Crystal form of nitrogen-containing heterocyclic derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to a crystal form of a nitrogen-containing heterocyclic derivative, and a preparation method therefor and the use thereof. In particular, the present invention relates to a crystal form of a compound represented by a general formula, and a preparation method therefor, a pharmaceutical composition containing a therapeutically effective amount of the crystal form, and the use thereof as an inhibitor in the treatment of cardiovascular diseases, cerebrovascular diseases, etc.

Description

Crystal forms, preparation methods and applications of a class of nitrogen-containing heterocyclic derivatives Technical Field

[0001] This invention belongs to the field of drug synthesis, specifically relating to the crystal form of a class of nitrogen-containing heterocyclic derivative inhibitors, their preparation methods, and applications. Background Technology

[0002] Cardiovascular disease (CVD) is a leading cause of death worldwide, and high levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor. The accumulation of LDL-C on the arterial walls leads to atherosclerosis and can trigger inflammatory responses, resulting in cardiovascular events such as heart attacks and strokes. Although statins can lower serum LDL-C and are currently the main lipid-lowering drugs in clinical practice, patients who are intolerant to statins or who fail to reach their treatment goals when receiving tolerated doses still face risks, such as patients with familial hypercholesterolemia. The discovery of PCSK9 inhibitors provides a more aggressive treatment option for homozygous and heterozygous familial hypercholesterolemia. The non-statin ezetimibe, when used in combination with statins, can lower LDL-C by 15%-20%, while PCSK9 inhibitors combined with statins can significantly lower LDL-C by 54%-74%. PCSK9 inhibitors can also overcome the intolerable side effects of statins, such as muscle pain.

[0003] PCSK9 (Proprotein convertase subtilisin kexin type 9) is a serine protease highly expressed in the liver. Loss-of-function mutations in the PCSK9 gene are associated with low LDL-C levels and reduced cardiovascular risk (Cohen, JC, 2006), and it has been clinically validated as a therapeutic target for hyperlipidemia. PCSK9 is synthesized as an enzyme precursor, and after synthesis, it undergoes autocatalytic cleavage within the cell. The propeptide binds to mature PCSK9 and is secreted extracellularly, thus blocking the catalytic activity of PCSK9.

[0004] PCSK9 is a major regulator of low-density lipoprotein receptor (LDLR) levels on the surface of hepatocytes and can inhibit the LDLR circulation pathway. LDLR function is crucial for maintaining cholesterol homeostasis, responsible for the uptake and degradation of low-density lipoprotein. Circulating LDL binds to the N-terminal ligand-binding domain of LDLR via apolipoprotein B100. The LDL / LDLR complex is internalized through receptor-mediated endocytosis. The low intracellular pH environment causes LDLR to release LDL, which then circulates back to the cell membrane. Intracellular free LDL is transported to lysosomes and degraded. Secreted PCSK9 interferes with LDLR circulation by binding to LDLR on the hepatocyte surface. After the PCSK9 / LDLR complex migrates through clathrin-encapsulated pits into the acidic endosomal chamber, a conformational change in LDLR leads to the formation of additional binding sites with PCSK9. Therefore, PCSK9 accompanies LDLR to lysosomes for degradation, preventing LDLR circulation and thus upregulating LDL-C levels.

[0005] Familial hypercholesterolemia (FH) is a hereditary disorder of low-density lipoprotein cholesterol metabolism, affecting approximately 1 in 250 people, characterized by significantly elevated LDL-c levels. Heterozygous FH patients have a 3-4 times higher risk of developing coronary artery disease (CAD) and often develop CAD an average of 10 years earlier than the general population. Statins lower LDL-C in heterozygous FH patients; studies by Besselin have shown that high-intensity statin therapy can reduce the risk of CAD and mortality by 44%. However, in many cases, the reduction in LDL-C is considered insufficient. The complementarity mechanism of statins involves upregulating sterol regulatory element-binding protein 2 (SREBP-2), thereby activating LDL receptors and PCSK9, increasing PCSK9 expression and secretion to bind LDLR, leading to elevated LDL-C levels in the blood. Therefore, while statins lower LDL by inhibiting HMGCoA, they counteract the effects of SREBP; adding a PCSK9 inhibitor to statin therapy can help overcome this mechanism. Considering that patients with familial hypercholesterolemia may not fully benefit from statin therapy, alternative treatments such as PCSK9 inhibitors are needed.

[0006] PCSK9 macromolecule inhibitors, such as the monoclonal antibody-based drugs Alirocumab and Evolocumab, selectively bind to extracellular PCSK9 and prevent its interaction with LDLR. They have been approved by the FDA for lowering LDL-C levels with a good safety profile. Studies have shown that in heterozygous FH patients who have not reached their LDL-C target after statin monotherapy, once-every-two-week injections of Alirocumab maximally reduce cardiovascular risk. Alirocumab has also shown a moderate increase in "good" cholesterol (HDL-C). Additionally, a PCSK9 siRNA drug, Inclisiran, is currently marketed. It lowers PCSK9 protein expression levels for long-term lipid reduction with a good safety profile. However, both of these drugs require injection and are expensive to produce. Currently, there are no marketed PCSK9 small molecule inhibitors, therefore there is a high demand for oral PCSK9 small molecule inhibitors.

[0007] There are already patent reports on PCSK9 small molecule inhibitors, such as WO2014170786 (Pfizer), WO2014150326 (Shifa), WO2020150473 (AZ), and WO2022133529 (Nyrada). Currently, AZD-0780 is the most advanced in Phase I clinical trials, while the others are in preclinical development. Several peptides have also been reported, with the most advanced being in Phase II clinical trials. This invention aims to develop an orally administered PCSK9 small molecule inhibitor.

[0008] PCT / CN2024 / 103568 discloses the structures of a series of nitrogen-containing heterocyclic compounds. In subsequent research and development, in order to facilitate the handling, filtration and drying of the products, and to seek suitable crystals that are easy to store and have long-term product stability, this invention has conducted a comprehensive study on the crystal forms of the above-mentioned compounds. Summary of the Invention

[0009] All contents relating to patent PCT / CN2024 / 103568 are incorporated herein by reference.

[0010] The object of this invention is to provide a crystal form of the compound of general formula (I) or its stereoisomers:

[0011] in:

[0012] Ring A is selected from 5-membered nitrogen-containing heterocyclic group, 6-membered nitrogen-containing heterocyclic group, 5-membered nitrogen-containing heteroaryl group, 6-membered nitrogen-containing heteroaryl group, 5-membered 6-membered bicyclic nitrogen-containing heterocyclic group, 6-membered 5-membered bicyclic nitrogen-containing heterocyclic group, 5-membered 6-membered bicyclic nitrogen-containing heteroaryl group, 6-membered 5-membered bicyclic nitrogen-containing heteroaryl group or 6-membered 6-membered bicyclic nitrogen-containing heteroaryl group;

[0013] R a Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy or C 1-6 Hydroxyalkyl;

[0014] R b Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy or C 1-6 hydroxyalkyl; and

[0015] x is 0, 1, 2, 3 or 4.

[0016] In a further technical solution of the present invention, ring A is selected from...

[0017] And / or, R a Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl;

[0018] Preferably, R a Selected from -H, -F, -O-CH3, -O-CHF2, -O-CF3, -CH2F, -CHF2, -CH3, -CF3, -CH2-CHF 23 Or -CH2-CF3;

[0019] And / or, R b Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl;

[0020] Preferably, R bSelected from -H, -D, -F, -Cl, -CN, -CH3, -CF3, -CH(CH3)2, -C(CH3)3, -C(CH3)2-OH, -C(CH3)2-CH2-OH, -O-CH3, -CH2-NH2, -CH2-OH or -OH.

[0021] A further technical solution of the present invention is the crystal form of compound 1, wherein compound 1 is 5'-fluoro-6'-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one.

[0022] Specifically, for crystal form A, its X-ray powder diffraction pattern shows a diffraction peak at 2θ of 9.4±0.2°; or at 18.7±0.2°; or at 23.4±0.2°; or at 20.0±0.2°; or at 19.0±0.2°; or at 17.7±0.2°; or at 24.3±0.2°. Alternatively, it may have a diffraction peak at 22.3±0.2°; or a diffraction peak at 21.2±0.2°; or a diffraction peak at 28.3±0.2°; preferably, it may include any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 diffraction peaks, more preferably, any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks. Crystal form B, its X-ray powder diffraction pattern shows a diffraction peak at 2θ of 18.1 ± 0.2°; or at 15.8 ± 0.2°; or at 27.6 ± 0.2°; or at 20.9 ± 0.2°; or at 26.5 ± 0.2°; or at 17.5 ± 0.2°; or at 24.9 ± 0.2°; or The diffraction peak is present at 7.6±0.2°; or at 16.3±0.2°; or at 19.9±0.2°; preferably, any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 diffraction peaks are included; more preferably, any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks are included.

[0023] Crystal form C exhibits the following X-ray powder diffraction patterns: a diffraction peak at 2θ = 16.1 ± 0.2°; or at 16.8 ± 0.2°; or at 21.2 ± 0.2°; or at 23.7 ± 0.2°; or at 28.3 ± 0.2°; or at 14.2 ± 0.2°; or at 22.1 ± 0.2°; or It has a diffraction peak at 13.6±0.2°; or a diffraction peak at 27.3±0.2°; or a diffraction peak at 25.8±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7, 8, 9 or 10 of them;

[0024] Crystal form D has an X-ray powder diffraction pattern showing a diffraction peak at 2θ = 24.1 ± 0.2°; or at 11.6 ± 0.2°; or at 6.4 ± 0.2°; or at 22.7 ± 0.2°; or at 24.4 ± 0.2°; or at 21.0 ± 0.2°; or at 27.3 ± 0.2°; or at... A diffraction peak is present at 19.0±0.2°; or at 15.7±0.2°; or at 26.0±0.2°; preferably, any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 diffraction peaks are included; more preferably, any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks are included.

[0025] A further technical solution of the present invention is that the X-ray powder diffraction pattern of crystal form A includes at least one or more diffraction peaks located at 2θ of 9.4±0.2°, 18.7±0.2°, and 23.4±0.2°, preferably two, more preferably three; optionally, it may further include at least one of the following: 2θ of 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, and 22.3±0.2°, preferably two, three, four, or five.

[0026] For example:

[0027] 9.4±0.2°, 18.7±0.2°;

[0028] 18.7±0.2°, 23.4±0.2°;

[0029] 9.4±0.2°, 23.4±0.2°;

[0030] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°;

[0031] 9.4±0.2°, 18.7±0.2°, 19.0±0.2°;

[0032] 18.7±0.2°, 23.4±0.2°, 19.0±0.2°;

[0033] 9.4±0.2°, 23.4±0.2°, 17.7±0.2°, 24.3±0.2°;

[0034] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 19.0±0.2°;

[0035] 18.7±0.2°, 23.4±0.2°, 17.7±0.2°, 24.3±0.2°, 22.3±0.2°;

[0036] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 19.0±0.2°, 17.7±0.2°.

[0037] The X-ray powder diffraction pattern of crystal form B contains at least one or more diffraction peaks located at 2θ of 18.1±0.2°, 15.8±0.2°, and 27.6±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 20.9±0.2°, 26.5±0.2°, 17.5±0.2°, 24.9±0.2°, and 7.6±0.2°, preferably two, three, four, or five.

[0038] The X-ray powder diffraction pattern of crystal form C includes at least one or more diffraction peaks located at 2θ of 16.1±0.2°, 16.8±0.2°, and 21.2±0.2°, preferably two, more preferably three; optionally, it may further include at least one of the following: 23.7±0.2°, 28.3±0.2°, 14.2±0.2°, 22.1±0.2°, and 13.6±0.2°, preferably two, three, four, or five.

[0039] The X-ray powder diffraction pattern of crystal form D contains at least one or more diffraction peaks located at 2θ of 24.1±0.2°, 11.6±0.2°, and 6.4±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 22.7±0.2°, 24.4±0.2°, 21.0±0.2°, 27.3±0.2°, and 19.0±0.2°, preferably two, three, four, or five.

[0040] In a further technical solution of the present invention, the X-ray powder diffraction pattern of crystal form A optionally includes one or more diffraction peaks located at 2θ of 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 25.8±0.2°, 15.8±0.2°, 37.9±0.2°, and 12.3±0.2°; preferably, it includes at least any 2-3 peaks, or 4-5 peaks, or 6-7 peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0041] For example:

[0042] 21.2±0.2°, 28.3±0.2°;

[0043] 21.2±0.2°, 37.6±0.2°;

[0044] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°;

[0045] 28.3±0.2°, 37.6±0.2°, 15.8±0.2°;

[0046] 21.2±0.2°, 37.6±0.2°, 25.8±0.2°, 12.3±0.2°;

[0047] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 25.8±0.2°;

[0048] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 25.8±0.2°, 15.8±0.2°;

[0049] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 37.9±0.2°, 12.3±0.2°;

[0050] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 25.8±0.2°, 15.8±0.2°, 37.9±0.2°;

[0051] 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 15.8±0.2°, 37.9±0.2°, 12.3±0.2°.

[0052] The X-ray powder diffraction pattern of crystal form B can also be selected to include those located at 2θ of 16.3±0.2°, 19.9±0.2°,

[0053] One or more diffraction peaks selected from 22.7±0.2°, 20.3±0.2°, 24.1±0.2°, 9.2±0.2°, and 16.9±0.2°; preferably including at least 2-3, 4-5, or 6-7 of these peaks; more preferably including any 2, 3, 4, 5, 6, or 7 of these peaks.

[0054] The X-ray powder diffraction pattern of crystal form C may optionally include one or more diffraction peaks located at 2θ of 27.3±0.2°, 25.8±0.2°, 9.2±0.2°, 22.9±0.2°, 24.0±0.2°, 14.6±0.2°, and 31.2±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0055] The X-ray powder diffraction pattern of crystal form D may optionally include one or more diffraction peaks located at 2θ of 15.7±0.2°, 26.0±0.2°, 20.5±0.2°, 20.0±0.2°, 28.6±0.2°, 23.3±0.2°, and 17.5±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0056] A further technical solution of the present invention is that the X-ray powder diffraction pattern of crystal form A includes one or more diffraction peaks located at 2θ of 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, 22.3±0.2°, 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, and 25.8±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks.

[0057] For example:

[0058] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°;

[0059] 9.4±0.2°, 18.7±0.2°, 20.0±0.2°, 19.0±0.2°;

[0060] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°;

[0061] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°;

[0062] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, 22.3±0.2°;

[0063] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 21.2±0.2°, 28.3±0.2°;

[0064] 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, 22.3±0.2°, 21.2±0.2°, 28.3±0.2°.

[0065] The X-ray powder diffraction pattern of crystal form B includes one or more diffraction peaks located at 2θ of 18.1±0.2°, 15.8±0.2°, 27.6±0.2°, 20.9±0.2°, 26.5±0.2°, 17.5±0.2°, 24.9±0.2°, 7.6±0.2°, 16.3±0.2°, 19.9±0.2°, 22.7±0.2°, and 20.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks.

[0066] The X-ray powder diffraction pattern of crystal form C includes one or more diffraction peaks located at 2θ of 16.1±0.2°, 16.8±0.2°, 21.2±0.2°, 23.7±0.2°, 28.3±0.2°, 14.2±0.2°, 22.1±0.2°, 13.6±0.2°, 27.3±0.2°, 25.8±0.2°, 9.2±0.2°, and 22.9±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks.

[0067] The X-ray powder diffraction pattern of crystal form D includes one or more diffraction peaks located at 2θ of 24.1±0.2°, 11.6±0.2°, 6.4±0.2°, 22.7±0.2°, 24.4±0.2°, 21.0±0.2°, 27.3±0.2°, 19.0±0.2°, 15.7±0.2°, 26.0±0.2°, 20.5±0.2°, and 20.0±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks.

[0068] A further technical solution of the present invention is the crystal form of compound 2, which is 6-(5-fluoro-6-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)aminopyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one, specifically,

[0069] Crystal form A has an X-ray powder diffraction pattern showing a diffraction peak at 2θ = 24.6 ± 0.2°; or at 26.2 ± 0.2°; or at 21.5 ± 0.2°; or at 14.4 ± 0.2°; or at 15.0 ± 0.2°; or at 29.8 ± 0.2°; or at 13.0 ± 0.2°; or It has a diffraction peak at 22.5±0.2°; or a diffraction peak at 18.7±0.2°; or a diffraction peak at 9.0±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7, 8, 9 or 10 of them;

[0070] Crystal form B has an X-ray powder diffraction pattern showing a diffraction peak at 2θ = 24.1 ± 0.2°; or at 12.7 ± 0.2°; or at 12.9 ± 0.2°; or at 7.7 ± 0.2°; or at 14.9 ± 0.2°; or at 11.8 ± 0.2°; or at 26.5 ± 0.2°; or at... A diffraction peak is present at 10.5±0.2°; or at 21.5±0.2°; or at 25.8±0.2°; preferably, any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks are included; more preferably, any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks are included.

[0071] Crystal form C exhibits the following X-ray powder diffraction patterns: a diffraction peak at 2θ = 24.7 ± 0.2°; or at 12.6 ± 0.2°; or at 18.6 ± 0.2°; or at 10.9 ± 0.2°; or at 19.5 ± 0.2°; or at 27.3 ± 0.2°; or at 15.0 ± 0.2°; or It has a diffraction peak at 8.4±0.2°; or a diffraction peak at 28.4±0.2°; or a diffraction peak at 25.6±0.2°; preferably, it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks;

[0072] Crystal form D has an X-ray powder diffraction pattern showing a diffraction peak at 2θ = 25.1 ± 0.2°; or at 14.0 ± 0.2°; or at 13.4 ± 0.2°; or at 16.6 ± 0.2°; or at 19.0 ± 0.2°; or at 9.6 ± 0.2°; or at 10.0 ± 0.2°; or at... A diffraction peak is present at 22.1±0.2°; or at 20.6±0.2°; or at 15.1±0.2°; preferably, any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 diffraction peaks are included; more preferably, any 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the above diffraction peaks are included.

[0073] A further technical solution of the present invention is that the X-ray powder diffraction pattern of crystal form A includes at least one or more diffraction peaks located at 2θ of 24.6±0.2°, 26.2±0.2°, and 21.5±0.2°, preferably two, more preferably three; optionally, it may further include at least one of the following: 2θ of 14.4±0.2°, 15.0±0.2°, 29.8±0.2°, 13.0±0.2°, and 22.5±0.2°, preferably two, three, four, or five.

[0074] The X-ray powder diffraction pattern of crystal form B contains at least one or more diffraction peaks located at 2θ of 24.1±0.2°, 12.7±0.2°, and 12.9±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 2θ of 7.7±0.2°, 14.9±0.2°, 11.8±0.2°, 26.5±0.2°, and 10.5±0.2°, preferably two, three, four, or five.

[0075] The X-ray powder diffraction pattern of crystal form C contains at least one or more diffraction peaks located at 2θ of 24.7±0.2°, 12.6±0.2°, and 18.6±0.2°, preferably two, more preferably three; optionally, it may further contain at least one diffraction peak located at 2θ of 10.9±0.2°, 19.5±0.2°, 27.3±0.2°, 15.0±0.2°, and 8.4±0.2°, preferably two, three, four, or five.

[0076] The X-ray powder diffraction pattern of crystal form D contains at least one or more diffraction peaks located at 2θ of 25.1±0.2°, 14.0±0.2°, and 13.4±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 2θ of 16.6±0.2°, 19.0±0.2°, 9.6±0.2°, 10.0±0.2°, and 22.1±0.2°, preferably two, three, four, or five.

[0077] In a further technical solution of the present invention, the X-ray powder diffraction pattern of crystal form A may optionally include one or more diffraction peaks located at 2θ of 18.7±0.2°, 9.0±0.2°, 5.3±0.2°, 19.3±0.2°, 27.0±0.2°, 11.1±0.2°, and 17.8±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0078] The X-ray powder diffraction pattern of crystal form B may optionally include one or more diffraction peaks located at 2θ of 21.5±0.2°, 25.8±0.2°, 28.1±0.2°, 25.0±0.2°, 19.1±0.2°, 16.0±0.2°, and 30.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0079] The X-ray powder diffraction pattern of crystal form C may optionally include one or more diffraction peaks located at 2θ of 28.4±0.2°, 25.6±0.2°, 22.7±0.2°, 17.1±0.2°, 29.6±0.2°, 23.2±0.2°, and 22.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0080] The X-ray powder diffraction pattern of crystal form D may optionally include one or more diffraction peaks located at 2θ of 20.6±0.2°, 15.1±0.2°, 23.6±0.2°, 19.5±0.2°, 30.8±0.2°, 11.7±0.2°, and 27.9±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

[0081] A further technical solution of the present invention is that the X-ray powder diffraction pattern of crystal form A includes one or more diffraction peaks located at 2θ of 24.6±0.2°, 26.2±0.2°, 21.5±0.2°, 14.4±0.2°, 15.0±0.2°, 29.8±0.2°, 13.0±0.2°, 22.5±0.2°, 18.7±0.2°, 9.0±0.2°, 5.3±0.2°, 19.3±0.2°, 27.0±0.2°, 11.1±0.2°, and 17.8±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks.

[0082] The X-ray powder diffraction pattern of crystal form B includes one or more diffraction peaks located at 2θ of 24.1±0.2°, 12.7±0.2°, 12.9±0.2°, 7.7±0.2°, 14.9±0.2°, 11.8±0.2°, 26.5±0.2°, 10.5±0.2°, 21.5±0.2°, 25.8±0.2°, 28.1±0.2°, 25.0±0.2°, 19.1±0.2°, 16.0±0.2°, and 30.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks.

[0083] The X-ray powder diffraction pattern of crystal form C includes one or more diffraction peaks located at 2θ of 24.7±0.2°, 12.6±0.2°, 18.6±0.2°, 10.9±0.2°, 19.5±0.2°, 27.3±0.2°, 15.0±0.2°, 8.4±0.2°, 28.4±0.2°, 25.6±0.2°, 22.7±0.2°, 17.1±0.2°, 29.6±0.2°, 23.2±0.2°, and 22.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks.

[0084] The X-ray powder diffraction pattern of crystal form D includes one or more diffraction peaks located at 2θ of 25.1±0.2°, 14.0±0.2°, 13.4±0.2°, 16.6±0.2°, 19.0±0.2°, 9.6±0.2°, 10.0±0.2°, 22.1±0.2°, 20.6±0.2°, 15.1±0.2°, 23.6±0.2°, 19.5±0.2°, 30.8±0.2°, 11.7±0.2°, and 27.9±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks.

[0085] A further technical solution of the present invention is characterized in that the compound or its stereoisomer is compound 1, whose crystal form A has the X-ray powder diffraction pattern shown in Figure 1; or has the DSC pattern shown in Figure 2; or has the TGA pattern shown in Figure 3;

[0086] Its crystal form B has the X-ray powder diffraction pattern shown in Figure 4; or the DSC pattern shown in Figure 5; or the TGA pattern shown in Figure 6.

[0087] Its crystal form C has the X-ray powder diffraction pattern shown in Figure 7; or the DSC pattern shown in Figure 8; or the TGA pattern shown in Figure 9;

[0088] Its crystal form D has the X-ray powder diffraction pattern shown in Figure 10; or the DSC pattern shown in Figure 11; or the TGA pattern shown in Figure 12;

[0089] The compound or its stereoisomer is compound 2.

[0090] Its crystal form A has the X-ray powder diffraction pattern shown in Figure 13; or the DSC pattern shown in Figure 14; or the TGA pattern shown in Figure 15;

[0091] Its crystal form B has the X-ray powder diffraction pattern shown in Figure 16; or the DSC pattern shown in Figure 17; or the TGA pattern shown in Figure 18;

[0092] Its crystal form C has the X-ray powder diffraction pattern shown in Figure 19; or the DSC pattern shown in Figure 20; or the TGA pattern shown in Figure 21;

[0093] Its crystal form D has the X-ray powder diffraction pattern shown in Figure 22; or the DSC pattern shown in Figure 23; or the TGA pattern shown in Figure 24;

[0094] The 2θ error between the positions of the top ten diffraction peaks with the highest relative peak intensities in the X-ray powder diffraction pattern and the corresponding positions of the diffraction peaks in the attached figure is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°.

[0095] A further technical solution of the present invention is characterized in that the crystal form is a solvate, hydrate or anhydrous; the number of water molecules is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5 or 3.

[0096] The present invention also includes a method for preparing the compound shown in the claims or its stereoisomer crystal form, specifically comprising the following steps:

[0097] Method 1:

[0098] 1) Weigh an appropriate amount of free base and dissolve it in a solvent at a certain temperature;

[0099] 2) Lower the temperature and stir to cool and crystallize;

[0100] 3) Dry to obtain a solid product;

[0101] The solvent is:

[0102] Water, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, acetonitrile, isopropyl acetate, methyl tert-butyl ether, 2-butanone, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, n-heptane, water / ethanol, methanol / water, ethyl acetate / ethanol, ethyl acetate / n-heptane, isopropyl ether, acetone / cyclohexane, methanol, toluene, dichloromethane.

[0103] Method 2:

[0104] 1) Weigh an appropriate amount of free base and dissolve it in a good solvent at a certain temperature;

[0105] 2) Add a poor solvent to the solution obtained above and stir until a solid precipitates;

[0106] 3) Stir, cool, and crystallize to obtain the target product;

[0107] in:

[0108] The benign solvent is selected from one or more of water, methanol, acetone, dichloromethane, and dimethyl sulfoxide; preferably dichloromethane / methanol, water / acetone, or dimethyl sulfoxide.

[0109] The undesirable solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, methyl tert-butyl ether, toluene, or isopropyl ether; preferably heptane, methyl tert-butyl ether, or water.

[0110] Method 3:

[0111] In both of the above methods, seed crystals can be added after step one, followed by stirring to induce crystallization.

[0112] Method 4:

[0113] 1) Weigh out an appropriate amount of free base and add a certain amount of solvent;

[0114] 2) Crystallization by stirring at room temperature;

[0115] 3) Dry to obtain a solid product;

[0116] The solvent is:

[0117] Water, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, acetonitrile, isopropyl acetate, methyl tert-butyl ether, 2-butanone, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, n-heptane, water / ethanol, water / isopropanol, methanol / water, ethyl acetate / ethanol, ethyl acetate / n-heptane, isopropyl ether, acetone / cyclohexane, methanol, tetrahydrofuran, toluene.

[0118] Method 5:

[0119] 1) Weigh an appropriate amount of free base and dissolve it in a solvent at a certain temperature;

[0120] 2) Curing occurs at room temperature;

[0121] 3) A solid product is obtained;

[0122] The solvents are: dimethyl sulfoxide, 1,4-dioxane, tetrahydrofuran, 10% water / methanol, 10% water / acetone, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile / methanol, tetrahydrofuran / ethanol, benzyl alcohol, dichloromethane / methanol / ethanol, dichloromethane / methanol / acetone, dichloromethane / methanol / ethyl acetate, (10% water / acetone) / methyl tert-butyl ether, (10% water / acetone) / ethanol, (10% water / acetone) / acetone, (10% water / acetone) / ethyl acetate.

[0123] Method Six:

[0124] 1) Weigh an appropriate amount of free alkali or its crystal form, add a poor solvent, and then add a certain proportion of acid organic solvent solution. Then, pulp the mixture at a certain temperature, preferably 0-60℃.

[0125] 2) Add an auxiliary material in proportion to the free alkali to the solid obtained after filtering the above solution, and then pulp it in water at a certain temperature, preferably 0-60℃;

[0126] Alternatively, the solid obtained after filtering the above solution can be pulped in water, preferably at a temperature of 0–60°C;

[0127] 3) Stir, cool, and crystallize to obtain the target product;

[0128] in:

[0129] The unsuitable solvent is selected from one or more of acetone, ethyl acetate, isopropyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, 2-methyltetrahydrofuran, or methanol; preferably one or more of ethanol, ethyl acetate, or acetone.

[0130] The acid is selected from: hydrochloric acid, methanesulfonic acid, and acetic acid;

[0131] The ratio of the free base to the acid is 1:1 to 1:2; wherein the preferred molar ratio of the acid is 1M.

[0132] The organic solvent is selected from one or more of methanol, ethanol, or ethyl acetate;

[0133] The excipients are selected from one or more of starch, HEC (hydroxyethyl cellulose), HPMC E5 (hydroxypropyl methyl cellulose E5), HPMC E50 (hydroxypropyl methyl cellulose E50), HPC LF (hydroxypropyl cellulose LF), and HPC EXF (hydroxypropyl cellulose EXF).

[0134] The present invention also provides a pharmaceutical composition comprising a therapeutically effective dose of the crystal form of the above-described compound, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0135] Further, the compound, and its stereoisomer crystal forms, constitute 0.1% to 95% by weight in the composition, preferably 0.5% to 85%, more preferably 1% to 60%, more preferably 2% to 50%, more preferably 2% to 40%, even more preferably 2-30%, even more preferably 2-20%, even more preferably 4% to 30%, even more preferably 5% to 30%, even more preferably 5% to 20%, specifically preferably 1%, 2%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (based on the total weight of the pharmaceutical composition).

[0136] The present invention further relates to the crystal form of the compound or its stereoisomer, or the use of the pharmaceutical composition thereof in the preparation of a PCSK9 inhibitor drug.

[0137] The present invention further relates to the crystal form of the compound or its stereoisomer, or the use of the pharmaceutical composition thereof in the preparation of an LDL-lowering drug.

[0138] The present invention further relates to the crystal forms of the compounds shown or their stereoisomers, or the pharmaceutical compositions described herein, in the treatment and / or prevention of cardiovascular diseases, cerebrovascular diseases, atherosclerosis, and / or their related diseases or symptoms; preferably, in the treatment and / or prevention of stroke, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome, and / or coronary artery disease.

[0139] The present invention also relates to a method for treating, preventing and / or treating stroke, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome and / or coronary artery disease, comprising administering to a patient a therapeutically effective dose of a crystalline form of the compound shown in the present invention or a stereoisomer thereof, or a pharmaceutical composition thereof. Attached Figure Description

[0140] Figure 1 shows the XRPD diagram of free base crystal form A of compound 1. Figure 2 shows the DSC diagram of free base crystal form A of compound 1. Figure 3 shows the TGA diagram of free base crystal form A of compound 1. Figure 4 shows the XRPD diagram of free base crystal form B of compound 1. Figure 5 shows the DSC diagram of free base crystal form B of compound 1. Figure 6 shows the TGA diagram of free base crystal form B of compound 1. Figure 7 shows the XRPD diagram of free base crystal form C of compound 1. Figure 8 shows the DSC diagram of free base crystal form C of compound 1. Figure 9 shows the TGA diagram of free base crystal form C of compound 1. Figure 10 shows the XRPD diagram of free base crystal form D of compound 1. Figure 11 shows the DSC diagram of free base crystal form D of compound 1. Figure 12 shows the TGA diagram of free base crystal form D of compound 1. Figure 13 shows the XRPD diagram of free base crystal form A of compound 2. Figure 14 shows the DSC diagram of free base crystal form A of compound 2. Figure 15 is a TGA diagram of the free base crystal form A of compound 2. Figure 16 is an XRPD diagram of the free base crystal form B of compound 2. Figure 17 is a DSC diagram of the free base crystal form B of compound 2. Figure 18 is a TGA diagram of the free base crystal form B of compound 2. Figure 19 is an XRPD diagram of the free base crystal form C of compound 2. Figure 20 is a DSC diagram of the free base crystal form C of compound 2. Figure 21 is a TGA diagram of the free base crystal form C of compound 2. Figure 22 is an XRPD diagram of the free base crystal form D of compound 2. Figure 23 is a DSC diagram of the free base crystal form D of compound 2. Figure 24 is a TGA diagram of the free base crystal form D of compound 2.

[0141] Detailed description of the invention

[0142] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0143] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight-chain or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 8 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. 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 their various branched isomers, etc. More preferably, lower alkyl groups containing 1 to 6 carbon atoms are used. 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, etc. Alkyl groups can be substituted or unsubstituted. When substituted, the substituent can be substituted at any usable connection point. The substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester groups. The present invention preferably uses methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuteralkyl, alkoxy-substituted alkyl, and hydroxy-substituted alkyl.

[0144] The term "heterocyclic group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, but excluding the ring portions of -OO-, -OS-, or -SS-, with the remaining ring atoms being carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, it contains 3 to 10 ring atoms; most preferably, it contains 3 to 8 ring atoms; further preferably, it contains 3-, 4-, 5-, 6-, 7-, or 8-membered heterocyclic groups containing 1 to 3 nitrogen atoms, optionally substituted with 1 to 2 oxygen atoms, sulfur atoms, or oxo groups, including nitrogen-containing monocyclic heterocyclic groups, nitrogen-containing spirocyclic groups, or nitrogen-containing fused heterocyclic groups; or, preferably, it contains 5 to 12 ring atoms, of which 1 to 4 are heteroatoms, further preferably, it contains 5-, 6-, 7-, 8-, 9-, 10-, 11-, or 12-membered heterocyclic groups containing 1 to 3 nitrogen and / or oxygen atoms.

[0145] Non-limiting examples of monocyclic heterocyclic groups include pyrrolyl, imidazoyl, tetrahydrofuranyl, tetrahydrothiophenyl, dihydroimidazoyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, acrylonitrile, 1,4-diazaheptanyl, pyranyl, etc., preferably pyrrolyl, morpholinyl, piperidinyl, acrylonitrile, 1,4-diazaheptanyl, and piperazinyl. Polycyclic heterocyclic groups include spirocyclic, fused-ring, and bridged-ring heterocyclic groups; wherein the spirocyclic, fused-ring, and bridged-ring heterocyclic groups involved are optionally connected to other groups by single bonds, or further cyclically linked to other cycloalkyl, heterocyclic, aryl, and heteroaryl groups by any two or more atoms on the ring.

[0146] The term "fused heterocyclic group" refers to a 5- to 20-membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with the other rings in the system. One or more rings may contain one or more double bonds, but none of the rings has a fully conjugated π-electron system. One or more ring atoms are selected from nitrogen, oxygen, or S(O). m (where m is an integer from 0 to 2) heteroatoms, with the remaining ring atoms being carbon. Preferably, they are 6 to 14 members, more preferably 7 to 10 members. Depending on the number of rings, they can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic fused heterocyclic groups, preferably bicyclic or tricyclic, more preferably 5-membered and 5-membered or 5-membered and 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include: wait.

[0147] The heterocyclic ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, wherein the ring connected to the parent structure is a heterocyclic group, and non-limiting examples include:

[0148] wait.

[0149] The heterocyclic group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl, or carboxylic acid ester group.

[0150] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., a ring sharing adjacent carbon atom pairs) group having a conjugated π-electron system, preferably 6- to 12-membered, such as phenyl and naphthyl. More preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclic, or cycloalkyl ring, including benzo5- to 10-membered heteroaryl, benzo3- to 8-membered cycloalkyl, and benzo3- to 8-membered heteroalkyl, preferably benzo5- to 6-membered heteroaryl, benzo3- to 6-membered cycloalkyl, and benzo3- to 6-membered heteroalkyl, wherein the heterocyclic group is a heterocyclic group containing 1-3 nitrogen, oxygen, and sulfur atoms; or may further include a ternary nitrogen-containing fused ring containing a benzene ring.

[0151] The ring connected to the parent structure is an aryl ring, and non-limiting examples include: wait.

[0152] The aryl group can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, oxo, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylic acid ester group.

[0153] The term "heteroaryl" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur, and nitrogen. The heteroaryl group is preferably 5 to 12-membered, more preferably 5- or 6-membered monocyclic heteroaryl or 8-12-membered bicyclic heteroaryl, such as imidazolyl, furanyl, thiophene, thiazolyl, pyrazolyl, oxazolyl, oxadiazolyl, pyrroleyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, thiadiazole, pyrazinyl, triazinyl, pyridazinyl, etc., preferably triazolyl, thiophene, imidazolyl, pyrazolyl, oxazolyl, pyrimidinyl, or thiazolyl; more preferably pyrazolyl, pyrroleyl, and oxazolyl.

[0154] The bicyclic heteroaryl group is preferably a 5-membered 5-membered bicyclic heteroaryl group, a 5-membered 6-membered bicyclic heteroaryl group, a 6-membered 5-membered bicyclic heteroaryl group, or a 6-membered 6-membered bicyclic heteroaryl group. Non-limiting examples include:

[0155] The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is a heteroaryl ring, and non-limiting examples include: wait.

[0156] The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups, independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, oxo, or carboxylic acid ester group.

[0157] The term "alkoxy" refers to -O- (alkyl) and -O- (unsubstituted cycloalkyl), where alkyl is defined as described above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentoxy, and cyclohexoxy. Alkoxy groups can be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or carboxylic acid ester group.

[0158] "Halogenated alkyl" refers to an alkyl group that has been substituted with one or more halogens, wherein the alkyl group is as defined above.

[0159] "Haloalkoxy" refers to an alkoxy group that has been substituted by one or more halogens, wherein the alkoxy group is as defined above.

[0160] "Hydroxyalkyl" refers to an alkyl group that has been replaced by a hydroxyl group, where the alkyl group is as defined above.

[0161] The different terms such as "X is selected from A, B, or C", "X is selected from A, B, and C", "X is A, B, or C", and "X is A, B, and C" all express the same meaning, that is, X can be any one or more of A, B, and C.

[0162] All hydrogen atoms described in this invention can be replaced by their isotope deuterium, and any hydrogen atom in the compounds of the embodiments of this invention can also be replaced by a deuterium atom.

[0163] "Optional" or "optionally" means that the event or environment described below may but does not have to occur, and the description includes the possibility or absence of such event or environment. For example, "optionally alkyl-substituted heterocyclic group" means that the alkyl group may but does not have to be present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0164] "Substituted" refers to one or more hydrogen atoms in a group, preferably up to five, more preferably one to three hydrogen atoms, which are independently substituted by the corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without much effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom having an unsaturated bond (such as an alkene).

[0165] "Pharmaceutical composition" means a mixture containing one or more of the compounds described herein or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0166] X-ray powder diffraction (XRPD) refers to the experimentally observed diffraction pattern or parameters derived from it, characterized by peak positions (x-axis) and peak intensities (y-axis). Those skilled in the art will understand that experimental errors depend on instrument conditions, sample preparation, and sample purity. In particular, it is known to those skilled in the art that X-ray diffraction patterns typically change with instrument conditions, and appropriate error tolerances for XRPD can be: 2θ ± 0.5°; 2θ ± 0.4°; 2θ ± 0.3°; 2θ ± 0.2°. It is particularly important to note that the relative intensities of the X-ray diffraction pattern can also vary with experimental conditions, so the order of peak intensities cannot be considered the sole or decisive factor. Furthermore, the influence of experimental factors such as sample height can cause an overall shift in peak angles, which is generally permissible. Therefore, those skilled in the art will understand that any crystal form with characteristic peaks identical or similar to those of the patterns of this invention falls within the scope of this invention.

[0167] "TGA" refers to thermogravimetric analysis (TGA) experiments.

[0168] "DSC" refers to the Differential Scanning Calorimetry (DSC) experiment. Detailed Implementation

[0169] The present invention is further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the present invention.

[0170] Example

[0171] The structures of the compounds of this invention were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer in the following solvents: deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or deuterated water (D2O), with tetramethylsilane (TMS) as the internal standard (if applicable).

[0172] LC-MS was performed using an Agilent 1200 Infinity Series mass spectrometer. HPLC was performed using an Agilent 1200DAD high-performance liquid chromatograph (Sunfire C18 150×4.6mm column) and a Waters 2695-2996 high-performance liquid chromatograph (Gimini C). 18 (150×4.6mm chromatographic column).

[0173] Thin-layer chromatography (TLC) uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The standard size for TLC is 0.15mm to 0.20mm, while the standard size for separating and purifying products using TLC is 0.4mm to 0.5mm. Column chromatography generally uses Yantai Huanghai 200-300 mesh silica gel as the carrier.

[0174] The starting materials used in the embodiments of the present invention are known and commercially available, or can be synthesized using or in accordance with methods known in the art.

[0175] Unless otherwise specified, all reactions in this invention are carried out under continuous magnetic stirring, in a dry nitrogen or argon atmosphere, using a dry solvent, and the reaction temperature is expressed in degrees Celsius.

[0176] The eluent system for silica gel column chromatography and the developing solvent system for thin-layer chromatography used in the intermediates and the purification compounds in the examples include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: dichloromethane and acetone system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and small amounts of basic or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0177] Unless otherwise specified, in the embodiments of the present invention, the ratio of the mobile phase in the HPLC chiral separation condition and the HPLC chiral analysis condition is a volume ratio.

[0178] Synthesis of Intermediate 1

[0179] (1S,3S)-N1-(7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine

[0180] Step 1: Under nitrogen protection, 2-bromo-7-fluoro-[1,2,4]triazolo[1,5-a]pyridine (500 mg, 2.31 mmol), N-[(1S,3S)-3-aminocyclopentyl]carbamate tert-butyl ester (510 mg, 2.55 mmol), cesium carbonate (1.51 g, 4.63 mmol), tris(dibenzylacetone)dipalladium (424 mg, 0.46 mmol), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (536 mg, 0.92 mmol) were dissolved in 1,4-dioxane (15 mL). The mixture was microwaved to 130 °C and stirred for 2 hours. The reaction solution was filtered, and the filtrate was concentrated. The residue was analyzed by silica gel column chromatography (elution system B) and preparative HPLC (formic acid system) to give N-[(1S,3S)-3-[(7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino]cyclopentyl]carbamate tert-butyl ester (240 mg), yield: 30.9%. MS m / z (ESI): 336.0 [M+H] + .

[0181] Step 2: The product from the previous step (202 mg, 0.60 mmol) was dissolved in methanol (2 mL) at room temperature and stirred. A solution of 1,4-dioxane hydrochloric acid (4 M, 5 mL) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was concentrated, and the residue was purified by preparative HPLC (ammonia system) to obtain (1S,3S)-N1-(7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine intermediate 1 (140 mg), yield: 98.6%. MS m / z (ESI): 236.2 [M+H] + .

[0182] Synthesis of intermediate 2

[0183] (1S,3S)-N1-(7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine

[0184] Step 1: 4-(trifluoromethyl)pyridin-2-amine (5 g, 30.84 mmol) and ethyl N-(thiomethylene)carbamate (4.85 g, 37.01 mmol) were dissolved in 1,2-dichloroethane (50 mL) at room temperature and stirred for 16 hours. The reaction solution was concentrated to obtain N-[[4-(trifluoromethyl)-2-pyridyl]aminomethylthiazolyl]carbamate ethyl ester (9.05 g). The product did not require purification and was used directly in the next reaction. MS m / z (ESI): 294.1 [M+H]+ .

[0185] Step 2: The product from the previous step (9 g, 30.69 mmol), hydroxylamine hydrochloride (10.66 g, 153.44 mmol), and N,N-diisopropylethylamine (11.90 g, 92.07 mmol) were dissolved in methanol (100 mL) at room temperature and stirred for 20 minutes. The mixture was then heated to 65 °C and stirred for 3 hours. The reaction solution was concentrated, and the residue was purified by silica gel column chromatography (elution system A) to give 7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine-2-amine (5.0 g), yield: 80.60%. MS m / z (ESI): 203.1 [M+H] + .

[0186] Step 3: The product from the previous step (5 g, 24.74 mmol) and copper bromide (5.52 g, 24.74 mmol) were dissolved in acetonitrile (50 mL), and tert-butyl nitrite (12.75 g, 123.68 mmol) was added. The reaction was stirred at room temperature for 0.5 hours, then heated to 70 °C and stirred for 2 hours. The reaction solution was concentrated, the residue was diluted with ethyl acetate (150 mL), filtered, the organic phase was washed with water (100 mL), concentrated, and the residue was purified by silica gel column chromatography (elution system A) to give 2-bromo-7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridine (5 g), yield 75.99%. MS m / z (ESI): 266.0, 268.0 [M+H] + .

[0187] Step 4: Under nitrogen protection, the product from the previous step (4.5 g, 16.92 mmol), N-[(1S,3S)-3-aminocyclopentyl]carbamate tert-butyl ester (3.39 g, 16.92 mmol), cesium carbonate (11.02 g, 33.83 mmol), tris(dibenzylacetone)dipalladium (2.32 g, 2.54 mmol), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (2.94 g, 5.07 mmol) were dissolved in 1,4-dioxane (120 mL). The reaction mixture was heated to 130 °C and stirred for 16 hours. The reaction solution was filtered and concentrated. The residue was purified by silica gel column chromatography (elution system A) to give N-[(1S,3S)-3-[[7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]amino]cyclopentyl]tert-butyl carbamate (3.7 g), yield: 56.76%. MS m / z (ESI): 386.2 [M+H] + .

[0188] Step 5: The product from the previous step (3.7 g, 9.60 mmol) and hydrochloric acid (4 M in dioxane, 36.00 mL) were dissolved in methanol (10 mL) at room temperature and stirred for one hour. The reaction solution was concentrated, and the residue was diluted with methanol and the pH was adjusted to 8-10 with saturated sodium bicarbonate solution. After concentration, the residue was purified by silica gel column chromatography (elution system A) to obtain (1S,3S)-N1-(7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine (2.74 g), yield: 100%. MS m / z (ESI): 286.2 [M+H] + .

[0189] Example 1

[0190] 5'-Fluoro-6'-(((1S,3S)-3-((7-Fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one

[0191] Step 1: Intermediate 1 (60 mg, 0.26 mmol), 5-bromo-2,3-difluoropyridine (99 mg, 0.51 mmol), and N,N-diisopropylethylamine (132 mg, 1.02 mmol) were dissolved in dimethyl sulfoxide (2 mL). The reaction mixture was heated to 100 °C and stirred for 16 hours. Saturated sodium chloride solution was added to the reaction mixture, and the aqueous phase was extracted with ethyl acetate (20 mL × 2). The organic phases were combined, dried, concentrated, and the residue was separated by silica gel column chromatography to obtain (1S,3S)-N 1 -(5-bromo-3-fluoropyridin-2-yl)-N 3 -(7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine 1a (75 mg), yield: 71.86%. MS m / z (ESI): 409.1 [M+H] + .

[0192] Step 2: Under nitrogen protection, 1a (75 mg, 0.18 mmol), 2-pyridone (52 mg, 0.55 mmol), cuprous iodide (35 mg, 0.18 mmol), trans-(1R,2R)-N,N'-dimethyl-1,2-cyclohexanediamine (26 mg, 0.18 mmol) and cesium carbonate (119 mg, 0.37 mmol) were dissolved in 1,4-dioxane (1.5 mL), and the reaction was heated to 120 °C and stirred for 16 hours. A saturated ammonium chloride solution was added to the reaction mixture. The aqueous phase was extracted with ethyl acetate (25 mL × 2). The organic phases were combined, dried, and concentrated. The residue was subjected to reversed-phase HPLC (formic acid system) to prepare 5'-fluoro-6'-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one 1 (27.9 mg), yield: 35.95%. MS m / z (ESI): 424.2 [M+H] + .

[0193] 1 H NMR(400MHz,DMSO-d6)δ8.71-8.61(m,1H),7.84(d,1H),7.68-7.62(m,1H),7 .58-7.53(m,1H),7.52-7.46(m,1H),7.30-7.24(m,1H),6.94(d,1H),6.90-6 .82(m,1H),6.75(d,1H),6.46(d,1H),6.32-6.25(m,1H),4.62-4.42(m,1H), 4.24-4.09(m,1H),2.22-2.07(m,2H),2.05-1.88(m,2H),1.69-1.48(m,2H).

[0194] Example 2

[0195] 6-(5-fluoro-6-(((1S,3S)-3-((7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)pyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one

[0196] Step 1: Intermediate 2 (150 mg, 0.53 mmol), 2,3-difluoro-5-nitro-pyridine (93 mg, 0.58 mmol), and cesium carbonate (428 mg, 1.31 mmol) were dissolved in N,N-dimethylformamide (3 mL), and the mixture was heated to 80 °C and stirred for 16 hours. The reaction mixture was filtered, and the filtrate was concentrated. The residue was purified by silica gel column chromatography (elution system B) to give (1S,3S)-N1-(3-fluoro-5-nitro-2-pyridyl)-N3-[7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopentane-1,3-diamine 2a (180 mg), yield: 80.5%. MS m / z (ESI): 426.1 [M+H] + .

[0197] Step 2: Under a hydrogen atmosphere, 2a (158 mg, 0.37 mmol) and palladium / carbon (40 mg, 0.037 mmol, purity: 10%) were dissolved in methanol (10 mL), and the reaction was stirred at room temperature for 2 hours. The reaction solution was filtered, and the filtrate was concentrated to give 3-fluoro-N2-[(1S,3S)-3-[[7-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyridin-2-yl]amino]cyclopentyl]pyridin-2,5-diamine 2b (131 mg), yield: 88.5%. MS m / z (ESI): 396.1 [M+H] + .

[0198] Step 3: 2b (131 mg, 0.33 mmol), methyl 3-(bromomethyl)pyridine-2-carboxylic acid (85 mg, 0.28 mmol), and N,N-diisopropylethylamine (107 mg, 0.83 mmol) were dissolved in a mixed solvent of n-butanol (6 mL) and N,N-dimethylformamide (0.5 mL). The mixture was heated to 40 °C and stirred for 11 hours, then heated to 110 °C and stirred for 5 hours. The reaction solution was filtered, the filtrate was concentrated, and the residue was purified by preparative HPLC (formic acid system) to give 2 (100.4 mg), yield: 70.7%. MS m / z (ESI): 513.2 [M+H] + .

[0199] 1H NMR(400MHz,DMSO-d6)δ8.82(d,1H),8.76(dd,1H),8.24(d,1H),8.12(dd,1H),8.03(dd,1H),7.85(s,1H),7.63(dd,1H),7.14 (dd,1H),7.01(d,1H),6.66(d,1H),4.96(s,2H),4.59–4.15(m,2H),2.24–2.10(m,2H),2.04–1.93(m,2H),1.68–1.51(m,2H).

[0200] Example 3

[0201] 6-(5-Fluoro-6-(((1S,3S)-3-((7-Fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)aminopyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one

[0202] Step 1: Under nitrogen protection, intermediate 1 (150 mg, 0.6 mmol), 2,3-difluoro-5-nitropyridine (123 mg, 0.7 mmol), and N,N-diisopropylethylamine (233 mg, 1.8 mmol) were dissolved in N,N-dimethylformamide (5 mL), and the reaction was stirred at 25 °C for 16 hours. The reaction mixture was filtered, the filtrate was concentrated, and the residue was separated by silica gel column chromatography (elution system A) to obtain (1S,3S)-N 1 -(3-Fluoro-5-nitropyridin-2-yl)-N 3 -(7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)cyclopentane-1,3-diamine 3a (200 mg), yield: 87%. MS m / z (ESI): 376.2 [M+H] + .

[0203] Step 2: Under a hydrogen atmosphere, 3a (200 mg, 0.5 mmol) and palladium on carbon (70 mg, 10%) were dissolved in methanol (5 mL), and the reaction was heated to 50°C and stirred for 2 hours. The reaction was filtered, and the filtrate was concentrated to obtain 3-fluoro-N. 2 -((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)pyridin-2,5-diamine 3b (170 mg), yield: 97%. MS m / z (ESI): 346.2 [M+H] + .

[0204] Step 3: Under nitrogen protection, 3b (90 mg, 0.25 mmol) and N,N-diisopropylethylamine (130 mg, 1 mmol) were dissolved in a mixture of N,N-dimethylformamide (2 mL) and tert-butanol (5 mL). Methyl 3-(bromomethyl)pyridinecarboxylate (50 mg, 0.23 mmol) was added with stirring. The reaction was stirred at 25 °C for 16 hours, and then heated to 85 °C and stirred for 16 hours. The reaction was filtered, the filtrate was concentrated, and the residue was separated by preparative HPLC (formic acid system) to obtain 6-(5-fluoro-6-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)aminopyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one 3 (20 mg), yield: 17%. MS m / z (ESI): 463.2 [M+H] + .

[0205] 1 H NMR(400MHz,DMSO-d6)δ8.76(dd,1H),8.65(dd,1H),8.24(d,1H),8.12(dd,1H),8.03(dd,1H),7.63(dd,1H),7.27(dd,1H),6.86 (td,1H),6.74(d,1H),6.67(d,1H),4.96(s,2H),4.49(q,1H),4.16(q,1H),2.20–2.10(m,2H),1.98(dt,2H),1.63–1.52(m,2H).

[0206] The synthesis method of the embodiments can be referred to the above embodiments.

[0207] The NMR characterization data of the relevant embodiments are shown in the table below:

[0208] Biological testing evaluation

[0209] The present invention will be further described and explained below with reference to test examples, but these embodiments are not intended to limit the scope of the present invention.

[0210] I. Combining experiments

[0211] Test Example 1: Determination of the binding ability of the compounds of the present invention to PCSK9 protein.

[0212] 1. Experimental objective: To detect the effect of compounds on the binding of PCSK9 protein using the HTRF method.

[0213] 2. Experimental Methods:

[0214] 1) Prepare a 1x experimental buffer solution with the following components: 20mM HEPES, 150mM NaCl, 1mM CaCl2, 0.01% Tween 20, and 0.01% BSA;

[0215] 2) Prepare a 2.5x final concentration PCSK9-His working solution (30nM) using 1x experimental buffer. Add 8uL of protein solution to each well of the 384-well plate except for the low control wells, and add 8uL of 1x experimental buffer to the low control wells.

[0216] 3) Preparation of compound working solution: First, the compound in the storage solution is uniformly serially diluted with DMSO (300 uM top, 3-fold, 10 doses). Then, 3.33 uL of each serially diluted compound is pipetted into 96.7 uL of 1x experimental buffer and mixed thoroughly to obtain the prepared compound working solution (10x).

[0217] 4) Pipette 2 μL of the compound into the corresponding well and incubate at 25°C for 10 minutes;

[0218] 5) Prepare a working solution of the probe compound at a final concentration of 4x using 1x experimental buffer (90 nM), mix thoroughly, add 5 μL to each well, and incubate at 25°C for 10 minutes;

[0219] 6) Prepare a 4x final concentration Anti-His Tb working solution (4x) using 1x experimental buffer, add 5 μL to each well, and incubate at 25°C for 2 hours;

[0220] 7) Envision reading HTRF665 / 615 program.

[0221] 3. Experimental data processing methods: XLfit four-parameter log(inhibitor) vs. response-variable slope (four parameters) was used to fit the compound concentration, corresponding inhibition rate, and nonlinearity, and IC was calculated. 50 .

[0222] 4. Experimental Results:

[0223] 5. Experimental conclusions: As can be seen from the data in the table, the compounds in the embodiments of this invention have a significant binding effect on the PCSK-9 protein.

[0224] II. Cell Function Experiments

[0225] Test Example 1: Determination of the effect of the compound of the present invention on the concentration of PCSK9 secreted by HepG2 cells.

[0226] 1. Experimental objective: To detect the inhibitory effect of the compound on PCSK9.

[0227] 2. Experimental Methods:

[0228] 1) HepG2 cell line was cultured in complete medium at 37°C with 5% CO2 until 70%–90% confluence.

[0229] 2) Digest and resuspend the cells in experimental culture medium, and seed 25,000 cells / well / 200μL into a 96-well cell culture plate and incubate at 37°C and 5% CO2 for 20-24 hours.

[0230] 3) Remove the culture medium from the cell culture plate and wash each well with 200 μL of experimental culture medium.

[0231] 4) Prepare positive control compound and test compound: Dilute positive control compound and test compound on compound plate.

[0232] 5) Add the diluted compound to the cell culture plate at 250 μL per well and incubate at 37°C with 5% CO2 for 48 hours.

[0233] 6) Collect 200 μL of cell culture medium per well and freeze at -80℃ for later use.

[0234] 7) Take the cell culture medium sample out of -80℃ to dissolve, vortex, centrifuge, and set aside.

[0235] 8) Prepare standard curves: Add the corresponding volume of dilution buffer to each standard tube in sequence. Take the corresponding volume of standard from the original tube or the previous concentration tube in the order of concentration 10, 5, 2.5, 1.25, 0.625, 0.313, 0.16, 0 ng / mL and dilute it in sequence.

[0236] 9) Prepare washing solution: Dilute 10x Wash buffer with Milli-Q to 1x and set aside.

[0237] 10) According to the plate map settings for standard curve wells and sample wells, add 100 μL of the corresponding standard and culture medium sample to each well, with 2 replicates. Seal with adhesive tape, place on a shaker at room temperature, gently shake to mix, and incubate for 1 hour.

[0238] 11) Place the plate on the plate washer, set the washing solution to 350 μL per well, repeat 4 times to wash the plate.

[0239] 12) Add 100 μL of HRP-conjugated detection antibody to each well, seal with adhesive tape, mix thoroughly on a shaker, and incubate for 1 hour.

[0240] 13) Place the plate on the plate washer, set the washing solution to 350 μL per well, repeat 4 times to wash the plate.

[0241] 14) Add 100 μL of Substrate reagent to each well, protect from light, seal with adhesive tape, mix thoroughly on a shaker, and incubate for 10-20 minutes.

[0242] 15) Add 100 μL of stop solution (1N H2SO4) to each well and mix well.

[0243] 16) Measure the optical density (OD) value of each well sequentially at a wavelength of 450 nm using an ELISA reader. Perform the detection within 30 minutes after the reaction is terminated.

[0244] 3. Experimental Data Processing Method: The OD values ​​read by the microplate reader were subtracted from the OD values ​​of the standard group (0 concentration) from those of the standard, control group, and sample to obtain the actual values ​​for each well. A standard curve was then plotted using GraphPad to calculate the sample concentration. If the sample was over-diluted, the final calculation needed to multiply by the corresponding dilution factor to obtain the actual sample concentration. Inhibition rate = (actual control concentration - actual sample concentration) / actual control concentration * 100. Based on the inhibition rates corresponding to different concentrations, the IC50 was plotted using GraphPad. 50 .

[0245] 4. Experimental Results:

[0246] 5. Experimental Conclusion: As can be seen from the data in the table, the compounds in the embodiments of this invention showed a strong inhibitory effect on the concentration of PCSK9 secreted by HepG2 cells in the experiment.

[0247] III. Pharmacokinetic Experiments

[0248] Test Example 1: Pharmacokinetic Determination in Mice

[0249] 1. Experimental objective: Using C57BL / 6J mice as test animals, to study the pharmacokinetic behavior of the compound of the present invention in mice (plasma) after oral and intravenous administration.

[0250] 2. Test Plan

[0251] 2.1 Test reagents: The compounds of this invention, prepared in-house;

[0252] 2.2 Test animals: C57 mice, male, purchased from Shanghai Bikai Laboratory Animal Co., Ltd., with animal production license number (SCXK(Shanghai)2013-0006 N0.311620400001794).

[0253] 2.3 Drug preparation: Oral administration drug preparation: 10% Solutol HS15

[0254] Weigh 10 g of Solutol HS15 solid, dissolve it in 90 mL of pure water, mix well and stir with ultrasound to form a clear solution. Weigh the compound of the present invention and dissolve it in this solution, shake well, and sonicate for 15 minutes to obtain a colorless clear solution with a concentration of 0.5 mg / mL. Intravenous administration drug preparation: 5% DMSO + 10% Solutol HS15 + 85% PBS. Weigh the compound of the present invention, first add 5% DMSO according to the proportion of the total administration volume, vortex and sonicate for 2 min to completely dissolve it; then add 10% Solutol HS15, vortex and sonicate for 2 min to completely dissolve it; finally add 85% PBS, vortex and sonicate for 5 min, and filter through a 0.22-μm filter membrane to obtain a colorless transparent clear solution with a concentration of 0.2 mg / mL.

[0255] 2.4 Administration: 3 male C57 mice; after fasting overnight, they were administered PO respectively, with a dose of 5 mg / kg and an administration volume of 10 mL / kg. 3 male C57 mice; after fasting overnight, they were administered IV respectively, with a dose of 1 mg / kg and an administration volume of 5 mL / kg.

[0256] 2.5 Sample collection: Before and 0.083 (iv), 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration to the mice, 0.04 mL of blood was collected from the orbital cavity, placed in an EDTA-K2 test tube, centrifuged at 6000 rpm for 6 min at 4°C to separate plasma, and stored at -80°C; the mice were allowed to eat 4 hours after administration.

[0257] 2.6 Measurement results: The final measurement results were obtained by using the LCMS / MS method.

[0258] 3. Experimental results: The main pharmacokinetic parameters were calculated using WinNonlin 6.1. [[ID=2^0]] [[ID=2^1]]

[0259] [[ID=2^2]]4. Experimental conclusion: The pharmacokinetic measurement results of C57BL / 6J mice showed that the compound of the present invention presented good PK advantages. [[ID=2^3]] [[ID=2^4]]

[0260] [[ID=2^5]]Test Example 2, Pharmacokinetic Determination of Cynomolgus Monkeys [[ID=2^6]] [[ID=2^7]]

[0261] 1. Research objective: To investigate the pharmacokinetic behavior of the compound of the present invention in cynomolgus monkeys (plasma) after oral administration at a dose of 5 mg / kg.

[0262] 2. Experimental Design:

[0263] 2.1 Experimental reagents: The compounds in the embodiments of this invention were prepared in-house.

[0264] 2.2 Experimental animals: 3 male cynomolgus monkeys per group, from Guangxi Xiongsen, Animal Production License No.: SCXK(Gui)2021-0004).

[0265] 2.3 Formulation: Oral administration drug preparation: 10% Solutol HS15 in water

[0266] Weigh 10g of solid Solutol HS15 and add it to 90ml of ddH2O in a 100ml volumetric flask. Vortex, mix, and sonicate to obtain a clear solution. Weigh the compound and add it to a 100mL glass bottle. Add the solution, vortex, and sonicate for 10 minutes to obtain a white suspension with a concentration of 1mg / mL.

[0267] 2.4 Administration: Three male cynomolgus monkeys were fasted overnight and then administered orally; the dose was 5 mg / kg, and the administration volume was 5 mL / kg.

[0268] 2.5 Sample collection: Blood collection: 0.3 mL of blood was collected from the forelimb veins of cynomolgus monkeys before and 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after drug administration. The blood was placed in EDTA-K2 anticoagulant tubes and centrifuged at 6000 rpm for 6 min at 4℃ to separate plasma. The plasma was stored at -80℃. The monkeys were fed 4 hours after drug administration.

[0269] 2.6 Sample preparation:

[0270] 1) Add 40 μL of plasma sample to 160 μL of acetonitrile to precipitate, mix, and centrifuge at 3500×g for 5–20 minutes.

[0271] 2) Take the supernatant solution after treatment and perform LC / MS / MS analysis to determine the concentration of the analyte. LC / MS / MS instrument: AB Sciex API 4000 Qtrap.

[0272] ●Liquid phase analysis: Liquid phase conditions: Shimadzu LC-20AD pump

[0273] ● Column: Agilent ZORBAX XDB-C18 (50×2.1mm, 3.5μm) Mobile phase: Solution A is 0.1% formic acid aqueous solution, Solution B is acetonitrile Flow rate: 0.4mL / min

[0274] ●Eluting time: 0-4.0 minutes, eluent as follows:

[0275] 3. Experimental results: The main pharmacokinetic parameters were calculated using WinNonlin 6.1.

[0276] 4. Experimental Conclusions: Pharmacokinetic analysis results in cynomolgus monkeys showed that the compounds of this invention exhibited good pharmacokinetic advantages, with the compound AUC being [missing information]. 0-∞ (ng / mL×h) ranges from 20,000 to 50,000, with the AUC of the dominant compound being... 0-∞ (ng / mL×h) is 30,000 to 50,000.

[0277] Test Example 3: In vitro metabolic stability study of the compounds of the present invention in mouse, rat and human liver microsomes

[0278] 1. Experimental objective: The objective of this experiment is to evaluate the metabolic stability of the compound in phase I and part of phase II in mouse, rat and human liver microsomes.

[0279] 2. Experimental Design

[0280] 2.1 Drug Preparation: The compounds of this invention were prepared into 10 mM stock solutions using DMSO (or other suitable solutions) and stored at -20°C for later use. The compounds of this invention were prepared in-house.

[0281] 2.2 Experimental Procedure

[0282] 1) Prepare buffer solution: Take 4.01 mL of 1M K2HPO4·3H2O (AR grade) and 0.99 mL of 1M KH2PO4 (AR grade), dissolve them in ultrapure water and bring the volume to 50 mL to prepare a phosphate buffer solution with a final concentration of 100 mM.

[0283] 2) Preparation of working solution: Add 2 μL of the compound stock solution to 998 μL of phosphate buffer to achieve a final concentration of 20 μM. The ratio and final concentration can be adjusted according to the properties of the compound.

[0284] 3) Prepare liver microsome working solution: 156.3 μL of 20 mg / mL microsomes, diluted to 5 mL with 100 mM phosphate buffer, mixed well, with a final concentration of 0.625 mg / mL.

[0285] 4) Prepare NADPH and UDPGA: Weigh 33.3 mg of NADPH and 25.8 mg of UDPGA, add 2 mL of 100 mM phosphate buffer, and the final concentration of both is 20 mM.

[0286] 5) Prepare the pore-drilling agent (Alamethicin): Weigh 1 mg of Alamethicin and add it to 200 μL of methanol to prepare a solution of 5 mg / mL. Then take 10 μL of this solution and add it to 990 μL of phosphate buffer (pH 7.4) to obtain a final concentration of 50 μg / mL.

[0287] 6) Prepare the reaction termination solution: Dilute the internal standard with acetonitrile (or other suitable solution) to prepare the termination solution and store it in a refrigerator at 2-8℃.

[0288] 7) Incubation Procedure: Add 400 μL of prepared liver microsomes, 25 μL of the compound working solution (20 μM), and 25 μL of Alamethicin (50 μg / mL) sequentially to a 96-well plate, and pre-incubate at 37°C for 10 min. Then add 50 μL of prepared NADPH / UDPGA to initiate the reaction and incubate at 37°C. The total reaction volume is 500 μL. The final concentrations of each component are as follows:

[0289] At time points of 0, 5, 15, 30, 60 and 120 min, 50 μL of each sample was taken and 200 μL of cold stop solution containing internal standard was added to terminate the reaction. The sample was centrifuged at 3500 rpm for 10 min and the supernatant was taken for LC-MS / MS analysis.

[0290] 2.4 Chromatographic Analysis

[0291] 1) Chromatographic conditions:

[0292] Instrument: Shimadzu LC-20AD; Column: Phenomenex C18 (50*4.6mm, 5μm particle size); Mobile phase: A: 0.1% formic acid aqueous solution, B: acetonitrile; Wash gradient: 0.2–1.6 min 5% A to 95% A, 3.0–3.1 min 95% A to 5% A; Flow rate: 1.0 ml / min; Run time: 4.0 min; Injection volume: 5 μL.

[0293] 2) Mass spectrometry conditions

[0294] Instrument: API4000 liquid chromatography-mass spectrometry system, AB Sciex; Ion source: electrospray ionization source (ESI); Dry gas: N2, temperature 500℃; Electrospray voltage: 5000V; Detection mode: positive ion detection; Scan mode: reaction monitoring (MRM); Scan time: 0.8401s.

[0295] 3. Data Processing: Calculate the raw data using the following formula:

[0296] Residual percentage % = (Peak area ratio of compound to internal standard at any time point) / (Peak area ratio of compound to internal standard at 0 minutes) × 100

[0297] T 1 / 2 =0.693 / Ke, where Ke represents the elimination rate constant.

[0298] In vitro intrinsic clearance rate of liver microsomes (CL) was calculated using Ke. int ) and hepatic intrinsic clearance (CL) int,liver )

[0299] CL int =0.693 / T 1 / 2 / Microsomal protein content (microsomal concentration during incubation, mg / mL)

[0300] CL int,liver =CL int × Liver microsomal protein content (mg / g) × Liver weight to body weight ratio

[0301] Based on the well-stirred model, the in vivo liver clearance rate (CL) was estimated. int,liver )

[0302] CL = (CL) int,liver ×fu×Qh) / (CL int,liver ×fu+Qh), where fu represents the free fraction in the blood, which is 1 by default. The parameters in the formula are shown in the table below.

[0303] 4. Experimental Results:

[0304] 5. Experimental conclusions: The results show that the compounds of the advantageous embodiments of the present invention exhibit stable metabolic effects in various liver microsomes, especially in human liver microsomes.

[0305] IV. Drug Efficacy Experiment

[0306] Test Example 1: In vivo pharmacodynamic study of the compound of the present invention in a B6-hPCSK9 transgenic mouse model of hyperlipidemia.

[0307] 1. Experimental objective: To evaluate the in vivo efficacy of the compound in a B6-hPCSK9 transgenic mouse model of hyperlipidemia.

[0308] 2 Experimental Operation and Data Processing

[0309] 2.1 Animals: B6-hPCSK9 transgenic C57 mice, 6-8 weeks old, male, purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

[0310] 2.2 Animal Model: After animals arrived at the barrier system, they were allowed to adapt for one week before being fed a high-fat diet. Animal weight and feed intake were recorded weekly.

[0311] 2.3 Grouping and Administration

[0312] a. Grouping is done using a random grouping method.

[0313] c. Based on the grouping results, begin administering the test drug (administration route: oral; administration volume: 10 mL / kg; administration frequency: once daily or single dose; administration period: 21 days; solvent: 10% Solutol HS 15 / 90% Saline). Test drug: the compound of this invention, prepared in-house.

[0314] d. After starting the test drug, weigh and feed twice a week, and collect blood once a week.

[0315] e. Process data using software such as Excel. Body weight change rate (BWC) (%) = (Weight at the end of treatment - Weight at the beginning of treatment) / Weight at the beginning of treatment × 100%; Feed intake (g / mice / day) = (Previous feed addition + Previous feed residue - Current feed residue) / Number of animals / Number of feeding days; Calculation of blood biochemical inhibition rate: Using the blood biochemical results of the Vehicle group tested in the same batch as a baseline, normalize the data of each treatment group, and then calculate the percentage of TC and LDL-C according to the formulas: TC change percentage (%) = (TC value after administration - TC value before administration) / TC value before administration * 100%; LDL-C change percentage (%) = (LDL-C value after administration - LDL-C value before administration) / LDL-C value before administration * 100%. Detect PCSK9 in plasma using ELISA.

[0316] 3. Experimental Results:

[0317] 4. Experimental conclusion: The compounds in the embodiments of this invention can effectively reduce LDL-C in the B6-hPCSK9 transgenic mouse model of hyperlipidemia.

[0318] Crystal form study of the examples

[0319] As is well known to those skilled in the art, the compounds in the above embodiments have been shown to effectively bind to the PCSK9 protein and exhibit good inhibitory effects on PCSK9 secreted by HepG2 cells. Their crystal forms often possess the same pharmacological and efficacy activities. Based on this, the inventors further investigated the physicochemical properties of the corresponding crystal forms. However, the preparation and characterization of the specific crystal forms described below do not constitute a limitation on the scope of protection of this invention. Those skilled in the art can use this invention as a basis to obtain more crystals of the compounds of this invention through conventional crystallization methods. These crystals are all schemes protected by this invention. Specifically, as follows:

[0320] 1.1 Experimental Apparatus

[0321] 1.1.1 Some parameters of physicochemical detection instruments

[0322] 1.2 Instruments and Liquid Chromatography Analysis Conditions

[0323] 1.2.1 Instruments and Equipment

[0324] 1.2.2 Chromatographic conditions

[0325] Chromatographic column: Waters Xbridge C18 (4.6mm*150mm, 3.5μm)

[0326] Flow rate: 1.0 mL / min; Column temperature: 37℃; Detection wavelength: 230 nm

[0327] Injection volume: 5 μL; Run time: 15 min; Diluent: Methanol

[0328] Mobile phase: A: 0.1% aqueous solution of phosphoric acid; B: acetonitrile

[0329] Crystal form of the compound

[0330] Example 1: Preparation of different crystal forms of (5'-fluoro-6'-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one)

[0331] 1.1 Preparation of Free Base Crystal Form A

[0332] Method 1: Take 6.5g of free alkali, add 20mL of methanol, slurry at 60℃ for 40min, slowly cool and then cool with ice water, and pump dry to obtain the product solid. The obtained solid was tested and XRPD was found to be free alkali crystal form A.

[0333] The characteristic X-ray diffraction peaks of crystal form A, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0334] After testing and analysis, it has the XRPD diagram shown in Figure 1, the DSC diagram shown in Figure 2, and the TGA diagram shown in Figure 3.

[0335] Method 2: Add 10 mg of compound in crystalline form A or amorphous form to 1 mL of solvent, stir at room temperature, centrifuge, and dry the solid under vacuum at 40°C to obtain a solid product. X-ray powder diffraction analysis confirmed that the product is crystalline form A.

[0336] The solvent is: water, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, acetonitrile, isopropyl acetate, methyl tert-butyl ether, 2-butanone, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, n-heptane, 7% water / ethanol (v / v), 10% water / isopropanol (v / v), methanol / water (1:1, v / v), ethyl acetate / ethanol (1:1, v / v), ethyl acetate / n-heptane (1:1, v / v), isopropyl ether, acetone / cyclohexane (1:4, v / v), methanol, toluene, or 10% water / ethanol (v / v).

[0337] Method 3: Dissolve 10 mg of compound A (crystalline form or amorphous form) in solvent 1 of the table below at room temperature or 40°C, add solvent 2 of the table below, stir at room temperature to induce crystallization, centrifuge, and vacuum dry the solid at 40°C to obtain the solid product. X-ray powder diffraction analysis shows that the product is crystalline form A.

[0338] Method 4: Dissolve 10 mg of compound A (crystalline form or amorphous form) in the solvent listed in the table below at room temperature or 40°C, and allow it to evaporate and solidify at room temperature to obtain a solid product. X-ray powder diffraction analysis confirmed that the product is crystalline form A.

[0339] Method 5: Dissolve 10 mg of compound A (crystalline form or amorphous form) in 1 ml of dichloromethane at 40 °C, stir at 5 °C to induce crystallization, centrifuge, and dry the solid under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis confirmed that the product is crystalline form A.

[0340] Method 6: Dissolve 150 mg of compound crystal form A in 2.5 mL of ethanol / water (10:1, v / v) at 85 °C, cool to 70 °C, add 7.5 mg of free crystal form A seed, cool and stir to precipitate crystals, stir overnight at 25 °C, filter, and dry the solid under vacuum at 40 °C to obtain the solid product.

[0341] 1.2 Preparation of Free Base Crystal Form B

[0342] Take 6.8 g of free alkali crystal form A, add 20 mL of ethanol, then add 20 mL of 1 M hydrochloric acid methanol solution, and slurry at 50 °C for 2 days. Filter, then slurry the resulting off-white solid with the excipient HPMC E50 in water at room temperature for 1 day. Filter, and the XRPD of the resulting off-white solid is identified as free alkali crystal form B.

[0343] The characteristic X-ray diffraction peaks of crystal form B, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0344] After testing and analysis, it has the XRPD diagram shown in Figure 4, the DSC diagram shown in Figure 5, and the TGA diagram shown in Figure 6.

[0345] 1.3 Preparation of Free Base Crystal Form C

[0346] Take 7.2 g of free alkali crystal form A, add 20 mL of acetone, then add 20 mL of 1 M hydrochloric acid methanol solution, and slurry at 50 °C for 2 days. Filter, then slurry the resulting off-white solid with the excipient HEC in water at room temperature for 1 day. Filter, and the XRPD of the resulting off-white solid is identified as free alkali crystal form C.

[0347] The characteristic X-ray diffraction peaks of crystal form C, expressed as 2θ angle and interplanar spacing d, are shown in the table below:

[0348] After testing and analysis, it has the XRPD diagram shown in Figure 7, the DSC diagram shown in Figure 8, and the TGA diagram shown in Figure 9.

[0349] 1.4 Preparation of Free Base Crystal Form D

[0350] Take 6.2 g of free alkali crystal form A, add 30 mL of acetone, then add 22 mL of 1 M methanesulfonic acid methanol solution, and stir at 50 °C for 2 days. Filter, then stir the resulting off-white solid in water at room temperature for 1 day. Filter, and the XRPD of the resulting off-white solid is identified as free alkali crystal form D.

[0351] The characteristic X-ray diffraction peaks of crystal form D, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0352] After testing and analysis, it has the XRPD diagram shown in Figure 10, the DSC diagram shown in Figure 11, and the TGA diagram shown in Figure 12.

[0353] Example 2 Preparation of different crystal forms of 6-(5-fluoro-6-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)aminopyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one

[0354] 2.1 Preparation of Free Alkali Crystal Form A

[0355] 10 mg of compound 2 was added to 240 μL of DMF and stirred at 60 °C until dissolved. This solution was then added to 1.0 mL of the unsuitable solvent listed in the table below, stirred overnight at room temperature, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis confirmed that the product was crystal form A.

[0356] The characteristic X-ray diffraction peaks of crystal form A, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0357] 2.2 Preparation of Free Base Crystal Form B

[0358] 10 mg of compound 2 was added to 0.2 mL of methanol, stirred at room temperature for 3 days, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis showed that the product had crystal form B.

[0359] The characteristic X-ray diffraction peaks of crystal form B, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0360] 2.3 Preparation of Free Base Crystal Form C

[0361] 10 mg of compound 2 was added to 0.2 mL of butyl formate, stirred at room temperature / 50 °C for 1–3 days, centrifuged, and the solid was dried under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis showed that the product was crystalline form C.

[0362] The characteristic X-ray diffraction peaks of crystal form C, expressed as 2θ angle and interplanar spacing d, are shown in the table below:

[0363] 2.4 Preparation of Free Alkali Crystal Form D

[0364] 12 mg of compound 2 was dissolved in 200 μL of NMP. The clear solution was placed in a H2O atmosphere and allowed to stand until a solid precipitated. The solid was centrifuged and dried under vacuum at 40 °C to obtain the solid product. X-ray powder diffraction analysis showed that the product was crystal form D.

[0365] The characteristic X-ray diffraction peaks of crystal form D, expressed in terms of 2θ angle and interplanar spacing d, are shown in the table below:

[0366] 3. Preparation of pharmaceutical compositions

[0367] 3.1 Raw Material Formula

[0368] 3.2 Preparation method

[0369] 3.2.1 Pretreatment of raw and auxiliary materials

[0370] The raw materials were pulverized using an air jet mill, with an inlet pressure range of 0.30–0.35 MPa and a pulverizing pressure range of 0.25–0.30 MPa. Colloidal silica and sodium bicarbonate were passed through a 40-mesh sieve.

[0371] 3.2.2 Premixed

[0372] The mixture is mixed using a hopper mixer and granulated using a vacuum pelletizer.

[0373] Premix I: Add microcrystalline cellulose, HS-10510, anhydrous lactose, low-substituted hydroxypropyl cellulose, and sodium bicarbonate to a hopper mixer and mix at a speed of 10 pm for 10 min.

[0374] Granulation: The premixed material I is granulated through a vacuum granulator with a screen aperture of 0.8mm and a rotation speed of 300-900rpm.

[0375] Premix II: Transfer the granulated material back to the hopper mixer, add the internally added magnesium stearate and mix at 10 rpm for 15 minutes.

[0376] 3.2.3 Dry Granulation

[0377] The dry granulation process is adopted, and the parameters of the dry granulator are set as follows: roller speed 2.0~4.0rpm, roller pressure 2.0~5.0MPa, and feeding speed 2.0~10.0rpm.

[0378] 3.2.4 Total Mixing

[0379] The mixture was mixed using a mixer at a speed of 10 rpm for 10 minutes.

[0380] 3.2.5 Tableting

[0381] Select the mold according to the specifications (5mg specification: Φ6.0mm shallow concave, 10mg specification: Φ7.0mm shallow concave; 50mg specification: Φ10.0mm shallow concave).

[0382] 3.2.6 Coating

[0383] The coating is performed using a high-efficiency coating machine.

[0384] ① Preparation of coating solution

[0385] Add the prescribed amount of purified water to a beaker, and while stirring, add the prescribed amount of film coating premix (gastric-soluble type). Stir for ≥60 minutes, and then pass the prepared coating solution through an 80-mesh sieve for later use.

[0386] ② Coating

[0387] Add the uncoated film to the coating pan. During the coating process, check the appearance of the film and record the weight gain of the coated film. When the weight gain reaches 2.0% to 4.0%, the coating is finished and drying continues for 5 minutes.

[0388] 4. Solid stability test

[0389] 4.1 Example 1: Stability Study of Crystal Form A

[0390] The free crystal form A was laid flat in the open, and the stability of the sample was investigated under the following conditions: high temperature (40℃ and 60℃), high humidity (RH 75% and RH 92.5%) and light. The sampling period was 30 days.

[0391] Free crystal forms B, C, and D were laid out in an open manner, and their stability was investigated under high temperature (40℃ and 60℃), high humidity (RH 75% and RH 92.5%), and light conditions, respectively. The sampling period was 30 days.

[0392] Conclusion: Free crystalline form A exhibits good physical and chemical stability for 30 days under the influencing factors of high temperature (40℃, 60℃), high humidity (75%RH, 92.5%RH) and light conditions; free crystalline forms B, C, and D also exhibit good physical and chemical stability for 30 days under the same conditions.

[0393] 4.2 Long-term / accelerated stability of crystal form A in Example 1 and long-term / accelerated stability of crystal forms B, C, and D in Example 1

[0394] The stability of free crystalline form A was investigated under conditions of 25℃ / 60%RH and 40℃ / 75%RH.

[0395] Conclusion: Long-term accelerated experiments show that free crystalline form A exhibits good physical and chemical stability under conditions of 25℃ / 60%RH and 40℃ / 75%RH for 2 months; free crystalline forms B, C, and D also exhibit good physical and chemical stability under the same conditions.

[0396] 4.3 Example 2: Long-term / accelerated stability of free alkali crystal form

[0397] Free crystal forms A, B, C, and D were laid out flat in the open, and the stability of the samples was investigated under the following conditions: high temperature (60℃, 70℃, and 80℃), high humidity (RH 26%, RH 41%, RH 75%, RH 79%, and RH 80%), and light irradiation. The sampling period was 30 days.

[0398] Conclusion: Free crystalline form A exhibits good physical and chemical stability over 30 days under influencing factors of high temperature (60℃, 70℃, and 80℃) and high humidity (RH 26%, RH 41%, RH 75%, RH 79%, and RH 80%). Free crystalline forms B, C, and D also exhibit good physical and chemical stability over 30 days under the same influencing factors.

[0399] 5. Hygroscopicity test

[0400] 5.1 Experimental Objective

[0401] The hygroscopicity of different crystal forms of free bases in Examples 1 and 2 under different relative humidity conditions was investigated.

[0402] 5.2 Experimental Procedure:

[0403] Different free alkali crystal forms from Examples 1 and 2 were placed in saturated water vapor with different relative humidities to achieve dynamic equilibrium between the crystal forms and the water vapor, and the percentage of moisture absorption and weight gain after equilibrium was calculated.

[0404] 5.3 Experimental Results:

[0405] In Example 1, after one cycle of moisture absorption and desorption under a relative humidity of 0-95%, the XRPD spectrum of the free alkali crystal form A in Example 1 did not change, that is, the crystal form did not change.

[0406] In Example 1, the XRPD spectrum of the free base crystal form C did not change, that is, the crystal form did not change.

[0407] In Example 2, after one cycle of moisture absorption and desorption under 0-95% relative humidity, the XRPD spectrum of the free alkali crystal form A in Example 2 did not change. That is, the crystal form did not change.

[0408] The other free base crystal forms in Examples 1 and 2 did not change.

[0409] 6. Solubility Experiment

[0410] Weigh approximately 1.0 mg of free radical crystalline form A from Example 1 and 3.0 mg of free radical crystalline form A from Example 2 into 2 mL glass bottles. Add 1 mL of different pH buffer solutions, fasting simulated intestinal fluid (FaSSIF), non-fasting simulated intestinal fluid (FeSSIF), fasting simulated gastric fluid (FaSSGF), and pure water, respectively. Shake overnight on a micromixer at 37°C. Filter the sample solutions through a 0.45 μm nylon filter membrane and collect the filtrate. Use the external standard method to determine the concentration using HPLC.

[0411] Other free base crystal forms from Examples 1 and 2 were tested using the same experimental method. The solubility of the compounds in different buffer solutions is as follows:

[0412] Experimental results: The solubility of free alkali crystal form A in Examples 1 and 2 is pH-dependent. Similarly, the solubility of other free alkali crystal forms in Examples 1 and 2 is pH-dependent.

[0413] 7. TGA of DSCs with different crystal forms

[0414] Experimental objective: To test all crystal forms of TGA and compare their stability up to 150°C.

[0415] Experimental procedure: Take about 5 mg of different crystal forms of free base and measure the weight change at 0-300℃ using a DSC instrument.

[0416] Experimental Results: The results show that crystal form A in Example 1 exhibits no weight loss before 150℃, indicating it is an amorphous form. Crystal forms B and C in Example 1 have low melting points and both show water peaks around 100℃. Crystal form D in Example 1 shows an endothermic crystallization peak at 168℃. Therefore, crystal form A is the more stable crystal form. Crystal forms A, B, C, and D in Example 2 exhibit no weight loss before 150℃, demonstrating good stability.

[0417] 8 Crystallization Process Experiment

[0418] 8.1 Solubility Test

[0419] Test method: Weigh 20mg of the compound, add a certain amount of organic solvent step by step, sonicate and observe the dissolution phenomenon.

[0420] Test results: The test results were obtained according to the test method. Conclusion: This product is readily soluble in DMSO, DMF and methanol / dichloromethane.

[0421] Solvent system selection: Based on solubility experimental data, the preferred solvents are DMSO, DMF and methanol / dichloromethane, and the preferred antisolvents are water and methyl tert-butyl ether.

[0422] 8.2 Antisolvent crystallization

[0423] Experimental objective: To investigate the crystal form, phenomena, residual solvent, and yield of the product by crystallization through the dropwise addition of an antisolvent.

[0424] Experimental design: Weigh a certain amount of free alkali raw material, add a certain volume of good solvent, dissolve it at room temperature, then add a certain volume of poor solvent, stir at room temperature for 2 hours. Filter, and vacuum dry at 50℃ overnight.

[0425] Experimental Results: The samples obtained in the DMSO / water, DMF / water, and methanol / dichloromethane / methyl tert-butyl ether solvent systems were all of crystal form A as described in Example 1, with yields of 85%, 80%, and 85% in the three mixed solvent systems, respectively. Similarly, the free base crystal forms B, C, and D of Example 1 and the free base crystal forms A, B, C, and D of Example 2 all showed good yields in the DMSO / water, DMF / water, and methanol / dichloromethane / methyl tert-butyl ether solvent systems.

[0426] Experimental data: The specific data is shown in the table below.

[0427] Experimental conclusions: The products obtained in the DMSO / water, DMF / water, and methanol / dichloromethane / methyl tert-butyl ether solvent systems were all crystal form A of Example 1. The free base crystal forms B, C, and D of Example 1 and the free base crystal forms A, B, C, and D of Example 2 did not change in the DMSO / water, DMF / water, and methanol / dichloromethane / methyl tert-butyl ether solvent systems.

[0428] 9. PK Study of Different Crystal Forms in Rats

[0429] 9.1 Experimental Objective

[0430] Using SD rats as test animals, the pharmacokinetic behavior of a single oral administration of free alkali crystals in rat plasma was studied.

[0431] 9.2 Experimental Procedure:

[0432] In Example 1, the free alkali crystal form A was suspended evenly in an aqueous solution containing 0.5% HPMC K4M and then administered to rats by gavage. Three parallel rats were administered the drug at a dose of 30 mpk.

[0433] The free alkali crystal forms B, C, and D of Example 1 were tested using the same experimental scheme as the free alkali crystal form A of Example 1.

[0434] In Example 2, the free alkali crystal forms A, B, C, and D were suspended evenly in an aqueous solution containing 0.5% CMC-Na and 1% Tween 80, and then administered to rats by gavage. Three parallel rats were administered the drugs at doses of 5 mg / kg, 15 mg / kg, 30 mg / kg, and 50 mg / kg, respectively.

[0435] 9.3 Experimental Results:

[0436] 9.4 Experimental Conclusions:

[0437] As can be seen from the data above, the free base crystal forms of Examples 1 and 2 of the present invention can meet the requirements for high exposure at low doses and have good pharmacokinetic properties.

Claims

1. A crystal form of a compound of general formula (I) or a stereoisomer thereof: in: Ring A is selected from 5-membered nitrogen-containing heterocyclic group, 6-membered nitrogen-containing heterocyclic group, 5-membered nitrogen-containing heteroaryl group, 6-membered nitrogen-containing heteroaryl group, 5-membered 6-membered bicyclic nitrogen-containing heterocyclic group, 6-membered 5-membered bicyclic nitrogen-containing heterocyclic group, 5-membered 6-membered bicyclic nitrogen-containing heteroaryl group, 6-membered 5-membered bicyclic nitrogen-containing heteroaryl group or 6-membered 6-membered bicyclic nitrogen-containing heteroaryl group; R a Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy or C 1-6 Hydroxyalkyl; R b Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Alkoxy, C 1-6 Halogenated alkoxy or C 1-6 hydroxyalkyl; and x is 0, 1, 2, 3 or 4.

2. The crystal form of the compound or its stereoisomers according to claim 1, characterized in that, Ring A is selected from And / or, R a Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl; Preferably, R a Selected from -H, -F, -O-CH3, -O-CHF2, -O-CF3, -CH2F, -CHF2, -CH3, -CF3, -CH2-CHF 23 Or -CH2-CF3; And / or, R b Selected from hydrogen, deuterium, halogen, amino, hydroxyl, cyano, nitro, C 1-3 Alkyl, C 1-3 Deuterated alkyl, C 1-3 Haloalkyl, C 1-3 Alkoxy, C 1-3 Halogenated alkoxy or C 1-3 Hydroxyalkyl; Preferably, R b Selected from -H, -D, -F, -Cl, -CN, -CH3, -CF3, -CH(CH3)2, -C(CH3)3, -C(CH3)2-OH, -C(CH3)2-CH2-OH, -O-CH3, -CH2-NH2, -CH2-OH or -OH.

3. The crystal form of the compound or its stereoisomers according to any one of claims 1-2, characterized in that, The compounds mentioned are selected from:

4. The crystal form of the compound or its stereoisomers according to any one of claims 1-3, characterized in that, It is the crystal form of compound 1, which is 5'-fluoro-6'-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)amino)-2H-[1,3'-bipyridine]-2-one, specifically, Crystal form A has an X-ray powder diffraction pattern showing a diffraction peak at 2θ of 9.4 ± 0.2°; or at 18.7 ± 0.2°; or at 23.4 ± 0.2°; or at 20.0 ± 0.2°; or at 19.0 ± 0.2°; or at 17.7 ± 0.2°; or at 24.3 ± 0.2°. Or it has a diffraction peak at 22.3±0.2°; or it has a diffraction peak at 21.2±0.2°; or it has a diffraction peak at 28.3±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form B exhibits the following diffraction peaks in its X-ray powder diffraction pattern: 18.1 ± 0.2° at 2θ; or at 15.8 ± 0.2°; or at 27.6 ± 0.2°; or at 20.9 ± 0.2°; or at 26.5 ± 0.2°; or at 17.5 ± 0.2°; or at 24.9 ± 0.2°. The peak; or has a diffraction peak at 7.6±0.2°; or has a diffraction peak at 16.3±0.2°; or has a diffraction peak at 19.9±0.2°; preferably includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form C exhibits the following diffraction peaks in its X-ray powder diffraction pattern: 16.1 ± 0.2° at 2θ; or at 16.8 ± 0.2°; or at 21.2 ± 0.2°; or at 23.7 ± 0.2°; or at 28.3 ± 0.2°; or at 14.2 ± 0.2°; or at 22.1 ± 0.2°. Or it has a diffraction peak at 13.6±0.2°; or it has a diffraction peak at 27.3±0.2°; or it has a diffraction peak at 25.8±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form D exhibits the following X-ray powder diffraction patterns: a diffraction peak at 2θ = 24.1 ± 0.2°; or at 11.6 ± 0.2°; or at 6.4 ± 0.2°; or at 22.7 ± 0.2°; or at 24.4 ± 0.2°; or at 21.0 ± 0.2°; or at 27.3 ± 0.2°. Or it has a diffraction peak at 19.0±0.2°; or it has a diffraction peak at 15.7±0.2°; or it has a diffraction peak at 26.0±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7 or 8 of them.

5. The crystal form of the compound or its stereoisomers according to claim 4, characterized in that, The X-ray powder diffraction pattern of crystal form A includes at least one or more diffraction peaks located at 2θ of 9.4±0.2°, 18.7±0.2°, and 23.4±0.2°, preferably two, more preferably three; optionally, it may further include at least one of the following: 2θ of 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, and 22.3±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form B contains at least one or more diffraction peaks located at 2θ of 18.1±0.2°, 15.8±0.2°, and 27.6±0.2°, preferably two, more preferably three; optionally, it may further contain at least one diffraction peak located at 2θ of 20.9±0.2°, 26.5±0.2°, 17.5±0.2°, 24.9±0.2°, and 7.6±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form C includes at least one or more diffraction peaks located at 2θ of 16.1±0.2°, 16.8±0.2°, and 21.2±0.2°, preferably two, more preferably three; optionally, it may further include at least one of the following: 23.7±0.2°, 28.3±0.2°, 14.2±0.2°, 22.1±0.2°, and 13.6±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form D contains at least one or more diffraction peaks located at 2θ of 24.1±0.2°, 11.6±0.2°, and 6.4±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 22.7±0.2°, 24.4±0.2°, 21.0±0.2°, 27.3±0.2°, and 19.0±0.2°, preferably two, three, four, or five.

6. The crystal form of the compound or its stereoisomers according to any one of claims 4-5, characterized in that, The X-ray powder diffraction pattern of crystal form A optionally includes one or more diffraction peaks located at 2θ of 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, 25.8±0.2°, 15.8±0.2°, 37.9±0.2°, and 12.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks. The X-ray powder diffraction pattern of crystal form B can also be selected to include those located at 2θ of 16.3±0.2°, 19.9±0.2°, One or more diffraction peaks selected from 22.7±0.2°, 20.3±0.2°, 24.1±0.2°, 9.2±0.2°, and 16.9±0.2°; preferably including at least 2-3, 4-5, or 6-7 of these peaks; more preferably including any 2, 3, 4, 5, 6, or 7 of these peaks. The X-ray powder diffraction pattern of crystal form C may optionally include one or more diffraction peaks located at 2θ of 27.3±0.2°, 25.8±0.2°, 9.2±0.2°, 22.9±0.2°, 24.0±0.2°, 14.6±0.2°, and 31.2±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks. The X-ray powder diffraction pattern of crystal form D may optionally include one or more diffraction peaks located at 2θ of 15.7±0.2°, 26.0±0.2°, 20.5±0.2°, 20.0±0.2°, 28.6±0.2°, 23.3±0.2°, and 17.5±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

7. The crystal form of the compound or its stereoisomers according to any one of claims 4-6, characterized in that, The X-ray powder diffraction pattern of crystal form A includes one or more diffraction peaks located at 2θ of 9.4±0.2°, 18.7±0.2°, 23.4±0.2°, 20.0±0.2°, 19.0±0.2°, 17.7±0.2°, 24.3±0.2°, 22.3±0.2°, 21.2±0.2°, 28.3±0.2°, 37.6±0.2°, and 25.8±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form B includes one or more diffraction peaks located at 2θ of 18.1±0.2°, 15.8±0.2°, 27.6±0.2°, 20.9±0.2°, 26.5±0.2°, 17.5±0.2°, 24.9±0.2°, 7.6±0.2°, 16.3±0.2°, 19.9±0.2°, 22.7±0.2°, and 20.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form C includes one or more diffraction peaks located at 2θ of 16.1±0.2°, 16.8±0.2°, 21.2±0.2°, 23.7±0.2°, 28.3±0.2°, 14.2±0.2°, 22.1±0.2°, 13.6±0.2°, 27.3±0.2°, 25.8±0.2°, 9.2±0.2°, and 22.9±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form D includes one or more diffraction peaks located at 2θ of 24.1±0.2°, 11.6±0.2°, 6.4±0.2°, 22.7±0.2°, 24.4±0.2°, 21.0±0.2°, 27.3±0.2°, 19.0±0.2°, 15.7±0.2°, 26.0±0.2°, 20.5±0.2°, and 20.0±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of these peaks.

8. The crystal form of the compound or its stereoisomers according to any one of claims 1-3, characterized in that, It is the crystal form of compound 2, which is 6-(5-fluoro-6-(((1S,3S)-3-((7-fluoro-[1,2,4]triazolo[1,5-a]pyridin-2-yl)amino)cyclopentyl)aminopyridin-3-yl)-5,6-dihydro-7H-pyrrolo[3,4-b]pyridin-7-one, specifically, Crystal form A has an X-ray powder diffraction pattern showing a diffraction peak at 2θ = 24.6 ± 0.2°; or at 26.2 ± 0.2°; or at 21.5 ± 0.2°; or at 14.4 ± 0.2°; or at 15.0 ± 0.2°; or at 29.8 ± 0.2°; or at 13.0 ± 0.2°. The peak; or has a diffraction peak at 22.5±0.2°; or has a diffraction peak at 18.7±0.2°; or has a diffraction peak at 9.0±0.2°; preferably includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form B exhibits the following diffraction peaks in its X-ray powder diffraction pattern: 2θ = 24.1 ± 0.2°; or 12.7 ± 0.2°; or 12.9 ± 0.2°; or 7.7 ± 0.2°; or 14.9 ± 0.2°; or 11.8 ± 0.2°; or 26.5 ± 0.2°. Or it has a diffraction peak at 10.5±0.2°; or it has a diffraction peak at 21.5±0.2°; or it has a diffraction peak at 25.8±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form C exhibits the following diffraction peaks in its X-ray powder diffraction pattern: 2θ = 24.7 ± 0.2°; or 12.6 ± 0.2°; or 18.6 ± 0.2°; or 10.9 ± 0.2°; or 19.5 ± 0.2°; or 27.3 ± 0.2°; or 15.0 ± 0.2°. The peak; or has a diffraction peak at 8.4±0.2°; or has a diffraction peak at 28.4±0.2°; or has a diffraction peak at 25.6±0.2°; preferably includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably includes any 2, 3, 4, 5, 6, 7 or 8 of them; Crystal form D exhibits the following X-ray powder diffraction patterns: a diffraction peak at 2θ = 25.1 ± 0.2°; or at 14.0 ± 0.2°; or at 13.4 ± 0.2°; or at 16.6 ± 0.2°; or at 19.0 ± 0.2°; or at 9.6 ± 0.2°; or at 10.0 ± 0.2°. Or it has a diffraction peak at 22.1±0.2°; or it has a diffraction peak at 20.6±0.2°; or it has a diffraction peak at 15.1±0.2°; preferably it includes any 2-3, 2-5, 3-5, 3-6, 3-8, 5-8, or 6-8 of the above diffraction peaks, more preferably it includes any 2, 3, 4, 5, 6, 7 or 8 of them.

9. The crystal form of the compound according to claim 8, characterized in that, The X-ray powder diffraction pattern of crystal form A contains at least one or more diffraction peaks located at 2θ of 24.6±0.2°, 26.2±0.2°, and 21.5±0.2°, preferably two, more preferably three; optionally, it may further contain at least one diffraction peak located at 2θ of 14.4±0.2°, 15.0±0.2°, 29.8±0.2°, 13.0±0.2°, and 22.5±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form B contains at least one or more diffraction peaks located at 2θ of 24.1±0.2°, 12.7±0.2°, and 12.9±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 2θ of 7.7±0.2°, 14.9±0.2°, 11.8±0.2°, 26.5±0.2°, and 10.5±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form C contains at least one or more diffraction peaks located at 2θ of 24.7±0.2°, 12.6±0.2°, and 18.6±0.2°, preferably two, more preferably three; optionally, it may further contain at least one diffraction peak located at 2θ of 10.9±0.2°, 19.5±0.2°, 27.3±0.2°, 15.0±0.2°, and 8.4±0.2°, preferably two, three, four, or five. The X-ray powder diffraction pattern of crystal form D contains at least one or more diffraction peaks located at 2θ of 25.1±0.2°, 14.0±0.2°, and 13.4±0.2°, preferably two, more preferably three; optionally, it may further contain at least one of the following: 2θ of 16.6±0.2°, 19.0±0.2°, 9.6±0.2°, 10.0±0.2°, and 22.1±0.2°, preferably two, three, four, or five.

10. The crystal form of the compound according to any one of claims 8-9, characterized in that, The X-ray powder diffraction pattern of crystal form A may optionally include one or more diffraction peaks located at 2θ of 18.7±0.2°, 9.0±0.2°, 5.3±0.2°, 19.3±0.2°, 27.0±0.2°, 11.1±0.2°, and 17.8±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks. The X-ray powder diffraction pattern of crystal form B may optionally include one or more diffraction peaks located at 2θ of 21.5±0.2°, 25.8±0.2°, 28.1±0.2°, 25.0±0.2°, 19.1±0.2°, 16.0±0.2°, and 30.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks. The X-ray powder diffraction pattern of crystal form C may optionally include one or more diffraction peaks located at 2θ of 28.4±0.2°, 25.6±0.2°, 22.7±0.2°, 17.1±0.2°, 29.6±0.2°, 23.2±0.2°, and 22.3±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks. The X-ray powder diffraction pattern of crystal form D may optionally include one or more diffraction peaks located at 2θ of 20.6±0.2°, 15.1±0.2°, 23.6±0.2°, 19.5±0.2°, 30.8±0.2°, 11.7±0.2°, and 27.9±0.2°; preferably, it includes at least 2-3, 4-5, or 6-7 of these peaks; more preferably, it includes any 2, 3, 4, 5, 6, or 7 peaks.

11. The crystal form of the compound according to any one of claims 8-10, characterized in that, The X-ray powder diffraction pattern of crystal form A includes one or more diffraction peaks located at 2θ of 24.6±0.2°, 26.2±0.2°, 21.5±0.2°, 14.4±0.2°, 15.0±0.2°, 29.8±0.2°, 13.0±0.2°, 22.5±0.2°, 18.7±0.2°, 9.0±0.2°, 5.3±0.2°, 19.3±0.2°, 27.0±0.2°, 11.1±0.2°, and 17.8±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form B includes one or more diffraction peaks located at 2θ of 24.1±0.2°, 12.7±0.2°, 12.9±0.2°, 7.7±0.2°, 14.9±0.2°, 11.8±0.2°, 26.5±0.2°, 10.5±0.2°, 21.5±0.2°, 25.8±0.2°, 28.1±0.2°, 25.0±0.2°, 19.1±0.2°, 16.0±0.2°, and 30.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form C includes one or more diffraction peaks located at 2θ of 24.7±0.2°, 12.6±0.2°, 18.6±0.2°, 10.9±0.2°, 19.5±0.2°, 27.3±0.2°, 15.0±0.2°, 8.4±0.2°, 28.4±0.2°, 25.6±0.2°, 22.7±0.2°, 17.1±0.2°, 29.6±0.2°, 23.2±0.2°, and 22.3±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks. The X-ray powder diffraction pattern of crystal form D includes one or more diffraction peaks located at 2θ of 25.1±0.2°, 14.0±0.2°, 13.4±0.2°, 16.6±0.2°, 19.0±0.2°, 9.6±0.2°, 10.0±0.2°, 22.1±0.2°, 20.6±0.2°, 15.1±0.2°, 23.6±0.2°, 19.5±0.2°, 30.8±0.2°, 11.7±0.2°, and 27.9±0.2°; preferably, it includes any 2, 3, 4, 5, 6, 8, 10, or 12 of these peaks.

12. The crystal form of the compound or its stereoisomers according to any one of claims 4-11, characterized in that, The compound mentioned is compound 1. Its crystal form A has the X-ray powder diffraction pattern shown in Figure 1; or the DSC pattern shown in Figure 2; or the TGA pattern shown in Figure 3; Its crystal form B has the X-ray powder diffraction pattern shown in Figure 4; or the DSC pattern shown in Figure 5; or the TGA pattern shown in Figure 6. Its crystal form C has the X-ray powder diffraction pattern shown in Figure 7; or the DSC pattern shown in Figure 8; or the TGA pattern shown in Figure 9; Its crystal form D has the X-ray powder diffraction pattern shown in Figure 10; or the DSC pattern shown in Figure 11; or the TGA pattern shown in Figure 12; The compound mentioned is compound 2. Its crystal form A has the X-ray powder diffraction pattern shown in Figure 13; or the DSC pattern shown in Figure 14; or the TGA pattern shown in Figure 15; Its crystal form B has the X-ray powder diffraction pattern shown in Figure 16; or the DSC pattern shown in Figure 17; or the TGA pattern shown in Figure 18; Its crystal form C has the X-ray powder diffraction pattern shown in Figure 19; or the DSC pattern shown in Figure 20; or the TGA pattern shown in Figure 21; Its crystal form D has the X-ray powder diffraction pattern shown in Figure 22; or the DSC pattern shown in Figure 23; or the TGA pattern shown in Figure 24; The 2θ error between the positions of the top ten diffraction peaks with the highest relative peak intensities in the X-ray powder diffraction pattern and the corresponding positions of the diffraction peaks in the attached figure is ±0.2° to ±0.5°, preferably ±0.2° to ±0.3°, and most preferably ±0.2°.

13. The crystal form of the compound or its stereoisomers according to any one of claims 1-12, characterized in that, The crystal form is a solvate, hydrate, or anhydrous; the number of water molecules is 0.2-3; preferably 0.2, 0.5, 1, 1.5, 2, 2.5, or 3.

14. A method for preparing the compound or its stereoisomer crystal form as described in any one of claims 1-13, specifically comprising the following steps: Method 1: 1) Weigh an appropriate amount of free base and dissolve it in a solvent at a certain temperature; 2) Lower the temperature and stir to cool and crystallize; 3) Dry to obtain a solid product; The solvent is: Water, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, acetonitrile, isopropyl acetate, methyl tert-butyl ether, 2-butanone, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, n-heptane, water / ethanol, methanol / water, ethyl acetate / ethanol, ethyl acetate / n-heptane, isopropyl ether, acetone / cyclohexane, methanol, toluene, dichloromethane; Method 2: 1) Weigh an appropriate amount of free base and dissolve it in a good solvent at a certain temperature; 2) Add a poor solvent to the solution obtained above and stir until a solid precipitates; 3) Stir, cool, and crystallize to obtain the target product; in: The benign solvent is selected from one or more of water, methanol, acetone, dichloromethane, and dimethyl sulfoxide; preferably dichloromethane / methanol, water / acetone, or dimethyl sulfoxide. The unsuitable solvent is selected from one or more of heptane, cyclohexane, n-hexane, n-pentane, water, methyl tert-butyl ether, toluene, or isopropyl ether; preferably heptane, methyl tert-butyl ether, or water. Method 3: In both of the above methods, seed crystals can be added after step one, and the mixture stirred to induce crystallization. Method 4: 1) Weigh out an appropriate amount of free base and add a certain amount of solvent; 2) Crystallization by stirring at room temperature; 3) Dry to obtain a solid product; The solvent is: Water, ethanol, isopropanol, n-propanol, acetone, ethyl acetate, acetonitrile, isopropyl acetate, methyl tert-butyl ether, 2-butanone, 2-methyltetrahydrofuran, methyl isobutyl ketone, cyclohexane, n-heptane, water / ethanol, water / isopropanol, methanol / water, ethyl acetate / ethanol, ethyl acetate / n-heptane, isopropyl ether, acetone / cyclohexane, methanol, tetrahydrofuran, toluene; Method 5: 1) Weigh an appropriate amount of free base and dissolve it in a solvent at a certain temperature; 2) Curing occurs at room temperature; 3) A solid product is obtained; The solvents are: dimethyl sulfoxide, 1,4-dioxane, tetrahydrofuran, 10% water / methanol, 10% water / acetone, N,N-dimethylformamide, N,N-dimethylacetamide, acetonitrile / methanol, tetrahydrofuran / ethanol, benzyl alcohol, dichloromethane / methanol / ethanol, dichloromethane / methanol / acetone, dichloromethane / methanol / ethyl acetate, (10% water / acetone) / methyl tert-butyl ether, (10% water / acetone) / ethanol, (10% water / acetone) / acetone, (10% water / acetone) / ethyl acetate; Method Six: 1) Weigh an appropriate amount of free alkali or its crystal form, add a poor solvent, and then add a certain proportion of acid organic solvent solution. Then, pulp the mixture at a certain temperature, preferably 0-60℃. 2) Add an auxiliary material in proportion to the free alkali to the solid obtained after filtering the above solution, and then pulp it in water at a certain temperature, preferably 0-60℃; Alternatively, the solid obtained after filtering the above solution can be pulped in water, preferably at a temperature of 0–60°C; 3) Stir, cool, and crystallize to obtain the target product; in: The unsuitable solvent is selected from one or more of acetone, ethyl acetate, isopropyl acetate, acetonitrile, ethanol, 88% acetone, tetrahydrofuran, 2-methyltetrahydrofuran, or methanol; preferably one or more of ethanol, ethyl acetate, or acetone. The acid is selected from: hydrochloric acid, methanesulfonic acid, and acetic acid; The ratio of the free base to the acid is 1:1 to 1:2; wherein the preferred molar ratio of the acid is 1M. The organic solvent is selected from one or more of methanol, ethanol, or ethyl acetate; The excipients are selected from one or more of starch, HEC (hydroxyethyl cellulose), HPMC E5 (hydroxypropyl methyl cellulose E5), HPMC E50 (hydroxypropyl methyl cellulose E50), HPC LF (hydroxypropyl cellulose LF), and HPC EXF (hydroxypropyl cellulose EXF).

15. A pharmaceutical composition comprising a therapeutically effective dose of the compound of any one of claims 1-13 or a stereoisomer thereof, and one or more pharmaceutically acceptable carriers, diluents or excipients.

16. The pharmaceutical composition according to claim 15, characterized in that, The effective dose of the compound or its stereoisomer crystal form comprises 0.1% to 95% by weight, preferably 1% to 60%, more preferably 2% to 50%, more preferably 2% to 40%, even more preferably 2-30%, even more preferably 2-20%, even more preferably 4% to 30%, even more preferably 5% to 30%, even more preferably 5% to 20%, and specifically preferably 1%, 2%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%.

17. Use of the compound or its stereoisomer crystal form according to any one of claims 1-13, or the pharmaceutical composition according to claim 15, in the preparation of a PCSK9 inhibitor medicament.

18. The use of the compound or its stereoisomer crystal form according to any one of claims 1-13, or the pharmaceutical composition according to claim 15, in the preparation of an LDL-lowering drug.

19. The use of the compound or its stereoisomer crystal form according to any one of claims 1-13, or the pharmaceutical composition according to claim 15, in the treatment and / or prevention of cardiovascular disease, cerebrovascular disease, atherosclerosis and / or related diseases or symptoms thereof; preferably, in the treatment and / or prevention of stroke, hypercholesterolemia, hyperlipidemia, hyperlipoproteinemia, hypertriglyceridemia, dyslipidemia, dyslipoproteinemia, atherosclerosis, hepatic steatosis, metabolic syndrome and / or coronary artery disease.

Images