Liposome of nitrogen-containing heterocyclic compound and use thereof

Abstract

A liposome of a nitrogen-containing heterocyclic compound, comprising the following components: a nitrogen-containing heterocyclic compound as shown in compound 3 below and a lipid component. The liposome of the present invention meets one or more of the following advantages: (1) small particle size and uniform distribution; (2) high API content; (3) high drug encapsulation efficiency; (4) good stability; (5) good anti-tumor activity; (6) good safety; and (7) exhibiting a long-acting effect.

Description

Liposome containing nitrogen heterocyclic compound and its application

[0001] This application claims priority to Chinese patent application No. 2023117212073, filed on December 13, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention relates to a liposome of a nitrogen-containing heterocyclic compound and application thereof. Background Art

[0003] RAS proteins are 21kDa guanine trinucleotide phosphate (GTP)-binding proteins located on the cell membrane, composed of 188 or 189 amino acids. The activity of RAS proteins influences cell growth, differentiation, the cytoskeleton, protein trafficking, and secretion. Their activity is regulated by binding to GTP or guanine dinucleotide phosphate (GDP). When bound to GDP, RAS proteins are in an "inactive" state. Upon stimulation by specific upstream growth factors, guanine nucleotide exchange factors (GEFs) catalyze the release of GDP from RAS proteins, allowing them to bind to GTP and enter an "activated" state. GTP-bound RAS proteins can activate downstream proteins and signaling pathways. RAS proteins possess weak GTPase activity, hydrolyzing GTP to GDP, thereby transitioning from an active to an inactive state. This hydrolysis process also requires the participation of GTPase-activating proteins (GAPs), which interact with RAS proteins and significantly enhance their ability to hydrolyze GTP to GDP. Any mutation in a RAS protein that affects its GTPase activity, its interaction with GAPs, or its ability to hydrolyze GTP to GDP will result in a prolonged activation of the RAS protein. This prolonged activation of the RAS protein continues to provide growth signals to downstream proteins, leading to continued cell growth and differentiation, and ultimately cancer. The RAS gene family consists of three members: KRAS, NRAS, and HRAS.

[0004] KRAS mutation is the most common oncogenic driver, present in a variety of tumors: lung adenocarcinoma (32%), colorectal cancer (41%), and pancreatic cancer (86%). KRAS mutation is most common with the G12 mutation at codon 12. For example, in KRAS-mutated lung adenocarcinoma, colorectal cancer, and pancreatic cancer, G12 mutations account for 85%, 68%, and 91%, respectively; G12 mutations include G12C, G12D, G12V, G12R and other mutation forms. Among patients with KRAS G12 mutations in lung adenocarcinoma, colorectal cancer, and pancreatic cancer, patients with KRAS G12D mutations accounted for 17%, 45%, and 45%, respectively, and patients with KRAS G12V mutations accounted for 23%, 30%, and 35%, respectively (for example, see Moore, AR et al. Nat Rev Drug Discov 19, 533 (2020)).

[0005] The KRAS G12D inhibitor MRTX1133 developed by Mitati Therapeutics, Inc. has been disclosed in WO2021041671. Its structure is as follows, and preclinical data has been made public.

[0006] Although some progress has been made in this field, there is currently no approved treatment for KRAS G12D and / or KRAS G12V mutations. Therefore, there is still a need to continue to develop effective, stable and safe small molecule KRAS G12D and / or KRAS G12V inhibitors for the treatment of diseases mediated by KRAS G12D and / or KRAS G12V mutations, such as cancer.

[0007] Liposomes are a common drug delivery system. (1) The structure of liposomes is very similar to that of cell membranes, making them highly biocompatible and biodegradable. Therefore, liposomes can protect drugs from being degraded by enzymes before they reach the lesion site. At the same time, drugs are "hidden" inside liposomes through physical encapsulation, which can improve drug stability, reduce drug toxicity, increase drug dosage, and achieve better therapeutic effects. (2) The bilayer surface formed by amphiphilic phospholipids can be structurally modified with ligands or other functional groups through physical or chemical means to make liposomes tissue-targeted.

[0008] However, the current state of the art lacks a wide variety of formulations for small-molecule KRAS G12D and / or KRAS G12V inhibitors, and improved formulations are crucial for improving the efficacy of preventive and therapeutic drugs. Therefore, there is an urgent need to develop dosage forms for these inhibitors. Summary of the Invention

[0009] To address the shortcomings of existing small molecule KRAS G12D and / or KRAS G12V inhibitors in insufficient dosage forms, the present invention provides liposomes containing nitrogen-containing heterocyclic compounds and their applications. The liposomes of the present invention meet one or more of the following advantages: (1) small and uniform particle size; (2) high API content; (3) high drug encapsulation efficiency; (4) good stability; (5) good anti-tumor activity; (6) good safety; and (7) long-lasting effects.

[0010] The present invention adopts the following technical solutions to solve the above technical problems:

[0011] The present invention provides a liposome of a nitrogen-containing heterocyclic compound, which comprises the following components: a nitrogen-containing heterocyclic compound (Compound 3) as shown below and a lipid component;

[0012] The mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:6.58 to 1:1.3;

[0013] In some embodiments, the mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:3 to 1:1.3; preferably 1:2 to 1:1.3; more preferably 1:1.31, 1:1.4, 1:1.46, 1:1.5, 1:1.65, 1:1.7, 1:1.86, 1:1.88 or 1:1.9.

[0014] In some embodiments, the lipid component is a conventional lipid component in the art, such as phospholipids, PEG lipids, and sterols.

[0015] In some embodiments, the phospholipid is a conventional phospholipid in the art, and the phospholipid is preferably distearoylphosphatidylcholine (DSPC) and / or hydrogenated soybean phosphatidylcholine (HSPC); for example, distearoylphosphatidylcholine (DSPC).

[0016] In some embodiments, the sterol is a conventional sterol in the art, and the sterol is preferably cholesterol (CHOL).

[0017] In some embodiments, the PEG lipid may be a lipid molecule having a polyethylene glycol hydrophilic end modified; preferably, PEG-modified phosphatidylethanolamine; for example, polyethylene glycol-modified phosphatidylethanolamine (MPEG2000-DSPE).

[0018] In some embodiments, the mass ratio of the phospholipid, the sterol, and the PEG lipid is preferably 1.4:0.46:0.024.

[0019] In some embodiments, the mass ratio of the nitrogen-containing heterocyclic compound to the phospholipid is 1:4.9 to 1:0.98, preferably 1:1.4.

[0020] In some embodiments, the mass ratio of the nitrogen-containing heterocyclic compound to the sterol is 1:1.6 to 1:0.32, preferably 1:0.46.

[0021] In some embodiments, the mass ratio of the nitrogen-containing heterocyclic compound to the PEG lipid is 1:0.09 to 1:0.017; preferably 1:0.024.

[0022] In some embodiments, the liposomes further comprise a buffer salt.

[0023] In some embodiments, the buffer salt is a conventional buffer salt in the art; the buffer salt is preferably a phosphate buffer salt; more preferably disodium hydrogen phosphate and / or sodium dihydrogen phosphate. The disodium hydrogen phosphate is more preferably disodium hydrogen phosphate heptahydrate; and the sodium dihydrogen phosphate is more preferably sodium dihydrogen phosphate monohydrate.

[0024] In some embodiments, the mass ratio of the sodium hydrogen phosphate heptahydrate to the sodium dihydrogen phosphate monohydrate is 1:0.35 to 1:0.18; preferably 1:0.37 or 1:0.17.

[0025] In some embodiments, the liposome further comprises a pH adjuster.

[0026] In some embodiments, the pH adjuster is a conventional pH adjuster in the art, preferably an inorganic acid or an inorganic base; the inorganic acid is preferably hydrochloric acid; the inorganic base is preferably sodium hydroxide. The pH adjuster can adjust the pH as needed, for example, to 7.3 or 7.4.

[0027] In some embodiments, the average particle size of the liposome is 50-160 nm, for example, 79 nm, 79.13 nm, 79.76 nm, 80 nm, 83 nm, 85 nm, 86 nm, 87 nm, 90 nm, 92 nm, 96 nm, 99 nm or 126 nm; preferably, 75-100 nm, for example, 79 nm, 79.13 nm, 79.76 nm, 80 nm, 83 nm, 85 nm, 86 nm, 87 nm, 90 nm, 92 nm, 96 nm or 99 nm.

[0028] In some embodiments, the PDI of the liposome is 0.07-0.2, for example, 0.072, 0.08, 0.083, 0.096, 0.106, 0.107, 0.1, 0.11, 0.12, 0.122, 0.1287, 0.13, 0.146, 0.15, 0.158, 0.16, 0.188 or 0.19; preferably 0.09-0.19, for example, 0.096, 0.106, 0.107, 0.1, 0.11, 0.12, 0.122, 0.1287, 0.13, 0.146, 0.15, 0.158, 0.16, 0.188 or 0.19.

[0029] In some embodiments, the liposomes further comprise a lyoprotectant and / or an osmotic pressure regulator.

[0030] In some embodiments, the lyoprotectant is a conventional lyoprotectant in the art, preferably trehalose and / or sucrose; more preferably trehalose.

[0031] In some embodiments, the osmotic pressure regulator is a conventional osmotic pressure regulator in the art, preferably trehalose and / or sucrose; more preferably trehalose.

[0032] In some embodiments, the average particle size of the liposome is 50 to 160 nm, preferably 75 to 100 nm, for example 80 nm, 81 nm, 82 nm, 89 nm, 89.4 nm, 90 nm, 92 nm or 92.1 nm.

[0033] In some embodiments, the liposomes have a PDI of 0.05 to 0.21; preferably 0.05 to 0.15; for example 0.1153, 0.117, 0.12, 0.122, 0.06, 0.061, 0.057, 0.056, 0.051, 0.097 or 0.1.

[0034] In some embodiments, the liposome is composed of any one of the following components: (1) the nitrogen-containing heterocyclic compound and the lipid component; (2) the nitrogen-containing heterocyclic compound, the lipid component and the buffer salt; (3) the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt and the pH adjuster; (4) the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt, and the lyoprotectant and / or osmotic pressure regulator; and (5) the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt, the pH adjuster, and the lyoprotectant and / or osmotic pressure regulator;

[0035] The mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:6.58 to 1:1.3.

[0036] In some embodiments, the liposomes can be prepared by the following raw materials, which are composed of any one of the following components: (1) the nitrogen-containing heterocyclic compound, the lipid component and the buffered salt solution; (2) the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution and the pH regulator; (3) the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution, and the lyoprotectant and / or osmotic pressure regulator; and (4) the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution, the pH regulator, and the lyoprotectant and / or osmotic pressure regulator;

[0037] The mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:6.58 to 1:1.3;

[0038] The lipid components (the phospholipids, the PEG lipids, the sterols), the pH regulator, the lyoprotectant and / or the osmotic pressure regulator are as described above; the buffered salt solution is an aqueous solution of the buffered salt described above.

[0039] In some embodiments, the liposome is prepared by the following method, comprising the steps of: (1) mixing the nitrogen-containing heterocyclic compound, the lipid component, and an organic solvent to obtain an organic phase solution;

[0040] (2) mixing the organic phase solution obtained in step (1) with the buffered saline solution 1 and concentrating to obtain an initial concentrated solution; performing displacement, filtration, and concentration with the buffered saline solution 2 to obtain a final concentrated solution;

[0041] The buffered saline solution 1 is an aqueous solution of the buffered saline described above; the buffered saline solution 2 is an aqueous solution of the buffered saline described above.

[0042] In some embodiments, in step (1), the organic solvent is a conventional organic solvent in the art, preferably an alcohol solvent, such as ethanol.

[0043] In some embodiments, in step (2), the buffered saline solution 1 is an aqueous solution of disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate.

[0044] In some embodiments, in step (2), the pH value of the buffered saline solution 1 is preferably 6-7; more preferably 6.5; the pH value of the buffered saline solution 1 can be adjusted by the pH regulator.

[0045] In some embodiments, in step (2), the concentration of the buffered saline solution 1 is preferably 5-30 mM; for example, 10 mM or 20 mM.

[0046] In some embodiments, in step (2), the buffered saline solution 2 is an aqueous solution of disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate.

[0047] In some embodiments, in step (2), the pH value of the buffered saline solution 2 is preferably 7.0 to 7.8; more preferably 7.4; the pH value of the buffered saline solution 2 can be adjusted by the pH regulator.

[0048] In some embodiments, in step (2), the concentration of the buffered salt solution 2 is preferably 5-30 mM; for example, 10 mM or 20 mM.

[0049] In some embodiments, in step (2), the mixing of the organic phase solution and the buffered saline solution 1 is a conventional mixing method in the art, preferably using a quaternary plunger pump for mixing; more preferably, the flow rate ratio of the organic phase solution: the buffered saline solution 1: the buffered saline solution 1: the buffered saline solution 1 is 2:2:8:8 or 1:1:9:9; for example, 2:2:8:8.

[0050] In some embodiments, in step (2), the concentration after mixing is performed by conventional methods in the art, preferably by hollow fiber column filtration. The transmembrane pressure of the concentration is more preferably 4 to 6.6 psi; for example, 4 to 4.5 psi or 5.8 to 6.53 psi. The volume of the initial concentrate is preferably 0.05 times the volume of the mixture of the organic phase solution and the buffered salt solution 1.

[0051] In some embodiments, in step (2), the volume of the buffered saline solution 2 may be 5 to 10 times the volume of the initial concentrated solution; preferably 7 times.

[0052] In some embodiments, in step (2), the concentration after displacement diafiltration is performed by a conventional concentration method in the art, preferably by hollow fiber column filtration concentration, and the transmembrane pressure of the concentration is more preferably 4 to 6.6 Psi; for example, 4 to 4.5 Psi or 5.8 to 6.53 Psi. The volume of the final concentrate is preferably 0.5 to 0.7 times the volume of the buffered saline solution 2, for example, 0.6 times or 0.67 times.

[0053] In some embodiments, when the liposomes are prepared by the above method, the method further comprises step (3): mixing the final concentrated solution obtained in step (2) with the lyoprotectant and / or osmotic pressure regulator.

[0054] In some embodiments, the lyoprotectant and / or osmotic pressure regulator is preferably present in the form of a solution, more preferably a solution formed by trehalose and the buffered saline solution 2; the mass volume concentration of trehalose in the solution formed by trehalose and the buffered saline solution 2 can be 37.2-40 g / 100 mL.

[0055] In some embodiments, the amount of the lyoprotectant and / or osmotic pressure regulator is the conventional amount in the art, and the volume ratio of the solution formed by the lyoprotectant and / or osmotic pressure regulator (such as trehalose) and the buffered salt solution 2 to the final concentrated solution in step (2) is preferably 1:3.

[0056] In some embodiments, in step (3), after the final concentrated solution obtained in step (2) is mixed with the lyoprotectant and / or osmotic pressure regulator, the mass volume concentration of the lyoprotectant and / or osmotic pressure regulator is 9.3 to 10 g / 100 ml, preferably 9.3 g / 100 ml.

[0057] In some embodiments, when the liposomes are prepared by the above method, the method further comprises a sterilization filtration step; preferably, pre-filtration is performed before the sterilization filtration step.

[0058] In some embodiments, when the liposomes are prepared by the above method, the method further comprises a freeze-drying step.

[0059] The present invention also provides an injection preparation comprising the liposome as described in any one of the above items.

[0060] In some embodiments, in the injection preparation, the liposomes are preferably in the form of a lyophilized powder; the injection preparation is more preferably a reconstituted liposome formed by the lyophilized powder of the liposome and a reconstitution solvent, and the reconstitution solvent is preferably water for injection, 0.9% sodium chloride injection, 5% glucose injection, water for injection and 5% glucose injection, or water for injection and 0.9% sodium chloride injection.

[0061] In some embodiments, the encapsulation efficiency of the reconstituted liposomes is 95% to 100%, such as 97%, 97.6%, 97.9%, 98% or 98.5%.

[0062] In some embodiments, the average particle size of the reconstituted liposomes is 100-300 nm; preferably 110-240 nm; for example, 115 nm, 130 nm, 140 nm, 153 nm, 153.6 nm, 154 nm, 166 nm, 167 nm or 224 nm.

[0063] In some embodiments, the PDI of the reconstituted liposomes is 0.05-0.3; preferably 0.1-0.25; for example 0.104, 0.11, 0.115, 0.12, 0.13, 0.132, 0.14, 0.16, 0.205 or 0.21.

[0064] The present invention also provides the use of the liposome or the injection preparation described above in the preparation of a medicament for treating and / or preventing RAS-mediated diseases, such as cancer.

[0065] The present invention also provides use of the liposome or the injection preparation described above in preparing a drug, which can be used to treat and / or prevent cancer.

[0066] In some embodiments, the RAS described above may be KRAS, such as a KRAS mutation, such as a KRAS G12D mutation or a KRAS G12V mutation.

[0067] In some embodiments, the cancer described above can be selected from one or more of colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, brain cancer, ovarian cancer, cervical cancer, testicular cancer, kidney cancer, head or neck cancer, bone cancer, skin cancer, rectal cancer, liver cancer, colorectal cancer, non-small cell lung cancer, small cell lung cancer, esophageal cancer, gastric cancer, thyroid cancer, bladder cancer, lymphoma, leukemia and melanoma.

[0068] In some embodiments, the cancer described above may be gastric cancer or pancreatic cancer.

[0069] Explanation of terms:

[0070] In addition to the foregoing, when used in the specification and claims of this application, the following terms have the meanings indicated below unless otherwise specifically stated.

[0071] The term "lipid component" refers to lipids that form the membrane material of the liposome bilayer, such as phospholipids, sterols, and PEG lipids. The phospholipids include, but are not limited to, DSPC and HSPC. The sterols include, but are not limited to, cholesterol. The PEG lipids include, but are not limited to, MPEG2000-DSPE.

[0072] The term "lyophilized powder" refers to the product obtained after freeze-drying.

[0073] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain preferred embodiments of the present invention.

[0074] The reagents and raw materials used in the present invention are commercially available.

[0075] The positive progress of the present invention is that the liposomes of the present invention meet one or more of the following advantages: (1) uniform particle size distribution; (2) high API content; (3) high drug encapsulation efficiency; (4) good stability; (5) good anti-tumor activity; (6) good safety; and (7) showing long-lasting effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG1 is a comparison of the release curves of Compound 3 liposomes for injection and Compound 3 in Example 3.

[0077] FIG2 is a curve showing the change in tumor volume in Test Example 2.

[0078] FIG3 is a weight change rate curve in Test Example 2. FIG.

[0079] FIG4 is a curve showing the change in tumor volume in Test Example 3.

[0080] FIG5 is a weight change curve in Test Example 3.

[0081] Figure 6 shows the expression of pERK in the tumor of BALB / c nude mice bearing AsPC-1 transplanted tumor model after administration of injection of compound 3 liposomes or compound A at different times in test example 4. T202 / Y204 Protein levels. DETAILED DESCRIPTION

[0082] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0083] The abbreviations used in the present invention are as follows:

[0084] DSPC: distearoylphosphatidylcholine;

[0085] HSPC: hydrogenated soy phosphatidylcholine;

[0086] CHOL: cholesterol;

[0087] MPEG2000-DSPE: phosphatidylethanolamine;

[0088] WFI: Water for injection.

[0089] Particle size detection method:

[0090] The ultra-performance liquid chromatography (UPLC) method (7 min) is shown in the table below:

[0091] I. Preparation of Compounds

[0092] Preparation of compound 3

[0093] (Palmitoyl<hexadecanoyl>oxy)methyl(1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-((isopropoxycarbonyl)oxy)naphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

[0094] Step 1: 4-(4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-5-chloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-7-yl)-5-ethynyl-6-fluoronaphthalen-2-ol (Compound A)

[0095] (Prepared with reference to Example 27 in WO 2023 / 274324)

[0096] MS m / z:637.2[M+H] +

[0097] Step 2: (Palmitoyl<hexadecanoyl>oxy)methyl(1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-hydroxynaphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

[0098] In a 100 mL reaction vial, 4-(4-((1R,5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-5-chloro-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-7-yl)-5-ethynyl-6-fluoronaphthalen-2-ol (86 mg, 0.13 mmol, 1.0 eq) was dissolved in acetonitrile (3 mL). Triethylamine (39 mg, 0.39 mmol, 3.0 eq) and (((4-nitrophenoxy)carbonyl)oxy)methyl palmitate (59 mg, 0.13 mmol, 1.0 eq) were added. The mixture was heated to 60°C and stirred for 30 minutes. The reaction was monitored by LCMS, and a small amount of starting material remained. The product was concentrated to give crude (palmitoyl<hexadecanoyl>oxy)methyl (1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-hydroxynaphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (123 mg) as a yellow solid.

[0099] MS m / z:949[M+H] +

[0100] Step 3: (Palmitoyl<hexadecanoyl>oxy)methyl(1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-((isopropoxycarbonyl)oxy)naphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

[0101] In a 100 mL reaction vial, (palmitoyl<hexadecanoyl>oxy)methyl (1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-hydroxynaphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (123 mg, 0.13 mmol, 1.0 eq) was dissolved in dichloromethane (3 mL). Triethylamine (26 mg, 0.26 mmol, 2.0 eq) and isopropyl chloroformate (18 mg, 0.15 mmol, 1.1 eq) were added under ice-cooling, and the mixture was stirred at room temperature for 30 minutes. The reaction was monitored for completion by LCMS. The mixture was washed sequentially with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The residue was separated and purified by preparative high-performance liquid chromatography to obtain (palmitoyl<hexadecanoyl>oxy)methyl(1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-((isopropoxycarbonyl)oxy)naphthalen-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy-d2)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (9.65 mg, 7% yield) as a white solid.

[0102] MS m / z:518[M / 2+H] +

[0103] 1 H NMR (400MHz, DMSO-d6) δ8.24(dd,J=9.2,5.8Hz,1H),8.14(d,J=2.6Hz,1H),7.69(t,J=9.0Hz,2H) ,5.75(s,2H),5.27(d,J=53.9Hz,1H),4.92(p,J=6.2Hz,1H),4.39(s,1H),4.10(s,1H),3.68(s,1 H),3.17-2.93(m,4H),2.82(q,J=8.3Hz,2H),2.36(t,J=7.3Hz,2H),2.15-1.92(m,4H),1.88-1.6 8(m,6H),1.52(q,J=7.4Hz,4H),1.34(d,J=6.3Hz,6H),1.28-1.08(m,24H),0.83(t,J=6.8Hz,3H).

[0104] HPLC separation conditions:

[0105] Column:SunFire Prep C18 OBDTM 5μm, 19*250mm column; flow rate: 25mL / min

[0106] Mobile phase A: Water (0.1% formic acid, v / v) Mobile phase B: ACN; Gradient: 20%-60% (v / v); Detection wavelength: 254 / 214 nm

[0107] Preparation of compound 8

[0108] (Palmitoyl<hexadecanoyl>oxy)methyl(1R,5S)-3-(5-chloro-7-(8-ethynyl-7-fluoro-3-((isopropoxycarbonyl)oxy)naphth-1-yl)-8-fluoro-2-(((2R,7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate

[0109] Compound 8 was prepared by referring to the method of steps 2 and 3 in “Preparation of Compound 3” using 4-(4-((1R, 5S)-3,8-diazabicyclo[3.2.1]octan-3-yl)-5-chloro-8-fluoro-2-(((2R, 7aS)-2-fluorotetrahydro-1H-pyrrolinazin-7a(5H)-yl)methoxy)pyrido[4,3-d]pyrimidin-7-yl)-5-ethynyl-6-fluoronaphthalen-2-ol (prepared according to Example 21 in WO 2023 / 274324) as a raw material.

[0110] MS m / z:1033.30[M+H]+

[0111] 1H NMR(400MHz,Chloroform-d)δ7.90(dd,J=9.1,5.7Hz,1H),7.82(m,1H),7.56(m,1H),7.36 (m,1H),5.83(s,2H),5.38–5.17(m,1H),5.01(p,J=6.3Hz,1H),4.43(m,2H),4.30–4.16(m, 2H),3.72(m,2H),3.32–3.10(m,3H),3.03–2.93(m,1H),2.85(m,1H),2.38(t,J=7.5Hz,2H ),2.34–2.08(m,3H),1.90(m,6H),1.65(m,2H),1.45–1.17(m,33H),0.87(t,J=6.8Hz,3H).

[0112] II. Liposome Formulation and Internal Aqueous Phase

[0113] 1. Lipid composition

[0114] (1) The screening of lipid components in liposomes was based on the screening of lipid components of compound 8, as follows:

[0115] Table 1 Composition of compound 8 and lipid components in the organic phase

[0116] Steps:

[0117] 1) Weigh a sample according to the organic phase composition in Table 1, add EtOH solution to compound 8 to dissolve compound 8 at a concentration of 1.00 mg / mL, and transfer the lipid component to the sample solution. This solution serves as the organic phase.

[0118] 2) 20 mM phosphate (pH 6.0) was used as the aqueous phase; the sample was mixed by MCM (microchannel mixing) and initially concentrated by TFF (tangential flow filtration), followed by displacement diafiltration and final concentration.

[0119] 3) The final concentrated sample was diluted to the target volume with the external aqueous phase (the theoretical content of compound 8 after dilution was 2 mg / mL). The content and particle size of the preparation after dilution were used as indicators. The test results are shown in Table 2.

[0120] Table 2 Test results and stability of formulations of prescription 1 and prescription 2

[0121] Note: PDI: Polydiseperse Index

[0122] The content of compound 8 was detected by UPLC method (7 min), and the particle size was detected by particle size detection method 1.

[0123] Results: The preparation prepared according to recipe 2 had a relatively good particle size distribution, an average particle size of 100 nm, a high compound content, and good stability within 28 days.

[0124] (2) Lipid component formulation of compound 3

[0125] According to the lipid component screening results of compound 8, referring to prescription 2 in Table 1, 1 mg / mL compound 8 was replaced with 1 mg / mL compound 3, and the organic solvent was 1 mL EtOH to develop the formulation of compound 3.

[0126] The operation steps are shown in Table 3 below:

[0127] Table 3 Experimental process of preparation

[0128] Results: The content, particle size, PDI, and yield were tested. The results are shown in Table 4. The particle size distribution was relatively uniform, the content and yield were high (the theoretical content of compound 3 after dilution and volume adjustment in step 4 was 2 mg / mL), and the sample was stable within 5 days.

[0129] Table 4 Preparation test results and their stability

[0130] Note: Yield = content / theoretical content (2.00 mg / mL).

[0131] The content of compound 3 was detected by UPLC method (7 min), and the particle size was detected by particle size detection method 1.

[0132] 2. Liposome drug-to-lipid ratio

[0133] Operation steps: Referring to the preparation process in Table 3, the content of each lipid component was kept unchanged and only the content of compound 3 was increased (increasing the drug-to-lipid ratio, the experimental formula is shown in Table 5). The content, yield and particle size of the preparation after constant volume were used as indicators to investigate the drug-to-lipid ratio range of the liposomes. The results are shown in Table 6.

[0134] Table 5 Experimental prescriptions with different drug-to-lipid ratios

[0135] Note: Drug-to-lipid ratio = Compound 3 dosage: (DSPC+CHOL+MPEG2000-DSPE) dosage; The content of each substance in the table is the theoretical content of each substance after dilution and constant volume.

[0136] Table 6 Test results of different drug-to-lipid ratios

[0137] Note: Yield = content * volume / (theoretical content * actual volume)%, not considering the loss of flushing pipeline; "Compound 3 content" in the table 1 " is the theoretical content of compound 3 after dilution and constant volume; "content 2 ” is the actual content of compound 3 after dilution and volume fixation.

[0138] The content of compound 3 was detected by UPLC method (7 min), and the particle size was detected by particle size detection method 1.

[0139] Results: The yield could reach above 90% when the drug-lipid ratio was between 1:1.88 and 1:1.46 (dosage of 7.00-9.00 mg / mL).

[0140] 3. Internal water phase

[0141] Procedure: Based on Prescription 2 in Table 5 and referring to the preparation process in Table 3, keep the formulation composition unchanged and only change the internal aqueous phase. Use the particle size and PDI of the preparation as indicators (see Table 7 for results) to examine the pH and ionic strength of the internal aqueous phase of the liposome.

[0142] Buffer preparation:

[0143] (1) Study on the pH value of the internal aqueous phase: The intermediate solution was prepared using an internal aqueous phase with a pH value of 6.0 to 7.0, and the pH value range of the internal aqueous phase was determined using particle size and PDI as the evaluation indicators.

[0144] (2) Study on the ionic strength of the internal aqueous phase buffer salt: 20 mM phosphate with a pH value of 6.5 and 10 mM phosphate with a pH value of 6.5 were used to prepare the intermediate solution, respectively. The particle size and PDI were used as the evaluation indicators to confirm the ionic strength of the internal aqueous phase buffer salt.

[0145] Table 7

[0146] Note: Particle size detection method 1 was used to detect the particle size.

[0147] 4. External water phase

[0148] Operation steps: According to prescription 2 in Table 5, refer to the preparation process in Table 3, keep the prescription composition unchanged, the inner aqueous phase solution is 10mM phosphate (pH 6.5), and the external aqueous phase with a pH value of 7.0 to 7.8 is used for replacement and concentration to obtain the final concentrated solution. The particle size and PDI are used as the evaluation indicators to confirm the pH value range of the external aqueous phase. The results are shown in Table 8.

[0149] Buffer preparation:

[0150] Table 8

[0151] Note: Particle size was determined using particle size detection method 1; PB refers to phosphate buffered saline.

[0152] Results: The final concentrated solution obtained by displacement and concentration with an external aqueous phase at pH 7.0-7.8 was relatively stable within 3 days.

[0153] 5. Types of osmotic pressure regulators and / or lyoprotectants

[0154] Procedure: Prepare the final concentrated solution after TFF according to Recipe 2 in Table 5 and the process in Table 3. Add trehalose and sucrose powders (the weight-volume concentrations of trehalose and sucrose in the liposome solution after addition are shown in Table 9). Filter, fill, and lyophilize. Reconstitute the lyophilized solution in 1 mL of WFI and analyze the results. See Table 9.

[0155] Table 9

[0156] Note: Particle size detection method 1 was used to detect the particle size.

[0157] 6. Osmotic pressure regulator and / or lyoprotectant concentration

[0158] Operation steps: According to prescription 2 in Table 5, the final concentrated solution after TFF was prepared referring to the process in Table 3. Trehalose powder or concentrated solution was added (the mass volume concentration of trehalose in the liposome solution after addition was as shown in Table 10). After filtration, the sample osmotic pressure and particle size were tested. The results are shown in Table 10.

[0159] Table 10

[0160] Note: Particle size detection method 1 was used to detect the particle size.

[0161] III. Liposome Preparation Process

[0162] Step 1: Solution preparation

[0163] (1) Preparation of organic phase solution

[0164] Compound 3 and lipid components (such as DSPC, CHOL, MPEG2000-DSPE) are added to an organic solvent (such as ethanol) and dissolved to obtain an organic phase solution.

[0165] (2) Preparation of internal aqueous phase solution

[0166] Phosphate buffer 1 (e.g., 5-30 mM, pH 6-7, pH can be adjusted with 1 mol / L sodium hydroxide or dilute hydrochloric acid solution) is prepared using disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate.

[0167] (3) Preparation of external aqueous phase solution

[0168] Phosphate buffer 2 (e.g., 5-30 mM, pH 7.0-7.8, pH can be adjusted with 1 mol / L sodium hydroxide or dilute hydrochloric acid solution) is prepared using disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate.

[0169] (4) Preparation of freeze-dried protective agent and / or osmotic pressure regulator solution

[0170] Take an appropriate amount of lyoprotectant and / or osmotic pressure regulator (such as trehalose), dissolve it in the external aqueous phase solution to obtain a lyoprotectant and / or osmotic pressure regulator solution (such as a trehalose concentrated solution with a mass volume concentration of 37.2-40g / 100ml).

[0171] Step 2: Mix

[0172] Connect the A, C, and D pumps of the quaternary plunger pump to the inner aqueous phase solution, and connect the B pump to the organic phase solution. Turn on the plunger pump and set the A, B, C, and D pumps to mix at a certain flow rate ratio (such as 1:1:9:9 or 2:2:8:8) to obtain a mixed solution.

[0173] Step 3: TFF Tangential Flow Filtration

[0174] The mixed solution was initially concentrated by passing it through a 50KD hollow fiber column, and then subjected to external aqueous phase displacement, filtration, and final concentration to obtain a final concentrated solution.

[0175] Step 4: Dilution and volume adjustment

[0176] Take an appropriate amount of lyoprotectant and / or osmotic pressure regulator solution (such as 37.2-40 g / 100 mL trehalose concentrated solution) and dilute the final concentrate after TFF at a certain volume ratio (such as 1:3) to a fixed volume (such as the mass volume concentration of trehalose in the fixed solution is 9.3-10 g / 100 ml).

[0177] Step 5: Filter

[0178] The fixed volume solution was pre-filtered with a 0.45 μm filter, and then the pre-filtered solution was sterilized twice with a 0.22 μm sterilizing filter.

[0179] Step 6: Filling, stoppering, freeze drying, and capping

[0180] The sterile filtered solution is filled according to the content results. After filling, the solution is half-plugged and sent to the freeze dryer for freeze drying to obtain the freeze-dried product, which is then capped to obtain the finished product.

[0181] IV. Examples

[0182] Example 1

[0183] Compound 3 (21 g), DSPC (29.34 g), CHOL (9.57 g), and MPEG2000-DSPE (0.51 g) were weighed and dissolved in ethanol at a drug-to-lipid ratio of 1:1.88. After complete dissolution, 6 L of an organic phase solution was obtained. The organic phase solution was mixed with an internal aqueous phase solution (10 mM phosphate buffer, pH 6.5) using a quaternary plunger pump (Hanbang) at a rate of 149 mL / min (organic phase): 149 mL / min (internal aqueous phase): 596 mL / min (internal aqueous phase): 596 mL / min (internal aqueous phase) to obtain a liposome solution with a particle size of 109.4 nm. After mixing, tangential flow filtration was performed using a mPES (modified polyethersulfone) hollow fiber column (Repligen, model K04-E050-05-N). The inlet end was controlled to have a flow rate of 6 to 8 L / min and a transmembrane pressure of 4.0 to 4.5 Psi for initial concentration by 20 times. The diafiltration was then performed using an external aqueous phase solution (10 mM phosphate buffer with a pH of 7.4) that was 7 times that of the initial concentrate. After diafiltration, the solution was further concentrated by approximately 1.5 times to obtain a concentrate with a liposome concentration of 11.37 mg / ml, a particle size of 79.13 nm, and a PDI of 0.19. The concentrate was diluted with a concentrated trehalose solution (trehalose powder was added to the external aqueous phase solution to form a trehalose mass-volume concentration of 37.2 g / 100 ml) at a volume ratio of 3:1, then sterile filtered to obtain a solution with a pre-filling liposome concentration of 5.88 mg / ml, a particle size of 89.40 nm, a PDI of 0.097, and a pH of 7.3. The solution was sterile filtered, filled, and lyophilized to obtain the final product (Compound 3 liposomes for injection). The final product was reconstituted with water for injection to obtain reconstituted liposomes with an average particle size of 153.6 nm, a PDI of 0.115, and an encapsulation efficiency of 98.5%.

[0184] Table 11 The main index test results of the final product and the reconstituted liposomes are as follows:

[0185] Note: The particle sizes in the examples were all measured using particle size detection method 1.

[0186] Example 2

[0187] At a drug-to-lipid ratio of 1:1.88, compound 3 (143.57 g), DSPC (195.51 g), CHOL (63.69 g), and MPEG2000-DSPE (3.50 g) were weighed and dissolved in ethanol to obtain 40 L of an organic phase solution after complete dissolution.

[0188] The organic phase solution and the internal aqueous phase solution (10 mM phosphate buffer, pH 6.5) were mixed using a quaternary high-pressure reciprocating plunger pump at a rate of 149 mL / min (organic phase): 149 mL / min (internal aqueous phase): 596 mL / min (internal aqueous phase): 596 mL / min (internal aqueous phase) to produce a liposome solution with a particle size of 69.18 nm. After mixing, the liposome solution was initially concentrated 20-fold using a modified polyethersulfone (mPES) hollow fiber column with a flow rate of 53 L / min and a transmembrane pressure of 0.4 bar. The diafiltration was then performed using an external aqueous phase solution (10 mM phosphate buffer, pH 7.4) seven times the initial concentrate. The diafiltration solution was further concentrated approximately 1.67-fold to obtain a liposome concentrate with a concentration of 10.47 mg / ml, a particle size of 79.76 nm, and a PDI of 0.1287. This concentrate was diluted with a concentrated trehalose solution (trehalose powder was added to the external aqueous phase solution to form a trehalose mass-volume concentration of 37.2g / 100ml) at a volume ratio of 3:1, then sterile filtered to obtain a solution with a pre-filling liposome concentration of 6.33mg / ml, a particle size of 92.1nm, a PDI of 0.1153, and a pH of 7.4. This solution was sterile filtered, filled, and lyophilized to obtain a bottled product (Compound 3 Liposomes for Injection) containing 42mg of Compound 3. The final product was reconstituted with water for injection to obtain reconstituted liposomes with an average particle size of 166nm, a PDI of 0.14, an encapsulation efficiency of 97.6%, and a total lipid content of 78.1mg / bottle.

[0189] Table 12 The main index test results of the final product and the reconstituted liposomes are as follows:

[0190] Note: The particle size before reconstitution is determined by particle size detection method 1; the particle size after reconstitution is determined by particle size detection method 2.

[0191] The final product obtained in Example 2 was reconstituted with water for injection and diluted with 5% glucose injection to obtain reconstituted liposomes with a concentration of 2.73 mg / mL of compound 3. The main indicators were detected, and the average particle size was 224 nm (the particle size was detected using particle size detection method 2), the polydispersity index PDI was 0.14, and the encapsulation efficiency was 97.9%.

[0192] The stability of the liposome samples of Compound 3 for injection in Example 2 was investigated at 5±3°C and 25±2°C / 60±5% RH, respectively. The results are shown in Tables 13 and 14 below, respectively.

[0193] Table 13

[0194] Note: Particle size detection method 2 was used to detect the particle size.

[0195] Table 14

[0196] Note: Particle size detection method 2 was used to detect the particle size.

[0197] Results: Under the conditions of 5±3℃, 25±2℃ / 60±5%RH, the quality test indicators of compound 3 liposomes for injection did not change significantly within 3 months, indicating good stability.

[0198] Example 3

[0199] The final product (Compound 3 liposomes for injection) was obtained by referring to the method of Example 2. After reconstitution with water for injection, the reconstituted liposomes were obtained. The average particle size was 154 nm, the PDI was 0.132, and the encapsulation efficiency was 100%.

[0200] Using a flow-through cell method with a 0.1% (w / v) sodium dodecyl sulfate solution (sodium dodecyl sulfate dissolved in 10 mM phosphate buffer, pH 7.4) as the release medium, the in vitro release curves of compound 3 liposomes for injection and compound 3 are shown in Figure 1. The results show that compared with compound 3, the release of compound 3 liposomes for injection was slower, and the time to reach the release plateau was longer than that of compound 3.

[0201] The stability of the liposome sample of Compound 3 for injection in Example 3 was investigated at 5±3°C. The results are shown in Table 15.

[0202] Table 15

[0203] Note: Particle size detection method 1 was used to detect the particle size.

[0204] Results: All quality test indicators of compound 3 liposome for injection showed no significant changes at 5±3℃, indicating good stability.

[0205] V. Biological Evaluation

[0206] Test Example 1: Whole Blood Stability Test

[0207] 1. Sample Preparation

[0208] a) Prepare human and rat whole blood;

[0209] b) Prepare a 10 mM stock solution of the compound in DMSO, take 10 μL and add 990 μL of 50% methanol solution to obtain a 100 μM working solution of the compound;

[0210] c) Add 10 μL of 100 μM compound working solution to 990 μL of whole blood to obtain a 1 μM incubation sample;

[0211] d) Incubate the sample in a water bath at 37°C for 0, 5, 30, 60, 120, 180, 240, and 300 minutes, respectively, and terminate the reaction with pre-cooled internal standard acetonitrile;

[0212] e) centrifuging the terminated sample at 12,000 rpm for 10 minutes;

[0213] f) The supernatant was transferred to a 96-well plate, and the sample was mixed with purified water (v / v=1:1).

[0214] 2. Determine the residual concentration of the compound after incubation and the peak time of the metabolite by LC-MS / MS

[0215] LC-MS / MS assay conditions:

[0216] Mobile phase A: 0.1% formic acid in water;

[0217] Mobile phase B: 0.1% formic acid in acetonitrile;

[0218] Column: ACQUITY UPLC BEH C18 1.7 μm 2.1*50 mm Column;

[0219] Flow rate: 0.50mL / min.

[0220] LC-MS / MS results:

[0221] Metabolite of compound 3 peak time: 0.70min; MS m / z: 637.2[M+H] + .

[0222] The stability results of the compounds after incubation are shown in the following table:

[0223] Note: T 1 / 2 represents the elimination half-life.

[0224] The results showed that compound 3 showed good stability in the whole blood stability test of humans and rats; the active product produced by compound 3 during the whole blood incubation process was compound A.

[0225] Test Example 2: Anti-tumor effect of test compound 3 in human pancreatic cancer HPAC subcutaneous xenograft model

[0226] This study was used to evaluate the antitumor effect of compound 3 in a subcutaneous xenograft BALB / c female nude mouse model of human pancreatic cancer HPAC (with KRAS G12D mutation).

[0227] Experimental animals: BALB / c nude mice, female, 7-8 weeks (age of mice at the time of tumor cell inoculation), weighing 16.8-21.1 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Shanghai Branch.

[0228] Animal modeling and random grouping: HPAC cells were cultured and expanded to 15 T225cm2 culture flasks. The cells were collected, resuspended in serum-free medium RPMI-1640, counted, and added with matrigel at a ratio of 1:1. 5×10 6 0.1 mL was inoculated subcutaneously in the right anterior region of BALB / c nude mice. When the average tumor volume reached approximately 230 mm 3 At 4 hr, the mice were randomly divided into two experimental groups according to tumor size, with 5 mice in each group. The day of grouping was defined as day 0, or D0.

[0229] Experimental plan: BALB / c nude mice were subcutaneously inoculated with HPAC cells to establish a cell line xenograft tumor model. The experiment was divided into a compound 3 (20 mg / kg) group and a vehicle group, with 5 mice in each group, and the drugs were administered by intraperitoneal injection at a volume of 10 μL / g. The vehicle group was given an equal amount of vehicle (10% dimethylacetamide + 5% polyethylene glycol (15)-hydroxystearate + 85% saline) twice daily for 4 weeks. Throughout the experiment, the tumor size and body weight of the mice were measured twice a week to observe any toxic reactions.

[0230] The calculation formula for tumor volume (TV) is: TV = 1 / 2 × a × b × b, where a and b represent the length and width of the tumor, respectively.

[0231] Body weight change rate (%) = (body weight / body weight on D0 - 1) × 100%.

[0232] After measurement and calculation, the mouse tumor volume results are shown in the following table:

[0233] After measurement and calculation, the weight change rate of mice is shown in the following table:

[0234] The tumor volume change curves of the two experimental groups are shown in Figure 2, and the body weight change rate curves are shown in Figure 3.

[0235] The results showed that compound 3 exhibited good anti-tumor effect in the human pancreatic cancer HPAC subcutaneous xenograft BALB / c female nude mouse animal model, with little effect on the body weight of nude mice, showing good safety.

[0236] Test Example 3: Pharmacodynamic evaluation of liposomes of compound 3 for injection in a BALB / c nude mouse subcutaneous transplant model of human metastatic pancreatic adenocarcinoma AsPC-1

[0237] This study was used to evaluate the anti-tumor effect of injectable compound 3 liposomes in a BALB / c female nude mouse model of subcutaneous xenograft of human metastatic pancreatic adenocarcinoma AsPC-1 (with KRAS G12D mutation).

[0238] Experimental animals: BALB / c nude mice, female, 6-8 weeks old (age of mice at the time of tumor cell inoculation), were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd.

[0239] Animal modeling and random grouping: AsPC-1 cells were cultured and expanded to a sufficient number. The cells were collected, resuspended in phosphate buffered saline, counted, and added to Matrigel at a ratio of 1:1. 5×10 6 0.1 mL was inoculated subcutaneously on the right back of BALB / c nude mice. When the average tumor volume reached about 280 mm 3 The grouping day is defined as the first day, that is, D1.

[0240] Experimental plan: BALB / c nude mice were subcutaneously inoculated with AsPC-1 cells to establish a cell line xenograft tumor model. The experiment was divided into 5 groups, with 6 animals in each group, namely Group-1 (solvent, 0.9% sodium chloride injection), Group-2~4 (Compound 3 liposomes for injection, 10 / 30 / 60 mg / kg), and Group-5 (positive control MRTX1133 (prepared with reference to Example 252 of WO2021041671), 10 mg / kg). Among them, MRTX1133 was administered by intraperitoneal injection twice a day for a total of 21 days. The animals in the other groups were all administered by tail vein injection once a week for a total of 3 times. During the entire experiment, the tumor size and body weight of the mice were measured twice a week to observe whether there was any toxic reaction. The solvent for Group-2~4 was water for injection / 0.9% sodium chloride injection. The solvent for Group-5 was 50mM citrate buffer (containing 10% sulfobutyl cyclodextrin) with a pH of 5.0.

[0241] The formula for calculating tumor volume is: tumor volume (mm 3 ) = 0.5 × (long diameter of tumor × short diameter of tumor 2 ).

[0242] The curve of tumor volume change is shown in Figure 4 , and the curve of body weight change is shown in Figure 5 .

[0243] The results showed that in the human pancreatic adenocarcinoma AsPC-1 subcutaneous transplant tumor model, the injectable compound 3 liposome exhibited good anti-tumor activity and had little effect on the body weight of nude mice. During the administration period, the animals tolerated it well and no animals died, showing good safety.

[0244] Test Example 4: PK / PD study of compound 3 liposomes for injection on BALB / c nude mice bearing human metastatic pancreatic adenocarcinoma AsPC-1 xenografts

[0245] 1. Test Purpose

[0246] The time-dependent pharmacokinetic (PK) and pharmacodynamic (PD) effects of compound 3 liposomes and compound A for injection were evaluated in a BALB / c nude mouse subcutaneous xenograft model bearing KRAS G12D mutant human metastatic pancreatic adenocarcinoma AsPC-1.

[0247] 2. Experimental Methods

[0248] AsPC-1 cells were cultured in 0.1 mL (5 × 10 6 A cell suspension of AsPC-1 cells (phosphate buffered saline: matrigel = 1:1, V / V) was inoculated subcutaneously on the right back of each mouse. 3 , grouping began (this day was marked as D1), 21 animals were selected, and the average tumor volume of the animals in the group was 435.8mm 3 The experiment was divided into 7 groups, with 3 mice in each group: Group 1 (vehicle, 0.9% sodium chloride injection), Groups 2 to 6 (Compound 3 liposome injection, 60 mg / kg), and Group 7 (Compound A, 10 mg / kg). Groups 2 to 6 received a single tail vein injection, while Group 7 received a single intraperitoneal injection.

[0249] At the corresponding time points, the experimental animals were euthanized and the tumor tissue was removed. The tumor tissue was divided into two parts (one large and one small (the smaller part was about 50 mg)) and weighed separately. One part (the larger part) was rinsed with cold saline to remove residual blood on the surface, weighed and placed in a pre-cooled tube on ice, and stored in a -80°C refrigerator for subsequent drug concentration analysis. The other part (the smaller part) was slightly dried on absorbent paper and cut in half, divided into two, and the two parts were placed in two cell cryopreservation tubes (weighed in advance) and weighed. After quick freezing with liquid nitrogen, it was temporarily stored in a container filled with dry ice, and then transferred to an ultra-low temperature refrigerator (≤-65°C) for storage in the dark, and subsequently subjected to protein immunoblotting.

[0250] The tumor tissue for drug concentration analysis was removed, and 4 volumes of pre-cooled phosphate buffered saline were added. The tumor tissue was ground into a tumor tissue homogenate using a tissue grinder, and the concentration of compound A was detected using LC-MS / MS.

[0251] The tumor tissue corresponding to the protein immunoblot analysis was removed, and an aliquot of the tumor tissue from the cell cryopreservation tube was transferred to a 5 mL dedicated tissue grinding tube on ice. 30 μL of 4°C / ice-cooled radioimmunoprecipitation lysis buffer (RIPA) (Strong) (Absin, Cat#Abs9229) [containing protease (Thermo, Cat#A32965) and phosphatase inhibitors (Thermo, Cat#A32957)] was added per mg of tumor tissue. Three grinding beads were added at the same time (sealed with sealing film, 30 s each time, 10 s interval, output frequency: 60 Hz, output speed: 1800 N, output current: 0.02 A) and ground (the grinding block was pre-cooled at 4°C overnight in advance) to fully homogenize the tumor tissue. After grinding, the tube was placed on ice for 10 to 20 minutes, all the liquid was aspirated, and the supernatant was collected after centrifugation at 14,000 rpm for 10 minutes at 4°C. 10 μL of the supernatant was diluted 10 times (using RIPA lysis buffer) for BCA quantification. After quantification, the concentration of the tumor homogenate was calculated. A portion of the homogenate was taken, 5× sodium dodecyl sulfate loading buffer was added, the final volume was adjusted with RIPA lysis buffer, and vortexed to mix. The sample was boiled in a 98°C metal bath for 15 minutes, then placed on ice for 2 minutes, separated briefly, and vortexed. Then, the sample was centrifuged at 12,000 rpm at room temperature for 1 minute and loaded for sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis. After sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis, wet transfer was performed. After blocking with 5% bovine serum albumin for 1.5 hours, β-Actin (used as an internal reference protein), p44 / 42MAPK (Erk1 / 2), and pERK were incubated respectively. T202 / Y204 [Phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204)] was incubated with gentle shaking on a shaker at 4°C overnight, and then incubated with secondary antibodies and developed with a chemiluminescent substrate reagent. The color development results were scanned and analyzed by the Tianneng 4600 fully automatic chemiluminescence image analysis system.

[0252] 3. Data Analysis

[0253] LC-MS / MS detection of intratumor compound A concentration:

[0254] Concentration of compound A in mouse tumors after intravenous administration of 60 mg / kg liposomes of compound 3

[0255] Concentration of compound A in mouse tumors after intraperitoneal injection of 10 mg / kg compound A

[0256] Western blotting to detect pERK in tumor tissues T202 / Y204 The protein levels are shown in Figure 6.

[0257] Conclusion: After a single injection of compound 3 liposomes, high drug concentrations and pERK expression can still be detected in the tumor 168 hours later. T202 / Y204 Compared with compound A, it showed a long-lasting effect.

Claims

1. A liposome of a nitrogen-containing heterocyclic compound, comprising the following components: a nitrogen-containing heterocyclic compound as shown in the following compound 3 and a lipid component; The mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:6.58 to 1:1.3; 2. The liposome according to claim 1, wherein It meets one or more of the following conditions: (1) The mass ratio of the nitrogen-containing heterocyclic compound to the lipid component is 1:3 to 1:1.3; preferably 1:2 to 1:1.3; more preferably 1:1.31, 1:1.4, 1:1.46, 1:1.5, 1:1.65, 1:1.7, 1:1.86, 1:1.88 or 1:1.9; (2) The lipid components are phospholipids, PEG lipids and sterols; the phospholipids are preferably distearoylphosphatidylcholine and / or hydrogenated soybean phosphatidylcholine, such as distearoylphosphatidylcholine; the sterol is preferably cholesterol; the PEG lipid is a lipid molecule with a polyethylene glycol-modified hydrophilic end, preferably PEG-modified phosphatidylethanolamine, such as hydrogenated phosphatidylethanolamine; (3) The liposomes may further comprise a buffer salt; the buffer salt is preferably a phosphate buffer salt; more preferably disodium hydrogen phosphate and / or sodium dihydrogen phosphate; the disodium hydrogen phosphate is, for example, disodium hydrogen phosphate heptahydrate; the sodium dihydrogen phosphate is, for example, sodium dihydrogen phosphate monohydrate; the mass ratio of the disodium hydrogen phosphate heptahydrate to the sodium dihydrogen phosphate monohydrate may be 1:0.35 to 1:0.18; preferably 1:0.37 or 1:0.17; and (4) The liposome is composed of the nitrogen-containing heterocyclic compound and the lipid component.

3. The liposome according to claim 2, wherein It meets one or more of the following conditions: (1) The mass ratio of the nitrogen-containing heterocyclic compound to the phospholipid is 1:4.9 to 1:0.98, preferably 1:1.4; (2) The mass ratio of the nitrogen-containing heterocyclic compound to the sterol is 1:1.6 to 1:0.32; preferably 1:0.46; (3) The mass ratio of the nitrogen-containing heterocyclic compound to the PEG lipid is 1:0.09 to 1:0.017; preferably 1:0.024; (4) The liposome further comprises a pH adjuster; the pH adjuster is preferably an inorganic acid or an inorganic base; the inorganic acid is preferably hydrochloric acid; the inorganic base is preferably sodium hydroxide; the pH adjuster can adjust the pH as needed, for example, to 7.3 or 7.4; (5) The liposome is composed of the nitrogen-containing heterocyclic compound, the lipid component and the buffer salt; and (6) The raw materials of the liposome are composed of the nitrogen-containing heterocyclic compound, the lipid component and a buffered salt solution; the buffered salt solution is an aqueous solution of the buffer salt as described in claim 2.

4. The liposome according to claim 3, wherein It meets one or more of the following conditions: (1) The average particle size of the liposome is 50 to 160 nm, for example, 79 nm, 79.13 nm, 79.76 nm, 80 nm, 83 nm, 85 nm, 86 nm, 87 nm, 90 nm, 92 nm, 96 nm, 99 nm or 126 nm; preferably 75 to 100 nm, for example, 79 nm, 79.13 nm, 79.76 nm, 80 nm, 83 nm, 85 nm, 86 nm, 87 nm, 90 nm, 92 nm, 96 nm or 99 nm; (2) The PDI of the liposome is 0.07 to 0.2, such as 0.072, 0.08, 0.083, 0.096, 0.106, 0.107, 0.1, 0.11, 0.12, 0.122, 0.1287, 0.13, 0.146, 0.15, 0.158, 0.16, 0.188 or 0.19; preferably 0.09 to 0.19, such as 0.096, 0.106, 0.107, 0.1, 0.11, 0.12, 0.122, 0.1287, 0.13, 0.146, 0.15, 0.158, 0.16, 0.188 or 0.19; (3) the liposome is composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt and the pH adjuster; and (4) The raw materials of the liposome are composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution and the pH adjuster.

5. The liposome according to any one of claims 1 to 4, wherein The liposome further comprises a freeze-drying protective agent and / or an osmotic pressure regulating agent.

6. The liposome according to claim 5, wherein The lyoprotectant is trehalose and / or sucrose, more preferably trehalose; the osmotic pressure regulator is preferably trehalose and / or sucrose, more preferably trehalose.

7. The liposome according to claim 6, wherein It meets one or more of the following conditions: (1) The average particle size of the liposome is 50 to 160 nm; preferably 75 to 100 nm; for example 80 nm, 81 nm, 82 nm, 89 nm, 89.4 nm, 90 nm, 92 nm or 92.1 nm; (2) The PDI of the liposome is 0.05 to 0.21; preferably 0.05 to 0.15; for example 0.1153, 0.117, 0.12, 0.122, 0.06, 0.061, 0.057, 0.056, 0.051, 0.097 or 0.1; (3) the liposome is composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt, and the lyoprotectant and / or the osmotic pressure regulator; or, the liposome is composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffer salt, the pH regulator, and the lyoprotectant and / or the osmotic pressure regulator; and (4) The raw materials of the liposomes are composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution, and the lyoprotectant and / or the osmotic pressure regulator; or, the raw materials of the liposomes are composed of the nitrogen-containing heterocyclic compound, the lipid component, the buffered salt solution, the pH regulator, and the lyoprotectant and / or the osmotic pressure regulator.

8. The liposome according to any one of claims 1 to 7, wherein The liposome is prepared by the following method, which comprises the following steps: (1) mixing the nitrogen-containing heterocyclic compound, the lipid component and an organic solvent to obtain an organic phase solution; (2) mixing the organic phase solution obtained in step (1) with the buffer salt solution 1, concentrating to obtain a primary concentrated solution; performing displacement, filtration and concentration with the buffer salt solution 2 to obtain a final concentrated solution; The buffered saline solution 1 is an aqueous solution of the buffered saline as claimed in claim 2; the buffered saline solution 2 is an aqueous solution of the buffered saline as claimed in claim 2.

9. The liposome according to claim 8, wherein It meets one or more of the following conditions: (1) In step (1), the organic solvent is an alcohol solvent, such as ethanol; (2) In step (2), the buffered saline solution 1 is an aqueous solution of disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate; (3) In step (2), the pH value of the buffered saline solution 1 is preferably 6 to 7; more preferably 6.5; the pH value of the buffered saline solution 1 can be adjusted by the pH adjusting agent as described in claim 3; (4) In step (2), the concentration of the buffered saline solution 1 is preferably 5 to 30 mM; for example, 10 mM or 20 mM; (5) In step (2), the buffered saline solution 2 is an aqueous solution of disodium hydrogen phosphate heptahydrate and sodium dihydrogen phosphate monohydrate; (6) In step (2), the pH value of the buffered salt solution 2 is preferably 7.0 to 7.8; more preferably 7.4; the pH value of the buffered salt solution 2 can be adjusted by the pH adjusting agent as described in claim 3; (7) In step (2), the concentration of the buffered salt solution 2 is preferably 5 to 30 mM; for example, 10 mM or 20 mM; (8) In step (2), the mixing of the organic phase solution and the buffer salt solution 1 is preferably performed using a quaternary piston pump; more preferably, the flow rate ratio of the organic phase solution: the buffer salt solution 1: the buffer salt solution 1: the buffer salt solution 1 is 2:2:8:8 or 1:1:9:9; for example, 2:2:8:8; (9) In step (2), the concentrated solution after mixing is preferably concentrated by filtration through a hollow fiber column, and the transmembrane pressure of the concentrated solution is more preferably 4 to 6.6 Psi; for example, 4 to 4.5 Psi or 5.8 to 6.53 Psi; the volume of the initial concentrated solution is preferably 0.05 times the volume of the mixed solution of the organic phase solution and the buffer salt solution 1; (10) In step (2), the volume of the buffered saline solution 2 may be 5 to 10 times the volume of the initial concentrated solution; preferably 7 times; (11) In step (2), the concentration after displacement diafiltration is preferably concentrated by hollow fiber column filtration, and the transmembrane pressure of the concentration is more preferably 4 to 6.6 Psi; for example, 4 to 4.5 Psi or 5.8 to 6.53 Psi; the volume of the final concentrate is preferably 0.5 to 0.7 times the volume of the buffered salt solution 2, for example, 0.6 times or 0.67 times; and (12) The method further comprises step (3): mixing the final concentrated solution obtained in step (2) with the lyoprotectant and / or osmotic pressure regulator; Preferably, the liposome satisfies one or more of the following conditions: (a) the lyoprotectant and / or osmotic pressure regulator is preferably present in the form of a solution, more preferably a solution formed by trehalose and the buffered saline solution 2; the mass volume concentration of trehalose in the solution formed by the trehalose and the buffered saline solution 2 may be 37.2 to 40 g / 100 mL; Preferably, the volume ratio of the solution formed by the trehalose and the buffered salt solution 2 to the final concentrated solution in step (2) is preferably 1:3; (b) in step (3), after the final concentrated solution obtained in step (2) is mixed with the lyoprotectant and / or osmotic pressure regulator, the mass volume concentration of the lyoprotectant and / or osmotic pressure regulator is 9.3 to 10 g / 100 mL, preferably 9.3 g / 100 mL; (c) the method further comprises a sterilizing filtration step; preferably, the sterilizing filtration step is preceded by a pre-filtration step; and (d) The method may further comprise a freeze-drying step.

10. An injection preparation, comprising the liposomes according to any one of claims 1 to 9; preferably, in the injection preparation, the liposomes are in the form of lyophilized powder; the injection preparation is more preferably a reconstituted liposome formed by the lyophilized powder of the liposomes and a reconstitution solvent, and the reconstitution solvent is preferably water for injection, 0.9% sodium chloride injection, 5% glucose injection, water for injection and 5% glucose injection, or water for injection and 0.9% sodium chloride injection.

11. The injection preparation according to claim 10, characterized in that It meets one or more of the following conditions: (1) the encapsulation efficiency of the liposome after reconstitution is 95% to 100%, for example 97%, 97.6%, 97.9%, 98% or 98.5%; (2) The average particle size of the liposomes after reconstitution is 100 to 300 nm; preferably 110 to 240 nm; for example, 115 nm, 130 nm, 140 nm, 153 nm, 153.6 nm, 154 nm, 166 nm, 167 nm or 224 nm; (3) The PDI of the reconstituted liposomes is 0.05 to 0.3; preferably 0.1 to 0.25; for example 0.104, 0.11, 0.115, 0.12, 0.13, 0.132, 0.14, 0.16, 0.205 or 0.

21.

12. Use of the liposome according to any one of claims 1 to 9 or the injection preparation according to claim 10 or 11 in the preparation of a drug, The drug is used to treat and / or prevent RAS-mediated diseases; or, the drug can be used to treat and / or prevent cancer; the RAS-mediated diseases are, for example, cancer.

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