Heterocyclic compounds capable of activating STING

Abstract

# imgabs0 #. The invention relates to a protein capable of activating an interferon gene stimulating protein (Stimulator of Interferon Gene; sTING) of formula (I). The invention further relates to pharmaceutical compositions comprising at least one compound of formula (I) and to the use of these compounds or pharmaceutical compositions as medicaments, for example for the treatment of canine or cat cancer or as vaccine adjuvants.

Description

Technical Field

[0001] The present invention relates to small molecules capable of activating STING (Stimulator of Interferon Gene) and salts thereof. Specifically, the present invention relates to heterocyclic compounds capable of activating STING. In addition, the present invention relates to pharmaceutical compositions and combinations comprising these compounds, and their use as medicaments. These compounds and pharmaceutical compositions comprising at least one of these compounds are applicable as medicaments, for example for the treatment of cancer (such as canine and / or feline cancer), and are applicable as vaccine adjuvants, for example for pigs. Accordingly, the present invention also relates to compounds and pharmaceutical compositions comprising at least one of these compounds for the treatment of feline or canine cancer.

Background Art

[0002] STING is one of the pattern recognition receptors (PRRPs) that play a central role in the innate immune system, which discriminates pathogens and host cells by detecting extracellular and intracellular danger signals, including damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). These recognition processes constitute the first line of defense against viral and bacterial infections as well as malignant cells. However, pathogens and cancer cells have evolved ways to evade immune system recognition. Therefore, the goal of immunotherapy is to initiate an antigen-specific immune response or reactivate an existing response against pathogen invaders or cancer cells in specific cell types of the immune system.

[0003] Among the PRRPs, STING (also known as TMEM173, MPYS, MITA, ERIS) belongs to the nucleic acid sensor family and is an adaptor protein for cytosolic DNA signaling. In mammalian cells in a healthy state, DNA is compartmentalized in the nucleus. In the case of pathogen infections (such as the invasion of DNA-containing pathogens) or in malignant cells, DNA is present in the cytoplasm. Here, STING is crucial for detecting the cytosolic DNA described above and inducing an immune response against pathogen events.

[0004] As a member of the innate immune defense mechanism, STING is expressed in almost all cell types, particularly endothelial cells, epithelial cells, and immune cells such as macrophages and dendritic cells. In dogs and cats, STING has been observed to be mainly expressed in the spleen, lung, blood, lymph nodes, and brain (Zhang et al., Microbial Pathogenesis, 113, 202 - 208, 2017; Zhang et al., Veterinary Immunology and Immunopathology 169, 54 - 62, 2016).

[0005] STING exists as a dimer in its basal state, with its N - terminal domain anchored in the ER and its C - terminal domain residing in the cytosol. Cyclic dinucleotides (CDNs) produced by the protein cyclic GMP - AMP synthase (cGAS) are the natural ligands for STING (Ablasser et al., Nature 498, 380 - 384, 2013). Binding of CDN to STING induces a conformational change, enabling the binding and activation of TANK - binding kinase (TBK1) and interferon regulatory factor 3 (IRF3), followed by re - localization from the ER to the perinuclear endosome (Liu et al., Science 347, issue 6227, 2630 - 1 - 2630 - 14, 2015). Phosphorylation of the transcription factors IRF3 and NF - kB by TBK1 leads to the expression of various cytokines, including type I interferons (IFNs).

[0006] Type I IFNs produced by antigen - presenting cells and other cell types are regarded as key events in activating T cells and thereby differentiating antigen - specific effector CD4 and CD8 T cells. It has been demonstrated that the absence of type I IFN results in a reduced T - cell - dependent immune response against viral infections or tumor cells (Zitvogel et al., Nature Reviews Immunology 15, 405 - 414, 2015). On the other hand, the presence of type I IFN signatures during cancer treatment is associated with an increased number of tumor - infiltrating T cells and potentially favorable clinical outcomes (Sistigu et al., Nature Medicine 20, 1301 - 1309, 2014).

[0007] The anti-tumor effects of type I interferons on feline and canine cancers have been studied in vitro and in vivo. Based on clinical studies of administering human recombinant interferon to dogs carrying tumors (lymphoma, fibrosarcoma, osteosarcoma, mycosarcoma, liposarcoma), delays or prevention of local recurrence and metastasis were recorded (Klotz et al., Veterinary Immunology and Immunopathology 191, 80-93, 2017).

[0008] The efficient secretion of type I IFN in the tumor microenvironment and the induction of T cell-dependent immune responses against cancer cells depend on the presence of STING, as demonstrated in recent studies in mice (Woo et al., Immunity 41, 5, 830-842, 2014; Corrales et al., Cell Reports 11, 1018-1030, 2015; Deng et al., Immunity 41, 5, 843-852, 2014). The absence of STING leads to reduced levels of type I IFN in the tumor microenvironment and reduced anti-tumor effects in several mouse tumor models, thereby highlighting the importance of the presence of type I IFN. On the other hand, specific activation of STING causes an increase in antigen-specific T cell immune responses against cancer cells.

[0009] Type I interferons can significantly enhance anti-tumor immune responses by inducing the activation of both acquired and innate immune cells.

[0010] Given the importance of type I IFN in several malignancies (including viral infections) and cancer treatment, strategies that allow for the specific activation of STING have therapeutic significance. STING activation can produce synergistic effects with various approved chemotherapeutic agents or other anti-cancer treatments (such as radiotherapy) (Wu et al., Med Res Rev May 2020; 40(3):1117-1141) or treatments for infectious diseases.

[0011] In the prior art, small molecule modulators of STING are described, for example, in WO 2020 / 075790.

Summary of the Invention

[0013] The compounds of the present invention are novel activators of STING, as demonstrated in an ex vivo system using canine whole blood.

[0014] On the one hand, the present invention relates to a compound of formula (I)

[0015]

[0016] wherein

[0017] B is a group selected from the following:

[0018] a 5- to 7-membered monocyclic heterocyclic group containing 1 or 2 N atoms,

[0019] a 6-membered bicyclic heterocyclic group containing 1 N atom,

[0020] a 7- to 11-membered bicyclic heterocyclic group containing 1 or 2 N atoms,

[0021] a 7-membered bicyclic heterocyclic group containing 1 N atom and 1 O atom,

[0022] a 6-membered monocyclic heterocyclic group containing 1 N atom and 1 heteroatom selected from O and S,

[0023] a 9-membered bicyclic heterocyclic group containing 3 heteroatoms, where 2 heteroatoms are N and the other is O,

[0024] a 9-membered bicyclic heterocyclic group containing 1 N atom and 1 S atom,

[0025] a 10-membered bicyclic heterocyclic group containing 3 N atoms, where 2 N atoms are substituted by C 1-6 alkyl,

[0026] phenyl,

[0027] a 9-membered bicyclic heteroaryl group containing 3 N atoms,

[0028] -C 1-4 alkylene-pyrimidine, and

[0029] -C 1-4 alkylene-O-C 1-3 alkyl;

[0030] D is a group selected from the following:

[0031] a 9-membered bicyclic heteroaryl group containing 2 N atoms,

[0032] a 10-membered bicyclic heteroaryl group containing 1 N atom, and

[0033] benzodioxole;

[0034] R 1 is selected from -H or -C 1-6 alkyl;

[0035] R 2 is selected from -H, halogen, and -C 1-6 alkyl, where the halogen is preferably fluorine or chlorine, more preferably fluorine;

[0036] R 3 is selected from -H, halogen, and -C 1-6 alkyl, where the halogen is preferably fluorine or chlorine, more preferably fluorine;

[0037] R 4a , R 4b and R 4c are each independently selected from -H, halogen and C 1-6 alkyl, the halogen is preferably fluorine or chlorine, more preferably fluorine, provided that R 4a , R 4b and R 4c At least one of is a halogen;

[0038] R 4d Selected from -C 1-6 Alkyl and C 3-6 Cycloalkyl;

[0039] R 5 Not present or selected from -H, -C 1-6 Alkyl, -S(O 2 )-C 1-6 Alkyl, -NH-S(O 2 )-C 1-6 Alkyl, =O, -C(O)-C 1-6 Alkyl, -C(O)H, -C(O)OH, -C(O)NH 2 、-C(O)OC 1-6 Alkyl, -NR 5.1 R 5.2 , -C 1-6 Alkylene -C(O)OH, -S(O 2 )-NH 2 , -pyrrolidin-2-one-1-yl, -tetrazolyl, and R having 1 or 2 heteroatoms selected from N and O 5.3 substituted 5-membered heteroaryl;

[0040] R 5.1 Selected from -H, -C 1-6 Alkyl, -C(O)-C 1-6 Alkyl and -C 1-6 Alkylene-OC 1-6 alkyl;

[0041] R 5.2 Selected from -H, -C 1-6 Alkyl, -C(O)-C 1-6 Alkyl and -C 1-6 Alkylene-OC 1-6 alkyl;

[0042] R 5.3 Selected from -H, -C 1-6 Alkyl and 6-membered heteroaryl having 1 or 2 heteroatoms selected from N and O;

[0043] R 6is absent or selected from -H, -C 1-6 alkyl, =O and -C(O)OH;

[0044] or a pharmaceutically acceptable salt thereof.

[0045] On the other hand, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0046] On the other hand, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which is used as a medicine.

[0047] On the other hand, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the same, which is used for treating feline or canine cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0049] The compounds of the present invention exhibit several advantageous properties, such as having favorable binding affinities for STING from various mammalian species (e.g., cats, mice, pigs, and dogs), particularly good binding affinity for canine STING, and favorable cellular activity measured by cell EC 50 (i.e., in canine whole blood).

[0050] Thus, on the other hand, the present invention provides new compounds of formula (I), including their salts, which activate STING and thus induce cytokine production in a STING-dependent manner in vitro and / or in vivo (e.g., in dogs), and have suitable pharmacological and pharmacokinetic properties for treatment (i.e., for use as a medicine).

[0051] The binding of a compound to a protein can be determined by known methods, such as surface plasmon resonance, scintillation proximity assay, isothermal titration calorimetry, or differential scanning fluorimetry. In the latter assay, the protein melting temperature (also known as the melting temperature T m ) is measured by the fluorescence change of a dye that binds to the hydrophobic part of the protein. After binding of a small molecule, the T m shift is related to the binding affinity of the small molecule. A high binding affinity of a STING agonist is reflected by a T m shift of >10 °C, preferably >13 °C, more preferably >15 °C. When measured by a binding assay, the compounds of the present invention preferably exhibit an interaction with canine STING (dSTING) that is reflected by a T m shift of >15 °C, more preferably >20 °C, and even more preferably >25 °C as determined by DSF.

[0052] Since STING has been shown to stimulate the production of type I interferons, such as interferon β (IFNβ), in myeloid cells and dendritic cells, the potency of STING agonists can be evaluated in a canine whole blood (cWB) assay, where IFNβ secretion is used as a readout. In this assay, freshly collected canine blood is incubated with a STING agonist, and the level of interferon β in the supernatant is quantified by ELISA. The compounds of the invention generally exhibit a cellular EC50 of less than 10 μM, preferably less than 5 μM, more preferably less than 1 μM, and most preferably less than 0.5 μM.

[0053] According to the invention, the combination of high dDSF and low dWB is particularly advantageous.

[0054] Compounds of general formula (I) of the invention

[0055]

[0056] wherein

[0057] B is a group selected from:

[0058] a 5- to 7-membered monocyclic heterocyclic group containing 1 or 2 N atoms,

[0059] a 6-membered bicyclic heterocyclic group containing 1 N atom,

[0060] a 7- to 11-membered bicyclic heterocyclic group containing 1 or 2 N atoms,

[0061] a 7-membered bicyclic heterocyclic group containing 1 N atom and 1 O atom,

[0062] a 6-membered monocyclic heterocyclic group containing 1 N atom and 1 heteroatom selected from O and S,

[0063] a 9-membered bicyclic heterocyclic group containing 3 heteroatoms, where 2 heteroatoms are N and the other is O,

[0064] a 9-membered bicyclic heterocyclic group containing 1 N atom and 1 S atom,

[0065] a 10-membered bicyclic heterocyclic group containing 3 N atoms, where 2 N atoms are substituted by C 1-6 alkyl,

[0066] phenyl,

[0067] a 9-membered bicyclic heteroaryl group containing 3 N atoms,

[0068] -C 1-4 alkylene-pyrimidine, and

[0069] -C 1-4 alkylene-O-C 1-3 alkyl;

[0070] D is a group selected from the following:

[0071] a 9-membered bicyclic heteroaryl containing 2 N atoms,

[0072] a 10-membered bicyclic heteroaryl containing 1 N atom, and

[0073] benzodioxolene;

[0074] R 1 is selected from -H or -C 1-6 alkyl;

[0075] R 2 is selected from -H, halogen and -C 1-6 alkyl, the halogen is preferably fluorine or chlorine, more preferably fluorine;

[0076] R 3 is selected from -H, halogen and -C 1-6 alkyl, the halogen is preferably fluorine or chlorine, more preferably fluorine;

[0077] R 4a 、R 4b and R 4c are each independently selected from -H, halogen and C 1-6 alkyl, the halogen is preferably fluorine or chlorine, more preferably fluorine, provided that at least one of R 4a 、R 4b and R 4c is halogen;

[0078] R 4d is selected from -C 1-6 alkyl and C 3-6 cycloalkyl;

[0079] R 5 is absent or is selected from -H, -C 1-6 alkyl, -S(O 2 )-C 1-6 alkyl, -NH-S(O 2 )-C 1-6 alkyl, =O, -C(O)-C 1-6 alkyl, -C(O)H, -C(O)OH, -C(O)NH 2 、-C(O)O-C 1-6 alkyl, -NR 5.1 R 5.2 、-C 1-6 alkylene-C(O)OH, -S(O 2 )-NH 2 、-pyrrolidin-2-one-1-yl, -tetrazolyl, and R-substituted with 1 or 2 heteroatoms selected from N and O 5.3Substituted 5-membered heteroaryl;

[0080] R 5.1 selected from -H, -C 1-6 alkyl, -C(O)-C 1-6 alkyl and -C 1-6 alkylene-O-C 1-6 alkyl;

[0081] R 5.2 selected from -H, -C 1-6 alkyl, -C(O)-C 1-6 alkyl and -C 1-6 alkylene-O-C 1-6 alkyl;

[0082] R 5.3 selected from -H, -C 1-6 alkyl and 6-membered heteroaryl having 1 or 2 heteroatoms selected from N and O;

[0083] R 6 absent or selected from -H, -C 1-6 alkyl, =O and -C(O)OH;

[0084] Or a pharmaceutically acceptable salt thereof is particularly suitable for treating pathophysiological processes associated with or regulated by STING, particularly suitable for treating cancer, such as feline or canine cancer, or used as a vaccine adjuvant, for example, in pigs.

[0085] Thus, on the other hand, the present invention further relates to a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one compound of formula (I), which is used as a drug.

[0086] Another aspect of the present invention relates to a compound of formula (I) as defined herein or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one compound of formula (I), which is used for treating feline or canine cancer.

[0087] For those skilled in the art, other aspects of the present invention will become obvious directly from the foregoing and the following description and examples.

[0088] Terms and definitions used

[0089] For terms not specifically defined herein, the meanings given to them by those skilled in the art in view of the present disclosure and the context should be given. However, as used in this specification, unless otherwise specified, the following terms have the indicated meanings and comply with the following conventions.

[0090] In the groups, radicals or moieties defined below, the number of carbon atoms is usually specified before the group, for example, C1-6 An alkyl group means an alkyl group or alkyl radical having 1 to 6 carbon atoms. Generally, in groups such as HO, H 2 N, (O)S, (O) 2 S, NC (cyano), HOOC, F 3 C, those skilled in the art can see one or more connection points of the group to the molecule from the free valence of the group itself. For a combined group containing two or more sub-groups, the last named sub-group is the group connection point. For example, the substituent "aryl-C 1-3 alkylene" means an aryl bonded to a C 1-3 alkyl-group, where the C 1-3 alkyl-group is bonded to the core or to the group to which the substituent is attached.

[0091] If the compounds of the present invention are depicted in chemical name and chemical formula form, in case of any inconsistency, the chemical formula shall prevail. An asterisk or a wavy line in a sub-formula can be used to indicate a bond connecting to the core molecule as defined.

[0092] For example, the term "3-carboxypropyl-group" represents the following substituent:

[0093]

[0094] where the carboxyl group is connected to the third carbon atom of the propyl group. The terms "1-methylpropyl-", "2,2-dimethylpropyl-" or "cyclopropylmethyl-" groups represent the following groups:

[0095]

[0096] A wavy line in a sub-formula can be used to indicate a bond connecting to the core molecule as defined. Alternatively, an asterisk in a sub-formula can be used to indicate a bond connecting to the core molecule as defined.

[0097] 1.1.1.1 Substituted terms

[0098] As used herein, the term "substituted" means that one or more hydrogens on a specified atom are replaced by a group selected from a defined group of substituents, provided that the normal valence of the specified atom is not exceeded and the substitution results in a stable compound. Similarly, the term "substituted" can be used in combination with chemical moieties rather than individual atoms, such as "substituted alkyl", "substituted aryl", etc. 1.1.1.2.

[0099] 1.1.1.2 Stereochemistry - Solvates - Hydrates

[0100] Unless otherwise indicated, throughout the specification and the appended claims, a given chemical formula or name shall encompass tautomers and all stereoisomers, optical isomers, and geometric isomers (e.g., enantiomers, diastereomers, E / Z isomers, etc.), their racemates, as well as mixtures of individual enantiomers in different ratios, mixtures of diastereomers, or mixtures in which such isomers and enantiomers are present in any of the foregoing forms, and their solvates, such as hydrates.

[0101] Unless otherwise indicated, "pharmaceutically acceptable salts" as more particularly defined hereinafter shall also encompass their solvates, such as hydrates.

[0102] 1.1.1.3 Stereoisomers

[0103] In general, substantially pure stereoisomers can be obtained according to synthetic principles known to those skilled in the art, for example, by separating the corresponding mixtures, by using stereochemically pure starting materials and / or by stereoselective synthesis. It is known in the art how to prepare optically active forms, such as by resolution of the racemic form or by synthesis, for example, starting from optically active starting materials and / or by using chiral reagents.

[0104] The enantiomerically pure compounds or intermediates of the present invention can be prepared via asymmetric synthesis, for example, by preparing and subsequently separating appropriate diastereomeric compounds or intermediates that can be separated by known methods (e.g., by chromatographic separation or crystallization), and / or by using chiral reagents (such as chiral starting materials, chiral catalysts or chiral auxiliaries).

[0105] In addition, it is known to those skilled in the art how to prepare enantiomerically pure compounds from the corresponding racemic mixtures, such as by chromatographic separation of the corresponding racemic mixture on a chiral stationary phase; or by using an appropriate resolving agent to resolve the racemic mixture, for example, by forming a diastereomeric salt of the racemic compound with an optically active acid or base, subsequently resolving the salt and releasing the desired compound from the salt; or by performing derivatization of the corresponding racemic compound with an optically active chiral auxiliary reagent, subsequently separating the diastereomers and removing the chiral auxiliary group; or by kinetic resolution of the racemate (e.g., by enzymatic resolution); by enantioselective crystallization from an aggregate of enantiomeric crystals under suitable conditions; or by (partial) crystallization from a suitable solvent in the presence of an optically active chiral auxiliary.

[0106] 1.1.1.4 Salts

[0107] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of sound medical judgment, are suitable for contact with the tissues of a mammal without excessive toxicity, irritation, allergic response, or other problems or complications and are commensurate with a reasonable benefit / risk ratio.

[0108] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by making its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines; alkali metal salts or organic salts of acidic residues such as carboxylic acids; and the like.

[0109] By way of example, such salts include salts from benzenesulfonic acid, benzoic acid, citric acid, ethanesulfonic acid, fumaric acid, gentisic acid, hydrobromic acid, hydrochloric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, 4-methyl-benzenesulfonic acid, phosphoric acid, salicylic acid, succinic acid, sulfuric acid, and tartaric acid.

[0110] Other pharmaceutically acceptable salts can be formed with cations from ammonia, L-arginine, calcium, 2,2'-iminobisethanol, L-lysine, magnesium, N-methyl-D-glucamine, potassium, sodium, and tris(hydroxymethyl)-aminomethane.

[0111] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. In general, such salts can be prepared by reacting the free acid or free base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile or mixtures thereof.

[0112] In addition to the salts mentioned above, salts of other acids (e.g., trifluoroacetate) used, for example, for purifying or isolating the compounds of the present invention also form part of the present invention.

[0113] 1.1.1.5 Halogens

[0114] The term halogen refers to fluorine, chlorine, bromine, and iodine.

[0115] 1.1.1.6 Heteroatoms

[0116] Heteroatoms can exist in all possible oxidation states. For example, sulfur can exist in the form of sulfoxide (R-S(O)-R') and sulfone (-R-S(O) 2 -R').

[0117] 1.1.1.7 Alkyl

[0118] The term "C 1-n"Alkyl" (where n is an integer selected from 2, 3, 4, 5, or 6, preferably 4, 5, or 6) represents an acyclic saturated branched or straight-chain hydrocarbon group having 1 to n C atoms. For example, the term C 1-5 alkyl encompasses the groups H 3 C-, H 3 C-CH 2 -, H 3 C-CH 2 -CH 2 -, H 3 C-CH(CH 3 )-, H 3 C-CH 2 -CH 2 -CH 2 -, H 3 C-CH 2 -CH(CH 3 )-, H 3 C-CH(CH 3 )-CH 2 -, H 3 C-C(CH 3 ) 2 -, H 3 C-CH 2 -CH 2 -CH 2 -CH 2 -, H 3 C-CH 2 -CH 2 -CH(CH 3 )-, H 3 C-CH 2 -CH(CH 3 )-CH 2 -, H 3 C-CH(CH 3 )-CH 2 -CH 2 -, H 3 C-CH 2 -C(CH 3 ) 2 -, H 3 C-C(CH 3 ) 2 -CH 2 -, H 3 C-CH(CH 3 )-CH(CH 3 )- and H 3 C-CH 2 -CH(CH 2 CH 3 )-.

[0119] 1.1.1.8 Alkylene

[0120] The term "C 1-n alkylene" (where n is an integer selected from 2, 3, 4, 5 or 6, preferably 4, 5 or 6), alone or in combination with another group, represents an acyclic saturated branched or straight-chain divalent alkyl group containing from 1 to n carbon atoms. By way of example, the term C 1-4 alkylene includes -CH 2 -, -CH 2 -CH 2 -, -CH(CH 3 )-, -CH 2 -CH 2 -CH 2 -, -C(CH 3 ) 2 -, -CH(CH 2 CH 3 )-, -CH(CH 3 )-CH 2 -, -CH 2 -CH(CH 3 )-, -CH 2 -CH 2 -CH 2 -CH 2 -, -CH 2 -CH 2 -CH(CH 3 )-, -CH(CH 3 )-CH 2 -CH 2 -, -CH 2 -CH(CH 3 )-CH 2 -, -CH 2 -C(CH 3 ) 2 -, -C(CH 3 ) 2 -CH 2 -, -CH(CH 3 )-CH(CH 3 )-, -CH 2 -CH(CH 2 CH 3 )-, -CH(CH 2 CH 3 )-CH 2 -, -CH(CH 2 CH 2 CH 3 )-, -CH(CH(CH 3 )) 2- and -C(CH 3 )(CH 2 CH 3 )-.

[0121] 1.1.1.9 Alkenyl

[0122] If at least two carbon atoms of the C 2-m alkyl group are bonded to each other by a double bond, the term "C 2-m alkenyl" is used for this group "C 2-m alkyl", where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6.

[0123] 1.1.1.10 Alkenylene

[0124] If at least two carbon atoms of the C 2-m alkylene group are bonded to each other by a double bond, the term "C 2-m alkenylene" is used for this group "C 2-m alkylene", where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6.

[0125] 1.1.1.11 Alkynyl

[0126] If at least two carbon atoms of the C 2-m alkyl group are bonded to each other by a triple bond, the term "C 2-m alkynyl" is used for this group "C 2-m alkyl", where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6.

[0127] 1.1.1.12 Alkynylene

[0128] If at least two of those carbon atoms of the C 2-m alkylene group are bonded to each other by a triple bond, the term "C 2-m alkynylene" is used for this group "C 2-m alkylene", where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6.

[0129] 1.1.1.13 Cycloalkyl

[0130] The term "C 3-k cycloalkyl" (where k is an integer selected from 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6), alone or in combination with another group, represents a cyclic saturated unbranched hydrocarbon group having 3 to k C atoms. For example, the term C 3-7 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[0131] 1.1.1.14 Cycloalkenyl

[0132] The term "C3-k "Cycloalkenyl" (where k is an integer selected from 3, 4, 5, 6, 7 or 8, preferably 4, 5 or 6) represents a cyclic unsaturated but non-aromatic unbranched hydrocarbon group having from 3 to k carbon atoms, wherein at least two carbon atoms are bonded to each other by a double bond. For example, the term C 3-7 Cycloalkenyl includes cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl and cycloheptatrienyl.

[0133] 1.1.1.15 Halo-(alkyl, alkylene or cycloalkyl)

[0134] The term "halo" added to "(alkyl, alkylene or cycloalkyl)" (saturated or unsaturated) defines such an alkyl, alkylene or cycloalkyl group in which one or more hydrogen atoms are replaced by a halogen atom selected from fluorine, chlorine or bromine, preferably fluorine and chlorine, particularly preferably fluorine. Examples include: H 2 FC-, HF 2 C-, F 3 C-.

[0135] 1.1.1.16 Carbocyclic group

[0136] The term "carbocyclic group" alone or in combination with another group means a monocyclic, bicyclic or tricyclic structure composed of 3 to 14 carbon atoms. The term "carbocyclic group" refers to fully saturated, partially saturated and aromatic ring systems. The term "carbocyclic group" encompasses fused, bridged and spiro ring systems.

[0137]

[0138]

[0139] 1.1.1.17 Aryl

[0140] As used herein, the term "aryl" alone or in combination with another group represents a carbocyclic aromatic monocyclic group containing 6 carbon atoms, which is optionally further fused with another five- or six-membered carbocyclic group that is aromatic, saturated or unsaturated. Aryl includes (but is not limited to) phenyl, indanyl, indenyl, naphthyl, anthryl, phenanthryl, tetrahydronaphthyl and dihydronaphthyl.

[0141] 1.1.1.18 Heterocyclic group

[0142] The term "heterocyclic group" means a saturated or unsaturated monocyclic or polycyclic system that optionally contains an aromatic ring, which contains one or more heteroatoms selected from N, O, S, SO, SO 2 and is composed of 3 to 14 ring atoms, wherein the heteroatoms are not part of an aromatic ring. The term "heterocyclic group" is intended to include all possible isomeric forms.

[0143] Accordingly, the term "heterocyclic group" includes the following exemplary structures (not shown in radical form since each form can be attached by a covalent bond to any atom so long as the appropriate valence is maintained):

[0144]

[0145]

[0146]

[0147] 1.1.1.19 Heteroaryl

[0148] The term "heteroaryl" means a monocyclic or polycyclic system containing at least one aromatic ring, which contains one or more heteroatoms selected from N, O, S, SO or SO 2 and consists of 5 to 14 ring atoms, wherein at least one of the heteroatoms is part of the aromatic ring, and wherein the resulting ring system must be chemically stable. The term "heteroaryl" is intended to include all possible isomeric forms.

[0149] Accordingly, the term "heteroaryl" includes the following exemplary structures (not shown in radical form since each form can be attached by a covalent bond to any atom so long as the appropriate valence is maintained):

[0150]

[0151]

[0152] Many of the terms given above may be used repeatedly to define chemical formulas or groups, and in each case independently have one of the meanings given above.

[0153] The term "bicyclic system" means a group consisting of 2 joined cyclic substructures, including spiro, fused and bridged ring systems.

[0154] Preferred Embodiments

[0155] A particularly preferred embodiment of the invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C 1-6 alkyl; and at least one of R 4a , R 4b and R 4c is fluorine or chlorine, and the remainder are -H or -C 1-3 alkyl; and R 4d is -C 1-6 alkyl.

[0156] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D is selected from:

[0157]

[0158] wherein R 4d is C 1-6 alkyl.

[0159] According to the present invention, unless otherwise indicated in the chemical formula, R 4a , R 4b , R 4c and / or R 4d can be attached at any position of the bicyclic structure.

[0160] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D is

[0161]

[0162] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D is selected from the following structures:

[0163]

[0164] wherein R 4a is -H; R 4b is halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0165] R 4a is halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and R 4d is methyl; or

[0166] R 4a is halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl;

[0167] or

[0168]

[0169] wherein R 4a is -H; R 4b is halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0170] R 4a is halogen, preferably chlorine or fluorine, more preferably fluorine; R 4bis -H; and R 4d is methyl; or

[0171] R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl;

[0172] or

[0173]

[0174] wherein R 4a is -H; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0175] R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and R 4d is methyl; or

[0176] R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is -methyl;

[0177] or

[0178]

[0179] wherein R 4b is -H or methyl; R 4c is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0180] R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4c is -H or methyl; R 4d is methyl; or

[0181] R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4c is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is -methyl.

[0182] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D is selected from the following structures:

[0183]

[0184] wherein R 4ais -H; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0185] R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and

[0186] R 4d is methyl; or

[0187] R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl;

[0188] or

[0189]

[0190] wherein R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or

[0191]

[0192] wherein R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is -methyl; or

[0193]

[0194] wherein R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4c is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is -methyl.

[0195] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein D is

[0196]

[0197] wherein R 4a is fluorine; R 4b is -H; and R 4d is methyl.

[0198] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R1 is methyl; R 2 is -H or a halogen; and R 3 is -H or a halogen.

[0199] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein

[0200] R 1 is methyl, R 2 is -H; and R 3 is -H; or

[0201] R 1 is methyl, R 2 is -H; and R 3 is fluorine; or

[0202] R 1 is methyl, R 2 is fluorine; and R 3 is -H.

[0203] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein B is selected from:

[0204]

[0205]

[0206] wherein R 7 is selected from H, -C 1-6 alkyl, -C 3-6 cycloalkyl and -OH; R 8 is (CH 2 ) n , where n is an integer from 1 to 3, preferably 1 or 2; R 9 is selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 10 is selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 11 is selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; X is CH or N; and Y is -O-, -S-, -S(O)-, -S(O) 2 -. In this embodiment, if X is N, then it is further preferred that R 7 is not -OH.

[0207] In another particularly preferred embodiment, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein B is selected from:

[0208]

[0209] wherein R 7 is selected from -H and -C 1-6 alkyl; R 9 is selected from -H and methyl, and preferably is -H; R 10 is selected from -H and methyl, and preferably is -H; R 11 is selected from -H and methyl, and preferably is -H; and Y is O.

[0210] Other particularly preferred compounds of the present invention are:

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] or a pharmaceutically acceptable salt thereof.

[0221] In one embodiment, the present invention relates to the compound of formula (I) in its non-salt form. In another embodiment, the present invention relates to the compound of formula (I) in a pharmaceutically acceptable salt form.

[0222] B, D, R as described above for formula (I) 1 , R 2 , R 3 , R 4a , R 4b , R 4c , R 4d , R 5 , R 5.1 , R 5.2 , R 5.3 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11Any one of the definitions of X and Y and each can be combined with each other.

[0223] On the one hand, the present invention relates to a pharmaceutical composition comprising at least one compound of formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0224] It has been found that the compound of formula (I) or a pharmaceutically acceptable salt thereof can be used for preventing and / or treating diseases and / or conditions in which modulating STING has a therapeutic benefit. Accordingly, on the other hand, the present invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one compound of formula (I) for use as a medicament. On the one hand, the present invention relates to a compound of formula (I), a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one of these compounds for treating feline or canine cancer.

[0225] On the other hand, the compounds of the present invention exhibit an interaction with canine STING (dSTING), which interaction is reflected by a T shift measured by DSF of > 15 °C, more preferably > 20 °C, and even more preferably > 25 °C. m shift.

[0226] In a preferred embodiment, the compounds of the present invention exhibit an interaction with dSTING as measured by DSF (dDSF) and also induce cytokine secretion in canine whole blood (dWB).

[0227] In a more preferred embodiment, the compounds of the present invention exhibit a combination of high dDSF and low dWB.

[0228] Therapeutic methods

[0229] On the one hand, the present invention relates to the use of a compound of formula (I), a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one of these compounds in a method of treating a disease.

[0230] Specifically, the compound of general formula (I) or a salt thereof is used for preventing and / or treating diseases and / or conditions in mammals (such as in cats, mice, pigs and dogs) in which modulating STING has a therapeutic benefit. Additionally, the compounds of the present invention are suitable as vaccine adjuvants due to their activity.

[0231] Diseases and conditions related to or modulated by STING include (but are not limited to) inflammatory, allergic or autoimmune diseases (such as allergic rhinitis or asthma), infectious diseases or cancer.

[0232] Autoimmune diseases include (but are not limited to) systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus (IDDM), dermatomyositis, and Sjogren's syndrome (SS).

[0233] The compounds of the present invention can be used to treat inflammation in any tissue and organ of the body, including (but not limited to) musculoskeletal inflammation, vascular inflammation, neural inflammation, digestive system inflammation, ocular inflammation, reproductive system inflammation, and other inflammations.

[0234] Examples of musculoskeletal inflammation that can be treated with the compounds of the present invention include: arthritis (including, for example, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, acute and chronic infectious arthritis, arthritis associated with gout and pseudogout, and juvenile idiopathic arthritis), tendinitis, synovitis, tenosynovitis, bursitis, fibrositis (muscle fiber pain), epicondylitis, myositis, and osteitis (including, for example, Paget's disease, osteitis pubis, and osteitis fibrosa cystica). Examples of ocular inflammation that can be treated with the compounds of the present invention include: blepharitis, blepharochalasis, conjunctivitis, dacryadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.

[0235] Examples of neural inflammation that can be treated with the compounds of the present invention include: encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis, and schizophrenia.

[0236] Examples of inflammation of the vascular or lymphatic system that can be treated with the compounds of the present invention include: joint stiffness, arthritis, phlebitis, vasculitis, and lymphangitis. Examples of inflammatory conditions of the digestive system that can be treated with the compounds of the present invention include: cholangitis, cholecystitis, enteritis, enterocolitis, gastritis, gastroenteritis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), ileitis, and proctitis.

[0237] Examples of inflammatory conditions of the reproductive system that can be treated with the compounds of the present invention include: cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis, and vulvodynia.

[0238] The compound can be used for treating autoimmune conditions having an inflammatory component. Such conditions include acute disseminated alopecia universalis, Behcet's disease, Chagas' disease, chronic fatigue syndrome, autonomic neuropathy, encephalomyelitis, ankylosing spondylitis, aplastic anemia, hidradenitis suppurativa, autoimmune hepatitis, autoimmune oophoritis, celiac disease, Crohn's disease, type 1 diabetes, giant cell arteritis, Goodpasture's syndrome, Grave's disease, Guillain-Barré syndrome, Hashimoto's disease, Henoch-Schonlein purpura, Kawasaki's disease, lupus erythematosus, microscopic colitis, microscopic polyarteritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, polyarteritis nodosa, polymyalgia, rheumatoid arthritis, Reiter's syndrome, Sjögren's syndrome, temporal arteritis, Wegener's granulomatosis, warm autoimmune haemolytic anemia, interstitial cystitis, Lyme disease, morphea, psoriasis, sarcoidosis, scleroderma, ulcerative colitis, and vitiligo.

[0239] The compound can be used for treating T cell-mediated hypersensitivity diseases having an inflammatory component. Such conditions include contact hypersensitivity, contact dermatitis (including contact dermatitis caused by poison ivy), urticaria, cutaneous allergy, respiratory allergy (hay fever, allergic rhinitis), and gluten-sensitive enteropathy (celiac disease).

[0240] Other inflammatory conditions treatable with the compounds include, for example, appendicitis, dermatitis, dermatomyositis, endocarditis, fibrositis, gingivitis, glossitis, hepatitis, hidradenitis suppurativa, iritis, laryngitis, mastitis, myocarditis, nephritis, otitis, pancreatitis, parotitis, pericarditis, peritonitis, pharyngitis, pleurisy, pneumonia, prostatitis, pyelonephritis, and stomatitis, transplant rejection (involving organs such as the kidney, liver, heart, lung, pancreas (e.g., islet cells), bone marrow, cornea, small intestine, skin allograft, skin homograft, and cardiac valve xenograft, serum sickness, and graft-versus-host disease), acute pancreatitis, chronic pancreatitis, acute respiratory distress syndrome, Sézary's syndrome, congenital adrenal hyperplasia, nonsuppurative thyroiditis, cancer-related hypercalcemia, pemphigus, bullous pemphigoid, severe erythema multiforme, exfoliative dermatitis, seborrheic dermatitis, seasonal or perennial allergic rhinitis, bronchial asthma, contact dermatitis, atopic dermatitis, drug hypersensitivity reactions, allergic conjunctivitis, keratitis, herpes zoster ophthalmicus, iritis, and iridocyclitis, choroidoretinitis, optic neuritis, symptomatic sarcoidosis, fulminant or disseminated tuberculosis chemotherapy, adult idiopathic thrombocytopenic purpura, adult secondary thrombocytopenia, acquired (autoimmune) hemolytic anemia, adult leukemia and lymphoma, childhood acute leukemia, regional enteritis, autoimmune vasculitis, multiple sclerosis, chronic obstructive pulmonary disease, solid organ transplant rejection, sepsis.

[0241] Preferred treatments include treatment of transplant rejection, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type 1 diabetes, asthma, inflammatory bowel disease, systemic lupus erythematosus, psoriasis, chronic lung disease, and inflammation associated with infectious conditions (e.g., sepsis).

[0242] On the one hand, the disease or condition to be treated with the compounds of the present invention is cancer. Examples of cancer diseases and conditions in which the compound of formula (I) or its salt or solvate may have potentially beneficial anti-tumor effects include (but are not limited to): lung cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, head cancer, neck cancer, uterine cancer, ovarian cancer, gastric cancer, colon cancer, colorectal cancer, breast cancer, esophageal cancer, small intestine cancer, intestinal cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, urethral cancer, prostate cancer, penile cancer, testicular cancer, ureteral cancer, bladder cancer, kidney cancer or liver cancer, cholangiocarcinoma; urothelial carcinoma; rectal cancer; anal area cancer; fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, renal pelvic cancer, renal cell carcinoma; sarcoma; soft tissue sarcoma; myxoma; rhabdomyoma; fibroma; lipoma; teratoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hemangioma; liver cancer; fibrosarcoma; chondrosarcoma; myeloma; chronic or acute leukemia; lymphocytic lymphoma; primary CNS lymphoma; CNS tumors; spinal axis tumors; squamous cell carcinoma; synovial sarcoma; malignant pleural mesothelioma; brainstem glioma; pituitary adenoma; bronchial adenoma; chondromatous hamartoma; mesothelioma; Hodgkin's Disease or a combination of one or more of the foregoing cancers.

[0243] Preferred cancers treatable with the compounds of the present invention are skin cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), liver cancer, pancreatic cancer, colon cancer, colorectal cancer, brain cancer, breast cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, cholangiocarcinoma, endometrial cancer, thyroid cancer, cervical cancer, gastric cancer, head cancer, neck cancer, sarcoma, soft tissue sarcoma, esophageal cancer, head and neck cancer, rectal and urothelial carcinoma, and lymphoma.

[0244] The novel compounds can be used for prophylactic, palliative, curative or semi-curative, short-term or long-term treatment of the diseases mentioned above, optionally also in combination with surgery, radiotherapy or other "state-of-the-art" compounds, such as cell growth inhibitors or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, antibodies, nanobodies, cancer targeting agents, viruses (including (but not limited to) oncolytic viruses) or immunogenic cell death inducers.

[0245] The novel compounds can also be used for the prophylactic, palliative, curative or semi-curative, short-term or long-term treatment of the diseases mentioned above by combining different routes of administration of the compounds (such as intravenous, intratumoral, subcutaneous, inhalation, oral, etc.), optionally also in combination with surgery, radiotherapy or other "state-of-the-art" compounds (such as cytostatic or cytotoxic substances, cell proliferation inhibitors, anti-angiogenic substances, steroids, antibodies, nanobodies, cancer targeting agents, viruses (including (but not limited to) oncolytic viruses) or immunogenic cell death inducers). Merely by way of example of surgery, partial or total tumor resection can be combined with the compounds of the present invention. Merely by way of example of radiotherapy, external beam radiotherapy can be combined with the compounds of the present invention.

[0246] In its role as an adjuvant, in certain embodiments the compounds and compositions of the present invention can be used as adjuvants in therapeutic or prophylactic strategies employing one or more vaccines. Thus, the compounds or salts thereof of the present invention can be used together with one or more vaccines selected to stimulate an immune response against one or more predetermined antigens. The compounds or salts thereof of the present invention can be provided together with such vaccines or provided as a supplement.

[0247] Such one or more vaccines can comprise inactivated or attenuated bacteria or viruses, which comprise the relevant antigens, purified antigens, live virus or bacterial delivery vectors that have been recombinantly engineered for the expression and / or secretion of antigens, antigen-presenting cell (APC) vectors comprising cells transfected with antigens or compositions comprising nucleic acids encoding antigens, liposomal antigen delivery agents or naked nucleic acid vectors encoding antigens. This list is not intended to be limiting. By way of example, such one or more vaccines can also comprise inactivated tumor cells or oncolytic viruses that express and secrete one or more of GM-CSF, CCL20, CCL3, IL-12p70, FLT-3 ligand, cytokines.

[0248] Accordingly, the present invention relates to a compound of general formula (I) for use as a medicament for treating cancer in cats or dogs, for example, or as a vaccine adjuvant for pigs, for example.

[0249] In a preferred aspect, the present invention relates to a compound of formula (I), a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one compound of formula (I) for treating cancer in cats or dogs.

[0250] In one embodiment, the present invention relates to a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one such compound for treating canine cancer, wherein the canine cancer is selected from osteosarcoma (OSA), oral melanoma, B-cell lymphoma, urothelial carcinoma (UC), angiosarcoma, mast cell tumor, soft tissue sarcoma, squamous cell carcinoma, T-cell lymphoma, mammary adenocarcinoma and anal sac carcinoma.

[0251] In another embodiment, the present invention relates to a compound or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one such compound, for use in the treatment of feline cancer, wherein the feline cancer is selected from B-cell and / or T-cell lymphoma, squamous cell carcinoma, mammary adenocarcinoma, mast cell tumor, and injection site sarcoma.

[0252] On the other hand, the present invention relates to a method for treating and / or preventing the diseases and conditions mentioned above, which comprises administering to an individual an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I).

[0253] In a particularly preferred embodiment, the present invention relates to a method for treating feline or canine cancer, which comprises administering to a cat or dog an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I).

[0254] In a more preferred embodiment, the present invention relates to a method for treating canine cancer, which comprises administering to a dog an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I), wherein the canine cancer is selected from osteosarcoma (OSA), oral melanoma, B-cell lymphoma, urothelial carcinoma (UC), angiosarcoma, mast cell tumor, soft tissue sarcoma, squamous cell carcinoma, T-cell lymphoma, mammary adenocarcinoma, and anal sac carcinoma.

[0255] In another more preferred embodiment, the present invention relates to a method for treating feline cancer, which comprises administering to a cat an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I), wherein the feline cancer is selected from B-cell and / or T-cell lymphoma, squamous cell carcinoma, mammary adenocarcinoma, mast cell tumor, and injection site sarcoma.

[0256] On the other hand, the present invention relates to a compound of general formula (I) for use in treating and / or preventing the cancers mentioned above before and / or after tumor resection and / or radiotherapy.

[0257] On the other hand, the present invention relates to the use of a compound of general formula (I) for the preparation of a medicament for treating and / or preventing the diseases and conditions mentioned above.

[0258] On the other hand, the present invention relates to a method for treating canine or feline cancer, which comprises administering to a dog or cat an effective amount of a compound of formula (I), a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising at least one compound of formula (I) in combination with radiotherapy.

[0259] Pharmaceutical composition

[0260] In another aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of the compounds mentioned above.

[0261] The pharmaceutical composition may be formulated in a manner suitable for administering a therapeutically effective amount of the compound. Suitable formulations for administering the compound of formula (I) will be apparent to those skilled in the art and include, for example, tablets, pills, capsules, suppositories, lozenges, troches, solutions, syrups, elixirs, cachets, injectable solutions (subcutaneous, intravenous, intramuscular, intraperitoneal, intratumoral and peritumoral), inhalants, infusions, elixirs, emulsions and powders. In addition, the compounds of the present invention may be administered via a targeted delivery platform, such as an antibody-drug conjugate, a nanobody-drug conjugate, a peptide-drug conjugate, a virus-like particle or a nanoparticle formulation.

[0262] Suitable tablets may be obtained, for example, by mixing one or more compounds of formula I with known excipients such as inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and / or lubricants.

[0263] For the purposes of the present invention, the pharmaceutical composition may be administered by a variety of means, including parenterally, non-parenterally, by inhalation spray, topically, nasally, orally or rectally in the form of a formulation containing a pharmaceutically acceptable carrier, adjuvant and vehicle. The pharmaceutical composition of the present invention may be administered in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension.

[0264] Combination therapy

[0265] The compounds of the present invention may be used alone or in combination with one or more additional therapeutic agents.

[0266] In another aspect, the present invention provides a method of treating a disease or condition in which modulating STING is beneficial, which comprises administering a therapeutically effective amount of a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent.

[0267] In another aspect, the present invention provides a method of treating an inflammatory, allergic or autoimmune disease, an infectious disease or cancer, which comprises administering a therapeutically effective amount of a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent.

[0268] Of course, the actual pharmaceutically effective amount or therapeutic dose will depend on factors known to those skilled in the art, such as the age and weight of the patient, the route of administration and the severity of the disease. In any case, the combination will be administered in a dose and manner that allows for the delivery of a pharmaceutically effective amount based on the patient's unique condition.

[0269] In certain embodiments, the compounds and compositions described herein are administered in combination with one or more additional compositions, the one or more additional compositions including vaccines intended to stimulate an immune response against one or more predetermined antigens; adjuvants; CTLA-4 and PD-1 pathway antagonists, lipids, liposomes, chemotherapeutic agents, immunomodulatory cell lines, cancer targeting agents, immunogenic cell death inducers, immunomodulators, where generally, immunomodulators can be understood as drugs of the general activation-regulatory type, and drugs that regulate and / or increase the frequency of a certain subtype of immune cells, etc.

[0270] The compounds and compositions described herein can be administered before, after, and / or simultaneously with additional therapeutic or prophylactic compositions or modalities.

[0271] The compounds, compositions of the invention (including any combination with one or more additional therapeutic agents) can be administered by mucosal (e.g., oral, sublingual, vaginal, nasal, cervical, etc.), intratumoral, intraperitoneal, peritumoral, percutaneous, inhaled, or parenteral (e.g., subcutaneous, intravenous, intramuscular, intraarterial, intradermal, intrathecal, and epidural administration) routes.

[0272] In addition, the compounds, compositions of the invention (including any combination with one or more additional therapeutic agents) can be administered via a targeted delivery platform, such as the targeted delivery platform can be an antibody-drug conjugate, a nanobody-drug conjugate, a peptide-drug conjugate, a virus-like particle, or a nanoparticle.

[0273] Among the possible administration methods, intraperitoneal, intratumoral, peritumoral, subcutaneous, inhaled, or intravenous administration is preferred. The compounds, compositions of the invention (including any combination with one or more additional therapeutic agents) can also be administered before, after, and / or simultaneously by a combination of different administration methods. By way of example only, intratumoral or peritumoral administration can be after inhaled or intravenous administration, or inhaled or intravenous administration can be after intratumoral or peritumoral administration. In addition, such administration of the compound via different routes can be before or after an additional therapeutic step, such as tumor resection or radiotherapy.

[0274] In a particularly preferred embodiment, the compounds of the invention, their pharmaceutically acceptable salts or pharmaceutical compositions comprising at least one compound of the invention are used in combination with radiotherapy. By way of example only, the compounds of the invention may be administered after radiotherapy. In addition, the compounds of the invention may be provided by intravenous administration after radiotherapy. In addition, the compounds of the invention may be provided by intravenous administration after tumor resection. In addition, the compounds of the invention may be provided by intratumoral administration after radiotherapy. In addition, the compounds of the invention may be provided by peritumoral administration after radiotherapy. In addition, the compounds of the invention may be provided by inhalation administration after tumor resection. In addition, the compounds of the invention may be provided by intravenous administration followed by intratumoral administration, and both administrations are carried out after radiotherapy. In addition, the compounds of the invention may be provided by intratumoral administration followed by intravenous administration, and both administrations are carried out after radiotherapy. In addition, the compounds of the invention may be provided by intravenous administration followed by peritumoral administration, and both administrations are carried out after radiotherapy. In addition, the compounds of the invention may be provided by peritumoral administration followed by intravenous administration, and both administrations are carried out after radiotherapy.

[0275] Methods for co-administering with additional therapeutic agents are well known in the art.

[0276] Due to the adjuvant properties of the compounds of the invention, they may also be used in combination with other therapeutic modalities, including other vaccines, adjuvants, antigens, antibodies and immunomodulators.

[0277] In addition to the compounds and their compositions of the invention described herein, the compositions or methods of the invention may further comprise one or more additional substances which, by their nature, can be used to stimulate or otherwise harness the immune system to respond to cancer antigens present on one or more targeted tumor cells.

[0278] The compounds of the invention may be used in combination with immune checkpoint inhibitors, such as immune checkpoint inhibitors selected from: CTLA-4 pathway antagonists, PD-1 pathway antagonists, Tim-3 pathway antagonists, Vista pathway antagonists, BTLA pathway antagonists, LAG-3 pathway antagonists or TIGIT pathway antagonists.

[0279] The compounds of the invention may be used in combination with immuno-oncology agonists, in combination with T cell receptor agonists or in combination with TNF receptor superfamily agonists or antagonists.

[0280] The compounds of the invention may be used in combination with therapeutic antibodies or therapeutic nanobodies. In some embodiments, the mechanism of action of the therapeutic antibody is antibody-dependent cell-mediated cytotoxicity (ADCC).

[0281] In other embodiments of the methods described herein, the compounds of the invention are used in combination with chemotherapeutic agents (e.g., small molecule drug compounds) known to those of skill in the art. Accordingly, the methods further comprise administering to the individual an effective amount of one or more chemotherapeutic agents as additional therapy or combination therapy.

[0282] It can also be used together / combinatorially with a compound of formula (I) or a pharmaceutically acceptable salt thereof (including all individual embodiments or general subsets of compound (I)), or one or more additional pharmacologically active substances used in pharmaceutical uses, uses, treatment and / or prevention methods as disclosed herein (above and below) include (but are not limited to): hormones, hormone analogs and antihormones (such as tamoxifen, toremifene, raloxifene, fulvestrant, megestrol acetate, flutamide, nilutamide, bicalutamide, aminoglutethimide, cyproterone acetate, finasteride, buserelin acetate, fludrocortisone, fluoxymesterone, medroxyprogesterone, octreotide); aromatase inhibitors (such as anastrozole, letrozole, liarozole, vorozole, exemestane, atamestane); LHRH agonists and antagonists (such as goserelin acetate, luprolide); inhibitors of growth factors and / or their corresponding receptors (growth factors such as: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), human epidermal growth factor (HER, such as HER2, HER3, HER4)) and / or their corresponding receptors;Inhibitors include, for example, (anti-)growth factor antibodies, (anti-)growth factor receptor antibodies, and tyrosine kinase inhibitors such as afatinib, dacomitinib, canertinib, neratinib, avitinib, poziotinib, AV412, PF-6274484, HKI 357, olmutinib, osimertinib, almonertinib, nazartinib, lazertinib, pelitinib, erlotinib, gefitinib, icotinib, sapitinib, lapatinib, varlitinib, vandetanib, TAK-285, AEE788, BMS599626 / AC-480, GW 583340, necitumumab, panitumumab, cetuximab, amivantanab, pertuzumab, trastuzumab, trastuzumab emtansine, or inhibitors of mutant EGFR, HER2 inhibitors with exon 20 mutations, and hepatocyte growth factor (HGF, c-MET, such as emibetuzumab, amivantanab, savolitinib, cabozantinib, foretinib); antimetabolites (such as methotrexate, raltitrexed, 5-fluorouracil (5-FU), capecitabine, floxuridine, gemcitabine, mercaptopurine, thioguanine, cladribine, pentostatin, cytarabine (ara-C), fludarabine, trifluridine, and the combination of tipiracil (=TAS102));Antitumor antibiotics (e.g., anthracyclins such as doxorubicin, doxil (pegylated liposomal doxorubicin hydrochloride), myocet (non-pegylated liposomal doxorubicin), daunorubicin, epirubicin, and idarubicin), mitomycin-C, bleomycin, dactinomycin, plicamycin, streptozocin); platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustin, meclorethamine, melphalan, chlorambucil, busulphan, dacarbazine); (dacarbazine), cyclophosphamide, ifosfamide, temozolomide, nitrosoureas such as carmustine and lomustine, thiotepa; antimitotic agents (e.g., Vinca alkaloids such as vinblastine, vindesine, vinorelbine, and vincristine; and taxanes such as paclitaxel, docetaxel, Abraxane); angiogenesis inhibitors (e.g., tasquinimod, bevacizumab), tubulin inhibitors; DNA synthesis inhibitors, PARP inhibitors, topoisomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrin, topotecan, irinotecan, mitoxantrone); serine / threonine kinase inhibitors (e.g., PDK 1 inhibitors, Raf inhibitors, A-Raf inhibitors, B-raf inhibitors, C-Raf inhibitors, mTOR inhibitors such as rapamycin, temsirolimus, everolimus, ridaforolimus, zotarolimus, sapanisertib, Torin 1, dactosilib, GDC-0349, vs-5584; vistusertib;AZD8055), mTORC1 / 2 inhibitors, PI3K inhibitors, PI3Kα inhibitors (such as alpelisib, serabelisib, GDC-0077, HH-CYH33, AMG 511, buparlisib, dactolisib, pictilisib, taselisib), dual mTOR / PI3K inhibitors, STK 33 inhibitors, AKT inhibitors, PLK 1 inhibitors, CDK4 / 6 inhibitors (such as palbociclib, ribociclib, abemaciclib, trilaciclib, PF-06873600), aurora kinase inhibitors; tyrosine kinase inhibitors (such as PTK2 / FAK inhibitors); protein-protein interaction inhibitors (such as IAP inhibitors / SMAC mimetics, MCL-1 (such as AZD-5991, AMG-176, AMG-397, S64315, S63845, A-1210477), MDM2, MDM2 / MDMX); MEK inhibitors (such as trametinib, cobimetinib, binimetinib, selumetinib, refametinib); SOS1-inhibitors (i.e., compounds that modulate / inhibit the GEF function of SOS1, for example, by binding to SOS1 and preventing the protein-protein interaction between SOS1 and (mutant) Ras proteins, such as KRAS; for example, BAY-293), inhibitors of GDP-loaded or GTP-loaded RAS and / or any of its mutants (i.e., compounds that modulate / inhibit the function of (mutant) RAS proteins, for example, by binding to GDP-loaded or GTP-loaded (mutant) RAS proteins, such as KRAS, NRAS, and / or HRAS, preferably KRAS); irreversible inhibitors of KRAS G12C (AMG-510, MRTX849, ARS-324, GDC-6036); reversible or irreversible binders for GDP-loaded (mutant) KRAS; reversible or irreversible binders for GTP-loaded (mutant) KRAS; ALK inhibitors (such as crizotinib, alectinib, entrectinib, brigatinib, ceritinib); ERK inhibitors; FLT3 inhibitors; BRD4 inhibitors; IGF-1R inhibitors; TRAILR2 agonists;Bcl-xL inhibitors; Bcl-2 inhibitors (such as venetoclax, obatoclax, navitoclax, oblimersen); Bcl-2 / Bcl-xL inhibitors; ErbB receptor inhibitors; BCR-ABL inhibitors; ABL inhibitors; Src inhibitors (such as dasatinib, ponatinib, bosutinib, vandetanib, KX-01, saracatinib, KX2-391, SU 6656, WH-4-023); rapamycin analogs (such as everolimus, temsirolimus, deforolimus, sirolimus); androgen synthesis inhibitors; androgen receptor inhibitors; DNMT inhibitors; HDAC inhibitors; ANG1 / 2 inhibitors; histone deacetylase inhibitors; IL6 inhibitors; inhibitors of JAK and / or any of its mutants; inhibitors of A-Raf and / or B-Raf and / or C-Raf and / or any of its mutants (encorafenib, dabrafenib, vemurafenib, PLX-8394, RAF-709 (=Example 131 in WO 2014 / 151616), LXH254, sorafenib, LY-3009120 (=Example 1 in WO 2013 / 134243), lifirafenib, TAK-632, agerafenib, CCT196969, RO5126766, RAF265); inhibitors of receptor tyrosine kinase (RTK) and / or any of its mutants; inhibitors of SHP2 and / or any of its mutants (such as SHP099, TNO155, RMC-4550, RMC-4630, IACS-13909); CYP17 inhibitors; radiopharmaceuticals; proteasome inhibitors (such as carfilzomib);Immunotherapeutic agents such as immune checkpoint inhibitors (e.g., CTLA4, PD1, PD-L1, PD-L2, LAG3, SIRPα-antibodies, and TIM3-binding molecules / immunoglobulins (ipilimumab, nivolumab, pembrolizumab, tislelizumab, atezolizumab, avelumab, durvalumab, pidilizumab, PDR-001 (= spartalizumab), AMG-404, ezabenlimab, sintilimab, camrelizumab, toribalimab, tislelizumab)); ADCC (antibody-dependent cell-mediated cytotoxicity) enhancers (e.g., anti-CD33 antibody, anti-CD37 antibody, anti-CD20 antibody); T cell engagers such as PSMA×CD3, B7H6 / CD3 (as disclosed in, for example, WO2021 / 064137), DLL3 / CD3 (as disclosed in, for example, WO2019 / 234220), e.g., bispecific T cell engagers; (such as, for example, CD3×BCMA, CD3×CD33, CD3×CD19), cancer vaccines, MDM2 inhibitors, oncolytic viruses, and various chemotherapeutic agents such as amifostin, anagrelid, clodronat, filgrastin, interferons, interferon α, leucovorin, procarbazine, levamisole, mesna, mitotane, pamidronate, and porfimer. The compounds of the present invention can be used in combination with the following: OX40 agonists, ICOS ligands, CD27 agonists, GITR agonists, Toll-like receptor agonists.

[0283] In a preferred embodiment, one or more additional pharmacologically active substances that can also be used in combination with the compound of formula (I) or a pharmaceutically acceptable salt thereof (including all individual embodiments or general subsets of compound (I)), or in pharmaceutical uses, uses, methods of treatment and / or prevention as disclosed herein (above and below) include: checkpoint inhibitors (ipilimumab, nivolumab, pembrolizumab, tislelizumab, atezolizumab, avelumab, durvalumab, pidilizumab, PDR-001 (= spartalizumab), AMG-404, epratuzumab, sintilimab, camrelizumab, tremelimumab, tislelizumab), taxanes (paclitaxel, docetaxel, albumin-bound paclitaxel (Abraxane)), T cell engagers (such as PSMA×CD3, B7H6 / CD3 (as disclosed in, for example, WO2021 / 604137), DLL3 / CD3 (as disclosed in, for example, WO2019 / 234220), for example bispecific T cell engagers (such as, for example, CD3×BCMA, CD3×CD33, CD3×CD19), cancer vaccines, MDM2 inhibitors and oncolytic viruses.

[0284] In other embodiments of the methods described herein, the compounds of the invention are used in combination with chemotherapeutic agents and / or additional drugs (such as cancer-targeted therapies) for treating the indications described in the methods herein. Accordingly, the methods further comprise administering to the individual an effective amount of one or more cancer-targeting agents as additional therapy or combination therapy.

[0285] In other embodiments of the methods described herein, the compounds of the invention are used in combination with chemotherapeutic agents and / or additional drugs and / or additional therapies (such as radiotherapy and / or tumor resection) for treating the indications described in the methods herein.

[0286] On the other hand, the present invention relates to a method of treating a STING-related or STING-regulated disease or condition in a patient, which comprises the step of administering to a patient in need of such treatment a therapeutically effective amount of a compound of the invention and a therapeutically effective amount of one or more of the additional therapeutic agents described above.

[0287] The combined use of the compounds of the invention with additional therapeutic agents can be carried out simultaneously or at staggered times.

[0288] The compounds of the invention and one or more additional therapeutic agents can be present together in one formulation or independently in two identical or different formulations, for example as a so-called kit-of-parts.

[0289] Accordingly, on the other hand, the present invention provides a combination comprising a compound of general formula (I) and at least one additional therapeutic agent.

[0290] On the other hand, the present invention provides a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent and one or more pharmaceutically acceptable excipients.

[0291] On the other hand, the present invention provides a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent for use in therapy.

[0292] On the other hand, the present invention provides a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent for use in the treatment of a disease or condition in which modulating STING is beneficial.

[0293] On the other hand, the present invention provides a combination comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent for use in the treatment of cancer (such as canine or feline cancer).

[0294] On the other hand, the present invention relates to a pharmaceutical composition comprising a compound of the present invention and one or more additional therapeutic agents described hereinabove, optionally together with one or more inert carriers and / or diluents.

[0295] Other features and advantages of the present invention will become apparent from the following more detailed examples, which illustrate the principles of the present invention by way of example.

[0296] Chemical synthesis

[0297] List of abbreviations

[0298]

[0299]

[0300] Other features and advantages of the present invention will become apparent from the following more detailed examples, which illustrate the principles of the present invention by way of illustration without limiting its scope.

[0301] Review

[0302] Unless otherwise stated, all reactions are carried out using common methods in a chemical laboratory in commercially available equipment. Starting materials sensitive to air and / or moisture are stored under a protective gas, and the corresponding reactions and operations are carried out under a protective gas (nitrogen or argon). The compounds of the present invention are named according to IUPAC guidelines. If a compound is represented by a structural formula and by its name, in case of conflict, the structural formula shall prevail.

[0303] Chromatography

[0304] Thin layer chromatography was carried out on ready-made TLC plates of silica gel 60 on glass (with fluorescent indicator F-254) manufactured by Merck.

[0305] The Biotage Isolera Four device was used together with an Interchim Puri Flash column (50 μm, 12 - 300 g) or a glass column packed with silica gel (Granula Silica Si-60A 35 - 70 μm) manufactured by Millipore for automated preparative NP chromatography.

[0306] Preparative RP HPLC was carried out using columns manufactured by Waters (Sunfire C18, 10 μm, 30×100 mm part number 186003971 or X-Bridge C18, 10 μm, 30×100 mm part number 186003930). Different gradients of H 2 O / acetonitrile or H 2 O / MeOH (where 0.1% TFA was added to water) were used, or an alkaline buffer aqueous solution (1 L of water contained 5 mL of ammonium bicarbonate solution (158 g / 1L H 2 O) and 2 mL of ammonia (solution of 7 mol / l in MeOH)) was used instead of the water-TFA-mixture to elute the compounds with different gradients.

[0307] Analytical HPLC (reaction monitoring) of intermediate compounds was carried out using columns manufactured by Waters and Phenomenex. In each case, the analytical device was also equipped with a mass detector.

[0308] HPLC mass spectrometry / UV spectrometry

[0309] The retention time / MS-ESI+ of the example compounds for characterizing the present invention was determined using, for example, an HPLC-MS device manufactured by Agilent (high performance liquid chromatography with a mass detector). The retention time tR of the compound eluted at the injection peak was 0.

[0310] Analytical HPLC method:

[0311] Acidic method

[0312] HPLC: Agilent 1260 Infinity II

[0313] MS: Agilent LC / MS (G6125B)

[0314] Column: Sunfire C18 2.5 μm, 3.0×30 mm

[0315] Eluent: A: H2O containing 0.1% TFA (v / v); B: MeCN (HPLC grade) 2 O; B: MeCN (HPLC grade)

[0316] Detection: MS: positive and negative modes

[0317] Column temperature: 60 °C

[0318] Gradient: 0.00 - 0.20 min: 3% B (flow rate 2.2 mL / min)

[0319] 0.20 - 1.20 min: 3% to 100% B (flow rate 2.2 mL / min)

[0320] 1.20 - 1.25 min: 100% B (flow rate 3.0 mL / min)

[0321] 1.25 - 1.40 min: 100% B (flow rate 3.0 mL / min)

[0322] Alkaline method

[0323] HPLC: Agilent 1260 Infinity II

[0324] MS: Agilent LC / MS (G6125B)

[0325] Column: X-Bridge C18, 2.5 μm, 3.0 × 30 mm

[0326] Eluent: A: H2O containing 0.1% NH4OH (v / v); B: MeCN (HPLC grade) 2 O; B: MeCN (HPLC grade)

[0327] Detection: MS: positive and negative modes

[0328] Column temperature: 60 °C

[0329] Gradient: 0.00 - 0.20 min: 3% B (flow rate 2.2 mL / min)

[0330] 0.20 - 1.20 min: 3% to 100% B (flow rate 2.2 mL / min)

[0331] 1.20 - 1.25 min: 100% B (flow rate 3.0 mL / min)

[0332] 1.25 - 1.40 min: 100% B (flow rate 3.0 mL / min)

[0333] Preparative HPLC method:

[0334] Acidic method

[0335] HPLC: Agilent 1260 Infinity II

[0336] MS: Agilent LC / MS (G6125B)

[0337] Column: Sunfire C18 10μm, 30×300mm

[0338] Eluent: A: H2O containing 0.1% TFA (v / v); B: MeCN (HPLC grade)

[0339] Detection: MS: positive mode and negative mode

[0340] Flow rate: 50 mL / min

[0341] Column temperature: 40 °C

[0342] Alkaline method

[0343] HPLC: Agilent 1260 Infinity II

[0344] MS: Agilent LC / MS (G6125B)

[0345] Column: X-Bridge C18, 10μm, 30×300mm

[0346] Eluent: A: H 2 O containing 0.1% NH4OH (v / v); B: MeCN (HPLC grade)

[0347] Detection: MS: positive mode and negative mode

[0348] Flow rate: 50 mL / min

[0349] Column temperature: 40 °C

[0350] Preparing the compounds of the present invention

[0351] The compounds of the present invention and their intermediates can be obtained using synthetic methods known to those skilled in the art and described in the organic synthesis literature. These methods are intended to be illustrative of the present invention and do not limit the scope of its subject matter and the claimed compounds. Preferably, the compounds are obtained in a manner similar to the preparation methods more fully explained below (specifically as described in the experimental section). In some cases, the order of performing the reaction steps may vary. Variations of the reaction methods known to those skilled in the art but not described in detail herein may also be used.

[0352] The general method for preparing the compounds of the present invention will be apparent to those skilled in the art studying the following routes. The starting materials can be prepared by the methods described in the literature or herein, or can be prepared in a similar or analogous manner. Any functional groups in the starting materials or intermediates can be protected using conventional protecting groups. These protecting groups can be cleaved at appropriate stages within the reaction sequence using methods familiar to those skilled in the art.

[0353] One method for preparing the compound of formula (I) is illustrated in Route I: Indazole B can be synthesized from o-methylaniline derivative A. Subsequent iodination gives 3-iodo-indazole C. Intermediate D can be obtained, for example, by Chan-Lam coupling using (6-fluoropyridin-3-yl)boronic acid. The conversion to intermediate F can be achieved, for example, via Suzuki coupling with intermediate E. Finally, the compound of formula (I) is synthesized, for example, by nucleophilic aromatic substitution. The product is separated by conventional means and preferably purified by chromatography.

[0354] Route I:

[0355]

[0356] A second method for preparing the compound of formula (I) is illustrated in Route II: Intermediate D can be converted to intermediate G via various methods, for example, by nucleophilic aromatic substitution. Intermediate G can be converted to the compound of formula (I), for example, via Suzuki coupling with intermediate E. The product is separated by conventional means and preferably purified by chromatography.

[0357] Route II:

[0358]

[0359] A third method for preparing the compound of formula (I) is illustrated in Route III: (6-fluoropyridin-3-yl)boronic acid can be converted to intermediate H via various methods, for example, by nucleophilic aromatic substitution. Intermediate G can be obtained from intermediate H, for example, by Chan-Lam coupling. Intermediate G can be converted to the compound of formula (I), for example, via Suzuki coupling with intermediate E. The product is separated by conventional means and preferably purified by chromatography.

[0360] Route III:

[0361]

[0362] Preparation of Intermediates

[0363] Intermediate 1

[0364] 4-Bromo-3-fluoro-2-methyl-2H-indazole

[0365]

[0366] To a solution of 4-bromo-2-methyl-2H-indazole (100 mg, 0.46 mmol) in MeCN (1 ml) was added 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane-bis-(tetrafluoroborate) (Selectfluor, 204 mg, 0.55 mmol), and the mixture was heated at 80 °C for 2 h using microwave. The crude reaction mixture was purified by preparative HPLC (acidic method). Two products were isolated, namely the title compound (4-bromo-3-fluoro-2-methyl-2H-indazole, 9 mg) and the regioisomer (4-bromo-7-fluoro-2-methyl-2H-indazole, 7 mg).

[0367] Alternatively, to a solution of 4-bromo-2-methyl-2H-indazole (1 g, 4.5 mmol) in anhydrous THF (20 ml) cooled to -78 °C was added LDA solution (2 M in THF, 5.23 ml, 10.5 mmol) and the mixture was stirred at this temperature for 30 min. Subsequently, N-fluorobenzenesulfonimide (NFSI, 2.93 g, 9.2 mmol) was added and the mixture was slowly warmed to room temperature and stirred for 2.5 h. The reaction was carefully quenched with water, diluted with DMF, acidified with TFA and purified by preparative HPLC (acidic method). The title compound (371 mg) was isolated.

[0368] 1H NMR (400 MHz, DMSO-d6) δ ppm 4.03 (d, J = 1.90 Hz, 3H) 7.15 (dd, J = 8.68, 7.16 Hz, 1H) 7.27 (d, J = 7.22 Hz, 1H) 7.50 (dd, J = 8.68, 1.84 Hz, 1H).

[0369] Intermediate 2

[0370] (3-Fluoro-2-methyl-2H-indazol-4-yl)boronic acid

[0371]

[0372] A mixture of intermediate 1 (445 mg, 1.94 mmol), bis-(neopentyl glycolato)diboron (543 mg, 2.33 mmol), and potassium acetate (763 mg, 7.77 mmol) in dioxane (10 ml) was degassed and maintained under a nitrogen atmosphere. [1,1'-Bis-(diphenylphosphino)-ferrocenyl]-dichloropalladium(II) DCM complex (79 mg, 0.097 mmol) was added to the mixture and it was heated at 85 °C for 2 hours. MeOH was added to the reaction mixture and then it was filtered. The filtrate was diluted with water and purified by preparative HPLC (acidic method) to give the title compound (200 mg).

[0373] 1H NMR (400 MHz, DMSO-d6) δ ppm 4.01 (d, J = 1.77 Hz, 3H), 7.12 (dd, J = 6.72, 2.79 Hz, 6H), 7.35 (dd, J = 8.81, 6.91 Hz, 6H), 7.53 (dd, J = 8.81, 1.71 Hz, 6H).

[0374] Intermediate 3

[0375] 1-(6-Fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0376]

[0377] To a stirred reaction mixture of 3-iodo-7-methyl-1H-indazole (16 g, 62 mmol) in DCM (200 ml) was added copper(II) acetate (16.89 g, 93 mmol), pyridine (9.80 g, 124 mmol), and 6-fluoropyridine-3-boronic acid (14.85 g, 105 mmol), and then the reaction mixture was stirred at room temperature for 72 hours. The reaction mixture was filtered through diatomaceous earth, the filtrate was concentrated and the crude product was purified by silica gel chromatography (EtOAc:hexane) to give the title compound (13 g).

[0378] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.09 (s, 3H), 7.25 (m, 1H), 7.34 (m, 1H), 7.42 (m, 2H), 8.28 (ddd, J = 8.65, 7.07, 2.79 Hz, 1H), 8.53 (d, J = 2.15 Hz, 1H).

[0379] Intermediate 4

[0380] (1R,5S,6R)-3-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester

[0381]

[0382] To a mixture of 1-(6-fluoro-3-pyridyl)-3-iodo-7-methyl-1H-indazole (Intermediate 3, 1 g, 2.83 mmol) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (1.037 g, 5.66 mmol) in NMP (6 ml) was added DIPEA (1.974 ml, 11.61 mmol). The reaction mixture was heated at 80 °C for 16 h. The reaction mixture was cooled and added to water. The precipitate formed was collected by filtration and dried under vacuum. The solid was purified by silica gel chromatography (cyclohexane:EtOAc) to give the title compound (1.14 g).

[0383] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.61 (t, J = 3.04 Hz, 1H), 2.08 (s, 3H), 2.26 (br s, 2H), 3.55 (dd, J = 9.19, 1.58 Hz, 2H), 3.63 (s, 3H), 3.83 (d, J = 10.90 Hz, 2H), 6.57 (d, J = 8.87 Hz, 1H), 7.18 (m, 1H), 7.25 (d, J = 6.84 Hz, 1H), 7.35 (d, J = 7.98 Hz, 1H), 7.67 (dd, J = 8.87, 2.66 Hz, 1H), 8.21 (d, J = 2.53 Hz, 1H).

[0384] Intermediate 5

[0385] (1R,5S,6R)-3-(5-{3'-Fluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester

[0386]

[0387] A mixture of Intermediate 4 (90 mg, 0.19 mmol), Intermediate 2 (39 mg, 0.2 mmol), and sodium carbonate (60 mg, 0.57 mmol) in dioxane (2 ml) and water (0.5 ml) was degassed and maintained under a nitrogen atmosphere. [1,1'-Bis-(diphenylphosphino)-ferrocenyl]-dichloropalladium(II) (PdCl 2 dppf, 6.9 mg, 0.009 mmol) was added to the mixture and the mixture was heated at 100 °C for 45 min. DMF was added to the reaction mixture, and then the reaction mixture was filtered. The filtrate was diluted with water, acidified with TFA and purified by preparative HPLC (acidic method) to give the title compound (46 mg).

[0388] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.65 (t, J = 3.04 Hz, 1H), 2.19 (s, 3H), 2.29 (br s, 2H), 3.64 (s, 4H), 3.60 (m, 2H), 3.86 (d, J = 11.03 Hz, 2H), 4.02 (d, J = 1.77 Hz, 3H), 6.69 (d, J = 9.00 Hz, 1H), 7.20 (m, 1H), 7.25 (m, 1H), 7.41 (m, 1H), 7.48 (m, 1H), 7.56 (dd, J = 8.74, 1.01 Hz, 1H), 7.83 (m, 2H), 8.33 (d, J = 2.41 Hz, 1H).

[0389] Intermediate 6

[0390] 3'-Fluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0391]

[0392] A mixture of intermediate 3 (365 mg, 1.03 mmol), intermediate 2 (200 mg, 1.03 mmol) and sodium carbonate (328 mg, 3.1 mmol) in dioxane (8 ml) and water (2 ml) was degassed and maintained under a nitrogen atmosphere. [1,1'-Bis-(diphenylphosphino)-ferrocenyl]-dichloropalladium(II) (PdCl 2 dppf, 38 mg, 0.05 mmol) was added to the mixture and the mixture was heated at 100 °C for 2.5 h. DMF was added to the reaction mixture, and then the reaction mixture was filtered. The filtrate was diluted with water, acidified with TFA and purified by preparative HPLC (acidic method) to give the title compound (260 mg).

[0393] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.07 (s, 1H), 2.18 (s, 3H), 4.03 (d, J = 1.65 Hz, 3H), 7.26 (m, 1H), 7.32 (m, 1H), 7.44 (m, 2H), 7.52 (d, J = 6.72 Hz, 1H), 7.59 (dd, J = 8.68, 1.33 Hz, 1H), 7.89 (d, J = 7.98 Hz, 1H), 8.35 (t, J = 7.81 Hz, 1H), 8.60 (d, J = 2.53 Hz, 1H).

[0394] Intermediate 7

[0395] (7-Fluoro-2-methyl-2H-indazol-4-yl)boronic acid

[0396]

[0397] Using the method described for Intermediate 2: The title compound (104 mg) was obtained from 4-bromo-7-fluoro-2-methyl-2H-indazole (100 mg).

[0398] Intermediate 8

[0399] 7'-Fluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0400]

[0401] Using the method described for Intermediate 5: The title compound (90 mg) was obtained from Intermediate 3 (120 mg) and Intermediate 7 (89 mg).

[0402] Intermediate 9

[0403] 2',7-Dimethyl-1H,2'H-3,4'-biindazole

[0404]

[0405] A mixture of 2-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (CAS: 845751-67-9; 5.5 g, 21.3 mmol) and K 2 CO 3 (6.69 g, 48.44 mmol) in EtOH (60 ml) was degassed and maintained under an argon atmosphere. Water (12.5 ml) was added to this mixture, followed by bis(di-tert-butyl(4-dimethylaminophenyl)phosphine)dichloropalladium(II) ((AmPhos)2*PdCl 2 : 343 mg, 0.49 mmol) and then 3-iodo-7-methyl-1H-indazole (CAS: 847906-27-8; 5 g, 19.4 mmol). The reaction mixture was heated at 75 °C for 1 h. Water (65 ml) containing N-acetyl-L-cysteine (3.16 g, 19.38 mmol) was added. The reaction mixture was cooled and the solid formed was collected by filtration to give the title compound (4.68 g).

[0406] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.60 (s, 3H), 4.25 (s, 3H), 7.14 (t, J = 7.17 Hz, 1H), 7.21 (d, J = 6.84 Hz, 1H), 7.39 (dd, J = 8.62, 6.97 Hz, 1H), 7.63 (d, J = 8.74 Hz, 1H), 7.73 (d, J = 6.59 Hz, 1H), 7.98 (d, J = 7.98 Hz, 1H), 8.70 (s, 1H), 13.39 (s, 1H).

[0407] Intermediate 10

[0408] 5'-Fluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0409]

[0410] To a solution of intermediate 9 (400 mg, 1.53 mmol) in dioxane (5 ml) and water (5 ml) was added NFSI (981 mg, 3.05 mmol) and the mixture was stirred at 80 °C for 16 h. DMF was added to the reaction mixture and then the mixture was filtered. The filtrate was diluted with water, acidified with TFA and purified by preparative HPLC (acidic method) to give the title compound (60 mg) and regioisomer (7'-fluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole, 30 mg) and difluorinated compound (5',7'-difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole, 40 mg).

[0411]

[0412] Intermediate 11

[0413] 5'-Fluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0414]

[0415] To a solution of intermediate 10 (60 mg, 0.21 mmol) and (6-fluoropyridin-3-yl)boronic acid (90.5 mg, 0.64 mmol) in DCM (5 ml) was added pyridine (51.8 μl, 0.64 mmol), then copper(II) acetate (58.3 mg, 0.32 mmol) and the mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with saturated NaHCO 3 (aqueous solution) and extracted with DCM, the organic layers were combined, dried over Na 2 SO 4Dry, filter and concentrate under reduced pressure. Purify the residue by preparative HPLC (acidic method) to afford the title compound (49 mg).

[0416] Intermediate 12

[0417] 5',7'-Difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0418]

[0419] Intermediate 12 was isolated from the synthesis of Intermediate 10 and the synthetic method is described herein and afforded the title compound (40 mg).

[0420] Alternatively,

[0421] To a solution of 7'-fluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole (960 mg, 3.43 mmol) in MeCN (30 mL) was added SelectFluor (971 mg, 2.74 mmol) and the mixture was stirred at room temperature for 45 minutes. An additional amount of SelectFluor (260 mg) was added with stirring at room temperature for 40 minutes. The reaction mixture was concentrated and redissolved in DMF, basified with aqueous NH 3 and purified by preparative HPLC (basic) to afford the desired product (100 mg, 10%).

[0422] The following by-product (8 mg, 3',5',7'-trifluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole) was also isolated.

[0423]

[0424] Intermediate 13

[0425] 5',7'-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0426]

[0427] Using the method described for Intermediate 11: The title compound (18 mg) was obtained from Intermediate 12 (40 mg) and (6-fluoropyridin-3-yl)boronic acid (38 mg).

[0428] Alternatively, SelectFluor (970.6 mg, 2.74 mmol) was added to a solution of intermediate 8 (960 mg, 3.425 mmol) in MeCN (30 ml) and stirred for 30 minutes. Another portion of SelectFluor (260 mg) was added and stirred for 40 minutes. The reaction mixture was concentrated and purified by preparative HPLC (basic) to afford the title compound (300 mg, 29%).

[0429] HPLC (basic): Rt = 1.182 min ((M+H)+ 299.0)

[0430] The following by-products were also isolated:

[0431] 5,5',7'-Trifluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole (100 mg, 10%)

[0432]

[0433] 3',5',7'-Trifluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole (80 mg, 8%)

[0434]

[0435] HPLC (acidic): Rt = 1.011 min ((M+H)+ 317.0)

[0436] Intermediate 14

[0437] 3'-Fluoro-2',7-dimethyl-1-[6-(piperazin-1-yl)pyridin-3-yl]-1H,2'H-3,4'-biindazole

[0438]

[0439] Using the method described for Example 2: The title compound (35 mg) was obtained from intermediate 6 (65 mg, 0.17 mmol) and piperazine (45.2 μl, 0.52 mmol) after preparative HPLC (basic method).

[0440] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.19 (s, 3H), 2.82 (m, 4H), 3.53 (m, 4H), 4.02 (d, J = 1.65 Hz, 3H), 6.95 (d, J = 9.00 Hz, 1H), 7.19 (m, 1H), 7.24 (m, 1H), 7.41 (m, 1H), 7.49 (m, 1H), 7.55 (d, J = 9.51 Hz, 1H), 7.76 (dd, J = 9.00, 2.66 Hz, 1H), 7.84 (d, J = 7.98 Hz, 1H), 8.32 (d, J = 2.66 Hz, 1H).

[0441] Intermediate 15

[0442] (1R,5S,6R)-Methyl 3-(5-iodopyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylate

[0443]

[0444] Heat a mixture of 2-fluoro-5-iodopyridine (6.0 g, 26.91 mmol), (1R,5S,6R)-methyl 3-azabicyclo[3.1.0]hexane-6-carboxylate hydrochloride (5.7 g, 32.29 mmol), and potassium carbonate (8.2 g, 59.20 mmol) in NMP (20 ml) under an argon atmosphere and stir at 120 °C for 16 h. Cool the reaction mixture, pour it into water, and stir for 1 h. Collect the solid formed by filtration, dry it under vacuum at 50 °C, and obtain the title compound (7.08 g).

[0445] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.52 (t, J = 3.04 Hz, 2H), 2.20 (m, 4H), 3.40 (dt, J = 10.96, 1.55 Hz, 4H), 3.61 (s, 6H), 3.69 (d, J = 10.77 Hz, 4H), 6.36 (d, J = 8.62 Hz, 2H), 7.72 (dd, J = 8.81, 2.34 Hz, 2H), 8.21 (d, J = 1.77 Hz, 2H)

[0446] Intermediate 16

[0447] 5-Fluoro-3-iodo-7-methyl-1H-indazole

[0448]

[0449] To a solution of 5-fluoro-7-methyl-1H-indazole (CAS: 1427377-45-4, 4 g, 26.64 mmol) in DMF (24 ml) and water (6 ml) was added potassium phosphate (8.48 g, 39.96 mmol). To this solution was slowly added portionwise iodine (7.44 g, 29.30 mmol). The reaction mixture was stirred at 30 °C for 1 h. Subsequently, a solution of sodium metabisulfite (7.6 g) in water (88 ml) was added to the reaction mixture and the mixture was stirred for 2.5 h. The solid formed was collected by filtration and the solid was washed with water. The solid was dried at 45 °C for 16 h.

[0450] 1H NMR (400 MHz, DMSO-d6) δ ppm 6.98 (dd, J = 8.62, 2.28 Hz, 2H), 7.16 (br d, J = 9.76 Hz, 2H), 13.69 (br s, 2H)

[0451] Intermediate 17

[0452] 3',5-difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0453]

[0454] Using the method described for Intermediate 5: The title compound (54 mg) was obtained from Intermediate 16 (100 mg, 0.33 mmol) and 3-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (111 mg, 0.39 mmol).

[0455] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.59 (s, 3H), 4.04 (d, J = 1.77 Hz, 3H), 7.14 (m, 1H), 7.38 (m, 2H), 7.46 (dd, J = 9.31, 2.09 Hz, 1H), 7.52 (m, 1H), 13.55 (m, 1H)

[0456] Intermediate 18

[0457] 6-fluoro-3-iodo-7-methyl-1H-indazole

[0458]

[0459] Using the method described for Intermediate 16: The title compound (7.55 g) was obtained from 6-fluoro-7-methyl-1H-indazole (CAS 1427395-91-2, 4.3 g, 28.64 mmol).

[0460] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.41 (m, 3H), 7.05 (t, J = 9.38 Hz, 1H), 7.27 (dd, J = 8.74, 4.82 Hz, 1H), 13.67 (s, 1H)

[0461] Intermediate 19

[0462] 6-Fluoro-1-(6-fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0463]

[0464] Using the method described for Intermediate 11: After recrystallization from Intermediate 18 (5 g, 17.39 mmol) and 2-fluoropyridine-5-boronic acid (3.75 g, 26.08 mmol) in EtOH, the title compound (3.35 g) was obtained.

[0465] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.95 (d, J = 1.77 Hz, 3H), 7.23 (t, J = 9.38 Hz, 1H), 7.44 (m, 2H), 8.30 (ddd, J = 8.68, 7.03, 2.79 Hz, 1H), 8.54 (d, J = 2.15 Hz, 1H)

[0466] Intermediate 20

[0467] (1R,5S,6R)-3-[5-(6-Fluoro-3-iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester

[0468]

[0469] Using the method described for Intermediate 4: From Intermediate 19 (2.0 g, 5.39 mmol) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (1.48 g, 8.08 mmol), the title compound (2.33 g) was obtained.

[0470] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.61 (t, J = 2.98 Hz, 1H), 1.95 (d, J = 1.27 Hz, 3H), 2.26 (br s, 2H), 3.55 (m, 2H), 3.63 (s, 3H), 3.83 (d, J = 10.90 Hz, 2H), 6.58 (d, J = 8.87 Hz, 1H), 7.17 (t, J = 9.24 Hz, 1H), 7.37 (dd, J = 8.81, 5.01 Hz, 1H), 7.69 (dd, J = 9.00, 2.66 Hz, 1H), 8.23 (d, J = 2.53 Hz, 1H)

[0471] Intermediate 21

[0472] 4-Bromo-7-fluoro-1H-indazole

[0473]

[0474] Stir a solution of 6-bromo-2,3-difluorobenzaldehyde (180 g, 0.82 mol, 1 equiv) in DME (900 mL) and hydrazine hydrate (900 mL) at 90 °C for 40 h. Concentrate the resulting mixture under reduced pressure. Grind the residue with n-hexane (500 mL). Filter the mixture and wash the cake with n-hexane (200 mL). This gives 4-bromo-7-fluoro-2H-indazole (140 g) as a pale yellow solid.

[0475] Intermediate 22

[0476] 4-Bromo-7-fluoro-2-methyl-2H-indazole

[0477]

[0478] Under a nitrogen atmosphere at 0 °C, add Me 3 OBF 4 (41.5 g, 280.4 mmol, 1.5 equiv) to a solution of 4-bromo-7-fluoro-2H-indazole (Intermediate 21, 40 g, 186.9 mmol, 1 equiv) in EtOAc (800 mL). Stir the mixture at room temperature for 16 h. Quench the reaction with water (500 mL). Extract the resulting mixture with EtOAc (3 × 500 mL). Wash the combined organic layers with brine (1 × 800 mL) and dry over anhydrous Na 2 SO 4 After filtration, concentrate the filtrate under reduced pressure. Purify the residue by silica gel column chromatography, eluting with petroleum ether:EtOAc (10:1 to 4:1), to give 4-bromo-7-fluoro-2-methyl-2H-indazole (30 g) as a white solid.

[0479] 1H NMR (300 MHz, DMSO-d6) δ 8.52 (d, J = 2.7 Hz, 1H), 7.21 (dd, J = 8.0, 3.7 Hz, 1H), 7.00 (dd, J = 11.5, 7.9 Hz, 1H), 4.22 (s, 3H).

[0480] Intermediate 23

[0481] 7-Fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0482]

[0483] To a solution of Intermediate 22 (500 mg, 2.14 mmol) and bis(pinacolato)diboron (832 mg, 3.21 mmol) in dioxane (5.0 ml) was added potassium acetate (840 mg, 8.56 mmol) and the mixture was degassed with argon. To the reaction mixture was added [1,1'-bis-(diphenylphosphino)-ferrocenyl]-dichloropalladium(II) (PdCl 2 dppf, 175 mg, 0.21 mmol) and the mixture was heated at 90 °C for 16 h. The reaction was cooled to room temperature. The reaction was diluted with water and the organics were extracted with EtOAc. The organic layers were combined, washed with brine, dried over MgSO 4 and filtered and concentrated under reduced pressure. The title compound (487 mg) was isolated after silica gel chromatography (cyclohexane:EtOAc).

[0484] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33 (s, 12H), 4.23 (s, 3H), 7.02 (m, 1H), 7.42 (dd, J = 7.35, 4.94 Hz, 1H), 8.45 (d, J = 2.91 Hz, 1H)

[0485] Intermediate 24

[0486] 5-Fluoro-1-(6-fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0487]

[0488] Using the method described for Intermediate 11: The title compound (3.3 g) was obtained after recrystallization from Intermediate 16 (10 g, 33.7 mmol) and 2-fluoropyridine-5-boronic acid (11.87 g, 84.22 mmol) in EtOH.

[0489] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.09 (s, 3H), 7.18 (m, 1H), 7.31 (dd, J = 9.89, 1.39 Hz, 1H), 7.43 (dd, J = 8.62, 3.04 Hz, 1H), 8.29 (m, 1H), 8.53 (d, J = 2.15 Hz, 1H)

[0490] Intermediate 25

[0491] (1R,5S,6R)-3-[5-(5-Fluoro-3-iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester

[0492]

[0493] Using the method described for Intermediate 4: The title compound (2.56 g) was obtained from Intermediate 24 (2.0 g, 5.39 mmol) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (1.97 g, 10.78 mmol).

[0494] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.61 (t, J = 3.04 Hz, 1H), 2.08 (s, 3H), 2.26 (br s, 2H), 3.55 (m, 2H), 3.63 (s, 3H), 3.83 (d, J = 10.90 Hz, 2H), 6.57 (d, J = 9.00 Hz, 1H), 7.10 (dd, J = 8.24, 2.03 Hz, 1H), 7.22 (dd, J = 9.89, 1.39 Hz, 1H), 7.68 (dd, J = 8.93, 2.72 Hz, 1H), 8.22 (d, J = 2.53 Hz, 1H)

[0495] Intermediate 26

[0496] 4-Bromo-3,7-difluoro-2-methyl-2H-indazole

[0497]

[0498] At room temperature, SelectFluor (30.93 g, 87.3 mmol, 2.0 equiv) was added to a stirred solution of 4-bromo-7-fluoro-2-methylindazole (10 g, 43.7 mmol, 1 equiv) in DMF (200 mL). The resulting mixture was stirred at 60 °C for 16 h. The mixture was purified by reverse phase flash chromatography under the following conditions: column, C18 silica gel; mobile phase, containing 0.5% NH 3 ·H 2Water of O, MeCN 50% to 75% gradient within 20 minutes; detector, UV 254 nm, to obtain 4-bromo-3,7-difluoro-2-methylindazole (3 g) in the form of a white solid.

[0499] 1H NMR (400 MHz, DMSO-d, ppm) δ: 7.21 (dd, J = 7.9, 3.5 Hz, 1H), 7.03 (dd, J = 11.6, 7.9 Hz, 1H), 4.06 (d, J = 2.0 Hz, 3H).

[0500] Intermediate 27

[0501] 3,7-Difluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2H-indazole

[0502]

[0503] Using the method described for Intermediate 23: The title compound (47 mg) was obtained from Intermediate 26 (35 mg, 0.14 mmol).

[0504] Intermediate 28

[0505] 5,5'-Difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0506]

[0507] To a solution of Intermediate 9 (1.2 g, 4.58 mmol) in dioxane (5 ml) and water (5 ml) was added N-fluorobenzenesulfonimide (4.42 g, 13.72 mmol) and the mixture was heated in a sealed container at 90 °C for 16 hours. Multiple fluorinated compounds were separated:

[0508] 40 mg

[0509] HPLC (acidic): Rt = 0.992 minutes ((M + H)+ 281.0)

[0510] 14 mg

[0511] HPLC (acidic): Rt = 1.022 minutes ((M + H)+ 281.0)

[0512] 30 mg

[0513] HPLC (acidic): Rt = 1.061 minutes ((M + H)+ 299.0)

[0514] 5 mg

[0515] HPLC (acidic): Rt = 1.061 min ((M+H)+ 317.0)

[0516] Title compound: 7 mg

[0517] HPLC (acidic): Rt = 1.061 min ((M+H)+ 299.0)

[0518] Intermediate 29

[0519] 5,5',7'-Trifluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0520]

[0521] The synthesis of this intermediate was as described for Intermediate 28, and 5 mg was isolated.

[0522] HPLC (acidic): Rt = 1.061 min ((M+H)+ 317.0)

[0523] Intermediate 30

[0524] 4-Bromo-6-fluoro-2-methyl-2H-indazole

[0525]

[0526] At 0 °C, trimethyloxonium tetrafluoroborate (3.38 g, 22.9 mmol, 1.2 eq) was added portionwise to a stirred solution of 4-bromo-6-fluoro-2H-indazole (4.1 g, 19.1 mmol, 1 eq) in EtOAc (40 mL). The reaction solution was stirred overnight at room temperature. The resulting mixture was diluted with water (40 mL) and extracted with EtOAc (3 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na 2 SO 4 and filtered. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / EtOAc (10:1) to give 4-bromo-6-fluoro-2-methylindazole (3.7 g) as a white solid.

[0527] 1H NMR (400 MHz, chloroform-d) δ 7.93 (s, 1H), 7.27 (ddd, J = 9.6, 2.0, 1H), 7.12 (dd, J = 8.8, 2.0 Hz, 1H), 4.22 (s, 3H).

[0528] Intermediate 31

[0529] 6-Fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0530]

[0531] At room temperature under a nitrogen atmosphere, potassium acetate (3.34 g, 34.053 mmol, 3 eq) and Pd(dppf)Cl 2 (0.83 g, 1.14 mmol, 0.1 eq) were added to a stirred solution of 4-bromo-6-fluoro-2-methylindazole (2.6 g, 11.4 mmol, 1 eq) and bis(pinacolato)diboron (4.32 g, 17.1 mmol, 1.5 eq) in 1,4-dioxane (26 mL). The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 4 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether / THF (15:1), to give the crude product. The crude product was further purified by trituration with n-hexane (5 mL) to give 6-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (1.56 g) as a white solid.

[0532] 1H NMR (400 MHz, chloroform-d) δ 8.26 (s, 1H), 7.44 - 7.38 (m, 2H), 4.24 (s, 3H), 1.41 (s, 12H).

[0533] Intermediate 32

[0534] 1-(6-Fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0535]

[0536] To a stirred solution of 3-iodo-7-methylindazole (5.0 g, 19.4 mmol, 1 eq) in DCM (75 mL) was added 2-(6-fluoropyridin-3-yl)boronic acid (6.83 g, 48.4 mmol), copper(II) acetate (5.28 g, 29.1 mmol) and pyridine (5.47 mL) and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was concentrated under reduced pressure and redissolved in EtOAc, and stirred with 2 mol / L aqueous NaOH (semi-saturated with NaCl) and the formed precipitate was removed by filtration. The EtOAc phase was washed with 2 mol / L aqueous NaOH (semi-saturated with NaCl), followed by washing with 2 × 2 mol / L aqueous HCl (semi-saturated with NaCl). The organic layer was dried over Na 2 SO 4Dry, filter and evaporate to dryness. Heat the crude product in 100 mL of EtOH (clear brown solution) under reflux and cool slowly to room temperature overnight. Collect the resulting precipitate by filtration, wash it three times with 5 mL of cold EtOH and dry it in a vacuum oven to obtain a white solid (3.80 g).

[0537] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.09 (s, 3H), 7.25 (m, 1H), 7.34 (m, 1H), 7.42 (m, 2H), 8.28 (m, 1H), 8.53 (d, J = 2.41 Hz, 1H).

[0538] Intermediate 33 and Intermediate 34

[0539] Methyl 2-[5-(3-iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2-azabicyclo[2.2.2]octane-5-carboxylate

[0540]

[0541] Add TFA (2 mL) to a solution of tert-butyl 2,5-dimethyl 2-azabicyclo[2.2.2]octane-2,5-dicarboxylate (723 mg, 2.55 mmol) and stir at room temperature for 30 minutes. Concentrate the reaction mixture under reduced pressure. Add a solution of Intermediate 32 (600 mg, 1.7 mmol, 1 equiv), DIPEA (780 μL, 4.59 mmol) in anhydrous NMP (8 mL) to the residue. Stir the resulting mixture at 140 °C for 16 hours. Acidify the resulting mixture with TFA and purify by preparative HPLC (acidic) to obtain a mixture of Intermediate 33 (240 mg) and Intermediate 34 (200 mg).

[0542] Intermediate 33

[0543] HPLC (acidic): Rt = 1.568 min ((M + H)+503)

[0544] Intermediate 34

[0545] HPLC (acidic): Rt = 1.596 min ((M + H)+503)

[0546] Intermediate 35 and Intermediate 36

[0547] (1R,4S,5R)-Methyl 2-[5-(3-iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2-azabicyclo[2.2.2]octane-5-carboxylate

[0548]

[0549] The chiral preparative HPLC was performed on Intermediate 33 (270 mg), and Intermediate 35 (93 mg) and Intermediate 36 (78 mg) were separated.

[0550] Chiral preparative HPLC

[0551] HPLC: Sepiatec PrepSFC100

[0552] Column: CHIRAL Cellulose-SC_20x 250mm_5μm

[0553] Eluent: A: 65% scCO2; B: 35% MeOH + 20 mM NH 3

[0554] Detection: UV: 254 nm

[0555] Column temperature: 40 °C

[0556] Flow rate: 60 mL / min

[0557] Gradient: Isocratic

[0558] Intermediate 37 and Intermediate 38

[0559] (1R,4S,5S)-2-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2-azabicyclo[2.2.2]octane-5-carboxylic acid methyl ester

[0560]

[0561] The chiral preparative HPLC was performed on Intermediate 34 (200 mg), and Intermediate 37 (51.6 mg) and Intermediate 38 (103 mg) were separated.

[0562] Chiral preparative HPLC

[0563] HPLC: Sepiatec PrepSFC100

[0564] Column: CHIRAL Cellulose-SC_20x 250mm_5μm

[0565] Eluent: A: 60% scCO2; B: 40% MeOH + 20 mM NH 3

[0566] Detection: UV: 254 nm

[0567] Column temperature: 40 °C

[0568] Flow rate: 60 mL / min

[0569] Gradient: isocratic

[0570] Intermediate 39

[0571] (1S,4S)-5-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0572]

[0573] To a stirred solution of Intermediate 32 (500 mg, 1.42 mmol, 1 equiv) in anhydrous NMP (5 mL) was added DIPEA (366 mg, 2.83 mmol) and (1S,4S)-2,5-diazabicyclo-[2.2.1]heptane-2-carboxylic acid tert-butyl ester (434 mg, 2.12 mmol). The resulting mixture was stirred at 80 °C for 15 h. The reaction mixture was diluted with 50 ml of water and extracted twice with EtOAc. The organic layers were combined and washed with water, 10% LiCl solution, saturated NaCl (aqueous solution), dried over MgSO 4 dried, filtered and concentrated under reduced pressure. The desired compound (607 mg, 81%) was isolated after silica gel chromatography (cyclohexane:EtOAc).

[0574] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 1.39 (m, 9H), 1.97 (br s, 2H), 2.10 (s, 3H), 3.24 (m, 1H), 3.38 (m, 2H), 3.57 (br d, J = 6.84 Hz, 1H), 4.49 (br d, J = 13.31 Hz, 1H), 4.89 (br s, 1H), 6.67 (br d, J = 8.87 Hz, 1H), 7.19 (m, 1H), 7.26 (d, J = 6.97 Hz, 1H), 7.35 (d, J = 7.98 Hz, 1H), 7.68 (dd, J = 8.87, 2.66 Hz, 1H), 8.22 (d, J = 2.53 Hz, 1H).

[0575] Intermediate 40

[0576] 1-{6-[(1S,4S)-2,5-Diazabicyclo[2.2.1]hept-2-yl]pyridin-3-yl}-3-iodo-7-methyl-1H-indazole trifluoroacetate

[0577]

[0578] To a stirred solution of intermediate 39 (632 mg, 1.19 mmol, 1 equiv) in anhydrous DCM (7 mL) was added TFA (3 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and used directly in the next step (513 mg, 79%).

[0579] Intermediate 41

[0580] 1-[(1S,4S)-5-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2,5-diazabicyclo[2.2.1]hept-2-yl]ethan-1-one

[0581]

[0582] To a stirred solution of intermediate 40 (510 mg, 0.935 mmol) in anhydrous MeTHF (5 mL) was added DIPEA (971 μL, 5.61 mmol) and acetic anhydride (133 μL, 1.4 mmol). The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with DCM and washed with 50 ml of water. The organic layer was dried over MgSO 4 dried, filtered and concentrated under reduced pressure. The desired compound was isolated after silica gel chromatography (MeOH:EtOAc).

[0583] HPLC (basic): Rt = 2.05 min ((M+H)+ 474.0)

[0584] Intermediate 42

[0585] (1R,4R)-5-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0586]

[0587] To a stirred solution of intermediate 32 (500 mg, 1.42 mmol, 1 equiv) in anhydrous NMP (5 mL) was added DIPEA (366 mg, 2.83 mmol) and (1R,4R)-2,5-diazabicyclo-[2.2.1]heptane-2-carboxylic acid tert-butyl ester (434 mg, 2.12 mmol). The resulting mixture was stirred at 80 °C for 15 h. The reaction mixture was diluted with 50 ml of water and extracted twice with EtOAc. The organic layers were combined and washed with water, 10% LiCl solution, saturated NaCl (aqueous solution), dried over MgSO 4Dry, filter and concentrate under reduced pressure. The desired compound (637 mg, 85%) was isolated after silica gel chromatography (cyclohexane: EtOAc).

[0588] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 1.39 (m, 9H), 1.97 (m, 2H), 2.10 (s, 3H), 3.25 (m, 1H), 3.36 (m, 2H), 3.58 (br d, J = 6.59 Hz, 1H), 4.49 (br d, J = 13.31 Hz, 1H), 4.89 (br s, 1H), 6.67 (br d, J = 8.74 Hz, 1H), 7.18 (t, J = 7.32 Hz, 1H), 7.26 (d, J = 6.97 Hz, 1H), 7.35 (d, J = 7.98 Hz, 1H), 7.68 (dd, J = 8.87, 2.66 Hz, 1H), 8.22 (d, J = 2.53 Hz, 1H).

[0589] Intermediate 43

[0590] (1R,4R)-5-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0591]

[0592] To a stirred solution of Intermediate 39 (632 mg, 1.19 mmol, 1 equiv) in anhydrous DCM (7 mL) was added TFA (3 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and used directly in the next step (513 mg, 79%).

[0593] Intermediate 44

[0594] (1R,4R)-5-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0595]

[0596] To a stirred solution of Intermediate 43 (510 mg, 0.935 mmol) in anhydrous MeTHF (5 mL) was added DIPEA (971 μL, 5.61 mmol) and acetic anhydride (133 μL, 1.4 mmol). The resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with DCM and washed with 50 ml of water. The organic layer was dried over MgSO 4Dry, filter and concentrate under reduced pressure. The desired compound was isolated after silica gel chromatography (MeOH:EtOAc).

[0597] HPLC (acidic): Rt = 0.874 min ((M+H)+474.0)

[0598] Intermediate 45

[0599] 5-Fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole

[0600]

[0601] To a stirred solution of 4-bromo-5-fluoro-2-methylindazole (WO2022221556 A1, 1.00 g, 4.32 mmol) and bis(pinacolato)diboron (1.7 g, 6.48 mmol) in dioxane (10 mL) was added KOAc (1.697 g, 17.29 mmol) and then the solution was degassed by bubbling argon through the solution. Subsequently, a complex of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) with DCM (1:1) (353 mg, 0.432 mmol) was added, and the reaction mixture was heated and stirred at 90 °C for 16 h. The reaction mixture was diluted with 50 ml of water and extracted with EtOAc. The organic layers were combined and washed with water, saturated NaCl (aqueous solution), dried over MgSO 4 Dry, filter and concentrate under reduced pressure. The desired compound (939 mg, 79%) was isolated after silica gel chromatography (cyclohexane:EtOAc).

[0602] HPLC (acidic): Rt = 1.022 min ((M+H)+277.2)

[0603] Intermediate 46 and Intermediate 47

[0604] (1S,4R,5R)-2-[5-(3-Iodo-7-methyl-1H-indazol-1-yl)pyridin-2-yl]-2-azabicyclo[2.2.1]heptane-5-carboxylic acid methyl ester

[0605]

[0606] To a stirred solution of intermediate 32 (260 mg, 0.74 mmol) in anhydrous NMP (3 mL) was added DIPEA (338 μL, 1.99 mmol) and (rel)-(1S,4R,5R)-methyl 2-azabicyclo[2.2.1]heptane-5-carboxylate hydrochloride (155 mg, 0.81 mmol). The resulting mixture was stirred at 95 °C for 15 h. The reaction mixture was acidified with TFA and purified by preparative HPLC (acidic) to give the title product, which was further purified by chiral HPLC chromatography to give intermediate 46 (110 mg) and intermediate 47 (112 mg).

[0607] Chiral preparative HPLC

[0608] HPLC: Sepiatec PrepSFC100

[0609] Column: Cellulose-3_21.2x 250mm_5μm

[0610] Eluent: A: 85% scCO2; B: 15% MeOH + 20 mM NH 3

[0611] Detection: UV: 254 nm

[0612] Column temperature: 40 °C

[0613] Flow rate: 60 mL / min

[0614] Gradient: Isocratic

[0615] Intermediate 48

[0616] 6'-Fluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0617]

[0618] To a stirred solution of intermediate 3 (1 g, 2.832 mmol) in dioxane (4 mL) and water (1 mL) was added 6-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (intermediate 31, 0.957 mg, 3.4 mmol) and K 2 CO 3 (1.174 g, 8.5 mmol) and the solution was degassed by bubbling argon through it. Subsequently, Pd(dppf)Cl 2(0.104 g, 0.14 mmol) and heat the reactants at 100 °C for 3 hours. Dilute the reaction mixture with EtOAc and filter through diatomaceous earth. Wash the remaining organic layer twice with 1 mol / L aqueous NaOH solution. Add 100 mg of activated carbon to the organic layer, and dry the organic layer over Na 2 SO 4 dry, filter and concentrate under reduced pressure. Separate the desired compound by silica gel chromatography (MeOH:EtOAc). Heat the crude product in 35 mL of iPrOH under reflux conditions and slowly cool to room temperature over 3 hours. Collect the precipitate by filtration (800 mg; 75%).

[0619] HPLC (acidic): Rt = 1.133 min ((M + H)+ 376.2)

[0620] Intermediate 49 and Intermediate 50

[0621] (1S,4R,5R)-2-Azabicyclo[2.2.1]heptane-2,5-dicarboxylic acid 2-tert-butyl ester 5-methyl ester and

[0622] (1R,4S,5S)-2-Azabicyclo[2.2.1]heptane-2,5-dicarboxylic acid 2-tert-butyl ester 5-methyl ester

[0623]

[0624] Add TEA (147 μL, 1.044 mmol) and Boc-anhydride (125.3 mg, 0.574 mmol) to a stirred mixture of (rel-1S,4R,5R)-methyl 2-azabicyclo[2.2.1]heptane-5-carboxylate hydrochloride (enamine, 100 mg, 0.522 mmol) in DCM (3 mL). Stir the resulting mixture at room temperature for 30 minutes. Concentrate the reaction mixture with DCM and obtain the title compound (105 mg, 79%) after preparative HPLC chromatography (basic).

[0625] Intermediate 51 and Intermediate 52

[0626] (1S,4R,5R)-2-[(tert-Butoxy)carbonyl]-2-azabicyclo[2.2.1]heptane-5-carboxylic acid and

[0627] (1R,4S,5S)-2-[(tert-Butoxy)carbonyl]-2-azabicyclo[2.2.1]heptane-5-carboxylic acid

[0628]

[0629] To a stirred solution of Intermediate 49 and Intermediate 50 (70 mg, 0.274 mmol) in DMF (1 mL) was added aqueous NaOH solution (4 M, 343 μL, 1.37 mmol). The resulting mixture was stirred at room temperature for 2 h. The reaction mixture was acidified with TFA and the title compound (44 mg, 67%) was isolated after preparative HPLC chromatography (acidic).

[0630] Intermediate 51 and Intermediate 52 (60 mg) were subjected to chiral preparative HPLC and Intermediate 51 (16 mg) and Intermediate 52 (26 mg) were isolated.

[0631] Chiral preparative HPLC

[0632] HPLC: Sepiatec PrepSFC100

[0633] Column: IG_10x 250mm_5μm

[0634] Eluent: A: 90% scCO2; B: 10% MeOH + 20 mM NH 3

[0635] Detection: UV: 220 nm

[0636] Column temperature: 40 °C

[0637] Flow rate: 15 mL / min

[0638] Gradient: Isocratic

[0639] Intermediate 51: 1 1H NMR (400 MHz, DMSO-d 6 ) δ ppm 1.40 (m, 11H), 1.79 (m, 2H), 2.50 (m, 2H), 2.69 (br s, 1H), 2.94 (d, J = 9.63 Hz, 1H), 3.13 (m, 1H), 4.07 (br d, J = 12.93 Hz, 1H), 12.21 (br s, 1H)

[0640] Intermediate 53

[0641] (1S,4R,5R)-2-Azabicyclo[2.2.1]heptane-5-carboxylic acid

[0642]

[0643] To a stirred solution of Intermediate 51 (12.3 mg, 0.051 mmol) in DCM (2 mL) was added TFA (500 μL) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and used directly in the next step.

[0644] Intermediate 54

[0645] (1R,4S,5S)-2-Azabicyclo[2.2.1]heptane-5-carboxylic acid

[0646]

[0647] To a stirred solution of Intermediate 52 (26 mg, 0.108 mmol) in DCM (2 mL) was added TFA (500 μL) and the mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure and used directly in the next step.

[0648] HPLC (acidic): Rt = 0.085 min ((M + H)+142.0)

[0649] Intermediate 55

[0650] (3-Bromo-2,5,6-trifluorophenyl)trimethylsilane

[0651]

[0652] Under nitrogen at -78 °C, LDA (1.14 L, 2.27 mol, 1.2 eq., 2 mol / L) was added dropwise to a stirred solution of 1-bromo-2,4,5-trifluorobenzene (400 g, 1.89 mol, 1 eq.) in THF (4000 mL). The resulting mixture was stirred at -78 °C for 2 hours. TMSCl (534 g, 4.914 mol, 2.6 eq.) was added dropwise to the mixture at -78 °C and the mixture was stirred at -78 °C for an additional 0.5 hour. The mixture was allowed to reach room temperature and stirred at room temperature for 0.5 hour. The mixture was quenched with ice water (2 L) and extracted with EtOAc (2 L × 3). The organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with PE:EA (10:1), to give (3-bromo-2,5,6-trifluorophenyl)trimethylsilane as a pale yellow oil (400 g, 74.48%).

[0653] Intermediate 56

[0654] 2-Bromo-3,5,6-trifluoro-4-(trimethylsilyl)benzaldehyde

[0655]

[0656] At -78 °C under nitrogen, LDA (851 mL, 1.7 mol, 1.2 eq., 2 mol / L) was added dropwise to a stirred solution of (3-bromo-2,5,6-trifluorophenyl)trimethylsilane (400 g, 1.418 mol, 1 eq.) in THF (4000 mL). The resulting mixture was stirred at -78 °C for 1 h. At -78 °C, DMF (517 g, 7.09 mol, 5 eq.) was added dropwise to the mixture and stirred at -78 °C for an additional 0.5 h. The resulting mixture was quenched with saturated NH 4 Cl (1 L) and extracted with EtOAc (2 L × 3). The organic phase was dried over Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography, eluting with petroleum ether:EtOAc (10:1), to give (3-bromo-2,5,6-trifluorophenyl)trimethylsilane as a pale yellow oil (350 g, 79.55%).

[0657] Intermediate 57

[0658] ([2-Bromo-3,5,6-trifluoro-4-(trimethylsilyl)phenyl]methylene}(methoxy)amine

[0659]

[0660] To a stirred solution of 2-bromo-3,5,6-trifluoro-4-(trimethylsilyl)benzaldehyde (300 g, 964.094 mmol, 1 eq.) and O-methylhydroxylamine hydrochloride (88.57 g, 1060.503 mmol, 1.1 eq.) in DME (3000 mL) was added K 2 CO 3 (199.86 g, 1446.141 mmol, 1.5 eq.). The resulting mixture was stirred at 50 °C for 2 h. The mixture was cooled to room temperature and diluted with EtOAc (1 L). The resulting mixture was washed with water (1 L). The organic layer was concentrated in vacuo to give the crude product (E)-([2-bromo-3,5,6-trifluoro-4-(trimethylsilyl)phenyl]methylene-(methoxy)amine (300 g, brown oil). The crude product was used directly in the next step without further purification.

[0661] Intermediate 58

[0662] 4-Bromo-5,7-difluoro-1H-indazole

[0663]

[0664] Stir a solution of (E)-([2-bromo-3,5,6-trifluoro-4-(trimethylsilyl)phenyl]methylene-(methoxy)amine (300 g, crude product) in hydrazine hydrate (1500 mL) and DME (1500 mL) at 80 °C for 16 h. Dilute the resulting mixture with EtOAc (1 L) and wash with water (1 L). Dry the organic layer over anhydrous Na 2 SO 4 4-Bromo-5,7-difluoro-2H-indazole (41 g, 19.95%) as a white solid was obtained by purifying the residue by silica gel column chromatography, eluting with petroleum ether:EtOAc (1:1).

[0665] Intermediate 59

[0666] 4-Bromo-5,7-difluoro-2-methyl-2H-indazole

[0667]

[0668] Add trimethyloxonium tetrafluoroborate (38.09 g, 257.493 mmol, 1.5 equiv) to a stirred solution of 4-bromo-5,7-difluoro-2H-indazole (40 g, 171.662 mmol, 1 equiv) in EA (800 mL) at 0 °C. Stir the resulting mixture at room temperature for 16 h. Dilute the reaction mixture with EtOAc (500 mL) and wash with water (500 mL). Concentrate the organic layer under reduced pressure. Purify the residue by silica gel column chromatography, eluting with petroleum ether:EtOAc (5:1) to give 4-bromo-5,7-difluoro-2-methylindazole (27 g, 63.67%) as a white solid and 4-bromo-5,7-difluoro-1-methyl-1H-indazole (1.5 g, 3.53%) as a white solid.

[0669] 1H NMR (400 MHz, chloroform-δ) δ 8.00 (d, J = 2.5 Hz, 1H), 6.90 (t, J = 9.7 Hz, 1H), 4.27 (s, 3H).

[0670] Intermediate 60

[0671] 5,7-Difluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane-2-yl)-2H-indazole

[0672]

[0673] To a solution of 4-bromo-5,7-difluoro-2-methylindazole (20 g, 80.958 mmol, 1 equiv) and bis(pinacolato)diboron (41.12 g, 161.916 mmol, 2 equiv) in dioxane (400 mL) was added KOAc (15.89 g, 161.916 mmol, 2 equiv) and Pd(dppf)Cl 2 .DCM (6.59 g, 8.096 mmol, 0.1 equiv). After stirring for 16 h at 90 °C under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with petroleum ether:EtOAc (5:1), to give 5,7-difluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (5.1336 g, 21.56%) as a white solid.

[0674] 1H NMR (400 MHz, chloroform-δ) δ 8.28 (d, J = 2.7 Hz, 1H), 6.79 (t, J = 10.3 Hz, 1H), 4.27 (s, 3H), 1.42 (s, 12H).

[0675] Intermediate 61

[0676] (1S,4S)-5-(5-{5',7'-Difluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0677]

[0678] To a mixture of intermediate 13 (15 mg, 0.038 mmol) and (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane (24 mg, 0.114 mmol) in MeCN (1.5 ml) was added DIPEA (24.6 mg, 0.191 mmol) and the mixture was heated at 105 °C for 16 h. The reaction was cooled and then concentrated and used directly in the next step without further purification.

[0679] HPLC (acidic): Rt = 1.075 min ((M + H)+ 572.2)

[0680] Intermediate 62

[0681] 1-{6-[(1S,4S)-2,5-Diazabicyclo[2.2.1]hept-2-yl]pyridin-3-yl}-5',7'-difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0682]

[0683] To a stirred solution of intermediate 61 (12.3 mg, 0.051 mmol) in DCM (2 mL) was added TFA (500 μL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and used directly in the next step.

[0684] HPLC (acidic): Rt = 0.877 min ((M+H)+ 472.2)

[0685] Intermediate 63

[0686] (1R,4R)-5-(5-{5',7'-Difluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester

[0687]

[0688] To a mixture of intermediate 13 (10 mg, 0.025 mmol) and (1R,4R)-2-Boc-2,5-diazabicyclo[2.2.1]heptane (15.6 mg, 0.076 mmol) in MeCN (1.5 ml) was added DIPEA (16.4 mg, 0.127 mmol) and the mixture was heated at 105 °C for 16 h. The reaction was cooled and then concentrated and used directly in the next step without further purification.

[0689] HPLC (acidic): Rt = 1.077 min ((M+H)+ 572.2)

[0690] Intermediate 64

[0691] 1-{6-[(1R,4R)-2,5-Diazabicyclo[2.2.1]hept-2-yl]pyridin-3-yl}-5',7'-difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0692] To a stirred solution of intermediate 63 (17.4 mg, 0.020 mmol) in DCM (2 mL) was added TFA (500 μL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and used directly in the next step.

[0693] HPLC (acidic): Rt = 0.871 min ((M+H)+ 472.2)

[0694] Intermediate 65

[0695] 1-(6-{2,5-Diazabicyclo[2.2.2]oct-2-yl}pyridin-3-yl)-5',7'-difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0696]

[0697] To a mixture of intermediate 13 (10 mg, 0.025 mmol) and 2,5-diazabicyclo[2.2.2]octane dihydrochloride (9.9 mg, 0.051 mmol) in NMP (1.5 ml) was added DIPEA (16.4 mg, 0.127 mmol) and the mixture was heated at 105 °C for 3 h. The reaction mixture was cooled and used directly in the next step without further purification.

[0698] HPLC (acidic): Rt = 0.904 min ((M + H)+ 486.2)

[0699] Intermediate 66

[0700] 3',7'-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0701]

[0702] Using the method described for intermediate 11: The title compound (435 mg) was obtained from intermediate 3 (500 mg) and intermediate 27 (517 mg).

[0703] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.18 (s, 3H), 4.07 (d, J = 1.90 Hz, 3H), 7.25 (m, 2H), 7.32 (m, 1H), 7.46 (m, 2H), 7.88 (d, J = 7.98 Hz, 1H), 8.34 (ddd, J = 8.68, 7.03, 2.79 Hz, 1H), 8.59 (d, J = 2.41 Hz, 1H).

[0704] Intermediate 67

[0705] 5-Fluoro-1-(6-fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0706]

[0707] Using the method described for Intermediate 11: The title compound (7.12 g, 57%) was obtained after recrystallization from Intermediate 16 (10 g, 35.86 mmol) and 2-fluoropyridine-5-boronic acid (12.89 g, 89.66 mmol) in EtOH.

[0708] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.09 (s, 3H), 7.18 (dd, J = 8.11, 2.28 Hz, 1H), 7.31 (dd, J = 9.89, 1.39 Hz, 1H), 7.43 (dd, J = 8.62, 3.04 Hz, 1H), 8.29 (m, 1H), 8.53 (d, J = 2.15 Hz, 1H).

[0709] Intermediate 68

[0710] 3',5-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0711]

[0712] Using the method described for Intermediate 11: The title compound (200 mg) was obtained after recrystallization from Intermediate 67 (1 g) and 3-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (920 mg) in EtOH.

[0713] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.18 (s, 3H), 4.03 (d, J = 1.77 Hz, 3H), 7.30 (dd, J = 9.76, 1.39 Hz, 1H), 7.41 (m, 1H), 7.46 (dd, J = 8.68, 2.98 Hz, 1H), 7.51 (d, J = 6.72 Hz, 1H), 7.58 (dd, J = 8.74, 1.27 Hz, 1H), 7.65 (dd, J = 8.74, 2.15 Hz, 1H), 8.36 (ddd, J = 8.65, 7.07, 2.79 Hz, 1H), 8.61 (d, J = 2.28 Hz, 1H).

[0714] Intermediate 69

[0715] Methyl 1-(5-{3',5-difluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)piperidine-4-carboxylate

[0716]

[0717] Using the method described for Intermediate 4: The title compound (60 mg) was obtained from Intermediate 68 (60 mg, 0.153 mmol) and methyl piperidine-4-carboxylate hydrochloride (67 mg, 0.458 mmol).

[0718] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 1.62 (m, 1H), 1.94 (br dd, J = 13.24, 3.10 Hz, 1H), 2.19 (s, 1H), 2.70 (m, 1H), 3.10 (m, 1H), 3.64 (s, 3H), 4.02 (d, J = 1.65 Hz, 3H), 4.30 (m, 1H), 7.03 (d, J = 9.13 Hz, 1H), 7.22 (dd, J = 9.82, 1.33 Hz, 1H), 7.40 (m, 1H), 7.47 (m, 1H), 7.57 (ddd, J = 15.18, 8.78, 1.52 Hz, 1H), 7.79 (dd, J = 9.06, 2.72 Hz, 1H), 8.34 (d, J = 2.66 Hz, 1H)

[0719] Intermediate 70

[0720] 3',6-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0721]

[0722] Using the method described for Intermediate 11: The title compound (766 mg) was obtained from Intermediate 19 (1 g) and 3-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (920 mg) after recrystallization from isopropanol.

[0723] 1 H NMR (400 MHz, DMSO-d 6)δ ppm 2.05 (m, 3H), 4.03 (d, J = 1.77 Hz, 3H), 7.23 (t, J = 9.38 Hz, 1H), 7.42 (m, 1H), 7.47 (dd, J = 8.62, 3.04 Hz, 1H), 7.51 (d, J = 6.59 Hz, 1H), 7.60 (dd, J = 8.68, 1.20 Hz, 1H), 7.91 (dd, J = 8.87, 4.94 Hz, 1H), 8.37 (ddd, J = 8.68, 7.03, 2.79 Hz, 1H), 8.62 (d, J = 2.28 Hz, 1H).

[0724] Intermediate 71

[0725] 5',5-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0726]

[0727] Using the method described for Intermediate 11: The title compound (73 mg) was obtained from Intermediate 67 (70 mg) and Intermediate 45 (90 mg) after preparative HPLC purification (acidic).

[0728] Intermediate 72

[0729] 5',6-Difluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0730]

[0731] Using the method described for Intermediate 11: The title compound (72 mg) was obtained from Intermediate 19 (70 mg) and Intermediate 45 (90 mg) after preparative HPLC purification (acidic).

[0732] HPLC (acidic): Rt = 1.127 min ((M + H)+ 394.2)

[0733] Intermediate 73

[0734] 5,5',7'-Trifluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0735]

[0736] This intermediate was isolated in the synthesis of Intermediate 13 - see the experiment for Intermediate 13.

[0737] Intermediate 74

[0738] 5,5',7'-Trifluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0739]

[0740] Using the method described for Intermediate 11: The title compound (140 mg) was obtained from Intermediate 73 (180 mg) and 2-fluoropyridine-5-boronic acid (200 mg) after preparative HPLC purification (acidic).

[0741] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.18 (s, 3H), 4.21 (s, 3H), 7.30 (br d, J = 9.89 Hz, 1H), 7.40 (m, 2H), 7.48 (dd, J = 8.68, 2.98 Hz, 1H), 8.46 (m, 1H), 8.64 (d, J = 2.79 Hz, 1H), 8.72 (d, J = 2.53 Hz, 1H).

[0742] Intermediate 75

[0743] 6,7'-Difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0744]

[0745] Using the method described for Intermediate 9: The title compound (965 mg) was obtained from Intermediate 18 (1000 mg) and Intermediate 23 (1078 mg) after preparative HPLC purification (acidic).

[0746] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.49 (d, J = 1.65 Hz, 3H), 4.27 (s, 3H), 7.07 (t, J = 9.31 Hz, 1H), 7.15 (dd, J = 11.53, 7.73 Hz, 1H), 7.64 (dd, J = 7.79, 4.12 Hz, 1H), 7.96 (dd, J = 8.87, 4.82 Hz, 1H), 8.78 (d, J = 2.79 Hz, 1H), 13.47 (s, 1H)

[0747] Intermediate 76

[0748] 5',6,7'-Trifluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0749]

[0750] To a solution of intermediate 75 (900 mg, 3.017 mmol) in MeCN (6 ml) and DMF (6 ml) was added SelectFluor (962 mg, 2.72 mmol) and the mixture was stirred for 90 minutes. The reaction mixture was concentrated and purified by silica gel chromatography (DCM:MeOH) to afford the title compound (180 mg, 19%).

[0751] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.49 (d, J = 1.77 Hz, 3H), 4.22 (s, 3H), 7.04 (t, J = 9.38 Hz, 1H), 7.33 (t, J = 11.15 Hz, 1H), 7.58 (dt, J = 8.84, 4.39 Hz, 1H), 8.54 (d, J = 2.79 Hz, 1H), 13.66 (s, 1H)

[0752] The following by-products were also isolated:

[0753] 3',6,7'-Trifluoro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole (35 mg, 4%)

[0754]

[0755] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.47 (m, 3H), 4.07 (d, J = 1.90 Hz, 3H), 7.04 (t, J = 9.31 Hz, 1H), 7.19 (dd, J = 11.53, 7.73 Hz, 1H), 7.34 (dd, J = 7.73, 4.18 Hz, 1H), 7.72 (dd, J = 8.87, 4.82 Hz, 1H), 13.53 (s, 1H)

[0756] Intermediate 77 and Intermediate 78

[0757] (1R,4S,5S)-2-(5-{3'-Fluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid methyl ester; (1S,4R,5R)-2-(5-{3'-Fluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid methyl ester

[0758]

[0759] Using the method described for Intermediate 4: The title compound (55 mg) was obtained from Intermediate 6 (45 mg, 0.12 mmol) and rac (1S,4R,5R)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid methyl ester hydrochloride (57.4 mg, 0.3 mmol).

[0760] HPLC (acidic): Rt = 0.909 min ((M+H)+ 511.2)

[0761] Chiral preparative HPLC was performed and Intermediate 77 (17.5 mg) and Intermediate 78 (15.7 mg) were separated.

[0762] Chiral preparative HPLC

[0763] HPLC: Sepiatec PrepSFC50_2

[0764] Column: CHIRAL Amylose-C_neo_20x 250mm_5μm

[0765] Eluent: A: 60% scCO2; B: 40% MeOH + 20 mM NH 3

[0766] Detection: UV: 220 nm

[0767] Column temperature: 40 °C

[0768] Flow rate: 12 mL / min

[0769] Gradient: Isocratic

[0770] Intermediate 79

[0771] (3,5-Difluoro-2-methyl-2H-indazol-4-yl)boronic acid

[0772]

[0773] To a solution of Intermediate 45 (100 mg, 0.355 mmol) in MeCN (6 mL) was added SelectFluor (962 mg, 2.72 mmol) and the mixture was stirred for 40 min. The reaction mixture was concentrated and purified by silica gel chromatography (DMC:MeOH) to give the title compound (13 mg, 17%).

[0774] HPLC (acidic): Rt = 0.547 min ((M+H)+ 213.0)

[0775] Intermediate 80

[0776] (3,6-Difluoro-2-methyl-2H-indazol-4-yl)boronic acid

[0777]

[0778] To a solution of Intermediate 31 (50 mg, 0.177 mmol) in MeCN (2 ml) was added SelectFluor (75.4 mg, 0.213 mmol) and the mixture was stirred for 30 minutes. The reaction mixture was concentrated and purified by silica gel chromatography (DCM:MeOH) to give the title compound (10 mg, 27%).

[0779] HPLC (acidic): Rt = 0.665 min ((M+H)+ 213.0)

[0780] Intermediate 81

[0781] 7'-Fluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0782]

[0783] Using the method described for Intermediate 9: The title compound (960 mg) was obtained after crystallization from water at 70 °C from 3-iodo-7-methyl-1H-indazole (1000 mg) and Intermediate 23 (1201 mg).

[0784] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.59 (s, 3H), 4.28 (s, 3H), 7.15 (m, 2H), 7.21 (m, 1H), 7.66 (dd, J = 7.79, 4.12 Hz, 1H), 7.95 (d, J = 8.11 Hz, 1H), 8.81 (d, J = 2.79 Hz, 1H), 13.37 (s, 1H).

[0785] Intermediate 82

[0786] 3',5',7'-Trifluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0787]

[0788] To a solution of intermediate 81 (300 mg, 3.425 mmol) in MeCN (30 ml) was added SelectFluor (970.6 mg, 2.74 mmol) and the mixture was stirred for 45 minutes. Another portion of SelectFluor (260 mg) was added and the mixture was stirred for 40 minutes. The reaction mixture was concentrated and purified by preparative HPLC (basic) to give the title compound (8 mg, 1%).

[0789] HPLC (acidic): Rt = 1.011 min ((M + H)+ 317.0)

[0790] The following were also isolated:

[0791] 5',7'-Difluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole (300 mg, 29%)

[0792]

[0793] 5,5',7'-Trifluoro-2',7-dimethyl-1H,2'H-3,4'-biindazole (100 mg, 10%)

[0794]

[0795] Intermediate 83

[0796] 5'-Chloro-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0797]

[0798] To a solution of intermediate 9 (300 mg, 1.144 mmol) in DMF (5 ml) was added N-chlorosuccinimide (170 mg, 1.258 mmol) and the mixture was stirred at 75 °C for 3.5 hours. The reaction mixture was concentrated and purified by preparative HPLC (basic) and a mixture of two regioisomers (195 mg) was obtained.

[0799] Preparative HPLC

[0800] HPLC: Torus-2-PIC

[0801] Column: Torus-2-PIC, 5 μm, 30 × 150 mm

[0802] Eluent: A: scCO2; B: MeOH + 20 mM NH 3

[0803] Detection: UV: 220 nm

[0804] Column temperature: 40 °C

[0805] Flow rate: 150 mL / min

[0806] Gradient: 20 - 80% MeOH in 4 minutes

[0807] Back pressure: 1886 psi

[0808] Intermediate 83 (67 mg, 33%).

[0809] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.60 (s, 3H), 4.13 (s, 3H), 7.05 (dd, J = 8.11, 6.97 Hz, 1H), 7.18 (d, J = 6.84 Hz, 1H), 7.35 (d, J = 8.11 Hz, 1H), 7.40 (d, J = 9.00 Hz, 1H), 7.71 (dd, J = 9.12, 0.89 Hz, 1H), 8.06 (s, 1H), 13.50 (s, 1H).

[0810] The following were also isolated:

[0811] 3'-Chloro-2',7-dimethyl-1H,2'H-3,4'-biindazole (74 mg, 37%)

[0812]

[0813] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.58 (s, 3H), 4.13 (s, 3H), 7.04 (dd, J = 8.05, 6.91 Hz, 1H), 7.17 (d, J = 6.97 Hz, 1H), 7.26 (dd, J = 6.84, 0.76 Hz, 1H), 7.42 (m, 2H), 7.70 (dd, J = 8.62, 0.76 Hz, 1H), 13.36 (s, 1H)

[0814] Intermediate 84

[0815] 5'-Chloro-1-(6-fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0816]

[0817] Using the method described for Intermediate 11: The title compound (80 mg) was obtained from Intermediate 83 (67 mg) and 2-fluoropyridine-5-boronic acid (79 mg) after preparative HPLC purification (acidic).

[0818] 1 H NMR (400 MHz, DMSO-d 6 ) δ ppm 2.20 (s, 3H), 4.15 (s, 3H), 7.21 (dd, J = 8.05, 7.03 Hz, 1H), 7.31 (d, J = 6.97 Hz, 1H), 7.46 (m, 2H), 7.51 (d, J = 7.98 Hz, 1H), 7.77 (dd, J = 9.12, 0.89 Hz, 1H), 8.26 (s, 1H), 8.46 (ddd, J = 8.62, 7.10, 2.79 Hz, 1H), 8.71 (d, J = 2.16 Hz, 1H) Example

[0819] Example 1

[0820] (1R,5S,6R)-3-(5-{3”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0821]

[0822] To a solution of intermediate 5 (55 mg, 0.11 mmol) in EtOH (4 ml) was added a solution of NaOH (aqueous solution, 4 M (aqueous solution), 110.8 μl, 0.44 mmol) and the mixture was stirred at 80 °C for 2.5 h. The reaction mixture was cooled and then concentrated. The residue was purified by preparative HPLC (acidic method) and the title compound (24 mg) was isolated.

[0823] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.49 (t, J = 3.04 Hz, 1H), 2.19 (s, 3H), 2.23 (br s, 2H), 3.58 (m, 2H), 3.85 (d, J = 10.90 Hz, 2H), 3.94 (s, 1H), 4.02 (d, J = 1.77 Hz, 3H), 6.67 (d, J = 9.00 Hz, 1H), 7.22 (m, 2H), 7.41 (m, 1H), 7.48 (m, 1H), 7.55 (dd, J = 8.68, 1.08 Hz, 1H), 7.80 (dd, J = 9.00, 2.66 Hz, 1H), 7.85 (d, J = 7.98 Hz, 1H), 8.32 (d, J = 2.53 Hz, 1H).

[0824] Example 2

[0825] 1-(5-{3”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)piperidine-4-carboxylic acid

[0826]

[0827] To a mixture of intermediate 6 (40 mg, 0.11 mmol) and methyl piperidinecarboxylate (46.7 mg, 0.32 mmol) in NMP (1.5 ml) was added DIPEA (55.1 mg, 0.43 mmol) and the mixture was heated at 140 °C for 16 h. The reaction was cooled. Subsequently, NaOH (4 M (aqueous solution), 0.5 ml) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was acidified by addition of TFA, diluted with DMF and purified by preparative HPLC (acidic method) to give the title compound (39 mg).

[0828] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.60 (m, 2H), 1.93 (br dd, J = 13.24, 3.10 Hz, 2H), 2.20 (s, 3H), 2.58 (m, 1H), 3.10 (m, 2H), 4.03 (d, J = 1.52 Hz, 3H), 4.30 (br d, J = 13.31 Hz, 2H), 7.03 (d, J = 9.12 Hz, 1H), 7.22 (m, 2H), 7.41 (m, 1H), 7.49 (m, 1H), 7.56 (d, J = 8.62 Hz, 1H), 7.79 (dd, J = 9.00, 2.66 Hz, 1H), 7.85 (d, J = 7.98 Hz, 1H), 8.34 (d, J = 2.66 Hz, 1H).

[0829] Example 3

[0830] (1R,5S,6R)-3-(5-{7”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0831]

[0832] Using the method described for Example 2: The title compound (15 mg) was obtained from intermediate 8 (40 mg) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (49 mg).

[0833] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.49 (t, J = 3.04 Hz, 1H), 2.18 (s, 3H), 2.24 (br s, 2H), 3.59 (m, 2H), 3.87 (d, J = 11.03 Hz, 2H), 4.09 (m, 1H), 4.23 (s, 3H), 6.69 (d, J = 9.00 Hz, 1H), 7.22 (m, 3H), 7.71 (dd, J = 7.79, 4.12 Hz, 1H), 7.86 (dd, J = 8.93, 2.60 Hz, 1H), 8.06 (d, J = 7.10 Hz, 1H), 8.39 (d, J = 2.53 Hz, 1H), 8.72 (d, J = 2.79 Hz, 1H).

[0834] Example 4

[0835] 1-(5-{7”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)piperidine-4-carboxylic acid

[0836]

[0837] Using the method described in Example 2: The title compound (41 mg) was obtained from intermediate 8 (40 mg) and methyl piperidinecarboxylate (47 mg).

[0838] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.62 (m, 2H), 1.94 (br dd, J = 13.24, 2.98 Hz, 2H), 2.06 (m, 1H), 2.19 (s, 3H), 2.59 (m, 1H), 3.11 (m, 2H), 4.24 (s, 3H), 4.25 (br s, 1H), 4.32 (brd, J = 13.31 Hz, 2H), 7.05 (d, J = 9.12 Hz, 1H), 7.22 (m, 3H), 7.71 (dd, J = 7.79, 4.12 Hz, 1H), 7.85 (dd, J = 9.06, 2.72 Hz, 1H), 8.06 (d, J = 7.35 Hz, 1H), 8.40 (d, J = 2.66 Hz, 1H), 8.74 (d, J = 2.79 Hz, 1H).

[0839] Example 5

[0840] (1R,5S,6S)-3-(5-{5”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0841]

[0842] Using the method described in Example 2: The title compound (16 mg) was obtained from intermediate 11 (24 mg) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (29 mg).

[0843] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.50 (t, J = 3.04 Hz, 1H), 2.20 (s, 3H), 2.24 (br s, 2H), 3.60 (br d, J = 10.52 Hz, 2H), 3.87 (d, J = 10.90 Hz, 2H), 4.17 (s, 3H), 6.70 (d, J = 9.00 Hz, 1H), 7.18 (m, 1H), 7.25 (m, 1H), 7.34 (dd, J = 10.84, 9.31 Hz, 1H), 7.67 (dd, J = 8.05, 4.37 Hz, 1H), 7.75 (dd, J = 9.31, 4.12 Hz, 1H), 7.91 (dd, J = 9.00, 2.66 Hz, 1H), 8.43 (d, J = 4.18 Hz, 2H), 8.42 (s, 1H).

[0844] Example 6

[0845] 1-(5-{5”-Fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)piperidine-4-carboxylic acid

[0846]

[0847] Using the method described in Example 2: The title compound (22 mg) was obtained from intermediate 11 (30 mg) and piperidinecarboxylic acid methyl ester (35 mg).

[0848] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.61 (m, 2H), 1.94 (br dd, J = 13.24, 2.98 Hz, 2H), 2.20 (m, 3H), 2.58 (m, 1H), 3.11 (m, 2H), 4.10 (m, 1H), 4.18 (m, 3H), 4.31 (br d, J = 13.18 Hz, 2H), 7.05 (d, J = 9.12 Hz, 1H), 7.18 (m, 1H), 7.25 (m, 1H), 7.34 (dd, J = 10.84, 9.31 Hz, 1H), 7.67 (dd, J = 7.92, 4.50 Hz, 1H), 7.75 (m, 1H), 7.88 (dd, J = 9.13, 2.66 Hz, 1H), 8.43 (m, 2H).

[0849] Example 7

[0850] (1R,5S,6S)-3-(5-{5”,7”-Difluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0851]

[0852] Using the method described in Example 2: The title compound (7.5 mg) was obtained from intermediate 13 (18 mg) and exo-3-azabicyclo[3.1.0]hexane-6-carboxylic acid methyl ester hydrochloride (21 mg).

[0853] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.45 (t, J = 2.98 Hz, 1H), 2.19 (m, 5H), 3.55 (br d, J = 10.52 Hz, 2H), 3.85 (d, J = 10.90 Hz, 2H), 4.20 (s, 3H), 6.61 (d, J = 8.87 Hz, 1H), 7.17 (m, 1H), 7.24 (m, 1H), 7.36 (t, J = 11.03 Hz, 1H), 7.65 (dd, J = 8.05, 4.37 Hz, 1H), 7.83 (dd, J = 8.87, 2.66 Hz, 1H), 8.39 (d, J = 2.54 Hz, 1H), 8.55 (d, J = 2.79 Hz, 1H).

[0854] Example 8

[0855] 1-[4-(5-{3”-Fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)piperazin-1-yl]ethan-1-one

[0856]

[0857] Using the method described in Example 2: The title compound (45 mg) was obtained from intermediate 6 (40 mg, 0.11 mmol) and 1-acetylpiperazine (41.4 mg, 0.32 mmol).

[0858] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.07 (s, 3H), 2.20 (s, 3H), 3.61 (s, 6H), 3.70 (m, 2H), 4.02 (m, 3H), 7.03 (d, J = 9.00 Hz, 1H), 7.20 (m, 1H), 7.25 (m, 1H), 7.41 (m, 1H), 7.49 (m, 1H), 7.56 (d, J = 8.74 Hz, 1H), 7.84 (m, 2H), 8.37 (d, J = 2.66 Hz, 1H).

[0859] Example 9

[0860] 4-(5-{3”-fluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)piperazine-1-carbaldehyde

[0861]

[0862] To a solution of intermediate 14 (30 mg, 0.068 mmol) in THF (3 ml) was added N-formylsaccharin (28.3 mg, 0.136 mmol) and the mixture was stirred at room temperature for 1 hour. Another portion of N-formylsaccharin (28.3 mg, 0.136 mmol) was added and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was diluted with a little water, acidified by adding TFA and purified by preparative HPLC (acidic method) to give the title compound (8 mg).

[0863] 1H NMR (400 MHz, DMSO-d6) δ ppm 2.19 (s, 3H), 3.53 (m, 4H), 3.67 (m, 4H), 4.02 (d, J = 1.65 Hz, 3H), 7.07 (d, J = 9.00 Hz, 1H), 7.20 (m, 1H), 7.25 (m, 1H), 7.41 (m, 1H), 7.49 (m, 1H), 7.56 (dd, J = 8.74, 1.01 Hz, 1H), 7.84 (m, 2H), 8.13 (s, 1H), 8.37 (d, J = 2.66 Hz, 1H)

[0864] Example 10

[0865] (1R,5S,6R)-3-(5-{3',5-difluoro-2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0866]

[0867] Intermediate 15 (62.6 mg, 0.18 mmol), intermediate 17 (50 mg, 0.12 mmol), potassium carbonate (20.1 mg, 0.15 mmol), copper(I) iodide (3.5 mg, 0.02 mmol), and 2-methylquinolin-8-ol (5.8 mg, 0.04 mmol) were heated in DMSO (1 ml) at 100 °C for 16 h. The reaction mixture was cooled. Subsequently, NaOH (4 M (aqueous solution), 0.5 ml) was added and the mixture was stirred at room temperature for 2 h. The reaction mixture was acidified by adding TFA, diluted with DMF, and purified by preparative HPLC (acidic method) to give the title compound (28 mg).

[0868] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.49 (t, J = 3.04 Hz, 1H), 2.19 (s, 3H), 2.23 (br s, 2H), 3.58 (m, 2H), 3.85 (br d, J = 10.90 Hz, 2H), 4.02 (d, J = 1.77 Hz, 3H), 6.67 (d, J = 9.00 Hz, 1H), 7.22 (dt, J = 9.76, 1.14 Hz, 1H), 7.40 (m, 1H), 7.47 (m, 1H), 7.57 (m, 2H), 7.81 (dd, J = 8.93, 2.60 Hz, 1H), 8.32 (d, J = 2.53 Hz, 1H)

[0869] Example 11

[0870] (1R,5S,6R)-3-(5-{3″,6-difluoro-2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0871]

[0872] A mixture of intermediate 20 (70 mg, 0.14 mmol), 3-fluoro-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)indazole (53 mg, 0.19 mmol), and sodium carbonate (45 mg, 0.43 mmol) in dioxane (2 ml) and water (0.5 ml) was degassed and maintained under a nitrogen atmosphere. [1,1'-Bis-(diphenylphosphino)-ferrocenyl]-dichloropalladium(II) (PdCl 2 dppf, 5.2 mg, 0.007 mmol) was added to the mixture and the mixture was heated at 100 °C for 1.5 h. DMF was added to the reaction mixture and then the mixture was filtered. The filtrate was diluted with water and purified by preparative HPLC (basic method) to give the title compound (56 mg).

[0873] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.47 (t, J = 3.04 Hz, 1H), 2.05 (d, J = 1.52 Hz, 3H), 2.21 (br s, 2H), 3.56 (m, 2H), 3.84 (d, J = 10.90 Hz, 2H), 4.02 (d, J = 1.65 Hz, 3H), 6.62 (d, J = 9.00 Hz, 1H), 7.15 (t, J = 9.38 Hz, 1H), 7.40 (m, 1H), 7.47 (m, 1H), 7.56 (dd, J = 8.62, 1.01 Hz, 1H), 7.76 (dd, J = 8.87, 2.66 Hz, 1H), 7.85 (dd, J = 8.81, 5.01 Hz, 1H), 8.31 (d, J = 2.53 Hz, 1H), 12.23 (br s, 1H)

[0874] Example 12

[0875] (1R,5S,6R)-3-(5-{5,7”-Difluoro-2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0876]

[0877] Using the method described for Example 11: The title compound (45 mg) was obtained from Intermediate 25 (73 mg) and Intermediate 23 (81.9 mg).

[0878] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.48 (t, J = 3.04 Hz, 1H), 2.17 (s, 3H), 2.24 (br s, 2H), 3.59 (m, 2H), 3.87 (d, J = 10.90 Hz, 2H), 4.23 (s, 3H), 6.68 (d, J = 9.00 Hz, 1H), 7.16 (dd, J = 11.47, 7.79 Hz, 2H), 7.22 (dd, J = 9.63, 1.27 Hz, 2H), 7.68 (dd, J = 7.86, 4.06 Hz, 2H), 7.84 (m, 3H), 8.39 (d, J = 2.53 Hz, 1H), 8.40 (m, 1H), 8.71 (d, J = 2.79 Hz, 1H)

[0879] Example 13

[0880] (1R,5S,6R)-3-(5-{3″,7″-Difluoro-2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0881]

[0882] Using the method described in Example 11: The title compound (15 mg) was obtained from Intermediate 4 (30 mg) and Intermediate 27 (18 mg).

[0883] HPLC (acidic): Rt = 0.907 min ((M + H)+501.2)

[0884] Example 14

[0885] (1R,5S,6R)-3-(5-{5,5″-Difluoro-2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0886]

[0887] Using the method described in Example 10: The title compound (5 mg) was obtained from Intermediate 15 (12 mg) and Intermediate 28 (7 mg).

[0888] HPLC (acidic): Rt = 0.941 min ((M + H)+501.0)

[0889] Example 15

[0890] (1R,5S,6R)-3-(5-{5,5″,7″-Trifluoro-2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-3-azabicyclo[3.1.0]hexane-6-carboxylic acid

[0891]

[0892] Using the method described in Example 10: The title compound (5 mg) was obtained from Intermediate 15 (8.1 mg) and Intermediate 29 (5 mg).

[0893] HPLC (acidic): Rt = 0.975 min ((M + H)+519.0)

[0894] The following examples were synthesized according to the methods described for Examples 1 to 15 above.

[0895] Table 1

[0896]

[0897]

[0898]

[0899]

[0900]

[0901]

[0902]

[0903]

[0904]

[0905]

[0906]

[0907]

[0908]

[0909]

[0910]

[0911]

[0912]

[0913]

[0914]

[0915]

[0916]

[0917]

[0918]

[0919]

[0920] Pharmacological activity

[0921] Biological examples

[0922] The compounds of the present invention were tested in two assays described below. That is, the compounds were tested in a canine differential scanning fluorimetry (DSF) assay and a canine whole blood assay. Representative results of the compounds of the present invention are compiled in Tables 2 and 3 below. Other assays used to test the compounds are also described below.

[0923] Canine differential scanning fluorimetry (DSF) assay

[0924] Canine STING (dSTING) protein production and purification

[0925] The protein used for biophysical experiments was recombinant canine STING protein, which contains its cytoplasmic cGAMP-binding ectodomain. A codon-optimized DNA sequence encoding amino acid residues 149 to 375 of canine STING (for expression in Escherichia coli) was synthesized by GeneArt (Regensburg, Germany) and inserted into the pET17b Escherichia coli expression vector. The protein construct encodes an N-terminal 8x His tag, followed by a tobacco etch virus protease (TEV) cleavage site and the above STING gene sequence. The resulting protein sequence of the canine STING protein (SEQ ID No. 1) used is listed below:

[0926] His-TEV—dSTING

[0927]

[0928] To express the above recombinant canine STING, the construct was transformed into Escherichia coli BL21 DE3 strain and grown in shake flasks in LB medium at 15 °C. Expression was induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 1 mM and the cultures were shaken overnight. The cell pellet was centrifuged and stored at -70 °C until further use. The protein was purified by thawing the cells in lysis buffer (20 mM TRIS-HCl, pH 8, 500 mM NaCl, 1 mM DTT, 0.5 mg / ml lysozyme, complete protease inhibitor (Roche) and Dnase (Roche)), followed by metal affinity purification using Ni-NTA resin and an elution buffer consisting of 20 mM TRIS-HCl, pH 8, 500 mM NaCl, 1 mM DTT, 300 mM imidazole. Cleavage of the His-tag with TEV protease was carried out overnight during dialysis in size exclusion buffer (20 mM TRIS-HCl, pH 8, 100 mM NaCl, 1 mM DTT). To further purify the target protein, a reverse nickel affinity column was used and the flow-through was applied to size exclusion chromatography. The peak fractions were collected and concentrated to 5 mg / mL.

[0929] Biophysical analysis - The stability of canine STING protein against thermal denaturation was determined by differential scanning fluorimetry (DSF).

[0930] The binding affinity of the compounds of the present invention was confirmed using a thermal shift assay, which measures the stability of canine STING in the presence of the compounds against thermal denaturation. In this assay, the melting temperature of the protein was monitored in the presence of a fluorescent dye that exhibits an affinity for the hydrophobic amino acids of the protein, which are buried in the folded state of the protein and gradually exposed during unfolding. The fluorescence of the dye is quenched in an aqueous environment and enhanced when the dye binds to the hydrophobic portion of the unfolded protein. A plot of fluorescence intensity versus temperature typically exhibits an S-shaped curve, which is interpreted by a two-state model of protein unfolding (differential scanning fluorimetry). The inflection point of the curve represents the "melting" temperature (Tm) of the protein, which is numerically calculated using the Boltzmann equation.

[0931] The thermal stability of canine STING protein was measured in an assay buffer containing 20 mM Tris, 150 mM NaCl at pH 7.5. The assay used a 384-well qPCR plate (catalog number 781358, BRAND), 'B' adhesive seal (catalog number MSB-1001, BIO-RAD), and operate on a CFX384 real-time system (Bio-Rad). Prepare a DMSO stock solution of SYPRO orange (SIGMA S5692-500UL). The compound stock solution (10 mM in DMSO) was diluted 1:2-fold in DMSO to an intermediate compound concentration of 5 mM, and then further diluted 1:40-fold in the assay buffer to obtain a compound concentration of 125 μM and 2.5% DMSO. Subsequently, the fluorescent dye stock solution (5000x SYPRO Orange) was mixed with the target protein and buffer to a concentration of 15 μM protein and 25x SYPRO Orange. 2 μl of this protein-dye mixture was added to 8 μl of the compound solution. The final volume was 10 μL. Wells 3 to 6 were used as negative controls (protein with 2% DMSO). Prepare the plate for replicate measurements and centrifuge at 1000 g for 2 minutes. In the measurement, use 160 cycles of 0.5 °C (temperature slope 15 seconds / cycle, 15 °C to 95 °C).

[0932] The final assay concentration for compound characterization was 100 μM compound, 3 μM target protein, 5x SYPRO Orange, and 2% DMSO in 10 μl. All dispensing steps were performed using a Hamilton Star pipetting robot (Hamilton).

[0933] Process the dissociation curve in Bio-Rad CFX Manager. The peak type was set to "negative". Assign compound codes for screening in the plate layout.

[0934] Take the average of the two TM measurement results and calculate the standard deviation. In the case of SD > 1.5 °C, repeat the measurement.

[0935] Subtract the melting temperature (Tm) obtained from the protein incubated alone with the ligand from the Tm obtained from the protein incubated with the ligand to generate a ΔTm value.

[0936] Canine whole blood analysis

[0937] To detect STING activation in the physiological environment, canine whole blood (dWB) was stimulated with the cyclic dinucleotide cGAMP or the test compound. Pathway activity was monitored by measuring IFNβ production.

[0938] The compound was delivered as a 10 mM DMSO solution, diluted and transferred using an Echo acoustic dispenser to 384-well assay plates (Greiner #781182), each well pre-filled with 10 μl of 1× HBSS (10× HBSS (+Ca / +Mg), #14065-049, Gibco). Typically, 8 concentrations were used, with the highest concentration in the final assay volume being 10 μM, followed by approximately 1:4 dilution steps. The DMSO concentration was set at 0.1% in the final assay volume. The 384-well assay plates contained 20 test compounds and DMSO, with DMSO in the control wells and cGAMP standard wells. Canine whole blood was collected as citrate blood (e.g., 3.8% concentration in Monovettes from Sarstedt) and kept at 4 °C overnight until used for analysis. 80 μl of the whole blood sample was transferred to each well of the 384-well assay plate filled with the compound / 1× HBSS. The blood plates were kept at room temperature for 60 minutes and continuously shaken at 450 rpm, covered with a lid but not sealed. The 10× cGAMP assay solution was diluted from a 2 mM stock solution in 1× HBSS immediately before use at room temperature. 10 μl of 10× cGAMP / HBSS was added to the high control wells, while only HBSS was added to all compound and low control wells. After covering the assay plates with a lid, the blood plates were incubated for 4 hours at 37 °C in an incubator without shaking. To detect IFNβ in canine plasma, an ELISA kit for canine interferon β (Biotrend #SEA222Ca) was used. The whole blood assay plates were centrifuged at 1000 g for 10 minutes at 8 °C. 40 μl of the supernatant was transferred from the 384-well whole blood plate to the corresponding 96-well ELISA plate, each well of which was pre-filled with 60 μl of assay diluent, by a 96-well pipetting robot. The plates were sealed with microplate seals and kept at 4 °C again overnight. The ELISA plates were brought to room temperature and then incubated for 1 hour at 37 °C in an incubator. The detection reagent A working solution was prepared by diluting the detection reagent A 1:100 in assay reagent A. Subsequently, the liquid was removed from the 96-well ELISA plates to add 100 μL of the detection reagent A working solution to each well. The ELISA plates were covered with a plate sealer and incubated for 1 hour at 37 °C in an incubator. By in H 2Prepare 1× wash buffer by diluting the 30× wash buffer concentrate in water. Prepare the working solution of Detection Reagent B by diluting Detection Reagent B 1:100 in Assay Reagent B. Wash the ELISA plate three times with 350 μl of wash buffer, then invert and blot dry with absorbent paper to remove any liquid. Add 100 μL of the working solution of Detection Reagent B to each well of the ELISA plate, then cover the ELISA plate with a plate sealer and incubate in an incubator at 37 °C for 30 minutes. After incubation, wash the ELISA assay plate five times with 350 μl of wash buffer, invert again and blot dry with absorbent paper to remove any residual liquid. Add 90 μL of TMB substrate to each well of the ELISA plate, then cover the ELISA plate with a plate sealer and incubate in an incubator at 37 °C for 15 minutes. Stop the reaction by adding 50 μL of stop solution and immediately measure the absorbance at 450 nm.

[0939] Data evaluation and calculation:

[0940] For data evaluation and calculation, the % control for each well is calculated based on the average of the high control (cGAMP-stimulated control) and the average of the low control (unstimulated control), using the following standard 4-parameter logistic regression formula:

[0941] [y = (a - d) / (1 + (x / c)^b) + d]

[0942] a = low value,

[0943] d = high value,

[0944] x = concentration M,

[0945] c = EC 50 M,

[0946] b = slope.

[0947] Canine liver microsome (dLM) assay

[0948] The metabolic degradation of the test compound was analyzed using the collected liver microsomes from dogs (beagles) at 37°C. The final incubation volume of 100 μl at each time point contained TRIS buffer pH 7.6 (0.1 M) at room temperature, magnesium chloride (5 mM), microsomal protein (1 mg / ml), and the test compound at a final concentration of 1 μM. After a short pre-incubation period at 37°C, the reaction was initiated by adding reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM), and the reaction was terminated by transferring aliquots to the solvent at different time points. Additionally, NADPH-independent degradation was monitored during incubation in the absence of NADPH and terminated at the last time point. The remaining test compound [%] after NADPH-independent incubation was reflected by the parameter c (control) (metabolic stability). The quenched incubation mixture was precipitated by centrifugation (10,000 g, 5 minutes).

[0949] The amount of the parent compound in the aliquots of the supernatant was analyzed by LC-MS / MS. The half-life (t1 / 2INVITRO) was determined by the slope of the semi-logarithmic plot of the concentration-time profile. The intrinsic clearance (CL_INTRINSIC) was calculated by taking into account the amount of protein in the incubation: CL_INTRINSIC [μl / min / mg protein] = (Ln 2 / (half-life [min] * protein content [mg / ml])) * 1000. For a more preferred cross-species comparison, the predicted clearance was expressed as a percentage of the hepatic blood flow rate [QH%] in each species. Generally, high stability of the compound across species (corresponding to low QH%) is desired.

[0950] Mouse liver microsome (mLM) analysis

[0951] The metabolic degradation of the test compound was analyzed using the collected liver microsomes from (male / female) mice (CD1) at 37°C. The final incubation volume of 100 μl at each time point contained TRIS buffer pH 7.6 (0.1 M) at room temperature, magnesium chloride (5 mM), microsomal protein (0.5 mg / ml), and the test compound at a final concentration of 1 μM. After a short pre-incubation period at 37°C, the reaction was initiated by adding reduced form of β-nicotinamide adenine dinucleotide phosphate (NADPH, 1 mM), and the reaction was terminated by transferring aliquots to the solvent at different time points. Additionally, NADPH-independent degradation was monitored during incubation in the absence of NADPH and terminated at the last time point. The remaining test compound [%] after NADPH-independent incubation was reflected by the parameter c (control) (metabolic stability). The quenched incubation mixture was precipitated by centrifugation (10,000 g, 5 minutes).

[0952] The amount of the parent compound in an aliquot of the supernatant was analyzed by LC-MS / MS. The half-life (t1 / 2INVITRO) was determined by the slope of the semi-logarithmic plot of the concentration-time profile. The intrinsic clearance (CL_INTRINSIC) was calculated by taking into account the amount of protein in the incubation: CL_INTRINSIC [μL / min / mg protein] = (Ln 2 / (half-life [min] * protein content [mg / ml])) * 1000. For a more preferred cross-species comparison, the predicted clearance is expressed as a percentage of the hepatic blood flow rate [QH%] in individual species. Generally, high stability of the compound across species (corresponding to low QH%) is desired.

[0953] Canine hepatocyte (dHep) assay

[0954] The metabolic degradation of the test compound was analyzed in a suspension of canine hepatocytes.

[0955] Incubation: Cryopreserved canine hepatocytes were incubated in a suitable buffer system (KHB buffer or a similar buffer or standard cell culture medium) containing 50% homologous serum. After an acclimation period (15 - 30 minutes) in an incubator (37 °C, 5 - 10% CO 2 , 85 - 95% humidity), the test compound was added to the hepatocyte suspension (pH 7.4, typical cell density of approximately 1 million cells / ml; final concentration of the test compound was 1 μM, final DMSO concentration < 0.05% v / v). The cells were incubated for up to 6 hours and samples were taken at 6 different time points. Subsequently, the samples were quenched with acetonitrile and precipitated by centrifugation. The remaining amount of the parent compound in the supernatant was then analyzed by HPLC-MS / MS.

[0956] Calculation: The elimination rate constant (ke) was calculated using the slope of the linear regression of the natural logarithm [substrate remaining % or substrate concentration] versus time [hours].

[0957] ke = (-1) * slope

[0958] ke = elimination rate constant [1 / hour]

[0959] The half-life was calculated from the elimination rate constant

[0960] t1 / 2 = ln(2) / ke

[0961] t1 / 2 = half-life [hours]

[0962] Calculation of intrinsic in vitro hepatic clearance:

[0963] CL_int_in vitro = ke * 1000 / (CD * 60)

[0964] CL_int_in vitro = In vitro intrinsic hepatic clearance [µL / min / million cells (Mio cells)]

[0965] CD = Cell density [million cells / mL]

[0966] Note: The above equation holds only when [substrate] << Km.

[0967] The calculated in vitro intrinsic hepatic clearance can be scaled up to the intrinsic in vivo hepatic clearance and used to predict the in vivo hepatic blood clearance (CL_ws) by using a liver model (well-stirred model).

[0968] CL_int_in vivo = (CL_int_in vitro * H * L) / 1000

[0969] CL_int_in vivo = In vivo intrinsic hepatic clearance [mL / min / kg]

[0970] H = Hepatocellularity [million cells / g liver]

[0971] L = Liver factor [g / kg body weight]

[0972] CL_ws = CL_int_in vivo * Q / (CL_int_in vitro + Q)

[0973] CL = Estimation of in vivo hepatic clearance [mL / min / kg]

[0974] Q = Hepatic blood flow rate [mL / min / kg]

[0975] ws = Well-stirred

[0976] QH% = CL_ws * 100 / Q

[0977] QH% = Clearance expressed as a percentage of hepatic blood flow rate

[0978] Concentration: Cell concentration at incubation time (10 6 / mL)

[0979] T_LAST: Terminal time point used (hours)

[0980] Mouse hepatocyte (mHep) analysis

[0981] The metabolic degradation of the test compound is analyzed in a suspension of mouse hepatocytes.

[0982] Incubation: Mouse hepatocytes stored at low temperature are incubated in an appropriate buffer system (KHB buffer or a similar buffer or standard cell culture medium) containing 50% species serum. After an environmental adaptation period (15 - 30 minutes) in an incubator (37°C, 5 - 10% CO2, 85 - 95% humidity), the test compound is added to the hepatocyte suspension (pH 7.4, typical cell density of approximately 1 million cells / ml; final concentration of the test compound is 1 μM, final DMSO concentration < 0.05% v / v). The cells are incubated for up to 6 hours and samples are taken at 6 different time points. Subsequently, the samples are quenched with acetonitrile and precipitated by centrifugation. The remaining amount of the parent compound in the supernatant is then analyzed by HPLC - MS / MS. Calculation: The elimination rate constant (ke) is calculated using the slope of the linear regression of the natural logarithm [substrate remaining % or substrate concentration] versus time [hours].

[0983] ke = (-1) * slope

[0984] ke = elimination rate constant [1 / hour]

[0985] The half - life is calculated from the elimination rate constant

[0986] t1 / 2 = ln(2) / ke

[0987] t1 / 2 = half - life [hours]

[0988] Calculation of intrinsic in vitro hepatic clearance:

[0989] CL_int_in vitro = ke * 1000 / (CD * 60)

[0990] CL_int_in vitro = intrinsic in vitro hepatic clearance [μl / minute / million cells]

[0991] CD = cell density [million cells / ml]

[0992] Note: The above equations are only valid when [substrate] << Km.

[0993] The calculated in vitro intrinsic hepatic clearance can be scaled up to the intrinsic in vivo hepatic clearance and used to predict the in vivo hepatic blood clearance (CL_ws) by using a liver model (well - stirred model).

[0994] CL_int_in vivo = (CL_int_in vitro * H * L) / 1000

[0995] CL_int_in vivo = intrinsic in vivo hepatic clearance [ml / minute / kg]

[0996] H = hepatocellularity [million cells / g liver]

[0997] L = Liver factor [g / kg body weight]

[0998] CL_ws = CL_int_in vivo * Q / (CL_int_in vitro + Q)

[0999] CL = Estimation of hepatic clearance in vivo [ml / min / kg]

[1000] Q = Hepatic blood flow rate [ml / min / kg]

[1001] ws = Well-stirred

[1002] QH% = CL_ws * 100 / Q

[1003] QH% = Clearance expressed as a percentage of hepatic blood flow rate

[1004] Concentration: Cell concentration at incubation time (10 6 / ml)

[1005] T_LAST: The terminal time point used (hours)

[1006] Mouse hepatocytes: 120 × 10e6 cells / g liver

[1007] Mouse liver factor: 55 g / kg body weight

[1008] Mouse blood flow rate: 90 ml / (min × kg).

[1009] Permeability analysis (MDCK-PGP)

[1010] This analysis provides information on the potential of a compound to cross the blood-brain barrier. Permeability measurements using a polarized confluent monolayer of MDCK-MDR1 cells grown on a permeable filter support are used as an in vitro absorption model.

[1011] The apparent permeability coefficient (PE) of a compound across an MDCK-MDR1 cell monolayer was measured in the top-to-bottom (AB) and bottom-to-top (BA) transport directions (pH 7.4, 37 °C). The AB permeability (PEAB) represents the drug absorption rate from blood to brain and the BA permeability (PEBA) represents the drug efflux rate from brain back to blood via passive permeation and active transport mechanisms, the active transport mechanisms being mediated by efflux and uptake transporters expressed on MDCK-MDR1 cells, mainly by overexpressed human MDR1 P-gp. Compounds were classified into permeability / absorption classes by comparing the AB permeability of the compound with that of a reference compound having known in vitro permeability and oral absorbability in humans. The same or similar permeability in both transport directions indicates passive permeation and a vector permeability indicates other active transport mechanisms. A PEBA higher than PEAB indicates involvement of active efflux mediated by MDR1 P-gp. Active transport is concentration-dependent and saturable.

[1012] MDCK-MDR1 cells (1 - 2×10 5 cells / 1 cm 2 area) were seeded on filter inserts (Costar transwell polycarbonate or PET filters, 0.4 μm pore size) and cultured (DMEM) for 7 days. Subsequently, MDR1 expression was enhanced by culturing the cells with 5 mM sodium butyrate in complete medium for 2 days. Compounds were dissolved in a suitable solvent (e.g., DMSO, 1 - 20 mM stock solution). The stock solution was diluted with HTP-4 buffer (128.13 mM NaCl, 5.36 mM KCl, 1 mM MgSO 4 4, 1.8 mM CaCl 2 2, 4.17 mM NaHCO 3 3, 1.19 mM Na 2 2HPO 4 4×7H 2 2O, 0.41 mM NaH 2 2PO 4 4×H 2 2O, 15 mM HEPES, 20 mM glucose, 0.25% BSA, pH 7.4) to prepare the transport solution (0.1 - 300 μM compound, final DMSO ≤ 0.5%). The transport solution (TL) was applied to the top or bottom outer donor side to measure A - B or B - A permeability, respectively (3 filters were repeated). The receiver side contained the same buffer as the donor side. Samples were collected from both the donor and the receiver sides at the start and end of the experiment and at various time intervals (up to 2 hours) for concentration measurement by HPLC-MS / MS or scintillation counting. The receiver volume after sampling was replaced with fresh receiver solution.

[1013] Result

[1014] Table 2: Interaction with canine STING measured by DSF (dDSF)

[1015]

[1016]

[1017]

[1018] Table 3: Cytokine secretion in canine whole blood (dWB) culture system measured by canine interferon-β (IFNb) ELISA

[1019]

[1020]

[1021] As demonstrated by the examples of the present invention, when measured by the binding assay, the compounds of the present invention exhibit an interaction with canine STING (dSTING), which is reflected by a T shift of > 15°C, more preferably > 20°C, and even more preferably > 25°C as measured by DSF. In addition, the compounds of the present invention induce cytokine secretion in canine whole blood (dWB). According to the present invention, the combination of high dDSF and low dWB is particularly advantageous. m ​

Claims

1. A compound of formula (I) wherein B is a group selected from: a 5- to 7-membered monocyclic heterocyclic group containing 1 or 2 N atoms, a 6-membered bicyclic heterocyclic group containing 1 N atom, a 7- to 11-membered bicyclic heterocyclic group containing 1 or 2 N atoms, a 7-membered bicyclic heterocyclic group containing 1 N atom and 1 O atom, a 6-membered monocyclic heterocyclic group containing 1 N atom and 1 heteroatom selected from O and S, a 9-membered bicyclic heterocyclic group containing 3 heteroatoms, wherein 2 heteroatoms are N and the other is O, a 9-membered bicyclic heterocyclic group containing 1 N atom and 1 S atom, A 10-membered bicyclic heterocyclic group containing 3 N atoms, wherein 2 N atoms are substituted by C 1-6 alkyl groups, phenyl, a 9-membered bicyclic heteroaryl group containing 3 N atoms, -C 1-4 alkylene-pyrimidine, and -C 1-4 alkylene-O-C 1-3 alkyl; D is a group selected from: a 9-membered bicyclic heteroaryl group containing 2 N atoms, a 10-membered bicyclic heteroaryl group containing 1 N atom, and benzodioxole; R 1 selected from -H or -C 1-6 alkyl; R 2 selected from -H, halogen and -C 1-6 alkyl, said halogen is preferably fluorine or chlorine, more preferably fluorine; R 3 selected from -H, halogen and -C 1-6 alkyl, wherein the halogen is preferably fluorine or chlorine, more preferably fluorine; R 4a 、 R 4b and R 4c each independently selected from -H, halogen and C 1-6 alkyl, wherein the halogen is preferably fluorine or chlorine, more preferably fluorine, provided that at least one of R 4a 、 R 4b and R 4c is halogen; R 4d selected from -C 1-6 alkyl and C 3-6 cycloalkyl; R 5 is absent or selected from -H, -C 1-6 alkyl, -S(O 2 )-C 1-6 alkyl, -NH-S(O 2 )-C 1-6 alkyl, =O, -C(O)-C 1-6 alkyl, -C(O)H, -C(O)OH, -C(O)NH 2 , -C(O)O-C 1-6 alkyl, -NR 5.1 R 5.2 , -C 1-6 alkylene-C(O)OH, -S(O 2 )-NH 2 , -pyrrolidin-2-one-1-yl, -tetrazolyl, and an R 5.3 -substituted 5-membered heteroaryl having 1 or 2 heteroatoms selected from N and O; R 5.1 selected from -H, -C 1-6 alkyl, -C(O)-C 1-6 alkyl and -C 1-6 alkylene-O-C 1-6 alkyl; R 5.2 selected from -H, -C 1-6 alkyl, -C(O)-C 1-6 alkyl and -C 1-6 alkylene-O-C 1-6 alkyl; R 5.3 selected from -H, -C 1-6 alkyl and 6-membered heteroaryl having 1 or 2 heteroatoms selected from N and O; R 6 is absent or selected from -H, -C 1-6 alkyl, =O and -C(O)OH; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein R 1 is -C 1-6 alkyl; and R 4a , R 4b and R 4c at least one of which is fluorine or chlorine, and the rest are -H or -C 1-3 alkyl; and R 4d is -C 1-6 alkyl; or a pharmaceutically acceptable salt thereof.

3. The compound according to any one of claims 1 to 2, wherein D is selected from: wherein R 4d is C 1-6 alkyl; or a pharmaceutically acceptable salt thereof.

4. The compound according to any one of claims 1 to 3, wherein D is or a pharmaceutically acceptable salt thereof.

5. The compound according to any one of claims 1 to 4, wherein D is selected from the following structures: wherein R 4a is -H; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and R 4d is methyl; or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or wherein R 4a is - H; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and R 4d is methyl; or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or wherein R 4a is -H; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is -H; and R 4d is methyl; or R 4a is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is - methyl; or wherein R 4b is -H or methyl; R 4c is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d is methyl; or R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4c is -H or methyl; and R 4d is methyl; or R 4b is a halogen, preferably chlorine or fluorine, more preferably fluorine; R 4c is a halogen, preferably chlorine or fluorine, more preferably fluorine; and R 4d -methyl; or a pharmaceutically acceptable salt thereof.

6. The compound according to any one of claims 1 to 5, wherein R 1 is methyl; R 2 is -H or halogen; and R 3 is -H or halogen; or a pharmaceutically acceptable salt thereof.

7. The compound according to any one of claims 1 to 6, wherein R 1 is methyl, R 2 is -H; and R 3 is -H; or R 1 is methyl, R 2 is -H; and R 3 is fluorine; or R 1 is methyl, R 2 is fluorine; and R 3 is -H; or a pharmaceutically acceptable salt thereof.

8. The compound according to any one of claims 1 to 7, wherein B is selected from: wherein R 7 is selected from H, -C 1-6 alkyl, -C 3-6 cycloalkyl and -OH; R 8 is (CH 2 ) n , where n is an integer from 1 to 3, preferably 1 or 2; R 9 selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 10 selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; R 11 selected from H, -C 1-6 alkyl and -C 3-6 cycloalkyl; X is CH or N; and Y is -O-, -S-, -S(O)-, -S(O) 2 -; or a pharmaceutically acceptable salt thereof.

9. The compound according to any one of claims 1 to 8, wherein B is selected from: wherein R 7 is selected from -H and -C 1-6 alkyl; R 9 Selected from -H and methyl, and preferably -H; R 10 selected from -H and methyl, and preferably -H; R 11 selected from -H and methyl, and preferably -H; and Y is O; or a pharmaceutically acceptable salt thereof.

10. A compound selected from the following structures: or a pharmaceutically acceptable salt thereof.

11. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

12. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 11, which is used as a medicine.

13. The compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to claim 11, which is used for treating feline or canine cancer.

14. The compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition for the use according to claim 13, wherein the canine cancer is selected from osteosarcoma (OSA), oral melanoma, B-cell lymphoma, urothelial carcinoma (UC), angiosarcoma, mast cell tumor, soft tissue sarcoma, squamous cell carcinoma, T-cell lymphoma, mammary adenocarcinoma, and anal sac carcinoma, or wherein the feline cancer is selected from B-cell and / or T-cell lymphoma, squamous cell carcinoma, mammary adenocarcinoma, mast cell tumor, and injection site sarcoma.

15. The compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition for the use according to claim 13 or 14, wherein the compound, the pharmaceutically acceptable salt thereof, or the pharmaceutical composition is used in combination with radiotherapy.

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