CN117500808A - Anthracene compounds, their preparation methods and medicinal uses - Google Patents

Abstract

The invention relates to an anthracene compound of a formula A, a preparation method and medical application thereof, wherein the anthracene compound has the effect of inhibiting the growth of tumor cells and has the potential of being used as an anti-tumor drug. The invention also relates to a method for quality control of a sample.

Description

Anthracene compounds, their preparation methods and medicinal uses Technical field

The present invention relates to the field of medical technology, specifically anthracene compounds with anti-tumor activity, their pharmaceutically acceptable salts and their preparation methods and uses.

Background technique

With the development of molecular biology technology and further understanding of pathogenesis at the cellular and molecular levels, the discovery of highly efficient and low-toxic anticancer drugs has become an important research direction by targeting key enzymes in the signaling pathways of tumor development and development. . Molecular targeted therapy of tumors is different from traditional tumor treatment methods. It is based on the study of tumor molecular biology and uses small molecule compounds to block key enzymes in the key pathways of tumor growth, thereby blocking the growth of tumor cells. role.

CN102378574A discloses azoanthracene derivatives as shown in formula B and their use in regulating glucagon-like peptide-1 receptors:

The inventor of the present application has studied the degradation process of such anthracene compounds and found that they are easily degraded in a suitable acidic environment, and their degradation products are usually more stable. CN102378574A points out that these azoanthracene derivatives have the effect of regulating glucagon-like peptide-1 (GLP-1) receptors. It is necessary to further study the activity of the degradation products of this type of compounds and discover their potential medicinal uses.

Contents of the invention

In one aspect, the invention provides an anthracene compound represented by Formula A:

and pharmaceutically acceptable salts thereof,

in:

X is independently selected from oxygen, hydrogen, and nitrogen;

R ₁ is selected from COOH or –COOCH ₃ ;

R ₂ is –Y ₁ –L–Y ₂ ;

L is selected from –(CH ₂ ) _a – or –O–, where a is 0 or 1;

Y ₁ is a phenylene group, and the phenylene group is a 1,3-phenylene group or a 1,4-phenylene group;

Y ₂ is selected from phenyl, pyridyl, diazaaryl or imidazolyl, and Y ₂ is optionally independently selected from hydrogen, halogen, C _1-4 alkyl, C _3-6 cycloalkyl, CF ₃ , – CH ₂ F, –CH ₂ CF ₃ , CN, –CH ₂ CN, –OCH ₃ , –O–C(CH ₃ ) ₃ , –OCH ₂ CH ₃ , –OCH(CH ₃ ) ₂ , –OCF ₃ , – OCH ₂ CF ₃ , NH ₂ , –SO ₂ –CH ₃ , –NHCH ₃ , –CO–NHCH ₃ , –NHCH ₂ CH ₃ , –N(CH ₃ ) ₂ , –CO–CH ₃ , –CO–NHCH ₃ Replaced 1-3 times;

R ₃ is selected from C _1-4 alkyl, C _3-6 cycloalkyl, phenyl, furyl, thienyl, oxazolyl, pyridyl, thiazolyl, pyrimidinyl, epoxy pentacyclic group, epoxy hexacyclic group Cyclic group, -sulfonyl-thiazolyl, -sulfonyl-oxazolyl, -sulfonyl-pyridyl, -carbonyl-oxazolyl, -carbonyl-pyridyl; which may be independently selected from halogen, amino, OH, COOH, CN, –CH ₂ CN, CF ₃ , –CH ₂ F, –CH ₂ CF, –C(CH ₃ ) ₃ , –O–C(CH ₃ ) ₃ , –OCH ₃ , C _1-4 alkyl , C _3-6 cycloalkyl, phenyl substituted 1-3 times.

In some embodiments, each X is independently oxygen.

In some embodiments, _R1 is COOH.

In some embodiments, L is selected from -( _CH2 ) _a- or -O-, where a is 0 or 1. When a is 0, L is a direct connection key. When a is 1, L is –CH ₂ –. In some preferred embodiments, L is a direct bond.

In some embodiments, Y ₁ is 1,4-phenylene.

In _some embodiments, _{when each} :

In some embodiments, when Y ₂ is phenyl, it is optionally substituted once with any one of H, F, Cl, CN, –CH ₂ CN, –OCH ₃ , CF ₃ and CH ₃ .

In some embodiments, when Y ₂ is pyridyl, it is optionally H, C _1-4 alkyl, –OCH ₃ , –N(CH ₃ ) ₂ , F, CN, NH ₂ , CH ₂ F, – Either CO–NHCH ₃ or –CO–CH ₃ is substituted 1-2 times. Preferably, Y ₂ is pyridyl substituted twice by methyl, preferably 4-pyridyl substituted twice by methyl, more preferably

In some embodiments, R ₃ is C _1-4 alkyl substituted by phenyl; preferably, R ₃ is 1-phenyl-propyl-, including (R)-1-phenyl-propyl- or (S)–1-phenyl–propyl–.

The present invention further provides compounds represented by the following 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]dioxino[2,3- g]isoquinoline-8-carboxamido)propionic acid, and pharmaceutically acceptable salts thereof:

The present invention also provides the preparation of the compound represented by 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]dioxino[2,3- g] The method of isoquinoline-8-carboxamido)propionic acid, the method includes the following steps:

a) Hydrolyze the compound represented by formula II to generate the compound of formula III. The specific reaction is as follows:

as well as

b) React compound III to prepare the compound of formula I. The specific reaction is as follows:

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

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 one of boron trifluoride, boron trichloride, aluminum trichloride or sulfur trioxide, Boron trichloride is preferred.

In some embodiments, the reaction of step b is performed in alkaline water.

In some embodiments, the reaction described in step a undergoes a post-treatment step before being put into the next reaction; the post-treatment step is selected from one of dilution, filtration, pH adjustment, washing, extraction, concentration, crystallization, and drying, or Various; the post-processing steps can be performed once or multiple times as needed.

In some embodiments, the post-treatment method of step a is: after preparing the compound of formula I, the crude compound I is obtained through pH adjustment, liquid separation, 2-methyltetrahydrofuran extraction, and concentration.

Pharmaceutically acceptable salts according to the present invention include acid addition salts and alkali salts.

The compounds provided by the present invention and their pharmaceutically acceptable salts may exist in a chiral form, that is, S configuration or R configuration. The compounds provided by the present invention and their pharmaceutically acceptable salts may exist in achiral forms. When the structure of a compound described in the present invention is exemplified in one configuration, it also means that the structure of another configuration or achiral form is disclosed.

The optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, in which the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the salt is formed by conventional methods known in the art. The method performs diastereoisomer resolution and then recovers the pure enantiomers. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g. amine generation from amines formate).

The compounds described in the present invention include stereoisomers of the compounds. The stereoisomers described in the present invention mean that when asymmetric carbon atoms exist in the compound represented by formula I, enantiomers will be produced; when a carbon-carbon double bond or cyclic structure exists in the compound, cis isomers will be produced. Anti-isomers; when ketones or oximes exist in compounds, tautomers will be produced; as a specific embodiment, the stereoisomers described in the present invention include but are not limited to: enantiomers, non- Enantiomers, racemates, cis-trans isomers, tautomers, geometric isomers, epimers and mixtures thereof.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereoisomers isomer, the (D)-isomer, the (L)-isomer, as well as their racemic mixtures and other mixtures, such as enantiomeric or diastereomerically enriched mixtures, all of which belong to the present invention. within the scope of the invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.

The pharmaceutically acceptable salts of the present invention may exist in unsolvated and solvated forms.

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

In another aspect, the present invention provides a pharmaceutical composition containing a compound represented by Formula A or a pharmaceutically acceptable salt thereof, and one or more other therapeutically active ingredients.

In another aspect, the present invention provides a pharmaceutical preparation, which contains a compound represented by formula A or a pharmaceutically acceptable salt thereof, and one or more pharmaceutical carriers. The pharmaceutical preparation is any clinically acceptable preparation dosage form.

The compounds provided by the present invention and their pharmaceutically acceptable salts can be formed into solid dosage forms, such as capsules, tablets, pills, lozenges, sugar-coated agents, granules, powders, ointments, creams, drops, etc.; the present invention The provided compounds and their pharmaceutically acceptable salts may be in liquid dosage forms, such as elixirs, syrups, emulsions, dispersions, suspensions, solutions, sprays, and the like.

The pharmaceutical carrier and/or pharmaceutical diluent that can be used in the pharmaceutical composition or pharmaceutical preparation of the present invention can be any conventional carrier and/or diluent in the field of pharmaceutical preparation.

In another aspect, the invention also provides a pharmaceutical composition containing (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]dioxino[2 ,3-g]isoquinoline-8-carboxamido)propionic acid or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, carriers, and excipients.

Pharmaceutical compositions of compounds of formula I of the present invention can be prepared according to methods well known in the art.

The dosage form of the compound of Formula I of the present invention or the pharmaceutical composition containing it may be a liquid dosage form or a solid dosage form. For example, liquid dosage forms can be true solutions, colloids, particulate dosage forms, emulsion dosage forms, or suspension dosage forms. Other dosage forms include tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, freeze-dried powder injections, etc.

The compound of formula I of the present invention or the pharmaceutical composition containing it can be made into ordinary preparations, or can be sustained-release preparations, controlled-release preparations, targeted preparations and various particulate drug delivery systems.

The inventors have discovered that compounds of formula I have anti-tumor activity and are able to inhibit the growth of, for example, cervical cancer cells.

Therefore, in another aspect, the present invention provides the use of a compound represented by formula A and a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.

In yet another aspect, the present invention provides the use of a compound of formula I and a pharmaceutically acceptable salt thereof for the preparation of anti-tumor drugs.

In some embodiments, the tumor is a malignant tumor, including but not limited to breast, cervical, rectal, pancreatic, prostate, renal, ovarian, bladder, fallopian tube, ovarian, peritoneal, Melanoma, glioma, or glioblastoma.

In yet another aspect, the present invention also provides a method of treating a disease, which method includes administering to a patient in need a therapeutically effective amount of a compound represented by 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.

Methods used for quality control

CN102378574A discloses 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]dioxino[2,3-g]isoquinoline-8-carbonyl] -Amino}-3-[4-(2,3-dimethyl-pyridin-4-yl)-phenyl]-propionic acid free base (hereinafter referred to as "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]dioxino[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 modulate GLP-1 receptors and have the potential to treat type 2 diabetes. The structures of OAD2 and OAD2·2HCl are as follows:

The researchers of the present invention found that during the preparation process of OAD2·2HCl, due to the strong acidity of the salt formation process and the influence of temperature during the drying process, it is easy to cause the ether bond to break and produce the compound of formula I described above. During the gradual scale-up process, the researchers found that as the batch size increased, the compounds of formula I tended to increase, but this trend did not appear in the previous small-scale production of OAD2·2HCl. The compound of formula I can be used as a reference compound for quality control of OAD2·2HCl and pharmaceutical compositions or preparations containing OAD2·2HCl.

In one aspect, the invention provides a method for quality control of a sample, wherein the sample is a raw material, a pharmaceutical composition or a formulation comprising said raw material related to a compound of Formula A, Formula A1 or Formula I described herein , the method includes: using the corresponding compound of Formula A, Formula A1 or Formula I as a reference compound, and measuring the level of the reference compound in the sample.

In some embodiments, the method optionally further includes comparing the measured level of a reference compound to an expected level of the reference compound in the sample.

The raw materials related to the compounds of formula A, formula A1 or formula I mean compounds capable of forming the compounds of formula A, formula A1 or formula I by degradation.

In some embodiments, the reference compound is the compound of Formula A. In some embodiments, the reference compound is the compound of Formula A1. In some preferred embodiments, the reference compound is the compound of Formula I.

In some embodiments, the sample is a starting material related to the compound of Formula I, preferably OAD2, preferably OAD2·2HCl. In some embodiments, the sample is a pharmaceutical composition or formulation comprising OAD2 or preferably OAD2·2HCl.

In some preferred embodiments, the invention provides a method for quality control of a sample that is OAD2, OAD2.2HCl, or a pharmaceutical composition or formulation containing OAD2 or OAD2.2HCl, the method comprising: The compound of formula I serves as a reference compound and the level of said reference compound in the sample is determined. In some embodiments, the method optionally further includes comparing the measured level of the reference compound to an expected level of the reference compound in the sample. In some such embodiments, the desired level of the compound of Formula I is less than 1.0%, such as less than 0.9%, or as low as the total weight of the compound of Formula I and OAD2 or preferably OAD2·2HCl. 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 lower than 0.08%, or lower than 0.07%, or lower than 0.06%, or lower than 0.05%, or lower than 0.04%, or lower than 0.03%, or lower than 0.02%, or lower than 0.01%. In other embodiments, in pharmaceutical compositions or formulations containing OAD2 or preferably OAD2·2HCl, the desired level of the compound of Formula I is less than 1.0%, based on the total weight of the pharmaceutical composition or formulation. , 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 low At 0.02%, or below 0.01%.

The method used to determine the level of the reference compound may be a method customarily used in the art, such as high performance liquid chromatography (HPLC).

definition

The compounds described in the present invention are named according to their chemical structural formulas. If the naming of a compound does not match the chemical structural formula when representing the same compound, the chemical structural formula shall prevail.

In the present invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, in order to better understand the present invention, definitions of some terms are provided below. When the definitions and explanations of terms provided in the present invention are different from the meanings commonly understood by those skilled in the art, the definitions and explanations of terms provided in the present invention shall prevail.

Unless otherwise stated, the terms "enantiomers" or "optical isomers" refer to stereoisomers that are mirror images of each other.

Unless otherwise stated, the term "cis-trans isomers" or "geometric isomers" refers to the inability of the double bonds or single bonds of the carbon atoms in the ring to rotate freely.

Unless otherwise stated, the term "diastereomer" refers to stereoisomers whose molecules have two or more chiral centers and are in a non-mirror image relationship between the molecules.

Unless otherwise stated, "(+)" means right-handed, "(-)" means left-handed, and "(±)" means racemic.

Unless otherwise stated, use wedge-shaped solid line keys and wedge-shaped dotted keys Represents the absolute configuration of a three-dimensional center, using a straight solid line key and straight dotted keys Indicates that the stereocenter is an absolute configuration, but it is not certain that it is a wedge-shaped solid line bond. Or a wedge-shaped dotted key?

The optically active (R)- and (S)-isomers as well as the D and L isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present disclosure is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliaries, where the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure of the desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amine group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the salt is formed by conventional methods known in the art. The method performs diastereoisomer resolution and then recovers the pure enantiomers. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally combined with chemical derivatization methods (e.g. amine generation from amines formate).

The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue, without undue toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" as used herein refers to a salt of a compound of the present disclosure prepared from a compound of the present disclosure having a specific substituent and a relatively non-toxic acid or base. When 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 pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts, etc. When 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 neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, including acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Similar acids such as fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid; also include salts of amino acids (such as arginine, etc.) , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups and thus can be converted into either base or acid addition salts.

The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid or basic groups. In general, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.

The term "optionally" or "optionally" as used herein means that the subsequently described event or condition may, but need not, occur, and that the description includes instances in which the stated event or condition occurs as well as instances in which the event or condition does not What happened.

The term "substituted" as used herein means that any one or more hydrogen atoms on a specific atom are replaced by a substituent, which may include deuterium and variants of hydrogen, as long as the valence state of the specific atom is normal and substituted The compound is stable.

The term "optionally substituted" as used herein means both "substituted" and "unsubstituted."

The term "halogen" as used herein means fluorine, chlorine, bromine and iodine.

The term "C _1-4 alkyl" herein refers to a straight-chain or branched alkyl group with 1 to 4 carbons, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, Isobutyl, sec-butyl, tert-butyl and 1-methylpropyl.

The term "C _{3 to 6} cycloalkyl" as used herein means a monovalent group obtained by removing any single hydrogen atom from a cyclic saturated aliphatic hydrocarbon having 3 to 6 carbons, that is, a cyclic ring of 3 to 6 carbons alkyl. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

Description of the drawings

Figure 1: MS pattern of compound I.

Figure 2: H-NMR pattern of compound I.

beneficial effects

The anthracene compounds of the present invention can be used as reference compounds for quality control of raw materials related thereto, pharmaceutical compositions or preparations containing the raw materials. Surprisingly, although the compounds from which they are derived have activity in modulating GLP-1 receptors and can therefore be used to treat type 2 diabetes, the anthracene compounds of the present invention have very different antitumor activities and have potential for use as antitumor agents. potential of the drug.

Detailed ways

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 do not limit the essence and scope of the present invention.

Experimental methods without specifying specific conditions in the examples of the present invention are usually conventional conditions, or conditions recommended by raw material or product manufacturers. Reagents with unspecified sources are usually conventional reagents that are commercially available.

Compound II of formula (S)-2-((3S,8S)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-7-((S) used in the present invention -1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g]isoquinoline-8-carboxamido) Methyl 3-(4-(2,3-dimethylpyridin-4-yl)phenyl)propionate can be prepared by referring to the method described in CN102378574A.

Example 1: (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]dioxino[2,3-g]isoquinoline-8- Preparation of carboxamido)propionic acid

Step a:

Add 30 grams of (S)-2-((3S,8S)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-7-((S) into a 1000 ml reaction bottle )-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g]isoquinoline-8-carboxamido )-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)propionic acid methyl ester (34.48mmol) and 350 ml of methylene chloride, stir to dissolve, protect with nitrogen, and cool to 0°C. Slowly add 150 ml of 1 mol/L boron trichloride solution in dichloromethane. After the addition is completed, keep 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 liquids. The aqueous phase was extracted with 250 ml of dichloromethane, combined with the dichloromethane phases, and concentrated under reduced pressure at 30°C to obtain 30 grams of (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 The crude product of -[1,4]dioxino[2,3-g]isoquinoline-8-carboxamido)propionic acid methyl ester was directly used in the next reaction.

Step b:

Add 30 grams of (S)-3-(4-(2,3-dimethylpyridin-4-yl)phenyl)-2-((3S,8S)-3-(4-) into a 500 ml reaction bottle Hydroxyphenyl)-7-((S)-1-phenylpropyl)-2,3,6,7,8,9-hexahydro-[1,4]dioxino[2,3-g ] Isoquinoline-8-carboxamido) crude methyl propionate and 150 ml of 2-methyltetrahydrofuran, stir to dissolve. Add 6 grams of sodium hydroxide in 75 ml of aqueous solution and stir the reaction at room temperature for 18 hours. After terminating the reaction, adjust pH=3-4 with 1N dilute hydrochloric acid. Separate the liquids, extract the aqueous phase with 100 ml*2 of 2-methyltetrahydrofuran, combine the organic phases, and concentrate under reduced pressure to obtain 12 grams 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-hexa Hydro-[1,4]dioxino[2,3-g]isoquinoline-8-carboxamido)propionic acid.

Example 2: Detection of Compounds of Formula I

The structure of the compound is determined by nuclear magnetic resonance (NMR) or/and mass spectrometry (MS); the NMR shift (δ) is measured in units of 10-6 (ppm); the NMR is measured with a Bruker DMX-500 nuclear magnetic instrument. The solvent was deuterated pyridine, and the internal standard was tetramethylsilane (TMS). MS was measured using a Bruker Esquire 3000plus mass spectrometer. Mass spectrometry (MS): Detection method: Bruker Esquire 3000plus mass spectrometer; ESI source; positive ion detection.

The result is as follows:

MS spectrum: HRMS(ESI)C ₄₃ H ₄₃ N ₃ O _6Theoretical value: 697.32+1.00[M+H], measured value: 698.32

¹ H-NMR spectrum: ¹ H-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,2H),3.08(dd,1H),2.60(s,3H),2.15( m,4H),2.01(m,1H),1.40(m,1H),0.75(t,3H).

Example 3: Evaluation of biological activity of compounds of formula I

Testing instruments and reagents

MTT method to investigate the anti-tumor activity of compounds of formula I

Weigh 0.5 grams of MTT (sigma), dissolve it in 100 ml of phosphate buffer saline (PBS), filter it with a 0.22 μm filter to remove bacteria in the solution, and store it at 4°C in the dark. During the preparation and storage process , the container is wrapped in aluminum foil, and it is protected from light as much as possible during the process of adding MTT solution.

experimental method:

1. Collect HeLa cells (from ATCC) in the logarithmic phase, adjust the concentration of cell suspension, seed the cells in a 96-well flat-bottomed plate, add 100 μl to each well, so that the cell density is approximately 1000-3000 cells/well (the edge wells are all Fill with sterile PBS) and incubate at 37°C for 12 hours in 5% CO ₂ .

2. Discard the original culture medium and add 200 μl of prepared culture medium containing different concentrations of the compound of formula I and blank control (the concentration gradient of the compound of formula I is 2.5, 5, 10, 20, 40, 80, 160 μmol/L; All culture media contain 1/1000 part dimethyl sulfoxide (DMSO), with 3-4 duplicate wells.

Continue to incubate for 48 hours at 3.5% CO ₂ and 37°C, and observe under an inverted microscope (Nikon Ti-S).

4. Discard the original medium, add 200 μl of medium without compounds and dimethyl sulfoxide and 20 μl of prepared MTT solution (5 mg/ml, 0.5% MTT solution) to each well, and continue incubating for 4 hours. Terminate culture.

5. Carefully aspirate the culture medium in the well, add 150 μl dimethyl sulfoxide to each well, place it on a shaker and shake at low speed for 10 minutes to fully dissolve the crystals, and read at OD490nm on a microplate reader (BioTek, Synergy H1). Measure the absorbance value of each well.

Experimental analysis: The dose response curve can be obtained by plotting different concentrations of the drug and the cell viability (OD ₄₉₀ ) of the cell line, from which the half inhibitory concentration (IC ₅₀ ) can be obtained.

Experimental results: The half inhibitory concentration (IC ₅₀ ) of the compound of formula I on cervical cancer cell lines is 27.24nM.

Example 4: Detection of the compound of formula I as a reference compound for OAD2·2HCl sample

Reagents and reagents: acetonitrile (HPLC), methanol (HPLC), trifluoroacetic acid (HPLC), purified water

Instruments and utensils: electronic analytical balance, liquid chromatograph, amidocetyl silica column, measuring flask, beaker

Chromatographic column: Supleco Ascentis Express RP-Amide, 3.0*150mm, 2.7um

Detection wavelength: 280nm

Flow rate: 0.425ml/min

Injection volume: 2μl

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

Mobile phase A: 0.05% trifluoroacetic acid aqueous solution

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

The elution gradient is as follows:

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

Determination method:

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

Take an appropriate amount of the compound of formula I and place it in a measuring bottle, add diluent (0.05% trifluoroacetic acid acetonitrile solution: 0.05% trifluoroacetic acid aqueous solution = 60:40 (V/V)) to dissolve, and dilute it to a solution of about 0.5 mg/ml. , shake well.

Precisely measure 10 μl of each solution prepared above, inject it into the liquid chromatograph, and record the chromatogram.

result:

The retention time of the compound of formula I is 3.567, the retention time of OAD2·2HCl is 22.059, and the relative retention time is: 0.161.

In light of the foregoing description, various modifications of the invention are intended to fall within the scope of the appended claims, in addition to those described herein. Each reference cited in this application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety.

Claims (21)

Compounds of formula A and their pharmaceutically acceptable salts: and pharmaceutically acceptable salts thereof, in: X is independently selected from oxygen, hydrogen, and nitrogen; R ₁ is selected from COOH or –COOCH ₃ ; R ₂ is –Y ₁ –L–Y ₂ ; L is selected from –(CH ₂ ) _a – or –O–, where a is 0 or 1; Y ₁ is a phenylene group, and the phenylene group is a 1,3-phenylene group or a 1,4-phenylene group; Y ₂ is selected from phenyl, pyridyl, diazaaryl or imidazolyl, and Y ₂ is optionally independently selected from hydrogen, halogen, C _1-4 alkyl, C _3-6 cycloalkyl, CF ₃ , – CH ₂ F, –CH ₂ CF ₃ , CN, –CH ₂ CN, –OCH ₃ , –O–C(CH ₃ ) ₃ , –OCH ₂ CH ₃ , –OCH(CH ₃ ) ₂ , –OCF ₃ , – OCH ₂ CF ₃ , NH ₂ , –SO ₂ –CH ₃ , –NHCH ₃ , –CO–NHCH ₃ , –NHCH ₂ CH ₃ , –N(CH ₃ ) ₂ , –CO–CH ₃ , –CO–NHCH ₃ Replaced 1-3 times; R ₃ is selected from C _1-4 alkyl, C _3-6 cycloalkyl, phenyl, furyl, thienyl, oxazolyl, pyridyl, thiazolyl, pyrimidinyl, epoxy pentacyclic group, epoxy hexacyclic group Cyclic group, -sulfonyl-thiazolyl, -sulfonyl-oxazolyl, -sulfonyl-pyridyl, -carbonyl-oxazolyl, -carbonyl-pyridyl; which may be independently selected from halogen, amino, OH, COOH, CN, –CH ₂ CN, CF ₃ , –CH ₂ F, –CH ₂ CF, –C(CH ₃ ) ₃ , –O–C(CH ₃ ) ₃ , –OCH ₃ , C _1-4 alkyl , C _3-6 cycloalkyl, phenyl substituted 1-3 times. The compound of claim 1, wherein X is independently oxygen. The compound of claim 1 or 2, wherein Y ₁ is 1,4-phenylene. The compound of any one of claims 1 to 3, wherein _R1 is COOH. The compound according to any one of claims 1 to 4, wherein L is a direct bond or _-CH2- , preferably a direct bond. The compound of any one of claims 1 to 5, wherein the compound has a structure represented by formula A ₁ : The compound according to any one of claims 1 to 6, wherein Y ₂ is phenyl, which is optionally substituted once with the following substituents: H, F, Cl, CN, –CH ₂ CN, –OCH ₃ , CF ₃ , CH ₃ . The compound according to any one of claims 1 to 6, wherein Y ₂ is 4-pyridyl, which is optionally substituted 1 to 2 times with the following substituents: H, CH ₃ , –OCH ₃ , –N (CH ₃ ) ₂ , F, CN, NH ₂ , –CH ₂ CH ₃ , –CH ₂ CH ₂ CH ₃ , –CH ₂ F, –CO–NHCH ₃ , –CO–CH ₃ ; preferably substituted by methyl 2 times 4-pyridyl, more preferably The compound according to any one of claims 1 to 8, R ₃ is -C _1-4 alkyl substituted by phenyl; preferably 1-phenyl-propyl-, including (R)-1-phenyl yl-propyl- or (S)-1-phenyl-propyl-. The compound according to claim 1 has the following structure shown in formula I: A method for preparing the compound of formula I as claimed in claim 10, comprising the following steps: a. React the compound shown in formula II to generate the compound of formula III: as well as b. React compound III to obtain the compound of formula I as shown in claim 10: The method of claim 11, wherein steps a and b are reacted in a suitable solvent, and the solvent is independently selected from the group consisting of tetrahydrofuran, 2-methyltetrahydrofuran, dioxane, acetone, dichloromethane, acetonitrile, and methanol or at least one of ethanol. The method of claim 11 or 12, wherein in step a, a hydrolysis reaction occurs between the compound of formula II and an acidic reagent, and the acidic reagent is selected from boron trifluoride, boron trichloride, aluminum trichloride or sulfur trioxide. kind of. The preparation method according to any one of claims 11 to 13, wherein the reaction of step b is carried out in alkaline water. Pharmaceutical composition, which contains an effective amount of the compound of formula I according to any one of claims 1 to 10 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients, carriers or excipients agent. Use of the compound according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 15 in the preparation of anti-tumor drugs. The use of claim 16, wherein the tumor is selected from the group consisting of breast cancer, cervical cancer, rectal cancer, pancreatic cancer, prostate cancer, kidney cancer, ovarian cancer, bladder cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, melanoma , glioma or glioblastoma. A method for quality control of a sample, wherein the sample is a raw material, a pharmaceutical composition or a preparation containing the raw material related to the compound of any one of claims 1 to 10, the method comprising: The corresponding compound according to any one of claims 1 to 10 is used as a reference compound, and the level of said reference compound in said sample is determined, and optionally the measured level of said reference compound Compare to the expected level of the reference compound in the sample. The method of claim 18, wherein the reference compound is a compound of formula A1 as claimed in claim 6, preferably a compound of formula I as claimed in claim 10. The method of claim 18 or 19, wherein the sample is OAD2 or preferably OAD2·2HCl, or a pharmaceutical composition or formulation comprising OAD2 or preferably OAD2·2HCl. The method of claim 20, wherein: The desired level of the compound of formula I is less than 1.0%, such as less than 0.9%, or less than 0.8%, or less than 0.7, based on the total weight of the compound of formula I and OAD2 or preferably OAD2·2HCl. %, or lower than 0.6%, or lower than 0.5%, or lower than 0.4%, or lower than 0.3%, or lower than 0.2%, or lower than 0.1%, or lower than 0.09%, or lower 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%; and/or The desired level of the compound of formula I is less than 1.0%, or less than 0.9%, or less than 0.8%, or less than 0.7 based on the total weight of the pharmaceutical composition or preparation containing OAD2 or preferably OAD2·2HCl. %, or lower than 0.6%, or lower than 0.5%, or lower than 0.4%, or lower than 0.3%, or lower than 0.2%, or preferably lower than 0.1%, or lower than 0.09%, or lower than 0.08% , or lower than 0.07%, or lower than 0.06%, or lower than 0.05%, or lower than 0.04%, or lower than 0.03%, or lower than 0.02%, or lower than 0.01%.