Abstract:
This patent of invention reports the method for the preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and derivatives with antitumoral properties: the sample denominated 37 compound was obtained with high yield and purity with ultrasonic technique presenting cytostatic activity (growth inhibition) in the concentrations evaluated and cytotoxic activity (cellular death) from the concentration of 0.25 mg/mL against nine different types of human cancer cell lines. This compound has a LD 50 , equals to 8.54 g/Kg. That means this product can be considered itself as practically nontoxic. Doxorubicin, anticarcinogen medicine used as reference in all these tests, is a product extremely toxic (LD 50  of 20 mg/Kg) and it does not inhibit the growth of Mama NCI-ADR cell line (the one that expresses the phenotype of resistance against multiple drugs), therefore, out product presented a strong cytostatic activity. Other derivatives also presented a strong cytostatic activity, especially the one denominated EHB1 compound.

Description:
TECHNICAL FIELD 
     The present patent of invention refers to a NEW METHOD FOR THE PREPARATION OF 1,5-BIS(4-HYDROXY-3-METHOXYPHENYL)-PENTA-1,4-DIEN-3-ONE AND DERIVATIVES WITH ANTITUMORAL ACTIVITIES. 
     BACKGROUND 
     Bibliographical background about 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one compound and derivatives and its method for the preparation. 
     The compound denominated 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one is known since the year 1927 since E. Glaser and E. Tramer for the first time reported its synthesis with a 60% yield (Journal für praktische Chemie, 116, 331-346, 1927) from the vanillin and acetone in the presence of concentrated hydrochloric acid, used as a catalyzer. 
     Further, P. Ramanan and M. Rao synthesized this product in 1989 (Indian Journal Pharm. Sci., 51, 207, 1989) from 4-O-methoxymethylvanillin and acetone in an alkaline medium, obtaining a yield of 42% after purifying the same using the thin-layer chromatograph (silica gel). 
     In 1997, a group integrated by S. Sardjiman, et al. (Eur. Journal Med. Chem. 32, 625-630, 1997) developed a new variant synthesis using equimolecular quantities of vanillin and acetone in the presence of concentrated hydrochloric acid, reporting a raw yield of 89% (without purifying). For this reason, the melting point indicated in this procedure was 58° C. less than the one reported by Glaser and Tramer. 
     M. Artico et al. also obtained this substance one year later (Journal Med. Chem. 41, 3948-3960, 1998) only obtaining a poor yield of 18%. The melting temperature (114-116° C.) is lower than the one reported by Glaser and Tramer, what is makes think that the compound was not obtained pure in despite of the use of the chromatograph of column. 
     Additionally, we can find in the bibliographic revision the article of the American patent (U.S. Pat. No. 4,521,629 of Jun. 4, 1985) of N. Cortese et al. entitled: “Method for the preparation of 1,5-bis-aryl-1,4-pentadien-3-ones.” This invention reports to a method of preparing certain bis-arylpentadienones containing fluorine, that were used as intermediate compounds for the preparation of insecticidal substituted amidinohydrazones, but it does not protect the products that appear in our patent request. 
     Furthermore, the following documents of other patents related to this family of organic compounds were found: 
     1-“Hair tonics containing bis(hydroxyphenyl)pentadienones.” Authors of the patent: Morita, Kazuyoshi; Hamada, Kazuto. Company: Kanebo, Ltd, Japan. Country: Jpn. Kokai Tokyo Koho, 7 pp. Idiom: Japanese. CA-Number: 134:183278. PI: JP 2001048756, A2 20010220 JP 1999-224982 19990809. 
     2-“Skin-lightening cosmetics containing distyryl ketones.” Author: Morita, Kazuyoshi. Company: Kanebo, Ltd., Japan. Country: Jpn. Kokai Tokyo Koho, 7 pp. Idiom: Japanese. CA-Number: 131:149078. PI: JP 11209235 A2 19990803 JP 1998-10414 19980122. 
     3-“Acidic planting baths and methods for electrodepositing bright and ductile zinc-nickel allows and additive composition for these baths.” Company: McGean-Rohco, Inc., USA. Author: Canaris, Valerie M. Country: U.S., 8 pp. Idiom: English. CA-Number: 111:183131. PI: US 4832802 A 19890523 US 1988-206017 19880610 EP 346161 A1 19891213 EP 1989-305925 19890612. 
     4-“Photopolymerizable compositions.” Compound: Eastman Kodak Co., USA. Authors: Noonan, John M.; McConkey, Robert C.; Arcesi, J. A.; Rauner: Frederick J. Country: Brit., 19 pp. Idiom: English PI: GB 1425476 A 19760218 GB 1973-3986 19730322 US 3748133 A 19730724 US 1972-237929 19720324. 
     None of these four patents, either directly or indirectly, are related to the antiproliferate properties shown by 1,5-bits(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and its derivatives. 
     Taking into account the bibliographic revision performed by the company Bypropind Patents and Trademarks based on the tracking in the Chemical Abstracts collection, of Lifescience, of Biomed of Fiocruz library and by us in the Beilstein collection, we have concluded the there is no patent of that compound and its derivatives applicable in the cancer treatment or even the ultrasonic technique in despite of the compound has been synthesized in the year of 1927 and so that the patent request is possible and legitimate. 
     SUMMARY OF THE INVENTION 
     The present patent of invention reports the antitumoral properties of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one and derivatives and its procedure of preparing. 
     The sample denominated 37/01 compound was obtained with high yield and purity by the ultrasonic technique presenting cytostatic activity (growth inhibition) in the concentrations evaluated and killed cell activity (cellular death) from the concentration of 0.25 μg/mL against nine different types of human cancer. This compound has a LD 50 , equals to 8.54 g/Kg. That means this product can be considered itself as practically nontoxic. Doxorubicin, anticarcinogen medicine used as reference in all of these tests, is a product extremely toxic (LD 50  of 20 mg/Kg) and its does not inhibit the growth of the Mama NCI-ADR cell lines (the one that expresses the phenotype of resistance against multiple drugs), therefore our product presented a strong cytostatic activity. 
     
       
                 
         
             
             
         
      
         
         37 :R 1 -CH 3 : R 2 -H; R 3 -H; R 4 -H; R 5 ═H; X-O 
         EHB1:R 1 ═CH 3 ; R 2 ═H 3 CCO; R 3 ═H; R 4 ═H; R 5 ═H; X═O 
         HB6:R 1 ═CH 3 ; R 2 ═CH 2 CH═C(CH 3 ) 2 ; R 3 ═H; R 4 ═H; R 5 -H; X-O 
         HBM1:R 1 ═CH 3 ; R 2 ═CH 3 ; R 3 ═H; R 4 ═H; R 5 ═H; X═O 
         HB5:R 1 ═CH 3 ; R 2 ═H; R 3 ═H; R 4 ═H; R 5 ═H; X═C(CN) 2    
         HB10:R 1 ═CH 3 ; R 2 ═H; R 3 ═CH 2 CH═C(CH 3 ) 2 ; R 4 ═H; R 5 ═H; X═O 
         HB11:R 1 ═CH 3 ; R 2 ═H; R 3 ═H; R 4 ═H; R 5 ═H; X═C(CN)CO 2 C 2 H 5    
         HB12:R 1 ═H; R 2 ═H; R 3 ═H; R 4 ═H; R 5 ═H; X═O 
         HB13:R 1 ═CH 3 ; R 2 ═H; R 3 ═Br; R 4 ═H; R 5 ═H; X═O 
         HB14:R 1 ═CH 3 : R 2 ═CH 2 CH═C(CH 3 ) 2 ; R 3 ═Br; R 4 ═H; R 5 -H; X═O 
         HB15:R 1 ═H; R 2 ═H; R 3 ═CH 2 CH═C(CH 3 ) 2 ; R 4 ═CH 2 CH═C(CH 3 ) 2 ; R 5 ═H; X═O 
       
    
     Antitumoral activity shown by synthesized compounds is shown in  FIG. 1 . 
     The following designations are utilized in  FIG. 1 : Legend: NCI460 (Lung tumor): UACC62 (Melanoma); MCF7 (Normal mamma tumor); NCIADR (Mamma tumor which expresses the phenotype resistance against multiple drugs) HT29 (Colon tumor); 786-O (Renal tumor); OVCAR-3 (Ovary tumor); PC-3 (Prostate tumor); K-562 (Leukemia); ED 50  (effective doses 50 expressed in micrograms by milliliters μg/mL). 
     Remarks: 
     All these results can be considered excellent, if we take into reference the ones published in the literature: 
     Banskota AH, et al. Chemical Constituents of Brazilian Propolis and their cytotoxic activities: J. Nat. Prod. 61, 896-900, 1998. 
     Banskota A H, et al. Two Novel Cytotoxic Benzofuran Derivatives from Brazilian propolis; J. Nat Prod. 63, 1277-1279, 2000 
     Kimoto T, et al. Apoptosis and Suppression of tumor growth by Artepillin C extracted from Brazilian Propolis; Cancer Detect, Prev. 22(6), 505-15, 1998. 
     It is observed that most of our products presented a strong antiproliferate action in concentration ranges (in ppm) much lower than the ones shown by some of the isolated compounds of the Brazilian Propolis. 
     Cytotoxicity of some isolated compounds of Brazilian Propolis by Banskota and Kimoto are shown in  FIG. 2 . 
     We want to point out that the obtained products by our team consist of raw material of our research project, from which we are obtaining new derivatives, guided by the principle of analogy and the results of TOPS-MODE predictions. 
     Curves Concentration response of the tested compounds is shown in  FIG. 3 . 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a table of data of antitumoral activity shown by synthesized compounds. 
         FIG. 2  is a table of data concerning the cytotoxicity of some isolated compounds of Brazilian Propolis by Banskota and Kimoto. 
         FIG. 3  is a graph plotting percentage of growth versus concentration of the antitumoral activity of the synthesized compounds in  FIG. 1 . 
         FIG. 4  is a graph plotting percentage of growth versus concentration for the antiproliferative activity of compound 37 or HB1. 
         FIG. 5  is a graph plotting percentage of growth versus concentration for the response of Doxorubicin. 
         FIG. 6  is a graph of the antitumoral activity of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1-4-dien-3-one. 
         FIG. 7  is a graph plotting percentage increase versus concentration for the antitumoral activity of HB6. 
         FIG. 8  is a graph plotting percentage increase versus concentration for the activity of HBMI. 
         FIG. 9  is a graph plotting percentage increase versus concentration for the activity of HB5. 
         FIG. 10  is a table showing the results of tests of compound 37 on an animal. 
         FIG. 11  is a graph detailing the evolution of an animal&#39;s body weight after receiving administration of 2.5 g of compound 37 per Kg of body weight. 
         FIG. 12  is a table of data describing change in body weight from tests of 2.5 g of compound 37 per Kg of animal body weight. 
         FIG. 13  is a graph of the changes in animal body weight resulting from administration of 3.0 g of compound 37 per Kg of animal body weight. 
         FIG. 14  is a table showing changes in animal body weight resulting from administration of 4.0 g of compound 37 per Kg of animal body weight. 
         FIG. 15  is a graph of the changes in animal body weight resulting from administration of 4.0 g of compound 37 per Kg of animal body weight. 
         FIG. 16  is a table showing changes in animal body weight resulting from administration of 5.0 g of compound 37 per Kg of animal body weight. 
         FIG. 17  is a graph of the changes in animal body weight resulting from administration of 5.0 g of compound 37 per Kg of animal body weight. 
         FIG. 18  is a table of cell lines used in the assessment assays. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     37 compound, obtained by means of an organic synthesis procedure, presented cytostatic activity (growth inhibition) for all cell lines and cytotoxic activity (cellular death) for NCI460 (Lung), UACC62 (Melanoma) and MCF7 (Mamma) and NCIADR (Mamma resistant) from 0.25 μg/mL in the first antitumoral tests performed into CPQBA, IJNICAMP, Sep. 24, 2001. 
     Later, these tests of antiproliferate activity were extended to the following cell liens: 
     Colon: Renal; Ovary; Prostate; Leukemia. A graph showing the results of these tests using compound 37 is shown in  FIG. 4 . 
     Curve Concentration response of 37. 
     The results of this compound were compared with Doxorubicin (commercial anticarcinogen used as a standard in these tests) being so similar and in some cases higher than this commercial anticarcinogen. A graph showing these results is shown in  FIG. 5 . 
     Curve concentration response Doxorubicin. 
     For example, our product inhibited the growth of the Mama NCI-ADR cell line (the one that presents the phenotype of resistance against multiple drugs). This result becomes itself very interesting since Doxorubicin, utilized as a positive control, did not inhibit the growth of this human cell line. 
     The derivatives of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta-1,4-dien-3-one shows the antitumoral activity identified in  FIG. 6 : 
     The sample denominated EHBI compound presented cytostatic activity for all cell lines from the concentration of 0.25 μg/mL and cytotoxic activity from the concentration of 25 μg/mL, excepting NCI-ADR cell line which had only its growth inhibition around 25%. The results showed this sample was not selective for the studied cell lines. 
     The sample denominated HB6 compound presented cytostatic activity for all cell lines from the concentration of 25 μg/mL and cytotoxic activity in the concentration of 250 μg/mL, for HT-29, 786-0, NCI-ADR and K562 cell lines. Furthermore, this sample presented cellular selectivity for HT-29 and 786-0 cell lines. Test data for HB6 is shown in  FIG. 7 . 
     The sample denominated HBMI compound presented cytostatic activity for all cell lines from the concentration of 0.25 μg/mL and only presented cytotoxic activity for PC-03 and OVCAR-3 cell lines in the concentration of 250 μg/mL. Test data for HBMI is shown in  FIG. 8 . 
     The sample denominated HB5 compound presented moderate cytostatic activity for all cell lines from the concentration of 0.25 μg/mL and did not present cytotoxic activity for any cell lines in the used concentrations. Test data for HB5 is shown in  FIG. 9 . 
     Also a toxicological test with the sample denominated 37 compound (Acute Toxicity I.D 50 , via intraperitoneal). The LD 50  value, evaluated by linear regression, was equal to 8.54 g/Kg, after 14 days of observation. That means this product can be considered itself, according to Loomis, in Principles of Toxicology, as practically nontoxic (compounds with LD 50  value between 5.0 and 15 g/Kg are considered practically nontoxic). 
     The table shown in  FIG. 10  indicates an animal&#39;s body weight that received 2.5 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The graph of  FIG. 11  describes the evolution of the animal&#39;s body weight that received 2.5 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The table of  FIG. 12  shows an animal&#39;s body weight that received 3.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The graph of  FIG. 13  describes the evolution of the animal&#39;s body weight that received 3.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The table of  FIG. 14  shows an animal&#39;s body weight that received 4.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The graph of  FIG. 15  shows the evolution of the animal&#39;s body weight that received 4.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The table of  FIG. 16  shows an animal&#39;s body weight that received 5.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     The graph of  FIG. 17  shows the evolution of the animal&#39;s body weight that received 5.0 g/Kg of animal body weight of the product denominated “37 COMPOUND,” administrated via intraperitoneal, in the beginning and ending of the acute toxicity test. 
     Further this invention is illustrated by means of the following examples of execution: 
     EXAMPLE 1 
     Preparation of 1,5-bis(4-hydroxy-3-methoxyphenyl)-penta 1,4-dien-3-one. (Method 1) 
     From vanillin and acetone in a 2:1 molar rate in acid medium, in temperatures that changes between 25 and 60° C., under ultrasonic irradiation conditions in a range from 25 to 40 KHz for a period of 1 to 3 hours further putting the reacted mixture into water/ice until producing the raw product, which dissolves in a solution of sodium hydroxide or potassium hydroxide (between 10-30%) being filtered; the filtrated is treated with hydrochloric acid or sulphuric acid from a concentration between 10-30%, the obtained product being filtered again, finally it is washed with distillated water until obtaining a neutral pH, this operation being repeated until the total purification of the product, not being necessary to perform a new purification of the compound using other procedures such as recrystallization or chromatographic column (the purity was determined by means of the HPLC technique): 
     Obtained 92% yield of the pure product. Melting point: 155-156° C. 
     Method of Preparation 2 
     A mixture formed of vanillin and acetone in a 2:1 molar rate in acid medium laid during 5-8 days, in temperatures that changes between −10 and 40° C., further putting the reacted mixture into water/ice until producing the raw product, which dissolves in a solution of sodium hydroxide or potassium hydroxide (between 10-30%) being filtered: the filtrated is treated with hydrochloric acid or sulphuric acid from a concentration between 10-30%, the obtained product being filtered again, finally it is washed with distillated water until obtaining a neutral pH. This operation is repeated until the total purification of the product, not being necessary to perform a new purification of the compound using other procedures such as recrystallization or chromatographic column (the purity was determined by means of the HPLC technique): 
     Obtained 89% yield of the pure product, melting point of 155-156° C. 
     EXAMPLE 2 
     Preparation of 1,5-bis(3-metoxy-4-acetoxyphenyl)penta-1,4-dien-3-one 1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one. 
     It is stirred with an excess of acetic anhydride and sodium acetate. It&#39;s heated in a range of temperature between 20 and 110° C. for a period of time between 30 minutes to 3 hours. The obtained product is put into distillated water with ice. The precipitate product is recrystallized with hot ethanol. Yield: 58%. Melting point: 150° C. 
     EXAMPLE 3 
     Preparation of 1,5-Bis[3-metoxy-4-(3-methyl-but-2-eniloxy)phenyl]penta-1,4-dien-3-one. 
     A mixture formed of 1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one (2 mmol) in 10 mL of dimethylformamide and potassium carbonate (6 mmol) is stirred in a range of temperature between 20-50° C. during a period of time from 20 to 60 minutes in inert atmosphere (argon or nitrogen). After that, 3 mmol of 3-methyl-but-2-enyl bromide with constant stirring. Further, the stir is kept for another period of 5-8 hours with a stream of inert gas pouring all the mixture content into water with ice. It&#39;s extracted with chloroform in three occasions with approximately 3 mL of this solvent. The organic phase is washed with a solution of NaRSO 4  and then with distillated water. The chloroform phase is dried with sodium sulfate anhydrous. After that, the solvent is filtered and rotoevaporated. The purification of the product is performed utilizing a chromatographic column filled with silica gel and using a mixture formed of toluene/ethyl acetate or n-Hexane/ethyl acetate as elution solvents in an appropriate rate. Yield: 53% of the oil liquid substance. 
     EXAMPLE 4 
     Preparation of 1,5-Bis(3,4-dimetoxyphenyl)-penta-1,4-dien-3-one 
     Method 1 
     The mixture of 3,4-dimethoxy-benzaldehyd and acetone in equimolecular rate condition in presence of hydrochloric acid was submitted to a ultrasonic bath in the frequence from 25 to 40 KHz between 10-60 minutes in a range of temperature between 25-60° C. Further the obtained product is put into distilled water and ice, filtering the precipitate and washing it with distillated water. The aqueous phase is extracted with chloroform and washing the chloroform phase with distillated water and then the chloroform phase is dried with sodium sulfate anhydrous, filtered and rotoevaporated. Yield: 87%. 
     Method 2 
     1,5-bis(4-hydroxy-3-metoxyphenyl)-penta-1,4-dien-3-one is stirred with an excess of dimethyl sulfate or methyl iodide in alkaline medium (KOH or NaOH), stirring it in a range of temperature between 25-50° C. during a period of time that varies between 5-24 hours. The formed mixture is put into cold water, filtering the formed precipitate, neutralizing it with HCl. Further, it is washed with water until neutral pH. The after purification of the product was not necessary. Yield: 85%. 
     EXAMPLE 5 
     Preparation of 1,5-Bis(3,4-dimetoxyphenyl)-penta-1,4-dien-3-ylidenmalonitrile 
     In a mixture formed of 1,5-Bis(3,4-dimethoxyphenyl)-penta-1,4-dien-3-one and malononitrile in equivmolecular rate condition, ammonium acetate, acetic acid and toluene are added following Cope&#39;s variant, heating in reflux for a period of time between 5-16 hours or following Knoevenagel&#39;s third variant using piperidine as catalyzer. The obtained product is put into distillated water and ice, filtering the precipitate and extracting the aqueous phase with chloroform and washing the chloroform phase with distillated water. Further, the chloroform phase is dried with sodium sulfate anhydrous, filtered and rotoevaporated. Yield: 76%. Melting point: 216° C. 
     Methodology utilized to perform the antitumoral tests: 
     REPORT OF THE ANIPROLIFERATE TEST IN TUMORAL HUMAN CELLS WITH THE SULFORRODAMINA B ESSAY 
     Cells 
     The cell lines used in the essays, described in Table 1, were maintained in recipients of 25 cm 2  (Nunc®), with 5 mL of culture medium RPMI 1640 supplemented with 5% bovine fetal serum (RPMI/SFB), at 37° C. in atmosphere of 5% of CO 2  and 100% of humidity. 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Cellular lineages used in the assessment essays of the antiproliferate 
               
               
                 activity 
               
             
          
           
               
                   
                 Cellular Type 
                 Code 
               
               
                   
                   
               
               
                   
                 Lung 
                 NCI460 
               
               
                   
                 Mama 
                 MCF-7 
               
               
                   
                   
                 NCI ADR* 
               
               
                   
                 Melanoma 
                 UACC-62 
               
               
                   
                 Colon 
                 HT 29 
               
               
                   
                 Renal 
                 786-0 
               
               
                   
                 Ovary 
                 OVCAR-3 
               
               
                   
                 Prostate 
                 PC-3 
               
               
                   
                 Leukemia 
                 K-562 
               
               
                   
                   
               
               
                   
                 *cell line that expresses the phenotype of resistance against multiple drugs. 
               
             
          
         
       
     
     All of the procedures described below were performed under sterile conditions (Laminar Flux Veco®, Class IIB2). 
     Experimental procedure 
     100 mL of cells in medium RPMI/SFB with 50 μg/mL of Gentamicin were inoculated in its respective densities of inoculation, in plates of 96 compartments and incubated for 24 hours at 37° C. in atmosphere of 5% of CO 2  and 100% of humidity. 
     After 24 hours, 100 mL of the test substance were added in the following concentrations of 250; 25; 2.5; 0.2 μg/mL diluted in RPMI/SFB/gentamicin. In this moment, the reading of one plate was performed for the determination of the T0 (cell control in the moment of adding the samples). The other plates were incubated for 48 hours. After this period, the experiment was paused by adding the trichlorine acetic acid to further determine the protein content by means of colorimetric essay with the sulforrodamina B. 
     Dilution of the samples 
     The stocked solutions were developed by diluting the samples in dimethyl sulfoxide (DMSO) in the concentration of 0.1 g/mL. That solution was diluted by 400 times in RPMI/SFB/gentamicina to be added in the plates of 96 compartments being obtained to the ideal concentration of DMSO (Skehan e cols. 1990). 
     Essay of the Sulforrodamina B (SRB) 
     At the end of the test, the plates of 96 compartments were centrifuged for 3 minutes at 2000 rpm, and they were fixed with 50 μL of a solution at 50% of trichloroacetic acid (TCA) at 4° C. To complete the cellular fixation, the plates were incubated for 1 hour at 4° C. 
     The plates were submitted to four consecutive washes with distillated water to remove the residues of TCA, medium, SFB and secondary metabolic and maintained at room temperature until completely dry. 
     A coloration was performed by the adding of 50 μL SRB at 0,4% (weight/volume) diluted in acetic acid at 1%, and maintained at 4° C. for 30 minutes. Further, they were washed by 4 consecutive times with a solution of acetic acid 1%. The residue of the washing of the solution was removed and the plates were dried again at room temperature. The colorant linked to the cellular protein was diluted with a solution of Trizma Base (Sigma®) in concentration of 10 ∞M and pH 10.5 for 5 minutes in ultrasonic bath. The spectrophotometric of absorbancy reading was performed in 560 nm in a micro plates lector (Labsystems Multiskan® MCC/340). 
     Analysis of the results 
     The absorbancy averages were assessed having discounted its respective nulls and by means of the formula shown below was determined the growth inhibition (IC) of each tested sample. 
     T&gt;C, the drug did not stimulate the growth, it does not present IG. 
     IF T≧T0 but &lt;C, the drug was cyostatic and the formula used is 100×[(T-To)/(C-T0)]. 
     If T&lt;T0, the drug was cytotoxic and the formula used is 100×[(T-T0)/(C-T0)]. 
     Considering that T is the absorbency average of the treated cell, C is the cell control and T0 is the cell control in the addition day. 
     The obtained result was subtracted from 100% so obtaining the percentage of growth inhibition. The samples were considered actives since they presented growth inhibition-dependent dose higher than 50% and selective lineage, that is, the preferential activity for only one type of tumoral cell or with cytostatic and/or killed cell effect well distinguished among the cell lines. 
     All the assays were performed in triplicates so that the presented results make reference to a representative experiment. The standard deflection from the average was always lower than 5%. 
     Methodology utilized to perform the toxicological tests: 
     Technique 
     * Ten albinic Swiss mice are used, of the male gender, weighing approximate 25 g, for each of the treated groups and control. 
     * Adaptation period: the animals are maintained in the test room for at least seven days before the beginning of the essay. 
     * The animals are submitted to a fast 12 hours before administering the test substance, done by gavage, at the time the animal&#39;s body weight is listed. 
     * After the administration, the animals are maintained in observation for a minimum period of 14 days. 
     * The number of dead animals for each one of the doses is listed and the LD 50  is assessed by Litchfield and Wilcoxon&#39;s (1949) method and the animal&#39;s body weight is listed at the end of the acute toxicity test.