Protease inhibitors. Part 12. Synthesis of potent matrix metalloproteinase and bacterial collagenase inhibitors incorporating sulfonylated N-4-nitrobenzyl-β-alanine hydroxamate moieties

Article abstract of DOI:10.1016/S0928-0987(00)00089-0

N-4-Nitrobenzyl-β-alanine was reacted with alkyl/arylsulfonyl halides, followed by conversion of the COOH to the CONHOH group. Structurally related compounds were obtained by reaction of N-4-nitrobenzyl-β-alanine with aryl isocyanates, arylsulfonyl isocyanates or benzoyl isothiocyanate, followed by similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was prepared from sulfanilyl- or metanilyl-4-nitrobenzyl-β-alanine by reaction with arylsulfonyl isocyanates, followed by the introduction of the hydroxamate moiety. The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2, MMP-8 and MMP-9, and of the Clostridium histolyticum collagenase (ChC). Some of the prepared hydroxamate derivatives proved to be very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety. Substitutions leading to the best inhibitors of MMP-1, a short-pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethyl-phenylsulfonyl at P(1') (K(I) of 3-5 nM). For MMP-2, MMP-8 and MMP-9 (deep-pocket enzymes), the best inhibitors were those containing perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-, 3- and 4-carboxy-phenylsulfonyl-, arylsulfonylureido- or arylsulfonylureido-sulfanilyl-/metanilyl moieties at P(1'). Bulkier groups in this position, such as 1- and 2-naphthyl-, substituted-naphthyl or quinoline-8-yl- moieties, among others, led to less effective MMP/ChC inhibitors. The best ChC inhibitors were again those containing pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl P(1') groups. This study demonstrates that the 4-nitrobenzyl moiety, investigated here for the first time, is an efficient P(2') anchoring moiety, whereas the β-alanyl scaffold can successfully replace the α-amino acyl one, for obtaining potent MMP/ChC inhibitors. Copyright (C) 2000 Elsevier Science B.V.

Full text of DOI:10.1016/S0928-0987(00)00089-0

European Journal of Pharmaceutical Sciences 11 (2000) 69–79
www.elsevier.nl/locate/ejps
Protease inhibitors
Part 12. Synthesis of potent matrix metalloproteinase and bacterial
collagenase inhibitors incorporating sulfonylated N-4-nitrobenzyl-b-alanine
hydroxamate moieties
Andrea Scozzafavaa, Marc A. Iliesb, Gheorghe Manolec, Claudiu T. Supurana ,
*
a
`
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121 Florence, Italy
bUniversity of Agricultural Sciences and Veterinary Medicine, Faculty of Biotechnologies, Department of Chemistry, B-dul Marasti nr. 59,
71331 Bucharest, Romania
cColentina Hospital, Department of Internal Medicine, Sos. Colentina 120, Bucharest, Romania
Received 3 December 1999; received in revised form 11 March 2000; accepted 15 March 2000
Abstract
N-4-Nitrobenzyl-b-alanine was reacted with alkyl/arylsulfonyl halides, followed by conversion of the COOH to the CONHOH group.
Structurally related compounds were obtained by reaction of N-4-nitrobenzyl-b-alanine with aryl isocyanates, arylsulfonyl isocyanates or
benzoyl isothiocyanate, followed by similar conversion of the COOH into the CONHOH moiety. Another subseries of derivatives was
prepared from sulfanilyl- or metanilyl-4-nitrobenzyl-b-alanine by reaction with arylsulfonyl isocyanates, followed by the introduction of
the hydroxamate moiety. The new compounds were assayed as inhibitors of four matrix metalloproteinases (MMPs), MMP-1, MMP-2,
MMP-8 and MMP-9, and of the Clostridium histolyticum collagenase (ChC). Some of the prepared hydroxamate derivatives proved to be
very effective collagenase/gelatinase inhibitors, depending on the substitution pattern at the sulfonamido moiety. Substitutions leading to
the best inhibitors of MMP-1, a short-pocket enzyme, were those involving pentafluorophenylsulfonyl or 3-trifluoromethyl-phenylsulfonyl
at P19 (KI of 3–5 nM). For MMP-2, MMP-8 and MMP-9 (deep-pocket enzymes), the best inhibitors were those containing
perfluoroalkylsulfonyl- and substituted-arylsulfonyl moieties, such as pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl-
, 3- and 4-carboxy-phenylsulfonyl-, arylsulfonylureido- or arylsulfonylureido-sulfanilyl-/metanilyl moieties at P19. Bulkier groups in this
position, such as 1- and 2-naphthyl-, substituted-naphthyl or quinoline-8-yl- moieties, among others, led to less effective MMP/ChC
inhibitors. The best ChC inhibitors were again those containing pentafluorophenylsulfonyl, 3- and 4-protected-aminophenylsulfonyl P19
groups. This study demonstrates that the 4-nitrobenzyl moiety, investigated here for the first time, is an efficient P29 anchoring moiety,
whereas the b-alanyl scaffold can successfully replace the a-amino acyl one, for obtaining potent MMP/ChC inhibitors.
2000
Elsevier Science B.V. All rights reserved.
Keywords: N-4-Nitrobenzyl-b-alanine; Hydroxamate; Sulfonyl halide; Matrix metalloproteinase; Bacterial collagenase; Enzyme inhibitor
1. Introduction
modeling (such as in the case of wound healing), bone
remodeling, apoptosis, cancer invasion and metastasis,
arthritis, atherosclerosis, aneurysm, breakdown of blood–
brain barrier, periodontal disease, skin ulceration, corneal
ulceration, gastric ulcers, and liver fibrosis, among others
(Bottomley et al., 1998; Johnson et al., 1998; Kahari and
Saarialho-Kere, 1999; Scozzafava and Supuran, 1999a,
2000; Williams et al., 1999). The matrix metalloprotein-
ases (MMPs), a family of zinc-containing endopeptidases
(also called matrixins), are thought to play a central role in
the above processes (Dioszegi et al., 1995; Tschesche,
1995; Johnson et al., 1998; Nagase and Woessner, 1999;
Whittaker et al., 1999).
The extracellular matrix (ECM) plays a crucial role for
the structure and integrity of various tissue types in higher
vertebrates (Johnson et al., 1998; Whittaker et al., 1999).
ECM turnover is involved in important physiological and
physiopathological events, such as embryonic develop-
ment, blastocyst implantation, nerve growth, ovulation,
morphogenesis, angiogenesis, tissue resorption and re-
*Corresponding author. Tel.: 139-55-275-7552; fax: 139-55-275-
7555.
E-mail address: cts@bio.chim.unifi.it (C.T. Supuran)
0928-0987/00/$ – see front matter
PII: S0928-0987(00)00089-0
2000 Elsevier Science B.V. All rights reserved.

70
A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
At least 20 members of this enzyme family, sharing
significant sequence homology, have been reported up to
now (Table 1) (Johnson et al., 1998; Nagase and Woes-
sner, 1999; Whittaker et al., 1999). They can be subdivided
(considering the macromolecular substrate requirements)
into: (i) collagenases (MMP-1, -8, -13 and -18); (ii)
gelatinases (MMP-2 and -9); (iii) stromelysins (MMP-3,
-10 and -11); and (iv) membrane-type MMPs (MT-MMPs)
(MMP-14, -15, -16 and -17). Recently, some new mem-
bers of the family have been evidenced, but little is known
for the moment regarding their properties, substrate spe-
cificity, or inhibition (Table 1).
Matrix metalloproteinases (MMPs) have recently be-
come interesting targets for drug design, in the search of
novel types of anticancer, antiarthritis or other pharmaco-
logical agents useful in the management of inflammatory
processes (Babine and Bender, 1997; Supuran and Scoz-
zafava, 2000a,b; Whittaker et al., 1999). It is thus possible
to envisage many medicinal chemistry applications by
inhibiting the activity of these enzymes, and several
pharmacological agents of the hydroxamate type, such as
batimastat (1), marimastat (2), trocade (3), or the sulfonyl
amino acid derivatives such as CGS 27023A (4a), or the
structurally related 4b are currently in advanced clinical
trials as antimetastasis, anticancer or antiarthritis drugs
Fig. 1. Structures 1–4.
(Babine and Bender, 1997; Supuran and Scozzafava,
2000a; Whittaker et al., 1999) (Fig. 1).
Literature data show (Grams et al., 1995a,b; Graff von
Roedern et al., 1998; Jeng et al., 1998; Krumme et al.,
1998; Hanessian et al., 1999; Kiyama et al., 1999; Supuran
et al., 2000) that MMP inhibitors have been studied
extensively in the last 15 years in order to find medicinal
chemistry applications. The same situation is not true for
inhibitors of the collagenases isolated from Clostridium
Table 1
Vertebrate MMPs, their molecular weights, substrates and preferred scissile amide bonds
Protein
MMP
MW
(kDa)
Principal substrate(s)
Preferred scissile
amide bond(s)
Collagenase 1
Gelatinase A
MMP-1
MMP-2
52
72
Fibrillar and nonfibrillar collagens
(types I, II, III, VI and X); gelatins
Basement membrane and nonfibrillar
collagens (types IV, V, VII, X);
fibronectin, elastin
Gly–Ile
Ala–Met
Stromelysin 1
MMP-3
57
Proteoglycan, laminin, fibronectin,
collagen (types III, IV, V, IX);
gelatins; pro-MMP-1
Gly–Leu
Matrilysin
Collagenase 2
Gelatinase B
MMP-7
MMP-8
MMP-9
28
64
92
Fibronectins; gelatins; proteoglycan
Fibrillar collagens (types I, II, III)
Basement membrane collagens
(types IV, V); gelatins
Ala–Ile
Gly–Leu; Gly–Ile
Gly–Ile; Gly–Leu
Stromelysin 2
MMP-10
54
Fibronectins; collagen (types III, IV);
Gelatins, pro-MMP-1
Gly–Leu
Stromelysin 3
Macrophage elastase
Collagenase 3
MMP-11
MMP-12
MMP-13
45
53
51.5
Serpin
Elastin
Ala–Met
Ala–Leu; Tyr–Leu
Gly–Ile
Fibrillar collagens (types I, II, III);
gelatins
MT1-MMP
MT2-MMP
MT3-MMP
MT4-MMP
MMP-14
MMP-15
MMP-16
MMP-17
66
61
55
58
Pro-72 kDa gelatinase
Not determined
Pro-72 kDa gelatinase
Not determined
Not determined
Not determined
Not determined
Ala–Gly
Collagenase 4
(Xenopus)
RASI 1
MMP-18
MMP-19
MMP-20
MMP-21
MMP-22
MMP-23
53
?
?
?
?
Not determined
Gelatin
Amelogenin (dentine); gelatin
Not determined
Not determined
Not determined
Gly–Ile
Not determined
Not determined
Not determined
Not determined
Not determined
Enamelysin
XMMP (Xenopus)
CMMP (chicken)
(No trivial name)
?

A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
71
1
histolyticum, which were much less investigated. This
collagenase (EC 3.4.24.3) is a 116 kDa protein belonging
to the M31 metalloproteinase family (Bond and Van Wart,
1984a,b; Rawlings and Barrett, 1995; Van Wart, 1998),
which is able to hydrolyze triple helical regions of collagen
under physiological conditions, as well as an entire range
of synthetic peptide substrates. In fact, the crude homoge-
nate of Clostridium histolyticum, which contains several
distinct collagenase isozymes (Bond and Van Wart,
1984a,b; Rawlings and Barrett, 1995; Van Wart, 1998), is
the most efficient system known for the degradation of
connective tissue, also being involved in the pathogenicity
of this and related clostridia, such as C. perfringens, which
causes human gas gangrene and food poisoning, among
others (Rood, 1998). Typically, these bacteria (and their
collagenases) cause so much damage and so quickly, that
antibiotics are ineffective. Thus, development of inhibitors
against these collagenases is also an interesting target for
drug design.
Similarly to the vertebrate MMPs, Clostridium his-
tolyticum collagenase (abbreviated as ChC) has the con-
served 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, and a water molecule/hydroxide ion
acting as nucleophile in the hydrolytic scission (Matsushita
et al., 1998, 1999; Jung et al., 1999). Similarly to the
MMPs, ChC is also a multiunit protein, consisting of four
segments, S1, S2a, S2b and S3, with S1 incorporating the
catalytic domain. Although the two types of enzymes
mentioned above (the MMPs 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 rather similar. One must also
mention that these enzymes could not be crystallized, for
X-ray crystallographic experiments, and in order to assist
the design of their inhibitors.
16PC FTIR spectrometer; H-NMR spectra, Varian Gem-
mini 200 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 for all the compounds reported here),
Carlo Erba Instrument CHNS Elemental Analyzer, Model
1106. All reactions were monitored by thin-layer chroma-
tography (TLC) using 0.25-mm precoated silica gel plates
(E. Merck). Preparative HPLC was performed on a
Dynamax-60A column (253250 mm) with a Beckman
EM-1760 instrument. The detection wavelength was 254
nm.
Amino acids (b-alanine), 4-nitrobenzyl chloride, sul-
fonyl chlorides, arylsulfonyl isocyanates, aryl isocyanates,
benzoyl isothiocyanate, triethylamine, carbodiimides, hy-
droxylamine, 5,59-dithiobis-(2-nitrobenzoic acid), FAL-
GPA, buffers and other reagents used in the syntheses were
commercially available compounds from Sigma, Acros or
Aldrich. The thioester MMP substrate, AcProLeuGly-S-
LeuLeuGlyOEt, was from Bachem.
2.1. Chemistry
2.1.1. Preparation of N-4-nitrobenzyl-b-alanine (7)
An amount of 7.8 g (0.10 M) of b-alanine (6) and a
stoichiometric amount of 4-nitrobenzyl chloride (16.1 g)
were suspended/dissolved in 150 ml of anhydrous acetoni-
trile and an equivalent amount of triethyl amine (0.10 mM,
14.7 ml) was added. The reaction mixture was stirred at
room temperature for 20 h, then the solvent was evapo-
rated in vacuo. The obtained reaction mixture was taken in
250 ml of water, the pH was brought to 7 with citric acid,
and crude product 7 precipitated by leaving the mixture
overnight at 48C. Recrystallization from ethanol afforded
the pure title compound in almost quantitative yield.
2.1.2. General procedure for the preparation of N-4-
nitrobenzyl-alkyl/arylsulfonyl b-alanines (A1–A35)
An amount of 2.10 g (10 mmol) of N-2-nitrobenzyl-b-
alanine (7) and 10 mmol of sulfonyl chloride were
suspended/dissolved in 100 ml of acetone125 ml of
water. A 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
solvent 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, compounds A1–
A35 were recrystallized from EtOH or MeOH. Yields were
around 75–90%.
In this paper we report the preparation of a series of
MMP and ChC inhibitors incorporating alkyl/arylsul-
fonamido-b-alanine
hydroxamate
as
well
as
arylsulfonylureido-/arylureido-b-alanine
hydroxamate
moieties in the molecule. Some of the new compounds,
assayed for the inhibition of purified collagenases (MMP-1
and MMP-8), gelatinases (MMP-2 and MMP-9) and of
ChC, showed high affinity for these enzymes (in the
nanomolar range), behaving as some of the best MMP/
ChC sulfonylated inhibitors reported up to now. SAR is
also discussed both for MMP inhibition as well as bacterial
collagenase inhibition with these new types of derivatives.
2.1.3. General procedure for the preparation of
2. Materials and methods
compounds B1–B35, D1–D5, F1–F6, H1–H3 and J1
An amount of 5 mM of carboxylic acid derivative
A1–A35, C1–C5, E1–E6, G1–G3 or I1 was dissolved/
suspended in 50 ml of anhydrous acetonitrile or acetone,
Melting points, heating plate microscope (not corrected);
IR spectra, KBr pellets, 400–4000 cm21 Perkin-Elmer

72
A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
and treated with 420 mg (6 mM) of hydroxylamine?HCl
and 1.10 g (6 mM) of EDCI?HCl or di-isopropyl-car-
bodiimide. The reaction mixture was magnetically stirred
at room temperature for 15 min, then 180 ml (12 mM) of
triethylamine were added and stirring was continued for 12
h at 48C. 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 (253
250 mm); 90% acetonitrile/10% methanol; flow rate 30
ml/min) afforded the pure hydroxamic acids.
2.1.7. N-4-Toluenesulfonyl-N-4-nitrobenzyl-b-alanine
(A13)
Pale yellow crystals, m.p. 211–2128C; 1H-NMR
(DMSO-d6), d, ppm: 2.52 (s, 3H, CH3C6H4), 3.62 (m, 4H,
]
CH2 of b-Ala), 4.36 (s, 2H, CH2 of benzyl); 7.25–7.64 (m,
]
]
4H, Hortho of CH3C6H41Hortho of O2NC6H4), 7.97 (d,
]
]
3JHH 5 8.1, 2H, Hmeta of CH3C6H4); 8.21 (d, 2H, Hmeta of
]
]
O2NC6H4), 11.69 (br s, 1H, COOH); 13C-NMR (DMSO-
d6), d, ppm: 26.0 (s, CH3C6H4), 36.5 (s, CH2 of b-Ala),
]
]
43.5 (s, CH2 of b-Ala), 44.7 (s, CH2 of benzyl), 123.8 (s,
]
]
Cmeta of O2NC6H4), 129.4 (Cortho of O2NC6H4), 130.5 (s,
Cmeta of CH3C6H4), 135.0 (s, Cortho of CH3C6H4), 144.5
(s, Cipso of O2NC6H4), 145.0 (s, Cipso of CH3C6H4), 147.8
(s, Cpara of O2NC6H4), 148.6 (s, Cpara of CH3C6H4),
177.3 (s, CO2H). Anal. found: C, 52.57; H, 4.76; N,
7.54%. C17H18N2O6S requires: C, 52.74; H, 4.43; N,
7.69%.
]
]
]
]
]
]
]
]
]
2.1.4. General procedure for the preparation of
compounds C1–C5, E1–E6 and I1
An amount of 2.20 g (10 mmol) of N-4-nitrobenzyl-b-
alanine (7) and a stoichiometric amount of arylsulfonyl
isocyanate (8), aryl isocyanate (9) or ben-
zoylisothiocyanate were suspended in 50 ml of anhydrous
acetonitrile 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 8) 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 quantita-
tive.
2.1.8. N-4-Toluenesulfonyl-N-4-nitrobenzyl-b-alanine
hydroxamate (B13)
Pale yellow crystals, m.p. 233–2348C; 1H-NMR
(DMSO-d6), d, ppm: 2.62 (s, 3H, CH3C6H4), 3.79 (m, 4H,
]
CH2 of b-Ala), 4.39 (s, 2H, CH2 of benzyl); 7.21–7.67 (m,
]
]
4H, Hortho of CH3C6H4 1Hortho of O2NC6H4), 8.00 (d,
]
]
3JHH 5 8.1, 2H, Hmeta of CH3C6H4); 8.21 (d, 2H, Hmeta of
]
]
O2NC6H4), 8.70 (br s, 1H, NHOH); 10.51 (br s, 1H,
]
NHOH); 13C-NMR (DMSO-d6), d, ppm: 26.5 (s,
]
CH3C6H4), 36.5 (s, CH2 of b-Ala), 44.6 (s, CH2 of
b-Ala), 45.1 (s, CH2 of benzyl), 123.8 (s, Cmeta of
O2NC6H4), 129.4 (Cortho of O2NC6H4), 130.7 (s, Cmeta of
CH3C6H4), 135.6 (s, Cortho of CH3C6H4), 144.3 (s, Cipso
of O2NC6H4), 145.3 (s, Cipso of CH3C6H4), 147.9 (s, Cpara
of O2NC6H4), 148.6 (s, Cpara of CH3C6H4), 174.0 (s,
CONHOH). Anal. found: C, 50.77; H, 4.39; N, 11.02%.
C17H19N3O6S requires: C, 50.65; H, 4.52; N, 11.08%.
]
]
]
]
]
2.1.5. General procedure for the preparation of
compounds G1–G3
]
]
]
]
The general procedure described above for the prepara-
tion of compounds A1–A35 was followed, except that
arylsulfenyl halides were used instead of alkyl/arylsul-
fonyl halides. The yields of the title sulfenamides were
around 60%.
]
]
]
]
2.1.9. N-4-Toluenesulfonylureido-N-4-nitrobenzyl-b-
alanine (C3)
2.1.6. General procedure for the preparation of
derivatives M,N(1–5)
Pale yellow crystals, m.p. 277–2788C; 1H-NMR
An amount of 10 mM of boc-protected derivative, A19
or A20, was treated with a small excess of TFA at room
temperature, under energetic magnetic stirring, for 30 min.
The excess TFA was evaporated in vacuo, the residue
taken in water and neutralized with sodium bicarbonate
until pH 5.5. The precipitated amino derivatives 10 and 11
were filtered and air dried. Reaction of these derivatives
with arylsulfonyl isocyanates 8 in acetone, as described
above, afforded the carboxylates K,L(1–5), which were
converted into the corresponding hydroxamates as de-
scribed above. Overall yields were around 60–63%.
All new compounds were characterized by 1H- and
13C-NMR spectroscopy and elemental analysis (60.4% of
the theoretical data). Data for a representative compound
of each series is provided below.
(DMSO-d6), d, ppm: 2.60 (s, 3H, CH3C6H4), 3.82 (m, 4H,
CH2 of b-Ala), 4.38 (s, 2H, CH2 of benzyl); 7.29–7.73 (m,
]
]
]
4H, Hortho of CH3C6H4 1Hortho of O2NC6H4), 7.99 (d,
]
]
3JHH 5 8.1, 2H, Hmeta of CH3C6H4); 8.16 (d, 2H, Hmeta of
O2NC6H4), 8.29 (br s, 2H, NHCONH); 11.73 (br s, 1H,
COOH); 13C-NMR (DMSO-d6), d, ppm: 26.5 (s,
CH3C6H4), 36.3 (s, CH2 of b-Ala), 43.9 (s, CH2 of
b-Ala), 45.3 (s, CH2 of benzyl), 123.9 (s, Cmeta of
O2NC6H4), 129.4 (Cortho of O2NC6H4), 131.9 (s, Cmeta of
CH3C6H4), 132.4 (s, NHCONH), 135.0 (s, Cortho of
CH3C6H4), 144.5 (s, Cipso of O2NC6H4), 145.7 (s, Cipso of
CH3C6H4), 147.8 (s, Cpara of O2NC6H4), 148.6 (s, Cpara
of CH3C6H4), 177.3 (s, CO2H). Anal. found: C, 50.34; H,
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
]
4.08; N, 10.15%. C17H17N3O7S requires: C, 50.12; H,
4.21; N, 10.31%.

A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
73
2.1.10. N-4-Toluenesulfonylureido-N-4-nitrobenzyl-b-
2.1.13. N-4-Nitrophenylsulfenyl-N-4-nitrobenzyl-b-
alaninehydroxamate (D3)
alanine (G1)
Pale yellow crystals, m.p. 273–2748C; 1H-NMR
Yellow crystals, m.p. 223–2248C; 1H-NMR (DMSO-
d6), d, ppm: 3.87 (m, 4H, CH2 of b-Ala), 4.37 (s, 2H, CH2
(DMSO-d6), d, ppm: 2.63 (s, 3H, CH3C6H4), 3.80 (m, 4H,
]
]
]
CH2 of b-Ala), 4.36 (s, 2H, CH2 of benzyl); 7.20–7.74 (m,
of benzyl); 6.75 (s, 1H, SNH), 7.21–7.62 (m, 4H, Hortho of
]
]
]
4H, Hortho of CH3C6H4 1Hortho of O2NC6H4), 7.99 (d,
CH3C6H4 1Hortho of O2NC6H4), 8.05 (d, 3JHH 5 8.3, 2H,
]
]
]
3JHH 5 8.1, 2H, Hmeta of CH3C6H4); 8.21 (d, 2H, Hmeta of
Hmeta of O2NC6H4); 8.21 (d, 2H, Hmeta of O2NC6H4),
]
]
]
]
11.68 (br s, 1H, COOH); 13C-NMR (DMSO-d6), d, ppm:
36.5 (s, CH2 of b-Ala), 44.0 (s, CH2 of b-Ala), 44.8 (s,
O2NC6H4), 8.32 (br s, 2H, NHCONH); 8.76 (br s, 1H,
NHOH); 10.59 (br s, 1H, NHOH); 13C-NMR (DMSO-d6),
]
]
]
]
d, ppm: 26.3 (s, CH3C6H4), 36.6 (s, CH2 of b-Ala), 44.9
CH2 of benzyl), 123.8 (s, Cmeta of O2NC6H4CH2), 129.4
]
]
]
]
]
(s, CH2 of b-Ala), 45.2 (s, CH2 of benzyl), 123.7 (s, Cmeta
(Cortho of O2NC6H4CH2), 130.3 (s, Cmeta of O2NC6H4S),
]
]
]
]
of O2NC6H4), 129.5 (Cortho of O2NC6H4), 131.3 (s, Cmeta
135.9 (s, Cortho of O2NC6H4S), 144.5 (s, Cipso of
]
]
]
]
of CH3C6H4), 132.4 (s, NHCONH), 135.0 (s, Cortho of
O2NC6H4CH2), 145.9 (s, Cipso of O2NC6H4S), 147.8 (s,
]
]
]
CH3C6H4), 144.5 (s, Cipso of O2NC6H4), 145.6 (s, Cipso of
Cpara of O2NC6H4CH2), 150.4 (s, Cpara of O2NC6H4S),
]
]
]
]
]
CH3C6H4), 147.7 (s, Cpara of O2NC6H4), 148.6 (s, Cpara
177.5 (s, CO2H). Anal. found: 49.45; H, 3.77; N,
]
]
of CH3C6H4), 174.6 (s, CONHOH). Anal. found: C,
11.24%.C16H15N3O6S requires: C, 49.58; H, 3.61; N,
11.56%.
]
48.09; H, 4.43; N, 13.12%. C18H20N4O7S requires: C,
48.34; H, 4.30; N, 13.26%.
2.1.14. N-4-Nitrophenylsulfenyl-N-4-nitrobenzyl-b-
alaninehydroxamate (H1)
2.1.11. N-4-Fluorophenylureido-N-4-nitrobenzyl-b-
alanine (E1)
Yellow crystals, m.p. 197–1988C; 1H-NMR (DMSO-
d6), d, ppm: 3.84 (m, 4H, CH2 of b-Ala), 4.38 (s, 2H, CH2
Yellow crystals, m.p. 238–2398C; 1H-NMR (DMSO-
d6), d, ppm: 3.80 (m, 4H, CH2 of b-Ala), 4.35 (s, 2H, CH2
]
]
of benzyl); 6.75 (s, 1H, SNH), 7.20–7.67 (m, 4H, Hortho of
]
CH3C6H4 1Hortho of O2NC6H4), 8.09 (d, 3JHH 5 8.2, 2H,
]
]
]
of benzyl); 7.18–7.66 (m, 4H, Hortho of CH3C6H4 1Hortho
]
]
Hmeta of O2NC6H4); 8.23 (d, 2H, Hmeta of O2NC6H4),
of O2NC6H4), 7.94 (d, 3JHH 5 8.0, 2H, Hmeta of FC6H4);
]
]
8.74 (br s, 1H, NHOH); 10.68 (br s, 1H, NHOH); 13C-
]
]
]
8.05 (br s, 2H, NHCONH); 8.22 (d, 2H, Hmeta of
]
NMR (DMSO-d6), d, ppm:, 36.5 (s, CH2 of b-Ala), 44.1
(s, CH2 of b-Ala), 44.6 (s, CH2 of benzyl), 123.8 (s, Cmeta
of O2NC6H4CH2), 129.6 (Cortho of O2NC6H4CH2), 130.6
(s, Cmeta of O2NC6H4S), 135.9 (s, Cortho of O2NC6H4S),
144.5 (s, Cipso of O2NC6H4CH2), 147.9 (s, Cpara of
O2NC6H4CH2), 148.5 (s, Cpara of O2NC6H4S), 150.3 (s,
O2NC6H4), 11.44 (br s, 1H, COOH); 13C-NMR (DMSO-
]
]
]
]
d6), d, ppm: 36.8 (s, CH2 of b-Ala), 44.3 (s, CH2 of
]
]
]
]
]
b-Ala), 44.6 (s, CH2 of benzyl), 123.8 (s, Cmeta of
]
]
]
O2NC6H4), 129.4 (Cortho of O2NC6H4), 131.4 (s, Cmeta of
FC6H4), 132.0 (s, NHCONH), 135.2 (s, Cortho of FC6H4),
144.5 (s, Cipso of O2NC6H4), 147.4 (s, Cpara of
]
]
]
]
]
]
]
]
]
Cipso of O2NC6H4S), 174.6 (s, CONHOH). Anal. found:
]
]
O2NC6H4), 148.9 (s, Cipso of FC6H4), 149.7 (s, Cpara of
]
C, 47.91; H, 3.65; N, 14.69%. C15H14N4O6S requires: C,
47.62; H, 3.73; N, 14.81%.
FC6H4), 177.8 (s, CO2H). Anal. found: C, 55.60; H, 4.13;
]
N, 12.00%. C17H16FN3O5 requires: C, 55.33; H, 4.06; N,
12.10%.
2.2. Enzymology
2.1.12. N-4-Fluorophenylureido-N-4-nitrobenzyl-b-
alaninehydroxamate (F1)
Human purified MMPs (MMP-1, MMP-2, MMP-8 and
MMP-9) were purchased from Calbiochem (Inalco,
Milano, Italy). They were activated (Nagase, 1997) in the
assay buffer by adding bovine trypsin (50 ml, 0.6 mg/ml)
to the proenzyme, followed by incubation at 378C for 10
min. The trypsin was then inactivated with aprotinin (50
ml, 1.2 mg/ml). Initial rates for the hydrolysis of the
thioester substrate AcProLeuGly-S-LeuLeuGlyOEt, cou-
pled to the reaction with 5,59-dithiobis-(2-nitrobenzoic
acid), were used for assessing the catalytic activity and
inhibition of the four MMPs mentioned above, by the
spectrophotometric method of Powers and Kam (1995) and
Johnson et al. (1999). The change of absorbance (´ 5
19,800 M21 cm21) at 405 nm was monitored continuously
at room temperature using a Cary 3 spectrophotometer
interfaced to a PC. A typical 100 ml reaction contained 50
mM MES, pH 6.0, 10 mM CaCl2, 100 mM substrate, 1
Yellow crystals, m.p. 200–2018C; 1H-NMR (DMSO-
d6), d, ppm: 3.78 (m, 4H, CH2 of b-Ala), 4.42 (s, 2H, CH2
]
]
of benzyl); 7.22–7.69 (m, 4H, Hortho of CH3C6H4 1Hortho
]
]
of O2NC6H4), 7.90 (d, 3JHH 5 8.2, 2H, Hmeta of FC6H4);
]
8.04 (br s, 2H, NHCONH); 8.24 (d, 2H, Hmeta of
]
O2NC6H4), 8.70 (br s, 1H, NHOH); 10.62 (br s, 1H,
]
NHOH); 13C-NMR (DMSO-d6), d, ppm: 36.9 (s, CH2 of
]
]
b-Ala), 44.1 (s, CH2 of b-Ala), 45.4 (s, CH2 of benzyl),
]
]
123.8 (s, Cmeta of O2NC6H4), 129.4 (Cortho of O2NC6H4),
130.7 (s, Cmeta of FC6H4), 131.7 (s, NHCONH), 135.5 (s,
Cortho of FC6H4), 144.7 (s, Cipso of O2NC6H4), 145.6 (s,
Cipso of FC6H4), 147.7 (s, Cpara of O2NC6H4), 148.9 (s,
Cpara of FC6H4), 174.8 (s, CONHOH). Anal. found: C,
52.84; H, 4.32; N, 15.40%. C17H16FN4O5 requires: C,
53.04; H, 4.17; N, 15.46%.
]
]
]
]
]
]
]
]
]
]

74
A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
mM 5,59-dithiobis-(2-nitrobenzoic acid) and 5 nM MMP.
For the KI determinations, DMSO solutions of the inhibitor
were included in the assay, resulting in a final con-
centration of 2% DMSO in the reaction mixture. Under
these conditions, KI values varied from 5 to 10% in
replicate experiments. KI were then determined using
Easson–Stedman (Bieth, 1995) plots and a linear regres-
sion program.
Clostridium histolyticum highly purified collagenase and
its substrate, FALGPA (furanacryloyl-leucyl-glycyl-prolyl-
alanine), were purchased from Sigma (Milano, Italy), and
their concentrations were determined from the absorbance
at 280 nm and the extinction coefficients furnished by the
supplier. The activity of such preparations was in the range
of 10 NIH units/mg solid. The potency of standard and
newly obtained inhibitors was determined from the inhibi-
tion of the enzymatic (amidolytic) activity of the collagen-
ase at 258C using FALGPA as substrate by the method of
Van Wart and Steinbrink (1981). The substrate was recon-
stituted 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 ´305 5 24,700 M21
cm21 in the above-mentioned reaction buffer. Measure-
ments were made using a Cary 3 spectrophotometer
interfaced to a PC. Initial velocities were thus estimated
using the direct linear plot-based procedure reported by
Van Wart and Steinbrink (1981). KI were then determined
according to Easson–-Stedman (Bieth, 1995) plots and a
linear regression program.
Scheme 1.
Scheme 2.
Supuran et al., 1999; Scozzafava et al., 1999a,b; Scoz-
zafava and Supuran, 2000).
By applying synthetic strategies related to those de-
scribed previously, the sulfenamido derivatives G1–G3
and H1–H3, as well as the thioureas I1 and J1 were also
obtained (Fig. 2).
3. Results
Non-exceptional routine synthetic procedures were used
for the preparation of the MMP inhibitors reported here,
related to those applied by MacPherson et al. (1997), Jeng
et al. (1998), Hanessian et al. (1999) and our group
(Scozzafava and Supuran, 1999b, 2000) for sulfonylated
amino acid hydroxamates. Reaction of 4-nitrobenzyl chlo-
ride 5 with b-alanine 6 afforded the key intermediate,
N-4-nitrobenzyl-b-alanine (7). Carboxylic acids A1–A35
were then prepared by reaction of alkyl/arylsulfonyl
chlorides with compound 7 (Scheme 1). Conversion of the
carboxylic acids A1–A35 into the corresponding hydroxa-
mates B1–B35 was done with hydroxylamine and diiso-
propyl carbodiimide (Scheme 1) (Tamura et al., 1998;
Borras et al., 1999; Supuran et al., 1999; Scozzafava et al.,
1999a,b).
Another approach for obtaining MMP and ChC in-
hibitors consisted of deprotection of the Boc-derivatives
A19 and A20 (with TFA), followed by reaction of the
Another series of derivatives (C1–C5, D1–D5 and E1–
E6, F1–F6) was obtained by reaction of arylsul-
fonylisocyanates 8 or aryl isocyanates 9 with the N-benzyl-
b-alanine derivative 7, followed by conversion of the
COOH moiety into the CONHOH one, as described above
(Scheme 2) (Tamura et al., 1998; Borras et al., 1999;
Fig. 2. Structures G1–G3, H1–H3, I1 and J1.

A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
75
fications. (i) The P1 substituent is absent in the compounds
designed by us, but the carbon atom chain was extended,
since in contrast to the previously reported compounds,
which are all a-amino acid derivatives, the inhibitors
reported here are b-alanine derivatives. (ii) The already
optimized (Jeng et al., 1998) benzyl group at the S29 site
was modified to the 4-nitrobenzyl moiety, as we have
shown previously that substituents at the benzyl moiety
with acidifying properties, such as 2-nitro, 2-chloro, etc.,
lead to better ChC inhibitors as compared to the corre-
sponding unsubstituted derivatives (Scozzafava and
Supuran, 1999b, 2000). (iii) A large number of diverse
alkyl/arylsulfonyl-, arylsulfonyl-ureido/arylureido-, or
arylsulfenyl moieties at S19, as these moieties are the major
determinants of activity/selectivity towards different
MMPs (Dioszegi et al., 1995; Tschesche, 1995; Johnson et
al., 1998; Nagase and Woessner, 1999; Whittaker et al.,
1999).
The new compounds reported here were obtained by
non-exceptional synthetic procedures, as outlined in
Schemes 1–3. These involved reaction of N-4-nitrobenzyl-
b-alanine (7) with alkyl/arylsulfonyl chlorides,
arylisocyanates, arylsulfonyl-isocyanates or benzoyl-iso-
thiocyanate, followed by conversion of the COOH moiety
to the hydroxamate one. Related synthetic strategies led to
some sulfenamides of type G and H, as well as to the
thioureas I and J. As it has recently been reported
(Hanessian et al., 1999; Whittaker et al., 1999) that
elongated moieties in P19 /P29 may lead to selectivity of the
obtained inhibitors for the deep-pocket enzymes, relative
to the short-pocket ones, we also studied an approach for
preparing inhibitors containing such moieties (Scheme 3).
Thus, the Boc-protected derivatives A19 and A20 were
treated with TFA in order to eliminate the protecting
group, and the amino derivatives thus obtained were
reacted with arylsulfonyl isocyanates 8. The carboxy
groups of derivatives K,L(1–5) were then converted to the
hydroxamate moieties by the standard procedure, as men-
tioned above, thus leading to derivatives M,N(1–5).
Inhibition data of Tables 2–4 show that the entire class
of sulfonylated b-alanine hydroxamate derivatives reported
here act as efficient metallo-peptidase inhibitors, but
important differences in activity were detected against the
diverse enzymes for the different substitution patterns of
the alkyl/arylsulfonamide moiety contained in the mole-
cules of the new inhibitors.
Scheme 3.
amino derivatives 10 and 11 with arylsulfonyl isocyanates,
and conversion of the COOH into the hydroxamate moiety,
thus leading to the carboxylates K,L(1–5) and the corre-
sponding hydroxamates M,N(1–5) (Scheme 3).
Inhibition data against four MMPs (MMP-1, MMP-2,
MMP-8 and MMP-9) and type II ChC with the compounds
reported in the present paper are shown in Tables 2 – 4.
4. Discussion
Sulfonylated amino acid hydroxamates were recently
discovered to act as efficient MMP inhibitors (MacPherson
et al., 1997; Bottomley et al., 1998; Almstead et al., 1999;
Cheng et al., 1999; Pavlovsky et al., 1999; Whittaker et al.,
1999; O’Brien et al., 2000). The first compound from this
class to be developed for clinical trials, type 4 (CGS
27023A), possesses the following structural features. (i)
An isopropyl substituent a to the hydroxamic acid moiety,
considered to slow down metabolism of the zinc-binding
function. It was shown to bind (by X-ray crystallography,
Babine and Bender, 1997; Pavlovsky et al., 1999, and
NMR spectroscopy, Moy et al., 1999) within the S1
subsite. (ii) A bulkier pyridyl-methyl (or benzyl, in struc-
turally related compounds) moiety substituting the amino
nitrogen atom, and binding within the S29 pocket of the
enzyme. (iii) A arylsulfonyl group, which occupies (but
generally does not fill) the specificity S19 pocket. For
reasons little explained for the moment, this group was a
4-methoxybenzenesulfonyl one in most of the investigated
derivatives (MacPherson et al., 1997; Jeng et al., 1998).
The promising MMP inhibition and clinical data of
some of these sulfonamide inhibitors prompted us to
investigate them in some detail. Our lead compound was 4,
to which we performed the following structural modi-
Thus, for MMP-1, an enzyme possessing a short spe-
cificity S19 pocket, aliphatic (B1–B6, B35), bulky aromatic
(B25, B30–B32, D1–D5, F5, F6, J1, M1–M5, N1–N5) or
heterocyclic (B34) moieties as P19 groups led to the least
effective hydroxamate inhibitors, with KI in the 30–80 nM
range. The carboxylates K,L(1–5) were also ineffective in
inhibiting this collagenase. Obviously, these data may be
explained by the restricted space available around the S19
subsite of MMP-1, which cannot accommodate bulky
groups in it, discriminating in this way between the bulky

76
A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
Table 2
Inhibition of MMPs and ChC with the hydroxamates B1–B35
a
R
Compound
KI (nM)
MMP-1b
MMP-2b
MMP-8b
MMP-9b
ChCc
CH3
CF3
CCl3
n-C4F9-
n-C8F17
Me2N-
C6H5-
PhCH2-
B1
B2
B3
B4
B5
B6
B7
B8
87
22
23
69
77
38
25
27
21
23
24
32
30
16
15
43
30
13
12
20
2
24
14
19
1.8
0.6
33
15
18
17
12
13
15
21
10
9
36
22
20
2.0
1.1
37
26
28
15
19
20
16
28
7
31
28
27
2.5
1.1
44
19
21
13
16
21
11
32
9
11
23
10
11
15
13
0.2
0.7
15
30
36
6
.100
95
78
14
9
88
53
56
40
48
42
36
44
15
14
34
15
13
18
8
4-F-C6H4-
B9
4-Cl-C6H4-
4-Br-C6H4-
4-I-C6H4-
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28d
B29d
B30
B31
B32
B33
B34
B35
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-MeO-C6H4-
2,4,6-Me3C6H2-
4-MeO-3-BocNH-C6H3-
2-HO-3,5-Cl2-C6H2-
3-HOOC-C6H4-
4-HOOC-C6H4-
1-Naphthyl
10
18
8
24
7
9
8
9
10
15
0.1
0.6
14
23
34
7
15
3.0
2.2
55
49
60
10
37
46
12
0.9
1.3
12
24
29
4
11
2.1
2.0
38
36
54
7
3
6
6
11
23
35
24
13
31
30
82
65
89
12
55
57
21
25
32
11
10
12
9
18
16
18
15
10
17
18
2.7
2.1
62
54
67
13
41
65
2-Naphthyl
5-Me2N-1-naphthyl-
2-Thienyl
Quinoline-8-yl
Camphor-10-yl
49
45
a KI values were obtained from Easson–Stedman (Bieth, 1995) plots using a linear regression program, from at least three different assays. Standard
errors were 5–10%.
b With the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.
c With FALGPA as substrate, spectrophotometrically.
d The C6H4-COOH moiety transformed into C6H4-CONHOH.
and less voluminous inhibitors. Inhibitors with KI in the
15–30 nM range against MMP-1 were also obtained.
These contained aromatic rings, substituted meta or para
with electron-attracting groups such as nitro, halogeno,
methoxy, etc. Efficient MMP-1 inhibitors (KI in the range
3–15 nM) were those containing the following moieties:
4-acetamido-phenyl- (B18), perfluorophenyl- (B21), 3-tri-
fluoromethyl-phenyl- (B22), 2,5-dichlorophenyl- (B23) or
2-hydroxy-3,5-dichlorophenyl- (B27), among others.
The deep-pocket enzymes MMP-2, MMP-8 and MMP-9
showed a more similar inhibition behavior, relatively
different from that of the previously discussed MMP-1.
Thus, again short aliphatic moieties at P19 led to the least
effective inhibitors, such as B1, B6 and B35. The same
was true for the bulky naphthyl-derived moieties, such as
in B30–B32, B34 and F6. All these derivatives possessed
KI . 20 nM against these three MMPs. More effective
inhibitors (KI in the range 5–20 nM) were the aromatic
derivatives, containing moieties such as 4-halogenophenyl,
nitrophenyl, boc-aminophenyl, etc. (B9–B12, B14–B20,
B23, B24, B27, D1–D5, F1–F5, H1–H3 and J1). Several
of the new inhibitors showed affinities in the 0.5–3.0 nM
range. These were: (i) the perfluoroalkyl/aryl-sulfonyl
compounds B4, B5 and B21/B22; (ii) the carboxyphenyl-
substituted derivatives B28 and B29; and (iii) the elon-
gated derivatives M1–M5 and N1–N5.
Inhibition of the bacterial collagenase ChC with the new
compounds reported here generally paralleled the MMP-1
inhibition data. Thus, potent inhibitors were obtained from
all classes of hydroxamates investigated here, such as the
alkyl/arylsulfonyl-4-nitrobenzyl-Gly derivatives (B5,
B20–B22, B26, B27, B29, B33, B34, etc.), the arylsul-

A. Scozzafava et al. / European Journal of Pharmaceutical Sciences 11 (2000) 69–79
77
Table 3
Inhibition of MMPs and ChC with hydroxamates of types D, F, H and J
a
R
Compound
KI (nM)
MMP-1b
MMP-2b
MMP-8b
MMP-9b
ChCc
Ph
D1
D2
D3
D4
D5
F1
F2
F3
F4
F5
F6
H1
H2
H3
J1
52
47
40
42
69
21
26
24
18
5
4
3.5
5
4
5
3.9
7
5
13
9
11
8
5
44
11
14
8
3
7
6
10
7
14
8
9
7
8
15
18
23
25
10
27
19
18
17
15
62
25
30
32
15
4-F-C6H4-
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-
–
8
10
13
10
6
36
5
.100
24
39
13
15
15
2
37
16
19
9
28
27
32
1.8
1.6
a KI values were obtained from Easson–Stedman (Bieth, 1995) plots using a linear regression program, from at least three different assays. Standard
errors were 5–10%.
b With the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.
c With FALGPA as substrate, spectrophotometrically.
fonylureas- and arylureas (such as D3, D5 and F5), the
sulfenamido-4-nitrobenzyl-Gly derivatives (such as H2 and
H3) or the thiourea J1. Thus, it seems that the S19-binding
moiety of the arylsulfonamide type, investigated for ob-
taining MMP inhibitors, can be efficiently substituted by
related moieties such as alkylsulfonyl-, arylsulfenyl-,
arylsulfonylureido-, arylureido- or benzoyl-thioureido,
without loss of the MMP/ChC inhibitory properties. In the
subseries of alkyl/arylsulfonamido derivatives (of types
B(1–35)) the best ChC inhibitory properties were corre-
lated with the presence of perfluoroalkylsulfonyl- (B4,
B5),
perfluorophenylsulfonyl-
(B21),
3-trifluoro-
methylphenylsulfonyl- (B22), 3-chloro-4-nitro-phenylsul-
fonyl- (B17), 3- or 4-protected-amino-phenylsulfonyl-
Table 4
Inhibition of MMPs and ChC with carboxylates and hydroxamates of types K, L, M and N
a
Compound
KI (nM)
MMP-1b
MMP-2b
MMP-8b
MMP-9b
ChCc
K1
K2
K3
K4
K5
L1
L2
L3
L4
L5
M1
M2
M3
M4
M5
N1
N2
N3
N4
N5
.100
.100
.100
.100
.100
.100
.100
.100
.100
.100
69
19
15
23
27
29
21
18
19
23
24
25
27
32
31
34
35
30
29
38
27
29
31
28
29
36
35
39
38
31
32
34
37
39
46
15
11
10
17
21
9
25
27
38
36
29
28
0.7
0.6
0.7
0.9
1.0
1.1
1.4
1.6
2.3
2.5
0.7
0.8
1.4
1.7
1.5
1.3
1.9
1.6
2.3
2.5
0.8
1.5
1.3
0.8
0.9
1.1
2.7
1.6
3.0
3.0
55
56
57
70
62
55
57
50
13
20
24
25
55
a KI values were obtained from Easson–Stedman (Bieth, 1995) plots using a linear regression program, from at least three different assays. Standard
errors were 5–10%.
b With the thioester substrate Ac-ProLeuGly-S-LeuLeuGlyOEt, spectrophotometrically.
c With FALGPA as substrate, spectrophotometrically.

78
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(B18–B20, B26), and 3- or 4-carboxy-phenylsulfonyl-
(B28, B29). All these derivatives possessed inhibition
constants in the range 5–10 nM against ChC. A second
group of sulfonamide inhibitors, containing moieties such
as 4-bromophenyl, 4-iodophenyl, 2-, 3- or 4-nitrophenyl,
2,5-dichlorophenyl-, 4-methoxyphenyl- or 2-thienyl sub-
stituting the N-4-nitrobenzyl-b-alanine hydroxamate, be-
haved as medium potency inhibitors, with affinities in the
12–30 nM range. The least active sulfonamides were those
containing methyl-, trihalomethyl-, dimethylamino-,
phenyl- and benzyl moieties. The arylsulfonylureido com-
pounds D1–D4 were more active than the corresponding
arylsulfonyl derivatives (compare, for instance, D1 with
B9, D2 and B10, etc.), acting as strong–medium potency
ChC inhibitors. Behaving similarly were the ureas of type
F, and the sulfenamides of type H. A very potent inhibitor
was the thiourea derivative J1. The elongated molecule
compounds of types K–N were slightly less effective as
compared to the previously mentioned derivatives.
In conclusion, we describe here a novel class of strong
inhibitors of the zinc proteases MMP-1, MMP-2, MMP-8,
MMP-9 and ChC (EC 3.4.24.3), a collagenase from
Clostridium histolyticum. The drug design has been real-
ized by utilizing X-ray data for the adduct of some MMPs,
with inhibitors of the sulfonyl amino acid hydroxamate
type. Reaction of N-4-nitrobenzyl-b-alanine with sulfonyl
chlorides, arylsulfonyl isocyanate, aryl isocyanates or
benzoyl isothiocyanate afforded the b-alanine derivatives
which were subsequently converted into the corresponding
hydroxamates. The best substitutions for obtaining high
affinity inhibitors (0.5–2.5 nM) involved hydrophobic
moieties at S19, such as perfluoroalkylsulfonyl-, sub-
stituted-arylsulfonyl, pentafluorophenylsulfonyl, 3- and 4-
carboxy-phenylsulfonyl-, 3-trifluoromethyl-phenylsulfonyl,
among others. Some of the new inhibitors possessing
elongated moieties at P19 also showed selectivity for the
deep-pocket enzymes (MMP-2, MMP-8 and MMP-9) over
MMP-1 and ChC.
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Acknowledgements
This research was financed by EU grant ERB CIPDCT
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