Cyclic amine derivative as well as composition and application thereof

Abstract

The present invention relates to a cyclic amine derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the cyclic amine derivative or the pharmaceutically acceptable salt thereof, and an application of the cyclic amine derivative or the pharmaceutically acceptable salt thereof or the composition thereof. The cyclic amine derivative disclosed by the invention has relatively strong apo (a) binding capacity, can effectively reduce the Lp (a) level, and is low in toxic and side effects.

Description

A cyclic amine derivative and its composition and application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on March 24, 2023, with application number 202310301299.3 and application name “A cyclic amine derivative, its composition and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to a cyclic amine derivative or a pharmaceutically acceptable salt thereof, a pharmaceutical composition comprising the cyclic amine derivative or a pharmaceutically acceptable salt thereof, and applications of the cyclic amine derivative or a pharmaceutically acceptable salt thereof or the composition thereof. Background Art

[0003] Hyperlipidemia is a major risk factor for atherosclerosis and is closely associated with cardiovascular disease, posing a serious threat to human health. In the human body, blood lipids must bind to apolipoproteins to form lipoproteins before they can be dissolved in the blood and transported to tissues for metabolism. Lipoproteins include chylomicrons (CM), very low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL), low-density lipoproteins (LDL), high-density lipoproteins (HDL), and lipoprotein (a).

[0004] Lp(a) readily deposits in blood vessel walls and can contribute to the formation of atherosclerotic lesions through various mechanisms. Its atherosclerotic potential is comparable to that of LDC-C. Furthermore, Lp(a) has thrombogenic effects. Lp(a) is synthesized in the liver and secreted into the circulation, primarily depositing in vascular tissue and the aortic valve leaflets. The lipid composition of Lp(a) is similar to that of LDL, but differs from Lp(a) in that it contains a specific apolipoprotein, apo(a), which is disulfide-bonded to apo B100. Apo(a) is a highly glycosylated, hydrophilic protein with structural polymorphism, accounting for 25% to 40% of the total Lp(a) protein and crucial for Lp(a)'s specific role in causing atherosclerotic cardiovascular disease (ASCVD).

[0005] Patent WO2020 / 247429A1 discloses a series of pharmaceutically acceptable compounds for lowering plasma Lp(a) levels, which can be used to prepare drugs for cardiovascular diseases. Although certain progress has been made in the field of drugs for treating cardiovascular diseases, there is still a need to further develop cardiovascular drugs that meet clinical needs.

[0006] Summary of the Invention

[0007] The present invention provides a cyclic amine derivative which has strong apo(a) binding ability, can effectively reduce Lp(a) levels and has low toxic and side effects.

[0008] Specifically, the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof:

[0009] in,

[0010] R is selected from aryl or heteroaryl, which may be further substituted;

[0011] R1, R2, R3 and R4 are each independently selected from H and alkyl, which may be further substituted;

[0012] x is 1 or 2, preferably 1;

[0013] y is 0 or 1;

[0014] In some embodiments, R is selected from C6-C20 aryl, preferably C6-C14 aryl, more preferably C6-C12 aryl, which may be further substituted;

[0015] In some embodiments, R1, R2, R3 and R4 are each independently selected from H and C1-6 alkyl;

[0016] In some embodiments, R1, R2, R3 and R4 are each independently selected from H and methyl, preferably H;

[0017] In some embodiments, R is selected from phenyl, benzo[d][1,3]dioxole, tetrahydroquinoline, tetrahydroisoquinoline, pyridine, quinoline or isoquinoline, which may be further substituted;

[0018] In some embodiments, R is selected from the following groups:

[0019] Wherein, n is 0, 1, 2 or 3;

[0020] R5 is each independently selected from hydrogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, alkylcarbonylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, aryl or heteroaryl; preferably alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, alkylcarbonylamino or halogen;

[0021] The R5 can be located at one, two or three of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th or 8th positions of the above-mentioned aryl or heteroaryl group.

[0022] In some embodiments, R5 is independently selected from C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, C1-6 alkylcarbonylamino, halogen or wherein x, y, and R4 are as defined above.

[0023] In some embodiments, R5 are each independently selected from trifluoromethyl, in Preferably Preferably

[0024] In some embodiments, R is selected from the following groups:

[0025] In the present invention, The carbon can be in R configuration or S configuration, preferably in R configuration;

[0026] In the present invention, the carbon atom to which the cyclic amine and the carboxyl group are connected may be in R configuration or S configuration, preferably in S configuration.

[0027] Specifically, the present invention also provides a compound of formula II or formula III or a pharmaceutically acceptable salt thereof:

[0028] in,

[0029] R, R2, R3, R4, x, and y are as defined above;

[0030] R6 are each independently selected from C1-6 alkyl; preferably methyl;

[0031] In the present invention, The carbon can be in R configuration or S configuration, preferably in R configuration;

[0032] In the present invention, the carbon atom to which the cyclic amine and the carboxyl group are connected may be in R configuration or S configuration, preferably in S configuration.

[0033] Specifically, the present invention also provides a compound of formula IV or a pharmaceutically acceptable salt thereof:

[0034] in,

[0035] Y is a trivalent group selected from

[0036] R, R3, R4, x, and y are as defined above;

[0037] In some embodiments, the compound of formula IV or a pharmaceutically acceptable salt thereof may be a compound of formula IV-1, formula IV-2, formula IV-3 or formula IV-4 or a pharmaceutically acceptable salt thereof:

[0038] R, R3, R4, x, and y are as defined above;

[0039] In the present invention, The carbon can be in R configuration or S configuration, preferably in R configuration;

[0040] In the present invention, the carbon atom to which the cyclic amine and the carboxyl group are connected may be in R configuration or S configuration, preferably in S configuration.

[0041] In some embodiments, the compound of the present invention is selected from:

[0042] The present invention also relates to a pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof.

[0043] In some embodiments, the pharmaceutical composition optionally comprises a pharmaceutically acceptable excipient.

[0044] The present invention also relates to a method for treating cardiovascular diseases, which comprises administering an effective amount of the compound of the present invention or a pharmaceutically acceptable salt thereof to a patient.

[0045] The present invention also relates to a method of treating a patient in need of treatment for elevated plasma levels of Lp(a), comprising administering to the patient an effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof.

[0046] The present invention also relates to a method for inhibiting the assembly of LDL particles and apo(a), comprising combining the compound of the present invention or a pharmaceutically acceptable salt thereof with apo(a).

[0047] The present invention also relates to the use of the compound of the present invention or its pharmaceutically acceptable salt or the above composition in preparing apo(a) binders.

[0048] In some embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof or the above composition is used to prepare an inhibitor for inhibiting the assembly of LDL particles and apo(a).

[0049] In some specific embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof or the above-mentioned composition is used to prepare an agent for reducing Lp(a) levels.

[0050] In some specific embodiments, the compound of the present invention or a pharmaceutically acceptable salt thereof or the above composition is used to prepare a drug for treating cardiovascular disease.

[0051] Detailed Description of the Invention

[0052] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described, as these may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[0053] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety. To the extent that definitions in this section are contrary to or inconsistent with definitions listed in patents, applications, and other publications incorporated by reference herein, the definitions in this section shall prevail over the definitions incorporated by reference herein.

[0054] In the present invention, "alkyl" refers to a saturated aliphatic hydrocarbon group, which may be a C1-20 alkyl group, preferably a C1-8 alkyl group, more preferably a C1-6 alkyl group, and most preferably a C1-3 alkyl group. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof.

[0055] In the present invention, "haloalkyl" refers to an alkyl group having one or more (preferably 1, 2, 3, 4, 5 or 6) halogen substituents, wherein alkyl is as defined above, and can be a C1-20 haloalkyl group, preferably a C1-8 haloalkyl group, more preferably a C1-6 haloalkyl group, and most preferably a C1-3 haloalkyl group. Non-limiting examples include trifluoromethyl, monofluoromethyl, difluoromethyl, trichloromethyl, pentafluoroethyl, etc.;

[0056] As used herein, "alkoxy" refers to an alkyl-O- group, wherein the alkyl group is as defined above;

[0057] In the present invention, "haloalkoxy" refers to haloalkyl-O-, wherein haloalkyl is as defined above;

[0058] In the present invention, "alkylamino" refers to alkyl-NH-, wherein alkyl is as defined above;

[0059] In the present invention, "alkylcarbonylamino" refers to alkyl-C(O)-NH-, wherein alkyl is as defined above;

[0060] In the present invention, "halogen" refers to fluorine, chlorine, bromine or iodine;

[0061] As used herein, "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, wherein the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, and more preferably 3 to 6 carbon atoms. Non-limiting examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like.

[0062] In the present invention, the alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, alkylcarbonylamino, cycloalkyl and the like may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available connection point. The substituent is preferably one or more of the following groups independently selected from alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylthio, alkylamino, alkylcarbonylamino, halogen, thiol, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate.

[0063] As used herein, "aryl" refers to an optionally substituted monocyclic, biaryl, or fused bicyclic or polycyclic ring system having the well-known characteristics of aromaticity, wherein at least one ring contains a completely conjugated π-electron system. Typically, an aryl group contains 6 to 20 carbon atoms ("C6-C20 aryl") as ring members, preferably 6 to 14 carbon atoms ("C6-C14 aryl") or more preferably 6 to 12 carbon atoms ("C6-C12 aryl"). Fused aryl groups can include an aryl ring fused to another aryl ring or to a saturated or partially unsaturated carbocyclic or heterocyclic ring. The point of attachment to the base molecule on such a fused aryl ring system can be a C atom of the aromatic portion of the ring system or a C or N atom of the non-aromatic portion. Examples of aryl groups include, but are not limited to, phenyl, biphenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, benzo[d][1,3]dioxole, and tetrahydronaphthyl.

[0064] In the present invention, " heteroaryl " refers to monocyclic, heterobiaryl or fused bicyclic or polycyclic ring system with well-known aromaticity feature, and it contains the ring atoms of specified number and comprises at least one heteroatom selected from N, O and S as the ring members in aromatic ring.Heteroatomic inclusion allows the aromaticity of 5-ring and 6-ring.Usually, heteroaryl contains 5-20 ring atoms (" 5-20 yuan heteroaryl "), preferably 5-14 ring atoms (" 5-14 yuan heteroaryl "), and more preferably 5-12 ring atoms (" 5-12 yuan heteroaryl ").Heteroaryl ring is connected to base molecule by the ring atoms of heteroaryl ring, thus keeps aromaticity.Therefore, 6 yuan heteroaryl rings can be connected to base molecule by ring C atom, and 5 yuan heteroaryl rings can be connected to base molecule by ring C or N atom. Examples of unsubstituted heteroaryl groups often include, but are not limited to, pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole, oxazole, isothiazole, thiazole, triazole, oxadiazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, benzofuran, benzothiophene, indole, benzimidazole, indazole, quinoline, isoquinoline, purine, triazine, naphthyridine, and carbazole.

[0065] In the present invention, the aryl or heteroaryl group may be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, alkylthio, alkylamino, alkylcarbonylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0066] As used herein, "elevated Lp(a) plasma levels" refers to plasma levels equal to or greater than about 50 mg / dL.

[0067] In the present invention, any isotope-labeled derivatives of the compounds of the present invention or their pharmaceutically acceptable salts are covered by the present disclosure. Atoms that can be isotopically labeled include but are not limited to hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, iodine, etc. They can be isotopically labeled. 2 H(D), 3 H. 11 C. 13 C. 14 C. 15 N. 18 F. 31 P. 32 P. 35 S. 36 Cl and 125Unless otherwise indicated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium at least 3000 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 45% deuterium incorporation).

[0068] In the present invention, "plurality" means two or more, for example, 2, 3, 4, 5 or 6.

[0069] As used herein, "optionally" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0070] In the present invention, "each independently" or "independently" means that substituents with the same selection range can be the same or different groups at each occurrence, and the group selection of the substituent at each occurrence is not affected by the selection of the substituent (or substituents with the same selection range) at other positions.

[0071] In the present invention, "pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity.

[0072] In this invention, the following abbreviations / terms are used:

[0073] Apo(a): Apolipoprotein(a)

[0074] Lp(a): lipoprotein(a)

[0075] TEA: triethylamine

[0076] THF: Tetrahydrofuran

[0077] LiHMDS: lithium hexamethyldisilazane salt

[0078] PE: Petroleum ether

[0079] EtOAc: ethyl acetate

[0080] MTBE: Methyl tert-butyl ether

[0081] 2-MeTHF: 2-methyltetrahydrofuran

[0082] DMAP: 4-dimethylaminopyridine

[0083] Boc2O: di-tert-butyl carbonate

[0084] HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate

[0085] DIEA: N,N-diisopropylethylamine

[0086] NMP: N-methylpyrrolidone

[0087] SPR: surface plasmon resonance

[0088] EDC: 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0089] NHS: N-hydroxysuccinimide

[0090] NaAc: sodium acetate

[0091] RU: Response Unit

[0092] BLI: Biolayer Interferometry

[0093] PBS: Phosphate buffered saline

[0094] BSA: bovine serum albumin

[0095] HEC: Hydroxyethylcellulose

[0096] EDTAK2: Ethylenediaminetetraacetic acid dipotassium Example

[0097] Example 1

[0098] first step:

[0099] Compound 1a (53.8 g, 249.94 mmol) was dissolved in THF (300 mL). TEA (63.23 g, 624.855 mmol) was added under ice-cooling conditions. The mixture was stirred under ice-cooling conditions for 5 minutes, followed by the addition of pivaloyl chloride (37.67 g, 312.43 mmol) and the mixture was stirred under ice-cooling conditions for 15 minutes. LiCl (13.24 g, 312.427 mmol) and (4S)-4-benzyl-1,3-oxazol-2-one (44.29 g, 249.94 mmol) were previously dissolved in THF (400 mL) and stirred until dissolved. The mixture was added to the reaction mixture containing compound 1a and stirred at room temperature for 24 hours. A white solid formed in the reaction mixture. 1M HCl was added until the solid disappeared. The organic layer was separated and washed once with 350 mL of 1M NaOH and once with saturated brine. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to yield the product as a yellow oil. 600 mL of MeOH:H2O (1:2) was added and the mixture was slurried overnight. The filter cake was filtered and dried to obtain a white solid 1b (68 g, yield 72.66%).

[0100] MS m / z(ESI):319.2[M-56] + .

[0101] Step 2:

[0102] Compound 1b (11 g, 29.38 mmol) was dissolved in THF (100 mL). LiHMDS (1 M in hexane, 41.13 mL) was added at -10°C. After incubation for 15 min, a THF solution (25 mL) of m-bromobenzyl bromide (8.08 g, 32.32 mmol) was added. The temperature was slowly raised to room temperature and the reaction was allowed to proceed overnight. The mixture was quenched with 20 mL of saturated ammonium chloride solution, water was added, and the mixture was extracted twice with EtOAc. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse-phase column chromatography (acetonitrile / water = 30%-100%) to afford product 1c (8 g, 50.11% yield).

[0103] MS m / z(ESI):487.1[M-56] + .

[0104] Step 3:

[0105] Compound 1c (8000 mg, 14.72 mmol) was dissolved in THF (100 mL). H₂O₂ (30% aqueous solution, 25.09 mL, 248 mmol) and an aqueous solution of lithium hydroxide monohydrate (926.51 mg, 22.08 mmol) (20 mL) were added and the mixture was allowed to react at room temperature for 2.5 h. A solution of sodium bisulfite (2.09 g, 29.74 mmol) in aqueous solution (100 mL) was added. The pH was adjusted to 10 with 2N NaOH aqueous solution, and the mixture was washed twice with TMBE. The aqueous phase was adjusted to pH 2-3 with 6M HCl and extracted three times with TMBE. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase purification (acetonitrile / water = 20%-100%) to afford product 1d (5650 mg, 99.88% yield). MS m / z (ESI): 384.1 [MH] - .

[0106] Step 4:

[0107] Compound 1d (5650 mg, 14.70 mmol) was dissolved in TMBE (100 mL), and NH3 / MeOH (7 M) (6 mL) was added. The mixture was stirred at room temperature overnight and concentrated to give 5900 mg of a white solid. This solid was dissolved in 2-MeTHF (100 mL), and O-tert-butyl-N,N'-diisopropylisourea (11781.18 mg, 58.81 mmol) was added. The mixture was reacted at 65°C overnight. Water was added, the mixture was extracted with EtOAc, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EtOAc / PE = 0-20%) to give the product 1e (5070 mg, 78.31% yield) as a colorless oil.

[0108] MS m / z(ESI):328.1[M-112] + .

[0109] Step 5:

[0110] Compound 1e (5070 mg, 11.51 mmol) was dissolved in THF (50 mL), and Pd(dppf)Cl2·CH2Cl2 (940.18 mg, 1.15 mmol), Cs2CO3 (11253.29 mg, 34.54 mmol), and potassium ethylene trifluoroborate (97% purity, 4626.41 mg, 33.50 mmol) were added. After nitrogen protection, the tube was sealed and reacted at 80°C overnight. Water was added, extracted with EtOAc, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EtOAc / PE = 0-20%) to give a colorless oily product 1f (3960 mg, yield 88.76%).

[0111] MS m / z(ESI):276.1[M-112] + .

[0112] Step 6:

[0113] Compound 1g (3960 mg, 10.22 mmol) was dissolved in THF (60 mL), and a solution of sodium periodate (4360.37 mg, 20.44 mmol) in water (30 mL) was added. OsO4 (4% in water, 4 mL) was added, and the mixture was allowed to react at room temperature overnight. The solid was removed by filtration, and water was added. The mixture was extracted with EtOAc. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EtOAc / PE = 0-30%) to afford the product 1g (1140 mg, 28.64% yield) as a colorless oil.

[0114] MS m / z(ESI):234.1[M-156] + .

[0115] Step 7:

[0116] Compound 1g (145 mg, 0.37 mmol) and 3-aminomethylpyridine (20 mg, 0.185 mmol) were dissolved in 5 mL of MeOH. Acetic acid (11.11 mg, 0.185 mmol) and 4A molecular sieves (20 mg) were added and allowed to react at room temperature for 3 h. Sodium cyanoborohydride (34.87 mg, 0.555 mmol) was added and allowed to react at room temperature overnight. The mixture was filtered, and the pH was adjusted to 8-9 by adding 2N aqueous NaOH. The solvent was then removed by spun off, and the mixture was separated and purified by preparative high-performance liquid chromatography (methanol / H₂O) to obtain Compound 1h (19 mg, 12% yield) as a colorless oil.

[0117] MS m / z(ESI):855.5[M+H] + .

[0118] Step 8:

[0119] Compound 1h (19.0 mg, 0.022 mmol) was dissolved in HCl / EtOAc (4 M, 2 mL) and allowed to react at room temperature overnight. The reaction system was cooled using a dry ice-ethanol bath. Purified water (5 mL) was slowly added to the reaction system and the temperature was continued to cool until the system was frozen. The reaction was then lyophilized to obtain a white solid, the hydrochloride salt of compound 1 (11.2 mg, yield: 78.1%).

[0120] MS m / z(ESI):543.3[M+H] + .

[0121] Example 2

[0122] first step:

[0123] Compound 1g (100 mg, 0.26 mmol) was dissolved in methanol (5 mL), and 3-aminomethylquinoline (20.33 mg, 0.13 mmol) was added. Five 4A molecular sieves were added, and the mixture was stirred at room temperature for 1 h. Glacial acetic acid (46.25 mg, 0.77 mmol) and sodium cyanoborohydride (48.4 mg, 0.77 mmol) were then added. The mixture was allowed to react at room temperature overnight. The reaction mixture was filtered and purified by reverse phase preparative (methanol / water = 80%-100%) to afford 2a (70 mg, 60% yield) as a white solid.

[0124] Step 2:

[0125] Compound 2a (70 mg, 0.077 mmol) was dissolved in HCl / EtOAc (4 M, 5 mL) and reacted overnight at 40° C. 10 mL of water was added, and the mixture was washed twice with EtOAc. The aqueous phase was lyophilized to obtain the hydrochloride salt of compound 2 (53.29 mg, 98.15%).

[0126] MS m / z(ESI):593.4[M+H] + .

[0127] Example 3

[0128] first step:

[0129] Compound 1g (100 mg, 0.26 mmol) and p-trifluoromethylbenzylamine (22.48 mg, 0.13 mmol) were dissolved in 5 mL of MeOH. Acetic acid (7.71 mg, 0.13 mmol) and 4A molecular sieves (20 mg) were added and allowed to react at room temperature for 3 h. Sodium cyanoborohydride (24.2 mg, 0.385 mmol) was added and allowed to react at room temperature overnight. The mixture was filtered, and the pH was adjusted to 8-9 by adding 2N aqueous NaOH. The solvent was then removed by spun off, and the product was separated and purified by preparative high-performance liquid chromatography (methanol / H₂O) to afford compound 3a (35 mg, yield: 29.5%) as a colorless oil.

[0130] Step 2:

[0131] Compound 3a (35 mg, 0.038 mmol) was dissolved in HCl / EtOAc (4 M, 2 mL) and allowed to react at room temperature overnight. The reaction system was cooled using a dry ice-ethanol bath. Purified water (5 mL) was slowly added to the reaction system and the temperature was continued to cool until the system was frozen. The reaction was then lyophilized directly. The hydrochloride salt of compound 3 was obtained by repeated lyophilization three times to obtain a white solid (27 mg, yield: 98.9%).

[0132] MS m / z(ESI):610.3[M+H] + .

[0133] Example 4

[0134] first step:

[0135] Compound 4a (22.81 g, 105.00 mmol) was dissolved in anhydrous tetrahydrofuran (240 mL), and ice-bathed under nitrogen protection. Triethylamine (25.30 g, 250.00 mmol) was added, and pivaloyl chloride (15.07 g, 125.00 mmol) was slowly added dropwise. After stirring at 10°C for 30 minutes, anhydrous lithium chloride (5.30 g, 125 mmol) and a solution of 4r (17.72 g, 100.00 mmol) in anhydrous tetrahydrofuran (240 mL) were added. The mixture was allowed to return to room temperature and stirred overnight. 1 M HCl aqueous solution (300 mL) was added under water bath, and the mixture was stirred for 1 minute and allowed to stand for stratification. The upper organic phase was separated and washed with 1 M The product was washed with aqueous NaOH (300 mL), then saturated brine (300 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and passed through silica gel (A / EtOAc = 100 / 0 to 80 / 20, A:PE / DCM = 1 / 1) to afford a colorless oil. The product was dissolved in anhydrous tetrahydrofuran (50 mL) and concentrated to afford 4b (26.00 g, 65.7% yield).

[0136] Step 2:

[0137] Compound 4b (26.00 g, 69.07 mmol) was dissolved in DMF (200 mL). Triethylamine (20.97 g, 207.20 mmol) and DMAP (0.84 g, 6.91 mmol) were added under a water bath. Boc2O (22.61 g, 103.60 mmol) was slowly added. The mixture was sealed with a nitrogen balloon. After stirring at room temperature overnight, the reaction was monitored by a plate to produce half of the product and half of the starting material. Additional triethylamine (13.97 g, 138.13 mmol) and Boc2O (15.07 g, 40.03 mmol) were added, and stirring was continued overnight. The mixture was diluted with ethyl acetate (600 mL), washed with water (200 mL × 3), concentrated, and passed through a silica gel column (PE / EtOAc = 100 / 0 to 84 / 16) to give a light yellow oily liquid 4c (24.00 g, yield 72.9%). The product was dissolved in anhydrous THF and concentrated, and the reaction was repeated twice for the next reaction.

[0138] Step 3:

[0139] Under nitrogen protection, compound 4c (9.70 g, 20.354 mmol) was added to anhydrous THF (80 mL), cooled to about -70 ° C, and LiHMDS / THF solution (1 M, 24.43 mL) was added dropwise. The mixture was stirred for 1 hour, and a solution of 4q (6.10 g, 24.43 mmol) in anhydrous THF (40 mL) was added dropwise. The mixture was returned to 0 ° C and stirred for 3 hours. After the reaction was completed by plate monitoring, saturated aqueous ammonium chloride solution (200 mL) was added at 0 ° C to quench the reaction. The organic phase was separated, washed with saturated brine (200 mL), dried over anhydrous sodium sulfate, filtered, concentrated, and slurried with petroleum ether to give a white solid (8.60 g). The mother liquor was passed through a silica gel column to give 2.00 g of a white solid. The two batches of product compound 4d were combined, totaling (10.60 g, yield 80%).

[0140] Step 4:

[0141] Compound 4d (10.20 g, 15.80 mmol) was dissolved in THF (60 mL). Hydrogen peroxide (2.687 mL, 23.70 mmol), LiOH (0.99 g, 23.70 mmol), and aqueous solution (24 mL) were added under ice-cooling. The mixture was stirred in an ice-cooling bath for 3 hours, and the reaction was monitored for completion by a plate. Saturated aqueous NaHSO₃ solution (5 mL) was added dropwise under ice-cooling. The mixture was returned to room temperature and stirred for 5 minutes. The pH was adjusted to 2-3 with 1 M HCl, and the mixture was extracted with ethyl acetate (100 mL × 2). The mixture was washed with saturated aqueous sodium chloride solution, concentrated, and reversed (water / acetonitrile, 62% peak). The mixture was concentrated and extracted with ethyl acetate (100 mL × 2). The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated to give 4e (6.00 g, 78% yield) as a colorless oil.

[0142] Step 5:

[0143] Compound 4e (6.00 g, 12.34 mmol) was dissolved in 2-methyltetrahydrofuran (50 mL), and triethylamine (2.50 g, 24.67 mmol) was added. After stirring at room temperature for 5 minutes, O-tert-butyl-N,N'-diisopropylisourea (8.65 g, 43.17 mmol) was added. The mixture was stirred at 65°C under a nitrogen balloon overnight, cooled to room temperature, filtered, and the filtrate was concentrated and passed through a silica gel column (PE / EtOAc = 10 / 1) to give a colorless oily liquid 4f (5.80 g, yield 86.67%).

[0144] Step 6:

[0145] Compound 4f (5.80 g, 10.69 mmol) was dissolved in a mixture of dioxane and water (30 / 10 mL). Potassium vinyl trifluoroborate (4.30 g, 32.07 mmol), CS2CO3 (10.45 g, 32.07 mmol), and Pd(dppf)Cl2·CH2Cl2 (0.44 g, 0.535 mmol) were added under a water bath. The system was purged with argon three times and stirred at 90°C overnight under argon. After cooling, the solution was concentrated to remove most of the dioxane. The solution was diluted with ethyl acetate (100 mL), washed with water (50 mL), concentrated, and passed through a silica gel column (PE / EtOAc = 5 / 1) to afford 4 g (4.80 g, 91.67% yield) of a colorless oil.

[0146] Step 7:

[0147] Compound 4g (4.80 g, 9.80 mmol) was dissolved in a mixture of THF / water (60 / 30 mL). An aqueous solution of OsO4 (0.98 mmol, 6.24 mL) was added under water bath, and the mixture was stirred for 15 minutes. Then, sodium periodate (6.29 g, 29.41 mmol) was added under water bath, and the mixture was stirred at 35°C overnight. The reaction was monitored for completion by TLC. A saturated aqueous solution of sodium thiosulfate (50 mL) was added under water bath, and the mixture was stirred for 5 minutes. The mixture was concentrated to remove most of the THF, and the mixture was diluted with ethyl acetate (150 mL). The mixture was washed with water (50 mL x 2), concentrated, and passed through a silica gel column (PE / EtOAc = 100 / 0 to 86 / 14) to afford 4h (3.6 g, 75% yield) as a colorless oily liquid.

[0148] Step 8:

[0149] Compound 4h (100 mg, 0.20 mmol) was dissolved in methanol (10 mL), and p-trifluoromethylbenzylamine (35.63 mg, 0.20 mmol) was added. 4A molecular sieves (20 mg, approximately 5 particles) were added and stirred for 2 h. Acetic acid (20 uL) was added, followed by sodium cyanoborohydride (63.91 mg, 1.02 mmol). The mixture was sealed and stirred at 70°C overnight. The solvent was removed by swirl, and H₂O (5 mL) was added. The pH was adjusted to 8-9 with 2N NaOH aqueous solution. The organic phase was extracted with ethyl acetate (5 mL x 3). The combined organic phases were dried by swirl, and then separated and purified by reverse phase preparative (methanol / H₂O) to afford Compound 4i (40 mg, yield: 30.22%) and Compound 4j (50 mg, yield: 21.82%) as white solids.

[0150] Step 9:

[0151] Compound 4j (38 mg) was added to a reaction flask, followed by EtOAc (2 mL) and 4N HCl / EtOAc (2 mL). The mixture was stirred for 2 h, and the reaction system was cooled in a dry ice-ethanol bath. Purified water (5 mL) was slowly added until the mixture was frozen. EtOAc (3 mL) was added for extraction, and the mixture was freeze-dried in a freeze dryer to obtain the hydrochloride salt of compound 4 (24.2 mg, yield: 97.89%) as a white flocculent solid.

[0152] MS m / z(ESI):614.3[M+H] + .

[0153] Example 5

[0154] first step:

[0155] Reactant 5a (5 g, 12.39 mmol) was added to tert-butyl alcohol (50 mL), water (50 mL), and NaClO₂ (11.21 g, 123.91 mmol) and stirred until dissolved. NaH₂PO₄ (8.92 g, 74.35 mmol) and 2-methyl-2-butene (10 mL) were then added and stirred overnight at 25°C. The solvent was evaporated, and EtOAc (80 mL) was added. The mixture was separated and the aqueous phase was extracted once with EtOAc (40 mL). The combined organic phases were washed with saturated NaCl solution (40 mL), separated, and dried. The filtrate was filtered and dried, and silica gel was added to the sample. The sample was purified by column chromatography (EtOAc / PE 0-50%) and dried to afford compound 5b (4.3 g, 82.72% yield).

[0156] MS m / z(ESI):320.3[M-100+H] + .

[0157] Step 2:

[0158] Reactant 5b (700 mg, 1.67 mmol) was dissolved in DMF (5 mL), and HATU (1008 mg, 2.65 mmol) and DIEA (685 mg, 5.3 mmol) were added. After stirring at room temperature for 15 min, a DMF solution of B31 (1400 mg, 1.77 mmol) (5 mL) was added and the mixture was reacted at 30°C overnight. Water was added, the mixture was extracted with EtOAc, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by reverse phase preparative purification (methanol / water) to afford 5c (1.5 g, 75.32% yield) as a solid.

[0159] MS m / z(ESI):497.4[M / 2-100] + .

[0160] Step 3:

[0161] Compound 5c (770 mg, 0.645 mmol) was dissolved in 4M HCl / EtOAc and reacted at 40°C overnight. Water was added and the mixture was washed twice with EtOAc. The aqueous phase was lyophilized to obtain the hydrochloride salt of compound 5 (510 mg, yield 94.76%).

[0162] MS m / z(ESI):726.5[M+H] + .

[0163] Example 6

[0164] first step:

[0165] Compound 5a (2000 mg, 4.96 mmol) was dissolved in methanol (100 mL), and NaBH4 (335.1 mg, 9.91 mmol) was added at -20°C. The mixture was allowed to react overnight at room temperature. After concentration, saturated aqueous sodium bicarbonate solution was added, and the mixture was extracted with EtOAc. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain product 6a (2000 mg, 99.5% yield) as a colorless oil.

[0166] MS m / z(ESI):306.2[M+H-100] + .

[0167] Step 2:

[0168] Compound 6a (1800 mg, 4.44 mmol) was dissolved in THF (100 mL), and PPh3 (2310 mg, 8.81 mmol) and NBS (900 mg, 5.06 mmol) were added at -20°C and reacted for 3 h. After filtration, the filtrate was added with 10 mL of water and extracted three times with EtOAc. The organic phases were combined, washed with saturated brine, filtered, concentrated, and purified by column chromatography (EtOAc / PE = 0-20%) to obtain the product 6b (1300 mg, 62.53% yield) as a colorless oil.

[0169] MS m / z(ESI):356.1[M-112] + .

[0170] Step 3:

[0171] Compound 6b (620.27 mg, 1.32 mmol) and 1,3,5-cyclohexanetriol (50 mg, 0.38 mmol) were dissolved in 5 mL of N-methylpyrrolidone. Sodium hydride (90.80 mg, 3.78 mmol) was added, and the atmosphere was purged with nitrogen three times. The reaction was allowed to react at room temperature for 16 h. The mixture was washed with H₂O (2 mL x 3) and the organic phase was extracted with ethyl acetate (3 mL x 3). The combined organic phases were spin-dried and purified using reverse phase preparative (methanol / H₂O) to afford compound 6c (90 mg, 18.3% yield) as a pale yellow solid.

[0172] MS m / z(ESI):520.0[(M-256) / 2+H] + .

[0173] Step 4:

[0174] Compound 6c (50 mg, 0.03 mmol) was dissolved in HCl / EtOAc (4 M, 3 mL) and allowed to react at room temperature for 16 h. Water (3 mL) was added to the reaction mixture, and the layers were separated. The aqueous phase was washed with ethyl acetate (3 mL x 3) and lyophilized to obtain the hydrochloride salt of compound 6 (19.75 mg, yield: 54.68%) as a pale yellow solid.

[0175] MS m / z(ESI):827.1[M+H] + .

[0176] Example 7

[0177] first step:

[0178] Compound 7a (3.00 g, 6.60 mmol) was dissolved in dioxane (26 mL), and pinacol boronate (2.51 g, 9.90 mmol), potassium acetate (1.29 g, 13.20 mmol), and Pd(dppf)Cl2·CH2Cl2 (0.27 g, 0.33 mmol) were added. The atmosphere was replaced with argon three times and stirred at 90°C overnight under argon protection. TLC monitoring indicated the disappearance of the starting material and the formation of a new spot. After cooling, the mixture was concentrated, diluted with ethyl acetate (50 mL), washed with water (50 mL), and concentrated to give 7b (3.31 g, 100% yield), a red liquid.

[0179] Step 2:

[0180] Compound 7b (3.31 g, 6.60 mmol) was dissolved in THF (50 mL) and a solution of NaOH (0.79 g, 19.80 mmol) in water (10 mL) was added. H2O2 (7.50 mL, 66.00 mmol) was slowly added dropwise under ice-bath conditions. After the addition was complete, the mixture was stirred at 25°C overnight. Saturated aqueous sodium thiosulfate solution (20 mL) was added under water bath to quench the hydrogen peroxide. The pH was adjusted to 3 with 1 M hydrochloric acid. The mixture was concentrated and most of the THF was removed. The mixture was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with water (50 mL), concentrated, and passed through a silica gel column (PE / EtOAc = 1 / 0 to 3 / 1) to obtain 7c (2.08 g, 80% yield) as a white solid.

[0181] Step 3:

[0182] Compound 7c (400 mg, 1.02 mmol) was dissolved in anhydrous THF (10 mL). 4A molecular sieves (50 mg) were added under an argon balloon. The mixture was stirred at room temperature for 10 minutes, then ice-cooled. Sodium hydride (40.88 mg, 1.02 mmol) was added. The mixture was returned to room temperature and stirred for 20 minutes. 7r (47.10 mg, 0.255 mmol) was added under ice-cooling, and the mixture was stirred at 40°C overnight. The formation of new spots was monitored by TLC. Saturated aqueous ammonium chloride (20 mL) was added under ice-cooling, and the mixture was extracted with ethyl acetate (20 mL x 2). The organic phases were combined, washed with water (50 mL x 1), concentrated, and passed through a silica gel column (PE / EtOAc = 3 / 1) to afford 7d (200.00 mg, 15.66% yield) as a white solid.

[0183] Step 4:

[0184] Compound 7d (40.00 mg, 0.03 mmol) was added to ethyl acetate hydrochloride (5 mL) in a single-necked flask and stirred at room temperature overnight. Deionized water (10 mL) was added at -20°C, the aqueous phase was separated, and the aqueous phase was back-extracted with ethyl acetate (10 mL×3). The aqueous phase was lyophilized, deionized water (10 mL) was added to dissolve the clear phase, and lyophilized again to obtain the hydrochloride salt of yellow solid compound 7 (27.03 mg, yield 94.88%).

[0185] Example 8

[0186] first step:

[0187] Compound 5b (94.67 mg, 0.23 mmol) was dissolved in DMF (5 mL), and HATU (104 mg, 0.27 mmol) and DIEA (53.03 mg, 0.41 mmol) were added. The mixture was allowed to react at room temperature for 15 min, followed by the addition of 8r (10 mg, 0.068 mmol). The reaction was allowed to react at room temperature overnight. Water was added, the mixture was extracted with EtOAc, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated. Preparation (methanol / water) afforded product 8a (87.75 mg, 95%).

[0188] MS m / z(ESI):474.4[M / 2-200] + .

[0189] Step 2:

[0190] Compound 8a (87.75 mg, 0.065 mmol) was dissolved in 4M HCl / EtOAc (5 mL) and reacted at 40°C overnight. 10 mL of water was added, and the mixture was washed twice with EtOAc. The aqueous phase was lyophilized to obtain the hydrochloride salt of product 8 (58.48 mg, yield 87.57%).

[0191] MS m / z(ESI):883.5[M+H] + .

[0192] Example 9

[0193] first step:

[0194] Reactant 7a (1.2 g, 2.64 mmol) was added to a reaction flask, NMP (2 mL) was added, and the reaction was stirred. Ammonia (5 mL) was added and the reaction was stirred. Cuprous oxide (0.38 g, 2.65 mmol) was added and water (2 mL) was added and the reaction was continued with stirring. The temperature was raised to 80°C and the reaction was stirred overnight. Water (20 mL) was added and stirred, EtOAc (20 mL) was added for extraction, the liquids were separated, the organic phase was dried by spin drying, and silica gel was added to mix the sample. The product was purified by column chromatography using PE / EtOAc (80:20) to obtain the product, which was dried to give solid compound 9a (726 mg, 70.4% yield).

[0195] Step 2:

[0196] Compound 6b (200 mg, 0.51 mmol) was dissolved in DMF (7 mL), and K2CO3 (212.35 mg, 1.54 mmol) and compound 9a (263.89 mg, 0.56 mmol) were added. The mixture was reacted at 60°C overnight. 9b (135 mg, 22.62% yield) was obtained by separation and purification using preparative HPLC (MeOH / H2O).

[0197] Step 3:

[0198] Compound 9b (120 mg, 0.10 mmol) was dissolved in 4M HCl / EtOAc (7 mL) and reacted at 40°C for 4 h. Water was added and the mixture was washed twice with EtOAc. The aqueous phase was lyophilized to obtain the hydrochloride salt of product 9 (64 mg, yield 73.77%).

[0199] MS m / z(ESI):698.4[M+H] + .

[0200] Example 10

[0201] first step:

[0202] 1,3,5-Tris(bromomethyl)benzene (37 mg, 0.10 mmol) and compound 10a (162 mg, 0.41 mmol) were dissolved in DMF (3 mL), and potassium carbonate (201 mg, 0.61 mmol) was added. The mixture was stirred at 60°C overnight and filtered. The filtrate was purified by reverse phase preparative chromatography (MeOH / H2O) to give 10b (70 mg, 52% yield) as a white solid.

[0203] Step 2:

[0204] Compound 10b (70 mg, 0.054 mmol) was dissolved in ethyl acetate (1 mL), and triethylsilane (0.1 mL) and HCl / EtOAc (4 mol / L, 3 mL) were added. The mixture was stirred at room temperature for 2 hours and concentrated. The resulting solid was rinsed three times with ethyl acetate, dissolved in 10 mL of pure water, and lyophilized to obtain the hydrochloride salt of compound 10 (36.18 mg, yield 72%) as a white solid.

[0205] Example 11

[0206] first step:

[0207] Compound 11a (60 mg, 0.154 mmol) was dissolved in methanol (5 mL), and 11b (110 mg, 0.139 mmol) and AcOH (37 mg, 0.616 mmol) were added. Five 4A molecular sieves were added to remove water. After reacting at room temperature for 1 h, AcOH (37 mg, 0.616 mmol) and NaBH3CN (21 mg, 0.621 mmol) were added and reacted at 70°C overnight. After filtering off the solids, the mixture was concentrated. Purification by preparative HPLC (methanol / water) afforded the product 11c (70 mg, 39% yield) as a colorless oil.

[0208] MS m / z(ESI):533.5[M / 2-50] + .

[0209] Step 2:

[0210] Compound 11c (70 mg, 0.26 mmol) was dissolved in 4M HCl / EtOAc (5 mL) and reacted at room temperature overnight. A solid was produced, which was washed twice with water and EtOAc. The aqueous phase was lyophilized twice to obtain the hydrochloride salt of product 11 (48 mg, yield 95%).

[0211] MS m / z(ESI):697.2[M+H] + .

[0212] Comparative Example 1

[0213] Comparative compound 1 can be synthesized with reference to patent WO2020 / 247429A1.

[0214] Biological test evaluation

[0215] Test Example 1: Using SPR to detect the affinity of the compound of the present invention to Apo(a)

[0216] 1. Experimental purpose: The purpose of this test is to test the affinity of the compound of the present invention to Apo(a)

[0217] 2. Experimental instruments and reagents:

[0218] Biomolecular interaction analyzer (Biacore 8K),

[0219] Apolipoprotein (a) was synthesized by Hangzhou Haoyang Biotechnology Co., Ltd.

[0220] NHS was purchased from Cytiva.

[0221] 3. Experimental methods:

[0222] Apolipoprotein(a) was covalently linked to a Series S Sensor Chip CM5 in a Biacore 8K instrument. Prior to injection, the activator was prepared by mixing 400 mM EDC and 100 mM NHS. The CM5 sensor chip was activated with the activator for 420 s at a flow rate of 10 μL / min. 60 μg / mL apolipoprotein(a) dissolved in 10 mM NaAc (pH 4.5) was injected into the Fc2 sample channel at a flow rate of 10 μL / min to achieve an immobilization level of approximately 10,000 RU. The Fc1 reference channel was inactivated for 420 s using 1 M ethanolamine hydrochloride-sodium hydroxide at a flow rate of 10 μL / min. The Fc1 reference channel was blocked using the same procedure as Fc2, but without the protein injection step. The compound was diluted to 100 nM in running buffer (1× PBS, pH 7.4, containing 0.005% Tween-20) and injected into channel Fc1-Fc2 at a flow rate of 30 μL / min. Binding was allowed to proceed for 90 s and dissociation for 210 s. Both binding and dissociation were performed in running buffer. The chip was regenerated by injecting 3 M magnesium chloride in running buffer for 30 s at a flow rate of 20 μL / min. The affinity constant, KD, reflects the binding strength of the interaction. When the compound concentration is KD, the equilibrium signal, Req, is half the Rmax. The results demonstrate that the compounds of this invention have strong binding affinity to human Apo(a) protein.

[0223] Test Example 2: BLI detection of affinity between the compound of the present invention and Apo(a)

[0224] 1. Experimental purpose: The purpose of this test is to test the affinity of the compound of the present invention to Apo(a)

[0225] 2. Experimental instruments and reagents:

[0226] Molecular interaction analyzer (ForteBio Octet red 96e),

[0227] SA sensor (ForteBio),

[0228] 96-well plates were purchased from Greine.

[0229] 3. Experimental methods:

[0230] Prepare two 96-well plates: one for the sample plate and one for the pre-wet plate. Add 200 μL of curing buffer per well to the pre-wet plate to pre-wet the SA sensor for at least ten minutes. Add all reagents and samples to the other black sample plate. After sample addition, place the sensor plate and sample plate into the ForteBio Octet Red 96e instrument. Set the assay program to run in sequence. Set the experimental temperature to 30°C and the acquisition frequency to 5.0 Hz. Run the biosensor in the first column in 1× PBS, pH 7.4, 0.02% Tween-20, and 0.1% BSA for 60 seconds. This serves as the baseline step. Dilute biotinylated apolipoprotein (a) to 40 μg / mL in 1× PBS, pH 7.4, 0.02% Tween-20, and 0.1% BSA and add it to the second column. Run the assay in this column for a period of time until the biotinylated apolipoprotein (a) reaches 2.0 nm on the sensor. The biosensor was run in 1× PBS, pH 7.4, 0.02% Tween-20, and 0.1% BSA in the third column for 60 seconds. This step served as the baseline step. The compound was diluted to 100 nM in 1× PBS, pH 7.4, 0.02% Tween-20, and 0.1% BSA and added to the fourth column, where the sensor was run for 60 seconds. The sensor was then run in 1× PBS, pH 7.4, 0.02% Tween-20, and 0.1% BSA in the third column for 120 seconds, during which time the compound dissociated from the sensor. The affinity constant, KD, reflects the binding capacity of the interaction. When the analyte concentration is equal to KD, the equilibrium signal, Req, is half the Rmax. The results demonstrate that the compounds of the present invention bind strongly to human Apo(a) protein.

[0231] Test Example 3: Mouse PK

[0232] 1. Experimental purpose: The purpose of this test is to test the PK of the compound of the present invention in mice

[0233] 2. Experimental animals:

[0234] mice (C57BL-6J),

[0235] 3. Experimental methods:

[0236] C57BL-6J mice were housed under a standard light cycle (12 h light / 12 h dark) at room temperature (18-26°C) and 40-70% relative humidity with free access to water and a normal diet. Five days prior to the study, mice were randomly divided into groups (n=3 / group) based on body weight. The dosing volume was calculated based on animal body weight on the day of dosing. A single oral gavage of 10 mL / kg was administered at a dose of 10 mg / kg in 1% HEC, 0.25% Tween 80 in water. Blood was collected from the submandibular vein or other appropriate vein at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after dosing. Approximately 0.03 mL of blood was collected per time point. Blood samples were placed on ice and centrifuged within 1 hour at 4°C, 6800 g, for 10 min to separate plasma for drug concentration determination.

[0237] Test Example 4: In vivo Lp(a) inhibition in cynomolgus monkeys

[0238] 1. Experimental purpose: The purpose of this test is to test the inhibitory effect of the compound of the present invention on Lp(a) in cynomolgus monkeys

[0239] 2. Experimental instruments and reagents:

[0240] Fully automatic biochemical analyzer (Hitachi 7600 model),

[0241] 3. Experimental methods:

[0242] Cynomolgus monkeys were housed under a standard light cycle (12 hours light / 12 hours dark) at room temperature (18-26°C) and 40-70% relative humidity. Drinking water was available 24 hours a day, and animals were fed twice daily, in the morning (approximately 10:30 AM) and afternoon (approximately 3:00 PM). Five days prior to the study, the monkeys were randomized (n=3 / group) based on body weight and baseline serum Lp(a) concentration. The dosing volume was calculated based on body weight on the first day of dosing, with a dose of 5 mL / kg. For five consecutive days, the drug was administered orally by gavage once daily at a dose of 3 mg / kg in 1% HEC and 0.25% Tween 80. During the acclimatization period (after an overnight fast), before dosing on Day 1, and 8 hours after dosing on Day 5, 1 mL of whole blood was collected from the cephalic or saphenous vein of the animals, allowed to stand at room temperature for 30 minutes, and serum was separated by centrifugation at 4°C, 3500 rpm, for 10 minutes. Serum Lp(a) levels were measured using an automated biochemical analyzer. The percentage reduction in Lp(a) in each group was determined by setting the average pre-dose Lp(a) level to 0% inhibition. The results demonstrate that the compounds of the present invention are effective in reducing plasma Lp(a) levels in vivo. The in vivo Lp(a) inhibition rates of the compounds of the present invention in cynomolgus monkeys are shown in the table below:

[0243] Test Example 5: Exposure determination in beagle dogs

[0244] 1. Experimental purpose: The purpose of this test is to test the exposure of the compound in beagle dogs

[0245] 2. Experimental methods:

[0246] Beagle dogs were housed under a standard light cycle (12 h light / 12 h dark) at room temperature (18-26°C) and 40-70% relative humidity, with free access to water and a normal diet. Five days prior to the study, beagle dogs were randomized by body weight into groups for oral administration (n=3 / group). The dose volume was calculated based on animal body weight on the day of dosing, with a single oral gavage of 5 mL / kg at a dose of 4.5 mg / kg in 1% HEC, 0.25% Tween 80 in water. Blood was collected from the forelimb vein or other suitable vein 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h after dosing. Approximately 1 mL of blood was collected for each sample, anticoagulated with K2-EDTA, placed on ice, and centrifuged within 1 h (2200 g, 10 min, 2-8°C) to separate plasma for plasma concentration measurement. Exposures in beagle dogs were calculated as shown in the following table:

[0247] Test Example 6: Bioavailability Determination in Cynomolgus Monkeys

[0248] 1. Experimental purpose: The purpose of this test is to test the bioavailability of the compound in cynomolgus monkeys

[0249] 2. Experimental methods:

[0250] Cynomolgus monkeys were housed under a standard light cycle (12 h light / 12 h dark) at room temperature (18-26°C) and 40-70% relative humidity with free access to water and a normal diet. Five days prior to the study, the monkeys were randomly divided into an intravenous or oral administration group (n=2 / group) based on body weight. The dosing volume was calculated based on the animal's body weight on the day of dosing. A single intravenous dose of 2 mL / kg was administered at a dose of 1 mg / kg in a vehicle consisting of 5% DMSO, 5% Solutol, and 90% Saline. Blood was collected via a forelimb vein or other suitable vein at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h post-dose. The dosing volume was calculated based on the animal's body weight on the day of dosing. A single oral gavage dose of 7.5 mg / kg was administered in a vehicle consisting of 1% HEC and 0.25% Tween 80 in water. Blood was collected from the forelimb vein or other suitable vein at 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h, and 72 h after administration. Approximately 1 mL of blood was collected for each sample, anticoagulated with K2-EDTA, and placed on ice. Plasma was centrifuged within 1 hour (centrifugation conditions: 2200 g, 10 min, 2-8°C) for plasma concentration determination. Bioavailability F was calculated as shown in the following table:

[0251] Test Example 7: hERG Experiment

[0252] 1. Experimental purpose: The purpose of this test is to test the inhibition rate of the compound on hERG potassium channel

[0253] 2. Experimental methods:

[0254] hERG currents were recorded using the whole-cell patch clamp technique. A suspension of HEK-293-hERG cells was placed in a small culture dish and placed on an inverted microscope stage. After the cells adhered, extracellular solution was perfused at a flow rate of 1–2 mL / min. Glass microelectrodes were pulled in two steps using a microelectrode puller. After filling the electrode with solution, the resistance to water was 2–5 MΩ. After establishing the whole-cell recording mode, the clamping potential was maintained at -80 mV. A depolarizing voltage of +60 mV was applied for 850 ms, followed by repolarization to -50 mV for 1275 ms to elicit the hERG tail current. This pulse sequence was repeated every 15 seconds throughout the experiment. After the current stabilized, the drug was administered by continuous extracellular perfusion from low to high concentrations. Starting from a low concentration, perfusion was continued until the drug effect stabilized, and then the next concentration was perfused. The inhibition rate of the compound on the hERG potassium channel was calculated as shown in the following table:

[0255] Test Example 8: Salmonella typhimurium reverse mutation test

[0256] Inoculate a single colony from the revived strain or the master plate into broth and incubate in a constant-temperature air-bath shaker at 100-120 rpm at 37±1°C for 10-16 hours. Label the plate and set aside. Pour a bottom agar medium (containing appropriate amounts of agar, Vogel-Bonner buffer, 20% glucose solution, and 20% magnesium sulfate solution) into a six-well plate, pouring approximately 20-25 mL into each well. Allow to cool and solidify naturally. Label each well and set aside. Three wells are tested in parallel for each group. Autoclave the top agar medium and incubate at approximately 65°C. To each test tube, add 0.1 mL of the negative control (DMSO), positive control, or test article, 0.1 mL of bacterial suspension, 0.5 mL of phosphate buffered saline (0.2M PBS) (-S9) or 0.5 mL of S9 mixture (+S9), and 2.5 mL of the top agar medium. Vortex to mix thoroughly and quickly spread evenly onto the petri dish containing the bottom agar medium. After natural cooling and solidification, the culture dish was inverted and placed in a 37°C incubator for 48 to 72 hours. After 48 to 72 hours of incubation, the culture dish was removed and the number of reverted colonies in each well was counted. The growth background was observed under a microscope to determine whether there was any antibacterial or bactericidal effect. The test results were recorded. The mutagenicity of the compounds of the present invention in various strains is shown in the following table:

[0257] Note:

[0258] In the "Metabolic Activation" column:

[0259] + indicates the addition of S9 mixed solution;

[0260] - indicates that no S9 mixture was added.

[0261] In the "Mutagenicity" column:

[0262] + indicates positive for mutagenicity;

[0263] - indicates negative for mutagenicity.

Claims

1. A compound of formula I, formula II, formula III or formula IV or a pharmaceutically acceptable salt thereof, wherein the structure of the compound of formula I, formula II, formula III or formula IV is as follows: in, R is selected from aryl or heteroaryl, which may be further substituted; R1, R2, R3 and R4 are each independently selected from H and alkyl, which may be further substituted; R6 are each independently selected from C1-6 alkyl; preferably methyl; Y is a trivalent group selected from x is 1 or 2, preferably 1; y is either 0 or 1.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound of formula IV is a compound of formula IV-1, formula IV-2, formula IV-3 or formula IV-4:

3. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, wherein R is selected from C6-20 aryl, preferably C6-14 aryl, more preferably C6-12 aryl, which may be further substituted.

4. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 are each independently selected from H and C1-6 alkyl, preferably H or methyl.

5. The compound according to claim 3 or a pharmaceutically acceptable salt thereof, wherein R is selected from phenyl, benzo[d][1,3]dioxole, tetrahydroquinoline, tetrahydroisoquinoline, pyridine, quinoline or isoquinoline, which may be further substituted.

6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein R is selected from the following groups: in, n is 0, 1, 2 or 3; R5 is each independently selected from hydrogen, alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, alkylcarbonylamino, halogen, hydroxy, nitro, cyano, cycloalkyl, aryl or heteroaryl; preferably alkyl, haloalkyl, alkoxy, haloalkoxy, alkylamino, alkylcarbonylamino or halogen; The R5 may be located at one, two or three of the 1, 2, 3, 4, 5, 6, 7 or 8 positions of the above-mentioned aryl or heteroaryl group.

7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein R5 is each independently selected from C1-6 alkyl, C3-6 cycloalkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, C1-6 alkylcarbonylamino, halogen or 8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein R5 is independently selected from trifluoromethyl, in Preferably Preferably 9. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein R is selected from the following groups:

10. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the following compounds:

11. A pharmaceutical composition comprising the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof.

12. Use of the compound according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof for preparing an agent for reducing Lp(a) levels.

13. The use according to claim 12, wherein the compound or a pharmaceutically acceptable salt thereof is used to prepare a drug for treating cardiovascular diseases.