Protease inhibitors. Part 8: Synthesis of potent Clostridium histolyticum collagenase inhibitors incorporating sulfonylated L-alanine hydroxamate moieties

Article abstract of DOI:10.1016/S0968-0896(99)00316-8

A series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-L-alanine, followed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other structurally related compounds were obtained by reaction of N-2-chlorobenzyl-L-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved to be 100-500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxy-phenylsulfonyl-; 3-trifluoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamido-carboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') sites, in order to achieve tight binding to the enzyme. This study also proves that the 2-chlorobenzyl moiety, investigated here for the first time, is an efficient P(2') anchoring moiety for obtaining potent ChC inhibitors. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00316-8

Bioorganic & Medicinal Chemistry 8 (2000) 637±645
Protease Inhibitors. Part 8: Synthesis of Potent
Clostridium histolyticum Collagenase Inhibitors Incorporating
Sulfonylated L-Alanine Hydroxamate Moieties
Andrea Scozzafava and Claudiu T. Supuran*
Á
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received 1 September 1999; accepted 18 November 1999
AbstractÐA series of hydroxamates was prepared by reaction of alkyl/arylsulfonyl halides with N-2-chlorobenzyl-l-alanine, fol-
lowed by conversion of the COOH moiety to the CONHOH group, with hydroxylamine in the presence of carbodiimides. Other
structurally related compounds were obtained by reaction of N-2-chlorobenzyl-l-alanine with aryl isocyanates, arylsulfonyl iso-
cyanates or benzoyl isothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety. The new com-
pounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a bacterial zinc metallo-peptidase
which degrades triple helical collagen as well as a large number of synthetic peptides. The prepared hydroxamate derivatives proved
to be 100±500 times more active collagenase inhibitors than the corresponding carboxylates. Substitution patterns leading to best
ChC inhibitors (both for carboxylates as well as for the hydroxamates) were those involving per¯uoroalkylsulfonyl- and sub-
stituted-arylsulfonyl moieties, such as penta¯uorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxy-
phenylsulfonyl-; 3-tri¯uoromethyl-phenylsulfonyl; as well as 1- and 2-naphthyl-, quinoline-8-yl- or substituted-arylsulfonylamido-
carboxyl moieties among others. Similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, ChC inhibitors of the
0
type reported here must incorporate hydrophobic moieties at the P2 and P3 sites, in order to achieve tight binding to the enzyme.
0
0
This study also proves that the 2-chlorobenzyl moiety, investigated here for the ®rst time, is an ecient P2 anchoring moiety for
obtaining potent ChC inhibitors. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
As amino acid and oligopeptide hydroxamates were
reported to act as powerful inhibitors for a large num-
ber of metallo-enzymes important as targets in the
drug design (such as the matrix metalloproteinases,
MMP's;6±9 thermolysin and ellastase;10 leucine amino-
peptidase;11 carboxypeptidase A;12 leukotriene A4
hydrolase;13 angiotensin I-converting enzyme;14 neuro-
tensin-degrading enzymes;15 endothelin-converting
enzyme;16 or UDP-3-O-(R-3-hydroxymyristoyl)-N-ace-
tylglucosamine deacetylase17±19 among others), we deci-
ded to investigate such derivatives as anti-bacterial
corneal keratitis agents. Some of the above-mentioned
derivatives were also shown to possess strong anti-
bacterial activity since by inhibiting some of the above
mentioned enzymes, they interfere with lipid A bio-
synthesis and inhibit the growth of Gram-negative
bacteria.17±19 Other compounds of this type were also
reported to act as anti-HIV agents in vitro.20 But the
most important applications in drug design of the
amino acid/oligopeptide hydroxamates is related to
their use for the development of MMP inhibitors as
anti-cancer21±25 or anti-arthritis7,26±28 drugs. The 23
During our studies in the design and synthesis of anti-
glaucoma agents with topical action,1±3 it was fre-
quently observed that experimental animals used in the
work (albino rabbits) developed eye infections due to
bacterial pathogens, among which Clostridium histolyti-
cum was the prevalent species. A literature survey
proved that bacterial corneal keratitis,4 a condition
leading to serious complications for which ecient cures
are dicult to envisage at the moment,4 has indeed been
reported to be associated with a highly increased col-
lagenase activity in the ocular tissues, in human patients
and experimental animals.5 Thus, it appeared of
interest to investigate in detail the design of bacterial
collagenase inhibitors with putative ophthalmologic
applications.
*Corresponding author. Tel.: +39-55-275-7552; fax: +39-55-275-
7555; e-mail: cts@bio.chim.uni®.it
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00316-8

638
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
MMP's presently known7,29 are involved in tissue
remodeling connected with tumor invasion and joint
destruction.6,7,22±26 Synthetic high anity inhibitors for
some of these enzymes, such as the four vertebrate col-
lagenases (MMP's 1, 8, 13 and 18), stromelysins 1 and 2
(MMP's 3 and 10, respectively) or the gelatinases A and
B (MMP's 2 and 9) were much investigated in the last
period, in order to develop novel pharmacological
agents of the hydroxamate type.6,7,25±29 Not the same
situation is true for the inhibitors of bacterial col-
lagenases, such as for instance, the enzyme isolated
from C. histolyticum,30±32 which was much less investi-
gated. This collagenase (EC 3.4.24.3) is a 116 kDa pro-
tein belonging to the M31 metalloproteinase family,29
being able to hydrolyze triple helical regions of collagen
under physiological conditions, as well as an entire
range of synthetic peptide substrates.30±32 In fact the
crude homogenate of Clostridium histolyticum, which
contains several distinct collagenase isozymes, is the
most ecient system known for the degradation of
connective tissue,31,32 being also involved in the patho-
genicity of this and related clostridia, such as C. per-
fringens, which causes human gas gangrene and food
poisoning among others.33
showed high anity for the enzyme (in the nanomolar
range), behaving as very potent inhibitors. The results
of the in vivo experiments as anti-corneal bacterial ker-
atitis agents with some of the compounds synthesized in
the present paper will be reported elsewhere.
Results
Synthesis
The key intermediate 3 for the synthesis of new col-
lagenase inhibitors reported here, was prepared by
reaction of 2-chlorobenzyl-chloride (1) with l-alanine
(2). Compounds A1±A34 were then obtained by the
reaction of 3 with alkyl/arylsulfonyl halides 4, as shown
in Scheme 1 and Table 1. Conversion of the carboxylic
acids A1±A34 into the corresponding hydroxamates
B1±B34 was done with hydroxylamine and diisopropyl
carbodiimide (Scheme 1).
Another series of derivatives (C1±C4; D1±D4 and E1±
E6; F1±F6) was obtained by reaction of arylsulfonyliso-
cyanates (5) or aryl isocyanates (6) with N-2-chloro-
benzyl-glycine (3), followed by the conversion of the
COOH moiety into the CONHOH one, as described
above (Schemes 2 and 3).
Similar to the vertebrate MMP's,7,29±35 C. histolyticum
collagenase (abbreviated as ChC) has the conserved
HExxH zinc-binding motif, which in this speci®c case is
His415ExxH, with the two histidines (His 415 and His
419) acting as Zn(II) ligands; whereas the third ligand
seems to be Glu 447,30±35 (in the case of the vertebrate
MMP's the zinc ion is coordinated by three histidines,
His 218, His 222 and His 228), and a water molecule/
hydroxide ion acting as a nucleophile in the hydrolytic
scission.5±9,30±35 Similar to the MMP's, ChC is also a
multidomain protein, consisting of four segments, S1,
S2a, S2b and S3,35 with S1 incorporating the catalytic
domain.
By applying synthetic strategies related to the previously
described ones, the sulfenamido derivatives G1±G3 and
H1±H3, as well as the thioureas I1 and J1 were also
obtained.
Thus, we hypothesized that amino acid hydroxamates
and some of their derivatives, which strongly inhibit
MMP's (collagenases, gelatinases, stromelysins, etc.),
would also act as potent ChC inhibitors. We also
hypothesized that the use of a collagen shield, soaked in
an antibiotic agent speci®c for the collagen-degrading
bacteria, would have a double bene®t for the patients
a€ected by bacterial corneal keratitis: (i) the collagenase
inhibitor would kill (or impair the growth of) bacteria
present on the cornea, improving and accelerating heal-
ing of the keratitis; (ii) the (protective) collagen shield
would acquire an augmented stability, as its degrada-
tion by the secreted collagenases would be delayed,
promoting/accelerating in this way the healing of the
wound. On the other hand, the topical application of
such a pharmacological agent would avoid serious sys-
temic toxicity problems.
Collagenase inhibition
Inhibition data against highly puri®ed ChC type VII,
with the newly prepared carboxylic and hydroxamic
acids are shown in Tables 1 and 2. The chromogenic
substrate FALGPA (2-furanacryloyl-l-Leu-Gly-l-Pro-
l-Ala) was used in the assay, with the spectro-
photometric method of Van Wart and Steinbrink.30
Inhibition data with some standard ChC inhibitors
In this paper we report the preparation of a series of
ChC inhibitors incorporating alkyl/arylsulfonamido-N-
2-chlorobenzyl-l-alanine hydroxamate as well as aryl-
sulfonylureido-/arylureido-N-2-chlorobenzyl-l-alanine
hydroxamate moieties in their molecule. Some of the new
compounds, assayed for the inhibition of puri®ed ChC,

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
639
Scheme 1.
Scheme 2.
Table 1. Inhibition of ChC with the carboxylic acids A1±A34 and the
corresponding hydroxamates B1±B34
R
Compound
KIa
(mM)
Compound
KIa
(nM)
CH3
CF3
CCl3
n-C4F9-
n-C8F17
Me2N-
C6H5-
PhCH2-
4-F-C6H4-
4-Cl-C6H4-
4-Br-C6H4-
4-I-C6H4-
4-CH3-C6H4
4-O2N-C6H4-
3-O2N-C6H4-
2-O2N-C6H4-
3-Cl-4-O2N-C6H3-
4-AcNH-C6H4-
4-BocNH-C6H4-
3-BocNH-C6H4-
C6F5-
3-CF3-C6H4
2,5-Cl2C6H3
4-CH3O-C6H4-
2,4,6-(CH3)3-C6H2-
4-CH3O-3-BocNH-C6H3-
2-HO-3,5-Cl2-C6H2-
3-HOOC-C6H4-
4-HOOC-C6H4-
1-Naphthyl
2-Naphthyl
5-Me2N-1-naphthyl-
2-Thienyl
A1
A2
A3
A4
A5
A6
A7
A8
21
B1
B2
B3
B4
B5
B6
B7
B8
92
76
71
11
8
3.3
5.2
3.0
1.5
40
24
16
12
13
77
58
54
36
35
33
30
45
15
11
16
10
11
9
8
5
6
15
22
17
8
9
8
A9
B9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
A34
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28b
B29b
B30
B31
B32
B33
B34
10
10
18
5.1
5.0
4.9
3.1
3.0
2.7
2.4
0.4
0.5
4.1
6.0
6.8
3.0
2.4
4.1
3.8
1.6
1.5
2.1
2.4
2.0
Scheme 3.
up to now.24,36±42 Since the ChC enzyme catalyzes
the cleavage of the Xaa-Gly peptide bond of the
repeating sequence of collagen: -Gly-Pro-Xaa-Gly-Pro-
Xaa- (Xaa=amino acid residue), it appears that the S3,
S2 and S1 subsites of the enzyme are occupied by Gly,
7
8
9
10
11
7
Pro and Xaa, respectively.30±36 Analogously, the S1 , S2
0
0
0
and S3 subsites are also occupied by Gly, Pro and Xaa,
respectively.30±36 Thus, many of the reported inhibitors
of ChC are aldehyde or ketone-type substrate analo-
gues, such as Pro6-Gly-l-Pro-GlyH (GlyH=glycine
aldehyde), with a KI of 340 mM;36 phosphoric and
phosphonic amides, such as iso-amylphosphonyl-
Gly-l-Pro-l-Ala (KI of 16 mM);37 thiols such as HS-
CH2CH2CO-Pro-Xaa (KI of the best compounds
around 0.2 mM);38±40 phosphonamide peptides of the
type p-nitrophenethyl-PO(OH)-Gly-Pro-Xaa (KI of the
best compound, with Xaa=2-aminohexanoic acid was
of 5 nM).41,42 As shown from the above data, either the
obtained inhibitors are relatively weak, or the high a-
nity ones are phosphorus based ligands which are not
suitable for the development of pharmaceutical agents,
due to their high toxicity.
Quinoline-8-yl
aKI-s values were obtained from Dixon plots using a linear regression
program, from at least three di€erent assays.
bThe C6H4-COOH moiety transformed into C6H4-CONHOH.
reported in the literature are also provided for compar-
ison in Table 3.
Discussion
A literature search showed that a relatively small number
of bacterial collagenase inhibitors have been reported

640
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
Table 2. Inhibition of ChC with the carboxylic acids of types C, E,
G, I and the corresponding hydroxamates of types D, F, H, J
MMP inhibitory properties of some arylsulfonyl±amino
hydroxamic acids of type 7a±7c recently reported by
Parker's26 and Hanessian's21 groups, we decided to use
such derivatives as lead molecules. In the ®rst of the
above mentioned study26 it was observed that best inhi-
bitors of type 7a (against mouse macrophage metallo-
elastase) were those incorporating: (i) Gly, Ala or Val as
spacers between the zinc-binding function (the hydro-
R
Compound KIa (mM) Compound KIa (nM)
4-F-C6H4-
C1
C2
C3
C4
E1
E2
E3
E4
E5
E6
G1
G2
G3
I1
3.6
3.4
4.1
4.0
7.4
7.6
6.8
5.5
5.8
5.0
6.0
6.1
6.1
0.9
D1
D2
D3
D4
F1
F2
F3
F4
F5
F6
H1
H2
H3
J1
9
8
4-Cl-C6H4-
4-CH3-C6H4-
2-CH3-C6H4-
4-F-C6H4-
3-Cl-C6H4-
4-Cl-C6H4-
2,4-F2-C6H3-
3,4-Cl2C6H3
1-Naphthyl
4-O2N-C6H4-
2-O2N-C6H4-
2,4-(O2N)2-C6H3-
Ð
11
10
18
17
15
14
15
11
13
12
11
6
0
xamic acid moiety) and the P2 site; (ii) benzyl or isobutyl
26
0
0
moieties at S2 , and (iii) arylsulfonyl moieties at S3 . It
should be noted that a relatively small number of
arylsulfonyl moieties were investigated in the above-
mentioned studies,21,26 the greatest number of the syn-
thesized inhibitors
7
containing the 4-methoxy-
benzenesulfonyl or biphenylsulfonyl moieties. The
proposed interaction sites between an inhibitor of type
7a and the active site of the enzyme is also shown sche-
matically in the formula of the inhibitor 7a.
aKI-s values were obtained from Dixon plots using a linear regression
program, from at least three di€erent assays.
Considering the above-mentioned ®ndings,21,26 we
opted for the following structural elements in the design
of the ChC inhibitors reported in the present study: (i)
a strong zinc-binding function (of the carboxylic acid,
or better, hydroxamic acid types);6,7 (ii) a relatively
compact spacer between this function and the rest of the
molecule, i.e. l-Ala;6,26 (iii) a variant of the already
Table 3. Inhibition of ChC with standard inhibitors
Compound
KIa (mM)
Pro6-Gly-Pro-GlyH
i-C5H11PO(OH)GlyProAla
p-O2NC6H4CH2CH2PO(OH)GlyPro-2AXb
340
16
0.005
optimized26 benzyl group at the S2 site, which has not
0
aKI-s values were obtained from Dixon plots using a linear regression
program, from at least three di€erent assays.
b2AX=2-aminohexanoic acid.
been investigated previously, i.e. the 2-chlorobenzyl
moiety. The presence of the halogen atom substituting
0
the S2 moiety was thought to be important for binding
of the inhibitor to the enzyme, due to its steric and
especially electronic e€ects that would clearly in¯uence
other properties of the inhibitor, such as the pKa value
of the hydroxamic acid group; (iv) variable alkyl/aryl-
sulfonyl-; arylsulfonyl-ureido/arylureido-; or arylsulfe-
0
nyl, moieties at S3 .
The new compounds reported here were obtained by
non-exceptional synthetic procedures, as outlined in
Schemes 1±3. These involved reaction of N-2-chloro-
benzyl-l-alanine (3) with alkyl/arylsulfonyl chlorides,43
arylisocyanates,44 arylsulfonyl-isocyanates45 or benzoyl-
isothiocyanate, followed by conversion of the COOH
moiety to the hydroxamate one.46 Related synthetic
strategies led to some sulfenamides of type G, H, as well
as to the thioureas I, J.
ChC inhibition data with the new compounds reported
here as well as other reported inhibitors are shown in
Tables 1±3.
The following should be noted regarding ChC inhibi-
tion data of Tables 1±3 with the new compounds and
standard inhibitors: (i) all hydroxamates were 100±500
times more active as ChC inhibitors as compared to the
corresponding carboxylic acids, probably due to the
enhanced Zn(II) coordinating properties of the CON-
HOH moiety (bidentate binding) as compared to the
COOH group (generally monodentate binding to the
zinc ion).6±9 (ii) Potent inhibitors were obtained from all
classes of hydroxamates investigated here, such as
the alkyl/arylsulfonyl-N-2-chlorobenzyl-Ala derivatives
(B5, B20±B22, B26, B27, B29, etc), the arylsulfonylureas-
Thus, the lead molecule that we used for designing the
novel ChC inhibitors reported here was not of the type
mentioned above. Taking into account the strong

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
641
and arylureas (such as D2, F5, F6), the sulfenamido-
benzyl-Ala derivatives (such as H2, H3) or the thiourea
0
J1. Thus, it seems that the S3 -binding moiety of the
arylsulfonamide type, previously investigated for the
obtaining of MMP inhibitors of type 7,21,26 can be e-
ciently substituted by related moieties such as alkylsul-
fonyl-; arylsulfenyl-; arylsulfonylureido-; arylureido- or
benzoyl-thioureido, without loss of the ChC inhibitory
properties. (iii) In the subseries of alkyl/arylsulfonamido
derivatives (of types A,B(1Ð34)) best ChC inhibitory
properties were correlated with the presence of per-
¯uoroalkylsulfonyl- (B4, B5), per¯uorophenylsulfonyl-
(B21), 3-tri¯uoromethylphenylsulfonyl- (B22); 3-chloro-
4-nitro-phenylsulfonyl- (B17); 3- or 4-protected-amino-
phenylsulfonyl- (B18±B20; B26); 3- or 4-carboxy-phe-
nylsulfonyl- (B28, B29), 1- or 2-naphthylsulfonyl as well
as 8-quinolinesulfonyl moieties (B30±B32; B34). All
these derivatives possessed inhibition constants in the
range of 5±12 nM against ChC, being among the most
potent such inhibitors ever reported. A second group of
sulfonamide inhibitors, containing moieties such as 4-
bromophenyl; 4-iodophenyl; 2-, 3- or 4-nitrophenyl; 2,5-
dichlorophenyl-; 2,4,6-trimethylphenyl-; 4-methoxy-
phenyl- or 2-thienyl, substituting the N-benzyl-glycine
hydroxamate, behaved as medium potency inhibitors,
with anities in the 15±30 nM range (Table 1). The least
active sulfonamides were those containing methyl-; tri-
halomethyl-; dimethylamino-; phenyl- and benzyl moi-
eties (Table 1). (iv) The arylsulfonylureido compounds
D1±D4 were more active than the corresponding aryl-
sulfonyl derivatives (compare for instance D1 with B9;
D2 and B10, etc), acting as strong ChC inhibitors.
Similarly behaved were the ureas of type F, and the
sulfenamides of type H, except for F5 and F6, as well as
H2 and H3, which are strong inhibitors. A very potent
inhibitor is the thiourea derivative J1 (Table 2). By
comparing the data of Tables 1±3, it is obvious that
the compounds reported in the present study are
among the best ChC inhibitors obtained up to now,
since other such derivatives usually had anities in the
micromolar range (except for the phosphonic acid
derivative mentioned in Table 3, which possessed an
anity of the same order of magnitude as that of our
compounds).
These studies8,9,47±49 showed that batimastat is bident-
ately coordinated to the Zn(II) ion of the enzyme,
through the hydroxamate OH moiety and hydroxamate
CO group. The OH and NH of the hydroxamate moiety
participate in supplementary interactions with the
enzyme, forming hydrogen bonds with Glu 198 and Ala
161.47 The hydrophobic residues in the P1 (isobutyl)
0
0
and P2 (benzyl) positions are also critical for the for-
mation of a strong E-I adduct: thus, the leucine side
0
0
chain of P1 extends into the S1 pocket, making hydro-
phobic contacts with several amino acid residues such as
His 197; Pro 217 and Val 194, whereas the phenyl ring
0
of P2 interacts with the side chains of Ile 159, Val 129
and Pro 217. Although the class III collagenase inhibi-
tors (in the classi®cation of Babine and Bender6) of the
sulfonamide type, to which the compounds reported
here presumably belong, were much less investigated
crystallographically, it is assumed that the general
binding mode illustrated above is also valid for them,
although some di€erences were also evidenced.6,21,26
Based on these observations, we propose a similar
binding mode of the sulfonamide inhibitors reported
here to ChC, as showed schematically in Figure 1.
The inhibitor probably coordinates bidentately to the
Zn(II) ion of ChC, whereas the hydrophobic moieties
0
0
Although ChC (or its catalytic domain) could not be
crystallized up to now (in our or in other laboratories),
and the precise binding of inhibitors cannot be inferred
from X-ray crystallographic data, several important
contributions in the ®eld of the MMP's have been
registered recently, and this might be useful in inter-
preting our inhibition data. Thus, Bode's, Moroder's
and Tschesche's groups reported8,9,47±49 several X-ray
crystallographic studies for the interaction of some
types of hydroxamate inhibitors with the catalytic
domain of MMP-8, a collagenase from vertebrates,
inhibited among others with a KI of 10 nM by one of the
collagenase inhibitors in clinical study, batimastat
(8).50,51 Another such derivative 9 (AG-3340), was
recently reported by the Agouron group to possess
broad antitumor and antiangiogenic activities in many
tumor models as well as in patients with advanced
malignancies of the lung and prostate.52±54
from the P2 and P3 sites (2-chlorobenzyl and penta-
¯uorophenyl, respectively) participate in hydrophobic
contacts which assure the strong anity of this inhibitor
for ChC (the inhibitor B21 for which the schematic
binding is shown above has a KI of 5 nM).
Figure 1. Proposed schematic binding of inhibitor B21 within the
active site of ChC.

642
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
Conclusion
General procedure for the preparation of N-2-chloroben-
zyl-alkyl/arylsulfonyl alanines A1±A34. An amount of
2.13 g (10 mmol) of N-2-chlorobenzyl-l-alanine (3) and
10 mMol of sulfonyl chloride (4) were suspended/
dissolved in 100 mL of acetone+25 mL of water. The
stoichiometric amount (10 mmol) of base (NaHCO3;
KHCO3, NaOH or Et3N) dissolved in a small amount
(20 mL) of water was added and the mixture stirred at
room temperature for 4±10 h (TLC control). The sol-
vent was evaporated, the reaction mixture was retaken
in 100 mL of water and the crude product extracted in
ethyl acetate. After evaporation of the solvent, the
compounds A1±A34 were recrystallized from EtOH or
MeOH. Yields were around 75±90%.
We describe here a novel class of strong inhibitors of the
zinc protease EC 3.4.24.3, a collagenase from C. histo-
lyticum. As no X-ray crystallographic structure of this
enzyme is available, the drug design has been realized by
utilizing X-ray data for the MMP's, related enzymes
which degrade the extracellular matrix in vertebrates.
Reaction of N-2-chlorobenzyl-l-alanine with sulfonyl
chlorides, arylsulfonyl isocyanate, aryl isocyanates or
benzoyl isothiocyanate a€orded the new derivatives
which were subsequently converted to the correspond-
ing hydroxamates. These latter derivatives were 100±500
times more inhibitory against ChC as compared to the
corresponding carboxylic acids. Best substitutions for
obtaining high anity inhibitors, involved hydrophobic
General procedure for the preparation of compounds B1±
B34, D1±D4, F1±F6, H1±H3 and J1. An amount of
5 mM of carboxylic acid derivative A1±A34, C1±C4,
E1±E6, G1±G3 or I1 was dissolved/suspended in 50 mL
of anhydrous acetonitrile or acetone, and treated with
0
moieties at S3 , such as per¯uoroalkylsulfonyl-; sub-
stituted-arylsulfonyl (penta¯uoro-phenylsulfonyl; 3- and
4-carboxy-phenylsulfonyl-; 3-tri¯uoromethyl-phenyl-
sulfonyl; or 1- and 2-naphthyl, etc.) moieties among
.
0
others. 2-Chlorobenzyl moieties at P2 seem to be a valid
420 mg (6 mM) of hydroxylamine HCl and 1.10 g
variant of the benzyl group, for obtaining tight-binding
collagenase inhibitors. This is the ®rst study reporting
nanomolar anity ChC inhibitors of the sulfonamide
type.
(6 mM) of EDCI. HCl or di-isopropyl-carbodiimide.
The reaction mixture was magnetically stirred at room
temperature for 15 min, then 180 mL (12 mM) of tri-
ethylamine were added and stirring was continued for
12 h at 4 ꢀC. The solvent was evaporated in vacuo and
the residue taken up in ethyl acetate (5 mL), poured into
a 5% solution of sodium bicarbonate (5 mL) and
extracted with ethyl acetate. The combined organic lay-
ers were dried over sodium sulfate and ®ltered, and the
solvent removed in vacuo. Preparative HPLC (Dyna-
max-60A column (25Â250 mM); 90% acetonitrile/10%
methanol; ¯ow rate of 30 mL/min) a€orded the pure
hydroxamic acids.
Experimental
Melting points (mp): heating plate microscope (not cor-
1
rected); IR spectra: KBr pellets, 400±4000cm Perkin±
Elmer 16PC FTIR spectrometer; 1H NMR spectra:
Varian 300CXP apparatus (chemical shifts are expres-
sed as d values relative to Me4Si as standard): Elemental
analysis (‹0.4% of the theoretical values, calculated for
the proposed formulasÐdata not shown): Carlo Erba
Instrument CHNS Elemental Analyzer, Model 1106. All
reactions were monitored by thin-layer chromatography
(TLC) using 0.25-mm precoated silica gel plates (E.
Merck). Preparative HPLC was performed on a Dyna-
max-60A column (25Â250 mM), with a Beckman EM-
1760 instrument. The detection wavelength was 254 nM.
General procedure for the preparation of compounds C1±
C4, E1±E6 and I1. An amount of 2.13 g (10 mmol) of
N-2-chlorobenzyl-alanine (3) and the stoichiometric
amount of arylsulfonyl isocyanate (5); aryl isocyanate
(6) or benzoyisothiocyanate were suspended in 50 mL of
anhydrous acetonitrile and 150 mL (10 mM) of triethyl-
amine were added. The reaction mixture was either
stirred at room temperature (in the case of derivatives
prepared from 5) or re¯uxed (for the other two types of
derivatives) for 2±6 h. The solvent was evaporated and
the reaction mixture worked up as described above. The
new compounds were recrystallized from ethanol.
Yields were almost quantitative.
Amino acids (l-Ala), sulfonyl chlorides, arylsulfonyl
isocyanates, aryl isocyanates, benzoyl isothiocyanate,
triethylamine, carbodiimides, hydroxylamine, 2-chlor-
obenzyl chloride, and other reagents/solvents used in
the syntheses were commercially available compounds
(from Sigma, Fluka, Acros or Aldrich).
General procedure for the preparation of compounds
G1±G3. The general procedure described above for the
preparation of compounds A1±A34 has been followed,
except that arylsulfenyl halides were used instead of
alkyl/arylsulfonyl halides. The yields in the title sulfe-
namides were around 75%.
Preparation of N-2-chlorobenzyl-L-alanine (3). An
amount of 9 g (0.10 M) of l-Ala (2) and the stoichio-
metric amount of 2-chlorobenzyl chloride (16.1 g,
12.6 mL) were suspended/dissolved in 50 mL of anhy-
drous acetonitrile and the equivalent amount of triethyl
amine (0.10 mM, 14.7 mL) was added. The reaction
mixture was heated at re¯ux for 20 h, then the solvent
was evaporated in vacuo. The obtained reaction mixture
was taken in 50 mL of water, the pH was brought to 7
with citric acid, and the crude product (3) precipitated
by leaving the mixture overnight at 4 ꢀC. Recrystalliza-
tion from ethanol a€orded the pure title compound in
almost quantitative yield.
1
The new compounds were characterized by H and 13C
NMR spectroscopy and elemental analysis. Data for a
representative compound of each series is provided
below.
N-4-Toluenesulfonyl-N-2-chlorobenzyl-L-alanine
A13.
White crystals, mp 197±199 ꢀC; H NMR (DMSO-d6),
1

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
643
d, ppm: 1.52 (d, 3JHH=6.5, 3H, CHCH3 of Ala), 2.53 (s,
3H, CH3C6H4), 3.79 (s, 2H, CH2 of benzyl); 3.90 (q, 1H,
CH of Ala); 7.22-7.59 (m, 6H, Hortho of CH3C6H4 and
Ala); 44.2 (s, CH2 of benzyl), 127.2 (s, C-5 of 2-Cl±
C6H4); 129.7 (C-4 of 2-Cl±C6H4); 129.9 (C-3 of 2-Cl±
C6H4); 130.4 (s, Cmeta of CH3C6H4), 130.8 (C-6 of 2-Cl±
C6H4); 132.7 (s, NHCONH), 134.2 (C-2 of 2-Cl±C6H4);
135.2 (C-1 of 2-Cl±C6H4); 135.5 (s, Cortho of CH3C6H4),
145.6 (s, Cipso of CH3C6H4), 148.5 (s, Cpara of CH3
C6H4), 174.7 (s, CONHOH). C18H20Cl3O5S (425.89);
calcd.: C, 50.76; H, 4.73; N, 9.87%; found: C, 50.85; H,
4.58; N, 9.69%.
3
Harom of 2-Cl-C6H4), 7.92 (d, JHH=8.1, 2H, Hmeta of
CH3C6H4); 11.54 (br s, 1H, COOH); 13C NMR
(DMSO-d6), d, ppm: 20.3 (s, CHCH3 of Ala); 26.1 (s,
CH3C6H4), 34.5 (s, CHCH3 of Ala); 43.5 (s, CH2 of
benzyl), 127.2 (s, C-5 of 2-Cl-C6H4); 129.7 (C-4 of 2-Cl-
C6H4); 129.9 (C-3 of 2-Cl-C6H4); 130.2 (s, Cmeta of
CH3C6H4), 130.8 (C-6 of 2-Cl-C6H4); 134.2 (C-2 of 2-
Cl-C6H4); 135.0 (C-1 of 2-Cl-C6H4); 135.7 (s, Cortho of
CH3C6H4), 145.5 (s, Cipso of CH3C6H4), 148.8 (s, Cpara of
CH3C6H4), 177.7 (s, CO2H). C17H18ClNO4S (360.85);
calcd. C, 55.10; H, 4.93; N, 3.81%; found: C, 55.16; H,
5.09; N, 3.75%.
N-4-Fluorophenylureido-N-2-chlorobenzyl-L-alanine E1.
White crystals, mp 176±177 ꢀC; H NMR (DMSO-d6),
1
d, ppm: 1.50 (d, 3JHH=6.5, 3H, CHCH3 of Ala), 3.78 (s,
2H, CH2 of benzyl); 3.92 (q, 1H, CH of Ala); 7.11±7.67
(m, 6H, Hortho of 4-FC6H4 and Harom of 2-Cl-C6H4),
3
7.95 (d, JHH=8.1, 2H, Hmeta of 4-FC6H4); 8.13 (br s,
N-4-Toluenesulfonyl-N-2-chlorobenzyl-L-alanine hydroxa-
mate B13. White crystals, mp 211±221 ꢀC; 1H NMR
(DMSO-d6), d, ppm: 1.54 (d, 3JHH=6.5, 3H, CHCH3 of
Ala), 2.62 (s, 3H, CH3C6H4), 3.79 (s, 2H, CH2 of ben-
2H, NHCONH); 11.42 (br s, 1H, COOH); 13C NMR
(DMSO-d6), d, ppm: 20.1 (s, CHCH3 of Ala); 34.5 (s,
CHCH3 of Ala); 43.5 (s, CH2 of benzyl), 127.2 (s, C-5 of
2-Cl-C6H4); 129.7 (C-4 of 2-Cl±C6H4); 129.9 (C-3 of 2-
Cl±C6H4); 130.0 (s, Cmeta of FC6H4), 130.8 (C-6 of 2-Cl±
C6H4); 132.3 (s, NHCONH), 134.2 (C-2 of 2-Cl±C6H4);
135.1 (s, Cortho of FC6H4), 135.3 (C-1 of 2-Cl±C6H4);
148.5 (s, Cipso of FC6H4), 149.5 (s, Cpara of FC6H4),
177.5 (s, CO2H). C17H16ClFN2O3 (350.78); calcd.: C,
58.21; H, 4.60; N, 7.99%; found: C, 58.42; H, 4.57; N,
8.00%.
zyl); 3.93 (q, 1H, CH of Ala); 7.24±7.65 (m, 6H, Hortho
3
of CH3C6H4 and Harom of 2-Cl-C6H4), 8.06 (d, JHH
=
8.1, 2H, Hmeta of CH3C6H4); 8.73 (br s, 1H, NHOH);
10.52 (br s, 1H, NHOH); 13C NMR (DMSO-d6), d,
ppm: 20.4 (s, CHCH3 of Ala); 26.5 (s, CH3C6H4), 34.4
(s, CHCH3 of Ala); 44.1 (s, CH2 of benzyl), 127.2 (s, C-5
of 2-Cl-C6H4); 129.7 (C-4 of 2-Cl-C6H4); 129.9 (C-3 of
2-Cl-C6H4); 130.6 (s, Cmeta of CH3C6H4), 130.8 (C-6 of
2-Cl-C6H4); 134.2 (C-2 of 2-Cl-C6H4); 135.0 (C-1 of 2-
Cl-C6H4); 135.3 (s, Cortho of CH3C6H4), 145.2 (s, Cipso of
CH3C6H4), 148.0 (s, Cpara of CH3C6H4), 174.9 (s,
CONHOH). C17H19ClN2O4S 8382.87) calcd. C, 53.33;
H, 5.00; N, 7.32%; found: C, 53.24; H, 5.07; N, 7.25%.
N - 4 - Fluorophenylureido - N - 2 - chlorobenzyl - L - alanine
hydroxamate F1. White crystals, mp 210±221 ꢀC; 1H
3
NMR (DMSO-d6), d, ppm: 1.56 (d, JHH=6.5, 3H,
CHCH3 of Ala), 3.82 (s, 2H, CH2 of benzyl); 3.93 (q,
1H, CH of Ala); 7.08±7.60 (m, 6H, Hortho of 4-FC6H4
and Harom of 2-Cl±C6H4), 7.90 (d, 3JHH=8.1, 2H, Hmeta
of 4-FC6H4); 8.12 (br s, 2H, NHCONH); 8.76 (br s, 1H,
NHOH); 10.69 (br s, 1H, NHOH); 13C NMR (DMSO-d6),
d, ppm: 20.3 (s, CHCH3 of Ala); 34.6 (s, CHCH3 of
Ala); 43.7 (s, CH2 of benzyl), 127.2 (s, C-5 of 2-Cl-
C6H4); 129.7 (C-4 of 2-Cl-C6H4); 129.9 (C-3 of 2-Cl-
C6H4); 130.0 (s, Cmeta of FC6H4), 130.8 (C-6 of 2-Cl-
C6H4); 132.1 (s, NHCONH), 134.3 (C-2 of 2-Cl±C6H4);
135.1 (s, Cortho of FC6H4), 135.3 (C-1 of 2-Cl±C6H4);
135.6 (s, Cortho of FC6H4), 145.8 (s, Cipso of FC6H4),
148.4 (s, Cpara of FC6H4), 174.5 (s, CONHOH).
C17H17ClFN3O3 (365.79); calcd.: C, 55.82; H, 4.68; N,
11.49%; found: C, 55.84; H, 4.76; N, 11.33%.
N-4-Toluenesulfonylureido-N-2-chlorobenzyl-L-alanine
ꢀ
1
C3. White crystals, mp 207±209 C; H NMR (DMSO-
3
d6), d, ppm: 1.51 (d, JHH=6.5, 3H, CHCH3 of Ala),
2.60 (s, 3H, CH3C6H4), 3.77 (s, 2H, CH2 of benzyl); 3.95
(q, 1H, CH of Ala); 7.12±7.68 (m, 6H, Hortho of
3
CH3C6H4 and Harom of 2-Cl-C6H4), 7.99 (d, JHH=8.1,
2H, Hmeta of CH3C6H4); 8.23 (br s, 2H, NHCONH);
11.70 (br s, 1H, COOH); 13C NMR (DMSO-d6), d,
ppm: 20.3 (s, CHCH3 of Ala); 26.6 (s, CH3C6H4), 34.5
(s, CHCH3 of Ala); 43.3 (s, CH2 of benzyl), 127.2 (s, C-5
of 2-Cl±C6H4); 129.7 (C-4 of 2-Cl±C6H4); 129.9 (C-3 of
2-Cl±C6H4); 130.4 (s, Cmeta of CH3C6H4), 130.8 (C-6 of
2-Cl-C6H4); 132.3 (s, NHCONH), 134.2 (C-2 of 2-Cl±
C6H4); 135.2 (C-1 of 2-Cl±C6H4); 135.6 (s, Cortho of
CH3C6H4), 145.1 (s, Cipso of CH3C6H4), 148.5 (s, Cpara
of CH3C6H4), 177.2 (s, CO2H). C18H19ClN2O5S
(410.88) calcd. C, 52.62; H, 4.66; N, 6.82%; found: C,
52.49; H, 4.51; N, 6.73%.
N-4-Nitrophenylsulfenyl-N-2-ꢀchlorobenzyl-L-alanine G1.
1
Yellow crystals, mp 216±217 C; H NMR (DMSO-d6),
3
d, ppm: 1.50 (d, JHH=6.5, 3H, CH3 of Ala), 3.75 (s,
2H, CH2 of benzyl); 3.93 (q, 1H, CH of Ala); 6.79 (s,
1H, SNH), 7.13±7.64 (m, 6H, Hortho of O2NC6H4 and
3
Harom of 2-Cl±C6H4), 8.05 (d, JHH=8.3, 2H, Hmeta of
N-4-Toluenesulfonylureido-N-2-chlorobenzyl-L-alanine
hydroxamate D3. White crystals, mp 221±222 ꢀC; 1H
NMR DMSO-d6), d, ppm: 1.54 (d, JHH=6.5, 3H,
O2NC6H4); 11.73 (br s, 1H, COOH); 13C NMR
(DMSO-d6), d, ppm: 20.1 (s, CHCH3 of Ala); 34.4 (s,
CHCH3 of Ala); 43.5 (s, CH2 of benzyl), 127.2 (s, C-5 of
2-Cl±C6H4); 129.7 (C-4 of 2-Cl±C6H4); 129.9 (C-3 of 2-
Cl±C6H4); 130.1 (s, Cmeta of O2NC6H4), 130.6 (C-6 of 2-
Cl±C6H4); 134.3 (C-2 of 2-Cl±C6H4); 135.2 (C-1 of 2-
Cl±C6H4); 135.5 (s, Cortho of O2NC6H4), 145.4 (s, Cipso
of O2NC6H4), 150.8 (s, Cpara of O2NC6H4), 177.7 (s,
CO2H). C16H15ClN2O4S (366.83); calcd.: C, 52.39; H,
4.12; N, 7.64; found: C, 52.50; H, 4.42; N, 7.51%.
3
CHCH3 of Ala), 2.64 (s, 3H, CH3C6H4), 3.79 (s, 2H,
CH2 of benzyl); 3.90 (q, 1H, CH of Ala); 7.11±7.62 (m,
6H, Hortho of CH3C6H4 and Harom of 2-Cl-C6H4), 7.97
3
(d, JHH=8.1, 2H, Hmeta of CH3C6H4); 8.23 (br s, 2H,
NHCONH); 8.78 (br s, 1H, NHOH); 10.57 (br s, 1H,
NHOH); 13C NMR (DMSO-d6), d, ppm: 20.5 (s,
CHCH3 of Ala); 26.3 (s, CH3C6H4), 34.7 (s, CHCH3 of

644
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. 8 (2000) 637±645
N-4-Nitrophenylsulfenyl-N-2-chlorobenzyl-L-alanine hy-
droxamate H1. Yellow crystals, mp 214±215 ꢀC; 1H
NMR (DMSO-d6), d, ppm: 1.55 (d, 3JHH=6.5, 3H, CH3
of Ala), 3.75 (s, 2H, CH2 of benzyl); 3.99 (q, 1H, CH of
Ala); 6.76 (s, 1H, SNH), 7.11±7.66 (m, 6H, Hortho of
O2NC6H4 and Harom of 2-Cl±C6H4), 8.17 (d, 3JHH=8.2,
2H, Hmeta of O2NC6H4); 8.75 (br s, 1H, NHOH); 10.68
(br s, 1H, NHOH); 13C NMR (DMSO-d6), d, ppm:, 20.1
(s, CHCH3 of Ala); 34.5 (s, CHCH3 of Ala); 43.6 (s,
CH2 of benzyl), 127.1 (s, C-5 of 2-Cl±C6H4); 129.7 (C-4
of 2-Cl±C6H4); 129.9 (C-3 of 2-Cl±C6H4); 130.2 (s, Cmeta
of O2NC6H4), 130.7 (C-6 of 2-Cl±C6H4); 134.5 (C-2 of
2-Cl±C6H4); 135.2 (C-1 of 2-Cl±C6H4); 135.9 (s, Cortho
of O2NC6H4), 145.6 (s, Cipso of O2NC6H4), 150.5 (s,
Cpara of O2NC6H4), 174.7 (s, CONHOH). C16H16
ClN3O4S (381.84); calcd.: C, 50.33; H, 4.22; N, 11.00;
found: C, 50.45; H, 4.21; N, 10.93%.
6. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
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Collagenase type VII (highly puri®ed) and FALGPA
were purchased from Sigma-Aldrich (Milano, Italy);
their concentrations were determined from the absor-
bance at 280 nM and the extinction coecients furn-
ished 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 deter-
mined 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.30
The substrate was reconstituted as 5 mM stock in
50 mM Tricine bu€er, 0.4 M NaCl, 10 mM CaCl2, pH
7.50. The rate of hydrolysis was determined from the
change in absorbance at 324 nM using an extinction
1
coecient for FALGPA E305=24,700 L mol 1 cm in
the above-mentioned reaction bu€er.30 Measurements
were made using a Perkin±Elmer spectrophotometer
interfaced with a PC. Initial velocities were estimated
using the direct linear plot-based procedure reported by
van Wart and Steinbrink.30 KI-s were then determined
according to Dixon, using a linear regression program.
The KI values determined are the means of at least three
determinations.
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Acknowledgement
This research was ®nanced in part by the EU grant ERB
CIPDCT 940051.
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