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    • 专利摘要:
    Compounds of pharmaceutical interest. The compounds of the present invention can be defined as derivatives of lithocolic acid and its metabolites. For this reason the compounds of the invention are named using the steroid numbering system. More particularly, it is directed to compounds derived from lithocolic acid, to the processes for obtaining them, to intermediate compounds of their synthesis, and to their applications. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2769905A1
    申请号:ES201831298
    申请日:2018-12-28
    公开日:2020-06-29
    发明作者:Ewa Marcinkowska；Klaudia Berkowska；Mosquera Antonio Mouriño；Ponte Rita Sigüeiro；Sunil Gaikwad
    申请人:Universidade de Santiago de Compostela；Univ Of Wroclaw；Uniwersytet Wroclawski；
    IPC主号:
    专利说明:

    [0001] Compounds of pharmaceutical interest
    [0003] Technical sector
    [0005] The present invention is directed to compounds of pharmaceutical interest. More particularly, it is directed to compounds derived from lithocolic acid, to the processes for obtaining them, to intermediate compounds of their synthesis, and to their applications.
    [0007] Background
    [0009] 1,25a-dihydroxyvitamin D 3 (1,25D) is the most active metabolite of vitamin D. It exerts its biological actions by specifically binding to its nuclear receptor, the vitamin D receptor (VDR). The endocrine system of vitamin D plays a fundamental role in the regulation of phosphorus-calcium metabolism, stimulating the intestinal absorption of these essential minerals and their mobilization in bone tissue. Although the actions on the metabolism of phosphate and calcium are the best known, epidemiological, biochemical, cellular, or molecular genetic studies have shown their involvement in other physiological processes, by inhibiting proliferation and inducing cellular differentiation, and pathological, such as psoriasis, diabetes, osteoporosis, autoimmune, degenerative, endocrine, cardiovascular, infectious, or tumor diseases.
    [0011] VDR also functions as a receptor for lithocolic acid (ACL), a hepatotoxic and potentially carcinogenic secondary bile acid (Masuno, et al., Journal of Lipid Research, 2013, volume 54, 2206-2213; Deluca, et al., PNAS, 2007, volume 104, 10006 10009; Belorusova, et al., Journal of Medicinal Chemistry, 2014, 57, 4710-4719). VDR is an order of magnitude more sensitive to LCA and its metabolites than other nuclear receptors. Activation of VDR by ACL or 1,25D induces in vivo expression of the enzyme cytochrome P450 CYP3A, which detoxifies ACL in the liver and intestine. These studies offer a mechanism that may explain the protective effects of vitamin D and its receptor against colon cancer ( Science, 2002 , 296, 1313-1316).
    [0013] One factor contributing to the damaging effects of a high-fat diet is an increase associated with the excretion of fecal bile acids, the most toxic of which is ACL, a secondary bile acid. Unlike primary bile acids, acid Chenodeoxycholic (CDCA) and cholic acid (CA), ACL is poorly reabsorbed in the enterohepatic circulation and enters the colon. At high concentrations, ACL breaks DNA strands, forming adducts with DNA, and inhibiting enzymes responsible for DNA repair. ACL can also promote colon cancer in animals, and its concentration is higher than other secondary bile acids in patients with colorectal cancer.
    [0015] In contrast to the direct correlation between diets high in fat, ACL, and colon cancer, intake of vitamin D and calcium reduces the incidence of colorectal cancer. Furthermore, the administration of vitamin D inhibits carcinogenesis of the colon induced by diets rich in fats or by the action of the accumulation of intrarectal ACL. One way to eliminate ACL is through its catabolism by the action of the enterohepatic cytochrome P450, CYP3A, a target gene for vitamin D.
    [0017] Thus, the development of new vitamin D analogues with the same properties of natural hormone, but also effective in inducing cell differentiation and in the expression of the VDR target gene, is an objective to be achieved for use in practice. clinic.
    [0019] Brief description of the invention
    [0021] The authors of the present invention have designed and obtained compounds derived from lithocolic acid that have affinity for the vitamin D receptor and also have activity in cell differentiation. This would allow its use for the treatment of certain diseases, in particular cancer, and more specifically breast, colon and prostate cancer.
    [0023] A further advantage of the invention is that, although the compounds of the invention are highly functionalized, the process for their preparation consists of few synthesis steps.
    [0025] Another additional advantage is that the intermediates obtained in this synthetic route have a high versatility regarding the nature of the substituents. More specifically, the functionality in C-3 and C-20 allows to easily prepare lithocolic acid derivatives with a wide variety of substituents. In particular, the compounds of the invention have substituents in the C-3 and C-20 position, so versatile that they can be tailored to the needs of your application and thus can also have R or S configuration in said positions as required. . The functionality in C-3 and C-20 also allows to prepare isotopically labeled derivatives in a simple and fast way.
    [0027] Thus, in one aspect the invention is directed to a compound of formula ( I ), its diastereoisomers or its enantiomers
    [0032] where R 1, R2 and R3 are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl, heteroaryl, arylalkyl, alkylacyl, arylacyl, alkoxy, aryloxy, alkylcarboxy, arylcarboxy, heterocycle, -OSiRaRbRc, where each of Ra, Rb, and Rc are selected from alkyl, aryl, arylalkyl, and heterocycle, and R1 and R2 may together form a methylene group substituted by a group selected from hydroxyalkyl, hydroxyalkenyl, and alkylcarboxy.
    [0034] Another aspect of the invention relates to a pharmaceutical composition comprising a compound of formula ( I ).
    [0036] Another aspect of the invention relates to the compound of formula ( I ) for use in medicine.
    [0037] In a particular embodiment, the invention is directed to the compounds of formula (I) for use in the treatment of disorders of calcium and phosphorus metabolism, osteoporosis, renal osteodystrophy, osteomalacia, hyperproliferative skin diseases, eczema, dermatitis, myopathy, leukemia, osteosarcomas, squamous carcinomas, melanomas, immune disorders and in the rejection of transplants. In another particular embodiment, the invention is directed to compounds of formula ( I ) for use for the treatment of cancer. More particularly, breast, colon and prostate cancer.
    [0038] In a particular embodiment, the invention is directed to compounds of formula ( I ) for use for the treatment of diseases of the immune system. In a more particular aspect, for use for the treatment of psoriasis.
    [0040] Description of the figures
    [0042] Figure 1. Shows the dose-response curves of the affinity of the VDR analogues. Serial dilutions of the compounds were prepared in 384-well plates. The VDR / Fluormone ™ VDR Red complex was then added to each sample well and the components to be tested were incubated for 4 hours at room temperature to reach equilibrium. The fluorescence polarization of each well was determined. Concentration-response curves were plotted using GraphPad Prism 7.
    [0044] Figure 2. Shows the nuclear location of the VDR protein in HL60 exposed to 1,25D or to the compounds of the invention tested. HL60 cells were exposed for 24 hours to 100nM 1.25D or the compounds of the invention tested. Nuclear extracts were separated by SDS-PAGE and electrodeposited on a PVDF membrane. The membrane was tested against VDR and HDAC2.
    [0046] Figure 3. Shows the dose-response curves of the expression of CD14 (Figure 3A) and CD11b (Figure 3B) induced by 1,25D or by the tested compounds of the invention. HL60 cells were exposed to 1,25D or to the compounds of the invention tested in a wide range of concentrations for 96h. The expression of the cell surface markers CD14 (A) and CD11b (B) were then determined by flow cytometry. The mean values (± SEM) of the percentages of antigen positive cells have been represented.
    [0048] Figure 4. Shows the expression of CYP24A1 in HL60 cells induced by 1,25D or by the tested compounds of the invention. HL60 cells were exposed to 1,25D or the compounds of the invention tested at concentrations of 1nM (figure 4A), 10nM (figure 4B) or 100nM (C) and after 96 h the expression of CYP24A1 mRNA was determined by PCR in real time. The bars in the plots show the average values (± SEM) of the orders of magnitude in the mRNA levels relative to the levels of the GAPDH mRNA . Values that are significantly higher for those obtained by the respective control cells are marked with asterisks (* p <0.05; ** p <0.01).
    [0049] Figure 5. Shows the expression of CD14 in HL60 cells induced by 1,25D or by tested compounds of the invention. HL60 cells were exposed to 1,25D or compounds of the invention at concentrations of 1nM (figure 5A), 10nM (figure 5B) or 100nM (figure 5C) and after 96 h the expression of CD14 mRNA was determined by PCR in real time. The bars in the plots show the average values (± SEM) of the orders of magnitude in the mRNA levels relative to the levels of the GAPDH mRNA . Values that are significantly higher for those obtained by the respective control cells are marked with asterisks (* p <0.05; ** p <0.01). Expressions that were significantly higher in cells exposed to 1.25D are marked with a hash (## p <0.01).
    [0051] Detailed description of the invention
    [0053] Definitions
    [0055] "Alkyl" refers to a linear or branched, cyclic or acyclic hydrocarbon chain consisting of unsaturated carbon and hydrogen atoms of 1 to 1 2 , preferably two to four carbon atoms, and which binds to the rest of the molecule by a single bond, which may optionally be isotopically labeled so that one or more hydrogens are replaced by deuterium (2 H) or tritium (3 H) and / or one or more carbons are replaced by carbon-11 (UC), carbon -13 ( 13 C) or carbon-14 ( 14 C), optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, a heteroalkyl group, a heterocyclic group or CF 3 , for example methyl, ethyl, "-propyl, / -propyl," -butyl, i-butyl, "-pentyl, cyclopropyl, etc.
    [0057] "Alkenyl" refers to a linear or branched, cyclic or acyclic hydrocarbon chain consisting of carbon and hydrogen atoms, containing at least one unsaturation, conjugated or not, from 2 to 12 , preferably from two to eight, more preferably from two to four carbon atoms, and which is attached to the rest of the molecule by a single bond and which may optionally be isotopically labeled so that one or more hydrogens are replaced by 2H or 3H and / or one or more carbons are replaced by 11 C, 13C or
    [0058] 14 C. Alkenyl radicals can be optionally substituted by one or more substituents such as a halogen atom, in a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, a heteroalkyl group, a heterocyclic group or CF 3 , for example, vinyl, allyl, butenyl (for example, 1-butenyl, 2-butenyl , 3-butenyl), or pentenyl (eg, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl).
    [0060] "Alkynyl" refers to a linear or branched, cyclic or acyclic hydrocarbon chain consisting of carbon and hydrogen atoms, containing at least one carbon-carbon triple bond, conjugated or otherwise, from two to twelve, preferably from two to eight, more preferably from two to four carbon atoms, and which is attached to the rest of the molecule by a single bond, such as -CCH, -CH 2 CCH, -CCCH 3 , -CH 2 CCCH 3 , and which may optionally be isotopically labeled as so that one or more hydrogens are replaced by H or 3 H 2 and / or one or more carbons are replaced by n C, 13 C or 14 C. the alkynyl radicals may be optionally substituted by one or more substituents such as an atom halogen, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, a heterocyclic group or CF 3 .
    [0062] "Aryl" refers to an aromatic hydrocarbon of 6 to 10 carbon atoms, such as phenyl or naphthyl, and which may optionally be isotopically labeled so that one or more hydrogens are replaced by 2 H or 3 H and / or one or more carbons are replaced by 11 C, 13 C or
    [0063] 14 C. The aryl radicals may be optionally substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, a cyano group, a nitro group, a thioalkoxy group, an alkyl or CF 3 group .
    [0065] "Arylalkyl" refers to one or more aryl groups attached to the rest of the molecule by an alkyl radical, for example, benzyl, 3- (phenyl) -propyl, etc.
    [0067] "Heterocycle" refers to a stable 3- to 15-membered ring consisting of carbon atoms and between 1 to 5 heteroatoms chosen from nitrogen, oxygen and sulfur, preferably a 4 to 8 membered ring consisting of one or more heteroatoms, and more preferably a 5-6 membered ring with one or more heteroatoms. For the purposes of this invention, heterocyclic groups can be monocyclic, bicyclic, or tricyclic systems, which can include fused rings; and the nitrogen or sulfur atom in the heterocyclic ring can be optionally oxidized; the nitrogen atom can optionally be quartearized; and the heterocyclic radical can be partial or fully saturated. Heterocyclic radicals can be aromatic (eg, they can have one or more aromatic rings) in which case they are considered "heteroaryls" for the purposes of the present invention. The heterocyclic ring may be substituted by one or more substituents selected from the group consisting of a halogen atom, a hydroxy group, a carboxy group, an alkoxy group, an alkyl group, a thioalkoxy group, a cyano group, a nitro group or CF 3 . Examples of such heterocycles include, for example, furan, thiophene, pyrrole, imidazole, triazole, isothiazole, benzothiophene, benzofuran, indole, benzoimidazole, tetrahydrofuran.
    [0069] "Alkoxy" refers to a radical of the formula -O-alkyl, for example methoxy, ethoxy, propoxy, etc.
    [0071] "Aryloxy" refers to a radical of the formula -O-aryl, for example phenoxy, benzyloxy, etc.
    [0072] "Alkylcarboxy" refers to an alkyl group that is attached to the rest of the molecule by a carboxy (-CO 2 -) group, such as EtOC (O).
    [0074] "Arylcarboxy" refers to an aryl group that is attached to the rest of the molecule by a carboxy group (-CO 2 -).
    [0076] "Alkylallo" refers to an alkyl group that is attached to the rest of the molecule by a carbonyl group (-CO-).
    [0078] "Arylacyl" refers to an aryl group that is attached to the rest of the molecule by a carbonyl group (-CO-).
    [0080] "Carboxyalkyl" refers to a carboxy (-CO 2 -) group that is attached to the rest of the molecule by an alkyl group. The carboxy group can be for example a carboxylic acid group or an alkyl ester such as for example ethylcarboxyalkyl.
    [0082] "Heteroalkyl" refers to an alkyl group in which one or more carbons are substituted by heteroatoms, preferably from 1 to 5, where the heteroatom can be selected from oxygen, sulfur, selenium, tellurium, nitrogen, phosphorous, arsenic.
    [0084] "Heteroalkenyl" refers to an alkenyl group in which one or more carbons are substituted by heteroatoms, preferably from 1 to 5, where the heteroatom can be selected from oxygen, sulfur, selenium, tellurium, nitrogen, phosphorous, arsenic.
    [0085] "Heteroalkynyl" refers to an alkynyl group in which one or more carbons are substituted by heteroatoms, preferably from 1 to 5, where the heteroatom can be selected from oxygen, sulfur, selenium, tellurium, nitrogen, phosphorous, arsenic.
    [0087] "Hydroxyalkyl" refers to a hydroxyl group (-OH) that is attached to the rest of the molecule by an alkyl chain, the alkyl chain can be branched or unbranched.
    [0088] "Hydroxyalkenyl" refers to a hydroxyl group (-OH) that is attached to the rest of the molecule by an alkenyl chain, the alkenyl chain may be branched or unbranched.
    [0090] "Methylene" refers to a group (CH 2 =).
    [0092] The compounds of the present invention can include diastereoisomers and / or enantiomers and their racemic mixtures, in terms of the presence of chiral centers, and isomers depending on the presence of multiple bonds (for example Z, E). Such isomers, diastereomers, enantiomers, and mixtures thereof are within the scope of the present invention.
    [0094] The compounds of the present invention can be defined as derivatives of lithocolic acid and its metabolites. For this reason the compounds of the invention are named using the steroid numbering system.
    [0096] In a particular embodiment, the invention is directed to compounds of formula (I) in which R1, R2 and R3 are selected from hydrogen, hydroxyalkyl, hydroxyalkenyl, carboxyalkyl and carboxyalkenyl, or R1 and R2 can together form a methylene group substituted by a group selected from hydroxyalkyl, hydroxyalkenyl, and alkylcarboxy.
    [0098] In a preferred embodiment, the invention is directed to compounds of formula (I) in which R2 is hydrogen and R1 is selected from hydroxyalkyl, heteroalkyl, arylalkyl, alkylacyl, arylacyl, carboxyalkyl, carboxyalkenyl, heteroalkenyl and hydroxyalkenyl.
    [0099] In a particular embodiment, the invention is directed to compounds of formula (I) in which R2 is hydrogen and R1 is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxy (tert-butyl), carboxymethyl, carboxy ethyl and carboxypropyl. In a PREFERRED EMBODIMENT The invention is directed to compounds of formula (I) in which R2 is hydrogen and R1 is hydroxy (tert-butyl).
    [0101] In a particular embodiment, the invention is directed to compounds of formula (I) in which R1 is hydrogen and R2 is selected from hydroxyalkyl, heteroalkyl, arylalkyl, alkylacyl, arylacyl, carboxyalkyl, carboxyalkenyl, heteroalkenyl and hydroxyalkenyl.
    [0102] In a particular embodiment, the invention is directed to compounds of formula (I) in which R1 is hydrogen and R2 is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxy (tert-butyl), carboxymethyl, carboxyethyl and carboxypropyl. In a particular embodiment the invention is directed to compounds of formula (I) in which R1 is hydrogen and R2 is hydroxy (tert-butyl).
    [0104] In a particular embodiment, the invention is directed to compounds of formula (I) in which R1 and R2 together form a methylene group substituted by a group selected from hydroxyalkyl, hydroxyalkenyl and alkylcarboxy. In a more particular embodiment, the invention is directed to compounds of formula (I) in which R1 and R2 together form a methylene group substituted by an alkylcarboxy group.
    [0106] In a particular embodiment, the invention is also directed to any of the compounds described above in which R3 is selected from alkyl, alkenyl, heteroalkyl, heteroalkenyl, aryl, heteroaryl, arylalkyl, hydroxyalkyl, and carboxyalkyl.
    [0108] In a particular embodiment the invention is directed to any of the following compounds:
    [0110] (JR) -4 - {(5JR, 8JR, 96 ', 10 ^, 13JR, 146', 17JR, Z) -3- (2-Ethoxy-2-oxoethylidene) -10,13-dimethylhexa decahydro- 1H- cyclopenta [a] phenanthren-17-yl} -pentanoic,
    [0112] (JR) -4 - {(5JR, 8JR, 96,, 10 ^, 13JR, 146 ', 17JR, £) -3- (2-Ethoxy-2-oxoethylidene) -10,13-dimethylhexa decahydro- 1H- cyclopenta [a] phenanthren-17-yl} -pentanoic,
    [0114] Acid (^) - 4 - [(3 ^, 5 ^, 8 ^, 9 ^, 10 ^, 13 ^, 14 ^, 17 ^) - 3- (2-Ethoxy-2-oxoethyl) -10,13- dimethylhexadeca-hydro-1H-cyclopenta [a] fenantren-17-yl] -pentanoic,
    [0116] Acid (JR) -4 - [(3JR, 5JR, 8JR, 96 ', 10 ^, 13JR, 146', 17R) -3- (2-hydroxy-2-methylpropyl) -10,13-dimethylhexadeca-hydro-1H cyclopenta [a] phenanthren-17-yl) pentanoic,
    [0117] (R) -4 - {((3S, 5JR, 8JR, 9S, 10S, 13JR, 14S, 17JR) -3- (2-ethoxy-2-oxoethyl) -10,13-dimethylhexadeca hydro-1H-cydopenta [a ] phenanthren-17-yl} -pentanoic, and
    [0119] (JR) -4 - [(3S, 5JR, 8JR, 9S, 10S, 13JR, 14S, 17JR) -3- (2-Hydroxy-2-methylpropyl) -10,13-dimethylhexadeca hydro-1H-cyclopenta [a ] phenanthren-17-yl] -pentanoic.
    [0121] The following examples illustrate the invention and should not be construed as limiting it:
    [0123] Example 1. Preparation of 3 »S, 5 ^, 8 ^, 9 ^, 10» S, 13 ^, 14 »S, 17 ^) - 17 - [(K) -5-Hydroxipentan-2-n] -10 , 13-dimethylhexadecahydro-1H-ridopenta [a] fenantren-3-ol (1)
    [0128] LiAlH 4 (2.02 g) was added portionwise over a solution of Litocolic Acid (4 g) in THF (100 mL) cooled to 0 ° C. After 15 min, the reaction mixture was removed from the bath and stirred at room temperature for 12 h. Hydrochloric acid (30 mL, 10%) was slowly added to the mixture cooled to 0 ° C and stirred for 30 min until the solution became clear. The mixture was extracted with AcOEt (3x25 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by crystallization. To the residue, 20 mL of a 1: 1 mixture of EtOAc / Hexanes was added. The resulting suspension was heated to 50 ° C until it becomes transparent. The mixture was allowed to come to room temperature and then cooled to 0 ° C, observing the formation of a white solid. The solid was filtered under vacuum to give alcohol 1 (3.79 g, 98%).
    [0130] Ref .: Arto Valkonen, Elina Sievanen, Satu Ikonen, Nikolai V. Lukashev, Pavel A. Donez, Alexej D. Averin, Manu Lahtinen, Erkki Kolehmainen. Novel lithocholaphanes: syntheses, NMR, MS and molecular modeling studies. J. Mol. Struc. 2007 , 846, 65-73.
    [0131] Example 2. Preparation of (3 »S, 5 ^, 8 ^, 9 ^, 10» S, 13 ^, 14 »S, 17 ^) - 17 - [(K) -5- (tert-Butndimetilsnnoxi) pentan- 2-yl | -10,13-dimethnhexadecahydro-1H-cyclopenta [a] fenantrenren-3-ol (2a)
    [0136] A suspension of diol 1 (2 g) in DMF (50 mL) was heated until dissolved. After 5 min, the solution was allowed to come to room temperature. Imidazole (0.375 g) and ferc-butyldimethylsilyl ether chloride (0.83 g) were successively added to the above solution. After stirring 15 min, the reaction was stopped by the addition of a saturated aqueous solution of NaCl (30 mL). The mixture was extracted with hexanes (3x20 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (10% EtOAc / hexanes) to give the monoprotected alcohol 2a (1.51 g, 57%, white foam) and the dioprotected diol 2b (0.287 g, 9% (16% BSMR), oil colorless), recovering starting material 1 (0.61 g, 1.68 mmol, 30%).
    [0138] Example 3. Preparation of (5 ^, 8 ^, 9 ^, 10A, 13 ^, 14 »S, 17 ^) - 17 - {(K) -5- (tert-ButndimethylsilUoxy) -pentan-2-n) - 10,13-dimethnhexadecahydro-3H-cyclopenta [a] fenantren-3-one (3)
    [0143] Pyridinium dichromate (PDC, 2.60 g) was added over a solution of the alcohol 2a (1.10 g) in CH 2 O 2 (45 mL). The mixture was stirred protected from light and at room temperature for 7 h. The mixture was diluted with MTBE (50 mL), stirred 15 min, and vacuum filtered over a celite pad, washing the solids with MTBE (3x20 mL). The filtrates were concentrated in vacuo and the residue was purified by flash column chromatography (2% EtOAc / Hexanes) to give ketone 3 (1.02 g 93%, white solid).
    [0144] Example 4. Preparation of (Z) -2 - {(5R, 8R, 9S, 10S, 13R, 14S, 17R) -17 - [(R) -5- (tert-ButyldimethylsilUoxy) pentan-2-yl | -10 , 13-dimethylhexa decahydro-3H-cyclopenta [a] phenanthrene-3-yliden} -ethyl acetate (4a) and (E) -2 - {(5R, 8R, 9S, 10S, 13R, 14S, 17R) -17 - [(R) -5- (tert-Butyldimethylsilyloxy) pentan-2-yl] -10,13 -dimethylhexa decahydro-3H-cyclopenta [a] fenenatren-3-yliden} ethyl acetate (4b)
    [0149] Triethylphosphonoacetate (3.2 mL) was dripped onto a suspension of NaH (0.640 g, 60% w / w) in THF
    [0150] (15 mL) cooled to 0 ° C. After 30 min, a solution of ketone 3 (1.5 g) in THF (15 mL) was added via cannula. After 5 min, the reaction mixture was removed from the bath and stirred at room temperature for 30 min. The reaction was stopped by slow addition of H 2 O (20 mL). The resulting mixture was extracted with EtOAc (3x25 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (5% EtOAc / Hexanes) to give the mixture of alkenes 4 (1.68 g, 98%). Esters 4 were subjected to preparative HPLC (Phenomenex Luna Silica 5D, 250x210 mm, 100 A; 0.5% EtOAc / Hexanes) obtaining 4a (0.746 g, 43%, colorless oil) and 4b (0.802 g, 47%, colorless oil) .
    [0152] Example 5. Preparation of (E) -2 - {(5R, 8R, 9S, 10S, 13R, 14S, 17R) -17 - [(R) -5-hydroxypentan-2-yl] -10,13-dimethyl hexadecahydro -3H-cyclopenta [a] fenantren-3-yliden} -ethyl acetate (13).
    [0153] A solution of TBAF in THF (1.1 mL, 1M) was added to a solution of ester 4a (0.40 g) in THF (10 mL). The reaction mixture was stirred at room temperature for 12h. The reaction was stopped by adding a saturated aqueous NaCl solution (15 mL). The resulting mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (10% EtOAc / hexanes) to give alcohol 13 (0.301 g, 95%, white solid).
    [0155] Example 6. Preparation of Acid (K) -4 - {(5 ^, 8 ^, 9 »S, 10» S, 13 ^, 14 »S, 17 ^ rZ) -3- (2-Ethoxy-2-oxoethylidene ) -10,13-dimethylhexa decahydro-1H-cidopenta [a] fenantren-17-yl} -pentanoic (A.11 or SUNIL5)
    [0160] Pyridinium dichromate (0.525 g) was added over a solution of the alcohol 13 (0.200 g) in CH 2 O 2 (15 mL). The mixture was stirred protected from light and at room temperature for 12 h. The mixture was diluted with MTBE (10 mL), vacuum filtered over a celite pad, and the solids were washed with MTBE (3x20 mL). The filtrates were concentrated in vacuo. The residue ( 14 ) was dissolved in DMF (5 mL) and Oxone® (0.106 g) was added to this solution. The mixture was stirred at room temperature for 3 h. The reaction was stopped by the addition of an aqueous HCl solution (10 mL, 10%). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (15% EtOAc / hexanes) to give A.11 (158 mg, 77%, colorless oil).
    [0162] Example 7. Preparation of (^) - 2 - {(5 ^, 8 ^, 9 ^, 10 ^, 13 ^, 14 ^, 17 ^) - 17 - [(^) - 5-hydroxypentan-2-yl] -10,13-dimethylhexadeca hydro-3H-cyclopenta [a] phenanthrenren-3-yliden} -ethyl acetate (15).
    [0165] A solution of TBAF in THF (0.55 mL, 1M) was added over a solution of ester 4b (0.200 g) in THF (10 mL). After 12 h, the reaction was stopped by the addition of an aqueous NaCl solution (15 mL). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by column chromatography (10% EtOAc / hexanes) to give the alcohol 15 (0.147 g, 93%, white solid).
    [0167] Example 8. Preparation of Acid (K) -4 - {(5 ^, 8 ^, 9 »S, 10» S, 13 ^, 14 »S, 17 ^, E) -3- (2-Ethoxy-2- oxoethylidene) -10,13-dimethylhexa decahydro-1H-cidopenta [a] fenentran-17-yl} -pentanoic (A.11 or SUNIL6)
    [0172] Pyridinium dichromate (0.525 g) was added to a solution of the alcohol 15 (0.20 g) in CH 2 O 2 (15 mL). The mixture was stirred protected from light and at room temperature for 12 h. The mixture was diluted with MTBE (10 mL), vacuum filtered over a celite pad, and the solids were washed with MTBE (3x20 mL). The filtrates were concentrated in vacuo and the resulting residue ( 16 , 0.180 g) was dissolved in DMF. Oxone® (0.085 g) was added to the previous solution. The mixture was stirred at room temperature for 3 h. The reaction was stopped by the addition of an aqueous HCl solution (10 mL, 10%) and the mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (20% EtOAc / hexanes) to give A.11 (137 mg, 66%, foamy solid).
    [0174] Example 9. Preparation of 2 - {(3 ^, 5 ^, 8 ^, 9 »S, 10» S, 13 ^, 14 »S, 17 ^) - 17 - [(K) -5- (tert-Butyldimethylsilyloxy ) ethyl pentan-2-yl] -10,13-dimethylhexadecahydro-1H-cyclopenta [a] fenantren-3-yl} acetate (5a).
    [0177] Pd / C (135 mg, 10% w / w) was added over a solution of 4a (0.690 g) in AcOEt (20 mL). The system was purged 3 times vacuum / hydrogen. The mixture was stirred under hydrogen atmosphere (H 2 balloon) for 12 h. The mixture was vacuum filtered over a celite pad washing AcOEt (20 mL). The filtrates were concentrated in vacuo and the resulting residue was purified by flash column chromatography (10% EtOAc / hexanes) to give the ester 5a (570 mg, 82%, colorless oil).
    [0179] Example 10. Preparation of 2 - {(3 ^, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 17 - [(^) - 5-hydroxypentan-2-yl] -10,13 -dimetnhexa decahydro-1H-cyclopenta [a] fenantren-3-yl} ethyl acetate (9)
    [0184] A solution of TBAF in THF (1.1 mL, 1M) was added over a solution of 5a (0.390 g) in THF (10 mL). After 12 h, the reaction was stopped by the addition of a saturated aqueous NaCl solution. The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (10% EtOAc / Hexanes) to give alcohol 9 (0.300 g, 0.699 mmol, 97%, white solid).
    [0186] Example 11. Preparation of Acid (^) - 4 - [(3 ^, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 3- (2-Ethoxy-2-oxoethyl) -10, 13-dimethylhexadeca-hydro-1H-cyclopenta [a] fenantren-17-yl] -pentanoic (A.9 or SUNIL3)
    [0189] Pyridinium dichromate (0.430 g) was added over a solution of alcohol 9 (0.165 g) in CH 2 CI 2 (25 mL). The mixture was stirred protected from light and at room temperature for 12 h. The mixture was diluted in MTBE (10 mL) and stirred for 15 min. The mixture was vacuum filtered over a celite pad, washing the solids with MTBE (3x20 mL) and the filtrates were concentrated. The residue ( 10 , 0.160 g) was dissolved in DMF (10 ml) and Oxone® (60 mg) was added to this solution. After 3 h, the reaction was stopped by adding a HCl solution (10 mL, 10%). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered and concentrated. The residue was purified by flash column chromatography (20% EtOAc / hexanes) to give acid A.9 (120 mg, 70%, white solid).
    [0191] Example 12. Preparation of 1 - {(3R, 5R, 8R, 9S, 10S, 13R, 14S, 17R) -17 - [(R) -5- (tert-Butndimethylsnnoxy) pentan-2-yl | -10,13 -dimetnhexadecahydro-1H-cyclopenta [a] phenanthrenren-3-yl} -2-methylpropan-2-ol (6)
    [0196] A solution of methylmagnesium bromide in THF (MeMgBr, 1.5 mL, 3M) was added to a solution of ester 5a (0.50 g) in THF (10 mL), cooled to 0 ° C. After 5 min, the mixture was removed from the bath and heated under reflux (65 ° C) for 3 h. The reaction was allowed to come to room temperature and was stopped by adding an aqueous HCl solution (15 mL, 10%). The mixture was extracted with AcOEt (3x15 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (5% EtOAc / hexanes) to give the alcohol 6 (0.479 g, 98%, colorless oil).
    [0197] Example 13. Preparation of (K) -4 - [(3 ^, 5 ^, 8 ^, 9 »S, 10» S, 13 ^, 14S, 17 ^) - 3- (2-Hydroxy-2-methpropyl) -10,13-dimethylhexa decahydro-1H-cyclopenta [a] fenantren-17-yl] pentan-1-ol (7)
    [0202] A solution of TBAF in THF (0.6 mL, 1M) was added over a solution of silicon ether 6 (0.226 g) in THF (1 mL). The mixture was stirred at room temperature for 12 h. The reaction was stopped by the addition of a saturated aqueous NaCl solution (5 mL). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash chromatography (15% EtOAc / hexanes) to give the alcohol 7 (0.167 g, 94%, white solid).
    [0204] Example 14. Preparation of Acid (K) -4 - [(3 ^, 5 ^, 8 ^, 9 »S, 10» S, 13 ^, 14 »S, 17 ^) - 3- (2-hydroxy-2 -methylpropyl) -10,13-dimethylhexadeca-hydro-1H-cyclopenta [a] phenanthrene-17-yl) pentanoic (A.5 or SUNIL1)
    [0209] Pyridinium dichromate (0.620 g) was added over a solution of the alcohol 7 (0.230 g) in CH 2 O 2
    [0210] (15 mL). The mixture was stirred protected from light and at room temperature for 7 h. The resulting mixture was diluted with MTBE (20 mL) and stirred 15 min. The mixture was then vacuum filtered over a celite pad, washing the solids with MTBE (3x20 mL). The filtrates were concentrated in vacuo and the residue (8, 0.229 g) was dissolved in DMF (10 ml). Oxone® (0.125 g) was added to the previous solution and the mixture was stirred at room temperature for 3 h. The reaction was stopped by addition of a saturated aqueous NaCl solution (10 mL) and the mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered and concentrated. The residue was purified by flash column chromatography (15% EtOAc / hexanes) to give acid 6 (0.180 g, 0.416 mmol, 76%, white solid).
    [0212] Example 15. Preparation of 2 - {(3A, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 17 - [(^) - 5-hydroxypentan-2-yl] -10,13- dimethylhexadecahydro-1H-ridopenta [a] phenentren-3-yl} -ethyl acetate (11)
    [0217] Poly (methylhydroxysiloxane) (PMHS, 0.53 mL) and a solution of potassium ferc-butoxide in THF (KOfBu, 0.41 ml) were added successively over a suspension of CuCl (38 mg) and (6) -tol-BINAP (45 mg ) in hexanes (8 mL). The mixture was sonic for 2h and then cooled to -20 ° C. Then, on top of the previous suspension, a solution of ester 4a (0.300 g) in z'PrOH (0.170 mL) and hexanes (2 mL) was added via cannula. After 3.5 h, the reaction mixture was removed from the bath and allowed to come to room temperature. The reaction was stopped by the addition of an aqueous HCl solution (15 mL, 10%). The mixture was extracted with AcOEt (3x20 mL). The combined organic phase was dried, filtered, and concentrated. The residue ( 5b and PMHS residues) was dissolved in THF (10 mL) and HF (0.5 mL, 48%) was added to this solution. After 12 h, the mixture was poured into a saturated aqueous NaHCO 3 solution (50 mL). The mixture was extracted with AcOEt (3x25 mL). The combined organic phase was washed with a saturated aqueous solution of NaCl (20 mL), dried, filtered and concentrated. The residue was purified by flash column chromatography (10% EtOAc / Hexanes) to give alcohol 11 (0.220 g, 0.508 mmol, 92%, white solid).
    [0219] Example 16. Preparation of Acid (K) -4 - {(3A, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 3- (2-ethoxy-2-oxoethyl) -10,13 -dimethylhexadeca hydro-1H-cyclopenta [a] fenantren-17-yl} -pentanoic (A.10 or SUNIL4)
    [0220] Pyridinium dichromate (0.430 g) was added over a solution of alcohol 11 (0.165 g) in CH 2 CI 2
    [0221] (10 mL). The mixture was stirred protected from light and at room temperature for 5 h. The reaction mixture was diluted with MTBE (10 mL), stirred for 15 min, and filtered through a pad of celite, washing the solids with MTBE (3x20 mL). The filtrates were concentrated and the resulting residue (12, 0.160 g) was dissolved in DMF (5 mL). Then Oxone® (0.060 g) was added and the mixture was stirred at room temperature for 3 h. The reaction was stopped by the addition of an aqueous HCl solution (10 mL, 10%). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (15% EtOAc / hexanes) to give A.10 (120 mg, 70%, white solid).
    [0223] Example 17. Preparation of (^) -4 - [(3A, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 3- (2-Hydroxy-2-methpropyl) -10,13- dimethylhexa decahydro-1H-cyclopenta [a] fenantren-17-yl] pentan-1-ol
    [0228] A solution of methylmagnesium bromide in THF (1 mL) was added to a solution, cooled to 0 ° C, of ester 11 (0.300 g) in THF (10 mL). After 5 min, the reaction mixture was removed from the bath and heated under reflux for 3 h. Then, the reaction was allowed to come to room temperature and was stopped by adding an aqueous HCl solution (15 mL, 5%). The mixture was extracted with AcOEt (3x15 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (15% EtOAc / hexanes) to give the alcohol 17 (0.276 g, 0.659 mmol, 95%, white solid).
    [0230] Example 18. Preparation of Acid (K) -4 - [(3A, 5 ^, 8 ^, 9A, 10A, 13 ^, 14A, 17 ^) - 3- (2-Hydroxy-2-methylpropyl) -10,13 -dimethylhexadeca hydro-1H-cyclopenta [a] fenantren-17-yl] -pentanoic (A.6 or SUNIL2)
    [0233] Pyridinium dichromate (PDC, 0.270 g) was added over a solution of alcohol 17 (0.150 g) in CH 2 Cl 2 (15 mL). The reaction mixture was stirred protected from light and at room temperature for 7 h. The mixture was then diluted with MTBE (50 mL), stirred 15 min, and vacuum filtered over a pad of celite, washing the solids with MTBE (3x20 mL). The filtrates were concentrated in vacuo and the resulting residue ( 18 , 0.149 g) was dissolved in DMF (10 mL). Oxone (0.054 g) was added to the above solution and the mixture was stirred at room temperature for 3 h. The reaction was stopped by the addition of a saturated aqueous NaCl solution (10 mL). The mixture was extracted with AcOEt (3x10 mL). The combined organic phase was dried, filtered, and concentrated. The residue was purified by flash column chromatography (15% EtOAc / hexanes) to give acid 6 (120mg, 0.277mmol, 77%, white solid).
    [0235] Example 19. In vitro tests
    [0237] 19.1. Methods
    [0239] 19.1.1. Compounds
    [0241] 1,25-Dihydroxyvitamin D3 (1,25D) was purchased from Cayman Europe (Tallinn, Estonia) and was dissolved in absolute ethanol to a concentration of 1 mM.
    [0243] Compounds of the invention to be tested: Compound A.9, prepared in Example 11, was dissolved in absolute ethanol at a concentration of 20 mM, and the remaining compounds tested (A.5 prepared in Example 14; A.6 prepared in Example 18; A.10 prepared in Example 16; A.11- (Z) prepared in Example 6; A.11- (E) prepared in Example 8) were dissolved in absolute ethanol at a concentration of 50 mM.
    [0245] 19.1.2. Cells
    [0247] HL60 cells were obtained from a local cell bank at the Institute of Immunology and Experimental Therapy in Wroclaw, Poland. The cells were grown in a medium RPMI 1640 supplemented with 10% fetal calf serum (FCS), penicillin 100 units / mL, and sterptomycin 100 pg / mL (Sigma, St. Louis, MO). Cells were grown in an atmosphere moistened with 95% air and 5% CO2 at 37 ° C. Cell number and viability were determined by counts on a hematocytometer and by trypan blue exclusion. The cells were seeded with a density of 2.5 x 105 cells / mL in a culture medium containing 1.25D, a compound of the present invention mentioned in section 19.1.1. or the equivalent volume of ethanol as a control vehicle.
    [0249] 19.1.3. VDR affinity test
    [0251] VDR affinity was determined using a competitive PolarScreen ™ vitamin D receptor assay kit following manufacturer's conditions (Invitrogen, Carlsbad, CA). 1,25D and the tested compounds were evaluated in the concentration range of 10-12-10 -5 M. The components of the test were incubated for 4h at room temperature to allow equilibrium to be reached. The polarized fluorescence of each tray was determined in triplicate using the Envision multiwell reader (PerkinElmer, Waltham, MA) and the average fluorescence polarization was calculated from these measurements. The entire test was repeated three times. IC50 values were calculated with the GraphPad Prism 7 program (GraphPad Software, San Diego CA) using the average of the values obtained.
    [0253] 19.1.4. Western blotting
    [0255] To obtain the cytosolic and nuclear extracts, 6x106 cells / sample were washed and disaggregated using NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific Inc., Worcester, MA) following the manufacturer's conditions. The cell lysates were denatured by adding the sample buffer five times (with a maximum volume corresponding to 1/4 of the volume of the lysate) and boiled for 5 min. 20 pL of each lysate was separated by SDS-PAGE and electrodeposited on a PVDF membrane. The membranes were dried and sequentially incubated with primary antibodies and secondary horseshoe fish conjugated peroroxidase antibodies. Protein bands were visualized by chemiluminescence. The membranes were then trimmed, dried again, and assayed for subsequent antibodies.
    [0256] 19.1.5. Cell differentiation and flow cytometry
    [0258] The cells were seeded with a density of 15 x 104 cells / mL in a culture medium containing 1.25D, a compound of the invention to be tested as indicated in 19.1.1. or the equivalent volume of ethanol as a control vehicle. After 96 h incubation, cells were washed with PBS / 0.1% BSA, and then incubated for 1 h, in an ice bath, with 1 | iL of CD14-PE and 2 | iL CD11b-APC (ImmunoTools, Friesoythe, Germany). Cells were washed and suspended in 350 ^ L PBS / 0.1% BSA prior to analysis on a Becton Dickinson Accuri C6 flow cytometer (San Jose, CA). Data analysis was performed using Becton Dickinson Accuri C6 programs. The test was repeated 3 to 5 times. The percentages of positive cells were printed on the charts and the EC50 values were calculated using the GraphPad Prism software.
    [0260] 19.1.6. CDNA synthesis and real-time PCR
    [0262] Total RNA was isolated using EXTRAzol reagent (BLIRT SA, Gdansk, Poland) following the manufacturer's conditions. The amount of RNA was determined using Nanodrop (Thermo Fisher Scientific Inc. Worcester, MA) and the quality of the RNA was verified by gel electrophoresis. RNA was transcribed into DNA using a "High Capacity cDNA Reverse Transcription" kit (Applied Biosystems, Foster City, CA). Real-time PCR was performed using the SensiFAST ™ SYBR Hi-ROX Kit (Bioline) in a "CFX Connect Real-Time PCR" detection system (Bio-Rad Laboratories, Inc.). The sequence of the CYP24A1 primers were (fp): 5'-CTCATGCTAAATACCCAGGTG-3 ', (rp): 5'-TCGCTGGCAAAACGCGATGGG-3' [1], the primers of CD14 were (fp): 5'-GTTCGGAAGACTTATCGACC ', (rp): 5'-CAGACGCAGCGGAAATCTTCATC-3' [2], while the GAPDH sequences were as follows: (fp): 5'-CAT GAG AAG TAT GAC AAC AGC CT-3 ', (rp): 5'-AGT CCT TCC ACG ATA CCA AAG T-3 '[3]. Magnitude changes in mRNA levels in the CYP24A1 or CD14 genes related to the GAPDH gene were calculated by relative quantitative analysis.
    [0264] 19.1.7. Analysis of results
    [0266] Microsoft Excel and Graphpad Prism 7 programs (San Diego, CA, USA) were used to analyze the results. Statistical analysis of Student's t of the differences between treated and untreated samples was used to determine independent samples. To verify if the samples treated with the compounds of the invention to be tested as indicated in 19.1.1., Were significantly different from the samples treated with 1.25D, a one-way ANOVA analysis followed by an adapted Dunnett analysis were used.
    [0268] 19.2. Results
    [0270] 19.2.1. Affinity of compounds for VDR
    [0272] The affinity of the compounds of the invention to be tested as indicated in 19.1.1. by the VDR receptor was determined by a competitive assay using fluorescence polarization. In this assay recombinant human VDR is added to a fluorescent VDR ligand to form a complex, resulting in a high value for fluorescence polarization. The compounds to be tested were then added to the complex in a 386-well tray. The tested compounds displace the fluorescent ligand from the complex, resulting in a lower polarization value. The affinity of the compounds for VDR was tested with a wide range of concentrations and was compared with 1.25D. It should be mentioned that the affinity for VDR could not be determined for those compounds of the invention applied at concentrations higher than 10-5 M. When the compounds were tested at high concentrations, the concentration response curve is U-shaped instead of being sigmoidal. The probable cause is due to the formation of colloidal association of the tested compounds. The concentration-response curve was plotted (presented in Figure 1), and IC50 values were calculated using GraphPad Prism 7.
    [0274] The affinity for VDR is compared to 1.25D, for which the relative binding affinity (RBA) is normalized to 100, and has been presented in Table 1.
    [0276] Table 1. Affinity of the compounds to the recombinant VDR.
    [0278]
    [0279] VDR affinity is expressed as IC50 and percentage activity. a The potency of 1,25D is normalized to 100. RBA: relative binding affinity. ND: not detected.
    [0281] 19.2.2. Nuclear translocation and accumulation of VDR in response to the compounds of the invention to be tested as indicated in 19.1.1.
    [0283] Since the nuclear accumulation of VDR and the pro-differentiating activity were correlated for previously tested analogs, it was studied as the compounds of the invention to be tested as indicated in 19.1.1. they influence the levels of VDR protein in HL60 cells exposed for 24h to 100 nM concentrations of analogs. VDR levels in nuclear fractions of cells were analyzed. The HDAC2 nuclear protein was used as a control, as a protein that does not change during cell differentiation from HL60. The amount of VDR in the cell nucleus had increased for 1,25D and for compounds A.5, A.6 and A.11- (Z) (Fig. 2).
    [0285] 19.2.3. HL60 cell differentiation
    [0287] HL60 cells were used to determine the pro-differentiating activity of the compounds of the invention to be tested as indicated in 19.1.1 .. After the initial scan, the concentration ranges were established for each compound. 1,25D was tested in concentrations between 0.032 nM - 100 nM, A.5, A.6 and A.11- (Z) were tested in concentrations 0.032 nM - 500 nM, while A.9, A.10 and A.11- (E) were applied in concentrations 4 ^ M - 50 ^ M. The cells were exposed to the compounds for 96 h and then the expression of the monocyte / macrophage differentiation marker CD14 and the monocyte / granulocyte differentiation marker CD11b were studied by flow cytometry. The percentages of CD14- and CD11b positive cells were recorded using the Becton Dickinson Accuri C6 software. The results are presented in Figures 3A and 3B. EC50 values were estimated from dose-response curves using GraphPad Prism 7 software. The effective molar ratios (EMR) for each analog compared to 1.25D for the CD14 antigen are depicted in Table 2 and for the antigen. for CD11b in Table 3.
    [0289] Table 2. Induction of CD14 expression by 1,25D and Sunil compounds.
    [0291]
    [0292]
    [0294] a EC 50 values were estimated from the dose-response curve using the GraphPad Prism 7 program. EC 50 values were used to calculate the effective molar ratio (EMR). EMR = EC 50 1.25D / EC 50 compound. ND - cannot be calculated (activity not detected).
    [0296] Table 3. Induction of CD11b expression by 1,25D and Sunil compounds.
    [0298]
    [0300] a EC 50 values were estimated from the dose-response curve using the GraphPad Prism 7 program. EC 50 values were used to calculate the effective molar ratio (EMR). EMR = EC 50 1.25D / EC 50 compound. ND - cannot be calculated (activity not detected).
    [0302] 19.2.4. Expression of CYP24A1 in HL60 cells in response to 1,25D and compounds of the invention to be tested as indicated in 19.1.1.
    [0304] HL60 cells were exposed to 1,25D or the compounds of the invention to be tested as indicated in 19.1.1. at 1 nM, 10 nM and 100 nM concentrations for 96h. Control cells were treated with an equivalent volume of ethanol (solvent for all compounds). The mRNA was then isolated from the cells, transcribed into cDNA, and the expression of CYP24A1, the most strongly regulated VDR target gene, was determined in real-time PCR relative to the GAPDH gene . The effects of exposure to 1 nM of the compounds is depicted in Figure 4A, to 10 nM in Figure 4B, and to 100 nM in Figure 4C. The expressions, which were significantly higher than those of the control cells, are marked with asterisks. The expressions, which were significantly higher than those of the cells exposed to 1.25D, are marked with a pad.
    [0306] 19.2.5. CD14 expression in HL60 cells in response to 1,25D and compounds of the invention.
    [0307] HL60 cells were exposed to 1,25D or the compounds of the invention as indicated in 19.1.1. at 1 nM, 10 nM and 100 nM concentrations for 96h. Control cells were treated with an equivalent volume of ethanol (solvent for all compounds). The mRNA was then isolated from the cells, transcribed into cDNA and the expression of CD14 was determined in a real-time PCR in relation to the GAPDH gene . The CD14 gene encodes a cell surface protein present in macrophages, which is a co-receptor for bacterial LPS. The effects of exposure to 1 nM of the compounds is depicted in Figure 5A, to 10 nM in Figure 5B, and to 100 nM in Figure 5C. The expressions, which were significantly higher than those of the control cells, are marked with asterisks.
    [0309] In view of the in vitro results obtained, we can conclude that compound A.5 or SUNIL1 is the most active of the group of lithocolic acid derivatives studied. This compound is especially effective in inducing cell differentiation and expression of the VDR target gene at a concentration of 10 nM.
    权利要求:
    Claims (21)
    [1]
    1.- Compound of formula (I), its diastereoisomers or its enantiomers

    [2]
    2. - Compounds of formula (I), according to claim 1, where R1, R2 and R3 are selected from hydrogen, hydroxyalkyl, hydroxyalkenyl, carboxyalkyl and carboxyalkenyl, or R1 and R2 can together form a methylene group substituted by a selected group between hydroxyalkyl, hydroxyalkenyl and alkylcarboxy.
    [3]
    3. - Compounds of formula (I), according to any of the preceding claims, wherein R2 is hydrogen and R1 is selected from hydroxyalkyl, heteroalkyl, arylalkyl, alkylacyl, arylacyl, carboxyalkyl, carboxyalkenyl, heteroalkenyl and hydroxyalkenyl.
    [4]
    4. - Compounds of formula (I), according to any of the preceding claims, wherein R2 is hydrogen and R1 is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxy (tert-butyl), carboxymethyl, carboxyethyl and carboxypropyl.
    [5]
    5. - Compounds of formula (I), according to any of the preceding claims, wherein R2 is hydrogen and R1 is hydroxy (tert-butyl).
    [6]
    6. - Compounds of formula (I), according to any of the preceding claims, where R1 is hydrogen and R2 is selected from hydroxyalkyl, heteroalkyl, arylalkyl, alkylacyl, arylacyl, carboxyalkyl, carboxyalkenyl, heteroalkenyl and hydroxyalkenyl.
    [7]
    7. - Compounds of formula (I), according to any of the preceding claims, where R1 is hydrogen and R2 is selected from hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxy (tert-butyl), carboxymethyl, carboxy ethyl and carboxypropyl.
    [8]
    8. - Compounds of formula (I), according to any of the preceding claims, where R1 is hydrogen and R2 is hydroxy (tert-butyl).
    [9]
    9. - Compounds of formula (I), according to any of the preceding claims, where R1 and R2 together form a methylene group substituted by a group selected from hydroxyalkyl, hydroxyalkenyl and alkylcarboxy.
    [10]
    10. - Compounds of formula (I), according to any of the preceding claims, wherein R1 and R2 together form a methylene group substituted by an alkylcarboxy group.
    [11]
    eleven.
    [12]
    12. - Compound of formula (I) according to any of the preceding claims selected from:
    (JR) -4 - {(5JR, 8JR, 96 ', 10 ^, 13JR, 146', 17JR, Z) -3- (2-Ethoxy-2-oxoethylidene) -10,13-dimethylhexa decahydro- 1H- cyclopenta [a] phenanthren-17-yl} -pentanoic,
    (JR) -4 - {(5JR, 8JR, 96,, 10 ^, 13JR, 146 ', 17JR, £) -3- (2-Ethoxy-2-oxoethylidene) -10,13-dimethylhexa decahydro- 1H- cyclopenta [a] phenanthren-17-yl} -pentanoic,
    Acid (^) - 4 - [(3 ^, 5 ^, 8 ^, 9 ^, 10 ^, 13 ^, 14 ^, 17 ^) - 3- (2-Ethoxy-2-oxoethyl) -10,13- dimethylhexadeca-hydro-1H-cyclopenta [a] fenantren-17-yl] -pentanoic,
    Acid (JR) -4 - [(3JR, 5JR, 8JR, 96 ', 10 ^, 13JR, 146', 17R) -3- (2-hydroxy-2-methylpropyl) -10,13-dimethylhexadeca-hydro-1H cyclopenta [a] phenanthren-17-yl) pentanoic,
    Acid (JR) -4 - {(36,, 5JR, 8JR, 9, S ,, 10) S ,, 13JR, 14) S ,, 17JR) -3- (2-ethoxy-2-oxoethyl) -10, 13-dimethylhexadeca hydro-1H-cidopenta [a] fenantren-17-yl} -pentanoic, and
    Acid (JR) -4 - [(36,, 5JR, 8JR, 9, S ,, 10) S ,, 13JR, 14) S ,, 17JR) -3- (2-Hydroxy-2-methylpropyl) -10, 13-dimethylhexadeca hydro-1H-cyclopenta [a] fenantren-17-yl] -pentanoic.
    [13]
    13. Pharmaceutical composition comprising a compound of formula ( I ) according to any of claims 1-12.
    [14]
    14. - Compound of formula ( I ), according to any of claims 1-12, for use in medicine.
    [15]
    fifteen.
    [16]
    16.
    [17]
    17. - Compound of formula (I), according to any of claims 1-12, for use in the treatment of breast, colon and prostate cancer.
    [18]
    18. - Compound of formula (I), according to any of claims 1-12, for use in the treatment of diseases of the immune system.
    [19]
    19.- Compound of formula (I), according to any of claims 1-12, for use in the treatment of psoriasis.
    [20]
    20. - Compound of formula (I), according to any of claims 1-12, for use in the treatment of diseases of the immune system.
    [21]
    twenty-one. - Compound of formula (I), according to any of claims 1-12, for use in the treatment of psoriasis.
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    WO2020136292A1|2020-07-02|
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    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2017101789A1|2015-12-14|2017-06-22|四川思路迪药业有限公司|Compound having anti-cancer effect, and method for preparation thereof and application thereof|
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