Pyridazinones and methods of use thereof

Abstract

Disclosed herein are therapeutic methods of using a compound of formula (A) in combination with a second therapeutic agent, for example, to treat renal disease.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 821,178, filed on March 20, 2019. Background Technology

[0003] Proteinuria is a condition in which excessive amounts of protein in the blood leak into the urine. It can progress from a loss of 30 mg of protein in the urine over a 24-hour period (called microalbuminuria) to >300 mg / day (called macroalbuminuria), and then to 3.5 grams or more of protein in a 24-hour period, or 25 times the normal amount. Proteinuria occurs when the glomeruli of the kidneys malfunction, leading to fluid accumulation in the body (edema). Long-term protein leakage has been shown to lead to kidney failure. Nephrotic syndrome (NS) accounts for approximately 12% of all end-stage renal disease cases and costs more than $3 billion annually in the United States. About 5 out of every 100,000 children are diagnosed with NS each year; today, 15 out of every 100,000 children have the disease. Relapse rates are also extremely high for patients who respond aggressively to treatment. 90% of children with nephrotic syndrome will respond to treatment; however, an estimated 75% will relapse. More effective treatments are needed for kidney diseases such as proteinuria or methods to reduce the risk of their progression.

[0004] Mammalian TRP channel proteins form six-transmembrane cation-permeable channels, which can be divided 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 believed to play an important role in the pathophysiology of many diseases. Many TRPs are expressed in different parts of the nephron in the kidney, and there is growing evidence that these channels are involved in both hereditary and acquired kidney disorders. TRPC6, TRPM6, and TRPP2 are associated with hereditary focal segmental glomerulosclerosis (FSGS), hypomagnesemia with secondary hypocalcemia (HSH), and polycystic kidney disease (PKD), respectively.

[0005] TRPC5 has also been reported to contribute to the underlying mechanisms of modulating innate fear responses. (J Neurosci, March 5, 2014; 34(10):3653–3667). Summary of the Invention

[0006] One aspect of the present invention is a method for treating kidney disease, the method comprising the steps of administering a TRPC5 inhibitory compound and a second therapeutic agent in combination to a subject in need. In some embodiments, the method of the present invention comprises the step of administering the following substances in combination to a subject in need:

[0007] a. A TRPC5 inhibitory compound having structural formula (A), or its tautomer or pharmaceutically acceptable salt:

[0008]

[0009] in

[0010] Each R is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, aryl, heterocyclic, heteroaryl, halogen, -OH, CN, cycloalkyl, -O-alkyl, -O-cycloalkyl, -O-aryl, -aryl-O-aryl, -CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl, -O-alkylene-O-alkyl, -heterocyclic-LR 4 and heteroaryl-LR 4 ;

[0011] R 4 The following groups are not present or are selected from: alkyl, cycloalkyl, polycyclic, aryl, heterocyclic, heteroaryl, -C(O)N(R) 5 )2 and CF3;

[0012] R 5 Independently H or alkyl;

[0013] R 6 Choose from the group consisting of: alkyl, cycloalkyl, aryl, heterocyclic, heteroaryl, alkylene-aryl, -C(O)N(R) 5 )2 and CF3;

[0014] L is absent or selected from the group consisting of the following: methylene, -C(O)-, -SO2-, -CH2N(Me)-, -N(R)-. 5 (R) 6 )-、-C(R 5 (R) 6 - and -OR 6 ;and

[0015] One and only one R is a -heterocyclic-LR group 4 or -heteroaryl-LR 4 ;as well as

[0016] b. A second therapeutic agent, selected from: immunomodulators, calcineurin inhibitors, renin-angiotensin-aldosterone system inhibitors, antiproliferative agents, alkylating agents, corticosteroids, angiotensin-converting enzyme inhibitors, adrenocorticotropic hormone stimulators, angiotensin receptor blockers, sodium-glucose transporter 2 inhibitors, double sodium-glucose transporter 1 / 2 inhibitors, nuclear factor-1 (erythroid-derived 2)-like 2 agonists, chemokine receptor 2 inhibitors, chemokine receptor 5 inhibitors, endothelin 1 receptor antagonists, beta-blockers, mineralocorticoid receptor antagonists, loop diuretics or thiazide diuretics, calcium channel blockers, statins, short-, intermediate-, or long-acting insulin, dipeptidyl peptidase-4 inhibitors, glucagon-like peptide-1 receptor agonists, sulfonylureas, apoptosis signal-regulated kinase-1, chymotrypsin inhibitors, selective glycation inhibitors, and renin inhibitors. Interleukin-33 inhibitors, farnesoid X receptor agonists, soluble guanylate cyclase stimulants, thromboxane receptor antagonists, xanthine oxidase inhibitors, erythropoietin receptor agonists, cannabinoid receptor type 1 inverse agonists, NADPH oxidase inhibitors, anti-vascular endothelial growth factor B, anti-fibrotic agents, enkephalinase inhibitors, dual CD80 / CD86 inhibitors, CD40 antagonists, cellular cholesterol and lipid blockers, PDGFR antagonists, Slit guide ligand 2, APOL1 inhibitors, Nrl2 activators / NF-κB inhibitors, somatostatin receptor agonists, PPARγ agonists, AMP-activated protein kinase stimulants, tyrosine kinase inhibitors, glucocorticoid synthase inhibitors, arginine vasopressin receptor 2 antagonists, xanthine oxidase inhibitors, and vasopressin receptor 2 antagonists.

[0017] In some implementations, the TRPC5 inhibitor and the second therapeutic agent are administered as separate dosage forms.

[0018] In an alternative implementation, the TRPC5 inhibitor and the second therapeutic agent are administered together as a fixed-dose combination (i.e., a single formulation).

[0019] In some implementations, the second therapeutic agent is an immunomodulator, a calcineurin inhibitor, a renin-angiotensin-aldosterone system inhibitor, an antiproliferator, a corticosteroid, an angiotensin-converting enzyme inhibitor, an angiotensin receptor blocker, a sodium-glucose transporter 2 inhibitor, a nuclear factor-1 (erythroid-derived 2)-like 2 agonist, a chemokine receptor 2 inhibitor, a chemokine receptor 5 inhibitor, or an endothelin 1 receptor antagonist.

[0020] In some implementations, the TRPC5 inhibitory compound is represented by the structural formula (AI), (A-II) or (A-III) or its tautomers or pharmaceutically acceptable salts;

[0021]

[0022] in

[0023] R 1 and R 3 Choose from the group consisting of: H, alkyl, alkenyl, alkynyl, aryl, heterocyclic, heteroaryl, halogen, -OH, -CN, -cycloalkyl, -O-alkyl, -O-cycloalkyl, -O-aryl, -aryl-O-aryl-CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl and -O-alkylene-O-alkyl, -heterocyclic-LR 4 and -heteroaryl-LR 4 ;

[0024] R 2 -heterocyclic-LR 4 ;

[0025] R 4 The following groups are not present or are selected from: alkyl, cycloalkyl, aryl, alkylene-aryl, alkylene-heteroaryl, heteroaryl, heterocyclic, -C(O)N(R) 5 )2 and CF3;

[0026] R 5 Independently H or alkyl;

[0027] R 6 Choose from the group consisting of: alkyl, cycloalkyl, aryl, heterocyclic, heteroaryl, alkylene-aryl, -C(O)N(R) 5 )2 and CF3;

[0028] L is absent or selected from the group consisting of the following: methylene, -C(O)-, -SO2-, -CH2N(Me)-, -N(R)-. 5 (R) 6 )-、-C(R 5 (R) 6 - and -OR 6 ;and

[0029] R 1 R 2 and R 3 One and only one of them is –heterocyclic-LR 4 or -heteroaryl-LR 4 .

[0030] In some implementations, the TRPC5 inhibitory compound has the structural formula (I):

[0031] Or its pharmaceutically acceptable salt; of which:

[0032] "---" indicates a single or double key.

[0033] X 1 For CH or N;

[0034] When "---" is a double key, X 2 For CH or N;

[0035] When "---" is a single key, X 2 For N(CH3),

[0036] When X 1 When it is CH, X 2 It is N or N(CH3);

[0037] Y is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropyl-1,1-diyl or -CH(CH3)-;

[0038] 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;

[0039] When "---" is a double bond, R 13 It is hydrogen, -CH2OH, -CH(OH)-CH2OH, -NH2, -CH(OH)CH3, -OCH3 or -NH-(CH2)2OH; and R 14 Does not exist; or

[0040] When "---" is a single key, R 13 and R 14 Together they form = O; and

[0041] R 5 and R 6 Each of them is independently hydrogen or -CH3.

[0042] In some embodiments, the TRPC5 inhibitory compound has structural formula (II):

[0043] Or its pharmaceutically acceptable salt; of which:

[0044] R 11 It can be chlorine, -CF3, -CHF2, or -CH3;

[0045] R 12 It is hydrogenated or fluorinated; and

[0046] R 13 It can be hydrogen, -NH2, -CH2OH or CH(OH)-CH2OH.

[0047] In some implementations, the immunomodulator is rituximab. In some implementations, the angiotensin-converting enzyme inhibitor is captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril, or cilazapril.

[0048] In some implementations, the angiotensin receptor blocker is losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan, or fimasartan.

[0049] In some implementations, the renin-angiotensin-aldosterone system inhibitor is aliskiren.

[0050] In some embodiments, the endothelin 1 receptor antagonist is ambrisentan, atrasentan, bosentan, or sparsentan. In other embodiments, the endothelin 1 receptor antagonist is macitentan.

[0051] In some embodiments, the antiproliferative agent is mycophenolate mofetil. In other embodiments, the antiproliferative agent is sodium mycophenolate or azathioprine.

[0052] In some embodiments, the SGLT2 inhibitor is canagliflozin, dapagliflozin, empagliflozin, a combination of empagliflozin and linagliptin, a combination of empagliflozin and metformin, or a combination of dapagliflozin and metformin. In some other embodiments, the SGLT2 inhibitor also inhibits SGLT1. In some aspects of these embodiments, the SGLT1 / 2 inhibitor is sotagliflozin.

[0053] In some embodiments, the calcineurin inhibitor is cyclosporine A or tacrolimus. In other embodiments, the calcineurin inhibitor is voclosporin.

[0054] In some implementations, the nuclear factor-1 (erythroid-derived 2)-like 2 agonist is bardoxolone or CXA-10.

[0055] In some embodiments, the chemokine receptor 2 inhibitor is PF-04136309 or ccx140. In other embodiments, the chemokine receptor 2 inhibitor is propagemanium (DMX-200).

[0056] In some implementations, the β-receptor blocker is metoprolol succinate, metoprolol tartrate, propranolol, or carvedilol.

[0057] In some implementations, the mineralocorticoid receptor antagonist is spironolactone, eplerenone, finerenone, esaxerenone, or apararenone.

[0058] In some implementations, the loop diuretic or thiazide diuretic is furosemide, bumetanide, torsemide, or bendroflumethiazide.

[0059] In some implementations, the calcium channel blocker is verapamil, diltiazem, amlodipine, or nifedipine.

[0060] In some implementations, statins are atorvastatin, pravastatin, fluvastatin, lovastatin, rosuvastatin, simvastatin, or pitavastatin.

[0061] In some implementations, short-acting, intermediate-acting, or long-acting insulin is NPH insulin ( Or biosimilars), lispro insulin Insulin glutathione, insulin glargine Detemir insulin Or insulin degludec

[0062] In some implementations, the dipeptidyl peptidase-4 inhibitor is sitagliptin, saxagliptin, linagliptin, or vildagliptin.

[0063] In some implementations, the glucagon-like peptide-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide, albiglutide, or semaglutide.

[0064] In some implementations, the sulfonylureas are glimepiride, glipizide, glyburide, glibenclamide, chlorpropamide, tolazamide, or tolbutamide.

[0065] In some implementations, apoptosis signal-regulated kinase-1 is selonsertib.

[0066] In some implementations, the chymotrypsin inhibitor is fulacimstat (BAY1142524).

[0067] In some implementations, the selective glycation inhibitor is GLY-230.

[0068] In some implementations, the renin inhibitor is SCO-272.

[0069] In some implementations, the interleukin-33 inhibitor is MEDI-3506.

[0070] In some implementations, the farnesoid X receptor agonist is nidufexor (LMB763).

[0071] In some implementations, the soluble guanylate cyclase stimulant is praliciguat, olinciguat, IW-6463, vericiguat, or riociguat.

[0072] In some implementations, the thromboxane receptor antagonist is SER150.

[0073] In some implementations, the xanthine oxidase inhibitor is TMX-049.

[0074] In some implementations, the erythropoietin receptor agonist is cibinetide (ARA-290).

[0075] In some implementations, the cannabinoid receptor type 1 inverse agonist is nimacimab, GFB-024, or CRB-4001.

[0076] In some implementations, the NADPH oxidase inhibitor is APX-115.

[0077] In some implementations, the anti-vascular endothelial growth factor B agent is CSL-346.

[0078] In some implementations, the anti-fibrotic agent is FT011.

[0079] In some implementations, the enkephalinase inhibitor is TD-1439, TD-0714, or sacubitril.

[0080] In some implementations, the dual CD80 / CD86 inhibitor is abatacept.

[0081] In some implementations, the CD40 antagonist is bleselumab (ASKP1240).

[0082] In some implementations, the cellular cholesterol and lipid blocker is VAR-200.

[0083] In some implementations, the PDGFR antagonist is ANG_3070.

[0084] In some implementations, Slit guide ligand 2 is PF-06730512.

[0085] In some implementations, the APOL1 inhibitor is VX-147.

[0086] In some implementations, the Nrl2 activator / NF-κB inhibitor is bardoxolone.

[0087] In some implementations, the somatostatin receptor agonist is lanreotide.

[0088] In some implementations, the PPARγ agonist is pioglitazone.

[0089] In some implementations, the AMP-activated protein kinase stimulator is metformin.

[0090] In some implementations, the tyrosine kinase inhibitor is tesevatinib.

[0091] In some implementations, the glucocorticoid synthase inhibitor is venglustat.

[0092] In some implementations, the arginine vasopressin receptor 2 antagonist is lixivaptan.

[0093] In some implementations, the xanthine oxidase inhibitor is oxypurinol.

[0094] In some implementations, the vasopressin receptor 2 antagonist is tolvaptan.

[0095] In some implementations, the second treatment agent is tacrolimus, cyclosporine A, rituximab, mycophenolate mofetil, corticosteroids, sparsentan, enalapril, or losartan.

[0096] In some implementations, the disease or condition is focal segmental glomerulosclerosis (FSGS), primary focal segmental glomerulosclerosis, hereditary focal segmental glomerulosclerosis, secondary focal segmental glomerulosclerosis, diabetic nephropathy, Allport syndrome, hypertensive nephropathy, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change disease, 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 nephropathy, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, or IgA nephropathy. In some implementations, the disease or condition is focal segmental glomerulosclerosis.

[0097] This method is effective for a variety of subjects, including mammals such as humans and other animals such as laboratory animals, such as mice, rats, rabbits, or monkeys, or domesticated and farm animals, such as cats, dogs, goats, sheep, pigs, cows, or horses. In some implementations, the subject is a human.

[0098] This invention offers several advantages. The preventive and therapeutic methods described herein are effective in treating kidney diseases such as proteinuria and have minimal side effects, if any. Furthermore, the methods described herein are effective in identifying compounds for treating kidney diseases, anxiety disorders, depression, or cancer, or for reducing the risk of developing these diseases.

[0099] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. While similar or equivalent methods and materials may be used in the practice or testing of the invention, the following description focuses on suitable methods and materials. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0100] Other features, objectives, and advantages of the invention will become apparent from the detailed description and claims. Attached Figure Description

[0101] Figure 1 Albumin excretion in PAN-injured rats treated with compound 100 or imidazoribine is shown.

[0102] Figure 2 The angiogenesis of human kidney organoids transplanted under the renal sac of rats is shown.

[0103] Figure 3 Oral administration of compound 100 resulted in drug exposure in implanted organoids.

[0104] Figure 4 The figure shows the effect of compound AO on albumin excretion in DOCA-type hypertensive rats.

[0105] Figures 5A-5F Confocal microscopy images of mouse foot cells pretreated with compounds AO or DMSO and subsequently damaged with sulfated glutenin (PS) are shown. Figure 5A , Figure 5B , Figure 5D , Figure 5E , Figure 5F ), and quantitative analysis of treated podocytes exhibiting actin-induced cytoplasmic collapse ( Figure 5C ).

[0106] Figures 6A-6F This image shows a confocal microscopy image of human iPSC-derived kidney organoids pretreated with compounds AO or DMSO and subsequently damaged with sulfated glutenin (PS). Figure 6A , 6B Quantitative analysis of the average phalloidin intensity for each organoid (6D, 6E, 6F), and quantification of the phalloidin intensity. Figure 6C ). Detailed Implementation

[0107] definition

[0108] The term "acyl" is recognized in the art and refers to a group represented by the general formula hydrocarbon C(O)-, preferably alkyl C(O)-.

[0109] The term "acylamino" is recognized in the art and refers to an amino group that has been substituted with an acyl group, and can be represented, for example, by the alkyl group C(O)NH-.

[0110] The term "acyloxy group" is recognized in the art and refers to a group represented by the general formula hydrocarbon C(O)O-, preferably alkyl C(O)O-.

[0111] The term "alkoxy" refers to an alkyl group linked to an oxygen atom, preferably a lower alkyl group. Representative alkoxy groups include methoxy, trifluoromethoxy, ethoxy, propoxy, tert-butoxy, etc.

[0112] The term "alkoxyalkyl" refers to an alkyl group that has been substituted with an alkoxy group, and can be represented by the general formula alkyl-O-alkyl.

[0113] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter referring to an alkenyl moiety having a hydrogen-substituted substituent on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons, including or not included in one or more double bonds. Furthermore, such substituents include all substituents considered for alkyl groups except where stability prohibits them, as discussed below. For example, consider the alkenyl group substituted with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups.

[0114] "Alkyl" or "alkane" refers to a fully saturated straight-chain or branched non-aromatic hydrocarbon. Typically, straight-chain or branched alkyl groups have 1 to about 20 carbon atoms, preferably 1 to about 10, unless otherwise defined. Examples of straight-chain and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups.

[0115] Furthermore, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter referring to an alkyl moiety having a hydrogen-substituted substituent on one or more carbons of the hydrocarbon backbone. Unless otherwise stated, such substituents may include, for example, halogens (e.g., fluorine), hydroxyl groups, carbonyl groups (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (such as thioesters, thioacetates, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonic acid groups, hypophosphonic acid groups, amino groups, amide groups, amidine, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate groups, sulfonic acid groups, aminosulfonyl, sulfonamide, sulfonyl, heterocyclic, aralkyl, or aromatic or heteroaromatic moiety. In a preferred embodiment, the substituent on the substituted alkyl group is selected from C10. 1-6 Alkyl, C 3-6 Cycloalkyl, halogen, carbonyl, cyano, or hydroxyl groups. In a more preferred embodiment, the substituent on the substituted alkyl group is selected from fluorinated, carbonyl, cyano, or hydroxyl groups. Those skilled in the art will understand that the portion substituted on the hydrocarbon chain itself may be substituted, if appropriate. For example, the substituents on the substituted alkyl group may include amino, azide, imino, amide, phosphoryl (including phosphonic and hypophosphonic acid groups), sulfonyl (including sulfate, sulfonamide, aminosulfonyl, and sulfonic acid groups), and silyl, as well as substituted and unsubstituted forms of ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylic acids, and esters), -CF3, -CN, etc. Exemplary substituted alkyl groups are described below. Cycloalkyl groups may be further substituted with alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl-substituted alkyl groups, -CF3, -CN, etc.

[0116] Unless otherwise stated, "alkylene" itself, or as part of another substituent, refers to a saturated straight-chain or branched divalent group having the stated number of carbon atoms and derived from the corresponding alkane by removing two hydrogen atoms. Examples of straight-chain and branched alkylenes 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-.

[0117] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "C" is used. x-y "This refers to groups containing x to y carbons in the chain. For example, the term "C" x-y "Alkyl" refers to a substituted or unsubstituted saturated hydrocarbon group, including straight-chain alkyl and branched alkyl groups containing x to y carbons in the chain, including haloalkyl. Preferred haloalkyl groups include trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, and pentafluoroethyl. CO alkyl indicates hydrogen when the group is at the terminal position, and a bond when it is internal. The term "C" indicates a bond. 2-y "Alkenyl" and "C" 2-y "Alkyne" refers to a substituted or unsubstituted unsaturated aliphatic group whose length and possible substitutions are similar to the alkyl groups mentioned above, but each contains at least one double or triple bond.

[0118] As used herein, the term "alkylamino" refers to an amino group that is substituted with at least one alkyl group.

[0119] 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-.

[0120] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond, and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl," wherein the latter refers to an alkynyl moiety having a hydrogen-substituted substituent on one or more carbons of the alkynyl group. Such substituents may be present on one or more carbons, including or not included in one or more triple bonds. Furthermore, such substituents include all substituents considered for alkyl groups as discussed above, except where stability prohibits them. For example, substitution of the alkynyl group with one or more alkyl, carbocyclic, aryl, heterocyclic, or heteroaryl groups is considered.

[0121] As used herein, the term "amide" refers to a group.

[0122]

[0123] Each RA Independently representing a hydrogen or hydrocarbon group, or two Rs A Together with the N atoms they are attached to, they form heterocycles with 4 to 8 atoms in the ring structure.

[0124] The terms "amine" and "amino" are recognized in the art and refer to unsubstituted and substituted amines and their salts, such as portions that can be represented by the following formula.

[0125]

[0126] Each R A Independently representing a hydrogen or hydrocarbon group, or two Rs A Together with the N atoms they are attached to, they form heterocycles with 4 to 8 atoms in the ring structure.

[0127] As used herein, the term "aminoalkyl" refers to an alkyl group that has been substituted with an amino group.

[0128] As used herein, the term "aralkyl" refers to an alkyl group that has been substituted with an aryl group.

[0129] As used herein, the term "aryl" includes a substituted or unsubstituted monocyclic aromatic group, wherein each atom of the ring is a carbon. Preferably, the ring is a 6- or 10-membered ring, more preferably a 6-membered ring. The term "aryl" also includes a polycyclic system having two or more rings, wherein two or more carbons are common to two adjacent rings, and wherein at least one ring is aromatic; for example, the other rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclic. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0130] The term "carbamate" is recognized in the art and refers to the following groups

[0131]

[0132] Each R A Independently representing hydrogen or a hydrocarbon group, such as an alkyl group, or two R groups. A Together with intermediary atoms, they form heterocycles with 4 to 8 atoms in the ring structure.

[0133] As used herein, the terms "carbocycle" and "carbocyclic" refer to a saturated or unsaturated ring in which each atom of the ring is carbon. The term "carbocycle" includes aromatic and non-aromatic carbocycles. Non-aromatic carbocycles include cycloalkane rings in which all carbon atoms are saturated and cycloalkene rings containing at least one double bond. "Carbocycle" includes 5-7 membered monocyclic rings and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. Carbocycles include bicyclic molecules in which one, two, three, or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each ring shares two adjacent atoms with another ring. Each ring of a fused carbocycle may be selected from a saturated ring, an unsaturated ring, and an aromatic ring. In exemplary embodiments, an aromatic ring (e.g., phenyl) may be fused with a saturated or unsaturated ring (e.g., cyclohexane, cyclopentane, or cyclohexene). Where valence permits, any combination of saturated bicyclic, unsaturated bicyclic, and aromatic bicyclic rings is included in the definition of a carbocyclic ring. Exemplary “carbocyclic rings” include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene, and adamantane. Exemplary fused carbocyclic rings include naphthane, 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 capable of carrying a hydrogen atom.

[0134] "Cycloalkyl" is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic compounds. Generally, unless otherwise defined, monocyclic cycloalkyl molecules have 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. The second ring of a bicyclic cycloalkyl molecule can be a saturated ring, an unsaturated ring, or an aromatic ring. Cycloalkyl molecules include bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl molecule in which each ring shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl molecule can be a saturated ring, an unsaturated ring, or an aromatic ring. "Cycloalkenyl" is a cyclic hydrocarbon containing one or more double bonds.

[0135] As used herein, the term "carbocyclic alkyl" refers to an alkyl group that has been substituted with a carbocyclic group.

[0136] The term "carbonate" is recognized in the art and refers to the group -OCO2-R. A , where R A It represents a hydrocarbon group.

[0137] As used herein, the term "carboxyl" refers to a group represented by the formula -CO2H.

[0138] As used herein, the term "ester" refers to the group -C(O)OR A, where R A It represents a hydrocarbon group.

[0139] As used herein, the term "ether" refers to a hydrocarbon group that is attached to another hydrocarbon group by an oxygen atom. Therefore, the ether substituent of a hydrocarbon group can be a hydrocarbon-O-. Ethers can be symmetrical or asymmetrical. Examples of ethers include, but are not limited to, heterocyclic-O-heterocycles and aryl-O-heterocycles. Ethers include an "alkoxyalkyl" group, which can be represented by the general formula alkyl-O-alkyl.

[0140] As used herein, the terms “halogen” and “halogen” refer to halogens and include chlorine, fluorine, bromine, and iodine.

[0141] As used herein, the terms “hetaralkyl” and “heteroaralkyl” refer to alkyl groups that have been substituted with heteroaryl groups.

[0142] As used herein, the term “heteroalkyl” refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent to each other.

[0143] The terms "heteroaryl" and "hetaryl" encompass substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structure contains at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, and wherein at least one ring is heteroaromatic; for example, the other rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclic groups. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0144] As used herein, the term "heteroatom" refers to an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.

[0145] The terms "heterocyclic group," "heterocyclic," and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3 to 10-membered rings, more preferably 3 to 7-membered rings, whose ring structure contains at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclic group" and "heterocyclic" also include polycyclic systems having two or more rings, wherein two or more carbons are common to two adjacent rings, and wherein at least one ring is heterocyclic; for example, the other rings may be cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclic groups. Heterocyclic groups include, for example, piperidine, piperazine, pyrrolidine, tetrahydropyran, tetrahydrofuran, morpholine, lactone, lactam, etc.

[0146] As used herein, the term "heterocyclic alkyl" or "heterocyclic alkyl" refers to an alkyl group that has been substituted with a heterocyclic group.

[0147] As used herein, the term "hydrocarbon group" refers to a group bonded by carbon atoms without =O or =S substituents and typically has at least one carbon-hydrogen bond and a predominantly carbon backbone, but may optionally contain heteroatoms. Therefore, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbon groups, but substituents such as acetyl (which has a =O substituent on the linking carbon) and ethoxy (which is linked by oxygen rather than carbon) are not considered hydrocarbon groups. Hydrocarbon groups include, but are not limited to, aryl, heteroaryl, carbocyclic, heterocyclic, alkyl, alkenyl, ynyl, and combinations thereof.

[0148] As used herein, the term "hydroxyalkyl" refers to an alkyl group that has been substituted with a hydroxyl group.

[0149] When used in conjunction with chemical moieties such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, the term "lower" is intended to include groups in which ten or fewer, preferably six or fewer, non-hydrogen atoms are present among the substituents. For example, "lower alkyl" refers to an alkyl group containing ten or fewer, preferably six or fewer carbon atoms. In some embodiments, 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, whether they appear alone or in combination with other substituents, such as in the description of hydroxyalkyl and aralkyl (in which case, for example, atoms within the aryl group are not counted when calculating the carbon atoms in the alkyl substituent).

[0150] The terms "polycyclic," "polycyclic," and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, aryl, heteroaryl, and / or heterocyclic) in which two or more atoms are shared by two adjacent rings; for example, the rings are "fused rings." Each ring of a polycyclic compound may be substituted or unsubstituted. In some embodiments, each ring of the polycyclic compound contains 3 to 10 atoms, preferably 5 to 7 atoms.

[0151] The term "silyl group" refers to a silicon moiety having three hydrocarbon groups attached to it.

[0152] The term "substituted" refers to a portion having a hydrogen-substituted substituent on one or more carbons of the main chain. It should be understood that "substituted" or "replaced by" includes the implicit condition that such substitution is based on the permissible valence of the substituted atom and the substituent, and that said substitution produces a stable compound, for example, which does not spontaneously undergo transformations such as rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is considered to include all permissible substituents in organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents in organic compounds. Permissible substituents can be one or more substituents and can be the same or different for a suitable organic compound. For the purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents in the organic compounds described herein that satisfy the valence of the heteroatom. Substituents may include any substituents described herein, such as halogens, hydroxyl groups, carbonyl groups (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl groups (such as thioesters, thioacetic acids, or thiocarbamates), alkoxy groups, phosphoryl groups, phosphate groups, phosphonic acid groups, hypophosphonic acid groups, amino groups, amide groups, amidine, imine, cyano, nitro, azide, mercapto, alkylthio, sulfate groups, sulfonic acid groups, sulfonamide groups, sulfinamide groups, sulfonyl groups, heterocyclic groups, aralkyl groups, or aromatic or heteroaromatic moieties. In a preferred embodiment, the substituents on the substituted alkyl group are selected from C10. 1-6 Alkyl, C 3-6 Cycloalkyl, halogen, carbonyl, cyano, or hydroxyl groups. In a more preferred embodiment, the substituent on the substituted alkyl group is selected from fluorinated, carbonyl, cyano, or hydroxyl groups. Those skilled in the art will understand that the substituent itself may be substituted where appropriate. Unless specifically stated as “unsubstituted,” references to the chemical portion herein should be understood to include substituted variants. For example, references to an “aryl” group or partly implicitly include both substituted and unsubstituted variants.

[0153] The term “sulfate” is recognized in the art and refers to the group -OSO3H or its pharmaceutically acceptable salt.

[0154] The term "sulfonamide" is recognized in the art and refers to a group represented by the following general formula.

[0155]

[0156] Each R A Independently representing hydrogen or a hydrocarbon group, such as an alkyl group, or two R groups. A Together with intermediary atoms, they form heterocycles with 4 to 8 atoms in the ring structure.

[0157] The term "sulfoxide" is recognized in the art and refers to the group -S(O)-R.A , where R A It represents a hydrocarbon group.

[0158] The term "sulfonate" is recognized in the art and refers to the group SO3H or its pharmaceutically acceptable salt.

[0159] The term "sulfone" is recognized in the art and refers to the group -S(O)2-R. A , where R A It represents a hydrocarbon group.

[0160] As used herein, the term "thioalkyl" refers to an alkyl group that has been substituted with a thiol group.

[0161] As used herein, the term "thioester" refers to the group -C(O)SR. A or -SC(O)R A , where R A It represents a hydrocarbon group.

[0162] As used in this article, the term "thioether" is equivalent to ether, in which oxygen is replaced by sulfur.

[0163] The term "urea" is recognized in the art and can be represented by the following general formula.

[0164]

[0165] Each R A Independently representing hydrogen or a hydrocarbon group, such as an alkyl group, or any R that appears. A Together with another intermediary atom, it forms a heterocycle with 4 to 8 atoms in the ring structure.

[0166] A "protecting group" is an atomic group that, when attached to a reactive functional group in a molecule, masks, reduces, or prevents the reactivity of that functional group. Typically, protecting groups can be selectively removed during synthesis as needed. 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”), triphenylmethyl and substituted triphenylmethyl, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro-veratroloxycarbonyl (“NVOC”), etc. Representative hydroxyl protecting groups include, but are not limited to, those in which the hydroxyl group is acylated (esterified) or alkylated, such as benzyl and triphenylmethyl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPS groups), ethylene glycol ethers such as ethylene glycol and propylene glycol derivatives, and allyl ethers.

[0167] As used herein, a therapeutic agent that “prevents” a disease, disorder, or condition or “reduces the risk of its development” refers to a compound that, in a statistically significant sample, reduces the incidence of a disease, disorder, or condition in a treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of a disorder or condition or reduces its severity relative to an untreated control sample.

[0168] The term "treatment" includes preventative and / or therapeutic treatment. The term "preventative or therapeutic" treatment is recognized in the art and includes administering one or more of the subject composition to the host. Treatment is preventative (i.e., it protects the host from developing the unwanted condition) if it is administered before the clinical manifestation of an unwanted ailment (e.g., a disease or other unwanted state in the host animal), and therapeutic (i.e., it is intended to alleviate, improve, or stabilize the existing unwanted ailment or its side effects) if it is administered after the manifestation of the unwanted ailment.

[0169] The phrases “combined administration” and “combined administration” refer to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., both compounds are effective simultaneously in the patient, which may include the synergistic effect of the two compounds). For example, the different therapeutic compounds may be administered simultaneously or sequentially in the same formulation or as separate formulations. In some embodiments, the different therapeutic compounds may be administered to each other within one hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or one week. Thus, the individual receiving this treatment may benefit from the combined effect of the different therapeutic compounds.

[0170] The term "prodrug" is intended to cover compounds that are converted into the therapeutically active agents of the present invention under physiological conditions. A common method for preparing a prodrug involves hydrolysis under physiological conditions to reveal one or more selected moieties of the 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 some embodiments, some or all of the compounds of the present invention in the formulations described above may be replaced with corresponding suitable prodrugs, such as where the hydroxyl group in the parent compound is presented as an ester or carbonate, or where the carboxylic acid present in the parent compound is presented as an ester.

[0171] As used herein, "small molecule" refers to an organic or inorganic small molecule with a molecular weight of less than about 3,000 Daltons. Typically, small molecules used in this invention have a molecular weight of less than 3,000 Daltons (Da). Small molecules can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 Da to about 2,500 Da, about 100 Da to about 2,000 Da, about 100 Da to about 1,750 Da, about 100 Da to about 1,500 Da, about 100 Da to about 1,250 Da, about 100 Da to about 1,000 Da, about 100 Da to about 750 Da, about 100 Da to about 500 Da, about 200 Da to about 1,500 Da, about 500 Da to about 1,000 Da, about 300 Da to about 1,000 Da, or about 100 Da to about 250 Da).

[0172] In some embodiments, "small molecule" refers to organic, inorganic, or organometallic compounds that typically have a molecular weight of less than about 1000. In some embodiments, a small molecule is an organic compound with a size of about 1 nm. In some embodiments, the small molecule pharmaceuticals of the present invention encompass oligopeptides and other biomolecules having a molecular weight of less than about 1000.

[0173] "Effective amount" is an amount sufficient to achieve a beneficial or desired outcome. For example, a therapeutic amount is an amount that achieves the desired therapeutic effect. This amount may be the same as or different from a preventive effective amount, which is the amount necessary to prevent the onset of disease or disease symptoms. An effective amount may be administered once or multiple times in an application or dose. The therapeutically effective amount of a composition depends on the composition chosen. The composition may be administered once or multiple times daily to once or multiple times weekly; including every other day. Those skilled in the art will understand that certain factors can influence the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease or disorder, prior treatment, the subject's general health condition and / or age, and any other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of the composition described herein may comprise a single treatment or a series of treatments.

[0174] The compounds of the present invention

[0175] One aspect of the present invention provides a method for treating kidney disease, the method comprising the steps of co-administering a TRPC5 inhibitory compound and a second therapeutic agent to a subject in need. In some embodiments, the TRPC5 inhibitory compound is a small molecule inhibitor of TRPC5.

[0176] small molecule inhibitors of TRPC5

[0177] In some implementations, the TRPC5 inhibitory compound is a compound of structural formula (A), or a tautomer thereof or a pharmaceutically acceptable salt thereof.

[0178]

[0179] in

[0180] Each R is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, aryl, heterocyclic, heteroaryl, halogen, -OH, CN, cycloalkyl, -O-alkyl, -O-cycloalkyl, -O-aryl, -aryl-O-aryl, -CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl, -O-alkylene-O-alkyl, -heterocyclic-LR 4 and heteroaryl-LR 4 ;

[0181] R 4 The following groups are not present or are selected from: alkyl, cycloalkyl, polycyclic, aryl, heterocyclic, heteroaryl, -C(O)N(R) 5 )2 and CF3;

[0182] R 5 Independently H or alkyl;

[0183] R6 Choose from the group consisting of: alkyl, cycloalkyl, aryl, heterocyclic, heteroaryl, alkylene-aryl, -C(O)N(R) 5 )2 and CF3;

[0184] L is absent or selected from the group consisting of the following: methylene, -C(O)-, -SO2-, -CH2N(Me)-, -N(R)-. 5 (R) 6 )-、-C(R 5 (R) 6 - and -OR 6 ;and

[0185] One and only one R is –heterocyclic-LR 4 or -heteroaryl-LR 4 .

[0186] In some implementations, the TRPC5 inhibitory compound is represented by the structural formula (AI), (A-II) or (A-III) or its tautomers or pharmaceutically acceptable salts;

[0187]

[0188] in

[0189] R 1 and R 3 Choose from the group consisting of: H, alkyl, alkenyl, alkynyl, aryl, heterocyclic, heteroaryl, halogen, -OH, -CN, -cycloalkyl, -O-alkyl, -O-cycloalkyl, -O-aryl, -aryl-O-aryl, -CF3, -C(H)F2, alkylene-CF3, alkylene-C(H)F2, -SO2-alkyl and -O-alkylene-O-alkyl, -heterocyclic-LR 4 and -heteroaryl-LR 4 ;

[0190] R 2 -heterocyclic-LR 4 ;

[0191] R 4 The following groups are not present or are selected from: alkyl, cycloalkyl, aryl, alkylene-aryl, alkylene-heteroaryl, heteroaryl, heterocyclic, -C(O)N(R) 5 )2 and CF3;

[0192] R 5 Independently H or alkyl;

[0193] R 6Choose from the group consisting of: alkyl, cycloalkyl, aryl, heterocyclic, heteroaryl, alkylene-aryl, -C(O)N(R) 5 )2 and CF3;

[0194] L is absent or selected from the group consisting of the following: methylene, -C(O)-, -SO2-, -CH2N(Me)-, -N(R5)(R6)-, -C(R5)(R6)-, and -OR. 6 ;and

[0195] R 1 R 2 and R 3 One and only one of them is –heterocyclic-LR 4 or -heteroaryl-LR 4 .

[0196] In some embodiments, the TRPC5 inhibitory compound is a compound disclosed in international patent application PCT / US18 / 51465, filed on September 18, 2018, the entire contents of which are incorporated herein by reference.

[0197] In some embodiments, the TRPC5 inhibitory compound is selected from any of the following compounds or a pharmaceutically acceptable salt thereof:

[0198]

[0199]

[0200]

[0201] In some implementations, the TRPC5 inhibitory compound has the structural formula (I):

[0202] Or its pharmaceutically acceptable salt;

[0203] in:

[0204] "---" indicates a single or double key.

[0205] X 1 For CH or N;

[0206] When "---" is a double key, X 2 For CH or N;

[0207] When "---" is a single key, X 2 For N(CH3),

[0208] When X 1 When it is CH, X 2 It is N or N(CH3);

[0209] Y is -O-, -N(CH3)-, -N(CH2CH2OH)-, cyclopropyl-1,1-diyl or -CH(CH3)-;

[0210] 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;

[0211] When "---" is a double bond, R 13 It is hydrogen, -CH2OH, -CH(OH)-CH2OH, -NH2, -CH(OH)CH3, -OCH3 or -NH-(CH2)2OH; and R 14 Does not exist; or

[0212] When "---" is a single key, R 13 and R 14 Together they form = O; and

[0213] R 15 and R 16 Each of them is independently hydrogen or -CH3. In some embodiments, if X 1 Let N, X 2 If N is N, Y is -O- or -N(CH3)-, and Q is 2-trifluoromethylphenyl, then R 13 R 15 and R 16 At least one of them is not hydrogen.

[0214] In some embodiments, the TRPC5 inhibitory compound has structural formula (II):

[0215] Or its pharmaceutically acceptable salt; of which:

[0216] R 11 It can be chlorine, -CF3, -CHF2, or -CH3;

[0217] R 12 It is hydrogenated or fluorinated; and

[0218] R 13 It can be hydrogen, -NH2, -CH2OH or CH(OH)-CH2OH.

[0219] In some implementation schemes, R11 It is -CHF2; and R 12 It is fluorinated.

[0220] In some embodiments, the TRPC5 inhibitory compound is selected from any of the following compounds or a pharmaceutically acceptable salt thereof:

[0221]

[0222]

[0223]

[0224]

[0225] In some embodiments, the TRPC5 inhibitory compound is a compound disclosed in U.S. Provisional Patent Application No. 62 / 732,728, filed September 18, 2018, or U.S. Provisional Patent Application No. 62 / 780,553, filed December 17, 2018, the entire contents of each of which are incorporated herein by reference.

[0226] In some embodiments, the TRPC5 inhibitory compound is selected from any of the following compounds or a pharmaceutically acceptable salt thereof:

[0227]

[0228]

[0229] In some implementations, the TRPC5 inhibitory compound is the following compound or a pharmaceutically acceptable salt thereof:

[0230]

[0231] Second treatment agent

[0232] In one aspect, the present invention relates to a method for treating kidney disease, the method comprising the steps of administering a TRPC5 inhibitory compound and a second therapeutic agent to a subject in need in combination. In some embodiments, the second therapeutic agent affects biological pathways other than the TRPC5-Rac1 pathway; thus, a subject receiving such treatment may benefit from the combined effect of the different therapeutic agents.

[0233] In some implementations, the second therapeutic agent is selected from immunomodulators, calcineurin inhibitors, renin-angiotensin-aldosterone system inhibitors, antiproliferators, corticosteroids, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, sodium-glucose transporter 2 inhibitors, nuclear factor-1 (erythroid-derived 2)-like 2 agonists, chemokine receptor 2 inhibitors, chemokine receptor 5 inhibitors, and endothelin 1 receptor antagonists.

[0234] In some implementations, the second therapeutic agent is further selected from alkylating agents, adrenocorticotropic hormone agonists, dual sodium-glucose transporter 1 / 2 inhibitors, beta-blockers (such as metoprolol succinate, metoprolol tartrate, propranolol, carvedilol), mineralocorticoid receptor antagonists (such as spironolactone, eplerenone, phenidone, esalidomide, or ampalidone), loop diuretics or thiazide diuretics (such as furosemide, bumetanide, torasemide, or benzfluthiazide), calcium channel blockers (such as verapamil, diltiazem, amlodipine, or nifedipine), statins (such as atorvastatin, pravastatin, fluvastatin, lovastatin, rosuvastatin, simvastatin, or pitavastatin), short-acting, intermediate-acting, or long-acting insulin (such as NPH insulin (Humulin R, Novolin)).R, biosimilars), insulin lispro (Humalog), insulin glutathione), insulin glargine (Basaglar, Lantus), insulin detemir (Levemir), insulin degludec (Tresiba), dipeptidyl peptidase-4 inhibitors (such as sitagliptin, saxagliptin, linagliptin, vildagliptin), glucagon-like peptide-1 receptor agonists (such as exenatide, liraglutide, dulaglutide, lixilatide, abiglutide, semaglutide), sulfonylureas (such as glimepiride, glipizide, glibenclamide, chlorpropamide, tolazoline, or tolbutamide), apoptosis signal-regulated kinase-1 (such as selenokinase). Chromoplastin inhibitors (such as vorasistat (BAY1142524), selective glycation inhibitors (such as GLY-230), renin inhibitors (such as SCO-272), interleukin-33 inhibitors (such as MEDI-3506), farnesoid X receptor agonists (such as nidufluoxetine (LMB763), soluble guanylate cyclase stimulators (such as palicidalis, oliquidioxin, IW-6463, velicidalis, liociguat), thromboxane receptor antagonists (such as SER150), xanthine oxidase inhibitors (TMX-049), erythropoietin receptor agonists (such as cibutide (ARA-29)). 0) Cannabinoid receptor type 1 inverse agonists (such as nimasizumab, GFB-024, CRB-4001), NADPH oxidase inhibitors (such as APX-115), anti-vascular endothelial growth factor B (such as CSL-346), anti-fibrotic agents (such as FT011), enkephalinase inhibitors (such as TD-1439, TD-0714, sacubitril), dual CD80 / CD86 inhibitors (such as abatacept), CD40 antagonists (such as bilesumab (ASKP1240), cellular cholesterol and lipid blockers (VAR-200), PDGFR antagonists (such as ANG_3070). Slit guide ligand 2 (such as PF-06730512), APOL1 inhibitors (such as VX-147), Nrl2 activators / NF-κB inhibitors (such as bardoxolone), somatostatin receptor agonists (such as lanreotide), PPARγ agonists (such as pioglitazone), AMP-activated protein kinase stimulators (such as metformin), tyrosine kinase inhibitors (such as tercivatinib), glucoceramide synthase inhibitors (such as venglucuronide malate), arginine vasopressin receptor 2 antagonists (such as ricivatin), xanthine oxidase inhibitors (such as hydroxypurine), or vasopressin receptor 2 antagonists (such as tolvaptan).

[0235] In some implementations, the immunomodulator is rituximab. Rituximab destroys both normal cells and malignant B cells with CD20 on their surface, and is therefore used to treat diseases characterized by B cell excess, B cell hyperactivity, or B cell dysfunction; such diseases include, but are not limited to, hematologic malignancies and autoimmune diseases.

[0236] In some implementations, the immunomodulator is mycophenolate mofetil. Administration of mycophenolate mofetil can impart beneficial effects, such as suppressing the immune system and preventing organ transplant rejection.

[0237] In some implementations, angiotensin-converting enzyme (ACE) inhibitors are captopril, zolfenpril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandopril, or cilazapril. ACE inhibitors are primarily used to treat hypertension and congestive heart failure. This group of drugs causes vasodilation and a decrease in blood volume, which leads to lower blood pressure and reduced myocardial oxygen demand. They inhibit angiotensin-converting enzyme, a key component of the renin-angiotensin system. They can also be used to treat other cardiovascular and renal diseases, including but not limited to acute myocardial infarction (heart attack), heart failure (left ventricular systolic dysfunction), and renal complications of diabetes (diabetic nephropathy).

[0238] In some implementations, the angiotensin receptor blocker is losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan, or femasartan. Uses of angiotensin receptor blockers include, but are not limited to, the treatment of hypertension (high blood pressure), diabetic nephropathy (kidney damage caused by diabetes), and congestive heart failure.

[0239] In some implementations, the renin-angiotensin-aldosterone system inhibitor is aliskiren. Inhibition of the renin-angiotensin-aldosterone system can confer beneficial effects such as lowering blood pressure and improving intraglomerular hemodynamics. Renin, the first enzyme in the renin-angiotensin-aldosterone system, plays a role in blood pressure control. It cleaves angiotensinogen into angiotensin I, which is then converted to angiotensin II by angiotensin-converting enzyme (ACE). Angiotensin II has both direct and indirect effects on blood pressure. It directly causes arterial smooth muscle contraction, leading to vasoconstriction and increased blood pressure. Angiotensin II also stimulates the adrenal cortex to produce aldosterone, which leads to increased sodium reabsorption in the renal tubules, followed by an increase in water, thereby increasing plasma volume and thus increasing blood pressure. Aliskiren binds to the S3bp binding site of renin, which is essential for its activity. Binding to this pocket prevents the conversion of angiotensinogen to angiotensin I. Aliskiren is also available as a combination therapy with hydrochlorothiazide.

[0240] In some implementations, the endothelin 1 receptor antagonist is ambrisentan, atrasentan, bosentan, macitentan, or sparsentan. Antagonism of the endothelin 1 receptor can confer beneficial effects such as lowering blood pressure and improving glomerular hemodynamics. Macitentan, ambrisentan, and bosentan are primarily used to treat pulmonary hypertension, which can have a multifactorial mechanism, including chronic renal failure.

[0241] In some implementations, the antiproliferative agent is mycophenolate mofetil, mycophenolate sodium, or azathioprine. Administration of mycophenolate mofetil, mycophenolate sodium, or azathioprine can impart beneficial effects such as suppressing the immune system and preventing organ transplant rejection.

[0242] In some implementations, SGLT2 inhibitors are canagliflozin, dapagliflozin, empagliflozin, combinations of empagliflozin and linagliflozin, combinations of empagliflozin and metformin, or combinations of dapagliflozin and metformin. Inhibition of SGLT2 can confer beneficial effects such as lowering glucose and improving intraglomerular hemodynamics. SGLT2 inhibitors, also known as gliflozin, are a class of drugs that inhibit glucose reabsorption in the kidneys and thus lower blood glucose. They act by inhibiting sodium-glucose transporter 2 (SGLT2). SGLT2 inhibitors are used to treat type 2 diabetes mellitus (T2DM). In addition to glycemic control, gliflozin has shown significant cardiovascular benefits in patients with T2DM. In studies of canagliflozin, the drug was found to enhance glycemic control as well as reduce weight and systolic and diastolic blood pressure. Glucose sodium cotransporter (SGLT) is a protein primarily found in the kidneys and plays an important role in maintaining glucose homeostasis in the blood. SGLT1 and SGLT2 are the two most well-known SGLTs in this family. SGLT2 is a major transporter protein that promotes the reabsorption of glucose filtered by the glomerulus back into the circulation and is responsible for approximately 90% of renal glucose reabsorption. SGLT2 is primarily expressed in the kidneys on the epithelial cells lining the first segment of the proximal convoluted tubule. By inhibiting SGLT2, glibenclamide prevents the kidneys from reuptakeing glucose from the glomerular filtrate, subsequently lowering blood glucose levels and promoting glucose excretion in the urine (glycosuria).

[0243] In some implementations, the SGLT2 inhibitor also inhibits SGLT1. In some aspects of these implementations, the SGLT1 / 2 inhibitor is sotagliflozin.

[0244] In some implementations, calcineurin inhibitors are cyclosporine A, vorticol, or tacrolimus. Calcineurin (CaN) is a calcium and calmodulin-dependent serine / threonine protein phosphatase (also known as protein phosphatase 3 and calcium-dependent serine-threonine phosphatase). It activates T cells of the immune system and can be blocked by drugs including, but not limited to, cyclosporine, vorticol, pimecrolimus, and tacrolimus. Calcineurin is activated by dephosphorylating nuclear factor (NFATc)—a transcription factor—in the cytoplasm of activated T cells. The activated NFAC then translocates to the nucleus, where it upregulates the expression of interleukin-2 (IL-2), which in turn stimulates the growth and differentiation of T cell responses. Calcineurin inhibitors such as tacrolimus are used to suppress the immune system of organ allogeneic transplant recipients to prevent rejection of transplanted tissues.

[0245] In some implementations, the nuclear factor-1 (erythroid-derived 2)-like 2 agonists are bardosulon or CXA-10. Agonism of nuclear factor-1 (erythroid-derived 2)-like 2 can confer beneficial effects such as anti-inflammatory activity. Nuclear factor-1 (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2, is a transcription factor encoded by the NFE2L2 gene in the human body. Nrf2 is a basic leucine zipper (bZIP) protein that regulates the expression of antioxidant proteins that protect against oxidative damage caused by injury and inflammation. Several drugs stimulating the NFE2L2 pathway are being investigated for the treatment of diseases caused by oxidative stress. Heme oxygenase-1 (HMOX1, HO-1) is an enzyme that catalyzes the breakdown of heme into the antioxidant biliverdin, the anti-inflammatory agent carbon monoxide, and iron. HO-1 is a target gene of Nrf2 and has been shown to protect against a variety of pathological conditions, including sepsis, hypertension, atherosclerosis, acute lung injury, kidney injury, and pain.

[0246] In some implementations, chemokine receptor 2 inhibitors are PF-04136309, ccx140, or propagidium (DMX-200). Inhibition of chemokine receptor 2 can confer beneficial effects such as suppression of the immune system. Chemokine receptor 2 (CCR2)-mediated recruitment of monocytes and other inflammatory cells is associated with the etiology of diabetic nephropathy, and inhibition of CCR2 can reduce albuminuria and prevent renal function decline in patients with diabetic nephropathy.

[0247] In some implementations, the second therapeutic agent is an NR12 activator / NF-κB inhibitor (such as bardoxazoline), a somatostatin receptor agonist (such as lanreotide), a PPARγ agonist (such as pioglitazone), an AMP-activated protein kinase stimulator (such as metformin), a tyrosine kinase inhibitor (such as tercivatinib), a glucocorticoid synthase inhibitor (such as venglucuronide malate), an arginine vasopressin receptor 2 antagonist (such as lixivatan), a xanthine oxidase inhibitor (such as hydroxypurine), or a vasopressin receptor 2 antagonist (such as tolvaptan). Each of these agents has been approved or is in human clinical trials for the treatment of polycystic kidney disease, particularly autosomal dominant polycystic kidney disease.

[0248] In some implementations, the second therapeutic agent is tacrolimus, cyclosporine A, rituximab, mycophenolate mofetil, corticosteroids (such as prednisone), sparsentan, enalapril, or losartan. In some implementations, the second therapeutic agent is vorciclosporine. In some implementations, the second therapeutic agent is enalapril, losartan, or cyclosporine A. Corticosteroids are a class of steroid hormones produced in the adrenal cortex of vertebrates, and synthetic analogues of these hormones. The two main classes of corticosteroids, glucocorticoids and mineralocorticoids, are involved in a wide range of physiological processes, including stress response, immune response and regulation of inflammation, carbohydrate metabolism, protein catabolism, blood electrolyte levels, and behavior. Mineralocorticoids such as aldosterone are involved in the regulation of electrolyte and water balance primarily by modulating ion transport in the renal tubular epithelial cells. Systemic corticosteroids are also used to treat diseases and conditions such as nephrotic syndrome, organ transplantation, adrenal insufficiency, and congenital adrenal hyperplasia.

[0249] In some embodiments, the compounds of the present invention may be racemic. In some embodiments, the compounds of the present invention may be enriched with one enantiomer. For example, the compounds of the present invention may have greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 95% or greater of ee.

[0250] The compounds of the present invention have more than one stereoisomer. Therefore, the compounds of the present invention can be enriched with one or more diastereomers. For example, the compounds of the present invention can have greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, or even 95% or greater of de. In some embodiments, the compounds of the present invention have essentially one isomer configuration at one or more stereoisomer centers and multiple isomer configurations at the remaining stereoisomer centers.

[0251] In some embodiments, the enantiomer excess of the stereocenter 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 greater.

[0252] As used in this article, the stereochemistry of compounds in which single bonds do not indicate their stereochemistry is drawn without stereochemistry.

[0253] As used herein, cleaving bonds or bold non-wedge bonds indicate relative rather than absolute stereochemical configurations (e.g., without distinguishing between enantiomers of a given diastereomer).

[0254] As used in this article, cleaving bonds or bold wedges indicate the absolute stereochemical configuration.

[0255] In some embodiments, the present invention relates to pharmaceutical compositions comprising the compounds of the present invention and pharmaceutically acceptable carriers. In some embodiments, therapeutic formulations or pharmaceutical compositions enriched with the compounds of the present invention may provide primarily one enantiomer of the compound. The enantiomer-enriched mixture may contain, for example, at least 60 mol%, or more preferably at least 75 mol%, 90 mol%, 95 mol%, or even 99 mol% of one enantiomer. In some embodiments, the compound enriched with one enantiomer is substantially free of the other enantiomer, wherein substantially free means that the substance in question accounts for less than 10%, or less than 5%, or less than 4%, or less than 3%, or less than 2%, or less than 1% of the other enantiomer compared to, for example, the amount in the composition or compound mixture. For example, if a composition or compound mixture contains 98 grams of a first enantiomer and 2 grams of a second enantiomer, then it can be said to contain 98 mol% of the first enantiomer and only 2% of the second enantiomer.

[0256] In some embodiments, a therapeutic formulation or pharmaceutical composition may be enriched to primarily provide a diastereomer of the compound of the present invention. The mixture enriched with diastereomers may contain, for example, at least 60 mol%, or more preferably at least 75 mol%, 90 mol%, 95 mol%, or even 99 mol% of a diastereomer.

[0257] Treatment

[0258] Non-selective Ca 2+-Permeable transient receptor potential (TRP) channels act as sensors, transducing 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). The dynamic rearrangement of the actin cytoskeleton depends on spatiotemporally controlled Ca2+. 2+ Inflow (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) and small GTPases RhoA and Rac1 act as key regulators of these changes (Etienne-Manneville and Hall, Nature 420, 629-635, 2002; Raftopoulou and Hall, Dev Biol 265, 23-32, 2004). RhoA induces the formation of stress fibers and adhesion plaques, while Rac1 mediates the formation of lamellar pseudopodia (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). Transient receptor potential (TRPC) cation channel subfamily C member 5 (TRPC5) works synergistically with TRPC6 to regulate Ca2+ influx, actin remodeling, and cell migration in renal podocytes and fibroblasts. TRPC5-mediated Ca2+ influx... 2+ Influx increases Rac1 activity, while TRPC6-mediated Ca2+ influx promotes RhoA activity. Gene silencing of the TRPC6 channel eliminates stress fibers and reduces focal contact, resulting in an active migratory cell phenotype. In contrast, gene silencing of the TRPC5 channel rescues stress fiber formation, resulting in a contractile cell phenotype. The results presented in this paper reveal a conserved signaling mechanism by which TRPC5 and TRPC6 channels control tightly regulated cytoskeleton dynamics homeostasis through differential coupling with Rac1 and RhoA.

[0259] Ca 2+--dependent actin-dependent cytoskeleton remodeling is a dynamic process driving cell migration (Wei et al., Nature 457, 901-905, 2009). RhoA and Rac1 act as switches responsible for cytoskeleton 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 active cell phenotype, while RhoA activity promotes the contractile phenotype (Etienne-Manneville and Hall, Nature 420, 629-635, 2002). 2+ It plays an important role in the regulation of small GTPases (Aspenstrom et al., Biochem J 377, 327-337, 2004). 2+ Spatially and temporally confined scintillation of Ca2+ accumulates near the front of migrating cells (Wei et al., Nature 457, 901-905, 2009). Therefore, the Ca2+ microdomains confer local bursts of Rac1 activity (Gardiner et al., Curr Biol 12, 2029-2034, 2002; Machacek et al., Nature 461, 99-103, 2009) as key events at the front. To date, the source of Ca2+ influx responsible for GTPase regulation remains largely elusive. Transient receptor potential (TRP) channels generate temporally and temporally confined Ca2+ associated with cell migration in fibroblasts and neuronal growth cones. 2+ Signal 0. Specifically, TRPC5 channels are known regulators of neuronal growth cone-guided neuronal pathways, and their activity in neurons depends on the activity of PI3K and Rac1 (Bezzerides et al., Nat Cell Biol 6, 709-720, 2004).

[0260] Podocytes are neuron-like cells that originate from the metarenal mesenchyme of the glomerulus in the kidney and are essential for the formation of renal filters (Somlo and Mundel, Nat Genet. 24, 333-335, 2000; Fukasawa et al., J Am Soc Nephrol 20, 1491-1503, 2009). Podocytes exhibit a highly precise cytoskeletal adaptation 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 are characterized by dysregulation of the 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 Ca2+ 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 onset of proteinuria, albumin loss into the Bowman's capsule, and eventual renal failure (Tryggvason and Wartiovaara, N Engl J Med 354, 1387-1401, 2006). The vasoactive hormone angiotensin II induces Ca2+ homeostasis in podocytes. 2+Ca2+ influx and prolonged treatment lead to stress fiber loss (Hsu et al., J Mol Med 86, 1379-1394, 2008). Although there is a recognized association between Ca2+ influx and cytoskeleton remodeling, the mechanisms by which podocytes sense and transduce extracellular cues that regulate cell shape and activity remain elusive. Mutations in the TRP canonical 6 (TRPC6) channel have been 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 regulating 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). TRPC5 channels antagonize TRPC6 channel activity to control tightly regulated cytoskeleton dynamics homeostasis through differential coupling with various small GTPases.

[0261] proteinuria

[0262] Proteinuria is a pathological condition in which protein is present in the urine. Albuminuria is a type of proteinuria. Microalbuminuria occurs when small amounts of albumin leak into the urine from the kidneys. In a normally functioning body, albumin is not normally present in urine because it is held in the bloodstream by the kidneys. Microalbuminuria can be diagnosed from 24-hour urine collections (20 μg / min to 200 μg / min) or, more commonly, from concentrations that rise at at least two points (30 mg / L to 300 mg / L). Microalbuminuria can be a precursor to diabetic nephropathy. Albumin levels exceeding these values ​​are called macroalbuminuria. Subjects with certain conditions, such as diabetic nephropathy, can progress from microalbuminuria to macroalbuminuria and reach nephrotic ranges (>3.5 g / 24 hours) when the kidney disease is advanced.

[0263] Causes of proteinuria

[0264] Proteinuria can be associated with a variety of diseases, including focal segmental glomerulosclerosis, IgA nephropathy, diabetic nephropathy, lupus nephritis, membranoproliferative glomerulonephritis, progressive (crescent) glomerulonephritis, and membranous glomerulonephritis.

[0265] A. Focal segmental glomerulosclerosis (FSGS)

[0266] Focal segmental glomerulosclerosis (FSGS) is a disease that attacks the kidney's filtration system (glomeruli), leading to severe scarring. FSGS is one of many causes of a condition known as nephrotic syndrome, which occurs when proteins in the blood leak into the urine (proteinuria). Primary FSGS, when the underlying cause cannot be found, usually presents as nephrotic syndrome. Secondary FSGS, when the underlying cause is identified, usually presents as kidney failure and proteinuria. FSGS can be hereditary; several known hereditary causes of FSGS exist.

[0267] For patients with FSGS, there are few available treatments. Many patients are treated with steroid regimens, most of which have very serious side effects. Some patients have shown positive responses to immunosuppressive drugs and blood pressure medications that have been shown to lower protein levels in urine. To date, there is no universally accepted effective treatment or cure, and there are no FDA-approved drugs for the treatment of FSGS. Therefore, there is a need for more effective methods to reduce or suppress proteinuria.

[0268] B. IgA nephropathy

[0269] IgA nephropathy (also known as IgA nephritis, IgAN, Bergey's disease, and pharyngitis-associated glomerulonephritis) is a form of glomerulonephritis (inflammation of the glomeruli in 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 associated with glomerular IgA deposition exist, the most common being Henscher's purpura (HSP), which is considered by many to be a systemic form of IgA nephropathy. Henscher's purpura presents with a characteristic purpuric rash, arthritis, and abdominal pain and is more common in young adults (16-35 years old). HSP is associated with a better prognosis than IgA nephropathy. In IgA nephropathy, 25%-30% of cases slowly progress to chronic renal failure within 20 years.

[0270] C. Diabetic nephropathy

[0271] Diabetic nephropathy, also known as Kimmelstiel-Wilson syndrome and intercapillary glomerulonephritis, is a progressive kidney disease caused by vascular lesions in the capillaries of the kidney glomeruli. It is characterized by nephrotic syndrome and diffuse glomerulosclerosis. This is due to 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 kidneys may begin to allow serum albumin in the urine to be higher than normal. As diabetic nephropathy progresses, more and more glomeruli are destroyed by nodular glomerulosclerosis and the amount of albumin excreted in the urine increases.

[0272] D. Lupus nephritis

[0273] Lupus nephritis is a kidney disorder and a complication of systemic lupus erythematosus (SLE). It occurs when antibodies and complement accumulate in the kidneys, causing inflammation. It typically causes proteinuria and can rapidly progress to kidney failure. Nitrogenous waste accumulates in the blood. SLE causes various diseases affecting the internal structures of the kidneys, including interstitial nephritis. Approximately 3 out of every 10,000 people are affected by lupus nephritis.

[0274] E. Membranoproliferative glomerulonephritis I / II / III

[0275] Membranoproliferative glomerulonephritis is a type of glomerulonephritis caused by deposits in the glomerular mesangium and thickening of the basement membrane, activation of complement, and damage to the glomeruli. Three types of membranoproliferative glomerulonephritis exist. Type I is caused by the deposition of immune complexes in the kidneys and is thought to be associated with the classical complement pathway. Type II is similar to Type I but is believed to be associated with the alternative complement pathway. Type III is very rare and is characterized by a mixture of subepithelial deposits and the typical pathological findings of Type I disease.

[0276] Based on immunofluorescence microscopy, two main types of MPGN exist: immune complex-mediated and complement-mediated. Hypocomplementemia is common in all types of MPGN. In immune complex-mediated MPGN, complement activation occurs via the classical pathway and is typically manifested as normal or slightly decreased serum C3 and low serum C4 concentrations. In complement-mediated MPGN, low serum C3 and normal C4 levels are usually present due to activation of the alternative pathway. However, normal serum C3 concentrations do not rule out complement-mediated MPGN, and it is not uncommon to find normal C3 concentrations in adults with dense deposit disease (DDD) or C3 glomerulonephritis (C3GN).

[0277] C3 glomerulonephritis (C3GN) presents as glomerulonephritis on light microscopy (LM), with bright C3 staining and the absence of C1q, C4, and immunoglobulins (Ig) on ​​immunofluorescence microscopy (IF), and shows mesangial and / or subendothelial electron-dense deposits on electron microscopy (EM). Intramembranous and subepithelial deposits are also occasionally present. The term "C3 glomerulonephropathy" is often used to include both C3GN and dense deposit disease (DDD), both caused by dysregulation of the complement alternative pathway (AP). C3GN and DDD can be difficult to distinguish from each other in LM and IF studies. However, EM shows mesangial and / or subendothelial, intramembranous, and subepithelial deposits in C3GN, while dense osmiophilic deposits are present along the glomerular basement membrane (GBM) and in the mesangium in DDD. Both C3GN and DDD differ from immune complex-mediated glomerulonephritis in the lack of immunoglobulin staining on IF. (Sethi et al., Kidney Int. (2012) 82(4): 465-473).

[0278] F. Progressive (crescentic) glomerulonephritis

[0279] Progressive (crescentic) glomerulonephritis (PG) is a kidney syndrome that, if left untreated, rapidly progresses to acute renal failure and death within months. In 50% of cases, PG is associated with underlying diseases such as Goodpasture's syndrome, systemic lupus erythematosus, or Wegener's granulomatosis; the remainder are idiopathic. Regardless of the underlying cause, PG involves severe damage to the glomeruli of the kidneys, many of which contain characteristic crescent-shaped scars. Patients with PG present with hematuria, proteinuria, and occasionally hypertension and edema. Although the degree of proteinuria may occasionally exceed 3 g / 24 hours, which is within the range associated with nephrotic syndrome, the clinical presentation is consistent with renal syndrome. Untreated disease may progress to decreased urine volume (oliguria), which is associated with impaired renal function.

[0280] G. Membranous glomerulonephritis

[0281] Membranous glomerulonephritis (MGN) is a slowly progressive kidney disease that primarily affects patients aged 30 to 50, typically Caucasians. It can develop into nephrotic syndrome. MGN is caused by circulating immune complexes. Current research indicates that most immune complexes are formed by antibodies binding to in situ antigens on the glomerular basement membrane. These antigens can be endogenous to the basement membrane or deposited from systemic circulation.

[0282] H. Allport syndrome

[0283] Allport syndrome is a genetic disorder affecting 1 in 5,000–10,000 children, characterized by glomerulonephritis, end-stage renal disease, and hearing loss. Allport syndrome can also affect the eyes, but these changes usually do not affect vision unless lens changes occur later in life. Hematuria is common. Proteinuria is characteristic of the progression of kidney disease.

[0284] I. Hypertensive Nephropathy

[0285] Hypertensive kidney disease (hypertensive nephrosclerosis (HN or HNS) or hypertensive nephropathy (HN)) is a medical condition involving kidney damage caused by chronic hypertension. HN can be classified into two types: benign and malignant. Benign nephrosclerosis is common in individuals over 60 years of age, while malignant nephrosclerosis is less common and affects 1%–5% of individuals with hypertension having a diastolic blood pressure exceeding 130 mmHg. Signs and symptoms of chronic kidney disease may include loss of appetite, nausea, vomiting, itching, somnolence or confusion, weight loss, and bad breath. Chronic hypertension damages kidney tissue; this includes small blood vessels, glomeruli, tubules, and interstitial tissue. The tissue hardens and thickens, a condition known as nephrosclerosis. Narrowing of blood vessels means less blood enters the tissue, and therefore less oxygen reaches the tissue, leading to tissue death (local ischemia).

[0286] J. Nephrotic syndrome

[0287] Nephrotic syndrome is a range of symptoms caused by kidney damage. These include protein in the urine, low blood albumin levels, high blood lipids, and significant swelling. Other symptoms may include weight gain, fatigue, and foamy urine. Complications may include blood clotting, infections, and high blood pressure. Causes include many kidney diseases such as focal segmental glomerulosclerosis, membranous nephropathy, and minimal change disease. It can also occur as a complication of diabetes or lupus. The underlying mechanism usually involves damage to the glomeruli of the kidneys. Diagnosis is usually based on urine tests and sometimes on kidney biopsy. It differs from nephritic syndrome in that there are no red blood cells in the urine. Nephrotic syndrome is characterized by massive proteinuria (>3.5 g / 1.73 m² body surface area / day, or >40 mg / m² body surface area / hour in children), hypoalbuminemia (<2.5 g / dL), hyperlipidemia, and edema starting in the face. Lipouria (lipids in the urine) may also occur, but is not necessary for the diagnosis of nephrotic syndrome. Hyponatremia can also occur due to low sodium excretion. Hereditary forms of nephrotic syndrome are often resistant to steroids and other immunosuppressive therapies. The goals of treatment are to control proteinuria and swelling, provide good nutrition to allow the child to grow, and prevent complications. Early and aggressive treatment is used to manage the condition.

[0288] K. Minimal Change Disease

[0289] Minimal change disease (MCD, minimal change glomerulonephropathy, and disease-free disease) is a kidney disease that causes nephrotic syndrome. Clinical signs of minimal change disease include proteinuria (abnormal excretion of protein, primarily albumin, into the urine), edema (swelling of soft tissues due to water retention), weight gain, and hypoalbuminemia (low serum albumin). These signs are collectively referred to as nephrotic syndrome. The first clinical sign of minimal change disease is usually edema, accompanied by associated weight gain. The swelling can be mild, but patients may present with edema in the lower half of the body, periorbital edema, scrotal / labial area swelling, and in more severe cases, generalized edema. In older adults, patients may also develop acute kidney injury (20%–25% of affected adults) and hypertension. Due to the disease course, patients with minimal change disease are also at risk of blood clotting and infection.

[0290] L. membranous nephropathy

[0291] Membranous nephropathy is characterized by the deposition of immune complexes on the glomerular basement membrane (GBM), resulting in GBM thickening. The cause is usually unknown (idiopathic), but secondary causes include medications, infections, autoimmune diseases, and cancer. Presenting symptoms include insidious onset of edema and severe proteinuria, accompanied by benign urinary sediment, normal renal function, and normal or elevated blood pressure. Membranous nephropathy is diagnosed via renal biopsy. Spontaneous remission is common. Treatment for patients at high risk of progression typically involves corticosteroids and cyclophosphamide or chlorambucil.

[0292] M. Post-infectious glomerulonephritis

[0293] Acute proliferative glomerulonephritis is a disorder of the glomeruli (nephropathy) or small blood vessels in the kidneys. It is a common complication of bacterial infections, typically caused by skin infections of Streptococcus types 12, 4, and 1 (impetigo), but it can also occur after streptococcal pharyngitis, hence it is also called post-infectious or post-streptococcal glomerulonephritis. It may be a risk factor for future albuminuria. In adults, signs and symptoms of infection may still be present when kidney problems occur, and terms such as infection-associated glomerulonephritis or bacterial infection-associated glomerulonephritis are also used. Acute glomerulonephritis caused 19,000 deaths in 2013, down from 24,000 worldwide in 1990. Acute proliferative glomerulonephritis (post-streptococcal glomerulonephritis) is caused by a streptococcal bacterial infection, usually three weeks after infection, typically in the pharynx or skin, taking into account the time required for antibody and complement protein production. The infection causes inflammation of the blood vessels in the kidneys, which impairs the kidney organ's ability to filter urine. [Citation needed] Acute proliferative glomerulonephritis is most common in children.

[0294] N. Thin basement membrane disease

[0295] Thin basement membrane disease (TBMD, also known as benign familial hematuria and thin basement membrane nephropathy or TBMN), along with IgA nephropathy, is the most common cause of hematuria without other symptoms. The only abnormal finding in this disease is thinning of the glomerular basement membrane in the kidneys. Its importance lies in its benign prognosis, with patients maintaining normal kidney function throughout their lives. Most patients with TBMD are discovered incidentally during urinalysis to have microscopic hematuria. Blood pressure, kidney function, and urinary protein excretion are usually normal. Mild proteinuria (less than 1.5 g / day) and hypertension are seen in a minority of patients. Frank hematuria and back pain should prompt an investigation for other causes, such as kidney stones or back pain-hematuria syndrome. Furthermore, the absence of systemic manifestations, therefore the presence of hearing or visual impairment, should prompt an investigation for hereditary nephritis, such as Allport syndrome. Some patients with TBMD are considered carriers of the gene that causes Allport syndrome.

[0296] O. Mesangial proliferative glomerulonephritis

[0297] Mesangial proliferative glomerulonephritis is a type of glomerulonephritis primarily associated with the mesangium. There is some evidence that interleukin-10 may inhibit it in animal models. [2] The World Health Organization (WHO) classifies it as lupus nephritis type II. Mesangial cells in the glomeruli absorb and degrade circulating immunoglobulins via endocytosis. This normal process stimulates mesangial cell proliferation and matrix deposition. Therefore, during periods of elevated circulating immunoglobulins (i.e., lupus and IgA nephropathy), one would expect to see an increase in the number of mesangial cells and matrix in the glomeruli. This is characteristic of nephritis syndromes.

[0298] P. Amyloidosis (primary)

[0299] Amyloidosis is a group of diseases in which abnormal proteins called amyloid fibrils accumulate in tissues. [4] Symptoms vary depending on the type and are usually variable. [2] They can include diarrhea, weight loss, feeling tired, swelling of the tongue, bleeding, numbness, weakness when standing, swelling of the legs, or splenomegaly. [2] There are about 30 different types of amyloidosis, each caused by the misfolding of a specific protein. [5] Some are hereditary, while others are acquired. [3] They are classified into local and systemic forms. [2] The four most common systemic types are light chain (AL), inflammatory (AA), dialysis (Aβ2M), and genetically related to old age (ATTR). Primary amyloidosis refers to amyloidosis in which no related clinical symptoms have been identified.

[0300] Q.c1q Nephropathy

[0301] C1q nephropathy is a rare glomerular disease characterized by mesangial C1q deposits observed under an immunofluorescence microscope. It is histologically defined and poorly understood. The optical microscopic features are heterogeneous, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), and proliferative glomerulonephritis. Clinical presentations are also diverse, ranging from asymptomatic hematuria or proteinuria in children and adults to Frankel's nephritis or nephrotic syndrome. Hypertension and renal insufficiency are common findings at diagnosis. Optimal treatment is unclear and is usually guided by the underlying optical microscopic lesions. Corticosteroids are the primary treatment, while immunosuppressants are reserved for steroid-resistant cases. The presence of nephrotic syndrome and FSGS appears to foreshadow adverse outcomes, contrary to the favorable outcomes in patients with MCD. (Devasahayam et al., “C1qNephropathy: The Unique Underrecognized Pathological Entity,” Analytical Cellular Pathology, Vol. 2015, Article ID 490413, 5 pages, 2015. https: / / doi.org / 10.1155 / 2015 / 490413.)

[0302] R. Anti-GBM disease

[0303] Antiglomerular basement membrane (GBM) disease, also known as Goodpasser's disease, is a rare condition that causes inflammation of small blood vessels in the kidneys and lungs. Antiglomerular basement membrane (GBM) antibodies primarily attack the kidneys and lungs, but systemic symptoms such as malaise, weight loss, fatigue, fever, and chills are also common, as are joint pain and aches. 60% to 80% of patients experience simultaneous lung and kidney involvement; 20%–40% have only kidney involvement, and less than 10% have only lung involvement. Lung symptoms usually precede kidney symptoms and typically include hemoptysis, chest pain (in less than 50% of cases), cough, and shortness of breath. Kidney symptoms typically include hematuria, protein in urine, unexplained swelling of the extremities or face, high blood urea levels, and hypertension. GPS induces abnormal production of antiGBM antibodies through plasma cells in the blood. AntiGBM antibodies attack the alveoli and glomerular basement membranes. These antibodies bind their reactive epitopes to the basement membrane and activate the complement cascade, leading to the death of labeled cells. It also involves T cells. It is generally considered a type II hypersensitivity reaction.

[0304] S. Polycystic kidney disease

[0305] Polycystic kidney disease (PKD) is a rare, progressive kidney disease and a leading cause of chronic kidney disease. PKD accounts for 7%–10% of patients with end-stage renal disease (ESRD). Approximately half of all PKD patients develop ESRD between the ages of 40 and 60. PKD affects all racial groups and is generally a slightly more progressive disease in men. There are two main types of PKD—autosomal dominant PKD (ADPKD) and autosomal recessive PKD (ARPKD). The former is more common, while the latter is often a pediatric condition with a more severe and accelerated course. Kidney cysts are a defining characteristic of PKD. Patients with PKD have an increased risk of hypertension, cardiovascular events (CV), aneurysms, liver cysts, pyelonephritis, and pain.

[0306] Measurement of urine protein levels

[0307] Protein levels in urine can be measured using methods known in the art. Until recently, accurate protein measurement required 24-hour urine collection. During this 24-hour collection, the patient urinates into a container that is kept refrigerated between bathroom breaks. The patient is instructed to begin collecting urine after their first bathroom break in the morning. Every drop of urine collected for the remainder of the day should be collected in the container. The next morning, the patient adds their first urination after waking up, completing the collection.

[0308] Recently, researchers have discovered that a single urine sample can provide the necessary information. In a newer technique, the amount of albumin in a urine sample is compared to the amount of creatinine, a waste product of normal muscle breakdown. The measurement is called the urine albumin-to-creatinine ratio (UACR). A urine sample containing more than 30 milligrams of albumin per gram of creatinine (30 mg / g) is a warning sign of a potential problem. If a lab test shows a level exceeding 30 mg / g, another UACR test should be performed after 1 to 2 weeks. If the second test also shows high levels of protein, the person has persistent proteinuria, a sign of declining kidney function, and additional tests to assess kidney function should be performed.

[0309] A test that measures blood creatinine levels also indicates whether a subject's kidneys are effectively removing waste. High blood creatinine levels are a sign of kidney damage. Physicians can use creatinine measurements to estimate how effectively the kidneys filter blood. This calculation is called estimating the glomerular filtration rate, or eGFR. Chronic kidney disease is present when the eGFR is less than 60 ml / min.

[0310] TRPC5

[0311] TRPCs are a family of transient receptor potential ion channels in animals. TRPC5 is a subtype of the TRPC family of transient receptor potential ion channels in mammals. Table 1 below highlights three examples of TRPC5.

[0312] Table 1

[0313]

[0314] Therefore, in some 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 to a subject in need a therapeutically effective amount of a compound of the present invention (e.g., a compound of structural formula I) or a pharmaceutical composition comprising said compound.

[0315] In some implementation schemes, the disease is kidney disease, anxiety disorder, depression, cancer, or diabetic retinopathy.

[0316] In some implementations, the disease or condition is selected from the following kidney diseases: focal segmental glomerulosclerosis (FSGS), diabetic nephropathy, Allport syndrome, hypertensive nephropathy, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change disease, 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 nephropathy, rapidly progressive GN, anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, or IgA nephropathy. In some implementations, the kidney disease is a proteinuric kidney disease. In some implementations, the kidney disease is a microalbuminuria or macroalbuminuria kidney disease.

[0317] In some implementation schemes, the disease or condition to be treated is pulmonary hypertension.

[0318] In some implementations, the disease or condition to be treated is pain selected from neuropathic pain and visceral pain.

[0319] In some implementations, the disease or condition is selected from chemoresistant breast cancer, doxorubicin-resistant breast cancer, chemoresistant colorectal cancer, medulloblastoma, and tumor angiogenesis cancer.

[0320] The present invention also provides a method for treating anxiety disorders, depression, or cancer or reducing the risk of their development, the method comprising administering to a subject in need a therapeutically effective amount of a compound of the present invention (e.g., a compound of formula I) or a pharmaceutical composition comprising said compound.

[0321] In some implementation schemes, the disease or condition to be 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, prediabetes, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).

[0322] Subjects awaiting treatment

[0323] In one aspect of the invention, subjects are selected based on whether they have kidney disease, pulmonary hypertension, anxiety disorder, depression, cancer, diabetic retinopathy, or pain, or are at risk of developing these diseases. In another aspect, subjects are selected based on whether they have kidney disease, anxiety disorder, depression, cancer, or diabetic retinopathy, or are at risk of developing these diseases. In yet another aspect of the invention, subjects are selected based on whether they have 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, prediabetes, metabolic syndrome, nonalcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), or are at risk of developing these diseases.

[0324] Subjects with proteinuria or at risk of developing proteinuria included those with diabetes, hypertension, or certain family history of the condition. In the United States, diabetes is the leading cause of end-stage renal disease (ESRD). In both type 1 and type 2 diabetes, albumin in the urine is one of the earliest 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 people with hypertension is an indicator of declining kidney function. If hypertension is not controlled, the person may progress to complete kidney failure. African Americans are more likely than white people to have hypertension and therefore develop kidney problems, even if their blood pressure is only slightly elevated. Other groups at risk of proteinuria include Native Americans, Hispanic / Latino Americans, Pacific Islanders, older adults, and overweight subjects.

[0325] In one aspect of the invention, subjects were selected based on whether they had proteinuria or were at risk of developing proteinuria. Subjects with proteinuria or at risk of developing proteinuria are those who exhibit one or more symptoms of the condition. Symptoms of proteinuria are known to those skilled in the art and include, but are not limited to, a large amount of protein in the urine, which may cause the urine to appear foamy in the bathroom. Significant protein loss can lead to edema, which may occur in the hands, feet, abdomen, or face. These are signs of significant protein loss and indicate that kidney disease has progressed. Laboratory testing is the only way to determine the presence of protein in a subject's urine before extensive kidney damage occurs.

[0326] This method is effective for a variety of subjects, including mammals such as humans and other animals such as laboratory animals like mice, rats, rabbits, or monkeys, or domesticated and farm animals 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.

[0327] Example

[0328] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0329] Example 1: Synthesis of Compound 100

[0330]

[0331] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0332] DBU (451.5 mg, 2.97 mmol, 2.00 equivalent) was added to a stirred solution of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (400 mg, 1.48 mmol, 1 equivalent) and 4-fluoro-2-(trifluoromethyl)phenol (400.6 mg, 2.22 mmol, 1.5 equivalent) in acetonitrile (10 mL). The resulting mixture was stirred at 80 °C for 2 h. 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 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 / EtOAc 2:1) to give 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (110 mg, 17.94%) as a brown solid.

[0333] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0334] TFA (1 mL, 13.46 mmol, 50.59 equivalents) was added to a stirred solution of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (110 mg, 0.27 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized 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 give 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (50 mg, 59.98%) as a brown solid.

[0335] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0336] At room temperature, 4,5-dichloro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (50 mg, 0.16 mmol, 1 equivalent) was added to a stirred solution of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (1.19 mg, 1.19 equivalent) in DIEA (2 mL). The resulting mixture was stirred at 100 °C for 2 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by preparative TLC (PE / EtOAc 2:1) to give 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (40 mg, 47.65%) as a brown solid.

[0337] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0338] TFA (1 mL, 13.46 mmol, 177.00 equivalent) was added dropwise to a stirred solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (40 mg, 0.08 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized 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: XBridgePrep OBD C18 column 30×150mm 5um; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (8.6 mg, 25.59%) as a white solid.

[0339] Example 2 Synthesis of Compound 140

[0340]

[0341] 4-Bromo-5,6,7,8-Tetrahydro-1,7-Naphthyl-7-carboxylic acid tert-butyl ester

[0342] Boc₂O (512.13 mg, 2.347 mmol, 2.00 equivalents) and TEA (474.90 mg, 4.693 mmol, 4 equivalents) were added to a solution of 4-bromo-5,6,7,8-tetrahydro-1,7-naphthylidine (250 mg, 1.173 mmol, 1 equivalent) in THF (10 mL, 123.430 mmol, 105.20 equivalents) 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 give tert-butyl 4-bromo-5,6,7,8-tetrahydro-1,7-naphthylidine-7-carboxylic acid (210 mg, 57.15%) as a pale yellow oil.

[0343] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyl-7-carboxylic acid tert-butyl ester

[0344] 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 to a solution of 4-bromo-5,6,7,8-tetrahydro-1,7-naphthyl-7-carboxylic acid tert-butyl ester (210 mg, 0.671 mmol, 1 equivalent) and 4-fluoro-2-(trifluoromethyl)phenol (241.52 mg, 1.341 mmol, 2 equivalents) in DMSO (10 mL). After stirring at 120 °C under a nitrogen atmosphere for 4 hours, the resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC and eluted with PE / EA (5 / 1) to give 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidine-7-carboxylic acid tert-butyl ester (100 mg, 36.17%) as a pale yellow solid.

[0345] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidine

[0346] TFA (414.75 mg, 3.637 mmol, 10 equivalents) was added to a solution of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthyl-7-carboxylic acid tert-butyl ester (150 mg, 0.364 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 432.46 equivalents) 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 for the next step.

[0347] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0348] A mixture of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidine (60 mg, 0.192 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (47.86 mg, 0.192 mmol, 1.00 equivalent) in DIEA (49.67 mg, 0.384 mmol, 2 equivalent) was stirred at 100 °C under a nitrogen atmosphere for 2 hours. The residue was purified by preparative TLC (PE / EA 1 / 1) to give 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 99.15%) as a pale yellow solid.

[0349] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidin-7-yl]-2,3-dihydropyridazin-3-one

[0350] TFA (217.23 mg, 1.905 mmol, 10.00 equivalent) was added to a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 0.191 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 825.67 equivalents). 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 min; 220 nm; Rt: 6.63 min) to obtain 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5,6,7,8-tetrahydro-1,7-naphthidin-7-yl]-2,3-dihydropyridazin-3-one (42.9 mg, 51.09%) as a white solid.

[0351] Example 3 Synthesis of Compound 120

[0352]

[0353] 2-Benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine

[0354] At room temperature, 4-fluoro-2-(trifluoromethyl)phenol (89.10 mg, 0.495 mmol, 0.6 equivalent) and CuI (94.22 mg, 0.495 mmol, 0.6 equivalent) were added to a stirred mixture of 2-benzyl-5-bromo-1,2,3,4-tetrahydro-2,6-naphthidine (250 mg, 0.825 mmol, 1 equivalent) and 2-(dimethylamino)acetic acid (170.05 mg, 1.649 mmol, 2.00 equivalent) in DMSO (5 mL). Then, Cs₂CO₃ (1074.59 mg, 3.298 mmol, 4 equivalent) was added at room temperature. The final reaction mixture was irradiated with microwave at 120 °C for 1 hour. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The crude product was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge Prep OBD C18 column 30×150mm 5um; mobile phase A: water (10mMNH4HCO3); mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 18% B to 35% B over 8 min; 220nm; Rt: 7.12min) to obtain 2-benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine (180mg, 54.25%) as a brown solid.

[0355] 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine

[0356] Pd / C (20 mg) was added to a stirred solution of 180 mg 2-benzyl-5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine in 10 mL MeOH 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 LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (DCM / MeOH 12:1) to give 100 mg 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine as a brown solid.

[0357] 4-Chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0358] Add 4,5-dichloro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine (100 mg, 0.320 mmol, 1 equivalent) to a stirred solution of 5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidine (100 mg, 0.320 mmol, 1 equivalent) in DIEA (0.1 mL) at room temperature. Stir the resulting mixture at 90 °C for 1 hour. Monitor the reaction by LCMS. Allow the mixture to cool to room temperature. The residue was purified by preparative TLC (DCM / MeOH; 12:1) to give 4-chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (130 mg, 77.34%) as a white solid.

[0359] 4-Chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidin-2-yl]-2,3-dihydropyridazin-3-one

[0360] TFA (1 mL) was added to a stirred solution of 4-chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidin-2-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (107 mg, 0.204 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction was monitored by LCMS. The mixture was alkalized 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 min; 220 nm; Rt: 7.55 min) to obtain 4-chloro-5-[5-[4-fluoro-2-(trifluoromethyl)phenoxy]-1,2,3,4-tetrahydro-2,6-naphthidin-2-yl]-2,3-dihydropyridazin-3-one (60 mg, 66.78%) as a white solid.

[0361] Example 4: Synthesis of Compound 118

[0362]

[0363] Ethyl 2-(benzylamino)propionate

[0364] TEA (7.63 g, 75.384 mmol, 1 equivalent) and NaBH(OAc)3 (31.95 g, 150.767 mmol, 2 equivalent) were added in portions to a stirred solution of benzaldehyde (8 g, 75.384 mmol, 1 equivalent) and TEA (7.63 g, 75.384 mmol, 1 equivalent) in DCE (100 mL, 1263.149 mmol, 16.76 equivalent) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at room temperature. The desired product was detected by LCMS. 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 give ethyl 2-(benzylamino)propionate (12 g, 76.80%) as a colorless oil.

[0365] Methyl 4-[benzyl(1-ethoxy-1-oxopropyl-2-yl)amino]butyrate

[0366] TEA (3.91 g, 38.596 mmol, 1 equivalent) and NaBH(OAc)3 (16.36 g, 77.193 mmol, 2 equivalents) were added in portions to a stirred solution of ethyl 2-(benzylamino)propionate (8 g, 38.596 mmol, 1 equivalent) and methyl 4-oxobutyrate (4.48 g, 38.596 mmol, 1.00 equivalent) in a DCE (120 mL, 1515.779 mmol, 39.27 equivalent) at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at room temperature. The desired product was detected by LCMS. 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-oxopropyl-2-yl)amino]butyrate (10 g, 84.29%), which is a colorless oil.

[0367] 1-Benzyl-2-methyl-3-oxoperidin-4-carboxylic acid methyl ester

[0368] t-BuOK (5.00 g, 52.051 mmol, 2 equivalents) was added in portions to a stirred solution of methyl 4-[benzyl(1-ethoxy-1-oxoprop-2-yl)amino]butyrate (8 g, 26.026 mmol, 1 equivalent) in toluene (100 mL) at room temperature under a nitrogen atmosphere. The mixture was stirred at 80 °C for 2 h. The desired product was detected by LCMS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (5:1 to 2:1) to give methyl 1-benzyl-2-methyl-3-oxopiperidin-4-carboxylic acid (6.5 g, 95.57%) as a white solid.

[0369] 7-Benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-ol

[0370] At room temperature under a nitrogen atmosphere, t-BuONa (4.41 g, 45.921 mmol, 2 equivalents) and formamidinium hydrochloride (3.70 g, 45.921 mmol, 2.00 equivalents) were added in portions to a stirred solution of methyl 1-benzyl-2-methyl-3-oxopiperidin-4-carboxylic acid (6 g, 22.960 mmol, 1 equivalent) in EtOH (80 mL, 1377.083 mmol, 59.98 equivalents). The mixture was stirred at 80 °C for 2 h. The desired product was detected by LCMS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (3:1 to 2:1) to give 7-benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-ol (5 g, 85.29%) as a white solid.

[0371] 4-Hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0372] Under a nitrogen atmosphere, 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 equivalents) were added to a solution of 7-benzyl-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-ol (5 g, 19.583 mmol, 1 equivalent) in EtOH (60 mL, 1032.812 mmol, 52.74 equivalents). The mixture was hydrogenated at room temperature under a hydrogen atmosphere for 2 h, filtered through a diatomaceous earth pad and concentrated under reduced pressure to give 4-hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (4.5 g, 86.61%), a white solid.

[0373] 4-Chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0374] CCl4 (5.22 g, 33.922 mmol, 2 equivalents) was added in portions to a stirred solution of 4-hydroxy-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (4.5 g, 16.961 mmol, 1 equivalent) and PPh3 (6.67 g, 25.442 mmol, 1.5 equivalents) in a DCE (60 mL, 0.606 mmol, 0.04 equivalents) under a nitrogen atmosphere at room temperature. The mixture was stirred at 70 °C for 2 hours. The desired product was detected by LCMS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EtOAc (7:1) to give 4-chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (4 g, 83.11%) as a white solid.

[0375] 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0376] K₂CO₃ (3.90 g, 28.193 mmol, 2 equivalents) was added in portions to a stirred solution of 4-chloro-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (4 g, 14.096 mmol, 1 equivalent) and 2-chloro-4-fluorophenol (2.07 g, 14.096 mmol, 1 equivalent) in DMF (50 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred at 70 °C for 1 h. The desired product was detected by LCMS. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography and eluted with PE / EtOAc (1:1) to give 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (4 g, 72.05%) as a white solid.

[0377] 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0378] TFA (4 mL) was added dropwise / partially to a stirred solution of 4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (4 g, 1 equivalent) in DCM (20 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred at room temperature for 2 h. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure to give 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.

[0379] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0380] 4,5-Dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (0.85 g, 3.404 mmol, 1 equivalent) was added in portions to a stirred solution of 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) at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at 100 °C. The desired product was detected by LCMS. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (1:1 to 1:2), to give 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (1 g, 58.01%) as a white solid.

[0381] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0382] TFA (2 mL) was added dropwise to a stirred solution of 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (1 g, 1 equivalent) in DCM (10 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred at room temperature for 1 h. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure to give 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (600 mg, 71.95%) as a white solid.

[0383] 4-Chloro-5-[(8R)-4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0384] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (250 mg, 1 equivalent) was subjected to preparative chiral HPLC (column: CHIRALPAK). IG, 20*250mm, 5um; mobile phase A: Hex:DCM=3:1 (0.1% FA)--HPLC, mobile phase B: EtOH--HPLC; flow rate: 20mL / min; gradient: 15B to 15B over 19min; 220 / 254nm; RT1:13.016; RT2:16.004) separation yielded 4-chloro-5-[(8R)-4-(2-chloro-4-fluorophenoxy)-8-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (144mg, 57.60%), a white solid.

[0385] Example 5 Synthesis of Compound 103

[0386]

[0387] 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0388] K₂CO₃ (1229.72 mg, 8.898 mmol, 3 equivalents) was added in portions to a stirred solution of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (800 mg, 2.966 mmol, 1 equivalent) and 2-(difluoromethyl)phenylacetic acid ester (1104.26 mg, 5.932 mmol, 2.00 equivalents) in DMF (20 mL) at 80 °C under a nitrogen atmosphere. The mixture was stirred for 2 hours. The reaction was monitored by LCMS. The reaction was quenched with water at room temperature. The mixture was extracted with EtOAc (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 reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of MeOH, in a 10% to 50% gradient over 10 min; detector, UV 254 nm, yielding 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (900 mg, 80.41%) as a grayish-white solid.

[0389] 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0390] At room temperature, tert-butyl 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid was added dropwise to a stirred solution of 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid (900 mg, 2.385 mmol, 1 equivalent) in DCM. The mixture was stirred for 1.5 h. The reaction was monitored by TLC (PE / EtOAc 10:1). The residue was alkalized 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 give 4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (329 mg, 49.75%) as a grayish-white solid.

[0391] 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0392] DIEA (175.43 mg, 1.357 mmol, 2.00 equivalent) was added in portions 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-dihydropyridazin-3-one (169.05 mg, 0.679 mmol, 1.00 equivalent) at 70 °C. The mixture was stirred at 70 °C for 2 hours. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of MeOH, in a 10% to 50% gradient over 10 min; detector, UV 254 nm, yielding 4-chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (328 mg, 56.60%) as a grayish-white solid.

[0393] 4-Chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0394] Trifluoroacetic acid (3 mL) was added dropwise to a stirred solution of 4-chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (328 mg, 0.670 mmol, 1 equivalent) in DCM (10 mL). The mixture was concentrated under vacuum. The product was purified by preparative HPLC to give 4-chloro-5-[4-[2-(difluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (256.4 mg, 94.38%) as a grayish-white solid.

[0395] Example 6 Synthesis of compounds 117 and 117a

[0396]

[0397] Ethyl 4-[(1-phenylethyl)amino]valerate

[0398] NaBH(OAc)3 (65.59 g, 309.449 mmol, 1.5 equivalent) was added in portions to a stirred solution of 1-phenylethyl-1-amine (25 g, 206.300 mmol, 1 equivalent) and ethyl 4-oxovalerate (29.74 g, 206.300 mmol, 1 equivalent) in a DCE (400 mL, 5052.598 mmol, 24.49 equivalent) at 25 °C under a nitrogen atmosphere. The solution was stirred at 25 °C for 2 hours. The reaction was quenched at 0 °C by adding H2O (400 mL). 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.

[0399] Ethyl 4-[(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]valerate

[0400] NaBH(OAc)3 (62.47 g, 294.762 mmol, 1.5 equivalents) was added in portions to a stirred solution of ethyl 4-[(1-phenylethyl)amino]valerate (49 g, 196.508 mmol, 1 equivalent) and ethyl 2-oxoacetate (40.12 g, 392.990 mmol, 2.00 equivalents) in a DCE (500 mL, 6315.747 mmol, 32.14 equivalents) at 25 °C under a nitrogen atmosphere. The solution was stirred at 25 °C for 2 hours. The reaction was quenched at 0 °C by adding H2O (400 mL). 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, ethyl 4-[(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]valerate (57 g, 86.47%), was used in the next step.

[0401] 2-Methyl-5-oxo-1-(1-phenylethyl)piperidine-4-carboxylic acid ethyl ester

[0402] t-BuOK (47.67 g, 424.810 mmol, 2.5 equivalents) was added in portions to a solution of ethyl 4-[(2-ethoxy-2-oxoethyl)(1-phenylethyl)amino]valerate (57 g, 169.924 mmol, 1 equivalent) in toluene (500 mL, 4699.452 mmol, 27.66 equivalents) at 0 °C. The mixture was stirred at 25 °C for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (50 / 1 to 10 / 1), to give ethyl 2-methyl-5-oxo-1-(1-phenylethyl)piperidine-4-carboxylic acid (29 g, 58.98%) as a yellow oil.

[0403] 7-(1-Cyclohexylethyl)-6-methyl-decahydropyrido[3,4-d]pyrimidin-4-ol

[0404] EtONa (5.88 g, 86.393 mmol, 2.50 equivalents) was added in portions to a solution of ethyl 2-methyl-5-oxo-1-(1-phenylethyl)piperidin-4-carboxylic acid (10 g, 34.557 mmol, 1 equivalent) and formamidinium hydrochloride (4.17 g, 51.836 mmol, 1.50 equivalents) in EtOH (100 mL, 1721.353 mmol, 49.81 equivalents). The mixture was stirred at 90 °C for 2 h. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (20 / 1 to 10 / 1) to give 7-(1-cyclohexylethyl)-6-methyl-decahydropyrido[3,4-d]pyrimidin-4-ol (3.4 g, 34.96%) as a yellow solid.

[0405] 4-Hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0406] Pd(OH)₂ / C (0.36 g, 2.599 mmol, 0.2 equivalents) was added to a solution of 6-methyl-7-(1-phenylethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-ol (3.5 g, 12.994 mmol, 1 equivalent), HCOONH₄ (4.10 g, 65.022 mmol, 5.00 equivalents), and Boc₂O (8.51 g, 38.983 mmol, 3 equivalents) in EtOH (50 mL, 860.677 mmol, 66.23 equivalents) under a nitrogen atmosphere. The mixture was hydrogenated at 70 °C for 2 h under a hydrogen atmosphere using a hydrogen balloon, filtered through a diatomaceous earth pad, and concentrated under reduced pressure. 4-Hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (1.8 g, 52.21%) was obtained as a yellow solid.

[0407] 4-Chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0408] CCl4 (3.13 g, 20.353 mmol, 3 equivalents) was added to a solution of 4-hydroxy-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (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). 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 4-chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (1.1 g, 57.14%) as a yellow solid.

[0409] 4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0410] K₂CO₃ (1.07 g, 7.753 mmol, 2 equivalents) was added to a solution of 4-chloro-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (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) 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 and eluted with PE / EA (10 / 1 to 5 / 1) to give 4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (1.2 g, 78.60%) as a yellow solid.

[0411] 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

[0412] 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-dihydropyridazin-3-one (678.42 mg, 2.724 mmol, 1.00 equivalent) in DIEA (704.01 mg, 5.447 mmol, 2 equivalent) was stirred at 100 °C under a nitrogen atmosphere for 16 hours. The residue was purified by preparative TLC (PE / EA 1 / 1) to give 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 (530 mg, 38.43%) as a pale yellow solid.

[0413] 4-Chloro-5-[(6R)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0414] TFA (1193.47 mg, 10.467 mmol, 10 equivalents) was added to a solution of 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 (530 mg, 1.047 mmol, 1 equivalent) in DCM (20 mL, 314.601 mmol, 300.57 equivalents). 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 min; 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: 20B to 20B over 15 min; 220 / 254 nm. Although the two isomers were separated by this technique, their absolute orientation was not determined. The compound obtained at 9.688 min was designated as 4-chloro-5-[(6S)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (60.9 mg, 13.78%), a white solid. The compound obtained at 11.813 min was designated as 4-chloro-5-[(6R)-4-(2-chloro-4-fluorophenoxy)-6-methyl-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (61.5 mg, 13.92%), a white solid.

[0415] Example 7 Synthesis of Compound 134

[0416]

[0417] 2-Chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0418] NaHCO3 (4.14 g, 49.282 mmol, 3.00 equivalent) was added to a stirred solution of 2-(difluoromethyl)-4-fluorophenol (5.33 g, 32.879 mmol, 2.00 equivalent) and 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (5 g, 16.438 mmol, 1 equivalent) in DMF (30 mL). The solution was stirred at 70 °C for 0.5 h. The mixture was then concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 10 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 70% B-95% B gradient over 100 min; detector: 254 nm. The fraction containing the desired product was collected at 92% B and concentrated under reduced pressure to give 2-chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (2.100 g), as a grayish-white solid.

[0419] 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2,7-dicarboxylic acid 7-tert-butyl-2-methyl ester

[0420] In a pressure vessel, Pd(PPh3)4 (107.54 mg, 0.093 mmol, 0.1 equivalent) was added to a solution of 2-chloro-4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (400 mg, 0.931 mmol, 1 equivalent) and TEA (188.34 mg, 1.861 mmol, 2 equivalent) in MeOH (15 mL, 370.484 mmol, 398.10 equivalent). The mixture was purged with nitrogen for 1 hour, then pressurized to 10 atm with carbon monoxide at 100 °C for 16 hours. The reaction mixture was cooled to room temperature and filtered to remove insoluble solids. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C 1820-40 μm, 330 g; Mobile phase A: water (with 10 mM NH4HCO3); Mobile phase B: acetonitrile; Flow rate: 80 mL / min; Gradient: 5%-5% B, 10 min, 35% B-65% B gradient over 20 min; Detector: 254 nm. The fraction containing the desired product was collected at 62% B and concentrated under reduced pressure to give 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-2,7-dicarboxylic acid 7-tert-butyl-2-methyl ester (100 mg, 23.70%) as a colorless oil.

[0421] 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0422] NaBH4 (16.69 mg, 0.441 mmol, 2 equivalents) was added to a stirred solution of 4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidinyl-2,7-dicarboxylic acid 7-tert-butyl-2-methyl ester (100 mg, 0.221 mmol, 1 equivalent) in t-BuOH (6 mL, 63.139 mmol, 286.29 equivalents). The solution was stirred at 70 °C for 3 hours. Water (3 mL) was then added to the mixture. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 10 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 45% B-80% B gradient over 20 min; detector: 254 nm. The fraction containing the desired product was collected at 74% B and concentrated under reduced pressure to give 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (35 mg, 37.30%) as a colorless oil.

[0423] [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]methanol

[0424] TFA (1 mg) was added to a stirred solution of 35 mg 4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (35 mg) in DCM (6 mg). The solution was stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 10 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, 25% B-55% B gradient over 20 min; detector: 254 nm. The fraction containing the desired product was collected under 41% B and concentrated under reduced pressure to give [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]methanol (20 mg), a colorless oil.

[0425] 4-Chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0426] Add [4-[2-(difluoromethyl)-4-fluorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-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) to the mixture at room temperature. Add DIEA (15.89 mg, 0.123 mmol, 2 equivalents) to the mixture at room temperature. Stir the mixture at 90 °C for 2 hours. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 10 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 35% B-70% B gradient over 20 min; detector: 254 nm. The fraction containing the desired product was collected at 65% B and concentrated under reduced pressure to give 4-chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (30 mg, 90.71%) as a colorless oil.

[0427] 4-Chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0428] TFA (1 mL) was added to a stirred solution of 30 mg 4-chloro-5-[4-[2-(difluoromethyl)-4-fluorophenoxy]-2-(hydroxymethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (5 mL) in DCM at room temperature. The solution was stirred at room temperature for 2 hours. The mixture was then 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: not limited, mobile phase B: not limited; flow rate: 60 mL / min; gradient: 20% B to 40% B over 8 min; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (8.7 mg), a white solid.

[0429] Compounds 128, 125, and 114 were prepared by means of the methods and schemes described in this embodiment, by using 2-trifluoromethylphenol, 4-fluoro-2-trifluoromethylphenol, and 4-fluoro-2-chlorophenol instead of 2-(difluoromethyl)-4-fluorophenol in the first step of synthesis.

[0430] Example 8 Synthesis of Compound 112

[0431]

[0432] 2-Chloro-4-(2-Chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0433] K₂CO₃ (726.99 mg, 5.260 mmol, 2.00 equivalent) was added in portions to a stirred mixture of 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (800 mg, 2.630 mmol, 1 equivalent) and 2-chloro-4-fluorophenol (578.16 mg, 3.945 mmol, 1.50 equivalent) in DMF (15 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 0.5 h. 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 silica gel column chromatography, eluted with PE / EtOAc (30 / 1 to 10 / 1), to give tert-butyl 2-chloro-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid (1 g, 91.78%) as a yellow oil.

[0434] 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0435] 1-(4-methoxyphenyl)methylamine (1159.02 mg, 8.449 mmol, 5.00 equivalent) was added in portions to a stirred mixture of 2-chloro-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (700 mg, 1.690 mmol, 1 equivalent) in THF (30 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 60 °C under a nitrogen atmosphere for 16 h. 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 reversed-phase rapid chromatography under the following conditions (column, C18 silica gel; mobile phase, aqueous acetonitrile, 60% to 95% gradient over 20 min; detector, UV 220 nm) to give 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (350 mg, 40.22%) as a yellow oil.

[0436] 4-(2-chloro-4-fluorophenoxy)-N-[(4-methoxyphenyl)methyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-amine

[0437] TFA (1 mL) was added dropwise to a stirred solution of 350 mg (1 equivalent) of 4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized 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 Shield RP18 OBD column, 5 μm, 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 over 1 min; 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]pyrimidin-2-amine (260 mg), which was a yellow oil.

[0438] 4-Chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0439] 4-(2-chloro-4-fluorophenoxy)-N-[(4-methoxyphenyl)methyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-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 under a nitrogen atmosphere for 2 hours. The residue was purified by reversed-phase rapid chromatography under the following conditions (column, C18 silica gel; mobile phase, aqueous acetonitrile, 50% to 85% gradient over 25 min; detector, UV 220 nm) to give 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (350 mg, 89.00%) as a yellow solid.

[0440] 5-[2-amino-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-4-chloro-2,3-dihydropyridazin-3-one

[0441] 200 mg of 4-chloro-5-[4-(2-chloro-4-fluorophenoxy)-2-[[(4-methoxyphenyl)methyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one was added to a stirred solution in TFA (8 mL, 107.704 mmol, 328.23 equivalents). The final reaction mixture was irradiated with microwave at 80 °C for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized 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 30×150mm 5um; mobile phase A: water (10mM NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 25% B to 40% B over 8 min; 220nm; Rt: 7.35min) to obtain 5-[2-amino-4-(2-chloro-4-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-4-chloro-2,3-dihydropyridazin-3-one (52.4 mg), a yellow solid.

[0442] Compounds 113, 116 and 102 were prepared by means of the methods and schemes described in this embodiment, by using 2-chlorophenol, 4-fluoro-2-trifluoromethylphenol and 2-trifluorophenol respectively in place of 2-chloro-4-fluorophenol in the first step of synthesis.

[0443] Example 9: Synthesis of compounds 129 and 130

[0444]

[0445] 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]ethyl-1-one

[0446] Pd(PPh3)4 (77.41 mg, 0.067 mmol, 0.05 equivalent) was added to a mixture of 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (600 mg, 1.340 mmol, 1 equivalent) and tributyl(1-ethoxyvinyl)stanane (967.80 mg, 2.680 mmol, 2.00 equivalent) in toluene (10 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 110 °C for 4 hours. The reaction was monitored by LCMS. This produces 2-(1-ethoxyvinyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (700 mg, 108.06%), as a yellow oil. The resulting crude mixture was used directly in the next step without further purification.

[0447] TFA (3.33 mL, 29.239 mmol, 21.70 equivalents) was added to a stirred solution of 2-(1-ethoxyvinyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (1 g, 2.068 mmol, 1 equivalent) in DCM (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture / residue was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 43% B-55% B gradient over 20 min; detector: 220 nm. The fraction containing the desired product was collected at 50% B and concentrated under reduced pressure to give 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]ethyl-1-one (750 mg, 102.06%) as a pale yellow solid.

[0448] 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0449] Add 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]ethyl-1-one (750 mg, 2.111 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (525.81 mg, 2.111 mmol, 1.00 equivalent) to the mixture. Add DIEA (818.47 mg, 6.333 mmol, 3.00 equivalent) to the mixture. Stir the mixture at 100 °C for 2 hours. Monitor the reaction by LCMS. Cool the mixture to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 60% B-85% B gradient over 20 min; detector: 220 nm. The fraction containing the desired product was collected at 80% B and concentrated under reduced pressure to give 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (230 mg, 19.18%) as a pale yellow oil.

[0450] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0451] NaBH4 (30.64 mg, 0.810 mmol, 2.00 equivalent) was added in portions to a stirred solution of 5-[2-acetyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (230 mg, 0.405 mmol, 1 equivalent) in MeOH (10 mL) under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EtOAc 1 / 1) to give 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 51.99%), as a pale yellow oil.

[0452] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1S)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1R)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0453] TFA (2.00 mL, 17.541 mmol, 127.89 equivalents) was added dropwise to a stirred solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-(1-hydroxyethyl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 0.211 mmol, 1 equivalent) in DCM (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The residue was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C 1820-40 μm, 330 g; Mobile phase A: water (with 5 mM NH4HCO3); Mobile phase B: acetonitrile; Flow rate: 80 mL / min; Gradient: 5%-5% B, 10 min, 40% B-80% B gradient over 25 min; Detector: 220 nm. Fractions containing the desired product were collected 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: Hex (0.1% FA)--HPLC, mobile phase B: EtOH--HPLC; flow rate: 16 mL / min; gradient: 30 B to 30 B over 33 min; 220 / 254 nm; RT1: 26.219; RT2: 29.589). Although two isomers were separated by this technique, absolute orientation was not determined. The compound obtained at 29.589 min was designated as 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1S)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (27.1 mg), a grayish-white solid. The compound obtained at 26.219 min was designated as 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-[(1R)-1-hydroxyethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (22.6 mg), a grayish-white solid.

[0454] Compound 119 was prepared using 2-chloro-4-[4-fluoro-2-chlorophenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester as a starting material, according to the method and scheme described in this embodiment.

[0455] Compounds 122 and 123 were prepared using 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester as a starting material, according to the methods and schemes described in this embodiment. Similarly, the absolute orientation of these isolated isomers was not determined, and designation as (S) or (R) is arbitrary.

[0456] Example 10 Synthesis of Compound 115

[0457]

[0458] 2-Chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0459] DBU (2.0 g, 13.15 mmol, 2 equivalents) was added to a stirred solution of 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (2 g, 6.58 mmol, 1 equivalent) and 2-(trifluoromethyl)phenol (1.6 g, 9.86 mmol, 1.5 equivalents) in acetonitrile (20 mL). 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 / EtOAc 10:1) to give 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (700 mg, 24.77%) as a colorless oil.

[0460] 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0461] (2-aminoethoxy)(tert-butyl)dimethylsilane (1019.89 mg, 5.816 mmol, 5.00 equivalent) was added to a solution of 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (500 mg, 1.163 mmol, 1 equivalent) in THF (15 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 50 °C for 16 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EtOAc 3 / 1) to give 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (440 mg, 66.51%), as a pale yellow oil.

[0462] 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]amino) ethanol-1-ol

[0463] TFA (3 mL, 40.389 mmol, 52.20 equivalents) was added to a stirred solution of 2-([2-[(tert-butyldimethylsilyl)oxy]ethyl]amino)-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (440 mg, 0.774 mmol, 1 equivalent) in DCM (10 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of ACN, gradient of 40% to 60% over 15 min; detector, UV 254 nm, to give 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]amino)ethanol-1-ol (220 mg), a pale yellow oil.

[0464] 4-Chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0465] Add 2-([4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]amino)ethanol-1-ol (220 mg, 0.621 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (154.66 mg, 0.621 mmol, 1.00 equivalent) to the mixture. Add DIEA (240.74 mg, 1.863 mmol, 3.00 equivalent) to the mixture. Stir the resulting mixture at 100 °C for 2 h. Monitor the reaction by LCMS. Cool the mixture to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: ACN; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 45% B-60% B gradient over 20 min; detector: 220 nm. The fraction containing the desired product was collected at 55% B and concentrated under reduced pressure to give 4-chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (210 mg, 59.66%) as a yellow solid.

[0466] 4-Chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0467] TFA (2 mL) was added to a stirred solution of 4-chloro-5-[2-[(2-hydroxyethyl)amino]-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (200 mg, 0.353 mmol, 1 equivalent) in DCM (5 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The mixture was alkalized 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 30×150mm 5um; mobile phase A: not limited, mobile phase B: not limited; flow rate: 60 mL / min; gradient: 25% B to 50% B over 8 min; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (106.3 mg), a white solid.

[0468] Example 11 Synthesis of compounds 138 and 139

[0469]

[0470] 7-Benzyl-2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0471] K₂CO₃ (1879.20 mg, 13.597 mmol, 2 equivalents) was added to a stirred solution of 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) in DMF (20 mL). The solution was stirred at 70 °C for 0.5 h. The mixture was then concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM TFA); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 70% B-95% B gradient over 20 min; detector: 254 nm. The fraction containing the desired product was collected at 95% B and concentrated under reduced pressure to give 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.

[0472] 7-Benzyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-one

[0473] A solution of 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) in HAc (10 mL, 174.515 mmol, 38.20 equivalent) and H2O (1 mL, 55.508 mmol, 12.15 equivalent) was stirred at 140 °C under a nitrogen atmosphere for 10 hours. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EA 1 / 1) to give 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.

[0474] 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-one

[0475] Pd / C (268.98 mg, 2.528 mmol, 2 equivalents) was added to a solution of 7-benzyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-one (530 mg, 1.264 mmol, 1 equivalent) in MeOH (10 mL, 246.989 mmol, 195.44 equivalents) under a nitrogen atmosphere. The mixture was hydrogenated at room temperature under a hydrogen atmosphere using a hydrogen balloon for 4 hours, filtered through a diatomaceous earth pad, and concentrated under reduced pressure. 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-one (430 mg, 103.34%) was given as a pale yellow solid.

[0476] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0477] A mixture of 4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-one (430 mg, 1.306 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (357.84 mg, 1.437 mmol, 1.1 equivalent) in DIEA (337.58 mg, 2.612 mmol, 2.00 equivalent) was stirred at 100 °C under a nitrogen atmosphere for 2 hours. The residue was purified by preparative TLC (PE / EA 1 / 1) to give 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (210 mg, 29.67%) as a pale yellow solid.

[0478] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one and 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0479] CH3I (47.15 mg, 0.332 mmol, 2.00 equivalent) was added dropwise to a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (90 mg, 0.166 mmol, 1 equivalent) and NaHCO3 (27.90 mg, 0.332 mmol, 2 equivalents) in DMF (10 mL, 129.218 mmol, 778.02 equivalents) under nitrogen atmosphere at 0 °C. 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 give 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg, 64.99%) and 4-chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (15 mg), as pale yellow solids.

[0480] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-1-methyl-2-oxo-1H,2H,5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0481] TFA (123.07 mg, 1.079 mmol, 10 equivalents) was added 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]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg, 0.108 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 1457.41 equivalents). 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 min; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (29.3 mg, 57.54%), as a white solid.

[0482] 4-Chloro-5-[4-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0483] TFA (30.77 mg, 0.270 mmol, 10 equivalents) was added to a solution of 4-chloro-5-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-2-methoxy-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (15 mg, 0.027 mmol, 1 equivalent) in DCM (5 mL, 78.650 mmol, 2914.83 equivalents). The resulting mixture was concentrated under reduced pressure. The crude product (20 mg) was purified by preparative HPLC under the following conditions (column: XBridgeShield 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 min; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (7.5 mg, 58.91%) as a white solid.

[0484] Example 12 Synthesis of Compound 110

[0485]

[0486] 2-Chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0487] DBU (2.0 g, 13.15 mmol, 2 equivalents) was added to a stirred solution of 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (2 g, 6.58 mmol, 1 equivalent) and 2-(trifluoromethyl)phenol (1.6 g, 9.86 mmol, 1.5 equivalents) in acetonitrile (20 mL). 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 / EtOAc 10:1) to give 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (700 mg, 24.77%) as a colorless oil.

[0488] 2-Methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0489] NaOMe (0.25 g, 0.005 mmol, 2 equivalents) was added to a solution of 2-chloro-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (1 g, 2.327 mmol, 1 equivalent) in MeOH (20 mL, 493.978 mmol, 212.32 equivalents). 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, eluting with PE / EA (10 / 1 to 1 / 1), to give 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (100 mg, 10.10%) as a pale yellow solid.

[0490] 2-Methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0491] TFA (268.03 mg, 2.351 mmol, 10 equivalents) was added to a solution of 2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (100 mg, 0.235 mmol, 1 equivalent) in DCM (10 mL) 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 min; 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.

[0492] 4-Chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0493] 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-dihydropyridazin-3-one (61.26 mg, 0.246 mmol, 1 equivalent) in DIEA (63.57 mg, 0.492 mmol, 2.00 equivalent) was stirred at 100 °C under a nitrogen atmosphere for 2 hours. The residue was purified by silica gel column chromatography, eluted with PE / EA (5 / 1 to 1 / 1), to give 4-chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 90.71%), as a pale yellow solid.

[0494] 4-Chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0495] TFA (254.36 mg, 2.231 mmol, 10.00 equivalent) was added to a solution of 4-chloro-5-[2-methoxy-4-[2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 0.223 mmol, 1 equivalent) in DCM (5 mL, 78.650 mmol, 352.56 equivalents). The resulting mixture was concentrated under reduced pressure. The crude product (150 mg) was purified by preparative HPLC under the following conditions (column: XBridgeShield 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 min; 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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (24.1 mg, 23.81%) as a white solid.

[0496] Example 13 Synthesis of Compound 108

[0497]

[0498] 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0499] K₂CO₃ (512.38 mg, 3.707 mmol, 2 equivalents) was added to a stirred solution of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (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). The resulting mixture was stirred at 70 °C for 1 h. The mixture was purified by reversed-phase rapid chromatography under the following conditions: column: (spnerical 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 min; 254 nm). The fraction containing the desired product was collected at 40% B and concentrated under reduced pressure. This produces 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (300 mg, 36.72%), a grayish-white solid.

[0500] 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0501] Pd(PPh3)4 (117.99 mg, 0.102 mmol, 0.1 equivalent) was added to a stirred solution of 4-(3-bromo-2-chlorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (450 mg, 1.021 mmol, 1 equivalent) and zinc dinitrile (143.87 mg, 1.225 mmol, 1.20 equivalent) in DMF (5 mL). The resulting mixture was stirred at 120 °C under a nitrogen atmosphere for 2 h. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: spnerical 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% over 55 min; 254 nm. The fraction containing the desired product was collected at 40% B and concentrated under reduced pressure. This yielded 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (280 mg, 70.89%), as a pale yellow solid.

[0502] 2-Chloro-3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]benzylnitrile

[0503] TFA (1 mL) was added to a stirred solution of 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (100 mg, 0.259 mmol, 1 equivalent) in DCM (3 mL). The resulting mixture was stirred at room temperature under air for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 7 with saturated NH4HCO3 (aqueous solution). The mixture was purified by reversed-phase rapid chromatography under the following conditions: column: spnerical C18, 20-40 μm, 180 g; mobile phase A: water (5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 30% B to 60% B over 30 min (254 nm). The fraction containing the desired product was collected at 45% B and concentrated under reduced pressure. This produces 2-chloro-3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]benzyl nitrile (60 mg, 80.95%), a pale yellow oil.

[0504] 2-Chloro-3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)benzylnitrile

[0505] 4-(2-chloro-3-cyanophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (60 mg, 0.155 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (38.63 mg, 0.155 mmol, 1.00 equivalent) were added to a stirred solution of DIEA (40.09 mg, 0.310 mmol, 2 equivalents). The resulting mixture was stirred at 100 °C under air for several hours. The residue was purified by preparative TLC (PE / EtOAc 1:1) to give 2-chloro-3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)benzyl nitrile (50 mg, 64.56%) as a pale yellow solid.

[0506] 2-Chloro-3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy]benzyl nitrile

[0507] TFA (1 mL) was added to a stirred solution of 2-chloro-3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)benzyl nitrile (50 mg, 0.100 mmol, 1 equivalent) in DCM (3 mL). The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 7 with saturated NH4CO3 (aqueous solution). The crude product was purified by preparative HPLC under the following conditions (column: XBridgePrep OBD C18 column 30×150mm 5um; mobile phase A: water (10mM NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 20% B to 42% B over 8 min; 220nm; Rt: 7.58min) to obtain 2-chloro-3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy]benzyl nitrile (14.5mg, 34.88%), as a grayish-white solid.

[0508] Example 14 Synthesis of Compound 111

[0509]

[0510] 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0511] Cs₂CO₃ (434.86 mg, 1.335 mmol, 2.00 equivalent) was added in portions to a stirred mixture of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (180 mg, 0.667 mmol, 1 equivalent) and 3-bromo-2-(trifluoromethyl)phenol (241.25 mg, 1.001 mmol, 1.50 equivalent) in DMF (10 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 0.5 h. 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 reversed-phase rapid chromatography under the following conditions (column, C18 silica gel; mobile phase, aqueous acetonitrile, 40% to 85% gradient over 30 min; detector, UV 220 nm) to give 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (150 mg, 47.39%) as a yellow oil.

[0512] 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0513] Pd(PPh3)4 (36.55 mg, 0.032 mmol, 0.1 equivalent) was added in portions to a stirred mixture of 4-[3-bromo-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (150 mg, 0.316 mmol, 1 equivalent) and Zn(CN)2 (111.43 mg, 0.949 mmol, 3.00 equivalent) in DMF (8 mL) under a nitrogen atmosphere at room temperature. The final reaction mixture was irradiated with microwave at 150 °C for 3 h. The reaction was monitored by LCMS. The residue was purified by reversed-phase rapid chromatography under the following conditions (column, C18 silica gel; mobile phase, aqueous acetonitrile, 40% to 95% gradient over 30 min; detector, UV 220 nm) to give 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (70 mg, 52.65%) as a yellow oil.

[0514] 3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]-2-(trifluoromethyl)benzylnitrile

[0515] TFA (1 mL) was added dropwise to a stirred solution of 70 mg tert-butyl 4-[3-cyano-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized 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 reversed-phase rapid chromatography under the following conditions (column, C18 silica gel; mobile phase, aqueous acetonitrile, 30% to 60% gradient over 20 min; detector, UV 220 nm) to obtain 3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]-2-(trifluoromethyl)benzyl nitrile (40 mg), a yellow oil.

[0516] 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)-2-(trifluoromethyl)benzylnitrile

[0517] 3-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]-2-(trifluoromethyl)benzylnitrile (40 mg, 0.125 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (62.22 mg, 0.250 mmol, 2.00 equivalent), and DIEA (48.42 mg, 0.375 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 under a nitrogen atmosphere for 16 hours. The residue was purified by preparative TLC (PE / EtOAc = 5 / 1) to give 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)-2-(trifluoromethyl)benzyl nitrile (50 mg, 75.12%) as a yellow oil.

[0518] 3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy]-2-(trifluoromethyl)benzylnitrile

[0519] TFA (1 mL) was added dropwise to a stirred solution of 3-([7-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy)-2-(trifluoromethyl)benzyl nitrile (50 mg) in DCM (10 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized 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 30×150mm 5um; mobile phase A: water (10mM NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 25% B to 45% B over 8 min; 220nm; Rt: 7.07min) to obtain 3-[[7-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]oxy]-2-(trifluoromethyl)benzyl nitrile (10.8 mg), a white solid.

[0520] Example 15 Synthesis of compounds 126 and 126a

[0521]

[0522] N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylene]-4-methylbenzene-1-sulfonylhydrazine

[0523] 4-Methylbenzene-1-sulfonylhydrazine (1.81 g, 9.719 mmol, 1.00 equivalent) was added in portions to a stirred solution of 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl-1-one (2 g, 9.702 mmol, 1 equivalent) in EtOH (40 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 6 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM AcOH); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, 45% B-70% B gradient over 20 min; detector: 220 nm. The fraction containing the desired product was collected at 60% B and concentrated under reduced pressure to give N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylene]-4-methylbenzene-1-sulfonylhydrazine (2.5 g, 68.83%), a white solid.

[0524] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]vinyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0525] Pd(acetonitrile)₂Cl₂ (72.14 mg, 0.278 mmol, 0.10 equivalent), Dppf (307.18 mg, 0.556 mmol, 0.2 equivalent), and t-BuOLi (489.71 mg, 6.117 mmol, 2.20 equivalent) were added in portions to a stirred mixture of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (750 mg, 2.781 mmol, 1 equivalent) and N-[(1E)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethylene]-4-methylbenzene-1-sulfonylhydrazine (2081.80 mg, 5.561 mmol, 2.00 equivalent) in 1,4-dioxane (20 mL) under a nitrogen atmosphere at room temperature. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with EtOAc (2 × 50 mL). The filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM AcOH); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 50% B-90% B gradient over 30 min; detector: 220 nm. The fraction containing the desired product was collected at 85% B and concentrated under reduced pressure to give 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]vinyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (800 mg, 67.95%) as a brown oil.

[0526] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0527] In a 100 mL round-bottom flask under a nitrogen atmosphere, Pd / C (10%, 30 mg) was added to a solution of 150 mg of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethenyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester in 30 mL MeOH. The mixture was hydrogenated at room temperature under a hydrogen atmosphere using a hydrogen balloon for 4 hours, filtered through a diatomaceous earth pad, and concentrated under reduced pressure. This yielded 150 mg of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester, a yellow oil.

[0528] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0529] TFA (1 mL) was added dropwise to a stirred solution of 150 mg of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with DCM (3 × 50 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. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (with 5 mM AcOH); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5%-5% B for 10 min, then 40% B-58% B gradient over 15 min; detector: 254 nm. The fraction containing the desired product was collected at 53% B and concentrated under reduced pressure to give 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (100 mg) as a yellow oil.

[0530] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0531] 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-dihydropyridazin-3-one (91.88 mg, 0.369 mmol, 1.20 equivalent), and DIEA (119.19 mg, 0.922 mmol, 3.00 equivalent) were added to a 50 mL round-bottom flask under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (with 5 mM AcOH); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5%-5% B for 10 min, then 40% B-60% B gradient over 15 min; detector: 220 nm. The fraction containing the desired product was collected at 53% B and concentrated under reduced pressure to give 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 72.57%) as a yellow oil.

[0532] 4-Chloro-5-[4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-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]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0533] TFA (1 mL) was added dropwise to a stirred solution of 200 mg 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 4 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized 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 Prep Phenyl OBD column 19×150mm 5um 13nm; mobile phase A: mobile phase B: flow rate: 60mL / min; gradient: 20% B to 37% B over 8 min; 220nm; Rt: 7.97min). Although two isomers were separated by this technique, their absolute orientation was not determined. The compound obtained at 1.819 min was designated as 4-chloro-5-[4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (11.8 mg), a grayish-white solid. The compound obtained at 2.470 min was designated as 4-chloro-5-[4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (13.5 mg), a white solid.

[0534] Example 16 Synthesis of Compound 133

[0535]

[0536] 4-[methyl[(3R,4R)-4-methylpiperidin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0537] Add (3R,4R)-1-benzyl-N,4-dimethylpiperidin-3-amine (2.43 g, 0.011 mmol, 1.50 equivalents) and 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (2 g, 0.007 mmol, 1 equivalent) to the mixture at room temperature. Add DIEA (1.92 g, 0.015 mmol, 2.00 equivalents) to the mixture at room temperature. Stir the mixture at 100 °C for 2 hours. The residue was purified by preparative TLC (PE / EtOAc 1:1) to give 4-[methyl[(3R,4R)-4-methylpiperidin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (670 mg, 25.00%), as a grayish-white solid.

[0538] (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]piperidin-3-amine

[0539] Trifluoroacetic acid (3 mL, 0.026 mmol, 6.00 equivalents) was added dropwise to a stirred solution of 4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (413 mg, 0.914 mmol, 1 equivalent) in 10 mL of DCM at 0 °C. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The solution was concentrated under reduced pressure. The crude product (362 mg) was purified by preparative HPLC under the following conditions (column: XBridgeShield 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 min; 220 nm; Rt: 21.65 min) to obtain (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]piperidine-3-amine (250 mg, 77.77%), which was a red oil.

[0540] 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0541] Add (3R,4R)-1-benzyl-N,4-dimethyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]piperidin-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) to the mixture at room temperature. Add DIEA (193.41 mg, 1.261 mmol, 2 equivalents) to the mixture at room temperature. Stir the mixture at 100 °C for 2 hours. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 45% B-95% B gradient over 30 min; detector: 254 nm. The fraction containing the desired product was collected at 85% B and concentrated under reduced pressure to give 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (245 mg, 58.04%) as a grayish-white solid.

[0542] 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-4-chloro-2,3-dihydropyridazin-3-one

[0543] Trifluoroacetic acid (3 mL, 0.026 mmol, 6.00 equivalent) was added dropwise to a stirred solution of 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-4-chloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (88 mg, 1 equivalent) in DCM (10 mL). The mixture was stirred at room temperature for 2 hours. The solution was then concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM TFA); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 33% B-95% B gradient over 30 min; detector: 254 nm. The fraction containing the desired product was collected at 90% B and concentrated under reduced pressure to give 5-(4-[[(3R,4R)-1-benzyl-4-methylpiperidin-3-yl](methyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-4-chloro-2,3-dihydropyridazin-3-one (33.5 mg, 44.74%) as a grayish-white solid.

[0544] Compound 133a was prepared by the method and scheme described in this embodiment, by using (3S,4S)-1-benzyl-N,4-dimethylpiperidine-3-amine instead of (3R,4R)-1-benzyl-N,4-dimethylpiperidine-3-amine.

[0545] Example 17 Synthesis of Compound 136

[0546]

[0547] 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0548] A mixture of 4-fluoro-2-(trifluoromethyl)aniline (6.64 g, 37.074 mmol, 2 equivalents), 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (5 g, 18.537 mmol, 1 equivalent), Pd(AcO)2 (0.83 g, 3.707 mmol, 0.2 equivalents), XantPhos (4.29 g, 7.415 mmol, 0.4 equivalents), and Cs2CO3 (12.08 g, 37.074 mmol, 2 equivalents) in 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 a crude product, which was purified by silica gel column chromatography by elution with PE:EA (20:1 to 1:2) to give 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (5.6 g, 73.26%) as a white solid.

[0549] 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0550] Cs₂CO₃ (4.74 g, 14.548 mmol, 2.00 equivalent) was added in portions to a stirred mixture of 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (3 g, 7.275 mmol, 1 equivalent) and methyl 2-bromoacetate (2.23 g, 14.578 mmol, 2.00 equivalent) in DMF (30 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was extracted with EtOAc (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 reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM TFA); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 55% B-85% B gradient over 30 min; detector: 220 nm. The fraction containing the desired product was collected at 79% B and concentrated under reduced pressure to give 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (500 mg, 14.19%) as a yellow solid.

[0551] 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0552] LiAlH4 (78.34 mg, 2.064 mmol, 2.00 equivalent) was added in portions to a stirred solution of 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-methoxy-2-oxoethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (500 mg, 1.032 mmol, 1 equivalent) in THF (50 mL) at -30 °C under a nitrogen atmosphere. The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The reaction was quenched at -30 °C by adding water (1 mL). The precipitated solid was collected by filtration and washed with MeOH (3 x 30 mL). The resulting mixture was concentrated under vacuum. The residue was purified by preparative TLC (PE / EA = 1 / 1) to give 4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (100 mg, 21.23%), as a yellow oil.

[0553] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0554] TFA (1 mL) was added dropwise to a stirred solution of 150 mg of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with DCM (3 × 50 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. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 40% B-58% B gradient over 15 min; detector: 220 nm. The fraction containing the desired product was collected at 53% B and concentrated under reduced pressure to give 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (100 mg) as a yellow oil.

[0555] 4-Chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0556] 2-[[4-fluoro-2-(trifluoromethyl)phenyl]([5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-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 under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5%-5% B for 10 min, then 40% B-60% B gradient over 15 min; detector: 220 nm. The fraction containing the desired product was collected at 55% B and concentrated under reduced pressure to give 4-chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (50 mg, 78.28%) as a yellow oil.

[0557] 4-Chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one

[0558] TFA (1 mL) was added dropwise to a stirred solution of 50 mg (4-chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one) in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (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 PrepOBD C18 column 30×150mm 5um; mobile phase A: not limited, mobile phase B: not limited; flow rate: 60mL / min; gradient: 30%B to 45%B over 8 min; 220nm; Rt: 7.6min) to obtain 4-chloro-5-(4-[[4-fluoro-2-(trifluoromethyl)phenyl](2-hydroxyethyl)amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one (6.2 mg), a white solid.

[0559] Example 18 Synthesis of Compound 132

[0560]

[0561] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0562] Me3SiI (472.57 mg, 2.362 mmol, 2.00 equivalent) was added dropwise to a stirred solution of t-BuONa (226.97 mg, 2.362 mmol, 2.00 equivalent) in DMSO (20 mL) at 40 °C under a nitrogen atmosphere. The resulting mixture was stirred at 40 °C under a nitrogen atmosphere for 0.5 h. Then, a solution of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]vinyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (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 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was extracted with EtOAc (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 reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH₄HCO₃); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 55% B-80% B gradient over 25 min; detector: 220 nm. The fraction containing the desired product was collected at 73% B and concentrated under reduced pressure to give 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (240 mg, 46.46%) as a yellow oil.

[0563] 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0564] TFA (1 mL, 13.463 mmol, 24.54 equivalents) was added dropwise to a stirred solution of 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (240 mg, 0.549 mmol, 1 equivalent) in DCM (10 mL). The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (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 reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 120 g; mobile phase A: water (with 5 mM AcOH); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5%-5% B for 10 min, then 33% B-45% B gradient over 20 min; detector: 254 nm. The fraction containing the desired product was collected at 40% B and concentrated under reduced pressure to give 4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (150 mg, 81.05%) as a yellow oil.

[0565] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0566] 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 equivalent), and DIEA (172.42 mg, 1.334 mmol, 3.00 equivalent) were added to a 50 mL round-bottom flask under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C for 2 hours under a nitrogen atmosphere. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20-40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5%-5% B for 10 min, then 40% B-60% B gradient over 15 min; detector: 220 nm. The fraction containing the desired product was collected at 54% B and concentrated under reduced pressure to give 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (200 mg, 81.78%) as a yellow oil.

[0567] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one

[0568] TFA (2 mL) was added dropwise to a stirred solution of 200 mg 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one in DCM (10 mL) at room temperature. The reaction mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (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 30×150mm 5um; mobile phase A: not limited, mobile phase B: not limited; flow rate: 60mL / min; gradient: 30%B to 55%B over 8 min; 220nm; Rt: 7.232min) to obtain 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]cyclopropyl]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one (39.2 mg), a grayish-white solid.

[0569] Example 19 Synthesis of Compound 109

[0570]

[0571] 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0572] K₂CO₃ (512.38 mg, 3.707 mmol, 2 equivalents) was added to a stirred solution of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (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). The resulting mixture was stirred at 70 °C for 0.5 h. The mixture was then cooled to room temperature. The reaction was quenched with water at room temperature. The resulting mixture was extracted with EtOAc (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 / EtOAc (5:1) to give 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (500 mg, 63.58%) as a white solid.

[0573] 4-(2-vinyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0574] K₂CO₃ (325.75 mg, 2.357 mmol, 2 equivalents) and Pd(PPh₃)₄ (68.09 mg, 0.059 mmol, 0.05 equivalents) were added to a solution of 4-(2-bromo-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (500 mg, 1.178 mmol, 1 equivalent) and pentamethyl-1,3,2-dioxane (334.72 mg, 2.357 mmol, 2.00 equivalents) in H₂O (2 mL) and 1,4-dioxane (16 mL). 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 / EtOAc (5:1) to give 4-(2-vinyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (250 mg, 57.12%), as a yellow oil.

[0575] 4-(2-Ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester

[0576] Pd / C (100 mg, 0.940 mmol, 1.40 equivalent) was added to a stirred solution of 4-(2-vinyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (250 mg, 0.673 mmol, 1 equivalent) in MeOH (10 mL). The resulting mixture was stirred at RT 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 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (210 mg, 0.08%) as a black oil.

[0577] 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine

[0578] TFA (1 mL) was added to a stirred solution of 210 mg (0.562 mmol, 1 equivalent) of 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (DCM) in 3 mL of DCM. The resulting mixture was stirred at room temperature under air for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The mixture was purified by reversed-phase rapid chromatography under the following conditions: column: spnerical 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 min (254 nm). The fraction containing the desired product was collected at 40% B and concentrated under reduced pressure. This produces 4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine (120 mg, 78.07%), a pale yellow oil.

[0579] 4-Chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0580] 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-dihydropyridazin-3-one (109.37 mg, 0.439 mmol, 1.00 equivalent) were added to a stirred solution of DIEA (113.49 mg, 0.878 mmol, 2 equivalents). The resulting mixture was stirred at 100 °C under air for 2 hours. The residue was purified by preparative TLC (PE / EtOAc 1:1) to give 4-chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 46.87%) as a pale yellow solid.

[0581] 4-Chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one

[0582] TFA (1 mL) was added to a stirred solution of 4-chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 0.206 mmol, 1 equivalent) in DCM (3 mL). The resulting mixture was stirred at room temperature for 2 hours. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 7 with saturated NH4HCO3 (aqueous solution). The crude product was purified by preparative HPLC under the following conditions (column: XBridgePrep OBD C18 column 30×150mm 5um; mobile phase A: water (10mM NH4HCO3), mobile phase B: acetonitrile; flow rate: 60mL / min; gradient: 30%B to 50%B over 8 min; 220nm; Rt: 7.27min) to obtain 4-chloro-5-[4-(2-ethyl-3-fluorophenoxy)-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2,3-dihydropyridazin-3-one (41.6mg, 50.31%) as a white solid.

[0583] Example 20 Synthesis of Compound 127

[0584]

[0585] 3-(methylamino)pyridine-4-carboxylic acid methyl ester

[0586] SOCl2 (43.01 g, 361.478 mmol, 5 equivalents) was added dropwise to a stirred 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) at 0 °C. The resulting mixture was stirred at 70 °C for 30 h. The reaction was monitored by LCMS. 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 alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (2 x 20 mL). The combined organic layers were washed with brine (1 x 30 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure to give methyl 3-(methylamino)pyridine-4-carboxylic acid (9 g, crude product) as a yellow solid.

[0587] 3-(N-methylacetamido)pyridine-4-carboxylic acid methyl ester

[0588] Pyridine (21.42 g, 270.791 mmol, 5 equivalents) and acetyl chloride (6.38 g, 81.237 mmol, 1.5 equivalents) were added dropwise to a stirred solution of methyl 3-(methylamino)pyridine-4-carboxylic acid (9 g, 54.158 mmol, 1 equivalent) in 100 mL of DCM. The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The solution was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: C18 column 330g; mobile phase A: water (10mM NH4HCO3), mobile phase B: acetonitrile; flow rate: 80mL / min; gradient: 10% B to 30% B over 25min; 254 / 220nm) to obtain methyl 3-(N-methylacetamido)pyridine-4-carboxylic acid (8g, 70.94%), which was a brown liquid.

[0589] 4-Hydroxy-1-methyl-1,2-dihydro-1,7-naphthid-2-one

[0590] t-BuOK (6.47 g, 57.632 mmol, 2 equivalents) was added to a stirred solution of methyl 3-(N-methylacetamido)pyridine-4-carboxylic acid (6 g, 28.816 mmol, 1 equivalent) in anhydrous 1,4-dioxane (100 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 1 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (10:1), to give 4-hydroxy-1-methyl-1,2-dihydro-1,7-naphthidin-2-one (4.5 g, 88.64%) as an orange solid.

[0591] 4-Chloro-1-methyl-1,2-dihydro-1,7-naphthid-2-one

[0592] POCl3 (3.92 g, 25.543 mmol, 1 equivalent) was added dropwise to a stirred solution of 4-hydroxy-1-methyl-1,2-dihydro-1,7-naphthid-2-one (4.5 g, 25.543 mmol, 1 equivalent) in anhydrous 1,4-dioxane (100 mL) at room temperature. The resulting mixture was stirred at 90 °C for 16 hours. The mixture was then cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with DCM / MeOH (10:1), to give 4-chloro-1-methyl-1,2-dihydro-1,7-naphthid-2-one (2 g, 40.23%) as a red solid.

[0593] 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthidium-2-one

[0594] Cs₂CO₃ (2.68 g, 8.221 mmol, 2 equivalents), 4-fluoro-2-(trifluoromethyl)aniline (1.47 g, 8.221 mmol, 2.00 equivalents), XantPhos (0.95 g, 1.644 mmol, 0.4 equivalents), and Pd(AcO)₂ (0.18 g, 0.822 mmol, 0.2 equivalents) were added to a stirred solution of 4-chloro-1-methyl-1,2-dihydro-1,7-naphthid-2-one (0.8 g, 4.111 mmol, 1 equivalent) in anhydrous 1,4-dioxane (15 mL) at room temperature under a nitrogen atmosphere. The final reaction mixture was irradiated with microwave at 110 °C for 4 h. The reaction was monitored by LCMS. The resulting mixture was extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with brine (1 x 100 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: C18 column 330g; mobile phase A: water (10mM AcOH), mobile phase B: acetonitrile; flow rate: 50mL / min; gradient: 20% B to 40% B over 40min; 254 / 220nm) to obtain 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthidin-2-one (1.1g, 79.34%) as a grayish-white solid.

[0595] 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthidium-2-one

[0596] PtO2 (67.33 mg, 0.296 mmol, 0.10 equivalent) was added to a stirred solution of 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2-dihydro-1,7-naphthid-2-one (1 g, 2.965 mmol, 1 equivalent) in THF (20 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 16 h. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with EtOAc (3 x 20 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography 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: 5% B to 20% B over 40 min; 254 / 220 nm). The fraction containing the desired product was collected under 16% B and concentrated under reduced pressure to give 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthid-2-one (750 mg, 74.11%) as a grayish-white solid.

[0597] 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-naphthidin-2-one]

[0598] DIPEA (568.00 mg, 4.395 mmol, 2 equivalents) was added to a stirred mixture of 4-[[4-fluoro-2-(trifluoromethyl)phenyl]amino]-1-methyl-1,2,5,6,7,8-hexahydro-1,7-naphthidium-2-one (750 mg, 2.197 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (1.09 g, 4.395 mmol, 2 equivalents) at room temperature. The resulting mixture was stirred at 100 °C for 2 hours. The reaction was monitored by LCMS. The residue was dissolved in DMF (10 mL). The solution was purified by reversed-phase rapid chromatography 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: 30% B to 50% B over 40 min; 254 / 220 nm). The fraction containing the desired product was collected at 44% B and concentrated under reduced pressure to give 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-naphthidium-2-one (1 g, 82.15%) as a yellow oil.

[0599] 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-naphthidin-2-one

[0600] Cs₂CO₃ (0.94 g, 2.888 mmol, 2 equivalents) and MeI (614.96 mg, 4.333 mmol, 3 equivalents) were added to a stirred solution of 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-naphthidium-2-one (800 mg, 1.444 mmol, 1 equivalent) in DMF (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction was monitored by LCMS. The mixture was purified by reversed-phase rapid chromatography 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: 40% B to 60% B over 40 min; 254 / 220 nm). The fraction containing the desired product was collected at 49% B and concentrated under reduced pressure to give 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-naphthidium-2-one (80 mg, 9.75%) as a yellow oil.

[0601] 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-naphthidin-2-one

[0602] TFA (0.5 mL, 6.732 mmol, 31.07 equivalents) was added dropwise to a stirred solution of 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-naphthid-2-one (80 mg, 0.141 mmol, 1 equivalent) in DCM (4.5 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The solution was rapidly purified by reverse-phase reaction to give 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-naphthidin-2-one (40 mg, 58.69%), a white solid.

[0603] Example 21 Synthesis of compounds 135 and 137

[0604]

[0605] 2-Chloro-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylic acid tert-butyl ester.

[0606] At room temperature, 4-fluoro-2-(trifluoromethyl)phenol (4.44 g, 24.66 mmol) and K₂CO₃ (3.41 g, 24.66 mmol) were added to a solution of 2,4-dichloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (5 g, 16.44 mmol) in DMF (50 mL). The resulting mixture was stirred at 70 °C for 1 hour. It was then cooled to room temperature. The mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: Spherical C18, 20–40 μm, 330 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 80 mL / min; gradient: 5% B over 10 min, 35% B to 45% B over 10 min; detector: 254 nm / 220 nm). The fraction containing the desired product was collected at 44% B and concentrated under reduced pressure to give 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (6.2 g, 85%) as a white solid).

[0607] 4-(4-fluoro-2-(trifluoromethyl)phenoxy)-2-vinyl-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylic acid tert-butyl ester.

[0608] To a solution of 2-chloro-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (500 mg, 1.12 mmol) in dioxane (10 mL), 2-vinyl-4,4,5,5-tetramethyl-1,3,2-dioxane (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 under a nitrogen atmosphere for 2 hours, the resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC and eluted with 17% ethyl acetate in petroleum ether to give 2-vinyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (490 mg, 99%) as a pale yellow solid.

[0609] 2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidine-7(6H)-carboxylic acid tert-butyl ester.

[0610] At room temperature, 4-hydroxy-4-methylmorpholine-4-onium (323 mg, 2.73 mmol) and K2OsO4·2H2O (34 mg, 0.091 mmol) were added to a solution of 2-vinyl-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (400 mg, 0.91 mmol) in DCM (20 mL). After stirring for another hour, the resulting mixture was concentrated under reduced pressure, and the residue was purified by reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20–40 μm, 120 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5% B for 10 min, 45% B to 65% B for 15 min; detector: 254 nm and 220 nm. The fraction containing the desired product was collected at 64% B and concentrated under reduced pressure to give 2-(1,2-dihydroxyethyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidine-7-carboxylic acid tert-butyl ester (280 mg, 65%) as a white solid.

[0611] 1-(4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,6,7,8-tetrahydropyridino[3,4-d]pyrimidin-2-yl)ethane-1,2-diol.

[0612] TFA (1 mL) was added to a stirred solution of 2-(1,2-dihydroxyethyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (280 mg, 0.59 mmol) in DCM (4 mL) 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 DCM (50 mL) and washed with a saturated aqueous solution of NaHCO3 (20 mL). 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 with 8% methanol in dichloromethane to give 1-[4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-2-yl]ethane-1,2-diol (180 mg, 82%), as a brown solid.

[0613] 4-Chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one.

[0614] 4,5-Dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (176 mg, 0.71 mmol) was added to a stirred solution of 2-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yloxy]benzaldehyde (180 mg, 0.71 mmol) in DIEA (0.5 mL) at room temperature. The resulting mixture was stirred at 90 °C for 1 hour. After cooling to ambient temperature, the mixture was concentrated under reduced pressure. The residue was purified by preparative TLC and eluted with 8% methanol in dichloromethane to give 4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)-2-(tetrahydro-2H-pyran-2-yl)pyridazin-3(2H)-one (140 mg, 43%) as a brown solid.

[0615] (S)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidin-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]pyrimidin-7(6H)-yl)pyridazin-3(2H)-one

[0616] TFA (1 mL) was added to a solution of 4-chloro-5-[2-(1,2-dihydroxyethyl)-4-[4-fluoro-2-(trifluoromethyl)phenoxy]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (150 mg, 0.27 mmol) in DCM (4 mL) 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 reversed-phase rapid chromatography under the following conditions: column: Spherical C18, 20–40 μm, 120 g; mobile phase A: water (with 5 mM NH4HCO3); mobile phase B: acetonitrile; flow rate: 45 mL / min; gradient: 5% B for 10 min, 45% B to 65% B for 15 min; detector: 254 nm and 220 nm. The fraction containing the desired product was collected at 64% B and concentrated under reduced pressure to give a racemic product (130 mg), which was then 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 min; detector: 254 / 220 nm. Although the two isomers were separated by this technique, their absolute orientation was not determined. The fraction containing the desired product was collected and concentrated under reduced pressure to give the following products: (S)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)pyridazin-3(2H)-one: retention time (4.97 min) (49.5 mg, 39%), as a white solid; and (R)-4-chloro-5-(2-(1,2-dihydroxyethyl)-4-(4-fluoro-2-(trifluoromethyl)phenoxy)-5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)pyridazin-3(2H)-one: retention time (8.05 min) (45.7 mg, 36%), as a white solid.

[0617] Example 22 Synthesis of Compound 131

[0618]

[0619] 4-[[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0620] Pd(AcO)₂ (83.24 mg, 0.371 mmol, 0.2 equivalents), Cs₂CO₃ (1207.95 mg, 3.707 mmol, 2.0 equivalents), and XantPhos (429.04 mg, 0.741 mmol, 0.4 equivalents) were added to a stirred mixture of 4-chloro-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (500 mg, 1.854 mmol, 1 equivalent) and 4-(trifluoromethyl)pyridin-3-amine (601.03 mg, 3.707 mmol, 2.0 equivalents) in 1,4-dioxane (5 mL) under a nitrogen atmosphere. The resulting mixture was stirred at 110 °C under a nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with DCM (3 × 2 mL). The filtrate was concentrated under reduced pressure. The resulting mixture was concentrated under reduced pressure. The crude product was purified by reversed-phase rapid chromatography 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 min; detector: 254 nm and 220 nm, collecting the desired product at 64% B) to give 4-[[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (600 mg, 81.86%) as a white solid.

[0621] 4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester

[0622] CH3I (0.95 g, 6.677 mmol, 2.0 equivalent) was added to a stirred mixture of 4-[[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (1.32 g, 3.339 mmol, 1 equivalent) and Cs2CO3 (2.18 g, 6.677 mmol, 2.0 equivalent) in DMF (10 mL) under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The crude product was purified by reversed-phase rapid chromatography 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 min; detector: 254 nm and 220 nm, collecting the desired product at 64% B) to give 4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (400 mg, 29.26%) as a brown solid.

[0623] N-Methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]-4-(trifluoromethyl)pyridine-3-amine

[0624] TFA (1 mL) was added to a stirred solution of 4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-carboxylic acid tert-butyl ester (220 mg, 0.537 mmol, 1 equivalent) in DCM (4 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized 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 give N-methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]-4-(trifluoromethyl)pyridin-3-amine (130 mg, 78.22%) as a brown solid.

[0625] 4-Chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0626] At room temperature, 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (104.69 mg, 0.420 mmol, 1 equivalent) was added to a stirred solution of N-methyl-N-[5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-4-yl]-4-(trifluoromethyl)pyridin-3-amine (130 mg, 0.420 mmol, 1 equivalent) in DIEA (0.5 mg). The resulting mixture was stirred at 90 °C for 1 h. The reaction was monitored by LCMS. 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 give 4-chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 45.58%) as a brown solid.

[0627] 4-Chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one

[0628] TFA (1 mL) was added to a stirred solution of 4-chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 0.192 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized 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 PrepPhenyl OBD column 19×150 mm 5 μm 13 nm; mobile phase A: water, 5 mM NH4HCO3; mobile phase B: acetonitrile; flow rate: 60 mL / min; gradient: 35% B to 55% B over 8 min; 220 nm; Rt: 7.13 min) to obtain 4-chloro-5-(4-[methyl[4-(trifluoromethyl)pyridin-3-yl]amino]-5H,6H,7H,8H-pyrido[3,4-d]pyrimidin-7-yl)-2,3-dihydropyridazin-3-one (52 mg, 61.99%) as a white solid.

[0629] Example 23 Synthesis of intermediates

[0630] A. 2-(difluoromethyl)-4-fluorophenylacetate

[0631]

[0632] 4-Fluoro-2-formylphenylacetate

[0633] Acetoacetate (14.57 g, 0.143 mmol, 2 equivalents) was added 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) at 25 °C. The solution was stirred at 25 °C for 30 min. The resulting solution was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EA (100 / 1 to 20 / 1) to give 4-fluoro-2-formylphenylacetate (12 g, 92.31%) as a pale yellow oil.

[0634] 2-(difluoromethyl)-4-fluorophenylacetate

[0635] DAST (21.24 g, 131.760 mmol, 2 equivalents) was added to a solution of 4-fluoro-2-formylphenylacetate (12 g, 65.880 mmol, 1 equivalent) in DCM (200 mL, 3146.009 mmol, 47.75 equivalents) at 0 °C. The solution was stirred at 25 °C for 4 hours. The resulting solution was quenched with water (100 mL). The resulting mixture was extracted with DCM (100 mL × 2). The combined organic layers were washed with saturated NaCl aqueous solution (100 mL × 2) and dried over anhydrous Na2SO4. After filtration, the filtrate 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 2-(difluoromethyl)-4-fluorophenylacetate (10 g, 74.35%) as a pale yellow oil.

[0636] B. 2-(difluoromethyl)-4-fluorophenol

[0637]

[0638] 1-Bromo-2-(difluoromethyl)-4-fluorobenzene

[0639] DAST (15.9 g, 98.52 mmol, 2 equivalents) was added to a stirred solution of 2-bromo-5-fluorobenzaldehyde (10 g, 49.26 mmol, 1 equivalent) in DCM (60 mL). The resulting mixture was stirred at -10 °C for 2 h. The reaction was quenched with water at -10 °C. The resulting mixture was extracted with EtOAc (4 × 30 mL). The combined organic layers were washed with brine (2 × 40 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (6:1), to give 1-bromo-2-(difluoromethyl)-4-fluorobenzene (8 g, 72.18%) as a pale yellow oil.

[0640] 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxacyclopentaborane

[0641] 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 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 equivalent) in 1,4-dioxane (300 mL, 3541.225 mmol, 25.70 equivalent) 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, eluting with PE / EA (10 / 1) to give 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxane (30 g, 80.03%), as a pale yellow oil. The reaction was monitored by TLC. The crude product was used directly in the next step.

[0642] 2-(difluoromethyl)-4-fluorophenol

[0643] H₂O₂ (30%) (50 mL, 2146.131 mmol, 11.68 equivalents) was added dropwise to a solution of 2-[2-(difluoromethyl)-4-fluorophenyl]-4,4,5,5-tetramethyl-1,3,2-dioxane (50 g, 183.776 mmol, 1 equivalent) in MeOH (300 mL, 7409.673 mmol, 40.32 equivalents) and H₂O (100 mL, 5550.837 mmol, 30.20 equivalents). 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), and the organic layer was washed with 3 × 200 mL of saturated NaCl (aqueous solution). The combined organic layers were dried with anhydrous Na2SO4 and concentrated under reduced pressure to give 2-(difluoromethyl)-4-fluorophenol (25 g, 83.91%), a pale yellow oil.

[0644] Example 24 Synthesis of compound MS

[0645]

[0646] 1-[1-(2-bromopyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester

[0647] 1-Azide-4-nitrobenzene (576.6 mg, 3.51 mmol, 1.40 equivalent) and Zn(OAc)₂ (460.5 mg, 2.51 mmol, 1.00 equivalent) were added in portions to a stirred mixture of 1-(2-bromopyridin-3-yl)ethyl-1-amine (1009.1 mg, 5.02 mmol, 2.00 equivalent) and 4-oxopiperidin-1-carboxylic acid tert-butyl ester (500 mg, 2.51 mmol, 1 equivalent) in DMF (10 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 60 °C under a nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge Shield RP18 OBD column, 20-40 μm, 19*150 mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 80 mL / min; gradient: 40% B to 80% B over 30 min; 220 nm; Rt: 7.08 min) to obtain 1-[1-(2-bromopyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (800 mg, 78.08%), as a yellow oil.

[0648] 1-[1-(2-vinylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester

[0649] Pd(PPh3)4 (226.4 mg, 0.20 mmol, 0.10 equivalent) and K2CO3 (812.4 mg, 5.88 mmol, 3.00 equivalent) were added in portions to a stirred mixture of 1-[1-(2-bromopyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (800 mg, 1.96 mmol, 1 equivalent) and 2-vinyl-4,4,5,5-tetramethyl-1,3,2-dioxane-pentaborane (301.8 mg, 1.96 mmol, 1.00 equivalent) in dioxane (30 mL) and H2O (6 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 90 °C under a nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (30 / 1 to 5 / 1) to give 1-[1-(2-vinylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (600 mg, 86.15%) as a yellow oil.

[0650] 1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester

[0651] In a 50 mL round-bottom flask under a nitrogen atmosphere, Pd / C (10%, 0.02 g) was added to a solution of 1-[1-(2-vinylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (300 mg, 0.84 mmol, 1 equivalent) in 20 mL MeOH. The mixture was hydrogenated at room temperature under a hydrogen atmosphere using a hydrogen balloon for 2 h, filtered through a diatomaceous earth pad, and concentrated under reduced pressure. This yielded 1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (260 mg, 86.18%) as a yellow oil.

[0652] 2-Ethyl-3-(1-[1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-1-yl]ethyl)pyridine

[0653] TFA (1 mL, 13.46 mmol, 18.51 equivalents) was added dropwise to a stirred solution of 1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (260 mg, 0.73 mmol, 1 equivalent) in a DCM (10 mL). The reaction mixture was stirred at room temperature for 16 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with CH2Cl2 (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 TLC (PE / EtOAc 1 / 1) to give 2-ethyl-3-(1-[1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-1-yl]ethyl)pyridine (150 mg, 80.14%) as a yellow oil.

[0654] 4-Chloro-5-[1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0655] 2-Ethyl-3-(1-[1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-1-yl]ethyl)pyridine (150 mg, 0.58 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (290.4 mg, 1.17 mmol, 2.00 equivalent), and DIEA (150.7 mg, 1.17 mmol, 2.00 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 under a nitrogen atmosphere for 16 h. The residue was purified by preparative TLC (PE / EtOAc = 1 / 1) to give 4-chloro-5-[1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxane-2-yl)-2,3-dihydropyridazin-3-one (145 mg, 52.93%) as a yellow oil.

[0656] 4-Chloro-5-[1-[(1R)-1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[1-[(1S)-1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one

[0657] TFA (1 mL, 13.46 mmol, 43.64 equivalents) was added dropwise to a stirred solution of 4-chloro-5-[1-[1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (145 mg, 0.31 mmol, 1 equivalent) in DCM (10 mL). The reaction mixture was stirred at room temperature for 4 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with CH2Cl2 (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 (75 mg) was purified by chiral preparative HPLC under the following conditions (column: CHIRALPAK IE, 2*25 cm, 5 μm; mobile phase: (Hex / DCM = 3 / 1) / EtOH = 80 / 20; flow rate: 20 mL / min; gradient: 20B to 20B over 20 min; 220 / 254 nm; RT1: 12.678; RT2: 16.738). At 1.380 min, 4-chloro-5-[1-[(1R)-1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (16.8 mg, 14.11%) was obtained as a white solid. At 1.832 min, 4-chloro-5-[1-[(1S)-1-(2-ethylpyridin-3-yl)ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (19.8 mg) was obtained as a white solid (E01224-021).

[0658] Synthesis of Example 25MX

[0659]

[0660] (4R)-4-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester

[0661] To a stirred solution of (2R)-2-methyl-4-oxopiperidin-1-carboxylic acid tert-butyl ester (1 g, 4.69 mmol, 1 equivalent) and 1-[2-(trifluoromethyl)phenyl]ethyl-1-amine (0.9 g, 4.76 mmol, 1.01 equivalent) in DMF (20 mL), 1-azido-4-nitrobenzene (1.1 g, 6.56 mmol, 1.4 equivalent) and Zn(OAc)₂ (0.9 g, 4.69 mmol, 1 equivalent) were added. The resulting mixture was stirred overnight at 60 °C. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of MeCN, 20% to 60% gradient over 40 min; detector, UV 254 nm. This produces (4R)-4-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (1.5 g, 77.94%), a grayish-white solid.

[0662] (4R)-4-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine

[0663] TFA (3 mL) was added to a stirred solution of (4R)-4-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine-5-carboxylic acid tert-butyl ester (1.5 g, 3.65 mmol, 1 equivalent) in DCM (9 mL). The resulting mixture was stirred at room temperature for 2 h. The mixture was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The solution was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of MeCN, 10% to 50% gradient over 30 min; detector, UV 254 nm. This produces (4R)-4-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine (1 g, 88.17%), as a yellow solid.

[0664] 4-Chloro-5-[(6R)-6-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0665] Add (6R)-6-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridine (200 mg, 0.64 mmol, 1 equivalent), 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (192.6 mg, 0.77 mmol, 1.2 equivalent), and DIEA (249.9 mg, 1.93 mmol, 3 equivalent) to a stirred solution. Stir the resulting mixture overnight at 100 °C. Purify the residue by reversed-phase rapid chromatography under the following conditions: column, C18 silica gel; mobile phase, aqueous solution of MeCN, 20% to 60% gradient over 40 min; detector, UV 254 nm. This produces 4-chloro-5-[(6R)-6-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (250 mg, 74.17%), a yellow solid.

[0666] 4-Chloro-5-[(6R)-6-methyl-1-[(1R)-1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[(6R)-6-methyl-1-[(1S)-1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one

[0667] TFA (2 mL) was added to a stirred solution of 4-chloro-5-[(6R)-6-methyl-1-[1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (240 mg, 0.46 mmol, 1 equivalent) in DCM (6 mL). The resulting mixture was stirred at room temperature for 2 h. The resulting mixture was concentrated under reduced pressure. The crude product was subjected to 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: ACN; Flow rate: 60 mL / min; Gradient: 27% B to 50% B over 7 min; 220 nm; Rt: 6.38 min) Purification yielded 4-chloro-5-[(6R)-6-methyl-1-[(1R)-1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5- [c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (22.4 mg, 11.12%), a yellow solid, and 4-chloro-5-[(6R)-6-methyl-1-[(1S)-1-[2-(trifluoromethyl)phenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (58.5 mg, 29.05%), a grayish-white solid.

[0668] Example 26 Synthesis of compound LY

[0669]

[0670] 3-(1-Chloroethyl)-2-ethylpyridine is prepared by using the corresponding pyridine through the methods and schemes described for 3-(1-chloropropyl)-2-ethylpyridine.

[0671]

[0672] 3-(1-Chloropropyl-1)-2-ethylpyridine

[0673] SOCl2 (432.0 mg, 3.63 mmol, 2.00 equivalents) was added dropwise to a stirred solution of 1-(2-ethylpyridin-3-yl)prop-1-ol (300 mg, 1.82 mmol, 1 equivalent) in DCM (20 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred for 16 h under a nitrogen atmosphere. The reaction was monitored by LCMS. The resulting mixture was concentrated under vacuum. This yielded 3-(1-chloropropyl)-2-ethylpyridine (350 mg, 104.95%) as a yellow oil.

[0674] Step 1.

[0675] 4-Chloro-5-[4-[1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0676] K₂CO₃ (92.5 mg, 0.67 mmol, 2.0 equivalent) and KI (111.1 mg, 0.67 mmol, 2.0 equivalent) were added in portions to a stirred mixture of 4-chloro-2-(oxan-2-yl)-5-(piperazin-1-yl)-2,3-dihydropyridazin-3-one (100 mg, 0.33 mmol, 1 equivalent) and 3-(1-chloroethyl)-2-ethylpyridine (68.1 mg, 0.40 mmol, 1.20 equivalent) in 10 mL of ACN in portions. The resulting mixture was stirred at 70 °C under nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with 2 × 30 mL of ACN. The filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (CH2Cl2 / MeOH 20 / 1) to give 4-chloro-5-[4-[1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 83.00%), as a yellow oil.

[0677] Step 2.

[0678] LY and LZ

[0679] 4-Chloro-5-[4-[(1S)-1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[4-[(1R)-1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2,3-dihydropyridazin-3-one

[0680] TFA (1 mL, 13.46 mmol, 48.46 equivalents) was added dropwise to a stirred solution of 4-chloro-5-[4-[1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 0.28 mmol, 1 equivalent) in DCM (10 mL). The reaction mixture was stirred at room temperature for 4 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NH4HCO3 (aqueous solution). The resulting mixture was extracted with CH2Cl2 (3 × 100 mL). The combined organic layers were washed with brine (1 × 50 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue (70 mg) was purified by chiral preparative HPLC under the following conditions (column: CHIRALPAKIE, 2*25 cm, 5 μm; mobile phase: MTBE / EtOH = 80 / 20; flow rate: 20 mL / min; gradient: 20B to 20B over 20 min; 220 / 254 nm; RT1: 12.678; RT2: 16.738). 4-chloro-5-[4-[(1S)-1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2,3-dihydropyridazin-3-one (9.5 mg, 9.83%) was obtained at 2.544 min as a pale yellow solid. At 2.984 min, 4-chloro-5-[4-[(1R)-1-(2-ethylpyridin-3-yl)ethyl]piperazin-1-yl]-2,3-dihydropyridazin-3-one (14.2 mg) was obtained as a pale yellow solid.

[0681] Example 27 Synthesis of LW

[0682]

[0683] Step 1.

[0684] 4-[(2-bromopyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester

[0685] AcOH (208.26 mg, 3.468 mmol, 1 equivalent) was added dropwise / partially to a stirred solution of 2-bromopyridin-3-amine (600 mg, 3.468 mmol, 1 equivalent) and 4-oxopiperidin-1-carboxylic acid tert-butyl ester (690.99 mg, 3.468 mmol, 1 equivalent) in a 20 mL DCM solution under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 2 h. NaBH(OAc)3 (1470.00 mg, 6.936 mmol, 2.00 equivalent) was added to the mixture at 0 °C. The mixture was stirred at room temperature overnight. The desired product was detected by LCMS. The reaction was quenched at 0 °C by adding water (40 mL). The aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The organic layer was concentrated under reduced pressure to obtain tert-butyl 4-[(2-bromopyridin-3-yl)amino]piperidine-1-carboxylic acid (800 mg, 64.75%), as a yellow solid.

[0686] Step 2.

[0687] 4-[(2-vinylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester

[0688] K₂CO₃ (931.03 mg, 6.737 mmol, 3 equivalents) and Pd(PPh₃)₄ (259.48 mg, 0.225 mmol, 0.1 equivalents) were added to a solution of 2-vinyl-4,4,5,5-tetramethyl-1,3,2-dioxane (691.71 mg, 4.491 mmol, 2 equivalents) and 4-[(2-bromopyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (800 mg, 2.246 mmol, 1 equivalent) in 1,4-dioxane (10 mL) and H₂O (2 mL). After stirring overnight at 80 °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 / EtOAc (5:1 to 3:1) to give 4-[(2-vinylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (600 mg, 88.07%) as a yellow solid.

[0689] Step 3.

[0690] 4-[(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester

[0691] In a 250 mL round-bottom flask under a nitrogen atmosphere, Pd / C (10%, 21.05 mg) was added to a solution of 4-[(2-vinylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (600 mg, 1.978 mmol, 1 equivalent) in 30 mL MeOH. The mixture was hydrogenated at room temperature under a hydrogen atmosphere using a hydrogen balloon for 3 h, filtered through a diatomaceous earth pad, and concentrated under reduced pressure to give 4-[(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (590 mg, 97.68%) as a yellow solid.

[0692] Step 4.

[0693] 2-Ethyl-N-(piperidin-4-yl)pyridine-3-amine

[0694] TFA (3 mL) was added dropwise to a stirred solution of 590 mg (1 equivalent) of 4-[(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester in DCM (15 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at room temperature for 1 h. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure to give 2-ethyl-N-(piperidine-4-yl)pyridin-3-amine (390 mg, 98.34%) as a white solid.

[0695] Step 5.

[0696] Compound LX

[0697] 4-Chloro-5-[4-[(2-ethylpyridin-3-yl)amino]piperidin-1-yl]-2,3-dihydropyridazin-3-one

[0698] DIEA (125.90 mg, 0.974 mmol, 2 equivalents) was added dropwise to a stirred solution of 2-ethyl-N-(piperidin-4-yl)pyridin-3-amine (100 mg, 0.487 mmol, 1 equivalent) and 4,5-dichloro-2,3-dihydropyridazin-3-one (80.35 mg, 0.487 mmol, 1.00 equivalent) in DMA (8 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at 100 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The crude product (60 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: ACN; flow rate: 60 mL / min; gradient: 18% B to 30% B over 6.5 min; 220 nm; Rt: 5.37 8.55 min) to obtain 4-chloro-5-[4-[(2-ethylpyridin-3-yl)amino]piperidin-1-yl]-2,3-dihydropyridazin-3-one (20 mg), a white solid, and 5-chloro-4-[4-[(2-ethylpyridin-3-yl)amino]piperidin-1-yl]-2,3-dihydropyridazin-3-one (7 mg), a white solid).

[0699] Step 6.

[0700] 4-[Ethyl(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester

[0701] AcOH (29.49 mg, 0.491 mmol, 1 equivalent) was added dropwise to a stirred solution of 4-[(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (150 mg, 0.491 mmol, 1 equivalent) and acetaldehyde (32.45 mg, 0.737 mmol, 1.5 equivalent) in a 10 mL DCM solution under a nitrogen atmosphere at 0 °C. The mixture was stirred at room temperature for 2 h. NaBH3CN (92.59 mg, 1.473 mmol, 3 equivalent) was added to the mixture at 0 °C. The mixture was stirred at room temperature overnight. The desired product was detected by LC-MS. The reaction was quenched at 0 °C by adding water (40 mL). The aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The organic layer was concentrated under reduced pressure to obtain 4-[ethyl(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester (150 mg, 91.59%), a white solid.

[0702] Step 8.

[0703] N,2-Diethyl-N-(piperidin-4-yl)pyridine-3-amine

[0704] TFA (2 mL) was added dropwise to a stirred solution of 150 mg (1 equivalent) of 4-[ethyl(2-ethylpyridin-3-yl)amino]piperidine-1-carboxylic acid tert-butyl ester in DCM (10 mL) at 0 °C under a nitrogen atmosphere. The mixture was stirred at room temperature for 2 h. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure to give N,2-diethyl-N-(piperidine-4-yl)pyridin-3-amine (100 mg, 95.27%) as a yellow solid.

[0705] Step 8.

[0706] compound LW

[0707] 4-Chloro-5-[4-[ethyl(2-ethylpyridin-3-yl)amino]piperidin-1-yl]-2,3-dihydropyridazin-3-one

[0708] DIEA (66.5 mg, 0.51 mmol, 2 equivalents) was added to a stirred solution of N,2-diethyl-N-(piperidin-4-yl)pyridin-3-amine (60 mg, 0.26 mmol, 1 equivalent) and 4,5-dichloro-2,3-dihydropyridazin-3-one (42.4 mg, 0.26 mmol, 1.00 equivalent) in DMA (5 mL, 53.78 mmol, 209.15 equivalents) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at 100 °C. The desired product was detected by LCMS. The 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 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 25% B to 40% B over 8 min; 220 nm; Rt: 7.58 min) to obtain 4-chloro-5-[4-[ethyl(2-ethylpyridin-3-yl)amino]piperidin-1-yl]-2,3-dihydropyridazin-3-one (24.3 mg), a white solid.

[0709] Example 28 Synthesis of OM

[0710] Compound OM The compound OK is prepared by using the corresponding amine, according to the methods and schemes described herein.

[0711]

[0712] OK preparation

[0713] 2-[[2-(difluoromethyl)phenyl]methyl]-2H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester

[0714] KI (752.04 mg, 4.530 mmol, 1 equivalent) and K₂CO₃ (1252.22 mg, 9.061 mmol, 2 equivalents) were added to a stirred solution of 1-(chloromethyl)-2-(difluoromethyl)benzene (800 mg, 4.530 mmol, 1 equivalent) and 1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (1337.96 mg, 4.530 mmol, 1.00 equivalent) in MeCN (15 mL) under a nitrogen atmosphere. The mixture was stirred overnight at 80 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (5:1 to 3:1) to give 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (500 mg, 25.34%) as a yellow solid, and 2-[[2-(difluoromethyl)phenyl]methyl]-2H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (200 mg, 10.14%) as a yellow solid.

[0715] 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester

[0716] TFA (2 mL, 26.926 mmol, 23.45 equivalents) was added dropwise to a stirred solution of 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (500 mg, 1.148 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 137.00 equivalents). The mixture was stirred at room temperature for 2 h. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure to give 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester (380 mg, 98.69%) as a yellow solid.

[0717] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester

[0718] 4,5-Dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (282.25 mg, 1.133 mmol, 1.00 equivalent) was added in portions to a stirred solution of 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester (380 mg, 1.133 mmol, 1 equivalent) in DIEA (292.90 mg, 2.266 mmol, 2 equivalent) at room temperature under a nitrogen atmosphere. The mixture was stirred overnight at 100 °C. The desired product was detected by LCMS. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (5:1 to 3:1), to give ethyl 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylate (500 mg, 80.52%) as a yellow solid.

[0719] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid

[0720] LiOH (100.51 mg, 4.197 mmol, 5 equivalents) was added in portions to a stirred solution of 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester (460 mg, 0.839 mmol, 1 equivalent) in THF (5 mL) and H₂O (5 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at 50 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: XBridgePrep OBD C18 column 30×150mm 5um; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 27% B to 55% B over 8 min; 220 nm; Rt: 7.82 min) to obtain 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (430 mg, 98.52%) as a colorless oil.

[0721] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-N,N-dimethyl-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide

[0722] CDI (37.4 mg, 0.23 mmol, 1.5 equivalents) was added in portions to a stirred solution of 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (80 mg, 0.15 mmol, 1 equivalent) in DMF (5 mL). The mixture was stirred at 50 °C for 2 h. Dimethylamine (13.9 mg, 0.31 mmol, 2.00 equivalents) was added to the mixture. The mixture was stirred at 50 °C overnight. The desired product was detected by LCMS. The resulting mixture was concentrated under vacuum to give 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-N,N-dimethyl-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (60 mg, 71.29%), as a yellow solid.

[0723] 5-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-1-[[2-(difluoromethyl)phenyl]methyl]-N,N-dimethyl-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide

[0724] TFA (2 mL) was added dropwise to a stirred solution of 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(difluoromethyl)phenyl]methyl]-N,N-dimethyl-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (50 mg, 1 equivalent) in 10 mL of DCM at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 1 h. The desired product was detected by LCMS. The resulting 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: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 23% B to 45% B over 7 min; 220 nm; Rt: 6.47 min) to obtain 5-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-1-[[2-(difluoromethyl)phenyl]methyl]-N,N-dimethyl-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (25 mg), a white solid.

[0725] Example 29 Synthesis of Compound OU

[0726]

[0727] Preparation of OU

[0728] 2-[[2-(difluoromethyl)phenyl]methyl]-2H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester

[0729] KI (752.04 mg, 4.530 mmol, 1 equivalent) and K₂CO₃ (1252.22 mg, 9.061 mmol, 2 equivalents) were added to a stirred solution of 1-(chloromethyl)-2-(difluoromethyl)benzene (800 mg, 4.530 mmol, 1 equivalent) and 1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (1337.96 mg, 4.530 mmol, 1.00 equivalent) in MeCN (15 mL) under a nitrogen atmosphere. The mixture was stirred overnight at 80 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (5:1 to 3:1) to give 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl 3-ethyl ester (500 mg, 25.34%) as a yellow solid, and 2-[[2-(difluoromethyl)phenyl]methyl]-2H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl 3-ethyl ester (200 mg, 10.14%) as a yellow solid.

[0730] 1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester

[0731] LiAlH4 (62.75 mg, 1.653 mmol, 1.2 equivalents) was added in portions to a stirred solution of 1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (600 mg, 1.378 mmol, 1 equivalent) in THF (10 mL, 123.430 mmol, 89.58 equivalents). The mixture was stirred at room temperature for 1 h. The desired product was detected by LCMS. The reaction was quenched at 0 °C by adding water (5 mL). The mixture was concentrated and purified by silica gel column chromatography (PE:EA = 2:1) to give 1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-5-carboxylic acid tert-butyl ester (500 mg, 92.24%) as a white solid.

[0732] (1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-yl)methanol

[0733] TFA (2 mL, 26.926 mmol, 21.19 equivalents) was added dropwise to a stirred solution of 1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-carboxylic acid tert-butyl ester (500 mg, 1.271 mmol, 1 equivalent) in DCM (10 mL, 157.300 mmol, 123.78 equivalents). The mixture was stirred at room temperature for 2 h. The desired product was detected by LCMS. The mixture was concentrated to give (1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-yl)methanol (370 mg, 99.26%) as a yellow solid.

[0734] 4-Chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0735] DIEA (326.06 mg, 2.523 mmol, 2 equivalents) was added dropwise to a stirred solution of (1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-3-yl)methanol (370 mg, 1.261 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (471.31 mg, 1.892 mmol, 1.5 equivalents) in DMA (1 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at 100 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (6:1 to 3:1), to give 4-chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (420 mg, 65.81%), as a white solid.

[0736] 4-Chloro-5-[3-(chloromethyl)-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0737] MsCl (108.68 mg, 0.949 mmol, 1.2 equivalents) was added dropwise to a stirred solution of 4-chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-(hydroxymethyl)-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (400 mg, 0.791 mmol, 1 equivalent) and TEA (160.00 mg, 1.581 mmol, 2 equivalents) in DCM (8 mL, 125.840 mmol, 159.17 equivalents) under a nitrogen atmosphere at 0 °C. The mixture was stirred overnight at room temperature. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (5:1), to give 4-chloro-5-[3-(chloromethyl)-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (400 mg, 96.48%), as a yellow solid.

[0738] 4-Chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-[(4-methylpiperazin-1-yl)methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0739] 1-Methylpiperazine (51.45 mg, 0.514 mmol, 5 equivalents) was added dropwise to a stirred solution of 4-chloro-5-[3-(chloromethyl)-1-[[2-(difluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg, 0.103 mmol, 1 equivalent) in MeCN (10 mL) under a nitrogen atmosphere at room temperature. The mixture was stirred overnight at 80 °C. The desired product was detected by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (5:1 to 1:1), to give 4-chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-[(4-methylpiperazin-1-yl)methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg, 99.31%), as a white solid.

[0740] 4-Chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-[(4-methylpiperazin-1-yl)methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2,3-dihydropyridazin-3-one

[0741] TFA (2 mL) was added dropwise to a stirred solution of 60 mg of 4-chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-[(4-methylpiperazin-1-yl)methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (60 mg) in DCM (10 mL). The mixture was stirred at room temperature for 2 h. The desired product was detected by LCMS. The reaction was quenched by adding 5 mL of saturated NaHCO3 (aqueous solution) at room temperature. The resulting mixture was concentrated under reduced pressure. The crude product (mg) was purified by preparative HPLC under the following conditions (column: XBridge PrepOBD C18 column 30×150mm 5um; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20% B to 35% B over 8 min; 220 nm; Rt: 7.25 min) to obtain 4-chloro-5-(1-[[2-(difluoromethyl)phenyl]methyl]-3-[(4-methylpiperazin-1-yl)methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridin-5-yl)-2,3-dihydropyridazin-3-one (30 mg), a white solid.

[0742] Example 30 Synthesis of compound OP

[0743]

[0744] Preparation of OP

[0745] 5-[[2-(trifluoromethyl)phenyl]methyl]-1H,2H,3H,4H,5H-cyclopenta[c]pyridine-2,7-dicarboxylic acid 2-tert-butyl-7-ethyl ester

[0746] At room temperature under a nitrogen atmosphere, K₂CO₃ (1.40 g, 10.158 mmol, 2 equivalents) and KI (0.84 g, 5.079 mmol, 1 equivalent) were added in portions to a stirred solution of 1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3,5-dicarboxylic acid 5-tert-butyl-3-ethyl ester (1.5 g, 5.079 mmol, 1 equivalent) and 1-(bromomethyl)-2-(trifluoromethyl)benzene (1.46 g, 6.095 mmol, 1.2 equivalents) in ACN (20 mL, 380.494 mmol) for 2 h. The resulting mixture was stirred at 80 °C under a nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The resulting mixture was extracted with EtOAc (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 resulting mixture was used directly in the next step without further purification (E00692-127).

[0747] 1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester

[0748] TFA (3 mL) was added in portions to a stirred solution of 5-[[2-(trifluoromethyl)phenyl]methyl]-1H,2H,3H,4H,5H-cyclopenta[c]pyridine-2,7-dicarboxylic acid 2-tert-butyl-7-ethyl ester (1 g, 2.215 mmol, 1 equivalent) in DCM (10 mL) at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The resulting mixture was used directly in the next step without further purification (E00692-129).

[0749] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid ethyl ester

[0750] DIEA was added in portions (5 mL) to a stirred solution of ethyl 1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylate (750 mg, 2.123 mmol, 1 equivalent) and 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (528.71 mg, 2.123 mmol, 1 equivalent) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred overnight at 100 °C under a nitrogen atmosphere to obtain a pure reaction. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was extracted with EtOAc (30 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (PE / EtOAc 1:1) to give ethyl 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylate (1 g, 83.24%), as a pale yellow oil.

[0751] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid

[0752] LiOH (0.21 g, 0.009 mmol, 5 equivalents) was added in portions to a stirred solution of ethyl 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylate (1 g, 1.767 mmol, 1 equivalent) in THF (5 mL) and H₂O (5 mL) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 50 °C under a nitrogen atmosphere for 3 h. The reaction was monitored by LCMS. The mixture was acidified to pH 6 with HCl (aqueous solution). The resulting mixture was extracted with EtOAc (30 mL). The combined organic layers were washed with brine (3 × 10 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with CH2Cl2 / MeOH (50:1 to 5:1) to give 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (700 mg, 73.65%), as a pale yellow oil.

[0753] 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide

[0754] CDI (316.51 mg, 1.952 mmol, 1.5 equivalents) was added in portions to a stirred solution of 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxylic acid (700 mg, 1.301 mmol, 1 equivalent) in 10 mL of DMF at room temperature under a nitrogen atmosphere. The resulting mixture was stirred at 50 °C under a nitrogen atmosphere for 1 h. NH4OAc (300.92 mg, 3.904 mmol, 3 equivalents) was added in portions to the mixture in portions over 5 min at 50 °C. The resulting mixture was stirred at 50 °C for another 2 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was extracted with EtOAc (30 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (20:1 to 5:1) to give 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (40 mg, 5.72%), as a pale yellow oil.

[0755] 5-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide

[0756] TFA (3 mL, 40.389 mmol, 542.16 equivalents) was added to a stirred solution of 5-[5-chloro-1-(oxan-2-yl)-6-oxo-1,6-dihydropyridazin-4-yl]-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (40 mg, 0.074 mmol, 1 equivalent) in DCM (10 mL). The resulting mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The crude product (30 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 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 20 mL / min; gradient: 24% B to 45% B over 7 min; 220 / 254 nm; Rt: 6.45 min) to obtain 5-(5-chloro-6-oxo-1,6-dihydropyridazin-4-yl)-1-[[2-(trifluoromethyl)phenyl]methyl]-1H,4H,5H,6H,7H-pyrazolo[4,3-c]pyridine-3-carboxamide (12.5 mg, 37.06%), as a white solid.

[0757] Example 31 Synthesis of compound NL

[0758]

[0759] Preparation of NK and NL

[0760] 1-Bromo-2-(difluoromethyl)-4-fluorobenzene

[0761] DAST (15.9 g, 98.64 mmol, 2.00 equivalents) was added to a stirred solution of 2-bromo-5-fluorobenzaldehyde (10 g, 49.26 mmol, 1 equivalent) in DCM (10 mL). The resulting mixture was stirred at -10 °C for 2 h. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluting with PE / EtOAc (5:1), to give 1-bromo-2-(difluoromethyl)-4-fluorobenzene (8.2 g, 73.98%) as a pale yellow oil.

[0762] 2-(difluoromethyl)-4-fluorobenzaldehyde

[0763] A solution of 1-bromo-2-(difluoromethyl)-4-fluorobenzene (8 g, 35.55 mmol, 1 equivalent) and n-BuLi (2.7 g, 42.15 mmol, 1.19 equivalent) in THF (150 mL) was stirred at -78 °C for 2 h. DMF (3.9 g, 53.33 mmol, 1.5 equivalent) was added to the mixture. The resulting mixture was stirred at -78 °C for 1 h. The reaction was quenched at -70 °C by adding water (50 mL). The solution was extracted with EtOAc (3 × 50 mL). The combined organic layers were washed with brine (2 × 30 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 / EtOAc (5:1) to give 2-(difluoromethyl)-4-fluorobenzaldehyde (3g, 48.46%), which was a pale yellow oil.

[0764] 1-[2-(difluoromethyl)-4-fluorophenyl]ethane-1-ol

[0765] CH3MgBr (25.84 mL, 25.84 mmol, 1.5 equivalent) was added dropwise to a stirred solution of 2-(difluoromethyl)-4-fluorobenzaldehyde (3 g, 17.23 mmol, 1 equivalent) in THF (30 mL, 416.06 mmol, 10 equivalent) under a nitrogen atmosphere at -30 °C. The resulting mixture was stirred at -10 °C under a nitrogen atmosphere for 2 h. The reaction was quenched with saturated NH4Cl (aqueous solution) at 0 °C. The mixture was extracted with EtOAc (3 × 300 mL). The combined organic layers were washed with brine (3 × 300 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc (100:1 to 50:1) to give 1-[2-(difluoromethyl)-4-fluorophenyl]ethyl-1-ol (2.68 g, 81.80%) as a red oil.

[0766] 1-(1-Chloroethyl)-2-(Difluoromethyl)-4-fluorobenzene

[0767] SO₂Cl₂ (6.7 g, 49.64 mmol, 3.52 equivalents) was added dropwise to a stirred solution / mixture of 1-[2-(difluoromethyl)-4-fluorophenyl]ethyl-1-ol (2.68 g, 14.09 mmol, 1 equivalent) in DCM (30 mL, 140.93 mmol, 10 equivalents) at 0 °C under air atmosphere. The resulting mixture was stirred at 20 °C for 2 h. The resulting oil was dried under vacuum to give 1-(1-chloroethyl)-2-(difluoromethyl)-4-fluorobenzene (2.36 g, 80.27%) as a red oil.

[0768] 1-[2-(difluoromethyl)-4-fluorophenyl]ethyl-1-amine

[0769] 1-(1-chloroethyl)-2-(difluoromethyl)-4-fluorobenzene (300 mg, 1.44 mmol, 1 equivalent) was added to a stirred solution of MeOH and NH3 (g) under a nitrogen atmosphere at room temperature. The resulting mixture was stirred at 70 °C under a nitrogen atmosphere for 20 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. This yielded 1-[2-(difluoromethyl)-4-fluorophenyl]ethyl-1-amine (130 mg, 47.78%) as a yellow oil. The resulting mixture was used directly in the next step without further purification.

[0770] 4-Chloro-5-[1-[(1S)-1-[2-(difluoromethyl)-4-fluorophenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[1-[(1R)-1-[2-(difluoromethyl)-4-fluorophenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one

[0771] At room temperature under a nitrogen atmosphere, 1-azido-4-nitrobenzene (78.9 mg, 0.48 mmol, 1.40 equivalent) and Zn(OAc)₂ (63.0 mg, 0.34 mmol, 1.00 equivalent) were added in portions to a stirred mixture of 1-[2-(difluoromethyl)-4-fluorophenyl]ethyl-1-amine (130.0 mg, 0.69 mmol, 2.00 equivalent) and 4-chloro-5-(4-oxopiridin-1-yl)-2,3-dihydropyridazin-3-one (78.2 mg, 0.34 mmol, 1 equivalent) in 10 mL of DMF. The resulting mixture was stirred at 60 °C under a nitrogen atmosphere for 16 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge Shield RP18 OBD column, 20-40µm, 19*150mm; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 80 mL / min; gradient: 30% B to 70% B over 30 min; 220 nm; Rt: 7.08 min) to obtain a mixture product. The residue (100 mg) was purified by chiral preparative HPLC under the following conditions: column: CHIRALPAK IF-3, 0.46*5 cm; 3µm; mobile phase: MtBE (0.1% DEA): EtOH = 80:20; detector: UV-254 nm. At 3.835 min, 4-chloro-5-[1-[(1S)-1-[2-(difluoromethyl)-4-fluorophenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (19.0 mg) was obtained as a grayish-white solid. At 3.185 min, 4-chloro-5-[1-[(1R)-1-[2-(difluoromethyl)-4-fluorophenyl]ethyl]-1H,4H,5H,6H,7H-[1,2,3]triazolo[4,5-c]pyridin-5-yl]-2,3-dihydropyridazin-3-one (33.8 mg) was obtained as a grayish-white solid.

[0772] Example 32 Synthesis of compound QM

[0773] Compound QM It is prepared by using the corresponding aniline according to the methods and schemes described in QL.

[0774]

[0775] Preparation of QL

[0776] 2-Vinyl-3-nitropyridine

[0777] Pd(PPh3)4 (0.73 g, 0.631 mmol, 1 equivalent) and 2-vinyl-4,4,5,5-tetramethyl-1,3,2-dioxane (1.00 equivalent) were added to a stirred mixture of 2-chloro-3-nitropyridine (2 g, 12.615 mmol, 1 equivalent) and Na2CO3 (2.67 g, 25.230 mmol, 2.0 equivalent) in 1,4-dioxane (20 mL) and H2O (1 mL) at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography and eluted with PE / EtOAc (10:1 to 5:1) to give 2-vinyl-3-nitropyridine (1.1 g, 58.08%) as a brown solid.

[0778] 2-Ethylpyridin-3-amine

[0779] Pd / C (100 mg, 0.266 mmol, 0.04 equivalent) was added to a stirred solution of 2-vinyl-3-nitropyridine (1.1 g, 7.327 mmol, 1 equivalent) in MeOH (10 mL) at room temperature under a hydrogen atmosphere. The resulting mixture was stirred at room temperature under a hydrogen atmosphere for 16 h. The reaction was monitored by LCMS. The resulting mixture was filtered, and the filter cake was washed with MeOH (2 × 10 mL). The filtrate was concentrated under reduced pressure. The residual product was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge PrepOBD C18 column 30×150mm 5um; mobile phase A: water (10 mmol / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20% B to 40% B over 11 min; 220 nm; Rt: 11.77 min) to obtain 2-ethylpyridine-3-amine (620 mg, 69.27%) as a white solid.

[0780] 2-[(2-ethylpyridin-3-yl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-carboxylic acid tert-butyl ester

[0781] Cs₂CO₃ (509.69 mg, 1.564 mmol, 2.0 equivalent) and Pd(AcO)₂ (35.12 mg, 0.156 mmol, 0.2 equivalent) were added to a stirred mixture of 2-chloro-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-carboxylic acid tert-butyl ester (200 mg, 0.782 mmol, 1 equivalent) and 2-ethyl-3-nitropyridine (238.02 mg, 1.564 mmol, 2.0 equivalent) in 1,4-dioxane (20 mL) under a nitrogen atmosphere. Then, XantPhos (181.03 mg, 0.313 mmol, 0.4 equivalent) was added under a nitrogen atmosphere at room temperature. The final reaction mixture was irradiated with microwave at 110 °C for 2 h. The reaction was monitored by LCMS. The mixture was then cooled to room temperature. The resulting mixture was filtered, and the filter cake was washed with CH2Cl2 (2 × 10 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by reversed-phase rapid chromatography 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: ACN; flow rate: 60 mL / min; gradient: 20% B to 40% B over 11 min; 220 nm; Rt: 11.77 min) to give 2-[(2-ethylpyridin-3-yl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl ester (250 mg, 93.62%) as a brown solid.

[0782] 2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-carboxylic acid tert-butyl ester

[0783] NaH (42.17 mg, 1.757 mmol, 2.0 equivalent) was added to a stirred solution of 2-[(2-ethylpyridin-3-yl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-carboxylic acid tert-butyl ester (300 mg, 0.879 mmol, 1 equivalent) in DMF (10 mL) under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred at 0 °C under a nitrogen atmosphere for 1 h. Then, CH3I (249.44 mg, 1.757 mmol, 2.00 equivalent) was added at 0 °C under a nitrogen atmosphere. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 1 h. The reaction was monitored by LCMS. The resulting mixture was diluted with water (2 mL). The crude product was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge Prep OBD C18 column 30×150mm 5um; mobile phase A: water (10 MMOL / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 20% B to 40% B over 11 min; 220 nm; Rt: 11.77 min) to obtain 2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidine-6-carboxylic acid tert-butyl ester (250 mg, 80.04%), as a brown solid.

[0784] N-(2-Ethylpyridin-3-yl)-N-methyl-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidin-2-amine

[0785] TFA (1 mL, 13.463 mmol, 22.98 equivalents) was added to a stirred solution of 2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-carboxylic acid tert-butyl ester (200 mg, 0.586 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The crude product was purified by reversed-phase rapid chromatography under the following conditions (column: XBridge Prep OBD C18 column 30×150mm 5um; mobile phase A: water (10 MMOL / L NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 18% B to 35% B over 8 min; 220 nm; Rt: 7.12 min) to obtain N-(2-ethylpyridin-3-yl)-N-methyl-6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine-2-amine (120 mg, 84.89%), as a brown solid.

[0786] 4-Chloro-5-[2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0787] At room temperature, 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyrrolo[3,4-d]pyrimidin-2-amine (120 mg, 0.497 mmol, 1 equivalent) was added to a stirred solution of N-(2-ethylpyridin-3-yl)-2,3-dihydropyridazin-3-one (99.10 mg, 0.398 mmol, 0.80 equivalent) in DIEA (0.1 mL). The resulting mixture was stirred at 90 °C for 1 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by preparative TLC (CH2Cl2 / MeOH 12:1) to give 4-chloro-5-[2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 42.97%), as a brown solid.

[0788] 4-Chloro-5-[2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-yl]-2,3-dihydropyridazin-3-one

[0789] TFA (1 mL) was added to a stirred solution of 4-chloro-5-[2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (100 mg, 0.214 mmol, 1 equivalent) in DCM (4 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The mixture was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The crude product (80 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: ACN; flow rate: 60 mL / min; gradient: 15% B to 35% B over 8 min; 220 nm; Rt: 6.65 min) to obtain 4-chloro-5-[2-[(2-ethylpyridin-3-yl)(methyl)amino]-5H,6H,7H-pyrrolo[3,4-d]pyrimidin-6-yl]-2,3-dihydropyridazin-3-one (67.4 mg, 82.17%) as a white solid.

[0790] Example 33 Synthesis of compounds MD and ME

[0791]

[0792] Step 1.

[0793] N-[(2R)-1-(2-chloroacetamido)propyl-2-yl]tert-butyl carbamate

[0794] A solution of Na₂CO₃ (3649.65 mg, 34.434 mmol, 2 equivalents) in H₂O (10 mL) was added to a stirred solution of N-[(2R)-1-aminopropyl-2-yl]carbamate tert-butyl (3 g, 17.217 mmol, 1 equivalent) in EA (50 mL). Then, a solution of 2-chloroacetyl chloride (3.89 g, 34.434 mmol, 2 equivalents) in EA (10 mL) was added dropwise at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The reaction was quenched with water at room temperature. The resulting mixture was extracted with EtOAc (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. This produces N-[(2R)-1-(2-chloroacetamido)prop-2-yl] tert-butyl carbamate (4.5 g, crude product), a white solid.

[0795] Step 2.

[0796] (5R)-5-methylpiperazin-2-one

[0797] A solution of TFA (10 mL, 134.630 mmol, 7.50 equivalents) in 10 mL of DCM was added dropwise to a stirred solution of N-[(2R)-1-(2-chloroacetamido)propyl-2-yl]carbamate (4.5 g, 17.948 mmol, 1 equivalent) in 30 mL of DCM at 0 °C. The resulting mixture was stirred at room temperature for 2 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. K₂CO₃ (4.96 g, 35.897 mmol, 2 equivalents) and KI (2.98 g, 17.948 mmol, 1 equivalent) were added to the mixture at room temperature. The resulting mixture was stirred again at 80 °C for 16 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with CH2Cl2 / MeOH (20:1 to 10:1) to give (5R)-5-methylpiperazin-2-one (2.5 g, crude product), which was a yellow oil.

[0798] Step 3.

[0799] 4-Chloro-5-[(2R)-2-methyl-5-oxoperpiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0800] Add 4,5-dichloro-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (5.46 g, 21.901 mmol, 1 equivalent) to a stirred solution of (5R)-5-methylpiperazin-2-one (2.5 g, 21.901 mmol, 1 equivalent) in DIEA (2 mL) at room temperature. Stir the resulting mixture at 100 °C for 16 h. Monitor the reaction by LCMS. Allow the mixture to cool to room temperature. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: C18, 330 g; mobile phase A: water / 0.05% NH4HCO3, mobile phase B: ACN; flow rate: 80 mL / min; gradient: 20% B to 30% B over 10 min; detector: 220 nm; monitor: 254 nm) to obtain 4-chloro-5-[(2R)-2-methyl-5-oxopiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (600 mg, 8.38%) as a yellow solid.

[0801] Step 4.

[0802] 4-Chloro-5-[(2R)-4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxoperpiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0803] t-BuONa (220.57 mg, 2.295 mmol, 1.5 equivalent) was added to a stirred mixture of 4-chloro-5-[(2R)-2-methyl-5-oxoperpiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (500 mg, 1.530 mmol, 1 equivalent) and 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl methanesulfonate (656.96 mg, 2.295 mmol, 1.5 equivalent) in ACN (20 mL) under a nitrogen atmosphere at room temperature. The final reaction mixture was irradiated with microwave at 110 °C for 3 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was concentrated under reduced pressure. The residue was purified by reversed-phase rapid chromatography under the following conditions (column: C18, 330 g; mobile phase A: water / 0.05% NH4HCO3, mobile phase B: ACN; flow rate: 80 mL / min; gradient: 55% B to 75% B over 15 min; detector: 220 nm; monitor: 254 nm) to obtain 4-chloro-5-[(2R)-4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxoperpiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 15.17%) as a yellow solid.

[0804] Step 5.

[0805] MD and ME

[0806] 4-Chloro-5-[(2R)-4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[(2R)-4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one

[0807] TFA (2 mL, 26.926 mmol, 115.99 equivalents) was added to a stirred solution of 4-chloro-5-[(2R)-4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxopiperazin-1-yl]-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (120 mg, 0.232 mmol, 1 equivalent) in DCM (8 mL). The resulting mixture was stirred at room temperature for 1 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under reduced pressure. The residue was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was concentrated under reduced pressure. The residue was subjected to reversed-phase rapid chromatography under the following conditions (column: XBridgePrep C18 OBD column 19 × 150 mm 5 μm; mobile phase A: water (10 mmol / L)). Purification was performed using NH4HCO3), mobile phase B: ACN; flow rate: 60 mL / min; gradient: 22% B to 51% B over 7 min; 254 / 220 nm; Rt: 6.4 min, yielding 4-chloro-5-[(2R)-4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one (16.3 mg, 16.22%) as a white solid, and 4-chloro-5-[(2R)-4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-2-methyl-5-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one (18.6 mg, 18.51%) as a white solid.

[0808] Example 34 Synthesis of compound MF

[0809]

[0810] Step 1.

[0811] 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethane-1-ol

[0812] A solution of MeMgBr in Et₂O (3 mol / L, 30 mL) was added dropwise to a stirred solution of 4-fluoro-2-(trifluoromethyl)benzaldehyde (3 g, 15.616 mmol, 1 equivalent) in THF (50 mL) under a nitrogen atmosphere at -30 °C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction was monitored by TLC. The reaction was quenched with saturated NH₄Cl (aqueous solution) at 0 °C. The resulting mixture was extracted with EtOAc (50 mL). The combined organic layers were washed with brine (3 × 50 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was used directly in the next step without further purification.

[0813] Step 2.

[0814] 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl methanesulfonate

[0815] MsCl (2.48 g, 21.618 mmol, 1.5 equivalent) was added dropwise to a stirred solution of 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl-1-ol (3 g, 14.412 mmol, 1 equivalent) and Et3N (2.92 g, 28.825 mmol, 2 equivalent) in DCM (60 mL) under a nitrogen atmosphere at 0 °C. The resulting mixture was stirred at room temperature under a nitrogen atmosphere for 2 h. The reaction was monitored by TLC. The reaction was quenched at 0 °C by adding saturated NH4Cl (water solution) (50 mL). The resulting mixture was extracted with EtOAc (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 to give 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl methanesulfonate (1.6 g, 38.78%) as a pale yellow oil.

[0816] Step 3.

[0817] 4-Chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-3-oxopiperazin-1-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one

[0818] Sodium 2,2-dimethylpropane-1-ol (563.43 mg, 5.116 mmol, 2 equivalents) was added in portions to a stirred solution of 4-chloro-2-(oxan-2-yl)-5-(3-oxopiperazin-1-yl)-2,3-dihydropyridazin-3-one (800 mg, 2.558 mmol, 1 equivalent) and 1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl methanesulfonate (878.63 mg, 3.070 mmol, 1.2 equivalents) in ACN (8 mL) under a nitrogen atmosphere at room temperature. The final reaction mixture was irradiated with microwave at 110 °C for 3 h. The reaction was monitored by LCMS. The mixture was cooled to room temperature. The resulting mixture was extracted with EtOAc (20 mL). The combined organic layers were washed with brine (3 × 10 mL) and dried over anhydrous Na₂SO₄. After filtration, the filtrate was concentrated under reduced pressure. The resulting mixture was used directly in the next step without further purification.

[0819] Step 4.

[0820] Compound MF

[0821] 4-Chloro-5-[4-[(1R)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-3-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one and 4-chloro-5-[4-[(1S)-1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-3-oxopiperazin-1-yl]-2,3-dihydropyridazin-3-one

[0822] TFA (3 mL) was added in portions to a stirred solution of 4-chloro-5-(4-[1-[4-fluoro-2-(trifluoromethyl)phenyl]ethyl]-3-oxopiperazin-1-yl)-2-(oxan-2-yl)-2,3-dihydropyridazin-3-one (110 mg, 0.219 mmol, 1 equivalent) in DCM (10 mL) at room temperature under nitrogen atmosphere. The resulting mixture was stirred at room temperature under nitrogen atmosphere for 3 h. The reaction was monitored by LCMS. The resulting mixture was concentrated under vacuum. The residue was alkalized to pH 8 with saturated NaHCO3 (aqueous solution). The resulting mixture was extracted with EtOAc (20 mL). The combined organic layers were washed with brine (3 × 20 mL) and dried over anhydrous Na2SO4. After filtration, the filtrate was concentrated under reduced pressure. The crude product (50 mg) was subjected to CHIRAL-HPLC under the following conditions (column: XBridge Prep OBD C18 column, 30 × 150 mm, 5 μm; mobile phase A: water (10 mmol / L)). Purification was performed...

Claims

1. Use of a TRPC5 inhibitory compound of structural formula (I) in the preparation of a medicament for use in combination with a second therapeutic agent to treat a disease or condition selected from kidney disease, anxiety, depression, pain or cancer, wherein: a. Formula (I) is represented by the following formula: or a pharmaceutically acceptable salt thereof; in: "---" 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 key, X 2 is N(CH3), When X 1 When it is CH, X 2 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-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; When "---" is a double bond, R 13 is hydrogen, -CH2OH, -CH(OH)-CH2OH, -NH2, -CH(OH)CH3, -OCH3 or -NH-(CH2)2OH; and R 14 does not exist; or When "---" is a single bond, R 13 and R 14 together form =O; and R 15 and R 16 Each of is independently hydrogen or -CH3; and b. The second therapeutic agent is selected from: an immunomodulator, a calcineurin inhibitor, a renin angiotensin aldosterone system inhibitor, an antiproliferative agent, a corticosteroid, an angiotensin converting enzyme inhibitor, an angiotensin receptor blocker, a sodium-glucose transporter 2 inhibitor, a nuclear factor-1 (erythroid-derived 2)-like 2 agonist, a chemokine receptor 2 inhibitor, a chemokine receptor 5 inhibitor, an endothelin 1 receptor antagonist and a SGLT2 inhibitor.

2. The method of claim 1, wherein the TRPC5 inhibitory compound has the structural formula (II): or a pharmaceutically acceptable salt thereof; wherein: R 11 is chlorine, -CF3, -CHF2 or -CH3; R 12 is hydrogen or fluorinated; and R 13 It is hydrogen, -NH2, -CH2OH or CH(OH)-CH2OH.

3. The use as claimed in claim 2, wherein R 11 is -CHF2; and R 12 It is fluorinated.

4. The use according to claim 1, wherein the TRPC5 inhibitory compound is selected from any one of the following compounds or a pharmaceutically acceptable salt thereof:

5. The use according to claim 1, wherein the TRPC5 inhibitory compound is selected from any one of the following compounds or a pharmaceutically acceptable salt thereof:

6. The use according to claim 1, wherein the TRPC5 inhibitory compound is the following compound or a pharmaceutically acceptable salt thereof:

7. The use according to claim 1, wherein the TRPC5 inhibitory compound is the following compound:

8. The use of any one of claims 1-7, wherein the second therapeutic agent is an immunomodulator, and the immunomodulator is rituximab.

9. The use of any one of claims 1 to 7, wherein the second therapeutic agent is an angiotensin converting enzyme inhibitor, and the angiotensin converting enzyme inhibitor is captopril, zofenopril, enalapril, ramipril, quinapril, perindopril, lisinopril, benazepril, imidapril, trandolapril or cilazapril.

10. The use of any one of claims 1-7, wherein the second therapeutic agent is an angiotensin receptor blocker, and the angiotensin receptor blocker is losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan or fimasartan.

11. The use according to any one of claims 1 to 7, wherein the second therapeutic agent is a renin angiotensin aldosterone system inhibitor, and the renin angiotensin aldosterone system inhibitor is aliskiren.

12. The use of any one of claims 1-7, wherein the second therapeutic agent is an endothelin 1 receptor antagonist, and the endothelin 1 receptor antagonist is ambrisentan, atrasentan, bosentan or sparsentan.

13. The use according to any one of claims 1 to 7, wherein the second therapeutic agent is an antiproliferative agent, and the antiproliferative agent is mycophenolate mofetil.

14. The use according to any one of claims 1 to 7, wherein the second therapeutic agent is a SGLT2 inhibitor, and the SGLT2 inhibitor is canagliflozin, dapagliflozin, empagliflozin, a combination of empagliflozin and linagliptin, a combination of empagliflozin and metformin, or a combination of dapagliflozin and metformin.

15. The use of any one of claims 1-7, wherein the second therapeutic agent is a calcineurin inhibitor, and the calcineurin inhibitor is cyclosporine A or tacrolimus.

16. The use of any one of claims 1-7, wherein the second therapeutic agent is a nuclear factor-1 (erythroid-derived 2)-like 2 agonist, and the nuclear factor-1 (erythroid-derived 2)-like 2 agonist is bardoxolone or CXA-10.

17. The use according to any one of claims 1 to 7, wherein the second therapeutic agent is a chemokine receptor 2 inhibitor, and the chemokine receptor 2 inhibitor is PF-04136309 or ccx140.

18. The use of any one of claims 1-7, wherein the second therapeutic agent is tacrolimus, cyclosporin A, rituximab, mycophenolate mofetil, corticosteroids, sparsentan, enalapril or losartan.

19. The use of claim 18, wherein the second therapeutic agent is enalapril, losartan or cyclosporin A.

20. The use of any one of claims 1-7, wherein the disease or disorder is focal segmental glomerulosclerosis (FSGS), primary focal segmental glomerulosclerosis, hereditary focal segmental glomerulosclerosis, secondary focal segmental glomerulosclerosis, diabetic nephropathy, Alport syndrome, hypertensive renal disease, nephrotic syndrome, steroid-resistant nephrotic syndrome, minimal change disease, 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, clq nephropathy, rapidly progressive glomerulonephritis (GN), anti-GBM disease, C3 glomerulonephritis, hypertensive nephrosclerosis, IgA nephropathy, autosomal recessive polycystic kidney disease, or autosomal dominant polycystic kidney disease.

21. The use according to any one of claims 1 to 7, wherein the disease or condition is pain.

22. The use of any one of claims 1-7, wherein the disease or disorder is anxiety.

23. The use of any one of claims 1-7, wherein the disease or disorder is depression.

24. The use of any one of claims 1-7, wherein the subject is a human.

25. The use of any one of claims 1-7, wherein the disease or disorder is cancer.

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