TW201927767A - Mitochondrial inhibitors for the treatment of proliferation disorders - Google Patents

Abstract

The invention provides compounds of formula I or pharmaceutically acceptable salt thereof Wherein B1, B2, B3 and B4 represent independently C(R3); X represents -CH(R5)-, -C(R5)= or -(C=O)-, and wherein when X represents -CH(R5)- or -(C=O)- 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, C1-C4alkyl, C1-C4alkoxy or -O-C1-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 proliferative disorders

The present invention relates to mitochondrial inhibitors and their use in treating proliferative disorders, especially cancer.

The powerhouse of mitochondrial cells because they produce most of the adenosine triphosphate (ATP), which is used as a chemical energy source (Campbell, NA, Williamson B., Heyden, RJBiology: Exploring Life [Biology: Exploring Life] 2006 edition, publisher: Pearson Prentice Hall, 2006). In addition, mitochondria are involved in other functions, such as cell signal transmission, differentiation, and death, and maintaining control of the cell cycle and cell growth (McBride HM et al., Curr. Biol. [Contemporary Biology], Vol. 16, No. 14, R551-60, 2006).

According to a phenomenon called aerobic glycolysis, cancer cells can reprogram their metabolism in a manner that facilitates glycolysis, regardless of the presence or absence of oxygen. This so-called "Warburg phenotype" involves high glucose uptake and high glycolytic activity (Warburg O., Science [Science], Vol. 123, No. 3191, pp. 309-314, 1956). However, cancer cells also rely on mitochondria to produce ATP through oxidative phosphorylation (OXPHOS) (Marchetti P. et al., International Journal of Cell Biology, Vol. 2015, pp. 1-17, 2015 And Solaini G. Et al., Biochim. Biophys. Acta [Journal of Biochemistry and Biophysics], Vol. 2, pp. 314-323, 2010).

Due to the metabolic characteristics of cancer cells, mitochondrial metabolism is now considered a potential target for anticancer agents. In fact, human cancer is associated with mitochondrial dysregulation, which promotes cancer cell survival, tumor progression and metastasis, and resistance to current anticancer drugs (Marchetti P. et al., International Journal of Cell Biology [International Journal of Cell Biology] ], Vol. 2015, pp. 1-17, 2015; Boland ML et al., Frontiers in Oncology [Vol. 3], 292, pp. 1-28, 2013, and Solaini G. et al., Biochim. Biophys. Acta [Journal of Biochemistry and Biophysics], Volume 1797, Pages 1171-1177, 2010). Metabolic reprogramming in cancer cells results in the maintenance of energy (ATP) production even under stressful conditions, thereby facilitating tumor growth and production of ATP by, for example, mitochondria using alternative carbon sources such as glutamine and fatty acids. Survival (Solaini G. et al., Biochim. Biophys. Acta [Journal of Biochemistry and Biophysics], vol. 1797, pp. 1171-1177, 2010). In fact, due to the separation of glycolytic flux and mitochondria, mitochondrial glutamine breakdown is preferentially used to generate ATP and thus contribute to the survival of cancer cells (DeBerardinis RJ et al., PNAS [Journal of the National Academy of Sciences], Volume 104, Issue 49, Pages 19345-19350, 2007), which is relevant for the development of certain tumor types (Strohecker AM et al. Cancer Discovery, Volume 3, Issue 11, 1272-1285 (2013) and anchor-independent growth (Weinberg F. et al., PNAS [Proceedings of the National Academy of Sciences], Volume 107, Issue 19, pages 8788-8793, 2010) are essential.

In addition, mitochondrial activity is also involved in the development of drug resistance. For example, chemotherapeutic drugs and targeted drugs (such as BRAF inhibitors) have been shown to induce metabolic metastases in cancer, leading to mitochondrial dependence (addiction), which is characterized by, for example, upregulating OXPHOS and mitochondrial biogenesis in viable cells (Marchetti P. Et al., International Journal of Cell Biology Journal of Cell Biology], Vol. 2015, pp. 1-17, 2015; and Vellinga T.T. et al., Clinical Cancer Research [Clinical Cancer Research], vol. 21, No. 12, pp. 2870-2879, 2015). In the case of BRAF inhibitors, acquired resistance is related to the maintenance of the OXPHOS phenotype regardless of the underlying resistance mechanism (Corazao-Rozas P. et al., Oncotarget [Volume Target], Volume 4, Issue 11 , Pp. 1986-1998, 2013), which indicates a potential metabolic platform that may be utilized at the therapeutic level. Taken together, the accumulated data provides strong evidence supporting mitochondria's involvement in cancer development and a strong theoretical basis for developing mitochondrial targeting agents against cancer.

Based on the increasing interest in using mitochondria as targets for cancer treatment, in recent years, some mitochondrial targeted research drugs have entered clinical development. For example, the antidiabetic drug metformin of OXPHOS is inhibited by inhibiting complex I of the mitochondrial respiratory chain (El-Mir et al., J. Biol. Chem. [Journal of Biochemistry], 275, pp. 223-228, 2000 And Wheaton WW et al., ELife Vol. 3, 2014) are currently being studied in some clinical trials for cancer patients (Chae YK et al., Oncotarget [Mormonic Target], March 19, 2016). The trials were encouraged by preclinical data in tumor models (Chae YK et al., Oncotarget, March 19, 2016) and the following observations: Patients with type 2 diabetes who have been treated with metformin have developed various types of cancer. Reduced risk (Quinn BJ, Kitagawa H., Memmott RM, et al. Trends Endocrinol. Metab. [Endocrinology and Metabolic Trends], Vol. 24, pp. 469-80, 2000, and Chae YK et al., Oncotarget [tumor target] , March 19, 2016). Subsequently, increased interest in this treatment has led to research on other complex 1 inhibitor classes (WO 2014/031928, WO 2014/031936, Ziegelbauer et al., Cancer Medicine [Cancer Medicine], Vol. 2, No. 5 Issue, pp. 611-624, 2013 and WO 2010/054763). Therefore, targeting mitochondrial metabolism is of great significance for the development of new therapeutic methods for cancer treatment.

Therefore, in a first aspect, the invention provides a compound of formula I and a pharmaceutically acceptable salt thereof Where B1, B2, B3, and B4 independently represent C (R3); X represents -CH (R5)-, -C (R5) = or -C (= O)-, and where X represents -CH (R5) -Or -C (= O)-, the dashed line represents a single bond, and when X represents -C (R5) =, the dashed line represents a double bond; R1 independently represents methyl, ethyl, and methoxy at each occurrence Or ethoxy; R2 represents halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, -N (R6a) (R6b) or -methylene-N (R6a) (R6b) ; R3 independently represents hydrogen, halogen, cyano, or methyl on each occurrence; R4a represents methyl or ethyl; R4b represents halogen, C1-C4 alkyl, C1-C4 alkoxy, or -O-C1- C4 alkylene-ring-Q; R5 represents hydrogen or methyl; R6a represents hydrogen or methyl; R6b represents hydrogen or methyl; ring-Q independently represents each time it is replaced by 1 to 3 R7 as needed R7 at each occurrence independently represents cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1 or 2; and q is 0, 1 or 2.

In another aspect, the invention provides a compound of formula I and a pharmaceutically acceptable salt thereof for use in the treatment of a proliferative disease, particularly cancer, in a subject selected from mammals, particularly humans.

In another aspect, the invention provides a compound of formula I and a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the treatment of a proliferative disease, particularly cancer, in a subject selected from mammals, particularly humans. In the use.

In another aspect, the invention provides a method of treating a proliferative disease, particularly cancer, in a subject selected from a mammal, particularly a human, the method comprising administering to the subject a compound of formula I or Its pharmaceutically acceptable salt.

In another aspect, the invention provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

Each alkyl moiety, either alone or as part of a larger group such as an alkoxy group, is straight or branched. Examples include methyl, ethyl, n-propyl, prop-2-yl, n-butyl, but-2-yl, 2-methyl-prop-1-yl or 2-methyl-prop-2-yl.

Each alkylene moiety is straight or branched and is, for example, -CH _2- , -CH ₂ -CH _2- , -CH (CH ₃ )-, -CH ₂ -CH ₂ -CH ₂ -,- CH (CH ₃ ) -CH ₂ -or -CH (CH ₂ CH ₃ )-.

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

Halogen is fluorine, chlorine, bromine or iodine.

When a group is said to be optionally substituted, it may be substituted or unsubstituted, such as being substituted with 1-3 substituents as needed.

Certain compounds of formula I may contain one or two or more chiral centers, and such compounds may be provided as pure mirror isomers or pure non-image isomers, What proportion of the mixture is provided. For example, when X represents -CH (R5)-and n is 2, or n is 1 and at least one R1 is different from H, H on the carbon atom connected to X by a dashed line may be in a vertical configuration or a flat configuration. And the present invention includes both isomers in any ratio. The compounds of the invention also include all cis / trans isomers (e.g., where the dashed lines are double bonds) and mixtures thereof in any proportion. The compounds of the invention also include all tautomeric forms of the compounds of formula I.

Compounds of formula I may also be solvated, especially hydrated, and this is also included in compounds of formula I. Solvation and hydration can occur during preparation.

References to compounds of the invention include pharmaceutically acceptable salts of the compounds. Such salts can also exist as hydrates and solvates. Examples of pharmacologically acceptable salts of compounds of formula (I) are physiologically acceptable salts of inorganic acids (e.g. hydrochloric acid, sulfuric acid and phosphoric acid), or organic acids (e.g. methanesulfonic acid, p-toluenesulfonic acid, lactic acid) , Acetic acid, trifluoroacetic acid, citric acid, succinic acid, fumaric acid, maleic acid and salicylic acid). Other examples of pharmacologically acceptable salts of compounds of formula (I) are alkali metal and alkaline earth metal salts (e.g. sodium, potassium, lithium, calcium or magnesium, ammonium) or salts of organic bases (E.g., methylamine salt, dimethylamine salt, triethylamine salt, piperidine salt, ethylenediamine salt, lysine salt, choline hydroxide salt, meglumine salt, Phosphonium or spermine).

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

A ring containing B1, B2, B3, and B4 as ring members can be represented by the group BI

Preferably, no more than two R3 substituents are not hydrogen.

An example of the structure of a ring containing B1, B2, B3, and B4 as ring members is represented by the group B-Ia, the group B-Ib, and the group B-Ic, where R3a is as defined for R3 but is not hydrogen:

Examples of rings containing B1, B2, B3, and B4 as ring members include the following groups:

Specific examples of X include -CH =, -CH _2- , and -C (= O)-.

Specific examples of R2 include chlorine, fluorine and cyano.

Specific examples of R3 include fluorine and chlorine.

R4b preferably represents halogen, methyl, ethyl, methoxy, ethoxy or -O-CH ₂ -ring-Q. R4b Specific examples include methyl, methoxy, ethoxy, chloro and -O-CH ₂ - phenyl.

A specific example of ring-Q is phenyl.

q is preferably 0.

n is preferably 1.

For the avoidance of doubt, R1 is not attached to a carbon atom bonded to X.

Any embodiment related to the chemical structure of the compounds of the present invention may be combined with any other embodiment where possible, including any example combination with the definitions of the substituents given above.

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

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

In another embodiment, X represents -C (= O)-.

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), a ring including B1, B2, B3, and B4 as a ring member is represented by a group B-Ia, a group B-Ib, or a group B-Ic: X represents -CH _2- , -CH = or -C (O)-; R2 represents halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, -N (R6a) (R6b) or -Methylene-N (R6a) (R6b); R3a independently represents halogen, cyano or methyl at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, C1-C4 alkyl, C1- C4 alkoxy or -O-C1-C4 alkylidene-cyclo-Q; R6a represents hydrogen or methyl; R6b represents hydrogen or methyl; ring-Q independently represents each time it appears to be 1 to 3 as needed R7 substituted phenyl; R7 at each occurrence independently represents cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1; and q is 0.

In another embodiment (Embodiment 2), a ring including B1, B2, B3, and B4 as a ring member is represented by a group B-Ia, a group B-Ib, or a group B-Ic: X represents -CH _2- , -CH = or -C (O)-; R2 represents halogen or cyano; R3a independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen or methyl , Ethyl, methoxy, ethoxy, or -O-CH ₂ -ring-Q; each ring-Q independently represents a phenyl group optionally substituted with 1 to 3 R7 at each occurrence; R7 at each occurrence When present, it independently represents cyano, methyl, halomethyl, methoxy, or halomethoxy; n is 1; and q is 0.

In another embodiment (Embodiment 3), a ring including B1, B2, B3, and B4 as a ring member is represented by a group B-Ia or a group B-Ib: X represents -CH =; R2 represents halogen or cyano; R3a independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -O-CH ₂ -phenyl; n is 1; and q is 0.

In another embodiment (Embodiment 4), a ring including B1, B2, B3, and B4 as a ring member is represented by a group B-Ia or a group B-Ib: X represents -CH =; R2 represents halogen or cyano; R3 independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, methyl, ethyl, methoxy, and ethoxy; n is 1; and q is 0.

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

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

The following compounds represent further embodiments of the present invention and their pharmaceutically acceptable salts: 4-[(4-chloro-2-fluoro-phenyl) methylene] -N- (2,3-dimethyl- 4-pyridyl) piperidine-1-carboxamide; 4-[(4-chloro-2-fluoro-phenyl) methylene] -N- (3-methoxy-2-methyl-4- Pyridyl) piperidine-1-carboxamide; N- (3-chloro-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) Methyl] piperidine-1-carboxamide; 4-[(4-cyano-2,6-difluoro-phenyl) methylene] -N- (3-ethoxy-2-methyl- 4-pyridyl) piperidine-1-carboxamide; N- (3-benzyloxy-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro- Phenyl) methylene] piperidine-1-carboxamide; 4-[(4-cyano-2,6-difluoro-phenyl) methylene] -N- (2-ethyl-3- Methyl-4-pyridyl) piperidine-1-carboxamide.

The invention also relates to pharmaceutical compositions comprising, for example, a therapeutically effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof, which compositions are particularly useful for treating proliferative disorders, especially cancer, As described herein. The composition may be formulated for parenteral administration, such as nasal, oral, rectal, lung, vaginal, sublingual, topical, transdermal, eye, ear, or especially for oral administration, such as in the form of an oral solid dosage form, E.g. granules, pills, powders, tablets, coated tablets (e.g. film-coated tablets or sugar-coated tablets), foamed tablets, hard and soft capsules or HPMC capsules (coated when applicable) , Orally disintegrating tablets, solutions, emulsions (such as lipid emulsions) or suspensions, or for parenteral administration, such as intravenous, intramuscular or subcutaneous, intrathecal, intradermal or epidural administration to mammals, especially Human, for example, by solution, In the form of a lipid emulsion or suspension containing microparticles or nanoparticle. Such compositions may contain the active ingredients alone, or preferably, together with a pharmaceutically acceptable carrier.

The compound of formula I or a pharmaceutically acceptable salt thereof can be processed with pharmaceutically inert inorganic or organic excipients for the production of oral solid dosage forms such as granules, pills, powders, tablets, coated tablets (e.g. Film-coated tablets or sugar-coated tablets), foamed tablets and hard capsules or HPMC capsules or orally disintegrating tablets. Fillers such as lactose, cellulose, mannitol, sorbitol, calcium phosphate, starch (e.g. corn starch) or derivatives thereof, binders such as cellulose, starch, polyvinylpyrrolidone or derivatives thereof, to aid flow Agents such as talc, stearic acid or salts thereof, and flowable agents such as fumed silica can be used as such excipients, for example for the preparation and manufacture of oral solid dosage forms such as granules, pills, powders, tablets, films Coated or sugar-coated tablets, foamed tablets, hard capsules or HPMC capsules or orally disintegrating tablets. Suitable excipients for soft capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols and the like.

Suitable excipients for preparing solutions (e.g. oral solutions), lipid emulsions or suspensions are, for example, water, alcohols, polyols, sucrose, invert sugar, glucose and the like.

Suitable excipients for parenteral preparations (such as injectable solutions) are, for example, water, alcohols, polyols, glycerol, vegetable oils, lecithin, surfactants and the like.

In addition, the pharmaceutical preparations may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts for changing osmotic pressure, buffers, masking agents, or antioxidants. Pharmaceutical preparations may also contain other therapeutically valuable substances.

Dosages can vary within wide limits and, of course, individual requirements are met in each particular case. Generally, a daily dose of about 1 mg to 1000 mg of a compound of general formula I (for example, 10 mg to 1000 mg of a compound of general formula I / person) should be appropriate, although the above upper limit (and similarly, the lower limit) can be exceeded if necessary. ).

The compound of formula I or a pharmaceutically acceptable salt thereof may also be used in combination with one or more other pharmaceutically active compounds, which preferably use different modes of action to effectively combat the same disease, or reduce or prevent Possible adverse side effects of compounds of formula I. Combination partners can be administered in this treatment, for example, by incorporating them into a single pharmaceutical formulation and administered simultaneously, or by administering two or more different dosage forms (each dosage form containing one or more than one combination partner) and Continuous application.

The compound of formula I or a pharmaceutically acceptable salt thereof according to the present invention as described above is particularly useful for treating proliferative disorders and / or diseases, such as cancer, in particular epithelial cancer, sarcoma, leukemia, myeloma and lymphoma, and Cancer of the brain and spinal cord, for example when administered in a therapeutically effective amount. Examples of such proliferative disorders and diseases include, but are not limited to, epithelial tumors, squamous cell tumors, basal cell tumors, transitional cell papillomas and carcinomas, adenomas and adenocarcinomas, appendage and skin attachment tumors, mucoepidermoid Tumors, cystic tumors, mucinous and serous tumors, catheters, lobular and medullary tumors, acinar cell tumors, complex epithelial tumors, specialized adenocarcinomas, paraganglioma and hemangiomas, nevi and melanoma , Soft tissue tumors and sarcomas, fibroid tumors, myxoma tumors, lipoma tumors, fibroid tumors, complex mixed and stromal tumors, fibroepithelial tumors, synovial tumors, mesothelioma, germ cell tumors , Trophoblastic tumors, mesangial tumors, hemangiomas, lymphangiomas, bone and chondroma tumors, giant cell tumors, hybrid bone tumors, odontogenic tumors, gliomas, neuroepithelial tumors and neuroendocrine tumors , Meningioma, schwannoma, granulosa cell tumor and alveolar soft tissue sarcoma, Hodgkin and non-Hodgkin lymphoma, B-cell lymphoma, T-cell lymphoma, hair cell lymphoma Tumors, Burkitts lymphoma, and other lymphoid reticulum tumors, plasmacytomas, mast cell tumors, immunoproliferative disorders, leukemias, heteromyelodysplastic disorders, lymphoproliferative disorders, and myelodysplastic syndromes .

Examples of cancers of affected organs and parts of the body include, but are not limited to, breast, cervix, ovary, 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, bone marrow) Neoplasm or lymphoma), bladder, urinary tract, kidney, skin, thyroid, brain, head, neck, prostate, and testes. Preferably, the cancer is selected from breast cancer, prostate cancer, cervical cancer, ovarian cancer, gastric cancer, colorectal cancer, pancreatic cancer, liver cancer, brain cancer, neuroendocrine cancer, lung cancer, kidney cancer, blood malignant tumor, melanin Tumors and sarcomas.

The term "treatment" as used herein in the context of treating a disease or disorder generally relates to the treatment and therapy of a human or animal (e.g., in veterinary applications) in which some desired therapeutic effect is obtained, for example, inhibiting the progression of the disease or disorder, And includes reducing the rate of progression, stopping the rate of progression, alleviating the symptoms of a disease or disorder, improving the disease or disorder, and curing the disease or disorder. Treatment (ie, prevention) is also included as a precautionary measure. For example, for patients who have not yet developed the disease or disorder but are at risk of developing the disease or disorder, the term "treatment" is encompassed. For example, treatment includes cancer prevention, reducing the incidence of cancer, and alleviating cancer symptoms.

As used herein, the term "therapeutically effective amount" refers to an amount of a compound, or material, composition or dosage form comprising a compound, effective to produce some desired therapeutic effect, with reasonable benefit when administered according to the desired treatment regimen / Risk ratio.

Compounds of formula I can be synthesized by the methods given below, by the methods given in the experimental section below, or by similar methods. The protocols described herein are not intended to present an exhaustive list of methods for preparing compounds of formula (I); rather, other techniques known to the skilled chemist can also be used for compound synthesis.

Those skilled in the art of organic synthesis will understand that the optimal 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 following reaction schemes and / or the order of the reaction steps may be changed to promote the reaction or avoid the formation of unwanted by-products. In addition, the functional groups present at various positions of the molecule must The reagents and reactions are compatible. Such restrictions on substituents compatible with the reaction conditions will be apparent to those skilled in the art, and alternative 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 a compound, and it is assumed that such a protecting group (PG) is present when necessary. Conventional protecting groups can be used according to standard practices well known in the art (see, for example, Greene TW, Wuts PGM, Protective Groups in Organic Synthesis, 5th edition, Publisher: John Wiley & Sons ( John Wiley & Sons), 2014). The protecting group may be removed at any convenient stage in the synthesis using conventional techniques well known in the art, or it may be removed during a subsequent reaction step or work-up.

In the general reaction sequence outlined below, unless otherwise stated, the abbreviations X, n, q, dashed lines, and common groups B1-4, R1, R2, R4a, and R4b are as defined for formula (I). Other abbreviations used herein are clearly defined or as defined in the experimental section.

The necessary starting materials for the synthetic methods as described herein (if not commercially available) can be prepared by procedures described in the scientific literature, or can be purchased from commercial sources using modifications to the methods reported in the scientific literature Preparation of compounds. For general guidance on reaction conditions and reagents, the reader may refer further to March J., Smith M., Advanced Organic Chemistry, 7th edition, publisher: John. Wiley & Sons, 2013.

The compounds according to the invention, their pharmaceutically acceptable salts, solvates and hydrates can be prepared according to the general reaction sequence outlined below, and then, if necessary, then: the substituents can be manipulated to obtain new end products. Such operations may include, but are not limited to, reduction, oxidation, alkylation, tritiation, substitution, coupling, including transition metal-catalyzed coupling and hydrolysis reactions known to those skilled in the art; removal of any protecting groups; formation of pharmaceutically acceptable Salt; or A pharmaceutically acceptable solvate or hydrate is formed.

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

When E2 is a leaving group such as imidazole, phenol, 4-nitrophenol, 2,2,2-trifluoro-ethanol or 1-hydroxypyrrolidin-2,5-dione, more preferably phenol or 4-nitro In the case of phenol, the coupling reaction between the compound of formula (3) and the compound of formula (4) is usually in various organic solvents such as tetrahydrofuran, dichloromethane, 1,2-dichloroethane, ether, ethyl acetate, Methylmethylene, N , N -dimethylformamide and acetonitrile, aqueous solvents and mixtures of these solvents under biphasic conditions (more commonly in N , N -dimethylformamide) This is done in the presence of an inorganic base such as sodium hydride, sodium carbonate or sodium bicarbonate or in the presence of an organic base such as triethylamine, pyridine and the like (more commonly triethylamine). The reactions are typically performed at -20 ° C to 80 ° C (typically at room temperature).

Compound of formula (4) (where E2 is a leaving group such as imidazole (can be activated by methylation before reaction), phenol, 4-nitrophenol, 2,2,2-trifluoro-ethanol or 1-hydroxypyrrole Pyridin-2,5-dione) is typically in the presence of a base such as sodium hydride, triethylamine, pyridine (diluted or pure), 4- (dimethylamino) pyridine in an aprotic solvent such as dichloromethane In chloroform, acetonitrile, tetrahydrofuran, ethyl acetate, the compound of formula (2) and 1,1'-carbonyldiimidazole, phenyl chloroformate, 4-nitrophenyl chloroformate, and chloroformic acid 2,2, respectively, Coupling reaction between 2-trifluoroethyl or N , N' -bissuccinimide carbonate. These reactions are typically performed at -10 ° C to 80 ° C.

Compounds of formula (4) where E2 is chlorine are usually obtained by the compound of formula (2) and phosgene or more commonly a phosgene analog (such as bis (trichloromethyl) carbonate or trichloromethylchloroformic acid). Esters). The reaction is typically in an aprotic and inert solvent such as dichloromethane, chloroform, acetonitrile, tetrahydrofuran, ethyl acetate (more commonly dichloromethane) in a base such as triethylamine, 4- (dimethylamino) It is carried out in the presence of pyridine or N , N -diisopropylethylamine. The reactions are typically carried out at -40 ° C to 50 ° C, usually at 0 ° C. The low stability of such intermediates generally does not allow isolation, and they are usually prepared in situ. The compound of formula (3) is then reacted with a compound of formula (4) to form the corresponding compound of formula (I).

Compounds of formula (2) can be obtained from commercial sources, or prepared according to procedures described in the literature or by procedures known to those skilled in the art.

plan 1

Similarly, as outlined in Scheme 2, a compound of formula (I) may be a compound of formula (5) (where E3 is a leaving group such as chlorine, imidazole, phenol, 4-nitrophenol, 2,2,2-tris Fluoroethanol or 1-hydroxypyrrolidin-2,5-dione, more preferably phenol or 4-nitrophenol) and a compound of formula (2) are prepared by a coupling reaction according to a similar procedure as previously described. Compounds of formula (5) can be prepared from compounds of formula (3) by a coupling reaction according to a similar procedure as described above.

Scenario 2

Alternatively, the compound of formula (I) can be generated from a compound of formula (6) and a compound of formula (7) via a transition metal-catalyzed coupling reaction (Scheme 3), where E4 is a halogen or a leaving group such as trifluoromethanesulfonate Sour. Typical catalysts include palladium (II) acetate, tris (dibenzylideneacetone) dipalladium (0), and the like. The reaction is typically carried out at a temperature of 0 ° C to 150 ° C, more commonly 100 ° C to 120 ° C. Generally, the reaction is performed on ligands such as di-tertiary butyl- [3,6-dimethoxy-2- (2,4,6-triisopropylphenyl) phenyl] phosphine, tertiary butyl -[2,3,4,5-tetramethyl-6- (2,4,6-triisopropylphenyl) phenyl] phosphine, 2- (dicyclohexylphosphino) biphenyl and the like and a base Such as tertiary sodium butoxide, cesium carbonate, potassium carbonate, and more commonly in the presence of cesium carbonate in various inert solvents such as toluene, tetrahydrofuran, , 1,2-dichloroethane, N , N -dimethylformamide, dimethylmethylene, water and acetonitrile or a mixture of solvents, more commonly in In progress.

Compounds of formula (6) can be obtained from compounds of formula (3) according to the procedures described in the literature or by procedures known to those skilled in the art. For example, a compound of formula (6) can be obtained by using a compound of formula (3) and trimethylsilyl isocyanate in an aprotic solvent such as acetonitrile, ethyl acetate, and chloroform. It is prepared by reacting triethylamine, 4- (dimethylamino) pyridine, N , N -diisopropylethylamine, and the like. The reaction can be carried out at a temperature of 0 ° C to 50 ° C, usually at room temperature.

Compounds of formula (7) can be obtained from commercial sources, or prepared according to procedures described in the literature or by procedures known to those skilled in the art.

Option 3

Compounds of formula (3) are usually obtained from commercial sources or prepared according to procedures described in the literature or by procedures known to those skilled in the art. For example, when X-C (R5) = (double bond Z , E, or Z / E ) (wherein formula R5 is hydrogen or methyl substituent), the compound of formula (3) can be represented by The compound of the formula (11-a) (wherein the PG-based amine protecting group) is prepared by deprotection of the amine protecting group. Amine protecting groups can be removed under standard conditions. For example, benzyl carbamate is deprotected by hydrogenolysis over a noble metal catalyst (such as palladium or palladium hydroxide on activated carbon) or other suitable catalyst such as Raleigh nickel. Under acidic conditions such as hydrochloric acid, in organic solvents such as methanol, The Boc group is removed either in ethyl acetate or in trichloroacetic acid, pure or diluted in a solvent such as dichloromethane. In palladium salts such as palladium acetate or tetrakis (triphenylphosphine) palladium (0) and allyl cation scavengers such as The Alloc group is removed in a solvent such as tetrahydrofuran, usually at a temperature of 0 ° C to 70 ° C, in the presence of chloroline, pyrrolidine, dimethylketone or tributylstannane. The N -benzyl-protected amine is deprotected by hydrogenolysis over a noble metal catalyst (such as palladium hydroxide on activated carbon) or other suitable catalyst such as Raleigh nickel. Under mild alkaline conditions (such as diluted in N, N -dimethylformamide or acetonitrile Or piperidine). The N -acetamidine-protected amine is deprotected by hydrolysis using an acidic or alkaline aqueous solution, usually at a temperature of 0 ° C to 100 ° C. Further general methods for removing amine protecting groups have been described in Greene TW, Wuts PGM, Protective Groups in Organic Synthesis, 5th edition, Press: John. Willie & Sons, 2014.

Compounds of formula (11-a) are usually obtained from commercial sources or prepared according to procedures described in the literature or by procedures known to those skilled in the art. Generally, a compound of formula (11-a) can be obtained from a compound of formula (10) and a compound of formula (9) respectively via Wittig or Horner-Wadsworth- Emmons), wherein E6 is a phosphonium salt (typically triphenylphosphonium salt) or a phosphonate (typically diethyl phosphonate). The Wittig reaction is a reaction of an aldehyde or a ketone with triphenylphosphonium ylide to obtain an olefin and triphenylphosphine oxide. Wittig reagents are usually prepared from phosphonium salts. To form a Wittig reagent, the phosphonium salt is suspended in a solvent such as diethyl ether or tetrahydrofuran, and a strong base such as n-butyllithium or lithium bis (trimethylsilyl) fluoramide is added.

With simple Yelide, the products are usually mainly Z -isomers, although smaller amounts of E -isomers are usually formed. If the E -isomer is the desired product, it can be modified using Schlosser. Alternatively, the Horner-Wadsworth-Emmons reaction mainly produces E-olefins. The Horner-Wadsworth-Emmons reaction is in the presence of a base such as sodium hydride or lithium bis (trimethylsilyl) fluoride in a solvent such as tetrahydrofuran or N , N -dimethylformamide. The stable phosphonate carboanions are usually condensed with aldehydes or ketones at temperatures between 0 ° C and 80 ° C. In contrast to the osmium ylide used in the Wittig reaction, the phosphonate-stabilized carboanions are more nucleophilic and more basic.

When the E6 is a sulfonium salt, the compound of formula (9) can be obtained, for example, by alkylation of triphenylphosphonium and a compound of formula (8) according to a well-known procedure, wherein E5 is a halogen.

When E6 is diethyl phosphonate, the compound of formula (9) can be obtained by the reaction of triethyl phosphite and compound of formula (8) according to a well-known procedure, in which E5 is a halogen.

Compounds of formula (8) can be obtained from commercial sources or prepared according to procedures described in the literature or by procedures known to those skilled in the art.

Compounds of formula (10) are usually obtained from commercial sources or prepared according to procedures described in the literature or by procedures known to those skilled in the art. An amine protecting group (PG) may be present in the starting material, or by bringing the corresponding free amine with allyl chloroformate, methyl ethyl chloroformate or benzyl chloroformate or with tertiary butyl dicarbonate It is introduced by reaction in the presence of a base such as sodium hydroxide, sodium bicarbonate, triethylamine, 4-dimethylaminopyridine or imidazole. Free amines can also be protected as N -benzyl derivatives by reaction with benzyl bromide or benzyl chloride in the presence of a base such as sodium carbonate or triethylamine. Alternatively, the N -benzyl derivative can be obtained by reductive amination in the presence of benzaldehyde. Free amines can also be protected as N -acetamidine derivatives by reaction with acetamidine chloride or acetic anhydride in the presence of a base such as sodium carbonate or trimethylamine. Further strategies for introducing other amine-protecting groups have been described in Greene TW, Wuts PGM, Protective Groups in Organic Synthesis, 5th edition, publisher: John. Willie & Sons, 2014.

Option 4

Alternatively, the compound of formula (11-a) can be synthesized as outlined in Scheme 5 from a compound of formula (14) and a compound of formula (15) via a cross-coupling reaction (i.e., Suzuki, Stille) , Negishi, etc.), wherein E10 is a halogen or a leaving group such as triflate. For example, when E9 is a boric acid or a borate, a compound of formula (14) may react with a compound of formula (15) via a Suzuki cross-coupling reaction to form a compound of formula (11-a). The Suzuki reaction is a palladium-catalyzed cross-coupling reaction between an organic boric acid or ester and an aryl or vinyl halide or triflate. Typical catalysts include palladium (II) acetate, tetra (triphenylphosphine) palladium (0), tris (dibenzylideneacetone) dipalladium (0), bis (triphenylphosphine) palladium (II) dichloride And [1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (II). This reaction can be performed in various organic solvents (including toluene, tetrahydrofuran, , 1,2-dichloroethane, N , N -dimethylformamide, dimethylmethylene, and acetonitrile), aqueous solution and neutral phase. The reactions are typically carried out under an inert atmosphere and at room temperature to 150 ° C, more commonly 90 ° C to 120 ° C. Additives such as cesium fluoride, potassium fluoride, potassium hydroxide, potassium carbonate, potassium acetate, potassium phosphate or sodium ethoxide often accelerate coupling. Instead of boric acid, potassium trifluoroborate and organoborane or borate can be used. Since there are many components in the Suzuki reaction, such as specific palladium catalysts, ligands, additives, solvents, and temperatures, many schemes have been identified. Those skilled in the art will be able to determine satisfactory solutions without undue experimentation.

Compounds of formula (15) can be obtained from commercial sources, or prepared according to procedures described in the literature or by procedures known to those skilled in the art.

The organoboric acid or ester of formula (14) is usually composed of a diboron reagent (such as bis (pinacitol) diboron or bisboronic acid) and a compound of formula (13) (where E8 is a halogen) on a palladium catalyst such as tris (dibenzylidene) Acetone) dipalladium (0) or chloro (2-dicyclohexylphosphino-2 ′, 4 ′, 6′-triisopropyl-1,1′-biphenyl) [2- (2'-amine -1,1'-biphenyl)] palladium (II) and a ligand such as triphenylphosphine or 2- (dicyclohexylphosphino) -2 ', 4', 6'-triisopropyl- Obtained in the presence of 1,1 biphenyl via Miyaura boride (Ishiyama T. et al., J. Org. Chem. [Journal of Organic Chemistry], vol. 60, pp. 7508-7510, 1995). This reaction can be performed in various organic solvents (including toluene, tetrahydrofuran, , 1,2-dichloroethane, N , N -dimethylformamide, dimethylmethylene, and acetonitrile), aqueous solution and neutral phase. The reactions are typically performed at room temperature to 150 ° C (more commonly at about 100 ° C). The key to the success of the boration reaction is the selection of a suitable base, as the strong activation of the product causes competitive Suzuki coupling to occur. Potassium acetate (Ishiyama et al., J. Org. Chem. [Journal of Organic Chemistry], vol. 60, pages 7508-7510, 1995) and potassium phenolate (Takagi J. et al., J. Am. Chem. Soc. [ The Journal of the American Chemical Society], Vol. 27, No. 27, 8001-8006, 2002) is actually the result of the Miyaura team's screening of different reaction conditions. Other bases such as potassium hydroxide, cesium carbonate, potassium carbonate, potassium phosphate or sodium ethoxide are also often used. For the Suzuki reaction, there are many components in the Miyaura boronization reaction, such as specific palladium catalysts, ligands, additives, solvents, temperatures, and many schemes have been identified. Those skilled in the art will be able to determine satisfactory solutions without undue experimentation.

The vinyl halide of formula (13) for preparing an organic boric acid or ester (14) can be prepared via a Wittig reaction between a compound of formula (10) and a compound of formula (12) according to the procedure described previously, wherein E7 is a triphenylphosphonium salt, and E8 is a halogen.

Option 5

In addition, compounds of formulae (11-a), (3), and (I) in which X is -C (R5) = (double bond Z, E, or Z / E) can be further reduced to produce formulas (11- b), (3) and (I) compounds, wherein X is -CH (R5)-, and formula R5 is hydrogen or methyl substituent. The reduction reaction is usually carried out on a precious metal catalyst such as palladium, palladium hydroxide on activated carbon (Trost BM et al., Chem. Eur. J. [European Journal of Chemistry], Vol. 5, No. 3, pp. 1055-1069. ), Platinum dioxide) or other combination Hydrogenation over a suitable catalyst. This hydrogenation step can be performed at any convenient stage during the synthesis.

When X is -C (O)-, the compound of formula (3) can be obtained from the compound of formula (11-c) by removing the amine protecting group (PG) according to the aforementioned procedure as outlined in Scheme 6.

The compound of the formula (11-c) can be obtained from a compound of the formula (15) (in which the E10-based halogen) is synthesized via a Weinreb ketone using a compound of the formula (16). This reaction is carried out in the presence of a strong base such as n-butyllithium or tertiary butyllithium in an organic solvent such as tetrahydrofuran under anhydrous conditions and usually at a temperature of -78 ° C to 60 ° C, preferably about 0 ° C. .

Compounds of formula (16) can be obtained from commercial sources, or prepared according to procedures described in the literature or by procedures known to those skilled in the art. For example, a compound of formula (16) can be prepared from the corresponding carboxylic acid and N, O -dimethylhydroxylamine via an amidine coupling reaction using methods well known in the art.

Alternatively, a compound of formula (11c) can be obtained from a compound of formula (15) (wherein the E10-based halogen) is reacted with a corresponding fluorenyl halide (for example, a compound of formula (18)) by Grignard . The Grignard reaction is typically performed under anhydrous conditions in an organic solvent such as tetrahydrofuran. The reaction is usually performed at -78 ° C to 60 ° C. Grignard reagents are generally used in the literature by the reaction of aryl halides of formula (15) with magnesium metal (Rogers HR et al., J. Am. Chem. Soc. [American Chemical Society], Vol. 102, No. 1 (Issues, pp. 217-226, 1980) or obtained by a magnesium halide exchange reaction using, for example, isopropylmagnesium chloride.

Option 6

Alternatively, a compound of formula (11-c) can be obtained from a compound of formula (17) and a compound of formula (18) by Friedel-Crafts amination (Scheme 7). The amine-protecting group (PG) is preferably an N -ethenyl group. The Friedel-Quarft tritium system uses a strong Lewis acid catalyst such as ferric chloride or aluminum chloride (more commonly aluminum chloride) to tritium the aromatic ring with tritium chloride. Friedel-Crafts dehydration can also be performed with anhydride. In general, a stoichiometric Lewis acid catalyst is required because the substrate and the product form a complex. The reaction is usually carried out under anhydrous conditions in an inert solvent such as acetonitrile, tetrahydrofuran, dichloromethane, 1,2-dichloroethane or in a pure mixture over a wide temperature range (for example, -20 ° C to 100 ° C). .

Compounds of formulae (17) and (18) can be obtained from commercial sources, or prepared according to procedures described in the literature or by procedures known to those skilled in the art.

Option 7

Whenever required, the substituents R1, R2, R3, R4a and / or R4b may be present as precursors in the starting material and / or may be transformed by additional conventional transformations during the synthetic pathways described herein . Such transformations can be performed at any convenient stage during the synthesis and can include, but are not limited to, the following list of reactions that are well known to those skilled in the art:

-Selective reduction of aryl-nitro groups using iron powder in the presence of an acidic aqueous solution (Bechamp reduction). Nitro groups can also be reduced by 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, in the case of X-C (R5) =, the reduction of R2 as a nitro group to an amine group can be selectively achieved by Becht reduction. Affects double bonds.

-Dealkylation of aromatic ethers in organic solvents such as dichloromethane using boron tribromide (Ilhyong R. et al., J. Am. Chem. Soc. [American Chemical Society Journal], 124 Volume, No. 44, pp. 12946-12947, 2002). This reaction can also be performed using trimethylbromosilane or trimethyliodosilane in an organic solvent such as acetonitrile and at a temperature of 0 ° C to 90 ° C. If necessary, sodium iodide can be used to support the reaction. For example, a compound of formula (I) in which R2 is a methoxy group can be converted into a compound of formula (I) in which R2 is a hydroxy group.

-Amido coupling reaction between carboxylic acid and amine. The reaction of an activating agent such as N, N under ethylcarbodiimide hydrochloride is present, as required - '- dicyclohexyl carbodiimide or N - (3- dimethylaminopropyl) - N' Addition of 1-hydroxybenzotriazole was performed. Other suitable coupling agents may be used, such as O- (7-azabenzotriazol-1-yl) -N , N , N ' , N' -tetramethylurea hexafluorophosphate, 2-ethoxy 1-ethoxycarbonyl-1,2-dihydroquinoline, carbonyldiimidazole or diethylphosphonium cyanide. If necessary, a base (such as triethylamine, N , N -diisopropylethylamine or pyridine) may be added for coupling. The amidine coupling is carried out at a temperature of, for example, -20 ° C to 80 ° C in an inert solvent, preferably an anhydrous aprotic solvent such as dichloromethane, acetonitrile, or N , N -dimethylformamide and chloroform. Alternatively, the carboxylic acid can be activated by conversion to its corresponding fluorenyl chloride or its corresponding activated ester, such as N -hydroxysuccinimide (Singh J. et al., Org. Process Res. Dev. [Research and Progress of Organic Methods], Vol. 6, No. 6, pp. 863-868, 2002) or benzothiazolyl thioester (Ishikawa T. et al., J. Antibiotics [Journal of Antibiotics], Vol. 53 No. 10, pp. 1071-1085, 2000). The resulting activated entity can be reacted, for example, at a temperature of -20 ° C to 80 ° C with an amine reagent in an aprotic solvent such as dichloromethane, chloroform, acetonitrile, N , N -dimethylformamide and tetrahydrofuran. If necessary, a base (such as triethylamine, N , N -diisopropylethylamine, pyridine, sodium hydroxide, sodium carbonate, potassium carbonate) can be added for coupling.

-A reductive amination reaction between an amine and an aldehyde or ketone. Reductive amination reactions that form intermediate imines between amines and aldehydes or ketones allow removal by physical or chemical means (such as distillation of solvent-water azeotrope, or the presence of desiccants such as molecular sieves, magnesium sulfate or sodium sulfate) The formed water is carried out in a solvent system. This solvent is typically toluene, n-hexane, tetrahydrofuran, dichloromethane, N, N -dimethylformamide, N, N -dimethylacetamide, acetonitrile, 1,2-dichloroethane, Or a solvent such as methanol or a mixture of 1,2-dichloroethane. The reaction can be catalyzed by a trace amount of acid, usually acetic acid. The intermediate imine is subsequently or simultaneously used with a suitable reducing agent (for example, sodium borohydride, sodium cyanoborohydride, sodium triethoxylate borohydride, see, for example, Hutchins MK, Comprehensive Organic Synthesis, Organic Press), publishing house: Editors Fleming; Pergamon Press, Volume 8, pages 25-78, 1991) or reduction by hydrogenation on a suitable catalyst such as palladium on activated carbon. This reaction is usually performed at -10 ° C to 110 ° C, preferably 0 ° C to 60 ° C. This reaction can also be performed in one pot. This reaction can also be performed in a protic solvent such as methanol or water in the presence of a pyridine-borane complex (Sato S. et al., Tetrahedron [tetrahedron], vol. 60, pp. 7899-7906, 2004). For example, reductive amination between a compound of formula (I) (where R2 is -CHO) and a compound of formula HN (R6a) (R6b) yields R2 is -CH ₂ -N (R6a) (R6b) and R6a and R6b is a compound of formula (I) as defined in the scope of the patent application.

- Substitution reaction. The substitution reaction can be in the presence of an inorganic base such as sodium hydride, potassium carbonate, cesium carbonate, etc. or an organic base such as triethylamine in various solvents such as acetonitrile, tetrahydrofuran or N , N -dimethylformamide, for example at -20 ° C. To 120 ° C. For example, a substitution reaction between a compound of formula (I) where R2 is -methylene-OH (which needs to be activated before the reaction) and a compound of formula HN (R6a) (R6b) gives R2 is -methylene-N ( R6a) (R6b) and R6a and R6b are compounds of formula (I) as defined in the scope of the patent application.

-Activate the hydroxyl groups before the substitution reaction. Hydroxyl groups can be converted to mesylate, tosylate or trifluoromethanesulfonate by: corresponding alcohols with methanesulfonyl chloride or methanesulfonic anhydride, p-toluenesulfonyl chloride, trifluoromethanesulfonium Chlorine or trifluoromethanesulfonic anhydride is reacted in the presence of a base such as triethylamine and the like in an anhydrous aprotic solvent such as pyridine, acetonitrile, tetrahydrofuran or dichloromethane, for example at a temperature of -30 ° C to 80 ° C.

-Reduction of alkyl esters (typically methyl or ethyl esters) to their corresponding alcohols. The reducing agent such as boron or aluminum hydride, lithium aluminum hydride, lithium borohydride, sodium borohydride is performed in a solvent such as tetrahydrofuran, methanol or ethanol, for example, at a temperature of -20 ° C to 80 ° C. Alternatively, the ester functional group uses an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide, or lithium hydroxide in water or in water with a polar protic or aprotic organic solvent such as two , Or a mixture of tetrahydrofuran or methanol, for example, at a temperature of -10 ° C to 80 ° C to hydrolyze to its corresponding carboxylic acid, or the ester functional group is hydrolyzed using an acidic aqueous solution. The obtained carboxylic acid is further reduced to the corresponding alcohol using a borane derivative such as a borane-tetrahydrofuran complex in a solvent such as tetrahydrofuran, for example, at a temperature of -10 ° C to 80 ° C.

-Oxidation of hydroxyl groups to ketones or aldehydes. The alcohol is converted to its corresponding aldehyde by oxidation under the conditions of Swern, Dess Martin, Sarett or Corey-Kim, or through NaOCl oxidation. Or ketone. Further methods are described in Larock RC, Comprehensive Organic Transformations. A guide to functional Group Preparation, 2nd edition, publishing house: Wiley-VCH (New York, Qi Just, Weinheim, Brisbane, Singapore, Toronto, 1999. Section aldehydes and ketones [sections of aldehydes and ketones], pp. 1235-1236 and 1238-1246. For example, R2-based compound of formula -CH ₂ OH (I) oxide by the use Daisimading can be converted to a reagent of formula R2 -CHO system (I) of the compound. The reaction is typically carried out in an aprotic solvent such as dichloromethane, for example at a temperature of 0 ° C to 50 ° C, and more often at room temperature.

Buchwald-Hartwig amination. Buchwald-Hartwig amination reaction (Surry DS and Buchwald SL, Chem. Sci. [Chemical Science], Vol. 2, pp. 27-50, 2011) are amines and aryl halides or trifluoro Cross-coupling reaction of mesylate with palladium. Typical catalysts include palladium (II) acetate or tris (dibenzylideneacetone) dipalladium chloroform complex. The reaction is typically carried out at a temperature of 0 ° C to 150 ° C. Generally, the reaction is at a ligand such as di-tertiary butyl- [3,6-dimethoxy-2- (2,4,6-triisopropylphenyl) phenyl] phosphine, 2- (di Cyclohexylphosphino) biphenyl etc. and bases such as sodium tert-butoxide, cesium carbonate, potassium carbonate in the presence of various inert solvents such as toluene, tetrahydrofuran, , 1,2-dichloroethane, N , N -dimethylformamide, dimethylmethylene and acetonitrile, neutralized in an aqueous solvent under biphasic conditions. Several versions of the reaction using a complex of copper and nickel instead of palladium have also been developed (Hartwig JF, Angew. Chem. Int. Ed. [Applied Chemistry International Edition], Volume 37, Issue 15, Issue 2046-2067 P. 1998). The reaction can be performed using microwave radiation. For example, R2 is obtained by a reaction between a compound of formula (I) of the R2 halogen (more commonly chlorine) and a compound of the formula HN (R6a) (R6b) via Buchwald-Hartwig amination. Compounds of the formula -I are -N (R6a) (R6b) and R6a and R6b are as defined in the scope of the patent application.

Nitrification of aromatic compounds. The nitration of aromatic compounds is a chemical process that introduces nitro groups into organic compounds. In the case of aromatic compound nitration, this process is an example of electrophilic aromatic substitution. The reaction is typically performed in a mixture of acids, which is usually nitric acid and another strong acid such as sulfuric acid or trifluoroacetic acid. The reaction can be carried out over a wide temperature range, for example from 0 ° C to 100 ° C.

Whenever an optically active form of a compound of the present invention is required, it can be obtained by performing one of the above procedures using pure mirror isomers or non-mirro isomers as starting materials, or resolution using standard procedures as the final product Or a mixture of enantiomers or non-enantiomers of an intermediate. Resolution of mirror image isomers can be achieved by chromatography on chiral stationary phases, such as REGIS® PIRKLE COVALENT (R-R) WHELK-02, 10 μm, 100 Å, 250 x 21.1 mm column. Alternatively, resolution of stereoisomers can be achieved by selective crystallization of a chiral intermediate or chiral product with a chiral acid such as camphorsulfonic acid or a non-mirromeric isomer salt of a chiral base such as phenethylamine Come to get. Alternatively, stereoselective synthetic methods may be used, such as by using a chiral variant of a protecting group, a chiral catalyst, or a chiral reagent in a reaction sequence, where appropriate.

Enzymatic techniques can also be used to prepare optically active compounds and / or intermediates.

The protocols and methods described herein are not intended to present an exhaustive list of methods for preparing compounds of Formula I; rather, other techniques known to the skilled chemist can also be used for 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 to more fully illustrate and disclose the present invention and the state of the art to which it belongs. Each of these references is incorporated herein by reference in its entirety to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

Specific embodiments of the present invention are described in the following examples, which are used to explain the present invention in more detail and should not be construed as limiting the present invention in any way.

Figures

Figure 1 shows the results of cell growth assays (crystal violet) in HeLa galactose and HeLa glucose cells treated with mitochondrial inhibitor antimycin A (Figure 1a) and Example 6 (Figure 1b) or the cytotoxic drug paclitaxel (Figure 1c). .

Examples

Preparation example

All reagents and solvents are usually used as received from commercial suppliers; under a nitrogen atmosphere, the reaction is routinely performed in dry glassware with anhydrous solvents; evaporation is carried out under reduced pressure by rotary evaporation, and by After removing residual solids by filtration, post-treatment routines are performed; all temperatures are given in degrees Celsius (° C) and are approximate; at room temperature (rt, typically in the range of 18 ° C to 25 ° C, unless otherwise noted) ); Unless otherwise stated, column chromatography (by rapid procedure) was used to purify the compounds and performed using Merck Silicone 60 (70-230 mesh ASTM); classic flash chromatography Methods are often replaced by automated systems. This does not change the separation process itself. Those skilled in the art will be able to replace classic flash chromatography with automated flash chromatography and vice versa. Typical automated systems can be used, such as those provided by Büchi or Isico (combiflash); the reaction mixture can usually be separated by preparative HPLC. Those skilled in the art will find suitable conditions for each separation; in some cases, the compounds are separated after purification as the corresponding trifluoroacetate (* 1) or the corresponding formate (* 2); Such compounds are labeled accordingly; reactions requiring higher temperatures are usually performed using conventional heating equipment; unless otherwise noted, microwave devices (e.g., CEM Explorer with 250W power) can also be used; hydrogenation or hydrogenolysis reactions Hydrogen in a balloon or Parr system or other suitable hydrogenation equipment can be used; unless otherwise noted, solution concentration and solid drying under reduced pressure; usually, the reaction process is tracked by TLC, HPLC or LC / MS, And the reaction time is given for the purpose of illustration only; the yield is given for the purpose of illustration only, and is not necessarily the maximum value available; usually, the structure of the final product of the present invention is confirmed by NMR and mass spectrometry techniques.

Proton NMR spectra were recorded on a Brucker 400 MHz spectrometer. The chemical shift (δ) is reported in ppm relative to Me ₄ Si as an internal standard, and the NMR coupling constant (J value) is expressed in Hertz (Hz). Each peak is represented as a broad singlet (br), singlet (s), doublet (d), triplet (t), quadruple (q), double doublet (dd), triplet of doublet Peak (td) or multiplet (m). A q-TofUltima (Waters AG or Thermo Scientific ^™ MSQ Plus ^™ ) mass spectrometer was used to generate mass spectra in positive ion ESI mode. The system is equipped with a standard Lockspray interface; each intermediate is purified to the standards required for subsequent stages and is fully characterized to confirm that the specified structure is correct; RP-C18-based columns are used for achiral phases Analytical and preparative HPLC; the following abbreviations can be used (for a comprehensive list of standard abbreviations, see also The Journal of Organic Chemistry Guidelines for Authors, 2017 ):

ACN Acetonitrile

Boc tertiary butoxycarbonyl group

BTC bis (trichloromethyl) carbonate

Cat.no. Catalog number

CDCl ₃ deuterated chloroform

DCM dichloromethane

DMAP 4-dimethylaminopyridine

DMF N, N -dimethylformamide

DMSO-d ⁶ deuterated dimethylsulfinium

EA ethyl acetate

ELSD evaporative light scattering detection

Ex. Examples

HATU O- (7-azabenzotriazol-1-yl) -N, N, N ’, N’-tetramethylurea hexafluorophosphate

c -Hex cyclohexane

n -Hex n-hexane

LAH lithium aluminum hydride

LC / MS

LHMDS Lithium bis (trimethylsilyl) fluoride

Me ₄ Si tetramethylsilane

MeOH methanol

nt not tested

Pd ₂ dba ₃ Tris (dibenzylideneacetone) dipalladium (0)

PE petroleum ether

t -BuOH tertiary butanol

TEA Triethylamine

TFAA trifluoroacetic anhydride

THF tetrahydrofuran

W Watt

X-Phos 2- dicyclohexyl phosphino-2 ', 4', 6 '- triisopropylbiphenyl

The following examples refer to compounds of formula ( I ) as shown in Table 1.

The examples listed in the table below can be prepared using the above procedures, and detailed synthetic methods are described in detail below. The instance numbers used in the far left column are used in the application text to identify the corresponding compounds.

Preparation Example 1: 4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (2,3- dimethyl-4-pyridyl) piperidine-1-Amides three Fluoroacetate:

Step 1-a: Preparation of tert-butyl 4- (bromomethylene) piperidine-1-carboxylic acid:

To a stirred suspension of (bromomethyl) triphenylphosphonium bromide (2 000 mg; 4.47 mmol) in THF (45 mL) cooled to -15 ° C over 5 minutes, a 1 M solution of LHMDS in THF (5.82 mL) was added dropwise. 5.82 mmol). The reaction mixture was stirred at -15 ° C for 15 minutes, and then treated with a solution of tert-butyl 4-oxopiperidine-1-carboxylic acid (1000 mg; 4.92 mmol) in THF (5 mL). The mixture was gradually warmed to room temperature and further stirred for 2 hours. The reaction with saturated aqueous NH ₄ Cl mixture was inactivated and then partitioned between EA and brine. , Dried over MgSO ₄ organic layer was separated, washed with brine, 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 obtain 4- (bromomethylene) piperidine-1 as a colorless oil. -Tert-butyl formate (960 mg).

¹ H-NMR (400 MHz, CDCl ₃ ) δ 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: 4-[(4,4,5,5-Tetramethyl-1,3,2-dioxolane-2-yl) methylene] piperidine-1-carboxylic acid tri Preparation of grade butyl ester:

A sealable tube was charged with tert-butyl 4- (bromomethylene) piperidine-1-carboxylate (700 mg; 2.51 mmol), potassium acetate (620 mg; 6.27 mol), and bis (pinar) at room temperature. Alcohol (pinacolato)) diboron (1040 mg; 4.01 mmol) and di (20 mL). Argon was bubbled into the reaction mixture for 10 minutes, and triphenylphosphine (70 mg; 0.25 mmol) and Pd ₂ dba ₃ (160 mg; 0.15 mmol) were added. The tube was purged with argon and sealed. The reaction mixture was then heated to 100 ° C and stirred for 4 hours. After cooling to room temperature, the reaction mixture was filtered and the filter 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 give 4-[(4,4,5,5-tetra as a pale yellow solid Methyl-1,3,2-dioxaborolan-2-yl) methylene] piperidine-1-carboxylic acid tert-butyl ester (720 mg).

¹ H-NMR (400MHz, CDCl ₃ ) δ 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 4-[(4-chloro-2-fluoro-phenyl) methylene] piperidine-1-carboxylic acid tert-butyl ester:

Under an argon atmosphere, X-Phos (750 mg; 1.53 mmol), 1-bromo-4-chloro-2-fluoro-benzene (1.93 mL; 15.31 mmol), 4-[(4,4,5,5- Tetramethyl-1,3,2-dioxolane-2-yl) methylene] piperidine-1-carboxylic acid tert-butyl ester (5 500 mg; 15.31 mmol), Pd ₂ dba ₃ (708 mg 0.76 mmol) and K ₃ PO ₄ (4 975 mg; 22.97 mmol) in H ₂ O (5 mL) and two The mixture in (100 mL) was heated to 100 ° C and stirred for 2 hours. After cooling to room temperature, H ₂ O and EA were added. , Dried over MgSO ₄ organic layer was separated, washed with brine, filtered and concentrated to dryness. The residue was purified by column chromatography (silica gel; PE: EA; 30: 1; v / v) to obtain 4-[(4-chloro-2-fluoro-phenyl) methylene] piperidine as a white solid. Tert-butyl-1-carboxylic acid (4000 mg).

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

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

Step 1-d: Preparation of 4-[(4-chloro-2-fluoro-phenyl) methylene] piperidine hydrochloride:

To a stirred solution of 4-[(4-chloro-2-fluoro-phenyl) methylene] piperidine-1-carboxylic acid tert-butyl ester (18200 mg; 55.2 mmol) in EA (50 mL) was added 2N of dropwise. HCl in EA (160 mL). The reaction mixture was stirred for 2 hours, and then concentrated to dryness to give 4-[(4-chloro-2-fluoro-phenyl) methylene] piperidine hydrochloride (12500 mg) as a white solid.

MS m / z (+ ESI): 226.2, 228.2 [M + H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ ppm: 9.10 (br, 2H), 7.47 (dd, J = 10.0, 2.1Hz, 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: 4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (2,3- dimethyl-4-pyridyl) piperidine-1-acyl-amine trifluoroacetic acid Preparation of salt:

To a solution of diphosgene (182 mg; 0.90 mmol) in THF (4 mL) at 0 ° C was added dropwise 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 (4 mL). The mixture was warmed to room temperature and stirred for 2 hours. 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 hours, the mixture was concentrated to dryness. By the residue purified by preparative HPLC to give a yellow solid, 4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (2,3- dimethyl-4-pyridyl) Piperidine-1-carboxamide trifluoroacetate (120 mg).

Preparation Example 2: 4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (3- methoxy-2-methyl-4-pyridyl) piperidine-1- Lamine:

4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (3- methoxy-2-methyl-4-pyridyl) piperidine-1-acyl-amine: To a cooled (-10 ° C) solution of 3-methoxy-2-methyl-pyridine-4-amine (35 mg; 0.26 mmol) and TEA (0.45 mL; 0.31 mmol) in anhydrous ACN (1 mL) was added chlorine Formic acid 2,2,2-trifluoroethyl (45 mg; 0.27 mmol). After the addition, the reaction mixture was warmed to room temperature and stirred for 24 hours. 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 hours, the reaction mixture was concentrated to dryness and the residue was purified by preparative HPLC, to give a pale brown solid 4 - [(4-chloro-2-fluoro-phenyl) - methylene] - N - (3-methoxy-2-methyl-4-pyridyl) piperidine-1-carboxamide (12 mg).

Preparation of Example 3: N- (3-chloro-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) methylene] piperidine-1 -Formamidine trifluoroacetate:

Step 1: Preparation of 3,5-difluoro-4- (4-piperidinylmethyl) benzonitrile hydrochloride:

Follow Schemes 5 and 4 and similar to the procedure described in Example 1 (steps 1-c and 1-d) using 4-[(4,4,5,5-tetramethyl-1,3,2-dioxa Cyclopentyl-2-yl) methylene] piperidine-1-carboxylic acid tert-butyl ester and 4-bromo-3,5-difluoro-benzonitrile were used as starting materials to prepare the title compound as a white solid.

MS m / z (+ ESI): 235.2 [M + H] ⁺ .

¹ H-NMR (400MHz, DMSO- d ₆ ) δ 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 = 5.6Hz, 2H), 2.28 (t, J = 5.6Hz, 2H).

Step 2: Preparation of phenyl N- (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 hours, and then concentrated to dryness to obtain crude N- (3-chloro-2-methyl-4-pyridyl) aminocarbamic acid phenyl ester (900 mg; 50% purity) as a yellow solid. Further purification was used in the next step.

MS m / z (+ ESI): 263.0, 265.0 [M + H] ⁺ .

Step 3: N- (3-Chloro-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) methylene] piperidine-1-methyl Preparation of ammonium trifluoroacetate:

To a solution of crude N- (3-chloro-2-methyl-4-pyridyl) carbamic acid phenyl ester (360 mg; 0.69 mmol: 50% purity) in DMF (6 mL) was added 3,5-difluoro-4 -(4-piperidinylmethyl) benzonitrile hydrochloride (220 mg; 0.82 mmol) and TEA (0.49 mL; 3.42 mmol). The reaction mixture was stirred for 2 hours and then diluted with ethyl acetate (100 mL). The solution was washed with water (3 x 30mL), dried over Na ₂ SO _4, and concentrated to dryness. Purification of the residue by prep-HPLC gave N- (3-chloro-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) as a white solid ) Methylene] piperidine-1-carboxamide (48 mg).

Preparation Example 4: 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- ethoxy-2-methyl-4-pyridyl) piperidine -1-formamidine trifluoroacetate:

Step 1-a: Preparation of 3-ethoxy-2-methyl-4-nitro-pyridine:

To a solution of 2-methyl-4-nitro-pyridin-3-ol (150 mg; 0.88 mmol) in DMF (4 mL) was added iodoethane (166 mg; 1.05 mmol) and K ₂ CO ₃ (245 mg; 1.75 mmol) . The reaction mixture was stirred for 16 hours and then diluted with EA (100 mL). The mixture was washed with H ₂ O (3 x 60 mL), the organic layer was dried over Na ₂ SO ₄ , filtered and concentrated to dryness to give 3-ethoxy-2-methyl-4- nitrate as a colorless oil. -Pyridine (76 mg).

MS m / z (+ ESI): 183.1 [M + H] ⁺ .

¹ H-NMR (400MHz, CDCl ₃ ) δ ppm: 8.43 (d, J = 5.2Hz, 1H), 7.49 (d, J = 5.2Hz, 1H), 4.12 (q, J = 7.2Hz, 2H), 2.68 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H).

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

A solution of 10% palladium on carbon (70 mg; 0.07 mmol) in 3-ethoxy-2-methyl-4-nitro-pyridine (84 mg; 0.42 mmol) in MeOH (3 mL) under a hydrogen atmosphere (1 bar) Stir for 2 hours. The insoluble matter was removed by filtration, and the filtrate was concentrated to dryness to obtain 3-ethoxy-2-methyl-pyridine-4-amine (58 mg) as a colorless oil.

MS m / z (+ ESI): 153.1 [M + H] ⁺ .

Step 1-c: Preparation of phenyl N- (3-ethoxy-2-methyl-4-pyridyl) carbamate:

To a solution of 3-ethoxy-2-methyl-pyridine-4-amine (64 mg; 0.38 mmol) in DCM (4 mL) was added TEA (0.27 mL; 1.91 mmol) and phenyl chloroformate (0.1 mL 0.80 mmol) . The reaction mixture was stirred for 16 hours, and then concentrated to dryness to obtain crude phenyl N- (3-ethoxy-2-methyl-4-pyridyl) aminocarbamate (100 mg; 80% purity) as a yellow solid, Used in the next step without further purification.

MS m / z (+ ESI): 273.2 [M + H] ⁺ .

Step 2: 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- ethoxy-2-methyl-4-pyridyl) piperidine -1 -Preparation of formamidine trifluoroacetate:

Following Scheme 1 and similar to Example 3 (Step 3) crude N- (3-ethoxy-2-methyl-4-pyridyl) carbamate and 3,5-difluoro-4- (4 -Piperidinylmethyl) benzonitrile hydrochloride as a starting material and, after purification by preparative HPLC, the title compound was obtained as a white solid.

Preparation of Example 5: N- (3-benzyloxy-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) methylene] piperidine -1-formamidine trifluoroacetate:

4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- hydroxy-2-methyl-4-pyridyl) piperidine-1-acyl-amine preparation: at -78 deg.] C boron tribromide (393mg; 1.57mmol) was slowly added 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- b A solution of oxy-2-methyl-4-pyridyl) piperidine-1-carboxamide (220 mg; 0.52 mmol) in anhydrous DCM (20 mL). The mixture was stirred at 0 ° C for 1 hour and then at room temperature for 24 hours. The reaction was deactivated by the addition of saturated NaHCO ₃ in H ₂ O. The aqueous layer was separated and extracted with 2-propanol: DCM (80 mL; 3: 1; v / v). The organic layers were combined, dried over Na ₂ SO _4, filtered, and concentrated to give a pale yellow solid, 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- Hydroxy-2-methyl-4-pyridyl) piperidine-1-carboxamide (170 mg).

MS m / z (+ ESI): 385.5 [M + H] ⁺ .

N- (3-benzyloxy-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro-phenyl) methylene] piperidine-1-carboxamidine amine trifluoroacetate: to a solution of 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (3- hydroxy-2-methyl-4-pyridyl) To a solution of piperidine-1-carboxamide (130 mg; 0.30 mmol) in anhydrous THF (10 mL) was 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 hours and then concentrated to dryness. The residue was purified by prep-HPLC to give N- (3-benzyloxy-2-methyl-4-pyridyl) -4-[(4-cyano-2,6-difluoro- Phenyl) methylene] piperidine-1-carboxamide trifluoroacetate (12 mg).

Preparation Example 6: 4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (2- ethyl-3-methyl-4-pyridyl) piperidin - 1-formamidine trifluoroacetate:

4 - [(4-cyano-2,6-difluoro-phenyl) - methylene] - N - (2- ethyl-3-methyl-4-pyridyl) piperidine-1-Amides Preparation of trifluoroacetate: To a solution of 2-ethyl-3-methyl-pyridine-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 hours, then 3,5-difluoro-4- (4-piperidinylmethyl) benzonitrile, trifluoroacetic acid (343 mg; 0.97 mmol) and TEA (0.41 mL; 2.92 mmol) in THF (3 mL) solution. After stirring for 18 hours, the mixture was concentrated to dryness, and the residue was purified by prep-HPLC to give 4-[(4-cyano-2,6-difluoro-phenyl) methylene]-as a white solid N- (2-ethyl-3-methyl-4-pyridyl) piperidine-1-carboxamide trifluoroacetate (150 mg).

Biological examples

Cell culture

HeLa (ATCC, CCL-2), a cervical tumor cell line, containing 10% fetal bovine serum (Sigma cat.no.F9665) and 1% penicillin / chain in 5% CO ₂ at 37 ° C. It was cultured in DMEM medium (Invitrogen cat.no. 11971, 4.5 g / L high glucose) of chlortetracycline (cat.no.P0781). HeLa galactose cells (i.e., HeLa cells grown in high concentrations of galactose) are derived from HeLa glucose cells (i.e., HeLa cells grown in high concentrations of glucose) by gradually increasing the presence of galactose as a sugar source. It is produced by changing the glucose in the medium to zero (50% galactose / 50% glucose medium for one week, then 75% galactose / 25% glucose medium for one week, and the third week is 100% galactose medium). The galactose medium (cat.no.11966 of Sigma) was supplemented with 10 mM galactose (cat.no.G5388 of Sigma).

Cell growth and proliferation assays of HeLa galactose and glucose cells

HeLa galactose cells and HeLa glucose cells were seeded at 2,000 cells / well and 1500 cells / well in 100 μL of complete medium in a 96-well plate (TPP, cat.no 92696). After overnight incubation, the cells were incubated in complete medium containing 0.001% DMSO or compound (final concentration of DMSO was 0.001%) for 72 hours. After removing the culture medium, the cells were fixed and stained by adding 50 μL of crystal violet staining (0.2% crystal violet in 50% methanol (Sigma-Aldrich cat.no.C0775)) to each well. The plate was incubated at room temperature for 1 hour. The stain was then decanted and the plate was washed 4 times with demineralized water. The plate was air-dried for 2 hours. 100 μL of a buffer solution (0.1M Tris pH 7.5, 0.2% SDS, 20% ethanol) was added to each well, and the plate was shaken to dissolve the stain. The absorbance at 590 nm was measured using a SpectraMax® 250 microplate reader (Molecular Devices). The IC50 antiproliferative / growth inhibition was calculated from the concentration-response curve using GraphPad Prism software.

Oxygen consumption measurement

Oxygen consumption is one of the most informative and direct measures of mitochondrial function, and can be measured, for example, using the MitoXpress® assay (Luxcel MX-2001, Luxcel Biosciences). MitoXpress® probes are part of a family of phosphorescent oxygen-sensitive probes. This assay utilizes the ability of oxygen to quench the excited state of the MitoXpress® probe. As the test material (ie, the cells) breathes, oxygen is depleted in the surrounding solution / environment, which increases the probe phosphorescence signal. Changes in oxygen consumption that reflect changes in mitochondrial activity are considered changes in MitoXpress® probe signals over time.

Cells were seeded at a density of 50'000 cells / well in a 96-well black plate (Greiner Bio-One Co. cat.no.655090) with a transparent bottom to a final volume of 100 μL. 24 hours After that, the incubation medium was removed, and 150 μL of fresh medium containing different concentrations of inhibitors was added to each well. Then, 10 μL of MitoXpress® and 150 μL of mineral oil were added to each well. Read from the top of the plate using a Synergy 4 microplate reader (BioTek) and 380 / 11nm excitation and 650 / 20nm emission or 665/40 emission (30 microsecond delay time, 100 microsecond integration time, set to medium or high Kinetic analysis of time-resolved fluorescence (TRF) wavelengths at 37 ° C for 5 hours at a gain or sensitivity setting. Compared to untreated cells, IC50 was calculated as the concentration (MitoXpress®) that inhibited the phosphorescent oxygen-sensitive probe signal by 50%.

Galactose cells are highly dependent on OXPHOS and are more sensitive to mitochondrial inhibitors than glucose cells (Gohil V.M. et al., Nat. Biotechnol. [Natural Biotechnology], Vol. 28, No. 3, pages 249-255, 2010). For example, the differential sensitivity of HeLa glucose and HeLa galactose cell growth is demonstrated by antimycin A (Sigma-Aldrich cat.no. A8674), which is an inhibitor of mitochondrial electron transport chain complex III. (Figure 1a), but not by cytotoxic compounds such as paclitaxel (CAS 33069-62-4) (Figure 1c). As shown in FIG. 1b (for example, Embodiment 6), the compounds of the present invention also show different sensitivities in HeLa glucose and HeLa galactose cell growth assays. In this way, HeLa galactose cells can be used to screen for mitochondrial inhibitors. In addition, as shown in Table 2, a compound having activity in HeLa galactose cells can be confirmed as a true mitochondrial inhibitor by testing oxygen consumption inhibition. The biological data are given in Table 2 below.

Claims (15)

A compound of formula I or a pharmaceutically acceptable salt thereof Where B1, B2, B3, and B4 independently represent C (R3); X represents -CH (R5)-, -C (R5) = or -C (= O)-, and where X represents -CH (R5) -Or -C (= O)-, the dashed line represents a single bond, and when X represents -C (R5) =, the dashed line represents a double bond; R1 independently represents methyl, ethyl, and methoxy at each occurrence Or ethoxy; R2 represents halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, -N (R6a) (R6b) or -methylene-N (R6a) (R6b) ; R3 independently represents hydrogen, halogen, cyano, or methyl on each occurrence; R4a represents methyl or ethyl; R4b represents halogen, C1-C4 alkyl, C1-C4 alkoxy, or -O-C1- C4 alkylene-ring-Q; R5 represents hydrogen or methyl; R6a represents hydrogen or methyl; R6b represents hydrogen or methyl; ring-Q independently represents each time it is replaced by 1 to 3 R7 as needed R7 at each occurrence independently represents cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1 or 2; and q is 0, 1 or 2. A compound or pharmaceutically acceptable salt thereof as claimed in claim 1, wherein n is 1. A compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, wherein X represents -CH =. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein the ring system containing B1, B2, B3, and B4 as ring members is composed of a group B-Ia, a group B-Ib, or Group B-Ic represents where R3a is as defined for R3, but not hydrogen: The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R 2 represents halogen or cyano. A compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, wherein R3 independently represents hydrogen or halogen at each occurrence. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, wherein R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -O-C1-C2alkylene- Ring-Q. A compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein q is 0. As in the compound of claim 1, the ring system containing B1, B2, B3 and B4 as ring members is represented by the group B-Ia, the group B-Ib or the group B-Ic: X represents -CH _2- , -CH = or -C (O)-; R2 represents halogen, cyano, hydroxy, methyl, ethyl, methoxy, ethoxy, -N (R6a) (R6b) or -Methylene-N (R6a) (R6b); R3a independently represents halogen, cyano or methyl at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, C1-C4 alkyl, C1- C4 alkoxy or -O-C1-C4 alkylene-cyclo-Q; R6a represents hydrogen or methyl; R6b represents hydrogen or methyl; ring-Q independently represents each time it appears to be 1 to 3 as needed R7 substituted phenyl; R7 at each occurrence independently represents cyano, methyl, halomethyl, methoxy or halomethoxy; n is 1; and q is 0. As in the compound of claim 1, the ring system containing B1, B2, B3 and B4 as ring members is represented by the group B-Ia, the group B-Ib or the group B-Ic: X represents -CH _2- , -CH = or -C (O)-; R2 represents halogen or cyano; R3a independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen or methyl , Ethyl, methoxy, ethoxy, or -O-CH ₂ -ring-Q; each ring-Q independently represents a phenyl group optionally substituted with 1 to 3 R7 at each occurrence; R7 at each occurrence When present, it independently represents cyano, methyl, halomethyl, methoxy, or halomethoxy; n is 1; and q is 0. As in the compound of claim 1, the ring system containing B1, B2, B3 and B4 as ring members is represented by the group B-Ia or the group B-Ib: X represents -CH =; R2 represents halogen or cyano; R3a independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, methyl, ethyl, methoxy, ethoxy or -O-CH ₂ -phenyl; n is 1; and q is 0. As in the compound of claim 1, the ring system containing B1, B2, B3 and B4 as ring members is represented by the group B-Ia or the group B-Ib: X represents -CH =; R2 represents halogen or cyano; R3a independently represents halogen at each occurrence; R4a represents methyl or ethyl; R4b represents halogen, methyl, ethyl, methoxy, and ethoxy; n is 1; and q is 0. A compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of a compound selected from mammals A proliferative disease in a subject, wherein the compound of formula I is as defined in any one of claims 1 to 12. A compound of formula I or a pharmaceutically acceptable salt thereof, as used in claim 13, wherein the disease is cancer. A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof as defined in any one of claims 1 to 12, and preferably comprising a pharmaceutically acceptable excipient.