Pyridazinone and its use

Abstract

The present invention provides a process for producing compounds for use in treating or reducing the risk of developing a disease or condition selected from kidney disease, pulmonary hypertension, anxiety, depression, cancer, diabetic retinopathy, or pain. [Solution] This is a process for preparing compound 100, shown by the following formula. JPEG2026090312000090.jpg76165

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 732,728, filed on September 18, 2018, and U.S. Provisional Patent Application No. 62 / 780,553, filed on December 17, 2018, each of which is incorporated herein by reference in its entirety.

Background Art

[0002] Proteinuria is a condition where an excess amount of protein in the blood leaks into the urine. Proteinuria can progress from a loss of 30 mg of protein into the urine in 24 hours (called microalbuminuria) to more than 300 mg / day (called overt albuminuria), and then reach a loss of more than 3.5 grams of protein in 24 hours or a level 25 times the normal amount. Proteinuria occurs when there is dysfunction in the glomeruli of the kidneys and causes fluid accumulation in the body (edema). Long - term protein leakage has been shown to lead to kidney failure. The nephrotic syndrome (NS) disease accounts for about 12% of widely recognized end - stage kidney disease cases, and the annual cost in the United States exceeds $3 billion. Approximately 5 out of every 100,000 children are diagnosed with NS each year, and today, 15 out of every 100,000 children have NS. In patients who respond positively to treatment, the recurrence rate is extremely high. 90% of children with nephrotic syndrome respond to treatment, but an estimated 75% relapse. There is a need for methods to more effectively treat kidney diseases such as proteinuria or reduce the risk of onset.

[0003] Mammalian TRP channel proteins form six-pass transmembrane cation-permeable channels that can be classified into six subfamilies (TRPC, TRPV, TRPM, TRPA, TRPP, and TRPML) based on amino acid sequence homology. Recent studies of TRP channels have shown that they are involved in many fundamental cellular functions and are thought to play a crucial role in the pathophysiology of many diseases. Many TRPs are expressed along different regions of the nephron in the kidney, and there is growing evidence suggesting that these channels are involved in both hereditary and acquired kidney damage. TRPC6, TRPM6, and TRPP2 are thought to be involved in hereditary focal segmental glomerulosclerosis (FSGS), hypomagnesemia with secondary hypocalcemia (HSH), and polycystic kidney disease (PKD), respectively.

[0004] TRPC5 has also been reported to contribute to the underlying mechanisms that control natural fear responses (J Neurosci. 2014 Mar 5;34(10):3653-3667).

[0005] Therefore, further inhibitors of TRPC5, TRPC4, or both are needed. [Overview of the Initiative]

[0006] This invention is at least in part based on the discovery that the activity of transient receptor potential cation channel subfamily C member 5 (TRPC5) eliminates actin stress fibers, reduces adhesion plaque formation, and results in a motile, migratory podocyte phenotype.

[0007] One aspect of the present invention is a compound that is an antagonist of TRPC5 or TRPC4 or both. In some embodiments, the compound of the present invention is the compound of structural formula I:

[0008] [ka]

[0009] (I) or a pharmaceutically acceptable salt thereof, wherein, "---" is a single bond or a double bond, X 1 is CH or N, when "---" is a double bond, X 2 is CH or N, when "---" is a single bond, X 2 is N(CH3), X 1 when X 2 is CH, X Y is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropane-1,1-diyl, or -CH(CH3)-, Q is 2-trifluoromethyl-4-fluorophenyl, 2-difluoromethyl-4-fluorophenyl, 2-trifluoromethylphenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2-chlorophenyl, 1-(benzyl)-4-methylpiperidin-3-yl, 4-trifluoromethylpyridin-3-yl, 2-trifluoromethyl-6-fluorophenyl, 2-trifluoromethyl-3-cyanophenyl, 2-ethyl-3-fluorophenyl, 2-chloro-3-cyanophenyl, 2-trifluoromethyl-5-fluorophenyl, or 2-difluoromethylphenyl, R 3 is hydrogen, -CH2OH, -CH(OH)-CH2OH, -NH2, -CH(OH)CH3, -OCH3, or -NH-(CH2)2OH; when "---" is a double bond, R 4 does not exist, when "---" is a single bond, R 3 and R 4 together form =O, R 5 and R 6 each is independently hydrogen or -CH3, provided that X 1 is N, X 2If is N, Y is -O- or -N(CH3), and Q is 2-trifluoromethylphenyl, then R 3 , R 5 , and R 6 The condition is that at least one of them is not hydrogen.

[0010] In some embodiments, the compound of the present invention has structural formula II:

[0011] [ka]

[0012] (II) It is represented by or a pharmaceutically acceptable salt thereof, in the formula, R 1 It is chloro, -CF3, -CHF2, or -CH3, R 2 is hydrogen or fluoro, R 3 This is hydrogen, -NH2, -CH2OH, or CH(OH)-CH2OH.

[0013] In one embodiment, the present invention relates to a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0014] In one embodiment, the present invention relates to a method for treating or reducing the risk of developing a disease or condition selected from renal disease, pulmonary hypertension, anxiety, depression, cancer, diabetic retinopathy, or pain, the method comprising administering a therapeutically effective amount of a compound or composition to a subject requiring such a dose. In some embodiments, the disease is renal disease, anxiety, depression, cancer, or diabetic retinopathy. In some embodiments, the disease or condition is a renal disease selected from focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive nephropathy, nephrotic syndrome, steroid-resistant nephropathy, minimal change syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immune complex-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, mesangial proliferative glomerulonephritis, primary amyloidosis, c1q nephritis, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, or IgA nephropathy. In some embodiments, the renal disease is proteinuria. In some embodiments, the renal disease is microalbuminuria or overt albuminuria. In some embodiments, the disease or condition being treated is pulmonary arterial hypertension. In some embodiments, the disease or condition to be treated is pain selected from neuropathic pain and visceral pain.

[0015] In some embodiments, the disease or condition is a cancer selected from chemotherapy-resistant breast cancer, adriamycin-resistant breast cancer, chemotherapy-resistant colorectal cancer, medulloblastoma, and tumor angiogenesis.

[0016] In some embodiments, the disease or condition being treated is transplant-associated FSGS, transplant-associated nephrotic syndrome, transplant-associated proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes mellitus, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

[0017] The method is effective in a variety of subjects, including mammals such as humans and other animals such as laboratory animals such as mice, rats, rabbits, or monkeys, or pets and livestock such as cats, dogs, goats, sheep, pigs, cows, or horses.

[0018] The present invention offers several advantages. The preventive and therapeutic methods described herein are effective, for example, in treating kidney diseases such as proteinuria, with minimal side effects, if any. Furthermore, the methods described herein are effective in identifying compounds that treat kidney disease, anxiety, depression, or cancer, or reduce the risk of developing them.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referred to herein are incorporated by reference in their entirety. In the event of any conflict, this specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are for illustrative purposes only and are not intended to limit the invention.

[0020] Other features, purposes, and advantages of the present invention will become apparent from the detailed description and claims. [Brief explanation of the drawing]

[0021] [Figure 1] This shows albumin excretion in PAN-impaired rats treated with compound 100 or mizoribine. [Figure 2] The urinary protein-creatinine ratio data from AT1R transgenic rats treated with compound 100 are shown in comparison to those treated with AngII injection and the solvent. [Figure 3]Figure 2 shows the urinary protein-creatinine ratio data, expressed as a baseline percentage (%). [Modes for carrying out the invention]

[0022] definition The term "acyl" is recognized in the relevant technical field and refers to a group represented by the general formula: hydrocarbyl C(O)-, preferably alkyl C(O)-.

[0023] The term "acylamino" is recognized in the relevant technical field and refers to an amino group substituted with an acyl group, which can be represented, for example, by the formula: hydrocarbyl C(O)NH-.

[0024] The term "acyloxy" is recognized in the relevant technical field and refers to a group represented by the general formula: hydrocarbyl C(O)O-, preferably alkyl C(O)O-.

[0025] The term "alkoxy" refers to an alkyl group to which oxygen is bonded, preferably a lower alkyl group. Typical alkoxy groups include methoxy, trifluoromethoxy, ethoxy, propoxy, and tert-butoxy.

[0026] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, and can be represented by the general formula: alkyl-O-alkyl.

[0027] As used herein, the term “alkenyl” refers to an aliphatic group containing at least one double bond, and includes both “unsubstituted alkenyls” and “substituted alkenyls,” the latter being an alkenyl moiety having a substituent that replaces one or more carbon atoms of the alkenyl group with hydrogen. Such substituents may be located on one or more carbon atoms that are not included in the one or more double bonds. Furthermore, such substituents include all those intended for alkyl groups, as described below, except where their use is unacceptable due to stability concerns. For example, substitution of an alkenyl group with one or more alkyl, carbocykryl, aryl, heterocyclyl, or heteroaryl groups is intended.

[0028] An "alkyl" group or "alkane" is a fully saturated linear or branched non-aromatic hydrocarbon. Generally, linear or branched alkyl groups have 1 to about 20 carbon atoms, preferably 1 to about 10, unless otherwise defined. Examples of linear and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. Linear or branched alkyl groups of C1 to C6 are also called "lower alkyl" groups.

[0029] Furthermore, the term “alkyl” (or “lower alkyl” as used throughout this specification, examples, and claims) includes both “unsubstituted alkyl” and “substituted alkyl,” the latter referring to an alkyl moiety having a substituent that substitutes one or more hydrogens on a hydrocarbon backbone. Unless otherwise specified, such substituents include, for example, halogens (e.g., fluoro), hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphine, amino, amide, amidine, imine, cyano, nitro, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. In preferred embodiments, the substituent of the substituted alkyl is C 1-6 Alkyl, C 3-6 The substituents are selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In a more preferred embodiment, the substituents of the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that, where appropriate, the substituted portion on the hydrocarbon chain can be substituted itself. For example, substituents on substituted alkyls include amino, azide, imino, amide, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonate), and silyl groups, as well as substituted and unsubstituted forms such as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates and esters), -CF3, and -CN. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl, -CF3, -CN, etc.

[0030] Unless otherwise specified, "alkylene" refers to a saturated linear or branched divalent group having the number of carbon atoms listed, either by itself or as part of another substituent, and derived by removing two hydrogen atoms from the corresponding alkane. Examples of linear and branched alkylene groups include -CH2-(methylene), -CH2-CH2-(ethylene), -CH2-CH2-CH2-(propylene), -C(CH3)2-, -CH2-CH(CH3)-, -CH2-CH2-CH2-CH2-, -CH2-CH2-CH2-CH2-CH2-(pentylene), -CH2-CH(CH3)-CH2-, and -CH2-C(CH3)2-CH2-.

[0031] "C x~y The term "acyl" means a group containing x to y carbon atoms in the chain, when used with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy. For example, "C x~y The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including haloalkyl groups, and linear and branched alkyl groups containing x to y carbon atoms in the chain. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. C0 alkyl groups indicate hydrogen when the group is at a terminal position and a bond when it is in the interior. 2~y "Alkenil" and "C 2~y The term "alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group that is similar in length and possible substitutions to the alkyl groups described above, but each contains at least one double or triple bond.

[0032] As used herein, the term "alkylamino" refers to an amino group substituted with at least one alkyl group.

[0033] As used herein, the term "alkylthio" refers to a thiol group substituted with an alkyl group, and can be represented by the general formula: alkylS-.

[0034] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond, and includes both "unsubstituted alkynyls" and "substituted alkynyls," the latter being an alkynyl moiety having a substituent that replaces one or more carbon atoms of the alkynyl group with hydrogen. Such substituents may be located on one or more carbon atoms that are included in or not included in the triple bond. Furthermore, such substituents include all those intended for alkyl groups as described above, except where their use is unacceptable due to stability concerns. For example, substitution of the alkynyl group with one or more alkyl, carbocykryl, aryl, heterocyclyl, or heteroaryl groups may be intended.

[0035] As used herein, the term "amide" is based on:

[0036] [ka]

[0037] This refers to each R A R independently represents a hydrogen atom or a hydrocarbyl group, or two R groups. A These atoms, along with the N atoms to which they are bonded, form a heterocycle containing 4 to 8 atoms in the ring structure.

[0038] The terms "amine" and "amino" are recognized in the relevant technical field, and include unsubstituted and substituted amines and their salts, for example,

[0039] [ka]

[0040] This refers to the part that can be represented by, where each R A R independently represents a hydrogen or a hydrocarbyl group, or two R A However, together with the N atoms to which they are bonded, they form a heterocycle with 4 to 8 atoms in the ring structure.

[0041] As used herein, the term "aminoalkyl" refers to an alkyl group substituted with an amino group.

[0042] As used herein, the term "aralkyl" refers to an alkyl group substituted with an aryl group.

[0043] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 6- or 10-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more adjacent rings share two or more carbon atoms, and at least one of the rings is aromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl and / or heterocyclyl. Examples of aryl groups include benzene, naphthalene, phenanthrene, phenol, and aniline.

[0044] The term "carbamate" is recognized in the relevant technical field, and

[0045] [ka]

[0046] This refers to each R A R independently represents a hydrogen or a hydrocarbyl group such as an alkyl group, or R A These atoms, along with intervening atoms, form a heterocycle containing 4 to 8 atoms in the ring structure.

[0047] As used herein, the terms “carbocyclic ring” and “carbocyclic formula” refer to saturated or unsaturated rings in which each atom of the ring is carbon. The term “carbocyclic ring” includes both aromatic and non-aromatic carbocyclic rings. Non-aromatic carbocyclic rings include both cycloalkane rings in which all carbon atoms are saturated, and cycloalkene rings having at least one double bond. “Carbocyclic rings” include monocyclic formulas with 5 to 7 members and bicyclic rings with 8 to 12 members. Each ring in a bicyclic carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. Carbocyclic rings include bicyclic molecules in which one, two, or three or more atoms are shared by two rings. The term “condensed carbocyclic ring” refers to a bicyclic carbocyclic ring in which each ring shares two adjacent atoms with the other ring. Each ring in a condensed carbocyclic ring can be selected from saturated, unsaturated, and aromatic rings. In one exemplary embodiment, an aromatic ring, such as phenyl, may be condensed with a saturated or unsaturated ring, such as cyclohexane, cyclopentane, or cyclohexane. Any combination of saturated, unsaturated, and aromatic bicyclic rings is included in the definition of a carbocyclic ring, as long as it is valence-wise possible. Examples of “carbocyclic rings” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octo-3-ene, naphthalene, and adamantane. Examples of condensed carbocyclic rings include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene, and bicyclo[4.1.0]hept-3-ene. A “carbocyclic ring” may be substituted at any one or more positions where hydrogen atoms may be present.

[0048] The "cycloalkyl" group is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Generally, monocyclic cycloalkyls have 3 to about 10 carbon atoms, more commonly 3 to 8 carbon atoms, unless otherwise defined. The second ring of a bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyls include bicyclic molecules in which one, two, or three or more atoms are shared by the two rings. The term "condensed cycloalkyl" refers to a bicyclic cycloalkyl in which each ring shares two adjacent atoms with the other ring. The second ring of a condensed bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. The "cycloalkenyl" group is a cyclic hydrocarbon having one or more double bonds.

[0049] As used herein, the term "carbocykylalkyl" refers to an alkyl group substituted with a carbocykyl group.

[0050] The term "carbonate" is recognized in the relevant technical field, and the base:-OCO2-R A (In the formula, R A (represents a hydrocarbyl group.)

[0051] As used herein, the term "carboxyl" refers to the group represented by the formula: -CO2H.

[0052] As used herein, the term "ester" refers to the base: -C(O)OR A (In the formula, R A (represents a hydrocarbyl group.)

[0053] As used herein, the term “ether” refers to a hydrocarbyl group bonded to another hydrocarbyl group via oxygen. Therefore, the ether substituent of a hydrocarbyl group can be hydrocarbyl-O-. Ethers may be symmetric or asymmetric. Examples of ethers, but not limited to, include heterocyclic-O-heterocyclic and aryl-O-heterocyclic groups. Ethers also include “alkoxyalkyl” groups, which can be represented by the general formula: alkyl-O-alkyl.

[0054] As used herein, the terms "halo" and "halogen" mean halogens, and include chloro, fluoro, bromo, and iodine.

[0055] As used herein, the terms "hetoaralkyl" and "heteroaralkyl" refer to alkyl groups substituted with hetoaryl groups.

[0056] As used herein, the term "heteroalkyl" refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, where no two heteroatoms are adjacent.

[0057] The terms "hetaryl" and "hetoaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, whose ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "hetaryl" and "hetoaryl" also include polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings, and at least one of the rings is heteroaromatic, for example, the other ring may be cycloalkyl, cycloalkenyl, aryl, heteroaryl and / or heterocyclyl. Examples of heteroaryl groups include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0058] As used herein, the term “heteroatom” means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0059] The terms "heterocyclyl," "heterocyclic," and "heterocyclic formula" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3-10 membered rings, more preferably 3-7 membered rings, whose ring structure contains at least one heteroatom, preferably 1 to 4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic formula" also include polycyclic ring systems having two or more rings in which two or more carbon atoms are common to two adjacent rings, where at least one of the rings is heterocyclic, and the other rings may be, for example, cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl. Examples of heterocyclyl groups include piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactone, lactam, and the like.

[0060] As used herein, the terms "heterocyclylalkyl" or "heterocycloalkyl" refer to alkyl groups substituted with heterocyclyl groups.

[0061] As used herein, the term "hydrocarbyl" refers to a group that has neither a substituent=O nor a substituent=S, and generally has at least one carbon-hydrogen bond and a carbon-based main chain, although it may contain a heteroatom, and is bonded via a carbon atom. Therefore, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyls for the purposes of this invention, but groups such as acetyl (which has a substituent=O on the bonded carbon) and ethoxy (which is bonded via oxygen, not carbon) are not considered hydrocarbyls. Examples of hydrocarbyl groups, but not limited to, include aryl, heteroaryl, carbocykryl, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0062] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxyl group.

[0063] When used with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term “lower” includes groups whose substituents contain 10 or fewer, preferably 6 or fewer, nonhydrogen atoms. “Lower alkyl” refers, for example, to alkyl groups containing 10 or fewer, preferably 6 or fewer carbon atoms. In certain embodiments, whether shown alone or in combination with other substituents, such as in descriptions like hydroxyalkyl and aralkyl (in which case, for example, atoms in the aryl group are not counted when counting the carbon atoms in the alkyl substituent), the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents as defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively.

[0064] The terms “polycyclyl,” “polycyclic,” and “polycyclic formula” refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclyl) in which two or more atoms are common to two adjacent rings (e.g., the rings are “fused rings”). Each of the rings in a polycyclic compound may be substituted or unsubstituted. In certain embodiments, each ring in a polycyclic compound contains 3 to 10, preferably 5 to 7, atoms within the ring.

[0065] The term "silyl" refers to the silicon portion formed by the bonding of three hydrocarbyl moieties.

[0066] The term “substituted” refers to a portion of the main chain having a group that substitutes a hydrogen on one or more carbon atoms. “Substitution” or “substituted with” implies that such substitution is based on the possible valencies of the substituted atom and the substituent, and that the substitution results in a stable compound (e.g., one that does not spontaneously change through rearrangement, cyclization, elimination, etc.). As used herein, the term “substituted” is intended to include all possible substituents of an organic compound. In a broader embodiment, possible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of an organic compound. There may be one or more possible substituents for a given organic compound, and they may be the same or different. For the purposes of this invention, a heteroatom such as nitrogen may have any possible substituent of the organic compound described herein that satisfies the hydrogen substituent and / or the valency of the heteroatom. Substituents may be any substituents described herein, for example, halogens, hydroxyls, carbonyls (e.g., carboxyls, alkoxycarbonyls, formyls, or acyls), thiocarbonyls (e.g., thioesters, thioacetates, or thioformates), alkoxyls, phosphoryls, phosphates, phosphonates, phosphinates, aminos, amides, amidines, imines, cyanos, nitros, azides, sulfhydryls, alkylthios, sulfates, sulfonates, sulfamoyls, sulfonamides, sulfonyls, heterocyclyls, aralkyls, or aromatic or heteroaromatic moieties. In preferred embodiments, the substituent of the substituted alkyl is C 1-6 Alkyl, C 3-6 The substituents are selected from cycloalkyl, halogen, carbonyl, cyano, or hydroxyl. In more preferred embodiments, the substituents of the substituted alkyl are selected from fluoro, carbonyl, cyano, or hydroxyl. Those skilled in the art will understand that, where appropriate, substituents can be substituted themselves. Unless specifically stated as “unsubstituted”, the term “chemical moiety” herein is understood to include substituted products. For example, the “aryl” group or moiety implicitly includes both substituted and unsubstituted products.

[0067] The term "sulfate" is recognized in the relevant technical field and refers to the base -OSO3H or its pharmaceutically acceptable salts.

[0068] The term "sulfonamide" is recognized in the relevant technical field, and its general formula is:

[0069] [ka]

[0070] This refers to the group represented by each R A This independently represents a hydrogen or an alkyl hydrocarbyl, or R A These atoms, along with intervening atoms, form a heterocycle containing 4 to 8 atoms in the ring structure.

[0071] The term "sulfoxide" is recognized in the relevant technical field, and the base is -S(O)-R A (In the formula, R A This refers to hydrocarbyl.

[0072] The term "sulfonate" is recognized in the relevant technical field and refers to the base SO3H or its pharmaceutically acceptable salts.

[0073] The term "sulfone" is recognized in the relevant technical field, and the base is -S(O)2-R A (In the formula, R A This refers to hydrocarbyl.

[0074] As used herein, the term "thioalkyl" refers to an alkyl group substituted with a thiol group.

[0075] As used herein, the term "thioester" refers to the base: -C(O)SR A or -SC(O)R A (In the formula, R AThis refers to hydrocarbyl.

[0076] As used herein, the term "thioether" is equivalent to ether, except that the oxygen is replaced by sulfur.

[0077] The term "urea" is recognized in the relevant technical field, and the general formula is:

[0078] [ka]

[0079] It can be represented by, and each R A R independently represents hydrogen or a hydrocarbyl such as an alkyl group, or any of the R groups present. A It forms a heterocycle with 4 to 8 atoms in the ring structure together with another RA and intervening atoms(s).

[0080] A "protecting group" refers to an atomic group that, when bonded to a reactive functional group within a molecule, blocks, reduces, or suppresses the reactivity of that functional group. Generally, protecting groups can be selectively removed as needed during the synthetic process. Examples of protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3rd Ed., 1999, John Wiley & Sons, NY, and Harrison et al., Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John Wiley & Sons, NY. Representative nitrogen protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ"), tert-butoxycarbonyl ("Boc"), trimethylsilyl ("TMS"), 2-trimethylsilyl-ethanesulfonyl ("TES"), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl ("FMOC"), and nitro-veratryloxycarbonyl ("NVOC"). Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group of benzyl and trityl ethers is acylated (esterified) or alkylated, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), glycol ethers (e.g., ethylene glycol and propylene glycol derivatives), and allyl ethers.

[0081] As used herein, a therapeutic agent that “prevents” or “reduces the risk of developing” a disease, disorder, or condition means a compound that, in a statistical sample, reduces the incidence of the disease or condition in a treated sample compared to a treated control sample, or delays the onset or reduces the severity of one or more symptoms of a disease, disorder, or condition compared to an untreated control sample.

[0082] The term “to treat” includes prophylactic and / or therapeutic measures. The term “prophylactic or therapeutic measure” is recognized in the art and includes administering one or more subject compositions to a host. A therapeutic agent is prophylactic (i.e., protects the host from the manifestation of the undesirable condition) if administered before the appearance of clinical symptoms of an undesirable condition (e.g., a disease or other undesirable condition in the host animal), whereas a therapeutic agent is therapeutic (i.e., aims to reduce, improve or stabilize an existing undesirable condition or its side effects) if administered after the appearance of the undesirable condition.

[0083] The expressions "co-administration" and "co-administered" refer to any form of administration of multiple different therapeutic compounds in which a second compound is administered while the previously administered therapeutic compound is still effective in the body (for example, the two compounds may be effective simultaneously in the patient's body, and a synergistic effect of the two compounds may be involved). For example, these different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or in separate formulations. In certain embodiments, different therapeutic compounds may be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other. Thus, an individual receiving such treatment may benefit from the combined effects of the different therapeutic compounds.

[0084] The term "prodrug" is intended to encompass compounds that are converted under physiological conditions to form compounds having the therapeutic activity of the present invention. One common method of producing a prodrug is to include one or more selected moieties that are hydrolyzed under physiological conditions to produce a desired molecule. In other embodiments, the prodrug is converted by the enzymatic activity of a host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds of the present invention in the formulations described above can be replaced with corresponding suitable prodrugs (e.g., giving a hydroxyl group in the parent compound as an ester or carboxylate, or giving a carboxylic acid present in the parent compound as an ester).

[0085] In this specification, "small molecule" refers to an organic or inorganic molecule with a molecular weight smaller than approximately 3000 daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3000 daltons (Da). These small molecules may be, for example, at least about 100 Da to about 3000 Da (e.g., about 100 to about 3000 Da, about 100 to about 2500 Da, about 100 to about 2000 Da, about 100 to about 1750 Da, about 100 to about 1500 Da, about 100 to about 1250 Da, about 100 to about 1000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0086] In some embodiments, "small molecule" generally refers to an organic, inorganic, or organometallic compound having a molecular weight of less than approximately 1000. In some embodiments, the small molecule is an organic compound having a size of around 1 nm. In some embodiments, the small molecule drugs of the present invention include oligopeptides and other biomolecules having a molecular weight of less than approximately 1000.

[0087] An "effective dose" is a quantity sufficient to produce a beneficial or desired result. For example, a therapeutic dose is the amount that produces the desired therapeutic effect. This amount may be the same as or different from the preventive effective dose, which is the amount required to prevent the onset of a disease or the symptoms of a disease. An effective dose can be administered in one or more doses, applications, or applications. The therapeutic effective dose of a composition is determined by the composition selected. A composition can be administered at least once a day to at least once a week, including once every other day. Those skilled in the art will recognize, but are not limited to, that certain factors, including the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other pre-existing conditions, may influence the dose and timing required to effectively treat the subject. Furthermore, the treatment of a subject with a composition described herein in terms of the therapeutic effective dose may consist of a single treatment or a series of treatments.

[0088] Compound of the present invention One aspect of the present invention provides a small molecule inhibitor of TRPC5.

[0089] In some embodiments, the compound of the present invention is a compound of structural formula I:

[0090] [ka]

[0091] (I) or a pharmaceutically acceptable salt thereof, During the ceremony, "---" indicates a single bond or a double bond. X 1 is CH or N, If "---" is a double bond, then X 2 is CH or N, If "---" is a single join, then X 2 It is N(CH3), X 1 If CH, then X 2 is N or N(CH3), Y is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropane-1,1-diyl, or -CH(CH3)-, Q is 2-trifluoromethyl-4-fluorophenyl, 2-difluoromethyl-4-fluorophenyl, 2-trifluoromethylphenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2-chlorophenyl, 1-(benzyl)-4-methylpiperidine-3-yl, 4-trifluoromethylpyridine-3-yl, 2-trifluoromethyl-6-fluorophenyl, 2-trifluoromethyl-3-cyanophenyl, 2-ethyl-3-fluorophenyl, 2-chloro-3-cyanophenyl, 2-trifluoromethyl-5-fluorophenyl, or 2-difluoromethylphenyl. R 3 R is hydrogen, -CH2OH, -CH(OH)-CH2OH, -NH2, -CH(OH)CH3, -OCH3, or -NH-(CH2)2OH, and if "---" is a double bond, 4 It does not exist. If "---" is a single bond, R 3 and R 4 Together with, they form =O, R 5 and R 6 Each of them is independently hydrogen or -CH3, However, X 1 N, X 2 If is N, Y is -O- or -N(CH3), and Q is 2-trifluoromethylphenyl, then R 3 , R 5 , and R 6 The condition is that at least one of them is not hydrogen.

[0092] In some embodiments, the compound of the present invention has structural formula II:

[0093] [ka]

[0094] (II) It is a compound represented by or a pharmaceutically acceptable salt thereof, in which, R 1 It is chloro, -CF3, -CHF2, or -CH3, R 2 is hydrogen or fluoro, R 3 This is hydrogen, -NH2, -CH2OH, or CH(OH)-CH2OH.

[0095] In some embodiments, R 1 If R is -CHF2, 2 It is not hydrogen.

[0096] In some embodiments, the compounds of the present invention are selected from any one of the following compounds or pharmaceutically acceptable salts thereof.

[0097] [ka]

[0098] [ka]

[0099] [ka]

[0100] [ka]

[0101] [ka]

[0102] [ka]

[0103] [ka]

[0104] [ka]

[0105] In some embodiments, the compounds of the present invention are selected from any one of the following compounds or pharmaceutically acceptable salts thereof.

[0106] [ka]

[0107] [ka]

[0108] In some embodiments, the compounds of the present invention are selected from any one of the following compounds or pharmaceutically acceptable salts thereof.

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] In certain embodiments, the compounds of the present invention may be racemic. In certain embodiments, the compounds of the present invention may be concentrated in one enantiomer. For example, the compounds of the present invention may have an enantiomer excess of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or even more than 95%.

[0113] The compounds of the present invention may have multiple stereocenters. Therefore, the compounds of the present invention may be concentrated with one or more diastereomers. For example, the compounds of the present invention may have a diastereomer excess of more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or even more than 95%. In certain embodiments, the compounds of the present invention have substantially one isomer configuration at one or more chiral centers and multiple isomer configurations at the remaining chiral centers.

[0114] In a particular embodiment, the enantiomer excess of the chiral center is at least 40%ee, 50%ee, 60%ee, 70%ee, 80%ee, 90%ee, 92%ee, 94%ee, 95%ee, 96%ee, 98%ee, or higher ee.

[0115] As used herein, single bonds drawn without stereochemistry do not represent the stereochemistry of the compound.

[0116] As used herein, hash lines or thick lines representing non-wedge bonds indicate a relative, not absolute, stereochemical configuration (e.g., they do not distinguish between enantiomers of specific diastereomers).

[0117] As used herein, hash lines or thick wedge bonds represent absolute stereochemistry.

[0118] In some embodiments, the present invention relates to pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically acceptable carriers. In certain embodiments, the therapeutic formulation or pharmaceutical composition of the present invention may be concentrated primarily with one enantiomer of the compound. The enantiomer-concentrated mixture may contain, for example, at least 60 mol%, or more preferably at least 75, 90, 95%, or even 99 mol%, of one enantiomer. In certain embodiments, the compound concentrated with one enantiomer is substantially free of the other enantiomer, where substantially free means that the substance in question constitutes less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1%, compared to the other enantiomer in the composition or mixture of compounds. For example, if the composition or mixture of compounds contains 98 grams of the first enantiomer and 2 grams of the second enantiomer, it is said to contain 98 mol percent of the first enantiomer and only 2% of the second enantiomer.

[0119] In certain embodiments, the therapeutic formulation or pharmaceutical composition may be concentrated to primarily give one diastereomer of the compound of the present invention. The diastereomer-concentrated mixture may contain, for example, at least 60 mol%, or more preferably at least 75, 90, 95%, or even 99 mol%, of one diastereomer.

[0120] Treatment method Non-selective Ca 2+Permeable transient receptor potential (TRP) channels function as sensors that transmit extracellular cues to the intracellular environment in various cellular processes, including actin remodeling and cell migration (Greka et al., Nat Neurosci 6, 837-845, 2003; Ramsey et al., Annu Rev Physiol 68, 619-647, 2006; Montell, Pflugers Arch 451, 19-28, 2005; Clapham, Nature 426, 517-524, 2003). Dynamic rearrangement of the actin cytoskeleton is regulated by spatiotemporal Ca 2+ Depending on the influx of (Zheng and Poo, Annu Rev Cell Dev Biol 23, 375-404, 2007; Brandman and Meyer, Science 322, 390-395, 2008; Collins and Meyer, Dev Cell 16, 160-161, 2009), the low molecular weight GTPases RhoA and Rac1 function as major modulators of these changes (Etienne-Manneville and Hall, Nature 420, 629-635, 2002; Raftopoulou and Hall, Dev Biol 265, 23-32, 2004). RhoA induces stress fiber and adhesion plaque formation, while Rac1 mediates lamellipodia formation (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). Transient receptor potential cation channel subfamily C member 5 (TRPC5) works in conjunction with TRPC6 to Ca 2+ It acts to regulate influx, actin remodeling, and cell motility of renal podocytes and fibroblasts. TRPC5-mediated Ca 2+ While influx increases Rac1 activity, TRPC6-mediated Ca 2+Influx promotes RhoA activity. Gene silencing of the TRPC6 channel eliminates stress fibers, reduces spot contact, and results in a motile, migratory cell phenotype. In contrast, gene silencing of the TRPC5 channel restores stress fiber formation and results in a contractile cell phenotype. The results described herein reveal a conserved signaling mechanism in which the TRPC5 and TRPC6 channels control a tightly regulated balance of cytoskeletal dynamics through differing bindings to Rac1 and RhoA.

[0121] Ca 2+ Actin-dependent remodeling of the actin cytoskeleton is a dynamic process that promotes cell migration (Wei et al., Nature 457, 901-905, 2009). RhoA and Rac1 function as switches involved in cytoskeletal rearrangement in migrating cells (Etienne-Manneville and Hall, Nature 420, 629-635, 2002; Raftopoulou and Hall, Dev Biol 265, 23-32, 2004). Rac1 activation mediates the motile cell phenotype, while RhoA activity promotes the contractile phenotype (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). Ca 2+ It plays a central role in the regulation of small molecule GTPases (Aspenstrom et al., Biochem J 377, 327-337, 2004). Ca 2+ The spatially and temporally restricted flicker is concentrated near the leading edge of cell migration (Wei et al., Nature 457, 901-905, 2009). Therefore, Ca 2+ The microdomain incorporates a local burst of Rac1 activity as a key event at its anterior end (Gardiner et al., Curr Biol 12, 2029-2034, 2002; Machacek et al., Nature 461, 99-103, 2009). To date, Ca, which is involved in GTPase regulation, has been identified. 2+The sources of influx remain largely unknown. Transient receptor potential (TRP) channels are linked to the cell migration of fibroblasts and neurons in the growth cone, and are temporally and spatially limited Ca 2+ It generates a signal. In detail, TRPC5 channels are known regulators of neuronal growth cone guidance 1, and their activity in neurons is dependent on PI3K and Rac1 activity (Bezzerides et al., Nat Cell Biol 6, 709-720, 2004).

[0122] Podocytes are neuron-like cells derived from the metanephrine mesenchymal system of the renal glomeruli and are essential for the formation of the renal filtration apparatus (Somlo and Mundel, Nat Genet. 24, 333-335, 2000; Fukasawa et al., J Am Soc Nephrol 20, 1491-1503, 2009). Podocytes possess a remarkably sophisticated repertoire of cytoskeletal adaptations to environmental cues (Somlo and Mundel, Nat Genet 24, 333-335, 2000; Garg et al., Mol Cell Biol 27, 8698-8712, 2007; Verma et al., J Clin Invest 116, 1346-1359, 2006; Verma et al., J Biol Chem 278, 20716-20723, 2003; Barletta et al., J Biol Chem 278, 19266-19271, 2003; Holzman et al., Kidney Int 56, 1481-1491, 1999; Ahola et al., Am J Pathol 155, 907-913, 1999; Tryggvason) and Wartiovaara, N Engl J Med 354,1387-1401,2006; Schnabel and Farquhar, J Cell Biol 111,1255-1263,1990; Kurihara et al., Proc Natl Acad Sci USA 89,7075-7079,1992). Early events of podocyte injury include uncontrolled actin cytoskeleton (Faul et al., Trends Cell Biol 17,428-437,2007; Takeda et al., J Clin Invest 108,289-301,2001; Asanuma et al., Nat Cell Biol 8,485-491,2006) and Ca 2+These changes are characterized by changes in homeostasis (Hunt et al., J Am Soc Nephrol 16, 1593-1602, 2005; Faul et al., Nat Med 14, 931-938, 2008). These changes are associated with the development of proteinuria, loss of albumin into the urinary space, and ultimately renal failure (Tryggvason and Wartiovaara, N Engl J Med 354, 1387-1401, 2006). Angiotensin II, a vasoactive hormone, is released into the Ca of podocytes. 2+ It induces inflow, and long-term treatment leads to the loss of stress fibers (Hsu et al., J Mol Med 86, 1379-1394, 2008). Ca 2+ While the link between cell influx and cytoskeletal rearrangement is recognized, the mechanism by which podocytes sense and transmit extracellular cues that regulate cell shape and motility remains unclear. Mutations in the TRP canonical 6 (TRPC6) channel have been shown to be associated with podocyte injury (Winn et al., Science 308, 1801-1804, 2005; Reiser et al., Nat Genet 37, 739-744, 2005; Moller et al., J Am Soc Nephrol 18, 29-36, 2007; Hsu et al., Biochim Biophys Acta 1772, 928-936, 2007), but little is known about the specific pathways that control this process. Furthermore, TRPC6 shares close homology with the other six members of the TRPC channel family (Ramsey et al., Annu Rev Physiol 68, 619-647, 2006; Clapham, Nature 426, 517-524, 2003). The TRPC5 channel antagonizes TRPC6 channel activity and regulates a tightly regulated balance of cytoskeletal dynamics through differences in binding to different small GTPases.

[0123] Proteinuria Proteinuria is a pathological condition in which protein is present in the urine. Albuminuria is a type of proteinuria. Microalbuminuria occurs when the kidneys leak small amounts of albumin into the urine. In a properly functioning body, albumin is normally not present in the urine because it is retained in the bloodstream by the kidneys. Microalbuminuria is diagnosed from a 24-hour urine sample (20-200 μg / min), or more commonly, from at least two elevated concentrations (30-300 mg / L). Microalbumin can be a precursor to diabetic nephropathy. Albumin levels higher than these values ​​are called overt albuminuria. For example, individuals with certain diseases such as diabetic nephropathy may progress from microalbuminuria to overt albuminuria, reaching the nephropathy range (over 3.5 g / 24 hours) when the kidney disease reaches its advanced stage.

[0124] Causes of proteinuria Proteinuria can be associated with several conditions, including focal segmental glomerulosclerosis, IgA nephropathy, diabetic nephropathy, lupus nephritis, membranoproliferative glomerulonephritis, progressive (crescentic) glomerulonephritis, and membranous glomerulonephritis.

[0125] A. Focal segmental glomerulosclerosis (FSGS) Focal segmental glomerulosclerosis (FSGS) is a disease that attacks the kidney's filtering system (glomeruli), causing severe scarring. FSGS is one of many causes of nephrotic syndrome (proteinuria), a condition in which proteins from the blood leak into the urine.

[0126] There are few treatment options available for patients with FSGS. Many patients are treated with steroid regimens, but many experience extremely serious side effects. Some patients have shown a positive response to immunosuppressants as well as blood pressure medications, which have been shown to lower levels of protein in the urine. To date, there are no generally accepted effective treatments or therapies, and there are no FDA-approved drugs for treating FSGS. Therefore, a more effective method to reduce or prevent proteinuria is desirable.

[0127] B. IgA nephropathy IgA nephropathy (also known as IgA nephritis, IgAN, Berger's disease, or glomerulonephritis with pharyngitis) is a form of glomerulonephritis (inflammation of the glomeruli of the kidneys). IgA nephropathy is the most common form of glomerulonephritis worldwide. Primary IgA nephropathy is characterized by the deposition of IgA antibodies in the glomeruli. Other diseases also involve IgA deposition in the glomeruli, the most common of which is Henoch-Schönlein purpura (HSP), considered a systemic form of IgA nephropathy. Henoch-Schönlein purpura presents with characteristic purpuric skin rash, arthritis, and abdominal pain, and is more commonly seen in young adults (16-35 years of age). HSP has a more benign prognosis than IgA nephropathy. In IgA nephropathy, 25-30% of cases experience slow progression to chronic renal failure over a 20-year period.

[0128] C. Diabetic nephropathy Diabetic nephropathy, also known as Kimmel-Steel-Wilson syndrome and intercapillary glomerulonephritis, is a progressive kidney disease caused by vascular damage to the glomeruli of the kidney. Diabetic nephropathy is characterized by nephrotic syndrome and diffuse glomerulosclerosis. Diabetic nephropathy is a result of long-term diabetes and is a major cause of dialysis. The earliest detectable change in the course of diabetic nephropathy is glomerular thickening. At this stage, the kidney may begin to leak more serum albumin than normal into the urine. As diabetic nephropathy progresses, the number of glomeruli destroyed by tuberous glomerulosclerosis increases, and the amount of albumin excreted in the urine increases.

[0129] D. lupus nephritis Lupus nephritis is a kidney disorder that is a complication of systemic lupus erythematosus (SLU). Lupus nephritis occurs when antibodies and complement accumulate in the kidneys, causing inflammation. Lupus nephritis often causes proteinuria and can rapidly progress to kidney failure. Nitrogenous waste products accumulate in the bloodstream. SLUPS causes various disorders of the internal structure of the kidneys, including interstitial nephritis. Lupus nephritis affects approximately 3 out of every 10,000 people.

[0130] E. Membranoproliferative glomerulonephritis I / II / III Membranoproliferative glomerulonephritis is a type of glomerulonephritis caused by the deposition of glomerular mesangia and thickening of the basement membrane, which activates complement and damages the glomeruli. There are three types of membranoproliferative glomerulonephritis. Type I is caused by immune complexes deposited in the kidney and is thought to be related to the classical complement pathway. Type II is similar to Type I but is thought to be related to the alternative complement pathway. Type III is extremely rare and is characterized by a combination of subepithelial deposits and the typical pathological findings of Type I disease.

[0131] F. Progressive (crescent-forming) glomerulonephritis Progressive (crescentic) glomerulonephritis (PG) is a renal syndrome that, if left untreated, rapidly progresses to acute renal failure and death within a few months. In 50% of cases, PG is associated with an underlying condition such as Goodpasture syndrome, systemic lupus erythematosus, or Wegener's granulomatosis, while the remaining cases are idiopathic. Regardless of the underlying cause, PG involves significant damage to the glomeruli of the kidney, many of which contain characteristic crescent-shaped scars. Patients with PG have hematuria, proteinuria, and sometimes hypertension and edema. Although the clinical presentation is consistent with nephrotic syndrome, the degree of proteinuria may exceed the range associated with nephrotic syndrome (3 g / 24 hours). Untreated disease may progress to decreased urine output (oliguria) due to declining renal function.

[0132] G. Membranephritis Membranoglionephritis (MGN) is a slowly progressive kidney disease that primarily affects patients between the ages of 30 and 50, usually Caucasians. Membranoglionephritis can develop into nephrotic syndrome. MGN is caused by circulating immune complexes. Current research indicates that the majority of immune complexes are formed by the in situ binding of antibodies to antigens to the glomerular basement membrane. These antigens can be endogenous in the basement membrane or deposited from the systemic circulation.

[0133] Measurement of urinary protein levels Urine protein levels can be measured using methods well known in the art. Until recently, accurate protein measurement required 24-hour urine collection. For 24-hour collection, the patient urinates into a container and stores it refrigerated each time they go to the toilet. The patient begins urine collection after their first toilet visit in the morning. All remaining urine for the day is collected in the container. The following morning, the patient adds their first urine after waking up, and collection is completed.

[0134] Recently, researchers have found that a single urine sample can provide the necessary information. A newer method compares the amount of albumin in a urine sample to the amount of creatinine, a waste product of normal muscle breakdown. This measurement is called the urinary albumin / creatinine ratio (UACR). A urine sample containing more than 30 milligrams of albumin per gram of creatinine (30 mg / g) is a warning sign that there may be a problem. If a laboratory test shows a level of more than 30 mg / g, another UACR test should be performed one to two weeks later. If the second test also shows high levels of protein, the subject has persistent proteinuria, a sign of impaired kidney function, and further tests are needed to assess kidney function.

[0135] A test that measures the amount of creatinine in the blood also indicates whether the subject's kidneys are efficiently removing waste products. Excessive creatinine in the blood is a sign that the subject has kidney damage. Doctors can use creatinine measurements to estimate how efficiently the kidneys are filtering blood. This calculation is called the estimated glomerular filtration rate, or eGFR. If the eGFR is less than 60 milliliters per minute (mL / min), it is considered chronic kidney disease.

[0136] TRPC5 TRPC is a family of transient receptor potential cation channels in animals. TRPC5 is a subtype of the TRPC family of transient receptor potential ion channels in mammals. Three examples of TRPC5 are shown in Table 1 below.

[0137] [Table 1]

[0138] Therefore, in certain embodiments, the present invention provides a method for treating or reducing the risk of developing a disease or condition selected from kidney disease, pulmonary hypertension, anxiety, depression, cancer, diabetic retinopathy, or pain, the method comprising administering a therapeutically effective amount of the compound of the present invention (e.g., the compound of structural formula I) or a pharmaceutical composition containing the compound to the subject of interest.

[0139] In some embodiments, the disease is renal disease, anxiety, depression, cancer, or diabetic retinopathy.

[0140] In some embodiments, the disease or condition is a renal disease selected from focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive nephropathy, nephrotic syndrome, steroid-resistant nephropathy, minimal change syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immune complex-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, mesangial proliferative glomerulonephritis, primary amyloidosis, c1q nephritis, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, or IgA nephropathy. In some embodiments, the renal disease is proteinuria. In some embodiments, the renal disease is microalbuminuria or overt albuminuria.

[0141] In a particular embodiment, the disease or condition being treated is pulmonary arterial hypertension.

[0142] In some embodiments, the disease or condition to be treated is pain selected from neuropathic pain and visceral pain.

[0143] In some embodiments, the disease or condition is a cancer selected from chemotherapy-resistant breast cancer, adriamycin-resistant breast cancer, chemotherapy-resistant colorectal cancer, medulloblastoma, and tumor angiogenesis.

[0144] The present invention also provides a method for treating anxiety, depression, or cancer, or for reducing the risk of developing them, which involves administering a compound of the present invention (e.g., a compound of formula I) or a pharmaceutical composition containing said compound to a subject in need.

[0145] In some embodiments, the disease or condition being treated is transplant-associated FSGS, transplant-associated nephrotic syndrome, transplant-associated proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes mellitus, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

[0146] Treatment targets In one aspect of the present invention, subjects are selected based on having or being at risk of developing renal disease, pulmonary hypertension, anxiety, depression, cancer, diabetic retinopathy, or pain. In another aspect, subjects are selected based on having or being at risk of developing renal disease, anxiety, depression, cancer, or diabetic retinopathy. In yet another aspect of the present invention, subjects are selected based on having or being at risk of developing pain, neuropathic pain, visceral pain, transplant-related FSGS, transplant-related nephrotic syndrome, transplant-related proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes mellitus, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

[0147] Individuals who have proteinuria, are at risk of developing it, or are at risk of developing it include those with diabetes, hypertension, or certain familial backgrounds. In the United States, diabetes is a leading cause of end-stage renal disease (ESRD). In both type 1 and type 2 diabetes, albumin in the urine is one of the first signs of declining kidney function. As kidney function declines, the amount of albumin in the urine increases. Another risk factor for developing proteinuria is hypertension. Proteinuria in patients with hypertension is an indicator of declining kidney function. If hypertension is not controlled, patients may progress to complete renal failure. African Americans are more prone to hypertension than Caucasians, and even a mild increase in blood pressure can easily lead to kidney damage. Other groups at risk of proteinuria include Native Americans, Hispanic / Latino Americans, Pacific Islander Americans, older adults, and overweight individuals.

[0148] In one aspect of the present invention, subjects are selected based on having proteinuria or being at risk of developing it. Subjects having proteinuria or being at risk of developing it are those who have one or more symptoms of the condition. The symptoms of proteinuria are well known in the art and include, but are not limited to, a large amount of protein in the urine, which can cause the urine to appear foamy in the toilet. The loss of large amounts of protein can lead to edema, causing swelling of the hands, feet, abdomen, or face. These are signs of massive protein loss and indicate that kidney disease is progressing. Laboratory testing is the only way to determine whether or not there is protein in a subject's urine before widespread kidney damage occurs.

[0149] The method is effective in a variety of subjects, including mammals, such as humans, and other animals, such as laboratory animals, such as mice, rats, rabbits, or monkeys, or pets and livestock, such as cats, dogs, goats, sheep, pigs, cows, or horses. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. [Examples]

[0150] The present invention will be further explained by the following examples, but these examples are not intended to limit the scope of the invention as described in the claims.

[0151] Example 1 Synthesis of Compound 100

[0152] [ka]

[0153] tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (400 mg, 1.48 mmol, 1 equivalent) and 4-fluoro-2-(trifluoromethyl)phenol (400.6 mg, 2.22 mmol, 1.5 equivalents) in acetonitrile (10 mL), DBU (451.5 mg, 2.97 mmol, 2.00 equivalents) was added at room temperature. The resulting mixture was stirred at 80 °C for 2 hours. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (3 × 100 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / SiO7 = 2:1) to obtain tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxelate (110 mg, 17.94%) as a brown solid.

[0154] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine To a stirred solution of tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (110 mg, 0.27 mmol, 1 equivalent) dissolved in DCM (4 mL), TFA (1 mL, 13.46 mmol, 50.59 equivalents) was added at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH = 12:1) to obtain 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (50 mg, 59.98%) as a brown solid.

[0155] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4,5-Dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (47.5 mg, 0.19 mmol, 1.19 equivalents) was added at room temperature to a stirred solution of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (50 mg, 0.16 mmol, 1 equivalent) dissolved in DIEA (2 mL). The resulting mixture was stirred at 100°C for 2 hours. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The residue was purified by preparative TLC (PE / siRNA=2:1) ​​to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (40 mg, 47.65%) as a brown solid.

[0156] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (40 mg, 0.08 mmol, 1 equivalent) was dissolved in DCM (4 mL) and stirred. TFA (1 mL, 13.46 mmol, 177.00 equivalents) was added dropwise at room temperature to this stirred solution. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The crude product (40 mg) was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mmol / L NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 18% B to 47% B over 7 minutes, 220 nm, Rt = 6.22 min) to obtain 4-chloro-5-[4-4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (8.6 mg, 25.59%) as a white solid.

[0157] Example 2 Synthesis of Compound 140

[0158] [ka]

[0159] Tert-butyl 4-bromo-5,6,7,8-tetrahydro-1,7-naphthylidene-7-carboxylate To a solution of 4-bromo-5,6,7,8-tetrahydro-1,7-naphthirizine (250 mg, 1.173 mmol, 1 equivalent) in THF (10 mL, 123.430 mmol, 105.20 equivalents), Boc2O (512.13 mg, 2.347 mmol, 2.00 equivalents) and TEA (474.90 mg, 4.693 mmol, 4 equivalents) were added at 25°C. The solution was stirred at 25°C for 2 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EA 5 / 1) to obtain tert-butyl 4-bromo-5,6,7,8-tetrahydro-1,7-naphthirizine-7-carboxylate (210 mg, 57.15%) as a pale yellow oil.

[0160] Tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthylidene-7-carboxylate To a DMSO solution (10 mL) of tert-butyl 4-bromo-5,6,7,8-tetrahydro-1,7-naphthyrizine-7-carboxylate (210 mg, 0.671 mmol, 1 equivalent) and 4-fluoro-2-(trifluoromethyl)phenol (241.52 mg, 1.341 mmol, 2 equivalents), Cs2CO3 (873.86 mg, 2.682 mmol, 4 equivalents), 2-(dimethylamino)acetic acid (41.46 mg, 0.402 mmol, 0.6 equivalents) and CuI (76.62 mg, 0.402 mmol, 0.60 equivalents) were added. After stirring at 120°C for 4 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC and eluted with PE / EA(5 / 1) to obtain tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthirizine-7-carboxelate (100 mg, 36.17%) as a pale yellow solid.

[0161] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthylidene To a DCM solution (10 mL, 157.300 mmol, 432.46 equivalents) of tert-butyl 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizine-7-carboxylate (150 mg, 0.364 mmol, 1 equivalent), TFA (414.75 mg, 3.637 mmol, 10 equivalents) was added at 25°C. The solution was stirred at 25°C for 2 hours. The resulting mixture was concentrated under reduced pressure. The residue was used in the next step.

[0162] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one A mixture of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthirizine (60 mg, 0.192 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (47.86 mg, 0.192 mmol, 1.00 equivalent) added to DIEA (49.67 mg, 0.384 mmol, 2 equivalents) was stirred at 100°C for 2 hours under an N2 atmosphere. The residue was purified by preparative TLC (PE / EA=1 / 1) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 99.15%) as a pale yellow solid.

[0163] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizin-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 0.191 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 825.67 equivalents), TFA (217.23 mg, 1.905 mmol, 10.00 equivalents) was added at 25°C. The solution was stirred at 25°C for 2 hours. The crude product (150 mg) was purified by preparative HPLC under the following conditions (column: XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 40% B over 7 minutes, 220 nm, Rt = 6.63 min) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyrizin-7-yl]-2,3-dihydropyridazine-3-one (42.9 mg, 51.09%) as a white solid.

[0164] Example 3 Synthesis of Compound 120

[0165] [ka]

[0166] 2-benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthylidene 2-benzyl-5-bromo-1,2,3,4-tetrahydro-2,6-naphthyrizine (250 mg, 0.825 mmol, 1 equivalent) and 2-(dimethylamino)acetic acid (170.05 mg, 1.649 mmol, 2.00 equivalents) were added to DMSO (5 mL) and stirred. 4-fluoro-2-(trifluoromethyl)phenol (89.10 mg, 0.495 mmol, 0.6 equivalents) and CuI (94.22 mg, 0.495 mmol, 0.6 equivalents) were then added at room temperature. Next, Cs2CO3 (1074.59 mg, 3.298 mmol, 4 equivalents) was added at room temperature. The final reaction mixture was irradiated with microwaves at 120 °C for 1 hour. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The crude product was purified by reverse-phase flush under the following conditions (column = XBridge Prep OBD C18 column 30 × 150 mm 5 μm; mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 18% B to 35% B over 8 minutes, 220 nm, Rt = 7.12 min) to obtain 2-benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthirizine (180 mg, 54.25%) as a brown solid.

[0167] 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthylidene 180 mg of 2-benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxyoxy]-1,2,3,4-tetrahydro-2,6-naphthirizine was dissolved in 10 mL of MeOH and stirred. Pd / C (20 mg) was added to this mixture at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 5 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH 12:1) to obtain 100 mg of 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthirizine as a brown solid.

[0168] 4-Chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthyrizin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthirizine (100 mg, 0.320 mmol, 1 equivalent) was dissolved in DIEA (0.1 mL) and stirred. To this stirred solution, 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (63.81 mg, 0.256 mmol, 0.8 equivalents) was added at room temperature. The resulting mixture was stirred at 90°C for 1 hour. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The residue was purified by preparative TLC (DCM / MeOH 12:1) to obtain 4-chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthyrizin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (130 mg, 77.34%) as a white solid.

[0169] 4-Chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthyridine-2-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthyrizin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (107 mg, 0.204 mmol, 1 equivalent) was dissolved in DCM (4 mL) and a stirred solution was prepared. TFA (1 mL) was added to this solution at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The mixture was basicized to pH 7 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The crude product (50 mg) was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 30% B to 50% B over 8 minutes, 220 nm, Rt = 7.55 min) to obtain 4-chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthyrizin-2-yl]-2,3-dihydropyridazine-3-one (60 mg, 66.78%) as a white solid.

[0170] Example 4 Synthesis of Compound 118

[0171] [ka]

[0172] Ethyl 2-(benzylamino)propanoate Benzaldehyde (8 g, 75.384 mmol, 1 equivalent) and TEA (7.63 g, 75.384 mmol, 1 equivalent) were dissolved in DCE (100 mL, 1263.149 mmol, 16.76 equivalents). To this stirred solution, TEA (7.63 g, 75.384 mmol, 1 equivalent) and NaBH(OAc)3 (31.95 g, 150.767 mmol, 2 equivalents) were added in small amounts at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at room temperature. The desired product was detected by LC-MS. The resulting mixture was extracted with DCM (2 × 150 mL). The combined organic extract was washed with brine (1 × 90 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to obtain ethyl 2-(benzylamino)propanoate (12 g, 76.80%) as a colorless oil.

[0173] Methyl 4-[benzyl(1-ethoxy-1-oxopropan-2-yl)amino]butanoate Ethyl 2-(benzylamino)propanoate (8 g, 38.596 mmol, 1 equivalent) and methyl 4-oxobutanoate (4.48 g, 38.596 mmol, 1.00 equivalent) were dissolved in DCE (120 mL, 1515.779 mmol, 39.27 equivalents). To this stirred solution, TEA (3.91 g, 38.596 mmol, 1 equivalent) and NaBH(OAc)3 (16.36 g, 77.193 mmol, 2 equivalents) were added in small amounts at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at room temperature. The desired product could be detected by LC-MS. The resulting mixture was extracted with DCM (2 × 150 mL). The combined organic layers were washed with brine (1 × 90 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure to obtain methyl 4-[benzyl(1-ethoxy-1-oxopropan-2-yl)amino]butanoate (10 g, 84.29%) as a colorless oil.

[0174] Methyl 1-benzyl-2-methyl-3-oxopiperidine-4-carboxylate Methyl 4-[benzyl(1-ethoxy-1-oxopropan-2-yl)amino]butanoate (8 g, 26.026 mmol, 1 equivalent) was dissolved in toluene (100 mL) and stirred. t-BuOK (5.00 g, 52.051 mmol, 2 equivalents) was added at room temperature. The mixture was stirred at 80°C for 2 hours. The desired product was detected by LC-MS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / siRNA (5:1 to 2:1) to obtain methyl 1-benzyl-2-methyl-3-oxopiperidine-4-carboxylate (6.5 g, 95.57%) as a white solid.

[0175] 7-benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-ol To a stirred solution prepared by dissolving methyl 1-benzyl-2-methyl-3-oxopiperidine-4-carboxylate (6 g, 22.960 mmol, 1 equivalent) in EtOH (80 mL, 1377.083 mmol, 59.98 equivalents), t-BuONa (4.41 g, 45.921 mmol, 2 equivalents) and methaneimidoamide hydrochloride (3.70 g, 45.921 mmol, 2.00 equivalents) were added in small amounts at room temperature under a nitrogen atmosphere. The mixture was stirred at 80°C for 2 hours. The desired product was detected by LC-MS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / siRNA (3:1 to 2:1) to obtain 7-benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-ol (5g, 85.29%) as a white solid.

[0176] tert-butyl 4-hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a solution of 7-benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-ol (5 g, 19.583 mmol, 1 equivalent) in EtOH (60 mL, 1032.812 mmol, 52.74 equivalents), Boc2O (8.55 g, 39.166 mmol, 2 equivalents), CH3COONa (1.81 g, 23.500 mmol, 1.2 equivalents), and Pd(OH)2 / C (275.01 mg, 1.958 mmol, 0.1 equivalent) were added under a nitrogen atmosphere. The mixture was hydrogenated at room temperature for 2 hours under a hydrogen atmosphere, filtered through a Celite pad, and concentrated under reduced pressure to obtain tert-butyl 4-hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (4.5 g, 86.61%) as a white solid.

[0177] tert-butyl 4-chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (4.5 g, 16.961 mmol, 1 equivalent) and PPh3 (6.67 g, 25.442 mmol, 1.5 equivalents) in DCE (60 mL, 0.606 mmol, 0.04 equivalents), CCl4 (5.22 g, 33.922 mmol, 2 equivalents) was added in small amounts at room temperature under a nitrogen atmosphere. The mixture was stirred at 70°C for 2 hours. The desired product was detected by LC-MS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / toluene (7:1) to obtain tert-butyl 4-chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (4g, 83.11%) as a white solid.

[0178] tert-butyl 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (4 g, 14.096 mmol, 1 equivalent) and 2-chloro-4-fluorophenol (2.07 g, 14.096 mmol, 1 equivalent) in DMF (50 mL), K2CO3 (3.90 g, 28.193 mmol, 2 equivalents) was added in small amounts at room temperature under a nitrogen atmosphere. The mixture was stirred at 70 °C for 1 hour. The desired product was detected by LC-MS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / toluene (1:1) to obtain tert-butyl 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (4g, 72.05%) as a white solid.

[0179] 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 4 g (1 equivalent) of tert-butyl 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was added to 20 mL of DCM and stirred. 4 mL of TFA was added dropwise at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 2 hours. The desired product was detected by LC-MS. The resulting mixture was concentrated under reduced pressure to obtain 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (2.7 g, 90.51%) as a grayish-white solid.

[0180] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4,5-Dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (0.85 g, 3.404 mmol, 1 equivalent) was added in small amounts at room temperature under a nitrogen atmosphere to a stirred solution prepared by dissolving 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (1 g, 3.404 mmol, 1 equivalent) in DIEA (1 mL). The mixture was stirred overnight at 100°C. The desired product was detected by LC-MS. The residue was purified by silica gel column chromatography and eluted with PE / SiO2 (1:1 to 1:2) to obtain 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (1g, 58.01%) as a white solid.

[0181] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (1 g, 1 equivalent) was dissolved in DCM (10 mL) and a stirred solution was added dropwise with TFA (2 mL) at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 1 hour. The desired product was detected by LC-MS. The resulting mixture was concentrated under reduced pressure to obtain 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (600 mg, 71.95%) as a white solid.

[0182] 4-Chloro-5-[(8R)-4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (250 mg, 1 equivalent) was subjected to preparative chiral HPLC (column = CHIRALPAK). Separation was performed using IG, 20 × 250 mm, 5 μm, mobile phase A = Hex:DCM = 3:1 (0.1% FA)-HPLC, mobile phase B = EtOH-HPLC, flow rate = 20 mL / min, gradient = 19 minutes (from 15B to 15B, 220 / 254 nm, RT1 = 13.016, RT2 = 16.004) to obtain 4-chloro-5-[(8R)-4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (144 mg, 57.60%) as a white solid.

[0183] Example 5 Synthesis of Compound 103

[0184] [ka]

[0185] tert-butyl 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (800 mg, 2.966 mmol, 1 equivalent) and 2-(difluoromethyl)phenylacetic acid (1104.26 mg, 5.932 mmol, 2.00 equivalent) in DMF (20 mL), K2CO3 (1229.72 mg, 8.898 mmol, 3 equivalents) was added in small amounts at 80°C under a nitrogen atmosphere. The mixture was stirred for 2 hours. The reaction was monitored by LC-MS. The reaction was stopped with water at room temperature. The mixture was extracted with RINKAN (3 × 50 mL). The combined organic layers were washed with brine (3 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = MeOH aqueous solution, gradient from 10% to 50% over 10 minutes, detector = UV254nm) to obtain tert-butyl 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (900 mg, 80.41%) as a grayish-white solid.

[0186] 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 900 mg, 2.385 mmol, 1 equivalent of tert-butyl 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (100 mg, 2.385 mmol, 1 equivalent) was dissolved in DCM and stirred. 3,3,3-trifluoropropanoic acid (3 mL, 6.00 equivalents) was added dropwise at room temperature to this stirred solution. The mixture was stirred for 1.5 hours. The reaction was monitored by TLC (PE / siRNA = 10:1). The residue was basicized to pH 8 with saturated NaHCO3 aqueous solution. The mixture was concentrated under reduced pressure. The crude product (100 mg) was purified by preparative HPLC under the following conditions to obtain 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (329 mg, 49.75%) as a grayish-white solid.

[0187] 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one DIEA (175.43 mg, 1.357 mmol, 2.00 equivalent) was added in small amounts at 70°C to a stirred solution of 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (328 mg, 1.183 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (169.05 mg, 0.679 mmol, 1.00 equivalent). The mixture was stirred at 70°C for 2 hours. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = MeOH aqueous solution, gradient from 10% to 50% over 10 minutes, detector = UV254nm) to obtain 4-chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (328 mg, 56.60%) as a grayish-white solid.

[0188] 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (328 mg, 0.670 mmol, 1 equivalent) was dissolved in DCM (10 mL) and stirred. Trifluoroacetic acid (3 mL) was added dropwise at room temperature to the mixture. The mixture was concentrated under vacuum. The product was purified by preparative HPLC to obtain 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (256.4 mg, 94.38%) as a grayish-white solid.

[0189] Example 6 Synthesis of Compounds 117 and 117a

[0190] [ka]

[0191] Ethyl 4-[1-phenylethyl]amino]pentanoate 1-phenylethane-1-amine (25 g, 206,300 mmol, 1 equivalent) and ethyl 4-oxopentanoate (29.74 g, 206,300 mmol, 1 equivalent) were dissolved in DCE (400 mL, 5052.598 mmol, 24.49 equivalents). To this stirred solution, NaBH(OAc)3 (65.59 g, 309.449 mmol, 1.5 equivalents) was added in small amounts at 25°C under a nitrogen atmosphere. The solution was stirred at 25°C for 2 hours. The reaction was stopped by adding H2O (400 mL) at 0°C. The resulting mixture was extracted with DCM (3 × 200 mL). The combined organic layers were washed with saturated NaCl aqueous solution (3 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product was used in the next step.

[0192] Ethyl 4-[(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]pentanoate Ethyl 4-[(1-phenylethyl)]pentanoate (49 g, 196.508 mmol, 1 equivalent) and 2-ethyl oxoacetate (40.12 g, 392.990 mmol, 2.00 equivalent) were dissolved in DCE (500 mL, 6315.747 mmol, 32.14 equivalents). To this stirred solution, NaBH(OAc)3 (62.47 g, 294.762 mmol, 1.5 equivalents) was added in small amounts at 25°C under a nitrogen atmosphere. The solution was stirred at 25°C for 2 hours. The reaction was stopped by adding H2O (400 mL) at 0°C. The resulting mixture was extracted with DCM (3 × 200 mL). The combined organic layer was washed with saturated NaCl aqueous solution (3 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product, ethyl 4-(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]pentanoate (57 g, 86.47%), was used in the next step.

[0193] Ethyl 2-methyl-5-oxo-1-(1-phenylethyl)piperidine-4-carboxylate To a solution of ethyl 4-(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]pentanoate (57 g, 169.924 mmol, 1 equivalent) in toluene (500 mL, 4699.452 mmol, 27.66 equivalents), t-BuOK (47.67 g, 424.810 mmol, 2.5 equivalents) was added in a port at 0°C. The mixture was stirred at 25°C for 2 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (50 / 1 to 10 / 1) to obtain ethyl 2-methyl-5-oxo-1-(1-phenylethyl)piperidine-4-carboxylate (29 g, 58.98%) as a yellow oil.

[0194] 7-(1-cyclohexylethyl)-6-methyl-decahydropyrido[3,4-d]pyrimidine-4-ol Ethyl 2-methyl-5-oxo-1-(1-phenylethyl)piperidine-4-carboxylate (10 g, 34.557 mmol, 1 equivalent) and methaneimidamide hydrochloride (4.17 g, 51.836 mmol, 1.50 equivalents) were dissolved in EtOH (100 mL, 1721.353 mmol, 49.81 equivalents). To this solution, EtONa (5.88 g, 86.393 mmol, 2.50 equivalents) was added in a port at 25°C. The mixture was stirred at 90°C for 2 hours. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (20 / 1 to 10 / 1) to obtain 7-(1-cyclohexylethyl)-6-methyl-decahydropyrido[3,4-d]pyrimidine-4-ol (3.4 g, 34.96%) as a yellow solid.

[0195] tert-butyl 4-hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a solution of 6-methyl-7-(1-phenylethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-ol (3.5 g, 12.994 mmol, 1 equivalent), HCOONH4 (4.10 g, 65.022 mmol, 5.00 equivalent), and Boc2O (8.51 g, 38.983 mmol, 3 equivalents) in EtOH (50 mL, 860.677 mmol, 66.23 equivalents), Pd(OH)2 / C (0.36 g, 2.599 mmol, 0.2 equivalents) was added under a nitrogen atmosphere. The mixture was hydrogenated at 70°C for 2 hours under a hydrogen atmosphere using a hydrogen balloon, filtered through a Celite pad, and concentrated under reduced pressure. tert-butyl4-hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.8 g, 52.21%) was obtained as a yellow solid.

[0196] tert-butyl 4-chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate tert-Butyl 4-hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.8 g, 6.784 mmol, 1 equivalent) and PPh3 (3.56 g, 13.569 mmol, 2 equivalents) in DCE (20 mL, 252.630 mmol, 37.24 equivalents) were added CCl4 (3.13 g, 20.353 mmol, 3 equivalents) at 25 °C. The mixture was stirred at 70 °C for 3 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (10 / 1 to 1 / 1) to give tert-butyl 4-chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.1 g, 57.14%) as a yellow solid.

[0197] tert-Butyl 4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate tert-Butyl 4-chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.1 g, 3.877 mmol, 1 equivalent) and 2-chloro-4-fluorophenol (0.85 g, 5.800 mmol, 1.50 equivalents) in DMF (15 mL, 193.826 mmol, 50.00 equivalents) were added K2CO3 (1.07 g, 7.753 mmol, 2 equivalents) at 25 °C. The mixture was stirred at 70 °C for 1 hour. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (10 / 1 to 5 / 1) to give tert-butyl 4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.2 g, 78.60%) as a yellow solid.

[0198] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one A mixture of 4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (800 mg, 2.724 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (678.42 mg, 2.724 mmol, 1.00 equivalent)) added to DIEA (704.01 mg, 5.447 mmol, 2 equivalents) was stirred at 100°C for 16 hours under a nitrogen atmosphere. The residue was purified by preparative TLC (PE / EA 1 / 1) to obtain 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (530 mg, 38.43%) as a pale yellow solid.

[0199] 4-Chloro-5-[(6R)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (530 mg, 1.047 mmol, 1 equivalent) in DCM (20 mL, 314.601 mmol, 300.57 equivalents), TFA (1193.47 mg, 10.467 mmol, 10 equivalents) was added at 25°C. The solution was stirred at 25°C for 2 hours. The resulting mixture was concentrated under reduced pressure. The crude product (600 mg) was purified by preparative HPLC under the following conditions (column = XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm; mobile phase A = water (10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 60 mL / min; gradient = 20% B to 40% B over 7 minutes, 220 nm; Rt = 6.63 min) to obtain a racemic mixture (200 mg). The residue (200 mg) was purified by chiral preparative HPLC under the following conditions (column = CHIRALPAK IE, 2 × 25 cm, 5 μm; mobile phase A = MTBE (0.1% FA)-HPLC; mobile phase B = IPA-HPLC; flow rate = 18 mL / min; gradient = 20 B to 20 B over 15 minutes, 220 / 254 nm). This method separated the two isomers, but the absolute orientation could not be determined. The compound specified as 4-chloro-5-[(6S)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (60.9 mg, 13.78%) was obtained as a white solid at 9.688 min. The compound specified as 4-chloro-5-[(6R)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (61.5 mg, 13.92%) was obtained as a white solid at 11.813 min.

[0200] Example 7 Synthesis of Compound 134

[0201] [ka]

[0202] tert-butyl 2-chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrimido[3,4-d]pyrimidine-7-carboxylate 2-(difluoromethyl)-4-fluorophenol (5.33 g, 32.879 mmol, 2.00 equivalents) and tert-butyl 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (5 g, 16.438 mmol, 1 equivalent) were dissolved in DMF (30 mL) and stirred. NaHCO3 (4.14 g, 49.282 mmol, 3.00 equivalents) was added at room temperature to the mixture. The solution was stirred at 70 °C for 0.5 hours. The mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 70% B to 95% B for 100 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 92% B and concentrated under reduced pressure to obtain tert-butyl 2-chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrimido[3,4-d]pyrimidine-7-carboxylate as a grayish-white solid (2.100 g).

[0203] 7-tert-butyl 2-methyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2,7-dicarboxylate To a solution of tert-butyl 2-chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (400 mg, 0.931 mmol, 1 equivalent) and TEA (188.34 mg, 1.861 mmol, 2 equivalents) in MeOH (15 mL, 370.484 mmol, 398.10 equivalents), Pd(PPh3)4 (107.54 mg, 0.093 mmol, 0.1 equivalent) was added in a pressure vessel. The mixture was purged with nitrogen for 1 hour and then pressurized with carbon monoxide at 100°C for 16 hours at 10 atmospheres. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 35% B to 65% B for 20 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 62% B and concentrated under reduced pressure to obtain 7-tert-butyl 2-methyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2,7-dicarboxylate (100 mg, 23.70%) as a colorless oil.

[0204] tert-butyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate 7-tert-butyl 2-methyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2,7-dicarboxylate (100 mg, 0.221 mmol, 1 equivalent) was dissolved in t-BuOH (6 mL, 63.139 mmol, 286.29 equivalents). To this stirred solution, NaBH4 (16.69 mg, 0.441 mmol, 2 equivalents) was added at room temperature. The solution was stirred at 70°C for 3 hours. Water (3 mL) was added to the mixture. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 45% B to 80% B for 20 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 74% B and concentrated under reduced pressure to obtain tert-butyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (35 mg, 37.30%) as a colorless oil.

[0205] [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]methanol 35 mg of tert-butyl 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 6 mg of DCM and stirred. TFA (1 mg) was added to this mixture at room temperature. The solution was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions: column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 25% B to 55% B for 20 minutes; detector = 254 nm. The fraction containing the desired product was recovered with 41% B and concentrated under reduced pressure to obtain [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]methanol (20 mg) as a colorless oil.

[0206] 4-Chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]methanol (20 mg, 0.061 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (15.31 mg, 0.061 mmol, 1 equivalent) were added to a 25 mL round-bottom flask at room temperature. DIEA (15.89 mg, 0.123 mmol, 2 equivalents) was added to the mixture at room temperature. The mixture was stirred at 90°C for 2 hours. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+10 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 35% B to 70% B for 20 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 65% B and concentrated under reduced pressure to obtain 4-chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (30 mg, 90.71%) as a colorless oil.

[0207] 4-Chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (30 mg) in DCM (5 mL), TFA (1 mL) was added at room temperature. The solution was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure. The crude product (30 mg) was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = unspecified, mobile phase B = unspecified, flow rate = 60 mL / min, gradient = 20% B to 40% B over 8 minutes, 220 nm, Rt = 7.22 min) to obtain 4-chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (8.7 mg) as a white solid.

[0208] Compounds 128, 125, and 114 were prepared by the method and scheme described in these examples, using 2-trifluoromethylphenol, 4-fluoro-2-trifluoromethylphenol, and 4-fluoro-2-chlorophenol, respectively, instead of 2-(difluoromethyl)-4-fluorophenol in the first step of synthesis.

[0209] Example 8 Synthesis of Compound 112

[0210] [ka]

[0211] tert-butyl 2-chloro-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred mixture prepared by dissolving tert-butyl 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (800 mg, 2.630 mmol, 1 equivalent) and 2-chloro-4-fluorophenol (578.16 mg, 3.945 mmol, 1.50 equivalents) in DMF (15 mL), K2CO3 (726.99 mg, 5.260 mmol, 2.00 equivalents) was added in small amounts at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 70°C for 0.5 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with ELISA (3 × 200 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / HCl (30 / 1 to 10 / 1) to obtain tert-butyl 2-chloro-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1 g, 91.78%) as a yellow oil.

[0212] tert-butyl 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate tert-Butyl 2-chloro-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (700 mg, 1.690 mmol, 1 equivalent) was dissolved in THF (30 mL), and 1-(4-methoxyphenyl)methanamine (1159.02 mg, 8.449 mmol, 5.00 equivalents) was added portionwise at room temperature under a nitrogen atmosphere to the stirred mixture. The resulting mixture was stirred at 60 °C for 16 h under a nitrogen atmosphere. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc (3 × 200 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = aqueous acetonitrile solution, gradient from 60% to 95% over 20 min, detector = UV 220 nm) to give tert-butyl 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (350 mg, 40.22%) as a yellow solid.

[0213] 4-(2-Chloro-4-fluorophenoxy)-N-[(4-methoxyphenyl)methyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-amine 350 mg (1 equivalent) of tert-butyl 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 10 mL of DCM and stirred. 1 mL of TFA was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with 3 × 100 mL of DCM. The combined organic layers were washed with brine (1 × 100 ml) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column = XBridge Shield RP18 OBD column, 5um, 19 × 150 mm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 25 mL / min, gradient = 2% B to 32% B in 1 minute, 220 / 254 nm, Rt = 7.08 min) to obtain 4-(2-chloro-4-fluorophenoxy)-N-[(4-methoxyphenyl)methyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-amine (260 mg) as a yellow solid.

[0214] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4-(2-chloro-4-fluorophenoxy)-N-[(4-methoxyphenyl)methyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-amine (260 mg, 0.627 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (156.11 mg, 0.627 mmol, 1.00 equivalent), and DIEA (242.99 mg, 1.880 mmol, 3.00 equivalent) were added to a 50 mL round-bottom flask at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 2 hours under a nitrogen atmosphere. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = aqueous acetonitrile solution, gradient from 50% to 85% over 25 minutes, detector = UV 220 nm) to obtain 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (350 mg, 89.00%) as a yellow solid.

[0215] 5-[2-amino-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-4-chloro-2,3-dihydropyridazine-3-one 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (200 mg) was dissolved in TFA (8 mL, 107.704 mmol, 328.23 equivalents) and mixed into a stirred solution. The final reaction mixture was irradiated with microwaves at 80°C for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with DCM (2 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 25% B to 40% B over 8 minutes, 220 nm, Rt = 7.35 min) to obtain 5-[2-amino-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-4-chloro-2,3-dihydropyridazine-3-one (52.4 mg) as a pale yellow solid.

[0216] Compounds 113, 116, and 102 were prepared by the method and scheme described in this example, using 2-chlorophenol, 4-fluoro-2-trifluoromethylphenol, and 2-trifluorophenol, respectively, instead of 2-chloro-4-fluorophenol in the first step of synthesis.

[0217] Example 9 Synthesis of Compounds 129 and 130

[0218] [ka]

[0219] 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]ethane-1-one A mixture of tert-butyl 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (600 mg, 1,340 mmol, 1 equivalent) and tributyl(1-ethoxyethenyl) stannane (967.80 g, 2,680 mmol, 2.00 equivalent) was added to toluene (10 mL). Pd(PPh3)4 (77.41 mg, 0.067 mmol, 0.05 equivalent) was added to this mixture under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 110 °C for 4 hours. The reaction was monitored by LC-MS. tert-butyl 2-(1-ethoxyethenyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (700 mg, 108.06%) was obtained as a yellow oily substance. The resulting crude mixture was used directly in the next step without further purification.

[0220] To a stirred solution of tert-butyl 2-(1-ethoxyethenyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1 g, 2.068 mmol, 1 equivalent) dissolved in DCM (5 mL), TFA (3.33 mL, 29.239 mmol, 21.70 equivalents) was added at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The mixture / residue was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 43% B to 55% B for 20 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 50% B and concentrated under reduced pressure to obtain 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]ethane-1-one (750 mg, 102.06%) as a pale yellow solid.

[0221] 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]ethane-1-one (750 mg, 2.111 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (525.81 mg, 2.111 mmol, 1.00 equivalent) were added to a 50 mL round-bottom flask at room temperature. DIEA (818.47 mg, 6.333 mmol, 3.00 equivalent) was added to the mixture. The resulting mixture was stirred at 100°C for 2 hours. The reaction was monitored by LC-MS. The mixture was allowed to cool to room temperature. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 60% B to 85% B for 20 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 80% B and concentrated under reduced pressure to obtain 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (230 mg, 19.18%) as a pale yellow oily substance.

[0222] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one To a 10 mL stirred solution of 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (230 mg, 0.405 mmol, 1 equivalent) in MeOH, NaBH4 (30.64 mg, 0.810 mmol, 2.00 equivalents) was added in small amounts at 0°C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / SiO=1:1) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (120 mg, 51.99%) as a pale yellow oily substance.

[0223] 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-2-[(1S)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one and 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-2-[(1R)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (120 mg, 0.211 mmol, 1 equivalent) was dissolved in DCM (5 mL) and stirred. TFA (2.00 mL, 17.541 mmol, 127.89 equivalents) was added dropwise at room temperature to this stirred solution. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The residue was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, gradient from 40% B to 80% B for 25 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 55% B and concentrated under reduced pressure. The crude product (50 mg) was purified by Chiral preparative HPLC under the following conditions (column = CHIRALPAK IE, 2 × 25 cm, 5 μm; mobile phase A = hexane (0.1% FA)-HPLC; mobile phase B = EtOH-HPLC; flow rate = 16 mL / min; gradient = 30 B to 30 B for 33 minutes, 220 / 254 nm; RT1 = 26.219, RT2 = 29.589). This method separated the two isomers, but their absolute orientation could not be determined. A compound designated as 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1S)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (27.1 mg) was obtained as a grayish-white solid at 29.589 min. A compound designated as 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1R)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one (22.6 mg) was obtained as a grayish-white solid at 26.219 min.

[0224] Compound 119 was prepared using tert-butyl 2-chloro-4-[4-fluoro-2-chlorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate as a starting material, according to the method and scheme described in this example.

[0225] Compounds 122 and 123 were prepared using tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate as a starting material, according to the method and scheme described in this example. Again, the absolute orientation of these separated isomers is not determined, and the designation of (S) or (R) is optional.

[0226] Example 10 Synthesis of Compound 115

[0227] [ka]

[0228] tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate 2 g, 13.15 mmol, 2 equivalents of DBU were added to a stirred solution prepared by dissolving tert-butyl 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (2 g, 6.58 mmol, 1 equivalent) and 2-(trifluoromethyl)phenol (1.6 g, 9.86 mmol, 1.5 equivalents) in acetonitrile (20 mL) at room temperature. The solution was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / siRNA = 10:1) to obtain tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (700 mg, 24.77%) as a colorless oil.

[0229] tert-butyl 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a solution of tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.163 mmol, 1 equivalent) in THF (15 mL), (2-aminoethoxy)(tert-butyl)dimethylsilane (1019.89 mg, 5.816 mmol, 5.00 equivalent) was added at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 50°C for 16 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / SiO₂=3 / 1) to obtain tert-butyl 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (440 mg, 66.51%) as a pale yellow oily substance.

[0230] 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]amino)ethane-1-ol To a stirred solution of tert-butyl 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (440 mg, 0.774 mmol, 1 equivalent) dissolved in DCM (10 mL), TFA (3 mL, 40.389 mmol, 52.20 equivalents) was added at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = ACN aqueous solution, gradient from 40% to 60% over 15 minutes, detector = UV254nm) to obtain 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]amino)ethane-1-ol (220 mg) as a pale yellow oily substance.

[0231] 4-Chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]amino)ethane-1-ol (220 mg, 0.621 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (154.66 mg, 0.621 mmol, 1.00 equivalent) were added to a 50 mL round-bottom flask at room temperature. DIEA (240.74 mg, 1.863 mmol, 3.00 equivalent) was added to the mixture. The resulting mixture was stirred at 100°C for 2 hours. The reaction was monitored by LC-MS. The mixture was allowed to cool to room temperature. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = ACN; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 45% B to 60% B for 20 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 55% B and concentrated under reduced pressure to obtain 4-chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (210 mg, 59.66%) as a yellow solid.

[0232] 4-Chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (200 mg, 0.353 mmol, 1 equivalent) was dissolved in DCM (5 mL) and a stirred solution was prepared. TFA (2 mL) was added to this solution at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = unspecified, mobile phase B = unspecified, flow rate = 60 mL / min, gradient = 25% B to 50% B over 8 minutes, 220 nm, Rt = 7.67 min) to obtain 4-chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (106.3 mg) as a white solid.

[0233] Example 11 Synthesis of Compounds 138 and 139

[0234] [ka]

[0235] 7-Benzyl-2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 4-Fluoro-2-(trifluoromethyl)phenol (1469.32 mg, 8.158 mmol, 1.20 equivalents) and 7-benzyl-2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (2000 mg, 6.799 mmol, 1 equivalent) were dissolved in DMF (20 mL) and stirred. K2CO3 (1879.20 mg, 13.597 mmol, 2 equivalents) was added at room temperature to the mixture. The solution was stirred at 70°C for 0.5 hours. The mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM TFA); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 70% B to 95% B for 20 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 95% B and concentrated under reduced pressure to obtain 7-benzyl-2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (2331 mg, 78.31%) as a grayish-white solid.

[0236] 7-Benzyl-4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-2-one 7-benzyl-2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (2 g, 4.568 mmol, 1 equivalent) was dissolved in HAc (10 mL, 174.515 mmol, 38.20 equivalents) and H2O (1 mL, 55.508 mmol, 12.15 equivalents). The solution was stirred at 140°C for 10 hours under an N2 atmosphere. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EA = 1 / 1) to obtain 7-benzyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-one (530 mg, 27.67%) as a pale yellow solid.

[0237] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-one 7-benzyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-one (530 mg, 1.264 mmol, 1 equivalent) was dissolved in MeOH (10 mL, 246.989 mmol, 195.44 equivalents), to which Pd / C (268.98 mg, 2.528 mmol, 2 equivalents) was added under a nitrogen atmosphere. The mixture was hydrogenated under a hydrogen atmosphere using a hydrogen balloon at room temperature for 4 hours, filtered through a Celite pad, and concentrated under reduced pressure. 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-one (430 mg, 103.34%) was obtained as a pale yellow solid.

[0238] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one A mixture of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-one (430 mg, 1.306 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (357.84 mg, 1.437 mmol, 1.1 equivalent)) added to DIEA (337.58 mg, 2.612 mmol, 2.00 equivalent) was stirred at 100°C for 2 hours under an N2 atmosphere. The residue was purified by preparative TLC (PE / EA=1 / 1) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (210 mg, 29.67%) as a pale yellow solid.

[0239] 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one and 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (90 mg, 0.166 mmol, 1 equivalent) and NaHCO3 (27.90 mg, 0.332 mmol, 2 equivalents) were dissolved in DMF (10 mL, 129.218 mmol, 778.02 equivalents). CH3I (47.15 mg, 0.332 mmol, 2.00 equivalents) was added dropwise at 0°C under a nitrogen atmosphere. The mixture was stirred at 25°C for 16 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EA=0 / 1) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg, 64.99%) and 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (15 mg) as pale yellow solids.

[0240] 4-Chloro-5-[4-[4-Fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (60 mg, 0.108 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 1457.41 equivalents), TFA (123.07 mg, 1.079 mmol, 10 equivalents) was added at 25°C. The resulting mixture was concentrated under reduced pressure. The crude product (100 mg) was purified by preparative HPLC under the following conditions (column: XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 40% B over 7 minutes, 220 nm, Rt = 6.63 min) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (29.3 mg, 57.54%) as a white solid.

[0241] 4-Chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (15 mg, 0.027 mmol, 1 equivalent) in DCM (5 mL, 78.650 mmol, 2914.83 equivalents), TFA (30.77 mg, 0.270 mmol, 10 equivalents) was added at 25°C. The resulting mixture was concentrated under reduced pressure. The crude product (20 mg) was purified by preparative HPLC under the following conditions (column: XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 40% B over 7 minutes, 220 nm, Rt = 6.63 min) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (7.5 mg, 58.91%) as a white solid.

[0242] Example 12 Synthesis of Compound 110

[0243] [ka]

[0244] tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate 2 g, 13.15 mmol, 2 equivalents of DBU were added to a stirred solution prepared by dissolving tert-butyl 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (2 g, 6.58 mmol, 1 equivalent) and 2-(trifluoromethyl)phenol (1.6 g, 9.86 mmol, 1.5 equivalents) in acetonitrile (20 mL) at room temperature. The solution was stirred at room temperature for 4 hours. The mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / siRNA = 10:1) to obtain tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (700 mg, 24.77%) as a colorless oil.

[0245] tert-butyl 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a solution of tert-butyl 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1 g, 2.327 mmol, 1 equivalent) in MeOH (20 mL, 493.978 mmol, 212.32 equivalents), NaOMe (0.25 g, 0.005 mmol, 2 equivalents) was added at 25°C. The mixture was stirred at 25°C for 4 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (10 / 1 to 1 / 1) to obtain tert-butyl 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (100 mg, 10.10%) as a pale yellow solid.

[0246] 2-Methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine To a 10 mL solution of tert-butyl 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (100 mg, 0.235 mmol, 1 equivalent) in DCM (10 mL), TFA (268.03 mg, 2.351 mmol, 10 equivalents) was added at 25°C. The solution was stirred at 25°C for 4 hours. The resulting mixture was concentrated under reduced pressure. The crude product (150 mg) was purified by preparative HPLC under the following conditions (column: XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 40% B over 7 minutes, 220 nm, Rt = 6.63 min) to obtain 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (80 mg, 104.62%) as a pale yellow solid.

[0247] 4-Chloro-5-[2-Methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one A solution of 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (80 mg, 0.246 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (61.26 mg, 0.246 mmol, 1 equivalent) in DIEA (63.57 mg, 0.492 mmol, 2.00 equivalents) was stirred at 100°C for 2 hours under a nitrogen atmosphere. The residue was purified by silica gel column chromatography and eluted with PE / EA (5 / 1 to 1 / 1) to obtain 4-chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (120 mg, 90.71%) as a pale yellow solid.

[0248] 4-Chloro-5-[2-Methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one To a solution of 4-chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (120 mg, 0.223 mmol, 1 equivalent) in DCM (5 mL, 78.650 mmol, 352.56 equivalents), TFA (254.36 mg, 2.231 mmol, 10.00 equivalents) was added at 25°C. The resulting mixture was concentrated under reduced pressure. The crude product (150 mg) was purified by preparative HPLC under the following conditions (column: XBridge Shield RP18 OBD column 30 × 150 mm, 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 40% B over 7 minutes, 220 nm, Rt = 6.63 min) to obtain 4-chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (24.1 mg, 23.81%) as a white solid.

[0249] Example 13 Synthesis of Compound 108

[0250] [ka]

[0251] tert-butyl 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.854 mmol, 1 equivalent) and 3-bromo-2-chlorophenol (461.46 mg, 2.224 mmol, 1.20 equivalents) in DMF (10 mL), K2CO3 (512.38 mg, 3.707 mmol, 2 equivalents) was added. The resulting mixture was stirred at 70°C for 1 hour. The mixture was purified by reverse-phase flash chromatography under the following conditions (spherical C18, 20-40 μm, 330 g, mobile phase A = water (5 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 80 mL / min, gradient = 20% B to 60% B over 55 minutes, 254 nm). The fraction containing the desired product was recovered with 40% B and concentrated under reduced pressure. This yielded tert-butyl 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (300 mg, 36.72%) as a grayish-white solid.

[0252] tert-butyl 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (450 mg, 1.021 mmol, 1 equivalent) and zinc dicarbonitride (143.87 mg, 1.225 mmol, 1.20 equivalents) in DMF (5 mL), Pd(PPh3)4 (117.99 mg, 0.102 mmol, 0.1 equivalent) was added. The resulting mixture was stirred under a nitrogen atmosphere at 120°C for 2 hours. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 180 g; mobile phase A = water (5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 10% B to 60% B over 55 minutes, = 254 nm). The fraction containing the desired product was recovered at 40% B and concentrated under reduced pressure. This yielded tert-butyl 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (280 mg, 70.89%) as a pale yellow solid.

[0253] 2-Chloro-3-[5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-4-yloxy]benzonitrile 1 mL of TFA was added to a stirred solution of tert-butyl 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (100 mg, 0.259 mmol, 1 equivalent) dissolved in 3 mL of DCM. The resulting mixture was stirred at room temperature under an air atmosphere for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 7 with saturated aqueous NH4HCO3. The mixture was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 180 g, mobile phase A = water (5 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 45 mL / min, gradient = gradient from 30% B to 60% B over 30 minutes, 254 nm). The fraction containing the desired product was recovered at 45% B and concentrated under reduced pressure. This yielded 2-chloro-3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yloxy]benzonitrile (60 mg, 80.95%) as a pale yellow oily substance.

[0254] 2-Chloro-3-([7-[5-Chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)benzonitrile A stirred solution was prepared by dissolving tert-butyl 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (60 mg, 0.155 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (38.63 mg, 0.155 mmol, 1.00 equivalent) in DIEA (40.09 mg, 0.310 mmol, 2 equivalents). The resulting mixture was stirred at 100°C for several hours under an air atmosphere. The residue was purified by preparative TLC (PE / siRNA=1:1) to obtain 2-chloro-3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)benzonitrile (50 mg, 64.56%) as a pale yellow solid.

[0255] 2-Chloro-3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazine-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy]benzonitrile 2-Chloro-3-([7-[5-Chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)benzonitrile (50 mg, 0.100 mmol, 1 equivalent) was dissolved in DCM (3 mL) and TFA (1 mL) was added to the stirred solution. The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 7 with saturated NH4CO3 aqueous solution. The crude product was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 20% B to 42% B over 8 minutes, 220 nm, Rt = 7.58 min) to obtain 2-chloro-3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy]benzonitrile (14.5 mg, 34.88%) as a grayish-white solid.

[0256] Example 14 Synthesis of Compound 111

[0257] [ka]

[0258] tert-butyl 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred mixture prepared by adding tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (180 mg, 0.667 mmol, 1 equivalent) and 3-bromo-2-(trifluoromethyl)phenol (241.25 mg, 1.001 mmol, 1.50 equivalents) to DMF (10 mL), Cs2CO3 (434.86 mg, 1.335 mmol, 2.00 equivalents) was added in small amounts at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 70°C for 0.5 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with RINKAN (3 × 200 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = aqueous acetonitrile solution, gradient from 40% to 85% over 30 minutes, detector = UV 220 nm) to obtain tert-butyl 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (150 mg, 47.39%) as a yellow oily substance.

[0259] tert-butyl 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred mixture prepared by adding tert-butyl 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (150 mg, 0.316 mmol, 1 equivalent) and Zn(CN)2 (111.43 mg, 0.949 mmol, 3.00 equivalents) to DMF (8 mL), Pd(PPh3)4 (36.55 mg, 0.032 mmol, 0.1 equivalent) was added in small amounts at room temperature under a nitrogen atmosphere. The final reaction mixture was irradiated with microwaves at 150 °C for 3 hours. The reaction was monitored by LC-MS. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = aqueous acetonitrile solution, gradient from 40% to 95% over 30 minutes, detector = UV 220 nm) to obtain tert-butyl 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (70 mg, 52.65%) as a yellow oily substance.

[0260] 3-[5H,6H,7H,8H-Pyrido[3,4-d]pyrimidine-4-yloxy]-2-(trifluoromethyl)benzonitrile 70 mg of tert-butyl 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 10 mL of dimethyl chlorine (DCM) and stirred. 1 mL of TFA was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with 3 × 100 mL of DCM. The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous sodium 2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = C18 silica gel, mobile phase = aqueous acetonitrile solution, gradient from 30% to 60% over 20 minutes, detector = UV 220 nm) to obtain 3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yloxy]-2-(trifluoromethyl)benzonitrile (40 mg) as a yellow oily substance.

[0261] 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)-2-(trifluoromethyl)benzonitrile 3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yloxy]-2-(trifluoromethyl)benzonitrile (40 mg, 0.125 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (62.22 mg, 0.250 mmol, 2.00 equivalent), and DIEA (48.42 mg, 0.375 mmol, 3.00 mmol, 3.00 equivalent) were added to a 25 mL round-bottom flask at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 16 hours under a nitrogen atmosphere. The residue was purified by preparative TLC (PE / siRNA=5 / 1) to obtain 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)-2-(trifluoromethyl)benzonitrile (50 mg, 75.12%) as a yellow oily substance.

[0262] 3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazine-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy]-2-(trifluoromethyl)benzonitrile 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy)-2-(trifluoromethyl)benzonitrile (50 mg) was dissolved in DCM (10 mL) and stirred. TFA (1 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column = XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 25% B to 45% B over 8 minutes, 220 nm, Rt = 7.07 min) to obtain 3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]oxy]-2-(trifluoromethyl)benzonitrile (10.8 mg) as a white solid.

[0263] Example 15 Synthesis of Compounds 126 and 126a

[0264] [ka]

[0265] N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylidene]-4-methylbenzene-1-sulfonohydrazide 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethane-1-one (2 g, 9.702 mmol, 1 equivalent) was dissolved in EtOH (40 mL) and stirred. To this stirred solution, 4-methylbenzene-1-sulfonohydrazide (1.81 g, 9.719 mmol, 1.00 equivalent) was added in small amounts at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 6 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM AcOH); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 45% B to 70% B for 20 minutes; detector = 220 nm). The fraction containing the desired product was recovered with 60% B and concentrated under reduced pressure to obtain N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylidene]-4-methylbenzene-1-sulfonohydrazide (2.5 g, 68.83%) as a white solid.

[0266] tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethenyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred mixture prepared by adding tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (750 mg, 2.781 mmol, 1 equivalent) and N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylidene]-4-methylbenzene-1-sulfonohydrazide (2081.80 mg, 5.561 mmol, 2.00 equivalents) to 1,4-dioxane (20 mL), Pd(acetonitrile) 2Cl2 (72.14 mg, 0.278 mmol, 0.10 equivalents), Dppf (307.18 mg, 0.556 mmol, 0.2 equivalents), and t-BuOLi (489.71 mg, 6.117 mmol, 2.20 equivalents) to a nitrogen atmosphere at room temperature, small amounts were added. The final reaction mixture was irradiated with microwaves at 100°C for 2 hours. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with ELISA (2 × 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM AcOH); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B over 10 minutes, then 50% B to 90% B over 30 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 85% B and concentrated under reduced pressure to obtain tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethenyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (800 mg, 67.95%) as a brown oily substance.

[0267] tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate In a 100 mL round-bottom flask, 150 mg of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethenyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was added to a 30 mL MeOH solution under a nitrogen atmosphere with Pd / C (10%, 30 mg). The mixture was hydrogenated at room temperature for 4 hours under a hydrogen atmosphere using a hydrogen balloon, filtered through a Celite pad, and concentrated under reduced pressure. This yielded 150 mg of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate as a yellow oily substance.

[0268] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 150 mg of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 10 mL of DCM and stirred. 1 mL of TFA was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with 3 × 50 mL of DCM. The combined organic layers were washed with brine (1 × 30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM AcOH); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% to 5% B for 10 minutes, then 40% B to 58% B for 15 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 53% B and concentrated under reduced pressure to obtain 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (100 mg) as a yellow oily substance.

[0269] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (100 mg, 0.307 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (91.88 mg, 0.369 mmol, 1.20 equivalents), and DIEA (119.19 mg, 0.922 mmol, 3.00 equivalents) were added to a 50 mL round-bottom flask at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM AcOH); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% to 5% B for 10 minutes, then 40% B to 60% B for 15 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 53% B and concentrated under reduced pressure to obtain 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (120 mg, 72.57%) as a yellow oily substance.

[0270] 4-Chloro-5-[4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one and 4-Chloro-5-[4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazin-3-one 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (200 mg) was dissolved in DCM (10 mL) and stirred. TFA (1 mL) was added dropwise at room temperature to this stirred solution. The reaction mixture was stirred at room temperature for 4 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with DCM (3 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by chiral preparative HPLC under the following conditions (column = XBridge preparative phenyl OBD column 19 × 150 mm 5 μm 13 nm, mobile phase A: mobile phase B: flow rate = 60 mL / min, gradient = 20% B to 37% B over 8 minutes, 220 nm, Rt = 7.97 min). This method separated the two isomers, but the absolute orientation could not be determined. The compound specified as 4-chloro-5-[4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (11.8 mg) was obtained as a grayish-white solid at 1.819 min. A compound designated as 4-chloro-5-[4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (13.5 mg) was obtained as a white solid in 2.470 minutes.

[0271] Example 16 Synthesis of Compound 133

[0272] [ka]

[0273] tert-butyl 4-[methyl[(3R,4R)-4-methylpiperidine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (3R,4R)-1-benzyl-N,4-dimethylpiperidine-3-amine (2.43 g, 0.011 mmol, 1.50 equivalents) and tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (2 g, 0.007 mmol, 1 equivalent) were added to a 25 mL round-bottom flask at room temperature. DIEA (1.92 g, 0.015 mmol, 2.00 equivalents) was added to the mixture at room temperature. The mixture was stirred at 100°C for 2 hours. The residue was purified by preparative TLC (PE / SiO=1:1) to obtain tert-butyl 4-[methyl[(3R,4R)-4-methylpiperidine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (670 mg, 25.00%) as a grayish-white solid.

[0274] (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]piperidine-3-amine 413 mg, 0.914 mmol, 1 equivalent of tert-butyl 4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (10 mL) was dissolved in 10 mL of DCM and stirred. Trifluoroacetic acid (3 mL, 0.026 mmol, 6.00 equivalents) was added dropwise at 0°C. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The solution was concentrated under reduced pressure. The crude product (362 mg) was purified by preparative HPLC under the following conditions (column = XBridge Shield RP18 OBD column 5 μm, 19 × 150 mm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 80 mL / min, gradient = 30% B to 80% B over 25 minutes, 220 nm, Rt = 21.65 min) to obtain (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]piperidine-3-amine (250 mg, 77.77%) as a red oily substance.

[0275] 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]piperidine-3-amine (263 mg, 0.748 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (186.38 mg, 0.748 mmol, 1.00 equivalent) were added to a 25 mL round-bottom flask at room temperature. DIEA (193.41 mg, 1.261 mmol, 2 equivalents) was added to the mixture at room temperature. The mixture was stirred at 100°C for 2 hours. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 45% B to 95% B for 30 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 85% B and concentrated under reduced pressure to obtain 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (245 mg, 58.04%) as a grayish-white solid.

[0276] 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-4-chloro-2,3-dihydropyridazine-3-one 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (88 mg, 1 equivalent) was dissolved in 10 mL of DCM and stirred. Trifluoroacetic acid (3 mL, 0.026 mmol, 6.00 equivalent) was added dropwise at 0°C. The mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM TFA); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 33% B to 95% B for 30 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 90% B and concentrated under reduced pressure to obtain 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidine-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-4-chloro-2,3-dihydropyridazine-3-one (33.5 mg, 44.74%) as a grayish-white solid.

[0277] Compound 133a was prepared by the method and scheme of this example, using (3S,4S)-1-benzyl-N,4-dimethylpiperidine-3-amine instead of (3R,4R)-1-benzyl-N,4-dimethylpiperidine-3-amine.

[0278] Example 17 Synthesis of Compound 136

[0279] [ka]

[0280] tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate A mixture of 4-fluoro-2-(trifluoromethyl)aniline (6.64 g, 37.074 mmol, 2 equivalents), tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (5 g, 18.537 mmol, 1 equivalent), Pd(AcO)2 (0.83 g, 3.707 mmol, 0.2 equivalents), xanthophos (4.29 g, 7.415 mmol, 0.4 equivalents), and Cs2CO3 (12.08 g, 37.074 mmol, 2 equivalents) added to 1,4-dioxane (80 mL) was stirred at 110 °C for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated to obtain the crude product, which was purified by silica gel column chromatography and eluted with PE:EA (20:1 to 1:2) to obtain tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (5.6 g, 73.26%) as a white solid.

[0281] tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (3 g, 7.275 mmol, 1 equivalent) and methyl 2-bromoacetate (2.23 mg, 14.578 mmol, 2.00 equivalent) were added to DMF (30 mL) and a stirred mixture was then gradually added Cs2CO3 (4.74 g, 14.548 mmol, 2.00 equivalent) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was extracted with RINKAN (3 × 400 mL). The combined organic layers were washed with brine (2 × 200 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM TFA); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 55% B to 85% B for 30 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 79% B and concentrated under reduced pressure to obtain tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 14.19%) as a yellow solid.

[0282] tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution of tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.032 mmol, 1 equivalent) dissolved in THF (50 mL), LiAlH4 (78.34 mg, 2.064 mmol, 2.00 equivalent) was added in small amounts at -30°C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 hours. The reaction was monitored by LC-MS. The reaction was stopped by adding water (1 mL) at -30°C. The precipitated solid was collected by filtration and washed with MeOH (3 × 30 mL). The resulting mixture was concentrated under vacuum. The residue was purified by preparative TLC (PE / SiO=1 / 1) to obtain tert-butyl 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (100 mg, 21.23%) as a yellow oily substance.

[0283] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 150 mg of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 10 mL of DCM and stirred. 1 mL of TFA was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with 3 × 50 mL of DCM. The combined organic layers were washed with brine (1 × 30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 40% B to 58% B for 15 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 53% B and concentrated under reduced pressure to obtain 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (100 mg) as a yellow oil.

[0284] 4-Chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 2-[[4-fluoro-2-(trifluoromethyl)phenyl]([5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl])amino]ethane-1-ol (40 mg, 0.112 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (55.92 mg, 0.224 mmol, 2.00 equivalent), and DIEA (43.53 mg, 0.337 mmol, 3.00 equivalent) were added to a 50 mL round-bottom flask at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% to 5% B for 10 minutes, then 40% B to 60% B for 15 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 55% B and concentrated under reduced pressure to obtain 4-chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (50 mg, 78.28%) as a yellow oily substance.

[0285] 4-Chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one 4-Chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (50 mg) was dissolved in DCM (10 mL) and stirred. TFA (1 mL) was added dropwise at room temperature to this stirred solution. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with ELISA (2 × 50 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = unspecified, mobile phase B = unspecified, flow rate = 60 mL / min, gradient = 30% B to 45% B over 8 minutes, 220 nm, Rt = 7.6 min) to obtain 4-chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one (6.2 mg) as a white solid.

[0286] Example 18 Synthesis of Compound 132

[0287] [ka]

[0288] tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution of t-BuONa (226.97 mg, 2.362 mmol, 2.00 equivalent) dissolved in DMSO (20 mL), Me3SiI (472.57 mg, 2.362 mmol, 2.00 equivalent) was added in small amounts at 40°C under a nitrogen atmosphere. The resulting mixture was stirred at 40°C under a nitrogen atmosphere for 0.5 hours. Next, a solution of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethenyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.181 mmol, 1 equivalent) in DMSO (5 mL) was added dropwise at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 hour. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was extracted with RINKAN (3 × 100 mL). The combined organic layers were washed with brine (2 × 100 ml) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH₄HCO₃); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 55% B to 80% B for 25 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 73% B and concentrated under reduced pressure to obtain tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (240 mg, 46.46%) as a yellow oil.

[0289] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 240 mg, 0.549 mmol, 1 equivalent of tert-butyl 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (10 mL) was dissolved in 10 mL of DCM and stirred. TFA (1 mL, 13.463 mmol, 24.54 equivalents) was added dropwise at room temperature to this stirred solution. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with ELISA (2 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM AcOH); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% to 5% B for 10 minutes, then 33% B to 45% B for 20 minutes; detector = 254 nm). The fraction containing the desired product was recovered at 40% B and concentrated under reduced pressure to obtain 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (150 mg, 81.05%) as a yellow oily substance.

[0290] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (150 mg, 0.445 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-1,2,3,6-tetrahydropyridazin-3-one (134.00 mg, 0.534 mmol, 1.20 equivalents), and DIEA (172.42 mg, 1.334 mmol, 3.00 equivalents) were added to a 50 mL round-bottom flask at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% to 5% B for 10 minutes, then 40% B to 60% B for 15 minutes; detector = 220 nm). The fraction containing the desired product was recovered at 54% B and concentrated under reduced pressure to obtain 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (200 mg, 81.78%) as a yellow oil.

[0291] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (200 mg) was dissolved in DCM (10 mL) and stirred. TFA (2 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NH4HCO3 aqueous solution. The resulting mixture was extracted with ELISA (2 × 100 mL). The combined organic layers were washed with brine (1 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC under the following conditions (column: XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = unspecified, mobile phase B = unspecified, flow rate = 60 mL / min, gradient = 30% B to 55% B over 8 minutes, 220 nm, Rt = 7.232 min) to obtain 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one (39.2 mg) as a grayish-white solid.

[0292] Example 19 Synthesis of Compound 109

[0293] [ka]

[0294] tert-butyl 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution prepared by dissolving tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.854 mmol, 1 equivalent) and 2-bromo-3-fluorophenol (424.87 mg, 2.224 mmol, 1.20 equivalents) in DMF (10 mL), K2CO3 (512.38 mg, 3.707 mmol, 2 equivalents) was added. The resulting mixture was stirred at 70°C for 0.5 hours. The mixture was cooled to room temperature. The reaction was stopped with water at room temperature. The resulting mixture was extracted with RINKAN (2 × 100 mL). The combined organic layers were washed with brine (2 × 100 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / RINKAN (5:1) to obtain tert-butyl 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 63.58%) as a white solid.

[0295] tert-butyl 4-(2-ethenyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a solution prepared by dissolving tert-butyl 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.178 mmol, 1 equivalent) and pentamethyl-1,3,2-dioxaborolane (334.72 mg, 2.357 mmol, 2.00 equivalent) in H2O (2 mL) and 1,4-dioxane (16 mL), K2CO3 (325.75 mg, 2.357 mmol, 2 equivalents) and Pd(PPh3)4 (68.09 mg, 0.059 mmol, 0.05 equivalent) were added. After stirring overnight at 90°C under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / siRNA (5:1) to obtain tert-butyl 4-(2-ethenyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (250 mg, 57.12%) as a yellow oily substance.

[0296] tert-butyl 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred solution of tert-butyl 4-(2-ethenyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (250 mg, 0.673 mmol, 1 equivalent) dissolved in MeOH (10 mL), Pd / C (100 mg, 0.940 mmol, 1.40 equivalents) was added. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 2 hours. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 10 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. This yielded tert-butyl 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (210 mg, 0.08%) as a black oily substance.

[0297] 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine 210 mg, 0.562 mmol, 1 equivalent of tert-butyl 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 3 mL of DCM and stirred. 1 mL of TFA was added to this stirred solution. The resulting mixture was stirred at room temperature under an air atmosphere for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 8 with saturated aqueous NH4HCO3. The mixture was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 180 g; mobile phase A = water (5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 25% B to 60% B over 40 minutes, 254 nm). The fraction containing the desired product was recovered at 40% B and concentrated under reduced pressure. This yielded 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (120 mg, 78.07%) as a pale yellow oily substance.

[0298] 4-Chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (120 mg, 0.439 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (109.37 mg, 0.439 mmol, 1.00 equivalent) were dissolved in DIEA (113.49 mg, 0.878 mmol, 2 equivalents) and stirred. The resulting mixture was stirred at 100°C for 2 hours under an air atmosphere. The residue was purified by preparative TLC (PE / siRNA=1:1) to obtain 4-chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 46.87%) as a pale yellow solid.

[0299] 4-Chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one 4-Chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 0.206 mmol, 1 equivalent) was dissolved in DCM (3 mL) and a stirred solution was added to it with TFA (1 mL). The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 7 with saturated NH4HCO3 aqueous solution. The crude product was purified by preparative HPLC under the following conditions (column = XBridge Prep OBD C18 column 30 × 150 mm 5 μm, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 30% B to 50% B over 8 minutes, 220 nm, Rt = 7.27 min) to obtain 4-chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2,3-dihydropyridazine-3-one (41.6 mg, 50.31%) as a white solid.

[0300] Example 20 Synthesis of Compound 127

[0301] [ka]

[0302] Methyl 3-(methylamino)pyridine-4-carboxylate To a solution of 3-(methylamino)pyridine-4-carboxylic acid (11 g, 72.296 mmol, 1 equivalent) in MeOH (500 mL, 12349.455 mmol, 170.82 equivalents), SOCl2 (43.01 g, 361.478 mmol, 5 equivalents) was added dropwise at 0°C. The resulting mixture was stirred at 70°C for 30 hours. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL). The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was extracted with siRNA (2 × 20 mL). The combined organic layers were washed with brine (1 × 30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to obtain methyl 3-(methylamino)pyridine-4-carboxylate (9 g, crude product) as a yellow solid.

[0303] Methyl 3-(N-methylacetamide)pyridine-4-carboxylate Methyl 3-(methylamino)pyridine-4-carboxylate (9 g, 54.158 mmol, 1 equivalent) was dissolved in 100 mL of DCM and stirred. Pyridine (21.42 g, 270.791 mmol, 5 equivalents) and acetyl chloride (6.38 g, 81.237 mmol, 1.5 equivalents) were added dropwise at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The solution was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flushing under the following conditions (column = spherical C18 column 330 g, mobile phase A = water (10 mM NH4HCO3), mobile phase B = acetonitrile, flow rate = 80 mL / min, gradient = gradient from 10%B to 30%B over 25 minutes, 254 / 220 nm) to obtain methyl 3-(N-methylacetamido)pyridine-4-carboxylate (8 g, 70.94%) as a brown liquid.

[0304] 4-Hydroxy-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one Methyl 3-(N-methylacetamido)pyridine-4-carboxylate (6 g, 28.816 mmol, 1 equivalent) was dissolved in anhydrous 1,4-dioxane (100 mL). To this stirred solution, t-BuOK (6.47 g, 57.632 mmol, 2 equivalents) was added at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90°C for 1 hour under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (10:1) to obtain 4-hydroxy-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one (4.5 g, 88.64%) as an orange solid.

[0305] 4-Chloro-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one 4-hydroxy-1-methyl-1,2-dihydro-1,7-naphthirizin-2-one (4.5 g, 25.543 mmol, 1 equivalent) was dissolved in 100 mL of anhydrous 1,4-dioxane and stirred. POCl3 (3.92 g, 25.543 mmol, 1 equivalent) was added to this stirred solution. The resulting mixture was stirred at 90°C for 16 hours. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with DCM / MeOH (10:1) to obtain 4-chloro-1-methyl-1,2-dihydro-1,7-naphthirizin-2-one (2 g, 40.23%) as a red solid.

[0306] 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one To a stirred solution prepared by dissolving 4-chloro-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one (0.8 g, 4.111 mmol, 1 equivalent) in anhydrous 1,4-dioxane (15 mL), Cs2CO3 (2.68 g, 8.221 mmol, 2 equivalents), 4-fluoro-2-(trifluoromethyl)aniline (1.47 g, 8.221 mmol, 2.00 equivalents), xanthophos (0.95 g, 1.644 mmol, 0.4 equivalents), and Pd(AcO)2 (0.18 g, 0.822 mmol, 0.2 equivalents) were added under a nitrogen atmosphere at room temperature. The final reaction mixture was irradiated with microwaves at 110 °C for 4 hours. The reaction was monitored by LC-MS. The resulting mixture was extracted with SiO2 (3 × 50 mL). The combined organic layers were washed with brine (1 × 100 ml) and dried on anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flush under the following conditions (column = spherical C18 column 330 g, mobile phase A = water (10 mM AcOH), mobile phase B = acetonitrile, flow rate = 50 mL / min, gradient = gradient from 20% B to 40% B over 40 minutes, 254 / 220 nm) to obtain 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthyridine-2-one (1.1 g, 79.34%) as a grayish-white solid.

[0307] 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthyrizin-2-one (1 g, 2.965 mmol, 1 equivalent) was dissolved in THF (20 mL) and a stirred solution was added to it under a nitrogen atmosphere at room temperature with PtO2 (67.33 mg, 0.296 mmol, 0.10 equivalent). The resulting mixture was stirred under a hydrogen atmosphere at room temperature for 16 hours. The reaction was monitored by LC-MS. The resulting mixture was filtered, and the filter cake was washed with ELISA (3 × 20 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flush under the following conditions (column = C18 column 330 g, mobile phase A = water (10 mM AcOH), mobile phase B = acetonitrile, flow rate = 80 mL / min, gradient = gradient from 5%B to 20%B over 40 minutes, 254 / 220 nm). The fraction containing the desired product was recovered at 16% and concentrated under reduced pressure to obtain 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyrizin-2-one (750 mg, 74.11%) as a grayish-white solid.

[0308] 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one (750 mg, 2.197 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (1.09 g, 4.395 mmol, 2 equivalents) were stirred together, and DIPEA (568.00 mg, 4.395 mmol, 2 iv) was added at room temperature. The resulting mixture was stirred at 100°C for 2 hours. The reaction was monitored by LC-MS. The residue was then dissolved in DMF (10 mL). The solution was purified by reverse-phase flash under the following conditions (column = C18 column 330 g, mobile phase A = water (10 mM FA), mobile phase B = acetonitrile, flow rate = 80 mL / min, gradient = gradient from 30% B to 50% B over 40 minutes, 254 / 220 nm). The fraction containing the desired product was recovered at 44% B and concentrated under reduced pressure to obtain 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one (1 g, 82.15%) as a yellow oily substance.

[0309] 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazine-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl](methyl)amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyrizin-2-one (800 mg, 1.444 mmol, 1 equivalent) was dissolved in DMF (20 mL) and a stirred solution was prepared. Cs2CO3 (0.94 g, 2.888 mmol, 2 equivalents) and MeI (614.96 mg, 4.333 mmol, 3 equivalents) were added at room temperature. The resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by LC-MS. The mixture was purified by reverse-phase flush under the following conditions (column = C18 column 120 g, mobile phase A = water (10 mM AcOH), mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = gradient from 40% B to 60% B over 40 minutes, 254 / 220 nm). The fraction containing the desired product was recovered at 49% B and concentrated under reduced pressure to obtain 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl](methyl)amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyrizin-2-one (80 mg, 9.75%) as a yellow oily substance.

[0310] 7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-4-[[4-fluoro-2-(trifluoromethyl)phenyl](methyl)amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyridine-2-one 7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-4-[[4-fluoro-2-(trifluoromethyl)phenyl](methyl)amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyrizin-2-one (80 mg, 0.141 mmol, 1 equivalent) was dissolved in DCM (4.5 mL) and stirred. TFA (0.5 mL, 6.732 mmol, 31.07 equivalents) was added dropwise at room temperature to this stirred solution. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The residue was basicized to pH 8 with saturated NaHCO3 aqueous solution. The solution was purified by reverse-phase flushing to obtain 7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-4-[[4-fluoro-2-(trifluoromethyl)phenyl](methyl)amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthyrizin-2-one (40 mg, 58.69%) as a white solid.

[0311] Example 21 Synthesis of Compounds 135 and 137

[0312] [ka]

[0313] tert-butyl 2-chloro-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylate To a solution of tert-butyl 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (5 g, 16.44 mmol) in DMF (50 mL), 4-fluoro-2-(trifluoromethyl)phenol (4.44 g, 24.66 mmol) and K2CO3 (3.41 g, 24.66 mmol) were added at room temperature. The resulting mixture was stirred at 70°C for 1 hour. After cooling to room temperature, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 330 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 80 mL / min; gradient = 5% B over 10 minutes, gradient from 35% B to 45% B over 10 minutes; detector = 254 nm / 220 nm). The fraction containing the desired product was recovered at 44% B and concentrated under reduced pressure to obtain tert-butyl 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (6.2 g, 85%) as a white solid.

[0314] tert-butyl 4-(4-fluoro-2-(trifluoromethyl)phenoxy)-2-vinyl-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylate To a solution of tert-butyl 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.12 mmol) in dioxane (10 mL), 2-ethenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (344 mg, 2.23 mmol), H2O (0.5 mL, 27.75 mmol), K2CO3 (309 mg, 2.23 mmol), and Pd(PPh3)4 (129 mg, 0.11 mmol) were added. After stirring at 95°C for 2 hours under a nitrogen atmosphere, the resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC and eluted in petroleum ether with 17% ethyl acetate to obtain tert-butyl 2-ethenyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (490 mg, 99%) as a pale yellow solid.

[0315] tert-butyl 2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylate To a 20 mL solution of tert-butyl 2-ethenyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (400 mg, 0.91 mmol) in DCM, 4-hydroxy-4-methylmorpholine-4-nium (323 mg, 2.73 mmol) and K2OsO4.2H2O (34 mg, 0.091 mmol) were added at room temperature. After stirring for a further hour, the resulting mixture was concentrated under reduced pressure, and the residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% B over 10 minutes, 45% B to 65% B over 15 minutes; detector = 254 nm and 220 nm). The fraction containing the desired product was recovered at 64% B and concentrated under reduced pressure to obtain tert-butyl 2-(1,2-dihydroxyethyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (280 mg, 65%) as a white solid.

[0316] 1-(4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine-2-yl)ethane-1,2-diol 280 mg, 0.59 mmol of tert-butyl 2-(1,2-dihydroxetyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate was dissolved in 4 mL of dichloride (DCM) and stirred. 1 mL of tfa (TFA) was added to this mixture at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was concentrated under vacuum. The residue was dissolved in 50 mL of DCM and washed with 20 mL of saturated aqueous NaHCO3 solution. The organic layer was separated and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by preparative TLC in dichloromethane with 8% methanol to obtain 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2-yl]ethane-1,2-diol (180 mg, 82%) as a brown solid.

[0317] 4-Chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)-2-(tetrahydro-2H-pyran-2-yl)pyridazine-3(2H)-one 180 mg, 0.71 mmol of 2-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yloxy]benzaldehyde (180 mg, 0.71 mmol) was dissolved in DIEA (0.5 mL) and stirred. 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (176 mg, 0.71 mmol) was added at room temperature to this mixture. The resulting mixture was stirred at 90°C for 1 hour. After cooling to ambient temperature, the resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC, and eluted in dichloromethane with 8% methanol to obtain 4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one (140 mg, 43%) as a brown solid.

[0318] (S)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)pyridazin-3(2H)-one and (R)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)pyridazin-3(2H)-one 4-Chloro-5-[2-(1,2-dihydroxyethyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (150 mg, 0.27 mmol) was dissolved in DCM (4 mL) and TFA (1 mL) was added at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The resulting mixture was concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography under the following conditions (column = spherical C18, 20-40 μm, 120 g; mobile phase A = water (+ 5 mM NH4HCO3); mobile phase B = acetonitrile; flow rate = 45 mL / min; gradient = 5% B over 10 minutes, gradient from 45% B to 65% B over 15 minutes; detector = 254 nm and 220 nm). The fraction containing the desired product was recovered at 64% B and concentrated under reduced pressure to obtain a racemic product (130 mg), which was separated by preparative chiral HPLC under the following conditions (column = XBridge Prep OBD C18 column 30 × 150 mm, 5 μm; mobile phase A = hexane; mobile phase B = EtOH; flow rate = 20 mL / min; gradient = 35% B over 10 minutes; detector = 254 / 220 nm). Although the two isomers were separated by this method, their absolute orientation could not be determined. The fraction containing the desired product was recovered and concentrated under reduced pressure to obtain the following products: a compound designated as (S)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)pyridazin-3(2H)-one (retention time (4.97 min) (49.5 mg, 39%)) as a white solid, and a compound designated as (R)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-yl)pyridazin-3(2H)-one (retention time (8.05 min) (45.7 mg, 36%)) as a white solid.

[0319] Example 22 Synthesis of Compound 131

[0320] [ka]

[0321] tert-butyl 4-[[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate To a stirred mixture prepared by adding tert-butyl 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (500 mg, 1.854 mmol, 1 equivalent) and 4-(trifluoromethyl)pyridine-3-amine (601.03 mg, 3.707 mmol, 2.0 equivalents) to 1,4-dioxane (5 mL), Pd(AcO)2 (83.24 mg, 0.371 mmol, 0.2 equivalents), Cs2CO3 (1207.95 mg, 3.707 mmol, 2.0 equivalents), and xanthophos (429.04 mg, 0.741 mmol, 0.4 equivalents) to a nitrogen atmosphere at room temperature, Pd(AcO)2 (83.24 mg, 0.371 mmol, 0.2 equivalents), Cs2CO3 (1207.95 mg, 3.707 mmol, 2.0 equivalents), and xanthophos (429.04 mg, 0.741 mmol, 0.4 equivalents) were added. The resulting mixture was stirred at 110°C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with DCM (3 × 20 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The crude product was purified by reverse-phase flash under the following conditions (column = C18, 120 g, mobile phase A = water / 0.05% NH4HCO3, mobile phase B = ACN, flow rate = 45 mL / min, gradient = 45% B to 65% B over 15 minutes, detector = 254 nm and 220 nm, desired product recovered at 64% B) to obtain tert-butyl 4-[[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (600 mg, 81.86%) as a white solid.

[0322] tert-butyl 4-[methyl[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate tert-butyl 4-[[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1.32 g, 3.339 mmol, 1 equivalent) and Cs2CO3 (2.18 g, 6.677 mmol, 2.0 equivalents) were added to DMF (10 mL) and stirred. CH3I (0.95 g, 6.677 mmol, 2.0 equivalents) was added under a nitrogen atmosphere at 0°C. The resulting mixture was stirred under a nitrogen atmosphere at room temperature for 2 hours. The reaction was monitored by LC-MS. The crude product was purified by reverse-phase flash under the following conditions (column = C18, 120 g, mobile phase A = water / 0.05% NH4HCO3, mobile phase B = ACN, flow rate = 45 mL / min, gradient = 45% B to 65% B over 15 minutes, detector = 254 nm and 220 nm, desired product recovered at 64% B) to obtain tert-butyl 4-[methyl[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (400 mg, 29.26%) as a brown solid.

[0323] N-methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]-4-(trifluoromethyl)pyridine-3-amine 220 mg, 0.537 mmol, 1 equivalent of tert-butyl 4-[methyl[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylate (1 equivalent) was dissolved in 4 mL of DCM and stirred. 1 mL of TFA was added to this stirred solution at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH = 12:1) to obtain N-methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]-4-(trifluoromethyl)pyridine-3-amine (130 mg, 78.22%) as a brown solid.

[0324] 4-Chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one N-methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-4-yl]-4-(trifluoromethyl)pyridine-3-amine (130 mg, 0.420 mmol, 1 equivalent) was dissolved in DIEA (0.5 mg) and stirred. To this stirred solution, 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (104.69 mg, 0.420 mmol, 1.0 equivalent) was added at room temperature. The resulting mixture was stirred at 90°C for 1 hour. The reaction was monitored by LC-MS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH = 12:1) to obtain 4-chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 45.58%) as a brown solid.

[0325] 4-Chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one 4-Chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazine-3-one (100 mg, 0.192 mmol, 1 equivalent) was dissolved in DCM (4 mL) and TFA (1 mL) was added to the stirred solution at room temperature. The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LC-MS. The resulting mixture was concentrated under reduced pressure. The mixture was basicized to pH 8 with saturated NaHCO3 aqueous solution. The resulting mixture was concentrated under reduced pressure. The crude product (100 mg) was purified by preparative HPLC under the following conditions (column: XBridge Prep Phenyl OBD column 19 × 150 mm, 5 μm, mobile phase A = water, 5 mM NH4HCO3, mobile phase B = acetonitrile, flow rate = 60 mL / min, gradient = 35% B to 55% B over 8 minutes, 220 nm, Rt = 7.13 min) to obtain 4-chloro-5-(4-[methyl[4-(trifluoromethyl)pyridine-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-yl)-2,3-dihydropyridazine-3-one (52 mg, 61.99%) as a white solid.

[0326] Example 23 Synthesis of the intermediate A. 2-(difluoromethyl)-4-fluorophenyl acetate

[0327] [ka]

[0328] 4-fluoro-2-formylphenyl acetate To a solution of 5-fluoro-2-hydroxybenzaldehyde (10 g, 71.371 mmol, 1 equivalent) in pyridine (100 mL, 1242.353 mmol, 17.41 equivalents), acetyl acetate (14.57 g, 0.143 mmol, 2 equivalents) was added at 25°C. The solution was stirred at 25°C for 30 minutes. The resulting solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (100 / 1 to 20 / 1) to obtain 4-fluoro-2-formylphenyl acetate (12 g, 92.31%) as a pale yellow oil.

[0329] 2-(difluoromethyl)-4-fluorophenyl acetate To a solution of 4-fluoro-2-formylphenyl acetate (12 g, 65.880 mmol, 1 equivalent) in DCM (200 mL, 3146.009 mmol, 47.75 equivalents), DAST (21.24 g, 131.760 mmol, 2 equivalents) was added at 0°C. The solution was stirred at 25°C for 4 hours. The reaction of the resulting solution was stopped with water (100 mL). The resulting mixture was extracted with DCM (100 mL x 2). The combined organic layers were washed with saturated NaCl aqueous solution (100 mL x 2) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA (10 / 1 to 5 / 1) to obtain 2-(difluoromethyl)-4-formylphenyl acetate (10 g, 74.35%) as a pale yellow oil.

[0330] B. 2-(difluoromethyl)-4-fluorophenol

[0331] [ka]

[0332] 1-Bromo-2-(difluoromethyl)-4-fluorobenzene 2-bromo-5-fluorobenzaldehyde (10 g, 49.26 mmol, 1 equivalent) was dissolved in DCM (60 mL) and stirred. DAST (15.9 g, 98.52 mmol, 2 equivalents) was added to the mixture. The resulting mixture was stirred at -10°C for 2 hours. The reaction was stopped with water at -10°C. The resulting mixture was extracted with siRNA (4 × 30 mL). The combined organic layers were washed with brine (2 × 40 mL) and dried over anhydrous sodium 2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / siRNA (6:1) to obtain 1-bromo-2-(difluoromethyl)-4-fluorobenzene (8 g, 72.18%) as a pale yellow oil.

[0333] 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane To a solution of 1-bromo-2-(difluoromethyl)-4-fluorobenzene (31 g, 137.773 mmol, 1 equivalent) and BPD (52.48 g, 206.664 mmol, 1.50 equivalents) in 1,4-dioxane (300 mL, 3541.225 mmol, 25.70 equivalents), AcOK (27.04 g, 275.546 mmol, 2 equivalents) and Pd(dppf)Cl2·CH2Cl2 (5.63 g, 6.889 mmol, 0.05 equivalents) were added under a nitrogen atmosphere at 25°C. The mixture was stirred at 90°C for 2 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EA(10 / 1) to obtain 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (30 g, 80.03%) as a pale yellow oil. The reaction was monitored by TLC. The crude residue was used directly in the next step.

[0334] 2-(difluoromethyl)-4-fluorophenol 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (50 g, 183.776 mmol, 1 equivalent) was dissolved in MeOH (300 mL, 7409.673 mmol, 40.32 equivalents) and H2O (100 mL, 5550.837 mmol, 30.20 equivalents). To this solution, H2O2 (30%) (50 mL, 2146.131 mmol, 11.68 equivalents) was added dropwise at 0°C. The solution was stirred at 25°C for 3 hours. The resulting solution was concentrated under reduced pressure. The residue was diluted with EA (500 mL). The organic layer was washed with 3 × 200 mL of saturated NaCl aqueous solution. The combined organic layers were dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain 2-(difluoromethyl)-4-fluorophenol (25 g, 83.91%) as a pale yellow oil.

[0335] Example 24: Assay of TRPC4 activity ICLN-1694 cells expressing TRPC4 (HEK-TREx hTRPC4) were prepared as follows: 24 hours before transfection using 2 mL of antibiotic-free cell medium (1 × DMEM / high glucose (hyclone#SH30022.02); 10% fetal bovine serum (Sigma), 2 mM sodium pyruvate, 10 mM HEPES), commercially available HekTrex-293 cells were placed in 1 × 6 well plates in a 0.7 × 10⁶ well configuration. 6Cells were seeded in wells. The human codon-optimized TRPC4 coding sequence was cloned into pcDNA5 / TO (Invitrogen, catalog no. V103320) using hygromycin as the resistance gene, and the plasmid (SEQ ID NO: 1) was grown using T-Rex-293 cells (Invitrogen, catalog no. R71007) according to the manufacturer's instructions. On day 2, a solution was prepared by adding 2 μg of plasmid DNA to 6 μl of Xtreme-GENE HP reagent in Optimem (total volume 200 μl) and incubating at room temperature for 15 minutes. Next, this plasmid solution was gently dropped onto each well and the plate was gently rotated for approximately 30 seconds to mix the medium and the complex. The transfected cells were incubated in a 10% CO2 incubator at 37°C for 24 hours. The transfected cells were harvested and transferred to a 2x150 mm dish containing antibiotic-free cell growth medium at 37°C.

[0336] The following day, selection was initiated by adding cell medium containing 150 μg / mL hygromycin and 5 μg / mL blastosidine to create a stable pool and promote cell growth. The medium containing the selecting agents was replaced every 1-2 days as needed to remove dead cells. After 7 days, the hygromycin concentration was reduced to 75 μg / mL and cell growth was continued.

[0337] Single clones were selected as follows: A stable pool was diluted to 10 cells / mL, seeded into 24 × 96 well plates (approximately 1 cell / well), and grown in cell medium for 7 days. Fresh medium (100 μl) was added, and the cells were grown for a further 1-2 weeks before being cryopreserved or used immediately.

[0338] Each compound was prepared or supplied as a 10 mM stock solution, generally using DMSO as the solvent. Ten dose-response curves were constructed using an Echo-550 acoustic dispenser. Source plates for each compound were prepared by serially diluting each compound stock in Echo-certified LDV plates to form 10 mM, 1 mM, and 0.1 mM solutions in DMSO. A four-fold dilution scheme was then prepared by sequentially spotting the 100% DMSO stock solution onto the source dose-response plates using the Echo. 100% DMSO was added to each spotted dose-response plate to a final volume of 5 μl. Next, 300 nl dose-response plates were spotted onto pre-incubation and stimulation assay plates. Then, 50 μl of pre-incubation buffer and 100 μl of stimulation buffer were added to each plate to achieve a final assay concentration range of 30 μM to 0.0001 μM with a final DMSO concentration of 0.3%.

[0339] ICLN-1694 cells (HEK-TREx hTRPC4) were plated into a 384-well black pdl-coated microplate and maintained in cell medium supplemented with 1 μg / mL tetracycline the day before use in the experiment. TRPC4 expression was induced by adding 1 μg / mL tetracycline at the time of plating. The medium was removed from the plate, and 10 μl of 4 μM Fluo-4AM (mixed with an equal volume of Pluronic F-127) was added to the cells in EBSS (NaCl (142 mM), KCl (5.4 mM), glucose (10 mM), CaCl2 (1.8 mM), MgCl2 (0.8 mM), HEPES (10 mM), pH 7.4). The cells were incubated at room temperature, protected from light, for 60–90 minutes. After the incubation period, the dye was removed and replaced with 10 μl of EBSS. Cells, pre-incubation, and stimulation plates were loaded into the FLIPR-II to initiate the assay. After measuring the baseline at 10 seconds with FLIPR, 10 μl of 2× compound (or control) was added. The fluorescence change was monitored for a further 5 minutes. After a 5-minute pre-incubation, 20 μl of 2× Englerin A (including 1× compound or control) was added to the cell plate. The final Englerin A stimulation concentration in the assay was 100 nM. After the addition of Englerin A, the fluorescence change was monitored for a further 5 minutes.

[0340] The compound-mediated modulation of the TRPC4 calcium response was determined as follows: After EnglerinA, fluorescence was monitored for 5 minutes. The maximum relative fluorescence response (control response with 1 μM of an internal control compound known to maximally block the TRPC4 calcium response, minus "REF INHIB" in the formula below) was captured and exported from FLIPR.

[0341] The effect of a compound is calculated as an inhibition rate (%) using the following formula.

[0342]

number

[0343] Here, "RFU" stands for Relative Fluorescence Unit.

[0344] The results of these assays are shown in Table 2 below, where "A" indicates IC50 nM or less. 50 "B" is an IC with a power of over 50nM and 500nM or less. 50 "C" is an IC with a mass greater than 500 nM and less than 1 μM. 50 "D" stands for IC with a size of 1 μm or larger. 50 "NT" indicates that the compound was not tested.

[0345] Example 25: Assay of TRPC5 activity ICLN-1633 cells expressing TRPC5 (HEK-TREx hTRPC5) were prepared as follows: 24 hours before transfection using 2 mL of antibiotic-free cell medium (1 × DMEM / high glucose (hyclone#SH30022.02); 10% fetal bovine serum (Sigma), 2 mM sodium pyruvate, 10 mM HEPES), commercially available HekTrex-293 cells were placed in 1 × 6 well plates in a 0.7 × 10⁶ format. 6 Cells were seeded in wells. The human TRPC5 coding sequence (NM_012471 with the silent T478C mutation) was cloned into pcDNA5 / TO (Invitrogen; catalog number V103320) using hygromycin as the resistance gene, and the plasmid (SEQ ID NO: 2) was grown using T-Rex-293 cells (Invitrogen; catalog number R71007) according to the manufacturer's instructions. On day 2, a solution was prepared by adding 2 μg of plasmid DNA to 6 μl of Xtreme-GENE HP reagent in Optimem (total volume 200 μl) and incubating at room temperature for 15 minutes. Next, this plasmid solution was gently dropped onto each well and the plate was gently rotated for about 30 seconds to mix the medium and the complex. The transfected cells were incubated in a 10% CO2 incubator at 37°C for 24 hours. The transfected cells were collected and transferred to a 2 x 150 mm dish containing antibiotic-free cell growth medium at 37°C.

[0346] The following day, selection was initiated by adding cell medium containing 150 μg / mL hygromycin and 5 μg / mL blastosidine to create a stable pool and promote cell growth. The medium containing the selecting agents was replaced every 1-2 days as needed to remove dead cells. After 7 days, the hygromycin concentration was reduced to 75 μg / mL and cell growth was continued.

[0347] A single clone was selected as follows: A stable pool was diluted to 10 cells / mL, seeded into 24 × 96 well plates (approximately 1 cell / well), and grown in cell medium for 7 days. Fresh medium (100 μl) was added, and the cells were grown for a further 1-2 weeks before being cryopreserved or used immediately.

[0348] Each compound was prepared or supplied as a 10 mM stock solution, generally using DMSO as the solvent. Ten dose-response curves were constructed using an Echo-550 acoustic dispenser. Source plates for each compound were prepared by serially diluting each compound stock in Echo-certified LDV plates to form 10 mM, 1 mM, and 0.1 mM solutions in DMSO. A four-fold dilution scheme was then prepared by sequentially spotting the 100% DMSO stock solution onto the source dose-response plates using the Echo. 100% DMSO was added to each spotted dose-response plate to a final volume of 5 μl. Next, 300 nl dose-response plates were spotted onto pre-incubation and stimulation assay plates. Then, 50 μl of pre-incubation buffer and 100 μl of stimulation buffer were added to each plate to achieve a final assay concentration range of 30 μM to 0.0001 μM with a final DMSO concentration of 0.3%.

[0349] Human ICLN-1633 cells expressing the gene were plated into a 384-well black PDL-coated microplate and maintained in TRPC5 medium the day before use in the experiment. TRPC5 expression was induced by adding 1 μg / mL tetracycline at the time of plating. The medium was removed from the plate and 10 μl of 4 μM Fluo-4AM (mixed with an equal volume of Pluronic F-127) was added to the cells in EBSS. The cells were incubated at room temperature, protected from light, for 60–90 minutes. After the incubation period, the dye was removed and replaced with 10 μl of EBSS. The cells, pre-incubated and stimulated plates were loaded into FLIPR-II and the assay was initiated. After measuring the baseline at 10 seconds in FLIPR, 10 μl of 2× compound (or control) was added. The change in fluorescence was monitored for a further 5 minutes. After a 5-minute pre-incubation, add 20 μl of 2× Riluzole (including 1× compound or control) to the cell plate. The final stimulating concentration of Riluzole in the assay should be 30 μM. Monitor the fluorescence changes for a further 5 minutes after the addition of Riluzole.

[0350] The compound-mediated modulation of the TRPC5 calcium response was determined as follows: After EnglerinA, fluorescence was monitored for 5 minutes. The maximum relative fluorescence response (control response with 1 μM of an internal control compound known to maximally block the TRPC5 calcium response, minus "REF INHIB" in the formula below) was captured and exported from FLIPR.

[0351] The effect of a compound is calculated as an inhibition rate (%) using the following formula.

[0352]

number

[0353] Here, "RFU" stands for Relative Fluorescence Unit.

[0354] The results of these assays are shown in Table 2 below. In the table, "A" represents an IC 50 of 50 nM or less, "B" represents an IC 50 of more than 50 nM and 500 nM or less, "C" represents an IC 50 of more than 500 nM and less than 1 μM, "D" represents an IC 50 of 1 μM or more, and "NT" indicates that the compound was not tested.

[0355]

Table 2

[0356] The 1 1H NMR and MS data of the selected compounds are shown in the following table.

[0357]

Chemical Structure

[0358]

Chemical Structure

[0359]

Chemical Structure

[0360]

Chemical Structure

[0361]

Chemical Structure

[0362]

Chemical Structure

[0363]

Chemical Structure

[0364] [ka]

[0365] [ka]

[0366] [ka]

[0367] [ka]

[0368] [ka]

[0369] Example 26: Effect of compound 100 on promycin aminonucleoside (PAN)-induced glomerular injury in rats. the purpose: The purpose of this experiment is to evaluate the dose-dependent effect of compound 100 on PAN-induced glomerular renal injury, as indicated by albuminuria.

[0370] method: Eighty male Sprague-Dawley rats, weighing approximately 125-150g and aged approximately 5-6 weeks, were obtained from Charles River. The rats were fed a standard diet (Harlan 8640), housed in cages under standard conditions, and allowed to acclimate for at least 5 days before the start of the experiment.

[0371] On the second day, the rats were divided into weight-matched treatment groups to balance the experiment, and each group was placed in a separate metabolic cage.

[0372] After collecting a 24-hour baseline (day 0) urine sample, baseline blood was collected by conscious tail vein puncture. Subsequently, the rats were administered the solvent or test substance.

[0373] Two hours after administration of the solvent or test agent on day 0, rats were administered (5 ml / kg subcutaneously) the solvent (sterile physiological saline) or promycin aminonucleoside (PAN, loading agent, 75 mg / kg) dissolved in the solvent.

[0374] Urine volume was measured intermittently (on days 4, 7, and 10) over 24 hours to obtain samples (4 samples / head / time point, 0.5 ml / sample). In addition, blood samples were collected intermittently (on days 4, 7, and 10) by conscious tail vein puncture 2 hours ± 1 minute after AM administration.

[0375] Immediately after the final blood draw, the rats were anesthetized with isoflurane, tissue was collected, and the animals were sacrificed. Kidney weight and other endpoints were obtained.

[0376] The urine samples were immediately flash-frozen with liquid nitrogen and stored at -80°C until analysis was performed.

[0377] Whole blood samples collected with K3EDTA were processed as appropriate for plasma preparation for PK measurement.

[0378] result: As shown in Figure 1, treatment with compound 100 at 30 mg / kg once daily (QD) or twice daily (BID) reduced urinary albumin excretion after PAN-induced injury. Significant reductions were observed on days 7 and 10 with BID administration of compound 100, and on day 10 with QD administration. Mizoribine served as a positive control compound and was also effective in reducing albuminuria.

[0379] Conclusion: Compound 100 is effective in reducing albuminuria in a PAN model of glomerular injury in rats.

[0380] Example 27 Compound 100 is effective in the AT1R transgenic rat model of FSGS. The FSGS AT1R transgenic rat model is characterized by podocyte-specific expression of human AT1R. Males have been shown to have significantly worse disease outcomes than females. The efficacy of TRPC5 inhibitors in the AT1R model has been demonstrated with tool compounds. See Zhou et al., Science (2017), vol.358(6368), 1332-1336.

[0381] In this experiment, pathophysiology in AT1R transgenic rats was enhanced by unilateral nephrectomy (UniNX) and AngII infusion via a minipump. Compound 100 was administered orally once daily at a dose of 3 mg / kg or 10 mg / kg, and the urinary protein-creatinine ratio was measured at weeks -1, 0, 1, 2, and 3 of treatment. Figure 2 shows the urinary protein-creatinine ratio throughout the experimental course for rats treated with compound 100 or rats treated with the solvent. UniNX, AngII initiation, and compound 100 initiation were performed at the indicated times. Figure 3 shows the same data as a baseline percentage (%).

[0382] These results demonstrate that compound 100 is effective in the AT1R transgenic rat model of FSGS.

[0383] Reference All U.S. patents, as well as U.S. patent application publications and international patent application publications cited herein, are incorporated herein by reference.

[0384] Equal portions The description in the above specification is sufficient to enable those skilled in the art to carry out the present invention. Each embodiment is intended as a single example of one aspect of the present invention, and since other functionally equivalent embodiments are also included in the scope of the present invention, the scope of the present invention should not be limited by the embodiments described. Various modifications of the present invention other than those shown and described herein will be apparent to those skilled in the art from the above description. These modifications are included in the scope of the appended "Claims". The advantages and objectives of the present invention are not necessarily encompassed in each embodiment of the present invention.

[0385] Sequence ID 1: TRPC4 plasmid sequence The DNA sequence of the TRPC4 plasmid used in Example 24 is shown below. The underlined nucleic acid represents the nucleic acid encoding human TRPC4.

[0386] [ka]

[0387] [ka]

[0388] [ka]

[0389] [ka]

[0390] [ka]

[0391] Sequence ID 2: TRPC5 plasmid sequence The DNA sequence of the TRPC5 plasmid used in Example 25 is shown below. The underlined nucleic acids represent the nucleic acids encoding human TRPC5.

[0392]

change

[0393]

change

[0394]

change

[0395]

change

[0396]

change

[0397]

change

Claims

1. Compounds with structural formula I: 【Chemistry 1】 (I) [In the formula, "---" indicates a single bond or a double bond. X １ is CH or N, If "---" is a double bond, then X ２ is CH or N, If "---" is a single bond, then X ２ N(CH) ３ ), X １ If is CH, then X ２ is N or N(CH ３ ) and Y is -O-, -N(CH ３ ), -N(CH ２ CH ２ OH)-, cyclopropane-1,1-diyl, or -CH(CH ３ ), and Q is 2-trifluoromethyl-4-fluorophenyl, 2-difluoromethyl-4-fluorophenyl, 2-trifluoromethylphenyl, 2-methyl-4-fluorophenyl, 2-chloro-4-fluorophenyl, 2-chlorophenyl, 1-(benzyl)-4-methylpiperidine-3-yl, 4-trifluoromethylpyridine-3-yl, 2-trifluoromethyl-6-fluorophenyl, 2-trifluoromethyl-3-cyanophenyl, 2-ethyl-3-fluorophenyl, 2-chloro-3-cyanophenyl, 2-trifluoromethyl-5-fluorophenyl, or 2-difluoromethylphenyl. R ３ is hydrogen, -CH ２ OH, -CH(OH)-CH ２ OH, -NH ２ , -CH(OH)CH ３ , -OCH ３ , or -NH-(CH ２ ) ２ If it is OH and "---" is a double bond, then R ４ It does not exist. If "---" is a single bond, R ３ and R ４ Together with, they form = O, R ５ and R ６ Each of them independently contains hydrogen or -CH ３ And, However, X １ N, X ２ If N, Y is -O- or -N(CH ３ ) and Q is 2-trifluoromethylphenyl, R ３ , R ５ , and R ６ [Provided that at least one of them is not hydrogen] or a pharmaceutically acceptable salt thereof.

2. Structural formula II: 【Chemistry 2】 (II) [In the formula, R １ is chloro, -CF ３ ,-CHF ２ , or -CH ３ And, R ２ is hydrogen or fluoro, R ３ is hydrogen, -NH ２ ien-CH ２ OH, or CH(OH)-CH ２ The compound according to claim 1, represented by [OH], or a pharmaceutically acceptable salt thereof.

3. R １ ga-CHF ２ If R ２ The compound according to claim 2, wherein the compound is not hydrogen.

4. A compound according to claim 1, selected from any one of the following compounds, or a pharmaceutically acceptable salt thereof: 【Chemistry 3-1】 【Chemistry 3-2】 【Chemistry 3-3】 [Chemistry 3-4] [Transformation 3-5] [Chemistry 3-6] 【Chemistry 3-7】 【Transformation 3-8】

5. A compound according to claim 4, selected from any one of the following compounds, or a pharmaceutically acceptable salt thereof: 【Chemistry 4-1】 【Chemistry 4-2】

6. A compound according to claim 5, selected from any one of the following compounds, or a pharmaceutically acceptable salt thereof: 【Chemistry 5-1】 【Chemistry 5-2】 【Chemistry 5-3】

7. A pharmaceutical composition comprising a compound according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. A method for treating or reducing the risk of developing a disease or condition selected from kidney disease, pulmonary hypertension, anxiety, depression, cancer, diabetic retinopathy, or pain, comprising administering a therapeutically effective amount of a compound according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, to a subject in need.

9. The method according to claim 8, wherein the disease or condition is a renal disease selected from focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Alport syndrome, hypertensive renal disease, nephrotic syndrome, steroid-resistant nephropathy, minimal change syndrome, membranous nephropathy, idiopathic membranous nephropathy, membranoproliferative glomerulonephritis (MPGN), immune complex-mediated MPGN, complement-mediated MPGN, lupus nephritis, post-infectious glomerulonephritis, thin basement membrane disease, mesangial proliferative glomerulonephritis, amyloidosis (primary), c1q nephritis, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, or IgA nephropathy.

10. The method according to claim 9, wherein the kidney disease is proteinuria.

11. The method according to claim 9, wherein the kidney disease is microalbuminuria or overt albuminuria.

12. The method according to claim 8, wherein the disease or condition being treated is pulmonary arterial hypertension.

13. The method according to claim 8, wherein the disease or condition to be treated is pain selected from neuropathic pain and visceral pain.

14. The method according to claim 8, wherein the disease or condition is a cancer selected from chemotherapy-resistant breast cancer, adriamycin-resistant breast cancer, chemotherapy-resistant colorectal cancer, medulloblastoma, and tumor angiogenesis.

15. The method according to claim 8, wherein the disease or condition is transplant-associated FSGS, transplant-associated nephrotic syndrome, transplant-associated proteinuria, cholestatic liver disease, polycystic kidney disease, autosomal dominant polycystic kidney disease (ADPKD), obesity, insulin resistance, type II diabetes mellitus, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

16. The method according to any one of claims 8 to 15, wherein the subject is a human.

Images