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    • 专利摘要:
    The present invention relates to zwitterions of saturated, monounsaturated, polyunsaturated carboxylic 2-phosphocholine acids and their acid derivatives with a structure described in the general formula (1) {IMAGE-01} and its derivatives, in which A is a carbon chain with a carbon number between 4 and 19, saturated, monounsaturated or polyunsaturated, mono or polyhydroxylated in different positions or not hydroxylated; and (R) can be any atom or group of atoms with a weight between 1 and 200 Da, where (R) is selected from: COOH, COOMe, COOEt, COOBut, COOBu, COOPri, COOBui, COOPr, COSEt, COSMe, COSPr, COSBu, COSPri, CSNR2 R 3, CONR2 R3, CN and COOM, where M is a cation derived from alkali metal or alkaline earth metal, such as lithium, sodium, potassium, cesium, magnesium or calcium, R2 and R3 can be the same or different and represent an alkyl radical, pharmaceutically acceptable, and their use against different types of carcinogenic cells . (Machine-translation by Google Translate, not legally binding)
    公开号:ES2739773A1
    申请号:ES201830802
    申请日:2018-08-02
    公开日:2020-02-03
    发明作者:Garcia Francisco Foubelo；Gil Jose Miguel Sansano；Salvador Jose Javier Soto；Segura Luis Botella；Serrano Jaime Jaisiel Melendez；Jimenez Daniel Garcia
    申请人:Universidad de Alicante；
    IPC主号:
    专利说明:

    [0001] ZWITTERIONIC COMPOUNDS OF CARBOXYLIC 2-PHOSPHOCOLINE ACIDS
    [0002]
    [0003] Zwitterionic compounds of 2-phosphocholine carboxylic acids and their use as cytotoxic agents
    [0004]
    [0005] FIELD OF THE INVENTION
    [0006]
    [0007] The present invention is framed both in the field of pharmaceutical chemistry and in medicine, more specifically it refers to compounds useful as cytotoxic agents, and more specifically, zwitterionic compounds derived from 2-phosphocholine.
    [0008]
    [0009] STATE OF THE PREVIOUS TECHNIQUE
    [0010]
    [0011] Phospholipids are structures derived from lipids formed by a hydrophobic zone, generally constituting two fatty acid chains, and a hydrophilic zone, formed by a phosphate group. Such structures mainly form cell membranes due to their amphiphilic nature.
    [0012]
    [0013] Phosphatidylcholines are a type of phospholipids in which the hydrophilic part is formed by a phosphocholine group, also forming biological membranes. Phosphocholine is formed by a phosphate group that supports a negative charge, linked to a choline unit, which supports a positive charge. Both phosphatidylcholines and sphingomyelins are the only phospholipid derivatives that do not contain glycerol as the main skeleton.
    [0014]
    [0015] Examples of phospholipid ester derivatives (US 2008/0175852 A1) or phosphorylpantoamide derivatives (DE 3728915), among others, have been found among the phospholipid structures.
    [0016]
    [0017] Another derivative of phosphatidylcholine are lysophosphatidylcholines, resulting from the loss of one of the fatty acids through partial hydrolysis, and their analogues. synthetic antitumor alkylsophospholipids (ALPs), generated by the exchange of the acyl group for the alkyl group, such as Edelfosine and Miltefosine. These two products have shown high metabolic stability and anticancer effects, by selective apoptosis of tumor cells, as well as immunomodulatory or antiprotozoal effectiveness ( CancerLetters, 2017, 388, 262-268.), ( International Journal of AntimicrobialAgents, 2017, 49, 465 -471) (Mollinedo et al., 1997. CancerRes . 57 : 1320 1328; Gajate et al., 2004. J Exp. Med. 200: 353-365). However, these compounds have drawbacks such as their poor water solubility or their high toxicity.
    [0018]
    [0019] US5049552A refers to the use of hexadecylphosphocholine (Miltefosine) in the treatment of cancer.
    [0020]
    [0021] EP1079838A1 discloses the use of Edelfosine in the treatment of brain tumors. WO2012095500A2 describes a pharmaceutical composition of oleyl phosphocholine useful in the treatment of cancer, or parasitic or skin diseases.
    [0022]
    [0023] JP55118494 describes phosphocholine derivatives with antitumor activity.
    [0024]
    [0025] In contrast, the present invention relates to compounds with a phosphocholine unit linked to the derivative of a saturated, monounsaturated or polyunsaturated fatty acid through the hydroxyl group located in the alpha position to the carbonyl group.
    [0026]
    [0027] As described in WO 2010/066931 these hydroxylated fatty acids in the position a (the position adjacent to the carboxyl), saturated, monounsaturated, or polyunsaturated, regulate the composition and structure of membrane lipids in all cells preventing or reversing diseases such as cancer, cardiovascular pathologies, neurodegenerative processes, obesity, metabolic disorders, inflammations or autoimmune diseases, among others. In these derivatives, both the double bond and the substituent located in the alpha carbon to the carbonyl carbon are essential for the proper functioning of the structure, since a greater curative or preventive activity has been observed with respect to structures without These two specific characteristics. In addition, it has been observed that the substituent in the 2 position blocks the degradation of the fatty acid or its derivative and allows it to remain prolonged, with respect to the fatty acid without said substituent, in the cellular medium, allowing its therapeutic action. WO 2010/066931 discloses the starting material of compound MCH-811 partially responsible for the activity. The synthesis of phosphatidylcholines has been addressed using different strategies such as the direct addition of phosphocholine or one of its salts or the step formation of phosphocholine on the substrate, among others, and different reagents or catalysts such as 2,4 chloride , 6-triisopropylbenzenesulfonyl or charge transfer complexes.
    [0028]
    [0029] Phosphatidic acid has also been used in the synthesis of phosphatidylcholines, using a condensing agent such as 2,4,6-triisopropylbenzenesulfonyl chloride, with the drawback of the difficulty in generating iodoform and its subsequent elimination of salt. choline, or choline tetraphenylborate ( Journal of LipidResearch. 1984, 25, 1140-1142.), used in conjunction with a condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride.
    [0030]
    [0031] Derivatives of p-nitrophenyl-O-phosphochinohydroxylalkyl esters (US2008 / 0175852 A1) have also been developed from alkyl esters with a good leaving group in the final methylene of the carbon chain and phosphocholine resulting from the corresponding phosphocholine alkanoate after treatment with a quaternary amine. This phosphocholine alkanoate is esterified or transesterified with depnitrophenyl trifluoroacetate to obtain the desired product. In this US patent application the use of the crown compounds is described and they also employ quite high temperature conditions, between 110 and 120 ° C. They are quite energetic reaction conditions that do not allow the synthesis of sensitive compounds. When there are double bonds the oxidation is very fast at that temperature, especially when in polyunsaturated fatty acids such as DHA, EPA or linoleic. However, our methodology works at room temperature at all times, mild conditions.
    [0032]
    [0033] In the present invention the synthesis of the presented product consists of two steps with mild reaction conditions, the first of addition of the dichlorophosphate of 2-Bromoethyl (or chloroethyl or iodoethyl) on the hydroxyl group located in alpha position to the carbonyl group and the second substitution step of the bromine, iodine or chlorine atom, by a trimethylamino group. These conditions improve the synthetic operation on a larger scale and the execution times are shorter compared to that described in US 2008/0175852 A1.
    [0034]
    [0035] It should be noted that cancer is, behind cardiovascular diseases, the second leading cause of death in the world, causing 14 million deaths in 2012 To treat this disease, anticancer agents capable of selectively attacking cancer cells are left leaving cells healthy unchanged. In this sense, lipid derivatives of different nature have demonstrated their antitumor efficacy, among other applications, as developed below.
    [0036]
    [0037] In recent years, phospholipid derivatives have been developed that have had different applications such as transdermal and transmembrane (US5,985,292 from Fournerou et al.), ( Journal of Controlled Release, 1998 , 51, 259-267), anticancer or for the treatment or prevention of atherosclerosis and other related disorders as well as inflammatory processes, autoimmune diseases and proliferative disorders among others.
    [0038]
    [0039] In the present invention activity has also been detected against different types of tumor cells.
    [0040]
    [0041] Therefore, it is necessary in the light of the above, to look for new chemical formulations, economical, synthetically simple to carry out and with a high activity against different tumor cell lines.
    [0042]
    [0043] BRIEF DESCRIPTION OF THE INVENTION
    [0044]
    [0045] The present invention refers to the Zwitterions of saturated, monounsaturated, polyunsaturated carboxylic 2-phosphocholine acids, and their pharmaceutically acceptable derivatives, with a structure as described in the general formula (1) and its derivatives, as detailed below.
    [0046] The present invention also refers to the synthesis of the compounds of the general formula (1), a synthesis consisting of 2 reaction steps (Schemes 1 and 2) where the corresponding 2-hydroxy derived from saturated, monounsaturated acid is transformed, polyunsaturated or its derivatives (esters, thioesters, amides, thioamides, nitriles or metal salts among others), in the Zwitterion of saturated, monounsaturated or polyunsaturated carboxylic acid 2-phosphocholine or one of its derivatives (esters, thioesters, amides, thioamides, nitriles or metal salts among others).
    [0047]
    [0048] DETAILED DESCRIPTION OF THE INVENTION
    [0049]
    [0050] Here the term "pharmaceutically acceptable derivative" refers for example to pharmaceutically acceptable salts, hydrates or polymorphs.
    [0051]
    [0052] Both the compounds of formula 1 defined in the first claim and the pharmaceutically acceptable derivatives may be in the crystalline, amorphous or polymorphic state.
    [0053]
    [0054] Here the term "polyunsaturated" refers to the compound comprising more than one double bond, or triple bond or more than one triple bond, or mixtures thereof. The present invention refers, in a first aspect, to a compound general formula (1)
    [0055]
    [0056]
    [0057]
    [0058] in which:
    [0059] - R can be any atom or group of atoms with a weight between 1 and 200 Da, - and A is a carbon chain that can have any number of carbon atoms between 4 and 19, saturated, monounsaturated or polyunsaturated, in which the double bonds C = C can have any type of configuration and be or not conjugated, and in which the chain A can be without hydroxylated or hydroxylated (mono or polyhydroxylated in different positions),
    [0060] and its pharmaceutically acceptable derivatives, such as salts, hydrates corresponding to said salts, or any polymorph.
    [0061]
    [0062] R is preferably selected from: COOH, COOMe, COOEt, COOBu *, COOBu, COOPr *, COOBui, COOPr, COSEt, COSMe, COSPr, COSBu, COSPr *, CN, CSNR2R3, CONR2R3, and COOM, where M is a cation derived from alkali or alkaline earth metal, such as lithium, sodium, potassium, cesium, magnesium or calcium, R2 and R3 can be the same or different and represent an alkyl radical, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl.
    [0063]
    [0064] In the above formula (1) saturated, monounsaturated, polyunsaturated carboxylic 2-phosphocholine acids and their acid derivatives such as salts, hydrates of said salts, thioesters, esters, amides, thioamides, nitriles are generally represented.
    [0065]
    [0066] Particular embodiments of the invention refer to a compound of formula (1), in which the hydrocarbon chain of A has a number of between 4 and 19 carbons. Further particular embodiments of the invention refer to a compound of formula (1), in which the hydrocarbon chain of A has a number of between 13 and 19 carbons.
    [0067]
    [0068] Additional particular embodiments of the invention refer to a compound of formula (1), in which A is a saturated, monounsaturated or polyunsaturated carbon chain, with any type of configuration in the double bonds C = C, mono or polyhydroxylated in different positions , or not hydroxylated.
    [0069]
    [0070] Preferred embodiments of the invention refer to a compound of formula (1), in which A is a fragment of fifteen carbon atoms containing a double bond C = C between C9-C10 carbons with Z configuration.
    [0071]
    [0072] Additional preferred embodiments of the invention refer to a compound of formula (1), in which R represents an ethyl ester.
    [0073] An especially preferred embodiment of the invention relates to a compound of formula (1), in which A is a fragment of fifteen carbon atoms containing a double bond C = C between the C9-C10 carbons with Z configuration and the group R represents a COOEt ethyl ester (it has been referred to as the MCH-811 molecule). The antitumor activity has been studied by measuring in vitro the IC50 index of different tumor cell lines.
    [0074]
    [0075] The characteristics of the compounds of formula (1) give the molecule a preventive or curative activity in the micromolar range for a wide variety of cancer cell lines (IC50 up to 7.06 ± 1.45 ^ M, see table 3), a lower degradation in the biological environment which implies a greater therapeutic effect, as well as a greater specificity towards the lipid bilayer of the cells and their action on them.
    [0076]
    [0077] Likewise, the present invention refers to the synthesis of said compounds of formula (1) through 2 reaction steps.
    [0078]
    [0079] According to Schemes 1 and 2, the corresponding 2-hydroxy derivative of saturated, monounsaturated or polyunsaturated acid is transformed into the zwitterion of saturated, monounsaturated, polyunsaturated 2-phosphocholine carboxylic acid or its derivative.
    [0080]
    [0081] The synthesis of the molecules of the present invention (1) consists of two simple steps performed under mild reaction conditions.
    [0082]
    [0083] The process for preparing a compound defined in one of the preceding claims, of formula (1) comprises:
    [0084] - generate an intermediate compound of general formula (2)
    [0085]
    [0086]
    [0087]
    [0088]
    [0089] wherein A and R have the meanings given for the compound of formula (1),
    [0090] by the addition of 2-bromoethyl dichlorophosphate of formula (3), or its chlorinated or iodinated analog (previously synthesized or in situ),
    [0091]
    [0092] on the hydroxyl group located in alpha position to the carbonyl group, of a compound of formula (4)
    [0093]
    [0094]
    [0095]
    [0096]
    [0097]
    [0098] wherein, A and R have the meanings given for the compound of formula (1), - substitution of the bromine atom, (chlorine or iodine) of the intermediate compound of formula (2) by a trimethylamino group and formation of the corresponding betaine .
    [0099]
    [0100] In the second step the product of the present invention (1) is obtained from the intermediate of the previous step (2) when reacting with trimethylamine dissolved in an alcohol, under mild conditions between 0 ° C and 60 ° C for 1-5 days and subsequent treatment with a mild inorganic base such as sodium acetate, sodium or potassium carbonate, sodium or potassium acid phosphate, and preferably sodium or potassium bicarbonate.
    [0101]
    [0102] According to a particular embodiment of the process, the first step of the synthesis comprises the addition of 2-bromoethyl dichlorophosphate (3) (or its chlorinated or iodinated analog) on the hydroxyl group located in the alpha position to the carbonyl group of the fatty acid, its derivative - monounsaturated or polyunsaturated (4) in the presence of an apolar solvent, such as toluene, xylene, diethyl ether, tert-butylmethyl ether (MTBE), tetrahydrofuran, heptane, hexane, a base, such as triethylamine, diisopropylethylamine (DIPEA), tributylamine , trimethylamine, N-methylpiperidine, 1,4-dimethylpiperazine, at room temperature (Scheme 1),
    [0103]
    [0104]
    [0105]
    [0106] Scheme 1
    [0107] where R and A, have the meanings given above.
    [0108]
    [0109] The second step consists in a substitution of the bromine atom (or chlorine or iodine) with a trimethylamino group, and subsequent formation of the corresponding zwitterion that is carried out on the product of the previous step (2) (Scheme 2). The reaction takes place in the presence of an apolar solvent, such as toluene, as a solvent at room temperature using a solution of trimethylamine as an aminating agent, in an alcohol, such as methanol, n-butanol, isopropanol, ethylene glycol, and preferably ethanol, followed of the sodium bicarbonate treatment to form the corresponding zwitterion.
    [0110]
    [0111]
    [0112]
    [0113]
    [0114]
    [0115] Scheme 2
    [0116]
    [0117] The present invention also relates to a pharmaceutically acceptable formulation comprising a compound of formula (1).
    [0118]
    [0119] The formulations of the compounds of the invention can be, for example, capsules with the following excipients: colloidal anhydrous silica, microcrystalline cellulose, lactose monohydrate, talc, magnesium stearate, gelatin, titanium dioxide, ferric oxide, purified water. They can also be oral formulations with the following excipients: hydroxypropyl cellulose, propylene glycol and purified water.
    [0120]
    [0121] It was observed that the molecule of general formula (1) shows in vitro activity against different types of carcinogenic cells, for example against tumor cell lines of: lung (non-small cell), pancreas, kidney, central nervous system (CNS) , prostate, colon, breast, melanoma, ovary, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, myeloma, immunoblastic large cell lymphoma, burkitt lymphoma, non-Hodgkin B-cell lymphoma, chronic lymphocytic leukemia, cell lymphoma mantle, multiple myeloma and acute T-cell leukemia (Tables 1,2 and 3).
    [0122] Furthermore, the present invention relates to the use of the compounds of formula (1) and their pharmaceutically acceptable derivatives as salts, hydrates or polymorphs, to be used as active species against different types of carcinogenic cells, preferably in prevention and / or treatment of cancers such as those mentioned in the preceding paragraph and in the claims.
    [0123]
    [0124] DETAILED DESCRIPTION OF PREPARATION EXAMPLES
    [0125]
    [0126] Example 1: Preparation of compound (2a) [(A = (CH2) 5- (Z) -CH = CH (CH2) 7CH3, (R) = COOEt] (MCH-811)
    [0127]
    [0128] In a 1 l balloon, ethyl 2-hydroxy oleate (37 g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Then, 2-bromoethyl dichlorophosphate (35.61 ml, 136 mmol) is added dropwise onto this solution and stirred overnight (~ 16 h) at room temperature (~ 25 ° C). After this time the reaction crude is washed with brine (2 X 250 ml) in a separatory funnel. Ethyl acetate (100 ml) is added to the aqueous phase to extract, and the two organic phases are combined, dried with MgSO4, filtered and concentrated in vacuo. 57.1 g (98% yield) of product are obtained in the form of brown oil.
    [0129]
    [0130] Example 1B: Preparation of compound (2a) [A = (CH2) 5- (Z) -CH = CH (CH2) 7CH3, (R) = COOEt] (MCH-811). In situ preparation of 2-bromoethylchlorophosphate
    [0131]
    [0132] In a 1 l balloon, phosphorus oxychloride (136 mmol), bromoethanol (136 mmol) and triethylamine (44 ml, 250 mmol) in toluene are added. (420 mL) and stir until homogeneous. Then over this solution, add the ethyl 2-hydroxy oleate (37 g, 113 mmol), and stir overnight (~ 16 h) at room temperature (~ 25 ° C). After this time the reaction crude is washed with brine (2 X 250 ml) in a separatory funnel. Ethyl acetate (100 ml) is added to the aqueous phase to extract, and the two organic phases are combined, dried with MgSO 4 , filtered and concentrated in vacuo. 57.1 g (98% yield) of product are obtained in the form of brown oil.
    [0133] Example 2: Preparation of compound (1a) [A = (CH2) 5- (Z) -CH = CH (CH2) 7CH3, (R) = COOEt] (MCH-811)
    [0134]
    [0135] The product of the previous reaction (2a, 57.1 g, 111 mmol) is dissolved with toluene (180 ml) in a 500 ml balloon. To this solution, a solution of trimethylamine in 4.2M ethanol (150 ml, 628 mmol) is added and stirred at room temperature (~ 25 ° C) for 3 days. After this time NaHCO3 (10 g, 119 mmol) and water (10 ml) are added and stirred for 2 hours. The crude is concentrated in vacuo and purified by chromatographic column (DCM / MeOH / NH3). 32 g (59% yield) of the pure product are obtained.
    [0136]
    [0137] Example 3: Preparation of compound (2b) [A = (CH2) 5- (Z) -CH = CHCH2CH (OH) (CH2) 5CH3, (R) = COOEt]
    [0138]
    [0139] In a 1 l balloon are added 12 - ((Terc-butyldimethylsilyl) oxy) 2-hydroxy ethyl oleate (51.62 g, 113 mmol). Continue as in example 1.
    [0140]
    [0141] Example 4: Preparation of compound (1b) [A = (CH2) 5- (Z) -CH = CHCH2CH (OH) (CH2) 5CH3, (R) = COOEt]
    [0142]
    [0143] With the product of the previous example 2b, we proceed as in example 2. Furthermore, in this case the protection of the TBDMS group should be continued and carried out using a fluorinated compound, such as tetrabutylammonium fluoride.
    [0144]
    [0145] Example 5: Preparation of compound (2c) [A = (CH2) 12CH3, (R) = COOEt]
    [0146]
    [0147] In a 1 l balloon the ethyl 2-hydroxy palmitate (34.0g, 113 mmol) is added, continued as in example 1.
    [0148]
    [0149] Example 6: Preparation of compound (1c) [A = (CH2) 12CH3, (R) = COOEt]
    [0150]
    [0151] With the product of the previous example 2c, proceed as in example 2.
    [0152] Example 7: Preparation of compound (2d) [A = (CH2) 14CH3, (R) = COOEt]
    [0153]
    [0154] In a 1 l balloon, ethyl 2-hydroxy stearate (37.1g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0155]
    [0156] Example 8: Preparation of compound (1d) [A = (CH2) 14CH3, (R) = COOEt]
    [0157]
    [0158] With the product of the previous example 2d, proceed as in example 2.
    [0159]
    [0160] Example 9: Preparation of compound (2e) [A = (CH2) 9- (Z) -CH = CH (CH2) 7CH3, (R) = COOEt]
    [0161]
    [0162] In a 1 l balloon, ethyl 2-hydroxyeuricate (43.23g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0163]
    [0164] Example 10: Preparation of compound (1e) [A = (CH2) 9- (Z) -CH = CH (CH2) 7CH3, (R) = COOEt]
    [0165]
    [0166] With the product of the previous example 2e, proceed as in example 2.
    [0167]
    [0168] Example 11: Preparation of compound (2f) [A = (CH2) 4- (Z, Z) -CH = CHCH2CH = CH (CH2) 5CH3, (R) = COOEt]
    [0169]
    [0170] In a 1 l balloon, ethyl 2-hydroxylininoleate (36.67 g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0171]
    [0172] Example 12: Preparation of compound (1f) [A = (CH2) 4- (Z, Z) -CH = CHCH2CH = CH (CH2) 5CH3, (R) = COOEt]
    [0173]
    [0174] With the product of the previous example 2f, proceed as in example 2.
    [0175] Example 13: Preparation of compound (2g) [A = (CH2) 2- (Z, Z, Z) -CH = CHCH2CH = CHCH2CH = CH (CH2) 4CH3, (R) = COOEt]
    [0176]
    [0177] In a 1 l balloon, ethyl 2-hydroxy-gamma-linoleate (36.44g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0178]
    [0179] Example 14: Preparation of compound (1g) [A = (CH2) 2- (Z, Z, Z) -CH = CHCH2CH = CHCH2CH = CH (CH2) 4CH3, (R) = COOEt]
    [0180]
    [0181] With the product of the previous example 2g, proceed as in example 2.
    [0182]
    [0183] Example 15: Preparation of compound (2h) [A = (CH2) 5- (Z, Z, Z) -CH = CHCH2CH = CHCH2CH = CHCH2CH3, (R) = COOEt]
    [0184]
    [0185] In a 1 l balloon, ethyl 2-hydroxy-alpha-linoleate (36.44 g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0186]
    [0187] Example 16: Preparation of compound (1h) [A = (CH2) 5- (Z, Z, Z) -CH = CHCH2CH = CHCH2CH = CHCH2CH3, (R) = COOEt]
    [0188]
    [0189] With the product of the previous example 2h, proceed as in example 2.
    [0190]
    [0191] Example 17: Preparation of compound (2i) [A = (CH2) 5- (Z, E, Z) -CH = CHCH = CHCH = CH (CH2) 3CH3, (R) = COOEt]
    [0192]
    [0193] In a 1 l balloon, ethyl 2-hydroxypunicate (36.44 g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0194]
    [0195] Example 18: Preparation of compound (1i) [A = (CH2) 5- (Z, E, Z) -CH = CHCH = CHCH = CH (CH2) 3CH3, (R) = COOEt]
    [0196] With the product of the previous example 2i, proceed as in example 2.
    [0197]
    [0198] Example 19: Preparation of compound (2j) [A = CH2- (Z, Z, Z, Z) - [CH = CHCH2] 4 (CH2) 4CH3, (R) = COOEt]
    [0199]
    [0200] In a 1 l balloon, the ethyl 2-hydroxyaraquidonate (39.38g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0201]
    [0202] Example 20: Preparation of compound (1j) [A = CH2- (Z, Z, Z, Z) - [CH = CHCH2] 4CH3, (R) = COOEt]
    [0203]
    [0204] With the product of the previous example 2j, proceed as in example 2.
    [0205]
    [0206] Example 21: Preparation of compound (2k) [A = CH2- (Z, Z, Z, Z, Z) - [CH = CHCH2] 5CH3, (R) = COOEt]
    [0207]
    [0208] In a 1 l balloon, ethyl 2-hydroxyeicosapentaenoate (39.16g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) are added and stirred until homogeneous. Continue as in example 1.
    [0209]
    [0210] Example 22: Preparation of compound (1k) [A = CH2- (Z, Z, Z, Z, Z) - [CH = CHCH2] 5CH3, (R) = COOEt]
    [0211]
    [0212] With the product of the previous example 2k, proceed as in example 2.
    [0213]
    [0214] Example 23: Preparation of compound (2l) [A = (Z, Z, Z, Z, Z, Z) - [CH = CHCH2] 6CH3, (R) = COOEt]
    [0215]
    [0216] In a 1 l balloon, add the ethyl 2-hydroxidecosahexaenoate (42.10g, 113 mmol), toluene (420 ml) and triethylamine (22 ml, 125 mmol) and stir until homogeneous. Continue as in example 1.
    [0217]
    [0218] Example 24: Preparation of compound (1l) [A = (Z, Z, Z, Z, Z, Z) - [CH = CHCH2] 6CH3, (R) = COOEt]
    [0219] With the product of the previous example 2l, proceed as in example 2.
    [0220] Scheme 3. Representation of the products of formula (1).
    [0221]
    [0222]
    [0223]
    [0224]
    [0225] Example 25: In vitro efficacy studies of MCH-811
    [0226] The cytotoxic activity of the molecule that describes the present invention was tested. using different human tumor lines to establish its antitumor efficacy. The in vitro activity test was performed as detailed below:
    [0227]
    [0228] Cells of the different tumor types were treated with compound MCH-811 with known concentrations, between 0.1 and 200 ^ M, for 72 hours. At the end of the incubation period, the alamarBlue® reagent (1/10 of the volume) was added directly to the cells in culture medium and 4 hours later the fluorescence was read, using an excitation wavelength of 485 nm and an emission wavelength of 595 nm.
    [0229]
    [0230] Cell viability was calculated by comparing the fluorescence values of each concentration with the fluorescence values of the untreated cell culture (100% viability). The IC50 values were estimated using GraphPadPrism software (v5).
    [0231]
    [0232] 3.1.- In vitro studies with human tumor cell lines of the pancreas (MiaPaca-2, PANC-1, BxPC-3)
    [0233]
    [0234] Cell proliferation assays were carried out with alamarBlue® at 72 h with different tumor lines of the pancreas. 750 MiaPaca-2 cells, 3,500 PANC-1 cells and 3,500 BxPC-3 cells were seeded per well in 96-well plates and the following IC50 values (50% inhibitory concentration, Mean ± SD) were obtained, calculated as the concentration of compound that causes 50% inhibition in cell proliferation (Table 1):
    [0235]
    [0236]
    [0237]
    [0238] Table 1. Efficacy of MCH-811 in different tumor cell lines of the pancreas.
    [0239] 3.2.- Studies in vitro with human tumor cell lines of lymphoma (SR, NAMALWA, DAUDI, DOHH2, JVM-2, KARPAS422)
    [0240]
    [0241] Cell proliferation assays were conducted with alamarBIue® at 72 h with different human tumor lines of various types of lymphomas. In 96-well plates, 6000 SR cells, 5000 NAMALWA and DAUDI cells and 10000 DOHH2, JVM-2 and KARPAS422 cells per well were seeded and the following 50% inhibitory concentration values (IC50, Mean ± SD) were obtained, calculated as The concentration of compound that causes 50% inhibition in cell proliferation (Table 2):
    [0242]
    [0243]
    [0244]
    [0245] Table 2. Efficacy of MCH-811 in different tumor cell lines of lymphoma. In brackets, the number of determinations is indicated when there are more than 2.
    [0246]
    [0247] 3.3.- Studies in vitro with other human tumor cell lines
    [0248] In vitro activity assays were performed as detailed above, adjusting the seeding density of the different tumor cell lines. The following table (Table 3) shows the IC50 values (concentration of product capable of inhibiting 50% of the tumor cell culture) expressed as Mean ± SD that were obtained for the different tumor lines studied.
    [0249]
    [0250]
    [0251]
    [0252]
    [0253] Table 3. Efficacy of MCH-811 in different human cell lines corresponding to other types of tumors.
    权利要求:
    Claims (22)
    [1]
    1. A 2-phosphocholine carboxylic acid zwitterionic compound of general formula (1)

    [2]
    2. The compound according to claim 1, wherein A has a number of carbon atoms between 13 and 19.
    [3]
    3. The compound according to one of the preceding claims, wherein R is selected from: COOH, COOMe, COOEt, COOBu *, COOBui, COOPri, COOBu, COOPr, COOPri, COSEt, COSMe, COSPr, COSBu, COSPH, CN , CSNR 2 R 3 , CONR 2 R 3 , and COOM, where M is a cation derived from alkali or alkaline earth metal, such as lithium, sodium, potassium, cesium, magnesium or calcium, R2 and R3 may be the same or different and they represent an alkyl radical, preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl or hexyl.4
    [4]
    4. The compound according to one of the preceding claims, wherein A is a saturated, monounsaturated or polyunsaturated carbon chain, with any type of configuration in the double bonds C = C, mono or polyhydroxylated in different positions, or not hydroxylated.
    [5]
    5. The compound according to one of the preceding claims, wherein R is an atom or group of atoms with a weight between 1 and 200 Da.
    [6]
    6. The compound according to one of the preceding claims, wherein:
    - "A" is a monounsaturated hydrocarbon chain.
    [7]
    7. The compound according to one of the preceding claims, wherein A is a fragment of fifteen carbon atoms containing a double bond C = C between the carbon atoms C9-C10 and with Z configuration.
    [8]
    8. The compound according to one of the preceding claims, wherein R represents an ethyl ester, R = COO-CH2-CH3.
    [9]
    9. The compound according to claim 1, wherein:
    -A is a fragment of fifteen carbon atoms containing a double bond C = C between C9-C10 carbon atoms with Z configuration.
    - R represents an ethyl ester, R = COO-CH2-CH3.
    [10]
    10. The compound according to claim 1, wherein the pharmaceutically acceptable derivative is a salt, a hydrate of said salt or a polymorph.
    [11]
    11. A process for preparing a compound defined in one of the preceding claims, of formula (1) comprising:
    - the addition of 2-bromoethyl dichlorophosphate of formula (3), or its chlorinated or iodinated analog
    OR
    CkII
    P ' Br / CI / l i
    Cl
    3
    on the hydroxyl group located in alpha position to the carbonyl group, of a compound of formula (4)
    in which:
    - A is a carbon chain with a number of carbon atoms between 4 and 19, saturated, monounsaturated or polyunsaturated, in which the configuration in the double bonds C = C is Z or E, mono or polyhydroxylated in different positions, or not hydroxylated, - R is an atom or group of atoms with a weight between 1 and 200 Da, generating an intermediate compound of general formula (2)

    [12]
    12. The method according to claim 11, wherein after substitution on the bromine, chlorine or iodine atom by a trimethylamino group, the corresponding betaine is formed by treatment with a base.
    [13]
    13. The process according to claim 11, wherein the base is sodium bicarbonate.
    [14]
    14. The process according to claim 11, wherein the addition of 2-bromoethyl, 2-chloroethyl or 2-iodoethyl dichlorophosphate, of formula (2) is carried out in the presence of an apolar solvent, and a base at room temperature.
    [15]
    15. The process according to claim 14, wherein the apolar solvent is toluene, the base is triethylamine and R is COOEt.
    [16]
    16. The method according to claim 11 wherein the compound of formula (2): -A is a fragment of fifteen carbon atoms containing a double bond C = C between C9-C10 Carbon atoms with Z configuration, and
    - R represents a COOEt group.
    [17]
    17. A pharmaceutically acceptable formulation comprising a compound of formula (1) defined in claim 1.
    [18]
    18. Use of a compound of formula (1) defined in claim 1 in therapy or disease prevention.
    [19]
    19. Use of a compound of formula (1) according to claim 18 as a cytotoxic agent.
    [20]
    20. Use of a compound of formula (1) according to claim 19, in the prevention or treatment of cancer.
    [21]
    21. Use according to claim 20, wherein the cancer is cancer selected from non-small cell lung cancer, pancreas, kidney, central nervous system (CNS), prostate, colon, breast, melanoma, ovary, acute lymphoblastic leukemia, acute promyelocytic leukemia, chronic myelogenous leukemia, myeloma, immunoblastic large cell lymphoma, Burkitt lymphoma, non-Hodgkin B-cell lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, multiple myeloma and acute T-cell leukemia.
    [22]
    22. Use according to claim 21, wherein the cancer is pancreatic cancer or lymphoma.
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    同族专利:
    公开号 | 公开日
    ES2739773B2|2020-11-17|
    WO2020025848A1|2020-02-06|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    EP0121088A1|1983-03-05|1984-10-10|A. Nattermann & Cie. GmbH|New o-acyl-alkanediol-phospholipids, processes for their preparation and pharmaceutical preparations containing them|
    AT393505B|1987-04-27|1991-11-11|Max Planck Gesellschaft|Medicinal products which contain alkylphosphoamines in combination with an alkylglycerol|
    WO2007071658A2|2005-12-19|2007-06-28|Æterna Zentaris Gmbh|Alkyl phospholipid derivatives with reduced cytotoxicity and uses thereof|
    WO2010050918A1|2008-10-31|2010-05-06|Qinghai Zhang|Branched chain detergents for membrane protein structural biology|
    WO2012095500A2|2011-01-14|2012-07-19|Dafra Pharma Research&Development Bvba|Pharmaceutical compositions for the treatment of parasitic diseases, cancer, or skin diseases by topical administration|
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