BRPI0803375A2 - compounds derived from 4-hydroxy-3-methoxy-benzaldehyde, process for obtaining, pharmaceutical composition, use of one or more compounds - Google Patents

Abstract

The present invention relates to compounds derived from vanillin (4-hydroxy-3-methoxy-benzaldehyde) and their synthetic methods of obtaining and purifying. Also provided by the invention are pharmaceutical compositions comprising one or more compounds described in the present invention. The invention further relates to the use of these compounds.

Description

Patent Descriptive Report for:

"4-HYDROXY-3-METOXY-BENZALDEHYDE DERIVATIVE COMPOUNDS, OBTAINING PROCESS, PHARMACEUTICAL COMPOSITION, USE OF ONE OTHER COMPOUND"

FIELD OF INVENTION

The present invention relates to compounds derived from vanillin (4-hydroxy-3-methoxy-benzaldehyde) and their synthetic and obtaining methods.

The present invention also relates to the in vitro and in vivo biological applications of these compounds as antitumor, antiparasitic and antioxidant agents.

In particular, it relates to the use of compounds for the manufacture of medicinal products for use in the treatment, prophylaxis or prevention of neoplastic diseases, metastatic, proliferative and / or degenerative lesions, immunosuppressive, immunodeficient, and degenerative pathologies and human and / or animal parasitic parasitic diseases. cosmetic products for use in the treatment and / or prevention of skin aging.

This invention further relates to pharmaceutical compositions containing said compounds.

BACKGROUND OF THE INVENTION

Compounds

There are several articles and patent documents related to the isolation, synthesis and diverse biological applications of curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) and its derivatives.

Toshiya Masuda et al. report, in their article published in the journal Phytochemistry, v.32 (6), pp. 1557-1560, 1993, the isolation of curcumin and 5 other compounds of analogous structure from domestic Curcuma, showing their antioxidant and anti-inflammatory properties.

Van der Goot et al. describes in his article published in the journal Eur. J. Med. Chem. v. 32, pp. 625-630, 1997, the antioxidant properties of cyclic dacurcumin analogs, which are comparable to dacurcumin activity.

Curcumin also has properties such as inhibition of HlV-Integrase, as described in the articles by Arctic and colleagues (J. Med. Chem., V.41, pp. 3948-3960,1998) and Mazumder and colleagues (Biochem. Pharmacol., v.49, pp. 1165-1170, 1995). These publications describe the synthesis of curcumin and derivatives according to the method described by Babu and Rajasekharan by condensing the corresponding arylaldehydes with acetylacetone and boric acid in the presence of low 1,2-3,4-tetrahydroquinoline. 10 to 55% (see KDV Babu and KN Rajasekharan OPPI Briefs, 26, 674-677, 1994).

On April 6, 1999, US Patent 5,891,924, entitled: "Curcumin (Differuloylmethane) Inhibition of NFkB Activation" was published. In this invention a method for inhibiting activation of NFkB transcription factor in animals by a pharmacologically effective dosing of curcumin is described.

In addition, US Patent 6,224,877 B1 of May 1, 2001, entitled "Process for Extraction of Curcuminoids from Curcuma species", has also protected the isolation method of this natural product.

The potent antitumor action of curcumin has been determined by Ramadasan et al., (Cancer Lett., V. 29 (2), pp. 197-202, 1985). Curcumin and its derivatives have shown a potent inhibitory effect on carcinogenesis caused by various types of chemical dyes.

The antioxidant mechanism of curcumin has been studied by Jovanovic et al. (J. Am. Chem. Soc. V. 121, pp. 9677-9681,1999) employing the methods of pulse radiolysis and flash-laser photolysis. The antioxidant mechanism dacurcumin has also been studied by Barclaye Vinqvist, described as published in the journal OrganicLetters, v. 2 (18), pp. 2841-2843, 2000. In this article it was concluded that the non-phenolic curcumin derivatives showed no antioxidant activity, emphasizing that acurcumin has the property of donating hydrogen atoms from the phenolic group and not from the CH2 group.

In U.S. Patent No. 6,228,365 B1, issued May 8, 2001, the inhibitory action of some synthetic and natural curcumin compounds on acarcinogenesis caused by chemicals has been described.

So-Young Parle and Darrick Kim found that several natural compounds obtained from "Curcuma longa", tested with PC12 cells, protected them from the action of Beta-amylase, which can prevent Alzheimer's-related diseases (J. Nat. Prod., V. 65 (9), pp. 1227-1231, 2002).

Curcumin and its derivatives have been found to more effectively protect PC12 cells (ED50 = 0.5-10} æg / mL) than Congo Red (ED50 = 37-39 æg / mL).

J.K. Buolamwini and H. Assefa reported the inhibitory activity against curcumin and its structural analogues against HIV-Tegrase (J. Med. Chem., V. 45, pp. 841-852,2002).

In 2002, the Cheng-Bu Liu-led collective (Organic Letters, v. 4 (17), pp. 2909-2911, 2002) confirmed the antioxidant mechanism of curcumin and its derivatives by methods supported by the theory of functional density. In this case, it was concluded that both acurcumin, methylcurcumin and vanylidenacetone present similar 0-H bond dissociation copies, indicating that phenolic groups are responsible for the hydrogenation of the atoms, which was also ratified by JS Wright in his article "Predicting". the antioxidantactivity of curcumin and curcuminoids "(J. MolecularStructure (THeochem), v. 59, pp. 207-217, 2002).

The synthesis and biological evaluation of novel dacurcumin analogues as anticancer and anti-angiogenic agents has been disclosed by Mamoru Shoji et al. (Bioorg. Med. Chem., V.12, pp. 3871-3883, 2004). In this paper it was concluded that the in vivo antitumor activity of curcumin is limited by the body's poor absorption of this compound, which would explain the low antitumor potency exhibited.

In 2004, K. M. Youssef et al. described the synthesis of curcumin structural analogs which show antioxidant forteactivity with values greater than 90% free radical inhibition (Arch. Pharm. Pharm. Med.Chem., v.337, pp. 42-54). The synthesis of these derivatives is based on reacting acetyl acetone (0.01 mol) with the corresponding aldehydes (0.02 mol) in the presence of alcoholic sodium hydroxide (50 mL, 10%) under stirring at room temperature for about 10 minutes, separating solids obtained and recrystallizing them from suitable solvents yields 45 to 77%.

D.L. Vander Jagt et al. (Bioorg. Med. Chem. V. 13, pp. 3811-3820, 2005) synthesized a series of alkylated derivatives and curcumin double coupling reduction products. One of these derivatives, 1,7-Bis (4-hydroxy-3-methoxyphenyl) -heptane-3,5-dione exhibited superior antioxidant activity than curcumin in the TRAP (Total Radical Trapping Anti-Oxidant Parameter) assay.

Thus, by conducting a survey of the available literature, it can be concluded that, to date, no synthesis of curcumin from vanillin and acetylacetone in acid medium has been reported using ultrasound. In addition, the antiproliferative properties,

corresponding metal antiparasitics, antioxidants and anti-inflammatory curcumin dosages such as: 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy- sodium phenolate and 3,5-dioxohepta-1,6-dienyl-bis (2-methoxyphenolate sodium) have not been reported either. No information on O-prenylated, acetylated and benzoylated derivatives is available either. dacurcumin.

Vanylidenacetone, also known as dehydrazingerone, was isolated from Zingiberofficinale by M. De Bernardi, G. Vidari and P. Vita-Finzi, (Phytochemistry, v.15, pp. 1785-1786, 1976), and their anti-inflammatory, antioxidant properties. Tumor and tumor cancers have been proven by Motohashi and co-workers (Cancer Lett. v. 134, pp. 37-42, 1998).

The synthesis of vanylidenacetone was carried out with 65% yield by M. N. A. Rao and G. Elias, from devaniline and acetone in basic medium (NaOH) and published in Eur. J. Med. Chem., V.23, p. 379-380, 1988. In this article, the syntheses of various substituted aryl derivatives which exhibit antiinflammatory properties are further described. It is noteworthy, however, that none of the obtained derivatives has an activity similar toibuprofen in the paw edema experiment.

HG Krishnamurty and S. Ghost (Ind. J. Chem., V.25 B, pp. 411-412, 1986) had previously employed vanylidenacetone derivatives as synthetic intermediates for obtaining the dihydrocurcumin natural product, component of Curcuma longa, by reaction between 4-O-benzyl dihydroferulic acid chloride and 4-O-benzylvanylidenacetone sodium hydride in tetrahydrofuran.

The antioxidant properties of vanilidenacetone were compared to those exhibited by curcumin and pelavitamin E, vanilidenacetone being less active than both (D. Rajakumar and M. N. Ra, Molecular and Cellular Biochemistry, v.140, pp. 73-79, 1994).

In another similar work developed by D.V.Rajakumar and M.N.A. Rao (Pharmazie, v. 49 (7), pp. 516-519,1994), vanylidenacetone derivatives have been synthesized, showing that 5-nitro-vanylidenacetone has a lipid peroxidation inhibition activity of 73.7% eIC50 equal to 2 0.1 µg / ml. However, vitamin E exhibited in this experiment an inhibition of 66.6% and an IC50 of 52.0 µg / ml.

E. Caballero et al. (Tetrahedron, v. 54, pp. 6111-6122, 1998) published the results of the synthesis of (±) -7- (3,4,5-Trimethoxyphenyl) -7-deoxidarubicinone, a new family of potentially antitumor anthracyclines from 3 4,5-trimethoxy-vanylideneacetone.

In the magazine Bioorg. Avg. Chem. Letters, v. 14, pp.1287-1289, 2004 A paper by D.SH.L.KIMJ.KIMJ.KIM has been described on the synthesis of davanylideneacetone derivatives with potential application against Alzheimer's disease. In this work 4- (4-hydroxy-3-methoxyphenyl) butan-2-one was synthesized by vanylidenacetone catalyzed hydrogenation in the presence of Pd-C / EtOH, HOAc, which is the raw material for the synthesis of 1- ( 4-hydroxy-3-methoxyphenyl) -oct-4-ene-3-one and structural analogs. 1- (4-Hydroxy-3-methoxyphenyl) -hexadec-4-ene-3-one product showed no cytotoxicity against PC12 and IMR32 cells, exhibiting cell protection (0.2 ± 0.1 µM ED50 for PC12 and 0.5 ± 0.2 æM) better than curcumin 17.1 ñ 5.7 æM for PC12 and 23.9 ñ 4.8 æM).

G.L. Silva et al. determined the antioxidant activity and free radical uptake of davanylidenacetone and the like, not finding very prominent activity of this product (Phytotherapy Research, v.19, pp. 1043-1047, 2005).

Twenty-eight compounds derived from vanilidenacetone, isoeugenol and 2-hydroxychalcona were synthesized by the J. Tatsuzaki team (J. Nat. Prod., V. 69, pp. 1445-1449, 2006), and their antiproliferative activity was evaluated in vitro against three cell lines (KB, KB-VCR and A549). Vanylidenacetone showed IC50 values greater than 10 µg / mL against the strains tested, however, 4- (3-ethoxy-2-hydroxy-phenyl) but-3-ene-2-one showed IC50 values of 2.0, 1.9 and 2.3 µg / mL, respectively. Also in this work, O-prenylated avanylideneacetone, 4- [3-methoxy-4- (3-methylbut-2-enyloxy) -phenyl] -but-3-ene-2-one was synthesized, and its antiproliferative activity was analyzed, showing IC50 values equal to 5.7, 3.5 and 3.8 µg / mL, respectively for the lines tested.

From the literature review we can conclude that neither the synthesis nor the antiproliferative, antiparasitic, antioxidant and antiinflammatory properties of sodium salt corresponding to vanylidenacetone, 2-methoxy-4- (3-oxo-but-1-enyl) phenolate is available . There was also no information on the existence of acetylated and benzoyl derivatives of this natural product.

Benzaldehyde cyanoacetoidrazones, methoxybenzaldehyde, salicylaldehyde, p-dimethylamino-benzaldehyde, furfural, crotonaldehyde, ethyl acetacetate, cyclohexanone, cinnamic aldehyde and vanillin, among others have already been described by J. Klosa (Archiv der Pharmazie Bd.287). 5, 302-304, 1954), showing the same tuberculostatic properties. Isonicotinonoylhydrazones were synthesized by D. Chakravarty, A. Bose and S. Bos, and the tuberculostatic properties of these compounds were also proved (J. Pharmaceutical Sciences, v. 53 (9), pp.1036-1039, 1964).

J. Quincoces and K. Peseke used furfural and acetofuran ascianoacetoidrazones to obtain 1,2-dihydropyridines (German Patent Document DE142549; July 02, 1980; see also article by the same authors entitled: "Synthese von substituierten 1-"). (Fur-2-yl) alkylidenamino] -1,2-dihydropyridin-3,5-dicarbonitrilen ", in Pharmazie, vol. 36, pages 534-535 (1981).

Cyanoacetohydrazide was used by the J. Quincoces team as a starting point for the synthesis of bioactive compounds (German Patent Documents DE 241 902 "Procedure for Obtaining Substituted Cyanoacetoidrazides"; DE 272 838 "Procedure for Obtaining Substituted Acrylides"; DE 273 059 "Procedure for obtaining acrylates"; DE 294 936 "Procedure for obtaining polycationic compounds (N '- [Bis (methylthio) methylen] -2,6,6-tricyano-3-methylthio-5-phenylhexa-3") , 5-dienhydrazides) "; DE 294 938" Procedure for obtaining pyrazolcarbohydrazides "; and DE 294 939" Procedure for obtaining 1,3-Dithietanes ").

The literature consulted did not reveal records of the synthesis and / or the antiproliferative, antiparasitic, antioxidant and anti-inflammatory properties of the corresponding salsodic vanillin cyanoacetoidrazone, 4- [2-cyanoacetyl) hydrazonomethyl] -2-methoxyphenolate desodium. Neither does information appear on the existence of the prenylated, acetylated and benzoylated derivatives of this compound, nor 3-bromo-4-hydroxy-5-methoxy-benzylidene-cyanoacetoidrazide, 4-hydroxy-3-methoxy-5-nitro-benzylidene-cyanoacetohydrazide corresponding sodium salts. No information related to the synthesis and biological properties of 2-cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide and its derivatives was found.

A literature review for avaniline and derivatives was also performed. Through this search, no information describing or related to the antitumor and antioxidant properties of this group of chemical substances was found in any of the existing media.

Cancer

One of the most important and promising forms of tumor treatment is chemotherapy, ie the use of single or combined chemical agents to control and remit the proliferation of these malignant tumors. This form of systemic treatment of the disease may be associated with older localized forms of treatment such as surgery and radiotherapy. The association of chemotherapy with other therapeutic forms for tumor treatment is standard practice. Its use allows the therapeutic synergism of pre- and postoperative tumor treatment or radiotherapy to promote the eradication of metastases.

Since the advent of cancer chemotherapy (1940), many of the ways in which cancer cells "escape" from the chemical have been identified. As cells develop drug resistance, they may also develop chemically and mechanically unrelated cross-resistance to other drugs in a phenomenon known as multidrug resistance (MDR).

Although current cancer therapy depends mainly on the use of surgery, radiation therapy and chemotherapy, progress in understanding the biology of malignant transformation and differences in control of normal and cancer cell proliferation has led to the discovery of new potential targets for cancer treatment. It is unlikely that new therapies will completely replace existing drugs, as in recent years these drugs have become more effective and their toxicities more treatable and predictable. However, a combination of existing drugs, new therapeutic approaches, and obtaining a molecular and genetic profile of each tumor would give each patient an individualized and specific treatment, increasing the effectiveness of treatment and decreasing the incidence of side effects.

Chagas disease

Chagas disease, caused by the protozoan Trypanosoma cruzi, is widely distributed in rural areas of Central and South America. It is one of the largest public health problems in the Americas, affecting about 16 to 18 million people, and an estimated 100 million people. People are still at risk for this disease. The life cycle of this parasite is characterized by the presence of different forms found in two hosts, one vertebrate and the other invertebrate. The epimastigote form (E) found inside the vector "barber" (Insects of the Reduviidae family) multiplies by binary division and differs in the metacyclic trypomastigote (TM) form.

Commonly used antiparasitic drugs, for example nifurtimox, a nitrofuran derivative (Bayer, previously marketed under the name of Lampit, recently discontinued), and benzonidazole, a nitroimidazole derivative (Roche, marketed under the name Radadil, Rochagam or Roganil). ), act through the generation of free radicals in their metabolism, being T. cruzimuito susceptible to damage caused by these radicals. It is believed that both drugs kill or inhibit the growth of the parasite due to the increase of its stroxidative. However, these drugs also cause many side effects in the patients being treated, mainly due to the effective concentration (500 µM) used (Maya et al, Biochem Comp. Physiol. A Mol.Integr. Physiol, 2006). Nifurtimox and benzonidazole act only in the acute and recent phase of T. cruzi infection and have major limitations due to their side effects due to their need for at least 60 days. On the other hand, allopurinol and ketoconazole, fluconazole and itraconazole antifungals, in clinical tests, have shown poor, null or controversial results for the treatment of the chronic phase of Chagas disease.

Recent studies of T. cruzi biochemistry have led to research on new drugs and the understanding of their mechanisms of action in an attempt to abolish the toxic systemic effect caused by the usual treatment drugs. Development of new drugs should target specific alboses of parasite structure and metabolism which should be studied, initially "in vitro". The target to be reached should be correlated with the parasitic activity of the drug and / or its derivatives, considering the least possible damage to the host cell (Coura et al., Mem. Inst. Oswaldo Cruz, v. 97 (1), pp. 3 -24, 2002).

On the other hand, studying the differential expression degenes in the different T. cruzi strains exposed to increasing concentrations of benzonidazole, it was verified the existence of benzonidazole resistant parasites. The emergence of parasite resistance to usual drug-treatment drugs is an additional but important problem. The combination of L-buthione (S, R) -sulfoximine (BSO) with nifurtimox and benzonidazole led to a decrease in the doses necessary to achieve the same clinical effect and, consequently, the minimization of side effects and / or duration of therapy. Nevertheless, the development of new, cheaper and easier-to-administer drugs is needed and urgent (Faundez et al., Antimicrob. Agents Chemother., V. 49 (1), pp. 126-130, 2005).

Antioxidant activity

Antioxidant agents are important for restricting harmful oxidative reactions to cells, which may predispose to the development of important clinical conditions such as heart disease and cancer (Diplock etal., Br. J. Nutr. 80 (1) pp. 77-112, 1998). There is growing evidence indicating that reactive oxygen species are not only toxic, but also play an important role in cellular signaling and in regulating gene expression.

Various biochemical and physiological stimuli, such as redox state tightness, impaired glucose and glycosylation deprivation, accumulation of polyunsaturated fatty acid peroxidation products, or oxidation and decomposition of cholesterol, can disrupt redox homeostasis and lead to stress accumulation, which may result in modified protein accumulation. Diseases such as Alzheimer's, Parkinson's and Huntington's are major neurological disorders associated with the production of these abnormal proteins.

Identifying new compounds with potent antioxidant activity can be extremely useful not only for the development of agents with anti-aging properties, but also for therapeutic agents with application in neoplastic and neurodegenerative diseases.

OBJECTIVES OF THE INVENTION

In view of the foregoing, the present invention has the object of providing new processes for obtaining vanillin-derived compounds (4-hydroxy-3-methoxy-benzaldehyde).

The present invention also provides novel vanillin-derived compounds.

It is another object of the present invention to provide pharmaceutical compositions which make use of the compounds described in the present invention.

It is a further object of the present invention to use the compound methods described in the present invention for the manufacture of medicaments for use in neoplastic diseases, metastatic, proliferative and / or degenerative lesions, immunosuppressive pathologies, edegenerative immunodeficients, and diverse human and / or animal parasites.

It is a further object of the present invention to use the compound methods described in the present invention for the manufacture of cosmetic products for use in treating and / or preventing skin aging.

It is a further object of the present invention to provide methods of treating and / or preventing neoplastic diseases, metastatic, proliferative and / or degenerative lesions, immunosuppressive, immunodeficient and degenerative disorders, and diverse parasites in humans and / or animals using the compounds described in the present invention.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to methods for obtaining vanillin (4-hydroxy-3-methoxy-benzaldehyde) derivatives.

In particular this invention relates to the preparation of decurcumin (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) and its alkylated derivatives from vanillin (4 -hydroxy-3-methoxy-benzaldehyde) or substituted vanillin, acetyl acetone and concentrated hydrochloric acid under ultrasonic irradiation conditions.

Another aspect of this invention is the process of transforming curcumin (1,7-bis- (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) into metal salts by the addition of a solution. of metal alkoxide and its corresponding alcohol in molar ratio 1: 1 or 2: 1, followed by evaporation of the alcohol in vacuo to a solid.

The present invention also relates to a process for preparing metal phenolate in which vanylidenacetone or a substituted vanylidenacetone is brought into common contact with metal alkoxide and an alcohol at 1: 1 molar ratio, followed by evaporation of the vacuum alcohol to the formation of a metal salt. in the solid state.

The invention also relates to a process for preparing a vanillin metal salt from 4-hydroxy-3-methoxy-benzaldehyde. This process comprises contacting 4-hydroxy-3-methoxy-benzaldehyde, a methoxy alkoxide (ROM) and a 1: 1 molar alcohol (ROH) followed by evaporation of the vacuum alcohol until a solid metal salt is formed. It further relates to a process for preparing acetylated vanillin derivatives or a substituted vanillin comprising mixing vanillin or a substituted vanillin, acetic anhydride and sodium acetate.

The invention also relates to a process for preparing 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde by mixing 4-hydroxy-3-methoxy-benzaldehyde, depotassium carbonate and dimethylformamide at 15 to 65 ° C. for 10 to 90 minutes, followed by the addition of prenyl bromide and stirring between 15 ° C and 60 ° C for 4 to 15 hours.

This invention also relates to a process for preparing cyanoacetoidrazones and derivatives of substituted devanilines wherein the substituted vanillins are mixed with cyanoacetohydrazide in the presence of an alcohol the mixture is heated to the formation of a precipitate.

The present invention also relates to a process for preparing 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide from devaniline, 4-hydroxymethyl 3-Methoxy-benzylidene-cyanoacetohydrazide, ammonium acetate and aceticoglacial acid.

This invention also relates to a process for preparing substituted 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazides with carbonyl compounds. in molar ratio 1: 1 under the conditions of Knoevenagel condensation and ultrasonic irradiation in the presence of ammonium acetate and acetic acid.

Another aspect of this invention is novel compounds of vanillin (4-hydroxy-3-methoxy-benzaldehyde) derivatives.

In particular this invention relates to the compound 1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione, or curcumin, obtained from vanillin, acetylacetone and hydrochloric acid under ultrasonic irradiation conditions.

This invention also relates to the compounds: sodium 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy-phenolate; 3,5-dioxohepta-1,6-dienyl-bis (2-methoxy-phenolate) disodium; Sodium 2-methoxy-4- (3-oxo-but-1-enyl) phenolate; 4-formyl-2-methoxy phenolate disodium; Phenyl 4-formyl-2-methoxy acetate; 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde; 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide; [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetoidrazide; (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetohydrazide and 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide. It is a further aspect of this invention to use the compounds for the manufacture of medicaments for use in the treatment, prophylaxis or prevention of neoplastic diseases, metastatic, proliferative and / or degenerative lesions, immunosuppressive pathologies, edegenerative immunodeficients, and diverse parasites in humans and / or animals other than manufacture. of cosmetic products for the treatment and / or prevention of skin aging.

This invention further relates to pharmaceutical compositions containing said compounds.

DEFINITIONS

Within the scope of this patent application abbreviations of the compounds described herein are often used.

For ease of reading the document below are the definitions of the various abbreviations as used in this request.

DB2 refers to the compound 1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione, or also known as curcumin.

DB3-A refers to 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy-phenolate disodium compound.

DB3-B refers to 3,5-dioxohepta-1,6-dienyl-bis (2-methoxyphenolate) disodium compound.R-6 refers to 4- (4-Hydroxy-3-methoxy compound) -phenyl) -but-3-en-2-one, also known asovanylidenacetone.

R 6 -B refers to sodium 2-methoxy-4- (3-oxo-but-1-enyl) phenolate.

RR-1 refers to the compound 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazide.

VI refers to 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde compound.

V4 refers to the 4-formyl-2-methoxy-phenylphenyl acetate compound.

V5 refers to the vanillin or 4-hydroxy-3-methoxy-benzaldehyde compound.

V6 refers to the 4-formyl-2-methoxy-phenolate disodium compound.

V10 refers to the compound 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide.

Vil refers to the compound [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetoidrazide.

V12 refers to the compound (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetoidrazide.

DESCRIPTION OF THE FIGURES

The following figures are part of this report and are included herein to illustrate certain aspects of the invention. The object of the present invention may be better understood with reference to one or more of these figures, in combination with the detailed description of the preferred embodiment herein.

Figure 1 shows the inhibitory effect of melanoma cell proliferation of compounds V4 and V6. Compounds V4e V6 at different concentrations (5, 10, 20, 40 and 80 (µg / ml)) were analyzed for the effect on proliferation of melan-a (ma) and melanoma (Tm5) melanocytes by MTT assay. Compound V4 showed dose dependent inhibitory effect of cell proliferation in both strains Compound V6 showed inhibitory effect dependent on melanoma cell proliferation, but not melanocytes.

Figure 2 shows linear regression curves depicting the viability of melanocyte melanocytes (ma) and murine melanoma cells (Tm5) incubated for 48h with different doses of compounds V4 and V6 by the MTT assay. The graphs show the ability of the compounds in inhibit (descending line, V4 and V6) or stimulate (ascending line, V6) cell proliferation, considering the initial cell number as 100%.

Figure 3 shows the potentiation of the cytotoxic effect of bleomycin by compound V6 on Tm demelanoma cells. Cells were incubated for 72h with: a) diluent (DMSO + phosphate buffer) (control experiment), b) compound V6 (40ug / mL), c) bleomycin (6mU / mL), and d) with a combination of compound V6 ( 40ug / ml) and bleomycin (6mU / ml). In the case of incubation with V6 and bleomycin, the first 24h the cells were incubated with V6 only and then bleomycin was added and the cells incubated for a further 48h. Relative cell number was estimated by colorimetric assay (MTT). Intense potentiation of the cytotoxic effect dableomycin was observed by compound V6.

Figure 4 shows the potentiation of carboplatin cytotoxic effect by compound V6 in Tm demelanoma cells. Cells were incubated for 72h with: a) diluent (DMSO + phosphate buffer), b) compound V6 (40ug / mL), c) carboplatin (25uM), and d) with a combination of compound V6 (40ug / mL) and carboplatin (25uM). In the case of incubation with V6 and carboplatin, in the first 24h cells were incubated with only V6 and then bleomycin was added and the cells were incubated for a further 48h. The relative number of cells was estimated by colorimetric assay (MTT). It was observed that there was intensification of the cytotoxic effect of carboplatin V6.

Figure 5 shows the effect of compounds V4 and V6 on growth of murine melanoma. C57B1 / 6 mice were subcutaneously inoculated with 2 x 105 melanomaTm5 cells and then treated with compounds V4 or V5 (1mg / day subcutaneously). The control group received daily injections of saline phosphate buffer (PBS) subcutaneously.

Figure 5A shows the evolution of murine domelanoma growth in animals treated with PBS, V4 and V5, respectively. Figure 5B shows the weight of the final tumor of the treatment period with PBS, V4 and V5 where significant inhibition of tumor growth is observed.

Figure 6 shows C57B1 / 6 mice inoculated subcutaneously with 2 x 105 Tm5 murine melanoma cells, treated daily with 100 µl subcutaneous saline phosphate buffer (PBS) and sacrificed 16 days after tumor cell injection. Red arrows indicate the presence of tumors in 3 different mice.

Figure 7 shows C57B1 / 6 mice subcutaneously inoculated with 2 x 105 Tm5 murine melanoma cells, treated 1 mg / day subcutaneously of compound V4, 16 days after tumor cell injection. Red plaques indicate the region of tumor cell inoculation in 3 different mice. Figure 8 shows C57B1 / 6 mice subcutaneously inoculated with 2 x 105 Tm5 murine melanoma cells, treated with 1 mg / day of subcutaneous compound V5, 16 days after tumor cell injection. Red signs indicate the region of tumor cell inoculation in 3 different mice.

Figure 9 shows the antioxidant activity of compounds V4 and V6 in melanocytes and melanoma cells. Melanocyte (melan-a; ma) and melanoma (Tm5) lines were treated or not with compounds V4 and V6 and analyzed for intracellular superoxide anion levels. Cells treated or not with the compounds were incubated with fluorophore DHE and the determination of intracellular superoxide anion level in melan-a and Tm5 cells was estimated after flow cytometer analysis (FacsCalibur; Becton & Dickinson).

Figure 10 shows experiments of VERO cell infection with Trypanosoma cruzi where the antiparasitic activity of the compounds DB3A, V4, V6, V10 and benzenzididazole on the intracellular doparasite amastigote forms was analyzed.

Figure 10A shows a VERO cell infected with Trypanosoma cruzi amastigote form and untreated (control experiment). The staining of the VERO cell nucleus and a large number of T.cruzid amastigotes disposed in the cytoplasm around the nucleus with the stained kinetoplast nuclei are observed.

Figure 10B shows a VERO cell infected with the Trypanosoma cruzi amastigote form and treated for 48 h with 5 µg / ml benzonidazole, currently used standard treatment drug. Effective antiparisitic action is observed.

Figure 10C shows a VERO cell infected with the Trypanosoma cruzi amastigote form and treated for 48 h with 10 µg / ml DB3A. Effective antiparisitic action similar to that of the standard drug is observed.

Figure 10D shows a VERO cell infected with the Trypanosoma cruzi amastigote form and treated for 48 h with 10 µg / ml V4.

Figure 10E shows a VERO cell infected with the Trypanosoma cruzi amastigote form and treated for 48 h with 10 µg (µg / ml V6.

Figure 10F shows a VERO amastigote form of Trypanosoma cruzi and treated for 48 h with 5 µg / ml V10.

Figure 11 shows in vivo experiments of mouse infection with Trypanosoma cruzi where antiparasitic activity of DB3A ebenzonidazole compounds was analyzed. The graphs show the isolated profile of parasitemia (parasites / mL of blood) in the animals for 18 days. (A) control (untreated); (B) combenzonidazole treatment; (C) treatment with DB3A. H means head, B means body, T means tail, HB means head and body and HBT means head and body. These are the identifications made to analyze the mice separately in the group.

Figure 12 shows the mean parasitemia (parasites / mL of blood) following in vivo infection with Trypanosoma cruzi in untreated (control) mice treated with benzonidazole or DB3A.

DETAILED DESCRIPTION OF THE INVENTION

Of the procurement processes

This invention relates to methods for obtaining vanillin (4-hydroxy-3-methoxy-benzaldehyde) derivatives.

In particular this invention relates to the preparation of decurcumin (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) and its alkylated derivatives of structure I:

<formula> formula see original document page 30 </formula> where

R 1, at each separate occurrence, is a hydrogen atom or an alkyl group;

R2, in each separate occurrence, is a hydrogen atom or an alkyl group.

R3, in each separate occurrence, is a hydrogen atom; CH 2 CH = C (CH 3) 2; C0CH3 or COPh.

on what

Z, in each separate occurrence, is a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group;

on what

US is ultrasonic irradiation.

In this process, curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) and its alkylated derivatives are obtained from vanillin (4-hydroxy-3). -methoxy-benzaldehyde) or substituted vanillin, acetyl acetone, and concentrated hydrochloric acid under ultrasonic irradiation conditions as presented in the above reaction and involves the following steps:

(a) contact vanillin or substituted vanillin; acetylacetone or alkylated derivatives of acetylacetone; and hydrochloric acid under ultrasonic irradiation conditions;

b) Leave the reaction mixture to stand;

c) Extract using sodium hydroxide or potassium hydroxide solution;

d) Filtering the reaction mixture under vacuum;

e) Acidifying the filtrate obtained with hydrochloric acid or sulfuric acid with the consequent formation of a precipitate;

f) Filtration and washing of the precipitate with distilled water;

g) drying the solid product.

Preferably the ultrasonic irradiation is performed in a range of 25 to 40 KHz for a time period ranging from 1 to 8 hours. The reaction mixture is preferably allowed to stand for a period of 8 to 24 hours over a temperature range of 10 to 60 ° C.

This invention also relates to the process of obtaining metal salts of curcumin or its derivatives (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) of structures II and III : <formula> formula see original document page 33 </formula>

on what

R1, at each separate occurrence, is a hydrogen atom or an alkyl group;

R 2, in each separate occurrence, is a hydrogen atom or an alkyl group;

R3, in each separate occurrence, is a hydrogen atom; CH 2 CH = C (CH 3) 2; CPCH3 or COPh;

M, in each separate occurrence, is a metal cation;

R, in each separate occurrence is CH3 or CH3CH2. In this process, a compound of structure I is placed in the presence of a metal alkoxide (ROM) solution and its corresponding alcohol (ROH) in 1: 1 or 2: 1 molar ratio as presented in the reactions. above and involves the following steps: a) placing a compound of structure I in the presence of a solution of metal alkoxide (ROM) and its corresponding alcohol (ROH) in molar ratio 1: 1 or 2: 1;

b) evaporating the alcohol in vacuo to the formation of a solid state salmetallic;

c) passing the metal salt obtained by sieves to obtain an easily soluble fine powder.

When the molar ratio between the metal alkoxide (ROM) and its corresponding alcohol (ROH) is 1: 1, a monometallic salt is obtained. When the molar ratio between the alkoxidometallic (ROM) and its corresponding alcohol (ROH) is 2: 1, a di-metallic salt is obtained according to the above reaction.

Preferably a sodium ethoxide ethanolic solution or a sodium methoxide methanolic solution may be used to obtain 4- [7- (4-hydroxy-3-methoxyphenyl) -3,5-dioxohepta-1,6 -dienyl] -2-methoxy-phenolate sodium or 3,5-dioxohepta-1,6-dienyl-bis (2-methoxy-phenolate) disodium.

The present invention also relates to a process for preparing vanylidenacetone metal phenolate of structure V: wherein:

R1 is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group;

M, is at each separate occurrence, is a stabilizing metal cation of the compound in the form of ion phenoxide.

In this process vanylidenacetone or a substituted general-purpose vanylidenacetone IV is brought into contact with a 1: 1 molar metal alkoxide (ROM) and an alcohol (ROH). The process involves the following steps:

(a) contacting vanylidenacetone or a vanylidenacetone of general structure IV, with alkoxide methyl (ROM) and an alcohol (ROH) at 1: 1 molar ratio;

b) evaporating the alcohol in vacuo to the formation of a solid state salmetallic;

c) passing the metal salt obtained by sieves to obtain an easily soluble fine powder.

Preferably 4- (4-Hydroxy-3-methoxy-phenyl) -but-3-en-2-one substituted vanylideneacetones may be used, in contact with a metal alkoxide (ROM) and an alcohol (ROH), in relation to molar ratio 1: 1 to obtain vanylidenacetone metal ofenolate, 2-Methoxy-4- (3-oxo-but-1-enyl) phenolate.

Preferably an ethanolic sodium ethoxide solution or a methanolic sodium methoxide solution may be used.

The invention further relates to a process for preparing a vanillin metal salt of structure VI from 4-hydroxy-3-methoxy-benzaldehyde.

<formula> formula see original document page 36 </formula>

R, is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group;

M, at each separate occurrence, is a stabilizing metal cation of the phenoxide ion compound;

This process comprises contacting 4-hydroxy-3-methoxy-benzaldehyde, a metal alkoxide (ROM) and an alcohol (ROH) in 1: 1 molar ratio and involves the following steps:

a) Bring 4-hydroxy-3-methoxy-benzaldehyde, a metal alkoxide (ROM) and an alcohol (ROH) into contact with a 1: 1 molar ratio.

b) evaporating the alcohol in vacuo to the formation of a solid state salmetallic;

c) passing the metal salt obtained by sieves to obtain an easily soluble fine powder.

Preferably 4-hydroxy-3-methoxy-benzaldehyde may be used to obtain the sodium vanylin-4-formyl-2-methoxy-phenolate metal salt.

Preferably a sodium ethoxide ethanolic solution or a sodium methoxide methanol solution may be used.

This invention further relates to a process for preparing acetylated vanillin derivatives or a substituted vanillin of structure VII.

<formula> formula see original document page 37 </formula>

Structure VII

on what

R is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group.

This process comprises mixing vanillin or substituted vanillin, acetic anhydride and sodium acetate and involves the following steps:

a) mix vanillin or a substituted vanillin, acetic anhydride and sodium acetate.

b) heating this mixture to a temperature between 80 ° C and 140 ° C under stirring and reflux for a time period of 1 to 10 hours;

c) pouring the mixture into ice water to give a precipitate.

Preferably the mixture should be heated to approximately 120 ° C under stirring and reflux for 2 hours and 30 minutes.

Preferably 4-hydroxy-3-methoxy-benzaldehyde, acetic anhydride and sodium acetate may be mixed to obtain phenyl 4-formyl-2-methoxy acetate.

The invention also relates to a process for preparing 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde of structure VIII.

<formula> formula see original document page 38 </formula>

which comprises the following steps:

a) mixing 4-hydroxy-3-methoxy-benzaldehyde, potassium carbonate and dimethylformamide at 15 to 65 ° C for 10 to 90 minutes;

b) adding prenyl bromide and stirring the reaction mixture at 15 ° C to 60 ° C for 4 to 15 hours;

c) pouring the obtained mixture into ice water yielding a white precipitate.

d) filter the obtained precipitate

Preferably 0.01 mol (1.52 g) of 4-hydroxy-3-methoxy-benzaldehyde (V5) with 0.03 mol (4.14 g) potassium carbonate and 10 ml of dimethylformamide at a constant temperature of 40 ° C should be used. C for 30 minutes.

Preferably 0.02 mol (2.98g, 2.32 mL) of prenyl bromide should be added and the reaction mixture should be stirred for 8 hours at 40 ° C.

This invention also relates to a process for preparing cyanoacetoidrazones and derivatives of structure X from substituted vanillins of structure IX.

Structure IX Structure X

<formula> formula see original document page 39 </formula>

on what

R is, at each separate occurrence, a hydrogen atom; CH 2 CH = C (CH 3) 2; C0CH3 or COPh.where

R1 is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group comprising the following steps:

(a) mixing substituted vanillins of structure IX with cyanoacetohydrazide in the presence of an ROH alcohol;

b) heating the mixture to the formation of a precipitate. Preferably, vanillin may be mixed and

cyanoacetohydrazide in the presence of ethanol to obtain 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide.

Preferably, 3-Methoxy-4- (3-methylbut-2-enyloxy) benzaldehyde and cyanoacetohydrazide may be mixed in the presence of ethanol to obtain [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetoidrazide .

Preferably, 3-Bromo-4-hydroxy-5-methoxy-benzaldehyde and cyanoacetohydrazide may be mixed in the presence of ethanol to obtain (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetohydrazide.

This invention also relates to a process for preparing substituted 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazides of structure XI <formula> formula see original document page 41 </formula>

on what

Z is, in each separate occurrence, a hydrogen atom; a metal cation; Eats; COC6H5;

CH 2 CH = C (CH 3) 2; methyl group; alkyl group.

on what

X is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group;

on what

Y is, in each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group;

comprising reacting CH-acid cyanoacetoidrazones substituted with carbonyl compounds at 1: 1 molar ratio under the conditions of Knoevenagel condensation and ultrasonic irradiation in the presence of acetic acid ammonium acetate. Preferably, ultrasonic irradiation occurs in a range of 25 to 40 KHz during it would take from 1 to 8 hours.

Preferably, the reaction takes place within a temperature range of between 20 and 60 ° C.

The present invention also relates to a process for preparing 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide comprising the following steps:

a) mixing vanillin, 4-hydroxy-3-methoxy-benzylidene-cyanoacetohydrazide, ammonium acetate and glacial acetic acid;

b) leave the mixture under ultrasonic radiation for an interval of 5 to 80 hours;

c) pouring the obtained mixture into ice water to give a yellow precipitate;

d) filter the precipitate.

Preferably the mixture is left under ultrasonic radiation for 30 hours.

This invention relates to novel compounds devaniline (4-hydroxy-3-methoxy-benzaldehyde).

In particular this invention relates to the compound 1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dioneobtide from vanillin, acetylacetone and hydrochloric acid under ultrasonic irradiation conditions .

This invention also relates to the compounds: sodium 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy-phenolate; 3,5-dioxohepta-1,6-dienyl-bis (2-methoxy-phenolate) disodium; Sodium 2-methoxy-4- (3-oxo-but-1-enyl) phenolate; 4-formyl-2-methoxy phenolate disodium; Phenyl 4-formyl-2-methoxy acetate; 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde; 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide; [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetoidrazide; (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetohydrazide and 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide.

This invention further relates to pharmaceutical compositions containing said compounds.

Properties and uses of the compounds derived from vanillin (4-hydroxy-3-methoxy-benzaldehyde):

The compounds of the present invention have the unexpected technical effect of having significant antitumor, antioxidant and antiparasitic activities that enable their use in a number of relevant biological, pharmaceutical and therapeutic applications.

a) Antitumor activity It is important to note that the compounds described in the present invention exhibit significant antitumor activity.

Namely, the compounds of the present invention induce a specific inflammatory response in human and animal primary tumors in vivo, preventing their growth and spread.

Accordingly, the compounds described herein prevent the diffusion and migration of tumor cells to internal organs, as well as formation of secondary tumors or metastases.

In particular, compounds V4 and V6 selectively inhibit tumor cell proliferation and tumor development.

On the other hand, the compounds DB2, DB3A, DB3B, V4 and V6 exhibit greater specific cytotoxic effect against several human and animal tumor lines, in vivo, and present 50% inhibitory concentration (IC50) values lower than the other tested compounds.

The compounds described herein also exhibit the ability to induce programmed cell death (apoptosis, anoikis) of human and animal proliferative diseases and thus inhibit growth and prevent migration of human and animal factors. This is a further aspect of this invention the use of the compounds of this invention. invention to treat or prevent or manufacture medicines for use in the treatment, prophylaxis or prevention of neoplastic, metastatic, proliferative and / or degenerative diseases. In particular, the compounds described herein may be used to treat or prevent or to manufacture medicaments for use in the treatment, prophylaxis or prevention of neoplastic diseases caused by lung cancer, multiple drug resistant breast and breast cancer, melanoma and melanoma pelvis cancers, lymphoid leukemias, Acute and chronic myeloids, erythroleukemia, myelodysplasias, colon cancers, ovaries, uterus, kidney, pancreas, prostate, soft tissue sarcomas, hepatocarcinomas, osteosarcomas, central nervous system, neuroblastomas, astrocytomas, oropharynx, thyroid, reproductive tract and prostate, male.

b) Co-adjuvant effect on chemotherapy treatment

The compounds of the present invention also have important properties as co-adjuvants in the treatment with chemotherapeutic agents.

Low doses of the compounds of the present invention when associated with low doses of bleomycin or carboplatin in a suitable molar ratio significantly inhibit the proliferation of tumor cells. The compounds of the present invention potentiate the antitumor effects of bleomycin or carboplatin to treat or prevent or manufacture medicaments for use in the treatment, prophylaxis or prevention of neoplastic diseases, metastatic, proliferative and / or degenerative lesions.

Accordingly, it is a further aspect of this invention a pharmaceutical composition comprising a compound or compounds of this invention and bleomycin or carboplatin.

Still another aspect of this invention is the compound of this invention in combination with bleomycin or carboplatin to treat or prevent or to manufacture medicaments for use in the treatment, prophylaxis or prevention of neoplastic diseases caused by lung cancer, multidrug-resistant breast and breast cancer, non-melanoma skin and melanomas, lymphoid leukemias, acute and chronic myeloids, erythroleukemia, myelodysplasias, colon, ovarian, uterine, kidney, pancreas, prostate cancer, soft tissue sarcomas, hepatocarcinomas, osteosarcomas, central nervous system, neuroblastomas, astropitomas, astrocitomas , thyroid, gastric, prostate, and male reproductive tract attachments. In addition, compounds V4 and V6 inhibit superoxide anion production, making tumor cells more susceptible to cell death by chemotherapeutic agents or ionizing radiation.

c) Immunostimulating effect

The compounds described herein also act as specific immunostimulating agents. Accordingly, it is another aspect of this invention the use of the water-written compounds to treat or prevent or to manufacture medicaments for use in the treatment, prophylaxis or prevention of human and animal immunosuppressive, immunodeficient and degenerative disorders. In particular the compounds of the present invention may be used to treat or prevent or to manufacture medicaments for use in the treatment, prophylaxis or prevention of neurodegenerative diseases such as Alzheimer's disease.

d) Antioxidant activity

The compounds V4 and V6 described in this invention exhibit potent antioxidant activity.

Accordingly, it is a further aspect of this invention to use the compounds described herein to treat or prevent to manufacture medicaments for use in the treatment, prophylaxis or prevention of neoplastic diseases, metastatic, proliferative and / or degenerative lesions, immunosuppressive depatologies, edegenerative immunodeficients and various parasitic diseases. humans and / or animals in addition to the manufacture of cosmetic products for the treatment and / or prevention of skin aging.

e) Antiparasitic activity

The compounds of the present invention exhibit action on proliferation of Trypanosoma cruzi epimastigote and trypomastigote forms of Trypanosoma cruzi in LIT culture medium on Trypanosoma cruzi parasite causing Chagas Disease, determined by the MTT method.

In addition, the compounds described herein, DB2, DB3A, V4, V6, V10 exhibit action on the viability of Trypanosoma cruzi blood tryptomastigotes and intracellular amastigotes in RPMI medium.

On the other hand, the compounds exhibit their action on intracellular amastigote forms of Trypanosoma cruzi, inducing apoptosis with parasite death without affecting the host cell, an important fact for the treatment of the chronic phase of Chagas disease without exhibiting undesirable toxic effects such as Nifurtimox and Benzonidazole.

Accordingly, it is a further aspect of this invention to use the compounds described herein to treat or prevent or to manufacture medicaments for use in the treatment, prophylaxis or prevention of parasites.

EXAMPLES

In order to allow a better understanding of the present invention and to clearly demonstrate the technical advances obtained, the results of the different assays performed with respect to this invention are now presented as examples.

Part 1: Synthesis, isolation and characterization of compounds EXAMPLE 1: Obtaining curcumin or 1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione (DB2)

<formula> formula see original document page 49 </formula>

0.01 mol of vanillin (4-hydroxy-3-methoxy-benzaldehyde) (1.52 g) and 2 mL of concentrated hydrochloric acid (38%) were placed under ultrasound conditions at 28 KHz.

Subsequently, 0.005 mol deacetyl acetone was added and then the mixture was subjected to ultrasonic irradiation at 28 KHz for 1 hour. The mixture was then allowed to stand for 24 hours at 30 ° C. Subsequently extraction was performed with sodium or potassium hydroxide solution and subsequent vacuum filtration. Then the mixture was acidified with hydrochloric or sulfuric acid, observing the formation of precipitate which was filtered off and washed with distilled water. The solid product was dried at room temperature and had a greenish brown color.

General Formula: C21H20O6. Molecular Mass: 368 Melting Point: 165-170 ° Yield: 2g (78%)

The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, DMSO): δ = 9.65 (s, 1H, OH-enolic); 8.27 (s, 1H, OH-phenolic); 7.63 (d, 1H, H -1); 7.34 (s, 1H, H-2 '); 7.19, (d, 1H, H-6); 6.82 (d, 1H, H-7); 7.17 (m, 2H, H-5 7H-6 '); 3,883 (s, 3H, CH 30).

13 C NMR (75 MHz, DMSO): δ = 188.10 (C = 0); 149.45 (C-3 '), 148.02 (C-4'); 142.83 (OI); 126.39 (OI '); 123.40 (C-2), 123.05 (C-6 '); 115.71 (C-5 '); 111.43 (C-2 '); 79.18 (C-4), 55.76 (CH30).

DEPT (DMSO): δ = 143.25 (OI); 123.82 (C-2); 123.46 (C-6 '), 116.13 (C-5'); 111.84 (C-2 '); 56.18 (CH30).

EXAMPLE 2: Obtaining Curcumin Metal Salts

2a) Obtaining sodium 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy-phenolate (DB3-A)

Sodium salt 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl] -2-methoxy-phenolate sodium was obtained from of 1,7-Bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione in the presence of disodium methoxide and methanol in a 1: 1 molar ratio.

1,7-Bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione was contacted with a 1: 1 molar ratio of sodium methoxide in methanol and then the solvent was evaporated in vacuo to a solid. The salt was obtained in high yield (94%). Once obtained, the phenolate was sieved to obtain a fine powder more easily solubilized in water. The product obtained is red in color.

General formula: C21H1906Na.

Molecular Mass: 390

Yield: 3.46g (94%)

UV-VIS: Absorbance = 0.962 (maximum absorption is X 395 nm in water)

Upon acidification with hydrochloric acid, the salt transforms to the corresponding 1,7-bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione which is extracted with ethyl ether, chloroform or ethyl acetate. The results of the structural characterization of the isolated compound are as follows:

1H NMR (300 MHz, DMSO): δ = 9.65 (s, 1H, OH-enolic); 8.27 (s, 1H, OH-phenolic); 7.63 (d, 1H, H -1); 7.34 (s, 1H, H-2 '); 7.19, (d, 1H, H-6); 6.82 (d, 1H, H-7); 7.17 (m, 2H, H-5 7H-6 '); 3,883 (s, 3H, CH 30).

13 C NMR (75 MHz, DMSO): δ = 188.10 (C = 0); 149.45 (C-3 '), 148.02 (C-4'); 142.83 (C-1); 126.39 (C-1 '); 123.40 (C-2), 123.05 (C-6 '); 115.71 (C-5 '); 111.43 (C-2 '); 79.18 (C-4), 55.76 (CH30).

DEPT (DMSO): δ = 143.25 (C-1); 123.82 (C-2); 123.46 (C-6 '), 116.13 (C-5'); 111.84 (C-2 '); 56.18 (CH30).

2b) Obtaining 3,5-dioxohepta-1,6-dienyl-bis (2-methoxy-phenolate) disodium (DB3-B)

<formula> formula see original document page 52 </formula>

The sodium salt 3,5-dioxohepta-1,6-dienyl-bis (2-methoxyphenolate) disodium was obtained from 1,7-Bis- (4-hydroxy-3-methoxy). (phenyl) hepta-1,6-dien-3,5-dione sodium methoxide and methanol in molar ratio 1: 2.

1,7-Bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione was contacted with a 1: 2 molar ratio of sodium methoxide in methanol. then, the solvent was evaporated in vacuo until a solid formed. The salt was obtained in high yield (93%). Once obtained, the phenolate was screened to obtain a more easily soluble powder. The product obtained has a red color.

General formula: C21H1806Na2

Molecular Mass: 412

Yield: 3.83g (93%)

UV-VIS: Absorbance = 0.141 (maximum absorption is Xmax. = 450 nm in water)

Upon acidification with hydrochloric acid, the salt transforms to the corresponding 1,7-bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione which is extracted with ethyl ether, chloroform or ethyl acetate. The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, DMSO): δ = 9.65 (s, 1H, OH-enolic); 8.27 (s, 1H, OH-phenolic); 7.63 (d, 1H, H -1); 7.34 (s, 1H, H-2 '); 7.19, (d, 1H, H-6); 6.82 (d, 1H, H-7); 7.17 (m, 2H, H-5 7H-6 '); 3,883 (s, 3H, CH 30).

13 C NMR (75 MHz, DMSO): δ = 188.10 (C = 0); 149.45 (C-3 '), 148.02 (C-4'); 142.83 (C-1); 126.39 (C-1 '); 123.40 (C-2), 123.05 (C-6 '); 115.71 (G-5 '); 111.43 (C-2 '); 79.18 (C-4), 55.76 (CH30).

DEPT (DMSO): δ = 143.25 (C-1); 123.82 (C-2); 123.46 (C-6 '), 116.13 (C-5'); 111.84 (C-2 '); 56.18 (CH30).

EXAMPLE 3: Obtaining Vanylidenacetone Derivatives

3a) Obtaining 4- (4-Hydroxy-3-methoxy-phenyl) -but-3-en-2-one (R-6)

<formula> formula see original document page 54 </formula>

4- (4-Hydroxy-3-methoxy-phenyl) -but-3-en-2-one was obtained from 0.01 mol (1.52g) of 4-hydroxy-3-methoxy benzaldehyde, dissolved in a hydroalcoholic solution (136 ml, 50% v / v), and the subsequent addition of 0.051 mol (3.6 ml) of acetone and 0.03 mol (12.0 ml) of a 10% hydroxide solution sodium. This reaction was allowed under constant stirring at room temperature for five days. However, every 24 hours, 0.03 mol (12.0 ml) of a 10% sodium hydroxide solution was added.

The mixture was poured into distilled water and ice. Following, extraction with ethyl ether was performed, followed by washing with water. The organic phase was dried with anhydrous sodium sulfate, and finally the solvent was evaporated in vacuo.

For purification of the obtained compound a silica gel filled chromatographic column was used, eluting with a toluene / ethyl acetate (7: 3) mixture.

Rf of product R-6 = 0.36 (Eluents: toluene, ethyl acetate 7: 3)

Rf of raw material = 0.45 (Eluents: toluene, ethyl acetate 7: 3)

General Formula: C11H1203.Molecular Mass: 192 Yield: 1.15g (60%)

The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, CDCl 3): δ = 7.451 (H-4, 1H, d), 6.587 (H-1H, d), aromatic protons: 6.933 (H-5 ', 1H, m), 7.056 ( H-6 ', 1H, m), 7.111 (H-2', 1H, m), 5.938 (OH, 1H, s), 3.938 (MeO, 3H, s), 2.367 (MeCO, 3H, s) .

3b) Obtaining 2-Methoxy-4- (3-oxo-but-1-enyl) phenol disodium (R 6 -B)

<formula> formula see original document page 56 </formula>

Sodium salt was obtained from 4- (4-hydroxy-3-methoxy-phenyl) -but-3-en-2-one in the presence of sodium methoxide and methanol in a 1: 1 molar ratio.

4- (4-Hydroxy-3-methoxy-phenyl) -but-3-en-2-onacoled in contact with a sodium methoxide in methanol solution and then the solvent was evaporated in vacuo to a solid. The salt was obtained in high yield (93%). Once obtained, the phenolate was sieved to obtain a fine powder more easily solubilized in water. The product obtained is red in color.

General formula: CllH1103Na.

Molecular Mass: 214

Yield 1.99g (93%)

Acidification with hydrochloric acid transforms to the corresponding vanylideneacetone which is extracted with ethyl ether, chloroform or ethyl acetate.

The results of the structural characterization of this compound coincide with the already known raw material data.

EXAMPLE 4: Obtaining 4-Hydroxy-3-methoxy-benzaldehyde derivatives

4a) Obtaining sodium 4-Formyl-2-methoxy-phenolate (V6)

<formula> formula see original document page 57 </formula>

This sodium salt was obtained from 4-hydroxy-3-methoxy-benzaldehyde (V5) in the presence of sodium methoxide and methanol in a 1: 1 molar ratio.

4-Hydroxy-3-methoxy-benzaldehyde was contacted with one of sodium methoxide in methanol and then the solvent was evaporated in vacuo until a solid formed. The salt was obtained in high yield (78%). Once obtained, the phenolate was screened to obtain a more easily soluble powder. The product obtained has a yellow color.

General formula: C8H703Na.

Molecular Mass: 174

Yield: 1.36 g (78%)

Upon acidification with hydrochloric acid, the salt becomes the corresponding 4-Hydroxy-3-methoxy-benzaldehyde (V5), which is extracted with ethyl ether, chloroform or ethyl acetate. The results of the structural characterization of this isolated compound are in accordance with its structure. 4b) Obtaining phenyl 4-Formyl-2-methoxy acetate (V4)

The reaction carried out was acetylation, which consisted of a mixture of 0.0062 mol (1.0 g) 4-hydroxy-3-methoxy benzaldehyde, 0.2 mol (20.4 g) acetic anhydride and 0.012 mol ( 1.0 g) sodium acetate. This mixture was heated at approximately 120 ° C under stirring and reflux for 2 hours and 30 minutes. The product obtained was poured into ice water, forming an orange precipitate.

General formula: C10H10O4

Molecular Mass: 194

Yield = 88%

The results of the structural characterization of the isolated compound are as follows:

1H-NMR (CDCl3): 9.8 (s, 1H, CHO); 7.30 (d, 2H, H-6); 7.18 (s, 1H, H2); 7.05 (dd, 1H, H5); 3.71 (s, 3H, MeO); 1.98 (s, 3H, CH 3) ppm.

13 C-NMR (CDCl 3): 190.6 (CHO); 168.0 (C = 0); 151.6 (C-3), 144.6 (C-4); 134.9 (C-1); 124.5 (C-5); 123.2 (C-6); 110.6 (C-2); 56.0 (CH30); 20.6 (CH 3) ppm.

MS (70 eV): 195 (M ++ + H); 43 (M + = 152.1) which corresponds to group C0CH3; 31 (152.1-121.2), which corresponds to the methoxy group.

4c) Obtaining (3-Methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde (VI)

<formula> formula see original document page 59 </formula>

0.01 mol (1.52 g) of 4-hydroxy-3-methoxy-benzaldehyde (V5) was mixed with 0.03 mol (4.14 g) of potassium carbonate and 10 ml of dimethylformamide at a constant temperature of 40 ° C per 30 minutes. Then 0.02 mol (2.98g, 2.32 mL) of prenyl bromide was added dropwise. The reaction mixture was stirred for 8 hours at 40 ° C and, soon after its completion, the mixture was poured into ice water and then waited until the next day until a white solid was filtered off and left to dry naturally.

General formula: C13H1603

Molecular Mass: 220

Yield: 95%

The results of the structural characterization of the isolated compound are as follows:

1H-NMR (CDCl3): 9.8 (s, 1H, CHO); 7.45 (d, 2H, H-6); 7.22 (s, 1H, H2); 6.90 (dd, 1H, H5); 5.51 (m, 1H, H2 '); 4.42 (d, 2H, H1 '); 3.91 (s, 3H, MeO); 1.80 (s, 6H, CH 3, C 4 ') 13 C-NMR (CDCl 3): 190.9 (CHO); 153.8 (C-4); 149.7 (C-3), 138.7 (C-3 '); 129.9 (C-1); 126.7 (C-6); 118.8 (C-2 '); 111.5 (C-2); 109.0 (C-5); 76.5 (C-1 '); 56.0 (CH30); 25.8 (C-4 '); 18.3 (CH 3) ppm.

DEPT-NMR (CDCl3): 190.95 (CHO); 126.79 (C-6); 118.90 (C-2 '); 111.58 (C-2); 109.01 (C-5); 65.94 (C-1 '); 56.00 (MeO); 25.87 (CH 3) ppm.

IR (Nujol): at 3076 (C sp 2 H); 2933, 2858 (C sp 3 H); 1681 (C = 0); 1585 (C = C); 1265 (C-O) cm -1.

EXAMPLE 5: Obtaining Cyanoacetoidrazones and Derivatives

<formula> formula see original document page 60 </formula>

General Technique:

Dissolve 0.01 mol vanillin or a corresponding vanillin in 5 ml ethanol, and 0.01 mol decianoacetohydrazide (0.99 g) in 5 ml ethanol. Both mixtures are heated at reflux for 0.5-10 hours until a precipitate forms.

5a) Obtaining 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide (V10)

<formula> formula see original document page 61 </formula>

Vanillin (0.01 mol) was dissolved in ethanol (5 ml), cyanoacetohydrazide (0.01 mol) in ethanol (5 ml). Both mixtures were heated at reflux for 3.5 hours until a precipitate formed. Melting point: 197-200 ° C Yield: 1.79 g (77%)

The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, DMSO): δ = 11.59 (s, 1H, OH); 9.52 (s, 1H, NH); 7.86 (s, 1H, CH = N); 7.27 (s, 1H, H-2 '); 7.06 (m, 1H, H6 '); 6.82 (m, 1H, H-5 '); 4.16 (s, 2H, CH 2); 3.79 (s, 3H, CH 30).

13 C NMR (75 MHz, DMSO): δ = 164.90 (C = 0); 158.94 (C-3 '), 149.68 (C-4'); 149.68 (CH = N); 125.69 (C-6 '); 122.09 (C-5 '), 116.31 (C-2'); 110.02 (CN); 56.06 (CH30); 24.72 (CH 2). DEPT (DMSO): δ = 145.22 (CH = N); 122.09 (C-6 '); 115.87 (C-5 '); 109.92 (C-2 '); 56.02 (CH30); 24.75 (CH 2).

5b) Obtaining [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetohydrazide (Vil)

<formula> formula see original document page 62 </formula>

0.01 mol of 3-methoxy-4- (3-methylbut-2-enyloxy) benzaldehyde was dissolved in 5 mL of ethanol, and 0.01 mol decianoacetohydrazide (0.99 g) in 5 mL of ethanol. Both mixtures were heated under reflux for 10 hours until a precipitate formed.

Melting point: 167-173 ° C Yield: 1.54 g (51.5%)

The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, DIVISO): δ = 8.05 (s, 1H, NH); 7.89 (s, 1H, CH = N); 7.30 (s, 1H, H-6 '); 7.27 (m, 1H, H2 '); 7.15 (m, 1H, H-5 '); 5.72 (= CH-prenyl); 4.53 (d, 2H, CH 2 -prenyl); 4.18 (CH 2 CN); 3.77 (s, 3H, CH 30); 1.72 (s, 3H, CH 3 -prenyl), 1.68 (s, 3H, CH 3 -prenyl).

13 C NMR (75 MHz, DMSO): δ = 165.02 (C = 0); 150.55 (C-4 '), 150.31 (C-3'); 144.93 (CH = N); 137.93 (= C (CH 3) 2); 126.92 (C-1 '); 122.42 (= CH-prenyl); 121.89 (C-6 '); 116.65 (CN); 113.14 (C-2 '); 109.23 (C-5 '); 65.37 (prenyl-OCH2); 55.91 (CH30); 25.89 (CH3-prenyl); 24.75 (CH 2 CN); 18.46 (CH 3 -prenyl).

DEPT (DMSO): δ = 144.91 (CH = N); 121.90 (= CH-prenyl); 120.14 (C-6 '); 113.04 (C-2 '); 109.10 (C-5 '); 65.32 (CH20-prenyl); 56.50 (CH30); 25.91 (CH3-prenyl); 25.23 (CH 2 CN); 18.47 (CH 3 -prenyl).

5c) Obtaining (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetohydrazide (V12)

<formula> formula see original document page 63 </formula>

0.01 mol of 3-bromo-4-hydroxy-5-methoxy-benzaldehyde was dissolved in 5 mL of ethanol, and 0.01 mol of decianoacetohydrazide (0.99 g) in 5 mL of ethanol. Both mixtures were heated at reflux for 0.5 hours until a precipitate formed.

Melting point: 222-226 ° C

Yield: 2.34 g (75%)

The results of the structural characterization of the isolated compound are as follows:

1 H NMR (300 MHz, DMSO): δ = 11.73 (s, 1H, OH); 9.99 (s, 1H, NH); 8.04 (s, 1H, CH = N); 7.41 (s, 1H, H-2 '); 7.28 (s, 1H, H6 '); 4.19 (CH 2 CN); 3.78 (s, 3H, CH 30).

13 C NMR (75 MHz, DMSO): δ = 165.17 (C = 0); 159.21 (C-5 '), 149.03 (C-4'); 143.77 (CH = N); 126.58 (C-1 '); 124.24 (C-2 '), 116.70 (CN); 109.41 (C-6 '); 109.71 (C-3 '); 56.73 (CH 30); 24.83 (CH 2 CN).

DEPT (DMSO): δ = 143.77 (CH = N); 124.24 (C-2 '); 109.41 (C-6 '); 56.72 (CH30); 24.83 (CH 2 CN).

EXAMPLE 6: Obtaining 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazide derivatives.

6a) Obtaining 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrydrazide (RR-1)

In a one-neck flask, 0.01 mol (1.52g) of vanillin was added; 0.01 mol (2.33g) of 4-hydroxy-3-methoxy-benzylidene-cyanoacetohydrazide; 0.05 mol (3.85g) ammonium acetate and 0.174 mol (10.49g) (10ml) aceticoglacial acid. The mixture was left under ultrasonic radiation (28 kHz) for 30 hours. After this time, the solution was poured into ice water until a yellow solid precipitate formed, and then the solid was filtered.

General Formula: C19H17N3O5Molecular Pasta: 367Yield = 80%

The results of the structural characterization of the isolated compound are as follows:

IR (Nujol): 3260 (OH); 3050 (C sp 2 H); 2925, 2850 (C sp 3 H); 2210 (CN); 1661 (C = 0); 1580 (C = C); 1260 (C-O) cm -1.

1 H NMR (300 MHz, DMSO): δ = 11.73 (s, 1H, OH); 11.69 (s, 1H, OH); 9.97 (s, 1H, NH); 8.00 (s, 1H, CH = N); 7.99 (s, 1H, H -3); 7.96 (s, 1H, CH = N); 7.03 (s, 1H, H-2 "); 6.99 (s, m, H-6"); 6.72 (m, 1H, H-5 '); 6.69 (m, 1H, H-6'); 6.64 (m, 1H, H-2 '); 6.57 (m, 1H, H, 5 '), 3.80 (s, 3H, CH 30), 3.78 (s, 3H, CH 30).

Part 2: Analysis of antitumor, antiparasitic and antioxidant activity of the compounds.

Part 2a: Antitumor Activity and Preclinical Assays

Cell cultivation. Demelanocyte cell lines, melan-a (Bennet et al., Int. J. Cancer, v.39 (3), pp. 414-418, 1987), and murine melanoma, Tm5 (Oba-Shinjo et al., Neoplasia, v. 8 (3), pp. 231-41, 2006; Corrêa et al., Int. J. Cancer, v.H4 (3), pp. 356-363,2005), are grown in RPMI medium, pH 6.9, containing 5% heat-inactivated fetal bovine desorption (Gibco BRL) and 5% CO2 humid atmosphere at 37 ° C. The culture medium used in the cultivation of melanocytes is added with 200 nM phorbol myristate acetate (PMA). Upon reaching subfluence, cells are removed from the culture bottles by the addition of 0.2% trypsin in phosphate buffered saline (PBS), pH 7.3, for a few seconds, followed by obliteration of proteolytic action by the addition of culture medium supplemented with fetal serum. bovine.

Animals. C57BL / 6 mice are kept in an appropriate environment with no dietary restrictions and 12-hour sleep-wake cycles. Tumorigenicity experiments used 6 to 8 week old female mice.

Dilution of compounds. The compounds studied are initially dissolved in DMSO at a concentration of 0.1 g / ml and then diluted in PBS at the concentrations to be tested in in vitro and in vivo assays.

In vitro proliferation assay. Cell proliferation in the presence or absence of compounds V4 and V6 was determined using the standard 3- (4,5-dimethylthiazol-2-yl) -2,5-dimethylthetrazolium bromide protocol (MTT;

Amresco). Cells from the melan-a murine melanocyte and Tm5 melanoma lineage were plated in 96-well plates (2 x 10 3 cells / well / 100 æl) in RPMI medium containing 5% fetal bovine desorption. After adhesion, the compounds to be tested at different concentrations (100u / well; 5, 10, 20, 40 and 80 p.g / mL) were added. After 0, 24, 48h and 72h of incubation, 20 µl of 5 mg / ml MTT was added well and incubated for 1h at 37 ° C in a 5% CO2 oven. The medium was removed and 100 µl of absolute isopropanol was added per well and incubated for 15 minutes at room temperature. The absorbance of the solution was determined by a plate spectrometer at 570 nm.

Adjuvant effect on chemotherapy treatment.

Tm5 melanoma cells were plated in 96-well plates (2 x 105 cells / well / 100. After adherence, the cells were treated or not with the compounds for 24 hours before treatment with bleomycin ecarboplatin chemotherapeutic agents for 48 hours at different concentrations (100 µl, After treatment, 20 µl of 5 mg / ml MTT were added per well After 1h at 37 ° C, the culture medium was removed and 100 µl of isopropanol was added to each well, followed by incubation for 15 minutes. absorbance of the solution was determined on a plate spectrometer at 570 nm. Inhibition percentages were calculated relative to the values obtained in the untreated controls. In vivo antitumor activity. C57BL / 6 combackground mice are maintained in the Department of the Department's Immunology Department. Microbiology and Immunology Laboratory in an appropriate environment, without restriction They are fed with 12-hour sleep-wake cycles. In the tumorigenicity experiments 6 to 8 weeks old female mice were used. Mice were inoculated subcutaneously with 2 x 105 syngeneic Tm5 melanoma cells in a 100 µl volume. Treatment with the compounds was initiated at the time of tumor cell inoculation. The mice received daily injections of the compounds at the delmg / mouse / day dose for 16 days. The mice were observed daily for the appearance of palpable tumors and the largest and smallest diameters were taken using a caliper. The tumor volume was calculated by the formula: smaller diameter2 x larger diameter / 2. When tumors reach about 100 mm in diameter, the animals are sacrificed and the tumor weights are determined after their surgical removal.

Results

Initially, the effects of compounds V4 and V6 on the proliferation of non-tumorigenic melanocytes melan-a (ma) and melanoma cells (Tm5) were evaluated using the MTT agent. As shown in Figure 1, cells were treated with the compounds at different times (24, 48.72h and 96h) at the following concentrations (0, 5, 10, 20, 40 and 80 (µg / mL). incubated with MTT followed by spectrophotometer analysis All four compounds had significant antiproliferative activity, and they preferentially inhibited melanoma cell proliferation Inhibitory effect of melanoma cell proliferation was dose dependent After 48 hours of culture in the If V4 or V6 compounds were present at different concentrations, the equation of the line and the linear regression curve were calculated using Graph Pad Prisma (Figure 2). Compound V4 presented IC50 of 150) j, g / mL for melanocytes melan-a and IC50. 96 µg / ml for Tm5 melanoma cells. Compound V6 showed IC50 of 85 µg / ml for Tm5 demelanoma cells and induced proliferation of melan-a melanocytes (TABLE 1).

TABLE 1: IC 50 values (æg / ml) of the compounds analyzed

<table> table see original document page 69 </column> </row> <table>

* Compound V6 stimulated proliferation of melanocyte melanocytes. Data in the literature show that vanillin is able to potentiate cytotoxicity of some DNA damaging agents (Tamai et al., Mutat. Res. V.268, pp. 231-237, 1992; Imanishi et al., Mutat, Res. V. 243, pp. 151-158, 1990), with sub-toxic vanillin concentrations resulting in dose-dependent sensitization of human tumor lines to cisplatin (Durant et al., Nucl. Acids Res. V .31 (19), pp. 5510-5512, 2003). Thus, we analyzed the ability of vanillin-derived compound V6 to potentiate the cytotoxic effect of two chemotherapeutic agents used in the clinic: bleomycin ecarboplatin. For this, murine melanoma cells were treated with compound V6 (40 ng / mL) for 24h, followed by chemotherapy treatment for 48h. The number of viable cells was estimated by the MTT assay and compared to the number of viable cells in untreated control groups, treated for 24 hours with V6 alone and treated for 48 hours with chemotherapy alone. Figures 3 and 4 show that compound V6 is able to sufficiently potentiate the cytotoxic effect of both chemotherapeutic bleomycin and carboplatin, respectively. It should be noted, for example, that treatment of V6 and bleomycin demelanoma cells eliminates almost all tumor cells (about 90%), which is not the case when these cells are treated with chemotherapy alone (it induced the death of about 20% of the cells). cells).

For analysis of the in vivo antitumor effect of vanillin-derived compounds V4 and V6, female C57BL / 6 mice were inoculated with melanoma cells ((2x105 cells; subcutaneous route) and daily treated with V4 and V6 compounds (1mg / day; Figure 5 shows an average inhibition of 90% in weight of V4-treated animal tumors and 67% in V6 animatractors after 16 days of tumor cell inoculation and initiation of treatment. The smallest diameter of the tumors was made daily after the appearance of the tumors in animals.We can see in Figure 5 a significant inhibition in the volume of tumors over time, both in animals treated with compound V4 and compound V6, compared with group control (untreated). Tumor volume was calculated by the formula: d2xD / 2. After the last measurement, the animals were sacrificed, the tumors removed and their weights determined.

Figures 6, 7 and 8 show pictures taken of the animals after 16 days of treatment, where the reduction in tumor size can be clearly seen in the animators with compounds V4 and V6 (Figures 7 and 8) compared to the untreated control group (Figure 6).

Evaluation Results of Cytotoxic Activity in Normal Human Tumor Cells and Fibroblasts

Culture of B16F10 Melanoma Tumor Lineages

The B16F10 murine melanoma tumor line was made by the Ludwig Institute of Switzerland. B16F10 cell adherent cell suspensions were obtained for all experimental procedures by treating the culture flasks with 0.2% trypsin for 5 minutes and inactivated with 10% fetal bovine serum. Dropped cells were centrifuged twice, resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum and 7ug Polymyxin-B (Sigma Chemical. Company, StLouis Mo-USA). The cell number was counted in a Malassez chamber, and the cell concentration was adjusted to 5x10 5 cells / mL.

Cell viability was determined by the Trypan Blue Exclusion test, with 95% of the cells viable. Cells were cultured in 96-well flat-bottomed plates (Corning) at a concentration of 2 x 105 cells maintained for 24 hours in a CO2 oven at 37 ° C.

After this time, the plates were centrifuged for 5 minutes at 2000 rpm at 4 ° C. The discarded supernatant was added with different concentrations of V4e and V6 compounds, which were diluted in supplemented RPMI-1640 culture medium and added with 7ug Polymyxin-B. (SigmaChemical Company). After incubation with the various diluted compounds in complete culture medium, cells were incubated with 0.5 mg / mL of 3- (4,5-dimethylazol-2-yl) -2,5-diphenyl tetrazolium bromide. .

Obtaining and culture of normal human skin fibroblasts

Skin biopsy fragments were obtained from routine diagnostic procedures of patients from the Dermatology Service of the Hospital das Clinicas da USP Medical School. Part of the edge of the surgery was immersed in Dulbecco's modified Eagle culture medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA) plus 10% fetal bovine serum (Cultilab Ltda., Campinas - SP), 125 mg. / mL ampicillin G and 50 ng / mL amphotericin B at 4 ° C and maintained at 4 ° C for up to 16 hours.

The fragments were washed three times in the same solution, reduced to approximately 1 mm3 fragments and transferred to 25 cm2 culture flasks, kept at 37 ° C, humid atmosphere containing 5% CO2. Cell growth was monitored daily, photocumented by inversion microscopy and culture medium changed every 2 or 3 days, according to cellular ometabolism.

After 7 days, the first change of medium cultivation of the flasks was performed. Once cell growth began, the culture medium was renewed every three days. 25 cm 2 plastic cultivable area vials were used, where an additional 3 mL of DME medium containing 10% fetal bovine serum and 1% antibiotic solution were added. antimicotic.

Colorimetric assay for cell viability determination (MTT)

The cell viability assay was performed to verify the effect of the various compounds on the various concentrations previously established: on the B16F10 tumor cell lines and normal human fibroblasts. The MTT method consists of a cell viability assay assaying mitochondrial dehydrogenase activity. MTT is a colorimetric method based on the ability of living cells to reduce 3- (4,5-dimethylazol-2-yl) -2,5-diphenyl tetrazolium bromide in the formazana product (Mosmann, 1983). After plating the cells (delx104 concentration per well of the culture plate) in RPMI-1640 and 10% SFB medium, the plates were kept at 37 ° C for 24, 48, 72 and 96 hours. Four hours before the set time was up, 20uL MTT (Sigma) (final concentration 10 µg / ml) was added. The plates were kept in the greenhouse for a further 4 hours. After the allotted time, 180uL of the supernatant was removed from each well and then 150uL of dimethyl sulfoxide (Sigma) was added. The mixture was homogenized for complete dissolution of the salt crystals formed by mitochondrial metabolism, thus resulting in a violet coloration.

The 96-well plate was read by spectrophotometer (Spectra MAX - 190) using the wavelength of 570nm. Results were analyzed by absorbing each well. The viability percentage was obtained by the formula: [(Absorbance of treated cells / Absorbance of untreated cells) x 100]. The experiments were performed in quadruplicates.

Sample Dilution

Samples of the synthetically obtained compounds were diluted in 10% DMSO solution in RPMI-1640 culture medium supplemented with 10% fetal bovine serum at concentrations of 250 to 2.5 µg / mL.

Compound V12 showed cytotoxic activity in B16F10 melanoma cells. After 24 hours of culture in the presence and absence of compost at various concentrations, the line equation and the linear regression curve were calculated using the Graph Pad Prism Instat program. Compound V12 showed IC50 of 138.90 ng / mL and high specificity (r 2 = 0.92) in B16F10 melanoma cells (Figure 9).

TABLE 2: Cytotoxic activity of some compost synthesized in B16F10 Melanoma cells

<table> table see original document page 76 </column> </row> <table>

TABLE 3: Cytotoxic activity of some compost synthesized in Normal Human Fibroblast cells.

<table> table see original document page 76 </column> </row> <table> <table> table see original document page 77 </column> </row> <table>

Compounds with high elective cytotoxic potential to induce toxicity and inhibit proliferation of tumor cells and not to harm normal cells were in increasing order respectively: DB-3A; DB-3B; R6-B.

PART 2b: Evaluation of Antioxidant Activity Methodology:

Intracellular levels of reactive deoxygen species in melanocytes melanocytes and melanomaTm5 cells were estimated using the dihydroetide compound (DHE) that fluoresces in the presence of superoxide anion inside the cell. Cells were cultured in culture dishes and treated for 24h with 40 µg / ml of the compounds to be analyzed. After washing with PBS, the cells were incubated for 30 minutes with 25ng / ml DHE at 37 ° C. Cells, after washing in PBS, were analyzed by flow cytometry.

Results:

The antioxidant activity of compounds V4 and V6 was purified using a compound that fluoresces in the presence of superoxide anion. Intracellular superoxide anion levels were determined in melan-a and eTm5 lines treated or not treated with compounds V4 and V6 (40 µg, ml / ml) by DHE fluorophore. The melanoma strain (Tm5) has high intracellular anion superoxide levels compared to the non-tumorigenic demelanocyte strain (melan-a) (Figure 10, upper; C). Treatment with compound V6, but not V4, resulted in a significant reduction in intracellular anion superoxide levels in both melanocyte and demelanoma lineage. Data from the literature show that intracellular superoxide anion levels may contribute to cell resistance to apoptosis, so their decrease in melanoma cells (as shown in Figure 10, V6) could make them more susceptible to death. In this case, this could be one of the mechanisms by which the V6 compound sensitizes melanoma cells to the cytotoxic effects of bleomycin and carboplatin chemotherapeutic agents.

The results obtained indicate an important antitumor capacity of these compounds derived from vanillin. Given the high metastatic and invasive capacity of melanoma and its tolerance to chemotherapy and radiotherapy, both compounds may be promising for use in therapies for low toxicity.

PART 2c: Results of Antiparasitic Tests

i) Evaluation of activity against trypanosomes

Evaluation of cytotoxic activity in VERO and anti-Txypanosoma cruzi cells of synthesized compounds.

It is of paramount importance to choose experimental models for the standardization of protocols for the use of new potentially therapeutic compounds in relation to Chagas disease. In vitro testing, the use of blood trypomastigote infectious forms, testing at 4 ° C (prophylaxis) and testing at 37 ° C (therapeutic), as compared to the violet crystal and the effect on intracellular amastigote forms, is of paramount importance. , compared with action of benzonidazole enifurtimox. Epimastigote forms are used in initial drug toxicity and sensitivity tests.

Regarding in vivo prototypes in the standardization of models for the acute and chronic phase of the disease, toxicity monitoring and cure are very importantCoura et al., Mem. Inst. Oswaldo Cruz, v. 97 (1), pp. 3-24,2002). Thus, the antiparasitic activity of synthetic substances was evaluated in intracellular amastigotes and trypomastigotes obtained from VERO cell cultures infected with metacyclic trypomastigotes, a form derived from the invertebrate barber host and disinfecting to the human vertebrate host (Pisco etal., Eur. J. Med. Chem., v.41 (3), pp. 401-407, 2006). Thus, the concentration of synthetic compounds (Pisco etal., Eur. J. Med. Chem., V.41 (3), pp. 401-407, 2006) capable of causing lysis in 50% of cells / parasites (IC50) on VERO cells and T. cruzi epimastigote forms, as well as antiparasitic activity on intracellular amastigote forms and human blood trypomastigotes were determined.

In vivo analyzes were performed by the administration of compounds with potential antiparasitic action preliminarily selected in invitro tests orally administered to mice experimentally infected with T. cruzi. Results are determined by analysis of parasitemia and survival rate.

For preclinical assays, the models used initially consist of VERO cells (VERO Africangreen monkey kidney cells-fibroblasts) - epitotoxicity assays), epimastigote forms, blood etripomastigote amastigotes (antiparasitic activity assays), tested on a composite and synthetic screening. bezonidazole. Then, as in vivo models, mice experimentally infected with T. cruzi and treated with in vitro selected compounds that show potentials for therapeutic studies are used.

Methodology

This work deals with the in vitro analysis of cytotoxic and antiparasitic activity of four compounds of synthetic origin (DB3A, V4, V6 and V10) at different concentrations.

Cytotoxic activity was analyzed in VERO eanti-Trypanosoma cruzi cells, strain CL, for compounds DB3, V4, V6, V10 and benzonidazole at concentrations 5, and 10 µg / mL, previously defined.

VERO cell culture

1x106 cells were placed in a 25 cm3 bottle with DMEM (Dulbecco's modified Eagle's medium-GibcoInvitrogen) supplemented with 10% Fetal Bovine Serum (SFB). This culture was kept in an oven at 37 ° C with 5% CO2 until semi-confluence of cells (approximately 24 to 36 h). The medium was removed and subsequently 1.5 ml trypsin (0.25%) was added and the culture was incubated until the cells detached. Then 3.5 mL of DMEM medium was added and the cells were transferred to a sterile Falcon tube. Then a centrifugation was performed at 3500 rpm for 5 minutes and the supernatant was discarded. The cells were resuspended in DMEM and counted in a hemocytometric chamber (from Neubauer), setting the concentration to 106 / mL. 105 cells / well were transferred to 24-well plates in a final 1 mL volume.

VERO cell treatment with synthetic compounds

Stock solutions of 2-5 mg / mL DB3A, VA, V6 and V10 were diluted in water, DMSO or ethanol according to their solubility. Increasing concentrations of the compounds (10, 25, 50, 100, 200 and 250 µg / ml) were added to the respective wells of the culture plate and incubated for 24 to 96 h at 37 ° C with 5% CO2.

Antiparasitic activity In vitro in cultures of T. cruz formapsimastigotes

In initial toxicity and compound sensitivity tests, the epimastigote forms of T.cruzi strain CL, grown in LIT medium (tryptose plus liver infusion) supplemented with 10% FBS for 24-48h, were used. Then 1 mL containing 10 6 parasites was placed in 24-well plates, and different concentrations (0.5 to 250 µg / mL) of compound were added to the respective wells, and the plates were cultured for 96 h at 28 ° C. Antiparasitic activity was evaluated by counting the number of parasites in the hemocytometric chamber. Two experiments were performed on different occasions with duplicates.

Culture of T. cruzi forms Metacyclic trypomastigotes

Metacyclic (infectious) trypomastigotes are obtained from cultures in LIT medium with 5% SFB and aged for 11 to 15 days. These forms are used for VERO cell culture infection and inoculation in mice in in vivo tests.

Obtaining Intracellular Amastigote Forms and Treatment with Compounds

VERO cells were diluted to a concentration of 103 µg / ml in DMEM medium with 10% SFB, and incubated in culture vials at 37 ° C for 24-48h with 5% CO2, allowing them to slide until they reached semi-confluent growth. They are then infected with 10 3 Trypanosoma cruzi trypomastigotes of CL strain. After 24 h incubation to allow invasion of the postropomastigote cells, the medium was changed and the infected cells incubated for 3-4 days to differentiate and multiply intracellular amastigotes. At this time, the cells were treated with medium containing 0.25% detripsin, collected in tube and 10 3 cells / mL, placed on 13 mm laminates in 24-well plates and cultured for 24 h at 37 ° C with 5% CO2. Then different concentrations of test substances were added to the culture. After 24 and 48 h of incubation, cultures were observed under an inverted microscope to evaluate the effect of compound concentration on parasitic cells. Then the laminates were washed with PBS, fixed with methanol, stained in Giembsa and mounted with glue Entelan (Invitrogen) on microscopy slides.

Analysis of compound activity

The laminates (in duplicates) were analyzed under an optical microscope and the activity of the compound measured considering: the number of VERO cells, VEROinfected cells, intra and extracellular etripomastigote intra and extracellular amastigotes. The Infection Index was determined as follows:% infected cells X intracellular amastigotes / infected cells. These data were plotted on graphs.

In vivo activity in mice

After in vitro activity analysis, compounds V4, V6 and V10 were selected for invivo testing, since they presented the best results. For this model Albino mice infected with metacyclic trypomastigote forms were used and treated with predetermined concentrations in in vitro experiments. For each experiment we used five animal groups that were treated daily with DB3A or orbenzonidazole orally. After 7 days, the parasitemia was confirmed with a domaterial tail blood slide to count the number of parasites. Parasitemia was assessed from day 8 on alternate days for 60 days.

Controls All procedures performed "invitro" and "in vivo" to evaluate the cytotoxic and antiparasitic activity of synthetic substances were compared with untreated controls and combenzonidazole treatment, the treatment drug currently used in T. cruzi.

In vitro tests:

Experiment 1: T. crazi infected VERO cells

Drug Free Control:

Control with usual treatment drug: Benzonidazole at concentrations 5, 10, 25 and 50 µg / mL

Tested Compounds:

DB3A (diluted with H2 O) at concentrations 5, 10, 25 and 50ug / mL

V4 (diluted in DMSO) at concentrations 5, 10, 25 and 50 µg / mL

V6 (diluted H20) at concentrations 5, 10, 25 and 50 µg / mL V10 (diluted DMSO) at concentrations 5, 10, 25 and 50ug / mL

All experiments were performed in duplicates.

Experiment 2: T. cruzi infected VERO cells

■ Drug-Free Control:

■ Control with usual treatment drug: Benzonidazole at concentrations 5 and 10 µg / mL

■ Tested Compounds:

DB3A (diluted in DMSO) at concentrations 5, 7.5, 10 and 15ug / mL

V4 (diluted in DMSO) at concentrations 5, 10, 25 and 50 μg / mL V6 (diluted in H20) at concentrations 5, 10, 25, 50, 100 and 200 μg / mL

V10 (diluted in DMSO) at concentrations 5, 10, 25 and 50ug / mL

The following table represents the summary of the experiments described above.

TABLE 4: T. cruzi infected VERO cells treated with different concentrations of compounds for 24 and 48 hours.

<table> table see original document page 86 </column> </row> <table> <table> table see original document page 87 </column> </row> <table>

All experiments were performed in duplicates.

Photos of the cells were elaborated for histological documentation of the cytotoxic and antiparasitic action obtained with the tested compounds, which are represented in Figure 11 (A, B, C, D, E, F).

The results of the in vitro experiments, both visual and culture visual identification identifying extracellular trypomastigote and amastigote forms, as well as histological analysis of VERO cells and amastigote forms show that:

1) Compound DB3A, at a concentration of 10 µg / mL anti-amastigote T-cruzi-like theme similar to the benzonidazole drug, followed by compounds V4 and V10, and compound V6 at that concentration has no parasitic activity.

2) Compound DB3A has cytotoxic activity for VERO cells at a concentration greater than 25 µg / mL and extracellular anti-trypomastigote activity at 10 µg / mL.3) Compound V6 is cytotoxic at concentrations of 50 µg / mL for VERO cells, showing discrete evidence of decreased numbers of infected cells in T. cruzi infected cell cultures. Compound V6 inhibits differentiation of intracellular amastigote forms into trypomastigotes at a concentration of 25 p.g / mL.

4) Compound V10 has no cytotoxic activity at the concentrations used in the experiments. At concentrations greater than 5 µg / mL, it appears to induce differentiation of intracellular amastigote forms into trypomastigotes.

5) Compound V4 at concentrations greater than 5 µg / ml induces differentiation of intracellular amastigote forms into trypomastigotes without reaching VERO cells.

In vivo tests:

Compound DB3A was used for in vivo studies. Albino mice, in groups of 5 animals, were treated with two doses on the 1st and 4th day after infection, with benzonidazole or DB3A, by the degassing technique.

DB3A and benzonidazole concentrations were calculated by ratios: in vitro dose | µg / mL X 10 X weight in kg of mouse. This calculation determined the concentrations of 2 mg, 2 mg and 4 mg per animal / dose for benzonidazole and DB3A, respectively. Parasitemia was evaluated on days 11, 13, 15 and 18. The graphs shown in Figure 12 show the isolated profile of parasitemia of animals in the treatment performed.

Conclusions:

Treatment data with DB3A by the oral route were similar to those for benzonidazole treatment, however, they have lower toxicity.

The animals had no change in their humidity during the procedures.

Finally we can conclude that the compounds used have good anti-Trypanosoma cruzi activity and that the DB3A compound has an evident efficiency against the parasite's extracellular forms, being a potential drug to treat the acute phase of Chagas disease.

Claims (64)

1. Process for obtaining curcumin (1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1,6-diene-3,5-dione) or its alkylated derivatives of structure I: <formula> formula see original document where R1, in each separate occurrence, is a hydrogen atom or an alkyl group, R2, in each separate occurrence, is a hydrogen atom or an alkyl group, where R3, in each separate occurrence, is an atom dehydrogen; CH 2 CH = C (CH 3) 2; C0CH3 or COPhA.where Z, in each separate occurrence, is a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group, wherein US is ultrasonic irradiation. characterized in that it comprises contacting vanillin, or substituted vanillin or alkylated derivatives of acetylacetone and hydrochloric acid under ultrasonic irradiation conditions. Process according to Claim 1, characterized in that it comprises the following steps: a) contacting vanillin or prenylated vanillin or acetylated vanillin or benzoylated vanillin; acetylacetone or alkylated acetylacetone derivatives; and hydrochloric acid under ultrasonic irradiation conditions b) leave the reaction mixture to stand c) perform an extraction using sodium hydroxide or potassium hydroxide solution d) filter the reaction mixture under vacuum d) acidify the filtrate obtained with acid hydrochloric acid or sulfuric acid with the consequent formation of a precipitate, e) filtration and washing of the precipitate with distilled water, f) drying of the solid product. Process according to either of claims 1 or 2, characterized in that the ultrasonic radiation is carried out in a range of 25 to 40KHz. Process according to any one of claims 1 to 3, characterized in that the ultrasonic radiation is performed for a period of time ranging from 1 to 8 hours. Process according to any one of Claims 1 to 4, characterized in that the reaction mixture is left to stand for a period of 8 to 24 hours. Process according to any one of Claims 1 to 5, characterized in that the reaction mixture is left to stand within a temperature range of 10 to 60 ° C. Process according to any one of claims 1 to 6, characterized in that they include contacting vanillin, acetyl acetone and hydrochloric acid under ultrasonic irradiation conditions to obtain 1,7-bis (4-hydroxy-3-methoxyphenyl) -hepta-1. 2,6-diene-3,5-dione. Process according to any one of claims 1 to 6, characterized in that vanillin, 0.0005 to 0.05 mol of acetylacetone and -0.02 to 20 ml of concentrated hydrochloric acid (10 to 60%) are placed under conditions. ultrasonic irradiation. Process for obtaining metal salts of curcumin or their derivatives of structures II or III: wherein R1, in each separate occurrence, is a hydrogen atom or an alkyl group; , in each separate occurrence, is a hydrogen atom or an alkyl group, wherein Z, in each separate occurrence, is a hydrogen atom; CH 2 CH = C (CH 3) 2; C0CH3 or COPh; M, in each separate occurrence, is a metal cation, wherein R, in each separate occurrence, is CH3 or CH3CH2 characterized in that it comprises placing a structure I compound in the presence of a metal alkoxide (ROM) solution and its alcohol (ROH) in molar ratio 1: 1 or 2: 1. Process according to Claim 9, characterized in that it comprises the following steps: a) placing a compound of structure I in the presence of a solution of metal alkoxide (ROM) and its corresponding alcohol (ROH) in 1: 1 molar ratio or 2: 1 (b) evaporate the alcohol in vacuo to the formation of a solid metal salt, (c) passage of the metal salt obtained by sieving to obtain an easily soluble fine powder. Process according to either of Claims 9 and 10, characterized in that a methanolic sodium methoxide solution is used. Process according to any one of claims 9 to 11, characterized in that it comprises 1,7-bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione in the presence of sodium methoxide and methanol in a 1: 1 molar ratio to give sodium salt 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-one sodium dienyl] -2-methoxy phenolate. Process according to any one of claims 9 to 11, characterized in that it comprises 1,7-bis- (4-hydroxy-3-methoxy-phenyl) hepta-1,6-dien-3,5-dione in the presence of sodium methoxide and methanol in a 1: 2 molar ratio to give the sodium salt, 3,5-dioxohepta-1,6-dienyl-bis (2-methoxyphenolate) disodium. Method for obtaining davanylidenacetone metal phenolate of structure V: wherein R1 is, in each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or, on each separate occurrence, M is a metal stabilizing cation of the compound in the form of an ion phenoxide, comprising contacting vanylidenacetone or a substituted general structure IV vanylidenacetone with a metal alkoxide (ROM) and an alcohol ( ROH), in molar ratio 1: 1. Process according to Claim 14, characterized in that it comprises the following steps: (a) contacting vanylidenacetone or a vanylidenacetone of general structure IV, with alkoxidometallic (ROM) and a 1: 1 molar alcohol (ROH); ) evaporating the alcohol in vacuo to the formation of a solid metal salt, (c) passing the metal salt obtained by sieving to obtain an easily soluble fine powder. Process according to either of claims 14 or 15, characterized in that a methanol solution of sodium methoxide is used. Process according to any one of claims 14 to 16, characterized in that it contacts 4- (4-Hydroxy-3-methoxy-phenyl) -but-3-en-2-one with sodium methoxide and methanol in a -1: 1 molar ratio to give sodium 2-Methoxy-4- (3-oxo-but-1-enyl) phenolate. 18. Process for obtaining a devaniline metal salt of structure VI from 4-hydroxy-3-methoxy-benzaldehyde <RTIgt; hydrogen atom; a bromine atom, a nitro group; an amino group; or M, is at each separate occurrence a metal stabilizing cation of the phenoxide ion compound comprising 4-hydroxy-3-methoxy-benzaldehyde, a metal alkoxide (ROM) and 1: 1 molar ratio (ROH). Process according to Claim 18, characterized in that it comprises the following steps: a) contacting 4-hydroxy-3-methoxy-benzaldehyde, a metal alkoxide (ROM) and an alcohol (ROH) in 1: 1 molar ratio (b) evaporating the alcohol in vacuo to the formation of a solid state salmetal, (c) passing the metal salt obtained by sieving to obtain an easily soluble fine powder. Process according to either of Claims 18 or 19, characterized in that a methanol solution of sodium methoxide is used. Process according to any one of Claims 18 to 20, characterized in that it contacts 4-hydroxy-3-methoxy-benzaldehyde with sodium methoxide and methanol in a 1: 1 molar ratio to yield 4-formyl-2. sodium methoxy phenolate. 22. Process for obtaining acetylated devaniline derivatives or a substituted vanillin of structure VII <formula> formula see original document page 98 </formula> wherein VII is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group, comprising mixing vanillin or substituted vanillin, acetic anhydride and sodium acetate. Process according to Claim 22, characterized in that it comprises the following steps: a) mixing vanillin or a substituted vanillin, acetic anhydride and sodium acetate. B) heating this mixture to a temperature between 80 ° C and-140 ° C. Under stirring and refluxing for a period of 1 to 10 hours, c) pouring the mixture into ice water to give a precipitate. Process according to any one of claims 22 or 23, characterized in that it comprises mixing 4-hydroxy-3-methoxy-benzaldehyde, acetic anhydride and sodium acetate to obtain 4-formyl-2-methoxy-acetate defenyl. Process according to claim 24, characterized in that it comprises mixing 0.0006 to 0.06 mol of 4-hydroxy-3-methoxy-benzaldehyde, 0.02 to 2 mol of anhydridoacetic and 0.0012 to 0, 12 mol sodium acetate. Process for preparing 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde of structure VIII <formula> formula see original document page 99 </formula> comprising the following steps: a) mixing 4-hydroxy-3-methoxy-benzaldehyde, potassium carbonate and dimethylformamide at 15 ° C to 65 ° C for 10 to 90 minutes, b) add prenyl bromide and stir the mixture daily at 15 ° C to 60 ° C for 4 minutes. c) pouring the obtained mixture into ice water to give a white precipitate d) filtering out the obtained precipitate A process according to claim 26, comprising 0.001 to 0.1 mold-4-hydroxy-3-methoxy-benzaldehyde with 0.003 to 0.3 mol of potassium carbonate, 1 to 100 ml of dimethylphenyl. ormamide and 0.002 to 0.2 mol prenyl bromide. A process for preparing edible cyanoacetohydrazones of structure X from vanillin or a substituted structure of vanillin of structure IX wherein R is, at each separate occurrence, a hydrogen atom; CH 2 CH = C (CH 3) 2; COCH3 or COPh wherein R1 is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group, comprising the following steps: (a) mixing vanillin or a substituted vanillin of structure X and cyanoacetohydrazide in the presence of an alcohol OH, b) heating the mixture to the formation of a precipitate. Process according to claim 28, characterized in that it comprises mixing vanylin ecianoacetohydrazide in the presence of ethanol to obtain 4-Hydroxy-3-methoxybenzylidene-cyanacetohydrazide. Process according to Claim 29, characterized in that it comprises mixing 0.001 to 0.1 mol devaniline in 0.5 to 50 ml ethanol, and 0.001 to 0.1 mol decianoacetohydrazide in 0.5 to 50 ml ethanol. Process according to claim 28, characterized in that you mix 3-methoxy-4- (3-methylbut-2-enyloxy) -benzaldehyde and cyanoacetohydrazide in the presence of ethanol to obtain [3-Methoxy-4- (3-methylbutyl). 2-Enyloxy) -benzylidene] -cyanoacetoidrazide. Process according to Claim 31, characterized in that it comprises 0.001 to 0.1 mol of -3-methoxy-4- (3-methylbut-2-enyloxy) -benzaldehyde in 0.5 to 50 ml of ethanol, and 0.001 to 0.1 mol of cyanoacetohydrazide in -0.5 to 50 ml of ethanol. Process according to claim 28, characterized in that it comprises mixing 3-Bromo-4-hydroxy-5-methoxy-benzaldehyde and cyanoacetohydrazide in the presence of ethanol to obtain 3-Bromo-4-hydroxy-5-methoxy-benzylidene). cyanacetohydrazide. Process according to claim 33, characterized in that it comprises 0.001 to 0.1 mol of 3-bromo-4-hydroxy-5-methoxy-benzaldehyde in 0.5 to 50 ml of ethanol and 0.001 to 0, 1 mol of cyanoacetohydrazide in 0.5 to 50mL ethanol. 35. Process for preparing substituted 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazides of structure XI <formula> formula see original document page 102 < wherein Z is, in each separate occurrence, a hydrogen atom; a metal cation; Eats; COC 6 H 5; CH 2 CH = C (CH 3) 2; methyl group; alkyl group wherein X is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group wherein Y is, at each separate occurrence, a hydrogen atom; a bromine atom, a nitro group; an amino group; or an acetylamino group characterized by the fact that CH-acid substituted cyanoacetoidrazones react with carbonyl compounds in 1: 1 molar relationship under the conditions of Knoevenagel condensation and ultrasonic irradiation in the presence of ammonium acetate and acetic acid. Process according to Claim 35, characterized in that the ultrasonic irradiation occurs in a range of 25 to 40 KHz during a time period ranging from 5 to 80 hours. Process according to either of claims 35 or 36, characterized in that the reaction takes place within a temperature range of 20 to -60 ° C. Process according to any one of Claims 35 to 37, characterized in that it comprises 0.001 to 0.1 mol of vanillin, 0.001 to 0.1 mol of 4-hydroxy-3-methoxy-benzylidene-cyanoacetohydrazide, 0.005 to -0, 5 mol ammonium acetate and 0.01 to 1 mol glacial acetic acid. 39. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is 4- [7- (4-hydroxy-3-methoxy-phenyl) -3,5-dioxohepta-1,6-dienyl ] -2-methoxyphenolate sodium. 40. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is 3,5-dioxohepta-1,6-dienyl-bis (2-methoxy-phenolate) disodium. 41. A compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is sodium 2-methoxy-4- (3-oxo-but-1-enyl) phenolate. 42. Compound derived from 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is sodium 4-formyl-2-methoxy-phenolate. 43. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is phenyl 4-formyl-2-methoxy acetate. 44. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is 3-methoxy-4- (3-methyl-but-2-enyloxy) benzaldehyde. 45. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is 4-Hydroxy-3-methoxy-benzylidene-cyanacetohydrazide. 46. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is [3-Methoxy-4- (3-methylbut-2-enyloxy) -benzylidene] -cyanoacetoidrazide. 47. 4-Hydroxy-3-methoxy-benzaldehyde derivative compound 4-Hydroxy-3-methoxy-benzaldehyde derivative characterized by the fact that it is (3-Bromo-4-hydroxy-5-methoxy-benzylidene) -cyanoacetoidrazide 48. Compound of 4-hydroxy-3-methoxy-benzaldehyde characterized in that it is 2-Cyano-3- (4-hydroxy-3-methoxy-phenyl) - (4-hydroxy-3-methoxy-benzylidene) -acrylhydrazide . Pharmaceutical composition comprising one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48, and one or more of a physiologically acceptable carrier, excipient, diluent or solvent. . Use of one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48, characterized in that it is for the manufacture of a medicament. Use according to claim 50, characterized in that it is for the manufacture of a medicament for use in the treatment, prophylaxis or prevention of neoplastic diseases, metastatic, proliferative / or degenerative lesions, immunosuppressive, immunodeficient, and degenerative pathologies. parasitic diseases in humans and / or animals. Use according to claim 51, characterized in that it is for the treatment or prevention of neoplastic diseases, prevention of metastatic, proliferative and / or degenerative lesions. Use according to claim 52, characterized in that it is for the treatment, prophylaxis or prevention of neoplastic diseases caused by lung cancer, multiple drug resistant breast and breast carcinoma, non-melanoma skin cancer, and melanoma, lymphoid leukemia. , acute screen myeloids, erythroleukemia, myelodysplasias, colon, ovarian, uterine, kidney, pancreas, prostate, soft-detached sarcomas, hepatocarcinomas, osteosarcomas, central nervous system, neuroblastomas, astrocytomas, oropharynx, thyroid, male prostate and gastro-appendage Use according to claim 51, characterized in that it is for the treatment, prophylaxis or prevention of immunosuppressive, immunodeficient and degenerative disorders. Use according to claim 54, characterized in that it is for the treatment, prophylaxis or prevention of neurodegenerative diseases. Use according to claim 55, characterized in that it is for the treatment, prophylaxis or prevention of Alzheimer's disease. Use according to claim 54, characterized in that it is for the treatment, prophylaxis or prevention of various parasitic diseases. Use according to claim 57, characterized in that it is for the treatment, prophylaxis or prevention of protozoan-induced human and / or animal parasites in the intestinal blood and protozoal tissue forms of the genera Leishmania, Plasmodium, Trypanosoma, Toxoplasma, Giardia, Entamoeba, as well as helminths belonging to the genera Taenia, Schistosoma, Ancylostoma, Necator, Ascaris, Enterobius, Wuchereria, in their different clinical manifestations. Use of one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48, characterized in that it is for applications as antioxidants and free radical scavengers . Use of one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48 characterized in that it is for the manufacture of a cosmetic product for treatment and / or prevention of skin aging. Pharmaceutical composition comprising one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 58, and carboplatin, and one or more of a carrier, excipient, diluent or physiologically acceptable solvents. Use of one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48 characterized in that the factor is for enhancing the antimural effects of carbarbatin. A pharmaceutical composition comprising one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48, and bleomycin, and one or more of a carrier, excipient, diluent or physiologically acceptable solvents. Use of one or more compounds obtained by the processes as defined in any one of claims 1 to 38 and / or as defined in any one of claims 39 to 48 characterized by the fact that it is to potentiate the antimural effects of bleomycin.