WO2021258173A1 - Phenolic binucleating ligands, binuclear metal compounds, medical-veterinary composition, processes for synthesizing binucleating ligands, process for synthesizing binuclear compounds, method of treatment of neoplasms and fungal diseases and use - Google Patents
Phenolic binucleating ligands, binuclear metal compounds, medical-veterinary composition, processes for synthesizing binucleating ligands, process for synthesizing binuclear compounds, method of treatment of neoplasms and fungal diseases and use Download PDFInfo
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- WO2021258173A1 WO2021258173A1 PCT/BR2021/050266 BR2021050266W WO2021258173A1 WO 2021258173 A1 WO2021258173 A1 WO 2021258173A1 BR 2021050266 W BR2021050266 W BR 2021050266W WO 2021258173 A1 WO2021258173 A1 WO 2021258173A1
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- copper
- complexes
- compounds
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
- C07D213/86—Hydrazides; Thio or imino analogues thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/10—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing aromatic rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F1/00—Compounds containing elements of Groups 1 or 11 of the Periodic System
- C07F1/08—Copper compounds
Definitions
- BINUCLEANT PHENOLIC BINDERS BINUCLEAR METALLIC COMPOUNDS, MEDICAL-VETERINARY COMPOSITION, BINUCLEANT BINDERS SYNTHESIS PROCESS, BINUCLEAR COMPOUNDS SYNTHESIS PROCESS, METHOD OF TREATMENT OF NEOPLES AND PHUGAL DISEASES
- the present invention relates to new metallodrugs containing a copper(II) binuclear coordination center and the non-symmetrical binding ligands 2-hydroxy-3- ⁇ [(2-hydroxybenzyl)(2-pyridylmethyl)amino] -methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L1 ) or 2-hydroxy-3- ⁇ bis[(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2) for treatment of neoplasms involving solid tumors and/or leukemias, as well as diseases of an opportunistic nature, such as diseases caused by pathogenic fungi.
- the present invention falls within the field of application of chemistry, pharmacy, medicine, biology and biochemistry, more specifically in the area of drug preparations containing active ingredients.
- the pharmaceutical composition described in this document deals with a group of macrophage inhibiting agents, among which one stands out. copper chelate.
- the Brazilian reference deals with cancer therapy (breast, lung and/or prostate) through the adjuvant use of copper chelates, but does not propose the use of metallo-drugs based on the metal itself.
- the intrinsic mechanism behind the antitumor action! is proposed to be improving the immune response or increasing the activity of an immune cell in a patient with a disease or condition.
- the compounds have been proposed as antitumor agents with preferential therapeutic use for the treatment of solid and blood tumors (eg leukemia).
- the tested copper mononuclear complex namely [Gu(4,7-dimethyl-1 ,10-phenanthroline)(glycinate)]NO 3 has a slightly higher activity than cisplatin, but lower than mitomycin, a reference drug used in cancer therapy
- the tests with experimental animals showed that only at higher doses of the compound was po It is possible to verify an increase in the survival of animals affected by the inoculation of lymphoid leukemia cells.
- krusei which are extremely important in situations of neoplasms and advanced tumors, and still others, such as Aspergillus spp, Cryptococcus spp, Scedosporium spp, Fusarium spp, Zygomycetes spp and Trichosporon spp, which are frequently found in immunocompromised patients due to chemotherapy.
- compositions have antiseptic properties, being active against gram-positive and gram-negative bacteria, fungi, viruses and spores, and may be applied over a wide range of temperatures.
- Methods of using the compositions of the present invention in the treatment and prevention of diseases caused by a variety of pathogens are described.
- the invention does not address their effectiveness against cancer cells and still makes reference to complexes mononuclear cells whose antimicrobial activity depends on association with other molecules.
- the present invention provides, in this way, new metallodrugs, synthesis process, method of treatment, use in the manufacture of drugs for the treatment of tumors and fungal diseases in humans and other mammals, as well as pharmaceutical and veterinary composition, based on binuclear copper(II) complexes, non-symmetric, with cytotoxic effect on cell lines of prostate, breast and lung cancer, which also show antifungal activity and low toxicity to healthy cells.
- a first mode of invention is the development of new technological alternatives, aimed at new compounds/binuclear copper complexes with antitumor activity, with potentially great impact on current cancer chemotherapy, whose arsenal of substances could be replaced or even optionally combined with the new compounds.
- biotechnological potential presented by these new complexes is not limited to antitumor activity, as they were also shown to be active against pathogenic fungi (eg Candida aibicans, Candida parapsiiosis, Candida tropicaiis and Candida krusei), showing great versatility of application for use in cancer therapy and opportunistic pathologies caused by fungi.
- pathogenic fungi eg Candida aibicans, Candida parapsiiosis, Candida tropicaiis and Candida krusei
- the present invention therefore provides, in this first modality, four metallodrugs derived from two new non-symmetric hydrazonic precursors, namely: 2-hydroxy-3- ⁇ [(2-hydroxybenzyl)(2-pyridylmethyl)amino]- methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L1), according to the structural formula below and illustrated in figure 1 , and 2-hydroxy-3- ⁇ bis[(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2), according to the structural formula below and illustrated in Figure 1 , belonging to the broad group of binucleating ligands, that is, capable of forming binuclear complexes by providing coordination environments suitable to accommodate two metallic centers in close proximity, such that a cooperative effect between them is possible, and even expected.
- Binuclear copper complexes generated from H 3 L1 and H 2 L2, both those with an exogenous hydroxide bridge (postulated as the active form) and those with exogenous acetate bridges (pro-drugs that would generate the biologically active form by reaction of hydrolysis in the organism), have unsaturated copper(II) centers or contain labile ligands, such as the water molecule, which enables their interaction with biological macromolecules such as, for example, DNA, RNA and/or proteins.
- a fifth and last modality would correspond to the use of synthesized compounds in the manufacture of drugs aimed at treating neopiasias. Drugs that present, concomitantly, little toxicity to healthy human cells, high efficacy against malignant cell lines of prostate, breast and lung, in addition to an antifungal effect.
- FIG. 4 Survival assay of cells from human tumor lines MCF-7, A549 and PC-3 treated for 24 h in the presence of complexes 1 (A) and 3 (B); human non-tumor lineage, MGF10A, treated with complexes 1 and 3 (C) and; cell lines MCF-7, A549, PC-3 and MCF10A submitted to treatment with the antitumor metallo drug cisplatin, used as reference.
- FIG. 9 Analysis of cell cycle inhibition (cell proliferation) during treatment with complexes 1 and 3 and the anti-tumor metallodrug cisplatin in MCF-7 (A and C) and A549 (B and C) cancer cells. Representative histograms (C) of the cell population distributed throughout the cell cycle, corresponding to the mean ⁇ standard error of 3 independent experiments.
- - Figure 11 Synthetic route used in the preparation of binuclear copper(ll) 1 and 3 complexes, including the synthesis conditions and yields obtained.
- the steps highlighted in the gray box correspond to the intrinsic processes related to the synthesis of new compounds (H 3 L1 ligand and complexes 1 and 3), and differentiate this synthetic route from others existing in the literature.
- - Figure 12 Synthetic route used in the preparation of binuclear copper(ll) 2 and 4 complexes, including the synthesis conditions and yields obtained.
- the steps highlighted in the gray box correspond to the intrinsic processes related to the synthesis of new compounds (H 2 L2 ligand and complexes 2 and 4), and differentiate this synthetic route from others existing in the literature.
- the present invention therefore describes "Binuclear copper(ii) complexes with potent antitumor activity, composition, chemical synthesis process and use in the preparation of drugs and in the treatment of cancer and infections by pathogenic fungi", which seek meet the growing demand for new treatments for neoplasms, both those involving solid tumors and leukemia, as well as fungal diseases.
- binuclear copper(ll) compounds different from those existing in the prior art, as they present changes in the chemical structure, toxic activity against human cancer cells and, at the same time, less toxicity to non- carcinogens in the body. Thus, they show greater efficacy and fewer side effects, offering patients undergoing chemotherapy for cancer a better quality of life and survival.
- the binuclear copper coordination compounds! i) and preferred non-symmetric linkers of the present invention are: 2-hydroxy-3- ⁇ [(2-hydroxybenzyl)(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L 1 ) as per formula structural below and illustrated in figure 1 and 2-hydroxy-3- ⁇ bis[(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2) as structural formula below and illustrated in figure 1 , as well as their respective binuclear copper(II) complexes containing exogenous acetate-type bridges: compound 1 and compound 2 according to the structural formulas below and shown in figure 2 or hydrox: compound 3 and compound 4 according to the structural formulas below and illustrated in figure 3 .
- Figure 5 graphically represents the induction of the production of reactive oxygen species (or ROS), a critical factor for the induction of the process endogenous apoptosis in cancer cells, during treatment with the new complexes 1 and 3, and with the chemotherapeutic used as control of the experiment, which is also applied in cancer therapy, doxorubicin.
- ROS reactive oxygen species
- Figure 5 shows the representative results of a series of at least three independent experiments performed on MCF-7 (A, B and C) and A549 (D, E and F) lines and represents the mean ⁇ standard error of the mean.
- the symbols (#) and ( *** ) represent a statistical difference in relation to the control treatments.
- Figure 6 graphically represents the alteration/loss of the mitochondrial membrane potential ( ⁇ m), target of reactive oxygen species and the first step in inducing the endogenous process of apoptosis, during the treatment of cancer cells with the new complexes copper 1 and 3, and the metallochemotherapeutic used in cancer therapy, cisplatin.
- This figure presents a graphical representation of the level of mitochondrial membrane potential, expressed as arbitrary fluorescence units in the MCF-7 ( Figure 6 ⁇ and C) and A549 ( Figure 6E and G) lines.
- Figure 7 graphically represents the activation of caspase enzymes 8 (Figure 7A and C) and 9 ( Figure 7B and D), which are key in the activation of the apoptosis process, during cell treatment of cancer with complexes 1 and 3 and the anti-tumor chemotherapy cisplatin.
- the results obtained are expressed as mean ⁇ standard error of the mean of at least three independent experiments and are shown as a relationship between the activity of cells treated with the novel complexes 1 and 3 or cisplatin and untreated cells.
- the symbol ( * ) represents statistical difference from cells not treated with copper or cisplatin compounds.
- Figure 8 graphically represents the induction of the apoptosis process through the analysis of DNA breakage / fragmentation of cancer cells by the TUNEL technique [Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling] during treatment with the new complexes 1 and 3, and the anti-tumor chemotherapy cisplatin.
- TUNEL Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling
- a and B representative TUNEL histograms can be seen showing the percentage of cells in apoptosis after treatment with cisplatin or copper(ll) complexes in the A549 (A) and MCF-7 (B) lines, respectively .
- Figure 8C represents the mean ⁇ standard error of the mean of at least three independent experiments with A549 and MCF-7 cells treated with cisplatin or copper(II) complexes.
- the symbol ( * ) represents statistical difference in relation to cells not treated with copper or cisplatin compounds.
- Figure 9 represents the analysis of cell cycle inhibition (cell proliferation) during treatment with complexes 1 and 3 in A549 and MCF-7 cancer cells.
- Figure 9 A and B
- Figure 10 graphically represents the toxicity of complex 1 and the chemotherapeutic used in cancer therapy, cisplatin in an alternative animal model, Galeria mellonei ⁇ a.
- the experiment was performed by injecting 1 or cisplatin, both at the same concentrations (1.0 mg/mL), directly into the hemocele of G. mellonei ⁇ a larvae.
- the complex did not show toxicity and did not interfere with the developmental stages (larva, pupa and moth) of the alternative animal model used in the study, showing a significantly different profile from that observed for treatment with the chemotherapy used in cancer treatment, cisplatin.
- binuclear complexes are the result of a very complex preparatory route (Figure 11), developed in the search for new agents with greater effectiveness to eliminate or reduce the proliferation of tumor cells.
- Figure 11 some works on binuclear copper compounds with a diverse range of applications have already been published in international scientific journals. However, in all of them, the structures presented are different from the ones we are proposing in this study. [041] From these works, we will highlight two references, discussed in more depth here, thus complementing the background of the invention. Although they may appear similar, these documents are essentially different in terms of the structure of the ligands used, effectiveness against human cancer cells, and selectivity.
- the compounds of the present invention have an advantage, which is characterized by the six-membered 4-pyridine ring, whose presence results from the use of isomyazide, known as an anti-tuberculosis agent, as a starting hydrazide in the preparation of new ligands.
- Proteomic data obtained by the group of the present study demonstrate that the direct activation of apoptosis occurs through the apoptosis-inducing factor (AIF), through the down-regulation of the RAS-ERK pathway, [049]
- AIF apoptosis-inducing factor
- casein kinase, calpain, well as the catalytic and regulatory subunits of the proteosome they also promote the activation of DNA degradation pathways.
- the down-regulation of the glycolytic enzyme pyruvate kinase indicates a down-regulation of glycolysis, forcing tumor cells to carry out respiratory metabolism through oxidative phosphorylation.
- This non-symmetric linker was prepared from the synthetic intermediate hbpamff, that is, from the compound 2-hydroxy-3- ⁇ [(2-hydroxybenzyl)(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde.
- a methanolic solution (5 ml) of the isonicotinic acid hydrazide, ssonsazide (0.138 g; 1 mmol) was dropped, under constant stirring, onto a solution of hbpamff (0.333 g, 0.9 mmol) in 10 ml of a mixture 1: 1 MeOH:Et 2 0, previously prepared in a 50 ml flask. The mixture was refluxed for 2 hours.
- This linker was prepared from the synthetic intermediate bpmamff, ie, 2-hydroxy-3- ⁇ bis[(2-pyridylmethyl)amino]-methyl ⁇ -5-methylbenzaldehyde. Isoniazid, previously dissolved in 5 ml of a mixture (v/v) 1:1 dichloromethane/MeOH (0.277 g, 2 mmol) was slowly dropped onto a solution containing bpmamff (0.703 g, 2 mmol) dissolved in 10 ml of the same mixing using ultrasound. The solution (50 ml flask) was then refluxed for 3 hours. Over the course of the reaction, the initially bright yellow mixture turned orange. The solvent was subsequently removed under reduced pressure and the oil obtained, left to stand in 1:1 MeCN/MeOH. After two weeks, light yellow microcrystalline needles were isolated.
- the non-symmetrical binuclear copper(li) complexes comprised in the present invention can be synthesized by the following process steps: 1. dissolution of an amount of the binucleating ligand, H 3 L1 or H 2 L2, in a pure organic solvent or mixture of solvents; 2. Slow addition of 2 equivalents of copper(II) salt, previously dissolved in methanol or a mixture of solvents.
- the counterion present in this salt can be coordinating (such as, for example, acetate, propionate or, more generally, a carboxylate anion capable of generating a prodrug) or not (as well as perchlorate, whale, among other capable counterions to generate a final drug in its active form);
- reaction mixture is then stirred, under moderate heating, for a period that may vary from 10 minutes to 24 hours - moderate heating is understood here as the use of a temperature lower than the boiling point of the solvent or mixture of solvents used;
- the solid obtained is filtered, washed with ice-cold organic solvent and dried in a vacuum.
- a greater mass of product and, eventually, crystals suitable for the process of structural determination by X-ray diffraction can be obtained by the slow evaporation of the mother liquor.
- Example 3 COMPLEXES synthesized using ACETATE
- the solution was filtered through paper and transferred to a 50 mL beaker, so that the solvent was completely evaporated by heating to 60 °C.
- the resulting solid was washed with ice-cold methanol, filtered on a Buchner funnel and stored under vacuum in a desiccator containing silica.
- the present study deals with the effects caused by a series of novel binuclear copper(ll) complexes ( Figures 2 and 3) in breast (MCF-7), lung (A549) and prostate (PC) cancer cell lines -3).
- the study carried out analyzed: (i) the survival (through the MTT assay) of the cancer cells; (ii) the intracellular production of reactive oxygen species (ROS) (through the DCFH2-DA probe assay); (iii) the mitochondrial membrane potential (via the assay with the fluorescent probe JC-1); (iv) the enzymatic activity for caspases 8 and 9; (v) DNA fragmentation (through the TUNNEL technique [Gravrieli et al, 1992 - Gavrieli Y, Sberman Y, Ben-Sasson SA, Identification of programmed death in situ via specific iabeling of nuciear DNA fragmentation.
- Cisplatin was used as a chemotherapy control because it is a drug widely used in the clinic and also because it is, until today, the reference for coordination compounds with antitumor activity.
- the cytotoxicity test was performed by the MTT method, which after metabolized by living cells is converted into purple formazan crystals. Cell survival was calculated as the percentage of living cells relative to cells not treated with any of the compounds.
- caspase 8 and 9 activities showed that only the activity of caspase 9 enzyme was altered, having its levels increased in cell lines treated with complexes 1 and 3. The result clearly indicates that complexes 1 and 3 activate the intrinsic pathway that triggers the apoptosis process in human cancer cells ( Figure 7).
- the metallodrugs of the present invention can constitute pharmaceutical compositions, being used alone or in combination with each other, adding them to pharmaceutically acceptable vehicles and excipients.
- Cancer therapy presents high treatment difficulty, due to its different origins, and may present an intrinsic and/or acquired resistance to available treatments. Furthermore, the excessive use of the same drug/medication can cause serious side effects to the patient.
- the metallodrugs described in the present invention can be combined/associated with any other drugs used in the treatment of neoplasms and related diseases.
- anti-tumor drugs such as copper chelators and chemotherapy drugs, particularly those based on platinum, such as cisplatin [c/s-diaminodichloroplatinum(ll)].
- Another possibility of combining the metallodrugs of the present invention comprises the one that can be performed with other copper complexes, however mononuclear. Different from those presented in this invention, the compounds may demonstrate synergistic effects during treatment.
- Treatment includes administering an amount of the complexes alone or in combination that is effective to alleviate, alter, remedy, ameliorate or affect the disease, disease symptoms or diseases resulting from opportunistic infections caused by pathogenic fungi, in order to prevent the aggravation of the main disease, cancer.
- the necessary dosages of the compounds and their compositions comprising the isolated complexes or in combination may vary in each case, being different for each individual treated.
- the definition of the ideal dosage will be determined by balancing the therapeutic benefit and any risk or side effects of the treatments described herein.
- the in vivo administration of these metallopharmaceuticals can be carried out in continuous dose or even intermittently (for example, in divided doses at appropriate intervals), throughout the course of treatment.
- the most effective administration protocols of chemotherapeutics used in the therapy of various types of cancer are well known to those skilled in the art and will vary with the formulation used for the therapy, the purpose of therapy, the target cell and the subject to be treated.
- An adequate dose of a platinum-based therapy may be a standard dose; a standard dosage of cisplatin for the treatment of testicular cancer is 20 mg/m 2 intravenously (IV) daily for 1 to 5 consecutive days every 3 weeks for 3-4 cycles of therapy when used in combination therapy; on the other hand, a standard dosage of cisplatin for the treatment of advanced ovarian carcinoma ranges from 75 to 100 mg/m 2 IV once every 3-4 weeks for 6 cycles when cisplatin is used in combination therapy. For the treatment of advanced bladder cancer, a standard dosage of cisplatin is 70 to 100 mg/m 2 IV once every 3-4 weeks for 4 cycles when cisplatin is used in combination therapy.
- a standard dosage for the treatment of recurrent or advanced head and neck cancer is 80 to 120 mg/m 2 IV once every 3 weeks or 20 to 100 mg/m 2 IV when used in combination chemotherapy regimens, with frequency of administration depending on the specific regime employed.
- a standard dosage of cisplatin for the treatment of cervical cancer is 40 mg/m 2 given. weekly for 6 weeks when combined with radiotherapy or 40 to 80 mg/m 2 every 3 weeks for 3-4 cycles when given in combination with other chemotherapy agents.
- a standard dosage of cispiatin in combination therapy is 75 to 100 mg/m 2 IV once every 3-4 weeks for 4-8 cycles, depending on the specific regimen used.
- a standard dosage of cispiatin is 50 to 120 mg/m 2 IV once every 3-4 weeks in combination chemotherapy regimens or 10 to 50 mg/m 2 IV every 3 weeks for 2 cycles when combined with radiation.
- a standard dosage of cispiatin is 50 to 100 mg/m 2 IV once every 3-4 weeks in single or combined chemotherapy regimens.
- the dose of the combination between the binuclear copper complexes of the present invention and another chemotherapeutic agent, such as cispiatin, but not limited exclusively to cispiatin, should be between 20 to 120 mg/m 2 of the drug's body surface antitumor, plus 10 to 480 mg/m 2 of body surface of binuclear copper(ll) complexes in a single intravenous infusion, every 3-4 weeks for the necessary number of cycles, obviously respecting the standard protocol used in specific chemotherapy for the cancer being treated.
- the dilution rates of the new composition will be comprised between 1 part of the antitumor drug: 0.5 - 4.0 parts of the binuclear copper complex or, alternatively, 0.5 part of the antitumor drug: 0.5 - 4.0 parts of the binuclear copper complex.
- composition is used in the preparation of an injectable solution or pharmaceutical formulation for oral use composed of both drugs and should be administered exclusively by intravenous infusion or orally.
- Binuclear copper(II) complexes can be given in combination with an agent or procedure to reduce any possible side effects (eg, diarrhea, nausea, and vomiting).
- antitumor agents examples include: pemetrexed, vinorelbine, gemcitabine, vinblastine, dacarbazine, temozolomide, 5FU (5-fluorouracil), cyclophosphamide , bleomycin, etoposide, ifosfamide, paclitaxel, methotrexate, doxorubicin, adriamycin, vincristine, mitomycin, docetaxel, and combinations of the above agents. These agents can also be administered in conjunction with performing surgical procedures and/or applying radiation.
Abstract
The present invention relates to novel metallodrugs containing a copper(II) binuclear coordination centre and the non-symmetrical binucleating ligands 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H3L1) or 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H2L2) suitable for the treatment of neoplasms involving solid tumours and/or leukaemias, and also opportunistic diseases, such as diseases caused by pathogenic fungi.
Description
LIGANTES BINUCLEÂNTES FENÓLICOS, COMPOSTOS METÁLICOS BINUCLEÂRES, COMPOSIÇÃO MÉDICO-VETERINÁRIA, PROCESSOS DE SÍNTESE DE LIGANTES BINUCLEÂNTES, PROCESSO DE SÍNTESE DE COMPOSTOS BINUCLEÂRES, MÉTODO DE TRATAMENTO DE NEOPLASIAS E DOENÇAS FÚNGICAS E USO BINUCLEANT PHENOLIC BINDERS, BINUCLEAR METALLIC COMPOUNDS, MEDICAL-VETERINARY COMPOSITION, BINUCLEANT BINDERS SYNTHESIS PROCESS, BINUCLEAR COMPOUNDS SYNTHESIS PROCESS, METHOD OF TREATMENT OF NEOPLES AND PHUGAL DISEASES
Campo da Invenção Field of Invention
[001] A presente invenção refere-se a novas metalodrogas contendo um centro de coordenação binuclear de cobre(ll) e os ligantes binucieantes não-simétricos 2-hidroxi-3-{[(2-hidroxibenzil)(2-piridilmetil)amino]-metil}-5-metilbenzaldeído isonicotinoíl hidrazona (H3L1 ) ou 2-hidroxi-3-{bis[(2-piridilmetil)amino]-metil}-5- metilbenzaldeído isonicotinoíl hidrazona (H2L2) para tratamento de neoplasias envolvendo tumores sólidos e/ou leucemias, bem como doenças de natureza oportunista, tal como as doenças causadas por fungos patogênicos. Em uma visão mais ampla, a presente invenção se insere no campo de aplicação da química, farmácia, medicina, biologia e bioquímica, mais especificamente na área de preparações de medicamentos contendo ingredientes ativos. [001] The present invention relates to new metallodrugs containing a copper(II) binuclear coordination center and the non-symmetrical binding ligands 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino] -methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L1 ) or 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2) for treatment of neoplasms involving solid tumors and/or leukemias, as well as diseases of an opportunistic nature, such as diseases caused by pathogenic fungi. In a broader view, the present invention falls within the field of application of chemistry, pharmacy, medicine, biology and biochemistry, more specifically in the area of drug preparations containing active ingredients.
Antecedentes da Invenção Background of the Invention
[002] O câncer está entre as principais causas de óbito no mundo. Assim, 0 desenvolvimento de estratégias quimioterapêuticas envolvendo novos agentes antitumorais tem sido uma das áreas de foco para tratamento da doença. [002] Cancer is among the leading causes of death worldwide. Thus, the development of chemotherapeutic strategies involving new antitumor agents has been one of the focus areas for the treatment of the disease.
[003] O pioneiro sucesso clínico da metalodroga cisplatina não só permitiu 0 desenvolvimento de fármacos à base de platina, mas também 0 uso de outros metais de transição que visam ser mais eficazes e eficientes quanto a: (i) a farmacologia; (ii) aumento da sobrevida dos pacientes; (iii) redução dos efeitos colaterais e; (iv) redução dos custos clínicos com 0 tratamento. [003] The pioneering clinical success of the metallodrug cisplatin not only allowed the development of drugs based on platinum, but also the use of other transition metals that aim to be more effective and efficient in terms of: (i) pharmacology; (ii) increased patient survival; (iii) reduction of side effects and; (iv) reduction of clinical costs with the treatment.
[004] Além disso, a síntese de tais compostos tem como objetivo aumentar a interação com 0 DNA, superando a resistência inerente ou adquirida à cisplatina. Em particular, grande foco tem sido dirigido para 0 desenvolvimento de compostos inéditos de cobre, cuja atividade anticâncer tem norteado muitas
pesquisas com o objetivo de descobrir novos agentes que sejam capazes de superar a resistência e os efeitos colaterais associados à cisplatina. Portanto, devido aos efeitos colaterais dos fármacos empregados na clínica atual, existe uma necessidade urgente em relação à síntese e ao desenvolvimento de novos fármacos quimioterápicos. [004] Furthermore, the synthesis of such compounds aims to increase the interaction with 0 DNA, overcoming the inherent or acquired resistance to cisplatin. In particular, great focus has been directed towards the development of novel copper compounds, whose anticancer activity has guided many research aimed at discovering new agents that are able to overcome the resistance and side effects associated with cisplatin. Therefore, due to the side effects of drugs used in the clinic today, there is an urgent need for the synthesis and development of new chemotherapy drugs.
[005] Até o presente momento, a totalidade das metalodrogas aprovadas para uso humano no tratamento do câncer apresentam a platina como elemento central. Isto acarreta uma série de desvantagens de ordem técnica, como a elevada toxicidade da platina, provocando uma gama de efeitos colaterais indesejáveis, o surgimento de linhagens celulares tumorais resistentes à terapia e económica, porque sendo a platina um metal relativamente raro, o custo dos reagentes de partida para a síntese das metalodrogas a base deste metal são muito caros, o que eleva sobremaneira o preço final dos fármacos. [005] To date, all metallo-drugs approved for human use in the treatment of cancer have platinum as a central element. This entails a series of technical disadvantages, such as the high toxicity of platinum, causing a range of undesirable side effects, the emergence of tumor cell lines resistant to therapy and economic, because platinum is a relatively rare metal, the cost of the reagents starting materials for the synthesis of metallodrugs based on this metal are very expensive, which greatly increases the final price of the drugs.
[006] Boa parte destes problemas são evitados pela aplicação do cobre na síntese, um metal barato, muito mais reativo que a platina (o que diminui o tempo de síntese das metalodrogas) e, ainda mais importante, um metal fisiológico que diminui de forma significativa os efeitos colaterais associados ao seu uso prolongado em ciclos quimioterápicos. [006] Most of these problems are avoided by the application of copper in the synthesis, a cheap metal, much more reactive than platinum (which decreases the synthesis time of metallodrugs) and, even more importantly, a physiological metal that decreases in shape side effects associated with its prolonged use in chemotherapy cycles.
[007] Existem estudos e desenvolvimentos que buscam esta transição entre a platina e o cobre, porém ainda não atingiram o resultado desejado. Por exemplo, o documento brasileiro, BR1120190241353, com data de prioridade 18/05/2017, intitulado de “TERAPIAS DE COMBINAÇÃO PARA O TRATAMENTO DE CÂNCER”, reporta um método para tratamento do câncer por meio de terapia de combinação envolvendo um agente inibidor da sinalização da polí[ADPríbose] po!imerase (PARP) e um segundo agente que regula a atividade dentro do microambiente tumoral, sendo um inibidor de células T reguladoras ( Treg ), um agente inibidor de macrófagos, um agente intensificador da resposta imune específica para o antígeno ou, ainda, uma combinação deles. Mais especificamente, a composição farmacêutica descrita neste documento trata de um grupo de agentes inibidores de macrófagos, dentre os quais destacam-se um
quelato de cobre. A referência brasileira trata de uma terapia contra o câncer (mama, pulmão e/ou próstata) por meio do uso coadjuvante de quelaios de cobre, mas não propõe o uso de metalodrogas baseadas no metal em si. Além disso, o mecanismo intrínseco por trás da ação antitumora! é proposto como sendo o melhoramento da resposta imune ou o aumento da atividade de uma célula imune em um paciente com uma doença ou condição. [007] There are studies and developments that seek this transition between platinum and copper, but they have not yet achieved the desired result. For example, the Brazilian document, BR1120190241353, with priority date 05/18/2017, entitled "COMBINATION THERAPIES FOR THE TREATMENT OF CANCER", reports a method for treating cancer through combination therapy involving a cancer-inhibiting agent. Poly[ADProbose] polymerase (PARP) signaling is a second agent that regulates activity within the tumor microenvironment, being an inhibitor of regulatory T cells ( Treg ), a macrophage inhibitory agent, a specific immune response enhancing agent for the antigen or even a combination of them. More specifically, the pharmaceutical composition described in this document deals with a group of macrophage inhibiting agents, among which one stands out. copper chelate. The Brazilian reference deals with cancer therapy (breast, lung and/or prostate) through the adjuvant use of copper chelates, but does not propose the use of metallo-drugs based on the metal itself. Also, the intrinsic mechanism behind the antitumor action! is proposed to be improving the immune response or increasing the activity of an immune cell in a patient with a disease or condition.
[008] Um outro exemplo ainda mais próximo seria o documento EP145711 , com data de prioridade 10/06/1983, o qual é intitulado de “CORRER COMPLEX FOR TREATING CÂNCER” e apresenta complexos de cobre(ll) exibindo atividades análogas à da enzima superóxido dismutase (SOD) em células de mamíferos. Tal propriedade é utilizada para o tratamento de câncer, visto que substitui a atividade SOD perdida ou significativamente reduzida em células cancerígenas. O tratamento proposto no documento EP145711 realmente apresenta vantagens quando comparado a outros métodos de seu estado da técnica, demonstrando que o cobre possui potencial para a redução do crescimento tumoral, aumento da sobrevida do organismo hospedeiro, diminuição das metástases tumorais ou ainda efeitos sobre a diferenciação morfológica das células cancerosas. Porém, o documento reporta compostos de cobre que incluem complexos de ligantes do tipo salicilato, seus solvatos, bem como suas misturas. Drogas estruturalmente simples, cuja eficácia pouco tem a ver com a natureza do ligante utilizado e sim com a atividade SOD inerente a complexos de diversos metais. Além disso, o mecanismo de ação proposto visa apenas uma das características das células tumorais, que é a recomposição da atividade SOD perdida nas células de câncer, criando desta maneira uma condição para interromper a divisão celular e, consequentemente, o crescimento do tumor. Como o modo de ação destes complexos não envolve o processo de morte celular, o que acaba por inviabilizar a associação do conceito de citotoxicidade (IC50) ao de atividade, torna-se difícil a comparação de sua eficácia com a de outros compostos. Assim, fica claro que o desenvolvimento racional de novos ligantes e a preparação de complexos que
exibam mecanismos alternativos, e de preferência múltiplos, de ação pode em muito melhorar a eficácia dos novos agentes antitumorais obtidos. [008] Another even closer example would be the document EP145711 , with priority date 10/06/1983, which is entitled "RUN COMPLEX FOR TREATING CANCER" and presents copper(ll) complexes exhibiting activities analogous to the enzyme superoxide dismutase (SOD) in mammalian cells. This property is used for cancer treatment, as it replaces lost or significantly reduced SOD activity in cancer cells. The treatment proposed in EP145711 really has advantages when compared to other methods of its state of the art, demonstrating that copper has the potential to reduce tumor growth, increase host organism survival, decrease tumor metastases or even effects on differentiation morphology of cancer cells. However, the document reports copper compounds that include complexes of salicylate-type ligands, their solvates, as well as their mixtures. Structurally simple drugs, whose effectiveness has little to do with the nature of the ligand used, but with the SOD activity inherent in complexes of different metals. Furthermore, the proposed mechanism of action targets only one of the characteristics of tumor cells, which is the recomposition of lost SOD activity in cancer cells, thus creating a condition to interrupt cell division and, consequently, tumor growth. As the mode of action of these complexes does not involve the cell death process, which ends up making the association of the concept of cytotoxicity (CI 50 ) with that of activity unfeasible, it is difficult to compare their effectiveness with that of other compounds. Thus, it is clear that the rational development of new ligands and the preparation of complexes that exhibiting alternative, and preferably multiple, mechanisms of action can greatly improve the efficacy of the new anti-tumor agents obtained.
[009] Uma classe importante de complexos de cobre propostos no âmbito da quimioterapia do câncer é descrita nos documentos americanos US5107005 e US5578326, datados de 21/04/1992 e 19/11/1996, respectivamente. Sob os títulos “Process to obtain new mixed copper aminoacidate complexos from phenylate phenanthrolines to be used as anticancerigenic agents” e “ Copper amino acidate diimine niirate compounds and their methyl derívatives and a process forpreparing them", essas invenções se referem à síntese de uma série de novos complexos ternários mononucleares de cobre(ll) contendo aminoácidos e fenantrolinas aromáticas como ligantes. Os compostos foram propostos como agentes antitumorais com uso terapêutico preferencial para o tratamento de tumores sólidos e do sangue (ex. leucemia). Os resultados de citotoxicidade mostraram que o complexo mononuclear de cobre testado, a saber [Gu(4,7-dimethyl-1 ,10-phenanthroline)(glycinato)]NO3 apresenta atividade sutilmente superior à cisplatina, porém inferior à mitomicina, drogas de referência empregadas na terapia do câncer. Os ensiaos com animais de experimentação (ratos da linhagem B6D2F1 ) mostraram que apenas para doses mais elevadas do composto foi possível verificar aumento da sobrevida dos animais afetados pela inoculação de células de leucemia linfoide. Contudo, a sua menor atividade em relação à droga de referência (mitomicina), a falta de um aprimorado teste estatístico para comparação dos resultados obtidos com a cisplatina, a falta de comparação dos ensaios em animais com as drogas de referência, a falta de definição com relação ao mecanismo de morte celular (ex. apoptose ou necrose) e o fato da invenção se referir apenas a complexos mononucleares de cobre deixam campo aberto para o aprimoramento dessas metalodrogas. [009] An important class of copper complexes proposed in the context of cancer chemotherapy is described in US documents US5107005 and US5578326, dated 21/04/1992 and 19/11/1996, respectively. Under the headings "Process to obtain new mixed copper aminoacidate complexes from phenylate phenanthrolines to be used as anticancerigenic agents" and "Copper amino acidate diimine niirate compounds and their methyl derivatives and a process forpreparing them", these inventions refer to the synthesis of a series of novel copper(II) mononuclear ternary complexes containing amino acids and aromatic phenanthrolines as ligands. The compounds have been proposed as antitumor agents with preferential therapeutic use for the treatment of solid and blood tumors (eg leukemia). the tested copper mononuclear complex, namely [Gu(4,7-dimethyl-1 ,10-phenanthroline)(glycinate)]NO 3 has a slightly higher activity than cisplatin, but lower than mitomycin, a reference drug used in cancer therapy The tests with experimental animals (rats of the B6D2F1 strain) showed that only at higher doses of the compound was po It is possible to verify an increase in the survival of animals affected by the inoculation of lymphoid leukemia cells. However, its lower activity compared to the reference drug (mitomycin), the lack of an improved statistical test to compare the results obtained with cisplatin, the lack of comparison of animal tests with the reference drugs, the lack of definition with regard to the mechanism of cell death (eg apoptosis or necrosis) and the fact that the invention refers only to mononuclear copper complexes leave an open field for the improvement of these metallodrugs.
[010] O documento europeu EP2407164, publicado em 18/01/2012, e intitulado de ‘‘Copper II complexes of phenanthroline and their use in câncer treatment” revela complexos ternários mono- ou binucleares de cobre(ll) em que o metal encontra-se ligado a uma das extremidades de um dicarboxilato aromático, não-
aromático ou alifático, o qual atua como ponte no caso dos complexos binucleares, e dois ligantes bidentados do tipo fenantrolina completam a esfera de coordenação dos centros cúpricos pentacoordenados. Eventualmente, um sexto ligante monodentado pode vir a estar presente. Os complexos são propostos como nucleases auto-ativadas para o tratamento de câncer e outras desordens proliferativas. Dentre os tipos de câncer abarcados pela tecnologia descrita no documento EP2407164 estão o câncer de colon, mama, próstata e ovário. Além disso, o mecanismo de ação está relacionado à clivagem controlada de DNA e RNA pelos complexos, os quais apresentam um importante avanço em relação à técnica anterior, pois inclui dois íons cobre(ll) em alguns dos compostos reivindicados. Se trata da principal referencia do atual estado da técnica. Estes complexos, contudo, apresentaram baixa eficácia geral em relação àqueles descritos na presente invenção: por exemplo, na linhagem de câncer de mama MCF-7, sob condições similares de teste, os compostos do documento EP2407164 foram -400 vezes menos ativos (compostos 1-3), ~40 vezes menos ativos (compostos 4 e 5) e -8 vezes menos ativo (composto 6) que os complexos 1 e 3 da presente invenção, descritos mais à frente. Além disso, nos complexos binucleares apresentados no documento EP2407164, a distância inter-metálica é demasiado grande como para permitir um efeito cooperativo entre ambos os centros cúpricos. Desta forma, ainda é preciso desenvolver metalodrogas que contenham cobre(il), que possam atuar também por meio da fragmentação do DNA, ou mesmo através de múltiplos mecanismos simultaneamente, mas com ligantes que as tornem ainda mais ativas e mais seletivas às células cancerígenas. [010] The European document EP2407164, published on 18/01/2012, and entitled ''Copper II complexes of phenanthroline and their use in cancer treatment” reveals mono- or binuclear copper(ll) ternary complexes in which the metal meets -if attached to one end of an aromatic dicarboxylate, not- aromatic or aliphatic, which acts as a bridge in the case of binuclear complexes, and two bidentate ligands of the phenanthroline type complete the sphere of coordination of the pentacoordinate cupric centers. Eventually, a monodentate sixth ligand may be present. The complexes are proposed as self-activated nucleases for the treatment of cancer and other proliferative disorders. Among the types of cancer covered by the technology described in EP2407164 are colon, breast, prostate and ovarian cancer. Furthermore, the mechanism of action is related to the controlled cleavage of DNA and RNA by the complexes, which present an important advance over the prior art, as it includes two copper(II) ions in some of the claimed compounds. It is the main reference of the current state of the art. These complexes, however, showed low overall efficacy compared to those described in the present invention: for example, in the breast cancer line MCF-7, under similar test conditions, the compounds of EP2407164 were -400 times less active (compounds 1 -3), ~40 times less active (compounds 4 and 5) and -8 times less active (compound 6) than complexes 1 and 3 of the present invention, described later. Furthermore, in the binuclear complexes presented in EP2407164, the intermetallic distance is too great to allow a cooperative effect between both cupric centers. Thus, it is still necessary to develop metallodrugs containing copper(il), which can also act through DNA fragmentation, or even through multiple mechanisms simultaneously, but with ligands that make them even more active and more selective to cancer cells.
[011] Com relação ao tratamento de infecções fúngicas, cabe citar o documento chinês CN102503901 , datado de 20/08/2012 e intitulado “Zole antífungai compounds, and preparation method and application thereof”. Assim como o pedido de patente WG2002Q39963, publicado em 23/05/2002 e intitulado de “Antífungai na/7 composition and method of use”, ambos tratam de tecnologias que utilizam compostos que contêm cobre para o tratamento de infecções por
fungos, dentre os quais destacam-se as seguintes espécies: Candida tropicaiis e Candida aibicans. No entanto, os documentos falham em propor um tratamento antifúngico a base de cobre aplicável a um espectro mais amplo de patógenos incluindo, por exemplo, os fungos C. parapsiíosis e C. krusei, de extrema importância em situações de neoplasias e tumores avançados, e ainda outros, tais como Aspergillus spp, Cryptococcus spp, Scedosporium spp, Fusarium spp , Zygomycetes spp e Trichosporon spp, os quais são frequentemente encontrados em pacientes imunocomprometidos por conta da quimioterapia. [011] Regarding the treatment of fungal infections, it is worth mentioning the Chinese document CN102503901 , dated 20/08/2012 and entitled “Zole antifungai compounds, and preparation method and application thereof”. As well as patent application WG2002Q39963, published on 23/05/2002 and entitled “Antífungal na/7 composition and method of use”, both deal with technologies that use copper-containing compounds for the treatment of infections by fungi, among which the following species stand out: Candida tropicaiis and Candida aibicans. However, the documents fail to propose a copper-based antifungal treatment applicable to a broader spectrum of pathogens including, for example, the fungi C. parapsiosis and C. krusei, which are extremely important in situations of neoplasms and advanced tumors, and still others, such as Aspergillus spp, Cryptococcus spp, Scedosporium spp, Fusarium spp, Zygomycetes spp and Trichosporon spp, which are frequently found in immunocompromised patients due to chemotherapy.
[012] Adicionalmente, no pedido de patente WO2Q02039963, é reportado um método de tratamento de micoses (onychomycosis) em unhas de humanos pela aplicação de uma composição contendo um sal de cobre. Não são apresentadas metalodrogas mais avançadas como as binucleares e nem ligantes específicos. [013] Por fim, o documento brasileiro PI0416387-7, publicado em 21/02/2007, intitulado de “Composição desinfetante e métodos de confecção e uso da mesma", descreve composição antimícrobiana que compreende um complexo único de metal com ligante monodentado, bidentado ou polidentado que exibe afinidade ao íon hidrogénio, um tensoativo ionogênico e um solvente, assim como um veículo farmaceuticamenfe aceitável. Essas composições têm propriedades antissépticas, sendo ativas contra bactérias gram-posítívas e gram- negativas, fungos, vírus e esporos, e podem ser aplicadas em um intervalo amplo de temperaturas. Métodos de uso das composições da presente invenção no tratamento e prevenção de doenças causadas por uma variedade de patógenos são descritos. Entretanto, a invenção não trata da sua eficácia contra células cancerígenas e ainda faz referência a complexos mononucleares cuja atividade antimicrobiana depende da associação com outras moléculas. [012] Additionally, in patent application WO2Q02039963, a method of treating mycoses (onychomycosis) in human nails by applying a composition containing a copper salt is reported. More advanced metallodrugs such as binuclear ones or specific ligands are not presented. [013] Finally, the Brazilian document PI0416387-7, published on 02/21/2007, entitled "Disinfectant composition and methods of making and using it", describes an antimicrobial composition comprising a single metal complex with a monodentate binder, bidentate or polydentate which exhibits affinity to hydrogen ion, an ionogenic surfactant, and a solvent, as well as a pharmaceutically acceptable carrier. These compositions have antiseptic properties, being active against gram-positive and gram-negative bacteria, fungi, viruses and spores, and may be applied over a wide range of temperatures. Methods of using the compositions of the present invention in the treatment and prevention of diseases caused by a variety of pathogens are described. However, the invention does not address their effectiveness against cancer cells and still makes reference to complexes mononuclear cells whose antimicrobial activity depends on association with other molecules.
[014] Diante do acima exposto, fica demonstrado que o uso do metal cobre já se mostra como uma alternativa tecnológica à platina, porém se baseia, até o presente momento, em composições mononucleares contendo o íon cobre(ll). Mesmo quando dois ions cobre(ll) são utilizados, em complexos binucleares voltados à atividade antitumoral, não se observa ainda o efeito cooperativo
esperado, o que resulta em baixa atividade. Adicionaimente, esses compostos não têm sido capazes de associar o tratamento concomitante ou mesmo isolado de fungos, o que possibilitaria uma quimioterapia do câncer que promovesse ao mesmo tempo o combate às doenças oportunistas causadas por fungos. [014] Given the above, it is demonstrated that the use of copper metal is already shown as a technological alternative to platinum, but it is based, to date, on mononuclear compositions containing the copper(ll) ion. Even when two copper(ll) ions are used, in binuclear complexes aimed at antitumor activity, the cooperative effect is not yet observed expected, which results in low activity. Additionally, these compounds have not been able to associate the concomitant or even isolated treatment of fungi, which would enable cancer chemotherapy that promotes at the same time the fight against opportunistic diseases caused by fungi.
[015] Assim, seria interessante se desenvolver novos compostos baseados em metais, metalodrogas com potente atividade antitumoral e antifúngica, na forma de complexos binucleares de cobre(li) contendo ligantes /V-acil-hidrazônicos. Como esperado, os novos agentes agem por meio de um mecanismo diferente de ação e bases moleculares próprias envolvidas no processo de morte celular. [015] Thus, it would be interesting to develop new compounds based on metals, metallodrugs with potent antitumor and antifungal activity, in the form of binuclear copper(li) complexes containing /V-acyl-hydrazonic ligands. As expected, the new agents act through a different mechanism of action and their own molecular bases involved in the cell death process.
Descrição Resumida da Invenção Brief Description of the Invention
[016] A presente invenção prevê, desta maneira, novas metalodrogas, processo de síntese, método de tratamento, uso na fabricação de medicamentos para o tratamento de tumores e doenças fúngicas em humanos e outros mamíferos, assim como composição farmacêutica e veterinária, baseada em complexos binucleares de cobre(ll), não-simétricos, com efeito citotóxico sobre linhagens celulares de câncer de próstata, mama e pulmão e que também venham a apresentar atividade antifúngica e baixa toxicidade para células saudáveis. [016] The present invention provides, in this way, new metallodrugs, synthesis process, method of treatment, use in the manufacture of drugs for the treatment of tumors and fungal diseases in humans and other mammals, as well as pharmaceutical and veterinary composition, based on binuclear copper(II) complexes, non-symmetric, with cytotoxic effect on cell lines of prostate, breast and lung cancer, which also show antifungal activity and low toxicity to healthy cells.
[017] Deste modo, uma primeira modalidade de invenção é o desenvolvimento de novas alternativas tecnológicas, voltadas aos novos compostos/complexos binucleares de cobre com atividade antitumoral, com potencial grande impacto sobre a atual quimioterapia do câncer, cujo arsenal de substâncias poderia ser substituído ou mesmo opcionalmente combinado aos novos compostos. [017] Thus, a first mode of invention is the development of new technological alternatives, aimed at new compounds/binuclear copper complexes with antitumor activity, with potentially great impact on current cancer chemotherapy, whose arsenal of substances could be replaced or even optionally combined with the new compounds.
[018] O potencial biotecnológico apresentado por estes novos complexos não se limita à atividade antitumoral, pois também se mostraram ativos contra fungos patogênícos (ex. Candida aíbicans, Candida parapsiiosis, Candida tropicaiis e Candida krusei), mosfrando grande versatilidade de aplicação para uso na terapia contra o câncer e patologias oportunistas causadas por fungos. [018] The biotechnological potential presented by these new complexes is not limited to antitumor activity, as they were also shown to be active against pathogenic fungi (eg Candida aibicans, Candida parapsiiosis, Candida tropicaiis and Candida krusei), showing great versatility of application for use in cancer therapy and opportunistic pathologies caused by fungi.
[019] Cabe ressaltar que as patologias causadas por fungos apresentam alfa incidência mundial e impactam, muito em particular, indivíduos com algum tipo de ímunodeficiência (celular ou humoral), tais como os pacientes em tratamento
quimioterápico contra o câncer. Por fim, além de serem empregados na terapia de neoplasias, bem como de doenças fúngicas, tais compostos poderão servir como plataforma para síntese de novos compostos modificados estruturalmenfe que aumentem a eficácia contra as células iumorais e/ou fungos pafogênicos. [020] A presente invenção provê, portanto, nesta primeira modalidade, quatro metalodrogas derivadas de dois novos precursores hidrazônicos não-simétricos, a saber: 2-hidroxi-3-{[(2-hidroxibenzil)(2-piridilmetil)amino]-metil}-5- metilbenzaldeído isonicotinoíl hidrazona (H3L1), conforme formula estrutural abaixo e ilustrado na figura 1 , e 2-hidroxi-3-{bis[(2-piridilmetil)amino]-metil}-5- metilbenzaldeído isonicotinoíl hidrazona (H2L2), conforme formula estrutural abaixo e ilustrado na figura 1 , pertencentes ao amplo grupo dos ligantes binucleantes, isto é, capazes de formar complexos binucleares por proporcionarem ambientes de coordenação apropriados para acomodar dois centros metálicos a uma proximidade, tal, que um efeito cooperativo entre ambos seja possível, e até mesmo esperado.
[021] A diferença entre esses ligantes está relacionada com sua porção não- hidrazônica: enquanto H3L1 apresenta grupos doadores terminais feno! e piridina (portanto, diferentes), H2L2 possui dois grupos piridina. Assim, como 0 número de átomos de oxigénio e nitrogénio disponíveis para doação de pares eletrónicos varia nesses ligantes, a dureza/maciez desse sítio de coordenação também se altera. Os complexos bínucleares de cobreíll) gerados a partir de H3L1 e H2L2, tanto aqueles com uma ponte exógena hidróxido (postulada como a forma ativa) quanto aqueles com pontes exógenas acetato (pró-drogas que gerariam a forma biologicamente ativa por reação de hidrólise no organismo), apresentam centros de cobre(ll) insaturados ou contendo ligantes lábeis, como a molécula de água, 0 que possibilita a interação dos mesmos com macromolécuias biológicas como, por exemplo, o DNA, RNA e/ou proteínas. [019] It is noteworthy that pathologies caused by fungi have an alpha incidence worldwide and impact, in particular, individuals with some type of immunodeficiency (cellular or humoral), such as patients under treatment chemotherapy against cancer. Finally, in addition to being used in the therapy of neoplasms, as well as fungal diseases, such compounds may serve as a platform for the synthesis of new structurally modified compounds that increase their effectiveness against limoral cells and/or paphogenic fungi. [020] The present invention therefore provides, in this first modality, four metallodrugs derived from two new non-symmetric hydrazonic precursors, namely: 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]- methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L1), according to the structural formula below and illustrated in figure 1 , and 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2), according to the structural formula below and illustrated in Figure 1 , belonging to the broad group of binucleating ligands, that is, capable of forming binuclear complexes by providing coordination environments suitable to accommodate two metallic centers in close proximity, such that a cooperative effect between them is possible, and even expected. [021] The difference between these ligands is related to their non-hydrazonic portion: while H 3 L1 has phen end donor groups! and pyridine (thus different), H 2 L2 has two pyridine groups. Thus, as the number of oxygen and nitrogen atoms available for electron pair donation varies in these ligands, the hardness/softness of this coordination site also changes. Binuclear copper complexes) generated from H 3 L1 and H 2 L2, both those with an exogenous hydroxide bridge (postulated as the active form) and those with exogenous acetate bridges (pro-drugs that would generate the biologically active form by reaction of hydrolysis in the organism), have unsaturated copper(II) centers or contain labile ligands, such as the water molecule, which enables their interaction with biological macromolecules such as, for example, DNA, RNA and/or proteins.
[022] Em uma segunda modalidade da presente invenção, temos composições farmacêuticas contendo as metalodrogas descritas acima, opcionalmente em conjunto com outras drogas antineoplásicas ou voltadas aos efeitos secundários do tratamento, e veículos farmaceuticamente aceitos. [022] In a second modality of the present invention, we have pharmaceutical compositions containing the metallodrugs described above, optionally together with other antineoplastic drugs or aimed at the side effects of treatment, and pharmaceutically acceptable vehicles.
[023] Em uma terceira modalidade da invenção temos o processo de produção de compostos com atividade anti-neopiásíca, preparados a partir dos ligantes H3L1 e H2L2 (Figura 1 ), constituídos por etapas de síntese dos complexos bínucleares de cobreíll): composto 1 e composto 2 conforme as formulas estruturais abaixo e ilustrados na figura 2, e composto 3 e composto 4 conforme as formulas estruturais abaixo e ilustrados na figura 3, podendo compreender após sua obtenção a associação de um ou mais destes complexos com outro fármaco antitumoral, preferencialmente, mas não se limitando, a cisplatina, para tratamento de neoplasias malignas de tumores sólidos ou leucemias.
[023] In a third modality of the invention we have the production process of compounds with anti-neoplastic activity, prepared from the ligands H 3 L1 and H 2 L2 (Figure 1), consisting of synthesis steps of binuclear copper complexes) : compound 1 and compound 2 according to the structural formulas below and illustrated in figure 2, and compound 3 and compound 4 according to the structural formulas below and illustrated in figure 3, which may include after obtaining the association of one or more of these complexes with another drug anti-tumor, preferably but not limited to cisplatin, for the treatment of malignant neoplasms of solid tumors or leukemias.
[024] Em uma quarta modalidade da invenção, temos o uso dos complexos no tratamento de neopiasías e de doenças causadas por fungos, associadas ou não ao tratamento do câncer, em que uma quantidade farmaceuticamente ativa é administrada aos pacientes durante o tratamento com quimioterápicos ou, ainda, no decorrer de outros tratamentos antitumorais. [024] In a fourth modality of the invention, we have the use of complexes in the treatment of neoplasms and diseases caused by fungi, associated or not with cancer treatment, in which a pharmaceutically active amount is administered to patients during treatment with chemotherapy or , also, in the course of other antitumor treatments.
[025] Uma quinta e última modalidade corresponderia ao uso dos compostos sintetizados na fabricação de medicamentos voltados ao tratamento de neopiasías. Medicamentos que apresentam de forma concomitante pouca toxicidade às células humanas saudáveis, alta eficácia contra linhagens celulares malignas de próstata, mama e pulmão, além de efeito antifúngico.
[025] A fifth and last modality would correspond to the use of synthesized compounds in the manufacture of drugs aimed at treating neopiasias. Drugs that present, concomitantly, little toxicity to healthy human cells, high efficacy against malignant cell lines of prostate, breast and lung, in addition to an antifungal effect.
[026] As figuras a seguir descritas servem de orientação sobre a presente invenção, as quais ilustram: [026] The figures described below serve as a guideline for the present invention, which illustrate:
- Figura 1 - Estruturas dos ligantes binucleantes inéditos H3L1 (A) e HsL2 (B). - Figure 1 - Structures of novel binucleant ligands H 3 L1 (A) and HsL2 (B).
- Figura 2 - Estruturas propostas para os novos complexos 1 (A) e 2 (B).- Figure 2 - Proposed structures for the new complexes 1 (A) and 2 (B).
- Figura 3 - Estruturas propostas para os novos complexos 3 (A) e 4 (B).- Figure 3 - Proposed structures for the new complexes 3 (A) and 4 (B).
- Figura 4 - Ensaio de sobrevivência das células de linhagens tumorais humanas MCF-7, A549 e PC-3 tratadas por 24 h na presença dos complexos 1 (A) e 3 (B); linhagem não tumoral humana, MGF10A, tratada com os complexos 1 e 3 (C) e; linhagens celulares MCF-7, A549, PC-3 e
MCF10A submetidas a tratamento com o metaloíármaco antitumoral cisplatina, usado como referência. - Figure 4 - Survival assay of cells from human tumor lines MCF-7, A549 and PC-3 treated for 24 h in the presence of complexes 1 (A) and 3 (B); human non-tumor lineage, MGF10A, treated with complexes 1 and 3 (C) and; cell lines MCF-7, A549, PC-3 and MCF10A submitted to treatment with the antitumor metallo drug cisplatin, used as reference.
- Figura 5 - Formação das EROs em células MCF-7 (A-C) e A549 (D-F) tratadas com os complexos 1 e 3, e a cisplatina. - Figure 5 - Formation of ROS in MCF-7 (A-C) and A549 (D-F) cells treated with complexes 1 and 3, and cisplatin.
- Figura 6 - Avaliação do potencial da membrana mitocondrial em células MCF-7 (A-D) e A549 (E-H) tratadas com os complexos 1 (A, B, E e F) e 3 (C, D, G e H), e com o metaloíármaco antitumoral cisplatina. - Figure 6 - Evaluation of mitochondrial membrane potential in MCF-7 (AD) and A549 (EH) cells treated with complexes 1 (A, B, E and F) and 3 (C, D, G and H), and with the antitumor metallo drug cisplatin.
- Figura 7 - Atividade caspase 8 e 9 em células A549 (A e B) e MCF-7 (C e D) tratadas com os complexos 1 e 3, e o metaloíármaco antitumoral cisplatina. - Figure 7 - Caspase 8 and 9 activity in A549 (A and B) and MCF-7 (C and D) cells treated with complexes 1 and 3, and the anti-tumor metallodrug cisplatin.
- Figura 8 - Análise da fragmentação do DNA (TÚNEL) em células MCF-7 (A) e A549 (B) tratadas com os complexos 1 e 3, e com a cisplatina. Histogramas (C) mostrando a população de células em apoptose. As barras correspondem à média ± erro padrão de 3 experiências independentes. - Figure 8 - Analysis of DNA fragmentation (TUNEL) in MCF-7 (A) and A549 (B) cells treated with complexes 1 and 3, and with cisplatin. Histograms (C) showing apoptotic cell population. Bars correspond to the mean ± standard error of 3 independent experiments.
- Figura 9 - Análise da inibição do ciclo celular (proliferação celular) durante o tratamento com os complexos 1 e 3 e o metaloíármaco antitumoral cisplatina em células de câncer MCF-7 (A e C) e A549 (B e C). Histogramas (C) representativos da população de células distribuídas pelo ciclo celular, correspondendo à média ± erro padrão de 3 experiências independentes. - Figure 9 - Analysis of cell cycle inhibition (cell proliferation) during treatment with complexes 1 and 3 and the anti-tumor metallodrug cisplatin in MCF-7 (A and C) and A549 (B and C) cancer cells. Representative histograms (C) of the cell population distributed throughout the cell cycle, corresponding to the mean ± standard error of 3 independent experiments.
- Figura 10 - A toxicidade do complexo 1 foi analisada em modelo invertebrado, Gaileria melionelía. (A) Curva de sobrevivência; (B) Número de larvas; (C) Número de pupas e (D) Número de mariposas. - Figure 10 - The toxicity of complex 1 was analyzed in an invertebrate model, Gaileria melionelía. (A) Survival curve; (B) Number of larvae; (C) Number of pupae and (D) Number of moths.
- Figura 11 - Rota sintética utilizada na preparação dos complexos binucleares de cobre(ll) 1 e 3, incluindo as condições de síntese e rendimentos obtidos. As etapas destacadas no quadro cinza correspondem aos processos intrínsecos referentes à síntese dos novos compostos (ligante H3L1 e complexos 1 e 3), e diferenciam a presente rota sintética de outras existentes na literatura.
- Figura 12 - Rota sintética utilizada na preparação dos complexos binucleares de cobre(ll) 2 e 4, incluindo as condições de síntese e rendimentos obtidos. As etapas destacadas no quadro cinza correspondem aos processos intrínsecos referentes à síntese dos novos compostos (ligante H2L2 e complexos 2 e 4), e diferenciam a presente rota sintética de outras existentes na literatura.
- Figure 11 - Synthetic route used in the preparation of binuclear copper(ll) 1 and 3 complexes, including the synthesis conditions and yields obtained. The steps highlighted in the gray box correspond to the intrinsic processes related to the synthesis of new compounds (H 3 L1 ligand and complexes 1 and 3), and differentiate this synthetic route from others existing in the literature. - Figure 12 - Synthetic route used in the preparation of binuclear copper(ll) 2 and 4 complexes, including the synthesis conditions and yields obtained. The steps highlighted in the gray box correspond to the intrinsic processes related to the synthesis of new compounds (H 2 L2 ligand and complexes 2 and 4), and differentiate this synthetic route from others existing in the literature.
[027] A presente invenção descreve, portanto, “Complexos binucleares de cobre(ii) com potente atividade antitumorai, composição, processo de síntese química e uso na preparação de medicamentos e no tratamento do câncer e de infecções por fungos patogênicos”, que buscam atender a crescente demanda por novos tratamentos para as neopiasias, tanto aquelas que envolvem tumores sólidos quanto leucemias, bem como de doenças fúngicas. [027] The present invention therefore describes "Binuclear copper(ii) complexes with potent antitumor activity, composition, chemical synthesis process and use in the preparation of drugs and in the treatment of cancer and infections by pathogenic fungi", which seek meet the growing demand for new treatments for neoplasms, both those involving solid tumors and leukemia, as well as fungal diseases.
[028] Maís especificamente, descreve novos compostos binucleares de cobre(ll), diferentes daqueles existentes no estado da técnica, pois apresentam alterações quanto à estrutura química, atividade tóxica contra células de câncer humanas e, ao mesmo tempo, menor toxicidade às células não cancerígenas do organismo. Assim, mostram uma maior eficácia e menores efeitos colaterais, oferecendo ao paciente submetido ao tratamento quimioterápico contra o câncer maior qualidade de vida e sobrevida. [028] More specifically, it describes new binuclear copper(ll) compounds, different from those existing in the prior art, as they present changes in the chemical structure, toxic activity against human cancer cells and, at the same time, less toxicity to non- carcinogens in the body. Thus, they show greater efficacy and fewer side effects, offering patients undergoing chemotherapy for cancer a better quality of life and survival.
[029] Os compostos de coordenação binucleares de cobrei! i) e ligantes não- simétricos preferidos da presente invenção, são: 2-hidroxi-3-{[(2-hidroxibenzil)(2- píridilmetil)amino]-metii}-5-metilbenzaldeído isonicotinoíl hidrazona (H3L1 ) conforme formula estrutural abaixo e ilustrado na figura 1 e 2-hidroxi-3-{bis[(2- piridilmetil)amino]-metil}-5-metilbenzaldeído isonicotinoíl hidrazona (H2L2) conforme formula estrutural abaixo e ilustrado na figura 1 , assim como os seus respectivos complexos binucleares de cobre(ll) contendo pontes exógenas do tipo acetato: composto 1 e composto 2 conforme as formulas estruturais abaixo e ilustrados na figura 2 ou hidroxo: composto 3 e composto 4 conforme as formulas estruturais abaixo e ilustrados na figura 3.
[029] The binuclear copper coordination compounds! i) and preferred non-symmetric linkers of the present invention are: 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L 1 ) as per formula structural below and illustrated in figure 1 and 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2) as structural formula below and illustrated in figure 1 , as well as their respective binuclear copper(II) complexes containing exogenous acetate-type bridges: compound 1 and compound 2 according to the structural formulas below and shown in figure 2 or hydrox: compound 3 and compound 4 according to the structural formulas below and illustrated in figure 3 .
[030] A escolha do metal teve como principal objetivo minimizar a toxicidade ao organismo, tratando-se o cobre, a diferença da platina, de um metal fisiológico encontrado em diversas metaloenzimas presentes nas células humanas. [030] The choice of metal had as main objective to minimize the toxicity to the organism, being copper, unlike platinum, a physiological metal found in several metalloenzymes present in human cells.
[031] Neste sentido, a Figura 4 representa graficamente a citotoxicidade dos complexos de cobre(ll) e cisplatina isolados nas linhagens celulares de câncer
de pulmão (A549), de mama (MCF-7) e de próstata (PC-3). A citotoxicidade foi avaliada através do ensaio MTT em células tumorais tratadas por 24 horas na presença dos novos complexos binucleares de cobre, 1 (A) e 3 (B), na linhagem MCF10A (controle de célula humana não tumoral) tratada com os complexos 1 e 3 (C), e nas células MCF-7, A549, PC-3 e MCF10A tratadas com cisplatina (D). Os resultados foram calculados a partir de experiências realizadas em triplicata (n=3) e são representados como média ± erro padrão da média, [031] In this sense, Figure 4 graphically represents the cytotoxicity of copper(ll) and cisplatin complexes isolated in cancer cell lines lung (A549), breast (MCF-7) and prostate (PC-3). Cytotoxicity was evaluated using the MTT assay in tumor cells treated for 24 hours in the presence of new binuclear copper complexes, 1 (A) and 3 (B), in the MCF10A (non-tumor human cell control) lineage treated with complexes 1 and 3 (C), and in MCF-7, A549, PC-3 and MCF10A cells treated with cisplatin (D). Results were calculated from experiments performed in triplicate (n=3) and are represented as mean ± standard error of the mean,
[032] Os resultados das curvas de sobrevivência foram usados para determinar a concentração necessária para matar 50% das células (IC50) tratadas com os complexos de cobre e cisplatina. A atividade citotóxica dos complexos para as células de origem tumoral foi cerca de 10-30 vezes superior às encontradas para a cisplatina (Figura 1 e Tabela 1), A linhagem MCF10A, célula epitelial de mama (célula não cancerígena) foi aproximadamente 7 e 8 vezes menos sensível aos tratamentos com os complexos de cobre, respectivamente, do que a linhagem de câncer de mama (MCF-7) (Figura 1 e Tabela 1), [032] The results of the survival curves were used to determine the concentration needed to kill 50% of the cells (IC50) treated with the copper and cisplatin complexes. The cytotoxic activity of the complexes for cells of tumor origin was about 10-30 times higher than those found for cisplatin (Figure 1 and Table 1), The MCF10A lineage, breast epithelial cell (non-cancer cell) was approximately 7 and 8 times less sensitive to treatments with copper complexes, respectively, than the breast cancer strain (MCF-7) (Figure 1 and Table 1),
Tabela 1. Valores de IC50 (μ M) nas linhagens MCF-7, A549, PC-3 e MCF10A tratadas com os complexos, doravante denominados, 1, 2, 3 e 4 por 24 h. As atividades do lígante H3L1 (presente nos complexos 1 e 3), assim como do sal de partida usado nas sínteses dos complexos e a da metalodroga comercial cisplatina foram incluídas para efeito de comparação.
[033] Em relação ao mecanismo de ação na qual se baseia a atividade destes novos complexos, observa-se que a Figura 5 representa graficamente a indução da produção de espécies reativas de oxigénio (ou EROs), um fator crítico para a indução do processo endógeno de apoptose em células de câncer, durante o tratamento com os novos complexos 1 e 3, e com o quimioterápico usado como controle do experimento, o qual também é aplicado na terapêutica do câncer, doxorubicina. O tratamento com a metade do valor de IC50 dos complexos 1 e 3 já é capaz de provocar o aumento da indução da produção das EROs. A Figura 5 mostra os resultados representativos de uma série de, no mínimo, três experimentos independentes realizados nas linhagens MCF-7 (A, B e C) e A549 (D, E e F) e representa a média ± erro padrão da média. Os símbolos (#) e (***) representam diferença estatística em relação aos tratamentos de controle. Table 1. IC 50 values (μ M) in MCF-7, A549, PC-3 and MCF10A strains treated with the complexes, hereinafter referred to as 1, 2, 3 and 4 for 24 h. The activities of the ligand H 3 L1 (present in complexes 1 and 3), as well as the starting salt used in the syntheses of the complexes and that of the commercial metallodrug cisplatin were included for comparison purposes. [033] Regarding the mechanism of action on which the activity of these new complexes is based, it is observed that Figure 5 graphically represents the induction of the production of reactive oxygen species (or ROS), a critical factor for the induction of the process endogenous apoptosis in cancer cells, during treatment with the new complexes 1 and 3, and with the chemotherapeutic used as control of the experiment, which is also applied in cancer therapy, doxorubicin. Treatment with half the IC 50 value of complexes 1 and 3 is already capable of increasing the induction of ROS production. Figure 5 shows the representative results of a series of at least three independent experiments performed on MCF-7 (A, B and C) and A549 (D, E and F) lines and represents the mean ± standard error of the mean. The symbols (#) and ( *** ) represent a statistical difference in relation to the control treatments.
[034] A Figura 6 representa graficamente a alteração/perda do potencial de membrana da mitocôndria (ΔΨm), alvo das espécies reativas de oxigénio e primeira etapa na indução do processo endógeno de apoptose, durante o tratamento de células de câncer com os novos complexos de cobre 1 e 3, e o metaloquimioterápico usado na terapêutica do câncer, cisplatina. Nessa figura, é apresentada uma representação gráfica do nível de potencial mitocondrial da membrana, expresso como unidades arbitrárias de fluorescência nas linhagens MCF-7 (Figura 6Â e C) e A549 (Figura 6E e G). Na Figura 6 também podemos observar, em forma de barras, a média ± erro padrão da média dos valores de DYhi em unidades arbitrárias de fluorescência de uma série de, no mínimo, três experimentos independentes realizados para as linhagens MCF-7 (Figura 6B e D) e A549 (Figura F e H). O símbolo (*) representa diferença estatística em relação aos tratamentos de controle. [034] Figure 6 graphically represents the alteration/loss of the mitochondrial membrane potential (ΔΨm), target of reactive oxygen species and the first step in inducing the endogenous process of apoptosis, during the treatment of cancer cells with the new complexes copper 1 and 3, and the metallochemotherapeutic used in cancer therapy, cisplatin. This figure presents a graphical representation of the level of mitochondrial membrane potential, expressed as arbitrary fluorescence units in the MCF-7 (Figure 6Â and C) and A549 (Figure 6E and G) lines. In Figure 6 we can also see, in bar form, the mean ± standard error of the mean of DYhi values in arbitrary fluorescence units from a series of at least three independent experiments performed for the MCF-7 lines (Figure 6B and D) and A549 (Figure F and H). The symbol ( * ) represents statistical difference in relation to control treatments.
[035] A Figura 7, por sua vez, representa graficamente a ativação das enzimas caspase 8 (Figura 7A e C) e 9 (Figura 7B e D), as quais são chave na ativação do processo de apoptose, durante o tratamento das células de câncer com os complexos 1 e 3 e o quimioterápico antitumoral cisplatina. Os resultados obtidos são expressos como média ± erro padrão da média de, no mínimo, três
experiências independentes e são mostrados como uma relação entre a atividade de células tratadas com os novos complexos 1 e 3 ou cisplatina e células não tratadas. O símbolo (*) representa diferença estatística em relação às células não tratadas com os compostos de cobre ou cisplatina. [035] Figure 7, in turn, graphically represents the activation of caspase enzymes 8 (Figure 7A and C) and 9 (Figure 7B and D), which are key in the activation of the apoptosis process, during cell treatment of cancer with complexes 1 and 3 and the anti-tumor chemotherapy cisplatin. The results obtained are expressed as mean ± standard error of the mean of at least three independent experiments and are shown as a relationship between the activity of cells treated with the novel complexes 1 and 3 or cisplatin and untreated cells. The symbol ( * ) represents statistical difference from cells not treated with copper or cisplatin compounds.
[036] A Figura 8 representa graficamente a indução do processo de apoptose através da análise da quebra / fragmentação do DNA das células de câncer pela técnica TUNEL [Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling] durante tratamento com os novos complexos 1 e 3, e o quimioterápico antitumoral cisplatina. Na Figura 8 (A e B) podem ser observados histogramas representativos TUNEL mostrando a porcentagem de células em apoptose após o tratamento com cisplatina ou com os complexos de cobre(ll) nas linhagens A549 (A) e MCF-7 (B), respectivamente. A Figura 8C, por sua vez, representa a média ± erro padrão da média de, no mínimo, três experiências independentes com células A549 e MCF-7 tratadas com cisplatina ou com os complexos de cobre(ll), O símbolo (*) representa diferença estatística em relação às células não tratadas com os compostos de cobre ou cisplatina. [036] Figure 8 graphically represents the induction of the apoptosis process through the analysis of DNA breakage / fragmentation of cancer cells by the TUNEL technique [Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling] during treatment with the new complexes 1 and 3, and the anti-tumor chemotherapy cisplatin. In Figure 8 (A and B) representative TUNEL histograms can be seen showing the percentage of cells in apoptosis after treatment with cisplatin or copper(ll) complexes in the A549 (A) and MCF-7 (B) lines, respectively . Figure 8C, in turn, represents the mean ± standard error of the mean of at least three independent experiments with A549 and MCF-7 cells treated with cisplatin or copper(II) complexes. The symbol ( * ) represents statistical difference in relation to cells not treated with copper or cisplatin compounds.
[037] Na Tabela 2, analisamos a competição pela ligação intercalativa ao DNA entre os complexos 1 e 3 e a sonda fluorescente iodeto de propídio, que é capaz de se intercalar às bases nucieotídicas do DNA. [037] In Table 2, we analyzed the competition for intercalative binding to DNA between complexes 1 and 3 and the fluorescent probe propidium iodide, which is capable of intercalating to the nucleotide bases of DNA.
Tabela 2, Competição entre 1 / 3 e iodeto de propídio (PI) na ligação ao DNA.
[038] A Figura 9, por outro lado, representa a análise da inibição do ciclo celular (proliferação celular) durante tratamento com os complexos 1 e 3 em células de câncer A549 e MCF-7. Na Figura 9 (A e B), apresentamos histogramas representativos da distribuição das fases do ciclo celular, demarcadas como Sub-G0/G1 , G0/G1 , S e G2/M (de esquerda para direita) em células A549 e MCF-7 tratadas com os complexos 1 e 3, e a cisplatina, respectivamente. Na Figura 9 (C), observamos a média ± erro padrão da média de, no mínimo, três experiências independentes de contagem da população de células em Sub- G0/G1 , fase que indica que as células estão em processo de apoptose causado pelo tratamento com cisplatina ou com os complexos. O símbolo (*) representa diferença estatisticamente significativa em relação ao controle, células não tratadas com cisplatina ou com os compostos de cobre. O símbolo (#) representa diferença estatística em relação aos tratamentos com cisplatina. Table 2, Competition between 1/3 and propidium iodide (PI) in DNA binding. [038] Figure 9, on the other hand, represents the analysis of cell cycle inhibition (cell proliferation) during treatment with complexes 1 and 3 in A549 and MCF-7 cancer cells. In Figure 9 (A and B), we present histograms representative of the distribution of cell cycle phases, demarcated as Sub-G0/G1 , G0/G1 , S and G2/M (from left to right) in A549 and MCF-7 cells treated with complexes 1 and 3, and cisplatin, respectively. In Figure 9 (C), we observe the mean ± standard error of the mean of at least three independent cell population count experiments in Sub-G0/G1, a phase that indicates that the cells are undergoing apoptosis process caused by the treatment. with cisplatin or with the complexes. The symbol ( * ) represents a statistically significant difference compared to control, cells not treated with cisplatin or copper compounds. The symbol (#) represents statistical difference in relation to treatments with cisplatin.
[039] A Figura 10 representa graficamente a toxicidade do complexo 1 e do quimioterápico usado na terapêutica do câncer, cisplatina em modelo animal alternativo, Galeria melloneiía. O experimento foi realizado pela injeção de 1 ou cisplatina, ambos nas mesmas concentrações (1 ,0 mg/mL), diretamente na hemocele das larvas de G. melloneiía. A notar, o complexo não apresentou toxicidade e também não interferiu nas fases de desenvolvimento (larva, pupa e mariposa) do modelo animal alternativo empregado no estudo, mostrando um perfil significativamente diferente daquele observado para o tratamento com o quimioterápico usado no tratamento do câncer, cisplatina. [039] Figure 10 graphically represents the toxicity of complex 1 and the chemotherapeutic used in cancer therapy, cisplatin in an alternative animal model, Galeria melloneiía. The experiment was performed by injecting 1 or cisplatin, both at the same concentrations (1.0 mg/mL), directly into the hemocele of G. melloneiía larvae. It should be noted that the complex did not show toxicity and did not interfere with the developmental stages (larva, pupa and moth) of the alternative animal model used in the study, showing a significantly different profile from that observed for treatment with the chemotherapy used in cancer treatment, cisplatin.
[040] Quanto ao processo de síntese, os complexos binucleares são resultado de uma rota preparatória bastante complexa (Figura 11), desenvolvida na busca por novos agentes com maior eficácia para eliminar ou reduzir a proliferação das células tumorais. Neste contexto, alguns trabalhos sobre compostos binucleares de cobre com uma diversa gama de aplicações já foram publicados em revistas científicas internacionais. No entanto, em todos eles, as estruturas apresentadas são diferentes das que estamos propondo no presente estudo.
[041] Destes trabalhos, iremos destacar duas referências, discutidas aqui de forma mais aprofundada, complementando assim os antecedentes da invenção. Embora possam parecer semelhantes, esses documentos sâo essencialmente distintos no que tange à estrutura dos ligantes utilizados, eficácia contra as células de câncer humano e seletividade. [040] As for the synthesis process, binuclear complexes are the result of a very complex preparatory route (Figure 11), developed in the search for new agents with greater effectiveness to eliminate or reduce the proliferation of tumor cells. In this context, some works on binuclear copper compounds with a diverse range of applications have already been published in international scientific journals. However, in all of them, the structures presented are different from the ones we are proposing in this study. [041] From these works, we will highlight two references, discussed in more depth here, thus complementing the background of the invention. Although they may appear similar, these documents are essentially different in terms of the structure of the ligands used, effectiveness against human cancer cells, and selectivity.
[042] A primeira delas é a referência publicada por Rey, et ai. em 2009 [J„ Inorg. Biochem, 103 (2009) 1323-1330], sendo o primeiro autor pertencente ao grupo de inventores da presente invenção. A publicação também explora um composto binuclear de cobre(il) contendo ponte exógena do tipo /i-hidroxo como agente antitumoral. Entretanto, existem importantes diferenças entre esse composto e aqueles alvos da presente invenção, diferenças essas responsáveis pela maior atividade dos novos compostos em comparação ao da publicação. [042] The first of these is the reference published by Rey, et al. in 2009 [Jn Inorg. Biochem, 103 (2009) 1323-1330], being the first author belonging to the group of inventors of the present invention. The publication also explores a binuclear copper(IL) compound containing exogenous i-hydroxy type bridge as an antitumor agent. However, there are important differences between this compound and those targets of the present invention, these differences being responsible for the greater activity of the new compounds compared to the publication.
[043] A maior diferença, e, portanto, a mais digna de nota, é certamente o caráter não~slmétrfco dos ligantes nos complexos a serem patenteados, em contraposição à base de Schsff simétrica derivada do ligante tridentado facial 6-amino-6-metilperidro~1 ,4-díazepína utilizada como precursor na publicação. [044] Ambos os ligantes descritos neste relatório técnico são não-simétricos e contêm um “braço pendente” hidrazônico como característica mais marcante. É precisamente essa característica estrutural diferenciada que torna os compostos de coordenação resultantes desta invenção de 10 até 30 vezes mais ativos que a císplatina, frente às linhagens celulares MCF-7, PC-3 e A549, enquanto que o anterior (publicação de 2009) é 37 vezes menos ativo que essa droga de referência na linhagem de leucemia testada, K562. [043] The biggest difference, and therefore the most noteworthy, is certainly the non-slmetric character of the ligands in the complexes to be patented, as opposed to the symmetric Schsff base derived from the 6-amino-6-facial tridentate ligand methylperhydro-1,4-diazepine used as precursor in the publication. [044] Both of the ligands described in this technical report are non-symmetric and contain a hydrazonic “pendant arm” as their most striking feature. It is precisely this differentiated structural characteristic that makes the coordination compounds resulting from this invention 10 to 30 times more active than cisplatin, against cell lines MCF-7, PC-3 and A549, while the previous one (publication of 2009) is 37 times less active than this reference drug in the tested leukemia strain, K562.
[045] Por outro lado, com relação à recente publicação [Inorg, Chem, 58 (2019) 8800-8819], também assinada pelo Prof. Nicolás A. Rey, destacamos: apesar de ambos os ligantes reportados neste artigo serem não-simétricos e possuírem também um “braço” hidrazônico, os grupos substituintes presentes na função hidrazona são diferentes dos presentes nos compostos da presente invenção. Enquanto na publicação (2019) têm-se os anéis substituintes heteroaromáticos de cinco membros 2-tiofeno e 2-furano, os compostos da presente invenção
apresentam uma vantagem, a qual é caracterizada pelo anel de seis membros 4-píridina, cuja presença decorre do uso da isoμíazida, conhecido agente anti- tuberculose, como bídrazida de partida no preparo dos novos ligantes. [045] On the other hand, with regard to the recent publication [Inorg, Chem, 58 (2019) 8800-8819], also signed by Prof. Nicolás A. Rey, we emphasize: although both ligands reported in this article are non-symmetric and also have a hydrazonic “arm”, the substituent groups present in the hydrazone function are different from those present in the compounds of the present invention. While in the publication (2019) the 5-membered heteroaromatic substituent rings 2-thiophene and 2-furan are given, the compounds of the present invention have an advantage, which is characterized by the six-membered 4-pyridine ring, whose presence results from the use of isomyazide, known as an anti-tuberculosis agent, as a starting hydrazide in the preparation of new ligands.
[046] Esta mudança estrutural causa um impacto direto sobre a atividade dos próprios ligantes orgânicos, assim como sobre o índice de seletividade dos complexos metálicos. Desta forma, sendo a isoniazída um fármaco comercial, a sua toxicidade é reiativamente baixa em relação àquelas das hidrazidas contendo anéis tiofênicos e furánicos. Por esse motivo, enquanto os ligantes derivados da isoniazida não se mostram muito ativos per se, os ligantes descritos no trabalho de 2019 são bastante citotóxicos contra diversas linhagens de células de câncer. Porém, essa atividade intrínseca dos ligantes tiofênicos e furánicos está associada a uma elevada toxicidade dos complexos metálicos, o que leva à queda no índice de seletividade. Desta maneira, os complexos binucleares de cobre(li) da presente invenção possuem índices de seletividade entre 5 e 8, enquanto aqueles publicados anteriormente têm índices da ordem de 1-2, o que constitui uma vantagem para os novos compostos. [046] This structural change causes a direct impact on the activity of the organic ligands themselves, as well as on the selectivity index of metal complexes. Thus, since isoniazid is a commercial drug, its toxicity is relatively low compared to that of hydrazides containing thiophenic and furanic rings. For this reason, while isoniazid-derived ligands are not very active per se, the ligands described in the 2019 work are quite cytotoxic against several cancer cell lines. However, this intrinsic activity of thiophenic and furanic ligands is associated with a high toxicity of metal complexes, which leads to a drop in the selectivity index. Thus, the binuclear copper(II) complexes of the present invention have selectivity indices between 5 and 8, while those previously published have indices of the order of 1-2, which is an advantage for the new compounds.
[047] Além disso, enquanto o artigo trata apenas de compostos com ponte exógena μ- bidroxo, o presente relatório técnico incorpora também complexos contendo uma ou duas pontes do tipo μ- acetato. Estas variantes podem ser consideradas pró-drogas, que manteriam a atividade em relação aos complexos com ponte μ-hidroxo mas seriam menos tóxicas para as células normais do organismo, uma vez que poderiam ser ativadas in situ, como pode ser visto nos resultados obtidos para a linhagem normal de mama humana MCF10A. [047] Furthermore, while the article deals only with exogenous μ-hydroxy bridged compounds, the present technical report also incorporates complexes containing one or two μ-acetate type bridges. These variants can be considered pro-drugs, which would maintain their activity in relation to μ-hydroxy bridged complexes but would be less toxic to normal cells of the organism, since they could be activated in situ, as can be seen in the results obtained for the normal human breast lineage MCF10A.
[048] Finalmente, nas publicações descritas acima, os autores não mostraram o mecanismo de ação pelo qual os compostos provocam a morte das células (ex. apoptose), o que poderia constituir uma desvantagem dos compostos publicados caso a morte celular induzida seja por necrose. Na presente invenção, devido à alta citotoxicidade e indução de grande produção de EROs e perda significativa do DYhi, acompanhada da ativação da caspase 9 e fragmentação de DNA, apresentamos compostos que causam claramente a
morte celular por apoptose, através da via intrínseca, dependente de sinais provenientes da mitocondria. Dados proteômicos obtidos pelo grupo do presente estudo demonstram que a ativação direta da apoptose ocorre através do fator indutor de apoptose (AIF), por meio da regulação negativa da via RAS-ERK, [049] A regulação negativa da caseína cinase, calpaína, bem como das subunidades catalíticas e reguladoras do proteossomo, também promovem a ativação das vias de degradação do DNÂ. Além disso, a regulação negativa da enzima glicolítica piruvato cinase, indica uma regulação negativa da glicólise, forçando as células tumorais a realizarem o metabolismo respiratório, através da fosforilação oxidativa. Estes resultados permitem considerar que 1 e 3 são potenciais agentes terapêuticos contra câncer de mama, pulmão e próstata. [050] De forma a melhor ilustrar as formas possíveis e preferidas da presente invenção, são apresentados exemplos dos diferentes processos de síntese envolvendo os ligantes binucleantes não-simétricos e os respectivos complexos de coordenação binucleares obtidos a partir deles.
[048] Finally, in the publications described above, the authors did not show the mechanism of action by which the compounds cause cell death (eg apoptosis), which could constitute a disadvantage of the published compounds if the cell death induced is by necrosis . In the present invention, due to the high cytotoxicity and induction of large ROS production and significant loss of DYhi, accompanied by caspase 9 activation and DNA fragmentation, we present compounds that clearly cause the cell death by apoptosis, through the intrinsic pathway, dependent on signals from the mitochondria. Proteomic data obtained by the group of the present study demonstrate that the direct activation of apoptosis occurs through the apoptosis-inducing factor (AIF), through the down-regulation of the RAS-ERK pathway, [049] The down-regulation of casein kinase, calpain, well as the catalytic and regulatory subunits of the proteosome, they also promote the activation of DNA degradation pathways. Furthermore, the down-regulation of the glycolytic enzyme pyruvate kinase indicates a down-regulation of glycolysis, forcing tumor cells to carry out respiratory metabolism through oxidative phosphorylation. These results allow us to consider that 1 and 3 are potential therapeutic agents against breast, lung and prostate cancer. [050] In order to better illustrate the possible and preferred forms of the present invention, examples are presented of the different synthesis processes involving the non-symmetrical binucleating ligands and the respective binuclear coordination complexes obtained from them.
[051] Este ligante não-simétrico foi preparado a partir do intermediário sintético hbpamff, isto é, do composto 2-hidroxi-3-{[(2-hidroxibenzil)(2-piridilmetil)amino]- metil}-5-metilbenzaldeído. Uma solução metanólica (5 mL) da hidrazida do ácido isonicotínico, ssonsazida (0,138 g; 1 mmoi) foi gotejada, sob agitação constante, sobre uma solução de hbpamff (0,333 g; 0,9 mmol) em 10 mL de uma mistura 1 :1 MeOH:Et20, previamente preparada num balão de 50 mL. A mistura foi refluxada por 2 horas. Durante esse período, a solução reacional foi adquirindo progressivamente uma coloração castanha. Ao término da reação, transferiu-se 0 conteúdo do balão para um béquer de 50 mL e deixou-se 0 mesmo em repouso para que, com a evaporação gradual do solvente, 0 produto pudesse precipitar. Dessa maneira, foi isolada uma grande quantidade de cristais castanhos. Este material foi filtrado em papel e lavado com éter etílico gelado.
[052] Obtiveram-se 0,398 g [~0,8 mmol; 481 ,6 g mol·'; rendimento de 90%) de produto, na forma de pequenos monocristais de cor castanha adequados ao processo de determinação estrutural por difraçâo de raios X. p.f.: 200 °C. Análise elementar - Porcentagens encontradas: C 89,6; H 5,9; N 14,5. Porcentagens calculadas com base na formula C28H27O3N5: C 89,8; H 5,7; N 14,5. A fórmula estrutura! correspondente ao ligante H3L1 pode ser observada na Figura 1A e a representação simplificada do processo de síntese, na Figura 11.
[051] This non-symmetric linker was prepared from the synthetic intermediate hbpamff, that is, from the compound 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde. A methanolic solution (5 ml) of the isonicotinic acid hydrazide, ssonsazide (0.138 g; 1 mmol) was dropped, under constant stirring, onto a solution of hbpamff (0.333 g, 0.9 mmol) in 10 ml of a mixture 1: 1 MeOH:Et 2 0, previously prepared in a 50 ml flask. The mixture was refluxed for 2 hours. During this period, the reaction solution gradually acquired a brown color. At the end of the reaction, the contents of the flask were transferred to a 50 mL beaker and left to rest so that, with the gradual evaporation of the solvent, the product could precipitate. In this way, a large amount of brown crystals was isolated. This material was filtered through paper and washed with ice-cold ethyl ether. [052] 0.398 g [~0.8 mmol; 481.6 g mol·'; 90% yield) of product, in the form of small brown colored single crystals suitable for the process of structural determination by X-ray diffraction. mp: 200 °C. Elementary analysis - Percentages found: C 89.6; H 5.9; No 14.5. Percentages calculated based on the formula C28H27O3N5: C 89.8; H 5.7; No 14.5. The structure formula! corresponding to the H 3 L1 ligand can be seen in Figure 1A and the simplified representation of the synthesis process in Figure 11.
[053] Este ligante foi preparado a partir do intermediário sintético bpmamff, isto é, 2-hidroxi-3-{bis[(2-piridilmetil)amino]-metil}-5-metilbenzaldeído. A isoniazida, previamente dissolvida em 5 mL de uma mixtura (v/v) 1 :1 diclorometano/MeOH (0,277 g; 2 mmol) foi lentamente gotejada sobre uma solução contendo bpmamff (0,703 g; 2 mmol) dissolvido em 10 mL da mesma mistura usando ultrassom. A solução (balão de 50 mL) foi então levada a refluxo por 3 horas. Ao longo da reação, a mistura, inicialmente amarela brilhante, tornou-se laranja. O solvente foi subsequentemente removido sob pressão reduzida e 0 óleo obtido, deixado em repouso em 1 :1 MeCN/MeOH. Após duas semanas, foram isoladas agulhas microcristalinas de cor amarelo-clara. [053] This linker was prepared from the synthetic intermediate bpmamff, ie, 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde. Isoniazid, previously dissolved in 5 ml of a mixture (v/v) 1:1 dichloromethane/MeOH (0.277 g, 2 mmol) was slowly dropped onto a solution containing bpmamff (0.703 g, 2 mmol) dissolved in 10 ml of the same mixing using ultrasound. The solution (50 ml flask) was then refluxed for 3 hours. Over the course of the reaction, the initially bright yellow mixture turned orange. The solvent was subsequently removed under reduced pressure and the oil obtained, left to stand in 1:1 MeCN/MeOH. After two weeks, light yellow microcrystalline needles were isolated.
[054] Obtiveram-se 0,957 g (~2 mmol; 466,53 g mol-1; rendimento quantitativo) do produto, na forma cristalina, p.f.: 133 °C. Análise elementar - Porcentagens encontradas: C 69,2; H 6,1 ; N 18,7, Porcentagens calculadas com base na formula C27H26O2N6: C 69,5; H 5,6; N 18,0. A fórmula estrutural correspondente ao ligante H2L2 encontra-se na Figura 1B e a representação simplificada do processo de síntese pode ser observada na Figura 12. [054] 0.957 g (~2 mmol; 466.53 g mol -1 ; quantitative yield) of the product was obtained in crystalline form, mp: 133 °C. Elementary analysis - Percentages found: C 69.2; H 6.1; N 18.7, Percentages calculated based on the formula C 27 H 26 O 2 N 6 : C 69.5; H 5.6; N 18.0. The structural formula corresponding to the H 2 L2 ligand is shown in Figure 1B and the simplified representation of the synthesis process can be seen in Figure 12.
[055] Os complexos binucleares não-simétricos de cobre(li) compreendidos na presente invenção podem ser sintetizados pelas seguintes etapas de processo: 1. dissolução de uma quantidade do ligante binucleante, H3L1 ou H2L2, em um solvente orgânico puro ou mistura de solventes;
2. adição lenta de 2 equivalentes de sal de cobre(ll), previamente dissolvido em metanol ou em uma mistura de solventes. O contra-íon presente neste sal pode ser coordenante (como, por exemplo, acetato, propionato ou, mais genericamente, um ânion carboxilato capaz de gerar uma pró-droga) ou não (assim como perclorato, baleio, dentre outros contra-íons capazes de gerar uma droga final na sua forma ativa); [055] The non-symmetrical binuclear copper(li) complexes comprised in the present invention can be synthesized by the following process steps: 1. dissolution of an amount of the binucleating ligand, H 3 L1 or H 2 L2, in a pure organic solvent or mixture of solvents; 2. Slow addition of 2 equivalents of copper(II) salt, previously dissolved in methanol or a mixture of solvents. The counterion present in this salt can be coordinating (such as, for example, acetate, propionate or, more generally, a carboxylate anion capable of generating a prodrug) or not (as well as perchlorate, whale, among other capable counterions to generate a final drug in its active form);
3. a mistura reacional é então agitada, sob aquecimento moderado, por um período que pode variar de 10 minutos até 24 horas - entende-se aqui por aquecimento moderado o uso de uma temperatura menor que o ponto de ebulição do solvente ou mistura de solventes utilizada; 3. the reaction mixture is then stirred, under moderate heating, for a period that may vary from 10 minutes to 24 hours - moderate heating is understood here as the use of a temperature lower than the boiling point of the solvent or mixture of solvents used;
4. caso o sal de cobre usado não possua como contra-íon uma espécie coordenante, adiciona-se uma quantidade adequada de base, dissolvida em água ou metanol, com o objetivo de favorecer a formação da ponte exógena do tipo m-hidroxo; e, por fim, 4. if the copper salt used does not have a coordinating species as a counterion, an adequate amount of base is added, dissolved in water or methanol, in order to favor the formation of an exogenous m-hydroxy type bridge; and finally,
5. o sólido obtido é filtrado, lavado com solvente orgânico gelado e seco no vácuo. Uma maior massa de produto e, eventualmente, cristais adequados ao processo de determinação estrutural por dif ração de raios X podem ser obtidos pela evaporação lenta da água-mãe. 5. the solid obtained is filtered, washed with ice-cold organic solvent and dried in a vacuum. A greater mass of product and, eventually, crystals suitable for the process of structural determination by X-ray diffraction can be obtained by the slow evaporation of the mother liquor.
Exemplo 3: COMPLEXOS sintetizados utilizando ACETATOExample 3: COMPLEXES synthesized using ACETATE
Síntese - [Cu2(μ- CH3COO)(OH2)(L1)]- 11/2 H2O, composto 1: Synthesis - [Cu 2 (μ- CH 3 COO)(OH 2 )(L1)]- 1 1 /2 H 2 O, compound 1:
[056] Num balão de reação de 50 mL, foram dissolvidos 0,127 g (0,26 mmol) de H3L1 em aproximados 10 mL de MeOH. A essa solução, adicionou-se, gota a gota, sob agitação constante, uma solução metanólica (20 mL) contendo 0,12 g (0,58 mmol) do sal acetato de cobre(ll) monoidratado, Cu(CH3COO)2-H20. O sistema reacional permaneceu em aquecimento por 40 minutos e, ao término desse período, 0 aquecimento foi interrompido, mantendo-se apenas a agitação por mais 20 minutos. Durante esse período, verificou-se que houve mudança de coloração, do amarelo inicial, passando pela cor laranja, verde e, por fim, verde escuro. A solução foi filtrada em papel e transferida para um bécher de 50 mL,
para que o solvente fosse completamente evaporado por aquecimento a 60 °C. O sólido resultante foi lavado com metanol gelado, filtrado em funil de Buchner e guardado sob vácuo em dessecador contendo sílica. [056] In a 50 mL reaction flask, 0.127 g (0.26 mmol) of H 3 L1 was dissolved in approximately 10 mL of MeOH. To this solution was added dropwise, under constant stirring, a methanolic solution (20 mL) containing 0.12 g (0.58 mmol) of the monohydrated copper(II) acetate salt, Cu(CH 3 COO) 2 -H 2 0. The reaction system remained under heating for 40 minutes and, at the end of this period, heating was interrupted, with only stirring being maintained for another 20 minutes. During this period, it was found that there was a change in color, from the initial yellow, passing through the color orange, green and, finally, dark green. The solution was filtered through paper and transferred to a 50 mL beaker, so that the solvent was completely evaporated by heating to 60 °C. The resulting solid was washed with ice-cold methanol, filtered on a Buchner funnel and stored under vacuum in a desiccator containing silica.
[057] Obtiveram-se 0,189 g (-0,25 mmol; 709,47 g mol·1; rendimento de 98%) do produto 1 , na forma de um sólido verde. Análise elementar - Porcentagens encontradas: C 49,4; H 4,4; N 8,9; Cu 19,8. Calculadas com base na fórmula
estrutura proposta para este composto encontra-se na Figura 2A. [057] 0.189 g (-0.25 mmol; 709.47 g mol· 1 ; 98% yield) of product 1 was obtained as a green solid. Elementary analysis - Percentages found: C 49.4; H 4.4; N 8.9; Cu 19.8. Calculated based on formula proposed structure for this compound is shown in Figure 2A.
Síntese » composto 2:
Synthesis » compound 2:
[058] Num balão de reação de 50 mL, foram dissolvidos 0,131 g (0,26 mmol) de H2L2 em aproximados 10 mL de uma mistura 1 :1 diclorometano/MeOH. A essa solução, adicionou-se, gota a gota, sob agitação constante, uma solução (15 mL; 1 :1 diclorometano/MeOH) contendo 0,106 g (0,53 mmol) de
Imediatamente após a adição do sal de cobre, 0 sistema, inicialmente amarelo claro, tornou-se verde escuro. A mistura permaneceu em aquecimento por 40 minutos. A solução foi filtrada em papel e 0 sólido verde obtido foi lavado com metano! gelado e seco a vácuo. O produto foi mantido em dessecador com sílica. [059] Obtiveram-se 0,218 g (0,25 mmol; 871 ,84 g mol'1; rendimento de 96%) de produto. Análise elementar - Porcentagens encontradas: C 42,6; H 5,7; N 10,9; Cu 15,0. Calculadas sobre
H 5,6; N 9,6; Cu 14,6. A estrutura proposta é mostrada na Figura 2B. [058] In a 50 mL reaction flask, 0.131 g (0.26 mmol) of H 2 L2 was dissolved in approximately 10 mL of a 1:1 dichloromethane/MeOH mixture. To this solution, a solution (15 ml; 1:1 dichloromethane/MeOH) containing 0.106 g (0.53 mmol) of Immediately after the addition of the copper salt, the initially light yellow system turned dark green. The mixture remained under heating for 40 minutes. The solution was filtered through paper and the green solid obtained was washed with methanol! chilled and vacuum dried. The product was kept in a desiccator with silica. [059] 0.218 g (0.25 mmol; 871.84 g mol- 1 ; yield 96%) of product were obtained. Elementary analysis - Percentages found: C 42.6; H 5.7; N 10.9; Cu 15.0. calculated on H 5.6; N 9.6; Cu 14.6. The proposed structure is shown in Figure 2B.
Exemplo 4: COMPLEXOS sintetizados utilizando HIDRÓXIDOExample 4: COMPLEXES synthesized using HYDROXIDE
Síntese - composto 3:
Synthesis - compound 3:
[969] Num balão de reação de 50 mL, foram dissolvidos 0,240 g (0,5 mmol) de HsL1 em 15 mL de MeOH. A essa solução de ligante, adicionou-se, gota a gota e sob agitação constante, outra solução, também metanólica (5 mL), contendo 0,37 g (1 ,0 mmol) de perclorato de cobre(ll) hexaidratado,
Após 10 minutos, foram lentamente gotejados 1 ,5 mL (1 ,5 mmol) de NaOH aquoso de concentração 1 mol L'1. O sistema reacional permaneceu em aquecimento por
40 minutos e, ao final do período, o aquecimento foi interrompido, mantendo-se apenas a agitação por mais 20 minutos. Verificou-se que ao longo da reação houve a formação progressiva de precipitado de coloração verde, a qual se intensificou próximo ao término da síntese. [969] In a 50 mL reaction flask, 0.240 g (0.5 mmol) of HsL1 was dissolved in 15 mL of MeOH. To this binder solution, another solution, also methanolic (5 mL), containing 0.37 g (1.0 mmol) of copper(II) perchlorate hexahydrate, was added dropwise and under constant stirring, After 10 minutes, were slowly dripped 1, 5 ml (1, 5 mmol) aqueous NaOH concentration of 1 mol L '1. The reaction system remained warming up for 40 minutes and, at the end of the period, heating was interrupted, with only stirring being maintained for another 20 minutes. It was found that throughout the reaction there was a progressive formation of a green colored precipitate, which intensified towards the end of the synthesis.
[061] Obtiveram-se 0,19 g (-0,25 mmol; 777,15 g mol'1; rendimento de 50%) do produto 3, na forma de um sólido verde, o qual foi filtrado em funil de Buchner, lavado com metanol gelado e guardado sob vácuo em um dessecador contendo sílica gel. Análise elementar - Porcentagens encontradas: C 43,6; H 3,7; N 9,1 ; Cu 17,3. Calculadas para
4,2; N 9,0; Cu 16,3. A estrutura proposta está esquematizada na Figura 3Â, na qual 0 contra-íon perclorato foi omitido para efeito de simplificação. [061] 0.19 g (-0.25 mmol; 777.15 g mol- 1 ; yield 50%) of product 3 were obtained as a green solid, which was filtered on a Buchner funnel. washed with chilled methanol and stored under vacuum in a desiccator containing silica gel. Elementary analysis - Percentages found: C 43.6; H 3.7; N 9.1; Cu 17.3. Calculated for 4.2; N 9.0; Cu 16.3. The proposed structure is outlined in Figure 3A, in which the perchlorate counterion was omitted for simplification.
Síntese - O, composto 4:
Synthesis - O, compound 4:
[062] Num balão de reação de 50 mL, foram dissolvidos 0,230 g (0,5 mmol) de H2L.2 em aproximados 20 mL de uma mistura 1 :1 diclorometano/MeOH. A essa solução, adicionou-se, gota a gota e sob agitação constante, 0,37 g (1 ,0 mmol) de
dissolvidos em 5 mL de MeOH. Após 40 minutos, 1 mL de uma solução metanólica de KOH (1 mmol; 1 mol L'1) foi lentamente gotejado sobre a mistura. O sistema permaneceu sob agitação por mais 20 minutos, imedíatamente após a interrupção da agitação, um sólido verde escuro foi filtrado em papel, lavado com metanol gelado e seco a vácuo. [062] In a 50 mL reaction flask, 0.230 g (0.5 mmol) of H2L.2 was dissolved in approximately 20 mL of a 1:1 dichloromethane/MeOH mixture. To this solution, 0.37 g (1.0 mmol) of dissolved in 5 ml of MeOH. After 40 minutes, 1 mL of a methanolic solution of KOH (1 mmol; 1 mol L' 1 ) was slowly dropped onto the mixture. The system remained under stirring for a further 20 minutes, immediately after stopping the stirring, a dark green solid was filtered on paper, washed with ice-cold methanol and dried in vacuo.
[063] Obtiveram-se 0,25 g (-0,25 mmol; 1017,08 g mol"1; rendimento de 50%) de produto, 0 qual foi guardado em um dessecador com sílica. Análise elementar - Porcentagens encontradas: C 28,4; H 3,5; N 8,7; Cu 12,2. Calculadas com base na fórmula
C 31 ,9; H 3,9; N 8,3; Cu 12,5. A estrutura proposta está esquematizada na Figura 3B. [063] 0.25 g (-0.25 mmol; 1017.08 g mol "1 ; yield 50%) of product were obtained, which was stored in a desiccator with silica. Elemental analysis - Percentages found: C 28.4; H 3.5; N 8.7; Cu 12.2. Calculated based on the formula C 31.9; H 3.9; N 8.3; 12.5 Cu. The proposed structure is outlined in Figure 3B.
ENSAIOS BIOLÓGICOS BIOLOGICAL TESTS
[064] O presente estudo lida com os efeitos causados por uma série de novos complexos binucleares de cobre(ll) (Figuras 2 e 3) em linhagens celulares de câncer de mama (MCF-7), pulmão (A549) e próstata (PC-3). O estudo realizado
analisou: (i) a sobrevivência (através do ensaio com MTT) das células de câncer; (ii) a produção intracelular de espécies reativas de oxigénio (EROs) (através do ensaio com a sonda DCFH2-DA); (iii) 0 potencial de membrana mitocondrial (através do ensaio com a sonda fluorescente JC-1 ); (iv) a atividade enzimáíica para caspases 8 e 9; (v) a fragmentação de DNA (através da técnica TÚNEL [Gravrieli et ai, 1992 - Gavrieli Y, Sberman Y, Ben-Sasson SA, Identification of programmed ceii death in situ via specific iabeling of nuciear DNA fragmentation. J Cell Biol. (1992) 119(3):493-501 ]); (vi) a competição pela ligação ao DNA entre os novos complexos de cobre e a sonda fluorescente iodeto de propidio; (vii) a parada do ciclo celular (através do ensaio com iodeto de propidio) e (viii) a toxicidade dos compostos em modelo animal alternativo Gaiieria meilonelía, [065] Para avaliar a atividade dos complexos de cobre(H), 1 x 104 células de câncer de mama (MCF-7), pulmão (A-549), próstata (PC-3) foram cultivadas em placas de 96 e após atingirem 100% de confluência nos poços foram expostas a concentrações crescentes (0,1 a 100 mM) dos complexos de cobre. A cisplatina foi usada como controle quimioterápico por se tratar de uma droga amplamente empregada na clínica e também por ser, até hoje, a referência de compostos de coordenação com atividade antitumoral. Após 24 h de tratamento, foi realizado 0 teste de citotoxicidade pelo método de MTT, que após metabolizado por células vivas é convertido em cristais de formazan de cor púrpura. A sobrevivência das células foi calculada como porcentagem de células vivas em relação às células não tratadas com nenhum dos compostos. [064] The present study deals with the effects caused by a series of novel binuclear copper(ll) complexes (Figures 2 and 3) in breast (MCF-7), lung (A549) and prostate (PC) cancer cell lines -3). The study carried out analyzed: (i) the survival (through the MTT assay) of the cancer cells; (ii) the intracellular production of reactive oxygen species (ROS) (through the DCFH2-DA probe assay); (iii) the mitochondrial membrane potential (via the assay with the fluorescent probe JC-1); (iv) the enzymatic activity for caspases 8 and 9; (v) DNA fragmentation (through the TUNNEL technique [Gravrieli et al, 1992 - Gavrieli Y, Sberman Y, Ben-Sasson SA, Identification of programmed death in situ via specific iabeling of nuciear DNA fragmentation. J Cell Biol. ( 1992) 119(3):493-501]); (vi) competition for DNA binding between the novel copper complexes and the fluorescent probe propidium iodide; (vii) the arrest of the cell cycle (through the propidium iodide assay) and (viii) the toxicity of the compounds in an alternative animal model Gaiieria meilonelia, [065] To assess the activity of copper(H) complexes, 1 x 10 4 breast (MCF-7), lung (A-549), prostate (PC-3) cancer cells were cultured in 96 plates and after reaching 100% confluence in the wells were exposed to increasing concentrations (0.1 to 100 mM) of the copper complexes. Cisplatin was used as a chemotherapy control because it is a drug widely used in the clinic and also because it is, until today, the reference for coordination compounds with antitumor activity. After 24 h of treatment, the cytotoxicity test was performed by the MTT method, which after metabolized by living cells is converted into purple formazan crystals. Cell survival was calculated as the percentage of living cells relative to cells not treated with any of the compounds.
[066] A eficiência do composto foi avaliada, através de regressão logarítmica não linear, calculando-se a concentração necessária para matar 50% das células tratadas (parâmetro IC50). Quando administrados às células de câncer humano, os compostos de cobre apresentaram citotoxicidade de 10 a 30 vezes superior às encontradas para a cisplatina (Tabela 1 ). É interessante notar que a linhagem MCF10A, de células epítelíais não tumorais, após tratamento com os complexos 1 e 3 foi aproximadamente 7-8 vezes menos suscetível aos compostos que a
linhagem de câncer MCF-7 (Tabela T). Isto indica uma seletividade dos complexos de cobre em relação às células de câncer, [066] The efficiency of the compound was evaluated, through non-linear logarithmic regression, calculating the concentration needed to kill 50% of treated cells (parameter IC50). When administered to human cancer cells, copper compounds exhibited 10 to 30-fold cytotoxicity than those found for cisplatin (Table 1). Interestingly, the non-tumor epithelial cell lineage MCF10A, after treatment with complexes 1 and 3 was approximately 7-8 times less susceptible to the compounds than the MCF-7 cancer lineage (Table T). This indicates a selectivity of copper complexes towards cancer cells,
[067] Para confirmar se a morte das células humanas tumoraís foi decorrente da apoptose, utilizou-se o método TUNEL, capaz de detectar a fragmentação do DNA pelo processo de apoptose. Nos resultados obtidos, é evidente que as células das linhagens de câncer humano de mama (MCF-7) e pulmão (A-549) estão morrendo por apoptose após tratamento com os complexos 1 e 3, uma vez que observa-se o aumento do número de células com fragmentação do DNA, em comparação àquelas células tratadas com cisplatina, demonstrando que tais compostos são capazes de induzir o processo de apoptose em células tumoraís (Figura 8). Para verificar se os complexos de cobre interagem com o DNA, empregamos um ensaio de competição entre os compostos e o iodeto de propídio (PI), Observa-se claramente que os complexos não competem com o PI pela intercalação ao DNA (Tabela 2). Em ambas as linhagens de células tratadas com os complexos de cobre, não é possível ver alterações significativas na intensidade da fluorescência em comparação com o controle, o que garante que os compostos possam não se ligar ao DNA como agentes intercalantes. [068] Ensaios de citometria de fluxo empregando a sonda fluorescente acetato de 2',7'-diclorodiidrofluoresceina (H2DCFDA) em células de câncer humano das linhagens MCF-7 e A549 tratadas com três diferentes concentrações: Vå c IC50 (0,5 μ M), IC50 (1 ,0 μ M) e 2 x IC50 (2,0 μ M) dos complexos 1 e 3 mostraram, visivelmente, que eles provocam 0 aumento na produção intracelular das EROs (Figura 5), No caso de todos os compostos testados, 0 aumento da produção das EROs foi maior que 0 observado quando se empregou como controle a droga antitumoral doxorubicina, usada na terapêutica de alguns tipos de câncer (Figura 5). Ressalta-se que a indução da produção de EROs constitui um dos mecanismos de ação de diversas drogas, tal como a doxorubicina, empregadas na terapia do câncer e que atuam como disparadores do processo de apoptose. [069] A perda de potencial de membrana mitocondrial constitui outro evento que desencadeia 0 processo de apoptose. A incubação durante 30 minutos com 0
JC-1 indicou que as células tratadas com os complexos 1 e 3 apresentaram uma redução ou uma perda significativa do potencial de membrana mitocondrial em comparação com as células controle (não tratadas) ou tratadas com cisplatina (Figura 6). Os resultados indicam uma redução acentuada na despolarização mitocondrial das células tumorais nas concentrações analisadas (Figura 6). [070] A determinação das atividades caspase 8 e 9 mostrou que somente a atividade da enzima caspase 9 foi alterada, tendo seus níveis aumentados nas linhagens celulares tratadas com os complexos 1 e 3. O resultado indica claramente que os complexos 1 e 3 ativam a via intrínseca que dispara o processo de apoptose em células humanas de câncer (Figura 7). [067] To confirm whether the death of human tumor cells was due to apoptosis, the TUNEL method was used, capable of detecting DNA fragmentation by the process of apoptosis. From the results obtained, it is evident that cells from the human breast (MCF-7) and lung (A-549) cancer lines are dying by apoptosis after treatment with complexes 1 and 3, since an increase in number of cells with DNA fragmentation, compared to those cells treated with cisplatin, demonstrating that such compounds are capable of inducing the apoptosis process in tumor cells (Figure 8). To verify whether copper complexes interact with DNA, we used a competition assay between the compounds and propidium iodide (PI). It is clearly observed that the complexes do not compete with PI for DNA intercalation (Table 2). In both cell lines treated with copper complexes, it is not possible to see significant changes in fluorescence intensity compared to the control, which ensures that the compounds cannot bind to DNA as intercalating agents. [068] Flow cytometry assays employing the fluorescent probe 2',7'-dichlorodihydrofluorescein acetate (H2DCFDA) in human cancer cells of MCF-7 and A549 lines treated with three different concentrations: Vå c IC 50 (0.5 μ M) IC50 (1, 0 μ M) and 2x IC 50 (2.0 μ M) of complex 1 and 3 showed visibly 0 they cause increase in intracellular production of ROS (Figure 5), where of all compounds tested, the increase in ROS production was greater than that observed when the antitumor drug doxorubicin, used in the treatment of some types of cancer, was used as a control (Figure 5). It is noteworthy that the induction of ROS production is one of the action mechanisms of several drugs, such as doxorubicin, used in cancer therapy and that act as triggers of the apoptosis process. [069] The loss of mitochondrial membrane potential is another event that triggers the process of apoptosis. Incubation for 30 minutes with 0 JC-1 indicated that cells treated with complexes 1 and 3 showed a significant reduction or loss of mitochondrial membrane potential compared to control (untreated) or cisplatin-treated cells (Figure 6). The results indicate a marked reduction in the mitochondrial depolarization of tumor cells at the concentrations analyzed (Figure 6). [070] The determination of caspase 8 and 9 activities showed that only the activity of caspase 9 enzyme was altered, having its levels increased in cell lines treated with complexes 1 and 3. The result clearly indicates that complexes 1 and 3 activate the intrinsic pathway that triggers the apoptosis process in human cancer cells (Figure 7).
[071] Devido à sua citotoxicidade e genotoxicidade, indução na produção das ERGs, perda do potencial de membrana mitocondrial e, em conjunto, ativação da caspase 9 e presença de quebra do DNA, concluímos que o tratamento com os complexos binucleares de cobre(il) provoca a morte das células tumorais humanas através do processo de apoptose, o qual é induzido pela via intrínseca. Os resultados permitem considerar os compostos de cobre deste estudo como potenciais agentes terapêuticos para diferentes tipos de câncer. Cabe ressaltar que os complexos 1 e 3 não apresentaram toxicidade para Galleria mellonella (Figura 10), um modelo invertebrado que tem sido bastante empregado como alternativa para substituir os modelos de experimentação animal. [071] Due to its cytotoxicity and genotoxicity, induction in the production of ERGs, loss of mitochondrial membrane potential and, together, activation of caspase 9 and presence of DNA breakage, we conclude that treatment with binuclear copper complexes(il ) causes the death of human tumor cells through the apoptosis process, which is induced by the intrinsic pathway. The results allow considering the copper compounds in this study as potential therapeutic agents for different types of cancer. It is noteworthy that complexes 1 and 3 did not show toxicity to Galleria mellonella (Figure 10), an invertebrate model that has been widely used as an alternative to replace animal experimentation models.
[072] Em pacientes com câncer, doenças oportunistas como doença invasiva ou não-invasiva causada por fungos patogênicos constituí uma complicação importante, provocando considerável mortalidade e morbidade. Além disso, os procedimentos de quimioterapia ou transplante costumam ser cancelados ou adiados em pacientes com doenças fúngicas invasivas, o que pode comprometer a sobrevída geral da pessoa acometida. Avaliamos a atividade antifúngica dos novos compostos binucleares de cobre e observamos que tanto o complexo 1 quanto o complexo 3 foram capazes de inibir o crescimento de quatro amostras de isolados clínicos de fungos patogênicos Candida spp (ex. Candida albicans , Candida parapsiiosis , Candida tropicaiis e Candida krusei) (Tabela 3).
[073] Os perfis de suscetibilidade microbiana aos compostos de cobre foram determinados de acordo com o protocolo descrito no documento M27-A3, publicado pelo Clinicai and Laboratory Standards Institutes (CLSI, 2008). [072] In cancer patients, opportunistic diseases such as invasive or non-invasive disease caused by pathogenic fungi constitute a major complication, causing considerable mortality and morbidity. In addition, chemotherapy or transplant procedures are often canceled or postponed in patients with invasive fungal diseases, which can compromise the affected person's overall survival. We evaluated the antifungal activity of the new binuclear copper compounds and observed that both complex 1 and complex 3 were able to inhibit the growth of four clinical isolates of Candida spp pathogenic fungi (eg Candida albicans , Candida parapsiiosis , Candida tropicaiis and Candida tropicaiis Candida krusei) (Table 3). [073] Microbial susceptibility profiles to copper compounds were determined according to the protocol described in document M27-A3, published by the Clinical and Laboratory Standards Institutes (CLSI, 2008).
Tabela 3. Inibição do crescimento através da determinação da concentração mínima inibitória (CMI) de amostras de isolados clínicos de fungos patogênicos.
Table 3. Growth inhibition by determining the minimum inhibitory concentration (MIC) of samples of clinical isolates of pathogenic fungi.
# Referersce: CLSI (2008). Clinicai and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptíbiliíy Testing of Yeasts: Approved Standard-3rd Edn M27-A3. Wayne, PA CLSI. # Referersce: CLSI (2008). Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard-3rd Edn M27-A3. Wayne, PA CLSI.
[074] As metalodrogas da presente invenção podem constituir composições farmacêuticas, sendo utilizadas de forma isolada ou em combinação entre si, adicionando-as a veículos e excipientes farmaceuticamente aceitáveis. [074] The metallodrugs of the present invention can constitute pharmaceutical compositions, being used alone or in combination with each other, adding them to pharmaceutically acceptable vehicles and excipients.
[075] A terapêutica do câncer apresenta elevada dificuldade de tratamento, devido a suas distintas origens, podendo apresentar uma resistência intrínseca e/ou adquirida aos tratamentos disponíveis. Além disso, o uso excessivo de uma mesma droga/medicamento pode provocar sérios efeitos colaterais ao paciente. Diante deste cenário, as metalodrogas descritas na presente invenção podem ser combinadas/associadas a quaisquer outras drogas utilizadas em tratamentos de neoplasias e doenças correlacionadas. Sobretudo a fármacos antitumorais, tais como quelantes de cobre e drogas quimioterápicas, principalmente aquelas baseadas na platina, tal como a cisplatina [c/s-diaminodicloroplatina(ll)]. [075] Cancer therapy presents high treatment difficulty, due to its different origins, and may present an intrinsic and/or acquired resistance to available treatments. Furthermore, the excessive use of the same drug/medication can cause serious side effects to the patient. Given this scenario, the metallodrugs described in the present invention can be combined/associated with any other drugs used in the treatment of neoplasms and related diseases. Especially anti-tumor drugs such as copper chelators and chemotherapy drugs, particularly those based on platinum, such as cisplatin [c/s-diaminodichloroplatinum(ll)].
[076] Uma outra possibilidade de combinação das metalodrogas da presente invenção compreende a que pode ser realizada com outros complexos de cobre, porém mononucleares. Sendo diferentes dos apresentados nesta invenção, os compostos podem demonstrar efeitos sinérgicos durante o tratamento. [076] Another possibility of combining the metallodrugs of the present invention comprises the one that can be performed with other copper complexes, however mononuclear. Different from those presented in this invention, the compounds may demonstrate synergistic effects during treatment.
[077] O tratamento inclui administrar uma quantidade dos complexos isolados ou em combinação que seja eficaz para aliviar, alterar, remediar, melhorar ou
afetar a doença, os sintomas da doença ou ainda de doenças decorrentes de infecções oportunistas causadas por fungos paiogênicos, de forma a impedir o agravamento da doença principal, o câncer. [077] Treatment includes administering an amount of the complexes alone or in combination that is effective to alleviate, alter, remedy, ameliorate or affect the disease, disease symptoms or diseases resulting from opportunistic infections caused by pathogenic fungi, in order to prevent the aggravation of the main disease, cancer.
[078] Será considerado que as dosagens necessárias dos compostos e suas composições compreendendo os complexos isolados ou em combinação podem variar em cada caso, sendo diferentes para cada indivíduo tratado. A definição da dosagem ideal será determinada através do equilíbrio entre o benefício terapêutico e qualquer risco ou efeitos colaterais dos tratamentos aqui descritos. [079] A administração in vivo destes metalofármacos pode ser efetuada em dose contínua ou, ainda, intermitentemente (por exemplo, em doses divididas em intervalos apropriados), ao longo do curso do tratamento. Os protocolos de administração mais eficazes dos quimioterápicos utilizados na terapêutica dos diversos tipos de câncer são bem conhecidos dos especialistas na técnica e variarão com a formulação utilizada para a terapia, o objetivo da terapia, a célula alvo e o sujeito a ser tratado. [078] It will be considered that the necessary dosages of the compounds and their compositions comprising the isolated complexes or in combination may vary in each case, being different for each individual treated. The definition of the ideal dosage will be determined by balancing the therapeutic benefit and any risk or side effects of the treatments described herein. [079] The in vivo administration of these metallopharmaceuticals can be carried out in continuous dose or even intermittently (for example, in divided doses at appropriate intervals), throughout the course of treatment. The most effective administration protocols of chemotherapeutics used in the therapy of various types of cancer are well known to those skilled in the art and will vary with the formulation used for the therapy, the purpose of therapy, the target cell and the subject to be treated.
[080] Uma dose adequada de uma terapêutica à base de platina pode ser uma dose padrão; uma dosagem padrão de cisplatina para o tratamento do câncer testicular é de 20 mg/m2 intravenoso (IV) diariamente por 1 a 5 dias consecutivos a cada 3 semanas por 3-4 ciclos de terapia quando usada em terapia combinada; por outro lado, uma dosagem padrão de cisplatina para o tratamento de carcinoma ovariano avançado varia de 75 a 100 mg/m2 IV uma vez a cada 3-4 semanas por 6 ciclos quando a cisplatina é usada em terapia combinada. Para o tratamento do câncer avançado de bexiga, uma dosagem padrão de cisplatina é de 70 a 100 mg/m2 IV uma vez a cada 3-4 semanas por 4 ciclos quando a cisplatina é usada em terapia combinada. Uma dosagem padrão para o tratamento de câncer de cabeça e pescoço recorrente ou avançado é de 80 a 120 mg/m2 IV uma vez a cada 3 semanas ou 20 a 100 mg/m2 IV quando usada em esquemas de quimioterapia combinada, com a frequência de administração dependendo do regime específico empregado. Uma dosagem padrão de cisplatina para o tratamento de câncer de colo do útero é 40 mg/m2 administrada
semanalmente por 6 semanas quando associada com radioterapia ou 40 a 80 mg/m2 a cada 3 semanas por 3-4 ciclos quando for administrada em combinação com outros quimioterápicos. Para o tratamento de carcinoma pulmonar de células não pequenas, uma dosagem padrão de cispiatina em terapia combinada é de 75 a 100 mg/m2 IV uma vez a cada 3-4 semanas por 4-8 ciclos, dependendo do regime específico utilizado. Para o tratamento do câncer de esófago avançado, uma dosagem padrão de cispiatina é de 50 a 120 mg/m2 IV uma vez a cada 3-4 semanas nos regimes de quimioterapia combinada ou 10 a 50 mg/m2 IV a cada 3 semanas por 2 ciclos quando combinada com radiação. Finalmente, para o tratamento do câncer de mama recorrente e metastático, uma dosagem padrão de cispiatina é de 50 a 100 mg/m2 IV uma vez a cada 3-4 semanas nos regimes de quimioterapia isolada ou combinada. [080] An adequate dose of a platinum-based therapy may be a standard dose; a standard dosage of cisplatin for the treatment of testicular cancer is 20 mg/m 2 intravenously (IV) daily for 1 to 5 consecutive days every 3 weeks for 3-4 cycles of therapy when used in combination therapy; on the other hand, a standard dosage of cisplatin for the treatment of advanced ovarian carcinoma ranges from 75 to 100 mg/m 2 IV once every 3-4 weeks for 6 cycles when cisplatin is used in combination therapy. For the treatment of advanced bladder cancer, a standard dosage of cisplatin is 70 to 100 mg/m 2 IV once every 3-4 weeks for 4 cycles when cisplatin is used in combination therapy. A standard dosage for the treatment of recurrent or advanced head and neck cancer is 80 to 120 mg/m 2 IV once every 3 weeks or 20 to 100 mg/m 2 IV when used in combination chemotherapy regimens, with frequency of administration depending on the specific regime employed. A standard dosage of cisplatin for the treatment of cervical cancer is 40 mg/m 2 given. weekly for 6 weeks when combined with radiotherapy or 40 to 80 mg/m 2 every 3 weeks for 3-4 cycles when given in combination with other chemotherapy agents. For the treatment of non-small cell lung carcinoma, a standard dosage of cispiatin in combination therapy is 75 to 100 mg/m 2 IV once every 3-4 weeks for 4-8 cycles, depending on the specific regimen used. For the treatment of advanced esophageal cancer, a standard dosage of cispiatin is 50 to 120 mg/m 2 IV once every 3-4 weeks in combination chemotherapy regimens or 10 to 50 mg/m 2 IV every 3 weeks for 2 cycles when combined with radiation. Finally, for the treatment of recurrent and metastatic breast cancer, a standard dosage of cispiatin is 50 to 100 mg/m 2 IV once every 3-4 weeks in single or combined chemotherapy regimens.
[081] A dose da combinação entre os complexos binucleares de cobre da presente invenção e outro agente quimioterápico, tal como a cispiatina, mas não se limitando exclusivamente à cispiatina, deverá estar compreendida entre 20 a 120 mg/m2 de superfície corporal do fármaco antitumoral, acrescida de 10 a 480 mg/m2 de superfície corporal dos complexos binucleares de cobre(ll) em infusão intravenosa única, a cada 3-4 semanas pelo número necessário de cicios, respeitando-se, obviamente, o protocolo padrão empregado na quimioterapia especifica para o câncer em tratamento. [081] The dose of the combination between the binuclear copper complexes of the present invention and another chemotherapeutic agent, such as cispiatin, but not limited exclusively to cispiatin, should be between 20 to 120 mg/m 2 of the drug's body surface antitumor, plus 10 to 480 mg/m 2 of body surface of binuclear copper(ll) complexes in a single intravenous infusion, every 3-4 weeks for the necessary number of cycles, obviously respecting the standard protocol used in specific chemotherapy for the cancer being treated.
[082] Assim, as taxas de diluição da nova composição estarão compreendidas entre 1 parte do fármaco antitumoral : 0,5 - 4,0 partes do complexo binuclear de cobre ou, alternativamente, 0,5 parte do fármaco antitumoral : 0,5 - 4,0 partes do complexo binuclear de cobre. [082] Thus, the dilution rates of the new composition will be comprised between 1 part of the antitumor drug: 0.5 - 4.0 parts of the binuclear copper complex or, alternatively, 0.5 part of the antitumor drug: 0.5 - 4.0 parts of the binuclear copper complex.
[083] A composição é utilizada no preparo de uma solução injetável ou formulação farmacêutica para uso oral composta por ambas as drogas e deve ser administrada exclusivamente por infusão intravenosa ou via oral. [083] The composition is used in the preparation of an injectable solution or pharmaceutical formulation for oral use composed of both drugs and should be administered exclusively by intravenous infusion or orally.
[084] Quando os complexos binucleares de cobre(ll) e o quimioterápico a base de platina são administrados em combinação com radiação, esta terapia
combinada pode ser empregada com doses mais baixas do agente administrado, reduzindo uma possível toxicidade ou complicação associada às várias terapias. [085] Os complexos binucleares de cobre(ll), isolados ou combinados com outros antitumorais, podem ainda ser administrados em combinação com um antimicrobiano ou antifúngico empregados na clinica médica e na dosagem padrão para tratamento de infecções bacterianas e fúngicas. [084] When binuclear copper(II) complexes and platinum-based chemotherapy are administered in combination with radiation, this therapy combined can be used with lower doses of the administered agent, reducing possible toxicity or complication associated with the various therapies. [085] Copper(ll) binuclear complexes, alone or combined with other antitumor agents, can still be administered in combination with an antimicrobial or antifungal used in the medical clinic and in standard dosage for the treatment of bacterial and fungal infections.
[086] Os complexos binucleares de cobre(ll), isolados ou em combinação, podem ser administrados em associação com um agente ou procedimento para reduzir qualquer possível efeito colateral (ex. diarréia, náusea e vómito). [086] Binuclear copper(II) complexes, alone or in combination, can be given in combination with an agent or procedure to reduce any possible side effects (eg, diarrhea, nausea, and vomiting).
[087] Exemplos de outros agentes antitumorais que podem ser administrados em associação com os complexos de cobre, isolados ou em combinação com a cisplatina, incluem: pemetrexedo, vinorelbina, gemcitabina, vinblastina, dacarbazina, temozolomida, 5FU (5-fluorouracila), ciclofosfamida, bleomicina, etoposídeo, ifosfamida, paclitaxel, metotrexato, doxorubicina, adriamicina, vincristina, mitomicina, docetaxel e ainda combinações dos agentes acima. Estes agentes também podem ser administrados em conjunto com a realização de processos cirúrgicos e / ou aplicação de radiação. [087] Examples of other antitumor agents that can be administered in combination with copper complexes, alone or in combination with cisplatin, include: pemetrexed, vinorelbine, gemcitabine, vinblastine, dacarbazine, temozolomide, 5FU (5-fluorouracil), cyclophosphamide , bleomycin, etoposide, ifosfamide, paclitaxel, methotrexate, doxorubicin, adriamycin, vincristine, mitomycin, docetaxel, and combinations of the above agents. These agents can also be administered in conjunction with performing surgical procedures and/or applying radiation.
[088] Exemplos de outros agentes antifúngicos que podem ser administrados em associação com os complexos de cobre, incluem os agentes das classes: poliênicos, azóis e equinocandinas. [088] Examples of other antifungal agents that can be administered in association with copper complexes include agents of the classes: polyenes, azoles and echinocandins.
[089] O escopo da presente invenção também abarca as diferentes formas de produção, composições, usos e aplicações das metalodrogas descritas. [089] The scope of the present invention also encompasses the different forms of production, compositions, uses and applications of the metallodrugs described.
[090] Os exemplos acima foram descritos para ilustrar as diferentes formas de produção e ensaios biológicos das composições / compostos que constam do presente documento, não devendo assim ser encarados como limitativos desta invenção, sabendo-se que pequenas variações do que fora acima descrito ainda farão parte do escopo da mesma.
[090] The examples above were described to illustrate the different forms of production and biological tests of the compositions / compounds contained in this document, and should therefore not be seen as limiting this invention, knowing that small variations of what was described above still will be part of its scope.
Claims
Reivindicações Claims
1. LIGANTES BINUCLEANTES FENÓLÍCOS, caracterizados por serem não- simétricos, possuindo um “braço pendente" hidrazônico com um anel de seis membros do tipo 4-piridina, decorrente da isoniazida, compreendendo ainda um número de átomos de oxigénio e nitrogénio derivados de feno! e/ou piridina disponíveis para doação de pares eletrónicos para variação de dureza/maciez dos sítios de coordenação, e sendo ainda capazes de formar metalodrogas antitumorais e antifúngicas. 1. PHENOLIC BINUCLEANT LINKERS, characterized by being non-symmetrical, having a hydrazonic "pendant arm" with a six-membered 4-pyridine ring, deriving from isoniazid, further comprising a number of oxygen and nitrogen atoms derived from phen! and/or pyridine available for donating electronic pairs to vary the hardness/softness of the coordination sites, and also being able to form antitumor and antifungal metallodrugs.
2. LIGANTES, de acordo com a reivindicação 1, caracterizado pelo ligante binucieante ser o 2-hidroxi~3-{[(2~hidroxibenzii)(2-piridilmetií)amino]-metii}-5- metilbenzaldeído isonícotinoíl hidrazona (H3L1 ) (1A), que apresenta grupos doadores terminais fenol e piridina (sítio “N2O”). 2. LINKERS, according to claim 1, characterized in that the binding linker is 2-hydroxy-3-{[(2-hydroxybenzyl)(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 3 L1 ) (1A), which has terminal phenol and pyridine donor groups (“N 2 O” site).
3. LIGANTES, de acordo com a reivindicação 1, caracterizado pelo ligante binucieante ser o 2-hidroxi-3-{bis[(2-piridilmetil)amino]-metil}-5- metilbenzaldeído isonícotinoíl hidrazona (H2L2) (1B), que possui 02 (dois) grupos doadores do tipo piridina como substituintes (sítio “N3”). 3. LINKERS, according to claim 1, characterized in that the binding linker is 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl}-5-methylbenzaldehyde isonicotinoyl hydrazone (H 2 L2) (1B) , which has 02 (two) pyridine-type donor groups as substituents ("N3" site).
4. COMPOSTOS METÁLICOS BiNUCLEARES, isto é, que acomodam dois centros metálicos, neste caso de cobre(li), caracterizados por serem metalodrogas antitumorais e antifúngicas, não-simétricas, para uso médico e veterinário, possuindo como precursor químico comum um dos dois ligantes hidrazônicos não-simétricos definidos nas reivindicações 1 a 3, e ligantes exógenos, voltados para a formação de uma pró-droga ou de complexos já na sua forma ativa, ditos compostos possuindo as estruturas:
4. BiNUCLEAR METALLIC COMPOUNDS, that is, they accommodate two metal centers, in this case copper(li), characterized by being non-symmetrical antitumor and antifungal metallodrugs for medical and veterinary use, having as a common chemical precursor one of the two ligands non-symmetric hydrazonics defined in claims 1 to 3, and exogenous ligands, aimed at the formation of a pro-drug or complexes already in their active form, said compounds having the structures:
5, COMPOSTOS, de acordo com a reivindicação 4, caracterizados por acomodarem dois centros metálicos de cobre(li) que interagem com macromolécuias biológicas tais como o DMA e/ou proteínas, levando à apoptose de células cancerígenas e fúngicas. 5, COMPOUNDS according to claim 4, characterized in that they accommodate two metallic copper(li) centers that interact with biological macromolecules such as DMA and/or proteins, leading to apoptosis of cancer and fungal cells.
8. COMPOSTOS, de acordo com a reivindicação 4, caracterizados por compreenderem ligantes externos capazes de formar pontes exógenas hidróxido, postulada como a forma ativa, ou pontes exógenas acetato, constituindo pró-drogas, e apresentarem centros de cobre(lí) insaturados ou contendo ligantes iábeis, corno a molécula de água para interação dos mesmos com macromolécuias biológicas como o DNA, RNA e/ou proteínas.
8. COMPOUNDS according to claim 4, characterized in that they comprise external ligands capable of forming exogenous hydroxide bridges, postulated as the active form, or exogenous acetate bridges, constituting prodrugs, and having unsaturated or containing copper(II) centers labile ligands, such as the water molecule for their interaction with biological macromolecules such as DNA, RNA and/or proteins.
7. COMPOSIÇÃO MÉDICO-VETERINÁRIA, caracterizada por compreender uma quantidade terapeuticamente eficaz das metalodrogas binudeares não- simétricas de cobre(ij) definidas nas reivindicações 4 ou 5, isoladas ou em combinação, e compreender ainda uma quantidade terapeuticamente eficaz de outras drogas adjuvantes, tais como as antineoplásicas, antifúngicas, antimicrobianas, drogas contra enjoos, vómitos, náuseas, diarreia e outras comorbidades e efeitos secundários, assim como uma quantidade determinada de agentes, excipientes e/ou veículos farmaceuticamente aceitáveis. 7. MEDICAL-VETERINARY COMPOSITION, characterized in that it comprises a therapeutically effective amount of the non-symmetrical copper(ij) bilayer metallodrugs defined in claims 4 or 5, alone or in combination, and further comprises a therapeutically effective amount of other adjuvant drugs, such as such as antineoplastics, antifungals, antimicrobials, drugs against nausea, vomiting, nausea, diarrhea and other comorbidities and side effects, as well as a certain amount of pharmaceutically acceptable agents, excipients and/or vehicles.
8. COMPOSIÇÃO, de acordo com a reivindicação 7, caracterizada pelas drogas adjuvantes antineoplásicas serem selecionadas dentre: quelantes de cobre, outros complexos mono- ou binudeares de cobre(ll), e/ou outros fármacos quimioterápicos, principalmente aqueles baseados na platina, tal como a cisplatina [c/s-diaminodicloroplatina(ll)]. 8. COMPOSITION, according to claim 7, characterized in that the antineoplastic adjuvant drugs are selected from among: copper chelators, other mono- or binude copper(ll) complexes, and/or other chemotherapeutic drugs, especially those based on platinum, such as such as cisplatin [c/s-diaminodichloroplatinum(ll)].
9. COMPOSIÇÃO, de acordo com qualquer uma das reivindicações 7 ou 8, caracterizada pelas drogas adjuvantes antineoplásicas que podem ser administrados em associação aos complexos de cobre definidos na reivindicação 4, isolados ou em combinação entre si ou com outras drogas, serem selecionadas dentre: pemetrexedo, vinorelbina, gemcitabina, vinblastina, dacarbazina, temozoiomida, 5FU (5-fiuorouraciia), ciclofosfamida, bleomicina, etoposídeo, ifosfamída, paclitaxel, metotrexato, doxorubicina, adriamieina, vincristina, mitomicina, docetaxel, ou ainda suas combinações. 9. COMPOSITION according to any one of claims 7 or 8, characterized in that the antineoplastic adjuvant drugs that can be administered in association with the copper complexes defined in claim 4, alone or in combination with each other or with other drugs, are selected from: pemetrexed, vinorelbine, gemcitabine, vinblastine, dacarbazine, temozoiomide, 5FU (5-fluorouraciia), cyclophosphamide, bleomycin, etoposide, ifosfamide, paclitaxel, methotrexate, doxorubicin, adriamiein, vincristine, mitomycin, docetate.
10. COMPOSIÇÃO, de acordo com a reivindicação 7, caracterizada pelos agentes antifúngicos ser selecionados preferencialmente dentre as classes dos poliênicos, azois e equinocandinas.
10. COMPOSITION, according to claim 7, characterized in that antifungal agents are preferably selected from the classes of polyenes, azoles and echinocandins.
11 . COMPOSIÇÃO, de acordo com a reivindicação 7, caracterizada pela taxa de diluição da composição estar compreendida entre 1 parte do fármaco antitumorai para 0,5 - 4,0 partes do complexo binuclear de cobre. 11 . COMPOSITION according to claim 7, characterized in that the rate of dilution of the composition is comprised between 1 part of the antitumor drug to 0.5 - 4.0 parts of the binuclear copper complex.
12. COMPOSIÇÃO, de acordo com a reivindicação 11 , caracterizada pela taxa de diluição da composição estar compreendida entre 0,5 parte do fármaco antitumorai para 0,5 - 4,0 partes do complexo binuclear de cobre. 12. COMPOSITION according to claim 11, characterized in that the rate of dilution of the composition is comprised between 0.5 part of the antitumor drug to 0.5 - 4.0 parts of the binuclear copper complex.
13. PROCESSO DE SÍNTESE DE LIGANTES BÍNUCLEANTES, caracterizado peia rota sintética para o !igante H3L1 seguir as seguintes etapas: a) partir do intermediário sintético hbpamff, 2-hidroxi~3-{[(2~ hidroxibenzil){2-piridiimeti!)amino]-metil}-5-metilbenzalde!do; b) uma solução metanóiica (5 ml) da hidrazida do ácido isonicotínico, ísoníazida (0,138 g; 1 mmol) ser gotejada, sob agitação constante, sobre uma solução de hbpamff (0,333 g; 0,9 mmol) em 10 mL de uma mistura 1 :1 MeOH:Et20, previamente preparada num balão de 50 mL; c) a mistura ser refluxada por 2 horas até adquirir progressivamente uma coloração castanha; d) ao término da reação, transferir o conteúdo do baião para um béquer de 50 mL; e e) deixar o mesmo em repouso para que, com a evaporação gradual do solvente, o produto precipitar, sendo isolada uma grande quantidade de cristais castanhos, o qual é filtrado e lavado com éter etílico gelado. 13. PROCESS OF SYNTHESIS OF BINUCLEANT LINKERS, characterized by the synthetic route for the linker H 3 L1 following the steps: a) from the synthetic intermediate hbpamff, 2-hydroxy-3-{[(2-hydroxybenzyl){2-pyridiimethyl !)amino]-methyl}-5-methylbenzaldehyde; b) a methanolic solution (5 ml) of isonicotinic acid hydrazide, isoniazid (0.138 g; 1 mmol) is dropped, under constant stirring, onto a solution of hbpamff (0.333 g, 0.9 mmol) in 10 ml of a mixture 1:1 MeOH:Et20, previously prepared in a 50 ml flask; c) the mixture is refluxed for 2 hours until it progressively acquires a brown color; d) at the end of the reaction, transfer the contents of the baião to a 50 mL beaker; ee) leaving it to rest so that, with the gradual evaporation of the solvent, the product precipitates, being isolated a large amount of brown crystals, which is filtered and washed with ice-cold ethyl ether.
14. PROCESSO DE SÍNTESE DE LIGANTES BÍNUCLEANTES, caracterizado peia rota sintética para o ligante H2L2 seguir as seguintes etapas: a) partir do intermediário sintético bpmamff, 2-hidroxi~3-{bis[(2~ piridiimetil)amino]-metil}-5-metilbenzaldeido; b) gotejar lentamente ssoniazida previamente dissolvida em 5 mL de uma mixtura (v/v) 1 :1 diclorometano/IVleOH (0,277 g; 2 mmol) foi sobre
uma solução contendo bpmamff (0,703 g; 2 mmoi) dissolvido em 10 ml da mesma mistura usando ultrassom; c) refluxo da solução (balão de 50 mL) por 3 horas; e d) remoção do solvente sob pressão reduzida, sendo o óleo obtido deixado em repouso em 1 :1 MeCN/MeOH, obtendo-se o ligante na forma cristalina. , PROCESSO DE SÍNTESE DE COMPOSTOS BÍNUCLEARES, caracíerizado por serem gerados a partir de H3L1 e H2L2 conforme definidos nas reivindicações 1 a 3, seguindo rotas de síntese para os complexos de fórmulas 1, 2, 3 e 4 definidas nas reivindicações 4 e 5, os quais possuem maior eficácia para eliminar ou reduzir a proliferação das células tumorais e fúngicas, dito processo compreendendo as seguintes etapas: a. dissolução de uma quantidade do ligante binucleante em um solvente orgânico puro ou mistura de solventes; b. adição lenia de 2 equivalentes de um sal de cobre(ll), previamente dissolvido em metanol ou em mistura de solventes, sendo o contra-íon presente neste sal coordenante, tais como acetato, propionato ou, mais genericamente, um ánion carboxilato, capazes de gerar uma pró- droga, ou não tal como perclorato, haleto, entre outros contra-íons capazes de gerar uma droga finai na sua forma ativa; c. a mistura reacionai sendo então agitada, sob aquecimento moderado, por um período que pode variar de 10 minutos até 24 horas; d. caso o sai de cobre usado não possua um contra-íon coordenante, adiciona-se uma quantidade adequada de base, dissolvida em água ou metanol, com o objetivo de favorecer a formação da ponte exógena do tipo m-hidroxo; e, por fim, e. o sólido obtido sendo filtrado, lavado com solvente orgânico gelado e seco no vácuo, sendo que uma maior massa de produto e, eventuaimente, cristais adequados ao processo de determinação
estrutural por difração de raios X podendo ser obtidos pela evaporação lenta da água-mãe. 14. PROCESS OF SYNTHESIS OF BINOCLEAN LINKERS, characterized by the synthetic route for the linker H 2 L2 to follow the following steps: a) from the synthetic intermediate bpmamff, 2-hydroxy-3-{bis[(2-pyridylmethyl)amino]-methyl }-5-methylbenzaldehyde; b) slowly dropping ssoniazid previously dissolved in 5 ml of a mixture (v/v) 1:1 dichloromethane/IVleOH (0.277 g, 2 mmol) was over a solution containing bpmamff (0.703 g; 2 mmol) dissolved in 10 ml of the same mixture using ultrasound; c) reflux of the solution (50 mL flask) for 3 hours; and d) removal of the solvent under reduced pressure, the oil obtained being left to stand in 1:1 MeCN/MeOH, obtaining the binder in crystalline form. , SYNTHESIS PROCESS OF BINOCLEAR COMPOUNDS, characterized by being generated from H 3 L1 and H 2 L2 as defined in claims 1 to 3, following synthesis routes for the complexes of formulas 1, 2, 3 and 4 defined in claims 4 and 5, which have greater efficacy to eliminate or reduce the proliferation of tumor and fungal cells, said process comprising the following steps: a. dissolving an amount of the binucleating ligand in a pure organic solvent or mixture of solvents; B. slow addition of 2 equivalents of a copper(II) salt, previously dissolved in methanol or a mixture of solvents, the counterion present in this coordinating salt, such as acetate, propionate or, more generally, a carboxylate anion, being capable of generate a prodrug, or not, such as perchlorate, halide, among other counterions capable of generating a final drug in its active form; ç. the reaction mixture is then stirred, under moderate heating, for a period which may vary from 10 minutes to 24 hours; d. if the copper salt used does not have a coordinating counterion, an adequate amount of base is added, dissolved in water or methanol, in order to favor the formation of an exogenous m-hydroxy type bridge; and, finally, e. the solid obtained being filtered, washed with cold organic solvent and dried in a vacuum, with a greater mass of product and, eventually, crystals suitable for the determination process structural by X-ray diffraction can be obtained by slow evaporation of the mother liquor.
16. MÉTODO DE TRATAMENTO DE NEOPLASIAS E DOENÇAS FÚNGICAS, caracterizado por compreender a administração dos complexos binucleares definidos nas reivindicações 4 a 6, de forma isolada ou em combinação entre eles, e/ou com outras drogas adjuvantes, ou ainda a administração das composições definidas nas reivindicações 7 a 12 em uma dose determinada, em um paciente humano ou animal. 16. METHOD OF TREATMENT OF NEOPLASMS AND FUNGI DISEASES, characterized in that it comprises the administration of the binuclear complexes defined in claims 4 to 6, alone or in combination between them, and/or with other adjuvant drugs, or the administration of the defined compositions in claims 7 to 12 at a determined dose, in a human or animal patient.
17. MÉTODO DE TRATAMENTO DE NEOPLASIAS E DOENÇAS FÚNGICAS, de acordo com a reivindicação 16, caracterizado por compreender a administração de uma dose compreendida entre 20 a 120 mg/m2, de superfície corporal, de um fármaco antitumorai acrescida de 10 a 480 mg/m2, de superfície corporal, dos complexos binucleares de cobre(ll) em infusão intravenosa ou a dose equivalente por via oral, a cada 3-4 semanas pelo número necessário de cicios, respeitando o protocolo padrão empregado na quimioterapia especifica para o tipo de câncer em tratamento. 17. METHOD OF TREATMENT OF NEOPLASMS AND FUNGI DISEASES, according to claim 16, characterized in that it comprises the administration of a dose comprised between 20 to 120 mg/m 2 , of body surface, of an antitumor drug plus 10 to 480 mg /m 2 , of body surface, of binuclear copper(ll) complexes in intravenous infusion or the equivalent dose orally, every 3-4 weeks for the necessary number of cycles, respecting the standard protocol used in chemotherapy specific for the type of cancer undergoing treatment.
18. MÉTODO DE TRATAMENTO DE NEOPLASIAS E DOENÇAS FÚNGICAS, de acordo com qualquer uma das reividicações 16 ou 17, caracterizado por compreender a administração de complexos binucleares de cobre(ll) 1 ,2, 3 4, isolados ou combinados entre si, podendo ser em combinação com antimicrobianos e/ou antifúngicos empregados na clínica médica e na dosagem padrão para tratamento de infecções bacterianas e fúngicas, em conjunto com cirurgia e/ou radiação, que seja eficaz para aliviar, alterar, remediar, melhorar ou afetar a doença, os sintomas da doença ou doenças decorrentes de infecções oportunistas causadas por fungos patogênicos, de forma a impedir o agravamento do câncer.
, USO, dos compostos binucieares das reivindicações 4 a 6, de forma isolada ou em combinação entre eles, e/ou com outras drogas adjuvantes, ou ainda das composições descritas nas reivindicações 7 a 12, caracterizado por ser para a fabricação de um medicamentos antitumorais e antifúngicos, para aliviar, alterar, remediar, melhorar ou afetar a doença neoplásica, os sintomas da doença ou doenças decorrentes de infecções oportunistas, como as causadas por fungos patogênicos, de forma a impedir o agravamento da doença primária, o câncer, ou ainda para tratamento de infecções de natureza bacteriana e/ou fúngica.
18. METHOD OF TREATMENT OF NEOPLASMS AND FUNGI DISEASES, according to any one of claims 16 or 17, characterized in that it comprises the administration of binuclear copper(ll) 1, 2, 3 4 complexes, isolated or combined with each other, which may be in combination with antimicrobials and/or antifungals used in clinical medicine and in the standard dosage for the treatment of bacterial and fungal infections, in conjunction with surgery and/or radiation, which is effective to alleviate, change, remedy, improve or affect the disease, disease symptoms or diseases resulting from opportunistic infections caused by pathogenic fungi, in order to prevent the cancer from worsening. USE, of the binary compounds of claims 4 to 6, alone or in combination with each other, and/or with other adjuvant drugs, or even of the compositions described in claims 7 to 12, characterized in that it is for the manufacture of an anti-tumor drug and antifungals, to alleviate, alter, remedy, improve or affect neoplastic disease, disease symptoms or diseases resulting from opportunistic infections, such as those caused by pathogenic fungi, in order to prevent the aggravation of the primary disease, cancer, or even for the treatment of infections of a bacterial and/or fungal nature.
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| BR102020013240A BR102020013240A8 (en) | 2020-06-26 | 2020-06-26 | Phenolic binucleant ligands, binuclear metallic compounds, medical-veterinary composition, binucleant ligand synthesis processes, binuclear compound synthesis process, and use |
| BR1020200132407 | 2020-06-26 |
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| WO (1) | WO2021258173A1 (en) |
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Non-Patent Citations (3)
| Title |
|---|
| MORAES RAFAELA DOS SANTOS: "Síntese e caracterização de ligantes binucleantes derivados da isoniazida e seus complexos binucleares de cobre (II)", MASTER DISSERTATION, 23 February 2011 (2011-02-23), Brazil, pages 1 - 106, XP055955750 * |
| MORAES, R. S.: "COMPLEXOS HOMO E HETEROBINUCLEARES DE LIGANTES DERIVADOS DA ISONIAZID/X COMO POTENCIES AGENTES ANTITUMORAIS", THESIS (DUTORADO EM QUIMICA -PROGRAMA DE POS GRADUATE EM QUIMICA DA PUC-RIO), 1 January 2016 (2016-01-01), Brazil, pages 1 - 163, XP009537922, DOI: 10.17771/PUCRio.acad.33320 * |
| RADA ARIAS, JESICA PAOLA: "Binucleating aroylhydrazonic ligands and their dicopper(II) complexes as new classes of potential anticancer agents syntheses, chemical characterization and biological activity", PHD THESIS, 16 August 2019 (2019-08-16), Brazil, pages 1 - 316, XP009538594 * |
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| BR102020013240A8 (en) | 2023-10-24 |
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