium. Mol. Hum. Reprod. 2000, 6, 68-74.
(13) Mori, K.; Ogawa, Y.; Ebihara, K.; Tamura, N.; Tashiro, K.;
Kuwahara, T.; Mukoyama, M.; Sugawara, A.; Ozaki, S.; Tanaka,
I.; Nakao, K. Isolation and characterization of CA XIV, a novel
membrane-bound carbonic anhydrase from mouse kidney. J .
Biol. Chem. 1999, 274, 15701-15705.
(14) Baird, T. T.; Waheed, A.; Okuyama, T.; Sly, W. S.; Fierke, C. A.
Catalysis and inhibition of human carbonic anhydrase IV.
Biochemistry 1997, 36, 2669-2678.
(15) Ivanov, S. V.; Kuzmin, I.; Wei, M. H.; Pack, S.; Geil, L.; J ohnson,
B. E.; Stanbridge, E. J .; Lerman, M. I. Down-regulation of
transmembrane carbonic anhydrase in renal cell carcinoma cell
lines by wild-type von Hippel-Lindau transgenes. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 12596-12601.
(16) Pocker, Y.; Meany, J . E. The catalytic versatility of carbonic
anhydrase from erythrocytes. The enzyme-catalyzed hydration
of acetaldehyde. J . Am. Chem. Soc. 1965, 87, 1809-1811.
(17) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. The enzyme-catalyzed hydrolysis of p-
nitrophenyl acetate. J . Am. Chem. Soc. 1965, 87, 5497-5498.
(18) Tu, C.; Thomas, H. G.; Wynns, G. C.; Silverman, D. N. Hydrolysis
of 4-nitrophenyl acetate catalyzed by carbonic anhydrase III from
bovine skeletal muscle. J . Biol. Chem. 1986, 261, 10100-10103.
(19) Kaiser, E. T.; Lo, K. W. The carbonic anhydrase catalyzed
hydrolysis of 2-hydroxy-5-nitro-ω-toluenesulfonic acid sultone.
J . Am. Chem. Soc. 1969, 91, 4912-4918.
(32) (a) Leung, D.; Abbenante, G.; Fairlie, D. P. Protease inhibitors:
Current status and future prospects. J . Med. Chem. 2000, 43,
305-341. (b) Babine, R. E.; Bender, S. L. Molecular recognition
of protein-ligand complexes: Applications to drug design. Chem.
Rev. 1997, 97, 1359-1472.
(33) (a) Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Synthesis of water-soluble, topically effective intraocular pres-
sure lowering aromatic/heterocyclic sulfonamides containing
cationic or anionic moieties: is the tail more important than the
ring? J . Med. Chem, 1999, 42, 2641-2650. (b) Borras, J .;
Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. Carbonic anhydrase inhibitors:
Synthesis of water-soluble, topically effective intraocular pres-
sure lowering aromatic/heterocyclic sulfonamides containing
8-quinoline-sulfonyl moieties: is the tail more important than
the ring? Bioorg. Med. Chem. 1999, 7, 2397-2406. (c) Scozzafava,
A.; Briganti, F.; Mincione, G.; Menabuoni, L.; Mincione, F.;
Supuran, C. T. Carbonic anhydrase inhibitors: Synthesis of
water-soluble, amino acyl/dipeptidyl sulfonamides possessing
long-lasting intraocular pressure-lowering properties via the
topical route. J . Med. Chem. 1999, 42, 3690-3700. (d) Supuran,
C. T.; Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G. Carbonic anhydrase inhibitors. Part 71. Synthesis
and ocular pharmacology of a new class of water-soluble,
topically effective intraocular pressure lowering sulfonamides
incorporating picolinoyl moieties. Eur. J . Pharm. Sci. 1999, 8,
317-328.
(34) Chufan, E. E.; Pedregosa, J . C.; Baldini, O. N.; Bruno-Blanch,
L. Anticonvulsant activity of analogues of acetazolamide. Far-
maco 1999, 54, 838-841.
(35) Bernhard, W. N.; Schalik, L. M.; Delaney, P. A.; Bernhard, T.
M.; Barnas, G. M. Acetazolamide plus low-dose dexamethasone
is better than acetazolamide alone to ameliorate symptoms of
acute mountain sickness. Aviat. Space Environ. Med. 1998, 69,
883-886.
(36) Puscas, I.; Supuran, C. T. Farmacologia clinica da ulcera peptica.
In Aparelho Digestivo; Coelho, J ., Ed.; MEDSI: Rio de J aneiro,
1996; pp 1704-1734.
(37) J ackson, E. K. Diuretics. In Goodman and Gilman’s The
Pharmacological Basis of Therapeutics, 9th ed.; Hardman, J . G.,
Limbird, L. E., Molinoff, P. B., Ruddon, R. W., Gilman, A. G.,
Eds.; McGraw-Hill: New York, 1996; pp 685-713.
(38) Supuran, C. T.; Briganti, F.; Tilli, S.; Chegwidden, W. R.;
Scozzafava, A. Carbonic anhydrase inhibitors: sulfonamides as
antitumor agents? Bioorg. Med. Chem. 2000, 8, in press.
(39) Chegwidden, W. R.; Spencer, I. M. Sulphonamide inhibitors of
carbonic anhydrase inhibit the growth of human lymphoma cells
in culture. Inflammopharmacology 1995, 3, 231-239.
(40) Kaneta, S.; Ishizuki, S.; Kasahara, M.; Nagao, S.; Takahashi,
H. Renal carbonic anhydrase activity in DBA/2FG-pcy/pcy mice
with inherited polycystic kidney disease. Exp. Anim. 1999, 48,
161-169.
(41) (a) Schmid, R. M.; Kloppel, G.; Adler, G.; Wagner, M. Acinar-
ductal-carcinoma sequence in transforming growth factor-R
transgenic mice. In Cell and Molecular Biology of Pancreatic
Carcinoma. Ann. N. Y. Acad. Sci. 1999, 880, 219-230. (b)
Nishimori, I.; Fujikawa-Adachi, K.; Onishi, S.; Hollingsworth,
M. A. Carbonic anhydrase in human pancreas: Hypotheses for
the pathophysiological roles of CA isozymes. In Cell and Molec-
ular Biology of Pancreatic Carcinoma. Ann. N. Y. Acad. Sci.
1999, 880, 5-16.
(42) Mincione, F.; Menabuoni, L.; Briganti, F.; Mincione, G.; Scoz-
zafava, A.; Supuran, C. T. Carbonic anhydrase inhibitors.
Inhibition of isozymes I, II and IV with N-hydroxysulfonamides
- A novel class of intraocular pressure lowering agents. J .
Enzyme Inhib. 1998, 13, 267-284.
(43) (a) Scozzafava, A.; Supuran, C. T. Protease inhibitors. Part 5.
Alkyl/arylsulfonyl- and arylsulfonylureido-/arylureido-glycine
hydroxamate inhibitors of Clostridium histolyticum collagenase.
Eur. J . Med. Chem. 2000, 35, 299-307. (b) Scozzafava, A.;
Supuran, C. T. Protease inhibitors. Part 9. Synthesis of Clostrid-
ium histolyticum collagenase inhibitors incorporating sulfonyl-
L-alanine hydroxamate moieties. Bioorg. Med. Chem. Lett. 2000,
10, 499-502.
(20) Pullan, L. M.; Noltmann, E. A. Specific arginine modification
at the phosphatase site of muscle carbonic anhydrase. Biochem-
istry 1985, 24, 635-640.
(21) Henkart, P.; Guidotti, G.; Edsall, J . T. Catalysis of the hydrolysis
of 1-fluoro-2,4-dinitrobenzene by carbonic anhydrase. J . Biol.
Chem. 1968, 243, 2447-2449.
(22) Malmstrom, B. G.; Nyman, P. O.; Strandberg, B.; Tilander, B.
Carbonic anhydrase. In Structure and Activity of Enzymes;
Goodwin, T. W., Harris, J . T., Hartley, B. S., Eds.; Academic
Press: New York, 1964; pp 121-137.
(23) Whitney, P. L.; Folsch, G.; Nyman, P. O.; Malmstrom, B. G.
Inhibition of human erythrocyte carbonic anhydrase
B by
chloroacetyl sulfonamides with labeling of the active site. J . Biol.
Chem. 1967, 242, 4206-4211.
(24) Supuran, C. T.; Conroy, C. W.; Maren, T. H. Is cyanate a carbonic
anhydrase substrate? Proteins 1997, 27, 272-278.
(25) Briganti, F.; Mangani, S.; Scozzafava, A.; Vernaglione, G.;
Supuran, C. T. Carbonic anhydrase catalyzes cyanamide hydra-
tion to urea: Is it mimicking the physiological reaction? J . Biol.
Inorg. Chem. 1999, 4, 528-536.
(26) (a) Christianson, D. W.; Ippolito, J . A. Structure-function
relationship between the carbonic anhydrases and the zinc
proteases. In Carbonic Anhydrase; Botre`, F., Gros, G., Storey,
B. T., Eds.; VCH: New York, 1991; pp 95-110. (b) Coleman, J .
E. Zinc enzymes. Curr. Opin. Chem. Biol. 1998, 2, 222-234.
(27) Supuran, C. T.; Scozzafava, A. Matrix metalloproteinase inhibi-
tors. In Proteinase and Peptidase Inhibition: Recent Potential
Targets for Drug Development; Smith, H. J ., Simons, C., Eds.;
Harwood Academic Press: London, 2000; in press.
(28) (a) Nagase, H.; Woessner, J . F., J r. Matrix metalloproteinases.
J . Biol. Chem. 1999, 274, 21491-21494. (b) Dioszegi, M.;
Cannon, P.; Van Wart, H. E. Vertebrate collagenases. Methods
Enzymol. 1995, 248, 413-431. (c) Tschesche, H. Human neu-
trophil collagenase. Methods Enzymol. 1995, 248, 431-449.
(29) J ohnson, L. L.; Dyer, R.; Hupe, D. J . Matrix metalloproteinases.
Curr. Opin. Chem. Biol. 1998, 2, 466-471.
(44) Supuran, C. T.; Briganti, F.; Mincione, G.; Scozzafava, A.
Protease inhibitors: Synthesis of L-alanine hydroxamate sul-
fonylated derivatives as inhibitors of Clostridium histolyticum
collagenase. J . Enzyme Inhib. 1999, 15, 111-128.
(45) Pocker, Y.; Stone, J . T. The catalytic versatility of erythrocyte
carbonic anhydrase. III. Kinetic studies of the enzyme-catalyzed
hydrolysis of p-nitrophenyl acetate. Biochemistry 1967, 6, 668-
678.
(30) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J . H. Design
and therapeutic application of matrix metalloproteinase inhibi-
tors. Chem. Rev. 1999, 99, 2735-2776.
(31) Bottomley, K. M.; J ohnson, W. H.; Walter, D. S. Matrix metal-
loproteinase inhibitors in arthritis. J . Enzymol. Inhib. 1998, 13,
79-102.

CA and MMP Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 20 3687
(46) (a) Powers, J . C.; Kam, C. M. Peptide thioester substrates for
serine peptidases and metalloendopeptidases. Methods Enzymol.
1995, 248, 3-18. (b) J ohnson, L. L.; Bornemeier, D. A.; J anowicz,
J . A.; Chen, J .; Pavlovsky, A. G.; Ortwine, D. F. Effect of species
differences on stromelysin-1 (MMP-3) inhibitor potency. J . Biol.
Chem. 1999, 274, 24881-24887.
(58) Scozzafava, A.; Supuran, C. T. Protease inhibitors. Synthesis of
potent bacterial collagenase and matrix metalloproteinase in-
hibitors incorporating N-4-nitrobenzylsulfonylglycine hydrox-
amate moieties. J . Med. Chem. 2000, 43, 1858-1865.
(59) Scozzafava, A.; Supuran, C. T. Protease inhibitors. Part 8.
Synthesis of potent Clostridium histolyticum collagenase inhibi-
(47) Van Wart, H. E.; Steinbrink, D. R. A continuous spectrophoto-
metric assay for Clostridium histolyticum collagenase. Anal.
Biochem. 1981, 113, 156-165.
L-alanine hydroxamate moieties.
tors incorporating sulfonylated
Bioorg. Med. Chem. 2000, 8, 637-645.
(60) (a) Marcotte, P. A.; Elmore, I. N.; Guan, Z.; Magoc, T. J .; Albert,
D. H.; Morgand, D. W.; Curtin, M. L.; Garland, R. B.; Guo, Y.;
Heyman, H. R.; Holms, J . H.; Sheppard, G. S.; Steinman, D. H.;
Wada, C. K.; Davidsen, S. K. Evaluation of the inhibition of other
metalloproteinases by matrix metalloproteinase inhibitors. J .
Enzyme Inhib. 1999, 14, 425-435. (b) Yamamoto, M.; Hirayama,
R.; Naruse, K.; Yoshino, K.; Shimada, A.; Inoue, S.; Kayagaki,
N.; Yagita, H.; Okumura, K.; Ikeda, S. Structure-activity
relationship of hydroxamate-based inhibitors on membrane-
bound Fas ligand and TNF-alpha processing. Drug Des. Discov.
1999, 16, 119-130.
(48) (a) Grams, F.; Crimmin, M.; Hinnes, L.; Huxley, P.; Pieper, M.;
Tschesche, H.; Bode, W. Structure determination and analysis
of human neutrophil collagenase complexed with a hydroxamate
inhibitor. Biochemistry 1995, 34, 14012-14020. (b) Grams, F.;
Reinemer, P.; Powers, J . C.; Kleine, T.; Pieper, M.; Tschesche,
H.; et al. X-ray structures of human neutrophil collagenase
complexed with hydroxamate and peptide thiols inhibitors.
Implications for substrate binding and rational drug design. Eur.
J . Biochem. 1995, 228, 830-841.
(49) Lovejoy, B.; Hassell, A. M.; Luther, M. A.; Weigl, D.; J ordan, S.
R. Crystal structures of recombinant 19-kDa human fibroblast
collagenase complexed to itself. Biochemistry 1994, 33, 8207-
8217.
(50) (a) Liljas, A.; Hakansson, K.; J onsson, B. H.; Xue, Y. Inhibition
and catalysis of carbonic anhydrase. Recent crystallographic
analyses. Eur. J . Biochem. 1994, 219, 1-10. (b) Briganti, F.;
Mangani, S.; Orioli, P.; Scozzafava, A.; Vernaglione, G.; Supuran,
C. T. Carbonic anhydrase activators: X-ray crystallographic and
spectroscopic investigations for the interaction of isozymes I and
II with histamine. Biochemistry 1997, 36, 10384-10392.
(51) (a) Supuran, C. T.; Clare, B. W. Carbonic anhydrase inhibitors.
Part 57. Quantum chemical QSAR of a group of 1,3,4-thiadiazole
and 1,3,4-thiadiazoline disulfonamides with carbonic anhydrase
inhibitory properties. Eur. J . Med. Chem. 1999, 34, 41-50. (b)
Clare, B. W.; Supuran, C. T. Carbonic anhydrase inhibitors. Part
61. Quantum Chemical QSAR of a group of benzenedisulfona-
mides. Eur. J . Med. Chem. 1999, 34, 463-474.
(52) (a) Brandstetter, H.; Engh, R. A.; Graf von Roedern, E.; Moroder,
L.; Huber, R.; Bode, W.; Grams, F. Structure of malonic acid-
based inhibitors bound to human neutrophil collagenase. A new
binding mode explains apparently anomalous data. Protein Sci.
1998, 7, 1303-1309. (b) Graff von Roedern, E.; Grams, F.;
Brandstetter, H.; Moroder, L. Design and synthesis of malonic
acid-based inhibitors of human neutrophil collagenase (MMP-
8). J . Med. Chem. 1998, 41, 339-345. (c) Pavlovsky, A. G.;
Williams, M. G.; Ye, Q. Z.; Ortwine, D. F.; Purchase, C. F.; White;
et al. X-ray structure of human stromelysin catalytic domain
complexed with nonpeptide inhibitors: Implications for inhibitor
selectivity. Protein Sci. 1999, 8, 1455-1462.
(61) Lindskog, S.; Behravan, G.; Engstrand, C.; Forsman, C.; J onsson,
B. H.; Liang, Z.; Ren, X.; Xue, Y. Structure-function relations
in human carbonic anhydrase II as studied by site-directed
mutagenesis. In Carbonic anhydrase - From biochemistry and
genetics to physiology and clinical medicine; Botre`, F., Gros, G.,
Storey, B. T., Eds.; VCH: Weinheim, 1991; pp 1-13.
(62) Behravan, G.; J onsson, B. H.; Lindskog, S. Fine-tuning of the
catalytic properties of carbonic anhydrase. Studies of a Thr200-
His variant of human isoenzyme II. Eur. J . Biochem. 1990, 190,
351-357.
(63) Khalifah, R. G.; Strader, D. J .; Bryant, S. H.; and Gibson, S. M.
Carbon-13 nuclear magnetic resonance probe of active site
ionization of human carbonic anhydrase B. Biochemistry 1977,
16, 2241-2247.
(64) Lindskog, S.; Coleman, J . E. The catalytic mechanism of carbonic
anhydrase. Proc. Natl. Acad Sci. U.S.A. 1964, 70, 2505-2508.
(65) Steiner, H.; J onsson, B. H.; Lindskog, S. The catalytic mecha-
nism of carbonic anhydrase. Hydrogen-isotope effects on the
kinetic parameters of the human C isoenzyme. Eur. J . Biochem.
1975, 59, 253-259.
(66) Maren, T. H.; Wynns, G. C.; Wistrand, P. J . Chemical properties
of carbonic anhydrase IV, the membrane-bound enzyme. Mol.
Pharmacol. 1993, 44, 901-906.
(67) Nagase, H. Activation mechanisms of matrix metalloproteinases.
Biol. Chem. 1997, 378, 151-160.
(68) Gao, J .; Qiao, S.; Whitesides, G. M. Increasing binding constants
of ligands to carbonic anhydrase by using “greasy tails”. J . Med.
Chem. 1995, 38, 2292-2301.
(69) (a) Briganti, F.; Pierattelli, A.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors. Part 37. Novel classes of isozymes
I and II inhibitors and their mechanism of action. Kinetic and
spectroscopic investigations on the native and cobalt-substituted
enzymes. Eur. J . Med. Chem. 1996, 31, 1001-1010. (b) Angleton,
E. L.; Van Wart, H. E. Preparation and reconstitution with
divalent metal ions of class I and class II Clostridium histolyti-
cum apocollagenase. Biochemistry 1988, 27, 7406-7412.
(70) Bertini, I.; Luchinat, C.; Scozzafava, A. Carbonic anhydrase: An
insight into the zinc binding site and into the active cavity
through metal substitution. Struct. Bonding 1982, 48, 45-92.
(71) (a) Eriksson, A. E.; Kylsten, P.; J ones, T. A.; Liljas, A. Crystal-
lographic studies of inhibitor binding sites in human carbonic
anhydrase II: A pentacoordinated binding of the SCN- ion to
the zinc. Proteins 1988, 4, 283-293. (b) Mangani, S.; Hakansson,
K. Crystallographic studies of the binding of protonated and
unprotonated inhibitors to carbonic anhydrase using hydrogen
sulphide and nitrate anions. Eur. J . Biochem. 1992, 210, 867-
871.
(53) Scolnick, L. R.; Clements, A. M.; Liao, J .; Crenshaw, L.; Hellberg,
M.; May, J .; Dean, T. R.; Christianson, D. W. Novel binding mode
of hydroxamate inhibitors to human carbonic anhydrase II. J .
Am. Chem. Soc. 1997, 119, 850-851.
(54) MacPherson, L. J .; Bayburt, E. K.; Capparelli, M. P.; Caroll, B.
J .; Goldstein, R.; J ustice, M. R.; et al. Discovery of CGS 27023A,
a nonpeptidic, potent, and orally active stromelysin inhibitor that
blocks cartilage degradation in rabbits. J . Med. Chem. 1997, 40,
2525-2532.
(55) (a) Alzuet, G.; Ferrer, S.; Borras, J .; Supuran, C. T. Complexes
of heterocyclic sulfonamides - a class of dual carbonic anhydrase
inhibitors. Roum. Chem. Quart. Rev. 1994, 2, 283-300. (b) Borja,
P.; Alzuet, G.; Server-Carrio`, J .; Borras, J .; Supuran, C. T. Zinc
complexes of carbonic anhydrase inhibitors. Crystal structure
of [Zn(5-amino-1,3,4-thiadiazole-2-sulfonamidate)2(NH3)]‚H2O.
Carbonic anhydrase inhibitory activity. Main Group Met. Chem.
1998, 21, 279-292. (c) Alzuet, G.; Casanova, J .; Borras, J .;
Garcia-Granda, S.; Gutie´rrez-Rodriguez, A.; Supuran, C. T.
Copper complexes modelling the interaction between benzola-
mide and Cu-substituted carbonic anhydrase. Crystal structure
of Cu(bz)(NH3)4 complex. Inorg. Chim. Acta 1998, 273, 334-338.
(56) Bieth, J . G. Theoretical and practical aspects of proteinase
inhibition kinetics. Methods Enzymol. 1995, 248, 59-84.
(57) (a) Itoh, M.; Hagiwara, D.; Kamiya, T. A new reagent for tert-
butoxycarbonylation: 2-tert-Butoxycarbonyloxyimino-2-phenyl-
acetonitrile. Org. Synth. 1980, 59, 95-101. (b) Supuran, C. T.;
Scozzafava, A. Carbonic anhydrase activators: amino acyl/
dipeptidyl histamine derivatives bind with high affinity to
isozymes I, II and IV, and act as efficient activators. Bioorg. Med.
Chem. 1999, 7, 2915-2924. (c) Mincione, G.; Menabuoni, L.;
Briganti, F.; Mincione, F.; Scozzafava, A.; Supuran, C. T.
Carbonic anhydrase inhibitors. Part 79. Synthesis of topically
acting sulfonamides incorporating GABA moieties in their
molecule, with long-lasting intraocular pressure-lowering prop-
erties. Eur. J . Pharm. Sci. 1999, 9, 185-199.
(72) Bertini, I.; Canti, G.; Luchinat, C.; Scozzafava, Characterization
of cobalt(II) bovine carbonic anhydrase and of its derivatives. J .
Am. Chem. Soc. 1978, 100, 4873-4877.
(73) Valee, B. L.; Auld, D. S. Zinc coordination, function and structure
of zinc enzymes and other proteins. Biochemistry 1990, 29,
5647-5659.
(74) Auld, D. S.; Bertini, I.; Donaire, A.; Messori, L.; Moratal, J . M.
pH-Dependent properties of cobalt(II) carboxypeptidase A -
inhibitor complexes. Biochemistry 1992, 31, 3840-3846.
(75) Larsen, K. S.; Zhang, K.; Auld, D. S. D-Phe complexes of zinc
and cobalt carboxypeptidase A. J . Inorg. Biochem. 1996, 64,
149-162.
(76) J ung, C. M.; Matsushita, O.; Katayama, S.; Minami, J .; Sakurai,
J .; Okabe, A. Identification of metal ligands in the Clostridium
histolyticum ColH collagenase. J . Bacteriol. 1999, 181, 2816-2822.
J M000027T