Non-chelating and non-reducing ferroptosis inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a compound shown in a formula (I) and a preparation method and application thereof, and the compound or pharmaceutically acceptable salt or stereoisomer of the compound is non-chelating and non-reducing and can be used as a ferroptosis inhibitor and used for preventing / treating ferroptosis related diseases. # imgabs0 #

Description

A non-chelating non-reductive ferroptosis inhibitor and its preparation method and use

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on June 23, 2022, with application number 202210716762.6 and invention name “A novel non-chelating non-reductive ferroptosis inhibitor, its preparation method and use”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention belongs to the technical field of chemical synthesis drugs, and specifically relates to a non-chelating non-reductive ferroptosis inhibitor, a preparation method thereof, and uses thereof. Background Art

[0003] Cell death is an irreversible phenomenon in life. Through in-depth research on cell death, Dixon et al. discovered a new type of cell death in 2012 and named it "ferroptosis." This new type of cell death is iron-dependent and is often accompanied by the accumulation of intracellular iron and lipid peroxides. Ferroptosis differs significantly from apoptosis, necrosis, and autophagy in terms of cellular morphology and function. Morphologically, ferroptosis is characterized by reduced mitochondrial volume, decreased mitochondrial membrane density, reduced or absent mitochondrial cristae, and rupture of the mitochondrial outer membrane.

[0004] In recent years, a series of studies have found that ferroptosis plays an important role in the occurrence and development of many diseases. It has become a research hotspot for the treatment and improvement of prognosis of related diseases such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, Alzheimer's disease, Parkinson's disease, acute kidney injury, doxorubicin (DOX)-induced cardiotoxicity, and acetaminophen-induced acute liver injury. Therefore, the development of novel ferroptosis inhibitors is a potential approach to treat these diseases.

[0005] Currently, most of the small molecule inhibitors of ferroptosis are antioxidants or iron chelators. They include Ferrostatin-1, Liproxstatin-1, Zileuton, CPO, DFO, Deferasirox and Deferiprone. The redox balance in the body's internal environment is crucial to health. Breaking the redox balance by non-selectively reducing the oxidative state may lead to diseases such as cancer and aging. For example, persistent reductive stress or lack of reactive oxygen species can hinder insulin signal transduction and glucose homeostasis, blunt the positive effects of exercise on insulin sensitivity, and induce cardiomyopathy, obesity or diabetes (Chinese Journal of Modern Applied Pharmacy, 2022, 39(01): 127-133.). Large clinical trials have confirmed that high-dose vitamin E / C (antioxidants) does not improve the clinical prognosis of patients, and higher doses of vitamin E may even lead to atherosclerosis and worsening vascular function, which may be related to the reductive stress caused by excessive antioxidants (Chinese Journal of Multi-Organ Diseases in the Elderly, 2019, 18(09): 713-716).

[0006] In addition, iron is an essential trace element in organisms and the active center of many biological enzymes. It is essential for normal cells, tumor cells, and microbial cells. Many molecules with chelating or metal-binding properties can affect all processes involving metal ions. Therefore, the use of iron chelators will inevitably lead to anemia (e.g., iron deficiency anemia) and side effects of many metabolic diseases. Therefore, the development of ferroptosis inhibitors that are neither antioxidants nor iron chelators is very necessary and of great significance.

[0007] Summary of the Invention

[0008] One of the objects of the present invention is to provide a non-chelating, non-reducing compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof;

[0009] One of the objects of the present invention is to provide a pharmaceutical preparation comprising the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof and a pharmaceutically common carrier;

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical preparation thereof;

[0011] Another object of the present invention is to provide the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical preparation thereof as a ferroptosis inhibitor;

[0012] Another object of the present invention is to provide the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical preparation thereof in the treatment and / or prevention of ferroptosis-related diseases, such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver damage, and other ferroptosis-related diseases such as diabetic complications.

[0013] To achieve the above objectives, the present invention provides the following technical solutions:

[0014] In some embodiments of the present invention, there is provided a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0015] in:

[0016] n is selected from 1 or 2;

[0017] R1 is independently selected from hydrogen, halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted benzene ring; or, two adjacent R1 are connected to form a benzene ring;

[0018] Ring A is selected from a substituted or unsubstituted five-membered or six-membered aryl group, a substituted or unsubstituted five-membered or six-membered heteroaryl group, a substituted or unsubstituted five-membered or six-membered heterocyclic group, or a substituted or unsubstituted N-containing di-fused cyclic group or tri-fused cyclic group; the substituents of Ring A are selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted five-membered or six-membered N-containing heterocyclic group;

[0019] Ring B is selected from a substituted or unsubstituted five-membered or six-membered aryl group, a substituted or unsubstituted five-membered or six-membered heteroaryl group, or a substituted or unsubstituted five-membered or six-membered heterocyclic group; the substituent of Ring B is selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, or substituted or unsubstituted C1-C6 alkyl;

[0020] The heteroatoms of the heterocyclic group and heteroaryl group are selected from one or more of N, O and S;

[0021] The substituents of the C1-C6 alkoxy group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy;

[0022] The substituents of the C1-C6 alkyl group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy;

[0023] The substituents of the benzene ring are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy;

[0024] The substituents of the five-membered or six-membered N-containing heterocyclic group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy.

[0025] Optionally, the heterocyclic group or heteroaryl group contains 1, 2 or 3 heteroatoms.

[0026] The heterocyclic group in the present invention is an aliphatic heterocyclic group.

[0027] In the present invention, the N-containing difused ring group is a group formed by condensing two five-membered or six-membered heteroaryl groups, or a group formed by condensing one five-membered or six-membered heteroaryl group and one five-membered or six-membered aryl group.

[0028] In the present invention, the N-containing tri-fused ring group is a group formed by condensing three five-membered or six-membered heteroaryl groups, or a group formed by condensing a five-membered or six-membered heteroaryl group and a five-membered or six-membered aryl group, and the total number of the heteroaryl groups and aryl groups is 3.

[0029] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably,

[0030] Ring A is phenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl or triazinyl; or is an N-containing fused ring formed by condensing any two or three of the above groups; and Ring A is substituted or unsubstituted.

[0031] Ring B is phenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrahydropyridinyl, tetrahydropyranyl, piperidinyl or piperazinyl; said ring B is substituted or unsubstituted.

[0032] The substituents of the above-mentioned ring A and ring B are the same as above and will not be repeated here.

[0033] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably,

[0034] Ring A is phenyl, pyrazolyl, pyridyl, indolyl, benzimidazolyl, benzothiazolyl or pyrrolopyridinyl; and the ring A is substituted or unsubstituted.

[0035] Ring B is phenyl, pyridyl, pyrimidinyl, pyrazolyl, tetrahydropyridyl or tetrahydropyranyl; and said ring B is substituted or unsubstituted.

[0036] The substituents of the above-mentioned ring A and ring B are the same as above and will not be repeated here.

[0037] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably,

[0038] The substituent of the ring A is selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, hexahydropyridyl and N-methylhexahydropyridyl;

[0039] The substituents of ring B are selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, n-propoxy, and n-butoxy.

[0040] In some embodiments of the present invention, there is provided a compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof:

[0041] in:

[0042] n is selected from 1 or 2;

[0043] R1 is independently selected from hydrogen, halogen, hydroxyl, nitro, C1-C6 alkoxy, C1-C6 alkyl, and benzene ring; or, two adjacent R1 are connected to form a benzene ring;

[0044] Ring A is selected from:

[0045] Ring B is selected from:

[0046] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably, the R1 is independently selected from hydrogen atom, halogen, hydroxyl, and C1-C6 alkyl.

[0047] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably,

[0048] The ring A is selected from:

[0049] The ring B is selected from:

[0050] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein preferably,

[0051] The ring B is selected from:

[0052] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein the compound can be selected from the following compounds:

[0053] In some embodiments of the present invention, a pharmaceutical preparation is provided, which is prepared using any of the above-mentioned compounds, or salts thereof, or stereoisomers thereof as active ingredients, and a pharmaceutically common carrier.

[0054] In some embodiments of the present invention, the provided pharmaceutical preparations are oral preparations and intravenous injection preparations.

[0055] In some embodiments of the present invention, there is provided the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the above-mentioned pharmaceutical preparation in the preparation of a ferroptosis inhibitor; the ferroptosis inhibitor is a targeted drug for inhibiting cell ferroptosis.

[0056] In one embodiment of the present invention, the use of the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the above-mentioned pharmaceutical preparation for treating and / or preventing diseases such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver damage, and diabetic complications is provided. The neurodegenerative disease is Parkinson's syndrome or Alzheimer's disease; and the stroke includes ischemic stroke and hemorrhagic stroke.

[0057] Definitions and General Terms:

[0058] The following terms and the like are used to describe the present invention. It should be understood that terms not specifically defined are given a meaning consistent with the use of the terms in the context of the present invention as understood by those of ordinary skill in the art.

[0059] The term "halogen" as used herein refers to fluorine, chlorine, bromine or iodine, preferably chlorine.

[0060] The term "C1-C6 alkyl" used in the present invention refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms, preferably a methyl group or an ethyl group.

[0061] The term "pharmaceutically acceptable salt" used in the present invention refers to a salt of the compound of the present invention, which is prepared by reacting the compound having specific substituents discovered in the present invention with a pharmaceutically acceptable acid or base.

[0062] The term "stereoisomers" as used herein refers to compounds that have identical chemical constitution but differ with regard to the arrangement of the atoms and groups in space. These include enantiomers, diastereomers, geometric isomers, atropisomers or conformational isomers.

[0063] The term "commonly used pharmaceutical carrier" as used herein refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc.

[0064] The term "non-chelating" as used in the present invention means that the lipid peroxidation caused by the Fenton reaction cannot be prevented by chelating iron.

[0065] The term "non-reducing" used in the present invention means that the protein has no antioxidant activity and cannot prevent damage to membrane lipids through a reducing mechanism, thereby inhibiting cell death.

[0066] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as deuterium (D), tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C) All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0067] Additionally, prodrugs and esters of the compounds of any of the above formulae are also encompassed within the scope of the present invention.

[0068] Through creative labor screening, the inventors have obtained a group of new non-chelating, non-reducing ferroptosis inhibitors, which address the potential risks of current chelators and antioxidants and provide a safer alternative for the clinical treatment of ferroptosis-related diseases. At the same time, the inventors also tested the inhibitory activity of representative compound 1 against 401 kinases, including PI3K. The results showed that compound 1 had no significant inhibitory effect on these targets, indicating that the compounds of the present invention are novel non-chelating, non-reducing ferroptosis inhibitors with good selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1. Reducing (A) and chelating (B) experiments of compounds 1 and 38 of the present invention.

[0070] Figure 2. Compound 1 of the present invention alleviates acetaminophen-induced acute liver injury. AH&E staining results. B and C. ALT and AST analysis in mouse serum. DETAILED DESCRIPTION

[0071] Example 1. Synthesis of 8-(3,6-dihydro-2H-pyran-4-yl)-2-phenyl-6-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)imidazo[1,2-a]pyrazine (Compound 1)

[0072] 1. Synthesis of 6,8-dibromo-2-phenylimidazo[1,2-a]pyrazine (Intermediate I)

[0073] The synthetic route of intermediate I is as follows:

[0074] 2-Amino-3,5-dibromopyrazine (1 g, 3.96 mmol, 1.00 eq) and 2-bromoacetylacetone (789.68 mg, 3.96 mmol, 1.00 eq) were placed in a 100 mL flask and dissolved in 1,4-dioxane (10 mL). The reaction was stirred at 120°C for 12 hours until a large amount of yellow solid gradually precipitated. The mixture was filtered, washed with 1,4-dioxane (5 mL x 2), and dried under vacuum to obtain Intermediate I as a yellow solid (1256.72 mg, 3.59 mmol, 90%).

[0075] 2. Synthesis of Intermediate II

[0076] The synthetic route of intermediate II is as follows:

[0077] Intermediate I (500 mg, 1.42 mmol, 1.00 eq), 3,6-dihydro-2H-pyran-4-boronic acid pyranose ester (300 mg, 1.42 mmol, 1.00 eq), Pd(dppf)Cl2 (104 mg, 0.14 mmol, 0.1 eq), and K3PO4 (591 mg, 4.28 mmol, 3.00 eq) were added to a two-necked flask and dissolved with 1,4-dioxane (10 mL). The argon atmosphere was replaced three times, and the reaction temperature was raised to 80°C. The reaction was monitored by TLC and completed after approximately 4 hours. The reaction was cooled to room temperature and extracted with water and ethyl acetate (3 x 20 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated. The organic layer was separated by column chromatography to yield Intermediate II (228 mg, 1.42 mmol, 45%).

[0078] 3. Synthesis of Intermediate III

[0079] The synthetic route of intermediate III is as follows:

[0080] Intermediate II (228 mg, 0.64 mmol, 1.00 eq), tert-butyl 4-[4-(4,4,5,5-tetramethyl-1,3,2-dioxybenzofuran-2-yl)-1H-pyrazol-1-yl]piperidine-1-carboxylate (242 mg, 0.64 mmol, 1.00 eq), Pd(dppf)Cl2 (46.78 mg, 0.064 mmol, 0.1 eq), and Cs2CO3 (264.96 mg, 1.92 mmol, 3.00 eq) were placed in a two-necked flask. The mixture was dissolved with 1,4-dioxane (10 mL). The argon atmosphere was replaced three times, and the reaction temperature was raised to 80°C. The reaction was monitored by TLC and completed after approximately 4 hours. The reaction was cooled to room temperature and extracted with water and ethyl acetate (3 x 20 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated. The organic layer was separated by column chromatography to obtain Intermediate III (209 mg, 1.42 mmol, 62%).

[0081] 4. Synthesis of Compound 1

[0082] The synthetic route of compound 1 is as follows:

[0083] 209 mg of intermediate III was added to 5 mL of dichloromethane and 3 mL of trifluoroacetic acid, reacted at room temperature for half an hour, then dried by rotation, dissolved in DCM, and purified by TLC using DCM:MeOH = 5:1 as the developing solvent to isolate the target product, Compound 1 (154 mg, 0.36 mmol, 91%).

[0084] Compound 1 1 H NMR and HRMS data are as follows:

[0085] 1 H NMR(400MHz,DMSO)δ8.76(s,1H),8.50(s,1H),8.39(s,1H),8.28(s,1H),8.07( d,J=7.2Hz,2H),8.00(s,1H),7.49(t,J=7.6Hz,2H),7.38(t,J=7.3Hz,1H),4.4 7(d,J=2.4Hz,2H),4.32(m,1H),3.92(t,J=5.4Hz,2H),3.13(d,J=12.5Hz,2H), 2.82(s,2H),2.69(t,J=11.3Hz,2H),2.01(m,3H),1.88(qd,J=12.9,3.9Hz,2H).

[0086] HRMS m / z(ESI)calcd for C 25 H 26 N6O[M+H] + 427.2241found:427.2243.

[0087] Example 2, Synthesis of Compound 2

[0088] Compound 2 was prepared using a similar synthesis method to Example 1 with a yield of 56%.

[0089] Compound 2 1 H NMR and HRMS data are as follows:

[0090] 1 H NMR (400MHz, DMSO) δ8.67(s,1H),8.50(s,1H),8.45(s,1H),8.05(d,J=7.3Hz,2H),7.83(s,1H),7.49(t,J=7.6Hz ,2H),7.38(t,J=7.3Hz,1H),4.47(d,J=2.3Hz,2H),3.92(t,J=5.4Hz,2H),3.80(s,3H),2.77(s,2H),2.61(s,3H).

[0091] HRMS m / z(ESI)calcd for C 22 H 21 N5O[M+H] + 372.1819found:372.1817.

[0092] Example 3, Synthesis of Compound 3

[0093] Compound 3 was prepared using a similar synthesis method to Example 1 with a yield of 53%.

[0094] Compound 3 1 H NMR and HRMS data are as follows:

[0095] 1H NMR (400MHz, DMSO) δ11.84(s,1H),9.23(s,1H),8.67(s,1H),8.51(s,1H),8.35(d,J=5.0Hz,1H),8.08(d,J=7.2Hz,2H),7.72(d,J=5.0Hz,1H),7.6 3(d,J=3.4Hz,1H),7.52(t,J=7.6Hz,2H),7.41(t,J=7.3Hz,1H),7.12(d, J=3.3Hz, 1H), 4.50 (d, J=2.3Hz, 2H), 3.96 (t, J=5.4Hz, 2H), 2.89 (s, 2H).

[0096] HRMS m / z(ESI)calcd for C 24 H 19 N5O[M+H] + 394.1662found:394.1663.

[0097] Example 4. Synthesis of Compound 4

[0098] Compound 4 was prepared using a similar synthesis method to Example 1 with a yield of 51%.

[0099] Compound 4 1 H NMR and HRMS data are as follows:

[0100] 1 H NMR (400MHz, DMSO) δ9.45(s,1H),9.22(s,1H),8.92(d,J=1.4Hz,1H),8.59(s,1H),8.47(s,1H),8.28(dd,J=8.6,1.6Hz,1H),8.22(d,J=8 .6Hz,1H),8.10(d,J=7.2Hz,2H),7.51(t,J=7.6Hz,2H),7.40(t,J=7.3Hz,1H),4.50(d,J=2.5Hz,2H),3.96(t,J=5.4Hz,2H),2.92(s,2H).

[0101] HRMS m / z(ESI)calcd for C 24 H 18 N4OS[M+H] + 411.1274found:411.1275.

[0102] Example 5. Synthesis of Compound 5

[0103] Compound 5 was prepared by a similar synthesis method as in Example 1 with a yield of 57%.

[0104] Compound 5 1 H NMR and HRMS data are as follows:

[0105] 1 H NMR (400MHz, DMSO) δ8.75(s,1H),8.50(s,1H),8.39(s,1H),8.22(s,1H),8.07(d,J=7.2Hz,2H),7.97(s,1H),7.5 0(t,J=7.6Hz,2H),7.39(t,J=7.3Hz,1H),4.47(d,J=2.5Hz,2H),3.93(d,J=5.5Hz,2H),3.91(s,3H),2.82(s,2H).

[0106] HRMS m / z(ESI)calcd for C 21 H 19 N5O[M+H] + 358.1662found:358.1664.

[0107] Example 6. Synthesis of Compound 6

[0108] Compound 6 was prepared by a similar synthesis method as in Example 1 with a yield of 42%.

[0109] Compound 6 1 H NMR and HRMS data are as follows:

[0110] 1 H NMR (400MHz, DMSO) δ13.05(s,1H),8.78(s,1H),8.49(s,1H),8.39(s,1H),8.26(s,1H),8.07(d,J=7.5Hz,2 H),7.49(t,J=7.7Hz,2H),7.38(t,J=7.4Hz,1H),4.47(d,J=2.1Hz,2H),3.92(t,J=5.3Hz,2H),2.83(s,2H).

[0111] HRMS m / z(ESI)calcd for C 20 H 17 N5O[M+H] + 344.1506found:344.1507.

[0112] Example 7, Synthesis of Compound 7

[0113] Compound 7 was prepared by a similar synthesis method as in Example 1 with a yield of 61%.

[0114] Compound 7 1 H NMR and HRMS data are as follows:

[0115] 1 H NMR (400MHz, DMSO) δ8.93(s,1H),8.62(s,1H),8.38(s,1H),8.30(d,J=1.9Hz,1H),8.06(dd,J=8.4,1.9Hz,1H),7.95(d,J=8.8Hz,2H),7.75 (d,J=8.4Hz,1H),7.06(d,J=8.9Hz,2H),4.48(s,2H),3.93(t,J=5.3Hz,2H),3.23(m,4H),2.83(s,2H),2.47(d,J=4.3Hz,4H),2.24(s,3H).

[0116] HRMS m / z(ESI)calcd for C 28 H 27 Cl2N5O[M+H] + 520.1665found:520.1667.

[0117] Example 8, Synthesis of Compound 8

[0118] Compound 8 was prepared using a similar synthesis method to that of Example 1 with a yield of 55%.

[0119] Compound 8 1 H NMR and HRMS data are as follows:

[0120] 1 H NMR (400MHz, CDCl3) δ8.47 (s, 1H), 8.26 (s, 1H), 8.08 (d, J = 8.3Hz, 2H), 7.96 (s,1H),7.91(d,J=8.8Hz,2H),7.68(dd,J=16.9,7.8Hz,4H),7.47(t,J=7.6 Hz,2H),7.37(t,J=7.3Hz,1H),7.03(d,J=8.8Hz,2H),4.59(d,J=2.5Hz,2H) ,4.05(t,J=5.4Hz,2H),3.31(m,4H),3.00(s,2H),2.61(m,4H),2.38(s,3H).

[0121] HRMS m / z(ESI)calcd for C 24 H 33 N5O[M+H] + 428.2758found:428.2759.

[0122] Example 9, Synthesis of Compound 9

[0123] Compound 9 was prepared using a similar synthesis method to Example 1 with a yield of 59%.

[0124] Compound 9 1 H NMR and HRMS data are as follows:

[0125] 1 H NMR (400MHz, DMSO) δ8.77(s,1H),8.63(s,1H),8.34(s,1H),8.31(m,2H),8.07(m,1H),8.01(s,1H),7.75(d,J=8.4Hz,1H),4.47(d,J=2.2Hz,2H),4 .28(m,1H),3.92(t,J=5.3Hz,2H),3.08(d,J=12.2Hz,2H),2.82(s,2H),2 .62(t,J=11.4Hz,2H),2.00(m,3H),1.84(ddd,J=16.9,13.1,5.1Hz,2H).

[0126] HRMS m / z(ESI)calcd for C 25 H 24 Cl2N6O[M+H] + 495.1461found:495.1462.

[0127] Example 10. Synthesis of Compound 10

[0128] Compound 10 was prepared using a similar synthesis method to that of Example 1 with a yield of 67%.

[0129] Compound 10 1 H NMR and HRMS data are as follows:

[0130] 1H NMR (400MHz, DMSO) δ8.95(s,1H),8.82(m,1H),8.74(s,1H),8.52(d,J=7.8Hz,1H),8.39(s,1H),8.23(dd,J=7.9,1.8Hz,1H),7.96(d,J=8.8Hz,2H ),7.80(t,J=8.0Hz,1H),7.07(d,J=8.9Hz,2H),4.50(s,2H),3.94(t,J=5 .2Hz,2H),3.23(m,4H),2.85(s,2H),2.47(d,J=5.2Hz,4H),2.24(s,3H).

[0131] HRMS m / z(ESI)calcd for C 28 H 28 N6O3[M+H] + 497.2296found:497.2297.

[0132] Example 11. Synthesis of Compound 11

[0133] Compound 11 was prepared using a similar synthesis method to Example 1 with a yield of 61%.

[0134] Compound 11 1 H NMR and HRMS data are as follows:

[0135] 1 H NMR (400MHz, DMSO) δ8.74(s,1H),8.49(s,1H),8.31(s,1H),8.22(s,1H),8.06(d,J=7.2Hz,2H),7. 97(s,1H),7.50(t,J=7.6Hz,2H),7.39(m,1H),3.92(s,3H),2.90(s,2H),2.77(s,2H),2.43(s,3H).

[0136] HRMS m / z(ESI)calcd for C 22 H 22 N6[M+H] + 371.1979found:371.1978.

[0137] Example 12. Synthesis of Compound 12

[0138] Compound 12 was prepared using a similar synthesis method to that of Example 1 with a yield of 61%.

[0139] Compound 12 1 H NMR and HRMS data are as follows:

[0140] 1 H NMR (400MHz, CDCl3) δ8.38(s,1H),8.21(s,1H),7.92(dd,J=15.8,8.8Hz,4H),7.83(s,1H),7.01(dd,J=10.2,9.0Hz,4H),3 .87(s,3H),3.40(d,J=3.1Hz,2H),3.30(m,4H),3.04(s,2H),2.77(t,J=5.7Hz,2H),2.61(m,4H),2.48(s,3H),2.37(s,3H).

[0141] HRMS m / z(ESI)calcd for C 30 H 34 N6O[M+H] + 495.2867found:495.2868.

[0142] Example 13. Synthesis of Compound 13

[0143] Compound 13 was prepared using a similar synthesis method to Example 1 with a yield of 61%.

[0144] Compound 13 1 H NMR and HRMS data are as follows:

[0145] 1 H NMR(400MHz,DMSO)δ8.90(s,1H),8.90(s,1H),8.51(s,1H),8.51(s,1H),8.32(s,2H),8.32(s,1 H),8.08(d,J=8.5Hz,3H),8.08(d,J=8.5Hz,2H),7.94(d,J=8.8Hz,3H),7.94(d,J=8.8Hz,2H),7 .55(d,J=8.5Hz,3H),7.55(d,J=8.5Hz,2H),7.06(d,J=8.9Hz,3H),7.06(d,J=8.9Hz,2H),3.27( s,2H),3.23(m,4H),2.87(s,2H),2.66(t,J=5.6Hz,2H),2.47(m,4H),2.35(s,3H),2.23(s,3H).

[0146] HRMS m / z(ESI)calcd for C 29 H31 ClN6[M+H] + 499.2371found:499.2371.

[0147] Example 14. Synthesis of Compound 14

[0148] Compound 14 was prepared using a similar synthesis method to that of Example 1 with a yield of 67%.

[0149] Compound 14 1 H NMR and HRMS data are as follows:

[0150] 1 H NMR (400MHz, DMSO) δ8.91(s,1H),8.62(s,1H),8.30(d,J=1.9Hz,2H),8.06(dd,J=8.4,2.0Hz,1H),7.95(d,J=8.9Hz,2H),7.76(d,J=8.4Hz,1H), 7.07(d,J=8.9Hz,2H),3.29(d,J=2.8Hz,2H),3.23(m,4H),2.88(s,2H), 2.67(t,J=5.5Hz,2H),2.47(d,J=5.4Hz,4H),2.36(s,3H),2.24(s,3H).

[0151] HRMS m / z(ESI)calcd for C 29 H 30 Cl2N6[M+H] + 533.1982found:533.1983.

[0152] Example 15. Synthesis of Compound 15

[0153] Compound 15 was prepared by a similar synthesis method as in Example 1 with a yield of 50%.

[0154] Compound 15 1 H NMR and HRMS data are as follows:

[0155] 1H NMR (400MHz, DMSO) δ8.88(s,1H),8.47(s,1H),8.38(s,1H),8.05(d,J=7.4Hz,2H),7.93(d,J=8.8Hz,2H),7.49(t,J=7.6Hz,2H),7.38(t ,J=7.3Hz,1H),7.05(d,J=8.9Hz,2H),3.63(d,J=1.5Hz,2H),3.22(m,4H),3.02(t,J=5.4Hz,2H),2.75(s,2H),2.47(m,4H),2.23(s,3H).

[0156] HRMS m / z(ESI)calcd for C 28 H 30 N6[M+H] + 451.2605found:451.2607.

[0157] Example 16. Synthesis of Compound 16

[0158] Compound 16 was prepared by a similar synthesis method as in Example 1 with a yield of 55%.

[0159] Compound 16 1 H NMR and HRMS data are as follows:

[0160] 1 H NMR (400MHz, CDCl3) δ8.29(s,1H),8.23(s,1H),8.11(s,1H),7.91(d,J=4.1Hz,2H),7.88(s,1H),7.82(d,J=7.8Hz,1H),7.52 (d,J=8.4Hz,1H),7.02(d,J=8.9Hz,2H),4.36(d,J=2.5Hz,2H),3.73(t,J=5.4Hz,2H),3.31(m,4H),3.00(s,2H),2.61(m,4H).

[0161] HRMS m / z(ESI)calcd for C 28 H 28 Cl2N6[M+H] + 519.1825found:519.1825.

[0162] Example 17. Synthesis of Compound 17

[0163] Compound 17 was prepared using a similar synthesis method to that of Example 1 with a yield of 55%.

[0164] Compound 17 1 H NMR and HRMS data are as follows:

[0165] 1 H NMR (400MHz, DMSO) δ8.99(s,1H),8.87(s,1H),8.57(s,1H),8.52(s,1H),8.16(d,J=7.3Hz,2H),8.00(d,J=8.8Hz,2H),7.52( t,J=7.6Hz,2H),7.40(t,J=7.3Hz,1H),7.07(d,J=8.9Hz,2H),4.04(s,3H),3.24(m,4H),2.47(d,J=4.9Hz,4H),2.24(s,3H).

[0166] HRMS m / z(ESI)calcd for C 27 H 27 N7[M+H] + 450.2401found:450.2403.

[0167] Example 18, Synthesis of Compound 18

[0168] Compound 18 was prepared using a similar synthesis method to Example 1 with a yield of 59%.

[0169] Compound 18 1 H NMR and HRMS data are as follows:

[0170] 1 H NMR (400MHz, DMSO) δ8.98(s,1H),8.85(s,1H),8.50(s,1H),8.10(d,J=7.5Hz,2H),7.94(d,J=8.7Hz,2H),7.51(t,J=7.6Hz,2 H),7.39(t,J=7.3Hz,1H),7.09(d,J=8.8Hz,2H),3.89(s,3H),3.24(m,4H),2.87(s,3H),2.47(d,J=4.8Hz,4H),2.24(s,3H).

[0171] HRMS m / z(ESI)calcd for C 28 H 29 N7[M+H] + 464.2557found:464.2559.

[0172] Example 19, Synthesis of Compound 19

[0173] Compound 19 was prepared using a similar synthesis method to that of Example 1 with a yield of 50%.

[0174] Compound 19 1 H NMR and HRMS data are as follows:

[0175] 1 H NMR (400MHz, DMSO) δ9.17(s,1H),8.92(m,2H),8.87(m,2H),8.65(s,1H),8.15(d,J=7.2Hz,2H),8.05(d,J=8.8Hz ,2H),7.54(t,J=7.6Hz,2H),7.43(t,J=7.4Hz,1H),7.11(d,J=8.9Hz,2H),3.26(m,4H),2.47(s,4H),2.24(s,3H).

[0176] HRMS m / z(ESI)calcd for C 28 H 26 N6[M+H] + 447.2292found:447.2292.

[0177] Example 20, Synthesis of Compound 20

[0178] Compound 20 was prepared using a similar synthesis method to that of Example 1 with a yield of 52%.

[0179] Compound 20 1 H NMR and HRMS data are as follows:

[0180] 1 H NMR (400MHz, DMSO) δ9.16(s,1H),8.91(d,J=5.0Hz,3H),8.86(d,J=4.9Hz,2H),8.65(s,1H),8.27(d,J=8.9Hz,1H),8.15(d ,J=7.9Hz,2H),7.54(t,J=7.6Hz,2H),7.43(t,J=7.3Hz,1H),6.96(d,J=9.0Hz,1H),3.52(m,4H),2.81(m,4H),1.25(m,1H).

[0181] HRMS m / z(ESI)calcd for C 26 H23 N7[M+H] + 434.2088found:434.2088.

[0182] Example 21, Synthesis of Compound 21

[0183] Compound 21 was prepared by a similar synthesis method to Example 1 with a yield of 51%.

[0184] Compound 21 1 H NMR and HRMS data are as follows:

[0185] 1 H NMR (400MHz, DMSO) δ9.01 (s, 1H), 8.95 (d, J = 6.0Hz, 2H), 8.86 (d, J = 5.9Hz, 2H) ,8.66(s,1H),8.47(s,1H),8.16(d,J=7.4Hz,2H),8.11(s,1H),7.54(t,J=7.6H z,2H),7.43(t,J=7.4Hz,1H),4.29(m,1H),3.08(d,J=12.6Hz,2H),2.62(t,J=1 1.6Hz, 2H), 2.02 (dd, J=11.3, 2.5Hz, 3H), 1.86 (ddd, J=23.9, 11.9, 3.7Hz, 2H).

[0186] HRMS m / z(ESI)calcd for C 25 H 23 N7[M+H] + 422.2088found:422.2089.

[0187] Example 22, Synthesis of Compound 22

[0188] Compound 22 was prepared using a similar synthesis method to Example 1 with a yield of 59%.

[0189] Compound 22 1 H NMR and HRMS data are as follows:

[0190] 1H NMR (400MHz, DMSO) δ9.92(d,J=2.1Hz,1H),9.15(dd,J=8.8,2.4Hz,1H),9.02(s,1H),8.59(s,1H),8.12(d,J=7.2Hz,2H),8.02(d,J=8.8Hz,2 H),7.53(t,J=7.6Hz,2H),7.41(t,J=7.3Hz,1H),7.09(d,J=8.6Hz,3H),4.00(s,3H),3.28–3.19(m,4H),2.48(d,J=5.4Hz,4H),2.24(s,3H).

[0191] HRMS m / z(ESI)calcd for C 29 H 28 N6O[M+H] + 477.2397found:477.2398.

[0192] Example 23. Synthesis of Compound 23

[0193] Compound 23 was prepared using a similar synthesis method to that of Example 1 with a yield of 58%.

[0194] Compound 23 1 H NMR and HRMS data are as follows:

[0195] 1 H NMR (400MHz, DMSO) δ9.17 (s, 1H), 8.85 (d, J = 2.0Hz, 1H), 8.70 (d, J = 4.8Hz, 1H), 8.65 (s, 1H), 8.21–8.15 (m, 1H), 8.03 (d, J = 7.3Hz, 2H), 7.9 5(d,J=8.8Hz,2H),7.49(t,J=7.5Hz,2H),7.39(t,J=7.3Hz,1H),7.08(d,J=8.9Hz,2H),3.27–3.21(m,4H),2.49–2.44(m,4H),2.24(s,3H).

[0196] HRMS m / z(ESI)calcd for C 28 H 25 FN6[M+H] + 465.2197found:465.2199.

[0197] Example 24, Synthesis of Compound 24

[0198] Compound 24 was prepared using a similar synthesis method to Example 1 with a yield of 54%.

[0199] Compound 24 1 H NMR and HRMS data are as follows:

[0200] 1 H NMR(400MHz,DMSO)δ9.20(s,1H),8.98–8.94(m,2H),8.92–8.88(m,2H),8.78(s,1H),8.71(s,1H),8.29(d,J=8.5Hz,1H),8.12–8 .04(m,4H),7.98(d,J=7.1Hz,1H),7.62–7.53(m,2H),7.12(d,J=8.9Hz,2H),3.29–3.23(m,4H),2.50–2.46(m,4H),2.25(s,3H).

[0201] HRMS m / z(ESI)calcd for C 32 H 28 N6[M+H] + 497.2448found:497.2447.

[0202] Example 25. Synthesis of Compound 25

[0203] Compound 25 was prepared using a similar synthesis method to that of Example 1 with a yield of 56%.

[0204] Compound 25 1 H NMR and HRMS data are as follows:

[0205] 1 H NMR (400MHz, DMSO) δ9.18(s,1H),8.88(s,3H),8.78(s,1H),8.40(d,J=1.9Hz,1H),8.15(dd,J=8.3,2.0Hz,2H),8 .06(d,J=8.9Hz,2H),7.80(d,J=8.4Hz,1H),7.11(d,J=8.9Hz,2H),3.29–3.23(m,4H),2.47(s,4H),2.25(s,3H).

[0206] HRMS m / z(ESI)calcd for C 28 H 24 Cl2N6[M+H] + 515.1512found:515.1511.

[0207] Example 26. Synthesis of Compound 26

[0208] Compound 26 was prepared using a similar synthesis method to Example 1 with a yield of 52%.

[0209] Compound 26 1 H NMR and HRMS data are as follows:

[0210] 1 H NMR (400MHz, DMSO) δ9.12(s,1H),8.76–8.68(m,3H),8.62(s,1H),8.13(d,J=7.3Hz,2H),8.03(d,J=8.8Hz,2H),7.54(t,J=7 .6Hz,2H),7.43(t,J=7.3Hz,1H),7.10(d,J=8.9Hz,2H),3.28–3.22(m,4H),2.67(s,3H),2.48(d,J=4.9Hz,4H),2.24(s,3H).

[0211] HRMS m / z(ESI)calcd for C 29 H 28 N6[M+H] + 461.2448found:461.2447.

[0212] Example 27, Synthesis of Compound 27

[0213] Compound 27 was prepared by a similar synthesis method as in Example 1 with a yield of 52%.

[0214] Compound 27 1 H NMR and HRMS data are as follows:

[0215] 1 H NMR(400MHz, CDCl3)δ8.92(dd,J=4.6,1.5Hz,2H),8.86(dd,J=4.7,1.4Hz,2H),8.41(s,1H),8.03(d,J=4.5Hz,2H), 8.01–7.94(m,3H),7.47(d,J=8.5Hz,2H),7.06(d,J=8.8Hz,2H),3.39–3.29(m,4H),2.67–2.57(m,4H),2.38(s,3H).

[0216] HRMS m / z(ESI)calcd for C 28 H25 ClN6[M+H] + 461.2448found:461.2447.

[0217] Example 28, Synthesis of Compound 28

[0218] Compound 28 was prepared using a similar synthesis method to Example 1 with a yield of 57%.

[0219] Compound 28 1 H NMR and HRMS data are as follows:

[0220] 1 H NMR (400MHz, CDCl3) δ8.96 (dd, J=4.6, 1.5Hz, 2H), 8.87 (dd, J=4.7, 1.5Hz, 2H), 8.44(s,1H),8.15(d,J=8.3Hz,2H),8.09(s,1H),7.98(d,J=8.8Hz,2H),7.74(d ,J=8.3Hz,2H),7.68(d,J=7.3Hz,2H),7.48(t,J=7.6Hz,2H),7.39(t,J=7.3Hz, 1H),7.07(d,J=8.8Hz,2H),3.38–3.30(m,4H),2.67–2.58(m,4H),2.39(s,3H).

[0221] HRMS m / z(ESI)calcd for C 24 H 30 N6[M+H] + 523.2605found:523.2606.

[0222] Example 29, Synthesis of Compound 29

[0223] Compound 29 was prepared using a similar synthesis method to that of Example 1 with a yield of 51%.

[0224] Compound 29 1 H NMR and HRMS data are as follows:

[0225] 1H NMR (400MHz, DMSO) δ9.94 (d, J=2.2Hz, 1H), 9.21 (dd, J=8.8, 2.3Hz, 1H), 8.87 (s, 1H), 8. 60(s,1H),8.46(s,1H),8.13(d,J=7.3Hz,2H),8.08(s,1H),7.53(t,J=7.6Hz,2H),7.42 (t,J=7.4Hz,1H),7.07(d,J=8.8Hz,1H),4.33–4.24(m,1H),3.09(d,J=12.5Hz,2H),2.6 4(t,J=11.4Hz,2H), 2.01(dd,J=17.5,9.7Hz,3H), 1.87(ddd,J=23.8,12.1,3.7Hz,2H).

[0226] HRMS m / z(ESI)calcd for C 26 H 25 N7O[M+H] + 452.2193found:452.2194.

[0227] Example 30, Synthesis of Compound 30

[0228] Compound 30 was prepared using a similar synthesis method to that of Example 1 with a yield of 50%.

[0229] Compound 30 1 H NMR and HRMS data are as follows:

[0230] 1 H NMR (400MHz, DMSO) δ8.99 (s, 1H), 8.78 (d, J = 5.1Hz, 1H), 8.72 (m, 2H), 8.65 (s, 1H) ,8.46(s,1H),8.15(d,J=7.3Hz,2H),8.11(s,1H),7.54(t,J=7.6Hz,2H),7.43(t,J =7.3Hz,1H),4.30(ddd,J=11.6,10.0,4.3Hz,1H),3.08(d,J=12.1Hz,2H),2.68(s ,3H),2.63(t,J=11.7Hz,2H),2.02(d,J=9.3Hz,3H),1.87(qd,J=11.8,3.6Hz,2H).

[0231] HRMS m / z(ESI)calcd for C 26 H 25 N7[M+H] +436.2244found:436.2245.

[0232] Example 31, Synthesis of Compound 31

[0233] Compound 31 was prepared using a similar synthesis method to that of Example 1 with a yield of 57%.

[0234] Compound 31 1 H NMR and HRMS data are as follows:

[0235] 1 H NMR (400MHz, DMSO) δ9.84(d,J=1.9Hz,1H),9.07(dd,J=8.8,2.2Hz,1H),8.95(s,1H),8.64(s,1H),8.30(s,1H),8.07(d,J=8.6Hz,1H ),7.99(d,J=8.7Hz,2H),7.75(d,J=8.4Hz,1H),7.06(d,J=8.8Hz,3H),3.99(s,3H),3.24(m,4H),2.48(d,J=5.0Hz,4H),2.25(s,3H).

[0236] HRMS m / z(ESI)calcd for C 29 H 26 Cl2N6O[M+H] + 545.1618found:545.1618.

[0237] Example 32, Synthesis of Compound 32

[0238] Compound 32 was prepared using a similar synthesis method to that of Example 1 with a yield of 54%.

[0239] Compound 32 1 H NMR and HRMS data are as follows:

[0240] 1 H NMR (400MHz, CDCl3) δ8.54(s,2H),8.40(s,1H),8.07(dd,J=10.3,3.2Hz,3H),7.97(d,J=8.8Hz,2H),7.51(t,J =7.5Hz,2H),7.41(t,J=7.4Hz,1H),7.07(d,J=8.9Hz,2H),3.33(m,4H),2.72(s,6H),2.62(m,4H),2.38(s,3H).

[0241] HRMS m / z(ESI)calcd for C 30 H 30 N6[M+H] + 475.2605found:475.2606.

[0242] Example 33. Synthesis of Compound 33

[0243] Compound 33 was prepared using a similar synthesis method as in Example 1 with a yield of 50%.

[0244] Compound 33 1 H NMR and HRMS data are as follows:

[0245] 1 H NMR (400MHz, DMSO) δ9.10(s,1H),8.61(s,1H),8.16(dd,J=6.0,4.7Hz,3H),8.10(s,1H),8.02(d,J=8.8Hz,2H),7.95(dd,J= 5.4,1.3Hz,1H),7.53(t,J=7.6Hz,2H),7.42(t,J=7.3Hz,1H),7.09(d,J=8.9Hz,2H),6.20(s,2H),3.25(m,4H),2.24(s,3H).

[0246] HRMS m / z(ESI)calcd for C 28 H 27 N7[M+H] + 462.2401found:462.2402.

[0247] Example 34. Synthesis of Compound 34

[0248] Compound 34 was prepared using a similar synthesis method to that of Example 1 with a yield of 59%.

[0249] Compound 34 1 H NMR and HRMS data are as follows:

[0250] 1H NMR (400MHz, DMSO) δ9.10(s,1H),8.70(dd,J=10.3,4.2Hz,3H),8.62(s,1H),8.34(d,J=1.8Hz,1H),8.11(dd,J=8.4,1.9Hz,1H),8 .02(d,J=8.7Hz,2H),7.78(d,J=8.4Hz,1H),7.09(d,J=8.9Hz,2H),3.25(m,4H),2.66(s,3H),2.48(d,J=5.0Hz,4H),2.24(s,3H).

[0251] HRMS m / z(ESI)calcd for C 29 H 26 Cl2N6[M+H] + 529.1669found:529.1668.

[0252] Example 35. Synthesis of Compound 35

[0253] Compound 35 was prepared using a similar synthesis method as in Example 1 with a yield of 59%.

[0254] Compound 35 1 H NMR and HRMS data are as follows:

[0255] 1 H NMR (400MHz, DMSO) δ10.23(d,J=1.3Hz,1H),9.54(dd,J=8.2,2.1Hz,1H),9.18(s,1H),8.66(s,1H),8.31(d,J=8.6Hz,1H),8.14(d,J=7.3H z,2H),8.04(d,J=8.8Hz,2H),7.54(t,J=7.4Hz,2H),7.43(t,J=7.5Hz,1H),7.10(d,J=8.9Hz,2H),3.26(m,4H),2.47(s,4H),2.24(s,3H).

[0256] HRMS m / z(ESI)calcd for C 29 H 25 N7[M+H] + 472.2244found:472.2246.

[0257] Example 36, Synthesis of Compound 36

[0258] Compound 36 was prepared using a similar synthesis method to that of Example 1 with a yield of 59%.

[0259] Compound 36 1 H NMR and HRMS data are as follows:

[0260] 1 H NMR (400MHz, DMSO) δ9.90 (d, J=2.3Hz, 1H), 9.15 (dd, J=8.8, 2.4Hz, 1H), 9.0 2(s,1H),8.59(s,1H),8.12(d,J=7.4Hz,2H),8.02(d,J=8.8Hz,2H),7.53(t, J=7.6Hz,2H),7.41(t,J=7.4Hz,1H),7.07(dd,J=11.6,9.0Hz,3H),4.46(q, J=7.0Hz,2H),3.25(m,4H),2.47(s,4H),2.24(s,3H),1.40(t,J=7.0Hz,3H).

[0261] HRMS m / z(ESI)calcd for C 30 H 30 N6O[M+H] + 491.2554found:491.2555.

[0262] Example 37, Synthesis of Compound 37

[0263] Compound 37 was prepared using a similar synthesis method to that of Example 1 with a yield of 63%.

[0264] Compound 37 1 H NMR and HRMS data are as follows:

[0265] 1 H NMR (400MHz, DMSO) δ10.05(s,2H),9.07(s,1H),8.62(s,1H),8.12(d,J=7.3Hz,2H),8.03(d,J=8.8Hz,2H),7.53(t ,J=7.6Hz,2H),7.42(t,J=7.3Hz,1H),7.08(d,J=9.0Hz,2H),4.07(s,3H),3.25(m,4H),2.47(s,4H),2.24(s,3H).

[0266] HRMS m / z(ESI)calcd for C 28 H 27 N7O[M+H]+ 478.2350found:478.2351.

[0267] Example 38, Synthesis of Compound 38

[0268] Compound 38 was prepared using a similar synthesis method to that of Example 1 with a yield of 57%.

[0269] Compound 38 1 H NMR and HRMS data are as follows:

[0270] 1 H NMR (400MHz, CDCl3-d) δ8.40(d,J=0.9Hz,1H),8.14(s,1H),7.91(d,J=7.8Hz,2H),7.86(d,J=7.9Hz,2H),7.75(s,1H),6.98(t,J=7 .0Hz,4H),4.55(s,2H),4.02(t,J=5.1Hz,2H),3.85(d,J=0.8Hz,3H),3.28(d,J=4.3Hz,4H),2.96(s,2H),2.60(m,4H),2.37(s,3H).

[0271] HRMS m / z(ESI)calcd for C 29 H 31 N5O2[M+H] + 482.2551found:482.2551.

[0272] Example 39, Synthesis of Compound 39

[0273] Compound 39 was prepared using a similar synthesis method to that of Example 1 with a yield of 52%.

[0274] Compound 39 1 H NMR and HRMS data are as follows:

[0275] 1H NMR (400MHz, DMSO-d6) δ9.71(s,1H),8.84(s,1H),8.39(s,1H),8.29(s,1H),7.88(dd,J=11.4,8.7Hz,4H),7.02(d,J=8.7H z,2H),6.88(d,J=8.4Hz,2H),4.44(s,2H),3.91(t,J=5.1Hz,2H),3.20(s,4H),2.81(s,2H),2.47(s,4H),2.23(s,3H).HRMS m / z(ESI)calcd for C 28 H 29 N5O2[M+H] + 468.2394found:468.2398.

[0276] Example 40, Synthesis of Compound 40

[0277] Compound 40 was prepared using a similar synthesis method as in Example 1 with a yield of 48%.

[0278] Compound 40 1 H NMR and HRMS data are as follows:

[0279] 1 H NMR(400MHz, CDCl3-d)δ8.43(s,1H),8.23(s,1H),7.89(m,5H),7.28(m,1H),7.25(m,1H),7.02(d,J=8.4Hz,2H),4 .58(d,J=2.1Hz,2H),4.04(t,J=5.3Hz,2H),3.30(m,4H),2.98(s,2H),2.61(m,4H),2.41(s,3H),2.37(s,3H).HRMS m / z(ESI)calcd for C 29 H 31 N5O[M+H] + 466.2601found:466.2602.

[0280] Example 41, Synthesis of Compound 41

[0281] Compound 41 was prepared using a similar synthesis method to that of Example 1 with a yield of 43%.

[0282] Compound 41 1 H NMR and HRMS data are as follows:

[0283] 1H NMR (400MHz, CDCl3-d) δ8.43(s,1H),8.23(s,1H),7.90(t,J=4.4Hz,3H),7.57(dd,J=10.2,5.0Hz,2H),7.37(t,J=7.9Hz,1H),7.02(d,J=8.9Hz,2H),6. 92(dd,J=8.2,1.8Hz,1H),4.57(d,J=2.6Hz,2H),4.04(t,J=5.4Hz,2H),3.9 1(s,3H),3.31(m,4H),2.98(d,J=1.4Hz,2H),2.61(m,4H),2.38(s,3H).HRMS m / z(ESI)calcd for C 33 H 31 N5O[M+H] + 482.2551found:482.2552.

[0284] Example 42, Synthesis of Compound 42

[0285] Compound 42 was prepared using a similar synthesis method to that of Example 1 with a yield of 59%.

[0286] Compound 42 1 H NMR and HRMS data are as follows:

[0287] 1 H NMR (400MHz, CDCl3) δ9.06(d,J=8.0Hz,2H),8.28(s,1H),8.07(d,J=7.9Hz,2H),7.97(m,3H),7.48(t,J=7.2Hz,2H),7.37(t,J =6.9Hz,1H),7.10(d,J=8.0Hz,2H),7.04(d,J=8.0Hz,2H),3.92(d,J=0.8Hz,3H),3.32(s,4H),2.63(s,4H),2.39(s,3H).HRMS m / z(ESI)calcd for C 30 H 29 N5O[M+H] + 476.2445found:476.2449.

[0288] Example 43. Synthesis of Compound 43

[0289] Compound 43 was prepared using a similar synthesis method to that of Example 1 with a yield of 47%.

[0290] Compound 43 1 H NMR and HRMS data are as follows:

[0291] 1 H NMR (400MHz, DMSO) δ10.00(s,1H),9.14(d,J=8.1Hz,1H),9.07(s,1H),8.62(s,1H),8.12(d,J=7.6Hz,2H),8.03(d,J=8.5H z,2H),7.53(m,3H),7.42(t,J=7.2Hz,1H),7.11(d,J=8.7Hz,2H),3.26(s,4H),2.62(s,3H),2.49(m,4H),2.26(s,3H).HRMS m / z(ESI)calcd for C 29 H 28 N6[M+H] + 461.2448found:461.2451.

[0292] Example 44. Synthesis of Compound 44

[0293] Compound 44 was prepared using a similar synthesis method to that of Example 1 with a yield of 47%.

[0294] Compound 44 1 H NMR and HRMS data are as follows:

[0295] 1 H NMR (400MHz, DMSO) δ9.92(s,1H),9.13(d,J=8.2Hz,1H),9.01(s,1H),8.57(s,1H),8.02(d,J=8.2Hz,2H),7.91(m,2H),7.41(t ,J=7.5Hz,1H),7.23(d,J=7.6Hz,1H),7.09(d,J=8.3Hz,3H),4.01(s,3H),3.26(s,4H),2.51(s,4H),2.44(s,3H),2.26(s,3H).

[0296] HRMS m / z(ESI)calcd for C 30 H 30 N6O[M+H] + 491.2554found:491.2556.

[0297] Example 45. Synthesis of Compound 45

[0298] Compound 45 was prepared using a similar synthesis method to that of Example 1 with a yield of 62%.

[0299] Compound 45 1 H NMR and HRMS data are as follows:

[0300] 1 H NMR (400MHz, DMSO) δ9.89(s,1H),9.12(m,1H),9.02(s,1H),8.68(s,1H),8.16(s,1H),8.09(d,J=7.6Hz,1H),8.03(d,J=8.4Hz,2H ),7.56(t,J=7.8Hz,1H),7.47(d,J=8.0Hz,1H),7.09(dd,J=8.7,4.0Hz,3H),4.01(s,3H),3.26(s,4H),2.47(m,4H),2.26(s,3H).

[0301] HRMS m / z(ESI)calcd for C 29 H 27 ClN6O[M+H] + 511.2008found:511.2006.

[0302] Example 46, Synthesis of Compound 46

[0303] Compound 46 was prepared using a similar synthesis method to Example 1 with a yield of 43%.

[0304] Compound 46 1 H NMR and HRMS data are as follows:

[0305] 1 H NMR (400MHz, DMSO) δ9.95 (s, 1H), 9.62 (s, 1H), 9.11 (m, 1H), 9.01 (s, 1H), 8.52 (s, 1H), 8.02 (d, J = 8.7Hz, 2H), 7.58 (s, 1H), 7.50 (d, J = 8. 0Hz,1H),7.31(t,J=7.7Hz,1H),7.09(d,J=8.9Hz,3H),6.80(d,J=8.0Hz,1H),4.00(s,3H),3.25(s,4H),2.47(m,4H),2.25(s,3H).HRMS m / z(ESI)calcd for C 29 H 28 N6O2[M+H] + 493.2347found:493.2348.

[0306] Example 47, Synthesis of Compound 47

[0307] Compound 47 was prepared using a similar synthesis method to Example 1 with a yield of 43%.

[0308] Compound 47 1 H NMR and HRMS data are as follows:

[0309] 1 H NMR (400MHz, DMSO-d6) δ10.05(s,2H),9.07(s,1H),8.62(s,1H),8.12(d,J=7.3Hz,2H),8.03(d,J=8.8Hz,2H),7.53(t HRMS m / z(ESI)calcd for C 28 H 27 N7O[M+H] + 478.2350found:478.2351.

[0310] Example 48, Synthesis of Compound 48

[0311] Compound 48 was prepared using a similar synthesis method as in Example 1 with a yield of 43%.

[0312] Compound 48 1 H NMR and HRMS data are as follows:

[0313] 1 H NMR (400MHz, CDCl3-d) δ9.02(m,2H),8.31(s,1H),8.07(m,2H),7.97(t,J=4.4Hz,3H),7.59(m,2H),7.53(m,1H), 7.48(t,J=7.5Hz,2H),7.38(t,J=7.4Hz,1H),7.04(d,J=8.9Hz,2H),3.31(m,4H),2.61(m,4H),2.38(s,3H).HRMS m / z(ESI)calcd for C 29 H 27 N5[M+H] + 446.2339found:446.2339.

[0314] Comparative Example 1, Synthesis of Comparative Example 1

[0315] Comparative Example 1 was prepared using a similar synthesis method to Example 1 with a yield of 52%.

[0316] Comparative Example 1 1 H NMR and HRMS data are as follows:

[0317] 1 H NMR (400MHz, DMSO) δ9.05(s,1H),8.83(m,4H),8.44(s,1H),8.20(s,1H),8.07(s,1H),7.92(s,1H),4.34(t,J=10.2 HRMS m / z(ESI)calcd for C 19 H 19 N7[M+H] + 346.1775found:346.1772.

[0318] Comparative Example 2, Synthesis of Comparative Example 2

[0319] A similar synthesis method to Example 1 was used to prepare the compound of Comparative Example 2 with a yield of 32%.

[0320] The 1H NMR and HRMS data of Comparative Example 2 are as follows:

[0321] 1H NMR (400MHz, DMSO) δ9.24(s,1H),9.02(s,1H),8.93(d,J=2.3Hz,1H),8.29(dd,J=8.9,2.4Hz,1H),8.22(s,1H),7.94(s,1H) ,7.75(d,J=2.9Hz,1H),7.07(dd,J=8.9,3.0Hz,1H),7.01(d,J=9.0Hz,1H),6.71(d,J=8.9Hz,1H),3.74(m,4H),3.56(m,4H).

[0322] HRMS m / z(ESI)calcd for C 20 H 18 N6O[M+H] + 359.1615 found:359.1617.

[0323] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0324] Test Example 1: Study on the Inhibition Rate of Ferroptosis by the Compounds of the Invention

[0325] The Ferroptosis screening model mainly uses the MTT cell viability assay. First, the fibrosarcoma cell line HT1080 is cultured in a dish, and the cells in the logarithmic growth phase are seeded in a specific number in a 96-well plate (3000 cells / well), 100 μL per well, and then placed in an incubator at 37°C and 5% CO2 to allow the cells to adhere. After 24 hours, 100 μL of a certain concentration of compound prepared using a specified culture medium and the Ferroptosis inducer Erastin (final concentration 10 μM) are added. Three replicates are set for each compound to ensure the accuracy of the results, and a negative control group, a positive control group (Fer-1 15 μM), a blank control group, and a solvent control group are set up. After adding the drug, the plate is placed in an incubator and cultured for 48 hours. On the day of the MTT experiment, prepare the MTT test solution in advance (5mg / mL MTT solution dissolved in physiological saline, stored at 4°C in the dark), add 20μL of MTT solution to each well, place in the incubator and continue to culture for 2-4h, then add 50μL of 20% SDS solution (dissolved in MiliiQ water, add 1% concentrated hydrochloric acid) to each well, place in the incubator overnight, and use a microplate reader to detect the absorbance at 570nm on the next day to calculate the drug's inhibition rate on Ferroptosis. The absorbance value of the general control group should be between 0.8-1.2 as a normal value. After obtaining the absorbance value data, calculate the average of the three replicate wells and use the following formula to calculate the inhibition rate:

[0326] Inhibition rate (IR) = [1-(A experimental group-A blank) / (A solvent-A blank)]*100%

[0327] GraphPad Prism 5 software was used to fit the inhibition rate curve and calculate the EC 50 .

[0328] EC analysis of the above 47 compounds 50 Test (EC 50 The test results are the average of three tests, Fer-1 is the positive control group), and the results are shown in Table 1 below:

[0329] Table 1 EC of the compounds of the present invention 50 value

[0330] As shown in Table 1, the EC values ​​of compounds 1-48 of the present invention are 50The values ​​are between 0.01-6.3 μM, all less than 10 μM, which proves that compounds 1-48 prepared by the present invention have good inhibitory effects on ferroptosis and can be used to prepare ferroptosis inhibitors. The EC values ​​of compounds 22 and 46 are 50 It was significantly better than the positive control Fer-1, showing excellent ferroptosis inhibition effect.

[0331] EC of Comparative Example 1 50 The value is greater than 10 μM. Compared with compound 21 of the present invention, the only difference is the presence or absence of benzene ring substitution. The absence of benzene ring substitution leads to a complete loss of ferroptosis inhibitory activity. Therefore, the benzene ring substitution in the core structure of the present invention is essential for the ferroptosis inhibitory activity.

[0332] In addition, the EC of compound 1 in ovarian cancer cell line ES-2 was tested. 50 The test method and operation steps are consistent with the above. The results are shown in Table 2 below:

[0333] Table 2 EC of the compounds of the present invention in ES-2 50 value

[0334] As shown in Table 2, compound 1 has a good inhibitory effect on ferroptosis and can be used to prepare ferroptosis inhibitors. Comparative Example 2 has no benzene ring substitution and loses its ferroptosis inhibitory activity. The benzene ring substitution in the core structure of the present invention is essential for the ferroptosis inhibitory activity.

[0335] Test Example 2: Kinase inhibitory activity of compound 1 (10 μM) of the present invention against 401 kinases

[0336] Kinase profiling was performed on compound 1 using various recombinant kinase targets at 10 μM ATP. The kinases tested included PI3K, FYN, MAPK, JAK, and CDK. Lower %Ctrl values ​​indicate stronger binding of the test compound to the kinase, calculated as follows:

[0337] The positive reference was a reference substance with a %Ctrl value of 0, and the negative reference was DMSO (%Ctrl value of 100). The results are shown in Table 3.

[0338] Table 3 Effects of Compound 1 of the present invention on the activity of the tested kinases

[0339] The results in Table 3 show that the "% Ctrl" values ​​of compound 1 of the present invention against 401 kinases including PI3K are all greater than 50, indicating no obvious inhibitory activity, indicating that the compound of the present invention has good selectivity as a ferroptosis inhibitor.

[0340] Test Example 3: Determination of the non-chelating and non-reducing properties of Compound 1 and Compound 38 of the present invention

[0341] Reducibility studies were performed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Compounds were prepared in methanol and pipetted into a 96-well plate, followed by the addition of the DPPH solution. The mixture was incubated at room temperature in the dark for 10 minutes. The absorbance at 517 nm was measured. Three independent replicates were performed, and the results are shown in Figure A.

[0342] The chelation properties of the compounds were studied by using the property of iron forming a colored complex with phenanthroline. The test compound solution, FeSO4 solution, and phenanthroline solution were added to 100 μL of distilled water and incubated at room temperature for 5 minutes. Finally, the CLARIOstar multifunctional microplate reader (BMG Labtech, Germany) was used to detect the presence of sample A. 562 At the same time, deferoxamine (DFO) and EDTA.2H2O were selected as positive controls, and DMSO was used as blank control. 562 Calculate sample well A for the standard value (100%) 562 The relative amounts of the results are shown in Figure B.

[0343] As shown in Figure 1: Figure A shows that compounds 1 and 38 of the present invention are non-reducing, while the positive control Fer-1 is reducing; Figure B shows that compounds 1 and 38 of the present invention do not have iron chelating properties, while deferoxamine and EDTA.2H2O have iron chelating properties.

[0344] In summary, the ferroptosis inhibitor prepared by the present invention is non-reducing and non-chelating, and can be used to prepare a new non-reducing and non-chelating ferroptosis inhibitor.

[0345] Test Example 4: Study on the compound 1 of the present invention in an animal model of liver injury.

[0346] Four-week-old male C57BL / 6J mice were bred for one week for adaptation. One day before the experiment, the mice were fasted for 12 hours. One hour after the mice were intraperitoneally injected with the compound solution, the experimental mice were injected with APAP (acetaminophen) solution (500 mg / kg) or normal saline. Six hours later, blood was collected from the orbits and the mouse serum samples were tested for ALT (alanine aminotransferase) and AST (aspartate aminotransferase). After euthanasia, the mice were dissected, the liver tissue was removed and washed with normal saline, and immediately fixed with neutral formalin for more than 24 hours and HE staining was performed. The blank control group refers to a blank control without the addition of drug or APAP, and the solvent control group refers to a solvent group in which 500 mg / kg of APAP was injected intraperitoneally into the mice. The results are shown in Figure 2.

[0347] As shown in Figure 2, Compound 1 significantly inhibited cell death and inflammation in the liver tissue of C57BL / J6 mice treated with APAP (Figure A). It also significantly reduced serum levels of aspartate aminotransferase (AST) (Figure B) and alanine aminotransferase (ALT) (Figure C). In an animal model of liver injury, Compound 1 prepared by the present invention exhibited a good therapeutic effect compared to the positive control Fer-1. Therefore, the drug of the present invention can be used to treat ferroptosis-related diseases.

[0348] In summary, the present invention synthesizes a non-chelating, non-reducing ferroptosis inhibitor. This compound has a strong inhibitory effect on ferroptosis and can be used to prepare ferroptosis inhibitors. It can also be used to prepare drugs for treating ferroptosis-related diseases such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver damage, and diabetic complications, and has broad market prospects.

[0349] The above examples are further explanations of the present application and provide implementation details of the present application. However, it should be noted that the above-described examples are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. It is still within the scope of protection of the present application for those skilled in the art to modify or replace them according to the prior art. The reagents used in the examples of the present application can all be commercially available.

Claims

1. A compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: in: n is selected from 1 or 2; R1 is independently selected from hydrogen, halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted benzene ring; or, two adjacent R1 are connected to form a benzene ring; Ring A is selected from a substituted or unsubstituted five-membered or six-membered aryl group, a substituted or unsubstituted five-membered or six-membered heteroaryl group, a substituted or unsubstituted five-membered or six-membered heterocyclic group, or a substituted or unsubstituted N-containing di-fused cyclic group or tri-fused cyclic group; the substituents of Ring A are selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, or a substituted or unsubstituted five-membered or six-membered N-containing heterocyclic group; Ring B is selected from a substituted or unsubstituted five-membered or six-membered aryl group, a substituted or unsubstituted five-membered or six-membered heteroaryl group, or a substituted or unsubstituted five-membered or six-membered heterocyclic group; the substituent of Ring B is selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, substituted or unsubstituted C1-C6 alkoxy, or substituted or unsubstituted C1-C6 alkyl; The heteroatoms of the heterocyclic group and heteroaryl group are selected from one or more of N, O, and S; the substituents of the C1-C6 alkoxy group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl, and C1-C6 alkoxy; The substituents of the C1-C6 alkyl group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy; The substituents of the benzene ring are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy; The substituents of the five-membered or six-membered N-containing heterocyclic group are independently selected from one or more of halogen, hydroxyl, nitro, amino, carboxyl, cyano, C1-C6 alkyl and C1-C6 alkoxy.

2. The compound according to claim 1, characterized in that The ring A is phenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl or triazinyl; or the ring A is an N-containing fused ring formed by condensing any two or three of the above groups; the ring A is substituted or unsubstituted; The ring B is selected from phenyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, tetrahydropyridinyl, tetrahydropyranyl, piperidinyl or piperazinyl; the ring B is substituted or unsubstituted; The number of heteroatoms contained in the heterocyclic group and heteroaryl group is 1, 2 or 3.

3. A compound represented by formula (I), or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof: in: n is selected from 1 or 2; R1 is independently selected from hydrogen, halogen, hydroxyl, nitro, C1-C6 alkoxy, C1-C6 alkyl, and benzene ring; or, two adjacent R1 are connected to form a benzene ring; Ring A is selected from: Ring B is selected from:

4. The compound according to claim 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The R1 is independently selected from hydrogen atom, halogen, hydroxyl, and C1-C6 alkyl.

5. The compound according to claim 3 or 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The ring A is selected from: The ring B is selected from:

6. The compound according to any one of claims 3 to 5, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The ring B is selected from:

7. The compound according to any one of claims 3 to 6, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, characterized in that: The compound is one of the following compounds:

8. A pharmaceutical preparation, characterized in that The preparation is prepared by using the compound according to any one of claims 1 to 7, or its pharmaceutically acceptable salt, or its stereoisomer as an active ingredient and adding a commonly used pharmaceutical carrier.

9. The pharmaceutical preparation according to claim 8, characterized in that The preparation is an oral preparation or an intravenous injection preparation.

10. Use of the compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the pharmaceutical preparation according to claim 8 or 9 in the preparation of a ferroptosis inhibitor.

11. Use of the compound according to any one of claims 1 to 7, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or the pharmaceutical preparation according to claim 8 or 9 in the preparation of a medicament for treating and / or preventing neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver damage, and diabetic complications.

Images