CN117777153A - 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives and preparation methods and applications thereof - Google Patents

Abstract

The invention belongs to the technical field of organic synthesis and medicine, and particularly relates to a 20 (S) -10, 11-difluoro methylenedioxy camptothecin derivative, and a preparation method and application thereof. The derivative is in the form of a compound with a structure shown in a formula (I), a stereoisomer and a pharmaceutically acceptable salt, and the novel 20 (S) -10, 11-difluoro methylenedioxy camptothecin derivative provided by the invention can be used for preparing camptothecins for preventing or treating tumors, has good anti-inflammatory activity, in-vivo and in-vitro anti-tumor activity and cell transmembrane transport capacity, and the preparation method is easy to operate, simple and convenient in post-treatment, low-cost and easily available in reaction initial raw materials, strong in reaction substrate designability, higher in reaction efficiency and stronger in practicability.

Description

20(S)-10,11-difluoromethylenedioxycamptothecin derivatives and their preparation methods and applications

Technical Field

The invention belongs to the technical field of organic synthesis and medicine, and specifically relates to a 20(S)-10,11-difluoromethylenedioxycamptothecin derivative and a preparation method and application thereof.

Background Art

Camptothecin (CPT) is a cytotoxic quinoline alkaloid that can inhibit DNA topoisomerase (TOPOI) and is a hot topic in the research and development of anti-tumor drugs. Camptothecin is a small molecule compound derived from natural products with excellent anti-tumor activity. Camptothecin derivatives are known as one of the three major discoveries of anti-cancer drugs in the 1990s, showing broad-spectrum anti-tumor activity and great research and application value. The mechanism of action of early camptothecin compounds is to form a ternary complex with TopI and DNA, blocking DNA replication and transcription, with disadvantages such as low selectivity and large toxic side effects. Due to the poor water solubility and toxicity of camptothecin under physiological conditions, it cannot be directly used in clinical practice. The derivatives obtained through structural modification, irinotecan, topotecan, and belotecan, have been successfully used in the treatment of colorectal cancer, ovarian cancer, prostate cancer, etc., and there are also more than a dozen drugs in the clinical research stage.

Chinese invention patent CN110590796A discloses camptothecin derivatives and their preparation methods and applications. The compounds provided by the invention are a class of camptothecin derivatives with novel structures, methylenedioxy groups introduced at positions 10 and 11 of the parent ring, and different substituent groups introduced at position 7. The raw materials for the preparation method are easily available, the synthesis method is simple, the purification method is simple and fast, and the compounds have excellent in vitro cytotoxic activity and excellent in vivo anti-tumor effects. Invention patents WO2005009347A2 and WO0149291 disclose methods for forming camptothecin compounds that are effective anti-tumor compounds, which can inhibit the enzyme topoisomerase I and can alkylate the DNA of the related topoisomerase I-DNA cleavable complex. However, the cell transmembrane transport ability of the above-mentioned compounds is not ideal, the cytotoxicity is high, the in vivo anti-tumor activity is poor, and the preparation method of the compounds has a complex reaction procedure when introducing substituents, low yield, high cost, and is not easy to prepare in large quantities. Since the synthesis route introduces substituents into the parent structure in the last step, steric hindrance and the conjugated system make it very difficult to form negative charge at the introduced position, so the types of substituents that can be introduced by the above method are very limited.

Camptothecin derivatives approved for anticancer use are susceptible to one or more of several mechanisms that cause cancer cells to be resistant to chemotherapy. There remains a need to develop new camptothecin derivatives, particularly more potent anticancer agents, and derivatives that are less toxic, more permeable and more soluble.

Summary of the invention

The present invention aims at the problems existing in the prior art and provides a 20(S)-10,11-difluoromethylenedioxycamptothecin derivative and a preparation method and application thereof.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

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

In formula (I), R is Where: X is n is an integer of 0 to 2; Z is selected from a substituted or unsubstituted ring structure, a substituted or unsubstituted C _1-10 alkenyl group, a substituted or unsubstituted C _1-10 alkynyl group, a substituted or unsubstituted octyl group, a substituted or unsubstituted decyl group, a substituted or unsubstituted C _7-10 alkoxy group, a substituted or unsubstituted C _7-10 alkylthio group, a substituted or unsubstituted C _7-10 alkylamino group, a substituted or unsubstituted C _7-10 alkylimino group, a substituted or unsubstituted C _7-10 alkenylamino group, a substituted or unsubstituted C _7-10 alkenylimino group, a substituted or unsubstituted C _7-10 alkylC _7-10 alkenylamino group, and a substituted or unsubstituted C _7-10 alkylC _7-10 alkenylimino group;

The substitution is mono-substitution or poly-substitution.

Preferably, the ring structure is selected from _C9-12 cycloalkyl, _C3-12 cycloalkenyl, aryl, _C2-11 ether ring or aromatic heterocyclic group, and the aromatic heterocyclic ring is selected from pyridine ring, furan ring, thiophene ring, pyrazole ring, indole ring, benzopyrazole ring, piperidine ring, morpholine ring, thiomorpholine ring, naphthalene ring or triazole ring.

Preferably, when it is a substituted structure, the substituted groups are each independently selected from halogen, C _1-10 alkyl, C _1-10 alkenyl, C _1-10 alkynyl, C _3-12 cycloalkyl, C 1-10 alkoxy, C _1-10 alkylthio, C _1-10 alkylsilyl, C _11-10 haloalkoxy, C _1-10 ester, 3-12 membered heterocyclyl, C _6-14 aryl, oxy C _6-14 aryl, oxy C _6-14 aromatic heteroyl, nitrogen C _6-14 aryl, nitrogen C _5-14 aromatic heteroyl, _5-14 membered heteroaryl, -CN, -NO ₂ , -CF ₂ H, -CF ₂ OH, -CF ₃ or -OCF ₃ .

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 haloalkoxy-substituted aryl, halogen atom-substituted aryl, unsubstituted C _2-11 ether ring substituents, C _2-11 ether ring substituents substituted by C _1-10 alkyl, C _2-11 ether ring substituents substituted by benzene ring, C _2-11 ether ring substituents substituted by halogen atom, unsubstituted aromatic heterocyclic substituents, C _1-10 alkyl-substituted aromatic heterocyclic substituents, halogen atom-substituted aromatic heterocyclic substituents, unsubstituted C _1-8 olefin substituents, or C _1-10 alkyl-substituted C _1-8 olefin substituents.

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

More preferably, Z is a substituted or unsubstituted C _2-11 ether ring substituent, and Z is selected from any one of the following groups:

More preferably, Z is a substituted or unsubstituted C _1-8 olefin substituent, and Z is selected from any one of the following groups:

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

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 can exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis- or trans-isomers.

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

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

(2) Salts with 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;

(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-methylglucamine salts), lower alkanolammonium 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, and lysine, respectively), or other conventional "prodrug" forms.

The present invention also provides a method for preparing the above derivative, comprising the following steps:

(1) reacting the raw material with triethylamine and a reducing agent to obtain an intermediate;

(2) reacting the intermediate, tricyclic ketone and a catalyst to obtain the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative.

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

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

Preferably, the raw material in step (1) is first dissolved in ethanol, and then a reducing agent is added, and triethylamine is added after hydrogen is introduced. The mass volume ratio of the raw material to triethylamine, iron powder or palladium 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.

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 bistrifluoromethanesulfonimide, magnesium bistrifluoromethanesulfonimide and p-toluenesulfonic acid hydrate.

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

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

Preferably, the reaction temperature in step (2) is 90-100° C., the reaction time is 8-12 h, and the pH is 5-6.

Preferably, n is 0, Z is a substituted aromatic group, and the preparation method of the raw material in step (1) comprises first subjecting 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde to a nitration reaction, then reacting it with a substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine, and finally reacting it with an oxidant.

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-difluorophenylboric acid, 3,4-difluorophenylboric acid, 4-fluorophenylboric acid, 4-trifluoromethylphenylboric acid and 4-methylthiophenylboric acid.

Preferably, the solvent for the nitration reaction includes fuming nitric acid and trifluoromethanesulfonic acid, the temperature for the nitration reaction is -75°C to -45°C, and the time for the nitration reaction is 2-3h.

Preferably, toluene is used as solvent during the reaction with substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine, the reaction temperature with substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine is 75-95° C., and the reaction time is 8-12 h.

Preferably, the oxidant is selected from any one of manganese dioxide, Dess-Martin periodinane, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetra-n-propylammonium perruthenate, sodium nitrite/acetic anhydride oxidant and aluminum tert-butoxide.

More preferably, the oxidant is a Dess-Martin periodinane.

Preferably, the solvent for the reaction with the oxidant is dichloromethane, the temperature for the reaction with the oxidant is 15-35° C., and the reaction time is 2-3 h.

Preferably, n is an integer of 0-2, Z is a substituted or unsubstituted C _2-11 ether ring substituent, a substituted or unsubstituted C _1-8 olefin substituent, or a substituted or unsubstituted aromatic heterocyclic substituent. The method for preparing the raw material in step (1) comprises reacting 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde with a Grignard reagent, then reacting with an oxidant, and finally performing a nitration reaction.

Preferably, the Grignard reagent is selected from any one of butylene magnesium bromide, hexylene magnesium bromide, 2-furylmethyl magnesium bromide, 4-pyranylmethyl magnesium bromide and heptylene magnesium bromide.

Preferably, before the reaction with the Grignard reagent, 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde is added to anhydrous tetrahydrofuran to dissolve, and the mixture is placed in an ice-water bath and stirred. The molar ratio of the 2,2-difluorobenzo[d][1,3]dioxole-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 progress is monitored by TLC. The developing solvent of the TLC is a mixture of petroleum ether and ethyl acetate in a volume ratio of 8-16:1-2. The temperature of the Grignard reagent reaction is 35-65° C., and the reaction time is 8-12 h.

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

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

More preferably, n is an integer of 0-2, Z is a substituted or unsubstituted aromatic heterocyclic substituent, and the nitration reaction is followed by a reaction with an aromatic heterocyclic formaldehyde.

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

Preferably, the reaction conditions with the aromatic heterocyclic formaldehyde are alkaline, the reaction temperature with the aromatic heterocyclic formaldehyde is 15-35° C., and the reaction time is 1-2 h.

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

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.

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

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

Preferably, the drying is carried out using anhydrous magnesium sulfate, and the purification is carried out using silica gel column chromatography.

Preferably, the filler of the silica gel column chromatography is 300-400 mesh silica gel.

Preferably, the eluent used for the 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.

More preferably, when n of the R group in the structural formula (I) is 0, and Z is a substituted aryl group, the synthetic route 1 for preparing 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives is as follows:

More preferably, when n of the R group in the structural formula (I) is an integer of 0-2, and Z is a substituted or unsubstituted C _2-11 ether ring substituent, a substituted or unsubstituted C _1-8 olefin substituent, the synthetic route 2 for preparing 20(S)-10,11-difluoromethylenedioxycamptothecin derivative is as follows:

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

The present invention also provides use of the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative in preparing a drug for preventing and/or treating cancer.

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

The cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, prostate cancer, leukemia and breast cancer.

The pharmaceutical composition can be prepared in the form of 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. The above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

If necessary, the pharmaceutical composition of the present invention may also be added with one or more pharmaceutically acceptable carriers, including conventional diluents, fillers, adhesives, wetting agents, absorption enhancers, surfactants, adsorption carriers and lubricants in the pharmaceutical field.

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

(1) The novel 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives provided by the present invention are used to prepare camptothecin drugs for preventing or treating tumors. The compounds have good solubility, anti-inflammatory activity, in vitro and in vivo anti-tumor activity and cell transmembrane transport ability.

(2) The preparation method of the present invention is easy to operate, the post-processing is simple, the reaction starting materials are cheap and easy to obtain, the reaction substrates are highly designable, the substrate functional group range is wide, the reaction efficiency is high, and 20(S)-10,11-difluoromethylenedioxycamptothecin derivatives with different substituents can be designed and synthesized according to actual needs, and the practicability is strong.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the synthesis route of the compound 20(S)-10,11-difluoromethylenedioxycamptothecin derivative of the present invention.

FIG2 shows the effect of compound 4 of the present invention on the PGE2 content in the supernatant of J774 cell culture.

FIG3 shows the effect of compound 8 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

FIG4 shows the effect of compound 10 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

FIG5 shows the effect of compound 11 of the present invention on the PGE2 content in the supernatant of J774 cell culture.

FIG6 shows the effect of compound 12 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

FIG7 shows the effect of compound 13 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

FIG8 shows the effect of compound 15 of the present invention on the PGE2 content in the supernatant of J774 cell culture.

FIG9 shows the effect of compound 19 of the present invention on the PGE2 content in the culture supernatant of J774 cells.

FIG. 10 is a curve showing the inhibition of HT-29 tumor growth by the experimental compound in mice.

DETAILED DESCRIPTION

The methods and techniques of the present invention are generally performed according to conventional methods known in the art, unless otherwise indicated. The nomenclature, experimental methods and techniques related to biology, pharmacology, medicine and medicinal chemistry described herein are those known and commonly used in the art. Chemical syntheses, chemical analyses, pharmaceutical preparations and formulations all employ standard techniques.

Unless otherwise specified, the scientific and technical terms used in this article shall have the meanings commonly understood by those of ordinary skill in the art. However, the following terms have the following definitions:

Cancer refers to a malignant tumor originating from epithelial tissue and is the most common type of malignant tumor. Correspondingly, malignant tumors originating from mesenchymal tissue are collectively called sarcomas. There are a few malignant tumors that are not named according to the above principles, such as Wilms tumor, malignant teratoma, etc.

"Cancer" is customarily used to refer to all malignant tumors, and refers to a large class of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Cancer has biological characteristics such as abnormal cell differentiation and proliferation, uncontrolled growth, invasiveness and metastasis. It is uncontrolled cell division and growth that lead to the formation of malignant tumors or cells. They invade adjacent tissues and can also be transferred to the distal parts of the body through the lymphatic system or blood flow. Its occurrence is a complex process with multiple factors and steps, which is divided into three processes: carcinogenesis, cancer promotion, and evolution. It is closely related to smoking, infection, occupational exposure, environmental pollution, unreasonable diet, and genetic factors. In the present invention, another equivalent description of "treating cancer" is "treating tumors" or "anti-cancer" or "anti-tumor". Cancer is a disease of uncontrolled cell growth that hinders the normal function of body organs and systems.

The products of the present invention can be used to treat a variety of cancers or subjects in danger 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, pharyngeal cancer, thyroid cancer, testicular cancer, brain cancer, bone cancer and blood cancer (such as leukemia, chronic lymphocytic leukemia) etc. Other cancers include, but are not limited to, basal cell carcinoma, biliary tract 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 lymphoma and non-Hodgkin 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.

A "therapeutically effective dose" is any amount of a drug as described below, which, when used alone or in combination with another therapeutic agent, promotes disease regression, which is manifested as a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of disorders or disabilities caused by illness. Therapeutically effective amounts or doses of drugs include "prophylactically effective amounts" or "prophylactically effective doses", which are any amount of a drug as described below, which, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of the disease, can inhibit the occurrence or recurrence of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of a disease can be assessed using various methods known to technicians, such as in an animal model system that can predict efficacy in humans or by measuring the activity of a compound agent in an in vitro assay system.

As an example, anticancer agents (pharmaceutical compositions for treating cancer) promote tumor regression in the subject. In a preferred embodiment, a therapeutically effective dose of a drug promotes the regression of cancer cells and even eliminates cancer. "Promoting cancer regression" means that a therapeutically effective amount of a drug administered alone or in combination with an anti-neoplastic agent (Anti-Neoplastic Agent) causes a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of the disease-free period, prevents obstacles or disability caused by illness, or otherwise improves the patient's disease symptoms. In addition, the terms "effective" and "effectiveness" for treatment include both pharmacological effectiveness and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote the regression of cancer in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and/or organism level caused by drug administration.

As an example of tumor treatment, in the most preferred embodiments, the therapeutically effective amount or dose of the drug is effective in inhibiting cell growth or tumor growth compared to an untreated animal model. The ability of a compound to inhibit tumor growth can be evaluated in an animal model system, which can predict efficacy in human tumors. Alternatively, this property of the composition can be assessed by examining the ability of the compound to inhibit cell growth, which can be measured in vitro by assays known to those skilled in the art. In a preferred embodiment of the invention, tumor regression can be clearly observed.

The present invention is further described in detail below in conjunction with embodiments.

The present invention synthesized and tested a series of novel camptothecin derivatives with difluoro-substituted methylenedioxy groups introduced at the 10 and 11 positions of the parent ring and different substituent groups introduced at the 7-position. The 20(S)-10,11-difluoromethylenedioxy camptothecin derivatives of the present invention can be listed as the structures of some compounds of the present invention shown in Table 1, but are not limited to the structures shown in Table 1 (the structures of all compounds are shown in the structures described in the Summary of the Invention):

Table 1

FIG1 shows a simplified synthesis route of the above compounds. The following is an explanation in conjunction with specific examples: A brief synthesis route 1 of compound 1-19 is as follows:

Example 1 Preparation of 20(S)-7-(3,4-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 1), the steps are as follows:

(1) Preparation of intermediate 2,2-difluoro-6-nitrobenzo[d][1,3]dioxole-5-carboxaldehyde (Compound B)

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

¹ H NMR (400MHz, Chloroform-d) δ10.17(s,1H),8.45(s,1H),7.95(s,1H).HR-MS:C ₈ H ₃ F ₂ NO ₅ for[M+H ] ⁺ ,cached 230.9979, found 231.0802.

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

¹ H NMR (400MHz, Chloroform-d) δ7.89 (s, 1H), 7.77 (s, 1H), 7.43 (s, 1H), 7.24 (d, J = 1.9 Hz, 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 F ₂ NO ₅ for[M+H] ⁺ ,caculated 337.0762, found 338.0591.

(3) Preparation of (2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)(3,4-dimethylphenyl)methanone (E2)

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

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 F ₂ NO ₅ for[M+H] ⁺ ,caculated 335.0605, found 336.0509.

(4) Preparation of (6-amino-2,2-difluorobenzo[d][1,3]dioxol-5-yl)(3,4-dimethylphenyl)methanone (F2)

At 25°C, the product of step (3) (2,2-difluoro-6-nitrobenz[d][1,3]dioxol-5-yl)(3,4-dimethylphenyl)methanone (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 a solvent, a hydrogen balloon was filled, and the mixture was stirred at 25°C for 8 h. The catalyst was removed by filtration and concentrated to obtain a yellow liquid, which was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: ethyl acetate = 80:1/50:1/20:1, v/v/), and concentrated to obtain a yellow solid product F2 of 116.4 mg. The yield of step (4) was 95.7%, and the purity was 97.67%.

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 F ₂ NO ₃ for [M+H] ⁺ , caculated 305.0863, found 306.0622.

(5) Preparation of 20(S)-7-(3,4-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 1)

The product (6-amino-2,2-difluorobenzo[d][1,3]dioxol-5-yl)(3,4-dimethylphenyl)methanone (F2) (116.0 mg, 1 mmol) obtained in step (4), 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, and then the pH value was adjusted (pH=5-6). The mixture was extracted three times, the organic phases were combined, washed once with saturated brine, dried over anhydrous magnesium sulfate and concentrated to obtain a yellow oily liquid, which was separated by silica gel column chromatography (eluent: 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 mixture was washed with methanol and filtered to obtain a pure white solid product (compound 1) on the filter cake, totaling 218.1 mg. The yield of step (5) was 73.3% and the purity was 99.1%.

¹ H 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＝14.7Hz,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 F ₂ N ₂ O ₆ for[M+H] ⁺ , caculated 532.1446, found 533.4.

Example 2 Preparation of 20(S)-7-(3,5-dimethylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 2), the steps are as follows:

3,5-dimethylphenylboric acid is used to replace the 3,4-dimethylphenylboric acid in step (2) of Example 1, manganese dioxide is used as the catalyst in step (3) instead of Dess-Martin oxidant, Raney nickel is used as the catalyst in step (4) instead of palladium carbon, and iodine is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate, and the remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1.

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

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 F ₂ NO ₅ for [M+H] ⁺ , caculated 337.0762, found 338.0621.

Step (3) obtained a total of 128.0 mg of the compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)(3,5-dimethylphenyl)methanone (E3) with a yield of 89.14% and a purity of 97.26%.

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 F ₂ NO ₅ for [M+H] ⁺ , caculated 335.0605, found 336.0527.

Step (4) obtained a total of 106.3 mg of the compound (6-amino-2,2-difluorobenzo[d][1,3]dioxol-5-yl)(3,5-dimethylphenyl)methanone (F3) with a yield of 91.2% and a purity of 97.66%.

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 F ₂ NO ₃ for [M+H] ⁺ ,cached 305.0863, found 306.0782.

Step (5) can finally prepare 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%.

¹ H 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 F ₂ N ₂ O ₆ for[M+H] ⁺ ,caculated 532.1446,found 533.1534.

Example 3 Preparation of 20(S)-7-(4-trimethylsilylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 3), the steps are as follows:

4-trimethylsilylphenylboronic acid is used to replace the 3,4-dimethylphenylboronic acid in step (2) of Example 1, pyridinium chlorochromate is used as the catalyst in step (3) instead of Dess-Martin oxidant, platinum carbon is used as the catalyst in step (4) instead of palladium carbon, and dodecyl sulfate is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 134.2 mg of a white solid product (compound 3) can be prepared. The yield of step (5) is 86.4% and the purity is 98.96%.

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＝16.3Hz,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 F ₂ N ₂ O ₆ Sifor[M+H] ⁺ , caculated 576.1528, found 577.3.

Example 4 Preparation of 20(S)-7-(3-methylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 4), the steps are as follows:

3-Methylphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (1) of Example 1, silver carbonate is used as the catalyst instead of Dess-Martin oxidant in step (3), zinc powder is used as the catalyst instead of palladium carbon in step (4), and ferric chloride hexahydrate is used as the catalyst instead of p-toluenesulfonic acid hydrate in step (5). The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 105.2 mg of a white solid product (compound 4) can be prepared. The yield of step (5) is 80.2% and the purity is 98.56%.

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),5.27(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 F ₂ N ₂ O ₆ for[M+H] ⁺ ,caculated 518.1289,found519.1338.

Example 5 Preparation of 20(S)-7-(4-ethylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 5), the steps are as follows:

4-ethylphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, N-bromosuccinimide is used as the catalyst in step (3) instead of Dess-Martin oxidant, rhodium powder is used as the catalyst in step (4) instead of palladium carbon, and aminosulfonic acid is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 128.2 mg of a white solid product (compound 5) can be prepared. The yield of step (5) is 87.6% and the purity is 98.20%.

¹ H 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 F ₂ N ₂ O ₆ for[M+ H] ⁺ ,caculated 532.1446,found 533.4.

Example 6 Preparation of 20(S)-7-(4-isopropylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 6), the steps are as follows:

4-isopropylphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, iodic acid is used as the catalyst in step (3) instead of Dess-Martin oxidant, stannous chloride is used as the catalyst in step (4) instead of palladium on carbon, 2,4,6-trichloro-1,3,5-triazine is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate, and the remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 107.8 mg of a white solid product (compound 6) can be prepared, and the yield of step (5) is 84.34% and the purity is 98.56%.

¹ H NMR (400MHz, Chloroform-d) δ7.83 (s, 1H), 7.65 (s, 1H), 7.48 (t, J = 6.6 Hz, 2H), 7.42 (s, 1H), 7.38 (d, J ＝7.3Hz,1H),7.32(d,J＝7.4Hz,1H),5.70(d,J＝16.3Hz,1H),5.27(d,J＝16.3Hz,1H),5.08(d,J＝3.3 Hz,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 F ₂ N ₂ O ₆ for[M+H] ⁺ ,caculated 546.1602, found 547.2.

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

3-Trifluoromethoxyphenylboric acid is used to replace the 3,4-dimethylphenylboric acid in step (2) of Example 1, hydrogen peroxide is used as the catalyst in step (3) instead of Dess-Martin oxidant, lithium aluminum tetrahydride is used as the catalyst in step (4) instead of palladium carbon, and bismuth trifluoromethylsulfonate is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 127.2 mg of a white solid product (compound 7) can be prepared. The yield of step (5) is 81.1% and the purity is 97.96%.

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 F ₅ N ₂ O ₇ for [M+H] ⁺ , caculated 588.0956, found 589.2.

Example 8 Preparation of 20(S)-7-(4-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 8), the steps are as follows:

4-methoxyphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, chromium trioxide is used as the catalyst in step (3) instead of Dess-Martin oxidant, sodium borohydride is used as the catalyst in step (4) instead of palladium carbon, and yttrium trifluoromethylsulfonate is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 120.0 mg of a white solid product (compound 8) can be prepared. The yield of step (5) is 83.69% and the purity is 97.69%.

¹ H 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 F ₂ N ₂ O ₇ for [M+H] ⁺ , caculated 534.1239, found 535.3.

Example 9 Preparation of 20(S)-7-(3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 9), the steps are as follows:

3-Methoxyphenylboric acid is used to replace 3,4-dimethylphenylboric acid in step (2) of Example 1, N-chlorosuccinimide is used as the catalyst in step (3) instead of Dess-Martin oxidant, lithium aluminum hydride is used as the catalyst in step (4) instead of palladium carbon, and lithium bistrifluoromethanesulfonyl imide is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1.

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

¹ H NMR (400MHz, Chloroform-d) δ7.73 (s, 1H), 7.52 (s, 1H), 7.28 (d, J = 8.2Hz, 1H), 6.92-6.80 (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 F ₂ NO ₆ for[M+H] ⁺ ,caculated 339.0554 , found 340.0472. HR-MS: C ₂₈ H ₂₀ F ₂ N ₂ O ₇ for [M+H] ⁺ , caculated 534.1239, found 535.3.

Step (3) obtained a total of 113.0 mg of the compound (2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)(3-methoxyphenyl)methanone (E3) with a yield of 95.5% and a purity of 95.63%.

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 H ₉ F ₂ NO ₆ for[M+H] ⁺ ,caculated337.0398,found 338.0257.

Step (4) obtained a total of 92.5 mg of the compound (6-amino-2,2-difluorobenzo[d][1,3]dioxol-5-yl)(3-methoxyphenyl)methanone (F3) with a yield of 89.9% and a purity of 94.65%.

¹ H NMR (400MHz, Chloroform-d) δ7.41-7.32 (m, 1H), 7.12 (d, J = 2.1Hz, 1H), 7.11 (q, J = 1.0Hz, 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 F ₂ NO ₄ for[M+H] ⁺ , caculated 307.0656, found 308.0508.

Step (5) can finally prepare a pure white solid product 20(S)-7-(3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 9) in a total of 106.7 mg, with a yield of 86.2% and a purity of 98.09%.

¹ H 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 F ₂ N ₂ O ₇ for[M+ H] ⁺ ,caculated 534.1239,found 535.3.

Example 10 The preparation of 20(S)-7-(3,5-dimethoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 10) is as follows:

3,5-dimethoxyphenylboric acid is used instead of 3,4-dimethylphenylboric acid in step (2) of Example 1, tetrapropylammonium perruthenate is used as the catalyst in step (3) instead of Dess-Martin oxidant, potassium borohydride is used as the catalyst in step (4) instead of palladium on carbon, and magnesium bistrifluoromethanesulfonyl imide is used as the catalyst in step (5) instead of p-toluenesulfonic acid hydrate. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 88.9 mg of a white solid product (compound 10) can be prepared. The yield of step (5) is 84.9% and the purity is 97.56%.

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＝16.4Hz,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 F ₂ N ₂ O ₈ for[M+H] ⁺ ,caculated 564.1344, found 565.4.

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

3-Fluoro-4-methoxyphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, sodium nitrite/acetic anhydride oxidant is used as the catalyst in step (3) instead of Dess-Martin oxidant, sodium sulfide is used as the catalyst in step (4) instead of palladium on carbon, and the remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 97.5 mg of a white solid product (Compound 11) can be prepared, and the yield of step (5) is 79.65%, and the purity is 98.10%.

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 F ₃ N ₂ O ₇ for[M+H] ⁺ ,caculated552.1144,found 553.0. Example 12 The preparation of 20(S)-7-(3-fluoro-5-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 12) is as follows:

3-Fluoro-5-methoxyphenylboronic acid is used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1, tert-butoxide aluminum is used as the catalyst in step (3) instead of Dess-Martin oxidant, and hydrogen sulfide is used as the catalyst in step (4) instead of palladium on carbon. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1, and a total of 106.5 mg of a white solid product (compound 12) can be prepared. The yield of step (5) is 86.3% and the purity is 95.65%.

¹ H 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 F ₃ N ₂ O ₇ for [M+H] ⁺ , caculated 552.1144, found 553.2.

Example 13 The preparation of 20(S)-7-(4-fluoro-3-methoxyphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 13) is as follows:

4-Fluoro-3-methoxyphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. Sodium disulfide was used as the catalyst in step (4) instead of palladium on carbon. The remaining catalysts, reagents and preparation methods were the same as those in steps (1) to (5) of Example 1. A total of 101.9 mg of a white solid product (Compound 13) was prepared. The yield of step (5) was 83.5% and the purity was 98.13%.

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 F ₃ N ₂ O ₇ for[M+H] ⁺ ,caculated 552.1144, found 533.4.

Example 14 The preparation of 20(S)-7-(3-fluoro-4-methylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 14) is as follows:

3-Fluoro-4-methylphenylboronic acid was used to replace the 3,4-dimethylphenylboronic acid in step (2) of Example 1. The remaining catalysts, reagents and preparation methods were the same as those in steps (1) to (5) of Example 1. A total of 88.2 mg of a white solid product (Compound 14) was obtained. The yield of step (5) was 87.5% and the purity was 98.05%.

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.

Example 15 The preparation of 20(S)-7-(2,4-difluorophenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 15) is as follows:

2,4-difluorophenylboric acid is used instead of 3,4-dimethylphenylboric acid in step (2) of Example 1, and ferrous sulfate is used as the catalyst in step (4) instead of palladium carbon. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1. A total of 77.8 mg of a white solid product (Compound 15) can be prepared. The yield of step (5) is 80.6% and the purity is 98.00%.

¹ H 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＝16.4Hz,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 F ₄ N ₂ O ₆ for[M+H] ⁺ , caculated 540.0944, found 541.1.

Example 16 The preparation of 20(S)-7-(3,4-difluorophenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 16) is as follows:

3,4-difluorophenylboric acid is used instead of 3,4-dimethylphenylboric acid in step (2) of Example 1, and sodium sulfite is used as the catalyst in step (4) instead of palladium carbon. The remaining required catalysts, reagents and preparation methods are the same as those in steps (1) to (5) of Example 1. A total of 87.2 mg of a white solid product (compound 16) can be prepared. The yield of step (5) is 85.9% and the purity is 97.95%.

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,4H),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 F ₄ N ₂ O ₆ for [M+H] ⁺ ,caculated 540.0944, found 541.1016.

Example 17 The preparation of 20(S)-7-(4-fluorophenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 17) is as follows:

4-Fluorobenzeneboronic acid was used instead of 3,4-dimethylboronic acid in step (2) of Example 1, sodium bisulfite was used as the catalyst in step (4) instead of palladium on carbon, and the remaining catalysts, reagents and preparation methods were the same as those in steps (1) to (5) of Example 1. A total of 100.4 mg of a white solid product (Compound 17) was prepared. The yield of step (5) was 88.4% and the purity was 97.69%.

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 F ₃ N ₂ O ₆ for [M+H] ⁺ , caculated 522.1039, found 523.1124.

Example 18 Preparation of 20(S)-7-(4-trifluoromethylphenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 18)

4-Trifluoromethylphenylboronic acid was used instead of 3,4-dimethylphenylboronic acid in step (2) of Example 1. The remaining catalysts, reagents and preparation methods were the same as those in steps (1) to (5) of Example 1. A total of 95.0 mg of a white solid product (Compound 18) was obtained. The yield of step (5) was 87% and the purity was 98.25%.

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]+,caculated 572.1007,found 579.1080.

Example 19 The preparation of 20(S)-7-(4-methylthiophenyl)-10,11-difluoromethylenedioxycamptothecin (Compound 19) is as follows:

4-methylthiophenylboronic acid was used to replace the 3,4-dimethylphenylboronic acid in step (2) of Example 1. Except that the reduction catalyst required in step (4) used iron powder instead of the previous reduction catalyst palladium carbon, the catalysts, reagents and preparation methods required for the remaining steps were the same as those in Example 1. A total of 89.4 mg of a white solid product (Compound 19) was prepared. The yield of step (5) was 87.2% and the purity was 97.96%.

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 F ₂ N ₂ O ₆ S for[M+H] ⁺ ,caculated 550.1010,found 551.1087.

The brief synthetic route 2 of compound 20-21 is as follows:

Example 20 The preparation of 20(S)-7-butylene-10,11-difluoromethylenedioxycamptothecin (Compound 20) is as follows:

(1) Preparation of 2,2-difluorobenzo[d][1,3]dioxole-5-enebutanol (G1)

The raw material 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde (A) (500 mg) was added to a three-necked flask, anhydrous THF was added to dissolve, and the mixture was placed in an ice-water bath and stirred. Under nitrogen protection, the Grignard reagent butylmagnesium bromide (385.4 mg) was added. After 10 minutes, the temperature was restored to 25°C and the reaction was continued for 12 hours. The reaction was monitored by TLC (developing solvent was a mixed solution of petroleum ether: ethyl acetate = 800 μL: 100 μL, v/v). After the reaction was completed, an appropriate amount of saturated NH ₄ The reaction was quenched with Cl solution to consume excess Grignard reagent. After stirring for 10 min, the mixed solution was transferred to a separatory funnel, extracted three times with dichloromethane (DCM), and the organic phases were combined and dried over anhydrous magnesium sulfate. The filtrate was filtered under reduced pressure and concentrated to obtain a light yellow liquid. The filtrate was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: ethyl acetate = 80:1, v/v). After concentration, 344.6 mg of yellow solid compound G1 was obtained with a yield of 88.3% and a purity of 95.68%.

HR-MS: C ₁₂ H ₁₂ F ₂ O ₃ for [M+H] ⁺ , caculated 242.0755, found 243.0726.

(2) Preparation of 2,2-difluorobenzo[d][1,3]dioxole-5-enebutanone (H1)

The intermediate compound 2,2-difluoro-6-nitrobenz[d][1,3]dioxole-5-enebutanol (G1) (334.6 mg) was placed in a round-bottom flask, and dichloromethane was added to dissolve it. Dess-Martin periodinane (DMP) (879.6 mg) was added and stirred at 25°C for 3 h. The reaction was monitored by TLC (developing solvent was a mixed solution of petroleum ether: ethyl acetate = 800 μL: 100 μL, v/v). After the reaction was completed, saturated Na ₂ SO ₃ solution and saturated NaHCO ₃ solution were added successively to quench the reaction. After stirring for about 20 min, the mixed solution was observed to be layered, and the DCM layer gradually clarified. The solution was transferred to a separatory funnel and saturated NaHCO ₃ solution was extracted three times, and then back-extracted with DCM three times. The organic phases were combined, dried over anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a light yellow liquid, which was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: ethyl acetate = 80:1, v/v). After concentration, 294.5 mg of light yellow liquid compound H1 was obtained with a yield of 88.7% and a purity of 97.65%.

HR-MS: C ₁₂ H ₁₀ F ₂ O ₃ for [M+H] ⁺ , caculated 240.0598, found 241.0556.

(3) Preparation of 2,2-difluoro-6-nitrobenzo[d][1,3]dioxole-5-enebutanone (I1)

The intermediate compound 2,2-difluorobenzo[d][1,3]dioxole-5-enebutanone (H1) (294.5 mg) was placed in a 50 mL round-bottom flask, 1 mL of concentrated sulfuric acid was added and the round-bottom flask was gently shaken to allow the concentrated sulfuric acid to fully activate the raw material along the flask wall, the round-bottom flask was placed in a low-temperature constant temperature stirring reaction bath at -15°C and stirred, fuming nitric acid was slowly added dropwise, 9 mL of nitric acid was added and placed at -15°C for 12 hours, TLC (developing solvent is a mixed solution of petroleum ether: ethyl acetate = 1500 μL: 100 μL) The reaction progress was monitored. After the reaction was completed, an appropriate amount of ice was added to the round-bottom flask to quench the reaction, and concentrated sulfuric acid was diluted to absorb 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 and dried over anhydrous magnesium sulfate, and then filtered under reduced pressure. The filtrate was concentrated to obtain a light yellow liquid, which was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: dichloromethane = 4:1, v/v). After concentration, 239.1 mg of the intermediate compound I1 was obtained, with a yield of 68.4% and a purity of 98.32%.

HR-MS: C ₁₂ H ₉ F ₂ NO ₅ for [M+H] ⁺ , caculated 285.0449, found 286.0413.

(4) Preparation of 6-amino-2,2-difluorobenzo[d][1,3]dioxole-5-enebutanone (J1)

The intermediate compound 2,2-difluoro-6-nitrobenz[d][1,3]dioxole-5-enebutanone (I1) (239.1 mg) was placed in a round-bottom flask, and Pd/C (36 mg), triethylamine (200 μL) and anhydrous ethanol were added in sequence. Hydrogen was introduced and stirred at 25°C for 12 h. The reaction progress was monitored by TLC (developing solvent was a mixed solution of petroleum ether: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 separated 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 spin-dried and then dry-loaded. The mixture was separated by silica gel column chromatography (eluent was a mixed solution of petroleum ether:ethyl acetate = 20:1, v/v). After concentration, 161.7 mg of the intermediate compound J1 was obtained with a yield of 75.6% and a purity of 97.64%.

HR-MS: C ₁₂ H ₁₁ F ₂ NO ₃ for [M+H] ⁺ , caculated 255.0707, found 256.0521.

(5) Preparation of 20(S)-7-butylene-10,11-difluoromethylenedioxycamptothecin (Compound 20)

The product 6-amino-2,2-difluorobenzo[d][1,3]dioxole-5-enepentanone (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) obtained in step (4) were added to a round-bottom flask, dissolved in glacial acetic acid, refluxed at 100°C under nitrogen protection for 12 h, and then the pH value was adjusted (pH = 5-6). The product was extracted with dichloromethane three times, the organic phases were combined, washed once with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain a yellow oily liquid. The product was separated by silica gel column chromatography (eluent: a mixed solution of dichloromethane: ethyl acetate = 9:1, v/v, with a small amount of glacial acetic acid), concentrated, washed with methanol, and filtered to obtain a pure white solid product (Compound 20) on the filter cake in a total of 195.3 mg, with a yield of 83.2% and a purity of 95.64%.

HR-MS: C ₂₅ H ₂₀ F ₂ N ₂ O ₆ for [M+H] ⁺ , caculated 482.1289, found 483.1256.

Example 21 The preparation of 20(S)-7-(4-pyranylmethyl)-10,11-difluoromethylenedioxycamptothecin (Compound 21) is as follows:

4-pyranylmethylmagnesium bromide is used to replace the butylmagnesium bromide in step (1) of Example 18, and sodium nitrite is used as the catalyst in step (4) instead of palladium on carbon. 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 a white solid product (Compound 24) can be prepared. The yield of step (5) is 84.4% and the purity is 97.25%.

HR-MS: C ₂₇ H ₂₄ F ₂ N ₂ O ₇ for [M+H] ⁺ , caculated 526.1552, found 527.1501.

The brief synthetic route 3 of compound 22-23 is as follows:

Example 22 The preparation of 20(S)-7-(5-methylfuran-2-ethyl)-10,11-difluoromethylenedioxycamptothecin (Compound 22) is as follows:

(1) Preparation of 2,2-difluorobenzo[d][1,3]dioxole-5-ethanol (Intermediate K)

The raw material 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde (A) (1 g) was added to a three-necked flask, anhydrous THF was added to dissolve, and the mixture was placed in an ice-water bath and stirred. Under nitrogen protection, Grignard reagent methylmagnesium bromide (961.0 mg) was added. After 10 minutes, the temperature was restored to 25°C and the reaction was continued for 12 hours. The reaction was monitored by TLC (developing solvent was a mixed solution of petroleum ether: ethyl acetate = 800 μL: 100 μL, v/v). After the reaction was completed, an appropriate amount of saturated NH ₄ The reaction was quenched with Cl solution to consume excess Grignard reagent. After stirring for ten minutes, the mixed solution was transferred to a separatory funnel, extracted three times with dichloromethane (DCM), and the organic phases were combined and dried over anhydrous magnesium sulfate. The filtrate was filtered under reduced pressure and concentrated to obtain a light yellow liquid. The filtrate was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: ethyl acetate = 80:1, v/v). After concentration, 882.0 mg of yellow solid compound K was obtained, with a yield of 81.2% and a purity of 97.88%.

HR-MS: C ₉ H ₈ F ₂ O ₃ for [M+H] ⁺ , caculated 202.0442, found 203.0386.

(2) Preparation of 2,2-difluorobenzo[d][1,3]dioxole-5-ethanone (Intermediate L)

The intermediate compound 2,2-difluoro-6-nitrobenzo[d][1,3]dioxole-5-ethanol (intermediate K) (882.0 mg) was placed in a round-bottom flask, and dichloromethane was added to dissolve it. Dess-Martin periodinant (DMP) (3024.4 mg) was added and stirred at 25°C for 3 h. The reaction was monitored by TLC (developing solvent was a mixed solution of petroleum ether: ethyl acetate = 800 μL: 100 μL, v/v). After the reaction was completed, saturated Na ₂ SO ₃ solution and saturated NaHCO ₃ solution were added successively to quench the reaction. After stirring for about 20 min, the mixed solution was observed to be layered, and the DCM layer gradually clarified. The solution was transferred to a separatory funnel and saturated NaHCO ₃ solution was extracted three times, and then back-extracted with DCM three times, the organic phases were combined, dried over anhydrous magnesium sulfate, and filtered under reduced pressure. The filtrate was concentrated to obtain a light yellow liquid, which was separated by silica gel column chromatography (eluent: a mixed solution of petroleum ether: ethyl acetate = 80:1, v/v) to obtain 734.7 mg of light yellow liquid compound L after concentration, with a yield of 82.6% and a purity of 98.31%.

HR-MS: C ₈ H ₅ F ₂ O ₃ for [M+H] ⁺ , caculated 187.0207, found 188.0146.

(3) Preparation of 2,2-difluoro-6-nitrobenzo[d][1,3]dioxole-5-ethanone (Intermediate M)

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

HR-MS: C ₈ H ₅ F ₂ O ₃ for [M+H] ⁺ , caculated 245.0136, found 246.0051.

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

The intermediate compound 2,2-difluoro-6-nitrobenz[d][1,3]dioxole-5-ethanone (intermediate M) (100 mg) was placed in a 50 mL round-bottom flask, and anhydrous ethanol solvent was added to dissolve it. Sodium hydroxide (19.6 mg) was added, and 5-methylfuran-2-carboxaldehyde (45 mg) was diluted and added. The mixture was stirred at 25° C. for 1 h. After the reaction, 10% hydrochloric acid was added to adjust the pH to neutral. The mixture was extracted with ethyl acetate for 3 times, and the upper layer was collected. The mixture was separated by silica gel column chromatography (eluent was a mixed solution of petroleum ether:ethyl acetate = 20:1, v/v). After concentration, 100.7 mg of the intermediate compound N1 was obtained, with a yield of 73.2% and a purity of 97.87%.

HR-MS: C ₁₅ H ₉ F ₂ NO ₆ for [M+H] ⁺ , caculated 337.0398, found 338.0301.

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

The intermediate compound 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-5-yl)-3-(5-methylfuran-2-yl)propenone (N1) (100.7 mg) was placed in a round-bottom flask, heated and dissolved with anhydrous ethanol as solvent, cooled to 25°C, Pd/C (15 mg) was added in sequence, hydrogen was introduced and triethylamine (100 μL) was added, and the mixture was stirred at 25°C for 12 h. TLC (developing solvent: petroleum ether: ethyl acetate = 800 The reaction progress was monitored by 3% 4% 4% 2-nitropropene (A), 2-nitropropene (A), 2-nitropropene (B), 2-nitropropene (C), 2-nitropropene (D), 2-nitropropene (E), 2-nitropropene (E), 2-nitropropene (D ...

HR-MS: C ₁₅ H ₁₃ F ₂ NO ₄ for [M+H] ⁺ , caculated 309.0813, found 310.0755.

(6) Preparation of 20(S)-7-(5-methylfuran-2-ethyl)-10,11-difluoromethylenedioxycamptothecin (Compound 25)

The product 1-(2,2-difluoro-6-nitrobenzo[d][1,3]dioxol-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) obtained in step (2) were added to a round-bottom flask, dissolved in glacial acetic acid, and refluxed at 95°C for 8 h under nitrogen protection, and the pH was adjusted to 5. value (pH = 5-6), extracted with dichloromethane three times, combined the organic phases, washed once with saturated brine, dried over anhydrous magnesium sulfate and concentrated to obtain a yellow oily liquid, which was separated by silica gel column chromatography (eluent: a mixed solution of dichloromethane: ethyl acetate = 9:1, v/v, with a small amount of glacial acetic acid), concentrated and washed with methanol and filtered to finally obtain 54.9 mg of a pure white solid product (compound 25) on the filter cake, with a yield of 55.3% and a purity of 97.62%.

HR-MS: C ₂₈ H ₂₂ F ₂ N ₂ O ₇ for [M+H] ⁺ , caculated 536.1395, found 537.1301.

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

5-Bromothiophene-2-carboxaldehyde was used instead of 5-methylfuran-2-carboxaldehyde in step (4) of Example 20, and ferrous carbonate was used as the catalyst in step (5) instead of palladium on carbon. The catalysts, reagents and preparation methods required for the remaining steps were the same as those in Example 20, and a total of 67.4 mg of a white solid product (Compound 27) was prepared. The yield of step (5) was 73.2% and the purity was 97.75%.

HR-MS: C ₂₇ H ₁₉ BrF ₂ N ₂ O ₆ S for [M+H] ⁺ , caculated 616.0115, found 617.0110.

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

¹ H 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).

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

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).

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

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).

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

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,1H),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).

Comparative Example 5: 20(S)-7-ethyl-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 81.8% and a purity of 97.42%.

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),5.19(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).

Comparative Example 6: 20(S)-7-propyl-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 85.3% and a purity of 98.22%.

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 = 16.3 Hz,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).

Comparative Example 7 20(S)-7-hexyl-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 83.4% and a purity of 98.28%.

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),1.90(ddt,J＝16.3,14.2,7.1Hz,2H),1.73(td,J＝ 9.5,8.6,5.1Hz,2H),1.49(p,J＝7.6Hz,2H),1.34(h,J＝3.4,2.7Hz,4H),1.03(t,J＝7.4Hz,3H),0.90( t,J＝7.0Hz,3H).

Comparative Example 8 20(S)-7-cyclohexyl-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 82.7% and a purity of 98.02%.

¹ H NMR (400MHz, Chloroform-d) δ7.80 (d, J = 9.8 Hz, 2H), 7.62 (s, 1H), 5.77 (d, J = 16.2 Hz, 1H), 5.42 (s, 2H), 5.32(d,J＝16.2Hz,1H),3.89(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).

Comparative Example 9: 20(S)-7-isopentyl-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 87.2% and a purity of 98.72%.

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 = 16.3 Hz,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).

Comparative Example 10 20(S)-10,11-difluoromethylenedioxycamptothecin was synthesized according to the method described in patent WO0149291, but the method was complicated, costly, low in yield, and low in purity of the final product. Therefore, according to the raw materials and proportions in the patent, the compound was prepared according to the synthetic route 2 of the present invention, with a final yield of 78.2% and a purity of 97.72%.

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).

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

HR-MS: C ₂₇ H ₁₉ BrF ₂ N ₂ O ₆ S for [M+H] ⁺ , caculated 527.1504, found 528.1411.

Experiment 1 In vitro antitumor activity test of the compounds of the present invention

Relying on the characteristics of rapid growth and unlimited reproduction of tumor cells, tumor cells metastasize and spread in the body by eroding normal cells, causing fatal damage to the body. Therefore, the primary condition for screening anti-tumor drugs is to have a significant lethal effect on tumor cells. This experiment uses the MTT experimental method to detect the lethality of the compound of the present invention and the control drug SN-38 to five 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).

Test Method:

(1) Preparation before the experiment: 10% complete culture medium: 10% serum + 1% penicillin-streptomycin mixture + DMEM culture medium; 0.5% MTT solution: weigh 250 mg of MTT and dissolve it in 50 mL of PBS buffer, filter it with a 0.22 μm sterile filter membrane to sterilize, and store the drug storage solution in a sealed container at 4°C in the dark.

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

(2) Cell culture: When the cells reached 80-90% confluence, they were digested with trypsin-EDTA (0.02%), counted with a hemacytometer and the cell density was adjusted to 5×10 ⁴ cells/mL based on the counting results. The cells were inoculated in a 96-well plate with 100 μL per well. 200 μL of PBS buffer was added to each well around the outermost circle of the 96-well plate and cultured in an incubator for 24 h.

(3) Adding drugs: When the cells reach 80% fusion, discard the supernatant, add 200 μL of different drug solutions to each experimental well. The drugs are the compounds of the present invention and SN-38 (topoisomerase I inhibitor, active metabolite of irinotecan), set up 3 parallel wells for each sample concentration, and add 200 μL of complete DMEM medium containing 1% FBS buffer as a blank control. Place the 96-well plate in an incubator at 37°C and 5% _CO2 and continue to culture for 72 hours. Add 20 μL of 0.5% MTT solution to each well, continue to culture for 4 hours, take out, carefully discard the supernatant, and add 150 μL of DMSO solution to each well to dissolve the blue-purple crystalline product. Place the 96-well plate on a micro-oscillator to shake and dissolve for 20 minutes, set the reference wavelength to 630 nm with an enzyme reader, and detect the absorbance OD value at 570 nm. Calculate the inhibition rate of drug on cell proliferation according to the following formula:

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

Wherein, 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 a drug-free medium.

Table 2 shows the IC ₅₀ values of compounds 1-23 and SN-38 of the present invention against Mia PaCa-2 cell line, A549 cell line, HT-29 cell line, A431 cell line and MDA-MB-231 cell line. From the data analysis of Table 2, it can be seen that:

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

② Compounds 4, 9, 12, 13, 15, 16 and 17 of the present invention showed excellent inhibitory activity against Mia PaCa-2 cells, among which the _IC50 value of compound 17 was less than 4.3 nM, far exceeding similar compounds, and also had higher inhibitory activity against the remaining four tumor cells than SN-38.

③ The compounds 4 and 13 of the present invention showed excellent inhibitory activity against A549 cells. The compounds of the present invention showed higher inhibitory activity against A549 cells than SN-38, and the IC ₅₀ values were much lower than that of the positive control SN-38.

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

⑤ The inhibitory activities of the compounds of the present invention on A431 cell lines are similar to those of SN-38, among which compounds 1, 4, 5, 16, and 17 have stronger inhibitory activities than SN-38.

⑥ The inhibitory activity of the compounds of the present invention on MDA-MB-231 cells is similar to that of SN-38.

The above in vitro tests show that the anti-tumor activity of the compound of the present invention is selective and has good anti-tumor activity, has anti-tumor advantages, and can be used to prepare drugs for treating cancer.

Table 2

Experiment 2 Test of Cell Transmembrane Transport Ability of the Compounds of the Invention

The cell membrane has barrier function, transmembrane material transport, transmembrane information transmission and energy conversion functions, which are determined by the chemical composition and molecular structure of the membrane. The transmembrane transport ability of drugs to tumor cells has a significant impact on drug efficacy and bioavailability. Therefore, the absorption and transport of the compounds in this study from the simulated intestinal cavity side (AP side) to the plasma side (BL side) and the secretion and excretion from the BL side to the AP side reflect the bidirectional transport situation, thereby evaluating the transbiological membrane transport ability of the compounds in this study.

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

(2) Cell model establishment: A Caco-2 cell monolayer membrane model was established according to the experimental methods known in the art. Caco-2 cell monolayer membranes with a transmembrane electrical resistance (TEER) > 200 Ω·cm ² and an apparent permeability coefficient (Papp) of fluorescent yellow transmembrane transport < 1×10 ^-6 cm/s were selected for the bidirectional transmembrane transport experiment of the compounds studied.

(3) Method for measuring absorption and transport from AP to BL: discard the original culture medium, add an appropriate amount of balanced salt solution (HBSS) for washing, then 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. Place the Transwell cell culture plate in an incubator for incubation. After 3 hours of incubation, draw 0.5 mL of transport sample solution from the BL side, and add the same volume of pre-warmed HBSS buffer to the BL side. Add equal volumes of acetonitrile to the transport sample solution, vortex and centrifuge (10000 rpm/min, 10 min). Pass through a 0.22 μm microporous filter membrane, take the supernatant, analyze by HPLC, and calculate the sample content.

(4) Determination of secretion and efflux from BL to AP: discard the original culture medium, add an appropriate amount of balanced salt solution (HBSS) for washing, then add 1.5 mL of 0.5 or 1 μM sample solution to the BL side, and add 0.5 mL of HBSS buffer to the AP side. Place the Transwell cell culture plate in an incubator for incubation. After 3 hours of incubation, draw 0.25 mL of transport sample solution from the AP side, and add the same volume of pre-warmed HBSS buffer to the AP side. Add equal volumes of acetonitrile to the transport sample solution, vortex and centrifuge (10000 rpm/min, 10 min). Pass through a 0.22 μm microporous filter membrane, take the supernatant, analyze by HPLC, and calculate the sample content.

(5) Calculation of Papp value and ER value:

The apparent permeability coefficient (Papp) value can be used to assess the ability of compounds to permeate Caco-2 cell monolayers.

Calculation formula: Papp = (V/(C ₀ ×A)) × (ΔQ/Δt).

Wherein, V represents the volume of the receiving solution (mL); A represents the area of the polycarbonate membrane, which is 1.12 cm ² ; C ₀ represents the initial concentration of the analyte (μg/mL); ΔQ/Δt represents the mass concentration of the analyte transported per unit time on the receiving side (μg/mL·s ^-1 ).

The efflux rate (ER) can reflect the efflux secretion of the tested compound to a certain extent.

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

Where Papp _(BL→AP) is the apparent permeability from BL to AP, and Papp _(AP→BL) is the apparent permeability from AP to BL.

According to the results in Table 3, the apparent permeability coefficients of most compounds in this study are higher than those of SN-38 and the comparative compounds, and the efflux rate is less than 1, which is not affected by the efflux pump, while the efflux rate of the control SN-38 and the comparative compounds is mostly greater than 1, which is affected by the efflux pump to a certain extent and is prone to drug resistance. Compared with the control group SN-38 and the comparative compounds, the compounds of the present invention have a relatively excellent cell transmembrane transport ability, which has a positive effect on the drug entering the cell to exert an anti-tumor effect.

Table 3

Experiment 3 In vitro anti-inflammatory activity test of the compounds of the present invention

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

Test Method:

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

Cell culture and administration: The J774 cells were cultured at a density of 2×10 ⁵ cells/mL, and the cells were inoculated in a 96-well plate, 200 μL per well, and placed in an incubator for 24 hours. Then the culture medium was collected, and the experimental groups of the compounds in this study were given drug solutions with different compound concentration gradients, and the LPS group and the control group were not given drugs. Both the experimental group and the LPS group were treated with 1 μg/mL LPS to activate the mouse macrophage J774 cell line, and the control group was not added with LPS. After 24 hours, centrifugation was performed. Finally, the concentration of PGE2 was determined using a commercially available ELISA kit.

To verify the anti-inflammatory activity of the compounds, the experimental results of the PGE2 content in the culture supernatant of J774 cells of compounds 4, 8, 10, 11, 12, 13, 15 and 19 of the present invention are shown in Figures 2 to 9. Compared with the LPS group, multiple compounds 4, 8, 10, 11, 12, 13, 15 and 19 of the present invention have certain anti-inflammatory activity on the activated mouse macrophage J774 cell line, among which compounds 4, 12, 13 and 19 have the most significant inhibitory activity on PGE2, and as the concentration of the compound increases, the level of PGE2 gradually decreases.

Experiment 4 In vivo antitumor activity test of the compounds of the present invention

Using the techniques known in the art, the in vivo antitumor activity of the compounds of the present invention was tested using a nude mouse HT-29 (colon cancer cell) xenograft tumor model. Based on the in vitro antitumor activity test results and the cell transmembrane transport ability test results, compounds 4, 12 and 13 of the present invention were selected for in vivo antitumor activity test, and the antitumor activity tests of three compounds of comparative examples 1, 10 and 11 were also performed. The positive control group was the marketed drug irinotecan.

Test method: Nude mice about five weeks old and weighing 18±2g were used in the experiment. Colon cancer cells (HT-29) were inoculated in the armpits of nude mice. When the tumor grew to about 50-100 mm ³ , the drug was administered. The experiment was divided into a saline group, an irinotecan control group, and an experimental drug group (including compounds 4, 12 and 13 of the present invention and comparative examples 1, 10 and 11), and each group consisted of 8 nude mice. Fasting for 2 hours before the experiment, oral administration, the experimental group was administered at a dose of 8 mg/kg, and the irinotecan control group was administered by intraperitoneal injection at a dose of 80 mg/kg. The administration frequency was once a week, three times on day 0, day 7 and day 14, respectively. The feeding period was 21 days, and the tumor volume and body weight were measured on day 0, day 4, day 7, day 11, day 14, day 18 and day 21.

The results are shown in Figure 10. Compounds 4, 12 and 13 of the present invention have good inhibitory activity against HT-29 tumors, and their activity is better than that of irinotecan and much higher than that of comparative examples 1, 10 and 11. The body weight of mice in the test groups of compounds 4, 12 and 13 synthesized by the present invention did not change significantly, and all physical signs of the mice were normal. The compounds of comparative examples 1, 10 and 11 have certain inhibitory activity against the tumor, but their inhibitory activity against tumor growth is not as good as that of irinotecan and the compounds synthesized by the present invention in the control group. After administration of the compound of comparative example 10, 2 mice died within 21 days. The body weight of mice in the other comparative example compounds decreased, and they had diarrhea to varying degrees, indicating that the toxicity was relatively large.

Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims (10)

1.20(S)-10,11-difluoromethylenedioxycamptothecin derivatives, characterized in that they are compounds having the structure shown in formula (I), their stereoisomers and pharmaceutically acceptable salt forms: In formula (I), R is Where: X is n is an integer of 0 to 2; Z is selected from a substituted or unsubstituted ring structure, a substituted or unsubstituted C _1-10 alkenyl group, a substituted or unsubstituted C _1-10 alkynyl group, a substituted or unsubstituted octyl group, a substituted or unsubstituted decyl group, a substituted or unsubstituted C _7-10 alkoxy group, a substituted or unsubstituted C _7-10 alkylthio group, a substituted or unsubstituted C _7-10 alkylamino group, a substituted or unsubstituted C _7-10 alkylimino group, a substituted or unsubstituted C _7-10 alkenylamino group, a substituted or unsubstituted C _7-10 alkenylimino group, a substituted or unsubstituted C _7-10 alkylC _7-10 alkenylamino group, and a substituted or unsubstituted C _7-10 alkylC _7-10 alkenylimino group; The substitution is mono-substitution or poly-substitution. 2. The 20(S)-10,11-difluoromethylenedioxycamptothecin derivative according to claim 1, characterized in that the ring structure is selected from C _9-12 cycloalkyl, C _3-12 cycloalkenyl, aryl, C _2-11 ether ring or aromatic heterocyclic group, and the aromatic heterocyclic group is selected from pyridine ring, furan ring, thiophene ring, pyrazole ring, indole ring, benzopyrazole ring, piperidine ring, morpholine ring, thiomorpholine ring, naphthalene ring or triazole ring; when it is a substituted structure, the substituted groups are each independently selected from halogen, C _1-10 alkyl, C _1-10 alkenyl, C _1-10 alkynyl, C _3-12 cycloalkyl, C 1-10 alkoxy, C _1-10 alkylthio, C _1-10 alkylsilyl, C _1-10 haloalkoxy, C _1-10 ester group, 3-12 _membered heterocyclic group, C _6-14 aryl, oxy C The invention can be selected from the group consisting of _{: -C 6-14} aryl, oxy C _6-14 aromatic heteroyl, nitrogen C _6-14 aryl, nitrogen C _5-14 aromatic heteroyl, 5-14 membered heteroaryl, -CN, -NO ₂ , -CF ₂ H, -CF ₂ OH, -CF ₃ or -OCF ₃ . 3. The 20(S)-10,11-difluoromethylenedioxycamptothecin derivative according to claim 1, characterized in that Z is selected from _C1-10 alkyl-substituted aryl, _C1-10 alkoxy-substituted aryl, C1-10 alkylthio-substituted aryl, _C1-10 alkylsilyl-substituted aryl, _C1-10 haloalkoxy-substituted aryl, halogen atom-substituted aryl, unsubstituted _C2-11 ether ring substituent, C1-10 _alkyl -substituted _C2-11 ether ring substituent, benzene ring-substituted _C2-11 ether ring substituent, halogen atom-substituted _C2-11 ether ring substituent, unsubstituted aromatic heterocyclic substituent, _C1-10 alkyl-substituted aromatic heterocyclic substituent, _halogen atom-substituted aromatic heterocyclic substituent, unsubstituted _C1-8 olefin substituent or _C1-10 alkyl-substituted _C1-8 olefin substituent; The Z is a substituted aryl group, and Z is selected from any one of the following groups: The Z is a substituted or unsubstituted C _2-11 ether ring substituent, and Z is selected from any one of the following groups: The Z is a substituted or unsubstituted C _1-8 olefin substituent, and Z is selected from any one of the following groups: The Z is a substituted or unsubstituted aromatic heterocyclic substituent, and Z is selected from any one of the following groups: 4. A method for preparing a 20(S)-10,11-difluoromethylenedioxycamptothecin derivative according to any one of claims 1 to 3, characterized in that it comprises the following steps: (1) reacting the raw material, triethylamine and a reducing agent to obtain an intermediate; (2) reacting the intermediate, tricyclic ketone and a catalyst to obtain the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative. 5. The preparation method according to claim 4, characterized in that the reducing agent described in step (1) is selected from any one of Raney nickel, palladium carbon, platinum carbon, zinc, rhodium, iron, stannous chloride, lithium aluminum tetrahydride, 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 raw material described in step (1) is first dissolved in ethanol, and then the reducing agent is added, and triethylamine is added after hydrogen is introduced, and the mass volume ratio of the raw material to triethylamine and the reducing agent is 100-240mg:100-800μL:15-36mg; the temperature of the reaction described in step (1) is 20-30°C, and the reaction time is 8-12h; 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 bistrifluoromethanesulfonyl imide, magnesium bistrifluoromethanesulfonyl imide and p-toluenesulfonic acid hydrate; the intermediate, tricyclic ketone and catalyst in step (2) 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 in step (2) is carried out under nitrogen protection, and the pH is adjusted after the reaction is completed. The reaction temperature is 90-100°C, the reaction time is 8-12h, and the pH is 5-6; n is 0, Z is a substituted aryl group, and the preparation method of the raw material in step (1) comprises first subjecting 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde to a nitration reaction, then reacting it with a substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine, and finally reacting it with an oxidant, wherein the solvent for the nitration reaction comprises fuming nitric acid and trifluoromethanesulfonic acid, the temperature for the nitration reaction is -75°C to -45°C, and the reaction time is 2-3h; the substituted phenylboronic acid is selected from 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 any one of phenylboronic acid, 3-fluoro-4-methylphenylboronic acid, 2,4-difluorophenylboric acid, 3,4-difluorophenylboric acid, 4-fluorophenylboric acid, 4-trifluoromethylphenylboric acid and 4-methylthiophenylboric acid; toluene is used as solvent when reacting with substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine; the temperature of the reaction with substituted phenylboric acid, palladium chloride, potassium carbonate and tri(1-naphthyl)phosphine is 75-95°C, and the reaction time is The reaction time is 8-12h; the oxidant is selected from any one of manganese dioxide, Dess-Martin oxidant, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetra-n-propylammonium perruthenate, sodium nitrite/acetic anhydride oxidant and aluminum tert-butoxide; the solvent for the reaction with the oxidant is dichloromethane, the temperature for the reaction with the oxidant is 15-35°C, and the reaction time is 2-3h. 6. The preparation method according to claim 4, characterized in that n is an integer of 0-2, Z is a substituted or unsubstituted C _2-11 ether ring substituent, a substituted or unsubstituted C _1-8 olefin substituents, substituted or unsubstituted aromatic heterocyclic substituents, the preparation method of the raw material in step (1) comprises reacting 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde with a Grignard reagent, then reacting with an oxidant, and finally performing a nitration reaction, wherein the Grignard reagent is selected from any one of butyl magnesium bromide, hexyl magnesium bromide, 2-furylmethyl magnesium bromide, 4-pyranylmethyl magnesium bromide and heptyl magnesium bromide; before the reaction with the Grignard reagent, 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde is added to anhydrous tetrahydrofuran to dissolve, and placed in an ice water bath and stirred, the molar ratio of the 2,2-difluorobenzo[d][1,3]dioxole-5-carboxaldehyde to the Grignard reagent is 1:5-15; when reacting with the Grignard reagent, the environment is anhydrous and oxygen-free , the reaction progress is monitored by TLC, the developing solvent of the TLC is a mixture of petroleum ether and ethyl acetate in a volume ratio of 8-16:1-2, the temperature of the Grignard reagent reaction is 35-65°C, and the reaction time is 8-12h; the oxidant is selected from any one of manganese dioxide, Dess-Martin oxidant, pyridinium chlorochromate, silver carbonate, N-bromosuccinimide, iodic acid, hydrogen peroxide, chromium trioxide, N-chlorosuccinimide, tetra-n-propylammonium perruthenate, sodium nitrite/acetic anhydride oxidant and aluminum tert-butoxide; the solvent for the reaction with the oxidant is dichloromethane, the temperature of the reaction with the oxidant is 15-35°C, and the reaction time is 2-3h; the solvent for the nitration reaction includes fuming nitric acid and concentrated sulfuric acid, the temperature of the nitration reaction is -20°C to -10°C, and the reaction time is 8-12h. 7. The preparation method according to claim 6, characterized in that n is an integer of 0-2, Z is a substituted or unsubstituted aromatic heterocyclic substituent, and the nitration reaction is followed by a reaction with an aromatic heterocyclic formaldehyde, wherein the aromatic heterocyclic formaldehyde is selected from any one of 5-methylfuran-2-carboxaldehyde, 5-bromothiophene-2-carboxaldehyde and thiophene-2-carboxaldehyde, the reaction condition with the aromatic heterocyclic formaldehyde is alkaline, the reaction temperature with the aromatic heterocyclic formaldehyde is 15-35° C., and the reaction time is 1-2 h. 8. The preparation method according to any one of claims 4 to 7, characterized in that the post-treatment of the reaction in each 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 silica gel column chromatography purification, the filler of the silica gel column chromatography is 300-400 mesh silica gel, and the eluent used in the 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. 9. Use of the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative according to any one of claims 1 to 3 or the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative prepared by the preparation method according to any one of claims 4 to 8 in the preparation of a drug for preventing and/or treating cancer. 10. A pharmaceutical composition comprising: an effective dose of the 20(S)-10,11-difluoromethylenedioxycamptothecin derivative according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier.