WO2019115709A1

WO2019115709A1 - Mitochondrial inhibitors for the treatment of proliferation disorders

Info

Publication number: WO2019115709A1
Application number: PCT/EP2018/084799
Authority: WO
Inventors: Florian Richalet; Mahmoud EL SHEMERLY; Heidi Lane
Original assignee: Basilea Pharmaceutica International AG
Priority date: 2017-12-14
Filing date: 2018-12-13
Publication date: 2019-06-20
Also published as: TW201927767A

Abstract

The invention provides compounds of formula I or pharmaceutically acceptable salt thereof wherein Bl, B2, B3 and B4 represent independently C(R3); X represents -CH(R5)-, -C(R5)= or -(C=0)-, and wherein when X represents -CH(R5)- or -(C=0)- the dotted line represents a single bond, and when X represents -C(R5)= the dotted line represents a double bond; R1 represents independently at each occurrence methy, ethyl, methoxy or ethoxy; R2 represents halogen, cyano, hydroxyl, methyl, ethyl, methoxy, ethoxy, -N(R6a)(R6b) or -methylene- N(R6a)(R6b); R3 represents independently at each occurrence hydrogen, halogen, cyano or methyl; R4a represents methyl or ethyl; R4b represents halogen, Cl-C4alkyl, Cl-C4alkoxy or -0-Cl-C4alkylene-Cycle-Q; R5 represents hydrogen or methyl; R6a represents hydrogen or methyl; R6b represents hydrogen or methyl; Cycle-Q represents independently at each occurrence phenyl optionally substituted by 1 to 3 R7; R7 represents independently at each occurrence cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1 or 2; and q is 0, 1 or 2; as well as methods of using the compounds to treat proliferation diseases, in particular cancer.

Description

Mitochondrial inhibitors for the treatment of proliferation disorders

The present invention relates to mitochondrial inhibitors and their use in the treatment of proliferation disorders, in particular cancer.

Mitochondria are the power house of the cell because they generate most of the adenosine triphosphate (ATP), used as a source of chemical energy (Campbell, N.A., Williamson B., Heyden, R.J. Biology: Exploring Life 2006^th Edition, Publisher: Pearson Prentice Hall, 2006). In addition, mitochondria are involved in other functions, such as cellular signaling, differentiation and death, as well as maintaining control of the cell cycle and cell growth (McBride H.M. et.al., Curr. Biol., vol. 16, no.14, R551-60, 2006). Cancer cells reprogram their metabolism in favour of glycolysis, regardless of oxygen presence, according to a phenomenon known as aerobic glycolysis. This so-called“Warburg phenotype” involves high glucose uptake and a high glycolytic activity (Warburg O., Science, vol. 123, no. 3191, pages 309- 314, 1956). Nevertheless, cancer cells are also dependent on mitochondria for ATP production through oxidative phosphorylation (OXPHOS) (Marchetti P. et al., International Journal of Cell Biology, vol. 2015, pages 1-17, 2015 and Solaini G. et al., Biochim. Biophys. Acta,, vol. 2, page: 314-323, 2010). Mitochondrial metabolism is now recognized as a potential target for anticancer agents due to the metabolic characteristic of cancer cells. Indeed, human cancer is associated with mitochondrial dysregulation, which promotes cancer cell survival, tumor progression and metastases as well as resistance to current anticancer drugs (Marchetti P. et al., International Journal of Cell Biology, vol. 2015, pages 1-17, 2015, Boland M.L. et al., Frontiers in Oncology, vol. 3, Article 292, pages 1-28, 2013 and Solaini G. et al., Biochim. Biophys. Acta, vol. 1797, pages 1171-1177, 2010). Metabolic reprogramming in cancer cells results in the maintenance of energy (ATP) production even under stressed conditions, contributing to tumor growth and survival through (for example) mitochondrial utilization of alternative carbon sources such as glutamine and fatty acids to generate ATP (Solaini G. et al., Biochim. Biophys. Acta, vol. 1797, pages 1171-1177, 2010). Indeed, as a result of the separation of glycolytic flux from mitochondria, mitochondrial glutaminolysis is preferentially used to produce ATP and, therefore, contribute to cancer cell survival (DeBerardinis R.J. et al., PNAS, vol. 104, no. 49, pages 19345-19350, 2007) being crucial for the development (Strohecker A.M. et al., Cancer Discovery, vol. 3, no. 11, pages 1272-1285, 2013) and anchorage-independent growth (Weinberg F. et al., PNAS, vol. 107, no. 19, pages 8788-8793, 2010) of certain tumor types.

Moreover, mitochondrial activity has also been associated with the development of drug resistance. For example, chemotherapeutic and targeted drugs (e.g. BRAF inhibitors) have been shown to induce a shift in cancer metabolism leading to mitochondrial dependency (addiction) characterized for example by upregulation of OXPHOS and mitochondrial biogenesis in surviving cells (Marchetti P. et al.,

International Journal of Cell Biology, vol. 2015, pages 1-17, 2015; and Vellinga T. T. et al., Clinical Cancer Research, vol. 21, no. 12, pages 2870-2879, 2015). In the case of BRAF inhibitors, acquired resistance was associated with maintenance of an OXPHOS phenotype regardless of the underlying resistance mechanism (Corazao-Rozas P. et al., Oncotarget, vol. 4, no. 11, pages 1986-1998, 2013), suggesting a potential metabolic arena that could be exploited on a therapeutic level. Hence, taken together, accumulating data provide convincing evidence supporting the involvement of mitochondria in cancer development and a strong rationale for developing mitochondrial targeted agents to fight cancer. Based on growing interest in mitochondria as therapeutic targets for cancer, in recent years a number of mitochondrial-targeting investigational agents have entered clinical development. For example, the anti diabetic medication metformin, which inhibits OXPHOS through inhibition of complex I of the mitochondrial respiratory chain (El-Mir et al., J. Biol. Chem. vol. 275, pages 223-228, 2000, and Wheaton W.W. et al., eLife vol. 3, 2014) is currently being investigated in a number of clinical trials in cancer patients (Chae Y.K. et al., Oncotarget, March 19, 2016). These trials were stimulated by prec finical data in tumor models (Chae Y.K. et al., Oncotarget, March 19, 2016) and the observation that type 2 diabetics treated with metformin had a decreased risk of developing various types of cancer (Quinn B.J., Kitagawa H., Memmott R.M., et al. Trends Endocrinol. Metab. vol. 24, pages 469-80, 2000 and Chae Y.K. et al., Oncotarget, March 19, 2016). Subsequently, increased interest in this therapeutic approach has led to other complex 1 inhibitor classes being investigated (WO2014/031928,

WO2014/031936, Ziegelbauer et al., Cancer Medicine, vol. 2 no. 5, pages 611-624, 2013 and

W02010/054763). Hence, targeting mitochondrial metabolism is of great interest for the development of novel therapeutic approaches for cancer treatment.

Accordingly in a first aspect the present invention provides compounds of formula I and pharmaceutically acceptable salts thereof

wherein

Bl, B2, B3 and B4 represent independently C(R3);

X represents -CH(R5)-, -C(R5)= or -C(=0)-, and wherein when X represents -CH(R5)- or -C(=0)- the dotted line represents a single bond, and when X represents -C(R5)= the dotted line represents a double bond;

Rl represents independently at each occurrence methyl, ethyl, methoxy or ethoxy;

R2 represents halogen, cyano, hydroxyl, methyl, ethyl, methoxy, ethoxy, -N(R6a)(R6b) or -methylene- N(R6a)(R6b);

R3 represents independently at each occurrence hydrogen, halogen, cyano or methyl;

R4a represents methyl or ethyl;

R4b represents halogen, Cl-C4alkyl, Cl-C4alkoxy or -0-Cl-C4alkylene-Cycle-Q;

R5 represents hydrogen or methyl; R6a represents hydrogen or methyl;

R6b represents hydrogen or methyl;

Cycle-Q represents independently at each occurrence phenyl optionally substituted by 1 to 3 R7;

R7 represents independently at each occurrence cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1 or 2; and

q is 0, 1 or 2.

In a further aspect the invention provides compounds of formula I and pharmaceutically acceptable salts thereof for use in the treatment of proliferation diseases, in particular cancer, in a subject selected from a mammal, in particular in a human.

In a further aspect the invention provides use of compounds of formula I and pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of proliferation diseases, in particular cancer, in a subject selected from a mammal, in particular in a human.

In a further aspect the invention provides methods of treating proliferation diseases, in particular cancer, in a subject selected from a mammal, in particular in a human, comprising administering the compound of formula I or pharmaceutically acceptable salt thereof to said subject.

In a further aspect the invention provides pharmaceutical compositions comprising a compound of formula I or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

Each alkyl moiety either alone or as part of a larger group such as alkoxy is a straight or branched chain. Examples include methyl, ethyl, «-propyl, prop-2-yl, «-butyl, but-2-yl, 2 -methyl-prop- l-yl or 2-methyl- prop-2-yl.

Each alkylene moiety is a straight or branched chain and is, for example, -CH₂-, -CH₂-CH₂-, -CH(CH₃)-, - CH₂-CH₂-CH₂-, -CH(CH₃)-CH₂-, or -CH(CH₂CH₃)-.

Each haloalkyl moiety either alone or as part of a larger group such as haloalkoxy is an alkyl group substituted by one or more of the same or different halogen atoms. Examples include difluoromethyl, trifluoromethyl and chlorodifluoromethyl.

Halogen is fluorine, chlorine, bromine, or iodine.

Where a group is said to be optionally substituted, it may be substituted or unsubstituted, for example optionally with 1-3 substituents.

Certain compounds of formula I may contain one or two or more centers of chirality and such compounds may be provided as pure enantiomers or pure diastereoisomers as well as mixtures thereof in any ratio.

For example, when X represents -CH(R5)- and n is 2, or n is 1 and at least one Rl is different than H, the H on the carbon atom connected to X by the dotted line may be in the axial or equatorial configuration and the invention includes both isomers in any ratio. The compounds of the invention also include all aWra/is-isomcrs (for example where the dotted line is a double bond) as well as mixtures thereof in any ratio. The compounds of the invention also include all tautomeric forms of the compounds of formula I. The compounds of formula I may also be solvated, especially hydrated, which are also included in the compounds of formula I. Solvation and hydration may take place during the preparation process.

Reference to compounds of the invention includes pharmaceutically acceptable salts of said compounds. Such salts may also exist as hydrates and solvates. Examples of pharmacologically acceptable salts of the compounds of formula (I) are salts of physiologically acceptable mineral acids, such as hydrochloric acid, sulfuric acid and phosphoric acid, or salts of organic acids, such as methane-sulfonic acid, p- toluenesulfonic acid, lactic acid, acetic acid, trifluoroacetic acid, citric acid, succinic acid, fumaric acid, maleic acid and salicylic acid. Further examples of pharmacologically acceptable salts of the compounds of formula (I) are alkali metal and alkaline earth metal salts such as, for example, sodium, potassium, lithium, calcium or magnesium salts, ammonium salts or salts of organic bases such as, for example, methylamine, dimethylamine, triethylamine, piperidine, ethylenediamine, lysine, choline hydroxide, meglumine, morpholine or arginine salts.

The following examples of substituent definitions may be combined in any combination.

The ring comprising Bl, B2, B3 and B4 as ring members may be represented by group B-I

Preferably no more than two R3 substituents are other than hydrogen.

Structural examples of the ring comprising Bl, B2, B3 and B4 as ring members are represented by group B-Ia, group B-Ib and group B-Ic, wherein R3a is as defined for R3 but is other than hydrogen:

Examples of the ring comprising B 1 , B2, B3 and B4 as ring members include the following groups:

Specific examples of X include -CH=, -CH₂- and -C(=0)-.

Specific examples of R2 include chloro, fluoro and cyano.

Specific examples of R3 include fluoro and chloro.

R4b preferably represents halogen, methyl, ethyl, methoxy, ethoxy, or -0-CH₂-Cycle-Q. Specific examples of R4b include methyl, methoxy, ethoxy, chloro and -0-CH₂-phenyl.

A specific example of Cycle-Q is phenyl

q is preferably 0.

n is preferably 1.

For the avoidance of doubt, Rl does not attach to the carbon atom bonded to X.

Any embodiment relating to the chemical structure of the compounds of the invention may be combined with any other embodiment where possible, including with any of the examples of substituent definitions given above.

In one embodiment X represents -CH(R5)-.

In another embodiment X represents -C(R5)=.

In another embodiment X represents -C(=0)-.

In another embodiment X represents -CH(R5)- or -C(R5)=.

In another embodiment n is 1.

In another embodiment n is 2.

In another embodiment q is 0.

In another embodiment q is 1 or 2. In another embodiment (Embodiment 1)

the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia, group B-Ib or group B-Ic:

(B-Ia) (B-Ib) (B-Ic);

X represents -CH₂-, -CH= or -C(=0)-;

R3a represents independently at each occurrence halogen, cyano or methyl;

R4a represents methyl or ethyl;

R4b represents halogen, Cl-C4alkyl, Cl-C4alkoxy or -0-Cl-C4alkylene-Cycle-Q;

R6a represents hydrogen or methyl;

R6b represents hydrogen or methyl;

R7 represents independently at each occurrence cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1 ; and

q is 0.

In another embodiment (Embodiment 2)

(B-Ia) (B-Ib) (B-Ic);

X represents -CH₂-, -CH= or -C(=0)-;

R2 represents halogen or cyano;

R3a represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -0-CH₂-Cycle-Q;

q is 0. In another embodiment (Embodiment 3)

the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia or group B-Ib:

(B-Ia) (B-Ib);

X represents -CH=;

R2 represents halogen or cyano;

R3a represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -0-CH₂-phenyl;

n is 1 ; and

q is 0.

In another embodiment (Embodiment 4)

(B-Ia) (B-Ib);

X represents -CH=;

R2 represents halogen or cyano;

R3 represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy;

n is 1 ; and

q is 0. In another embodiment the compound of formula I is a compound of formula la

wherein R2, R3, R4a and R4b are as defined for the compound of formula I, including preferred definitions thereof. For example, R2, R3, R4a and R4b may be as defined in Embodiment 1 (wherein R3 is R3a) or for example R2, R3, R4a and R4b may be as defined in Embodiment 2 (wherein R3 is R3a), or for example R2, R3, R4a and R4b may be as defined in Embodiment 3 (wherein R3 is R3a), or for example R2, R3, R4a and R4b may be as defined in Embodiment 4 (wherein R3 is R3a).

In another embodiment the compound of formula I is a compound of formula lb

wherein R2, R3, R4a and R4b are as defined for the compound of formula I, including preferred definitions thereof. For example, R2, R3, R4a and R4b may be as defined in Embodiment 1 (wherein R3 is R3a), or for example R2, R3, R4a and R4b may be as defined in Embodiment 2 (wherein R3 is R3a).

In another embodiment the compound of formula I is a compound of formula Ic

wherein R2, R3, R4a and R4b are as defined for the compound of formula I, including preferred definitions thereof. For example, R2, R3, R4a and R4b may be as defined in Embodiment 1 (wherein R3 is R3a), or for example R2, R3, R4a and R4b may be as defined in Embodiment 2 (wherein R3 is R3a). The following compounds represent further embodiments of the invention, as well as pharmaceutically acceptable salts thereof:

4-[(4-chloro-2-fluoro-phenyl)methylene]-N-(2,3-dimethyl-4-pyridyl)piperidine-l -carboxamide;

4-[(4-chloro-2-fluoro-phenyl)methylene]-N-(3-methoxy-2-methyl-4-pyridyl)piperidine-l -carboxamide; N-(3-chloro-2-methyl-4-pyridyl)-4-[(4-cyano-2,6-difluoro-phenyl)methylene]piperidine-l -carboxamide; 4-[(4-cyano-2,6-difluoro-phenyl)methylene]-N-(3-ethoxy-2-methyl-4-pyridyl)piperidine-l -carboxamide; N-(3-benzyloxy-2-methyl-4-pyridyl)-4-[(4-cyano-2,6-difluoro-phenyl)methylene]piperidine-l- carboxamide;

4-[(4-cyano-2,6-difluoro-phenyl)methylene]-N-(2-ethyl-3-methyl-4-pyridyl)piperidine-l -carboxamide.

The present invention relates also to pharmaceutical compositions that comprise a compound of formula I as active ingredient or or pharmaceutically acceptable salt thereof, e.g. present in a therapeutically- effective amount, which can be used especially in the treatment of the proliferation disorders, in particular cancer, as described herein. Compositions may be formulated for non-parenteral administration, such as nasal, buccal, rectal, pulmonary, vaginal, sublingual, topical, transdermal, ophthalmic, otic or, especially, for oral administration, e.g. in the form of oral solid dosage forms, e.g. granules, pellets, powders, tablets, coated tablets (e.g. film or sugar coated), effervescent tablets, hard and soft gelatine or HPMC capsules, coated as applicable, orally disintegrating tablets, solutions, emulsions (e.g. lipid emulsions) or suspensions, or for parenteral administration, such as intravenous, intramuscular or subcutaneous, intrathecal, intradermal or epidural administration, to mammals, especially humans, e.g. in the form of solutions, lipid emulsions or suspensions containing microparticles or nanoparticles. The compositions may comprise the active ingredient alone or, preferably, together with a pharmaceutically acceptable carrier.

The compounds of formula I or pharmaceutically acceptable salts thereof can be processed with pharmaceutically inert, inorganic or organic excipients for the production of oral solid dosage forms, e.g. granules, pellets, powders, tablets, coated tablets (e.g. film or sugar coated), effervescent tablets and hard gelatin or HPMC capsules or orally disintegrating tablets. Fillers e.g. lactose, cellulose, mannitol, sorbitol, calcium phosphate, starch (e.g. com starch) or derivatives thereof, binders e.g. cellulose, starch, polyvinylpyrrolidone, or derivatives thereof, glidants e.g. talcum, stearic acid or its salts, flowing agents e.g. fumed silica, can be used as such excipients e.g. for formulating and manufacturing of oral solid dosage forms, such as granules, pellets, powders, tablets, film or sugar coated tablets, effervescent tablets, hard gelatine or HPMC capsules, or orally disintegrating tablets. Suitable excipients for soft gelatin capsules are e.g. vegetable oils, waxes, fats, semisolid and liquid polyols etc.

Suitable excipients for the manufacture of solutions (e.g. oral solutions), lipid emulsions or suspensions are e.g. water, alcohols, polyols, saccharose, invert sugar, glucose etc.

Suitable excipients for parenteral formulations (e.g. injection solutions) are e.g. water, alcohols, polyols, glycerol, vegetable oils, lecithin, surfactants etc..

Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants. They can also contain still other therapeutically valuable substances. The dosage can vary within wide limits and will, of course, be fitted to the individual requirements in each particular case. In general, a daily dosage of about 1 to 1000 mg, e.g. 10 to 1000 mg per person of a compound of general formula I should be appropriate, although the above upper limit (and likewise the lower limit) can also be exceeded when necessary.

The compounds of formula I or pharmaceutically acceptable salts thereof can also be used in combination with one or more other pharmaceutically active compounds, which are either effective against the same disease, preferably using a different mode of action, or which reduce or prevent possible undesired side effects of the compounds of formula I. The combination partners can be administered in such a treatment either simultaneously, e.g. by incorporating them into a single pharmaceutical formulation, or consecutively by administration of two or more different dosage forms, each containing one or more than one of the combination partners.

Compounds of formula I according to the invention as described above or pharmaceutically acceptable salts thereof are particularly useful for the treatment of proliferation disorders and/or diseases such as cancer, in particular carcinoma, sarcoma, leukemia, myeloma and lymphoma and cancers of the brain and spinal cord, e.g. when administered in therapeutically effective amounts. Examples of such proliferation disorders and diseases include, but are not limited to, epithelial neoplasms, squamous cell neoplasms, basal cell neoplasms, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, adnexal and skin appendage neoplasms, mucoepidermoid neoplasms, cystic neoplasms, mucinous and serous neoplasms, ducal-, lobular and medullary neoplasms, acinar cell neoplasms, complex epithelial neoplasms, specialized gonadal neoplasms, paragangliomas and glomus tumours, naevi and melanomas, soft tissue tumours and sarcomas, fibromatous neoplasms, myxomatous neoplasms, lipomatous neoplasms, myomatous neoplasms, complex mixed and stromal neoplasms, fibroepithelial neoplasms, synovial like neoplasms, mesothelial neoplasms, germ cell neoplasms, trophoblastic neoplasms, mesonephromas, blood vessel tumours, lymphatic vessel tumours, osseous and chondromatous neoplasms, giant cell tumours, miscellaneous bone tumours, odontogenic tumours, gliomas,

neuroepitheliomatous and neuroendocrine neoplasms, meningiomas, nerve sheath tumours, granular cell tumours and alveolar soft part sarcomas, Hodgkin's and non-Hodgkin's lymphomas, B-cell lymphoma, T- cell lymphoma, hairy cell lymphoma, Burkitts lymphoma and other lymphoreticular neoplasms, plasma cell tumours, mast cell tumours, immunoproliferative diseases, leukemias, miscellaneous

myeloproliferative disorders, lymphoproliferative disorders and myelodysplastic syndromes.

Examples of cancers in terms of the organs and parts of the body affected include, but are not limited to, the breast, cervix, ovaries, colon, rectum (including colon and rectum i.e. colorectal cancer), lung (including small cell lung cancer, non-small cell lung cancer, large cell lung cancer and mesothelioma), endocrine system, bone, adrenal gland, thymus, liver, stomach (gastric cancer), intestine, pancreas, bone marrow, hematological malignancies (such as lymphoma, leukemia, myeloma or lymphoid malignancies), bladder, urinary tract, kidneys, skin, thyroid, brain, head, neck, prostate and testis. Preferably the cancer is selected from the group consisting of breast cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer, hematological malignancies, melanoma and sarcomas.

The term "treatment" or“treating” as used herein in the context of treating a disease or disorder, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the disease or disorder, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviation of symptoms of the disease or disorder, amelioration of the disease or disorder, and cure of the disease or disorder. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the disease or disorder, but who are at risk of developing the disease or disorder, is encompassed by the term "treatment." For example, treatment includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc..

The term "therapeutically-effective amount," as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

The compounds of formula I can be synthesized by methods given below, by methods given in the experimental part below or by analogous methods. The schemes described herein are not intended to present an exhaustive list of methods for preparing the compounds of formula (I); rather, additional techniques of which the skilled chemist is aware may be also used for the compound synthesis.

It is understood by one skilled in the art of organic synthesis that optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by routine optimization procedures. In some cases, the order of performing the following reaction schemes, and/or reaction steps, may be varied to facilitate the reaction or to avoid the formation of unwanted side products. In addition, the functionality present at various positions of the molecule must be compatible with the reagents and reactions proposed. Such restrictions to the substituents, which are compatible with the reaction conditions, will be readily apparent to one skilled in the art and alternate methods must then be used. Furthermore in some of the reactions mentioned herein it may be necessary or desirable to protect any sensitive groups in compounds and it will be assumed that such protecting groups (PG) as necessary are in place. Conventional protecting groups may be used in accordance with standard practice, well known in the art (for illustration see Greene T.W, Wuts P.G.M, Protective Groups in Organic Synthesis, 5th Edition, Publisher: John Wiley & Sons, 2014) The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the art, or they may be removed during a later reaction step or work-up. In the general sequence of reactions outlined below, the abbreviations X, n, q, the dotted line and the generic groups Bl-4, Rl, R2, R4a and R4b are as defined for formula (I), unless otherwise specified. Other abbreviations used herein are explicitly defined, or are as defined in the experimental section.

The necessary starting materials for the synthetic methods as described herein, if not commercially available, may be made by procedures which are described in the scientific literature, or may be made from commercially available compounds using adaptations of processes reported in the scientific literature. The reader is further referred to March J., Smith M., Advanced Organic Chemistry, 7th Edition Publisher: John Wiley & Sons, 2013 for general guidance on reaction conditions and reagents.

The compounds according to the present invention, pharmaceutically acceptable salts, solvates, and hydrates thereof can be prepared according to the general sequence of reactions outlined below, followed, if necessary, by:

manipulation of substituents to give a new final product. These manipulations may include, but are not limited to, reduction, oxidation, alkylation, acylation, substitution, coupling, including transition-metal catalyzed coupling and hydrolysis reactions which are commonly known by those skilled in the art;

removing any protecting groups;

forming a pharmaceutically acceptable salt; or

forming a pharmaceutically acceptable solvate or hydrate.

Generally, compounds of formula (I) can be obtained by the coupling reaction of a compound of formula (3) and a compound of formula (4), wherein E2 is a halogen or a leaving group such as imidazole, phenol, 4-nitrophenol, 2,2,2-trifluoro-ethanol or l-hydroxypyrrolidine-2,5-dione (Scheme 1).

When E2 is a leaving group such as imidazole, phenol, 4-nitrophenol, 2,2,2-trifluoro-ethanol or 1 - hydroxypyrrolidine-2,5-dione, more preferably a phenol or 4-nitrophenol, the coupling reaction between a compound of formula (3) and a compound of formula (4) is generally performed in a variety of organic solvents such as tetrahydrofuran, dichloromethane, l,2-dichloroethane, diethylether, ethyl acetate, dimethylsulfoxide, AyV-dimcthylformamidc, and acetonitrile, aqueous solvents and a mixture of theses solvents under biphasic conditions (more frequently in /V, N- d i m c t hy 1 fo r m a m i d c ) in a presence of an inorganic base such as sodium hydride, sodium carbonate or sodium hydrogen carbonate or in the presence of an organic base such as triethylamine, pyridine or alike (more frequently triethylamine). Reactions are typically run from -20 °C to 80 °C (generally at room temperature).

The compounds of formula (4), wherein E2 is a leaving group such as imidazole (which can be activated by methylation prior to the reaction), phenol, 4-nitrophenol, 2,2,2-trifluoro-ethanol or 1 - hydroxypyrrolidine-2,5-dione, are typically obtained by the coupling reaction of a compound of formula (2) and 1 , G-carbonyldiimidazole, phenyl chloro formate, 4-nitrophenyl chloro formate, 2,2,2-trifluoroethyl chloroformate or NN'- D i sued n i mi dy 1 carbonate, respectively, in presence of a base, such as sodium hydride, triethylamine, pyridine (diluted or neat), 4-(dimethylamino)pyridine in aprotic solvents such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate. Reactions are typically run from -10 °C to 80 °C.

The compounds of formula (4), for which E2 is a chlorine are generally prepared in situ by the reaction of a compound of formula (2) and phosgene or more frequently a phosgene analogue (such as

bis(trichloromethyl) carbonate or trichloromethyl chloroformate). The reaction is typically performed in aprotic and inert solvents such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate (more frequently dichloromethane) in presence of a base such as triethylamine, 4-(dimethylamino)- pyridine or V, V-diisopropylethylamine. Reactions are typically run from -40 °C to 50 °C, generally 0 °C. The low stability of such intermediates does often not allow isolation and they are generally prepared in situ. A compound of Formula (3) is allowed to react subsequently with a compound of formula (4) to generate the corresponding compound of Formula (1).

Compounds of formula (2) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art.

(3) (I)

Scheme 1 Similarly, as outlined in scheme 2, compounds of formula (1) can be prepared from a compound of formula (5), wherein E3 is a leaving group such as chlorine, imidazole, phenol, 4-nitrophenol, 2,2,2- trifluoro-ethanol or l-hydroxypyrrolidine-2,5-dione, more preferably a phenol or 4-nitrophenol and a compound of formula (2) by a coupling reaction, following similar procedures previously described. Compounds of formula (5) can be prepared from a compound of formula (3) by a coupling reaction following similar procedures as described above.

Scheme 2

Alternatively, compounds of formula (I) can be generated from a compound of formula (6) and a compound of formula (7), wherein E4 is a halogen or a leaving group such as a triflate, via a transition- metal catalyst reaction coupling (scheme 3). Typical catalysts include palladium(II) acetate,

tris(dibenzylideneacetone)dipalladium(0) or alike. The reaction is typically run at a temperature from 0 °C to 150 °C, more frequently from 100 °C to 120 °C. Usually the reaction is performed in the presence of a ligand such as di-fert-butyl-[3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]phosphane, di -tert- butyl-[2,3,4,5-tetramethyl-6-(2,4,6-triisopropylphenyl)phenyl]phosphane, 2-

(dicyclohexylphosphino)biphenyl or the like and a base such as sodium feri-butylate, cesium carbonate, potassium carbonate, more frequently cesium carbonate in a large variety of inert solvents such as toluene, tetrahydrofuran, dioxane, 1 ,2-dichloroethane, /V, ,V- d i m c t by 1 fo r m a m i dc , dimethylsulfoxide, water and acetonitrile, or a mixture of solvents, more frequently in dioxane.

Compounds of formula (6) can be obtained from compounds of formula (3) following procedures described in literature, or by procedures known by a person skilled in the art. For example, a compound of formula (6) can be prepared by the reaction of a compound of formula (3) with isocyanatotrimethylsilane in aprotic solvents such as acetonitrile, ethyl acetate, chloroform and more frequently in dichloromethane in a presence of an organic base such as triethylamine, 4-(dimethylamino)pyridine, NN- diisopropylethylamine or alike. The reaction can be run at a temperature from 0 °C to 50 °C, generally at room temperature.

Compounds of formula (7) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art.

Scheme 3

Compounds of formula (3) are generally obtained from commercial sources, or prepared following procedures described in literature, or by procedures known by a person skilled in the art. For example, when X is -C(R5)= (double bond Z, E or Z/E ), in which formulae R5 is a hydrogen or a methyl substituent, compounds of formula (3) can be prepared from a compound of formula (1 l-a), wherein PG is an amino protecting group, by deprotection of the amino protecting group, as outlined in scheme 4. The amino protecting group can be removed under standard conditions. For example the benzyl carbamates are deprotected by hydrogenolysis over a noble metal catalyst (e.g. palladium or palladium hydroxide on activated carbon) or other suitable catalyst e.g. Raney-Ni. The Boc group is removed under acidic conditions such as hydrochloric acid in an organic solvent such as methanol, dioxane or ethyl acetate, or trifluoroacetic acid neat or diluted in a solvent such as dichloromethane. The Alloc group is removed in presence of a palladium salt such as palladium acetate or tetrakis(triphenylphosphine)palladium(0) and an allyl cation scavenger such as morpholine, pyrrolidine, dimedone or tributylstannane generally at temperatures from 0 °C to 70 °C in a solvent such as tetrahydrofuran. The N- benzyl protected amines are deprotected by hydrogenolysis over a noble metal catalyst (e.g. palladium hydroxide on activated carbon) or other suitable catalyst e.g. Raney-Ni. The Fmoc protecting group is removed under mild basic conditions such as diluted morpholine or piperidine in /V, N- d i m c t by 1 fo r m a m i dc or acetonitrile. The N- acetyl protected amines are deprotected by hydrolysis using either acidic or basic aqueous solution generally at temperatures from 0 °C to 100 °C. Further general methods to remove amine protecting groups have been described in Greene T.W, Wuts P.G.M, Protective Groups in Organic Synthesis, 5th Edition, Publisher: John Wiley & Sons, 2014.

Compounds of formula (1 l-a) are generally obtained from commercial sources, or prepared following procedures described in literature, or by procedures known by a person skilled in the art. Generally, compounds of formula (1 l-a) can be prepared from a compound of formula (10) and a compound of formula (9), wherein E6 is a phosphonium salt (typically triphenylphosphonium salt) or a phosphonate (typically diethyl phosphonate) via a Wittig or Homer-Wadsworth-Emmons reaction, respectively. The Wittig reaction is the reaction of an aldehyde or ketone with a triphenyl phosphonium ylide to afford an alkene and triphenylphosphine oxide. The Wittig reagent is usually prepared from a phosphonium salt. To form the Wittig reagent, the phosphonium salt is suspended in a solvent such as diethyl ether or tetrahydrofuran and a strong base such as «-butyl lithium or lithium bis(trimethylsilyl)amide is added. With simple ylides, the product is usually mainly the Z-isomer, although a lesser amount of the T-isomcr also is often formed. If the T-isomcr is the desired product, the Schlosser modification may be used. Alternatively the Homer-Wadsworth-Emmons reaction produces predominantly E-alkenes. The Homer- Wadsworth-Emmons reaction is the condensation of stabilized phosphonate carbanions with aldehydes or ketones in presence of a base such as sodium hydride or lithium bis(trimethylsilyl)amide in a solvent such as tetrahydrofuran or /V, N- d i m c t by 1 fo r ma m i d c , generally at temperatures from 0 °C to 80 °C. In contrast to phosphonium ylides used in the Wittig reaction, phosphonate-stabilized carbanions are more nucleophilic and more basic.

When E6 is a phosphonium salt, a compound of formula (9) can be for example obtained by alkylation of triphenylphosphine and a compound of formula (8), wherein E5 is a halogen, following well-known procedures.

When E6 is a diethyl phosphonate, a compound of formula (9) can be obtained by the reaction of triethylphosphite and a compound of formula (8), wherein E5 is a halogen, following well-known procedures.

Compounds of formula (8) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art.

Compounds of formula (10) are generally obtained from commercial sources, or prepared following procedures described in literature, or by procedures known by a person skilled in the art. The amino protecting groups (PG) can be present in the starting material or introduced by reacting the corresponding free amine with allyl, fluorenylmethyl or benzyl chloroformate, or with di-fert-butyl dicarbonate in presence of a base such as sodium hydroxide, sodium hydrogen carbonate, triethylamine, 4- dimethylaminopyridine or imidazole. The free amine can also be protected as /V-bcnzyl derivatives by reaction with benzyl bromide or chloride in presence of a base such as sodium carbonate or triethylamine. Alternatively, /V-bcnzyl derivatives can be obtained through reductive amination in presence of benzaldehyde. The free amine can also be protected as /V- acetyl derivatives by reaction with acetyl chloride or acetic anhydride in presence of a base such as sodium carbonate or trimethylamine. Further strategies to introduce other amino protecting groups have been described in Greene T.W, Wuts P.G.M, Protective Groups in Organic Synthesis, 5th Edition, Publisher: John Wiley & Sons, 2014.

Scheme 4

Alternatively, compounds of formula (1 l-a) can be prepared from a compound of formula (14) and a compound of formula (15), wherein E10 is a halogen or a leaving group such as triflate via cross-coupling reaction (i.e. Suzuki, Stille, Negishi, etc), as outlined in scheme 5. For example, when E9 is a boronic acid or a boronic ester, a compound of formula (14) can react with a compound of formula (15) to form a compound of formula (1 l-a) via Suzuki cross-coupling reaction. The Suzuki reaction is a palladium- catalyzed cross-coupling reaction between organoboronic acids or esters and aryl or vinyl halides or triflates. Typical catalysts include palladium(II) acetate, tetrakis(triphenylphosphine)palladium(0), tris(dibenzylideneacetone)dipalladium(0), bis(triphenylphosphine)palladium(II) dichloride and

[1,1’bis(diphenylphosphino)ferrocene]dichloropalladium(II). The reaction can be carried out in a variety of organic solvents including toluene, tetrahydrofuran, dioxane, 1 ,2-dichloroethane, N,N- dimethylformamide, dimethylsulfoxide and acetonitrile, aqueous solvents and under biphasic conditions. Reactions are typically run under inert atmosphere from room temperature to 150 °C, more frequently from 90 °C to 120 °C. Additives such as cesium fluoride, potassium fluoride, potassium hydroxide, potassium carbonate, potassium acetate, potassium phosphate or sodium ethylate frequently accelerate the coupling. Potassium trifluorob orates and organoboranes or boronate esters may be used in place of boronic acids. As there are numerous components in the Suzuki reaction such as the particular palladium catalyst, the ligand, additives, solvent, temperature, numerous protocols have been identified. One skilled in the art will be able to identify a satisfactory protocol without undue experimentation.

Compounds of formula (15) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art. Organoboronic acids or esters of formula (14) are generally obtained from diboron reagents (such as bis(pinacolato)diboron or bis-boronic acid) and a compound of Formula (13), wherein E8 is halogen, via Miyaura borylation (Ishiyama T. et al., J. Org. Chem., vol. 60, pages 7508-7510, 1995) in presence of a palladium catalyst such as tris(dibenzylideneacetone)dipalladium(0) or chloro(2-dicyclohexylphosphino- 2',4',6'-triisopropyl-l,l '-biphenyl)[2-(2'-amino-l,l '-biphenyl)]palladium(II) and a ligand such as triphenylphosphine or 2-(dicyclohexylphosphino)-2',4',6’-tri-isopropyl-l,r-biphenyl. The reaction can be carried out in a variety of organic solvents including toluene, tetrahydrofuran, dioxane, 1 ,2- dichloroethane, V, V-dimethylformamide, dimethylsulfoxide and acetonitrile, aqueous solvents and under biphasic conditions. Reactions are typically run from room temperature to 150 °C (more frequently at around 100 °C). Crucial for the success of the borylation reaction is the choice of an appropriate base, as strong activation of the product enables the competing Suzuki coupling. The use of potassium acetate (Ishiyama et al., J. Org. Chem., vol. 60, pages 7508-7510, 1995) and potassium phenolate (Takagi J. et al., J. Am Chem Soc., vol. 27, no. 27, pages 8001-8006, 2002) is actually the result of a screening of different reaction conditions by the Miyaura group. Other bases such as potassium hydroxide, cesium carbonate, potassium carbonate, potassium phosphate or sodium ethylate are frequently used as well. As for the Suzuki reaction, there are numerous components in the Miyaura borylation reaction such as the particular palladium catalyst, the ligand, additives, solvent, temperature and numerous protocols have been identified. One skilled in the art will be able to identify a satisfactory protocol without undue experimentation.

Vinyl halides of formula (13) used for the preparation of organoboronic acids or esters (14) can be prepared via a Wittig reaction between a compound of formula (10) and a compound of formula (12), wherein E7 is a triphenylphosphonium salt and E8 is a halogen, following procedures previously described.

(14) (15) (11 )

Scheme 5 ln addition, the compounds of formulae (11-a), (3) and (1), wherein X is -C(R5)= (double bond Z, E or Z/E), can further be reduced to generate compounds of formulae (11 -b), (3) and (1), respectively, wherein X is -CH(R5)-, in which formulae R5 is a hydrogen or a methyl substituent. The reduction reaction is usually performed by hydrogenation over a noble metal catalyst (e.g. palladium, palladium hydroxide on activated carbon (Trost B.M., et al., Chem Eur. J., vol. 5, no. 3, page 1055-1069, platinum dioxide) or other suitable catalysts. This hydrogenation step can be performed at any convenient stage during the synthesis.

When X is -C(O)-, compounds of formula (3) can be obtained from a compound of formula (1 l-c) as outlined in scheme 6, by removal of the amino protecting group (PG), following procedure previously described.

Compounds of formula (1 l-c) can be obtained from a compound of formula (15), wherein E10 is a halogen, via Weinreb Ketone Synthesis with a compound of formula (16). The reaction takes place in the presence of a strong base such, «-butyl lithium or ί-butyl lithium under anhydrous conditions in an organic solvent such as tetrahydrofuran and generally at temperatures from -78 °C to 60 °C (around 0 °C is preferred).

Compounds of formula (16) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art. For example, compounds of formula (16) can be prepared from the corresponding carboxylic acid and N,0- dimethylhydroxylamine via amide coupling reaction using methods well known in the art.

Alternatively, compounds of formula (1 lc) can be obtained from a compound of formula (15), wherein El 0 is a halogen, via Grignard reaction with a corresponding acyl halogenide (e.g. a compound of formula (18)). The Grignard reaction is typically performed under anhydrous conditions in an organic solvent such as tetrahydrofuran. The reaction is usually run from -78 °C to 60 °C. The Grignard reagent is generally obtained from the reaction of an aryl halide of formula (15) and magnesium metal using classical methods widely described in literature (Rogers H.R. et al., J. Am. Chem. Soc., vol. 102, no. 1, pages 217-226, 1980) or by magnesium-halide exchange reaction using e.g. isopropylmagnesium chloride.

Scheme 6

Alternatively, compounds of formula (11 -c) can be obtained from a compound of formula (17) and a compound of formula (18) by Friedel-Crafts acylation (scheme 7). The amino protecting group (PG) is preferentially an /V- acetyl group. Friedel-Crafts acylation is the acylation of aromatic rings with an acyl chloride using a strong Lewis acid catalyst such as ferric chloride or aluminium chloride (more frequently aluminium chloride). Friedel-Crafts acylation is also possible with acid anhydrides. Normally, a stoichiometric amount of the Lewis acid catalyst is required, because both the substrate and the product form complexes. The reaction is generally performed under anhydrous conditions in an inert solvent such as acetonitrile, tetrahydrofuran, dichloromethane, l,2-dichloroethane or in neat mixture at a wide range of temperatures (e.g. from -20 °C to 100 °C).

Compounds of formulae (17) and (18) can be obtained from commercial sources, or are prepared following procedures described in literature, or by procedures known by a person skilled in the art.

(17) (18) (11 -c)

Scheme 7

Whenever required, the substituents Rl, R2, R3, R4a and / or R4b can be present as precursors in the starting material, and / or can be transformed by additional routine transformations during the synthetic pathways described herein. These transformations might be carried out at any convenient stage during the synthesis and may include, but are not limited to the following lists of reactions, which are commonly known by those skilled in the art: - Selective reduction of the aryl-nitro group (Bechamp reduction) using iron powder in the presence of aqueous acidic solution. The nitro group can also be reduced via catalytic hydrogenolysis over a noble metal catalyst (such as palladium on activated carbon) or other suitable hydrogenation catalyst. For example, when R2 is a nitro group, reduction of the nitro group to R2 is an amino group can be selectively achieved by Bechamp reduction without affecting the double bond, when X is -C(R5)=.

- Dealkylation of aromatic ether using boron tribromide or alike in an organic solvent such as dichloromethane (Ilhyong R. et al., J. Am. Chem. Soc., vol. 124, no. 44, pages 12946-12947, 2002). The reaction can also be performed using trimethylsilyl bromide or iodide in an organic solvent such as acetonitrile and at a temperature e.g. from 0 °C to 90 °C. Optionally, sodium iodide can be used to support the reaction. For example, a compound of formula (I), wherein R2 is a methoxy group can be converted to a compound of formula (I), wherein R2 is a hydroxyl group.

- Amide coupling reaction between a carboxylic acid and an amine. The reaction takes place in the presence of an activating agent such as /V, /V’ - d i cy c 1 o h c x y 1 c a r b o d i i m i d c o r /V- ( 3 - d i m c t h y 1 a m i n o p r o py 1 ) - X’-ethylcarbodiimide hydrochloride, with the optional addition of l-hydroxybenzotriazole. Other suitable coupling agents may be utilized such as, 0-(7-azabenzotriazol-l-yl)-A,A,A’,A’-tetramethyluronium hexafluorophosphate, 2-ethoxy- 1 -ethoxycarbonyl- 1 ,2-dihydroquinoline, carbonyldiimidazole or diethylphosphorylcyanide. Optionally, a base like triethylamine, /V, /V- d i i s o p ro p y 1 c t h y 1 a m i n c or pyridine can be added to perform the coupling. The amide coupling is conducted at a temperature e.g. from -20 °C to 80 °C, in an inert solvent, preferably a dry aprotic solvent like dichloromethane, acetonitrile or N,N- dimethylformamide and chloroform. Alternatively, the carboxylic acid can be activated by conversion into its corresponding acid chloride or its corresponding activated ester, such as the N- hydroxysuccinimidyl ester (Singh J., et al., Org. Process Res. Dev., vol. 6, no. 6, pages 863-868, 2002) or the benzothiazolyl thioester (Ishikawa T. et al., J. Antibiotics, vol. 53, no. 10, pages 1071-1085, 2000). The generated activated entity can react e.g. at a temperature from -20 °C to 80 °C with the amine reagent in an aprotic solvent like dichloromethane, chloroform, acetonitrile, /V, N- di m c t by 1 fo r m a m i d c and tetrahydrofuran. Optionally, a base like triethylamine, /V, ,V- d i i s o p ro p y 1 c t h y 1 a m i n c , pyridine, sodium hydroxide, sodium carbonate, potassium carbonate can be added to perform the coupling.

- Reductive amination reaction between an amine and an aldehyde or a ketone. The reductive amination reaction between the amine and the aldehyde or the ketone to form an intermediate imine is conducted in a solvent system allowing the removal of the formed water through physical or chemical means (e.g. distillation of the solvent-water azeotrope or presence of drying agents such as molecular sieves, magnesium sulfate or sodium sulfate). Such solvent is typically toluene, n-hcxanc, tetrahydrofuran, dichloromethane /V, N- d i m c t by 1 fo r m a m i d c , /V, ,V- d i m c t h y 1 ac et a m i d c , acetonitrile, 1 ,2-dichloroethane or mixture of solvents such as methanol or l,2-dichloroethane. The reaction can be catalyzed by traces of acid (usually acetic acid). The intermediate imine is reduced subsequently or simultaneously with a suitable reducing agent (e.g. sodium borohydride, sodium cyanoborohydride, sodium

triacetoxyborohydride, see e.g. Hutchins M.K., Comprehensive Organic Synthesis, Publisher: Fleming, Eds; Pergamon Press, vol. 8, pages. 25-78, 1991) or through hydrogenation over a suitable catalyst such as palladium on activated carbon. The reaction is usually carried out from -10 °C to 110 °C, preferably from 0 °C to 60 °C. The reaction can also be carried out in one pot. ft can also be performed in protic solvents such as methanol or water in presence of a picoline-borane complex (Sato S. et al, Tetrahedron, vol. 60, pages 7899-7906, 2004). For example, the reductive amination reduction between a compound of formula (1), wherein R2 is -CHO and a compound of formula HN(R6a)(R6b) leads to a compound of formula (1), wherein R2 is -CH₂-N(R6a)(R6b) and R6a and R6b are as defined by the claims.

- Substitution reaction. The substitution reaction can be performed in presence of an inorganic base such as sodium hydride, potassium carbonate, cesium carbonate or the like or an organic base such as triethylamine or the like in a wide variety of solvents such as acetonitrile, tetrahydrofuran or N,N- dimethylformamide e.g. at a temperature from -20 °C to 120 °C. For example, the substitution reaction between a compound of formula (1), wherein R2 is -methylene-OH (which needs to be activated prior to the reaction), and a compound of formula HN(R6a)(R6b) leads to a compound of formula (1), wherein R2 is -methylene-N(R6a)(R6b) and R6a and R6b are as defined by the claims.

- Activation of the hydroxyl group prior to substitution reaction. Hydroxyl group can be transformed to a mesylate, a tosylate or a triflate by reacting the corresponding alcohol with methanesulfonyl chloride or methanesulfonic anhydride, p-toluenesulfonyl chloride, trifluoromethanesulfonyl chloride or

trifluoromethanesulfonic anhydride, respectively, in presence of a base such as triethylamine or the like in a dry aprotic solvent such as pyridine, acetonitrile, tetrahydrofuran or dichloromethane e.g. at a temperature from -30 °C to 80 °C.

- Reduction of alkyl ester (typically methyl or ethyl esters) into their corresponding alcohols. This reduction is performed with a reducing agent like boron or aluminium hydride, lithium aluminium hydride, lithium borohydride, sodium borohydride in a solvent such as tetrahydrofuran, methanol or ethanol from e.g. at a temperature from -20 °C to 80 °C. Alternatively, the ester function is hydrolyzed into its corresponding carboxylic acid using an alkali hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide in water or in a mixture of water with polar protic or aprotic organic solvents such as dioxane, tetrahydrofuran or methanol e.g. at a temperature from -10 °C to 80 °C or the ester function is hydrolyzed using aqueous acidic solution. The resulting carboxylic acid is further reduced into the corresponding alcohol using a borane derivative such as borane-tetrahydrofuran complex in a solvent such as tetrahydrofuran e.g. at a temperature from -10 °C to 80 °C.

- Oxidation of hydroxyl group to ketone or aldehyde. The alcohol is transformed into its corresponding aldehyde or ketone through oxidation under Swem, Dess-Martin, Sarett or Corey-Kim conditions respectively, or via NaOCl oxidation. Further methods are described in Larock R.C., Comprehensive Organic Transformations. A guide to functional Group Preparation, 2nd Edition, Publisher: Wiley-VCH; New York, Chichester, Weinheim, Brisbane, Singapore, Toronto, 1999. Section aldehydes and ketones, p.1235-1236 and 1238-1246. For example, a compound of formula (1), wherein R2 is -CH₂OH can be converted to a compound of formula (1), wherein R2 is -CHO by oxidation using Dess-Martin reagent. The reaction is typically run in an aprotic solvent such as dichloromethane e.g. at a temperature from 0 °C to 50 °C, more frequently at room temperature.

- Buchwald-Hartwig animation. The Buchwald-Hartwig amination reaction (Surry D.S. and Buchwald S.L., Chem. Sci., vol. 2, pages 27-50, 2011) is a palladium-catalyzed cross-coupling reaction of amines and aryl halides or triflates. Typical catalysts include palladium(II) acetate, or

tris(dibenzylideneacetone)dipalladium chloroform complex. The reaction is typically run at a temperature from 0 °C to 150 °C. Usually the reaction is performed in the presence of a ligand such as di-feri-butyl- [3,6-dimethoxy-2-(2,4,6-triisopropylphenyl)phenyl]-phosphane, 2-(dicyclohexylphosphino)biphenyl or the like and a base such as sodium feri-butylate, cesium carbonate, potassium carbonate in a large variety of inert solvents such as toluene, tetrahydrofuran, dioxane, 1 ,2-dichloroethane, V, V-dimethylformamide, dimethylsulfoxide and acetonitrile, aqueous solvents and under biphasic conditions. Several versions of the reaction, employing complexes of copper and nickel rather than palladium, have also been developed (Hartwig J.F., Angew. Chem. Int. Ed., vol. 37, no. 15, pages 2046-2067, 1998). The reaction can be performed using microwave irradiation. For example, the reaction between a compound of formula (I), wherein R2 is a halogen (more frequently a chlorine) and a compound of formula HN(R6a)(R6b) via Buchwald-Hartwig amination leads to a compound of formula (I), wherein R2 is -N(R6a)(R6b) and R6a and R6b are as defined by the claims.

- Nitration of aromatic compounds. The nitration of aromatic compounds is the chemical process for the introduction of a nitro group into an organic compound ln the case of the nitration of aromatic compounds, this process is one example of the electrophilic aromatic substitution. The reaction is typically run in a mixture of acids, usually nitric acid and another strong acid, such as sulfuric acid or trifluoroacetic acid. The reaction can be performed in a wide range of temperature (e.g. from 0 °C to 100 °C).

Whenever an optically active form of a compound of the invention is required, it may be obtained by carrying out one of the above procedures using a pure enantiomer or diastereomer as a starting material, or by resolution of a mixture of the enantiomers or diastereomers of the final product or intermediate using a standard procedure. The resolution of enantiomers may be achieved by chromatography on a chiral stationary phase, such as for example REG1S® P1RKLE COVALENT (R-R) WHELK-02, 10 pm, 100 A, 250 x 21.1 mm column. Alternatively, resolution of stereoisomers may be obtained by preparation and selective crystallization of a diastereomeric salt of a chiral intermediate or chiral product with a chiral acid, such as camphorsulfonic acid or with a chiral base such as phenylethylamine. Alternatively a method of stereoselective synthesis may be employed, for example by using a chiral variant of a protecting group, a chiral catalyst or a chiral reagent where appropriate in the reaction sequence.

Enzymatic techniques may also be used for the preparation of optically active compounds and/or intermediates. The schemes and processes described herein are not intended to present an exhaustive list of methods for preparing the compounds of formula I; rather, additional techniques of which the skilled chemist is aware of may be also used for the compound synthesis.

All aspects and embodiments of the invention described herein may be combined in any combination where possible.

A number of publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Particular embodiments of the invention are described in the following examples, which serve to illustrate the invention in more detail and should not be construed as limiting the invention in any way.

Figures

Figure 1 shows the results of the cell growth assays (crystal violet) in HeLa galactose and HeLa glucose cells treated with mitochondrial inhibitors Antimycin A (Figure la) and Example 6 (Figure lb) or the cytotoxic drug Paclitaxel (Figure lc).

Examples

Preparation Examples

All reagents and solvents are generally used as received from the commercial supplier;

reactions are routinely performed with anhydrous solvents in well-dried glassware under nitrogen atmosphere;

evaporations are carried out by rotary evaporation under reduced pressure and work-up procedures are carried out after removal of residual solids by filtration;

all temperatures are given in degree Celcius (°C) and are approximate temperatures; unless otherwise noted, operations are carried out at room temperature (rt), that is typically in the range 18 - 25 °C;

column chromatography (by the flash procedure) is used to purify compounds and is performed using Merck silica gel 60 (70-230 mesh ASTM) unless otherwise stated;

classical flash chromatography is often replaced by automated systems. This does not change the separation process per se. A person skilled in the art will be able to replace a classical flash

chromatography process by an automated one, and vice versa. Typical automated systems can be used, as they are provided by Biichi or lsco (combiflash) for instance;

reaction mixture can often be separated by preparative HPLC. A person skilled in the art will find suitable conditions for each separation; in some cases the compounds are isolated after purification in a form of the corresponding trifluoroacetic salt (*l), or the respective formic acid salt (*2); such compounds are marked accordingly;

reactions, which required higher temperature, are usually performed using classical heating instruments; but can also be performed using microwave apparatus (e.g. CEM Explorer at a power of 250 W), unless otherwise noted;

hydrogenation or hydrogenolysis reactions can be performed using hydrogen gas in balloon or using Pc/ff-apparatus system or other suitable hydrogenation equipment;

concentration of solutions and drying of solids was performed under reduced pressure unless otherwise stated;

in general, the course of reactions is followed by TLC, HPLC, or LC/MS and reaction times are given for illustration only; yields are given for illustration only and are not necessarily the maximum attainable; the structure of the final products of the invention is generally confirmed by NMR and mass spectral techniques.

Proton NMR spectra were recorded on a Brucker 400 MHz spectrometer. Chemical shifts (d) are reported in ppm relative to Me₄Si as internal standard, and NMR coupling constants (J values) are in Hertz (Hz). Each peak is denoted as a broad singlet (br), singlet (s), doublet (d), triplet (t), quadruplet (q), doublet of doublets (dd), triplet of doublets (td) or multiplet (m). Mass spectra were generated using a q-Tof Ultima (Waters AG or Thermo Scientific™ MSQ Plus™) mass spectrometer in the positive ESI mode. The system was equipped with the standard Lockspray interface;

each intermediate is purified to the standard required for the subsequent stage and is characterized in sufficient detail to confirm that the assigned structure is correct;

analytical and preparative HPLC on non-chiral phases are performed using RP-C18 based columns; the following abbreviations may be used (reference can also be made to The Journal of Organic Chemistry Guidelines for Authors, 2017 for a comprehensive list of standard abbreviations):

ACN Acetonitrile

Boc fert-butoxy carbonyl group

BTC Bis(trichloromethyl)carbonate

Cat. no. Catalog number

CDCfi Deuterated chloroform

DCM Dichloromethane

DMAP 4-Dimethylaminopyridine

DMF N ,V- Dimethyl formamidc

DMSO-d⁶ Deuterated dimethyl sulfoxide

EA Ethyl acetate

ELSD Evaporative light scattering detection Ex. Example

HATU 0-(7-azabenzotriazol- 1 -yl)-N,N,N’ ,N’ -tetramethyluronium hexafluorophosphate c-Hex Cyclohexane

«-Flex n-Hexane

LAH Lithium aluminum hydride

LC/MS Liquid chromatography coupled to mass spectroscopy

LHMDS Lithium bis(trimethylsilyl)amide

Me₄Si T etramethylsilane

MeOH Methanol

nt Not Tested

Pd₂dba₃ Tris(dibenzylideneacetone)dipalladium(0)

PE Petroleum Ether

i-BuOH fert-butanol

TEA Triethylamine

TFAA Trifluoroacetic anhydride

THF T etrahydrofuran

W Watt

X-Phos 2-Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl The following Examples refer to the compounds of formula (I) as indicated in Table 1.

The Examples listed in the following table can be prepared using procedures described above, and detailed synthesis methodology is described in detail below. The Example numbers used in the leftmost column are used in the application text for identifying the respective compounds.

Table 1: Exemplified compounds t

t

Preparation of Example 1: 4-[(4-chloro-2-fluoro-phenvDmethylenel-/V-(2,3-dimethyl-4- nyritlyl)nineritline-l -carboxamide, trifluoroacetic acid:

Step 1-a: Preparation of tert- butyl 4-(biOmomcthylcnc)nincridinc- 1 -carboxylatc:

To a stirred suspension of (bromomethyl)triphenylphosphonium bromide (2 000 mg; 4.47 mmol) in THF (45 mL) cooled to -15 °C was added dropwise LHMDS solution, 1M in THF (5.82 mL; 5.82 mmol) over 5 min. The reaction mixture was stirred for 15 min at -15 °C and then treated with a solution of ieri-butyl 4-oxopiperidine-l-carboxylate (1 000 mg; 4.92 mmol) in THF (5 mL). The mixture was allowed to warm up gradually to rt and further stirred for 2 h. The reaction mixture was deactivated with a saturated aqueous solution of NH₄C1 and then partitioned between EA and brine. The organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated to dryness. The residue was purified by column chromatography (silica gel; c-Hex : EA; 1 :0 to 4:1 ; v/v) to afford ieri-butyl 4-(bromomethylene)- piperidine-l-carboxylate (960 mg) as a colorless oil.

‘H-NMR (400 MHz, CDC1₃) d ppm: 6.02 (s, 1H), 3.48 - 3.42 (m, 4H), 2.42 (m, 2H), 2.27 (m, 2H), 1.49 (s, 9H).

Step 1-b: Preparation of ieri-butyl 4-r(4.4.5.5-tetramethyl-l.3.2-dioxaborolan-2-yl)methylene1piperidine- l-carboxylate:

A sealable tube was charged with ieri-butyl 4-(bromomethylene)piperidine-l-carboxylate (700 mg; 2.51 mmol), potassium acetate (620 mg; 6.27 mmol), bis(pinacolato)diboron (1 040 mg; 4.01 mmol) and dioxane (20 mL) at rt. Argon was bubbled in the reaction mixture for 10 min and triphenylphosphine (70 mg; 0.25 mmol) and Pd dba₃ (160 mg; 0.15 mmol) were added. The tube was flushed with argon and sealed. The reaction mixture was then heated to 100 °C and stirred for 4 h. After cooling to rt, the reaction mixture was filtered and the cake was washed with EA. The filtrate was finally concentrated to dryness. The residue was then purified by column chromatography (silica gel; c-Hex : EA; 1 :0 to 4:1 ; v/v) to afford ieri-butyl 4-[(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)methylene]piperidine-l-carboxylate (720 mg) as a light yellow solid.

‘H-NMR (400 MHz, CDCI₃) d ppm: 5.17 (s, 1H), 3.48 - 3.42 (m, 4H), 2.62 (m, 2H), 2.28 (m, 2H), 1.49 (s, 9H), 1.28 (s, 12H).

Step 1-c: Preparation of ieri-butyl 4-r(4-chloro-2-fluoro-phenyl)methylene1piperidine-l -carboxylate: Under argon atmosphere, a mixture of X-Phos (750 mg; 1.53 mmol), l-bromo-4-chloro-2-fluoro-benzene (1.93 mL; 15.31 mmol), ieri-butyl 4-[(4, 4,5, 5-tetramethyl-l, 3, 2-dioxaborolan-2-yl)methylene]piperidine- 1 -carboxylate (5 500 mg;, 15.31 mmol), Pd dba₃ (708 mg; 0.76 mmol) and K₃P0₄ (4 975 mg; 22.97 mmol) in a mixture of H₂0 (5 mL) and dioxane (100 mL) was heated to 100 °C and stirred for 2 h. After cooling down to rt, H₂0 and EA were added. The organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated to dryness. The residue was purified by column chromatography (silica gel;PE: EA; 30:1 ; v/v) to afford feri-butyl 4-[(4-chloro-2-fluoro-phenyl)methylene]piperidine-l- carboxylate (4 000 mg) as a white solid.

MS xn/z (+ESI): 311.1, 313.1 [M-/-Bu+HCOOH]⁺.

‘H-NMR (400 MHz, CDC1₃) d ppm: 7.10 - 7.06 (m, 3H), 6.20 (s, 1H), 3.51 (t, J= 5.6 Hz, 2H), 3.41 (t, J = 5.6 Hz, 2H), 2.36 - 2.30 (m, 4H), 1.48 (s, 9H).

Step 1-d: Preparation of 4-r(4-chloro-2-fluoro-phenyl)methylene1piperidine. hydrochloride:

To a stirred solution of tert- butyl 4-[(4-chloro-2-fluoro-phenyl)methylene]piperidine-l-carboxylate (18 200 mg; 55.2 mmol) in EA (50 mL) was added dropwise HC1 solution, 2 N in EA (160 mL). The reaction mixture was stirred for 2 h and then concentrated to dryness to afford 4-[(4-chloro-2-fluoro- phenyl)methylene]piperidine, hydrochloride (12 500 mg) as a white solid.

MS xn/z (+ESI): 226.2, 228.2 [M+H]⁺.

‘H-NMR (400 MHz, DMSO-ί ή) d ppm: 9.10 (br, 2H), 7.47 (dd, J= 10.0, 2.1 Hz, 1H), 7.42 - 7.24 (m, 2H), 6.35 (s, 1H), 3.16 (t, J= 6.0 Hz, 2H), 3.08 (t, J= 6.0 Hz, 2H), 2.63 - 2.56 (m, 2H), 2.51 - 2.46 (m, 2H).

Step 2: Preparation of 4-r(4-chloro-2-fluoro-nhcnyl )mcthylcnc1-/V-(2.3-dimcthyl-4-nyridyl )nincridinc- 1 - carboxamide, trifluoroacetic acid:

To a solution of diphosgene (182 mg; 0.90 mmol) in THF (4 mL) was added dropwise a solution of 4- amino-2,3-dimethylpyridine (187 mg; 1.50 mmol), DMAP (19 mg; 0.15 mmol) and TEA (0.43 mL; 3.00 mmol) in THF (4mL) at 0 °C. The mixture was allowed to warm up to rt and stirred for 2 h. The mixture was then treated with a solution of 4-[(4-chloro-2-fluoro-phenyl)methylene]piperidine, hydrochloride (390 mg; 1.50 mmol) and TEA (0.43 mL; 3.00 mmol) in THF (4 mL). After stirring for 16 h, the mixture was concentrated to dryness. The residue was purified by preparative HPLC to afford 4-[(4-chloro-2- fluoro-phenyl)methylene] -A-(2,3 -dimethyl-4-pyridyl)piperidine- 1 -carboxamide, trifluoroacetic acid (120 mg) as a yellow solid.

Preparation of Example 2: 4-|(4-chloro-2-fluoro-phen_\ l)meth_\ lenel- V-(3-methox_\ -2-meth_\ l-4- p\ ridyl)piperidine-l -carboxamide:

Preparation of 4- r(4-chloro-2-fluoro-phenyl)methylene1 -N-(3 -methoxy-2-methyl-4-pyridyl)pip eridine- 1 - carboxamide:

To a cooled (-10 °C) solution of 3-methoxy-2-methyl-pyridin-4-amine (35 mg; 0.26 mmol) and TEA (0.45 mL; 0.31 mmol) in dry ACN (1 mL) was added 2,2,2-trifluoroethyl chloroformate (45 mg; 0.27 mmol). Upon addition, the reaction mixture was allowed to warm to rt and stirred for 24 h. 4-[(4-chloro- 2-fluoro-phenyl)methylene]piperidine, hydrochloride (70 mg; 0.26 mmol) and TEA (0.09 mL; 0.65 mmol) were added and the reaction mixture was heated to 70 °C. After stirring for 24, the reaction mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford 4-[(4- chloro-2-fluoro-phenyl)methylene] -N-(3 -methoxy-2-methyl-4-pyridyl)piperidine- 1 -carboxamide (12 mg) as a light brown solid.

Preparation of Example 3: A_'-(3-chloro-2-methyl-4-pyridyl)-4-|(4-cvano-2,6-difluoro- phenyl) methylenel piperidine- 1 -carboxamide, trifluoroacetic acid:

Step 1: Preparation of 3.5-difluoro-4-(4-piperidylidenemethyl)benzonitrile. hydrochloride:

The title compound was prepared as a white solid following schemes 5 and 4 and in analogy to procedures described in Example 1 (step l-c and l-d) using tot-butyl 4-[(4,4,5,5-tetramethyl-l,3,2- dioxaborolan-2-yl)methylene]piperidine-l-carboxylate and 4-bromo-3,5-difluoro-benzonitrile as starting materials.

MS m/z (+ESI): 235.2 [M+H]⁺.

‘H-NMR (400 MHz, DMSO-ί ή) d ppm: 8.70 (br, 2H), 7.87 - 7.82 (m, 2H), 6.20 (s, 1H), 3.20 (m, 2H), 3.07 (m, 2H), 2.61 (t, J= 5.6 Hz, 2H), 2.28 (t, J= 5.6 Hz, 2H).

Step 2: Preparation of phenyl iV-(3-chloro-2-methyl-4-pyridyl)carbamate:

To a solution of 4-amino-3-chloro-2-methylpyridine (500 mg; 3.44 mmol) in pyridine (20 mL) was added phenyl chloroformate (0.88 mL; 6.87 mmol). The reaction mixture was stirred for 16 h and then concentrated to dryness to afford crude phenyl V-(3-chloro-2-methyl-4-pyridyl)carbamate (900 mg; 50 % purity) as a yellow solid, which was used in the next step without further purification.

MS m/z (+ESI): 263.0, 265.0 [M+H]⁺.

Step 3: Preparation of iV-(3-chloro-2-methyl-4-pyridyl)-4-r(4-cvano-2.6-difluoro- phenyl)methylene1piperidine-l -carboxamide, trifluoroacetic acid:

To a solution of crude phenyl iV-(3-chloro-2-methyl-4-pyridyl)carbamate (360 mg; 0.69 mmol: 50 % purity) in DMF (6 mL) were added 3,5-difluoro-4-(4-piperidylidenemethyl)benzonitrile, hydrochloride (220 mg; 0.82 mmol) and TEA (0.49 mL; 3.42 mmol). The reaction mixture was stirred for 2 h and then diluted with ethyl acetate (100 mL). The solution was washed with water (3 x 30 mL), dried over Na₂S04, and concentrated to dryness. The residue was purified by preparative HPLC to afford ,V-(3-chloro-2- methyl-4-pyridyl)-4-[(4-cyano-2,6-difluoro-phenyl)methylene]piperidine-l -carboxamide (48 mg) as a white solid.

Preparation of Example 4: 4-|(4-cvano-2,6-difluoro-phenyl)methylenel-Y-(3-ethoxy-2-methyl-4- pyridyl)piperidine-l -carboxamide, trifluoroacetic acid:

Step 1-a: Preparation of 3 -cthoxy-2-mcthyl-4-nitro- pyridine: To a solution of 2-methyl-4-nitro-pyridin-3-ol (150 mg; 0.88 mmol) in DMF (4 mL) were added iodoethane (166 mg; 1.05 mmol) and K₂CO₃ (245 mg; 1.75 mmol). The reaction mixture was stirred for 16 h and then diluted with EA (100 mL). The mixture was washed with H₂0 (3 x 60 mL) and the organic layer was dried over Na₂S04, filtered and concentrated to dryness to afford 3-ethoxy-2-methyl-4-nitro- pyridine (76 mg) as a colorless oil.

MS m/z (+ESI): 183.1 [M+H]⁺.

‘H-NMR (400 MHz, CDCI₃) d ppm: 8.43 (d, J= 5.2 Hz, 1H), 7.49 (d, J= 5.2 Hz, 1H), 4.12 (q, J= 12 Hz, 2H), 2.68 (s, 3H), 1.47 (t, J= 12 Hz, 3H).

Step 1-b: Preparation of 3-ethoxy-2-methyl-pyridin-4-amine:

A solution of 3-ethoxy-2-methyl-4-nitro-pyridine (84 mg; 0.42 mmol) in MeOH (3 mL) containing palladium, 10 % on charcoal (70 mg; 0.07 mmol) was stirred under hydrogen atmosphere (1 bar) for 2 h. Insolubles were removed by filtration and the filtrate was concentrated to dryness to afford 3-ethoxy-2- methyl-pyridin-4-amine (58 mg) as a colorless oil.

MS m/z (+ESI): 153.1 [M+H]⁺.

Step 1-c: Preparation of phenyl ,V-(3-cthoxy-2-mcthyl-4-nvndyl)carbamatc:

To a solution of 3-ethoxy-2-methyl-pyridin-4-amine (64 mg; 0.38 mmol) in DCM (4mL) were added TEA (0.27 mL; 1.91 mmol) and phenyl chloroformate (0.1 mL 0.80 mmol). The reaction mixture was stirred for 16 h and then concentrated to dryness to afford crude phenyl ,V-(3-cthoxy-2-mcthyl-4- pyridyl)carbamate (100 mg; 80 % purity) as a yellow solid, which was used in the next step without further purification.

MS m/z (+ESI): 273.2 [M+H]⁺.

Step 2: Preparation of 4-r(4-cyano-2.6-difluoro-o hcnyl )mcthylcnc1-.V-(3 -cthoxy-2- mcthyl-4- pyridyl)piperidine-l -carboxamide, trifluoroacetic acid:

The title compound was prepared as a white solid following scheme 1 and in analogy to Example 3 (step 3) using crude phenyl /V-(3-cthoxy-2-mcthyl-4-pyridyl)carbamatc and 3,5-difluoro-4-(4- piperidylidenemethyl)benzonitrile, hydrochloride as starting materials and after purification by preparative HPLC.

Preparation of Example 5: V-(3-benzyloxy-2-methyl-4-pyridyl)-4-|(4-cvano-2,6-difluoro- phenyl) methylenel piperidine- 1 -carboxamide, trifluoroacetic acid:

Preparation of 4- r(4-cvano-2.6-difluoro-phenyl)methylene1 -N-(3 -hvdroxy-2-methyl-4-pyridyl)piperidine- 1 -carboxamide: Boron tribromide (393 mg; 1.57 mmol) was added slowly into a solution of 4-[(4-cyano-2,6-difluoro- phenyl)methylene]-iV-(3-ethoxy-2-methyl-4-pyridyl)piperidine-l-carboxamide (220 mg; 0.52 mmol) in dry DCM (20 mL) at -78 °C. The mixture was stirred for 1 h at 0° C and then for 24 h at rt. The reaction was deactivated by the addition of saturated NaHCOs in H₂0. The aqueous layer was separated and was extracted with a mixture 2-propanol : DCM (80 mL; 3:1 ; v/v). The organic layers were combined, dried over Na₂S04, filtered and concentrated to afford 4-[(4-cyano-2,6-difluoro-phenyl)methylene]-iV-(3- hydroxy-2-methyl-4-pyridyl)piperidine-l -carboxamide (170 mg) as a light yellow solid.

MS m/z (+ESI): 385.5 [M+H]⁺.

Preparation of iV-(3-benzyloxy-2-methyl-4-t)yridyl)-4-r(4-cvano-2.6-difluoro- phenyl)methylene1piperidine-l -carboxamide, trifluoroacetic acid:

To a solution of 4-[(4-cyano-2,6-difluoro-phenyl)methylene]-iV-(3-hydroxy-2-methyl-4- pyridyl)piperidine-l -carboxamide (130 mg; 0.30 mmol) in dry THF (10 mL) were added benzyl bromide (0.04 mL; 0.30 mmol), tetrabutylammonium bromide (21 mg; 0.06 mmol) and potassium hydroxide (26 mg; 0.40 mmol). The solution was stirred for 24 h and then concentrated to dryness. The residue was purified by preparative HPLC to afford V-(3-benzyloxy-2-methyl-4-pyridyl)-4-[(4-cyano-2,6-difluoro- phenyl)methylene]piperidine-l -carboxamide, trifluoroacetic acid (12 mg) as a white solid.

Preparation of Example 6: 4-|(4-cvano-2,6-difluoro-phenyl)methylenel- Y-(2-ethyl-3-methyl-4- pyridyl)piperidine-l -carboxamide, trifluoroacetic acid:

Preparation of 4-r(4-cvano-2.6-difluoro-phenyl)methylene1-iY-(2-ethyl-3-methyl-4-pyridyl)piperidine-l- carboxamide. trifluoroacetic acid:

To a solution of 2-ethyl-3-methyl-pyridin-4-amine (170 mg; 0.97 mmol) in THF (10 mL) was added TEA (0.41 mL; 2.92 mmol) and BTC (145 mg; 0.49 mmol). The mixture was stirred for 6 h and then treated with a solution 3,5-difluoro-4-(4-piperidylidenemethyl)benzonitrile, trifluoroacetic acid (343 mg; 0.97 mmol) and TEA (0.41 mL; 2.92 mmol) in THF (3 mL). After stirring for 18 h, the mixture was concentrated to dryness and the residue was purified by preparative HPLC to afford 4-[(4-cyano-2,6- difluoro-phcnyl)mcthylcnc]-/V-(2-cthyl-3-mcthyl-4-pyridyl)pipcridinc- 1 -carboxamide, trifluoroacetic acid (150 mg) as a white solid.

Biological Examples

Cell culture

The cervical tumor cell line HeLa (ATCC, CCL-2) was cultivated in DMEM medium (Invitrogen cat. no.11971, 4.5 g/L high glucose) containing 10% fetal calf serum (Sigma cat. no. F9665) and 1%

Penicillin/Streptomycin (Sigma cat. no. P0781) at 37 °C in 5% C0₂. HeLa galactose cells (i.e. HeLa cells that grow in high concentrations of galactose) were generated from HeLa glucose cells (i.e. HeLa cells that grow in high concentrations of glucose) by gradually changing the amount of glucose in the media to zero glucose in the presence of galactose as a sugar source (50% galactose /50% glucose media for one week, then 75% galactose /25% glucose media for one week, to 100% galactose media in the third week). Galactose media (Invitrogen cat. no. 11966) was supplemented with 10 mM galactose (Sigma cat. no. G5388).

Cell growth and proliferation assay of HeLa Galactose and Glucose Cells

HeLa galactose cells and HeLa glucose cells were seeded in 96 well plates (TPP, cat.no 92696) at 2000 and 1500 cells/well, respectively, in 100 pL of complete medium. After overnight incubation the cells were incubated for 72 hours in complete medium containing 0.001% DMSO or compounds (final concentration of DMSO 0.001%). After the medium was removed, cells were fixed and stained by adding 50 pL crystal violet staining (0.2 % crystal violet (Sigma- Aldrich cat. no. C0775) in 50% methanol) per well. The plates were incubated for 1 hour at room temperature. Subsequently the stain was decanted and plates were washed 4 times with de-mineralized water. Plates were air-dried for 2 hours. The stain was dissolved by adding 100 pL buffer (0.1 M Tris pH 7.5, 0.2% SDS, 20% ethanol) per well and shaking the plates. Absorbance at 590 nm was measured using a SpectraMax® 250 plate reader (Molecular Devices). Antiproliferative / growth inhibition IC50s were calculated from concentration-response curves using GraphPad Prism software.

Oxygen consumption assay

Oxygen consumption is one of the most informative and direct measures of mitochondrial function and can be measured, for example, by using the MitoXpress® assay (Luxcel MX-2001, Luxcel Biosciences). The MitoXpress® probe is one of a family of phosphorescent oxygen sensitive probes. The assay exploits the ability of oxygen to quench the excited state of the MitoXpress® probe. As the test material respires (i.e. the cells), oxygen is depleted in the surrounding solution/environment, which increases the probe phosphorescence signal. Changes in oxygen consumption reflecting changes in mitochondrial activity are seen as changes in the MitoXpress® probe signal over time.

Cells were seeded in 96 well black plates with transparent bottoms (Greiner Bio-One cat. no. 655090) at a density of 50Ό00 cells/well in a final volume of 100 pL. After 24 hours the incubation media was removed and 150 pL of fresh media containing inhibitors at different concentrations was added to each well. Then, 10 pL of MitoXpress® and 150 pL mineral oil were added per well. Reading from the top of the plate, kinetic analysis was performed at 37 °C for 5 hours using a Synergy 4 plate reader (BioTek) and Time-resolved Fluoresence (TRF) wavelengths of 380/11 nm excitation and 650/20 nm emission or 665/40 emission (30 microsecond delay time, 100 microsecond integration time, gain or sensitivity settings set at either medium or high). IC50s were calculated as the concentration that inhibits 50% of the phosphorescent oxygen sensitive probe signal (MitoXpress®) as compared to untreated cells. Galactose cells are highly dependent on OXPHOS and more sensitive to mitochondrial inhibitors than glucose cells (Gohil V.M. et al., Nat. Biotechnol., vol. 28, no. 3, pages 249-255, 2010). For example, a differential sensitivity in HeLa glucose versus HeLa galactose cell growth is exhibited by Antimycin A (Sigma- Aldrich cat. no. A8674), an inhibitor of complex III of the electron transport chain of the mitochondria (Figure la), but not by a cytotoxic compound such as Paclitaxel (CAS 33069-62-4) (Figure lc). Compounds of the invention also exhibit differential sensitivity in HeLa glucose versus HeLa galactose cell growth assays as shown in Figure lb, for example Example 6. As such HeLa galactose cells can be used to screen for mitochondrial inhibitors. Moreover, compounds with activity in HeLa galactose cells can be confirmed as true mitochondrial inhibitors by testing Oxygen consumption inhibition as shown in Table 2. Biological data are given below in Table 2.

Table 2:

Claims

1. A compound of formula I or pharmaceutically acceptable salt thereof

wherein

Bl, B2, B3 and B4 represent independently C(R3);

R4a represents methyl or ethyl;

R4b represents halogen, Cl-C4alkyl, Cl-C4alkoxy or -0-Cl-C4alkylene-Cycle-Q;

R5 represents hydrogen or methyl;

R6a represents hydrogen or methyl;

R6b represents hydrogen or methyl;

q is 0, 1 or 2.

2. A compound according to claim 1 or pharmaceutically acceptable salt thereof, wherein n is 1.

3. A compound according to claim 1 or claim 2 or pharmaceutically acceptable salt thereof, wherein X represents -CH=.

4. A compound according to any one of claims 1 to 3 or pharmaceutically acceptable salt thereof, wherein the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia, group B- Ib or group B-lc, wherein R3a is as defined for R3 but is other than hydrogen:

5. A compound according to any one of claims 1 to 4 or pharmaceutically acceptable salt thereof, wherein R2 represents halogen or cyano.

6. A compound according to any one of claims 1 to 5 or pharmaceutically acceptable salt thereof, wherein R3 represents independently at each occurrence hydrogen or halogen.

7. A compound according to any one of claims 1 to 6 or pharmaceutically acceptable salt thereof, wherein R4b represents halogen, methyl, ethyl, methoxy, ethoxy, or -0-Cl-C2alkylene-Cycle-Q.

8. A compound according to any one of claims 1 to 7 or pharmaceutically acceptable salt thereof, wherein q is 0.

9. A compound according to claim 1, wherein the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia, group B-Ib or group B-lc:

X represents -CH₂-, -CH= or -C(=0)-;

R3a represents independently at each occurrence halogen, cyano or methyl;

R4a represents methyl or ethyl;

R4b represents halogen, Cl-C4alkyl, Cl-C4alkoxy or -0-Cl-C4alkylene-Cycle-Q;

R6a represents hydrogen or methyl;

R6b represents hydrogen or methyl;

q is 0.

10. A compound according to claim 1, wherein the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia, group B-Ib or group B-lc:

(B-Ia) (B-Ib) (B-Ic);

X represents -CH₂-, -CH= or -C(=0)-;

R2 represents halogen or cyano;

R3a represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -0-CH₂-Cycle-Q;

q is 0.

11. A compound according to claim 1, wherein the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia or group B-Ib:

(B-Ia) (B-Ib);

X represents -CH=;

R2 represents halogen or cyano;

R3a represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -0-CH₂-phenyl;

n is 1 ; and

q is 0.

12. A compound according to claim 1, wherein the ring comprising Bl, B2, B3 and B4 as ring members is represented by group B-Ia or group B-Ib:

(B-Ia) (B-Ib);

X represents -CH=;

R2 represents halogen or cyano;

R3a represents independently at each occurrence halogen;

R4a represents methyl or ethyl;

R4b represents halogen, methyl, ethyl, methoxy, ethoxy;

n is 1 ; and

q is 0.

13. A compound of formula I or pharmaceutically acceptable salt thereof for use in the treatment of proliferative diseases, in a subject selected from a mammal, wherein the compound of formula I is as defined in any one of claims 1 to 12.

14. A compound of formula I or pharmaceutically acceptable salt thereof, for use according to claim 13, wherein the disease is cancer.

15. A pharmaceutical composition comprising a compound of formula I as defined in any one of claims 1 to 12, or pharmaceutically acceptable salt thereof and preferably a pharmaceutically acceptable excipient.