20(S)-10,11-difluoromethylenedioxy camptothecin derivatives, processes for their preparation and use

Abstract

The application belongs to the technical field of organic synthesis and medicine, and particularly relates to 20(S)-10,11-difluoromethylenedioxy camptothecin derivatives, a preparation method and application thereof. The derivative is a compound with a structure shown in formula (I), stereoisomers and pharmaceutically acceptable salt forms. The novel 20(S)-10,11-difluoromethylenedioxy camptothecin derivative can be used for preparing and synthesizing camptothecin drugs for preventing or treating tumors. The compound has good anti-inflammatory activity, in-vivo and in-vitro anti-tumor activity and cell transmembrane transport capacity. The preparation method is easy to operate, the post-treatment is simple, the reaction starting material is cheap and easy to obtain, the reaction substrate has strong designability, the reaction efficiency is high, and the practicability is strong.

Description

Technical Field

[0001] This invention belongs to the fields of organic synthesis and pharmaceutical technology, specifically relating to 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives, their preparation methods, and applications. Background Technology

[0002] Camptothecin (CPT) is a cytotoxic quinoline alkaloid that inhibits DNA topoisomerase (TOPOI) and is a hot topic in antitumor drug development. Camptothecin is derived from small molecule compounds with excellent antitumor activity found in natural products. Camptothecin derivatives are hailed as one of the three major discoveries in anticancer drugs in the 1990s, demonstrating broad-spectrum antitumor activity and significant research and application value. Early camptothecin compounds acted by forming a ternary complex with TopoI and DNA, blocking DNA replication and transcription, but suffered from low selectivity and significant toxic side effects. Due to its poor water solubility and toxicity under physiological conditions, camptothecin could not be directly applied clinically. Derivatives obtained through structural modification, such as irinotecan, topotecan, and beloteccan, have been successfully used to treat colorectal cancer, ovarian cancer, and prostate cancer, and more than ten other drugs are currently in clinical trials.

[0003] Chinese invention patent CN110590796A discloses camptothecin derivatives, their preparation methods, and applications. The compounds provided by this invention are a novel class of camptothecin derivatives with methylenedioxy groups introduced at positions 10 and 11 of the parent ring and different substituents introduced at position 7. The preparation method uses readily available raw materials, is simple in synthesis, and is convenient and rapid in purification. Furthermore, the compounds exhibit excellent in vitro cytotoxic activity and superior in vivo antitumor effects. Invention patents WO2005009347A2 and WO0149291 disclose methods for forming camptothecin compounds that are effective antitumor compounds. These compounds can inhibit topoisomerase I and can alkylate the DNA of the related topoisomerase I-DNA cleavable complex. However, the above compounds do not have ideal transmembrane transport capabilities, high cytotoxicity, and poor in vivo antitumor activity. Furthermore, the preparation methods of these compounds involve complex reaction procedures, low yields, high costs, and difficulty in large-scale preparation when introducing substituents. Since the synthetic route involves introducing substituents into the parent structure in the final step, steric hindrance and the conjugated system make it very difficult to form negative charges at the introduction site. Therefore, the types of substituents that can be introduced by the above methods are very limited.

[0004] Camptothecin derivatives approved for anticancer use are susceptible to one or more of the mechanisms that enable cancer cells to tolerate chemotherapy. Further development of new camptothecin derivatives is needed, particularly more potent anticancer agents and derivatives with lower toxicity, better permeability, and better solubility. Summary of the Invention

[0005] This invention addresses the problems existing in the prior art by providing a 20(S)-10,11-difluoromethylenedioxycamptothecin derivative, its preparation method, and its applications.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives, compounds having the structure shown in formula (I), their stereoisomers, and pharmaceutically acceptable salt forms:

[0008]

[0009] In equation (Ⅰ), R is Where: X is n is an integer between 0 and 2; Z is selected from substituted or unsubstituted ring structures, substituted or unsubstituted C 1-10 alkenyl, substituted or unsubstituted C 1-10 Alkynyl, substituted or unsubstituted octyl, substituted or unsubstituted decyl, substituted or unsubstituted C 7-10 alkoxy, substituted or unsubstituted C 7-10 alkylthio, substituted or unsubstituted C 7-10 Alkylamine, substituted or unsubstituted C 7-10 alkylimine group, substituted or unsubstituted C 7-10 alkenylamine, substituted or unsubstituted C 7-10 alkenyl imine, substituted or unsubstituted C 7-10 Alkyl C 7-10 alkenylamine and substituted or unsubstituted C 7-10 Alkyl C 7-10 alkenylimine group;

[0010] The substitution can be a single substitution or multiple substitutions.

[0011] Preferably, the ring structure is selected from C 9-12 cycloalkyl, C 3-12 Cycloalkenyl, aryl, C 2-11 The ether ring group or the aromatic heterocyclic group, wherein the aromatic heterocyclic ring is selected from the pyridine ring, furan ring, thiophene ring, pyrazole ring, indole ring, benzopyrazole ring, piperidine ring, morpholine ring, thiomorpholine ring, naphthalene ring or triazole ring.

[0012] Preferably, when it is a substituted structure, the substituted groups are each independently selected from halogens, C 1-10 Alkyl, C 1-10 alkenyl, C 1-10 alkynyl group, C 3-12 cycloalkyl, C 1-10 Alkoxy, C1-10 Alkylthio, C 1-10 Alkylsilyl, C 11-10 Halogenated alkoxy groups, C 1-10 Ester group, 3-12 membered heterocyclic group, C 6-14 aryl, oxygen C 6-14 Aryl, oxygen C 6-14 Aromatic heteroyl, nitrogen C 6-14 Aryl, nitrogen C 5-14 Aromatic heteroyl, 5-14 heteroaryl, -CN, -NO2, -CF2H, -CF2OH, -CF3 or -OCF3.

[0013] Preferably, Z is selected from C. 1-10 Alkyl-substituted aryl, C 1-10 Alkoxy-substituted aryl, C 1-10 Alkylthio-substituted aryl, C 1-10 Alkylsilyl-substituted aryl, C 1-10 aryl groups substituted with alkoxy groups, aryl groups substituted with halogen atoms, and unsubstituted C groups. 2-11 ether ring substituents, C 1-10 Alkyl-substituted C 2-11 C-rings with ether ring substituents and benzene ring substituents 2-11 ether ring substituents, halogen atom-substituted C 2-11 Ether ring substituents, unsubstituted aromatic heterocyclic substituents, C 1-10 Alkyl-substituted aromatic heterocyclic substituents, halogen-substituted aromatic heterocyclic substituents, and unsubstituted C 1-8 olefin substituents or C 1-10 Alkyl-substituted C 1-8 Olefin substituents.

[0014] More preferably, Z is a substituted aryl group, and Z is selected from any one of the following groups:

[0015]

[0016] More preferably, Z is a substituted or unsubstituted C. 2-11 The ether ring substituent, Z, is selected from any of the following groups:

[0017]

[0018] More preferably, Z is a substituted or unsubstituted C. 1-8 The olefin substituent, Z, is selected from any of the following groups:

[0019]

[0020] More preferably, Z is a substituted or unsubstituted aromatic heterocyclic substituent, and Z is selected from any one of the following groups:

[0021]

[0022] The stereoisomers include conformational isomers, optical isomers (such as enantiomers and diastereomers), and geometric isomers (such as cis-trans isomers). These isomers or combinations thereof may exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, or cis or trans isomers.

[0023] The pharmaceutically acceptable salts referred to are salts formed by the reaction of the above-mentioned derivatives with inorganic acids, organic acids, alkali metals, or alkaline earth metals. These salts include (but are not limited to):

[0024] (1) Salts formed with the following inorganic acids: such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid or phosphoric acid;

[0025] (2) Salts formed with the following organic acids, such as acetic acid, lactic acid, citric acid, succinic acid, fumaric acid, gluconic acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, oxalic acid, succinic acid, tartaric acid, maleic acid or arginine.

[0026] (3) Other salts, including salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium), ammonium salts or water-soluble amine salts (such as N-methylglucosamine salts), lower alkanol ammonium salts and other pharmaceutically acceptable amine salts (such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively), or other conventional forms of "prodrugs".

[0027] The present invention also provides a method for preparing the above-mentioned derivatives, comprising the following steps:

[0028] (1) The raw material reacts with triethylamine and a reducing agent to obtain an intermediate;

[0029] (2) The intermediate, tricyclic ketone and catalyst are reacted to obtain the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative.

[0030] Preferably, the reducing agent in step (1) is selected from any one of Raney nickel, palladium on carbon, platinum on carbon, zinc, rhodium, iron, stannous chloride, lithium aluminum hydride, sodium borohydride, lithium aluminum hydride, potassium borohydride, sodium sulfide, hydrogen sulfide, sodium disulfide, ferrous sulfate, sodium sulfite, sodium bisulfite, sodium nitrite, and ferrous dicarbonate.

[0031] More preferably, the reducing agent in step (1) is iron powder or palladium on carbon.

[0032] Preferably, in step (1), the raw material is first dissolved in ethanol, then a reducing agent is added, hydrogen gas is introduced, and then triethylamine is added. The mass-volume ratio of the raw material to triethylamine, iron powder, or palladium on carbon is 100-240 mg: 100-800 μL: 15-36 mg. The reaction temperature in step (1) is 20-30°C, and the reaction time is 8-12 h.

[0033] Preferably, the catalyst in step (2) is selected from any one of iodine, dodecyl sulfate, ferric chloride hexahydrate, aminosulfonic acid, 2,4,6-trichloro-1,3,5-triazine, bismuth trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, lithium bis(trifluoromethanesulfonyl)imide, magnesium bis(trifluoromethanesulfonyl)imide, and p-toluenesulfonic acid hydrate.

[0034] More preferably, the catalyst in step (2) is p-toluenesulfonic acid hydrate.

[0035] Preferably, the intermediate, tricyclic ketone and catalyst described in step (2) are dissolved in glacial acetic acid in a molar ratio of 1-1.5:1:0.4-0.6. The reaction described in step (2) is carried out under nitrogen protection, and the pH is adjusted after the reaction is completed.

[0036] Preferably, the reaction temperature in step (2) is 90-100℃, the reaction time is 8-12h, and the pH is 5-6.

[0037] Preferably, n is 0 and Z is a substituted aryl group. The preparation method of the raw material in step (1) includes first nitrifying 2,2-difluorobenzo[d][1,3]dioxane-5-carboxaldehyde, then reacting it with substituted phenylboronic acid, palladium chloride, potassium carbonate and tris(1-naphthyl)phosphine, and finally reacting it with an oxidant.

[0038] Preferably, the substituted phenylboronic acid is selected from any one of 3,4-dimethylphenylboronic acid, 3,5-dimethylphenylboronic acid, 4-trimethylsilylphenylboronic acid, 3-methylphenylboronic acid, 4-ethylphenylboronic acid, 4-isopropylphenylboronic acid, 3-trifluoromethoxyphenylboronic acid, 4-methoxyphenylboronic acid, 3-methoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 3-fluoro-4-methoxyphenylboronic acid, 3-fluoro-5-methoxyphenylboronic acid, 4-fluoro-3-methoxyphenylboronic acid, 3-fluoro-4-methylphenylboronic acid, 2,4-difluorophenylboronic acid, 3,4-difluorophenylboronic acid, 4-fluorophenylboronic acid, 4-trifluoromethylphenylboronic acid, and 4-methylthiophenylboronic acid.

[0039] Preferably, the solvent for the nitration reaction includes fuming nitric acid and trifluoromethanesulfonic acid, the temperature of the nitration reaction is -75℃ to -45℃, and the nitration reaction time is 2-3 hours.

[0040] Preferably, toluene is used as the solvent when reacting with substituted phenylboronic acid, palladium chloride, potassium carbonate and tris(1-naphthyl)phosphine, and the reaction temperature is 75-95°C and the reaction time is 8-12 h.

[0041] Preferably, the oxidant is selected from any one of manganese dioxide, Desmartin oxidant, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetrapropylammonium perruthenate, sodium nitrite / acetic anhydride oxidant, and aluminum tert-butoxide.

[0042] More preferably, the oxidant is a Desmartin oxidant.

[0043] Preferably, the solvent used in the reaction with the oxidant is dichloromethane, the reaction temperature is 15-35°C, and the reaction time is 2-3 hours.

[0044] Preferably, n is an integer between 0 and 2, and Z is the substituted or unsubstituted C. 2-11 Ether ring substituents, substituted or unsubstituted C 1-8 The preparation method of the raw material in step (1) includes reacting 2,2-difluorobenzo[d][1,3]dioxane-5-carboxaldehyde with a Grignard reagent, then reacting it with an oxidant, and finally carrying out a nitration reaction.

[0045] Preferably, the Grignard reagent is selected from any one of allyl magnesium bromide, allyl magnesium bromide, 2-furanmethyl magnesium bromide, 4-pyranmethyl magnesium bromide, and allenyl magnesium bromide.

[0046] Preferably, before reacting with the Grignard reagent, 2,2-difluorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde is dissolved in anhydrous tetrahydrofuran and stirred in an ice-water bath. The molar ratio of 2,2-difluorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde to the Grignard reagent is 1:5-15. During the reaction with the Grignard reagent, the environment is anhydrous and oxygen-free. The reaction process is monitored by TLC. The developing solvent of the TLC is a mixture of petroleum ether and ethyl acetate with a volume ratio of 8-16:1-2. The reaction temperature of the Grignard reagent is 35-65℃, and the reaction time is 8-12 h.

[0047] Preferably, the oxidant is selected from any one of manganese dioxide, Desmartin oxidant, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetra-n-propylperruthenium peroxide, sodium nitrite / acetic anhydride oxidant, and aluminum tert-butoxide, wherein the oxidant is most preferably Desmartin oxidant, the solvent for the reaction with the oxidant is dichloromethane, the reaction temperature with the oxidant is 15-35°C, and the reaction time is 2-3 hours.

[0048] Preferably, the solvent for the nitration reaction includes fuming nitric acid and concentrated sulfuric acid, the temperature of the nitration reaction is -20℃ to -10℃, and the reaction time is 8-12h.

[0049] More preferably, n is an integer from 0 to 2, Z is a substituted or unsubstituted aromatic heterocyclic substituent, and the nitration reaction is followed by a reaction with aromatic heterocyclic formaldehyde.

[0050] Preferably, the aromatic heterocyclic formaldehyde is selected from any one of 5-methylfuran-2-carboxaldehyde, 5-bromothiophene-2-carboxaldehyde, and thiophene-2-carboxaldehyde.

[0051] Preferably, the reaction conditions with aromatic heterocyclic formaldehyde are alkaline, the reaction temperature is 15-35℃, and the reaction time is 1-2 hours.

[0052] Preferably, all reactions in each step further include quenching, extraction, washing, concentration, and purification.

[0053] Preferably, the quenching solvent is selected from one or more of ice water, saturated ammonium chloride solution, saturated sodium sulfite solution, and saturated sodium bicarbonate solution.

[0054] Preferably, the solvent used for extraction is selected from dichloromethane, ethyl acetate, or saturated sodium bicarbonate solution, and the extraction is performed 2-6 times.

[0055] Preferably, the solvent for the water washing is a saturated sodium chloride solution or a saturated sodium bicarbonate solution, and the water washing is performed 1-3 times.

[0056] Preferably, the drying is performed using anhydrous magnesium sulfate, and the purification is performed by silica gel column chromatography.

[0057] Preferably, the packing material for the silica gel column chromatography is 300-400 mesh silica gel.

[0058] Preferably, the eluent used for purification is selected from a mixture of petroleum ether and ethyl acetate in a volume ratio of 20-150:1, a mixture of dichloromethane and ethyl acetate in a volume ratio of 2-10:1, a mixture of petroleum ether and dichloromethane in a volume ratio of 2-10:1, or a mixture of dichloromethane, methanol, and glacial acetic acid in a volume ratio of 50-400:1:0.1.

[0059] More preferably, when n of the R group in structural formula (Ⅰ) is 0 and Z is a substituted aryl group, the synthetic route 1 for preparing the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative is shown below:

[0060]

[0061] More preferably, when n of the R group in structural formula (Ⅰ) is an integer between 0 and 2, and Z is a substituted or unsubstituted C group... 2-11 Ether ring substituents, substituted or unsubstituted C 1-8 Synthetic route 2 for preparing 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives by olefin substituents is shown below:

[0062]

[0063] More preferably, when n of the R group in structural formula (Ⅰ) is an integer between 0 and 2, and Z is a substituted or unsubstituted aromatic heterocyclic substituent, the synthetic route 3 for preparing the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative is shown below:

[0064]

[0065] The present invention also provides the use of the above-mentioned 20(S)-10,11-difluoromethylenedioxycamptothecin derivative in the preparation of medicaments for the prevention and / or treatment of cancer.

[0066] The present invention also provides a pharmaceutical composition comprising: an effective dose of the above-described 20(S)-10,11-difluoromethylenedioxycamptothecin derivative, and a pharmaceutically acceptable carrier.

[0067] The cancers mentioned are selected from pancreatic cancer, lung cancer, colon cancer, prostate cancer, leukemia, and breast cancer.

[0068] The pharmaceutical composition can be formulated into liquid preparations such as tablets, capsules, powders, granules, lozenges, suppositories, oral liquids, or sterile parenteral suspensions, as well as injections such as large or small volume injections and lyophilized powders. All of the above dosage forms can be prepared using conventional methods in the pharmaceutical field.

[0069] If necessary, the pharmaceutical compositions of the present invention may also incorporate one or more pharmaceutically acceptable carriers, including diluents, fillers, binders, humectants, absorption enhancers, surfactants, adsorbents, and lubricants conventional in the pharmaceutical field.

[0070] Compared with the prior art, the present invention has the following beneficial effects:

[0071] (1) The novel 20(S)-10,11-difluoromethylenedioxycamptothecin derivative provided by the present invention is used to prepare and synthesize camptothecin-based drugs for the prevention or treatment of tumors. These compounds have good solubility, anti-inflammatory activity, in vitro and in vivo antitumor activity and cell transmembrane transport ability.

[0072] (2) The preparation method of the present invention is easy to operate, the post-processing is simple, the starting raw materials are cheap and readily available, the reaction substrates are highly designable, the substrate functional groups are wide-ranging, the reaction efficiency is high, and 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives with different substituents can be designed and synthesized according to actual needs, which is highly practical. Attached Figure Description

[0073] Figure 1 This is a simplified synthetic route diagram of the compound 20(S)-10,11-difluoromethylenedioxycamptothecin derivative of the present invention.

[0074] Figure 2 The effect of compound 4 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0075] Figure 3 The effect of compound 8 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0076] Figure 4 The effect of compound 10 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0077] Figure 5 The effect of compound 11 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0078] Figure 6 The effect of compound 12 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0079] Figure 7 The effect of compound 13 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0080] Figure 8 The effect of compound 15 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0081] Figure 9 The effect of compound 19 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

[0082] Figure 10 The curves show the inhibitory effects of the experimental compounds on HT-29 tumor growth in mice. Detailed Implementation

[0083] The methods and techniques of this invention are generally performed according to conventional methods known in the art, unless otherwise stated. The nomenclature, experimental methods, and techniques related to biology, pharmacology, and medicine and pharmaceutical chemistry described herein are known and commonly used in the art. Chemical synthesis, chemical analysis, pharmaceutical preparation, and formulation methods all employ standard techniques.

[0084] Unless otherwise stated, the scientific and technical terms used herein shall have the meanings commonly understood by one of ordinary skill in the art. However, the following terms shall have the following definitions:

[0085] Cancer refers to malignant tumors originating from epithelial tissue and is the most common type of malignant tumor. Correspondingly, malignant tumors originating from mesenchymal tissue are collectively called sarcomas. A few malignant tumors are not named according to the above principles, such as nephroblastoma and malignant teratoma.

[0086] "Cancer" is a general term encompassing all malignant tumors, referring to a large category of diseases characterized by the uncontrolled growth of abnormal cells within the body. Cancer is characterized by abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness, and metastasis. It is caused by uncontrolled cell division and growth leading to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream. Its occurrence is a complex, multi-factorial, and multi-step process, divided into three stages: carcinogenesis, tumor promotion, and progression. It is closely related to smoking, infection, occupational exposure, environmental pollution, unhealthy diet, and genetic factors. In this invention, "treating cancer" can also be equivalently described as "treating tumors," "anti-cancer," or "anti-tumor." Cancer is a disease characterized by uncontrolled cell growth that impairs the normal function of bodily organs and systems.

[0087] The products of this invention can be used to treat subjects with a variety of cancers or at risk of developing cancer. Examples of such cancers include pancreatic cancer, breast cancer, prostate cancer, lung cancer, ovarian cancer, cervical cancer, skin cancer, melanoma, colon cancer, stomach cancer, liver cancer, esophageal cancer, kidney cancer, throat cancer, thyroid cancer, testicular cancer, brain cancer, bone cancer, and blood cancers (such as leukemia and chronic lymphocytic leukemia). Other cancers include, but are not limited to, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system (CNS) cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, intraepithelial neoplasia, laryngeal cancer, lung cancer (small cell, large cell), lymphoma (including Hodgkin's lymphoma and non-Hodgkin's lymphoma); melanoma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth and pharynx); retinoblastoma; rhabdomyosarcoma; respiratory system cancer; sarcoma; uterine cancer; urinary system cancer; and other cancers and sarcomas.

[0088] A “therapeutic effective dose” is any amount of a drug, as described below, that, when used alone or in combination with another therapeutic agent, promotes disease regression, manifested as a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of impairment or disability resulting from the disease. A therapeutically effective dose or amount of a drug includes a “preventive effective dose,” which is any amount of a drug, as described below, that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering from a relapse of the disease, inhibits the onset or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit disease progression or recurrence can be assessed using various methods known to those skilled in the art, such as in animal model systems where efficacy in humans can be predicted or by measuring the activity of the compound reagent in an in vitro assay system.

[0089] As an example, an anticancer agent (a pharmaceutical composition for treating cancer) promotes tumor regression in a subject. In a preferred embodiment, a therapeutically effective dose of the drug promotes cancer cell regression and even the elimination of cancer. "Promoting cancer regression" means that administration, alone or in combination with an anti-neoplastic agent, of a therapeutically effective dose of the drug results in reduced tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods, prevention of impairment or disability caused by the disease, or otherwise improves the patient's disease symptoms. Furthermore, the terms "effective" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or biological level due to drug administration.

[0090] As an example of tumor treatment, in the most preferred embodiment, a therapeutically effective amount or dose of the drug can effectively inhibit cell or tumor growth compared to an untreated animal model. The ability of the compound to inhibit tumor growth can be evaluated in animal model systems, which can predict its efficacy in human tumors. Alternatively, this property of the composition can be assessed by testing the compound's ability to inhibit cell growth, such inhibition being measured in vitro using assays known to those skilled in the art. In a preferred embodiment of the invention, tumor regression can be clearly observed.

[0091] The present invention will be further described in detail below with reference to the embodiments.

[0092] This invention synthesizes and tests a series of novel camptothecin derivatives with difluoro-substituted methylenedioxy groups at the 10 and 11 positions of the parent ring and different substituent groups at the 7- position. The 20(S)-10,11-difluoromethylenedioxy camptothecin derivatives described in this invention can be listed as some of the compounds in Table 1, but are not limited to the structures shown in Table 1 (all compound structures are described in the invention description).

[0093] Table 1

[0094]

[0095]

[0096] Figure 1 A simplified synthetic route diagram for the above compounds is shown. The following specific examples illustrate the simplified synthetic route 1 for compounds 1-19:

[0097]

[0098]

[0099] Example 1: Preparation of 20(S)-7-(3,4-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 1), the steps of which are as follows:

[0100] (1) Preparation of intermediate 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-carboxaldehyde (compound B)

[0101] At 25°C, trifluoromethanesulfonic acid (2823.52 μL) and dichloromethane (11764.8 μL) were mixed and allowed to stand for separation. The upper layer was taken into a round-bottom flask, fuming nitric acid (882.34 μL) was added, and the mixture was stirred for 5 min. The reaction flask was then cooled to -65°C, and raw material A (2 g) was slowly added dropwise. After 3 h, the reaction was complete. The reaction was quenched with ice water, and the mixture was extracted three times with dichloromethane. The organic phases were combined, dried with anhydrous magnesium sulfate, and concentrated to obtain a yellow oily liquid. The liquid was separated by silica gel column chromatography (eluent was a mixed solution of petroleum ether and dichloromethane = 10:1 / 2:1, v / v). After concentration, a total of 1785.9 mg of yellow solid compound B was obtained. The yield of step (1) was 71.9%, and the purity was 97.76%.

[0102] 1 H NMR(400MHz,Chloroform-d)δ10.17(s,1H),8.45(s,1H),7.95(s,1H).HR-MS:C8H3F2NO5 for[M+H] + , caculated 230.9979, found 231.0802.

[0103] (2) Preparation of (2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-yl)(3,4-dimethylphenyl)methanol (D2): The product of step (1), 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-carboxaldehyde (compound B) (100 mg), was used as a raw material. 3,4-dimethylphenylboronic acid (97.4 mg), tris(1-naphthyl)phosphine (8.92 mg), palladium chloride (3.84 mg), and anhydrous carbonic acid were added. Potassium (179.5 mg) was dissolved in a round-bottom flask with toluene (5 mL) as solvent. The mixture was stirred at 88 °C for 7 h. After concentration, it was dissolved in dichloromethane. The catalyst was removed by filtration. After concentration, a preliminary product was obtained. The product was separated by silica gel column chromatography (eluent was a mixed solution of petroleum ether and ethyl acetate = 80:1 / 50:1 / 20:1, v / v). After concentration, a total of 140.4 mg of solid product D2 was obtained. The yield of step (2) was 96.2%, and the purity was 98.16%.

[0104] 1 H NMR(400MHz,Chloroform-d)δ7.89(s,1H),7.77(s,1H),7.43(s,1H),7.24(d,J =1.9Hz,1H),6.61(d,J=2.7Hz,1H),2.91(d,J=3.8Hz,1H),2.41(s,7H).HR-MS:C 16 H 13 F2NO5 for [M+H] + , caculated 337.0762, found 338.0591.

[0105] (3) Preparation of (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3,4-dimethylphenyl) methyl ketone (E2)

[0106] At 25°C, the product (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3,4-dimethylphenyl)methanol (D2) (140 mg) and Desmartin oxidant (264.3 mg) from step (2) were dissolved in dichloromethane (5 mL). After stirring at 25°C for 3 h, saturated sodium sulfite solution (15 mL) and saturated sodium bicarbonate solution (15 mL) were added and stirred for 30 min to quench the reaction. The mixture was then extracted three times with dichloromethane, and the organic phases were combined. The mixture was washed once with saturated sodium bicarbonate, dried with anhydrous magnesium sulfate, and concentrated to obtain the preliminary product. The product was separated by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate = 150:1 / 100:1 / 50:1, v / v / ). After concentration, a total of 133.5 mg of solid product E2 was obtained. The yield of step (3) was 96.0%, and the purity was 97.22%.

[0107] 1 H NMR(400MHz,Chloroform-d)δ7.97(s,1H),7.55(s,1H),7.41(d,J=9.7Hz,1H),7 .20(d,J=7.9Hz,1H),7.14(s,1H),2.31(d,J=13.0Hz,6H),1.59(s,1H).HR-MS:C 16 H 11 F2NO5 for [M+H] + , caculated 335.0605, found 336.0509.

[0108] (4) Preparation of (6-amino-2,2-difluorobenzo[d][1,3]dioxolane-5-yl)(3,4-dimethylphenyl) methyl ketone (F2)

[0109] At 25°C, the product of step (3) (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3,4-dimethylphenyl) methyl ketone (E2) (133.5 mg), triethylamine (800 μL), and palladium on carbon (20.0 mg) were placed in a round-bottom flask, methanol (5 mL) was added as solvent, a hydrogen balloon was filled, and the mixture was stirred at 25°C for 8 h. The catalyst was removed by filtration, and the product was concentrated to obtain a yellow liquid. The liquid was separated by silica gel column chromatography (eluent was a mixed solution of petroleum ether and ethyl acetate = 80:1 / 50:1 / 20:1, v / v / ). After concentration, a total of 116.4 mg of yellow solid product F2 was obtained. The yield of step (4) was 95.7%, and the purity was 97.67%.

[0110] 1 H NMR (400MHz, Chloroform-d) δ7.37 (s, 1H), 7.31 (d, J = 7.7Hz, 1H), 7.21 (d, J = 7.7Hz, 1H), 7.14 (s, 1H), 6.42 (s, 1H), 2.33 (d, J = 5.1Hz, 5H). HR-MS: C 16 H 13 F2NO3 for [M+H] + , caculated 305.0863, found 306.0622.

[0111] (5) Preparation of 20(S)-7-(3,4-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 1)

[0112] The products obtained in step (4), namely (6-amino-2,2-difluorobenzo[d][1,3]dioxolane-5-yl)(3,4-dimethylphenyl) methyl ketone (F2) (116.0 mg, 1 mmol), tricyclic ketone (99.6 mg, 1 mmol), and p-toluenesulfonic acid hydrate (43.4 mg, 0.6 mmol), were added to a round-bottom flask, dissolved in glacial acetic acid (5 mL), and refluxed at 90 °C for 8 h under nitrogen protection. The pH was then adjusted to 5-6, and dichloromethane was added. The extract was repeated three times, the organic phases were combined, washed once with saturated brine, dried with anhydrous magnesium sulfate, and concentrated to obtain a yellow oily liquid. The liquid was separated by silica gel column chromatography (the eluent was a mixed solution of dichloromethane:methanol:glacial acetic acid = 200:1:0.1 / 50:1:0.1 / 400:1:0.1, v / v / v). After concentration, the liquid was washed with methanol and filtered. Finally, 218.1 mg of pure white solid product (compound 1) was obtained on the filter cake. The yield of step (5) was 73.3%, and the purity was 99.1%.

[0113] 1H NMR(400MHz,Chloroform-d)δ7.82(d,J=5.7Hz,1H),7.65(d,J=4.1Hz,1H),7.37(t,J=7.3Hz,1H),7.16-7.10(m,1H),5.69(d,J=14.7 Hz,1H),5.27(d,J=16.4Hz,1H),5.16-5.03(m,2H),2.38(d,J=4.0Hz,3H),1.98-1.81(m,J=7.3Hz,2H),1.03(t,J=7.4Hz,3H).HR-MS:C 29 H 22 F2N2O6 for [M+H] + , caculated 532.1446, found 533.4.

[0114] Example 2: Preparation of 20(S)-7-(3,5-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 2), the steps of which are as follows:

[0115] The 3,4-dimethylphenylboronic acid in step (2) of Example 1 is replaced with 3,5-dimethylphenylboronic acid. In step (3), manganese dioxide is used instead of Desmartin oxidant as catalyst. In step (4), Raney nickel is used instead of palladium on carbon as catalyst. In step (5), iodine is used instead of p-toluenesulfonic acid hydrate as catalyst. The other required catalysts, reagents and preparation methods are the same as in steps (1)-(5) of Example 1.

[0116] Step (2) yielded 144.5 mg of compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3,5-dimethylphenyl)methanol (D3), with a yield of 99.0% and a purity of 98.44%.

[0117] 1 H NMR (400MHz, Chloroform-d) δ7.73 (s, 1H), 7.56 (s, 1H), 6.92 (d, J = 13.1Hz, 3H), 6.42 (d, J = 3.1Hz, 1H), 2.76 (d, J = 3.9Hz, 1H), 2.29 (d, J = 0.7Hz, 6H). HR-MS: C 16 H 13 F2NO5 for [M+H] + , caculated 337.0762, found 338.0621.

[0118] Step (3) yielded 128.0 mg of compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3,5-dimethylphenyl) methyl ketone (E3), with a yield of 89.14% and a purity of 97.26%.

[0119] 1 H NMR (400MHz, Chloroform-d) δ7.91 (s, 1H), 7.26 (s, 2H), 7.18 (d, J = 5.6Hz, 1H), 7.07 (s, 1H), 2.27 (s, 6H). HR-MS: C 16 H 11 F2NO5 for [M+H] + , caculated 335.0605, found336.0527.

[0120] Step (4) yielded 106.3 mg of compound (6-amino-2,2-difluorobenzo[d][1,3]dioxolane-5-yl)(3,5-dimethylphenyl) methyl ketone (F3), with a yield of 91.2% and a purity of 97.66%.

[0121] 1 H NMR(400MHz,Chloroform-d)δ7.16(s,3H),7.12(s,1H),6.42(s,1H),2.37(s,6H).HR-MS:C 16 H 13 F2NO3 for [M+H] + , caculated 305.0863, found 306.0782.

[0122] Step (5) finally yields 116.5 mg of pure white solid 20(S)-7-(3,5-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 2), with a yield of 81.7% and a purity of 98.87%.

[0123] 1H NMR(400MHz,Chloroform-d)δ7.84(s,1H),7.66(s,1H),7.39(s,1H),7.21(s,1H),7.01(d,J=19.8Hz,2H),5.70(d,J=16.4Hz,1H),5.27 (d,J=16.4Hz,1H),5.15-4.99(m,2H),4.01(s,1H),2.44(d,J=2.7Hz,6H),1.88(dq,J=14.0,7.1Hz,2H),1.03(t,J=7.4Hz,3H).HR-MS:C 29 H 22 F2N2O6 for [M+H] + , caculated 532.1446, found 533.1534.

[0124] Example 3: Preparation of 20(S)-7-(4-trimethylsilylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 3), the steps of which are as follows:

[0125] The 3,4-dimethylphenylboronic acid in step (2) of Example 1 was replaced with 4-trimethylsilylphenylboronic acid, the catalyst in step (3) was replaced with pyridinium chlorochromate instead of Desmartin oxidant, the catalyst in step (4) was replaced with platinum on carbon instead of palladium on carbon, and the catalyst in step (5) was replaced with dodecyl sulfate instead of p-toluenesulfonic acid hydrate. The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 134.2 mg of white solid product (compound 3) was obtained. The yield of step (5) was 86.4% and the purity was 98.96%.

[0126] 1 H NMR(400MHz,Chloroform-d)δ7.84(s,1H),7.76(t,J=6.6Hz,2H),7.65(s,1H),7.43(d,J=8.1Hz,1H),5.70(d,J=16.3 Hz,1H),5.27(d,J=16.4Hz,1H),5.07(d,J=3.0Hz,2H),1.99-1.79(m,2H),1.03(t,J=7.4Hz,3H),0.38(s,8H).HR-MS:C 30 H 26 F2N2O6Sifor[M+H] + , caculated 576.1528, found 577.3.

[0127] Example 4: Preparation of 20(S)-7-(3-methylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 4), the steps of which are as follows:

[0128] 3-Methylphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (1) of Example 1. Silver carbonate was used instead of Desmartin oxidant in step (3). Zinc powder was used instead of palladium on carbon in step (4). Ferric chloride hexahydrate was used instead of p-toluenesulfonic acid hydrate in step (5). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 105.2 mg of white solid product (compound 4) was obtained. The yield of step (5) was 80.2% and the purity was 98.56%.

[0129] 1 H NMR(400MHz,Chloroform-d)δ7.84(d,J=5.0Hz,1H),7.66(d,J=3.6Hz,1H),7.51(q,J=7.2Hz,1H),7.40(d,J=7.7Hz,1H),7.18(d,J=7.4Hz,1H),5.2 7(d,J=16.4Hz,1H),5.08(d,J=7.0Hz,2H),3.98(d,J=1.6Hz,1H),2.49(d, J=3.7Hz,3H),1.99-1.79(m,J=7.2Hz,2H),1.03(t,J=7.4Hz,3H).HR-MS:C 28 H 20 F2N2O6 for [M+H] + , caculated 518.1289, found519.1338.

[0130] Example 5: Preparation of 20(S)-7-(4-ethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 5), the steps of which are as follows:

[0131] 4-Ethylphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. N-bromosuccinimide was used instead of Desmartin oxidant in step (3). Rhodium powder was used instead of palladium on carbon in step (4). Aminosulfonic acid was used instead of p-toluenesulfonic acid hydrate in step (5). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 128.2 mg of white solid product (compound 5) was obtained. The yield of step (5) was 87.6% and the purity was 98.20%.

[0132] 1H NMR (400MHz, Chloroform-d) δ7.83 (s, 1H), 7.65 (s, 1H), 7.46 (t, J = 7.4Hz, 2H), 7.43-7.28 (m, 3H), 5.69 (d, J = 16.4Hz, 1H), 5.26 (d, J = 16. 4Hz,1H),5.15-4.99(m,2H),3.99(s,1H),2.81(q,J=7.6Hz,2H),1.99-1.79(m,2H),1.37(t,J=7.6Hz,3H),1.03(t,J=7.4Hz,3H).HR-MS:C 29 H 22 F2N2O6 for [M+H] + , caculated 532.1446, found 533.4.

[0133] Example 6: Preparation of 20(S)-7-(4-isopropylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 6), the steps of which are as follows:

[0134] The catalyst used in step (2) of Example 1 was replaced with 4-isopropylphenylboronic acid instead of 3,4-dimethylphenylboronic acid. The catalyst used in step (3) was iodic acid instead of Desmartin oxidant. The catalyst used in step (4) was stannous chloride instead of palladium on carbon. The catalyst used in step (5) was 2,4,6-trichloro-1,3,5-triazine instead of p-toluenesulfonic acid hydrate. The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 107.8 mg of white solid product (compound 6) was obtained. The yield of step (5) was 84.34% and the purity was 98.56%.

[0135] 1 H NMR(400MHz,Chloroform-d)δ7.83(s,1H),7.65(s,1H),7.48(t,J=6.6Hz,2H),7.4 2(s,1H),7.38(d,J=7.3Hz,1H),7.32(d,J=7.4Hz,1H),5.70(d,J=16.3Hz,1H),5.2 7(d,J=16.3Hz,1H),5.08(d,J=3.3Hz,2H),3.97(s,1H),3.06(hept,J=7.0Hz,1H), 1.88(dh,J=14.3,7.2Hz,2H),1.37(d,J=6.9Hz,6H),1.03(t,J=7.3Hz,3H).HR-MS:C 30 H 24 F2N2O6 for [M+H] +, caculated 546.1602, found 547.2.

[0136] Example 7 Preparation of 20(S)-7-(3-trifluoromethoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 7), the steps are as follows:

[0137] The catalyst used in Example 1 was 3-trifluoromethoxyphenylboronic acid instead of 3,4-dimethylphenylboronic acid in step (2), hydrogen peroxide was used instead of Desmartin oxidant in step (3), lithium aluminum hydride was used instead of palladium on carbon in step (4), and bismuth trifluoromethanesulfonate was used instead of p-toluenesulfonic acid hydrate in step (5). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 127.2 mg of white solid product (compound 7) was obtained. The yield of step (5) was 81.1% and the purity was 97.96%.

[0138] 1 H NMR(400MHz, DMSO-d6)δ8.20(d,J=2.5Hz,1H),7.84(td,J=8.1,4.5Hz,1H),7.75-7.64(m,3H),7.56(s,1H),7.33 (s,1H),6.54(s,1H),5.40(s,2H),5.16-4.94(m,2H),1.83(q,J=7.3,6.9Hz,2H),0.87(t,J=7.3Hz,3H).HR-MS:C 28 H 17 F5N2O7 for [M+H] + , caculated 588.0956, found 589.2.

[0139] Example 8: Preparation of 20(S)-7-(4-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 8), the steps of which are as follows:

[0140] 4-Methoxyphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. Chromium trioxide was used instead of Desmartin oxidant in step (3). Sodium borohydride was used instead of palladium on carbon in step (4). Yttrium trifluoromethanesulfonate was used instead of p-toluenesulfonic acid hydrate in step (5). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 120.0 mg of white solid product (compound 8) was obtained. The yield of step (5) was 83.69% and the purity was 97.69%.

[0141] 1H NMR(400MHz,Chloroform-d)δ7.83(s,1H),7.64(s,1H),7.46-7.31(m,3H),7.14(d,J=8.1Hz,2H),5.71(d,J=16.3Hz,1H),5 .28(d,J=16.3Hz,1H),5.09(s,2H),3.94(s,3H),3.84(s,1H),1.88(dq,J=14.5,7.3Hz,2H),1.04(t,J=7.5Hz,3H).HR-MS:C 28 H 20 F2N2O7 for [M+H] + , caculated 534.1239, found 535.3.

[0142] Example 9: Preparation of 20(S)-7-(3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 9), the steps of which are as follows:

[0143] 3-Methoxyphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. N-chlorosuccinimide was used instead of Desmartin oxidant in step (3). Lithium aluminum hydride was used instead of palladium on carbon in step (4). Lithium bis(trifluoromethanesulfonyl)imide was used instead of p-toluenesulfonic acid hydrate in step (5). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1.

[0144] Step (2) yielded 119.1 mg of compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3-methoxyphenyl)methanol (D10), with a yield of 81.1% and a purity of 95.65%.

[0145] 1 H NMR(400MHz,Chloroform-d)δ7.73(s,1H),7.52(s,1H),7.28(d,J=8.2Hz,1H),6.9 2-6.80(m,3H),6.47(d,J=3.3Hz,1H),3.80(s,3H),2.79(d,J=4.0Hz,1H).HR-MS:C 15 H 11 F2NO6 for [M+H] + , caculated 339.0554, found 340.0472. HR-MS:C 28 H 20 F2N2O7 for [M+H] +, caculated 534.1239, found 535.3.

[0146] Step (3) yielded a total of 113.0 mg of compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxolane-5-yl)(3-methoxyphenyl) methyl ketone (E3), with a yield of 95.5% and a purity of 95.63%.

[0147] 1 H NMR(400MHz,Chloroform-d)δ7.98(s,1H),7.40(dd,J=2.6,1.6Hz,1H),7.34(dd,J=8.3,7.6Hz,1H),7.20-7.11(m,3H),3.86(s,3H).HR-MS:C 15 H9F2NO6 for [M+H] + ,caculated337.0398,found 338.0257.

[0148] Step (4) yielded a total of 92.5 mg of compound (6-amino-2,2-difluorobenzo[d][1,3]dioxolane-5-yl)(3-methoxyphenyl) methyl ketone (F3), with a yield of 89.9% and a purity of 94.65%.

[0149] 1 H NMR(400MHz,Chloroform-d)δ7.41-7.32(m,1H),7.12(d,J=2.1Hz,1H),7.11(q,J=1.0 Hz,1H),7.07(ddd,J=8.2,2.4,1.1Hz,1H),6.43(d,J=4.3Hz,2H),3.85(s,3H).HR-MS:C 15 H 11 F2NO4 for [M+H] + , caculated 307.0656, found 308.0508.

[0150] Step (5) ultimately yields 106.7 mg of a pure white solid product, 20(S)-7-(3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 9), with a yield of 86.2% and a purity of 98.09%.

[0151] 1H NMR(400MHz,Chloroform-d)δ7.84(s,1H),7.65(s,1H),7.54(q,J=7.8Hz,1H),7.16-7.08(m,1H),7.03-6.87(m,3H),5.70(d,J=16.4Hz, 1H),5.27(d,J=16.4Hz,1H),5.08(t,J=3.1Hz,2H),3.93(s,1H),3.89(d,J=4.2Hz,3H),1.94-1.84(m,2H),1.03(t,J=7.3Hz,3H).HR-MS:C 28 H 20 F2N2O7 for [M+H] + , caculated 534.1239, found 535.3.

[0152] Example 10: Preparation of 20(S)-7-(3,5-dimethoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 10), the steps of which are as follows:

[0153] 3,5-Dimethoxyphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. In step (3), tetra-n-propylammonium perruthenate was used instead of Desmartin oxidant as catalyst. In step (4), potassium borohydride was used instead of palladium on carbon as catalyst. In step (5), magnesium bis(trifluoromethanesulfonylimide) was used instead of p-toluenesulfonic acid hydrate as catalyst. The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 88.9 mg of white solid product (compound 10) was obtained. The yield of step (5) was 84.9% and the purity was 97.56%.

[0154] 1 H NMR(400MHz,Chloroform-d)δ7.84(s,1H),7.65(s,1H),7.43(s,1H),6.64(s,1H),6.50(d,J=17.4Hz,2H),5.71(d,J=16.4 Hz,1H),5.28(d,J=16.3Hz,1H),5.09(s,2H),3.89(s,1H),3.86(s,6H),1.88(h,J=7.8Hz,2H),1.08-0.99(m,3H).HR-MS:C 29 H 22 F2N2O8 for [M+H] + , caculated 564.1344, found 565.4.

[0155] Example 11 Preparation of 20(S)-7-(3-fluoro-4-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 11), the steps are as follows:

[0156] By replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 3-fluoro-4-methoxyphenylboronic acid, replacing the Desmartin oxidant with sodium nitrite / acetic anhydride oxidant in step (3), replacing the palladium on carbon with sodium sulfide oxidant in step (4), and using the same catalysts, reagents and preparation methods as in steps (1)-(5) of Example 1, a total of 97.5 mg of white solid product (compound 11) can be prepared. The yield of step (5) is 79.65% and the purity is 98.10%.

[0157] 1 H NMR (400MHz, DMSO-d6) δ8.11(s,1H),7.61(s,2H),7.48(d,J=14.7Hz,2H),7.26(s,1H),6.52(s,1H),5.37(s, 2H),5.06(d,J=19.2Hz,1H),4.89(t,J=16.5Hz,1H),3.99(s,3H),1.77(s,2H),0.85(t,J=7.4Hz,3H).HR-MS:C 28 H 19 F3N2O7 for [M+H] + ,calculated 552.1144,found 553.0. Example 12 Preparation of 20(S)-7-(3-fluoro-5-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 12), the steps are as follows:

[0158] By replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 3-fluoro-5-methoxyphenylboronic acid, replacing Desmartin oxidant with aluminum tert-butoxide as catalyst in step (3), replacing palladium on carbon with hydrogen sulfide as catalyst in step (4), and using the same catalysts, reagents and preparation methods as in steps (1)-(5) of Example 1, a total of 106.5 mg of white solid product (compound 12) can be prepared. The yield of step (5) is 86.3% and the purity is 95.65%.

[0159] 1H NMR (400MHz, DMSO-d6) δ8.16(s,1H),7.63(s,1H),7.32(s,1H),7.16-7.04(m,3H),6.54(s,1H),5.40(s,2H),5.09(dd,J=18 .8,11.5Hz,1H),4.97(dd,J=18.8,4.3Hz,1H),3.89(d,J=10.1Hz,3H),1.82(q,J=7.3Hz,2H),0.87(t,J=7.3Hz,3H).HR-MS:C 28 H 19 F3N2O7 for [M+H] + , caculated 552.1144, found 553.2.

[0160] Example 13 Preparation of 20(S)-7-(4-fluoro-3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 13), the steps are as follows:

[0161] By replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 4-fluoro-3-methoxyphenylboronic acid, and replacing palladium on carbon with sodium disulfide as the catalyst in step (4), the remaining required catalysts, reagents and preparation methods are the same as in steps (1)-(5) of Example 1, a total of 101.9 mg of white solid product (compound 13) can be prepared. The yield of step (5) is 83.5% and the purity is 98.13%.

[0162] 1 H NMR(400MHz,Chloroform-d)δ7.86(d,J=4.5Hz,1H),7.67(d,J=3.6Hz,1H),7.35(s,2H),7.26(s,1H),7.08-6.89(m,2H),5.68(d,J=16.4Hz,1H),5. 26(d,J=16.4Hz,1H),5.16-5.00(m,2H),4.07(d,J=2.6Hz,1H),3.93(d,J= 13.6Hz, 3H), 1.89 (dh, J=14.2, 7.3Hz, 2H), 1.02 (t, J=7.3Hz, 3H). HR-MS: C 28 H 19 F3N2O7 for [M+H] + , caculated 552.1144, found 533.4.

[0163] Example 14 Preparation of 20(S)-7-(3-fluoro-4-methylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 14), the steps are as follows:

[0164] Replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 3-fluoro-4-methylphenylboronic acid, and using the same catalysts, reagents and preparation methods as in steps (1)-(5) of Example 1, a total of 88.2 mg of white solid product (compound 14) can be obtained. The yield of step (5) is 87.5% and the purity is 98.05%.

[0165] 1H NMR(400MHz,Chloroform-d)δ7.85(d,J=2.6Hz,1H),7.66(s,1H),7.46(q,J=8.2Hz,1H),7.36(s,1H),7.15(t,J=6.9Hz,1H),7.11-7.03(m,1H) ,5.69(d,J=16.4Hz,1H),5.26(d,J=16.4Hz,1H),5.13-5.01(m,2H),4.06(s,1H),2.44(s,3H),1.88(hept,J=7.1Hz,2H),1.02(t,J=7.4Hz,3H). HR-MS:C28H19F3N2O6 for[M+H]+,caculated536.1195,found537.1105.

[0166] Example 15: Preparation of 20(S)-7-(2,4-difluorophenyl)-10,11-difluoromethylenedioxycamptothecin (compound 15), the steps of which are as follows:

[0167] 2,4-Difluorophenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, and ferrous sulfate was used instead of palladium on carbon in step (4). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 77.8 mg of white solid product (compound 15) was obtained. The yield of step (5) was 80.6% and the purity was 98.00%.

[0168] 1H NMR(400MHz,Chloroform-d)δ7.88(s,1H),7.67(s,1H),7.38(p,J=7.8,7.2Hz,1H),7.24(s,2H),7.16(q,J=8.9Hz,2H),5.71(d,J=1 6.4Hz,1H),5.28(d,J=16.4Hz,1H),5.15-4.98(m,2H),3.89(d,J=7.8Hz,1H),1.89(p,J=7.5Hz,2H),1.04(t,J=7.5Hz,3H).HR-MS:C 27 H 16 F4N2O6 for [M+H] + , caculated 540.0944, found 541.1.

[0169] Example 16 Preparation of 20(S)-7-(3,4-difluorophenyl)-10,11-difluoromethylenedioxycamptothecin (compound 16), the steps are as follows:

[0170] 3,4-Difluorophenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, and sodium sulfite was used instead of palladium on carbon in step (4). The other required catalysts, reagents and preparation methods were the same as in steps (1)-(5) of Example 1. A total of 87.2 mg of white solid product (compound 16) was obtained. The yield of step (5) was 85.9% and the purity was 97.95%.

[0171] 1 H NMR(400MHz,Chloroform-d)δ7.87(s,4H),7.66(s,4H),7.46(p,J=8.2Hz,5H),7.31(s,6H),7.23(s,8H),5.70(d,J=16.4Hz,4 H),5.27(d,J=16.4Hz,4H),5.08(s,7H),4.84(s,1H),3.93(s,4H),1.89(hept,J=7.0Hz,9H),1.03(t,J=7.3Hz,12H).HR-MS:C 27 H 16 F4N2O6 for [M+H] + , caculated 540.0944, found 541.1016.

[0172] Example 17 Preparation of 20(S)-7-(4-fluorophenyl)-10,11-difluoromethylenedioxycamptothecin (compound 17), the steps are as follows:

[0173] By replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 4-fluorophenylboronic acid, and replacing palladium on carbon with sodium bisulfite as the catalyst in step (4), the remaining required catalysts, reagents and preparation methods are the same as in steps (1)-(5) of Example 1, a total of 100.4 mg of white solid product (compound 17) can be prepared. The yield of step (5) is 88.4% and the purity is 97.69%.

[0174] 1 H NMR(400MHz,Chloroform-d)δ7.86(s,1H),7.67(s,1H),7.50-7.45(m,1H),7.43-7.33(m,3H),7.32(s,1H),5.69(d,J=16.4Hz ,1H),5.27(d,J=16.4Hz,1H),5.07(d,J=4.6Hz,2H),4.00(s,1H),1.88(dq,J=14.4,7.2Hz,2H),1.03(t,J=7.4Hz,3H).HR-MS:C 27 H 17 F3N2O6 for [M+H] + , caculated 522.1039, found 523.1124.

[0175] Example 18 Preparation of 20(S)-7-(4-trifluoromethylphenyl)-10,11-difluoromethylenedioxycamptothecin (compound 18)

[0176] By replacing 3,4-dimethylphenylboronic acid in step (2) of Example 1 with 4-trifluoromethylphenylboronic acid, and using the same catalysts, reagents and preparation methods as in steps (1)-(5) of Example 1, a total of 95.0 mg of white solid product (compound 18) can be obtained. The yield of step (5) is 87% and the purity is 98.25%.

[0177] 1H NMR(400MHz,Chloroform-d)δ7.96-7.86(m,3H),7.68(d,J=2.2Hz,1H),7.59(dd,J=19.4,8.0Hz,2H),7.27(d,J=2.2Hz,2H),5.70(dd,J=16. 4,2.3Hz,1H),5.28(d,J=16.6Hz,1H),5.06(s,2H),3.91(s,1H),1.90(hept,J=8.3,7.6Hz,2H),1.04(t,J=7.4Hz,3H).HR-MS:C28H17F5N2O6 for[M+H]+,cached 572.1007,found 579.1080.

[0178] Example 19 Preparation of 20(S)-7-(4-methylthiophenyl)-10,11-difluoromethylenedioxycamptothecin (compound 19), the steps are as follows:

[0179] 4-Methylthiophenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. Except for the iron powder used as the reduction catalyst in step (4), which is different from the previous reduction catalyst palladium on carbon, the catalysts, reagents and preparation methods required for the other steps are the same as in Example 1. A total of 89.4 mg of white solid product (compound 19) can be prepared. The yield of step (5) is 87.2% and the purity is 97.96%.

[0180] 1 H NMR(400MHz,Chloroform-d)δ7.84(s,1H),7.64(s,1H),7.47(d,J=8.0Hz,2H),7.43-7.29(m,3H),5.71(d,J=16.8Hz,1H) ,5.28(d,J=16.0Hz,1H),5.09(s,2H),3.83(s,1H),2.61(s,3H),1.89(hept,J=7.2Hz,2H),1.04(t,J=7.4Hz,3H).HR-MS:C 28 H 20 F2N2O6S for [M+H] + , caculated 550.1010, found 551.1087.

[0181] The simplified synthetic route 2 for compounds 20-21 is as follows:

[0182]

[0183] Example 20: Preparation of 20(S)-7-enbutyl-10,11-difluoromethylenedioxycamptothecin (compound 20), the steps are as follows:

[0184] (1) Preparation of 2,2-difluorobenzo[d][1,3]dioxane-5-enbutanol (G1)

[0185] 500 mg of 2,2-difluorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde (A) was added to a three-necked flask and dissolved in anhydrous THF. The flask was stirred in an ice-water bath, and 385.4 mg of Grignard reagent allyl magnesium bromide was added under nitrogen protection. After 10 min, the temperature was restored to 25 °C and the reaction was allowed to proceed for 12 h. The reaction was monitored by TLC (using a mixture of petroleum ether and ethyl acetate in a ratio of 800 μL to 100 μL, v / v). After the reaction was complete, an appropriate amount of saturated nitrogen was added. The reaction was quenched with H4Cl solution, consuming excess Grignard reagent. After stirring for 10 min, the mixture was transferred to a separatory funnel and extracted three times with dichloromethane (DCM). The organic phases were combined, dried with anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a pale yellow liquid, which was separated by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 80:1, v / v). After concentration, a total of 344.6 mg of yellow solid compound G1 was obtained, with a yield of 88.3% and a purity of 95.68%.

[0186] HR-MS:C 12 H 12 F₂O₃ for [M+H] + , caculated 242.0755, found 243.0726.

[0187] (2) Preparation of 2,2-difluorobenzo[d][1,3]dioxane-5-enbutanone (H1)

[0188] The intermediate compound 2,2-difluoro-6-nitrobenzo[d][1,3]dioxanepenten-5-enbutanol (G1) (334.6 mg) was added to a round-bottom flask, dissolved in dichloromethane, and then Dysmartin oxidant (DMP) (879.6 mg) was added. The mixture was stirred at 25 °C for 3 h. The reaction was monitored by TLC (using a mixture of petroleum ether and ethyl acetate, 800 μL: 100 μL, v / v). After the reaction was completed, saturated Na2SO3 solution and saturated NaHCO3 solution were added successively to quench the reaction. After stirring for about 20 minutes, the mixture was observed to separate into layers, with the DCM layer gradually becoming clear. The solution was transferred to a separatory funnel and extracted three times with saturated NaHCO3 solution, followed by three back-extractions with DCM. The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a pale yellow liquid, which was then separated by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a ratio of 80:1, v / v). After concentration, a total of 294.5 mg of the pale yellow liquid compound H1 was obtained, with a yield of 88.7% and a purity of 97.65%.

[0189] HR-MS:C 12 H 10 F₂O₃ for [M+H] + , caculated 240.0598, found 241.0556.

[0190] (3) Preparation of 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-enbutanone (I1)

[0191] The intermediate compound 2,2-difluorobenzo[d][1,3]dioxacyclopenten-5-enbutanone (H1) (294.5 mg) was added to a 50 mL round-bottom flask. 1 mL of concentrated sulfuric acid was added, and the flask was gently shaken to allow the sulfuric acid to fully activate the reactants along the flask wall. The flask was then placed in a -15°C constant-temperature stirred reaction bath with stirring. Fuming nitric acid was slowly added dropwise. After adding 9 mL of nitric acid, the mixture was placed at -15°C for 12 h. TLC was performed (using a petroleum ether:ethyl acetate mixture of 1500 μL: 100 μL as the developing solvent). The reaction was monitored. After the reaction was completed, an appropriate amount of ice was added to the round-bottom flask to quench the reaction and dilute the concentrated sulfuric acid while absorbing heat. After the ice melted, the solution was transferred to a 100 mL separatory funnel and extracted three times with DCM. The organic phases were combined, dried with anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a pale yellow liquid, which was separated by silica gel column chromatography (eluent was a mixture of petroleum ether and dichloromethane = 4:1, v / v). After concentration, 239.1 mg of intermediate compound I1 was obtained, with a yield of 68.4% and a purity of 98.32%.

[0192] HR-MS:C12 H9F2NO5 for [M+H] + , caculated 285.0449, found 286.0413.

[0193] (4) Preparation of 6-amino-2,2-difluorobenzo[d][1,3]dioxane-5-enbutanone (J1)

[0194] The intermediate compound 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-enbutanone (I1) (239.1 mg) was placed in a round-bottom flask, and Pd / C (36 mg), triethylamine (200 μL), and anhydrous ethanol were added sequentially. Hydrogen gas was introduced and the mixture was stirred at 25 °C for 12 h. The reaction progress was monitored by TLC (developing solvent was a mixed solution of petroleum ether and ethyl acetate = 800 μL: 100 μL, v / v). After the reaction was completed, the mixture was washed with DCM and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography. The concentrate was dissolved in DCM and an appropriate amount of silica gel was added. The solvent was evaporated to dryness and the sample was loaded onto the silica gel column for separation by silica gel column chromatography (eluent was a mixed solution of petroleum ether and ethyl acetate = 20: 1, v / v). After concentration, 161.7 mg of intermediate compound J1 was obtained, with a yield of 75.6% and a purity of 97.64%.

[0195] HR-MS:C 12 H 11 F2NO3 for [M+H] + , caculated 255.0707, found 256.0521.

[0196] (5) Preparation of 20(S)-7-enbutyl-10,11-difluoromethylenedioxycamptothecin (compound 20)

[0197] The products obtained in step (4), 6-amino-2,2-difluorobenzo[d][1,3]dioxane-5-enpentanone (J1) (161.7 mg, 1.5 mmol), tricyclic ketone (99.7 mg, 1 mmol), and p-toluenesulfonic acid hydrate (43.4 mg, 0.4 mmol), were added to a round-bottom flask, dissolved in glacial acetic acid, and refluxed at 100 °C for 12 h under nitrogen protection. The pH was adjusted (pH = 5-6), and the mixture was extracted three times with dichloromethane. The organic phases were combined, washed once with saturated brine, dried with anhydrous magnesium sulfate, and concentrated to obtain a yellow oily liquid. The liquid was separated by silica gel column chromatography (eluent was a mixture of dichloromethane and ethyl acetate in a ratio of 9:1, v / v, with a small amount of glacial acetic acid). After concentration, the liquid was washed with methanol and filtered. Finally, 195.3 mg of pure white solid product (compound 20) was obtained on the filter cake, with a yield of 83.2% and a purity of 95.64%.

[0198] HR-MS:C 25 H 20 F2N2O6 for [M+H] + , caculated 482.1289, found 483.1256.

[0199] Example 21 Preparation of 20(S)-7-(4-pyranmethyl)-10,11-difluoromethylenedioxycamptothecin (compound 21), the steps are as follows:

[0200] Using 4-pyranmethylmagnesium bromide instead of allyl magnesium bromide in step (1) of Example 18, and using sodium nitrite instead of palladium on carbon as the catalyst in step (4), the catalysts, reagents, and preparation methods required for the remaining steps are the same as those in Example 18. A total of 205.8 mg of white solid product (compound 24) can be prepared. The yield of step (5) is 84.4%, and the purity is 97.25%.

[0201] HR-MS:C 27 H 24 F2N2O7 for [M+H] + , caculated 526.1552, found 527.1501.

[0202] The simplified synthetic routes for compounds 22-23 are as follows:

[0203]

[0204] Example 22 Preparation of 20(S)-7-(5-methylfuran-2-ethyl)-10,11-difluoromethylenedioxycamptothecin (compound 22), the steps are as follows:

[0205] (1) Preparation of 2,2-difluorobenzo[d][1,3]dioxane-5-ethanol (intermediate K)

[0206] 1 g of 2,2-difluorobenzo[d][1,3]dioxacyclopentene-5-carboxaldehyde (A) was added to a three-necked flask and dissolved in anhydrous THF. The flask was stirred in an ice-water bath, and 961.0 mg of Grignard reagent methyl magnesium bromide was added under nitrogen protection. After 10 min, the temperature was restored to 25 °C and the reaction was allowed to proceed for 12 h. The reaction was monitored by TLC (using a mixture of petroleum ether and ethyl acetate in a ratio of 800 μL to 100 μL, v / v). After the reaction was complete, an appropriate amount of saturated nitrogen was added. The reaction was quenched with H4Cl solution, consuming excess Grignard reagent. After stirring for ten minutes, the mixture was transferred to a separatory funnel and extracted three times with dichloromethane (DCM). The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a pale yellow liquid, which was separated by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 80:1, v / v). After concentration, a total of 882.0 mg of yellow solid compound K was obtained, with a yield of 81.2% and a purity of 97.88%.

[0207] HR-MS: C9H8F2O3 for [M+H] + , caculated 202.0442, found 203.0386.

[0208] (2) Preparation of 2,2-difluorobenzo[d][1,3]dioxacyclopentene-5-ethyl ketone (intermediate L)

[0209] The intermediate compound 2,2-difluoro-6-nitrobenzo[d][1,3]dioxanepentene-5-ethanol (intermediate K) (882.0 mg) was added to a round-bottom flask, dissolved in dichloromethane, and then Dysmartin oxidant (DMP) (3024.4 mg) was added. The mixture was stirred at 25 °C for 3 h. The reaction was monitored by TLC (using a mixture of petroleum ether and ethyl acetate, 800 μL: 100 μL, v / v). After the reaction was completed, saturated Na₂SO₃ solution and saturated NaHCO₃ solution were added successively to quench the reaction. The reaction was extinguished, and after stirring for about 20 minutes, the mixture was observed to separate into layers. The DCM layer gradually became clear. The solution was transferred to a separatory funnel and extracted three times with saturated NaHCO3 solution, followed by three back-extractions with DCM. The organic phases were combined, dried with anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a pale yellow liquid, which was separated by silica gel column chromatography (eluent was a mixture of petroleum ether and ethyl acetate in a ratio of 80:1, v / v). After concentration, a total of 734.7 mg of the pale yellow liquid compound L was obtained, with a yield of 82.6% and a purity of 98.31%.

[0210] HR-MS: C8H5F2O3 for [M+H] + , caculated 187.0207, found 188.0146.

[0211] (3) Preparation of 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-ethyl ketone (intermediate M)

[0212] The intermediate compound 2,2-difluorobenzo[d][1,3]dioxane-5-ethyl ketone (intermediate L) (734.7 mg) was added to a 50 mL round-bottom flask. 1 mL of concentrated sulfuric acid was added, and the flask was gently shaken to allow the sulfuric acid to fully activate the reactants along the flask wall. The flask was then placed in a -15°C constant-temperature stirring bath with stirring. Fuming nitric acid was slowly added dropwise. After adding 9 mL of nitric acid, the mixture was placed at -15°C for 12 h. TLC was performed (the developing solvent was a mixture of petroleum ether and ethyl acetate = 1500 μL: 100 μL, v / v) Monitor the reaction progress. After the reaction is complete, add an appropriate amount of ice to the round-bottom flask to quench the reaction, dilute the concentrated sulfuric acid and absorb heat at the same time. After the ice melts, transfer the solution to a 100mL separatory funnel and extract three times with DCM. Combine the organic phases, dry with anhydrous magnesium sulfate, filter under reduced pressure, concentrate the filtrate to obtain a pale yellow liquid, and separate it by silica gel column chromatography (eluent is a mixed solution of petroleum ether and dichloromethane = 4:1, v / v). After concentration, 618.9 mg of intermediate compound M is obtained, with a yield of 64.3% and a purity of 97.98%.

[0213] HR-MS: C8H5F2O3 for [M+H] + , caculated 245.0136, found 246.0051.

[0214] (4) Preparation of 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)-3-(5-methylfuran-2-yl)propenone (N1)

[0215] The intermediate compound 2,2-difluoro-6-nitrobenzo[d][1,3]dioxane-5-ethyl ketone (intermediate M) (100 mg) was added to a 50 mL round-bottom flask, dissolved in anhydrous ethanol, and then sodium hydroxide (19.6 mg) was added. 5-methylfuran-2-carboxaldehyde (45 mg) was diluted and added. The mixture was stirred at 25 °C for 1 h. After the reaction was complete, the pH was adjusted to neutral with 10% hydrochloric acid. The mixture was extracted three times with ethyl acetate, and the supernatant was collected. Separation was performed by silica gel column chromatography (eluent was a 20:1 mixture of petroleum ether and ethyl acetate, v / v). After concentration, 100.7 mg of intermediate compound N1 was obtained, with a yield of 73.2% and a purity of 97.87%.

[0216] HR-MS:C 15 H9F2NO6 for [M+H] +, caculated 337.0398, found 338.0301.

[0217] (5) Preparation of 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)-3-(5-methylfuran-2-yl)acetone (O1)

[0218] The intermediate compound 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)-3-(5-methylfuran-2-yl)propenone (N1) (100.7 mg) was placed in a round-bottom flask and dissolved in anhydrous ethanol by heating. After cooling to 25°C, Pd / C (15 mg) was added sequentially, followed by the introduction of hydrogen gas and the addition of triethylamine (100 μL). The mixture was stirred at 25°C for 12 h. TLC was performed using petroleum ether:ethyl acetate = 800 μL as the developing solvent. The reaction was monitored by mixing a 100 μL:100 μL solution (v / v). After the reaction was complete, the solution was washed with DCM and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography. The concentrate was dissolved in DCM, and an appropriate amount of silica gel was added. The solvent was evaporated to dryness and the sample was loaded onto the silica gel column for separation by eluent (a 20:1 mixture of petroleum ether and ethyl acetate, v / v). After concentration, 57.8 mg of intermediate compound O1 was obtained, with a yield of 62.6% and a purity of 98.51%.

[0219] HR-MS:C 15 H 13 F2NO4 for [M+H] + , caculated 309.0813, found 310.0755.

[0220] (6) Preparation of 20(S)-7-(5-methylfuran-2-ethyl)-10,11-difluoromethylenedioxycamptothecin (compound 25)

[0221] The products obtained in step (2), 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxacyclopenten-5-yl)-3-(5-methylfuran-2-yl)acetone (O1) (57.8 mg, 1.3 mmol), tricyclic ketone (48.5 mg, 1 mmol), and p-toluenesulfonic acid hydrate (17.8 mg, 0.5 mmol), were added to a round-bottom flask, dissolved in glacial acetic acid, and reacted under nitrogen protection at 95 °C for 8 h. The pH was then adjusted. The solution was extracted three times with dichloromethane at pH 5-6. The organic phases were combined, washed once with saturated brine, dried with anhydrous magnesium sulfate, and concentrated to obtain a yellow oily liquid. Separation was achieved by silica gel column chromatography (eluent: a mixture of dichloromethane and ethyl acetate in a ratio of 9:1, v / v, with a small amount of glacial acetic acid). After concentration, the solution was washed with methanol and filtered to obtain 54.9 mg of pure white solid product (compound 25) on the filter cake, with a yield of 55.3% and a purity of 97.62%.

[0222] HR-MS:C 28 H 22 F2N2O7 for [M+H] + , caculated 536.1395, found 537.1301.

[0223] Example 23 Preparation of 20(S)-7-(5-bromothiophene-2-ethyl)-10,11-difluoromethylenedioxycamptothecin (compound 23), the steps are as follows:

[0224] 5-Bromothiophene-2-carboxaldehyde was used instead of 5-methylfuran-2-carboxaldehyde in step (4) of Example 20. Ferrous dicarbonate was used instead of palladium on carbon in step (5). The catalysts, reagents and preparation methods required for the remaining steps were the same as those in Example 20. A total of 67.4 mg of white solid product (compound 27) was obtained. The yield of step (5) was 73.2% and the purity was 97.75%.

[0225] HR-MS:C 27 H 19 BrF₂N₂O₆S for [M+H] + , caculated 616.0115, found 617.0110.

[0226] Comparative Example 1 synthesized 20(S)-7-(4-trifluoromethylphenyl)-10,11-methylenedioxycamptothecin according to the method described in patent EP3852760, with a final yield of 36.5% and a purity of 94.02%.

[0227] 1H NMR(600MHz,Chloroform-d)δ7.92(m,2H),7.66(s,1H),7.57(m,3H),6.91(s,1H),6.18(d,J=3.6Hz,2H),5.71(d ,J=16.1Hz,1H),5.28(d,J=16.2Hz,1H),4.99(d,J=11.9Hz,2H),3.85(s,1H),1.89(m,2H),1.05(t,J=7.2Hz,3H).

[0228] Comparative Example 2 synthesized 20(S)-7-(3,5-dimethylphenyl)-10,11-methylenedioxycamptothecin according to the method described in patent EP3852760, with a final yield of 30.6% and a purity of 91.00%.

[0229] 1 H NMR(600MHz,Chloroform-d)δ7.60(s,1H),7.50(s,1H),7.14(s,1H),7.02(s,1H),6.98(s,1H),6.94(s,1H),6.11(q,J=1.2Hz,2H), 5.69(d,J=16.1Hz,1H),5.25(d,J=16.1Hz,1H),5.00(m,2H),3.75(s,1H),2.41(d,J=1.4Hz,6H),1.90(m,2H),1.06(t,J=7.4Hz,3H).

[0230] Comparative Example 3 synthesized 20(S)-7-(3-furan)-10,11-methylenedioxycamptothecin according to the method described in patent CN110590796A, with a final yield of 60.6% and a purity of 92.03%.

[0231] 1 H NMR (400MHz, CF3COOD) δ8.10(s,1H),8.02(s,1H),7.80(s,1H),7.75(s,1H),7.60(d,J=12.2Hz,1H),6.82(s, 1H), 6.38 (d, J = 24.8Hz, 2H), 5.84 (d, J = 16.6Hz, 1H), 5.56 (dd, J = 50.1, 25.2Hz, 3H), 2.08 (s, 2H), 1.09 (s, 3H).

[0232] Comparative Example 4 synthesized 20(S)-7-(3-thiophene)-10,11-methylenedioxycamptothecin according to the method described in patent CN110590796A, with a final yield of 46.6% and a purity of 92.80%.

[0233] 1 H NMR(400MHz,CF3COOD)δ8.10(s,1H),7.87(s,1H),7.80(s,1H),7.62(s,1H),7.60(s,1H),7.40(d,J=4.6Hz,1 H), 6.36 (s, 2H), 5.86 (d, J = 17.1Hz, 1H), 5.51 (d, J = 15.6Hz, 2H), 2.11 (q, J = 7.2Hz, 2H), 1.10 (t, J = 7.3Hz, 3H).

[0234] Comparative Example 5 synthesized 20(S)-7-ethyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared according to synthetic route 2 of this invention, achieving a final yield of 81.8% and a purity of 97.42%.

[0235] 1 H NMR(400MHz,DMSO-d6)δ8.16(s,1H),8.03(s,1H),7.25(s,1H),6.50(s,1H),5.40(s,2H),5.1 9(s,2H),2.70(t,J=8.0Hz,3H),1.82(p,J=8.0,7.6Hz,2H),1.25(m,2H)0.86(d,J=7.6Hz,3H).

[0236] Comparative Example 6 synthesized 20(S)-7-propyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared by synthetic route 2 according to the present invention, achieving a final yield of 85.3% and a purity of 98.22%.

[0237] 1 H NMR(400MHz,Chloroform-d)δ7.80(s,1H),7.63(d,J=7.3Hz,2H),5.74(d,J=16.4Hz,1H),5.30(d,J=16.3Hz,1H),5. 25(s,2H),3.93(s,1H),3.09(dd,J=8.9,6.8Hz,2H),1.96-1.76(m,4H),1.10(t,J=7.3Hz,3H),1.03(t,J=7.4Hz,3H).

[0238] Comparative Example 7 synthesized 20(S)-7-hexyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared according to synthetic route 2 of this invention, achieving a final yield of 83.4% and a purity of 98.28%.

[0239] 1 H NMR (400MHz, Chloroform-d) δ7.80 (s, 1H), 7.62 (d, J = 4.6Hz, 2H), 5.74 (d, J = 16. 3Hz,1H),5.34-5.26(m,1H),5.24(s,2H),3.92(s,1H),3.10(t,J=8.0Hz,2H),1.9 0(ddt,J=16.3,14.2,7.1Hz,2H),1.73(td,J=9.5,8.6,5.1Hz,2H),1.49(p,J=7.6 Hz, 2H), 1.34 (h, J = 3.4, 2.7Hz, 4H), 1.03 (t, J = 7.4Hz, 3H), 0.90 (t, J = 7.0Hz, 3H).

[0240] Comparative Example 8 synthesized 20(S)-7-cyclohexyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared according to synthetic route 2 of the present invention, achieving a final yield of 82.7% and a purity of 98.02%.

[0241] 1 H NMR(400MHz,Chloroform-d)δ7.80(d,J=9.8Hz,2H),7.62(s,1H),5.77(d,J=16.2Hz,1H),5.42(s,2H),5.32(d,J=16.2Hz,1H),3.8 9(s,1H),2.04(d,J=20.6Hz,4H),1.92(ddd,J=16.5,14.2,7.0Hz,4H),1.57-1.37(m,2H),1.31-1.23(m,1H),1.06(t,J=7.3Hz,3H).

[0242] Comparative Example 9 synthesized 20(S)-7-isopentyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared according to synthetic route 2 of the present invention, achieving a final yield of 87.2% and a purity of 98.72%.

[0243] 1 H NMR(400MHz,Chloroform-d)δ7.81(s,1H),7.61(d,J=8.4Hz,2H),5.74(d,J=16.3Hz,1H),5.29(d,J=16.3Hz,1H),5.23(s, 2H),3.98(s,1H),3.12-3.03(m,2H),1.86(dtt,J=34.3,13.2,6.9Hz,3H),1.57(dt,J=12.0,6.8Hz,2H),1.10-0.98(m,9H).

[0244] Comparative Example 10 synthesized 20(S)-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291. However, this method is complex, costly, has low yield, and results in low purity of the final product. Therefore, according to the raw materials and proportions in that patent, the compound was prepared according to synthetic route 2 of the present invention, achieving a final yield of 78.2% and a purity of 97.72%.

[0245] 1 H NMR (400MHz, DMSO-d6) δ8.67(s,1H),8.09(s,2H),7.30(s,1H),6.53(s,1H),5.41(s,2H),5.25(s,2H),1.90-1.80(m,2H),0.88(t,J=7.4Hz,3H).

[0246] Comparative Example 11 synthesized 20(S)-(2-methylaminoacetyl)-7-ethyl-10,11-difluoromethylenedioxycamptothecin according to the method described in patent WO0149291, with a final yield of 49.3% and a purity of 96.31%.

[0247] HR-MS:C 27 H 19 BrF₂N₂O₆S for [M+H] + , caculated 527.1504, found528.1411.

[0248] Experiment 1: In vitro antitumor activity test of the compounds of this invention

[0249] Given the rapid growth and unlimited proliferation of tumor cells, they metastasize and spread within the body by invading normal cells, causing fatal damage. Therefore, a primary condition for screening anti-tumor drugs is their significant lethal effect on tumor cells. This experiment used the MTT assay to detect the lethality of the compound of this invention and the control drug SN-38 against five types of tumor cells, including Mia PaCa-2 (human pancreatic cancer cells), A549 cells (human non-small cell lung cancer cell line), HT-29 (human colon cancer cells), A431 (human epidermal cancer cells), and MDA-MB-231 (human breast cancer cells).

[0250] Test method:

[0251] (1) Preparation before the experiment: 10% complete culture medium: 10% serum + 1% penicillin and streptomycin mixture + DMEM culture medium; 0.5% MTT solution: weigh 250mg MTT and dissolve it in 50mL PBS buffer, filter it through a 0.22μm sterile filter membrane for sterilization, and store the drug storage solution in a sealed container at 4℃ away from light.

[0252] The experimental compounds (compounds 1-23 and SN-38 of this invention (a topoisomerase I inhibitor, the active metabolite of irinotecan)) were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. Before administration, the solutions were diluted with complete DMEM medium containing 1% FBS buffer to prepare drug solutions with concentration gradients of 6.25, 12.5, 25, 50, 100, and 200 nM.

[0253] (2) Cell culture: When the cells reach 80-90% confluence, digest them with trypsin-EDTA (0.02%), count them using a hemocytometer, and adjust the cell density to 5×10⁶ cells / year based on the counting results. 4 The fractions were seeded at a rate of 100 μL / mL into each well of a 96-well plate. 200 μL of PBS buffer was added to each well around the outermost edge of the 96-well plate, and the plates were incubated for 24 h.

[0254] (3) Drug addition: When the cells reach 80% confluence, discard the supernatant and add 200 μL of different drug solutions to each well. The drugs are the compounds of this invention and SN-38 (a topoisomerase I inhibitor, the active metabolite of irinotecan). Three parallel wells are set for each sample concentration. Add 200 μL of complete DMEM medium containing 1% FBS buffer as a blank control. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 72 h. Add 20 μL of 0.5% MTT solution to each well and incubate for another 4 h. Remove the plate, carefully discard the supernatant, and add 150 μL of DMSO solution to each well to dissolve the blue-purple crystals. Shake the 96-well plate on a microplate shaker for 20 min to dissolve the crystals. Set the reference wavelength to 630 nm using a microplate reader and detect the absorbance (OD) value at 570 nm. Calculate the inhibition rate of cell proliferation by the drug according to the following formula:

[0255] Proliferation inhibition rate % = (OD blank group - OD experimental group) / OD blank group × 100%.

[0256] In the formula, OD experimental group is the absorbance value of the drug-treated group; OD blank group is the absorbance value of the cell group cultured normally in drug-free medium.

[0257] Table 2 shows the IC50 values ​​of compounds 1-23 and SN-38 of the present invention against Mia PaCa-2, A549, HT-29, A431, and MDA-MB-231 cell lines. 50 Values. Analysis of the data in Table 2 shows that:

[0258] ①Most of the compounds of this invention have better inhibitory activity against the above five cell lines than the positive control SN-38.

[0259] ② Compounds 4, 9, 12, 13, 15, 16 and 17 of this invention exhibit excellent inhibitory activity against Mia PaCa-2 cells, among which compound 17 has the highest IC50 value. 50 The value is less than 4.3 nM, far exceeding that of similar compounds, and it also has higher inhibitory activity against the other four types of tumor cells compared to SN-38.

[0260] ③ Compounds 4 and 13 of this invention exhibit excellent inhibitory activity against A549 cells. Both compounds of this invention show higher inhibitory activity against A549 cells than SN-38, with an IC50 value of [missing value]. 50 The values ​​were all much lower than those of the positive control SN-38.

[0261] ④ Compounds 4, 8, 9, 10, 11, 12, 13, 16, 17 and 21 of the present invention exhibit superior inhibitory activity against HT-29 cells compared to SN-38.

[0262] ⑤ The inhibitory activity of the compounds in this invention against the A431 cell line is similar to that of SN-38, among which compounds 1, 4, 5, 16 and 17 have stronger inhibitory activity than SN-38.

[0263] ⑥ The inhibitory activity of the compounds in this invention on MDA-MB-231 cells is similar to that of SN-38.

[0264] The above in vitro experiments show that the compounds of the present invention have selective and good antitumor activity, possess antitumor advantages, and can be used to prepare drugs for treating cancer.

[0265] Table 2

[0266]

[0267]

[0268] Experiment 2: Testing the transmembrane transport capacity of the compound of this invention in cells.

[0269] The cell membrane serves as a barrier, facilitating transmembrane transport, transmembrane information transmission, and energy conversion, functions determined by its chemical composition and molecular structure. The transmembrane transport capacity of drugs to tumor cells significantly impacts drug efficacy and bioavailability. Therefore, this study evaluates the transmembrane transport capacity of the compound by observing its absorption and transport from the simulated intestinal lumen (AP side) to the plasma (BL side) and its secretion and efflux from the BL side to the AP side.

[0270] (1) Preparation of sample solutions: Based on the results of in vitro antitumor activity tests, compounds 1, 4, 5, 8, 9, 10, 11, 12, 13, 16, 17 and 21 of the present invention with good in vitro antitumor activity and comparative examples 1, 4, 5, 8, 10 and 11 were selected for cell transmembrane transport ability tests. The positive control drug SN-38 was used in this experiment. 1 μM of each sample required for this experiment was accurately weighed and placed in 1 mL volumetric flasks. The samples were dissolved and diluted to volume with DMSO to obtain the sample stock solution (1 mM). The stock solution was placed in a refrigerator at 4 °C for later use. Before use, the stock solution was diluted with HBSS buffer to 0.5 μM and 1 μM.

[0271] (2) Cell model establishment: A Caco-2 cell monolayer membrane model was established according to well-known experimental methods in the field, selecting cells with a transmembrane resistance (TEER) value > 200 Ω·cm. 2 Furthermore, the apparent permeability coefficient (Papp) of fluorescein transmembrane transport... <l×10 -6 The bidirectional transmembrane transport of the compounds studied was carried out using a Caco-2 cell monolayer membrane at a speed of cm / s.

[0272] (3) Assay method for AP to BL side absorption and transport: Discard the original culture medium and wash with an appropriate amount of balanced salt solution (HBSS). Add 0.5 mL of 0.5 or 1 μM sample solution to the AP side, and add 1.5 mL of HBSS buffer to the BL side. Incubate the Transwell cell culture plate in an incubator. After 3 hours of incubation, aspirate 0.5 mL of the transport sample solution from the BL side and add the same volume of pre-warmed HBSS buffer to the BL side. Add an equal volume of acetonitrile to the transport sample solution, vortex, and centrifuge (10000 rpm / min, 10 min). Filter through a 0.22 μm microporous membrane, collect the supernatant, analyze by HPLC, and calculate the sample content.

[0273] (4) Method for measuring secretion efflux from BL to AP side: Discard the original culture medium and wash with an appropriate amount of balanced salt solution (HBSS). Add 1.5 mL of 0.5 or 1 μM sample solution to the BL side, and simultaneously add 0.5 mL of HBSS buffer to the AP side. Incubate the Transwell cell culture plate in an incubator. After 3 hours of incubation, aspirate 0.25 mL of the transported sample solution from the AP side, and simultaneously add the same volume of pre-warmed HBSS buffer to the AP side. Add an equal volume of acetonitrile to the transported sample solution, vortex, and centrifuge (10000 rpm / min, 10 min). Filter through a 0.22 μm microporous membrane, collect the supernatant, analyze by HPLC, and calculate the sample content.

[0274] (5) Calculation of Papp value and ER value:

[0275] Apparent permeability coefficient (Papp) values ​​can be used to assess the ability of compounds to penetrate a Caco-2 cell monolayer.

[0276] Calculation formula: Papp=(V / (C0×A))×(ΔQ / Δt).

[0277] In the formula, V represents the volume of the solution on the receiving side (mL); A represents the area of ​​the polycarbonate membrane, which is 1.12 cm². 2 C0 represents the initial concentration of the analyte (μg / mL); ΔQ / Δt represents the mass concentration of the analyte transported per unit time at the receiving side (μg / mL·s). -1 ).

[0278] Efflux rate (ER) can reflect the efflux and secretion of the tested compound to some extent.

[0279] Calculation formula: ER = Papp (BL→AP) / Papp (AP→BL) ×100%.

[0280] In the formula, Papp (BL→AP)Papp represents the apparent permeability coefficient from the BL side to the AP side. (AP→BL) Let be the apparent permeability coefficient from the AP side to the BL side.

[0281] According to the results in Table 3, most of the compounds in this study had higher apparent permeability coefficients than SN-38 and the comparative compounds, and their efflux rates were less than 1, indicating they were not affected by efflux pumps. In contrast, the efflux rates of the control compound SN-38 and the comparative compounds were mostly greater than 1, indicating they were affected by efflux pumps to some extent and were more prone to developing drug resistance. Compared with the control compound SN-38 and the comparative compounds, the compounds of this invention possess superior transmembrane transport capabilities, which positively influences drug entry into cells to exert its antitumor effect.

[0282] Table 3

[0283]

[0284]

[0285] Experiment 3: In vitro anti-inflammatory activity test of the compound of the present invention

[0286] COX-2 has been recognized as an upstream agonist of inflammatory mediators (especially prostaglandin PGE2). This invention utilizes techniques known in the art to analyze the in vitro anti-inflammatory activity of the compounds of this invention by measuring the concentration of PGE2 secreted by COX-2 (cyclooxygenase-2) in the supernatant of LPS (serum lipase) activated mouse macrophage J774 cell line.

[0287] Test method:

[0288] Drug solution preparation: The drug stock solution was stored in a sealed container at 4°C, protected from light. Compounds 4, 8, 10, 11, 12, 13, 15, and 19 of this invention were initially dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 mM. Before administration, the stock solution was diluted with complete DMEM medium containing 1% FBS (fetal bovine serum) to prepare four different concentrations of drug solutions (6.25, 50, 125, and 150 nM / mL).

[0289] Cell culture and drug administration: J774 cells were cultured at a density of 2 × 10⁶ cells / year. 5 Cells were seeded at a concentration of 200 μL per well in 96-well plates and incubated for 24 h. The culture medium was then collected. In this study, the experimental groups were treated with drug solutions of different compound concentration gradients, while the LPS group and control group received no drug. Both the experimental and LPS groups were treated with 1 μg / mL LPS to activate the J774 mouse macrophage cell line. The control group received no LPS. After 24 h, the cells were centrifuged. Finally, the concentration of PGE2 was determined using a commercially available ELISA kit.

[0290] To verify the anti-inflammatory activity of the compounds, the experimental results of PGE2 content in the J774 cell culture supernatant of compounds 4, 8, 10, 11, 12, 13, 15 and 19 of this invention are as follows: Figures 2-9 As shown. Compared with the LPS group, multiple compounds 4, 8, 10, 11, 12, 13, 15 and 19 in this invention have certain anti-inflammatory activities on the activated mouse macrophage J774 cell line. Among them, compounds 4, 12, 13 and 19 have the most significant inhibitory activity on PGE2, and the level of PGE2 gradually decreases with increasing concentration of the compounds.

[0291] Experiment 4: In vivo antitumor activity test of the compound of the present invention

[0292] Using techniques known in the art, the in vivo antitumor activity of the compounds of this invention was tested using a nude mouse HT-29 (colon cancer cell) xenograft model. Based on the results of in vitro antitumor activity tests and cell transmembrane transport capacity tests, compounds 4, 12, and 13 of this invention were selected for in vivo antitumor activity testing. Simultaneously, the antitumor activity of comparative compounds 1, 10, and 11 was tested. The positive control group was the marketed drug irinotecan.

[0293] Test method: Nude mice approximately five weeks old and weighing 18±2g were used in the experiment. Colon cancer cells (HT-29) were inoculated into the armpits of the nude mice, and the tumors grew to 50-100mm. 3 Drug administration began around day 21. The experiment was divided into a saline group, an irinotecan control group, and an experimental drug group (including compounds 4, 12, and 13 of this invention and comparative examples 1, 10, and 11), with 8 nude mice in each group. Mice were fasted for 2 hours before the experiment and administered the drugs orally. The experimental group received 8 mg / kg of the drug, while the irinotecan control group received 80 mg / kg of the drug via intraperitoneal injection. Administration was once a week, administered three times a week on day 0, day 7, and day 14. The feeding period was 21 days. Tumor volume and body weight were measured on day 0, day 4, day 7, day 11, day 14, day 18, and day 21.

[0294] See results Figure 10Compounds 4, 12, and 13 of this invention exhibit good inhibitory activity against HT-29 tumors, with activity superior to irinotecan and significantly higher than comparative examples 1, 10, and 11. Mice in the test groups using compounds 4, 12, and 13 synthesized in this invention showed no significant change in body weight, and all vital signs remained normal. Comparative examples 1, 10, and 11 showed some inhibitory activity against this tumor, but their tumor growth-inhibiting activity was less than that of the control group irinotecan and the compounds synthesized in this invention. Two mice died within 21 days after administration of comparative example 10, and mice using other comparative examples showed a decrease in body weight and varying degrees of diarrhea, indicating significant toxicity.

[0295] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. 20(S)-10,11-difluoromethylenedioxycamptothecin derivative, characterized in that, Selected from any of the following compounds: 。 2. A method for preparing the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative as described in claim 1, characterized in that, Includes the following steps: (1) First, 2,2-difluorobenzo[d][1,3]dioxane-5-carboxaldehyde is nitrated, then reacted with substituted phenylboronic acid, palladium chloride, potassium carbonate and tris(1-naphthyl)phosphine, and finally reacted with an oxidizing agent to obtain the raw material; the raw material is dissolved in ethanol, then a reducing agent is added, hydrogen gas is introduced and then triethylamine is added to react to obtain the intermediate; (2) The intermediate, tricyclic ketone and catalyst are reacted to obtain the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative.

3. The preparation method according to claim 2, characterized in that, In step (1), the solvent for the nitration reaction includes fuming nitric acid and trifluoromethanesulfonic acid, the temperature of the nitration reaction is -75℃ to -45℃, and the reaction time is 2-3 h; In step (1), the substituted phenylboronic acid is selected from any one of 3,4-dimethylphenylboronic acid, 3-methylphenylboronic acid, 3-methoxyphenylboronic acid, 3,5-dimethoxyphenylboronic acid, 3-fluoro-4-methoxyphenylboronic acid, 3-fluoro-5-methoxyphenylboronic acid, 4-fluoro-3-methoxyphenylboronic acid, 3,4-difluorophenylboronic acid, and 4-fluorophenylboronic acid; toluene is used as the solvent when reacting with the substituted phenylboronic acid, palladium chloride, potassium carbonate, and tris(1-naphthyl)phosphine; the reaction temperature with the substituted phenylboronic acid, palladium chloride, potassium carbonate, and tris(1-naphthyl)phosphine is 75-95℃, and the reaction time is 8-12 h. In step (1), the oxidant is selected from any one of manganese dioxide, Desmartin oxidant, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetrapropylammonium perruthenate, sodium nitrite / acetic anhydride oxidant, and aluminum tert-butoxide; the solvent for the reaction with the oxidant is dichloromethane, the reaction temperature is 15-35℃, and the reaction time is 2-3 h; In step (1), the reducing agent is selected from any one of Raney nickel, palladium on carbon, platinum on carbon, zinc, rhodium, iron, stannous chloride, lithium aluminum hydride, sodium borohydride, lithium aluminum hydride, potassium borohydride, sodium sulfide, hydrogen sulfide, sodium disulfide, ferrous sulfate, sodium sulfite, sodium bisulfite, sodium nitrite, and ferrous dicarbonate; the mass-volume ratio of the raw material to triethylamine and the reducing agent is 100-240 mg: 100-800 μL: 15-36 mg; the reaction temperature in step (1) is 20-30℃, and the reaction time is 8-12 h. In step (2), the catalyst is selected from any one of iodine, dodecyl sulfate, ferric chloride hexahydrate, aminosulfonic acid, 2,4,6-trichloro-1,3,5-triazine, bismuth trifluoromethanesulfonate, yttrium trifluoromethanesulfonate, lithium bis(trifluoromethanesulfonyl)imide, magnesium bis(trifluoromethanesulfonyl)imide, and p-toluenesulfonic acid hydrate; the intermediate, tricyclic ketone, and catalyst are dissolved in glacial acetic acid, and the molar ratio of the intermediate, tricyclic ketone, and catalyst is 1-1.5:1:0.4-0.6; the reaction is carried out under nitrogen protection, and the pH is adjusted after the reaction is completed; the reaction temperature is 90-100℃, the reaction time is 8-12 h, and the pH is 5-6.

4. The preparation method according to claim 2 or 3, characterized in that, The post-processing of each reaction step includes quenching, extraction, washing, concentration, and purification. The quenching solvent is selected from one or more of ice water, saturated ammonium chloride solution, saturated sodium sulfite solution, and saturated sodium bicarbonate solution. The extraction solvent is selected from dichloromethane, ethyl acetate, or saturated sodium bicarbonate solution. The washing solvent is saturated sodium chloride solution or saturated sodium bicarbonate solution. The purification is carried out by silica gel column chromatography. The silica gel column is packed with 300-400 mesh silica gel. The eluent used for purification is selected from a mixture of petroleum ether and ethyl acetate in a volume ratio of 20-150:1, a mixture of dichloromethane and ethyl acetate in a volume ratio of 2-10:1, a mixture of petroleum ether and dichloromethane in a volume ratio of 2-10:1, or a mixture of dichloromethane, methanol, and glacial acetic acid in a volume ratio of 50-400:1:0.

1.

5. The use of the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of cancer.

6. A pharmaceutical composition comprising: an effective dose of the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative of claim 1, and a pharmaceutically acceptable carrier.

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