Protease inhibitors - Part 5. Alkyl/arylsulfonyl- and arylsulfonylureido-/arylureido- glycine hydroxamate inhibitors of Clostridium histolyticum collagenase

Article abstract of DOI:10.1016/S0223-5234(00)00127-6

Reaction of alkyl/arylsulfonyl halides with glycine afforded a series of derivatives which were first N-benzylated by treatment with benzyl chloride, and then converted to the corresponding hydroxamic acids with hydroxylamine in the presence of carbodiimide derivatives. Other derivatives were obtained by reaction of N-benzyl-glycine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by conversion of their COOH group into the CONHOH moiety, as mentioned above. The 90 new compounds reported here were assayed as inhibitors of the Clostridium histolyticum collagenase (EC 3.4.24.3), a zinc enzyme which degrades triple helical regions of native collagen. The prepared hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized hydroxamates, substitution patterns leading to the best inhibitors were those involving perfluoroalkylsulfonyl- and substituted- arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4- carboxyphenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl or 1- and 2-naphthyl among others. Thus, it seems that similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, Clostridium histolyticum collagenase inhibitors should incorporate hydrophobic moieties at the P₁, and P₂, sites, whereas the α-carbon substituent may be a small and compact moiety (such as H. for the Gly derivatives reported here). Such compounds might lead to the design of collagenase inhibitor-based drugs useful as anti-cancer, anti-arthritis or anti-bacterial agents for the treatment of corneal keratitis. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00127-6

Eur. J. Med. Chem. 35 (2000) 299−307
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001276/FLA
299
Original article
Protease inhibitors – Part 5.
Alkyl/arylsulfonyl- and arylsulfonylureido-/arylureido- glycine hydroxamate
inhibitors of Clostridium histolyticum collagenase
Andrea Scozzafava, Claudiu T. Supuran*
Università degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received 3 June 1999; revised 14 September 1999, accepted 14 September 1999
Abstract – Reaction of alkyl/arylsulfonyl halides with glycine afforded a series of derivatives which were first N-benzylated by treatment with
benzyl chloride, and then converted to the corresponding hydroxamic acids with hydroxylamine in the presence of carbodiimide derivatives.
Other derivatives were obtained by reaction of N-benzyl-glycine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate,
followed by conversion of their COOH group into the CONHOH moiety, as mentioned above. The 90 new compounds reported here were
assayed as inhibitors of the Clostridium histolyticum collagenase (EC 3.4.24.3), a zinc enzyme which degrades triple helical regions of native
collagen. The prepared hydroxamate derivatives were generally 100–500 times more active than the corresponding carboxylates. In the series
of synthesized hydroxamates, substitution patterns leading to the best inhibitors were those involving perfluoroalkylsulfonyl- and
substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-carboxyphenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl or 1-
and 2-naphthyl among others. Thus, it seems that similarly to the matrix metalloproteinase (MMP) hydroxamate inhibitors, Clostridium
histolyticum collagenase inhibitors should incorporate hydrophobic moieties at the P1≠ and P2≠ sites, whereas the α-carbon substituent may
be a small and compact moiety (such as H, for the Gly derivatives reported here). Such compounds might lead to the design of collagenase
inhibitor-based drugs useful as anti-cancer, anti-arthritis or anti-bacterial agents for the treatment of corneal keratitis. © 2000 Éditions
scientifiques et médicales Elsevier SAS
collagenase / Clostridium histolyticum / glycine hydroxamate / zinc metalloproteinase / sulfonamide
1. Introduction
growth of Gram-negative bacteria [12–14]. Other com-
pounds of this type were reported to act as anti-HIV
agents in vitro [15]. But the most important applications
in drug design of the amino acid/oligopeptide hydroxam-
ates is related to their use for the development of MMP
inhibitors as anti-cancer [16–20] or anti-arthritis [2,
21–23] drugs. The 20 MMP’s presently known [2, 24] are
involved in tissue remodelling connected with tumour
invasion and joint destruction [1, 2, 17–21]. Synthetic
high affinity inhibitors for some of these enzymes, such
as the four vertebrate collagenases (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 [1, 2,
20–23]. The same situation is not true for the inhibitors of
other collagenases, such as the enzyme isolated from
Clostridium histolyticum [25–27], which were much less
Amino acid and oligopeptide hydroxamates were re-
ported to act as powerful inhibitors for a large number of
metallo-enzymes important as targets for the drug design,
such as the matrix metalloproteinases, MMP’s [1–4],
thermolysin and ellastase [5], leucine aminopeptidase [6],
carboxypeptidase A [7], leukotriene A4 hydrolase [8],
angiotensin I-converting enzyme [9], neurotensin-
degrading enzymes [10], endothelin-converting enzyme
[11], or UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglu-
cosamine deacetylase [12–14] among others. Some of
these derivatives possess strong antibacterial activity,
since by inhibiting some of the above mentioned enzymes
they interfere with lipid A biosynthesis and inhibit the
*Correspondence and reprints: cts@bio.chim.unifi.it

300
investigated. This collagenase (EC 3.4.24.3) is a 116 kDa
protein belonging to the M31 metalloproteinase family
[24], which is able to hydrolyse triple helical regions of
collagen under physiological conditions, as well as an
entire range of synthetic peptide substrates [25–27]. In
fact, the crude homogenate of Clostridium histolyticum,
which contains several distinct collagenase isozymes, is
the most efficient system known for the degradation of
connective tissue, [26, 27], being also involved in the
pathogenicity of this and related clostridia, such as C.
perfringens, which causes human gas gangrene and food
poisoning among others [28].
Similarly to the vertebrate MMP’s [2, 24], Clostridium
histolyticum collagenase (abbreviated as ChC) has the
conserved HExxH zinc-binding motif, which in this
specific 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 [29, 30] (in the case of
the vertebrate MMP’s the zinc ion is co-ordinated by
three histidines, His 218, His 222 and His 228), and a
water molecule/hydroxide ion acting as a nucleophile in
the hydrolytic scission [1–4]. Similarly to the MMP’s,
ChC is also a multiunit protein, consisting of four
segments, S1, S2a, S2b and S3 [30], with S1 incorporat-
ing the catalytic domain. Although the two types of
collagenases mentioned above (the MMP’s type and the
bacterial ChC) are relatively different, it is generally
considered that their mechanism of action for the hy-
drolysis of proteins and synthetic substrates is quite
similar [1–4, 24, 29, 30]. Thus, we hypothesized that
amino acid hydroxamates and some of their derivatives
which strongly inhibit MMP’s (collagenases, gelatinases,
etc.) would also act as potent ChC inhibitors. Our interest
in this type of enzyme inhibitor is related to the devel-
opment of pharmacological agents for the treatment of
bacterial corneal keratitis [31], a condition leading to
serious complications for which efficient cures are diffi-
cult to envisage [32]. It was in fact recently reported [31]
that collagen shields applied to the cornea of patients with
bacterial keratitis degrade rapidly, due to the collagenases
secreted by the pathogen bacterial species, but these
shields also protect to some extent the corneal collagen
degradation and thus the ocular surface [31, 33–35]. The
use of such a shield impregnated with an antibiotic agent
specific for the collagen-degrading bacteria would thus
have a double benefit for the patient: i) the collagenase
inhibitor would kill (or impair the growth of) bacteria
present on the cornea, improving and accelerating healing
of the keratitis; ii) the protective collagen shield would
possess an augmented stability, as its degradation by the
secreted collagenases would be delayed, promoting/
accelerating in this way the healing of the wound.
Figure 1. Synthesis of compounds A1–A33 and B1–B33.
In this paper we report the preparation of a series of
ChC inhibitors incorporating alkyl/arylsulfonamido-
glycine hydroxamate as well as arylsulfonylureido-/
arylureido-glycine hydroxamate moieties in their mol-
ecule. Some of the new compounds, assayed for the
inhibition of purified ChC, showed nanomolar affinity for
the enzyme, behaving as the most potent ChC inhibitors
reported up to now.
2. Chemistry
Compounds A1–A33 were prepared by reaction of
alkyl/arylsulfonyl chlorides 1 with glycine 2, followed by
treatment with benzyl chloride, as shown in figure 1 and
table I. Conversion of the carboxylic acids A1–A33 into
the corresponding hydroxamates B1–B33 was done with
hydroxylamine and diisopropyl carbodiimide (figure 1).
Another series of derivatives (C1–C4, D1–D4 and
E1–E6, F1–F6) was obtained by reaction of arylsulfo-
nylisocyanates 4 or aryl isocyanates 6 with N-benzyl-
glycine 5, followed by the conversion of the COOH
moiety into the CONHOH one, as described above
(figures 2 and 3).
Finally, 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 (figure 4).
3. Pharmacology
Inhibition data against highly purified ChC type VII,
with the newly prepared carboxylic and hydroxamic
acids, are shown in tables I and II. The chromogenic
substrate FALGPA (2-furanacryloyl-L-Leu-Gly-L-Pro-L-
Ala) was used in the assay, with the spectrophotometric

301
Table I. Inhibition of ChC with the carboxylic acids A1–A33 and
the corresponding hydroxamates B1–B33.
R
Compound Kia Compound Kia
(µM)
(nM)
CH3
CF3
CCl3
n-C4F9-
A1
A2
A3
A4
25 B1
112
52
51
13
9
5
6
3
2
B2
B3
B4
B5
n-C8F17
A5
Me2N-
C6H5-
PhCH2-
A6
A7
A8
A9
47 B6
24 B7
19 B8
15 B9
14 B10
14 B11
13 B12
17 B13
94
54
48
51
45
39
30
44
16
15
24
12
13
10
9
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-
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
6
5
7
3
4
3
2
B14
B15
B16
B17
B18
B19
B20
Figure 2. Synthesis of compounds C1–C4 and D1–D4.
Analogously, the S1', S2' and S3' subsites are also occu-
pied by Gly, Pro and Xaa, respectively [36]. Thus, many
of the reported inhibitors of ChC are aldehyde or ketone-
type substrate analogues, such as Pro6-Gly-L-Pro-GlyH
(GlyH = glycine aldehyde), with a KI of 340 µM [36];
phosphoric and phosphonic amides, such as iso-
amylphosphonyl-Gly-L-Pro-L-Ala (KI of 16 µM) [37];
thiols such as HS-CH2CH2CO-Pro-Xaa (KI of the best
compounds around 0.2 µM) [38–40]; phosphonamide
peptides of the type p-nitrophenethyl-PO(OH)-Gly-Pro-
Xaa (KI of the best compound, with Xaa = 2-amino-
hexanoic acid was of 5 nM) [41, 42]. As shown from the
0.7 B21
0.7 B22
6
7
21
27
15
8
3-CF3-C6H4
2,5-Cl2C6H3
4-CH3O-C6H4-
2,4,6-(CH3)3-C6H2-
5
7
7
3
4
5
5
4
4
5
4
B23
B24
B25
B26
B27
B28
B29
B30
B31
B32
B33
4-CH3O-3-BocNH-C6H3- A26
2-HO-3,5-Cl2-C6H2-
3-HOOC-C6H4-
4-HOOC-C6H4-
1-Naphthyl
2-Naphthyl
5-Me2N-1-naphthyl-
2-thienyl
A27
A28
A29
A30
A31
A32
A33
9
b
b
11
12
13
16
aKI values were obtained from Dixon plots using a linear regres-
sion program [51], from at least three different assays. bThe
C6H4–COOH moiety transformed into C6H4–CONHOH.
method of van Wart and Steinbrink [25]. Inhibition data
with some standard ChC inhibitors reported in the litera-
ture are also provided for comparison in table III.
4. Results and discussion
Few ChC (or for other bacterial collagenases) inhibi-
tors were reported up to now in the literature [36–42]. As
the ChC enzyme catalyses 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, Pro and Xaa, respectively [36].
Figure 3. Synthesis of compounds E1–E6 and F1–F6.

302
Table III. Inhibition of ChC with standard inhibitors.
Compound
Kia (µM)
Pro6-Gly-Pro-GlyH
340
16
0.005
i-C5H11PO(OH)GlyProAla
p-O2NC6H4CH2CH2PO(OH)GlyPro-2AXb
aKI values were obtained from Dixon plots using a linear regres-
sion program [51], from at least three different assays. b2AX =
2-aminohexanoic acid.
droxamic acids of type 7 recently reported by Parker’s
group [21], we decided to use such derivatives as lead
molecules. In the above mentioned study it was observed
that best inhibitors of type 7 (against mouse macrophage
metallo-elastase) were those incorporating: i) Gly, Ala or
Val as spacers between the zinc-binding function (the
hydroxamic acid moiety) and the P1' site; ii) benzyl or
isobutyl moieties at S2', and iii) arylsulfonyl moieties at
S3' [21] (figure 5). It should be noted that a relatively
small number of arylsulfonyl moieties were investigated
in the above-mentioned study [21], the greatest number
of the synthesized inhibitors 7 containing the 4-methoxy-
benzenesulfonyl moiety. The proposed interaction sites
between an inhibitor of type 7 and the active site of the
enzyme is shown schematically in figure 5.
Taking into account the above-mentioned findings that
led to strong mouse macrophage metallo-elastase inhibi-
tors [21], 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 hydroxamic acid type) [1, 2]; ii) a small (com-
pact) group in α to it [1, 21]; iii) the already optimized
[21] benzyl group at the S2' site, and iv) variable
alkyl/arylsulfonyl-, arylsulfonyl-ureido/arylureido-, or ar-
ylsulfenyl moieties at S3'.
Figure 4. Structures of compounds G1–G3, H1–H3, I1 and
J1.
above data, either the obtained inhibitors are relatively
weak, or the high affinity ones are phosphorus based
ligands which are not suitable for the development of
pharmaceutical agents, due to their high toxicity.
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 MMP
inhibitory properties of some arylsulfonyl-glycine hy-
Table II. Inhibition of ChC with the carboxylic acids of types C,
E, G and I and the corresponding hydroxamates of types D, F, H
and J.
R
Compound
Kia
(µM)
Compound
Kia
(nM)
The new compounds reported here were obtained by
unexceptional synthetic procedures, as outlined in figures
4-F-C6H4-
C1
C2
C3
C4
E1
E2
E3
E4
E5
E6
G1
G2
G3
I1
10
7
9
10
12
10
9
8
6
5
8
D1
D2
D3
D4
F1
F2
F3
F4
F5
F6
H1
H2
H3
21
15
16
17
36
39
34
30
12
13
17
15
10
6
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-
—
6
7
0.7 J1
aKI values were obtained from Dixon plots using a linear regres-
sion program [51], from at least three different assays.
Figure 5. Interaction sites between inhibitor 7 and the enzyme.

303
1–3. These involved reaction of glycine or N-benzyl-
glycine with alkyl/arylsulfonyl chlorides [43], arylisocy-
anates [44], arylsulfonyl-isocyanates [45] 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 and H, as
well as to the thioureas I and J.
The following should be noted regarding the ChC
inhibition data of tables I–III with the new compounds
and standard inhibitors: i) all hydroxamates were
100–500 times more active as ChC inhibitors as com-
pared to the corresponding carboxylic acids, probably
due to the enhanced Zn(II) co-ordinating properties of the
CONHOH moiety (bidentate binding) as compared to the
COOH group (generally monodentate binding to the zinc
ion) [1–3]; ii) potent inhibitors were obtained from all
classes of hydroxamates investigated here, such as the
alkyl/arylsulfonyl-benzyl-Gly derivatives (B5, B20–B22,
B26, B27, B29, etc.), the arylsulfonylureas- and arylureas
(such as D2, F5 and F6), the sulfenamido-benzyl-Gly
derivatives (such as H2 and H3) or the thiourea J1. Thus,
it seems that the S2'-binding moiety of the arylsulfona-
mide type, previously investigated for obtaining MMP
inhibitors of type 7 [21], can be efficiently substituted by
related moieties such as alkylsulfonyl-, 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 (1–33)) best ChC inhibitory properties were corre-
lated with the presence of perfluoroalkylsulfonyl- (B4,
B5), perfluorophenylsulfonyl- (B21), 3-trifluoromethyl-
phenylsulfonyl- (B22), 3-chloro-4-nitro-phenylsulfonyl-
(B17), 3- or 4-protected-amino-phenylsulfonyl- (B18–
B20 and B26), 3- or 4-carboxy-phenylsulfonyl- (B28 and
B29) and 1- or 2-naphthylsulfonyl moieties (B30–B32).
All these derivatives possessed inhibition constants in the
range of 6–13 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 affinities in the 15–30 nM range (table I). The least
active sulfonamides were those containing methyl-,
trihalomethyl-, dimethylamino-, phenyl- and benzyl moi-
eties (table I); iv) the arylsulfonylureido compounds
D1–D4 were more active than the corresponding arylsul-
fonyl derivatives (compare for instance D1 with B9; D2
and B10, etc), acting as strong, medium potency ChC
inhibitors. Type F ureas behaved similarly, and the
sulfenamides of type H, except for F5 and F6, as well as
Figure 6. Batimastat 8.
H2 and H3, which are strong inhibitors. A very potent
inhibitor is the thiourea derivative J1 (table II). By
comparing the data of tables I–III, 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 affinities in the micromolar range
(except for the phosphonic acid derivative mentioned in
table III, which possessed an affinity of the same order of
magnitude as that of our compounds).
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 con-
tributions in the field of the MMP’s have been registered
recently, and this might be useful in interpreting our
inhibition data. Thus, Bode’s and Tschesche’s groups
reported [3, 4, 47–49] several X-ray crystallographic
studies for the interaction of some types of hydroxamate
inhibitors with the catalytic domain of MMP-8, a colla-
genase from vertebrates, inhibited among others with a KI
of 10 nM by the collagenase inhibitor in clinical study,
batimastat 8 [50] (figure 6).
These studies [3, 4, 47–49] showed that batimastat is
bidentately co-ordinated 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 en-
zyme, forming hydrogen bonds with Glu 198 and Ala 161
[47]. The hydrophobic residues in the P1' (isobutyl) and
P2' (benzyl) positions are also critical for the formation of
a strong E-I adduct: thus, the leucine side chain of P1'
extends into the S1' pocket, making hydrophobic contacts
with several amino acid residues such as His 197, Pro 217
and Val 194, whereas the phenyl ring of P2' interacts with
the side chains of Ile 159, Val 129 and Pro 217. Although
the class collagenase III inhibitors (in the classification of
Babine and Bender [1]) of the sulfonamide type, to which
the compounds reported here presumably belong, were
much less investigated crystallographically, it is assumed

304
6. Experimental section
6.1. Chemistry
Melting points: heating plate microscope (not cor-
rected); IR spectra: KBr pellets, 400–4 000 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 (± 0.4% of the theoretical values, calculated for
the proposed formulas, data 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.
Figure 7. Schematic binding of the inhibitor B21 within the
active site of ChC.
Merck). Preparative HPLC was performed on
a
that the general binding mode illustrated above is also
valid for them, although some differences were also
evidenced [1, 21]. Based on these observations we
propose a similar binding mode of the sulfonamide
inhibitors reported here to ChC, as shown schematically
in figure 7.
The inhibitor probably co-ordinates bidentately to the
Zn(II) ion of ChC, whereas the hydrophobic moieties
from the P2' and P3' sites (benzyl and pentafluorophenyl,
respectively) participate in hydrophobic contacts which
assure the strong affinity of this inhibitor for ChC (the
inhibitor B21 for which the schematic binding is shown
above has a KI of 6 nM).
Dynamax-60A column (25 × 250 mm), with a Beckman
EM-1760 instrument. The detection wavelength was
254 nm.
Amino acids (Gly, N-benzyl-Gly), sulfonyl chlorides,
arylsulfonyl isocyanates, aryl isocyanates, benzoyl-
isothiocyanate, triethylamine, carbodiimides, hydroxyl-
amine, and other reagents used in the syntheses were
commercially available compounds (from Sigma, Acros
or Aldrich).
6.1.1. General procedure for the preparation
of alkyl/arylsulfonyl glycines 3
An amount of 1.50 g (20 mmol) of glycine 2 and
20 mmol of sulfonyl chloride 1 were suspended/dissolved
in 100 mL of acetone + 25 mL of water. The stoichiomet-
ric amount (20 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 solvent was evaporated,
the reaction mixture was re-taken in 100 mL of water and
the crude 3 extracted in ethyl acetate. After evaporation of
the solvent the products were recrystallized from EtOH or
MeOH. Yields were around 85–95%.
5. Conclusion
We describe here a novel class of strong inhibitors of
the zinc protease EC 3.4.24.3, a collagenase from
Clostridium histolyticum. 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 extracellular matrix in
vertebrates. Reaction of glycine or N-benzyl-glycine with
sulfonyl chlorides, arylsulfonyl isocyanate, aryl isocyan-
ates or benzoyl isothiocyanate afforded the glycine de-
rivatives which were subsequently converted into the
corresponding hydroxamates. These latter derivatives
were 100–500 times more inhibitory against ChC as
compared to the corresponding carboxylic acids. Best
substitutions for obtaining high affinity inhibitors in-
volved hydrophobic moieties at S3', such as
perfluoroalkylsulfonyl-, substituted-arylsulfonyl (penta-
fluoro-phenylsulfonyl, 3- and 4-carboxy-phenylsulfonyl-,
3-trifluoromethyl-phenylsulfonyl, or 1- and 2-naphthyl)
moieties among others. This is the first study reporting
nanomolar affinity ChC inhibitors of the sulfonamide
type.
6.1.2. General procedure for the preparation
of derivatives A1–A33
An amount of 10 mM of alkyl/arylsulfonyl-Gly 3 and
the stoichiometric amount of benzyl chloride were
suspended/dissolved in 50 mL of anhydrous acetonitrile
and the stoichiometric amount (10 mM) of triethyl amine
(10 mM, 1.47 mL) was added. The reaction mixture was
heated at reflux for 2 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 carboxylic acids A1–A33 extracted in ethyl
acetate. Recrystallization from methanol/water afforded
the pure title compounds in almost quantitative yield.

305
6.1.3. General procedure for the preparation
(s, CH2 of Gly), 42.7 (s, CH2 of benzyl), 130.1 (s, Cmeta
of CH3C6H4), 131.8 (s, Cpara of Ph), 133.6 (s, Cmeta of
Ph), 134.5 (s, Cortho of Ph), 135.0 (s, Cortho of CH3C6H4),
141.5 (s, Cipso of Ph), 145.0 (s, Cipso of CH3C6H4), 148.6
(s, Cpara of CH3C6H4), 177.3 (s, CO2H). Anal.
C16H17NO4S (C, H, N).
of compounds B1–B33, D1–D4, F1–F6, H1–H3 and J1
An amount of 5 mM of carboxylic acid derivative
A1–A33, C1–C4, E1–E6, G1–G3 or I1 was dissolved/
suspended in 50 mL of anhydrous acetonitrile or acetone,
and treated with 420 mg (6 mM) of hydroxylamine HCl
and 1.10 g (6 mM) of EDCI. HCl or di-isopropyl-
carbodiimide. The reaction mixture was magnetically
stirred at room temperature for 15 min, then 180 µL
(12 mM) of triethylamine 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
layers were dried over sodium sulfate and filtered, and the
solvent removed in vacuo. Preparative HPLC (Dynamax-
60A column (25 × 250 mm); 90% acetonitrile/10%
methanol; flow rate of 30 mL/min) afforded the pure
hydroxamic acids.
6.1.5.2. N-4-Toluenesulfonyl-N-benzyl-glycine
hydroxamate B13
1
White crystals, m.p. 233–235 °C; H-NMR (DMSO-
d6), δ, ppm: 2.62 (s, 3H, CH3C6H4), 3.71 (s, 2H, CH2 of
Gly), 3.77 (s, 2H, CH2 of benzyl); 7.24–7.66 (m, 7H,
H
ortho of CH3C6H4 and Harom of Ph), 8.04 (d, 3JHH = 8.1,
2H, Hmeta of CH3C6H4); 8.70 (br s, 1H, NHOH); 10.48
(br s, 1H, NHOH); 13C-NMR (DMSO-d6), δ, ppm: 26.5
(s, CH3C6H4), 40.6 (s, CH2 of Gly), 42.1 (s, CH2 of
benzyl), 130.7 (s, Cmeta of CH3C6H4), 131.8 (s, Cpara of
Ph), 133.5 (s, Cmeta of Ph), 134.8 (s, Cortho of Ph), 135.6
(s, Cortho of CH3C6H4), 141.5 (s, Cipso of Ph), 145.3 (s,
Cipso of CH3C6H4), 148.6 (s, Cpara of CH3C6H4), 174.0
(s, CONHOH). Anal. C16H18N2O4S (C, H, N).
6.1.4. General procedure for the preparation
of compounds C1–C4, E1–E6 and I1
An amount of 1.65 g (10 mmol) of N-benzyl-glycine 5
and the stoichiometric amount of arylsulfonyl isocyanate
4, aryl isocyanate 6 or benzoylisothiocyanate were sus-
pended in 50 mL of anhydrous acetonitrile and 150 µL
(10 mM) of triethylamine were added. The reaction
mixture was either stirred at room temperature (in the
case of derivatives prepared from 4) or refluxed (for the
other two types of derivatives) for 2–6 h. The solvent was
evaporated and the reaction mixture worked up as de-
scribed at 6.1.1. The new compounds were recrystallized
from ethanol. Yields were almost quantitative.
6.1.5.3. N-4-Toluenesulfonylureido-N-benzyl-glycine C3
White crystals, m.p. 217–218 °C; H-NMR (DMSO-
d6), δ, ppm: 2.60 (s, 3H, CH3C6H4), 3.67 (s, 2H, CH2 of
1
Gly), 3.75 (s, 2H, CH2 of benzyl); 7.29–7.58 (m, 7H,
H
ortho of CH3C6H4 and Harom of Ph), 7.99 (d, 3JHH = 8.1,
2H, Hmeta of CH3C6H4); 8.24 (br s, 2H, NHCONH);
11.76 (br s, 1H, COOH); 13C-NMR (DMSO-d6), δ, ppm:
26.1 (s, CH3C6H4), 40.8 (s, CH2 of Gly), 42.3 (s, CH2 of
benzyl), 130.9 (s, Cmeta of CH3C6H4), 131.5 (s, Cpara of
Ph), 132.4 (s, NHCONH), 133.7 (s, Cmeta of Ph), 134.6 (s,
C
ortho of Ph), 135.0 (s, Cortho of CH3C6H4), 141.5 (s, Cipso
of Ph), 145.0 (s, Cipso of CH3C6H4), 148.6 (s, Cpara of
CH3C6H4), 177.3 (s, CO2H). Anal. C17H19N3O5S (C, H,
N).
6.1.5. General procedure for the preparation
of compounds G1–G3
The general procedure described above for the prepa-
ration of compounds A1–A33 was followed, except that
N-benzyl-glycine 5 was used instead of glycine 2, and
arylsulfenyl halides instead of alkyl/arylsulfonyl halides.
The yields in the title sulfenamides were around 90%.
6.1.5.4. N-4-Toluenesulfonylureido-N-benzyl-glycine
hydroxamate D3
1
White crystals, m.p. 279–281 °C; H-NMR (DMSO-
1
d6), δ, ppm: 2.63 (s, 3H, CH3C6H4), 3.69 (s, 2H, CH2 of
Gly), 3.75 (s, 2H, CH2 of benzyl); 7.29–7.62 (m, 7H,
Hortho of CH3C6H4 and Harom of Ph), 7.99 (d, 3JHH = 8.1,
2H, Hmeta of CH3C6H4); 8.24 (br s, 2H, NHCONH); 8.76
(br s, 1H, NHOH); 10.54 (br s, 1H, NHOH); 13C-NMR
(DMSO-d6), δ, ppm: 26.3 (s, CH3C6H4), 40.9 (s, CH2 of
Gly), 42.2 (s, CH2 of benzyl), 130.7 (s, Cmeta of
CH3C6H4), 131.6 (s, Cpara of Ph), 132.4 (s, NHCONH),
133.9 (s, Cmeta of Ph), 134.7 (s, Cortho of Ph), 135.0 (s,
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.
6.1.5.1. N-4-Toluenesulfonyl-N-benzyl-glycine A13
1
White crystals, m.p. 192–194 °C; H-NMR (DMSO-
d6), δ, ppm: 2.50 (s, 3H, CH3C6H4), 3.62 (s, 2H, CH2 of
Gly), 3.75 (s, 2H, CH2 of benzyl); 7.22–7.59 (m, 7H,
H
ortho of CH3C6H4 and Harom of Ph), 7.97 (d, 3JHH = 8.1,
Cortho of CH3C6H4), 141.5 (s, Cipso of Ph), 145.0 (s, Cipso
2H, Hmeta of CH3C6H4); 11.61 (br s, 1H, COOH);
of CH3C6H4), 148.6 (s, Cpara of CH3C6H4), 174.6 (s,
CONHOH). Anal. C17H20N4O5S (C, H, N).
13C-NMR (DMSO-d6), δ, ppm: 26.0 (s, CH3C6H4), 40.4

306
6.1.5.5. N-4-Fluorophenylureido-N-benzyl-glycine E1
(s, Cipso of O2NC6H4), 174.6 (s, CONHOH). Anal.
C15H15N3O4S (C, H, N).
1
White crystals, m.p. 135–136 °C; H-NMR (DMSO-
d6), δ, ppm: 3.60 (s, 2H, CH2 of Gly), 3.73 (s, 2H, CH2
of benzyl); 7.21–7.54 (m, 7H, Hortho of FC6H4 and Harom
of Ph), 7.94 (d, 3JHH = 8.0, 2H, Hmeta of FC6H4); 8.05 (br
s, 2H, NHCONH); 11.44 (br s, 1H, COOH); 13C-NMR
(DMSO-d6), δ, ppm: 40.3 (s, CH2 of Gly), 42.6 (s, CH2
of benzyl), 130.1 (s, Cmeta of FC6H4), 131.6 (s, Cpara of
Ph), 132.0 (s, NHCONH), 133.9 (s, Cmeta of Ph), 134.5 (s,
Cortho of Ph), 135.2 (s, Cortho of FC6H4), 141.4 (s, Cipso of
Ph), 148.9 (s, Cipso of FC6H4), 149.7 (s, Cpara of FC6H4),
177.8 (s, CO2H). Anal. C16H15FN2O3 (C, H, N).
6.2. Pharmacology
Collagenase type VII (highly purified) and FALGPA
were purchased from Sigma Chemical Co. (St. Louis,
MO, USA); their concentrations were determined from
the absorbance at 280 nm and the extinction coefficients
furnished by the supplier. The activity of such prepara-
tions 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 (amido-
lytic) activity of the collagenase, at 23 °C, using FALGPA
as substrate, by the method of van Wart and Steinbrink
[25] 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 absorbance at 324 nm using an extinction
coefficient for FALGPA e305 = 24 700 L. mol–1. cm–1 in
the above-mentioned reaction buffer [25]. 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 [25]. KI’s were then determined according
to Dixon, using a linear regression program [51]. The KI
values determined are the means of at least three deter-
minations.
6.1.5.6. N-4-Fluorophenylureido-N-benzyl-glycine
hydroxamate F1
1
White crystals, m.p. 212–213 °C; H-NMR (DMSO-
d6), δ, ppm: 3.72 (s, 2H, CH2 of Gly), 3.79 (s, 2H, CH2
of benzyl); 7.23–7.60 (m, 7H, Hortho of FC6H4 and Harom
of Ph), 7.90 (d, 3JHH = 8.2, 2H, Hmeta of FC6H4); 8.04 (br
s, 2H, NHCONH); 8.71 (br s, 1H, NHOH); 10.59 (br s,
1H, NHOH); 13C-NMR (DMSO-d6), δ, ppm: 40.1 (s,
CH2 of Gly), 42.4 (s, CH2 of benzyl), 130.1 (s, Cmeta of
FC6H4), 131.3 (s, Cpara of Ph), 131.7 (s, NHCONH),
133.8 (s, Cmeta of Ph), 134.7 (s, Cortho of Ph), 135.5 (s,
Cortho of FC6H4), 141.7 (s, Cipso of Ph), 145.6 (s, Cipso of
FC6H4), 148.9 (s, Cpara of FC6H4), 174.8 (s, CONHOH).
Anal. C16H16FN3O3 (C, H, N).
6.1.5.7. N-4-Nitrophenylsulfenyl-N-benzyl-glycine G1
1
Yellow crystals, m.p. 175–177 °C; H-NMR (DMSO-
d6), δ, ppm: 3.61 (s, 2H, CH2 of Gly), 3.75 (s, 2H, CH2
Acknowledgements
of benzyl); 6.75 (s, 1H, SNH), 7.20–7.56 (m, 7H, Hortho
3
of O2NC6H4 and Harom of Ph), 8.05 (d, JHH = 8.3, 2H,
This research was financed in part by the EU grant
ERB CIPDCT 940051. Special thanks are addressed to
Prof. Osamu Matsushita (Kagawa Medical University,
Japan) for critically reading and correcting our manu-
script.
Hmeta of O2NC6H4); 11.68 (br s, 1H, COOH); 13C-NMR
(DMSO-d6), δ, ppm: 40.0 (s, CH2 of Gly), 42.8 (s, CH2
of benzyl), 130.3 (s, Cmeta of O2NC6H4), 131.8 (s, Cpara
of Ph), 133.7 (s, Cmeta of Ph), 134.8 (s, Cortho of Ph),
135.9 (s, Cortho of O2NC6H4), 141.2 (s, Cipso of Ph), 145.6
(s, Cipso of O2NC6H4), 150.4 (s, Cpara of O2NC6H4),
177.5 (s, CO2H). Anal. C15H14N2O4S (C, H, N).
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