WO2013004871A1 - Amides of 2-amino-1,3-propanediols and use thereof as ceramidase inhibitors - Google Patents

Amides of 2-amino-1,3-propanediols and use thereof as ceramidase inhibitors Download PDF

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WO2013004871A1
WO2013004871A1 PCT/ES2012/070485 ES2012070485W WO2013004871A1 WO 2013004871 A1 WO2013004871 A1 WO 2013004871A1 ES 2012070485 W ES2012070485 W ES 2012070485W WO 2013004871 A1 WO2013004871 A1 WO 2013004871A1
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compound
formula
dihydroxyoctadecan
alkyl
acetamide
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PCT/ES2012/070485
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Spanish (es)
French (fr)
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José Luis ABAD SAIZ
Luz Del Carmen Camacho Castillo
Josefina Casas Brugulat
Gemma Fabrias Domingo
María GARRIDO MARTÍNEZ
Timothy Thomson Okatsu
Óscar MECA CORTÉS
Antonio Delgado Cirilo
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Consejo Superior De Investigaciones Científicas (Csic)
Universidad De Barcelona
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Publication of WO2013004871A1 publication Critical patent/WO2013004871A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/17—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/18—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/74—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/76—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of an unsaturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C247/00—Compounds containing azido groups
    • C07C247/02—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C247/04—Compounds containing azido groups with azido groups bound to acyclic carbon atoms of a carbon skeleton being saturated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/03—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C311/04—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms to acyclic carbon atoms of hydrocarbon radicals substituted by singly-bound oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C311/00—Amides of sulfonic acids, i.e. compounds having singly-bound oxygen atoms of sulfo groups replaced by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C311/01—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms
    • C07C311/02—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton
    • C07C311/03—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C311/05—Sulfonamides having sulfur atoms of sulfonamide groups bound to acyclic carbon atoms of an acyclic saturated carbon skeleton having the nitrogen atoms of the sulfonamide groups bound to hydrogen atoms or to acyclic carbon atoms to acyclic carbon atoms of hydrocarbon radicals substituted by nitrogen atoms, not being part of nitro or nitroso groups

Definitions

  • Ceramidases are hydrolases that catalyze the hydrolysis of ceramides in sphingosine and fatty acids in mammals, bacteria and fungi. According to their optimum pH, ceramidases are grouped into acidic, neutral and alkaline. To date, five different ceramidases have been cloned and expressed in a functional way: acid ceramidase, neutral and three alkaline.
  • Compound B13 ((1 R, 2R) -2 - (/ V-tetradecanoylamino) -1- (4-nitrophenyl) -1, 3- propanediol) is another acid ceramidase inhibitor. It is selective for this ceramidase, since it does not modify the activity of neutral and alkaline ceramidases. B13 induces the accumulation of ceramide and the death of SW403 cells (human adenocarcinoma), meianoma, and LNCaP prostate cells. In addition, B13 prevents the growth of tumors in vivo and sensitizes prostate tumors against radiation-induced apoptosis.
  • B13 is a neutral and lipophilic molecule, it is not very suitable to reach and accumulate in the lysosome, the acidic compartment in which the acidic ceramidase is found. Therefore, various structural analogues have been designed to improve the ability to reach the biological target. From these works, three families of analogues with different specificity regarding the intracellular organelle have emerged: a) lysosomotropic alkylamines (for example, LCL204); b) mitochondrotic cationic analogs (such as LCL85); c) neutral analogs without selectivity with respect to the compartment in which they accumulate (LCL15).
  • lysosomotropic alkylamines for example, LCL204
  • mitochondrotic cationic analogs such as LCL85
  • neutral analogs without selectivity with respect to the compartment in which they accumulate (LCL15).
  • LCL204 is able to selectively localize in lysosomes and induce apoptosis in prostate cancer cells and Fas-induced apoptosis in squamous cancer cells.
  • LCL204 (or AD2646) causes lysosomal destabilization and rapid degradation, dependent on cathepsin, of ceramidase acid, suggesting a lack of tumor specificity.
  • desipramine which is capable of decreasing the activity of acid ceramidase by stimulating its proteolytic degradation dependent on cathepsin, as well as for other amphiphilic compounds (chloropromazine, chioroquine), although not for other lysosomotropic agents ( ammonium chloride, bafilomycin A1).
  • LCL464 stands out, capable of inhibiting acid ceramidase both in vivo and in vitro, but without the ability to induce its proteolytic degradation.
  • compound LCL464 causes an increase in caspase-dependent apoptosis in various types of cancer.
  • Patent application WO200650264 describes ceramide conjugates with pyridinium salts and their use in the treatment of cancer.
  • This patent application also describes the compound I-B2 (2-bromo-N ⁇ ((2S, 3R) -1,3-dihydroxyheptadecan-2-yl) acetamide) as an intermediate for obtaining the compounds for the treatment of Cancer.
  • this prior art document does not disclose that compound I-B2 can be used as an anticancer.
  • Short-length N-acyl chain 3-ketoamides have been described as capable of inducing apoptosis in leukemia cells (Azuma et al. Bioorg Med Chem. 2007, 15, 2860-2867).
  • the present invention provides new ceramide derivatives acid. Likewise, the present invention also provides a family of compounds with antiproliferative and cytotoxic activity by inhibiting acid ceramidasa activity. In particular, the present invention provides a family of compounds for the treatment of cancer.
  • the present invention relates to a compound of formula (I):
  • Z is selected from H and OH
  • n is an integer selected from 0 to 1
  • Ri is selected from alkyl (CrC 3 o), alkenyl (C 2 -C 3 o), and alkynyon (C 2 - C 3 o),
  • B is selected from -H, -N 3 and -C CH,
  • R 2 is selected from -NHR 3 and maleimide, where
  • R 3 is selected from -CO-R4, -CO-CO-R4 and -SO2-R4, where
  • R 4 is selected from alkyl (Ci-Ci6) alkyl, (C2-Ci6), alkynyl (C2 ⁇ Ci 6), epoxide and aziridine, wherein the alkyl, alkenyl or alquiniio of R1 and R4 may independently be , optionally substituted by one or more substituents independently selected from halogen, OH, OR, OCF 3 , NH 2 , NO 2 , NRR ', NHCOR; CONRR, CHO, COOH, COOR, OCOR and CN, where R and R 'are alkyl or alkenyl; with the condition of
  • R 3 is different from -COR 4 with R 4 being alkyl (CrCi 6 ) unsubstituted or substituted by halogen or hydroxyl;
  • alkyl refers, in the present invention, to non-cyclic, linear or branched hydrocarbon chain radicals, which bind to the rest of the molecule through a single bond, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ferc-butyl, sec-butyl, n-pentyl, n-hexy, decyl or dodecyl.
  • the alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, carbonite, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro.
  • alkenyl refers to radicals of non-cyclic, linear or branched hydrocarbon chains, which contain one or more double carbon-carbon bonds, preferably contain a single carbon-carbon double bond, and which are attached to the rest of the molecule by a single bond, for example, vinyl, 1-propene, allyl, isoprenyl, 2-butenyl or 1,3-butadiene.
  • Alkenium radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, carbonyl, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro.
  • alkynyl refers to radicals of non-cyclic, linear or branched hydrocarbon chains, which have one or more triple carbon-carbon bonds, preferably contain a single carbon-carbon triple bond, and which are attached to the rest of the molecule by a simple link, for example, ethiny or 1-propyne.
  • the alkynyl group may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxy, carbon, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro.
  • substituents such as halogen, hydroxyl, azido, alkoxy, carboxy, carbon, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro.
  • the present invention encompasses all possible stereoisomers not only their racemic mixtures but also their optically active isomers. Obtaining a single enantiomer can be achieved by any of the procedures commonly used, for example, by resolution of the racemic mixture through recrystallization techniques, chiraine synthesis, enzymatic resolution, biotransformation or chromatographic resoiu tangible.
  • pharmaceutically acceptable salt means a salt that retains an efficacy and biological properties similar to the free base or free acid and which is not bothersome in the biological sense or in any other.
  • Pharmaceutically acceptable salts may include acid addition salts, such as mesylates, smokers, hydrochlorides, citrates, maleates or tartrates.
  • Physiologically acceptable salts can also be formed with inorganic acids such as sulfuric or phosphoric acids.
  • basic type salts of an alkali metal, such as sodium, or an alkaline earth metal, for example calcium or magnesium can be formed.
  • solvate in the present patent application means an aggregate resulting from the ionic or molecular association between molecules of one or more solvents and molecules of one of the compounds object of this invention.
  • the solvate may comprise, for example, water molecules, alcohols, ketones, acetates or mixtures.
  • the solvate may comprise molecules of water, ethanol, isopropanol, acetone or mixtures.
  • the solvates object of the present invention can be obtained by methods known to a person skilled in the art, for example, by crystallization under controlled conditions.
  • the compound of formula (I) of the present invention is characterized in that Ri is selected from alkyl (CrC 30 ) and alkenyl (C 2 -C 3 0 ), preferably R is selected from alkyl (CC 3 or ) and alkenyl (C 2 -C 3 o) unsubstituted, more preferably Ri is selected from (C8-Cie) alkyl and alkenium (C8-Ci6) unsubstituted.
  • the compound of formula (I) object of the present patent application is characterized in that n can be equal to zero.
  • the compound of formula (I) object of the present patent application is characterized in that R 3 is -CO-R4, where R4 can be alkyl (CrCi6), and B can be selected from -N 3 and -CECH.
  • R 4 may be a (Ci-Ci 6 ) alkyl substituted with at least one halogen atom, more preferably the halogen may be a fluorine or bromine atom, even more preferably the halogen is bromine. Even more preferably, R 4 may be an alkyl (Ci-C5) substituted with at least one bromine atom.
  • R 4 may be an alkyl (CrC 6) substituted with at least one halogen atom, more preferably the halogen may be a fluorine or bromine atom, even more preferably the halogen is bromine.
  • R 4 may be an alkyl (Ci-C5) substituted with at least one bromine atom.
  • the compound of formula (I) object of the present patent application is characterized in that R3 is -CO-R4, where R4 can be epoxide.
  • the compound of formula (I) object of the present patent application where R3 is -CO-R4, where F can be epoxy, is also characterized in that n can be 1 and Z can be OH.
  • the compound of formula (I) object of the present patent application is characterized in that R3 is -CO-R4, where R 4 can be alkenyl (C2-C e) or alkyne (C2-C) and).
  • R4 can be a aiquenilo (C2-Ci6) substituted with at least one halogen atom, more preferably, R4 can be a alqueniio (C 2 Ci 6) substituted with at least one fluorine atom or bromine, in an even more preferred R4 may be a (C2-Ci6) alkenyl substituted with at least one bromine atom.
  • the compound of formula (I) object of the present patent application is characterized in that R 3 is -CO-R4, where R4 is alkenyl (C 2 -Ci 6 ) or alkynyl (C 2 -Ci6), and may be substituted by a! minus a group -CHO or - COOH.
  • the compound of formmuia (I) object of the present patent application is characterized in that f3 ⁇ 4 is -CO-CO-R4, where R4 can be alkyl (CrCi 6 ) and B can be selected from - N 3 and -CECH.
  • the compound of formula (I) object of the present patent application is characterized in that R 3 can be -SO2-R4, where R 4 can be selected from alkyl (Ci-Ci6), alkenyl ( C2-Ci6), alkynyl (C2-C e), epoxide and aziridine.
  • R 4 may be alkyl (CrCi 6 ), more preferably (C C8) alkyl.
  • the compound of formula (I) object of the present patent application is characterized in that R3 can be -SO2-R4, where R 4 can be selected from alkyl (CrCi 6 ), alkenyl (C 2 -Ci 6 ), aiquinyl (C 2 -Ci 6 ), epoxide and aziridine, n can be 1 and Z can be OH.
  • R 4 may be (Ci-Ci6) alkyl, more preferably (d-C8) alkyl.
  • the compound of formula (I) object of the present patent application is characterized in that R2 can be maieimide.
  • the compound of formula (I) as defined above is selected from the list consisting of:
  • the compound of formula (I) as defined above in this patent application is selected from the list consisting of:
  • the compound of formula (I) when n is 1 as defined above is selected from the list consisting of:
  • the compounds of formula (I) can be prepared following different methods known to any person skilled in the field of organic synthesis, in particular by the general procedures described below.
  • the starting materials are commercially available or can be prepared by methods of the literature.
  • a Cat. Grubbs (second generation); b: 1) H 2 , Pd / C; 2) NaN3 / DMF; c: NaN 3 / DMF; d: NaH / THF; e: 1) Li-C ⁇ C-TMS; 2) Bu 4 NF; f: HCI / MeOH; g: 1) cat. TsOH / MeOH; 2) NaOH / EtOH; h: 1) PCC / CH 2 CI 2 ; 2) cat. TsOH / MeOH.
  • compound 1 (Herold, Helv. Chim. Acta. 1988, 71, 354-62) is reacted with a terminal oiefin (such as 2a or 2b) in a cross-metathesis reaction in the presence of second generation Grubbs catalyst (Schmidt, Angew. Chem. Int. Ed. 2003, 42, 4996-9) obtaining compounds 3.
  • a terminal oiefin such as 2a or 2b
  • second generation Grubbs catalyst Schot, Angew. Chem. Int. Ed. 2003, 42, 4996-9
  • bromoderivative 3a is reacted with sodium azide in dimethylformamide, obtaining 3c, followed by acid hydrolysis of the oxazolidine ring and the Boc group.
  • mesylates 4 are obtained, which can be hydrogenated to 5.
  • reaction of 4 or 5 with lithium trimethylsilylacetyl followed by deprotection of acetylide with tetrabutylammonium fluoride, terminal alkynes 6 and 7, respectively. Its transformation into the corresponding IIG and IID amines is achieved by sequential hydrolysis of the isopropylidene and carbamate groups.
  • the above amino alcohols, as well as sphingosine or dihydrosphingosine, can be acylated by reaction with an acid chloride in the presence of a base.
  • an acid anhydride such as maleic anhydride, or a carboxylic acid can also be used in the presence of a suitable coupling agent such as, for example, 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), the ⁇ , ⁇ '- diisopropylcarbodiimide (DIC), O- (7-azabenzotriazol-1- il) hexafluoro phosphate - / V, A /, A / 'A /' - tetramethyluronium (HATU) or benzotriazol-1-yloxy -tris pyrroiidinophosphonium (PyBOP) and an activator such as 1-hydroxybenzotriazole (HOBt), in the presence of a
  • the sulfonamides can be prepared by coupling the corresponding amines with ethanesuifonyl chloride in tetrahydrofuran solution.
  • compositions comprising a compound of general formula (I) or one of its pharmaceutically acceptable stereoisomers, salts or solvates as defined in this patent application and at least one pharmaceutically acceptable excipient.
  • the pharmaceutical composition It can be presented in a form adapted to parenteral, oral, sublingual, nasai, intrathecai, bronchial, lymphatic, rectal, transdermal or inhaled administration.
  • both the compound of general formula (I) or a stereoisomer, a pharmaceutically acceptable salt or soivate thereof as defined in the present patent application as a compound chosen from 2,2-dibromo-N- ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) Acetamide or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds are useful for the treatment of a disease associated with the inhibition of acid ceramidase, and therefore are useful in the treatment and / or prevention of a disease. which studies with cellular hyperproliferation.
  • compositions of a compound of general formula (I) or a stereoisomer, a sai or a pharmaceutically acceptable soivate thereof as defined in the present patent application are useful for the treatment and prevention of a disease that occurs with cell hyperproliferation .
  • the compound or composition for use in the treatment or prevention of a disease that occurs with cellular hyperproliferation as defined above in the present application for patent can be used for the treatment of a disease chosen from cancer, metastasis, inflammation, asthma and arteriosclerosis.
  • the compound or composition for use in the treatment or prevention of a disease that occurs with cell hyperproliferation as defined in the present patent application can be used in the treatment of prostate cancer, pancreas, brain , colon, lung, breast, head and neck, ovary, larynx, urinary bladder, uterus, skin, sarcomas, lymphomas or leukemia.
  • prostate or lung cancer Preferably, prostate or lung cancer.
  • the present invention also relates to a method for the prevention or treatment of an individual who suffers from or is susceptible to suffering from disease that occurs with cellular hyperproliferation, in particular the treatment of cancer and more preferably the treatment of prostate or lung cancer, which comprises administering to said individual a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof together with sufficient amounts of pharmaceutically acceptable excipients.
  • the present invention also relates to the use of both a compound of general formula (I) or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof as defined in the present patent application, and the use of a compound chosen between 2,2-dibromo- N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxioctadecan-2-yl ) acetamide or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds, to make a pharmaceutical composition useful in the treatment of a disease associated with the inhibition of acid ceramidase, and therefore useful in the treatment and / or prevention of a disease that occurs with cellular hyperproliferation.
  • said composition can be used for the treatment of a disease caused by cancer, metastasis, inflammation, asthma and arteriosclerosis. More preferably, the tai composition as described above can be used in the treatment of prostate cancer, pancreas, brain, colon, lung, breast, head and colium, ovary, larynx, urinary bladder, uterus, skin, sarcomas , lymphomas or leukemia.
  • prostate or lung cancer Preferably, prostate or lung cancer.
  • the compounds used in the present invention can be used alone or with other drugs to provide a combination therapy.
  • the other drugs can be part of the same composition, or they can be supplied as a separate composition for administration at the same time or at a different time.
  • a compound of formula generates!
  • composition of a compound of formula (I) as defined above in the present patent application or the composition comprising a compound chosen from 2,2-dibromo-N- ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a sai o a pharmaceutically acceptable soivate of one of these compounds and at least one excipient can be used in combination with another therapy for the treatment of a disease as defined above.
  • the therapeutic agents may be tamoxifen, daunorubicin, etoposide, pacitax, dacarbazide, temozoomomide, temsirolimus, fenretinide, resveratrol, borinostat, sorafenib, imatinib, bortezomib, gemcitabine or cispiatin.
  • Figure 1 Effect of the compounds on the acid ceramidasa activity.
  • the tests were performed on transduced Farber cells to overexpress acid ceramidase, either intact (white bars) or in lysates (gray bars). Incubation was carried out by adding together the inhibitor (16 ⁇ ) with the phylogenic substrate (16 ⁇ ) for 3 h. The procedure was followed as detailed in example 1 of the section on examples of biological tests (Bedia et al. Chembiochem 2007, 8, 642). The y axis indicates the percentage of enzymatic activity with respect to the control.
  • Figure 2 Effect of the compounds on the acid ceramidase activity.
  • the tests were carried out on human lung adenocarcinoma cells A549. The intact cells were incubated with the inhibitor (16 ⁇ ) for 24 h and then the phylogenic substrate (16 ⁇ ) was added, which was incubated for 3 h. Hydrolysis was determined by measuring the fluorescence generated after proceeding as indicated in example 1 of the section on examples of biological tests (Bedia et al. Chemb ⁇ ochem 2007, 8, 642). The axis and indicates the percentage of enzymatic activity with respect to the control.
  • Figure 3 Effect of the compounds I-B2, I-B17 and I-B9 on the acidic ceramidase activity of PC-3Mc prostate cancer cells.
  • the assays were carried out by incubation of the cells with the inhibitor. The cells were seeded in 96-well plates at a density of 10,000 cells / ml and the compounds were added 24 h after seeding. Three doses of each inhibitor, 1 ⁇ , 5 ⁇ and 10 ⁇ , were used in incubations for 48 h and the tests were performed in triplicate. After this incubation period, the medium was eimiminated and fresh medium containing the fluorogenic substrate (16 ⁇ ) was added and incubated for 3 h.
  • Hydrolysis is determined by measuring the fluorescence generated after proceeding as indicated in example 1 of the section on examples of biological tests (Bedia et al. Chemb ⁇ ochem 2007, 8, 642). The axis and indicates the percentage of enzymatic activity with respect to the control.
  • FIG. 5 Effect of compounds I-B2, I-B17 and I-B9 on the sphingolipidoma of the prostate cancer cells PC-3Mc.
  • the assays were carried out by incubation of the cells with the compounds. The cells were seeded in 6-well plates at a density of 250,000 cells / ml and the compounds were added 24 h after seeding. Three doses of each inhibitor, 1 ⁇ , 5 ⁇ and 10 ⁇ , were used in incubations for 48 h and the Trials were performed in triplicate. Three doses of each compound, 1 ⁇ , 5 ⁇ and 10 ⁇ , were used in incubations for 48 h. After this incubation period, the medium is removed, the cells are harvested and processed as detailed in example 2 of examples of biological tests.
  • A ceramides; B, dihydroceramides; C, sphingomyeliins; D dihydrosphingomyelins; E, glucosylceramides.
  • Figure 6 Effect of the compounds on the viability of A cells, lung adenocarcinoma A549 (black) and leukemia Jurkat A3 (gray) and B, prostate cancer PC3 / Mc.
  • the cells were seeded at a density of 200,000 cells per milliliter and, 24 hours after planting, they were incubated with the compounds for 24 h (A) or 72 h (B), after which the number of viable cells was determined through the MTT reduction test, as specified in example 3 of examples of biological tests.
  • Figure 7 Effect of the compounds on the growth of PC-3Mc cells on plastic substrate. 500 cells were seeded in each well of 96-well plates, leaving to adhere to the plastic for 24 h, followed by treatment with the compounds at a final concentration of 5 ⁇ . The effect of incubations with these compounds on cell growth was determined by quantifying the number of cells present at 24 h, 48 h, 72 h, 120 h, 144 h and 168 h after treatment with the compounds. The y-axis indicates the relative proportion of the number of cells with respect to day 1 and the x-axis refers to the time (t) in days.
  • Figure 8 Effect of inhibitors on the formation of cell colonies in semi-solid medium.
  • the PC-3Mc cells seeded in complete culture medium containing 3% agar, were treated with the test compounds at final concentrations of 1 ⁇ or 5 ⁇ , reacting them with a periodicity of 3 days, coinciding with the addition of new crop The number of colonies was counted 3 weeks after treatment.
  • the y axis indicates the number of colonies (No. Col.)
  • FIG. 9 Effect of ios inhibitors on cell invasion.
  • PC-3Mc cells were treated with the test compounds at a final concentration of 5 ⁇ during the 48 hours prior to the invasiveness test. After that treatment time, the cells were collected, resuspended in complete medium, and seeded on the upper chambers of the Transwell inserts coated with Matrigei (10 mg / mL). The test compounds were added to both the upper and lower chamber, at a concentration of 5 ⁇ , this treatment being maintained throughout the entire duration of the test. Each condition was performed in triplicate. The y-axis indicates the number of cells (No.) Figure 10. Formulas of the compounds synthesized in the examples.
  • 3C-NMR (101 MHz, CDCI 3 ): ⁇ 133.51, 128.29, 94.58, 81.20, 74.25, 65.08, 62.44, 51.61, 32.53, 29.55, 29.51, 29.45, 29.31, 29.28, 29.24, 28.97, 28.51, 26.84, 26.38, 24.74.
  • Step 1 On a solution of the 4 or 5 mesylates (2 mmol) in 8 mL of a 1: 1 THF / HMPA (hexamethiphosphotriamide) mixture cooled to -30 ° C, 4.5 mL of a solution was added dropwise 0.5 M of lithium trimethisysilylacetylide in THF (equivalent to 2.25 mmol). The reaction mixture was kept under stirring while slowly heating to room temperature. After consumption of the starting product (about 1 h, by TLC analysis), the reaction was stopped by adding 5 mL of saturated NH 4 Ci solution and the mixture was extracted with hexane (3 x 10 mL). The combined organic extracts were washed with water and brine, dried over anhydrous MgSO 4 and evaporated under reduced pressure to obtain the corresponding intermediate trimethylsilyl acetylides, which were used in the next step without purification.
  • THF / HMPA hexamethiphosphotriamide
  • Step 2 A solution of trimethylsilyl acetylide (1.5 mmol) in anhydrous THF (5 mL) was treated with 2 mL of a 1 M solution of Bu 4 NF in THF under Ar atmosphere. After stirring for 30 min at room temperature, the reaction mixture was treated with H 2 0 (0.5 mL), dried over anhydrous MgS0 4 and evaporated under reduced pressure, obtaining acetylenes 6 and 7.
  • Step 1 A solution of 460 mg (1 mmol) of compound 3a in ethanol (10 mL) was subjected to hydrogenation, at atmospheric pressure, in the presence of 10 mg of 10% Pd / C. After 12 h of stirring at room temperature, the reaction mixture was filtered over Celite and the filtrate obtained was evaporated to dryness to give a crude which was subjected to the following reaction.
  • Step 2 A solution of 100 mg (1.54 mmol) of sodium azide in anhydrous DMF (10 mL) was added, dropwise, over a solution of the crude from the previous reaction in 5 mL of DMF, under Ar atmosphere at room temperature. After the addition, the reaction mixture was heated to 65 ° C and kept under stirring for 12h. Then, the mixture was diluted with 40 mL of water and extracted with Et 2 0 (3 x 15 mL). The organic phases were washed with brine (2 x 10 mL), dried over anhydrous MgSO 4 , filtered and evaporated to give a residue that was purified by flash chromatography (Hexane / EtOAc 7: 3).
  • Step 1 A solution of 0.6 mmol of 6 or 7 and TsOH (12 mg, 0.06 mmol) in MeOH (10 mL) was stirred at 25 ° C for 6 h. After this time, the solvent was removed under reduced pressure and the resulting residue was dissolved in EtOAc The organic solution was washed, successively, with a saturated solution of NaHC03 and brine and dried. Evaporation of the solvent provides a residue that was used directly in the next stage of synthesis.
  • Step 2 A solution of the above crude in 2N NaOH (30 mL) and EtOH (30 mL) was heated at 80 ° C for 3 h. The reaction mixture was then cooled, concentrated, under reduced pressure, to a quarter of the volume and extracted with Et2Ü (3 x 15 mL). The combined organic phases were dried over MgSO 4 , filtered and evaporated to give the corresponding final product.
  • Step 1 A solution of 550 mg (1.30 mmol) of 8 in 30 mL of CH 2 CI 2 was treated with pyridinium chlorochromate (PCC) (420 mg, 1.90 mmol). The reaction mixture was kept under stirring at room temperature for 1 h, after which another 420 mg of PCC was added and kept under stirring for an additional 12 h. Then the reaction mixture was filtered. on Celite and the filtrate was evaporated under reduced pressure to give a crude that was used directly in the next step.
  • PCC pyridinium chlorochromate
  • Step 2 A solution of the above crude in 20 mL of MeOH was treated with 1.2 mL of acetiium chloride at 25 ° C for 6 h. After this time, the solvent was removed under reduced pressure to give the final product as the corresponding hydrochloride.
  • Example I-B A / -f (2S, 3R) -1, 3-dihydroxyoctadecan-2-ylletanosuifonamide
  • Example 1-B 2-Bromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide. According to the synthesis procedure described in patent application WO200650264. Example 1.
  • Example 1-B5 A / -f (2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] propioiamide
  • Example 1-B6 A / -r (2S, 3f?) - 1, 3-dihydroxyoctadecan-2-yl1but-2-inamide
  • 3C-NMR (101 MHz, CD 3 OD): ⁇ 141.0, 126.3, 72.5, 62.3, 56.9, 35.0, 33.1, 30.9, 30.8, 30.5, 26.9, 23.8, 18.0, 14.6.
  • Example 1-B A / -f (2S, 3ff) -1, 3-dihydroxyoctadecan-2-yl1methacryamide
  • Example 1-B10 A -r (2S.3R) -N-1.3-dihydroxyoctadecan-2-yl-1-3-methyl-2-butenamide
  • the inhibitory activity of ceramidases by the compounds of the present invention was tested on fibroblasts of a Farber patient transduced for overexpression of ia ceramidase and on the lines of lung cancer A549 (American Type Culture Colection) and prostate cancer PC-3 / Mc.
  • the A549 line was maintained in the middle of HAM F12 supplemented with 2 mM glutamine, the Farber line was grown in DMEM medium and the PC-3 / Mc line was maintained in RPMI1640 medium.
  • the medium is supplemented with 10% serum of bovine fetus, 100 units / mL of penicillin and 10 ⁇ g / mL of streptomycin and the cultures were maintained at 37 O in an atmosphere of 5% CO 2 95% air and a humidity of 90%.
  • the cells were seeded in 96-well plates at a density of
  • the medium was removed and replaced with fresh medium (100 microliters per well) containing the fluorogenic substrate (Bedia et al. Chembiochem 2007, 8, 642) and the inhibitor at a concentration of 16 ⁇ .
  • the substrate can also be added directly without replacing the culture medium. In any case, it was incubated at 37 ° C for 3 hours, the cells were lysed and the enzymatic activity was determined following the procedure described (Bedia et ai. Chembiochem 2007, 8, 642). In some cases, the inhibitor and the substrate were added together and incubated for 3 h before proceeding with the cell's analysis.
  • the inhibitory effect of the compounds on the acidic ceramidase activity of the transduced Farber cells for the overexpression of the acidic ceramidase was determined.
  • the cells were incubated together with substrate and test compound for 3 h and after this time, the enzymatic activity was determined as described above.
  • the compounds I-B2, I-B8, I-B9 and I-B17 were the most active in intact cells, although only I-B2, I-B9 and I-B17 were also active in vitro.
  • the inhibitory effect of the compounds on the acidic ceramidase activity of A549 lung adenocarcinoma cells was determined.
  • the cells were incubated with the test compounds for 24 h and after this time the substrate was added, incubated for 3 h and continued as described above.
  • the compounds ID2, IE2 IF2 IG2 and IH2 they were the most active, presenting an inhibition of the acid ceramidasa superior to 95% with respect to the control.
  • the effect of compounds I-B2, I-9B and I-B17 on the acidic ceramidase activity of PC-3 c prostate cancer cells was determined.
  • the cells were seeded in 96-well plates at a density of 10,000 cells / ml and the test compounds were added 24 hours after seeding.
  • Figure 3 graphically represents the results obtained.
  • Compounds I-B17 and I-B2 cause a clear inhibition of acidic ceramidase activity at all doses used, with greater inhibitory effect at higher doses.
  • the cells were seeded in 6-well plates at a density of 250,000 cells / ml.
  • the compounds were added 24 h after seeding and incubated for different periods of time. After the treatments, the cells were washed with PBS and transferred to glass vials, where lipid extracts were prepared following the procedure described (Merrill et al Methods, 2005, 36, 207).
  • the analyzes were carried out by ultra-liquid liquid chromatography coupled to an accelerated flight time mass detector, which allows the identification of the compounds based on their exact mass, by electrospray ionization in positive mode.
  • the chromatographic and analytical conditions are those described in previous works (Munoz-Olaya et al ChemMedChem, 2008, 3, 946 and Canals et al Bioorg. Med. Chem., 2009, 17, 235).
  • the effects of the inhibitors I-B17, I-B9 and I-B2 on the sphingolipidoma of the prostate cancer cell line PC-3 c are detailed.
  • the sphingolipid content was determined after 48 h of incubation with the inhibitors at two different doses, 1 ⁇ or 5 ⁇ . Ceramides, dihydroceramides, sphingomyelins, dihydrosphingomyelins and glucosylceramides levels were determined.
  • Figure 5 graphically illustrates the changes in the levels of the different sphingolipids after incubating the PC-3Mc cells with the inhibitors.
  • I-B2 treatments caused a significant accumulation of ceramides and dihydroceramides at both 1 ⁇ and 5 ⁇ .
  • Treatment with I-B17 also caused an accumulation of ceramides and dihydroceramides, more pronounced in treatments with an inhibitor concentration of 5 ⁇ .
  • cytotoxicity was determined by measuring mitochondrial dehydrogenase activity with 3- (4,5-dimethithiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) after 24-72 h incubation of the cells with the problem compounds at different concentrations.
  • Figure 6A illustrates the effect of the test compounds on the viability of A549 lung cancer cells and dermal fibroblasts after 24 h of incubation.
  • Figure 6B graphically illustrates the effects of inhibitors I-B17, I-B9 and I-B2 on the viability of PC-3Mc cells after 72 h of incubation.
  • PC-3Mc cells grown in medium with 10% fetal bovine serum, were exposed to the test compounds, at a final concentration of 5 ⁇ .
  • 500 cells were seeded in each well of 96-well plates, allowed to adhere to the plastic for 24 h, followed by treatment with the compounds. Each treatment was performed in triplicate wells.
  • Figure 7 graphically illustrates the effect of incubations with these compounds on cell growth, quantified as the number of cells present at 24 h, 48 h, 72 h, 120 h, 144 h and 168 h of treatment with the compounds.
  • Compounds I-B17 and I-B2 caused an almost total inhibition of the growth of PC-3Mc cells during the first 6 days of treatment.
  • compound I-B9 did not cause significant differences over the number of cells present over 7 days of treatment, compared to control cells, exposed only to the solvent used for the compounds.
  • Example 5 Effect of inhibitors on the formation of cell colonies in semi-solid medium.
  • the ability of PC-3Mc cells to grow forming three-dimensional colonies in semi-suspension was determined.
  • the cells are resuspended at temperatures that preserve cell viability in a complete culture medium containing 0.6% agar, subsequently allowing to solidify at room temperature, on a bed of medium with 3% agar.
  • the cells seeded in this arrangement are incubated for 2 or 3 weeks at 37 ° C in an atmosphere of 5% CO2 / 95% air and 90% humidity.
  • the problem compounds were added at final concentrations of 1 ⁇ or 5 ⁇ , reacting them with a periodicity of 3 days, coinciding with the addition of new culture medium.
  • the colonies are fixed with 0.5% glutardehyde, stained with violet crystal and visualized under a microscope.
  • the colonies of a diameter greater than or equal to 0.2 mm is provided with the ImageJ 1.43u program (NIH, USA).
  • Figure 8 illustrates how, at the concentration of 1 ⁇ , none of the compounds affected the ability of PC-3Mc cells to form colonies, while at 5 ⁇ , compounds I-B17 and I-B2 inhibit colony formation below 25% compared to the control.
  • Example 6 Effect of inhibitors on cell invasiveness.
  • the test known as invasiveness in Boyden chambers was used.
  • the commercial version that has been used of these chambers is called Transweli (from the Corning house), and consists of relatively inert polyester membranes, with pores of 8.0, Lim, placed in a plastic support that is inserted into 96-well plates. wells. These membranes are coated with components of the extraceiular matrix (Matrigel Growth Factor Reduced, from Becton-Dickinson).
  • the cells are deposited with medium lacking fetal bovine serum in the upper chamber, and the same medium is placed in the lower chamber (the 96-well plate well). Invasion of the cells from the upper chamber into the lower chamber is allowed for 48 hours under the usual culture conditions described in previous examples.
  • PC-3Mc cells were treated with the test compounds at a final concentration of 5 ⁇ during the 48 hours prior to the invasiveness test. After that treatment time, the cells were detached from the culture plates by incubation with tripisin (25 units / mL) and EDTA (0.1 mM) for 5 minutes, resuspended in complete medium, and seeded on the upper chambers of the Transweli inserts. previously covered with Matrigel to a concentration of 10 mg / mL. The test compounds were added to both the upper and lower chamber, at a concentration of 5 mM, this treatment being maintained throughout the entire duration of the test. Each condition was performed in triplicate. Figure 9 illustrates how, under these conditions, both I-B17 and I-B2 significantly inhibit the invasiveness of PC-3Mc cells.

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Abstract

The invention relates to a compound having formula (I) or a stereoisomer, a salt or a solvate, in which: A is -CH(OH)- or -C(=0)-; Z is H or OH; n is 0 or 1; R1 is alkyl(C1-C30), alkenyl(C2-C30) or alkynyl(C2-C30); B is -H, -N3 or -CΞCH; R2 is -NHR3 or maleimide; R3 is -COR4, -COCOR4 or -SO2R4; and R4 is alkyl(C1-C16), alkenyl(C2-C16), alkynyl(C2-C16), epoxide or aziridine, on the condition that: a) when A is -CH(OH) and B is H, R3 is different from -COR4, R4 being alkyl(C1-C16); b) when A is -CH(OH), B is H and R3 is -COCOR4, R4 being alkyl(C6), R1 is different from -CH=CH2-alkyl(C12), -CΞCH-alkyl(C12) or alkyl(C13-C15); or c) when A is -C(=O), R1 is alkenyl(C2-C30), B is H and n is 0, R3 is different from -COR4, R4 being alkyl(C1C16). The invention also relates to the use of the compound having formula (I), 2,2-dibromo-N-((2S,3R)-1,3-dihydroxyoctadecan-2-il)acetamide or 2-bromo-N-((2S,3R)-1,3-dihydroxyoctadecan-2-il)acetamide in the treatment or prevention of a disease associated with cellular hyperproliferation, either alone or in combination.

Description

Amidas de 2-amino-1,3-propanodioies y su uso como inhibidores de ceramidasas  Amides of 2-amino-1,3-propanedioies and their use as ceramidase inhibitors
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
Las ceramidasas son hidrolasas que catalizan la hidrólisis de las ceramidas en esfingosina y ácidos grasos en mamíferos, bacterias y hongos. Según su pH óptimo, las ceramidasas se agrupan en ácidas, neutras y alcalinas. Hasta ia fecha, se han clonado y expresado de forma funcional cinco ceramidasas distintas: la ceramidasa ácida, ia neutra y tres alcalinas.  Ceramidases are hydrolases that catalyze the hydrolysis of ceramides in sphingosine and fatty acids in mammals, bacteria and fungi. According to their optimum pH, ceramidases are grouped into acidic, neutral and alkaline. To date, five different ceramidases have been cloned and expressed in a functional way: acid ceramidase, neutral and three alkaline.
Existen evidencias que ponen de manifiesto ei papel importante que desempeñan las ceramidasas, especialmente la ceramidasa ácida, en el desarrollo y la progresión del cáncer, así como en la respuesta de los tumores a la terapia. La ceramidasa ácida está sobreexpresada en diversas líneas celulares y tejidos cancerosos, io que parece contribuir a la disminución de los niveles de ceramida y al aumento de los de esfíngosína-1 -fosfato, con el consiguiente aumento de la proliferación celular y resistencia a la muerte celular. En muchos casos, la inhibición de la ceramidasa ácida conduce a apoptosis. En numerosos trabajos se confirma la relación entre el aumento de la actividad ceramidasa ácida y la resistencia a la radío y quimioterapia, así como el interés del uso de inhibidores de la ceramidasa ácida como fármacos anticancerosos, tanto solos como en combinación con otras terapias. En células de glioblastoma resistentes a la radiación se observaron niveles elevados en la expresión de ceramidasa ácida. El tratamiento de dichas células con /V-oleoíletanolamina aumentó su sensibilidad frente a la radiación, con el consiguiente aumento de los niveles de ceramida, activación de caspasas y apoptosis. There is evidence that highlights the important role that ceramidases, especially acid ceramidase, play in the development and progression of cancer, as well as in the response of tumors to therapy. Acid ceramidase is overexpressed in various cell lines and cancerous tissues, which seems to contribute to the decrease in ceramide levels and to the increase in sphingngine-1-phosphate levels, with the consequent increase in cell proliferation and resistance to death. mobile. In many cases, the inhibition of acid ceramidase leads to apoptosis. Numerous studies confirm the relationship between the increase in acid ceramidase activity and resistance to radium and chemotherapy, as well as the interest in the use of acid ceramidase inhibitors as anticancer drugs, both alone and in combination with other therapies. In radiation resistant glioblastoma cells, elevated levels of acid ceramidase expression were observed. The treatment of said cells with / V-oleoylethanolamine increased their sensitivity to radiation, with the consequent increase in ceramide levels, activation of caspases and apoptosis.
Respecto a ía ceramidasa neutra, no se ha examinado con tanto detalle su papel en el cáncer. Sin embargo, Wu y col [Biochim Biophys Acta 2009, 1791, 730-739] comprobaron que la disminución de actividad ceramidasa neutra inducida por gemcitabína da fugar a una parada de cicio celular en la fase G(0)/G(1 ) en un tipo particular de células endoteliales murinas. Por último, el aumento de la muerte celular inducida por el inhibidor de la ceramidasa neutra DMAPP es otro de ios ejemplos que sustancia el papel de la ceramidasa neutra en el desarrollo y progresión del cáncer. Regarding neutral ceramidase, its role in cancer has not been examined in such detail. However, Wu et al [Biochim Biophys Acta 2009, 1791, 730-739] found that the decrease in ceramidase activity Gemcitabine-induced neutral eludes a stoppage of cellular cycle in the G (0) / G (1) phase in a particular type of murine endothelial cells. Finally, the increase in cell death induced by the neutral ceramidase inhibitor DMAPP is another of the examples that substantiate the role of neutral ceramidase in the development and progression of cancer.
La búsqueda de inhibidores de la ceramidasa ácida ha recibido una mayor atención dado su interés como posibles fármacos antiproliferativos y citostáticos en la quimioterapia del cáncer. Uno de los primeros inhibidores descritos fue la /V-oleoíletanolamina, usado solamente como herramienta farmacológica, puesto que su escasa potencia hace inviable su uso terapéutico. Se ha descrito que la /V-oleoiletanoiamina inhibe las ceramidasas neutra y alcalina de queratinocitos, así como la glucosilación de ias ceramidas naturales en células CHP-100 de neuroepítelíoma a concentraciones no tóxicas, proceso que va acompañado de un aumento de ceramidas y la inducción de la apoptosis. The search for acid ceramidase inhibitors has received increased attention given its interest as possible antiproliferative and cytostatic drugs in cancer chemotherapy. One of the first inhibitors described was / V-oleoylethanolamine, used only as a pharmacological tool, since its low potency makes its therapeutic use unfeasible. It has been described that / V-oleoyletanoiamina inhibits the neutral and alkaline ceramidases of keratinocytes, as well as the glycosylation of natural ceramides in CHP-100 neuroepítelíoma cells at non-toxic concentrations, a process that is accompanied by an increase in ceramides and induction of apoptosis.
El compuesto B13 ((1 R,2R)-2-(/V-tetradecanoílamino)-1-(4-nitrofenil)-1 ,3- propanodíol) es otro inhibidor de la ceramidasa ácida. Es selectivo de esta ceramidasa, ya que no modifica la actividad de ias ceramidasas neutra y alcalinas. El B13 induce la acumulación de ceramida y la muerte de células SW403 (adenocarcinoma humano), meianoma, y células LNCaP de próstata. Además, el B13 previene el crecimiento de tumores in vivo y sensibiliza tumores de próstata frente a ia apoptosis inducida por radiación. Puesto que el B13 es una molécula neutra y lipófiía, no es muy adecuada para alcanzar y acumularse en el lisosoma, el compartimento ácido en el que se encuentra la ceramidasa ácida. Por ello, se han diseñado diversos análogos estructurales con objeto de mejorar la capacidad de alcanzar la diana biológica. De estos trabajos han surgido tres familias de análogos con diferente especificidad respecto al orgánulo íntracelular: a) alquilaminas lísosomotrópicas (por ejemplo, LCL204); b) análogos catiónicos mitocondriotrópicos (como el LCL85); c) análogos neutros sin selectividad respecto al compartimiento en el que se acumulan (LCL15). Entre los análogos lísosomotrópicos, el LCL204 es capaz de localizarse selectivamente en lisosomas y de inducir apoptosis en células de cáncer de próstata y apoptosis inducida por Fas en células escamosas de cáncer. Sin embargo, el LCL204 (o AD2646) provoca la desestabilización lisosomal y una rápida degradación, dependiente de catepsina, de la ceramidasa acida, lo que sugiere una falta de especificidad tumoral. Un efecto similar ha sido descrito para la desipramina, que es capaz de disminuir la actividad de la ceramidasa ácida por estimulación de su degradación proteolítica dependiente de catepsina, así como para otros compuestos anfifílicos (cloropromazina, cioroquina), aunque no para otros agentes lísosomotrópicos (cloruro amónico, bafilomicina A1 ). Compound B13 ((1 R, 2R) -2 - (/ V-tetradecanoylamino) -1- (4-nitrophenyl) -1, 3- propanediol) is another acid ceramidase inhibitor. It is selective for this ceramidase, since it does not modify the activity of neutral and alkaline ceramidases. B13 induces the accumulation of ceramide and the death of SW403 cells (human adenocarcinoma), meianoma, and LNCaP prostate cells. In addition, B13 prevents the growth of tumors in vivo and sensitizes prostate tumors against radiation-induced apoptosis. Since B13 is a neutral and lipophilic molecule, it is not very suitable to reach and accumulate in the lysosome, the acidic compartment in which the acidic ceramidase is found. Therefore, various structural analogues have been designed to improve the ability to reach the biological target. From these works, three families of analogues with different specificity regarding the intracellular organelle have emerged: a) lysosomotropic alkylamines (for example, LCL204); b) mitochondrotic cationic analogs (such as LCL85); c) neutral analogs without selectivity with respect to the compartment in which they accumulate (LCL15). Among the lysosomotropic analogs, LCL204 is able to selectively localize in lysosomes and induce apoptosis in prostate cancer cells and Fas-induced apoptosis in squamous cancer cells. However, LCL204 (or AD2646) causes lysosomal destabilization and rapid degradation, dependent on cathepsin, of ceramidase acid, suggesting a lack of tumor specificity. A similar effect has been described for desipramine, which is capable of decreasing the activity of acid ceramidase by stimulating its proteolytic degradation dependent on cathepsin, as well as for other amphiphilic compounds (chloropromazine, chioroquine), although not for other lysosomotropic agents ( ammonium chloride, bafilomycin A1).
Se ha descrito una nueva generación de inhibidores lisosomotrópicos de la ceramidasa ácida desprovistos de la capacidad desestabilizadora del íisosoma del LCL204. Este tipo de inhibidores muestran un grupo ω-aminoacilo junto con ia combinación de elementos estructurales del B13 y del LCL204 (AD2646). Dentro de este grupo destacan el LCL464, capaz de inhibir la ceramidasa ácida tanto in vivo como in vitro, pero sin capacidad para inducir su degradación proteolítica. Además, el compuesto LCL464 provoca un aumento de la apoptosis dependiente de caspasas en diversos tipos de cáncer. A new generation of lysosomotropic acid ceramidase inhibitors devoid of the destabilizing capacity of the LCL204 ileosome has been described. This type of inhibitor shows a ω-aminoacyl group together with the combination of structural elements of B13 and LCL204 (AD2646). Within this group, LCL464 stands out, capable of inhibiting acid ceramidase both in vivo and in vitro, but without the ability to induce its proteolytic degradation. In addition, compound LCL464 causes an increase in caspase-dependent apoptosis in various types of cancer.
De forma paralela al desarrollo de análogos del B13, se introdujeron modificaciones estructurales similares en la estructura del DMAPP. Este es un compuesto utilizado frecuentemente por sus propiedades inhibidoras de las ceramidasas neutra y alcalinas, tai y como se indica en el trabajo de [Bielawska y col Bioorganic and Medicinal Chemistry 2007, 16, 1032-1045]. Se han publicado estructuras de análogos de ceramidas diferentes a los compuestos de la presente invención como moduladores de la apoptosis (Chang et al., J Am Chem Soc. 2002 124, 1856-1857), inhibidores de la producción de interleuquina 4 (Park et al. Bioorg Med Chem. 2005, 13, 2589- 2595.) e inhibidores de la activación de la protein quinasa C para el tratamiento de enfermedades relacionadas con el sistema nervioso (US5519007). La solicitud de patente WO200650264 describe conjugados de ceramidas con sales de piridinio y su uso en el tratamiento del cáncer. Esta solicitud de patente también describe el compuesto I-B2 (2-bromo-N~((2S,3R)- 1 ,3-dihidroxiheptadecan-2-yl)acetamida) como un intermedio para ia obtención de los compuestos para el tratamiento de cáncer. Sin embargo, este documento del estado de la técnica no divulga que el compuesto I-B2 pueda utilizarse como anticancerigeno. Otras patentes describen otros análogos de ceramidas diferentes a los de la presente invención como inhibidores de ceramidasas (WO2003005965 y WO2005051891 ), para el tratamiento del cáncer (EP1580187) y para uso en el tratamiento de la inflamación (WO2004064823). Parallel to the development of analogs of B13, similar structural modifications were introduced in the DMAPP structure. This is a compound frequently used for its neutral and alkaline ceramidases inhibiting properties, tai and as indicated in the work of [Bielawska et al Bioorganic and Medicinal Chemistry 2007, 16, 1032-1045]. Ceramide analog structures other than the compounds of the present invention have been published as apoptosis modulators (Chang et al., J Am Chem Soc. 2002 124, 1856-1857), inhibitors of the production of interleukin 4 (Park et al. Bioorg Med Chem. 2005, 13, 2589-2595.) and inhibitors of the activation of protein kinase C for the treatment of diseases related to the nervous system (US5519007). Patent application WO200650264 describes ceramide conjugates with pyridinium salts and their use in the treatment of cancer. This patent application also describes the compound I-B2 (2-bromo-N ~ ((2S, 3R) -1,3-dihydroxyheptadecan-2-yl) acetamide) as an intermediate for obtaining the compounds for the treatment of Cancer. However, this prior art document does not disclose that compound I-B2 can be used as an anticancer. Other patents describe other ceramide analogs other than those of the present invention as ceramidase inhibitors (WO2003005965 and WO2005051891), for the treatment of cancer (EP1580187) and for use in the treatment of inflammation (WO2004064823).
Se han descrito 3-cetoamidas de cadena de N-acilo de corta longitud con capacidad de inducir apoptosís en células de leucemia (Azuma et al. Bioorg Med Chem. 2007, 15, 2860-2867). Short-length N-acyl chain 3-ketoamides have been described as capable of inducing apoptosis in leukemia cells (Azuma et al. Bioorg Med Chem. 2007, 15, 2860-2867).
También se han publicado diversas famiiias de inhibidores de la ceramidasa ácida con estructura de análogos de ceramidas o de aminoetanoles sustituidos en C2 [Bedia et al., Org. Biomol. Chem. 2005 3, 3707-12. Grijalvo et al., Chem. Phys. Lipids. 2006 144, 69-84. Bedia et al., Chem. Phys. Lipids. 2008 156, 33-40]. Una de estas familias posee estructura de tioéter, aunque presenta poca actividad citotóxica frente a células tumoraíes. Various families of acid ceramidase inhibitors with structure of ceramide analogs or C2 substituted aminoethane have also been published [Bedia et al., Org. Biomol Chem. 2005 3, 3707-12. Grijalvo et al., Chem. Phys. Lipids. 2006 144, 69-84. Bedia et al., Chem. Phys. Lipids. 2008 156, 33-40]. One of these families has a thioether structure, although it has little cytotoxic activity against tumor cells.
Descripción de la invención Description of the invention
La presente invención proporciona nuevos derivados de ceramida ácida. Así mismo, la presente invención también proporciona una familia de compuestos con actividad antiproliferativa y citotóxica mediante la inhibición de la actividad ceramídasa ácida. En particular, la presente invención proporciona una familia de compuestos para el tratamiento del cáncer. The present invention provides new ceramide derivatives acid. Likewise, the present invention also provides a family of compounds with antiproliferative and cytotoxic activity by inhibiting acid ceramidasa activity. In particular, the present invention provides a family of compounds for the treatment of cancer.
En un primer aspecto, la presente invención se refiere a un compuesto de fórmula (I): In a first aspect, the present invention relates to a compound of formula (I):
Figure imgf000007_0001
) o un estereoisómero, una sai o un solvato farmacéuticamente aceptable de éste; donde:
Figure imgf000007_0001
) or a stereoisomer, a sai or a pharmaceutically acceptable solvate thereof; where:
A se selecciona entre -CH(OH)- y -C(=0)-,  A is selected from -CH (OH) - and -C (= 0) -,
Z se selecciona entre H y OH,  Z is selected from H and OH,
n es un número entero seleccionado entre 0 y 1 ,  n is an integer selected from 0 to 1,
R-i se selecciona entre aíquilo(CrC3o), alquenilo(C2-C3o), y alquiniío(C2- C3o), Ri is selected from alkyl (CrC 3 o), alkenyl (C 2 -C 3 o), and alkynyon (C 2 - C 3 o),
B se selecciona entre -H, -N3 y -C CH, B is selected from -H, -N 3 and -C CH,
R2 se selecciona entre -NHR3 y maleimida, donde R 2 is selected from -NHR 3 and maleimide, where
R3 se selecciona entre -CO-R4, -CO-CO-R4 y -SO2-R4, donde R 3 is selected from -CO-R4, -CO-CO-R4 and -SO2-R4, where
R4 se selecciona entre alquilo(Ci-Ci6), alquenilo(C2-Ci6), alquinilo(C-2~ Ci6), epóxído y aziridina, donde los grupos alquilo, alquenilo o alquiniio de R1 y R4 pueden estar, independientemente, opcionalmente sustituidos por uno o varios sustituyentes elegidos independientemente entre halógeno, OH, OR, OCF3, NH2, NO2, NRR', NHCOR; CONRR , CHO, COOH, COOR, OCOR y CN, donde R y R' son alquilo o alquenilo; con la condición de que R 4 is selected from alkyl (Ci-Ci6) alkyl, (C2-Ci6), alkynyl (C2 ~ Ci 6), epoxide and aziridine, wherein the alkyl, alkenyl or alquiniio of R1 and R4 may independently be , optionally substituted by one or more substituents independently selected from halogen, OH, OR, OCF 3 , NH 2 , NO 2 , NRR ', NHCOR; CONRR, CHO, COOH, COOR, OCOR and CN, where R and R 'are alkyl or alkenyl; with the condition of
a) cuando A es -CH(OH) y B es H, R3 es diferente de -COR4 siendo R4 alquilo(CrCi6) sin sustituir o sustituido por halógeno o hidroxilo; a) when A is -CH (OH) and B is H, R 3 is different from -COR 4 with R 4 being alkyl (CrCi 6 ) unsubstituted or substituted by halogen or hydroxyl;
b) cuando A es -CH(OH), B es H y R3 es -COCOR4 siendo R4 alquilo- Ce, R1 es diferente de -CH=CH2- alquilo(Ci2), -C≡CH-aiquilo(Ci2) o alquilo(Ci3-Ci5); o b) when A is -CH (OH), B is H and R 3 is -COCOR4 where R 4 is alkyl-Ce, R1 is different from -CH = CH 2 -alkyl (Ci 2 ), -C≡CH-aiquyl ( Ci 2 ) or (Ci3-Ci 5 ) alkyl; or
c) cuando A es -C(=O), R1 es alquenilo(C2-C3o), B es H y n es 0, R3 es diferente de -COR4 siendo R4 alquílo(CrCi6). c) when A is -C (= O), R1 is alkenyl (C 2 -C3o), B is H and n is 0, R3 is different from -COR 4 R 4 being alkyl (CrCi6).
El término "alquilo" se refiere, en la presente invención, a radicales de cadenas hidrocarbonadas no cíclicas, lineales o ramificadas, que se unen al resto de la molécula medíante un enlace sencillo, por ejemplo, metilo, etilo, n- propilo, /-propilo, n-butilo, ferc-butilo, sec-butilo, n-pentilo, n-hexiio, decilo o dodeciio. Los grupos alquilo pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como halógeno, hidroxilo, azido, alcoxilo, carboxilo, carbonita, ciano, carbaldehido, alcoxicarbonilo, amino o nitro. The term "alkyl" refers, in the present invention, to non-cyclic, linear or branched hydrocarbon chain radicals, which bind to the rest of the molecule through a single bond, for example, methyl, ethyl, n-propyl, / -propyl, n-butyl, ferc-butyl, sec-butyl, n-pentyl, n-hexy, decyl or dodecyl. The alkyl groups may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, carbonite, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro.
El término "alquenilo" se refiere a radicales de cadenas hidrocarbonadas no cíclicas, lineales o ramificadas, que contienen uno o más enlaces carbono-carbono dobles, preferentemente contiene un único doble enlace carbono-carbono, y que están unidos al resto de la molécula por un enlace simple, por ejemplo, vinilo, 1-propeniio, alilo, isoprenilo, 2-butenilo o 1 ,3-butadíenilo. Los radicales alqueniíos pueden estar opcionalmente sustituidos por uno o más sustituyentes tales como halógeno, hidroxilo, azido, alcoxilo, carboxilo, carbonilo, ciano, carbaldehido, alcoxicarbonilo, amino o nitro. Eí término "alquínilo" se refiere a radicales de cadenas hidrocarbonadas no cíclicas, lineales o ramificadas, que tienen uno o más tripies enlaces carbono-carbono, preferentemente contiene un único triple enlace carbono- carbono, y que están unidos ai resto de la molécula por un enlace simple por ejemplo, etinüo o 1-propinílo. El grupo alquinilo puede estar opcionaimente sustituido por uno o más sustituyentes tales como halógeno, hidroxilo, azido, alcoxilo, carboxiio, carbonüo, ciano, carbaldehido, alcoxicarbonílo, amino o nitro. Algunos de los compuestos de formula (I) de ia presente invención pueden tener uno o más centros estereogénicos. La presente invención abarca todos los posibles estereoisómeros no sólo sus mezclas racémicas sino también sus isómeros ópticamente activos. La obtención de un único enantiómero puede conseguirse mediante alguno de los procedimientos comúnmente empleados, por ejemplo, por resolución de ia mezcla racémica medíante técnicas de recristaiización, síntesis quíraí, resolución enzimática, biotransformación o resoiución cromatográfica. The term "alkenyl" refers to radicals of non-cyclic, linear or branched hydrocarbon chains, which contain one or more double carbon-carbon bonds, preferably contain a single carbon-carbon double bond, and which are attached to the rest of the molecule by a single bond, for example, vinyl, 1-propene, allyl, isoprenyl, 2-butenyl or 1,3-butadiene. Alkenium radicals may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxyl, carbonyl, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro. The term "alkynyl" refers to radicals of non-cyclic, linear or branched hydrocarbon chains, which have one or more triple carbon-carbon bonds, preferably contain a single carbon-carbon triple bond, and which are attached to the rest of the molecule by a simple link, for example, ethiny or 1-propyne. The alkynyl group may be optionally substituted by one or more substituents such as halogen, hydroxyl, azido, alkoxy, carboxy, carbon, cyano, carbaldehyde, alkoxycarbonyl, amino or nitro. Some of the compounds of formula (I) of the present invention may have one or more stereogenic centers. The present invention encompasses all possible stereoisomers not only their racemic mixtures but also their optically active isomers. Obtaining a single enantiomer can be achieved by any of the procedures commonly used, for example, by resolution of the racemic mixture through recrystallization techniques, chiraine synthesis, enzymatic resolution, biotransformation or chromatographic resoiución.
El término "sal farmacéuticamente aceptable" significa una sal que conserva una eficacia y propiedades biológicas similares a la base libre o dei ácido libre y que no es molesta en sentido biológico ni en ningún otro. The term "pharmaceutically acceptable salt" means a salt that retains an efficacy and biological properties similar to the free base or free acid and which is not bothersome in the biological sense or in any other.
Las sales farmacéuticamente aceptables pueden incluir ias sales de adición de ácidos, tales como mesilatos, fumaratos, clorhidratos, citratos, maleatos o tartratos. También pueden formarse sales fisiológicamente aceptables con ácidos inorgánicos como son los ácidos sulfúrico o fosfórico. Asimismo, pueden formarse sales de tipo básico de un metal alcalino, como por ejemplo el sodio, o de un metal alcalinotérreo, por ejemplo calcio o magnesio. Puede haber más de un catión o anión dependiendo del número de funciones con carga y de la valencia de los cationes y aniones. El término "solvato" en la presente solicitud de patente significa un agregado resultante de la asociación iónica o molecular entre moléculas de uno o más solventes y moléculas de uno de los compuestos objeto esta invención. El solvato puede comprender, por ejemplo, moléculas de agua, alcoholes, cetonas, acetatos o mezclas. En particular, el solvato puede comprender moléculas de agua, etanol, isopropanol, acetona o mezclas. Los solvatos objeto de la presente invención se pueden obtener por métodos conocidos por un experto en la materia, por ejemplo, por cristalización en condiciones controladas. Pharmaceutically acceptable salts may include acid addition salts, such as mesylates, smokers, hydrochlorides, citrates, maleates or tartrates. Physiologically acceptable salts can also be formed with inorganic acids such as sulfuric or phosphoric acids. Likewise, basic type salts of an alkali metal, such as sodium, or an alkaline earth metal, for example calcium or magnesium, can be formed. There may be more than one cation or anion depending on the number of charged functions and the valence of the cations and anions. The term "solvate" in the present patent application means an aggregate resulting from the ionic or molecular association between molecules of one or more solvents and molecules of one of the compounds object of this invention. The solvate may comprise, for example, water molecules, alcohols, ketones, acetates or mixtures. In particular, the solvate may comprise molecules of water, ethanol, isopropanol, acetone or mixtures. The solvates object of the present invention can be obtained by methods known to a person skilled in the art, for example, by crystallization under controlled conditions.
De acuerdo con una realización preferente, el compuesto de fórmula (I) de la presente invención se caracteriza porque Ri se selecciona entre alquilo(CrC30) y alquenilo(C2-C30), preferentemente R se selecciona entre alquilo(C C3o) y alquenílo(C2-C3o) sin sustituir, más preferentemente Ri se selecciona entre alquilo(C8-Cie) y alqueniio(C8-Ci6) sin sustituir. According to a preferred embodiment, the compound of formula (I) of the present invention is characterized in that Ri is selected from alkyl (CrC 30 ) and alkenyl (C 2 -C 3 0 ), preferably R is selected from alkyl (CC 3 or ) and alkenyl (C 2 -C 3 o) unsubstituted, more preferably Ri is selected from (C8-Cie) alkyl and alkenium (C8-Ci6) unsubstituted.
De acuerdo con una realización preferente adicional, el compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque n puede ser igual a cero. According to a further preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that n can be equal to zero.
De acuerdo con otra realización preferente adicional, el compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R3 es -CO-R4, donde R4 puede ser alquilo(CrCi6), y B se puede seleccionar entre -N3 y -CECH. According to another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R 3 is -CO-R4, where R4 can be alkyl (CrCi6), and B can be selected from -N 3 and -CECH.
Preferentemente, R4 puede ser un alquilo(Ci-Ci6) sustituido con al menos un átomo de halógeno, más preferentemente el halógeno puede ser un átomo de flúor o bromo, aún más preferentemente el halógeno es bromo. De forma aún más preferente, R4 puede ser un alquílo(Ci-C5) sustituido con al menos un átomo bromo. De acuerdo con otra realización preferente adicional, el compuesto de fórmula (I) objeto de la presente solicitud de patente caracterizado porque R3 es -CO-R4, donde R4 puede ser aiquilo(Ci-Ci6), y donde B se puede seleccionar entre -N3 y -CECH tal como se ha indicado anteriormente, también se caracteriza porque A es -C(=0). Preferably, R 4 may be a (Ci-Ci 6 ) alkyl substituted with at least one halogen atom, more preferably the halogen may be a fluorine or bromine atom, even more preferably the halogen is bromine. Even more preferably, R 4 may be an alkyl (Ci-C5) substituted with at least one bromine atom. According to another additional preferred embodiment, the compound of formula (I) object of the present patent application characterized in that R 3 is -CO-R 4 , where R 4 can be alkyls (Ci-Ci 6 ), and where B is You can select between -N 3 and -CECH as indicated above, it is also characterized in that A is -C (= 0).
Preferentemente, R4 puede ser un alquilo(CrC 6) sustituido con al menos un átomo de halógeno, más preferentemente el halógeno puede ser un átomo de flúor o bromo, aún más preferentemente el halógeno es bromo. Preferably, R 4 may be an alkyl (CrC 6) substituted with at least one halogen atom, more preferably the halogen may be a fluorine or bromine atom, even more preferably the halogen is bromine.
De forma aún más preferente, R4 puede ser un alquílo(Ci-C5) sustituido con al menos un átomo bromo. De acuerdo con otra realización preferente adicional, el compuesto de fórmula (I) objeto de ía presente solicitud de patente se caracteriza porque R3 es -CO-R4, donde R4 puede ser epóxido. Even more preferably, R 4 may be an alkyl (Ci-C5) substituted with at least one bromine atom. According to another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R3 is -CO-R4, where R4 can be epoxide.
Preferentemente, eí compuesto de fórmula (I) objeto de la presente solicitud de patente donde R3 es -CO-R4, donde F puede ser epóxido, también se caracteriza porque n puede ser 1 y Z puede ser OH. Preferably, the compound of formula (I) object of the present patent application where R3 is -CO-R4, where F can be epoxy, is also characterized in that n can be 1 and Z can be OH.
De acuerdo con otra realización preferente adicional, ei compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R3 es -CO-R4, donde R4 puede ser aiquenilo(C2-C e) o alquiniio(C2-C e). According to another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R3 is -CO-R4, where R 4 can be alkenyl (C2-C e) or alkyne (C2-C) and).
Preferentemente, R4 puede ser un aiquenilo(C2-Ci6) sustituido con al menos un átomo de halógeno, más preferentemente, R4 puede ser un alqueniio(C2-Ci6) sustituido con al menos un átomo de flúor o bromo, de forma aún más preferente R4 puede ser un alquenilo(C2-Ci6) sustituido con al menos un átomo de bromo. De acuerdo con otro modo de realización especialmente preferente, el compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R3 es -CO-R4, donde R4 es alquenilo(C2-Ci6) o aíquínilo(C2-Ci6), y puede estar sustituido por a! menos un grupo -CHO o - COOH. Preferably, R4 can be a aiquenilo (C2-Ci6) substituted with at least one halogen atom, more preferably, R4 can be a alqueniio (C 2 Ci 6) substituted with at least one fluorine atom or bromine, in an even more preferred R4 may be a (C2-Ci6) alkenyl substituted with at least one bromine atom. In accordance with another particularly preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R 3 is -CO-R4, where R4 is alkenyl (C 2 -Ci 6 ) or alkynyl (C 2 -Ci6), and may be substituted by a! minus a group -CHO or - COOH.
De acuerdo con otra realización preferente adicional, el compuesto de fórmuia (I) objeto de ia presente solicitud de patente se caracteriza porque f¾ es -CO-CO-R4, donde R4 puede ser alquilo(CrCi6) y B se puede seleccionar entre -N3 y -CECH. According to another additional preferred embodiment, the compound of formmuia (I) object of the present patent application is characterized in that f¾ is -CO-CO-R4, where R4 can be alkyl (CrCi 6 ) and B can be selected from - N 3 and -CECH.
De acuerdo con otra realización preferente adicional, el compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R3 puede ser -SO2-R4, donde R4 se puede seleccionar entre alquilo(Ci-Ci6), alquenilo(C2-Ci6), alquinilo(C2-C e), epóxido y aziridína . Preferentemente R4 puede ser alquiio(CrCi6), más preferentemente alquilo(C C8). According to another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R 3 can be -SO2-R4, where R 4 can be selected from alkyl (Ci-Ci6), alkenyl ( C2-Ci6), alkynyl (C2-C e), epoxide and aziridine. Preferably R 4 may be alkyl (CrCi 6 ), more preferably (C C8) alkyl.
De acuerdo con otra realización preferente adicional, el compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R3 puede ser -SO2-R4, donde R4 se puede seleccionar entre alquilo(CrCi6), alquenilo(C2-Ci6), aiquinilo(C2-Ci6), epóxido y aziridina, n puede ser 1 y Z puede ser OH. Preferentemente R4 puede ser alquilo(Ci-Ci6), más preferentemente alquilo(d-C8). According to another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R3 can be -SO2-R4, where R 4 can be selected from alkyl (CrCi 6 ), alkenyl (C 2 -Ci 6 ), aiquinyl (C 2 -Ci 6 ), epoxide and aziridine, n can be 1 and Z can be OH. Preferably R 4 may be (Ci-Ci6) alkyl, more preferably (d-C8) alkyl.
De acuerdo con otra realización preferente adicional, ei compuesto de fórmula (I) objeto de la presente solicitud de patente se caracteriza porque R2 puede ser maieimida. De acuerdo con otro modo de realización preferente adicional, el compuesto de fórmula (I) tal como se ha definido anteriormente se selecciona de la lista que consiste en: In accordance with another additional preferred embodiment, the compound of formula (I) object of the present patent application is characterized in that R2 can be maieimide. In accordance with another additional preferred embodiment, the compound of formula (I) as defined above is selected from the list consisting of:
1- [(2S,3R)-1 ,3-díhidroxioctadecan-2-il]-1 W-pirrol-2,5-díona,  1- [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] -1 W-pyrrole-2,5-done,
A/-[(2S,3R,E)-1 ,3-díhídroxioctadec-4-en-2-íl]etanosulfonamida, A / - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-2-yl] ethanesulfonamide,
N-[(2S,3 :?)-1 ,3-dihidroxioctadecan-2-il]etanosulfonamida, N - [(2S, 3?) - 1, 3-dihidroxioctadecan-2-yl] methanesulfonamide,
A/-[(2S,3R)-1 ,3-dihidroxíoctadec-17-in-2-íl]etanosulfonamida, A / - [(2S, 3R) -1, 3-dihydroxyoctadec-17-in-2-yl] ethanesulfonamide,
A/-[(2S,3R)-14-azido-1 ,3-dihidroxitetradecan-2-ii)etanosulfonamida,  A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-ii) ethanesulfonamide,
2- bromo-/V-[(2S,3R)-1 ,3-dihidroxioctadec-17-in-2-il]acetamida,  2- bromo- / V - [(2S, 3R) -1, 3-dihydroxyoctadec-17-in-2-yl] acetamide,
A/-[(2S,3f?)-14-azido-1 ,3-dihidroxitetradecan-2-il]bromoacetamida, A / - [(2S, 3f?) - 14-azido-1, 3-dihydroxytetradecan-2-yl] bromoacetamide,
N-[(2S,3R, E)- 14-azido- 1 , 3-d i h id roxitetradec-4-en-2-i l]bromoacetam ida , 2-bromo-A/-[(2S,3R,£)-1 ,3-dihidroxioctadec-4-en-17-in-2-il]acetamida, N - [(2S, 3R, E) - 14-azido- 1, 3-dih id roxitetradec-4-en-2-yl] bromoacetam ida, 2-bromo-A / - [(2S, 3R, £) - 1,3-dihydroxyoctadec-4-en-17-in-2-yl] acetamide,
(S)-/V-(14-azido-1-hidroxi-3-oxotetradecan-2-ii)-2-bromoacetamida, (S) - / V- (14-azido-1-hydroxy-3-oxotetradecan-2-ii) -2-bromoacetamide,
(RS)-A/-[(2S,3R,E)-1 ,3-dihidroxioctadec-4-en-2-il]oxirano-2-carboxamida, (RS)-A/-[(2S,3R)-1 ,3-dihidroxioctadecan-2-ii]oxirano-2-carboxamida, (RS) -A / - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-2-yl] oxirane-2-carboxamide, (RS) -A / - [(2S, 3R) - 1,3-dihydroxyoctadecan-2-ii] oxirane-2-carboxamide,
(RS)-/V-[(2S,3R,E)-1 ,3-dihídroxioctadec-4-en-17-in-2-íl]oxírano-2- carboxamida,  (RS) - / V - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-17-in-2-yl] oxirane-2-carboxamide,
(RS)-A/-[(2S,3R)-14-azido-1 ,3-díhídroxitetradecan-2-íl]oxírano-2-carboxamída, A/-[(2S,3f?)-1 ,3-dihidroxioctadecan-2-il]propiolamida,  (RS) -A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl] oxirane-2-carboxamide, A / - [(2S, 3f?) - 1, 3-dihydroxyoctadecan -2-il] propiolamide,
/V-[(2S,3R)-1 ,3-dihidroxíoctadecan-2-íl3but-2-inamida, / V - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl3but-2-inamide,
A/-[(2S,3R)-1 ,3-dihidroxioctadecan-2-il3acrilamida, A / - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl3-acrylamide,
(£)-/V-[(2S,3R)-1 ,3-dihidroxioctadecan-2-ii]-2-butenamida, (£) - / V - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-ii] -2-butenamide,
A/-[(2S,3R)-1 ,3-dihídroxioctadecan-2-il3metacrílamida, A / - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl3methacrylamide,
N-[(2S,3 :?)-N-1 ,3-dihidroxyoctadecan-2-il]-3-metil-2-butenamida, N - [(2S, 3?) - N-1, 3-dihidroxyoctadecan-2-yl] -3-methyl-2-butenamide,
(2E,4E)-N-[(2S,3R)-1 ,3-díhidroxioctadecan-2-il]hexa-2,4-dienamida, (2E, 4E) -N - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] hexa-2,4-dienamide,
Ácido (E)-4-[(2S,3/:?)-1 ,3-dihidroxioctadecan-2-iiamino]-4-oxo-2-butenoico, (Z)-2,3-dibromo-N-[(2S,3/:?)-1 ,3-dihidroxíoctadecan-2-íl3-4-oxo-2-butenamida, (2S,3R)-2-(bromometii)-N-(1 ,3-dihidroxioctadecan-2-il)acrilamida, (E) -4 - [(2S, 3 /?) - 1, 3-2-ylamino dihidroxioctadecan-] -4-oxo-2-butenoic acid, (Z) -2,3-dibromo-N - [( 2S, 3 /?) - 1, 3-dihidroxíoctadecan-2-oxo-2-íl3-4-butenamide, (2S, 3R) -2- (bromometii) -N- (1, 3-dihidroxioctadecan-2yl ) acrylamide,
(E,2S,3R)-/V-(1 ,3-díhidroxi-2-octadecil)-2-metíl-2-butenamída, (E, 2S, 3R) - / V- (1,3-dihydroxy-2-octadecyl) -2-methyl-2-butenamide,
(2S,3R)-N-(1 ,3-dihidroxi-17-octadecin-2-il)-2-oxooctanamida y (2S, 3R) -N- (1,3-dihydroxy-17-octadecin-2-yl) -2-oxooctanamide and
(2S,3R)-N-(14-azido-1 ,3-dihidroxi-2-tetradecií)-2-oxooctanamída, (2S, 3R) -N- (14-azido-1, 3-dihydroxy-2-tetradecyl) -2-oxooctanamide,
o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de uno de estos compuestos . Preferentemente, el compuesto de fórmula (I) tal como se ha definido anteriormente en esta solicitud de patente se selecciona de la lista que consiste en: or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds. Preferably, the compound of formula (I) as defined above in this patent application is selected from the list consisting of:
2-bromo-A/-[(2S,3R)-1 ,3-díhidroxioctadec-17~in-2-il]acetamida, 2-Bromo-A / - [(2S, 3R) -1, 3-dihydroxyoctadec-17 ~ in-2-yl] acetamide,
A/-[(2S,3R)-14-azido-1 ,3-dihídroxitetradecan-2-íi]bromoacetamida, A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl] bromoacetamide,
A/-[(2S,3R,E)-14-azido-1 ,3-dihidroxitetradec-4-en-2-il]bromoacetamida, A / - [(2S, 3R, E) -14-azido-1, 3-dihydroxytetradec-4-en-2-yl] bromoacetamide,
2-bromo-/V-[(2S,3R,E)-1 ,3-dihidroxíoctadec-4-en-17-ín-2-il]acetamida y 2-Bromo- / V - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-17-ín-2-yl] acetamide and
(S)-A/-(14-azído-1-hidroxí-3-oxotetradecan-2-il)-2-bromoacetamída, (S) -A / - (14-azido-1-hydroxy-3-oxotetradecan-2-yl) -2-bromoacetamide,
o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos. or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds.
De acuerdo con otro modo de realización preferente adicional, el compuesto de fórmula (I) cuando n es 1 tal como se ha definido anteriormente se selecciona de la lista que consiste en: According to another additional preferred embodiment, the compound of formula (I) when n is 1 as defined above is selected from the list consisting of:
A/-[(2S,3S,4R)-1 ,3,4-tríhídroxioctadecan-2-il]etanosulfonamida y  A / - [(2S, 3S, 4R) -1, 3,4-trídroxioctadecan-2-yl] ethanesulfonamide and
(RS)-N-[(2S,3S,4R)-1 ,3,4-trihidroxioctadecan-2-il]oxirano-2-carboxamida, o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos. (RS) -N - [(2S, 3S, 4R) -1, 3,4-trihydroxyoctadecan-2-yl] oxirane-2-carboxamide, or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds.
Los compuestos de fórmula (I) pueden prepararse siguiendo distintos métodos conocidos para cualquier persona experta en el campo de la síntesis orgánica, en particular por los procedimientos generales que se describen a continuación. Los materiales de partida están disponibles comercialmente o bien se pueden preparar mediante métodos de la literatura. The compounds of formula (I) can be prepared following different methods known to any person skilled in the field of organic synthesis, in particular by the general procedures described below. The starting materials are commercially available or can be prepared by methods of the literature.
Los compuestos de fórmula (I) pueden obtenerse a partir de los métodos y esquemas descritos a continuación: Esquema 1. The compounds of formula (I) can be obtained from the methods and schemes described below: Scheme 1.
Figure imgf000015_0001
Figure imgf000015_0001
a: Cat. Grubbs (segunda generación); b: 1 ) H2, Pd/C; 2) NaN3/DMF; c: NaN3/DMF; d: NaH/THF; e: 1 ) Li-C≡C-TMS; 2) Bu4NF; f: HCI/MeOH; g: 1 ) cat. TsOH/MeOH; 2) NaOH/EtOH; h: 1 ) PCC/CH2CI2; 2) cat. TsOH/MeOH. a: Cat. Grubbs (second generation); b: 1) H 2 , Pd / C; 2) NaN3 / DMF; c: NaN 3 / DMF; d: NaH / THF; e: 1) Li-C≡C-TMS; 2) Bu 4 NF; f: HCI / MeOH; g: 1) cat. TsOH / MeOH; 2) NaOH / EtOH; h: 1) PCC / CH 2 CI 2 ; 2) cat. TsOH / MeOH.
En primer lugar, se hace reaccionar el compuesto 1 (Herold, Helv. Chim. Acta. 1988, 71, 354-62) con una oiefina terminal (como, por ejemplo, 2a o 2b) en una reacción de metátesis cruzada en presencia del catalizador de Grubbs de segunda generación, (Schmidt, Angew. Chem. Int. Ed. 2003, 42, 4996-9) obteniéndose los compuestos 3. Por hídrogenación del intermedio 3a seguida de sustitución nucleófila del átomo de bromo por azida sódica se llega a los intermedios 8, que por hidrólisis de los grupos protectores conducen a los aminodíoles H-E. Por otra parte, por oxidación de 8 y posterior hidrólisis de los grupos protectores se obtienen las aminas ll-H. Para obtener las aminas ll-F, ei bromoderivado 3a se hace reaccionar con azida sódica en dimetilformamida, obteniéndose 3c, seguido de hidrólisis ácida del anillo de oxazolidina y del grupo Boc. Mediante reacción de los intermedios 3b con hídruro de sodio en dimetilformamida se obtienen los mesílatos 4, que pueden hidrogenarse a 5. Mediante reacción de 4 ó 5 con trimetilsililacetiluro de litio seguida de desproteccíón del acetíluro con fluoruro de tetrabutilamonio se obtienen los alquinos terminales 6 y 7, respectivamente. Su transformación en las correspondientes aminas IIG y IID se consigue mediante hidrólisis secuencial de los grupos isopropilideno y carbamato. First, compound 1 (Herold, Helv. Chim. Acta. 1988, 71, 354-62) is reacted with a terminal oiefin (such as 2a or 2b) in a cross-metathesis reaction in the presence of second generation Grubbs catalyst (Schmidt, Angew. Chem. Int. Ed. 2003, 42, 4996-9) obtaining compounds 3. By hydrogenation of intermediate 3a followed by nucleophilic substitution of the bromine atom by sodium azide, intermediates 8, which by hydrolysis of the protective groups lead to the aminodiols HE. On the other hand, by oxidation of 8 and subsequent hydrolysis of protective groups are obtained ll-H amines. To obtain the ll-F amines, bromoderivative 3a is reacted with sodium azide in dimethylformamide, obtaining 3c, followed by acid hydrolysis of the oxazolidine ring and the Boc group. By reacting intermediates 3b with sodium hydride in dimethylformamide, mesylates 4 are obtained, which can be hydrogenated to 5. By reaction of 4 or 5 with lithium trimethylsilylacetyl followed by deprotection of acetylide with tetrabutylammonium fluoride, terminal alkynes 6 and 7, respectively. Its transformation into the corresponding IIG and IID amines is achieved by sequential hydrolysis of the isopropylidene and carbamate groups.
Los anteriores amino alcoholes, asi como la esfingosína o la dihidroesfingosina, pueden acilarse por reacción con un cloruro de ácido en presencia de una base. Alternativamente, también puede emplearse un anhídrido de ácido, como el anhídrido maleico, o un ácido carboxílico en presencia de un agente de acoplamiento adecuado como, por ejemplo, la 1- etil-3-(3-dimetilaminopropil)carbodiimida (EDC) , la Ν,Ν'- díisopropilcarbodiimida (DIC), el hexafluoro fosfato de O-(7-azabenzotriazol-1- il)-/V,A/,A/'A/'-tetrametiluronio (HATU) o el benzotriazol-1-iloxi-tris pirroiidinofosfonío (PyBOP) y un activador como el 1 -hidroxibenzotriazol (HOBt), en presencia de una base como trietilamína, en un disolvente como diciorometano y bajo atmosfera inerte (E. Vaieur, et al.,Chem Soc Rev 2009, 38, 606-31 ) Las sulfonamidas pueden prepararse por acoplamiento de las aminas correspondientes con cloruro de etanosuifonilo en disolución de tetrahídrofurano. The above amino alcohols, as well as sphingosine or dihydrosphingosine, can be acylated by reaction with an acid chloride in the presence of a base. Alternatively, an acid anhydride, such as maleic anhydride, or a carboxylic acid can also be used in the presence of a suitable coupling agent such as, for example, 1- ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), the Ν, Ν'- diisopropylcarbodiimide (DIC), O- (7-azabenzotriazol-1- il) hexafluoro phosphate - / V, A /, A / 'A /' - tetramethyluronium (HATU) or benzotriazol-1-yloxy -tris pyrroiidinophosphonium (PyBOP) and an activator such as 1-hydroxybenzotriazole (HOBt), in the presence of a base such as triethylamine, in a solvent such as dichloromethane and under an inert atmosphere (E. Vaieur, et al., Chem Soc Rev 2009, 38 , 606-31) The sulfonamides can be prepared by coupling the corresponding amines with ethanesuifonyl chloride in tetrahydrofuran solution.
Otro aspecto de la presente invención se refiere a una composición farmacéutica que comprende un compuesto de fórmula general (I) o uno de sus estereisómeros, sales o solvatos farmacéuticamente aceptables tal como se define en esta solicitud de patente y al menos un excipiente farmacéuticamente aceptable. Preferentemente, ia composición farmacéutica se puede presentar en una forma adaptada a la administración parenteral, oral, sublingual, nasai, intratecai, bronquial, linfática, rectal, transdérmica o inhalada. De acuerdo con un aspecto adicional de la presente invención, tanto el compuesto de fórmula general (I) o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de éste tal como se ha definido en la presente solicitud de patente, como un compuesto elegido entre 2,2-dibromo-N- ((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamida y 2-bromo-N-((2S,3R)-1 ,3- dihidroxioctadecan-2-il)acetamida o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de uno de estos compuestos son útiles para el tratamiento de una enfermedad asociada con la inhibición de la ceramidasa ácida, y por lo tanto son útiles en el tratamiento y/o prevención de una enfermedad que cursa con híperproliferación celular. Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of general formula (I) or one of its pharmaceutically acceptable stereoisomers, salts or solvates as defined in this patent application and at least one pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition It can be presented in a form adapted to parenteral, oral, sublingual, nasai, intrathecai, bronchial, lymphatic, rectal, transdermal or inhaled administration. According to a further aspect of the present invention, both the compound of general formula (I) or a stereoisomer, a pharmaceutically acceptable salt or soivate thereof as defined in the present patent application, as a compound chosen from 2,2-dibromo-N- ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) Acetamide or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds are useful for the treatment of a disease associated with the inhibition of acid ceramidase, and therefore are useful in the treatment and / or prevention of a disease. which studies with cellular hyperproliferation.
De acuerdo con otro aspecto adicional de la presente invención, tanto las composiciones de un compuesto de fórmula general (I) o un estereoisómero, una sai o un soivato farmacéuticamente aceptable de éste tal como se ha definido en la presente solicitud de patente, como fas composiciones que comprenden un compuesto que se puede elegir entre 2,2- dibromo-N-((2S,3R)-1 ,3-díhídroxioctadecan-2-il)acetamida y 2-bromo-N- ((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamída o un estereoisómero, una sai o un soivato farmacéuticamente aceptable de uno de estos compuestos y al menos un excipiente farmacéuticamente aceptable, son útiles para el tratamiento y prevención de una enfermedad que cursa con híperproliferación celular. In accordance with another additional aspect of the present invention, both the compositions of a compound of general formula (I) or a stereoisomer, a sai or a pharmaceutically acceptable soivate thereof as defined in the present patent application, as fas Compositions comprising a compound that can be chosen from 2,2-dibromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N- ((2S, 3R) - 1,3-dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a sai or a pharmaceutically acceptable soivate of one of these compounds and at least one pharmaceutically acceptable excipient, are useful for the treatment and prevention of a disease that occurs with cell hyperproliferation .
Preferentemente, el compuesto o composición para utilizar en el tratamiento o prevención de una enfermedad que cursa con híperproliferación celular tal como se han definido anteriormente en la presente solicitud de patente, se puede utilizar para el tratamiento de una enfermedad elegida entre cáncer, metástasis, inflamación, asma y arteriesclerosis. Preferably, the compound or composition for use in the treatment or prevention of a disease that occurs with cellular hyperproliferation as defined above in the present application for patent, can be used for the treatment of a disease chosen from cancer, metastasis, inflammation, asthma and arteriosclerosis.
De forma más preferente, el compuesto o composición para utilizar en el tratamiento o prevención de una enfermedad que cursa con hiperproliferación celular tal como se han definido en la presente solicitud de patente, se puede utilizar en el tratamiento del cáncer de próstata, páncreas, cerebro, colon, pulmón, mama, cabeza y cuello, ovario, laringe, vejiga urinaria, útero, piel, sarcomas, línfomas o leucemia. Preferentemente, cáncer de próstata o pulmón. More preferably, the compound or composition for use in the treatment or prevention of a disease that occurs with cell hyperproliferation as defined in the present patent application, can be used in the treatment of prostate cancer, pancreas, brain , colon, lung, breast, head and neck, ovary, larynx, urinary bladder, uterus, skin, sarcomas, lymphomas or leukemia. Preferably, prostate or lung cancer.
La presente invención también se refiere a un método para la prevención o el tratamiento de un individuo que padece o es susceptible de padecer enfermedad que cursa con hiperproliferación celular, en particular el tratamiento del cáncer y más preferentemente el tratamiento del cáncer de próstata o pulmón, que comprende la administración a dicho individuo de una cantidad terapéuticamente efectiva de un compuesto de fórmula (I) o de una sal farmacéuticamente aceptable del mismo junto con cantidades suficientes de excipientes farmacéuticamente aceptables. The present invention also relates to a method for the prevention or treatment of an individual who suffers from or is susceptible to suffering from disease that occurs with cellular hyperproliferation, in particular the treatment of cancer and more preferably the treatment of prostate or lung cancer, which comprises administering to said individual a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof together with sufficient amounts of pharmaceutically acceptable excipients.
La presente invención también se refiere al uso tanto de un compuesto de fórmula general (I) o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de éste tal como se ha definido en la presente solicitud de patente, como al uso de un compuesto elegido entre 2,2-dibromo- N-((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamída y 2-bromo-N-((2S,3R)-1 ,3- dihidroxioctadecan-2-il)acetamida o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos, para fabricar una composición farmacéutica útil en el tratamiento de una enfermedad asociada con la inhibición de la ceramidasa ácída, y por lo tanto útil en el tratamiento y/o prevención de una enfermedad que cursa con hiperproliferación celular. Preferentemente, dicha composición se puede utilizar para el tratamiento de una enfermedad eiegida entre cáncer, metástasis, infiamación, asma y arteriesclerosis. De forma más preferente, la composición tai como se ha descrito anteriormente se puede utilizar en el tratamiento del cáncer de próstata, páncreas, cerebro, colon, pulmón, mama, cabeza y cuelio, ovario, laringe, vejiga urinaria, útero, piel, sarcomas, linfomas o leucemia. Preferentemente, cáncer de próstata o pulmón. The present invention also relates to the use of both a compound of general formula (I) or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof as defined in the present patent application, and the use of a compound chosen between 2,2-dibromo- N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxioctadecan-2-yl ) acetamide or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds, to make a pharmaceutical composition useful in the treatment of a disease associated with the inhibition of acid ceramidase, and therefore useful in the treatment and / or prevention of a disease that occurs with cellular hyperproliferation. Preferably, said composition can be used for the treatment of a disease caused by cancer, metastasis, inflammation, asthma and arteriosclerosis. More preferably, the tai composition as described above can be used in the treatment of prostate cancer, pancreas, brain, colon, lung, breast, head and colium, ovary, larynx, urinary bladder, uterus, skin, sarcomas , lymphomas or leukemia. Preferably, prostate or lung cancer.
Los compuestos usados en la presente invención pueden usarse solos o con otros fármacos para proporcionar una terapia de combinación. Los otros fármacos pueden formar parte de ia misma composición, o se pueden suministrar en forma de composición separada para la administración al mismo tiempo o en un momento diferente. Así, de acuerdo con otro aspecto adicional de la presente invención, un compuesto de fórmula genera! (I) tal como se ha definido en la presente solicitud de patente o un compuesto elegido entre 2,2-díbromo-N-((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamida y 2-bromo-N-((2S,3R)-1 ,3-dihidroxioctadecan-2-ií)acetamída o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de uno de estos compuestos, se puede utilizar en combinación con otra terapia para el tratamiento de una enfermedad tal como se ha definido anteriormente. The compounds used in the present invention can be used alone or with other drugs to provide a combination therapy. The other drugs can be part of the same composition, or they can be supplied as a separate composition for administration at the same time or at a different time. Thus, according to another additional aspect of the present invention, a compound of formula generates! (I) as defined in the present patent application or a compound chosen from 2,2-dibromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo- N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-i) acetamide or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds, can be used in combination with another therapy for the treatment of a disease as defined above.
De acuerdo con otro aspecto adicional de la presente invención, la composición de un compuesto de fórmula (I) tal como se ha definido anteriormente en la presente solicitud de patente o la composición que comprende un compuesto elegido entre 2,2-dibromo-N-((2S,3R)-1 ,3- dihidroxioctadecan-2-il)acetamida y 2-bromo-N-((2S,3R)-1 ,3- dihidroxioctadecan-2-il)acetamída o un estereoisómero, una sai o un soivato farmacéuticamente aceptable de uno de estos compuestos y al menos un excipiente, se puede utilizar en combinación con otra terapia para el tratamiento de una enfermedad tal como se ha definido anteriormente. In accordance with a further aspect of the present invention, the composition of a compound of formula (I) as defined above in the present patent application or the composition comprising a compound chosen from 2,2-dibromo-N- ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a sai o a pharmaceutically acceptable soivate of one of these compounds and at least one excipient can be used in combination with another therapy for the treatment of a disease as defined above.
Otros tipos de terapia pueden ser quimioterapia o radioterapia. A título de ejemplo, los agentes terapéuticos pueden ser tamoxífen, daunorubicina, etoposido, pacíitaxel, dacarbacida, temozoíomída, temsirolimus, fenretínida, resveratrol, borinostat, sorafenib, imatinib, bortezomib, gemcitabina o cispiatin. Other types of therapy may be chemotherapy or radiotherapy. By way of example, the therapeutic agents may be tamoxifen, daunorubicin, etoposide, pacitax, dacarbazide, temozoomomide, temsirolimus, fenretinide, resveratrol, borinostat, sorafenib, imatinib, bortezomib, gemcitabine or cispiatin.
A io largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para ios expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de ía práctica de ía invención. Los siguientes ejemplos y dibujos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. Throughout the description and claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figura 1. Efecto de los compuestos sobre la actividad ceramídasa ácida. Los ensayos se efectuaron sobre células de Farber transducidas para que sobreexpresen la ceramidasa ácida, ya sea intactas (barras blancas) o en lisados (barras grises). La incubación se llevó a cabo añadiendo conjuntamente el inhibidor (16 μΜ) con el sustrato fiuorogénico (16 μΜ) durante 3 h. A continuación se procedió tal como se detalla en el ejemplo 1 del apartado de ejemplos de ensayos biológicos (Bedia et al. Chembiochem 2007, 8, 642). El eje y indica el porcentaje de actividad enzimática respecto al control. Figure 1. Effect of the compounds on the acid ceramidasa activity. The tests were performed on transduced Farber cells to overexpress acid ceramidase, either intact (white bars) or in lysates (gray bars). Incubation was carried out by adding together the inhibitor (16 μΜ) with the phylogenic substrate (16 μΜ) for 3 h. The procedure was followed as detailed in example 1 of the section on examples of biological tests (Bedia et al. Chembiochem 2007, 8, 642). The y axis indicates the percentage of enzymatic activity with respect to the control.
Figura 2. Efecto de los compuestos sobre la actividad ceramidasa ácida. Los ensayos se efectuaron sobre células de adenocarcinoma de pulmón humano A549. Las células intactas se incubaron con el inhibidor (16 μΜ) durante 24 h y luego se añadió el sustrato fiuorogénico (16 μΜ), que se incubó durante 3 h. La hidrólisis se determinó midiendo la fluorescencia generada después de proceder tal como se indica en ei ejemplo 1 dei apartado de ejemplos de ensayos biológicos (Bedia et al. Chembíochem 2007, 8, 642). El eje y índica el porcentaje de actividad enzimática respecto al control. Figure 2. Effect of the compounds on the acid ceramidase activity. The tests were carried out on human lung adenocarcinoma cells A549. The intact cells were incubated with the inhibitor (16 μΜ) for 24 h and then the phylogenic substrate (16 μΜ) was added, which was incubated for 3 h. Hydrolysis was determined by measuring the fluorescence generated after proceeding as indicated in example 1 of the section on examples of biological tests (Bedia et al. Chembíochem 2007, 8, 642). The axis and indicates the percentage of enzymatic activity with respect to the control.
Figura 3. Efecto de ios compuestos I-B2, I-B17 e I-B9 sobre la actividad ceramidasa ácida de las células de cáncer de próstata PC-3Mc. Los ensayos se efectuaron por incubación de las células con el inhibidor. Las células se sembraron en placas de 96 pocilios a una densidad de 10.000 células/ml y los compuestos se añadieron 24 h después de la siembra. Se usaron tres dosis de cada inhibidor, 1 μΜ, 5 μΜ y 10 μΜ, en incubaciones durante 48 h y los ensayos se realizaron por triplicado. Tras este período de incubación, se eíimínó el medio y se añadió medio fresco conteniendo el eí sustrato fluorogénico (16 μΜ) y se incubó durante 3 h. La hidrólisis se determina midiendo la fluorescencia generada después de proceder tal como se índica en el ejemplo 1 del apartado de ejemplos de ensayos biológicos (Bedia et al. Chembíochem 2007, 8, 642). Eí eje y indica el porcentaje de actividad enzimática respecto al control. Figura 4. Efecto de los compuestos sobre el ceramidoma. Los ensayos se efectúan sobre células de adenocarcinoma de pulmón humano A549. Las céluías intactas se incubaron con el inhibidor (16 μΜ) durante 24 h y íuego se recolectaron las células y se procesaron tal como se detalla en ei ejemplo 2 de ejempíos de ensayos biológicos. Figure 3. Effect of the compounds I-B2, I-B17 and I-B9 on the acidic ceramidase activity of PC-3Mc prostate cancer cells. The assays were carried out by incubation of the cells with the inhibitor. The cells were seeded in 96-well plates at a density of 10,000 cells / ml and the compounds were added 24 h after seeding. Three doses of each inhibitor, 1 μΜ, 5 μΜ and 10 μΜ, were used in incubations for 48 h and the tests were performed in triplicate. After this incubation period, the medium was eimiminated and fresh medium containing the fluorogenic substrate (16 μΜ) was added and incubated for 3 h. Hydrolysis is determined by measuring the fluorescence generated after proceeding as indicated in example 1 of the section on examples of biological tests (Bedia et al. Chembíochem 2007, 8, 642). The axis and indicates the percentage of enzymatic activity with respect to the control. Figure 4. Effect of the compounds on the ceramidoma. The tests are carried out on human lung adenocarcinoma cells A549. The intact cells were incubated with the inhibitor (16 μΜ) for 24 h and then the cells were collected and processed as detailed in example 2 of biological test specimens.
Figura 5. Efecto de los compuestos I-B2, I-B17 e I-B9 sobre el esfingolipidoma de ias células de cáncer de próstata PC-3Mc. Los ensayos se efectuaron por incubación de las células con los compuestos. Las células se sembraron en placas de 6 pocilios a una densidad de 250.000 células/ml y los compuestos se añadieron 24 h después de la siembra. Se usaron tres dosis de cada inhibidor, 1 μ , 5 μΜ y 10 μΜ, en incubaciones durante 48 h y los ensayos se realizaron por triplicado. Se usaron tres dosis de cada compuesto, 1 μΜ, 5 μΜ y 10 μΜ, en incubaciones durante 48 h. Tras este período de incubación, se elimina el medio se recolectan las células y se procesan tal como se detalla en el ejemplo 2 de ejemplos de ensayos biológicos. A, ceramidas; B, dihidroceramídas; C, esfingomieiinas; D dihidroesfingomielinas; E, glucosilceramidas. Figure 5. Effect of compounds I-B2, I-B17 and I-B9 on the sphingolipidoma of the prostate cancer cells PC-3Mc. The assays were carried out by incubation of the cells with the compounds. The cells were seeded in 6-well plates at a density of 250,000 cells / ml and the compounds were added 24 h after seeding. Three doses of each inhibitor, 1 μ, 5 μΜ and 10 μΜ, were used in incubations for 48 h and the Trials were performed in triplicate. Three doses of each compound, 1 μΜ, 5 μΜ and 10 μΜ, were used in incubations for 48 h. After this incubation period, the medium is removed, the cells are harvested and processed as detailed in example 2 of examples of biological tests. A, ceramides; B, dihydroceramides; C, sphingomyeliins; D dihydrosphingomyelins; E, glucosylceramides.
Figura 6. Efecto de los compuestos sobre la viabilidad de las células de A, adenocarcinoma de pulmón A549 (negro) y de leucemia Jurkat A3 (gris) y B, cáncer de próstata PC3/Mc. Las células se sembraron a una densidad de 200.000 células por mililitro y, 24 h después de la siembra, se incubaron con los compuestos durante 24 h (A) o 72 h (B), tras las cuales se determinó el número de células viables medíante el ensayo de reducción del MTT, tal como se específica en el ejemplo 3 de ejemplos de ensayos biológicos. El eje y índica el porcentaje del numero de células respecto al control. Figure 6. Effect of the compounds on the viability of A cells, lung adenocarcinoma A549 (black) and leukemia Jurkat A3 (gray) and B, prostate cancer PC3 / Mc. The cells were seeded at a density of 200,000 cells per milliliter and, 24 hours after planting, they were incubated with the compounds for 24 h (A) or 72 h (B), after which the number of viable cells was determined through the MTT reduction test, as specified in example 3 of examples of biological tests. The axis and indicates the percentage of the number of cells with respect to the control.
Figura 7. Efecto de los compuestos sobre el crecimiento de las células PC- 3Mc sobre sustrato plástico. Se sembraron 500 células en cada pocilio de placas de 96 pocilios, dejándose adherir al plástico durante 24 h, seguido de tratamiento con los compuestos a una concentración final de 5 μΜ. El efecto de las incubaciones con estos compuestos sobre el crecimiento celular de determinó cuantificado el número de células presentes a las 24 h, 48 h, 72 h, 120 h, 144 h y 168 h después del tratamiento con los compuestos. El eje y índica la proporción relativa del número de células respecto al día 1 y el eje x se refiere al tiempo (t) en días. Figure 7. Effect of the compounds on the growth of PC-3Mc cells on plastic substrate. 500 cells were seeded in each well of 96-well plates, leaving to adhere to the plastic for 24 h, followed by treatment with the compounds at a final concentration of 5 μΜ. The effect of incubations with these compounds on cell growth was determined by quantifying the number of cells present at 24 h, 48 h, 72 h, 120 h, 144 h and 168 h after treatment with the compounds. The y-axis indicates the relative proportion of the number of cells with respect to day 1 and the x-axis refers to the time (t) in days.
Figura 8. Efecto de los inhibidores sobre la formación de colonias celulares en medio semísóíido. Las células PC-3Mc, sembradas en medio de cultivo completo que contiene agar al 3%, se trataron con los compuestos problema a concentraciones finales de 1 μΜ o 5 μΜ, reañadiéndolos con una periodicidad de 3 días, coincidiendo con la adición de medio de cultivo nuevo. El número de colonias se contó 3 semanas después del tratamiento. El eje y indica el número de colonias (N° Col.) Figure 8. Effect of inhibitors on the formation of cell colonies in semi-solid medium. The PC-3Mc cells, seeded in complete culture medium containing 3% agar, were treated with the test compounds at final concentrations of 1 μΜ or 5 μΜ, reacting them with a periodicity of 3 days, coinciding with the addition of new crop The number of colonies was counted 3 weeks after treatment. The y axis indicates the number of colonies (No. Col.)
Figura 9. Efecto de ios inhibidores sobre la invasividad celuiar. Las células PC-3Mc fueron tratadas con los compuestos problema a una concentración final de 5 μΜ durante las 48 h previas al ensayo de invasividad. Tras ese tiempo de tratamiento, las células fueron recolectadas, resuspendidas en medio completo, y sembradas sobre las cámaras superiores de los insertos Transwell recubiertos con Matrigei (10 mg/mL). Los compuestos problema se añadieron tanto a la cámara superior como a la inferior, a una concentración de 5 μΜ, manteniéndose este tratamiento a io largo de todo el período de duración del ensayo. Cada condición se realizó por triplicado. El eje y indica el número de células (N°) Figura 10. Fórmulas de ios compuestos sintetizados en los ejemplos. Figure 9. Effect of ios inhibitors on cell invasion. PC-3Mc cells were treated with the test compounds at a final concentration of 5 μΜ during the 48 hours prior to the invasiveness test. After that treatment time, the cells were collected, resuspended in complete medium, and seeded on the upper chambers of the Transwell inserts coated with Matrigei (10 mg / mL). The test compounds were added to both the upper and lower chamber, at a concentration of 5 μΜ, this treatment being maintained throughout the entire duration of the test. Each condition was performed in triplicate. The y-axis indicates the number of cells (No.) Figure 10. Formulas of the compounds synthesized in the examples.
EJEMPLOS EXAMPLES
A continuación se ilustrará la invención mediante unos ensayos realizados por los inventores, que pone de manifiesto la especificidad y efectividad de los inhibidores de ceramidasas de la presente invención. The invention will now be illustrated by tests carried out by the inventors, which shows the specificity and effectiveness of the ceramidase inhibitors of the present invention.
Ejemplos de síntesis química Síntesis de ios precursores de las bases esfinqoides H(D-H) Examples of chemical synthesis Synthesis of the precursors of the sphincter bases H (D-H)
Reacción de metátesis entre el alcohol 1 y las oiefinas terminales 2a y 2b: síntesis de 3a y de 3b Metathesis reaction between alcohol 1 and terminal oiefins 2a and 2b: synthesis of 3a and 3b
A una disolución desgasada del alcohol 1 (5 mmol) y de la olefina terminal 2a o 2b (20 mmol) en diclorometano (80 mL) se añadieron 400 mg (aproximadamente 0.5 mmol) de catalizador de Grubbs de segunda generación. La mezcla se agitó a la temperatura de reflujo bajo atmósfera de Ar durante 5 h. Tras la evaporación dei disolvente, el residuo se purificó por cromatografía flash (hexano / EtOAc 1 :3). (S)-4-i( E)-12-bromo-1 -hidroxidodec-2-enil1-2,2-dimetiioxazoiidina-3- carboxilato de terc-butiio (3a) To a worn solution of alcohol 1 (5 mmol) and terminal olefin 2a or 2b (20 mmol) in dichloromethane (80 mL) was added 400 mg (approximately 0.5 mmol) of second Grubbs catalyst generation. The mixture was stirred at reflux temperature under Ar atmosphere for 5 h. After evaporation of the solvent, the residue was purified by flash chromatography (hexane / EtOAc 1: 3). (S) -4-i (E) -12-Bromo-1-hydroxidedec-2-enyl1-2,2-dimethioxazoiidine-3-carboxylic acid tert-butyl ester (3a)
Rendimiento: 85% Yield: 85%
1H-RMN (400 MHz, CDCi3): δ 5.58 (m, 1 H); 5.45 (m, 1 H); 4.20-3.70 (m, 4H); 3.35 (t, 2H); 1.95 (m, 2H); 1.78 (m, 2H); 1.65-1 .35 (m, 12H); 1.45 (s, 9H); 1 .18 (s, 6H), 1 H-NMR (400 MHz, CDCi 3 ): δ 5.58 (m, 1 H); 5.45 (m, 1 H); 4.20-3.70 (m, 4H); 3.35 (t, 2H); 1.95 (m, 2H); 1.78 (m, 2 H); 1.65-1 .35 (m, 12H); 1.45 (s, 9H); 1 .18 (s, 6H),
13C-RMN (101 MHz, CDCI3): δ 154.32, 133.94, 133.40, 129.26, 128.25, 94.51 , 81 .13, 74.16, 65.03, 62.36, 34.17, 32.90, 32.48, 29.46, 29.36, 29.26, 29.19, 28.96, 28.84, 28.79, 28.47, 28.42, 28.24, 26.35, 24.71. (S)-4-r(RE)-1-hidroxi-14-(metiisuifoniloxi)tetradec-2-enil]-2,2- dimetiioxazoiidina-3-carboxiiato de tere-butilo (3b) 13 C-NMR (101 MHz, CDCI 3 ): δ 154.32, 133.94, 133.40, 129.26, 128.25, 94.51, 81 .13, 74.16, 65.03, 62.36, 34.17, 32.90, 32.48, 29.46, 29.36, 29.26, 29.19, 28.96 , 28.84, 28.79, 28.47, 28.42, 28.24, 26.35, 24.71. (S) -4-r (RE) -1-hydroxy-14- (methisisiphenyloxy) tetradec-2-enyl] -2,2- dimethioxazoiidine-3-carboxyterate tere-butyl (3b)
Rendimiento: 89%Yield: 89%
H-RMN (400 MHz, CDCí3): δ 5.82-5.75 (m, 1 H); 5.45-5.30 (m, 1 H); 4.22 (t, 2H); 4.30-3.80 (m, 4H); 3.05 (s, 3H); 2.10-2.05 (m, 2H); 1.75-1.70 (m, 2H); 1.55-1.45 (s ancho, 15H); 1.70-1.15 (m, 12H) H-NMR (400 MHz, CDCi 3 ): δ 5.82-5.75 (m, 1 H); 5.45-5.30 (m, 1 H); 4.22 (t, 2H); 4.30-3.80 (m, 4H); 3.05 (s, 3H); 2.10-2.05 (m, 2H); 1.75-1.70 (m, 2H); 1.55-1.45 (wide s, 15H); 1.70-1.15 (m, 12H)
(S)-4-f(RE)-12-azido-1-hidroxidodec-2-enil1-2,2-dimetiioxazoiidina-3- carboxilato de terc-butiio (3c) (S) -4-f (RE) -12-azido-1-hydroxidedec-2-enyl1-2,2-dimethioxazoiidine-3-tert-butyl carboxylate (3c)
Una disolución de 122 mg (1.87 mmol) de azida sódica en DMF anhidro (10 mL) se añadió, gota a gota, sobre una disolución del bromuro 3a en 5 mL de DMF, bajo atmósfera de Ar a temperatura ambiente. Finalizada la adición, la mezcla de reacción se calentó a 65° C y se mantuvo en agitación durante 12h. A continuación, ia mezcla se diluyó con 40 mL de agua y se extrajo con Et20 (3 x 15 mL). Las fases orgánicas se lavaron con salmuera (2 x 10 mL), se secaron sobre MgS04 anhidro, se filtraron y se evaporaron para dar un residuo que se purificó por cromatografía flash (Hexano / EtOAc 7:3). Rendimiento: 91 % A solution of 122 mg (1.87 mmol) of sodium azide in anhydrous DMF (10 mL) was added, dropwise, onto a solution of bromide 3a in 5 mL of DMF, under an atmosphere of Ar at room temperature. After the addition, the reaction mixture was heated to 65 ° C and kept under stirring for 12h. Then, the mixture was diluted with 40 mL of water and extracted with Et 2 0 (3 x 15 mL). The organic phases were washed with brine (2 x 10 mL), dried over anhydrous MgSO 4 , filtered and evaporated to give a residue that was purified by flash chromatography (Hexane / EtOAc 7: 3). Yield: 91%
1H-RMN (400 MHz, CDCi3): δ 5.82 - 5.66 (m, 1 H), 5.48 - 5.36 (m, 1 H), 4.22 - 3.75 (m, 4H), 3.25 (t, J = 7.0 Hz, 2H), 2.11 - 1.96 (m, 2H), 1.45 (s, 9H); 1.32 (s, 6H); 1.55-1.35 (m, 18H). 1 H-NMR (400 MHz, CDCi 3 ): δ 5.82 - 5.66 (m, 1 H), 5.48 - 5.36 (m, 1 H), 4.22 - 3.75 (m, 4H), 3.25 (t, J = 7.0 Hz , 2H), 2.11-1.96 (m, 2H), 1.45 (s, 9H); 1.32 (s, 6H); 1.55-1.35 (m, 18H).
3C-RMN (101 MHz, CDCI3): δ 133.51 , 128.29, 94.58, 81.20, 74.25, 65.08, 62.44, 51.61 , 32.53, 29.55, 29.51 , 29.45, 29.31 , 29.28, 29.24, 28.97, 28.51 , 26.84, 26.38, 24.74. 3C-NMR (101 MHz, CDCI 3 ): δ 133.51, 128.29, 94.58, 81.20, 74.25, 65.08, 62.44, 51.61, 32.53, 29.55, 29.51, 29.45, 29.31, 29.28, 29.24, 28.97, 28.51, 26.84, 26.38, 24.74.
Metanosu!fonato de (E)-13-r(1R7aS)-5.5-dimetil-3-oxo-tetrahidro-1 H- oxazolof3,4-c]oxazol-1 -il]tridec-12-enilo (4) Methanesu! (E) -13-r (1R7aS) -5.5-dimethyl-3-oxo-tetrahydro-1 H- oxazolof3,4-c] oxazol-1-yl] tridec-12-enyl (4)
Sobre una disolución de 505 mg (1 mmol) de 3b en THF anhidro (10 ml_), enfriada en un baño de hielo, se añadieron 80 mg (2 mmol) de NaH. Tras agitación a temperatura ambiente durante 18h, ia mezcla de reacción se enfrió en un baño de hielo, se trató con una disolución acuosa saturada de NaHC03 y se extrajo con Et20 (3 x 5 ml_). Los extractos orgánicos reunidos se lavaron con salmuera y se secaron sobre MgS04 anhidro. Tras la evaporación del disolvente se obtuvo el compuesto 4, que se sometió a la etapa posterior sin purificación. On a solution of 505 mg (1 mmol) of 3b in anhydrous THF (10 ml_), cooled in an ice bath, 80 mg (2 mmol) of NaH was added. After stirring at room temperature for 18h, the reaction mixture was cooled in an ice bath, treated with a saturated aqueous solution of NaHC0 3 and extracted with Et 2 0 (3 x 5 ml_). The combined organic extracts were washed with brine and dried over anhydrous MgSO4. After evaporation of the solvent, compound 4 was obtained, which was subjected to the subsequent step without purification.
Rendimiento: 85% Yield: 85%
1H-RMN (400 MHz, CDCI3): δ 5.90-5.85 (m, 1 H); 5.80-5.75 (m, 1 H); 5.05-4.95 (m, 1 H); 4.55 (ancho, 1 H); 4.20 (t, 2H); 3.90-3.75 (m, 2H); 3.15 (s, 3H); 2.05 (m, 2H); 1.55 (s, 3H); 1.48 (s, 3H); 1.50-1.35 (ancho, 18H). 1 H-NMR (400 MHz, CDCI 3 ): δ 5.90-5.85 (m, 1 H); 5.80-5.75 (m, 1 H); 5.05-4.95 (m, 1 H); 4.55 (width, 1 H); 4.20 (t, 2H); 3.90-3.75 (m, 2H); 3.15 (s, 3H); 2.05 (m, 2H); 1.55 (s, 3 H); 1.48 (s, 3 H); 1.50-1.35 (width, 18H).
Metanosuífonato de 13-ΓΠ R7aS)-5,5-dimetil-3-oxo-tetrahidro-1 H-oxazolof3,4- cloxazol-1-illtrideciio (5) 13-ΓΠ R7aS) -5,5-dimethyl-3-oxo-tetrahydro-1 H-oxazolof3,4-cloxazol-1-illtrideciium methanesuifonate (5)
Una disolución de 430 mg (1 mmoi) del compuesto 4 en etanol (10 ml_) se sometió a hidrogenación, a presión atmosférica, en presencia de 10 mg de Pd/C al 10 %. Tras 12 h de agitación a temperatura ambiente, la mezcla de reacción se filtró sobre Celíte y el filtrado obtenido se evaporó a sequedad proporcionando el compuesto 5.  A solution of 430 mg (1 mmoi) of compound 4 in ethanol (10 ml_) was subjected to hydrogenation, at atmospheric pressure, in the presence of 10 mg of 10% Pd / C. After 12 h of stirring at room temperature, the reaction mixture was filtered over Celite and the filtrate obtained was evaporated to dryness to provide compound 5.
Rendimiento: 98% H-RMN (400 MHz, CDCI3): δ 4.62 (m, 1 H); 4.30 (m, 1 H); 4.20 (t, 2H); 3.75- 3.55 (m, 2H); 2.85 (s, 3H); 1.75-1.65 (m, 2H); 1 .55-1.50 (m, 2H); 1.61 (s, 3H); 1.48 (s, 3H); 1.55-1 .25 (ancho, 20H). Síntesis de ios acetilenos 6 y 7 a partir de los mesilatos 4 y 5 Yield: 98% H-NMR (400 MHz, CDCI 3 ): δ 4.62 (m, 1 H); 4.30 (m, 1 H); 4.20 (t, 2H); 3.75-3.55 (m, 2H); 2.85 (s, 3 H); 1.75-1.65 (m, 2H); 1.55-1.50 (m, 2H); 1.61 (s, 3H); 1.48 (s, 3 H); 1.55-1 .25 (width, 20H). Synthesis of ios acetylenes 6 and 7 from mesylates 4 and 5
Etapa 1 : Sobre una disolución de ios mesilatos 4 ó 5 (2 mmol) en 8 mL de una mezcla 1 :1 THF/HMPA (hexametiifosfotríamída) enfriada a -30 °C, se añadieron, gota a gota, 4.5 mL de una disolución 0.5 M de trimetiisililacetiluro de litio en THF (equivalentes a 2.25 mmol). La mezcla de reacción se mantuvo en agitación mientras se calentó lentamente hasta alcanzar la temperatura ambiente. Tras el consumo del producto de partida (alrededor de 1 h, por análisis por TLC), la reacción se detuvo por adición de 5 mL de disolución saturada de NH4Ci y la mezcla se extrajo con hexano (3 x 10 mL). Los extractos orgánicos reunidos se lavaron con agua y salmuera, se secaron sobre MgS04 anhidro y se evaporaron a presión reducida para obtener los correspondientes acetiluros de trimetilsílílo intermedios, que se usaron en la etapa siguiente sin purificación. Step 1: On a solution of the 4 or 5 mesylates (2 mmol) in 8 mL of a 1: 1 THF / HMPA (hexamethiphosphotriamide) mixture cooled to -30 ° C, 4.5 mL of a solution was added dropwise 0.5 M of lithium trimethisysilylacetylide in THF (equivalent to 2.25 mmol). The reaction mixture was kept under stirring while slowly heating to room temperature. After consumption of the starting product (about 1 h, by TLC analysis), the reaction was stopped by adding 5 mL of saturated NH 4 Ci solution and the mixture was extracted with hexane (3 x 10 mL). The combined organic extracts were washed with water and brine, dried over anhydrous MgSO 4 and evaporated under reduced pressure to obtain the corresponding intermediate trimethylsilyl acetylides, which were used in the next step without purification.
Etapa 2: Una disolución del acetíluro de trímetilsililo (1 .5 mmol) en THF anhidro (5 mL) se trató con 2 mL de una disolución 1 M de Bu4NF en THF bajo atmósfera de Ar. Tras agitación durante 30 mín a temperatura ambiente, la mezcla de reacción se trató con H20 (0.5 mL), se secó sobre MgS04 anhidro y se evaporó a presión reducida, obteniéndose los acetilenos 6 y 7. (1 7aS,E)-5,5-dimetil-1 -(pentadec-1 -en-14-inil)-dihidro-1 /-/-oxazoio[3,4- cloxazol-3(5H)-ona (6) Step 2: A solution of trimethylsilyl acetylide (1.5 mmol) in anhydrous THF (5 mL) was treated with 2 mL of a 1 M solution of Bu 4 NF in THF under Ar atmosphere. After stirring for 30 min at room temperature, the reaction mixture was treated with H 2 0 (0.5 mL), dried over anhydrous MgS0 4 and evaporated under reduced pressure, obtaining acetylenes 6 and 7. (1 7aS, E) -5,5-dimethyl-1 - (pentadec-1-en-14-inyl) -dihydro-1 / - / - oxazoium [3,4- cloxazol-3 (5H) -one (6)
Rendimiento: 75% Yield: 75%
1H-RMN (400 MHz, CDCI3): δ 5.95-5.80 (m, 1 H); 5.75-5.70 (m, 1 H); 5.15-5.05 (m, 1 H); 4.65 (ancho, 1 H); 3.85-3.65 (m, 2H); 2.15-1.95 (m, 4H); 1.85 (t, 1 H); 1.50 (s, 3H); 1.45 (s, 3H); 1.50-1.25 (ancho, 18H). (1 RJa$)-5,5-dimetil-1 -(pentadec-14-inil)-dihidro-1 H-oxazoioí3,4-c]oxazoi- 3(5H)-ona (7) 1 H-NMR (400 MHz, CDCI 3 ): δ 5.95-5.80 (m, 1 H); 5.75-5.70 (m, 1 H); 5.15-5.05 (m, 1 H); 4.65 (width, 1 H); 3.85-3.65 (m, 2H); 2.15-1.95 (m, 4H); 1.85 (t, 1 H); 1.50 (s, 3H); 1.45 (s, 3H); 1.50-1.25 (width, 18H). (1 RJa $) - 5,5-dimethyl-1 - (pentadec-14-inyl) -dihydro-1 H-oxazoioí3,4-c] oxazoi- 3 (5H) -one (7)
Rendimiento: 79%Yield: 79%
H-RMN (400 MHz, CDCi3): δ 5.15-5.05 (m, 1 H); 4.65 (ancho, 1 H); 3.85-3.65 (m, 2H); 2.05-1 .95 (ancho, 2H); 1 .85 (t, 1 H); 1 .50 (s, 3H); 1 .45 (s, 3H); 1 .50- 1 .45 (m, 4H); 1 .35-1 .25 (ancho, 20H) H-NMR (400 MHz, CDCi 3 ): δ 5.15-5.05 (m, 1 H); 4.65 (width, 1 H); 3.85-3.65 (m, 2H); 2.05-1 .95 (width, 2H); 1.85 (t, 1 H); 1.50 (s, 3H); 1.45 (s, 3 H); 1 .50-1 .45 (m, 4H); 1 .35-1 .25 (width, 20H)
(S)-4-f(f?)-12-azido-1 -hidroxidodecill-2,2-dimetiloxazolidina-3-carboxilato de terc-butilo (8) (S) -4-f (f?) - 12-azido-1-hydroxydecyl-2,2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester (8)
Etapa 1 : Una disolución de 460 mg (1 mmol) del compuesto 3a en etanol (10 mL) se sometió a hidrogenación, a presión atmosférica, en presencia de 10 mg de Pd/C al 10 %. Tras 12 h de agitación a temperatura ambiente, la mezcla de reacción se filtró sobre Celite y el filtrado obtenido se evaporó a sequedad proporcionando un crudo que se sometió a la reacción siguiente. Step 1: A solution of 460 mg (1 mmol) of compound 3a in ethanol (10 mL) was subjected to hydrogenation, at atmospheric pressure, in the presence of 10 mg of 10% Pd / C. After 12 h of stirring at room temperature, the reaction mixture was filtered over Celite and the filtrate obtained was evaporated to dryness to give a crude which was subjected to the following reaction.
Etapa 2: Una disolución de 100 mg (1 .54 mmol) de azida sódica en DMF anhidro (10 mL) se añadió, gota a gota, sobre una disolución del crudo de la reacción anterior en 5 mL de DMF, bajo atmósfera de Ar a temperatura ambiente. Finalizada la adición, la mezcla de reacción se calentó a 65° C y se mantuvo en agitación durante 12h. A continuación, la mezcla se diluyó con 40 mL de agua y se extrajo con Et20 (3 x 15 mL). Las fases orgánicas se lavaron con salmuera (2 x 10 mL), se secaron sobre MgS04 anhidro, se filtraron y se evaporaron para dar un residuo que se purificó por cromatografía flash (Hexano / EtOAc 7:3). Step 2: A solution of 100 mg (1.54 mmol) of sodium azide in anhydrous DMF (10 mL) was added, dropwise, over a solution of the crude from the previous reaction in 5 mL of DMF, under Ar atmosphere at room temperature. After the addition, the reaction mixture was heated to 65 ° C and kept under stirring for 12h. Then, the mixture was diluted with 40 mL of water and extracted with Et 2 0 (3 x 15 mL). The organic phases were washed with brine (2 x 10 mL), dried over anhydrous MgSO 4 , filtered and evaporated to give a residue that was purified by flash chromatography (Hexane / EtOAc 7: 3).
Rendimiento global : 72 % Overall yield: 72%
1H-RMN (400 MHz, CDCI3): δ 4.15-3.95 (m, 2H); 3.90-3.70 (m, 2H); 3.25 (t, 2H); 1 .45 (s, 9H); 1 .65-1 .60 (m, 4H); 1 .40-1 .20 (m, 22H) 1 H-NMR (400 MHz, CDCI 3 ): δ 4.15-3.95 (m, 2H); 3.90-3.70 (m, 2H); 3.25 (t, 2H); 1.45 (s, 9H); 1.65-1 .60 (m, 4H); 1 .40-1 .20 (m, 22H)
13C-RMN (101 MHz, CDCI3): δ 154.1 , 94.3, 73.0, 64.8, 62.6, 51 .5, 34.5, 33.9, (29.7-28.4), 26.8, 26.5, 26.1 , 24.3. Síntesis de bases esfinqoides il(D-H) 13 C-NMR (101 MHz, CDCI 3 ): δ 154.1, 94.3, 73.0, 64.8, 62.6, 51 .5, 34.5, 33.9, (29.7-28.4), 26.8, 26.5, 26.1, 24.3. Synthesis of sphinctoid bases il (DH)
(2S,3R)-2-amino-14-azidotetradecano-1 ,3-diol (li-E) y (2S,3 E)-2-amino-14- azidotetradec-4-eno-1 ,3-dioi (H-F) (2S, 3R) -2-amino-14-azidotetradecane-1, 3-diol (li-E) and (2S, 3 E) -2-amino-14-azidotetradec-4-eno-1, 3-dioi ( HF)
Una disolución de (0.5 mmol) de 8 o de 3c en MeOH (10 mL) se trató con cloruro de acetilo (0.6 mL). La mezcla de reacción se agitó a temperatura ambiente durante 6 h, transcurridas las cuales se evaporó el disolvente a presión reducida. El residuo obtenido se disuelve en agua, se alcalinizó con una disolución de NaOH 1 N y se extrajo con EtOAc (3 x 5 mL). Las fases orgánicas reunidas se secaron (MgSO4) y se evaporaron a sequedad y el residuo se purificó por cromatografía flash (hexano / EtOAc 7:3) para obtener el producto deseado. A solution of (0.5 mmol) of 8 or 3c in MeOH (10 mL) was treated with acetyl chloride (0.6 mL). The reaction mixture was stirred at room temperature for 6 h, after which the solvent was evaporated under reduced pressure. The obtained residue is dissolved in water, made alkaline with a solution of 1 N NaOH and extracted with EtOAc (3 x 5 mL). The combined organic phases were dried (MgSO 4 ) and evaporated to dryness and the residue was purified by flash chromatography (hexane / EtOAc 7: 3) to obtain the desired product.
(li-E): Rendimiento a partir de 8 : 82 % (li-E): Yield from 8: 82%
1H-RMN (400 MHz, CDCI3): δ 3.70 (m, 2H); 3.60 (m, 1 H); 3.25 (t, 2H); 2.85 (m, 1 H); 1.65-1.55 (m, 2H); 1.53-1.45 (m, 3H); 1.40-1.20 (m, 15H). 1 H-NMR (400 MHz, CDCI 3 ): δ 3.70 (m, 2H); 3.60 (m, 1 H); 3.25 (t, 2H); 2.85 (m, 1 H); 1.65-1.55 (m, 2H); 1.53-1.45 (m, 3H); 1.40-1.20 (m, 15H).
13C-RMN (101 MHz, CDCI3): δ 74.61 , 63.50, 55.80, 51.57, 33.95, 29.80, 29.71 , 29.69, 29.63, 29.60, 29.58, 29.26, 28.93, 26.81 , 26.23. (H-F): Rendimiento a partir de 3c : 85 % 1 3 C-NMR (101 MHz, CDCI 3 ): δ 74.61, 63.50, 55.80, 51.57, 33.95, 29.80, 29.71, 29.69, 29.63, 29.60, 29.58, 29.26, 28.93, 26.81, 26.23. (HF): Performance from 3c: 85%
1H-RMN (400 MHz, CDCI3): δ 5.80-5.65 (m, 1 H); 5.50-5.35 (m. 1 H); 4.05 (m, 1 H); 3.70-3.55 (m, 2H); 3.25 (t, 2H); 2.85-2.75 (m, 1 H); 2.05 (m, 2H); 1.55 (m, 2H); 1.40-1.20 (m, 13H). 1 H-NMR (400 MHz, CDCI 3 ): δ 5.80-5.65 (m, 1 H); 5.50-5.35 (m. 1 H); 4.05 (m, 1 H); 3.70-3.55 (m, 2H); 3.25 (t, 2H); 2.85-2.75 (m, 1 H); 2.05 (m, 2H); 1.55 (m, 2H); 1.40-1.20 (m, 13H).
13C-RMN (101 MHz, CDCI3): δ 134.63, 129.37, 75.20, 63.81 , 56.25, 51.57, 32.45, 29.52, 29.46, 29.40, 29.30, 29.24, 28.93, 26.80. 13 C-NMR (101 MHz, CDCI 3 ): δ 134.63, 129.37, 75.20, 63.81, 56.25, 51.57, 32.45, 29.52, 29.46, 29.40, 29.30, 29.24, 28.93, 26.80.
(2S,3R)-2-aminooctadec-17-in-1.3-diol (H-P) y (2S,3 E)-2-aminooctadec-4- en-17-in-1 ,3-diol (li-G) por hidrólisis de 6 y 7 (2S, 3R) -2-aminooctadec-17-in-1.3-diol (HP) and (2S, 3 E) -2-aminooctadec-4- in-17-in-1, 3-diol (li-G) by hydrolysis of 6 and 7
Etapa 1. Una disolución de 0.6 mmol de 6 ó 7 y TsOH (12 mg, 0.06 mmol) en MeOH (10 mL) se agitó a 25 °C durante 6 h. Transcurrido este tiempo, se eliminó el disolvente a presión reducida y el residuo resultante se disolvió en EtOAc. La disolución orgánica se lavó, sucesivamente, con una disolución saturada de NaHC03 y salmuera y se secó. La evaporación del disolvente proporciona un residuo que se usó directamente en la siguiente etapa de síntesis. Step 1. A solution of 0.6 mmol of 6 or 7 and TsOH (12 mg, 0.06 mmol) in MeOH (10 mL) was stirred at 25 ° C for 6 h. After this time, the solvent was removed under reduced pressure and the resulting residue was dissolved in EtOAc The organic solution was washed, successively, with a saturated solution of NaHC03 and brine and dried. Evaporation of the solvent provides a residue that was used directly in the next stage of synthesis.
Etapa 2. Una disolución del crudo anterior en 2N NaOH (30 mL) y EtOH (30 mL) se calentó a 80 °C durante 3 h. La mezcla de reacción se enfrió a continuación, se concentra, a presión reducida, hasta una cuarta parte del volumen y se extrajo con Et2Ü (3 x 15 mL). Las fases orgánicas reunidas se secaron sobre MgS04, se filtraron y se evaporaron para dar el correspondiente producto final. Step 2. A solution of the above crude in 2N NaOH (30 mL) and EtOH (30 mL) was heated at 80 ° C for 3 h. The reaction mixture was then cooled, concentrated, under reduced pressure, to a quarter of the volume and extracted with Et2Ü (3 x 15 mL). The combined organic phases were dried over MgSO 4 , filtered and evaporated to give the corresponding final product.
(li-D): Rendimiento global a partir de 7: 70%(li-D): Overall yield from 7: 70%
H-RMN (400 MHz, CDCI3): δ 3.990-3.65 (m, 2H); 3.45 (m, 1 H); 2.82 (m, 1 H); 2.01 (m, 2H); 1.82 (t, 1 H); 1.45 (m, 4H); 1.29-1.26 (m, 20H). H-NMR (400 MHz, CDCI 3 ): δ 3,990-3.65 (m, 2H); 3.45 (m, 1 H); 2.82 (m, 1 H); 2.01 (m, 2H); 1.82 (t, 1 H); 1.45 (m, 4H); 1.29-1.26 (m, 20H).
13C-RMN (101 MHz, CDCI3): δ 83.5, 75.1 , 62.5, 61.1 , 33.1 , (29.9-28.2), 23.5, 21.4. 13 C-NMR (101 MHz, CDCI 3 ): δ 83.5, 75.1, 62.5, 61.1, 33.1, (29.9-28.2), 23.5, 21.4.
(Il-G): Rendimiento global a partir de 6: 73% (Il-G): Overall yield from 6: 73%
1H-RMN (400 MHz, CDCI3): δ 5.69-5.65 (m, 2H); 4.15 (m, 1 H); 3.90-3.65 (m, 2H); 2.85 (m, 1 H); 2.05-1.95 (m, 4H); 1.85 (t, 1 H); 1.45-1.30 (m, 18H).1 H-NMR (400 MHz, CDCI 3 ): δ 5.69-5.65 (m, 2H); 4.15 (m, 1 H); 3.90-3.65 (m, 2H); 2.85 (m, 1 H); 2.05-1.95 (m, 4H); 1.85 (t, 1 H); 1.45-1.30 (m, 18H).
3C-RMN (101 MHz, CDCI3): δ 131.1 , 129.7, 83.5, 75.5, 68.9, 62.3, 56.8, 35.1 , (29.9-28.4), 21.2. Hidrocioruro de (S)-2-amino-14-azido-1-hidroxitetradecan-3-ona (li-H) 3C-NMR (101 MHz, CDCI 3 ): δ 131.1, 129.7, 83.5, 75.5, 68.9, 62.3, 56.8, 35.1, (29.9-28.4), 21.2. (S) -2-amino-14-azido-1-hydroxytetradecan-3-one (li-H) hydrochloride
Etapa 1. Una disolución de 550 mg (1.30 mmol) de 8 en 30 mL de CH2CI2 se trató con clorocromato de piridinio (PCC) (420 mg, 1.90 mmol). La mezcla de reacción se mantuvo en agitación a temperatura ambiente durante 1 h, transcurrida la cual se añadieron otros 420 mg de PCC y se mantuvo en agitación durante 12 h más. A continuación, la mezcla de reacción se filtró sobre Celite y el filtrado se evaporó a presión reducida para dar un crudo que se utilizó directamente en la siguiente etapa. Step 1. A solution of 550 mg (1.30 mmol) of 8 in 30 mL of CH 2 CI 2 was treated with pyridinium chlorochromate (PCC) (420 mg, 1.90 mmol). The reaction mixture was kept under stirring at room temperature for 1 h, after which another 420 mg of PCC was added and kept under stirring for an additional 12 h. Then the reaction mixture was filtered. on Celite and the filtrate was evaporated under reduced pressure to give a crude that was used directly in the next step.
Etapa 2. Una disolución del crudo anterior en 20 mL de MeOH se trató con 1.2 mL de cloruro de acetiio a 25 °C durante 6 h. Transcurrido este tiempo, se eliminó el disolvente a presión reducida para dar el producto final en forma del correspondiente hídrocloruro. Step 2. A solution of the above crude in 20 mL of MeOH was treated with 1.2 mL of acetiium chloride at 25 ° C for 6 h. After this time, the solvent was removed under reduced pressure to give the final product as the corresponding hydrochloride.
Rendimiento global a partir de 8: 89 %Overall yield from 8: 89%
H-RMN (400 MHz, CD3OD): δ 4.15 (t ancho, 1 H); 4.10 (dd, 1 H); 3.95 (dd, 1 H), 3.3 (t, 2H); 2.65 (t, 2H); 1.60-1.55 (m, 2H); 1.45-1.25 (ancho, 16H). H-NMR (400 MHz, CD 3 OD): δ 4.15 (broad t, 1 H); 4.10 (dd, 1 H); 3.95 (dd, 1 H), 3.3 (t, 2H); 2.65 (t, 2H); 1.60-1.55 (m, 2H); 1.45-1.25 (width, 16H).
13C-RMN (101 MHz, CD3OD): δ 205.19, 62.19, 60.26, 52.44, 39.62, 30.62, 30.60, 30.57, 30.52, 30.27, 30.09, 29.92, 27.82, 24.18. 13 C-NMR (101 MHz, CD 3 OD): δ 205.19, 62.19, 60.26, 52.44, 39.62, 30.62, 30.60, 30.57, 30.52, 30.27, 30.09, 29.92, 27.82, 24.18.
Obtención de los compuestos de fórmula (I). Obtaining the compounds of formula (I).
Ejemplo 1-B4. 1-K2S.3ffl-1 ,3-dihidroxioctadecan-2-ii1-1H-pirrol-2,5-diona Example 1-B4. 1-K2S.3ffl-1, 3-dihydroxyoctadecan-2-ii1-1H-pyrrole-2,5-dione
Una disolución de esfinganina (30 mg, 0,10 mmol) en CH2CI2 (1 mL) se adicionó, gota a gota, sobre 1 mL de una disolución de anhídrido maleíco 0.1 M en CH2CI2, enfriada exteriormente con un baño de hielo. A continuación, se retiró el baño y la mezcia se mantuvo en agitación a temperatura ambiente durante 16 h, transcurridas las cuales se adicionó cloruro de oxaliío (8.5 pL, 0,1 mmoi) y una gota de DMF, manteniendo ia agitación durante otras 8 h. Al final de este periodo, la mezcla de reacción se trató con 0,5 mL de una disolución 0,2 M de Et3N en de CH2CI2 y se agitó a temperatura ambiente. Al cabo de 1 h, ía mezcia de reacción se íavó con agua (3 x 2,5 mL) y ia fase orgánica se secó sobre MgS04 anhidro y se evaporó a presión reducida. El crudo resultante se purificó por cromatografía flash (hexano/EtOAc 4:1 ), obteniéndose 32 mg (81 % rendimiento) del compuesto I-B4. A solution of sphinganine (30 mg, 0.10 mmol) in CH 2 CI 2 (1 mL) was added dropwise over 1 mL of a 0.1 M maleic anhydride solution in CH 2 CI 2 , cooled externally with a ice bath Then, the bath was removed and the mixture was kept under stirring at room temperature for 16 h, after which oxaliium chloride (8.5 pL, 0.1 mmoi) and a drop of DMF were added, maintaining the stirring for another 8 h. At the end of this period, the reaction mixture was treated with 0.5 mL of a 0.2 M solution of Et 3 N in CH 2 CI 2 and stirred at room temperature. After 1 h, the reaction mixture was washed with water (3 x 2.5 mL) and the organic phase was dried over anhydrous MgSO 4 and evaporated under reduced pressure. The resulting crude was purified by flash chromatography (hexane / EtOAc 4: 1), yielding 32 mg (81% yield) of compound I-B4.
1H-RMN (400 MHz, CDCI3): δ 6.94 (s, 2H), 3.75 - 3.68 (m, 1 H), 3.87 - 3.80 (m, 3H), 1.65 - 1.45 (m, 4H), 1.25 (ancho, 24H), 0.85 (t ancho, 3H). 3C-RMN (101 MHz, CDCI3): 170.25, 135.84, 69.06, 58.21 , 51.53, 33.71 , 32.13, (29.90 - 29.60), 26.04, 22.91 , 14.35. 1H-NMR (400 MHz, CDCI 3 ): δ 6.94 (s, 2H), 3.75 - 3.68 (m, 1 H), 3.87 - 3.80 (m, 3H), 1.65 - 1.45 (m, 4H), 1.25 (wide , 24H), 0.85 (wide t, 3H). 3 C-NMR (101 MHz, CDCI 3 ): 170.25, 135.84, 69.06, 58.21, 51.53, 33.71, 32.13, (29.90 - 29.60), 26.04, 22.91, 14.35.
Método genera! para ¡a preparación de sulfonamidas por acoplamiento con cloruro de etanosuifoniio Method generates! for the preparation of sulfonamides by coupling with ethanesuifoniium chloride
Sobre una disolución de la correspondiente base esfingoide II (0,10 mmol) y Na2C03 (21 mg, 0,20 mmol) en THF (1 ml_) y agua (2 ml_) se adicionaron, gota a gota, 14 μΙ_, (0,15 mmol) de cloruro de etanosuifonilo. La mezcla de reacción se mantuvo en agitación durante 18h a temperatura ambiente. A continuación, se diluyó con 5 mL de disolución saturada de NaCI, se extrajo con CH2CI2 (3 x 5 mL), se secó sobre MgS04 anhidro y se evaporó a presión reducida. El residuo resultante se purificó por cromatografía flash (hexano/EtOAc 4:1 ) para proporcionar el compuesto deseado. Ejemplo I-A1. A/-f(2S,3 E)-1 ,3-dihidroxioctadec-4-en-2-¡netanosulfonamida Rendimiento: 75% On a solution of the corresponding sphingoid base II (0.10 mmol) and Na 2 C0 3 (21 mg, 0.20 mmol) in THF (1 ml_) and water (2 ml_) were added dropwise, 14 μΙ_ , (0.15 mmol) of ethanesuifonyl chloride. The reaction mixture was kept under stirring for 18h at room temperature. It was then diluted with 5 mL of saturated NaCl solution, extracted with CH 2 CI 2 (3 x 5 mL), dried over anhydrous MgSO 4 and evaporated under reduced pressure. The resulting residue was purified by flash chromatography (hexane / EtOAc 4: 1) to provide the desired compound. Example I-A1 A / -f (2S, 3 E) -1, 3-dihydroxyoctadec-4-en-2-netanesulfonamide Yield: 75%
1H-RMN (400 MHz, CDCI3): δ 5.69 (m, 1 H); 5.65 (m, 1 H); 4.15 (m, 1 H); 3.80-3.60 (m, 2H); 3.50 (q, 2H); 2.90 (m, 1 H); 2.01 (m, 2H); 1.29 (s, 3H); 1.35-1.26 (m, 22H); 0.86 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 5.69 (m, 1 H); 5.65 (m, 1 H); 4.15 (m, 1 H); 3.80-3.60 (m, 2H); 3.50 (q, 2H); 2.90 (m, 1 H); 2.01 (m, 2H); 1.29 (s, 3 H); 1.35-1.26 (m, 22H); 0.86 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 131.5, 129.9, 72.1 , 59.5, 54.3, 48.8, 34.1 , 32.2, (30.0-29.5), 22.7, 14.1 , 2.3. 1 3 C-NMR (101 MHz, CDCI 3 ): δ 131.5, 129.9, 72.1, 59.5, 54.3, 48.8, 34.1, 32.2, (30.0-29.5), 22.7, 14.1, 2.3.
Ejemplo I-B1. A/-f(2S,3R)-1 ,3-dihidroxioctadecan-2-illetanosuifonamida Example I-B1. A / -f (2S, 3R) -1, 3-dihydroxyoctadecan-2-ylletanosuifonamide
Rendimiento: 77% Yield: 77%
1H-RMN (400 MHz, CDCI3): δ 3.45 (q ancho, 2H), 3.97 - 3.70 (m, 3H), 2.78 (m, 1 H), 1.68 - 1.41 (m, 4H), 1.28 (t ancho, 3H), 1.23 (ancho, 24H), 0.85 (t ancho, 3H). 1H-NMR (400 MHz, CDCI 3 ): δ 3.45 (q width, 2H), 3.97 - 3.70 (m, 3H), 2.78 (m, 1 H), 1.68 - 1.41 (m, 4H), 1.28 (t wide , 3H), 1.23 (width, 24H), 0.85 (t width, 3H).
13C-RMN (101 MHz, CDCI3): δ 75.06, 60.61 , 54.83, 47.56, 33.91 , 31.93, (29.95 - 26.04), 22.81 , 14.25, 2.35. Ejemplo 1-C1. N-\(2S,3SAR)- ,3,4-trihidroxioctadecan-2-il]etanosulfonamida Rendimiento: 79% 13 C-NMR (101 MHz, CDCI 3 ): δ 75.06, 60.61, 54.83, 47.56, 33.91, 31.93, (29.95 - 26.04), 22.81, 14.25, 2.35. Example 1-C1. N - \ (2S, 3SAR) -, 3,4-trihydroxyoctadecan-2-yl] ethanesulfonamide Yield: 79%
1H-RMN (400 MHz, CDCI3): δ 3.90-3.60 (m, 2H); 3.50 (m, 1 H); 3.45 (q, 2H); 3.18 (m, 1 H); 2.75 (m, 1 H); 1.51 (m, 2H); 1.30 (s, 3H); 1.30-1.26 (s ancho, 24H); 0.86 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 3.90-3.60 (m, 2H); 3.50 (m, 1 H); 3.45 (q, 2H); 3.18 (m, 1 H); 2.75 (m, 1 H); 1.51 (m, 2H); 1.30 (s, 3H); 1.30-1.26 (wide s, 24H); 0.86 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 76.5, 71.3, 59.6, 48.4, 47.2, 31.9, 31.2, (29.9- 29.3), 23.7, 22.6, 14.2, 2.3. 13 C-NMR (101 MHz, CDCI 3 ): δ 76.5, 71.3, 59.6, 48.4, 47.2, 31.9, 31.2, (29.9-29.3), 23.7, 22.6, 14.2, 2.3.
Ejemplo I-D1. A/-f(2S,3 ?)-1 ,3-dihidroxioctadec-17-in-2-illetanosulfonamida Rendimiento: 84% Example I-D1. A / -f (2S, 3?) - 1, 3-dihydroxyoctadec-17-in-2-ylletanesulfonamide Yield: 84%
1H-RMN (400 MHz, CDCi3): δ 3.90-3.70 (m, 3H); 3.45 (q, 2H); 2.80 (m, 1 H); 2.03 (m, 2H); 1 .85 (t, J = 2.5, 1 H); 1 .28 (s, 3H); 1.20-1.40 (s ancho, 24H). 1 H-NMR (400 MHz, CDCi 3 ): δ 3.90-3.70 (m, 3H); 3.45 (q, 2H); 2.80 (m, 1 H); 2.03 (m, 2H); 1.85 (t, J = 2.5, 1 H); 1.28 (s, 3 H); 1.20-1.40 (wide s, 24H).
3C-RMN (101 MHz, CDCi3): δ 83.5; 72.5; 47.6, 69.1 ; 60.2; 58.4; 32.9; (29.5-28.5); 23.4; 21.3, 2.3. 3C-NMR (101 MHz, CDCi 3 ): δ 83.5; 72.5; 47.6, 69.1; 60.2; 58.4; 32.9; (29.5-28.5); 23.4; 21.3, 2.3.
Ejemplo I-E1. /V-K2S.3ffl-14-azido-1 ,3-dihidroxitetradecan-2- iDetanosulfonamida Example I-E1. /V-K2S.3ffl-14-azido-1, 3-dihydroxytetradecan-2- iDetanesulfonamide
Rendimiento: 71 %Yield: 71%
H-RMN (400 MHz, CDCI3): δ 3.80-3.60 (m, 2H); 3.45 (m, 1 H); 3.40 (q, 2H); 2.80 (m, 1 H); 1.45 (m, 2H); 1.30-1.25 (m, 20H). H-NMR (400 MHz, CDCI 3 ): δ 3.80-3.60 (m, 2H); 3.45 (m, 1 H); 3.40 (q, 2H); 2.80 (m, 1 H); 1.45 (m, 2H); 1.30-1.25 (m, 20H).
13C-RMN (101 MHz, CDCi3): δ 71.5, 60.1 , 55.3, 50.2, 48.7, 33.1 , 30.2, (29.9- 29.2); 27.1 , 23.2, 2.3. 13 C-NMR (101 MHz, CDCi 3 ): δ 71.5, 60.1, 55.3, 50.2, 48.7, 33.1, 30.2, (29.9-29.2); 27.1, 23.2, 2.3.
Método general para ia preparación de amidas por acopiamiento con ácidos carboxílicos General method for the preparation of amides by coupling with carboxylic acids
Sobre una disolución de la correspondiente base esfingoide li (0,10 mmol) y Et3N (30 μΙ_, aproximadamente 0,20 mmoí) en CH2CI2 (1 ml_) bajo atmósfera de argón, se añadió, gota a gota, una disolución del correspondiente ácido carboxílico (0,10 mmoí), HOBt (18 mg, 0,13 mmol) y EDC (20 mg, 0,13 mmoí) en CH2CI2 (1 ml_). La mezcla de reacción se agitó a temperatura ambiente durante 1 h, se lavó con agua (3 x 0,5 ml_) y se concentró a presión reducida. El crudo resultante se purificó por cromatografía fíash, utilizando un gradiente CH2CÍ2/MeOH (de 0 a 5%), obteniéndose las correspondientes amidas con ios rendimientos que se indican en cada ejemplo Acoplamiento con ácido bromoacético On a solution of the corresponding sphingoid base li (0.10 mmol) and Et 3 N (30 μΙ_, approximately 0.20 mmoi) in CH2CI2 (1 ml_) under argon, a solution of the solution was added dropwise corresponding carboxylic acid (0.10 mmoi), HOBt (18 mg, 0.13 mmol) and EDC (20 mg, 0.13 mmoi) in CH2CI2 (1 ml_). The reaction mixture was stirred at room temperature for 1 h, washed with water (3 x 0.5 ml_) and concentrated under reduced pressure. The resulting crude was purified by fiash chromatography using a gradient CH2 CI2 / MeOH (0 to 5%) to give the corresponding amides with ios yields indicated in each example Coupling with bromoacetic acid
Ejemplo 1-D2. 2-bromo-A/-f(2S,3/:?)-1 ,3-dihidroxioctadec-17-in-2-iilacetamida Rendimiento: 72% Example 1-D2. 2-bromo-A / -f (2S, 3 /?) - 1, 3-dihidroxioctadec-17-yn-2-iilacetamida Yield: 72%
1H-RMN (400 MHz, CDCI3): δ 8.05 (d ancho, 1 H); 4.21 (s, 2H); 3.90-3.70 (m, 4H); 2.03 (m, 2H); 1.85 (t, J = 2.5, 1 H); 1.20-1.40 (s ancho, 24H). 1 H-NMR (400 MHz, CDCI 3 ): δ 8.05 (broad d, 1 H); 4.21 (s, 2H); 3.90-3.70 (m, 4H); 2.03 (m, 2H); 1.85 (t, J = 2.5, 1 H); 1.20-1.40 (wide s, 24H).
13C-RMN (101 MHz, CDCI3): δ 177.5; 83.5; 72.5; 69.1 ; 60.2; 58.4; 33.1 ; (29.5-28.5); 23.4; 21.3 1 3 C-NMR (101 MHz, CDCI 3 ): δ 177.5; 83.5; 72.5; 69.1; 60.2; 58.4; 33.1; (29.5-28.5); 23.4; 21.3
Ejemplo A/-í(2S,3ffl-14-azido-1.3-dihidroxitetradecan-2- illbromoacetamida Example A / -i (2S, 3ffl-14-azido-1,3-dihydroxytetradecan-2- illbromoacetamide
Rendimiento: 77% Yield: 77%
1H-RMN (400 MHz, CDCI3): δ 7.7 (d ancho, 1 H); 4.10 (s, 2H); 3.90-3.75 (m, 4H); 3.25 (t, 2H); 1 .3-1.25 (ancho, 20H). 1 H-NMR (400 MHz, CDCI 3 ): δ 7.7 (broad d, 1 H); 4.10 (s, 2H); 3.90-3.75 (m, 4H); 3.25 (t, 2H); 1 .3-1.25 (width, 20H).
3C-RMN (101 MHz, CDCI3): δ 166.3, 74.2, 62.2, 53.9, 51.6, 42.8, 34.6, 29.6, 29.3, 28.9, 26.8, 26.0. 3C-NMR (101 MHz, CDCI 3 ): δ 166.3, 74.2, 62.2, 53.9, 51.6, 42.8, 34.6, 29.6, 29.3, 28.9, 26.8, 26.0.
Ejemplo I-F2. A/-f(2S,3 £)-14-azido-1 ,3-dihidroxitetradec-4-en-2- illbromoacetamida Example I-F2. A / -f (2S, £ 3) -14-azido-1, 3-dihydroxytetradec-4-en-2- illbromoacetamide
Rendimiento: 73%  Yield: 73%
1H-RMN (400 MHz, CDCI3): δ 7.72 (d ancho, 1 H), 5.81 (m, 1 H); 5.53 (m, 1 H); 4.35 (m, 1 H); 4.10 (s, 2H); 4.01 (dd, 1 H); 3.90 (m, 1 H), 3.73 (dd, 1 H); 3.25 (t, 2H); 2.10 (m, 2H); 1.65 (m, 2H); 1.40-1.25 (s ancho, 14H). 1 H-NMR (400 MHz, CDCI 3 ): δ 7.72 (broad d, 1 H), 5.81 (m, 1 H); 5.53 (m, 1 H); 4.35 (m, 1 H); 4.10 (s, 2H); 4.01 (dd, 1 H); 3.90 (m, 1 H), 3.73 (dd, 1 H); 3.25 (t, 2H); 2.10 (m, 2H); 1.65 (m, 2H); 1.40-1.25 (wide s, 14H).
3C-RMN (101 MHz, CDCI3): δ 166.6, 134.8, 128.6, 77.5, 77.2, 76.8, 74.2, 62.0, 54.8, 51 .6, 42.8, 32.4, 29.5, 29.5, 29.2, 29.2, 28.9, 26.8. Ejemplo I-G2. 2-bromo-A/-f(2S.3RE)-1 ,3-dihidroxioctadec-4-en-17-in-2- illacetamida 3C-NMR (101 MHz, CDCI 3 ): δ 166.6, 134.8, 128.6, 77.5, 77.2, 76.8, 74.2, 62.0, 54.8, 51 .6, 42.8, 32.4, 29.5, 29.5, 29.2, 29.2, 28.9, 26.8. Example I-G2. 2-Bromo-A / -f (2S.3RE) -1, 3-dihydroxioctadec-4-en-17-in-2- illacetamide
Rendimiento: 84% Yield: 84%
H-RMN (400 MHz, CDCi3): δ 8.10 (d ancho, 1 H); 5.71 -5.68 (m, 2H); 4.60 (m, 1 H); 3.90-3.70 (m, 3H); 2.10-1.95 (m, 4H); 1 .82 (t, J = 2.5, 1 H); 1 .45-1 .30 (m, 18H). H-NMR (400 MHz, CDCi 3 ): δ 8.10 (broad d, 1 H); 5.71-5.68 (m, 2H); 4.60 (m, 1 H); 3.90-3.70 (m, 3H); 2.10-1.95 (m, 4H); 1.82 (t, J = 2.5, 1 H); 1 .45-1 .30 (m, 18H).
13C-RMN (101 MHz, CDCI3): δ 177.5; 131 .1 ; 129.7; 83.7; 72.8; 69.1 ; 60.0; 53.7; 34.1 ; (30.0-28.5); 21 .4 Ejemplo 1-H2. ( S)-N-( 14-azido-1 -hidroxi-3-oxotetradecan-2-i0-2- bromoacetamida 13 C-NMR (101 MHz, CDCI 3 ): δ 177.5; 131 .1; 129.7; 83.7; 72.8; 69.1; 60.0; 53.7; 34.1; (30.0-28.5); 21 .4 Example 1-H2. (S) -N- (14-azido-1-hydroxy-3-oxotetradecan-2-i0-2- bromoacetamide
Rendimiento: 81 %Yield: 81%
H-RMN (400 MHz, CDCI3): δ 4.20 (s, 2H), 4.16 (d, J = 3.8 Hz, 2H), 4.10 (dd, J = 12.0, 4.3 Hz, 2H), 3.97 (dd, J = 12.0, 3.4 Hz, 2H), 2.64 (t, J = 7.2 Hz, 4H), 1 .60 (dd, J = 16.4, 8.9 Hz, 9H), 1 .32 (s, 34H). H-NMR (400 MHz, CDCI 3 ): δ 4.20 (s, 2H), 4.16 (d, J = 3.8 Hz, 2H), 4.10 (dd, J = 12.0, 4.3 Hz, 2H), 3.97 (dd, J = 12.0, 3.4 Hz, 2H), 2.64 (t, J = 7.2 Hz, 4H), 1 .60 (dd, J = 16.4, 8.9 Hz, 9H), 1 .32 (s, 34H).
13C-RMN (101 MHz, CDCI3): δ 205.2, 178.5, 62.19, 60.26, 52.44, 39.62, 30.62, 30.60, 30.57, 30.52, 30.27, 30.09, 28.7, 29.4, 29.92, 27.82, 24.18. 13 C-NMR (101 MHz, CDCI 3 ): δ 205.2, 178.5, 62.19, 60.26, 52.44, 39.62, 30.62, 30.60, 30.57, 30.52, 30.27, 30.09, 28.7, 29.4, 29.92, 27.82, 24.18.
Ejemplo 1-B2. 2-bromo-N-((2S,3R)-1 ,3-dihidroxíoctadecan-2-íl)acetamida. Según procedimiento de síntesis descrito en la solicitud de patente WO200650264. Ejemplo 1. Example 1-B2. 2-Bromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide. According to the synthesis procedure described in patent application WO200650264. Example 1.
Acoplamiento con ácido dibromoacético Dibromoacetic Acid Coupling
Ejemplo 1-B17. 2,2-dibromo-N-((2S,3R)-1 ,3-dihidroxioctadecan-2-i0acetamida. Rendimiento: 77%  Example 1-B17. 2,2-dibromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan-2-10-acetamide. Yield: 77%
1H-RMN (400 MHz, CDCi3): δ 6.23 (s, 1 H), 3.80-3.70 (m, 3H), 3.65 (t ancho, 1 H), 1 .55 (m. 2H), 1 .40-1 .20 (s ancho, 26H), 0.91 (t, 3H). 1 H-NMR (400 MHz, CDCi 3 ): δ 6.23 (s, 1 H), 3.80-3.70 (m, 3H), 3.65 (wide t, 1 H), 1.55 (m. 2H), 1. 40-1 .20 (wide s, 26H), 0.91 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 166.9, 71 .9, 61 .7, 57.6, 37.9, 34.9, 33.1 , 30.9-30.6, 30.5, 26.6, 23.7, 14.4. Acopiamiento con ácido qiicídico 1 3 C-NMR (101 MHz, CDCI 3 ): δ 166.9, 71 .9, 61 .7, 57.6, 37.9, 34.9, 33.1, 30.9-30.6, 30.5, 26.6, 23.7, 14.4. Coupling with qiicidic acid
Ejemplo 1-A3. (f?S)-/V-f(2S,3R£)-1.3-dihidroxioctadec-4-en-2-illoxirano-2- carboxamida  Example 1-A3. (f? S) - / V-f (2S, £ 3R) -1.3-dihydroxyoctadec-4-en-2-illoxyran-2-carboxamide
Rendimiento: 77% Yield: 77%
H-RMN (400 MHz, CDCI3): δ 8.10 (d ancho, 1 H); 5.69-5.67 (m, 2H); 4.62 (m, 1 H); 3.80-3.60 (m, 3H); 3.50 (m, 1 H); 2.90-2.60 (m, 2H); 2.01 (m, 2H); 1.33- 1.26 (m, 22H); 0.85 (t, 3H). H-NMR (400 MHz, CDCI 3 ): δ 8.10 (broad d, 1 H); 5.69-5.67 (m, 2H); 4.62 (m, 1 H); 3.80-3.60 (m, 3H); 3.50 (m, 1 H); 2.90-2.60 (m, 2H); 2.01 (m, 2H); 1.33-1.26 (m, 22H); 0.85 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 173.5, 131.1 , 129.7, 73.1 , 60.2, 56.2, 55.4, 48.1 , 35.1 , 32.2, (29.9-29.3), 22.8, 14.2. 13 C-NMR (101 MHz, CDCI 3 ): δ 173.5, 131.1, 129.7, 73.1, 60.2, 56.2, 55.4, 48.1, 35.1, 32.2, (29.9-29.3), 22.8, 14.2.
Ejemplo 1-B3. (RS)-/V-Í(2S,3R)-1 ,3-dihidroxioctadecan-2-illoxirano-2- carboxamida Example 1-B3. (RS) - / V-Í (2S, 3R) -1, 3-dihydroxyoctadecan-2-illoxyran-2-carboxamide
Rendimiento: 79% Yield: 79%
H-RMN (400 MHz, CDCI3): δ 8.10 (d ancho, 1 H); 3.80-3.60 (m, 4H); 3.52 (m, 1 H); 2.90-2.60 (m, 2H); 1.50 (m, 2H); 1.30-1.25 (m, 26H); 0.85 (t, 3H). H-NMR (400 MHz, CDCI 3 ): δ 8.10 (broad d, 1 H); 3.80-3.60 (m, 4H); 3.52 (m, 1 H); 2.90-2.60 (m, 2H); 1.50 (m, 2H); 1.30-1.25 (m, 26H); 0.85 (t, 3H).
13C-RMN (101 MHz, CDCí3): δ 175.5, 72.4, (59.9-59.8), 56.2, 48.4, 32.8, 31.7, (29.6-29.3), 23.4, 22.8, 14.1. 13 C-NMR (101 MHz, CDCí 3 ): δ 175.5, 72.4, (59.9-59.8), 56.2, 48.4, 32.8, 31.7, (29.6-29.3), 23.4, 22.8, 14.1.
Ejemplo 1-C3. (RS)-N-r(2S,3S,4 )-1 ,3,4-trihidroxioctadecan-2-il1oxirano-2- carboxamida Example 1-C3. (RS) -N-r (2S, 3S, 4) -1, 3,4-trihydroxyoctadecan-2-yl-oxoxyran-2-carboxamide
Rendimiento: 72% Yield: 72%
H-RMN (400 MHz, CDCi3): δ 8.05 (d ancho, 1 H); 3.95-3.60 (m, 4H); 3.51 (m, 1 H); 3.20 (m, 1 H); 2.90-2.65 (m, 2H); 1.45 (m, 2H); 1.30-1.25 (m, 24H); 086 (t, 3H). H-NMR (400 MHz, CDCi 3 ): δ 8.05 (broad d, 1 H); 3.95-3.60 (m, 4H); 3.51 (m, 1 H); 3.20 (m, 1 H); 2.90-2.65 (m, 2H); 1.45 (m, 2H); 1.30-1.25 (m, 24H); 086 (t, 3H).
13C-RMN (101 MHz, CDCí3): δ 172.5, 77.5, 71.2, 60.3, 56.2, 53.2, 47.6, (31.8- 31.6), (29.9-29.3), 23.8, 22.6, 14.2. 1 3 C-NMR (101 MHz, CDCí 3 ): δ 172.5, 77.5, 71.2, 60.3, 56.2, 53.2, 47.6, (31.8-31.6), (29.9-29.3), 23.8, 22.6, 14.2.
Ejemplo 1-D3. (RS)-A/-f(2S,3RE)-1 ,3-dihidroxioctadec-4-en-17-in-2-ii]oxirano-Example 1-D3. (RS) -A / -f (2S, 3RE) -1, 3-dihydroxyoctadec-4-en-17-in-2-ii] oxirane-
2-carboxamida 2-carboxamide
Rendimiento: 73% H-RMN (400 MHz, CDCI3): δ 8.05 (d ancho, 1 H); 3.90-3.70 (m, 4H); 3.51 (m, 1 H); 2.90-2.65 (m, 2H); 2.05 (m, 2H); 1.85 (t, J = 2.5, 1 H); 1.46-1.42 (m, 4H); 1.29-1.26 (s ancho, 20H).Yield: 73% H-NMR (400 MHz, CDCI 3 ): δ 8.05 (broad d, 1 H); 3.90-3.70 (m, 4H); 3.51 (m, 1 H); 2.90-2.65 (m, 2H); 2.05 (m, 2H); 1.85 (t, J = 2.5, 1 H); 1.46-1.42 (m, 4H); 1.29-1.26 (wide s, 20H).
3C-RMN (101 MHz, CDCI3): δ 172.5, 83.5, 72.3, 69.1 , 59.9, 59.7, 56.2, 48.2, 32.6, 23.5, 21.4, (29.9-28.5). 3C-NMR (101 MHz, CDCI 3 ): δ 172.5, 83.5, 72.3, 69.1, 59.9, 59.7, 56.2, 48.2, 32.6, 23.5, 21.4, (29.9-28.5).
Ejemplo l-E3. (RS)-/V-í(2S,3f?)-14-azido-1 ,3-dihidroxitetradecan-2-illoxirano-2- carboxamida Example l-E3. (RS) - / V-í (2S, 3f?) - 14-azido-1, 3-dihydroxytetradecan-2-illoxyran-2-carboxamide
Rendimiento: 79%  Yield: 79%
1H-RMN (400 MHz, CDCI3): δ 8.15 (d ancho, 1 H); 3.90-375 (m, 4H); 3.51 (m, 1 H); 2.90-2.65 (m, 2H); 1.45-1.30 (s ancho, 22H). 1 H-NMR (400 MHz, CDCI 3 ): δ 8.15 (broad d, 1 H); 3.90-375 (m, 4H); 3.51 (m, 1 H); 2.90-2.65 (m, 2H); 1.45-1.30 (wide s, 22H).
13C-RMN (101 MHz, CDC!3): δ 173.3, 72.4, (59.9-59.8), 55.4, 51.2, 47.6, 33.1 , (30.0-29.3), 26.7, 23.3. Otras reacciones de acopiamiento 13 C-NMR (101 MHz, CDC! 3 ): δ 173.3, 72.4, (59.9-59.8), 55.4, 51.2, 47.6, 33.1, (30.0-29.3), 26.7, 23.3. Other stockpile reactions
Ejemplo 1-B5: A/-f(2S,3R)-1 ,3-dihidroxioctadecan-2-il]propioiamida  Example 1-B5: A / -f (2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] propioiamide
Rendimiento: 90%Yield: 90%
H-RMN (400 MHz, CDCI3): δ 7.77 (d ancho, 1 H), 4.23 - 4.03 (m, 1 H), 3.97 - 3.70 (m, 3H), 2.72 - 2.49 (m, 2H), 1 .68 - 1.41 (m, 4H), 1.23 (ancho, 24H), 0.85 (t ancho, 3H). H-NMR (400 MHz, CDCI 3 ): δ 7.77 (broad d, 1 H), 4.23 - 4.03 (m, 1 H), 3.97 - 3.70 (m, 3H), 2.72 - 2.49 (m, 2H), 1 .68 - 1.41 (m, 4H), 1.23 (width, 24H), 0.85 (t width, 3H).
13C-RMN (101 MHz, CDC!3): δ 145.9, 84.5, 77.8, 73.06, 59.98, 57.53, 33.91 , 32.75, (29.90 - 29.58), 26.04, 22.98, 14.42. 13 C-NMR (101 MHz, CDC! 3 ): δ 145.9, 84.5, 77.8, 73.06, 59.98, 57.53, 33.91, 32.75, (29.90 - 29.58), 26.04, 22.98, 14.42.
Ejemplo 1-B6. A/-r(2S,3f?)-1 ,3-dihidroxioctadecan-2-il1but-2-inamida Example 1-B6. A / -r (2S, 3f?) - 1, 3-dihydroxyoctadecan-2-yl1but-2-inamide
Rendimiento: 91 % Yield: 91%
1H-RMN (400 MHz, CDCI3): δ 8.01 (d ancho, 1 H), 4.23 - 4.03 (m, 1 H), 3.97 - 3.70 (m, 3H), 2.72 - 2.49 (m, 2H), 1.80 (s, 3H), 1.68 - 1.41 (m, 4H), 1.23 (ancho, 24H), 0.85 (t ancho, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 8.01 (broad d, 1 H), 4.23 - 4.03 (m, 1 H), 3.97 - 3.70 (m, 3H), 2.72 - 2.49 (m, 2H), 1.80 (s, 3H), 1.68 - 1.41 (m, 4H), 1.23 (wide, 24H), 0.85 (wide t, 3H).
13C-RMN (101 MHz, CDCI3): δ 145.2, 91.2, 78.8, 72.0, 59.6, 58.53, 35.1 , 32.9, (29.9-29.5), 26.0, 22.9, 14.3, 2.7. Ejemplo 1-B7. A/-r(2S,3R)-1 ,3-dihidroxioctadecan-2-il1acrilamida 13 C-NMR (101 MHz, CDCI 3 ): δ 145.2, 91.2, 78.8, 72.0, 59.6, 58.53, 35.1, 32.9, (29.9-29.5), 26.0, 22.9, 14.3, 2.7. Example 1-B7. A / -r (2S, 3R) -1, 3-dihydroxyoctadecan-2-yl1-acrylamide
Rendimiento: 89% Yield: 89%
1H-RMN (400 MHz, CDCI3): δ 6.33 (d, J = 17.0, 1 H), 6.17 (dd, J = 10.2, 16.9, 1 H), 5.69 (d, J = 1 1.1 , 1 H), 4.16 - 3.99 (m, 1 H), 3.96 - 3.71 (m, 3H), 1.68 - 1 .46 (m, 4H), 1.25 (s ancho, 24H), 0.88 (t, J = 6.8, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 6.33 (d, J = 17.0, 1 H), 6.17 (dd, J = 10.2, 16.9, 1 H), 5.69 (d, J = 1 1.1, 1 H ), 4.16 - 3.99 (m, 1 H), 3.96 - 3.71 (m, 3H), 1.68 - 1 .46 (m, 4H), 1.25 (wide s, 24H), 0.88 (t, J = 6.8, 3H) .
13C-RMN (101 MHz, CDCI3): δ 165.7, 131 .0, 127.3, 74.5, 62.4, 54.0, 34.7, 32.1 , 30.0, 29.7, 29.5, 26.3, 22.8, 14.2. 13 C-NMR (101 MHz, CDCI 3 ): δ 165.7, 131 .0, 127.3, 74.5, 62.4, 54.0, 34.7, 32.1, 30.0, 29.7, 29.5, 26.3, 22.8, 14.2.
Ejemplo I-B8. (E)-/V-f(2S,3fí)-1 ,3-dihidroxioctadecan-2-illbut-2-enamida Example I-B8. (E) - / V-f (2S, 3fí) -1, 3-dihydroxioctadecan-2-illbut-2-enamide
Rendimiento: 86% Yield: 86%
1H-RMN (400 MHz, CDCI3): δ 6.88 (dq, J = 6.7, 13.3, 1 H), 6.40 (d, J = 7.6, 1 H), 5.87 (d, J = 14.3, 1 H), 4.04 (d ancho, 1 H), 3.92 - 3.74 (m, 3H), 1.87 (d, J = 6.6, 3H), 1.70-1.46 (m, 4H), 1.25 (ancho, 24 H), 0.88 (t, J = 6.5, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 6.88 (dq, J = 6.7, 13.3, 1 H), 6.40 (d, J = 7.6, 1 H), 5.87 (d, J = 14.3, 1 H) , 4.04 (d wide, 1 H), 3.92 - 3.74 (m, 3H), 1.87 (d, J = 6.6, 3H), 1.70-1.46 (m, 4H), 1.25 (wide, 24 H), 0.88 (t , J = 6.5, 3H).
3C-RMN (101 MHz, CD3OD): δ 141.0, 126.3, 72.5, 62.3, 56.9, 35.0, 33.1 , 30.9, 30.8, 30.5, 26.9, 23.8, 18.0, 14.6. 3C-NMR (101 MHz, CD 3 OD): δ 141.0, 126.3, 72.5, 62.3, 56.9, 35.0, 33.1, 30.9, 30.8, 30.5, 26.9, 23.8, 18.0, 14.6.
Ejemplo 1-B9. A/-f(2S,3ff)-1 ,3-dihidroxioctadecan-2-il1metacriiamidaExample 1-B9. A / -f (2S, 3ff) -1, 3-dihydroxyoctadecan-2-yl1methacryamide
H-RMN (400 MHz, CDCI3): δ 8.22 (ancho, 1 H), 5.89 (s ancho, 1 H), 5.49 (s ancho, 1 H), 3.88-3.63 (m, 2H), 3.83 (m, 1 H), 3.75 (m, 1 H), 1.93 (s ancho, 1 H), 1.75-1.50 (m, 4H), 1.27 (ancho, 24 H), 0.85 (t, J = 6.5, 3H) H-NMR (400 MHz, CDCI 3 ): δ 8.22 (width, 1 H), 5.89 (width s, 1 H), 5.49 (width s, 1 H), 3.88-3.63 (m, 2H), 3.83 (m , 1 H), 3.75 (m, 1 H), 1.93 (wide s, 1 H), 1.75-1.50 (m, 4H), 1.27 (wide, 24 H), 0.85 (t, J = 6.5, 3H)
13C-RMN (101 MHz, CD3OD): δ 168.6, 141.3, 124.0, 72.3, 59.9, 59.8, 32.7, 31.8, 29.9-29.3, 23.4, 22.7, 14.1 13 C-NMR (101 MHz, CD 3 OD): δ 168.6, 141.3, 124.0, 72.3, 59.9, 59.8, 32.7, 31.8, 29.9-29.3, 23.4, 22.7, 14.1
Ejemplo 1-B10. A -r(2S.3R)-N-1.3-dihidroxioctadecan-2-il1-3- metil-2-butenamida Example 1-B10. A -r (2S.3R) -N-1.3-dihydroxyoctadecan-2-yl-1-3-methyl-2-butenamide
1H-RMN (400 MHz, CDCI3): δ 6.26 (ancho, 1 H), 6.64 (s, 1 H), 4.1 (m, 1 H), 3.85 (m, 3H); 2.16 (s, 3H), 1.86 (s, 3H), 1.54 (m, 2H), 1.25 (ancho, 26H), 0.88 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 6.26 (width, 1 H), 6.64 (s, 1 H), 4.1 (m, 1 H), 3.85 (m, 3H); 2.16 (s, 3H), 1.86 (s, 3H), 1.54 (m, 2H), 1.25 (width, 26H), 0.88 (t, 3H).
13C-RMN (101 MHz, CDCi3): δ 167.4, 151.2, 1 18.5, 74.3, 62.7, 53.9, 34.7, 32.0, 29.8-29.5, 27.3, 26.1 , 22.8, 19.9, 14.2. Ejemplo 1-B11. (2E,4a-N-f(2S.3R)-1.3-dihidroxioctadecan-2-il]hexa-2,4- dienamida 13 C-NMR (101 MHz, CDCi 3 ): δ 167.4, 151.2, 1 18.5, 74.3, 62.7, 53.9, 34.7, 32.0, 29.8-29.5, 27.3, 26.1, 22.8, 19.9, 14.2. Example 1-B11. (2E, 4a-Nf (2S.3R) -1.3-dihydroxyoctadecan-2-yl] hexa-2,4-dienamide
1H-RMN (400 MHz, CDCI3): δ 7.1 1 (dd, J=14.8 Hz, J'=10 Hz, 1 H), 6.90 (d ancho, 1 H), 6.05 (m, 2H), 5.78 (d, J=15.2 Hz, 1 H), 3.95 (m, 2H), 3.62 (m, 1 H), 1.78 (d, J=6Hz, 3H), 1.45 (ancho, 2H), 1.19 (ancho, 26H), 0.82 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 7.1 1 (dd, J = 14.8 Hz, J '= 10 Hz, 1 H), 6.90 (broad d, 1 H), 6.05 (m, 2H), 5.78 (d, J = 15.2 Hz, 1 H), 3.95 (m, 2H), 3.62 (m, 1 H), 1.78 (d, J = 6Hz, 3H), 1.45 (width, 2H), 1.19 (width, 26H ), 0.82 (t, 3H).
13C-RMN (101 MHz, CDCI3,CD3OD): δ 167.3, 141 .7, 138.2, 129.7, 121.3, 73.3, 61.9, 54.5, 34.3, 31.9, 29.7-29.3, 26.0, 22.7, 18.5, 14.1. 13 C-NMR (101 MHz, CDCI 3 , CD 3 OD): δ 167.3, 141 .7, 138.2, 129.7, 121.3, 73.3, 61.9, 54.5, 34.3, 31.9, 29.7-29.3, 26.0, 22.7, 18.5, 14.1 .
Ejemplo 1-B12. Ácido Example 1-B12. Acid
(E)-4-r(2S,3R)-1 ,3-dihidroxioctadecan-2-ilaminol-4-oxo-2-butenoico (E) -4-r (2S, 3R) -1, 3-dihydroxyoctadecan-2-ylaminol-4-oxo-2-butenoic
1H-RMN (400 MHz, CDCI3): δ 7.8 (ancho, 1 H), 6.38 (dd ancho, 2H), 4.12 (d ancho, 1 H), 3.88 (ancho, 3H), 1.45 (ancho, 2H), 1.25 (ancho, 26H), 0.87 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 7.8 (width, 1 H), 6.38 (dd width, 2H), 4.12 (d width, 1 H), 3.88 (width, 3H), 1.45 (width, 2H ), 1.25 (width, 26H), 0.87 (t, 3H).
3C-RMN (101 MHz, CDCI3): 5166.4, 166.2, 135.5, 132.5, 73.6, 61.2, 54.7, 34.5, 32.1 , 29.8-29.5, 26.1 , 22.8, 14.2 3C-NMR (101 MHz, CDCI 3 ): 5166.4, 166.2, 135.5, 132.5, 73.6, 61.2, 54.7, 34.5, 32.1, 29.8-29.5, 26.1, 22.8, 14.2
Ejemplo 1-B13. (Z)-2,3-dibromo-N-r(2S,3R)-1 ,3-dihidroxioctadecan-2-il1-4- οχο-2-butenamidaExample 1-B13. (Z) -2,3-dibromo-N-r (2S, 3R) -1, 3-dihydroxyoctadecan-2-yl1-4- οχο-2-butenamide
H-RMN (400 MHz, CDC!3): δ 9.70 (s, 1 H), 8.22 (ancho, 1 H), 3.85 (m, 2H), 3.75 (m, 1 H), 3.63 (m, 1 H), 1.45 (m, 2H), 1.28 (ancho, 26H), 0.85 (t, 3H). H-NMR (400 MHz, CDC! 3 ): δ 9.70 (s, 1 H), 8.22 (width, 1 H), 3.85 (m, 2H), 3.75 (m, 1 H), 3.63 (m, 1 H ), 1.45 (m, 2H), 1.28 (width, 26H), 0.85 (t, 3H).
13C-RMN (101 MHz, CDC!3): δ 192.3, 166.2, 143.2, 134.5, 72.5, 60.2, 58.9, 32.8, 31.8, 29.9-29.3, 23.5, 22.6, 14.1. 13 C-NMR (101 MHz, CDC! 3 ): δ 192.3, 166.2, 143.2, 134.5, 72.5, 60.2, 58.9, 32.8, 31.8, 29.9-29.3, 23.5, 22.6, 14.1.
Ejemplo 1-B14. (2S,3f?)-2-(bromometil)-N-(1 ,3-dihidroxioctadecan-2- iPacrilamida Example 1-B14. (2S, 3f?) - 2- (bromomethyl) -N- (1,3-dihydroxyoctadecan-2- iPacrylamide
1H-RMN (400 MHz, CDCi3): δ 8.22 (ancho, 1 H), 6.02 (d, J=2.5 Hz, 1 H), 5.81 (d, J=2.5 Hz, 1 H), 4.12 (s, 2H), 3.85-3.53 (m, 4H), 1.45 (m, 2H), 1.29 (ancho, 26H), 0.85 (t, 3H). 1 H-NMR (400 MHz, CDCi 3 ): δ 8.22 (width, 1 H), 6.02 (d, J = 2.5 Hz, 1 H), 5.81 (d, J = 2.5 Hz, 1 H), 4.12 (s , 2H), 3.85-3.53 (m, 4H), 1.45 (m, 2H), 1.29 (width, 26H), 0.85 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 169.1 , 142.5, 127.2, 71.2, 59.9, 59.7, 36.5, 33.1 , 31.5, 29.9-29.1 , 23.5, 22.6, 14.1. Ejemplo 1-B15. (E,2S.3R)-A/-(1 ,3-dihidroxi-2-octadecil)-2-methil-2-butenamida 1H-RMN (400 MHz, CDCI3): δ 6.62 (ancho, 1 H), 6.50 (ancho, 1 H), 4.01 (m, 1 H), 3.85-3.75 (m, 3H), 1.87 (s ancho, 3H), 1 .77 (d, J=6.8 Hz, 3H), 1.54 (m, 2H), 1.25 (ancho, 26H), 0.87 (t, 3H). 13 C-NMR (101 MHz, CDCI 3 ): δ 169.1, 142.5, 127.2, 71.2, 59.9, 59.7, 36.5, 33.1, 31.5, 29.9-29.1, 23.5, 22.6, 14.1. Example 1-B15. (E, 2S.3R) -A / - (1,3-dihydroxy-2-octadecyl) -2-methyl-2-butenamide 1 H-NMR (400 MHz, CDCI 3 ): δ 6.62 (width, 1 H) , 6.50 (width, 1 H), 4.01 (m, 1 H), 3.85-3.75 (m, 3H), 1.87 (s width, 3H), 1.77 (d, J = 6.8 Hz, 3H), 1.54 ( m, 2H), 1.25 (width, 26H), 0.87 (t, 3H).
3C-RMN (101 MHz, CDCI3): δ 169.8, 131.7, 131.6, 74.2, 62.5, 54.1 , 34.6, 32.1 ,29.8-29.5, 26.1 , 22.8, 14.3, 14.1 , 12.5. 3C-NMR (101 MHz, CDCI 3 ): δ 169.8, 131.7, 131.6, 74.2, 62.5, 54.1, 34.6, 32.1, 29.8-29.5, 26.1, 22.8, 14.3, 14.1, 12.5.
Ejemplo 1-D16. (2S,3R)-N-(1 ,3-dihidroxi-17-octadecin-2-in-2-oxooctanamida H-RMN (400 MHz, CDCI3): δ 8.25 (ancho, 1 H), 3.80-3.60 (m, 4H), 2.40 (t ancho, 2H), 2.03 (m, 2H), 1.85 (t, J=2.7 Hz, 1 H), 1.65 (m, 2H), 1.45 (m, 4H), 1.35-1.25 (m, 26H), 0.97 (t, 3H). Example 1-D16. (2S, 3R) -N- (1,3-dihydroxy-17-octadecin-2-in-2-oxoctanamide H-NMR (400 MHz, CDCI 3 ): δ 8.25 (width, 1 H), 3.80-3.60 ( m, 4H), 2.40 (wide t, 2H), 2.03 (m, 2H), 1.85 (t, J = 2.7 Hz, 1 H), 1.65 (m, 2H), 1.45 (m, 4H), 1.35-1.25 (m, 26H), 0.97 (t, 3H).
13C-RMN (101 MHz, CDCI3): δ 197.6, 159.9, 83.5, 72.4, 68.1 , 59.9, 59.7, 32.8-31.5, 30.1-28.5, 23.5-21.5, 14.2. Ejemplo 1-E16. (2S,3R)-N-(14-azido-1.3-dihidroxi-2-tetradecin-2- oxooctanamida 13 C-NMR (101 MHz, CDCI 3 ): δ 197.6, 159.9, 83.5, 72.4, 68.1, 59.9, 59.7, 32.8-31.5, 30.1-28.5, 23.5-21.5, 14.2. Example 1-E16. (2S, 3R) -N- (14-azido-1,3-dihydroxy-2-tetradecin-2- oxooctanamide
1H-RMN (400 MHz, CDCI3): δ 8.25 (ancho, 1 H), 3.85-3.62 (m, 4H), 2.45 (t ancho, 2H), 1.60-1.45 (m, 4H), 1.35-1.25 (ancho, 26H), 0.95 (t, 3H). 1 H-NMR (400 MHz, CDCI 3 ): δ 8.25 (width, 1 H), 3.85-3.62 (m, 4H), 2.45 (t wide, 2H), 1.60-1.45 (m, 4H), 1.35-1.25 (width, 26H), 0.95 (t, 3H).
3C-RMN (101 MHz, CDC!3): δ 197.5, 160.2, 73.1 , 59.9, 59.7, 51.2, 32.6, 32.4, 31.8, 30.0-28.5, 26.8, 23.5, 23.1 , 22.9, 14.2. 3C-NMR (101 MHz, CDC! 3 ): δ 197.5, 160.2, 73.1, 59.9, 59.7, 51.2, 32.6, 32.4, 31.8, 30.0-28.5, 26.8, 23.5, 23.1, 22.9, 14.2.
Ejemplos de ensayos biológicos Examples of biological tests
Ejemplo 1. Actividad inhibidora de las ceramidasas  Example 1. Ceramidases inhibitory activity
La actividad inhibidora de ias ceramidasas por ios compuestos de la presente invención se ensayó sobre fibroblastos de un enfermo de Farber transducidos para ia sobreexpresion de ia ceramidasa ácida y sobre las iíneas de cáncer de pulmón A549 (American Type Culture Coílection) y de cáncer de próstata PC-3/Mc. La línea A549 se mantuvo en medio de HAM F12 supiementado con glutamina 2 mM, la linea de Farber se cultivó en medio DMEM y la línea PC-3/Mc se mantuvo en medio RPMI1640. En todos los casos, ei medio se suplemento con un 10% de suero de feto bovino, 100 unidades/mL de penicilina y 10 μg/mL de estreptomicina y los cultivos se mantuvieron a 37 O en atmósfera de 5% CO 2 95% aire y una humedad del 90%. Las células se sembraron en placas de 96 pocilios a una densidad deThe inhibitory activity of ceramidases by the compounds of the present invention was tested on fibroblasts of a Farber patient transduced for overexpression of ia ceramidase and on the lines of lung cancer A549 (American Type Culture Colection) and prostate cancer PC-3 / Mc. The A549 line was maintained in the middle of HAM F12 supplemented with 2 mM glutamine, the Farber line was grown in DMEM medium and the PC-3 / Mc line was maintained in RPMI1640 medium. In all cases, the medium is supplemented with 10% serum of bovine fetus, 100 units / mL of penicillin and 10 μg / mL of streptomycin and the cultures were maintained at 37 O in an atmosphere of 5% CO 2 95% air and a humidity of 90%. The cells were seeded in 96-well plates at a density of
10.000 células por mililitro y después de 24-48 h, ei medio se eliminó y se sustituyó por medio nuevo (100 microlitros por pocilio) conteniendo el sustrato fluorogénico (Bedia et al. Chembiochem 2007, 8, 642) y el inhibidor a una concentración de 16 μΜ. También se puede añadir el sustrato directamente sin reemplazar el medio de cultivo. En cualquier caso, se incubó a 37 °C durante 3 horas, se procedió a la lísis de las células y se determinó la actividad enzimática siguiendo ei procedimiento descrito (Bedia et ai. Chembiochem 2007, 8, 642). En algunos casos, el inhibidor y el sustrato se añadieron conjuntamente y se incubaron durante 3 h antes de proceder a la iisis de las células. 10,000 cells per milliliter and after 24-48 h, the medium was removed and replaced with fresh medium (100 microliters per well) containing the fluorogenic substrate (Bedia et al. Chembiochem 2007, 8, 642) and the inhibitor at a concentration of 16 μΜ. The substrate can also be added directly without replacing the culture medium. In any case, it was incubated at 37 ° C for 3 hours, the cells were lysed and the enzymatic activity was determined following the procedure described (Bedia et ai. Chembiochem 2007, 8, 642). In some cases, the inhibitor and the substrate were added together and incubated for 3 h before proceeding with the cell's analysis.
En un ejemplo concreto se determinó el efecto inhibidor de los compuestos sobre la actividad ceramidasa ácída de las células de Farber transducidas para la sobreexpresion de la ceramidasa ácida. Es este caso, las células se incubaron conjuntamente con sustrato y compuesto problema durante 3 h y después de este tiempo, se determinó la actividad enzimática tal como se describe más arriba. Tal como se ilustra en la Figura 1 , ios compuestos I-B2, I-B8, I-B9 y I-B17, y fueron los más activos en células intactas, aunque sólo I-B2, I-B9 y I-B17 fueron también activos in vitro. In a specific example, the inhibitory effect of the compounds on the acidic ceramidase activity of the transduced Farber cells for the overexpression of the acidic ceramidase was determined. In this case, the cells were incubated together with substrate and test compound for 3 h and after this time, the enzymatic activity was determined as described above. As illustrated in Figure 1, the compounds I-B2, I-B8, I-B9 and I-B17, and were the most active in intact cells, although only I-B2, I-B9 and I-B17 were also active in vitro.
En otro ejemplo concreto se determinó el efecto inhibidor de los compuestos sobre la actividad ceramidasa ácída de las células de adenocarcinoma de pulmón A549. Las células se incubaron con los compuestos problema durante 24 h y después de este tiempo se añadió el sustrato, se incubó durante 3 h y se continuó tal como se describe más arriba. Tal como se muestra en ia Figura 2, ios compuestos ID2, IE2 IF2 IG2 y IH2 fueron los más activos, presentando una inhibición de la ceramídasa ácida superior al 95% respecto al control. In another specific example, the inhibitory effect of the compounds on the acidic ceramidase activity of A549 lung adenocarcinoma cells was determined. The cells were incubated with the test compounds for 24 h and after this time the substrate was added, incubated for 3 h and continued as described above. As shown in Figure 2, the compounds ID2, IE2 IF2 IG2 and IH2 they were the most active, presenting an inhibition of the acid ceramidasa superior to 95% with respect to the control.
En otro ejemplo se determinó el efecto de los compuestos I-B2, I-9B y I- B17 sobre la actividad ceramidasa ácida de las células de cáncer de próstata PC-3 c. Las células se sembraron en placas de 96 pocilios a una densidad de 10.000 céíulas/ml y los compuestos problema se añadieron 24 horas después de la siembra. Se usaron tres dosis de cada inhibidor, 1 μΜ, 5 μΜ y 10 μΜ, en incubaciones durante 48 h. La Figura 3 representa gráficamente los resultados obtenidos. Los compuestos I-B17 y I-B2 causan una clara inhibición de la actividad ceramidasa ácida a todas las dosis empleadas, con mayor efecto inhibidor a mayor dosis. In another example, the effect of compounds I-B2, I-9B and I-B17 on the acidic ceramidase activity of PC-3 c prostate cancer cells was determined. The cells were seeded in 96-well plates at a density of 10,000 cells / ml and the test compounds were added 24 hours after seeding. Three doses of each inhibitor, 1 μΜ, 5 μΜ and 10 μΜ, were used in incubations for 48 h. Figure 3 graphically represents the results obtained. Compounds I-B17 and I-B2 cause a clear inhibition of acidic ceramidase activity at all doses used, with greater inhibitory effect at higher doses.
Ejemplo 2. Efecto de los inhibidores sobre el ceramidoma Example 2. Effect of inhibitors on ceramidoma
En estos experimentos, las células se sembraron en placas de 6 pocilios a una densidad de 250.000 células/mí. Los compuestos se añadieron 24 h después de la siembra y se incubaron durante distintos períodos de tiempo. Después de los tratamientos, las células se lavaron con PBS y se transfirieron a viales de vidrio, donde se prepararon ios extractos lipidíeos siguiendo el procedimiento descrito (Merrill et al Methods, 2005, 36, 207). Los análisis se llevaron a cabo por cromatografía líquida de ultrarresolución acoplada a un detector de masas de tiempo de vuelo acelerado, que permite la identificación de los compuestos en base a su masa exacta, mediante ionización en electrospray en modo positivo. Las condiciones cromatográficas y analíticas son las descritas en trabajos anteriores (Munoz-Olaya et al ChemMedChem, 2008, 3, 946 y Canals et al Bioorg. Med. Chem., 2009, 17, 235).  In these experiments, the cells were seeded in 6-well plates at a density of 250,000 cells / ml. The compounds were added 24 h after seeding and incubated for different periods of time. After the treatments, the cells were washed with PBS and transferred to glass vials, where lipid extracts were prepared following the procedure described (Merrill et al Methods, 2005, 36, 207). The analyzes were carried out by ultra-liquid liquid chromatography coupled to an accelerated flight time mass detector, which allows the identification of the compounds based on their exact mass, by electrospray ionization in positive mode. The chromatographic and analytical conditions are those described in previous works (Munoz-Olaya et al ChemMedChem, 2008, 3, 946 and Canals et al Bioorg. Med. Chem., 2009, 17, 235).
En un ejemplo concreto se detallan los efectos de los compuestos problema sobre la producción de ceramídas totales en la línea de cáncer de pulmón A549 después de 24 h de incubación. Los resultados se ilustran en la Figura 4. Los compuestos que producen un mayor aumento de los niveles de ceramidas son ID2, IF2, IG2 y IH2, con las cuales las ceramidas son 6 veces superiores a las de las células tratadas con vehículo. In a specific example, the effects of the test compounds on the production of total ceramides in the A549 lung cancer line after 24 h of incubation are detailed. The results are illustrated in Figure 4. Compounds that produce a greater increase in levels of Ceramides are ID2, IF2, IG2 and IH2, with which ceramides are 6 times higher than those of vehicle treated cells.
En otro ejemplo concreto se detallan los efectos de ios inhibidores I-B17, I-B9 y I-B2 sobre el esfingolipidoma de ia línea celular de cáncer de próstata PC-3 c. El contenido en esfingolípidos se determinó tras 48 h de incubación con los inhibidores a dos dosis diferentes, 1 μΜ o 5 μ . Se determinaron niveles de ceramidas, dihidroceramídas, esfingomielinas, dihidroesfingomielinas y glucosilceramidas. La Figura 5 ilustra gráficamente los cambios en ios niveles de los diferentes esfingolípidos tras incubar las células PC-3Mc con los inhibidores. Los tratamientos con I-B2 causaron, tanto a 1 μΜ como a 5 μΜ, una acumulación significativa de ceramidas y dihidroceramídas. Ei tratamiento con I-B17 también causó una acumulación de ceramidas y dihidroceramídas, más acusada en tratamientos con una concentración del inhibidor de 5 μΜ. In another specific example, the effects of the inhibitors I-B17, I-B9 and I-B2 on the sphingolipidoma of the prostate cancer cell line PC-3 c are detailed. The sphingolipid content was determined after 48 h of incubation with the inhibitors at two different doses, 1 μΜ or 5 μ. Ceramides, dihydroceramides, sphingomyelins, dihydrosphingomyelins and glucosylceramides levels were determined. Figure 5 graphically illustrates the changes in the levels of the different sphingolipids after incubating the PC-3Mc cells with the inhibitors. I-B2 treatments caused a significant accumulation of ceramides and dihydroceramides at both 1 μΜ and 5 μΜ. Treatment with I-B17 also caused an accumulation of ceramides and dihydroceramides, more pronounced in treatments with an inhibitor concentration of 5 μΜ.
Ejemplo 3. Efecto de los inhibidores sobre la viabilidad celular Example 3. Effect of inhibitors on cell viability
La citotoxícidad se determinó por medida de la actividad deshídrogenasa mitocondríal con el bromuro de 3-(4,5-dimetiítíazol-2-il)-2,5-difeniltetrazolío (MTT) después de 24-72 h de incubación de las células con los compuestos problema a distintas concentraciones. La Figura 6A iiustra el efecto de los compuestos problema sobre la viabilidad de las células de cáncer de pulmón A549 y de fibroblastos dérmicos después de 24 h de incubación. La Figura 6B ilustra gráficamente los efectos de los inhibidores I-B17, I-B9 y I-B2 sobre la viabilidad de las células PC-3Mc después de 72 h de incubación. El análisis de la curva generada con los datos representados proporciona unos valores de CC-50 sobre las células PC-3Mc de 13,7 μΜ para el compuesto I-B17, de 37,2 μΜ para el compuesto I-B9 y de más de 100 μΜ para el compuesto I-B2. Es decir, la citotoxícidad relativa de los tres compuestos es, de mayor a menor, la causada por I-B17, I-B9 y I-B2. Ejemplo 4. Efecto de los inhibidores sobre el crecimiento celuiar sobre sustrato plástico. The cytotoxicity was determined by measuring mitochondrial dehydrogenase activity with 3- (4,5-dimethithiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) after 24-72 h incubation of the cells with the problem compounds at different concentrations. Figure 6A illustrates the effect of the test compounds on the viability of A549 lung cancer cells and dermal fibroblasts after 24 h of incubation. Figure 6B graphically illustrates the effects of inhibitors I-B17, I-B9 and I-B2 on the viability of PC-3Mc cells after 72 h of incubation. The analysis of the curve generated with the represented data provides CC-50 values on PC-3Mc cells of 13.7 μΜ for compound I-B17, 37.2 μΜ for compound I-B9 and more than 100 μΜ for compound I-B2. That is, the relative cytotoxicity of the three compounds is, from highest to lowest, that caused by I-B17, I-B9 and I-B2. Example 4. Effect of inhibitors on cell growth on plastic substrate.
Las células PC-3Mc, cultivadas en medio con un 10% de suero fetal bovino, fueron expuestas a los compuestos problema, a una concentración final de 5 μΜ. Para realizar este ejemplo, se sembraron 500 células en cada pocilio de placas de 96 pocilios, dejándose adherir al plástico durante 24 h, seguido de tratamiento con los compuestos. Cada tratamiento se realizó en pocilios triplicados. La Figura 7 ilustra gráficamente el efecto de las incubaciones con estos compuestos sobre el crecimiento celular, cuantificado como número de células presentes a las 24 h, 48 h, 72 h, 120 h, 144 h y 168 h de tratamiento con los compuestos. Los compuestos I-B17 y I-B2 causaron una casi total inhibición del crecimiento de las células PC-3Mc durante los primeros 6 días de tratamiento. Por el contrario, el compuesto I-B9 no causó diferencias significativas sobre el número de células presentes a lo largo de los 7 días del tratamiento, en comparación con células control, expuestas únicamente al disolvente utilizado para los compuestos.  PC-3Mc cells, grown in medium with 10% fetal bovine serum, were exposed to the test compounds, at a final concentration of 5 μΜ. To perform this example, 500 cells were seeded in each well of 96-well plates, allowed to adhere to the plastic for 24 h, followed by treatment with the compounds. Each treatment was performed in triplicate wells. Figure 7 graphically illustrates the effect of incubations with these compounds on cell growth, quantified as the number of cells present at 24 h, 48 h, 72 h, 120 h, 144 h and 168 h of treatment with the compounds. Compounds I-B17 and I-B2 caused an almost total inhibition of the growth of PC-3Mc cells during the first 6 days of treatment. In contrast, compound I-B9 did not cause significant differences over the number of cells present over 7 days of treatment, compared to control cells, exposed only to the solvent used for the compounds.
Ejemplo 5. Efecto de los inhibidores sobre la formación de colonias celulares en medio semisólido. Example 5. Effect of inhibitors on the formation of cell colonies in semi-solid medium.
En este ejempio, se determinó la capacidad de las células PC-3Mc para crecer formando colonias tridimensionales en semisuspensíón. En el ensayo utilizado, las células se resuspenden a temperaturas que preservan la viabilidad celular en medio de cultivo completo que contiene agar al 0,6%, dejando posteriormente solidificar a temperatura ambiente, sobre un lecho de medio con 3% de agar. Las células sembradas en esta disposición se incuban durante 2 o 3 semanas a 37 °C en una atmósfera del 5% de CO2 / 95% aire y humedad del 90%. Una vez sembradas, se añadieron los compuestos problemas a concentraciones finales de 1 μΜ o 5 μΜ, reañadiéndolos con una periodicidad de 3 días, coincidiendo con la adición de medio de cultivo nuevo. Después de 3 semanas, las colonias se fijan con glutardehído al 0.5%, se tiñen con cristal de violeta y se visualizan al microscopio. Las colonias de un diámetro mayor o igual que 0.2 mm se cuentan con el programa ImageJ 1.43u (NIH, USA). In this example, the ability of PC-3Mc cells to grow forming three-dimensional colonies in semi-suspension was determined. In the assay used, the cells are resuspended at temperatures that preserve cell viability in a complete culture medium containing 0.6% agar, subsequently allowing to solidify at room temperature, on a bed of medium with 3% agar. The cells seeded in this arrangement are incubated for 2 or 3 weeks at 37 ° C in an atmosphere of 5% CO2 / 95% air and 90% humidity. Once seeded, the problem compounds were added at final concentrations of 1 μΜ or 5 μΜ, reacting them with a periodicity of 3 days, coinciding with the addition of new culture medium. After 3 weeks, the colonies are fixed with 0.5% glutardehyde, stained with violet crystal and visualized under a microscope. The colonies of a diameter greater than or equal to 0.2 mm is provided with the ImageJ 1.43u program (NIH, USA).
La Figura 8 ilustra cómo, a la concentración de 1 μΜ, ninguno de ios compuestos afectó la capacidad de las células PC-3Mc para formar colonias, mientras que a 5 μΜ, los compuestos I-B17 y I-B2 inhiben la formación de colonias por debajo de un 25% respecto al control. Figure 8 illustrates how, at the concentration of 1 μΜ, none of the compounds affected the ability of PC-3Mc cells to form colonies, while at 5 μΜ, compounds I-B17 and I-B2 inhibit colony formation below 25% compared to the control.
Ejemplo 6. Efecto de los inhibidores sobre la invasividad celular. Example 6. Effect of inhibitors on cell invasiveness.
En la realización de este ejemplo, se utilizó el ensayo conocido como de invasividad en cámaras de Boyden. La versión comercial que se ha usado de estas cámaras se llama Transweli (de la casa Corning), y consiste en membranas relativamente inertes de políéster, con poros de 8.0 ,Lim, colocadas en un soporte plástico que se inserta en pocilios de placas de 96 pocilios. Estas membranas se recubren con componentes de la matriz extraceiular (Matrigel Growth Factor Reduced, de la casa Becton-Dickinson). Las células se depositan con medio carente de suero fetal bovino en la cámara superior, y en la cámara inferior (el pocilio de la placa de 96 pocilios) se coloca el mismo medio. Se permite la invasión de las células desde la cámara superior hacia la cámara inferior durante 48 h en las condiciones habituales de cultivo descritas en anteriores ejemplos. Al cabo de esa incubación, se procede ai recuento de las células que han pasado a la cámara inferior. Para la realización de este ejemplo, las células PC-3Mc fueron tratadas con los compuestos problema a una concentración final de 5 μΜ durante las 48 h previas al ensayo de invasividad. Tras ese tiempo de tratamiento, las células fueron despegadas de las placas de cultivo medíante incubación con tripisína (25 unidades / mL) y EDTA (0.1 mM) durante 5 minutos, resuspendidas en medio completo, y sembradas sobre las cámaras superiores de los insertos Transweli previamente cubiertos con Matrigel a una concentración de 10 mg/mL. Los compuestos problema se añadieron tanto a la cámara superior como a la inferior, a una concentración de 5 mM, manteniéndose este tratamiento a lo largo de todo el periodo de duración del ensayo. Cada condición se realizó por triplicado. La Figura 9 ilustra cómo, en estas condiciones, tanto el I-B17 como el I-B2 inhiben significativamente la invasividad de las células PC-3Mc. In carrying out this example, the test known as invasiveness in Boyden chambers was used. The commercial version that has been used of these chambers is called Transweli (from the Corning house), and consists of relatively inert polyester membranes, with pores of 8.0, Lim, placed in a plastic support that is inserted into 96-well plates. wells. These membranes are coated with components of the extraceiular matrix (Matrigel Growth Factor Reduced, from Becton-Dickinson). The cells are deposited with medium lacking fetal bovine serum in the upper chamber, and the same medium is placed in the lower chamber (the 96-well plate well). Invasion of the cells from the upper chamber into the lower chamber is allowed for 48 hours under the usual culture conditions described in previous examples. After this incubation, the cells that have passed to the lower chamber are counted. For the realization of this example, PC-3Mc cells were treated with the test compounds at a final concentration of 5 μΜ during the 48 hours prior to the invasiveness test. After that treatment time, the cells were detached from the culture plates by incubation with tripisin (25 units / mL) and EDTA (0.1 mM) for 5 minutes, resuspended in complete medium, and seeded on the upper chambers of the Transweli inserts. previously covered with Matrigel to a concentration of 10 mg / mL. The test compounds were added to both the upper and lower chamber, at a concentration of 5 mM, this treatment being maintained throughout the entire duration of the test. Each condition was performed in triplicate. Figure 9 illustrates how, under these conditions, both I-B17 and I-B2 significantly inhibit the invasiveness of PC-3Mc cells.

Claims

REIVINDICACIONES: CLAIMS:
1.- Un compuesto de fórmula (I) 1.- A compound of formula (I)
Figure imgf000046_0001
Figure imgf000046_0001
Fórmula (I) Formula (I)
o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de éste, or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof,
A se selecciona entre -CH(OH)- y -C(=0)-, A is selected from -CH (OH) - and -C (= 0) -,
Z se seíeccíona entre H y OH,  Z is selected between H and OH,
n es un número entero seleccionado entre 0 y 1 ,  n is an integer selected from 0 to 1,
R-i se selecciona entre aíquilo(C-i-C3o), alquenilo(C2-C30) y alquiniío(C C30), Ri is selected from alkyl (Ci-C3o), alkenyl (C 2 -C 30 ) and alkyne (C C30),
B se selecciona entre -H, -N3 y -CECH, B is selected from -H, -N 3 and -CECH,
R2 se selecciona entre -NHR3 y maleimida, donde R 2 is selected from -NHR 3 and maleimide, where
R3 se selecciona entre -COR4, -COCOR4 y -SO2R4, donde R 3 is selected from -COR 4 , -COCOR4 and -SO2R4, where
R4 se selecciona entre alquilo(Ci-Ci6), alquenilo(C2-Ci6), alquinilo(C C e), epóxido y aziridina; donde los grupos alquilo, aiqueniio o alquinilo de R1 y R4 pueden estar, independientemente, opcionalmente sustituidos por uno o varios sustituyentes elegidos independientemente entre halógeno, OH, OR, OCF3, NH2, NO2, NRR', NHCOR; CONRR , CHO, COOH, COOR, OCOR y CN, donde R y R' son alquilo o aiqueniio con la condición de que R 4 is selected from alkyl (Ci-Ci 6 ), alkenyl (C 2 -Ci 6 ), alkynyl (CC e), epoxide and aziridine; wherein the alkyl, aiqueniium or alkynyl groups of R 1 and R 4 may be independently substituted optionally by one or more substituents independently selected from halogen, OH, OR, OCF 3 , NH 2 , NO 2 , NRR ', NHCOR; CONRR, CHO, COOH, COOR, OCOR and CN, where R and R 'are alkyl or aiqueniium with the condition of
a) cuando A es -CH(OH) y B es H, R3 es diferente de -COR4 siendo R4 aiquilo(CrCi6) sin sustituir o sustituido por halógeno o hidroxilo; a) when A is -CH (OH) and B is H, R 3 is different from -COR 4 with R 4 being alkyls (CrCi 6 ) unsubstituted or substituted by halogen or hydroxyl;
b) cuando A es -CH(OH), B es H,y R3 es -COCOR4 siendo R4 alquilo(C6), R es diferente de
Figure imgf000047_0001
-C≡CH-aiquilo(C 2) o alquilo(Ci3-Ci5); o b) when A is -CH (OH), B is H, and R 3 is -COCOR 4 where R4 is alkyl (C 6 ), R is different from
Figure imgf000047_0001
-C≡CH-aiquilo (C 2) alkyl or (Ci-Ci3 5); or
c) cuando A es -C(=0), R-i es alquenilo(C2-C30), B es H y n es 0, R3 es diferente de -COR4 siendo R4 aiquilo(d-Ci6). c) when A is -C (= 0), Ri is alkenyl (C 2 -C 30 ), B is H and n is 0, R 3 is different from -COR4 with R 4 being aiquyl (d-Ci6).
2. - Un compuesto de fórmula (I) según ia reivindicación 1 , caracterizado porque R1 se selecciona entre alquílo(CrC3o) y alquenilo(C2-C3o) 2. - A compound of formula (I) according to claim 1, characterized in that R1 is selected from alkyl (CrC 3 o) and alkenyl (C 2 -C 3 o)
3. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 o 2, caracterizado porque n es 0. 3. - A compound of formula (I) according to any one of claims 1 or 2, characterized in that n is 0.
4. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3, caracterizado porque R3 es -CO-R4 siendo R4 alquílo(CrCi6), y B se selecciona entre ~N3 y -CECH. 4. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R 3 is -CO-R 4 where R 4 is alkyl (CrCi 6 ), and B is selected from ~ N 3 and -CECH.
5. - Un compuesto de fórmula (I) según la reivindicación 4, caracterizado porque R4 es un alquilo(CrCi6) sustituido con ai menos un átomo de halógeno. 5. - A compound of formula (I) according to claim 4, characterized in that R 4 is an alkyl (CrCi 6 ) substituted with at least one halogen atom.
6.- Un compuesto de fórmula (I) según ía reivindicación 5, caracterizado porque R4 es un alquilo(Ci-Ci6) sustituido con al menos un átomo de flúor o bromo. 6. A compound of formula (I) according to claim 5, characterized in that R 4 is a (Ci-Ci 6 ) alkyl substituted with at least one fluorine or bromine atom.
7.- Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 4 a 6, caracterizado porque A es -C(=0). 7. A compound of formula (I) according to any one of claims 4 to 6, characterized in that A is -C (= 0).
8. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3 , caracterizado porque R3 es -CO-R4 siendo R4 epóxído. 8. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R3 is -CO-R 4 with R4 being epoxy.
9. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 o 2, caracterizado porque R3 es -CO-R4 siendo R4 epóxido, n es 1 y Z es9. - A compound of formula (I) according to any one of claims 1 or 2, characterized in that R3 is -CO-R 4 being R 4 epoxide, n is 1 and Z is
OH. OH
10. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3, caracterizado porque R3 es -CO-R4 y R4 se selecciona entre alquenilo(C2-Ci6)y alquinilo(C2-Ci6). 10. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R3 is -CO-R4 and R4 is selected from alkenyl (C 2 -Ci 6 ) and alkynyl (C 2 -Ci 6 ) .
11. - Un compuesto de fórmula (I) según la reivindicación 10, caracterizado porque R4 es un alquenilo(C2-C 6) sustituido con al menos un átomo de halógeno. 11. - A compound of formula (I) according to claim 10, characterized in that R 4 is a (C 2 -C 6 ) alkenyl substituted with at least one halogen atom.
12. - Un compuesto de fórmula (I) según la reivindicación 11 , caracterizado porque R4 es un alquenilo{C2-Ci6) sustituido con al menos un átomo de flúor o bromo. 12. - A compound of formula (I) according to claim 11, characterized in that R 4 is an alkenyl {C 2 -Ci 6 ) substituted with at least one fluorine or bromine atom.
13.- Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 10 a 12, caracterizado porque R4 es un alquenílo(C2-Ci6) o alquinilo(C2-Ci6) sustituido con al menos un grupo -CHO o - COOH. 13. A compound of formula (I) according to any one of claims 10 to 12, characterized in that R 4 is an alkenyl (C 2 -Ci 6 ) or alkynyl (C 2 -Ci 6 ) substituted with at least one group - CHO or - COOH.
14. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3, caracterizado porque R3 es -CO-CO-R4 siendo R4 aíquilo(Ci-C-i6) y B se selecciona entre -N3 y -C CH. 14. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R3 is -CO-CO-R4 where R 4 is alkyl (Ci-C-i6) and B is selected from -N 3 and -C CH.
15. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3, caracterizado porque R3 es -SO2-R4, donde R4 se selecciona entre alquilo(CrCi6), alquenilo(C2-Ci6), alquínílo(C2-C 6), epóxido y azíridina. 15. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R 3 is -SO 2 -R4, wherein R 4 is selected from alkyl (CrCi6), alkenyl (C 2 -Ci6), alkynyl (C 2 -C 6), epoxide and azyridine.
16. - Un compuesto de fórmula (I) según la reivindicación 15, caracterizado porque f¾ es alquiio(Ci-Ci6). 16. - A compound of formula (I) according to claim 15, characterized in that f¾ is alkyl (Ci-Ci6).
17. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 2, caracterizado porque R3 es -SO2-R4, donde R4 se selecciona entre alquilo(d-Ci6), alquenilo(C-2-Ci6), alquínilo(C-2-Ci6), epóxido y aziridina, n es 1 y Z es OH. 17. - A compound of formula (I) according to any one of claims 1 to 2, wherein R3 is -SO2-R4, wherein R4 is selected from alkyl (d-Ci6) alkyl, (C-2-Ci6) , alkynyl (C-2-Ci6), epoxide and aziridine, n is 1 and Z is OH.
18. - Un compuesto de fórmula (I) según la reivindicación 17, caracterizado porque R4 es alquiio(CrCi6). 18. - A compound of formula (I) according to claim 17, characterized in that R4 is alkyl (CrCi 6 ).
19. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 3, caracterizado porque R2 es maleimida. 19. - A compound of formula (I) according to any one of claims 1 to 3, characterized in that R 2 is maleimide.
20.- Un compuesto de fórmula (I) según una cualquiera de ias reivindicaciones 1 a 3, caracterizado porque dicho compuesto se selecciona de la lista que consiste en: 20. A compound of formula (I) according to any one of claims 1 to 3, characterized in that said compound is selected from the list consisting of:
1 -[(2S,3R)-1 ,3-dihidroxioctadecan-2-il]-1 H-pirroí-2,5-diona,  1 - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] -1 H-pyrroi-2,5-dione,
A/-[(2S,3 E)-1 ,3-dihidroxioctadec-4-en-2-il]etanosulfonamida, A / - [(2S, 3 E) -1, 3-dihydroxyoctadec-4-en-2-yl] ethanesulfonamide,
A/-[(2S,3R)-1 ,3~díhídroxioctadecan-2-il]etanosulfonamída, A / - [(2S, 3R) -1, 3 ~ dihydroxyoctadecan-2-yl] ethanesulfonamide,
A/-[(2S,3R)-1 ,3-dihídroxioctadec-17-ín-2-il]etanosulfonamída, A / - [(2S, 3R) -1, 3-dihydroxyoctadec-17-ín-2-yl] ethanesulfonamide,
A/-[(2S,3R)-14-azido-1 ,3-dihidroxitetradecan-2-il)etanosulfonamida, A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl) ethanesulfonamide,
2-bromo-A/-[(2S,3R)-1 ,3-díhidroxioctadec-17~in-2-il]acetamida, 2-Bromo-A / - [(2S, 3R) -1, 3-dihydroxyoctadec-17 ~ in-2-yl] acetamide,
A/-[(2S,3R)-14-azido-1 ,3-dihídroxitetradecan-2-íi]bromoacetamida, A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl] bromoacetamide,
A/-[(2S,3R,E)-14-azido-1 ,3-díhídroxitetradec-4-en-2-il]bromoacetamida, 2-bromo-A/-[(2S,3 £)-1 ,3-dihidroxioctadec-4-en-17-in-2-il]acetamida, A / - [(2S, 3R, E) -14-azido-1, 3-dihydroxytetradec-4-en-2-yl] bromoacetamide, 2-bromo-A / - [(2S, £ 3) -1, 3 -dihydroxyoctadec-4-en-17-in-2-yl] acetamide,
(S)-A/-(14-azído-1-hidroxí-3-oxotetradecan-2-il)-2-bromoacetamída, (S) -A / - (14-azido-1-hydroxy-3-oxotetradecan-2-yl) -2-bromoacetamide,
(RS)-/V-[(2S,3R,E)-1 ,3-dihidroxioctadec-4-en-2-ii]oxirano-2-carboxamida, (f?S)-A/-[(2S,3R)-1 ,3-dihidroxioctadecan-2-il]oxirano-2-carboxamida, (RS) - / V - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-2-ii] oxirane-2-carboxamide, (f? S) -A / - [(2S, 3R ) -1, 3-dihydroxyoctadecan-2-yl] oxirane-2-carboxamide,
(RS)-A/-[(2S,3 E)-1 ,3-dihidroxioctadec-4-en-17-in-2-il]oxirano-2- carboxamida, (RS)-/V-[(2S,3R)-14-azido-1 ,3-dihidroxitetradecan-2-il]oxirano-2-carboxamida, A/-[(2S,3R)-1 ,3-díhidroxioctadecan-2-il]propiolamida, (RS) -A / - [(2S, 3 E) -1, 3-dihydroxyoctadec-4-en-17-in-2-yl] oxirane-2-carboxamide, (RS) - / V - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl] oxirane-2-carboxamide, A / - [(2S, 3R) -1, 3-dihydroxyoctadecan- 2-il] propiolamide,
/V-[(2S,3R)-1 ,3-dihídroxioctadecan-2-il]but-2-inamida, / V - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] but-2-inamide,
A/-[(2S,3R)-1 ,3-dihidroxioctadecan-2-il]acniamida, A / - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl] acniamide,
(E)~A/-[(2S,3R)~1 ,3-díhídroxioctadecan~2-il]-2-butenamida, (E) ~ A / - [(2S, 3R) ~ 1,3-dihydroxyoctadecan ~ 2-yl] -2-butenamide,
A/-[(2S,3R)-1 ,3-dihidroxioctadecan-2-il3metacrilamida, A / - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yl3methacrylamide,
A/-[(2Sl3R)-N-1 ,3-dihidroxyoctadecan-2-il]-3-metil-2-butenamida, A / - [(2S l 3R) -N-1, 3-dihydroxyoctadecan-2-yl] -3-methyl-2-butenamide,
(2E,4E)-N-[(2S,3R)-1 ,3-dihidroxioGtadecan-2-il]hexa-2,4-dienamida, (2E, 4E) -N - [(2S, 3R) -1, 3-dihydroxyGtadecan-2-yl] hexa-2,4-dienamide,
Ácido (E)-4-[(2S,3R)-1 ,3-dihidroxioctadecan-2-iíamino]-4-oxo-2-butenoico, (Z)-2,3-dibromo-N-[(2S,3R)-1 ,3-dihidroxioctadecan-2-ii]-4-oxo-2-butenamida, (2S,3 ?)-2-(bromometi!)-N-(1 ,3-dihidroxioctadecan-2-il)acrilamida, (E) -4 - [(2S, 3R) -1, 3-dihydroxyoctadecan-2-yiamino] -4-oxo-2-butenoic acid, (Z) -2,3-dibromo-N - [(2S, 3R ) -1, 3-dihydroxyoctadecan-2-ii] -4-oxo-2-butenamide, (2S, 3?) - 2- (bromometi!) - N- (1,3-dihydroxyoctadecan-2-yl) acrylamide,
(E,2S,3R)-/V-(1 ,3-dihidroxi-2-octadeGil)-2-metil-2-butenamida, (E, 2S, 3R) - / V- (1,3-dihydroxy-2-octadeGil) -2-methyl-2-butenamide,
(2S,3R)-N-(1 ,3-dihidroxi-17-octadecin-2-il)-2-oxooctanamida y (2S, 3R) -N- (1,3-dihydroxy-17-octadecin-2-yl) -2-oxooctanamide and
(2S,3R)-N-(14-azido-1 ,3-díhidroxi-2-tetradecíl)-2-oxooctanamida, (2S, 3R) -N- (14-azido-1, 3-dihydroxy-2-tetradecyl) -2-oxooctanamide,
o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos. or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds.
21. - Un compuesto de fórmula (I) según la reivindicación 20, caractenzado porque dicho compuesto se selecciona de ía lista que consiste en: 21. - A compound of formula (I) according to claim 20, characterized in that said compound is selected from the list consisting of:
2-bromo-/V-[(2S,3R)-1 ,3-dihidroxioctadec-17-ín-2-il]acetamida, 2-Bromo- / V - [(2S, 3R) -1, 3-dihydroxioctadec-17-ín-2-yl] acetamide,
A/-[(2S,3R)-14-azido-1 ,3-dihídroxitetradecan-2-íl]bromoacetamída, A / - [(2S, 3R) -14-azido-1, 3-dihydroxytetradecan-2-yl] bromoacetamide,
A/-[(2S,3R;E)-14-azido-1 ,3-dihidroxitetradec-4-en-2-il]bromoacetamida, 2-bromo-A/-[(2S,3R,E)-1 ,3-dihidroxioctadec-4-en-17-in-2-il]acetamida y A / - [(2S, 3R ; E) -14-azido-1, 3-dihydroxytetradec-4-en-2-yl] bromoacetamide, 2-bromo-A / - [(2S, 3R, E) -1, 3-dihydroxyoctadec-4-en-17-in-2-yl] acetamide and
(S)-A/-(14-azido-1-hidroxi-3-oxotetradecan-2-ii)-2-bromoacetamida, (S) -A / - (14-azido-1-hydroxy-3-oxotetradecan-2-ii) -2-bromoacetamide,
o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos. or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds.
22. - Un compuesto de fórmula (I) según una cualquiera de las reivindicaciones 1 a 2, caracterizado porque dicho compuesto se selecciona de la lista que consiste en: 22. - A compound of formula (I) according to any one of claims 1 to 2, characterized in that said compound is selected from the list consisting of:
/V-[(2S,3S,4R)-1 ,3,4-trihidroxioctadecan-2-il]etanosulfonamida y (RS)-N-[(2S,3Sl4R)-1 ,3,4-trihidroxioctadecan-2-il]oxirano-2-carboxamida, o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos. / V - [(2S, 3S, 4R) -1, 3,4-trihydroxyoctadecan-2-yl] ethanesulfonamide and (RS) -N - [(2S, 3S 1 4R) -1, 3,4-trihydroxyoctadecan-2-yl] oxirane-2-carboxamide, or a pharmaceutically acceptable stereoisomer, salt or solvate of one of these compounds.
23.- Composición farmacéutica que comprende un compuesto de fórmula general (I) o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de éste tal como se ha definido en una cualquiera de las reivindicaciones 1 a 22, y ai menos un excipiente farmacéuticamente aceptable. 23. Pharmaceutical composition comprising a compound of general formula (I) or a stereoisomer, a pharmaceutically acceptable salt or solvate thereof as defined in any one of claims 1 to 22, and at least one pharmaceutically acceptable excipient. .
24. - Un compuesto de fórmula general (I) tai como se ha definido en una cualquiera de las reivindicaciones 1 a 22 o un compuesto elegido entre 2,2- dibromo-N-((2S,3R)-1 ,3-dihidroxioctadecan-2-ii)acetamida y 2-bromo-N- ((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamída o un estereoisómero, una sal o un solvato farmacéuticamente aceptable de uno de estos compuestos, para utilizar en el tratamiento o prevención de una enfermedad que cursa con hiperproliferación celular. 24. - A compound of general formula (I) tai as defined in any one of claims 1 to 22 or a compound selected from 2,2-dibromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan -2-ii) acetamide and 2-bromo-N- ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a pharmaceutically acceptable salt or solvate of one of these compounds, for use in the treatment or prevention of a disease that occurs with cellular hyperproliferation.
25. - Una composición tal como se define en la reivindicación 23 o una composición que comprende un compuesto elegido entre 2,2-dibromo-N-25. - A composition as defined in claim 23 or a composition comprising a compound chosen from 2,2-dibromo-N-
((2S,3R)-1 ,3-díhidroxioctadecan-2-il)acetamida y 2-bromo-N-((2S,3R)-1 ,3- dihidroxioctadecan-2-il)acetamida o un estereoisómero, una sai o un solvato farmacéuticamente aceptable de uno de estos compuestos y al menos un excipiente, para utilizar en el tratamiento o prevención de una enfermedad que cursa con hiperproliferación celular. ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2-bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a sai o a pharmaceutically acceptable solvate of one of these compounds and at least one excipient, for use in the treatment or prevention of a disease that occurs with cellular hyperproliferation.
26. - Un compuesto según la reivindicación 24 o una composición según la reivindicación 25, para utilizar en el tratamiento o prevención de una enfermedad cursada con hiperproliferación celular elegida entre cáncer, metástasis, inflamación, asma y arteríosclerosis. 26. - A compound according to claim 24 or a composition according to claim 25, for use in the treatment or prevention of a disease carried out with cellular hyperproliferation chosen from cancer, metastasis, inflammation, asthma and arteriosclerosis.
27. - Un compuesto o una composición según la reivindicación 26, para utilizar en el tratamiento de un tipo de cáncer elegido entre cáncer de próstata, de páncreas, de cerebro, de colon, de pulmón, de mama, de cabeza y cuello, de ovario, de laringe, de vejiga urinaria, de útero, de piel, sarcomas, linfomas y leucemia. 27. - A compound or a composition according to claim 26, for use in the treatment of a type of cancer chosen from prostate, pancreas, brain, colon, lung, breast, head and neck cancer, of ovary, larynx, urinary bladder, uterus, skin, sarcomas, lymphomas and leukemia.
28. - Un compuesto o una composición según la reivindicación 27, para utilizar en el tratamiento de un tipo de cáncer elegido entre cáncer de próstata y cáncer de pulmón. 28. - A compound or a composition according to claim 27, for use in the treatment of a type of cancer chosen from prostate cancer and lung cancer.
29. - Un compuesto de fórmula general (I) tai como se ha definido en una cualquiera de las reivindicaciones 1 a 22 o un compuesto elegido entre 2,2- dibromo-N-((2S,3R)-1 ,3-dihidroxioctadecan-2-ii)acetamida y 2-bromo-N- ((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamída o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de uno de estos compuestos, para utilizar en combinación con otra terapia para el tratamiento de una enfermedad tal como se define en una cualquiera de las reivindicaciones 24, 26 a 28. 29. - A compound of general formula (I) tai as defined in any one of claims 1 to 22 or a compound selected from 2,2-dibromo-N - ((2S, 3R) -1, 3-dihydroxyoctadecan -2-ii) acetamide and 2-bromo-N- ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds, for use in combination with another therapy for the treatment of a disease as defined in any one of claims 24, 26 to 28.
30.- Una composición tai como se define en la reivindicación 23 o una composición que comprende un compuesto elegido entre 2,2-dibromo-N- ((2S,3R)-1 ,3-dihidroxioctadecan-2-il)acetamida y 2-bromo-N-((2S,3R)-1 ,3- díhidroxioctadecan~2-íl)acetamida o un estereoisómero, una sal o un soivato farmacéuticamente aceptable de uno de estos compuestos al menos un excipiente, para utilizar en combinación con otra terapia para el tratamiento de una enfermedad tal como se define en una cualquiera de las reivindicaciones 25, 26 a 28. 30. A tai composition as defined in claim 23 or a composition comprising a compound selected from 2,2-dibromo-N- ((2S, 3R) -1, 3-dihydroxyoctadecan-2-yl) acetamide and 2 -bromo-N - ((2S, 3R) -1, 3- dihydroxyoctadecan ~ 2-yl) acetamide or a stereoisomer, a pharmaceutically acceptable salt or soivate of one of these compounds at least one excipient, to be used in combination with another therapy for the treatment of a disease as defined in any one of claims 25, 26 to 28.
PCT/ES2012/070485 2011-07-01 2012-06-29 Amides of 2-amino-1,3-propanediols and use thereof as ceramidase inhibitors WO2013004871A1 (en)

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