Anthracene compounds, their preparation methods and pharmaceutical uses

Abstract

This invention relates to anthracene compounds of formula A, their preparation methods, and pharmaceutical uses. These anthracene compounds have the effect of inhibiting tumor cell growth and have the potential to be used as antitumor drugs. The invention also relates to methods for quality control of samples.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to anthracene-like compounds with antitumor activity, their pharmaceutically acceptable salts, and their preparation methods and uses. Background Technology

[0002] With the development of molecular biology techniques and a deeper understanding of pathogenesis at the cellular and molecular levels, the discovery of highly effective and low-toxicity anticancer drugs targeting key enzymes in tumor signaling pathways has become an important research direction. Molecular targeted therapy for tumors differs from traditional tumor treatment methods. It is based on research in tumor molecular biology, using small molecule compounds to block key enzymes in critical pathways of tumor growth, thereby inhibiting tumor cell growth.

[0003] CN102378574A discloses an azoanthracene derivative as shown in Formula B and its use in regulating glucagon-like peptide-1 receptors:

[0004]

[0005] The inventors of this application studied the degradation process of such anthracene compounds and found that they are readily degraded under suitable acidic conditions, and their degradation products are generally more stable. CN102378574A indicates that these azoanthracene derivatives have a regulatory effect on glucagon-like peptide-1 (GLP-1) receptors. Further activity studies on the degradation products of these compounds are necessary to discover their potential pharmaceutical uses. Summary of the Invention

[0006] In one aspect, the present invention provides anthracene compounds as shown in Formula A:

[0007]

[0008] and its pharmaceutically acceptable salts,

[0009] in:

[0010] X is independently selected from oxygen, hydrogen, and nitrogen;

[0011] R1 is selected from COOH or –COOCH3;

[0012] R2 is –Y1–L–Y2;

[0013] L is selected from –(CH2) a – or –O–, where a is 0 or 1;

[0014] Y1 is a phenylene, wherein the phenylene is 1,3-phenylene or 1,4-phenylene;

[0015] Y2 is selected from phenyl, pyridyl, diazaaryl, or imidazolyl, and Y2 is optionally independently converted by hydrogen, halogen, or C. 1-4 Alkyl, C 3-6 Cycloalkyl, CF3, –CH2F, –CH2CF3, CN, –CH2CN, –OCH3, –O–C(CH3)3, –OCH2CH3, –OCH(CH3)2, –OCF3, –OCH2CF3, NH2, –SO2–CH3, –NHCH3, –CO–NHCH3, –NHCH2CH3, –N(CH3)2, –CO–CH3, –CO–NHCH3 are substituted 1-3 times;

[0016] R3 is selected from C 1-4 Alkyl, C 3-6 Cycloalkyl, phenyl, furanyl, thiophene, oxazolyl, pyridyl, thiazolyl, pyrimidinyl, epoxypentacycloyl, epoxyhexaneyl, -sulfonyl-thiazolyl, -sulfonyl-oxazolyl, -sulfonyl-pyridinyl, -carbonyl-oxazolyl, -carbonyl-pyridinyl; optionally independently represented by halogen, amino, OH, COOH, CN, -CH2CN, CF3, -CH2F, -CH2CF, -C(CH3)3, -O-C(CH3)3, -OCH3, C 1-4 Alkyl, C 3-6 Cycloalkyl and phenyl substitutions 1-3 times.

[0017] In some implementations, each X is oxygen independently.

[0018] In some implementations, R1 is COOH.

[0019] In some implementations, L is selected from –(CH2). a – or –O–, where a is 0 or 1. When a is 0, L is a direct linker. When a is 1, L is –CH2–. In some preferred embodiments, L is a direct linker.

[0020] In some implementations, Y1 is 1,4-phenylene.

[0021] In some embodiments, when each X is independently O, R1 is COOH, L is a direct linker, and Y1 is 1,4-phenylene, the compound has a structure as shown in Formula A1:

[0022]

[0023] In some embodiments, when Y2 is phenyl, it may be substituted once with any one of H, F, Cl, CN, –CH2CN, –OCH3, CF3, or CH3.

[0024] In some implementations, when Y2 is pyridyl, it may optionally be H or C. 1-4 The alkyl group, –OCH3, –N(CH3)2, F, CN, NH2, CH2F, –CO–NHCH3, and –CO–CH3 are substituted once or twice. Preferably, Y2 is a pyridyl group substituted twice with a methyl group, more preferably a 4-pyridyl group substituted twice with a methyl group, and even more preferably...

[0025] In some embodiments, R3 is a C substituted with a phenyl group. 1-4 Alkyl; preferably, R3 is 1-phenyl-propyl-, including (R)-1-phenyl-propyl- or (S)-1-phenyl-propyl-.

[0026] The present invention further provides compounds of Formula I: (2S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)propionic acid, and pharmaceutically acceptable salts thereof:

[0027]

[0028] This invention also provides a method for preparing the compound (2S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)propionic acid as shown in Formula I, the method comprising the following steps:

[0029] a) The compound shown in Formula II is hydrolyzed to generate the compound of Formula III, and the specific reaction is as follows:

[0030] as well as

[0031] b) Compound III is reacted to prepare compound I, and the specific reaction is as follows:

[0032]

[0033] In some embodiments, steps a and b are reacted in a corresponding solvent; said solvent is independently selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, acetone, dichloromethane, acetonitrile, methanol, or ethanol.

[0034] In some embodiments, the compound of formula II in step a undergoes a hydrolysis reaction with an acidic reagent; the acidic reagent is selected from boron trifluoride, boron trichloride, aluminum trichloride, or sulfur trioxide, preferably boron trichloride.

[0035] In some implementations, the reaction in step b is carried out in alkaline water.

[0036] In some implementations, the reaction described in step a is followed by a post-processing step before being fed into the next reaction; the post-processing step is selected from one or more of dilution, filtration, pH adjustment, washing, extraction, concentration, crystallization, and drying; the post-processing step may be performed once or multiple times as needed.

[0037] In some implementations, the post-processing of step a is as follows: after obtaining the compound of formula I, the crude product of compound I is obtained by pH adjustment, separation, extraction with 2-methyltetrahydrofuran, and concentration.

[0038] The pharmaceutically acceptable salts described in this invention include acid addition salts and base salts.

[0039] The compounds and pharmaceutically acceptable salts provided by this invention may exist in chiral forms, i.e., S-configuration or R-configuration. The compounds and pharmaceutically acceptable salts provided by this invention may also exist in achiral forms. When the compounds described in this invention are illustrated with one configuration, it also indicates the disclosure of another configuration or achiral form.

[0040] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated, and the auxiliary group is cleaved to provide the desired enantiomer in pure form. Alternatively, when the molecule contains a basic functional group (such as an amine) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to form carbamates).

[0041] The compounds described in this invention include stereoisomers of the compounds. The stereoisomers described in this invention refer to the enantiomers produced when the compound as shown in Formula I contains an asymmetric carbon atom; the cis-trans isomers produced when the compound contains a carbon-carbon double bond or a cyclic structure; and the tautomers produced when the compound contains a ketone or oxime. As a specific embodiment, the stereoisomers described in this invention include, but are not limited to: enantiomers, diastereomers, racemic isomers, cis-trans isomers, tautomers, geometric isomers, epimers, and mixtures thereof.

[0042] The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

[0043] The pharmaceutically acceptable salts described in this invention can exist in both non-solventized and solvated forms.

[0044] The compounds provided by this invention and their pharmaceutically acceptable salts can be used alone or in combination with at least one other therapeutic agent in treatment.

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of formula A or a pharmaceutically acceptable salt thereof, and one or more other therapeutically active ingredients.

[0046] In another aspect, the present invention provides a pharmaceutical preparation comprising a compound of formula A or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical carriers. The pharmaceutical preparation is any clinically acceptable dosage form.

[0047] The compounds and pharmaceutically acceptable salts provided by this invention can be formulated into solid dosage forms, such as capsules, tablets, pills, lozenges, sugar-coated tablets, granules, powders, ointments, creams, drops, etc.; the compounds and pharmaceutically acceptable salts provided by this invention can be formulated into liquid dosage forms, such as elixirs, syrups, emulsions, dispersants, suspensions, solutions, sprays, etc.

[0048] The pharmaceutical carriers and / or pharmaceutical diluents used in the pharmaceutical compositions or pharmaceutical formulations of the present invention can be any conventional carriers and / or diluents in the field of pharmaceutical formulations.

[0049] In another aspect, the present invention also provides a pharmaceutical composition comprising (2S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxin[2,3-g]isoquinoline-8-carboxamido)propionic acid or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, carriers, and excipients.

[0050] The pharmaceutical composition of compound I of the present invention can be prepared according to methods known in the art.

[0051] The dosage form of the compound of Formula I of this invention or the pharmaceutical composition containing it can be a liquid dosage form or a solid dosage form. For example, liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, etc.

[0052] The compounds of Formula I of the present invention or pharmaceutical compositions containing them can be formulated into ordinary formulations, or into sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0053] The inventors have discovered that compounds of formula I have antitumor activity and can inhibit the growth of, for example, cervical cancer cells.

[0054] Therefore, in another aspect, the present invention provides the use of compounds as shown in Formula A and pharmaceutically acceptable salts thereof in the preparation of medicaments for treating tumors.

[0055] In another aspect, the present invention provides the use of compounds of Formula I and pharmaceutically acceptable salts thereof in the preparation of antitumor drugs.

[0056] In some implementations, the tumor is a malignant tumor, including but not limited to breast cancer, cervical cancer, rectal cancer, pancreatic cancer, prostate cancer, kidney cancer, ovarian cancer, bladder cancer, fallopian tube tumors, ovarian tumors, peritoneal tumors, melanoma, glioma, or glioblastoma.

[0057] In another aspect, the present invention also provides a method of treating a disease, the method comprising administering to a patient in need a therapeutically effective amount of a compound of formula A, preferably formula I, and a pharmaceutically acceptable salt thereof. In some embodiments, the disease is a tumor, particularly a malignant tumor, including but not limited to breast cancer, cervical cancer, rectal cancer, pancreatic cancer, prostate cancer, kidney cancer, ovarian cancer, bladder cancer, fallopian tube tumors, ovarian tumors, peritoneal tumors, melanoma, glioma, and glioblastoma.

[0058] Methods for quality control

[0059] CN102378574A discloses the free base of the azoanthracene derivative (S)-2-{[(3S,8S)-3-[4-(3,4-dichloro-benzyloxy)-phenyl]-7-((S)-1-phenyl-propyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxano[2,3-g]isoquinoline-8-carbonyl]-amino}-3-[4-(2,3-dimethyl-pyridin-4-yl)-phenyl]-propionic acid (hereinafter referred to as "OA"). OAD2 and its dihydrochloride (S)-2-{[(3S,8S)-3-[4-(3,4-dichloro-benzyloxy)-phenyl]-7-((S)-1-phenyl-propyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbonyl]-amino}-3-[4-(2,3-dimethyl-pyridin-4-yl)-phenyl]-propionic acid·2HCl (hereinafter referred to as "OAD2·2HCl"). They have the function of regulating GLP-1 receptors and have the potential to treat type 2 diabetes. The structures of OAD2 and OAD2·2HCl are as follows:

[0060]

[0061]

[0062] The researchers of this invention discovered that during the preparation of OAD2·2HCl, the strong acidity of the salt formation process and the temperature-dependent effects of the drying process easily lead to the breakage of ether bonds, resulting in compounds of Formula I as described above. During scale-up, the researchers found that the production of compounds of Formula I tended to increase with increasing batch size, a trend not observed in previous small-scale production of OAD2·2HCl. The compounds of Formula I can be used as reference compounds for the quality control of OAD2·2HCl and pharmaceutical compositions or formulations containing OAD2·2HCl.

[0063] In one aspect, the present invention provides a method for quality control of a sample, wherein the sample is a raw material, a pharmaceutical composition or formulation comprising a compound of formula A, formula A1 or formula I described herein, the method comprising: using a corresponding compound of formula A, formula A1 or formula I as a reference compound and determining the level of the reference compound in the sample.

[0064] In some embodiments, the method optionally further includes comparing the measured level of the reference compound with the expected level of the reference compound in the sample.

[0065] The raw materials associated with the compounds of formula A, A1, or I refer to compounds that can be degraded to form the compounds of formula A, A1, or I.

[0066] In some embodiments, the reference compound is a compound of formula A. In some embodiments, the reference compound is a compound of formula A1. In some preferred embodiments, the reference compound is a compound of formula I.

[0067] In some embodiments, the sample is a raw material associated with a compound of Formula I, preferably OAD2, more preferably OAD2·2HCl. In some embodiments, the sample is a pharmaceutical composition or formulation comprising OAD2 or preferably OAD2·2HCl.

[0068] In some preferred embodiments, the present invention provides a method for quality control of a sample, said sample being OAD2, OAD2·2HCl, or a pharmaceutical composition or formulation containing OAD2 or OAD2·2HCl, said method comprising: using a compound of formula I as a reference compound, and determining the level of said reference compound in the sample. In some embodiments, said method optionally further comprises comparing the determined level of said reference compound with a desired level of said reference compound in the sample. In some such embodiments, the desired level of the compound of Formula I, based on the total weight of the compound and OAD2 or preferably OAD2·2HCl, is less than 1.0%, for example less than 0.9%, or less than 0.8%, or less than 0.7%, or less than 0.6%, or less than 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%, or less than 0.1%, or less than 0.09%, or less than 0.08%, or less than 0.07%, or less than 0.06%, or less than 0.05%, or less than 0.04%, or less than 0.03%, or less than 0.02%, or less than 0.01%. In other embodiments, in a pharmaceutical composition or formulation containing OAD2 or preferably OAD2·2HCl, the desired level of the compound of Formula I, based on the total weight of the pharmaceutical composition or formulation, is less than 1.0%, or less than 0.9%, or less than 0.8%, or less than 0.7%, or less than 0.6%, or less than 0.5%, or less than 0.4%, or less than 0.3%, or less than 0.2%, or preferably less than 0.1%, or less than 0.09%, or less than 0.08%, or less than 0.07%, or less than 0.06%, or less than 0.05%, or less than 0.04%, or less than 0.03%, or less than 0.02%, or less than 0.01%.

[0069] The method used to determine the level of the reference compound can be a method conventionally used in the art, such as high performance liquid chromatography (HPLC).

[0070] definition

[0071] The compounds described in this invention are named according to their chemical structural formulas. If the name of the compound differs from its chemical structural formula when referring to the same compound, the chemical structural formula shall prevail.

[0072] Unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, definitions of some terms are provided below. When the definitions and interpretations of terms provided in this invention differ from the meanings commonly understood by those skilled in the art, the definitions and interpretations provided in this invention shall prevail.

[0073] Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

[0074] Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

[0075] Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

[0076] Unless otherwise stated, "(+)" indicates right-handed rotation, "(-)" indicates left-handed rotation, and "(±)" indicates racemic rotation.

[0077] Unless otherwise specified, use wedge-shaped solid line keys. and wedge-shaped dashed key The absolute configuration of the center of a solid is represented by a straight solid line key. and straight dashed key This indicates that the center of the solid is an absolute configuration, but it is uncertain whether it is a wedge-shaped solid line key. or wedge-shaped dashed key

[0078] Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of this disclosure, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide a pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution using conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography employing a chiral stationary phase and optionally combined with chemical derivatization (e.g., from amines to form carbamates).

[0079] The term "pharmaceutically acceptable" in this article means that, with respect to compounds, materials, compositions, and / or dosage forms, they are suitable for use in contact with human and animal tissues, within the bounds of reliable medical judgment, without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0080] The term "pharmaceutically acceptable salt" as used herein refers to a salt of the compounds disclosed herein, prepared by reacting a compound of the present disclosure having specific substituents with a relatively non-toxic acid or base. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, etc. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; salts of amino acids (such as arginine); and salts of organic acids such as glucuronic acid. Certain compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

[0081] The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or basic groups by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in their free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

[0082] The terms “optional” or “optionally” in this document refer to events or conditions that may, but are not required, to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

[0083] The term “substituted” in this article means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable.

[0084] The term "optionally replaced" in this article refers to both "replaced" and "not replaced" scenarios.

[0085] In this article, the term "halogen" refers to fluorine, chlorine, bromine, and iodine.

[0086] The term "C" in this article 1～4 "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 4 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, and 1-methylpropyl.

[0087] The term "C" in this article 3～6 "Cycloalkyl" refers to a monovalent group obtained by removing any single hydrogen atom from a cyclic saturated aliphatic hydrocarbon having 3 to 6 carbons, i.e., a cycloalkyl group with 3 to 6 carbons. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. Attached Figure Description

[0088] Figure 1 MS chromatogram of compound I.

[0089] Figure 2 : H-NMR spectrum of compound I.

[0090] Beneficial effects

[0091] The anthracene compounds of the present invention can be used as reference compounds for quality control of related raw materials, pharmaceutical compositions or formulations containing said raw materials. Surprisingly, although their source compounds have GLP-1 receptor-modulating activity and are therefore suitable for treating type 2 diabetes, the anthracene compounds of the present invention exhibit very different antitumor activities and have the potential to be used as antitumor drugs. Detailed Implementation

[0092] The present invention will be explained in more detail below with reference to embodiments or experimental examples. The embodiments or experimental examples of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the nature and scope of the present invention.

[0093] Experimental methods in this invention, where specific conditions are not specified, are generally under standard conditions or according to the conditions recommended by the raw material or product manufacturer. Reagents whose source is not specified are generally commercially available conventional reagents.

[0094] The compound of formula II used in this invention, (S)-2-((3S,8S)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)propionate, can be prepared according to the method described in CN102378574A.

[0095] Example 1: Preparation of (2S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)propionic acid

[0096] Step a:

[0097] In a 1000 mL reaction flask, add 30 g (34.48 mmol) of methyl (S)-2-((3S,8S)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carboxamido)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)propionate and 350 mL of dichloromethane. Stir to dissolve, under nitrogen protection, and cool to 0 °C. Slowly add 150 mL of a 1 mol / L boron trichloride solution in dichloromethane. After the addition is complete, maintain the reaction at 0 °C for 18 hours. Add 150 mL of dichloromethane and 500 mL of 5% sodium bicarbonate solution, stir for 30 minutes, and separate the layers. The aqueous phase was extracted with 250 mL of dichloromethane. The dichloromethane phases were combined and concentrated under reduced pressure at 30 °C to obtain 30 g of crude (S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((3S,8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)propionate, which was used directly in the next step of the reaction.

[0098] Step b:

[0099] Add 30 g of crude (S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((3S,8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxinro[2,3-g]isoquinoline-8-carbamate)propionate and 150 mL of 2-methyltetrahydrofuran to a 500 mL reaction flask and stir to dissolve. Add 75 mL of an aqueous solution of 6 g of sodium hydroxide and stir at room temperature for 18 hours. After terminating the reaction, adjust the pH to 3-4 with 1N dilute hydrochloric acid. The aqueous phase was extracted with 100 mL of 2-methyltetrahydrofuran, and the organic phases were combined and concentrated under reduced pressure to obtain 12 g of (2S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((8S)-3-(4-hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxin[2,3-g]isoquinoline-8-carbamoyl)propionic acid.

[0100] Example 2: Detection of Compound I

[0101] The structure of the compound was determined by nuclear magnetic resonance (NMR) and / or mass spectrometry (MS); NMR shifts (δ) were expressed in units of 10⁻⁶ (ppm); NMR measurements were performed using a Bruker DMX-500 NMR spectrometer with deuterated pyridine as the solvent and tetramethylsilane (TMS) as the internal standard. MS measurements were performed using a Bruker Esquire 3000plus mass spectrometer. Mass spectrometry (MS): Detection method: Bruker Esquire 3000plus mass spectrometer; ESI source; positive ion detection.

[0102] The results are as follows:

[0103] MS Map: HRMS(ESI)C 43 H 43 Theoretical N3O6 concentration: 697.32 + 1.00 [M + H], Measured value: 698.32

[0104] 1 H-NMR spectrum: 1H-NMR (400MHz, Py-d5): δ8.71(d,1H),8.55(d,1H),7.18-7.59(m,13H),7.13(s,1H ),7.06(s,1H),7.04(d,1H),4.55(m,1H),5.24(d,1H),4.45(dd,1H),4.11(dd,1H) ,4.09(d,1H),3.94(dd,1H),3.78(dd,1H),3.66(dd,1H),3.56(dd,1H),3.47(dd,2 H),3.08(dd,1H),2.60(s,3H),2.15(m,4H),2.01(m,1H),1.40(m,1H),0.75(t,3H).

[0105] Example 3: Evaluation of the bioactivity of compound I

[0106] Testing instruments and reagents

[0107]

[0108] The antitumor activity of compound I was investigated using the MTT assay.

[0109] Weigh 0.5 g of MTT (sigma) and dissolve it in 100 ml of phosphate-buffered saline (PBS). Filter the solution through a 0.22 μm filter to remove bacteria. Store at 4 °C in the dark. During preparation and storage, wrap the container with aluminum foil and try to avoid light when adding the MTT solution.

[0110] Experimental methods:

[0111] 1. Collect HeLa cells in the logarithmic growth phase (from ATCC), adjust the cell suspension concentration, and seed the cells into 96-well plates. Add 100 μl to each well to make the cell density approximately 1000-3000 cells / well (fill the edge wells with sterile PBS). Incubate at 5% CO2 and 37°C for 12 hours.

[0112] 2. Discard the original culture medium and add 200 μl of the prepared culture medium containing different concentrations of Formula I compound and blank control (the concentration gradient of Formula I compound is 2.5, 5, 10, 20, 40, 80, 160 μmol / L; all culture media contain 0.1% dimethyl sulfoxide), and set 3-4 replicates.

[0113] Continue incubation at 3.5% CO2 and 37°C for 48 hours, and observe under an inverted microscope (Nikon Ti-S).

[0114] 4. Discard the original culture medium, add 200 μl of culture medium free of compounds and dimethyl sulfoxide and 20 μl of prepared MTT solution (5 mg / ml, i.e., 0.5% MTT solution) to each well, continue incubation for 4 hours, and then terminate the culture.

[0115] 5. Carefully aspirate the culture medium from the wells, add 150 μl of dimethyl sulfoxide to each well, and place on a shaker at low speed for 10 minutes to fully dissolve the crystals. Measure the absorbance of each well at OD490 nm using a microplate reader (BioTek, Synergy H1).

[0116] Experimental analysis: The effects of different drug concentrations on cell line viability (OD) were investigated. 490 Plotting the dose-response curve yields the half-maximal inhibitory concentration (IC50), from which the IC50 can be determined. 50 ).

[0117] Experimental results: The half-maximal inhibitory concentration (IC50) of compound I against cervical cancer cell lines was... 50 The value is 27.24 nM.

[0118] Example 4: Detection reagents and test reagents for compound I as a reference compound in OAD2·2HCl sample: acetonitrile (HPLC), methanol (HPLC), trifluoroacetic acid (HPLC), purified water.

[0119] Instruments and equipment: electronic analytical balance, liquid chromatograph, amide-hexadecyl silica column, volumetric flasks, beakers

[0120] Chromatographic column: Supleco Ascentis Express RP-Amide column (3.0*150mm, 2.7um)

[0121] Detection wavelength: 280nm

[0122] Flow rate: 0.425 ml / min

[0123] Injection volume: 2 μl

[0124] Diluent: 0.05% trifluoroacetic acid aqueous solution - 0.05% trifluoroacetic acid acetonitrile solution (40:60)

[0125] Mobile phase A: 0.05% trifluoroacetic acid aqueous solution

[0126] Mobile phase B: 0.05% trifluoroacetic acid acetonitrile solution: 0.05% trifluoroacetic acid methanol solution = 1:2 (V / V)

[0127] The elution gradient is as follows:

[0128] time Mobile phase A (%) Mobile phase B (%) 0 50 50 10 45 55 35 15 85 36 1 99 38 1 99 38.1 50 50 45 50 50

[0129] Determination method:

[0130] Take an appropriate amount of OAD2·2HCl and place it in a volumetric flask. Add diluent (0.05% trifluoroacetic acid acetonitrile solution: 0.05% trifluoroacetic acid aqueous solution = 60:40 (V / V)) to dissolve it and dilute it to a solution of about 0.5 mg / ml. Shake well.

[0131] Take an appropriate amount of compound of formula I and place it in a volumetric flask. Add diluent (0.05% trifluoroacetic acid acetonitrile solution: 0.05% trifluoroacetic acid aqueous solution = 60:40 (V / V)) to dissolve it and dilute it to a solution of about 0.5 mg / ml. Shake well.

[0132] Accurately measure 10 μl of each of the above-prepared solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0133] result:

[0134] The retention time of compound I is 3.567, the retention time of OAD2·2HCl is 22.059, and the relative retention time is 0.161.

[0135] In addition to those described herein, various modifications of the invention are also intended to fall within the scope of the appended claims, based on the foregoing description. All references cited in this application (including all patents, patent applications, journal articles, books, and any other disclosures) are incorporated herein by reference in their entirety.

Claims

1. A compound and pharmaceutically acceptable salts thereof, wherein the compound has the following structure as shown in Formula I: （I）。 2. A method of preparing the compound of Formula I according to claim 1, comprising the steps of: a. reacting a compound as shown in Formula II to produce a compound of Formula III: and b. reacting the compound III to produce the compound of Formula I according to claim 1: ； wherein steps a and b are reacted in a suitable solvent independently selected from at least one of tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, acetone, dichloromethane, acetonitrile, methanol or ethanol; wherein the compound of Formula II in step a is hydrolyzed with an acidic reagent selected from one of boron trifluoride, boron trichloride, aluminum trichloride or sulfur trioxide; and wherein the reaction of step b is carried out in aqueous base.

3. A pharmaceutical composition comprising an effective amount of the compound of Formula I according to claim 1 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable adjuvant, carrier or excipient.

4. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 3 in the preparation of an anti-tumor medicament, wherein the tumor is cervical cancer.

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