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 cancers (such as canine and / or feline cancers), 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 cancers.

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 trigger 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 cytoplasmic 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 cytoplasmic 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, especially 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 that enables 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, no. 6227, 2630 - 1 - 2630 - 14, 2015). Phosphorylation of the transcription factors IRF3 and NF - kB by TBK1 leads to the expression of multiple 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 lack 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 results in 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, the specific activation of STING leads to enhanced 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 adaptive 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 have a synergistic effect 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] R1 is -C 1-6 alkyl or -C 3-6 cycloalkyl;

[0018] R 2a is selected from -H or -C 1-6 alkyl;

[0019] R 2b is selected from -H, -C 1-6 alkyl, -C 1-6 alkylene-OH, -C(O)OH, -C(O)O-C 1-6 alkyl and -pyrazolyl-C 1-6 alkyl;

[0020] R 2c is -H or -C 1-6 alkyl;

[0021] R 3 is selected from (* indicates the point of attachment):

[0022]

[0023] R 4 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0024] R 5 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0025] R 6 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0026] R 7 is selected from H, -C 1-6 alkyl, C 3-6 cycloalkyl and -OH;

[0027] R 8 is (CH 2 ) n , where n is an integer from 1 to 3, preferably 1 or 2;

[0028] X is CH or N; and

[0029] Y is -O-, -S-, -S(O)-, -S(O) 2 -;

[0030] or a pharmaceutically acceptable salt thereof.

[0031] 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.

[0032] 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.

[0033] 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

[0035] 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).

[0036] Thus, on the other hand, the present invention provides novel 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).

[0037] 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 test, 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.

[0038] 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 the STING agonist, and the level of interferon β in the supernatant is quantified by ELISA. 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.

[0039] According to the invention, the combination of high dDSF and low dWB is particularly advantageous.

[0040] Compounds of general formula (I) of the invention

[0041]

[0042] wherein

[0043] R 1 is -C 1-6 alkyl or -C 3-6 cycloalkyl;

[0044] R 2a is selected from -H or -C 1-6 alkyl;

[0045] R 2b is selected from -H, -C 1-6 alkyl, -C 1-6 alkylene-OH, -C(O)OH, -C(O)O-C 1-6 alkyl and -pyrazolyl-C 1-6 alkyl;

[0046] R 2c is -H or -C 1-6 alkyl;

[0047] R 3 is selected from (* indicates the point of attachment):

[0048]

[0049] R 4 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0050] R 5 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0051] R 6 is selected from H, -C 1-6Alkyl and C 3-6 Cycloalkyl;

[0052] R 7 selected from H, -C 1-6 alkyl, C 3-6 cycloalkyl and -OH;

[0053] R 8 is (CH 2 ) n , where n is an integer from 1 to 3, preferably 1 or 2;

[0054] X is CH or N; and

[0055] Y is -O-, -S-, -S(O)-, -S(O) 2 -;

[0056] Or a pharmaceutically acceptable salt thereof is particularly suitable for treating pathophysiological processes related to or regulated by STING, particularly suitable for treating cancers, such as feline or canine cancers, or used as a vaccine adjuvant, for example, in pigs.

[0057] Accordingly, 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.

[0058] 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 cancers.

[0059] For those skilled in the art, other aspects of the present invention will become apparent directly from the foregoing and the following description and examples.

[0060] Terms and definitions used

[0061] 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.

[0062] In the groups, radicals or moieties defined below, the number of carbon atoms is usually specified before the group, for example, C 1-6 alkyl means an alkyl group or alkyl radical having 1 to 6 carbon atoms. Generally, in such as HO, H 2 N, (O)S, (O) 2 S, NC (cyano), HOOC, F3 In groups such as C etc., those skilled in the art can see one or more connection points between the group and 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 an 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.

[0063] If the compounds of the present invention are depicted in chemical names and chemical formulas, in case of any inconsistency, the chemical formula shall prevail. An asterisk or a wavy line in the sub-formula can be used to indicate a bond connecting to the core molecule as defined.

[0064] For example, the term "3-carboxypropyl-group" represents the following substituent:

[0065]

[0066] 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:

[0067]

[0068] A wavy line in the sub-formula can be used to indicate a bond connecting to the core molecule as defined. Alternatively, an asterisk in the sub-formula can be used to indicate a bond connecting to the core molecule as defined.

[0069] 1.1.1.1 Substituted terms

[0070] 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.

[0071] 1.1.1.2 Stereochemistry - Solvates - Hydrates

[0072] 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, and mixtures of individual enantiomers, mixtures of diastereomers, or mixtures in which such isomers and enantiomers are present in any of the foregoing forms, in different proportions, as well as their solvates, such as hydrates.

[0073] Unless otherwise indicated, “pharmaceutically acceptable salts” as more particularly defined hereinafter shall also encompass their solvates, such as hydrates.

[0074] 1.1.1.3 Stereoisomers

[0075] 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.

[0076] The enantiomerically pure compounds or intermediates of the present invention can be prepared via asymmetric synthesis, for example by preparing and subsequently separating suitable 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).

[0077] 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 a suitable 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 carrying out the 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.

[0078] 1.1.1.4 Salts

[0079] The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms that, within the scope of reasonable medical judgment, are suitable for contact with the tissues of mammals without excessive toxicity, irritation, allergic response, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0080] As used herein, "pharmaceutically acceptable salts" refer 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.

[0081] For 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.

[0082] 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.

[0083] The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compounds containing basic or acidic moieties by conventional chemical methods. Generally, 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 a mixture thereof.

[0084] In addition to the salts mentioned above, salts of other acids (e.g., trifluoroacetate) used, for example, for the purification or isolation of the compounds of the present invention also form part of the present invention.

[0085] 1.1.1.5 Halogen

[0086] The term halogen refers to fluorine, chlorine, bromine, and iodine.

[0087] 1.1.1.6 Heteroatom

[0088] 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').

[0089] 1.1.1.7 Alkyl

[0090] The term "C" alone or in combination with another group 1-n"Alkyl" (where n is an integer selected from 2, 3, 4, 5, or 6, preferably 4, 5, or 6) means 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 )-.

[0091] 1.1.1.8 Alkylene

[0092] 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 )-.

[0093] 1.1.1.9 Alkenyl

[0094] 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.

[0095] 1.1.1.10 Alkenylene

[0096] 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.

[0097] 1.1.1.11 Alkynyl

[0098] 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.

[0099] 1.1.1.12 Alkynylene

[0100] 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.

[0101] 1.1.1.13 Cycloalkyl

[0102] 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.

[0103] 1.1.1.14 Cycloalkenyl

[0104] 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 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.

[0105] 1.1.1.15 halo-(alkyl, alkylene or cycloalkyl)

[0106] 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-.

[0107] 1.1.1.16 carbocyclic group

[0108] 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.

[0109]

[0110] 1.1.1.17 aryl

[0111] 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, anthracenyl, phenanthryl, tetrahydronaphthyl and dihydronaphthyl.

[0112] 1.1.1.18 heterocyclic group

[0113] 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.

[0114] 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 provided the appropriate valence is maintained):

[0115]

[0116]

[0117]

[0118] 1.1.1.19 Heteroaryl

[0119] 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, where at least one of the heteroatoms is part of the aromatic ring, and the resulting ring system must be chemically stable. The term "heteroaryl" is intended to include all possible isomeric forms.

[0120] 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 provided the appropriate valence is maintained):

[0121]

[0122]

[0123] 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.

[0124] The term "bicyclic system" means a group consisting of 2 joined cyclic substructures, including spiro, fused and bridged ring systems.

[0125] Preferred Embodiment

[0126] One preferred embodiment of the present invention relates to a compound of formula (I)

[0127]

[0128] or a pharmaceutically acceptable salt thereof, wherein

[0129] R 1 is -C 1-6 alkyl or -C 3-6 cycloalkyl;

[0130] R 2a is selected from -H or -C 1-6 alkyl;

[0131] R 2b is selected from -H, -C 1-6 alkyl, -C 1-6 alkylene-OH, -C(O)OH, -C(O)O-C 1-6 alkyl and -pyrazolyl-C 1-6 alkyl;

[0132] R 2c is -H or -C 1-6 alkyl;

[0133] R 3 is selected from the following (* indicates the point of attachment):

[0134]

[0135] R 4 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0136] R 5 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0137] R 6 is selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl;

[0138] R 7 is selected from H, -C 1-6 alkyl, C 3-6 cycloalkyl and -OH;

[0139] R 8 is (CH 2 ) n , where n is an integer from 1 to 3, preferably 1 or 2;

[0140] X is CH or N; and if X is N, then R 7 is not -OH; and

[0141] Y is -O-, -S-, -S(O)-, -S(O) 2 -.

[0142] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C 1-6 alkyl, preferably methyl.

[0143] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2a is -C1-6 an alkyl group, preferably a methyl group; R 2b is -H or -C 1-6 alkyl group; and R 2c is -H or -C 1-6 alkyl group.

[0144] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C 1-6 alkyl group, preferably a methyl group, R 2a is -C 1-6 alkyl group, preferably a methyl group; R 2b is -H or -C 1-6 alkyl group; and R 2c is -H or -C 1-6 alkyl group.

[0145] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 2a is a methyl group; R 2b is -H; and R 2c is -H.

[0146] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 is -C 1-6 alkyl group, preferably a methyl group, R 2a is a methyl group; R 2b is -H; and R 2c is -H.

[0147] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0148]

[0149] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0150]

[0151] wherein R 7 is -C 1-6 alkyl group, preferably a methyl group.

[0152] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0153]

[0154] and R 1 is -C 1-6 alkyl, preferably methyl.

[0155] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0156]

[0157] and R 2a is -C 1-6 alkyl, preferably methyl; R 2b is -H or -C 1-6 alkyl; and R 2c is -H or -C 1-6 alkyl.

[0158] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0159]

[0160] R 1 is -C 1-6 alkyl, preferably methyl; R 2a is -C 1-6 alkyl, preferably methyl; R 2b is -H or -C 1-6 alkyl; and R 2c is -H or -C 1-6 alkyl.

[0161] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0162] and R 1 is -C 1-6 alkyl, preferably methyl, R 2a is methyl; R 2b is -H; and R 2c is -H.

[0163] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0164] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0165]

[0166] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0167] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0168] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0169] R 4 is -H or methyl, preferably -H; R 5 and R 6 are independently -H or methyl, preferably R 5 and R 6 are both -H; and Y is O. Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0170]

[0171] R 1 is methyl; R 4 is -H or methyl, preferably -H; R 5 and R 6 are independently -H or methyl, preferably R 5 and R 6 are both -H; Y is O.

[0172] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0173]

[0174] R 2a is -C 1-6 alkyl, preferably methyl; R 2b is -H or -C 1-6 alkyl; and R 2c is -H or -C 1-6 alkyl; R 4 is -H or methyl, preferably -H; R 5 and R 6 are independently -H or methyl, preferably R 5 and R 6All are -H; Y is O.

[0175] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 3 is

[0176]

[0177] R 1 is methyl; R 2a is -C 1-6 alkyl, preferably methyl; R 2b is -H or -C 1-6 alkyl; and R 2c is -H or -C 1-6 alkyl; R 4 is -H or methyl, preferably -H; R 5 and R 6 are independently -H or methyl, preferably R 5 and R 6 are both -H; Y is O.

[0178] Another particularly preferred embodiment relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein R 1 is methyl; R 2a is methyl; R 2b and R 2c are -H; R 3 is

[0179]

[0180] R 4 , R 5 and R 6 are -H; and Y is O.

[0181] Particularly preferred compounds of the present invention are selected from the following structures:

[0182]

[0183] or a pharmaceutically acceptable salt thereof.

[0184] Furthermore, particularly preferred compounds of the present invention are:

[0185]

[0186]

[0187]

[0188] or a pharmaceutically acceptable salt thereof.

[0189] In one embodiment, the present invention relates to compounds of formula (I) in their non-salt form. In another embodiment, the present invention relates to compounds of formula (I) in their pharmaceutically acceptable salt form.

[0190] R as described above for formula (I) 1 、R 2a 、R 2b 、R 2c 、R 3 、R 4 、R 5 、R 6 、R 7 、R 8 Any one of the definitions of R, R, R, R, R, R, R, R, R, R, R, R, X and Y and each can be combined with each other.

[0191] 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.

[0192] It has been found that the compounds of formula (I) or their pharmaceutically acceptable salts can be used for the prevention and / or treatment of 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 the treatment of feline or canine cancer.

[0193] On the other hand, the compounds of the present invention exhibit an interaction with canine STING (dSTING), which is reflected by a T shift measured by DSF of >15 °C, more preferably >20 °C, and even more preferably >25 °C. m offset.

[0194] 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).

[0195] In a more preferred embodiment, the compounds of the present invention exhibit a combination of high dDSF and low dWB.

[0196] Treatment methods

[0197] 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 for treating a disease.

[0198] Specifically, the compounds of general formula (I) or salts thereof are 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.

[0199] Diseases and conditions associated with 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.

[0200] Autoimmune diseases include (but are not limited to) systemic lupus erythematosus, psoriasis, insulin-dependent diabetes mellitus (IDDM), dermatomyositis, and Sjogren's syndrome (SS).

[0201] 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, neuroinflammation, digestive system inflammation, ocular inflammation, reproductive system inflammation, and other inflammations.

[0202] 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, dacryoadenitis, keratitis, keratoconjunctivitis sicca (dry eye), scleritis, trichiasis, and uveitis.

[0203] Examples of neuroinflammation that can be treated with the compounds of the present invention include: encephalitis, Guillain-Barre syndrome, meningitis, neuromyotonia, narcolepsy, multiple sclerosis, myelitis, and schizophrenia.

[0204] Examples of inflammation of the vascular or lymphatic system that can be treated with the compounds of the present invention include: arthralgia, 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 diseases (such as Crohn's disease and ulcerative colitis), ileitis, and proctitis.

[0205] Examples of inflammatory conditions of the reproductive system treatable with the compounds of the present invention include: cervicitis, chorioamnionitis, endometritis, epididymitis, omphalitis, oophoritis, orchitis, salpingitis, tubo-ovarian abscess, urethritis, vaginitis, vulvitis and vulvodynia.

[0206] The compounds may be used to treat autoimmune conditions having an inflammatory component. Such conditions include acute disseminated alopecia areata, Behcet's disease, Chagas' disease, chronic fatigue syndrome, autonomic dysfunction, 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 rheumatica, 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.

[0207] The compounds may be used to treat 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).

[0208] Other inflammatory conditions treatable with the available 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 heart 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 herpetiform dermatitis, 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.

[0209] Preferred treatments include the 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).

[0210] 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 a potentially beneficial anti-tumor effect 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.

[0211] 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.

[0212] 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 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.

[0213] The novel compound can also be used for prophylactic, palliative, curative or semi-curative, short-term or long-term treatment of the diseases mentioned above by the following means: combining different administration routes of the compound (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). Only as an example of surgery, partial or complete tumor resection can be combined with the compounds of the present invention. Only as an example of radiotherapy, external beam radiotherapy can be combined with the compounds of the present invention.

[0214] 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.

[0215] Such one or more vaccines can include inactivated or attenuated bacteria or viruses, which contain relevant antigens, purified antigens, live virus or bacterial delivery vectors that are recombinantly engineered for the expression and / or secretion of antigens, antigen-presenting cell (APC) vectors containing cells transfected with antigens or compositions containing nucleic acids encoding antigens, liposomal antigen delivery agents, or naked nucleic acid vectors encoding antigens. This list is not intended to be restrictive. By way of example, such one or more vaccines can also include inactivated tumor cells or oncolytic viruses that express and secrete one or more of GM-CSF, CCL20, CCL3, IL-12p70, FLT-3 ligand, cytokines.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 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 the treatment of feline cancer, wherein said feline cancer is selected from B-cell and / or T-cell lymphoma, squamous cell carcinoma, mammary adenocarcinoma, mast cell tumor, and injection site sarcoma.

[0220] 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).

[0221] 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).

[0222] 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 said 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.

[0223] 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 said feline cancer is selected from B-cell and / or T-cell lymphoma, squamous cell carcinoma, mammary adenocarcinoma, mast cell tumor, and injection site sarcoma.

[0224] On the other hand, the present invention relates to a compound of general formula (I) for treating and / or preventing the cancers mentioned above before and / or after tumor resection and / or radiotherapy.

[0225] 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.

[0226] 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.

[0227] Pharmaceutical composition

[0228] In another aspect of the present invention, there is provided a pharmaceutical composition comprising at least one of the compounds mentioned above.

[0229] 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 can 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.

[0230] Suitable tablets can 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.

[0231] For the purposes of the present invention, the pharmaceutical composition can 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 can be administered in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension.

[0232] Combination therapy

[0233] The compounds of the present invention can be used alone or in combination with one or more additional therapeutic agents.

[0234] 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.

[0235] 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.

[0236] 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 permits delivery of a pharmaceutically effective amount based on the patient's unique condition.

[0237] 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 a vaccine intended to stimulate an immune response against one or more predetermined antigens; an adjuvant; 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, as well as drugs that regulate and / or increase the frequency of a certain immune cell subtype, etc.

[0238] The compounds and compositions described herein can be administered before, after, and / or simultaneously with additional therapeutic or prophylactic compositions or treatment modalities.

[0239] The compounds, compositions of the present 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.

[0240] In addition, the compounds, compositions of the present 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.

[0241] Among the possible administration methods, intraperitoneal, intratumoral, peritumoral, subcutaneous, inhaled, or intravenous administration is preferred. The compounds, compositions of the present 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. Only as an example, 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.

[0242] 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.

[0243] Methods for co - administration with additional therapeutic agents are well known in the art.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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).

[0249] 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 skilled 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.

[0250] 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, treatments and / or prophylactic methods as disclosed herein (above and below) include (but are not limited to): hormones, hormone analogs and antihormones (such as tamoxifen, toremifene, raloxifene, fulvestrant, medroxyprogesterone 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, Torin1, 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, inhibitors of CDK4 / 6 (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, SU6656, 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 WO2013 / 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 antibodies, anti-CD37 antibodies, anti-CD20 antibodies); T cell engagers, such as PSMA×CD3, B7H6 / CD3 (as disclosed, for example, in WO2021 / 064137), DLL3 / CD3 (as disclosed, for example, in 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: OX40 agonists, ICOS ligands, CD27 agonists, GITR agonists, Toll-like receptor agonists.

[0251] In a preferred embodiment, one or more additional pharmacologically active substances that can also be used in combination with a 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 (e.g., PSMA×CD3, B7H6 / CD3 (as disclosed, for example, in WO2021 / 604137), DLL3 / CD3 (as disclosed, for example, in WO2019 / 234220), e.g., bispecific T cell engagers (such as, for example, CD3×BCMA, CD3×CD33, CD3×CD19), cancer vaccines, MDM2 inhibitors, and oncolytic viruses.

[0252] 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 targeting 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.

[0253] 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.

[0254] On the other hand, the present invention relates to a method for treating a STING-related or STING-modulated 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 present invention and a therapeutically effective amount of one or more of the additional therapeutic agents described above.

[0255] The combined use of the compounds of the invention with additional therapeutic agents can be carried out simultaneously or at staggered times.

[0256] 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.

[0257] 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.

[0258] Another aspect of 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.

[0259] 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.

[0260] 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 modulation of STING is beneficial.

[0261] 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).

[0262] 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 as described hereinabove, optionally together with one or more inert carriers and / or diluents.

[0263] 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.

[0264] Chemical synthesis

[0265] List of abbreviations

[0266]

[0267]

[0268] 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 and not limitation.

[0269] Review

[0270] Unless otherwise stated, all reactions are carried out using conventional methods in a chemical laboratory in commercially available equipment. Starting materials that are 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.

[0271] Chromatography

[0272] Thin layer chromatography was carried out on ready-to-use TLC plates of silica gel 60 on glass (with fluorescent indicator F-254) manufactured by Merck.

[0273] The Biotage Isolera Four device was used together with an Interchim Puri Flash column (50 μm, 12 - 300 g) or a glass column filled with silica gel (Granula Silica Si-60A 35 - 70 μm) manufactured by Millipore for automated preparative NP chromatography.

[0274] 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 / 1 L 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.

[0275] Analytical HPLC (reaction monitoring) of intermediate compounds was carried out using columns manufactured by Waters and Phenomenex. In each case, the analytical equipment also had a mass detector.

[0276] HPLC mass spectrometry / UV spectrometry

[0277] The retention time / MS-ESI+ of the example compounds used to characterize 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.

[0278] Analytical HPLC method:

[0279] Acidic method

[0280] HPLC: Agilent 1260 Infinity II

[0281] MS: Agilent LC / MS (G6125B)

[0282] Column: Sunfire C18 2.5 μm, 3.0×30 mm

[0283] Eluent: A: H₂O containing 0.1% TFA (v / v); B: MeCN (HPLC grade) 2 O; B: MeCN (HPLC grade)

[0284] Detection: MS: positive mode and negative mode

[0285] Column temperature: 60 °C

[0286] Gradient: 0.00 - 0.20 min: 3% B (flow rate 2.2 mL / min)

[0287] 0.20 - 1.20 min: 3% to 100% B (flow rate 2.2 mL / min)

[0288] 1.20 - 1.25 min: 100% B (flow rate 3.0 mL / min)

[0289] 1.25 - 1.40 min: 100% B (flow rate 3.0 mL / min)

[0290] Alkaline method

[0291] HPLC: Agilent 1260 Infinity II

[0292] MS: Agilent LC / MS (G6125B)

[0293] Column: X-Bridge C18, 2.5 μm, 3.0 × 30 mm

[0294] Eluent: A: H₂O containing 0.1% NH₄OH (v / v); B: MeCN (HPLC grade) 2 O; B: MeCN (HPLC grade)

[0295] Detection: MS: positive mode and negative mode

[0296] Column temperature: 60 °C

[0297] Gradient: 0.00 - 0.20 min: 3% B (flow rate 2.2 mL / min)

[0298] 0.20 - 1.20 min: 3% to 100% B (flow rate 2.2 mL / min)

[0299] 1.20 - 1.25 min: 100% B (flow rate 3.0 mL / min)

[0300] 1.25 - 1.40 min: 100% B (flow rate 3.0 mL / min)

[0301] Preparative HPLC method:

[0302] Acidic method

[0303] HPLC: Agilent 1260 Infinity II

[0304] MS: Agilent LC / MS (G6125B)

[0305] Column: Sunfire C18 10μm, 30×300 mm

[0306] Eluent: A: H2O containing 0.1% TFA (v / v); B: MeCN (HPLC grade)

[0307] Detection: MS: positive mode and negative mode

[0308] Flow rate: 50 mL / min

[0309] Column temperature: 40 °C

[0310] Alkaline method

[0311] HPLC: Agilent 1260 Infinity II

[0312] MS: Agilent LC / MS (G6125B)

[0313] Column: X-Bridge C18, 10μm, 30×300 mm

[0314] Eluent: A: H 2 O containing 0.1% NH4OH (v / v); B: MeCN (HPLC grade)

[0315] Detection: MS: positive mode and negative mode

[0316] Flow rate: 50 mL / min

[0317] Column temperature: 40 °C

[0318] Preparation of the compounds of the present invention

[0319] 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 the 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 be varied. Variations of the reaction methods known to those skilled in the art but not described in detail herein may also be used.

[0320] The general methods 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.

[0321] 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 isolated by conventional means and preferably purified by chromatography.

[0322] Route I:

[0323]

[0324] Preparation of intermediates

[0325] Intermediate 1

[0326] 1-(6-Fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole

[0327]

[0328] 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 (16.889 g; 93 mmol), pyridine (9.796 g; 124 mmol) and 6-fluoropyridine-3-boronic acid (14.851 g; 105 mmol). The reaction mixture was then stirred at room temperature for 72 h. The reaction was filtered through Celite, the filtrate was concentrated, and the crude product was purified by silica chromatography (EtOAc:hexane) to give the title compound (13 g).

[0329] 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).

[0330] Intermediate 2

[0331] 1-(6-Fluoropyridin-3-yl)-2',7-dimethyl-1H,2'H-3,4'-biindazole

[0332] Stir a mixture of 1-(6-fluoropyridin-3-yl)-3-iodo-7-methyl-1H-indazole (Intermediate 1, 1.0 g, 2.8 mmol), 2-methyl-4-(tetramethyl-1,3,2-dioxaborolan-2-yl)-2H-indazole (1.1 g, 4.2 mmol), [1,1'-bis-(diphenylphosphino)ferrocene]dichloropalladium(II) (0.21 g, 0.28 mmol), and Na 2 CO 3 (0.9 g, 8.5 mmol) in dioxane (20 ml) and water (5 ml) under an argon atmosphere at 110 °C for 2 h. At room temperature, add water (100 ml) and extract the mixture with EtOAc. Dry the combined organic layers over MgSO4, concentrate in vacuo, and purify the product by RP HPLC to give the title compound (0.8 g). Example

[0333] Example 1

[0334] 2-[(3R)-1-(5-{2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)pyrrolidin-3-yl]acetic acid

[0335]

[0336] Add (R)-pyrrolidine-3-acetic acid methyl ester hydrochloride (106 mg, 0.56 mmol) to a solution of Intermediate 2 (80 mg, 0.22 mmol) in anhydrous NMP (2 ml), then add DIPEA (145 mg, 1.12 mmol) and heat the mixture at 140 °C for 7 h. Cool the reaction mixture, add NaOH (aqueous solution) (4 M, 300 μl, 1.2 mmol), and stir it at room temperature for 1 h. Acidify the reaction mixture with TFA and purify by preparative HPLC (acidic method) to give the title compound (87 mg).

[0337] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.76 (dq, J = 12.29, 8.45 Hz, 1H), 2.21 (s, 3H), 2.34 (m, 1H), 2.48 (m, 2H), 2.67 (m, 1H), 3.20 (dd, J = 10.65, 7.73 Hz, 1H), 3.51 (m, 1H), 3.66 (m, 1H), 3.81 (dd, J = 10.58, 7.29 Hz, 1H), 4.06 (br d, J = 31.05 Hz, 1H), 4.20 (s, 3H), 6.78 (br d, J = 9.12 Hz, 1H), 7.25 (m, 2H), 7.42 (dd, J = 8.62, 6.97 Hz, 1H), 7.67 (d, J = 8.62 Hz, 1H), 7.77 (d, J = 6.72 Hz, 1H), 7.94 (dd, J = 9.13, 2.28 Hz, 1H), 8.09 (d, J = 7.10 Hz, 1H), 8.41 (d, J = 2.53 Hz, 1H), 8.61 (s, 1H)

[0338] HPLC (acidic): Rt = 0.802 min ((M + H)+481.2)

[0339] Example 2

[0340] 2-[(3S)-1-(5-{2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)pyrrolidin-3-yl]acetic acid

[0341]

[0342] Using the method described for Example 1: The title compound (19.6 mg) was obtained from intermediate 2 (50 mg) and (S)-methyl pyrrolidine-3-acetate hydrochloride (65 mg).

[0343] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.75 (dq, J = 12.32, 8.40 Hz, 1H), 2.20 (s, 4H), 2.47 (d, J = 7.35 Hz, 2H), 2.65 (dquin, J = 14.93, 7.36, 7.36, 7.36, 7.36 Hz, 1H), 3.17 (dd, J = 10.58, 7.67 Hz, 1H), 3.48 (m, 1H), 3.63 (m, 1H), 3.79 (dd, J = 10.52, 7.35 Hz, 1H), 4.20 (s, 3H), 6.64 (d, J = 9.00 Hz, 1H), 7.24 (m, 1H), 7.42 (dd, J = 8.62, 6.97 Hz, 1H), 7.66 (d, J = 8.62 Hz, 1H), 7.77 (d, J = 6.59 Hz, 1H), 7.82 (dd, J = 8.93, 2.60 Hz, 1H), 8.09 (dd, J = 7.60, 1.01 Hz, 1H), 8.37 (d, J = 2.53 Hz, 1H), 8.61 (s, 1H)

[0344] HPLC (acidic): Rt = 0.865 min ((M + H)+467)

[0345] Example 3

[0346] 2-[4-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)morpholin-2-yl]acetic acid

[0347]

[0348] Using the method described in Example 1: The title compound (57.6 mg) was obtained from intermediate 2 (50 mg) and methyl 2-(morpholin-2-yl)acetate hydrochloride (72 mg) after preparative HPLC.

[0349] HPLC (acidic): Rt = 0.947 min ((M + H)+483.2)

[0350] Example 4

[0351] 2-(5-{2”,7-dimethyl-1H,2”H-[3,4”-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid

[0352]

[0353] Using the method described in Example 1: The title compound (69 mg) was obtained from Intermediate 2 (50 mg) and methyl 2-azabicyclo[2.2.1]heptane-5-carboxylate (57 mg) after preparative HPLC.

[0354] HPLC (acidic): Rt = 0.811 min ((M+H)+ 479.2)

[0355] Example 5

[0356] (1S,4R,5R)-2-(5-{2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid and (1R,4S,5S)-2-(5-{2′,7-dimethyl-1H,2′H-[3,4′-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid

[0357] Using the method described in Example 1: The title compound (56 mg) was obtained from Intermediate 2 (50 mg) and (1S,4R,5R)-methyl 2-azabicyclo[2.2.1]heptane-5-carboxylate (57 mg).

[0358] 1H NMR (400 MHz, DMSO-d6) δ ppm 1.68 (m, 1H), 1.76 (m, 1H), 1.99 (m, 3H), 2.22 (s, 5H), 2.66 (m, 1H), 2.93 (br s, 2H), 3.25 (br d, J = 9.63 Hz, 2H), 3.52 (dd, J = 9.82, 3.49 Hz, 1H), 4.20 (s, 3H), 4.70 (br s, 1H), 6.79 (br d, J = 7.10 Hz, 2H), 7.25 (m, 3H), 7.42 (dd, J = 8.62, 6.97 Hz, 2H), 7.66 (d, J = 8.74 Hz, 2H), 7.76 (d, J = 6.59 Hz, 2H), 7.91 (dd, J = 8.93, 2.09 Hz, 2H), 8.08 (d, J = 7.10 Hz, 2H), 8.39 (d, J = 2.53 Hz, 2H), 8.61 (s, 2H)

[0359] HPLC (acidic): Rt = 0.811 min ((M+H)+ 479.2)

[0360] Example 6

[0361] 2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.2]octane-5-carboxylic acid

[0362] (Mixture of stereoisomers)

[0363]

[0364] Using the method described in Example 1: The title compound (42 mg) was obtained from Intermediate 2 (50 mg) and 2-azabicyclo[2.2.2]octane-5-carboxylic acid (0.35 mmol) after preparative HPLC.

[0365] HPLC (acidic): Rt = 0.839 min ((M+H)+493.2)

[0366] Examples 7 to 10

[0367] Chiral preparative HPLC separation of Example 6 was carried out using the conditions described below to give the following stereoisomers:

[0368] (1S,4R,5R)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.2]octane-5-carboxylic acid (single stereoisomer)

[0369] (1R,4S,5S)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.2]octane-5-carboxylic acid (single stereoisomer)

[0370] (1S,4R,5S)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.2]octane-5-carboxylic acid (single stereoisomer)

[0371] (1R,4S,5R)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.2]octane-5-carboxylic acid (single stereoisomer)

[0372] Chiral preparative HPLC

[0373] HPLC: Sepiatec PrepSFC50

[0374] Column: CHIRAL Cellulose-SJ_10x 250mm_5μm

[0375] Eluent: A: 80% scCO 2 ; B: 20% MeOH + 20 mM NH 3

[0376] Detection: UV: 220 nm

[0377] Column temperature: 40 °C

[0378] Flow rate: 15 mL / min

[0379] Gradient: Isocratic

[0380] Example 11

[0381] 1-[(1R,4R)-5-(5-{2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl]ethan-1-one (single stereoisomer)

[0382]

[0383] Heat a mixture of Intermediate 2 (50 mg, 0.14 mmol), (1R,4R)-2,5-diazabicyclo[2.2.1]heptane (30 mg, 0.31 mmol) and DIPEA (90.4 mg, 0.7 mmol) in anhydrous NMP (2 ml) at 140 °C for 15 h. Cool the mixture to room temperature and add acetic anhydride (57 mg, 0.56 mmol) and DIPEA (90 mg, 0.7 mmol), and stir it at room temperature for 1 h. Purify the product by preparative HPLC (acidic) to give the title compound (20.5 mg).

[0384] HPLC (acidic): Rt = 0.859 min ((M + H)+478.2)

[0385] Example 12

[0386] 1-[(1S,4S)-5-(5-{2″,7-dimethyl-1H,2″H-[3,4″-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.1]hept-2-yl]ethan-1-one (single stereoisomer)

[0387]

[0388] Using the method described in Example 11: The title compound was obtained from Intermediate 2 and (1S,4S)-2,5-diazabicyclo[2.2.1]heptane after preparative HPLC.

[0389] Example 13

[0390] 1-[5-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.2]oct-2-yl]ethan-1-one (mixture of stereoisomers)

[0391]

[0392] Using the method described in Example 11: The title compound (23 mg) was obtained from Intermediate 2 (50 mg) and 2,5-diazabicyclo[2.2.2]octane dihydrochloride (68 mg) after preparative HPLC.

[0393] HPLC (acidic): Rt = 0.896 min ((M+H)+492.2)

[0394] Examples 14 to 15

[0395] Using the conditions described below for chiral preparative HPLC separation of Example 13, the following stereoisomers were obtained:

[0396] 1-[(1R,4R)-5-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.2]oct-2-yl]ethan-1-one (single stereoisomer)

[0397] 1-[(1S,4S)-5-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.2]oct-2-yl]ethan-1-one (single stereoisomer)

[0398] Chiral preparative HPLC

[0399] HPLC: Sepiatec PrepSFC50

[0400] Column: CHIRAL Amylose_10x 250mm_5μm

[0401] Eluent: A: 60% scCO 2 ; B: 40% MeOH + 20 mM NH 3

[0402] Detection: UV: 220 nm

[0403] Column temperature: 40 °C

[0404] Flow rate: 15 mL / min

[0405] Gradient: Isocratic

[0406] Example 14

[0407] 1-[(1R,4R)-5-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.2]oct-2-yl]ethan-1-one (single stereoisomer)

[0408] Example 15

[0409] 1-[(1S,4S)-5-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2,5-diazabicyclo[2.2.2]oct-2-yl]ethan-1-one (single stereoisomer)

[0410] Example 16

[0411] 2-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (mixture of stereoisomers)

[0412] Using the method described in Example 1: The title compound (55 mg) was obtained from Intermediate 2 (50 mg) and methyl 2-azabicyclo[2.2.1]heptane-5-carboxylate (57.1 mg, 0.35 mmol) after preparative HPLC (acidic).

[0413] HPLC (acidic): Rt = 0.846 min ((M+H)+ 479.0)

[0414] Example 17

[0415] (1S,4R,5R)-2-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid and (1R,4S,5S)-2-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (mixture of stereoisomers)

[0416]

[0417] Using the method described in Example 1: Intermediate 2 (200 mg) and methyl cis-2-azabicyclo[2.2.1]heptane-5-carboxylate (215 mg, 1.12 mmol) gave the title compound (55 mg) after preparative HPLC (acidic).

[0418] HPLC (acidic): Rt = 0.859 min ((M+H)+ 479.2)

[0419] Examples 18 to 19

[0420] Chiral preparative HPLC separation of Example 16 was carried out using the conditions described below to give the following stereoisomers:

[0421] (1R,4S,5S)-2-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (single stereoisomer)

[0422] (1S,4R,5R)-2-(5-{2',7-dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (single stereoisomer)

[0423] Chiral preparative HPLC

[0424] HPLC: Sepiatec PrepSFC100

[0425] Column: CHIRAL Amylose-C_neo_20x 250nm_5μm

[0426] Eluent: A: 60% scCO 2 ; B: 40% MeOH + 20 mM NH 3

[0427] Detection: UV: 220 nm

[0428] Column temperature: 40 °C

[0429] Flow rate: 60 mL / min

[0430] Gradient: Isocratic

[0431] Example 18

[0432] (1R,4S,5S)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (single stereoisomer)

[0433]

[0434] 1 H NMR(400MHz,DMSO-d 6 )δ ppm 1.72(m,2H),1.99(m,2H),2.22(s,3H),2.66(dd,J=8.49,5.45Hz,1H),2.94(br s,1H),3.26(br d,J=9.76Hz,1H),3.53(dd,J=9.76,3.42Hz,1H),4.20(s,3H),4.71(br s,1H),6.82(br d,J=8.24Hz,1H),7.25(m,2H),7.42(dd,J=8.62,6.97Hz,1H),7.67(d,J=8.62Hz,1H),7.76(d,J=6.84Hz,1H),7.93(dd,J=9.12,2.15Hz,1H),8.09(d,J=7.35Hz,1H),8.40(d,J=2.53Hz,1H),8.61(s,1H).

[0435] Example 19

[0436] (1S,4R,5R)-2-(5-{2',7-Dimethyl-1H,2'H-[3,4'-biindazol]-1-yl}pyridin-2-yl)-2-azabicyclo[2.2.1]heptane-5-carboxylic acid (single stereoisomer)

[0437] 1 H NMR(400MHz,DMSO-d 6)δ ppm 1.72 (m, 2H), 1.99 (m, 2H), 2.23 (s, 3H), 2.66 (dd, J = 8.43, 5.64 Hz, 1H), 2.94 (br s, 1H), 3.26 (br d, J = 9.63 Hz, 1H), 3.53 (dd, J = 9.76, 3.42 Hz, 1H), 4.20 (s, 3H), 4.71 (br s, 1H), 6.82 (br d, J = 7.10 Hz, 1H), 7.25 (m, 2H), 7.42 (dd, J = 8.62, 6.97 Hz, 1H), 7.67 (d, J = 8.62 Hz, 1H), 7.76 (d, J = 6.72 Hz, 1H), 7.93 (dd, J = 9.06, 2.22 Hz, 1H), 8.09 (d, J = 7.35 Hz, 1H), 8.40 (d, J = 2.53 Hz, 1H), 8.61 (s, 1H).

[0438] Pharmacological activity

[0439] Biological examples

[0440] 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 assay and a canine whole blood assay. Representative results of the compounds of the present invention are compiled in Tables 1 and 2 below. Other assays used to test the compounds are also described below.

[0441] Canine differential scanning fluorimetry (DSF) assay

[0442] Canine STING (dSTING) protein production and purification

[0443] The protein used for biophysical experiments was recombinant canine STING protein, which contains its cytoplasmic cGAMP-binding extracellular domain. 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:

[0444] His-TEV—dSTING

[0445]

[0446] To express the recombinant canine STING described above, the construct was transformed into Escherichia coli strain BL21DE3 and grown in a shake flask 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 culture was 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 effluent was applied to size exclusion chromatography. The peak fractions were collected and concentrated to 5 mg / mL.

[0447] Biophysical analysis - Differential scanning fluorimetry (DSF) was used to determine an increased stability of the canine STING protein against thermal denaturation.

[0448] The binding affinity of the compounds of the invention was confirmed using a thermal shift assay that measures the stability of the appropriate protein species of canine STING against thermal denaturation in the presence of the compound. In this assay, the melting temperature of the protein is 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 are gradually exposed during unfolding. The dye fluorescence 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.

[0449] The thermal stability of the 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), 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 (SIGMAS5692-500UL). 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 repeated measurements and centrifuge at 1000 g for 2 minutes. In the measurement, use 160 cycles of 0.5 °C (temperature slope 15 s / cycle, 15 °C to 95 °C).

[0450] 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).

[0451] Process the dissociation curve in Bio-Rad CFX Manager. The peak type was set to "negative". Assign the compound codes for screening in the plate layout.

[0452] 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.

[0453] Subtract the melting temperature (Tm) obtained from the STING protein alone from the T obtained from the protein incubated with the ligand to generate a ΔTm value.

[0454] Canine whole blood analysis

[0455] To detect STING activation in the physiological environment, canine whole blood (dWB) was stimulated with the cyclic dinucleotide cGAMP or a test compound. Pathway activity was monitored by measuring IFNβ production.

[0456] The compound was delivered as a 10 mM DMSO solution, diluted and transferred using an Echo acoustic dispenser to a 384-well assay plate (Greiner #781182), with 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 plate 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., at a concentration of 3.8% 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 plate was 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 plate with a lid, the blood plate was incubated in an incubator at 37 °C for 4 hours without shaking. To detect IFNβ in canine plasma, an ELISA kit for canine interferon β (Biotrend #SEA222Ca) was used. The whole blood assay plate was 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 using a 96-well pipetting robot, with each well of the 96-well ELISA plate pre-filled with 60 μl of assay diluent. The plate was sealed with a microplate seal and kept at 4 °C overnight again. The ELISA plate was brought to room temperature and then incubated in an incubator at 37 °C for 1 hour. 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 plate to add 100 μL of the detection reagent A working solution to each well. The ELISA plate was covered with a plate sealer and incubated in an incubator at 37 °C for 1 hour. 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.

[0457] Data evaluation and calculation:

[0458] For data evaluation and calculation, the % control for each well is calculated based on the mean of the high control (cGAMP-stimulated control) and the mean of the low control (unstimulated control), using the following standard 4-parameter logistic regression formula:

[0459] [y = (a - d) / (1 + (x / c)^b) + d]

[0460] a = low value,

[0461] d = high value,

[0462] x = concentration M,

[0463] c = EC 50 M,

[0464] b = slope.

[0465] Canine liver microsome (dLM) assay

[0466] 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 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).

[0467] The amount of the parent compound in 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 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.

[0468] Mouse liver microsome (mLM) analysis

[0469] 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 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).

[0470] 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 from 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. In general, high stability of the compound across species (corresponding to low QH%) is desired.

[0471] Canine hepatocyte (dHep) assay

[0472] The metabolic degradation of the test compound was analyzed in a suspension of canine hepatocytes.

[0473] Incubation: Cryopreserved canine hepatocytes were incubated in an appropriate buffer system (KHB buffer or a similar buffer or standard cell culture medium) containing 50% homologous serum. After an equilibration 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.

[0474] 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].

[0475] ke = (-1) * slope

[0476] ke = elimination rate constant [1 / hour]

[0477] The half-life was calculated from the elimination rate constant

[0478] t1 / 2 = ln(2) / ke

[0479] t1 / 2 = half-life [hours]

[0480] Calculation of intrinsic in vitro hepatic clearance:

[0481] CL_int_in vitro = ke * 1000 / (CD * 60)

[0482] CL_int_in vitro = In vitro intrinsic hepatic clearance [µL / min / 10^6 cells (Mio cells)]

[0483] CD = Cell density [10^6 cells / mL]

[0484] Note: The above equation is only valid when [substrate] << Km.

[0485] 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).

[0486] CL_int_in vivo = (CL_int_in vitro * H * L) / 1000

[0487] CL_int_in vivo = In vivo intrinsic hepatic clearance [mL / min / kg]

[0488] H = Hepatocellularity [10^6 cells / g liver]

[0489] L = Liver factor [g / kg body weight]

[0490] CL_ws = CL_int_in vivo * Q / (CL_int_in vitro + Q)

[0491] CL = Estimated in vivo hepatic clearance [mL / min / kg]

[0492] Q = Hepatic blood flow rate [mL / min / kg]

[0493] ws = Well-stirred

[0494] QH% = CL_ws * 100 / Q

[0495] QH% = Clearance expressed as a percentage of hepatic blood flow rate

[0496] Concentration: Cell concentration at incubation time (10 6 / mL)

[0497] T_LAST: Terminal time point used (hours)

[0498] Mouse hepatocyte (mHep) analysis

[0499] The metabolic degradation of the test compound is analyzed in a suspension of mouse hepatocytes.

[0500] Incubation: Incubate cryopreserved mouse hepatocytes 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), add the test compound 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). Incubate the cells for up to 6 hours and take samples at 6 different time points. Subsequently, quench the samples with acetonitrile and precipitate by centrifugation. Then analyze the remaining amount of the parent compound in the supernatant by HPLC - MS / MS. Calculation: Calculate the elimination rate constant (ke) using the slope of the linear regression of the natural logarithm [substrate remaining % or substrate concentration] versus time [hours].

[0501] ke = (-1) * slope

[0502] ke = elimination rate constant [1 / hour]

[0503] The half - life is calculated from the elimination rate constant

[0504] t1 / 2 = ln(2) / ke

[0505] t1 / 2 = half - life [hours]

[0506] Calculation of intrinsic in vitro hepatic clearance:

[0507] CL_int_in vitro = ke * 1000 / (CD * 60)

[0508] CL_int_in vitro = intrinsic in vitro hepatic clearance [μl / minute / million cells]

[0509] CD = cell density [million cells / ml]

[0510] Note: The above equations are only valid when [substrate] << Km.

[0511] The calculated intrinsic in vitro 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).

[0512] CL_int_in vivo = (CL_int_in vitro * H * L) / 1000

[0513] CL_int_in vivo = intrinsic in vivo hepatic clearance [ml / minute / kg]

[0514] H = hepatocellularity [million cells / g liver]

[0515] L = Liver factor [g / kg body weight]

[0516] CL_ws = CL_int_in vivo * Q / (CL_int_in vitro + Q)

[0517] CL = Estimation of in vivo hepatic clearance [ml / min / kg]

[0518] Q = Hepatic blood flow rate [ml / min / kg]

[0519] ws = Well-stirred

[0520] QH% = CL_ws * 100 / Q

[0521] QH% = Clearance expressed as a percentage of hepatic blood flow rate

[0522] Concentration: Cell concentration at incubation time (10 6 / ml)

[0523] T_LAST: Terminal time point used (hours)

[0524] Mouse hepatocytes: 120 × 10e6 cells / g liver

[0525] Mouse liver factor: 55 g / kg body weight

[0526] Mouse blood flow rate: 90 ml / (min × kg).

[0527] Permeability analysis (MDCK-PGP)

[0528] 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.

[0529] The apparent permeability coefficient (PE) of a compound across a monolayer of MDCK-MDR1 cells (pH 7.4, 37 °C) was measured in the top-to-bottom (AB) and bottom-to-top (BA) transport directions. 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 diffusion and active transport mechanisms, the active transport mechanisms being mediated by efflux and uptake transporters expressed on MDCK-MDR1 cells, predominantly 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 with known in vitro permeability and oral absorption in humans. The same or similar permeability in both transport directions indicates passive diffusion, and a vectorial permeability indicates other active transport mechanisms. A PEBA higher than PEAB indicates active efflux mediated by MDR1 P-gp. Active transport is concentration-dependent and saturable.

[0530] 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). Stock solutions were 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 transport solutions (0.1 - 300 μM compound, final DMSO ≤ 0.5%). The transport solutions (TL) were 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.

[0531] Result

[0532] Table 1: Interaction with canine STING measured by DSF (dDSF)

[0533]

[0534]

[0535] Table 2: Cytokine secretion in canine whole blood (dWB) culture system measured by canine interferon-β (IFNb) ELISA

[0536] Example Number <![CDATA[EC 50 (nM)]]> Example 1 307.9 Example 2 212.0 Example 3 3063.5 Example 4 703.9 Example 5 529.0 Example 6 416.8 Example 9 651.6 Example 10 184.9 Example 11 619.7 Example 13 281.6 Example 14 2918.4 Example 15 95.1 Example 18 519.2 Example 19 278.3

[0537] As demonstrated by the examples of the present invention, when measured by a binding assay, the compounds of the present invention exhibit an interaction with canine STING (dSTING), which is reflected by a T shift measured by DSF of >15°C, more preferably >20°C, and even more preferably >25°C. m 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.

Claims

1. A compound of formula (I) wherein R 1 is -C 1-6 alkyl or -C 3-6 cycloalkyl; R 2a selected from -H or -C 1-6 alkyl; R 2b selected from -H, -C 1-6 alkyl, -C 1-6 alkylene-OH, -C(O)OH, -C(O)O-C 1-6 alkyl and -pyrazolyl-C 1-6 alkyl; R 2c is -H or -C 1-6 alkyl; R 3 Selected from (* indicates connection point): R 4 selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl; R 5 selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl; R 6 selected from H, -C 1-6 alkyl and C 3-6 cycloalkyl; R 7 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; X is CH or N; and Y is -O-, -S-, -S(O)-, -S(O) 2 -; or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein R 1 is -C 1-6 alkyl, preferably methyl; or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1 or 2, wherein R 2a is -C 1-6 alkyl, preferably methyl; R 2b is -H or -C 1-6 alkyl; and R 2c is -H or -C 1-6 alkyl; or a pharmaceutically acceptable salt thereof.

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

5. The compound according to any one of claims 1 to 4, wherein R 3 is wherein R 7 is -C 1-6 alkyl, preferably methyl; or a pharmaceutically acceptable salt thereof.

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

7. The compound according to any one of claims 1 to 4 or 6, wherein R 3 For R 4 is -H or methyl, preferably -H; R 5 and R 6 are independently -H or methyl, preferably R 5 and R 6 are both -H; and Y is O; or a pharmaceutically acceptable salt thereof.

8. The compound according to any one of claims 1 to 4 or 6, wherein R 1 is methyl; R 2a is methyl; R 2b and R 2c is -H; R 3 For R 4 、R 5 and R 6 are -H; and Y is O; or a pharmaceutically acceptable salt thereof.

9. A compound selected from the following structures: 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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