Small molecule drug conjugate based on anisamide targeting human prostate cancer cells as well as preparation method and application of small molecule drug conjugate

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a small molecule drug conjugate model, in particular to a small molecule drug conjugate based on anisamide targeting human prostate cancer cells as well as a preparation method and application thereof. The micromolecular drug conjugate is used as a prodrug, and is composed of four parts, namely a PEG-modified anisidine target head, a drug connexon composed of a glucuronic acid trigger and a self-closed loop, maleimide and an SN38 cytotoxic drug. According to the prodrug, the targeting capacity of the drug to prostate cancer cells is improved through a PEG-modified anisidine target head, a good drug release system is constructed by a drug linker composed of a glucuronic acid trigger and a self-closed loop, the water solubility and stability of the drug are improved by taking albumin as a carrier, and long-acting circulation in vivo is realized. The compound also shows a remarkable curative effect in the aspect of treating prostatic cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a small molecule drug conjugate model, in particular to a small molecule drug conjugate based on anisamide targeting human prostate cancer cells, a preparation method thereof and an application thereof. Background Art

[0002] In the current medical field, many diseases still lack effective treatments, and cancer is one of them. Prostate cancer is the second most common cancer among men worldwide, and its treatment strategies include surgery, radiotherapy, chemotherapy, and targeted therapy. Drug therapy plays an important role in the treatment of prostate cancer. Therefore, it is particularly important to find new drug compounds to improve the treatment of prostate cancer. Among many drugs, conjugate drugs (XDC) have become a hot topic for many scientists, such as antibody-drug conjugates (ADC), small molecule drug conjugates (SMDC), etc. Although this type of drug has made some progress in treatment, the following problems still exist:

[0003] 1. The off-target toxicity caused by immunogenicity and low internalization efficiency of ADC drugs still limit its development.

[0004] 2. Even though prostate cancer patients receive radical chemotherapy, about 20% still have residual tumor after surgery. This incomplete tumor removal may lead to recurrence of the disease or recurrence after treatment.

[0005] 3. SMDC is easily cleared by the kidneys because of its small molecule size, and the drug is not easy to work in the body.

[0006] 7-Ethyl-10-hydroxycamptothecin (SN38) is a cytotoxic drug with strong toxicity and significant anti-tumor effect. However, SN38 has extremely poor solubility and lacks targeting, which seriously restricts its clinical application.

[0007] According to Thomas, JD, et al. Sigma1 Targeting to Suppress AberrantAndrogenReceptor Signaling in Prostate Cancer. Cancer Res 1May 2017; 77(9): 2439-2452. According to the literature, a 26kDa endoplasmic reticulum membrane protein is called sigma receptor. After nearly half a century of in-depth research on it, pharmacologists have proposed this receptor as a therapeutic target for cancer and mental illness. Interestingly, this protein is overexpressed in many cancer cells, such as prostate cancer, lung cancer, breast cancer, melanoma, etc. This also shows that sigma receptors have great potential and feasibility as targeted therapeutic targets. According to Banerjee, R., et al. Anisamide-targeted stealth liposomes: A potent carrier for targeting doxorubicin to human prostate cancer cells. International Journal of Cancer 112 (4): 693-700. According to literature records, anisamide has a high affinity for sigma receptors, which can meet the drug's targeting needs for tumors. However, the existing anisamide targeting sigma receptors is fat-soluble, and when the fragment is connected to a prodrug fragment with poor water solubility, the entire drug will be difficult to dissolve in water, which is not convenient for administration. And the drug targeting effect of some anisamide target heads is not very excellent. Summary of the invention

[0008] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a small molecule drug conjugate based on anisamide targeting human prostate cancer cells, a preparation method thereof and its application. The inventor designed a prodrug model, and then successfully synthesized a drug compound with a unique structure through organic chemical synthesis and biological characterization, and found that it showed significant potential in the treatment of prostate cancer.

[0009] After detailed chemical experimental research and verification at the biological level, the inventor found that the synthesized drug has good activity. The drug compound targets prostate cancer cells through anisamide ligands, and delivers the overall prodrug to tumor cells and tumor microenvironment. A large number of glucuronidases in the microenvironment act as a pair of scissors to cut the glucosidic bond, causing 1,6 elimination, self-closed ring self-cleavage, and release anti-tumor active drugs to achieve the purpose of precise targeted therapy. At the same time, because of the limitations of small molecule conjugated drugs themselves, that is, small molecular weight, easy to be metabolized by the kidneys, and short drug efficacy, the inventor introduced maleimide into the prodrug to bind to albumin in the human body, which can achieve the long-term circulation of the prodrug in the body, increase the drug action time, and improve the treatment efficiency. Thereby achieving the treatment of prostate cancer. This discovery will provide a new approach for the treatment of prostate cancer and bring significant clinical value.

[0010] In a first aspect of the present invention, a small molecule drug conjugate targeting human prostate cancer cells based on anisamide is provided.

[0011] The small molecule drug conjugate of the present invention is a prodrug having a structure as shown in general formula I:

[0012]

[0013] Wherein, n1 and n2 both represent the number of repeating units; n1 = 1, 2, 6 or 10; n2 = 1, 2 or 3;

[0014] Preferably, in the general formula I, n1=1; n2=1, and the structural formula of the small molecule drug conjugate is as follows:

[0015]

[0016] The drug conjugate of the present invention is a prodrug, which is composed of a PEG-modified anisamide target head, a glucuronic acid trigger and a linker consisting of a self-closed ring, an SN38 cytotoxic drug, and maleimide. The functions of each part are described as follows:

[0017] 1. Effects of PEG-modified anisamide target heads: 1) The water solubility of anisamide target heads can be increased through intermediate PEGylation modification; 2) The anisamide part has an alkynyl group, which can directly combine the target head with the glucuronic acid linker of the linker through a click reaction, and a one-step reaction can stabilize the connection and avoid the occurrence of other side reactions. 3) The present invention increases the water solubility of the drug by introducing PEG chains (i.e., 1, 2, 6 or 10). The general formula of the prodrug of the present invention supports multivalent anisamide (i.e., n2=1, 2 or 3), which can theoretically increase the targeting.

[0018] 2. Linker composed of glucuronic acid trigger and self-closed ring: The tumor microenvironment and lysosomes of tumor cells contain a large number of glucuronidases, which can act on the glucuronic acid trigger, thereby causing the self-closed ring to cyclize and quickly release cytotoxic drugs.

[0019] 3. SN38 cytotoxic drug: Topoisomerase I (Top1) inhibitor, the active ingredient in the prodrug that kills cancer cells.

[0020] 4. Maleimide: Maleimide can bind to albumin in the human body, achieving long-term circulation of the prodrug in the body and increasing the duration of drug action.

[0021] In the second aspect of the present invention, a method for preparing the drug conjugate as described in the first aspect is provided.

[0022] The preparation method comprises three steps:

[0023] S1: Preparation of functionalized PEG-modified anisamide small molecule ligand;

[0024] S2: preparing a compound comprising a glucuronic acid trigger and a prodrug linker consisting of a self-closing ring and a toxic payload SN38;

[0025] S3: First connect the anisamide small molecule ligand of S1 and the compound containing the prodrug linker and the toxic payload of S2, and then connect maleimide to obtain the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect.

[0026] Said S1: preparation of functionalized PEG-modified anisamide small molecule ligand, the preparation route thereof is as follows:

[0027]

[0028] n1 and n2 in the reaction scheme are as defined in the first aspect.

[0029] S1 specifically includes the following sub-reaction steps:

[0030] 1a: Compound 1 was dissolved in DMSO, 4M NaOH solution was added, and then tert-butyl acrylate was added, and the mixture was reacted at 25°C for 24 hours to obtain compound 1-1; the molar ratio of compound 1 to tert-butyl acrylate was 1:n2*1.1;

[0031] 1b: Disperse compound 1-2, i.e. 5-hexynoic acid, in anhydrous dichloromethane, add EDCI and NHS, react at 25°C for 6h, then add compound 1-1 and DIEA, and continue to react at 25°C overnight to obtain compound 1-3; the molar ratio of compound 1-1 to compound 1-2, i.e. 5-hexynoic acid, is 1:1.1; the molar ratio of compound 1-1 to EDCI is 1:1.3; the molar ratio of compound 1-1 to NHS is 1:1.3; the molar ratio of compound 1-1 to DIEA is 1:2.5;

[0032] 1c: Dissolve compound 1-3 in anhydrous dichloromethane, add trifluoroacetic acid in a volume equal to that of anhydrous dichloromethane, and react at 25°C for 3h to obtain compound 1-4;

[0033] 1d: Compound 1-4 was dissolved in N,N-dimethylformamide, and compound 1-5, EDCI, HOBT and DIEA were added, and the mixture was reacted overnight at 25°C to obtain compound 1-6; the molar ratio of compound 1-4 to compound 1-5 was 1:n2*1.1; the molar ratio of compound 1-4 to EDCI was 1:n2*1.3; the molar ratio of compound 1-4 to HOBT was 1:n2*1.3; the molar ratio of compound 1-4 to DIEA was 1:n2*3;

[0034] 1e: Dissolve compound 1-6 in dichloromethane, add TFA trifluoroacetic acid in a volume equal to that of dichloromethane, and react at 25°C for 3h to obtain compound 1-7;

[0035] 1f: Compound 1-7 is dissolved in anhydrous dichloromethane, compound 1-8, i.e., p-methoxybenzoyl chloride and DIEA are added, and the mixture is reacted overnight at 25°C to obtain compound 1-9; the molar ratio of compound 1-7 to compound 1-8, i.e., p-methoxybenzoyl chloride, is 1:n2*1.2; the molar ratio of compound 1-7 to DIEA is 1:n2*4;

[0036] S2: Preparation of a compound comprising a glucuronic acid trigger, a prodrug linker consisting of a self-closed ring and a toxic payload SN38:

[0037]

[0038] S2 specifically includes the following sub-reaction steps:

[0039] 2a: Compound A, i.e., γ-aminobutyric acid, is dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1, 1H-imidazole-1-sulfonyl azide hydrochloride, copper sulfate pentahydrate and potassium carbonate are added, and the mixture is reacted at 25° C. for 12 hours to obtain compound B; the molar ratio of compound A to 1H-imidazole-1-sulfonyl azide hydrochloride is 1:1.1; the molar ratio of compound A to copper sulfate pentahydrate is 1:0.03; and the molar ratio of compound A to potassium carbonate is 1:2;

[0040] 2b: Compound B, i.e., 4-azidobutyric acid, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25° C. for 12 h to obtain compound 2-1; the molar ratio of the compound B to NHS was 1:1.1; the molar ratio of the compound B to EDCI was 1:1.05;

[0041] 2c: Compound 2-2, i.e. Boc-L-lysine, and compound 2-1 were added to anhydrous acetonitrile, and then DIEA was added, and the mixture was reacted at 25°C for 10 hours to obtain compound 2-3; the molar ratio of compound 2-1 to compound 2-2, i.e. Boc-L-lysine, was 1:1; the molar ratio of compound 2-1 to DIEA was 1:2.5;

[0042] 2d: Compound 2-3, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25°C for 12 hours to obtain compound 2-4; the molar ratio of the compound 2-3 to NHS was 1:1.1; the molar ratio of the compound 2-3 to EDCI was 1:1.03;

[0043] 2e: Under nitrogen protection, compound C, i.e., AD-glucuronic acid methyl ester, was dissolved in anhydrous acetonitrile, and 4-hydroxy-3-nitrobenzaldehyde and silver oxide were added respectively, and the mixture was reacted at 25° C. in the dark for 12 h to obtain compound D; the molar ratio of compound C to 4-hydroxy-3-nitrobenzaldehyde was 1:1.1; the molar ratio of compound C to silver oxide was 1:1.4;

[0044] 2f: At 0°C, compound D, i.e. (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, was dissolved in a mixed solvent of V i-PrOH:V CHCl3 with a volume ratio of 1:5, and then NaBH4 and silica gel were added respectively, and the mixture was reacted at 25°C for 2.5h to obtain compound E; the molar ratio of compound D to NaBH4 was 1:3;

[0045] 2g: Under a hydrogen atmosphere, compound E, i.e. (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate and platinum dioxide were dissolved in ethyl acetate and reacted at 25°C for 6h to obtain compound 2-5; the mass ratio of compound E to platinum dioxide was 1:0.2;

[0046] 2h: Compound 2-5 was dissolved in anhydrous dichloromethane, and compound 2-6, i.e. Boc-beta-alanine and EEDQ were added respectively, and the mixture was reacted at 25°C for 12h to obtain compound 2-7; the molar ratio of compound 2-5 to compound 2-6, i.e. Boc-beta-alanine, was 1:1.05; the molar ratio of compound 2-5 to EEDQ was 1:1.5;

[0047] 2i: At 0°C, compound 2-7 and carbon tetrabromide are added to anhydrous tetrahydrofuran, triphenylphosphine is slowly added dropwise to the reaction system, and the mixture is reacted at 25°C in the dark for 14 hours to obtain compound 2-8; the molar ratio of compound 2-7 to carbon tetrabromide is 1:1.5; the molar ratio of compound 2-7 to triphenylphosphine is 1:1.5;

[0048] 2j: Under nitrogen protection, compound 2-8, compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, and silver oxide were added to anhydrous acetonitrile, and reacted at 25°C in the dark for 12 hours to obtain compound 2-10; the molar ratio of the compound 2-8 to the compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, was 1:1.05; the molar ratio of the compound 2-8 to silver oxide was 1:1.4;

[0049] 2k: Under nitrogen protection, compound 2-10 was dissolved in anhydrous tetrahydrofuran in an ice-salt bath, 2.0 M borane dimethyl sulfide was slowly added, stirred for 30 min, the ice-salt bath was removed, and the reaction was carried out at 25 °C for 3 h to obtain compound 2-11; the molar ratio of compound 2-10 to borane dimethyl sulfide was 1:2.8;

[0050] 2l: Under nitrogen protection, compound 2-11 was dissolved in anhydrous dichloromethane in an ice bath, triphosgene was added, DIEA was slowly added dropwise, and then the ice bath was removed, and the reaction was carried out at 25°C for 15 minutes to obtain compound 2-12; the molar ratio of the compound 2-11 to triphosgene was 1:0.5; the molar ratio of the compound 2-11 to DIEA was 1:4;

[0051] 2m: Under nitrogen protection, compound 2-12 and DMAP were added to the reaction system, followed by SN38, and the reaction was carried out at 25°C for 12 hours to obtain compound 2-13; the molar ratio of compound 2-12 to DMAP was 1:1.2; the molar ratio of compound 2-12 to SN38 was 1:1.5;

[0052] 2n: Compound 2-13 was dissolved in dichloromethane, 4M HCl-dioxane solution was added, and the mixture was reacted at 25°C for 4 hours to obtain compound 2-14;

[0053] 2o: Compound 2-14 and compound 2-4 were added to anhydrous dichloromethane, DIEA was added, and the mixture was reacted at 25°C for 14 hours to obtain compound 2-15; the molar ratio of compound 2-14 to compound 2-4 was 1:1; the molar ratio of compound 2-14 to DIEA was 1:2.5;

[0054] 2p: Dissolve compound 2-15 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-16;

[0055] 2q: Compound 2-16 was dissolved in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1, and a 0.3M lithium hydroxide aqueous solution was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at 25°C for 30 minutes to obtain compound 2-17;

[0056] Said S3: first connect the anisamide small molecule ligand of S1 and the compound containing the prodrug linker and the toxic payload of S2, and then connect maleimide to obtain the small molecule drug conjugate based on anisamide targeting human prostate cancer cells as described in the first aspect. The reaction route is as follows:

[0057]

[0058] n1 and n2 in the reaction scheme are as defined in the first aspect.

[0059] S3 specifically includes the following sub-reaction steps:

[0060] 3a: Under strict nitrogen protection, the compound 2-17 prepared by S2 and the compound 1-9 prepared by S1 were dissolved in a mixed solution of MeOH and H2O with a volume ratio of 1:1, CuSO4·5H2O and sodium ascorbate were added, and the mixture was reacted at 25°C for 3h to obtain compound 3-2; the molar ratio of the compound 2-17 to the compound 1-9 was 1:1.2; the molar ratio of the compound 2-17 to CuSO4·5H2O was 1:2; the molar ratio of the compound 2-17 to sodium ascorbate was 1:2;

[0061] 3b: Compound 3-2 and compound 3-3, 6-(maleimido)hexanoic acid succinimidyl ester, were dissolved in anhydrous DMF, DIEA was added to adjust the pH to 7-8, and the mixture was reacted at 25° C. for 4 h to obtain compound 3-4. The molar ratio of compound 3-2 to compound 3-3 was 1:1.4.

[0062] Preferably, n1 and n2 in the general formula are both 1, and the preparation method of the drug conjugate is as follows:

[0063] 1) Prepare a functionalized triethylene glycol-modified anisamide small molecule ligand, the reaction route is as follows:

[0064]

[0065] The specific process is as follows:

[0066] A: Disperse compound 1-2, i.e. 5-hexynoic acid, in anhydrous dichloromethane, add EDCI and NHS, react at 25°C for 6h, then add compound 1-1 and DIEA, and continue to react at 25°C overnight to obtain compound 1-3; the molar ratio of compound 1-1 to compound 1-2, i.e. 5-hexynoic acid, is 1:1.1; the molar ratio of compound 1-1 to EDCI is 1:1.3; the molar ratio of compound 1-1 to NHS is 1:1.3; the molar ratio of compound 1-1 to DIEA is 1:2.5;

[0067] B: Dissolve compound 1-3 in anhydrous dichloromethane, add trifluoroacetic acid in a volume equal to that of anhydrous dichloromethane, and react at 25°C for 3h to obtain compound 1-4;

[0068] C: Compound 1-4 was dissolved in N,N-dimethylformamide, and compound 1-5, EDCI, HOBT and DIEA were added, and the mixture was reacted overnight at 25°C to obtain compound 1-6; the molar ratio of compound 1-4 to compound 1-5 was 1:1.1; the molar ratio of compound 1-4 to EDCI was 1:1.3; the molar ratio of compound 1-4 to HOBT was 1:1.3; the molar ratio of compound 1-4 to DIEA was 1:3;

[0069] D: Dissolve compound 1-6 in dichloromethane, add TFA trifluoroacetic acid in a volume equal to that of dichloromethane, and react at 25°C for 3 hours to obtain compound 1-7;

[0070] E: Compound 1-7 is dissolved in anhydrous dichloromethane, compound 1-8 (p-methoxybenzoyl chloride) and DIEA are added, and the mixture is reacted at 25°C overnight to obtain compound 1-9; the molar ratio of compound 1-7 to compound 1-8 (p-methoxybenzoyl chloride) is 1:1.2; the molar ratio of compound 1-7 to DIEA is 1:4;

[0071] 2) Prepare a compound comprising a glucuronic acid trigger and a prodrug linker consisting of a self-closed ring and a toxic payload SN38, the reaction route is as follows:

[0072]

[0073] The specific process is as follows:

[0074] A: Compound 2-2, i.e. Boc-L-lysine, and compound 2-1 were added to anhydrous acetonitrile, and then DIEA was added, and the mixture was reacted at 25°C for 10 hours to obtain compound 2-3; the molar ratio of compound 2-1 to compound 2-2, i.e. Boc-L-lysine, was 1:1; the molar ratio of compound 2-1 to DIEA was 1:2.5;

[0075] B: Compound 2-3, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25°C for 12 hours to obtain compound 2-4; the molar ratio of compound 2-3 to NHS was 1:1.1; the molar ratio of compound 2-3 to EDCI was 1:1.03;

[0076] C: Compound 2-5 was dissolved in anhydrous dichloromethane, and then compound 2-6, i.e. Boc-beta-alanine and EEDQ were added respectively, and the mixture was reacted at 25°C for 12 hours to obtain compound 2-7; the molar ratio of compound 2-5 to compound 2-6, i.e. Boc-beta-alanine, was 1:1.05; the molar ratio of compound 2-5 to EEDQ was 1:1.5;

[0077] D: At 0°C, compound 2-7 and carbon tetrabromide are added to anhydrous tetrahydrofuran, triphenylphosphine is slowly added dropwise to the reaction system, and the mixture is reacted at 25°C in the dark for 14 hours to obtain compound 2-8; the molar ratio of compound 2-7 to carbon tetrabromide is 1:1.5; the molar ratio of compound 2-7 to triphenylphosphine is 1:1.5;

[0078] E: Under nitrogen protection, compound 2-8, compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, and silver oxide were added to anhydrous acetonitrile, and reacted at 25°C in the dark for 12 hours to obtain compound 2-10; the molar ratio of the compound 2-8 to the compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, was 1:1.05; the molar ratio of the compound 2-8 to silver oxide was 1:1.4;

[0079] F: Under nitrogen protection, compound 2-10 was dissolved in anhydrous tetrahydrofuran in an ice-salt bath, 2.0 M borane dimethyl sulfide was slowly added, stirred for 30 min, the ice-salt bath was removed, and the reaction was carried out at 25°C for 3 h to obtain compound 2-11; the molar ratio of compound 2-10 to borane dimethyl sulfide was 1:2.8;

[0080] G: Under nitrogen protection, compound 2-11 was dissolved in anhydrous dichloromethane in an ice bath, triphosgene was added, DIEA was slowly added dropwise, and then the ice bath was removed, and the reaction was carried out at 25°C for 15 minutes to obtain compound 2-12; the molar ratio of the compound 2-11 to triphosgene was 1:0.5; the molar ratio of the compound 2-11 to DIEA was 1:4;

[0081] H: Under nitrogen protection, compound 2-12 and DMAP were added to the reaction system, followed by SN38, and the reaction was carried out at 25°C for 12 hours to obtain compound 2-13; the molar ratio of compound 2-12 to DMAP was 1:1.2; the molar ratio of compound 2-12 to SN38 was 1:1.5;

[0082] I: Dissolve compound 2-13 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-14;

[0083] J: Compound 2-14 and compound 2-4 were added to anhydrous dichloromethane, DIEA was added, and the mixture was reacted at 25°C for 14 hours to obtain compound 2-15; the molar ratio of compound 2-14 to compound 2-4 was 1:1; the molar ratio of compound 2-14 to DIEA was 1:2.5;

[0084] K: Dissolve compound 2-15 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-16;

[0085] L: Compound 2-16 was dissolved in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1, and a 0.3M lithium hydroxide aqueous solution was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at 25°C for 30 minutes to obtain compound 2-17;

[0086] 3) The anisamide small molecule ligand of S1 and the drug linker of S2 are combined to prepare the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect. The reaction scheme is as follows:

[0087]

[0088] The specific steps are as follows:

[0089] A: Under strict nitrogen protection, the compound 2-17 prepared by S2 and the compound 1-9 prepared by S1 were dissolved in a mixed solution of MeOH and H2O with a volume ratio of 1:1, and CuSO4·5H2O and sodium ascorbate were added, and the reaction was carried out at room temperature for 3 hours to obtain compound 3-2; the molar ratio of the compound 2-17 to the compound 1-9 was 1:1.2; the molar ratio of the compound 2-17 to CuSO4·5H2O was 1:2; the molar ratio of the compound 2-17 to sodium ascorbate was 1:2;

[0090] B: Compound 3-2 and compound 3-3, 6-(maleimido)hexanoic acid succinimidyl ester, were dissolved in anhydrous DMF, DIEA was added to adjust the pH to 7-8, and the mixture was reacted at 25° C. for 4 h to obtain compound 3-4. The molar ratio of compound 3-2 to compound 3-3 was 1:1.4.

[0091] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising a small molecule drug conjugate based on anisamide targeting human prostate cancer cells as described in the first aspect and a pharmaceutically acceptable macromolecular carrier thereof, wherein the macromolecular carrier is human serum albumin.

[0092] In the fourth aspect of the present invention, there is provided the use of the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect in the preparation of an anti-prostate tumor drug.

[0093] The terms and abbreviations involved in the present invention are explained as follows:

[0094] Drug conjugates: Drug conjugates are a type of drug that is made by coupling molecules with specific targeting properties (such as antibodies, peptides, small molecules, etc.) with cytotoxic drugs (payloads) through chemical linkers.

[0095] Prodrug: Prodrug refers to a drug that can only exert its pharmacological effect after metabolic transformation in the body. Prodrugs themselves usually do not have pharmacological activity, but through metabolic processes in the body, such as enzymatic hydrolysis, hydrolysis, oxidation-reduction reactions, they are converted into biologically active parent drugs or active metabolites, thereby exerting therapeutic effects.

[0096] EDCI: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

[0097] NHS: N-hydroxysuccinimide

[0098] DIEA: N,N-diisopropylethylamine

[0099] HOBT: 1-Hydroxybenzotriazole

[0100] TFA: trifluoroacetic acid

[0101] EEDQ: 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline

[0102] DMAP:4-dimethylaminopyridine

[0103] The present invention has the following technical effects:

[0104] 1) The prodrug compounds of the present invention have good water solubility and are easy to administer.

[0105] 2) The prodrug of the present invention has good stability. As can be seen from Example 4 of the specific implementation method, the inventors conducted an in vitro stability test on the compound, and judged the stability of the prodrug in PBS (pH = 7.4) and ABS (pH = 5.4), respectively. The test results showed that the release of SN38 was not detected in the compound within 7 days, indicating that the prodrug has good stability.

[0106] 3) The prodrug of the present invention has good drug release ability. As can be seen from Example 5 of the specific embodiment, the inventors conducted an in vitro drug release experiment on the compound. In PBS (pH = 7.4) solution, the prodrug completely released SN38 within 3 hours. In ABS (pH = 5.4) solution, the two prodrugs completely released SN38 in nearly 2 hours. This shows that the prodrug has good drug release ability.

[0107] 4) After the additional addition of β-glucuronidase, the drug activity of the prodrug of the present invention is greatly improved. As can be seen from Example 6 of the specific embodiment, at the cellular level, the prodrug was subjected to an in vitro cytotoxicity experiment. It can be observed that in 4 tumor cell lines, the prodrug still showed strong cytotoxicity without the additional addition of β-glucuronidase. After the additional addition of β-glucuronidase, the drug activity was greatly improved. This also confirms the successful application of the prodrug molecule at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1 This is the H NMR spectrum characterization diagram of the prodrug of Example 3.

[0109] Figure 2 This is the NMR carbon spectrum characterization diagram of the prodrug of Example 3.

[0110] Figure 3 This is the high-resolution mass spectrum of the prodrug of Example 3.

[0111] Figure 4 , 5 This is the purity test of the prodrug of Example 3.

[0112] Figure 6The results of the in vitro drug release experiment in Example 5 under PBS (pH=7.4) conditions.

[0113] Figure 7 The results of the in vitro drug release experiment in Example 5 under ABS (pH=5.4) conditions.

[0114] Figure 8 The schematic diagram of the structure of the small molecule drug conjugate based on anisamide targeting human prostate cancer cells of the present invention, wherein the blue part is the maleimide part, the black part connected to the blue part is the glucuronidase trigger, the green part connected to the black part is the self-closed loop, the red part connected to the self-closed loop is SN38, and the purple part connected to the glucuronidase trigger is the anisamide target head. DETAILED DESCRIPTION

[0115] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0116]

[0117] Compound 1-1 used in Example 1: can be purchased directly or prepared by yourself. The preparation method is as follows: Compound 1, i.e., ethanolamine (20 g, 0.33 mol) is dissolved in DMSO, 4M NaOH solution is added, and then tert-butyl acrylate (46 g, 0.36 mol) is added, and the mixture is reacted at 25°C for 24 hours. After the reaction system is concentrated, it is purified by column chromatography to obtain a colorless oily liquid, i.e., Compound 1-1.

[0118] The above-mentioned compound 1 can be purchased directly (purchased from Anergy), so during preparation, compound 1 can be used as a starting material to prepare compound 1-1.

[0119] Compound 2-1 used in Example 2 can be purchased directly or prepared by yourself. The preparation method is as follows:

[0120] 1) Compound A (γ-aminobutyric acid) (2.12 g, 20.6 mmol) was dissolved in methanol: water = 10:1, 1H-imidazole-1-sulfonyl azide hydrochloride (4.76 g, 22.67 mmol), copper sulfate pentahydrate (0.154 g, 0.62 mmol), potassium carbonate (5.69 g, 41.2 mmol) were added and reacted at 25°C for 12 h. TLC detected that the reaction was complete, filtered, the filtrate was collected, and concentrated to obtain a crude product, which was diluted with ethyl acetate (20 mL), washed three times with saturated ammonium chloride solution (10 mL), and the organic phase was separated, dried over anhydrous sodium sulfate for 2 h, and concentrated under reduced pressure to obtain a colorless liquid compound B.

[0121] 2) Compound B (4-azidobutyric acid) (4 g, 31 mmol), NHS (3.92 g, 34.09 mmol), EDCI (6.22 g, 32.6 mmol) were weighed into an eggplant-shaped bottle, anhydrous acetonitrile was added thereto, and the mixture was reacted at 25°C for 12 h. The reaction was complete by TLC detection, and the crude product was obtained by concentration. After dilution with dichloromethane:methanol = 10:1 (20 mL), the mixture was washed three times with pure water (10 mL), the organic phase was separated, dried over anhydrous sodium sulfate for 2 h, and concentrated under reduced pressure to obtain a white solid compound 2-1.

[0122] The above compounds A and B can be purchased directly (from Bidex Pharmaceuticals), so during the preparation, either compound A or B can be used as a starting material to prepare compound 2-1.

[0123] Compound 2-5 used in Example 2 can be purchased directly or prepared by yourself. The preparation method is as follows:

[0124] 1) Under nitrogen protection, compound C, i.e. AD-glucuronic acid methyl ester (30 g, 76 mmol) was dissolved in 270 mL of anhydrous acetonitrile, and then 4-hydroxy-3-nitrobenzaldehyde (13.9 g, 83 mmol) and silver oxide (24.8 g, 110 mmol) were added respectively, and the reaction was carried out at 25°C in the dark for 12 h. After TLC monitoring, the reaction was completed and the solvent was concentrated under reduced pressure. Dilute with dichloromethane, filter with diatomaceous earth, collect the filtrate, and concentrate to obtain a crude product. Purify by column chromatography (PE, PE:EA:DCM=5:1:1 to 2:1:1) to obtain compound D (32.4 g, yield 88.5%) as a white fluffy solid.

[0125] 2) At 0°C, compound D, i.e. (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (32.4 g, 67.1 mmol), was dissolved in 540 mL (V i-PrOH:VCHCl3=1:5), and NaBH4 (7.66 g, 0.2 mol) and silica gel (57.5 g) were added respectively, and the mixture was reacted at 25°C for 2.5 h. After TLC monitoring, the reaction was completed, 1M HCl was added to quench the reaction, and the mixture was filtered through diatomaceous earth. The filtrate was collected and concentrated to obtain a crude product. Compound E (22.75 g, 70% yield) was obtained as a white fluffy solid by column chromatography purification (DCM, DCM:MeOH=200:1 to 150:1).

[0126] 3) Under hydrogen atmosphere, compound E, i.e. (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetic acid triester (22.75 g, 46.9 mmol) and platinum dioxide (2.2 g, 9.7 mmol) were dissolved in 230 mL of ethyl acetate. The reaction was carried out at 25°C for 6 h. After TLC monitoring, the reaction solution was filtered after the reaction was completed. The solvent was concentrated under reduced pressure to obtain 21.91 g of compound 2-5.

[0127] The above compounds C, D, and E can all be purchased directly (from Bidex Pharmaceuticals), so during preparation, compounds C, D, or E can be used as starting materials to prepare compounds 2-5.

[0128] Example 1

[0129] The functionalized triethylene glycol-modified monovalent anisamide small molecule ligand was prepared by the following reaction scheme:

[0130]

[0131] The specific steps are as follows:

[0132] 1) Synthesis of compound 1-3: Compound 1-2, i.e. 5-hexynoic acid (10.83 g, 97 mmol), was dispersed in 100 mL of anhydrous dichloromethane, EDCI (20.24 g, 110 mmol) and NHS (12.65 g, 110 mmol) were added, and the reaction was carried out at 25°C for 6 h. The acid was completely activated by TLC monitoring. Compound 1-1, i.e. amino-polyethylene glycol-tert-butyl propionate (16.61 g, 88 mmol) and DIEA (28.4 g, 220 mmol) were then added, and the reaction was continued overnight at 25°C. The reaction was completed by TLC detection. The reaction system was diluted with dichloromethane and washed with saturated sodium bicarbonate solution, water, and saturated sodium chloride solution in sequence. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography to obtain a colorless oily liquid, i.e. compound 1-3.

[0133] 2) Synthesis of compound 1-4: Compound 1-3 was dissolved in anhydrous dichloromethane, trifluoroacetic acid (the volume was the same as that of anhydrous dichloromethane) was added, and the reaction was carried out at 25°C for 3 hours. The reaction was complete when detected by TLC. The reaction system was directly decompressed to completely remove the solvent to obtain a colorless viscous liquid, namely compound 1-4.

[0134] 3) Synthesis of compound 1-6: Compound 1-4 (2 g, 8.81 mmol) was dissolved in 20 mL of N,N-dimethylformamide, and compound 1-5 (2.4 g, 9.7 mmol), EDCI (2.2 g, 11.45 mmol), HOBT (1.55 g, 11.45 mmol), and DIEA (3.42 g, 26.43 mmol) were added. The reaction was allowed to proceed overnight at 25°C, and the reaction was complete when detected by TLC. The reaction system was completely decompressed to remove the solvent, and the reaction was washed with water and a saturated sodium chloride aqueous solution. The crude product was purified by column chromatography to obtain a light yellow oily liquid, namely compound 1-6.

[0135] 4) Synthesis of compound 1-7: Compound 1-6 was dissolved in dichloromethane, trifluoroacetic acid (the volume was the same as that of dichloromethane) was added, and the reaction system was decompressed to completely remove the solvent. A small amount of petroleum ether was added during the decompression to remove part of the trifluoroacetic acid to obtain a yellow viscous liquid, namely compound 1-7.

[0136] 5) Synthesis of compound 1-9: Compound 1-7 (1.77 mg, 5.0 mmol) was dissolved in anhydrous dichloromethane, and compound 1-8, i.e., p-methoxybenzoyl chloride (1 g, 5.9 mmol) and DIEA (2.58 g, 20 mmol) were added. The reaction was allowed to proceed overnight at 25°C, and the reaction was complete when detected by TLC. The reaction system was decompressed to completely remove the solvent, and the reaction was washed with water and a saturated sodium chloride aqueous solution. The crude product was purified by column chromatography to obtain a light yellow oily liquid, i.e., compound 1-9.

[0137] Example 2

[0138] The synthesis of the drug linker consisting of a glucuronic acid trigger and a self-closed ring, the reaction route is as follows:

[0139]

[0140] The specific steps are as follows:

[0141] 1) Synthesis of compound 2-3: Add compound 2-2, i.e. Boc-L-lysine (6.28 g, 25.5 mmol), compound 2-1 (4-azidobutyric acid-N-hydroxysuccinimide ester) (5.76 g, 25.49 mmol) to a reaction flask, add anhydrous acetonitrile (60 mL), and add DIEA (8.24 g, 63.76 mmol). React at 25°C for 10 h. TLC detection shows that the reaction is complete, and the crude product is obtained by concentration. After dilution with dichloromethane (30 mL), the product is washed three times with saturated ammonium chloride solution (15 mL), the organic phase is separated, dried over anhydrous sodium sulfate for 2 h, and purified by column chromatography (DCM, DCM:MeOH=150:1 to 30:1) to obtain compound 2-3 as a white solid (4.26 g, yield 46.9%)

[0142] 2) Synthesis of compound 2-4: Compound 2-3 (4.26 g, 11.93 mmol), NHS (1.51 g, 13.13 mmol), EDCI (2.35 g, 12.3 mmol) were weighed into an eggplant-shaped bottle, anhydrous acetonitrile was added thereto, and the mixture was reacted at 25°C for 12 h. The reaction was complete by TLC detection, and the crude product was obtained by concentration. After dilution with dichloromethane (30 mL), the mixture was washed three times with pure water (15 mL), the organic phase was separated, dried over anhydrous sodium sulfate for 2 h, and concentrated under reduced pressure to obtain a white solid compound 2-4.

[0143] 3) Synthesis of compound 2-7: Compound 2-5 (21.91 g, 48 mmol) was dissolved in 250 mL of anhydrous dichloromethane, and compound 2-6, i.e. Boc-beta-alanine (9.59 g, 50 mmol) and EEDQ (17.78 g, 72 mmol) were added respectively, and the mixture was reacted at 25°C for 12 h. After TLC monitoring, the reaction was completed and the solvent was concentrated under reduced pressure. The mixture was purified by column chromatography (DCM, DCM: MeOH = 200: 1 to 60: 1) to obtain a light yellow solid compound 2-7 (20.13 g, yield 66.8%).

[0144] 4) Synthesis of compound 2-8: At 0°C, compound 2-7 (20.13 g, 32.2 mmol) and carbon tetrabromide (16.11 g, 48.7 mmol) were placed in a three-necked flask, nitrogen was replaced, and anhydrous tetrahydrofuran was injected into the flask. Triphenylphosphine (12.89 g, 49.2 mmol) was slowly added dropwise to the reaction system, and the reaction was carried out at 25°C in the dark for 14 hours. After TLC monitoring, the reaction was completed, and the solvent was concentrated under reduced pressure to obtain a crude product. Purification by column chromatography (PE, PE:DCM=2:1 to 1:3, DCM:EA=30:1 to 10:1) gave a light yellow solid compound 2-8 (21.6 g, yield 97.6%)

[0145] 5) Synthesis of compound 2-10: Under nitrogen protection, compound 2-8 (25.6 g, 37.2 mmol), compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide (7.11 g, 39 mmol) and silver oxide (12 g, 52 mmol) were weighed into a three-necked flask, injected with 320 mL of anhydrous acetonitrile, and reacted at 25 ° C in the dark for 12 hours. TLC monitoring, the reaction was completed, and the solvent was concentrated under reduced pressure. Dilute with dichloromethane, filter with diatomaceous earth, collect the filtrate, and concentrate to obtain a crude product. Purify by column chromatography (DCM, DCM: MeOH = 200: 1 to 30: 1) to obtain compound 2-10 (14.4 g, yield 49.1%) as a light yellow solid.

[0146] 6) Synthesis of compound 2-11: Under nitrogen protection, compound 2-10 (7.57 g, 9.6 mmol) was dissolved in anhydrous tetrahydrofuran (100 mL) in an ice-salt bath, and borane dimethyl sulfide (2.0 M, dissolved in tetrahydrofuran) (14.4 mL, 26.99 mmol) was slowly added. After stirring for 30 min, the ice-salt bath was removed and the reaction was carried out at 25°C for 3 h. After the reaction was completed by TLC monitoring, the unreacted borane dimethyl sulfide was quenched with methanol in an ice bath. The reaction solution was concentrated under reduced pressure to obtain compound 2-11 as a yellow solid, which was directly used for the next step without purification.

[0147] 7) Synthesis of compound 2-12: Under nitrogen protection, compound 2-11 (3.5 g, 3.48 mmol) was dissolved in anhydrous dichloromethane (100 mL) in an ice bath, triphosgene (0.66 g, 1.74 mmol) was added, DIEA (2.33 g, 13.92 mmol) was slowly added dropwise, and the ice bath was removed, and the reaction was carried out at 25°C for 15 min. Compound 2-12 was obtained and directly used for the next step without treatment.

[0148] 8) Synthesis of compound 2-13: Under nitrogen protection, compound 2-12 (3.78 g, 4.51 mmol) and DMAP (0.66 g, 5.41 mmol) were added to the reaction system, followed by SN38 (2.65 g, 6.76 mmol), and the reaction was carried out at 25°C for 12 h. After TLC monitoring, the reaction was completed and the solvent was concentrated under reduced pressure. The reaction solution was concentrated, diluted with dichloromethane (30 mL), and then washed three times with saturated ammonium chloride solution (10 mL). The organic phase was separated, dried over anhydrous sodium sulfate for 2 h, and concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (DCM, DCM: MeOH = 200: 1 to 50: 1) to obtain compound 2-13 as a yellow solid (3.99 g, yield 66.6%)

[0149] 9) Synthesis of compound 2-14: Compound 2-13 (3.99 g, 3.34 mmol) was dissolved in 50 mL of dichloromethane, and 30 mL of 4M HCl-dioxane solution was added, and the mixture was reacted at 25°C for 4 h. The reaction was complete when detected by TLC, and the reaction system was directly decompressed to completely remove the solvent to obtain viscous solid compound 2-14.

[0150] 10) Synthesis of compound 2-15: Compound 2-14 (2.29 g, 2.09 mmol) and compound 2-4 (0.95 g, 2.09 mmol) were added to a reaction flask, anhydrous dichloromethane (50 mL) and DIEA (0.68 g, 5.26 mmol) were added. The reaction was carried out at 25°C for 14 h. The reaction was complete by TLC detection and the crude product was obtained by concentration. After dilution with dichloromethane (30 mL), the product was washed three times with saturated ammonium chloride solution (15 mL), the organic phase was separated, dried over anhydrous sodium sulfate for 2 h, and purified by column chromatography (DCM, DCM: MeOH = 150: 1 to 25: 1) to obtain a yellow solid compound 2-15 (2.2 g, yield 73.3%)

[0151] 11) Synthesis of compound 2-16: Compound 2-15 (0.6 g, 0.42 mmol) was dissolved in 6 mL of dichloromethane, and 0.6 mL of 4 M HCl-dioxane solution was added, and the mixture was reacted at 25°C for 4 h. The reaction was complete when detected by TLC, and the reaction system was directly decompressed to completely remove the solvent to obtain viscous solid compound 2-16.

[0152] 12) Synthesis of compound 2-17: Compound 2-16 (0.56 g, 0.42 mmol) was dissolved in 30 mL of a mixed solution of dichloromethane: acetonitrile = 1:1, and lithium hydroxide aqueous solution (0.25 M, 18.3 mL) was added dropwise to the reaction system under ice bath conditions and reacted at 25°C for 30 min. LC-MS detected that the reaction was complete, 1 M HCl was added to adjust the pH to 3-4, and the crude product was concentrated. The crude product was purified by reverse phase column chromatography to obtain a light yellow solid compound 2-17.

[0153] Example 3

[0154] The functionalized triethylene glycol-modified monovalent anisamide small molecule ligand prepared in Example 1 (i.e., compound 1-9) is combined with the glucuronic acid trigger and the self-closed ring-forming Linker + active drug + maleimide drug linker (i.e., compound 2-17) in Example 2. The reaction route is as follows:

[0155]

[0156] The specific steps are as follows:

[0157] 1) Synthesis of compound 3-2: Under strict nitrogen protection, compound 3-1, i.e. compound 2-17 (152 mg, 0.127 mmol) obtained in Example 2 and compound 1-9 (69 mg, 0.141 mmol) obtained in Example 1 were dissolved in a mixed solution of MeOH:H2O=1:1, and CuSO4·5H2O (64 mg, 0.25 mmol) and sodium ascorbate (51 mg, 0.25 mmol) were added, and the mixture was reacted at room temperature for 3 h. The reaction solution was purified by reverse phase column chromatography to obtain compound 3-2;

[0158] 2) Synthesis of compound 3-4: Compound 3-2 (136 mg, 0.081 mmol) and compound 3-3, 6-(maleimido)hexanoic acid succinimidyl ester (33 mg, 0.11 mmol) were dissolved in anhydrous DMF, DIEA was added to adjust the pH to 7-8, and the reaction was carried out at room temperature for 4 hours. The reaction solution was purified by reverse phase column chromatography to obtain compound 3-4, which was numbered as prodrug L1878.

[0159] Figure 1 This is the nuclear magnetic hydrogen spectrum characterization diagram of the prodrug L1878 in the example. Figure 2 This is the NMR carbon spectrum characterization diagram of the prodrug L1878 in the example. Figure 3 This is a high-resolution mass spectrum of the prodrug L1878 of the example. Figure 4 , 5 is the purity test of the prodrug L1878 of the embodiment. Figure 1-5 It can be seen that the purity of the prodrug L1878 was tested by HPLC and the purity of L1878 was measured to be 97.91%.

[0160] Example 4 ABS and PBS stability test of the compound

[0161] In order to study the prodrug L1878 prepared in Example 3 in PBS (pH=7.4) simulating the physiological environment and ABS (pH=5.4) simulating the tumor microenvironment, HPLC analysis was used to detect the release ratio of SN38 to evaluate the stability of the drug.

[0162] The specific operation is as follows: dissolve L1878 in DMSO, the solution concentration is (10mM), take 40μL of the mother solution and add it to 360μL of PBS solution or ABS solution, and incubate at 37℃ with constant temperature shaking. Use a pipette to aspirate 30μL of the test solution at different time points (1, 3, 5, 7 days) and add it to 120μL of acetonitrile and mix well. Use HPLC to analyze the diluted test solution. (Each group of experiments is repeated three times)

[0163] The HPLC detection method is as follows: Phase A is 0.1% trifluoroacetic acid aqueous solution, and Phase B is acetonitrile. The elution gradient is (B) 0-1min: 5%; 1-16min: 5%-95%; 16-18min: 95%; 18-20min: 95%-5%; 21min: end the method.

[0164] The test results are shown in Table 1: L1878 has excellent stability in both ABS solution and PBS solution, and the release of SN38 was not detected until the seventh day.

[0165] Table 1 Release of SN38 from S1878 in PBS and ABS

[0166]

[0167] In vitro drug release experiment of Example 5 compound

[0168] In order to verify that the prodrug L1878 prepared in Example 3 can release SN38 under the action of β-glucuronidase, this study used HPLC to detect the release amount of SN38, thereby studying the in vitro release properties of the drug.

[0169] The specific operation is as follows: dissolve L1878 in DMSO to prepare a mother solution with a concentration of 20mM. Take 15μL of the mother solution and dilute it to 1500μL with PBS solution (pH=7.4) or ABS solution (pH=5.4). Add β-glucuronidase (250U) to the diluted solution, divide the solution into three equal parts and incubate at 37°C with shaking. Take out the sample (100μL) at a specific time point (15min, 30min, 60min, 90min, 120min, 180min, 240min, 300min), and immediately add pre-cooled acetonitrile (100μL) to terminate the enzymatic reaction. Centrifuge the sample at 4°C for half an hour, take the supernatant for HPLC, and detect the release amount of SN38. (Each group of experiments was repeated three times)

[0170] The HPLC detection method is as follows: Phase A is 0.1% trifluoroacetic acid aqueous solution, and Phase B is acetonitrile. The elution gradient is (B) 0-1min: 5%; 1-16min: 5%-95%; 16-18min: 95%; 18-20min: 95%-5%; 21min: end the method.

[0171] Test results such as Figure 6 and Figure 7As shown, in PBS (pH = 7.4) solution, L1878 completely released SN38 within 3 hours. In ABS (pH = 5.4) solution, L1878 completely released SN38 in nearly 2 hours. This shows that the drug release performance is better in the acidic environment simulating the tumor microenvironment.

[0172] Example 6 Experimental study on the proliferation inhibition activity of three experimental groups of compounds SN38, L1878, and L1878+β on 22RV1, PC-3, BXPC-3, and 4T1 cell lines (22RV1 and PC-3 are prostate cancer cells), i.e., cytotoxicity experiment

[0173] In order to evaluate the effect of the L1878 prodrug compound prepared in Example 3 on the proliferation ability of tumor cells, the MTT method was used to evaluate the cytotoxicity of SN38, L1878, and L1878+β on four cell lines: 22RV1, PC-3, BXPC-3, and 4T1.

[0174] Dissolve SN38 and L1878 in a small amount of DMSO, and dilute the stock solution in the complete culture medium corresponding to different cells (DMSO content ≤ 2%), with a maximum experimental concentration of 0.1mM. Take the cultured 22RV1, PC-3, BXPC-3, 4T1 cells, and inoculate their single cell suspension in a 96-well plate (6000 cells / well). Place the 96-well plate in a constant temperature sterile cell culture incubator for 24 hours. After the cells adhere to the wall, add a series of concentrations of SN38, L1878, and L1878+β solutions (drug concentrations of 0.01, 0.1, 1, 10, 100, 1000, 10000, 100000nM, respectively) to the cell culture wells in the 96-well plate, and set up 3 replicates for each drug concentration. At the same time, set up a blank group with only complete culture medium, MTT and triple solution, and a control group with cells, complete culture medium, MTT and triple solution but no drug administration. After administration, the 96-well plate was placed in a cell culture incubator for 72 hours. After 72 hours, 100 μL of culture solution containing MTT (MTT concentration was 0.5 mg / mL) was added to each well. After the addition was completed, the 96-well plate was placed in the incubator for 4 hours. 100 μL of the triple solution was added. After the addition was completed, it was placed in a cell culture incubator overnight and the absorbance OD value was measured with an enzyme marker. The data was fitted with GrapHPad Prism 8 software to calculate the cell survival rate.

[0175] The test results are shown in Table 2. Table 2 shows the results of the proliferation inhibition activity of the cytotoxic drug SN38, the example prodrug compound L1878, and L1878 with additional β-glucuronidase on 22RV1, PC-3, BXPC-3, and 4T1 cell lines.

[0176] Table 2 In vitro cytotoxicity data of SN38, S1878, and S1878+β-Glu

[0177]

[0178] As can be seen from Table 2: In the four tumor cell lines, the prodrug L1878 still showed strong cytotoxicity without the additional addition of β-glucuronidase (the strongest toxicity was in the prostate cancer 22RV1 cell line). After the additional addition of β-glucuronidase, the drug activity was greatly improved, and the activity of the prodrug L1878 after cleavage by β-glucuronidase was comparable to that of SN38. This also confirms the successful application of the prodrug molecule at the cellular level.

[0179] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A small molecule drug conjugate based on anisamide targeting human prostate cancer cells, wherein the small molecule drug conjugate is a prodrug, characterized in that: It has a structure as shown in general formula I: Wherein, n1 and n2 both represent the number of repeating units; n1=1, 2, 6 or 10; n2=1, 2 or 3.

2. The small molecule drug conjugate according to claim 1, characterized in that: In the general formula I, n1=1; n2=1, and the structural formula of the small molecule drug conjugate is as follows:

3. The method for preparing a small molecule drug conjugate according to claim 1 or 2, characterized in that: The preparation method comprises three steps: S1: Preparation of functionalized PEG-modified anisamide small molecule ligand; S2: preparing a compound comprising a glucuronic acid trigger and a prodrug linker consisting of a self-closing ring and a toxic payload SN38; S3: firstly connect the anisamide small molecule ligand of S1 and the compound containing the prodrug linker and the toxic payload of S2, and then connect maleimide to obtain the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect; Said S1: preparation of functionalized PEG-modified anisamide small molecule ligand, the preparation route thereof is as follows: S1 specifically includes the following sub-reaction steps: 1a: Compound 1 was dissolved in DMSO, 4M NaOH solution was added, and then tert-butyl acrylate was added, and the mixture was reacted at 25°C for 24 hours to obtain compound 1-1; the molar ratio of compound 1 to tert-butyl acrylate was 1:n2*1.1; 1b: Disperse compound 1-2, i.e., 5-hexynoic acid, in anhydrous dichloromethane, add EDCI and NHS, react at 25°C for 6h, then add compound 1-1 and DIEA, and continue to react at 25°C overnight to obtain compound 1-3; the molar ratio of compound 1-1 to compound 1-2, i.e., 5-hexynoic acid, is 1:1.1; the molar ratio of compound 1-1 to EDCI is 1:1.3; the molar ratio of compound 1-1 to NHS is 1:1.3; the molar ratio of compound 1-1 to DIEA is 1:2.5; 1c: Dissolve compound 1-3 in anhydrous dichloromethane, add trifluoroacetic acid in a volume equal to that of anhydrous dichloromethane, and react at 25°C for 3h to obtain compound 1-4; 1d: Compound 1-4 was dissolved in N,N-dimethylformamide, and compound 1-5, EDCI, HOBT and DIEA were added, and the mixture was reacted overnight at 25°C to obtain compound 1-6; the molar ratio of compound 1-4 to compound 1-5 was 1:n2*1.1; the molar ratio of compound 1-4 to EDCI was 1:n2*1.3; the molar ratio of compound 1-4 to HOBT was 1:n2*1.3; the molar ratio of compound 1-4 to DIEA was 1:n2*3; 1e: Dissolve compound 1-6 in dichloromethane, add TFA trifluoroacetic acid in a volume equal to that of dichloromethane, and react at 25°C for 3h to obtain compound 1-7; 1f: Compound 1-7 is dissolved in anhydrous dichloromethane, compound 1-8, i.e., p-methoxybenzoyl chloride and DIEA are added, and the mixture is reacted overnight at 25°C to obtain compound 1-9; the molar ratio of compound 1-7 to compound 1-8, i.e., p-methoxybenzoyl chloride, is 1:n2*1.2; the molar ratio of compound 1-7 to DIEA is 1:n2*4; S2: Preparation of a compound comprising a glucuronic acid trigger, a prodrug linker consisting of a self-closed ring and a toxic payload SN38: S2 specifically includes the following sub-reaction steps: 2a: Compound A, i.e., γ-aminobutyric acid, is dissolved in a mixed solvent of methanol and water in a volume ratio of 10:1, 1H-imidazole-1-sulfonyl azide hydrochloride, copper sulfate pentahydrate and potassium carbonate are added, and the mixture is reacted at 25° C. for 12 hours to obtain compound B; the molar ratio of compound A to 1H-imidazole-1-sulfonyl azide hydrochloride is 1:1.1; the molar ratio of compound A to copper sulfate pentahydrate is 1:0.03; and the molar ratio of compound A to potassium carbonate is 1:2; 2b: Compound B, i.e., 4-azidobutyric acid, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25° C. for 12 h to obtain compound 2-1; the molar ratio of the compound B to NHS was 1:1.1; the molar ratio of the compound B to EDCI was 1:1.05; 2c: Compound 2-2, i.e. Boc-L-lysine, and compound 2-1 were added to anhydrous acetonitrile, and then DIEA was added, and the mixture was reacted at 25°C for 10 hours to obtain compound 2-3; the molar ratio of compound 2-1 to compound 2-2, i.e. Boc-L-lysine, was 1:1; the molar ratio of compound 2-1 to DIEA was 1:2.5; 2d: Compound 2-3, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25°C for 12 hours to obtain compound 2-4; the molar ratio of the compound 2-3 to NHS was 1:1.1; the molar ratio of the compound 2-3 to EDCI was 1:1.03; 2e: Under nitrogen protection, compound C, i.e., AD-glucuronic acid methyl ester, was dissolved in anhydrous acetonitrile, and 4-hydroxy-3-nitrobenzaldehyde and silver oxide were added respectively, and the mixture was reacted at 25° C. in the dark for 12 h to obtain compound D; the molar ratio of compound C to 4-hydroxy-3-nitrobenzaldehyde was 1:1.1; the molar ratio of compound C to silver oxide was 1:1.4; 2f: At 0°C, compound D, i.e. (2S,3R,4S,5S,6S)-2-(4-formyl-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, was dissolved in a mixed solvent of V i-PrOH:V CHCl3 with a volume ratio of 1:5, and then NaBH4 and silica gel were added respectively, and the mixture was reacted at 25°C for 2.5h to obtain compound E; the molar ratio of compound D to NaBH4 was 1:3; 2g: Under a hydrogen atmosphere, compound E, i.e. (2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-2-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triacetate and platinum dioxide were dissolved in ethyl acetate and reacted at 25°C for 6h to obtain compound 2-5; the mass ratio of compound E to platinum dioxide was 1:0.2; 2h: Compound 2-5 was dissolved in anhydrous dichloromethane, and compound 2-6, i.e. Boc-beta-alanine and EEDQ were added respectively, and the mixture was reacted at 25°C for 12h to obtain compound 2-7; the molar ratio of compound 2-5 to compound 2-6, i.e. Boc-beta-alanine, was 1:1.05; the molar ratio of compound 2-5 to EEDQ was 1:1.5; 2i: At 0°C, compound 2-7 and carbon tetrabromide are added to anhydrous tetrahydrofuran, triphenylphosphine is slowly added dropwise to the reaction system, and the mixture is reacted at 25°C in the dark for 14 hours to obtain compound 2-8; the molar ratio of compound 2-7 to carbon tetrabromide is 1:1.5; the molar ratio of compound 2-7 to triphenylphosphine is 1:1.5; 2j: Under nitrogen protection, compound 2-8, compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, and silver oxide were added to anhydrous acetonitrile, and reacted at 25°C in the dark for 12 hours to obtain compound 2-10; the molar ratio of the compound 2-8 to the compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, was 1:1.05; the molar ratio of the compound 2-8 to silver oxide was 1:1.4; 2k: Under nitrogen protection, compound 2-10 was dissolved in anhydrous tetrahydrofuran in an ice-salt bath, 2.0 M borane dimethyl sulfide was slowly added, stirred for 30 min, the ice-salt bath was removed, and the reaction was carried out at 25 °C for 3 h to obtain compound 2-11; the molar ratio of compound 2-10 to borane dimethyl sulfide was 1:2.8; 2l: Under nitrogen protection, compound 2-11 was dissolved in anhydrous dichloromethane in an ice bath, triphosgene was added, DIEA was slowly added dropwise, and then the ice bath was removed, and the reaction was carried out at 25°C for 15 minutes to obtain compound 2-12; the molar ratio of the compound 2-11 to triphosgene was 1:0.5; the molar ratio of the compound 2-11 to DIEA was 1:4; 2m: Under nitrogen protection, compound 2-12 and DMAP were added to the reaction system, followed by SN38, and the reaction was carried out at 25°C for 12 hours to obtain compound 2-13; the molar ratio of compound 2-12 to DMAP was 1:1.2; the molar ratio of compound 2-12 to SN38 was 1:1.5; 2n: Compound 2-13 was dissolved in dichloromethane, 4M HCl-dioxane solution was added, and the mixture was reacted at 25°C for 4 hours to obtain compound 2-14; 2o: Compound 2-14 and compound 2-4 were added to anhydrous dichloromethane, DIEA was added, and the mixture was reacted at 25°C for 14 hours to obtain compound 2-15; the molar ratio of compound 2-14 to compound 2-4 was 1:1; the molar ratio of compound 2-14 to DIEA was 1:2.5; 2p: Dissolve compound 2-15 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-16; 2q: Compound 2-16 was dissolved in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1, and a 0.3M lithium hydroxide aqueous solution was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at 25°C for 30 minutes to obtain compound 2-17; The S3 described above: firstly connect the anisamide small molecule ligand of S1 and the compound containing the prodrug linker and the toxic payload of S2, and then connect maleimide to obtain the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect; the reaction route is as follows: S3 specifically includes the following sub-reaction steps: 3a: Under strict nitrogen protection, the compound 2-17 prepared by S2 and the compound 1-9 prepared by S1 were dissolved in a mixed solution of MeOH and H2O with a volume ratio of 1:1, CuSO4·5H2O and sodium ascorbate were added, and the mixture was reacted at 25°C for 3h to obtain compound 3-2; the molar ratio of the compound 2-17 to the compound 1-9 was 1:1.2; the molar ratio of the compound 2-17 to CuSO4·5H2O was 1:2; the molar ratio of the compound 2-17 to sodium ascorbate was 1:2; 3b: Compound 3-2 and compound 3-3, ie, 6-(maleimido)hexanoic acid succinimidyl ester, were dissolved in anhydrous DMF, DIEA was added to adjust the pH to 7-8, and the mixture was reacted at 25° C. for 4 h to obtain compound 3-4; the molar ratio of compound 3-2 to compound 3-3 was 1:1.

4.

4. The method for preparing a small molecule drug conjugate according to claim 2, characterized in that: The steps include: 1) Prepare a functionalized triethylene glycol-modified anisamide small molecule ligand, the reaction route is as follows: The specific process is as follows: A: Disperse compound 1-2, i.e. 5-hexynoic acid, in anhydrous dichloromethane, add EDCI and NHS, react at 25°C for 6h, then add compound 1-1 and DIEA, and continue to react at 25°C overnight to obtain compound 1-3; the molar ratio of compound 1-1 to compound 1-2, i.e. 5-hexynoic acid, is 1:1.1; the molar ratio of compound 1-1 to EDCI is 1:1.3; the molar ratio of compound 1-1 to NHS is 1:1.3; the molar ratio of compound 1-1 to DIEA is 1:2.5; B: Dissolve compound 1-3 in anhydrous dichloromethane, add trifluoroacetic acid in a volume equal to that of anhydrous dichloromethane, and react at 25°C for 3h to obtain compound 1-4; C: Compound 1-4 was dissolved in N,N-dimethylformamide, and compound 1-5, EDCI, HOBT and DIEA were added, and the mixture was reacted overnight at 25°C to obtain compound 1-6; the molar ratio of compound 1-4 to compound 1-5 was 1:1.1; the molar ratio of compound 1-4 to EDCI was 1:1.3; the molar ratio of compound 1-4 to HOBT was 1:1.3; the molar ratio of compound 1-4 to DIEA was 1:3; D: Dissolve compound 1-6 in dichloromethane, add TFA trifluoroacetic acid in a volume equal to that of dichloromethane, and react at 25°C for 3 hours to obtain compound 1-7; E: Compound 1-7 is dissolved in anhydrous dichloromethane, compound 1-8 (p-methoxybenzoyl chloride) and DIEA are added, and the mixture is reacted at 25°C overnight to obtain compound 1-9; the molar ratio of compound 1-7 to compound 1-8 (p-methoxybenzoyl chloride) is 1:1.2; the molar ratio of compound 1-7 to DIEA is 1:4; 2) Prepare a compound comprising a glucuronic acid trigger and a prodrug linker consisting of a self-closed ring and a toxic payload SN38, the reaction route is as follows: The specific process is as follows: A: Compound 2-2, i.e. Boc-L-lysine, and compound 2-1 were added to anhydrous acetonitrile, and then DIEA was added, and the mixture was reacted at 25°C for 10 hours to obtain compound 2-3; the molar ratio of compound 2-1 to compound 2-2, i.e. Boc-L-lysine, was 1:1; the molar ratio of compound 2-1 to DIEA was 1:2.5; B: Compound 2-3, NHS and EDCI were added to anhydrous acetonitrile and reacted at 25°C for 12 hours to obtain compound 2-4; the molar ratio of compound 2-3 to NHS was 1:1.1; the molar ratio of compound 2-3 to EDCI was 1:1.03; C: Compound 2-5 was dissolved in anhydrous dichloromethane, and then compound 2-6, i.e. Boc-beta-alanine and EEDQ were added respectively, and the mixture was reacted at 25°C for 12 hours to obtain compound 2-7; the molar ratio of compound 2-5 to compound 2-6, i.e. Boc-beta-alanine, was 1:1.05; the molar ratio of compound 2-5 to EEDQ was 1:1.5; D: At 0°C, compound 2-7 and carbon tetrabromide are added to anhydrous tetrahydrofuran, triphenylphosphine is slowly added dropwise to the reaction system, and the mixture is reacted at 25°C in the dark for 14 hours to obtain compound 2-8; the molar ratio of compound 2-7 to carbon tetrabromide is 1:1.5; the molar ratio of compound 2-7 to triphenylphosphine is 1:1.5; E: Under nitrogen protection, compound 2-8, compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, and silver oxide were added to anhydrous acetonitrile, and reacted at 25°C in the dark for 12 hours to obtain compound 2-10; the molar ratio of the compound 2-8 to the compound 2-9, i.e., 2-hydroxy-5-nitrophenylcarboxamide, was 1:1.05; the molar ratio of the compound 2-8 to silver oxide was 1:1.4; F: Under nitrogen protection, compound 2-10 was dissolved in anhydrous tetrahydrofuran in an ice-salt bath, 2.0 M borane dimethyl sulfide was slowly added, stirred for 30 min, the ice-salt bath was removed, and the reaction was carried out at 25°C for 3 h to obtain compound 2-11; the molar ratio of compound 2-10 to borane dimethyl sulfide was 1:2.8; G: Under nitrogen protection, compound 2-11 was dissolved in anhydrous dichloromethane in an ice bath, triphosgene was added, DIEA was slowly added dropwise, and then the ice bath was removed, and the reaction was carried out at 25°C for 15 minutes to obtain compound 2-12; the molar ratio of the compound 2-11 to triphosgene was 1:0.5; the molar ratio of the compound 2-11 to DIEA was 1:4; H: Under nitrogen protection, compound 2-12 and DMAP were added to the reaction system, followed by SN38, and the reaction was carried out at 25°C for 12 hours to obtain compound 2-13; the molar ratio of compound 2-12 to DMAP was 1:1.2; the molar ratio of compound 2-12 to SN38 was 1:1.5; I: Dissolve compound 2-13 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-14; J: Compound 2-14 and compound 2-4 were added to anhydrous dichloromethane, DIEA was added, and the mixture was reacted at 25°C for 14 hours to obtain compound 2-15; the molar ratio of compound 2-14 to compound 2-4 was 1:1; the molar ratio of compound 2-14 to DIEA was 1:2.5; K: Dissolve compound 2-15 in dichloromethane, add 4M HCl-dioxane solution, and react at 25°C for 4h to obtain compound 2-16; L: Compound 2-16 was dissolved in a mixed solution of dichloromethane and acetonitrile in a volume ratio of 1:1, and a 0.3M lithium hydroxide aqueous solution was added dropwise to the reaction system under ice bath conditions, and the reaction was carried out at 25°C for 30 minutes to obtain compound 2-17; 3) The anisamide small molecule ligand of S1 and the drug linker of S2 are combined to prepare the anisamide-based small molecule drug conjugate targeting human prostate cancer cells as described in the first aspect. The reaction scheme is as follows: The specific steps are as follows: A: Under strict nitrogen protection, the compound 2-17 prepared by S2 and the compound 1-9 prepared by S1 were dissolved in a mixed solution of MeOH and H2O with a volume ratio of 1:1, and CuSO4·5H2O and sodium ascorbate were added, and the reaction was carried out at room temperature for 3 hours to obtain compound 3-2; the molar ratio of the compound 2-17 to the compound 1-9 was 1:1.2; the molar ratio of the compound 2-17 to CuSO4·5H2O was 1:2; the molar ratio of the compound 2-17 to sodium ascorbate was 1:2; B: Compound 3-2 and compound 3-3, ie, 6-(maleimido)hexanoic acid succinimidyl ester, were dissolved in anhydrous DMF, DIEA was added to adjust the pH to 7-8, and the reaction was carried out at 25° C. for 4 hours to obtain compound 3-4; the molar ratio of compound 3-2 to compound 3-3 was 1:1.

4.

5. A pharmaceutical composition, characterized in that It comprises a small molecule drug conjugate based on anisamide targeting human prostate cancer cells and a pharmaceutically acceptable macromolecular carrier thereof as claimed in claim 1 or 2, characterized in that the macromolecular carrier is human serum albumin.

6. Use of a small molecule drug conjugate based on anisamide targeting human prostate cancer cells as claimed in claim 1 or 2 in the preparation of anti-prostate tumor drugs.

7. Use of anisamide-based small molecule drug conjugate targeting human prostate cancer cells prepared by the method of claim 3 or 4 in the preparation of anti-prostate tumor drugs.

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