Anti-tumor drug prodrug, pharmaceutical composition and application in tumor targeted therapy

Abstract

The present application relates to a kind of antitumor prodrugs, pharmaceutical composition and application in the field of tumor targeted therapy.In order to provide a kind of antitumor prodrug with tumor targeting specific recognition and aggregation effect, the present application provides the antitumor prodrug as shown in formula I or formula II: the antitumor drug modification mode described above can effectively improve the antitumor activity of antitumor drug, and a kind of synergistic antitumor administration mode is also provided, with good clinical application value.

Description

[0001] This case is a divisional application of case No. 202210719332.X (application date: June 23, 2022, name of the invention: an anti-tumor drug prodrug, a pharmaceutical composition and its application in the field of tumor targeted therapy). Technical Field

[0002] The present invention belongs to the technical field of anti-tumor targeted drugs, and specifically relates to an anti-tumor drug prodrug, a pharmaceutical composition containing the prodrug, and its application in the field of tumor targeted therapy. Background Art

[0003] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0004] Cancer, also known as a malignant tumor, is a disease caused by a complex combination of factors, characterized by the uncontrolled proliferation of malignant cells. According to the World Health Organization, cancer kills millions of people worldwide each year, posing a serious threat to human health. Currently, chemotherapy remains the primary treatment. However, toxic side effects on normal cells, low bioavailability, and drug resistance remain key bottlenecks in chemotherapy.

[0005] By leveraging the specific recognition and aggregation of an endogenous carrier by tumor tissue, the carrier is covalently or non-covalently linked to an anti-tumor drug in vitro. This allows the carrier-linked anti-tumor drug to effectively reach the tumor site, enhancing tumor targeting. The prototype drug is released under the action of various hydrolytic enzymes in the body, killing tumor cells, increasing the effective concentration of chemotherapy drugs, and reducing toxic side effects on normal cells.

[0006] Prodrugs are compounds that are inactive or have minimal activity after chemically modifying the original drug. Once an anti-tumor prodrug enters tumor tissue, it can be converted into the active original drug via enzymatic or non-enzymatic pathways in vivo, thereby increasing the effective concentration of the active ingredient in the tumor tissue and reducing toxic side effects on normal tissues, ultimately achieving targeted therapy. Among the many endogenous small molecule carriers, long-chain lipids are an ideal carrier for constructing anti-tumor prodrugs. Summary of the Invention

[0007] Based on the above technical background, the purpose of the present invention is to provide an anti-tumor prodrug with tumor-targeted specific recognition and aggregation effects. This type of prodrug can not only reduce the toxicity of the original anti-tumor drug, but also increase the effective therapeutic concentration of the drug, thereby achieving the effect of tumor-targeted treatment and having good clinical application prospects.

[0008] To achieve the above objectives, the present invention uses endogenous long-chain fats in the human body as a targeting carrier, which is non-covalently linked to various anti-tumor drugs to form prodrugs. This design is based on the following facts:

[0009] 1) Long-chain fats are important components of fat, hormones, and proteins. They can provide the energy required for cell growth and play a very important role in maintaining the normal physiological functions of cells. They are a type of endogenous substance that is harmless to the human body.

[0010] 2) Rapidly proliferating tumor cells require large amounts of long-chain fats as a source of energy and metabolism, and they also provide lipids for cell membrane formation. This results in a significantly higher content of long-chain fats in tumor cells than in normal cells. Therefore, prodrugs based on long-chain fats can enhance tumor targeting and enhance translocation to target organs.

[0011] 3) Fatty acids, especially long-chain fatty acids, easily bind to human serum albumin and can serve as conjugate carriers. Therefore, long-chain fatty acids can more efficiently target the drugs they carry to the tumor site by binding to albumin in the body. It is well known that human serum albumin is also a nutrient source required by tumor tissue, and the demand is much higher than that of normal cells. When drugs conjugated with fatty acids bind to serum proteins, on the one hand, they can reduce oxidation of drugs transported in the circulatory system, and on the other hand, the introduction of fatty chains increases lipophilicity, which can prolong the drug's action time in the body. Therefore, the introduction of groups containing long-chain fatty acids can simulate the non-covalent binding of fatty acids to human serum proteins to form a tumor-targeted drug delivery system.

[0012] 4) The introduction of long fatty chains can also adjust the lipid-water partition coefficient of the drug, greatly improve the bioavailability of anticancer drugs, reduce toxic side effects, and improve the drug's drugability.

[0013] Specifically, the present invention provides the following technical solutions:

[0014] In a first aspect, the present invention provides an anti-tumor drug prodrug, wherein the anti-tumor drug and the targeting modifying group are connected via a linker, and the prodrug structure is as shown in Formula I or Formula II:

[0015]

[0016]

[0017] Wherein, drug means antitumor drug;

[0018] R is a targeting modification group, which is one of, but not limited to, a methyl group, a carboxyl group, an amino group, an ester group, a hydroxamic acid group, a hydrazine group, and an amide group containing a substituent;

[0019] A is a linker, which includes but is not limited to one or a combination of disulfide bonds, amide bonds, ester bonds, polyamines or aliphatic groups (such as heterocycles, aromatic heterocycles, aromatic rings, and aliphatic chains).

[0020] Preferably, the anti-tumor drugs include but are not limited to cytotoxic drugs, hormone drugs, biological response modifiers, and monoclonal antibody drugs. As long as they can be modified in the above manner, they meet the requirements of the prodrug provided in the first aspect. In the structure shown in Formula II, the two drugs are the same drug or different drugs. In a preferred embodiment, the two drugs are two different anti-tumor drugs for the purpose of synergistic enhancement.

[0021] Preferably, the anti-tumor drug is 5-fluorouracil, and the prodrug structure is shown in the following formula III or formula IV:

[0022]

[0023]

[0024] Wherein, n=a natural number between 4 and 20.

[0025] In a specific embodiment, the prodrug represented by formula III is selected from any one of the following compounds:

[0026] N-(4-(5-Fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)hexanamide (code name: FA-5FU-B-6C);

[0027] N-(4-(5-Fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)octanamide (code name: FA-5FU-B-8C);

[0028] N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)decanamide (code name FA-5FU-B-10C);

[0029] N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)dodecanoamide (code name FA-5FU-B-12C);

[0030] N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)tetradecanoic acid amide (code name FA-5FU-B-14C);

[0031] N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)palmitamide (code name FA-5FU-B-16C);

[0032] N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)phenyl)stearic amide (Code FA-5FU-B-18C);

[0033] N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)phenyl)stearic amide (Code FA-5FU-B-18C);

[0034] N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)phenyl)stearic amide (Code FA-5FU-B-18C);

[0035] The synthetic route of the compound of Formula III is as follows:

[0036]

[0037] The specific synthesis method is as follows:

[0038] After adding Et3N and K2CO3 to the DMSO solution of 5-Fu and reacting for a period of time, the DMSO solution of 1-fluoro-4-nitrobenzene is added dropwise; after the above reaction system is reacted at 55-65°C under N2 atmosphere for 4-6 hours, it is cooled, the yellow precipitate is collected, and Pd-C catalytic hydrogenation is performed to obtain a yellow oil; the yellow oil is dissolved in dichloromethane, different carbon chain length n-alkanoic acid, HOBt and EDCI are added under ice bath conditions and reacted for 10-20 min, N,N-diisopropylethylamine is added and the reaction is continued at room temperature for 4-5 h, then the crude product is washed and dried, and the crude product is purified by column chromatography with ethyl acetate / petroleum ether to obtain the target compound of Formula III.

[0039] In a specific embodiment, the prodrug of Formula IV is selected from any one of the following compounds:

[0040] (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)methyl 4-(tetradecyloxy)benzoate (Code LFC14-Ben-5FU);

[0041] (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)methyl 4-(tetradecyloxy)benzoate (Code LFC14-Ben-5FU);

[0042] (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)methyl 4-(tetradecyloxy)benzoate (Code LFC14-Ben-5FU).

[0043] The synthetic route of the compound of Formula IV is as follows:

[0044]

[0045] The specific synthesis method is as follows:

[0046] 5-Fu was mixed with a 30-40% formaldehyde aqueous solution, acetonitrile was added, the temperature was raised to 55-65°C, and the mixture was stirred until the solid dissolved, and the reaction was continued for 40-50 minutes. The formaldehyde, water, and acetonitrile in the reaction system were removed to obtain a colorless, transparent, viscous liquid product, 1,3-dihydroxymethyl-5-fluorouracil. 1,3-dihydroxymethyl-5-fluorouracil, p-alkoxybenzoic acids of different carbon chains, DCC, and DMAP were dissolved in anhydrous acetonitrile, the reaction was carried out under ice bath conditions for 0.5-1.5 hours, the temperature was raised to room temperature, and the reaction was continued for 22-26 hours. The filtrate was partially concentrated, and the residue was washed, dried, and purified by column chromatography with ethyl acetate / petroleum ether (1:1, v:v) to obtain a white solid, which is the compound represented by formula IV.

[0047] Preferably, the anti-tumor drug is paclitaxel, and the prodrug structure is shown in the following formula V:

[0048]

[0049] (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-9-((2R,3S)-3-benzamido-2-(4-(dodecylamino)-4-oxobutanoyl)oxy)-3-phenylpropanoyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxyl-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methylcyclodeca[3,4]benzo[1,2-b]oxy-6,12b-diacetate (code name: FA-Taxol);

[0050] The synthetic route of the compound represented by the above formula V is as follows:

[0051]

[0052] The specific synthesis steps are as follows:

[0053] Paclitaxel is dissolved in anhydrous pyridine, stirred, and then succinic anhydride is added. The reaction is carried out at room temperature for 3 to 5 hours, and then the solvent is removed. Water is added and the pH of the reaction system is adjusted to 2 to 3. The reaction is extracted with ethyl acetate, and the organic phase is partially washed and dried. After removing the solvent, succinyl paclitaxel is obtained. Succinyl paclitaxel is dissolved in anhydrous THF, isobutyl chloroformate and TEA are added, and after stirring for 4 to 6 minutes, long-chain alkylamines of different carbon chains are added. The reaction is continued for 0.8 to 1.2 hours until the reaction is complete. After removing the solvent, ethyl acetate is added for extraction, and the organic phase is washed with water and saturated brine, respectively. The organic phase is collected and concentrated, dried over anhydrous sodium sulfate, and the crude product is purified by column chromatography using petroleum ether / acetone to obtain the compound represented by formula V.

[0054] Preferably, the anti-tumor drug is methotrexate, and the prodrug structure is shown in the following formulas VI-1 to VI-4:

[0055]

[0056] The synthesis method of the compounds represented by formula VI-1 to VI-4 is as follows:

[0057] Dissolve methotrexate in anhydrous THF, add isobutyl chloroformate and triethylamine, stir evenly, then add alkylamines of different carbon chain lengths and continue the reaction for 0.8 to 12 hours. After removing the solvent, add ethyl acetate to extract the product and purify it by column chromatography.

[0058] Preferably, the anti-tumor drug is temozolomide, and the prodrug structure is shown in the following formula VII:

[0059] Wherein, n is 11, 13 or 15;

[0060] In a specific embodiment, the prodrug represented by formula VII is selected from any one of the following compounds:

[0061] Dodecyl-3-methyl-4-oxy-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylate;

[0062] Tetradecyl-3-methyl-4-oxy-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylate;

[0063] Hexadecyl-3-methyl-4-oxy-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxylate.

[0064] The synthesis method of the compound represented by formula VII is as follows:

[0065] Temozolomide is dissolved in concentrated sulfuric acid, and a NaNO2 aqueous solution is added dropwise under an ice bath and reacted for 0.8 to 1.2 hours. The reaction is continued at room temperature for 0.8 to 1.2 hours. The temperature is raised to 45 to 55°C and the reaction is continued for 1 to 2 hours. The reaction system is cooled to obtain a precipitate, which is the intermediate. The intermediate, DCC and DMAP are dissolved in DMF, and after a period of reaction, alkyl alcohols of different carbon chain lengths are added. The reaction is continued at room temperature for 45 to 50 hours until the reaction is complete. Ethyl acetate is added to the reaction solution for extraction, and the organic phase is washed with water and saturated brine, respectively. The concentrated organic phase is collected and dried over anhydrous sodium sulfate, and the crude product is purified by column chromatography with ethyl acetate / petroleum ether = 3:1 (v:v) to obtain the compound represented by formula VII.

[0066] In a second aspect, the present invention provides a pharmaceutical composition comprising the anti-tumor drug prodrug described in the first aspect.

[0067] Preferably, in the pharmaceutical composition, the anti-tumor drug prodrug should be in a therapeutically effective dose, or be capable of releasing a therapeutically effective dose of the anti-tumor drug. The "therapeutically effective dose" refers to an amount effective to achieve the desired therapeutic or preventive effect at the necessary dose and time period, including eliminating, reducing, delaying, minimizing, or preventing the adverse effects of the disease. The content of the active ingredient corresponding to the effective dose can be determined by conventional means based on the subject being treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the anti-tumor drug prodrug can range from about 0.01 to 99%, 0.1 to 70%, 1 to 30%, 0.01 to 0.05%, 0.05 to 0.1%, 0.1 to 0.3%, 0.3 to 0.5%, 0.5 to 1%, 1 to 3%, 3 to 5%, 5 to 10%, 10 to 20%, 20 to 30%, 30 to 50%, 50 to 70%, or 70 to 99%.

[0068] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier in the art, and the dosage of the pharmaceutically acceptable carrier should be harmless to the subject, specifically including but not limited to buffers, antioxidants, preservatives, bactericides, hydrophilic polymers, amino acid monosaccharides, disaccharides and other carbohydrates, chelating agents, tension regulators, surfactants, salt-forming counterions, metal complexes and / or non-ionic surfactants.

[0069] Preferably, in the pharmaceutical composition, the anti-tumor drug prodrug is administered as a single active ingredient, or in combination with other active ingredients; in a specific embodiment, in the pharmaceutical composition, the compound represented by formula III is administered in combination with the tumor suppressor protein p53.

[0070] In addition, when the above-mentioned pharmaceutical composition is used to prepare a pharmaceutical preparation for in vivo administration, the pharmaceutical preparation should be sterile. The sterility of the pharmaceutical preparation can be achieved by conventional methods in the art, such as filtration through a sterile filter membrane.

[0071] The third aspect of the present invention provides the use of the anti-tumor drug prodrug described in the first aspect and the pharmaceutical composition described in the second aspect in the field of tumor targeted therapy.

[0072] Preferably, the application in the field of tumor targeted therapy includes but is not limited to any of the following:

[0073] (1) Used in the preparation and development of anti-tumor preparations;

[0074] (2) Used for the treatment of patients with drug-resistant tumors or patients receiving combination therapy.

[0075] In the above aspect (1), the anti-tumor preparation includes but is not limited to anti-tumor drugs or anti-tumor model agents; further, the anti-tumor drug is an oral preparation and a parenteral preparation, such as a tablet, pill, capsule or injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0077] Figure 1 To demonstrate the in vitro anti-tumor growth effects of the FA-5FU-B series of compounds;

[0078] Figure 1 (A) The effects of LFC-B-5FU series compounds on SJSA-1 cells;

[0079] Figure 1 (B) shows the effects of FA-B-5-FU series compounds on MDA-MB-231 cells;

[0080] Figure 1 (C) shows the effects of FA-B-5-FU series compounds on MCF-7 cells;

[0081] Figure 2 This is a graph showing the in vivo tumor inhibition results of the compound FA-Taxol in Example 2;

[0082] Figure 3 For Example 5 compound LFC 12 -Ben-5FU and LFC 14 - In vivo tumor inhibition results of Ben-5FU;

[0083] Figure 4 for LFC 12 -MTX, FA-MTX-C, FA-MTX-A, LFC 12 -MTZ and FA-B-12C synergistic antitumor activity with the tumor suppressor protein p53. DETAILED DESCRIPTION

[0084] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0085] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0086] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0087] Example 1 Preparation of 5-fluorouracil (5FU) prodrug (3)

[0088] The compound of formula I of the present invention can be prepared by the following reaction route:

[0089]

[0090] The reagents used in the above preparation process are: a) Et3N, K2CO3, DMSO; then 10% Pd / C, H2; b) EDCI, HOBt, DIPEA, CH2Cl2.

[0091] Specifically, the present invention provides the preparation process of some illustrative compounds and effect verification data in the examples.

[0092] Preparation of Intermediate 1-(4-aminophenyl)-5-fluoropyrimidine-2,4(1H,3H)-dione (2)

[0093] 5-Fu (3 g, 23 mmol) was dissolved in 10 mL of dry DMSO and heated to 60 °C until 5-Fu was completely dissolved. Et3N (2.3 g, 23 mmol) and K2CO3(1.59 g, 11.5 mmol) were added successively. After 10 min of further reaction, 1-fluoro-4-nitrobenzene (3.2 g, 23 mmol) dissolved in 5 mL of dry DMSO was added dropwise. The reaction was continued at 60 °C under N2atmosphere for about 5 h until the reaction was completed as monitored by TLC. Ice water was added and the yellow precipitate was collected and recrystallized from ethyl acetate / petroleum ether (1 : 1, v:v) and dried. The solid was dissolved in dry methanol and hydrogenated using 10% Pd-C until the reaction was completed. The crude product obtained was recrystallized from dry methanol to give a yellow oil with a two-step yield of 62% which was used directly in the next step.

[0094] Preparation of N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)phenyl) dodecanamide (3d, code FA-5FU-B-12C)

[0095] Compound 2 (250 mg, 1.13 mmol) was dissolved in dichloromethane (15 mL) and placed in an ice bath at 0 °C. Dodecanoic acid (230 mg, 1.13 mmol), HOBt (370 mg, 2.26 mmol) and EDCI (430 mg, 2.26 mmol) were added successively. The reaction mixture was allowed to react at 0 °C for 15 min and then N,N-diisopropylethylamine (530 mg, 3.39 mmol) was added. The ice bath was removed and the reaction was allowed to continue at room temperature for 4-5 h. The reaction mixture was washed with 5% KHSO4(3 x 5 mL), saturated NaHCO3(3 x 5 mL) and saturated brine (25 mL) successively. The combined organic phase was dried over anhydrous MgSO4and the organic solvent was evaporated. The crude product was purified by column chromatography using ethyl acetate / petroleum ether (1 : 1, v:v) to give the target compound as a white solid with a yield of 45% and a melting point of 150-152 °C. ESI-MS (m / z): 404.2 [M+H] + ; 426.2 [M+Na] + ; 442.2 [M+K] + ; C 22 H 30 FN3O3(403.23).

[0096] Preparation of N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)phenyl) hexanamide (3a, code FA-5FU-B-6C)

[0097] Prepared in a similar manner to compound 3d, except that n-dodecanoic acid was replaced by n-hexanoic acid. The product was a white solid, yield 55.2%, m.p. 149-151 °C. ESI-MS m / z: 318.3 [M-H] - ; C 16 H 18 FN3O3(319.13).

[0098] Preparation of N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)phenyl)octanamide (3b, code FA-5FU-B-8C)

[0099] Prepared in a similar manner to compound 3d, except that n-dodecanoic acid was replaced by the corresponding reactant with the number of carbon atoms. The product was a white solid, yield 57.1%, m.p. 153-155 °C. ESI-MS m / z: 348.2 [M+H] + ; C 18 H 22 FN3O3(347.16).

[0100] Preparation of N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)phenyl)decanamide (3c, code FA-5FU-B-10C)

[0101] Prepared in a similar manner to compound 3d, except that n-dodecanoic acid was replaced by the corresponding reactant with the number of carbon atoms. The product was a white solid, yield 34.5%, m.p. 153-155 °C. ESI-MS m / z: 376.2 [M+H] + ; C 20 H 26 FN3O3(375.20).

[0102] Preparation of N-(4-(5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)phenyl)tetradecanamide (3e, code FA-5FU-B-14C)

[0103] Prepared in a similar manner to compound 3d, except that n-dodecanoic acid was replaced by the corresponding reactant with the number of carbon atoms. The product was a white solid, yield 44.7%, m.p. 158-160 °C. ESI-MS m / z: 437.3 [M+H] + ; C 24 H 34 FN3O3(431.26).

[0104] Preparation of N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)palmitamide (3f, code FA-5FU-B-16C)

[0105] This product was prepared by a method similar to compound 3d, except that the n-dodecanoic acid in the above preparation method was replaced with a reactant with the corresponding number of carbon atoms. The product was a white solid with a yield of 23.7% and a melting point of 157-158°C. ESI-MS m / z: 460.3 [M+H] + ; C 26 H 38 FN3O3(459.29).

[0106] Preparation of N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)stearamide (3 g, code FA-5FU-B-18C)

[0107] This product was prepared by a method similar to compound 3d, except that the n-dodecanoic acid in the above preparation method was replaced with a reactant with the corresponding number of carbon atoms. The product was a white solid with a yield of 40.2%. The melting point was 163-164°C. ESI-MS m / z: 488.4 [M+H] + ; C 28 H 42 FN3O3(487.32).

[0108] Preparation of N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)eicosatriamide (3h, code FA-5FU-B-20C)

[0109] Prepared by a method similar to compound 3d, replacing n-dodecanoic acid with reactants corresponding to the number of carbon atoms in the above preparation method. The product is a white solid with a yield of 39.2%. Melting point: 164-166°C. ESI-MS m / z: 514.6 [MH] - ; C 30 H 46 FN3O3(515.35).

[0110] Preparation of N-(4-(5-fluoro-2,4-dioxy-3,4-dihydropyrimidin-1(2H)-yl)phenyl)docosapentaenoic acid amide (3i, code FA-5FU-B-22C)

[0111] Prepared by a method similar to compound 3d, replacing n-dodecanoic acid with reactants corresponding to the number of carbon atoms in the above preparation method. The product is a white solid with a yield of 25.3%. Melting point: 161-162°C. ESI-MS m / z: 542.6 [MH] - ; C32 H 50 FN3O3(543.38).

[0112] Example 2 Preparation of paclitaxel (Taxol) prodrug (Compound 6, codenamed FA-Taxol)

[0113]

[0114] The reagents used in the above preparation process are: a) succinic anhydride, anhydrous pyridine; b) dodecylamine; isobutyl chloroformate; triethylamine; anhydrous THF

[0115] Synthesis of 4-((1S,2R)-1-benzamide-3-((2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-6,12b-diacetoxy-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxy-2a,3,4,4a,5,6,9,11,12,12a,12b-dodecahydro-1H-7,11-methylcyclodeca[3,4]benzo[1,2-b]oxy-9-yl)oxy)-3-oxy-1-phenylpropan-2-yl)oxy)-4-oxobutanoic acid (Intermediate 5)

[0116] Paclitaxel (4,500 mg, 0.585 mmol) was dissolved in anhydrous pyridine (5 mL) and stirred for five minutes. Succinic anhydride (730 mg, 7.295 mmol) was then added. After stirring at room temperature for four hours, the reaction was complete by TLC. The solvent was removed under reduced pressure, and 10 mL of water was added. After stirring for two hours, the pH was adjusted to 2-3 with 1 mol / L HCl. The mixture was extracted three times with ethyl acetate (3 × 50 mL). The organic phases were combined, washed three times with 0.2 mol / L NaHCO₃ (3 × 50 mL), washed once with saturated sodium chloride (50 mL), dried over anhydrous MgSO₄, filtered, and the solvent removed under reduced pressure to yield a white solid, which was directly used in the next reaction.

[0117] Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-9-((2R,3S)-3-benzamido-2-(4-(dodecylamino)-4-oxobutanoyl)oxy)-3-phenylpropanoyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxyl-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methylcyclodeca[3,4]benzo[1,2-b]oxy-6,12b-diacetate (Compound 6, codenamed FA-Taxol)

[0118] Succinylpaclitaxel (5,78 mg, 0.08 mmol) was weighed and dissolved in 4 mL of anhydrous THF. Isobutyl chloroformate (16 μL, 0.12 mmol) and TEA (22 μL, 0.16 mmol) were added, at which point the reaction system became turbid. After stirring for five minutes, dodecylamine (22 mg, 0.12 mmol) was added and the reaction continued for 1 hour until the reaction was complete as determined by TLC. The THF was evaporated to dryness, diluted with ethyl acetate (10 mL), washed three times with water (3×15 mL), and once with saturated NaCl (15 mL). The aqueous phase was extracted twice with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, evaporated to dryness under reduced pressure, and then column chromatography was performed. The product 6 (43 mg) was obtained by column chromatography using a petroleum ether:acetone ratio of 2:1 (v:v) as the eluent. The yield was 46.32%. ESI-MS m / z:1121.6 [M+H] + ; C 63 H 80 N2O 16 (1120.55). 1 H NMR (400MHz, CDCl3) δ8.19-8.08(m,2H,ArH),7.87-7.76(m,2H,ArH),7.65-7.28(m,11H,ArH),7.16(d,J=8.8Hz,1H,NH),6.29(s,1H,H 10 ),6.21(t,J=8.0Hz,1H,H 13 ),5.93(dd,J=8.7,3.7Hz,1H,NH),5.68(d,J=7.0Hz,1H,H3'),5.50-5.44(m,2H,H2 and H2'),4.97(d,J=7.8Hz,1H,H5),4.40-4.47(m,1H,H7),4.30(dd,J=8.4Hz,1H,H20),4.20(d,J=8.4Hz,1H,H20),3.80(d,J=7.0Hz,1H,H 3),3.19-3.09(m,2H,H6”),2.76(t,J=6.9Hz,2H,H3”),2.58-2.52(m,1H,7-OH),2.46(m,2H,H2”),2.43(s,3H,CH3),2.27-2.35(m,1H,H 6a ),2.22(s,3H,CH3),2.04-2.14(m,1H,H 14a ),1.92(s,3H,H19),1.83-1.90(m,1H,H 14b ),1.71-1.76(m,1H,H 6b), 1.68 (s, 3H, H18), 1.48-1.39 (m, 2H, H7”), 1.27 (m, 18H, H8”-H16”), 1.23 (s, 3H, H17), 1.13 (s, 3H, H16), 0.88 (t, J = 6.8 Hz, 3H, H17”). 13 C NMR (101 MHz, CDC13) δ 203.82, 171.86, 171.18, 170.70, 169.85, 168.12, 167.22, 167.01, 142.77, 137.17, 133.70, 133.60, 132.82, 131.89, 130.23, 129.27, 129.04, 128.72, 128.60, 128.47, 127.31, 126.73, 84.46, 81.06, 79.13, 76.43, 75.62, 75.16, 74.36, 72.10, 71.82, 58.53, 53.17, 45.62, 43.19, 39.77, 35.56, 31.90, 30.96, 29.59, 29.32, 29.26, 26.88, 26.81, 22.66, 22.12, 20.79, 14.77, 14.09, 9.61.

[0119] Example 3 Preparation of methotrexate (MTX) prodrugs

[0120]

[0121] The reagents used in the above preparation scheme are as follows: a) dodecylamine; isobutyl chloroformate, triethylamine, anhydrous THF; b) EDCI, HOAt, DIPEA, THF

[0122] (R)-2-(4-((5,7-diaminopyrido[3,4-b]pyrazin-3-yl)methyl)(methyl)amino)benzamide)-5- (dodecylamino)-5-oxopentanoic acid (Compound 7, code LFC 12 Preparation of (R)-2-(4-((5,7-diaminopyrido[3,4-b]pyrazin-3-yl)methyl)(methyl)amino)benzamide)-5- (dodecylamino)-5-oxopentanoic acid (Compound 7, code LFC

[0123] Compound 7 was obtained in a yield of 55.1% using a similar amide condensation method as that of Compound 6. ESI-MS m / z: 621.55 [M+H] + ; C 33 H 48 N8O4 (620.38).

[0124] Preparation of 2-(4-((2,4-diaminopterin-6-yl)methyl)(methyl)amino)benzoyl)-5,14,17-trioxy-23-tetradecyl-9,10-disulfanyl-6,13,18-triazine tetrachloride-1,24-dicarboxylic acid (Compound 8, code name FA-MTX-AC)

[0125] Using an amide condensation method similar to compound 6, the yield of compound 8 was 35.1%. ESI-MS m / z: 1027.6 [M+H] + ; C 48 H 74 N 12 O9S2(1026.51).

[0126] Preparation of (R)-5-((S)-1-amino-1-oxo-6-palmitoylhexacan-2-yl)amino)-2-(4-((2,4-diaminopterin-6-yl)methyl)(methyl)amino)benzoyl)-5-oxopentanoic acid (Compound 9, code name FA-MTX-A)

[0127] Using an amide condensation method similar to compound 6, the yield of compound 9 was 31.6%. ESI-MS m / z: 820.7 [M+H] + ; C 42 H 65 N 11 O6(819.51).

[0128] Preparation of (3R,8S)-8-carbamoyl-3-carboxy-1-(4-((2,4-diaminopterin-6-yl)methyl)(methyl)amino)phenyl)-1,6,14-trioxy-15-thia-2,7,13-triazatriaza-31-carboxylic acid (Compound 10, code name FA-MTX-C)

[0129] Using an amide condensation method similar to compound 6, the yield of compound 10 was 27.6%. ESI-MS m / z: 896.5 [M+H] + ; C 43 H 65 N 11 O8S(895.47).

[0130] Example 4 Preparation of Temozolomide (MTZ) Prodrug

[0131]

[0132] The reagents used in the above preparation process are: a) NaNO2; H2SO4; H2O; b) dodecanol; DCC, DMAP, DMF

[0133] Preparation of 3-methyl-4-oxo-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxylic acid (Intermediate 11)

[0134] Temozolomide (0.99 g, 5 mmol) was dissolved in 5 mL of concentrated sulfuric acid. A solution of NaNO2 (0.69 g, 10 mmol) in water (4 mL) was added dropwise under an ice bath. After the addition was complete, the reaction was continued at 0°C for 1 hour, then at room temperature for 1 hour, and then at 50°C for 1.5 hours until TLC indicated completion. The reaction solution was poured into ice water while hot. A precipitate formed under stirring. The precipitate was filtered, washed with water, and dried to obtain intermediate 11 (0.9 g, 92%). ESI-MS m / z: 196.1 [M+H] + ; C6H5N5O3(195.04).

[0135] Dodecyl-3-methyl-4-oxy-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxylate (Compound 12a, code name LFC 12 Preparation of -MTZ)

[0136] Intermediate 11 (100 mg, 0.51 mmol), DCC (310 mg, 1.5 mmol), and DMAP (10 mg, 0.08 mmol) were dissolved in 15 mL of DMF at room temperature. After 30 minutes, dodecanol (190 mg, 1 mmol) was added and the reaction continued at room temperature for 48 hours until TLC indicated completion. After filtration, the reaction solution was added with 50 mL of ethyl acetate and extracted with 1N HCl (3 × 30 mL), 5% NaHCO₃ solution (30 mL), and saturated brine (30 mL). The product was dried over anhydrous MgSO₄, evaporated to dryness under reduced pressure, and then column chromatography was performed. The product 12a (170 mg) was obtained in a 91.8% yield by column chromatography using a mixture of petroleum ether:ethyl acetate (v:v) as the eluent. ESI-MS m / z: 364.4 [M+H] + ; C 18 H 29 N5O3(363.23). 1 H NMR(400MHz, CDCl3)δ8.46(s,1H,CH),4.47(t,J=6.8Hz,2H,O-CH2),4.06(s,3H,N-CH3)1.86-1.80(m,2 H,CH2),1.45(dd,J=12.9,6.8Hz,2H,CH2),1.29(s,2H,CH2),1.26(s,14H,CH2),0.89-0.86(m,3H,CH3).

[0137] Tetradecyl-3-methyl-4-oxy-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxylate (Compound 12b, code name LFC 14 Preparation of -MTZ)

[0138] Using a similar method to compound 12a, the yield of compound 12b was 89.1%. ESI-MS m / z: 392.4 [M+H] + ; C 20 H 33 N5O3(391.26). 1 H NMR(400MHz, CDCl3) δ8.46(s,1H,CH),4.46(t,J=6.8Hz,2H,O-CH2),4.05(s,3H,N-CH3),1.86-1.80(m ,2H,CH2),1.45(t,J=8.6Hz,2H,CH2),1.28(s,2H,CH2),1.24(s,18H,CH2),0.86(d,J=7.0Hz,3H,CH3).

[0139] Hexadecyl-3-methyl-4-oxy-3,4-dihydroimidazole[5,1-d][1,2,3,5]tetrazine-8-carboxylate (Compound 12c, code name LFC 16 Preparation of -MTZ)

[0140] Using a similar method to compound 12a, the yield of compound 12c was 86.7%. ESI-MS m / z: 420.6 [M+H] + ; C 22 H 37 N5O3(419.29). 1 H NMR (400MHz, CDCl3) δ8.45 (s, 1H, CH), 4.46 (t, J = 6.8Hz, 2H, O-CH2), 4.05 (s, 3H, N-CH3), 1.84-1.79(m,2H,CH2),1.56(s,2H,CH2),1.24(s,24H,CH2),0.86(d,J=7.1Hz,3H,CH3).

[0141] Example 5 Preparation of 5-fluorouracil (5FU) prodrug (Compound 14)

[0142]

[0143] Preparation of N,N'-1,3-dihydroxymethyl-5-fluorouracil (Intermediate 13)

[0144] Into a 50 ml beaker, 5-Fu (4 mmol, 0.52 g) and 37% aqueous formaldehyde solution (0.712 g, 0.648 ml, 8.8 mmol) were added, 5 ml of anhydrous dry acetonitrile was added, a stirring bar was added, heated and stirred and the temperature was raised to 60 °C, until the solid was completely dissolved, the reaction was continued for 50 min, then the excess formaldehyde, water and acetonitrile were removed under reduced pressure using a rotary evaporator, obtaining a colorless transparent viscous liquid product, 1,3-dihydroxymethyl-5-fluorouracil 0.78 g, with a yield of 95.1%. The product was used directly in the next reaction without isolation. ESI-MS m / z: 189.2 [M-H] - ; 1 H NMR (600 MHz, DMSO-d6): δ 6.17-6.53 (2 x OH, 2H, m), 5.06-5.34 (2H, NC H 2OH, m), 4.77-4.84 (2H, NC H 2OH, m), 3.22-3.28 (CH, s, 1H) ppm.

[0145] Methyl (5-fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl) 4- (dodecyloxy)benzoate (Compound 14a, Code LFC 12 -Ben-5FU) Preparation

[0146] Into a 50 ml beaker, 5-Fu (4 mmol, 0.52 g) and 37% aqueous formaldehyde solution (0.712 g, 0.648 ml, 8.8 mmol) were added, 5 ml of anhydrous dry acetonitrile was added, a stirring bar was added, heated and stirred and the temperature was raised to 60 °C, until the solid was completely dissolved, the reaction was continued for 50 min, then the excess formaldehyde, water and acetonitrile were removed under reduced pressure using a rotary evaporator, obtaining a colorless transparent viscous liquid product, 1,3-dihydroxymethyl-5-fluorouracil 0.78 g, with a yield of 95.1%. The product was used directly in the next reaction without isolation. ESI-MS m / z: 189.2 [M-H] + ; C 24 H 33 FN2O5 (448.24). 1H NMR (400 MHz, CDC13) δ: 8.38 ~ 8.37 (d, 1H, NH), 8.01 ~ 7.99 (d, J = 8 Hz, 2H, 2CH), 7.81 ~ 7.80 (d, J = 4 Hz, 1H, CH), 6.93 ~ 6.91 (d, J = 8 Hz, 2H, 2CH), 5.86 (s, 2H, CH2), 4.03 ~ 4.00 (t, J = 8 Hz, 2H, CH2), 1.83 ~ 1.76 (m, 2H, CH2), 1.47 ~ 1.42 (m, 2H, CH2), 1.32 ~ 1.26 (m, 16H, C8H 16 ), 0.90 ~ 0.86 (t, J = 8 Hz, 3H, CH3).

[0147] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)methyl 4-(tetradecyloxy)benzoate (Compound 14b, Code LFC 14 -Ben-5FU) was prepared

[0148] Compound 14b was obtained in 66.7% yield using a similar method to Compound 14a. ESI-MS m / z: 477.5 [M+H] + ; C 26 H 37 N2O5 (476.27). 1 H-NMR (400 MHz, CDC13) δ 8.53 ~ 8.52 (d, 1H, NH), 8.01 ~ 7.99 (d, J = 8 Hz, 2H, 2CH), 7.81 ~ 7.79 (d, J = 4 Hz, 1H, CH), 6.93 ~ 6.91 (d, J = 8 Hz, 2H, 2CH), 5.86 (s, 2H, CH2), 4.03 ~ 4.00 (t, J = 8 Hz, 2H, CH2), 1.83 ~ 1.76 (m, 2H, CH2), 1.49 ~ 1.42 (m, 2H, CH2), 1.37 ~ 1.23 (m, 20H, C 10 H 20 ), 0.90 ~ 0.86 (t, J = 8 Hz, 3H, CH3).

[0149] (5-Fluoro-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)methyl 4-(tetradecyloxy)benzoate (Compound 14b, Code LFC 16 -Ben-5FU) was prepared

[0150] Compound 14c was obtained in 55.8% yield using a similar method to Compound 14a. ESI-MS m / z: 505.5 [M+H] + ; C 28 H41 FN2O5(504.30). 1 H NMR (400MHz, CDCl3) δ8.47~8.4(d,1H,NH),8.01~7.99(d,J=8Hz,2H,2CH),7.80~7.79(d,J=4Hz,1H,CH),6.93~6.9 1(d,J=8Hz,2H,2CH),5.86(s,2H,CH2),4.03~4.00(t,J=8Hz,2H,CH2),1.82~1.76(m,2H,CH),1.47~1.26(m,24H,C 12 H 24 ),0.90~0.86(t,J=8Hz,3H,CH3).

[0151] Example 6: In vitro inhibition of tumor cell proliferation by the compound of Example 1

[0152] (1) Experimental method: Tumor cells in the logarithmic growth phase were obtained and digested with trypsin to prepare a single cell suspension. The cells were counted, diluted to 5000 cells per well, and evenly seeded on a 96-well plate, with 100 μL per well. A blank control was set up: only the same volume of culture medium; a negative control: only the cell suspension was added without drug. After 72 hours, the culture medium in the 96-well plate was aspirated, and 100 μL of the test compound at different concentrations was added to the experimental wells. Four replicate wells were set up for each concentration, and 100 μL of culture medium was added to the negative control wells and the blank control wells, respectively. After incubation in a 37°C, 5% CO2 incubator for 24 hours, 100 μL was discarded from each well, and 100 μL of culture medium containing 10% CCK8 was added. After incubation in a 37°C, 5% CO2 incubator for 1.5 hours, the OD value at 450 nm was measured using a multifunctional microplate reader (BioTek), and then data processing was performed.

[0153] (2) Calculation of inhibition rate: inhibition rate = (OD value of negative control group - OD value of experimental group) / (OD value of negative control group - OD value of blank group), IC was calculated using Graphpadprism 8 software. 50 value.

[0154] (3) Experimental results: The anti-proliferative effects of the compounds listed in the examples of the present invention on three tumor cell lines: SJSA-1, MDA-MB-231 and MCF-7 are as follows: Figure 1 All the compounds of Example 1 showed significant anti-tumor proliferation effects, especially the compounds modified with 12 carbon atoms, 14 carbon atoms and 16 carbon atoms showed the best anti-tumor proliferation effects.

[0155] Example 7: In vivo antitumor activity of the compound of Example 2 (codenamed FA-Taxol)

[0156] (1) Experimental method: Nude mice with athymic tumors inoculated with SKOV-3 (ovarian cancer cells) were divided into three groups, with 3 mice in each group. When the tumor grew to 100 mm 3 At 37 ℃, paclitaxel and the compound of Example 2 (FA-Taxol) were administered by intraperitoneal injection, and the administration days were as follows: Figure 2 Indicated by the middle arrow. Under constant temperature of 37℃, each nude mouse was intraperitoneally injected with drugs twice a week. The injection dose was calculated based on the molecular weight and weight, and the injection was completed within 30 minutes each time. The tumor volume was measured twice a week and the tumor volume ± SEM (mm 3 ) to record the results.

[0157] (2) Experimental results: Figure 2 It can be seen that the in vivo anti-tumor effect of the drug FA-Taxol modified by LFC is much higher than that of the original drug Taxol.

[0158] Example 8: Compound of Example 5 (code LFC 12 -Ben-5FU and LFC 14 -Ben-5FU) in vivo antitumor activity

[0159] The MDA-MB-231 (breast cancer cell) nude mouse transplant tumor model was used, and the experimental method was the same as in Example 7. The results are shown in FIG. Figure 3 As shown. It can be seen that the 5FU derivative LFC modified by LFC 12 -Ben-5FU and LFC 14 -Ben-5FU has significantly higher in vivo tumor inhibition activity than the original drug 5FU.

[0160] Example 9: Compound LFC 12 Synergistic in vivo antitumor activity of MTX, FA-MTX-C, FA-MTX-A, LFC12-MTZ, and FA-B-12C with the tumor suppressor protein p53

[0161] The SJSA-1 (human osteosarcoma cell) nude mouse transplant tumor model was used, and the experimental method was the same as in Example 7. The results are shown in FIG. Figure 4 As shown. It can be seen that the MTX derivative LFC modified by LFC 12 -MTX, FA-MTX-C and FA-MTX-A; LFC-modified MTZ derivatives LFC 12The in vivo tumor-suppressing activity of 5FU-MTZ was significantly higher than that of their respective parent drugs, MTX and MTZ. Furthermore, we also investigated the synergistic antitumor effect of the LFC-modified 5FU derivative FA-5FU-B-12C, which exhibits excellent in vitro activity, and the tumor suppressor protein p53. As shown in the figure, FA-5FU-B-12C exhibited a strong synergistic antitumor effect with p53, surpassing that of the tumor suppressor protein p53.

[0162] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An anti-tumor drug prodrug, characterized in that: The anti-tumor drug is paclitaxel, and the prodrug structure is shown in the following formula V: Formula V.

2. A method for preparing the anti-tumor drug prodrug according to claim 1, characterized in that: The synthetic route of the compound shown in formula V is as follows: The synthesis steps are as follows: Paclitaxel is dissolved in anhydrous pyridine, stirred, and then succinic anhydride is added. The reaction is carried out at room temperature for 3 to 5 hours, and then the solvent is removed. Water is added and the pH of the reaction system is adjusted to 2 to 3. Extraction is performed with ethyl acetate, and the organic phase is partially washed and dried. The solvent is removed to obtain succinyl paclitaxel. Succinyl paclitaxel is dissolved in anhydrous THF, isobutyl chloroformate and TEA are added, and the reaction is continued for 0.8 to 1.2 hours until the reaction is complete. After removing the solvent, the mixture is separated and purified by column chromatography using petroleum ether / acetone to obtain the compound represented by formula V.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the anti-tumor drug prodrug according to claim 1; In the pharmaceutical composition, the anti-tumor drug prodrug is in a therapeutically effective dose, or is capable of releasing a therapeutically effective dose of the anti-tumor drug; Alternatively, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier in the art; Alternatively, the pharmaceutical composition is sterile when used to prepare a pharmaceutical preparation for in vivo administration; Alternatively, in the pharmaceutical composition, the anti-tumor drug prodrug is administered as a single active ingredient, or can be administered in combination with other active ingredients.

4. Use of the anti-tumor drug prodrug according to claim 1 and the pharmaceutical composition according to claim 3 in the preparation of drugs in the field of tumor targeted therapy, characterized in that: The application method is selected from any of the following: (1) For the preparation and development of anti-tumor preparations; (2) Used for the treatment of patients with tumor resistance or patients receiving combination therapy; In the above aspect (1), the anti-tumor preparation is an anti-tumor drug or an anti-tumor model agent; the anti-tumor drug is an oral preparation or a parenteral preparation.

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