Sn38-fatty alcohol prodrugs, self-assembled nanoparticles thereof, and uses thereof

Abstract

The application relates to a kind of SN38-fatty alcohol prodrugs and its self-assembly nanoparticles and application, belong to the field of new excipients and new dosage forms of pharmaceutical preparations, and relates to a kind of 7-ethyl-10-hydroxy camptothecin prodrug antitumor preparation, specifically to the construction of SN38-fatty alcohol prodrug and its self-assembly nanoparticles containing the prodrug, and its application as a drug delivery system in the preparation of antitumor drugs.The structure general formula (I) of SN38-fatty alcohol prodrug or its pharmaceutically acceptable salt is as follows: wherein, n, R, X are described in the claims and the specification.The SN38-fatty alcohol prodrug of the application can be self-assembled into nanoparticles, can effectively improve curative effect, reduce its toxic side effect.

Description

Technical Field

[0001] This invention belongs to the field of new excipients and dosage forms for pharmaceutical preparations, and relates to an SN38-fatty alcohol prodrug, its self-assembled nanoparticles, and its applications. Specifically, it relates to the construction of the SN38-fatty alcohol prodrug and the self-assembled nanoparticles containing the prodrug, and its application in the preparation of antitumor drugs. Background Technology

[0002] In recent years, the incidence of malignant tumors has been gradually increasing, seriously threatening human health. Chemotherapy is one of the most effective strategies in cancer treatment. Camptothecin (CPT) belongs to the class of alkaloid antitumor drugs, acting on DNA topoisomerases to selectively kill proliferating tumor cells. Studies have found that introducing a hydroxyl group at the 10 position of the camptothecin parent ring can increase its antitumor activity by 20 times, and adding steric hindrance at the 7 position can improve blood stability. Therefore, the camptothecin derivative 7-ethyl-10-hydroxycamptothecin (SN38) has become a hot topic in the development of camptothecin-based antitumor drugs. However, camptothecin drugs have extremely poor water solubility and can cause serious adverse reactions, such as bone marrow suppression, diarrhea, vomiting, and hematuria, which limit the clinical application of camptothecin drugs.

[0003] Prodrugs are drugs that are chemically modified to have little or no activity in vitro, but release their active form in vivo through enzymatic or non-enzymatic conversion. Prodrug strategies are an effective way to improve the delivery efficiency of chemotherapy drugs. The commercially available formulation Campto, also known as irinotecan hydrochloride injection, is a prodrug irinotecan obtained by modifying the structure of SN38 with a bispiperidine group. Intravenous infusion of irinotecan effectively improves the problems of poor solubility and high toxicity of SN38. However, the conversion rate of active SN38 in vivo is only 0.1-1%, limiting its antitumor activity. Nanoparticle drug delivery systems can effectively prolong the circulation time of drugs in vivo and enhance antitumor effects. Based on this, self-assembled nanoparticles based on prodrug strategies, as nanoparticle drug delivery systems, combine the advantages of nanotechnology and prodrug strategies. With their high drug loading capacity, good stability, and low toxicity, they have become a hot topic in chemotherapy drug delivery research in recent years.

[0004] Prodrugs typically consist of three parts: a parent drug, a linker chain, and a side chain, with the parent drug and side chain linked together by the linker chain. To construct prodrugs with self-assembly capabilities, most existing SN38 prodrugs use fatty acids as side chains. Fatty acid side chains can increase the structural flexibility of the prodrug molecule, balance intermolecular forces, and promote prodrug self-assembly. Meanwhile, the length and structure of the side chain, the connection position of the side chain, and the carbon chain length of the side chain can all affect the self-assembly capability, formulation properties, in vivo fate, and antitumor efficacy of prodrug self-assembled nanoparticles. Therefore, finding a prodrug self-assembled nanoparticle that can improve self-assembly capability is a key research focus.

[0005] As linking chains, different linking chains have different elemental compositions, chain lengths, or linking positions, resulting in varying redox sensitivities. Therefore, SN38 prodrugs with different linking chains and side chain modifications also exhibit different formulation properties, in vivo fate, and antitumor effects.

[0006] Whether for prodrug delivery or nanomedicine delivery systems, intelligently triggering the selective release of drugs at the target site is crucial for the efficacy and safety of formulations. Therefore, intelligent drug delivery systems based on tumor microenvironment-stimulated-response release have become a research hotspot in recent years. Compared to normal cells, tumor cells contain higher concentrations of reactive oxygen species (ROS) and glutathione (GSH). This unique redox microenvironment has been widely used to design intelligent responsive drug delivery systems to achieve tumor-specific drug release while reducing toxic side effects on normal organs and tissues. Monosulfide bonds, disulfide bonds, monoselenoses, and diselenoses all possess redox-sensitive properties, enabling them to intelligently respond to the high redox state within tumor cells and release drugs. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides an SN38-fatty alcohol prodrug, its self-assembled nanoparticles, and its applications. Specifically, it comprises an SN38-fatty alcohol prodrug and the self-assembled nanoparticles formed therefrom. The SN38-fatty alcohol prodrug is selected from SN38-linear fatty alcohol prodrugs or SN38-branched fatty alcohol prodrugs, containing side chains with different linking chains and carbon chain lengths. The self-assembled nanoparticles formed from this SN38-fatty alcohol prodrug have advantages such as small and uniform particle size distribution, high drug loading, good stability, good antitumor effect, and good safety. Furthermore, the SN38-branched fatty alcohol prodrug more effectively disrupts the tight packing of prodrug molecules, further enhancing the self-assembly ability of the prodrug. Using the self-assembled nanoparticles as a nanomedicine delivery system can target antitumor activity. This invention provides more options for developing new prodrugs and their self-assembled nanoparticles as nanomedicine delivery systems, meeting the urgent clinical need for highly effective and low-toxicity chemotherapy agents.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides an SN38-fatty alcohol prodrug of general structural formula (I) or a pharmaceutically acceptable salt thereof:

[0010]

[0011] Where n = 1 to 3;

[0012] R is a straight-chain or branched C3-C30 hydrocarbon group;

[0013] X is one of the following: disulfide bond, monosulfide bond, diselenide bond, or monoselenide bond.

[0014] Furthermore, when R is a branched C3-C30 hydrocarbon group, the branch is selected from one or more of C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

[0015] Preferably, R is a straight-chain or branched C3-C24 hydrocarbon group;

[0016] When R is a branched C3-C24 hydrocarbon group, the branch is selected from one or more of C1-C10 alkyl, C2-C10 alkenyl or C2-C10 alkynyl.

[0017] Preferably, R is a straight-chain or branched C10-C24 hydrocarbon group;

[0018] When R is a branched C10-C24 hydrocarbon group, the branch is selected from one or more of C6-C10 alkyl, C6-C10 alkenyl or C6-C10 alkynyl.

[0019] Preferably, when R is a branched C10-C24 alkyl group, the branch is a C6-C10 alkyl group.

[0020] Preferably, R is a straight-chain or branched C16-C24 hydrocarbon group. When R is a branched C16-C24 hydrocarbon group, the branch is a C6-C10 alkyl group.

[0021] More preferably, R is a branched C19-C21 alkyl group, and the branch is a C9-C11 alkyl group.

[0022] When R is an unsaturated hydrocarbon group, the number of alkenyl groups, alkynyl groups, or the sum of alkenyl and alkynyl groups contained in the unsaturated hydrocarbon group is 1-5.

[0023] When R is a branched hydrocarbon group, the branched fatty alcohol hydroxyl group used in R is substituted at the 1st to 28th carbon of the branched fatty alcohol.

[0024] Preferably, the branched fatty alcohol hydroxyl group in R is substituted at carbons 1-15 of the branched fatty alcohol, more preferably at carbons 9-11 of the branched fatty alcohol hydroxyl group.

[0025] Furthermore, the branched substitution position in R is the carbon where the branched fatty alcohol hydroxyl group is located.

[0026] Specifically, when the fatty alcohol in the SN38-fatty alcohol prodrug of the present invention is a branched fatty alcohol, the branched fatty alcohol is one of 2-hexyl-octanol, 1-heptyl-octanol, 2-hexyl-decanol, 1-butyl-dodecylol, 1-heptyl-nonanol, 1-octyl-nonanol, 2-octyl-decanol, 2-heptyl-undecanol, 1-nonyl-decanol, 2-octyl-dodecylol, 2-decyl-tetradecylol, 2-dodecyl-tetradecylol, 8-pentadecanol, 9-heptadecylol, 10-nonadecanol, or 11-tetradecylol.

[0027] Preferably, the branched fatty alcohol is 8-pentadecanol, 9-heptadecylol, 10-nonadecanol, or 11-tetradecylol.

[0028] In the SN38-fatty alcohol prodrug, SN38 is linked to the fatty alcohol via a diacid as a linking chain. The diacid is a monothiodiacid, monoselenodiacid, dithiodiacid, or diselenodiacid. Specifically, the monothiodiacid is monothiodiacetic acid, monothiodipropionic acid, or monothiodibutyric acid; the monoselenodiacid is monoselenodiacetic acid, monoselenodipropionic acid, or monoselenodibutyric acid; the dithiodiacid is 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, or 4,4'-dithiodibutyric acid; and the diselenodiacid is 2,2'-diselenodiacetic acid, 3,3'-diselenodipropionic acid, or 4,4'-diselenodibutyric acid.

[0029] Specifically, the present invention provides SN38-fatty alcohol prodrugs preferably SN38-8-pentadecanol prodrug, SN38-9-heptadecylol prodrug, SN38-10-nonadecanol prodrug, and SN38-11-tetradecylol prodrug;

[0030] Using 4,4'-dithiodibutyric acid as the linking chain, the corresponding prodrugs were named SN38-SS-C15, SN38-SS-C17, SN38-SS-C19, and SN38-SS-C21, respectively, with the following structural formulas:

[0031]

[0032] SN38-8-pentadecanol prodrug SN38-SS-C15 with 4,4'-dithiodibutyric acid as the linking chain;

[0033]

[0034] SN38-9-heptadecyl alcohol prodrug SN38-SS-C17 with 4,4'-dithiodibutyric acid as the linking chain;

[0035]

[0036] SN38-10-Ninetetrahydric acid prodrug SN38-SS-C19, with 4,4'-dithiodibutyric acid as the linking chain;

[0037]

[0038] SN38-11-dodecanoic acid as a linking chain for the SN38-SS-C21 prodrug of 4,4'-dithiodibutyric acid;

[0039] This invention also provides an SN38-11-hexaenoic acid prodrug using monothiodiacetic acid as the linking chain, named SN38-S-C21, with the following structural formula:

[0040]

[0041] Monothiodiacetic acid as a linking chain SN38-11-hexaenoic acid prodrug SN38-S-C21;

[0042] This invention also provides an SN38-11-dodecanoic acid prodrug using monoselenodiacetic acid and 4,4'-diselenodibutyric acid as linking chains, the corresponding prodrugs being named SN38-Se-C21 and SN38-SeSe-C21, respectively, with the following structural formulas:

[0043]

[0044] Monoselenodiacetic acid as a linking chain SN38-11-hexaenoic acid prodrug SN38-Se-C21;

[0045]

[0046] 4,4'-diselenodibutyric acid as the linking chain of the SN38-11-dodecanoic acid prodrug SN38-SeSe-C21.

[0047] The method for synthesizing the SN38-fatty alcohol prodrug of the present invention includes the following steps:

[0048] Step 1: After dissolving the diacid into a diacid anhydride, it is esterified with a fatty alcohol to obtain a fatty alcohol-diacid monoester intermediate. The molar ratio of fatty alcohol to diacid anhydride is (1-10):(5-15). The diacid is a monothiodiacid, a monoselenodiacid, a dithiodiacid, or a diselenodiacid.

[0049] Step 2: The fatty alcohol-diacid monoester undergoes an esterification reaction with SN38 to obtain the final product SN38-fatty alcohol prodrug, wherein the molar ratio of fatty alcohol-diacid monoester to SN38 is 1:(0.5-10), and the reaction equation route is one of the following:

[0050]

[0051] Where n and R are as described above.

[0052] The synthesis method of the above-mentioned SN38-fatty alcohol prodrug specifically includes the following steps:

[0053] (1) Dissolve the dicarboxylic acid in acetic anhydride (AC2O) and stir at room temperature for 2-4 hours to react the dicarboxylic acid into dicarboxylic anhydride. After the reaction is complete, add toluene and remove toluene and acetic anhydride by rotary evaporation under reduced pressure.

[0054] (2) Take fatty alcohol and 4-dimethylaminopyridine (DMAP), and dissolve them together with the diacid anhydride obtained in step (1) in dichloromethane. Stir at room temperature for 12-18 hours, and separate the intermediate product by chromatography column: fatty alcohol-diacid monoester.

[0055] (3) Take 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt) and 4-dimethylaminopyridine (DMAP), and dissolve them together with the intermediate fatty alcohol-diacid monoester in anhydrous dichloromethane. Stir in an ice bath for 2-4 hours, then add SN38, stir at room temperature for 24-48 hours, and then purify by preparative liquid phase separation to obtain the final product: SN38-fatty alcohol prodrug.

[0056] The synthesis method of the SN38-fatty alcohol small molecule prodrug is carried out under nitrogen protection throughout the reaction process.

[0057] In step (1), the dicarboxylic acid is monothiodiacetic acid, monothiodipropionic acid, monothiodibutonic acid, monoselenodiacetic acid, monoselenodipropionic acid, monoselenodibutonic acid, 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, 4,4'-dithiodibutonic acid, 2,2'-diselenodiacetic acid, 3,3'-diselenodipropionic acid, or 4,4'-diselenodibutonic acid.

[0058] In step (1), the ratio of dicarboxylic acid to acetic anhydride is 1 mmol:(1-10) mL, preferably 1 mmol:(1-2) mL.

[0059] In step (2), the fatty alcohol is a C3-C30 straight-chain or branched fatty alcohol. When it is a branched fatty alcohol, the branch is one or more of C1-C18 alkyl, C2-C18 alkenyl or C2-C18 alkynyl.

[0060] In step (2), the molar ratio of DMAP: fatty alcohol: dicarboxylic anhydride is 1:(1-10):(2-15), preferably 1:(2-5):(10-15).

[0061] In step (3), the molar ratio of the intermediate product fatty alcohol-diacid monoester: HOBt: EDCI: DMAP: SN38 is 1:(1-10):(2-6):(0.2-5):(0.5-10), preferably 1:(1-2):(2-4):(0.3-2):(0.8-2).

[0062] In step (3), the SN38-fatty alcohol prodrug prepared has a purity of over 99%.

[0063] This invention also provides self-assembled nanoparticles of SN38-fatty alcohol prodrugs, wherein the self-assembled nanoparticles of SN38-fatty alcohol prodrugs are non-PEGylated SN38-fatty alcohol prodrug self-assembled nanoparticles, PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles, actively targeted modified SN38-fatty alcohol prodrug self-assembled nanoparticles, SN38-fatty alcohol prodrug self-assembled nanoparticles loaded with hydrophobic fluorescent substances, or SN38-fatty alcohol prodrug self-assembled nanoparticles loaded with drugs.

[0064] The method for preparing the self-assembled nanoparticles of the SN38-fatty alcohol prodrug includes the following steps:

[0065] When the SN38-fatty alcohol prodrug self-assembled nanoparticles are non-PEGylated, the preparation method is as follows: the SN38-fatty alcohol prodrug is dissolved in an organic solvent, and the solution is slowly added dropwise to water while stirring. The SN38-fatty alcohol prodrug spontaneously forms uniform nanoparticles. The organic solvent in the formulation is removed by vacuum rotary evaporation to obtain a nanocolloidal solution without any organic solvent, which is the non-PEGylated SN38-fatty alcohol prodrug self-assembled nanoparticles.

[0066] When the self-assembled nanoparticles are PEG-modified SN38-fatty alcohol prodrugs or actively targeted SN38-fatty alcohol prodrugs, the preparation method is as follows: A PEG modifier or an actively targeted modifier and an SN38-fatty alcohol prodrug are dissolved in an organic solvent. While stirring, the solution is slowly added dropwise to water, and the SN38-fatty alcohol prodrug spontaneously forms uniform nanoparticles. The organic solvent in the formulation is removed by rotary evaporation under reduced pressure to obtain a nanocolloidal solution free of any organic solvent, which is the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticle or actively targeted SN38-fatty alcohol prodrug self-assembled nanoparticle. The mass ratio of SN38-fatty alcohol prodrug to PEG modifier or actively targeted modifier is 1:(0.1-1). The PEG modifier is an amphiphilic polymer or a targeting group, more preferably DSPE-PEG, TPGS, PEG-PLGA, PEG-PE, or DSPE-PEG-FA. The actively targeted modifier is a substance capable of targeting specific tissues, preferably an antibody, sugar residue, hormone, receptor, or ligand.

[0067] When the self-assembled nanoparticles are SN38-fatty alcohol prodrugs loaded with hydrophobic fluorescent substances or SN38-fatty alcohol prodrugs loaded with drugs, the preparation method is as follows: PEG modifier, hydrophobic fluorescent substance or drug, and SN38-fatty alcohol prodrug are dissolved in an organic solvent. The solution is slowly added dropwise to water while stirring, and the SN38-fatty alcohol prodrug spontaneously forms uniform nanoparticles. The organic solvent in the preparation is removed by rotary evaporation under reduced pressure to obtain a nanocolloidal solution without organic solvent, which is the self-assembled nanoparticles of SN38-fatty alcohol prodrugs loaded with hydrophobic fluorescent substances or SN38-fatty alcohol prodrugs loaded with drugs. The mass ratio of SN38-fatty alcohol prodrug:PEG modifier:hydrophobic fluorescent substance or drug is 1:(0.1-1):(0.1-1).

[0068] The self-assembled nanoparticles of the SN38-fatty alcohol prodrug have a particle size of 110-130 nm, a particle size distribution of less than 0.2, and a drug loading of 34-40%.

[0069] The application of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in the preparation of antitumor drugs.

[0070] The application of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in the preparation of injection, oral or topical drug delivery systems.

[0071] The application of the SN38-fatty alcohol prodrug or the self-assembled nanoparticles of the SN38-fatty alcohol prodrug in the preparation of antitumor drugs can improve efficacy and reduce toxicity.

[0072] The beneficial effects of this invention are:

[0073] (1) This invention designs and synthesizes SN38-fatty alcohol prodrugs containing different fatty alcohol side chains, and the synthesis method is simple and easy to implement; and prepares self-assembled nanoparticles of SN38-fatty alcohol prodrugs with small particle size and uniform particle size distribution, and the preparation method is simple and easy to implement; (2) The effects of branched fatty alcohol side chains of different lengths on the formulation properties and antitumor activity of the self-assembled nanoparticles of the prodrugs were investigated. The results show that the self-assembled nanoparticles of SN38-fatty alcohol prodrugs can effectively improve the efficacy of SN38 and reduce its toxic side effects; different side chains have a significant impact on the formulation properties and antitumor activity of the self-assembled nanoparticles of SN38-fatty alcohol prodrugs; when 11-hexaenoic acid is used as a side chain, the self-assembled nanoparticles of the prodrugs have the best safety. This invention provides a new strategy and option for developing highly effective and low-toxicity chemotherapy agents. Attached Figure Description

[0074] Figure 1 This is the mass spectrum of the SN38-fatty alcohol prodrug with monothiodiacetic acid as the linking chain in Example 5 of the present invention.

[0075] Figure 2 This is a graph showing the tumor volume change during the in vivo antitumor experiment of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles of Example 8 of the present invention.

[0076] ns: P≥0.05*: P<0.05 (both are two-tailed t-tests)

[0077] Figure 3 This is a graph showing the weight change of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles in an in vivo antitumor experiment according to Example 8 of the present invention.

[0078] ns: P≥0.05 (all two-tailed t-tests)

[0079] Figure 4 This is a tumor load diagram in an in vivo antitumor experiment of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles of Example 8 of the present invention.

[0080] ns: P≥0.05***: P<0.001****: P<0.0001 (all two-tailed t-tests)

[0081] Figure 5 This is a graph showing the routine biochemical indicators of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles in an in vivo antitumor experiment according to Example 8 of the present invention.

[0082] Figure 6This is a graph showing routine blood parameters in an in vivo antitumor experiment of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles of Example 8 of the present invention.

[0083] Figure 7 This is a graph showing the tumor volume change during the in vivo antitumor experiment of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles of Example 9 of the present invention.

[0084] Figure 8 This is a graph showing the tumor volume change at the same administration concentration in an in vivo antitumor experiment using PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles from Example 9 of the present invention.

[0085] *: P < 0.05 **: P < 0.01 (both are two-tailed t-tests)

[0086] Figure 9 This is a graph showing the weight change of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles in an in vivo antitumor experiment according to Example 9 of the present invention.

[0087] ****: P < 0.0001 (all two-tailed t-tests)

[0088] Figure 10 This is a tumor load diagram in an in vivo antitumor experiment of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles of Example 9 of the present invention.

[0089] ns: P≥0.05***: P<0.001****: P<0.0001 (all two-tailed t-tests)

[0090] Figure 11 This is a graph showing the change in mouse body weight during the tolerance test of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles in Example 10 of this invention.

[0091] Figure 12 This is a graph showing the survival rate of the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles in the tolerance experiment of Example 10 of the present invention. Detailed Implementation

[0092] The present invention will be further described in detail below with reference to the embodiments.

[0093] Example 1:

[0094] Synthesis of SN38-8-pentadecanol prodrug with 4,4'-dithiodibutyric acid as the linking chain

[0095] 4,4'-Dithiodibutyric acid was dissolved in acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, the solution was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL round-bottom flask. Three times the volume of toluene was added, and the toluene and acetic anhydride were removed by rotary evaporation under reduced pressure to obtain dithiodibutyric anhydride. In the reaction, the ratio of 4,4'-dithiodibutyric acid to acetic anhydride was 1:1, with units of mmol:mL.

[0096] Dithiodibutyric anhydride was dissolved in dichloromethane, followed by a dichloromethane solution of 8-pentadecanol, and then a dichloromethane solution of 4-dimethylaminopyridine (DMAP) was slowly added dropwise at a rate of 1-2 mL / min. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate crude 8-pentadecanol-dithiodibutyric acid monoester. The purified 8-pentadecanol-dithiodibutyric acid monoester was obtained by column chromatography using a cyclohexane-acetone elution system. The molar ratio of DMAP:8-pentadecanol:dithiodibutyric anhydride was 0.4:2:1.

[0097] The purified 8-pentadecanol-dithiodibutyric acid monoester was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP), and activated in an ice bath at 0°C for 2 hours. Then, a dichloromethane solution of SN38 was added, and the mixture was stirred at 25°C for 48 hours. After the reaction was completed, the product was separated by the preparation solution to obtain 4,4'-dithiodibutyric acid as the SN38-8-pentadecanol prodrug with the linking chain. The molar ratio of 8-pentadecanol-dithiodibutyric acid monoester: HOBt: EDCI: DMAP: SN38 = 1:1:2:0.4:0.8.

[0098] use 1 The structure of the product was confirmed by H-NMR. The spectral analysis results are as follows:

[0099] 1H NMR (400MHz, Chloroform-d) δ8.24 (d, J=9.2Hz, 1H, Ar-H), 7.85 (d, J=2.5Hz, 1H, Ar-H),7.65(s,1H,Ar-H),7.56(dd,J=9.2,2.5Hz,1H,Ar-H),5.35–5.24(m,3H,-OH,-O-CH2-), 4.88(p,J=6.4Hz,1H,-CH-),3.71(s,2H,-N-CH2)3.17(q,J=7.7Hz,2H,-Ar-CH2),2.91–2.70 (m,6H,-SS-(CH2)3-),2.44(t,J=7.3Hz,2H,-SS-CH2-),2.23(p,J=7.1Hz,2H,-SS-CH2-), 2.06(p,J=7.2Hz,2H,-SS-CH2-),1.97–1.84(m,2H,-SS-CH2-),1.51(d,J=6.0Hz,4H,-CH2- ), 1.41 (t, J = 7.7Hz, 3H, -CH3), 1.27 (d, J = 7.0Hz, 20H, -CH2-), 1.05 (t, J = 7.4Hz, 3H, -CH3), 0.88 (d, J = 6.3Hz, 6H, -CH3).

[0100] Example 2:

[0101] Synthesis of SN38-9-heptadecyl prodrug with 4,4'-dithiodibutyric acid as the linking chain

[0102] 4,4'-Dithiodibutyric acid was dissolved in acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, the solution was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL round-bottom flask. Three times the amount of toluene was added, and the toluene and acetic anhydride were removed by rotary evaporation under reduced pressure to obtain dithiodibutyric anhydride. The ratio of 4,4'-dithiodibutyric acid to acetic anhydride was 1:1, with units of mmol:mL.

[0103] Dithiodibutyric anhydride, formed by dissolving in dichloromethane, was added, followed by a dichloromethane solution of 9-heptadecyl alcohol. A solution of 4-dimethylaminopyridine (DMAP) dissolved in dichloromethane was then slowly added dropwise at a rate of 1-2 mL / min. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate product, crude 9-heptadecyl alcohol-dithiodibutyric acid monoester. The purified 9-heptadecyl alcohol-dithiodibutyric acid monoester was obtained by column chromatography using a cyclohexane-acetone elution system. The molar ratio of DMAP:9-heptadecyl alcohol:dithiodibutyric anhydride was 0.4:2:1.

[0104] The purified 9-heptadecyl-dithiodibutyric acid monoester was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP), and activated in an ice bath at 0°C for 2 hours. Then, a dichloromethane solution of SN38 was added, and the mixture was stirred at 25°C for 48 hours. After the reaction was completed, the product was separated by the preparation solution to obtain 4,4'-dithiodibutyric acid as the SN38-9-heptadecyl prodrug with the linking chain. The molar ratio of 9-heptadecyl-dithiodibutyric acid monoester:HOBt:EDCI:DMAP:SN38 = 1:1:2:0.4:0.8.

[0105] use 1 The structure of the product was confirmed by H-NMR. The spectral analysis results are as follows:

[0106] 1 H NMR (400MHz, Chloroform-d) δ8.25 (d, J=9.2Hz, 1H, Ar-H), 7.85 (d, J=2.4Hz, 1H, Ar-H),7.65(s,1H,Ar-H),7.57(dd,J=9.1,2.5Hz,1H,Ar-H),,5.35–5.24(m,3H,-OH,-O-CH2- ),4.88(dt,J=12.6,6.2Hz,1H,-CH-),3.71(s,2H,-N-CH2-),3.17(q,J=7.7Hz,2H,-Ar-CH2-), 2.89–2.73(m,6H,-SS-(CH2)3-),2.44(t,J=7.3Hz,2H,-SS-CH2-),2.23(p,J=7.0Hz,2H,-S- S-CH2-),2.05(p,J=7.1Hz,2H,-SS-CH2-),1.90(ddt,J=21.6,14.2,7.2Hz,2H,-CH2-),1.53- 1.48(m,4H,-CH2-),1.44–1.20(m,27H,-(CH2) 12 ,-CH3), 1.05(t,J=7.4Hz,3H,-CH3), 0.87(t,J=6.8Hz,6H,-CH3).

[0107] Example 3:

[0108] Synthesis of SN38-10-nonadecanol prodrug with 4,4'-dithiodibutyric acid as the linking chain

[0109] 4,4'-Dithiodibutyric acid was dissolved in acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, the solution was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL round-bottom flask. Three times the volume of toluene was added, and the toluene and acetic anhydride were removed by rotary evaporation under reduced pressure to obtain dithiodibutyric anhydride. The ratio of 4,4'-dithiodibutyric acid to acetic anhydride was 1:1, in mmol:mL.

[0110] Dithiodibutyric anhydride, formed by dissolving in dichloromethane, was added, followed by a dichloromethane solution of 10-nonadecanol. A solution of 4-dimethylaminopyridine (DMAP) dissolved in dichloromethane was then slowly added dropwise at a rate of 1-2 mL / min. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate crude 10-nonadecanol-dithiodibutyric acid monoester. The purified 10-nonadecanol-dithiodibutyric acid monoester was obtained by column chromatography using a cyclohexane-acetone elution system. The molar ratio of DMAP: 10-nonadecanol: dithiodibutyric anhydride was 0.4:2:1.

[0111] The purified 10-nonadecanol-dithiodibutyric acid monoester was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP), and activated in an ice bath at 0°C for 2 hours. Then, a dichloromethane solution of SN38 was added, and the mixture was stirred at 25°C for 48 hours. After the reaction, the product was separated by the preparation solution to obtain 4,4'-dithiodibutyric acid as the SN38-10-nonadecanol prodrug with the linking chain. The molar ratio of 10-nonadecanol-dithiodibutyric acid monoester:HOBt:EDCI:DMAP:SN38 = 1:1:2:0.4:0.8.

[0112] use 1 The structure of the product was confirmed by 1H-NMR. The spectral analysis results are as follows:

[0113] 1H NMR(400MHz,Chloroform-d)δ8.24(d,J=9.2Hz,1H,Ar-H),7.85(d,J=2.4Hz,1H, Ar-H),7.65(s,1H,Ar-H),7.56(dd,J=9.2,2.5Hz,1H,Ar-H),5.37–5.23(m,3H,-OH,-O-CH2-), 4.88(p,1H,-CH-),3.72(s,2H,-N-CH2-),3.17(q,J=7.6Hz,2H,-Ar-CH2-),2.89–2.73(m,6H,- SS-(CH2)3-),2.44(t,J=7.3Hz,2H,-SS-CH2-),2.22(p,J=7.1Hz,2H,-SS-CH2-),2.05(p,J= 7.2Hz,2H,-SS-CH2-),1.95–1.83(m,2H,-CH2-),1.51(d,J=5.7Hz,4H,-CH2-),1.41(t,J=7.7 Hz,3H,-CH3),1.25(s,28H,-(CH2) 14 ), 1.05 (t, J = 7.4Hz, 3H, - CH3), 0.87 (t, J = 6.8Hz, 6H, - CH3).

[0114] Example 4:

[0115] Synthesis of SN38-11-dodecanoic acid prodrug with 4,4'-dithiodibutyric acid as the linking chain

[0116] 4,4'-Dithiodibutyric acid was dissolved in acetic anhydride in a 25 mL round-bottom flask. After complete dissolution, the solution was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL round-bottom flask. Three times the volume of toluene was added, and the toluene and acetic anhydride were removed by rotary evaporation under reduced pressure to obtain dithiodibutyric anhydride. The ratio of 4,4'-dithiodibutyric acid to acetic anhydride was 1:1, in mmol:mL.

[0117] Dithiodibutyric anhydride, formed by dissolving in dichloromethane, was added, followed by a dichloromethane solution of 11-eicosyl alcohol. A solution of 4-dimethylaminopyridine (DMAP) dissolved in dichloromethane was then slowly added dropwise at a rate of 2 mL / min. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate crude 11-eicosyl alcohol-dithiodibutyric acid monoester. The purified 11-eicosyl alcohol-dithiodibutyric acid monoester was obtained by column chromatography using a cyclohexane-acetone elution system. The molar ratio of DMAP:11-eicosyl alcohol:dithiodibutyric anhydride was 0.4:2:1.

[0118] The purified 1,4'-dienoyl-dithiodibutyric acid monoester was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP). The mixture was activated in an ice bath at 0°C for 2 hours, followed by the addition of a dichloromethane solution of SN38. After the reaction, the product was separated from the preparation solution to obtain 4,4'-dithiodibutyric acid as the SN38-1,1-dienoyl prodrug with the linking chain. The molar ratio of 1,1-dienoyl-dithiodibutyric acid monoester:HOBt:EDCI:DMAP:SN38 was 1:1:2:0.4:0.8.

[0119] use 1 The structure of the product was confirmed by 1H-NMR. The spectral analysis results are as follows:

[0120] 1 H NMR (400MHz, Chloroform-d) δ8.25 (d, J=9.1Hz, 1H, Ar-H), 7.85 (d, J=2.2Hz, 1H, Ar-H),7.65(s,1H,Ar-H),7.56(dd,J=9.1,2.3Hz,1H,Ar-H),5.34–5.25(m,3H,R-OH,-O-CH2- ),4.89–4.86(m,1H,-CH-),3.71(s,2H,-N-CH2-),3.17(q,J=7.6Hz,2H,-Ar-CH2-),2.83–2.71 (m,6H,-SS-(CH2)3-),2.43(s,2H,-SS-CH2-),2.27–2.19(m,2H,-SS-CH2-),2.04(s,2H,-SS- CH2-),1.94–1.84(m,2H,-CH2-),1.51(d,J=5.0Hz,4H,-CH2-),1.41(t,J=7.6Hz,3H,-CH3), 1.26(s,32H,-(CH2) 14 -), 1.04 (d, J = 7.4Hz, 3H, -CH3), 0.88 (d, J = 2.7Hz, 6H, -CH3).

[0121] Example 5:

[0122] Synthesis of SN38-11-hexaenoic acid prodrug with monothiodiacetic acid as the linking chain

[0123] Monothiodiacetic acid was dissolved in acetic anhydride and placed in a 25 mL round-bottom flask. After complete dissolution, the mixture was magnetically stirred at 25 °C for 2 hours and then transferred to a 100 mL round-bottom flask. Three times the volume of toluene was added, and the toluene and acetic anhydride were removed by rotary evaporation under reduced pressure to obtain dithiodibutyric anhydride. The ratio of monothiodiacetic acid to acetic anhydride was 1:1, with units of mmol:mL.

[0124] The monothiodiacetic anhydride formed by dissolving in dichloromethane was added, followed by a dichloromethane solution of 11-eicosyl alcohol, and a dichloromethane solution of 4-dimethylaminopyridine (DMAP) was slowly added dropwise. The mixture was magnetically stirred at 25°C for 12 hours to obtain the intermediate crude 11-eicosyl alcohol-monothiodiacetic acid monoester. The purified 11-eicosyl alcohol-monothiodiacetic acid monoester was obtained by column chromatography using a cyclohexane-acetone elution system. The molar ratio of DMAP:11-eicosyl alcohol:monothiodiacetic anhydride was 0.4:2:1.

[0125] The purified 11-eicosyl-monothiodiacetic acid monoester was added to a dichloromethane solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP). The mixture was activated in an ice bath at 0°C for 2 hours. Then, a dichloromethane solution of SN38 was added, and the mixture was stirred at 25°C for 48 hours. After the reaction, the product was separated from the preparation solution to obtain monothiodiacetic acid as the SN38-11-eicosyl-monothiodiacetic acid prodrug for the linker chain. The molar ratio of 11-eicosyl-monothiodiacetic acid monoester:HOBt:EDCI:DMAP:SN38 was 1:1:2:0.4:0.8.

[0126] The structure of the product was confirmed by mass spectrometry, and the mass spectrum is shown below. Figure 1 As shown, MS(ESI)m / z for C 47 H 66 N₂O₈S[M+H] + :819.4688.

[0127] Example 6: Preparation of PEG-modified small molecule prodrug self-assembled nanoparticles

[0128] Accurately weigh 0.4 mg of DSPE-PEG. 2K 2 mg of the prodrug from Examples 1-5 was dissolved in 200 μL of acetone. The ethanol solution was then slowly added dropwise to 2 mL of deionized water with stirring, spontaneously forming PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles with uniform particle size. Acetone was removed by rotary evaporation under reduced pressure to obtain a colloidal nanoparticle solution free of organic reagents. As shown in Table 1, the particle size of each group of nanoparticles was within 120 nm, the particle size distribution was less than 0.2 mm, and the surface charge was between -20 and -30 mV.

[0129] Table 1. Particle size, particle size distribution, surface charge, and drug loading of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles

[0130]

[0131] The results showed that SN38-fatty alcohol prodrugs with different branched fatty alcohol side chains could form self-assembled nanoparticles. The PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles formed by disulfide-bridged SN38-branched fatty alcohol prodrugs all had particle sizes around 120 nm, while the SN38-S-C21 NPs bridged by monosulfide bonds had smaller particle sizes. The particle size distribution of all prodrug nanoparticles was very uniform, around 0.1 nm, which helps the nanoparticles achieve targeted accumulation in solid tumors through their high permeability and retention effect. The surface charge of the nanoparticles was around -20 to -30 mV, which helps prevent nanoparticle aggregation through charge repulsion.

[0132] Example 7: Cytotoxicity of self-assembled nanoparticles of PEG-modified SN38-fatty alcohol prodrug

[0133] The cytotoxicity of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles on mouse colon cancer (CT26) cells was investigated using the MTT assay. First, healthy cells were digested and diluted with culture medium to a concentration of 2 × 10⁻⁶. 4 After homogenizing the cells, 100 μL of cell suspension was added to each well of a 96-well plate and incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion. Once adherent, SN38 solution, Campto, or the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles prepared in Example 5 were added. In this experiment, the drug solution and the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticle formulation were prepared and diluted using the corresponding culture media and aseptically filtered through a 0.22 μm filter membrane. 100 μL of the test solution was added to each well, with three parallel wells for each concentration. The control group, i.e., without the test drug solution, only 100 μL of culture medium was added, and the plates were incubated together with the cells. Forty-eight hours after drug administration, the 96-well plate was removed, and 20 μL of 5 mg / mL MTT solution was added to each well. The plate was incubated for 4 hours, then the plate was shaken. After thoroughly blotting the remaining liquid onto filter paper, 200 μL of DMSO was added to each well, and the plate was shaken for 10 minutes to fully dissolve the blue-purple crystals. Well A1 (containing only 200 μL of DMSO) was designated as the zeroing well. The absorbance of each well after zeroing was measured at 570 nm using a microplate reader.

[0134] The results of the cytotoxicity experiments were analyzed, and the half-maximal inhibitory concentrations (IC50) of the solution and prodrug nanoparticles were calculated.50 Compared to the SN38 solution, Campto and the prodrug nanoparticles exhibited reduced cytotoxicity. This is because SN38 requires a certain amount of time to be released from the prodrug nanoparticles. Campto showed the lowest cytotoxicity, indicating a lower efficiency in releasing active SN38. In contrast, the prodrug nanoparticles were more cytotoxic; among the four prodrug nanoparticles, SN38-SS-C15NPs showed higher cytotoxicity against tumor cells.

[0135] Table 2. Cytotoxicity of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles

[0136]

[0137] Example 8: In vivo antitumor experiment of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles

[0138] Mouse colon cancer cell suspension (4T1, 5x10) 6 (100 μL / cell) was injected subcutaneously on the dorsal side of male BALB / c mice. The tumor was allowed to grow to 160 mm². 3 At that time, tumor-bearing mice were randomly divided into 6 groups of 7 mice each: phosphate-buffered saline (PBS), SN38 solution, SN38-SS-C15 NPs, SN38-SS-C17 NPs, SN38-SS-C19 NPs, and SN38-SS-C21 NPs. The nanoparticles used for drug administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5, and the dosage was 2.5 mg / kg (calculated based on SN38 concentration). Administration was once every other day for 5 consecutive days. After administration, the survival status and weight changes of the mice were monitored daily, and tumor volume was measured. The results are as follows: Figure 2 and Figure 3 As shown. Mice were sacrificed one day after the last administration, and organs and tumors were harvested for further analysis and evaluation. Figure 2 This indicates that in the PBS group, tumor volume grew rapidly, reaching 2000 mm on day 10. 3 Around 800 mm. In contrast, the prodrug nanoparticle group significantly inhibited tumor growth (<800 mm). 3 However, there were no significant differences between the groups; Figure 3 This indicates that the weight of the SN38 solution and the self-assembled nanoparticles showed no significant change; the tumor burden map is shown below. Figure 4 See the graph for routine biochemical indicators. Figure 5 See the blood routine index chart. Figure 6 ; Figure 4 This indicates that the tumor burden of the prodrug nanoparticle group was lower than that of the SN38 solution group; Figure 5This indicates that the liver function index alanine aminotransferase in the SN38-SS-C15 NPs group was higher than the standard value, indicating poor safety. Figure 6 The results indicate that SN38 solution, SN38-SS-C15 NPs, and SN38-SS-C17 NPs caused abnormalities in blood cell markers, which are manifestations of acute leukemia; SN38-SS-C19 NPs and SN38-SS-C21 NPs showed better safety. In conclusion, at lower doses, SN38-SS-C19 NPs and SN38-SS-C21 NPs, with disulfide bonds as chemical links, exhibit potent antitumor effects without causing significant systemic toxicity, making them a safe and effective chemotherapy drug delivery system.

[0139] Example 9: In vivo antitumor experiment of PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticles

[0140] Mouse colon cancer cell suspension (4T1, 5x10) 6 (100 μL / cell) was injected subcutaneously on the dorsal side of male BALB / c mice. The tumor was allowed to grow to 130 mm². 3 At that time, tumor-bearing mice were randomly divided into 7 groups of 7 mice each: phosphate-buffered saline (PBS), Camptothecin 3 mg / kg, 6 mg / kg, SN38-SS-C19 NPs 3 mg / kg, 6 mg / kg, and SN38-SS-C21 NPs 3 mg / kg, 6 mg / kg. The dosage was calculated based on the SN38 concentration. The nanoparticles used for administration were PEG-modified small molecule prodrug self-assembled nanoparticles prepared in Example 5. Administration was once every other day for 5 consecutive days. After administration, the survival status and weight changes of the mice were monitored daily, and tumor volume was measured. The results are as follows: Figure 7 , Figure 8 , Figure 9 and Figure 10 As shown. Figure 7 This indicates that in the PBS group, tumor volume grew rapidly, reaching 1800 mm on day 10. 3 Around 800 mm, tumor growth was significantly inhibited in both the Campto group and the prodrug self-assembled nanoparticle group (<800 mm). 3 ). Figure 8 The results showed that, at the same dosage, the tumor volume in the SN38-SS-C21 NPs and SN38-SS-C19 NPs groups was smaller than that in the Campto group; Figure 9 The results showed that at a dose of 6 mg / kg, the SN38-SS-C19 NPs group experienced significant weight loss, indicating poor safety. Figure 10The results indicate that the prodrug self-assembled nanoparticle group has a lower tumor burden than the PBS group and the Campto group. At the same administration concentration, SN38-SS-C21 NPs and SN38-SS-C19 NPs have better tumor inhibition effects than Campto, and SN38-SS-C21 NPs have better safety, indicating that prodrugs with 11-dodecanool as the side chain have a greater advantage in safety.

[0141] Example 10: Tolerance test of self-assembled nanoparticles of PEG-modified SN38-fatty alcohol prodrug

[0142] Male BALB / c mice were randomly divided into 7 groups of 6 mice each. Two groups received intravenous injections of SN38 solution and Campto, respectively. The other four groups received intravenous injections of SN38-SS-C15 NPs, SN38-SS-C17 NPs, SN38-SS-C19 NPs, and SN38-SS-C21 NPs, respectively. One group served as a control group. Administered the drugs every 24 hours at a dose of 5 mg / kg, calculated based on the SN38 concentration, for a total of 12 administrations. Body weight changes and survival status were observed after each administration. Results are as follows: Figure 11 and Figure 12 As shown, mice died after the fourth dose of the SN38 solution. The safety results of the four prodrug nanoparticles were: SN38-SS-C21 NPs > SN38-SS-C19 NPs > SN38-SS-C17 NPs > SN38-SS-C15 NPs. The results indicate that the length of the linker chain affects the safety of the prodrug; SN38-SS-C21 NPs, containing a longer linker chain, showed better safety, suggesting that 11-hexaenoic acid (11-hexaenoic acid) has a greater safety advantage as a linker chain.

Claims

1. A self-assembled nanoparticle of an SN38-fatty alcohol prodrug, characterized in that, It is a PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticle, wherein the SN38-fatty alcohol prodrug is SN38-10-nonadecanol prodrug and SN38-11-tetradecanool prodrug. The aforementioned SN38-10-nonadecanol prodrug is named SN38-SS-C19, and its structural formula is: The SN38-11-dodecanool prodrug is named SN38-SS-C21, and its structural formula is: The method for synthesizing the SN38-fatty alcohol prodrug is shown in the following formula: Where n and R correspond to the corresponding positional structures of SN38-SS-C19 and SN38-SS-C21.

2. A method for preparing self-assembled nanoparticles of the SN38-fatty alcohol prodrug according to claim 1, characterized in that, Includes the following steps: The PEG modifier and SN38-fatty alcohol prodrug were dissolved in acetone. While stirring, the solution was slowly added dropwise to water, causing the SN38-fatty alcohol prodrug to spontaneously form uniform nanoparticles. The acetone in the formulation was removed by rotary evaporation under reduced pressure, yielding a nanocolloidal solution free of any organic solvents, which is the PEG-modified SN38-fatty alcohol prodrug self-assembled nanoparticle. The mass ratio of SN38-fatty alcohol prodrug to PEG modifier was 1:(0.1-1), and the PEG modifier was DSPE-PEG. 2K The obtained nanoparticles have a particle size of 110~130nm, a particle size distribution of less than 0.2, and a drug loading of 34~40%.

3. The application of the self-assembled nanoparticles of the SN38-fatty alcohol prodrug according to claim 1 in the preparation of anti-colon cancer drugs.

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