[1,3]Thiazolo[4,5-d]-pyrimidin-7-ones as NOX4 inhibitors

Abstract

The present disclosure provides [1,3]thiazolo[4,5-d]pyrimidon-7-ones that are inhibitors of NOX4 and are therefore useful in treating diseases treatable by inhibition of NOX4. Also provided are pharmaceutical compositions containing the same and methods for preparing the same. JPEG2026508289000148.jpg6571

Description

[Technical Field]

[0001] The present disclosure provides certain thiazolo-pyrimidinone derivatives that are inhibitors of NOX4 and are therefore useful for treating diseases treatable by inhibition of NOX4. The present invention further relates to pharmaceutical compositions and combinations comprising these compounds, as well as the use of the compounds in the treatment of diseases associated with or modulated by NOX4. In particular, the pharmaceutical compositions of the present invention are suitable for treating interstitial lung diseases, such as idiopathic lung diseases, and may also be suitable for treating fibrosis, allergic diseases, and inflammatory diseases. [Background technology]

[0002] Redox signaling is a crucial part of various cell signaling pathways involved in the control of cell growth, differentiation, metabolism, immune regulation, and other physiological functions. Redox signaling is characterized by redox reactions or the formation of covalent adducts between sensors and second messengers. In this regard, oxidation of sensor proteins can modulate their activity, structure, or susceptibility to degradation. In the case of redox signaling, second messengers consist of reactive oxygen species and reactive nitrogen species (ROS and RNS). These are generated as by-products during metabolic reactions in mitochondria and also by specialized enzymes such as NADPH-oxidases (NOXes). At the same time, complex antioxidant systems have evolved to protect cells from damage caused by ROS and RNS. This ensures a tight balance between ROS / RNS formation and degradation. When this balance is disrupted, oxidative stress occurs, resulting in increased protein oxidation and aberrant redox signaling. Therefore, oxidative stress is associated with various pathophysiological conditions, such as interstitial lung disease, cancer, and inflammatory diseases.

[0003] Over the past decade, multiple studies have demonstrated that redox balance is disrupted in interstitial lung diseases such as idiopathic pulmonary fibrosis (IPF). 1Furthermore, NOX4 is specifically upregulated in the lungs of IPF patients. 1 It has been shown that patients with IPF experience a decline in lung function and blood oxygenation, which is caused by remodeling of the lung parenchyma in combination with stiffening of the lung tissue due to increased matrix deposition. When the epithelium is damaged, for example, by infection or air pollutants, fibroblasts are activated, allowing migration of immune cells and epithelial progenitor cells to the damaged site. Once the damage is resolved, fibroblasts and immune cells undergo apoptosis, allowing the tissue to fully recover. In IPF, abnormal repair occurs, resulting in progressive remodeling of the lung parenchyma. 2 Recent studies using single-cell sequencing have enabled the identification and comparison of different cell types in lung tissue from IPF patients or healthy controls. These studies showed an increased number of fibroblasts and a strong differentiation of epithelial cell populations.

[0004] NOX4 is part of the NADPH oxidase enzyme family. NOXes are membrane-bound, multisubunit protein complexes that transport electrons across the plasma membrane, generating ROS. While the activity of all other family members (NOX1, NOX2, NOX3, and NOX5) is tightly regulated and inducible, NOX4 is the only family member that is constitutively active. 1 NOX4 is localized to the plasma membrane, perinuclear vesicles, endoplasmic reticulum (ER), mitochondria, and nuclear membrane. NOX4-mediated generation of hydrogen peroxide (H2O2) leads to reversible oxidation and / or S-glutathionylation of cysteine ​​thiol groups. 1 Thereby, NOX4 regulates TGFβ signaling and other important pathways involved in disease progression. 1 .

[0005] In addition to hyperplastic alveolar cells, fibroblasts from IPF show higher NOX4 expression compared with fibroblasts isolated from healthy tissue. The role of NOX4 in fibroblast activation and proliferation was investigated in lung fibroblasts. 1Fibroblast activation, as well as fibroblast proliferation, are known processes that contribute to disease progression. Immunohistochemistry of control and IPF lung samples confirmed increased NOX4 staining in fibroblasts, with stronger staining in bronchial and alveolar epithelial cells. 1 .

[0006] Acute respiratory distress syndrome (ARDS) and acute lung injury (ALI) are characterized by pulmonary infiltration and edema. NOX4 is elevated in patients, and animal models have shown that NOX4 plays an important role in cell recruitment. 4 .

[0007] Nonalcoholic steatohepatitis (NASH) is characterized by increased hepatic fat mass and inflammation. A chronic feedback loop of lipotoxicity, increased inflammation, and cell death leads to liver fibrosis. Through this process, hepatocytes, i.e., stellate cells, dedifferentiate into an activated phenotype, one of the hallmarks of which is the induction of profibrotic and proinflammatory expression patterns. In a major redox-mediated signaling pathway, the NOX family, particularly NOX4, plays a key role in a feedback loop. NOX4-dependent signaling directly leads to the transcriptional induction of NFkB and MAPk, resulting in the induction of gene products such as smooth muscle actin (SMA), collagen, and tumor necrosis factor (TNF).

[0008] Published literature has shown that NOX4 is expressed in astrocytes and is expressed throughout the process of astrocyte activation. Conversely, inhibition of NOX4 attenuates the expression of astrocyte activation markers. In addition to in vitro analyses, studies of NOX4 knockout mice have shown reduced fibrosis and inflammation in NASH-related models.

[0009] Apart from preventing the onset and progression of fibrosis, NOX4 plays an important role in the integrity of the vascular endothelium. In this regard, NOX4 activity promotes the production of nitric oxide (NO), leading to relaxation of the vascular network and reduced inflammation in the vascular endothelium. Subsequently, inhibition of NOX4 has a beneficial effect on portal hypertension.

[0010] In cancer, deregulation of ROS contributes to tumor initiation, progression, and metastasis. In some instances, enhanced ROS generated by various oncogenic perturbations is required for tumorigenicity, leading to further DNA damage and genomic instability in cancer cells. 5 Increased ROS occurs through various mechanisms, such as increased expression of NOX proteins or NOX activators, or downregulation of ROS control systems. NOX4 itself is reported to be elevated in many types of tumors, leading to enhanced proliferation, migration, and apoptosis. 5 Tumor-associated macrophages are affected by ROS and can acquire an immunosuppressive phenotype within tumors in response to ROS. 5 Furthermore, NOX4 is upregulated in tumors when fibroblasts are educated to become cancer-associated fibroblasts (CAFs), leading to the elimination of CD8+ T cells and immunosuppression. In multiple mouse models of cancer, deletion or inhibition of NOX4 reversed this immunosuppression and restored immunotherapeutic responses to anti-PD-1 (programmed cell death protein 1, PD1) therapy. 5 Therefore, specific inhibition of NOX proteins in tumor cells themselves or in stromal cells represents an amenable target for antitumor therapy.

[0011] A list of the references cited above can be found here. 1. Veith, C., Boots, A. W., Idris, M., Schooten, F.-J. van & Vliet, A. van der. Redox Imbalance in Idiopathic Pulmonary Fibrosis: A Role for Oxidant Cross-Talk Between NADPH Oxidase Enzymes and Mitochondria. Antioxid Redox Sign 31, 1092-1115 (2019). 2. Hinz, B. & Lagares, D. Evasion of apoptosis by myofibroblasts: a hallmark of fibrotic diseases. Nat Rev Rheumatol 16, 11-31 (2020). 3. Habermann, A. C. et al. Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis. Sci Adv 6, eaba1972 (2020). 4. Lee, S. H. et al. NADPH oxidase 4 signaling in a ventilator-induced lung injury mouse model. Respir Res 23, 73 (2022). 5. Cheung, E. C. & Vousden, K. H. The role of ROS in tumour development and progression. Nat Rev Cancer 22, 280-297 (2022).

Summary of the Invention

[0012] Detailed Description of the Invention The present invention discloses novel thiazolo-pyrimidinone derivatives that are NOX4 inhibitors, which have suitable pharmacological and pharmacokinetic properties that allow them to be used as pharmaceuticals for the treatment of conditions and / or diseases treatable by inhibition of NOX4.

[0013] In the prior art, WO2016207785 discloses benzoxazole- and benzothiazole-based NOX4 inhibitors having the following general structural formula, and in Examples 1, 15, and 80 thereof, they show the following IC values ​​in an assay using modified human embryonic kidney (HEK) cells using Amplex Red as a detection reagent: 50 Values ​​are reported.

[0014] [ka]

[0015] Table A TIFF2026508289000003.tif95157

[0016] When tested in a metabolic stability assay as described herein below, Examples 1, 15 and 80 exhibit clearance in human hepatocytes of 52% QH, 29% QH, and 46% QH, respectively.

[0017] Another patent application, WO2005049613, discloses modulators of the vanilloid 1 receptor (VR1) based on bicyclic pyrimidin-4-(3H)-ones of the following general structure: [ka]

[0018] Examples 19, 20, 30, 59, and 63 of WO2005049613 were tested for their NOX4 inhibitory activity using the assays described herein below, and showed the following IC 50 The value is obtained.

[0019] Table B TIFF2026508289000005.tif217130 TIFF2026508289000006.tif60129

[0020] As is evident from the above data, the tested compounds of WO2005049613 show poor activity as NOX4 inhibitors. Surprisingly, replacing the para-chloro substituent in Example 63 of WO2005049613 with a para-hydroxy substituent resulted in an IC 2000 as tested in the NOX4 inhibition assay described herein below. 50 It was found that NOX4 inhibition was enhanced by increasing the concentration of HCl from 55 μM to 0.66 μM, as shown in the table below by Example 1 of the present invention.

[0021] Table C TIFF2026508289000007.tif59148

[0022] The effect of a hydroxy group at this position on the NOX4 inhibitory activity of other compounds of the invention is also shown in the table below.

[0023] Table D TIFF2026508289000008.tif223150 TIFF2026508289000009.tif54145

[0024] It is therefore an object of the present invention to provide thiazolo-pyrimidinones that are potent, metabolically stable and selective NOX4 inhibitors.

[0025] The compounds of the invention according to general formula (I) [ka] where X 1 , X 2 , X 4 are, independently of each other, N or CR 1 and; X 3 is N or CR 1.1 and; X 1 , X 2 , X 3 , X 4 provided that no more than two of are simultaneously N; R 1 is selected from the group consisting of —H and halogen; R 1.1 is selected from the group consisting of -H, halogen, and -CN; R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group; The compounds, or salts thereof, exhibit several advantageous properties, such as high potency, high metabolic stability, and high selectivity for NOX4, but not for NOX1, NOX2, NOX3, and NOX5.

[0026] Compounds of the present invention typically have an IC value of less than 600 nM, preferably less than 400 nM, more preferably less than 200 nM, and most preferably less than 100 nM. 50 values ​​indicate NOX4 inhibition (see assay descriptions below and Table 1). High potency allows for lower doses to be used to achieve a pharmacological effect. A lower dose has the advantages of lower "drug load" or "drug burden" (parent drug and its metabolites) for the patient, reducing the likelihood of side effects, and lowering the production costs of the drug product.

[0027] In a further aspect of the invention, the compounds according to the invention are selective NOX4 inhibitors, exhibiting selectivity over NOX1, NOX2, NOX3 and NOX5. Preferably, the IC 50 However, the IC 50More preferably, the IC50 for NOX4 is 10 times lower than that of the compound. 50 However, the IC 50 Most preferably, the compound has an IC value for NOX4 that is 30-fold lower than that of the compound. 50 However, the IC 50 This compound is 100-fold lower than the ATP-dependent agonist (see assay description and Table 2 herein below).

[0028] Furthermore, the compounds of the present invention are metabolically stable, as demonstrated in human hepatocytes. In this regard, metabolic stability in human hepatocytes is defined as 45% QH or less, preferably 30% QH or less, and more preferably 20% QH or less (see the assay descriptions and Table 3 hereinbelow, and the definition of the method for calculating % QH = hepatic blood flow hereinbelow). Accordingly, compounds of the present invention are expected to have favorable in vivo clearance in humans and, therefore, favorable long-term effects. In the context of selecting and / or designing drugs with favorable pharmacokinetic properties, stability in human hepatocytes indicates the compound's susceptibility to biotransformation, since the liver is the primary site of metabolism for many drugs. Because human hepatocytes contain cytochrome P450 (CYPs) and additional enzymes for phase II metabolism (e.g., phosphatases and sulfatases), they represent a model system for studying how drugs are metabolized in vitro. Hepatocyte stability is associated with several advantages, including improved bioavailability and half-life, which allows for lower and less frequent administration to patients, and is thus a desirable attribute for compounds used as drugs in the treatment of disease.

[0029] Terms and definitions used General definition Terms not specifically defined herein should be given the meaning that one of ordinary skill in the art would give them in light of the disclosure and context, except that as used herein, unless otherwise specified, the following terms have the indicated meanings and are subject to the following conventions:

[0030] In the groups, radicals, or moieties defined below, the number of carbon atoms is often specified preceding the radical, e.g., C 1-6 -Alkyl means an alkyl group having from 1 to 6 carbon atoms. In general, for groups such as HO, H2N, (O)S, (O)2S, NC (cyano), HOOC, F3C, etc., the point of attachment of the group to the molecule will be known to one skilled in the art from the free valence of the group itself. For combined groups containing two or more subgroups, the last named subgroup is the point of attachment of the group, e.g., the substituent "aryl-C 1-3 -Alkylene" is C 1-3 - means an aryl group bonded to an alkyl group, where the alkyl group is bonded to the core or group to which the substituent is attached.

[0031] When compounds of the invention are depicted in the form of a chemical name and a chemical formula, the formula shall prevail in the event of any discrepancy. A wavy line may be used in a sub-formula to represent the bond connecting the defined core molecule.

[0032] For example, the term "3-carboxypropyl group" represents the following substituent: TIFF2026508289000011.tif2643 where the carboxy group is attached to the third carbon atom of the propyl group. The term "1-methylpropyl-", "2,2-dimethylpropyl-" or "cyclopropylmethyl-" represents the following group: TIFF2026508289000012.tif22150 Wavy lines may be used in subformulas to represent bonds that connect to the defined core molecule.

[0033] The term substituted As used herein, the term "substituted" means that one or more hydrogens on a designated atom have been replaced with a group selected from a defined group of substituents, provided that the replacement does not exceed the normal valence of the designated atom and that the substitution results in a stable compound. Similarly, the term "substituted" may be used in conjunction with chemical moieties in place of a single atom, such as, for example, "substituted alkyl," "substituted aryl," etc.

[0034] Unless otherwise specified, throughout this specification and the appended claims, a given chemical formula or name is intended to encompass tautomers and all stereoisomers, optical isomers, and geometric isomers (e.g., enantiomers, diastereomers, E / Z isomers, etc.) and their racemates, as well as mixtures of different proportions of separate enantiomers, mixtures of diastereomers, or mixtures of any of the aforementioned forms in which such isomers and enantiomers exist, as well as solvates thereof, e.g., hydrates, etc. Unless otherwise specified, "pharmaceutically acceptable salts" are also intended to include solvates thereof, such as, for example, hydrates, as defined in more detail below.

[0035] salt As used herein, the phrase "pharmaceutically acceptable" is used to refer to compounds, substances, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human tissue without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0036] As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by forming acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like.

[0037] 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. Further 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.

[0038] The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base form of these compounds with a sufficient amount of the appropriate base or acid in water or an organic diluent such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile, or in a mixture thereof. For example, salts of acids other than those mentioned above (eg, trifluoroacetates), which are useful for purifying or isolating the compounds of this invention, also form part of this invention.

[0039] halogen The term halogen refers to fluorine, chlorine, bromine and iodine.

[0040] Alkyl "C 1-n The term "alkyl" (where n is an integer selected from 2, 3, 4, 5 or 6, preferably 4, 5 or 6), either alone or in combination with other radicals, denotes an acyclic, saturated, branched or straight chain hydrocarbon radical having 1 to n carbon atoms. For example, C 1-5The term alkyl encompasses the radicals HC-, HC-CH-, HC-CH-CH-, HC-CH(CH)-, HC-CH-CH-CH-, HC-CH(CH)-, HC-CH(CH)-CH-, HC-C(CH)-, HC-CH-CH-CH-, HC-CH-CH-CH-, HC-CH-CH-CH(CH)-, HC-CH-CH(CH)-, HC-CH-CH(CH)-CH-, HC-CH(CH)-CH-, HC-CH-C(CH)-, HC-C(CH)-CH-, HC-CH(CH)-CH(CH)-, and HC-CH-CH(CHCH)-.

[0041] Alkylene "C 1-n The term "alkylene" (where n is an integer selected from 2, 3, 4, 5 or 6, preferably 4, 5 or 6), either alone or in combination with other radicals, denotes an acyclic, saturated, branched or straight chain divalent alkyl radical containing 1 to n carbon atoms. For example, C 1-4 The term alkylene includes -CH-, -CH-CH-, -CH(CH)-, -CH-CH-CH-, -C(CH)-, -CH(CHCH)-, -CH(CH)-CH-, -CH-CH(CH)-, -CH-CH-CH-CH-, -CH-CH-CH(CH)-, -CH(CH)-CH-CH-, -CH-CH(CH)-CH-, -CH-C(CH)-, -C(CH)-CH-, -CH(CH)-CH(CH)-, -CH-CH(CHCH)-, -CH(CHCH)-CH-, -CH(CHCH)-CH-, -CH(CHCH)-CH-, -CH(CHCHCH)-, -CH(CHCHCH)-, -CH(CHCHCH)-, and -C(CH)(CHCH)-.

[0042] Alkenyl "C 2-m The term alkenyl refers to the 2-m "m" is used for "alkyl" groups, where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6, if at least two carbon atoms of said group are joined to each other by a double bond.

[0043] Alkenylene "C 2-m The term alkenylene refers to "C 2-m alkylene groups, where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6, if at least two carbon atoms of said group are joined to each other by a double bond.

[0044] Alkynyl "C 2-m The term alkynyl refers to the 2-m "m" is used for "alkyl" groups, where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6, if at least two carbon atoms of said group are joined to each other by a triple bond.

[0045] Alinylene "C 2-m The term alkynylene refers to "C 2-m alkylene groups, where m is an integer selected from 3, 4, 5 or 6, preferably 4, 5 or 6, if at least two of the carbon atoms of said group are joined to each other by a triple bond.

[0046] cycloalkyl "C 3-k The term "cycloalkyl" (where k is an integer selected from 3, 4, 5, 7 or 8, preferably 4, 5 or 6), either alone or in combination with other radicals, denotes a cyclic, saturated, unbranched hydrocarbon radical having from 3 to k carbon atoms. For example, C 3-7 The term cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0047] cycloalkenyl "C 3-kThe term "cycloalkenyl" (where k is an integer selected from 3, 4, 5, 7 or 8, preferably 4, 5 or 6), either alone or in combination with other radicals, denotes a cyclic, unsaturated but non-aromatic, unbranched hydrocarbon radical having from 3 to k carbon atoms, at least two of which are connected to each other by a double bond. For example, C 3-7 The term cycloalkenyl includes cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cycloheptadienyl, and cycloheptatrienyl.

[0048] halo-(alkyl, alkylene, or cycloalkyl) The term "halo" appended to an "alkyl," "alkylene," or "cycloalkyl" group (saturated or unsaturated) defines an alkyl, alkylene, or cycloalkyl group in which one or more hydrogen atoms have been replaced by fluorine, chlorine, or bromine, preferably a halogen atom selected from among fluorine and chlorine, particularly preferably fluorine. Examples include F3C-, H2FC-, HF2C.

[0049] Carbocyclyl The term "carbocyclyl," either alone or in combination with other radicals, means a monocyclic, bicyclic, or tricyclic ring structure of 3 to 14 carbon atoms. The term "carbocyclyl" refers to fully saturated, partially saturated, and aromatic ring systems. The term "carbocyclyl" encompasses fused, bridged, and spiro ring systems.

[0050] TIFF2026508289000013.tif69170

[0051] Heterocyclyl The term "heterocyclyl" means a saturated or unsaturated monocyclic or polycyclic ring system of 3 to 14 ring atoms, optionally including an aromatic ring, containing one or more heteroatoms selected from N, O, S, SO, SO2, none of which are part of an aromatic ring. The term "heterocyclyl" is intended to include all possible isomeric forms.

[0052] Thus, the term "heterocyclyl" includes the following exemplary structures (not depicted as radicals, as each form may optionally be attached to any atom via a covalent bond as long as appropriate valence is maintained):

[0053] TIFF2026508289000014.tif19170 TIFF2026508289000015.tif22170 TIFF2026508289000016.tif25170 TIFF2026508289000017.tif22170 TIFF2026508289000018.tif26170 TIFF2026508289000019.tif26170 TIFF2026508289000020.tif19170 TIFF2026508289000021.tif21170 TIFF2026508289000022.tif22170 TIFF2026508289000023.tif18170 TIFF2026508289000024.tif22170 TIFF2026508289000025.tif22170 TIFF2026508289000026.tif20170 TIFF2026508289000027.tif20170 TIFF2026508289000028.tif22170 TIFF2026508289000029.tif22170 TIFF2026508289000030.tif25170 TIFF2026508289000031.tif24170 TIFF2026508289000032.tif17170

[0054] Heteroaryl The term "heteroaryl" refers to a monocyclic or polycyclic ring system of 5 to 14 heteroatoms containing at least one aromatic ring and containing one or more heteroatoms selected from N, O, S, SO, or SO, wherein at least one of the heteroatoms is part of an aromatic ring. The term "heteroaryl" is intended to include all possible isomeric forms.

[0055] Thus, the term "heteroaryl" includes the following exemplary structures (not depicted as radicals, as each form may optionally be attached to any atom via a covalent bond as long as appropriate valence is maintained):

[0056] TIFF2026508289000033.tif39155 TIFF2026508289000034.tif47155 TIFF2026508289000035.tif40155 TIFF2026508289000036.tif59155

[0057] Many of the above terms are used repeatedly in the definitions of formulae or groups and can in each case, independently of one another, have one of the above meanings. The term "bicyclic ring system" means a group consisting of two linked ring moieties, including spiro, fused, and bridged ring systems.

[0058] Preferred Embodiments In another embodiment, the present invention provides a compound of formula (I): JPEG2026508289000037.jpg6774(I) where X 1 , X 2 , X 3 , X 4 At least one X, provided that no more than two of them are N at the same time. 1 , X 2 , X 3 , X 4 is -N; and where R 1 is —H or halogen; and where R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0059] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 , X 3 , X 4 At least one X, provided that no more than two of them are N at the same time. 1 , X 2 , X 3 , X 4 is -N; and where R 1 is —H or halogen; and where R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0060] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X2 , X 3 , X 4 At least one X, provided that no more than two of them are N at the same time. 1 , X 2 , X 3 , X 4 is -N; and where R 1 is -H or -F; and where R 1.1 is selected from the group consisting of -H, -F, and -CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CF3, and a methyl group, or a salt thereof.

[0061] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 , X 3 , X 4 At least one X, provided that no more than two of them are N at the same time. 1 , X 2 , X 3 , X 4 is -N; and where R 1 is -H; and where R 1.1 is -F; and where R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CF3, and a methyl group, or a salt thereof.

[0062] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 , X 3 , X 4 At least one X, provided that no more than two of them are N at the same time. 1 , X2 , X 3 , X 4 is -N; and where R 1 is -H; and where R 1.1 is -F; and where R 2 , R 3 , R 4 , R 5 , R 6 are each independently -H or -F, or a salt thereof.

[0063] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0064] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0065] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CF3, and a methyl group, or a salt thereof.

[0066] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0067] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CF3, and a methyl group, or a salt thereof.

[0068] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -F, -Cl, -Br, -CF3, and a methyl group, or a salt thereof.

[0069] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 is N, X 1 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R2 , R 4 , and R 6 are each independently -H or -F, or a salt thereof.

[0070] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , and R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0071] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0072] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, -Cl, -Br, -F, -CF3, and a methyl group, or a salt thereof.

[0073] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0074] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H or -F; and where R 1.1is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -Cl, -Br, -F, -CF3, and a methyl group, or a salt thereof.

[0075] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -F, -CF3, and a methyl group, or a salt thereof.

[0076] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 , X 2 and X 4 is CR 1 where X 3 is CR 1.1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 is -H or -F, or a salt thereof.

[0077] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0078] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0079] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 2 , R 3 , R 4 , R 5 , R 6are each independently selected from the group consisting of -H, -Cl, -Br, -F, -CF3, and a methyl group, or a salt thereof.

[0080] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is -H or -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0081] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is -H or -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -Cl, -Br, -F, -CF3, and a methyl group, or a salt thereof.

[0082] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1is -H or -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently -H or -F, or a salt thereof.

[0083] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is -H; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -F, -CF3, and a methyl group, or a salt thereof.

[0084] In another embodiment, the present invention provides a compound of formula (I), wherein X 2 and X 3 is N; and where X 1 and X 4 is CR 1 and where R 1 is -H; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently -H or -F, or a salt thereof.

[0085] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4is CR 1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0086] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is selected from the group consisting of -H, -F, and -Cl; and wherein R 1.1 is selected from the group consisting of —H, halogen, and —CN; and 2 , R 3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0087] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 2 , R3 , R 4 , R 5 , R 6 are each independently selected from the group consisting of -H, -F, -CF3, and a methyl group, or a salt thereof.

[0088] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, halogen, -CF3, and a methyl group, or a salt thereof.

[0089] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is -H or -F; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -Cl, -Br, -F, -CF3, and a methyl group, or a salt thereof.

[0090] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently selected from the group consisting of -H, -F, -CF3, and a methyl group, or a salt thereof.

[0091] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 2 is N; and where X 3 is CR 1.1 and X 4 is CR 1 and where R 1 is -H; and where R 1.1 is -F; and where R 3 and R 5 is -H; and where R 2 , R 4 , and R 6 are each independently -H or -F, or a salt thereof.

[0092] In another embodiment, the present invention provides a compound of formula (I), wherein X 1 and X 3 is N; and where X 2 and X 4 is CR 1 and wherein X3 is N and X4 is C-R1; and wherein R 1 is -H; and R 3 and R 5is -H; and where R 2 , R 4 , and R 6 are each independently -H or -F, or a salt thereof.

[0093] In another embodiment, the present invention provides JPEG2026508289000038.jpg189135 JPEG2026508289000039.jpg204135 JPEG2026508289000040.jpg205141 JPEG2026508289000041.jpg194144 JPEG2026508289000042.jpg152145, or a pharmaceutically acceptable salt thereof.

[0094] In another embodiment, the present invention provides TIFF2026508289000043.tif20981 JPEG2026508289000044.jpg21581 JPEG2026508289000045.jpg5770, or a pharmaceutically acceptable salt thereof.

[0095] In another embodiment, the present invention provides JPEG2026508289000046.jpg20487 JPEG2026508289000047.jpg21988, or a pharmaceutically acceptable salt thereof.

[0096] Biological assays Assays for determining NOX4 inhibition Establishment of cell lines for NOX4 inhibition assay The synthetic gene phNOX4_DNA3_1_Zeo (Accession: AAF68973) is assembled from synthetic oligonucleotides and / or PCR products. The fragment is cloned into pcDNA3.1_Zeo_A011 using the NheI and XhoI cloning sites. Plasmid DNA is purified from transformed bacteria, and its concentration is determined by UV spectroscopy. The final construct is verified by sequencing. The plasmid is combined with Nucleofector Kit V and transfected by electroporation (Amaxa electroporation device). NOX4 overexpression is selected using the selective antibiotic Zeocin.

[0097] NOX4 Inhibition Assay (HyPerBlu™) The inhibitory activity of the exemplified compounds of the present invention is determined by the following procedure: NOX4 inhibition was assessed using HEK293 cells stably overexpressing human NOX4 (hNOX4), which constitutively generate high levels of hydrogen peroxide (HO). Cells were cultured in 4.5 g / L glucose-containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 250 μg / mL Zeocin in a 37°C, 5% CO2 incubator. For the assay, cells were seeded into 384-well plates. After 24 h, cells were washed and treated with several concentrations (10 nM–30 μM) of test compound (diluted in DMSO) or 100 μM diphenyleneiodonium chloride (DPI, used as a positive control) in assay buffer consisting of phosphate-buffered saline (PBS) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The cells were then incubated for 2 h at 24°C in a humidified incubator.

[0098] After incubation, Lumigen HyPerBlu™ is added and the cells are incubated for an additional 30 minutes at 24°C in a humidified incubator. Luminescence is then measured using an Envision Multimode Plate reader to determine the level of H2O2 produced in the wells. The results are shown in Table 1.

[0099] [Table 1] Table E JPEG2026508289000048.jpg211147

[0100] Assays to determine selectivity for NOX1, NOX2, NOX3, and NOX5 Establishment of cell lines Plasmids are purchased from Vectorbuilder. All subunits required for HEK-hNOX1 were cloned as follows: ·pLV-CMV-hNOX1-puro (NM_007052.5) ·pLV-CMV-hNOXO1-blas (NM_172168.3) ·pLV-CMV-hp22phox-hygro (NM_000101.4) ·pLV-CMV-hNOXA1-neo (NM_006647.2) For HEK-hNOX3 ·pLV-CMV-hNOX3-puro (NM_015718.3)

[0101] Third-generation lentiviral particles are produced in HEK293 suspension cells by transient transfection of three helper and expression plasmids. The primary lentiviral supernatant is collected, clarified by filtration, and concentrated by sedimentation. Lentiviral titers are determined in transducing units (TU / mL) by transduction of HT1080 cells and colony formation assays. Parental HEK293 cells purchased from CLS GmbH are transduced with replication-deficient third-generation lentiviral particles (MOI 2) and grown for at least two weeks before cryopreservation of test lots. Culture medium is changed three times a week, and cells are subcultured at least once a week. Selection antibiotics are added according to the plasmid (1 μg / ml puromycin, 1.5 μg / ml blasticidin, 200 μg / ml G418, and 100 μg / ml hygromycin).

[0102] For HEK-hNOX5 The synthetic gene hNOX5 (Accession: Q96PH1) is cloned into pcDNA3.1_Zeo. Plasmid DNA is purified from transformed bacteria and its concentration is determined by UV spectroscopy. The final construct is verified by sequencing. The plasmid is combined with Nucleofector Kit V and transfected by electroporation (Amaxa electroporation device). NOX5 overexpression is selected using the selective antibiotic Zeocin.

[0103] NOX1 Inhibition Assay (L-012) The evaluation of NOX4 inhibitors on NOX1 activity is determined in human NOX1 (hNOX1) stably overexpressing HEK293 cells.

[0104] Cells were cultured in 4.5 g / L glucose-containing DMEM medium supplemented with 10% fetal calf serum (FCS), 1 μg / mL puromycin, 1.5 μg / mL blasticidin, 200 μg / mL geneticin (G418), and 100 μg / mL hygromycin in a 37°C, 5% CO2 incubator. For the assay, cells were seeded into 384-well plates in 10% FCS-containing DMEM medium. After 24 hours, cells were washed and treated with several concentrations (30 nM–100 μM) of test compound for 30 minutes. They were then stimulated with 1 μM phorbol myristate acetate (PMA) to induce NOX1-dependent ROS production (except for wells used as positive controls). Finally, 8-amino-5-chloro-2,3-dihydro-7-phenyl-pyrido[3,4-d]pyridazine-1,4-dione sodium salt (L-012, CAS 143556-24-5) was added to all wells at 400 μM, and the cells were incubated for an additional 3 hours in an incubator. Luminescence was then measured using a SpectraMax Paradigm Microplate Reader. The data are shown in Table 2.

[0105] NOX2 Inhibition Assay (L-012) The evaluation of NOX4 inhibitors on NOX2 activity is determined in human blood-derived granulocytes.

[0106] Different concentrations of test compounds (30 nM–100 μM), DMSO (as a negative control), or diphenyliodonium chloride (DPI; 30 μM, as a positive control) prepared in assay buffer (PBS containing CaCl2 and MgCl2 + 0.1% bovine serum albumin (BSA)) were added to a 384-well plate. Freshly isolated granulocytes were then added to the wells containing the compound-containing assay buffer and incubated at 37°C for 1 hour. Next, N-formylmethionine-leucine-phenylalanine (fMLP; 730 nM), phorbol myristate acetate (PMA, 100 nM) (a stimulator of ROS production), and L-012 (200 μM, an ROS indicator) were added to the wells, and the cells were incubated for an additional 30 minutes at 37°C. Luminescence was then measured using a SpectraMax M5 microplate reader. The data are shown in Table 2.

[0107] NOX3 Inhibition Assay (L-012) The evaluation of NOX4 inhibitors on NOX3 is determined in human NOX3 (hNOX3) stably overexpressing HEK293 cells.

[0108] Cells were cultured in 4.5 g / L glucose-containing DMEM medium supplemented with 10% fetal calf serum (FCS), 1 μg / mL puromycin, and 1.5 μg / mL blasticidin in a 37°C, 5% CO2 incubator. Cells were seeded in 384-well plates for 2 days. For the assay, cells were washed and treated with several concentrations (10 nM–30 μM) of test compound (diluted in DMSO) or 100 μM DPI (in assay buffer consisting of PBS, as a positive control) and incubated for 2 hours at 24°C in a humidified incubator. Cells were then stimulated with 0.1 μM PMA to induce NOX3-dependent ROS production. Finally, L-012 was added at 100 μM and incubated for 2 hours at 24°C in a humidified incubator. Luminescence was measured using a PHERAstar multimode reader. Data are shown in Table 2.

[0109] NOX5 Inhibition Assay (L-012) Evaluation of NOX4 inhibitors on NOX5 activity is determined in human NOX5 (hNOX5) stably overexpressing HEK293 cells.

[0110] Cells were cultured in 4.5 g / L glucose-containing DMEM medium supplemented with 10% fetal bovine serum and 250 μg / mL Zeocin in a 37°C, 5% CO2 incubator. Cells were seeded overnight in 384-well plates. For the assay, cells were washed and treated with several concentrations (10 nM–30 μM) of test compound (diluted in DMSO and added to assay buffer consisting of PBS) or 100 μM DPI as a positive control, and incubated for 2 hours at 24°C in a humidified incubator. Cells were then stimulated with 0.1 μM PMA to induce NOX5-dependent ROS production. Finally, L-012 was added at 100 μM and incubated for 2 hours at 24°C in a humidified incubator. Luminescence was measured using a PHERAstar multimode reader. Data are shown in Table 2.

[0111] [Table 2] JPEG2026508289000049.jpg47159 JPEG2026508289000050.jpg239149 JPEG2026508289000051.jpg109142 *:N / A=Not available

[0112] Assessment of hepatocyte clearance The metabolic degradation of test compounds is assayed in suspension hepatocytes. Hepatocytes (cryopreserved) are incubated in Dulbecco's modified Eagle's medium containing 5% human serum (supplemented with glucagon 3.5 μg / 500 mL, insulin 2.5 mg / 500 mL, and hydrocortisone 3.75 mg / 500 mL).

[0113] Following a 30 min pre-incubation in an incubator (37 °C, 10% CO), 5 μl of test compound solution (80 μM; diluted 1:25 in culture medium from a 2 mM DMSO stock solution) is added to 395 μl of suspended hepatocytes (cell density ranging from 0.25 to 5 μM cells / mL, typically 1 μM cells / mL; final test compound concentration 1 μM, final DMSO concentration 0.05%).

[0114] The cells are incubated (incubator, orbital shaker) for 6 hours, and samples (25 μl) are taken at 0, 0.5, 1, 2, 4, and 6 hours. The samples are transferred to acetonitrile and pelleted by centrifugation (5 minutes). The supernatant is transferred to a new 96-deep-well plate, evaporated under nitrogen, and resuspended.

[0115] The depletion of the parent compound is analyzed by HPLC-MS / MS. CL int is calculated as follows: CL_INTRINSIC=Dose / AUC=(C0 / CD) / (AUD+clast / k)x 1000 / 60 C0: initial concentration in incubation [μM], CD: cell density of viable cells [10 6 cells / mL], AUD: area under the data [µM x h], clast: concentration of the final data point [µM], k: slope of the regression line of the reduction of the parent [h-1].

[0116] The calculated in vitro hepatic intrinsic clearance can be scaled up to the intrinsic in vivo hepatic clearance and, using a well-stirred liver model, can be used to predict the hepatic in vivo blood clearance (CL).

[0117] CL_INTRINSIC_INVIVO[ml / min / kg]=(CL_INTRINSIC[μL / min / 10 6 cell] x hepatocellular [10 6 cells / g liver] x liver factor [g / kg body weight] / 1000

[0118] CL[ml / min / kg]=CL_INTRINSIC_INVIVO[ml / min / kg] x hepatic blood flow [ml / min / kg] / (CL_INTRINSIC_INVIVO[ml / min / kg]+hepatic blood flow [ml / min / kg])

[0119] Qh[%]=CL[ml / min / kg] / hepatic blood flow[ml / min / kg])

[0120] Hepatocellular, human: 120 x 10 6 cells / g liver Liver factor, human: 25.7 g / kg body weight Blood flow, human: 21 ml / (min x kg)

[0121] The resulting data is shown in Table 3. [Table 3] Table F TIFF2026508289000052.tif189136

[0122] Treatment methods The present invention relates to compounds of general formula (I) useful for the prevention and / or treatment of diseases and / or conditions associated with or modulated by NOX4 activity, including, but not limited to, the treatment and / or prevention of chronic liver disease, portal hypertension, viral infection, cancer, interstitial lung disease, retinopathy, acute and chronic inflammation, and fibrotic diseases. In particular, the pharmaceutical composition of the present invention is suitable for the treatment of interstitial lung diseases, such as idiopathic lung diseases, and may also be suitable for the treatment of fibrotic diseases, allergic and inflammatory diseases.

[0123] The compounds of general formula (I) are useful for the prevention and / or treatment of: vascular inflammation, arteriosclerosis, interstitial lung disease (e.g., idiopathic pulmonary fibrosis, progressive pulmonary fibrosis), hepatic fibrosis, pulmonary hypertension, portal hypertension, cirrhosis, acute onset of chronic liver failure (ACLF), sepsis, multiple organ failure, diabetic retinopathy, wet age-related macular degeneration (AMD), dry AMD, cardiovascular disease, NOX4+ cancer-associated fibroblast-rich tumors (pancreatic, lung, breast, colon, head and neck tumors), systemic sclerosis, inflammatory bowel disease, Duchenne muscular dystrophy, COVID-19, acute respiratory distress syndrome, and influenza.

[0124] Thus, the present invention relates to compounds of general formula (I) for use as medicines.

[0125] Furthermore, the present invention relates to the use of compounds of general formula (I) for the treatment and / or prevention of diseases and / or conditions associated with or modulated by NOX4 activity.

[0126] Furthermore, the present invention relates to the use of compounds of general formula (I) for the treatment and / or prevention of chronic liver diseases, viral infections, cancer, interstitial lung diseases, retinopathy, acute and chronic inflammation, and fibrotic diseases.

[0127] In particular, the pharmaceutical compositions of the present invention are suitable for the treatment of interstitial lung diseases, such as idiopathic lung diseases, and may additionally be suitable for the treatment of fibrotic, allergic and inflammatory diseases.

[0128] Furthermore, the present invention relates to the use of compounds of general formula (I) for the treatment and / or prevention of: vascular inflammation, arteriosclerosis, interstitial lung diseases such as idiopathic pulmonary fibrosis, progressive pulmonary fibrosis, hepatic fibrosis, pulmonary hypertension, portal hypertension, cirrhosis, acute onset of chronic liver failure (ACLF), sepsis, multiple organ failure, diabetic retinopathy, wet age-related macular degeneration (AMD), dry AMD, cardiovascular diseases, NOX4+ cancer-associated fibroblast-rich tumors (pancreatic, lung, breast, colon, and head and neck tumors), systemic sclerosis, inflammatory bowel disease, Duchenne muscular dystrophy, COVID-19, acute respiratory distress syndrome, influenza, pulmonary hypertension.

[0129] In a further aspect, the present invention relates to compounds of general formula (I) for use in the treatment and / or prevention of the aforementioned diseases and conditions.

[0130] In a further aspect, the present invention relates to the use of compounds of general formula (I) for the preparation of medicaments for the treatment and / or prevention of the aforementioned diseases and conditions.

[0131] In a further aspect, the present invention relates to a method for the treatment or prevention of the above-mentioned diseases and conditions, which method comprises the administration to a human of an effective amount of a compound of general formula (I).

[0132] The daily dose range of the compound of general formula (I) that can be administered is usually 0.00001 to 100 mg / kg body weight, for example 0.00001 to 10 mg / kg body weight of the patient. Each dosage unit can conveniently contain 0.001 to 1000 mg, for example 0.001 to 100 mg.

[0133] The actual pharmaceutically effective amount or therapeutic dose will generally 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 event, the compound will be administered at a dosage and in a manner such that a pharmacologically effective amount is delivered, based on the patient's specific condition.

[0134] Pharmaceutical Composition Suitable formulations for administering a 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, sachets, injectables, inhalants, and powders.

[0135] Suitable tablets can be obtained, for example, by mixing one or more compounds according to formula (I) with known excipients, such as inert diluents, carriers, disintegrants, adjuvants, surfactants, binders and / or lubricants.

[0136] Combination therapy Certain compounds of the present invention can further be combined with one or more, preferably one, additional therapeutic agent, according to embodiments, selected from the group of therapeutic agents useful in the treatment of diseases or conditions previously described herein, particularly those associated with chronic liver disease, viral infections, cancer, interstitial lung disease, retinopathy, acute and chronic inflammation, and fibrotic diseases.

[0137] According to other embodiments, the additional therapeutic agent is selected from the group of therapeutic agents useful for the treatment of diseases or conditions described herein above, particularly those associated with vascular inflammation, arteriosclerosis, interstitial lung disease (e.g., idiopathic pulmonary fibrosis, progressive pulmonary fibrosis), hepatic fibrosis, pulmonary hypertension, portal hypertension, cirrhosis, acute onset of chronic liver failure (ACLF), sepsis, multiple organ failure, diabetic retinopathy, wet age-related macular degeneration (AMD), dry AMD, cardiovascular disease, NOX4+ cancer-associated fibroblast-rich tumors (pancreatic, lung, breast, colon, head and neck tumors), systemic sclerosis, inflammatory bowel disease, Duchenne muscular dystrophy, COVID-19, acute respiratory distress syndrome, and influenza.

[0138] Suitable additional therapeutic agents for such combinations include, in particular, those that enhance the therapeutic effect of one or more active ingredients and / or allow for a reduction in the dosage of one or more active ingredients, e.g., for one of the aforementioned indications.

[0139] Thus, the compounds of the present invention may be combined with one or more additional therapeutic agents selected from the group consisting of: antifibrotic agents (e.g., Ofev, PDE4i); immunotherapeutic agents (e.g., PD-1, aCTLA-4); sGC activators; ATX inhibitors; SGLT2 inhibitors (e.g., dapagliflozin, empagliflozin); THRb inhibitors; GLP1 agonists and combinations of GLP1 agonists; FGF analogues such as FGF21 or FGF19; KRAS-G12C inhibitors (e.g., sotorasib); KRAS-G12D inhibitors; MDM2-p53 antagonists; Her2 inhibitors; platinum derivatives (e.g., cisplatin, oxaliplatin, carboplatin); alkylating agents (e.g., estramustine, mechlorethamine, melphalan, chlorambucil, busulfan, dacarbazine, cyclophosphamide, ifosfamide, temozolomide, nitrosoureas, e.g., carmustine and lomustine, thiotepa; antimitotic agents (e.g., vinca alkaloids, e.g., vinblastine, vindesine, vinorelbine, and vincristine); taxanes, e.g., paclitaxel, docetaxel, nab-paclitaxel (Abraxane); angiogenesis inhibitors (e.g., tascuinimod, bevacizumab); tubulin inhibitors; DNA synthesis inhibitors; PARP inhibitors; topoisomerase inhibitors; Isomerase inhibitors (e.g., epipodophyllotoxins such as etoposide and etopophos, teniposide, amsacrine, topotecan, irinotecan, mitoxantrone); antimetabolites (e.g., methotrexate, raltitrexed, 5-fluorouracil (5-FU), capecitabine, floxuridine, gemcitabine, mercaptopurine, thioguanine, cladribine, pentostatin, cytarabine (ara) C), fludarabine, a combination of trifluridine and tipiracil (=TAS102)); antitumor antibiotics (e.g., doxorubicin, Doxil (polyethylene glycol-modified liposomal doxorubicin hydrochloride), Myoset (non-polyethylene glycol-modified liposomal doxorubicin), anthracyclines such as daunorubicin, epirubicin and idarubicin, mitomycin C, bleomycin, dactinomycin, plicamycin, streptozocin); inhibitors of vascular endothelial growth factor. Furthermore, the compounds of the present invention can be used in combination with radiation therapy regimens.

[0140] Therefore, in another aspect, the present invention relates to the use of the compounds of the present invention in combination with one or more additional therapeutic agents as described above and below for the treatment of diseases or conditions that may be affected by or are mediated by NOX4, in particular the diseases or conditions described above and below.

[0141] In a further aspect, the present invention relates to a method for treating a disease or condition in a patient that can be affected by inhibition of NOX4, the method comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with a therapeutically effective amount of one or more additional therapeutic agents.

[0142] In a further aspect, the present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in combination with one or more additional therapeutic agents for the treatment of a disease or condition that can be affected by the inhibition of NOX4 in a patient in need thereof.

[0143] In yet another aspect, the present invention relates to a method for treating a disease or condition mediated by NOX4 activity in a patient, the method comprising administering to a human patient in need of such treatment a therapeutic amount of a compound of the present invention in combination with a therapeutically effective amount of one or more additional therapeutic agents described above and below.

[0144] The combined use of the compounds of the present invention and the additional therapeutic agent may be simultaneous or staggered. The compound of the present invention and one or more additional therapeutic agents may be present together in one formulation, e.g., a tablet or capsule, or may be present separately in two identical or different formulations, e.g., as a so-called kit of parts.

[0145] Thus, in another aspect, the present invention relates to a pharmaceutical composition comprising a compound according to the present invention and one or more additional therapeutic agents as described above and below, optionally together with one or more inert carriers and / or diluents.

[0146] Other features and advantages of the present invention will become apparent from the following more detailed embodiments which illustrate, by way of example, the principles of the invention.

[0147] preparation The compounds according to the present invention and their intermediates can be obtained using synthetic methods known to those skilled in the art and disclosed in the organic synthesis literature. Preferably, the compounds are obtained by methods similar to the preparation methods described more fully below, especially those described in the Examples section. In some cases, the order of the reaction steps carried out can be changed. Variations of the reaction methods known to those skilled in the art but not described in detail here can also be used.

[0148] General methods for preparing the compounds of the present invention will be apparent to those skilled in the art upon examination of the following reaction schemes. Any functional groups in the starting materials or intermediates may be protected using conventional protecting groups. These protecting groups may be cleaved again at an appropriate stage in the reaction sequence using methods well known to those skilled in the art.

[0149] The compounds of the present invention are prepared by the synthetic methods described below, in which the substituents of the general formula have the previously defined meanings. These methods are illustrative of the present invention, and do not limit the scope of the present invention and the claimed compounds to these examples. Where the preparation of starting compounds is not described, they are commercially available or can be prepared analogously to known compounds or methods described herein. Materials described in the literature are prepared according to published synthetic methods. Abbreviations are defined in the Examples section.

[0150] Scheme 1: JPEG2026508289000053.jpg65147

[0151] Intermediate II (Step 1, Intermediate I → Intermediate II) can be prepared by treating Intermediate I with a suitable thiocarbonylating agent, such as 1,1′-thiocarbonyl-bis(pyridin-2(1H)-one) or 1,1-thiocarbonyldiimidazole (TCDI), in a suitable solvent, such as acetonitrile (Scheme 1). The preferred reaction temperature is between room temperature and 75° C. The formation of the thiourea (Step 2, Intermediate II → Intermediate III) can be achieved by reacting Intermediate II with aniline in a suitable solvent, such as acetonitrile, 2-methyltetrahydrofuran, or N,N-dimethylformamide, in the presence or absence of a suitable base, such as triethylamine or N,N-diisopropylethylamine. The cyclization (Step 3, Intermediate III → Intermediate IV) can be achieved by treating Intermediate III with a suitable base, such as aqueous sodium hydroxide or lithium hydroxide, in a suitable solvent, such as methanol, ethanol, tetrahydrofuran, or water. Compounds of Invention V (Step 4, Intermediate IV → Compounds of Invention V) can be prepared by reacting Intermediate IV with a benzyl halide and a suitable base such as N,N-diisopropylethylamine or triethylamine in a suitable solvent such as N,N-dimethylacetamide, N,N-dimethylformamide, methanol, or tetrahydrofuran.

[0152] Scheme 2: JPEG2026508289000054.jpg3199

[0153] Alternatively, compounds of invention V can be obtained directly by reacting intermediate III with a benzyl halide, such as benzyl chloride or benzyl bromide, in a suitable solvent, such as N,N-dimethylformamide or tetrahydrofuran, and a suitable base, such as N,N-diisopropylethylamine or aqueous lithium hydroxide, at a temperature between room temperature and 40° C. (Scheme 2). [Example]

[0154] preparation 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 literature on organic synthesis, for example, "Comprehensive Organic Transformations," Second Edition, Richard C. Larock, John Wiley & Sons, 2010, and "March's Advanced Organic Chemistry," Seventh Edition, Michael B. Smith, John Wiley & Sons, 2013. Preferably, the compounds are obtained by methods similar to the preparation methods described more fully below, especially in the experimental section. In some cases, the procedures implemented in carrying out the reaction schemes can be modified. Reaction variants not described in detail herein but known to those skilled in the art can also be used. General processes for the preparation of the compounds of the present invention will be apparent to those skilled in the art upon review of the following schemes. The starting compounds are commercially available or can be prepared by methods described in the literature or herein, or by methods analogous or similar thereto. Prior to carrying out the reaction, conventional protecting groups can be used to protect the corresponding functional groups in the starting compounds. These protecting groups can be removed again at an appropriate stage in the reaction sequence using methods and literature well known to those skilled in the art, such as those described in "Protecting Groups", 3rd Edition, Philip J. Kocienski, Thieme, 2005, and "Protective Groups in Organic Synthesis", 4th Edition, Peter G. M. Wuts, Theodora W. Greene, John Wiley & Sons, 2006. The terms "ambient temperature" and "room temperature" are used interchangeably and refer to a temperature of about 20° C., for example, between 19° C. and 24° C.

[0155] Abbreviation: JPEG2026508289000055.jpg99146 JPEG2026508289000056.jpg177147

[0156] nomenclature The names of the compounds described in the experimental section were generated using PerkinElmer ChemDraw® software (Version 22.2.0.330).

[0157] Preparation of starting materials Unless otherwise stated, starting materials are commercially available and used without further purification.

[0158] The following starting materials are prepared as described in the cited literature: 5-((4-Methoxybenzyl)oxy)pyrazin-2-amine: M. Yamamoto, M. Takadoi, Y. Fukuda, Y. Asahina, Cyclopentylacrylic acid amide derivatives, WO 2009133687, April 2009

[0159] Preparation of intermediates Intermediate 1 Ethyl 4-isothiocyanatothiazole-5-carboxylate TIFF2026508289000057.tif25111

[0160] 1,1'-Thiocarbonylbis(pyridin-2(1H)-one) (28.3 g, 122 mmol, 1.05 equiv.) is added to a solution of ethyl 4-aminothiazole-5-carboxylate (20.0 g, 116 mmol, 1 equiv.) in acetonitrile (110 mL), and the reaction mixture is heated to 75 °C. After 3 h, the reaction mixture is poured into ice water, and the mixture is extracted with tert-butyl methyl ether. The phases are separated, and the organic phase is washed with water and dried over sodium sulfate. All volatile substances are removed under reduced pressure, and the residue is purified by flash column chromatography (silica gel, gradient from cyclohexane to cyclohexane / ethyl acetate 70:30) to give the product.

[0161] C7H6N2O2S2 (M = 214.0 g / mol) ESI 215 [M+H] + R t (HPLC) 0.96 min (Method A)

[0162] Intermediate 2 Ethyl 4-(3-(4-hydroxyphenyl)thioureido)thiazole-5-carboxylate TIFF2026508289000058.tif30129

[0163] 4-Aminophenol (102 mg, 993 μmol, 1 equiv.) is added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 200 mg, 933 μmol, 1 equiv.) in acetonitrile (2 mL), and the mixture is stirred at room temperature. After 18 h, the precipitate is filtered and dried to give the desired product.

[0164] C 13 H 13 N3O3S2 (M = 323.0 g / mol) ESI 324 [M+H] + R t (HPLC) 0.93 min (Method A)

[0165] Intermediate 3 6-(4-hydroxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000059.tif41123

[0166] Sodium hydroxide solution (4 M in water, 662 μL, 2.65 mmol, 4.00 equiv.) is added to a mixture of ethyl 4-(3-(4-hydroxyphenyl)thioureido)thiazole-5-carboxylate (Intermediate 2, 214 mg, 662 μmol, 1 equiv.) in methanol (3 mL). After 5 h, the reaction mixture is neutralized with aqueous hydrochloric acid (4 M, 662 μL), and the resulting precipitate is filtered and dried to give the desired product.

[0167] C 11 H7N3O2S2 (M = 277.0 g / mol) ESI 278[M+H] + R t (HPLC) 0.61 min (Method A)

[0168] Intermediate 4 Ethyl 4-(3-(3,5-difluoro-4-methoxyphenyl)thioureido)thiazole-5-carboxylate TIFF2026508289000060.tif35120

[0169] 3,5-Difluoro-4-methoxyaniline x H2SO4 (2.40 g, 9.33 mmol, 1 equiv.) is added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 2.00 g, 9.33 mmol, 1 equiv.) and triethylamine (1.30 mL, 9.33 mmol, 1 equiv.) in acetonitrile (10 mL). After 4 h, the reaction mixture is diluted with diisopropyl ether, and the precipitate is filtered and dried to give the desired product.

[0170] C 14 H 13F2N3O3S2 (M = 373.0 g / mol) ESI 374 [M+H] + R t (HPLC) 1.20 min (Method B)

[0171] Intermediate 5 6-(3,5-Difluoro-4-methoxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000061.tif41103

[0172] Sodium hydroxide solution (2 M in water, 5.00 mL, 10.0 mmol, 2 equiv.) is added to a mixture of ethyl 4-(3-(3,5-difluoro-4-methoxyphenyl)thioureido)thiazole-5-carboxylate (Intermediate 4, 1.87 g, 5.00 mmol, 1 equiv.) in tetrahydrofuran (50 mL). After 4 h, aqueous potassium bisulfate is added, and the resulting precipitate is filtered and dried to give the desired product.

[0173] C 12 H7F2N3O2S2 (M = 327.0 g / mol) ESI 328 [M+H] + R t (HPLC) 0.91 min (Method A)

[0174] Intermediate 6 6-(3,5-difluoro-4-hydroxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000062.tif4180

[0175] A solution of boron tribromide (1 M in CHCl, 19.9 mL, 19.9 mmol, 5 equiv.) is added to a mixture of 6-(3,5-difluoro-4-methoxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 5, 1.30 g, 3.97 mmol, 1 equiv.) in dichloromethane (20 mL) at −10° C. After the addition is complete, the reaction mixture is heated to 50° C. After 18 h, the reaction mixture is cooled to room temperature and poured into ice water, and the resulting precipitate is filtered and dried to give the desired product.

[0176] C 11 H5F2N3O2S2 (M = 313.0 g / mol) ESI 314 [M+H] + R t (HPLC) 0.74 min (Method B)

[0177] Intermediate 7 Methyl 4-(3-(5-hydroxypyrimidin-2-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000063.tif30143

[0178] Intermediate 7 can be prepared by procedures similar to those described for Intermediate 2 using appropriate starting materials.

[0179] C 10 H9N5O3S2 (M = 311.0 g / mol) ESI 312 [M+H] + R t (HPLC) 0.76 min (Method B)

[0180] Intermediate 8 6-(5-hydroxypyrimidin-2-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000064.tif41106

[0181] Methyl 4-(3-(5-hydroxypyrimidin-2-yl)thioureido)thiazole-5-carboxylate (Intermediate 7, 540 mg, 1.73 mmol, 1 equiv.) is added portionwise to a mixture of aqueous sodium hydroxide (1 M, 3.47 mL, 3.47 mmol, 2.00 equiv.) and water (25 mL), and the mixture is stirred at room temperature. After 30 min, aqueous hydrochloric acid (1 M, 650 μL) is added, and the precipitate is filtered, washed with water, and dried to give the product.

[0182] C9H5N5O2S2 (M = 279.0 g / mol) ESI 280 [M+H] + R t (HPLC) 0.58 min (Method F)

[0183] Intermediate 9 Ethyl 4-(3-(6-methoxypyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000065.tif30143

[0184] 1,1'-Thiocarbonylbis(pyridin-2(1H)-one) (8.09 g, 34.8 mmol, 1.20 equiv.) is added to a solution of ethyl 4-aminothiazole-5-carboxylate (5.00 g, 29.0 mmol, 1 equiv.) in acetonitrile (100 mL), and the mixture is heated to 70°C. After 18 h, the reaction mixture is cooled to ambient temperature and treated with 6-methoxypyridin-3-amine (5.41 g, 43.6 mmol, 1.50 equiv.). After 30 min, water is added, and the resulting precipitate is filtered and dried to give the product.

[0185] C 13 H 14 N4O3S2 (M = 338.1 g / mol) ESI 339 [M+H] + R t (HPLC) 1.00 min (Method A)

[0186] Intermediate 10 6-(6-Methoxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000066.tif41107

[0187] Aqueous sodium hydroxide (4 M, 3.92 mL, 15.7 mmol, 2.00 equiv.) is added to a mixture of ethyl 4-(3-(6-methoxypyridin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 9, 2.65 g, 7.83 mmol, 1 equiv.) and ethanol (30 mL). After 30 min, aqueous hydrochloric acid (4 M, 3.92 mL, 15.7 mmol, 2 equiv.) is added, and the resulting precipitate is filtered and dried to give the desired product.

[0188] C 11 H8N4O2S2 (M = 292.0 g / mol) ESI 293 [M+H] + R t (HPLC) 0.70 min (Method A)

[0189] Intermediate 11 5-((4-chloro-2-fluorobenzyl)thio)-6-(6-methoxypyridin-3-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000067.tif41123

[0190] 4-Chloro-1-(bromomethyl)-2-fluorobenzene (30.6 mg, 171 μmol, 1 equiv.) is added to a solution of 6-(6-methoxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 10, 50 mg, 171 μmol, 1 equiv.) and triethylamine (73.3 μL, 529 μmol, 3.10 equiv.) in N,N-dimethylacetamide (2 mL). After 1 hour, the reaction mixture is concentrated, and the residue is triturated with water, filtered, and dried to give the desired product.

[0191] C 18 H 12 ClFN4O2S2 (M = 434.0 g / mol) ESI 435 [M+H] + R t (HPLC) 1.04 min (Method A)

[0192] The following intermediates are prepared using the appropriate starting materials in a manner similar to that described for Intermediate 11. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0193] Table G TIFF2026508289000068.tif198159 TIFF2026508289000069.tif186159

[0194] Intermediate 20 Ethyl 4-(3-(5-fluoro-6-hydroxypyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000070.tif30129

[0195] Ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 2.00 g, 9.37 mmol, 1 equiv.) is added to a solution of 5-amino-3-fluoropyridin-2-ol (1.50 g, 9.37 mmol, 1 equiv.) in 2-methyltetrahydrofuran (15 mL). After 18 h, tert-butyl methyl ether is added, and the precipitate is filtered to give the desired product.

[0196] C 12 H 11 FN4O3S2 (M = 342.0 g / mol) ESI 343 [M+H] + R t (HPLC) 0.89 min (Method B)

[0197] Intermediate 21 6-(5-Fluoro-6-hydroxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000071.tif41109

[0198] Ethyl 4-(3-(5-fluoro-6-hydroxypyridin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 20, 525 mg, 1.53 mmol, 1 equiv.) is added portionwise to aqueous sodium hydroxide (0.1 M, 12 mL, 1.53 mmol, 1 equiv.) and the mixture is stirred at room temperature. After 30 minutes, the reaction mixture is poured into aqueous potassium bisulfate, and the precipitate is filtered and washed with water to give the product.

[0199] C 10 H5FN4O2S2 (M = 296.0 g / mol) ESI 297 [M+H] + R t (HPLC) 0.60 min (Method B)

[0200] Intermediate 22 2-Methoxy-5-(7-oxo-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-6(7H)-yl)-nicotinonitrile TIFF2026508289000072.tif41133

[0201] 5-Amino-2-methoxynicotinonitrile (15.7 mg, 100 μmol, 1 equiv.) was added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 21.4 mg, 100 μmol, 1 equiv.) and N,N-diisopropylethylamine (30.0 μL, 173 μmol, 2.00 equiv.) in N,N-dimethylformamide (2 mL). After 18 hours, aqueous lithium hydroxide (2 M, 100 μL, 200 μM, 2.00 equiv.) and 2-(bromomethyl)-1,3,5-trifluorobenzene (22.0 mg, 98.0 μmol, 1 equiv.) were added. After 18 hours, the reaction mixture was filtered, and the filtrate was purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0202] C 19 H 10 F3N5O2S2 (M = 461.0 g / mol) ESI 462 [M+H] + R t (HPLC) 1.10 min (Method A)

[0203] Intermediate 23 Ethyl 4-(3-(5-chloro-6-hydroxypyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000073.tif30129

[0204] Intermediate 23 is prepared by procedures similar to those described for intermediate 20 using appropriate starting materials.

[0205] C 12 H11 ClN4O3S2 (M = 358.0 g / mol) ESI 359 [M+H] + R t (HPLC) 0.50 min (Method C)

[0206] Intermediate 24 6-(5-chloro-6-hydroxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000074.tif41109

[0207] Intermediate 24 is prepared by procedures similar to those described for Intermediate 3 using appropriate starting materials.

[0208] C 10 H5ClN4O2S2 (M = 312.0 g / mol) ESI 313 [M+H] + R t (HPLC) 0.27 min (Method C)

[0209] Intermediate 25 Methyl 4-(3-(4,6-difluoropyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000075.tif51126

[0210] 4,6-Difluoropyridin-3-amine (250 mg, 1.86 mmol, 1 equiv.) is added to a solution of methyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 53, 373 mg, 1.86 mmol, 1 equiv.) in N,N-dimethylacetamide (2 mL). After 18 hours, tert-butyl methyl ether and water are added, and the precipitate is filtered to give the product.

[0211] C 11 H8F2N4O2S2 (M = 330.0 g / mol) ESI 331 [M+H] + R t (HPLC) 1.00 min (Method B)

[0212] Intermediate 26 6-(4,6-difluoropyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000076.tif51128

[0213] Methyl 4-(3-(4,6-difluoropyridin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 25, 250 mg, 757 μmol, 1 equiv.) is added to a solution of 2-(bromomethyl)-1,3,5-trifluorobenzene (170 mg, 757 μmol, 1 equiv.) and N,N-diisopropylethylamine (131 μL, 757 μmol, 1 equiv.) in tetrahydrofuran (5 mL). After 2 h, additional 2-(bromomethyl)-1,3,5-trifluorobenzene (170 mg, 757 μmol, 1 equiv.) and N,N-diisopropylethylamine (131 μL, 757 μmol, 1 equiv.) are added, the mixture is heated to 40° C., and after 18 h, the reaction mixture is purified directly by flash column chromatography (silica gel, gradient cyclohexane / ethyl acetate 90:10 to cyclohexane / ethyl acetate 50:50) to give the desired product.

[0214] C 17 H7F5N4OS2 (M = 442.0 g / mol) ESI 443 [M+H] + R t (HPLC) 1.02 min (Method F)

[0215] Intermediate 27 6-(4-Fluoro-6-(2-(trimethylsilyl)ethoxy)pyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)-thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000077.tif51143

[0216] Sodium hydride (55% dispersion in mineral oil, 19.7 mg, 452 μmol, 1 equiv.) was added to a solution of 2-(trimethylsilyl)ethan-1-ol (66.1 μL, 452 μmol, 1 equiv.) in tetrahydrofuran (4 mL). After 10 min, 6-(4,6-difluoropyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo-[4,5-d]pyrimidin-7(6H)-one (Intermediate 26, 200 mg, 452 μmol, 1 equiv.) was added. After 18 hours, the reaction mixture is filtered and the residue is purified by preparative reverse-phase HPLC (Waters XBridge™-C18, gradient of acetonitrile in water, 0.1% NH3) and then by flash column chromatography (silica gel, gradient of cyclohexane / ethyl acetate 80:20 to cyclohexane / ethyl acetate 50:50) to give the desired product.

[0217] C 22 H 20 F4N4O2S2Si (M=540.0 g / mol) ESI 541 [M+H] + R t (HPLC) 1.27 min (Method F)

[0218] Intermediate 28 6-(2-Fluoro-6-methoxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000078.tif41117

[0219] Intermediate 28 is prepared analogously to that described in Example 20 using appropriate starting materials.

[0220] C 11 H7FN4O2S2 (M = 310.0 g / mol) ESI 311 [M+H] + R t (HPLC) 0.87 min (Method B)

[0221] Intermediate 29 6-(2-Fluoro-6-methoxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000079.tif41129

[0222] 2-(Bromomethyl)-1,3,5-trifluorobenzene (167 mg, 741 μmol, 1 equiv.) is added to a solution of 6-(2-fluoro-6-methoxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 28, 230 mg, 741 μmol, 1 equiv.) and N,N-diisopropylethylamine (128 μL, 741 μmol, 1 equiv.) in N,N-dimethylacetamide (2.3 mL). After 2 hours, water is added, and the precipitate is filtered and washed with water to give the product.

[0223] C 18 H 10 F4N4O2S2 (M = 454.0 g / mol) ESI 455 [M+H] + R t (HPLC) 1.13 min (Method B)

[0224] Intermediate 30a 2,5,6-trifluoropyridin-3-amine TIFF2026508289000080.tif2575

[0225] A mixture of 2,3,6-trifluoro-5-nitropyridine (400 mg, 2.13 mmol, 1 equiv.) and Raney nickel (30 mg) in methanol (5 mL) is stirred under a hydrogen atmosphere (3 bar). After 24 h, the mixture is filtered and the filtrate is concentrated to give the product.

[0226] C5H3F3N2 (M = 148.0 g / mol) ESI 149 [M+H] + R t (HPLC) 0.67 min (Method A)

[0227] Intermediate 30b Ethyl 4-(3-(2,5,6-trifluoropyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000081.tif51121

[0228] Intermediate 30b is prepared by procedures similar to those described for Intermediate 2 using appropriate starting materials.

[0229] C 12 H9F3N4O2S2 (M = 362.0 g / mol) ESI 363 [M+H] + R t (HPLC) 1.11 min (Method A)

[0230] Intermediate 31 6-(2,5-difluoro-6-methoxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000082.tif51140

[0231] 2-(Bromomethyl)-1,3,5-trifluorobenzene (280 mg, 1.21 mmol, 1 equiv.) is added to a solution of ethyl 4-(3-(2,5,6-trifluoropyridin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 30, 568 mg, 1.25 mmol, 1 equiv.) and N,N-diisopropylethylamine (420 μL, 2.43 mmol, 1.90 equiv.) in N,N-dimethylformamide (2 mL), and the mixture is stirred for 2 h. Water and dichloromethane are added, and the layers are separated. The aqueous layer is extracted with dichloromethane, and the combined organic layers are filtered through a phase transfer filter. The filtrate is concentrated under reduced pressure, and the residue is triturated with a mixture of water and ethanol and dried. The residue is treated with a mixture of methanol (2 mL) and aqueous ammonium hydroxide (33%, 1 mL). After 48 hours, water is added and the precipitate is filtered and washed with dichloromethane to give the product.

[0232] C 18 H9F5N4O2S2 (M = 472.0 g / mol) ESI 473 [M+H] + R t (HPLC) 1.10 min (Method A)

[0233] Intermediate 32 4,5-Difluoro-6-methoxynicotinic acid TIFF2026508289000083.tif30113

[0234] A solution of n-butyllithium in hexane (2.5 M, 2.22 mL, 5.55 mmol, 1 equiv.) is added to a mixture of lithium tetramethylpiperidide (2.56 g, 16.9 mmol, 3.00 equiv.) and tetrahydrofuran (5 mL) at −64° C. After 5 minutes, a mixture of 5-fluoro-6-methoxynicotinic acid (1.00 g, 5.55 mmol, 1 equiv.) and tetrahydrofuran (10 mL) is added, and the reaction mixture is warmed to −40° C. After 1.5 hours, N-fluorobenzenesulfonimide (5.41 g, 16.7 mmol, 3.00 equiv.) and tetrahydrofuran (10 mL) is added. After 40 minutes, dichloromethane and water are added, and the reaction mixture is warmed to room temperature. The layers are separated, and the organic layer is extracted with water. The combined aqueous layers are concentrated and the residue is purified by reverse phase HPLC (Waters Sunfire™-C18, gradient of acetonitrile in water, 0.1% TFA) to give the desired product.

[0235] C7H5F2NO3 (M = 189.0 g / mol) ESI 190 [M+H] + R t (HPLC) 0.73 min (Method A)

[0236] Intermediate 33 tert-Butyl (4,5-difluoro-6-methoxypyridin-3-yl)carbamate TIFF2026508289000084.tif2594

[0237] A mixture of 4,5-difluoro-6-methoxynicotinic acid (Intermediate 32, 273 mg, 1.44 mmol, 1 equiv.), diphenylphosphoryl azide (401 mg, 1.46 mmol, 1 equiv.), and triethylamine (204 μL, 1.45 mmol, 1 equiv.) in tert-butanol (6 mL) is heated to 90 °C. After 3.5 h, water is added, and the mixture is extracted with dichloromethane. The combined organic layers are filtered through a phase transfer filter, and the filtrate is concentrated under reduced pressure. The residue is purified by flash column chromatography (silica gel, gradient from cyclohexane to cyclohexane / ethyl acetate 90:10) to give the desired product.

[0238] C 11 H 14 F2N2O3 (M = 260.0 g / mol) ESI 261 [M+H] + R t (HPLC) 0.98 min (Method A)

[0239] Intermediate 34 4,5-Difluoro-6-methoxypyridin-3-amine TIFF2026508289000085.tif2591

[0240] A solution of hydrochloric acid in 1,4-dioxane (4 M, 2 mL) is added to tert-butyl (4,5-difluoro-6-methoxypyridin-3-yl)carbamate (Intermediate 33, 220 mg, 845 μmol, 1 equiv.) After 18 h, the precipitate is filtered and dried to give the desired product.

[0241] C6H6F2N2O (M=160.0 g / mol) ESI 161 [M+H] + R t (HPLC) 0.64 min (Method A)

[0242] Intermediate 35 Ethyl 4-(3-(4,5-difluoro-6-methoxypyridin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000086.tif51119

[0243] 4,5-Difluoro-6-methoxypyridin-3-amine (Intermediate 34, 73.4 mg, 373 μmol, 1 equiv.) was added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 80.0 mg, 373 μmol, 1 equiv.) and triethylamine (130 μL, 933 μmol, 2.50 equiv.) in N,N-dimethylformamide (2 mL). After 1.5 h, water was added, and the precipitate was filtered and dried to give the desired product.

[0244] C 13 H 12 F2N4O3S2 (M = 374.0 g / mol) ESI 375 [M+H] + R t (HPLC) 1.07 min (Method A)

[0245] Intermediate 36 6-(4,5-difluoro-6-methoxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000087.tif51140

[0246] 2-(Bromomethyl)-1,3,5-trifluorobenzene (29.0 mg, 125 μmol, 1.20 equiv.) was added to a solution of ethyl 4-(3-(4,5-difluoro-6-methoxypyridin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 35, 40.0 mg, 107 μmol, 1 equiv.) and N,N-diisopropylethylamine (36.0 μL, 208 μmol, 1.90 equiv.) in N,N-dimethylformamide (2 mL). After 1.5 h, water was added, and the precipitate was filtered and dried to give the desired product.

[0247] C 18 H9F5N4O2S2 (M = 472.0 g / mol) ESI 473 [M+H] + R t (HPLC) 1.10 min (Method A)

[0248] Intermediate 37 Ethyl 4-(3-(2-methoxypyrimidin-5-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000088.tif35155

[0249] 2-Methoxypyrimidin-5-amine (615 mg, 4.67 mmol, 1 equiv.) is added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 1.00 g, 4.67 mmol, 1 equiv.) in acetonitrile (10 mL). After 1 h, the precipitate is filtered and dried to give the desired product.

[0250] C 12 H 13 N5O3S2 (M = 339.0 g / mol) ESI 340 [M+H] + R t (HPLC) 0.90 min (Method A)

[0251] Intermediate 38 6-(2-Methoxypyrimidin-5-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000089.tif41110

[0252] Sodium hydroxide solution (4 M in water, 2.11 mL, 8.45 mmol, 2 equiv.) is added to a mixture of ethyl 4-(3-(2-methoxypyrimidin-5-yl)thioureido)thiazole-5-carboxylate (Intermediate 37, 1.43 g, 4.23 mmol, 1 equiv.) in methanol (15 mL). After 10 min, the precipitate is filtered and dried to give the desired product.

[0253] C 10 H7N5O2S2 (M = 293.0 g / mol) ESI 294 [M+H] + R t (HPLC) 0.65 min (Method A)

[0254] Intermediate 39 5-((4-chloro-2-fluorobenzyl)thio)-6-(2-methoxypyrimidin-5-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000090.tif41132

[0255] 4-Chloro-1-(chloromethyl)-2-fluorobenzene (30.5 mg, 170 μmol, 1 equiv.) was added to a solution of 6-(2-methoxypyrimidin-5-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 38, 50 mg, 170 μmol, 1 equiv.) and triethylamine (73.0 μL, 527 μmol, 3.10 equiv.) in N,N-dimethylacetamide (2 mL). After 1 h, water was added, and the precipitate was filtered and dried to give the desired product.

[0256] C 17 H 11 ClFN5O2S2 (M = 435.0 g / mol) ESI 436 [M+H] + R t (HPLC) 0.97 min (Method A)

[0257] The following compounds are prepared using the appropriate starting materials in a manner similar to that described for intermediate 39. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0258] Table H TIFF2026508289000091.tif205166 TIFF2026508289000092.tif147162

[0259] Intermediate 47 Ethyl 4-(3-(6-methoxypyridazin-3-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000093.tif30133

[0260] 6-Methoxypyridazin-3-amine (291 mg, 2.33 mmol, 1 equiv.) is added to a solution of ethyl 4-isothiocyanatothiazole-5-carboxylate (Intermediate 1, 500 mg, 2.33 mmol, 1 equiv.) in acetonitrile (5 mL). After 1 h, the precipitate is filtered and dried to give the desired product.

[0261] C 12 H 13 N5O3S2 (M = 339.0 g / mol) ESI 340 [M+H] + R t (HPLC) 0.97 min (Method A)

[0262] Intermediate 48 6-(6-Methoxypyridazin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000094.tif41112

[0263] Aqueous sodium hydroxide (4 M, 803 μL, 3.21 mmol, 2.00 equiv.) is added to a mixture of ethyl 4-(3-(6-methoxypyridazin-3-yl)thioureido)thiazole-5-carboxylate (Intermediate 47, 545 mg, 1.61 mmol, 1 equiv.) in methanol (4 mL). After 18 h, aqueous hydrochloric acid (4 M, 803 μL, 2.00 equiv.) and water are added, and the precipitate is filtered and dried to give the desired product.

[0264] C 10 H7N5O2S2 (M = 293.0 g / mol) ESI 294 [M+H] + R t (HPLC) 0.63 min (Method A)

[0265] Intermediate 49 6-(6-hydroxypyridazin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000095.tif4195

[0266] Trimethylsilyl chloride (604 μL, 4.77 mmol, 4.50 equiv.) was added to a mixture of 6-(6-methoxypyridazin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 48, 386 mg, 1.05 mmol, 1 equiv.) and potassium iodide (157 mg, 947 μmol, 0.900 equiv.) in acetonitrile (10 mL). After 18 h, water was added, and the precipitate was filtered and dried to give the desired product.

[0267] C9H5N5O2S2 (M = 279.0 g / mol) ESI 280 [M+H] + R t (HPLC) 0.47 min (Method A)

[0268] Intermediate 50 Ethyl 4-(3-(5-methoxypyrazin-2-yl)thioureido)thiazole-5-carboxylate TIFF2026508289000096.tif30130

[0269] Intermediate 50 is prepared by procedures similar to those described for Intermediate 2 using appropriate starting materials.

[0270] C 19 H 19 N5O4S2 (M = 445.1 g / mol) ESI 446 [M+H] + R t (HPLC) 1.13 min (Method A)

[0271] Intermediate 51 6-(5-((4-methoxybenzyl)oxy)pyrazin-2-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one TIFF2026508289000097.tif41108

[0272] Intermediate 51 is prepared by procedures similar to those described for Intermediate 3 using appropriate starting materials.

[0273] C 17 H 13 N5O3S2 (M = 399.0 g / mol) ESI 400 [M+H] + R t (HPLC) 0.92 min (Method A)

[0274] Intermediate 52 6-(5-((4-methoxybenzyl)oxy)pyrazin-2-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]-pyrimidin-7(6H)-one TIFF2026508289000098.tif41133

[0275] Intermediate 52 is prepared by procedures similar to those described for Intermediate 11 using appropriate starting materials.

[0276] C 24 H 16 F3N5O3S2 (M = 543.1 g / mol) ESI 544 [M+H] + R t (HPLC) 1.09 min (Method A)

[0277] Intermediate 53 Methyl 4-isothiocyanatothiazole-5-carboxylate TIFF2026508289000099.tif25121

[0278] Intermediate 53 is prepared in a similar manner to intermediate 1 by substituting methyl 4-aminothiazole-5-carboxylate for ethyl 4-aminothiazole-5-carboxylate as the starting material.

[0279] C6H4N2O2S2 (M=200.0 g / mol) ESI 201 [M+H] + R t (HPLC) 0.86 min (Method A)

[0280] Preparation of final compounds Example 1 5-((3-fluorobenzyl)thio)-6-(4-hydroxyphenyl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000100.tif41133

[0281] 1-(Bromomethyl)-3-fluorobenzene (34.1 mg, 180 μmol, 1 equiv.) is added to a solution of 6-(4-hydroxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 3, 50.0 mg, 180 μmol, 1 equiv.) and triethylamine (77.2 μL, 557 μmol, 3.10 equiv.) in N,N-dimethylacetamide (1 mL). After 18 hours, the reaction mixture is filtered, and the filtrate is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0282] C 18 H 12 FN3O2S2 (M = 385.0 g / mol) ESI 386 [M+H] + R t (HPLC) 0.91 min (Method A)

[0283] Example 2 5-((4-chloro-2-fluorobenzyl)thio)-6-(3,5-difluoro-4-hydroxyphenyl)thiazolo[4,5-d]-pyrimidin-7(6H)-one TIFF2026508289000101.tif41133

[0284] 4-Chloro-1-(chloromethyl)-2-fluorobenzene (14.3 mg, 80.0 μmol, 1 equiv.) was added to a solution of 6-(3,5-difluoro-4-hydroxyphenyl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 6, 25.1 mg, 80.0 μmol, 1 equiv.) and N,N-diisopropylethylamine (25.0 μL, 145 μmol, 1.80 equiv.) in N,N-dimethylformamide (2 mL). After 18 hours, the reaction mixture was filtered, and the filtrate was purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0285] C 18 H9ClF3N3O2S2 (M = 455.0 g / mol) ESI 456 [M+H] + R t (HPLC) 0.92 min (Method G)

[0286] The following compounds are prepared using the appropriate starting materials by procedures analogous to the general procedures described in Example 2. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0287] Table I TIFF2026508289000102.tif203162 TIFF2026508289000103.tif146164

[0288] Example 10 6-(2,5-difluoro-4-hydroxyphenyl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000104.tif41134

[0289] A mixture of 4-amino-2,5-difluorophenol (72.6 mg, 500 μmol, 1 equiv.), methyl 4-isothiocyanatothiazole-5-carboxylate (100 mg, 500 μmol, 1 equiv.), and N,N-diisopropylethylamine (150 μL, 867 μmol, 1.70 equiv.) in N,N-dimethylformamide (5 mL) is heated to 55° C. After 3 hours, the reaction mixture is cooled to ambient temperature, and aqueous lithium hydroxide (2 M, 500 μL, 1.00 mmol, 2.00 equiv.) is added. After 2 hours, 2-(bromomethyl)-1,3,5-trifluorobenzene (110 mg, 500 μmol, 1 equiv.) is added. After 30 minutes, the reaction mixture is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0290] C 18 H8F5N3O2S2 (M = 457.0 g / mol) ESI 458 [M+H] + R t (HPLC) 1.05 min (Method A)

[0291] The following compounds are prepared using the appropriate starting materials by procedures similar to those described in Example 10. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0292] Table J TIFF2026508289000105.tif201161 TIFF2026508289000106.tif192161 TIFF2026508289000107.tif101167

[0293] Example 21 6-(5-hydroxypyrimidin-2-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000108.tif41132

[0294] Example 21 is prepared by procedures similar to those described in Example 1 using the appropriate starting materials.

[0295] C 16 H8F3N5O2S2 (M = 423.0 g / mol) ESI 424 [M+H] + R t (HPLC) 0.64 min (Method F)

[0296] Example 22 5-((4-chloro-2-fluorobenzyl)thio)-6-(6-hydroxypyridin-3-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000109.tif41121

[0297] Trimethylsilyl chloride (79.3 μL, 624 μmol, 4.50 equiv.) is added to a mixture of 5-((4-chloro-2-fluorobenzyl)thio)-6-(6-hydroxypyridin-3-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one (Intermediate 11, 60.0 mg, 138 μmol, 1 equiv.) and potassium iodide (20.6 mg, 124 μmol, 0.9 equiv.) in acetonitrile (2 mL). After 18 hours, the mixture is diluted with N,N-dimethylacetamide, and the mixture is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0298] C 17 H 10 ClFN4O2S2 (M = 420.0 g / mol) ESI 421 [M+H] + R t (HPLC) 0.72 min (Method E)

[0299] The following compounds are prepared using the appropriate starting materials in a manner similar to that described in Example 22. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0300] Table K TIFF2026508289000110.tif202150 TIFF2026508289000111.tif192156

[0301] Example 31 (General Procedure A) 6-(5-Fluoro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000112.tif41130

[0302] Triethylamine (72.0 μL, 522 μmol, 3.10 equiv.) is added to a solution of 6-(5-fluoro-6-hydroxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 21, 50 mg, 169 μmol, 1 equiv.) and 2-(bromomethyl)-1,3,5-trifluorobenzene (38.0 mg, 170 μmol, 1 equiv.) in N,N-dimethylacetamide (2 mL). After 40 minutes, the reaction mixture is filtered, and the filtrate is purified by reverse-phase HPLC (Waters XBridge™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0303] C 17 H8F4N4O2S2 (M = 440.0 g / mol) ESI 441 [M+H] + R t (HPLC) 0.48 min (Method C)

[0304] Example 32 (General Procedure B) 6-(5-Fluoro-6-hydroxypyridin-3-yl)-5-((4-(trifluoromethyl)benzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000113.tif41133

[0305] Triethylamine (423 μL, 3.04 mmol, 3.00 equiv.) is added to a solution of 6-(5-fluoro-6-hydroxypyridin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 21, 300 mg, 1.01 mmol, 1 equiv.) and 1-(bromomethyl)-4-(trifluoromethyl)benzene (242 mg, 1.01 mmol, 1 equiv.) in methanol (20 mL). After 1 h, the reaction mixture is poured into water and acidified with hydrochloric acid. The resulting precipitate is filtered and purified by flash column chromatography (silica gel, gradient of dichloromethane to dichloromethane / methanol 90:10) to give the desired product.

[0306] C 18 H 10 F4N4O2S2 (M = 454.0 g / mol) ESI 455 [M+H] + R t (HPLC) 0.54 min (Method C)

[0307] The following compounds are prepared using the appropriate starting materials by procedures analogous to those described in Examples 31 or 32, General Procedures A or B. As will be appreciated by those skilled in the art, analogous examples may include variations on the general reaction conditions.

[0308] Table L TIFF2026508289000114.tif204156 TIFF2026508289000115.tif189159 TIFF2026508289000116.tif232170 TIFF2026508289000117.tif51165

[0309] Example 47 2-Hydroxy-5-(7-oxo-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-6(7H)-yl)nicotinonitrile TIFF2026508289000118.tif41129

[0310] Trimethylsilyl chloride (20.0 μL, 158 μmol, 4.30 equiv.) is added to a mixture of 2-methoxy-5-(7-oxo-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-6(7H)-yl)nicotinonitrile (Intermediate 22, 17.0 mg, 37.0 μmol, 1 equiv.) and sodium iodide (4.97 mg, 33.0 μmol, 0.9 equiv.) in acetonitrile (0.5 mL). After 5 hours, trimethylsilyl chloride (20.0 μL, 158 μmol, 4.30 equiv.) is added and the mixture is heated to 45° C. After 18 hours, the mixture is diluted with N,N-dimethylacetamide, and the mixture is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient of acetonitrile in water, 0.1% TFA) to give the desired product.

[0311] C 18 H8F3N5O2S2 (M = 447.0 g / mol) ESI 448 [M+H] + R t (HPLC) 0.70 min (Method E)

[0312] Example 48 6-(5-chloro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000119.tif41124

[0313] Example 48 is prepared analogously to the procedure described for Example 31 using the appropriate starting materials.

[0314] C 17 H8ClF3N4O2S2 (M=456.0 g / mol) ESI 457 [M+H] + R t (HPLC) 0.53 min (Method C)

[0315] Example 49 6-(4-Fluoro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000120.tif46121

[0316] Trifluoroacetic acid (214 μL, 2.78 mmol, 50.0 equiv.) is added to a mixture of 6-(4-fluoro-6-(2-(trimethylsilyl)ethoxy)pyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one (Intermediate 27, 30.0 mg, 55.0 μmol, 1 equiv.) in dichloromethane (1.5 mL). After 1 hour, the mixture is concentrated under reduced pressure, and the residue is triturated with water, filtered, and dried to give the desired product.

[0317] C 17 H8F4N4O2S2 (M = 440.0 g / mol ESI 441 [M+H] + R t (HPLC) 0.71 min (Method F)

[0318] Example 50 6-(2-Fluoro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]-pyrimidin-7(6H)-one TIFF2026508289000121.tif41132

[0319] A solution of boron tribromide in dichloromethane (1 M, 22.5 mL, 22.5 mmol, 37.9 equiv.) is added to neat 6-(2-fluoro-6-methoxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo-[4,5-d]pyrimidin-7(6H)-one (Intermediate 29, 270 mg, 594 μmol, 1 equiv.), and the reaction mixture is heated to 50° C. After 18 h, the mixture is diluted with dichloromethane and poured into ice water. The layers are separated, and the organic layer is washed with water and concentrated under reduced pressure. The residue is purified by flash column chromatography (silica gel, gradient cyclohexane / ethyl acetate 90:10 to cyclohexane / ethyl acetate 20:80) to give the desired product.

[0320] C 17 H8F4N4O2S2 (M = 440.0 g / mol) ESI 441 [M+H] + R t (HPLC) 1.00 min (Method B)

[0321] Example 51 6-(2,5-difluoro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]-pyrimidin-7(6H)-one TIFF2026508289000122.tif41129

[0322] Example 51 is prepared analogously to the procedure described for Example 50 using the appropriate starting materials.

[0323] C 17 H7F5N4O2S2 (M = 458.0 g / mol) ESI 459 [M+H] + R t (HPLC) 0.95 min (Method A)

[0324] Example 52 6-(4,5-difluoro-6-hydroxypyridin-3-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]-pyrimidin-7(6H)-one TIFF2026508289000123.tif41129

[0325] Example 52 is prepared analogously to the procedure described for Example 47 using the appropriate starting materials.

[0326] C 17 H7F5N4O2S2 (M = 458.0 g / mol) ESI 459 [M+H] + R t (HPLC) 0.89 min (Method A)

[0327] Example 53 5-((4-chloro-2-fluorobenzyl)thio)-6-(2-hydroxypyrimidin-5-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000124.tif41131

[0328] Trimethylsilyl chloride (92.2 μL, 727 μmol, 4.50 equiv.) is added to a mixture of 5-((4-chloro-2-fluorobenzyl)thio)-6-(2-methoxypyrimidin-5-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one (Intermediate 39, 70.0 mg, 161 μmol, 1 equiv.) and potassium iodide (23.0 mg, 145 μmol, 0.90 equiv.) in acetonitrile (2 mL). After 18 hours, the mixture is concentrated, and the residue is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0329] C 16H9ClFN5O2S2 (M = 421.0 g / mol) ESI 422 [M+H] + R t (HPLC) 0.67 min (Method E)

[0330] The following compounds are prepared using the appropriate starting materials in a manner similar to that described in Example 53. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0331] Table M TIFF2026508289000125.tif202155 TIFF2026508289000126.tif148159

[0332] Example 61 5-((4-chloro-2-fluorobenzyl)thio)-6-(6-hydroxypyridazin-3-yl)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000127.tif41126

[0333] 4-Chloro-1-(chloromethyl)-2-fluorobenzene (14.3 mg, 80.0 μmol, 1 equiv.) was added to a solution of 6-(6-hydroxypyridazin-3-yl)-5-thioxo-5,6-dihydrothiazolo[4,5-d]pyrimidin-7(4H)-one (Intermediate 49, 22.3 mg, 80.0 μmol, 1 equiv.) and N,N-diisopropylethylamine (25.0 μL, 145 μmol, 1.80 equiv.) in N,N-dimethylformamide (2 mL). After 18 hours, the reaction mixture was filtered, and the filtrate was purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient acetonitrile in water, 0.1% TFA) to give the desired product.

[0334] C 16 H9ClFN5O2S2 (M = 421.0 g / mol) ESI 422 [M+H]+ R t (HPLC) 0.74 min (Method G)

[0335] The following compounds are prepared using the appropriate starting materials in a manner similar to that described in Example 61. As will be appreciated by those skilled in the art, analogous examples may involve variations in the general reaction conditions.

[0336] Table N TIFF2026508289000128.tif208164 TIFF2026508289000129.tif146165

[0337] Example 69 6-(5-hydroxypyrazin-2-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]pyrimidin-7(6H)-one TIFF2026508289000130.tif41115

[0338] Trifluoroacetic acid (1.00 mL, 13.0 mmol, 235 equiv) is added to a solution of 6-(5-((4-methoxybenzyl)oxy)pyrazin-2-yl)-5-((2,4,6-trifluorobenzyl)thio)thiazolo[4,5-d]-pyrimidin-7(6H)-one (Intermediate 52, 30.0 mg, 55.0 μmol, 1 equiv) in dichloromethane (2 mL). After 1 hour, the reaction mixture is concentrated and the residue is purified by reverse-phase HPLC (Waters Sunfire™-C18, gradient of acetonitrile in water, 0.1% TFA) to give the desired product.

[0339] C 16 H8F3N5O2S2 (M = 423.0 g / mol) ESI 424 [M+H] + R t (HPLC) 0.69 min (Method E)

[0340] Analytical HPLC method Method A TIFF2026508289000131.tif41141 Instrument description: Agilent 1200; analytical column: Sunfire (Waters) C18 3.0 x 30 mm 2.5 μm; column temperature: 60°C

[0341] Method B TIFF2026508289000132.tif41141 Instrument description: Agilent 1200; analytical column: Sunfire (Waters) C18 3.0 x 30 mm 2.5 μm; column temperature: 60°C

[0342] Method C TIFF2026508289000133.tif36141 Instrument description: Waters Acquity; Analytical column: Xbridge (Waters) BEH C18 2.1 x 30 mm 1.7 μm; Column temperature: 60°C

[0343] Method D TIFF2026508289000134.tif31120 Instrument description: Waters Acquity; analytical column: XBridge (Waters) C18 3.0 x 30 mm 2.5 μm; column temperature: 60°C

[0344] Method E TIFF2026508289000135.tif36141 Instrument description: Waters Acquity; analytical column: Sunfire (Waters) C18 3.0 x 30 mm 2.5 μm; column temperature: 60°C

[0345] Method F TIFF2026508289000136.tif41141 Instrument description: Agilent 1200; analytical column: Xbridge (Waters) C18 3.0 x 30 mm 2.5 μm; column temperature: 60°C

[0346] Method G TIFF2026508289000137.tif36141 Instrument description: Waters Acquity; analytical column: Sunfire C18 (Waters) 3.0 x 30 mm, 2.5 μm; column temperature: 60°C

Claims

1. A compound of formula (I), （I） where: X 1 , X 2 , X 4 are each independently N or C—R 1 and X 3 is N or C-R 1.1 and X 1 , X 2 , X 3 , X 4 provided that no more than two of are simultaneously N; R 1 is —H or halogen; R 1.1 is selected from the group consisting of —H, halogen, and —CN; R 2 , R 3 , R 4 , R 5 , R 6 are each independently —H, a halogen, or —CF 3 and a methyl group; A compound or a salt thereof.

2. X 1 , X 2 , X 3 , X 4 The compound according to claim 1, or a salt thereof, wherein at least one of the following is -N.

3. X 1 , X 2 , X 4 is C-R 1 and X 3 is C-R 1.1 and R 1 is —H, —F, or —Cl; R 1.1 is —H, —F, or —Cl; The compound according to claim 1, or a salt thereof.

4. X 2 is N; X 1 and X 4 Ga-C-R 1 and X 3 is C-R 1.1 and R 1 is —H, —F, or —Cl; R 1.1 is —H, —F, or —Cl; 3. The compound according to claim 1 or 2, or a salt thereof.

5. X 2 and X 3 is N; X 1 and X 4 Ga-C-R 1 and R 1 is —H, —F, or —Cl; 3. The compound according to claim 1 or 2, or a salt thereof.

6. X 1 and X 2 is N; X 3 is C-R 1.1 and X 4 is C-R 1 and R 1 is —H, —F, or —Cl; R 1.1 is —H, —F, or —Cl; 3. The compound according to claim 1 or 2, or a salt thereof.

7. R 1 is —H or —F; R 1.1 is —H or —F; R 3 and R 5 is —H; R 2 , R 4 , R 6 are each independently —H, a halogen, or —CF 3 and methyl groups; The compound according to any one of claims 1 to 6, or a salt thereof.

8. 、 、 、 、 、 、 、 、 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:

9. A compound according to any one of claims 1 to 8 in salt-free form.

10. A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for use as a medicine.

11. Chronic liver disease, portal hypertension, viral infections, cancer, interstitial lung disease, retinopathy, acute and chronic inflammation, and fibrotic diseases.

10. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for use in the treatment and / or prevention of a disease selected from the group consisting of interstitial lung diseases.

12. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt thereof, for use in the treatment and / or prevention of a disease selected from the group consisting of vascular inflammation, arteriosclerosis, interstitial lung disease (e.g., idiopathic pulmonary fibrosis, progressive pulmonary fibrosis), hepatic fibrosis, pulmonary hypertension, portal hypertension, cirrhosis, acute onset of chronic liver failure (ACLF), sepsis, multiple organ failure, diabetic retinopathy, wet age-related macular degeneration (AMD), dry AMD, cardiovascular disease, NOX4+ cancer-associated fibroblast-rich tumors (pancreatic, lung, breast, colon, head and neck tumors), systemic sclerosis, inflammatory bowel disease, Duchenne muscular dystrophy, COVID-19, acute respiratory distress syndrome, and influenza.

13. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

14. 10. A pharmaceutical combination comprising one or more compounds according to one or more of claims 1 to 9 or pharmaceutically acceptable salts thereof, and further comprising as active ingredient a substance selected from the group consisting of antifibrotic agents, immunotherapeutic agents, sGC activators, ATX inhibitors, SGLT2 inhibitors, THRb inhibitors, GLP1 agonists and combinations of GLP1 agonists, FGF analogues, KRAS-G12C inhibitors, KRAS-G12D inhibitors, MDM2-p53 antagonists, Her2 inhibitors, and chemotherapeutic agents.

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