WO2009060114A1 - Inhibitors of enzyme o6-alkylguanine-dna-methyltransferase for cancer treatment - Google Patents
Inhibitors of enzyme o6-alkylguanine-dna-methyltransferase for cancer treatment Download PDFInfo
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- WO2009060114A1 WO2009060114A1 PCT/ES2008/070190 ES2008070190W WO2009060114A1 WO 2009060114 A1 WO2009060114 A1 WO 2009060114A1 ES 2008070190 W ES2008070190 W ES 2008070190W WO 2009060114 A1 WO2009060114 A1 WO 2009060114A1
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- Prior art keywords
- methyl
- compound
- compounds
- hagt
- dna
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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
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4704—2-Quinolinones, e.g. carbostyril
-
- 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
- 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
- C07D401/06—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 only aliphatic carbon atoms
Definitions
- the present invention is framed in the field of medical chemistry, and more specifically in that of enzyme inhibitor compounds for therapeutic use applicable in particular as adjuvants of chemotherapy cancer treatment, by decreasing the negative effects of said therapy.
- the treatment of most tumors is based on a multiple approach that includes surgical resection and radiotherapy, aimed at the main tumor, and chemotherapy, which acts throughout the body to prevent and combat the appearance of tumors in other locations. (metastasis).
- the main compounds used in chemotherapy are alkylating agents (cyclophosphamide, mechlorethamine), antimetabolites (6-mercaptopurine, 5-fluoruracil), plant-derived alkaloids (vinblastine, vincristine), and various biological molecules (antitumor antibiotics, enzymes, hormones, etc. .).
- Nitrosoureas and related compounds are similar to alkylating agents and their therapeutic effectiveness is attributed, in large part, to the cytotoxic potential of DNA-induced lesions [1]. Tumor tissues, given the higher proliferation rate of their cells, synthesize more DNA, so they are the most affected by these lesions.
- said compounds cause methylations of oxygen in position 6 of the guanine molecules of DNA, causing O 6 -methylguanine (0 6 -mG). Methylation of guanine in this position causes it to mate with a thymine (T) instead of with a cytosine (C), and can also cause GC cross-linking.
- the replication of the 0 6 -mG chain causes alterations in the new DNA chain that finally produce the inhibition of both the replication and transcription of the DNA, leading in both cases to cell death by apoptosis [1].
- the cells have mechanisms to repair the lesions of their DNA produced under normal physiological conditions, either by environmental factors or by internally generated metabolites.
- O 6 -alkylguanine-DNA-alkyltransferase the enzyme in humans is known as hAGT, from the English “human O 6 -alkylguanine-DNA alkyltransferase", which transfers the methyl group of 0 6 -mG to an amino acid of its active center (a cysteine at position 145), thus restoring the original guanine [1,2].
- hAGT the enzyme in humans
- the synthesis of hAGT is increased in the majority of tumor cells, so tumors are often resistant to chemotherapy with alkylating agents [3, 4].
- hAGT is the main cause of resistance to chemotherapy in glioblastoma, a type of malignant tumor that constitutes approximately 50% of brain tumors. Therefore, the use of hAGT inhibitors as adjuvants for chemotherapy treatments has been studied, since the effect of the alkylating agents could be reduced, their therapeutic dose could be reduced, which in turn would reduce their side effects.
- O 6 -benzylguanine (O 6 -bG) and its analogues inactivate hAGT in vitro and in vivo [2], but it has been shown in clinical trials that O 6 -BG improves the efficacy of chemo-therapeutic agents [5]
- O 6 -BG improves the efficacy of chemo-therapeutic agents [5]
- hAGT has more affinity for 0 6 -mG than for O 6 -bG, so it preferentially binds to DNA to present O 6 -mG, so that its repair activity is more favored than the inhibition by O 6 -bG.
- the inhibitors of the DNA repair function of the hAGT described so far are not sufficiently effective.
- the authors of the present invention therefore applied the methodology of drug design based on the three-dimensional structure of therapeutic targets to solve this technical problem. For this, they analyzed the structure of the hAGT [8] and were based on the hypothesis that DNA binding does not involve a significant conformational change in this enzyme, as it has been found for Ada protein (which also repairs DNA alkylations ). This hypothesis also fits with the mechanism of inversion of the rented base that is proposed for this type of enzymes [9]. The application of this methodology, at the point of confluence between Ia
- the present invention relates to the use of piperadinyl methyl tetrazol quinolinone derivatives and diphenyl triazole pyrimidine derivatives as inhibitors of DNA repair action performed by
- CGRID generates a three-dimensional mesh on the active center of the protein and allows calculating both electrostatic and van der Waals interactions between each of the center atoms active of the protein with each of the main atoms (carbon, nitrogen, oxygen, sulfur, hydrogen, phosphorus, fluorine, chlorine, bromine, iodine) of the molecules of the chemical library used.
- the edges of the three-dimensional mesh for the hAGT were located at a minimum distance of 5 A from any atom of the methylated nucleotide (such as 0 6 -mG) on which the active center of the enzyme will act, and the spacing between each point of the mesh was 0.5 A.
- CDOCK a program of analysis of the molecular fit or docking whose function of assigning values is based on the meshes calculated with CGRID.
- Three probes prototype molecules were used: benzene to detect hydrophobic zones, and water and methanol to detect areas capable of forming hydrogen bonds.
- Said compounds demonstrated hAGT inhibitory capacity in vitro and in vivo, in the latter case resulting in greater efficacy of alkylating agents to control cell proliferation (Example 2).
- the identified compounds can be grouped into two groups according to their chemical structure: compounds with the formula I (derivatives of piperadinyl methyl-tetrazol-quinolinone) and compounds with the formula Il (derived from diphenyl-triazolo-pyrimidine), hereinafter "compounds of The invention.
- a first aspect of the present invention refers to the use of a compound capable of inhibiting the action of O 6 -alkylguanine-DNA-alkyltransferase characterized in that it is a derivative of piperadinyl-methyl-tetrazol-quinolone with the general formula (in below formula I):
- - X is a carbon (C) or nitrogen (N) atom, interchangeably;
- R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkylethyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl , imidazolylpropyl, R 7 , methyl-R 7 , ethyl-R 7 , propyl-R 7 , methoxy-R 7 , ethoxy-R 7 , propoxy-R 7 , where R 7 is an aryl in which each of its five Remaining positions are substituted by a group independently selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atom
- X is nitrogen
- R 1 is 2,5-dimethylphenyl
- R 2 is hydrogen
- R 3 is methoxy
- R 4 is phenyl methyl, causing 3 - [[4- (2,5 -dimethylphenyl) -1 -piperazinyl] [1 - (phenylmethyl) -i H-tetrazol-5-yl] methyl] -6-methoxy-2 (1 / - /) - quinolinone (hereinafter, compound Ia or Pibetine, depending on denomination given by the inventors), with the formula:
- X is carbon
- R 1 is phenyl methyl
- R 2 is methyl
- R 3 is hydrogen
- R 4 is phenyl methyl
- a second aspect of the present invention refers to the use of a compound capable of inhibiting the action of O 6 -alkylguanine-DNA-alkyltransferase characterized in that it is a diphenyl-triazolo-pyrimidine derivative with the general formula (hereinafter, formula II ):
- each of the groups R and R is independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl, imidazolylpropyl, R 8 , methyl-R 8 , ethyl-R 8 , propyl-R 8 , methoxy-R 8 , ethoxy-R 8 , propoxy-R 8 , where R 8 is an aryl in which each of its five remaining positions is replaced by a independently selected group between hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atoms), alkylcarbonyl, alkylamino, and sulfonylamino.
- R 5 is phenylmethoxy and R 6 is ethyl, originating the compound called 7- [4 - (benzyloxy) phenyl] -5- (4-ethylphenyl) -4,5,6,7-tetrahydro - [1, 2,4] triazolo [1,5-a] pyrimidine (hereinafter, lia compound), with the formula:
- R 5 is phenylmethoxy and R 6 is ethoxy, causing the compound called 7- [4- (benzyloxy) phenyl] -5- (4-ethoxyphenyl) -4,5,6,7- tetrahydro- [1, 2,4] triazolo [1, 5- a] pyrimidine (hereinafter, compound Mb) with the formula:
- Example 2 the treatment of cell cultures derived from carcinomas with said compounds enhanced the therapeutic action of carmustine (1,3-Bis (2-chloroethyl) 1-nitrosourea), a frequently used alkylating agent to combat multiple myeloma, brain tumors, Hodgkin's disease, and non-Hodgkin's lymphomas, showing the therapeutic potential of the use of the compounds of the invention as co-adjuvants of chemo-therapeutic agents in the treatment of tumors.
- carmustine (1,3-Bis (2-chloroethyl) 1-nitrosourea
- a frequently used alkylating agent to combat multiple myeloma
- brain tumors a frequently used alkylating agent to combat multiple myeloma
- Hodgkin's disease Hodgkin's disease
- non-Hodgkin's lymphomas showing the therapeutic potential of the use of the compounds of the invention as co-adjuvants of chemo-therapeutic agents
- a third aspect of the present invention includes the use of isomers or mixtures of isomers of the compounds of the invention, including the isomers of the compounds Ia, Ib, Ma and Mb described herein. invention, as adjuvants of chemo-therapeutic agents.
- Such isomers comprise:
- a fourth aspect of the present invention refers to the use of prodrugs of the compounds of the invention as adjuvants of chemo-therapeutic agents.
- prodrug comprises any compound derived from formulas I or Il as defined in the present invention (for example, esters, carbamates, amides etc.), which, when administered to a individual, is able to provide, directly or indirectly, one or more of the compounds of the invention.
- said prodrug is a compound that, in comparison with the compounds of the invention, increases its bioavailability when administered to an individual, or that enhances the release of the compounds of the invention in a biological compartment (for example, in the brain or the particular tissue affected by the tumor or metastasis).
- the preparation of said prodrug can be carried out by conventional methods known to those skilled in the art, which can be found by example , in "Textbook of Drug Design and Discovery" (Krogsgaard-Larsen P, Liljefors T and U Madsen, publisher Taylor and Francis, 2002, 3rd Edition).
- a fifth aspect of the present invention refers to the use of the crystalline forms of the compounds of the invention in the free state or as solvates, as adjuvants of chemo-therapeutic agents.
- solvate includes both solvates that can be used in the composition of a medicament, and solvates useful in the preparation of said medicament. Solvates can be obtained by conventional solvation methods well known to those skilled in the art.
- a sixth aspect of the present invention refers to the use of pharmaceutically acceptable salts of the compounds of the invention as adjuvants of chemo-therapeutic agents.
- the salts of the compounds of the invention that are not pharmaceutically acceptable may also be useful in the preparation of drugs, so that all salts of the compounds of the invention, whether or not pharmaceutically acceptable, are included in the scope of the present invention. .
- a seventh aspect of the present invention relates to the use as adjuvants of chemo-therapeutic agents, of compounds of the invention containing one or more isotopically enriched atoms.
- the present invention relates to pharmaceutical compositions with a therapeutically effective amount of one or more compounds of the invention, their isomers, prodrugs, solvates or salts, both present in essentially pure form and in admixture with auxiliary agents, additives or pharmaceutically acceptable carriers and, eventually, other therapeutic agents.
- auxiliary agents, additives or carriers must be compatible with the other components of the composition and not cause damage to their receptors.
- the pharmaceutical compositions include those that are suitable for oral, rectal, nasal, topical administration (including oral, sublingual and epicutaneous), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal).
- compositions as well as the auxiliary agents, additives or carriers (diluents, solvents, disintegrants, emulsifiers, absorbents, antioxidants, lubricants, colorants, sweeteners, humectants, etc.) are well known in the pharmacy technique, and examples of both can be found in "Textbook of Drug Design and Discovery” (Krogsgaard- Larsen P, Liljefors T and U Madsen, publisher Taylor and Francis, 2002, 3rd Edition).
- the compounds of the invention can be used together with other drugs to provide combination therapy, either as part of the same pharmaceutical composition or as separate compositions, administered simultaneously or not simultaneously.
- the expression "therapeutically effective amount” refers to the amount of one or more compounds of the invention, their isomers, prodrugs, solvates or salts, capable of developing in the receptor the therapeutic effect mediated by Ia inhibition of the DNA repair activity of hAGT. That amount or dose will be determined, among other causes, by the characteristics of the compounds and their isomers, prodrugs, solvates or salts, as well as by the pharmaceutical composition containing them, the route and frequency of administration, and the age and condition of the individual subject to which it is administered.
- a particular embodiment of the present invention constitutes the use of a compound of the invention of formula I or II, its isomers, prodrugs, solvates or salts, or mixtures thereof, in the preparation of a drug or pharmaceutical composition for the treatment of cancer.
- carcinomas including those of prostate, breast, lung, pancreas, colorectal, gastric, esophageal, laryngeal, thyroid, hepatic, urinary bladder, renal, uterine, and cervical
- any type of sarcomas including osteosarcomas, soft tissue sarcomas and angiosarcomas
- any type of hematopoietic tumor including leukemia and lymphomas
- nervous system tumor including neuroblastomas, glioblastomas and astrocytomas
- dermatological cancer including melanomas, basal cell carcinomas and squamous cell carcinomas.
- said use is carried out in combination with other antitum
- Figure 1 Methodologies for the identification of small molecules that inhibit the activity of the enzyme O 6 -alkylguanine-DNA-methyl-transferase.
- the process began in two parallel ways, consisting of the virtual analysis, by computer means, of Ia three-dimensional structure, more energy-stable conformations and distribution of charges, radii and charges of the atoms that are on the surface of the molecules of a compound library (ZINC) and the active center of the enzyme.
- ZINC compound library
- the data resulting from said analysis for the more than three million compounds of the library were stored in a relational database (BBDD) in a MySQL environment.
- BBDD relational database
- FIG. 3 Formulas of specific embodiments of compounds of the invention.
- the chemical formulas of four compounds that constitute specific embodiments of the invention are shown: two compounds having the general formula I and two compounds having the general formula II.
- Compound Ia 3 - [[4- (2,5-dimethylphenyl) -1 -piperazinyl] [1 - (phenylmethyl) -1H-tetrazol-5-yl] methyl] -6-methoxy-2 (1 / - /) -quinolinone (called Pibetine by the inventors);
- compound Ib 3 - [(4-benzyl-1-piperadinyl) - (1-benzyl-1 H- 5-tetrazolyl)] methyl-7-methyl-2 (1 / - /) quinolinone compound Ma, (benzyloxy) phenyl ] - 5- (4-ethylphenyl) -4,5,6,7-tetrahydro- [1, 2,4] triazolo [1,
- FIG. 4 Structural models of the molecular fit between compounds of the invention and the active center of the hAGT enzyme.
- the illustrations show representations of the surface of hAGT, together with rod models of compounds Ia, Ib, Ma and Mb of the invention, illustrating the optimum fit between the active center of the hAGT and said compounds according to results obtained through the CDOCK program.
- the upper figure shows the remaining activity of the hAGT against the different concentrations of the compound Ia (black rhombus, ⁇ ) and Ib (gray rhombus, ⁇ ).
- the lower figure shows the remaining activity of the hAGT against the different concentrations of compound Ma (black triangle, A) and Mb (gray triangle, ⁇ ).
- the effect of different compounds of the invention is shown in concentrations in the range of 0 to 100 ⁇ M on the formation of colonies of human colorectal adenocarcinoma tumor cells untreated with the carmustine chemotherapeutic agent (black columns) or treated with a concentration of 40 ⁇ M of said agent (gray columns).
- Example 1 Inhibition of DNA repair activity of hAGT in vitro by compounds Ia, Ib, Ma and Mb.
- E. coli cells were transformed with vectors derived from plasmids pet21 and pet28, including in the first case a sequence of the hAGT (plasmid pet21-hAGT) and in the second the mutant sequence (originating the vector pet28-hAGT- ⁇ C177- C145S).
- Transformed bacteria were selected by growth in LB-agar medium with ampicillin (100 ⁇ g / ml) to select bacterial colonies containing the pet21-hAGT vector or with kanamycin (50 ⁇ g / ml) to select those containing the pet28-hAGT- vector T- ⁇ C177-C145S.
- the cell rupture was carried out in a Fisher Bioblock Scientific 75042 sonicator, after which the solutions were centrifuged at 185844 g for 45 min. The supernatant was filtered through cellulose acetate filters with pores 45 ⁇ M in diameter (Sarstedt) and the filtrate was applied to a HiTrap TM FF column (Amersham Biosciences).
- the protein fractions obtained were concentrated by centrifugation with Amicon Ultra-15 devices (Millipore) and purified by molecular sieve chromatography on a Superdex 75 16/60 column (Amersham Biosciences), using as a 150 mM NaCI buffer solution, 10 mM dithiothreitol ( DTT) and 0.1 mM ethylene diamino-tetra-acetate (EDTA).
- fractions corresponding to the eluted protein of this column were again concentrated by Amicon Ultra-15 devices and were taken to a concentration of 2 mg / ml in the same buffer in the presence of 40% glycerol, for preservation at -2O 0 C until later use.
- DMSO dimethyl sulfoxide
- the reaction was initiated by the addition of substrate, [3 H] -methyl-DNA, and incubated at 37 0 C for 90 minutes.
- the final volume of the reactions was 200 ⁇ l, the final concentrations of the hAGT and the [ 3 H] -methyl-DNA being 0.5 ⁇ M and 50 ⁇ M, respectively. All reactions were stopped by the addition of 400 ⁇ l of a 13% aqueous solution of trichloroacetic acid (TCA, Sigma).
- TCA trichloroacetic acid
- the radioactivity corresponding to the H-methyl group 3 incorporated into the hAGT during the enzymatic DNA repair reaction was measured with a liquid scintillation counter (Wallac 1414), using the Optiphase HiSafe 3 scintillation cocktail (Perkin Elmer).
- This remaining activity of the hAGT after the preincubation with the compounds of the invention was calculated in relation to the positive controls, in which the enzyme was pre-incubated only with the DMSO solvent (with no inhibitor compound).
- As a negative control tests were used with the enzyme hAGT-C145S, mutant without activity. All tests were performed in duplicate, and in each of them the different concentrations of the compounds were performed in quadruplicate. The results are expressed by the percentage of remaining hAGT activity in relation to that determined for the positive controls (in which there is no inhibition of the repair activity of said enzyme).
- Example 2 Optimization of the antitumor effect of carmustine (alkylating agent) by inhibition by compounds Ia, Ma and Mb of the hAGT activity in vivo.
- Colony formation test 15,000 cells were seeded in each of the 6 wells of culture plates (Falcon) using RPMI 1640 (Genycell) as a medium supplemented with 10% fetal bovine serum (Biowhittaker). After an incubation for 48 hours at 37 ° C, the medium was replaced and different concentrations of the compounds Ia, Ma and Mb (0.5, 10, 50 and 100 ⁇ M) were added to the new medium.
- hAGT The efficacy of hAGT to inhibit cell growth was determined by counting the colonies formed using cresyl violet staining (violet crystal, Merck). After incubation in the staining solution for 30 minutes at room temperature, the colonies were washed several times with distilled and deionized water (MiIIiQ) and allowed to dry at room temperature. Finally, they were resuspended with a 10% aqueous solution of acetic acid and the absorbance of the resuspensions at 590 nm was measured.
- Active and alkylated human AGT structures a novel zinc site, inhibitor and extrahelical base binding. Embo J, 19 (7), 1719-1730. (2000).
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Abstract
The invention relates to the use of compounds derived from piperadinyl-methyl-tetrazole-quinolinone and derived from diphenyl-triazolo- pyrimidine as inhibitors of the reparative action of the DNA produced by enzyme O6-alkylguanine-DNA-methyltransferase, and to pharmaceutical compositions containing same, for the preparation of coadjuvant drugs for use in antitumour therapy using alkylating agents.
Description
INHIBIDORES DE LA ENZIMA O6-ALQUILGUANINA-ADN-METIL- TRANSFERASA PARA EL TRATAMIENTO DEL CÁNCER.INHIBITORS OF ENZYME OR 6- ALKYLGUANINE-DNA-METHYL-TRANSFER FOR THE TREATMENT OF CANCER.
SECTOR DE LA TÉCNICASECTOR OF THE TECHNIQUE
La presente invención se enmarca en el campo de Ia química médica, y más concretamente en el de compuestos inhibidores de enzimas para uso terapéutico aplicables en particular como adyuvantes del tratamiento del cáncer por quimioterapia, al disminuir los efectos negativos de dicha terapia.The present invention is framed in the field of medical chemistry, and more specifically in that of enzyme inhibitor compounds for therapeutic use applicable in particular as adjuvants of chemotherapy cancer treatment, by decreasing the negative effects of said therapy.
ESTADO DE LA TÉCNICASTATE OF THE TECHNIQUE
El tratamiento de Ia mayor parte de los tumores se basa en una aproximación múltiple que incluye Ia resección quirúrgica y Ia radioterapia, dirigidas al tumor principal, y Ia quimioterapia, que actúa en todo el organismo para prevenir y combatir Ia aparición de tumores en otras localizaciones (metástasis). Los principales compuestos usados en quimioterapia son los agentes alquilantes (ciclofosfamida, mecloretamina), antimetabolitos (6-mercaptopurina, 5-fluoruracilo), alcaloides derivados de plantas (vinblastina, vincristina), y diversas moléculas biológicas (antibióticos antitumorales, enzimas, hormonas, etc.).The treatment of most tumors is based on a multiple approach that includes surgical resection and radiotherapy, aimed at the main tumor, and chemotherapy, which acts throughout the body to prevent and combat the appearance of tumors in other locations. (metastasis). The main compounds used in chemotherapy are alkylating agents (cyclophosphamide, mechlorethamine), antimetabolites (6-mercaptopurine, 5-fluoruracil), plant-derived alkaloids (vinblastine, vincristine), and various biological molecules (antitumor antibiotics, enzymes, hormones, etc. .).
Las nitrosoureas y los compuestos relacionados (procarbazina, estreptozina, temozolomida) son similares a los agentes alquilantes y su efectividad terapéutica se atribuye, en buena parte, al potencial citotóxico de las lesiones que inducen en el ADN [1]. Los tejidos tumorales, dada Ia mayor tasa de proliferación de sus células, sintetizan más ADN, por Io que son los más afectados por estas lesiones. En particular dichos compuestos provocan metilaciones del oxigeno en posición 6 de las moléculas de guanina del ADN, originando O6-metilguanina (06-mG). La metilación de Ia guanina en esta posición provoca que se aparee con una timina (T) en vez de con una citosina (C), y también puede provocar entrecruzamientos G-C
por Io que Ia replicación de Ia cadena con 06-mG origina alteraciones en Ia nueva cadena de ADN que finalmente producen Ia inhibición tanto de Ia replicación como de Ia transcripción del ADN, conduciendo en ambos casos a muerte celular por apoptosis [1]. Sin embargo las células presentan mecanismos para reparar las lesiones de su ADN producidas en condiciones fisiológicas normales, ya sea por factores medioambientales o por metabolitos generados internamente. En particular Ia resistencia a los compuestos alquilantes de O6 está mediada por Ia enzima O6-alquilguanina-ADN-alquiltransferasa (Ia enzima en humanos es conocida como hAGT, del inglés "human O6- alkylguanine-DNA alkyltransferase"), que transfiere el grupo metilo de Ia 06-mG a un aminoácido de su centro activo (un cisteína en posición 145), restaurando así Ia guanina original [1 , 2]. La síntesis de Ia hAGT se encuentra aumentada en Ia mayoría de las células tumorales, por Io que a menudo los tumores son resistentes a Ia quimioterapia con agentes alquilantes [3, 4]. Así por ejemplo, Ia acción de Ia hAGT es Ia causa principal de Ia resistencia a quimioterapia en el glioblastoma, un tipo de tumor maligno que constituye aproximadamente el 50% de los tumores cerebrales. Se ha estudiado por tanto el uso de inhibidores de Ia hAGT como coadyuvantes para los tratamientos de quimioterapia, ya que al potenciarse el efecto de los agentes alquilantes podría reducirse su dosis terapéutica, Io que a su vez supondría una disminución de sus efectos secundarios. La O6-benzilguanina (O6-bG) y sus análogos inactivan hAGT in vitro e in vivo [2], pero aunque se ha demostrado en ensayos clínicos que Ia O6-BG mejora Ia eficacia de agentes quimio-terapéuticos [5], existen limitaciones considerables para su uso terapéutico: su toxicidad sobre Ia mielina de las células nerviosas, su escasa solubilidad en agua y su rápida degradación en el plasma celular [6]. Además, Ia hAGT tiene más afinidad por Ia 06-mG que por Ia O6-bG, por Io que se une preferentemente al ADN
que presente O6-mG, de modo que su actividad reparadora está más favorecida que Ia inhibición por O6-bG.Nitrosoureas and related compounds (procarbazine, streptozine, temozolomide) are similar to alkylating agents and their therapeutic effectiveness is attributed, in large part, to the cytotoxic potential of DNA-induced lesions [1]. Tumor tissues, given the higher proliferation rate of their cells, synthesize more DNA, so they are the most affected by these lesions. In particular, said compounds cause methylations of oxygen in position 6 of the guanine molecules of DNA, causing O 6 -methylguanine (0 6 -mG). Methylation of guanine in this position causes it to mate with a thymine (T) instead of with a cytosine (C), and can also cause GC cross-linking. Therefore, the replication of the 0 6 -mG chain causes alterations in the new DNA chain that finally produce the inhibition of both the replication and transcription of the DNA, leading in both cases to cell death by apoptosis [1]. However, the cells have mechanisms to repair the lesions of their DNA produced under normal physiological conditions, either by environmental factors or by internally generated metabolites. In particular, resistance to the alkylating compounds of O 6 is mediated by the enzyme O 6 -alkylguanine-DNA-alkyltransferase (the enzyme in humans is known as hAGT, from the English "human O 6 -alkylguanine-DNA alkyltransferase"), which transfers the methyl group of 0 6 -mG to an amino acid of its active center (a cysteine at position 145), thus restoring the original guanine [1,2]. The synthesis of hAGT is increased in the majority of tumor cells, so tumors are often resistant to chemotherapy with alkylating agents [3, 4]. Thus, for example, the action of hAGT is the main cause of resistance to chemotherapy in glioblastoma, a type of malignant tumor that constitutes approximately 50% of brain tumors. Therefore, the use of hAGT inhibitors as adjuvants for chemotherapy treatments has been studied, since the effect of the alkylating agents could be reduced, their therapeutic dose could be reduced, which in turn would reduce their side effects. O 6 -benzylguanine (O 6 -bG) and its analogues inactivate hAGT in vitro and in vivo [2], but it has been shown in clinical trials that O 6 -BG improves the efficacy of chemo-therapeutic agents [5] There are considerable limitations for its therapeutic use: its toxicity on the myelin of nerve cells, its poor water solubility and its rapid degradation in cellular plasma [6]. In addition, hAGT has more affinity for 0 6 -mG than for O 6 -bG, so it preferentially binds to DNA to present O 6 -mG, so that its repair activity is more favored than the inhibition by O 6 -bG.
Algunos estudios recientes han mostrado que los oligodesoxirribonucleótidos que contienen O6-bG inactivan más eficazmente a Ia hAGT [7]. En concreto, se ha demostrado el potencial inhibidor in vitro de moléculas de 11 desoxirribonucleótidos que contienen O6-bG modificadas con enlaces metilfosfonato terminales para protegerlas de Ia degradación por nucleasas. Sin embargo, en modelos celulares el potencial inhibidor de Ia hAGT por estas cadenas de desoxirribonucleótidos se redujo en una escala de unas 1000 veces en comparación a Io observado in vitro, sugiriendo que Ia incorporación de estas moléculas por las células es un factor limitante de su acción terapéutica.Some recent studies have shown that oligodeoxyribonucleotides containing O 6 -bG inactivate hAGT more effectively [7]. Specifically, the potential in vitro inhibitor of 11 deoxyribonucleotide molecules containing O 6 -bG modified with terminal methylphosphonate bonds to protect them from nuclease degradation has been demonstrated. However, in cellular models the potential inhibitor of hAGT by these deoxyribonucleotide chains was reduced on a scale of about 1000 times compared to what was observed in vitro, suggesting that the incorporation of these molecules by the cells is a limiting factor of their therapeutic action
Así pues, los inhibidores de Ia función reparadora de ADN de Ia hAGT descritos hasta ahora no son suficientemente efectivos. Los autores de Ia presente invención aplicaron por tanto Ia metodología del diseño de fármacos basado en Ia estructura tridimensional de las dianas terapéuticas para solventar este problema técnico. Para ello analizaron Ia estructura de Ia hAGT [8] y se fundamentaron en Ia hipótesis de que Ia unión del ADN no comporta un cambio conformacional significativo en esta enzima, al igual que se ha encontrado para Ia proteína Ada (que también repara alquilaciones del ADN). Esta hipótesis encaja asimismo con el mecanismo de inversión de Ia base alquilada que se plantea para este tipo de enzimas [9]. La aplicación de esta metodología, en el punto de confluencia entre IaThus, the inhibitors of the DNA repair function of the hAGT described so far are not sufficiently effective. The authors of the present invention therefore applied the methodology of drug design based on the three-dimensional structure of therapeutic targets to solve this technical problem. For this, they analyzed the structure of the hAGT [8] and were based on the hypothesis that DNA binding does not involve a significant conformational change in this enzyme, as it has been found for Ada protein (which also repairs DNA alkylations ). This hypothesis also fits with the mechanism of inversion of the rented base that is proposed for this type of enzymes [9]. The application of this methodology, at the point of confluence between Ia
Química, Ia Biología y Ia Bioinformática ha permitido identificar dos familias de compuestos con gran afinidad por el centro activo de Ia proteína hAGT y que poseen Ia capacidad de inhibir su actividad biológica de forma relevante.
DESCRIPCIÓN DETALLADA DE LA INVENCIÓNChemistry, Biology and Bioinformatics has allowed us to identify two families of compounds with great affinity for the active center of the hAGT protein and that have the ability to inhibit their biological activity in a relevant way. DETAILED DESCRIPTION OF THE INVENTION
La presente invención se relaciona con el uso de derivados de piperadinil-metil-tetrazol-quinolinona y derivados de difenil-triazolo- pirimidina como inhibidores de Ia acción reparadora del ADN realizada porThe present invention relates to the use of piperadinyl methyl tetrazol quinolinone derivatives and diphenyl triazole pyrimidine derivatives as inhibitors of DNA repair action performed by
Ia enzima O6-alquilguanina-ADN-alquiltransferasa y con composiciones farmacéuticas que los contienen, para Ia preparación de medicamentos coadyuvantes de Ia terapia antitumoral basada en agentes alquilantes.The enzyme O 6 -alkylguanine-DNA-alkyltransferase and with pharmaceutical compositions containing them, for the preparation of adjuvant drugs of the antitumor therapy based on alkylating agents.
El procedimiento para identificar familias de dichos compuestos se inició con el cribado virtual de Ia base de datos de compuestos químicos (quimioteca) ZINC [10], que contiene más de dos millones de compuestos. En dicho cribado se buscaron candidatos a interaccionar con el centro activo de Ia hAGT, particularmente compuestos que encajaran en el surco hidrofóbico que presenta Ia superficie de dicho centro activo y que está definido por los aminoácidos Metionina 134, Prolina 140 y el "lazo" del centro activo (comprendido entre los aminoácidos Valina 155 y Glicina 160, inclusive). La interacción o encaje molecular ("docking") de los compuestos de Ia quimioteca en dicho surco se analizó virtualmente según el proceso esquematizado en Ia Figura 1. La estructura inicial de Ia hAGT se obtuvo de Ia base de datos PDBThe procedure to identify families of said compounds began with the virtual screening of the database of chemical compounds (chemical library) ZINC [10], which contains more than two million compounds. In said screening, candidates were sought to interact with the active center of the hAGT, particularly compounds that fit into the hydrophobic groove that presents the surface of said active center and which is defined by the amino acids Methionine 134, Proline 140 and the "loop" of the active center (between the amino acids Valine 155 and Glycine 160, inclusive). The interaction or molecular fit ("docking") of the compounds of the library in said groove was analyzed virtually according to the process outlined in Figure 1. The initial structure of the hAGT was obtained from the PDB database
(RCSB Protein Data Bank, disponible en www.rcsb.org/pdb/ [11]). De dicha base se tomó un modelo de Ia proteína, del que se descartaron las moléculas de agua y ADN que aparecían interaccionando con Ia misma. A este modelo se Ie añadieron los átomos de hidrógeno con el programa protonate del paquete AMBER [12], y se Ie asignaron cargas y radios atómicos a todos los átomos de Ia proteína utilizando el mismo paquete. A continuación, se caracterizó energéticamente el centro activo mediante Ia combinación de tres programas propios desarrollados por los inventores:(RCSB Protein Data Bank, available at www.rcsb.org/pdb/ [11]). From this base a model of the protein was taken, from which the water and DNA molecules that appeared interacting with it were discarded. To this model hydrogen atoms were added with the protonate program of the AMBER package [12], and atomic charges and radii were assigned to all the atoms of the protein using the same package. Next, the active center was energetically characterized by the combination of three proprietary programs developed by the inventors:
• CGRID [13], genera una malla tridimensional sobre el centro activo de Ia proteína y permite calcular tanto las interacciones electrostáticas como las de tipo van der Waals entre cada uno de los átomos de centro
activo de Ia proteína con cada uno de los átomos principales (carbono, nitrógeno, oxígeno, azufre, hidrógeno, fósforo, flúor, cloro, bromo, yodo) de las moléculas de Ia quimioteca utilizada. Los bordes de Ia malla tridimensional para Ia hAGT se situaron a un mínimo de distancia de 5 A de cualquier átomo del nucleótido metilado (tal como Ia 06-mG) sobre el que actuará el centro activo de Ia enzima, y el espaciado entre cada punto de Ia malla fue de 0.5 A.• CGRID [13], generates a three-dimensional mesh on the active center of the protein and allows calculating both electrostatic and van der Waals interactions between each of the center atoms active of the protein with each of the main atoms (carbon, nitrogen, oxygen, sulfur, hydrogen, phosphorus, fluorine, chlorine, bromine, iodine) of the molecules of the chemical library used. The edges of the three-dimensional mesh for the hAGT were located at a minimum distance of 5 A from any atom of the methylated nucleotide (such as 0 6 -mG) on which the active center of the enzyme will act, and the spacing between each point of the mesh was 0.5 A.
• CDOCK [13], un programa de análisis del encaje molecular o docking cuya función de asignación de valores se basa en las mallas calculadas con CGRID. Se utilizaron 3 sondas (moléculas prototipo): benceno para detectar zonas hidrofóbicas, y agua y metanol para detectar zonas susceptibles de formar puentes de hidrógeno.• CDOCK [13], a program of analysis of the molecular fit or docking whose function of assigning values is based on the meshes calculated with CGRID. Three probes (prototype molecules) were used: benzene to detect hydrophobic zones, and water and methanol to detect areas capable of forming hydrogen bonds.
• GAGA [14], que comprime Ia información obtenida por CDOCK para una sonda en una serie de funciones gaussianas que actúan como puntos farmacofóricos.• GAGA [14], which compresses the information obtained by CDOCK for a probe in a series of Gaussian functions that act as pharmacoforic points.
La "preparación" de los compuestos de Ia quimioteca ZINC para su cribado basado en el posible encaje molecular con el centro activo de hAGT se inició con el cálculo de las coordenadas tridimensionales de todos los compuestos de esta quimioteca mediante el programa CORINA [Corina Molecular Networks, Erlangen, Alemania]. Este programa permite además generar todas las combinaciones tridimensionales de un determinado compuesto de Ia quimioteca a través de las diferentes conformaciones de sus anillos y estereoisómeros. Con las coordenadas tridimensionales se realizó un análisis conformacional de todos estas combinaciones mediante el programa ALFA, desarrollado por los autores de Ia presente invención. El análisis permitió obtener las conformaciones más estables energéticamente de cada compuesto, asignándoles a su vez radios tipo AMBER a todos sus átomos [12]. Por último, se calcularon las cargas parciales de cada átomo mediante el paquete MOPAC [15]. Este cálculo se realizó para cada una de las estructuras tridimensionales
obtenidas con el programa CORINA. Toda Ia información que se fue generando (estructuras tridimensionales de las conformaciones con sus radios y cargas) se almacenó en una base de datos relacional en MySQL [disponible en www.mysql.com]. En total se obtuvo información de 2.288.455 compuestos en formato bidimensional (SMILES, Daylight Chemical Information Systems, Inc.), que fue procesada e incluida en Ia base de datos relacional.The "preparation" of the compounds of the ZINC library for screening based on the possible molecular fit with the active center of hAGT began with the calculation of the three-dimensional coordinates of all the compounds of this library through the CORINA program [Corina Molecular Networks , Erlangen, Germany]. This program also allows generating all three-dimensional combinations of a certain compound of the library through the different conformations of its rings and stereoisomers. With the three-dimensional coordinates, a conformational analysis of all these combinations was carried out using the ALFA program, developed by the authors of the present invention. The analysis allowed to obtain the most stable conformations of each compound, in turn assigning AMBER radii to all its atoms [12]. Finally, the partial charges of each atom were calculated using the MOPAC package [15]. This calculation was performed for each of the three-dimensional structures obtained with the CORINA program. All the information that was generated (three-dimensional structures of the conformations with their radii and loads) was stored in a relational database in MySQL [available at www.mysql.com]. In total, 2,288,455 compounds were obtained in two-dimensional format (SMILES, Daylight Chemical Information Systems, Inc.), which was processed and included in the relational database.
Las posiciones más probables de estos compuestos en su encaje con el centro activo se obtuvieron después de Ia utilización de dos programas de filtros. El programa DOCK [16] se utilizó para asignar a cada geometría centro activo - compuesto un valor (ZScore) en función de los contactos entre ambas moléculas. Se seleccionaron aquellos compuestos que presentaban geometrías con valores de ZScore iguales o superiores a 5 para ser analizados exhaustivamente con el programa CDOCK [17]. De los 976 millones de conformaciones de los compuestos de ZINC se seleccionaron 4.787 para su cribado exhaustivo con CDOCK. Todos los resultados obtenidos, tanto del filtrado inicial como del filtrado exhaustivo se almacenaron en Ia base de datos relacional. Para Ia identificación final de compuestos con potencial inhibidor de Ia hAGT se llevó a cabo una actualización de los valores energéticos de los resultados obtenidos con CDOCK incorporando una componente coulómbica más refinada y los términos necesarios para Ia desolvatación del centro activo y del compuesto (puesto que ambas moléculas inicialmente se encuentran en solución en Ia célula, deben dejar de reaccionar con el solvente en las regiones en las que interaccionan entre sí). La actualización se llevó a cabo mediante Ia resolución de Ia ecuación de Poisson implementada en el paquete DelPhi (http://wiki.c2b2. columbia.edu/honiglab_public/index.php/Software). También se añadió Ia componente no polar, calculada como un valor proporcional al área de superficie accesible al solvente (S>4S>4) [18]. Así
pues, fue posible realizar una reclasificación de los resultados en función de Ia suma de las energías de van der Waals, coulómbica, desolvatación del centro activo y del compuesto potencialmente inhibidor y componente no polar. A partir de esta nueva clasificación se inspeccionaron visualmente los resultados de los compuestos que presentaban los 100 mejores resultados y se seleccionaron 21 compuestos dentro de dicho grupo que mostraban Ia mayor afinidad frente al centro activo de Ia hAGT.The most probable positions of these compounds in their fit with the active center were obtained after the use of two filter programs. The DOCK program [16] was used to assign each active center geometry - compound a value (ZScore) based on the contacts between both molecules. Those compounds that presented geometries with ZScore values equal to or greater than 5 were selected to be thoroughly analyzed with the CDOCK program [17]. Of the 976 million conformations of the ZINC compounds, 4,787 were selected for thorough screening with CDOCK. All the results obtained, both from the initial filtering and the exhaustive filtering were stored in the relational database. For the final identification of compounds with potential hAGT inhibitor, an update of the energy values of the results obtained with CDOCK was carried out incorporating a more refined coulombic component and the terms necessary for the desolvation of the active center and the compound (since both molecules are initially in solution in the cell, they must stop reacting with the solvent in the regions in which they interact with each other). The update was carried out by means of the resolution of the Poisson equation implemented in the DelPhi package (http: //wiki.c2b2. Columbia.edu/honiglab_public/index.php/Software). The non-polar component was also added, calculated as a value proportional to the surface area accessible to the solvent (S>4S> 4) [18]. So therefore, it was possible to reclassify the results based on the sum of van der Waals energies, coulombic, desolvation of the active center and the potentially inhibitory compound and non-polar component. From this new classification, the results of the compounds that presented the 100 best results were visually inspected and 21 compounds were selected within said group that showed the highest affinity against the active center of the hAGT.
Dichos compuestos demostraron capacidad inhibidora de Ia hAGT in vitro e in vivo, resultando en el último caso en una mayor eficacia de los agentes alquilantes para controlar Ia proliferación celular (Ejemplo 2). Los compuestos identificados pueden agruparse en dos grupos según su estructura química: compuestos con Ia fórmula I (derivados de piperadinil- metil-tetrazol-quinolinona) y compuestos con Ia fórmula Il (deridados de difenil-triazolo-pirimidina), en adelante "compuestos de Ia invención". Así, un primer aspecto de Ia presente invención se refiere al uso de un compuesto capaz de inhibir Ia acción de Ia O6-alquilguanina-ADN- alquiltransferasa caracterizado porque es un derivado de piperadinil-metil- tetrazol-quinolona con Ia fórmula general (en adelante fórmula I):Said compounds demonstrated hAGT inhibitory capacity in vitro and in vivo, in the latter case resulting in greater efficacy of alkylating agents to control cell proliferation (Example 2). The identified compounds can be grouped into two groups according to their chemical structure: compounds with the formula I (derivatives of piperadinyl methyl-tetrazol-quinolinone) and compounds with the formula Il (derived from diphenyl-triazolo-pyrimidine), hereinafter "compounds of The invention. " Thus, a first aspect of the present invention refers to the use of a compound capable of inhibiting the action of O 6 -alkylguanine-DNA-alkyltransferase characterized in that it is a derivative of piperadinyl-methyl-tetrazol-quinolone with the general formula (in below formula I):
en Ia que:in which:
- X es un átomo de carbono (C) o nitrógeno (N), indistintamente;
- R1, R2, R3 y R4 son iguales o diferentes y están independientemente seleccionados del grupo constituido por: hidrógeno, alquilo lineal, alquilo ramificado, cicloalquilmetil, cicloalquiletil, aminocarbonilalquil, alquiloxi lineal, alquiloxi ramificado, furilmetil, imidazolilmetil, imidazoliletil, imidazolilpropil, R7, metil-R7, etil-R7, propil-R7, metoxi-R7, etoxi-R7, propoxi- R7, donde R7 es un arilo en el que cada una de sus cinco posiciones restantes está sustituida por un grupo independientemente seleccionado entre hidrógeno, flúor, cloro, bromo, trifluorometilo, hidroxi, alcoxi (alquilóxido de 1 a 4 átomos de C), alquilcarbonil, alquilamino, y sulfonilamino.- X is a carbon (C) or nitrogen (N) atom, interchangeably; - R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkylethyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl , imidazolylpropyl, R 7 , methyl-R 7 , ethyl-R 7 , propyl-R 7 , methoxy-R 7 , ethoxy-R 7 , propoxy-R 7 , where R 7 is an aryl in which each of its five Remaining positions are substituted by a group independently selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atoms), alkylcarbonyl, alkylamino, and sulfonylamino.
En una realización particular de Ia presente invención X es nitrógeno, R1 es 2,5-dimetilfenil, R2 es hidrógeno, R3 es metoxi y R4 es fenil-metil, originando Ia 3-[[4-(2,5-dimetilfenil)-1 -piperazinil][1 -(fenilmetil)-i H-tetrazol- 5-il]metil]-6-metoxi-2(1/-/)-quinolinona (en adelante, compuesto Ia o Pibetina, según denominación dada por los inventores), con Ia fórmula:In a particular embodiment of the present invention X is nitrogen, R 1 is 2,5-dimethylphenyl, R 2 is hydrogen, R 3 is methoxy and R 4 is phenyl methyl, causing 3 - [[4- (2,5 -dimethylphenyl) -1 -piperazinyl] [1 - (phenylmethyl) -i H-tetrazol-5-yl] methyl] -6-methoxy-2 (1 / - /) - quinolinone (hereinafter, compound Ia or Pibetine, depending on denomination given by the inventors), with the formula:
En otra realización particular de Ia presente invención, X es carbono, R1 es fenil-metil, R2 es metil, R3 es hidrógeno y R4 es fenil-metil, originando Ia 3-[(4-bencil-1-piperadinil)-(1-bencil-1H-5-tetrazolil)]metil-7-metil- 2(1/-/)quinolinona (en adelante compuesto Ib), con Ia fórmula:
In another particular embodiment of the present invention, X is carbon, R 1 is phenyl methyl, R 2 is methyl, R 3 is hydrogen and R 4 is phenyl methyl, causing 3 - [(4-benzyl-1-piperadinyl) ) - (1-Benzyl-1H-5-tetrazolyl)] methyl-7-methyl- 2 (1 / - /) quinolinone (hereinafter compound Ib), with the formula:
Un segundo aspecto de Ia presente invención se refiere al uso de un compuesto capaz de inhibir Ia acción de Ia O6-alquilguanina-ADN- alquiltransferasa caracterizado porque es un derivado de difenil-triazolo- pirimidina con Ia fórmula general (en adelante, fórmula II):A second aspect of the present invention refers to the use of a compound capable of inhibiting the action of O 6 -alkylguanine-DNA-alkyltransferase characterized in that it is a diphenyl-triazolo-pyrimidine derivative with the general formula (hereinafter, formula II ):
en Ia que cada uno de los grupos R y R está independientemente seleccionado del grupo constituido por: hidrógeno, alquilo lineal, alquilo ramificado, cicloalquilmetil, cicloalquiletil, aminocarbonilalquil, alquiloxi lineal, alquiloxi ramificado, furilmetil, imidazolilmetil, imidazoliletil, imidazolilpropil, R8, metil-R8, etil-R8, propil-R8, metoxi-R8, etoxi-R8, propoxi- R8, donde R8 es un arilo en el que cada una de sus cinco posiciones restantes está sustituida por un grupo independientemente seleccionado
entre hidrógeno, flúor, cloro, bromo, trifluorometilo, hidroxi, alcoxi (alquilóxido de 1 a 4 átomos de C), alquilcarbonil, alquilamino, y sulfonilamino.in which each of the groups R and R is independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl, imidazolylpropyl, R 8 , methyl-R 8 , ethyl-R 8 , propyl-R 8 , methoxy-R 8 , ethoxy-R 8 , propoxy-R 8 , where R 8 is an aryl in which each of its five remaining positions is replaced by a independently selected group between hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atoms), alkylcarbonyl, alkylamino, and sulfonylamino.
En otra realización particular de Ia presente invención R5 es fenilmetoxi y R6 es etil, originando el compuesto denominado 7-[4 -(benciloxi)fenil]-5- (4-etilfenil)-4,5,6,7-tetrahidro-[1 ,2,4]triazolo[1 ,5-a]pirimidina (en adelante, compuesto lia), con Ia fórmula:In another particular embodiment of the present invention R 5 is phenylmethoxy and R 6 is ethyl, originating the compound called 7- [4 - (benzyloxy) phenyl] -5- (4-ethylphenyl) -4,5,6,7-tetrahydro - [1, 2,4] triazolo [1,5-a] pyrimidine (hereinafter, lia compound), with the formula:
En una cuarta realización particular de Ia presente invención R5 es fenilmetoxi y R6 es etoxi, originando el compuesto denominado 7-[4- (benciloxi)fenil]-5-(4-etoxifenil)-4,5,6,7-tetrahidro-[1 ,2,4]triazolo[1 ,5- a]pirimidina (en adelante, compuesto Mb) con Ia fórmula:
In a fourth particular embodiment of the present invention R 5 is phenylmethoxy and R 6 is ethoxy, causing the compound called 7- [4- (benzyloxy) phenyl] -5- (4-ethoxyphenyl) -4,5,6,7- tetrahydro- [1, 2,4] triazolo [1, 5- a] pyrimidine (hereinafter, compound Mb) with the formula:
Los modelos tridimensionales de unión entre los compuestos de Ia invención descritos en las realizaciones particulares (compuestos Ia - o Pibetina- , Ib, Ma y Mb) con el centro activo de Ia hAGT se muestran en las Figuras 6, 7, 8 y 9. El análisis de los efectos de dichos compuestos sobre Ia hAGT demostró que los mismos son capaces de inhibir competitivamente Ia actividad reparadora de esta enzima en presencia de su sustrato natural, Ia cadena de ADN metilada.The three-dimensional models of union between the compounds of the invention described in the particular embodiments (compounds Ia - or Pibetina-, Ib, Ma and Mb) with the active center of the hAGT are shown in Figures 6, 7, 8 and 9. The analysis of the effects of said compounds on hAGT showed that they are capable of competitively inhibiting the repair activity of this enzyme in the presence of its natural substrate, the methylated DNA chain.
Asimismo, según se muestra en el Ejemplo 2, el tratamiento de cultivos de células derivadas de carcinomas con dichos compuestos potenció Ia acción terapéutica de Ia carmustina (1 ,3-Bis(2-cloroetil)1-nitrosourea), un agente alquilante empleado frecuentemente para combatir el mieloma múltiple, tumores del cerebro, enfermedad de Hodgkin's, y linfomas no Hodgkin's, mostrando el potencial terapéutico del uso de los compuestos de Ia invención como co-adyuvantes de agentes quimio-terapéuticos en el tratamiento de tumores.Also, as shown in Example 2, the treatment of cell cultures derived from carcinomas with said compounds enhanced the therapeutic action of carmustine (1,3-Bis (2-chloroethyl) 1-nitrosourea), a frequently used alkylating agent to combat multiple myeloma, brain tumors, Hodgkin's disease, and non-Hodgkin's lymphomas, showing the therapeutic potential of the use of the compounds of the invention as co-adjuvants of chemo-therapeutic agents in the treatment of tumors.
Un tercer aspecto de Ia presente invención incluye el uso de isómeros o mezclas de isómeros de los compuestos de Ia invención, incluyendo los isómeros de los compuestos Ia, Ib, Ma y Mb descritos en Ia presente
invención, como coadyuvantes de agentes quimio-terapéuticos. Dichos isómeros comprenden:A third aspect of the present invention includes the use of isomers or mixtures of isomers of the compounds of the invention, including the isomers of the compounds Ia, Ib, Ma and Mb described herein. invention, as adjuvants of chemo-therapeutic agents. Such isomers comprise:
- isómeros en disposición Z o E definidos por Ia posición de los sustituyentes respecto a los enlaces múltiples que presentan los compuestos,- isomers in Z or E arrangement defined by the position of the substituents with respect to the multiple bonds that the compounds have,
- isómeros ópticos o enantiómeros, originados por Ia presencia de centros quirales (átomos unidos a cuatro sustituyentes diferentes) que provocan Ia diferenciación entre enantiómeros respecto a su actividad óptica (rotación de Ia luz polarizada al pasar a través de una solución del enantiómero),- optical isomers or enantiomers, caused by the presence of chiral centers (atoms attached to four different substituents) that cause the differentiation between enantiomers with respect to their optical activity (rotation of the polarized light when passing through a solution of the enantiomer),
- diastereoisómeros, isómeros con al menos dos centros quirales, estando los sustituyentes de uno de dichos centros dispuestos igual en ambos diastereoisómeros y los sustituyentes de al menos otro de los centros dispuestos de forma distinta entre ambos.- diastereoisomers, isomers with at least two chiral centers, the substituents of one of said centers being arranged equally in both diastereoisomers and the substituents of at least one of the centers arranged differently between the two.
Un cuarto aspecto de Ia presente invención se refiere al uso de profármacos de los compuestos de Ia invención como coadyuvantes de agentes quimio-terapéuticos. El término "profármaco", tal como aquí se utiliza comprende cualquier compuesto derivado de las fórmulas I ó Il tal y como se definen en Ia presente invención (por ejemplo, esteres, carbamatos, amidas etc.), que, cuando se administra a un individuo, es capaz de proporcionarle, directa o indirectamente, uno o varios de los compuestos de Ia invención.A fourth aspect of the present invention refers to the use of prodrugs of the compounds of the invention as adjuvants of chemo-therapeutic agents. The term "prodrug", as used herein, comprises any compound derived from formulas I or Il as defined in the present invention (for example, esters, carbamates, amides etc.), which, when administered to a individual, is able to provide, directly or indirectly, one or more of the compounds of the invention.
En una realización preferente de Ia presente invención, dicho profármaco es un compuesto que, en comparación con los compuestos de Ia invención aumenta su biodisponibilidad cuando se administra a un individuo, o que potencia Ia liberación de los compuestos de Ia invención en un compartimiento biológico (por ejemplo, en el cerebro o el tejido particular afectado por el tumor o Ia metástasis). La preparación de dicho profármaco puede llevarse a cabo mediante métodos convencionales conocidos por los expertos en Ia materia, que pueden encontrarse por
ejemplo en "Textbook of Drug Design and Discovery" (Krogsgaard-Larsen P, Liljefors T y Madsen U; editorial Taylor and Francis, 2002, 3a Edición).In a preferred embodiment of the present invention, said prodrug is a compound that, in comparison with the compounds of the invention, increases its bioavailability when administered to an individual, or that enhances the release of the compounds of the invention in a biological compartment ( for example, in the brain or the particular tissue affected by the tumor or metastasis). The preparation of said prodrug can be carried out by conventional methods known to those skilled in the art, which can be found by example , in "Textbook of Drug Design and Discovery" (Krogsgaard-Larsen P, Liljefors T and U Madsen, publisher Taylor and Francis, 2002, 3rd Edition).
Un quinto aspecto de Ia presente invención se refiere al uso de las formas cristalinas de los compuestos de Ia invención en estado libre o como solvatos, como coadyuvantes de agentes quimio-terapéuticos. El término "solvato", tal como aquí se utiliza, incluye tanto solvatos que puedan ser utilizados en Ia composición de un medicamento, como solvatos útiles en Ia preparación de dicho medicamento. Los solvatos pueden obtenerse por métodos convencionales de solvatación bien conocidos por los técnicos en Ia materia.A fifth aspect of the present invention refers to the use of the crystalline forms of the compounds of the invention in the free state or as solvates, as adjuvants of chemo-therapeutic agents. The term "solvate", as used herein, includes both solvates that can be used in the composition of a medicament, and solvates useful in the preparation of said medicament. Solvates can be obtained by conventional solvation methods well known to those skilled in the art.
Un sexto aspecto de Ia presente invención se refiere al uso de sales farmacéuticamente aceptables de los compuestos de Ia invención como coadyuvantes de agentes quimio-terapéuticos. Las sales de los compuestos de Ia invención que no sean farmacéuticamente aceptables pueden ser útiles asimismo en Ia preparación de fármacos, por Io que todas las sales de los compuestos de Ia invención, sean o no farmacéuticamente aceptables están incluidas en el ámbito de Ia presente invención.A sixth aspect of the present invention refers to the use of pharmaceutically acceptable salts of the compounds of the invention as adjuvants of chemo-therapeutic agents. The salts of the compounds of the invention that are not pharmaceutically acceptable may also be useful in the preparation of drugs, so that all salts of the compounds of the invention, whether or not pharmaceutically acceptable, are included in the scope of the present invention. .
Un séptimo aspecto de Ia presente invención se refiere al uso como coadyuvantes de agentes quimio-terapéuticos, de compuestos de Ia invención que contengan uno o más átomos isotópicamente enriquecidos. Por ejemplo, compuestos con una de las fórmulas I ó Il en las que uno o varios átomos de hidrogeno han sido sustituidos por átomos de deuterio o de tritio, uno o varios átomos de de carbono han sido sustituidos por átomos de 13C o 14C o uno o varios átomos de nitrógeno han sido sustituidos por 15N.
En un octavo aspecto, Ia presente invención se relaciona con composiciones farmacéuticas con una cantidad terapéuticamente efectiva de uno o más compuestos de Ia invención, sus isómeros, profármacos, solvatos o sales, tanto presentes en forma esencialmente pura como en mezcla con agentes auxiliares, aditivos o portadores farmacéuticamente aceptables y, eventualmente, otros agentes terapéuticos. Los agentes auxiliares, aditivos o portadores deben ser compatibles con los demás componentes de Ia composición y no causar perjuicios a sus receptores. Las composiciones farmacéuticas incluyen aquéllas que son adecuadas para administración oral, rectal, nasal, tópica (incluidas Ia bucal, sublingual y epicutánea), o parenteral (incluidas Ia subcutánea, intramuscular, intravenosa e intradérmica). Los métodos para Ia preparación de dichas composiciones así como los agentes auxiliares, aditivos o portadores (diluyentes, solventes, disgregantes, emulgentes, absorbentes, antioxidantes, lubricantes, colorantes, edulcorantes, humectantes, etc) son bien conocidos en Ia técnica de Ia farmacia, y ejemplos de ambos pueden encontrarse en "Textbook of Drug Design and Discovery" (Krogsgaard- Larsen P, Liljefors T y Madsen U; editorial Taylor and Francis, 2002, 3a Edición). Los compuestos de Ia invención, sus isómeros, profármacos, solvatos o sales, así como las composiciones farmacéuticas que los contienen pueden ser utilizados junto con otros fármacos para proporcionar terapia de combinación, ya sea formando parte de Ia misma composición farmacéutica o como composiciones separadas, administradas simultánea o no simultáneamente.A seventh aspect of the present invention relates to the use as adjuvants of chemo-therapeutic agents, of compounds of the invention containing one or more isotopically enriched atoms. For example, compounds with one of the formulas I or Il in which one or more hydrogen atoms have been replaced by deuterium or tritium atoms, one or several carbon atoms have been substituted by atoms of 13 C or 14 C or one or several nitrogen atoms have been replaced by 15 N. In an eighth aspect, the present invention relates to pharmaceutical compositions with a therapeutically effective amount of one or more compounds of the invention, their isomers, prodrugs, solvates or salts, both present in essentially pure form and in admixture with auxiliary agents, additives or pharmaceutically acceptable carriers and, eventually, other therapeutic agents. The auxiliary agents, additives or carriers must be compatible with the other components of the composition and not cause damage to their receptors. The pharmaceutical compositions include those that are suitable for oral, rectal, nasal, topical administration (including oral, sublingual and epicutaneous), or parenteral (including subcutaneous, intramuscular, intravenous and intradermal). The methods for the preparation of said compositions as well as the auxiliary agents, additives or carriers (diluents, solvents, disintegrants, emulsifiers, absorbents, antioxidants, lubricants, colorants, sweeteners, humectants, etc.) are well known in the pharmacy technique, and examples of both can be found in "Textbook of Drug Design and Discovery" (Krogsgaard- Larsen P, Liljefors T and U Madsen, publisher Taylor and Francis, 2002, 3rd Edition). The compounds of the invention, their isomers, prodrugs, solvates or salts, as well as the pharmaceutical compositions containing them can be used together with other drugs to provide combination therapy, either as part of the same pharmaceutical composition or as separate compositions, administered simultaneously or not simultaneously.
En el sentido utilizado en esta descripción, Ia expresión "cantidad terapéuticamente efectiva" se refiere a Ia cantidad de uno o más compuestos de Ia invención, sus isómeros, profármacos, solvatos o sales, capaz de desarrollar en el receptor el efecto terapéutico mediado por Ia inhibición de Ia actividad reparadora de ADN de Ia hAGT. Dicha cantidad o
dosis vendrá determinada, entre otras causas, por las características propias de los compuestos y sus isómeros, profármacos, solvatos o sales, así como por Ia composición farmacéutica que los contengan, Ia vía y frecuencia de administración, y Ia edad y condición del sujeto individual al que se Ie administra.In the sense used in this description, the expression "therapeutically effective amount" refers to the amount of one or more compounds of the invention, their isomers, prodrugs, solvates or salts, capable of developing in the receptor the therapeutic effect mediated by Ia inhibition of the DNA repair activity of hAGT. That amount or dose will be determined, among other causes, by the characteristics of the compounds and their isomers, prodrugs, solvates or salts, as well as by the pharmaceutical composition containing them, the route and frequency of administration, and the age and condition of the individual subject to which it is administered.
Una realización particular de Ia presente invención Io constituye el uso de un compuesto de Ia invención de fórmula I ó II, sus isómeros, profármacos, solvatos o sales, o sus mezclas, en Ia elaboración de un medicamento o composición farmacéutica para el tratamiento del cáncer, incluyendo, pero no limitado, al tratamiento de carcinomas incluyendo los de próstata, mama, pulmón, páncreas, colorrectal, gástrico, esofágico, laríngeo, tiroideo, hepático, de vejiga urinaria, renal, uterino, y de cérvix, cualquier tipo de sarcomas incluyendo osteosarcomas, sarcomas de partes blandas y angiosarcomas, cualquier tipo de tumor hematopoyético incluyendo leucemias y linfomas, cualquier tipo de tumor del sistema nervioso incluyendo neuroblastomas, glioblastomas y astrocitomas, cualquier cáncer dermatológico incluyendo melanomas, carcinomas de células básales y carcinomas de células escamosas. En una realización preferente de Ia presente invención, dicho uso se lleva a cabo en combinación con otros tratamientos antitumorales, sean éstos mediante Ia administración de medicamentos, sustancias radiactivas, radioterapia, quimioterapia, cirugía o cualquier procedimiento médico.A particular embodiment of the present invention constitutes the use of a compound of the invention of formula I or II, its isomers, prodrugs, solvates or salts, or mixtures thereof, in the preparation of a drug or pharmaceutical composition for the treatment of cancer. , including, but not limited to the treatment of carcinomas including those of prostate, breast, lung, pancreas, colorectal, gastric, esophageal, laryngeal, thyroid, hepatic, urinary bladder, renal, uterine, and cervical, any type of sarcomas including osteosarcomas, soft tissue sarcomas and angiosarcomas, any type of hematopoietic tumor including leukemia and lymphomas, any type of nervous system tumor including neuroblastomas, glioblastomas and astrocytomas, any dermatological cancer including melanomas, basal cell carcinomas and squamous cell carcinomas. In a preferred embodiment of the present invention, said use is carried out in combination with other antitumor treatments, be they by means of the administration of medications, radioactive substances, radiotherapy, chemotherapy, surgery or any medical procedure.
DESCRIPCIÓN DEL CONTENIDO DE LAS FIGURASDESCRIPTION OF THE CONTENT OF THE FIGURES
Figura 1. Metodologías para Ia identificación de pequeñas moléculas inhibidoras de Ia actividad de Ia enzima O6-alquilguanina- ADN-metil-transferasa. El proceso se inició por dos vías paralelas, consistentes en el análisis virtual, mediante medios informáticos, de Ia
estructura tridimensional, conformaciones más estables energéticamente y distribución de cargas, radios y cargas de los átomos que se encuentran en Ia superficie de las moléculas de una quimioteca de compuestos (ZINC) y del centro activo de Ia enzima. Los datos resultantes de dicho análisis para los más de tres millones de compuestos de Ia quimioteca se almacenaron en una base de datos (BBDD) relacional en entorno MySQL. Las posiciones más probables de los compuestos en su encaje con el centro activo de Ia enzima se obtuvieron asignando a cada par geométrico centro activo - compuesto un valor (ZScore) en función de los contactos entre ambas moléculas, calculado mediante un programa informático (DOCK). Las parejas geométricas con valores de ZScore superiores a 5 se re-analizaron con el programa CDOCK, diseñado específicamente por los autores de Ia invención. Posteriormente se llevo a cabo una reclasificación de los resultados en función de Ia suma de las energías de van der Waals, componente coulómbica, desolvataciones del centro activo y del compuesto potencialmente inhibidor (pérdida de interacción con moléculas del disolvente para que puedan interaccionar entre sí) y componente no polar. A partir de esta nueva clasificación se inspeccionaron visualmente los resultados y se seleccionaron 21 compuestos que mostraban Ia mayor probabilidad de interacción estable con el centro activo de Ia hAGT.Figure 1. Methodologies for the identification of small molecules that inhibit the activity of the enzyme O 6 -alkylguanine-DNA-methyl-transferase. The process began in two parallel ways, consisting of the virtual analysis, by computer means, of Ia three-dimensional structure, more energy-stable conformations and distribution of charges, radii and charges of the atoms that are on the surface of the molecules of a compound library (ZINC) and the active center of the enzyme. The data resulting from said analysis for the more than three million compounds of the library were stored in a relational database (BBDD) in a MySQL environment. The most probable positions of the compounds in their fit with the active center of the enzyme were obtained by assigning to each geometric pair active center - compound a value (ZScore) based on the contacts between both molecules, calculated by means of a computer program (DOCK) . Geometric pairs with ZScore values greater than 5 were re-analyzed with the CDOCK program, specifically designed by the authors of the invention. Subsequently, a reclassification of the results was carried out based on the sum of van der Waals energies, coulombic component, desolvatations of the active center and the potentially inhibitory compound (loss of interaction with solvent molecules so that they can interact with each other) and non-polar component. From this new classification, the results were visually inspected and 21 compounds were selected that showed the greatest probability of stable interaction with the active center of the hAGT.
Figura 2. Fórmulas generales de los compuestos de Ia invención.Figure 2. General formulas of the compounds of the invention.
Se muestran las fórmulas generales de los compuestos de Ia invención (fórmulas I y II).The general formulas of the compounds of the invention (formulas I and II) are shown.
Figura 3. Fórmulas de realizaciones específicas de compuestos de Ia invención. Se muestran las fórmulas químicas de cuatro compuestos que constituyen realizaciones específicas de Ia invención: dos compuestos que tienen Ia fórmula general I y dos compuestos que tienen Ia fórmula general II. Compuesto Ia, 3-[[4-(2,5-dimetilfenil)-1 -piperazinil][1 -(fenilmetil)- 1H-tetrazol-5-il]metil]-6-metoxi-2(1/-/)-quinolinona (denominado Pibetina
por los inventores); compuesto Ib, 3-[(4-bencil-1 -piperadinil)-(1 -bencil-1 H- 5-tetrazolil)]metil-7-metil-2(1/-/)quinolinona compuesto Ma, (benciloxi)fenil]- 5-(4-etilfenil)-4,5,6,7-tetrahidro-[1 ,2,4]triazolo[1 ,5-a]pirimidina; y compuesto llb,7-[4-(benciloxi)fenil]-5-(4-etoxifenil)-4,5,6,7-tetrahidro-[1 ,2,4]triazolo[1 ,5- a]pirimidina.Figure 3. Formulas of specific embodiments of compounds of the invention. The chemical formulas of four compounds that constitute specific embodiments of the invention are shown: two compounds having the general formula I and two compounds having the general formula II. Compound Ia, 3 - [[4- (2,5-dimethylphenyl) -1 -piperazinyl] [1 - (phenylmethyl) -1H-tetrazol-5-yl] methyl] -6-methoxy-2 (1 / - /) -quinolinone (called Pibetine by the inventors); compound Ib, 3 - [(4-benzyl-1-piperadinyl) - (1-benzyl-1 H- 5-tetrazolyl)] methyl-7-methyl-2 (1 / - /) quinolinone compound Ma, (benzyloxy) phenyl ] - 5- (4-ethylphenyl) -4,5,6,7-tetrahydro- [1, 2,4] triazolo [1,5-a] pyrimidine; and compound llb, 7- [4- (benzyloxy) phenyl] -5- (4-ethoxyphenyl) -4,5,6,7-tetrahydro- [1,4,4] triazolo [1,5- a] pyrimidine.
Figura 4. Modelos estructurales del encaje molecular entre compuestos de Ia invención y el centro activo de Ia enzima hAGT. Las ilustraciones muestran representaciones de Ia superficie de hAGT, junto a modelos de varillas de compuestos Ia, Ib, Ma y Mb de Ia invención, ilustrando el encaje óptimo entre el centro activo de Ia hAGT y dichos compuestos según resultados obtenidos mediante el programa CDOCK.Figure 4. Structural models of the molecular fit between compounds of the invention and the active center of the hAGT enzyme. The illustrations show representations of the surface of hAGT, together with rod models of compounds Ia, Ib, Ma and Mb of the invention, illustrating the optimum fit between the active center of the hAGT and said compounds according to results obtained through the CDOCK program.
Figura 5. Inhibición por los compuestos Ia, Ib, Ma y Mb de Ia actividad reparadora de ADN de Ia enzima hAGT in vitro. La actividad remanente de Ia hAGT tras pre-incubarse con distintas concentraciones en el intervalo de 0 a 100 μM de los compuestos de Ia invención se representa como el porcentaje de dicha actividad durante Ia posterior incubación de Ia mezcla utilizando [3H]-metil-ADN como sustrato, respecto a Ia actividad medida en el control positivo. Como control negativo de Ia reacción se utilizó proteína hAGT muíante (C145S, representado por un círculo, O), y como control positivo se sustituyeron los compuestos por Ia misma concentración del disolvente utilizado para su disolución, el DMSO (representado por un cuadrado negro, "). La figura superior muestra Ia actividad remanente de Ia hAGT frente a las distintas concentraciones del compuesto Ia (rombo negro, ^) y Ib (rombo gris, ^). La figura inferior muestra Ia actividad remanente de Ia hAGT frente a las distintas concentraciones del compuesto Ma (triángulo negro, A ) y Mb (triángulo gris, ^ ).
Figura 6. Inhibición por los compuestos Ia, Ma y Mb de Ia actividad reparadora de ADN de Ia enzima hAGT in vivo. Se muestra el efecto de distintos compuestos de Ia invención en concentraciones en el intervalo de 0 a 100 μM sobre Ia formación de colonias de células tumorales de adenocarcinoma colorectal humano sin tratar con el agente quimioterapeútico carmustina (columnas en negro) o tratadas con una concentración de 40 μM de dicho agente (columnas en gris). El número de colonias se representa como porcentaje de las mismas respecto al número observado en el control negativo: células incubadas con el medio utilizado para disolver los distintos compuestos de Ia invención (concentración = 0 μM) y no tratadas con carmustina (columnas en negro en el extremo izquierdo de todas las gráficas).Figure 5. Inhibition by compounds Ia, Ib, Ma and Mb of the DNA repair activity of the hAGT enzyme in vitro. The remaining activity of the hAGT after pre-incubation with different concentrations in the range of 0 to 100 μM of the compounds of the invention is represented as the percentage of said activity during the subsequent incubation of the mixture using [ 3 H] -methyl- DNA as a substrate, with respect to the activity measured in the positive control. As a negative control of the reaction, mutant hAGT protein (C145S, represented by a circle, O) was used, and as a positive control the compounds were substituted by the same concentration of the solvent used for its dissolution, the DMSO (represented by a black square, "). The upper figure shows the remaining activity of the hAGT against the different concentrations of the compound Ia (black rhombus, ^) and Ib (gray rhombus, ^). The lower figure shows the remaining activity of the hAGT against the different concentrations of compound Ma (black triangle, A) and Mb (gray triangle, ^). Figure 6. Inhibition by compounds Ia, Ma and Mb of the DNA repair activity of the hAGT enzyme in vivo. The effect of different compounds of the invention is shown in concentrations in the range of 0 to 100 μM on the formation of colonies of human colorectal adenocarcinoma tumor cells untreated with the carmustine chemotherapeutic agent (black columns) or treated with a concentration of 40 μM of said agent (gray columns). The number of colonies is represented as a percentage of them with respect to the number observed in the negative control: cells incubated with the medium used to dissolve the different compounds of the invention (concentration = 0 μM) and not treated with carmustine (black columns in the left end of all the graphs).
EJEMPLOS DE MODOS DE REALIZACIÓN DE LA INVENCIÓNEXAMPLES OF EMBODIMENTS OF THE INVENTION
Ejemplo 1. Inhibición de Ia actividad reparadora de ADN de Ia hAGT in vitro por los compuestos Ia, Ib, Ma y Mb.Example 1. Inhibition of DNA repair activity of hAGT in vitro by compounds Ia, Ib, Ma and Mb.
El análisis computacional descrito en Ia presente invención permitió identificar dos compuestos particulares para cada una de las fórmulas generales I y Il (compuestos Ia y Ib, y Ma y Mb, respectivamente) que encajaban con buena afinidad en el centro activo de Ia enzima hAGTThe computational analysis described in the present invention allowed to identify two particular compounds for each of the general formulas I and Il (compounds Ia and Ib, and Ma and Mb, respectively) that fit with good affinity in the active center of the hAGT enzyme
(veáse Ia Figura 4) y por tanto podían interferir competitivamente con Ia interacción entre dicha enzima y su sustrato, el ADN metilado. Para demostrarlo experimentalmente, se analizó Ia capacidad de dichos compuestos de inhibir Ia actividad reparadora de ADN de Ia enzima hAGT mediante un ensayo in vitro utilizando hAGT sintetizada en cultivos de E. coli recombinantes. Como control negativo en dicho ensayos se utilizó hAGT de un muíante, el hAGT-ΔC177-C145S (en adelante, hAGT-C145S) donde Ia cisteína 145 responsable de Ia actividad reparadora de esta
enzima es sustituida por una serina, provocando Ia pérdida de su actividad.(see Figure 4) and therefore could competitively interfere with the interaction between said enzyme and its substrate, methylated DNA. To demonstrate it experimentally, the ability of said compounds to inhibit the DNA repair activity of the hAGT enzyme was analyzed by an in vitro assay using hAGT synthesized in recombinant E. coli cultures. As a negative control in said tests hAGT of a mutant was used, hAGT-ΔC177-C145S (hereinafter, hAGT-C145S) where cysteine 145 responsible for the repair activity of this Enzyme is replaced by a serine, causing the loss of its activity.
Síntesis y purificación de hAGT y hAGT-C145S. Se transformaron células de E. coli con vectores derivados de los plásmidos pet21 y pet28, incluyendo en el primer caso una secuencia de Ia hAGT (plásmido pet21- hAGT) y en el segundo Ia secuencia muíante (originando el vector pet28- hAGT-ΔC177-C145S). Se seleccionaron las bacterias transformadas mediante crecimiento en medio LB-agar con ampicilina (100 μg/ml) para seleccionar las colonias bacterianas conteniendo el vector pet21-hAGT o con kanamicina (50 μg/ml) para seleccionar aquellas conteniendo el vector pet28-hAGT- T-ΔC177-C145S. Se transfirieron colonias individuales a 0,6 litros de medio LB, conteniendo el antibiótico correspondiente a las mismas concentraciones antes indicadas y se incubaron en agitación a 37°C durante 16 h. A continuación se realizaron diluciones de 150 ml_ de dicho cultivo en 1 ,5 litros de LB conteniendo el antibiótico correspondiente, pero a una concentración dos veces menor que Ia inicial. Estas diluciones de los cultivos se incubaron de nuevo en agitación a 37°C hasta que alcanzaron Ia fase de crecimiento exponencial (D.O.βoo nm ~ 0,6), momento en que Ia temperatura se redujo a 3O C. Cuando se estabilizó esta temperatura para todo el cultivo se Ie añadió 1 mM de isopropil α-D- tiogalactopiranósido (Calbiochem A) para inducir Ia expresión de Ia hATG. Tras 4 horas de incubación se centrifugaron los cultivos durante 15 min, resuspendiendo los sedimentos en una solución tampón de 50 mM Tris pH=8,0 y 20% sacarosa. Estas resuspensiones se congelaron por inmersión en nitrógeno líquido y se conservaron a -80°C hasta Ia purificación de Ia hAGT.Synthesis and purification of hAGT and hAGT-C145S. E. coli cells were transformed with vectors derived from plasmids pet21 and pet28, including in the first case a sequence of the hAGT (plasmid pet21-hAGT) and in the second the mutant sequence (originating the vector pet28-hAGT-ΔC177- C145S). Transformed bacteria were selected by growth in LB-agar medium with ampicillin (100 μg / ml) to select bacterial colonies containing the pet21-hAGT vector or with kanamycin (50 μg / ml) to select those containing the pet28-hAGT- vector T-ΔC177-C145S. Individual colonies were transferred to 0.6 liters of LB medium, containing the corresponding antibiotic at the same concentrations indicated above and incubated with shaking at 37 ° C for 16 h. Subsequently, dilutions of 150 ml of said culture were made in 1.5 liters of LB containing the corresponding antibiotic, but at a concentration twice lower than the initial one. These dilutions of the cultures were incubated again with stirring at 37 ° C until they reached the exponential growth phase (DOβoo nm ~ 0.6), at which time the temperature was reduced to 3 ° C. When this temperature was stabilized for all The culture was added 1 mM of isopropyl α-D-thiogalactopyranoside (Calbiochem A) to induce the expression of hATG. After 4 hours of incubation, the cultures were centrifuged for 15 min, the sediments being resuspended in a 50 mM Tris buffer solution pH = 8.0 and 20% sucrose. These resuspensions were frozen by immersion in liquid nitrogen and kept at -80 ° C until the purification of the hAGT.
Para dicha purificación se descongelaron las resuspensiones celulares y se les añadió solución tampón para Ia lisis celular: 350 mM cloruro de sodio; 20 mM Imidazol; 0,2% IGEPAL; 1 μl β-mercaptoetanol, 20% sacarosa, y 50 mM Tris pH=8,0. La rotura celular se llevó a cabo en un sonicador Fisher Bioblock Scientific 75042, tras Io que las disoluciones se
centrifugaron a 185844 g durante 45 min. El sobrenadante se filtró a través de filtros de acetato de celulosa con poros de 45 μM de diámetro (Sarstedt) y se aplicó el filtrado a una columna HiTrap™ FF (Amersham Biosciences). La proteína unida específicamente a dicha columna a través de Ia cola de histidinas fue eluída mediante el flujo de una solución tampón de Imidazol en gradiente de 10 mM a 500 mM, y 350 mM NaCI, 20 mM Tris pH=8,0 y 1 mM β-mercaptoetanol. Las fracciones proteicas obtenidas se concentraron mediante centrifugación con dispositivos Amicon Ultra-15 (Millipore) y se purificaron mediante cromatografía de tamiz molecular en una columna Superdex 75 16/60 (Amersham Biosciences), utilizando como solución tampón 150 mM NaCI, 10 mM ditiotreitol (DTT) y 0,1 mM etilen- diamino-tetra-acetato (EDTA). Las fracciones correspondientes a Ia proteína eluída de esta columna se volvieron a concentrar mediante dispositivos Amicon Ultra-15 y se llevaron a una concentración de 2 mg/ml en el mismo tampón en presencia de 40% de glicerol, para su conservación a -2O0C hasta su posterior utilización.For said purification, cell resuspensions were thawed and buffer solution for cell lysis was added: 350 mM sodium chloride; 20 mM Imidazole; 0.2% IGEPAL; 1 μl β-mercaptoethanol, 20% sucrose, and 50 mM Tris pH = 8.0. The cell rupture was carried out in a Fisher Bioblock Scientific 75042 sonicator, after which the solutions were centrifuged at 185844 g for 45 min. The supernatant was filtered through cellulose acetate filters with pores 45 μM in diameter (Sarstedt) and the filtrate was applied to a HiTrap ™ FF column (Amersham Biosciences). The protein specifically bound to said column through the histidine tail was eluted by the flow of a gradient Imidazole buffer solution from 10 mM to 500 mM, and 350 mM NaCI, 20 mM Tris pH = 8.0 and 1 mM β-mercaptoethanol. The protein fractions obtained were concentrated by centrifugation with Amicon Ultra-15 devices (Millipore) and purified by molecular sieve chromatography on a Superdex 75 16/60 column (Amersham Biosciences), using as a 150 mM NaCI buffer solution, 10 mM dithiothreitol ( DTT) and 0.1 mM ethylene diamino-tetra-acetate (EDTA). The fractions corresponding to the eluted protein of this column were again concentrated by Amicon Ultra-15 devices and were taken to a concentration of 2 mg / ml in the same buffer in the presence of 40% glycerol, for preservation at -2O 0 C until later use.
Preparación del ADN metilado. Se metilo ADN de timo de ternera (Sigma) con [3H]-metilnitrosourea (Amersham Biosciences) siguiendo el protocolo descrito en [19]. Se resuspendió 1 mg de ADN en 1 mi de solución tampón de 10 mM cacodilato sódico pH=7,0 durante 24 horas a 4°C y a continuación se Ie añadieron 7 μl de [3H]-metilnitrosourea, incubándose Ia mezcla a 37°C durante 2 h. Finalizada Ia reacción de metilación, el ADN metilado se precipitó con acetato sódico y etanol. El precipitado obtenido, conteniendo el [3H]-metil-ADN se lavó de forma exhaustiva con etanol y se resuspendió finalmente en solución tampón 50 mM Tris pH=7,8, 1 mM DTT y 5 mM EDTA, conservándose a -20°C hasta su uso en los ensayos de actividad.Preparation of methylated DNA. Calf thymus DNA (Sigma) was mixed with [ 3 H] -methylnitrosourea (Amersham Biosciences) following the protocol described in [19]. 1 mg of DNA was resuspended in 1 ml of 10 mM sodium cacodylate buffer solution pH = 7.0 for 24 hours at 4 ° C and then 7 µl of [ 3 H] -methylnitrosourea was added, the mixture was incubated at 37 ° C for 2 h. After the methylation reaction, the methylated DNA was precipitated with sodium acetate and ethanol. The precipitate obtained, containing the [ 3 H] -methyl-DNA, was thoroughly washed with ethanol and finally resuspended in 50 mM Tris buffer solution pH = 7.8, 1 mM DTT and 5 mM EDTA, stored at -20 ° C until use in activity tests.
Ensayo de Ia actividad de Ia hAGT in vitro. La actividad reparadora del ADN de Ia hAGT y su inhibición por los compuestos de Ia invención se determinó siguiendo básicamente el método descrito en [20]. Brevemente, Ia enzima hAGT se preincubó durante 30 min a 37°C con diferentes
concentraciones de los compuestos Ia, Ib (Asinex), Ma ó Mb (Chemdiv, Inc.) (0, 25, 50, 75 o 100 μM) disueltos en dimetilsufóxido (DMSO), usando como medio de reacción una solución tampón 50 mM Tris pH=7,8, 1 mM DTT y 5 mM EDTA. Tras esta pre-incubación se inició Ia reacción mediante Ia adición del substrato, [3H]-metil-ADN, y se incubó a 370C durante 90 minutos. El volumen final de las reacciones fue de 200 μl, siendo las concentraciones finales de Ia hAGT y del [3H]-metil-ADN 0.5 μM y 50 μM, respectivamente. Todas las reacciones se detuvieron por Ia adición de 400 μl de una solución acuosa de ácido tricloroacético (TCA, Sigma) al 13%. El [3H]-metil-ADN restante, no reparado por Ia acción de Ia hAGT se hidrolizó calentando Ia mezcla de reacción hasta alcanzar 90°C durante 30 min. La hAGT se precipitó por centrifugación a 1610O g durante 10 minutos y se lavaron los precipitados resultantes con una solución acuosa de TCA al 5%. Tras dichos lavados se resuspendieron en 200 μl de solución tampón 0.2 M de Tris pH=8,0. La radioactividad correspondiente al grupo 3H-metil incorporado a Ia hAGT durante Ia reacción enzimática de reparación del ADN se midió con un contador de centelleo líquido (Wallac 1414), utilizando el cóctel de centelleo Optiphase HiSafe 3 (Perkin Elmer). Esta actividad remanente de Ia hAGT tras Ia preincubación con los compuestos de Ia invención se calculó en relación a los controles positivos, en los que Ia enzima se preincubó sólo con el disolvente DMSO (con ningún compuesto inhibidor). Como control negativo se usaron ensayos con Ia enzima hAGT-C145S, muíante sin actividad. Todos los ensayos se efectuaron por duplicado, y en cada uno de ellos las diferentes concentraciones de los compuestos se realizaron por cuadriplicado. Los resultados se expresan mediante el porcentaje de actividad remanente de hAGT en relación a Ia determinada para los controles positivos (en los que no hay inhibición de Ia actividad reparadora de dicha enzima). Los resultados de este ensayo se ilustran en Ia Figura 5, donde se puede observar que los compuestos Ia, Ib, Ma y Mb inhiben Ia actividad
reparadora del ADN metilado catalizada por Ia hAGT, siendo mayor Ia eficacia inhibidora para los compuestos de fórmula Il que para los de fórmula I, y dentro de éstos, siendo mayor Ia eficacia inhibidora del compuesto Ib que Ia del Ia (compárense las IC50, concentración de compuesto necesaria para reducir al 50% Ia actividad reparadora de ADN de Ia hAGT).Test of the activity of hAGT in vitro. The repair activity of hAGT DNA and its inhibition by the compounds of the invention was determined basically following the method described in [20]. Briefly, the hAGT enzyme was pre-incubated for 30 min at 37 ° C with different concentrations of compounds Ia, Ib (Asinex), Ma or Mb (Chemdiv, Inc.) (0, 25, 50, 75 or 100 μM) dissolved in dimethyl sulfoxide (DMSO), using as a reaction medium a 50 mM Tris buffer solution pH = 7.8, 1 mM DTT and 5 mM EDTA. After this pre-incubation the reaction was initiated by the addition of substrate, [3 H] -methyl-DNA, and incubated at 37 0 C for 90 minutes. The final volume of the reactions was 200 μl, the final concentrations of the hAGT and the [ 3 H] -methyl-DNA being 0.5 μM and 50 μM, respectively. All reactions were stopped by the addition of 400 µl of a 13% aqueous solution of trichloroacetic acid (TCA, Sigma). The remaining [ 3 H] -methyl-DNA, not repaired by the action of the hAGT, was hydrolyzed by heating the reaction mixture until reaching 90 ° C for 30 min. The hAGT was precipitated by centrifugation at 1610O g for 10 minutes and the resulting precipitates were washed with a 5% aqueous TCA solution. After these washes, they were resuspended in 200 µl of 0.2 M Tris buffer solution pH = 8.0. The radioactivity corresponding to the H-methyl group 3 incorporated into the hAGT during the enzymatic DNA repair reaction was measured with a liquid scintillation counter (Wallac 1414), using the Optiphase HiSafe 3 scintillation cocktail (Perkin Elmer). This remaining activity of the hAGT after the preincubation with the compounds of the invention was calculated in relation to the positive controls, in which the enzyme was pre-incubated only with the DMSO solvent (with no inhibitor compound). As a negative control, tests were used with the enzyme hAGT-C145S, mutant without activity. All tests were performed in duplicate, and in each of them the different concentrations of the compounds were performed in quadruplicate. The results are expressed by the percentage of remaining hAGT activity in relation to that determined for the positive controls (in which there is no inhibition of the repair activity of said enzyme). The results of this test are illustrated in Figure 5, where it can be seen that compounds Ia, Ib, Ma and Mb inhibit the activity repair of methylated DNA catalyzed by hAGT, the inhibitory efficacy being greater for compounds of formula I than for those of formula I, and within these, the inhibitory efficacy of compound Ib being greater than that of Ia (compare IC50, concentration of compound necessary to reduce to 50% the DNA repair activity of the hAGT).
Ejemplo 2. Optimización del efecto antitumoral de Ia carmustina (agente alquilante) mediante inhibición por los compuestos Ia, Ma y Mb de Ia actividad hAGT in vivo.Example 2. Optimization of the antitumor effect of carmustine (alkylating agent) by inhibition by compounds Ia, Ma and Mb of the hAGT activity in vivo.
Dado que Ia acción de Ia hAGT es Ia base de uno de los mecanismos de supervivencia de las células frente a Ia lesión en su ADN provocada por su metilación, puede concluirse que Ia inhibición de dicha enzima debe provocar una mayor sensibilidad por parte de las células a compuestos que, como los agentes alquilantes usados en quimioterapia, inducen dichas lesiones. Este es un efecto positivo desde el punto de vista terapéutico puesto que optimiza Ia acción antitumoral de dichos agentes. Los autores de Ia presente invención demostraron este efecto en cultivos de células tumorales proveniente de adenocarcinoma colorectal humano (código ATCC: HTB 38) tratadas con carmustina. En particular se empleó el ensayo de formación de colonias, previamente utilizado en el estudio de agentes adyuvantes de quimioterapia en células tumorales [21].Since the action of the hAGT is the basis of one of the survival mechanisms of the cells against the injury in their DNA caused by their methylation, it can be concluded that the inhibition of said enzyme should cause greater sensitivity on the part of the cells to compounds that, such as alkylating agents used in chemotherapy, induce such lesions. This is a positive effect from the therapeutic point of view since it optimizes the antitumor action of said agents. The authors of the present invention demonstrated this effect in tumor cell cultures from human colorectal adenocarcinoma (ATCC code: HTB 38) treated with carmustine. In particular, the colony formation assay was used, previously used in the study of chemotherapy adjuvant agents in tumor cells [21].
Ensayo de formación de colonias. Se sembraron 15000 células en cada uno de los 6 pocilios de placas de cultivo (Falcon) utilizando como medio el RPMI 1640 (Genycell) suplementado con 10% de suero fetal bovino (Biowhittaker). Tras una incubación durante 48 horas a 37°C se reemplazó el medio y se añadió al nuevo medio diferentes concentraciones de los compuestos Ia, Ma y Mb (0, 5, 10, 50 y 100 μM). Tras una pre-incubación durante 6 horas con los compuestos de Ia invención, se añadió a los cultivos carmustina (1 ,3-Bis(2-cloroetil)1- nitrosourea, también conocido como BCNU, Sigma) disuelta en una
solución 1 :1 de tampón fosfato salino pH=7,6 y etanol para llegar a una concentración final de 40 μM en el medio de cultivo. Asimismo se efectuaron controles positivos para cada uno de los cultivos con las diferentes concentraciones de compuestos de Ia invención en los que se añadía únicamente Ia solución 1 :1 de tampón fosfato salino pH=7,6 y etanol sin contener carmustina. Se incubaron todos los cultivos 2 horas y se reemplazó el medio de cultivo por medio RPMI suplementado con suero fetal bovino y que contenía las respectivas concentraciones de los compuestos de Ia invención. Tras un periodo de incubación de 16 horas se lavaron las células y se resembraron 2000 células por pocilio en nuevas placas de cultivo, creciéndose los cultivos durante 12 días a 37°C.Colony formation test. 15,000 cells were seeded in each of the 6 wells of culture plates (Falcon) using RPMI 1640 (Genycell) as a medium supplemented with 10% fetal bovine serum (Biowhittaker). After an incubation for 48 hours at 37 ° C, the medium was replaced and different concentrations of the compounds Ia, Ma and Mb (0.5, 10, 50 and 100 μM) were added to the new medium. After a pre-incubation for 6 hours with the compounds of the invention, carmustine (1,3-Bis (2-chloroethyl) 1- nitrosourea, also known as BCNU, Sigma) dissolved in a 1: 1 solution of phosphate buffered saline pH = 7.6 and ethanol to reach a final concentration of 40 μM in the culture medium. Likewise, positive controls were carried out for each of the cultures with the different concentrations of compounds of the invention in which only the 1: 1 solution of phosphate buffered saline pH = 7.6 and ethanol without carmustine was added. All cultures were incubated for 2 hours and the culture medium was replaced by RPMI medium supplemented with fetal bovine serum and containing the respective concentrations of the compounds of the invention. After an incubation period of 16 hours the cells were washed and 2000 cells were reseeded per well in new culture plates, the cultures being grown for 12 days at 37 ° C.
La eficacia de Ia hAGT para inhibir el crecimiento celular se determinó mediante el recuento de las colonias formadas usando tinción con violeta de cresilo (cristal violeta, Merck). Tras una incubación en Ia solución de tinción durante 30 minutos a temperatura ambiente se lavaron las colonias varias veces con agua destilada y desionizada (MiIIiQ) y se dejaron secar a temperatura ambiente. Finalmente, se resuspendieron con una solución acuosa de ácido acético al 10% y se midió Ia absorbancia de las resuspensiones a 590 nm. El número de colonias se representó como porcentaje de las mismas respecto al valor observado en el control negativo: células pre-incubadas e incubadas sólo con el medio utilizado para disolver los distintos compuestos de Ia invención (concentración = 0 μM) y no tratadas con carmustina (columnas en negro en el extremo izquierdo de todas las gráficas). Los resultados de este ensayo se ilustran en Ia Figura 6. Se observó que los compuestos ensayados potenciaban el efecto inhibidor de Ia proliferación celular o formación de colonias mediado por Ia carmustina. El compuesto Ia reforzó el efecto de Ia carmustina a concentraciones iguales o superiores a 10 μM de dicho compuesto, mientras que los compuestos Ma y Mb requirieron concentraciones iguales o superiores a 50 μM para mostrar un refuerzo de Ia acción de Ia carmustina.
BIBLIOGRAFÍAThe efficacy of hAGT to inhibit cell growth was determined by counting the colonies formed using cresyl violet staining (violet crystal, Merck). After incubation in the staining solution for 30 minutes at room temperature, the colonies were washed several times with distilled and deionized water (MiIIiQ) and allowed to dry at room temperature. Finally, they were resuspended with a 10% aqueous solution of acetic acid and the absorbance of the resuspensions at 590 nm was measured. The number of colonies was represented as a percentage of them with respect to the value observed in the negative control: cells pre-incubated and incubated only with the medium used to dissolve the different compounds of the invention (concentration = 0 μM) and not treated with carmustine (columns in black at the left end of all graphs). The results of this test are illustrated in Figure 6. It was observed that the compounds tested potentiated the inhibitory effect of cell proliferation or colony formation mediated by carmustine. Compound Ia reinforced the effect of carmustine at concentrations equal to or greater than 10 μM of said compound, while compounds Ma and Mb required concentrations equal to or greater than 50 μM to show a reinforcement of the action of carmustine. BIBLIOGRAPHY
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Claims
1.- Uso de un compuesto derivado de piperadinil-metil-tetrazol- quinolinona, de fórmula I1.- Use of a compound derived from piperadinyl-methyl-tetrazol-quinolinone, of formula I
- X es un átomo de carbono (C) o nitrógeno (N), indistintamente;- X is a carbon (C) or nitrogen (N) atom, interchangeably;
- R1, R2, R3 y R4 son iguales o diferentes y están independientemente seleccionados del grupo constituido por: hidrógeno, alquilo lineal, alquilo ramificado, cicloalquilmetil, cicloalquiletil, aminocarbonilalquil, alquiloxi lineal, alquiloxi ramificado, furilmetil, imidazolilmetil, imidazoliletil, imidazolilpropil, R7, metil-R7, etil-R7, propil-R7, metoxi-R7, etoxi-R7, propoxi- R7, donde R7 es un arilo en el que cada una de sus cinco posiciones restantes está sustituida por un grupo independientemente seleccionado entre hidrógeno, flúor, cloro, bromo, trifluorometilo, hidroxi, alcoxi (alquilóxido de 1 a 4 átomos de C), alquilcarbonil, alquilamino, y sulfonilamino; o bien un isómero, profármaco, solvato o sal farmacéuticamente aceptables de dicho compuesto, en Ia elaboración de una composición farmacéutica o medicamento destinado al tratamiento del cáncer. - R 1 , R 2 , R 3 and R 4 are the same or different and are independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkylethyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl , imidazolylpropyl, R 7 , methyl-R 7 , ethyl-R 7 , propyl-R 7 , methoxy-R 7 , ethoxy-R 7 , propoxy-R 7 , where R 7 is an aryl in which each of its five Remaining positions are substituted by a group independently selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atoms), alkylcarbonyl, alkylamino, and sulfonylamino; or a pharmaceutically acceptable isomer, prodrug, solvate or salt of said compound, in the preparation of a pharmaceutical composition or medicament for the treatment of cancer.
2.- Uso de un compuesto según Ia reivindicación 1 donde2. Use of a compound according to claim 1 wherein
- X es nitrógeno,- X is nitrogen,
- R1 es 2,5-dimetilfenil,- R 1 is 2,5-dimethylphenyl,
- R2 es hidrógeno, - R3 es metoxi,- R 2 is hydrogen, - R 3 is methoxy,
- R4 es fenil-metil,- R 4 is phenyl methyl,
Compuesto denominado 3-[[4-(2,5-dimetilfenil)-1-piperazinil][1- (fenilmetil)-1/-/-tetrazol-5-il]metil]-6-metoxi-2(1/-/)-quinolinona.Compound called 3 - [[4- (2,5-dimethylphenyl) -1-piperazinyl] [1- (phenylmethyl) -1 / - / - tetrazol-5-yl] methyl] -6-methoxy-2 (1 / - /) - quinolinone.
3.- Uso de un compuesto según Ia reivindicación 1 donde3. Use of a compound according to claim 1 wherein
- X es carbono,- X is carbon,
- R1 es fenil-metil,- R 1 is phenyl methyl,
- R2 es metil,- R 2 is methyl,
- R3 es hidrógeno, - R4 es fenil-metil, compuesto denominado 3-[(4-bencil-1-piperadinil)-(1-bencil-1/-/-5- tetrazolil)]metil-7-metil-2(1/-/)quinolinona.- R 3 is hydrogen, - R 4 is phenyl methyl, a compound called 3 - [(4-benzyl-1-piperadinyl) - (1-benzyl-1 / - / - 5- tetrazolyl)] methyl-7-methyl- 2 (1 / - /) quinolinone.
4.- Uso de un compuesto derivado de difenil-triazolo-pirimidina, de fórmula Il 4.- Use of a compound derived from diphenyl-triazolo-pyrimidine, of formula Il
en Ia que cada uno de los grupos R y R está independientemente seleccionado del grupo constituido por: hidrógeno, alquilo lineal, alquilo ramificado, cicloalquilmetil, cicloalquiletil, aminocarbonilalquil, alquiloxi lineal, alquiloxi ramificado, furilmetil, imidazolilmetil, imidazoliletil, imidazolilpropil, R8, metil-R8, etil-R8, propil-R8, metoxi-R8, etoxi-R8, propoxi- R8, donde R8 es un arilo en el que cada una de sus cinco posiciones restantes está sustituida por un grupo independientemente seleccionado entre hidrógeno, flúor, cloro, bromo, trifluorometilo, hidroxi, alcoxi (alquilóxido de 1 a 4 átomos de C), alquilcarbonil, alquilamino, y sulfonilamino; o bien un isómero, profármaco, solvato o sal farmacéuticamente aceptables de dicho compuesto, en Ia elaboración de una composición farmacéutica o medicamento destinado al tratamiento del cáncer.in which each of the groups R and R is independently selected from the group consisting of: hydrogen, linear alkyl, branched alkyl, cycloalkylmethyl, cycloalkyl, aminocarbonylalkyl, linear alkyloxy, branched alkyloxy, furylmethyl, imidazolylmethyl, imidazolylethyl, imidazolylpropyl, R 8 , methyl-R 8 , ethyl-R 8 , propyl-R 8 , methoxy-R 8 , ethoxy-R 8 , propoxy-R 8 , where R 8 is an aryl in which each of its five remaining positions is replaced by a group independently selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, hydroxy, alkoxy (alkyloxide of 1 to 4 C atoms), alkylcarbonyl, alkylamino, and sulfonylamino; or a pharmaceutically acceptable isomer, prodrug, solvate or salt of said compound, in the preparation of a pharmaceutical composition or medicament for the treatment of cancer.
5.- Uso de un compuesto según Ia reivindicación 4 donde5. Use of a compound according to claim 4 wherein
- R5 es fenilmetoxi,- R 5 is phenylmethoxy,
- R6 es etil, compuesto denominado 7-[4 -(benciloxi)fenil]-5-(4-etilfenil)-4, 5,6,7- tetrahidro-[1 ,2,4]triazolo[1 ,5-a]pirimidina. - R 6 is ethyl, compound called 7- [4 - (benzyloxy) phenyl] -5- (4-ethylphenyl) -4, 5,6,7-tetrahydro- [1, 2,4] triazolo [1, 5- a] pyrimidine.
6.- Uso de un compuesto según Ia reivindicación 4 donde6. Use of a compound according to claim 4 wherein
- R5 es fenilmetoxi,- R 5 is phenylmethoxy,
- R6 es etoxi, compuesto denominado 7-[4-(benciloxi)fenil]-5-(4-etoxifenil)-4, 5,6,7- tetrahidro-[1 ,2,4]triazolo[1 ,5-a]pirimidina.- R 6 is ethoxy, a compound called 7- [4- (benzyloxy) phenyl] -5- (4-ethoxyphenyl) -4, 5,6,7-tetrahydro- [1, 2,4] triazolo [1, 5- a] pyrimidine.
7.- Uso de un compuesto según cualquiera de las reivindicaciones 1 a 6, caracterizado porque Ia composición farmacéutica o medicamento está destinado al tratamiento de un cáncer seleccionado del grupo que comprende: carcinoma de próstata, mama, pulmón, páncreas, esófago, laringe, tiroides, hígado, vejiga urinaria, riñon, útero, cerviz, carcinoma cólorrectal, gástrico, osteosarcoma, sarcoma de partes blandas y angiosarcoma; tumor hematopoyético (incluyendo leucemias y linfomas), neuroblastomas, glioblastomas y astrocitomas, melanoma, carcinoma dérmico de células básales y carcinoma dérmico de células escamosas.7. Use of a compound according to any of claims 1 to 6, characterized in that the pharmaceutical composition or medicament is intended for the treatment of a cancer selected from the group comprising: prostate carcinoma, breast, lung, pancreas, esophagus, larynx, thyroid, liver, urinary bladder, kidney, uterus, cervix, chlorectal carcinoma, gastric, osteosarcoma, soft tissue sarcoma and angiosarcoma; hematopoietic tumor (including leukemia and lymphomas), neuroblastomas, glioblastomas and astrocytomas, melanoma, dermal basal cell carcinoma and dermal squamous cell carcinoma.
8.- Composiciones farmacéuticas y medicamentos útiles para el tratamiento del cáncer por sí solos o en combinación con otras composiciones farmacéuticas o medicamentos, caracterizados porque comprenden al menos:8.- Pharmaceutical compositions and medicaments useful for the treatment of cancer alone or in combination with other pharmaceutical compositions or medicaments, characterized in that they comprise at least:
- un compuesto de fórmula I, o- a compound of formula I, or
- un compuesto de fórmula II, o- a compound of formula II, or
- una mezcla de ambos, o- a mixture of both, or
- un isómero, profármaco, solvato o sal farmacéuticamente aceptables de uno de dichos compuestos.- a pharmaceutically acceptable isomer, prodrug, solvate or salt of one of said compounds.
9.- Composiciones farmacéuticas y medicamentos según Ia reivindicación 8 en Ia que el compuesto de fórmula I o de fórmula Il está seleccionado del grupo constituido por: • 3-[[4-(2,5-dimetilfenil)-1 -piperazinil][1 -(fenilmetil)-i H-tetrazol-5- il]metil]-6-metoxi-2(1/-/)-quinolinona, • 3-[(4-bencil-1 -piperadinil)-(1 -bencil-1 /-/-5-tetrazolil)]metil-7-metil- 2(1/-/)quinolinona,9. Pharmaceutical compositions and medicaments according to claim 8 wherein the compound of formula I or formula Il is selected from the group consisting of: • 3 - [[4- (2,5-dimethylphenyl) -1 -piperazinyl] [ 1 - (phenylmethyl) -i H-tetrazol-5- yl] methyl] -6-methoxy-2 (1 / - /) - quinolinone, • 3 - [(4-Benzyl-1-piperadinyl) - (1-benzyl-1 / - / - 5-tetrazolyl)] methyl-7-methyl-2 (1 / - /) quinolinone,
• 7-[4 -(benciloxi)fenil]-5-(4-etilfenil)-4,5,6,7-tetrahidro- [1 ,2,4]triazolo[1 ,5-a]pirimidina, y • 7-[4-(benciloxi)fenil]-5-(4-etoxifenil)-4,5,6,7-tetrahidro-• 7- [4 - (benzyloxy) phenyl] -5- (4-ethylphenyl) -4,5,6,7-tetrahydro- [1, 2,4] triazolo [1,5-a] pyrimidine, and • 7 - [4- (benzyloxy) phenyl] -5- (4-ethoxyphenyl) -4,5,6,7-tetrahydro-
[1 ,2,4]triazolo[1 ,5-a]pirimidina. [1, 2,4] triazolo [1,5-a] pyrimidine.
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| ES200702958A ES2330495B1 (en) | 2007-11-07 | 2007-11-07 | INHIBITORS OF ENZYME O6-ALQUILGUANINA-ADN-METIL-TRANSFERASA FOR THE TREATMENT OF CANCER. |
| ESP200702958 | 2007-11-07 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2624837A2 (en) * | 2010-10-04 | 2013-08-14 | Institute For Hepatitis And Virus Research | Novel inhibitors of secretion of hepatitis b virus antigens |
| US11377452B2 (en) * | 2017-01-16 | 2022-07-05 | Drexel University | Glutamate transporter activators and methods using same |
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| US6593342B1 (en) * | 1998-07-15 | 2003-07-15 | Laboratoire L. Lafon | Pharmaceutical compositions comprising 2-quinolones |
| US20070032518A1 (en) * | 2002-02-07 | 2007-02-08 | Amgen Inc. | Compounds and methods of use |
-
2007
- 2007-11-07 ES ES200702958A patent/ES2330495B1/en not_active Withdrawn - After Issue
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- 2008-10-20 WO PCT/ES2008/070190 patent/WO2009060114A1/en active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6593342B1 (en) * | 1998-07-15 | 2003-07-15 | Laboratoire L. Lafon | Pharmaceutical compositions comprising 2-quinolones |
| US20070032518A1 (en) * | 2002-02-07 | 2007-02-08 | Amgen Inc. | Compounds and methods of use |
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| DATABASE CHEMCATS [online] EE.UU.; 9 April 2007 (2007-04-09), "Zelinsky Screening Library.", accession no. STN Database accession no. 2034097371 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2624837A2 (en) * | 2010-10-04 | 2013-08-14 | Institute For Hepatitis And Virus Research | Novel inhibitors of secretion of hepatitis b virus antigens |
| EP2624837A4 (en) * | 2010-10-04 | 2014-03-26 | Inst Hepatitis & Virus Res | Novel inhibitors of secretion of hepatitis b virus antigens |
| US8921381B2 (en) | 2010-10-04 | 2014-12-30 | Baruch S. Blumberg Institute | Inhibitors of secretion of hepatitis B virus antigens |
| US9533990B2 (en) | 2010-10-04 | 2017-01-03 | Drexel University | Inhibitors of secretion of hepatitis B virus antigens |
| US11377452B2 (en) * | 2017-01-16 | 2022-07-05 | Drexel University | Glutamate transporter activators and methods using same |
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| ES2330495A1 (en) | 2009-12-10 |
| ES2330495B1 (en) | 2010-09-20 |
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