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19. Derivatives 9 were prepared as described in: Scozzafava,
A.; Supuran, C. T. Eur. J. Med. Chem. 2000, 35, 299, whereas
derivatives 10 as described in: Scozzafava, A.; Supuran, C. T.
J. Med. Chem. 2002, 45, 284. A typical procedure for the
synthesis of one such compound is described below: an
amount of 252 mg (1 mmol) of Phe-Ala (Sigma-Aldrich,
Milan, Italy) was suspended in 30 mL of anhydrous acetone
and the equivalent amount (185mg, 1 mmol) of 4-tosyl-iso-
cyanate (Sigma-Aldrich, Milan, Italy) dissolved in a small
volume of the same solvent was added dropwise. The mixture
was stirred for 1 h at room temperature, the solvent was eva-
porated in vacuo and the obtained sulfonylurea 10 crystallized
from 120 mL of ethanol–water (1:1, v/v). The yield was quan-
titative. The obtained compound 10 was then suspended in 50
mL of anhydrous acetonitrile together with the equivalent
amount of 5-amino-2-mercapto-1,3,4-thiadiazole (133 mg),
and 1 mmol (126 mg=98 mL) of diisopropylcarbodiimide was
added dropwise. The mixture was magnetically stirred at room
temperature for 12 h (TLC control), the solvent was evapo-
rated in vacuo, the raw product washed with 2Â10 mL of
water for removing the diisopropylurea formed in the reaction
and the desired product 13d was recrystallized from 70 mL of
methanol-water (2:1, v/v). The yield was of 83%. Pale yellow
crystals, mp 212–213 ꢁC (dec.); IR (KBr), cmꢀ1: 1150 (SOs2ym),
1283 (amide III), 1375(SO 2as), 1584 (amide II), 1609 (C¼N of
1
thiadiazole); 1710 (amide I), 3060 (NH); H NMR (300 MHz,
DMSO-d6), d, ppm: 2.37 (d, 3H, CH3 of Ala); 2.50 (s, 3H,
CH3C6H4); 3.33 (m, 2H, CH2CH of Phe), 3.62 (q, 1H, CH of
13. (a) Gavuzzo, E.; Pochetti, G.; Mazza, F.; Gallina, C.;
Gorini, B.; D’Alessio, S.; Pieper, M.; Tschesche, H.; Tucker,
P. A. J. Med. Chem. 2000, 43, 3377. (b) Pavlovsky, A. G.;
Williams, M. G.; Ye, Q.; Ortwine, D. F.; Purchase, C. F., II;
White, A. D.; Dhanaraj, V.; Roth, B. D.; Johnson, L. L.;
Hupe, D.; Humblet, C.; Blundell, T. L. Protein Sci. 1999, 8,
1455. (c) Tamura, Y.; Watanabe, F.; Nakatani, T.; Yasui, K.;
Fuji, M.; Komurasaki, T.; Tsuzuki, H.; Maekawa, R.; Yosh-
ioka, T.; Kawada, K.; Sugita, K.; Ohtani, M. J. Med. Chem.
1998, 41, 640.
3
3
Ala), 4.10 (dd, JHH=5.0, JHH=7.8, 1H, CH2CH of Phe),
7.11–7.35(m, H5, Harom of Phe); 7.38 (2H, Hortho of
3
CH3C6H4); 7.72 (d, JHH=8.2, 2H, Hmeta of CH3C6H4); 13C
NMR (75MHz, DMSO- d6), d, ppm: 22.1 (s, CH3 of Ala); 26.0
(s, CH3C6H4), 34.5(s, CHCH3 of Ala); 41.7 (s, CH2CH of
Phe), 59.3 (s, CH2CH of Phe), 132.3 (s, Cmeta of CH3C6H4),
132.7 (s, NHCONH), 133.8 (s, Cmeta of Phe), 134.4 (s, Cortho of
Phe), 135.1 (s, Cortho of CH3C6H4), 141.5(s, Cipso of Phe), 145.0
(s, Cipso of CH3C6H4), 148.6 (s, Cpara of CH3C6H4), 154.5 (C-2
of thiadiazole), 166.9 and 172.3 (CONH), 183.1 (C¼S). Anal.,
found: C, 48.37; H, 4.66; N, 15.14; S, 17.82%; C22H24N6O5S3
requires C, 48.16; H, 4.41; N, 15.32; S, 17.53%..
14. (a) Clare, B. W.; Scozzafava, A.; Supuran, C. T. J. Med.
Chem. 2001, 44, 2253. (b) Scozzafava, A.; Supuran, C. T. J.
Med. Chem. 2000, 43, 1858. (c) Scozzafava, A.; Supuran, C. T.
J. Med. Chem. 2000, 43, 3677.
20. Bieth, J. G. Meth. Enzymol. 1995, 248, 5 9.
15. (a) Scozzafava, A.; Supuran, C. T. Eur. J. Med. Chem.
2000, 35, 299. (b) Supuran, C. T.; Scozzafava, A. Eur. J.
Pharm. Sci. 2000, 10, 67. (c) Supuran, C. T.; Briganti, F.;
Mincione, G.; Scozzafava, A. J. Enzyme Inhib. 2000, 15, 111.
(d) Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem. 2000,
8, 637. (e) Scozzafava, A.; Supuran, C. T. Bioorg. Med. Chem.
Lett. 2000, 10, 499. (f) Scozzafava, A.; Supuran, C. T. Eur. J.
Pharm. Sci. 2000, 11, 69.
16. (a) Jacobsen, E. J.; Mitchell, M. A.; Hendges, S. K.;
Belonga, K. L.; Skaletzky, L. L.; Stelzer, L. S.; Lindberg, T. J.;
Fritzen, E. L.; Schostarez, H. J.; O’Sullivan, T. J.; Maggiora,
L. L.; Stuchly, C. W.; Laborde, A. L.; Kubicek, M. F.; Poor-
man, R. A.; Beck, J. M.; Miller, H. R.; Petzold, G. L.; Scott,
P. S.; Truesdell, S. E.; Fallace, T. L.; Wilks, J. W.; Fisher, C.;
Goodman, L. V.; Staples, D. J.; Baldwin, E. T.; Finzel, B. C.
J. Med. Chem. 1999, 42, 1525. (b) Stockman, B. J.; Waldon,
D. J.; Gates, J. A.; Scahill, T. A.; Kloosterman, D. A.; Mizsak,
S. A.; Jacobsen, E. J.; Belonga, K. L.; Mitchell, M. A.; Mao,
B.; Petke, J. D.; Goodman, L. V.; Powers, E. A.; Ledbetter,
S. R.; Kaytes, P. S.; Vogali, G.; Marshall, V. P.; Petzold, G. L.;
Poorman, R. A. Protein Sci. 1998, 7, 2281.
21. (a) Powers, J. C.; Kam, C. M. Meth. Enzymol. 1995, 248,
3. (b) Johnson, L. L.; Bornemeier, D. A.; Janowicz, J. A.;
Chen, J.; Pavlovsky, A. G.; Ortwine, D. F. J. Biol. Chem.
1999, 274, 24881.
22. Van Wart, H. E.; Steinbrink, D. R. Anal. Biochem. 1981,
113, 156.
23. Human purified MMPs (MMP-1, MMP-2, MMP-8 and
MMP-9) were from Calbiochem (Inalco, Milan, Italy). They
were activated in the assay buffer by adding bovine trypsin (50
mL, 0.6 mg/mL) to the proenzyme, followed by incubation at
37 ꢁC for 10 min. The trypsin was then inactivated with apro-
tinin (50 mL, 1.2 mg/mL). Initial rates for the hydrolysis of the
thioester substrate AcProLeuGly-S-LeuLeuGlyOEt, coupled
to the reaction with 5,50-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,21a modified by Johnson et al.21b
The change of absorbance (e=19,800 Mꢀ1 cmꢀ1 21
at 405nm
)
was monitored continuously at room temperature, using a
Cary 3 spectrophotometer interfaced with a PC. A typical 100
mL reaction contained 50 mM MES, pH 6.0, 10 mM CaCl2,