TW202523322A - Gemcitabine anticancer derivatives and their anticancer pharmaceutical uses and preparation methods - Google Patents

Abstract

The present invention provides a gemcitabine anti-cancer derivative with the following formula H11, the anti-cancer pharmaceutical use thereof, and a preparation method therefor. The gemcitabine anti-cancer derivative has the potential to be developed into an anti-cancer drug activated by means of AKR1C3. Moreover, the gemcitabine anti-cancer derivative can be the pharmaceutically acceptable salt, prodrug, solvate or isotope variant, wherein the salt is a basic salt or an acid salt, preferably an ammonium salt, a carbonate, a sulfate, a phosphate or a hydrochloride, the solvate is a hydrate or an alcoholate, and the alcoholate is an ethanolate, the prodrug is selected from an ester and an amide.

Description

Gemcitabine anticancer derivatives and their anticancer pharmaceutical uses and preparation methods

The present invention relates to the further development of gemcitabine to obtain a gemcitabine anticancer derivative with targeted AKR1C3 enzyme activation, and belongs to the field of cancer treatment drug research and development.

Invention by Jian-xin Duan et al.: Nitrobenzyl derivative anticancer agent, PCT application number PCT/US2016/025665, publication number WO2016/161342A1, corresponding to Chinese application number 201680020013.2, publication number CN108136214A discloses a series of nitrobenzyl derivatives: In fact, the nitrobenzyl structure is connected to the anticancer drug residue through ^L1 to obtain the AKR1C3 activated anticancer drug, and the linker ^L1 is limited to have different structures for different anticancer drug residues D.

In particular, in this invention application, the inventors designed a derivative of gemcitabine combined with a nitrobenzyl structure having the following structure: . And three specific compounds: , , .

The TH3057 compound was further synthesized and tested for its IC ₅₀ value of cell proliferation inhibition activity on the H460 cell line that overexpresses the AKR1C3 enzyme. When an AKR1C3 enzyme inhibitor was added, the IC 50 values of the compound's cell proliferation inhibition activity on the _H460 cell line that overexpresses the AKR1C3 enzyme were 0.2 μmol/L and 5 μmol/L, respectively.

The inventors continued to study and found that the gemcitabine-nitrobenzyl derivatives designed in the above invention are not all anticancer drugs activated by AKR1C3, or compounds similar to TH3057. The compounds obtained by connecting the gemcitabine residue and the nitrobenzyl structure of ¹ are not all AKR1C3-activated anticancer drugs. Tests have shown that not all of these compounds can be activated by the AKR1C3 enzyme or have an extremely low activation ratio. Therefore, the inventors designed and synthesized a series of gemcitabine anticancer derivatives that are confirmed to be activated by the AKR1C3 enzyme or have a higher activation ratio based on the above-mentioned compounds. Moreover, these compounds may have better potential to be developed as AKR1C3-activated anticancer drugs than TH3057, especially compound H11 therein. Therefore, the present invention provides a new possibility or idea for developing AKR1C3-activated anticancer drugs with a higher specific activation ratio.

The present invention provides a novel structure of gemcitabine anticancer derivatives, which are AKR1C3 activated anticancer drugs.

A gemcitabine anticancer derivative having structural formula III, or a pharmaceutically acceptable salt or prodrug or solvate or isotopic variant thereof: (III) wherein _R1 is selected from H, halogen, means that _R1 can replace any 4 positions on the benzene ring and the number of replacements is 1, 2, 3 or 4; _R2 is selected from phenyl, halogen-substituted phenyl, C1-C3 alkyl or halogen-substituted C1-C3 alkyl; _R3 is selected from C1-C6 alkyl or halogen-substituted C1-C6 alkyl, benzyl. Halogen is selected from F, Cl, Br and I. Alkyl includes linear and branched alkyl and cycloalkyl.

Preferably, R ₁ is selected from H or mono-substituted F; Preferably, R ₂ is selected from phenyl, fluorophenyl, methyl or trifluoromethyl; Preferably, R ₃ is selected from methyl, ethyl, propyl, butyl, benzyl, or bromo-substituted methyl, ethyl, propyl, butyl; More preferably, R ₁ is selected from H or mono-substituted F, R ₂ is selected from phenyl, fluorophenyl, methyl or trifluoromethyl, R ₃ is selected from methyl, ethyl, propyl, butyl, benzyl, or bromo-substituted methyl, ethyl, propyl, butyl.

Further preferably, the above structural formula III is selected from gemcitabine anticancer derivatives having the following structural formula, or pharmaceutically acceptable salts or prodrugs or solvates or isotopic variants thereof: (H11).

In this application, gemcitabine is defined in a broad sense, including the following compounds contained in the marketed drug gemcitabine (GEMZAR, ZEFY): It also includes stereoisomers and racemates thereof. Wherein, the salt is a alkali salt (specifically a salt produced by reaction with a base) or an acid salt (specifically a salt produced by reaction with an acid).

That is, the present invention provides a pharmaceutically acceptable salt of a compound, and the salt may be an alkaline salt, including a salt formed by the compound and an inorganic base (e.g., alkali metal hydroxide, alkali earth metal hydroxide, etc.) or an organic base (e.g., monoethanolamine, diethanolamine, or triethanolamine, etc.). Alternatively, the salt may be an acidic salt, including a salt formed by the compound and an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, perchloric acid, sulfuric acid, or phosphoric acid, etc.) or an organic acid (e.g., methanesulfonic acid, trifluoromethanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, fumaric acid, oxalic acid, maleic acid, citric acid, etc.).

The selection and preparation of pharmaceutically acceptable salts and solvates of compounds are well known in the art. For gemcitabine and its derivatives, since they contain primary and secondary amines in their structures, the preferred salts are those generated by reaction with acids, such as hydrochlorides and monophosphates.

The compounds described herein may also be used in the form of a solvent complex, such as a hydrate, an alcohol complex, etc., and the alcohol complex includes an ethanol complex.

The prodrug is selected from esters or amides.

Since the gemcitabine anticancer derivative with structural formula III contains a hydroxyl group, it can form corresponding esters with carboxylic acids, sulfonic acids, acyl chlorides, etc.; since the structure contains primary and secondary amines, it can form corresponding amides with carboxylic acids, sulfonic acids, acyl chlorides, etc.

The concepts of isotopic variants or isomers in the present invention are based on the concepts of isotopic variants and chiral isomers in patent application PCT/US2016/062114, publication number WO2017087428, corresponding to Chinese application number 2016800446081, publication number CN108290911A.

Since the anticancer derivatives of gemcitabine with structural formula III have two key metabolic sites: Therefore, the inventors speculated that the possible structures of deuterium isotope substituted compounds are as follows: .

The present invention also provides a medicine or preparation containing the above compound.

The present invention also provides the use of the gemcitabine anticancer derivatives having the structural formula III and their pharmaceutically acceptable salts or prodrugs or solvent compounds or isotopic variants in the preparation of drugs for treating cancer patients or tumor patients or diseases or cell proliferative diseases caused by cancer or tumor.

Preferably, the drug or preparation is used to treat a patient's tumor or cancer disease, wherein the tumor or cancer includes: Lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, bone cell carcinoma, epithelial carcinoma, bile duct carcinoma, choriocarcinoma, embryonal carcinoma, spermatogonial carcinoma, Wilms' carcinoma, colloid carcinoma, astrocytoma, neuromedulloblastoma, craniopharyngioma, ependymoma, pinealoma, hematoblastoma, vocal cord Neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromyxoma, fibroostoma, fibromyxosarc ... tumor, chordoma, chorioadenoma, chorioepithelioma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, osteofibroma, osteofibrosarcoma, angiosarcoma, angiosarcoma, angiolipoma, angiochondroma, angioblastoma, angiokeratoma, angioneuroma, angioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipomyoma, angiomyoneuroma, angiomyoma, angiomyoneuroma, angiomyoma, angiomyoneuroma, lymphangiosarcoma, lymphangio blastoma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, endothelioma, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoma, leiomyofibroma, rhabdomyomas, rhabdomyosarcomas, rhabdomyomyxoma, acute lymphatic leukemia, acute myeloid leukemia, anemia of chronic disease, polycythemia vera, lymphoma, endometrial carcinoma, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma.

The cancer or tumor is a primary brain cancer, a brain tumor, or a metastatic cancer or tumor that has metastasized to the brain.

Preferably, the drug or preparation is used alone or in combination to treat non-small cell lung cancer, pancreatic cancer, ovarian cancer, and breast cancer.

The relevant information of gemcitabine pharmaceutical preparations that have been marketed is as follows.

Gemcitabine hydrochloride for injection in China, Zefei, national medicine standard H20030105, its indications are described as being used to treat the following diseases: Locally advanced or metastatic non-small cell lung cancer, including single-agent and combined with cisplatin; Locally advanced or metastatic pancreatic cancer, including single-agent; Gemcitabine combined with paclitaxel can be used to treat relapsed, unresectable, locally recurrent or metastatic breast cancer after adjuvant/neoadjuvant chemotherapy. Unless there are clinical contraindications, anthracycline antibiotics should have been used in previous chemotherapy.

The indications of GEMZAR in the United States are as follows: GEMZAR® is a nucleoside metabolic inhibitor indicated: • in combination with carboplatin, for the treatment of advanced ovarian cancer that has relapsed at least 6 months after completion of platinum-based therapy. (1.1) • in combination with paclitaxel, for first-line treatment of metastatic breast cancer after failure of prior anthracycline-containing adjuvant chemotherapy, unless anthracyclines were clinically contraindicated. (1.2) • in combination with cisplatin, for the treatment of non-small cell lung cancer. (1.3) • as a single agent for the treatment of pancreatic cancer. (1.4) Translated into Chinese as follows: GEMZAR® is a nucleoside metabolic inhibitor indicated for: • In combination with carboplatin, for the treatment of advanced ovarian cancer that has recurred at least 6 months after completion of platinum-based therapy. • In combination with paclitaxel, for the first-line treatment of metastatic breast cancer after failure of prior adjuvant chemotherapy containing anthracyclines, unless anthracyclines are clinically contraindicated. • In combination with cisplatin, for the treatment of non-small cell lung cancer. • Alone, for the treatment of pancreatic cancer.

The anticancer derivative of Citabine with structural formula III and the drug or preparation prepared therefrom are gemcitabine derivatives, and metabolize gemcitabine after activation of AKR1C3 to exert anti-tumor effects. Therefore, the inventors preliminarily speculate that they may have similar indications to the marketed drug gemcitabine.

The present invention also provides a method for treating cancer or tumors, which comprises the steps of applying the above-mentioned drug or preparation; and measuring the AKR1C3 reductase content or expression level of the patient's cancer cells or tissues. If the AKR1C3 reductase content or expression level is equal to or greater than a predetermined value, the above-mentioned drug or preparation is administered to the patient.

Preferably, AKR1C3 antibody can be used to measure the AKR1C3 reductase content or expression level.

The present invention also provides another method for treating cancer or tumors, which comprises a step of applying the above-mentioned drug or preparation; and a step of regulating the content or expression level of AKR1C3 reductase. When the regulation makes the content or expression level of AKR1C3 reductase equal to or greater than a predetermined value, the above-mentioned drug or preparation is administered to the patient.

Methods that can be used to measure the AKR1C3 reductase content include ELISA method, IHC method, etc.

Commercial human aldehyde-keto reductase 1C3 (AKR1C3) ELISA test kits can be used to directly test liquid samples such as plasma and blood, while other samples can be tested after processing.

IHC, or immunohistochemistry, is suitable for testing solid tumor samples.

Detecting the expression level of AKR1C3 refers to detecting the expression level of the corresponding mRNA. Studies have shown that the AKR1C3 enzyme content and AKR1C3 RNA expression level of cancer cells of different solid tumors are significantly correlated, and the AKR1C3 enzyme content and AKR1C3 RNA expression level of different blood cancer cell lines are significantly correlated. Therefore, by detecting the ARKR1C3 RNA content (or expression level), the enzyme content can also be predicted or characterized, and then the medication can be guided. q-PCR technology can be used to detect the RNA (messenger RNA, i.e., mRNA) content or expression level of AKR1C3.

For methods for detecting AKR1C3 enzyme content levels or q-PCR expression, please refer to the applicant's patent application publications WO2022183483A1, WO2022048492A1 or related research papers (Reddi D, Seaton BW, Woolston D, et al. AKR1C3 expression in T acute lymphoblastic leukemia/lymphoma for clinical use as a biomarker. Sci Rep . 2022;12(1):5809. Published 2022 Apr 6. doi:10.1038/s41598-022-09697-6).

The predetermined value in this application needs to be obtained through clinical trials. Taking the similar AKR1C3 enzyme-activated anticancer prodrug AST-3424 (OBI-3424) as an example, its predetermined value is IHC test method H-score 135 (Tsimberidou, A.M., Verschraegen, C.F., Wesolowski, R. et al. Phase 1 dose-escalation study evaluating the safety, pharmacokinetics, and clinical activity of OBI-3424 in patients with advanced or metastatic solid tumors. Br J Cancer 129, 266–274 (2023). https://doi.org/10.1038/s41416-023-02280-4).

Studies have shown that the AKR1C3 enzyme content in the tumor tissue of head and neck cancer patients after radiotherapy increases. Therefore, irradiating the patient's tumor tissue with radiation from radiotherapy may increase the expression level of the AKR1C3 enzyme. Radiation includes α, β, and γ rays produced by radioactive isotopes and x-rays, electron beams, proton beams, and other particle beams produced by various x-ray therapy machines or accelerators.

Studies have shown that certain chemical ingredients can also promote the expression of AKR1C3 enzyme in the human body, such as kukui (scientific name: Aleurites moluccana L. Willd.) kernel oil (Japanese patent application JP2021145582A, invention name AKR1C1, AKR1C2 and AKR1C3 production promoter) and sodium butyrate (Frycz BA, Murawa D, Borejsza-Wysocki M, et al. Transcript level of AKR1C3 is down-regulated in gastric cancer. Biochem Cell Biol . 2016;94(2):138-146. doi:10.1139/bcb-2015-0096).

This method is mainly aimed at patients with low AKR1C3 reductase levels. Through certain regulatory treatment/drug administration processes, the patient's AKR1C3 reductase level is adjusted to an appropriate level.

Regarding the medicaments or preparations described herein, the prepared medicaments contain the indicated compound or its salt or solvent complex within a specific dosage range, and/or the prepared medicaments are administered in a specific dosage form and a specific administration method.

The medicament or preparation herein may also contain a pharmaceutically acceptable excipient or formulation. The medicament may be in any dosage form for clinical use, such as tablets, suppositories, dispersible tablets, enteric disintegrating tablets, chewable tablets, orodisintegrating tablets, capsules, sugar-coated tablets, granules, dry powders, oral solutions, small needles for injection, freeze-dried powder injections for injection, or large infusions. Depending on the specific dosage form and administration method, the pharmaceutically acceptable excipients or formulations in the drug may include one or more of the following: diluents, solubilizers, disintegrants, suspending agents, lubricants, binders, fillers, flavor enhancers, sweeteners, antioxidants, surfactants, preservatives, encapsulating agents, and pigments, etc.

"Applying" or "administering" a drug should be to achieve a "therapeutically effective amount", which means an amount that, when administered to a cancer patient, will have a predetermined therapeutic effect, such as alleviating, improving, relieving or eliminating one or more manifestations of the patient's cancer. The therapeutic effect may not occur when a single dose is administered, and may only occur after a series of doses are administered. Therefore, a therapeutically effective amount can be administered by one or more administrations.

"Treating" a condition or patient means taking steps to obtain beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, relief or improvement of one or more symptoms of cancer; reduction in the extent of the disease; delay or slowing of the progression of the disease; improvement, palliation, or stabilization of the disease state; or other beneficial results. In some cases, treatment of cancer can result in a partial response or stabilization of the disease.

The subject of "treating cancer or tumor" refers to any appropriate species: mammals, fish, such as mammals, such as rodents, dogs, cats, horses or humans. Preferably, the patient is a mammal, more preferably a human.

Also provided is a method for preparing a gemcitabine derivative H11, comprising hydrolyzing a compound H11-D under acidic conditions and alkalizing the compound: .

This reaction is a hydrolysis reaction to remove the Boc protecting group. The hydrolysis reaction is carried out under acidic conditions. The acid generally used is a non-oxidizing inorganic acid or organic acid. The reaction is often carried out in an organic solvent to facilitate the precipitation of the salt generated in the reaction. Commonly used acid and reaction solvent systems include: hydrochloric acid-dioxane, trifluoroacetic acid-dichloromethane, etc. The reaction conditions are relatively mild, generally room temperature.

The acidic condition is provided by TFA, preferably by introducing H11-D into a mixed system of TFA and DCM.

Since the product H11 has an _-NH2 group, it exists in the form of a salt under acidic conditions and therefore needs to be alkalized, i.e., the salt after acidic hydrolysis is converted into a free amine using a corresponding base. The alkalization operation process includes: adding a base to the reaction system to make the system alkaline. Considering the acid-base stability of H11, the base used should not be too strong, and the pH value after the final alkalization should not be too low. The base used in the alkalization operation includes alkaline metal hydroxides, bicarbonates, carbonates, such as NaOH, KOH, _NaHCO3 , _KHCO3 , _K2CO3 , _Na2CO3 , _etc. It is preferred that the pH of the reaction system is 8 when the aqueous solution of the _above base is added to the reaction system after hydrolysis.

A pH of 8 refers to the measurement using pH test paper (general pH test paper, not precision pH test paper). Due to the visual observation error in the test paper colorimetry process, the actual value may be greater than 7 and less than 10. Therefore, if the measured pH is greater than 7 and less than 10, it should be considered to be equivalent to the pH of 8 in the above reaction system.

Also provided is a preparation method of gemcitabine derivative H11-D, comprising the following operations: Operation 1, stirring and reacting H11-A1 with POX ₃ and organic amine; Operation 2, after the reaction is complete, adding H11-A2 and organic amine and stirring and reacting; Operation 3, after the reaction is complete, adding isopropylamine and organic amine and stirring and reacting; Operation 4, adding water or acidic salt solution for post-treatment, , Wherein, X is Cl or Br.

The organic amine may be triethylamine, trimethylamine, diethylamine, dimethylamine, or the like.

Acidic salts are salts whose aqueous solutions are acidic, such as some weak alkaline strong acid salts (salts formed by weak bases and strong acids): ammonium sulfate and ammonium chloride.

Preferably, the organic amine in operations 1, 2 and 3 is triethylamine, and the reaction solvent is dichloromethane.

Preferably, the adding of isopropylamine in operation 3 is the adding of its tetrahydrofuran solution, and the acidic salt solution in operation 4 is a saturated NH ₄ Cl solution.

The terms not specifically explained or described in this application document shall be based on the textbooks of medicinal chemistry, organic chemistry, biochemistry, and pharmacology.

The present invention is described below with reference to specific embodiments. Those skilled in the art will appreciate that these embodiments are only used to illustrate the present invention and do not limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagent materials, etc. used in the following examples are commercially available products unless otherwise specified.

1. H460 cancer cell inhibition test

In vitro cytotoxicity assays were performed using human tumor cell lines.

In vitro proliferation data for the H460 non-small cell lung cancer human tumor cell line (AKR1C3 high expressing cell line) are reported in the compound table below.

The _IC50 value is obtained by the following steps:

Specifically, exponentially growing cells were plated in 96-well plates at a density of 2×10 ³ cells/100 µL/well and incubated at 37°C in 5% CO ₂ , 95% air and 100% relative humidity for 24 hours. Test compound alone: 24 hours after cell plating, compound alone was applied, 99 µL of growth medium was added to each well, and then 1µL of 200X prepared compound alone was added, gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO ₂ incubator. After further incubation for 72 hours, the number of viable cells was detected using CTG. The CTG detection method is as follows: Place the cell test plate at room temperature for 30 minutes, discard 100 µL of culture medium from each well. Add 25 µL of CTG reagent (CelltiterGlo reagent kit) to each well, place it on a fast oscillator for 2 minutes, and place it at room temperature away from light for 30 minutes. Read the chemiluminescent signal value with a multifunctional enzyme marker, and the reading time is 1000ms. Use computer software to calculate the drug concentration that causes 50% growth inhibition (IC ₅₀ ), and the results are listed in Table 1 to obtain the corresponding value of IC ₅₀ (nM).

Similarly, to further verify whether the compounds are activated by human AKR1C3 (aldosterone reductase family 1 member C3), some compounds were tested for H460 cancer cell proliferation under the condition of pretreatment with the specific AKR1C3 enzyme inhibitor AST-3021 (3 µM). The specific experimental steps are as follows: 24 hours after cell plating, 98 µL of growth medium was added to each well, and then 1 µL of 200X compound AST-3021 was added to each well, and gently shaken to ensure uniform mixing. After placing in the incubator for 2 hours, 1 µL of 200X prepared test compound was added, and gently shaken to ensure uniform mixing, and then placed in a 37°C, 5% CO ₂ incubator. The inhibitor used was compound 36 from Flanagan et al., Bioorganic and Medicinal Chemistry (2014) pp. 967-977. , referred to as AST-3021 in this application. The values tested in this case are the values corresponding to +AST-3021 IC ₅₀ (nM) in Table 1.

The specific activation ratio is obtained by dividing the value corresponding to +AST-3021 IC ₅₀ (nM) by the value corresponding to IC ₅₀ (nM). The relative size of the specific activation ratio can be used to evaluate whether the compound is a cytotoxic compound activated by human AKR1C3 (aldosterone reductase family 1 member C3): when the specific activation ratio is less than 1, it is considered that the compound is not activated by human AKR1C3 (aldosterone reductase family 1 member C3); when the specific activation ratio is greater than 1, it is considered that the compound is activated by human AKR1C3 (aldosterone reductase family 1 member C3), and the larger the value, the stronger the degree of activation and the better the selectivity of AKR1C3.

Table 1: Comparison of IC ₅₀ values and specific activation ratios (+AST-3021 IC ₅₀ /IC ₅₀ ) of each compound ± AST-3021

The experimental results show that human AKR1C3 can activate the above compounds H2~H11, that is, the series of compounds are AKR1C3-activated anticancer gemcitabine prodrugs. In particular, by comparing the compounds on the left side of the above table with the comparative compounds D1~D8 on the right side, it can be seen that the compound of structural formula (I) obtained by the N atom connected to R ₁ being an imine structure (-NH-) is higher in vitro cytotoxic than the compound of structural formula (Comparison I) obtained by the N atom connected to R 1 being a tertiary amine group ₍ comparison under the condition that the number of carbon atoms connected to N is close and the structure is similar): Generally, the values corresponding to IC ₅₀ (nM) in the table and the values corresponding to +AST-3021 IC ₅₀ (nM) in the table are both lower (that is, the compound of structural formula I has stronger cytotoxicity).

If the compound on the left in the above table is represented by the following general formula compound (I), and the compound on the right is represented by the following general formula compound (Comparison I) or (Comparison II), then compound (I) has a higher AKR1C3 specific activation ratio than compound (Comparison I). （I） (Comparison I), (Contrast II).

In the above structure, _R1 to _R7 refer to broad substituents and are not defined in this application. They are only used to illustrate the different situations in the phosphate linking groups in the compounds of this application and the comparative compounds: (corresponding to compound III of this application), (Comparison I), (Comparison II) Differences in the activities of the compounds formed.

Furthermore, experiments also confirmed that the compound of structural formula (Comparison II) was not activated by AKR1C3 at all, and it may not be a substrate of AKR1C3 enzyme.

That is to say, the present invention corrects the nitrobenzyl derivative anticancer agent of the invention application, PCT application number PCT/US2016/025665, publication number WO2016/161342A1, corresponding to the nitrobenzyl derivative indicated in the Chinese application number 201680020013.2, publication number CN108136214A: , which are all inaccurate views on anticancer drugs activated by AKR1C3. That is, it is clarified that for gemcitabine-nitrobenzyl derivatives, the compound of structural formula (Comparison II) is not activated by AKR1C3 at all, and through comparison of examples, it is found that the tumor cell proliferation inhibitory activity of the compound of structural formula (I) is higher than that of the compound of structural formula (Comparison I).

2. AKR1C3 activation-dependent cell proliferation inhibitory activity experiment

2.1 Inhibitory test of gemcitabine and compound H11 on H460 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021

The experimental protocol is as follows: 1) Add H460 cell suspension to a 96-well plate, 100 µL per well, with a cell density of 2000/well. 2) Incubate cells overnight in a 37°C, 5% CO ₂ incubator. 3) Compound treatment and combination therapy: 24 hours after cell plating, add 98 µL of growth medium to each well. Add 1 µL of AST-3021 to each well (final concentration 3 µM), shake gently to ensure even mixing, place in the incubator for 2 hours, add 1 µL of test compound at different concentrations, shake gently to ensure even mixing, and then place in a 37°C, 5% CO ₂ incubator. Single drug application: 24 hours after cell plating, add 99 µL of growth medium to each well. Add 1 µL of test compound of different concentrations, shake gently to ensure uniform mixing, and then place in a 37°C, 5% CO ₂ incubator. 4) Place the cell plate in the incubator for 72 hours. 5) Place the cell plate to be tested at room temperature for 30 minutes, and discard 100 µL of medium from each well. 6) Add 25 µL of CTG reagent to each well, place on a fast oscillator for 2 minutes, and place at room temperature away from light for 30 minutes. 7) Read the chemiluminescent signal value with a multifunctional enzyme labeler, and the reading time is 1000ms. 8) GraphPad Prism 5 software was used to calculate IC ₅₀ , and the following nonlinear fitting formula was used to obtain the IC ₅₀ (half maximal inhibitory concentration) of the compound.

To further verify that the compound is specifically activated by human AKR1C3 (aldosterone reductase family 1 member C3), the cells were pretreated with the specific AKR1C3 enzyme inhibitor AST-3021 (3 μM) and then the compound was added to detect whether the proliferation of H460 tumor cells was affected by the presence or absence of AST-3021.

The IC ₅₀ data of the inhibitory effects of compounds gemcitabine and H11 on H460 tumor cells in the presence or absence of the AKR1C3 inhibitor AST-3021 are shown in Tables 2 and 3. The IC ₅₀ curve corresponding to gemcitabine is shown in Figure 1. In Table 3, the IC ₅₀ curve of compound H11 corresponding to Experiment No. 3 is shown in Figure 2.

Table 2: _IC50 data of gemcitabine inhibition on H460 cancer cells in the presence or absence of AKR1C3 inhibitors Compound IC ₅₀ (nmol/L) Specific activation ratio AST-3021 not added Add AST-3021 Gecitabine 13.19 14.21 1.08

Table 3: _IC50 data of the inhibitory effect of compound H11 on H460 cancer cells in the presence or absence of AKR1C3 inhibitors Experiment number Compound IC ₅₀ (nmol/L) Specific activation ratio AST-3021 not added Add AST-3021 1 H11 16.25 1473.00 90.65 2 H11 30.29 2348.00 77.52 3 H11 35.70 2942.00 80.41

The test results show that the IC ₅₀ value of gemcitabine is close with or without the addition of AST-3021, and the specific activation ratio is only 1.08, indicating that the activity of gemcitabine in inhibiting cancer cells is not dependent on the expression of AKR1C3. However, the IC ₅₀ value of compound H11 with or without the addition of AST-3021 is quite different. The results of three different time tests all show that its specific activation ratio is more than 75 times. Combined with the relationship between the IC ₅₀ specific activation ratio of gemcitabine with or without the addition of AST-3021, it shows that compound H11 is an anticancer compound that depends on AKR1C3 activation, and its in vitro activity is closely related to the expression of the AKR1C3 enzyme.

2.2 Inhibitory test of compound H11 on HepG2 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021

We selected HepG2 cells with high expression of human AKR1C3 and used the same test method in 2.1 to test the _IC50 data of compound H11 on HepG2 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021, as shown in Table 4. The _IC50 curve corresponding to Experiment No. 5 is shown in Figure 3.

Table 4: _IC50 data of the inhibitory effect of compound H11 on HepG2 cancer cells in the presence or absence of AKR1C3 inhibitors Experiment number Compound IC ₅₀ (nmol/L) Specific activation ratio AST-3021 not added Add AST-3021 4 H11 42.85 3350.00 78.18 5 H11 39.82 1582.00 39.73 6 H11 80.89 1660.00 20.52

The test results showed that the IC ₅₀ value of compound H11 varied greatly with or without the addition of AST-3021. The results of three tests at different time points all showed that its specific activation ratio was more than 20 times, indicating that compound H11 was an anticancer compound that depended on AKR1C3 activation for HepG2 cancer cells, and its in vitro activity was closely related to the expression of the AKR1C3 enzyme.

2.3 In vitro proliferation inhibition experiment of compound H11 on different pancreatic cancer cell lines/different leukemia cancer cell lines

Referring to the single-drug operation process in the above Example 2.1, the in vitro proliferation inhibition effect of different pancreatic cancer cell lines/different leukemia cancer cell lines was tested. The specific IC ₅₀ results are shown in Tables 15 and 16 below.

Table 15: In vitro proliferation inhibition results of compound H11 on 13 pancreatic cancer cell lines with different AKR1C3 RNA expression levels

Table 16: In vitro proliferation inhibition results of compound H11 on 15 leukemia cell lines with different AKR1C3 RNA/protein expression levels

In the table, AKR1C3 Expression refers to AKR1C3 RNA Expression, and AKR1C3 RNA is expressed in LOG2 (FPKM).

FPKM, Fragments Per Kilobase of exon model per Million mapped fragments.

The Normalized AKR1C3 Protein Expression in the table was measured by Western blot experiment, and the ratio of the gray value of the AKR1C3 protein band of each cancer cell to the gray value of the reference protein β-tubulin band was used as the relative value of protein expression (Normalized AKR1C3 Protein Expression).

According to the in vitro proliferation inhibitory activity data of the above-mentioned compound H11 in different pancreatic cancer and different leukemia cancer cells, the cell proliferation inhibitory activity of the H11 compound is highly correlated with the AKR1C3 RNA content and the strength of AKRC3 protein expression, showing dependence.

3. Comparison of in vivo efficacy of compound H11 and gemcitabine in pancreatic cancer PA1222 model

Experimental description: Tumor tissues were collected from HuPrime® pancreatic cancer xenograft model PA1222 (AKR1C3 high expression pancreatic cancer PDX model, H-score=204.28, AKR1C3 RNA Log2 FPKM=7.386) tumor-bearing mice, cut into tumors with a diameter of 2-3 mm and implanted subcutaneously in the right anterior shoulder blade of BALB/c nude mice. When the average tumor volume ^was about 102 mm3, the mice were randomly divided into groups according to the tumor size. Table 5 shows the experimental design of the anti-tumor effect of the test drug in the HuPrime® pancreatic cancer PA1222 tumor model.

Table 5: Experimental design for the anti-tumor effect of the test drug in the HuPrime® pancreatic cancer PA1222 tumor model Group Number of animals Medication group Dosage (mg/kg) How to give medicine Dosing cycle 1 5 10% anhydrous ethanol + 10% polyoxyethylene (35) castor oil + 80% glucose injection D5W (pH 7.7-8.0) - caudal vein iv Q2D×3 weeks 2 5 Gecitabine 80 Intraperitoneal injection Q3Dx4 3 5 H11 240 Gavage QD×21 4 5 H11 160 Intraperitoneal injection QD×5, 2 days off, 2 weeks off; QD×5 5 5 H11 160 caudal vein iv Q2D×3 weeks Note: 1. The dosage volume is 10 μL/g. 2. Q3Dx4: Administer once every three days for 4 consecutive times; Q2Dx3: Administer once every two days for 3 consecutive weeks; QDx21: Administer once a day for 21 consecutive days; QD×5: 2 days off, 2 weeks off; QD×5: Administer once a day for 5 consecutive days, stop for 2 days, stop for 2 weeks, then administer for 5 consecutive days, once a day.

On the 32nd day after the first administration, i.e., Day 32, the second, third, fourth, and fifth groups were terminated, tumors were removed, tumor weights were weighed, tumor tissues were photographed, and tumor tissues were rapidly frozen. On Day 57, the first group was terminated, tumors were removed, tumor weights were weighed, tumor tissues were photographed, and tumor tissues were rapidly frozen. The efficacy was evaluated based on the relative tumor inhibition rate TGI (%), and the safety was evaluated based on the weight changes and deaths of the animals.

The efficacy evaluation criteria and calculation methods are as follows: Relative tumor proliferation rate, T/C%, that is, the percentage of the relative tumor volume or tumor weight of the treatment group and the control group at a certain time point. The calculation formula is as follows: T/C% = _TRTV / _CRTV × 100% ( _TRTV : average RTV of the treatment group; _CRTV : average RTV of the vehicle control group; RTV= _Vt / _V0 , _V0 is the tumor volume of the animal at the time of grouping, and _Vt is the tumor volume of the animal after treatment); Relative tumor inhibition rate, TGI (%), the calculation formula is as follows: TGI% = (1-T/C) × 100%. (T and C are the average relative tumor volume (RTV) of the treatment group and the control group at a specific time point, respectively).

The efficacy of each group in the pancreatic cancer PA1222 model was analyzed on the 32nd day after the first administration, and the results are shown in Table 6. The average tumor volume on different days was recorded and the average value was taken, and the results are shown in Table 7. The average tumor volume in the first 32 days after the start of drug administration was plotted into a curve, as shown in Figure 4.

Table 6: Efficacy analysis of each group in HuPrime® pancreatic cancer PA1222 model Experimental group 32 days after the first dose (i.e. Day 32, 10/4/2021) Tumor volume ( ±S) Relative tumor volume ( ±S） TGI (%) T/C (%) P Value (compared to control group) Group 1 629.85±136.87 6.21±1.36 - - - Group 1 1247.07±307.89 (Day 56) 12.15±2.89 - - - Group 2 6.79±4.33 0.06±0.04 99.03% 0.97% <0.001 Group 3 44.61±22.14 0.47±0.27 92.35% 7.65% <0.001 Group 4 202.58±45.40 2.01±0.41 67.62% 32.38% <0.05 Group 5 0.00±0.00 0.00±0.00 100.00% 0.00% <0.001 Note: 1. Data are expressed as “mean ± standard error”; 2. T/C % = T _RTV / C _RTV × 100%; TGI% = (1-T/C) × 100%;

Table 7: Changes in tumor volume of mice in each group with treatment time in HuPrime® pancreatic cancer PA1222 model Tumor volume (mm ³ ) ( ±S) time Group 1 Group 2 Group 3 Group 4 Group 5 0 days after the start of medication 102.69±6.88 102.95±6.86 102.83±6.64 101.91±5.96 102.11±6.00 4 days after starting medication 144.96±13.68 104.07±9.22 123.16±3.48 105.94±8.67 98.46±3.72 7 days after starting medication 177.74±24.98 74.43±5.22 90.07±4.65 96.63±11.34 79.97±3.69 11 days after the start of medication 243.12±34.25 38.92±4.90 54.28±5.75 116.07±22.87 40.67±3.20 14 days after starting medication 299.90±51.57 24.66±3.94 41.73±7.84 139.61±22.61 22.37±2.70 18 days after the start of medication 358.17±54.38 17.66±4.82 34.60±7.08 153.86±27.60 11.52±4.90 21 days after the start of medication 399.99±56.79 12.13±3.15 29.52±12.59 188.69±33.45 11.56±3.08 25 days after the start of medication 490.19±78.73 10.23±3.05 30.71±15.00 198.68±40.97 6.73±2.81 28 days after the start of medication 540.46±97.48 7.97±3.70 30.59±12.12 195.90±41.71 0.00±0.00 32 days after the start of medication 629.85±136.87 6.79±4.33 44.61±22.14 202.58±45.40 0.00±0.00 35 days after the start of medication 669.64±145.73 / / / / 39 days after the start of medication 799.46±184.86 / / / / 42 days after the start of medication 912.51±196.70 / / / / 46 days after the start of medication 1004.17±295.28 / / / / 49 days after the start of medication 1064.65±273.52 / / / / 53 days after the start of medication 1154.21±310.62 / / / / 56 days after the start of medication 1247.07±307.89 / / / /

In order to verify the effect of the test drug on the weight change of the experimental animals, based on the above experimental method, the weight change of the experimental animals was recorded at different time points, as shown in Table 8, and the weight growth rate was calculated and plotted into a curve graph, as shown in Figure 5.

Table 8: Average weight of mice on different days (g) Days 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Group 1 22.2 / 22.4 / 22.4 / / 22.9 22.9 / 22.6 22.9 23.0 / 22.4 / / / 22.8 / Group 2 21.1 / / 21.2 21.0 / 21.2 21.4 / 21.5 / 21.3 / / 21.9 / / / 22.1 / Group 3 21.8 21.5 21.5 21.6 21.9 22.3 21.7 21.6 21.7 22.5 21.6 21.9 22.0 21.9 21.6 21.6 22.1 21.8 21.9 21.8 Group 4 21.4 22.0 21.5 22.0 21.7 / / 22.3 / / / 22.5 / / 22.9 / / / 22.7 / Group 5 21.6 / 21.8 / 21.4 / / 20.9 21.6 / 21.3 22.1 22.2 / 22.0 / 22.3 / 22.1 / Days 20 twenty one twenty two twenty three twenty four 25 26 28 32 33 35 39 40 42 46 47 49 53 56 57 Group 1 22.6 23.0 / / / 23.6 / 23.1 23.4 23.5 23.3 23.7 24.1 23.6 24.1 24.2 23.5 24.2 23.7 22.9 Group 2 / 22.6 / / / 22.3 / 22.1 23.1 / / / / / / / / / / / Group 3 21.9 21.8 / / / 22.6 / 22.4 22.9 / / / / / / / / / / / Group 4 / 22.9 22.6 22.5 22.6 22.5 / 22.6 23.3 / / / / / / / / / / / Group 5 22.1 22.3 / / / 22.6 / 22.5 23.2 / / / / / / / / / / /

The above experimental data show that: The test drug gemcitabine (80 mg/kg, Group 2), H11 240 mg/kg (Group 3, QD×21) and H11 160 mg/kg, i.v. (Group 5, Q2D×3weeks) treatment groups had extremely significant tumor inhibition effects on Day 32 after the first administration, with relative tumor inhibition rates TGI (%) of 99.03%, 92.35% and 100%, respectively, and complete tumor inhibition rates of 60%, 20% and 100%, respectively. The H11 160 mg/kg, i.p. (Group 4, QD×5, 2 days off, 2 weeks off; QD×5) treatment group had a significant tumor inhibition effect on Day 32 after the first administration, with a relative tumor inhibition rate TGI (%) of 67.62%. At the same time, the tumor inhibition effect of the test drug H11 160 mg/kg, i.v. (Group 5, Q2D×3weeks) was better than that of H11 160 mg/kg, i.p. (Group 4, QD×5, 2 days off, 2 weeks off; QD×5), and the statistical comparison between the two groups (Group 5 and Group 4) was significantly different (p < 0.001). The anti-tumor effect of the test drug H11 160 mg/kg, i.v. (Group 5, Q2D×3weeks) was better than that of H11 240 mg/kg, p.o. (Group 3, QD×21), and there was a significant difference in the statistical comparison between the two groups (Group 5 and Group 3) (p < 0.05). The anti-tumor effect of the test drug H11 160 mg/kg, i.v. (Group 5, Q2D×3weeks) and H11 240 mg/kg, p.o. (Group 3, QD×21) was not significantly different from that of the test drug gemcitabine (80 mg/kg, Group 2) treatment group, and there was no significant difference in the statistical comparison between the two groups (Group 5 vs Group 2 and Group 3 vs Group 2) (p values were all greater than 0.05).

The mice in the test drug gemcitabine 80 mg/kg treatment group, H11 240 mg/kg (Group 3, QD×21) treatment group, H11 160 mg/kg, i.p. (Group 4, QD×5, 2 days off, 2 weeks off; QD×5) treatment group, test drug H11 160 mg/kg, i.v. (Group 5, Q2D×3weeks) treatment group, and control group did not show any obvious weight loss, and the treatment was well tolerated.

4. Metabolic stability evaluation experiment

4.1 Metabolic stability study of compound H11 and positive control verapamil in liver microsomes

Experimental plan: (I) Solution preparation Prepare the main solution according to Table 9 below: Table 9: Preparation of the main solution Reagent Material concentration Volume Final concentration Phosphate buffer 100 mM 216.25 μL 100 mM Microsomes 20 mg/mL 6.25 μL 0.5 mg/mL (ii) Two groups of experiments were performed separately: a) Cofactor (NADPH): Add 25 μL of 10 mM NADPH to the culture medium. The final concentration of liver microsomes was 0.5 mg/mL, and the final concentration of NADPH was 1 mM. b) No cofactor (NADPH): Add 25 μL of 100 mM phosphate buffer. The final concentration of liver microsomes was 0.5 mg/mL. Preheat the mixture at 37°C for 10 minutes. (iii) Add 2.5 μL of 100 μM control compound or test compound solution to start the reaction. Verapamil was used as a positive control in this study. The final concentrations of the test compound and the control compound were both 1 μM. The culture medium was cultured in water at 37°C. (iv) Take 30 μL aliquots from the reaction solution at 0.5, 5, 15, 30 and 60 min. The reaction was stopped by adding 5 volumes of cold acetonitrile and IS (internal standard, containing 100 nM alprazolam, 200 nM caffeine and 100 nM tolbutamide). The sample was centrifuged at 3,220 g for 40 min. Take 100 μL of the supernatant and mix with 100 μL ultrapure water, aliquot and perform LC-MS/MS analysis. (v) Data analysis All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. The slope value k was determined by the natural logarithm linear regression of the parent drug residual percentage versus incubation time curve. (1) The in vitro half-life (in vitro t _1/2 ) was determined from the slope value: in vitro t _1/2 = -0.693/k. (2) The in vitro t _1/2 (min) was converted to in vitro intrinsic clearance (in vitro CL _int , µL/min/1×10 ⁶ cells) using the following formula (mean of replicates): CL _int = - kV/N, where V = culture volume (0.2 mL) and N = number of hepatocytes per well (0.1×10 ⁶ cells). (3) Scale-up CL _int (mL/min/kg), hepatic clearance (Predicted Hepatic CL _H) (mL/min/kg) and hepatic extraction ratio (ER) were calculated using the following formula: Scale-up CLint = (0.693/T _1/2 )×(1/(hepatocyte concentration (0.5×10 ⁶ cells/ml)))×scaling factor (Table 10); Predicted Hepatic CL _H = (QH×Scale-up CL _int ×fub) / (QH + Scale-up CL _int ×fub); ER = Predicted Hepatic CL _H /QH; where QH is the hepatic blood flow (mL/min/kg) in Table 10, and fub is the fraction of unbound drug in plasma, which is assumed to be 1.

Table 10: Scaling Factors for Intrinsic Clearance Prediction in Human, Monkey, Dog, Rat and Mouse Hepatocytes Species Liver microsomal protein Liver weight per kg body weight Scaling Factor Hepatic Blood Flow (mL/min/kg) Per gram of liver Human 40 25.7 1028 twenty one monkey 40 30 1200 44 dog 55 32 1760 31 Rat 61 40 2440 68 Mouse 47 88 4136 90 Note: Scaling Factors = (microsomal protein per gram of liver) × (liver weight per kilogram of body weight).

Experimental results: The above experimental scheme was used to detect and calculate the metabolic stability data of compound H11 and the positive control verapamil in human, monkey, dog, rat and mouse liver microsomes. Table 11 shows the residual percentage change data of compound H11 and verapamil in human, monkey, dog, rat and mouse liver microsomes over time. Table 12 shows the metabolic stability data of compound H11 and verapamil in human, monkey, dog, rat and mouse liver microsomes.

Table 11: Data on the percentage of compound H11 and verapamil remaining in human, monkey, dog, rat and mouse liver microsomes over time Compound Species Whether to add cofactors Remaining percentage (%) 0.5 minutes 5 minutes 15 minutes 30 minutes 60 minutes Verapamil people Add NADPH 100.00 40.59 9.10 2.75 BLOD No NADPH added 100.00 - - - 104.85 monkey Add NADPH 100.00 5.34 BLOD BLOD BLOD No NADPH added 100.00 - - - 104.77 dog Add NADPH 100.00 31.34 5.52 0.66 BLOD No NADPH added 100.00 - - - 93.24 Rat Add NADPH 100.00 27.74 2.94 BLOD BLOD No NADPH added 100.00 - - - 103.79 Mouse Add NADPH 100.00 10.79 BLOD BLOD BLOD No NADPH added 100.00 - - - 98.85 H11 people Add NADPH 100.00 86.00 63.82 43.87 26.14 No NADPH added 100.00 - - - 100.86 monkey Add NADPH 100.00 46.01 13.27 0.59 0.09 No NADPH added 100.00 - - - 105.87 dog Add NADPH 100.00 90.20 71.99 52.53 32.12 No NADPH added 100.00 - - - 92.14 Rat Add NADPH 100.00 85.67 56.17 29.55 13.20 No NADPH added 100.00 - - - 103.20 Mouse Add NADPH 100.00 85.55 55.03 36.05 16.85 No NADPH added 100.00 - - - 93.32 Note: BLOD means below the limit of detection.

Table 12: Metabolic stability data of compound H11 and verapamil in liver microsomes of humans, monkeys, dogs, rats and mice Compound Species In vitro half-life t _1/2 (min) In vitro intrinsic clearance CL _int (μL/min/mg) Scale-up CL _int (mL/min/Kg) Predicted Hepatic CL _H (mL/min/kg) Hepatic Extraction Ratio (ER) Verapamil people 3.46 400.78 412.00 19.98 0.95 monkey 1.06 1304.60 1565.52 42.80 0.97 dog 2.69 515.70 907.63 29.98 0.97 Rat 2.43 569.96 1390.69 64.83 0.95 Mouse 1.40 989.53 4092.70 88.06 0.98 H11 people 31.01 44.71 45.97 14.41 0.69 monkey 5.06 274.95 329.94 38.81 0.88 dog 36.33 38.16 67.17 21.21 0.68 Rat 20.11 68.99 168.33 48.43 0.71 Mouse 23.30 59.52 246.18 65.90 0.73

Experimental conclusion: The remaining percentage of the positive control verapamil during the incubation process decreased significantly with the extension of the incubation time (Table 12). The data results are consistent with the characteristics of the positive drug. Therefore, the activity of the liver microsomes in this experiment is normal, and the experimental results for the detection of drug H11 are credible.

The experimental results showed that the intrinsic clearance of H11 after incubation in human, monkey, dog, rat and mouse liver microsomes was 44.71μL/min/10 ⁶ , 274.95μL/min/10 ⁶ , 38.16μL/min/10 ⁶ , 68.99μL/min/10 ⁶ and 59.52μL/min/10 ⁶ cells, respectively. H11 had a higher clearance rate in monkey liver microsomes and poorer stability; it had moderate clearance in human, dog, rat and mouse liver microsomes and had a certain degree of stability.

4.2 Plasma stability study of compound H11

Experimental plan: (i) Solution preparation Prepare a 1 mM working solution of the test compound in DMSO. Prepare a 1 mM working solution of propantheline in acetonitrile. Prepare a 1 mM working solution of lovastatin in DMSO. Propantheline is used as a positive control for plasma stability tests in humans, monkeys, dogs and mice. Lovastatin is used as a positive control for rat plasma stability tests. (ii) Experimental process A. Add 398 µL of plasma per cell to the culture plate and preheat the culture plate at 37°C for 15 minutes. B. After pre-incubation, add 2 µL of working solution (test solution or control solution) to the above 398 µL plasma, and the final concentration is 5 µM. The final concentration of organic solvent was 0.5%. The test was performed in duplicate. C. The reaction samples were incubated at 37°C. D. 50 µL was extracted from the reaction samples at 0, 15, 30, 60 and 120 min. The reaction was stopped by adding 450 µL of cold acetonitrile as internal standard. E. All samples were vortexed for 10 min and then centrifuged at 3,220 g for 30 min to precipitate the protein. 100 µL of the supernatant was transferred to a new plate. Based on the LC-MS signal response and peak shape, the supernatant was diluted with ultrapure water. (III) Data analysis Samples were analyzed by LC-MS/MS and all calculations were performed using Microsoft Excel. Peak area ratios were determined from the extracted ion chromatograms. (1) The percentage of the remaining compound at each time point is calculated by the following formula: Remaining percentage t min (%) = peak area ratio t min / peak area ratio 0 min × 100, where peak area ratio t min is the peak area ratio of the control compound to the test compound at t min, and peak area ratio 0 min is the peak area ratio of the control compound to the test compound at time zero. (2) The in vitro half-life (in vitro t _1/2 ) is determined by the slope value: t _1/2 = -0.693/k, where the slope value k is determined by the natural logarithm linear regression of the parent drug remaining percentage and incubation time curve.

Experimental results: The above experimental scheme was used to detect and calculate the metabolic stability data of compound H11 and positive reference substances propantheline and lovastatin in the plasma of humans, monkeys, dogs, rats and mice. Table 13 shows the residual percentage change data of compound H11 and positive reference substances propantheline and lovastatin in the plasma of humans, monkeys, dogs, rats and mice over time.

Table 13: Data on the percentage changes of compound H11, propantheline and lovastatin in plasma over time Compound Species Remaining percentage (%) t _1/2 (min) 0 minutes 15 minutes 30 minutes 60 minutes 120 minutes Propantheline people 100.00 81.24 59.99 32.76 7.75 36.64 monkey 100.00 90.53 77.57 70.39 49.22 121.02 dog 100.00 101.04 87.98 85.64 66.64 202.71 Lovastatin (Mevinolin) Rat 100.00 5.27 0.52 0.45 0.34 3.53 Propantheline Mouse 100.00 69.89 43.11 15.28 2.03 21.83 H11 people 100.00 95.74 94.17 102.77 99.46 ∞ monkey 100.00 100.61 92.99 103.66 98.48 ∞ dog 100.00 104.14 103.37 102.42 103.04 ∞ Rat 100.00 108.59 105.39 100.74 109.53 ∞ Mouse 100.00 102.19 96.83 104.70 97.53 ∞

Experimental conclusion: The experimental data of the positive control propantheline and lovastatin are consistent with the characteristics of the positive drug. Therefore, the activity of the different plasmas used in this experiment is normal, and the experimental results of the test drug H11 are credible.

Research data show that H11 is stable in the plasma of mice, rats, dogs, monkeys, and humans when incubated at 37°C for at least 2 hours, with no apparent metabolism.

5. Maximum tolerance dose (MTD) experiment

Experimental process: Mice were divided into groups and administered drugs according to the research scheme in Table 14 below. The drug concentration and dosage levels are shown in Table 14. On days 1 to 5 and days 14 to 19, different dosage levels of vehicle control or compound H11 were intraperitoneally injected, and the drug volume was 10 ml/kg. Animals in groups 1, 3, and 4 were killed and autopsied on day 22, and animals in group 2 were killed and autopsied on day 16. Table 14: Research scheme Group Handling of medicines Dosage level (mg/kg/day) Concentration (mg/ml) Number of mice (male/female) 1 Blank carrier ^a / / 2/2 2 H11 80 8 3/3 3 H11 160 16 3/3 4 H11 320 32 3/3 Note: a. Conventional 10% polyoxyethylene (35) castor oil and 10% ethanol are dissolved in 5% glucose injection.

Parameters assessed included cage observations, detailed clinical observations and postdose observations (approximately 1-2 hours after dosing), body weight and weight change, food intake, and gross pathology.

Experimental results: (1) Animal mortality During the experiment, all experimental animals survived until the planned autopsy. (2) Clinical symptoms No abnormal clinical manifestations related to the compound were observed. (3) Effects on animal body weight and food intake No abnormal changes in body weight and food intake related to the compound were observed. (4) Pathological examination results Compound-related pathological examinations mainly include splenomegaly at ≥160mg/kg, and white changes in the renal capsule in the 320mg/kg dose group. No other abnormalities were observed.

Experimental conclusion: Under the experimental conditions, the mice were intraperitoneally injected with compound H11 once a day for 5 consecutive days, for a total of two rounds. The animals were safe. Except for the splenomegaly and renal capsule changes in the 320 mg/kg dose group found in the autopsy, no other abnormalities were found. Therefore, the maximum tolerated dose under the experimental conditions can reach 320 mg/kg (intraperitoneal injection, once a day, for 5 consecutive days), while the literature reported that the MTD of gemcitabine is 120 mg/kg (intraperitoneal injection, once every 3 days, for 4 consecutive times) (Cancer Chemotherapy and Pharmacology 38(4):335-42). Obviously, as a prodrug of gemcitabine, H11 has a larger tolerated dose than gemcitabine and is safer.

VI. Study on the drug resistance mechanism of compound H11 and gemcitabine

Gemcitabine (dFdC) is hydrophilic and is transported into cells through various nucleoside transporters (NTs), including human concentrating nucleoside transporters (hCNTs) and human equilibrative nucleoside transporters (hENTs). It is known that gemcitabine is transported into cells through five human nucleoside transporters, hCNT1, hCNT2, hCNT3, and hENT1 and hENT2, among which hENT1 protein can bidirectionally transport gemcitabine into or out of cells to maintain the concentration balance of gemcitabine in cells (Reference 1, Reference 2).

Studies have shown that the loss of hENT1 protein significantly inhibits the transport of gemcitabine into cells, and low expression of hENT1 protein can serve as a clinical indicator of poor prognosis for patients treated with gemcitabine (Reference 3).

After entering the cell, gemcitabine is phosphorylated into gemcitabine monophosphate (dFdCMP) by deoxycytidine kinase (dCK), and then gradually phosphorylated into gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) by pyrimidine nucleoside monophosphate kinase (NMPK, also known as UMP/CMP) and nucleoside diphosphate kinase (NDPK), and finally enters the cell nucleus to inhibit DNA synthesis. In the above-mentioned gemcitabine activation process, the dCK-mediated formation of monophosphorylated dFdCMP from dFdC is the main rate-limiting step, which determines the subsequent phosphorylation of gemcitabine and whether it can be completely activated, and finally enters the cell nucleus to exercise the function of inhibiting DNA synthesis (Reference 1, Reference 4, Reference 5, Reference 6).

At the same time, a large amount of gemcitabine that enters the cell will be deaminated by cytidine dehydrogenase (CDA) to form inactive 2’,2’-difluorodeoxyuridine (dFdU) and be excreted from the cell membrane.

In summary, due to the above metabolic characteristics of gemcitabine, it may produce drug resistance in the process of inhibiting tumor cell proliferation. The drug resistance of tumor cells to gemcitabine mainly involves three aspects: 1. The transport function of hENT1 protein on gemcitabine entering cells; 2. The rate-limiting process of dCK-mediated phosphorylation of dFdC to form dFdCMP; 3. CDA-mediated deamination of gemcitabine.

The series of compounds developed by the present invention are small molecule prodrugs of gemcitabine. According to the experiment in Example 2, it can be seen that the prodrug is specifically activated by the AKR1C3 enzyme.

In order to further study whether the series of gemcitabine small molecule prodrugs developed by the present invention that are specifically activated by the AKR1C3 enzyme also have similar metabolic pathways or drug resistance as gemcitabine, the inventors further conducted the following mechanism studies: 1. Using the nucleoside transporter inhibitor Dipyridamole to conduct a cytotoxic study of the nucleoside transporter (NT)-dependent cytotoxicity of compound H11 and gemcitabine in H460 cells; 2. Using the dCK inhibitor 2’-deoxycytidine to conduct a dCK-dependent cytotoxicity study of compound H11 and gemcitabine in H460 cells; 3. Tetrahydrouridine, an inhibitor of cytidine dehydrogenase (CDA), was used to study the CDA-dependent cytotoxicity of compound H11 in H460 cells.

6.1 Study on the cytotoxicity of compound H11 and gemcitabine in H460 cells in a nucleoside transporter-dependent manner

Referring to the combined drug and single drug operation process in the above Example 2.1, the in vitro proliferation inhibition effect of compound H11 and gemcitabine alone or in combination with the nucleoside transporter inhibitor Dipyridamole (CAS No.: 58-32-2) was tested. The specific _IC50 results are shown in Table 17 below, and the inhibition curve is shown in Figure 6.

Table 17: In vitro proliferation inhibition test results of compound H11 and gemcitabine alone or in combination with nucleoside transporter inhibitor Dipyridamole Test compound IC ₅₀ (nM)/fold difference Give medicine alone + 4 µM Dipyridamole + 8 µM Dipyridamole + 16 µM Dipyridamole Gemcitabine 13.39/1 250.9/18.74 340.3/25.41 518.5/38.72 H11 33.79/1 11.98/0.35 12.66/0.37 12.71/0.38

In addition, the applicant's single-drug test of Dipyridamole on H460 cells found that the IC ₅₀ value was around 30µM, which was quite different from the above-mentioned combination concentration. Moreover, as shown in Figure 6, the inhibition rate curve hardly changed after the combination of different concentrations of Dipyridamole, which means that the above-mentioned concentration of Dipyridamole has almost no effect on the proliferation of H460 cells.

The above experimental results show that in H460, gemcitabine exhibits nucleoside transporter-dependent cytotoxicity, while compound H11 exhibits non-nucleoside transporter-dependent cytotoxicity. Gemcitabine needs to rely on the transport of nucleoside transporters to enter cells and exert anti-tumor effects, while H11 does not rely on nucleoside transporters to enter cells and exert anti-tumor effects.

The series of gemcitabine prodrug compounds developed by the present invention are all gemcitabine phosphate prodrugs formed by connecting the nitrobenzyl group and the hydroxymethyl group on the gemcitabine molecule through a phosphate group. Therefore, the series of gemcitabine prodrugs developed by the present invention are likely to overcome the drug resistance of gemcitabine caused by the impaired transport function of nucleoside transporters.

6.2 Deoxycytidine kinase (dCK)-dependent cytotoxicity studies of compound H11 and gemcitabine in H460 cells

Referring to the combined drug and single drug operation procedures in the above Example 2.1, the in vitro proliferation inhibition effect of compound H11 and gemcitabine alone or in combination with the deoxycytidine kinase (dCK) inhibitor 2'-deoxycytidine (CAS No.: 951-77-9) was tested. The specific _IC50 results are shown in Table 18 below, and the inhibition curve is shown in Figure 7.

Table 18: In vitro proliferation inhibition test results of compound H11 alone or in combination with gemcitabine and deoxycytidine kinase inhibitor 2'-deoxycytidine Test compound IC ₅₀ (nM) Fold Difference Give medicine alone + 10 µM 2'-deoxycytidine Gemcitabine 16.63 1316 79.09 H11 45.68 68.43 1.5

In addition, the applicant tested the IC ₅₀ value of 2'-deoxycytidine on H460 cells as a single drug, which was around 600µM, which was significantly different from the above-mentioned combination concentration, meaning that the above-mentioned concentration of Dipyridamole had almost no effect on H460 cell proliferation.

The above experimental results show that in H460, gemcitabine exhibits dCK-dependent cytotoxicity, while compound H11 exhibits dCK-independent cytotoxicity. This further indicates that the activation product of H11 can avoid the dCK-mediated phosphorylation process, effectively reducing the phosphorylation step of the H11 activation product.

The series of gemcitabine prodrug compounds developed by the present invention are all gemcitabine phosphate prodrugs formed by connecting nitrobenzyl and hydroxymethyl on gemcitabine molecules through phosphate groups, and can directly release gemcitabine monophosphate (dFdCMP) through AKR1C3 enzyme activation, thereby effectively improving the specific tumor targeted killing ability while avoiding the rate-limiting process of dCK-mediated phosphorylation of dFdC to form dFdCMP. Therefore, the series of gemcitabine prodrug compounds developed by the present invention can increase the specific targeted killing ability of tumor cells through AKR1C3 highly expressed in tumor cells, and can effectively overcome the drug resistance of tumor cells to gemcitabine.

6.3 Cytotoxicity study of compound H11 in H460 cells in a cytidine dehydrogenase (CDA)-dependent manner

Referring to the combined drug and single drug operation procedures in the above Example 2.1, the in vitro proliferation inhibition effect of compound H11 and gemcitabine alone or in combination with cytidine dehydrogenase (CDA) inhibitor Tetrahydrouridine (CAS No.: 18771-50-1) was tested, and the specific IC ₅₀ results are shown in Figure 8 below.

In addition, the applicant's single-drug test of 2'-deoxycytidine on H460 cells found that the IC ₅₀ value was above 900µM, which is significantly different from the above-mentioned combination concentration, meaning that the above-mentioned concentration of Dipyridamole has almost no effect on H460 cell proliferation.

The above experimental results show that in H460, compound H11, like gemcitabine, exhibits non-CDA-dependent cytotoxicity. CDA cannot deaminate the activation product of H11, and therefore cannot inhibit the cytotoxicity of H11. The series of gemcitabine prodrug compounds developed by the present invention release the same activation products after being specifically activated by the AKR1C3 enzyme. Therefore, the series of gemcitabine prodrugs developed by the present invention will not be inhibited by CDA and can exert anti-tumor effects.

The above research results all indicate that the series of gemcitabine prodrug compounds developed by the present invention are small molecule prodrugs specifically activated by AKR1C3, and their cancer cell proliferation inhibition activity depends on the expression level of AKR1C3 (including RNA and protein).

In the process of highly specific tumor killing, it can effectively avoid multiple steps of gemcitabine inactivation in cells. In other words, the compound provided by the present invention can increase the specific targeted killing ability of tumor cells through AKR1C3 highly expressed in tumor cells, and at the same time has the possibility of effectively overcoming the drug resistance of tumor cells to gemcitabine.

Furthermore, animal models (PDX) also preliminarily demonstrated that the H11 compound has a pancreatic cancer treatment effect that is superior to or equivalent to gemcitabine.

In terms of safety, as a prodrug of gemcitabine, H11 has a larger tolerable dose than gemcitabine and is safer: the MTD of the H11 compound is 320 mg/kg, while that of gemcitabine is 120 mg/kg.

In terms of stability, experiments using human liver microsomes and blood showed that H11 has good stability and has the potential to be developed into a human drug.

VII. Synthesis of Compounds

The reagents and instruments used are explained as follows: DCM, dichloromethane; TEA, triethylamine; TFA, trifluoroacetic acid; LCMS, liquid chromatography-mass spectrometry. Chemical reagents and drugs whose sources are not specified in the synthesis process are all analytically pure or chemically pure and are purchased from commercial reagent companies. Other English abbreviations that appear are subject to the explanations in the field of organic chemistry.

Synthesis of compound H11

Under N ₂ protection, H11-A1 (6.0 g, 12.9 mmol) and POCl ₃ (1.2 mL, 12.9 mmol) were added to dry DCM (60 mL), and a solution of TEA (2.2 mL, 15.5 mmol) in DCM (10 mL) was added dropwise at -10 ^° C and stirred. H11-A1 was purchased or synthesized according to other research literature or patents (PCT application number PCT/US2016/025665, publication number WO2016/161342A1, corresponding to Chinese application number 201680020013.2, publication number CN108136214A).

After a period of time, the reaction was complete, and a DCM solution of H11-A2 (4.6 g, 12.9 mmol, synthesized according to reference or purchased) and TEA (2.2 mL, 15.5 mmol) was added dropwise and stirred.

Add tetrahydrofuran solution of isopropylamine (2 M, 13.2 mL, 25.9 mmol) dropwise to the system, then add TEA (5.4 mL, 10.1 mmol) and stir at room temperature. Add saturated NH ₄ Cl solution dropwise, filter, separate the filtrate, extract the aqueous phase with DCM, wash the organic phase with brine, dry over Na ₂ SO ₄ , concentrate and analyze by reverse phase flash column chromatography to obtain H11-D (2.0 g (85% content or more) and 2.0 g (50% content), total yield 25.2%) as a light yellow solid. MS: Calculated 923.8, found 924.2 ([M+H] ⁺ ).

Under _N2 protection, H11-D (1.5 g, 1.62 mmol) was added to DCM (15 mL), TFA (18.7 g, 165.2 mmol) was added, and the reaction was monitored for completion after stirring at room temperature. The pH was adjusted to 8 with saturated _NaHCO3 aqueous solution, extracted with DCM, the organic phase was washed with brine, dried with _{Na2SO4 ,} concentrated, and separated by HPLC neutral preparation (the mobile phase used was adjusted to neutral by adding acid or base) to obtain H11 (550 mg, yield of 47.0%) as a white solid.

¹ H-NMR (400 MHz, CD ₃ OD) δ 8.04 (dd, J = 8.4, 3.6 Hz, 1H), 7.60-7.48 (m, 4H), 7.39 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.18 (t, J = 8.8 Hz, 2H), 6.94-6.91 (m, 2H), 6.26-6.13 (m, 1H), 5.84 (dd, J = 7.6, 4.4 Hz, 1H), 5.12-5.09 (m, 2H), 4.34-4.17 (m, 3H), 4.04-4.01 (m, 1H), 3.37-3.31 (m, 1H), 1.15-1.12 (m, 6H). MS: Calculated MS, 723.2, found, 724.0 ([M+H] ⁺ ).

Synthesis of other compounds

Similar to the method for synthesizing compound H11, isopropylamine is replaced with organic amines of other structures, such as methylamine, ethylamine, n-propylamine, 2-bromoethylamine, isobutylamine, 2-methyl-2-propylamine, 2,2-dimethyl-1-propylamine, benzylamine, cyclohexylamine, cyclopentylamine, cyclobutylamine, cyclopropylamine, 2,2,2-trifluoroethylamine; H11-A2 is replaced with other corresponding raw materials, as shown in the following structure: , wherein the substituents R ₂ , R ₆ , R ₇ and The structure corresponds.

Other similar compounds can be obtained by referring to the synthesis steps of compound H11 above. The spectral data of other compounds and H11 are given below:

Compound H2 was prepared and separated by HPLC neutral chromatography to obtain the product (27.6 mg, 11.7%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD)δ8.06-8.03(m,1H),7.55-7.49(m,4H),7.42-7.39(m,1H),7.31(s,1H),7.21-7.15(m,2H),6.95-6.91(m,2H),6.23-6.17(m,1H ),5.85-5.83(m1H),5.12-5.10(m,2H),4.34-4.16(m,3H),4.04-4.02(m,1H),2.97-2.89(m,2H),1.13-1.08(m,3H).MS:Calculated 709.5,found 710.2([M+H] ⁺ ).

Compound H3 was prepared and separated by HPLC neutral chromatography to obtain the product (71 mg, 29.2%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.06-8.03(m,1H),7.56-7.41(m,5H),7.31(s,1H),7.18(t, J =8.8Hz,2H),6.94-6.91(m,2H),6.23-6.17(m,2H),5.85-5.82(m,2H),5.12-5.10(m,2H),4.34-4.21 (m,4H),4.03-4.02(m,1H),2.87-2.81(m,2H),1.51-1.45(m,2H),0.90-0.86(m,3H).MS:Calculated 723.2,found 724.0([M+H] ⁺ ).

Compound H4 was prepared and separated by HPLC neutral chromatography to obtain the product (53.2 mg, 16.7%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.04 (dd, J =8.4,3.2Hz,1H),7.61-7.46(m,4H),7.42-7.40(m,1H),7.32(s,1H),7.20-7.16(m,2H),6.94-6.91(m,2H),6.24-6.20(m,1H),5.87(d, J =7.6Hz,1H),5.14(d, J =8.4Hz,2H),4.38-4.18(m,3H),4.06-4.03(m,1H),3.42-3.38(m,2H),3.28-3.25(m,2H).MS:Calculated 787.1,found 788.0 and 790.0([M+H] ⁺ ).

Compound H6 was prepared and separated by HPLC to obtain the product (17.1 mg, 13.2%) as a white solid. ¹ H-NMR (300MHz, CD ₃ OD) δ8.07(d, J =8.7Hz,1H),7.69(d, J =8.7Hz,2H),7.61-7.58(m,1H),7.45(d, J =8.2Hz,1H),7.30(s,1H),7.16(d, J =8.1Hz,2H),6.23-6.22(m,1H),5.85(d, J =7.5Hz,1H),5.11(d, J =8.4Hz,2H),4.32-4.20(m,Hz,3H),4.06-4.05(m,1H),2.96-2.90(m,2H),1.11(t,J=6.7Hz,3H).MS:Calculated 665.1,found 666.0([M+H] ⁺ ).

Compound H7 was prepared and separated by HPLC (50.0 mg, 15.7%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.03 (dd, J =8.4, 3.2Hz, 1H), 7.59-7.41 (m, 5H), 7.32 (s, 1H), 7.18 (t, J =8.8Hz,2H),6.93-6.91(m,2H),6.21-6.16(m,1H),5.86-5.84(m,1H),5.11(d, J =8.4Hz,2H),4.30-4.24(m,3H),4.05-4.0(s,1H),2.72-2.68(m,2H),1.74-1.61(m,1H),0.88-0.86(m,6H).MS:Calculated 737.2,found 738.2([M+H] ⁺ ).

Compound H8 was prepared and separated by HPLC neutral chromatography to obtain the product (49.5 mg, 15.3%) as a white solid. ¹ H-NMR (400 MHz, CD ₃ OD) δ8.00 (dd, J =8.4, 3.2 Hz, 1H), 7.56-7.43 (m, 4H), 7.33-7.14 (m, 9H), 6.92-6.88 (m, 2H), 6.21-6.16 (m, 1H), 5.80-5.78 (m, 1H), 5.05-5.02 (m, 2H), 4.34-4.00 (m, 6H). MS: Calculated 771.2, found 772.1 ([M+H] ⁺ ).

Compound H9 was prepared and separated by HPLC neutral chromatography to obtain the product (80.0 mg, yield: 25.5%) as a white solid. ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.03 (dd, J = 8.4, 3.2 Hz, 1H), 7.67 (d, J = 8.4 Hz, 2H), 7.54 (dd, J = 16.0, 7.6 Hz, 1H), 7.37-7.35 (m, 1H), 7.31 – 7.20 (m, 6H), 7.12 (d, J = 8.0 Hz, 2H), 6.22 – 6.17 (m, 1H), 5.80 (t, J = 8.0 Hz, 1H), 5.08 -5.02 (m, 2H), 4.35 -4.10 (m, 3H), 4.10-4.01 (m, 3H). MS: Calculated MS, 727.1, found, 728.1 ([M+H] ⁺ ).

Compound H10 was prepared and separated by HPLC neutral chromatography to obtain the product (65.1 mg, 21.3%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.06 (dd, J =8.4, 3.2Hz, 1H), 7.69 (d, J =8.7Hz, 2H), 7.61-7.60 (m, 1H), 7.44 (d, J =8.5Hz, 1H), 7.31 (s, 1H), 7.15 (d, J =8.4Hz,2H),6.23-6.19(m,1H),5.86(dd, J =7.6,2.4Hz,1H),5.11(d, J =8.0Hz,2H),4.35-4.16(m,3H),4.05-4.04(m,1H),2.72-267m,2H),1.73-1.62(m,1H),0.89-0.86(m,6H).MS:Calculated 693.2, found 694.1([M+H] ⁺ ).

Compound H11 was prepared and separated by HPLC neutral chromatography to obtain the product (550 mg, 47.0 %) as a white solid. ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.04 (dd, J = 8.4, 3.6 Hz, 1H), 7.60-7.48 (m, 4H), 7.39 (d, J = 8.8 Hz, 1H), 7.31 (s, 1H), 7.18 (t, J = 8.8 Hz, 2H), 6.94-6.91 (m, 2H), 6.26-6.13 (m, 1H), 5.84 (dd, J = 7.6, 4.4 Hz, 1H), 5.12-5.09 (m, 2H), 4.34-4.17 (m, 3H), 4.04-4.01 (m, 1H), 3.37-3.31 (m, 1H), 1.15-1.12 (m, 6H).MS: Calculated MS, 723.2, found, 724.0 ([M+H] ⁺ ).

Compound D1 was prepared and separated by HPLC to obtain the product (15 mg, yield: 9.6%) as a white solid. ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.05 (dd, J = 8.4, 6.4 Hz, 1H), 7.61-7.50 (m, 4H), 7.45-7.40 (m, 1H), 7.30 (d, J = 2.0 Hz, 1H), 7.18 (t, J = 8.8 Hz, 2H), 6.96-6.90 (m, 2H), 6.27-6.16 (m, 1H), 5.88-5.83 (m, 1H), 5.19 (dd, J = 8.8, 2.4 Hz, 2H), 4.43-4.30 (m, 2H), 4.28-4.13 (m, 3H), 4.07-4.02 (m, 1H), 1.38-1.27 (m, 3H). MS: Calculated MS, 710.1 found, 710.7 ([M+H] ⁺ ).

Compound D2 was prepared and separated by HPLC neutral chromatography to obtain the product (4.5 mg, 4.1%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.06-8.03(m,1H),7.56-7.39(m,5H),7.28(s,1H),7.17(t, J =8.8Hz,2H),6.95-6.92(m,2H),6.23-6.15(m,1H),5.85(t, J =7.2Hz,1H),5.12-5.05(m,2H),4.31-4.14(m,3H),4.05-4.03(m,1H),3.12-3.06(m,4H),1.11-107(m,6H).MS:Calculated 737.2,found 738.0([M+H] ⁺ ).

Compound D3 was prepared and separated by HPLC neutral chromatography to obtain the product (6.6 mg, yield 21.1%) as a white solid. ¹ H-NMR (300MHz, CD ₃ OD) δ8.05 (d, J =7.8Hz, 1H), 7.61-7.39 (m, 5H), 7.30 (s, 1H), 7.18 (t, J =8.7Hz,2H),6.95-6.92(m,2H),6.25-6.16(m,1H),5.86-5.82(m,1H),5.10(d, J =8.7Hz,2H),4.28-4.14(m,3H),4.04(m,1H),2.68(d, J =10.2Hz,6H).MS:Calculated 709.2,found 710.0([M+H] ⁺ ).

Compound D4 was prepared and separated by HPLC (14.2 mg, 6.9%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.05(t, J =8.0Hz,1H),7.60-7.47(m,4H),7.40(t, J =7.2Hz,1H),7.30(d, J =4.4Hz,1H),7.18(t, J =8.8Hz,2H),6.96-6.92(m,2H),6.22-6.20(m,1H),5.87-5.84(m,1H),5.24-5 .20(m,2H),4.55-4.33(m,2H),4.24–4.15(m,1H),4.05-4.03(m,1H),2.22(d, J =16.0Hz,4H).MS:Calculated 707.2,found 707.9([M+H] ⁺ ).

Compound D5 was prepared and separated by HPLC neutral chromatography to obtain the product (53.5 mg, 26.7%) as a white solid. ¹ H-NMR (400MHz, CD ₃ CD3OD) δ8.07(dd, J =8.4,3.2Hz,1H),7.70(d, J =8.8Hz,2H),7.60(t, J =7.2Hz,1H),7.45(d, J =8.4Hz,1H),7.29(s,1H),7.17(d, J =7.6Hz,2H),6.25-6.18(m,1H),5.87-5.84(m,1H),5.12-5.08(m,2H),4.30-4.23(m,3H),4.06-4.05(m,1H),2.70-2.66(m,6H).MS:Calculated 665.1,found 666.0([M+H] ⁺ ).

Compound D6 was prepared and separated by HPLC neutral chromatography to obtain the product (45.0 mg, 19.1%) as a white solid. ¹ H-NMR (400 MHz, CD ₃ OD) δ8.04-8.00 (m, 1H), 7.55-7.16 (m, 13H), 6.92-6.97 (m, 2H), 6.24-6.17 (m, 1H), 5.80-5.76 (m, 1H), 5.32-5.28 (m, 2H), 4.56-4.42 (m, 2H), 4.23-4.15 (m, 1H), 4.07-4.05 (m, 1H). MS: Calculated 758.1, found 759.1 ([M+H] ⁺ ).

Compound D7 was prepared and separated by HPLC (92.0 mg, 29.3%) as a white solid. ¹ H-NMR (400MHz, CD ₃ OD) δ8.04(dd, J =8.4,2.8Hz,1H),7.67(d, J =8.8Hz,2H),7.43-7.33(m,4H),7.25-7.17(m,4H),7.12(d, J =8.0Hz,2H),6.23-6.16(m,1H),5.78(t, J =8.0Hz,1H),5.30(dd, J =9.2,6.4Hz,2H),4.56-4.43(m,2H),4.23-4.16(m,1H),4.07-4.06(m,1H).MS:Calculated 714.1,found 715.0([M+H] ⁺ ).

Compound D8 was unstable after synthesis and was not tested.

without

FIG1 shows the inhibition rate curve of different concentrations of gemcitabine on H460 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021, wherein the horizontal coordinate conc.Log(nM) represents the logarithm value of the concentration value in nmol/L with base 10 as the base, and the vertical coordinate inhibition% represents the inhibition rate percentage.

FIG2 shows the inhibition rate curves of H460 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021 at different concentrations of compound H11, wherein the horizontal coordinate conc.Log(nM) represents the logarithm of the concentration value in nmol/L with base 10 as the unit, and the vertical coordinate inhibition% represents the inhibition rate percentage.

FIG3 shows the inhibition rate curves of HepG2 cancer cells in the presence or absence of AKR1C3 inhibitor AST-3021 at different concentrations of compound H11, wherein the horizontal coordinate conc.Log(nM) represents the logarithm of the concentration value in nmol/L with base 10 as the base, and the vertical coordinate inhibition% represents the inhibition rate percentage.

FIG4 is a curve showing the changes in tumor volume on different days in the in vivo efficacy experiment of compound H11 and gemcitabine in the pancreatic cancer PA1222 model.

FIG5 is a curve showing the weight change rate of mice on different days in the in vivo efficacy experiment of compound H11 and gemcitabine in the pancreatic cancer PA1222 model.

FIG6 shows the inhibition rate curves of compound H11 and gemcitabine alone or in combination with a nucleoside transporter inhibitor on H460 cancer cells, wherein the horizontal coordinate conc.Log(nM) represents the logarithm of the concentration value in nmol/L with base 10 as the base, and the vertical coordinate inhibition% represents the inhibition rate percentage.

FIG7 is a curve showing the inhibition rate of compound H11 and gemcitabine alone or in combination with a deoxycytidine kinase inhibitor on H460 cancer cells, wherein the horizontal coordinate conc.Log(nM) represents the logarithm of the concentration value in nmol/L with base 10 as the base, and the vertical coordinate inhibition% represents the inhibition rate percentage.

FIG8 is a curve showing the inhibition rate of compound H11 and gemcitabine alone or in combination with a cytidine dehydrogenase inhibitor on H460 cancer cells, wherein the horizontal coordinate conc.Log(nM) represents the logarithm of the concentration value in nmol/L with base 10 as the base, and the vertical coordinate inhibition% represents the inhibition rate percentage.

Claims (15)

A gemcitabine anticancer derivative having structural formula III, or a pharmaceutically acceptable salt or prodrug or solvate or isotopic variant thereof: (III) wherein _R1 is selected from H, halogen, It means that _R1 can replace any 4 positions on the benzene ring and the number of replacements is 1, 2, 3 or 4; _R2 is selected from phenyl, halogen-substituted phenyl, C1-C3 alkyl or halogen-substituted C1-C3 alkyl; and _R3 is selected from C1-C6 alkyl or halogen-substituted C1-C6 alkyl, benzyl. The gemcitabine anticancer derivative as described in claim 1, wherein: R ₁ is selected from H or mono-substituted F; or R ₂ is selected from phenyl, fluorophenyl, methyl or trifluoromethyl; or R ₃ is selected from methyl, ethyl, propyl, butyl, benzyl, or bromo-substituted methyl, ethyl, propyl, butyl; or R ₁ is selected from H or mono-substituted F, R ₂ is selected from phenyl, fluorophenyl, methyl or trifluoromethyl, and R ₃ is selected from methyl, ethyl, propyl, butyl, benzyl, or bromo-substituted methyl, ethyl, propyl, butyl. The gemcitabine anticancer derivative as described in claim 1 is selected from gemcitabine anticancer derivatives having the following structural formula, or pharmaceutically acceptable salts or prodrugs or solvates or isotopic variants thereof: (H11). The anticancer derivative of gemcitabine as described in claim 1, wherein, the salt is an alkaline salt or an acidic salt, preferably an ammonium salt, a carbonate, a sulfate, a phosphate, or a hydrochloride; the solvent complex is a hydrate or an alcohol complex, and the alcohol complex is an ethanol complex; the prodrug is selected from an ester or an amide. A medicine or preparation containing the gemcitabine anticancer derivative or a pharmaceutically acceptable salt or prodrug or solvate or isotope variant thereof as described in any one of claims 1 to 4. Use of a gemcitabine anticancer derivative or a pharmaceutically acceptable salt or prodrug or solvate or isotope variant thereof as described in any one of claims 1 to 4 in the preparation of a medicament for treating cancer patients or tumor patients or a disease or cell proliferative disease caused by cancer or tumor. The drug or preparation as described in claim 5 is used to treat a patient's tumor or cancer disease, wherein the tumor or cancer includes: Lung cancer, non-small cell lung cancer, liver cancer, pancreatic cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, melanoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, bone cell carcinoma, epithelial carcinoma, bile duct carcinoma, choriocarcinoma, embryonal carcinoma, spermatogonial carcinoma, Wilms' carcinoma, colloid carcinoma, astrocytoma, neuromedulloblastoma, craniopharyngioma, ependymoma, pinealoma, hematoblastoma, vocal cord Neuroma, meningioma, neuroblastoma, optic neuroblastoma, retinoblastoma, neurofibroma, fibrosarcoma, fibroblastoma, fibroma, fibroadenoma, fibrochondroma, fibrocystoma, fibromyxoma, fibroostoma, fibromyxosarc ... tumor, chordoma, chorioadenoma, chorioepithelioma, chorioblastoma, osteosarcoma, osteoblastoma, osteochondrofibroma, osteochondrosarcoma, osteochondroma, osteocystoma, osteodentinoma, osteofibroma, osteofibrosarcoma, angiosarcoma, angiosarcoma, angiolipoma, angiochondroma, angioblastoma, angiokeratoma, angioneuroma, angioendothelioma, angiofibroma, angiomyoma, angiolipoma, angiolymphangioma, angiolipomyoma, angiomyoneuroma, angiomyoma, angiomyoneuroma, angiomyoma, angiomyoneuroma, lymphangiosarcoma, lymphangio blastoma, lymphangioma, lymphoma, lymphomyxoma, lymphosarcoma, lymphangiofibroma, lymphocytoma, lymphoepithelioma, lymphoblastoma, endothelioma, endothelioma, synovioma, synovial sarcoma, mesothelioma, connective tissue tumor, Ewing's tumor, leiomyoma, leiomyosarcoma, leiomyoma, leiomyofibroma, rhabdomyomas, rhabdomyosarcomas, rhabdomyomyxoma, acute lymphatic leukemia, acute myeloid leukemia, anemia of chronic disease, polycythemia vera, lymphoma, endometrial carcinoma, glioma, colorectal cancer, thyroid cancer, urothelial carcinoma, or multiple myeloma. The drug or preparation as described in claim 5, the drug or preparation is used alone or in combination to treat non-small cell lung cancer, pancreatic cancer, ovarian cancer, and breast cancer. The medicament or formulation of claim 5, wherein the cancer or tumor is primary brain cancer, brain tumor, or metastatic cancer or tumor that has metastasized to the brain. A method for preparing a gemcitabine derivative H11, comprising hydrolyzing a compound H11-D under acidic conditions and alkalizing the compound H11-D: . In the preparation method as described in claim 10, the acidic condition is provided by TFA, preferably by introducing H11-D into a mixed system of TFA and DCM. As described in claim 10, the alkalization operation process includes: adding alkali to the reaction system to make the system alkaline, wherein the alkali used includes hydroxides, bicarbonates, and carbonates of alkaline metals, which are added in the form of aqueous solutions, and after hydrolysis, the aqueous solution of the alkali is added to the reaction system at a pH of 8. A preparation method of gemcitabine derivative H11-D comprises the following operations: Operation 1, stirring and reacting H11-A1 with POX ₃ and organic amine; Operation 2, adding H11-A2 and organic amine after the reaction is complete and stirring and reacting; Operation 3, adding isopropylamine and organic amine after the reaction is complete and stirring and reacting; and Operation 4, adding water or acidic salt solution for post-treatment. ; Wherein, X is Cl or Br. The preparation method as described in claim 13, wherein the organic amine in operations one, two and three is triethylamine, and the reaction solvent is dichloromethane. The preparation method as described in claim 13, wherein the isopropylamine added in operation three is a tetrahydrofuran solution thereof, and the acidic salt solution in operation four is a saturated NH ₄ Cl solution.