Long-circulating pharmaceutical preparation, and preparation method therefor and pharmaceutical use thereof

Abstract

A long-circulating pharmaceutical preparation, and a preparation method therefor and the pharmaceutical use thereof. The preparation has the advantages of a high encapsulation efficiency, a good stability, and a high particle size uniformity, and significantly improves the anti-tumor effect and safety of active ingredients.

Description

A long-circulating drug formulation, its preparation method and medical uses

[0001] This disclosure is based on and claims priority to Chinese patent application No. 202510035706.X, filed on January 9, 2025, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to the field of pharmaceutical preparations, and more specifically, to a long-circulating pharmaceutical preparation, its preparation method, and its pharmaceutical uses. Background Technology

[0003] Camptothecin (CPT) is an alkaloid extracted from the bark of the camptotheca acuminata tree. It possesses DNA topoisomerase inhibitory activity and can be used for anti-tumor purposes. However, its structural instability and adverse reactions to the digestive and urinary systems limit its application. Researchers both domestically and internationally have performed various structural modifications on camptothecin, obtaining numerous derivatives. Currently, the main derivatives used clinically include irinotecan (CPT-11), topotecan (TPT), belotacan, and other novel camptothecin-like compounds. However, these compounds generally have extremely low water solubility, which limits their clinical application prospects.

[0004] Liposomes are a class of drug delivery systems composed of cholesterol and phospholipid bilayers that encapsulate drugs into microvesicles. They possess excellent biocompatibility and are a safe and effective drug carrier. However, liposomal formulations of camptothecin drugs generally suffer from poor stability and low encapsulation efficiency, resulting in safety and efficacy that fail to meet clinical application requirements. Therefore, developing liposomal formulations of camptothecin drugs with good stability, high encapsulation efficiency, and safety and efficacy has long been a problem that needs to be solved in the biopharmaceutical field. Summary of the Invention

[0005] Through extensive experimentation and repeated exploration, the inventors of this application unexpectedly discovered that liposomes containing DNA topoisomerase inhibitors with a specific composition possess advantages such as high encapsulation efficiency, good stability, and high particle size uniformity, and significantly improve the antitumor efficacy and safety of the active ingredient. Therefore, this application relates to pharmaceutical compositions possessing the above advantages, methods for their preparation, and uses.

[0006] In a first aspect, this application relates to a pharmaceutical composition comprising a pharmaceutically active ingredient and one or more ingredients selected from phospholipids, cholesterol, and polyethylene glycol derivatives; said pharmaceutically active ingredient being a DNA topoisomerase inhibitor.

[0007] In some embodiments, the pharmaceutical composition comprises a pharmaceutically active ingredient, phospholipids, cholesterol, and a polyethylene glycol derivative; the pharmaceutically active ingredient is a DNA topoisomerase inhibitor.

[0008] In some embodiments, the DNA topoisomerase inhibitor is selected from compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, solvates, isotopically labeled compounds, polymorphs, metabolites, or prodrugs thereof:

[0009] Among them, R1 is selected from C 1-6 Alkyl or -(C 1-6 (alkylene)-NR 11 R 12 The alkyl or alkylene group may be optionally replaced by halogen, OH, CN, NO2, -NH2, or -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, C 1-4 Halogenated alkyl groups and C 1-4 One or more substituents in the haloalkoxy group are substituted;

[0010] R2 is selected from H, halogens, OH, CN, NO2, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-4 Alkyl, or C 1-4 Alkoxy;

[0011] R 11 R 12 Each is independently selected from H and C. 1-6 Alkyl or R 21 Substituted acyl or sulfonyl group, R 21 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl.

[0012] In some implementations, R1 is selected from C 1-6 Alkyl or -(C 1-6 (alkylene)-NR 11 R 12 R 11 R 12 Each is independently selected from H and C. 1-6 Alkyl, C 1-6 alkyl acyl or C 1-6 Alkyl sulfonyl group.

[0013] In some implementations, R1 is selected from C 1-6 Alkyl or -(C 1-6 (alkylene)-NR 11 R 12 R 11 R 12 Each is independently selected from C 1-6 Alkyl, acetyl, or methanesulfonyl.

[0014] In some implementations, R1 is selected from C 1-6 Alkyl or -(C 1-3 (alkylene)-NR 11 R 12 R 11 R 12 Each is independently selected from C 1-6 Alkyl or methanesulfonyl.

[0015] In some embodiments, R1 is ethyl or -ethylidene-NR 11 R 12 R 11 It is isopropyl, R 12 It can be acetyl or methanesulfonyl.

[0016] In some implementations, R2 is selected from H, halogen, OH, CN, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-4 Alkyl, or C 1-4 Alkyl group.

[0017] In some implementations, R2 is H or OH.

[0018] In some embodiments, the DNA topoisomerase inhibitor structure is selected from the following compounds or their pharmaceutically acceptable salts, stereoisomers, solvates, isotopically labeled compounds, polymorphs, metabolites, or prodrugs:

[0019] In some embodiments, the phospholipid is selected from one, two, or more of egg yolk lecithin, soybean lecithin, or various animal-derived phospholipids, hydrogenated egg yolk lecithin, hydrogenated soybean phosphatidylcholine, dipalmitoyl phosphatidylcholine, myristoyl phosphatidylcholine, distearate phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. In some specific embodiments, the phospholipid is egg yolk lecithin. In one specific embodiment, the phospholipid is egg yolk lecithin E80.

[0020] In some embodiments, the polyethylene glycol derivative is a phospholipid-polyethylene glycol complex.

[0021] In some embodiments, the phospholipid in the phospholipid-polyethylene glycol complex is selected from one or more of oleoylphosphatidylcholine (DOPC), stearoylphosphatidylcholine (DSPC), stearoylphosphatidylethanolamine (DSPE), myristoylphosphatidylcholine (DMPC), palmitoylphosphatidylcholine (DPPC), palmitoylphosphatidylglycerol (DPPG), and stearoylphosphatidylglycerol (DSPG). In one specific embodiment, the phospholipid in the phospholipid-polyethylene glycol complex is DSPE.

[0022] In some embodiments, the polyethylene glycol in the polyethylene glycol derivative has a molecular weight of 1000-10000 Da. In some specific embodiments, the polyethylene glycol in the polyethylene glycol derivative has a molecular weight of 1000-5000 Da. In one specific embodiment, the polyethylene glycol in the polyethylene glycol derivative has a molecular weight of 2000 Da.

[0023] In some embodiments, the phospholipid-polyethylene glycol complex is selected from one or more of phosphatidylcholine-polyethylene glycol (PC-PEG), phosphatidylethanolamine-polyethylene glycol (PE-PEG), stearoylphosphatidylcholine-polyethylene glycol (DSPC-PEG), and stearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG). In some specific embodiments, the phospholipid-polyethylene glycol complex is stearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

[0024] In one specific embodiment, the polyethylene glycol derivative is DSPE-PEG2000.

[0025] In some embodiments, the weight ratio of the active pharmaceutical ingredient to the total amount of the phospholipids, cholesterol, and polyethylene glycol derivatives is 0.1:100 to 10:100. In some specific embodiments, the weight ratio of the active pharmaceutical ingredient to the total amount of the phospholipids, cholesterol, and polyethylene glycol derivatives is 1:100 to 10:100. In some specific embodiments, the weight ratio of the active pharmaceutical ingredient to the total amount of the phospholipids, cholesterol, and polyethylene glycol derivatives is 1:100 to 1:25; for example, 1:100, 2:100, 3:100, or 4:100.

[0026] In some embodiments, the weight ratio of phospholipids to cholesterol is 94.9:0.1 to 1:2. In some embodiments, the weight ratio of phospholipids to cholesterol is 82:13 to 1:1. In some specific embodiments, the weight ratio of phospholipids to cholesterol is 76:19 to 48:47. In some specific embodiments, the weight ratio of phospholipids to cholesterol is 50:45 to 70:25. In some specific embodiments, the weight ratio of phospholipids to cholesterol is 53:42, 54:41, 55:40, 56:39, 57:38, or 58:37. In one specific embodiment, the weight ratio of phospholipids to cholesterol is 56:39.

[0027] In some embodiments, the polyethylene glycol derivative accounts for 0.1% to 10% of the total weight of the phospholipids, cholesterol, and polyethylene glycol derivative. In some specific embodiments, the polyethylene glycol derivative accounts for 0.5% to 8% of the total weight of the phospholipids, cholesterol, and polyethylene glycol derivative. In some specific embodiments, the polyethylene glycol derivative accounts for 5% to 8% of the total weight of the phospholipids, cholesterol, and polyethylene glycol derivative. In some specific embodiments, the polyethylene glycol derivative accounts for 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8% of the total weight of the phospholipids, cholesterol, and polyethylene glycol derivative. In some specific embodiments, the polyethylene glycol derivative accounts for 5% of the total weight of the phospholipids, cholesterol, and polyethylene glycol derivative.

[0028] In some embodiments, the pharmaceutical composition is a long-circulating formulation.

[0029] In some embodiments, the pharmaceutical composition is a liposome.

[0030] In some embodiments, the pharmaceutical composition is a long-circulating liposome.

[0031] In some embodiments, the pharmaceutical composition is an injection.

[0032] In some embodiments, the pharmaceutical composition contains 1 to 10 parts by weight of a pharmaceutically active ingredient, 20 to 80 parts by weight of phospholipids, 20 to 60 parts by weight of cholesterol, and 1 to 10 parts by weight of a polyethylene glycol derivative; the pharmaceutically active ingredient is a DNA topoisomerase inhibitor.

[0033] In some embodiments, the pharmaceutical composition contains 1 to 5 parts by weight of the compound of formula (I), 45 to 65 parts by weight of egg yolk lecithin, 30 to 50 parts by weight of cholesterol, and 3 to 7 parts by weight of a polyethylene glycol derivative.

[0034] In some embodiments, the pharmaceutical composition contains 1 to 5 parts by weight of compound 1, 45 to 65 parts by weight of egg yolk lecithin, 30 to 50 parts by weight of cholesterol, and 3 to 7 parts by weight of a polyethylene glycol derivative.

[0035] In some embodiments, the pharmaceutical composition contains 3 parts by weight of compound 1, 56 parts by weight of egg yolk lecithin, 39 parts by weight of cholesterol, and 5 parts by weight of a polyethylene glycol derivative.

[0036] In some embodiments, the pharmaceutical composition comprises: 1 to 5 parts by weight of a compound of formula (I), 45 to 65 parts by weight of egg yolk lecithin, 30 to 50 parts by weight of cholesterol, and 3 to 7 parts by weight of a polyethylene glycol derivative, and purified water.

[0037] In some embodiments, the pharmaceutical composition contains 1 to 5 parts by weight of compound 1, 45 to 65 parts by weight of egg yolk lecithin E80, 30 to 50 parts by weight of cholesterol, and 3 to 7 parts by weight of a polyethylene glycol derivative.

[0038] In some embodiments, the pharmaceutical composition contains 3 parts by weight of compound 1, 56 parts by weight of egg yolk lecithin E80, 39 parts by weight of cholesterol, and 5 parts by weight of a polyethylene glycol derivative.

[0039] In some embodiments, the pharmaceutical composition further comprises water. In some embodiments, the water is water suitable for liposome formulations, such as purified water, water for injection, or sterile water for injection.

[0040] In some embodiments, the average particle size of the pharmaceutical composition is 5–1000 nm. In some specific embodiments, the average particle size of the pharmaceutical composition is 10–500 nm. In some specific embodiments, the average particle size of the pharmaceutical composition is 10–200 nm.

[0041] In some embodiments, the polydispersity index (PDI) of the pharmaceutical composition is below 0.8. In some specific embodiments, the PDI of the pharmaceutical composition is below 0.5. In some specific embodiments, the PDI of the pharmaceutical composition is below 0.3.

[0042] In some embodiments, the zeta potential of the pharmaceutical composition is 0 to ±50 mV. In some specific embodiments, the zeta potential of the pharmaceutical composition is ±5 to ±30 mV. In some specific embodiments, the zeta potential of the pharmaceutical composition is -5 to -30 mV. In some specific embodiments, the zeta potential of the pharmaceutical composition is -5.0±1, -5.5±1, -6.0±1, -6.5±1, -7.0±1, -7.5±1, -8.0±1, -8.5±1, -9.0±1, -9.5±1, -10.0±1, -10.5±1, -11.0±1, -11.5±1, -12.0±1, -12.5±1, -13.0±1, -13.5±1, -14.0±1, -14.5±1, -15.0±1, -15.5±1, -16.0±1, or -16.5±1.

[0043] In a second aspect, this disclosure also provides a method for preparing any of the pharmaceutical compositions described above, comprising the following steps:

[0044] a) Weigh the active pharmaceutical ingredient, phospholipids, cholesterol, and polyethylene glycol derivatives and dissolve them in an organic solvent;

[0045] b) Remove organic solvents;

[0046] c) Add water to hydrate the membrane;

[0047] d) Homogenize.

[0048] In some embodiments, the organic solvent in step a) is selected from one or more of methanol, ethanol, isopropanol, acetone, butanone, ethyl acetate, butyl acetate, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, cyclohexane, and n-hexane. In one specific embodiment, the organic solvent in step a) is dichloromethane.

[0049] In some embodiments, step b) removes the organic solvent by rotary evaporation or thin-film evaporation.

[0050] Rotary evaporation is a process in which a lipid-organic solution is descaled and rotated to remove the solvent, forming a uniform lipid film on the inner wall of a container. It can be performed under standard experimental conditions in the field. Thin-film evaporation refers to the efficient vaporization and separation of the solvent by heating the tube wall to form a lipid film.

[0051] In some embodiments, step b) removes the organic solvent by rotary evaporation.

[0052] In some implementations, step c) involves hydrating the thin film using ultrasound.

[0053] Thin-film hydration refers to contacting the lipid film prepared by the aforementioned method with an aqueous medium, and then using temperature-controlled stirring / oscillation to allow the lipids to self-assemble into a liposome suspension, which can then be further processed to obtain liposomes of a specific particle size.

[0054] In some embodiments, the ultrasound time in step c) is 0.5 to 5 minutes. In some specific embodiments, the ultrasound time in step c) is 1 to 3 minutes.

[0055] In some embodiments, the ultrasonic power of step c) is 100–600 W. In some specific embodiments, the ultrasonic power of step c) is 180–540 W. In some specific embodiments, the ultrasonic power of step c) is 240–480 W. In one specific embodiment, the ultrasonic power of step c) is 300 W, 320 W, 340 W, 360 W, 380 W, 400 W, or 420 W.

[0056] In some embodiments, the hydration temperature of step c) is 15–50°C. In some specific embodiments, the hydration temperature of step c) is 25–45°C. In one specific embodiment, the hydration temperature of step c) is 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0057] In some embodiments, step d) involves homogenization using ultrasound, high-pressure homogenization, or dynamic high-pressure microfluidics. In some specific embodiments, homogenization is performed using ultrasound. In one specific embodiment, homogenization is performed using probe ultrasound.

[0058] In some embodiments, step d) involves homogenization by probe ultrasound for 1 to 60 minutes. In some specific embodiments, the probe ultrasound time is 3 to 15 minutes. In some specific embodiments, the probe ultrasound time is 8, 9, 10, 11, or 12 minutes.

[0059] In some embodiments, the ultrasonic power of the probe in step d) is 100–500 W. In some specific embodiments, the ultrasonic power of the probe in step d) is 180–300 W. In one specific embodiment, the ultrasonic power of the probe in step d) is 230 W, 240 W, 250 W, 260 W, or 270 W.

[0060] In a third aspect, this disclosure also provides liposomes prepared by the method described in the second aspect.

[0061] In a fourth aspect, this disclosure also provides a pharmaceutical formulation comprising the pharmaceutical composition described in the first aspect of this disclosure or the liposomes described in any of the third aspects, and a pharmaceutically acceptable carrier and / or excipient.

[0062] In a fifth aspect, this disclosure also provides an article of manufacture comprising a pharmaceutical composition, liposome, or pharmaceutical preparation of the present disclosure, and a container for containing the pharmaceutical composition, liposome, or pharmaceutical preparation. In some embodiments, the container is a glass bottle, a metal alloy container, or a pre-filled syringe.

[0063] In a fifth aspect, this application relates to the use of any of the foregoing pharmaceutical compositions, liposomes, pharmaceutical preparations, or articles in the preparation of a medicament, wherein the medicament is used for:

[0064] (a) Prevention and / or treatment of cancer or tumors;

[0065] (b) As an adjunct therapy for cancer or tumors;

[0066] (c) Diagnosing cancer or tumor;

[0067] Any combination of (d)(a)-(c).

[0068] In some embodiments, this application relates to the use of any of the foregoing pharmaceutical compositions, liposomes, pharmaceutical formulations, or articles in the preparation of a medicament, said medicament being used for:

[0069] (a) Prevention and / or treatment of cancer;

[0070] (b) Adjunctive therapy for tumors;

[0071] (c) Diagnosing tumors;

[0072] Any combination of (d)(a)-(c).

[0073] In some embodiments, the drug is administered separately, in combination, simultaneously, or sequentially with another pharmaceutically active agent; optionally, the other pharmaceutically active agent is a chemotherapy drug.

[0074] In some embodiments, this application provides a pharmaceutical composition, liposome, pharmaceutical formulation, or article of any of the above, for use in:

[0075] (a) Prevention and / or treatment of cancer or tumors;

[0076] (b) As an adjunct therapy for cancer or tumors;

[0077] (c) Diagnosing cancer or tumor;

[0078] Any combination of (d)(a)-(c).

[0079] In some embodiments, this application provides a pharmaceutical composition, liposome, pharmaceutical formulation, or article of any of the above, for use in:

[0080] (a) Prevention and / or treatment of cancer;

[0081] (b) Adjunctive therapy for tumors;

[0082] (c) Diagnosing tumors;

[0083] Any combination of (d)(a)-(c).

[0084] In some embodiments, this application provides a method for preventing, treating, adjuvant treating, and / or diagnosing cancer or tumors, the method comprising administering a therapeutically effective amount of any of the above-described pharmaceutical compositions, liposomes, pharmaceutical preparations, or articles to an individual in need.

[0085] In some embodiments, this application provides a method for preventing, treating, adjuvant treating, and / or diagnosing tumors, the method comprising administering a therapeutically effective amount of any of the above-described pharmaceutical compositions, liposomes, pharmaceutical preparations, or articles to an individual in need.

[0086] In some embodiments, the cancer or tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer or squamous cell carcinoma of the lung, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, bile duct cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, glioma, melanoma, leukemia, and lymphoma.

[0087] In some embodiments, the tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer or squamous cell carcinoma of the lung, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, bile duct cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, glioma, melanoma, leukemia, and lymphoma. Attached Figure Description

[0088] Figure 1 shows the in vitro release curve of liposome A in Experiment Example 3.

[0089] definition

[0090] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to technical terms herein refer to techniques commonly understood in the art, including variations or equivalent substitutions of techniques that are obvious to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to better explain the invention.

[0091] As used herein, the terms “including,” “comprising,” “having,” “containing,” or “involving,” and their other variations herein, are inclusive or open-ended and do not exclude other unlisted elements or method steps.

[0092] Unless otherwise stated, the following definitions shall apply as used herein. For the purposes of this invention, chemical elements are defined according to the periodic table of elements, CAS edition, and the Chemical Reagents Handbook, 75th edition, 1994. Furthermore, general principles of organic chemistry can be found in “Organic Chemistry,” Thomas Sorrell, University Science Books, Sausalito: 1999, and “March's Advanced Organic Chemistry,” by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

[0093] As used herein, the term "alkyl" refers to a 1-20 carbon atom saturated straight-chain or branched aliphatic hydrocarbon group, wherein the alkyl group may be independently and optionally substituted by one or more substituents described in this disclosure. As used herein, the term "C" refers to... 1-6 "Alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 6 carbon atoms. The term "C"... 1-4 "Alkyl" refers to a saturated straight-chain or branched aliphatic hydrocarbon group having 1 to 4 carbon atoms, optionally substituted with one or more (such as 1 to 4) suitable substituents such as halogens. Examples of alkyl groups include, but are not limited to, methyl (Me, -CH3), ethyl (Et, -CH2CH3), n-propyl (n-Pr, -CH2CH2CH3), isopropyl (i-Pr, -CH(CH3)2), n-butyl (-Bu, -CH2CH2CH2CH3), 2-methylpropyl or isobutyl ( i -Bu, -CH2CH(CH3)2), 1-methylpropyl or sec-butyl ( s-Bu, -CH(CH3)CH2CH3), tert-butyl (t-Bu, -C(CH3)3), n-pentyl (-CH2CH2CH2CH2CH3), 2-pentyl (-CH(CH3)CH2CH2CH3), 3-pentyl (-CH(CH2CH3)2), 2-methyl-2-butyl (-C(CH3)2CH2CH3), 3-methyl-2-butyl (-CH(CH3)CH(CH3)2), 3-methyl-1-butyl (-CH2CH2CH(CH3)2), 2-methyl-1-butyl (-CH2CH(CH3)CH2CH3), n-hexyl (-CH2CH2CH2CH2CH2CH3), 2-hexyl (-CH(CH3)CH2CH2C) H2CH3), 3-hexyl (-CH(CH2CH3)(CH2CH2CH3)), 2-methyl-2-pentyl (-C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (-CH(CH3)CH(CH3)CH2CH3), 4-methyl-2-pentyl (-CH(CH3)CH2CH(CH3)2), 3-methyl-3-pentyl (-C(CH3)(CH2CH3)2), 2-methyl-3-pentyl (-CH(CH2CH3)CH(CH3)2), 2,3-dimethyl-2-butyl (-C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (-CH(CH3)C(CH3)3), n-heptyl, n-octyl, etc. The term "alkyl" and its prefix "alkane" are used here, both encompassing straight-chain and branched saturated aliphatic hydrocarbon carbon chains.

[0094] In this document, the term "alkoxy" refers to a group formed by the attachment of an alkyl group to an oxygen group (alkyl-O-). The term "alkyl" is as defined above. For example, the term "C"... 1-12 "Alkoxy" refers to "C 1-12 The alkoxy group, labeled "alkyl-O-", contains 1-12 carbon atoms. In one embodiment, the alkoxy group contains 1-6 carbon atoms. In another embodiment, the alkoxy group contains 1-4 carbon atoms. In yet another embodiment, the alkoxy group contains 1-3 carbon atoms. Such embodiments include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, or n-hexoxy.

[0095] In this document, the term "cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic, including spirocyclic, fused or bridged systems, such as bicyclic [1.1.1]pentyl, bicyclic [2.2.1]heptyl, bicyclic [3.2.1]octyl or bicyclic [5.2.0]nonyl, decahydronaphthyl, etc.), optionally substituted by one or more (such as 1 to 3) suitable substituents. For example, the term "C 3-10 "Cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (including fused, bridged, or spirocyclic structures) hydrocarbon ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) having 3 to 10 cyclic carbon atoms, optionally substituted with one or more (such as 1 to 3) suitable substituents, wherein the substituents may be, but are not limited to, oxo (=O), fluorine, chlorine, bromine, iodine, hydroxyl, amino, -C (=O)-NH2, carboxyl, -S (=O). t OH, -OS (=O) t -H, -S (=O) t NH2, triazolyl, tetrazolyl, -(CR3R3') t -NH2, alkyl, alkyl-S (=O) t -, haloalkyl, hydroxyalkyl, alkoxy, alkylamino, alkathiol, haloalkoxy, amino, aryl, heteroaryl, alkenyl, alkynyl, heterocyclic, thiol, nitro, aryloxy, hydroxyalkoxy, alkanoyl, benzyl, cyclopropyl, phenyl, alkyl-C(=O)-, alkyl-C(=O)-NH-, formamido or alkoxyalkyl, etc., where R3 and R3' are independently hydrogen or alkyl, and t is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, C 3-8 cycloalkyl, C 3-6 Cycloalkyl.

[0096] Examples of cycloalkyl groups further include, but are by no means limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopentyl-1-enyl, 1-cyclopentyl-2-enyl, 1-cyclopentyl-3-enyl, cyclohexyl, 1-cyclohexyl-1-enyl, 1-cyclohexyl-2-enyl, 1-cyclohexyl-3-enyl, cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, adamantyl, etc.

[0097] As used herein, the term "halogen" is defined as including fluorine, chlorine, bromine, or iodine.

[0098] As used in this article, the term "halogenation" refers to the substitution of one or more (such as 1 to 3) identical or different halogen atoms.

[0099] As used herein, the term "haloalkyl" refers to an alkyl group substituted with one or more (such as 1 to 3) identical or different halogen atoms. For example, the term "C" 1-6 "Halogenated alkyl" refers to alkyl halogroups having 1 to 6 carbon atoms, such as -CF3, -C2F5, -CHF2, -CH2F, -CH2CF3, -CH2Cl, or -CH2CH2CF3.

[0100] As used herein, the term "haloalkoxy" refers to an alkoxy group substituted with one or more (such as 1 to 3) identical or different halogen atoms. For example, the term "C 1-6 "Haloalkoxy" refers to haloalkoxy groups having 1 to 6 carbon atoms, such as -O-CF3, -O-C2F5, -O-CHF2, -O-CH2F, -O-CH2CF3, -O-CH2Cl, or -O-CH2CH2CF3, etc.

[0101] As used herein, the term "alkenyl" refers to an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and in which one hydrogen atom is replaced by a bond. The alkenyl group can be straight-chain or branched and contains from about 2 to about 15 carbon atoms. In one embodiment, the alkenyl group contains from about 2 to about 12 carbon atoms. In another embodiment, the alkenyl group contains from about 2 to about 6 carbon atoms. Non-limiting examples of alkenyl groups include vinyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl, and decenyl. The alkenyl group can be unsubstituted or substituted with one or more identical or different substituents, each substituent being independently selected from halogens, alkenyl, alkynyl, aryl, cycloalkyl, cyano, hydroxyl, -O-alkyl, -O-aryl, -alkylene-O-alkyl, alkylthio, -NH2, -NH(alkyl), -N(alkyl)2, -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O-alkyl. The term "C..." 2-6 "Alkenyl" refers to an alkenyl group with 2 to 6 carbon atoms.

[0102] As used herein, the term "heterocyclic" or "heterocyclic group" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic group, for example, having 2, 3, 4, 5, 6, 7, 8, or 9 carbon atoms in the ring and one or more (e.g., 1, 2, 3, or 4) independently selected from N, O, or S(O). tHeteroatoms (where t is 0, 1, or 2), such as 3-12 membered heterocyclic groups, 3-10 membered heterocyclic groups, 3-9 membered heterocyclic groups, 3-8 membered heterocyclic groups, 3-7 membered heterocyclic groups, 3-6 membered heterocyclic groups, 5-6 membered heterocyclic groups, etc. Representative examples of heterocyclic groups include, but are not limited to, ethylene oxide, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydropyrrolyl, hexahydro-1H-pyrrolline, pyrrolidone, imidazoalkyl, pyrazolyl, tetrahydropyranyl, tetrahydropyridinyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazine, etc.

[0103] As used herein, the term "aryl" or "aromatic ring" refers to a fully carbon monocyclic or fused-ring polycyclic aromatic group having a conjugated π-electron system. For example, the term "C 6-10 "Aryl" or "C" 6-10 "Aromatic ring" refers to an aromatic group containing 6 to 10 carbon atoms, such as phenyl (ring) or naphthyl (ring). The aryl or aromatic ring may optionally be substituented by one or more (such as 1 to 3) suitable substituents (e.g., halogen, -OH, -CN, -NO2, C). 1-6 Alkyl groups, etc., are substituted.

[0104] In this document, the term "heteroaryl" refers to an aromatic cyclic group in which at least one ring atom is a heteroatom, such as a nitrogen atom, an oxygen atom, a boron atom, or a sulfur atom. Optionally, the ring atom (e.g., a carbon atom, a nitrogen atom, or a sulfur atom) in the cyclic structure may be oxidized. Specific examples include, but are not limited to, 5-10-membered heteroaryl, 6-10-membered heteroaryl, 5-10-membered nitrogen-containing heteroaryl, 6-10-membered oxygen-containing heteroaryl, 6-8-membered nitrogen-containing heteroaryl, 5-8-membered oxygen-containing heteroaryl, etc., such as furanyl, thiophene, pyrrole, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, oxadiazolyl, imidazole, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3, 4-Oxadiazolyl, pyridyl, 2-pyridoneyl, 4-pyridoneyl, pyrimidinyl, 1,4-dioxazadienyl, 2H-1,2-oxazinyl, 4H-1,2-oxazinyl, 6H-1,2-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl, 4H-1,4-oxazinyl, pyridazinyl, 1,2,3-triazinyl, 1,3,5-triazinyl, 1,2,4,5-tetraazinyl, aziridine-heptanetrienyl, 1,3-diazacycloheptanetrienyl, aziridine-octatetraenyl, etc.

[0105] The hydrogen in the groups involved in this disclosure can be replaced by isotopes such as protium, deuterium, and tritium.

[0106] The term "substitution" refers to the selective replacement of one or more (e.g., 1, 2, 3, or 4) hydrogen atoms on a specified atom by a designated group, provided that the substitution does not exceed the normal valence of the specified atom in the present case and the substitution forms a stable compound. Combinations of substituents and / or variables are permitted only if such combinations form a stable compound.

[0107] If a substituent is described as “optionally substituted with…”, then the substituent may be (1) unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the carbon (to the extent that any hydrogens are present) may be substituted individually and / or together with independently selected substituents or not substituted. If the nitrogen of the substituent is described as being optionally substituted with one or more of the substituents in the list, then one or more hydrogens on the nitrogen (to the extent that any hydrogens are present) may each be substituted with independently selected substituents or not substituted.

[0108] If a substituent is described as being "independently selected" from a group of groups, then each substituent is selected independently of the others. Therefore, each substituent may be the same as or different from another (other) substituent.

[0109] As used herein, the term "one or more" means one or more under reasonable conditions, such as two, three, four, five, six, seven, eight, nine, or ten.

[0110] The text used This is represented as a connection key.

[0111] Unless otherwise specified, as used herein, the connection point of a substituent may be derived from any suitable location of the substituent.

[0112] When the bond of a substituent is such that it passes through the ring and connects two atoms, then such a substituent can be bonded to any cyclic atom in the substituted ring.

[0113] This disclosure also includes all pharmaceutically acceptable isotopically labeled compounds identical to those of this disclosure, except that one or more atoms are replaced by atoms having the same atomic number but with an atomic mass or mass number different from the dominant atomic mass or mass number in nature. Examples of isotopes suitable for inclusion in the compounds of this disclosure include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H, deuterium (D), tritium (T); carbon isotopes (e.g., H, deuterium (D), tritium (T)); 11 C 13 C and 14 C); isotopes of chlorine (e.g.) 37 Cl); isotopes of fluorine (e.g., Cl); 18F); isotopes of iodine (e.g., F); 123 I and 125 I); nitrogen isotopes (e.g.) 13 N and 15 N); isotopes of oxygen (e.g., N); 15 O、 17 O and 18 O); isotopes of phosphorus (e.g., O); phosphorus isotopes (e.g., O); 32 P); and isotopes of sulfur (e.g., ... 35 S). Certain isotope-labeled compounds of this disclosure (e.g., those doped with radioisotopes) can be used in drug and / or substrate tissue distribution studies (e.g., analysis). Radioisotope tritium (i.e. 3 H) and carbon-14 (i.e. 14 C) It is particularly suitable for this purpose due to its ease of incorporation and monitoring. Using positron-emitting isotopes (e.g.) 11 C 18 F, 15 O and 13 Substitution of N) can be used in positron emission tomography (PET) studies to examine substrate acceptor occupancy. Isotopically labeled compounds of this disclosure can be prepared by methods similar to those described in the accompanying routes and / or examples and preparations, by replacing previously used unlabeled reagents with appropriate isotopically labeled reagents. Pharmaceutically acceptable solvates of this disclosure include those in which the crystallization solvent can be isotopically substituted, for example, D2O, acetone-d6, or DMSO-d6.

[0114] The term "stereoisomer" refers to an isomer formed due to at least one asymmetric center. In compounds having one or more (e.g., 1, 2, 3, or 4) asymmetric centers, racemic mixtures, single enantiomers, diastereomer mixtures, and individual diastereomers can be produced. Specific individual molecules can also exist as geometric isomers (cis / trans). Similarly, the compounds of this disclosure can exist as mixtures of two or more structurally different forms in rapid equilibrium (commonly referred to as tautomers). Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, etc. It is to be understood that the scope of this application covers all such isomers or mixtures thereof in any proportion (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

[0115] This disclosure covers all possible crystalline forms or polymorphs of the compounds disclosed herein, which may be a single polymorph or a mixture of more than one polymorph in any proportion.

[0116] It should also be understood that certain compounds of this disclosure may exist in their free form for therapeutic purposes, or, where appropriate, in their pharmaceutically acceptable derivative forms. In this disclosure, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites, or prodrugs that, upon administration to a patient in need, can directly or indirectly provide the compounds of this disclosure or their metabolites or residues. Therefore, when referring herein to “compounds of this disclosure,” it is also intended to encompass the various derivative forms of the compounds described above.

[0117] Pharmaceutically acceptable salts of the compounds disclosed herein include their acid addition salts and base addition salts. Suitable acid addition salts are formed by acids that form pharmaceutically acceptable salts. Suitable base addition salts are formed by bases that form pharmaceutically acceptable salts. A review of suitable salts can be found in Stahl and Wermuth's "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds disclosed herein are known to those skilled in the art.

[0118] The compounds disclosed herein may exist as solvates (preferably hydrates), wherein the compounds of this disclosure contain a polar solvent as a structural element of the compound's crystal lattice. The amount of the polar solvent, particularly water, may be stoichiometric or non-stoichiometric.

[0119] Those skilled in the art will understand that not all nitrogen-containing heterocycles can form N-oxides because nitrogen requires available lone pairs of electrons to be oxidized into oxides; those skilled in the art will identify nitrogen-containing heterocycles that can form N-oxides. Those skilled in the art will also recognize that tertiary amines can form N-oxides. Synthetic methods for preparing N-oxides of heterocycles and tertiary amines are well known to those skilled in the art, including the oxidation of heterocycles and tertiary amines with peroxy acids such as peracetic acid and m-chloroperoxybenzoic acid (MCPBA), hydrogen peroxide, alkyl peroxides such as tert-butyl peroxide, sodium perborate, and dioxiranes such as dimethyldioxirane. These methods for preparing N-oxides have been extensively described and reviewed in the literature, see, for example: T.L. Gilchrist, Comprehensive Organic Synthesis, vol. 7, pp. 748-750; A.R. Katritzky and A.J. Boulton, Eds., Academic Press; and G.W. H. Heeseman and E.S. G. Wierstiuk, Advances in Heterocyclic Chemistry, vol. 22, pp. 390-392, A.R. Katritzky and A.J. Boulton, Eds., Academic Press.

[0120] The scope of this disclosure also includes metabolites of the compounds of this disclosure, i.e., substances formed in the body upon administration of the compounds of this disclosure. Such products can be generated, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, enzymatic hydrolysis, etc., of the administered compound. Therefore, this disclosure includes metabolites of the compounds of this disclosure, including compounds prepared by methods that expose the compounds of this disclosure to mammals for a time sufficient to produce their metabolites.

[0121] This disclosure further includes, within its scope, prodrugs of the compounds of this disclosure, which are certain derivatives of the compounds of this disclosure that may themselves have little or no pharmacological activity, and which, when administered to or onto the body, can be converted, for example, by hydrolysis and cleavage into the compounds of this disclosure having the desired activity. Typically, such prodrugs are functional group derivatives of the compounds that are readily converted in vivo into the compounds with the desired therapeutic activity. Further information regarding the use of prodrugs can be found in “Pro-drugs as Novel Delivery Systems,” Vol. 14, ACS Symposium Series (T. Higuchi and V. Stella) and “Bioreversible Carriers in Drug Design,” Pergamon Press, 1987 (EB Roche, editor, American Pharmaceutical Association). Prodrugs of this disclosure can be prepared, for example, by replacing suitable functional groups present in the compounds of this disclosure with certain portions known to those skilled in the art as “pro-moiety” (e.g., as described in “Design of Prodrugs,” H. Bundgaard (Elsevier, 1985)).

[0122] The term “about” means within ±10% of the stated value, preferably within ±5%, and more preferably within ±2%.

[0123] Unless otherwise specified, percentages in this application refer to weight percentages, and parts refer to weight parts.

[0124] As used herein, the term “particle size” refers to the size of a particle, and “average particle size” (Z-average Size) refers to the average particle size obtained by means of light scattering, which can be measured by conventional particle size measurement techniques and instruments well known to those skilled in the art, such as the Malvern nanoparticle size analyzer.

[0125] As used in this article, the term "PDI" refers to particle size distribution. The larger the PDI, the wider the particle size distribution; the smaller the PDI, the narrower the particle size distribution and the more uniform the particle size.

[0126] As used in this article, the term "homogenization" refers to the process of micronizing and homogenizing the dispersions in a suspension system, which simultaneously reduces the size of the dispersions and improves the uniformity of their distribution.

[0127] This application is by no means limited to the methods and materials described herein. In the event that one or more of the referenced documents, patents and similar materials differ from or contradict this application (including but not limited to the definition of terminology, application of terminology, described techniques, etc.), the description and accompanying structural formulas of this application shall prevail.

[0128] All technical features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive technical features and / or steps. Detailed Implementation

[0129] The present disclosure is now described with reference to the following illustrative and not limiting embodiments. Unless otherwise specified, the experiments and methods described in the embodiments are generally performed according to conventional methods well known in the art and described in various references. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will appreciate that the embodiments are described by way of example and are not intended to limit the scope of protection claimed in this disclosure. All disclosed patents and other references mentioned herein are incorporated herein by reference in their entirety.

[0130] 1. Instruments involved in the embodiments

[0131] High-performance liquid chromatograph (Agilent 1260 Infinity, USA); Zetasizer Nano ZS90 laser particle size analyzer (Malvern, UK); Ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology Co., Ltd.)

[0132] 2. Cell lines and animal sources involved in the examples

[0133] Mouse breast cancer 4T1 cell line (ATCC); mouse colon cancer cell line CT26 (ATCC); human non-small cell lung cancer cell line A549 (ATCC); human breast cancer cell line MDA-MB-231 (ATCC); mouse Lewis lung cancer cell line LLC (ATCC); Balb / c mice (Speford (Beijing) Biotechnology Co., Ltd.)

[0134] 3. Source of drugs or reagents involved in the examples

[0135] Egg yolk lecithin E80 (Lipoid, Germany); 1,2-distearate-sn-glycerol-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG, Fanshuo Biotechnology Co., Ltd.); Cholesterol (Shanghai Maclean Biochemical Technology Co., Ltd.); (S)-N-(2-(4-ethyl-4-hydroxy-3,14-dione-3,4,12,14-tetrahydro-1H-pyrano[3',4',6,7]indolazido[1,2-b]quinoline-11-yl)ethyl)-N-isopropylmethanesulfonamide (self-made by Sichuan Kelun Biotech Co., Ltd.); 7-ethyl-10-hydroxycamptothecin (SN38, Dalian Meilun Biotechnology Co., Ltd.).

[0136] 4. Detection methods involved in the examples

[0137] For detection methods not specifically specified in the examples, they should be performed under conventional conditions or by referring to existing literature in the field (e.g., Xue Yuan, Liang Yi, Wang Leilei, et al. Preparation and in vitro properties of liposomes co-loaded with dihydroporphyrin and perfluorohexane [J]. West China Journal of Pharmaceutical Sciences, 2021, 36(02):143-147; Zhou Ting, Li Chunyan, Li Yun, et al. Optimization of formulation process of hydroxycamptothecin liposomes by orthogonal design [J]. Chinese Herbal Medicines, 2019, 42(09):2128-2132; Sun Li, Sun Kaoxiang, Chen Lingli. Preparation, quality evaluation and preliminary pharmacodynamic study of SN-38 liposomes [J]. China Pharmaceutical Industry Journal, 2019, 50(02):204-209.).

[0138] (1) High-performance liquid chromatography analysis and testing methods

[0139] A Kromasll C18 column (150 mm × 4.6 mm, 5 μm) packed with octadecylsilane-bonded silica gel was used. Mobile phase A consisted of an aqueous solution containing 10% (v / v) acetonitrile (containing 0.05% trifluoroacetic acid), and mobile phase B consisted of acetonitrile (containing 0.03% trifluoroacetic acid). The elution program is shown in Table 1. The detection wavelength was 240 nm; the column temperature was 40 °C; the flow rate was 1.0 mL / min; and the injection volume was 20 μL. Acetonitrile was used as the sample diluent.

[0140] Table 1 Elution Procedures for High Performance Liquid Chromatography

[0141] (2) Particle size determination: Dynamic light scattering method was used.

[0142] Example 1-1: Properties of different formulation liposomes

[0143] Compound 1 and lipids (egg yolk lecithin E80 or soybean lecithin S100) were weighed according to the amounts specified in Table 2A. They were dissolved in dichloromethane and transferred to a flask. The flask was then rotary evaporated at 37°C in a water bath at 100 rpm until the dichloromethane was completely removed. 1–10 ml of purified water was added, and the mixture was sonicated in a water bath at 20–50°C with a power of 100–500 W for 0–30 min. The mixture was then homogenized by sonication in an ice bath with a probe at a power of 100–500 W for 1–60 min. The average particle size and polydispersity index (PDI) of the liposomes were measured, and the results are shown in Table 2B.

[0144] Table 2A: Dosage of each component in liposomes

[0145] Table 2B Results of the investigation of liposome properties

[0146] As shown in Table 2B, liposome 1-1 using egg yolk lecithin E80 exhibits better particle size uniformity and a smaller average particle size compared to liposome 1-2 using soybean lecithin S100. However, the stability of both liposomes needs improvement; solids clearly precipitated at the bottom after standing overnight.

[0147] Examples 1-2: Properties of different formulation liposomes

[0148] Compound 1 and total lipids (including egg yolk lecithin E80: cholesterol: DSPE-PEG2000 = 56:39:5 (m:m:m)) were weighed according to the amounts specified in Table 3A. Liposomes were prepared according to the method in Example 1-1, and the average particle size, encapsulation efficiency, and stability of each liposome were determined. The results are shown in Table 3B.

[0149] Table 3A: Dosage of each component in liposomes

[0150] Table 3B shows the results of the investigation of each liposome.

[0151] The results above show that liposomes 1-3, 1-4, and 1-5 all have ideal encapsulation efficiency, and the particle size does not change significantly over a period of up to 8 days, demonstrating good stability.

[0152] Example 2: Investigation of the properties of liposomes with different formulations

[0153] Compound 1, egg yolk lecithin E80 in different proportions, cholesterol, and DSPE-PEG2000 were weighed separately, with 3 mg of compound 1 and 5 mg of DSPE-PEG2000. The amounts of egg yolk lecithin E80 and cholesterol are shown in Table 4. The compounds were dissolved in dichloromethane and transferred to flasks. The mixtures were then rotary evaporated at 37°C in a water bath at 100 rpm until the dichloromethane was completely removed. 1–10 ml of purified water was added, and the mixture was sonicated in a water bath at 20–50°C for 0–30 min. The mixtures were then homogenized by sonication at 100–500 W using an ultrasonic probe in an ice bath for 1–60 min. The average particle size, encapsulation efficiency, and stability of each liposome were measured. The results are shown in Table 5.

[0154] Table 4. Amounts of phospholipids and cholesterol in liposomes

[0155] Table 5. Results of the investigation of each liposome.

[0156] The results above show that liposomes 2-3, 2-4, and 2-5 all exhibited ideal encapsulation efficiency. Furthermore, the particle size of liposome 2-4 did not change significantly over an period of up to 8 days, demonstrating excellent stability.

[0157] Example 3: Properties of different formulation liposomes

[0158] Compound 1, egg yolk lecithin E80, cholesterol, and DSPE-PEG2000 were weighed separately, with 3 mg of Compound 1, 56 mg of egg yolk lecithin E80, and 39 mg of cholesterol. The amounts of DSPE-PEG2000 are shown in Table 6 below. After dissolving in dichloromethane, the solutions were transferred to flasks and rotary evaporated at 37°C in a water bath at 100 rpm until the dichloromethane was completely removed. 1–10 ml of purified water was added, and the solutions were sonicated in a water bath at 20–50°C for 0–30 min. The solutions were then homogenized by sonication at 100–500 W using an ultrasonic probe in an ice bath for 1–60 min. The average particle size, encapsulation efficiency, and stability of each liposome were measured. The results are shown in Table 7.

[0159] Table 6. Dosage of DSPE-PEG2000 in liposomes

[0160] Table 7 Results of the study on the amount of DSPE-PEG2000 added

[0161] The results above show that liposomes 3-2 to 3-4 all have good stability. In particular, the particle size of liposomes 3-3 and 3-4 did not change significantly over a period of up to 8 days, indicating good stability.

[0162] Example 4: Properties of liposomes prepared by different processes

[0163] Compound 1, egg yolk lecithin E80, cholesterol, and DSPE-PEG2000 were weighed separately, with 3 mg of compound 1, 56 mg of lecithin, 39 mg of cholesterol, and 5 mg of DSPE-PEG2000. These were dissolved in dichloromethane and transferred to a flask. The mixture was then rotary evaporated at 37°C in a water bath at 100 rpm until the dichloromethane was completely removed. 1–10 ml of purified water was added, and the mixture was sonicated in a water bath at 20–50°C for 0–30 min. The mixture was then homogenized by sonication in an ice bath. The sonication power and homogenization time are shown in Table 8. Except for the sonication power and homogenization time, the preparation conditions for each liposome were consistent. The average particle size, encapsulation efficiency, and stability of each liposome were measured. The results are shown in Table 9.

[0164] Table 8. Homogenization parameters for liposomes

[0165] Table 9. Results of the investigation of liposomes prepared by different processes.

[0166] The results above show that liposomes 4-2 to 4-6 all have low PDI, indicating that the liposomes have high uniformity in particle size distribution.

[0167] Example 5: Properties of liposomes prepared by different processes

[0168] Compound 1, egg yolk lecithin E80, cholesterol, and DSPE-PEG2000 were weighed separately, with 3 mg of compound 1, 56 mg of lecithin, 39 mg of cholesterol, and 5 mg of DSPE-PEG2000. These were dissolved in dichloromethane and transferred to a flask. The mixture was then rotary evaporated at 37°C in a water bath at 100 rpm until the dichloromethane was completely removed. 1–10 ml of purified water was added, and the mixture was ultrasonically sonicated in a water bath for 0–30 min at the temperatures shown in Table 10. The mixture was then homogenized by ultrasonication at a power of 100–500 W in an ice bath for 1–60 min. Except for the water bath ultrasonication temperature, the preparation conditions for each liposome were consistent. The average particle size, encapsulation efficiency, and stability of each liposome were measured. The results are shown in Table 11.

[0169] Table 10 Ultrasonic water bath temperature for liposomes

[0170] Table 11 Results of the study on liposomes

[0171] The results above show that liposomes 5-1 to 5-3, prepared at different water bath temperatures, all exhibit good particle size uniformity and encapsulation efficiency, with liposomes 5-1 and 5-2 achieving an encapsulation efficiency as high as 90%.

[0172] Experimental Example 1: Particle size, potential, and morphology of liposomes

[0173] Weigh 3 mg of compound 1, 56 mg of egg yolk lecithin E80, 39 mg of cholesterol, and 5 mg of DSPE-PEG2000, dissolve them in 3 ml of dichloromethane, and transfer them to a 25 ml round-bottom flask; remove the dichloromethane completely by rotary evaporation at 100 r / min for 5 min in a 37 °C water bath; add 3 ml of purified water and sonicate in a 37 °C water bath for 2 min to hydrate the membrane; homogenize by sonication at 250 W power in an ice bath for 10 min to obtain liposome A.

[0174] Liposome B was prepared by replacing compound 1 with SN38 (structure shown below) according to the above dosage and process.

[0175] The particle size, potential, and polydispersity index (PDI) of liposome A were determined using a laser particle size analyzer. Based on the dynamic light scattering measurements, the average diameter, PDI, and zeta potential of liposome A were (160±20) nm, 0.23±0.05, and (-10.5±1) mV, respectively, indicating that liposome A has a small particle size and uniform distribution.

[0176] Experimental Example 2: Encapsulation efficiency and drug loading of liposomes

[0177] Using purified water as the eluent, the free compound 1 and the liposome-bound compound 1 in liposome A prepared in Experimental Example 1 were separated by dextran gel column chromatography. The content of compound 1 in the separated liposome-bound compound 1 was quantitatively determined using the high-performance liquid chromatography analysis method described above, and the result was taken as m. 脂 Liposome A prepared in Example 1 without dextran column chromatography was directly taken, and the content of compound 1 in it was determined as m. 总药 ; liposome A prepared in Experiment 1 without dextran column chromatography was placed in a pre-weighed dry vial, dried, and then weighed. The weight of the liposome was determined to be m. 总 The drug loading and encapsulation efficiency are calculated using the following formula: DL (Drug Loading) = m 脂 / m 总 ×100%; EE (Encapsulation efficiency)%=m 脂 / m 总药×100%, the drug loading of liposome A was determined to be 2.84±0.02% and the encapsulation efficiency was 90.43±1.44%.

[0178] Experimental Example 3: In vitro release of liposomes

[0179] The in vitro drug release behavior of liposome A prepared in Experimental Example 1 was investigated using the dialysis bag method in a phosphate buffer solution containing 2% sodium dodecyl sulfate (SDS). As shown in the release curves in Figure 1, free drug compound 1 was released relatively quickly, almost completely released after 4 hours; while liposome A was released more slowly, with approximately 10% released after 2 hours, approximately 50% cumulatively released after 24 hours, and approximately 80% cumulatively released after 48 hours. This indicates that liposome A achieved a prolonged release effect.

[0180] Experimental Example 4: In vitro antitumor efficacy of liposomes

[0181] The inhibitory effects of liposomes A, B, and free drugs on the activity of breast cancer cell line 4T1, lung cancer cell line A549, colon cancer cell line CT26, and breast cancer cell line MDA-MB-231 were investigated using the Cell Counting Kit-8 (CCK8) reagent. Different cell lines were divided into groups of 5 × 10⁻⁶ cells. 3 Cells were seeded at a concentration of [number] cells / well in 96-well plates and cultured for 12 hours. Once cells were fully adherent, the culture medium was discarded. 100 μl of liposome solution and free drug solution of different concentrations were added to each well. After drug administration, the cells were incubated at 37°C for another 24 hours. The culture medium was then discarded, and 100 μl of CCK8 solution was added to each well. The cells were incubated at 37°C for another 4 hours. The absorbance A was measured at 450 nm using a microplate reader and recorded as A. test Meanwhile, cells treated with a drug-free culture medium served as a control group, denoted as A. control The blank solvent was used as a blank control group, denoted as A. blank The formula for calculating cell viability is: Cell viability (%) = (A test -A blank ) / (A control -A blank *100%. The half-maximal inhibitory concentration (IC50) for each drug administration group was calculated using Graphpad Prism. 50 (μg·ml -1 The results of the antitumor activity study of liposomes are shown in Table 12. Additionally, the free compound 1 showed an effect on the IC50 of 4T1 cells. 50 It was 4.18 ± 0.28 μg·ml - 1 Free compound SN38 for IC50 in 4T1 cells 50 It was 5.123 ± 0.23 μg·ml-1 .

[0182] Table 12 Results of cytotoxicity experiments with free drugs and liposomes

[0183] The above data indicate that both liposomes A and B exhibit antitumor activity against multiple tumor cell lines, with liposome A showing stronger antitumor activity. Furthermore, for both compound 1 and SN38, liposome formulation significantly enhanced the antitumor activity.

[0184] Experimental Example 5: In vivo antitumor efficacy of liposomes

[0185] Forty healthy female Balb / c mice were divided into two groups, and ectopic subcutaneous colon cancer CT26 model and orthotopic breast cancer 4T1 model were established respectively according to the following methods.

[0186] Establishment of a mouse subcutaneous colon cancer (CT26) model: CT26 colon cancer cells from mice in the logarithmic growth phase were collected, washed with pre-cooled PBS, and diluted to 1×10⁻⁶. 6 Cells / ml were mixed thoroughly and stored on ice for use in mouse tumor inoculation. Healthy Balb / c mice were randomly selected and 100 μL of cell suspension was inoculated into the right back of the mouse.

[0187] Establishment of a mouse orthotopic breast cancer (4T1) model: Logarithmic growth phase mouse breast cancer cells 4T1 were collected, washed with pre-cooled PBS, and diluted to 1×10⁻⁶. 6 Cells / ml were mixed thoroughly and stored on ice for use in mouse tumor inoculation. Healthy Balb / c mice were randomly selected, and 100 μl of 4T1 cell suspension was inoculated under the right thoracic fat pad of the second pair of nipples.

[0188] After tumor growth, mice were randomly divided into three groups according to tumor size: liposome A group, compound 1 group, and saline group. The mice were administered the drug at a dose of 2 mg / kg every two days for a total of five administrations. From the first administration, tumor volume (V = 0.5 × length × width) was measured every other day. 2 The mouse tumors were monitored and their weight was recorded. After the experiment, the tumors were dissected, photographed, and weighed. The tumor growth inhibition rate (TGI) was calculated using the following formula: TGI% = (W saline -W 给药组 ) / W saline ×100%, W saline W represents the average tumor weight in the saline group. 给药组 The tumor weight is for the treatment group.

[0189] After the experiment, the average tumor weight and average tumor inhibition rate of each group are shown in Tables 13-1 and 13-2 below. In the ectopic subcutaneous colon cancer CT26 model, the weight of mice in each treatment group showed the same trend as that of mice in the saline group; in the orthotopic breast cancer 4T1 model, the weight of mice in compound 1 group decreased by 14% during the administration period and returned to normal after the administration period, while the weight fluctuation trend of mice in the other treatment groups was consistent with that of the saline group.

[0190] Table 13-1 Tumor volume and TGI in the CT26 colon cancer model after the experiment.

[0191] Table 13-2 Tumor volume and TGI at the end of the experiment in the 4T1 breast cancer model

[0192] The above results indicate that liposome A has a superior tumor-suppressing effect compared to compound 1, and its safety is significantly higher than that of compound 1.

[0193] The embodiments described above are merely exemplary depictions of several implementations of this disclosure, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of protection of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this disclosure, and these all fall within the scope of protection of this disclosure. The scope of protection of this disclosure should be determined by the appended claims.

Claims

A pharmaceutical composition comprising a pharmaceutically active ingredient and one or more ingredients selected from phospholipids, cholesterol, and polyethylene glycol derivatives; said pharmaceutically active ingredient being a DNA topoisomerase inhibitor; Preferably, the DNA topoisomerase inhibitor is selected from compounds of formula (I) or pharmaceutically acceptable salts, stereoisomers, solvates, isotopically labeled compounds, polymorphs, metabolites, or prodrugs thereof: in, R1 is selected from C 1-6 Alkyl or -(C 1-6 (alkylene)-NR 11 R 12 The alkyl or alkylene group may be optionally replaced by halogen, OH, CN, NO2, -NH2, or -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-4 Alkyl, C 1-4 Alkoxy, C 1-4 Hydroxyalkyl, C 1-4 Halogenated alkyl groups and C 1-4 One or more substituents in the haloalkoxy group are substituted; R2 is selected from H, halogens, OH, CN, NO2, -NH2, -NH(C) 1-6 alkyl), -N(C) 1-6 Alkyl)2, C 1-4 Alkyl, or C 1-4 Alkoxy; R 11 R 12 Each is independently selected from H and C. 1-6 Alkyl or R 21 Substituted acyl or sulfonyl group, R 21 Selected from C 1-6 Alkyl, Halogenated C 1-6 Alkyl, 6-10 aryl and 5-12 heteroaryl. According to claim 1, the pharmaceutical composition wherein the DNA topoisomerase inhibitor structure is selected from the following compounds or their pharmaceutically acceptable salts, stereoisomers, solvates, isotopically labeled compounds, polymorphs, metabolites, or prodrugs: The pharmaceutical composition according to claim 1 or 2 has one or more of the following characteristics: (1) The phospholipid is selected from one, two or more of the following: egg yolk lecithin, soybean lecithin or animal-derived phospholipids, hydrogenated egg yolk lecithin, hydrogenated soybean lecithin, dipalmitoylphosphatidylcholine, myristoylphosphatidylcholine, distearate phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin; preferably egg yolk lecithin (e.g., egg yolk lecithin E80); (2) The polyethylene glycol derivative is a phospholipid-polyethylene glycol complex; preferably, the phospholipid-polyethylene glycol complex is selected from one or more of phosphatidylcholine-polyethylene glycol (PC-PEG), phosphatidylethanolamine-polyethylene glycol (PE-PEG), stearoylphosphatidylcholine-polyethylene glycol (DSPC-PEG), and stearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG); preferably stearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and more preferably DSPE-PEG2000; (3) The weight ratio of the active pharmaceutical ingredient to the total amount of the phospholipid, cholesterol and polyethylene glycol derivative is 0.1:100 to 10:100; more preferably 1:100 to 10:100; more preferably 1:100 to 1:25; for example, 1:100, 2:100, 3:100 or 4:100; (4) The weight ratio of phospholipids to cholesterol is 94.9:0.1 to 1:2; more preferably 82:13 to 1:1; more preferably 76:19 to 48:47; more preferably 50:45 to 70:25; for example, 53:42, 54:41, 55:40, 56:39, 57:38, or 58:37; (5) The weight of the polyethylene glycol derivative accounts for 0.1% to 10% of the total weight of the phospholipid, cholesterol and polyethylene glycol derivative, preferably 0.5% to 8%, more preferably 1% to 5%, for example 1%, 2%, 3%, 4% or 5%; (6) The pharmaceutical composition is a liposome. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition contains 1 to 10 parts by weight of a pharmaceutically active ingredient, 20 to 80 parts by weight of phospholipids, 20 to 60 parts by weight of cholesterol, and 1 to 10 parts by weight of a polyethylene glycol derivative; wherein the pharmaceutically active ingredient is a DNA topoisomerase inhibitor. Preferably, the pharmaceutical composition contains 1 to 5 parts by weight of the compound of formula (I), 45 to 65 parts by weight of egg yolk lecithin, 30 to 50 parts by weight of cholesterol, and 3 to 7 parts by weight of a polyethylene glycol derivative. The method for preparing the pharmaceutical composition according to any one of claims 1 to 4 comprises the following steps: a) Weigh the active pharmaceutical ingredient, phospholipids, cholesterol, and polyethylene glycol derivatives and dissolve them in an organic solvent; b) Remove organic solvents; c) Add water to hydrate the membrane; d) Homogenize. The preparation method according to claim 5 has one or more of the following characteristics: (1) The organic solvent in step a) is selected from one or more of methanol, ethanol, isopropanol, acetone, butanone, ethyl acetate, butyl acetate, chloroform, dichloromethane, tetrahydrofuran, dimethyl sulfoxide, cyclohexane, and n-hexane, preferably dichloromethane; (2) Step b) removes the organic solvent by rotary evaporation or thin-film evaporation; (3) Step c) involves hydrating the thin film using ultrasound; (4) The hydration temperature in step c) is preferably 15-50℃, preferably 25-45℃, for example 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, or 40℃. (5) In step d), homogenization is performed by ultrasonic method, high pressure homogenization method or dynamic high pressure micro-jet method, preferably by ultrasonic method, and even more preferably by probe ultrasonic method. (6) Step d) Homogenization is performed by probe ultrasound for 1 to 60 minutes; more preferably, the probe ultrasound time is 3 to 15 minutes, for example 8, 9, 10, 11, or 12 minutes. (7) The ultrasonic power of the probe in step d) is 100-500w, more preferably 180-300w, for example 230w, 240w, 250w, 260w or 270w. A liposome prepared by the method of claim 5 or 6. A pharmaceutical formulation comprising the pharmaceutical composition of any one of claims 1 to 4 or the liposome of claim 7, and a pharmaceutically acceptable carrier and / or excipient. An article comprising a pharmaceutical composition according to any one of claims 1 to 4, a liposome according to claim 7, or a pharmaceutical preparation according to claim 8, and a container for containing the pharmaceutical composition, liposome, or pharmaceutical preparation. Use of the pharmaceutical composition according to any one of claims 1 to 4, the liposome according to claim 7, the pharmaceutical formulation according to claim 8, or the article according to claim 9 in the preparation of a medicament, wherein the medicament is used for: (a) Prevention and / or treatment of cancer or tumors; (b) As an adjunct therapy for cancer or tumors; (c) Diagnosing cancer or tumor; Any combination of (d)(a)-(c); Preferably, the cancer or tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer or squamous cell carcinoma of the lung, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, bile duct cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, glioma, melanoma, leukemia, and lymphoma. The pharmaceutical composition according to any one of claims 1 to 4, the liposome according to claim 7, the pharmaceutical formulation according to claim 8, or the article according to claim 9, is used for: (a) Prevention and / or treatment of cancer or tumors; (b) As an adjunct therapy for cancer or tumors; (c) Diagnosing cancer or tumor; Any combination of (d)(a)-(c); Preferably, the cancer or tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer or squamous cell carcinoma of the lung, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, bile duct cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, glioma, melanoma, leukemia, and lymphoma. A method for the prevention, treatment, adjuvant therapy and / or diagnosis of tumors, the method comprising administering to an individual in need a therapeutically effective amount of the pharmaceutical composition of any one of claims 1 to 4, the liposome of claim 7, the pharmaceutical formulation of claim 8 or the article of claim 9; Preferably, the cancer or tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer or squamous cell carcinoma of the lung, liver cancer, stomach cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, bile duct cancer, nasopharyngeal carcinoma, head and neck squamous cell carcinoma, glioma, melanoma, leukemia, and lymphoma.

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