WO2014013117A1 - Chromenopyrazole diones as cannabinoid quinone derivatives having anti-tumour activity - Google Patents
Chromenopyrazole diones as cannabinoid quinone derivatives having anti-tumour activity Download PDFInfo
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- WO2014013117A1 WO2014013117A1 PCT/ES2013/070499 ES2013070499W WO2014013117A1 WO 2014013117 A1 WO2014013117 A1 WO 2014013117A1 ES 2013070499 W ES2013070499 W ES 2013070499W WO 2014013117 A1 WO2014013117 A1 WO 2014013117A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
- C07D491/04—Ortho-condensed systems
- C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
- C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
Definitions
- the present invention is encompassed in the field of pharmacology. Specifically, the present invention relates to chromene-derived compounds [4,3-c] pyrazol-6,9-diones, their use for the manufacture of a medicament, and the use of this medicament for an antitumor treatment.
- Quinones are compounds of natural or synthetic origin. Several of them have some physiological functions such as the vitamin K necessary for blood clotting. However, many of them are cytotoxic, which makes them candidates for antitumor drugs. The antineoplastic effects of quinones have been widely described in the literature (C. Asche, Min ⁇ -Rev. Med. Chem. 2005, 5, 449-467). For example, anthracyclines that are antibiotics from bacteria of the genus Streptomyces, are used in the treatment of many types of cancers.
- Cannabinoids act on the receptors of the endocannabinoid system and can provide therapeutic benefits in various pathologies and in particular they are useful in the treatment of cancer. On the one hand, they have palliative effects: antiemetics and analgesics (RG Pertwee, Br. J. Pharmacol. 2009, 156 (3), 397- 411), and on the other hand, show antitumor properties by themselves (M. Guzman, Nat. Rev. Cancer 2003, 3, 745-765; S. Sarfaraz et al, Cancer Res. 2008, 68, 330-342). Cannabinoids have been shown to inhibit the growth of various tumor cells in laboratory animals, for example glioma (I.
- Chromenopyrazole structures such as cannabinoids developed by the research group of the authors of the present invention have been the subject of a patent application (WO2010109050), of which their activity as analgesics has been disclosed, without having been attributed antitumor properties .
- the present invention faces the problem of providing new compounds useful for the treatment of cancer. Due to the potential of quinones as anticancer and cannabinoids as anticancer, antiemetic and analgesic, it is of great interest to have molecules that can combine the characteristics of a quinone and a cannabinoid in the same structure. Preferably, said compounds should preserve and even improve the cytotoxic activity of known compounds while avoiding part of the associated adverse side effects.
- the authors of the present invention have found that the compounds of formula (I) act as antitumor agents.
- the compounds of formula (I) claimed in the present invention consist of a series of chromenopyrazoldiones that, in addition to having quinone structures, have in their structure a pyrazole ring that proved necessary to maintain a certain affinity for cannabinoid receptors , and that differentiates them from other quinone structures known in the state of the art, such as those previously described by Mechoulam et al. ⁇ Tetrahedron 1968, 24, 5615-5624; W02005 / O 67917; WO2008 / 107878).
- the present invention relates to a compound of general formula (I),
- - R 1 is independently selected from hydrogen or alkyl
- R 2 is independently selected from hydrogen, alkyl ⁇ -
- - R 3 is independently selected from hydrogen or alkyl
- alkyl refers, in the present invention, to aliphatic, linear or branched, saturated or unsaturated chains, having 1 to 18 carbon atoms, preferably 1 to 9 carbon atoms.
- alkyl refers, in the present invention, to aliphatic, linear or branched, saturated or unsaturated chains, having 1 to 18 carbon atoms, preferably 1 to 9 carbon atoms.
- the alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl or carboxylic acid.
- Said substituted alkyl groups are for example, but not limited to, 1- (1, 3-dithiolane) hexyl, 1-hydroxyethyl, hydroxymethyl, etc.
- aryl refers, in the present invention, to single or multiple aromatic rings, having between 5 and 18 links in which a proton has been removed from the ring.
- the aryl groups are for example, but not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthracil.
- the aryl group has 5 to 7 carbon atoms and more preferably the aryl group is a phenyl.
- Aryl radicals may be optionally substituted by one or more substituents such as alkyl (Ci-C 6), halogen, hydroxyl or carboxylic acid.
- Substituted aryl radicals are, for example, but not limited to 2,4-dichlorophenyl, 1,3-dichlorophenyl, 3,5-difluorophenyl, 3- methoxyphenyl.
- halogen refers, in the present invention, to bromine, chlorine, iodine or fluorine. Preferably chlorine.
- the size of the substituents R 1 to R 3 is important for the functionality of the compounds of formula (I), such as when they are intended to modify, among others, their pharmacokinetic, bioavailability, solubility or stability properties.
- possible carbon chain substitutions in R 3 are those in which said substituent does not exceed 5 carbon atoms, while in the case of the substituents of R 1 and R 2 the number of atoms can be quite greater .
- the positions defined by the substituents R 1 and R 2 can be functionalized with long carbon chains, such as fatty acids, where the most commonly used include chains of up to 18 carbon atoms.
- substituents such as 1,1-dimethylheptyl (of 9 carbon atoms) constitute a good example of an intermediate carbon chain between the maximum and minimum values contemplated herein.
- the compounds of the invention also include compounds that differ only in the presence of one or more isotopically enriched atoms.
- the compounds having the present structures, except for the replacement of a hydrogen with a deuterium or tritium, or the replacement of a carbon with a carbon enriched in 13 C or 14 C or a nitrogen enriched in 15 N are within the scope of this invention.
- tautomer or “tautomeric form”, as used in the present invention, refers to structural isomers of different energies that are interconvertible via a low energy barrier.
- protonic tautomers also known as prototropic tautomers
- Valencia tautomers include interconversions by reorganization of some bond electrons.
- pharmaceutically acceptable salts, solvates, prodrugs refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound that, when administered to a receptor, is capable of providing (directly or indirectly) a compound as described in This document.
- pharmaceutically acceptable salts are also within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts.
- the preparation of salts, prodrugs and derivatives can be carried out by methods known in the art.
- salts of compounds provided herein are synthesized by conventional chemical methods from an original compound containing a basic or acidic moiety.
- such salts are prepared, for example, by reacting the free acid or base forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mix of the two.
- non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
- acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iodhydrate, sulfate, nitrate, phosphate and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.
- mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iodhydrate, sulfate, nitrate, phosphate
- organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate.
- base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and salts of organic bases such as, for example, ethylenediamine, ethanolamine, N, - dialkylene ethanolamine, triethanolamine, glucamine and basic amino acid salts.
- Particularly preferred derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by making a compound administered orally more easily absorbed by blood), or that enhance the release of the original compound in a biological compartment (for example, the brain or lymphatic system) in relation to the original species.
- prodrug is used in its broadest sense and encompasses those derivatives that are converted in vivo into the compounds of the invention. Such derivatives will be apparent to those skilled in the art, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the compounds present: esters, amino acid esters, phosphate esters, salts sulfonate esters metallic, carbamates, and amides.
- the compounds of formula (I) may be in crystalline form as free compounds or as solvates and it is intended that both forms are within the scope of the present invention.
- the methods of Solvation are generally known within the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.
- the compounds of formula (I) or their salts or solvates are preferably in a pharmaceutically acceptable or substantially pure form.
- Pharmaceutically acceptable means, among others, that they have a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels.
- the purity levels for the active ingredient are preferably greater than 50%, more preferably, greater than 70%, more preferably, greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of its salts, solvates or prodrugs.
- the compounds of general formula (I) can be found as tautomers of formula (II) or formula (III). However, in the examples described herein, where R 3 is hydrogen, they have been obtained as compounds of formula (III) when R 2 is also hydrogen, and as compounds of formula (II) when R 2 is an alkyl group, as they are the methyl or ethyl groups.
- the present invention relates to a compound of general formula (II),
- R 1 is independently selected from hydrogen or Ci Ci _ 8;
- R 2 is independently selected from hydrogen, ⁇ -Cis alkyl or C 5 -Ci 8 aryl;
- R 3 is independently selected from hydrogen or C1-C5 alkyl.
- the present invention relates to a compound of general formula (III),
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen.
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 1 is a 1,1-dimethylheptyl group.
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 2 is a group selected from hydrogen, methyl or ethyl.
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen and R 1 is a 1,1-dimethylheptyl group .
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen and R 2 is a group selected from hydrogen, methyl or ethyl. Even more preferred, it is a compound of formula (II) when R 2 is methyl or ethyl.
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 1 is a 1,1-dimethylheptyl group and R 2 is a group selected from hydrogen, methyl or ethyl.
- the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen, R 1 is a 1,1-dimethylheptyl group and R 2 is a group selected from hydrogen, methyl or ethyl.
- the compound of formula (I) or any of its previous preferred embodiments and examples is selected from one of the group consisting of: a) 7- (1 ', 1' -dimethylheptyl) - 1, -dihydro-4, -dimethylchromen [4,3- c] pyrazol-6, 9-dione; b) 7- (1 ', 1' -dimethylheptyl) -2, 4-dihydro-2, 4, 4-trimethylchromen [4,3- c] pyrazol-6, 9-dione; c) 7- (1 ', 1' -dimethylheptyl) -2-ethyl-2, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione; or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof.
- Said compound defined in section a) above consists of a compound of general formula (I) that responds to the structure of formula (II) where R 1 is 1,1-dimethylheptyl, R 2 is hydrogen and R 3 is hydrogen.
- Said compound defined in section b) above consists of a compound of general formula (I) that responds to the structure of formula (III) where R 1 is 1,1-dimethylheptyl, R 2 is methyl and R 3 is hydrogen.
- Said compound defined in section c) above consists of a compound of general formula (I) that responds to the structure of formula (III) where R 1 is 1,1-dimethylheptyl, R 2 is ethyl and R 3 is hydrogen.
- any of the compounds defined above ie those compounds that respond to the general formula (I) (including the compounds of general formulas (II) or (III), or any of the preferred embodiments or examples), they may also be referred to herein as "compound or compounds of the invention".
- the compounds of the invention have antiproliferative activity.
- the cannabinoid activity of the compounds of formula (I) is described based on in vitro tests of binding to human CB1 or CB2 receptors.
- the compounds of formula (I) and their pharmaceutically acceptable salts, prodrugs or solvates thereof, can therefore be used in the prevention and / or treatment of a disorder or a proliferative disease.
- compositions containing a therapeutically effective amount of a compound of formula (I), its pharmaceutically acceptable salts, prodrugs or solvates thereof, together with pharmaceutically acceptable carriers constitute an additional aspect of the present invention That is, a second aspect of the invention relates to a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound of the invention.
- said pharmaceutical composition may also be referred to as "pharmaceutical composition of the invention”.
- vehicle refers to a diluent, adjuvant or excipient with which the active substance is administered.
- Such pharmaceutical vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
- water or aqueous solutions of saline solution and aqueous solutions of dextrose and glycerol are used as vehicles, particularly for injectable solutions.
- Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences” by EW Martin, 1995.
- the vehicles of the invention are approved by the regulatory agency of a state government or a federal government, or are listed in the United States Pharmacopoeia, in the European Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.
- the amount of compound of the invention, its pharmaceutically acceptable salts, prodrugs or solvates thereof, therapeutically effective to be administered (also referred to herein as therapeutically effective or effective amount), as well as its dosage to treat a pathological state with said Compounds will depend on numerous factors, including age, patient status, disease severity, route and frequency of administration, modulator compound to be used, etc.
- the compounds and pharmaceutical compositions of this invention can be used alone or together with other drugs to provide a combination therapy.
- the other drugs can form part of the same pharmaceutical composition, or be provided as a separate pharmaceutical composition, for administration at the same time or at a different time.
- Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) for oral, topical or parenteral administration.
- a third aspect of the present invention relates to the use of a compound of the invention for the manufacture of a pharmaceutical composition.
- the compounds of formula (I) have an antiproliferative activity.
- antiproliferative activity refers to the proliferation of malignant tumors.
- malignant tumors refer to blastoma, carcinoma, glioma, leukemia, lymphoma, melanoma.
- a fourth aspect of the invention thus relates to a compound of the invention, or a pharmaceutical composition of the invention, for use in the treatment of a cancer or a malignant tumor, where preferably the cancer or malignant tumor is selected from blastoma. , carcinoma, glioma, leukemia, lymphoma and melanoma.
- the cancer or malignant tumor is selected from blastoma. , carcinoma, glioma, leukemia, lymphoma and melanoma.
- their use in the treatment of cancers or malignant tumors may be advantageous because they combine said cancer or tumor treatment with additional effects derived from their cannabinoid activity (e.g. antiemetic and / or analgesic effects), where such additional effects may be beneficial for the patient by reducing side effects of a cancer treatment such as emesis and / or pain.
- said cancer or malignant tumor is selected from a malignant liver tumor (eg a hepatocarcinoma) or a malignant prostate tumor (eg a prostate cancer).
- a malignant liver tumor eg a hepatocarcinoma
- a malignant prostate tumor eg a prostate cancer
- the compound of the invention, or the pharmaceutical composition of the invention, for use in the treatment of a cancer or a malignant tumor can obviously be understood as a method of treating said cancer or tumor, which comprises administration to a subject of a therapeutically effective amount of said compound or pharmaceutical composition of the invention.
- the present invention also relates to a method of treating a cancer or a malignant tumor (preferably selected from blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma; and even more preferably, between a hepatocarcinoma or a cancer prostate), which comprises administering to a subject the compound of the invention in a therapeutically effective amount, or a pharmaceutical composition of the invention comprising the compound of the invention in a therapeutically effective amount.
- a cancer or a malignant tumor preferably selected from blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma; and even more preferably, between a hepatocarcinoma or a cancer prostate
- this aspect of the present invention also contemplates the use of the compound of the invention, or of the pharmaceutical composition of the invention, for the manufacture of a pharmaceutical composition, for the treatment of said cancers or malignant tumors.
- a compound of the invention for the manufacture of a pharmaceutical composition for the treatment of a cancer or a malignant tumor where the cancer or malignant tumor is preferably selected from blastoma, carcinoma, is also the object of the invention.
- glioma, leukemia, lymphoma and melanoma is also the object of the invention.
- said cancer or tumor is a hepatocarcinoma or a prostate cancer.
- a fifth aspect of the invention relates to a process for obtaining a compound of general formula (I), which comprises an oxidation with [bis (trifluoroacetoxy) iodine] benzene of a compound of general formula (IV),
- R 1 is independently selected from hydrogen or Ci Ci _ 8;
- R 2 is independently selected from hydrogen, ⁇ -Cis alkyl or C 5 -Ci 8 aryl;
- R is independently selected from hydrogen or C1-C5 alkyl.
- FIG. 1 Effect of the compounds of examples 1, 2 and 3 of the present description on cell viability.
- the HepG2 cell lines (derived from hepatocellular carcinoma), LNCaP (derived from androgen dependent prostate cancer) and PC-3 (derived from androgen independent prostate cancer), were treated with increasing doses of the compounds of examples 1, 2 and 3 for 48 hours after which the viability with MT was determined.
- the data are averages of two experiments performed in triplicate + D.E. (Standard deviation)
- FIG. 1 Effect of the compound of example 1 of the present description on the cell cycle.
- the LNCaP and PC-3 cell lines were treated for 24 hours with the doses of the compound of the Example 1 to which a 50% decrease in cell viability was obtained.
- A After treatment, the cell cycle was analyzed by flow cytometry.
- B After the treatment, the cells were stained with Annexin V and with propidium iodide, determining the different cell populations by flow cytometry in order to detect the cells in apoptosis. The image is representative of two independent tests performed in duplicate.
- FIG. 3 In vivo antitumor effect of the compound of example 1 of the present description on prostate cancer lines.
- Nu / nu atomic mice were injected subcutaneously with LNCaP cells (A) or PC-3 cells (B).
- A LNCaP cells
- B PC-3 cells
- a 15-day treatment with vehicle (Control) or 2 mg / Kg of the example 1 compound administered intraperitoneally was started.
- the tumor was measured daily.
- the figure shows the growth curves of the tumors from the beginning of the treatment. The results are expressed as both by one of the initial tumor size and correspond to the mean ⁇ SE (Standard Error) of 6 mice per group.
- ⁇ SE Standard Error
- 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4, 3-c] pyrazol-9- has been used as starting products corresponding oles.
- the 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4, 3- c] pyrazole-9-starting olives can be obtained by a combination of synthetic reactions known in the state of the art such as those mentioned in the article by Cumella J. et al. ChemMedChem 2012, 7, 452-463.
- the 4,4-dimethyl dihydrochromen [4,3-c] pyrazol-6,9-diones of formula (I) of the present invention are obtained by oxidation of the 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4,3-c] pyrazol-9-oles corresponding. This oxidation occurs regiospecifically due to the use of [bis (trifluoroacetoxy) iodine] benzene (BTIB) which allows to obtain the desired para-quinone.
- BTIB [bis (trifluoroacetoxy) iodine] benzene
- Example 1 Preparation and obtaining 7- (1 ', 1' -dimethylheptyl) -1, 4- dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione
- reaction mixture is neutralized with a saturated solution of NaHC0 3 and extracted with diethyl ether.
- organic phase is washed with water and dried over anhydrous MgSO 4 .
- the solvent is evaporated in vacuo and the resulting mixture is purified by chromatographic column using as eluent Hex / AcOEt (1: 2). Rto. : 21% Red solid, mp: 85.7 ° C.
- Example 2. Preparation and obtaining 7- (1 ', 1' -dimethylheptyl) -2, 4- dihydro-2, 4, 4-trimethylchromen [4, 3-c] pyrazol-6, 9-dione
- the cannabinoid activity of the compounds of formula (I) was assessed by carrying out in vitro tests of displacement of the cannabinoid radioligand [ 3 H] -CP55940 (10 ⁇ ) and [ 3 H] -WIN 55,212-2 (50 ⁇ ) in cells transfected by human CB1 or CB2 receptors.
- the affinity constants of the compound of example 1 of the present invention are given below by way of illustration: 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3- c] pyrazol-6, 9-dione (Example 1) 324 nM (CB1), 134 nM (CB2).
- LNCaP and PC-3 cells were routinely grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU / ml sodium penicillin G, 100 g / ml streptomycin sulfate and 0.25 g / ⁇ amphotericin B (Invitrogen, Paisley, UK). The cells were kept in a humid environment, at 37 ° C and with 5% C0 2 .
- FBS fetal bovine serum
- 100 IU / ml sodium penicillin G 100 g / ml streptomycin sulfate
- 0.25 g / ⁇ amphotericin B Invitrogen, Paisley, UK.
- Annexin V-FITC apoptosis detection kit (BD, New Jersey) commercial kit was used.
- the cells were seeded in six-well plates with a density of 5,000 cells / cm 2 . Once the treatments were carried out, the cells were trypsinized and centrifuged at 400 g for 5 minutes. Subsequently, the cells were incubated 15 minutes in the dark with 5 ⁇ of Annexin V, conjugated with fluorescein isothiocyanate (FITC) and 5 ⁇ of propidium iodide dissolved in 100 ⁇ of binding buffer (0.1 M Hepes pH 7.4 , 1.4 M NaCl, 25 mM CaCl 2 ). After the incubation time, 400 ⁇ of binding buffer was added and analyzed with a FACSCalibur cytometer.
- FITC fluorescein isothiocyanate
- the Mice were injected subcutaneously in the right flank, with 0.1 ml of PBS buffer with 10 million PC-3 cells or LNCaP.
- the tumor size (which is calculated with the formula 0.523xW 2 xL; W being the width of the tumor and L the length thereof) was controlled and calculated daily using a calibrator. After fifteen days of treatment the animals were sacrificed and the tumors were removed calculating the weight and size of the tumor at the end of the treatment.
- the effect of the compounds examples 1, 2 and 3 on cell viability was analyzed using three cell lines derived from human tumors.
- the HepG2 cell line comes from a differentiated hepatocellular carcinoma
- the LNCaP cell line comes from an androgen-dependent prostate tumor
- the PC-3 cell line comes from an androgen-independent and therefore more aggressive prostate cancer.
- the three compounds were more effective in prostate cell lines than in hepatocellular carcinoma, highlighting the example 1 compound of the present patent as the most potent in all cell lines with an IC50 of 30 ⁇ in HepG2 and 15 ⁇ cells in LNCaP and PC-3 cells.
- Antitumor activity in vivo Illustratively, the antitumor activity of 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione (example 1) is presented ) in vivo. It was analyzed by subcutaneous induction of xenograph tumors in immunocompromised mice administering the example compound of the present patent (2 mg / Kg) dissolved in olive oil, intraperitoneally for 15 days consecutively. Control animals were given the same volume of olive oil
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Abstract
The invention relates to a compound of general formula (I) or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, wherein R1 is independently selected from hydrogen or C1-C18 alkyl; R2 is independently selected from hydrogen, C1-C18 alkyl or C5-C18 aryl; and R3 is independently selected from hydrogen or C1-C5 alkyl. The invention also relates to the use of said compound of formula (I) for treating cancer or a malignant tumor, such as a blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma, and to a method for treating said cancer or tumour, comprising the administration of a therapeutically effective quantity of the compound of formula (I). Consequently, the invention further relates to a pharmaceutical composition comprising a therapeutically effective quantity of a compound of general formula (I) and at least one pharmaceutically acceptable excipient. In addition, the invention relates to the method for producing said compound of general formula (I) by means of oxidation with [bis(trifluoroacetoxy)iode]benzene of the corresponding precursor phenolic derivative.
Description
CROMENOPIRAZOLDIONAS COMO DERIVADOS CANNABINOIDES DE QUINONAS CON CHROMENOPIRAZOLDIONAS AS CANNABINOID DERIVATIVES OF QUINONAS WITH
ACTIVIDAD ANTITUMORAL ANTITUMORAL ACTIVITY
DESCRIPCION DESCRIPTION
Sector de la Técnica Technical Sector
La presente invención se engloba en el campo de la farmacología. Específicamente, la presente invención se refiere a compuestos derivados de cromeno [ 4 , 3-c] pirazol-6, 9-dionas , su uso para la fabricación de un medicamento, y el uso de este medicamento para un tratamiento antitumoral. The present invention is encompassed in the field of pharmacology. Specifically, the present invention relates to chromene-derived compounds [4,3-c] pyrazol-6,9-diones, their use for the manufacture of a medicament, and the use of this medicament for an antitumor treatment.
Estado de la Técnica Anterior Prior State of the Art
A pesar de los recientes avances en la quimioterapia y la radiación, el cáncer sigue siendo una de las principales causas de muerte a cualquier edad en todo el mundo. Se han hecho y se continúan realizando esfuerzos enormes con el fin de obtener nuevos agentes antitumorales activos y seguros para administrarlos a pacientes que padecen cáncer. Despite recent advances in chemotherapy and radiation, cancer remains one of the leading causes of death at any age worldwide. Huge efforts have been made and continue to be made in order to obtain new active and safe antitumor agents to administer to patients suffering from cancer.
Las quinonas son compuestos de origen natural o sintético. Varias de ellas poseen algunas funciones fisiológicas tal como sucede con la vitamina K necesaria para la coagulación de la sangre. Sin embargo, muchas de ellas son citotóxicas, lo que las convierte en candidatos a fármacos antitumorales. Los efectos antineoplásicos de quinonas han sido ampliamente descritos en la literatura (C. Asche, Míní-Rev. Med. Chem. 2005, 5, 449-467) . Por ejemplo, las antraciclinas que son antibióticos procedentes de bacterias del género Streptomyces, se usan en el tratamiento de muchos tipos de cánceres. Quinones are compounds of natural or synthetic origin. Several of them have some physiological functions such as the vitamin K necessary for blood clotting. However, many of them are cytotoxic, which makes them candidates for antitumor drugs. The antineoplastic effects of quinones have been widely described in the literature (C. Asche, Miní-Rev. Med. Chem. 2005, 5, 449-467). For example, anthracyclines that are antibiotics from bacteria of the genus Streptomyces, are used in the treatment of many types of cancers.
Los cannabinoides actúan sobre los receptores del sistema endocannabinoide y pueden proporcionar beneficios terapéuticos en diversas patologías y en particular son útiles en el tratamiento del cáncer. Por un lado, tienen efectos paliativos: antieméticos y analgésicos (R. G. Pertwee, Br. J. Pharmacol. 2009, 156 (3) , 397-
411), y por otro lado, muestran propiedades antitumorales por ellos mismos (M. Guzman, Nat. Rev. Cáncer 2003, 3, 745-765; S. Sarfaraz et al, Cáncer Res. 2008, 68, 330-342) . Se ha mostrado que los cannabinoides inhiben el crecimiento de diversas células tumorales en animales de laboratorio, por ejemplo de glioma (I. Galve-Roperh et al, Nat. Med. 2000, 6, 313-319), de melanoma, de mama y de próstata (I. Diaz-Laviada, Nat. Rev. ürol . 2011, 8(10), 553-561) . Sin embargo, se ha descrito que un cannabinoide, el (9)- tetrahidrocannabinol potencia el crecimiento de cáncer de mama y metástasis específicamente en células que expresan bajos niveles de receptores de cannabinoides, lo que sugiere que la exposición a cannabinoides puede incrementar la incidencia de cáncer de mama, así como otros cánceres que no expresan receptores cannabinoides (S. Sarfaraz, V. M. Adhami, D, N. Syed, F. Afaq, H. Muhktar, Cáncer Res. 2008, 68, 339-342) . Cannabinoids act on the receptors of the endocannabinoid system and can provide therapeutic benefits in various pathologies and in particular they are useful in the treatment of cancer. On the one hand, they have palliative effects: antiemetics and analgesics (RG Pertwee, Br. J. Pharmacol. 2009, 156 (3), 397- 411), and on the other hand, show antitumor properties by themselves (M. Guzman, Nat. Rev. Cancer 2003, 3, 745-765; S. Sarfaraz et al, Cancer Res. 2008, 68, 330-342). Cannabinoids have been shown to inhibit the growth of various tumor cells in laboratory animals, for example glioma (I. Galve-Roperh et al, Nat. Med. 2000, 6, 313-319), melanoma, breast and of prostate (I. Diaz-Laviada, Nat. Rev. ürol. 2011, 8 (10), 553-561). However, it has been described that a cannabinoid, (9) - tetrahydrocannabinol, enhances the growth of breast cancer and metastasis specifically in cells that express low levels of cannabinoid receptors, suggesting that exposure to cannabinoids may increase the incidence of breast cancer, as well as other cancers that do not express cannabinoid receptors (S. Sarfaraz, VM Adhami, D, N. Syed, F. Afaq, H. Muhktar, Cancer Res. 2008, 68, 339-342).
Existen múltiples procesos biológicos involucrados en el desarrollo de células tumorales y por ello, se suelen utilizar terapias combinadas en el tratamiento contra el cáncer. El concepto de "designed múltiple ligands" para abordar la terapia anticancerosa consiste en desarrollar una sola molécula capaz de actuar sobre varias dianas terapéuticas. Sin embargo, conseguir este tipo de moléculas es muy difícil porque los motivos farmacofóricos están asociados a dianas distintas. El planteamiento más usado consiste en la unión de dichos farmacoforos mediante un espaciador. Es mucho menos frecuente conseguir una sola entidad molecular capaz de actuar sobre dos dianas diferentes. There are multiple biological processes involved in the development of tumor cells and therefore, combined therapies are often used in cancer treatment. The concept of "designed multiple ligands" to address anticancer therapy is to develop a single molecule capable of acting on several therapeutic targets. However, getting this type of molecules is very difficult because the pharmacophoric motifs are associated with different targets. The most commonly used approach consists in the union of said pharmacoforos by means of a spacer. It is much less frequent to achieve a single molecular entity capable of acting on two different targets.
Estructuras de cromenopirazoles como cannabinoides desarrolladas por el grupo de investigación de los autores de la presente invención han sido el objeto de una solicitud de patente (WO2010109050 ) , de las cuales se ha divulgado su actividad como analgésicos, sin que se les haya atribuido propiedades antitumorales . Chromenopyrazole structures such as cannabinoids developed by the research group of the authors of the present invention have been the subject of a patent application (WO2010109050), of which their activity as analgesics has been disclosed, without having been attributed antitumor properties .
Quinonas derivadas de estructuras cannabinoides con propiedades antitumorales han sido descritas por primera vez por R. Mechoulam {Tetrahedron 1968, 24, 5615-5624). De hecho tres de estas
estructuras fueron objeto de una solicitud de patente (WO2005/067917 ) . Se trata de para-quinonas derivadas del cannabidiol, del A8-tetrahidrocannabinol y del cannabinol. Sin embargo, estas quinonas de naturaleza triciclica, a pesar de mostrar propiedades antiproliferativas en lineas celulares de cáncer humano, no se unen al receptor cannabinoide CB1 según precisan sus autores, y por lo tanto, excluyen un mecanismo antitumoral de tipo cannabinoide. Más recientemente, otras quinonas derivadas del cannabidiol y su uso en el tratamiento de cánceres han sido reivindicadas en otra solicitud de patente (WO2008/107878 ) donde se describe la actividad anti-proliferativa pero en ningún caso tampoco se ha encontrado que presenten actividad cannabinoide y/o de unión a los receptores cannabinoides . Es decir, que introducir una quinona en este tipo de estructura cannabinoide condujo a la pérdida de propiedades cannabinoides. Quinones derived from cannabinoid structures with antitumor properties have been described for the first time by R. Mechoulam {Tetrahedron 1968, 24, 5615-5624). In fact three of these structures were the subject of a patent application (WO2005 / 067917). It is para-quinones derived from cannabidiol, of A 8 -tetrahydrocannabinol and cannabinol. However, these quinones of tricyclic nature, in spite of showing antiproliferative properties in human cancer cell lines, do not bind to the CB1 cannabinoid receptor as their authors specify, and therefore, exclude a cannabinoid-type antitumor mechanism. More recently, other quinones derived from cannabidiol and their use in the treatment of cancers have been claimed in another patent application (WO2008 / 107878) where anti-proliferative activity is described but in no case have they been found to have cannabinoid activity and / or cannabinoid receptor binding. That is, introducing a quinone into this type of cannabinoid structure led to the loss of cannabinoid properties.
Por tanto, la presente invención se enfrenta con el problema de proporcionar nuevos compuestos útiles para el tratamiento del cáncer. Debido a la potencialidad de las quinonas como anticancerosos y de los cannabinoides como anticancerosos, antieméticos y analgésicos, resulta de gran interés disponer de moléculas que puedan reunir en una misma estructura las características de una quinona y de un cannabinoide. Preferiblemente, dichos compuestos deberían conservar e incluso mejorar la actividad citotóxica de los compuestos conocidos a la vez que evitan parte de los efectos secundarios adversos asociados . Therefore, the present invention faces the problem of providing new compounds useful for the treatment of cancer. Due to the potential of quinones as anticancer and cannabinoids as anticancer, antiemetic and analgesic, it is of great interest to have molecules that can combine the characteristics of a quinone and a cannabinoid in the same structure. Preferably, said compounds should preserve and even improve the cytotoxic activity of known compounds while avoiding part of the associated adverse side effects.
Descripción de la invención Description of the invention
Los autores de la presente invención han encontrado que los compuestos de fórmula (I) actúan como antitumorales . Los compuestos de fórmula (I) reivindicados en la presente invención, consisten en una serie de cromenopirazoldionas que, además de tener estructuras de quinonas, tienen presente en su estructura un anillo de pirazol que resultó ser necesario para mantener una cierta afinidad por los receptores cannabinoides, y que los diferencia de otras estructuras de quinona conocidas en el estado de la técnica, como por ejemplo
las descritas previamente por Mechoulam et al. {Tetrahedron 1968, 24, 5615-5624; W02005 /O 67917 ; WO2008/107878 ) . The authors of the present invention have found that the compounds of formula (I) act as antitumor agents. The compounds of formula (I) claimed in the present invention, consist of a series of chromenopyrazoldiones that, in addition to having quinone structures, have in their structure a pyrazole ring that proved necessary to maintain a certain affinity for cannabinoid receptors , and that differentiates them from other quinone structures known in the state of the art, such as those previously described by Mechoulam et al. {Tetrahedron 1968, 24, 5615-5624; W02005 / O 67917; WO2008 / 107878).
En un primer aspecto, la presente invención se refiere a un compuesto de fórmula general (I), In a first aspect, the present invention relates to a compound of general formula (I),
Fórmula (I) o un tautómero, sal farmacéuticamente aceptable, profármaco o solvato del mismo, donde: Formula (I) or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, where:
- R1 se selecciona independientemente entre hidrógeno o alquilo- R 1 is independently selected from hydrogen or alkyl
Ci_Ci8 ; Ci _ Ci 8 ;
- R2 se selecciona independientemente entre hidrógeno, alquilo Οχ-- R 2 is independently selected from hydrogen, alkyl Οχ-
Cis o arilo C5-Ci8; Cis or aryl C 5 -Ci 8 ;
- R3 se selecciona independientemente entre hidrógeno o alquilo- R 3 is independently selected from hydrogen or alkyl
Ci-C5. Ci-C 5 .
El término "alquilo" se refiere, en la presente invención, a cadenas alifáticas, lineales o ramificadas, saturadas o insaturadas, que tienen de 1 a 18 átomos de carbono, preferiblemente entre 1 y 9 átomos de carbono. Por ejemplo, pero sin limitarse a, metilo, etilo, n-propilo, i-propilo, n-butilo, tere-butilo, sec-butilo, n-pentilo, n-hexilo, n-heptilo, 1' , 1' -dimetilheptilo, 1, 2-dimetiheptilo, Ι',Ι'- dimetiletilo, adamantilo, ciclohexilo, 1-ciclopentilhexilo, etc. Los grupos alquilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como halógeno, hidroxilo o ácido carboxilico.
Dichos grupos alquilo sustituidos son por ejemplo, pero sin limitarse a, 1- ( 1 , 3-ditiolano ) hexilo, 1-hidroxietilo, hidroximetilo , etc . The term "alkyl" refers, in the present invention, to aliphatic, linear or branched, saturated or unsaturated chains, having 1 to 18 carbon atoms, preferably 1 to 9 carbon atoms. For example, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, tere-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, 1 ', 1' - dimethylheptyl, 1,2-dimethheptyl, Ι ', Ι'-dimethylethyl, adamantyl, cyclohexyl, 1-cyclopentylhexyl, etc. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl or carboxylic acid. Said substituted alkyl groups are for example, but not limited to, 1- (1, 3-dithiolane) hexyl, 1-hydroxyethyl, hydroxymethyl, etc.
El término "arilo" se refiere, en la presente invención, a anillos aromáticos sencillos o múltiples, que tienen de entre 5 a 18 eslabones en los que se ha eliminado un protón del anillo. Los grupos arilo son por ejemplo, pero sin limitarse a, fenilo, naftilo, difenilo, indenilo, fenantrilo o antracilo . Preferiblemente el grupo arilo tiene de 5 a 7 átomos de carbono y más preferiblemente el grupo arilo es un fenilo. Los radicales arilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como alquilo (Ci-C6) , halógeno, hidroxilo o ácido carboxilico. Radicales arilo sustituidos son por ejemplo, pero sin limitarse a, 2,4- diclorofenilo, 1, 3-diclorofenilo, 3, 5-difluorofenilo, 3- metoxifenilo . The term "aryl" refers, in the present invention, to single or multiple aromatic rings, having between 5 and 18 links in which a proton has been removed from the ring. The aryl groups are for example, but not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthracil. Preferably the aryl group has 5 to 7 carbon atoms and more preferably the aryl group is a phenyl. Aryl radicals may be optionally substituted by one or more substituents such as alkyl (Ci-C 6), halogen, hydroxyl or carboxylic acid. Substituted aryl radicals are, for example, but not limited to 2,4-dichlorophenyl, 1,3-dichlorophenyl, 3,5-difluorophenyl, 3- methoxyphenyl.
El término "halógeno" se refiere, en la presente invención, a bromo, cloro, yodo o flúor. Preferentemente a cloro. The term "halogen" refers, in the present invention, to bromine, chlorine, iodine or fluorine. Preferably chlorine.
Según la invención, el tamaño de los sustituyentes R1 a R3 es importante para la funcionalidad de los compuestos de fórmula (I), como por ejemplo cuando se pretenden modificar entre otras sus propiedades farmacocinéticas , de biodisponibilidad, de solubilidad o de estabilidad. De este modo, sustituciones de cadena carbonada posibles en R3 son aquellas en las cuales dicho sustituyente no exceda de 5 átomos de carbono, mientras que en el caso de los sustituyentes de R1 y de R2 el número de átomos puede ser bastante mayor. De hecho, las posiciones definidas por los sustituyentes R1 y R2 pueden ser funcionalizadas con largas cadenas carbonadas, como por ejemplo ácidos grasos, donde los más habitualmente empleados incluyen cadenas de hasta 18 átomos de carbono. No obstante, sustituyentes como el 1 , 1-dimetilheptilo (de 9 átomos de carbono) constituye un buen ejemplo de cadena carbonada intermedia entre los valores máximo y mínimo aquí contemplados . menos que se indique lo contrario, los compuestos de la invención
también incluyen compuestos que difieren sólo en la presencia de uno o más átomos isotópicamente enriquecidos. Por ejemplo, los compuestos que tienen las presentes estructuras, a excepción de la sustitución de un hidrógeno por un deuterio o por tritio, o la sustitución de un carbono por un carbono enriquecido en 13C o 14C o un nitrógeno enriquecido en 15N, están dentro del alcance de esta invención . According to the invention, the size of the substituents R 1 to R 3 is important for the functionality of the compounds of formula (I), such as when they are intended to modify, among others, their pharmacokinetic, bioavailability, solubility or stability properties. Thus, possible carbon chain substitutions in R 3 are those in which said substituent does not exceed 5 carbon atoms, while in the case of the substituents of R 1 and R 2 the number of atoms can be quite greater . In fact, the positions defined by the substituents R 1 and R 2 can be functionalized with long carbon chains, such as fatty acids, where the most commonly used include chains of up to 18 carbon atoms. However, substituents such as 1,1-dimethylheptyl (of 9 carbon atoms) constitute a good example of an intermediate carbon chain between the maximum and minimum values contemplated herein. Unless otherwise indicated, the compounds of the invention they also include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, the compounds having the present structures, except for the replacement of a hydrogen with a deuterium or tritium, or the replacement of a carbon with a carbon enriched in 13 C or 14 C or a nitrogen enriched in 15 N, are within the scope of this invention.
El término "tautómero" o "forma tautomérica", tal y como se usa en la presente invención, se refiere a isómeros estructurales de diferentes energías que son interconvertibles vía una barrera de baja energía. Por ejemplo, tautómeros protónicos (también conocidos como tautómeros prototrópicos ) que incluyen interconversiones mediante la migración de un protón, como por ejemplo isomerizaciones ceto-enólicas o imina-enamina . Los tautómeros de valencia incluyen interconversiones por reorganización de algunos electrones de enlace . The term "tautomer" or "tautomeric form", as used in the present invention, refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, protonic tautomers (also known as prototropic tautomers) that include interconversions by migration of a proton, such as keto-enolic or imine-enamine isomerizations. Valencia tautomers include interconversions by reorganization of some bond electrons.
El término "sales, solvatos, profármacos farmacéuticamente aceptables" se refiere a cualquier sal, éster, solvato farmacéuticamente aceptable, o cualquier otro compuesto que, cuando se administra a un receptor es capaz de proporcionar (directamente o indirectamente) un compuesto según se describe en el presente documento. Sin embargo, se apreciará que las sales farmacéuticamente no aceptables también están dentro del alcance de la invención ya que éstas pueden ser útiles en la preparación de sales farmacéuticamente aceptables. La preparación de sales, profármacos y derivados puede llevarse a cabo mediante métodos conocidos en la técnica . The term "pharmaceutically acceptable salts, solvates, prodrugs" refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound that, when administered to a receptor, is capable of providing (directly or indirectly) a compound as described in This document. However, it will be appreciated that pharmaceutically acceptable salts are also within the scope of the invention since these may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by methods known in the art.
Por ejemplo, sales farmacéuticamente aceptables de compuestos previstos en el presente documento, se sintetizan mediante métodos químicos convencionales a partir de un compuesto original que contiene un resto básico o ácido. Generalmente, tales sales se preparan, por ejemplo, haciendo reaccionar las formas de ácido o base libre de los compuestos con una cantidad estequiométrica de la base o ácido apropiado en agua o en un disolvente orgánico o en una
mezcla de los dos. Generalmente, se prefieren medios no acuosos como éter, acetato de etilo, etanol, isopropanol o acetonitrilo . Ejemplos de sales de adición de ácidos incluyen sales de adición de ácido mineral tales como, por ejemplo, clorhidrato, bromhidrato, yodhidrato, sulfato, nitrato, fosfato y sales de adición de acido orgánico tales como, por ejemplo, acetato, maleato, fumarato, citrato, oxalato, succinato, tartrato, malato, mandelato, metanosulfonato y p-toluenosulfonato . Ejemplos de sales de adición de bases incluyen sales inorgánicas tales como, por ejemplo, sales de sodio, potasio, calcio, amonio, magnesio, aluminio y litio, y sales de bases orgánicas tales como, por ejemplo, etilenodiamina, etanolamina, N, -dialquilenetanolamina, trietanolamina, glucamina y sales de aminoácidos básicos . For example, pharmaceutically acceptable salts of compounds provided herein are synthesized by conventional chemical methods from an original compound containing a basic or acidic moiety. Generally, such salts are prepared, for example, by reacting the free acid or base forms of the compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mix of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, iodhydrate, sulfate, nitrate, phosphate and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate and p-toluenesulfonate. Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum and lithium salts, and salts of organic bases such as, for example, ethylenediamine, ethanolamine, N, - dialkylene ethanolamine, triethanolamine, glucamine and basic amino acid salts.
Los derivados o profármacos particularmente favoritos son aquellos que aumentan la biodisponibilidad de los compuestos de esta invención cuando se administran tales compuestos a un paciente (por ejemplo, haciendo que un compuesto administrado por vía oral se absorba más fácilmente por la sangre), o que potencian la liberación del compuesto original en un compartimento biológico (por ejemplo, el cerebro o el sistema linfático) con relación a la especie original . Particularly preferred derivatives or prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a patient (for example, by making a compound administered orally more easily absorbed by blood), or that enhance the release of the original compound in a biological compartment (for example, the brain or lymphatic system) in relation to the original species.
Cualquier compuesto que es un profármaco de un compuesto de fórmula (I) está dentro del alcance de la invención. El término "profármaco" se usa en su sentido más amplio y abarca aquellos derivados que se convierten in vivo en los compuestos de la invención. Tales derivados serán evidentes para aquellos expertos en la técnica, e incluyen, dependiendo de los grupos funcionales presentes en la molécula y sin limitación, los siguientes derivados de los compuestos presentes: esteres, esteres de aminoácido, esteres de fosfato, esteres de sulfonato de sales metálicas, carbamatos, y amidas . Any compound that is a prodrug of a compound of formula (I) is within the scope of the invention. The term "prodrug" is used in its broadest sense and encompasses those derivatives that are converted in vivo into the compounds of the invention. Such derivatives will be apparent to those skilled in the art, and include, depending on the functional groups present in the molecule and without limitation, the following derivatives of the compounds present: esters, amino acid esters, phosphate esters, salts sulfonate esters metallic, carbamates, and amides.
Los compuestos de fórmula (I) pueden estar en forma cristalina como compuestos libres o como solvatos y se pretende que ambas formas están dentro del alcance de la presente invención. Los métodos de
solvatación se conocen generalmente dentro de la técnica. Los solvatos adecuados son solvatos farmacéuticamente aceptables. En una realización particular, el solvato es un hidrato. The compounds of formula (I) may be in crystalline form as free compounds or as solvates and it is intended that both forms are within the scope of the present invention. The methods of Solvation are generally known within the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.
Los compuestos de fórmula (I) o sus sales o solvatos están preferiblemente en una forma farmacéuticamente aceptable o sustancialmente pura. Por forma farmacéuticamente aceptable se entiende, entre otros, que tienen un nivel de pureza farmacéuticamente aceptable excluyendo los aditivos farmacéuticos normales tales como diluyentes y portadores, y no incluyendo material considerado tóxico a niveles de dosificación normales. Los niveles de pureza para el principio activo son preferiblemente superiores al 50%, más preferiblemente, superiores al 70%, más preferiblemente, superiores al 90%. En una realización preferida, son superiores al 95% del compuesto de fórmula (I), o de sus sales, solvatos o profármacos . The compounds of formula (I) or their salts or solvates are preferably in a pharmaceutically acceptable or substantially pure form. Pharmaceutically acceptable means, among others, that they have a pharmaceutically acceptable level of purity excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels. The purity levels for the active ingredient are preferably greater than 50%, more preferably, greater than 70%, more preferably, greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of its salts, solvates or prodrugs.
Los compuestos de fórmula general (I) pueden encontrarse como tautómeros de fórmula (II) o fórmula (III) . Sin embargo, en los ejemplos aquí descritos, donde R3 es hidrógeno, se han obtenido como compuestos de fórmula (III) cuando R2 es también hidrógeno, y como compuestos de fórmula (II) cuando R2 es un grupo alquilo, como son los grupos metilo o etilo. The compounds of general formula (I) can be found as tautomers of formula (II) or formula (III). However, in the examples described herein, where R 3 is hydrogen, they have been obtained as compounds of formula (III) when R 2 is also hydrogen, and as compounds of formula (II) when R 2 is an alkyl group, as they are the methyl or ethyl groups.
De acuerdo con una realización preferida, la presente invención se refiere a un compuesto de fórmula general (II), According to a preferred embodiment, the present invention relates to a compound of general formula (II),
Fórmula (II)
o un tautómero, sal farmacéuticamente aceptable, profármaco o solvato del mismo, donde R1, R2 y R3 se definen como anteriormente: Formula (II) or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, wherein R 1 , R 2 and R 3 are defined as above:
- R1 se selecciona independientemente entre hidrógeno o alquilo Ci_Ci8 ; - R 1 is independently selected from hydrogen or Ci Ci _ 8;
- R2 se selecciona independientemente entre hidrógeno, alquilo Οχ- Cis o arilo C5-Ci8; - R 2 is independently selected from hydrogen, Οχ-Cis alkyl or C 5 -Ci 8 aryl;
- R3 se selecciona independientemente entre hidrógeno o alquilo C1-C5. - R 3 is independently selected from hydrogen or C1-C5 alkyl.
En otra realización preferida, la presente invención se refiere a un compuesto de fórmula general (III), In another preferred embodiment, the present invention relates to a compound of general formula (III),
Fórmula (III) o un tautómero, sal farmacéuticamente aceptable, profármaco o solvato del mismo, donde R1, R2 y R3 se definen como anteriormente. Formula (III) or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, wherein R 1 , R 2 and R 3 are defined as above.
En un ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R3 es un hidrógeno. In a preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen.
En otro ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R1 es un grupo 1 , 1-dimetilheptilo . In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 1 is a 1,1-dimethylheptyl group.
En otro ejemplo preferido, el compuesto de fórmula ( I ) , asi como cualquiera de sus formas tautoméricas de fórmula (II) ó ( III ) , es un
compuesto donde R2 es un grupo seleccionado entre hidrógeno, metilo o etilo . In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 2 is a group selected from hydrogen, methyl or ethyl.
En otro ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R3 es un hidrógeno y R1 es un grupo 1,1- dimetilheptilo . In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen and R 1 is a 1,1-dimethylheptyl group .
En otro ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R3 es un hidrógeno y R2 es un grupo seleccionado entre hidrógeno, metilo o etilo. Aún más preferido, es un compuesto de fórmula (II) cuando R2 es metilo o etilo. In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen and R 2 is a group selected from hydrogen, methyl or ethyl. Even more preferred, it is a compound of formula (II) when R 2 is methyl or ethyl.
En otro ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R1 es un grupo 1 , 1-dimetilheptilo y R2 es un grupo seleccionado entre hidrógeno, metilo o etilo. In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 1 is a 1,1-dimethylheptyl group and R 2 is a group selected from hydrogen, methyl or ethyl.
En otro ejemplo preferido, el compuesto de fórmula (I), asi como cualquiera de sus formas tautoméricas de fórmula (II) ó (III), es un compuesto donde R3 es un hidrógeno, R1 es un grupo 1 , 1-dimetilheptilo y R2 es un grupo seleccionado entre hidrógeno, metilo o etilo. In another preferred example, the compound of formula (I), as well as any of its tautomeric forms of formula (II) or (III), is a compound where R 3 is a hydrogen, R 1 is a 1,1-dimethylheptyl group and R 2 is a group selected from hydrogen, methyl or ethyl.
De acuerdo con un modo de realización preferido, el compuesto de formula (I) o cualquiera de sus realizaciones y ejemplos preferidos anteriores, se selecciona entre uno del grupo que consiste en: a) 7- (1' , 1' -dimetilheptil ) -1, -dihidro- 4, -dimetilcromeno [4,3- c]pirazol-6, 9-diona; b) 7- (1' , 1' -dimetilheptil) -2, 4-dihidro-2, 4, 4-trimetilcromeno [4,3- c] pirazol-6, 9-diona; c) 7- (1' , 1' -dimetilheptil) -2-etil-2, 4-dihidro-4, 4- dimetilcromeno [4, 3-c]pirazol-6, 9-diona;
o un tautómero, sal farmacéuticamente aceptable, profármaco o solvato de los mismos . According to a preferred embodiment, the compound of formula (I) or any of its previous preferred embodiments and examples, is selected from one of the group consisting of: a) 7- (1 ', 1' -dimethylheptyl) - 1, -dihydro-4, -dimethylchromen [4,3- c] pyrazol-6, 9-dione; b) 7- (1 ', 1' -dimethylheptyl) -2, 4-dihydro-2, 4, 4-trimethylchromen [4,3- c] pyrazol-6, 9-dione; c) 7- (1 ', 1' -dimethylheptyl) -2-ethyl-2, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione; or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof.
Dicho compuesto definido en el apartado a) anterior, consiste en un compuesto de fórmula general (I) que responde a la estructura de fórmula (II) donde R1 es 1, 1-dimetilheptilo, R2 es hidrógeno y R3 es hidrógeno . Said compound defined in section a) above, consists of a compound of general formula (I) that responds to the structure of formula (II) where R 1 is 1,1-dimethylheptyl, R 2 is hydrogen and R 3 is hydrogen.
Dicho compuesto definido en el apartado b) anterior, consiste en un compuesto de fórmula general (I) que responde a la estructura de fórmula (III) donde R1 es 1, 1-dimetilheptilo, R2 es metilo y R3 es hidrógeno . Said compound defined in section b) above, consists of a compound of general formula (I) that responds to the structure of formula (III) where R 1 is 1,1-dimethylheptyl, R 2 is methyl and R 3 is hydrogen.
Dicho compuesto definido en el apartado c) anterior, consiste en un compuesto de fórmula general (I) que responde a la estructura de fórmula (III) donde R1 es 1, 1-dimetilheptilo, R2 es etilo y R3 es hidrógeno . Said compound defined in section c) above, consists of a compound of general formula (I) that responds to the structure of formula (III) where R 1 is 1,1-dimethylheptyl, R 2 is ethyl and R 3 is hydrogen.
Según la presente memoria, cualquiera de los compuestos definidos anteriormente, es decir aquellos compuestos que responden a la fórmula general (I) (incluyendo los compuestos de fórmulas generales (II) o (III), o cualquiera de las realizaciones o ejemplos preferidos), pueden ser igualmente referidos en esta memoria como "compuesto o compuestos de la invención". According to the present specification, any of the compounds defined above, ie those compounds that respond to the general formula (I) (including the compounds of general formulas (II) or (III), or any of the preferred embodiments or examples), they may also be referred to herein as "compound or compounds of the invention".
En la presente invención se ha comprobado que los compuestos de la invención presentan actividad antiproliferativa . Y además, se describe la actividad cannabinoide de los compuestos de fórmula (I) en base a ensayos in vi tro de unión a los receptores humanos CB1 o CB2. Los compuestos de fórmula (I) y sus sales farmacéuticamente aceptables, profármacos o solvatos de los mismos, pueden ser utilizados, por tanto, en la prevención y/o el tratamiento de un trastorno o de una enfermedad proliferativa . Las composiciones farmacéuticas que contienen una cantidad terapéuticamente eficaz de un compuesto de fórmula (I), sus sales farmacéuticamente aceptables, profármacos o solvatos del mismo, junto con los vehículos farmacéuticamente aceptables, constituyen un aspecto adicional de la
presente invención. Es decir, un segundo aspecto de la invención se refiere a una composición farmacéutica que comprende al menos un vehículo farmacéuticamente aceptable y una cantidad terapéuticamente eficaz de al menos un compuesto de la invención. En adelante, dicha composición farmacéutica puede ser igualmente referida como "composición farmacéutica de la invención". In the present invention it has been found that the compounds of the invention have antiproliferative activity. And in addition, the cannabinoid activity of the compounds of formula (I) is described based on in vitro tests of binding to human CB1 or CB2 receptors. The compounds of formula (I) and their pharmaceutically acceptable salts, prodrugs or solvates thereof, can therefore be used in the prevention and / or treatment of a disorder or a proliferative disease. Pharmaceutical compositions containing a therapeutically effective amount of a compound of formula (I), its pharmaceutically acceptable salts, prodrugs or solvates thereof, together with pharmaceutically acceptable carriers, constitute an additional aspect of the present invention That is, a second aspect of the invention relates to a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a therapeutically effective amount of at least one compound of the invention. Hereinafter, said pharmaceutical composition may also be referred to as "pharmaceutical composition of the invention".
El término "vehículo" se refiere a un diluyente, adyuvante o excipiente con el que se administra el principio activo. Tales vehículos farmacéuticos pueden ser líquidos estériles, tales como agua y aceites, incluyendo aquellos de origen del petróleo, animal, vegetal o sintético, tales como aceite de cacahuete, aceite de soja, aceite mineral, aceite de sésamo y similares. Se emplean preferiblemente como vehículos agua o disoluciones acuosas de solución salina y disoluciones acuosas de dextrosa y glicerol, particularmente para las disoluciones inyectables. Vehículos farmacéuticos adecuados se describen en "Remington' s Pharmaceutical Sciences" por E. W. Martin, 1995. Preferiblemente, los vehículos de la invención están aprobados por la agencia reguladora de un gobierno de estado o un gobierno federal, o están enumerados en la Farmacopea Estadounidense, en la Farmacopea Europea u otra farmacopea reconocida en general para su uso en animales, y más particularmente en humanos . The term "vehicle" refers to a diluent, adjuvant or excipient with which the active substance is administered. Such pharmaceutical vehicles may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Preferably water or aqueous solutions of saline solution and aqueous solutions of dextrose and glycerol are used as vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles are described in "Remington's Pharmaceutical Sciences" by EW Martin, 1995. Preferably, the vehicles of the invention are approved by the regulatory agency of a state government or a federal government, or are listed in the United States Pharmacopoeia, in the European Pharmacopoeia or other pharmacopoeia generally recognized for use in animals, and more particularly in humans.
La cantidad de compuesto de la invención, sus sales farmacéuticamente aceptables, profármacos o solvatos del mismo, terapéuticamente eficaz que debe administrarse (también referida en la presente descripción como cantidad terapéuticamente eficaz o efectiva) , así como su dosificación para tratar un estado patológico con dichos compuestos, dependerá de numerosos factores, entre los que se encuentra la edad, el estado del paciente, la severidad de la enfermedad, la ruta y frecuencia de administración, el compuesto modulador a utilizar, etc. The amount of compound of the invention, its pharmaceutically acceptable salts, prodrugs or solvates thereof, therapeutically effective to be administered (also referred to herein as therapeutically effective or effective amount), as well as its dosage to treat a pathological state with said Compounds will depend on numerous factors, including age, patient status, disease severity, route and frequency of administration, modulator compound to be used, etc.
Los compuestos y composiciones farmacéuticas de esta invención pueden ser empleados solos o junto con otros fármacos para proporcionar una terapia combinada . Los otros fármacos pueden formar
parte de la misma composición farmacéutica, o ser proporcionados como una composición farmacéutica separada, para su administración al mismo tiempo o en un momento diferente. Ejemplos de composiciones farmacéuticas incluyen cualquier composición sólida (comprimidos, pildoras, cápsulas, gránulos, etc.) o liquida (disoluciones, suspensiones o emulsiones) para la administración oral, tópica o parenteral . The compounds and pharmaceutical compositions of this invention can be used alone or together with other drugs to provide a combination therapy. The other drugs can form part of the same pharmaceutical composition, or be provided as a separate pharmaceutical composition, for administration at the same time or at a different time. Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) for oral, topical or parenteral administration.
Un tercer aspecto de la presente invención se refiere al uso de un compuesto de la invención para la fabricación de una composición farmacéutica . A third aspect of the present invention relates to the use of a compound of the invention for the manufacture of a pharmaceutical composition.
De acuerdo con una realización preferida, los compuestos de formu (I) tienen una actividad antiproliferativa . According to a preferred embodiment, the compounds of formula (I) have an antiproliferative activity.
De acuerdo con las características de algunas realizaciones, la actividad antiproliferativa se refiere a la proliferación de tumores malignos . According to the characteristics of some embodiments, antiproliferative activity refers to the proliferation of malignant tumors.
De acuerdo con las características de algunas realizaciones, los tumores malignos se refieren a blastoma, carcinoma, glioma, leucemia, linfoma, melanoma. According to the characteristics of some embodiments, malignant tumors refer to blastoma, carcinoma, glioma, leukemia, lymphoma, melanoma.
Un cuarto aspecto de la invención se refiere por tanto a un compuesto de la invención, o una composición farmacéutica de la invención, para su uso en el tratamiento de un cáncer o un tumor maligno, donde preferentemente el cáncer o tumor maligno se selecciona entre blastoma, carcinoma, glioma, leucemia, linfoma y melanoma. Teniendo en cuenta las propiedades de los compuestos de la invención, su uso en el tratamiento de cánceres o tumores malignos puede resultar ventajoso debido a que combinan dicho tratamiento del cáncer o tumor con efectos adicionales derivados de la actividad cannabinoide de estos (p. ej . efectos antieméticos y/o analgésicos), donde dicho efectos adicionales pueden ser beneficiosos para el paciente al disminuir efectos secundarios propios de un tratamiento contra el cáncer como son la emesis y/o el dolor. En una realización preferida, dicho cáncer o tumor maligno es seleccionado entre un
tumor maligno de hígado (p. ej . un hepatocarcinoma) o un tumor maligno de próstata (p. ej . un cáncer de próstata) . Según la presente descripción, el compuesto de la invención, o la composición farmacéutica de la invención, para su uso en el tratamiento de un cáncer o un tumor maligno, puede ser obviamente entendido como un método de tratamiento de dicho cáncer o tumor, que comprende la administración a un sujeto de una cantidad terapéuticamente efectiva de dicho compuesto o composición farmacéutica de la invención. Dicho en otras palabras, la presente invención se refiere asimismo a un método de tratamiento de un cáncer o un tumor maligno (preferentemente seleccionado entre blastoma, carcinoma, glioma, leucemia, linfoma y melanoma; y aún más preferentemente, entre un hepatocarcinoma o un cáncer de próstata) , que comprende administrar a un sujeto el compuesto de la invención en una cantidad terapéuticamente efectiva, o una composición farmacéutica de la invención que comprenda el compuesto de la invención en una cantidad terapéuticamente efectiva. A fourth aspect of the invention thus relates to a compound of the invention, or a pharmaceutical composition of the invention, for use in the treatment of a cancer or a malignant tumor, where preferably the cancer or malignant tumor is selected from blastoma. , carcinoma, glioma, leukemia, lymphoma and melanoma. Taking into account the properties of the compounds of the invention, their use in the treatment of cancers or malignant tumors may be advantageous because they combine said cancer or tumor treatment with additional effects derived from their cannabinoid activity (e.g. antiemetic and / or analgesic effects), where such additional effects may be beneficial for the patient by reducing side effects of a cancer treatment such as emesis and / or pain. In a preferred embodiment, said cancer or malignant tumor is selected from a malignant liver tumor (eg a hepatocarcinoma) or a malignant prostate tumor (eg a prostate cancer). According to the present description, the compound of the invention, or the pharmaceutical composition of the invention, for use in the treatment of a cancer or a malignant tumor, can obviously be understood as a method of treating said cancer or tumor, which comprises administration to a subject of a therapeutically effective amount of said compound or pharmaceutical composition of the invention. In other words, the present invention also relates to a method of treating a cancer or a malignant tumor (preferably selected from blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma; and even more preferably, between a hepatocarcinoma or a cancer prostate), which comprises administering to a subject the compound of the invention in a therapeutically effective amount, or a pharmaceutical composition of the invention comprising the compound of the invention in a therapeutically effective amount.
Asimismo, este aspecto de la presente invención también contempla el uso del compuesto de la invención, o de la composición farmacéutica de la invención, para la fabricación de una composición farmacéutica, para el tratamiento de dichos cánceres o tumores malignos. Esto quiere decir, que es asimismo objeto de la invención el uso de un compuesto de la invención para la fabricación de una composición farmacéutica para tratamiento de un cáncer o un tumor maligno, donde el cáncer o tumor maligno preferentemente se selecciona entre blastoma, carcinoma, glioma, leucemia, linfoma y melanoma; y más preferentemente dicho cáncer o tumor es un hepatocarcinoma o un cáncer de próstata. Also, this aspect of the present invention also contemplates the use of the compound of the invention, or of the pharmaceutical composition of the invention, for the manufacture of a pharmaceutical composition, for the treatment of said cancers or malignant tumors. This means that the use of a compound of the invention for the manufacture of a pharmaceutical composition for the treatment of a cancer or a malignant tumor, where the cancer or malignant tumor is preferably selected from blastoma, carcinoma, is also the object of the invention. glioma, leukemia, lymphoma and melanoma; and more preferably said cancer or tumor is a hepatocarcinoma or a prostate cancer.
Un quinto aspecto de la invención se refiere a un procedimiento de obtención de un compuesto de fórmula general (I), que comprende una oxidación con [bis ( trifluoroacetoxi ) yodo ] benceno de un compuesto de fórmula general (IV),
A fifth aspect of the invention relates to a process for obtaining a compound of general formula (I), which comprises an oxidation with [bis (trifluoroacetoxy) iodine] benzene of a compound of general formula (IV),
Fórmula (IV) donde los sustituyentes R1, R2 y R3 se definen como anteriormente: Formula (IV) where the substituents R 1 , R 2 and R 3 are defined as above:
R1 se selecciona independientemente entre hidrógeno o alquilo Ci_Ci8 ; R 1 is independently selected from hydrogen or Ci Ci _ 8;
R2 se selecciona independientemente entre hidrógeno, alquilo Οχ- Cis o arilo C5-Ci8; R 2 is independently selected from hydrogen, Οχ-Cis alkyl or C 5 -Ci 8 aryl;
R se selecciona independientemente entre hidrógeno o alquilo C1-C5. R is independently selected from hydrogen or C1-C5 alkyl.
Breve descripción de las figuras Brief description of the figures
Figura 1. Efecto de los compuestos de los ejemplos 1, 2 y 3 de la presente descripción sobre la viabilidad celular. Las lineas celulares HepG2 (derivada de carcinoma hepatocelular ) , LNCaP (derivada de cáncer de próstata dependiente de andrógenos) y PC-3 (derivada de cáncer de próstata independiente de andrógenos), fueron tratadas con dosis crecientes de los compuestos de los ejemplos 1, 2 y 3 durante 48h tras lo cual se determinó la viabilidad con MT . Los datos son medias de dos experimentos realizados en triplicado + D.E. (Desviación Estándar) Figure 1. Effect of the compounds of examples 1, 2 and 3 of the present description on cell viability. The HepG2 cell lines (derived from hepatocellular carcinoma), LNCaP (derived from androgen dependent prostate cancer) and PC-3 (derived from androgen independent prostate cancer), were treated with increasing doses of the compounds of examples 1, 2 and 3 for 48 hours after which the viability with MT was determined. The data are averages of two experiments performed in triplicate + D.E. (Standard deviation)
Figura 2. Efecto del compuesto del ejemplo 1 de la presente descripción sobre el ciclo celular. Las lineas celulares LNCaP y PC- 3 fueron tratadas durante 24 horas con las dosis del compuesto del
ejemplo 1 a las que se obtuvo un 50% de disminución de la viabilidad celular. A, Después del tratamiento se analizó el ciclo celular por citometria de flujo. B, Después del tratamiento las células se tiñeron con Anexina V y con ioduro de propidio determinándose las distintas poblaciones celulares mediante citometria de flujo con el fin de detectar las células en apoptosis. La imagen es representativa de dos ensayos independientes realizados por duplicado . Figure 2. Effect of the compound of example 1 of the present description on the cell cycle. The LNCaP and PC-3 cell lines were treated for 24 hours with the doses of the compound of the Example 1 to which a 50% decrease in cell viability was obtained. A, After treatment, the cell cycle was analyzed by flow cytometry. B, After the treatment, the cells were stained with Annexin V and with propidium iodide, determining the different cell populations by flow cytometry in order to detect the cells in apoptosis. The image is representative of two independent tests performed in duplicate.
Figura 3. Efecto antitumoral in vivo del compuesto del ejemplo 1 de la presente descripción sobre las lineas de cáncer de próstata. Se inyectaron subcutáneamente ratones atimicos nu/nu con células LNCaP (A) o células PC-3 (B) . Cuando los tumores alcanzaron un tamaño de 150 mm3, se comenzó un tratamiento de 15 días de duración con vehículo (Control) o 2 mg/Kg del compuesto ejemplo 1 administrado por vía intraperitoneal . El tumor se midió diariamente. En la figura se representan las curvas de crecimiento de los tumores desde el comienzo del tratamiento. Los resultados se expresan como tanto por uno del tamaño inicial del tumor y corresponden a la media ± E.E. (Error Estándar) de 6 ratones por grupo. A la derecha de cada gráfica se muestra una imagen representativa de un tumor control y uno tratado con el compuesto del ejemplo 1 de cada una de las dos líneas celulares utilizadas Figure 3. In vivo antitumor effect of the compound of example 1 of the present description on prostate cancer lines. Nu / nu atomic mice were injected subcutaneously with LNCaP cells (A) or PC-3 cells (B). When the tumors reached a size of 150 mm 3 , a 15-day treatment with vehicle (Control) or 2 mg / Kg of the example 1 compound administered intraperitoneally was started. The tumor was measured daily. The figure shows the growth curves of the tumors from the beginning of the treatment. The results are expressed as both by one of the initial tumor size and correspond to the mean ± SE (Standard Error) of 6 mice per group. To the right of each graph is shown a representative image of a control tumor and one treated with the compound of example 1 of each of the two cell lines used
Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención . The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
EJEMPLOS EXAMPLES
Para la síntesis de los compuestos de fórmula (I) de la presente invención se ha utilizado como productos de partida, los 4,4- dimetil-7- (H o alquil ) -dihidrocromeno [ 4 , 3-c] pirazol-9-oles correspondientes. Los 4, 4-dimetil-7- (H o alquil ) -dihidrocromeno [ 4 , 3- c] pirazol-9-oles de partida pueden obtenerse mediante una combinación de reacciones sintéticas conocidas en el estado del arte
tales como las mencionadas en el articulo de Cumella J. et al. ChemMedChem 2012, 7, 452-463. For the synthesis of the compounds of formula (I) of the present invention, 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4, 3-c] pyrazol-9- has been used as starting products corresponding oles. The 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4, 3- c] pyrazole-9-starting olives can be obtained by a combination of synthetic reactions known in the state of the art such as those mentioned in the article by Cumella J. et al. ChemMedChem 2012, 7, 452-463.
Procedimiento general para la síntesis de para-q inonas derivados de cromeno [4, 3-cJpirazol . General procedure for the synthesis of para-q inones derived from chromene [4,3-cJpirazol.
Los 4 , 4-dimetil-dihidrocromeno [ 4 , 3-c] pirazol-6, 9-dionas de fórmula (I) de la presente invención se obtienen por oxidación de los 4,4- dimetil-7- (H o alquil ) -dihidrocromeno [ 4 , 3-c] pirazol-9-oles correspondientes. Esta oxidación transcurre de forma regioespecifica debido al empleo de [bis (trifluoroacetoxi) yodo] benceno (BTIB) que permite obtener la para-quinona deseada. The 4,4-dimethyl dihydrochromen [4,3-c] pyrazol-6,9-diones of formula (I) of the present invention are obtained by oxidation of the 4,4-dimethyl-7- (H or alkyl) -dihydrochromen [4,3-c] pyrazol-9-oles corresponding. This oxidation occurs regiospecifically due to the use of [bis (trifluoroacetoxy) iodine] benzene (BTIB) which allows to obtain the desired para-quinone.
Este procedimiento se resume en el siguiente esquema (I) This procedure is summarized in the following scheme (I)
ESQUEMA (I) SCHEME (I)
Ejemplo 1.- Preparación y obtención de 7- (1 ', 1' -dimetilheptil) -1 , 4- dihidro-4 , 4-dimetilcromeno [4 , 3-c]pirazol-6, 9-diona Example 1.- Preparation and obtaining 7- (1 ', 1' -dimethylheptyl) -1, 4- dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione
A una disolución de 7- ( 1' , 1 ' -dimetilheptil ) -1 , 4-dihidro-4 , 4- dimetilcromeno [ 4 , 3-c] pirazol-9-ol (0,130 g, 0,38 mmol) en CH3CN/ H20 (6:1, 2,5 mL) se añade gota a gota una disolución de [bis (trifluoroacetoxi) yodo] benceno (BTIB) (0,490 g, 1,14 mmol) en 2 mi de CH3CN/ H20 (6:1) . La reacción se agita durante 15 minutos a temperatura ambiente observando que la disolución adquiere un color rojo intenso. La mezcla de reacción se neutraliza con una disolución saturada de NaHC03 y se extrae con dietiléter. La fase orgánica se lava con agua y se seca sobre MgS04 anhidro. El disolvente se evapora a vacio y la mezcla resultante se purifica mediante columna cromatográfica utilizando como eluyente Hex/AcOEt (1:2). Rto . : 21% Sólido rojo, P.f.: 85,7 °C. 1H-RMN (CDC13, 300 MHz) δ: 8,41 (sa, 1H,
H) ; 7,40 (s, 1H, 3-H); 6,69 (s, 1H, 8-H); 1,59 (sa, 6H, OC(CH3)2); 1,55-1,48 (m, 2H, 2'-H); 1,30 (s, 6H, C(CH3)2); 1,27-1,23 (m, 6H, 3'- H, 4'-H, 5'-H); 1,19 (sa, 2H, 6'H); 0,86-0,82 (m, 3H, 7'-H) ppm. 13C- RMN (CDC13) 5:184,1 (6-C); 180,9 (9-C); 160,2 (5a-C); 161,2 (7-C); 137,8 (9b-C); 132,0 (8-C); 130,4 (9a-C); 129,5 (3-C); 113,8 (3a-C); 78,6 (OC(CH3)2); 43,3 (2'-C); 30,9 (l'-C); 29,6; 28,7 y 27,4 (3'-C, 4'-C y 5'-C); 25,1 (C(CH3)2); 23,2 (OC(CH3)2); 21,8 (6'-C); 14,0 (7'- C) ppm. HPLC/MS*: Gradiente: 70% A a 100% A; EM (ES+) m/z: 357 (98%) [M+H]+; tR (tiempo de retención) : 3,37 min. Anal.: C2iH28N203 (356, 46 g/mol) Teórico: C, 70,76%; H, 7,92%. Hallado: C, 71,03%; H, 8,24%. To a solution of 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-9-ol (0.1330 g, 0.38 mmol) in CH 3 CN / H 2 0 (6: 1, 2.5 mL) a solution of [bis (trifluoroacetoxy) iodine] benzene (BTIB) (0.490 g, 1.14 mmol) in 2 mL of CH 3 is added dropwise CN / H 2 0 (6: 1). The reaction is stirred for 15 minutes at room temperature observing that the solution acquires an intense red color. The reaction mixture is neutralized with a saturated solution of NaHC0 3 and extracted with diethyl ether. The organic phase is washed with water and dried over anhydrous MgSO 4 . The solvent is evaporated in vacuo and the resulting mixture is purified by chromatographic column using as eluent Hex / AcOEt (1: 2). Rto. : 21% Red solid, mp: 85.7 ° C. 1 H-NMR (CDC1 3 , 300 MHz) δ: 8.41 (sa, 1H, H); 7.40 (s, 1 H, 3-H); 6.69 (s, 1H, 8-H); 1.59 (sa, 6H, OC (CH 3 ) 2 ); 1.55-1.48 (m, 2H, 2'- H); 1.30 (s, 6H, C (CH 3) 2); 1.27 to 1.23 (m, 6H, 3 '- H, 4'-H, 5'-H); 1.19 (sa, 2H, 6'H); 0.86-0.82 (m, 3H, 7'-H) ppm. 13 C-NMR (CDC1 3 ) 5: 184.1 (6-C); 180.9 (9-C); 160.2 (5a-C); 161.2 (7-C); 137.8 (9b-C); 132.0 (8-C); 130.4 (9a-C); 129.5 (3-C); 113.8 (3a-C); 78.6 (OC (CH 3 ) 2 ); 43.3 ( 2'- C); 30.9 (l '-C); 29.6; 28.7 and 27.4 (3 '-C 4' -C and 5 '-C); 25.1 (C (CH 3 ) 2 ); 23.2 (OC (CH 3 ) 2 ); 21.8 (6' - C); 14.0 (7 '- C) ppm. HPLC / MS *: Gradient: 70% A to 100% A; MS (ES + ) m / z: 357 (98%) [M + H] + ; t R (retention time): 3.37 min. Anal .: C 2 iH 28 N 2 0 3 (356, 46 g / mol) Theoretical: C, 70.76%; H, 7.92%. Found: C, 71.03%; H, 8.24%.
Ejemplo 2.- Preparación y obtención de 7- (1' , 1' -dimetilheptil) -2 , 4- dihidro-2 , 4 , 4-trimetilcromeno [4 , 3-c]pirazol-6, 9-diona Example 2.- Preparation and obtaining 7- (1 ', 1' -dimethylheptyl) -2, 4- dihydro-2, 4, 4-trimethylchromen [4, 3-c] pyrazol-6, 9-dione
La síntesis de este compuesto se basa en el procedimiento descrito en el ejemplo 1 siguiendo las condiciones de reacción detalladas a continuación : 7- ( 1' , 1 ' -dimetilheptil ) -2, 4-dihidro-2, 4, 4- trimetilcromeno [ 4 , 3-c] pirazol-9-ol (0,035 g, 0,1 mmol),The synthesis of this compound is based on the procedure described in Example 1 following the reaction conditions detailed below: 7- (1 ', 1' -dimethylheptyl) -2, 4-dihydro-2, 4, 4-trimethylchromen [ 4, 3-c] pyrazol-9-ol (0.035 g, 0.1 mmol),
[bis (trifluoroacetoxi) yodo] benceno (BTIB) (0,129 g, 0,3 mmol). Eluyente Hex/AcOEt (1:4). Rto . : 33 % Sólido rojo, P.f.: 138,2 °C. 1H- RMN (CDCI3, 300 MHz) δ: 7,36 (s, 1H, 3-H); 6,61 (s, 1H, 8-H); 3,95[bis (trifluoroacetoxy) iodine] benzene (BTIB) (0.129 g, 0.3 mmol). Eluent Hex / AcOEt (1: 4). Rto. : 33% Red solid, mp: 138.2 ° C. 1 H-NMR (CDCI 3 , 300 MHz) δ: 7.36 (s, 1H, 3-H); 6.61 (s, 1H, 8-H); 3.95
(s, 3H, NCH3, ) ; 1,84 (s, 6H, OC(CH3)2); 1,70 (sa, 2H, 2'-H); 1,33 (s, 6H, C(CH3)2); 1,26 (sa, 6H, 3'-H, 4'-H, 5'-H); 1,16-1,13 (m, 2H, 6'H); 0,85 (t, J= 6,9 Hz, 3H, 7'-H) ppm. 13C-RMN (CDC13) 5:186,2 (6- C); 181,4 (9-C); 160,2 (5a-C); 155,0 (7-C); 139,6 (9b-C); 135,4 (8- C); 129,3 (9a-C); 126,9 (3-C); 115,7 (3a-C); 81,5 (OC(CH3)2); 43,0(s, 3H, NCH 3 ,); 1.84 (s, 6H, OC (CH 3 ) 2 ); 1.70 (sa, 2H, 2'-H); 1.33 (s, 6H, C (CH 3 ) 2 ); 1.26 (sa, 6H, 3'-H, 4'-H, 5'-H); 1.16-1.13 (m, 2H, 6'H); 0.85 (t, J = 6.9 Hz, 3H, 7'-H) ppm. 13 C-NMR (CDC1 3 ) 5: 186.2 (6- C); 181.4 (9-C); 160.2 (5a-C); 155.0 (7-C); 139.6 (9b-C); 135.4 (8-C); 129.3 (9a-C); 126.9 (3-C); 115.7 (3a-C); 81.5 (OC (CH 3 ) 2 ); 43.0
(NCH3) ; 40,9 (2'-C); 31,7 (l'-C); 30,8; 29,3 y 28,1 (3'-C, 4 ' -C y 5'- C) ; 27,5 (C(CH3)2); 23,4 (OC(CH3)2); 22,9 (6'-C); 14,6 (7'-C) ppm. HPLC/MS*: Gradiente: 80% A a 100% A; EM (ES+) m/z: 371 (100%) [M+H]+; tR: 2,43 min. Anal.: C22H3oN203 (370, 49 g/mol) Teórico: C, 71,32%; H, 8,16%. Hallado: C, 70,98%; H, 8,31%. (NCH 3 ); 40.9 (2'-C); 31.7 (l'-C);30.8; 29.3 and 28.1 (3'-C, 4'-C and 5'-C); 27.5 (C (CH 3 ) 2 ); 23.4 (OC (CH 3 ) 2 ); 22.9 (6'-C); 14.6 (7'-C) ppm. HPLC / MS *: Gradient: 80% A to 100% A; MS (ES + ) m / z: 371 (100%) [M + H] + ; t R : 2.43 min. Anal .: C 22 H 3 oN 2 0 3 (370, 49 g / mol) Theoretical: C, 71.32%; H, 8.16%. Found: C, 70.98%; H, 8.31%.
Ejemplo 3.- Preparación y obtención de 7- (1' , 1' -dimetilheptil) -2- etil-2 , 4-dihidro-4 , 4-dimetilcromeno [4 , 3-c]pirazol-6 , 9-diona Example 3.- Preparation and obtaining 7- (1 ', 1' -dimethylheptyl) -2-ethyl-2, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione
La síntesis de este compuesto se basa en el procedimiento descrito en el ejemplo 1 siguiendo las condiciones de reacción detalladas a
continuación: 7- (1' , 1' -dimetilheptil ) -2-etil-2, -dihidro-4, 4- dimetilcromeno [ 4 , 3-c] pirazol-9-ol (0,061 g, 0,16 mmol) ,The synthesis of this compound is based on the procedure described in Example 1 following the reaction conditions detailed in continued: 7- (1 ', 1' -dimethylheptyl) -2-ethyl-2, -dihydro-4, 4- dimethylchromen [4, 3-c] pyrazol-9-ol (0.061 g, 0.16 mmol),
[bis (trifluoroacetoxi) yodo] benceno (BTIB) (0,212 g, 0,49 mmol). Eluyente Hex/AcOEt (2:3) . Rto . : 36 % Sólido rojo, P.f.: 83,9 °C. 1H- RMN (CDC13, 300 MHz) δ: 7,15 (s, 1H, 3-H); 6,50 (s, 1H, 8-H); 4,28[bis (trifluoroacetoxy) iodine] benzene (BTIB) (0.212 g, 0.49 mmol). Eluent Hex / AcOEt (2: 3). Rto. : 36% Red solid, mp: 83.9 ° C. 1 H-NMR (CDC1 3 , 300 MHz) δ: 7.15 (s, 1H, 3-H); 6.50 (s, 1H, 8-H); 4.28
(c, J= 7,4 Hz, 2H, NCfí2CH3, ) ; 1,72 (s, 6H, OC(CH3)2); 1,61-1,58 (m, 2H, 2'-H); 1,49 (t, J= 7,4 Hz, 3H, NCH2Cfí3) ; 1,30 (s, 6H, C(CH3)2); 1,23-1,10 (m, 8H, 3'-H, 4'-H, 5'-H y 6'-H); 0,89 (t, J" = 6,9 Hz, 3H, 7'-H) ppm. 13C-RMN (CDC13) 5:183,4 (6-C); 180,5 (9-C); 152,4 (5a-C); 151,4 (7-C); 137,2 (9b-C); 133,1 (8-C); 122,1 (3-C); 120,9 (3a-C); 110,6 (9a-C); 79,9 (OC(CH3)2); 46,6 (NCH2CH3) ; 39,7 (2'-C); 37,6 (1'- C) ; 31,6; 29,7 y 28,7 (3'-C, 4 ' -C y 5'-C); 26,6 (OC(CH3)2); 24,1(c, J = 7.4 Hz, 2H, NCfí 2 CH 3 ,); 1.72 (s, 6H, OC (CH 3 ) 2 ); 1.61-1.58 (m, 2H, 2'-H); 1.49 (t, J = 7.4 Hz, 3H, NCH 2 Cfí 3 ); 1.30 (s, 6H, C (CH 3 ) 2 ); 1.23-1.10 (m, 8H, 3'-H, 4'-H, 5'-H and 6'-H); 0.89 (t, J " = 6.9 Hz, 3H, 7'-H) ppm. 13 C-NMR (CDC1 3 ) 5: 183.4 (6-C); 180.5 (9-C) ; 152.4 (5a-C); 151.4 (7-C); 137.2 (9b-C); 133.1 (8-C); 122.1 (3-C); 120.9 ( 3a-C); 110.6 (9a-C); 79.9 (OC (CH 3 ) 2 ); 46.6 (NCH 2 CH 3 ); 39.7 (2'-C); 37.6 ( 1'- C); 31.6; 29.7 and 28.7 (3'-C, 4'-C and 5'-C); 26.6 (OC (CH 3 ) 2 ); 24.1
(C(CH3)2); 21,5 (6'-C); 14,6 (NCH2CH3) ; 13,0 (7'-C) ppm. HPLC/MS*: Gradiente: 70% A a 100% A; EM (ES+) m/z: 385 (96%) [M+H]+; tR: 3,86 min. Anal.: C23H32N203 (384, 51 g/mol) Teórico: C, 71,84%; H, 8,39%. Hallado: C, 72,01%; H, 8,62%. (C (CH 3 ) 2 ); 21.5 (6'-C); 14.6 (NCH 2 CH 3 ); 13.0 (7'-C) ppm. HPLC / MS *: Gradient: 70% A to 100% A; MS (ES + ) m / z: 385 (96%) [M + H] + ; t R : 3.86 min. Anal .: C 23 H 32 N 2 0 3 (384, 51 g / mol) Theoretical: C, 71.84%; H, 8.39%. Found: C, 72.01%; H, 8.62%.
* Para todos los datos de HPLC/MS las fases móviles empleadas son: A (MeCN + 0,08% ácido fórmico) y B (H20 + 1% ácido fórmico). El gradiente utilizado viene indicado en cada caso, en 5 minutos a un flujo de 0,25 mL/min y λ = 254 nm. * For all HPLC / MS data the mobile phases used are: A (MeCN + 0.08% formic acid) and B (H 2 0 + 1% formic acid). The gradient used is indicated in each case, in 5 minutes at a flow of 0.25 mL / min and λ = 254 nm.
C. Ensayos biológicos C. Biological tests
C .1. Afinidad por los receptores cannabinoides . C. 1. Affinity for cannabinoid receptors.
En la presente invención se valoró la actividad cannabinoide de los compuestos de fórmula (I) llevando a cabo ensayos in vi tro de desplazamiento del radioligando cannabinoide [3H]-CP55940 (10μΜ) y [3H] -WIN 55,212-2 (50μΜ) en células transfectadas por los receptores humanos CB1 o CB2. A continuación se proporciona las constantes de afinidad del compuesto del ejemplo 1 de la presente invención a modo de ilustración: 7- ( 1' , 1 ' -dimetilheptil ) -1 , 4-dihidro-4 , 4- dimetilcromeno [ 4 , 3-c] pirazol-6, 9-diona (Ejemplo 1) 324 nM (CB1), 134 nM (CB2) . In the present invention, the cannabinoid activity of the compounds of formula (I) was assessed by carrying out in vitro tests of displacement of the cannabinoid radioligand [ 3 H] -CP55940 (10μΜ) and [ 3 H] -WIN 55,212-2 (50μΜ ) in cells transfected by human CB1 or CB2 receptors. The affinity constants of the compound of example 1 of the present invention are given below by way of illustration: 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3- c] pyrazol-6, 9-dione (Example 1) 324 nM (CB1), 134 nM (CB2).
C.2. Efecto antiproliferativo in vitro e in vivo.
El efecto antiproliferativo de la 7- (1' , 1' -dimetilheptil) -1, 4- dihidro-4, -dimetilcromeno [4, 3-c] pirazol-6, 9-diona (ejemplo 1), de la 7- ( 1' , 1 ' -dimetilheptil ) -2 , 4-dihidro-2, 4, 4-trimetilcromeno [4,3- c] pirazol-6, 9-diona (ejemplo 2) y de la 7- (1' , 1' -dimetilheptil) -2- etil-2, 4-dihidro-4, 4-dimetilcromeno [4, 3-c] pirazol-6, 9-diona (ej emplo 3) de la presente invención fue estudiado in vitro en lineas celulares derivadas de carcinoma hepatocelular y de cáncer de próstata utilizando los siguientes métodos. C.2. Antiproliferative effect in vitro and in vivo. The antiproliferative effect of 7- (1 ', 1' -dimethylheptyl) -1, 4- dihydro-4, -dimethylchromen [4, 3-c] pyrazol-6, 9-dione (example 1), of 7- (1 ', 1' -dimethylheptyl) -2, 4-dihydro-2, 4, 4-trimethylchromen [4,3- c] pyrazol-6, 9-dione (example 2) and 7- (1 ', 1'-dimethylheptyl) -2- ethyl-2, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione (eg emplo 3) of the present invention was studied in vitro in lines Cells derived from hepatocellular carcinoma and prostate cancer using the following methods.
Mantenimiento y cultivo celular. Las células LNCaP y PC-3 fueron crecidas de manera rutinaria en medio RPMI 1640 suplementado con un 10% de suero fetal bovino (FBS), 100 IU/ml penicilina G sódica, 100 g/ml de sulfato de estreptomicina y 0,25 g/μΐ de anfotericina B (Invitrogen, Paisley, UK) . Las células se mantuvieron en ambiente húmedo, a 37 °C y con un 5% de C02. Maintenance and cell culture. LNCaP and PC-3 cells were routinely grown in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 IU / ml sodium penicillin G, 100 g / ml streptomycin sulfate and 0.25 g / μΐ amphotericin B (Invitrogen, Paisley, UK). The cells were kept in a humid environment, at 37 ° C and with 5% C0 2 .
Test de proliferación celular. Tras el tratamiento de las células con los diferentes compuestos, éstas se incubaron con 0,3 mg/ml de bromuro de 3- ( 4 , 5-dimetiltiazol-2-il ) 2 , 5-difenil tetrazolio (MTT) a 37°C en oscuridad durante lh. Posteriormente los cristales se solubilizaron con 500 μΐ de isopropanol ácido y agitación suave. Una vez que la coloración era homogénea se valoraron las distintas absorbancias a 570 nm con un espectrofotómetro de placa (ELX 800 Bio-Tek Instruments, INC) . La medida de absorbancia obtenida en el pocilio control se consideró como el 100% de células vivas, y por lo tanto los valores obtenidos en los diferentes tratamientos fueron referidos a este dato. Cell proliferation test. After treatment of the cells with the different compounds, they were incubated with 0.3 mg / ml of 3- (4,5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide (MTT) at 37 ° C In darkness for lh. Subsequently the crystals were solubilized with 500 μΐ of acid isopropanol and gentle agitation. Once the coloration was homogeneous, the different absorbances at 570 nm were evaluated with a plate spectrophotometer (ELX 800 Bio-Tek Instruments, INC). The absorbance measurement obtained in the control well was considered as 100% of living cells, and therefore the values obtained in the different treatments were referred to this data.
Análisis del ciclo celular mediante citometria de flujo. Para realizar esta técnica las células fueron sembradas, tratadas durante el tiempo estimado y posteriormente levantadas con tripsina. Después se centrifugaron a 400 g durante 5 minutos y se resuspendieron en etanol al 70%. La suspensión celular se mantuvo durante 30 minutos a -20°C para que la células quedaran fijadas y permeabilizadas . Transcurrido ese tiempo las células se volvieron a centrifugar para
retirar el etanol y poder ser lavadas con PBS . Por último se resuspendieron en PBS con 20 μg/μl de ioduro de propidio y 0,2 mg/ml de RNAsa (con el fin de degradar el RNA para que no interfiera en la medida de la florescencia) . Las células se analizaron con un citómetro FACSCalibur (Becton Dickinson Labware, New Yersey, USA) . Para la cuantificación de los porcentajes de células en las diferentes fases del ciclo celular se utilizó el programa WinMDI 2.8, siguiendo las instrucciones del mismo. Analysis of the cell cycle by flow cytometry. To perform this technique the cells were seeded, treated for the estimated time and subsequently lifted with trypsin. They were then centrifuged at 400 g for 5 minutes and resuspended in 70% ethanol. The cell suspension was maintained for 30 minutes at -20 ° C so that the cells were fixed and permeabilized. After that time the cells were centrifuged again to remove the ethanol and be able to be washed with PBS. Finally, they were resuspended in PBS with 20 μg / μl of propidium iodide and 0.2 mg / ml of RNAse (in order to degrade the RNA so that it does not interfere with the extent of flowering). Cells were analyzed with a FACSCalibur cytometer (Becton Dickinson Labware, New Yersey, USA). For the quantification of the percentages of cells in the different phases of the cell cycle, the WinMDI 2.8 program was used, following its instructions.
Unión de ioduro de propidio y Anexina V-FITC. Se empleó el kit comercial Annexín V-FITC apoptosís detection kit (BD, Nueva Jersey). Las células se sembraron en placas de seis pocilios con una densidad de 5.000 células/cm2. Una vez realizados los tratamientos, se tripsinizaron las células y se centrifugaron a 400 g durante 5 minutos. Posteriormente, se incubaron las células 15 minutos en oscuridad con 5 μΐ de Anexina V, conjugada con isotiocianato de fluoresceina (FITC) y 5 μΐ de ioduro de propidio disueltos en 100 μΐ de tampón de unión (Hepes 0,1 M pH 7,4, NaCl 1,4 M, CaCl2 25 mM) . Después del tiempo de incubación, se añadieron 400 μΐ de tampón de unión y se analizaron con un citómetro FACSCalibur. Union of propidium iodide and Annex V-FITC. The Annexin V-FITC apoptosis detection kit (BD, New Jersey) commercial kit was used. The cells were seeded in six-well plates with a density of 5,000 cells / cm 2 . Once the treatments were carried out, the cells were trypsinized and centrifuged at 400 g for 5 minutes. Subsequently, the cells were incubated 15 minutes in the dark with 5 μΐ of Annexin V, conjugated with fluorescein isothiocyanate (FITC) and 5 μΐ of propidium iodide dissolved in 100 μΐ of binding buffer (0.1 M Hepes pH 7.4 , 1.4 M NaCl, 25 mM CaCl 2 ). After the incubation time, 400 μΐ of binding buffer was added and analyzed with a FACSCalibur cytometer.
El efecto antiproliferativo in vivo de la 7- ( 1' , 1 ' -dimetilheptil ) - 1, 4-dihidro-4, 4-dimetilcromeno [4, 3-c] pirazol-6, 9-diona (ejemplo 1) fue realizado según el método descrito a continuación, a modo de ilustración . The in vivo antiproliferative effect of 7- (1 ', 1'-dimethylheptyl) -1,4-dihydro-4,4-dimethylchromen [4,3-c] pyrazol-6,9-dione (example 1) was performed according to the method described below, by way of illustration.
Estudios in vivo. Se utilizaron ratones macho atimicos nu/nu ( BALB/c01aHsd-Foxnlnu) de cuatro semanas de edad (Harían Ibérica, Barcelona, España) . Los animales fueron mantenidos en una zona libre de gérmenes patógenos (S.P.F, space patogen free) en el Centro de Experimentación Animal de la Universidad de Alcalá, con ciclos constantes de luz/oscuridad de 12 horas. In vivo studies Four-week-old male nu / nu (BALB / c01aHsd-Foxnl nu ) male mice (Harían Ibérica, Barcelona, Spain) were used. The animals were kept in a pathogen-free zone (SPF) in the Animal Experimentation Center of the University of Alcalá, with constant light / dark cycles of 12 hours.
Los estudios fueron realizados en concordancia con la regulación española para el uso y cuidados de los animales de experimentación y según las directrices de la Unión Europea relacionadas con la investigación animal (Real Decreto 1201/2005 del 10 de Octubre) . Los
ratones fueron inyectados subcutáneamente en el flanco derecho, con 0,1 mi de Tampón PBS con 10 millones células PC-3 o LNCaP . Aproximadamente una semana después, cuando los tumores alcanzaron un tamaño de 150 mm3, los animales fueron divididos en dos grupos (n=8) que fueron tratados diariamente con vehículo (control) o con 1,5 mg/Kg de compuesto objeto de la presente patente administrados de manera intraperitoneal . El tamaño de tumor (que se calcula con la fórmula 0,523xW2xL; siendo W el ancho del tumor y L la longitud del mismo) fue controlado y calculado a diario mediante el uso de un calibrador. Tras quince días de tratamiento los animales fueron sacrificados y se extrajeron los tumores calculándose el peso y tamaño del tumor al finalizar el tratamiento. The studies were carried out in accordance with the Spanish regulation for the use and care of experimental animals and according to the guidelines of the European Union related to animal research (Royal Decree 1201/2005 of October 10). The Mice were injected subcutaneously in the right flank, with 0.1 ml of PBS buffer with 10 million PC-3 cells or LNCaP. Approximately one week later, when the tumors reached a size of 150 mm 3 , the animals were divided into two groups (n = 8) that were treated daily with vehicle (control) or with 1.5 mg / Kg of compound object of the This patent administered intraperitoneally. The tumor size (which is calculated with the formula 0.523xW 2 xL; W being the width of the tumor and L the length thereof) was controlled and calculated daily using a calibrator. After fifteen days of treatment the animals were sacrificed and the tumors were removed calculating the weight and size of the tumor at the end of the treatment.
Resultados biológi Biological Results
Actividad antiproliferativo in vitro. Se presentan a continuación y a modo ilustrativo, los resultados de los ensayos antiproliferativos in vitro de la 7- ( 1' , 1 ' -dimetilheptil ) -1 , 4-dihidro-4 , 4- dimetilcromeno [ 4 , 3-c] pirazol-6, 9-diona (ejemplo 1), de la 7-(l',l'- dimetilheptil ) -2 , 4-dihidro-2 , 4 , 4-trimetilcromeno [4, 3-c]pirazol-6, 9- diona (ejemplo 2) y de la 7- ( 1' , 1 ' -dimetilheptil ) -2-etil-2 , 4- dihidro-4, 4-dimetilcromeno [4, 3-c] pirazol-6, 9-diona (ejemplo 3) de la presente invención sobre la proliferación celular. In vitro antiproliferative activity. The results of the in vitro antiproliferative assays of 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazole- are presented below and by way of illustration 6, 9-dione (example 1), of 7- (l ', l'-dimethylheptyl) -2, 4-dihydro-2, 4, 4-trimethylchromen [4, 3-c] pyrazole-6, 9- dione (example 2) and 7- (1 ', 1' -dimethylheptyl) -2-ethyl-2, 4- dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione ( Example 3) of the present invention on cell proliferation.
El efecto de los compuestos ejemplos 1, 2 y 3 sobre la viabilidad celular se analizó utilizando tres líneas celulares derivadas de tumores humanos. La línea celular HepG2 procede de un carcinoma hepatocelular diferenciado, la línea celular LNCaP procede de un tumor de próstata dependiente de andrógenos y la línea celular PC-3 procede de un cáncer de próstata independiente de andrógenos y por lo tanto más agresivo. Como se observa en la figura 1, los tres compuestos fueron más efectivos en las líneas celulares de próstata que en la de carcinoma hepatocelular destacando el compuesto ejemplo 1 de la presente patente como el más potente en todas las líneas celulares con una IC50 de 30 μΜ en las células HepG2 y de 15 μΜ en las células LNCaP y PC-3. Con el fin de caracterizar el efecto de la quinona ejemplo 1 de la presente patente sobre el ciclo celular se
trataron las células con la IC50 de dicho compuesto (15 μΜ) durante 24 horas realizándose a continuación un análisis de ciclo mediante citometria de flujo. Los resultados mostrados en la figura 2A indican que el compuesto PM49 produce un aumento de la población celular subGO correspondiente a las células que han sufrido apoptosis. Este aumento es mayor en las células LNCaP que en las PC- 3. Asimismo, el análisis mediante citometria de las células teñidas con Anexina V y ioduro de propidio reveló que el compuesto ejemplo 1 inducía apoptosis tanto en la línea celular LNCaP como en las células PC-3, ya que aumentaban las células doblemente teñidas (cuadrante superior derecho de cada figura) . De nuevo el efecto fue superior en las células LNCaP, en las que la población de células en apoptosis temprana (cuadrante inferior derecho) se cuadruplicó y las células en apoptosis tardía (cuadrante superior derecho) se triplicó (Figura 2B) . The effect of the compounds examples 1, 2 and 3 on cell viability was analyzed using three cell lines derived from human tumors. The HepG2 cell line comes from a differentiated hepatocellular carcinoma, the LNCaP cell line comes from an androgen-dependent prostate tumor and the PC-3 cell line comes from an androgen-independent and therefore more aggressive prostate cancer. As can be seen in Figure 1, the three compounds were more effective in prostate cell lines than in hepatocellular carcinoma, highlighting the example 1 compound of the present patent as the most potent in all cell lines with an IC50 of 30 μΜ in HepG2 and 15 μΜ cells in LNCaP and PC-3 cells. In order to characterize the effect of the quinone example 1 of the present patent on the cell cycle, The cells were treated with the IC50 of said compound (15 μΜ) for 24 hours, followed by a cycle analysis by flow cytometry. The results shown in Figure 2A indicate that compound PM49 produces an increase in the subGO cell population corresponding to cells that have undergone apoptosis. This increase is greater in LNCaP cells than in PC-3s. Likewise, cytometry analysis of cells stained with Annexin V and propidium iodide revealed that the example 1 compound induced apoptosis both in the LNCaP cell line and in the cells. PC-3, as doubly stained cells increased (upper right quadrant of each figure). Again, the effect was superior in LNCaP cells, in which the population of cells in early apoptosis (lower right quadrant) quadrupled and cells in late apoptosis (upper right quadrant) tripled (Figure 2B).
Actividad antitumoral in vivo. A modo ilustrativo se presenta la actividad antitumoral de la 7- ( 1' , 1 ' -dimetilheptil ) -1 , 4-dihidro-4 , 4- dimetilcromeno [ 4 , 3-c] pirazol-6, 9-diona (ejemplo 1) in vivo. Se analizó mediante la inducción subcutánea de tumores xenógrafos en ratones inmunodeprimidos administrándose el compuesto ejemplo 1 de la presente patente (2 mg/Kg) disuelto en aceite de oliva, por vía intraperitoneal durante 15 días de forma consecutiva. A los animales control se les administró el mismo volumen de aceite de olivaAntitumor activity in vivo. Illustratively, the antitumor activity of 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione (example 1) is presented ) in vivo. It was analyzed by subcutaneous induction of xenograph tumors in immunocompromised mice administering the example compound of the present patent (2 mg / Kg) dissolved in olive oil, intraperitoneally for 15 days consecutively. Control animals were given the same volume of olive oil
(vehículo) que a los animales tratados. Como se observa en la figura 3, la administración del compuesto ejemplo 1 de la presente patente redujo el crecimiento del tumor en casi un 40% en las células PC-3 mientras que en las células LNCaP bloqueó casi totalmente el crecimiento tumoral, permaneciendo los tumores al mismo tamaño que al inicio del tratamiento. Las imágenes de los tumores diseccionados al final del tratamiento (a la derecha de cada gráfica) muestran la reducción del tamaño de los tumores de los animales tratados con 7-(vehicle) than to treated animals. As seen in Figure 3, administration of the compound example 1 of the present patent reduced tumor growth by almost 40% in PC-3 cells while in LNCaP cells almost completely blocked tumor growth, the tumors remaining at the same size as at the beginning of the treatment. The images of the dissected tumors at the end of the treatment (to the right of each graph) show the reduction in the size of the tumors of the animals treated with 7-
(1' , 1 ' -dimetilheptil ) -1 , 4-dihidro-4 , 4-dimetilcromeno [4, 3-c]pirazol- 6, 9-diona (ejemplo 1) en comparación con los animales control. (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione (example 1) compared to control animals.
Estos resultados indican que las quinonas derivadas de cannabinoides tienen un efecto antitumoral sobre diversas líneas celulares. Este
efecto es especialmente evidente como lo ilustramos con el compuesto ejemplo 1 de la presente patente y la linea celular derivada de cáncer de próstata LNCaP, una de las lineas generalmente resistente a diversos compuestos. Por lo tanto los resultados aquí mostrados indican que los compuestos reivindicados en la presente patente podrían tener aplicaciones terapéuticas para el tratamiento de diferentes tumores siendo particularmente efectivas para los tumores de próstata dependientes de andrógenos.
These results indicate that cannabinoid-derived quinones have an antitumor effect on various cell lines. This The effect is especially evident as we illustrate with the compound example 1 of the present patent and the cell line derived from prostate cancer LNCaP, one of the lines generally resistant to various compounds. Therefore the results shown here indicate that the compounds claimed in the present patent could have therapeutic applications for the treatment of different tumors being particularly effective for androgen-dependent prostate tumors.
Claims
REIVINDICACIONES
1 . Un compuesto de fórmula general (I), one . A compound of general formula (I),
Fórmula (I) o un tautómero, sal farmacéuticamente aceptable, profármaco o solvato del mismo, donde: Formula (I) or a tautomer, pharmaceutically acceptable salt, prodrug or solvate thereof, where:
- R1 se selecciona independientemente entre hidrógeno o alquilo- R 1 is independently selected from hydrogen or alkyl
Ci_Ci8 ; Ci _ Ci 8 ;
- R2 se selecciona independientemente entre hidrógeno, alquilo Οχ-- R 2 is independently selected from hydrogen, alkyl Οχ-
Cis o arilo C5-Ci8; Cis or aryl C 5 -Ci 8 ;
- R3 se selecciona independientemente entre hidrógeno o alquilo Ci-C5. - R 3 is independently selected from hydrogen or Ci-C 5 alkyl.
2. Un compuesto según la reivindicación 1 de fórmula general (II), 2. A compound according to claim 1 of general formula (II),
Fórmula (II)
o un tautómero, sal farmacéuticamente aceptable, profármaco solvato del mismo, donde R1, R2 y R3 se definen como en reivindicación 1. Formula (II) or a tautomer, pharmaceutically acceptable salt, solvate prodrug thereof, wherein R 1 , R 2 and R 3 are defined as in claim 1.
3 . Un compuesto según la reivindicación 1 de fórmula general (III) 3 . A compound according to claim 1 of general formula (III)
Fórmula (III) o un tautómero, sal farmacéuticamente aceptable, profármaco solvato del mismo, donde R1, R2 y R3 se definen como en reivindicación 1. Formula (III) or a tautomer, pharmaceutically acceptable salt, solvate prodrug thereof, wherein R 1 , R 2 and R 3 are defined as in claim 1.
Un compuesto según una de las reivindicaciones 1 a 3, donde un hidrógeno A compound according to one of claims 1 to 3, wherein a hydrogen
5 . Un compuesto según una de las reivindicaciones 1 a 4, donde R1 es un grupo 1 , 1-dimetilheptilo . 5 . A compound according to one of claims 1 to 4, wherein R 1 is a 1,1-dimethylheptyl group.
6 . Un compuesto según una de las reivindicaciones 1 a 5, donde R2 es un grupo seleccionado entre hidrógeno, metilo o etilo. 6. A compound according to one of claims 1 to 5, wherein R 2 is a group selected from hydrogen, methyl or ethyl.
Un compuesto según una de las reivindicaciones 1 a 6, que se selecciona del grupo que consiste en: a) 7- (1' , 1' -dimetilheptil ) -1 , 4-dihidro- 4, 4-dimetilcromeno [4,3- c] pirazol- 6, 9-diona;
b) 7- (1' , 1' -dimetilheptil ) -2, -dihidro-2, 4, -trimetilcromeno [4,3- c] pirazol-6, 9-diona; c) - (1' , 1' -dimetilheptil) -2-etil-2, 4-dihidro-4, 4- dimetilcromeno [4, 3-c]pirazol-6, 9-diona. A compound according to one of claims 1 to 6, which is selected from the group consisting of: a) 7- (1 ', 1' -dimethylheptyl) -1, 4-dihydro-4, 4-dimethylchromen [4,3- c] pyrazole-6,9-dione; b) 7- (1 ', 1' -dimethylheptyl) -2, -dihydro-2,4, -trimethylchromen [4,3- c] pyrazol-6, 9-dione; c) - (1 ', 1' -dimethylheptyl) -2-ethyl-2, 4-dihydro-4, 4-dimethylchromen [4, 3-c] pyrazol-6, 9-dione.
8 . Una composición farmacéutica caracterizada porque comprende a menos un excipiente farmacéuticamente aceptable y una cantida terapéuticamente eficaz de al menos un compuesto de fórmula genera (I) definido en una cualquiera de las reivindicaciones 1 a 7. 8. A pharmaceutical composition characterized in that it comprises at least one pharmaceutically acceptable excipient and a therapeutically effective amount of at least one compound of formula generates (I) defined in any one of claims 1 to 7.
9 . Uso de un compuesto definido en una cualquiera de las reivindicaciones 1 a 7 para la fabricación de una composición farmacéutica . 9. Use of a compound defined in any one of claims 1 to 7 for the manufacture of a pharmaceutical composition.
10. Uso de un compuesto definido en una cualquiera de las reivindicaciones 1 a 7 para la fabricación de una composición farmacéutica para tratamiento de un cáncer o un tumor maligno. 10. Use of a compound defined in any one of claims 1 to 7 for the manufacture of a pharmaceutical composition for treating a cancer or a malignant tumor.
11 . Uso de un compuesto según la reivindicación 10, donde el cáncer o tumor maligno se selecciona entre blastoma, carcinoma, glioma, leucemia, linfoma y melanoma. eleven . Use of a compound according to claim 10, wherein the cancer or malignant tumor is selected from blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma.
12 . Uso de un compuesto según la reivindicación 10, donde el cáncer o tumor maligno se selecciona entre un hepatocarcinoma o un cáncer de próstata . 12. Use of a compound according to claim 10, wherein the cancer or malignant tumor is selected from a hepatocarcinoma or a prostate cancer.
13 . Un compuesto definido en una cualquiera de las reivindicaciones 1 a 7, o una composición farmacéutica definida en la reivindicación 8, para uso en el tratamiento de un cáncer o un tumor maligno. 13. A compound defined in any one of claims 1 to 7, or a pharmaceutical composition defined in claim 8, for use in the treatment of a cancer or a malignant tumor.
14 . Un compuesto o una composición farmacéutica según la reivindicación 13, donde el cáncer o tumor maligno se selecciona entre blastoma, carcinoma, glioma, leucemia, linfoma y melanoma.
14. A compound or pharmaceutical composition according to claim 13, wherein the cancer or malignant tumor is selected from blastoma, carcinoma, glioma, leukemia, lymphoma and melanoma.
15 . Un compuesto o una composición farmacéutica según la reivindicación 13, donde el cáncer o tumor maligno es leccionado entre un hepatocarcinoma o un cáncer de próstata. fifteen . A compound or a pharmaceutical composition according to claim 13, wherein the cancer or malignant tumor is selected between a hepatocarcinoma or a prostate cancer.
16 . Un método de tratamiento de un cáncer o un tumor maligno que comprende administrar a un paciente afectado de dicho cáncer o tumor maligno una cantidad terapéuticamente efectiva de un compuesto de una de las reivindicaciones 1 a 7. 16. A method of treating a cancer or a malignant tumor comprising administering to a patient affected by said cancer or malignant tumor a therapeutically effective amount of a compound of one of claims 1 to 7.
17 . Un procedimiento de obtención de un compuesto de fórmula general (I), caracterizado porque comprende una oxidación con [bis ( trifluoroacetoxi ) yodo ] benceno de un compuesto de fórmula general ( IV) , 17. A process for obtaining a compound of general formula (I), characterized in that it comprises an oxidation with [bis (trifluoroacetoxy) iodine] benzene of a compound of general formula (IV),
Fórmula (IV) donde los sustituyentes R1, R2 y R3 se definen como en la reivindicación 1.
Formula (IV) wherein the substituents R 1 , R 2 and R 3 are defined as in claim 1.
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