Knigth's Move in the Periodic Table, From Copper to Platinum, Novel Antitumor Mixed Chelate Copper Compounds, Casiopeinas, Evaluated by an in Vitro Human and Murine Cancer Cell Line Panel

We synthesized a novel anticancer agents based on mixed chelate copper (II) complexes, named Casiopeínas® has of general formula [Cu(N-N)(N-O)H2O]NO3 (where, N-N = diimines as 1,10- phenanthroline, 2,2-bipyridine, or substituted and N-O=aminoeidate or [Cu(N-N)(O-O)H2O]NO3 (where NN= diimines as 10-phenanthroline, 2,2-bipyridine or substituted Casiopeínas I, II, IV, V, VI, VII VIII and O-O=acetylacetonate, salicylaldehidate Casiopínas III). We evaluated the in vitro antitumor activity using a human cancer cell panel and some nurine cancer cells. Eleven Casiopeinas are evaluated in order to acquire some structure-activity correlations and some monodentated Casiopeinäs analogues; cisplatinum was used as control drug. The 50% growth inhibition observed is, in all cases reach with concentrations of Casiopeina's 10 or 100 times lower than cisplatinum. In a previous work we reported the induction of apoptosis by Casiopeina II. The results indicate that Casiopeinass are a promising new anticancer drug candidates to be developed further toward clinical trials.

The general formula of Casiopeinas is [Cu(N-N)(N-O)Hg_O]NO3 (where, N-N diimines as 1,10phenanthroline, 2,2-bipyridine, or substituted and N-O=aminocidate or [Cu(N-N)(O-O)H20]NO3 (vhere, N-N=diimines as 1,10-phenanthroline, 2,2obipyridine or substituted Casiopeinas I, II, IV, V, VI, VII VIII and O-O=acetylacetonate, salicylaldehidate Casiopehaas III). The design of the molecules was based in three main factors: the comlx)unds should contain an essential metal for diminish toxici.ty; contain chelates that favor the cis-configuration around the metal ion and the mixed chelates that contain different level of hydrophobicity. Casiopeinas were design to have antitumor activity, based on previous works in cisplatinum and other transition metal series. These reported compounds are proposed to present some degree of DNA-interaction.
A preliminary report of antineoplastic activity was presented (7), also SOD like activity and the induction of apoptosis has been reported (8,9). Casiopefnas have shown cytotoxicity in several munne tumoral cell lines and strong in vivo antitumor activity in routine tumoral models in our preliminar results (10). qqae present study was designed to evaluate the in vitro antitumor activity of several Casiopeinas against various human and routine tumoral cell lines and to observe a correlation of activity as a function of the peripherical substituents on the ligands. The in vitro test is one of the most adequate methods to evaluate anticancer activity in a large range of compounds. In Figm'e the structure of Casiopema III-I is shown as the perchlorate salt (6).
Lena Ruiz-Ramirez et (1)..Equimolecular soluuon of the CuNO3 and the coesponding diimine are mixed tggether followed by an equimglecular aqueous solution of die charged ligand, previously. deprotonated. The resulting solution is concentrated and the solid obtained is filtered anal recristallized several times.

CELL LINES
Cancer Cell Line Panel. To evaluate drags tbr the cell growth inhibition profile, we established a human cancer cell line or munne tumoral cell-line from the panel described (11). With this system we have examined the antiproliferative effect of the Casiopefnas, andbasal contror drugs mentioned above.
Human: HeLa (adenocarcinoma Stage IV A), SiHa (carcinoma Stae IIIB), CaS.ki The details of measuring cell grovJa inhibition are described elsewhere (citas26 del articulo). Briefly, the cells were plated at proper density in 96-well plates with DMEM and 10% of FBS and allow to attach for 24 hr.. The cells were exposed to drugs (Casiopeinas, Blanks and conlrol &'ug) for 24 hr. Then the cell growth was determined according to the sulforhodamine B assay, described by Skehan (28del articulo). Data calculations were made according to the method described previously (26 de art). Absorbance for the control well (C) and the test well (T) were measured at 564 nm. Moreover, at time 0 (addition of the drugs), absorbance for the test well (To) was also measured. Using these measurements, cell growth inhibition (percentage of growth) by each concentration of drug was calculated as: % growth =100 X [(T-T0)/(C-To)], when T > To mad % growth=100 X [(T-To)/T], when T < To. By using the computer to process % growth values, the 50% ga'owlh inhibition parameter (Gls0) was determined. The GI50 was calculated as 100 X [(T-To)/(C-To)] 50 (15). RESULTS

AND DISCUSSION
The general project for de 'eloping new drugs is showed in the flow diagram Diagram I.
In this work we present the in vitro evaluation over Human and Murine Tumoral Cell Lines.
The Casiopeinas chosen tbr this studv are those that are selection of the different diim, ines with the porpuse of observe the effedt of the same charge ligand, as glycine with several diimines, then we kept the same aminoacidate and observe the effect of the diimine. Also we have synthesized and tested some monochelates copper complexes in order to observe if the diss6ciated complex may present some activity.
The results on GI50 (Growth Inhibition) prod,ucedby the several Casiopeinas over the Uterine Cervix Human Tumoral Lines are sho n in Table I The Casiopeinas showed antineoplastic activity in all the different human and murine cell lines, however, this activity is higher for the cervic-uterine human tumoral cell lines.
The Casiopeinas with symmetric phenanthrolines, especially with substituents at 4, 7 positions showed more activity than those with substitution in 5 position. Regarding to the O-O donor, the higher activity was showed m those Casiopeinas with glycine. Platinum, N-vel Antitumor Mixed "helate C'opper (,ompounds -rl'hc antincoplastlc aztivity of the Cm;iopeinas and Cisplatin is low for highly mestastatic cell lines like lcwis Lung Carcinoma and t316 Melanoma --'l'he tnonochelate copper complexes shown a very low activity comared with those of the Casiopeinas, these results clearcly indicafcd that c activity of C.asiopeinas is due to the whole moleculc. -Casiopeinas keep being a promising resource for the treatment of neoplastic diseases, because have shown higher activity than the conm.)l da-ug (Cisplatin).