Phospholipid derivative, liposome thereof and application thereof

Abstract

The present application relates to a kind of phospholipid derivatives, its liposome and application.The phospholipid derivative of the present application has special phospholipid membrane damage effect under the degradation of sPLA2, the generation of this effect is based on the biphase of surface amphiphilic molecule in phospholipid membrane and the characteristics of sPLA2 substrate extraction.In the present application, low proportion of phospholipid derivative can be incorporated into the phospholipid membrane of liposome, and the degradation of phospholipid derivative by enzyme will cause strong membrane interference effect, which greatly improves the responsiveness of liposome to enzyme, so a small amount of phospholipid derivative can be used, and the sensitivity to enzyme is still maintained without causing toxic reaction, which well avoids the safety problem faced by current preparation of sPLA2 sensitive liposome, so the new drug release system constructed by the phospholipid derivative has good application prospect.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a phospholipid derivative, its liposomes, and its applications. Background Technology

[0002] In the field of cancer treatment, liposomes are the most successful and widely used drug carriers to date. Liposomes are generally spherical particles composed of phospholipids, exhibiting a structure where a bilayer phospholipid membrane encapsulates a watery core, with sizes ranging from tens of nanometers to several micrometers. The potential of liposomes as drug carriers was proposed as early as the 1970s, and by the mid-1990s, the technology had reached maturity, marked by the FDA approval of the liposome-encapsulated anticancer drug Doxil in 1996. Currently, liposome-encapsulated anticancer drugs marketed internationally include doxorubicin, daunorubicin, vincristine, cytarabine, and irinotecan. [1] The domestic market also includes paclitaxel. Liposomes function by altering the distribution and metabolism of anticancer drugs in the body, increasing drug accumulation at tumor sites, and thus reducing the drug's toxic side effects. After injection and entry into the circulatory system, most drug molecules are encapsulated by liposomes, preventing contact with blood cells; similarly, the amount of toxic drug entering normal tissues is relatively reduced. Normal vascular endothelial cells typically have adjacent pores between 1-3 nanometers, while some tumor vascular endothelial cells have pores ranging from 100 to several thousand nanometers. [2] Increased permeability allows liposomes around 100 nanometers in size to pass intact through the tumor's blood vessel walls and enter the tumor site. Simultaneously, because tumors lack a fully functional lymphatic system, the liposomes deposit within the tumor site. [3] .

[0003] Although liposomes have been shown to be effective in reducing the toxic side effects of anticancer drugs in the field of antitumor therapy, their clinical efficacy, especially in improving survival rates, is still unsatisfactory. Literature has tracked the clinical performance of liposomal anticancer drug formulations that have been marketed or are in clinical trials. [4] The study concluded that liposomes are highly effective in reducing toxic side effects, such as the cardiotoxicity of anthracyclines and the nephrotoxicity and neurotoxicity of cisplatin. However, in terms of efficacy as a survival indicator, compared with traditional chemotherapy without drug carriers (including bare drugs or combinations of several bare drugs), there was either no significant difference or only a very mild prolongation of survival (a few months), and some liposome-encapsulated drugs even showed no efficacy in clinical trials. The most well-known example is the liposome-encapsulated cisplatin anticancer drug SPI-077, which showed a significant reduction in toxic side effects in clinical trials, but essentially no anticancer activity. [4] .

[0004] So what causes liposomes to perform poorly in terms of therapeutic efficacy? This is likely due to the unique physiological conditions of the tumor site and the inability of liposomes to release drugs at specific points within the tumor. In the tumor microenvironment, the extracellular matrix fluid is saturated with collagen, glycoproteins, and proteoglycans, making it highly viscous. Simultaneously, the tumor interior has hydrostatic pressure and lacks a lymphatic system. In this unique environment, only small drug molecules can diffuse freely within the tumor site, while large molecules, including liposomes, have difficulty moving towards the tumor center and can only deposit in the periphery of the tumor. [5] This makes it difficult for liposomes to make extensive contact with tumor cells and release drugs through endocytosis. Liposome formulations primarily release small-molecule drugs around the tumor site, which then diffuse freely to various parts of the tumor to exert their effects. This makes drug release at the tumor site crucial to efficacy. If release is ineffective or the release rate is suboptimal, the therapeutic effect will be compromised. However, in designing liposomes, to prevent premature leakage of drug molecules from the drug carrier in the circulatory system and the resulting toxicity, high-phase-transition-temperature phospholipids (saturated phospholipids with long fatty chains) and high cholesterol content are commonly used in liposome formulations. The combination of these two components forms a thick, compact phospholipid membrane that stably encapsulates drug molecules, but this also makes it difficult for drug molecules to escape from this membrane once the liposome reaches the tumor site. Taking the standard spatially stabilized liposome (SSL) Doxil as an example, the formulation contains HSPC / Chol / DSPE-PEG2k (molar ratio 56:38:5), where HSPC is saturated hydrogenated soybean phosphatidylcholine with a phase transition temperature of 50℃, Chol is cholesterol, and DSPE-PEG2k is distearate phosphatidylethanolamine-polyethylene glycol. [1] The aforementioned SPI-077 also uses a similar liposome formulation. Studies have shown that SPI-077 can accumulate at the tumor site, but because cisplatin cannot be released from the liposomes, it is ineffective in treating tumors. [6] Compared to SPI-077, the anthracycline doxorubicin encapsulated in Doxil can be passively released from liposomes. This is presumably related to the encapsulation of doxorubicin into the liposomes via an ammonium sulfate gradient. At the tumor site, the loss of the ammonium sulfate gradient allows doxorubicin to slowly infiltrate through the phospholipid membrane. Therefore, Doxil's efficacy is comparable to or even better than that of bare doxorubicin or other traditional chemotherapy drugs. [7,8] However, it is worth mentioning that this passive drug release mechanism of anthracycline doxorubicin cannot be imitated by other classes of anticancer drugs, including cisplatin, and the passive drug release rate of Doxil is also likely not the optimal rate.

[0005] In conclusion, identifying liposomes capable of targeted and active drug release at tumor sites has become a pressing task and a key to improving the overall efficacy of liposome formulations, especially for platinum-based antitumor drugs such as cisplatin and oxaliplatin. Platinum-based antitumor drugs are widely used clinically, with approximately 50% of cancer treatment regimens including at least one. [9] Currently, liposome formulations encapsulating platinum-based antitumor drugs are still lacking in clinical practice. [10-12] This is due to the lack of a safe and effective drug release mechanism.

[0006] To achieve targeted drug delivery to tumor sites, liposomes sensitive to the overexpression of secretory phospholipase A2 (sPLA2) in the tumor microenvironment can be constructed. The key to designing such enzyme-responsive drug carriers lies in a deep understanding of the enzyme's structure and function, and then exploring whether various aspects of enzyme degradation can enhance its responsiveness. This includes substrate preference, substrate processing, and the specific movement of degradation products. From this perspective, it can be seen that previous studies on the design of sPLA2-responsive liposomes have primarily focused on substrate preference, i.e., adding enzyme-preferred phospholipids to the formulation. [13-17] However, the latter two factors—substrate treatment and the specific movement of degradation products—were not properly utilized. Therefore, the design of this invention utilizes these latter two factors to significantly enhance the sensitivity of liposomes to sPLA2.

[0007] sPLA2 is a family of enzymes that are ubiquitous in the human body, distributed in plasma, intercellular fluid, and other body fluids, with a molecular weight between 14-16 kDa (except for sPLA2 III, 55 kDa). [18,19] sPLA2 is highly expressed in bodily fluids such as tears and pancreatic fluid, and is also highly expressed in pathological conditions such as inflammation, bacterial infection, asthma, atherosclerosis, and tumors. [18-20] Within the sPLA2 family, sPLA2 IIa has been found to be overexpressed in various cancers, including gastric cancer, pancreatic cancer, breast cancer, lung cancer, rectal cancer, liver cancer, and prostate cancer. [21-26] Various surveys and studies have found that the sPLA2 positivity rate in tumor tissue increases sequentially among rectal cancer, pancreatic cancer, esophageal cancer, small bowel cancer, and prostate cancer, ranging from 55% to 91%. [26,27] A typical sPLA2 is a kidney-shaped protein with a highly conserved His / Asp catalytic center and a lipid-soluble channel leading to it. [28,29] As a membrane-active phospholipase, sPLA2 exhibits activity against aggregated phospholipid structures such as liposomes. It catalyzes the cleavage of glycerol phospholipids at the sn-2 position, producing one molecule of lysophospholipid and one molecule of fatty acid. [28,29] ( Figure 1 ).

[0008] sPLA2-responsive liposomes have been extensively studied. [13-17] The variability in enzyme levels at different tumor sites in different patients (range 9.9–188 ng / mL in tumor-associated exudate, mean 55 ± 44.3 ng / mL) dictates that an ideal liposome system must be hypersensitive to sPLA2 to overcome its variability and achieve adequate drug release.

[21] sPLA2 at the tumor site has a strong affinity for negatively charged phosphatidylglycerol (PG). [15-18] The current strategy for developing sPLA2-responsive liposomes for clinical application is to add a large amount of PG phospholipids to the formulation (PG accounts for 25-40 mol% of the formulation). [16,17,30] However, this strategy has led to safety issues with the formulation in vivo. For example, LiPlacis, a liposome formulation developed by a foreign pharmaceutical company specifically targeting sPLA2 to encapsulate cisplatin, has a liposome formulation of DSPC:DSPG:DSPE-PEG2000 (70:25:5). DSPC is a saturated octadecanoyl phosphatidylcholine, DSPG is a saturated octadecanoyl phosphatidylglycerol, and DSPE-PEG is distearate phosphatidylethanolamine-polyethylene glycol. This formulation utilizes the affinity of the anionic phospholipid DSPG for secretory phospholipid A2 to accelerate liposome degradation and drug release. However, in a 2009 clinical trial, this formulation was found to have excessive PG phospholipid activation of the complement system, leading to a high incidence of acute injection reactions and nephrotoxicity, thus the clinical trial was terminated.

[30] Between 2017 and 2019, LiPlacis underwent Phase I / II clinical trials in breast and prostate cancer, yielding positive results. However, to date, there has been no news of this formulation progressing to Phase III clinical trials. Animal studies have also demonstrated that liposome formulations with high levels of prostaglandins (PGs) can cause severe weight loss, possibly due to premature leakage of the encapsulated drug in the liver. [16,17] All of this suggests that overuse of PG phospholipids is not a good strategy for constructing sPLA2-responsive liposomes.

[0009] Although PG phospholipids are endogenous phospholipids, aside from surfactants in the lungs, other tissues and fluids in the human body, including blood, contain only trace amounts. Therefore, if a drug carrier contains a high percentage of PG phospholipids, it is highly likely to trigger host defense. Several early studies have shown that liposomes containing ≥20 mol% PG phospholipids can trigger the complement system. Two studies in mouse models have shown that 20–30 mol% PG phospholipids in polyethylene glycol liposome formulations lead to rapid clearance of encapsulated drugs or a shortened formulation circulation time. In general, liposomes containing a high percentage of PG phospholipids enhance the sensitivity of liposomes to sPLA2 and their interaction with tumor cells, but at the expense of the stability of liposomes in vivo circulation.

[0010] Further investigation into the enzymatic hydrolysis process of common phospholipids revealed that the resulting membrane interference effect is relatively weak. When hydrolysis begins, sPLA2 anchors on the phospholipid membrane surface, covering 30-40 phospholipid molecules. One of these molecules, located beneath the membrane, enters the active site via the enzyme's lipophilic channel. This active site is approximately 1.5 nm above the phospholipid membrane. Since the thickness of the phospholipid bilayer is 1.85-2.03 nm (the thickness of membranes constructed from pure dipalmitoylphosphatidylcholine (DPPC) and distearate phosphatidylcholine (DSPC), respectively), most phospholipid molecules are pulled out of the membrane. After hydrolysis, the metabolites lysophospholipids and fatty acids, both possessing a long lipophilic carbon chain, reintegrate into the phospholipid membrane, initiating another hydrolysis cycle. Because the two carbon chains of common phospholipids are generally saturated straight chains, occupying little space, the membrane interference caused by their removal and reintegration is minimal. Therefore, if membrane defects are to be created to achieve drug release, large-scale enzymatic hydrolysis of ordinary phospholipids is necessary, which is also inefficient.

[0011] As can be seen from the above, the current design strategies for sPLA2-responsive liposomes have shortcomings. One is the problem of in vivo stability and safety due to the excessive use of sPLA2-preferred PG phospholipids. The other is the problem of insufficient membrane interference capacity during enzymatic hydrolysis of ordinary phospholipids, resulting in insufficient sensitivity of the liposome formulation to enzymes. Therefore, it is necessary to find new strategies to construct sPLA2-responsive liposomes.

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[0042] 30.de Jonge,MJ,Slingerland,M.,Loos,WJ,Wiemer,EA,Burger,H.,Mathijssen,RH,Kroep,JR,den Hollander,MA,van der Biessen,D.,Lam,MH,etal.Early cessation of the clinical development of LiPlaCis,a liposomal cisplatin formulation.Eur.J.Cancer 2010,46,3016-3021. Summary of the Invention

[0043] The present invention aims to solve the technical problems existing in the prior art. To this end, the present invention proposes a novel phospholipid derivative, its liposomes, and its applications.

[0044] As mentioned above, past construction of sPLA2-sensitive liposomes focused solely on the enzyme's substrate preference, neglecting substrate processing and the specific movement of degradation products during enzymatic degradation. Therefore, to explore whether the latter two can enhance the enzymatic interference with phospholipid membranes, this invention turns its attention to amphiphilic molecules (FAs), such as bile acids. As amphiphilic molecules, bile acids have four nonpolar rings on one side and one or more polar hydroxyl groups and a carboxyl group on the other. Figure 2 A, 2B). Amphiphilic molecules tend to lie flat on the phospholipid membrane surface. At relatively high bile acid / phospholipid ratios, these molecules can form dimers within the membrane. Figure 2 C). Studies have shown that the conversion of amphiphilic molecules from docking at the membrane surface to dimers can interfere with phospholipid membranes, leading to the release of liposome contents. Inspired by this, this invention hypothesizes that, in the opposite direction, enzyme-mediated conversion of amphiphilic molecules from dimers to docking at the membrane surface can also lead to drug release from liposomes.

[0045] This invention introduces an amphiphilic molecule, such as lithocholic acid (LCA), at the sn-2 position of phospholipids to form sn-1 stearic acid sn-2 lithocholic acid phosphatidylcholine (LCA-PC) and sn-1 stearic acid sn-2 lithocholic acid phosphatidylglycerol (LCA-PG). Figure 2 D). Assuming that sPLA2's substrate extraction capability can extract amphiphilic molecule-modified phospholipids from the phospholipid membrane, and that the amphiphilic molecule at the sn-2 position can be degraded by sPLA2, the conversion of the amphiphilic molecule from the membrane to the membrane surface can be completed, resulting in drug release.

[0046] Taking lithocholic acid-modified phospholipids as an example, this invention designed and synthesized a series of novel modified phospholipids with lithocholic acid, chenodeoxycholic acid, or their derivatives introduced at the sn-2 position, which can be degraded by secretory phospholipase A2, for the preparation of liposomes. After performing an enzymatic luciferin release test on liposomes constructed using phospholipids modified with cholic acid or its derivatives, this invention discovered that the liposomes exhibit strong membrane pore-opening activity upon degradation by secretory phospholipase A2, leading to a large release of the contents within the liposomes. This new mechanism is named the SECRIS effect. Its mechanism is as follows: Figure 3 As shown, a novel liposome formulation containing sn-2-lithocholic acid-modified phospholipids, compared to an older formulation targeting secretory phospholipase A2 containing high levels of PG phospholipids, exhibits a more efficient drug release mechanism. Only 10% of the novel PG phospholipids is needed to achieve high sPLA2 responsiveness, avoiding the complement activation and subsequent acute injection reactions caused by excessive PG phospholipid use. Furthermore, it may improve the stability of the formulation in systemic circulation. Related studies have shown that liposomes containing 10 mol% PG phospholipids do not activate the complement system. A study in a mouse model also showed that a polyethylene glycol liposome formulation containing 10 mol% PG and encapsulating cisplatin was more stable in systemic circulation than a formulation containing 20-30% PG. This invention is expected to optimize existing clinical liposome formulations, leading to significant advancements in antitumor efficacy, and expand the application of liposomes to more antitumor drugs, such as cisplatin and oxaliplatin.

[0047] Specifically, one object of the present invention is to provide a faceted amphiphilic molecule-modified phospholipid capable of producing the SECRIS effect, comprising:

[0048] a) Design of general-purpose sn-2 cholic acid phosphatidylcholine: Phosphatidylcholine (PC) is a neutral phospholipid with good internal stability and is a basic component of liposome formulations. Cholic acid derivatives, such as lithocholic acid or its natural metabolite glycine lithocholic acid, are introduced into the sn-2 position of phosphatidylcholine. The above compounds are mainly based on stearoyl groups at the sn-1 position, but cholic acid derivatives can also be attached to the sn-1 position to form bisubstituted products.

[0049] b) Design of sn-2 bile acid phospholipids preferentially degraded by tumor-secreted phospholipase A2: Tumor-mediated secreted phospholipase A2 has a strong affinity for phosphatidylglycerol (PG), therefore, a bile acid derivative is introduced at its sn-2 position. This type of compound is preferentially degraded by tumor-mediated secreted phospholipase A2, releasing the bile acid derivative. The preferential release of the bile acid derivative leads to faster liposome degradation and drug release. DSPE-PEG (distearylphosphatidylethanolamine-polyethylene glycol) is an essential component of standard SSL formulations. Polyethylene glycol can form a hydration film on the liposome surface, reducing protein adsorption and thus prolonging the liposome's lifespan in vivo. PEG phospholipids also have a strong affinity for secreted phospholipase A2, and because they occupy a prominent position on the phospholipid membrane, they are preferentially attacked by secreted phospholipase A2. This invention introduces a bile acid derivative at the sn-2 position of PEG phospholipids and investigates the effect of the compound on the fluorescence release rate of liposomes under the action of sPLA2.

[0050] Specifically, the phospholipid derivatives according to the present invention are represented by the following general chemical formula I:

[0051]

[0052] X is selected from O and NR5;

[0053] L represents a linker, which is a saturated or unsaturated, substituted or unsubstituted straight-chain or branched hydrocarbon group containing 0-2 -O-, -NR5-, -C(=O)-O- or -C(=O)-NH- groups on the main chain;

[0054] R1 is selected from various cholic acid and cholic acid derivative residues or saturated or unsaturated, straight-chain or branched or cyclic, substituted or unsubstituted acyl or alkyl groups, wherein one or more hydrogen atoms on the acyl or alkyl group may optionally be substituted with halogen atoms, hydroxyl, alkoxy, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, amino, carboxyl, ester, dithio, cyano or nitro groups.

[0055] R3 is selected from hydrophilic groups, which are groups containing hydrogen bonds to form donors or receivers, including hydroxyl, aldehyde, carboxyl, mercapto, amino, optionally substituted amino, and ester, amide, and alkoxy groups substituted by the above hydrophilic groups; the optionally substituted amino is selected from alkyl-substituted amino, unsaturated cyclic amino, and saturated cyclic amino.

[0056] The R3 substituent can be in the α or β conformation;

[0057] Alternatively, two R3 atoms and the same carbon atom attached to them can form a carbon-carbon double bond, and the two R3 atoms can represent an oxo group (=O).

[0058] In (R3)n, n is selected from natural numbers from 1 to 4;

[0059] Furthermore, the A / B rings in the structural formula are in a cis conformation;

[0060] R4 is selected from the following groups: H, Wherein, m1 is selected from an integer of 2-4; m2 is selected from an integer of 1-3; m3 is selected from an integer of 1-3; m4 is selected from an integer of 0-5; m5 is independently selected from an integer of 1-200; and the group may optionally be replaced by a targeting agent selected from folic acid, bile acid, monoclonal antibody, amino acid fragment, polypeptide or protein, or polysaccharide.

[0061] R5 is selected from hydrogen, alkyl, alkenyl, cycloalkyl, and aryl.

[0062] According to a preferred embodiment of the present invention, in R3, the alkyl-substituted amino group is selected from methylamino, dimethylamino, ethylamino, diethylamino, and propylamino; the unsaturated cyclic amino group is selected from imidazolyl and triazolyl; and the saturated cyclic amino group is selected from pyrrolidinyl, piperidinyl, and piperazine.

[0063] According to a preferred embodiment of the present invention, wherein R3 is selected from hydroxyl, optionally substituted ester group, and optionally substituted amide group; the substituents on the ester group and amide group are selected from amino, aldehyde, mercapto, carboxyl, pyrrolyl, piperidinyl, imidazole, and triazolyl; preferably, in R3, the alkyl-substituted amino group is selected from methylamino, dimethylamino, ethylamino, diethylamino, and propylamino, the unsaturated cyclic amino group is selected from imidazole and triazolyl, and the saturated cyclic amino group is selected from pyrrolyl, piperidinyl, and piperazine.

[0064] According to a preferred embodiment of the present invention, the two R3s together represent an oxo group.

[0065] According to a preferred embodiment of the present invention, n is selected from an integer from 1 to 3; more preferably, n is selected from 1 or 2.

[0066] According to a preferred embodiment of the present invention, wherein: 1) when n=4 in (R3)n, the four R3s represent two oxo groups, which may be distributed on rings A and B respectively or concentrated on one of the rings; 2) when n=3 in (R3)n, two R3s together represent one oxo group, and the oxo group and the other R3 may be distributed on rings A and B respectively or concentrated on one of the rings; or, 3) when n=1 or 2 in (R3)n, R3 is selected from hydroxyl, aldehyde, carboxyl, mercapto, amino, methylamino, dimethylamino, ethylamino, diethylamino, imidazolyl, triazolyl, pyrrolyl, piperidinyl, piperazine, etc. Or two R3s together represent an oxo group; wherein Z is selected from O, NNH or an optionally substituted amino group; Y is selected from hydroxyl, aldehyde, carboxyl, mercapto, amino, methylamino, dimethylamino, ethylamino, diethylamino, imidazolyl, triazolyl, pyrrolyl, piperidinyl, piperazine.

[0067] According to a preferred embodiment of the present invention, R1 is selected from cholic acid and its derivative residues or C 1-50 The acyl or alkyl group is a straight-chain, branched, or cyclic group; more preferably, R1 is selected from cholic acid residues, lithocholic acid residues, 3-ketolithocholic acid residues, or C. 12-18 The straight-chain acyl group; most preferably, the R1 is selected from stearyl acyl.

[0068] According to a preferred embodiment of the present invention, wherein R4 is selected from the following groups: H, Wherein, m1 is selected from integers 2-4; m2 is selected from integers 1-3; m3 is selected from integers 1-3; m4 is selected from integers 1-4; m5 is independently selected from integers 1-200; more preferably, m5 is independently selected from integers 30-80.

[0069] According to a preferred embodiment of the present invention, wherein R4 is selected from the following groups: Where m1 and m3 are selected from integers from 1 to 3; m5 = 44-45.

[0070] According to a preferred embodiment of the present invention, the phospholipid derivative is selected from the following compounds:

[0071]

[0072]

[0073] The present invention also provides a method for preparing the above-mentioned phospholipid derivatives, comprising the following steps: esterification reaction of various cholic acids or their derivatives and the sn-2 hydroxyl group of phospholipid compounds under the conditions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and a base as a catalyst.

[0074] Preferably, the base is selected from 4-dimethylaminopyridine, pyridine, triethylamine or a combination thereof; more preferably, the base is 4-dimethylaminopyridine.

[0075] Preferably, in the preparation method, the reaction temperature is 0-25℃ and the reaction time is 2-60h; more preferably, the reaction temperature is 0-7℃ and the reaction time is 45-60h.

[0076] Preferably, the preparation method is carried out in an organic solvent, which is preferably chloroform (trichloromethane), dichloromethane, carbon tetrachloride, ethyl acetate, tetrahydrofuran (THF), toluene, methanol, ethanol, propanol, butanol, acetonitrile, DMF, DMSO, pyridine, benzene, toluene, xylene, or a mixture thereof.

[0077] It should be understood that, according to the present invention, when a numerical range is given, unless otherwise expressly stated herein, each smaller range between any of the stated values ​​is included within the scope of the invention. The upper and lower limits of these smaller ranges may independently include or exclude the range, and each range, whether any or none of the upper and lower limits are included in these smaller ranges, is also within the scope of the invention.

[0078] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While similar or equivalent methods and materials described herein may be used to practice or test the invention, some possible and exemplary methods and materials are described hereafter. Any and all publications mentioned herein are incorporated herein by reference in order to disclose and describe methods and / or materials in conjunction with the publications cited. It should be understood that in the event of conflict, any content of the included publications shall replace the content of this document.

[0079] According to an example of the present invention, the term "hydrocarbon group" used alone or in combination refers to a group containing carbon and hydrogen elements, including straight-chain, branched, saturated or unsaturated chain groups, such as alkyl, alkenyl, and ynyl groups; and saturated or unsaturated cyclic groups, such as cycloalkyl and cycloalkenyl groups; preferably, the cycloalkenyl group is selected from aromatic groups.

[0080] The term "alkyl" used alone or in combination refers to a saturated hydrocarbon group, preferably a lower alkyl group with less than fifty carbon atoms, that is, a saturated straight-chain or branched hydrocarbon group containing one to fifty carbon atoms.

[0081] The term "acyl" refers to a group terminated with a carbonyl group, preferably a straight-chain or branched C-chain group. 1-50 Alkyl acyl group.

[0082] The term "(Cx-y)alkyl" (where x and y are each integers) refers to an alkyl group as previously defined, containing x to y carbon atoms. For example, (C... 1-6 Alkyl groups contain one to six carbon atoms. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, 3-methyl-butyl, 2,2-dimethyl-propyl, and 3,3-dimethyl-butyl. To avoid any ambiguity, if a group is referred to as, for example, propyl or butyl, it means n-propyl or n-butyl, respectively.

[0083] The term "alkoxy" refers to R a O- group, where R aThe alkoxy group refers to an alkyl group, preferably a lower-order alkyl group with fewer than twenty carbon atoms; that is, according to the present invention, the alkoxy groups specifically include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentoxy, isopentoxy, tert-pentoxy, neopentoxy, hexoxy, isohexoxy, tert-hexoxy, neohexoxy, palmoxy, stearoxy, eicosoxy, etc.

[0084] The term "ester group" refers to R a COO- group, where R a It refers to alkyl groups, preferably lower-order alkyl groups with fewer than twenty carbons.

[0085] The term "amide group" refers to R a (C=O)NH- group, where R a It refers to alkyl groups, preferably lower-order alkyl groups with fewer than twenty carbons.

[0086] The term "cycloalkyl" used alone or in combination refers to a saturated monocyclic hydrocarbon ring. For example, cycloalkyl contains three to eight carbon atoms. Examples of cycloalkyl are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.

[0087] The term "aryl" used alone or in combination refers to phenyl, naphthyl, anthraceneyl, phenanthryl, and preferably phenyl. The aryl groups mentioned above are either unsubstituted or substituted.

[0088] The term "heteroaryl," used alone or in combination, refers to a 5- to 10-membered monocyclic or bicyclic aromatic ring containing one to a maximum of four heteroatoms (each independently selected from oxygen, nitrogen, and sulfur). Examples of such heteroaryls include 5-membered heteroaryls such as furanyl, oxazolyl, isoxazolyl, oxadiazolyl, thienyl, thiazolyl, isothiazolyl, thiadiazolyl, pyrroleyl, imidazolyl, pyrazolyl, triazolyl, and tetrazolyl; 6-membered heteroaryls such as pyridyl, pyrimidinyl, pyridazinyl, and pyrazinyl; and 8- to 10-membered bicyclic heteroaryls such as indolyl, isoyindolyl, benzofuranyl, isobenzofuranyl, benzothiopheneyl, and indolyl. Azolyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisoxahiazolyl, benzotriazolyl, benzoxadiazolyl, benzothiadiazolyl, thienopyridyl, quinolinyl, isoquinolinyl, naphridyl, cinolinyl, quinazolinyl, quinoxalolinyl, phthalazinyl, pyrrolopyridyl, pyrazolopyridyl, pyrazolopyrimidinyl, pyrazolopyrazinyl, imidazopyridyl, imidazopyridinyl, and imidazothiazolyl. The heteroaryl groups mentioned above are either unsubstituted or substituted as explicitly defined.

[0089] The term "alkenyl" refers to an unsaturated hydrocarbon group containing at least one carbon-carbon double bond, preferably C. 2-6 Alkenyl groups, specifically, examples of alkenyl groups according to the present invention include: vinyl, propenyl, allyl, 1-butenyl, 2-butenyl, isobutenyl, pentenyl, and hexenyl.

[0090] The term "alkynyl" refers to an unsaturated hydrocarbon group containing at least one carbon-carbon triple bond, preferably C. 2-6 The alkynyl group, specifically, examples of alkynyl groups according to the present invention include: ethynyl, n-propynyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, pentylyl, and hexynyl.

[0091] The term "halogen" means fluorine, chlorine, bromine or iodine; preferably fluorine, chlorine or bromine; more preferably fluorine or chlorine.

[0092] The term "hydroxyl group" refers to the -OH group.

[0093] The term "thiol" refers to the -SH group.

[0094] The term "disulfide" refers to the group -SS-.

[0095] The term "cyano" refers to the -CN group.

[0096] The term "amino" refers to the -NH2 group.

[0097] The term "nitro" refers to the group -NO2.

[0098] The term "carboxyl group" refers to the group -COOH.

[0099] The term "aldehyde" refers to the group -CHO.

[0100] The term "oxo" refers to a group that is equal to O.

[0101] the term This indicates that the group is connected to the general formula structure through this bond.

[0102] The "derivatives" of the compounds of this invention include their salts, esters, enol ethers, enol esters, acetals, hydrazones, ketals, orthoesters, hemiacetals, acids, bases, solvates, hydrates, or prodrugs. Such derivatives can be readily prepared by those skilled in the art using known derivatization methods.

[0103] Another aspect of the invention relates to a phospholipid liposome prepared from a phospholipid derivative comprising the aforementioned phospholipid derivative according to the invention.

[0104] According to a preferred embodiment of the present invention, the phospholipid liposomes are prepared from phospholipid derivatives and other phospholipid compounds according to the present invention.

[0105] According to a preferred embodiment of the present invention, the molar ratio of the phospholipid derivative and other phospholipid compounds of the present invention is 1:4-19; more preferably, the molar ratio is 1:9-15; and most preferably, the molar ratio is about 1:9. Herein, "about" refers to ±2%.

[0106] According to a preferred embodiment of the present invention, the preparation method is preferably a freeze-thaw method.

[0107] According to a preferred embodiment of the present invention, the other phospholipid compounds are mainly selected from DPPC, DPPG, DSPC, DSPG, HSPC (hydrogenated soybean phospholipids), DSPE-PEG, or combinations thereof.

[0108] Another aspect of the invention relates to the use of phospholipid derivatives or phospholipid liposomes according to the invention in the field of drug delivery.

[0109] According to a preferred embodiment of the present invention, the drug is a drug for treating cancer; the cancer is preferably a cancer that overexpresses secretory phospholipase A2. Furthermore, secretory phospholipase A2 is also highly expressed in tears, digestive juices secreted by the pancreas, and in inflammatory sites under pathological conditions, bacterial infections, asthma, and atherosclerosis; the drug can also be a drug for treating diseases and conditions of the corresponding organs.

[0110] According to the present invention, the medicine can be a variety of different substances, which are applicable to a variety of different applications, including but not limited to: pharmaceutical, nutritional, cosmetic and diagnostic fields. Exemplary substances include, but are not limited to: bisphosphonates, lobaplatin, carboplatin, nedaplatin, cisplatin, oxaloplatin, carmustine, camptothecin, ciprofloxacin, chloromethane, cyclophosphamide, cycloparin, cytarabine, dacarbazine, retinoids, deoxyfluorouridine, fluoroortic acid, galdromycin, vinorelbine, gemcitabine, gossypol, isophosphamide, hydroxytamoxifen, irinotecan, phytic acid, protein kinase inhibitors, paclitaxel, resveratrol, taxane, methylselenocysteine, methotrexate, 6-thioguanine, tyrosine phosphorylation inhibitors, baicalin, etoposide, antisense oligonucleotides, siRNA, chemically modified RNA, citrate, 1,2,3,4-butanetetracarboxylic acid, and octasulfate. Sucrose, polyphosphates, ciprofloxacin, morphine, hydroxymorphone, buprenorphine, and methadone. These agents can be encapsulated individually or in the same liposome with another agent (e.g., one or more agents; two or more agents) to provide synergistic effects.

[0111] Drugs of particular interest include, but are not limited to: anticancer chemotherapeutic agents (e.g., doxorubicin, danorubicin, camptothecin, cisplatin, etc.), antibiotics (e.g., antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, etc.), analgesics, anesthetics, antiacne agents, biomolecules (e.g., nucleic acids (e.g., RNA, DNA, siRNA, etc.), polypeptides (e.g., peptides, including recombinant polypeptides and peptides, including natural or chemically modified polypeptides and peptides (e.g., PEGylated polypeptides)), antibodies, etc.), antigenic substances (e.g., which may be components of vaccines), anticoagulants, and drugs for the treatment of neuroregenerative diseases. Compounds that can load ion gradients into liposomes, such as benzocaine, chloroprocaine, cocaine, procaine, tetracaine, bupivacaine, lidocaine, mepivacaine, fentanyl, and trimethaine; analgesics such as diclofenac; and molecules that can load ion gradients into liposomes, such as ammonium sulfate, triethylamine sulfate, triethylamine salt of sucrose octasulfate, triethylamine polyphosphate, ammonium salt of phytic acid, sodium acetate, triethylamine or ammonium salt, sodium oxalate, triethylamine or ammonium salt, and propionic acid. Sodium, triethylamine or ammonium salts of succinic acid, sodium, triethylamine or ammonium salts of 1,2,3,4-butanetetracarboxylic acid, sodium, triethylamine or ammonium salts of pyridine-2,3,5,6-tetracarboxylic acid, sodium, triethylamine or ammonium salts of 1,2,4,5-benzenetetracarboxylic acid, sodium, triethylamine or ammonium salts of 1,2,4,5-cyclohexanetetracarboxylic acid, sodium, triethylamine or ammonium salts of 1,3,5-benzenetricarboxylic acid, and acetates of polyamines such as spermine, spermidine, triaminoethylamine, etc.

[0112] According to a preferred embodiment of the present invention, cosmetic agents (e.g., flavonoids, antioxidants such as gamma-linolenic acid, beta-carotene, anthocyanins, beta-sitosterol) and dietary additives (e.g., vitamins) can also be used as drugs loaded in the liposomes of the present invention.

[0113] Exemplary cosmetic agents that can be used as loadings in this invention may include hydrating agents, proteins (e.g., collagen), vitamins, phytochemicals, enzymes, antioxidants, essential oils, UV protectants (e.g., oxybenzone), cleansers, pigments, fragrances, etc. (e.g., for use in cosmetics, makeup products, fragrances, perfumes, skin care products, and cosmetic auxiliaries). Retinoic acid is particularly useful, especially for sterol-modified amphiphilic lipid emulsions containing retinoic acid in skin care products.

[0114] Diagnostic agents include detectable markers, which can be radiolabels, fluorophores, luminescent compounds, MRI contrast agents such as gadolinium, positron emission tomography markers, etc. In some embodiments, liposomes themselves can be used as diagnostic agents, for example, as microbubbles in ultrasound diagnostics.

[0115] Of particular interest are drug delivery vehicles that can be used for toxic drugs (e.g., various cancer chemotherapy agents, doxorubicin, daunorubicin, camptothecin, etc.) and / or drugs with poor water solubility (e.g., amphotericin B, retinoic acid, etc.). Drug delivery vehicles containing amphiphilic molecule-modified phospholipids for use as vaccine delivery vehicles are also of particular interest, especially those that exhibit storage stability (e.g., at ambient temperature and / or 4°C).

[0116] The drug-loaded compounds of the present invention can be formulated into pharmaceutical preparations for use in a variety of routes of administration, including parenteral, enteral, nasal, and pulmonary administration, and may contain one or more excipients. Exemplary formulations include topical, transdermal, injectable (e.g., intravenous, intramuscular, subcutaneous), aerosol, and oral preparations, and can be formulated into pharmaceutical articles, cosmetic articles, nutritional articles, etc. The drug-loaded compounds of the present invention can be lyophilized or stored in solution.

[0117] Therefore, acceptable dosage forms of formulations of the pharmaceutically loaded compounds of the present invention include, but are not limited to, aqueous solutions, suspensions, dispersions, lotions, washes, creams, ointments, balms, and ointments. The pharmaceutically loaded compounds of the present invention can also be administered via tablets or capsules (e.g., dissolved in the digestive tract) and suppositories. The loaded compounds can also be provided in devices such as patches, bandages, etc.

[0118] In addition to the compounds of this invention and the desired effective load, topical formulations may comprise a pharmaceutically acceptable local delivery vehicle suitable for application to the skin or mucous membranes of an animal. Topical formulations can be provided in a variety of suitable dosage forms, including, but not limited to, lotions, gels, ointments, creams, balms, ointments, etc. These compositions may be in the form of aqueous solutions or emulsions, such as oil and water emulsions (e.g., oil-in-water emulsions or water-in-oil emulsions). If desired, topical formulations may contain penetration enhancers or other agents that facilitate transdermal delivery.

[0119] The dosage forms considered in this article can usually be formulated using physiologically acceptable carriers, excipients, stabilizers, etc., and these dosage forms can be sustained-release or timed-release dosage forms. Acceptable carriers, excipients, and diluents for therapeutic applications are well-known in the pharmaceutical field and described, for example, in Remington's Pharmaceutical Science (ARGennaro, ed., Mack Publishing Co., 1985). These substances are non-toxic to the recipient at the doses and concentrations used and contain buffers such as phosphates, citrates, acetates, and other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) peptides such as polyarginine; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as poly(vinylpyrrolidone); amino acids such as glycine, glutamic acid, aspartic acid, and arginine; monosaccharides; disaccharides and other carbohydrates, including cellulose and its derivatives, glucose, mannose, and dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; and cationic and nonionic surfactants such as Tween, PLURONICS, and PEG in topical formulations.

[0120] Preparations used for therapeutic administration must be sterile. Sterility can be readily achieved through sterile membrane filtration or other conventional methods, such as radiation or treatment with gas, heat, or autoclave. The pH of the preparations of the present invention is typically 3-11, more preferably 5-9. The optimal dose can be administered to the subject requiring treatment with the preparations of the present invention (typically a mammal) to provide the best therapeutic effect. The dosage and method of administration may vary depending on the subject and factors such as the type of host being treated; in animals, for example, this includes sex, weight, diet, current treatment, overall clinical condition, the specific hydrophobic compound used, the specific purpose of these compounds, and other factors known to those skilled in the art. Those skilled in the art can determine the effective dose level, i.e., the dose level at which the desired result is obtained.

[0121] Formulations can be prepared to be stored under conditions suitable for preserving the activity of any effective load and maintaining the integrity of the compounds of the present invention and any other liposomes that may be present. Liposomes according to the present invention are stable at 4°C for one year or longer; therefore, storage at 4°C is suitable for long-term preservation of the compounds described herein.

[0122] Compounds of the present invention How to use

[0123] The compounds of this invention can be used in a variety of pharmaceutical, cosmetic, diagnostic, and biomedical fields. Exemplary applications are shown below.

[0124] For example, pharmaceutical, nutritional, and cosmetic applications typically involve administering a composition containing the compounds of the present invention to an animal subject. Administration is usually achieved by exposing the animal to the composition via any suitable route (e.g., parenteral, intestinal, nasal, pulmonary, etc.). In these applications, the animal is a subject requiring treatment because the subject needs treatment or can benefit from such treatment. In the context of treatment methods, "animal subject" as used herein generally refers to a subject requiring treatment, and / or, in the context of diagnostic methods, refers to a subject suspected of having a disease detectable by that diagnostic method. Subjects include animals, including mammals such as humans, livestock, pets, etc.

[0125] This article generally uses the term "therapeutic" and the like to indicate the attainment of a desired pharmacological and / or physiological effect. This effect can be preventative in terms of the complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of the site or complete stabilization or cure of a disease and / or reversal of the adverse effects caused by that disease. As used herein, "therapeutic" encompasses any treatment of disease in mammals, particularly humans, including: (a) preventing the occurrence of disease or symptoms in subjects predisposed to develop the disease or symptoms but not yet diagnosed with the disease or symptoms; (b) suppressing disease symptoms, i.e., halting their development; or (c) alleviating disease symptoms, i.e., causing the disease or symptoms to subside.

[0126] The composition containing sterol-modified amphiphilic lipids and its effective loading can be selected according to the patient and condition to be treated and the benefits of administration.

[0127] The compounds of this invention can also be used in diagnostic methods. Such methods include diagnosing diseases by detecting analytes in biological samples of animal subjects.

[0128] As used herein, “diagnosis” generally includes determining a subject’s susceptibility to a disease or condition, determining whether a subject is currently affected by a disease or condition, determining the prognosis of a subject affected by a disease or condition, and assessing the effectiveness of therapeutic agents (e.g., monitoring a subject’s condition to provide information on the efficacy or effectiveness of treatment). The term “biological sample” includes various types of samples obtained from an organism that can be used for diagnostic or monitoring tests. This term includes biologically derived blood and other fluid samples, solid tissue samples such as biopsy samples or tissue cultures or cells derived therefrom and their progeny. This term includes samples that have been obtained and manipulated in various ways, such as by treatment with reagents, dissolution, or enrichment of certain components. This term includes clinical samples, and also includes cells in cell cultures, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.

[0129] Diagnostic methods utilizing loaded compounds of the present invention typically involve detecting the presence or absence of an analyte in a fluid. This method includes contacting the liquid with a composition containing the compound of the present invention and detecting changes in the compound, wherein the changes in the compound indicate the presence or absence of the analyte and even changes in its concentration. Changes in the lipid composition can be, for example, color changes (e.g., due to changes in the emission wavelength of the encapsulated fluorophore), changes in the size or integrity of the liposomes containing the compound of the present invention, etc. Therefore, changes in the liposome composition can be detected by evaluating the properties of the lipid composition, such as optical properties (e.g., reflectivity), phase transitions, etc.

[0130] Other applications

[0131] Compositions containing the compounds of the present invention also have various applications in fields not directly related to treatment or diagnosis. For example, compositions containing the compounds of the present invention facilitate the delivery of payloads (e.g., nucleic acids, peptides) into cells. In this method, cultured animal cells (e.g., cultured mammalian cells, such as human cells) are contacted with the compound of the present invention containing the payload of interest, thereby facilitating the delivery of the payload into the cell. Such methods can be used to introduce nucleic acids, peptides, or other compounds that do not readily cross animal cell membranes.

[0132] The compounds of this invention can also be used as artificial bilayers, and therefore can be used in devices such as biosensors. Biosensors utilizing artificial bilayers are well known in the art. They are particularly suitable for situations where the lipid surface is in direct contact with blood, plasma, serum, or other bodily fluids containing cells, proteins, or lipids.

[0133] Reagent Kits and Systems

[0134] The kits and systems provided facilitate the production and / or utilization of the compositions described herein. The kits considered herein may contain one or more compounds of the present invention, the reagent of interest to be delivered, and other excipients, etc. They may be provided in different containers or typically in sterile containers as a single composition.

[0135] In addition, the kit may contain instructions for use of the components of the kit, particularly the compositions of the present invention contained in the kit.

[0136] According to other aspects of the invention, the invention provides a method of administering a composition comprising a compound of the invention to an animal, the method comprising contacting the animal with the compound of the invention.

[0137] In other respects, the present invention provides a method for administering a composition comprising amphiphilic molecule-modified phospholipids to cells, the method comprising contacting the cells with the amphiphilic molecule-modified phospholipid composition of the present invention.

[0138] In other aspects, the present invention provides a method for detecting the presence or absence of an analyte in a fluid, comprising contacting the fluid with a composition containing a compound of the present invention and detecting at least one change in a detectable property of the liposome composition or the fluid. In a related embodiment, the fluid is a biological fluid. In a further related embodiment, detection is performed by evaluating the properties of the liposome composition.

[0139] This invention demonstrates the following beneficial effects:

[0140] This invention designs and synthesizes a series of novel modified phospholipids with cholic acid or its derivative introduced at the sn-2 position, which can be degraded by secretory phospholipase A2 for the preparation of liposomes. The novel modified phospholipids exhibit a strong membrane perforation effect when degraded by secretory phospholipase A2, known as the SECRIS effect. The mechanism is as follows: specific lithocholic acid-modified phospholipid molecules are extracted by the enzyme into its hydrophobic channels and then reach the active site for hydrolysis. At this point, the entire phospholipid molecule migrates at least 1.5 nm outside the phospholipid membrane, while the lithocholic acid residue, which is 1.4 nm long and originally inserted into the hydrophobic portion of the 1.3 nm thick (monolayer) DPPC phospholipid membrane, is completely extracted from the hydrophobic region and enters the hydrophilic region (0.55 nm thick). The 3α-OH in the lithocholic acid residue quickly forms hydrogen bonds in the hydrophilic region. After hydrolysis, the product lysophospholipid re-insertes into the phospholipid membrane, while the other product, lithocholic acid, rotates, pushing aside surrounding phospholipids and lying flat on the membrane surface. This process causes membrane perforation, leading to the leakage of luciferin (CF). Enzymatic CF and oxaliplatin release assays showed that this effect resulted in a large release of CF and oxaliplatin, much faster than the clinical trial formulation LiPlaCis liposomes (DSPC:DSPG:DSPE-PEG2k 70:25:5) and its improved version DPPG25 (DPPC:DPPG:DSPE-PEG2k 70:25:5), which did not exhibit this effect. This indicates that this effect significantly enhances the liposome's responsiveness to secretory phospholipase A2.

[0141] In cell experiments, liposomes loaded with oxaliplatin and containing the compound LCA-PG exhibited similar anti-tumor cell effects to DPPG25 in the presence of sPLA2 (75 ng / mL). In nude mouse tumor-bearing experiments, the liposome formulation containing 10% LCA-PG showed better tumor growth inhibition and lower toxicity compared to DPPG25, resulting in longer survival in the animal group.

[0142] A novel liposome formulation containing sn-2 lithocholic acid-modified phospholipids, compared to an older formulation targeting secretory phospholipase A2 with high levels of PG phospholipids, exhibits a more efficient drug release mechanism. High sPLA2 responsiveness can be achieved using only 10% of the novel PG phospholipids, avoiding complement activation, subsequent acute injection reactions, and decreased liposome stability in systemic circulation caused by excessive PG phospholipid use. Related studies have shown that liposomes containing 10 mol% PG phospholipids do not activate the complement system. A study in a mouse model also showed that a polyethylene glycol liposome formulation containing 10 mol% PG and encapsulating cisplatin was more stable in systemic circulation than a formulation containing 20-30% PG. This invention is expected to optimize existing clinical liposome formulations, leading to advancements in antitumor efficacy, and expand the application of liposomes to more antitumor drugs, such as cisplatin and oxaliplatin. Attached Figure Description

[0143] Figure 1 Secretory phospholipase A2 cleaves the ester bond at the sn-2 site of phospholipids to produce lysophospholipids and free fatty acids.

[0144] Figure 2 Two modes of lithocholic acid presence in phospholipid membranes and the design of novel lithocholic acid-modified phospholipids. (A) Amphiphilic characteristics of lithocholic acid; (B) Front and side views of the lithocholic acid structure; (C) Two modes of lithocholic acid-phospholipid presence in phospholipid membranes: amphiphilic properties allow lithocholic acid to adhere to the phospholipid membrane surface, or amphiphilic properties allow lithocholic acid to form a dimer within the membrane; (D) Phospholipids with lithocholic acid and 3-deoxylithocholic acid linked at the sn-2 position: Compound 1, sn-1-stearic acid-sn-2-lithocholic acid phosphatidylcholine (LCA-PC); Compound 2, sn-1-stearic acid-sn-2-lithocholic acid phosphatidylglycerol (LCA-PG); Compound 3, sn-1-stearic acid-sn-2-3'-deoxylithocholic acid phosphatidylcholine (dLCA-PC).

[0145] Figure 3 Enzymatic hydrolysis of LCA-PC produces the SECRIS effect. Left: Chemical structure of LCA-PC; Middle 1: LCA-modified phospholipids form dimers within the phospholipid membrane; Middle 2: sPLA2 extracts LCA-PC from the phospholipid membrane to its active site for hydrolysis. This process extracts LCA-PC from the hydrophobic region of the DPPC phospholipid membrane, exposing the 3α-hydroxyl groups of the LCA portion to a hydrophilic environment; Right: After hydrolysis, lysophospholipids reinserte into the phospholipid membrane, while the degraded LCA rotates and lies flat on the phospholipid membrane surface.

[0146] Figure 4The phase transition temperature of different liposomes was determined by differential scanning calorimetry. DPPG25, DPPC:DPPG:DSPE-PEG2k (70:25:5); LCA-PG10, DPPC:LCA-PG:DSPE-PEG2k (85:10:5); LCA-PG15, DPPC:LCA-PG:DSPE-PEG2k (80:15:5); LCA-PG (10), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:25:10:5 mol%); LCA-PG (15), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:20:15:5 mol%).

[0147] Figure 5 Cryo-electron microscopy images of LCA-PG10 and LCA-PG15. LCA-PG10, DPPC:LCA-PG:DSPE-PEG2k (85:10:5); LCA-PG15, DPPC:LCA-PG:DSPE-PEG2k (80:15:5); the ratios are molar ratios.

[0148] Figure 6Liposome fluorescein release assay under the action of bee venom type (Bv)sPLA2 (100 ng / mL). (A) 1-hour luciferin release rate of DPPC liposomes with 10 mol% cholic acid and derivative-modified phospholipids (compounds 1-7) under Bv sPLA2 treatment; (B) 1-hour luciferin release rate of DPPC liposomes containing DSPG25, DPPG25, and 10 mol% cholic acid and derivative-modified phospholipids (compounds 1-7) under Bv sPLA2 treatment; (C) 24-hour luciferin release rate of DSPC:DSPE-PEG2K (95:5) liposomes with 5 mol% LCA-PEG2K (compound 8) under Bv sPLA2 treatment; (D) Release curves of DPPC:DSPE-PEG formulation with 2.5 mol% biLCA-PC (compound 9) added, and ordinary DPPC:DSPE-PEG formulation under sPLA2 treatment. The reaction temperature was 25-30℃. Data are presented as mean ± standard deviation, and each test was performed three times.Abbreviations: DPPC: 100% DPPC; 10% LCA-PC, DPPC: LCA-PC (9: 1 mol%); 10% LCA-PG, DPPC: LCA-PG (9: 1 mol%); 10% kLCA-PC, DPPC: kLCA-PC (9:1 mol%); 10% kLCA-PG, DPPC: kLCA-PG (9:1 mol%); 10% CDCA-PC, DPPC: CDCA-PC (9:1 mol%); 10 %7kCDCA-PC, DPPC:7kCDCA-PC (9:1mol%); 10%bkCDCA-PC, DPPC:bkCDCA-PC (9:1mol%); DSPG25, DSPC:DSPG:DSPE-PEG2k (with the same phospholipid ratio as cisplatin liposome LiPlaCis triggered by tumor site overexpression via Phase II clinical trials, 70:25:5mol%); DPPG25, DPPC:DPPG :DSPE-PEG2k (70:25:5mol%); LCA-PC10, DPPC: LCA-PC: DSPE-PEG2k (85:10:5mol%); LCA-PG10, DPPC: LCA-PG :DSPE-PEG2k (85:10:5mol%); kLCA-PC10, DPPC: kLCA-PC: DSPE-PEG2k (85:10:5mol%); kLCA-PG10, DPPC:kLC A-PG: DSPE-PEG2k (85:10:5mol%); CDCA-PC10, DPPC: CDCA-PC: DSPE-PEG2k (85:10:5mol%); 7kCDCA-PC10, DP PC:7kCDCA-PC:DSPE-PEG2k (85:10:5mol%); bkCDCA-PC10, DPPC: bkCDCA-PC:DSPE-PEG2k (85:10:5mol%); 5% DSPC: DSPE-PEG2k, DSPC: DSPE-PEG2k (95:5 mol%); 5% LCA-PEG2k, DSPC: LCA-PEG2k (95:5 mol%).

[0149] Figure 7Liposome luciferin release assay under the action of human tear (HTear) sPLA2 (100 ng / mL). The luciferin release of liposomes containing DSPG25, DPPG25, and DPPC:DSPE-PEG2k liposomes containing 10 mol% cholic acid and derivative-modified phospholipids (compounds 1-4) was measured after 24 hours under the action of HTear sPLA2. The experiment was conducted at room temperature. Data are expressed as mean ± standard deviation, and each experiment was performed three times. Abbreviations: DSPG25,DSPC:DSPG:DSPE-PEG2k (70:25:5 mol%); DPPG25,DPPC:DPPG:DSPE-PEG2k (70:25:5 mol%); LCA-PC10,DPPC:LCA-PC:DSPE-PEG2k (85:10:5 mol%); LCA-PG10,DPPC:LCA-PG:DSPE-PEG2k (85:10:5 mol%); LCA-P G15, DPPC:LCA-PG:DSPE-PEG2k (80:15:5mol%); kLCA-PC10, DPPC:kLCA-PC:DSPE-PEG2k (85:10:5mol%); kLC A-PG10, DPPC:kLCA-PG:DSPE-PEG2k (85:10:5mol%); kLCA-PG15, DPPC:kLCA-PG:DSPE-PEG2k (80:15:5mol%).

[0150] Figure 8Liposome luciferin release assay under the action of Colo205 tumor cells sPLA2 (100 ng / mL). The luciferin release of liposomes secreted by Colo205 cells after 24 hours was measured using DSPG25, DPPG25, and DSPE-PEG2k liposomes containing 10% or 15% bile acid and derivative-modified phospholipids (compounds 1–4). The experiment was conducted at room temperature. Data are expressed as mean ± standard deviation, and each assay was performed three times. Abbreviations: DSPG25, DSPC:DSPG:DSPE-PEG2k (70:25:5mol%); DPPG25, DPPC:DPPG:DSPE-PEG2k (70:25:5mol%); LCA-PC10, DPPC:LCA-PC:DSPE-PEG2 k(85:10:5mol%); LCA-PG10, DPPC:LCA-PG:DSPE-PEG2k (85:10:5mol%); LCA-PG15, DPPC:LCA-PG:DSPE-PEG2k (80:15:5mol%); kLCA-PC10, D PPC: kLCA-PC: DSPE-PEG2k (85:10:5 mol%); kLCA-PG10, DPPC: kLCA-PG: DSPE-PEG2k (85:10:5 mol%); kLCA-PG15, DPPC: kLCA-PG: DSPE-PEG2k (80:15:5mol%); LCA-PG (10), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:25:10:5); LCA-PG (15), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:20:15:5).

[0151] Figure 9 Release curves of oxaliplatin (L-OHP) from DPPG25 and DSPE-PEG2k liposomes containing 10 or 15 mol% LCA-PG under the action of Colo205 sPLA2. The total phospholipids of the liposome formulations were 500 μM, and the experiments were conducted at 37 °C. Drug release data of the liposome formulations under the action of Colo205 sPLA2 at (A) 100 ng / mL and (B) 25 ng / mL are expressed as mean ± standard deviation, and each experiment was measured three times. Abbreviations: DPPG25, DPPC:DPPG:DSPE-PEG2k (70:25:5 mol%); LCA-PG (10), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:25:10:5); LCA-PG (15), HSPC:DPPC:LCA-PG:DSPE-PEG2k (60:20:15:5).

[0152] Figure 10 LCA-PG liposomes showed sPLA2-dependent anti-HT-29 cell proliferation effects. (A) Inhibitory effects of oxaliplatin, and drug-loaded liposomes DPPG25, LCA-PG(10) and LCA-PG(15) on HT-29 cells that do not secrete sPLA2, with and without Colo205 sPLA2 (75 ng / mL); (B) Inhibitory effects of blank liposomes DPPG25, LCA-PG(10) and LCA-PG(15) on HT-29 cells that do not secrete sPLA2, with and without Colo205 sPLA2 (75 ng / mL); The cytotoxicity of blank liposomes under enzymatic action may be due to lysophospholipids and LCA generated by enzymatic degradation of phospholipids; Data are presented as mean ± standard deviation, and each experiment was performed three times.

[0153] Figure 11 .Diocytosis assay of DiO-labeled liposomes. Flow cytometry showed the fluorescence of (A) Colo205 or (B) HT-29 cells after co-incubation with DiO-labeled liposomes; the efficiency of internalization of (C) Colo205 or (D) HT-29 cells by DiO-labeled liposomes was further quantified and compared; measurements were taken after 2 h of co-incubation with DiO-labeled liposomes (3 μM DiO); data are expressed as mean ± standard deviation, and each assay was performed three times. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data were calculated using one-way ANOVA.

[0154] Figure 12 In vivo anticancer activity of LCA-PG-phospholipid cryptic liposomes in a female nude mouse model of subcutaneous Colo205 tumor formation. The dosing regimen was as follows: administration began on day 13 of subcutaneous tumor formation, administered every 4 days for a total of 3 doses (4q4d), at a concentration of 5 mg / kg. The dosing dates are shown by the arrows in Figure B. (A) Tumor size growth curve (n=9 mice / group), (B) Nude mouse body weight change curve (n=9 mice / group), (C) Nude mouse survival curve (n=9 mice / group). Data were analyzed using the log-rank (Mantel-Cox) test. **p<0.01, ****p<0.0001, and ns showed no significant difference. Detailed Implementation

[0155] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0156] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0157] Terminology Explanation

[0158] Dipalmitoylphosphatidylcholine (DPPC)

[0159] Distearate phosphatidylcholine (DSPC)

[0160] Dipalmitoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2k)

[0161] Distearatelphosphatidylglycerol (DSPG)

[0162] Dipalmitoylphosphatidylglycerol (DPPG)

[0163] Hydrogenated soybean lecithin (HSPC)

[0164] Raw materials and instruments

[0165] Oxaliplatin was purchased from Shanghai Bid Technology Co., Ltd.; Dipalmitoylphosphatidylcholine (DPPC), distearylphosphatidylcholine (DSPC), distearylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG2k), distearylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylglycerol (DPPG), 18:0 Lyso PC, 18:0 Lyso PG, and 16:0 Lyso PC were purchased from Avanti Polar. Lipids, Inc.; phosphate standard solution and fluorescein were purchased from Sigma-Aldrich Shanghai Trading Co., Ltd.; lithocholic acid and tris(hydroxymethyl)aminomethane (>99%) were purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; human tears sPLA2 were obtained from a classmate's tears; sPLA2 secreted by Colo205 cells were obtained from Colo205 cell culture medium; bee venom type sPLA2-IIA enzyme and sPLA2 (human type IIA) ELISA kit were purchased from Cayman's distributor, Shenzhen Xinbosheng Biotechnology Co., Ltd.; HT-29 cells were purchased from McCoy's. 5A (cat:CM2002) medium and Colo205 tumor cells were purchased from Guangzhou Saiku Biotechnology Co., Ltd.; penicillin / antibiotic (15140122, Gibco) and fetal bovine serum (FBS, 10099141C, Gibco) were purchased from Life Sciences Co., Ltd.; RPMI-1640 medium (C11875500BT) was purchased from Thermo Fisher Scientific Co., Ltd.; MTT was purchased from VWR; and laboratory animals were purchased from Guangdong Provincial Animal Experiment Center. Other reagents, unless otherwise specified, were purchased from suppliers and used directly without further processing.

[0166] Microplate reader (Bio Tek or Synergy Neo2 Bio Tek, Vermont, USA); Laser particle size analyzer (Microtrac Nanotrac Wave II, Microtrac Inc., USA); Liposome extractor (LiposoFast-Basic, Ottawa, Canada or MD25-100,000, Esquin, USA); Centrifuge (Thermo Fisher, NY, USA); Vortex mixer (Vortex, QL-901); Differential scanning calorimeter (DSC 214 Polyma, Netzsch, Germany); Cryogenic sample preparation equipment (Vitrobot Mark IV, Thermo Fisher Scientific); Cryo-transmission electron microscope (300kV TitanKrios, Thermo Fisher Scientific).

[0167] Example 1

[0168] Synthesis of compound 1 (LCA-PC): 1-stearoyl-2-lithocholic acid-sn-glycerol-3-phosphocholine.

[0169]

[0170] Synthesis of Compound 13

[0171] Compound 12 (lithocholic acid, 500 mg, 1.33 mmol) and triethylamine (404 mg, 3.99 mmol) were dissolved in 10 mL of anhydrous tetrahydrofuran. Tert-butyldimethylsilyltrifluoromethanesulfonate (TBSOTf, 703 mg, 2.66 mmol) was added dropwise under ice bath conditions. After the addition was complete, the reaction temperature was allowed to rise naturally to room temperature. The reaction progress was monitored by TLC. After the reactants had reacted completely, approximately 3-6 hours later, the reaction was stopped. The reaction solution was evaporated to dryness, redissolved in 100 mL of ethyl acetate, and the organic phase was washed three times with water and once with saturated brine. The solution was then dried over anhydrous sodium sulfate. The organic phase was evaporated to dryness using a rotary evaporator under reduced pressure. The crude product was purified by silica gel column chromatography (silica gel, 200-300 mesh, petroleum ether:ethyl acetate = 5:1) to give compound 13 (640 mg, 98%). TLC: R f =0.24 (petroleum ether: ethyl acetate = 5:1). 1H NMR (500MHz, CDCl3) δ (ppm): 3.57 (tt, J=10.9, 4.6Hz, 1H, 3β-H), 2.40 (ddd, J=15.5, 10.3, 5.1Hz, 1H,23-H),2.25(ddd,J=15.9,9.8,6.4Hz,1H,23-H),1.93(dt,J=12.3,3.2Hz,1H,5-H),1.89-1.77 (m,6H,4-H,7-H,22-H),1.59-1.02(m,19H,1-H,2-H,6-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H, 17-H,20-H),0.93-0.88(m,15H,19-H,21-H,C(CH3)3),0.63(s,3H,18-H),0.06(s,6H,Si(CH3)2).

[0172] Synthesis of Compound 14

[0173] Compound 13 (294 mg, 0.60 mmol) was dissolved in anhydrous chloroform. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 328 mg, 1.71 mmol) was added under ice bath conditions. After reacting for 2 hours, 4-dimethylaminopyridine (DMAP, 209 mg, 1.71 mmol) was added under ice bath conditions. After reacting for 10 minutes, 18:0 lyso-PC (298 mg, 0.57 mmol) was added under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature. After reacting for 48 hours, the reaction mixture was evaporated to dryness. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 5:1, dichloromethane:methanol:water = 100:25:4) to give compound 14 (500 mg, 88%). TLC (dichloromethane:methanol:water = 100:25:4): R f =0.75. 1 H NMR (400MHz, CDCl3) δ (ppm): 5.19 (s, 1H, OCH), 4.46-3.68 (m, 8H, CH2PO4CH2CH2N, COOC H2 CH),3.57(dq,J=10.4,5.2,4.5Hz,1H,3β-H),3.32(s,9H,N(CH3)3),2.39-2.15(m,4H,COOC H2CH2,23-H),1.94(d,J=11.0Hz,1H,5-H),1.78-1.07(m,55H,1-H,2-H,4-H ,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15 ),0.89(d,J=4.1Hz,18H,19-H,21-H,C(CH3)3,CH2C H3 ),0.63(s,3H,18-H),0.05(s,6H,Si(CH3)2).

[0174] Synthesis of Compound 1

[0175] Compound 14 (632 mg, 0.62 mmol) and p-toluenesulfonic acid (53 mg, 0.31 mmol) were dissolved in anhydrous methanol:anhydrous tetrahydrofuran = 2:1 solution and reacted at room temperature for 10 hours. The reaction solution was then evaporated. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 5:1, dichloromethane:methanol:water = 100:25:4) to give compound 1 (455 mg, 87%). TLC (dichloromethane:methanol = 10:1): R f =0.17. 1 H NMR (400MHz, CDCl3) δ (ppm): 5.18 (tq, J=5.7, 2.8Hz, 1H, OCH), 4.46-3.68 (m, 8H, CH2PO4CH2CH2N, COOC H2 CH),3.60(ddd,J=15.6,10.1,4.1Hz,1H,3β-H),3.35(s,9H,N(CH3)3),2.40-2.09(m,4H,COOC H2 CH2,23-H),2.02-1.89(m,1H,5-H),1.81-1.04(m,55H,1-H,2-H,4-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15 ),0.93-0.83(m,9H,19-H,21-H,CH2C H3 ),0.63(s,3H,18-H). ESI-Ultra-LCMS:calculated for C 50 H 92 NO9P + ([M+H)) + )at 882.65,found at 882.45.

[0176] Example 2

[0177] Synthesis of compound 2 (LCA-PG): 1-stearoyl-2-lithocholic acid-sn-glycerol-3-phospho-(1'-rac-glycerol)).

[0178]

[0179] Synthesis of Compound 16

[0180] Compound 15 (18:0 Lyso PG, 212 mg, 0.397 mmol) and 2,2-dimethoxypropane (124 mg, 1.191 mmol) were dissolved in anhydrous acetone:anhydrous chloroform = 2:1. Trifluoroacetic acid (TFA, 22 mg, 0.199 mmol) was added, and the mixture was reacted at 45 °C for 16 hours. After the reaction was complete, the mixture was evaporated to dryness under reduced pressure to ensure complete removal of trifluoroacetic acid. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 8:1, dichloromethane:methanol = 3:1) to give compound 16 (210 mg, 96%). TLC (dichloromethane:methanol = 10:1): R f =0.33. 1 H NMR (500MHz, CDCl3) δ (ppm): 4.32 (br, J=5.9Hz, 1H, OH), 4.27-3.62 (m, 10H, COOCH2CH, CH2PO4CH2CHCH2), 2.33 (t, J=7.6Hz, 2H, COOC H2 CH2),1.60(br,J=7.6Hz,2H,COOCH2C H2 CH2),1.43(s,3H,CCH3),1.36(s,3H,CCH3),1.28(d,J=6.6Hz,28H,(CH2) 14 ),0.90(t,J=6.8Hz,3H,CH2C H3 ESI-HRMS: calculated for C 27 H 52 O9P - ([M] - )at 551.33544, found at 551.33459.

[0181] Synthesis of Compound 17

[0182] Compound 13 (232 mg, 0.472 mmol) was dissolved in 10 mL of anhydrous chloroform. EDCI (226 mg, 1.18 mmol) was added under ice bath conditions. After reacting for 2 hours, DMAP (144 mg, 1.18 mmol) was added under ice bath conditions. After reacting for 10 minutes, compound 16 (217 mg, 0.394 mmol) was added under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature. After reacting for 48 hours, the reaction mixture was evaporated to dryness. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 15:1, dichloromethane:methanol:water = 100:25:4), followed by purification using a Sephadex LH-20 gel column with dichloromethane / methanol (1:1) as the eluent, yielding compound 17 (382 mg, 95%). TLC (dichloromethane:methanol:water = 100:25:4): R f =0.77. 1 H NMR (500MHz, CDCl3) δ (ppm): 5.26 (s, 1H, OCH), 4.41-3.82 (m, 9H, COOC H2 CH,CH2PO4CH2CHCH2),3.57(tt,J=10.5,4.5Hz,1H,3β-H),2.36-2.20(m,4H,23-H,COOC H2 CH2),1.93(d,J=11.6Hz,1H,5-H),1.88-1.67(m,6H,4-H,7-H,22-H),1.62-0.99 (m,55H,1-H,2-H,6-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,(CH2) 15 ,CCH3,CCH3),0.89(d,J=3.8Hz,18H,19-H,21-H,CH2C H3 ,C(CH3)3),0.62(s,3H,18-H),0.05(s,6H,Si(CH3)2). ESI-HRMS:calculated for C 57 H 104 O 11 P - Si([M] - )at1023.70910, found at 1023.70721.

[0183] Synthesis of Compound 2

[0184] Compound 17 (202 mg, 0.197 mmol) was dissolved in anhydrous methanol:anhydrous tetrahydrofuran = 2:1. p-Toluenesulfonic acid (17 mg, 0.0985 mmol) was added, and the mixture was reacted at room temperature for 24 hours. The reaction solution was evaporated, and the crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 10:1, dichloromethane:methanol:water = 65:25:4). It was then purified again by a Sephadax LH-20 gel column with dichloromethane:methanol = 1:1 as the eluent, yielding compound 2 (130 mg, 76%). TLC (dichloromethane:methanol = 10:1): R f =0.17. 1 H NMR (500MHz, CDCl3) δ (ppm): 5.23 (s, 1H, OCH), 4.51-3.39 (m, 10H, CH2PO4CH2CHCH2, COOC H2 CH,3β-H),2.39-2.15(m,4H,23-H,COOC H2 CH2),1.93(dt,J=12.4,3.1Hz,1H,5-H),1.86-1.01(m,55H,1-H,-H,4-H, 6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15 ),1.02-0.83(m,9H,19-H,21-H,CH2C H3 ),0.62(s,3H,18-H). ESI-HRMS:calculated for C 48 H 86 O 11 P - ([M] - )at 869.59023, found at 869.58972.

[0185] Example 3

[0186] Synthesis of compound 3 (kLCA-PC): 1-stearoyl-2-3'-ketolithocholic acid-sn-glycerol-3-phosphocholine.

[0187]

[0188] Synthesis of Compound 18

[0189] Lithocholic acid (800 mg, 2.12 mmol) was dissolved in 15 mL of acetone. Jones' reagent (0.811 mL, concentration: 2.67 mol / L) was added under ice bath conditions. The reaction was stopped after 15 min, and the Jones' reagent was removed by filtration through a short silica gel column. The filtrate was evaporated to dryness to obtain the crude product. The crude product was purified by silica gel column chromatography using petroleum ether:ethyl acetate = 4:1 as the eluent to give compound 18 (685 mg, 85.7%). TLC (petroleum ether:ethyl acetate = 4:1): R f =0.24. 1 H NMR (500MHz, CDCl3) δ (ppm): 2.43-2.02 (m, 6H, 2-H, 4-H, 23-H), 1.91-1.07 (m, 22H, 1-H, 5-H, 6-H, 7-H, 8-H, 9-H, 11 -H,12-H,14-H,15-H,16-H,17-H,20-H,22-H),1.01(s,3H,19-H),0.93(d,J=6.6Hz,3H,21-H),0.68(s,3H,18-H).

[0190] Synthesis of Compound 3

[0191] Compound 18 (200 mg, 0.53 mmol) was dissolved in anhydrous chloroform. EDCI (290 mg, 1.52 mmol) was added under ice bath conditions, and the reaction proceeded for 4 hours. DMAP (185 mg, 1.52 mmol) was then added under ice bath conditions, and the reaction proceeded for 10 minutes. Finally, 18:0 lyso-PC (265.61 mg, 0.53 mmol) was added under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature, and the reaction proceeded for 48 hours. The reaction mixture was then evaporated to dryness. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol:water = 65:25:4), followed by a second purification using a Sephadex LH-20 gel column with dichloromethane:methanol = 1:1 as the eluent, yielding compound 3 (294 mg, 65%). TLC (dichloromethane:methanol = 10:1): R f =0.21. 1 H NMR (500MHz, CDCl3) δ (ppm): 5.18 (tt, J=6.4, 3.0Hz, 1H, OCH), 4.28-3.85 (d, J=7.2Hz, 6H, CH2PO4CH2, COOC H2 CH),3.81-3.74(m,2H,NCH2),3.34(d,J=6.8Hz,9H,N(CH3)3),2.34-2.00(m,8H,2-H,4-H,23-H,COOC H2CH2),1.90-1.22(m,52H,1-H,5-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15 ),1.00(s,3H,CH2C H3 ),0.92-0.85(m,6H,19-H,21-H),0.67(s,3H,18-H). ESI-Ultra-LCMS:calculated for C 50 H 90 NO9P - [M+H] + at 880.6, found at 880.3.

[0192] Example 4

[0193] Synthesis of compound 4 (kLCA-PG): 1-stearoyl-2-3'-ketolithocholic acid-sn-glycerol-3-phospho-(1'-rac-glycerol)).

[0194]

[0195] Synthesis of Compound 19

[0196] Compound 18 (178 mg, 0.472 mmol) was dissolved in 10 mL of anhydrous chloroform. EDCI (260 mg, 1.36 mmol) was added under ice bath conditions. After reacting for 4 hours, DMAP (166 mg, 1.36 mmol) was added under ice bath conditions. After reacting for 10 minutes, compound 16 (250 mg, 0.453 mmol) was added under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature. After reacting for 48 hours, the reaction mixture was evaporated to dryness. The crude product was purified once using a Sephadax LH-20 gel column with dichloromethane:methanol as the eluent to give compound 19 (214 mg, 52%). TLC (dichloromethane:methanol = 7:1): R f =0.59. 1 H NMR (500MHz, CDCl3) δ (ppm): 5.26-5.16 (m, 1H, OCH), 4.43-3.78 (m, 9H, CH2PO4CH2CHCH2, COOC H2 CH),2.40-2.10(m,8H,2-H,4-H,23-H,COOC H2CH2),1.91-1.19(m,58H,1-H,5-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15, CCH3,CCH3),1.00(s,3H,19-H),0.93-0.83(m,6H,21-H,CH2C H3 ),0.66(s,3H,18-H).

[0197] Synthesis of Compound 4

[0198] Compound 19 (100 mg, 0.112 mmol) was dissolved in anhydrous methanol:anhydrous tetrahydrofuran = 2:1. p-Toluenesulfonic acid (16 mg, 0.0985 mmol) was added, and the reaction was carried out at room temperature for 10 hours. All solvents were evaporated, and the crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 10:1) to give compound 4 (61 mg, 60%). TLC (dichloromethane:methanol = 8:1): R f =0.35. 1 H NMR (500MHz, CDCl3) δ (ppm): 5.22 (s, 1H, OCH), 4.50-3.44 (m, 9H, CH2PO4CH2CHCH2, COOC H2 CH),2.26(qdd,J=41.4,33.4,29.2,13.6Hz,4H,23-H,COOC H2 CH2),2.08-1.02(m,56H,1-H,2-H,4-H,5-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 15 ,),1.02-0.83(m,9H,19-H,21-H,CH2C H3 ),0.65(d,J=21.1Hz,3H,18-H). ESI-HRMS:calculated for C 48 H 84 O 11 P - ([M] - )at 867.57457, found at 867.57410.

[0199] Example 5

[0200] Synthesis of compound 5 (CDCA-PC): 1-stearoyl-2-chenodeoxycholic acid-sn-glycerol-3-phosphocholine.

[0201]

[0202] Synthesis of Compound 21

[0203] Compound 20 (300 mg, 0.76 mmol) was dissolved in THF (6 mL) under nitrogen protection. Triethylamine (318 μL, 2.29 mmol) and TBSOTf (527 μL, 2.29 mmol) were added sequentially at 0 °C. The mixture was stirred in an ice bath for 15 min, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The reaction mixture was extracted with ethyl acetate / water three times. The organic phase was collected, washed twice with saturated brine, and dried over Na₂SO₄. The crude product was purified by silica gel column chromatography (PE / EA, 10:1–3:1) with a gradient elution to give a white solid 21 (259 mg, 67%). 1 H NMR(500MHz, CDCl3)δ3.83(q,J=3.1Hz,1H),3.43(tt,J=10.9,4.5Hz,1H),2.29–2.15(m,2H ), 2.00–1.73 (m, 7H), 1.55–1.17 (m, 19H), 0.88 (d, J = 3.2Hz, 15H), 0.65 (s, 3H), 0.04 (s, 6H).

[0204] Synthesis of Compound 22

[0205] Compound 21 (340 mg, 0.67 mmol) was dissolved in CHCl3 (6 mL). 1-Ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (256 mg, 1.34 mmol) was added at 0 °C, and the mixture was stirred in an ice bath for 30 min. Then, 4-(dimethylamino)pyridine (DMAP) (164 mg, 1.34 mmol) was added in an ice bath, and the mixture was stirred for 5 min. Next, 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphocholine (18:0 Lyso PC) (234 mg, 0.45 mmol) was added in an ice bath. After the reaction temperature was raised to room temperature, the reaction was continued for 53 h. The reaction was then stopped, and the reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by Sephadax LH-20 gel column chromatography (CH2Cl2 / MeOH, 1:1), followed by gradient elution by silica gel column chromatography (CH2Cl2 / MeOH, 5:1, CH2Cl2 / MeOH / H2O, 100:25:4) to give a colorless oil 23 (225 mg, 33%). 1 H NMR (500MHz, CDCl3) δ5.25–5.11(m,1H),4.47–3.71(m,10H),3.34(s,9H),2.26(tdt,J=31 .0,24.2,10.2Hz,5H),1.79–1.17(m,54H),0.92–0.84(m,18H),0.64(s,3H),0.06(s,6H).

[0206] Synthesis of Compound 5

[0207] Compound 22 (300 mg, 0.30 mmol) was dissolved in a MeOH / THF (6 mL, 2:1, V / V) mixed solvent, and p-toluenesulfonic acid (41 mg, 0.24 mmol) was added. The reaction was carried out at room temperature for 12 h, and the reaction solution was evaporated to dryness under reduced pressure. The solution was purified by Sephadex LH-20 gel column chromatography (CH2Cl2 / MeOH, 1:1) and silica gel column chromatography (CH2Cl2 / MeOH / H2O, 100:25:4) to give a white solid 5 (170 mg, 64%). 1 H NMR(500MHz, CDCl3)δ5.20(t,J=5.5Hz,1H),4.38–3.69(m,10H),3.32(s,9H),2.37 –2.20(m,4H),1.77–1.17(m,56H),0.95–0.86(m,9H),0.64(s,3H).LRMScalculated for C 50 H 92 NO 10 P[M+H] + 898.5, found at 898.6.

[0208] Example 6

[0209] Synthesis of compound 6 (7kCDCA-PC): 1-stearoyl-2-7'-ketochenodeoxycholic acid-sn-glycerol-3-phosphocholine.

[0210]

[0211] Synthesis of Compound 24

[0212] Compound 23 (3α-hydroxy-7-oxo-5β-cholic acid, 500 mg, 1.28 mmol) was dissolved in THF (8 mL) under nitrogen protection. Triethylamine (530 μL, 3.84 mmol) and TBSOTf (350 μL, 1.54 mmol) were added sequentially at 0 °C. The mixture was stirred in an ice bath for 15 min, then the ice bath was removed, and the reaction was allowed to proceed at room temperature for 3 h. The reaction mixture was then evaporated to dryness under reduced pressure. The reaction mixture was extracted with ethyl acetate / water three times. The organic phase was collected, washed twice with saturated brine, and dried over Na₂SO₄. The crude product was purified by silica gel column chromatography (PE / EA, 2:1) to give a white solid 24 (585 mg, 91%). 1 H NMR(500MHz, CDCl3)δ3.54(tt,J=10.9,4.8Hz,1H),2.86–2.75(m,1H),2.44–2.31(m,2H),2.30–2.15(m,2H),1.99 –1.75(m,7H),1.60–1.52(m,2H),1.46–1.22(m,10H),1.16(s,3H),0.98–0.80(m,15H),0.64(s,3H),0.02(s,6H).

[0213] Synthesis of Compound 25

[0214] Compound 24 (234 mg, 0.46 mmol) was dissolved in CHCl3 (6 mL). 1-Ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (178 mg, 0.93 mmol) was added at 0 °C, and the mixture was stirred in an ice bath for 30 min. Then, 4-(dimethylamino)pyridine (DMAP) (84 mg, 0.69 mmol) was added in an ice bath, and the mixture was stirred for 5 min. Next, 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphocholine (18:0 Lyso PC) (113 mg, 0.92 mmol) was added in an ice bath. After the reaction temperature was raised to room temperature, the reaction was continued for 48 h. The reaction was then stopped, and the reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by Sephadex LH-20 gel column chromatography (CH2Cl2 / MeOH, 1:1), followed by gradient elution by silica gel column chromatography (CH2Cl2 / MeOH, 5:1, CH2Cl2 / MeOH / H2O, 100:25:4) to give a colorless oil 25 (118 mg, 18%). 1 H NMR (500MHz, CDCl3) δ5.17 (qd, J=6.2, 4.0, 2.9Hz, 1H), 4.37 (ddd, J=12.3, 5.5, 2.9Hz, 1H), 4.29 (d, J=6.9Hz,2H),4.11(dd,J=12.1,7.2Hz,1H),3.92(dtt,J=17.6,10.9,6.1Hz,2H),3.75(dd,J=5.8,3. 2Hz,2H),3.54(tt,J=10.5,4.7Hz,1H),3.32(s,9H),3.16(qd,J=7.2,5.2Hz,1H),2.41–2.20(m,7H) ,1.86–1.73(m,5H),1.48–1.19(m,45H),1.17(s,3H),0.92–0.83(m,15H),0.63(s,3H),0.02(s,6H).

[0215] Synthesis of Compound 6

[0216] Compound 25 (500 mg, 0.49 mmol) was dissolved in a MeOH / THF (8 mL, 2:1, V / V) mixed solvent, and p-toluenesulfonic acid (68 mg, 0.40 mmol) was added. The reaction was carried out at room temperature for 4 h, and the reaction solution was evaporated to dryness under reduced pressure. The solution was purified by Sephadex LH-20 gel column chromatography (CH2Cl2 / MeOH, 1:1) and silica gel column chromatography (CH2Cl2 / MeOH / H2O, 100:25:4) to give a white solid 6 (373 mg, 84%). 1H NMR (500MHz, CDCl3) δ5.19(s,1H),4.33(d,J=34.5Hz,4H),4.13(t,J=9.4Hz,2H),3.57–3.51(m,1H),3.31(s,9H),3.14(d,J= 8.6Hz,1H),2.85(d,J=16.7Hz,2H),2.46–2.15(m,7H),1.52–1.20(m,51H),0.95–0.85(m,9H),0.64(s,3H).LRMScalculated for C 50 H 90 NO 10 P[M+H] + 896.4, found at 896.5.

[0217] Example 7

[0218] Synthesis of compound 7 (bkCDCA-PC): 1-stearoyl-2-3',7'-biketochenodeoxycholic acid-sn-glycerol-3-phosphocholine.

[0219]

[0220] Synthesis of Compound 26

[0221] Compound 23 (3α-hydroxy-7-oxo-5β-cholic acid, 300 mg, 0.77 mmol) was dissolved in acetone (6 mL), and Jones' reagent (293 μL, 76.47 mmol) was added at 0 °C. The mixture was stirred in an ice bath for 10 min, and the reaction was stopped. The reaction mixture was then evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (PE / EA, 2:1) to give a white solid 26 (188 mg, 63%). 1 H NMR(500MHz, CDCl3) δ2.49(t,J=11.3Hz,1H),2.43–2.32(m,2H),2.28(ddq,J=12.7,6.2,3.6,3.1Hz,3H),2.07(ddt,J=16.6,13.0,3.3Hz,3 H),1.98–1.80(m,7H),1.65–1.45(m,10H),1.42(d,J=2.2Hz,1H),0.94(d,J=6.5Hz,3H),0.88(s,3H),0.69(s,3H).HRMS(ESI):calculated for C 24 H36 NO4[M+H] + 389.26853, found at 389.26867.

[0222] Synthesis of Compound 7

[0223] Compound 26 (133 mg, 0.34 mmol) was dissolved in CHCl3 (5 mL). 1-Ethyl-3-(3-dimethylpropylamine)carbodiimide (EDCI) (131 mg, 0.68 mmol) was added at 0 °C, and the mixture was stirred in an ice bath for 30 min. Then, 4-(dimethylamino)pyridine (DMAP) (84 mg, 0.69 mmol) was added in an ice bath, and the mixture was stirred for 5 min. Next, 1-stearoyl-2-hydroxy-sn-glycerol-3-phosphocholine (18:0 Lyso PC) (120 mg, 0.23 mmol) was added in an ice bath. After the reaction temperature was raised to room temperature, the reaction was continued for 48 h. The reaction was then stopped, and the reaction mixture was evaporated to dryness under reduced pressure. The crude product was purified by Sephadax LH-20 gel column chromatography (CH2Cl2 / MeOH, 1:1), followed by gradient elution by silica gel column chromatography (CH2Cl2 / MeOH, 5:1, CH2Cl2 / MeOH / H2O, 100:25:4) to give a colorless oil 7 (43 mg, 14%). 1 H NMR (500MHz, CDCl3) δ5.18 (qt, J=5.5, 3.7, 2.9Hz, 1H), 4.37 (dt, J=12.0, 3.0Hz, 1H), 4.2 7(d,J=7.7Hz,2H),4.12(ddd,J=10.9,7.6,3.2Hz,1H),3.92(h,J=5.3Hz,2H),3.72–3.67( m,2H),3.27(s,9H),2.33–2.22(m,5H),1.98–1.19(m,53H),1.12(dt,J=10.3,8.4Hz,3H) ,0.92(dd,J=6.5,2.8Hz,3H),0.87(t,J=6.9Hz,3H),0.69(s,3H).HRMS(ESI):calculated for C 50 H 88 NO 10 P[M+H] + 894.62204, found at 894.62341.

[0224] Example 8

[0225] Synthesis of compound 8 (LCA-PEG2k): 1-stearoyl-2-lithocholic acid-sn-glycerol-3-phosphoethanolamine-N-methoxy(polyethylene glycol)-2000.

[0226]

[0227] Synthesis of Compound 29

[0228] Compound 27 (polyethylene glycol monomethyl ether 2000, 700 mg, 2000 g / mol, 0.350 mmol) and triethylamine (71 mg, 101.19 g / mol, 0.700 mmol) were dissolved in anhydrous acetonitrile, and compound 28 (di(N-succinimide) carbonate, 135 mg, 256.17 g / mol, 0.525 mmol) was added. The reaction was carried out at room temperature for 24 h. After the reaction was completed, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by Sephadex LH-20 gel column chromatography with dichloromethane / methanol (1:1, v / v) as the eluent to give 689 mg of compound 29, with a yield of 92%. 1 H NMR (400MHz, CDCl3) δ (ppm): 4.47 (dq, J=4.6, 2.3Hz, 2H, COOCH2), 3.65 (d, J=2.0Hz, 184H, (OCH2CH2O) 44-46 ,3.39(s,3H,OCH3),2.85(d,J=1.8Hz,4H,CO(CH2)2CO).

[0229] Synthesis of Compound 31

[0230] Compound 30 (18:0 lyso-PE, 200 mg, 481.60 g / mol, 0.415 mmol) and triethylamine (218 mg, 101.19 g / mol, 2.159 mmol) were dissolved in 15 mL of chloroform / methanol / water (40:20:1.5, v / v / v). Benzyl chloroformate (276 mg, 170.59 g / mol, 1.62 mmol) was added at 0 °C, and the reaction was allowed to proceed to room temperature for 2 h. The solvent was removed by vacuum distillation to obtain the crude product, which was then purified by silica gel column chromatography using dichloromethane / methanol (15:1, v / v) as the eluent, yielding 245 mg of compound 31 in 97% yield. 1H NMR(400MHz,DMSO)δ(ppm):7.46–7.18(m,5H,Ph),5.86(s,1H,NH),4.92(s,2H,CH2Ph),3.96–3.78(m,2H,COOCH2),3.78–3.37(m,5H,CH2PO4CH2,CHOH),3. 08(d,J=5.8Hz,2H,NHCH2),2.19(t,J=7.4Hz,2H,COOCH2),1.41(d,J=7.1Hz,2H,COOCH2CH2),1.15(d,J=1.7Hz,28H,(CH2)14),0.83–0.70(m,3H,CH3CH2).

[0231] Synthesis of Compound 32

[0232] Compound 13 (286 mg, 490.80 g / ml, 0.583 mmol) and compound 31 (342 mg, 615.73 g / ml, 0.555 mmol) were dissolved in 12 ml of anhydrous chloroform. EDCl (379 mg, 191.70 g / ml, 1.982 mmol) and DMAP (203 mg, 122.17 g / mol, 1.665 mmol) were added under ice bath conditions. The reaction temperature was raised to room temperature, and after 24 h, the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using gradient elution with petroleum ether / ethyl acetate (1:1, v / v) and dichloromethane / methanol (12:1, v / v), respectively, to give 530 mg of compound 32, with a yield of 97%. 1 H NMR (400MHz, CDCl3) δ (ppm): 7.29 (d, J = 6.4Hz, 5H), 6.15 (s, 1H), 5.22 (s, 1H), 5.09 (d, J = 11.0Hz, 2H), 4.43–3.85 (m, 6H), 3.57 (tt, J = 10.5, 4.6Hz, 1H),3.49–3.15(m,2H),2.22(s,4H),1.95–1.15(m,56H,1-H,2-H,4-H,5- H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH 2)14 ,COOCH2CH2),0.89(d,J=4.1Hz,18H,m,9H,19-H,21-H,C(CH3)3),0.90–0.83(m ,18H,19-H,21-H,C(CH3)3,CH2CH3),0.63(s,3H,18-H),0.05(s,6H,Si(CH3)2).

[0233] Synthesis of Compound 33

[0234] Compound 32 (500 mg, 1088.53 g / mol, 0.459 mmol) was dissolved in 10 mL of tetrahydrofuran, and ammonium formate (434 mg, 63.06 g / mol, 6.885 mmol) dissolved in 1.5 mL of water was added. Then, 130 mg of Pd / C was added, and the reaction was carried out at room temperature for 8 h. The solid residue was removed by filtration, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography using gradient elution with petroleum ether / ethyl acetate (1:1, v / v), dichloromethane / methanol / water (120:10:1, v / v / v), and dichloromethane / methanol / water (100:25:4, v / v / v), respectively, to obtain 357 mg of compound 33, with a yield of 82%. 1 H NMR (400MHz, CDCl3) δ (ppm): 5.21 (t, J=5.2Hz, 1H, OCH), 4.51–3.83 (m, 6H, CH2PO4CH2, COOCH2), 3.58 (dt, J=11.0, 6.0Hz, 1H, 3β-H), 3.17 (s, 2H, NH2CH2), 2.29(t,J=7.8Hz,6H,23-H,COOCH2,NH2),1.97–1.18(m,56H,1-H,2-H,4-H,5 -H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 14 ,COOCH2CH2),0.89(d,J=4.1Hz,18H,19-H,21-H,C(CH3)3,CH2CH3),0.63(s,3H,18-H),0.05(s,6H,Si(CH3)2).

[0235] Synthesis of Compound 34

[0236] Compound 33 (500 mg, 954.39 g / mol, 0.524 mmol) and compound 29 (1.178 g, 2141.09 g / mol, 0.550 mmol) were dissolved in 15 mL of chloroform, and then triethylamine (159 mg, 101.19 g / mol, 1.572 mmol) was added. The mixture was reacted at room temperature for 24 h. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by Sephadax LH-20 gel column chromatography with dichloromethane / methanol (1:1, v / v) as the eluent to give 1.35 g of compound 34, with a yield of 71%. 1H NMR (400MHz, CDCl3) δ (ppm): 5.20 (s, 1H, OCH), 4.42–4.12 (m, 6H, CH2PO4CH2, COOCH2), 3.64 (s, 191H, 3β-H, (OCH2CH2O) 44-46 ,OCH3,NHCH2),2.28(t,J=7.8Hz,4H,23-H,COOCH2),1.93(d,J=11.0Hz,1H,5-H),1.25(m,5 5H,1-H,2-H,4-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 14 ,COOCH2CH2),0.88(d,J=4.5Hz,18H,19-H,21-H,C(CH3)3,CH2CH3),0.63(s,3H,18-H),0.05(s,6H,Si(CH3)2).

[0237] Synthesis of Compound 8

[0238] Cerium ammonium nitrate (CAN, 1.48 g, 548.22 g / mol, 0.201 mmol) was dissolved in isopropanol / methanol / tetrahydrofuran / water (1:1:1:0.02, v / v / v) and reacted for four days until the brown color disappeared and the oxidizing property of CAN was eliminated. Compound 34 (1.35 g, 2980 g / ml, 0.45 mmol) was added, and the reaction was carried out at room temperature for 24 h. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by Sephadex LH-20 gel column chromatography with dichloromethane / methanol (1:1, v / v) as the eluent to give 531 mg of compound 8, with a yield of 41%. 1 HNMR (500MHz, CDCl3) δ (ppm): 6.14 (s, 1H, NH), 5.19 (d, J = 5.1Hz, 1H, OCH), 4.38–3.36 (m, 197H, 3β-H, (OCH2CH2O) 44-46 ,OCH3,NHCH2,CH2PO4CH2,COOCH2),2.37–2.18(m,4H,23-H,COOCH2),1.97–1.91(m,1H,5-H),1.89– 1.09(m,55H,1-H,2-H,4-H,6-H,7-H,8-H,9-H,11-H,12-H,14-H,15-H,16-H,17-H,20-H,22-H,(CH2) 14,COOCH2CH2),0.97–0.83(m,9H,19-H,21-H,CH2CH3),0.63(s,3H,18-H).

[0239] Example 9

[0240] Synthesis of compound 9 (biLCA-PC): 1-lithocholic acid-2-lithocholic acid-sn-glycerol-3-phosphocholine.

[0241]

[0242] Synthesis of Compound 36

[0243] Compound 35, glycerol phosphatidylcholine (GPC) (514.5 mg, 2 mmol), was dissolved in 25 mL of methanol. Cadmium chloride (366.7 mg, 2 mmol) was slowly added to the solution in ice water, and the mixture was stirred at 0 °C for 4 h. The mixture was filtered, and the filter cake was dried under infrared radiation to obtain compound 36 (GPC-CdCl2, approximately 700 mg). Compound 36 was directly introduced into the next reaction step without NMR identification.

[0244] Synthesis of Compound 37

[0245] Compound 13 (440 mg, 0.896 mmol, 490.8 g / mol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 230 mg, 1.2 mmol), 4-dimethylaminopyridine (DMAP, 146.6 mg, 1.2 mmol), and compound 36 (GPC-CdCl2, 98.7 mg, 0.224 mmol) were dissolved in anhydrous chloroform under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature, and the reaction mixture was allowed to react for 120 hours. The reaction solution was then evaporated to dryness. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 85:15, dichloromethane:methanol:water = 100:25:4) to give compound 37 (1202.86 g / mol, 281.7 mg, 104.5%). 1 H NMR (400MHz, CDCl3) δ (ppm): 5.19 (s, 1H, OCH), 4.46-3.8 (m, 8H, CH2PO4CH2CH2N, COOC H2 CH),3.6(dq,J=10.4,5.2,4.5Hz,2H,3β-H),3.4(s,9H,N(CH3)3),2.45-2.1(m,4H,COOC H2CH2),1.94-1.0(multiple peaks),0.89(d,J=4.1Hz,31H,19-H,21-H,C(CH3)3),0.63(s,6H,18-H),0.05(s,12H,Si(CH3)2).

[0246] Synthesis of Compound 9

[0247] Compound 37 (40 mg, 1202.86 g / mol, 0.03325 mmol), tetrabutylammonium fluoride (1 M in THF, 261.47 g / mol, 0.698 mL, 0.698 mmol), and glacial acetic acid (60.05 g / mol, 0.698 mmol, 4 μL) were dissolved in 1.0 mL of anhydrous tetrahydrofuran solution and reacted at room temperature for 120 hours. The reaction solution was then evaporated. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 85:15, dichloromethane:methanol:water = 100:25:4) to give compound 9 (20 mg, 974.34 g / mol, 62%). 1 H NMR (400MHz, CDCl3) δ (ppm): 5.25 (s, 1H, OCH), 4.5-3.75 (m, 8H, CH2PO4CH2CH2N, COOC H2 CH),3.6(dq,J=10.4,5.2,4.5Hz,2H,3β-H),3.4(s,9H,N(CH3)3),2.45-2.1(m,4H,COOC H2 CH2), 1.94-1.0 (multiple peaks), 0.89 (21H, 19-H, 21-H), 0.63 (s, 6H, 18-H). ESI-Ultra-LCMS:calculated for C 56 H 97 NO 10 P + ([M+H)) + )at 974.34,found at 974.68.

[0248] Example 10

[0249] Synthesis of compound 10 (bikLCA-PC): 1-3'-ketolithocholic acid-2-3'-ketolithocholic acid-sn-glycerol-3-phosphocholine.

[0250]

[0251] Synthesis of Compound 10

[0252] Compound 18 (340 mg, 0.908 mmol, 374.57 g / mol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 174 mg, 0.908 mmol), 4-dimethylaminopyridine (DMAP, 111 mg, 0.908 mmol), and compound 36 (GPC-CdCl2, 100 mg, 0.227 mmol) were dissolved in anhydrous chloroform under ice bath conditions. The reaction temperature was allowed to rise naturally to room temperature, and the reaction mixture was allowed to react for 120 hours. The reaction solution was then evaporated to dryness. The crude product was purified by silica gel column chromatography (silica gel, 200–300 mesh, dichloromethane:methanol = 85:15, dichloromethane:methanol:water = 100:25:4) to give compound 10 (970.3 g / mol, 50 mg, 22.7%). 1 H NMR (400MHz, CDCl3) δ (ppm): 5.19 (s, 1H, OCH), 4.46-3.8 (m, 8H, CH2PO4CH2CH2N, COOC H2 CH),3.4(s,9H,N(CH3)3),2.45-2.1(m,12H,CH2COCH2,COOC H2 CH2),2.05-1.0(multiple peaks),1.0-0.75(12H,19-H,21-H),0.63(s,6H,18-H). ESI-Ultra-LCMS:calculated for C 56 H 92 NO 10 P + ([M+H)) + )at970.65,found at 974.65.

[0253] Example 11

[0254] Synthesis of compound 11 (LCAgly-PC): 1-stearoyl-2-Lithocholylglycine-sn-glycerol-3-phosphocholine.

[0255]

[0256] Synthesis of Compound 38

[0257] Glycine benzyl ester hydrochloride (2.420 g, 201.65 g / mol, 12 mmol) was dissolved in approximately 20 mL of dichloromethane. Triethylamine (2 g, 101.19 g / mol, 20 mmol) was added, and the mixture was stirred at room temperature for 30 min. Then, compound 12 (3.770 g, 376.58 g / mol, 10.00 mmol), EDCl (2.8800 g, 191.70 g / mol, 15.00 mmol), and 1-hydroxybenzotriazole (HOBT) were added. 2.030 g (135.12 g / mol, 15.00 mmol) was stirred overnight at room temperature, and the reaction progress was monitored by TLC plate (dichloromethane:methanol = 20:1, v / v). After the reaction was completed, the reaction solution was poured directly into about 100 mL of water, extracted three times with dichloromethane, the organic phases were combined, washed with saturated brine, and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation, and then recrystallized from dichloromethane and petroleum ether to give 4.519 g of white blocky solid compound 38, with a yield of 86.3%. 1 H NMR (400MHz, CDCl3) δ7.43–7.29(m,5H),5.95(s,1H),5.19(s,2H),4.09(d,J=5.1Hz,2H),3.67–3.57(m,1H),0.92(t,J=3.1Hz,6H),0.64(s,3H); 13 C NMR(101MHz, CDCl3)δ173.63(s),170.00(s),135.09(s),128.70–128.22(m),71.81(s) ,67.17(s),56.44(s),55.94(s),42.69(s),42.05(s),41.33(s),40.38(s),40.13(s),3 6.42(s),35.80(s),35.35(d,J=10.8Hz),34.52(s),33.21(s),31.53(s),30.51(s),28 .17(s),27.14(s),26.36(s),24.16(s),23.32(s),20.77(s),18.32(s),12.00(s); HRMS for C 33 H 50 O4N([M+H)) + )Calcd:524.3734,Found:524.3738.

[0258] Synthesis of Compound 39

[0259] Compound 38 (3.150 g, 523.73 g / mol, 6.00 mmol) was dissolved in 25 mL of anhydrous tetrahydrofuran, and 1.80 mL of triethylamine was added. Under nitrogen protection and an ice bath, TBSOTf (2.379 g, 264.33 g / mol, 9 mmol) was injected. After stirring for 10 min, the ice bath was removed, and the mixture was stirred at room temperature for 3 h. The reaction progress was monitored by TLC (petroleum ether: ethyl acetate = 3:1, v / v). After the reaction was completed, the solvent was removed by vacuum distillation, and an appropriate amount of ethyl acetate was added to dissolve the product. The organic phase was then washed twice with water and once with saturated brine. The product was dried over anhydrous sodium sulfate for 2 h, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography, and the product was eluted with eluent (petroleum ether: ethyl acetate = 5:1, v / v) to obtain a pale yellow transparent viscous liquid. After vacuum drying for 2 days, 3.8140 g of white solid compound 39 was obtained, with a yield of 99.7%. 1 H-NMR (400MHz, CDCl3) δ7.42–7.31(m,5H),5.94(t,J=4.8Hz,1H),5.19(s,2H),4 .09(d,J=5.2Hz,2H),3.58(s,1H),0.93–0.88(m,15H),0.63(s,3H),0.06(s,6H); 13 C-NMR(101MHz, CDCl3)δ173.79(s),169.97(s),135.18(s),128.66–128.14(m),72.74(s),67.0 2(s),56.33(s),55.95(s),42.66(s),42.22(s),41.29(s),40.14(d,J=7.3Hz),36.88(s),35.8 0(s),35.46(d,J=14.6Hz),34.52(s),33.09(s),31.55(s),30.99(s),28.17(s),27.25(s),26. 36(s),25.94(s),24.17(s),23.36(s),20.77(s),18.29(d,J=8.9Hz),12.00(s),-4.61(s); HRMS for C 39 H 64 O4NSi([M+H) + )Calcd:638.4599Found:638.4598.

[0260] Synthesis of Compound 40

[0261] Compound 39 (3.4126 g, 5.35 mmol) was dissolved in 30 mL of tetrahydrofuran, 600 mg of Pd / C was added, hydrogen gas was bubbled through, and the mixture was stirred overnight at room temperature. The reaction progress was monitored by TLC (dichloromethane:methanol = 10:1, v / v). After the reaction was complete, Pd / C was removed by filtration, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography, and the solution was eluted with eluent (dichloromethane:methanol = 10:1, v / v) to give 2.784 g of white solid compound 40, with a yield of 95.0%. 1 H-NMR(400MHz, CDCl3)3.97(s,2H),3.58(s,1H),0.93–0.88(m,15H),0.63(s,3H),0.05(s,6H); 13 C-NMR (101MHz, DMSO) δ173.27(s),171.83(s),72.37(s),56.27(d,J=26.5Hz),42.67(s),41.87(s),40 .96(s),40.58(s),40.26(d,J=21.0Hz),39.95(s),39.74(s),39.53(s),39.32(s),36.96(s),35.76(s) ,35.32(d,J=10.9Hz),34.50(s),32.50(s),31.87(s),31.15(s),28.11(s),27.20(s),26.33(d,J=29.4 Hz),26.15–25.96(m),24.23(s),23.59(s),20.86(s),18.69(s),18.14(s),12.28(s),-4.22(s).; HRMS for C 32 H 58 O4NSi([M+H) + )Calcd:548.4130Found:548.4125.

[0262] Preparation of compound 41

[0263] Compound 40 (129.0 mg, 0.235 mmol) was dissolved in 5 mL of chloroform, followed by the addition of compound 3 (lyso-PC, 100.2 mg, 0.191 mmol), EDCl (116.0 mg, 0.605 mmol), and DMAP (70.9 mg, 0.573 mmol). The reaction was stirred at room temperature, and the reaction progress was monitored by TLC (dichloromethane:methanol:water = 100:25:4, v / v / v). After 48 h, the reaction was stopped, and silica gel column chromatography was performed. The sample was first washed with eluent (dichloromethane:methanol = 5:1, v / v) to remove impurities. DMAP was removed, and unreacted TBS-glycine benzyl ester lithocholic acid was washed with eluent (dichloromethane:methanol = 5:2, v / v). Finally, the product and some water-soluble impurities were washed with eluent (dichloromethane:methanol:water = 100:25:4, v / v / v) to obtain the product. The solvent was removed by vacuum distillation to obtain the crude product. An appropriate amount of water was added to the crude product, and it was extracted with eluent (dichloromethane:methanol = 2:1, v / v) until the aqueous phase was clear. The organic phase was collected, and the solvent was removed by vacuum distillation. The product was dried under vacuum for 3 hours to obtain 90.4 mg of a pale yellow semi-solid compound 41, with a yield of 45.0%. 1 H-NMR (400MHz, CDCl3) δ7.58(s,1H),5.23(s,1H),4.33(d,J=48.1Hz,4H),3.98(d,J=36.1Hz,4H),3.79 (s,2H),3.58(s,1H),3.33(s,9H),2.29(t,J=7.3Hz,2H),0.94–0.85(m,18H),0.63(s,3H),0.05(s,6H); 13C-NMR(101MHz, CDCl3)δ174.25(s),173.52(s),170.03(s),72.73(s),71.87(s),66.13(s),63.61(s),62.34(s),59.24(s), 56.25(d,J=10.8Hz),54.17(s),42.65(s),42.20(s),41.12(s),40.13(s),36.85(s),35.79(s),35.52(s),34.50(s),33.92 (s),32.95(s),31.68(d,J=30.8Hz),30.96(s),29.60(dd,J=9.9,4.0Hz),29.24(t,J=7.9Hz),28.21(s),27.22(s),26.34(s) ),25.88(s),24.76(s),24.16(s),23.31(s),22.60(s),20.74(s),18.28(d,J=12.7Hz),14.03(s),12.00(s),-4.69(s); HRMS forC 58 H 110 O 10 N2PSi([M+H) + )Calcd:1053.7662Found:1053.7662.

[0264] Synthesis of Compound 11

[0265] Compound 41 (50.0 mg, 1054 g / mol, 0.0475 mmol) was dissolved in eluent (isopropanol:methanol:tetrahydrofuran = 1:1:1, v / v / v), and 100 mg of cerium ammonium nitrate (CAN, 548.22 g / mol, 0.182 mmol) was added. Then, one drop of water was added, and the mixture was stirred at room temperature. The reaction progress was monitored by TLC plate and eluent (dichloromethane:methanol:water = 100:25:4, v / v / v). After 4 hours, the reaction was stopped, and an appropriate amount of dichloromethane was added to precipitate CAN and its derivatives. The mixture was filtered, and the filtrate was collected. The solvent was removed by vacuum distillation, and the mixture was subjected to silica gel column chromatography. Small molecule impurities were first washed with eluent (dichloromethane:methanol = 5:2, v / v / v), and then the product was separated with eluent (dichloromethane:methanol:water = 125:25:4, v / v / v) to obtain 8.3 mg of white solid. HRMS forC 52 H 96 O 10 NP([M+H] + )Calcd939.6797 Found:939.6797.

[0266] Example 12 Effect Experiment

[0267] Routine procedures for liposome preparation

[0268] Liposomes were prepared using a thin-film method. In short, phospholipids were first dissolved in chloroform to prepare a phospholipid stock solution. The phospholipid concentration was determined by Bartlett's assay and stored at -20°C for later use. Different phospholipids were precisely measured using a syringe in different proportions (as shown in Table 1) and placed in round-bottom flasks. After evaporating a large amount of organic solvent by rotary evaporation at 45°C under reduced pressure for 15 minutes, the solution was heated to 65°C and rotary dried for 30 minutes to form a thin film of phospholipids on the flask wall. The flasks were then dried overnight in a vacuum drying oven to remove trace amounts of organic solvent. An aqueous solution containing the drug to be encapsulated, at a temperature 15°C higher than the liposome phase transition temperature, was added to the round-bottom flask containing the phospholipid film to hydrate the film. The solution was vortexed every 5 to 10 minutes until the solid completely disappeared. After hydration, the liposomes underwent at least six freeze-thaw cycles using liquid nitrogen and a water bath at a temperature 15°C higher than the liposome phase transition temperature to further stabilize the structure. The resulting liposome suspension is passed through two polycarbonate membranes with a pore size of 100 nm, resulting in a uniform liposome size distribution of approximately 70-110 nm. Unencapsulated free drug molecules are removed through a specific purification process to obtain liposomes with high loading rates. These liposomes are then passed through a 0.22 μm membrane on a clean bench to remove bacteria and prevent them from affecting liposome stability. All prepared liposomes are stored at 4°C and must not be placed in a freezer below 0°C, as freezing will damage the phospholipid membrane and cause drug leakage.

[0269] Preparation of CF liposomes

[0270] Preparation of 100 mM CF solution: CF was dissolved in Tris-HCl buffer (5 mM, pH 7.4), and the solution was sonicated to dissolve the CF. The pH was adjusted to 7.4 using 1 M NaOH solution. After preparing the liposome membrane using standard procedures, the phospholipid membrane was hydrated using the 100 mM CF solution. After freeze-thaw cycles, 1 ml of liposomes was extruded through a polycarbonate bilayer 23 times using a LiposoFast-Basic liposome extruder. The hydration, membrane extrusion, and freeze-thaw cycles were all performed at temperatures 15°C above the liposome phase transition temperature. Free CF was removed using a gel G75 (Pharmacia, GE Healthcare) with Tris-HCl buffer as the mobile phase.

[0271] Preparation of oxaliplatin liposomes

[0272] Preparation of 7 mg / ml oxaliplatin aqueous solution: Oxaliplatin was dissolved in HEPES buffer (10 mM HEPES, 5% glucose, pH 7.4) and stirred at 65°C for 0.5 hours to dissolve. After preparing liposome membranes using standard procedures, the phospholipid membranes were hydrated using 7 mg / ml oxaliplatin aqueous solution. After freeze-thaw cycles, the liposomes were extruded through a bilayer polycarbonate membrane five times using a pneumatic liposome extruder (MD25-100) in the liposome tank chamber to obtain liposomes with uniform particle size. Unencapsulated free oxaliplatin liposomes were removed by dialyzing through a 100 kDa dialysis bag for 48 hours. 2 ml of liposome suspension required 600 ml of HEPES buffer as the dialysis medium. The dialysis medium was changed at least three times to ensure complete dialysis: once after 7 hours, once after 14 hours, and once after 27 hours. After dialysis, the liposomes were sterilized by membrane transfiltration and stored at 4°C for in vitro cytotoxicity tests and in vivo anticancer activity tests in nude mice.

[0273] Determination of liposome particle size distribution

[0274] The particle size distribution of liposomes was determined using a Nanotrac Wave II nanoparticle size analyzer based on dynamic light scattering technology and zeta potential. Specific procedures included: diluting liposomes with water to an appropriate concentration for particle size determination (approximately 30-fold dilution of 40 mg / ml phospholipid); selecting volume mode for particle size distribution in the software; and testing each sample three times independently. The particle size distribution results for the liposomes are shown in Table 1.

[0275] Table 1. Liposome composition and particle size distribution (n=3, mean ± standard deviation)

[0276]

[0277]

[0278] Determination of liposome phase transition temperature

[0279] Liposome phase transition temperatures were determined and analyzed using differential scanning calorimetry (DSC). Specific procedures included: a total sample concentration of 0.6 to 0.8 μg; a lidded aluminum crucible as the reference; a scanning mode of 5 to 8 cycles of heating / cooling between 0 and 70 °C to allow the liposomes to reach thermal equilibrium; and stopping the detection when the liposome phase transition temperature stabilized at a certain value. The temperature change rate was 10 °C / min. Excessive heating (above 15 °C) reduced sensitivity, causing characteristic peaks to drift at higher temperatures and making adjacent peaks less distinct; excessive heating broadened the endothermic peaks. Data were analyzed using Origin 9 software. Different liposome phase transition temperatures are shown below. Figure 4 As shown.

[0280] Analysis of liposome morphology

[0281] Liposome cryopreservation samples were prepared using a Vitrobot Mark IV cryopreservation instrument: 4 μl of liposome suspension with a phospholipid concentration of approximately 40 mg / ml was transferred using a 10 μl pipette and placed on the front side of a 100-well 300-mesh carbon membrane copper mesh (Quantifoil, R1.2 / 1.3) that had undergone hydrophilic treatment. After removing excess liposome suspension with filter paper for 3 seconds, the copper mesh was placed in liquid ethane for rapid freezing of the liposomes. The sample preparation environment was maintained at 10°C and 100% humidity. After cryopreservation, the samples were stored in liquid nitrogen for later analysis. A 300 kV Titan Krios cryo-transmission electron microscope was used, equipped with a direct electron detection camera (FEI Falcon IV detector) and a Gatan BioQuantum energy filter. The transmission electron magnification was ×29000, and the size of one pixel was [missing information]. Liposome morphology analysis results as follows Figure 5 As shown.

[0282] Analysis of liposomal phospholipid concentration

[0283] The concentration of liposomal phospholipids was determined by analyzing phosphoric acid (P) content using the Bartlett assay. In the Bartlett assay, phospholipids in the sample are hydrolyzed by acid into inorganic phosphates. These inorganic phosphates react with the phosphomolybdate ion of phosphomolybdic acid to produce a blue color, and the intensity of this color development is linearly related to the P concentration within a certain range. The absorbance of the test samples was measured using a Synergy Neo2 microplate reader (Bio Tek, Vermont, USA) at a wavelength of 830 nm.

[0284] Determination of oxaliplatin encapsulation efficiency in liposomes

[0285] Free oxaliplatin and liposomes were separated using a G-50 gel column (Pharmacia, GE Healthcare, 12mm × 305mm). 300 μl of liposome suspension was added to the top of the gel column, using HEPES buffer (10 mM HEPES, 5% glucose, pH 7.4) as the eluent. A 50 ml volumetric flask was used to collect the oxaliplatin liposome suspension. After the milky blue liposomes had eluted completely, a 100 ml volumetric flask was used to collect the free oxaliplatin solution. 300 μl of 10% Triton was added to the liposome suspension, and the liposome phospholipid membrane was disrupted in a 65°C water bath to release the oxaliplatin drug. The volume was then adjusted to 50 ml. After the free oxaliplatin had eluted completely, the volume was adjusted to 100 ml, mixed thoroughly, and ready for analysis and quantification. The mass of free oxaliplatin and the mass of oxaliplatin encapsulated in the liposomes were determined. The liposome loading rate was calculated using Formula 2.

[0286]

[0287] W eca W represents the mass of oxaliplatin loaded in liposomes. free. This indicates the mass of free oxaliplatin.

[0288] Detection of oxaliplatin concentration

[0289] Oxaliplatin was quantitatively analyzed using high-performance liquid chromatography-ultraviolet (HPLC-UV) detection. Preparation of standards: Oxaliplatin was dissolved in HEPES buffer to prepare 1 mg / mL solutions. Different volumes were then diluted to 1 mL, resulting in a series of standards with concentrations of 10, 40, 100, 200, 320, and 400 μg / mL. HPLC conditions: Gradient elution with 5% methanol and 95% water for 7 min; C18 reversed-phase column (XBridgex ○4.6×250 mm, 5 μm PN:186003117); flow rate 1 mL / min; injection volume 20 μL. UV detection wavelength was 250 nm.

[0290] Release of oxaliplatin (L-OHP) from liposomes

[0291] After preparing oxaliplatin liposomes, they were diluted with HEPES buffer (pH 7.4). 20 mM CaCl2 and Colo205sPLA2 (if needed) were added to the buffer, resulting in a total phospholipid concentration of 500 μM and a CaCl2 concentration of 1 mM. The samples were incubated in an incubator (Binder, Tuttlingen, Germany) at 37°C for 48 h. At 0, 0.5, 2, 4, 6, 12, 24, and 48 h, 200 μL of the sample was transferred to an ultrafiltration tube (MWCO = 100 kDa) and centrifuged to separate free L-OHP and L-OHP-loaded liposomes. The free L-OHP (Mf) was detected by HPLC-UV. The total L-OHP concentration (Mtol) was determined by adding 10% Triton X-100 (v / v) to the liposome sample. The release of L-OHP from liposomes was calculated using the formula Mf / (Mtol)×100%.

[0292] Collection and concentration measurement of sPLA2 secreted by Colo205 cells

[0293] Colo205 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-drug antibodies at 37°C with 5% CO2 and adequate humidity. Cells were cultured in T-75 cell culture dishes until the total cell density reached 80%-90% in whole culture medium. The medium was then converted to empty culture medium, and after 48 hours of culture, the cell culture was collected in 50ml centrifuge tubes. Cells were removed by ultracentrifugation at 4,000 rpm and 4°C for 10 minutes. The supernatant was transferred to 50ml 10kDa ultrafiltration centrifuge tubes at 4,000 rpm and 4°C until concentrated to a suitable sPLA2 concentration. The concentrate was then transferred to centrifuge tubes and stored at -20°C for later use. The sPLA2 concentration was detected using an sPLA2 (human Type IIa) ELISA kit, with a limit of detection of 15.6 pg / mL. The sPLA2 standard was prepared by diluting the sample 2-fold from 2,000 pg according to the instructions to obtain a series of standard concentrations. After color development using the sPLA2 kit, the absorbance was measured using a microplate reader at 450 nm. The concentration of sPLA2 in the test sample was obtained by standard curve analysis. The concentration of sPLA2 before concentration was 58 ng / mL, and the concentration of sPLA2 after ultrafiltration concentration was 1.75 μg / mL.

[0294] Collection and concentration measurement of sPLA2 in human tears

[0295] Tears secreted during the viewing of touching TV dramas were collected using disposable sterile pipettes and stored at 20°C for later use. The concentration of sPLA2 secreted in human tears was determined to be 147 μg / mL under the above sPLA2 assay conditions.

[0296] Measurement of sPLA2 concentration secreted by Colo205 cells in 96-well plates

[0297] Colo205 cells were cultured in 96-well plates (Corning, NO 3599) at a density of 10,000 cells per well. The cell culture medium was RPMI-1640 containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. Culture conditions were 37°C, 5% CO2, and a controlled humidity environment. After 24 hours of cell attachment, the supernatant was removed, and 100 μl of fresh whole culture medium was added. Cell culture was collected from three wells at 24, 48, and 72 hours to determine the concentration of sPLA2 secreted by Colo205 cells at different time points. Data are expressed as mean ± standard deviation, and each sample was tested three times. Data were processed and analyzed using GraphPadPrism 8 software.

[0298] This study investigated the release of CF from liposomes containing amphiphilic molecule-modified phospholipids under the influence of sPLA2.

[0299] The control group was the one without sPLA2, and the experimental group was the one with sPLA2. The control group only added liposomes and CaCl2, while the experimental group additionally added Bv sPLA2 derived from bee venom, sPLA2 secreted from human tears, and sPLA2 secreted by Colo205 cells. Finally, Tris-HCl buffer was added to adjust the concentrations of phospholipids, CaCl2, and sPLA2 to 50 nmol / ml, 1 mM, and 100 ng / ml, respectively. The reaction solution was reacted at room temperature (25–30 °C) in the dark. At 0, 1, and 24 hours, 150 μl of the solution was added to 96-well plates, and the absorbance was read using a microplate reader. The excitation wavelength was 480 nm, and the emission wavelength was 510 nm. 10 μl of 10% Triton was added to disrupt the phospholipid membrane, and the total absorbance was measured. The CF release rate of the liposomes with amphiphilic molecule-modified phospholipids was calculated using Equation 1.

[0300]

[0301] F t F represents the absorbed light intensity at time t, and F0 represents the absorbed light intensity at time 0. triton This represents the absorbed light intensity after adding 10% Triton. Data are expressed as mean ± standard deviation, with each sample tested independently three times.

[0302] Liposome in vitro fluorescein and oxaliplatin release assay

[0303] Liposomes constructed from synthetic sn-2-cholic acid-modified phospholipids (comprising 10%-15% of the total molar amount of liposomal phospholipids) were coated with 5(6)-carboxyfluorescein. The release of the fluorescently labeled liposomal marker was tested by adding 100 ng / ml of sPLA2 to the suspension. The sPLA2 used included samples derived from bee venom, human tears, and tumor cells. The prepared liposomes are divided into two categories. One category is a simple formulation, which is prepared by mixing DPPC (dispalmitoylphosphatidylcholine) or DSPC (distearylphosphatidylcholine) with novel phospholipids. The other category is a complex formulation, which contains distearylphosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2k). This PEG-modified lipid can form a steric hindrance layer on the liposome, which significantly weakens the interaction between the liposome and the protein (i.e., opsonization), thereby reducing the probability of it being recognized and taken up by MPS. In vivo, it exhibits a lower clearance rate and a longer circulation time, and is called PEG liposome.

[0304] When testing PEG liposomes, a formulation with the same or similar liposome composition as LiPlaCis was used as a control group. LiPlaCis is a liposome for sPLA2 enzyme-targeted drug release in clinical trials. The control group DSPG25 (DSPC:DSPG:DSPE-PEG2k 70:25:5) and the LiPlaCis formulation had the exact same phospholipid composition and ratio, both containing 18-carbon phospholipids. Furthermore, the upgraded version of DSPG25 used in this invention, DPPG25, has a formulation of DPPC:DPPG:DSPE-PEG2k 70:25:5, in which all phospholipids except DSPE-PEG2k are 16-carbon phospholipids. Since sPLA2 prefers to degrade short-carbon phospholipids, DPPG25 is more sensitive to the enzyme than DSPG25.

[0305] 1. Results of fluorescence emission assay on bee venom sPLA2 (compounds 1-9 tested)

[0306] 10 mol% of novel phospholipids (compounds 1-7) were added to the DPPC formulation, and their release under the action of sPLA2 was measured. Within one hour, liposomes containing compounds 1-4 released all CF, three times faster than the pure DPPC liposome formulation, representing a 65% increase in release. Figure 6 A). Its release mechanism is as follows: Figure 3 As shown, LCA-modified phospholipids enter the hydrophobic channel of the enzyme to reach the active site for hydrolysis. The entire phospholipid molecule migrates at least 1.5 nm outside the membrane during this process, while the LCA residues, which are 1.4 nm long and originally inserted into the hydrophobic region of a 1.3 nm thick (monolayer) phospholipid membrane, are completely extracted from the hydrophobic region and enter the hydrophilic region (0.55 nm thick). The 3α-OH in LCA quickly forms hydrogen bonds in the hydrophilic region. After hydrolysis, the product lysophospholipid re-insertes into the phospholipid membrane, while the other product, LCA, rotates, pushing aside surrounding phospholipids and lying flat on the membrane surface. This process causes membrane pores, leading to CF leakage. This specific action (Substrate Extraction, Cleavage, Rotation, Interface Settling) assisted by sPLA2 in modifying phospholipids and their metabolites is currently called the SECRIS effect. Similarly, from... Figure 6 As can be seen from A, the SECRIS effect caused by compounds 5-7 is much smaller than that caused by compounds 1-4. The steroid nucleus of compounds 1-4 has only one hydrophilic group (hydroxyl or ketone), while the steroid nucleus of compounds 5-7 has two hydrophilic groups. It is very likely that the multiple hydrophilic groups caused the weakening of the SECRIS effect. The underlying reason may be that the increased hydrogen bonds between the dimers of compounds 5-7 reduced the efficiency of enzymatic hydrolysis or prevented them from maintaining a flat state on the membrane surface after enzymatic hydrolysis.

[0307] In determining the formulation of PEG liposomes, as described above, this invention uses DSPC25 and DPPC25 as references and compares them with formulations containing compounds 1-7. PEG phospholipids (DSPE-PEG2k) are located in protruding positions on the liposome phospholipid membrane, so sPLA2 preferentially attacks them because sPLA2 can detect protruding sites on the phospholipid membrane. This leads to a delay in the degradation of other phospholipids, including modified phospholipids. For example... Figure 6 As shown in Figure B, when exposed to bee venom-type sPLA2, PEG liposomes containing 10% LCA-PG released only 30% of the fluorescein CF within 1 hour, while the non-PEG liposome formulation containing 10% LCA-PG released all of it within 1 hour. Figure 6 Similar results were observed with kLCA-PG liposomes. However, under the action of bee venom-type sPLA2, PEG liposomes containing LCA-PC released 96% of the luciferin within 1 hour, indicating that bee venom-type sPLA2 preferred to degrade LCA-modified phospholipids containing PC phospholipid heads compared to LCA-modified phospholipids containing PG phospholipid heads. PEG liposomes containing kLCA-PC also exhibited excellent release curves. Compared to the control group, liposomes containing 10% LCA-PC released the drug 10 times faster than the control group DSPG25 liposomes and 3 times faster than DPPG25 liposomes. Compounds 5-7 performed very poorly on PEG liposomes. Figure 6 .B).

[0308] In this invention, 5 mol% of compound 8 was added to the DSPC formulation and compared with a formulation containing 5 mol% DSPE-PEG. Their release under the action of sPLA2 was measured. The results are as follows: Figure 6 As shown in Figure C, the addition of LCA-PEG2k caused instability in the formulation. Fluorescein leakage was rapid in the absence of the enzyme, while the release of fluorescein decreased after the enzyme was added. The specific mechanism is not yet clear. Furthermore, this experiment only measured LCA-PEG2k alone; its effects when combined with other novel phospholipids such as LCA-PC and LCA-PG in the formulation are still unknown.

[0309] Similarly, in this invention, 2.5 mol% biLCA-PC (compound 9) was added to the DPPC:DSPE-PEG formulation and compared with the ordinary DPPC:DSPE-PEG formulation. The release curves of these compounds under the action of sPLA2 were measured. The results are as follows: Figure 6 As shown in Figure D, the addition of the disubstituted compound biLCA-PC accelerates the release of luciferin during enzymatic hydrolysis, resulting in a faster release compared to the standard formulation.

[0310] 2. Fluorescence release assay results on human tear sPLA2 (compounds 1-4 tested)

[0311] Human tears were collected from researchers, and human-derived sPLA2 preferred to degrade PG phospholipids. Under the action of human tear sPLA2, increasing the amount of PG phospholipid compounds 2 and 4 (LCA-PG and kLCA-PG) increased release. Compared with DSPG25, LCA-PG10 and LCA-PG15 increased CF release by more than 2 times and 3 times, respectively. Figure 7 Similarly, kLCA-PG showed similar performance. This confirms that liposome formulations containing LCA-PG and kLCA-PG, under the influence of sPLA2 in human tears, release fluorescein much faster than the DSPG25 formulation and its enhanced version, DPPG25.

[0312] 3. Results of fluorescence release assay on tumor sPLA2 (compounds 1-4 tested)

[0313] Colo205 tumor cells secrete sPLA2, which is also of human origin and also prefers to degrade PG phospholipids. Compared to DPPG25, LCA-PG15 increased CF release by more than 3 times. Figure 8 The luciferin release performance of kLCA-PG is inferior to that of LCA-PG.

[0314] Adding 10%-15% amphiphilic phospholipids to the DPPC:DSPE-PEG2k liposome formulation appears to lower the principal phase transition temperature of the formulation (40-41 degrees Celsius compared to 45 degrees Celsius for DPPG25). Figure 4 This would prevent the formulation from stably encapsulating the drug at 37 degrees Celsius. To increase temperature stability, this invention incorporates 60 mol% hydrogenated soybean phosphatidylcholine (HSPC) to prepare two formulations, LCA-PG (10) and LCA-PG (15), both with a phase transition temperature of 50 degrees Celsius. Figure 4 Both formulations showed superior luciferin release under enzyme induction compared to DPPG25, approximately twice that of the DPPG25 formulation. Figure 8 ).

[0315] 4. Oxaliplatin release assay (testing compound 2)

[0316] Oxaliplatin is a first-line anticancer drug for the treatment of metastatic rectal cancer. It was encapsulated in three liposomal formulations: DPPG25, LCA-PG (10), and LCA-PG (15). Drug release curves were measured under tumor sPLA2 stimulation. It was observed that at both high (100 ng / mL) and low (25 ng / mL) enzyme concentrations, the drug release of the LCA-PG-containing liposomal formulation was superior to that of the DPPG25 formulation. Figure 9Especially at relatively low sPLA2 concentrations (25 ng / mL), DPPG25 essentially lost its responsiveness to the enzyme, while the two formulations containing LCA-PG released 93-100% of oxaliplatin within 48 hours. Figure 9 B). Drug-loaded and blank liposome in vitro cytotoxicity assays (test compound 2)

[0317] Colo205 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-drug antibiotics at 37°C with 5% CO2 and appropriate humidity. HT-29 cells were cultured in McCoy's 5a medium containing 10% FBS and 1% penicillin-drug antibiotics at 37°C with 5% CO2 and appropriate humidity. Both Colo205 and HT-29 cells were passaged every 2–3 days. Cytotoxicity was determined using the MTT assay. HT-29 cell suspension was added to 96-well plates at 5,000 cells per well. After 24 hours of cell culture and cell attachment, the supernatant was aspirated, and 3% FBS, or free oxaliplatin containing 3% FBS, or oxaliplatin liposomes containing 3% FBS were added. Colo205 sPLA2 could be added if necessary. After 24 hours of incubation, the culture medium was removed, replaced with fresh culture medium, and incubated for another 48 hours. Cell growth was measured using the CCK-8 assay. Data are presented as mean ± standard deviation, and each sample was tested three times. Data were processed and analyzed using GraphPad Prism 8 software.

[0318] To investigate whether the accelerated release effect of liposomes containing LCA-PG could translate into better in vitro antitumor efficacy, this invention loaded oxaliplatin (L-OHP) into liposomes and determined the cytotoxicity of various formulations against HT-29 cells lacking sPLA2 secretion capacity, with and without the addition of Colo205 sPLA2. Oxaliplatin is a first-line anticancer drug for the treatment of metastatic rectal cancer. The reason for not directly using Colo205 cells for toxicity experiments in this invention is that they secrete sPLA2 at a very slow rate; by the time Colo205 cells produce a sufficiently high level of the enzyme to enter the solvent, the cell density in the cell culture wells would already be too high.

[0319] Liposomes containing LCA-PG exhibited sPLA2-dependent anti-tumor cell growth ability against Ht-29 cells. Figure 10 A). When 75 ng / mL Colo205 sPLA2 was added to the sample, the IC50 of LCA-PG liposomes containing LCA-PG(10) / (15) was [value missing]. 50The values ​​were between 12 and 17 μM. In contrast, without enzymes, even at drug concentrations as high as 100 μM, the inhibition of tumor cell growth by 50% was still not achieved. The DPPG25 formulation loaded with oxaliplatin showed good anti-Ht-29 cell growth ability and appeared to be unaffected by enzymes. This suggests that DPPG25 may be released in different ways. Data from this invention indicate that DPPG25 has better cell fusion or endocytosis properties than LCA-PG(10) / (15). Figure 11 It appears that the main mechanism of action of DPPG25 is through cell fusion or endocytosis and intracellular drug release.

[0320] The drug carrier itself may also affect the growth of cancer cells in vitro. This invention found that, under the action of 75 ng / mL LCA205 sPLA2, blank liposomes containing LCA-PG showed a stronger Ht-29 tumor cell killing effect than the DPPG25 carrier. Figure 10 The underlying reason may be that LCA-PG is degraded by enzymes to produce cytotoxic LCA.

[0321] Overall, LCA-PG-containing liposomes, when in an environment with relatively high sPLA2 concentrations, can produce anti-tumor cell effects similar to DPPG25, although they may exert their effects using different drug release mechanisms.

[0322] Tumor-bearing experiment in nude mice (testing compound 2)

[0323] Animal experiments have been applied for and passed the ethical review of the Animal Management and Use Committee (IACUC) of Southern Medical University. The subjects for the anticancer activity experiment were 6-week-old female nude mice. Tumors were created subcutaneously in the right back region near the leg, and 200 μl of Colo205 cells were inoculated at a cell concentration of 6 × 10⁻⁶. 6 Tumors per milliliter. By day 13, the average tumor volume had grown to 100 mm. 3 At that time, the mice were randomly divided into 5 groups of 9 mice each, based on tumor volume. The drug was administered via tail vein injection at a concentration of 5 mg / ml, once every four days for a total of 4 doses. The 5 groups of nude mice were administered the following drugs: a) isotonic glucose HEPES buffer (blank control group); b) free oxaliplatin (positive control group); c) oxaliplatin DPPG25 (positive control group); d) oxaliplatin LCA-PG (10); e) oxaliplatin LCA-PG (15). Tumor size was measured 2-3 times per week using calipers, and tumor volume was calculated using Formula 3. Tumor weight was measured 2-3 times per week to observe the health status of the nude mice and the degree of drug toxicity. When the tumor volume exceeded 1500 mm², the tumor was considered a tumor growth factor. 3When a nude mouse exhibited these symptoms, such as loss of 20% of its body weight or lethargy, it was euthanized to reduce pain, in accordance with the animal experiment ethics review guidelines of the Animal Management and Use Committee (IACUC) of Southern Medical University. Data were analyzed using GraphPad Prism 8 software. Survival analysis (Kaplan-Meierplots) was performed based on the above-mentioned observation termination criteria. In the survival analysis curves, the median survival time of the experimental groups and the significance comparison were analyzed using the Log-rank (Mantel-Cox) test. Significance criteria were established: ns > 0.05 indicated no significance, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0324]

[0325] a represents the longest side of the tumor, and b represents the shortest side of the tumor.

[0326] Based on the above series of studies, this invention selected drug-loaded liposomes containing LCA-PG for further animal tumor-bearing experiments. HEPES buffer, naked oxaliplatin, and DPPG25 formulations encapsulating oxaliplatin served as control groups. In this model, Colo205 cells transplanted into nude mice continuously secreted sPLA2. According to one study, the average concentration of sPLA2 in the interstitial fluid of Colo205 tumor cells was 79.42 ± 63.16 ng / mL. Figure 12 As shown in Figure A, LCA-PG(10) exhibited the best tumor growth inhibition effect before 40 days, followed by the two liposome formulations LCA-PG(15) and DPPG25. Although in vitro experiments showed that DPPG25 was far more efficient than LCA-PG(10) / (15) in cell fusion or endocytosis. Figure 11 However, results showed that in animal models, the responsiveness of the formulation to sPLA2 may be more important for tumor suppression. In in vitro experiments, DPPG25 liposomes can contact all cancer cells, thus maximizing their effectiveness through cell fusion or endocytosis and intracellular drug release. However, in in vivo experiments, liposomes deposit on the periphery of the tumor, with only a small fraction of tumor cells able to directly contact them. Therefore, intracellular drug release is limited, and sPLA2-mediated release may determine the tumor-suppressive effect. Of course, in vivo efficacy is not only determined by drug release from liposomes, but also by the stability of the formulation in the circulatory system and the proportion of liposomes that deposit on the tumor.

[0327] Besides tumor suppression, the safety of the formulation should also be considered. The DPPG25 group showed high toxicity, manifested as significant weight loss and subcutaneous hemorrhage in nude mice; 5 out of 9 nude mice were euthanized due to weight loss exceeding 20%. Figure 12 .B, as shown in Table 2. Some nude mice in the LCA-PG(15) and bare-drug groups also showed similar toxic reactions. However, none of the nine nude mice in the LCA-PG(10) group experienced severe weight loss throughout the entire course of treatment (Table 2). The median survival time in the LCA-PG10 group was 57 days, which was significantly better than the 36 days in the DPPG25 group and the 40 days in the bare-drug group. Figure 12 As shown in .C. The severe toxicity observed in the DPPG25 group was likely related to hepatotoxicity, caused by premature release of oxaliplatin encapsulated in liposomes in the liver due to excessive use of PG phospholipids (containing 25% PG phospholipids). It appears that LCA-PG10, using only 10% PG phospholipids, did not cause this toxicity. Table 2. Analysis and comparison of the anticancer activity of oxaliplatin and oxaliplatin liposomes in a nude mouse model with Colo205 colon cancer (n = 9 mice / group).

[0328]

[0329] Intravenous administration was performed on days 13, 17, 21, and 25 after tumor initiation. The dose of oxaliplatin was 5 mg / kg per intravenous injection.

[0330] Based on the above experimental results, this invention discovers a novel phospholipid derivative and the liposomes constructed from it. This phospholipid derivative exhibits a unique phospholipid membrane disruption effect under the action of sPLA2, termed the SECRIS effect. This effect arises from the properties of amphiphilic molecules and sPLA2. Data from this invention shows that low-ratio (10-15%) amphiphilic molecule-modified phospholipids can be incorporated into the liposome phospholipid membrane, while degradation of these phospholipids leads to a strong membrane interference effect. This effect significantly improves the responsiveness of the liposomes, allowing for the use of small amounts of PG-modified phospholipids while maintaining enzyme sensitivity without causing toxic reactions. This effectively avoids the safety issues currently faced in preparing sPLA2-sensitive liposomes.

Claims

1. A phospholipid derivative selected from the following compounds: 、 、 。 2. A phospholipid liposome, characterized in that, It is prepared from the phospholipid derivatives described in claim 1.

3. The phospholipid liposome according to claim 2, characterized in that, It is prepared from the phospholipid derivatives of claim 1 and other phospholipid compounds; wherein the molar ratio of the phospholipid derivatives of claim 1 to other phospholipid compounds is 1:4-19.

4. The use of the phospholipid derivative of claim 1 or the phospholipid liposome of any one of claims 2-3 in the preparation of a pharmaceutical product; in, The drug is used under physiological or pathological conditions of secretory phospholipase A2 overexpression, such as tears, digestive juices secreted by the pancreas, sites of inflammation, bacterial infection, asthma, and atherosclerosis.

5. The application according to claim 4, wherein the drug is a drug for treating cancers that overexpress secretory phospholipase A2.

6. The application according to claim 5, characterized in that, The drug is selected from carboplatin, nedaplatin, cisplatin, oxaloplatin, lobaplatin, carmustine, camptothecin, doxorubicin, daunorubicin, irinotecan, cytarabine, paclitaxel, vinorelbine, gemcitabine, siRNA, mRNA, or combinations thereof.

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