Protease inhibitors: Synthesis of a series of bacterial collagenase inhibitors of the sulfonyl amino acyl hydroxamate type

Article abstract of DOI:10.1021/jm010087e

A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzylL-alanine was prepared. Related compounds were obtained by reaction of N-2-nitrobenzyl-LAla with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the conversion of the COOH into the CONHOH moiety. The new compounds were assayed as inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in the degradation of extracellular matrix. Many of the obtained hydroxamates proved to be effective bacterial collagenase inhibitors, the main contributor to activity being the substitution pattern at the sulfonamido moiety. The best ChC inhibitors were those containing pentafluorophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P₁ groups among others, with affinities in the low nanomolar range. This study also proves that the 2-nitrobenzyl- moiety, similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2000, 43, 1858-1865) is an efficient P₂ anchoring moiety for obtaining potent bacterial collagenase inhibitors.

Full text of DOI:10.1021/jm010087e

J . Med. Chem. 2001, 44, 2253-2258
2253
P r otea se In h ibitor s: Syn th esis of a Ser ies of Ba cter ia l Colla gen a se In h ibitor s of
th e Su lfon yl Am in o Acyl Hyd r oxa m a te Typ e
Brian W. Clare,*,† Andrea Scozzafava,‡ and Claudiu T. Supuran*,‡
Department of Chemistry, The University of Western Australia, W.A. Australia 6009, and Universita´ degli Studi,
Laboratorio di Chimica Inorganica e Bioinorganica, Via Gino Capponi 7, I-50121, Florence, Italy
Received February 27, 2001
A series of sulfonyl amino acyl hydroxamates incorporating alkyl/arylsulfonyl-N-2-nitrobenzyl-
L-alanine was prepared. Related compounds were obtained by reaction of N-2-nitrobenzyl-L-
Ala with aryl isocyanates, arylsulfonyl isocyanates, or benzoyl isothiocyanate, followed by the
conversion of the COOH into the CONHOH moiety. The new compounds were assayed as
inhibitors of the Clostridium histolyticum collagenase (ChC), a bacterial protease involved in
the degradation of extracellular matrix. Many of the obtained hydroxamates proved to be
effective bacterial collagenase inhibitors, the main contributor to activity being the substitution
pattern at the sulfonamido moiety. The best ChC inhibitors were those containing pentafluo-
rophenylsulfonyl and 3- and 4-protected-aminophenylsulfonyl P1′ groups among others, with
affinities in the low nanomolar range. This study also proves that the 2-nitrobenzyl- moiety,
similarly to the 4-nitrobenyl one previously investigated (Scozzafava, A.; Supuran, C. T. J .
Med. Chem. 2000, 43, 1858-1865) is an efficient P2′ anchoring moiety for obtaining potent
bacterial collagenase inhibitors.
In tr od u ction
substrates. In fact the crude homogenate of Clostridium
histolyticum, which contains several distinct collagenase
isozymes,11,12 is the most efficient system known for the
degradation of connective tissue, being also involved in
the pathogenicity of this and related clostridia, such as,
among others, C. perfringens, which causes human gas
gangrene and food poisoning.13 Typically, these bacteria
(and their collagenases) cause so much damage and so
quickly, that common antibiotics now available are
ineffective. Thus, development of inhibitors against
these collagenases represents an interesting target for
drug design. Similarly to the vertebrate MMPs, Clostrid-
ium histolyticum collagenase (abbreviated as ChC)
contains the conserved HExxH zinc-binding motif,
which in this specific case is constituted by 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.14,15 Like the
MMPs, ChC is a multiunit protein, consisting of four
segments, S1, S2a, S2b, and S3, with S1 incorporating
the catalytic domain.14 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.11-15
One must also mention that this bacterial collagenase
has not been crystallized to the present, and no X-ray
crystallographic/NMR structures are available in order
to assist the design of active site directed inhibitors,
although much effort has been spent in our and other
laboratories for this task.
Protease inhibitors proved recently to be of high
clinical value in the management of many diseases,
especially considering the extensively investigated viral
proteases of the human immunodeficiency virus (HIV)
or human hepatitis B virus (HBV) among others.1 In
contrast to such viral proteases, bacterial proteases have
only marginally been studied for the development of
specific inhibitors, although such agents would consti-
tute a new generation of antibiotics,2 free (at least in
the beginning) of the pathogen resistance to antibiotics
problem, observed with almost all the bacterial cell wall
biosynthesis inhibitors used nowadays as antibacterial
agents.1-3
Another class of proteolytic enzymes much investi-
gated ultimately for the development of specific inhibi-
tors with pharmaceutical applications is constituted by
the matrix metalloproteinases (MMPs). Inhibitors of
these zinc enzymes were extensively studied in recent
years, as these proteases are involved in many crucial
physiologic and pathologic processes connected with
extracellular matrix (ECM) degradation.4-10 The same
situation is not true for the inhibitors of related enzymes
that degrade ECM, such as the bacterial collagenases
isolated from Clostridium histolyticum, which were
much less investigated. This collagenase (EC 3.4.24.3)
is a 116 kDa protein belonging to the M31 metallopro-
teinase family,11,12 being able to efficiently hydrolyze
triple helical regions of collagens under physiological
conditions as well as an entire range of synthetic peptide
* Corresponding authors. B.W.C.: (address) Department of Chem-
istry, The University of Western Australia, 35 Stirling Highway,
Crawley, WA 6009 Australia; (phone) +61-8-9337 7824; (fax) +61-8-
9380-1005; (e-mail) bwc@crystal.uwa.edu.au. C.T.S.: (fax) +39-055-
2757555; (e-mail) claudiu.supuran@unifi.it.
In previous contributions16-19 we have shown that
potent inhibitors of ChC of the sulfonylated amino acid
hydroxamate type can be developed, in analogy with the
same type of compounds reported to act as MMP
inhibitors.19-22 In this paper we extend our previous
† The University of Western Australia.
‡ Universita´ degli Studi.
10.1021/jm010087e CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/24/2001

2254 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Clare et al.
Ta ble 2. Inhibition of ChC with the Carboxylic Acids of
Types C, E, G, I and the Corresponding Hydroxamates of
Types D, F , H, J
Ta ble 1. Inhibition of Clostridium Histolyticum Collagenase
(ChC) with the Carboxylic Acids A1-A35 and the
Corresponding Hydroxamates B1-B35
R
compd KIa (µM) compd KIa (nM)
CH3
CF3
CCl3
n-C4F9-
n-C8F17
Me2N-
Al
A2
A3
A4
A5
A6
A7
A8
22
5.1
5.0
2.1
1.7
B1
B2
B3
B4
B5
B6
B7
B8
86
70
64
10
8
79
59
55
30
32
33
34
41
13
10
11
9
12
9
8
10
5
5
14
19
21
9
10
8
43
C6H5-
27
18
12
11
10
11
17
PhCH2-
4-F-C6H4-
4-CI-C6H4-
4-Br-C6H4-
4-I-C6H4-
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
B1l
B12
B13
B14
B15
B16
B17
B18
B19
B20
B21
B22
B23
B24
B25
B26
B27
B28
B29b
B30b
B31
B32
B33
B34
B35
R
compd
KIa (µM)
compd
KIa (nM)
4-CH3-C6H4-
4-O2N-C6H4-
Ph
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-
C1
C2
C3
C4
C5
E1
E2
E3
E4
E5
E6
G1
G2
G3
I1
3.6
3.0
2.7
3.1
3.0
6.2
6.6
6.8
6.5
5.3
5.0
6.0
6.2
5.2
0.7
D1
D2
D3
D4
D5
F1
F2
F3
F4
F5
F6
H1
H2
H3
J 1
12
8
7
10
9
15
17
19
12
14
10
13
12
9
5.0
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
5.2
4.4
3.1
3.3
2.6
2.5
2.0
0.3
0.4
3.4
5.7
6.0
2.5
2.7
2.1
1.9
1.6
1.8
2.1
2.0
2.0
8
a
KI-s values were obtained from Dixon plots using a linear
regression program, from at least three different assays. Errors
were around (10% (from at least three determinations).
7
6
8
9
10
9
Sch em e 1
quinoline-8-yl
a
KI-s values were obtained from Dixon plots using a linear
regression program, from at least three different assays. Errors
b
were around (10% (from at least three determinations). The
C6H4-COOH moiety transformed into C6H4-CONHOH.
work in the design of bacterial protease inhibitors16-19
and report the preparation of a series of ChC inhibitors
incorporating alkyl/arylsulfonamido-L-alanine hydrox-
amate as well as arylsulfonylureido/arylureido-L-alanine
hydroxamate moieties in their molecule. Some of the
new compounds, assayed for the inhibition of ChC,
showed high affinity for this enzyme (in the nanomolar
range), behaving as some of the best ChC sulfonylated
inhibitors reported up to now. The compounds prepared
and tested in this study are presented in Tables 1
and 2.
tion of alkyl/arylsulfonyl chlorides with the L-alanine
derivative 3 (Scheme 1). Conversion of the carboxylic
acids A1-A35 into the corresponding hydroxamates
B1-B35 was done with hydroxylamine and diisopropyl
carbodiimide (Scheme 1).9,23-26
Resu lts a n d Discu ssion
Syn th esis. The compounds reported here were ob-
tained as shown in Schemes 1-3, as for similar deriva-
tives previously reported by our or other groups.16-22
Reaction of 2-nitrobenzyl chloride 1 with L-alanine 2
afforded the key intermediate, N-2-nitrobenzyl-L-Ala 3.
Carboxylic acids A1-A35 were then prepared by reac-
Other derivatives (C1-C5; D1-D5 and E1-E6; F 1-
F 6) were obtained by reaction of arylsulfonylisocyanates
4 or aryl isocyanates 5 with N-2-nitrobenzyl-L-Ala 3,
followed by the conversion of the COOH moiety into the
CONHOH one, as described above (Schemes 2 and
3).25,27

Synthesis of Sulfonyl Amino Acyl Hydroxamates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2255
Sch em e 2
observed that, in addition to acting as strong MMP
inhibitors,4,20 compounds of this type are also effective
bacterial collagenase (ChC) inhibitors, with potencies
in the nanomolar range in some cases.16-19 It appeared
thus of interest to extend the study of this type of
protease inhibitors as well as the relationship governing
structure-activity correlations in this class of biologi-
cally active compounds.
The new inhibitors reported here were obtained by
routine procedures involving the reaction of amino acids
with benzyl/alkyl/arylsulfonyl halides, followed by con-
version of the carboxy moiety to the hydroxamate one
(Scheme 1).16-20 Derivatives A1-A35 and B1-B35 were
obtained in this way. Alternatively, derivatization of the
amino acid derivative 3 with arylsulfonyl isocyanates
4 or aryl isocyanates 5 afforded urea derivatives which
were then similarly transformed into the corresponding
hydroxamic acids D1-D5 and F 1-F 6 (Schemes 2 and
3).
Sch em e 3
Ch C In h ibitor y Activity. Inhibition data against
type II ChC with the compounds reported in the present
paper are shown in Tables 1 and 2. The potency of
standard and newly obtained inhibitors 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.28
The following should be noted regarding ChC inhibi-
tion data of Tables 1 and 2: (i) all hydroxamates were
50-500 more active as ChC inhibitors as compared to
the corresponding carboxylic acids, probably due to the
enhanced Zn(II) coordinating properties of the CON-
HOH moiety (bidentate binding) as compared to the
COOH group (generally monodentate binding to the zinc
ion);6-9 (ii) potent inhibitors were obtained from all
classes of hydroxamates investigated here, such as the
alkyl/arylsulfonyl-N-2-nitrobenzyl-Ala derivatives (B5;
B15-B24; B27, B29-B35), the arylsulfonylureas- and
arylureas (such as D2-D5, F 5, F 6), the sulfenamido-
benzyl-Ala derivatives (such as H3), or the thiourea J 1.
Thus, it seems that the S1′-binding moiety of the
arylsulfonamide type, previously investigated for the
obtaining of MMP inhibitors,9,10,21 can be efficiently
substituted by related moieties such as alkylsulfonyl-;
arylsulfenyl-; arylsulfonylureido-; arylureido-; or ben-
zoyl-thioureido, without loss of the ChC/MMP inhibitory
properties; (iii) in the subseries of alkyl/arylsulfonamido
derivatives (of types A,B(1-35)) the best ChC inhibitory
properties were correlated with the presence of perfluo-
roalkylsulfonyl- (B4, B5), perfluorophenylsulfonyl- (B22);
3-trifluoromethylphenylsulfonyl- (B23); 3-chloro-4-ni-
trophenylsulfonyl- (B17); 3- or 4- protected-aminophen-
ylsulfonyl- (B18-B20; B27); 3- or 4-carboxyphenylsul-
fonyl- (B29, B30); and 1- or 2-naphthylsulfonyl as well
as 8-quinolinesulfonyl moieties (B30-B35). All these
derivatives possessed inhibition 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-fluorophenyl (B9), 4-nitrophenyl (B14), 2,5-dichloro-
phenyl- (B24), 4-methoxyphenyl- (B25), or 2,4,6-tri-
methylphenyl- (B26) substituting the N-2-nitrobenzyl-
L-Ala hydroxamate, behaved as medium potency inhibi-
tors, with affinities in the 13-30 nM range (Table 1).
The least active sulfonamides were those containing
By applying synthetic strategies related to the previ-
ously described ones, the sulfenamido derivatives G1-
G3 and H1-H3 as well as the thioureas I1 and J 1 were
also prepared.
MacPherson et al.20a originally reported that sulfo-
nylated amino acid hydroxamates act as powerful MMP
inhibitors, and this type of compounds was then further
explored by other researchers.9,16-19,20b It was thus

2256 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Clare et al.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
C1-C5; E1-E6; a n d I1. An amount of 2.10 g (10 mMol) of
N-2-nitrobenzyl-L-Ala 3 and the stoichiometric amount of
arylsulfonyl isocyanate 4, aryl isocyanate 5, or benzoyl isothio-
cyariate was suspended in 50 mL of anhydrous acetone, and
150 µL (10 mM) of triethylamine was 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.
methyl-; trihalomethyl-; dimethylamino-; phenyl-; and
benzyl moieties (Table 1); (iv) the arylsulfonylureido
compounds D1-D5 were more active than the corre-
sponding arylsulfonyl derivatives (compare for instance
D2 with B9; D3 and B10, etc.), acting as strong ChC
inhibitors. The ureas of type F and the sulfenamides of
type H behaved similarly, except for F 2 and F 3, which
are somehow weaker inhibitors. A potent inhibitor is
the thiourea derivative J 1 (Table 2).
Exp er im en ta l Section
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
G1-G3. The general procedure described above for the
preparation of compounds A1-A35 has been followed, except
that arylsulfenyl halides were used instead of alkyl/arylsul-
fonyl halides. The yields in the title sulfenamides were around
70%.
Gen er a l Meth od s. Melting points: heating plate micro-
scope (not corrected); IR spectra: KBr pellets, 400-4000 cm-1
Perkin-Elmer 16PC FTIR spectrometer; 1H NMR spectra:
Varian Gemmini 200 apparatus (chemical shifts are expressed
as δ values relative to Me4Si as standard); elemental analysis
((0.4% of the theoretical values, calculated for the proposed
formulas for all the compounds reported here): Carlo Erba
Instrument CHNS Elemental Analyzer, Model 1106. All reac-
tions were monitored by thin-layer chromatography (TLC)
using 0.25-mm precoated silica gel plates (E. Merck). Prepara-
tive HPLC was performed on a Dynamax-60A column (25 ×
250 mm), with a Beckman EM-1760 instrument. The detection
wavelength was 254 nm.
Amino acids (L-Ala), 4-nitrobenzyl chloride, sulfonyl chlo-
rides, arylsulfonyl isocyanates, aryl isocyanates, benzoyl isothio-
cyanate, triethylamine, carbodiimides, hydroxylamine, FAL-
GPA, buffers, and other reagents used in the syntheses were
commercially available compounds, from Sigma, Acros, or
Aldrich.
P r ep a r a tion of N-2-Nitr oben zyl-L-a la n in e 3. An amount
of 8.6 g (0.10 M) of L-Ala 2 and the stoichiometric amount of
2-nitrobenzyl chloride (16.1 g) were suspended/dissolved in 150
mL of anhydrous acetonitrile, and the equivalent amount of
triethylamine (0.10 mM, 14.7 mL) was added. The reaction
mixture was stirred at room temperature for 10 h, and then
the solvent was evaporated in vacuo. The obtained reaction
mixture was taken in 200 mL of water, the pH was brought
to 7 with citric acid, and the crude product 3 precipitated by
leaving the mixture overnight at 4 °C. Recrystallization from
ethanol afforded the pure title compound in almost quantita-
tive yield.
Gen er a l P r oced u r e for th e P r ep a r a tion of N-2-Ni-
t r ob en zyl-a lk yl/a r ylsu lfon yl L-Ala n in es A1-A35. An
amount of 2.10 g (10 mMol) of N-2-nitrobenzyl-L-Ala 3 and 10
mmol of sulfonyl chloride were suspended/dissolved in 100 mL
of acetone + 25 mL of water. The stoichiometric amount (10
mmol) of base (NaHCO3, KHCO3, NaOH, or Et3N) dissolved
in a small amount (20 mL) of water was added, and the
mixture was 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 was
extracted in ethyl acetate. After evaporation of the solvent,
the compounds A1-A35 were recrystallized from EtOH or
MeOH. Yields were around 75-90%.
1
All the new compounds were characterized by H- and 13C
NMR spectroscopy and elemental analysis. Data for a repre-
sentative compound of each series is provided below.
N-4-Tolu en esu lfon yl-N-2-n itr oben zyl-L-alan in e A13. Pale
1
yellow crystals, mp 171-2 °C; H NMR (DMSO-d6), δ, ppm:
1.53 (d, 3J HH ) 6.5, 3H, CHCH3 of Ala), 2.55 (s, 3H, CH3C6H4),
3.73 (s, 2H, CH2 of benzyl); 3.90 (q, 1H, CH of Ala); 7.20-7.59
(m, 6H, Hortho of CH3C6H4 and Harom of 2-O2N-C6H4), 7.92 (d,
3J HH ) 8.1, 2H, Hmeta of CH3C6H4); 11.54 (br s, 1H, COOH);
13C NMR (DMSO-d6), δ, ppm: 20.1 (s, CHCH3 of Ala); 26.4 (s,
CH3C6H4), 34.5 (s, CHCH3 of Ala); 43.0 (s, CH2 of benzyl), 127.2
(s, C-5 of 2-O2N-C6H4); 129.2 (C-4 of 2- O2N-C6H4); 129.8 (C-3
of 2-O2N-C6H4); 130.1 (s, Cmeta of CH3C6H4), 130.9 (C-6 of 2-
O2N-C6H4); 134.0 (C-2 of 2- O2N-C6H4); 135.4 (C-1 of 2- O2N-
C6H4); 135.7 (s, Cortho of CH3C6H4), 145.5 (s, Cipso of CH3C6H4),
148.8 (s, Cpara of CH3C6H4), 177.3 (s, CO2H). Anal. Found: C,
52.48; H, 4.61; N, 7.67%. C16H16N2O6S requires: C, 52.74; H,
4.43; N, 7.69%.
N-4-Tolu en esu lfon yl-N-2-n itr oben zyl-L-alan in e h ydr ox-
a m a te B13. Pale yellow crystals, mp 213-5 °C; 1H NMR
3
(DMSO-d6), δ, ppm: 1.53 (d, J HH ) 6.5, 3H, CHCH3 of Ala),
2.65 (s, 3H, CH3C6H4), 3.79 (s, 2H, CH2 of benzyl); 3.98 (q, 1H,
CH of Ala); 7.14-7.68 (m, 6H, Hortho of CH3C6H4 and Harom of
2- O2N-C6H4), 8.10 (d, 3J HH ) 8.1, 2H, Hmeta of CH3C6H4); 8.78
(br s, 1H, NHOH); 10.56 (br s, 1H, NHOH); 13C NMR (DMSO-
d6), δ, ppm: 20.4 (s, CHCH3 of Ala); 26.1 (s, CH3C6H4), 34.3
(s, CHCH3 of Ala); 44.7 (s, CH2 of benzyl), 127.0 (s, C-5 of
2-O2N-C6H4); 129.5 (C-4 of 2-O2N-C6H4); 129.9 (C-3 of 2-
O2N-C6H4); 130.6 (s, Cmeta of CH3C6H4), 130.8 (C-6 of 2- O2N-
C6H4); 134.7 (C-2 of 2- O2N-C6H4); 135.3 (C-1 of 2- O2N-C6H4);
135.8 (s, Cortho of CH3C6H4), 145.2 (s, Cipso of CH3C6H4), 148.0
(s, Cpara of CH3C6H4), 175.6 (s, CONHOH). Anal. Found: C,
50.69; H, 4.87; N, 10.98%. C16H17N3O6S requires: C, 50.65;
H, 4.52; N, 11.08%.
N -4-Tolu e n e su lfon ylu r e id o-N -2-n it r ob e n zyl-L -a la -
n in e C3. Pale yellow crystals, mp 274-5 °C; 1H NMR (DMSO-
3
d6), δ, ppm: 1.52 (d, J HH ) 6.5, 3H, CHCH3 of Ala), 2.57 (s,
3H, CH3C6H4), 3.79 (s, 2H, CH2 of benzyl); 3.95 (q, 1H, CH of
Ala); 7.17-7.68 (m, 6H, Hortho of CH3C6H4 and Harom of 2-O2N-
3
C6H4), 7.99 (d, J HH ) 8.2, 2H, Hmeta of CH3C6H4); 8.25 (br s,
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
B1-B35; D1-D5; F 1-F 6; H1-H3; a n d J 1. 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 and treated with 420 mg (6 mM) of
hydroxylamine‚HCI and 1.10 g (6 mM) of EDCI‚HCl or
diisopropyl-carbodiimide. The reaction mixture was magneti-
cally stirred at room temperature for 15 min, then 180 µL (12
mM) of triethylamine was added, and stirring was continued
for 12 h at 4 °C. The solvent was evaporated in vacuo, and
the residue was 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 was removed in
vacuo. Preparative HPLC (Dynamax-60A column (25 × 250
mm); 90% acetonitrile/10% methanol; flow rate of 30 mL/min)
afforded the pure hydroxamic acids.
2H, NHCONH); 11.73 (br s, 1H, COOH); 13C NMR (DMSO-
d6), δ, ppm: 20.1 (s, CHCH3 of Ala); 26.4 (s, CH3C6H4), 34.7
(s, CHCH3 of Ala); 43.8 (s, CH2 of benzyl), 127.0 (s, C-5 of
2-O2N-C6H4); 129.3 (C-4 of 2-O2N-C6H4); 129.9 (C-3 of
2-O2N-C6H4); 130.5 (s, Cmeta of CH3C6H4), 130.9 (C-6 of 2-
O2N-C6H4); 132.7 (s, NHCONH), 134.1 (C-2 of 2- O2N-C6H4);
135.0 (C-1 of 2-O2N-C6H4); 135.6 (s, Cortho of CH3C6H4), 145.8
(s, Cipso of CH3C6H4), 148.6 (s, Cpara of CH3C6H4), 177.9 (s,
CO2H). Anal. Found: C, 50.09; H, 4.35; N, 10.21%. C17H17N3O7S
requires: C, 50.12; H, 4.21; N, 10.31%.
N -4-Tolu e n e su lfon ylu r e id o-N -2-n it r ob e n zyl-L -a la -
n in e Hyd r oxa m a te D3. Pale yellow crystals, mp 229-30 °C;
3
1H NMR (DMSO-d6), δ, ppm: 1.54 (d, J HH ) 6.5, 3H, CHCH3
of Ala), 2.61 (s, 3H, CH3C6H4), 3.82 (s, 2H, CH2 of benzyl); 3.95
(q, 1H, CH of Ala); 7.12-7.60 (m, 6H, Hortho of CH3C6H4 and
3
Harom of 2- O2N-C6H4), 7.99 (d, J HH ) 8.3 Hz, 2H, Hmeta of

Synthesis of Sulfonyl Amino Acyl Hydroxamates
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2257
CH3C6H4); 8.29 (br s, 2H, NHCONH); 8.75 (br s, 1H, NHOH);
10.66 (br s, 1H, NHOH); 13C NMR (DMSO-d6), δ, ppm: 20.6
(s, CHCH3 of Ala); 26.7 (s, CH3C6H4), 34.4 (s, CHCH3 of Ala);
44.5 (s, CH2 of benzyl), 127.0 (s, C-5 of 2-O2N-C6H4); 129.3
(C-4 of 2-O2N-C6H4); 129.9 (C-3 of 2- O2N-C6H4); 130.5 (s,
Cmeta of CH3C6H4), 130.9 (C-6 of 2- O2N-C6H4); 132.8 (s,
NHCONH), 134.0 (C-2 of 2- O2N-C6H4); 135.1 (C-1 of 2- O2N-
C6H4); 135.6 (s, Cortho of CH3C6H4), 145.6 (s, Cipso of CH3C6H4),
148.9 (s, Cpara of CH3C6H4), 174.3 (s, CONHOH). Anal. Found:
C, 48.60; H, 4.13; N, 13.21%. C17H18N4O7S requires C, 48.34;
H, 4.30; N, 13.26%.
En zym e P r ep a r a tion s. Clostridium histolyticum highly
purified collagenase and its substrate FALGPA (furanacryloyl-
leucyl-glycyl-prolyl-alanine) were purchased from Sigma Chemi-
cal Co. (Milano, Italy), and their concentrations were deter-
mined 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 deter-
mined from the inhibition of the enzymatic (amidolytic) activity
of the collagenase, at 25 °C, using FALGPA as substrate, by
the method of van Wart and Steinbrink.28 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 ꢀ305 ) 24 700 M-1 cm-1
in the above-mentioned reaction buffer.28 Measurements were
made using a Cary 3 spectrophotometer interfaced with a PC.
Initial velocities were thus estimated using the direct linear
plot-based procedure reported by van Wart and Steinbrink.28
KI-s were then determined according to Dixon plots and a
linear regression program.
N-4-Flu or oph en ylu r eid o-N-2-n itr oben zyl-L-a la n in e E1.
1
Pale yellow crystals, mp 169-70 °C; H NMR (DMSO-d6), δ,
ppm: 1.54 (d, 3J HH ) 6.5, 3H, CHCH3 of Ala), 3.78 (s, 2H, CH2
of benzyl); 3.96 (q, 1H, CH of Ala); 7.11-7.69 (m, 6H, Hortho of
3
4-FC6H4 and Harom of 2-O2N-C6H4), 7.95 (d, J HH ) 8.1, 2H,
Hmeta of 4-FC6H4); 8.15 (br s, 2H, NHCONH); 11.47 (br s, 1H,
COOH); 13C NMR (DMSO-d6), δ, ppm: 20.2 (s, CHCH3 of Ala);
34.5 (s, CHCH3 of Ala); 43.8 (s, CH2 of benzyl), 127.8 (s, C-5 of
2-O2N-C6H4); 129.7 (C-4 of 2-O2N-C6H4); 129.9 (C-3 of 2-
O2N-C6H4); 130.1 (s, Cmeta of FC6H4), 130.8 (C-6 of 2- O2N-
C6H4); 132.6 (s, NHCONH), 134.1 (C-2 of 2- O2N-C6H4); 135.0
(s, Cortho of FC6H4), 135.7 (C-1 of 2-O2N-C6H4); 148.9 (s, Cipso
of FC6H4), 149.8 (s, Cpara of FC6H4), 177.9 (s, CO2H). Anal.
Found: C, 55.29; H, 4.24; N, 12.06%. C16H14FN3O5 requires:
C, 55.33; H, 4.06; N, 12.10%.
Ack n ow led gm en t. This research was financed in
part by the EU grant ERB CIPDCT 940051 and by a
grant from the Italian CNR (Target Project Biotechnol-
ogy).
N-4-Flu or oph en ylu r eido-N-2-n itr oben zyl-L-alan in e Hy-
d r oxa m a te F 1. Pale yellow crystals, mp 219-20 °C; 1H NMR
Refer en ces
3
(DMSO-d6), δ, ppm: 1.56 (d, J HH ) 6.5, 3H, CHCH3 of Ala),
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3.80 (s, 2H, CH2 of benzyl); 3.97 (q, 1H, CH of Ala); 7.10-7.62
(m, 6H, Hortho of 4-FC6H4 and Harom of 2- O2N-C6H4), 7.93 (d,
3J HH ) 8.1, 2H, Hmeta of 4-FC6H4); 8.16 (br s, 2H, NHCONH);
8.79 (br s, 1H, NHOH); 10.64 (br s, 1H, NHOH); 13C NMR
(DMSO-d6), δ, ppm: 20.2 (s, CHCH3 of Ala); 34.7 (s, CHCH3
of Ala); 43.7 (s, CH2 of benzyl), 127.0 (s, C-5 of 2- O2N-C6H4);
129.5 (C-4 of 2- O2N-C6H4); 129.9 (C-3 of 2- O2N-C6H4); 130.3
(s, Cmeta of FC6H4), 130.8 (C-6 of 2- O2N-C6H4); 132.6 (s,
NHCONH), 134.4 (C-2 of 2- O2N-C6H4); 135.1 (s, Cortho of
FC6H4), 135.7 (C-1 of 2- O2N-C6H4); 135.6 (s, Cortho of FC6H4),
145.7 (s, Cipso of FC6H4), 148.5 (s, Cpara of FC6H4), 174.5 (s,
CONHOH). Anal. Found: C, 52.90; H, 4.13; N, 15.33%. C16H15
-
FN4O5 requires: C, 53.04; H, 4.17; N, 15.46%.
N-4-Nitr oph en ylsu lfen yl-N-2-n itr oben zyl-L-alan in e G1.
1
Yellow crystals, mp 228-9 °C; H NMR (DMSO-d6), δ, ppm:
1.55 (d, 3J HH ) 6.5, 3H, CH3 of Ala), 3.79 (s, 2H, CH2 of benzyl);
3.90 (q, 1H, CH of Ala); 6.75 (s, 1H, SNH), 7.10-7.66 (m, 6H,
Hortho of 4-O2N-C6H4 and Harom of 2- O2N-C6H4), 8.09 (d,
3J HH ) 8.3, 2H, Hmeta of 4-O2N-C6H4); 11.78 (br s, 1H, COOH);
13C NMR (DMSO-d6), δ, ppm: 20.4 (s, CHCH3 of Ala); 34.3 (s,
CHCH3 of Ala); 43.8 (s, CH2 of benzyl), 127.1 (s, C-5 of 2-O2N-
C6H4); 129.8 (C-4 of 2-O2N-C6H4); 129.9 (C-3 of 2-O2N-C6H4);
130.0 (s, Cmeta of 4- O2N-C6H4), 130.3 (C-6 of 2-O2N-C6H4);
134.1 (C-2 of 2- O2N-C6H4); 135.2 (C-1 of 2- O2N-C6H4); 135.5
(s, Cortho of 4-O2N-C6H4), 145.0 (s, Cipso of 4-O2N-C6H4), 150.8
(s, Cpara of 4-O2N-C6H4), 177.6 (s, CO2H). Anal. Found: 49.62;
H, 3.70; N, 11.49%.C15H13N3O6S requires: C, 49.58; H, 3.61;
N, 11.56%.
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N-4-Nitr oph en ylsu lfen yl-N-2-n itr oben zyl-L-alan in e Hy-
d r oxa m a te H1. Yellow crystals, mp 248-9 °C; 1H NMR
(DMSO-d6), δ, ppm: 1.55 (d, 3J HH ) 6.5, 3H, CH3 of Ala), 3.78
(s, 2H, CH2 of benzyl); 3.96 (q, 1H, CH of Ala); 6.83 (s, 1H,
SNH), 7.15-7.68 (m, 6H, Hortho of 4-O2N-C6H4 and Harom of
3
2-O2N-C6H4), 8.17 (d, J HH ) 8.2, 2H, Hmeta of 4-O2N-C6H4);
8.75 (br s, 1H, NHOH); 10.70 (br s, 1H, NHOH); 13C NMR
(DMSO-d6), δ, ppm:, 20.1 (s, CHCH3 of Ala); 34.5 (s, CHCH3
of Ala); 43.8 (s, CH2 of benzyl), 127.1 (s, C-5 of 2-O2N-C6H4);
129.6(C-4 of 2- O2N-C6H4); 129.9 (C-3 of 2- O2N-C6H4); 130.2
(s, Cmeta of 4- O2N-C6H4), 130.5 (C-6 of 2-O2N-C6H4); 134.5
(C-2 of 2-O2N-C6H4); 135.0 (C-1 of 2-O2N-C6H4); 135.9 (s,
Cortho of 4- O2N-C6H4), 145.1 (s, Cipso of 4- O2N-C6H4), 150.5
(s, Cpara of 4-O2N-C6H4), 174.2 (s, CONHOH). Anal. Found:
C, 47.54, H, 3.85; N, 14.73%. C15H14N4O6S requires: C, 47.62,
H, 3.73; N, 14.81%.

2258 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
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