Protease inhibitors: Synthesis of Clostridium histolyticum collagenase inhibitors incorporating sulfonyl-L-alanine hydroxamate moieties

Article abstract of DOI:10.1016/S0960-894X(00)00026-3

A series of hydroxamates was obtained by the reaction of N-(4-nitrobenzyl)-L-alanine with alkyl/arylsulfonyl halides, followed by conversion of the COOH group into CONHOH. Structurally-related compounds were prepared similarly by using arylsulfonyl isocyanates, aryl isocyanates or arylsulfenyl halides instead of the sulfonyl halides. Many of the new compounds showed nanomolar affinity for the bacterial collagenase isolated from the pathogen Clostridium histolyticum. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00026-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 499±502
Protease Inhibitors: Synthesis of Clostridium histolyticum
Collagenase Inhibitors Incorporating Sulfonyl-L-alanine
Hydroxamate Moieties
Andrea Scozzafava and Claudiu T. Supuran*
Á
Universita degli Studi, Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, 50121 Florence, Italy
Received 20 September 1999; accepted 13 January 2000
AbstractÐA series of hydroxamates was obtained by the reaction of N-(4-nitrobenzyl)-l-alanine with alkyl/arylsulfonyl halides,
followed by conversion of the COOH group into CONHOH. Structurally-related compounds were prepared similarly by using
arylsulfonyl isocyanates, aryl isocyanates or arylsulfenyl halides instead of the sulfonyl halides. Many of the new compounds
showed nanomolar anity for the bacterial collagenase isolated from the pathogen Clostridium histolyticum. # 2000 Elsevier
Science Ltd. All rights reserved.
Introduction
bacterial corneal keratitis, a condition leading to serious
complications for which ecient cures are dicultly
envisageable.12±14 It was in fact recently reported12±14
that collagen shields applied to the cornea of patients
with bacterial keratitis degrade rapidly, due to the col-
lagenases secreted by the pathogen bacterial species, but
these shields also protect, to some extent, the corneal
collagen degradation and thus the ocular surface.12±14
The use of such a shield impregnated with an antibiotic
agent speci®c for the collagen-degrading bacteria would
thus have a double bene®t for the patient: (i) the col-
lagenase inhibitor would kill (or impair the growth of)
bacteria present on the cornea, improving and accel-
erating healing of the keratitis; (ii) the protective col-
lagen 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.12±14
Clostridium histolyticum collagenase (EC 3.4.24.3) is a
116 kDa zinc-protein belonging to the M9 metallo-
proteinase family,1 being able to hydrolyze triple helical
regions of collagen under physiological conditions, as
well as an entire range of synthetic peptide substrates,2±8
being also involved in the pathogenicity of this and
related clostridia, such as Clostridium perfringens, which
causes human gas gangrene and food poisoning among
others.2±8
Similar to the vertebrate MMP's,9,10 C. histolyticum
collagenase (abbreviated as ChC) has the conserved
HExxH zinc-binding motif, which in this speci®c case is
His415ExxH, with two histidine residues (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
cleavage.2±10 Similarly to the MMP's, ChC is also a
multiunit protein, consisting of four segments, S1, S2a,
S2b and S3,5,6 with S1 incorporating the catalytic
domain. Amino acid hydroxamates act as ecient inhi-
bitors of MMPs,11 and presumably also of ChC. Thus,
we hypothesized that this class of compounds which
strongly inhibit MMPs (collagenases, gelatinases, etc.)
would also act as potent ChC inhibitors. Our interest in
this type of enzyme inhibitors is related to the develop-
ment of pharmacological agents for the treatment of
Chemistry
In this paper we report the preparation of a series of
ChC inhibitors incorporating alkyl/arylsulfonamido-l-
alanine hydroxamate as well as arylsulfonylureido-/ary-
lureido-l-alanine hydroxamate moieties in their mole-
cule. As the ChC enzyme catalyzes the cleavage of the
Xaa-Gly peptide bond of the repeating sequence of
collagen: -Gly-Pro-Xaa-Gly-Pro-Xaa- (Xaa=amino acid
residue), it appears that the S3, S2 and S1 subsites of the
enzyme are occupied by Gly, Pro and Xaa, respec-
*Corresponding author. Tel.: +39-55-2757555; fax: +39-55-2757555;
e-mail: cts@bio.chim.uni®.it
tively.15 Analogously, the S1 , S2 and S3 subsites are
0
0
0
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00026-3

500
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 10 (2000) 499±502
also occupied by Gly, Pro and Xaa, respectively. Thus,
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); (ii) a small (compact)
group in a to it; (iii) the already optimized benzyl16±18
of per¯uoroalkylsulfonyl- (B4, B5), per¯uorophenyl-
sulfonyl- (B21), 3-tri¯uoromethylphenylsulfonyl- (B22);
3-chloro-4-nitro-phenylsulfonyl- (B17); 3- or 4-pro-
tected-amino-phenylsulfonyl- (B18±B20; B26); 3- or 4-
carboxy-phenylsulfonyl- (B28, B29), 1- or 2-naphthyl-
sulfonyl as well as 8-quinolinesulfonyl moieties (B30±
B32; B34). All these derivatives possessed inhibition
constants in the range of 5±12 nM against ChC, being
among the most potent such inhibitors ever reported. A
second group of sulfonamide inhibitors, containing
moieties such as 4-bromophenyl; 4-iodophenyl; 2-, 3- or
4-nitrophenyl; 2,5-dichlorophenyl-; 2,4,6-trimethylphe-
nyl-; 4-methoxyphenyl- or 2-thienyl, substituting the N-
benzyl-glycine hydroxamate, behaved as medium
potency inhibitors, with anities in the 15±30 nM range
(Table 1). The least active sulfonamides were those
0
group at the S2 site, converted to a 4-nitrobenzyl one;
and (iv) variable alkyl/arylsulfonyl-; arylsulfonyl-ureido/
0
arylureido-; or arylsulfenyl, moieties at S1 .16±18 The new
inhibitors were obtained as shown in Scheme 1.
The key intermediate 3 was obtained by alkylation of L-
alanine with 4-nitrobenzyl chloride. Its reaction with
sulfonyl halides 4,19 arylsulfonyl isocyanates,20 aryl iso-
cyanates,20 sulfenyl halides,21 gave the ChC inhibitors
shown in Tables 1 and 2 after the conversion of the
COOH into the CONHOH, moiety.23,24
Table 1. Inhibition22 of Clostridium histolyticum collagenase (ChC)
with the carboxylic acids A1±A35 and the corresponding hydrox-
amates B1±B35
Enzyme Inhibition
The following should be noted regarding ChC inhibi-
tion data of Tables 1 and 2 with the new compounds
reported here: (i) all hydroxamates were 100±500 more
active as ChC inhibitors as compared to the corre-
sponding carboxylic acids, probably due to the
enhanced Zn(II) coordinating properties of the CON-
HOH moiety (bidentate binding) as compared to the
COOH group (generally monodentate binding to the
zinc ion);9±16 (ii) potent inhibitors were obtained from
all classes of hydroxamates investigated here, such as
the alkyl/arylsulfonyl-N-4-nitrobenzyl-Ala derivatives
(B5; B20±B22; B26, B27, B29, etc.), the arylsulfonylur-
eas- and arylureas (such as D2, F5, F6), the sulfena-
mido-4-nitrobenzyl-Ala derivatives (such as H2, H3) or
R
Compound
KIa
(mM)
Compound
KIa
(nM)
CH3
CF3
CCl3
n-C4F9-
n-C8F17
Me2N-
C6H5-
PhCH2-
4-F-C6H4-
4-Cl-C6H4-
4-Br-C6H4-
4-I-C6H4-
4-CH3-C6H4-
4-O2N-C6H4-
3-O2N-C6H4-
2-O2N-C6H4-
3-Cl-4-O2N-C6H3-
4-AcNH-C6H4-
4-BocNH-C6H4-
3-BocNH-C6H4-
4-Ac-C6H4-
C6F5-
3-CF3-C6H4
2,5-Cl2C6H3
4-CH3O-C6H4-
2,4,6-(CH3)3-C6H2-
4-CH3O-3-BocNH-C6H3-
2-HO-3,5-Cl2-C6H2-
3-HOOC-C6H4-
4-HOOC-C6H4-
1-Naphthyl
2-Naphthyl
5-Me2N-1-naphthyl-
2-Thienyl
A1
A2
A3
A4
A5
A6
A7
A8
18
B1
B2
B3
B4
B5
B6
B7
B8
76
75
52
11
7
69
54
50
35
32
30
37
36
10
12
13
9
4.7
5.1
2.3
1.8
36
21
15
10
9
10
12
13
5.3
5.5
4.8
3.3
3.1
2.4
2.6
2.1
0.4
0.3
3.6
5.2
6.0
2.1
2.5
2.0
1.4
1.2
1.3
1.1
2.1
1.2
0
the thiourea J1. Thus, it seems that the S1 -binding
moiety of the arylsulfonamide type, previously investi-
gated for the obtaining of MMP inhibitors of type 7,16±19
can be eciently substituted by related moieties such as
alkylsulfonyl-; arylsulfenyl-; arylsulfonylureido-; arylur-
eido- or benzoyl-thioureido, without loss of the ChC
inhibitory properties; (iii) in the subseries of alkyl/aryl-
sulfonamido derivatives (of types A,B(1-35)) best ChC
inhibitory properties were correlated with the presence
A9
B9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
A33
A34
A35
B10
B11
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
B29b
B30b
B31
B32
B33
B34
B35
11
10
8
9
5
6
13
21
17
8
12
9
6
7
8
7
11
8
Quinoline-8-yl
aKI-s values were obtained from Dixon plots using a linear regression
program,4 from at least three di€erent assays. Errors were around
‹10% (from at least three determinations).
bCOOH transformed into -CONHOH.
Scheme 1.

A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 10 (2000) 499±502
501
Table 2. Inhibition22 of ChC with the carboxylic acids of types C, E, G, I and the corresponding hydroxamates of types D, F, H, J
R
Compound
KIa (mM)
Compound
Kia (nM)
4-F-C6H4-
C1
C2
C3
C4
E1
E2
E3
E4
E5
E6
G1
G2
G3
I1
3.3
2.4
2.5
3.1
5.4
6.1
6.5
6.2
5.0
4.6
5.5
6.3
5.1
0.9
D1
D2
D3
D4
F1
F2
F3
F4
F5
F6
H1
H2
H3
J1
9
7
12
8
12
14
18
15
13
10
11
7
4-Cl-C6H4-
4-CH3-C6H4-
2-CH3-C6H4-
4-F-C6H4-
3-Cl-C6H4-
4-Cl-C6H4-
2,4-F2-C6H3-
3,4-Cl2C6H3
1-Naphthyl
4-O2N-C6H4-
2-O2N-C6H4-
2,4-(O2N)2-C6H3-
Ð
9
6
aKI-s values were obtained from Dixon plots using a linear regression program, from at least three di€erent assays.4 Errors were around ‹10%
(from at least three determinations).
containing methyl-; trihalomethyl-; dimethylamino-;
phenyl- and benzyl moieties (Table 1); (iv) the arylsul-
fonylureido compounds D1±D4 were more active than
the corresponding arylsulfonyl derivatives (compare for
instance D1 with B9; D2 and B10, etc.), acting as strong
ChC inhibitors. Similarly behaved are 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).
References and Notes
1. Van Wart, H. E. Clostridium collagenases. In Handbook of
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2. Bond, M. D.; Van Wart, H. E. Biochemistry 1984, 23, 3077.
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1992, 18, 59.
8. Groos, E. B. In Cornea: Fundamentals of Cornea and
External Disease; Krachmer, J. H.; Mannis, M. J.; Holland,
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105±142.
The new class of hydroxamate inhibitors reported here
presumably binds bidentately to the Zn(II) ion of the
enzyme, as the structurally related MMP inhibitors.
Probably the methyl group provening from Ala occu-
0
pies the S1 site, the nitrobenzyl moiety the S2 site,
0
whereas the arylsulfonyl group the S1 site.9,10 These
interactions would thus explain the very strong ChC
inhibitory properties of the new compounds.
9. Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H.
Chem. Rev. 1999, 99, 2735.
Acknowledgement
10. Babine, R. E.; Bender, S. L. Chem. Rev. 1997, 97, 1359.
11. Bottomley, K. M.; Johnson, W. H.; Walter, D. S. J.
Enzyme Inhib. 1998, 13, 79±101.
This research was ®nanced in part by the EU grant ERB
CIPDCT 940051.

502
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12. Reidy, J. J.; Gebhardt, B. M.; Kaufman, H. E. Cornea
1990, 9, 196.
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19. Borras, J.; Scozzafava, A.; Menabuoni, L.; Mincione, F.;
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purchased from Sigma-Aldrich (Milan, Italy); their con-
centrations were determined from the absorbance at 280 nM
and the extinction coecients 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 inhibi-
tors was determined from the inhibition of the enzymatic
(amidolytic) activity of the collagenase, at 25 ꢀC, using
FALGPA as substrate, by the method of van Wart and
Steinbrink.4 The substrate was reconstituted as 5 mM stock in
50 mM Tricine bu€er, 0.4 M NaCl, 10 mM CaCl2, pH 7.50.
The rate of hydrolysis was determined from the change in
absorbance at 324 nM using an extinction coecient for
FALGPA E305=24,700 L mol 1 cm 1 in the above-mentioned
reaction bu€er.4 Measurements were made using a Perkin±
Elmer spectrophotometer interfaced with a PC. Initial velo-
cities were estimated using the direct linear plot-based proce-
dure reported by van Wart and Steinbrink.4 KI-s were then
determined according to Dixon, using a linear regression pro-
gram. The KI values determined are the means of at least three
determinations.
20. Scozzafava, A.; Supuran, C. T. J. Enzyme Inhib. 1999, 14,
343.
21. Briganti, F.; Scozzafava, A.; Supuran, C. T. Bioorg. Med.
Chem. Lett. 1999, 9, 2043.
22. Collagenase type VII (highly puri®ed) and its substrate,
FALGPA (furanacryloyl-leucyl-glycyl-prolyl-alanine) were
23. Scozzafava, A.; Menabuoni, L.; Mincione, F.; Briganti, F.;
Mincione, G.; Supuran, C. T. J. Med. Chem. 1999, 42, 2641.
24. Scozzafava, A.; Briganti, F.; Mincione, G.; Menabuoni,
L.; Mincione, F.; Supuran, C. T. J. Med. Chem. 1999, 42,
3690.