Liposomal compositions of compounds having sting agonistic activity and methods of making and uses thereof

Abstract

The present application relates to liposome compositions of compounds having STING agonistic activity and methods of making and using the same. In particular, the present application relates to liposomes of a compound of Formula I comprising a liposomal membrane and an internal aqueous phase encapsulated inside the liposomal membrane, wherein the internal aqueous phase comprises a complex of a metal ion and an ionized compound of Formula I. The liposomes can improve tissue targeting of the compound of Formula I, enhance anti-tumor efficacy, prolong its blood circulation time, enhance patient compliance and / or reduce the risk of clinical administration,

Description

TECHNICAL FIELD

[0001] The present application relates to the field of preparations, in particular to liposome compositions of compounds (especially Compound A) having STING agonistic activity and methods of preparation and uses thereof. BACKGROUND

[0002] Interferon gene stimulator (STING) is a protein encoded by TMEM173 gene, containing 379 amino acids. STING is targeted on endoplasmic reticulum through its N-terminal region, and the C-terminal contains an action / activation region, which can be activated by 2'3'-cGAMP in cytoplasm or cytoplasmic DNA sensor, and is one of the key factors for mediating innate immune signaling. STING is highly expressed in immune cells. Once activated, STING receptor will start a cascade of innate immune response through multiple pathways, including stimulating immune cells to secrete various cytokines, and then inducing adaptive immune response. Studies have found that the main result of STING activation is the activation of TBK1 / IRF3 and the downstream transcription of type I interferon genes, and type I interferon is a key conductor for initiating innate and adaptive T cell responses. Type I interferon can stimulate the maturation and differentiation of antigen-presenting cells and enhance the activation of T cells. After the activation of systemic tumor antigen-specific T cells, the anti-tumor effect can be exerted. Studies have shown that after the activation of STING signaling pathway by STING agonists, a large number of immune CD8 T cells infiltrate the tumor site and release cytokines, and a large number of tumor cells die, causing tumor shrinkage or even complete regression, and the anti-tumor effect is obvious and continuous.

[0003] As a new generation of tumor immunotherapy, STING agonists can be nucleic acids, polypeptides, proteins, small molecule compounds, and the most studied STING agonists include cyclic dinucleotides and their derivatives, as well as small molecules optimized by high-throughput screening. Currently, Aduro Biotech's ADU-S100 / MIW815 (the earliest developed cyclic dinucleotide human STING agonist) and Merck's MK-1454 have entered the clinical stage. In addition, the compound of formula I is a new type of STING agonist (PCT / CN2020 / 085525), which has high affinity with human STING protein, can effectively activate STING protein, activate related cellular immune activity, and play a therapeutic role:

[0004]

[0005]

[0006] Although preclinical results show that STING agonists directly injected into tumors (including a variety of refractory, metastatic solid tumors) cause tumor disappearance, and the growth and metastasis of tumors in other parts of the body are also significantly inhibited, its clinical application is still limited. This is mainly due to the limited indications, high difficulty and safety risk of intratumoral injection administration route, so the optimization of the administration route for such compounds is still a hot and difficult research topic today, and the development of new drug delivery systems is of great significance to improve patient compliance, reduce clinical risk, expand the scope of indications and improve therapeutic effect. SUMMARY

[0007] To solve the above problems, the present application prepares a liposome composition of the compound of formula I, aiming to prolong the in vivo residence time, improve tissue targeting, enhance anti-tumor efficacy, increase patient compliance, and reduce the risk of clinical drug use.

[0008] In one aspect, the present application provides a liposome comprising a liposome membrane and an internal aqueous phase encapsulated inside the liposome membrane, wherein the internal aqueous phase contains a complex of a metal ion and an ionized compound of formula I;

[0009] The compound of formula I has the following structure:

[0010]

[0011] wherein L 3 is selected from a covalent bond and -(C(R 9 )2) q -;

[0012] Each R 9 is independently selected from H, halogen, cyano, -OR a , -SR a , -NR a R b , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, each of which C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl is optionally substituted with one or more substituents independently selected from halogen, C 1-6 alkyl, C 1-6 haloalkyl, cyano, hydroxyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, C 1-6 alkoxy and -OR a ; or two R 9 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 3-10 membered heterocyclyl; or any one R 9 and R 5 together with the atoms between them form a 3-10 membered heterocyclyl ring;

[0013] R 2 and R 3 are the same or different and each is independently selected from the group consisting of H, halogen, cyano, -OR a , -SR a , -S(O)R a , -S(O)2R a , -NR a R b , -C(O)-NR a R b , -NR a -C(O)-R a , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, each of which C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, C 1-6 alkyl, C 1-6 haloalkyl, cyano, hydroxy and C 1-6 alkoxy;

[0014] R 5 is selected from the group consisting of H, C 1-6 alkyl, C 3-10 cycloalkyl and 3-10 membered heterocyclyl, each of which C 1-6 alkyl, C 3-10 cycloalkyl and 3-10 membered heterocyclyl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, cyano, hydroxy, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, -NRa R b , -CO2R a , and -S(O)2R a ;

[0015] R 6 is selected from H, C 1-6 1-6alkyl, C 3-10 3-10cycloalkyl, 3-10 membered heterocyclyl, C 6-10 6 aryl, and 5-10 membered heteroaryl, said C a 1-6alkyl, C 7 3-10cycloalkyl, 3-10 membered heterocyclyl, C 1-6 6 aryl, and 5-10 membered heteroaryl is optionally substituted with one or more R 3-10 ;

[0016] each occurrence of R 6-10 is selected from halogen, cyano, hydroxy, -NR c R c , -C(O)2-R a , C b 1-6alkyl, C a 1-6alkoxy, and C 1-6 haloalkoxy, wherein said C 1-6 1-6alkyl, C 1-6 1-6alkoxy, and C 1-6 haloalkoxy is optionally substituted with one or more substituents independently selected from cyano, -OR 1-6 , -NR 1-6 R a , -C(O)2-R a , C b 1-6alkyl, C a 1-6alkoxy, and -SO2R 1-6 ;

[0017] R a is selected from H, C 7 1-6alkyl, C 1-6 3-10cycloalkyl, and 3-10 membered heterocyclyl, said C 3-10 1-6alkyl, C 1-6 3-10cycloalkyl, and 3-10 membered heterocyclyl is optionally substituted with one or more substituents independently selected from halogen, cyano, hydroxy, 3-10 membered heterocyclyl, -NR 3-10 R a , -C(O)2-R b , C a 1-6alkyl, C 1-6 1-6alkoxy, and -SO2R a ;

[0018] each occurrence of R a and Rb each independently selected from H, C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 alkoxy, said C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 alkoxy are each optionally substituted with one or more substituents independently selected from the group consisting of hydroxy, halogen and C 1-6 alkyl;

[0019] or R a , R b together with the nitrogen atom to which they are attached form a 3-7 membered heterocyclyl;

[0020] X 2 is selected from C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 1-6 alkylene-X 4 and C 1-6 alkylene-X 4 -C 1-6 alkylene, said C 1-6 alkylene, C 2-6 alkenylene, C 2-6 alkynylene, C 3-6 cycloalkyl and 3-6 membered heterocyclyl are each optionally substituted with one or more substituents independently selected from the group consisting of halogen, cyano, hydroxy, C 1-6 alkyl and C 1-6 alkoxy;

[0021] X 4 is selected from -O-, -S-, -NR a -, -C(O)-, -C(O)-NR a -, -S(O)-, -S(O)2-, -S(O)2-NR a -, -O-C(O)-NR a -, -NR a -C(O)-NR a - and -NR a -S(O)2-NR a -;

[0022] m and n are each independently selected from 0, 1, 2 and 3; and

[0023] q is selected from 1, 2 and 3.

[0024] In another aspect, the present application provides a composition comprising said liposome, wherein said liposome is exposed to an external aqueous phase.

[0025] In another aspect, the present application provides a pharmaceutical composition comprising the liposome or the liposome composition, optionally, further comprising a pharmaceutically acceptable carrier in the pharmaceutical composition.

[0026] In another aspect, the present application provides a method for preparing the liposome composition, comprising the following steps:

[0027] (1) preparing blank liposome: dissolving phospholipid and cholesterol in organic solvent to obtain oil phase solution, and forming primary emulsion with inner aqueous phase buffer by injection method, thin film hydration method, reverse evaporation method or pipeline emulsification method, granulating to obtain blank liposome, wherein the inner aqueous phase buffer is a buffer with pH of 3-8 containing metal ions as described above;

[0028] (2) constructing ion gradient: removing metal ions in the outer aqueous phase of the blank liposome with outer aqueous phase buffer as displacement medium, so that the inner aqueous phase and the outer aqueous phase have a metal ion gradient (ΔM≥1) of not less than 1 unit and a H + gradient (ΔpH=0-4) of 0-4 units, to obtain a liposome intermediate; preferably, the inner aqueous phase and the outer aqueous phase of the liposome intermediate have a metal ion gradient (ΔM≥2) of not less than 2 units and a H + gradient (ΔpH=0.5-3) of 0.5-3 units;

[0029] (3) drug loading: dissolving the compound of formula I in the same buffer as the outer aqueous phase buffer to obtain a drug solution, and adding it to the liposome intermediate, incubating at 40-70°C for 2-60 min, and cooling to obtain the liposome composition;

[0030] Optionally, the long-circulating material described herein is added to the oil phase solution of step (1) or the drug solution of step (3).

[0031] In another aspect, the present application provides the liposome, the liposome composition or the pharmaceutical composition for activating the STING signaling pathway.

[0032] In another aspect, the present application provides the liposome, the liposome composition or the pharmaceutical composition for preventing or treating STING-mediated related diseases.

[0033] In another aspect, the present application provides the use of the liposome, the liposome composition or the pharmaceutical composition in the preparation of a medicament for preventing or treating STING-mediated related diseases.

[0034] In another aspect, the present application provides a method of preventing or treating a STING-mediated related disease, comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of the liposome, the liposome composition or the pharmaceutical composition of the present application.

[0035] In an embodiment of the present application, the STING-mediated related disease is a tumor, preferably, the STING-mediated related disease is a cancer (e.g. colorectal cancer). DETAILED DESCRIPTION

[0037] Definitions

[0038] In the present application, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art, unless otherwise indicated. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0039] As used herein, the term "liposome" refers to a self-assembled structure comprising one or more bilayers of amphipathic lipids, each bilayer comprising two monolayers of amphipathic lipids oriented in opposite directions. An amphipathic lipid comprises a polar (hydrophilic) head group covalently linked to one or two or more nonpolar (hydrophobic) acyl or alkyl chains. The very unfavorable contact between the hydrophobic acyl chains and the surrounding aqueous medium causes the amphipathic lipid molecules themselves to arrange so that the polar head groups are oriented toward the surface of the bilayer and the acyl chains are oriented toward the interior of the bilayer, effectively preventing the acyl chains from contacting the aqueous environment.

[0040] The liposomes used herein can have a single bilayer of lipids surrounding or encapsulating an aqueous compartment (unilamellar liposomes) or multiple bilayers of lipids (multilamellar liposomes). Various forms of liposomes are described, for example, in Cullis et al. Biochim. Biophys Acta, 559:399-420 (1987).

[0041] As used herein, the term "metal ion gradient (AM)" is calculated according to the following formula: where Min is the concentration of metal ions in the internal aqueous phase and Mout is the concentration of free metal ions in the external aqueous phase.

[0042] As used herein, the term "H + Gradient (ApH) refers to the difference between the pH of the external aqueous phase and the pH of the internal aqueous phase.

[0043] As used herein, the term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0044] As used herein, the term "C 1-6"alkyl" means a straight or branched chain alkyl group containing from 1 to 6 carbon atoms, including C 1-5 "alkyl", C 1-4 "alkyl", C 1-3 "alkyl" and C 1-2 "alkyl", etc. Typical examples are methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, 2-methylbutyl, neopentyl, 1-ethylpropyl, n-hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, 1,2-dimethylpropyl, and the like.

[0045] As used herein, the term "C 1-6 "alkylene" means C 1-6 "alkylene" means C 1-5 "alkylene", C 1-4 "alkylene", C 1-3 "alkylene" and C 1-2 "alkylene", etc. Typical examples are methylene, ethylene, 1,3-propylene, 1,4-butylene, and the like.

[0046] As used herein, the term "C 2-6 "alkenyl" means a straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond and from 2 to 6 carbon atoms, including C 2-4 "alkenyl", etc. Typical examples are ethenyl, propenyl, 2-propenyl, butenyl, 2-butenyl, butadienyl, pentenyl, 2-methyl-but-enyl, 3-methyl-but-enyl, 1,3-pentadienyl, 1,4-pentadienyl, hexenyl, 2-ethyl-but-enyl, 3-methyl-pentenyl, 4-methyl-pentenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, and the like.

[0047] As used herein, the term "C 2-6 "alkenylene" means C 2-6 "alkenylene" means C 2-4 "alkenylene", etc. Typical examples are ethenylene, 1,3-propenylene, and the like.

[0048] As used herein, the term "C 2-6 "alkynyl" means a straight or branched chain hydrocarbon group containing at least one carbon-carbon triple bond and from 2 to 6 carbon atoms, including C 2-4 "alkynyl", etc. Typical examples are ethynyl, propynyl, 2-propynyl, butynyl, and the like.

[0049] As used herein, the term "C 2-6 Alkynyl refers to a straight or branched chain hydrocarbon group having from 2 to 20 carbon atoms, and more preferably 2 to 10 carbon atoms, and most preferably 2 to 6 carbon atoms, which contains at least one carbon- carbon triple bond. Typical examples include ethynyl, 1-propynyl, 2-propynyl, and the like. 2-6 Alkynyl refers to a straight or branched chain hydrocarbon group having from 2 to 20 carbon atoms, and more preferably 2 to 10 carbon atoms, and most preferably 2 to 6 carbon atoms, which contains at least one carbon- carbon triple bond. Typical examples include ethynyl, 1-propynyl, 2-propynyl, and the like. 2-4 Alkynyl refers to a straight or branched chain hydrocarbon group having from 2 to 20 carbon atoms, and more preferably 2 to 10 carbon atoms, and most preferably 2 to 6 carbon atoms, which contains at least one carbon- carbon triple bond. Typical examples include ethynyl, 1-propynyl, 2-propynyl, and the like.

[0050] As used herein, the term "C 1-6 Haloalkyl refers to a straight or branched chain alkyl group as previously described further substituted by at least one halogen, including C 1-6 Haloalkyl refers to a straight or branched chain alkyl group as previously described further substituted by at least one halogen, including C 1-4 Haloalkyl, C 1-3 Haloalkyl, C 1-2 Haloalkyl. Typical examples include trifluoromethyl, chloromethyl, and the like.

[0051] As used herein, the term "C 1-6 Alkoxy refers to a radical formed by the removal of a hydrogen atom from an -OH group, with the remainder of the molecule, including C 1-6 Alkoxy, C 1-6 Alkoxy, C 1-4 Alkoxy, C 1-3 Alkoxy, C 1-2 Alkoxy. Typical examples include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy, and the like.

[0052] As used herein, the term "C 1-6 Haloalkoxy refers to a straight or branched chain alkoxy group as previously described further substituted by at least one halogen, including C 1-6 Haloalkoxy, C 1-4 Haloalkoxy, C 1-3 Haloalkoxy, C 1-2 Haloalkoxy. Typical examples include trifluoromethoxy, and the like.

[0053] As used herein, the term "C 3-10 Cycloalkyl refers to a monocyclic or bicyclic saturated or partially unsaturated non-aromatic hydrocarbon ring having from 3 to 10 carbon atoms. Typical examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[0054] As used herein, the term "C 3-10 Cycloalkyl refers to a monocyclic or bicyclic saturated or partially unsaturated non-aromatic hydrocarbon ring having from 3 to 10 carbon atoms. Typical examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. 3-10 Cycloalkyl refers to a monocyclic or bicyclic saturated or partially unsaturated non-aromatic hydrocarbon ring having from 3 to 10 carbon atoms. Typical examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. 3-8 Cycloalkyl, C 3-6 Cycloalkyl, C 5-6Cycloalkylene. Typical examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, cycloheptylene, cyclooctylene, and the like.

[0055] As used herein, the term "C6- 10 Aryl" means an aromatic group containing 6 to 10 ring-forming carbon atoms, such as phenyl or naphthyl.

[0056] As used herein, the term "5-10 membered heteroaryl" means an aromatic group containing 5-10 ring members, and at least 1 (e.g., 1, 2, 3, or 4) of the ring members is a heteroatom selected from N, O, and S, including 5-6 membered heteroaryl. The heteroaryl group can be a monocyclic heteroaryl, a bicyclic heteroaryl, or a polycyclic heteroaryl. Typical examples are furyl, thienyl, pyrrolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, isoxazolyl, imidazolyl, 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, pyrimidinyl, pyridazinyl, pyrazinyl, 1,2,3-triazinyl, and the like.

[0057] As used herein, the term "3.10 membered heterocyclyl" means a saturated or partially saturated 3-10 membered cyclic hydrocarbon group containing at least one (e.g., containing 1, 2, or 3) heteroatom selected from N, O, and S, including 3-8 membered heterocyclyl, 3-7 membered heterocyclyl, 3-6 membered heterocyclyl, and 5-6 membered heterocyclyl. Typical examples are oxiranyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, imidazolinyl, dihydropyrazolyl, pyrazolidinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, oxazinyl, and the like.

[0058] As used herein, the term "3-10 membered heterocyclyl" means a saturated or partially saturated 3-10 membered cyclic hydrocarbon group containing at least one (e.g., containing 1, 2, or 3) heteroatom selected from N, O, and S, including 3-8 membered heterocyclyl, 3-7 membered heterocyclyl, 3-6 membered heterocyclyl, and 5-6 membered heterocyclyl. Typical examples are oxiranyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, imidazolinyl, dihydropyrazolyl, pyrazolidinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, oxazinyl, and the like.

[0059] As used herein, the term "one or more" means 1 or more (e.g., 2, 3, 4, 5, or 10) under reasonable conditions.

[0060] When a group is described as being "optionally substituted" with one or more substituents, then the group can be (1) unsubstituted or (2) substituted. If a carbon on a group is described as being optionally substituted with one or more substituents selected from a group, then one or more hydrogens on the carbon (to the extent there are any hydrogens present) can be replaced, individually and / or together, with an independently selected optional substituent. If a nitrogen on a group is described as being optionally substituted with one or more substituents, then one or more hydrogens on the nitrogen (to the extent there are any hydrogens present) can each be replaced with an independently selected optional substituent.

[0061] The term "effective amount" as used herein refers to an amount that is sufficient to achieve the desired therapeutic effect, e.g., an amount that achieves a reduction in the symptoms associated with the disease being treated.

[0062] The term "treatment" as used herein is intended to mean the reduction or elimination of the disease state or condition being addressed. A subject is successfully "treated" if the subject exhibits an observable and / or detectable reduction in or improvement in one or more indicators and symptoms, following administration of a therapeutic amount of the crystalline form or pharmaceutical composition thereof according to the methods described herein. It should also be understood that the treatment of a disease state or condition includes a partial but significant treatment, as well as complete treatment.

[0063] Liposomes

[0064] In a first aspect, the present application provides a liposome comprising a liposomal membrane and an internal aqueous phase encapsulated inside the liposomal membrane, wherein the internal aqueous phase comprises a complex of a metal ion and an ionized compound of Formula I,

[0065] The compound of Formula I has the following structure:

[0066]

[0067] wherein L 3 is selected from the group consisting of a covalent bond and.(C(R 9 )2) q -;

[0068] each R 9 is independently selected from the group consisting of H, halogen, cyano, -OR a , -SR a , -NR a R b , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5.10 membered heteroaryl, said C1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, each optionally substituted with one or more substituents independently selected from halo, C 1-6 alkyl, C 1-6 haloalkyl, cyano, hydroxy, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, C 1-6 alkoxy and -OR a ; or two R 9 together with the carbon atom to which they are attached form a C 3-10 cycloalkyl or 3-10 membered heterocyclyl; or any one R 9 and R 5 together with the atoms between them form a 3-10 membered heterocyclyl ring;

[0069] R 2 and R 3 are the same or different and each is independently selected from H, halo, cyano, -OR a , -SR a , -S(O)R a , -S(O)2R a , -NR a R b , -C(O)-NR a R b , -NR a -C(O)-R a , C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, said C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 3-6 cycloalkyl, 3-6 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl, each optionally substituted with one or more substituents independently selected from halo, C 1-6 alkyl, C 1-6 haloalkyl, cyano, hydroxy and C 1-6 alkoxy;

[0070] R 5 is selected from H, C 1-6 alkyl, C 3-10cycloalkyl and 3-10 membered heterocyclyl, said C 1-6 alkyl, C 3-10 cycloalkyl and 3-10 membered heterocyclyl are each optionally substituted with one or more substituents independently selected from the group consisting of halo, cyano, hydroxy, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 1-6 haloalkyl, C 1-6 alkoxy, C 1-6 haloalkoxy, -NR a R b , -CO2R a , and -S(O)2R a ;

[0071] R 6 is selected from the group consisting of H, C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl, 5-10 membered heteroaryl, -OR a , and -C(O)2R 7 , said C 1-6 alkyl, C 3-10 cycloalkyl, 3-10 membered heterocyclyl, C 6-10 aryl and 5-10 membered heteroaryl are optionally substituted with one or more R c ;

[0072] R c is each independently selected from the group consisting of halo, cyano, hydroxy, -NR a R b , -C(O)2-R a , C 1-6 alkyl, C 1-6 alkoxy and C 1-6 haloalkoxy, wherein said C 1-6 alkyl, C 1-6 alkoxy and C 1-6 haloalkoxy are each optionally substituted with one or more substituents independently selected from the group consisting of cyano, -OR a , -NR a R b , -C(O)2-R a , C 1-6 alkoxy and -SO2R a ;

[0073] R 7 is selected from the group consisting of H, C 1-6 alkyl, C 3-10 cycloalkyl and 3-10 membered heterocyclyl, said C 1-6 alkyl, C 3-10The cycloalkyl group and the 3-10 membered heterocyclic group are each optionally substituted by one or more substituents independently selected from the following: halogen, cyano, hydroxyl, 3-10 membered heterocyclic group, -NR. a R b -C(O)2-R a C 1-6 Alkoxy and -SO2R a ;

[0074] When it occurs, R a and R b Each is independently selected from H and C. 1-6 Alkyl, C 3-6 cycloalkyl and C 1-6 Alkoxy, the C 1-6 Alkyl, C 3-6 cycloalkyl and C 1-6 Each alkoxy group is optionally selected by one or more independently chosen from hydroxyl, halogen, and C. 1-6 Alkyl substituents;

[0075] Or R a R b Together with the nitrogen atom attached to it, they form a 3-7 membered heterocyclic group;

[0076] X 2 Selected from C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-6 cycloalkyl, 3-6 membered heterocyclic, C 1-6 Alkylene-X 4 and C 1-6 Alkylene-X 4 -C 1-6 Alkylene, the C 1-6 Alkylene, C 2-6 imidene group, C 2-6 Ethyne group, C 3-6 The cycloalkyl group and the 3-6 membered heterocyclic group are each optionally substituted by one or more substituents independently selected from the following: halogen, cyano, hydroxyl, C 1-6 Alkyl and C 1-6 Alkoxy;

[0077] X 4 Selected from -O-, -S-, -NR a -、-C(O)-、-C(O)-NR a -, -S(O)-, -S(O)2-, -S(O)2-NR a -、-OC(O)-NR a -、-NR a -C(O)-NR a- and -NR a - S(O)2-NR a -;

[0078] m and n are each independently selected from 0, 1, 2 and 3; and

[0079] q is selected from 1, 2 and 3.

[0080] In some embodiments, the compound of formula I is Compound A as shown below:

[0081]

[0082] The liposomes of the present application have an average particle size of 30-300 nm, preferably 30-200 nm, for example 50-150 nm, and more preferably 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or 150 nm.

[0083] In embodiments of the present application, when the compound of formula I contains an acidic group such as a carboxylic acid group, the following ionization equilibrium exists in the presence of an internal aqueous phase environment, (for ease of illustration, the compound of formula I containing a carboxylic acid group is represented by IC00H), wherein the ionized compound of formula I (ICOO ) can bind to a metal ion having strong coordination to form a water-insoluble or poorly soluble complex, such as a precipitate or a colloidal precipitate. In some embodiments, the ionized compound of formula I binds to the metal ion via an anionic group (e.g., -COO - or -SO3 - ).

[0084] In the present application, a polyvalent metal ion (e.g., divalent or trivalent) is preferred, such as Ca 2+ , Zn 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Fe 3+ or Al 3+ . In some embodiments, the concentration of the metal ion in the internal aqueous phase is 10-500 mM, more preferably 20-300 mM, for example, 20 mM, 50 mM, 80 mM, 100 mM, 120 mM, 150 mM, 200 mM, 250 mM or 300 mM.

[0085] In some embodiments, the molecules constituting the liposome membrane include phospholipids and cholesterol. The phospholipids can be selected from the group consisting of lecithin, cephalin, soybean phospholipid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin, and cardiolipin, etc. Studies have found that the stability of phospholipids can be further increased by converting unsaturated bonds into saturated bonds through hydrogenation. Therefore, in some embodiments, the phospholipids in the present application also include hydrogenated phospholipids, such as hydrogenated soybean phospholipid (HSPC), distearoylphosphatidylcholine (DSPC), and hydrogenated sphingomyelin (HSM).

[0086] Studies have found that the addition of a small amount of negatively charged phospholipids in the liposome membrane material can form a negative charge layer on the surface of the liposome membrane, which can not only reduce the transmembrane leakage of positive ions or protons, but also maintain the ion gradient for a long time, and prevent the aggregation between liposomes. Therefore, in some embodiments, the phospholipids constituting the liposome membrane can include negatively charged phospholipids, such as phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), or phosphatidylserine (PS), etc.

[0087] The cholesterol, as a hydrophobic molecule, can be combined with the phospholipids, embedded in the membrane, prevent the phospholipids from condensing into a crystal structure, and adjust the membrane structure fluidity. The cholesterol as a component of the liposome membrane can play a role in stabilizing the liposome.

[0088] Long-circulating liposomes

[0089] The liposome membrane can be modified by polyethylene glycol (PEG) or its derivatives to enhance the hydrophilicity of the liposome membrane, reduce the interaction between plasma proteins and the liposome membrane, prevent the aggregation and fusion of the liposomes, avoid the uptake of the liposomes by the reticuloendothelial system cells, and prolong the in vivo circulation time of the liposomes.

[0090] Thus, in some embodiments, the liposomes of the present application can be further modified with a functional long-circulating material to form long-circulating liposomes. The functional long-circulating material is selected from polyethylene glycol (PEG) and its derivatives selected from one or more of polyethylene glycol-vitamin E succinate (PEG-TPGS), polyethylene glycol-cholesterol (PEG-Chol), polyethylene glycol modified distearoyl phosphatidyl ethanolamine (MPEG-DSPE), polyethylene glycol modified dimyristoyl phosphatidyl ethanolamine (MPEG-DMPE), polyethylene glycol modified dipalmitoyl phosphatidyl ethanolamine (MPEG-DPPE). In some embodiments, the molecular weight of the polyethylene glycol is from 500 to 10,000 daltons, for example, from 1,000 to 7,000 daltons, preferably from 2,000 to 5,000 daltons. In some preferred embodiments, the liposomes are further modified with MPEG2000-DSPE.

[0091] In some embodiments, the weight ratio of the compound of Formula I, the phospholipid, the cholesterol and the long-circulating material in the long-circulating liposomes is 1:(1-300):(0.2-150):(0.01-150), preferably 1:(2-200):(1-100):(0.05-100).

[0092] Liposome compositions

[0093] In another aspect, the present application provides a liposome composition comprising the liposomes of the present application, and the liposomes are exposed to an external aqueous phase.

[0094] In some embodiments, the pH of the external aqueous phase is from 6 to 8, preferably from 6.0 to 7.5. In some embodiments, the external aqueous phase contains a buffer salt, for example, sucrose. histidine solution, Tris-HCl buffer. In addition, chelating agents such as EDTA are beneficial to obtain the ion concentration difference between the internal and external aqueous phases of the liposomes, to ensure a higher pH gradient, and can reduce the adsorption of ions on the surface of the liposome membrane, thus in some embodiments, the liposome composition of the present application further contains a chelating agent, for example, EDTA, in the external aqueous phase. A catalytic amount of chelating agent can achieve good results.

[0095] Methods of preparation

[0096] In the preparation of liposome drugs, according to the way and mechanism of drug loading, there are mainly passive loading and remote loading. Passive loading refers to dissolving the drug in the water phase or organic phase, and then preparing drug-containing liposomes by appropriate methods. This method is suitable for drugs with strong water solubility or fat solubility. For amphiphilic drugs, such as some weak acids and weak bases, the oil-water partition coefficient is greatly affected by the pH and ionic strength of the medium. The encapsulation efficiency of liposomes prepared by passive loading is low. Remote loading is achieved by the principle that the neutral form of weak acid or weak base can cross the phospholipid bilayer, while the ionized form cannot. By forming an ion or compound gradient across the membrane between the inside and outside of the liposome, a pH gradient or ion gradient is finally established between the inside and outside of the liposome, so that the drug in the water phase outside the liposome spontaneously aggregates inside the liposome to achieve drug loading.

[0097] In one aspect, the present application provides a method for preparing the liposome composition by active loading, which mainly includes the following three steps: (1) preparing blank liposomes; (2) constructing an ion gradient; and (3) loading drugs.

[0098] Preparation of blank liposomes:

[0099] Blank liposomes can be prepared by, for example, injection method, thin film hydration method, reverse evaporation method or pipeline emulsification method.

[0100] The injection method is divided into ether injection method and ethanol injection method. In some technical solutions, blank liposomes are prepared by ethanol injection method, and the specific steps include dissolving the liposome membrane material in an appropriate amount of ethanol, slowly injecting it into the buffer through a fine needle or pipe, and forming liposomes.

[0101] The operation steps of the thin film hydration method include: dissolving the membrane material in an organic solvent such as chloroform, then rotating evaporation in a glass bottle to form a thin film on the inner wall of the bottle, and then adding buffer to the bottle while stirring to obtain liposomes.

[0102] The operation steps of the reverse evaporation method include: dissolving the membrane material in an organic solvent, adding an aqueous solution of the drug to be encapsulated, and ultrasonic oscillation for a short time until a stable W / O emulsion is formed, then evaporating the organic solvent under reduced pressure, adding buffer dropwise after the gel is detached from the wall of the vessel, and then evaporating under reduced pressure to prepare a liposome aqueous suspension.

[0103] The operation steps of the pipeline emulsification method include: dissolving the membrane material in the same or different organic solvents, mixing it with the continuously flowing water phase in the pipeline under high-speed shearing, and then mixing the material through shearing, extrusion, etc. to form liposomes by instantaneous and uniform fine dispersion of the organic phase and the water phase.

[0104] The prepared nascent liposomes can be subjected to size adjustment by a shearing machine, a homogenizer or an extrusion device to obtain liposomes with desired particle size. In some embodiments, the average particle size of the liposomes is in the range of 30-300 nm, preferably 30-200 nm, for example 50-150 nm, and for example 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or 150 nm.

[0105] In some embodiments, the method for preparing the blank liposomes comprises the following steps:

[0106] The phospholipid and cholesterol are dissolved in an organic solvent to obtain an oil phase solution, and the nascent liposomes are prepared by injection method, thin film hydration method, reverse evaporation method or pipeline emulsification method with an internal aqueous phase buffer solution, size adjustment, to obtain blank liposomes, wherein the internal aqueous phase buffer solution is a buffer solution with pH of 3-8 containing metal ions as described above;

[0107] Optionally, the long-circulating material as described above can also be added to the oil phase solution to prepare long-circulating liposomes.

[0108] In some embodiments, the internal aqueous phase buffer solution is an acetate buffer solution, for example calcium acetate-acetic acid buffer solution.

[0109] Constructing ion gradient:

[0110] During the hydration to prepare the blank liposomes, the buffer solution used is the internal aqueous phase buffer solution, and in order to construct the transmembrane gradient, the external aqueous phase needs to be replaced after the blank liposomes are prepared. The method for replacing the external aqueous phase is known in the art, for example, the external aqueous phase can be replaced or most of the free state metal ions in the external aqueous phase are removed by means of complexation, dialysis, ultrafiltration or centrifugation.

[0111] In some embodiments, the ion gradient is constructed by the following method:

[0112] The metal ions in the external aqueous phase of the blank liposomes are removed with the replacement medium of the external aqueous phase buffer solution, so that the internal aqueous phase and the external aqueous phase have a metal ion gradient of not less than 1 unit (ΔM≥1), to obtain a liposome intermediate. In some embodiments, the internal aqueous phase and the external aqueous phase of the liposome intermediate have a metal ion gradient of not less than 2 units (ΔM≥2).

[0113] Optionally, the pH gradient is constructed at the same time as the ion gradient.

[0114] In some embodiments, the ion gradient and the pH gradient are constructed by the following method:

[0115] The metal ions in the external aqueous phase of the blank liposome are removed by using the external aqueous phase buffer as the displacement medium, so that the internal and external aqueous phases have a metal ion gradient (ΔM≥I) of not less than 1 unit and a H + gradient (ΔpH=0-4) to obtain a liposome intermediate.

[0116] In some embodiments, the internal and external aqueous phases of the liposome intermediate have a metal ion gradient (ΔM≥2) of not less than 2 units and a H + gradient (ΔpH=0.5-3).

[0117] In some embodiments, the external aqueous phase buffer is a sucrose-histidine solution or a Tris-HCl buffer.

[0118] Drug loading:

[0119] The compound of formula I has a dissociation equilibrium in the aqueous phase, In the external aqueous phase, the undissociated compound I can diffuse across the liposome membrane to the internal aqueous phase under a concentration gradient; in the internal aqueous phase, the dissociated drug molecules increase, bind with metal ions to form complexes (such as precipitates or complexes). Due to the concentration difference of the undissociated compound I on both sides of the membrane, it continuously enters the internal aqueous phase from the external aqueous phase and forms a complex with metal ions, and finally achieves the purpose of stable encapsulation of the drug. In this process, organic acids such as acetate can combine with hydrogen ions to form organic acids such as acetic acid, which overflow from the inside of the liposome to the outside of the membrane, which is the driving force for the continuous diffusion of compound I from the outside of the liposome to the inside of the liposome to form a precipitate or a complex.

[0120] In some embodiments, drug loading can be carried out by the following method:

[0121] The compound of formula I is dissolved in the same buffer as the external aqueous phase buffer to obtain a drug solution, and is added to the liposome intermediate, incubated at 40-70°C for 2-60 min, and cooled to obtain the liposome composition; preferably, incubated at 40-70°C for 5-50 min; optionally, the long-circulating material described above can also be added to the drug solution to prepare long-circulating liposomes.

[0122] The phospholipids that make up the membrane of the liposome are temperature sensitive. As the temperature increases, the acyl side chains of the phospholipid molecules change from an ordered arrangement to a disordered arrangement, causing a change in the properties of the membrane: from a gel crystalline state to a liquid crystalline state, an increase in the membrane cross section, a decrease in the bilayer thickness, an increase in the membrane fluidity. The temperature at which this transition occurs is called the phase transition temperature. Each phospholipid has its own unique phase transition temperature. If the phase transition temperature of the phospholipid membrane is low, the energy barrier to be overcome for the transport across the membrane is low, and the loading of the drug can be completed at room temperature. Conversely, the loading process must be completed at a higher temperature. When the incubation temperature is greater than the phase transition temperature of the liposome, the permeability of the phospholipid membrane increases, which can facilitate the loading of the drug, but the increase in permeability also causes the destruction of the transmembrane gradient during loading. The incubation temperature suitable for use in the present application is between 40 and 70 °C, for example 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C or 70 °C.

[0123] The quality of the liposomes can be evaluated by appearance, particle size distribution, encapsulation efficiency and stability of the liposomes, among others. The encapsulation efficiency is an important indicator of the intrinsic quality of the liposomes. It refers to the ratio of the encapsulated drug to the total amount of drug in the formulation. Methods for determining the encapsulation efficiency are known in the art. It can generally be determined by microcolumn centrifugation, ultracentrifugation, dialysis, gel column chromatography, ultrafiltration, protamine precipitation, etc. In some embodiments, the compound of Formula I or other therapeutic agent, if any, is present in the aqueous phase of the liposome or attached to the liposome. In some embodiments, the encapsulation efficiency of the liposome is > 70%, preferably > 80%, more preferably > 90%, more preferably > 95%.

[0124] Pharmaceutical compositions and methods of treatment

[0125] In another aspect, the liposomes of the present application can be provided as a pharmaceutical composition containing the liposomes or liposome composition of the present application, and a pharmaceutically acceptable carrier.

[0126] The pharmaceutically acceptable carrier can be, for example, physiological saline, isotonic glucose, isotonic sucrose, Ringer's solution and Hanks' solution. Buffering substances can be added to obtain a storage-stable preferred pH, for example, a pH between 6.0 and 8.0. In some embodiments, a pH of 6.0 to 7.5 is preferred, at which the liposome membrane has better stability, as well as stability of the encapsulation of the drug.

[0127] The pharmaceutical composition can also contain a stabilizer and / or an antioxidant. In some embodiments, the stabilizer is selected from one or more of ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium salt, and ethylenediaminetetraacetic acid calcium salt. In some embodiments, the stabilizer is added in an amount of 0-0.5% / v%. In some embodiments, the antioxidant is a water-soluble or oil-soluble antioxidant. In some embodiments, the water-soluble antioxidant is selected from one or more of ascorbic acid, sodium bisulfite, sodium sulfite, sodium metabisulfite, L-cysteine. In some embodiments, the oil-soluble antioxidant is selected from one or more of alpha-tocopherol, alpha-tocopherol succinate, alpha-tocopherol acetate. In some embodiments, the antioxidant is added in an amount of 0-0.5% / v%.

[0128] The dosage of the pharmaceutical composition will depend on the amount of drug encapsulated in the liposome, the disease state to be treated, and the dosage form and judgment of the practitioner. Generally, the pharmaceutical composition is administered in a dosage sufficient to deliver to the subject a therapeutically effective amount of the drug.

[0129] The amount of the pharmaceutical composition necessary to deliver a therapeutically effective amount of the dosage can be determined by routine in vivo and in vitro methods in the art of drug testing. See, e.g., Handbook of Anticancer Drug’s Development, D. B. Budman, A. H. Calvert, E. K. Rowinsky (eds.), LWW, 2003. Typically, the dosage of the pharmaceutical composition of the present application is in the range of about 0.001 mg to about 500 mg of drug per kilogram of body weight, usually in the range of about 0.01 mg to about 100 mg of drug per kilogram of body weight.

[0130] Typically, the pharmaceutical composition of the present application can be prepared as a topical or injectable solution, either as a liquid solution or suspension. Solid forms suitable for dissolution or suspension in liquid vehicles prior to injection can also be prepared. Enteric coated tablets or gel capsules can also be formulated according to methods known in the art.

[0131] The liposomes of the present application can be administered in any pharmaceutically acceptable manner depending on the disease to be treated. Possible routes of administration include injection, parenteral routes, for example intramuscular, subcutaneous, intravenous, intra-arterial, intraperitoneal, intra-articular, intrameningeal, intrathecal, or other routes, for example oral, nasal, ocular, rectal, vaginal or pulmonary, for example by inhalation. For delivery of liposome drugs formulated according to the present application to tumors of the central nervous system, it is particularly advantageous to slowly and continuously infuse the liposomes directly intracranially into the tumor (convection enhanced delivery or CED). See Saito, et al., Cancer Research, Vol. 64, p. 2572-2679, 2004; Mamot, et al., J. Neuro-Oncology, Vol. 68, p. 1-9, 2004. The liposomes of the present application can also be applied directly to tissue surfaces. Sustained release administration, pH dependent release administration or other specific chemical or environmental condition mediated release administration are also expressly included in the present application, for example by means such as depot injection or bioerodible implant.

[0132] In another aspect, the present application provides the liposomes, liposome compositions or pharmaceutical compositions for use in activating the STING signaling pathway.

[0133] In another aspect, the present application provides the liposomes, liposome compositions or pharmaceutical compositions for use in preventing or treating STING-mediated related diseases.

[0134] In another aspect, the present application provides the use of the liposomes, liposome compositions or pharmaceutical compositions of the present application in the manufacture of a medicament for preventing or treating STING-mediated related diseases.

[0135] Another object of the present application is to provide a method for preventing or treating STING-mediated related diseases, comprising administering to an individual in need thereof a prophylactically or therapeutically effective amount of the liposomes, liposome compositions or pharmaceutical compositions of the present application.

[0136] According to some embodiments of the present application, the STING-mediated related diseases are tumors, preferably, the diseases are cancers (e.g. colorectal cancer).

[0137] Advantages of the Invention

[0138] The present application provides liposomes of the compound of Formula I, and compositions and pharmaceutical compositions thereof. The liposomes of the present application can significantly prolong the blood half-life of the encapsulated compound when used in the treatment of tumors, and achieve better anti-tumor effects. Attached Figure Description

[0139] Figure 1 The exposure levels of the liposome composition and compound solution in various tissues are shown in the graph.

[0140] Figure 2 The graph shows the changes in tumor volume in a mouse CT-26 syngeneic xenograft model caused by the liposome composition and compound solution. Detailed Implementation

[0141] The following embodiments are provided to further illustrate the present invention, but the invention is not limited thereto. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but all such modifications or improvements are within the scope of the invention as long as they do not depart from its basic idea.

[0142] Unless otherwise specified, the experiments and methods described in the examples were generally performed according to conventional methods well known in the art and described in various references. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0143] Compound examples and test examples

[0144] The structure of the compound was determined by nuclear magnetic resonance (NMR). 1 It was determined by 1H NMR or mass spectrometry (MS). 1 The 1H NMR was performed using a JEOL Eclipse 400 NMR spectrometer. The solvents used were deuterated methanol (CD3OD), deuterated chloroform (CDCl3), or hexadeuterated dimethyl sulfoxide (DMSO-d6). The internal standard was tetramethylsilane (TMS). Chemical shifts (δ) were given in parts per million (ppm).

[0145] The instrument used for MS measurements was an Agilent (ESI) mass spectrometer, manufacturer: Agilent, model: Agilent 6120B.

[0146] Preparation method of high performance liquid chromatograph:

[0147] Instrument model: Agilent 1260; Column: Waters SunFire Prep C18 OBD (19mm×150mm×5.0μm); Column temperature: 25℃; Flow rate: 20.0mL / min; Detection wavelength: 214nm; Elution gradient: (0min: 10%A, 90%B; 16.0min: 90%A, 10%B); Mobile phase A: Acetonitrile; Mobile phase B: 0.05% formic acid aqueous solution.

[0148] Thin layer chromatography silica gel plates (TLC) were used Merck aluminum plates (20 x 20 cm) and thin layer chromatography separation and purification was performed using Yantai GF 254 (1 mm).

[0149] The reaction was monitored by thin layer chromatography (TLC) or LC-MS; the developing solvent systems used included dichloromethane and methanol, n-hexane and ethyl acetate, and petroleum ether and ethyl acetate, and the volume ratio of the solvents was adjusted according to the polarity of the compound or adjusted by adding triethylamine, etc.

[0150] Column chromatography generally used 200-300 mesh silica gel as the carrier. The eluent system included dichloromethane and methanol, and petroleum ether and ethyl acetate, and the volume ratio of the solvents was adjusted according to the polarity of the compound, or a small amount of triethylamine was added for adjustment.

[0151] Unless otherwise specified in the examples, the reaction temperature was room temperature (20-35°C);

[0152] The reagents used in the present application were purchased from Acros Organics, Aldrich Chemical Company, and TCI Chemicals, etc.

[0153] In the conventional synthesis method and the examples, and the intermediate synthesis examples, the meanings of each abbreviation are as shown below.

[0154]

[0155]

[0156] Preparation Example 1: Preparation of 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4- oxobutanoic acid ethyl ester (Int-A) and 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4- oxobutanoic acid ethyl ester (Int-B)

[0157]

[0158] First Step: Preparation of 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid methyl ester

[0159] To a solution of 6-fluoro-veratraldehyde (10.0 g, 54.3 mmol) in N,N- dimethylformamide (200 mL) was added methyl mercaptoacetate (6.9 g, 65.2 mmol) and potassium carbonate (22.5 g, 162.9 mmol) and heated to 60 °C for 15 h. The reaction was slowly poured into water (1000 mL) and stirred for 2 h, filtered and the solid washed with water (500 mL) and dried under vacuum at 60 °C to give the title compound of this step (12.0 g, yield: 87.6 %). MS m / z (ESI): 253.0 [M+H] + .

[0160] Second step: Preparation of 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid

[0161] To a solution of methyl 5,6-dimethoxybenzo[b]thiophene-2-carboxylate (12.0 g, 47.6 mmol) in methanol (100 mL) and water (20 mL) was added sodium hydroxide (3.8 g, 95.1 mmol) and stirred at room temperature for 4 h. The reaction was concentrated under reduced pressure at 40 °C to remove some of the methanol and the remaining material was added to water (500 mL) and the pH adjusted to 3 with dilute hydrochloric acid. The solid was filtered and washed with water (500 mL) and dried under vacuum at 60 °C to give the title compound (8.0 g, yield: 70.6 %). MS m / z (ESI): 239.0 [M+H] + .

[0162] Third step: Preparation of N,5,6-trimethoxy-N-methylbenzo[b]thiophene-2- carboxamide

[0163] To a solution of 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (8.2 g, 34.4 mmol) and hydroxylamine hydrochloride (4.1 g, 41.3 mmol) in dichloromethane (100 mL) was added HATU (13.1 g, 34.4 mmol) followed by DIPEA (8.9 g, 68.8 mmol) and stirred at room temperature for 4 h. The reaction was poured into water (300 mL) and extracted with dichloromethane (50 mL) three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate concentrated under reduced pressure to give the title compound of this step (9.5 g, yield: 98.1 %). MS m / z (ESI): 282.1 [M+H] + .

[0164] Fourth step: Preparation of 1-(5,6-dimethoxybenzo[b]thiophen-2-yl)ethanone

[0165] N,5,6-trimethoxy-N-methylbenzo[b]thiophene-2-carboxamide (10.0 g, 35.6 mmol) was dissolved in tetrahydrofuran (200 mL) and methylmagnesium bromide (106.6 mmol) in tetrahydrofuran (35.6 mL) was added slowly at 0 °C and the reaction was allowed to warm to room temperature slowly over 4 h. The reaction was poured into saturated aqueous ammonium chloride solution (600 mL) and extracted with ethyl acetate (100 mL) three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound of this step (7.9 g, yield: 94.1 %). MS m / z (ESI): 237.1 [M+H] + .

[0166] Fifth Step: Preparation of 2-bromo-l-(5,6-dimethoxybenzo[b]thiophen-2-yl)ethanone

[0167] l-(5,6-dimethoxybenzo[b]thiophen-2-yl)ethanone (2.0 g, 8.5 mmol) and copper bromide (5.7 g, 25.4 mmol) were added to ethyl acetate (60 mL) and the reaction was allowed to warm to 80 °C for 8 h. The reaction was filtered and the filtrate was concentrated under reduced pressure. To the residue was added sodium sulfite (1.8 g, 14.2 mmol), acetonitrile (15 mL), water (15 mL) and acetic acid (8 mL) and stirred at room temperature for 2 h. The reaction was poured into water (150 mL) and extracted with ethyl acetate (30 mL) three times. The organic phases were combined, washed with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound of this step (1.5 g, yield: 67.0 %). MS m / z (ESI): 315.0 [M+H] + .

[0168] Sixth Step: Preparation of 2-(2-(5,6-dimethoxybenzo[b]thiophen-2-yl)-2-oxoethyl)propanedioic acid di-tert-butyl ester

[0169] Di-tert-butyl propanedioate (23.8 g, 110.4 mmol) was dissolved in tetrahydrofuran (200 mL) and sodium hydride (60 %) (4.2 g, 110.4 mmol) was added slowly at 0 °C and stirred at 0 °C for 1 h. To the reaction was added 2-bromo-l-(5,6-dimethoxybenzo[b]thiophen-2-yl)ethanone (17.4 g, 55.2 mmol) in tetrahydrofuran (20 mL) slowly and the reaction was allowed to warm to room temperature slowly over 2 h. The reaction was poured into saturated aqueous ammonium chloride solution (500 mL) and extracted with ethyl acetate (100 mL) three times. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure. The concentrate was purified by preparative high performance liquid chromatography to give the title compound of this step (22.5 g, yield: 90.5 %).

[0170] Step 7: Preparation of 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4- oxobutanoic acid

[0171] Di-tert-butyl 2-(2-(5,6-dimethoxybenzo[b]thiophen-2-yl)-2-oxoethyl)malonate (8.0 g, 17.8 mmol) was dissolved in hydrogen chloride solution in 1,4-dioxane (80 mL, 4 mol / L) and stirred at 120 °C for 18 hours. The reaction was poured into water (200 mL) and extracted with ethyl acetate (50 mL) three times. The organic phase was combined and washed with saturated aqueous sodium chloride solution three times, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound of this step (4.7 g, yield: 89.9%). MS m / z (ESI): 295.1 [M+H] + .

[0172] Step 8: Preparation of ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4- oxobutanoate

[0173] Ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (4.0 g, 13.6 mmol) was dissolved in ethanol (80 mL) and concentrated sulfuric acid (1 mL) was added with stirring at room temperature. The temperature was raised to 80 °C and the reaction was carried out for 3 hours. The reaction was poured into water (200 mL) and extracted with ethyl acetate (50 mL) three times. The organic phase was combined and washed with saturated aqueous sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound of this step (3.9 g, yield: 89.0%). MS m / z (ESI): 323.1 [M+H] + .

[0174] Step 9: Preparation of ethyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4- oxobutanoate (Int-A) and ethyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4- oxobutanoate (Int-B)

[0175] Ethyl 4-(5,6-dimethoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (0.73 g, 2.3 mmol) was dissolved in dichloromethane (8 mL), and aluminum trichloride (4.2 g, 22.7 mmol) was slowly added while being cooled in an ice bath, and then it was allowed to rise to room temperature and stirred for 24 hours. The reaction solution was poured into water (100 mL) and adjusted to pH = 2 with dilute hydrochloric acid, and extracted with ethyl acetate (30 mL) three times, and the combined organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure, and the concentrate was purified with a preparative high-performance liquid chromatograph to obtain ethyl 4-(6-hydroxy-5-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Int-A: 493 mg, yield: 71.4%) and ethyl 4-(5-hydroxy-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Int-B: 170 mg, yield: 24.3%).

[0176] Int-A:

[0177] MS m / z (ESI): 309.1 [M+H] + ;

[0178] 1 H-NMR (400 MHz, CDC13) δ: 7.87 (s, 1H), 7.33 (s, 1H), 7.22 (s, 1H), 6.08 (s, 1H), 4.17 (q, J = 8.0 Hz, 2H), 3.97 (s, 3H), 3.32-3.29 (m, 2H), 2.80-2.76 (m, 2H), 1.27 (t, J = 8.0 Hz, 3H).

[0179] Int-B:

[0180] MS m / z (ESI): 309.1 [M+H] + ;

[0181] 1 H-NMR (400 MHz, CDC13) δ: 7.84 (s, 1H), 7.32 (s, 1H), 7.22 (s, 1H), 5.85 (s, 1H), 4.17 (q, J = 8.0 Hz, 2H), 3.98 (s, 3H), 3.33-3.30 (m, 2H), 2.79-2.76 (m, 2H), 1.27 (t, J = 8.0 Hz, 3H).

[0182] Intermediate Preparation Example 2: Preparation of ethyl 4-(5-(3-hydroxypropoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Int D)

[0183]

[0184] Ethyl 4-(5-hydroxy-6-methoxybenzo[b]thiophene-2-yl)-4-oxobutanoate (Int-B, 250.0 mg, 0.81 mmol) was dissolved in acetonitrile (20 mL), potassium carbonate (223.8 mg, 1.62 mmol), potassium iodide (27.0 mg, 0.16 mmol) and 3-bromo-1-propanol (169.0 mg, 1.22 mmol) were added and heated to 60 °C for 12 h. The reaction was concentrated and poured into water (50 mL) and extracted with ethyl acetate (25 mL) three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure to give the title compound of this step (Int D, 200.0 mg, yield: 64.8%). MS m / z (ESI): 367.1 [M+H] + .

[0185] Intermediate Preparation Example 3: Preparation of ethyl 3-(N-methyl-6-hydroxy-5-methoxybenzo[b]thiophene-2-carboxamido)propanoate (Int-E) and ethyl 3-(N-methyl-5-hydroxy-6-methoxybenzo[b]thiophene-2-carboxamido)propanoate (Int-F)

[0186]

[0187] First Step: Preparation of ethyl 3-(N-methyl-5,6-dimethoxybenzo[b]thiophene-2-carboxamido)propanoate

[0188] Ethyl 3-(methylamino)propanoate (389 mg, 2.32 mmol) and 5,6-dimethoxybenzo[b]thiophene-2-carboxylic acid (500 mg, 2.10 mmol) were dissolved in tetrahydrofuran (15 mL), HATU (1.6 g, 4.2 mmol) and DIPEA (814 mg, 6.3 mmol) were added and heated to 60 °C for 4 h. The reaction was poured into water (100 mL) and extracted with ethyl acetate (30 mL) three times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, the concentrate was purified by preparative high performance liquid chromatography to give the title compound (492 mg, yield: 66.8%). MS m / z (ESI): 352.1 [M+H]+. 1 H-NMR (400 MHz, CDC13) δ: 7.47 (s, 1H), 7.25 (s, 1H), 7.21 (s, 1H), 4.16 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.88-3.85 (m, 2H), 3.28 (s, 3H), 2.74-2.71 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H).

[0189] Step 2: Preparation of 3-(N-methyl-6-hydroxy-5-methoxybenzo[b]thiophene-2- formamido)propionic acid ethyl ester (Int-E) and 3-(N-methyl-5-hydroxy-6- methoxybenzo[b]thiophene-2-formamido)propionic acid ethyl ester (Int-F)

[0190] Ethyl 3-(N-methyl-5,6-dimethoxybenzo[b]thiophene-2-formamido)propionate (520 mg, 1.48 mmol) was dissolved in dichloromethane (25 mL), and aluminum chloride (1.97 g, 14.80 mmol) was slowly added under ice bath, and then the temperature was raised to room temperature, and stirred for 24 hours. The reaction solution was poured into water (100 mL), and adjusted to pH = 2 with dilute hydrochloric acid, extracted with ethyl acetate (30 mL) three times, the organic phase was combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain ethyl 3-(N-methyl-6-hydroxy-5-methoxybenzo[b]thiophene-2-formamido)propionate (Int-E, 305 mg, yield: 61.2%) and ethyl 3-(N-methyl-5-hydroxy-6-methoxybenzo[b]thiophene-2-formamido)propionate (Int-F, 174 mg, yield: 34.9%).

[0191] Int-E:

[0192] MS m / z (ESI): 338.1 [M+H] + ;

[0193] 1 H-NMR (400 MHz, DMSO-d6) δ: 9.55 (s, 1H), 7.62 (s, 1H), 7.38 (s, 1H), 7.27 (s, 1H), 4.07 (q, J = 8.0 Hz, 2H), 3.85-3.83 (m, 3H), 3.73-3.65 (m, 2H), 3.21-3.18 (m, 3H), 2.68-2.65 (m, 2H), 1.17 (t, J = 8.0 Hz, 3H).

[0194] Int-F:

[0195] MS m / z (ESI): 338.1 [M+H] + ;

[0196] 1H-NMR (400 MHz, DMSO-d6) δ: 9.25 (s, 1H), 7.58 (s, 1H), 7.50 (s, 1H), 7.23 (s, 1H), 4.07 (q, J = 8.0 Hz, 2H), 3.85-3.82 (m, 3H), 3.73-3.68 (m, 2H), 3.21-3.18 (m, 3H), 2.68-2.65 (m, 2H), 1.17 (t, J = 8.0 Hz, 3H).

[0197] Preparation of intermediate: Preparation of ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Compound 5)

[0198]

[0199] Ethyl 4-(5-(3-hydroxypropoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Int-D, 110 mg, 300.2 μmol), 3-(N-methyl-6-hydroxy-5-methoxybenzo[b]thiophene-2-carboxamido)propanoic acid ethyl ester (Int-E, 101.2 mg, 300.2 μmol) and triphenylphosphine (157.5 mg, 600.4 μmol) were dissolved in tetrahydrofuran (10 mL), and after adding diisopropyl azodicarboxylate (121.4 mg, 600.4 μmol) under nitrogen protection, the reaction was stirred at room temperature for 12 hours. The reaction was concentrated under reduced pressure at 40 °C to remove tetrahydrofuran, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound (102.3 mg, yield: 49.7%).

[0200] MS m / z (ESI): 686.2 [M+H] + .

[0201] 1 H-NMR (400 MHz, DMSO-d6) δ: 8.21-8.15 (m, 1H), 7.66-7.65 (m, 1H), 7.62-7.60 (m, 2H), 7.56-7.48 (m, 1H), 7.42 (s, 1H), 4.27-4.20 (m, 4H), 4.09-4.03 (m, 4H), 3.86-3.82 (m, 6H), 3.77-3.74 (m, 2H), 3.30-3.26 (m, 2H), 3.33 (s, 3H), 2.68-2.65 (m, 4H), 2.30-2.27 (m, 2H), 1.24-1.10 (m, 6H).

[0202] Preparation of Compound: Preparation of 4-(5-(3-((2-((2-carboxyethyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoic acid (Compound A)

[0203]

[0204] Ethyl 4-(5-(3-((2-((3-ethoxy-3-oxopropyl)(methyl)carbamoyl)-5-methoxybenzo[b]thiophen-6-yl)oxy)propoxy)-6-methoxybenzo[b]thiophen-2-yl)-4-oxobutanoate (Compound 5, 50.0 mg, 72.9 μmol) was dissolved in ethanol (6 mL) and water (2 mL), after adding sodium hydroxide (8 mg, 200.0 μmol), stirring at room temperature for 12 hours. The reaction solution was poured into water (50 mL), adjusted to pH = 2 with dilute hydrochloric acid, extracted with ethyl acetate (15 mL) three times, the organic phase was combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated under reduced pressure, and the concentrate was purified by preparative high performance liquid chromatography to obtain the title compound (8 mg, yield: 16.6%).

[0205] MS m / z (ESI): 630.1 [M+H] + .

[0206] 1 H-NMR (400 MHz, DMSO-d6) δ: 12.28 (s, 2H), 8.20-8.14 (m, 1H), 7.66-7.60 (m, 3H), 7.56-7.48 (m, 1H), 7.42 (s, 1H), 4.27-4.17 (m, 4H), 3.92-3.80 (m, 6H), 3.71 (s, 2H), 3.27-3.24 (m, 2H), 3.18 (s, 3H), 2.62-2.59 (m, 4H), 2.30-2.27 (m, 2H).

[0207] Example 1 of Compound Pharmacodynamic Test In vivo pharmacodynamics of the compound in a MC38 mouse colon cancer syngeneic transplantation tumor model

[0208] In this experimental example, after intratumoral injection (i.t.) of Compound A, the tumor volume and tumor weight changes of the MC38 mouse colon cancer transplantation tumor model mice were measured and recorded, so as to test the pharmacodynamics of each test product.

[0209] (1) Experimental cell strains and experimental animals

[0210] Experimental cell strain: Mouse colon cancer MC38 cells (Nanjing Kebai) were cultured at 37°C in a 5% CO2 incubator (culture medium: RPMI-1640 (Hyclone) containing 10% fetal bovine serum (Gibco)). Routine digestion was performed with trypsin-EDTA (Hyclone) for passage. When the cells were in the exponential growth phase, with a saturation degree of 80%-90%, the cells were collected and counted.

[0211] Experimental animals: C57BL / 6J mice, 6-8 weeks old, female, weighing 18-20 grams, all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and raised in a specific pathogen-free (SPF) level animal room.

[0212] (2) Tumor cell inoculation and grouping

[0213] MC38 cells were resuspended in phosphate buffered saline (PBS) at a density of 5 x 10 6 cells / mL. 0.1 mL of PBS containing 5 x 10 5 MC38 cells was subcutaneously inoculated into the right and left dorsal scapular regions of each mouse, and when the average volume of the left and right tumors reached 100 mm 3 , the mice were randomly grouped according to the tumor volume.

[0214] (3) Experimental method

[0215] When the average volume of the subcutaneously transplanted tumors reached 100 mm 3 , the mice were randomly grouped according to the tumor volume, with 8 mice per group, and intratumoral injection of the drug was started on the right tumors, with no drug given to the left tumors. The drug was given at a frequency of BIW x 2, for a total of 2 weeks, and the specific drug administration scheme is shown in Table 1. After drug administration, the left and right tumor volumes were measured twice per week, and the animal mortality was observed daily.

[0216] 4. Experimental indicators and statistical analysis

[0217] The antitumor efficacy of the test product was evaluated using the tumor volume inhibition rate TGI 体积 (%). The tumor volume was measured using a vernier caliper. The calculation formula for the tumor volume was V = 0.5 x a x b 2 , where a and b represent the long diameter and short diameter of the tumor, respectively. The tumor volume inhibition rate TGI 体积 (%) = [(1-(the average tumor volume at the end of drug administration in the drug administration group - the average tumor volume at the start of drug administration in the drug administration group) / (the average tumor volume at the end of drug administration in the vehicle control group - the average tumor volume at the start of drug administration in the vehicle control group)] x 100%; when the tumor volume at the end of drug administration is less than the tumor volume at the start of drug administration (i.e., tumor regression), TGI 体积Tumor volume growth inhibition rate TGI (%) = [(1 - (average tumor volume at the end of administration of a certain administration group - average tumor volume at the beginning of administration of the administration group) / (average tumor volume at the end of administration of the vehicle control group - average tumor volume at the beginning of administration of the vehicle control group))] x 100%; when tumor regression occurs (i.e. tumor volume at the end of administration is less than tumor volume at the beginning of administration), TGI (%) = [1 - (average tumor volume at the end of administration of a certain administration group - average tumor volume at the beginning of administration of the administration group) / average tumor volume at the beginning of administration of the administration group] x 100%.

[0218] Statistical analysis was performed using Graphpad Prism software based on tumor volume at the end of the experiment. Comparison between two groups was analyzed using Student's t-test. P < 0.05 was considered to be significantly different.

[0219] Table 1 Administration schedule of test substances

[0220]

[0221] 5. Test results

[0222] After 2 weeks of administration, the body weight of animals in each administration group increased, and no animal died. The test results of tumor volume and tumor inhibition effect of mice are shown in Table 2.

[0223] The experimental results showed that intratumoral injection of Compound A at a unit dose of 10 μg (i.t., BIW x 2) had a significant tumor inhibition effect on the administration side tumor of the model, and the tumor volume TGI on the 18th day after administration was 106.24% (P = 0.0007), and also had a tumor inhibition effect on the non-administration side tumor, and the tumor volume TGI on the non-administration side was 51.09% (P = 0.055).

[0224] Table 2 Tumor inhibition effect (tumor volume) of Compound A administration for different days on MC38 syngeneic tumor model

[0225]

[0226] Note: a. Tumor volume (mm 3 ) = mean ± standard error (SEM), n = 8.

[0227] b. Tumor volume growth inhibition rate TGI 体积 (%) = [(1 - (average tumor volume at the end of administration of a certain administration group - average tumor volume at the beginning of administration of the administration group) / (average tumor volume at the end of administration of the vehicle control group - average tumor volume at the beginning of administration of the vehicle control group))] x 100%; when tumor regression occurs (i.e. tumor volume at the end of administration is less than tumor volume at the beginning of administration), TGI (%) = [1 - (average tumor volume at the end of administration of a certain administration group - average tumor volume at the beginning of administration of the administration group) / average tumor volume at the beginning of administration of the administration group] x 100%. 体积 (%) = [1 - (average tumor volume at the end of administration of a certain administration group - average tumor volume at the beginning of administration of the administration group) / average tumor volume at the beginning of administration of the administration group] x 100%.

[0228] Liposome examples and test examples

[0229] In the following examples, the particle size of the liposomes is determined by dynamic light scattering method, unless otherwise specified. The specific steps of the dynamic light scattering method are as follows: an appropriate amount of liposome sample is diluted with water, and then the particle size is determined by using a Malvern particle size analyzer (Malvern Nano-ZS90). The particle size is expressed in intensity.

[0230] In the following examples, the encapsulation efficiency of the liposomes is determined by gel column method, unless otherwise specified. The specific steps of the gel column method are as follows: a PD-10 Desalting column is preloaded with one column, and 25 mL of deionized water is used for rinsing and equilibration. 0.2 mL of sample is loaded onto the column, and after 1-2 min, it is eluted with 0.9% sodium chloride solution. The first 6 mL of eluate is collected in a 25 mL volumetric flask, diluted with solvent to constant volume, and used as the drug sample. The subsequent 24 mL of eluate is collected in a 25 mL volumetric flask, diluted with solvent to constant volume, and used as the free drug sample. Another 0.2 mL of sample is taken in a 25 mL volumetric flask, diluted with solvent to constant volume, and used as the non-column sample. The ultraviolet-visible spectrophotometry is used for determination, and the encapsulation efficiency is calculated according to the following formula: encapsulation efficiency = W / (W+Wf) x 100%. Wherein, W is the amount of encapsulated drug, and Wf is the amount of free drug.

[0231] Example 1 of liposomes

[0232] (1) Preparation of blank liposomes

[0233] Hydrogenated soybean lecithin 15 g and cholesterol 5 g are dissolved in 100 ml of anhydrous ethanol, incubated to 55°C, and an oil phase solution is obtained. The oil phase solution is injected into 800 ml of an aqueous phase solution containing zinc ions that has been incubated to 55°C (the composition of the aqueous phase is: 120 mM ZnCl2 solution, and acetic acid is added to adjust the pH to 4.5), and after the injection is completed, the stirring is continued for 30 min to obtain a liposome initial milk. The blank liposomes are obtained by high-pressure microfluidization to about 100 nm.

[0234] (2) Construction of Zn 2+ Gradient and pH gradient: after the whole particle is completed, the blank liposomes obtained in step (1) are subjected to dialysis treatment using tangential flow ultrafiltration device with sucrose-histidine-EDTA solution (pH 6.0) as the replacement medium, until the Zn 2+ concentration of the outer aqueous phase is 0.05 mM and the pH is 6.0. That is, the Zn 2+ gradient of the inner aqueous phase is 3.4 (i.e. ΔM=3.4) and the pH gradient is 1.5 (ΔpH=1.5), and the liposome intermediate containing zinc ions in the inner aqueous phase is obtained.

[0235] (3) Drug loading

[0236] Compound A 20 mg and MPEG2000-DSPE 30 mg were weighed, dissolved in sucrose-histidine solution (pH 6.0) at 55°C, and then added to 5 g of the intermediate obtained in step (2) which had been incubated at 55°C. After 5 min of continued heating and stirring, the mixture was cooled in an ice-water bath to obtain the liposomes. The particle size of the obtained liposomes was 100 nm, and the encapsulation efficiency of the compound was 95.1%.

[0237] Liposome Example 2

[0238] (1) Preparation of blank liposomes

[0239] Phosphatidylserine 5 g and cholesterol 1 g were dissolved in 10 ml of chloroform, and the solution was incubated at 50°C until it was dissolved. The chloroform was removed by rotary evaporation to obtain a liposome membrane. 100 mM calcium acetate (pH 4.5) was incubated at 55°C, and then added to the liposome membrane. The mixture was rotated for 5 min, and then placed in a 55°C water bath for 30 min. The mixture was homogenized by high-pressure microjet to a particle size of 101 nm to obtain the blank liposomes.

[0240] (2) Construction of Ca 2+ Gradient and pH gradient: The blank liposomes obtained in step (1) were dialyzed against sucrose-histidine solution (pH 6.5) by tangential flow ultrafiltration until the Ca 2+ concentration of the external water phase was 1 mM. The Ca 2+ gradient was 2 (ΔM=2) and the pH gradient was 2 (ΔpH=2) to obtain a liposome intermediate containing Ca 2+ in the internal water phase.

[0241] (3) Drug loading

[0242] Compound A 20 mg was weighed, dissolved in water at 60°C, and then added to 4 g of the intermediate obtained in step (2) which had been incubated at 60°C. After 30 min of continued heating and stirring, the mixture was cooled in ice water to obtain the liposome composition. The average particle size of the obtained liposomes was 105 nm, and the encapsulation efficiency of the compound was 92.9%.

[0243] Liposome Example 3

[0244] (1) Preparation of drug-loaded liposomes

[0245] Hydrogenated soybean lecithin 15 g, cholesterol 5 g, and MPEG2000-DSPE 5 g were dissolved in 100 ml of anhydrous ethanol, and the solution was incubated at 60°C until it was dissolved to obtain an oil phase solution. 10 g of a drug was added to 500 ml of water, and the solution was incubated at 60°C until it was dissolved to obtain an aqueous phase solution. The oil phase solution was injected into the aqueous phase solution, and the mixture was stirred for 30 min to obtain a liposome initial emulsion. The liposome initial emulsion was homogenized by high-pressure microjet to a particle size of 95 nm to obtain the drug-loaded liposomes.

[0246] (2) Removing free drug: The drug-loaded liposomes obtained in step (1) were subjected to dialysis treatment with a tangential flow ultrafiltration device using sucrose-histidine solution (pH 6.5) as the replacement medium, and were concentrated and recovered by ultrafiltration for 15 times with equal volume. The obtained liposomes had a particle size of 96.3 nm, and the compound encapsulation rate was 92.1%.

[0247] Liposome Example 4

[0248] (1) Preparation of blank liposomes

[0249] Hydrogenated soybean lecithin 15 g, cholesterol 5 g, and MPEG2000-DSPE 5 g were weighed and dissolved in 100 ml of anhydrous ethanol, and incubated to dissolve at 60°C to obtain an oil phase solution. The oil phase was injected into 500 ml of an aqueous phase solution (water phase composition: 100 mM calcium acetate solution, and add acetic acid to adjust the pH to 5.0) that had been incubated to 60°C containing calcium ions at a certain speed. After injection, stirring was continued for 30 min to obtain a liposome primary milk. The high-pressure microfluidizer was used for homogenization to 90 nm to obtain the blank liposomes.

[0250] (2) Construction of Ca 2+ Gradient and pH gradient: The blank liposomes obtained in step (1) were subjected to dialysis treatment with a tangential flow ultrafiltration device using sucrose-histidine solution (pH 6.5) as the replacement medium until the Ca 2+ concentration of the outer aqueous phase was 1 mM and the pH was 6.5. That is, the Ca 2+ gradient of the inner and outer aqueous phases was 2 (i.e., ΔM=2) and the pH gradient was 1.5 (ΔpH=1.5), so that the intermediate liposomes containing Ca 2+ in the inner aqueous phase were obtained.

[0251] (3) Drug loading

[0252] Compound A 15.5 mg was weighed and dissolved in water at 60°C, and 3.3 g of the intermediate obtained in step (2) was added, and heating and stirring were continued for 30 min, and then ice water was added for cooling to obtain the liposome composition. The average particle size of the obtained liposomes was 95.2 nm, and the compound encapsulation rate was 96.1%.

[0253] Stability investigation of liposome Example 5

[0254] The prepared liposome composition was placed at room temperature (25°C) and refrigerated (2-8°C) conditions for 0, 1, 3, 6 months, respectively, and the particle size and encapsulation efficiency of the liposome composition under different storage conditions and time were determined (as shown in Table 3 below). According to the results, the liposome composition prepared in Example 3 was stable in particle size distribution at 25°C and 2-8°C, but slowly leaked with time and the encapsulation efficiency decreased. The liposome composition prepared in Example 4 could be stored stably at room temperature and refrigerated environment for 6 months, and the particle size and encapsulation efficiency remained almost unchanged within 6 months.

[0255] Table 3 Stability study of liposome composition

[0256]

[0257] Liposome Example 6

[0258] (1) Preparation of blank liposome

[0259] DSPC 12 g and cholesterol 4 g were dissolved in 100 ml of anhydrous ethanol, incubated to 60°C to obtain an oil phase solution. The oil phase was injected into a 500 ml aqueous phase solution (water phase composition: 120 mM calcium acetate solution, pH 6.5 adjusted with acetic acid) containing calcium ions which had been incubated to 60°C at a certain speed. After injection, the mixture was continuously stirred for 30 min to obtain the initial milk of liposome. The nano-homogenizer and extruder were used for particle size adjustment. After the initial milk of liposome was homogenized to 100 nm by high-pressure homogenizer, the particle size was adjusted to 65 nm by extruder with 0.08 μm and 0.050 μm in sequence, to obtain the blank liposome.

[0260] (2) Construction of ion gradient: The blank liposome obtained in step (1) was dialyzed by tangential flow ultrafiltration device with Tris-HCl (pH 6.5) as the displacement medium until the Ca 2+ concentration of the outer water phase was 0.03 mM, so that the Ca 2+ gradient between the inner and outer water phases was 3.6 (i.e. ΔM = 3.6), to obtain the liposome intermediate containing Ca 2+ in the inner water phase.

[0261] (3) Preparation of liposome composition

[0262] Compound A 15.5 mg and MPEG2000-DSPE 25.5 mg were weighed, heated to 60°C and dissolved in 10% sucrose-histidine solution (pH 6.5), then 3.5 g of the intermediate obtained in step (2) was added, and the mixture was continuously heated and stirred for 15 min, and then cooled with ice water to obtain the liposome composition. The average particle size of the obtained liposome composition was 75 nm, and the encapsulation efficiency of the compound was 98.5%.

[0263] Pharmacokinetic test of liposome test example 1 compound A and compound A liposome composition in mouse colon cancer cell CT-26 mouse subcutaneous transplanted tumor model (tumor model)

[0264] (1) Model construction: 3 x 10 5 Personal colon cancer cell CT-26 cells, when the tumor grows to 200-300 mm 3 , randomly grouped and dosed.

[0265] (2) Dosing method and dose: intravenous injection, liposome composition 0.2 mg / kg, compound solution 20 mg / kg. The liposome composition refers to liposome example 1, and the compound solution is compound dissolved in normal saline.

[0266] (3) Sampling detection: animals were taken blood at 0.5, 1, 2, 4, 8, 24, 48 h and 2 min, 15 min, 0.5 h, 1 h, 2 h, 4 h, 8 h, 24 h after administration to detect the content of free and total compound. And collect the relevant tissues of the animals: heart, liver, spleen, lung, kidney, pancreas, uterus, ovary, bone marrow, tumor, brain, homogenate and detected by LC-MS / MS method.

[0267] (4) The pharmacokinetic results are shown in the following table:

[0268] Table 4 Pharmacokinetic parameters

[0269]

[0270] According to the pharmacokinetic parameter results, the plasma half-life of the liposome composition is 8.5 h, and the compound solution is 0.58 h. Compared with the compound solution, the half-life of the liposome composition is significantly prolonged by about 14.6 times, indicating that the prepared liposome composition has the effect of significantly prolonging the blood half-life.

[0271] The exposure of each tissue is shown in Figure 1 , the exposure of the liposome in the tumor is significantly higher than that of the compound solution at a dose of only 1 / 100 of the compound, indicating that the liposome has a certain tumor targeting property, which provides a material basis for improving the efficacy.

[0272] Liposome test example 2 Compound A and Compound A liposome composition mouse CT-26 colon and rectal cancer syngeneic tumor model pharmacodynamic test (tumor pharmacodynamics)

[0273] (1) Model construction: 1 x 10 6 CT-26 cells of mouse colon and rectal cancer cell line were transplanted into the right abdomen of female nude mice to form subcutaneous tumors.

[0274] (2) Administration method and dosage: when the tumor volume is 200-300mm 3 The mice were grouped and administered. The compound (20mg / kg and 30mg / kg), the liposome composition (1mg / kg), the blank liposome, and the physiological saline were injected intravenously once a week, for a total of 2 injections. The tumor volume was evaluated, and the change in tumor volume was observed. The anti-tumor effects of the compound solution and the liposome composition at different dosages were compared. The liposome composition was as in Liposome Example 4. The blank liposome was an intermediate without the compound, and was prepared in the same manner as in Liposome Example 4, except that no compound A was added in step (3) of the preparation of the liposome composition, and only MPEG2000-DSPE was added. The other processes were the same.

[0275] (3) Results

[0276] The change in tumor volume is shown in Table 1. Figure 2 Definitions Liposomes Long-circulating liposomes Liposome compositions Methods of preparation Pharmaceutical compositions and methods of treatment Figure 1 Figure 2 Compound examples and test examples Liposome examples and test examples Figure 1 Figure 2 Compared with the compound A solution (30mg / kg), the liposome composition had similar or even better anti-tumor effects (the same trend in tumor volume change and P values less than 0.001) at a dosage of compound 1 / 30. Compared with the compound A solution (20mg / kg), the liposome composition had significantly improved anti-tumor effects (P<0.01 vs P<0.05). The P values were statistical values of the liposome composition or the compound at different dosages compared with the blank liposome or the physiological saline. P<0.05 (*) indicates a statistical difference, and P<0.01 (**) and P<0.001 (***) indicate significant and very significant differences, respectively.

[0277] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and such changes are within the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. Liposomes, which contain a liposome membrane and an internal aqueous phase encapsulated within the liposome membrane, wherein, The internal aqueous phase contains a complex formed by metal ions and an ionized compound of formula I. Wherein, the compound of formula I is compound A as shown below: ； The metal ion is Ca. 2+ Zn 2+ Cu 2+ Mg 2+ , or Mn 2+ The concentration of the metal ions is 10-500 mM, and the metal ions form a water-insoluble or poorly soluble complex with the ionized compound of formula I.

2. The liposomes of claim 1, wherein the concentration of the metal ion is 20-300 mM.

3. The liposomes of claim 1, wherein: The metal ions react with the ionized compound of formula I to form a precipitate.

4. The liposomes of claim 1, wherein: The metal ions react with the ionized compound of formula I to form a colloidal precipitate.

5. The liposomes of claim 1, wherein, The components constituting the liposome membrane include phospholipids and cholesterol; the phospholipids are selected from one or more of lecithin, cephalin, soybean phospholipids, phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), sphingomyelin, hydrogenated soybean phosphatidylcholine (HSPC), distearate phosphatidylcholine (DSPC), and hydrogenated sphingomyelin.

6. The liposomes of claim 1, wherein: The components constituting the liposome membrane include phospholipids and cholesterol; the phospholipids are cardiophospholipids.

7. The liposomes of claim 5, wherein: The liposome membrane is modified with a functional long-circulating material.

8. The liposomes of claim 7, wherein: The functional long-cycle material is polyethylene glycol (PEG).

9. The liposomes of claim 7, wherein: The functional long-cycle material is selected from one or more of polyethylene glycol-vitamin E succinate, polyethylene glycol-cholesterol, polyethylene glycol-modified distearate phosphatidylethanolamine, polyethylene glycol-modified dimyristoyl phosphatidylethanolamine, and polyethylene glycol-modified dipalmitoyl phosphatidylethanolamine.

10. The liposomes of claim 9, wherein: The long-cycle material is polyethylene glycol-vitamin E succinate or polyethylene glycol-modified distearate phosphatidylethanolamine.

11. The liposomes of claim 10, wherein: The long-cycle material is polyethylene glycol-modified distearate phosphatidylethanolamine.

12. The liposome of claim 7, wherein the weight ratio of the compound of formula I, phospholipid, cholesterol and long-circulating material is 1:(1~300):(0.2~150):(0.01~150).

13. The liposome of claim 12, wherein the weight ratio of the compound of formula I, phospholipid, cholesterol and long-circulating material is 1:(2~200):(1~100):(0.05~100).

14. The liposomes of claim 1, wherein the average particle size is 30-300 nm.

15. The liposomes of claim 14, wherein the average particle size is 30-200 nm.

16. The liposomes of claim 15, wherein the average particle size is 50-150 nm.

17. A liposome composition comprising the liposomes of any one of claims 1-16, wherein the liposomes are exposed to an external aqueous phase.

18. The liposome composition of claim 17, wherein, The external aqueous phase contains buffer salts.

19. The liposome composition of claim 18, wherein, The external aqueous phase contains a sucrose-histidine solution or a Tris-HCl buffer solution.

20. The liposome composition of claim 19, wherein, The external aqueous phase also contains a chelating agent.

21. The liposome composition of claim 20, wherein, The chelating agent is EDTA.

22. A pharmaceutical composition comprising a liposome according to any one of claims 1-16 or a liposomal composition according to any one of claims 17-21, optionally comprising a pharmaceutically acceptable carrier.

23. A method for preparing the liposome composition according to any one of claims 17-21, comprising the following steps: (1) Preparation of blank liposomes: Phospholipids and cholesterol are dissolved in an organic solvent to obtain an oil phase solution, and a primary emulsion is formed with an inner aqueous phase buffer by injection, thin film hydration, reverse evaporation or pipeline emulsification, and the liposomes are granulated to obtain blank liposomes, wherein the inner aqueous phase buffer is a buffer containing metal ions as defined in claim 1 with a pH of 3-8. (2) Constructing an ion gradient: Using an external aqueous buffer as the replacement medium, metal ions in the in vitro aqueous phase of the blank lipid were removed, so that the internal and external aqueous phases had a metal ion gradient of not less than 1 unit and an H+ gradient of 0-4 units. + Gradient preparation of liposome intermediates; (3) Drug loading: Dissolve the compound of Formula I in the same buffer solution as the external aqueous buffer to obtain a drug solution, and add it to the liposome intermediate. Incubate at 40~70°C for 2~60 min, and cool to obtain the liposome composition; Optionally, add the long-circulating material as defined in any one of claims 7-11 to the oil phase solution in step (1) or the drug solution in step (3).

24. The method of claim 23, wherein, The second step is as follows: Constructing an ion gradient: Using an external aqueous buffer as the replacement medium, metal ions in the in vitro aqueous phase of the blank liposome intermediate are removed, resulting in a metal ion gradient of at least 2 units and a H+ gradient of 0.5–3 units between the in vivo and external aqueous phases. + gradient.

25. The method of claim 23, wherein, The third step is as follows: The compound of Formula I is dissolved in the same buffer solution as the external aqueous phase buffer to obtain a drug solution, which is then added to the liposome intermediate and incubated at 40-70°C for 5-50 minutes. After cooling, the liposome composition is obtained.

26. Use of the liposomes according to any one of claims 1-16, the liposome composition according to any one of claims 17-21, or the pharmaceutical composition according to claim 22 in the preparation of a medicament for treating colorectal cancer.

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