Pharmaceutical composition containing a KRAS G12D inhibitor

Abstract

The present disclosure relates to a pharmaceutical composition comprising a KRAS G12D inhibitor. Specifically, the present disclosure relates to a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a lipid. The pharmaceutical composition can be used more clinically favorably. 【Chemical 1】 JPEG2025524799000032.jpg55110

Description

Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing a KRAS G12D inhibitor and a method for preparing the same.

Background Art

[0002] RAS is one of the oncogenic genes with the highest mutation rate in tumors, and approximately 30% of human malignant tumors are associated with mutations in the RAS gene. The RAS family includes KRAS, NRAS, and HRAS. Among them, KRAS mutations are the most common, accounting for approximately 85%. KRAS mutations are frequently found in solid tumors, and high-frequency mutations exist in lung cancer (17%), colorectal cancer (33%), and pancreatic cancer (61%), which are the three major lethal cancers in humans. Among the gene mutations of KRAS, 97% are those in which the 12th or 13th amino acid residue is mutated, and among them, G12D is an important mutation. Data analysis of the European and American populations has shown that in pancreatic cancer, colorectal cancer, and non-small cell lung cancer, G12D mutations account for 36%, 12%, and 4% of patients, respectively.

[0003] After KRAS is activated, various functions such as cell proliferation, survival, migration, and metabolism are regulated by many downstream signaling pathways represented by RAF-MEK-ERK, PI3K-AKT-mTOR, and TIAM1-RAc. After the KRAS gene mutates, the protein is continuously in an activated state, so that the downstream signaling pathway is continuously activated to promote the occurrence of tumors.

[0004] The KRAS protein has no small molecule binding site in the traditional sense on its surface and has an extremely high affinity for guanine acid and is extremely difficult to inhibit. Therefore, for a long time, it has been regarded as a drug target that is impossible to develop drugs against. However, due to the importance and universality of the abnormal activation of KRAS in cancer progression, KRAS has always been, and still is, a target that has received great attention in drug development. Currently, in addition to KRAS G12C inhibitors, there is still a lack of effective KRAS inhibitors for other mutations, so most patients with KRAS mutations still have no therapeutic drugs. Since G12D is a variant that is widely highly expressed in various tumors, the development of inhibitors targeting it has important clinical significance.

[0005] WO2022268051 provides a new KRAS G12D inhibitor compound (Formula I), and this compound has relatively good pharmaceutical activity.

[0006]

Chemical formula

Summary of the Invention

[0007] The present disclosure aims to provide a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0008] One aspect of the present disclosure provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and a lipid.

[0009]

Chemical formula

[0010] In some embodiments, the lipid comprises at least one phospholipid.

[0011] In some embodiments, the pharmaceutical composition described in the present disclosure is a liposome.

[0012] Another aspect of the present disclosure provides liposomes comprising a compound of Formula I or a pharmaceutically acceptable salt thereof.

[0013] The lipids described in the present disclosure may include fully neutral or negatively charged phospholipids. The term "phospholipid" refers to a hydrophobic molecule containing at least one phosphorus group and may be natural or synthetic. For example, a phospholipid may include a phosphorus-containing group and a saturated or unsaturated alkyl group optionally substituted with an OH, COOH, oxo, amine, or substituted or unsubstituted aryl group. Phospholipids differ from one another in the length and degree of unsaturation of their acyclic chains.

[0014] In some embodiments, the phospholipid includes one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidic acid, and phosphatidylinositol. The term "phosphatidylcholine" refers to phosphatidylcholine and its derivatives. Examples applicable to the phospholipids of the present disclosure include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC), dioleoylphosphatidylcholine (DOPC), dieicoylphosphatidylcholine (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), dipalmitoylphosphatidylethanolamine (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), dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidylinositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidylinositol (DOPI), dimyristoyl phosphatidylinositol (DMPI), distearoyl phosphatidylinositol (DSPI) and derivatives thereof (e.g., derivatives modified with hydrophilic polymers) are included.,

[0015] The phospholipids modified with the above hydrophilic polymers include, but are not limited to, distearoyl phosphatidylethanolamine modified with polyethylene glycol (PEG-DSPE), distearoyl phosphatidylglycerol modified with polyethylene glycol (PEG-DSPG), and cholesterol modified with polyethylene glycol (PEG-CHOL). The molecular weight of PEG is 200 to 10,000 daltons, and may be, for example, 1,000 to 5,000 daltons, or 2,000 to 5,000 daltons.,

[0016] In some embodiments, the phospholipids described in the present disclosure are selected from dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dielaidoyl phosphatidylcholine (DEPC), dilauroyl phosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylcholine (DMPC) and derivatives thereof modified with polyethylene glycol.,

[0017] In some embodiments, the liposomes comprise distearoyl phosphatidylethanolamine modified with polyethylene glycol. In some embodiments, the distearoyl phosphatidylethanolamine modified with polyethylene glycol has a polyethylene glycol molecular weight of 200 to 10,000 daltons. In some embodiments, the distearoyl phosphatidylethanolamine modified with polyethylene glycol has a polyethylene glycol molecular weight of 1,000 to 5,000 daltons. In some embodiments, the distearoyl phosphatidylethanolamine modified with polyethylene glycol has a polyethylene glycol molecular weight of 2,000 to 5,000 daltons.

[0018] In some embodiments, the distearoyl phosphatidylethanolamine modified with polyethylene glycol is selected from polyethylene glycol 2000 - distearoyl phosphatidylethanolamine (mPEG2000 - DSPE).

[0019] In some embodiments, based on the amount of the total lipid mixture, the molar content of the phospholipid is selected from 10% to 80%, including 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 numbers, but not limited thereto, for example, it may be 30% - 70%.

[0020] The present disclosure may also include other neutral lipids, cationic lipids and / or anionic lipids.

[0021] Examples that can be used for other neutral lipids according to the present disclosure include one or more of steroids such as cholesterol and its derivatives, lecithin, soybean phospholipid, cephalin, sphingomyelin, and hydrogenated soybean phospholipid. In some embodiments, the molar percentage of the steroid does not exceed 90% with respect to the amount of the total lipid mixture.

[0022] In some embodiments, the lipid contains at least one phospholipid and cholesterol. The molar percentage of cholesterol may be 0.1% - 90% with respect to the amount of the total lipid mixture, for example, 10% - 80%, or 20% - 70%.

[0023] In some embodiments, the phospholipid is selected from dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dilauroyl phosphatidylcholine (DLPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylcholine (DMPC), and derivatives thereof modified with polyethylene glycol.

[0024] In some embodiments, the lipid contains hydrogenated soybean phosphatidylcholine, cholesterol, and polyethylene glycol 2000 - distearoyl phosphatidylethanolamine.

[0025] In some embodiments, the content of the phospholipid derivative modified with the hydrophilic polymer (mole fraction relative to the total liposome membrane components) is selected from 0.1% to 50%, including 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 numbers, but not limited thereto. For example, it may be 0.5% to 20%.

[0026] In some embodiments, the pharmaceutical composition further comprises a scavenger. In some embodiments, the inner aqueous phase of the liposome contains a scavenger.

[0027] The method for preparing the liposome can adopt the active drug loading method. The active drug loading method is a drug loading method that delivers a therapeutic agent from an external medium across the bilayer membrane of the liposome into the internal aqueous space by a polyatomic ion-gradient. These gradients are created by embedding at least one polyatomic ion as a scavenger in the internal aqueous space of the liposome and then replacing the external medium of the liposome by known techniques such as column separation, dialysis or centrifugation with an additional medium having a relatively low polyatomic ion concentration, such as pure water, sucrose solution or physiological saline. To capture the therapeutic agent in the internal aqueous space of the liposome, a polyatomic ion gradient is created between the internal aqueous space and the external medium of the liposome.

[0028] The scavengers described in the present disclosure include, but are not limited to, sulfates, sulfites, gluconates, phosphates, hydrogen phosphates, molybdates, carbonates, nitrates, etc. Exemplary scavengers include ammonium sulfate, ammonium phosphate, ammonium molybdate, ammonium sucrose octasulfate, triethylammonium sucrose octasulfate, copper gluconate, ammonium ethyl sulfate, ammonium ethylenediaminetetraacetate, ammonium chloride, ammonium hydroxide, ammonium acetate, ammonium dextran sulfate or triethylammonium dextran sulfate, and combinations thereof, but are not limited thereto.

[0029] In some embodiments, the scavenger is ammonium sulfate or copper gluconate.

[0030] In some embodiments, the internal aqueous phase of the liposome has a scavenger concentration selected from 10 - 400 mM, which may 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, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM, 305 mM, 310 mM, 315 mM, 320 mM, 325 mM, 330 mM, 335 mM, 340 mM, 345 mM, 350 mM, 355 mM, 360 mM, 365 mM, 370 mM, 375 mM, 380 mM, 385 mM, 390 mM, 395 mM, 400 mM, or any value between any two of these numbers, for example, it may be 20 - 400 mM.

[0031] In some embodiments, the pharmaceutical composition is a liposome comprising a compound of Formula I or a pharmaceutically acceptable salt thereof, a lipid comprising at least one phospholipid and a steroid, and a scavenger.

[0032] In some embodiments, the phospholipid is selected from dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dilauroyl phosphatidylcholine (DLPC), hydrogenated soy phosphatidylcholine (HSPC), distearoyl phosphatidylethanolamine (DSPE), dimyristoyl phosphatidylcholine (DMPC), and derivatives thereof modified with polyethylene glycol, and the steroid is cholesterol.

[0033] In some embodiments, the inner aqueous phase of the liposome described in the present disclosure further comprises at least one pH adjuster, and the pH adjuster may be selected from amino acids such as arginine, histidine, glycine, acids such as ethylenediaminetetraacetic acid, pentetic acid, hydrochloric acid, phosphoric acid, malic acid or sulfuric acid, salts such as sodium salts, potassium salts, ammonium salts of the above acids, basic compounds (bases) such as aqueous ammonia, sodium hydroxide, potassium hydroxide, triethanolamine, etc. For example, it may be sodium hydroxide, hydrochloric acid, aqueous ammonia, phosphoric acid, malic acid, histidine, triethanolamine. Also, the pH adjuster can be used in combination with two or more of the above ammonium salts.

[0034] In some embodiments, the concentration of the pH adjuster in the inner aqueous phase of the liposome is selected from 1 to 300 mM, and may 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, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM or any value between any two numbers, for example, it may be 20 to 300 mM.

[0035] 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, and may 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 / mL, 0.85 mg / mL, 0.90 mg / mL, 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, 100 mg / mL or any value between any two numerical values, for example, it may be 0.05 to 10 mg / mL.

[0036] In another aspect, in order to improve the encapsulation rate of the compound of formula I or a pharmaceutically acceptable salt thereof in liposomes, the ionic strength can be increased by adding a solution containing electrolytes (salt solution) to the outer phase of the liposomes, thereby increasing the encapsulation rate. The electrolytes (salts) contained in the outer phase of the liposomes are not particularly limited, and for example, they may be sodium chloride, potassium chloride, for example, sodium chloride. Also, physiological saline may be used. Further, the outer phase of the liposomes such as a liposome dispersion may contain sugar, electrolytes, and / or amino acids or salts thereof, and may contain sugar or electrolytes, and amino acids or salts thereof. As an example, the sugar may be selected from sucrose, trehalose or glucose, the electrolyte may be selected from physiological saline, sodium chloride or potassium chloride, and the amino acid or its salt may be selected from histidine or its salt.

[0037] In some embodiments, the outer phase of the liposome contains sodium chloride and histidine or a salt thereof.

[0038] In some embodiments, the outer phase of the liposome has a concentration of the amino acid selected from 1 to 300 mM, which may 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, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, 200 mM, 205 mM, 210 mM, 215 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 255 mM, 260 mM, 265 mM, 270 mM, 275 mM, 280 mM, 285 mM, 290 mM, 295 mM, 300 mM or any value between any two numerical values, for example, it may be 5 to 200 mM.

[0039] In some embodiments, the pharmaceutical composition is a liposome comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a lipid comprising at least one phospholipid and a steroid. The inner aqueous phase of the liposome contains a scavenger and a pH adjuster, and the outer aqueous phase of the liposome contains one or more selected from sugars, electrolytes and amino acids or salts thereof.

[0040] In some embodiments, for the liposome described in the present disclosure, the pH of the inner aqueous phase is 5 to 8, and the pH of the outer phase is 5 to 11. Preferably, the pH of the inner aqueous phase is 5 to 8, and the pH of the outer phase is 5 to 8.

[0041] The present disclosure further provides a method for preparing a liposome, which includes the steps of preparing a blank liposome and introducing a compound of formula I or a pharmaceutically acceptable salt thereof into the inner aqueous phase of the blank liposome.

[0042] The present disclosure can prepare blank liposomes by thin film dispersion method, reverse phase evaporation method, direct hydration method, surfactant removal method, ethanol injection method and improved ethanol injection method.

[0043] In some embodiments, blank liposomes having a desired particle size can be obtained by processing with devices such as probe ultrasonic, high pressure homogenizer, microfluidizer, extruder and the like.

[0044] Also, various parameters (such as the amount of membrane components, temperature, etc.) in the preparation process of liposomes can be appropriately adjusted and determined according to the liposome preparation method or the composition and particle size of the target liposomes.

[0045] In another aspect, the substitution or dilution operation according to the present disclosure can be performed by methods such as dialysis, centrifugation, gel filtration, etc. Also, by substituting or diluting the outer phase of the liposome, the active substance can be effectively encapsulated in the inner aqueous phase of the liposome.

[0046] Dialysis, centrifugation and gel filtration are all common operations in this field. For example, dialysis may be performed with a dialysis membrane. As the dialysis membrane, a membrane made of polyethersulfone material may be selected.

[0047] In some embodiments, the pharmaceutical composition is a liquid preparation. The liposome composition obtained from the above-introduced step may be directly used as the final liposome composition (pharmaceutical composition), or the final liposome composition (pharmaceutical composition) may be obtained by adjusting (such as substituting) the outer phase of the liposomes in the obtained liposome composition.

[0048] In some other embodiments, the pharmaceutical composition is a solid preparation, and the liposome composition obtained from the above-introduced step may be dried to obtain a final solid liposome composition (pharmaceutical composition). The methods for drying the liposome composition include, but are not limited to, freeze-drying and spray-drying.

[0049] In another aspect, the pharmaceutical composition is a solid preparation, and the pharmaceutical composition may be used as a liquid preparation by dissolving or suspending it in a suitable solvent. The solvent may be appropriately set according to the purpose of use of the liposome composition, etc. For example, when the pharmaceutical composition is an injection, the solvent is preferably sterile distilled water.

[0050] The present disclosure further provides a freeze-dried composition obtained by freeze-drying the above pharmaceutical composition, or a freeze-dried composition obtained after redissolving in a liquid medium, wherein the liquid medium used for redissolving is selected from physiological saline, water for injection, glucose injection, or glucose sodium chloride injection.

[0051] The present disclosure further provides the use of the above pharmaceutical composition, liposome, freeze-dried composition or redissolved solution in the preparation of a medicament for treating and / or preventing a disease or medical condition, wherein the disease or medical condition is cancer.

[0052] The diseases or medical 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, gastric cancer, lung cancer, liver cancer, kidney cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, 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.

[0053] In some embodiments, the above diseases or medical conditions are selected from pancreatic cancer, colorectal cancer, and non-small cell lung cancer.

[0054] The pharmaceutical composition described in the present disclosure has good stability, a high liposome encapsulation rate, a large drug loading capacity, a low allergic reaction to liposomes, and can be better used clinically.

[0055] The "mass / volume percentage" (w / v) described in the present disclosure refers to the mass (unit: g) of the above components contained per 100 mL of the liquid system, that is, g / 100 mL.

[0056] The content (including the content percentage) of various substances described in the present disclosure and their mutual ratios are all allowed an error of ±5%. For example, "the content of the compound of formula I or a pharmaceutically acceptable salt thereof in the composition is 5 to 20 mg / mL" means that the content of the compound of formula I or a pharmaceutically acceptable salt thereof in the composition is 4.75 to 21 mg / mL, and all belong to the scope of the present disclosure. "The weight ratio of the compound of formula I or a pharmaceutically acceptable salt thereof to the above lipid is selected from 1:1 to 1:100" means that the weight ratio of the compound of formula I or a pharmaceutically acceptable salt thereof to the above lipid is selected from 1:0.95 to 1:105, and all belong to the scope of the present disclosure.

[0057] The "mixing" and "blending" in the present disclosure mean that the addition order of components is not limited. For example, mixing A into B may mean adding A to B, or may mean adding B to A. Mixing A and B may mean adding A to B and then mixing, or may mean adding B to A and then mixing.

[0058] Liposome refers to a fine lipid membrane vesicle composed of bilayer phospholipids or any similar amphiphilic lipid encapsulating an internal aqueous medium. The liposomes of the present disclosure may be multilamellar vesicles (MLV), large unilamellar vesicles (LUV), or small unilamellar vesicles. The size is usually 30 nm to 200 nm. Also, the structure of the liposome membrane in the present disclosure is not specifically limited.

[0059] The liposome membrane is a bilayer phospholipid that separates the internal aqueous medium from the external aqueous medium.

[0060] The inner aqueous phase of a liposome refers to the aqueous region surrounded by the lipid bilayer of the liposome, and is used synonymously with "inner aqueous phase" and "the inner aqueous phase of the liposome". When the liposome is dispersed in a liquid, the "outer phase of the liposome" refers to the region not surrounded by the lipid bilayer of the liposome (i.e., the region other than the inner aqueous phase and the lipid bilayer).

[0061] A blank liposome refers to one that does not contain an active ingredient to be carried in the inner aqueous phase of the liposome, for example, a compound of Formula I according to the present disclosure.

[0062] The encapsulation rate refers to the percentage of the substance to be coated (e.g., a certain drug) in the total amount of the drug in the liposome suspension. It is one of the important indicators for the quality control of liposomes and nanoparticles, and reflects the degree to which the drug is encapsulated in the carrier.

[0063] The encapsulation rate includes the measurement of the encapsulation percentage and the coating volume. In general literature, mainly the encapsulation percentage (Encapsulation percentage, EN%) is considered. It is expressed by the formula EN% = (1 - Cf / Ct) × 100%. In the formula, Cf is the amount of free drug, and Ct is the total amount of the drug in the liposome suspension.

[0064]

Number

[0065] In the present disclosure, the "polymer molecular weight" adopts the number average molecular weight, which refers to the statistical average value of the molecular weights of all polymer chains in the sample, and its definition is as follows. Among them, M i represents the single-chain molecular weight, and N i represents the number of chains having the corresponding molecular weight. M n is predicted by the polymerization mechanism and can be determined by measuring the number of molecules in a sample of a predetermined mass, for example, by a method of monodispersity such as end-group analysis. Mn When characterizing the molecular weight distribution, the same number of molecules are distributed on both sides of M n 「Particle size distribution」 or the term 「PSD」 refers to the particle size distribution in the liposome composition measured by the dynamic light scattering technique well known to those skilled in the art, for example, using a Malvern Mastersizer

[0066] 2000. 「d(0.1)」 described in the present disclosure refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 10%. 「d(0.5)」 refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 50%. 「d(0.9)」 refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 90%. TM 2000 is used to measure the particle size distribution in the liposome composition. 「d(0.1)」 described in the present disclosure refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 10%. 「d(0.5)」 refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 50%. 「d(0.9)」 refers to the particle size corresponding when the cumulative particle size distribution percentage of one sample reaches 90%.

Brief Description of the Drawings

[0067]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0068] The present disclosure will be further described in detail by the following examples. These examples are used for illustrative purposes only and do not limit the scope of the present disclosure. The present disclosure incorporates all the contents in PCT / CN2022 / 100016.

[0069] The structure of the compound is determined by nuclear magnetic resonance (NMR) or / and mass spectrometry (MS). The NMR shift (δ) is 10 -6It is expressed in the unit of (ppm). For NMR measurement, a nuclear magnetic resonance spectrometer Bruker AVANCE-400 or Bruker AVANCE NEO 500M is used. The measurement solvents are deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl3), and deuterated methanol (CD3OD), and the internal standard is tetramethylsilane (TMS).

[0070] For MS measurement, a liquid chromatograph mass spectrometer Agilent 1200 / 1290 DAD-6110 / 6120 Quadrupole MS (manufacturer: Agilent, MS model number: 6110 / 6120 Quadrupole MS) was used.

[0071] waters ACQuity UPLC-QD / SQD (manufacturer: waters, MS model number: waters ACQuity Qda Detector / waters SQ Detector) THERMO Ultimate 3000-Q Exactive (manufacturer: THERMO, MS model number: THERMO Q Exactive) For high performance liquid chromatography (HPLC) analysis, high performance liquid chromatographs Agilent HPLC 1200DAD, Agilent HPLC 1200VWD, and Waters HPLC e2695-2489 were used.

[0072] For chiral HPLC analysis measurement, a high performance liquid chromatograph Agilent 1260 DAD was used.

[0073] For high performance preparative liquid chromatography, preparative chromatographs Waters 2545-2767, Waters 2767-SQ Detecor2, Shimadzu LC-20AP, and Gilson GX-281 were used.

[0074] For chiral preparation, a preparative chromatograph Shimadzu LC-20AP was used.

[0075] As the CombiFlash high-speed fractionation chromatograph, Combiflash Rf200 (TELEDYNE ISCO) was used.

[0076] As the silica gel plate for thin-layer chromatography, Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates were used. The specifications of the silica gel plates used for thin-layer chromatography (TLC) were 0.15 mm to 0.2 mm, and the specifications for the separation and purification of products by thin-layer chromatography were 0.4 mm to 0.5 mm.

[0077] For silica gel column chromatography, generally, silica gel with a mesh size of 200 - 300 made by Yantai Huanghai silica gel was used as the carrier.

[0078] Kinase average inhibition rate and IC 50 For the measurement of the value, a plate reader NovoStar (BMG Labtech, Germany) was used.

[0079] The known starting materials according to the present disclosure may be synthesized by known methods in this field or according thereto, or may be purchased from companies such as ABCR GmbH & Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc, and Darui Chemicals.

[0080] In the examples, unless otherwise specified, all reactions can be carried out in an argon atmosphere or a nitrogen gas atmosphere.

[0081] An argon atmosphere or a nitrogen gas atmosphere means that an argon or nitrogen gas balloon with a volume of about 1 L is connected to the reaction flask.

[0082] A hydrogen atmosphere means that a hydrogen balloon with a volume of about 1 L is connected to the reaction flask.

[0083] For the hydrogenation reaction under pressure, a Parr 3916EKX type hydrogenation apparatus and a Qinglan QL-500 type hydrogen generator or an HC2-SS type hydrogenation apparatus were used.

[0084] Generally, the operation of evacuating and filling with hydrogen was repeated three times for the hydrogenation reaction.

[0085] For the microwave reaction, a CEM Discover-S 908860 type microwave reactor was used.

[0086] In the examples, unless otherwise specified, the solution refers to an aqueous solution.

[0087] In the examples, unless otherwise specified, the reaction temperature is room temperature of 20°C to 30°C.

[0088] For monitoring the progress of the reaction in the examples, thin layer chromatography (TLC) was used. The developing solvent used for the reaction, the eluent system for column chromatography for purifying the compound, and the developing solvent system for thin layer chromatography include A: dichloromethane / methanol system, B: n-hexane / ethyl acetate. The volume ratio of the solvents may be adjusted according to the polarity of the compound, or may be adjusted by adding a small amount of basic or acidic reagents such as triethylamine and acetic acid.

[0089] When the compounds in the examples contain two or more chiral centers, the relative stereochemistry of these compounds was identified by NMR studies and / or X-ray diffraction. In such cases, the prefix "rel" was used, and these compounds were identified by the R / S nomenclature. In this case, R / S does not indicate the absolute stereochemistry but only provides relative stereochemical information. For example,

Chemical formula

Chemical formula

[0090] Example 1 (±)-rel-(1R,2R,5S)-2-(Hydroxymethyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl 1 g [Chemical formula] Step 1 (±)-Methyl 5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylate 1 b (±)-Methyl 2-pyrrolidone-5-carboxylate 1 a (100 g, 698.61 mmol, Shanghai Bide), dimethyl sulfate (110 g, 872.10 mmol) were mixed and reacted at 60 °C for 16 h. The reaction solution was cooled to room temperature and put into a solution of triethylamine (100 g) and methyl tert-butyl ether (150 mL) under an ice bath, extracted with methyl tert-butyl ether (300 mL × 6), and then concentrated under reduced pressure to obtain the crude title compound 1 b (90 g, yield: 81.9%). The product was used in the next reaction without purification. MS m / z (ESI): 158.1 [M+1].[[]END]]

[0091] Step 2 (±)-Methyl 5-(2-methoxy-1-nitro-2-oxoethylidene)pyrrolidine-2-carboxylate 1 c The crude product compound 1 b (90 g, 572.64 mmol) and methyl nitroacetate (68.18 g, 572.63 mmol) were mixed, heated to 60 °C and reacted with stirring for 30 h. After the reaction solution was cooled to room temperature, ethyl acetate (300 mL) was added, stirred for 0.5 h and then filtered. After the filter cake was dried, the title compound 1 c (70 g, yield: 50%) was obtained. The product was used in the next reaction without purification. MS m / z (ESI): 245.1 [M+1].[[]END]]

[0092] Step 3 Methyl 4-oxo-3,8-diazabicyclo[3.2.1]octane-2-carboxylate (diastereomer mixture) 1 d The crude product compound 1c (14 g, 57.3 mmol) was dissolved in 600 mL of methanol, 10% palladium on carbon catalyst (wet) (14 g) was added, the mixture was purged with hydrogen gas three times, and the reaction was carried out with stirring for 48 hours. The reaction solution was filtered through diatomaceous earth, and the filtrate was concentrated to obtain the crude product of the title compound 1d (10 g, yield: 94.6%). The product was used in the next reaction without purification. MS m / z (ESI): 185.2 [M+1].

[0093] Step 4 8-(tert-Butyl) 2-methyl (±)-rel-(1R,2R,5S)-4-oxo-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 1e The crude product 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 in an ice bath, and the reaction was carried out with stirring 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 obtain the title compound 1e (3.3 g, yield: 21.3%). MS m / z (ESI): 285.2 [M+1]. HPLC analysis: retention time 1.02 minutes, purity: 98.5% (column: ACQUITY UPLC® BEH, C18, 1.7 μm, 2.1×50 mm, mobile phase: water (10 mM ammonium bicarbonate), acetonitrile, gradient composition ratio: acetonitrile 10% - 95%).

[0094] Step 5 8-(tert-Butyl) 2-methyl (±)-rel-(1R,2R,5S)-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 1f Compound 1e (400 mg, 1.4 mmol) was dissolved in 2 mL of tetrahydrofuran, 3.5 mL of a 2 M borane dimethyl sulfide complex tetrahydrofuran solution was added, and the reaction was carried out with stirring for 14 hours. Methanol was added to the reaction solution to quench it, 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%). MS m / z (ESI): 271.2 [M+1].

[0095] Step 6 (±)-rel-(1R,2R,5S)-2-(Hydroxymethyl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl 1g Compound 1f (1 g, 3.69 μmol) was dissolved in 15 mL of tetrahydrofuran, 4.4 mL of a 1 M lithium aluminum hydride tetrahydrofuran solution was added, and the reaction was carried out with stirring at 0 °C for 1 hour. 0.2 mL of water, 0.2 mL of a 15% aqueous sodium hydroxide solution, and 0.4 mL of water were sequentially added to the reaction solution. Anhydrous sodium sulfate was further added and stirred for 10 minutes, then filtered. After the filtrate was concentrated, the title compound 1g (430 mg, yield: 47.9%) was obtained and used directly in the next reaction without purification of the product. MS m / z (ESI): 243.1 [M+1].

[0096] Example 2 2,6-Dichloro-3-fluoropyridin-4-amine 2a

Chemical Structure

[0097] Example 3 5-Ethyl-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 3-p1 5-Ethyl-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 3-p2

Chemical Structure

[0098] Step 2 4-((tert-Butoxycarbonyl)amino)-2,6-dichloro-5-fluoronicotinate 3b Compound 2a (1.8 g, 9.94 mmol) was dissolved in tetrahydrofuran (50 mL), 20 mL of a 2M sodium bis(trimethylsilyl)amide tetrahydrofuran solution was added in an ice bath, and the reaction was carried out with stirring for 0.5 hour. Then, di-tert-butyl dicarbonate (6.5 g, 29.7 mmol) was added, and the reaction was carried out with stirring for 14 hours. A saturated ammonium chloride aqueous solution was added to the reaction solution to quench it, and the mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and then the filtrate was concentrated under reduced pressure. The residue was purified with eluent system B to obtain the title compound 3b (1 g, yield: 26.3%), and the product was used directly in the next reaction without purification. MS m / z(ESI): 381.1[M + 1].

[0099] Step 3 4-Amino-2,6-dichloro-5-fluoronicotinate 3c Compound 3b (1 g, 2.62 mmol) was dissolved in ethyl acetate (8 mL), 3 mL of 4 M hydrochloric acid dioxane solution was added, and the reaction was carried out with stirring for 2 hours. The pH was adjusted to neutral with 4 M aqueous sodium hydroxide solution in an ice bath, and extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, the desiccant was removed by filtration, and then the filtrate was concentrated under reduced pressure. The residue was purified with eluent system B to obtain the crude title compound 3c (500 mg, yield: 67.8%). MS m / z (ESI): 281.1 [M+1].

[0100] Step 4 tert-Butyl 2,6-dichloro-5-fluoro-4-(3-(2,2,2-trichloroacetyl)ureido)nicotinate 3d The crude product compound 3c (500 mg, 1.77 mmol) was dissolved in tetrahydrofuran (10 mL), trichloroacetyl isocyanate (670 mg, 3.55 mmol) was added, and the reaction was carried out with stirring for 30 minutes. After the reaction solution was concentrated under reduced pressure, the crude title compound 3d (835 mg, yield: 99.7%) was obtained, and the product was used in the next reaction without purification. MS m / z (ESI): 467.9 [M+1].

[0101] Step 5 5,7-Dichloro-8-fluoro-pyrido[4,3-d]pyrimidine-2,4-diol 3e The crude product compound 3d (835 mg, 1.77 mmol) was dissolved in 7 M ammonia methanol solution (10 mL), and the reaction was carried out with stirring for 1 hour. The reaction solution was concentrated under reduced pressure, methyl tert-butyl ether (10 mL) was added to the residue, and after stirring for 0.5 hour, it was filtered. After the filter cake was dried, the crude title compound 3e (400 mg, yield: 89.9%) was obtained, and the product was used in the next reaction without purification. MS m / z (ESI): 249.9 [M+1].

[0102] Step 6 2,4,5,7-Tetrachloro-8-fluoro-pyrido[4,3-d]pyrimidine 3f The crude product compound 3e (300 mg, 1.19 mmol) was dissolved in phosphorus oxychloride (6 mL), N,N-diisopropylethylamine (800 mg, 6.19 mmol) was added, and the reaction was carried out with stirring at 110 °C for 3 hours. After the reaction solution was cooled to room temperature, it was concentrated under reduced pressure to obtain the crude title compound 3f (344 mg, yield: 97.7%). The product was used in the next reaction without purification. MS m / z (ESI): 285.8 [M+1].

[0103] Step 7 (±)-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 3g 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, and the reaction was carried out with stirring at the same temperature for 1 hour. Then, the temperature was returned to room temperature and the reaction was carried out for 16 hours. The reaction solution was concentrated under reduced pressure, and the residue was purified with eluent system B to obtain the crude title compound 3g (1.56 g, yield: 73.7%). MS m / z (ESI): 606.2 [M+1].

[0104] Step 8 (1S,2S,5R)-2-(((tert-butyldimethylsilyl)oxy)methyl)-3-(5,7-dichloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl 3h-1 and (1R,2R,5S)-2-(((tert-Butyldimethylsilyl)oxy)methyl)-3-(5,7-dichloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl diastereomeric mixture of 3h-1 and 3h-2 Compound 3g (1.4 g, 2.3 mmol) was dissolved in 1,4-dioxane (20 mL), ((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methanol (650 mg, 4.08 mmol, Pharm. Spec.), N,N-diisopropylethylamine (1.5 g, 11.6 mmol), and 4A molecular sieve (1.4 g) were added, and the reaction was carried out with stirring at 105 °C for 6 hours. After the reaction solution was cooled to room temperature, it was filtered and concentrated under reduced pressure to obtain a diastereomeric mixture of the title compounds 3h-1 and 3h-2 (1.68 g, yield: 99.8%). The product was used in the next reaction without purification. MS m / z (ESI): 729.2 [M+1]

[0105] Step 9 (5aS,6S,9R)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptane-14-carboxylic acid tert-butyl 3i-1 and (5aR,6R,9S)-2-chloro-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptane-14-carboxylic acid tert-butyl diastereomeric mixture of 3i-2 A diastereomeric mixture of crude products 3h-1 and 3h-2 (1.68 g, 2.3 mmol) was added to tetrabutylammonium fluoride (2.59 g, 11.51 mmol), stirred at room temperature for 16 h, 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%). MS m / z(ESI): 579.2[M+1].

[0106] Step 10 (5aS,6S,9R)-2-(8-Ethyl-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptane-14-carboxylic acid tert-butyl 3j-1 and (5aR,6R,9S)-2-(8-Ethyl-3-(methoxymethoxy)naphthalen-1-yl)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptane-14-carboxylic acid tert-butyl 3j-2, a 1:1 diastereomeric mixture A diastereomeric mixture 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 by the method disclosed in Intermediate 21 on page 111 of the specification in 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 a mixed solution of 36 mL of 1,4-dioxane and water (V:V = 5:1). Under a nitrogen gas atmosphere, the reaction was carried out at 100 °C for 6 hours. After concentrating the reaction solution under reduced pressure, a crude 1:1 mixture of diastereomers of the title compounds 3j-1 and 3j-2 (2.5 g, yield: 100%) was obtained. MS m / z(ESI): 759.2[M+1].

[0107] Step 11 5-Ethyl-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 3p-1 5-Ethyl-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 3p-2 A diastereomeric mixture of crude compounds 3j-1 and 3j-2 (2.4 g, 3.16 mol) was dissolved in ethyl acetate (40 mL), 17 mL of a 4 M dioxane solution of hydrochloric acid was added, and 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 obtain a 1:1 mixture of diastereomers of the title compounds 3p-1 and 3p-2 (620 mg, yield: 32.6%). MS m / z(ESI): 615.2[M+1]. 1 H NMR (500 MHz, CD3OD): δ 7.63 (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, 1H), 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).

[0108] The diastereomeric mixture of compounds 3p-1 and 3p-2 was separated by chiral column (Shimadzu LC-20AP, column: DAICEL CHIRALPAK® IC, 25×250 mm, 10 μm, mobile phase A: n-hexane, mobile phase B: ethanol (0.1% 7M NH3 in MeOH)) and gradient ratio: A:B: 30:70, flow rate: 30 mL / min) to obtain the title compound 3p-1 (50 mg, yield: 35.7%) and 3p-2 (50 mg, yield: 35.7%).

[0109] Single configuration compound (relatively short retention time) 3p-2: (50 mg, yield: 35.7%).

[0110] MS m / z (ESI): 615.2 [M+1].

[0111] Chiral HPLC analysis: retention time 9.85 minutes, purity: 99% (column: DAICEL CHIRALPAK® IC, 250×4.6 mm, 5 μm, mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0 mL / min).

[0112] 1 H NMR (500 MHz, CD3OD): δ 7.61 - 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, 1H), 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 - 0.88 m, 3H).

[0113] Compound with a single configuration (relatively long retention time) 3p-1: (50 mg, yield: 35.7%).

[0114] MS m / z (ESI): 615.2 [M+1].

[0115] Chiral HPLC analysis: retention time 16.0 minutes, purity: 99% (column: DAICEL CHIRALPAK® IC, 250×4.6 mm, 5 μm, mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0 mL / min).

[0116] 11H NMR (500 MHz, CD3OD): δ 7.61-7.59 (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), 0.99-0.88 m, 3H).

[0117] Example 4 5-Ethyl-6-fluoro-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 4p-1 5-Ethyl-6-fluoro-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 4p-2

Chemical Structure

[0118] Step 11 5-Ethyl-6-fluoro-4-((5aS,6S,9R)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 4p-1 5-Ethyl-6-fluoro-4-((5aR,6R,9S)-1-fluoro-12-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolidin-7a(5H)-yl)methoxy)-5a,6,7,8,9,10-hexahydro-5H-4-oxa-3,10a,11,13,14-pentaaza-6,9-methanonaphtho[1,8-ab]heptan-2-yl)naphthalen-2-ol 4p-2 The diastereomeric mixture of crude products Compound 4a-1 and 4a-2 (160 mg, 205.9 μmol) was dissolved in ethyl acetate (5 mL), 1 mL of 4 M hydrochloric acid dioxane solution was added, and 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 obtain a 1:1 mixture of diastereomers of the title compound 4p-1 (i.e., the compound represented by Formula I) and 4p-2 (10 mg, yield: 7.2%). MS m / z (ESI): 633.2 [M+1]. 1H NMR (500 MHz, CD3OD): δ 7.67 (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).

[0119] The diastereomeric mixture of compounds 4p-1 and 4p-2 was separated on a Chimera column (Shimadzu LC-20AP, column: DAICEL CHIRALPAK® IC, 25 × 250 mm, 10 μm, mobile phase A: n-hexane, mobile phase B: ethanol (0.1% of 7 M NH in MeOH)), with a gradient ratio of A:B:40:60, flow rate: 30 mL / min, to give the title compounds 4p-1 (26 mg, yield: 43.3%) and 4p-2 (26 mg, yield: 43.3%).

[0120] Single configuration compound (relatively short retention time) 4p-2: (26 mg, yield: 43.3%).

[0121] MS m / z(ESI): 633.2 [M+1].

[0122] Chimera HPLC analysis: retention time 7.89 min, purity: 99% (column: DAICEL CHIRALPAK® IC, 250 × 4.6 mm, 5 μm, mobile phase: n-hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0 mL / min).

[0123] 11H NMR (500 MHz, CD3OD): δ 7.67 (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).

[0124] Single - configuration compound (relatively long retention time) 4p - 1: (26 mg, yield: 43.3%).

[0125] MS m / z (ESI): 633.2 [M + 1].

[0126] Chiral HPLC analysis: retention time 13.8 minutes, purity: 99% (column: DAICEL CHIRALPAK® IC, 250×4.6 mm, 5 μm, mobile phase: n - hexane and ethanol (containing 0.2% diethylamine), flow rate: 1.0 mL / min).

[0127] 1 1H NMR (500 MHz, CD3OD): δ 7.67 (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).

[0128] Example 5

Table 1

[0129] Copper gluconate was weighed, an appropriate amount of water was added, and it was stirred and dissolved uniformly. The pH value was adjusted to 7.4 with triethanolamine, and the volume was fixed to 180 mL to obtain aqueous solution 1. Histidine hydrochloride and sodium chloride were weighed, dissolved in an appropriate amount of water, and then the pH was adjusted to 7.4 with sodium hydroxide and / or hydrochloric acid, and the volume was fixed to 2400 mL to obtain aqueous solution 2.

[0130] Hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine were weighed. After adding absolute ethanol, it was heated and stirred to dissolve to obtain a lipid solution. The lipid solution was added to the above aqueous solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then continuously hydrated and most of the ethanol was removed under reduced pressure, and it was extruded as a whole by an extruder to obtain blank liposomes. The blank liposomes were treated with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous solution 2. The compound of formula I was dissolved in an ethanol solution, an appropriate amount of hydrochloric acid was added to obtain a drug solution of 120 mg / mL. The drug solution was slowly added to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then rapidly cooled to room temperature, the product was concentrated to the target concentration by a tangential flow system, the remaining ethanol was further removed, the volume was fixed, and a liposome product was obtained. The characterization information of the product is as shown in the following table.

[0131]

Table 2

[0132] Example 6

Table 3

[0133] Weighed ammonium sulfate and phosphoric acid, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 5.85 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous solution 2.

[0134] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of phospholipids, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded the whole through an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of phospholipids, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0135]

Table 4

[0136] Example 7

Table 5

[0137] Weighed ammonium sulfate and phosphoric acid, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 7.0 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous solution 2.

[0138] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, then heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded the whole through an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0139]

Table 6

[0140] Example 8

Table 7

[0141] Weighed ammonium sulfate and citric acid monohydrate, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 7.0 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous phase solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous phase solution 2.

[0142] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous phase solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of the phospholipid, continued to hydrate and removed most of the ethanol under reduced pressure, and extruded it entirely with an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous phase solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0143]

Table 8

[0144] Example 9

Table 9

[0145] Weighed ammonium sulfate and malic acid, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 7.0 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous phase solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous phase solution 2.

[0146] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, then heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous phase solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded it entirely with an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous phase solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0147]

Table 10

[0148] Example 10

Table 11

[0149] Weighed ammonium sulfate and histidine hydrochloride, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 7.0 with ammonia water, made up the volume to 180 mL to obtain aqueous solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 7.5 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous solution 2.

[0150] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded the whole through an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of the phospholipid, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0151]

Table 12

[0152] Example 11

Table 13

[0153] Weighed ammonium sulfate and phosphoric acid, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 5.85 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved them in an appropriate amount of water, then adjusted the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous solution 2.

[0154] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, heated with stirring to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of phospholipids, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded the whole through an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of phospholipids, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0155]

Table 14

[0156] Example 12

Table 15

[0157] Weighed ammonium sulfate and phosphoric acid, added an appropriate amount of water, stirred to dissolve uniformly, adjusted the pH value to 5.85 with aqueous ammonia, made up the volume to 180 mL to obtain aqueous phase solution 1. Weighed histidine hydrochloride and sodium chloride, dissolved with an appropriate amount of water, then adjusted the pH to 6.35 with sodium hydroxide and / or hydrochloric acid, made up the volume to 2400 mL to obtain aqueous phase solution 2.

[0158] Weighed hydrogenated soy phosphatidylcholine, cholesterol, and cultured phosphatidylethanolamine, added absolute ethanol, heated and stirred to dissolve to obtain a lipid solution. Added the lipid solution to the above aqueous phase solution 1, stirred for about 15 minutes under conditions higher than the phase transition temperature of phospholipids, then continued to hydrate and removed most of the ethanol under reduced pressure, and extruded through an extruder to obtain blank liposomes. Treated the blank liposomes with a tangential flow filtration system to replace the outer aqueous phase of the liposomes with aqueous phase solution 2. Dissolved the compound of formula I in an ethanol solution, added an appropriate amount of hydrochloric acid to obtain a drug solution of 120 mg / mL. Slowly added the drug solution to the blank liposomes, incubated for 15 minutes under conditions higher than the phase transition temperature of phospholipids, then rapidly cooled to room temperature, concentrated the product to the target concentration by a tangential flow system, further removed the remaining ethanol, made up the volume to obtain a liposome product, and the characterization information of the product is as shown in the following table.

[0159]

Table 16

[0160] Biological evaluation Test Example 1: Biological evaluation of the AGS cell ERK phosphorylation inhibition experiment (HTRF method) I. Purpose of the test This experiment detected the inhibitory effect of the compound represented by formula I on cell ERK phosphorylation, and evaluated the inhibitory effect of the compound on the KRAS target according to the magnitude of IC 50 .

[0161] II. Experimental method AGS cells (Nanjing Kebai, CBP60476) were cultured in RPMI 1640 (Hyclone, SH30809.01) complete medium containing 10% fetal bovine serum. On the first day of the experiment, AGS cells were seeded in a 96-well plate at a density of 40,000 cells / well with 190 μL of cell suspension per well and placed in a cell incubator at 37 °C and 5% CO2 for overnight culture.

[0162] On the next day, 10 μL of the compound to be measured, which was serially diluted with the complete medium, was added to each well. The final concentrations of the compound were nine concentration points with a 5-fold serial dilution starting from 10 μM. A blank control containing 0.5% DMSO was set up. The well plate was placed in a cell incubator at 37 °C and 5% CO2 for 1 hour of incubation. After incubation, the 96-well cell culture plate was taken out, the medium was aspirated and removed, and 200 μL of PBS (Shanghai Yuanpei Biotechnology Co., Ltd., B320) was added to each well for one wash. The PBS was aspirated and removed, and 50 μL of lysis buffer (lysis buffer, Cisbio, 64KL1FDF) containing a blocking reagent (blocking reagent, Cisbio, 64KB1AAC) was added to each well. The well plate was placed on a shaker and shaken at room temperature for 40 minutes for lysis. After lysis, pipetting was performed with a pipette to mix uniformly, and 16 μL of the lysate was transferred from each well to two HTRF 96-well detection plates (Cisbio, 66PL96100) respectively. Then, 4 μL of the pre-mixed phosphorylated ERK1 / 2 antibody solution (Cisbio, 64AERPEG) or 4 μL of the pre-mixed total ERK1 / 2 antibody solution (Cisbio, 64NRKPEG) was added to the two plates respectively. The microplate was sealed with a plate sealing film, centrifuged in a microplate centrifuge for 1 minute, and incubated overnight in the dark at room temperature.

[0163] On the third day, the fluorescence values emitted at wavelengths of 665 nm and 620 nm, which were excited at a wavelength of 337 nm, were read using an ENVISION multifunctional plate reader (PerkinElmer, ENVISION).

[0164] III. Data Analysis and Results Using the software Graphpad Prism, the IC value of the inhibitory activity of the compound was calculated based on the concentration of the compound and the ratio of phosphorylated ERK / total ERK. Please refer to Table 1 below for the results. 50

[0165]

Table 17

[0166] Conclusion: The compound represented by Formula I has a relatively good inhibitory effect on ERK phosphorylation in AGS cells.

[0167] Test Example 2: Biological Evaluation of the Inhibitory Effect of GP2d and AGS Cell 3D Proliferation I. Test Objective By testing the inhibitory effect of the compound represented by Formula I on the 3D proliferation of GP2d and AGS cells, the inhibitory effect of the compound represented by Formula I on the KRAS target was evaluated.

[0168] II. Experimental Method GP2d cells (Nanjing Kebai, CBP60010) were cultured in complete medium, i.e., DMEM / high glucose medium (Hyclone, SH30243.01) containing 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, GP2d cells were seeded in a 96-well low-attachment plate (Corning, CLS7007-24EA) at a density of 1000 cells / well with 90 μL of cell suspension per well. After centrifugation at 2000 rpm for 5 minutes at room temperature, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured overnight.

[0169] ​AGS cells (Nanjing Kebo, CBP60476) were cultured in complete medium, namely RPMI 1640 medium (Hyclone, SH30809.01) containing 10% fetal bovine serum (Corning, 35-076-CV). On the first day of the experiment, AGS cells were seeded into a 96-well low-attachment plate (Corning, CLS7007-24EA) at a density of 1000 cells / well with 90 μL of cell suspension per well. After centrifugation at 2000 rpm for 5 minutes at room temperature, the cells were placed in a cell incubator at 37°C and 5% CO2 and cultured overnight.

[0170] On the next day, 10 μL of the compound to be measured, which was serially diluted in complete medium, was added to each well. The final concentrations of the compound for GP2d cells were nine concentration points serially diluted three-fold from 1 μM, and those for AGS cells were nine concentration points serially diluted three-fold from 10 μM. A blank control containing 0.5% DMSO was set up in each case. The well plates were placed in a cell incubator at 37°C and 5% CO2 and incubated for 120 hours. On the seventh day, the 96-well cell culture plates were taken out, 50 μL of CellTiter-Glo® 3D reagent (Promega, G9682) was added to each well, and the plates were shaken at room temperature for 25 minutes, then pipetted to mix uniformly. Then, 50 μL was taken out and transferred to a white opaque 96-well plate (PE, 6005290), and the luminescence signal values were read using a multifunctional microplate reader (PerkinElmer, ENVISION).

[0171] III. Data Analysis and Results The IC 50 values of the inhibitory activities of the compounds were calculated using the software Graphpad Prism. For the results, please refer to Table 2 below.

[0172]

Table 18

[0173] Conclusion: The compounds represented by Formula I have relatively good inhibitory effects on the 3D growth of AGS and GP2d cells.

[0174] Test Example 3: Detection of the Affinity of Compounds for KRAS Protein Isoform G12D or WT by SPR Method First, biotinylated Avi-KRAS-WT or Avi-KRAS-G12D was diluted to 20 μg / mL with 1×HBS-P+ (Cat.#BR1006-71) buffer containing 100 mM MgCl2, and SA (Cat.#BR1005-31) biosensor chip channel 2 was flowed for 420 s to obtain a coupling level of about 5000 - 7000 RU. Further, compound samples were injected in order from high to low for 120 s and then dissociated for 720 s. The test adopted a single-cycle kinetics mode. The reaction signal was detected in real time by a Biacore 8K instrument to obtain a binding-dissociation curve. After the test, data analysis was performed by Biacore 8K evaluation software, and the data was fitted with a 1:1 model to obtain affinity data. Please refer to Table 3 below for the results.

[0175]

Table 19

[0176] Conclusion: The compound represented by Formula I has relatively good affinity for KRAS protein isoform G12D or WT.

Images