Protease inhibitors. Part 7: Inhibition of Clostridium histolyticum collagenase with sulfonylated derivatives of L-valine hydroxamate

Article abstract of DOI:10.1016/S0928-0987(99)00090-1

Sulfonylated L-valine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with the title amino acid, followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group. Other derivatives were obtained by reaction of N-benzyl-L- valine with arylisocyanates, arylsulfonylisocyanates or benzoylisothiocyanate, followed by the similar conversion of the COOH into the CONHOH moiety, with hydroxylamine in the presence of carbodiimides. The obtained compounds were assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical collagen. The hydroxamate derivatives were generally 100-500 times more active than the corresponding carboxylates. In the series of synthesized derivatives, substitution patterns leading to best ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-; 3-trifluoromethylphenylsulfonyl; or 1- and 2- naphthyl among others. Similarly to the matrix metalloproteinase hydroxamate inhibitors, ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P(2') and P(3') subsites, in order to achieve tight binding to the enzyme. Such compounds might lead to drugs useful in the treatment corneal bacterial keratitis. (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S0928-0987(99)00090-1

European Journal of Pharmaceutical Sciences 10 (2000) 67–76
www.elsevier.nl/locate/ejps
Protease inhibitors. Part 7
Inhibition of Clostridium histolyticum collagenase with sulfonylated
derivatives of L-valine hydroxamate
*
Claudiu T. Supuran , Andrea Scozzafava
`
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received 6 August 1999; received in revised form 21 October 1999; accepted 23 November 1999
Abstract
Sulfonylated L-valine hydroxamate derivatives were obtained by reaction of alkyl/arylsulfonyl halides with the title amino acid,
followed by treatment with benzyl chloride, and conversion of the COOH moiety to the CONHOH group. Other derivatives were obtained
by reaction of N-benzyl-L-valine with arylisocyanates, arylsulfonylisocyanates or benzoylisothiocyanate, followed by the similar
conversion of the COOH into the CONHOH moiety, with hydroxylamine in the presence of carbodiimides. The obtained compounds were
assayed as inhibitors of the Clostridium histolyticum collagenase, ChC (EC 3.4.24.3), a zinc enzyme which degrades triple helical
collagen. The hydroxamate derivatives were generally 100–500 times more active than the corresponding carboxylates. In the series of
synthesized derivatives, substitution patterns leading to best ChC inhibitors were those involving perfluoroalkylsulfonyl- and substituted-
arylsulfonyl moieties, such as pentafluorophenylsulfonyl; 3- and 4-protected-aminophenylsulfonyl-; 3- and 4-carboxyphenylsulfonyl-;
3-trifluoromethylphenylsulfonyl; or 1- and 2-naphthyl among others. Similarly to the matrix metalloproteinase hydroxamate inhibitors,
ChC inhibitors of the type reported here must incorporate hydrophobic moieties at the P29 and P39 subsites, in order to achieve tight
binding to the enzyme. Such compounds might lead to drugs useful in the treatment corneal bacterial keratitis.
B.V. All rights reserved.
2000 Elsevier Science
Keywords: Collagenase; Clostridium histolyticum; L-Valine hydroxamate; Zinc metalloproteinase; Sulfonamide
1. Introduction
collagen degradation and thus the ocular tissue (Schiff et
al., 1992). Taking into account our interest in the develop-
ment of topically effective ophthalmologic drugs (Scoz-
zafava et al., 1999a,b; Supuran et al., 1999), we hypoth-
esize here that the use of a collagen shield impregnated
with an antibiotic agent specific for the collagen-degrading
bacteria would have a double benefit for the patients
affected by bacterial corneal keratitis: (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
acquire an augmented stability, as its degradation by the
secreted collagenases would be delayed, promoting/ac-
celerating in this way the healing of the wound.
Since amino acid and oligopeptide hydroxamates were
reported to act as powerful inhibitors for a large number of
metalloenzymes important as targets in the drug design
[such as the matrix metalloproteinases, MMPs (Babine and
Bender, 1997; Bottomley et al., 1998; Krumme et al.,
1998); thermolysin and elastase (Grobelny et al., 1992);
carboxypeptidase A (Kim and Jin, 1999); leukotriene A4
Bacterial resistance to antibiotics has become a major
medical problem, with the emergence of strains difficult to
control by the clinically available compounds and the
urgent need to develop novel antibacterial drugs (Bartoloni
et al., 1990; Onishi et al., 1996; Heinemann, 1999).
Among the different such conditions reported in the
literature, bacterial corneal keratitis represents a serious
case, leading to complications for which efficient cures are
difficult to envisage at the moment (Schiff et al., 1992;
Groos, 1997). It has been reported (Schiff et al., 1992) that
this infection is associated with a highly increased col-
lagenase activity in the ocular tissues. Furthermore, col-
lagen shields applied to the cornea of patients affected by
this disease degrade rapidly, due to the collagenases
secreted by the pathogen bacterial species, although such
shields were shown to protect to some extent the corneal
*Corresponding author. Fax: 139-055-275-7555.
E-mail address: cts@bio.chim.unifi.it (C.T. Supuran)
0928-0987/00/$ – see front matter
PII: S0928-0987(99)00090-1
2000 Elsevier Science B.V. All rights reserved.

68
C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
hydrolase (Hogg et al., 1995); angiotensin I-converting
enzyme (Harris et al., 1983); neurotensin-degrading en-
zymes (Bourdel et al., 1996); endothelin-converting en-
zyme (Bihovsky et al., 1995); or UDP-3-O-(R-3-hydroxy-
myristoyl)-N-acetylglucosamine deacetylase (Jackmann et
al., 1999) among others], we decided to investigate such
derivatives as possible antibacterial corneal keratitis
agents. Some of the above-mentioned derivatives were
shown to possess strong antibacterial activity since by
inhibiting some of the above mentioned enzymes, they
interfere with lipid A biosynthesis and inhibit the growth
of Gram-negative bacteria (Onishi et al., 1996). 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 anticancer (Woj-
towicz-Praga et al., 1996; Johnson et al., 1998; Levy et al.,
1998; Lozonschi et al., 1999) or antiarthritis (Bottomley et
al., 1998; Jeng et al., 1998; Groneberg et al., 1999) drugs.
The 23 MMPs presently known (Rawlings and Barrett,
1995; Bottomley et al., 1998) are involved in tissue
remodeling connected with tumor invasion and joint
destruction (Bottomley et al., 1998; Jeng et al., 1998;
Groneberg et al., 1999). Synthetic high affinity inhibitors
for some of these enzymes, such as the four vertebrate
collagenases (MMPs 1, 8, 13 and 18), stromelysins 1 and 2
(MMPs 3 and 10, respectively) or the gelatinases A and B
(MMPs 2 and 9) were much investigated in the last period,
in order to develop novel pharmacological agents of the
hydroxamate type (Bottomley et al., 1998; Graf von
Roedern et al., 1998; Jeng et al., 1998; Yamamoto et al.,
1998; Groneberg et al., 1999). The same situation is not
true for the inhibitors of bacterial collagenases, such as, for
instance, the enzyme isolated from Clostridium his-
tolyticum (Bond and Van Wart, 1984a,b), which were much
less investigated. This collagenase (ChC; EC 3.4.24.3) has
recently been studied in detail by Matsushita’s group, who
showed that it is a 116 kDa protein belonging to the M31
metalloproteinase family (Rawlings and Barrett, 1995). It
is able to hydrolyze triple helical regions of collagen under
physiological conditions, as well as an entire range of
synthetic peptide substrates (Bond and Van Wart, 1984a,b;
Matsushita et al., 1998, 1999). In fact the crude homoge-
nate of C. histolyticum, which contains several distinct
collagenase isozymes (Matsushita et al., 1999), is the most
efficient system known for the degradation of connective
tissue (Bond and Van Wart, 1984a,b), being also involved
in the pathogenicity of this and related clostridia, such as
Clostridium perfringens, which causes human gas gan-
grene and food poisoning among others (Rood, 1998).
Thus, we hypothesized that amino acid hydroxamates
and some of their derivatives which strongly inhibit MMPs
(collagenases, gelatinases, stromelysins, etc) would also act
as potent ChC inhibitors. In this paper we report the
preparation of a series of ChC inhibitors incorporating
alkyl/arylsulfonamido-L-valine hydroxamate as well as
arylsulfonylureido-/arylureido-L-valine hydroxamate moi-
eties in their molecule. Some of the new compounds,
assayed for the inhibition of purified ChC, showed high
affinity for the enzyme (in the nanomolar range), behaving
as some of the best ChC inhibitors reported up to now. It is
thus quite probable that compounds of the type reported in
this paper also inhibit the collagenase activity of bacterial
species involved in corneal bacterial keratitis, but the
results of the in vivo experiments will be reported else-
where.
2. Materials and methods
Melting points, heating plate microscope (not corrected);
1H-NMR spectra, Varian 300CXP apparatus (chemical
shifts are expressed as d values relative to Me4Si as
standard); elemental analysis (60.4% of the theoretical
values, calculated for the proposed formulas), 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 (Merck).
Preparative HPLC was performed on a Dynamax-60A
column (253250 mm), with a Beckman EM-1760 instru-
ment. The detection wavelength was 254 nm.
Amino acids (L-valine), sulfonyl chlorides, arylsul-
fonylisocyanates, arylisocyanates, benzoylisothiocyanate,
triethylamine, carbodiimides, hydroxylamine, and other
reagents/solvents used in the syntheses were commercially
available compounds (from Sigma, Acros or Aldrich).
N-Benzyl-valine was prepared by benzylation of valine
with benzyl chloride.
2.1. Chemistry
2.1.1. General procedure for the preparation of alkyl/
arylsulfonyl valines 3
An amount of 2.16 g (20 mM) of L-valine 2 and 20 mM
of sulfonyl chloride 1 were suspended/dissolved in 100 ml
of acetone130 ml of water. The stoichiometric amount (20
mM) of base (NaHCO3; KHCO3, NaOH or Et3N) dis-
solved 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 retaken 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 71–83% (Scheme 1).
2.1.2. General procedure for the preparation of
derivatives A1–A33
An amount of 10 mM of alkyl/arylsulfonyl-L-valine 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

C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
69
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 (253250 mm); 90% acetonitrile–10% metha-
nol; flow-rate of 30 ml/min] afforded the pure hydroxamic
acids (Schemes 2–4).
2.1.4. General procedure for the preparation of
compounds C1–C4; E1–E6 and I1
An amount of 2.14 g (10 mM) of N-benzyl-valine 5 and
the stoichiometric amount of arylsulfonylisocyanate 4;
arylisocyanate 6 or benzoyisothiocyanate were suspended
in 50 ml of anhydrous acetone and 150 ml (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 described above. The new
compounds were recrystallized from ethanol. Yields were
almost quantitative (Schemes 2–4).
Scheme 1.
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 (Scheme 1).
2.1.3. General procedure for the preparation of
2.1.5. General procedure for the preparation of
compounds G1–G3
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
The general procedure described above for the prepara-
tion of compounds A1–A33 has been followed, except that
N-benzyl-valine 5 was used instead of valine 2, and
arylsulfenyl halides instead of alkyl/arylsulfonyl halides.
The yields in the title sulfenamides were around 70%
(Scheme 4).
and 1.10
g
(6 mM) of EDCI?HCl or diiso-
propylcarbodiimide. The reaction mixture was magnetical-
ly stirred at room temperature for 15 min, then 180 ml (12
mM) of triethylamine were added and stirring was con-
tinued for 12 h at 48C. The solvent was evaporated in
The new compounds were characterized by 1H- and
13C-NMR spectroscopy and elemental analysis. Data for a
representative compound of each series is provided below.
Scheme 2.

70
C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
Scheme 3.
N-4-Toluene-sulfonyl-N-benzyl-L-valine A13, white
134.4 (s, Cortho of Ph), 135.7 (s, Cortho of CH3C6H4), 141.8
]
]
crystals, m.p. 165–68C; 1H-NMR (dimethyl sulfoxide
(s, Cipso of Ph), 145.5 (s, Cipso of CH3C6H4), 148.8 (s,
]
]
[DMSO]-d6), d, ppm: 1.13 (d, 3JHH56.7, 6H, CH(CH3)2 of
Cpara of CH3C6H4), 175.1 (s, CO2H). Anal. found: C,
]
]
]
62.98; H, 6.59; N, 3.76%; C19H23NO4S requires: C, 63.14;
H, 6.41; N, 3.87%.
valine), 2.23–2.50 (m, 1H, CH(CH3)2 of valine), 2.53 (s,
]
3H, CH3C6H4), 3.75 (s, 2H, CH2 of benzyl); 3.74 (d,
]
]
3JHH54.5, 1H, NHCH of valine); 7.22–7.59 (m, 7H, Hortho
N-4-Toluene-sulfonyl-N-benzyl-L-valine hydroxamate
]
]
1
3
B13, white crystals, m.p. 212–38C; H-NMR (DMSO-d6),
of CH3C6H4 and Harom of Ph), 7.97 (d, JHH58.1, 2H,
]
d, ppm: 1.12 (d, 3JHH56.7, 6H, CH(CH3)2 of valine),
Hmeta of CH3C6H4); 11.61 (br s, 1H, COOH); 13C-NMR
]
]
2.23–2.55 (m, 1H, CH(CH3)2 of valine), 2.62 (s, 3H,
(DMSO-d6), d, ppm: 17.8 (s, CH(CH3)2 of valine); 26.1 (s,
]
]
3
CH3C6H4), 30.3 (s, CH(CH3)2 of valine); 42.1 (s, CH2 of
CH3C6H4), 3.79 (s, 2H, CH2 of benzyl); 3.77 (d, JHH5
]
]
]
]
]
]
]
benzyl), 61.5 (s, NCH of valine); 130.2 (s, Cmeta of
4.4, 1H, NCH of valine); 7.24–7.60 (m, 7H, Hortho of
]
]
CH3C6H4), 131.9 (s, Cpara of Ph), 133.4 (s, Cmeta of Ph),
CH3C6H4 and Harom of Ph), 8.02 (d, 3JHH58.1, 2H, Hmeta
]
]
]
]
Scheme 4. Structures G1–G3; H1–H3 and I1, J1.

C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
71
of CH3C6H4); 8.70 (br s, 1H, NHOH); 10.49 (br s, 1H,
Cmeta of FC6H4), 131.8 (s, Cpara of Ph), 132.3 (s,
]
]
]
NHOH); 13C-NMR (DMSO-d6), d, ppm: 17.7 (s,
NHCONH), 133.9 (s, Cmeta of Ph), 134.6 (s, Cortho of Ph),
]
]
]
]
135.1 (s, Cortho of FC6H4), 141.9 (s, Cipso of Ph), 148.5 (s,
CH(CH3)2 of valine); 26.5 (s, CH3C6H4), 30.1 (s,
]
]
]
]
Cipso of FC6H4), 149.5 (s, Cpara of FC6H4), 175.0 (s,
CO2H). Anal. found: C, 66.03; H, 6.39; N, 8.07%;
C19H21FN2O3 requires: C, 66.27; H, 6.15; N, 8.13%.
CH(CH3)2 of valine); 42.1 (s, CH2 of benzyl), 61.5 (s,
]
]
]
]
NHCH of valine); 130.7 (s, Cmeta of CH3C6H4), 131.8 (s,
]
]
]
Cpara of Ph), 133.5 (s, Cmeta of Ph), 134.8 (s, Cortho of Ph),
]
]
]
135.3 (s, Cortho of CH3C6H4), 141.6 (s, Cipso of Ph), 145.2
N-4-Fluorophenylureido-N-benzyl-L-valine hydroxamate
]
]
1
(s, Cipso of CH3C6H4), 148.1 (s, Cpara of CH3C6H4), 174.6
F1, white crystals, m.p. 215–78C; H-NMR (DMSO-d6),
]
]
]
d, ppm: 1.11 (d, 3JHH56.7, 6H, CH(CH3)2 of valine),
(s, CONHOH). Anal. found: C, 60.75; H, 6.52; N, 7.32%;
]
C19H24N2O4S requires: C, 60.62; H, 6.43; N, 7.44%.
2.20–2.51 (m, 1H, CH(CH3)2 of valine), 3.71 (s, 2H, CH2
]
]
of benzyl); 3.80 (d, 3JHH54.5, 1H, NCH of valine); 7.19–
N-4-Toluene-sulfonylureido-N-benzyl-L-valine
C3,
]
1
white crystals, m.p. 188–908C; H-NMR (DMSO-d6), d,
7.61 (m, 7H, Hortho of FC6H4 and Harom of Ph), 7.90 (d,
]
]
ppm: 1.15 (d, 3JHH56.7, 6H, CH(CH3)2 of valine), 2.22–
3JHH58.1, 2H, Hmeta of FC6H4); 8.07 (br s, 2H,
]
]
2.51 (m, 1H, CH(CH3)2 of valine), 2.60 (s, 3H,
NHCONH); 8.75 (br s, 1H, NHOH); 10.63 (br s, 1H,
]
]
3
NHOH); 13C-NMR (DMSO-d6), d, ppm: 17.6 (s,
CH3C6H4), 3.77 (s, 2H, CH2 of benzyl); 3.76 (d, JHH5
]
]
]
4.5, 1H, NCH of valine); 7.29–7.58 (m, 7H, Hortho of
CH(CH3)2 of valine); 30.4 (s, CH(CH3)2 of valine); 42.8
(s, CH2 of benzyl), 61.6 (s, NHCH of valine); 130.0 (s,
Cmeta of FC6H4), 131.5 (s, Cpara of Ph), 131.4 (s,
]
]
]
]
CH3C6H4 and Harom of Ph), 7.99 (d, 3JHH58.1, 2H, Hmeta
of CH3C6H4); 8.25 (br s, 2H, NHCONH); 11.72 (br s, 1H,
COOH); 13C-NMR (DMSO-d6), d, ppm: 17.8 (s,
CH(CH3)2 of valine); 26.4 (s, CH3C6H4), 30.4 (s,
CH(CH3)2 of valine); 42.3 (s, CH2 of benzyl), 61.5 (s,
NHCH of valine); 130.4 (s, Cmeta of CH3C6H4), 131.5 (s,
Cpara of Ph), 132.1 (s, NHCONH), 133.6 (s, Cmeta of Ph),
134.6 (s, Cortho of Ph), 135.0 (s, Cortho of CH3C6H4), 141.5
(s, Cipso of Ph), 145.4 (s, Cipso of CH3C6H4), 148.7 (s,
Cpara of CH3C6H4), 175.0 (s, CO2H). Anal. found: C,
]
]
]
]
]
]
NHCONH), 133.9 (s, Cmeta of Ph), 134.3 (s, Cortho of Ph),
]
]
]
135.5 (s, Cortho of FC6H4), 141.0 (s, Cipso of Ph), 145.8 (s,
]
]
]
]
Cipso of FC6H4), 148.4 (s, Cpara of FC6H4), 174.1 (s,
CONHOH). Anal. found: C, 63.80; H, 6.12; N, 11.62%;
C19H22FN3O3 requires: C, 63.50; H, 6.17; N, 11.69%.
N-4-Nitrophenylsulfenyl-N-benzyl-L-valine G1, yellow
crystals, m.p. 169–708C; 1H-NMR (DMSO-d6), d, ppm:
]
]
]
]
]
]
]
]
]
]
]
]
]
]
3
1.14 (d, JHH56.7, 6H, CH(CH3)2 of valine), 2.23–2.55
]
]
]
59.30; H, 5.87; N, 6.91%; C20H24N2O5S requires: C,
59.39; H, 5.98; N, 6.93%.
(m, 1H, CH(CH3)2 of valine), 3.70 (s, 2H, CH2 of benzyl);
]
]
3.78 (d, 3JHH54.3, 1H, NCH of valine); 6.78 (s, 1H, SNH),
]
]
N-4-Toluene-sulfonylureido-N-benzyl-L-valine hydroxa-
7.20–7.56 (m, 7H, Hortho of O2NC6H4 and Harom of Ph),
]
]
1
3
mate D3, white crystals, m.p. 220–18C; H-NMR (DMSO-
8.05 (d, JHH58.3, 2H, Hmeta of O2NC6H4); 11.68 (br s,
]
d6), d, ppm: 1.11 (d, 3JHH56.7, 6H, CH(CH3)2 of valine),
1H, COOH); 13C-NMR (DMSO-d6), d, ppm: 17.8 (s,
]
2.23–2.51 (m, 1H, CH(CH3)2 of valine), 2.62 (s, 3H,
CH(CH3)2 of valine); 30.2 (s, CH(CH3)2 of valine); 42.7
]
]
]
]
3
CH3C6H4), 3.75 (s, 2H, CH2 of benzyl); 3.73 (d, JHH5
(s, CH2 of benzyl), 61.3 (s, NHCH of valine); 130.1 (s,
]
]
]
4.3, 1H, NCH of valine); 7.23–7.61 (m, 7H, Hortho of
Cmeta of O2NC6H4), 131.8 (s, Cpara of Ph), 133.6 (s, Cmeta
]
]
]
]
]
CH3C6H4 and Harom of Ph), 7.98 (d, 3JHH58.1, 2H, Hmeta
of Ph), 134.3 (s, Cortho of Ph), 135.5 (s, Cortho of
O2NC6H4), 141.6 (s, Cipso of Ph), 145.7 (s, Cipso of
O2NC6H4), 150.8 (s, Cpara of O2NC6H4), 175.2 (s,
CO2H). Anal. found: C, 60.08; H, 5.63; N, 7.53%;
]
]
]
]
of CH3C6H4); 8.26 (br s, 2H, NHCONH); 8.75 (br s, 1H,
]
]
NHOH); 10.54 (br s, 1H, NHOH); 13C-NMR (DMSO-d6),
]
]
]
d, ppm: 17.7 (s, CH(CH3)2 of valine); 26.3 (s, CH3C6H4),
30.3 (s, CH(CH3)2 of valine); 42.0 (s, CH2 of benzyl),
61.4 (s, NHCH of valine); 130.5 (s, Cmeta of CH3C6H4),
131.6 (s, Cpara of Ph), 132.8 (s, NHCONH), 133.9 (s, Cmeta
]
]
]
C18H20N2O4S requires: C, 59.98; H, 5.59; N, 7.77.
]
]
N-4-Nitrophenylsulfenyl-N-benzyl-L-valine hydroxamate
]
]
1
H1, yellow crystals, m.p. 181–28C; H-NMR (DMSO-d6),
]
]
]
of Ph), 134.6 (s, Cortho of Ph), 135.3 (s, Cortho of
d, ppm: 1.13 (d, 3JHH56.7, 6H, CH(CH3)2 of valine),
]
]
]
CH3C6H4), 141.7 (s, Cipso of Ph), 145.4 (s, Cipso of
2.23–2.52 (m, 1H, CH(CH3)2 of valine), 3.71 (s, 2H, CH2
]
]
]
]
CH3C6H4), 148.5 (s, Cpara of CH3C6H4), 174.5 (s,
of benzyl); 3.79 (d, 3JHH54.5, 1H, NCH of valine); 6.74 (s,
]
]
CONHOH). Anal. found: C, 57.38; H, 5.87; N, 10.00%;
1H, SNH), 7.21–7.66 (m, 7H, Hortho of O2NC6H4 and
]
]
]
C20H25N3O5S requires: C, 57.26; H, 6.01; N, 10.02%.
N-4-Fluorophenylureido-N-benzyl-L-valine E1, white
crystals, m.p. 143–48C; 1H-NMR (DMSO-d6), d, ppm:
Harom of Ph), 8.12 (d, 3JHH58.2, 2H, Hmeta of O2NC6H4);
]
]
8.73 (br s, 1H, NHOH); 10.65 (br s, 1H, NHOH); 13C-
]
]
NMR (DMSO-d6), d, ppm:, 17.8 (s, CH(CH3)2 of valine);
30.4 (s, CH(CH3)2 of valine); 42.6 (s, CH2 of benzyl),
61.2 (s, NHCH of valine); 130.4 (s, Cmeta of O2NC6H4),
131.2 (s, Cpara of Ph), 133.6 (s, Cmeta of Ph), 134.7 (s,
Cortho of Ph), 135.6 (s, Cortho of O2NC6H4), 141.4 (s, Cipso
of Ph), 148.5 (s, Cpara of O2NC6H4), 150.3 (s, Cipso of
]
3
1.13 (d, JHH56.7, 6H, CH(CH3)2 of valine), 2.23–2.54
]
]
]
(m, 1H, CH(CH3)2 of valine), 3.70 (s, 2H, CH2 of benzyl);
]
]
]
]
3.77 (d, 3JHH54.3, 1H, NCH of valine); 7.21–7.54 (m, 7H,
]
]
]
Hortho of FC6H4 and Harom of Ph), 7.97 (d, 3JHH58.1, 2H,
]
]
]
]
]
]
Hmeta of FC6H4); 8.06 (br s, 2H, NHCONH); 11.40 (br s,
]
]
1H, COOH); 13C-NMR (DMSO-d6), d, ppm: 17.5 (s,
O2NC6H4), 174.3 (s, CONHOH). Anal. found: C, 57.48;
]
CH(CH3)2 of valine); 30.1 (s, CH(CH3)2 of valine); 42.5
H, 5.76; N, 11.11%; C18H21N3O4S requires: C, 57.58; H,
5.64; N, 11.19.
]
]
]
(s, CH2 of benzyl), 61.5 (s, NHCH of valine); 130.0 (s,
]

72
C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
2.2. Enzymology
Yiotakis et al., 1988; Dive et al., 1990; Yiotakis et al.,
1994). 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– (Xaa5amino
acid residue), it appears that the S3, S2 and S1 subsites of
the enzyme are occupied by Gly, Pro and Xaa, respectively
(Galardy and Grobelny, 1983). Analogously, the S19, S29
and S39 subsites are also occupied by Gly, Pro and Xaa,
respectively (Galardy and Grobelny, 1983). Thus, many of
the reported inhibitors of ChC are aldehyde or ketone-type
substrate analogues, such as Pro6 –Gly-L-Pro–GlyH
(GlyH5glycine aldehyde), with a KI of 340 mM (Galardy
and Grobelny, 1983); phosphoric and phosphonic amides,
such as iso-amylphosphonyl-Gly–L-Pro–L-Ala (KI of 16
mM) (Grobelny and Galardy, 1985); thiols such as HS-
CH2CH2CO–Pro–Xaa (KI of the best compounds around
0.2 mM) (Vencill et al., 1985; Yiotakis and Dive, 1986;
Yiotakis et al., 1988); phosphonamide peptides of the type
p-nitrophenethyl-PO(OH)–Gly–Pro–Xaa (KI of the best
compound, with Xaa52-aminohexanoic acid was of 5 nM)
(Dive et al., 1990; Yiotakis et al., 1994). As shown from
the 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.
Collagenase type VII (highly purified) and its substrate,
FALGPA
(2-furanacryloyl-L-leucylglycyl-L-prolyl-L-
alanine) were purchased from Sigma-Aldrich (Milano,
Italy); 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 (National Institute of Health)
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
258C, using FALGPA as substrate, by the method of Van
Wart and Steinbrink (1981). The collagenase cleaves only
the Leu–Gly bond of FALGPA, with formation of
furanacryloyl-leucine and Gly–Pro–Ala (van Wart and
Steinbrink, 1981). 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 e305524 700 l/mol/cm
in the above-mentioned reaction buffer (van Wart and
Steinbrink, 1981). Measurements were made using a
Perkin-Elmer spectrophotometer interfaced with a personal
computer. Initial velocities were estimated using the direct
linear plot-based procedure reported by van Wart and
Steinbrink (1981). KI (inhibition constant) values were
then determined according to Dixon (1953), using a linear
regression program. The KI values determined are the
means of at least three determinations.
3. Results and discussion
Similarly to the vertebrate MMPs, C. 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 (Matsushita et al., 1998; Jung et al., 1999), (in
the case of the vertebrate MMPs the zinc ion is coordi-
nated by three histidines, His 218, His 222 and His 228),
and a water molecule/hydroxide ion acting as nucleophile
in the hydrolytic scission. Similarly to the MMPs, ChC is
also a multidomain protein, consisting of four segments,
S1, S2a, S2b and S3 (Matsushita et al., 1998; Jung et al.,
1999), with S1 incorporating the catalytic domain. Al-
though the two types of collagenases mentioned above (the
MMPs type and the bacterial ChC) are relatively different,
it is generally considered that their mechanism of action
for the hydrolysis of proteins and synthetic substrates is
quite similar (Matsushita et al., 1998; Jung et al., 1999).
A literature search showed that few ChC (or for other
bacterial collagenases) inhibitors were reported up to now
(Galardy and Grobelny, 1983; Grobelny and Galardy,
1985; Vencill et al., 1985; Yiotakis and Dive, 1986;
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 hydrox-
amic acids of type 7 recently reported by Parker’s group
(Jeng et al., 1998), 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 metalloelastase) were those incorporating: (i)
Gly, Ala or Val as spacers between the zinc-binding
function (the hydroxamic acid moiety) and the P29 site; (ii)
benzyl or isobutyl moieties at S29; and (iii) arylsulfonyl
moieties at S39 (Jeng et al., 1998). It should be noted that a
relatively small number of arylsulfonyl moieties were
investigated in the above-mentioned study (Jeng et al.,

C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
73
Table 1
1998), the greatest number of the synthesized inhibitors 7
containing the 4-methoxybenzenesulfonyl moiety. The
proposed interaction sites between the inhibitor and the
active site of the enzyme is also shown schematically in
the formula of the inhibitor 7.
Considering the above-mentioned findings (Jeng et al.,
1998), 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); (ii) a relatively
compact spacer between this function and the rest of the
molecule, i.e., L-valine; (iii) the already optimized (Jeng et
al., 1998) benzyl group at the S29 site; and (iv) variable
alkyl/arylsulfonyl-; arylsulfonylureido/arylureido-; or
arylsulfenyl moieties at S39.
The new compounds reported here were obtained by
nonexceptional synthetic procedures, as outlined in
Schemes 1–3. These involved reaction of L-alanine or
N-benzyl-L-alanine with alkyl/arylsulfonyl chlorides
(Supuran et al., 1998a,b), arylisocyanates (Scozzafava and
Supuran, 1999), arylsulfonylisocyanates (Scozzafava and
Supuran, 1999) or benzoylisothiocyanate, followed by
conversion of the COOH moiety to the hydroxamate one.
Related synthetic strategies led to some sulfenamides of
type G, H, as well as to the thioureas I, J (Scheme 4).
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 inhibition
data of Tables 1–3 with the new compounds and standard
inhibitors: (i) all hydroxamates were 100–500 more active
as ChC inhibitors as compared to the corresponding
carboxylic acids, probably due to the enhanced Zn(II)
coordinating properties of the CONHOH moiety (bidentate
binding) as compared to the COOH group (generally
monodentate binding to the zinc ion); (ii) potent inhibitors
were obtained from all classes of hydroxamates investi-
gated here, such as the alkyl/arylsulfonyl-benzyl-valine
derivatives (B5; B20–B22; B26, B27, B29, etc), the
arylsulfonylureas- and arylureas (such as D2, F5, F6), the
sulfenamido-benzyl-valine derivatives (such as H2, H3) or
the thiourea J1. Thus, it seems that the S39-binding moiety
of the arylsulfonamide type, previously investigated for the
obtaining of MMP inhibitors of type 7 (Jeng et al., 1998),
can be efficiently substituted by related moieties such as
Inhibition of ChC with the carboxylic acids A1–A33 and the corre-
sponding hydroxamates B1–B33
a
a
R
Compound
KI
Compound
KI
(mM)
(nM)
CH3
CF3
CCl3
n-C4F9-
n-C8F17
Me2N-
C6H5-
PhCH2-
A1
A2
A3
A4
A5
A6
A7
A8
A9
18
B1
B2
B3
B4
B5
B6
B7
B8
B9
100
47
52
10
8
3.3
5.6
3.2
1.9
41
20
15
12
11
65
54
40
42
44
36
30
33
15
11
18
11
10
8
7
5
6
11
17
18
7
7
9
4-F-C6H4-
4-Cl-C6H4-
4-Br-C6H4-
4-I-C6H4-
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28b
B29b
B30
B31
B32
B33
10
10
15
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
5.0
5.1
5.3
3.0
3.5
2.4
2.3
0.4
0.6
4.0
6.3
6.1
3.3
3.2
4.5
2.4
3.3
1.4
2.0
2.1
8
9
6
8
2-Naphthyl
5-Me2N-1-naphthyl-
2-Thienyl
10
a KI values were obtained from Dixon plots using a linear regression
program, from at least three different assays.
b The C6H4-COOH moiety transformed into C6H4-CONHOH.
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
a
a
R
Compound
KI
Compound
KI
(mM)
(nM)
4-F-C6H4-
C1
C2
C3
C4
E1
E2
E3
E4
E5
E6
G1
G2
G3
I1
7.5
6.9
7.0
8.0
9.1
10
8.3
7.4
4.2
5.6
6.0
6.2
7.0
0.6
D1
D2
D3
D4
F1
F2
F3
F4
F5
F6
H1
H2
H3
J1
19
15
13
12
31
33
30
21
9
12
13
12
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-
–
alkylsulfonyl-;
arylsulfenyl-;
arylsulfonylureido-;
arylureido- or benzoylthioureido, without loss of the ChC
inhibitory properties; (iii) in the subseries of alkyl/arylsul-
fonamido derivatives [of types A,B (1–33)) best ChC
inhibitory properties were correlated with the presence of
perfluoroalkylsulfonyl- (B4, B5), perfluorophenylsulfonyl-
(B21), 3-trifluoromethylphenylsulfonyl- (B22); 3-chloro-4-
nitrophenylsulfonyl- (B17); 3- or 4-protected-amino-
phenylsulfonyl- (B18–B20; B26); 3- or 4-carbox-
yphenylsulfonyl- (B28, B29), 1- or 2-naphthylsulfonyl
moieties (B30–B32). All these derivatives possessed inhi-
6
a KI values were obtained from Dixon plots using a linear regression
program, from at least three different assays.

74
C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
Table 3
the precise binding of inhibitors cannot be inferred from
X-ray crystallographic data, several important contribu-
tions in the field of the MMPs have been registered
recently, and this might be useful in interpreting our
inhibition data. Thus, Moroder’s and Tschesche’s groups
reported (Graf von Roedern et al., 1998; Krumme et al.,
1998) several X-ray crystallographic studies for the inter-
action of some types of hydroxamate inhibitors with the
catalytic domain of MMP-8, a collagenase from verte-
brates, inhibited among others with a KI of 10 nM by the
collagenase inhibitor in clinical study, batimastat 8 (Woj-
towicz-Praga et al., 1996).
Inhibition of ChC with standard inhibitors
a
Compound
KI
(mM)
Pro6 –Gly–Pro–GlyH
340
16
0.005
i-C5H11PO(OH)–Gly–Pro–Ala
p-O2NC6H4CH2CH2PO(OH)–Gly–Pro–2AXb
a KI values were obtained from Dixon plots using a linear regression
program, from at least three different assays.
b 2AX52-aminohexanoic acid.
bition constants in the range of 5–12 nM against ChC,
being among the most potent such inhibitors reported up to
now. 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-methoxyphenyl- or 2-thienyl, substituting the N-benzyl-
valine hydroxamate, behaved as medium potency in-
hibitors, with affinities in the 15–30 nM range (Table 1).
The least active sulfonamides were those containing
methyl-; trihalomethyl-; dimethylamino-; phenyl- and
benzyl moieties (Table 1); (iv) the arylsulfonylureido
compounds D1–D4 were more active than the corre-
sponding arylsulfonyl derivatives (compare for instance D1
with B9; D2 and B10, etc), acting as strong–medium
potency ChC inhibitors. Similarly behaved 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 affinities in the micromolar range
(except for the phosphonic acid derivative mentioned in
Table 3, which possessed an affinity of the same order of
magnitude as that of our compounds).
The above mentioned studies (Wojtowicz-Praga et al.,
1996) showed that batimastat is bidentately 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 (Graf von Roedern et al., 1998;
Krumme et al., 1998). The hydrophobic residues in the P19
(isobutyl) and P29 (benzyl) positions are also critical for
the formation of a strong enzyme–inhibitor adduct: thus,
the leucine side chain of P19 extends into the S19 pocket,
making hydrophobic contacts with several amino acid
residues such as His 197; Pro 217 and Val 194, whereas
the phenyl ring of P29 interacts with the side chains of Ile
159, Val 129 and Pro 217. Although the class III collagen-
ase inhibitors (in the classification of Babine and Bender,
1997) of the sulfonamide type, to which the compounds
reported here presumably belong, were much less investi-
gated crystallographically, it is assumed that the general
binding mode illustrated above is also valid for them,
although some differences were also evidenced (Babine
and Bender, 1997). Based on these observations, we
propose a similar binding mode of the sulfonamide in-
hibitors reported here to ChC, as shown schematically in
Fig. 1.
Although ChC (or its catalytic domain) could not be
crystallized up to now (in our or in other laboratories), and
The inhibitor probably coordinates bidentately to the
Zn(II) ion of ChC, whereas the hydrophobic moieties from
the P29 and P39 sites (benzyl and pentafluorophenylsulfonyl,
respectively) participate in hydrophobic contacts which
Fig. 1. Proposed schematic binding of inhibitor B21 within the active site
of ChC.

C.T. Supuran, A. Scozzafava / European Journal of Pharmaceutical Sciences 10 (2000) 67–76
75
inhibitors of neurotensin-degrading enzymes. Synthesis and enzyme
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Dive, V., Yiotakis, A., Nicolaou, A., Toma, F., 1990. Inhibition of
Clostridium histolyticum collagenases by phosphonamide peptide
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Dixon, M., 1953. Determination of enzyme-inhibitor constants. Biochem.
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Galardy, R.E., Grobelny, D., 1983. Inhibition of collagenase from
Clostridium histolyticum by phosphoric and phosphonic amides.
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Graf von Roedern, E., Brandstetter, H., Engh, R.A., Bode, W., Grams, F.,
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inhibitor B21 for which the schematic binding is shown
above has a KI of 5 nM).
In conclusion, we describe here a novel class of strong
inhibitors of the zinc protease EC 3.4.24.3, a collagenase
from C. histolyticum. As no X-ray crystallographic struc-
ture of this enzyme is available, the drug design has been
realized by utilizing X-ray data for the MMPs, related
enzymes which degrade extracellular matrix in vertebrates.
Reaction of valine or N-benzyl-valine with sulfonyl chlo-
rides, arylsulfonylisocyanate, arylisocyanates or ben-
zoylisothiocyanate afforded the new valine derivatives
which were subsequently converted to the corresponding
hydroxamates. These sulfonamide-based hydroxamic acid
derivatives were 100–500 times more inhibitory against
ChC as compared to the corresponding carboxylic acids,
similar to structurally-related compounds recently reported
as metalloelastase inhibitors by Jeng et al. (1998). Best
substitutions for obtaining high affinity inhibitors, involved
hydrophobic moieties at S39, such as perfluoroalkylsul-
fonyl-;
substituted-arylsulfonyl
(pentafluorophenyl-
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good inhibitors of the angiotensin I-converting enzyme. Biochem.
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Acknowledgements
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 manuscript.
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