Pharmaceutical composition containing KRAS G12D inhibitor

Abstract

The present disclosure relates to a pharmaceutical composition comprising a KRAS G12D inhibitor. In particular, the present disclosure relates to a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt and lipid thereof. The pharmaceutical composition can be better applied clinically. # imgabs0 #

Description

A pharmaceutical composition comprising a KRAS G12D inhibitor Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition comprising a KRAS G12D inhibitor and a preparation method thereof. Background Art

[0002] RAS is one of the most frequently mutated oncogenes in tumors, with approximately 30% of human malignancies linked to RAS gene mutations. The RAS family includes KRAS, NRAS, and HRAS, with KRAS mutations being the most common, accounting for approximately 85%. KRAS mutations are common in solid tumors and are highly prevalent in the three most lethal human cancers: lung cancer (17%), colorectal cancer (33%), and pancreatic cancer (61%). Of the KRAS gene mutations, 97% involve mutations at amino acid residues 12 or 13, with G12D being a key mutation. Analysis of data from European and American populations shows that G12D mutations occur in 36%, 12%, and 4% of patients with pancreatic cancer, colorectal cancer, and non-small cell lung cancer, respectively.

[0003] Once activated, KRAS regulates multiple aspects of cell proliferation, survival, migration, and metabolism through numerous downstream signaling pathways, including RAF-MEK-ERK, PI3K-AKT-mTOR, and TIAM1-RAc. KRAS mutations cause the protein to remain in an activated state, leading to persistent activation of downstream signaling pathways and promoting tumorigenesis.

[0004] Because the KRAS protein lacks traditional small molecule binding sites on its surface and has an extremely high affinity for guanylate, making it extremely difficult to inhibit, it has long been considered an undruggable drug target. However, given the importance and prevalence of abnormal KRAS activation in cancer progression, KRAS has been and remains a target of great interest in drug development. Currently, with the exception of KRAS G12C inhibitors, there is still a lack of KRAS inhibitors that are effective against other mutations, leaving most patients with KRAS mutations without treatment. G12D, as a mutant that is widely and highly expressed in various tumors, has important clinical significance for the development of inhibitors against it.

[0005] WO2022268051 provides a new KRAS G12D inhibitor compound (Formula I) having good pharmaceutical activity.

[0006] Summary of the Invention

[0007] The present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a lipid.

[0009] In some embodiments, the lipid comprises at least one phospholipid.

[0010] In some embodiments, the pharmaceutical compositions described herein are liposomes.

[0011] Another aspect of the present disclosure provides a liposome comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0012] The lipids described in the present disclosure can include completely neutral or negatively charged phospholipids. The term "phospholipid" refers to a hydrophobic molecule containing at least one phosphorus group, which can be natural or synthetic. For example, a phospholipid can include a phosphorus-containing group and a saturated or unsaturated alkyl group that is optionally substituted with OH, COOH, oxo, amine, or substituted or unsubstituted aryl groups. Phospholipids differ from each other in the length and degree of unsaturation of their acyclic chains.

[0013] In some embodiments, the phospholipids include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and phosphatidylinositol. The term "phosphatidylcholine" refers to phosphatidylcholine and its derivatives. Examples of phospholipids suitable for use in the present disclosure include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), egg yolk phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), l-myristoyl-2-palmitoylphosphatidylcholine (MPPC), l-palmitoyl-2-myristoylphosphatidylcholine (PMPC), l-palmitoyl-2-stearoylphosphatidylcholine (PSPC), l-stearoyl-2-palmitoylphosphatidylcholine (SPPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylcholine Ethanolamine (DPPE), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dipalmitoylglycerophosphoglycerol (DPPG), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoylphosphatidylinositol (DMPI), distearoylphosphatidylinositol (DSPI), and derivatives thereof (e.g., hydrophilic polymer-modified derivatives).

[0014] The hydrophilic polymer-modified phospholipids include, but are not limited to, polyethylene glycol-modified distearoylphosphatidylethanolamine (PEG-DSPE), polyethylene glycol-modified distearoylphosphatidylglycerol (PEG-DSPG), and polyethylene glycol-modified cholesterol (PEG-CHOL). The molecular weight of PEG is 200-10,000 daltons, for example, 1,000-5,000 daltons, or 2,000-5,000 daltons.

[0015] In some embodiments, the phospholipids described in the present disclosure are selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine (DSPE) and dimyristoylphosphatidylcholine (DMPC) and their polyethylene glycol-modified derivatives.

[0016] In some embodiments, the liposomes contain polyethylene glycol-modified distearoylphosphatidylethanolamine. In some embodiments, the polyethylene glycol in the polyethylene glycol-modified distearoylphosphatidylethanolamine has a molecular weight of 200-10,000 daltons. In some embodiments, the polyethylene glycol in the polyethylene glycol-modified distearoylphosphatidylethanolamine has a molecular weight of 1,000-5,000 daltons. In some embodiments, the polyethylene glycol in the polyethylene glycol-modified distearoylphosphatidylethanolamine has a molecular weight of 2,000-5,000 daltons.

[0017] In some embodiments, the polyethylene glycol-modified distearoylphosphatidylethanolamine is selected from polyethylene glycol 2000-distearoylphosphatidylethanolamine (mPEG2000-DSPE).

[0018] In some embodiments, the molar content of phospholipids relative to the amount of the total lipid mixture is selected from 10 to 80%, including but not limited to 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80% or any value between any two values, for example, it can be 30-70%.

[0019] The present disclosure may also comprise other neutral lipids, cationic lipids and / or anionic lipids.

[0020] Examples of other neutral lipids that can be used in the present disclosure include: one or more of steroids such as cholesterol and its derivatives, phosphatidylcholine, soybean lecithin, cephalin, sphingomyelin, and hydrogenated soybean lecithin. In some embodiments, the molar percentage of steroids relative to the total lipid mixture is no more than 90%.

[0021] In some embodiments, the lipid comprises at least one phospholipid and cholesterol. Relative to the total lipid mixture, the molar percentage of cholesterol may be 0.1%-90%, for example, 10%-80%, or 20%-70%.

[0022] In some embodiments, the phospholipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine (DSPE) and dimyristoylphosphatidylcholine (DMPC) and their polyethylene glycol-modified derivatives.

[0023] In some embodiments, the lipid comprises hydrogenated soy phosphatidylcholine, cholesterol, and polyethylene glycol 2000-distearoylphosphatidylethanolamine.

[0024] In some embodiments, the content of the hydrophilic polymer-modified phospholipid derivative (relative to the total liposome membrane components, molar fraction) is selected from 0.1 to 50%, including but not limited to 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%. , 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or any value between any two values, for example, it can be 0.5~20%.

[0025] In some embodiments, the pharmaceutical composition further comprises a capture agent. In some embodiments, the aqueous phase within the liposome comprises a capture agent.

[0026] The preparation method of the liposomes can adopt an active drug loading method. The active drug loading method is a drug loading method in which the therapeutic agent is transported from the external medium across the bilayer membrane of the liposome to the internal aqueous space through a polyatomic ion gradient. These gradients are generated by embedding at least one polyatomic ion as a capture agent in the internal aqueous space of the liposome and replacing the external medium of the liposome with an additional medium with a lower polyatomic ion concentration, such as pure water, sucrose solution or normal saline, through known techniques such as column separation, dialysis or centrifugation. A polyatomic ion gradient is created between the internal aqueous space of the liposome and the external medium to capture the therapeutic agent in the internal aqueous space of the liposome.

[0027] The capture agents disclosed herein include, but are not limited to, sulfates, sulfites, gluconates, phosphates, hydrogen phosphates, molybdates, carbonates, and nitrates. Exemplary capture agents include, but are not limited to, ammonium sulfate, ammonium phosphate, ammonium molybdate, ammonium sucrose octasulfate, triethylammonium sucrose octasulfate, copper gluconate, ethylammonium sulfate, ammonium salt of ethylenediaminetetraacetic acid, ammonium chloride, ammonium hydroxide, ammonium acetate, dextran sulfate ammonium, triethyl dextran sulfate ammonium, and combinations thereof.

[0028] In some embodiments, the capture agent is ammonium sulfate or copper gluconate.

[0029] In some embodiments, the concentration of the capture agent in the aqueous phase within the liposome is selected from 10 to 400 mM, and can be 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 5mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220m M, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM, 305mM, 310mM, 315mM, 320mM, 325mM, 330mM, 335mM, 340mM, 345mM, 350mM, 355mM, 360mM, 365mM, 370mM, 375mM, 380mM, 385mM, 390mM, 395mM, 400mM or any value between any two values, for example, 20-400mM.

[0030] In some embodiments, the pharmaceutical composition is a liposome comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, a lipid, and a capture agent, wherein the lipid comprises at least one phospholipid and a steroid.

[0031] In some embodiments, the phospholipid is selected from dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine (DSPE) and dimyristoylphosphatidylcholine (DMPC) and their polyethylene glycol-modified derivatives; and the steroid is cholesterol.

[0032] In some embodiments, the aqueous phase within the liposomes described herein further comprises at least one pH adjuster. The pH adjuster can be selected from amino acids such as arginine, histidine, and glycine; acids such as ethylenediaminetetraacetic acid, pentetic acid, hydrochloric acid, phosphoric acid, malic acid, or sulfuric acid; salts such as sodium, potassium, and ammonium salts of the aforementioned acids; and alkaline compounds (bases) such as ammonia, sodium hydroxide, potassium hydroxide, and triethanolamine. Examples of such pH adjusters include sodium hydroxide, hydrochloric acid, ammonia, phosphoric acid, malic acid, histidine, and triethanolamine. Furthermore, a combination of two or more of the aforementioned ammonium salts can be used.

[0033] In some embodiments, the concentration of the pH regulator in the aqueous phase within the liposome is selected from 1 to 300 mM, and can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM or any value between any two values, for example, 20 to 300mM.

[0034] In some embodiments, the concentration of the compound of formula I or a pharmaceutically acceptable salt thereof is selected from 0.01 to 100 mg / mL, which can be 0.01 mg / mL, 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, 0.35 mg / mL, 0.40 mg / mL, 0.45 mg / mL, 0.50 mg / mL, 0.55 mg / mL, 0.60 mg / mL, 0.65 mg / mL, 0.70 mg / mL, 0.75 mg / mL, 0.80 mg

[0014] The dosage form may be any value between 0.05 and 10 mg / mL, for example, 0.05 to 10 mg / mL, 0.15 to 0.25 mg / mL, 0.25 to 0.35 mg / mL, 0.40 to 0.50 mg / mL, 0.60 to 0.75 mg / mL, 0.80 to 0.95 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, or 100 mg / mL, or any value between any two values, for example, 0.05 to 10 mg / mL.

[0035] On the other hand, in order to improve the encapsulation efficiency of the compound of formula I or its pharmaceutically acceptable salt in liposomes, a solution containing an electrolyte (salt solution) can be added to the liposome external phase to increase the ionic strength, thereby increasing the encapsulation efficiency. The electrolyte (salt) contained in the liposome external phase is not particularly limited, for example, it can be sodium chloride, potassium chloride, for example, sodium chloride. In addition, normal saline can also be used. In addition, the liposome external phase as a liposome dispersion, etc., can contain sugars, electrolytes, and / or amino acids or their salts, and can also contain sugars or electrolytes, and amino acids or their salts. As an example, sugars are selected from sucrose or trehalose or glucose, electrolytes are selected from normal saline or sodium chloride or potassium chloride, and amino acids or their salts can be selected from histidine or its salts.

[0036] In some embodiments, the liposome external phase contains sodium chloride and histidine or a salt thereof.

[0037] In some embodiments, the concentration of the amino acid in the liposome external phase is selected from 1 to 300 mM, and can be 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM , 145mM, 150mM, 155mM, 160mM, 165mM, 170mM, 175mM, 180mM, 185mM, 190mM, 195mM, 200mM, 205mM, 210mM, 215mM, 220mM, 225mM, 230mM, 235mM, 240mM, 245mM, 250mM, 255mM, 260mM, 265mM, 270mM, 275mM, 280mM, 285mM, 290mM, 295mM, 300mM or any value between any two values, for example, 5-200mM.

[0038] In some embodiments, the pharmaceutical composition is a liposome, which comprises a compound of Formula I or a pharmaceutically acceptable salt thereof and a lipid, wherein the lipid comprises at least one phospholipid and a steroid, the inner aqueous phase of the liposome comprises a capture agent and a pH adjuster, and the outer aqueous phase of the liposome comprises one or more selected from sugars, electrolytes, and amino acids or their salts.

[0039] In some embodiments, the pH of the internal aqueous phase of the liposomes described herein is 5-8, and the pH of the external phase is 5-11. Preferably, the pH of the internal aqueous phase is 5-8, and the pH of the external phase is 5-8.

[0040] The present disclosure also provides a method for preparing liposomes, comprising the steps of preparing blank liposomes, and introducing the compound of formula I or a pharmaceutically acceptable salt thereof into the aqueous phase within the blank liposomes.

[0041] The present disclosure can prepare blank liposomes by using a thin film dispersion method, a reverse phase evaporation method, a direct hydration method, a surfactant removal method, an ethanol injection method, and a modified ethanol injection method.

[0042] In some embodiments, probe ultrasound, high-pressure homogenizer, microfluidizer, extruder, and other devices can be used to obtain blank liposomes with the desired particle size.

[0043] In addition, various parameters during the preparation of liposomes (such as the amount of membrane components, temperature, etc.) can be appropriately adjusted and determined according to the liposome preparation method or the target liposome composition, particle size, etc.

[0044] On the other hand, the replacement or dilution operation of the present disclosure can be performed by dialysis, centrifugation, gel filtration, etc. In addition, by replacing or diluting the liposome external phase, the active substance can also be effectively encapsulated in the liposome aqueous phase.

[0045] Dialysis, centrifugation and gel filtration are all conventional operations in the art. For example, dialysis can be performed using a dialysis membrane, which can be made of polyethersulfone.

[0046] In some embodiments, the pharmaceutical composition is a liquid preparation, and the liposome composition obtained by the above-mentioned introduction steps can be directly used as the final liposome composition (pharmaceutical composition), or the liposomal external phase of the liposome composition obtained can be adjusted (replaced, etc.) to obtain the final liposome composition (pharmaceutical composition).

[0047] In other embodiments, the pharmaceutical composition is a solid preparation, and the liposome composition obtained by the above-mentioned introduction step can be dried to form a final solid liposome composition (pharmaceutical composition). The method for drying the liposome composition can include but is not limited to freeze drying and spray drying.

[0048] On the other hand, the pharmaceutical composition is a solid preparation. The pharmaceutical composition can be used as a liquid preparation by dissolving or suspending it in an appropriate solvent. The solvent can be appropriately set according to the purpose of use of the liposome composition. For example, when the pharmaceutical composition is used as an injection, the solvent is preferably sterile distilled water.

[0049] The present disclosure also provides a lyophilized composition, which is obtained by freeze-drying the aforementioned pharmaceutical composition, or the aforementioned pharmaceutical composition is obtained by reconstituted the lyophilized composition with a liquid medium, wherein the liquid medium used for reconstitution is selected from physiological saline, water for injection, glucose injection or glucose and sodium chloride injection.

[0050] The present disclosure further provides use of the aforementioned pharmaceutical composition, liposome, lyophilized composition or reconstituted solution in the preparation of a medicament for treating and / or preventing a disease or condition, wherein the disease or condition is cancer.

[0051] The diseases or conditions described in the present disclosure are selected from brain cancer, thyroid cancer, head and neck cancer, nasopharyngeal cancer, pharyngeal cancer, oral cancer, salivary gland cancer, esophageal cancer, stomach cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, small intestine cancer, gastrointestinal stromal tumors, urothelial cancer, urethral cancer, bladder cancer, breast cancer, vaginal cancer, ovarian cancer, endometrial cancer, cervical cancer, fallopian tube cancer, testicular cancer, prostate cancer, hemangioma, leukemia, lymphoma, myeloma, skin cancer, lipoma, bone cancer, soft tissue sarcoma, neurofibroma, glioma, neuroblastoma and glioblastoma.

[0052] In some embodiments, the disease or condition is selected from pancreatic cancer, colorectal cancer, and non-small cell lung cancer.

[0053] The pharmaceutical composition disclosed herein has good stability, high liposome encapsulation efficiency, large drug loading capacity, and low liposome allergic reaction, and can be better applied in clinical practice.

[0054] The “mass volume percentage” (w / v) described in the present disclosure refers to the mass (in g) of the component contained in every 100 mL of liquid system, that is, g / 100 mL.

[0055] The contents (including percentages) of the various substances described in the present disclosure and the ratios therebetween are allowed to have an error of ±5%. For example, "the content of the compound of formula I or its pharmaceutically acceptable salt in the composition is 5-20 mg / mL" means that the content of the compound of formula I or its pharmaceutically acceptable salt in the composition is 4.75-21 mg / mL, all of which are within the scope of the present disclosure; "the weight ratio of the compound of formula I or its pharmaceutically acceptable salt to the lipid is selected from 1:1 to 1:100" means that the weight ratio of the compound of formula I or its pharmaceutically acceptable salt to the lipid is selected from 1:0.95 to 1:105, all of which are within the scope of the present disclosure.

[0056] The terms "mixed" and "mixed" in the present disclosure do not limit the order in which the components are added. For example, mixing A into B can mean either adding A to B or adding B to A. Mixing A and B can mean either adding A to B or adding B to A.

[0057] Liposomes are microscopic lipid vesicles composed of a bilayer of phospholipids or any similar amphiphilic lipids that encapsulate an internal aqueous medium. The liposomes disclosed herein may be multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs), or small unilamellar vesicles. They typically range in size from 30 nm to 200 nm. Furthermore, there are no specific limitations on the membrane structure of the liposomes disclosed herein.

[0058] The liposome membrane is a double layer of phospholipids that separates the inner aqueous medium from the outer aqueous medium.

[0059] The intraliposomal aqueous phase refers to the aqueous region surrounded by the lipid bilayer of the liposome and is used synonymously with "internal aqueous phase" and "internal aqueous phase of the liposome." When liposomes are dispersed in a liquid, the "extraliposomal phase" refers to the region not surrounded by the lipid bilayer of the liposome (i.e., the region excluding the internal aqueous phase and the lipid bilayer).

[0060] Blank liposomes refer to liposomes in which the aqueous phase does not contain the active ingredient to be loaded, such as the compound of formula I disclosed herein.

[0061] Encapsulation efficiency refers to the percentage of the encapsulated substance (e.g., a drug) in the total drug content of the liposome suspension. It is an important indicator for liposome and nanoparticle quality control, reflecting the degree to which the drug is encapsulated by the carrier.

[0062] Encapsulation efficiency includes both percent encapsulation and encapsulation volume. Literature generally focuses on encapsulation percentage (EN%). The formula is EN% = (1 - Cf / Ct) × 100%. Where Cf is the amount of free drug, and Ct is the total amount of drug in the liposome suspension.

[0063] The encapsulation efficiency can be determined by conventional methods for determining the encapsulation efficiency, such as the HPLC method.

[0064] In this disclosure, the term "polymer molecular weight" refers to the number average molecular weight, which is the statistical average of the molecular weights of all polymer chains in a sample and is defined as follows: i Represents the molecular weight of a single chain, N i Represents the number of chains with the corresponding molecular weight. M n It can be predicted by the polymerization mechanism and determined by measuring the number of molecules in a given mass of sample; for example, colligative methods such as end group analysis. n To characterize the molecular weight distribution, M n There are equal numbers of molecules distributed on both sides.

[0065] The term "particle size distribution" or "PSD" refers to the particle size distribution determined by dynamic light scattering techniques well known to those skilled in the art, such as using a Malvern Mastersizer TM The particle size distribution of the liposome composition is measured using a 2000 μg / ml particle size distribution analyzer. As used herein, "d(0.1)" refers to the particle size at which the cumulative particle size distribution percentage of a sample reaches 10%. "d(0.5)" refers to the particle size at which the cumulative particle size distribution percentage of a sample reaches 50%. "d(0.9)" refers to the particle size at which the cumulative particle size distribution percentage of a sample reaches 90%. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] FIG1 is a cryo-scanning electron microscopy (Cryo-TEM) image of liposomes in Example 5;

[0067] FIG2 is a cryo-scanning electron microscopy (Cryo-TEM) image of the liposomes of Example 6. DETAILED DESCRIPTION

[0068] The present disclosure is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure. The present disclosure is incorporated herein by reference in its entirety by reference to PCT / CN2022 / 100016.

[0069] The structures of the compounds were determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS). -6 The unit of ppm is given. NMR measurements were performed using a Bruker AVANCE-400 NMR spectrometer or a Bruker AVANCE NEO 500M NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), or deuterated methanol (CD3OD), and tetramethylsilane (TMS) was used as the internal standard.

[0070] MS was measured using an Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS liquid spectrometer-mass spectrometer (manufacturer: Agilent, MS model: 6110 / 6120 Quadrupole MS).

[0071] waters ACQuity UPLC-QD / SQD (Manufacturer: waters, MS model: waters ACQuity Qda Detector / waters SQ Detector)

[0072] THERMO Ultimate 3000-Q Exactive (Manufacturer: THERMO, MS model: THERMO Q Exactive)

[0073] High performance liquid chromatography (HPLC) analysis was performed using Agilent HPLC 1200DAD, Agilent HPLC 1200VWD and Waters HPLC e2695-2489 high pressure liquid chromatographs.

[0074] Chiral HPLC analysis was performed using an Agilent 1260 DAD high performance liquid chromatograph.

[0075] High performance liquid chromatography (HPLC) was performed using Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 preparative chromatographs.

[0076] Chiral preparations were performed using a Shimadzu LC-20AP preparative chromatograph.

[0077] The CombiFlash rapid preparation instrument used was Combiflash Rf200 (TELEDYNE ISCO).

[0078] The thin layer chromatography silica gel plate uses Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plate. The specification of the silica gel plate used in thin layer chromatography (TLC) is 0.15mm~0.2mm, and the specification used for thin layer chromatography separation and purification products is 0.4mm~0.5mm.

[0079] Silica gel column chromatography generally uses Yantai Huanghai Silica Gel 200-300 mesh silica gel as the carrier.

[0080] Average kinase inhibition rate and IC 50 The values ​​were determined using a NovoStar microplate reader (BMG, Germany).

[0081] The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, Darui Chemicals, and other companies.

[0082] Unless otherwise specified in the examples, all reactions can be carried out under an argon atmosphere or a nitrogen atmosphere.

[0083] Argon atmosphere or nitrogen atmosphere means that the reaction bottle is connected to an argon or nitrogen balloon with a capacity of about 1 L.

[0084] Hydrogen atmosphere means that the reaction bottle is connected to a hydrogen balloon with a capacity of about 1L.

[0085] The pressurized hydrogenation reaction uses a Parr 3916EKX hydrogenator and a Qinglan QL-500 hydrogen generator or an HC2-SS hydrogenator.

[0086] The hydrogenation reaction is usually carried out by evacuating the chamber and filling it with hydrogen, and the operation is repeated three times.

[0087] A CEM Discover-S 908860 microwave reactor was used for the microwave reaction.

[0088] Unless otherwise specified in the examples, the solution refers to an aqueous solution.

[0089] Unless otherwise specified in the examples, the reaction temperature is room temperature, 20°C to 30°C.

[0090] The reaction progress in the examples was monitored by thin layer chromatography (TLC). The developing solvent used in the reaction, the eluent system for column chromatography used to purify the compound, and the developing solvent system for thin layer chromatography included: A: dichloromethane / methanol system, B: n-hexane / ethyl acetate. The volume ratio of the solvents was adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid could also be added for adjustment.

[0091] Where the compounds in the examples contain two or more chiral centres, the relative stereochemistry of these compounds was determined by NMR studies and / or X-ray diffraction. In these cases, the prefix "rel" followed by the R / S nomenclature is used to identify these compounds, where the R / S only provides relative stereochemical information and does not indicate absolute stereochemistry. For example, express A 1:1 mixture of the two compounds is called a racemate.

[0092] Example 1

[0093] (±)-rel-(1R,2R,5S)-2-(hydroxymethyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 1g

[0094] first step

[0095] (±)-5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylic acid methyl ester 1b

[0096] Methyl (±)-2-pyrrolidone-5-carboxylate 1a (100 g, 698.61 mmol, Shanghai Bidex) and dimethyl sulfate (110 g, 872.10 mmol) were mixed and reacted at 60°C for 16 hours. The reaction solution was cooled to room temperature and poured into a solution of triethylamine (100 g) and methyl tert-butyl ether (150 mL) under an ice bath. After extraction with methyl tert-butyl ether (300 mL × 6), the mixture was concentrated under reduced pressure to obtain the crude title compound 1b (90 g, yield: 81.9%), which was used directly in the next step without purification.

[0097] MS m / z(ESI):158.1[M+1].

[0098] Step 2

[0099] (±)-5-(2-methoxy-1-nitro-2-oxoethylidene)pyrrolidine-2-carboxylic acid methyl ester 1c

[0100] The crude compound 1b (90 g, 572.64 mmol) and methyl nitroacetate (68.18 g, 572.63 mmol) were mixed, heated to 60°C and stirred for 30 hours. After the reaction solution was cooled to room temperature, ethyl acetate (300 mL) was added, stirred for 0.5 hours, and filtered. The filter cake was dried to obtain the title compound 1c (70 g, yield: 50%), which was used directly in the next step without purification.

[0101] MS m / z(ESI):245.1[M+1].

[0102] Step 3

[0103] 4-Oxo-3,8-diazabicyclo[3.2.1]octane-2-carboxylic acid methyl ester (diastereomeric mixture) 1d

[0104] Crude compound 1c (14 g, 57.3 mmol) was dissolved in 600 mL of methanol, and 10% palladium-on-carbon catalyst (wet) (14 g) was added. The atmosphere was replaced with hydrogen three times, and the reaction was stirred for 48 hours. The reaction solution was filtered through celite, and the filtrate was concentrated to obtain the crude title compound 1d (10 g, 94.6% yield), which was used directly in the next step without purification.

[0105] MS m / z(ESI):185.2[M+1].

[0106] Step 4

[0107] 8-(tert-Butyl)2-methyl(±)-rel-(1R,2R,5S)-4-oxo-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 1e

[0108] The crude compound 1d (10 g, 54.2 mmol) was dissolved in 300 mL of dichloromethane. Triethylamine (16 g, 158.12 mmol) and di-tert-butyl dicarbonate (11 g, 50.4 mmol, Shanghai Shaoyuan) were added under ice-cooling. The reaction was stirred for 14 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent system B to give the title compound 1e (3.3 g, yield: 21.3%).

[0109] MS m / z(ESI):285.2[M+1].

[0110] HPLC analysis: retention time 1.02 minutes, purity: 98.5% (chromatographic column: ACQUITY C18, 1.7 μm, 2.1*50 mm; mobile phase: water (10 mM ammonium bicarbonate), acetonitrile, gradient ratio: acetonitrile 10%-95%).

[0111] Step 5

[0112] 8-(tert-Butyl)2-methyl(±)-rel-(1R,2R,5S)-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 1f

[0113] Compound 1e (400 mg, 1.4 mmol) was dissolved in 2 mL of tetrahydrofuran, and 3.5 mL of a 2 M solution of borane dimethyl sulfide complex in tetrahydrofuran was added. The mixture was stirred and reacted for 14 hours. Methanol was added to the reaction solution to quench the reaction, and the reaction was continued at 50°C for 14 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent System A to obtain the title compound 1f (176 mg, yield: 46.2%).

[0114] MS m / z(ESI):271.2[M+1].

[0115] Step 6

[0116] (±)-rel-(1R,2R,5S)-2-(hydroxymethyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 1g

[0117] Compound 1f (1 g, 3.69 μmol) was dissolved in 15 mL of tetrahydrofuran, and 4.4 mL of 1 M lithium aluminum hydride solution in tetrahydrofuran was added. The mixture was stirred at 0°C for 1 hour. 0.2 mL of water, 0.2 mL of 15% aqueous sodium hydroxide solution, and 0.4 mL of water were added to the reaction solution in sequence. Anhydrous sodium sulfate was then added and stirred for 10 minutes. The mixture was filtered and the filtrate was concentrated to give 1 g of the title compound (430 mg, yield: 47.9%). The product was used directly in the next step without purification.

[0118] MS m / z(ESI):243.1[M+1].

[0119] Example 2

[0120] 2,6-Dichloro-3-fluoropyridin-4-amine 2a

[0121] Compound 1h (5 g, 30.6 mmol) was dissolved in 20 mL of N,N-dimethylformamide and 20 mL of acetonitrile, and 1-chloromethyl-4-fluoro-1,4-diazabicyclo[2.2.2]octane bis(tetrafluoroborate) salt (13 g, 36.8 mmol) was added. The mixture was reacted at 80°C for 0.5 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography with eluent System B to give the title compound 3a (2.2 g, yield: 39.6%).

[0122] MS m / z(ESI):180.9[M+1].

[0123] Example 3

[0124] 5-Ethyl-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptacyclo[2-yl]naphtho[1,8-ab]heptacyclo[2-yl]naphtho[3-ol]

[0125] 5-Ethyl-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl)naphthalen-2-ol 3-p2

[0126] first step

[0127] (±)-rel-(1R,2R,5S)-tert-butyl 2-(((tert-butyldimethylsilyl)oxy)methyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate 3a

[0128] Compound 1g (8.8g, 36.3mmol), tert-butyldimethylsilyl chloride (16g, 106.1558mmol), and 4-dimethylaminopyridine (4g, 32.4739mmol) were dissolved in 200mL of dichloromethane. Triethylamine (15g, 148.23mmol, 21.4286mL) was added and stirred for 16 hours. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography with eluent System B to give the title compound 3a (8g, yield: 61.7%).

[0129] MS m / z(ESI):357.1[M+1].

[0130] Step 2

[0131] tert-Butyl 4-((tert-Butoxycarbonyl)amino)-2,6-dichloro-5-fluoronicotinate 3b

[0132] Compound 2a (1.8 g, 9.94 mmol) was dissolved in tetrahydrofuran (50 mL). 20 mL of a 2 M solution of sodium bistrimethylsilylamide in tetrahydrofuran was added under ice-cooling. The mixture was stirred for 0.5 hour, and then di-tert-butyl dicarbonate (6.5 g, 29.7 mmol) was added. The mixture was stirred for 14 hours. Saturated aqueous ammonium chloride was added to the reaction solution for quenching. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified with eluent System B to give the title compound 3b (1 g, yield: 26.3%), which was used in the next step without further purification.

[0133] MS m / z(ESI):381.1[M+1].

[0134] Step 3

[0135] tert-Butyl 4-amino-2,6-dichloro-5-fluoronicotinate 3c

[0136] Compound 3b (1 g, 2.62 mmol) was dissolved in ethyl acetate (8 mL), and 3 mL of 4 M hydrochloric acid in dioxane was added. The reaction was stirred for 2 hours. The pH was adjusted to neutral with 4 M aqueous sodium hydroxide solution under ice bath, and extracted with ethyl acetate (10 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was purified with eluent System B to give the crude title compound 3c (500 mg, yield: 67.8%).

[0137] MS m / z(ESI):281.1[M+1].

[0138] Step 4

[0139] tert-Butyl 2,6-dichloro-5-fluoro-4-(3-(2,2,2-trichloroacetyl)ureido)nicotinate 3d

[0140] The crude compound 3c (500 mg, 1.77 mmol) was dissolved in tetrahydrofuran (10 mL), and trichloroacetyl isocyanate (670 mg, 3.55 mmol) was added. The mixture was stirred for 30 minutes, and the reaction solution was concentrated under reduced pressure to give the crude title compound 3d (835 mg, yield: 99.7%), which was used directly in the next step without purification.

[0141] MS m / z(ESI):467.9[M+1].

[0142] Step 5

[0143] 5,7-Dichloro-8-fluoro-pyrido[4,3-d]pyrimidine-2,4-diol 3e

[0144] The crude compound 3d (835 mg, 1.77 mmol) was dissolved in 7 M ammonia methanol solution (10 mL) and stirred for 1 hour. The reaction solution was concentrated under reduced pressure, and methyl tert-butyl ether (10 mL) was added to the residue. After stirring for 0.5 hour, the mixture was filtered and the filter cake was dried to give the crude title compound 3e (400 mg, yield: 89.9%), which was used directly in the next step without purification.

[0145] MS m / z(ESI):249.9[M+1].

[0146] Step 6

[0147] 2,4,5,7-Tetrachloro-8-fluoro-pyrido[4,3-d]pyrimidine 3f

[0148] The crude compound 3e (300 mg, 1.19 mmol) was dissolved in phosphorus oxychloride (6 mL), and N,N-diisopropylethylamine (800 mg, 6.19 mmol) was added. The reaction was stirred at 110°C for 3 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure to obtain the crude title compound 3f (344 mg, yield: 97.7%). The product was used directly in the next step without purification.

[0149] MS m / z(ESI):285.8[M+1].

[0150] Step 7

[0151] (±)-rel-(1R,2R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-(2,5,7-trichloro-8-fluoropyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester 3g

[0152] Compound 3f (1.0 g, 3.48 mmol) and N,N-diisopropylethylamine (0.9 g, 6.9 mmol) were dissolved in 15 mL of dichloromethane. 3a (1.25 g, 3.5 mmol) was added at -78°C. The reaction was stirred for 1 hour and then returned to room temperature for 16 hours. The reaction solution was concentrated under reduced pressure and the residue was purified with eluent system B to give the crude title compound 3g (1.56 g, yield: 73.7%).

[0153] MS m / z(ESI):606.2[M+1].

[0154] Step 8

[0155] tert-Butyl (1S,2S,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-(5,7-dichloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolazin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate 3h-1

[0156] and

[0157] A diastereomeric mixture of tert-butyl (1R,2R,5S)-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-(5,7-dichloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate 3h-2

[0158] Compound 3g (1.4 g, 2.3 mmol) was dissolved in 1,4-dioxane (20 mL), and ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizine-7a(5H)-yl)methanol (650 mg, 4.08 mmol, Yao Ming), N,N-diisopropylethylamine (1.5 g, 11.6 mmol), and 4A molecular sieves (1.4 g) were added. The reaction was stirred at 105°C for 6 hours. The reaction solution was cooled to room temperature, filtered, and concentrated under reduced pressure to obtain a crude diastereomeric mixture of the title compounds 3h-1 and 3h-2 (1.68 g, yield: 99.8%). The product was used directly in the next step without purification.

[0159] MS m / z(ESI):729.2[M+1]

[0160] Step 9

[0161] (5aS,6S,9R)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl-14-carboxylic acid tert-butyl ester 3i-1

[0162] and

[0163] A diastereomeric mixture of (5aR,6R,9S)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl-14-carboxylic acid tert-butyl ester 3i-2

[0164] Tetrabutylammonium fluoride (2.59 g, 11.51 mmol) was added to the diastereomeric mixture of crude compounds 3h-1 and 3h-2 (1.68 g, 2.3 mmol) and stirred at room temperature for 16 hours; the reaction solution was concentrated under reduced pressure, and the residue was purified with eluent system B to obtain a diastereomeric mixture of the title compounds 3i-1 and 3i-2 (1.0 g, yield: 75.0%).

[0165] MS m / z(ESI):579.2[M+1].

[0166] Step 10

[0167] (5aS,6S,9R)-2-(8-ethyl-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl-14-carboxylic acid tert-butyl ester 3j-1

[0168] and

[0169] A 1:1 mixture of diastereomers of (5aR,6R,9S)-2-(8-ethyl-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptane-14-carboxylic acid tert-butyl ester 3j-2

[0170] A mixture of diastereomers of compounds 3i-1 and 3i-2 (1.9 g, 3.28 mmol), 2-(8-ethyl-3-(methoxymethoxy)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.5 g, 4.38 mmol, prepared using the method disclosed for Intermediate 21 on page 111 of the specification of patent application "WO2021 / 041671"), tetrakis(triphenylphosphine)palladium (1.16 g, 1 mmol, adamas), and cesium carbonate (4.7 g, 14.4 mmol) were dissolved in 36 mL of a mixture of 1,4-dioxane and water (V:V = 5:1). The mixture was reacted at 100°C under a nitrogen atmosphere for 6 hours. The reaction solution was then concentrated under reduced pressure to obtain a crude 1:1 mixture of diastereomers of the title compounds 3j-1 and 3j-2 (2.5 g, 100% yield).

[0171] MS m / z(ESI):759.2[M+1].

[0172] Step 11

[0173] 5-Ethyl-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptan-2-yl)naphthalen-2-ol 3p-1

[0174] 5-Ethyl-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptacyclo[2-yl]naphtho[1,8-ab]heptacyclo[2-yl]naphtho[2-yl]ol 3p-2

[0175] The crude diastereomeric mixture of compounds 3j-1 and 3j-2 (2.4 g, 3.16 mol) was dissolved in ethyl acetate (40 mL), and 17 mL of 4 M hydrochloric acid-dioxane solution was added. The reaction was carried out at 0°C for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Waters-2545, column: SharpSil-T C18, 30*150 mm, 5 μm; mobile phase: aqueous phase (10 mmol / L ammonium bicarbonate) and acetonitrile, gradient ratio: acetonitrile 38%-45%, flow rate: 30 mL / min) to give a 1:1 mixture of diastereoisomers of the title compounds 3p-1 and 3p-2 (620 mg, yield: 32.6%).

[0176] MS m / z(ESI):615.2[M+1].

[0177] 1 H NMR (500MHz, CD3OD): δ7.63(d,1H),7.37(q,1H),7.29(q,1H),7.18(dd,1H),7.08(t,1H),5.48-5.29(m,2H),4.55-4.28(m,4H),4.17(dd,1 H),3.84-3.75(m,1H),3.70(t,1H),3.47(q,1H),3.31-3.07(m,3H),2.57-2.17(m,5H),2.15-1.79(m,7H),1.63(t,1H),1.08-0.87(m,3H).

[0178] The diastereomeric mixture of compounds 3p-1 and 3p-2 was separated by chiral column (Shimadzu LC-20AP, column: DAICEL 25*250mm, 10μm; mobile phase A: n-hexane, mobile phase B: ethanol (0.1% 7M NH3in MeOH)) and, gradient ratio: A:B: 30:70, flow rate: 30mL / min) to obtain the title compounds 3p-1 (50mg, yield: 35.7%) and 3p-2 (50mg, yield: 35.7%).

[0179] Single configuration compound (shorter retention time) 3p-2: (50 mg, yield: 35.7%).

[0180] MS m / z(ESI):615.2[M+1].

[0181] Chiral HPLC analysis: retention time 9.85 minutes, purity: 99% (chromatographic column: DAICEL 250*4.6mm, 5μm; mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0mL / min).

[0182] 1H NMR (500MHz, CD3OD): δ7.61-7.59(m,1H),7.36-7.32(m,1H),7.26-7.25(m,1H),7.18-7.13(m 1H),7.05-6.95(m,1H),5.37-5.22(m,2H),5.09-5.00(m,1H),4.61-4.56( m,1H),4.49-4.41(m,1H),4.30(dd,1H),4.24-4.18(m,1H),4.16-4.09(m,1 H),3.72(dd,1H),3.62(dd,1H),3.27-3.17(m,3H),3.01(td,1H),2.48(dt, 1H),2.40–2.10(m,5H),1.99(td,2H),1.94-1.75(m,4H),0.99-0.88m,3H).

[0183] Single configuration compound (longer retention time) 3p-1: (50 mg, yield: 35.7%).

[0184] MS m / z(ESI):615.2[M+1].

[0185] Chiral HPLC analysis: retention time 16.0 minutes, purity: 99% (chromatographic column: DAICEL 250*4.6mm, 5μm; mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0mL / min).

[0186] 1 H NMR (500MHz, CD3OD): δ7.61-7.59(m,1H),7.36-7.32(m,1H),7.26-7.25(m,1H),7.18-7.13(m 1H),7.05-6.94(m,1H),5.36-5.33(m,2H),5.10-5.01(m,1H),4.61-4.56(m,1H ),4.49-4.41(m,1H),4.30(dd,1H),4.24-4.22(m,1H),4.16-4.10(m,1H),3.73- 3.72(m,1H),3.64-3.61(m,1H),3.26-3.20(m,3H),3.04-2.99(m,1H),2.48(dt, 1H),2.38-2.17(m,5H),2.03-1.96(m,2H),1.93-1.78(m,4H),0.99-0.88m,3H).

[0187] Example 4

[0188] 5-Ethyl-6-fluoro-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptacyclo-2-yl)naphthalen-2-ol 4p-1

[0189] 5-Ethyl-6-fluoro-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl)naphthalen-2-ol 4p-2

[0190] Step 10

[0191] (5aS,6S,9R)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl-14-carboxylic acid tert-butyl ester 4a-1

[0192] and

[0193] A 1:1 mixture of diastereomers of (5aR,6R,9S)-2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptane-14-carboxylic acid tert-butyl ester 4a-2

[0194] A diastereomeric mixture of compounds 3i-1 and 3i-2 (300 mg, 518.1 μmol), 2-(8-ethyl-7-fluoro-3-(methoxymethoxy)naphthalen-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (280 mg, 777.2 μmol, prepared by the method disclosed in Intermediate 18 on page 104 of the specification of patent application "WO2021 / 041671"), tetrakis(triphenylphosphine)palladium (120 mg, 103.8 μmol), and cesium carbonate (506 mg, 1.55 mmol) were dissolved in 6 mL of a mixed solution of 1,4-dioxane and water (V:V=5:1). The reaction was carried out at 100°C for 6 hours under nitrogen atmosphere. The reaction solution was concentrated under reduced pressure to give a crude diastereoisomer mixture of the title compounds 4a-1 and 4a-2 in a ratio of 1:1 (400 mg). The product was used directly in the next step without purification.

[0195] MS m / z(ESI):777.2[M+1].

[0196] Step 11

[0197] 5-Ethyl-6-fluoro-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptacyclo-2-yl)naphthalen-2-ol 4p-1

[0198] 5-Ethyl-6-fluoro-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolizin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methano[1,8-ab]heptyl)naphthalen-2-ol 4p-2

[0199] The crude diastereomeric mixture of compounds 4a-1 and 4a-2 (160 mg, 205.9 μmol) was dissolved in ethyl acetate (5 mL), and 1 mL of 4 M hydrochloric acid-dioxane solution was added. The reaction was carried out at 0°C for 2 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified by high performance liquid chromatography (Waters-2545, column: SharpSil-T C18, 30*150 mm, 5 μm; mobile phase: aqueous phase (10 mmol / L ammonium bicarbonate) and acetonitrile, gradient ratio: acetonitrile 38%-45%, flow rate: 30 mL / min) to give the title compound 4p-1 (i.e., the compound of Formula I) and a 1:1 diastereomeric mixture of 4p-2 (10 mg, yield: 7.2%).

[0200] MS m / z(ESI):633.2[M+1].

[0201] 1 H NMR (500MHz, CD3OD): δ7.67(ddd,1H),7.32-7.21(m,2H),7.11-7.01(m,1H),5 .38-5.35(m,2H),5.11-5.03(m,1H),4.64-4.59(m,1H),4.52-4.46(m,1H),4. 34-4.29(m,1H),4.25(dd,1H),4.18-4.12(m,1H),3.74(br,1H),3.65(br,1H) ,3.26-3.23(m,3H),3.05-3.01(m,1H),2.61-1.81(m,12H),0.94-0.82(m,3H).

[0202] The diastereomeric mixture of compounds 4p-1 and 4p-2 was separated by chiral column (Shimadzu LC-20AP, column: DAICEL 25*250mm, 10μm; mobile phase A: n-hexane, mobile phase B: ethanol (0.1% 7M NH3in MeOH)) and, gradient ratio: A:B: 40:60, flow rate: 30mL / min) to obtain the title compounds 4p-1 (26mg, yield: 43.3%) and 4p-2 (26mg, yield: 43.3%).

[0203] Single configuration compound (shorter retention time) 4p-2: (26 mg, yield: 43.3%).

[0204] MS m / z(ESI):633.2[M+1].

[0205] Chiral HPLC analysis: retention time 7.89 minutes, purity: 99% (chromatographic column: DAICEL 250*4.6mm, 5μm; mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0mL / min).

[0206] 1 H NMR (500MHz, CD3OD): δ7.67(ddd,1H),7.32-7.21(m,2H),7.11-7.01(m,1H), 5.38-5.27(m,2H),5.11-5.03(m,1H),4.64-4.59(m,1H),4.52-4.44(m,1H), 4.33(d,1H),4.24(dd,1H),4.18-4.12(m,1H),3.75(br,1H),3.66(br,1H),3 .27-3.18(m,3H),3.05-3.03(m,1H),2.60-1.81(m,12H),0.93-0.82(m,3H).

[0207] Single configuration compound (longer retention time) 4p-1: (26 mg, yield: 43.3%).

[0208] MS m / z(ESI):633.2[M+1].

[0209] Chiral HPLC analysis: retention time 13.8 minutes, purity: 99% (chromatographic column: DAICEL 250*4.6mm, 5μm; mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0mL / min).

[0210] 1 H NMR (500MHz, CD3OD): δ7.67(ddd,1H),7.32-7.21(m,2H),7.11-7.01(m,1H),5 .38-5.35(m,2H),5.11-5.03(m,1H),4.64-4.59(m,1H),4.52-4.46(m,1H),4. 34-4.29(m,1H),4.25(dd,1H),4.18-4.12(m,1H),3.74(br,1H),3.65(br,1H) ,3.26-3.23(m,3H),3.05-3.01(m,1H),2.61-1.81(m,12H),0.94-0.82m,3H).

[0211] Example 5

[0212] Weigh copper gluconate, add an appropriate amount of water, and stir to dissolve evenly. Adjust the pH to 7.4 with triethanolamine, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 7.4 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0213] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0214] Example 6

[0215] Weigh ammonium sulfate and phosphoric acid, add an appropriate amount of water, and stir to dissolve evenly. Adjust the pH to 5.85 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0216] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0217] Example 7

[0218] Weigh ammonium sulfate and phosphoric acid, add an appropriate amount of water, stir and dissolve evenly, adjust the pH to 7.0 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0219] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0220] Example 8

[0221] Weigh ammonium sulfate and citric acid monohydrate, add an appropriate amount of water, stir and dissolve evenly, adjust the pH to 7.0 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0222] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0223] Example 9

[0224] Weigh ammonium sulfate and malic acid, add an appropriate amount of water, stir and dissolve evenly, adjust the pH to 7.0 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0225] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0226] Example 10

[0227] Weigh ammonium sulfate and histidine hydrochloride, add an appropriate amount of water, stir and dissolve evenly, adjust the pH to 7.0 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0228] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0229] Example 11

[0230] Weigh ammonium sulfate and phosphoric acid, add an appropriate amount of water, and stir to dissolve evenly. Adjust the pH to 5.85 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0231] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0232] Example 12

[0233] Weigh ammonium sulfate and phosphoric acid, add an appropriate amount of water, and stir to dissolve evenly. Adjust the pH to 5.85 with aqueous ammonia, and dilute to 180 ml to obtain aqueous solution 1. Weigh histidine hydrochloride and sodium chloride, dissolve them in an appropriate amount of water, adjust the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, and dilute to 2400 ml to obtain aqueous solution 2.

[0234] Weigh hydrogenated soybean phosphatidylcholine, cholesterol, and phosphatidylethanolamine, add anhydrous ethanol and heat with stirring to dissolve to obtain a lipid solution. The lipid solution is added to the above-mentioned aqueous solution 1, stirred for about 15 minutes under the condition higher than the phospholipid phase transition temperature, then continued to be hydrated and decompressed to remove most of the ethanol, and the whole granules are squeezed out by an extruder to obtain blank liposomes. The blank liposomes are processed with a tangential flow filtration system, and the extraliposomal aqueous phase is replaced with an aqueous solution 2. The compound of formula I is dissolved in an ethanolic solution, and an appropriate amount of hydrochloric acid is added to obtain a drug solution of 120mg / ml. The drug solution is slowly added to the blank liposomes, incubated for 15 minutes under the condition higher than the phospholipid phase transition temperature, rapidly cooled to room temperature, concentrated to the target concentration with a tangential flow system and further removed to remove residual ethanol, constant volume, to obtain a liposome product, and product characterization information is as shown in the following table.

[0235] Biological evaluation

[0236] Test Example 1: Biological Evaluation of ERK Phosphorylation Inhibition Experiment in AGS Cells (HTRF Method)

[0237] 1. Test Purpose

[0238] This experiment was conducted to detect the inhibitory effect of the compound shown in formula I on ERK phosphorylation in cells. 50 The size of the compound was evaluated to determine its inhibitory effect on the KRAS target.

[0239] 2. Experimental Methods

[0240] AGS cells (Nanjing Kebai, CBP60476) were cultured in RPMI1640 (Hyclone, SH30809.01) complete medium supplemented with 10% fetal bovine serum. On the first day of the experiment, AGS cells were seeded at a density of 40,000 cells / well in a 96-well plate using complete medium, with 190 μL of cell suspension per well. The cells were incubated overnight at 37°C in a 5% CO2 incubator.

[0241] The next day, 10 μL of a serial dilution of the test compound prepared in complete medium was added to each well. The final concentration of the compound was a five-fold serial dilution starting from 10 μM, with nine concentration points. A blank control containing 0.5% DMSO was set up. The plate was incubated in a cell culture incubator at 37°C and 5% CO2 for 1 hour. After incubation, the 96-well cell culture plate was removed, the medium was aspirated, and 200 μL of PBS (Shanghai Yuanpei Biotechnology Co., Ltd., B320) was added to each well for washing. The PBS was aspirated, and 50 μL of lysis buffer (Cisbio, 64KL1FDF) containing blocking reagent (Cisbio, 64KB1AAC) was added to each well. The plate was shaken on a shaker at room temperature for 40 minutes. After lysis, pipette to mix thoroughly. Transfer 16 μL of lysate per well to two HTRF 96-well assay plates (Cisbio, 66PL96100). Then, add 4 μL of premixed phosphorylated ERK1 / 2 antibody solution (Cisbio, 64AERPEG) or 4 μL of premixed total ERK1 / 2 antibody solution (Cisbio, 64NRKPEG) to each plate. Seal the plates with sealing film, centrifuge in a microplate centrifuge for 1 minute, and incubate overnight at room temperature in the dark.

[0242] On the third day, the fluorescence values ​​were read using an ENVISION multifunctional microplate reader (PerkinElmer, ENVISION) with excitation at 337 nm and emission at 665 nm and 620 nm.

[0243] 3. Data Analysis and Results

[0244] Graphpad Prism software was used to calculate the IC of the inhibitory activity of the compound based on the compound concentration and the ratio of phosphorylated ERK / total ERK. 50 The results are shown in Table 1 below.

[0245] Table 1 Data on ERK phosphorylation inhibitory activity in AGS cells

[0246] Conclusion: The compound represented by formula I has a good inhibitory effect on ERK phosphorylation in AGS cells.

[0247] Test Example 2: Biological Evaluation of GP2d and AGS Cell 3D Proliferation Inhibition Experiment

[0248] 1. Test Purpose

[0249] The inhibitory effect of the compound represented by Formula I on the KRAS target was evaluated by testing the 3D proliferation inhibitory effect of the compound represented by Formula I on GP2d and AGS cells.

[0250] 2. Experimental Methods

[0251] GP2d cells (Nanjing Kebai, CBP60010) were cultured in complete medium (DMEM / high glucose medium (Hyclone, SH30243.01) supplemented with 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, GP2d cells were seeded at a density of 1000 cells / well in a 96-well low attachment plate (Corning, CLS7007-24EA) using complete medium. 90 μL of cell suspension was added to each well, centrifuged at 2000 rpm for 5 minutes at room temperature, and then incubated overnight in a 37°C, 5% CO2 cell incubator.

[0252] AGS cells (Nanjing Kebai, CBP60476) were cultured in complete medium (RPMI1640 medium (Hyclone, SH30809.01) supplemented with 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, AGS cells were seeded at a density of 1000 cells / well in a 96-well low attachment plate (Corning, CLS7007-24EA) using complete medium. 90 μL of cell suspension was added to each well, centrifuged at 2000 rpm for 5 minutes at room temperature, and then incubated overnight at 37°C in a 5% CO2 incubator.

[0253] On the second day, 10 μL of a serial dilution of the test compound prepared in complete culture medium was added to each well. The final concentration of the compound for GP2d cells was 9 concentration points of a 3-fold serial dilution starting from 1 μM, and the final concentration of the compound for AGS cells was 9 concentration points of a 3-fold serial dilution starting from 10 μM. A blank control containing 0.5% DMSO was set up for both. The well plate was placed in a cell culture incubator at 37°C and 5% CO2 for 120 hours. On the seventh day, the 96-well cell culture plate was removed and 50 μL of the test compound was added to each well. 3D reagent (Promega, G9682) was shaken at room temperature for 25 minutes, then pipetted to mix thoroughly and 50 μL was transferred to a white opaque 96-well plate (PE, 6005290). The luminescent signal was read using a multi-function microplate reader (PerkinElmer, ENVISION).

[0254] 3. Data Analysis and Results

[0255] The IC of the inhibitory activity of the compounds was calculated using Graphpad Prism software 50 The results are shown in Table 2 below.

[0256] Table 2 3D proliferation inhibitory activity data of AGS and GP2d cells

[0257] Conclusion: The compound represented by formula I has a good inhibitory effect on the 3D proliferation of AGS and GP2d cells.

[0258] Test Example 3: SPR method to detect the affinity of compounds to KRAS protein isoforms G12D or WT

[0259] Biotinylated Avi-KRAS-WT or Avi-KRAS-G12D was diluted to 20 μg / mL in 1× HBS-P+ (Cat.#BR1006-71) buffer containing 100 mM MgCl2 and then passed through channel 2 of an SA (Cat.#BR1005-31) biosensor chip for 420 s, achieving a coupling level of approximately 5000-7000 RU. Compound samples were then injected sequentially from high to low concentration for 120 s, followed by a 720 s dissociation period. The experiment was performed in single-cycle kinetic mode. The reaction signal was detected in real time on a Biacore 8K instrument to generate binding and dissociation curves. Data analysis was performed using Biacore 8K evaluation software, with a 1:1 model used for data fitting and affinity data. The results are shown in Table 3 below.

[0260] Conclusion: The compound represented by formula I has good affinity to KRAS protein isoform G12D or WT.

Claims

1. A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a lipid, 2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is a liposome.

3. The pharmaceutical composition according to claim 1 or 2, wherein the lipid comprises at least one phospholipid, preferably dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), dioleoylphosphatidylcholine (DOPC), dierucoylphosphatidylcholine (DEPC), egg yolk phosphatidylcholine (EPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soybean phosphatidylcholine (HSPC), l-myristoyl-2-palmitoylphosphatidylcholine (MPPC), l- Palmitoyl-2-myristoylphosphatidylcholine (PMPC), l-palmitoyl-2-stearoylphosphatidylcholine (PSPC), l-stearoyl-2-palmitoylphosphatidylcholine (SPPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dipalmitoylglycerophosphatidylglycerol (DPPG), di Palmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dipalmitoylphosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoylphosphatidylinositol (DMPI), distearoylphosphatidylinositol (DSPS), Acylphosphatidyl inositol (DSPI), and their derivatives, more preferably dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dieucoylphosphatidylcholine (DEPC), dilauroylphosphatidylcholine (DLPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylethanolamine (DSPE) and dimyristoylphosphatidylcholine (DMPC) and their polyethylene glycol-modified derivatives, most preferably hydrogenated soybean phosphatidylcholine (HSPC) and polyethylene glycol 2000-distearoylphosphatidylethanolamine (mPEG2000-DSPE).

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the molar percentage of the phospholipids relative to the total lipid mixture is selected from 10% to 80%, preferably 30% to 70%.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the lipid further comprises a steroid, preferably cholesterol.

6. The pharmaceutical composition according to claim 5, wherein the molar percentage of the steroid relative to the amount of the total lipid mixture is selected from 0.1% to 90%, preferably 10% to 80%, more preferably 20% to 70%.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising a capturing agent, wherein the capturing agent is preferably sulfate, sulfite, gluconate, phosphate, hydrogen phosphate, molybdate, carbonate and nitrate, more preferably ammonium sulfate, ammonium phosphate, ammonium molybdate, sucrose octasulfate ammonium, sucrose octasulfate triethylammonium, copper gluconate, ethylammonium sulfate, edetate ammonium salt, ammonium chloride, ammonium hydroxide, ammonium acetate, dextran sulfate ammonium or triethyl dextran sulfate ammonium and combinations thereof.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the aqueous phase within the liposome further contains at least one pH regulator, preferably sodium hydroxide, hydrochloric acid, ammonia water, phosphoric acid, malic acid, histidine, or triethanolamine.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the concentration of the compound of formula I or a pharmaceutically acceptable salt thereof is selected from 0.01 to 100 mg / mL, preferably 0.05 to 10 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the liposome external phase contains one or more selected from sugars, electrolytes, amino acids or their salts, preferably sodium chloride and histidine or its salts.

11. A freeze-dried composition obtained by freeze-drying or spray-drying the pharmaceutical composition according to any one of claims 1 to 10.

12. A freeze-dried composition, which can be reconstituted to obtain the pharmaceutical composition according to any one of claims 1 to 10. 13 . A reconstituted solution obtained by reconstituted the freeze-dried composition according to claim 11 .

14. A liposome comprising a compound of formula I or a pharmaceutically acceptable salt thereof, 15. A method for preparing the pharmaceutical composition according to any one of claims 2 to 10 or the liposome according to claim 14, the method comprising: The invention relates to a step of preparing blank liposomes and introducing the compound of formula I or a pharmaceutically acceptable salt thereof into the aqueous phase of the blank liposomes.

16. Use of the pharmaceutical composition of any one of claims 1 to 10, the lyophilized composition of claim 11 or 12, the reconstituted solution of claim 13, or the liposome of claim 14 in the preparation of a medicament for treating a disease or condition, wherein the disease or condition is preferably cancer, more preferably selected from brain cancer, thyroid cancer, head and neck cancer, nasopharyngeal cancer, pharyngeal cancer, oral cancer, salivary gland cancer, esophageal cancer, gastric cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, colorectal cancer, small intestine cancer, gastrointestinal stromal tumor, urothelial cancer, urethral cancer, bladder cancer, breast cancer, vaginal cancer, ovarian cancer, endometrial cancer, cervical cancer, fallopian tube cancer, testicular cancer, prostate cancer, hemangioma, leukemia, lymphoma, myeloma, skin cancer, lipoma, bone cancer, soft tissue sarcoma, neurofibroma, glioma, neuroblastoma and glioblastoma.

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