1864 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Scozzafava and Supuran
benzyl), 7.29-7.73 (m, 4H, Hortho of CH3C6H4 + Hortho of
O2NC6H4), 7.99 (d, 3J HH ) 8.1 Hz, 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) δ 26.1 (s, CH3C6H4),
40.8 (s, CH2 of Gly), 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.
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, Cipso of O2NC6H4S), 174.6 (s, CON-
HOH). Anal. Found: C, 47.91, H, 3.65; N, 14.69. C15H14N4O6S
Requires: C, 47.62, H, 3.73; N, 14.81.
En zym e P r ep a r a tion s. Human purified MMPs (MMP-1,
MMP-2, MMP-8 and MMP-9) were purchased from Calbiochem
(Inalco, Milano, Italy). They were activated26 in the assay
buffer by adding bovine trypsin (50 µL, 0.6 mg/mL) to the
proenzyme, followed by incubation at 37 °C for 10 min. The
trypsin was then inactivated with aprotinin (50 µL, 1.2 mg/
mL). Initial rates for the hydrolysis of the thioester substrate
AcProLeuGly-S-LeuLeuGlyOEt coupled to the reaction with
5,5′-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 Kam24a
N-4-Tolu en esu lfon ylu r eid o-N-4-n itr oben zylglycin e h y-
d r oxa m a te, D3: white crystals, mp 273-4 °C; 1H NMR
(DMSO-d6) δ 2.63 (s, 3H, CH3C6H4), 3.69 (s, 2H, CH2 of Gly),
4.36 (s, 2H, CH2 of benzyl), 7.20-7.74 (m, 4H, Hortho of CH3C6H4
3
+ Hortho of O2NC6H4), 7.99 (d, J HH ) 8.1 Hz, 2H, Hmeta of
CH3C6H4), 8.21 (d, 2H, Hmeta of O2NC6H4), 8.32 (br s, 2H,
NHCONH), 8.76 (br s, 1H, NHOH), 10.59 (br s, 1H, NHOH);
13C NMR (DMSO-d6) δ 26.3 (s, CH3C6H4), 40.9 (s, CH2 of Gly),
45.2 (s, CH2 of benzyl), 123.7 (s, Cmeta of O2NC6H4), 129.5 (Cortho
of O2NC6H4), 131.3 (s, Cmeta of CH3C6H4), 132.4 (s, NHCONH),
135.0 (s, Cortho of CH3C6H4), 144.5 (s, Cipso of O2NC6H4), 145.6
(s, Cipso of CH3C6H4), 147.7 (s, Cpara of O2NC6H4), 148.6 (s, Cpara
of CH3C6H4), 174.6 (s, CONHOH). Anal. Found: C, 48.09; H,
4.43; N, 13.12. C17H18N4O7S Requires: C, 48.34; H, 4.30; N,
13.26.
and J ohnson et al.24b The change of absorbance (ꢀ ) 19 800
24a
M-1‚cm-1
)
at 405 nm was monitored continuously at room
temperature, using a Cary 3 spectrophotometer interfaced with
a PC. A typical 100-µL reaction contained 50 mM MES, pH
6.0, 10 mM CaCl2, 100 µM substrate, 1 mM 5,5′-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 concentration of 2% DMSO in the reaction
mixture. In these conditions, KI values varied from 5% to 10%
in replicate experiments. KI’s were then determined by using
Easson-Stedman23 plots and a linear regression program.
N-4-F lu or op h en ylu r eid o-N-4-n itr oben zylglycin e, E1:
1
white crystals, mp 194-6 °C; H NMR (DMSO-d6) δ 3.60 (s,
2H, CH2 of Gly), 4.35 (s, 2H, CH2 of benzyl), 7.18-7.66 (m,
3
C. histolyticum highly purified collagenase and its substrate
FALGPA (furanacryloyl-leucyl-glycyl-prolyl-alanine) were pur-
chased from Sigma Chemical Co. (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 inhibition of the enzymatic
(amidolytic) activity of the collagenase, at 25 °C, using FAL-
GPA as substrate, by the method of van Wart and Steinbrink.25
The substrate was reconstituted as 5 mM stock in 50 mM
Tricine buffer, 0.4 M NaCl, 10 mM CaCl2, pH 7.50. The rate
of hydrolysis was determined from the change in absorbance
4H, Hortho of CH3C6H4 + Hortho of O2NC6H4), 7.94 (d, J HH
)
8.0 Hz, 2H, Hmeta of FC6H4), 8.05 (br s, 2H, NHCONH), 8.22
(d, 2H, Hmeta of O2NC6H4), 11.44 (br s, 1H, COOH); 13C NMR
(DMSO-d6) δ 40.3 (s, CH2 of Gly), 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 O2NC6H4), 148.9 (s, Cipso
of FC6H4), 149.7 (s, Cpara of FC6H4), 177.8 (s, CO2H). Anal.
Found: C, 55.60; H, 4.13; N, 12.00. C16H14FN3O5 Requires: C,
55.33; H, 4.06; N, 12.10.
N-4-F lu or op h en ylu r eid o-N-4-n itr oben zylglycin e h y-
d r oxa m a te, F 1: white crystals, mp 201-3 °C; 1H NMR
(DMSO-d6) δ 3.72 (s, 2H, CH2 of Gly), 4.36 (s, 2H, CH2 of
benzyl), 7.22-7.69 (m, 4H, Hortho of CH3C6H4 + Hortho of
at 324 nm using an extinction coefficient for FALGPA ꢀ305
)
24 700 M-1‚cm-1 in the above-mentioned reaction buffer.25
Measurements were made using a Cary 3 spectrophotometer
interfaced with a PC. Initial velocities were thus estimated
using the direct linear plot-based procedure reported by van
Wart and Steinbrink.25 KI’s were then determined according
to Easson-Stedman23 plots and a linear regression program.
3
O2NC6H4), 7.90 (d, J HH ) 8.2 Hz, 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)
δ 40.1 (s, CH2 of Gly), 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. C16H15FN4O5 Requires: C,
53.04; H, 4.17; N, 15.46.
Ack n ow led gm en t. This research was financed by
the EU Grant ERB CIPDCT 940051. Special thanks are
addressed to Dr. M. A. Ilies for expert technical as-
sistance.
N-4-Nitr op h en ylsu lfen yl-N-4-n itr oben zylglycin e, G1:
1
yellow crystals, mp 223-4 °C; H NMR (DMSO-d6) δ 3.61 (s,
2H, CH2 of Gly), 4.37 (s, 2H, CH2 of benzyl), 6.75 (s, 1H, SNH),
7.21-7.62 (m, 4H, Hortho of CH3C6H4 + Hortho of O2NC6H4), 8.05
Refer en ces
3
(d, J HH ) 8.3 Hz, 2H, Hmeta of O2NC6H4), 8.21 (d, 2H, Hmeta of
(1) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J . H. Design
and therapeutic application of matrix metalloproteinase inhibi-
tors. Chem. Rev. 1999, 99, 2735-2776.
(2) Bottomley, K. M.; J ohnson, W. H.; Walter, D. S. Matrix metal-
loproteinase inhibitors in arthritis. J . Enzyme Inhib. 1998, 13,
79-102.
(3) (a) Babine, R. E.; Bender, S. L. Molecular recognition of protein-
ligand complexes: Applications to drug design. Chem. Rev. 1997,
97, 1359-1472. (b) Zook, S. E.; Dagnino, R., J r.; Deason, M. E.;
Bender, S. L. PCT Int. Appl. WO 97/20824.
O2NC6H4), 11.68 (br s, 1H, COOH); 13C NMR (DMSO-d6) δ 40.0
(s, CH2 of Gly), 44.8 (s, CH2 of benzyl), 123.8 (s, Cmeta of
O2NC6H4CH2), 129.4 (Cortho of O2NC6H4CH2), 130.3 (s, Cmeta of
O2NC6H4S), 135.9 (s, Cortho of O2NC6H4S), 144.5 (s, Cipso of
O2NC6H4CH2), 145.9 (s, Cipso of O2NC6H4S), 147.8 (s, Cpara of
O2NC6H4CH2), 150.4 (s, Cpara of O2NC6H4S), 177.5 (s, CO2H).
Anal. Found: 49.45; H, 3.77; N, 11.24. C15H13N3O6S Re-
quires: C, 49.58; H, 3.61; N, 11.56.
N-4-Nitr op h en ylsu lfen yl-N-4-n itr oben zylglycin e h y-
d r oxa m a te, H1: yellow crystals, mp 182-3 °C; 1H NMR
(DMSO-d6) δ 3.70 (s, 2H, CH2 of Gly), 4.38 (s, 2H, CH2 of
benzyl), 6.75 (s, 1H, SNH), 7.20-7.67 (m, 4H, Hortho of CH3C6H4
(4) Supuran, C. T.; Scozzafava, A. Matrix metalloproteinase inhibi-
tors. In Proteinase and Peptidase Inhibition: Recent Potential
Targets for Drug Development; Smith, H. J ., Simons, C., Eds.;
Harwood Academic Press: London, 2000; in press.
(5) (a) Nagase, H.; Woessner, J . F., J r. Matrix metalloproteinases.
J . Biol. Chem. 1999, 274, 21491-21494. (b) Dioszegi, M.;
Cannon, P.; Van Wart, H. E. Vertebrate collagenases. Methods
Enzymol. 1995, 248, 413-431. (c) Tschesche, H. Human neu-
trophil collagenase. Methods Enzymol. 1995, 248, 431-449.
3
+ Hortho of O2NC6H4), 8.09 (d, J HH ) 8.2 Hz, 2H, Hmeta of
O2NC6H4), 8.23 (d, 2H, Hmeta of O2NC6H4), 8.74 (br s, 1H,
NHOH), 10.68 (br s, 1H, NHOH); 13C NMR (DMSO-d6) δ,
40.5 (s, CH2 of Gly), 44.6 (s, CH2 of benzyl), 123.8 (s, Cmeta of