1120
A. Scozzafava, C. T. Supuran / Bioorg. Med. Chem. Lett. 10 (2000) 1117±1120
10. Smith, G. E. P.; Alliger, G.; Carr, E. L.; Young, K. C. J.
Org. Chem. 1949, 14, 935.
11. Supuran, C. T.; Briganti, F.; Scozzafava, A. J. Enz. Inhib.
1997, 12, 175.
16. Tureci, O.; Sahin, U.; Vollmar, E.; Siemer, S.; Gottert, E.;
Seitz, G.; Parkkila, A. K.; Shah, G. N.; Grubb, J. H.; Pfreund-
schuh, M.; Sly, W. S. Proc. Natl. Acad. Sci. USA 1998, 95, 7608.
17. Mori, K.; Ogawa, Y.; Ebihara, K.; Tamura, N.; Tashiro,
K.; Kuwahara, T.; Mukoyama, M.; Sugawara, A.; Ozaki, S.;
Tanaka, I.; Nakao, K. J. Biol. Chem. 1999, 274, 15701.
18. Chegwidden, W. R.; Spencer, I. M. In¯ammopharmacol.
1995, 3, 231.
19. Teicher, B. A.; Liu, S. D.; Liu, J. T.; Holden, S. A.; Her-
man, T. S. Anticancer Res. 1993, 13, 1549.
20. Puscas, I., Coltau, M., Farcau, D., Puscas, C., Supuran, C.
T. In Carbonic Anhydrase and Modulation of Physiologic and
Pathologic Processes in the Organism; Puscas, I. Ed.; Helicon:
Timisoara (Roumania), 1994; pp 551±557.
21. Mohamadi, F.; Spees, M. M.; Grindey, G. B. J. Med.
Chem. 1992, 35, 3012.
22. Scozzafava, A.; Supuran, C. T. J.Enz. Inhib. 1999, 14, 343.
23. Inhibition of tumor cell growth was determined as follows:
stock solutions of inhibitor (1 mM) were prepared in DMSO, and
dilutions up to 10 nM prepared with distilled deionized water.
12. For example, by using sodium hypochlorite as oxidizing
agent, the followed procedure was: an amount of 10 mMols
sulfonamide 1±20 was treated with 100 mL of a solution con-
taining 0.8 g (20 mMols) sodium hydroxide (KOH could also
be used). After dissolution of the sulfonamide, into the mag-
netically stirred reaction mixture were slowly (1±2 h) and con-
comitantly dropped the following two aqueous solutions: (i)
50 mL of a solution containing 10 mMols of sodium/potas-
sium N-morpholyldithiocarbamate 21; and (ii) 50 mL of
NaClO solution, containing the stoichiometric amount (10
mMols) of oxidizing agent. The temperature was maintained
in the range of 25±30 ꢀC (generally cooling of the reaction
mixture had to be done), with strong magnetic stirring, for 1±3
h (TLC control; the thiocarbamylsulfenamide and the thiuram
22 started to precipitate immediately after the addition of the
oxidizing agent). A double±2.5-fold amount of initial sulfon-
amide could be used in the synthesis (for relatively inexpensive
raw materials, such as 3±6, for example), case in which the
yields in thiocarbamylsulfenamides were increased. In order to
purify the obtained thiocarbamylsulfenamides, the obtained
crude precipitate was ®ltered and treated with an excess (100±
150 mL) of a 0.03±0.05 M NaOH (or KOH) and magnetically
stirred for 30 min at 4 ꢀC. The insoluble thiuram was ®ltered
and the sodium/potassium salts of the thiocarbamylsulfen-
amides acidi®ed with a 10% HCl solution (till pH 5.5), when
the pure A(1±20) precipitated. They were then ®ltered, thor-
oughly washed with water and air dried. Yields were generally
in the range of 25±40%, but for some derivatives they were
much higher (around 70±75 % for A3, A5 and A6).
The percentage growth (PG) of the cell lines in the presence of ®ve
M) of inhibitor was calculated
4
concentrations (10 8±10
according to one of the following two expressions (1) or (2):
PG 100 Â ꢀMean OD test Mean OD0=
ꢀMean OD ctrl Mean OD0;
ꢀ1
when ꢀMean OD test Mean OD0 ꢁ 0;
PG 100 Â ꢀMean OD test Mean OD0=
ꢀMean OD0; when ꢀMean OD test Mean OD0 < 0;
ꢀ2
13. Pocker, Y.; Stone, J. T. Biochemistry 1967, 6, 668. Initial
rates of 4-nitrophenyl acetate hydrolysis catalysed by dierent
CA isozymes were monitored spectrophotometrically, at 400
nm, 25 ꢀC, with a Cary 3 instrument interfaced with an IBM
compatible PC. A molar absorption coecient E of 18,400
where Mean OD0=the average optical density measure-
ments of sulforhodamine B (SRB)-derived color just before
exposure of cells to the test compounds; Mean ODtest=the
average optical density measurements of SRB-derived
color after 48 h exposure of cells to the test compounds;
Mean ODctrl=the average optical density measurements
of SRB-derived color after 48 h with no exposure of cells to
the test compounds. GI50 represents the molarity of inhi-
1
1
M
cm
was used for the 4-nitrophenolate formed by
hydrolysis, in the conditions of the experiments (pH 7.40).
Non-enzymatic hydrolysis rates were subtracted from the
observed rates. Stock solutions of inhibitor (1 mM) were pre-
pared in DMSO and dilutions up to 0.1 nM were done there-
after with distilled deionized water. Inhibitor and enzyme
solutions were preincubated together for 15 min at room tem-
perature prior to assay, in order to allow for the complete
formation of the E±I complex. The inhibition constant Ki was
determined as described in ref 8. Enzyme concentrations were
3.0 nM for hCA II, 9 nM for hCA I and 28 nM for bCA IV.
14. Maren, T. H.; Wynns, G. C.; Wistrand, P. J. Mol. Phar-
macol. 1993, 44, 901.
bitor producing a 50% inhibition of growth of the tumor
4
cells after 48 h exposure to variable concentrations (10
-
8
10 M) of the test compound, measured as outlined
before, and this parameter was obtained by interpolation.
GI50 is in fact the molarity of inhibitor at which PG=50%.
The standard sulforhodamine B (SRB) protein assay has
been used to estimate cell viability or growth (cf. Teicher,
B. A. (Ed.). Anticancer Drug Development Guide: Pre-
clinical Screening, Clinical Trials, and Approval; Humana
Press Inc.: Totowa, NJ, 1997; pp 7±125).
15. Pastorekova, S.; Parkkila, S.; Parkkila, A. K.; Opa-
versusky, R.; Zelnik, V.; Saarnio, J.; Pastorek, J. Gastro-
enterology 1997, 112, 398.