Compounds and methods for the treatment of cancer

Abstract

The present invention relates to compounds that derive from bile acids, such as ursodeoxycholic acid and to their use in the prevention and / or treatment of cancer, in particular of cholangiocarcinoma.

Description

[0001] COMPOUNDS AND METHODS FOR THE TREATMENT OF CANCER

[0002] FIELD OF THE INVENTION

[0003]

[0001] The present invention relates to compounds that derive from bile acids such as ursodeoxycholic acid and to their use in the prevention and / or treatment of cancer, in particular of cholangiocarcinoma.

[0004] BACKGROUND

[0005]

[0002] Cholangiocarcinoma, also commonly referred to as CCA or bile duct cancer in the art, is an aggressive malignancy originating from the epithelial cells of the bile ducts, named cholangiocytes, which can give rise to the obstruction of the bile ducts. They are anatomically classified into intrahepatic CCA (iCCA), perihilar CCA (pCCA) and distal CCA (dCCA). This disease accounts for approximately 15% of primary liver cancers and 3% of gastrointestinal cancers globally. Its incidence is rising, with higher rates observed in Southeast Asia due to liver fluke infections (>6 case per 100,000 inhabitants), while in Western countries, the annual incidence is around 0.5-2 cases per 100,000 population.

[0006]

[0003] CCA is highly lethal due to its asymptomatic growth, aggressiveness, late diagnosis, and high chemoresistance. Surgical removal of the tumor or liver transplantation are presented as the only potentially curative options, although they are only indicated in a small number of patients who meet strict clinical criteria, and the chances of tumor recurrence are high. The first-line pharmacological treatment for advanced cancer is the triple combination of gemcitabine, cisplatin and immunotherapy (i.e., durvalumab or pembrolizumab), which only increases the median survival of patients by 3 months and is therefore considered palliative. Other chemotherapies include the use of capecitabine, oxaliplatin and 5-fluorouracil. Chemotherapeutic agents may be used in combination therapies.

[0007]

[0004] Treatment of CCA depends on disease stage. Surgery, combined with adjuvant capecitabine, remains the only potentially curative option but is feasible for less than 30% of patients due to late-stage diagnosis. For advanced or inoperable cases, systemic chemotherapy using cisplatin and gemcitabine has long been the standard. Recent advancements include targeted therapies for specific genetic alterations (e.g., IDH1, FGFR2, HER2) and immunotherapies such as immune checkpoint inhibitors. The combination of chemotherapy with immunotherapeutic agents such as durvalumab or pembrolizumab has become a new first-line treatment, offering modest survival benefits.

[0005] CCA develops through a multifactorial process involving chronic biliary tract inflammation, genetic mutations, and significant epigenetic dysregulation. Epigenetic alterations, such as aberrant DNA methylation, histone modifications, and dysregulated non-coding RNAs expression, disrupt gene expression by silencing tumor suppressor genes and activating oncogenes. Histones together with DNA form the fundamental units of the dynamic chromatin structure, called nucleosomes. Histone deacetylases (HDACs) are a family of hydrolase enzymes that catalyse the deacetylation of lysine residues located in different proteins, including histones, favouring the packaging of chromatin or heterochromatin. Among them, several isoforms of the HDAC (nuclear) family are considered epigenetic regulators, as their catalytic activity leads to changes in chromatin structure, thus modulating the ability of transcription factors to reach their target genes and exert their functions. HDACs are essential for the correct functioning of a wide range of cellular processes, such as differentiation, cell cycle regulation, modulation of cytoskeleton dynamics, metabolic processes, autophagy and apoptosis, among many others. Importantly, alterations in the expression and / or activity of different HDACs have been described in several types of cancer, including CCA.

[0008]

[0006] Class I Histone deacetylases (HDACs), which comprise HDAC1, 2, 3, and 8 play a central role in controlling cell cycle regulation, cell differentiation, and tissue development. These enzymes exert their function by deacetylating histones and a growing number of non-histone proteins, thereby regulating gene expression and several other cellular processes. While pan-HDAC inhibitors such as vorinostat and panobinostat have been commercialized, their clinical utility is frequently restricted by high toxicity, low efficacy, and the development of drug resistance due to their nonselective nature. In response to these limitations, researchers have focused on the development of isoform-selective inhibitors, particularly targeting Class I HDACs like HDAC1, HDAC2, and HDAC8, which can potentially reduce side effects compared to pan-inhibition. Class I selective inhibitors are in this regard highly desirable.

[0009]

[0007] Several HDAC inhibitors have been disclosed in the art as being useful for the prevention and / or treatment of CCA, e.g. vorinostat (Kwak TW, et al. Int J Nanomedicine. 2017; 12:7669-7680), romidepsin (Li P. et al. Technol Cancer Res Treat. 2020 Oct 14; 19: 1533033820960754), MS-275 (Shankar, S. et al. Adv. Exp. Med. Biol. 2008, 615, 261-298), MI-192 (Yin, Y. et al. Cell Death Dis. 2017, 8, e2856), the natural products KK4 and ICG15042 (Saenglee, S. et al. Biomed. Pharmacother. 2018, 98, 233-24), ACY1215 and tubastatin-A (Gradilone, S. A. et al. Cancer Res. 2013, 73, 2259-2270). In addition, HDAC inhibitors may actsynergistically with chemotherapeutic agents. In this regard the combination of SAHA (suberoylanilide hydroxamic acid) or TSA (trichostatin A) with cisplatin (Asgar, M. A. et al. Int. J. Oncol. 2016, 48, 409-420) or 5-fluorouracil have been reported (Sriraksa, R. et al. Asian Pac. J. Cancer Prev. 2013, 14, 2503-2508). Similary, Jung D. E. et al. Sci. Rep. 2017, 7, 1-13 the combination of CG200745 with either of cisplatin, 5-fluorouracil or oxaliplatin for decreasing CCA cell viability.

[0010]

[0008] Ursodeoxycholic acid (UDCA) has been suggested in the art as adjuvant or palliative chemotherapeutic agent and as a therapeutic combination option that enforces the effect of other antitumor agents in CCA (Lee, J. et al. Oncol Lett. 2022 Oct 26;24(6):448). The state of the art is however silent about the use of UDCA derivatives for the prevention and / or treatment of cholangiocarcinoma.

[0011]

[0009] International patent application WO 2019 / 129913 A1 discloses derivatives of UDCA useful for the treatment and / or prevention of polycystic diseases, such as polycystic liver disease by selective inhibition of HDAC6. This document discloses in particular the following compounds:

[0012]

[0013] This document does not disclose neither hints that any of the disclosed compounds could be particularly useful for the treatment of cancer.

[0014]

[0010] From what is disclosed in the art, it derives that there is still a need for HDAC inhibitors useful in the prevention and / or treatment of cancer.

[0015] SUMMARY OF THE INVENTION

[0016]

[0011] After exhaustive research, the inventors have found that certain UDCA derivatives bearing an aromatic 1,2-diamine group are particularly useful in the prevention and / or treatment of cancer, in particular of cholangiocarcinoma.

[0017]

[0012] The experimental results shown on file support the fact that the compounds developed by the inventors are suitable inhibitors of at least one of HDAC1, HDAC2, HDAC3, HDAC8 and HDAC11, which is not expected in view of the prior art disclosing UDCA derivatives as HDAC6 inhibitors.

[0018]

[0013] Class-l-selective HDAC inhibition provides unexpectedly superior therapeutic outcomes compared with both single-isoform inhibition and pan-HDAC inhibition. Class-I HDAC isoforms (HDAC1, HDAC2, HDAC3, and HDAC8) operatein overlapping co-repressor complexes, and simultaneous suppression of these isoforms results in coordinated reactivation of tumor-suppressor pathways, yielding stronger antiproliferative and pro-apoptotic effects than inhibition of any single HDAC isoform alone. At the same time, restricting inhibitory activity to the Class-1 subgroup avoids the broad off-target effects characteristic of pan-HDAC inhibitors, which disrupt multiple HDAC classes and can lead to undesirable toxicities and non-specific biological effects due to widespread perturbation of gene-regulatory networks. Class-l-selective inhibition thus achieves a balanced and therapeutically advantageous modulation of epigenetic activity, offering enhanced efficacy over single-isoform inhibition and improved safety and specificity over pan-HDAC inhibition.

[0019]

[0014] Accordingly, the compounds of the invention allow inhibiting a plurality of Class-I HDACs with high selectivity, which advantageously allows achieving high therapeutic activity with minimalized side effects.

[0020]

[0015] The compounds according to the invention have also been proven to efficiently:

[0021] (i) inhibit the growth of cancer cell lines of different types of cancer, including leukemia, lung, colon, central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, bile duct cancer and breast cancer,

[0022] (ii) decreasing the cell viability of bile duct cancer cell lines,

[0023] (iii) inducing apoptosis of bile duct cancer cell lines,

[0024] (iv) decreasing the relative size of tridimensional spheroids formed on bile duct cancer cell lines and bile duct cancer cell lines resistant to cisplatin,

[0025] (v) decrease the migration capacity of bile duct cancer cells.

[0026]

[0016] Thus, a first aspect of the invention relates to a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use in the treatment and / or prevention of cancer

[0027] R4

[0028]

[0029] (I)

[0030] wherein:

[0031] X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;

[0032] n is an integer of from 1 to 2;

[0033] each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0034] Y is a group selected from the groups of formula Y1 and Y2,

[0035]

[0036] R2

[0037] Y1Y2

[0038] wherein:

[0039] R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring or a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro; and

[0040] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0041]

[0017] The invention also relates to novel derivatives of bile acids. Thus, a second aspect of the invention relates to a compound of formula (I’)

[0042] R4

[0043]

[0044] (I’)

[0045] wherein:

[0046] X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;

[0047] R1 is selected from the group consisting of hydrogen, a (Ci-Cs)alkyl chain and a (C1-Cs)haloalkyl chain;

[0048] n is an integer of from 1 to 2;each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0049] Y is a group selected from the groups of formula Y1 and Y2,

[0050] NH2<

[0051] R9

[0052] TR3 R8

[0053] R2

[0054]

[0055] Y1Y2

[0056] wherein:

[0057] R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; and

[0058] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro;

[0059] or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof.

[0060]

[0018] In a third aspect, the invention relates to a compound according to the second aspect of the invention for use in medicine.

[0061]

[0019] In a fourth aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I’) or a pharmaceutically acceptable salt thereof according to the second aspect of the invention and at least one pharmaceutically acceptable excipient, vehicle or carrier.

[0062]

[0020] In a fifth aspect, the invention relates to a process for the preparation of a compound of formula (I’) according to the second aspect of the invention comprising the step of causing a bile acid of formula (II) that is selected from the group consisting of ursodeoxycholic acid, deoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, chenodeoxycholic acid, hyodeoxycholic acid and dehydrocholic acid or a stereoisomer thereof to react with a compound of formula (III)

[0063] R4

[0064]

[0065] (III)wherein R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;

[0066] n is an integer of from 1 to 2;

[0067] each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0068] Y is a group selected from the groups of formula Y1 and Y2,

[0069]

[0070] R2

[0071] Y1Y2

[0072] wherein:

[0073] R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; and

[0074] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0075]

[0021] The sixth aspect of the invention relates to a composition for use in the prevention and / or treatment of cancer, said composition comprising a therapeutically effective amount of (i) a compound of formula (I) according to the first aspect of the invention and (ii) a chemotherapeutic agent suitable for the prevention and / or treatment of cancer.

[0076] BRIEF DESCRIPTION OF THE DRAWINGS

[0077]

[0022] Fig. 1 describes a comparative immunoblot measuring the respective levels of acetylation of H3K9 (substrate for nuclear HDACs) and a-tubulin (substrate for HDAC6) in cholangiocarcinoma cell line EG1 incubated during 24 hours in presence of a 10 pM solution of a ursodeoxycholic acid analogue as shown.

[0078]

[0023] Fig. 2 describes a comparative immunoblot measuring the respective levels of acetylation of H3K9 (substrate for nuclear HDACs) and a-tubulin (substrate for HDAC6) in cholangiocarcinoma cell lines EG1 and HUCCT1 incubated during 24 hours (top) and 48 hours (bottom) in the presence of a solution of a compoundaccording to the invention (compound (la)) and a comparative example (compound (le)) having a concentration of 2 pM or 10 pM.

[0079]

[0024] Fig. 3 shows the dosis-response curve of the inhibiting effect of the compound of formula (la) on HDAC1, HDAC2, HDAC3, HDAC8 and HDAC11.

[0080]

[0025] Fig. 4 shows the effect of incubating a 10 pM solution of one of the compounds (la)-(lj) on the proliferation (top) and apoptosis (bottom) of cholangiocarcinoma cell line EG1 during 48 hours, as measured by flow cytometry.

[0081]

[0026] Fig. 5 shows representative bar diagrams quantifying the respective effects of solutions of the compounds of formula (la) and (le) having a concentration of 2 pM or 10 pM on the proliferation of cholangiocarcinoma cell lines EG1, HUCCT1 and WITT during 48 hours, as measured by flow cytometry.

[0082]

[0027] Fig. 6 shows representative bar diagrams quantifying the respective effects of solutions of the compounds of formula (la) and (le) having a concentration of 10 pM or 20 pM on the apoptosis of cholangiocarcinoma cell lines EG1 and HUCCT1 during 48 hours, as measured by flow cytometry.

[0083]

[0028] Fig. 7 shows: (A - top) fluorescence microscopy images of spheroids formed on cholangiocarcinoma EG1 cells incubated during 48 hours with a solution of (la) at a concentration of 0 pM, 2 pM, 10 pM or 20 pM and (B - bottom) quantification of relative spheroid area and death after 48 hours incubation of EG1 cells with a solution of (la) or (le) at a concentration of 0 pM, 2 pM, 10 pM or 20 pM.

[0084]

[0029] Fig. 8 shows the growth inhibition, expressed as a percentage, of several cancer cell lines of the National Cancer Institute 60 Cell Line Screening Program exposed to a dosis of 10 pM of the compound of formula (la) (bottom) or of formula (le) (top). Each point in the graphic represents a different cell line of a determined cancer type.

[0085]

[0030] FIG. 9 shows (A - top) fluorescence microscopy images of spheroids formed on cholangiocarcinoma EG1 cells resistant to cisplatin (EG1-R) incubated during 48 hours with a solution of (la) at a concentration of 0 pM, 10 pM or 20 pM or a 30 pM solution of cisplatin and (B - bottom) quantification of relative spheroid area after 48 hours incubation of EG1-R with a solution of (la) or (le) at a concentration of 0 pM, 10 pM or 20 pM or with a solution of cisplatin at a concentration of 30 pM.

[0086]

[0031] FIG. 10 shows (top) microscopy pictures of the transwell assays carried out with a population of EG1 cells incubated during 24 hours with a solution in bovin fetal serum of a compound of formula (la) having a concentration of 0 pM, 10 pM or 20 pM and (bottom) the quantified relative amount of migrated cells after 24 hours of incubation.

[0087] DETAILED DESCRIPTION

[0032] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0088]

[0033] For the purposes of the invention, any ranges given include both the lower and the upper end-points of the range. Ranges given, such as temperatures, times, molar ratio, volume ratio and the like, should be considered approximate (i.e. with a 5% margin of variation around indicated point), unless specifically stated.

[0089]

[0034] In the context of the invention, the term “salt” should be understood to mean an ionic compound formed by a cation and an anion. In the case of acidic products, the product will form the anion together with a cationic counterion, and in the case of basic products the product will form the cation together with an anionic counterion. Examples of anionic counterions are acid anions, whether inorganic (such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, and phosphate, inter alia) or organic (such as, for example, acetate, trifluoroacetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, and p-toluenesulfonate, inter alia). Examples of cationic counterions are cations of bases such as alkali metals, preferably sodium or potassium, alkali-earth metals, preferably calcium and magnesium and cations of organic nitrogenous bases such as primary, secondary, or tertiary amines.

[0090]

[0035] In the context of the present invention, the term “solvate”, when used in the context of a solvate of a compound, refers to the presence of a stoichiometric or non-stoichiometric amount of one or more molecules of one or more solvents in the crystal structure of said compound.

[0091]

[0036] An "effective amount" or "therapeutically effective amount" of a drug or pharmacologically active agent means a non-toxic but sufficient amount of the drug or agent to provide the desired effect. The amount that is "effective" will vary from subject to subject, depending on the age and general condition of the individual, the particular active agent or agents, and the like. Therefore, it is not always possible to specify an exact "effective quantity". However, an appropriate "effective" amount in any individual case can be determined by the skilled person using routine experimentation.

[0092]

[0037] In the context of the invention, the term “acyl” refers to a group that is attached to the remainder of the molecule via a C=O group.

[0093]

[0038] In the context of the invention, several bile acids are referred to, said bile acids being well known in the art. These bile acids are ursodeoxycholic acid,deoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, chenodeoxycholic acid, hyodeoxycholic acid and dehydrocholic acid:

[0094]

[0095] cholic acid chenodeoxycholic acidlithocholic acid

[0096]

[0097] obeticholic acidhyodeoxycholic acid dehydrocholic acid

[0098]

[0039] As used herein, the term “treatment” or derivations thereof include the eradication, removal, reversion, alleviation, modification, or control of a disease or disorder.

[0099]

[0040] As used herein, the term “prevention” or derivations thereof refer to the avoiding or minimizing of the onset or development of a disease or disorder.

[0100]

[0041] The term “excipient” refers to components of a drug compound other than the active ingredient. They preferably include a "carrier, adjuvant and / or vehicle". Carriers are forms to which substances are incorporated to improve the delivery and the effectiveness of drugs. Drug carriers are used in drug-delivery systems such as the controlled-release technology to prolong in vivo drug actions, decrease drug metabolism, and reduce drug toxicity. Carriers are also used in designs to increase the effectiveness of drug delivery to the target sites of pharmacological actions. Adjuvant is a substance added to a drug product formulation that affects the action of the active ingredient in a predictable way. Vehicle is an excipient or a substance, preferably without therapeutic action, used as a medium to give bulk for the administration of medicines. Such pharmaceutical carriers, adjuvants or vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal,vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, excipients, disgregants, wetting agents or diluents.

[0101]

[0042] In the context of the present invention, the term “cancer” refers to a group of diseases characterized by the uncontrolled growth and proliferation of abnormal cells, which can invade surrounding tissues and spread to distant sites within the body (metastasis). Cancer typically arises as the result of genetic and / or epigenetic alterations that disrupt normal cellular regulatory mechanisms governing proliferation, differentiation, apoptosis, and repair processes. These alterations enable tumor cells to evade growth suppressors, resist cell death, sustain angiogenesis, and promote invasion and metastasis, ultimately leading to the formation of malignant tumors. Examples of cancer diseases are well known in the art and will become apparent to the skilled person. Those include, among others, leukemia, lung, colon, central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, bile duct cancer and breast cancer.

[0102]

[0043] In the context of the present invention, the term “alkyl” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims. For example, a (Ci-Ce)alkyl group refers to a linear or branched saturated hydrocarbon chain having from one to six carbon atoms. Examples of alkyl groups include methyl, ethyl, n-propyl, / -propyl, n-butyl, n-pentyl and n-hexyl.

[0103]

[0044] In the context of the present invention, the term “haloalkyl” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims and wherein at least one hydrogen is replaced with a halogen atom. In particular embodiments, said halogen atom is a fluorine atom. Examples of haloalkyl groups include, in a non-limiting manner, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, chloroethyl, amddicholorethyl.

[0104]

[0045] In the context of the present invention, the term “5- to 6- membered heteroaryl ring” refers to an aromatic ring comprising from 5 to 6 ring members, said ring members being selected from C, CH, N, NH, O and S, at least one of said members being selected from N, NH, O and S. Said heteroaryl ring may further be optionally substituted at any available position as disclosed herein. Examples of such 5- to 6-membered heteroaryl ring include, in a non limiting way, pyrrole, furan, thiophene, imidazole, thiazole, oxazole, pyrazole, pyridine, pyridazine, pyrimidine, pyrazine, 1,3,5-triazine, and 1,2,4-triazine.

[0105]

[0046] In the context of the present invention, the term “halo” refers to a halogen group, such as fluoro, chloro, bromo or iodo.

[0047] In the context of the present invention, the term “alkyloxy” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims that is connected to the remaining part of the molecule with an oxy -0- diradical. Examples of alkyloxy groups include methyloxy, ethyloxy, n-propyloxy, / -propyloxy, n-butyloxy, n-pentyloxy and n-hexyloxy.

[0106]

[0048] In the context of the present invention, the term “alkylamino” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims that is connected to the remaining part of the molecule with an amino -NH- diradical. Examples of alkylamino groups include methylamino, ethylamino, n-propylamino, / -propylamino, n-butylamino, n-pentylamino and n-hexylamino.

[0107]

[0049] In the context of the present invention, the term “alkylcarbonyl” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims that is connected to the remaining part of the molecule with a carbonyl -CO- diradical. Examples of alkylcarbonyl groups include methylcarbonyl, ethylcarbonyl, n-propylcarbonyl, / -propylcarbonyl, n-butylcarbonyl, n-pentylcarbonyl and n-hexylcarbonyl.

[0108]

[0050] In the context of the present invention, the term “alkyloxycarbonyl” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims that is connected to the remaining part of the molecule with an oxycarbonyl -OCO diradical. Examples of alkyloxycarbonyl groups include methyloxycarbonyl, ethyloxycarbonyl, n-propyloxycarbonyl, / -propyloxycarbonyl, n-butyloxycarbonyl, n-pentyloxycarbonyl and n-hexyloxycarbonyl.

[0109]

[0051] In the context of the present invention, the term “alkylcarbonyloxy” refers to a linear or branched saturated hydrocarbon chain having the number of carbon atoms specified in the description and in the claims that is connected to the remaining part of the molecule with a carbonyloxy -COO- diradical. Examples of alkylcarbonyloxy groups include methylcarbonyloxy, ethylcarbonyloxy, n-propylcarbonyloxy, / -propylcarbonyloxy, n-butylcarbonyloxy, n-pentylcarbonyloxy and n-hexylcarbonyloxy.

[0110]

[0052] As defined above, a first aspect of the invention relates to a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use in the treatment and / or prevention of cancer

[0111]

[0112] wherein:

[0113] X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;

[0114] R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;

[0115] n is an integer of from 1 to 2;

[0116] each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0117] Y is a group selected from the groups of formula Y1 and Y2,

[0118] Y1Y2

[0119]

[0120] wherein:

[0121] R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring or a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro; and

[0122] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0123]

[0053] In an embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein n is 1.

[0054] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Ri is selected from the group consisting of hydrogen, methyl and trifluoromethyl.

[0124]

[0055] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Ri is hydrogen.

[0125]

[0056] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein n is 1 and Ri is hydrogen.

[0126]

[0057] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, halo, cyano and nitro.

[0127]

[0058] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein each one of R4, R5, R6and R7is hydrogen.

[0128]

[0059] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0129] - n is 1;

[0130] - R1 is hydrogen; and

[0131] - each one of R4, R5, R6and R7is hydrogen.

[0132]

[0060] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein X is a ursodeoxycholic acyl group.

[0133]

[0061] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Y is a group of formula Y1.

[0134]

[0062] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring or a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from halo, cyano and nitro.

[0135]

[0063] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring, said phenyl ring being optionally substituted at anyavailable position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0136]

[0064] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Yi, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring, said phenyl ring being optionally substituted at any available position with a halo group.

[0137]

[0065] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2and R3together with the carbon atoms to which they are attached form a phenyl ring optionally substituted at one or more available position with a fluorine group.

[0138]

[0066] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, 4-fluorophenyl, 5-fluorophenyl and 4,5-difluorophenyl.

[0139]

[0067] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro. Said thiophenyl, furanyl and pyrrolyl rings ar epreferably thiophen-2-yl, furan-2-yl and pyrrol-2-yl.

[0140]

[0068] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionally substituted at any available position with a halo group. Said thiophenyl, furanyl and pyrrolyl rings ar epreferably thiophen-2-yl, furan-2-yl and pyrrol-2-yl.

[0141]

[0069] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionally substituted at one or more available positionwith a fluorine group. Said thiophenyl, furanyl and pyrrolyl rings are preferably thiophen-2-yl, furan-2-yl and pyrrol-2-yl.

[0142]

[0070] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0143] - n is 1;

[0144] - R1 is hydrogen; and

[0145] - Y is a group of formula Yi wherein R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl - preferably a phenyl ring - said ring being optionally substituted at one or more available position with a fluorine group.

[0146]

[0071] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0147] - n is 1;

[0148] - R1 is hydrogen;

[0149] - each one of R4, Rs, Re and R7 is hydrogen; and

[0150] - Y is a group of formula Y1 wherein R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl - preferably a phenyl ring - said ring being optionally substituted at one or more available position with a fluorine group.

[0151]

[0072] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0152] - X is a ursodeoxycholic acyl group;

[0153] - n is 1;

[0154] - R1 is hydrogen; and

[0155] - Y is a group of formula Y1 wherein R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl - preferably a phenyl ring - said ring being optionally substituted at one or more available position with a fluorine group.

[0156]

[0073] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0157] - n is 1;

[0158] - R1 is hydrogen;

[0159] - Y is a group of formula Y1 wherein R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl - preferably a phenyl ring - said ringbeing optionally substituted at one or more available position with a fluorine group; and

[0160] - each one of R4, R5, R6and R7is hydrogen.

[0161]

[0074] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein:

[0162] - X is a ursodeoxycholic acyl group;

[0163] - n is 1;

[0164] - R1 is hydrogen;

[0165] - Y is a group of formula Y1 wherein R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring optionally substituted at one or more available position with a fluorine group; and

[0166] - each one of R4, R5, R6and R7is hydrogen.

[0167]

[0075] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is selected from the group consisting of the compounds of formula (la), (lb), (lc) and (Id)

[0168]

[0169] (lc) (Id)

[0170]

[0076] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is the compound of formula (la).

[0171]

[0077] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Y is a group of formula Y2.

[0172]

[0078] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Y isa group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furanyl, thiophenyl and pyrrolyl that is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

[0173]

[0079] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl that is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

[0174]

[0080] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl.

[0175]

[0081] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is one wherein - n is 1;

[0176] - R1 is hydrogen;

[0177] - Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl; and - each one of R4, R5, R6and R7is hydrogen.

[0178]

[0082] In a further embodiment of the first aspect of the invention, the compound of formula (I) for use in the prevention and / or treatment of cancer is selected from the group consisting of:

[0179] N-(2-aminophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0180] N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0181] N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino-4,5-difluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-amino-1H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(thiophen-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(1H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-aminophenyl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino4,5-difluorophenyl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0182]

[0183] N-(3-amino-1H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(thiophen-2-yl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(1H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-amino-1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0184] N-(thiophen-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0185] N-(1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0186] N-(2-aminophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0187] N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0188] N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0189] N-(2-amino-4,5-difluorophenyl)-4-(((R)-4- ((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0190] N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0191] N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0192] N-(3-amino-1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0193] N-(thiophen-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0194] N-(1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-aminophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0195] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0196] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0197] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0198] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0199] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0200] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0201] N-(thiophen-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0202] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0203] N-(2-aminophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0204] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0205] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0206] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0207] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0208] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0209] N-(thiophen-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0210] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0211] N-(2-aminophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0212] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0213] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0214] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0215] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0216] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0217] N-(thiophen-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0218] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0219] N-(2-aminophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0220] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0221] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0222] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0223] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0224] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0225] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0226] N-(thiophen-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0227] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-aminophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0228] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0229] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0230] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0231] N-(3-aminofuran-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0232] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0233] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0234] N-(thiophen-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide and

[0235] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide.

[0236]

[0083] The first aspect of the invention also relates to stereoisomers, pharmaceutically acceptable salts and solvates of the compounds of formula (I) as described above for use in the prevention and / or treatment of cancer.

[0237]

[0084] In a further embodiment, the first aspect of the invention relates to a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof as defined above for use in the prevention and / or treatment of cancer wherein said cancer is selected from the group consisting of leukemia, lung, colon, central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, bile duct cancer and breast cancer.

[0238]

[0085] In a further embodiment, the first aspect of the invention relates to a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptablesalt or solvate thereof as defined above for use in the prevention and / or treatment of cancer wherein said cancer is bile duct cancer or cholangiocarcinoma.

[0239]

[0086] The first aspect of the invention may also relate to the use of a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof as defined above in the preparation of a medicament for the prevention and / or treatment of cancer.

[0240]

[0087] The first aspect of the invention may also relate to a method for the treatment and / or prevention of cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof as defined above.

[0241]

[0088] As mentioned above, the invention also relates in a second aspect to novel compounds of formula (I’)

[0242] R4

[0243] k.. R5

[0244] R7O

[0245]

[0246] wherein:

[0247] X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;

[0248] R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;

[0249] n is an integer of from 1 to 2;

[0250] each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0251] Y is a group selected from the groups of formula Y1 and Y2,

[0252] l2

[0253] I R

[0254] R2R:I

[0255]

[0256] wherein:R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; and

[0257] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro;

[0258] or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof.

[0259]

[0089] In an embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein n is 1.

[0260]

[0090] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein R1 is selected from the group consisting of hydrogen, methyl and trifluoromethyl.

[0261]

[0091] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein R1 is hydrogen.

[0262]

[0092] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein n is 1 and R1 is hydrogen.

[0263]

[0093] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, halo, cyano and nitro.

[0264]

[0094] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein each one of R4, R5, R6and R7is hydrogen.

[0265]

[0095] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein:

[0266] - n is 1;

[0267] - R1 is hydrogen; and

[0268] - each one of R4, R5, R6and R7is hydrogen.

[0269]

[0096] In a further embodiment of the first aspect of the invention, the compound of formula (I’) is one wherein X is a ursodeoxycholic acyl group.

[0270]

[0097] In a further embodiment of the first aspect of the invention, the compound of formula (I’) is one wherein Y is a group of formula Y1.

[0271]

[0098] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at anyavailable position with a group selected from halo, cyano and nitro; or (ii) a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from halo, cyano and nitro.

[0272]

[0099] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Yi, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0273]

[0100] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring substituted at any available position with a group selected from a halo group.

[0274]

[0101] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring substituted at one or more available position with a fluorine group.

[0275]

[0102] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of 4-fluorophenyl, 5-fluorophenyl and 4,5-difluorophenyl.

[0276]

[0103] In a further embodiment of the first aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro. Said thiophenyl, furanyl and pyrrolyl rings ar epreferably thiophen-2-yl, furan-2-yl and pyrrol-2-y I.

[0277]

[0104] In a further embodiment of the first aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionally substituted at any available position with a halo group. Said thiophenyl, furanyl and pyrrolyl rings ar epreferably thiophen-2-yl, furan-2-yl and pyrrol-2-yl.

[0278]

[0105] In a further embodiment of the first aspect of the invention, the compound of formula (I’) is one wherein, in the group of formula Y1, R2 and R3 together with the carbon atoms to which they are attached form a ring selected from the group consisting of phenyl, thiophenyl, furanyl and pyrrolyl, said ring being optionallysubstituted at one or more available position with a fluorine group. Said thiophenyl, furanyl and pyrrolyl rings are preferably thiophen-2-yl, furan-2-yl and pyrrol-2-yl.

[0279]

[0106]

[0280]

[0107] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein:

[0281] - n is 1;

[0282] - R1 is hydrogen; and

[0283] - Y is a group of formula Yi wherein R2 and R3 together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at one or more available position with a fluorine group or (ii) a ring selected from the group consisting of thiophenyl, furanyl and pyrrolyl - preferably a thiophen-2-yl, a furan-2-yl or a 1 H-pyrrol-2yl ring - said ring being optionally substituted at one or more available position with a fluorine group.

[0284]

[0108] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein:

[0285] - n is 1;

[0286] - R1 is hydrogen;

[0287] - R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring substituted at one or more available position with a fluorine group; and

[0288] - each one of R4, R5, R6and R7is hydrogen.

[0289]

[0109] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein:

[0290] - X is a ursodeoxycholic acyl group;

[0291] - n is 1;

[0292] - R1 is hydrogen;

[0293] - Y is a group of formula Y1 wherein R2 and R3 together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at one or more available position with a fluorine group or (ii) a ring selected from the group consisting of thiophenyl, furanyl and pyrrolyl - preferably a thiophen-2-yl, a furan-2-yl or a 1 H-pyrrol-2yl ring - said ring being optionally substituted at one or more available position with a fluorine group; and

[0294] - each one of R4, R5, R6and R7is hydrogen.

[0295]

[0110] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is selected from the group consisting of the compounds of formula (I’b), (I’c) and (I’d)

[0296]

[0297]

[0111] The molecular formulae of the compounds of formula (I’b), (I’c) and (I’d) are respectively the same as the molecular formulae of the compounds of formula (lb), (Ic) and (Id).

[0298]

[0112] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein Y is a group of formula Y2.

[0299]

[0113] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furanyl, thiophenyl and pyrrolyl that is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

[0300]

[0114] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl that is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

[0301]

[0115] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- memberedheteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl.

[0302]

[0116] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is one wherein

[0303] - n is 1;

[0304] - R1 is hydrogen;

[0305] - Y is a group of formula Y2 wherein Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring selected from the group consisting of furan-2-yl, thiophen-2-yl and pyrrol-2-yl; and - each one of R4, R5, R6and R7is hydrogen.

[0306]

[0117] In a further embodiment of the second aspect of the invention, the compound of formula (I’) is selected from the group consisting of:

[0307] N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0308] N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0309] N-(2-amino-4,5-difluorophenyl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0310] N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0311] N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0312] N-(3-amino-1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0313] N-(thiophen-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0314] N-(1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-aminophenyl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0315] N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0316] N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0317] N-(2-amino4,5-difluorophenyl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0318] N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0319] N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0320] N-(3-amino-1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0321] N-(thiophen-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0322] N-(1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3, 12-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0323] N-(2-aminophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0324] N-(2-amino-4-fluorophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7,12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0325] N-(2-amino-5-fluorophenyl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)- 3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0326] N-(2-amino-4,5-difluorophenyl)-4-(((R)-4- ((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0327] N-(3-aminofuran-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0328] N-(3-aminothiophen-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0329] N-(3-amino-1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0330] N-(thiophen-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0331] N-(1 H-pyrrol-2-yl)-4-(((R)-4-((3R,5S,7R,8R,9S, 10S, 12S, 13R, 14S, 17R)-3,7, 12-trihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0332] N-(2-aminophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0333] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0334] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0335] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0336] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0337] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,7R,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0338] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(thiophen-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0339] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,7R,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0340] N-(2-aminophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0341] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0342] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0343] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0344] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0345] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0346] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0347] N-(thiophen-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0348] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,8R,9S, 10S, 13R, 14S, 17R)-3-hydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0349] N-(2-aminophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0350] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0351] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0352] N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0353] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0354] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0355] N-(thiophen-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0356] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6R,7R,8S,9S, 10S, 13R, 14S, 17R)-6-ethyl-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0357] N-(2-aminophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0358] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0359] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0360] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(3-aminofuran-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0361] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0362] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0363] N-(thiophen-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0364] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((3R,6S,8S,9S, 10R, 13R, 14S, 17R)-3,6-dihydroxy- 10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0365] N-(2-aminophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0366] N-(2-amino-4-fluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0367] N-(2-amino-5-fluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0368] N-(2-amino-4,5-difluorophenyl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0369] N-(3-aminofuran-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0370] N-(3-aminothiophen-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;

[0371] N-(3-amino-1 H-pyrrol-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl- 3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide;N-(thiophen-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide and

[0372] N-(1 H-pyrrol-2-yl)-4-(((4R)-4-((8R,9S, 10S, 13R, 14S, 17R)-10, 13-dimethyl-3,7, 12-trioxohexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide.

[0373]

[0118]

[0374]

[0119] The second aspect of the invention also relates to stereoisomers, pharmaceutically acceptable salts and solvates of the compounds of formula (I’) as described above.

[0375]

[0120] As mentioned above, the third aspect of the invention relates to a compound of formula (I’) or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to the second aspect of the invention for use in medicine.

[0376]

[0121] In an embodiment, the third aspect of the invention relates to a compound of formula (I’) or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof as defined in any of the embodiments described above defining the compound of formula (I’) for use in medicine.

[0377]

[0122] The third aspect of the invention may also relate to the use of a compound of formula (I’) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof as defined above in the preparation of a medicament.

[0378]

[0123] The third aspect of the invention may also relate to use of a compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof as defined above as a medicament.

[0379]

[0124] The fourth aspect of the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I’) or a pharmaceutically acceptable salt thereof according to the second aspect of the invention and at least one pharmaceutically acceptable excipient, vehicle or carrier.

[0380]

[0125] Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, granules, etc.) or liquid composition (solutions, suspensions, or emulsions) for oral, topical, intravenous or parenteral administration.

[0381]

[0126] In a preferred embodiment, the pharmaceutical compositions are oral compositions. The dosage forms suitable for oral administration may include tablets and capsules and contain conventional excipients known in the art, such as binding agents, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example, lactose, sugar, cornstarch, calcium phosphate, sorbitol, or glycine; lubricants for preparing tablets, for example, magnesium stearate; disintegrants, for example, starch, polyvinylpyrrolidone,sodium starch glycolate, or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.

[0382]

[0127] The solid oral compositions can be prepared by means of conventional methods of mixing, filling, or preparing tablets. Repeated mixing operations can be used to distribute the active ingredient throughout all the compositions using large amounts of fillers. Such operations are conventional in the art. The tablets can be prepared, for example, by means of dry or wet granulation, and they can optionally be coated according to methods that are well known in normal pharmaceutical practice, particularly with an enteric coating.

[0383]

[0128] The pharmaceutical compositions can also be adapted for parenteral or intravenous administration, such as sterile solutions, suspensions, or lyophilized products in the form of suitable unit dose. Suitable excipients such as bulking agents, buffering agents, or surface-active agents can be used.

[0384]

[0129] The mentioned formulations will be prepared using common methods such as those described or referred to in the Spanish and United States Pharmacopeias and similar reference texts.

[0385]

[0130] In general, the effective administered amount of a compound of the invention will depend on the relative efficacy of the chosen compound, the severity of the disorder being treated, and the weight of the patient. However, the active compounds will usually be administered one or more times a day, for example, 1, 2, 3, or 4 times a day, with typical total daily doses in the range of 0.01 to 1000 mg / kg / day.

[0386]

[0131] The fifth aspect of the invention relates to a process for the preparation of a compound of formula (I’) according to the second aspect of the invention comprising the step of causing a bile acid of formula (II) that is selected from the group consisting of ursodeoxycholic acid, deoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, chenodeoxycholic acid, hyodeoxycholic acid and dehydrocholic acid or a stereoisomer thereof to react with a compound of formula (III)

[0387]

[0388] wherein R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;

[0389] n is an integer of from 1 to 2;

[0390] each one of R4, Rs, Re and R7 is independently selected from the group consisting of hydrogen, (Ci-Ce)alkyl, (Ci-Ce)haloalkyl, (Ci-Ce)alkyloxy, (Ci-C6)alkylamino, (C1-Ce)alkylcarbonyl, (Ci-C6)alkyloxycarbonyl, (Ci-C6)alkylcarbonyloxy, halo, cyano and nitro;

[0391] Y is a group selected from the groups of formula Y1 and Y2,

[0392]

[0393] R2

[0394] Y1Y2

[0395] wherein:

[0396] R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; and

[0397] Rs and R9 together with the carbon atoms to which they are attached form a 5- to 6-membered heteroaryl ring optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro.

[0398]

[0132] In embodiments of the fifth aspect of the invention, the compound of formula (III) is one wherein each one of n, R1, R2, R3, R4, Rs, Re, R7, Rs and R9 is independently as defined in any of the embodiments of the second aspect of the invention detailed above defining these groups.

[0399]

[0133] Adequate conditions for forming a compound of formula (I’) from the compounds of formulae (II) and (III) are well established in the art and will become apparent to the skilled person upon reduction to practice of the invention. Such conditions have been disclosed for instance in WO 2019 / 129913 A1, methods A, B and C, p. 20 I. 11 to p. 22 I. 7, the content of which is incorporated herein by reference.

[0400]

[0134] The process of the fifth aspect of the invention is in particular carried out in the presence of a peptidic coupling agent, an organic or inorganic base and a tertiary amine compound. Suitable peptidic coupling agents, organic or inorganic bases andtertiary amine compounds will independently become apparent to the skilled person on the basis of common general knowledge and the Examples disclosed below.

[0401]

[0135] As mentioned above, the sixth aspect of the invention relates to a composition for use in the prevention and / or treatment of cancer, said composition comprising a therapeutically effective amount of (i) a compound of formula (I) according to the first aspect of the invention and (ii) a chemotherapeutic agent suitable for the prevention and / or treatment of cancer.

[0402]

[0136] In an embodiment of the sixth aspect of the invention, the compound of formula (I) is as defined in any embodiment of the first aspect of the invention defining said compound detailed above.

[0403]

[0137] In a further embodiment of the sixth aspect of the invention, the composition is for use in the treatment of cancer wherein said cancer is selected from the group consisting of leukemia, lung, colon, central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, bile duct cancer and breast cancer.

[0404]

[0138] In a further embodiment of the sixth aspect of the invention, the composition is for use in the treatment of cancer wherein said cancer is bile duct cancer or cholangiocarcinoma.

[0405]

[0139] Suitable chemotherapeutic agents are selected depending on their therapeutic or prophylactic activity towards the targeted cancer disease and its stage of development.

[0406]

[0140] For instance, the chemotherapeutic agent may be selected from the group consisting of abraxane, actinomycin, alitretinoin, all-trans retinoic acid, altretamine, azacitidine, azathioprine, belotecan, bendamustine, bexarotene, bleomycin, bortezomib, busulfan, cabazitaxel, camptothecin, carboplatin, carboquone, carmustine, capecitabine, cisplatin, chlorambucil, chlormethine, chlorozotocin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, erlotinib, etoposide, exatecan, fludarabine, fluorouracil, fotemustine, gefitinib, gemcitabine, gimatecan, hydroxyurea, idarubicin, ifosfamide, imatinib, irinotecan, ixabepilone, larotaxel, lomustine, melphalan, melphalanflufenamide, mercaptopurine, methotrexate, mitobronitol, mitomycinc, mitoxantrone, nelarabine, nimustine, nitrosoureas, oxaliplatin, paclitaxel, pemetrexed, pipobroman, ranimustine, romidepsin, semustine, streptozotocin, tafluposide, taxotere, temozolomide, tesetaxel, teniposide, thiotepa, tioguanine, topotecan, treosulfan, tretinoin, triaziquone, triethylenemelamine, valrubicin, vemurafenib, vinblastine, vincristine, vindesine, vinorelbine, vismodegib, vorinostat and combinations thereof.

[0407]

[0141] In a further embodiment, the chemotherapeutic agent may be selected from the group consisting of gemcitabine, cisplatin, capecitabine, oxaliplatin, 5-fluorouracil and combinations thereof. This is particularly the case when the cancer to be treated comprises cholangiocarcinoma or bile duct cancer.

[0408]

[0142] Each of the compound of formula (I) and the chemotherapeutic agent may be present in said combination in a therapeutically or prophylactically effective amount and / or in a manner that the combination composition is therapeutically or prophylactically effective.

[0409]

[0143] The sixth aspect of the invention may also relate to the use of a a composition comprising a therapeutically effective amount of (i) a compound of formula (I) according to the first aspect of the invention and (ii) a chemotherapeutic agent suitable for the prevention and / or treatment of cancer as defined above in the preparation of a medicament for the prevention and / or treatment of cancer.

[0410]

[0144] The sixth aspect of the invention may also relate to a method for the treatment and / or prevention of cancer comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising a (i) a compound of formula (I) according to the first aspect of the invention and (ii) a chemotherapeutic agent suitable for the prevention and / or treatment of cancer.

[0411]

[0145] Throughout the description and claims the word “comprises" and variations of the word, are not intended to exclude other technical features, additives, components or steps. Furthermore, the word “comprise” encompasses the cases of “consist of” and “consists essentially of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention.

[0412] EXAMPLES

[0413] List of abbreviations

[0414] DMF: N, N-dimethylformamide

[0415] EDCI: 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide

[0416] TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate DCM: dichloromethane

[0417] EtOAc: ethyl acetate

[0418] IR: Infrared spectroscopy

[0419] NMR: Nuclear Magnetic Resonance spectroscopy

[0420] DMAP: 4-(Dimethylamino)pyridine

[0421] HRMS: High Resolution Mass Spectrometry

[0422] ESI: Electrospray IonizationBSA: bovine serum albumin

[0423] DPBS: Dulbecco’s Phosphate-Buffered Saline

[0424] DMSO: Dimethyl sulfoxide

[0425] dH2O: Distilled water

[0426] DMEM / F12: Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 GlutaMAX: L-alanyl-L-glutamine

[0427] P / S: Penicillin-Streptomycin

[0428] EGF: Epidermal Growth Factor

[0429] B27: B27 Supplement (a serum-free supplement containing vitamins, hormones, and antioxidants)

[0430] FBS: Fetal Bovine Serum

[0431]

[0146] methyl 4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoate was prepared according to the procedure for the preparation of the compound 3a disclosed in Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference.

[0432]

[0147] 4-(((R)-4-((3R,5S,7S,8R,9S,1 OS, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecanohydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoic acid was prepared according to the procedure for the preparation of the compound 4a disclosed in Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference.

[0433]

[0148] 4-((4-(((R)-4-((3R,5S,7S,8R,9S,1 OS, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzoic acid was prepared by hydrolysis of methyl 4-((4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzoate following the procedure of Method B of Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference. Methyl 4-((4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzoate was prepared according to the procedure for the preparation of the compound 6a disclosed in Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference.

[0434]

[0149] 6-((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoic acid was prepared by hydrolysis of methyl 6-((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate following the procedure of Method B of Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference. Methyl 6-((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13- dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoate was prepared according to the procedure for the preparation of the compound 3f disclosed in Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference.

[0435]

[0150] 5-(((R)-4-((3R,5S,7S,8R,9S,1 OS, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)furan-2- carboxylic acid was prepared by hydrolysis of methyl 5-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)furan-2- carboxylate following the procedure of Method B of Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis in Experimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference. Methyl 5-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)furan-2- carboxylate was prepared according to the procedure for the preparation of the compound 3g disclosed in Caballero-Camino et al. Synthetic Conjugates of Ursodeoxycholic Acid Inhibit Cystogenesis inExperimental Models of Polycystic Liver Disease. Hepatology 73(1 ):p 186-203, January 2021 (DOI: 10.1002 / hep.31216), the content of which is incorporated herein by reference.

[0436] Example 1 Preparation of N-(2-aminophenyl)-4-(((R)-4- ((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadeca-hydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzamide (Ia)

[0437] o

[0438]

[0439]

[0151] To a solution of 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecanohydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoic acid (206 mg, 0.38 mmol) in DMF (2.5 mL), o-phenylenediamine (41.1 mg, 0.38 mmol) and TBTII (148 mg, 0.46 mmol) were added. The solution was brought to 0 °C in an inert atmosphere and a solution of EtsN (0.2 mL) in DMF (0.24 mL) with a concentration of 3.26 M was added dropwise. The mixture was stirred for 3 hours at 0 °C. The reaction was monitored by thin layer chromatography. The solvent was then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (7.5 mL) and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL) and saturated NaCI (1 x 5 mL). The organic phase was dried over MgSCU and the evaporated under reduced pressure. The solid obtained was purified by column chromatography and the compound (la) was isolated as a white solid. Yield 39%.

[0440]

[0152] Melting point 168-170 °C;

[0441]

[0153] IR: 3294, 2927, 2862, 1647, 1505, 1048, 745 cm’1;

[0442]

[0154] 1H NMR (400 MHz, DMSO-de) 69.61 (s, 1 H), 8.38 (t, J = 6.0 Hz, 1H), 7.92 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.16 (d, J = 7.9 Hz, 1 H), 7.04 - 6.92 (m, 1 H), 6.78 (dd, J = 8.0, 1.4 Hz, 1 H), 6.69 - 6.49 (m, 1 H), 4.88 (s, 2H), 4.42 (d, J = 4.6 Hz, 1 H), 4.37 - 4.25 (m, 2H), 3.86 (d, J = 6.8 Hz, 1 H), 3.29 (2H), 2.25 - 2.01 (m, 2H), 1.98 - 1.58 (m, 6H), 1.56 - 1.27 (m, 9H), 1.27 - 0.94 (m, 5H), 0.94 - 0.82 (m, 10H), 0.62 (s, 3H);

[0443]

[0155] 13C NMR (101 MHz, DMSO-de) 6 172.63, 165.05, 143.38, 143.11, 132.99, 127.74, 126.81, 126.66, 126.43, 123.32, 116.23, 116.11, 69.70, 69.46, 55.87, 54.75,43.08, 43.01, 42.15, 41.71, 38.71, 37.71, 37.26, 34.91, 34.82, 33.75, 32.39, 31.69, 30.24, 28.19, 26.72, 23.31, 20.84, 18.45, 12.03;

[0444]

[0156] HRMS (ESI) for C38H54N3O4, calculated [M+H]+: 616.4114. Obtained: 616.4110.

[0445] Example 2: Preparation of (4R)-N-(4-((2-amino-4-fluorophenyl)amino)benzyl)-4-((3S,7S,8R,9S, 10S, 13R, 14S)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-l H-cyclopenta[a]phenanthren-17-yl)pentanamide (lb)

[0446] o

[0447]

[0448]

[0157] To a solution of 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecanohydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoic acid (500 mg, 0.95 mmol) in DMF (5 mL), 4,5- tert-Butyl (2-amino-5-fluorophenyl)carbamate (214.9 mg, 0.95 mmol) and TBTII (365.9 mg, 1.14 mmol) were added. The solution was brought to 0 °C in an inert atmosphere and a solution of EtsN (0.2 mL) in DMF (0.24 mL) with a concentration of 3.26 M was added dropwise. The mixture was stirred for 3 hours at 0 °C. The reaction was monitored by thin-layer chromatography. The solvent was then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (7.5 mL) and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL), and saturated NaCI (1 x 5 mL). The organic phase was dried over MgSCU and the evaporated under reduced pressure. The compound was solubilized in DCM (10 mL) and then 20 equiv. of TFA was added. The reaction mixture was stirred for 14h. Finally, the solvent was eliminated under vacuum. The solid obtained was purified by column chromatography, and the compound (lb) was isolated as a white solid. Yield 33%

[0449]

[0158] Melting point 169-170°C

[0450] Example 3: Preparation of (4R)-N-(4-((2-amino-5-fluorophenyl)amino)benzyl)-4-((3S,7S,8R,9S, 10S, 13R, 14S)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-l H-cyclopenta[a]phenanthren-17-yl)pentanamide (Ic)

[0451]

[0452]

[0159] To a solution of 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecanohydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoic acid (500 mg, 0.95 mmol) in DMF (5 mL), tert-Butyl 2-amino-4-fluorophenylcarbamate (214.9 mg, 0.95 mmol) and TBTII (365.9 mg, 1.14 mmol) were added. The solution was brought to 0 °C in an inert atmosphere and a solution of EtsN (0.2 mL) in DMF (0.24 mL) with a concentration of 3.26 M was added dropwise. The mixture was stirred for 3 hours at 0 °C. The reaction was monitored by thin-layer chromatography. The solvent was then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (7.5 mL) and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL), and saturated NaCI (1 x 5 mL). The organic phase was dried over MgSCU and the evaporated under reduced pressure. The compound was solubilized in DCM (10 mL) and then 20 equiv. of TFA was added. The reaction mixture was stirred for 14h. Finally, the solvent was eliminated under vacuum. The solid obtained was purified by column chromatography, and the compound (Ic) was isolated as a white solid. Yield 48%

[0453]

[0160] Melting point 175-176°C

[0454] Example 4: Preparation of (4R)-N-(4-((2-amino-4,5-difluorophenyl)amino)benzyl)-4 ((3S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide (Id)

[0455]

[0456]

[0161] To a solution of 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecanohydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoic acid (500 mg, 0.95 mmol) in DMF (5 mL), 4,5-Difluoro-1,2-phenylenediamine (137.2 mg, 0.95 mmol) and TBTU (369.8 mg, 1.15 mmol) were added. The solution was brought to 0 °C in an inert atmosphere and a solution of EtsN (0.2 mL) in DMF (0.24 mL) with a concentration of 3.26 M was added dropwise. The mixture was stirred for 3 hours at 0 °C. The reaction was monitored by thin layer chromatography. The solvent was then evaporated under reduced pressure, and the residue was dissolved in ethyl acetate (7.5 mL) and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL) and saturated NaCI (1 x 5 mL). The organic phase was dried over MgSCU and the evaporated under reduced pressure. The solid obtained was purified by column chromatography and the compound (Id) was isolated as a white solid. Yield 55%

[0457]

[0162] Melting point 168-169°C

[0458] Comparative Example 1: Preparation of 4-((2-((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadeca hydro-1 H-cyclopenta[alphenanthren-17-yl)pentanamide)acetamido)methyl)-N-hydroxybenzamide (le)

[0459]

[0460]

[0163] A suspension of sodium methoxide in methanol (previously prepared 2.0 g, 37 mmol solution) was added dropwise to a solution containing hydroxylamine hydrochloride (694.9 mg, 10 mmol) and phenolphthalein (1 mg) under inert atmosphere and at 0 °C until a permanent color change from white to pink was observed. Next, methyl 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamide)methyl)benzoate (539.4 mg, 1 mmol) dissolved in dry methanol (1 ml) was added. The reaction mixture was left to reach room temperature, and its progression was monitored by thin layer chromatography. The initial reagents were consumed after 90 hours. After this time has lapsed, distilled water (10 ml) was added to the reaction medium, and it was acidified with glacial acetic acid. The product was then extracted with diethyl ether (3 x 20 ml). The combined organic fractions were dried on MgSCU and evaporated under reduced pressure. The product thus obtained was dissolved again in methanol (1 ml) and precipitated withwater. This precipitate was filtered and the solvent was evaporated. The title product was thereby obtained as a white solid. Yield 61 %.

[0461]

[0164] Melting point 170-172°C

[0462]

[0165] IR 3275, 2927, 2862, 1638, 1535, 1012 cm’1

[0463]

[0166] 1H NMR (400 MHz, DMSO-de) 5 11.17 (s, 1H), 9.01 (s, 1H), 8.34 (t, J = 6.0 Hz, 1 H), 7.69 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 8.1 Hz, 2H), 4.44 (d, J = 4.5 Hz, 1 H), 4.28 (dd, J = 5.9, 3.6 Hz, 2H), 3.87 (d, J = 6.8 Hz, 1H), 3.29 (2H). 2.24 - 2.01 (m, 2H), 1.99- 1.59 (m, 6H), 1.56- 1.26 (m, 10H), 1.26 - 0.94 (m, 7H), 0.94 - 0.84 (m, 7H), 0.62 (s, 3H)

[0464]

[0167] 13C NMR (101 MHz, DMSO-de) 6 172.63, 163.99, 143.06, 131.18, 126.92, 126.83, 69.73, 69.47, 55.88, 54.76, 43.09, 43.02, 42.18, 41.71, 39.85, 38.73, 37.73, 37.27, 34.93, 34.84, 33.77, 32.38, 31.67, 30.25, 28.21, 26.73, 23.33, 20.86, 18.44, 12.04; HRMS (ESI) for C32H47N2O4, calculated [[M + H] + [-H2O]]+: 523.3536. Obtained: 523.3536.

[0465] Comparative Example 2: Preparation of N-(3-aminonaphthalen-2-yl)-4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13 dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamide (If)

[0466]

[0467]

[0168] To a solution of 4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13- dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzoic acid (290 mg, 0.90 mmol) in DMF (4.9 mL), 2,3-diaminonaphthalene (120 mg, 0.74 mmol) and TBTU (290 mg, 0.90 mmol) were added. The solution was stirred for 24 hours at room temperature. The solvent was then evaporated under reduced pressure. The product was precipitated in EtOAc and the formed white solid was filtered and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL) and saturated NaCI (1 x 5 mL). The solid was then dissolved in a minimal amount of MeOH and precipitated with diethyl ether at 0 °C, then filtered. The residue was purified over silica gel chromatography(MeOH / DCM 1:15) and the compound (If) was isolated as a reddish brown solid. Yield 54%.

[0468]

[0169] Melting point 164 °C;

[0469]

[0170] IR: 3304, 2925, 2857, 1645, 1532, 1284, 1015, 743 cm’1;

[0470]

[0171] 1H NMR (400 MHz, DMSO-de) 6 9.78 (s, 1H), 8.41 (t, J = 6.0 Hz, 1H), 7.99 (d, J = 7.8 Hz, 2H), 7.87 (s, 1 H), 7.68 (d, J = 8.1 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 7.9 Hz, 2H), 7.30 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1 H), 7.10 (s, 1H), 5.25 (s, 2H), 4.45 (d, J = 4.4 Hz, 1H), 4.35 (t, J = 4.9 Hz, 2H), 3.88 (d, J = 6.7 Hz, 1 H), signal corresponding to 2H overlapped with broad signal of water at 3.34 ppm, 2.26- 2.02 (m, 2H), 2.02-1.60 (m, 6H), 1.56-1.26 (m, 10H), 1.25-0.97 (m, 8H), 0.95 (s, 7H), 0.63 (s, 3H);

[0471]

[0172] 13C NMR (101 MHz, DMSO-de) 6 173.15, 166.17, 144.06, 142.66, 133.45, 133.26, 128.31, 127.63, 127.34, 126.92, 126.84, 125.93, 125.27, 124.56, 122.16, 109.20, 70.19, 69.94, 56.35, 55.23, 43.57, 43.49, 42.64, 42.21, 39.19, 38.19, 37.74, 35.41, 35.30, 34.23, 32.88, 32.18, 30.72, 28.68, 27.21, 23.79, 21.32, 18.93, 12.52;

[0472]

[0173] HRMS (ESI) for C42H55N3O4H, calculated [M + H]+: 666.4193. Obtained: 666.4267.

[0473] Comparative Example 3: Preparation of N-(2-aminophenyl)-4-((4-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzamide (lg)

[0474]

[0475]

[0174] To a solution of 4-((4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13- dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzoic acid (110 mg, 0.17 mmol) in DMF (0.6 mL), o-phenylenediamine (20 mg, 0.17 mmol) and TBTU (70 mg, 0.21 mmol) were added. The solution was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The product was precipitated in EtOAc and the formed white solid was filtered and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCOs (2 x 5 mL) and saturated NaCI (1 x 5 mL). Thesolid was then dissolved in a minimal amount of MeOH and precipitated with diethyl ether at 0°C, then filtered. The residue was purified over silica gel chromatography (MeOH / DCM 1:10) and the compound (Ig) was isolated as an off-white solid. Yield 34%.

[0476]

[0175] Melting point 180 °C;

[0477]

[0176] IR: 3273, 2925, 2860,1637, 1527, 1502, 1451, 1304, 1046, 1016, 746 cm’1;

[0478]

[0177] 1H NMR (400 MHz, DMSO-de) 69.65 (s, 1H), 9.08 (t, J = 6.3 Hz, 1H), 8.37 (t, J = 6.0 Hz, 1H), 7.94 (d, J = 7.9 Hz, 2H), 7.86 (d, J = 7.9 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 7.9 Hz, 1H), 6.98 (t, J = 6.9 Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H), 6.63 (t, J = 7.5 Hz, 1H), 4.55 (d, J = 6.0 Hz, 2H), 4.39-4.25 (m, 2H), signal corresponding to 2H overlapped with broad signal of water at 3.34 ppm, 2.24-2.14 (m, 1H), 2.14-2.03 (m, 1H), 1.99-1.91 (m, 1H), 1.87-1.64 (m, 5H), 1.49-1.29 (m, 10H), 1.24-1.01 (m, 8H), 0.93-0.86 (m, 7H), 0.62 (s, 3H);

[0479]

[0178] 13C NMR (101 MHz, DMSO-de) 6 172.65, 166.07, 165.14, 143.30, 133.07, 132.64, 127.83, 127.26, 126.92, 126.65, 126.45, 123.56, 116.57, 116.33, 69.72, 69.47, 55.87, 54.75, 43.10, 43.02, 42.41, 42.17, 41.71, 38.72, 37.72, 37.27, 34.92, 34.83, 33.76, 32.39, 31.68, 30.25, 28.20, 26.73, 23.32, 20.85, 18.46, 12.04;

[0480]

[0179] HRMS (ESI) for C46H60N4O5, calculated [M + H]+: 749.4564. Obtained: 749.4635.

[0481] Comparative Example 4: Preparation of N-(3-aminonaphthalen-2-yl)-4-((4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7- dihydroxy-10,13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzamide (Ih)

[0482]

[0483]

[0180] To a solution of 4-((4-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13- dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)benzamido)methyl)benzoic acid (400 mg, 0.60 mmol) in DMF (2.1 mL), 2,3-diaminonaphthalene (100 mg, 0.61 mmol) and TBTU (230 mg, 0.73 mmol) were added. A solution of EtsN (0.7 mL, 5.19 mmol) in DMF (0.6 mL)was added slowly. The mixture was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The product was precipitated in EtOAc and the formed white solid was filtered and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCO₃ (2 x 5 mL) and saturated NaCl (1 x 5 mL). The solid was then dissolved in a minimal amount of MeOH and precipitated with diethyl ether at 0 °C, then filtered. The residue was purified over silica gel chromatography (MeOH / DCM 1:10) and the compound (Ih) was isolated as an orange solid. Yield 13%.

[0484]

[0181] Melting point 217 °C;

[0485]

[0182] IR: 3309, 2924, 2862, 1854, 1636, 1541, 1448, 1282, 1015, 743 cm’1;

[0486]

[0183] 1H NMR (400 MHz, DMSO-d6) 69.76 (s, 1H), 9.10 (t, J = 6.1 Hz, 1H), 8.37 (t, J = 6.0 Hz, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.88 (s, 1H), 7.86 (d, J = 2.7 Hz, 2H), 7.67 (d, J = 8.1 Hz, 1 H), 7.56 (d, J = 8.3 Hz, 1 H), 7.47 (d, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.29 (t, J = 7.5 Hz, 1 H), 7.15 (t, J = 7.5 Hz, 1 H), 7.09 (s, 1H), 5.25 (s, 2H), 4.57 (d, J = 5.9 Hz, 2H), 4.45 (d, J = 4.5 Hz, 1H), 4.32 (t, J = 4.8 Hz, 2H), 4.04 (s, 2H), 3.88 (d, J = 6.7 Hz, 1H), 3.32 (s, 2H), 2.24-2.15 (m, 1H), 2.12-2.06 (m, 1H), 1.98-1.91 (m, 1H), 1.87-1.64 (m, 5H), 1.50-1.29 (m, 10H), 1.24- 1.02 (m, 8H), 0.94-0.85 (m, 7H), 0.62 (s, 3H);

[0487]

[0184] 13C NMR (101 MHz, DMSO-d₆) δ 173.14, 166.57, 166.23, 143.95, 143.80, 142.66, 133.57, 133.27, 133.12, 128.38, 127.74, 127.65, 127.44, 126.92, 126.85, 125.95, 125.28, 124.53, 122.17, 109.20, 70.19, 69.94, 56.34, 55.22, 43.56, 43.48, 42.89, 42.63, 42.17, 39.19, 38.19, 37.74, 35.39, 34.23, 32.85, 32.14, 30.72, 28.66, 27.19, 23.79, 21.32, 18.92, 12.51;

[0488]

[0185] HRMS (ESI) for C50H62N4O5, calculated [M + H]+: 799.4720. Obtained: 799.4791.

[0489] Comparative Example 5: Preparation of N-(3-aminonaphthalen-2-yl)-6-((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanamide (li)

[0490]

[0491]

[0186] DMAP (80 mg, 0.63 mmol), EDCI (180 mg, 0.95 mmol), and 6-((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)hexanoic acid (400 mg, 0.79 mmol) were dissolved in DCM (2.4 mL). A solution of 2,3-diaminonaphthalene, (250 mg, 1.58 mmol) in DCM (1.6 mL) was added dropwise. The mixture was stirred overnight at room temperature. The solvent was evaporated under reduced pressure. The product was precipitated in EtOAc and the formed white solid was filtered and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCO₃ (2 x 5 mL) and saturated NaCl (1 x 5 mL). The solid was then dissolved in a minimal amount of MeOH and precipitated with diethyl ether at 0°C, then filtered. The residue was purified over silica gel chromatography (MeOH / DCM 1:15) and the compound (li) was isolated as an orange solid. Yield 13%.

[0492]

[0187] Melting point 152 °C;

[0493]

[0188] IR: 3302, 2927, 2861, 1636, 1541, 1453, 1365, 1284, 1047, 743, 608, 475 cm-1;

[0494]

[0189] 1H NMR (400 MHz, DMSO-d6) 69.19 (s, 1H), 7.91 (s, 1 H), 7.76 (t, J = 5.5 Hz, 1H), 7.61 (d, J = 7.5 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.25 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 5.21 (s, 2H), 4.45 (d, J = 4.6 Hz, 1H), 4.04 (s, 1H), 3.87 (d, J = 6.9 Hz, 1H), 3.32 (s, 2H), 3.04 (t, J = 6.6 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.11-2.03 (m, 1H), 1.97-1.90 (m, 2H), 1.70-1.61 (m, 5H), 1.49-1.28 (m, 17H), 1.19-1.08 (m, 6H), 0.89-0.86 (m, 6H), 0.60 (s, 3H);

[0495]

[0190] 13C NMR (101 MHz, DMSO-d₆) δ 172.25, 171.62, 140.85, 132.16, 126.99, 126.45, 126.38, 125.07, 124.69, 121.84, 121.61, 108.37, 69.70, 69.58, 69.45, 69.33, 55.82, 54.68, 43.01, 42.15, 38.69, 38.16, 37.71, 37.25, 35.93, 34.94, 34.81, 33.74, 32.46, 31.70, 30.22, 29.01, 28.15, 26.69, 26.13, 24.95, 23.29, 20.82, 18.45, 11.99;

[0496]

[0191] HRMS (ESI) for C40H59N3O4H, calculated [M + H]+: 646.4506. Obtained: 646.4580.

[0497] Comparative Example 6: Preparation of N-(2-aminophenyl)-5-(((R)-4-((3R,5S,7S,8R,9S, 10S, 13R, 14S, 17R)-3,7-dihydroxy-10, 13-dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)furan-2- carboxamide (li)

[0498]

[0499]

[0192] To a solution of 5-(((R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10, 13- dimethylhexadecahydro-1 H-cyclopenta[a]phenanthren-17-yl)pentanamido)methyl)furan-2- carboxylic acid (200 mg, 0.47 mmol) in DMF (2.1 mL), o-phenylenediamine (42 mg, 0.39 mmol) and TBTU (151 mg, 0.47 mmol) were added. A solution of Et₃N (0.6 mL, 4.0 mmol) in DMF (0.5 mL) was added slowly. The mixture was stirred overnight at room temperature. The solvent was then evaporated under reduced pressure. The crude was partially dissolved in EtOAc and the remaining white solid was filtered and washed successively with 1 N HCI (3 x 5 mL), saturated NaHCO₃ (2 x 5 mL) and saturated NaCl (1 x 5 mL). The solid was then dissolved in a minimal amount of MeOH and precipitated with diethyl ether at 0°C, then filtered. The residue was purified over silica gel chromatography (MeOH / DCM 1:10) and the compound (ij) was isolated as a yellow solid. Yield 37%.

[0500]

[0193] Melting point 150-152 °C;

[0501]

[0194] IR: 3322, 2926, 2862, 1651, 1540, 1454, 1314, 1048, 1014, 748 cm’1;

[0502]

[0195] 1H NMR (400 MHz, DMSO-d6) 6 9.45 (s, 1H), 8.37 (t, J = 5.7 Hz, 1H), 7.23 (d, J = 3.4 Hz, 1H), 7.13 (dd, J = 7.9, 1.6 Hz, 1H), 7.02-6.92 (m, 1H), 6.78 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.4, 1.4 Hz, 1H), 6.39 (d, J = 3.4 Hz, 1H), 4.88 (s, 2H), 4.45 (d, J = 4.6 Hz, 1 H), 4.32 (d, J = 5.5 Hz, 2H), 3.87 (d, J = 6.9 Hz, 1 H), 3.32-3.26 (m, 2H), 2.22-2.12 (s, 1H), 2.11-2.00 (m, 1H), 1.98- 1.90 (m, 1H), 1.86-1.63 (m, 5H), 1.47-1.28 (m, 9H), 1.25-1.03 (m, 9H), 0.91-0.86 (m, 6H), 0.61 (s, 3H);

[0503]

[0196] 13C NMR (101 MHz, DMSO-d6) δ 173.11, 156.85, 155.87, 147.00, 143.56, 127.03, 122.97, 116.82, 116.66, 115.46, 109.05, 70.18, 69.93, 65.38, 56.33, 55.18, 43.54, 43.47, 42.63, 39.18, 38.19, 37.73, 36.17, 35.39, 35.29, 34.23, 32.69, 32.01, 30.70, 28.64, 27.18, 23.78, 21.30, 18.92, 15.63, 12.50;

[0504]

[0197] HRMS (ESI) for C31H47N3O6, calculated [M + H]+: 605.2829. Obtained: 605.3885.

[0505] Compound activity assays

[0506] Immunoblot experiments

[0198] Procedure: Cells were seeded at a density of approximately 2-3x105cells / well in 6-well plates (Corning) coated with a thin layer of collagen type I. After overnight incubation at 37 °C and 5% CO2, cells were incubated with the indicated compounds of formula (I) at a concentration of 2 or 10 pM in complete media (0.1% DMSO) and were maintained for 24 or 48 hours at appropriate incubation conditions. Following the incubation period, the cells underwent two washes using 1X DPBS (Gibco - Thermo Fisher Scientific). Subsequently, they were lysed by adding 80 pL of cold radio-immunoprecipitation assay (RIPA) lysis buffer and assisted by scraping. The composition of the RIPA buffer includes: 150 mM NaCI, 50 mM Tris pH 7.5, 0.1% SDS, 1% Triton X100, 0.5% sodium deoxycholate, protease inhibitors (1 tablet / 50 mL, Complete; Roche), and phosphatase inhibitors (1 mM orthovanadate, 10 mM NaF, 100 mM [3-glycerophosphate) (all from Sigma-Aldrich). Whole-cell lysates from the cultured cells were collected and then frozen at -80 °C to facilitate the disruption of cell membranes. Upon thawing, the samples were subjected to centrifugation at 14,000 rpm for 10 minutes at 4 °C. The supernatant was collected, and the total amount of protein was quantified using Pierce™ BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific). Briefly, a 1:5 dilution of each protein sample was prepared in a 96-well plate (Coming) using dH₂O. Simultaneously, a calibration curve spanning concentrations from 0 to 2 mg / mL of BSA was prepared. Additionally, the vehicle solution (i.e., RIPA) was included in the plate as a blank sample. Afterwards, A and B reagents of the BCA Kit were mixed (in a 1:50 proportion) and 200 pL of the mixture were added to each well. The plate was then placed in a 37 °C incubator for 30 minutes. Following the incubation period, the absorbance was measured at 570 nm using a Halo LED 96® microplate reader (Dynamica Scientific Ltd., UK).

[0507]

[0199] Protein acetylation levels were analyzed by immunoblotting using 30 pg of cell culture protein extract. Samples were denaturalized by adding 5X Protein Loading Buffer [250 mM Tris pH 6.8, 10% SDS, 50% glycerol (all three from Applichem Panreac), 0.05% bromophenol blue (Probus) and 500 mM 2-mercaptoethanol (Sigma-Aldrich)] and heating the samples at 95 °C for 5 minutes. Then, proteins were separated by electrophoresis in 12.5% SDS-PAGE acrylamide gels and electro-transferred onto nitrocellulose membranes (BioRad, Hercules, CA). After blocking with 5% Bovine Serum Albumin (BSA) / Tris-buffered saline with 0.1% Tween® 20 (TBS-T) for 1 hour at room temperature, membranes were incubated overnight at 4 °C with primary antibodies diluted in the blocking solution (5% BSA / TBS-T)[(Ac-a-tubulin (Sigma); a-tubulin (Abeam); Ac-H3K9 (Cell Signaling)]. Membranes were then washed with TBS-T and incubated with Horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling) at 1:5,000dilution for 1 hour at room temperature in blocking solution. Afterwards, membranes were washed with TBS to remove unbound secondary antibody, incubated with Novex® ECL HRP Chemiluminescent Substrate Reagent Kit (Invitrogen) and visualized in an iBright Imaging System (Thermo Fisher Scientific).

[0508]

[0200] Results: Different immunoblot experiments have been carried out with compounds (la)-(lj) using CCA cell lines EGI and HUCCT1 after 24 or 48 hours incubation at different concentrations (2 pM or 10 pM), the results of which are shown on Figures 1 and 2. H3K9 is a substrate of nuclear HDACs, while a-tubulin is a substrate of HDAC6. Results show that after an incubation of 24 hours with the compounds of formula (la), (lb), (Ic) and (Id) at 10 pM, these compounds show high inhibitory activity and selectivity towards nuclear HDACs, thus increasing the acetylation levels of Lys9 in histone 3 (H3K9) in both studied cell lines of CCA, including in concentrations as low as 2 pM in the case of compound (la). Remarkably, compounds (le)-(lj) do not show such activity and selectivity. Compound (le) is selective for HDAC6, as already known from WO 2019 / 129913 A1. The experiments of Figure 2 after 24 hours of incubation further show a marked dosis dependence for both compounds, a-tubulin protein levels were used as loading controls.

[0509] HDAC inhibitory activity

[0510]

[0201] Procedure: The HDAC activity assays were performed using acetylated peptide substrates labelled with 7-amino-4-methylcoumarin (AMC) at Reaction Biology Corporation (Pennsylvania, USA). All assays were performed in a solution buffer (50 mM Tris-HCI, pH 8.0; 137 mM NaCI; 2.7 mM KCI; 1 mM MgCl2; supplemented with 1 mg / mL of BSA for dilution; BioMol Cat. # KI-143). Briefly, 50 pL of peptide substrate and an optimal concentration of the corresponding enzyme were incubated in the assay buffer at a final DMSO concentration of 1% in the presence of increased concentrations of the compounds of formula (I) at 30°C for 2 hours. The reactions were carried out in 96-well fluorimeter microplates in a final reaction volume of 50 pL. After the deacetylation reaction, Fluor-de-Lys-Developer (BioMol Cat. # KI-105) was added to each well to digest the deacetylated substrate according to manufacturer instructions, thereby producing the fluorescent signal. The reaction was carried out for 45 min at 30°C with 5% CO2. Then, the fluorescence signal was measured using an excitation wavelength of 360 nm and an emission wavelength at 460 nm in a fluorimeter (GeminiXS, Molecular Devices, Sunnyvale, CA). All the experiments were performed in triplicate. The IC50 values were calculated by fitting the experimental data with Graphpad Prism 6 software usingthe equation log(inhibitor) vs. normalized response with variable slope. DMSO was used as a negative control.

[0511]

[0202] Results: Figure 3 shows the dosis-response curve of the inhibiting effect of the compound of formula (la) on HDAC1, HDAC2, HDAC3, HDAC8 and HDAC11. Table 1 below provides the values of IC50 for the compound of formula (la) over the different isoforms of HDAC

[0512] Table 1

[0513] HDAC isoform number IC50 of compound (la) (M)

[0514] 1 3.70 x IO’7

[0515] 2 5.95 x IO’7

[0516] 3 2.16 x IO’6

[0517] 4

[0518] 5

[0519] No inhibitory effect at assayed concentrations 6

[0520] 7

[0521] 8 1.39 x 10-5

[0522] 9

[0523] No inhibitory effect at assayed concentrations 10

[0524] 11 5.78 x 10-6

[0525]

[0203] The results of Table 1 show that the compound of formula (la) is an efficient inhibitor of HDAC isoforms 1, 2, 3, 8 and 11, while the inhibitory effect on other isoforms is more moderate. The inventors have found in particular by analyzing the expression of the different forms of HDACs in four international cohorts of patients (Copenhague (GSE26566); The Cancer Genome Atlas (TCGA: doi:10.7908 / C17W6BJS); TIGER-LC (GSE76297); Job (E-MTAB-6389)) that HDACs 1, 2, 8 and 11 are particularly overexpressed in tumoral CCA tissues if compared with adjacent non-tumoral tissues. These results thus suggest that the compound of formula (la) is particularly suitable for the treatment of CCA.

[0526] Flow cytometry

[0527]

[0204] Procedure for cancer cell proliferation evaluation: CCA cells in culture were stained with CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) according to manufacturer’s protocol and seeded them at a density of 3x104cells / well in complete media in collagen type I coated 12-well plates. After overnight incubation at 37°C and 5% CO2, cells were incubated with the indicated compounds of formula(I) in complete media (0.1 % DMSO) and were maintained for 48 hours at appropriate incubation conditions. Finally, cells were trypsinized and fluorescence intensities of individual cells were measured by flow cytometry using a Guava® easyCyte 8HT (Merck Millipore). Cell population was gated by size and complexity using forward and side scattering. Fluorescence threshold was established at the fluorescence value in which 50% of control cells were above the threshold and 50% of control cells were below the threshold. Proliferation rates were calculated by measuring the proportion of cells bellow the fluorescence threshold for each condition. Results are represented as percentage relative to the vehicle-treated or control cells (100% of proliferation).

[0528]

[0205] Procedure for cancer cell apoptosis evaluation: Cancer cell apoptosis was evaluated using FITC Annexin V (BioLegend) and TO-PROTM-3 iodide (Invitrogen Thermo Fisher Scientific) by flow cytometry. Cells were seeded at a density of 1.2x104cells per well in a collagen-coated 24-well plate. After 24 hours, cells were incubated with the indicated compounds of formula (I), or the vehicle solution (DMSO) for 48 hours. Subsequently, cells were collected and stained with FITC Annexin V (BioLegend) for 15 minutes at room temperature and TO-PROTM-3 iodide (Invitrogen - Thermo Fisher Scientific) for 15 minutes at 4°C. Fluorescence was measured by flow cytometry using the Guava easyCyte 8HT Flow Cytometer (Merck Millipore). Puromycin (2 pg / mL) was used as a positive control for cell death. Results are shown as relative to vehicle-incubated or control cells.

[0529]

[0206] Results: Fig. 4 shows the effect of incubating a 10 pM solution of one of the compounds (la)-(lj) on the proliferation (A) and apoptosis (B) of cholangiocarcinoma cell line EG1 during 48 hours, as measured by flow cytometry. Fig. 5 shows representative bar diagrams quantifying the respective effects of solutions of the compounds of formula (la) and (le) having a concentration of 2 pM or 10 pM on the proliferation of cholangiocarcinoma cell lines EG1, HUCCT1 and WITT during 48 hours, as measured by flow cytometry. Fig. 6 shows representative bar diagrams quantifying the respective effects of solutions of the compounds of formula (la) and (le) having a concentration of 10 pM or 20 pM on the apoptosis of cholangiocarcinoma cell lines EG1 and HUCCT1 during 48 hours, as measured by flow cytometry.

[0530]

[0207] The results of Figs. 4-6 show that the compounds according to the invention, e.g. compounds (la)-(ld), are suitable for reducing cell proliferation and inducing apoptosis when in contact with the CCA cell line EGI1 and are more efficient in this regard than compounds described in the art, such as compound (le). This superior performance of (la) over (le) is also observed for CCA cell lines HUCCT1 and WITT. Formation of tridimensional spheroids

[0208] Procedure: CCA cells were stained with CellTrace™ CFSE Cell Proliferation Kit (Invitrogen) according to manufacturer’s protocol and seeded in a 96-well U-bottom plate (Greiner bio-one) in 50 pL / well of complete spheroid medium (DMEM F12 + Glutamax + P / S + EGF + B27 + Insulin) at a density of 3,000 cells / well. The plate was then centrifuged at 400 g for 10 minutes and maintained at 37°C with 5% CO2 for 24 hours to allow spheroid formation. The following day, 50 pL of normal medium (DMEM-F12 + P / S + Glutamax) was added to each well. Subsequently, the different treatments with compounds of formula (I) were added in 100 pL of medium to each well. After 48 hours of treatment, CCA spheroids were stained with TO-PRO™-3 iodide (Invitrogen - Thermo Fisher Scientific) for 15 minutes at 4 °C. Photographs were taken at baseline (0 hours) and 48 hours after treatment using the Axio Observer 7 microscope. The size of the CCA spheroids was measured using ImageJ software version 1.50 (NIH, Bethesda, MA, USA), and TO-PRO™-3 iodide fluorescence was quantified with ZEN Blue software (Zeiss).

[0531]

[0209] Results: Fig. 7 shows: (A - top) fluorescence microscopy images of spheroids formed on cholangiocarcinoma EG1 cells incubated during 48 hours with a solution of (la) at a concentration of 0 pM, 2 pM, 10 pM or 20 pM and (B - bottom) quantification of spheroid growth relative to baseline (0 hours) and cell death after 48 hours incubation of EG1 cells with a solution of (la) or (le) at a concentration of 0 pM, 2 pM, 10 pM or 20 pM. The results of Figure 7 show that the compound of formula (la) is more efficient than the compound of formula (le) in reducing the growth of formed tridimensional spheroids (CFSE) and causing cell death (TO-PRO-3).

[0532]

[0210] FIG. 9 shows (A - top) fluorescence microscopy images of spheroids formed on cholangiocarcinoma EG1 cells resistant to cisplatin (EG1-R) incubated during 48 hours with a solution of (la) at a concentration of 0 pM, 10 pM or 20 pM or a 30 pM solution of cisplatin and (B - bottom) quantification of relative spheroid area after 48 hours incubation of EG1 cells resistant to cisplatin with a solution of (la) or (le) at a concentration of 0 pM, 10 pM or 20 pM or with a solution of cisplatin at a concentration of 30 pM. The results of Figure 9 show that the compound of formula (la) is suitable for reducing the area of formed tridimensional spheroids in CCA cell lines resistant to cisplatin, even at concentrations as low as 2 pM.

[0533] Cancer cell line screening

[0534]

[0211] Procedure: The sixty human tumor cell lines of the cancer screening panel consisted of the following cell lines:

[0535] Breast Cancer: MCF7, MDA-MB-231, HS 578T, BT-549, T-47D,Central Nervous System (CNS) Cancer: SF-268, SF-295, SF-539, SNB-19, SNB-75, U251,

[0536] Colon Cancer: COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12, SW- 620,

[0537] Leukemia: CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226, SR, Lung Cancer: A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522,

[0538] Melanoma: LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK- MEL-28, SK-MEL-5, UACC-257, UACC-62,

[0539] Ovarian Cancer: IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK- OV-3,

[0540] Prostate Cancer: PC-3, DU-145,

[0541] Renal Cancer: 786-0, A498, ACHN, CAKI-1, RXF-393, SN12C, TK-10, UO- 31

[0542] Cell lines were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. 40 pl of cells were inoculated into the wells of white 384-well microtiter plates at plating densities ranging from 250 to 2500 cells / well, depending on the doubling time of individual cell lines. After inoculation, the microtiter plates were incubated at 37°C with 5% CO2 and 95% relative humidity for 24 h before the addition of controls and test compounds. The test compounds and controls are solubilized in dimethyl sulfoxide (DMSO) at 400-fold the final test concentration (10 pM) and stored frozen in polypropylene 384-well microtiter plates.

[0543]

[0212] Twenty-four hours after cell line inoculation, acoustic dispensing was used to transfer 100 nl of DMSO (0.25% (v / v), final) into the wells of microtiter plates, each containing a single cell line. Subsequently, 40 pl of CellTiter-Glo were dispensed into the wells, according to the manufacturer's protocol. Luminescence was measured to assess cell viability at the time of drug addition (time zero, Tz). Acoustic dispensing was also used to transfer 100 nl of controls and test compounds (400-fold dilution, final) into duplicate microtiter plates for each cell line to achieve technical replicates. Controls in each microtiter plate included vehicle, 100% cytotoxicity (1 pM Staurosporin NSC755774 and 3 pM Gemcitabine NSC613327 [n = 8]), and five concentrations of doxorubicin (NSC123127, 25 pM [n = 2], 2.5 pM [n = 1], 250 nM [n = 2], 25 nM [n = 1], 2.5 nM [n = 2]). Following the delivery of controls and test compounds, the microtiter plates were incubated for 72 h at 37 °C with 5 % CO2 and 95% relative humidity. After 72 h of exposure, 40 pl of CellTiter-Glo were dispensed into the wells of the microtiter plates and luminescence was measured, according to the manufacturer's protocol, to assess cell viability. Using the various measurements (time zero [Tz], vehicle control growth [C], and growth in thepresence of test agent at the five concentrations [Ti]), the percentage growth (%G) was calculated at each of the test compound concentrations.

[0544]

[0213] Results: Compounds (la) and (le) were tested at a concentration of 10 pM according to the above procedure. Fig. 8 shows the growth inhibition, expressed as a percentage, of several cancer cell lines of the National Cancer Institute 60 Cell Line Screening Program exposed to a dosis of 10 pM of the compound of formula (la) (bottom) or of formula (le) (top). Each point in the graphic represents a different cell line of a determined cancer type. The results of Figure 8 suggest that the compound of formula (la) is surprisingly particularly suitable for the prevention and / or treatment of cancer; in particular of leukemia, lung cancer, colon cancer, central nervous system cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and breast cancer, while the compound of formula (le) appears to be much less active in inhibiting the growth of cancer cells at the tested concentration.

[0545] Migration and invasion assays

[0546]

[0214] Procedure: CCA cells (EGI-1 cell lines) were seeded at a density of 30,000 cells in the upper side of 6.5 mm inserts with polycarbonate membranes of 8 pm pore size without coating (migration assay) or coated with Matrigel (invasion assay) (Corning). Cells were maintained overnight at 37°C and 5% CO2 in DMEM F12 + Glutamax + 1% P / S + 1% FBS at both sides of the transwell membrane. The next day, culture media was removed, and membranes were rinsed at both sides with PBS. Subsequently, treatments with solutions of (la) (10 pM or 20 pM) were added in DMEM F12 + Glutamax + 1% P / S on the upper side of the membrane and DMEM F12 + Glutamax + 1% P / S + 10% FBS on the bottom side to promote cell migration. Cells were maintained at 37°C and 5% CO2 for 24 hours in culture at the described conditions. Finally, cell culture media was removed, and cells were fixed and stained with a solution of crystal violet in 4% paraformaldehyde. Photographs were taken in an Axio Observer 7 microscope and area of migrated cells was quantified with ZEN Blue software (Zeiss).

[0547]

[0215] Results: FIG. 10 shows (top) microscopy pictures of the transwell assays carried out with a population of EGI-1 cells incubated during 24 hours with a solution in bovine fetal serum of a compound of formula (la) having a concentration of 0 pM, 10 pM or 20 pM and (bottom) the quantified relative amount of migrated cells after 24 hours of incubation. The results of Fig. 10 show that, after 24 hours of incubation with different doses of the compound of formula (la), the migratory ability of EGI-1 CCA cells is reduced in a dosis-dependent manner.

Claims

CLAIMS1. A compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use in the treatment and / or prevention of cancer(I)wherein:X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;n is an integer of from 1 to 2;each one of R4, R5, R6and R7is independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkyloxy, (C1-C6)alkylamino, (C1-C6)alkylcarbonyl, (C1-C6)alkyloxycarbonyl, (C1-C6)alkylcarbonyloxy, halo, cyano and nitro;Y is a group selected from the groups of formula Y1 and Y2,Y1Y2wherein:R2 and R3 together with the carbon atoms to which they are attached form a phenyl ring or a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (Ci-C3)alkyl, (Ci-C3)haloalkyl, halo, cyano and nitro; andR8and R9together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

2. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to claim 1 wherein Ri is hydrogen.

3. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 2 wherein n is 1.

4. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 3 wherein each one of R4, R5, R6and R7is hydrogen.

5. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 4 wherein X is a ursodeoxycholic acyl group.

6. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 5 wherein Y is a group of formula Y1.

7. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 6 wherein, in the group of formula Y1, R2and R3together with the carbon atoms to which they are attached form a phenyl ring, said phenyl ring being optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

8. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 7 wherein, in the group of formula Y1, R2and R3together with the carbon atoms to which they are attached form a phenyl ring optionally substituted at one or more available position with a fluorine group.

9. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 8 that is selected from the group consisting of the compounds of formula (la), (lb), (Ic) and (Id)(lc) (Id)10. The compound of formula (I) or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof for use according to any one of claims 1 to 9 wherein said cancer is bile duct cancer or cholangiocarcinoma.

11. A compound of formula (I’)(I’)wherein:X is an acyl group selected from the group consisting of ursodeoxycholic acyl, deoxycholic acyl, lithocholic acyl, obeticholic acyl, cholic acyl, chenodeoxycholic acyl, hyodeoxycholic acyl and dehydrocholic acyl;R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;n is an integer of from 1 to 2;each one of R4, R5, R6and R7is independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkyloxy, (C1-C6)alkylamino, (C1-C6)alkylcarbonyl, (C1-C6)alkyloxycarbonyl, (C1-C6)alkylcarbonyloxy, halo, cyano and nitro;Y is a group selected from the groups of formula Y1 and Y2,Y1Y2wherein:R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; andR8and R9together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof.

12. The compound of formula (I’) according to claim 11 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein R1 is hydrogen.

13. The compound of formula (I’) according to any one of claim 11 or 12 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein n is 1.

14. The compound of formula (I’) according to any one of claim 11 to 13 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein each one of R4, R5, R6and R7is hydrogen.

15. The compound of formula (I’) according to any one of claim 11 to 14 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein X is a ursodeoxycholic acyl group.

16. The compound of formula (I’) according to any one of claim 11 to 15 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein Y is a group of formula Y1.

17. The compound of formula (I’) according to any one of claim 11 to 16 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein in the group of formula Y1, R2and R3together with the carbon atoms to which they are attached form a phenyl ring optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

18. The compound of formula (I’) according to any one of claim 11 to 17 or a stereoisomer thereof or a pharmaceutically acceptable salt or solvate thereof wherein in the group of formula Y1, R2and R3together with the carbon atoms towhich they are attached form a phenyl ring optionally substituted at one or more available position with a fluorine group.

19. The compound of formula (I’) according to any one of claim 11 to 18 or that is selected from the compounds of formula (lb), (Ic) and (Id) as defined in claim 9 or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof.

20. Compound of formula (I’) or a stereoisomer or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 11 to 19 for use in medicine.

21. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I’) or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 19 and at least one pharmaceutically acceptable excipient, vehicle or carrier.

22. Process for the preparation of a compound of formula (I’) according to any one of claims 11 to 19 comprising the step of causing a bile acid of formula (II) that is selected from the group consisting of ursodeoxycholic acid, deoxycholic acid, lithocholic acid, obeticholic acid, cholic acid, chenodeoxycholic acid, hyodeoxycholic acid and dehydrocholic acid or a stereoisomer thereofto react with a compound of formula (III)(HI)wherein R1is selected from the group consisting of hydrogen, a (C1-C6)alkyl chain and a (C1-C6)haloalkyl chain;n is an integer of from 1 to 2;each one of R4, R5, R6and R7is independently selected from the group consisting of hydrogen, (C1-C6)alkyl, (C1-C6)haloalkyl, (C1-C6)alkyloxy, (C1-C6)alkylamino, (C1-C6)alkylcarbonyl, (C1-C6)alkyloxycarbonyl, (C1-C6)alkylcarbonyloxy, halo, cyano and nitro;Y is a group selected from the groups of formula Y1 and Y2,Y2wherein:R2and R3together with the carbon atoms to which they are attached form (i) a phenyl ring substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro or (ii) a 5- to 6- membered heteroaryl ring, wherein each of said phenyl ring and said 5- to 6- membered heteroaryl ring is optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro; andR8and R9together with the carbon atoms to which they are attached form a 5- to 6- membered heteroaryl ring optionally substituted at any available position with a group selected from (C1-C3)alkyl, (C1-C3)haloalkyl, halo, cyano and nitro.

23. Composition for use in the prevention and / or treatment of cancer, said composition comprising a therapeutically effective amount of (i) a compound of formula (I) according to any one of claims 1 to 10 and (ii) a chemotherapeutic agent suitable for the prevention and / or treatment of cancer.

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