CN111233809A - Millepachine-CA-4 derivative and preparation method and application thereof - Google Patents

Abstract

The invention discloses a Millepachine-CA-4 derivative, a preparation method and application thereof, the derivative has good stability, high anti-tumor activity and small toxic and side effects, effectively overcomes the defects that CA-4 has cardiovascular toxicity under high dosage, and CA-4 is unstable, is easy to photolyze, is easy to generate cis-trans isomerization, has poor water solubility and the like, and can be used for preparing anti-tumor medicaments.

Description

Millepachine-CA-4 derivative and preparation method and application thereof

Technical Field

The invention relates to the field of pharmaceutical chemistry, in particular to a Millepachine-CA-4 derivative, a preparation method thereof and application thereof in preparing anticancer drugs.

Background

Microtubules widely exist in eukaryotic cells and serve as key components of cytoskeleton, and the microtubules (Tubulin) are protein dimers consisting of α Tubulin (α -Tubulin) and β Tubulin (β -Tubulin), so that the inhibition of Tubulin aggregation can prevent the formation of spindle, prevent tumor cell mitosis from normally progressing, block cell cycle, and inhibit tumor cell growth.

Tubulin inhibitors (TPIs) are a class of anticancer drugs that act on Tubulin (Tubulin) to prevent cell proliferation. The natural products are always important resources for research and development of antitumor drugs, and the current TIPs clinically used for first-line antitumor mainly comprise taxanes, epothilones, camptothecins and vinblastines, and Eribulin (Eribulin) which is successfully researched and developed in Japan and is recently sold in China. However, these TPIs often have the disadvantages of precious sources, complex molecular structures and difficulty in large-scale synthesis, so natural pharmaceutical workers have been looking for finding TIPs with relatively simple structures, easy chemical synthesis and excellent therapeutic effects.

Millepachine is a natural product which is extracted from Millettia crassipes and has the anti-tumor activity of a benzofuran heterocyclic structure, has the characteristic of relatively simple structure, and the anti-tumor effect needs to be further improved by structural modification. Combretastatin A-4(CA-4) is a cis-stilbene 'star small molecule natural product' with simple structure acting on Tubulin, and is one of the most active compounds in the currently known TPIs. The disodium phosphate form CA-4P has already finished the clinical trial of stage III, the one that is proposed as antithyroid cancer is on the market in Europe; however, the results of subsequent clinical trials show that CA-4P has cardiovascular toxicity at high dosage and CA-4 has the disadvantages of instability, photolysis and cis-trans isomerization, and the clinical trials have been stopped by the FDA at present. Since the discovery of CA-4, researchers have been working on the structural modification, and medicinal chemists have designed and synthesized about 280000 compounds in order to find lead compounds with better activity and better pharmaceutical prospect, wherein the derivatives of CA-4 with stilbene structure, namely Ombrabulin (AVE8062), the derivatives of CA-4 with benzophenone, namely BNC105P12, and the like all enter the clinical research stage.

Therefore, the Millepachine and the CA-4 have the advantages of simple structure and excellent antitumor activity, but also have the defects of instability, poor bioavailability, cardiovascular toxicity and the like. Based on the early-stage basis, the invention designs a series of Millepachine-CA-4 derivatives with novel structures by mixing benzofuran heterocycles in two natural products, namely Millepachine, and a stilbene structure in CA-4.

Disclosure of Invention

The invention aims to provide a Millepachine-CA-4 derivative which has good stability, high antitumor activity and small toxic and side effects, so as to overcome the defects that CA-4 has cardiovascular toxicity under high dosage, and CA-4 is unstable, is easy to photolyze, is easy to generate cis-trans isomerization, has poor water solubility and the like.

Another object of the present invention is to provide a process for producing the above Millepachine-CA-4 derivative.

The invention also aims to provide an anti-tumor pharmaceutical composition which takes millepaine-CA-4 derivatives as active ingredients.

The invention further aims to provide application of the Millepachine-CA-4 derivative in preparing antitumor drugs.

The purpose of the invention is realized by the following technical scheme:

a Millepachine-CA-4 derivative has a structural formula shown in formula I:

wherein Ar is A, B or C three different substituted benzopyran heterocycles, X is O, S, NCH₃Or Se; y is C or O; r₁Is halogen, hydroxy, alkoxy, amino or alkylamino; r₂、R₃、R₄、R₅Independently selected from hydrogen, hydroxy, amino, halogen, trifluoromethyl, alkoxy or disodium phosphate.

The alkoxy is preferably C1-C4 linear or branched chain alkoxy; more preferably OCH₃、OCH₂CH₃。

The alkylamino is preferably a linear or branched alkylamino of C1-C3; more preferably NHCH₃、NCH₂CH₃、N(CH₃)₂。

The preparation method of the Millepachine-CA-4 derivative comprises the following steps:

(1) synthesis of benzopyran bromo intermediate: respectively carrying out nucleophilic substitution reaction on 3-chloro-3-methyl-1-butyne and different substituted bromobenzene 1/bromobenzene 4/bromobenzene 7 in the presence of 1, 8-diazabicycloundecene-7-ene (DBU) and cuprous chloride to respectively obtain an intermediate 2/an intermediate 5/an intermediate 8; under the conditions of 60-80 ℃ and pyridine existence, the intermediate 2/the intermediate 5/the intermediate 8 are subjected to intramolecular ring closure reaction to respectively obtain a benzopyran bromo-intermediate 3/an intermediate 6/an intermediate 9;

(2) in the presence of n-butyllithium, reacting the benzopyran bromo-intermediate 3/intermediate 6/intermediate 9 with different substituted benzaldehydes 10 respectively to obtain a benzhydryl alcohol intermediate 11; then oxidizing by 2-iodoxybenzoic acid (IBX) to obtain a benzophenone intermediate 12; in the presence of n-butyl lithium (n-BuLi), performing a witting reaction on the benzophenone intermediate 12 and methyl triphenyl phosphonium bromide to obtain an intermediate 13;

(3) under the catalytic condition of palladium carbon, carrying out hydrogenation reduction reaction on the benzophenone intermediate 12 or the intermediate 13 to obtain an aniline intermediate 14; reacting the aniline intermediate 14 with a haloacetyl halide, preferably bromoacetyl bromide or chloroacetyl chloride, in the presence of potassium carbonate to provide the target compound 15; the target compound 15 reacts with different substituted sodium alkoxides, ammonia water or different substituted amines respectively to obtain a target compound 16/a target compound 17/a target compound 18 with different substituted amide side chains respectively.

In the step (1), the molar use ratio of 3-chloro-3-methyl-1-butyne to bromobenzene is (1-3): 1, the molar use ratio of DBU to bromobenzene is (1-3): and 1, stirring for 5-10 hours at room temperature under the reaction condition.

In the step (1), the condition of intramolecular ring closure reaction of the intermediate 2/the intermediate 5/the intermediate 8 is reflux reaction at 60-80 ℃ for 8-12 hours.

In the step (2), the specific preparation steps of the benzhydrol intermediate 11 are as follows: under the protection of argon, 2-3 parts by mole of benzopyran bromo-intermediate 3/intermediate 6/intermediate 9 are dissolved in anhydrous Tetrahydrofuran (THF), the solution is cooled to-60 to-80 ℃, then 2-3 parts by mole of n-butyl lithium is slowly added, the stirring and balancing are carried out for 0.5 to 1 hour, 1 part by mole of THF solution of different substituted benzaldehydes is added, the stirring is continued for 12 to 24 hours, after the reaction is finished, a saturated ammonium chloride solution is added for quenching reaction, ethyl acetate is extracted to obtain an organic phase, and after the reaction is finished, a crude product is subjected to reduced pressure drying and column chromatography separation to obtain a benzhydryl alcohol intermediate 11.

In the step (2), the preparation of the benzophenone intermediate 12 comprises the following steps: dissolving 1 molar part of the benzhydryl alcohol intermediate 11 in a THF solution, adding 2-5 molar parts of 2-iodoxybenzoic acid (IBX) powder under the stirring condition, stirring for 5-10 hours, filtering out a precipitate after TLC monitoring reaction is finished, concentrating the filtrate to obtain a crude product, and performing column chromatography separation to obtain the benzophenone intermediate 12.

In the step (2), the preparation steps of the intermediate 13 are as follows: under the protection of argon, dissolving 2-3 molar parts of methyl triphenyl phosphonium bromide in anhydrous THF, cooling the solution to-60-80 ℃, slowly dropwise adding 2-3 molar parts of n-BuLi, gradually increasing to 20-25 ℃, stirring for 0.5-1 hour, cooling the mixture to-60-80 ℃, slowly dropwise adding 1 molar part of a THF solution of a benzophenone intermediate 12, stirring for 0.5-1 hour, gradually increasing to 20-25 ℃, stirring for 12-24 hours, monitoring the reaction by TLC, adding a saturated ammonium chloride solution, quenching the reaction, extracting with ethyl acetate to obtain an organic phase, and separating a crude product after decompression column chromatography drying to obtain an intermediate 13.

In the step (3), the aniline intermediate 14 is prepared by the following steps: adding 10-15% (g/g) palladium carbon into 1 molar part of a THF solution of a benzophenone intermediate 12 or an intermediate 13 under an ice bath condition, reacting for 6-12 hours under a hydrogen condition, filtering to remove the palladium carbon, drying a filtrate under reduced pressure, and performing column chromatography separation on a crude product to obtain an aniline intermediate 14.

In step (3), the target compound 15 is prepared as follows: dissolving 1 molar part of aniline intermediate 14 in Dichloromethane (DCM) solution, adding 8-10 molar parts of potassium carbonate, stirring at room temperature for 0.5-1 hour, dropwise adding 5-10 molar parts of halogenated acetyl halogen (preferably bromoacetyl bromide or chloroacetyl chloride solution) at the temperature of 0-4 ℃ in an ice bath, continuing to react for 4-8 hours, after TLC monitoring reaction is finished, extracting with ethyl acetate to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 15.

In step (3), the target compound 16 is prepared as follows: dissolving 1 molar part of target compound 15 in methanol, adding 2-5 molar parts of sodium alkoxide (preferably C1-C4 sodium alkoxide) with different chain lengths, refluxing and stirring for 5-12 hours at 50-80 ℃, extracting with ethyl acetate to obtain an organic phase after TLC monitoring reaction is finished, and performing column chromatography separation on a crude product after decompression drying to obtain the target compound 16.

In step (3), the target compound 17 is prepared as follows: dissolving 1 molar part of target compound 15 in ethanol, dropwise adding 5-10 molar parts of ammonia water solution, stirring at room temperature for 1-6 hours, performing TLC monitoring reaction, extracting with ethyl acetate to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain the target compound 17.

In step (3), the target compound 18 is prepared as follows: dissolving 1-2 parts by mole of a target compound 15 in acetone, adding 2-4 parts by mole of potassium carbonate under stirring, then adding 1-2 parts by mole of amine hydrochloride with different chain lengths, stirring and reacting at 50-80 ℃ for 5-10 hours, after TLC monitoring reaction is finished, adding ethyl acetate for extraction to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 18.

The reaction formula of the step (1) is as follows:

the reaction formula of the step (2) is as follows:

the reaction formula of the step (3) is as follows:

in the above formula, X' is halogen; 16a-16c R₁Is hydroxy or alkoxy; 17a-17c R₁Is amino; 18a-18c R₁Is alkylamino.

An anti-tumor pharmaceutical composition contains a therapeutically effective amount of the Millepachine-CA-4 derivative or a pharmaceutically acceptable prodrug, salt, hydrate, solvate, crystal form or diastereoisomer thereof as an active ingredient, and one or more pharmaceutically acceptable carriers. The various dosage forms of the pharmaceutical composition may be prepared according to conventional manufacturing methods in the pharmaceutical art, such as by mixing the active ingredient with one or more carriers and then formulating the same into the desired dosage form.

The pharmaceutically acceptable carrier refers to a pharmaceutical carrier conventional in the pharmaceutical field, and includes an excipient, a disintegrant, a binder, a lubricant, an antioxidant, a coating agent, a colorant, a fragrance, or a surfactant.

The Millepachine-CA-4 derivative and the pharmaceutical composition are used for preparing antitumor drugs. The antitumor drug is mainly used for treating lung cancer, small cell lung cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, breast cancer, prostate cancer, breast cancer, cervical cancer, ovarian cancer, melanoma, squamous cell carcinoma, long fiber cell tumor, medulloblastoma, acute lymphatic leukemia, multiple myeloma and the like.

The amount of Millepachine-CA-4 derivative administered will vary depending on the route of administration, the age, weight, disease and severity of the condition being treated, etc.

Compared with the prior art, the invention has the following advantages and effects:

(1) the Millepachine-CA-4 derivative with a benzofuran heterocyclic structure is a novel tumor tubulin inhibitor, and is a novel small molecular compound which is obtained by performing mixed transformation on structures of two natural products to improve the anti-tumor activity and obviously improve the defect of CA-4.

(2) The derivative has obvious inhibition effect on various tumor cells, and the anti-proliferation inhibition activity of the derivative on a human lung cancer cell strain A549 is improved by nearly 250 times compared with that of Millepachine.

(3) The derivative has small toxic and side effects on normal human cells, has higher selectivity on various normal human cell strains, and reduces the toxicity on normal cells by about 40 times compared with CA-4P.

(4) Herg cardiotoxicity evaluation tests show that the derivative of the invention has almost no cardiotoxicity, and the safety is obviously improved compared with a positive drug CA-4P.

(5) The metabolic stability test of the liver microsome shows that the derivative has better metabolic stability in the liver microsome, and the length of t1/2 is more than 4 hours and is about 8 times of that of the positive drug CA-4P.

(6) The tumor inhibition rate of the derivative is 78 percent, the tumor inhibition rate of the positive drug CA-4P is 38.5 percent, and the tumor inhibition rate of the control compound Millepachine is only 15.1 percent, and the result shows that the antitumor activity of the newly designed Millepachine-CA-4 derivative is obviously improved.

Drawings

FIG. 1 is a graph of mice sacrificed after completion of the experiment.

FIG. 2 is a block diagram of a tumor of a mouse dissected after the end of an experiment.

FIG. 3 is a graph of tumor volume growth in groups of mice during dosing.

FIG. 4 is a graph showing the weight of the exfoliated tumor mass in each group.

FIG. 5 is a body weight graph of each group of animals.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The synthesis method of A, B, C three series antitumor compounds provided by the invention comprises the following steps:

EXAMPLE 1 preparation of Compound A-1

(1) Intermediate 3: preparation of 5-methoxy-2, 2-dimethyl-2H-pyran-benzaldehyde.

Commercial raw material 1(1mmol) was dissolved in 10mL of acetonitrile, DBU (2mmol), 3-chloro-3-methyl-1-butyne (1.5mmol), and iminoketone chloride (0.1mmol) were added in this order with stirring under ice-bath conditions, and after stirring for 8 hours, diluted hydrochloric acid was added to adjust pH 2, followed by extraction with ethyl acetate. The organic phase is washed with saturated common salt water, dried over anhydrous sodium sulfate, the solvent is removed under reduced pressure to obtain a crude product, and the crude product is separated by column chromatography (petroleum ether: ethyl acetate, 10:1) to obtain an intermediate 2.

Intermediate 2(1mmol) was dissolved in 10mL pyridine, reacted at 70 ℃ for 10 hours and then removed under reduced pressureThe solvent was removed to give a crude product, which was separated by column chromatography (petroleum ether: ethyl acetate, 10:1) to give intermediate 3. Yellow oil, yield 68%,¹H NMR(400MHz,CDCl₃)δ10.31(s,1H),7.69(d,J＝8.8Hz,1H),6.64(d,J＝10.1Hz,1H),6.49(d,J＝8.8Hz,1H),5.64(d,J＝10.1Hz,1H),3.89(s,3H),1.48(s,6H)。

(2) intermediate 12: preparation of (5-methoxy-2, 2-dimethyl-2H-pyran) (4-methoxy-2-nitro) benzophenone

Intermediate 3(2mmol) was dissolved in anhydrous THF (20mL) under argon protection, and the solution was then cooled to-78 ℃; n-BuLi (2mmol) was then slowly added dropwise to the solution and stirred for 0.5h, followed by addition of a commercial solution of starting material 10(1mmol) in THF. After stirring for 20h, adding saturated ammonium chloride solution to quench the reaction, extracting with ethyl acetate, combining the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, and removing the solvent under reduced pressure to obtain a crude product, namely an intermediate 11.

Intermediate 11(1mmol) was dissolved in THF (10mL) and IBX (3mmol) was added portionwise. Stirring at room temperature for 8h, and filtering out precipitate; the filtrate was concentrated to give crude product, which was separated by column chromatography (petroleum ether: ethyl acetate, 3: 1) to give intermediate 12 as an orange solid in 85% yield.¹H NMR(400MHz,Chloroform-d)δ7.87(d,J＝7.5Hz,1H),7.60(d,J＝7.5Hz,1H),7.51(s,1H),7.27(dd,J＝7.5,2.0Hz,1H),6.52(d,J＝10.8Hz,1H),6.45(d,J＝7.5Hz,1H),5.20(d,J＝10.8Hz,1H),3.91(s,3H),3.81(s,3H),1.49(s,6H)。

(3) Preparation of intermediate (2-amino-4-methoxy) (5-methoxy-2, 2-dimethyl-2H-pyran) benzophenone (14)

Dissolving intermediate 12(500mg) in THF solution (3:1,60mL), adding 10% (g: g) palladium-carbon powder under stirring in ice bath, reacting at room temperature for 10 hours under hydrogen protection, and monitoring the reaction completion by TCL dot plateAfter completion, the palladium-carbon was removed by filtration, the filtrate was subjected to vacuum to remove the solvent to obtain a crude product, which was subjected to column chromatography (petroleum ether: ethyl acetate, 10:1) to obtain intermediate 14. The yield is 89 percent,¹H NMR(400MHz,Chloroform-d)δ7.60(d,J＝7.5Hz,1H),7.40(d,J＝7.5Hz,1H),6.86(s,2H),6.72(dd,J＝7.5,2.0Hz,1H),6.53(d,J＝10.8Hz,1H),6.48(d,J＝7.5Hz,1H),5.77(s,0H),5.21(d,J＝11.0Hz,1H),3.91(s,3H),3.81(s,3H),1.51(s,6H)。

(4) preparation of target Compound A-1

Intermediate 14(1mmol) was dissolved in 10mL of dichloromethane, potassium carbonate powder (8mmol) was added with stirring under ice-bath conditions, and after stirring for 5min bromoacetyl bromide (8mmol) was slowly added dropwise. Slowly heating the system to room temperature for reaction for 5 hours, then quenching by using a saturated sodium bicarbonate solution, and extracting by using DCM for three times; and (3) combining organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, removing the solvent under reduced pressure to obtain a crude product, and performing column chromatography separation (petroleum ether: ethyl acetate, 10:1) to obtain the target compound A-1. White solid, yield 78%,¹H NMR(400MHz,Chloroform-d)δ9.90(s,1H),7.52(t,J＝7.7Hz,2H),7.29(s,1H),6.85(dd,J＝7.5,2.0Hz,1H),6.66–6.35(m,2H),5.19(d,J＝11.0Hz,1H),4.24(s,2H),3.91(s,3H),3.81(s,3H),1.46(s,6H).¹³C NMR(101MHz,Chloroform-d)δ197.96,166.17,159.04,157.58,155.45,140.93,130.28,127.03,126.10,122.91,117.66,116.28,111.37,108.73,107.61,106.65,77.48,56.01,55.56,38.48,27.87。

the compound B-1 and the compound C-1 have the same synthesis method as the compound A-1, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-1, a white solid, 85% yield.¹H NMR(400MHz,Chloroform-d)δ9.09(s,1H),7.47(d,J＝7.5Hz,1H),7.37(d,J＝1.9Hz,1H),7.21(d,J＝7.5Hz,1H),7.05(d,J＝7.5Hz,1H),6.69-6.55(m,1H),6.35(d,J＝11.0Hz,1H),5.42(d,J＝10.8Hz,1H),4.05(s,2H),3.95(s,3H),3.90(s,3H),1.58(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.38,166.17,155.01,148.82,141.48,140.93,131.77,130.82,125.74,124.95,122.91,120.11,111.75,108.73,106.65,76.73,56.47,56.10,29.72,27.67.

Compound C-1, white solid, yield 83%,¹H NMR(400MHz,Chloroform-d)δ9.10(s,1H),7.85(d,J＝1.9Hz,1H),7.35(d,J＝1.8Hz,1H),6.83(d,J＝9.0Hz,1H),6.28(d,J＝10.8Hz,1H),5.64(d,J＝11.0Hz,1H),3.92(s,2H),3.89(s,3H),3.59(s,3H),1.51(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.99,166.17,158.98,146.10,144.75,141.00,133.30,131.51,130.28,123.23,122.88,122.35,120.26,109.31,108.75,106.75,77.53,56.28,55.56,29.71,28.07.

EXAMPLE 2 preparation of Compound A-2

Dissolving the compound A-1(2mmol) in dry methanol, adding sodium methoxide (6mmol) under stirring at room temperature, refluxing for 8h, and after the reaction is finished, cooling the system to room temperature. Ethyl acetate was added for extraction, and the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and then the solvent was removed under reduced pressure. And purifying the crude product by column chromatography to obtain a target product A-2. White solid, 73% yield.¹HNMR(400MHz,Chloroform-d)δ9.24(s,1H),7.57(s,2H),7.27(d,J＝7.5Hz,1H),6.85(d,J＝7.5,1H),6.66(d,J＝1.9Hz,1H),6.47(d,J＝7.5Hz,1H),5.23(d,J＝8.8Hz,1H),4.10(d,J＝4.9Hz,2H),3.95(s,3H),3.87(s,3H),2.87(t,J＝5.0Hz,1H),1.44(s,6H).¹³C NMR(101MHz,Chloroform-d)δ197.80,171.54,159.04,155.17,152.77,140.60,134.73,127.03,126.10,122.91,117.66,115.30,111.37,108.73,107.61,106.65,77.48,61.00,56.27,55.93,27.87。

The synthesis methods of the compounds B-2 and C-2 are the same as the synthesis method of the compound A-2, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-2, yellow solid, yield 72%,¹H NMR(400MHz,Chloroform-d)δ9.50(s,1H),8.65(s,1H),7.33(d,J＝7.5Hz,1H),6.93(d,J＝7.3Hz,1H),6.69(dd,J＝7.5,2.0Hz,1H),6.63(d,J＝7.5Hz,1H),6.26(d,J＝10.8Hz,1H),5.22(d,J＝11.0Hz,1H),4.16(s,2H),3.93(d,J＝11.1Hz,6H),2.34(t,J＝4.9Hz,1H),1.50(s,6H).¹³C NMR(101MHz,Chloroform-d)δ197.90,170.74,159.04,151.86,151.04,148.82,139.09,133.74,132.84,125.74,124.95,122.91,118.32,111.72,106.22,102.89,77.38,61.77,56.32,55.56,27.67。

compound C-2, white solid, 73% yield,¹H NMR(400MHz,Chloroform-d)δ9.30(s,1H),7.84(s,1H),7.45(d,J＝7.5Hz,1H),7.33(s,1H),7.09(d,J＝1.9Hz,1H),6.92(d,J＝8.3Hz,1H),6.31(d,J＝9.9Hz,1H),5.13(d,J＝10.8Hz,1H),4.20(d,J＝2.9Hz,2H),3.98(s,3H),3.91(s,3H),3.34(t,J＝5.0Hz,1H),1.52(s,6H).¹³C NMR(101MHz,Chloroform-d)δ199.02,171.86,158.98,152.81,147.57,145.79,137.12,135.11,133.02,123.023,122.88,122.35,120.26,110.61,107.26,104.85,77.53,60.88,56.28,55.56,28.07.

EXAMPLE 3 preparation of Compound A-3

Compound A-1(2mmol) was dissolved in ethanol (15mL), and 25% NH was added with stirring₃·H₂O (8mmol), and reacted at room temperature for 3 hours. After the reaction, ethyl acetate was added to the system to extract, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate and then the solvent was removed under reduced pressure. And purifying the crude product by column chromatography to obtain a target compound A-3. White solid, yield 65%.¹HNMR(400MHz,Chloroform-d)δ9.26(s,1H),7.92(s,2H),7.59(d,J＝7.5Hz,1H),7.26(d,J＝7.5Hz,1H),6.72(d,J＝9.5Hz,1H),6.47(d,J＝8.6Hz,1H),5.27(d,J＝10.8Hz,1H),3.85(s,2H),3.83(s,3H),3.81(s,3H),1.54(s,2H),1.47(s,6H).¹³C NMR(101MHz,Chloroform-d)δ194.7,168.48,159.04,155.45,150.13,140.93,132.53,128.36,127.71,123.34,117.66,116.75,111.37,109.43,106.65,103.37,76.48,56.01,55.56,44.32,27.82。

The synthesis methods of the compounds B-3 and C-3 are the same as the synthesis method of the compound A-3, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-3, yellow solid, 80% yield,¹H NMR(500MHz,Chloroform-d)δ9.31(s,1H),8.62(s,1H),7.42(d,J＝8.2Hz,1H),6.75(d,J＝7.5Hz,1H),6.64(d,J＝7.3Hz,1H),6.36(d,J＝4.4Hz,1H),6.18(d,J＝11.0Hz,1H),5.12(d,J＝11.0Hz,1H),3.90(s,2H),3.85(s,6H),1.82(s,2H),1.69(s,6H).¹³C NMR(101MHz,Chloroform-d)δ195.35,169.46,159.04,150.65,148.33,141.13,132.03,131.31,129.81,125.74,124.15,122.01,119.05,116.30,109.66,107.06,77.38,56.32,56.12,45.41,27.63.

compound C-3, white solid, yield 69%,¹H NMR(400MHz,Chloroform-d)δ9.24(s,1H),8.05(s,1H),7.49-7.47(m,1H),7.09(d,J＝1.9Hz,1H),6.80(d,J＝1.9Hz,1H),6.77(d,J＝7.5,1.9Hz,1H),6.37(d,J＝11.2Hz,1H),5.57(d,J＝10.8Hz,1H),3.96(s,3H),3.94(s,2H),3.92(s,3H),1.62(s,2H),1.54(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.73,168.90,158.98,146.71,143.82,140.24,134.26,131.20,130.49,123.52,122.88,121.75,120.77,109.31,107.90,105.25,78.03,56.28,55.20,44.74,28.31.

example 4 preparation of Compounds A-4, A-5, A-6

Compound a-1(2mmol) was dissolved in acetone, potassium carbonate (4mmol) was added with stirring, and then the corresponding methylamine hydrochloride/ethylamine hydrochloride/dimethylamine hydrochloride (2mmol) were added, respectively. Stirring and reacting for 3h at 70 ℃, cooling to room temperature after the reaction is finished, and adding ethyl acetate for extraction. Washing the organic phase with saturated salt water, drying with anhydrous sodium sulfate, removing the solvent under reduced pressure, and purifying the crude product by column chromatography to obtain corresponding target compounds A-4, A-5 and A-6.

Compound A-4, a yellow solid, 86% yield.¹H NMR(400MHz,Chloroform-d)δ9.55(s,1H),7.84(d,J＝1.9Hz,2H),7.52-7.50(m,1H),7.40(d,J＝7.5Hz,1H),6.72(d,J＝7.7Hz,1H),6.56(d,J＝8.9Hz,1H),5.34(d,J＝8.8Hz,1H),3.94(s,3H),3.91(s,3H),3.25(s,2H),2.82(s,1H),2.53(s,3H),1.47(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.64,169.71,159.73,158.47,156.47,141.90,130.63,127.45,126.59,123.43,118.05,116.80,111.87,109.27,107.98,107.09,77.95,56.40,55.78,52.73,26.98,27.82。

Compound A-5, a yellow solid, yield 79%.¹H NMR(400MHz,Chloroform-d)δ9.38(s,1H),8.33(s,2H),7.56(d,J＝7.5Hz,1H),7.40(d,J＝7.5Hz,1H),6.74(dd,J＝7.5,1.9Hz,1H),6.62(d,J＝11.0Hz,1H),5.28(d,J＝10.8Hz,1H),3.95(s,3H),3.86(s,3H),3.55(s,2H),2.67(q,J＝8.0Hz,2H),1.51(s,6H),1.13(t,J＝8.0Hz,3H).¹³C NMR(101MHz,Chloroform-d)δ194.60,170.90,159.97,158.23,156.42,141.55,131.17,127.54,126.61,123.44,118.08,116.92,112.43,109.48,108.17,107.24,78.09,56.99,56.24,52.44,43.79,27.27,16.20。

Compound A-6, a yellow solid, 81% yield.¹H NMR(400MHz,Chloroform-d)δ9.45(s,1H),8.06(d,J＝1.9Hz,2H),7.56(d,J＝7.5Hz,1H),7.38(d,J＝7.5Hz,1H),6.75(dd,J＝7.4,1.9Hz,1H),6.47(d,J＝10.8Hz,1H),5.27(d,J＝11.0Hz,1H),3.94(s,3H),3.87(s,3H),3.14(s,2H),2.58(s,6H),1.59(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.80,169.35,159.79,158.29,156.01,141.67,130.89,127.71,126.57,123.67,118.24,116.74,112.13,109.43,108.19,107.08,77.69,63.61,56.52,55.81,46.65,28.14。

The synthesis methods of the compounds B-4, B-5, B-6, C-4, C-5 and C-6 are the same as the synthesis methods of the compounds A-4, A-5 and A-6, the structural formulas are shown in the table 1, and the characterization data are as follows:

compound B-4, yellow solid, 73% yield,¹H NMR(400MHz,Chloroform-d)δ9.26(s,1H),8.13(d,J＝2.0Hz,1H),7.39(d,J＝7.5Hz,1H),6.97(d,J＝7.5Hz,1H),6.90(d,J＝7.5Hz,1H),6.74-6.72(m,1H),6.35(d,J＝10.8Hz,1H),5.22(d,J＝11.0Hz,1H),3.94(s,3H),3.86(s,3H),3.26(s,2H),2.63(s,3H),1.51(s,6H).¹³C NMR(101MHz,Chloroform-d)δ191.56,169.68,159.66,149.36,141.92,141.34,132.33,131.33,130.53,126.30,125.36,123.41,120.47,112.10,109.11,107.24,77.92,56.71,56.03,52.50,36.50,27.91.

compound B-5, yellow solid, 76% yield,¹H NMR(400MHz,Chloroform-d)δ9.32(s,1H),8.16(s,1H),7.41(d,J＝7.5Hz,1H),7.06(d,J＝3.8Hz,1H),6.97(d,J＝3.8Hz,1H),6.77(dd,J＝7.5Hz,2.0Hz,1H),6.38(d,J＝11.0Hz,1H),5.22(d,J＝10.8Hz,1H),3.92(s,3H),3.83(s,3H),3.52(s,2H),2.74(q,J＝8.0Hz,2H),1.65(s,6H),1.22(t,J＝8.0Hz,3H).¹³CNMR(101MHz,Chloroform-d)δ198.84,170.81,159.70,149.95,141.73,141.14,132.04,131.13,130.54,126.00,125.18,123.13,120.49,111.97,109.72,107.52,78.23,56.28,55.94,52.24,43.83,27.87,15.73.

compound B-6, yellow solid, yield 79%,¹H NMR(400MHz,Chloroform-d)δ9.39(s,1H),8.09(d,J＝2.0Hz,1H),7.39(d,J＝7.5Hz,1H),6.97(d,J＝7.5Hz,1H),6.91(d,J＝7.5Hz,1H),6.73(dd,J＝7.5,2.0Hz,1H),6.36(d,J＝10.8Hz,1H),5.20(d,J＝10.8Hz,1H),3.91(s,3H),3.82(s,3H),3.13(s,2H),2.80(s,6H),1.65(s,6H).¹³C NMR(101MHz,Chloroform-d)δ198.37,169.14,159.04,149.34,141.83,141.23,132.26,131.33,130.62,126.19,125.36,123.50,120.82,112.68,109.28,106.18,77.81,63.63,56.74,56.03,46.50,28.03。

compound C-4, brown solid, 77% yield,¹H NMR(400MHz,Chloroform-d)δ9.35(s,1H),8.12(s,1H),7.50(d,J＝7.4Hz,2H),7.13(d,J＝1.7Hz,1H),6.75(dd,J＝7.4,1.8Hz,1H),6.54(d,J＝11.3Hz,1H),5.33(d,J＝10.8Hz,1H),3.91(s,3H),3.89(s,3H),3.21(s,2H),2.56(s,3H),1.53(s,6H).¹³C NMR(101MHz,Chloroform-d)δ195.08,169.32,159.39,146.53,145.10,141.61,134.11,132.08,130.72,123.62,122.57,121.90,120.44,109.64,108.98,107.08,78.72,57.30,55.97,53.23,37.53,28.72.

compound C-5, yellow solid, 77% yield,¹H NMR(400MHz,Chloroform-d)δ9.47(s,1H),7.89(s,1H),7.48(d,J＝10.8Hz,2H),7.10(d,J＝2.0Hz,1H),6.74(dd,J＝7.4,1.9Hz,1H),6.53(d,J＝10.9,1.0Hz,1H),5.26(d,J＝11.0Hz,1H),3.99(s,3H),3.86(s,3H),3.49(s,2H),2.65(q,J＝8.0Hz,2H),1.64(s,6H),0.96(t,J＝8.0Hz,3H).¹³C NMR(101MHz,Chloroform-d)δ198.02,171.56,159.81,147.11,145.34,141.71,133.73,132.11,130.87,123.45,123.11,122.61,120.50,109.62,108.97,107.11,78.54,56.57,55.76,53.00,44.25,28.84,15.94.

compound C-6, brown solid, 77% yield,¹H NMR(400MHz,CDCl₃)δ9.54(s,1H),8.02(s,1H),7.50(d,J＝1.2Hz,2H),7.05(s,1H),6.76(d,J＝7.9Hz,1H),6.38(d,J＝9.9Hz,1H),5.21(d,J＝9.9Hz,1H),3.93(s,3H),3.81(s,3H),3.26(s,2H),2.81(s,6H),1.63(s,6H).¹³CNMR(101MHz,Chloroform-d)δ199.68,169.09,159.38,146.59,144.96,141.61,133.76,131.91,130.80,123.52,123.08,122.58,120.41,109.56,108.95,106.98,77.74,63.48,56.62,55.97,46.31,28.50.

EXAMPLE 5 preparation of Compound A-7

(1) Preparation of intermediate 5-methoxy-2- (1- (5-methoxy-2, 2-dimethyl-2H-pyran)) aniline (14)

Preparation of intermediate 13: methyl triphenyl phosphonium bromide (2mmol) was dissolved in anhydrous THF (30mL) under argon protection, and then the reaction was cooled to-78 ℃. n-BuLi (2mmol) was added slowly to the mixture, warmed to room temperature and stirred for 0.5 h. The mixture was cooled again to-78 ℃ and a solution of intermediate 12(1mmol) in THF (2mL) was added dropwise, stirred for 0.5h, then warmed to room temperature and stirred for 18 h; after the reaction is finished, adding saturated ammonium chloride solution for quenching, and extracting by ethyl acetate. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give a crude product, which was separated by column chromatography (petroleum ether: ethyl acetate, 3: 1) to give intermediate 13.

Preparation of intermediate 14: the intermediate 13 is used as a raw material to synthesize the intermediate 14, and the specific synthesis method is the same as the corresponding step (3) of the example 1. Yellow solid, yield 87%,¹H NMR(400MHz,Chloroform-d)δ7.15(d,J＝7.5Hz,1H),7.10(d,J＝7.5Hz,1H),6.55–6.48(m,1H),6.42(d,J＝7.5Hz,1H),5.60(s,1H),5.48(s,2H),5.44(d,J＝1.1Hz,1H),5.30(s,1H),5.24(d,J＝10.8Hz,1H),3.91(s,3H),3.81(s,3H),1.49(s,6H).

(2) preparation of target Compound A-7

The specific reaction procedure was the same as in the corresponding procedure (4) of example 1.

Compound A-7, a yellow solid, in 72% yield,¹H NMR(400MHz,Chloroform-d)δ9.90(s,1H),7.41–7.14(m,2H),7.03(d,J＝7.5Hz,1H),6.62(dd,J＝7.5,2.0Hz,1H),6.49(d,J＝11.0Hz,1H),6.37(d,J＝7.5Hz,1H),5.45(s,1H),5.34–5.23(m,2H),4.24(s,2H),3.91(s,3H),3.81(s,3H),1.66(s,6H).¹³C NMR(101MHz,Chloroform-d)δ166.17,158.01,155.15,153.39,140.32,139.09,128.64,127.61,127.03,120.59,118.20,117.66,115.70,111.09,109.88,108.23,106.47,77.48,56.01,55.56,38.48,27.87.

the synthesis methods of the compounds B-7 and C-7 are the same as the synthesis method of the compound A-7, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-7, yellow solid, 69% yield,¹H NMR(400MHz,Chloroform-d)δ9.90(s,1H),8.57(s,1H),7.20(d,J＝7.5Hz,1H),6.88–6.72(m,2H),6.61(dd,J＝7.5,2.0Hz,1H),6.43(d,J＝10.8Hz,1H),5.70(s,1H),5.55(s,1H),5.09(d,J＝11.0Hz,1H),4.24(s,2H),3.90(s,3H),3.81(s,3H),1.55(s,6H).¹³C NMR(101MHz,Chloroform-d)δ166.17,158.01,146.95,146.79,141.41,139.09,130.82,128.73,128.64,127.51,124.00,120.59,120.11,119.33,110.21,109.88,106.47,77.38,56.32,55.56,38.48,27.67.

compound C-7, yellow solid, 65% yield,¹H NMR(400MHz,Chloroform-d)δ9.90(s,1H),8.49(s,1H),7.23(d,J＝1.0Hz,1H),7.05(d,J＝7.5Hz,1H),6.68–6.56(m,2H),6.54(dd,J＝10.9,1.0Hz,1H),5.62(d,J＝1.1Hz,1H),5.37(s,1H),5.20(d,J＝10.8Hz,1H),4.24(s,2H),3.90(s,3H),3.81(s,3H),1.62(s,6H).¹³C NMR(101MHz,Chloroform-d)δ166.17,157.93,146.19,145.61,142.89,139.11,133.13,131.51,128.74,122.35,121.20,119.85,116.88,113.16,109.93,106.52,77.53,56.28,55.56,38.48,28.07.

EXAMPLE 6 preparation of Compound A-8

Compound A-8 was prepared using Compound A-1 as the starting material, the procedure for preparation being as in example 2.

Compound A-8, a white solid, 67% yield,¹H NMR(400MHz,Chloroform-d)δ9.14(s,1H),8.16(s,1H),7.28(d,J＝7.5Hz,1H),7.04(d,J＝7.5Hz,1H),6.59(d,J＝7.5Hz,1H),6.54-6.47(m,2H),5.64(d,J＝11.0Hz,1H),5.59(s,1H),5.42(s,1H),4.07(s,2H),3.91(s,3H),3.81(s,3H),3.32(t,J＝4.9Hz,1H),1.39(s,6H).¹³C NMR(101MHz,Chloroform-d)δ171.54,155.34,151.24,146.46,145.70,144.59,136.15,130.18,128.70,123.09,118.29,116.17,113.66,112.76,110.42,109.52,101.85,75.55,61.77,56.01,55.56,27.87.

the synthesis methods of the compounds B-8 and C-8 are the same as the synthesis method of the compound A-8, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-8, a white solid, in 59% yield,¹H NMR(400MHz,Chloroform-d)δ8.62(s,1H),7.72(s,1H),7.01(d,J＝7.5Hz,1H),6.67(d,J＝7.5Hz,1H),6.60-6.53(m,2H),6.21(d,J＝9.9Hz,1H),5.70(s,1H),5.30(d,J＝9.9Hz,1H),5.26(s,1H),4.06(s,2H),3.87(d,J＝5.0Hz,6H),3.31(t,J＝5.0Hz,1H),1.45(s,6H).¹³C NMR(101MHz,Chloroform-d)δ171.76,159.16,152.06,151.94,147.89,141.93,138.39,131.34,128.74,127.04,126.27,123.10,122.99,119.33,110.19,109.88,106.47,76.89,62.15,56.61,55.99,27.57.

compound C-8, white solid, 73% yield,¹H NMR(400MHz,Chloroform-d)δ8.84(s,1H),7.73(s,1H),7.06(s,1H),6.76(d,J＝8.3Hz,1H),6.62(s,2H),6.27(d,J＝7.5Hz,1H),5.87(d,J＝9.4Hz,1H),5.29(d,J＝1.8Hz,2H),4.05(s,2H),3.91(s,3H),3.82(s,3H),3.33(t,J＝4.9Hz,1H),1.49(s,6H).¹³C NMR(101MHz,Chloroform-d)δ172.92,153.66,147.85,146.42,145.61,141.79,135.13,133.13,131.51,122.48,121.20,120.36,119.08,116.88,112.53,112.15,106.52,76.75,61.77,56.28,55.56,28.07.

EXAMPLE 7 preparation of Compound A-9

Compound A-9 was prepared using compound A-8 as the starting material, the procedure for preparation being as in example 3.

Compound A-9, a white solid, 89% yield.¹H NMR(400MHz,Chloroform-d)δ9.42(s,1H),8.60(s,1H),7.20(d,J＝2.9Hz,1H),6.86(d,J＝8.3Hz,1H),6.53(d,J＝10.0Hz,2H),6.48(d,J＝8.6Hz,1H),5.63(s,1H),5.40(s,1H),5.28(d,J＝10.0Hz,1H),3.94(s,3H)3.88(s,2H),3.82(s,3H),2.52(s,2H),1.42(s,6H).¹³C NMR(101MHz,Chloroform-d)δ168.60,157.99,155.25,153.55,139.80,138.70,128.34,127.30,126.52,120.06,117.86,117.32,115.19,110.48,109.26,107.44,105.24,77.53,56.01,55.56,44.13,27.82.

The synthesis methods of the compounds B-9 and C-9 are the same as the synthesis method of the compound A-9, the structural formula is shown in the table 1, and the characterization data are as follows:

compound B-9, a white solid, 62% yield,¹H NMR(400MHz,Chloroform-d)δ9.52(s,1H),7.73(s,1H),6.97(d,J＝1.9Hz,1H),6.70(d,J＝7.5Hz,2H),6.54(dd,J＝7.5,2.1Hz,1H),6.32(d,J＝11.0Hz,1H),5.67(s,1H),5.32(s,1H),5.13(d,J＝10.8Hz,1H),3.91(s,3H),3.86(s,2H),3.83(s,3H),1.97(s,2H),1.53(s,6H).¹³C NMR(101MHz,Chloroform-d)δ159.43,147.13,146.34,141.58,139.49,131.08,128.74,128.64,127.74,125.29,121.69,120.36,119.78,110.78,109.88,106.88,77.38,56.58,55.85,44.74,27.94.

compound C-9, white solid, yield 86%,¹H NMR(400MHz,CDCl₃)δ9.43(s,1H),7.73(s,1H),7.75(s,1H),6.93(d,J＝4.5Hz,1H),6.77(d,J＝6.8Hz,2H),6.57(dd,J＝11.8,2.5Hz,1H),6.36(d,J＝11.7Hz,1H),5.65(s,1H),5.28(s,1H),5.24(d,J＝11.8Hz,1H),3.92(s,3H),3.87(s,2H),3.82(s,3H),2.00(s,2H),1.58(s,6H).¹³C NMR(101MHz,Chloroform-d)δ168.94,158.51,146.82,145.61,143.39,139.77,133.72,131.98,129.20,123.31,121.65,120.40,117.76,141.14,110.35,107.11,77.89,56.71,55.94,44.58,28.36.

example 8 preparation of Compounds A-10, A-11, A-12

Compounds A-10, A-11 and A-12 were prepared using Compound A-7 as the starting material, and the specific preparation procedure was the same as in example 4.

Compound A-10, white solid, 89% yield,¹H NMR(400MHz,CDCl₃)δ9.48(s,1H),7.76(s,1H),7.07(d,J＝7.5Hz,1H),6.98(d,J＝10.0Hz,1H),6.59(dd,J＝7.5,1.8Hz,1H),6.51(d,J＝10.8Hz,1H),6.40(d,J＝11.0Hz,1H),5.53(s,1H),5.34(s,1H),5.28(d,J＝11.0Hz,1H),3.92(s,3H),3.86(s,3H),3.25(s,2H),2.5(s,3H),1.63(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.87,156.97,154.52,152.27,141.63,139.93,129.39,128.17,126.82,121.07,118.86,117.20,115.46,111.51,110.29,108.63,107.05,77.85,56.56,55.65,51.60,36.83,27.75.

compound A-11, white solid, yield 75%,¹H NMR(400MHz,Chloroform-d)δ9.50(s,1H),8.56(s,1H),7.36(d,J＝4.2Hz,1H),7.20(d,J＝8.8Hz,1H),6.59-6.56(m,2H),6.44(d,J＝7.5Hz,1H),5.56(s,1H),5.43(s,1H),5.38(d,J＝8.8Hz,1H),3.95(s,3H),3.89(s,3H),3.46(s,2H),2.58(dd,J＝14.5,7.2Hz,2H),1.62(s,6H),1.0(t,J＝7.5Hz,3H).¹³C NMR(101MHz,Chloroform-d)δ170.28,157.85,154.91,153.11,140.54,139.32,128.96,127.85,126.68,120.69,118.34,117.24,115.23,111.53,110.02,108.33,106.11,77.75,56.27,55.81,51.75,43.67,28.33,15.34.

compound A-12, a white solid, yield 78%,¹H NMR(400MHz,Chloroform-d)9.54(s,1H),7.96(s,1H),7.09(d,J＝8.5Hz,1H),6.98(d,J＝8.2Hz,1H),6.64(dd,J＝7.9,2.4Hz,1H),6.51(d,J＝9.9Hz,1H),6.19(s,1H),5.56(d,J＝10.8Hz,1H),5.37(s,1H),5.28(d,J＝10.8Hz,1H),3.81(d,J＝2.1Hz,6H),3.17(s,2H),2.84(s,6H),1.66(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.32,158.63,156.07,154.32,146.21,144.71,139.09,130.48,129.01,121.40,118.20,116.84,115.70,113.40,110.64,109.88,106.47,78.03,63.26,56.05,55.63,46.00,27.41.

the compounds B-10, B-11, B-12, C-10, C-11 and C-12 have the same synthesis method as the compounds A-10, A-11 and A-12, the structural formula is shown in Table 1, and the characterization data are as follows:

compound B-10, white solid, 92% yield,¹H NMR(400MHz,CDCl₃)δ9.42(s,1H),7.80(s,1H),7.12(d,J＝2.2Hz,1H),6.58(dd,J＝7.5,1.7Hz,1H),6.48(m,2H),6.43(d,J＝10.8Hz,1H),5.69(s,1H),5.51(s,1H),5.19(d,J＝11.0Hz,1H),3.98(s,3H),3.92(s,3H),3.19(s,2H),2.49(s,3H),1.61(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.02,157.60,147.11,146.60,141.76,138.70,131.12,129.09,128.35,126.85,123.36,120.78,119.82,118.89,110.59,109.40,106.72,77.88,56.32,55.35,51.49,37.23,28.81.

compound B-11, white solid, yield 85%¹H NMR(400MHz,Chloroform-d)δ9.43(s,1H),7.69(s,1H),7.15(d,J＝8.8Hz,1H),6.76(d,J＝7.5Hz,1H),6.66(dd,J＝11.0,1.9Hz,2H),6.48(d,J＝7.5Hz,1H),5.65(s,1H),5.47(s,1H),5.19(d,J＝8.6Hz,1H),3.91(s,3H),3.81(s,3H),3.46(s,2H),2.67(dd,J＝13.5,4.9Hz,2H),1.57(s,6H),1.18(t,J＝8.0Hz,3H).¹³CNMR(101MHz,Chloroform-d)δ170.40,158.85,147.95,146.79,142.62,138.06,132.34,130.02,129.57,127.16,125.13,121.14,119.76,118.86,110.47,109.70,106.71,77.67,56.26,55.80,51.65,41.62,29.72,15.34.

Compound B-12, white solid, yield 81%,¹H NMR(400MHz,Chloroform-d)δ9.45(s,1H),8.61(s,1H),6.96(d,J＝2.8Hz,1H),6.74(s,2H),6.46(dd,J＝8.8,2.1Hz,1H),6.29(d,J＝10.8Hz,1H),5.76(s,1H),5.54(s,1H),5.21(d,J＝11.0Hz,1H),3.90(s,3H),3.84(s,3H),3.36(s,2H),2.83(s,6H),1.50(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.21,158.34,147.03,146.92,141.57,139.23,131.17,129.02,128.54,127.67,124.18,120.76,119.85,118.93,110.50,109.59,107.04,78.11,61.89,56.78,56.11,46.06,27.22。

compound C-10, white solid, yield 79%,¹H NMR(500MHz,Chloroform-d)δ9.45(s,1H),7.83(s,1H),6.98(d,J＝7.4Hz,1H),6.77(s,1H),6.65(d,J＝1.8Hz,1H),6.56(dd,J＝8.8,1.9Hz,1H),6.36(d,J＝9.9,1H),5.64(s,1H),5.35(s,1H),5.13(d,J＝10.8Hz,1H),3.91(s,3H),3.91(s,3H),3.22(s,2H),2.28(s,3H),1.53(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.52,158.69,146.47,145.56,143.06,138.81,133.53,131.04,128.25,122.97,120.58,119.47,117.51,113.73,109.93,107.78,106.52。

compound C-11, white solid, 73% yield,¹H NMR(400MHz,Chloroform-d)δ9.54(s,1H),7.88(s,1H),7.14(d,J＝7.5Hz,1H),6.95(s,1H),6.67(dd,J＝10.8,2.2Hz,1H),6.43(d,J＝7.2Hz,2H),5.50(s,1H),5.23(s,1H),4.97(d,J＝8.6Hz,1H),3.94(s,3H),3.90(s,3H),3.56(s,2H),2.59(dd,J＝11.8,2.6Hz,2H),1.53(s,6H),1.10(t,J＝8.8Hz,3H).¹³C NMR(101MHz,Chloroform-d)δ170.52,158.50,146.47,145.52,142.68,138.93,131.99,131.14,128.55,123.23,120.77,119.47,116.51,112.64,109.45,108.28,106.14,77.81,56.36,56.20,52.01,43.67,28.30,15.42。

compound C-12, white solid, yield 68%,¹H NMR(400MHz,Chloroform-d)δ9.54(s,1H),7.92(s,1H),7.06(d,J＝3.5Hz,1H),6.90(s,1H),6.64(dd,J＝11.8,2.5Hz,1H),6.53(s,1H),6.40(d,J＝7.5Hz,1H),5.62(s,1H),5.33(s,1H),5.13(d,J＝10.8Hz,1H),3.98(s,3H),3.91(s,3H),3.25(s,2H),2.60(s,6H),1.52(s,6H).¹³C NMR(101MHz,Chloroform-d)δ169.37,158.40,147.11,145.28,142.70,138.92,133.81,130.83,128.25,123.04,120.68,119.54,117.90,112.48,109.39,108.34,107.14,77.83,63.68,56.55,55.31,45.93,28.30。

test example 1: cell culture:

the cell lines used in the present invention include: a549 (human lung cancer cell strain), Bel-7402 (human liver cancer cell strain), Hela (human cervical cancer cell strain), MCF-7 (human breast cancer cell strain), and MGC-803 (human gastric cancer cell strain). The five cells were cultured in DMEM high-sugar medium containing 10% heat-inactivated fetal calf serum, 100U/mL penicillin and 100. mu.g/mL streptomycin at 37 ℃ with 5% CO₂Culturing in an incubator with saturated humidity.

Test example 2: antiproliferative inhibition activity test method:

in the invention, MTT method is adopted to test the proliferation inhibition activity of the synthesized compound on five human cancer cell lines, and IC is calculated₅₀The value is obtained. The cell lines used were: a549 (human lung cancer cell line), A2780 (human ovarian cancer cell line), Hela (human cervical cancer cell line), MCF-7 (human breast cancer cell line), and HL-60 (human leukemia cell line). The specific operation steps are as follows: digesting the cells in logarithmic growth phase with pancreatinAt 5X 10⁴And paving the medium in a 96-well plate at the density of each well, continuously culturing for 24h, then removing the old medium by suction, adding the medium containing the medicines with different concentrations, setting six concentrations for each medicine, and setting three multiple wells for each concentration. Adding reagent and culturing for 48h, adding 5mg/mL MTT aqueous solution into each hole, absorbing and removing the culture medium after culturing for 4h, adding 150 mu L dimethyl sulfoxide (DMSO) into each hole, keeping the hole on a horizontal shaking table in the dark for 15min to dissolve crystal violet, and reading the absorbance value at 570nM by using an enzyme labeling instrument. From the absorbance values, the cell growth inhibition at each concentration was calculated using the following formula: inhibition rate%_Dosing-A_{Blank space})/(A_control-A_{Blank space}) 100% (note: a. the_Dosing，A_{Blank space}，A_controlAbsorbance values for each well, blank, control) and then the IC of each compound was determined using GraphPad Prism 5 software₅₀The value is obtained.

Test example 3: inhibition of tubulin aggregation activity assay methods:

the test principle of the tubulin inhibition experiment used in the present invention is that when tubulin is polymerized into microtubules, the aggregation degree is detected by the inserted fluorescent probe 4,6-diamidino-2-phenyl-indole (DAPI), and the higher the aggregation degree, the stronger the fluorescence. The assay was performed using a commercial tubulin aggregation test kit (cyto-skelton, cat. # BK 011P). Before the experiment, the parameters of a fluorescence microplate reader are set according to the instruction of the kit, and a compound to be detected with corresponding concentration (the final concentration of DMSO is 1%) is prepared, and 20 muL of GTPstock, 1.5mL of Buffer 1, Tubulin glycol Buffer and 1-tube Tubulin stock solution in the kit are unfrozen on ice. In the experiment, 205. mu.L of Buffer 1, 4.4. mu.L of GTP stock, 150. mu.L of Tubulin glycol Buffer and 85. mu.L of Tubulin stock solution were added to the reaction solution, mixed and placed on ice. Adding 5 μ L of compounds to be tested with different concentrations into a 96-well plate, adding equal amount of blank solvent into a blank control group, preheating at 37 deg.C for 1min, and rapidly adding 55 μ L of the prepared reaction solution. Dynamically detecting the polymerization of the whole microtube by a fluorescence microplate reader under the conditions that the emission wavelength is 410nm, the excitation wavelength is 340nm and the incubation temperature is 37 DEG CThe reaction was continued for 90min, with readings every 1 min. After the reading is finished, the inhibition rate at each concentration is calculated according to the reading, and the half Inhibition Concentration (IC) is obtained by GraphPad Prism 5 according to the result of the concentration-inhibition rate₅₀). The experimental results were repeated three times or more, and the average was taken as the final result.

Test example 4: evaluation of cardiotoxicity in hERG Potassium channel inhibition experiments

CA-4P is found to have cardiotoxicity in clinical trials, and the compounds designed by the invention are CA-4 derivatives, so the work in this section adopts hERG experiments to evaluate the cardiotoxicity of candidate molecules. CHO-hERG recombinant cells grown in log phase were taken and digested with cell dissociation reagent Detachin. After complete digestion, adding a culture medium to adjust the cell density to 2-5 multiplied by 10⁶Adding the cell suspension to a Qpatch instrument to complete the processes of single cell high-impedance sealing and whole cell mode formation, clamping the cells at-80 mV after obtaining a whole cell recording mode, giving 40mV depolarization stimulation, giving a pre-voltage of-50 mV for 50ms, then repolarizing to-50 mV to maintain for 5s, and then returning to-80 mV. This voltage stimulus was applied every 15s, and after 2min of recording, extracellular fluid was given for 1min, and then compounds were given at different concentrations, starting from the lowest test concentration, for 1min each, at least 3 groups of cells were tested at each concentration, and the experimental data were statistically analyzed by XLFit software.

Test example 5: LC/MS/MS detection of metabolic stability of candidate molecules in liver microsomes

The commercialized rat and human liver microsomes are selected for experiments, and β -NADPNa is prepared₂Regeneration system, preparation final concentration is 1.3mM β -NADPNa respectively₂3.3mM glucose-6-phosphate solution, 0.4unit/mL glucose-6-phosphate dehydrogenase solution, 3.3mM MgCl₂The solution and 1mg/mL liver microsome form a mixed system, a certain volume of the compound (final concentration is 100 mu M) of the invention is added into the mixed system, and the mixture is evenly mixed and incubated at 37 ℃ for different times. After the incubation was completed, the reaction was terminated by adding 500. mu.L of glacial acetonitrile to the system and vortexing for 30 seconds. Centrifuging at 4 deg.C and 12,500rpm/min for 10min, and subjecting the supernatant to HPLC and LC-MS/MS on-machine quantitative analysisThe remaining concentration of the compound.

Test example 6: antitumor activity of in vivo nude mouse transplantation tumor

Balb/c-nu nude mice (5 weeks old, 18-20g) were kept in an SPF barrier environment with free access to water and food. Collecting cells cultured to 3-5 generations and growing in logarithmic phase, digesting with pancreatin, and preparing into 1.0 × 10 with serum-free culture medium⁷Cell suspension at individual/mL concentration. The cell suspension is inoculated into subcutaneous tissues of the right limb of a nude mouse close to the armpit, and each injection is 0.1 mL. When the tumor mass in the body of the mouse grows to about 1000mm³In the meantime, the mice were sacrificed by cervical dislocation, tumor tissue pieces were peeled off, connective tissues and necrotic parts on the tissues were removed by surgical scissors on ice, and the tumor pieces were cut into pieces of uniform size of about 4-6mm³The tissue blocks of (4) were subcutaneously inoculated in the right axillary limb of nude mice. The length of the tissue block to be treated is as long as 100-200mm³The administration is divided into groups. Mice were randomly divided into a blank control group, a group administered with the compound of the present invention, and a group administered with the positive drug CA-4P, each group consisting of 10 mice. Intraperitoneal injection administration is carried out, a blank group is given with the same volume of physiological saline solution, administration is carried out once every other day, the length and the short diameter of the tumor tissue and the weight of a mouse are measured and recorded, and the volume of the tumor tissue is calculated according to the following formula: tumor volume (mm)³) 0.5 x long diameter x short diameter². One month after dosing, mice were sacrificed by decapitation, tumor tissue mass was dissected off, tumor mass weight was recorded, and tumor inhibition rate of the administered compound was calculated.

The results of the pharmacological activities of the above test examples are as follows

Results 1: the compounds prepared in the above examples have inhibitory activity on proliferation of human cancer cell lines and inhibitory activity on tubulin aggregation

As shown in Table 1, the compound of the invention has better anti-tumor cell proliferation activity and IC (Integrated Circuit) on five human cancer cell lines₅₀The value reaches a submicron molar level, the inhibition activity on target spot tubulin and the anti-proliferation inhibition activity on five human cancer cell lines are far better than that of a natural product Millepachine before modification, and the activities of partial preferred compounds are respectively improved by about 10 times and 80 times (see table 1).

TABLE 1 partial compounds of the invention inhibit tubulin aggregation activity in vitro and anti-proliferation inhibitory activity against five human cancer cell lines

Results 2: evaluation of toxicity of Compounds prepared in the above examples on human Normal cell lines

As shown in Table 2, part of the compounds show lower cytotoxicity to human normal cell strains, which indicates that the derivatives have small toxic and side effects.

TABLE 2 Selectivity of some of the compounds of the invention for human normal cell lines

Results 3: evaluation of early Heart safety toxicity of Compounds prepared in the above examples

In the early safety evaluation stage of new drug development, the compound inhibits IC on hERG potassium ion channel₅₀>Compounds were considered to have weak or no hERG inhibitory activity at 10 μ M; when the IC is₅₀<At 1 μ M, the compounds are considered to have strong hERG inhibitory activity, and the compounds must be evaluated before entering the clinic with emphasis on the risk of inducing cardiotoxicity. CA-4P showed cardiotoxicity in clinical trials at high doses, as shown in the table3 shows that preferred compounds of the invention inhibit hERG potassium channel inhibition IC₅₀The content of the compounds is more than 10 mu M, which indicates that the compounds have better safety.

TABLE 3 summary of hERG potassium channel inhibitory activity of the compounds of the invention

Results 4: metabolic stability results for the compounds prepared in the above examples

One of the biggest drawbacks of CA-4 is the instability of biological activity due to the cis-structural instability of the carbon-carbon double bond in the structure, and therefore the present invention uses a carbonyl structure instead of the carbon-carbon double bond in the CA-4 structure. After the compound is incubated with the human liver microsome for a certain time, the residual amount of the compound is tested by using LC-MS/MS, so that t1/2 of the compound is calculated, as shown in Table 4, the compounds prepared by the invention have better metabolic stability, and t1/2 is more than 8 times of that of a positive drug CA-4P, which indicates that the derivative has good stability.

TABLE 4 Metabolic stability of Compounds of the invention in human liver microsomes

Results 5: results of in vivo antitumor Activity of Compounds of the invention

Established by a nude mouse transplanted tumor model, one month after administration, mice were sacrificed (as shown in fig. 1), tumor tissues of the mice were exfoliated (as shown in fig. 2), weights of various tumor masses were recorded, a trend of change in tumor volume of each mouse of each group was recorded during administration (as shown in fig. 3), and a tumor inhibition rate after administration was calculated from the exfoliated tumor mass weight after the end of administration (as shown in fig. 4). The results showed that at the end of the experiment, the mean tumor volume of the control group, to which no drug treatment was administered, was 1586.88mm³The average weight was 1.809 g. The mean tumor volume of the modified natural product Millepachine administration group was 1347.93mm³The average weight was 1.492g, and the tumor inhibition rate was 15.1%. Positive control drugMean tumor volume of 976.02mm in CA-4P-treated group³The average weight was 1.092g, and the tumor inhibition rate was 38.5%. The tumor volumes and average weights of the treatment groups of the compounds A-8, B-8 and C-8 prepared in this example were 421.07mm, respectively³(0.469g)、366.07mm³(0.399g)、501.45mm³(0.556g), the tumor inhibition rates were 74.1%, 77.9%, and 69.3%, respectively. The mean body weight of each group was recorded at the end of the experiment, as shown in figure 5; there was no significant difference in body weight between the mice in each group, indicating no significant toxicity.

The above description is only an example of the present invention, but the present invention is not limited to the above example, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention and are equivalent to each other are included in the protection scope of the present invention.

Claims (9)

1. A Millepachine-CA-4 derivative is characterized in that the structural formula is shown as a formula I:

wherein Ar is A, B or C three different substituted benzopyran heterocycles, X is O, S, NCH₃Or Se; y is C or O; r₁Is halogen, hydroxy, alkoxy, amino or alkylamino; r₂、R₃、R₄、R₅Independently selected from hydrogen, hydroxy, amino, halogen, trifluoromethyl, alkoxy or disodium phosphate.

2. The Millepachine-CA-4 derivative according to claim 1, wherein: the alkoxy is C1-C4 straight chain or branched chain alkoxy; the alkylamino is C1-C3 straight-chain or branched-chain alkylamino.

3. A method for preparing the Millepachine-CA-4 derivative as described in claim 1 or 2, which comprises the steps of:

(1) synthesis of benzopyran bromo intermediate: respectively carrying out nucleophilic substitution reaction on 3-chloro-3-methyl-1-butyne and different substituted bromobenzene 1/bromobenzene 4/bromobenzene 7 in the presence of 1, 8-diazabicycloundecene-7-ene and cuprous chloride to respectively obtain an intermediate 2/an intermediate 5/an intermediate 8; under the conditions of 60-80 ℃ and pyridine existence, the intermediate 2/the intermediate 5/the intermediate 8 are subjected to intramolecular ring closure reaction to respectively obtain a benzopyran bromo-intermediate 3/an intermediate 6/an intermediate 9;

(2) in the presence of n-butyllithium, reacting the benzopyran bromo-intermediate 3/intermediate 6/intermediate 9 with different substituted benzaldehydes 10 respectively to obtain a benzhydryl alcohol intermediate 11; then oxidizing by 2-iodoxybenzoic acid to obtain a benzophenone intermediate 12; in the presence of n-butyl lithium, performing a witting reaction on the benzophenone intermediate 12 and methyl triphenyl phosphonium bromide to obtain an intermediate 13;

(3) under the catalytic condition of palladium carbon, carrying out hydrogenation reduction reaction on the benzophenone intermediate 12 or the intermediate 13 to obtain an aniline intermediate 14; in the presence of potassium carbonate, reacting the aniline intermediate 14 with halogen acetyl halide to obtain a target compound 15; the target compound 15 reacts with different substituted sodium alkoxides, ammonia water or different substituted amines respectively to obtain a target compound 16/a target compound 17/a target compound 18 with different substituted amide side chains respectively.

4. The method for preparing Millepachine-CA-4 derivatives according to claim 3, wherein: in the step (1), the molar use ratio of 3-chloro-3-methyl-1-butyne to bromobenzene is (1-3): the molar use ratio of the 1, 1, 8-diazabicycloundecen-7-ene to bromobenzene is (1-3): 1.

5. the method for preparing Millepachine-CA-4 derivatives according to claim 3, wherein: in the step (2), the preparation of the benzhydrol intermediate 11 comprises the following steps: under the protection of argon, dissolving 2-3 molar parts of benzopyran bromo-intermediate 3/intermediate 6/intermediate 9 in anhydrous tetrahydrofuran, cooling to-60-80 ℃, then slowly adding 2-3 molar parts of n-butyl lithium, stirring and balancing for 0.5-1 hour, then adding 1 molar part of THF (tetrahydrofuran) solution of different substituted benzaldehydes, continuously stirring for 12-24 hours, after the reaction is finished, adding saturated ammonium chloride solution to quench the reaction, extracting ethyl acetate to obtain an organic phase, and performing column chromatography separation on a crude product after reduced pressure drying to obtain a benzhydryl alcohol intermediate 11;

the preparation steps of the benzophenone intermediate 12 are as follows: dissolving 1 molar part of the benzophenone intermediate 11 in a THF solution, adding 2-5 molar parts of 2-iodoxybenzoic acid powder under the stirring condition, stirring for 5-10 hours, filtering out a precipitate after TLC monitoring reaction is finished, concentrating the filtrate to obtain a crude product, and performing column chromatography separation to obtain a benzophenone intermediate 12;

the preparation steps of the intermediate 13 are as follows: under the protection of argon, dissolving 2-3 molar parts of methyl triphenyl phosphonium bromide in anhydrous THF, cooling the solution to-60-80 ℃, slowly dropwise adding 2-3 molar parts of n-BuLi, gradually increasing to 20-25 ℃, stirring for 0.5-1 hour, cooling the mixture to-60-80 ℃, slowly dropwise adding 1 molar part of a THF solution of a benzophenone intermediate 12, stirring for 0.5-1 hour, gradually increasing to 20-25 ℃, stirring for 12-24 hours, monitoring the reaction by TLC, adding a saturated ammonium chloride solution, quenching the reaction, extracting with ethyl acetate to obtain an organic phase, and separating a crude product after decompression column chromatography drying to obtain an intermediate 13.

6. The method for preparing Millepachine-CA-4 derivatives according to claim 3, wherein: in the step (3), the aniline intermediate 14 is prepared by the following steps: adding 10-15% (g/g) palladium carbon into 1 molar part of a THF solution of a benzophenone intermediate 12 or an intermediate 13 under an ice bath condition, reacting for 6-12 hours under a hydrogen condition, filtering to remove the palladium carbon, drying a filtrate under reduced pressure, and performing column chromatography separation on a crude product to obtain an aniline intermediate 14;

the preparation procedure of target compound 15 was as follows: dissolving 1 mol part of aniline intermediate 14 in a dichloromethane solution, adding 8-10 mol parts of potassium carbonate, stirring at room temperature for 0.5-1 hour, dropwise adding 5-10 mol parts of halogenated acetyl halogen at the temperature of 0-4 ℃ in an ice bath, continuously reacting for 4-8 hours, extracting with ethyl acetate after TLC monitoring reaction is finished to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 15.

7. The method for preparing Millepachine-CA-4 derivatives according to claim 3, wherein: in step (3), the target compound 16 is prepared as follows: dissolving 1 molar part of target compound 15 in methanol, adding 2-5 molar parts of sodium alkoxide with different chain lengths, refluxing and stirring for 5-12 hours at 50-80 ℃, extracting with ethyl acetate to obtain an organic phase after TLC monitoring reaction is finished, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 16;

the procedure for the preparation of target compound 17 was as follows: dissolving 1 molar part of target compound 15 in ethanol, dropwise adding 5-10 molar parts of ammonia water solution, stirring at room temperature for 1-6 hours, performing TLC monitoring reaction, extracting with ethyl acetate to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 17;

the preparation of target compound 18 was as follows: dissolving 1-2 parts by mole of a target compound 15 in acetone, adding 2-4 parts by mole of potassium carbonate under stirring, then adding 1-2 parts by mole of amine hydrochloride with different chain lengths, stirring and reacting at 50-80 ℃ for 5-10 hours, after TLC monitoring reaction is finished, adding ethyl acetate for extraction to obtain an organic phase, and performing column chromatography separation on a crude product after decompression drying to obtain a target compound 18.

8. An anti-tumor pharmaceutical composition, which is characterized in that: comprising as an active ingredient a therapeutically effective amount of a Millepachine-CA-4 derivative according to claim 1 or 2 or a pharmaceutically acceptable prodrug, salt, hydrate, solvate, crystalline form or diastereomer thereof, together with one or more pharmaceutically acceptable carriers.

9. Use of a Millepachine-CA-4 derivative as claimed in claim 1 or 2, wherein: application in preparing antitumor drugs.

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