WO2016110237A1

WO2016110237A1 - Α-carboline compound , preparation method therefor and uses thereof

Info

Publication number: WO2016110237A1
Application number: PCT/CN2016/070110
Authority: WO
Inventors: 杨春皓; 缪泽鸿; 张小飞; 衣君玫; 贺茜; 宦霞娟; 宋姗姗; 王迎庆; 陈奕; 丁健
Original assignee: 中国科学院上海药物研究所
Priority date: 2015-01-05
Filing date: 2016-01-05
Publication date: 2016-07-14
Also published as: CN105820165B; CN105820165A

Abstract

The present invention provides anα-carboline compound, a preparation method therefor and uses thereof. Specially provided is anα-carboline compound represented by the following general formula (I), wherein each group is defined as in the specification. Said compound of formula I has dual effects on inhibiting topoisomerase II and microtubulin, and can inhibit the proliferation of cancer cells.

Description

咔-porphyrin compound, preparation method and use thereof

Technical field

The invention belongs to the fields of medicinal chemistry and medical treatment. Specifically, it relates to a kind of mother core compound containing α-carboline (9H-pyrido[2,3-b]indole), a pharmaceutically acceptable salt thereof and a hydrate thereof, and a preparation method thereof and their treatment and topological difference The use of enzyme II and drugs for tubulin-related diseases such as tumors.

Background technique

As a carrier of genetic information, DNA plays a very important role in the entire life process. In DNA transcription, replication, and gene expression, DNA topoisomerase regulates and maintains the complex topology of DNA. Since J.C. Wang discovered the first ω protein that can change the DNA topology in 1971, researchers have studied the DNA topoisomerase in detail. DNA topoisomerases are classified into topoisomerase I and topoisomerase II, depending on the mechanism of action and biological structure. The eukaryotic topoisomerase II is a homodimer comprising two subtypes: TopoIIα and TopoIIβ. The physiological functions of the two are quite different: TopoIIα tends to act on positive supercoiled DNA, while TopoIIβ does not show specific selectivity to supercoiled DNA conformation; TopoIIα plays an important role in cell proliferation, in cell fast The high proliferative phase can be used as a marker to judge the degree of cell proliferation; TopoIIβ is relatively stable in cells and has no correlation with cell proliferation status and cell cycle. TopoI produces a single-strand break on the DNA duplex, allowing another single strand to pass through the gap, altering the situation of DNA supercoil or helix. TopoII differs from TopoI in its mechanism of cleavage, which produces a double-strand break on the DNA backbone, allowing another double-stranded DNA to pass through the gap. In addition to performing all TopoI functions, TopoII also separates sister chromatids that crosslink each other after DNA replication is complete. DNA topoisomerase II inhibitors disrupt enzyme activity by affecting various stages of the TopoII enzyme action process. It can act directly on DNA, or on topoisomerase II, and can also act on DNA topoisomerase II-DNA cleavage complex to complete the inhibition of topoisomerase II activity and ultimately lead to cell death. Die. Studies have shown that the cytotoxicity of most DNA topoisomerase II inhibitors is through the formation of DNA-enzyme-drug ternary complexes, the last step in blocking the reaction between enzymes and DNA, ie the re-spinning of single-stranded or double-stranded DNA at the incision site. Bonded to achieve. The role of the inhibitor is actually to transform the topoisomerase II, which functions normally in the cell, into a causative agent that causes DNA strand breaks. The ultimate cause of cell death may be due to the erroneous repair of DNA strand breaks or due to breakable complexes. The formation and stable presence of the substance activates a series of processes in the cell that cause programmed cell death. Based on the understanding of the key role of TopoII in cells, the study of topoisomerase II inhibitors has been one of the hot spots in the development of anti-tumor drugs. Currently available topoisomerase II inhibitors are etoposide, teniposide, doxorubicin, idarubicin, epirubicin and mitoxantrone.

Microtubules are the main components of the cytoskeleton. They are composed of α-tubulin and β-tubulin heterodimer and have the characteristics of hollow tubular structure. In addition, there is a gamma tubulin, which is not a component of microtubules but participates in the assembly of microtubules. Microtubules have the kinetic properties of polymerization and depolymerization and play an important role in maintaining cell morphology, cell division, signal transduction and material transport. Microtubules polymerize into spindles in the early stage of cell division, and the spindle in the mitosis pulls the chromosomes into the two poles and moves into the two daughter cells to complete cell proliferation. Since microtubules play an extremely important role in cell division, they have become one of the important targets for antitumor drug research. Tubulin inhibitors acting on microtubule systems have also become an effective class of antitumor drugs. Tubulin inhibitors are classified into two types depending on the mechanism of action: a tubulin depolymerization inhibitor that inhibits tubulin polymerization and a tubulin polymerization agent that promotes tubulin polymerization. At the same time, according to the different sites of tubulin inhibitors and tubulin, they can be divided into three categories: tubulin inhibitors acting on the colchicine site, and tubulin inhibitors acting on the vinblastine site. And acting on yew Tubulin inhibitor at the alcohol site. A large number of structurally diverse tubulin inhibitors have been synthesized, and some compounds have shown strong anti-tumor effects.

Clinically, DNA topoisomerase II inhibitors and tubulin inhibitors are often used in combination to treat tumors. Common doxorubicin, etoposide combined with vincristine and paclitaxel are used to treat leukemia, lung cancer, breast cancer and stomach cancer. . Combination therapy has many advantages. For example, the combination of paclitaxel and doxorubicin not only synergizes the treatment of cancer, but also reduces side effects caused by drug toxicity. In addition, combination therapy can also slow tumor resistance to a certain extent. Clearly, dual inhibitors of DNA topoisomerase II and tubulin will be easier and easier to control than combination therapy. However, there are no reports of DNA topoisomerase II and tubulin dual inhibitors at this stage.

The α-carboline (9H-pyrido[2,3-b]indole) is one of the porphyrin compounds. In the 1960s and 1970s, when the porphyrin alkaloid, chloramphenicol, was found in the seeds of Peganum harmala, the porphyrin derivative had a variety of biological activities, which led to the study of porphyrin derivatives. The porphyrin is a pyridoindole structure. According to the difference of the pyridine nitrogen atom, the Greek letters are different in the porphyrin prefix. Among them, the β-carboline compound is the most common in the natural product, and the α-porphyrin compound. There are relatively few studies. Most of the natural products having an α-porphyrin structure have excellent biological activity, and exhibit excellent antiviral and anticancer activities due to their binding activity to DNA or inhibitory activity against Topo-II. The α-carboline compounds also have antidepressant and antipsychotic effects. Alpha-carboline derivatives are also useful as kinase inhibitors, such as cell cycle dependent kinase inhibitors, tyrosine kinase inhibitors and the like. In addition, it has been reported that α-carboline compounds have antimalarial activity.

In summary, there is an urgent need in the art to develop new α-carboline derivatives having antiviral and anticancer activities.

Summary of the invention

It is an object of the present invention to provide a novel α-carboline derivative having antiviral and anticancer activities.

In a first aspect of the invention, there is provided a class of compounds having the structure of formula I:

among them,

R ₁ , R ₂ , R ₃ , R ₄ are each independently selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, substituted or unsubstituted C1-C4 straight or branched alkyl, substituted or unsubstituted Substituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C4 linear or branched alkoxy group, substituted or unsubstituted C1-C4 linear or branched alkylene-amino group, substituted Or an unsubstituted C1-C4 linear or branched amine group, and a substituted or unsubstituted saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R _{5 is} selected from the group consisting of hydrogen, a substituted or unsubstituted C1-C4 linear or branched alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C1-C4 linear chain. Or a branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched alkylene-hydroxy group, a substituted or unsubstituted C1-C4 linear or branched amine group, and a substituted or unsubstituted a saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R _{6 is} selected from the group consisting of hydrogen, halogen, hydroxy, amino, methylamino, dimethylamino, substituted or unsubstituted C1-C4 straight or branched alkoxy, substituted or unsubstituted C1-C4 straight a branched or branched amino group, a substituted or unsubstituted C1-C4 linear or branched amino-oxy group;

R _{7 is} selected from the group consisting of hydrogen, halogen, hydroxy, amino, methyl, methoxy, methylamino, dimethylamino, -OAc, methoxycarbonyl, acetylamino, substituted or unsubstituted C1-C4 straight chain Or a branched alkyl group, a substituted or unsubstituted C1-C4 linear or branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched amine group;

R _{8 is} selected from the group consisting of hydrogen, halogen, hydroxy, amino, methyl, methoxy, methylamino, dimethylamino, methoxycarbonyl, acetylamino, methylsulfonyl, substituted or unsubstituted C1-C4 straight a branched or branched alkyl group, a substituted or unsubstituted C1-C4 linear or branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched amine group;

X is a linking group selected from the group consisting of methylene, oxygen, sulfur, carbonyl, sulfoxide, sulfone,

Wherein said one or more hydrogen atoms on the substituent group are substituted with a substituent selected from the group consisting of halogen, C1-C8 alkyl-amino group, hydroxyl group, methylamino group, amino group, C1-C8 Alkyl-oxyl.

In another preferred embodiment, the substituted alkyl group is substituted with a substituent selected from the group consisting of an oxygen-containing group, a nitrogen-containing group, or a fluorine atom.

In another preferred embodiment, R _{5 is} selected from the group consisting of hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R _{6 is} selected from the group consisting of hydrogen, hydroxy, -NH-CH ₃ , -NH ₂ , -N(CH ₃ ) ₂ , -O(CH ₂ ) ₂ OH, -O(CH ₂ ) ₂ N(CH ₃ ) ₂ ;

R _{7 is} selected from the group consisting of hydrogen, fluorine, hydroxyl, methyl, methoxy, acetylamino, -OAc;

R _{8 is} selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, methoxy, acetylamino, dimethylamino, methylsulfonate;

X is selected from the group consisting of oxygen, sulfur,

Carbonyl, sulfoxide, sulfone,

In another preferred embodiment, R ₁ is hydrogen or halogen;

R ₂ is hydrogen, halogen, methoxy, dimethylamino, morpholinyl;

R ₃ is hydrogen, halogen, methoxy;

R ₄ is hydrogen, halogen or methoxy;

R ₅ is hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R ₆ is H, -OH, -O(CH ₂ ) ₂ OH, -O(CH ₂ ) ₂ N(CH ₃ ) ₂ , -NH-CH ₃ , -N(CH ₃ ) ₂ , -NH ₂ ;

R ₇ is H, halogen, -OCH ₃ , -OH, -OAc;

R ₈ is H, halogen, methyl, -OCH ₃ , -SO ₂ -CH ₃ , -N(CH ₃ ) ₂ , -NHAc.

In another preferred embodiment, R ₁ is hydrogen or fluorine;

R ₂ is hydrogen, fluorine, chlorine, methoxy, dimethylamino, morpholinyl;

R ₃ is hydrogen, fluorine, chlorine or methoxy;

R ₄ is hydrogen, fluorine or methoxy;

R ₅ is hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R ₆ is hydrogen, hydroxy, amino, methylamino, dimethylamino, -O(CH ₂ ) ₂ OH, -O(CH ₂ ) ₂ N(CH ₃ ) ₂ ;

R ₇ is hydrogen, fluorine, hydroxyl, methoxy, -OAc;

R ₈ is hydrogen, fluorine, chlorine, bromine, methoxy, acetylamino, dimethylamino, methylsulfonate;

X is a linked sulfur, methylamino, carbonyl, sulfoxide, sulfone group,

In another preferred embodiment, in the compound, any one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , R ₈ and X is a specific compound described in the examples. The corresponding group in the middle.

In another preferred embodiment, the compound of formula I has a structure selected from the group consisting of:

In another preferred embodiment, the compound is a compound selected from the group consisting of I-4, I-11, I-16, I-23, I-28, I-32, I-33, I-37. , I-36, I-34, I-41.

According to a second aspect of the invention, there is provided a process for the preparation of a compound according to the first aspect of the invention, comprising the step (b) and optionally (c), (c1) and/or (c2):

(b) a ring closure reaction with a compound of formula 3 in the presence of a reducing agent in an organic solvent to provide a compound of formula Ia;

(c) subjecting a compound of formula Ia to R ₅ Y in an organic solvent in the presence of a base to give a compound of formula Ib;

(c1) preparing a compound of formula Ib' with a compound of formula Ib;

(c2) preparing a compound of formula I using a compound of formula Ib';

Preferably, the method further comprises the steps of:

(a) reacting a compound of formula 1 with a compound of formula 2 in an organic solvent to provide a compound of formula 3;

Wherein Y is selected from the group consisting of chlorine, iodine, bromine, mesylate, p-toluenesulfonate, triflate; the remainder of each group is as defined in the first aspect of the invention Said.

In another preferred embodiment, the step (a) is carried out in the presence of acetic anhydride and/or sodium acetate.

Preferably, the molar ratio of the compound of formula 1 to sodium acetate is from 1:0.8 to 1.2.

Preferably, the molar ratio of the compound of formula 1 to the compound of formula 2 is from 1:0.8 to 1.2.

In another preferred embodiment, the step (a) is carried out in the presence of titanium tetrachloride and pyridine (preferably, the molar ratio of the compound of the formula 1 to titanium tetrachloride is 1:1 to 3).

In another preferred embodiment, in the step (a), the organic solvent is selected from the group consisting of acetic anhydride, pyridine, tetrahydrofuran, dioxane, DMF, N-methylpyrrolidone, dichloromethane, Chloroform, or a combination thereof.

In another preferred embodiment, in the step (a), the reaction temperature is 70-100 °C.

In another preferred embodiment, in the step (b), the reducing agent is selected from the group consisting of hydrogen, iron powder, zinc powder, sulfide; more preferably iron powder or zinc powder.

In another preferred embodiment, the step (b) is carried out under reflux of acetic acid.

In another preferred embodiment, in the step (b), the organic solvent is selected from the group consisting of acetic acid, tetrahydrofuran, dioxane, N,N-dimethylformamide, and N-methylpyrrolidone. , or a combination thereof.

In another preferred embodiment, in the step (b), the reaction temperature is from 90 to 130 °C.

In another preferred embodiment, in the step (c), the base is selected from the group consisting of sodium hydride, potassium t-butoxide, sodium t-butoxide, or a combination thereof; preferably sodium hydride.

In another preferred embodiment, in the step (c), the reaction temperature is 10 to 40 ° C (preferably room temperature).

In another preferred embodiment, in the step (c), the organic solvent is selected from the group consisting of N,N-dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, tetrahydrofuran, or Its combination.

According to a third aspect of the invention, there is provided a process for the preparation of a compound according to the first aspect of the invention, which comprises the step (d):

(d) reacting a compound of formula 5 with a compound of formula 6 in an inert solvent in the presence of a base to provide a compound of formula Ic Object

Or the method comprises the step (e) and the optional step (f):

(e) reacting a compound of formula 5 with a compound of formula 7 in the presence of a base in an inert solvent to provide a compound of formula Id;

(f) reacting a compound of formula Id with an oxidizing agent in the presence of an oxidizing agent in an inert solvent to provide a compound of formula Ie;

In another preferred embodiment, in the step (d), the base is selected from the group consisting of sodium t-butoxide, and/or potassium t-butoxide.

In another preferred embodiment, in the step (d), the reaction is carried out in the presence of a transition metal, preferably Pd or Cu.

In another preferred embodiment, in the step (d), the reaction is carried out in the presence of a catalyst; preferably, the catalyst is selected from the group consisting of zero-valent palladium, a phosphine ligand, or a combination thereof. .

In another preferred embodiment, in the step (d), the reaction temperature is from 100 to 120 °C.

In another preferred embodiment, in the step (d), the inert solvent is t-butanol.

In another preferred embodiment, in the step (e), the base is selected from the group consisting of methyl lithium, n-butyl lithium, t-butyl lithium, or a combination thereof; preferably methyl lithium, uncle Butyl lithium, or a combination thereof.

In another preferred embodiment, in the step (e), the reaction temperature is -80 ° C to room temperature (10 to 40 ° C).

In another preferred embodiment, in the step (e), the inert solvent is anhydrous tetrahydrofuran.

In another preferred embodiment, in the step (f), the oxidizing agent is selected from the group consisting of chromium trioxide/pyridine, chromium trioxide/sulfuric acid, Dess-Martin oxidizing agent or manganese dioxide, or a combination thereof; Preferred is the Dess-Martin oxidant.

In another preferred embodiment, in the step (f), the inert solvent is dichloromethane.

In another preferred embodiment, in the step (f), the reaction temperature is 10 to 40 ° C (preferably room temperature).

In another preferred embodiment, the method further comprises an optional step (d1) of preparing a compound of formula Ic' with a compound of formula Ic;

Wherein X' is selected from the group consisting of methylene, oxygen, sulfur, carbonyl, sulfoxide, sulfone,

And X' is different from X.

A fourth aspect of the invention provides a use of a compound of formula I according to the first aspect of the invention, or a pharmaceutically acceptable salt or hydrate thereof, for (a) non-therapeutic inhibition of tumors in vitro Cell growth; (b) preparation of a pharmaceutical composition for treating tumor; (c) non-therapeutic inhibition of topoisomerase II activity in vitro; (d) non-therapeutic inhibition of tubulin activity in vitro; (e) in vitro Non-therapeutic depolymerization of cell microtubules; (f) non-therapeutic inhibition of proliferation of tumor cells in vitro; (g) preparation of a pharmaceutical composition having topoisomerase II and tubulin dual inhibitory activity.

In another preferred embodiment, the tumor cells are selected from the group consisting of oral cancer cells, lung cancer cells, liver cancer cells, leukemia cells, gastric cancer cells, cervical cancer cells, ovarian cancer cells, breast cancer cells, colon cancer cells, prostate cells. Cancer cells, or a combination thereof.

In another preferred embodiment, when the compound of formula I is used to inhibit tumor cell proliferation growth, the inhibitory activity has an IC ₅₀ value of from 1 to 2000 nmol, preferably from 2 to 1000 nmol, more preferably from 10 to 500 nmol.

According to a fifth aspect of the invention, there is provided a topoisomerase II activity inhibitor comprising an inhibitory effective amount of a compound according to the first aspect of the invention, or a pharmaceutical thereof An acceptable salt or hydrate.

According to a sixth aspect of the invention, a cell microtubule depolymerizing agent comprising a depolymerization effective amount of a compound according to the first aspect of the invention, or a pharmaceutically acceptable thereof, is provided Salt or hydrate.

According to a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt or hydrate thereof.

In another preferred embodiment, the pharmaceutical composition is a pharmaceutical composition for treating a tumor.

In another preferred embodiment, the pharmaceutical composition is for inhibiting the activity of topoisomerase II.

In another preferred embodiment, the pharmaceutical composition is for inhibiting the activity of tubulin.

In another preferred embodiment, the pharmaceutical composition of the pharmaceutical composition comprises an oral preparation, an injection, and an external preparation.

In an eighth aspect of the invention, there is provided a topoisomerase II and a tubulin dual inhibitor, the inhibitor comprising a compound according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof or Hydrate.

In another preferred embodiment, the dual inhibitor is used to treat a tumor.

In another preferred embodiment, the dual inhibitor inhibits expression of a protein selected from the group consisting of MCL-1 (myeloid cell leukemia-1), cIAP1 (cellular inhibitor of apoptosis protein-1) or XIAP (X- Linked inhibitor of apoptosis protein).

A ninth aspect of the invention provides a method of treating a tumor, the method comprising the steps of: administering to a subject in need thereof a safe and effective amount of a compound as described in the first aspect of the invention or as in the seventh aspect of the invention Said pharmaceutical composition.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is an antitumor spectrum of Compound I-4;

Figure 2 is the effect of Compound I-4 on cell microtubules (the scale length in the figure indicates 10 μM);

Figure 3 is a graph showing the inhibitory effect of Compound I-4 on Top2 enzyme activity;

Figure 4 shows the difference between Compound I-4 and the combination in HeLa cells.

detailed description

The inventors have conducted long-term and intensive studies to provide a class of compounds having the structure shown in Formula I, which inhibits the kDNA helicase activity of topoisomerase II and promotes the depolymerization of cellular tubulin. In turn, the growth of tumor cells is inhibited. Based on the above findings, the inventors completed the present invention.

the term

As used herein, unless otherwise specified, the term "substituted" means that one or more hydrogen atoms on the group are substituted with a substituent selected from the group consisting of C ₁ -C ₁₀ alkyl, C ₃ -C ₁₀ naphthenic a group, a C ₁ -C ₁₀ alkoxy group, a halogen, a hydroxyl group, a carboxyl group (-COOH), a C ₁ -C ₁₀ aldehyde group, a C ₂ -C ₁₀ acyl group, a C ₂ -C ₁₀ ester group, an amino group, a phenyl group; The phenyl group includes an unsubstituted phenyl group or a substituted phenyl group having 1 to 3 substituents selected from the group consisting of halogen, C ₁ -C ₁₀ alkyl, cyano, OH, nitro, C ₃ ～C ₁₀ cycloalkyl, C ₁ -C ₁₀ alkoxy, amino.

Unless otherwise specified, among all compounds of the present invention, each chiral carbon atom may optionally be in the R configuration or the S configuration, or a mixture of the R configuration and the S configuration.

The term "C ₁ -C ₄ alkyl" refers to a straight or branched alkyl group having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec. Base, tert-butyl, or the like.

The term "C ₃ -C ₆ cycloalkyl" refers to a cycloalkyl group having 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or the like.

The term "C ₁ -C ₄ alkoxy" refers to a straight or branched alkoxy group having from 1 to 4 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy , isobutoxy, sec-butoxy, tert-butoxy, or the like.

The term "halogen" refers to F, Cl, Br and I.

The term "C ₁ -C ₄ amine group" means a group having a structure of "C ₁ -C ₄ alkyl-NH-" or "(alkyl) ₂ -N- (total number of carbon atoms 1-4)", for example CH ₃ NH-, C ₂ H ₅ NH-, C ₃ H ₇ NH-, (CH ₃ ) ₂ N-, or the like. Wherein, the definition of C ₁ -C ₄ alkyl is as defined above.

The term "C ₁ -C ₄ alkylene-amino" refers to having "C ₁ -C ₄ alkylene-NH ₂ ", "alkyl-N-alkylene- (total number of carbon atoms 1-4)" Or a group of "(alkyl) ₂ -N-alkylene-(the total number of carbon atoms is 1-4)" structure, such as -CH ₂ NH ₂ , -C ₂ H ₅ NH ₂ , -C ₃ H ₇ NH ₂ , -C ₂ H ₄ N(CH ₃ ) ₂ , or the like. Here, the C ₁ -C ₄ alkylene group is a group in which a C ₁ -C ₄ alkyl group loses one hydrogen atom, and the C ₁ -C ₄ alkyl group has the same meaning as defined above.

Compound of formula I

SUMMARY OF THE INVENTION It is an object of the present invention to provide a compound of the formula I which contains a mother nucleus of α-carboline (9H-pyrido[2,3-b]indole) or a pharmaceutically acceptable salt or hydrate thereof.

among them,

R ₁ , R ₂ , R ₃ , R ₄ are hydrogen or halogen, C1-C4 linear or branched alkyl, C3-C6 cycloalkyl, C1-C4 containing oxygen, nitrogen or fluorine. a linear or branched alkyl group, an alcohol or an amine, and a saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R ₅ is hydrogen, a C1-C4 linear or branched alkyl group, a C3-C6 cycloalkyl group, an oxygen or nitrogen-containing C1-C4 linear or branched alkyl group, an alcohol or an amine, and an oxygen or a saturated five- or six-membered heterocyclic group of nitrogen;

R ₆ is hydrogen, halogen, hydroxy, amino, methylamino, dimethylamino, a C1-C4 linear or branched alkyl group, an alcohol or an amine containing oxygen or nitrogen;

R ₇ is hydrogen, halogen, hydroxy, amino, methoxy, methylamino, dimethylamino, methoxycarbonyl, acetylamino, a C1-C4 linear or branched alkyl group, an alcohol or an amine containing oxygen or nitrogen;

R ₈ is hydrogen, halogen, hydroxy, amino, methoxy, methylamino, dimethylamino, methoxycarbonyl, acetylamino, methylsulfonyl, a C1-C4 linear or branched alkyl group containing oxygen or nitrogen, Alcohol or amine;

X is a linking group and may be a methylene group, an oxygen group, a sulfur group, a carbonyl group, a sulfoxide group, a sulfone group,

Preferably, R ₁ , R ₂ , R ₃ , R ₄ are hydrogen, halogen, C1-C4 linear or branched alkyl, C3-C6 cycloalkyl, C1 containing oxygen, nitrogen or fluorine. a linear or branched alkyl group, an alcohol or an amine of -C4, and a saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R ₅ is hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R ₇ is hydrogen, fluorine, hydroxyl, methoxy, methoxycarbonyl, acetylamino;

X is the link of oxygen, sulfur,

Carbonyl, sulfoxide, sulfone,

More preferably,

R ₁ is hydrogen, fluorine;

R ₂ is hydrogen, fluorine, chlorine, methoxy, dimethylamino, morpholinyl;

R ₃ is hydrogen, fluorine, chlorine or methoxy;

R ₄ is hydrogen, fluorine, methoxy;

R ₅ is hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R ₆ is hydrogen, hydroxy, amine, methylamino, dimethylamino, -O(CH ₂ ) ₂ OH, -O(CH ₂ ) ₂ N(CH ₃ ) ₂ ;

R ₇ is hydrogen, fluorine, hydroxyl, methoxy or methoxycarbonyl;

X is a linked sulfur, methylamino, carbonyl, sulfoxide, sulfone group,

Most preferably, the invention provides a compound as shown below:

Preparation of compounds of formula I

Another object of the present invention is to provide a process for the preparation of a compound of the formula I, the synthesis route being as shown in Scheme 1 or 2:

plan 1

a) Compound 1 (3-aldehyde chromone) and compound 2 (o-nitrophenylacetonitrile) are condensed under certain conditions to form compound 3 under the conditions of acetic anhydride/sodium acetate system or titanium tetrachloride/pyridine system;

b) Compound 3 is heated under a reducing agent (hydrogen, iron powder, zinc powder, sulfide) to form a compound 4/I.

c) Compound 4 is substituted with a base (sodium hydride, potassium t-butoxide, sodium tert-butoxide) to further form compound I.

Preferably, the step a) is that the compound 1 and the compound 2 are dissolved in acetic anhydride, and the reaction is stirred overnight at 70-100 ° C under the action of sodium acetate, and the reaction is complete by thin layer chromatography.

The condition of the step b) is that the compound 3 is refluxed under the reduction of iron powder or zinc powder, and the reaction is completed by thin layer chromatography.

The condition of the step c) is that the compound 4 is substituted with room temperature to form the compound I under the action of sodium hydride.

Scenario 2

d) catalytically coupling compound 5 with compound 6 in the presence of a base (Pd or Cu) / ligand to form compound I;

e) compound 5 and aromatic aldehyde 7 under strong base (methyl lithium, n-butyl lithium or tert-butyl lithium) to form compound 8 / I;

f) Part of Compound 8 is further oxidized to the final production I under the action of an oxidizing agent (chromium trioxide/pyridine, chromium trioxide/sulfuric acid, Dess-Martin oxidizing agent or manganese dioxide).

Preferably, the conditions of the step d) are: compound 7 and compound 8 are treated with t-butanol as a solvent, sodium tert-butoxide as a base, 0-valent palladium and a phosphine ligand as a catalyst, and reacted at 100-120 ° C to a thin layer chromatography. The reaction is complete.

The conditions of the step e) are: anhydrous tetrahydrofuran as a solvent, methyl lithium, t-butyllithium as a base, and reacted at -80 ° C to room temperature.

The condition of the step f) is: dichloromethane as a solvent, the oxidant is a Dess-martin oxidant, and reacted to a thin room at room temperature. The reaction was confirmed by layer chromatography.

Use of the compound of formula I

The inventors designed to synthesize a class of topoisomerase II and tubulin dual inhibitors containing an alpha-carboline parent core as shown in Formula I. The compounds of formula I have a well-defined structure-activity relationship, and some of the compounds show strong cell proliferation inhibition, such as compounds I-4, I-32, I-33 and the like. Such new structures of α-carboline compounds are expected to become novel antitumor drugs, including oral cancer, lung cancer, liver cancer, leukemia, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer and prostate cancer.

In particular, the compound of formula I exhibits a mechanism of action different from that of a topoisomerase II inhibitor (such as etoposide) and a tubulin inhibitor (such as vincristine) in an apoptosis-inducing assay. In particular, the dual inhibitor inhibits the expression of a protein selected from the group consisting of MCL-1 (myeloid cell leukemia-1), cIAP1 (cellular inhibitor of apoptosis protein-1) or XIAP (X-linked inhibitor). Of apoptosis protein, and the above-mentioned protein was not observed to be inhibited in the combination.

Pharmaceutical composition and method of administration

Since the compound of the present invention has excellent dual inhibitory activity against topoisomerase II and tubulin, the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates, And a pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for the treatment, prevention, and alleviation of diseases associated with topoisomerase II and/or tubulin activity or expression, particularly suitable for topoisomerase II A disease associated with both tubulin activity or expression. According to the prior art, the compounds of the present invention are useful for the treatment of diseases such as oral cancer, lung cancer, liver cancer, leukemia, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer and prostate cancer.

The pharmaceutical compositions of the present invention comprise a safe or effective amount of a compound of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. By "safe and effective amount" it is meant that the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical compositions contain from 1 to 2000 mg of the compound of the invention per agent, more preferably from 5 to 200 mg of the compound of the invention per agent. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition are capable of intermingling with the compounds of the invention and with each other without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid). , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifier (such as

), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.

The mode of administration of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative modes of administration include, but are not limited to, oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. .

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (c) humectants, For example, glycerin; (d) a disintegrant such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent such as paraffin; (f) Absorption Speeding agents, for example, quaternary amine compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, stearin Calcium acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or a mixture thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active compound or compound in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active compound, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.

In addition to these inert diluents, the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and flavoring agents.

In addition to the active compound, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.

Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

Dosage forms for the compounds of the invention for topical administration include ointments, powders, patches, propellants and inhalants. The active ingredient is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or, if necessary, propellants.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

When a pharmaceutical composition is used, a safe and effective amount of a compound of the invention is administered to a mammal (e.g., a human) in need of treatment wherein the dosage is a pharmaceutically effective effective dosage, for a 60 kg body weight, The dose to be administered is usually from 1 to 2000 mg, preferably from 5 to 500 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

The main advantages of the present invention over the prior art include:

(1) A novel structure of a compound having both inhibitory activity of topoisomerase II and tubulin inhibitory activity is provided.

(2) Providing a compound having tumor suppressing activity, which compound can be used for the preparation of a medicament for treating a tumor.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

The structure and preparation method of the compound of the present invention and the activity of inhibiting tumor cells in vitro will be further described below with reference to the examples without restricting the present invention.

In all the examples, starting compound 1, compound 2, compound 5 compound 6 and compound 7 were purchased from Shanghai Shuiya Pharmaceutical Technology Co., Ltd., Haoyuan Technology (Shanghai) Co., Ltd., Beijing Bailingwei Technology Co., Ltd., Shanghai Jingchun Biochemical Technology Co., Ltd. Co., Ltd., Shanghai Titan Technology Co., Ltd. The palladium catalyst and phosphine ligand were purchased from Beijing Belling Technology Co., Ltd. Unless otherwise specified, other starting reagents, solvents, and materials are all sourced from Sinopharm Reagent Group. ¹ H NMR was recorded by a Brucher AM-400 or GEMINI-300 nuclear magnetic resonance apparatus, and chemical shifts were expressed in δ (ppm). Mass spectra were recorded on an Agilent 1200-6110 single quadrupole liquid chromatography mass spectrometer. Separation 200-300 mesh silica gel was supplied by Qingdao Ocean Chemical Plant. The chemical reagents represented by the English abbreviation are as follows:

DMF N,N-dimethylformamide

PPA polyphosphoric acid

THF tetrahydrofuran

mCPBA m-chloroperoxybenzoic acid

DCM dichloromethane

Dba dibenzylideneacetone

Xphos 2-bicyclohexylphosphine-2',4',6'-triisopropylbiphenyl

TMS trimethylsilyl

Preparation and synthesis of compounds

Example 1 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3a)

In a 25 mL round bottom flask, add compound 3-aldehyde-chromone 1a 100 mg (0.574 mmol), o-nitrophenylacetonitrile 93 mg (0.574 mmol), sodium acetate 47 mg (0.574 mmol), acetic anhydride 5 mL, oil at 90 ° C Stir in a bath and heat overnight. The next day, the temperature was lowered to room temperature, and the reaction was quenched by pouring into 150 mL of ice water. 50 mL of DCM was extracted three times, and the organic phase was combined and washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness on a rotary evaporator. The crude product was purified by silica gel column chromatography ( petroleum ether / ethyl acetate = 4:1) The yellow solid was 162 mg in a yield of 88%.

^{1 H NMR (300MHz, Chloroform-} d) δ9.03 (s, 1H), 8.27 (d, J = 9.4Hz, 1H), 8.18 (d, J = 8.1Hz, 1H), 7.82-7.41 (m, 8H ).

LC-MS: 319 (M+1).

Preparation of (2-hydroxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-1)

Into a 25 mL round bottom flask, 5 mL of acetic acid, Compound 3a 100 mg (0.314 mmol), and iron powder 106 mg (1.89 mmol) were added, and the mixture was heated under reflux for 2 hours. After cooling to room temperature, the reaction system was poured into 200 mL of water, and extracted with 50 mL of DCM. The organic phase was combined, then washed with 50 mL of brine, dried over anhydrous sodium sulfate and evaporated. Petroleum ether / ethyl acetate = 5:1) yielded 58 mg of pale yellow solid, yield 64%.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.30 (s, 1H), 10.32 (s, 1H), 8.88 (s, 1H), 8.73 (s, 1H), 8.29 (d, J = 7.9Hz, 1H), 7.62–7.38 (m, 4H), 7.28 (t, J = 7.9 Hz, 1H), 7.10–6.91 (m, 2H).

LC-MS: 289 (M+1).

Example 2 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(6-methyl-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3b)

95 mg (0.5 mmol) of 3-aldehyde-based 6-methyl chromone 1b was used as a starting material, and 147 mg of a pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 9.01 (s, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.04 (s, 1H), 7.79 - 7.71 (m, 1H), 7.69 - 7.52 (m, 4H), 7.46 (d, J = 8.6 Hz, 1H), 2.49 (s, 3H).

LC-MS: 333 (M+1).

Preparation of (2-hydroxy-5-methylphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-2)

100 mg (0.3 mmol) of 3b was used as a starting material, and a compound of the compound I-1 was obtained.

^{1 H NMR (300MHz, DMSO-} d 6) δ13.62 (s, 1H), 12.11 (s, 1H), 8.68 (d, J = 7.7Hz, 1H), 8.19 (d, J = 8.7Hz, 1H) , 8.05–7.95 (m, 1H), 7.91 (s, 1H), 7.57–7.42 (m, 2H), 7.33–7.21 (m, 1H), 7.11 (d, J=6.8 Hz, 1H), 6.85 (d) , J = 7.9 Hz, 1H), 2.32 (s, 3H).

LC-MS: 303 (M + 1).

Example 3 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(7-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3c)

103 mg (0.5 mmol) of 3-aldehyde 7-methoxyxanone 1c was used as a starting material, and a pale yellow solid (125 mg) was obtained with a yield of 72%.

¹ H NMR (300 MHz, Chloroform-d) δ 8.96 (s, 1H), 8.22 - 8.13 (m, 2H), 7.79 - 7.58 (m, 3H), 7.54 (s, 1H), 7.04 (dd, J = 8.9, 2.4 Hz, 1H), 6.93 (d, J = 2.3 Hz, 1H), 3.95 (s, 3H).

LC-MS: 349 (M+1).

Preparation of (2-hydroxy-4-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-3)

105mg (0.3mmol) 3c was used as the starting material, and the synthesis process of the compound I-1 gave a white solid, 74 mg, yield 78%.

^{1 H NMR (300MHz, DMSO-} d 6) δ13.30 (s, 1H), 12.10 (s, 1H), 8.67 (d, J = 8.4Hz, 1H), 8.18 (d, J = 8.8Hz, 1H) , 8.02 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 2.4 Hz, 2H), 7.56 - 7.42 (m, 2H), 7.25 (t, J = 7.4 Hz, 1H), 6.95 - 6.82 ( m, 2H), 3.79 (s, 3H).

LC-MS: 319 (M+1).

Example 4 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3d)

103 mg (0.5 mmol) of 6-methoxy-3-aldehyde-chromone 1d was used as a starting material, and 152 mg of pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 8.86 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 7.81 - 7.49 (m, 5H), 7.10 (d, J = 8.4 Hz, 1H) ), 6.89 (d, J = 9.4 Hz, 1H), 4.00 (s, 3H).

LC-MS: 349 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-4)

105 mg (0.3 mmol) of 3d was used as a starting material. With reference to the synthesis of compound I-1, a white solid (yield: 57 mg, yield: 60%).

^{1 H NMR (300MHz, DMSO-} d 6) δ12.31 (s, 1H), 9.73 (s, 1H), 8.89 (s, 1H), 8.73 (s, 1H), 8.30 (d, J = 8.1Hz, 1H), 7.61 - 7.45 (m, 2H), 7.28 (t, J = 8.2 Hz, 1H), 7.12 - 7.03 (m, 1H), 6.99 - 6.87 (m, 2H), 3.72 (s, 3H).

LC-MS: 319 (M+1).

Example 5 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(6-fluoro-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3e)

97 mg (0.5 mmol) of 6-fluoro-3-aldehyde-chromone 1e was used as a starting material, and 152 mg of a pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (s, 1H), 8.19 (dd, J = 8.1, 1.3 Hz, 1H), 7.90 (dd, J = 8.0, 3.0 Hz, 1H), 7.76 (td) , J = 7.5, 1.4 Hz, 1H), 7.70 - 7.56 (m, 3H), 7.53 - 7.45 (m, 2H).

LC-MS: 337 (M+1).

Preparation of (2-hydroxy-5-fluorophenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-5)

101 mg (0.3 mmol) of 3e was used as a starting material, and a white solid of 60 mg was obtained with reference to the compound I-1.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 13.68 (s, 1H), 12.17 (s, 2H), 8.74 - 8.65 (m, 1H), 8.21. (d, J = 7.7 Hz, 1H), 8.05 ( d, J = 8.4 Hz, 1H), 7.97 (d, J = 13.8 Hz, 1H), 7.60 - 7.42 (m, 2H), 7.28 (t, J = 7.4 Hz, 1H), 7.20 - 7.11 (m, 1H) ), 7.02–6.89 (m, 1H).

LC-MS: 307 (M+1).

Example 6 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(6-chloro-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3f)

105 mg (0.5 mmol) of 6-chloro-3-aldehyde-chromone 1f was used as a starting material, and 162 mg of pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (s, 1H), 8.23 (d, J = 2.6 Hz, 1H), 7.80 - 7.57 (m, 5H), 7.55 (s, 1H), 7.53 - 7.48 (m, 1H).

LC-MS: 353 (M+1).

Preparation of (2-hydroxy-5-chlorophenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-6)

106 mg (0.3 mmol) of 3f was used as a starting material, and a compound of the compound I-1 was obtained.

^{1 H NMR (300MHz, DMSO-} d 6) δ13.90 (s, 1H), 12.18 (s, 1H), 8.71 (d, J = 8.4Hz, 1H), 8.31-8.14 (m, 2H), 8.09 ( d, J = 9.2 Hz, 1H), 7.63 - 7.44 (m, 2H), 7.42 - 7.21 (m, 2H), 6.99 (d, J = 8.8 Hz, 1H).

LC-MS: 323 (M + 1).

Example 7 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(6-bromo-4-oxo-4H-chromone-3-substituted) acrylonitrile (compound 3g)

127 mg (0.5 mmol) of 6-bromo-3-aldehyde-chromone 1 g was used as a starting material, and 173 mg of a pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (s, 1H), 8.39 (d, J = 2.4 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.90 - 7.55 (m, 4H) ), 7.54–7.43 (m, 2H).

LC-MS: 397 (M+1), 399 (M+3).

Preparation of (2-hydroxy-5-bromophenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-7)

120 mg (0.3 mmol) of 3 g was used as a starting material, and the synthesis of the compound I-1 gave 63 mg of a yellow solid.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.31 (s, 1H), 10.33 (s, 1H), 8.88 (s, 1H), 8.73 (s, 1H), 8.30 (d, J = 8.4Hz, 1H), 7.65–7.46 (m, 4H), 7.34–7.23 (m, 1H), 6.97 (d, J=8.6 Hz, 1H).

LC-MS: 367 (M+1), 369 (M+3).

Example 8 Preparation of (E/Z)-2-(2-nitrophenyl)-3-(7-fluoro-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3h)

97 mg (0.5 mmol) of 7-fluoro-3-aldehyde-chromone 1h was used as a starting material, and the synthesis of the compound 3a gave 151 mg of a pale yellow solid.

¹ H NMR (300 MHz, Chloroform-d) δ 8.99 (s, 1H), 8.29 (dd, J = 8.9, 6.2 Hz, 1H), 8.19 (d, J = 8.3 Hz, 1H), 7.80 - 7.71 (m) , 1H), 7.70–7.56 (m, 2H), 7.50 (s, 1H), 7.26–7.14 (m, 2H).

LC-MS: 337 (M+1).

Preparation of (2-hydroxy-4-fluorophenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-8)

101 mg (0.3 mmol) of 3 h was used as a starting material. With reference to the synthesis of compound I-1, a yellow solid was obtained (yield: 45%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.16 (s, 1H), 8.70 (d, J = 8.4 Hz, 1H), 8.18 (t, J = 8.9 Hz, 2H), 7.97 (d, J = 8.5 Hz, 1H), 7.60–7.42 (m, 2H), 7.27 (t, J = 8.0 Hz, 1H), 6.88–6.71 (m, 2H).

LC-MS: 307 (M+1).

Example 8 (E/Z)-2-(2-nitrophenyl)-3-(7-hydroxy-6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3i Preparation

110 mg (0.5 mmol) of 7-hydroxy-6-methoxy-3-aldehyde-chromone 1i was used as a starting material, and a pale yellow solid was obtained (yield: 65%).

¹ H NMR (300 MHz, Chloroform-d) δ 9.01 (s, 1H), 8.19 (d, J = 8.8 Hz, 1H), 7.85 - 7.57 (m, 4H), 7.52 (s, 1H), 7.33 (s) , 1H), 3.96 (s, 3H), 2.38 (s, 3H).

LC-MS: 365 (M+1).

Preparation of 5-hydroxy-2-methoxy-4-(9H-pyrido[2,3-b]indole-3-carbonyl)phenylacetate (Compound I-9)

100 mg (0.274 mmol) of 3i was used as a starting material. With reference to the synthesis of compound I-1, a yellow solid was obtained (yield: 44%).

^{1 H NMR (300MHz, DMSO-} d 6) δ12.29 (s, 1H), 10.10 (s, 1H), 8.92 (s, 1H), 8.76 (s, 1H), 8.40-8.24 (m, 2H), 7.62–7.43 (m, 1H), 7.35–7.23 (m, 1H), 7.16 (s, 1H), 6.77 (s, 1H), 3.73 (s, 3H), 2.30 (s, 3H).

LC-MS: 377 (M+1).

Example 9 Preparation of (2,4-dihydroxy-5-methoxyphenyl)(9H-pyrido[2,3-b]indol-3-substituted)methanone (Compound I-10)

5 mL round bottom flask, add 90 mg (0.239 mmol) I-9, 95.6 mg (2.39 mmol) NaOH, 4 mL THF, 1 mL water, stir at room temperature overnight, dilute 150 mL water next time, extract three times with 50 mL DCM, combine organic phase, 50 mL The mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.23 (s, 1H), 11.68 (s, 2H), 8.90 (s, 1H), 8.75 (s, 1H), 8.32 (d, J = 7.3Hz, 1H), 7.63–7.46 (m, 2H), 7.37–7.20 (m, 1H), 7.08 (s, 1H), 6.47 (s, 1H), 3.68 (s, 3H).

LC-MS: 335 (M+1).

Example 11 (E/Z)-2-(2-nitrophenyl)-3-(6,7-dimethoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3j) Preparation

200 mg (0.854 mmol) of 6,7-dimethoxy-3-aldehyde-chromone 1j was used as a starting material, and 142 mg of a pale yellow solid was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 8.98 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 7.81 - 7.53 (m, 5H), 6.95 (s, 1H), 4.03 (s) , 3H), 4.00 (s, 3H).

LC-MS: 379 (M+1).

Preparation of (2-hydroxy-4,5-dimethoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-11)

135 mg (0.357 mmol) of 3j was used as a starting material, and the synthesis of the compound I-1 gave a pale yellow solid (yield: 68%).

^{1 H NMR (300MHz, DMSO-} d 6) δ12.24 (s, 1H), 11.58 (s, 1H), 8.91 (s, 1H), 8.76 (s, 1H), 8.32 (d, J = 8.0Hz, 1H), 7.62–7.45 (m, 2H), 7.35–7.22 (m, 1H), 7.09 (s, 1H), 6.67 (s, 1H), 3.87 (s, 3H), 3.67 (s, 3H).

LC-MS: 349 (M+1).

Example 12 (E/Z)-2-(4,5-Dimethoxynitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile Preparation of (Compound 3k)

102 mg (0.5 mmol) of 1 d, 111 mg (0.5 mmol) of 2b was used as a starting material, and the synthesis of the compound 3a gave 150 mg of a yellow solid, yield 74%.

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (s, 1H), 7.76 (s, 1H), 7.60 (d, J = 3.2 Hz, 1H), 7.54 - 7.44 (m, 2H), 7.39 - 7.31 (m, 1H), 6.90 (s, 1H), 4.04 (s, 3H), 4.01 (s, 3H), 3.92 (s, 3H).

LC-MS: 409 (M+1).

Preparation of (6,7-dimethoxy-9H-pyrido[2,3-b]indole-3-substituted)(2-hydroxy-5methoxyphenyl)methanone (Compound I-12)

100 mg (0.245 mmol) of 3k was used as a starting material, and a white solid of 54 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, Chloroform-} d) δ11.46 (s, 1H), 9.67 (s, 1H), 8.80 (s, 1H), 8.62 (s, 1H), 7.53 (s, 1H), 7.24-7.16 (m, 2H), 7.12–7.02 (m, 2H), 4.02 (s, 3H), 4.01 (s, 3H), 3.74 (s, 3H).

LC-MS: 379 (M+1).

Example 13 (E/Z)-2-(4-methoxynitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 31) Preparation

102 mg (0.5 mmol) of 1 d, 96 mg (0.5 mmol) of 2c was used as a starting material, and a white solid of 122 mg was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 8.99 (s, 1H), 7.67 (d, J = 2.9 Hz, 1H), 7.60 (d, J = 3.0 Hz, 1H), 7.53 - 7.45 (m, 3H) ), 7.33 (dd, J = 9.2, 3.0 Hz, 1H), 7.23 (d, J = 2.6 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H).

LC-MS: 379 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(7-methoxy-9H-pyrido[2,3-b]indol-3-substituted)methanone (Compound I-13)

100 mg (0.265 mmol) of 3 l was used as a starting material, and a white solid of 50 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.18 (s, 1H), 9.72 (s, 1H), 8.75 (s, 1H), 8.63 (s, 1H), 8.17 (d, J = 8.8Hz, 1H), 7.12–6.81 (m, 5H), 3.86 (s, 3H), 3.72 (s, 3H).

LC-MS: 349 (M+1).

Example 14 (E/Z)-2-(4-Chloro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (compound 3 m Preparation

102 mg (0.5 mmol) of 1 d, 98 mg (0.5 mmol) of 2d was used as a starting material, and a white solid of 132 mg was obtained with reference to compound 3a.

^{1 H NMR (300MHz, Chloroform-} d) δ9.02 (s, 1H), 8.17 (s, 1H), 7.73 (dd, J = 8.5,2.3Hz, 1H), 7.62-7.46 (m, 4H), 7.34 (dd, J=9.2, 2.9 Hz, 1H), 3.93 (s, 3H).

LC-MS: 383 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(7-chloro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-14)

100 mg (0.262 mmol) of 3 m was used as a starting material, and a white solid of 63 mg was obtained with reference to the compound I-1.

LC-MS: 353 (M+1).

Example 15 (E/Z)-2-(4-Fluoro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3n Preparation

102 mg (0.5 mmol) of 1 d, 90 mg (0.5 mmol) of 2e was used as a starting material, and the synthesis of the compound 3a gave 170 mg of a yellow solid, yield 93%.

¹ H NMR (300 MHz, Chloroform-d) δ 9.01 (s, 1H), 7.92 (dd, J = 8.1, 2.7 Hz, 1H), 7.63 - 7.55 (m, 2H), 7.55 - 7.45 (m, 3H) , 7.40–7.31 (m, 1H), 3.93 (s, 3H).

LC-MS: 367 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(7-fluoro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-15)

100 mg (0.273 mmol) of 3 n was used as a starting material, and a white solid of 55 mg was obtained with reference to the compound I-1.

¹ H NMR (300 MHz, Chloroform-d) δ 9.01 (s, 1H), 7.92 (dd, J = 8.1, 2.7 Hz, 1H), 7.63 - 7.55 (m, 1H), 7.55 - 7.45 (m, 1H) , 7.40–7.31 (m, 4H), 3.93 (s, 3H).

LC-MS: 337 (M+1).

Example 16 (E/Z)-2-(5-fluoro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3o) Preparation

102mg (0.5mmol) 1d, 90mg (0.5mmol) 2f as raw material, with reference to the synthesis process of compound 3a, The white solid was 135 mg in a yield of 74%.

^{1 H NMR (300MHz, DMSO-} d 6) δ9.01 (s, 1H), 8.38-8.26 (m, 1H), 7.81-7.58 (m, 5H), 7.50 (d, J = 6.0Hz, 1H), 3.89 (s, 3H).

LC-MS: 367 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(6-fluoro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-16)

100 mg (0.273 mmol) of 3o was used as a starting material, and a white solid of 58 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.34 (s, 1H), 9.73 (s, 1H), 8.95 (s, 1H), 8.75 (s, 1H), 8.22 (d, J = 11.9Hz, 1H), 7.61–7.46 (m, 1H), 7.46–7.27 (m, 1H), 7.18–7.02 (m, 1H), 6.99–6.88 (m, 2H), 3.73 (s, 3H).

LC-MS: 337 (M+1).

Example 17 (E/Z)-2-(6-fluoro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (compound 3p Preparation

102 mg (0.5 mmol) of 1 d, 90 mg (0.5 mmol) of 2 g was used as a starting material, and the synthesis of the compound 3a gave 165 mg of a white solid, yield 90%.

¹ H NMR (300 MHz, Chloroform-d) δ 9.06 (s, 1H), 7.99 (d, J = 9.4 Hz, 1H), 7.68 - 7.47 (m, 5H), 7.38 - 7.30 (m, 1H), 3.92 (s, 3H).

LC-MS: 367 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(5-fluoro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-17)

130 mg (0.355 mmol) of 3p was used as a starting material, and a white solid of 60 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.63 (s, 1H), 9.75 (s, 1H), 8.81 (s, 1H), 8.63 (s, 1H), 7.60-7.36 (m, 2H), 7.16–6.89 (m, 4H), 3.72 (s, 3H).

LC-MS: 337 (M+1).

Example 18 (E/Z)-2-(3-Methoxy-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile ( Preparation of compound 3q)

102 mg (0.5 mmol) of 1 d, 97 mg (0.5 mmol) of 2 h was used as a starting material, and a white solid of 120 mg was obtained with reference to compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 8.89 (s, 1H), 7.64 - 7.45 (m, 4H), 7.37 - 7.29 (m, 1H), 7.15 (t, J = 7.8 Hz, 2H), 3.96 (s, 3H), 3.91 (s, 3H).

LC-MS: 379 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(8-methoxy-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-18)

100 mg (0.265 mmol) of 3q was used as a starting material, and a white solid of 48 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.43 (s, 1H), 9.73 (s, 1H), 8.85 (s, 1H), 8.73 (s, 1H), 7.86 (d, J = 7.6Hz, 1H), 7.28–6.86 (m, 5H), 3.99 (s, 3H), 3.72 (s, 3H).

LC-MS: 349 (M+1).

Example 19 (E/Z)-2-(5-Chloro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3r Preparation

102 mg (0.5 mmol) of 1 d, 97 mg (0.5 mmol) of 2i was used as a starting material, and the synthesis of the compound 3a gave 168 mg of a yellow solid.

¹ H NMR (300 MHz, Chloroform-d) δ 9.03 (s, 1H), 8.15 (d, J = 9.1 Hz, 1H), 7.66 - 7.48 (m, 5H), 7.41 - 7.31 (m, 1H), 3.93 (s, 3H).

LC-MS: 383 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(6-chloro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-19):

145 mg (0.265 mmol) of 3r was used as a starting material, and a white solid 82 mg was obtained according to the synthesis of the compound I-1. The yield was 61%.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.45 (s, 1H), 9.76 (s, 1H), 8.99 (s, 1H), 8.78 (s, 1H), 8.48 (s, 1H), 7.63- 7.43 (m, 2H), 7.15 - 7.05 (m, 1H), 7.02 - 6.86 (m, 2H), 3.74 (s, 3H).

LC-MS: 353 (M+1).

Example 20 (E/Z)-2-(3-Fluoro-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile (Compound 3s) Preparation

102 mg (0.5 mmol) of 1 d, 91 mg (0.5 mmol) of 2j was used as a starting material, and a white solid 135 mg was obtained with reference to the compound 3a.

¹ H NMR (300 MHz, Chloroform-d) δ 8.95 (s, 1H), 7.68 - 7.56 (m, 2H), 7.54 - 7.45 (m, 1H), 7.46 - 7.30 (m, 3H), 3.92 (s, 3H).

LC-MS: 367 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(8-fluoro-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-20)

115 mg (0.314 mmol) of 3 s was used as a starting material, and a white solid of 33 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.82 (s, 1H), 9.76 (s, 1H), 8.95 (s, 1H), 8.79 (s, 1H), 8.16 (d, J = 7.8Hz, 1H), 7.48–7.35 (m, 1H), 7.33–7.20 (m, 1H), 7.12–7.04 (m, 1H), 7.02–6.90 (m, 2H), 3.73 (s, 3H).

LC-MS: 337 (M+1).

Example 21 (E/Z)-2-(5-Methoxy-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile ( Preparation of compound 3t)

102 mg (0.5 mmol) of 1 d, 97 mg (0.5 mmol) of 2k was used as a starting material, and the synthesis of the compound 3a was carried out as a light yellow solid (158 mg, yield: 84%).

^{1 H NMR (300MHz, Chloroform-} d) δ9.02 (d, J = 0.9Hz, 1H), 8.24 (d, J = 9.1Hz, 1H), 7.60 (d, J = 3.1Hz, 1H), 7.53- 7.48 (m, 2H), 7.34 (dd, J = 9.2, 3.1 Hz, 1H), 7.05 (dd, J = 9.1, 2.8 Hz, 1H), 6.99 (d, J = 2.7 Hz, 1H), 3.97 (s) , 3H), 3.93 (s, 3H).

LC-MS: 379 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(6-methoxy-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-21)

125 mg (0.331 mmol) of 3t was used as a starting material, and a white solid of 52 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.13 (s, 1H), 9.74 (s, 1H), 8.90 (s, 1H), 8.74 (s, 1H), 7.93 (d, J = 2.5Hz, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.21 - 7.03 (m, 2H), 7.03 - 6.90 (m, 2H), 3.85 (s, 3H), 3.74 (s, 3H).

LC-MS: 349 (M+1).

Example 22 (E/Z)-3-(6-(Methylsulfonyl)-4-oxo-4H-chromone-3-substituted)-2-(2-nitrophenyl)acrylonitrile (Compound 3u) preparation

100 mg (0.397 mmol) of 1k was used as a starting material, and the synthesis of the compound 3a gave a pale yellow solid (yield: 82%).

^{1 H NMR (300MHz, Chloroform-} d) δ9.04 (d, J = 1.0Hz, 1H), 8.87 (d, J = 2.3Hz, 1H), 8.30 (dd, J = 8.8,2.4Hz, 1H), 8.21 (dd, J=8.0, 1.3 Hz, 1H), 7.80–7.75 (m, 2H), 7.62–7.58 (m, 2H), 7.48–7.46 (m, 1H), 3.15 (s, 3H).

LC-MS: 397 (M+1).

Preparation of (2-hydroxy-5-(methylsulfonyl)phenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-22)

100 mg (0.253 mmol) of 3u was used as a starting material, and a white solid 32 mg was obtained with reference to the compound I-1.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.37 (s, 1H), 11.23 (s, 1H), 8.93 (s, 1H), 8.76 (s, 1H), 8.32 (d, J = 7.7Hz, 1H), 8.04–7.88 (m, 2H), 7.64–7.48 (m, 2H), 7.37–7.14 (m, 2H), 3.23 (s, 3H).

LC-MS: 367 (M+1).

Example 23 (E/Z)-2-(5-Dimethylamino-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile ( Preparation of compound 3v)

102 mg (0.5 mmol) of 1 d, 103 mg (0.5 mmol) of 2 l was used as a starting material, and the synthesis of Reference Compound 3a was carried out as a pale yellow solid (yield: 62%).

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (d, J = 1.0 Hz, 1H), 8.21. (d, J = 9.4 Hz, 1H), 7.60 (d, J = 3.0 Hz, 1H), 7.53 - 7.48 (m, 1H), 7.44 (d, J = 1.0 Hz, 1H), 7.34 (dd, J = 9.2, 3.0 Hz, 1H), 6.68 (dd, J = 9.5, 2.8 Hz, 1H), 6.56 (d) , J = 2.9 Hz, 1H), 3.92 (s, 3H), 3.17 (s, 6H).

LC-MS: 392 (M+1).

Preparation of (2-hydroxy-5-methoxyphenyl)(6-dimethylamino-9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-23)

100 mg (0.256 mmol) of 3v was used as a starting material, and the synthesis of the compound I-1 gave 39 mg of a red solid, yield 42%.

^{1 H NMR (300MHz, DMSO-} d 6) δ11.92 (s, 1H), 9.71 (s, 1H), 8.83 (s, 1H), 8.67 (s, 1H), 7.65 (s, 1H), 7.39 ( d, J = 8.7 Hz, 1H), 7.15 - 7.03 (m, 2H), 6.99 - 6.86 (m, 2H), 3.72 (s, 3H), 2.94 (s, 6H).

LC-MS: 362 (M+1).

Example 24 (E/Z)-2-(5-morpholinyl-2-nitrophenyl)-3-(6-methoxy-4-oxo-4H-chromone-3-substituted) acrylonitrile ( Preparation of compound 3w)

102 mg (0.5 mmol) of 1 d, 124 mg (0.5 mmol) of 2 m was used as a starting material, and the synthesis of Reference Compound 3a was carried out as a white solid 177 mg, yield 82%.

¹ H NMR (300 MHz, Chloroform-d) δ 9.02 (s, 1H), 8.21 (d, J = 9.3 Hz, 1H), 7.60 (d, J = 3.1 Hz, 1H), 7.53 - 7.43 (m, 2H) ), 7.34 (dd, J = 9.2, 3.1 Hz, 1H), 6.89 (dd, J = 9.3, 2.8 Hz, 1H), 6.79 (d, J = 2.8 Hz, 1H), 3.92 (s, 3H), 3.91 –3.85 (m, 4H), 3.48–3.39 (m, 4H).

LC-MS: 434 (M + 1).

Preparation of (2-hydroxy-5-methoxyphenyl)(6-morpholinyl-9H-pyrido[2,3-b]indol-3-substituted)methanone (Compound I-24)

100 mg (0.231 mmol) of 3w was used as a starting material, and a white solid of 52 mg was obtained with reference to the compound I-1.

^{1 H NMR (600MHz, DMSO-} d 6) δ12.05 (s, 1H), 9.72 (s, 1H), 8.86 (s, 1H), 8.72 (s, 1H), 7.88 (s, 1H), 7.44 ( d, J = 8.8 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H), 7.06 (dd, J = 8.8, 2.9 Hz, 1H), 6.98 - 6.92 (m, 2H), 3.79 (s, 4H) ), 3.73 (s, 3H), 3.14 (s, 4H).

LC-MS: 404 (M+1).

Example 25 Preparation of N-(3-methoxyphenyl)-9H-pyrido[2,3-b]indol-3-amine (Compound I-26)

In a 25 mL round bottom flask, 5 mL of tert-butanol, 100 mg (0.404 mmol) of 3-bromo-9H-pyrido[2,3-b]indole (Compound 5), 65 mg (0.526 mmol) 3-methoxyl was added. Aniline (Compound 6a), 30 mg (0.042 mmol) of Pd ₂ dba ₃ , 31 mg (0.084 mmol) of Xphos, 117 mg (1.21 mmol) of sodium tert-butoxide, protected with nitrogen, and stirred at 100 ° C for 12 hours. After the reaction was completed, the system was cooled to room temperature, poured into 150 mL of ice water and quenched, extracted with 50 mL of DCM three times. The organic phase was combined, then washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate and evaporated to dryness Purification (petroleum ether / ethyl acetate = 2:1) gave a pale yellow solid (yield: 44%).

^{1 H NMR (300MHz, Chloroform-} d) δ9.09 (s, 1H), 8.35 (d, J = 2.2Hz, 1H), 8.21 (d, J = 2.3Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.55 - 7.45 (m, 2H), 7.31 - 7.20 (m, 1H), 7.16 (t, J = 8.0 Hz, 1H), 6.53 (dd, J = 8.9, 1.0 Hz, 1H), 6.49 –6.40 (m, 1H), 3.77 (s, 3H).

LC-MS: 290 (M+1).

Example 26 Preparation of N-(3-methoxyphenyl)-N-methyl-9H-pyrido[2,3-b]indol-3-amine (Compound I-27)

100 mg (0.404 mmol) of the compound 5, 73 mg (0.526 mmol) of the compound 6b was used as a starting material, and a yellow solid 55 mg was obtained with reference to the compound I-26.

^{1 H NMR (300MHz, Chloroform-} d) δ10.16 (s, 1H), 8.51 (s, 1H), 8.45-8.30 (m, 2H), 8.20 (s, 1H), 8.04 (dd, J = 22.8, 9.8 Hz, 1H), 7.61–7.39 (m, 3H), 7.23–7.03 (m, 1H), 6.45–6.27 (m, 2H), 3.74 (s, 3H), 3.41 (s, 3H).

LC-MS: 304 (M+1).

Example 27 Preparation of 3-((3-methoxyphenyl)-thio)-9H-pyrido[2,3-b]indole (Compound I-28)

100 mg (0.404 mmol) of compound 5, 85 mg (0.606 mmol) of compound 6c was used as a starting material, and a yellow solid 55 mg was obtained with reference to compound I-26.

^{1 H NMR (300MHz, Chloroform-} d) δ9.53 (s, 1H), 8.63 (d, J = 2.1Hz, 1H), 8.50 (d, J = 2.1Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.54–7.50 (m, 2H), 7.34–7.27 (m, 1H), 7.16 (t, J=7.9 Hz, 1H), 6.78–6.67 (m, 3H), 3.72 (s, 3H) .

LC-MS: 307 (M+1).

Example 28 Preparation of 3-((3-methoxyphenyl)-sulfoxide)-9H-pyrido[2,3-b]indole (Compound I-29)

In a 25 mL round bottom flask, 20 mg (0.065 mmol) of compound I-28, 11 mg (0.07 mmol) of mCPBA, 5 mL of DCM were added, and the reaction was stirred at room temperature for 3 hours, then poured into 150 mL of DCM, followed by 50 mL of saturated sodium thiosulfate solution. The mixture was washed with 50 mL of brine, dried over anhydrous sodium sulfate, and evaporated to dryness.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.24 (s, 1H), 8.86 (s, 1H), 8.73 (s, 1H), 8.28 (d, J = 9.0Hz, 1H), 7.69 (d, J = 11.9 Hz, 1H), 7.52 (s, 2H), 7.50 - 7.33 (m, 1H), 7.29 (m, 2H), 7.04 (d, J = 8.2 Hz, 1H), 3.80 (s, 3H).

LC-MS: 323 (M + 1).

Example 29 Preparation of 3-((3-methoxyphenyl)-sulfone)-9H-pyrido[2,3-b]indole (Compound I-30)

20 mg (0.065 mmol) of compound I-28, 22 mg (0.14 mmol) of mCPBA was used as a starting material, and a pale yellow solid (yield:

^{1 H NMR (300MHz, DMSO-} d 6) δ12.50 (s, 1H), 9.18 (s, 1H), 8.98 (s, 1H), 8.37 (d, J = 9.1Hz, 1H), 7.65-7.47 ( m, 5H), 7.41 - 7.29 (m, 1H), 7.22 (d, J = 9.9 Hz, 1H), 3.84 (s, 3H).

LC-MS: 339 (M+1).

Example 30 Preparation of 2-(methylaminophenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-31)

In a 25 mL round bottom flask, 35 mg (0.122 mmol) of compound 1-1, 5 mL of pyridine was added, and 42 mg (0.146 mmol) of trifluoromethanesulfonic anhydride was added dropwise at room temperature, and the reaction was stirred at room temperature overnight. The reaction system was evaporated to dryness under reduced pressure on the next day, and 23 mg (0.122 mmol) of cuprous iodide and 15 mL of a solution of methylamine in ethanol were added, and the reaction was sealed at 100 ° C overnight. After cooling to room temperature on the next day, pour into 150 mL of water, extract three times with 50 mL of DCM, wash the organic phase, wash with 50 mL of saturated brine, dry over anhydrous sodium sulfate, and evaporate on a rotary evaporator. Purify by silica gel column chromatography ( petroleum ether / acetic acid The ester = 3:1) gave an orange-red solid of 35 mg in a yield of 94%.

¹ H NMR (300 MHz, Chloroform-d) δ 9.29 (s, 1H), 8.35 (s, 1H), 8.21 (s, 1H), 8.08 (d, J = 8.1 Hz, 1H), 7.74 (s, 1H) ), 7.61 - 7.49 (m, 3H), 7.38 - 7.29 (m, 1H), 7.04 (d, J = 7.7 Hz, 1H), 6.92 - 6.82 (m, 1H), 6.70 - 6.59 (m, 1H), 3.28(s,3H).

LC-MS: 302 (M+1).

Preparation of Example 31 (2-Amino-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound 1-32)

71 mg (0.222 mmol) of compound I-4 was used as a starting material, and the preparation method of the compound I-31 was carried out, and ammonia solution of ammonia was used instead of the ethanol solution of methylamine to obtain 18 mg of a yellow solid in a yield of 26%.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.20 (s, 1H), 8.83 (s, 1H), 8.68 (s, 1H), 8.30 (d, J = 8.8Hz, 1H), 7.59-7.47 ( m, 2H), 7.32 - 7.23 (m, 1H), 7.05 (dd, J = 8.3, 3.0 Hz, 1H), 6.94 - 6.83 (m, 1H), 6.51 (s, 1H), 3.58 (s, 3H) .

LC-MS: 318 (M + 1).

Preparation of Example 32 (2-Methylamino-5-methoxyphenyl)(9H-pyrido[2,3-b]indol-3-substituted)methanone (Compound I-33)

Using 71 mg (0.222 mmol) of compound I-4 as a starting material, the title compound was obtained by the procedure of Compound I-31 to give a yellow solid (yield: 68%).

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.13 (s, 1H), 8.55 (s, 1H), 8.35 (s, 1H), 8.21. (d, J = 8.1 Hz, 1H), 7.80 - 7.40 ( m, 3H), 7.26 (s, 1H), 6.94 (m, 2H), 6.29 (s, 1H), 3.46 (s, 3H), 3.21 (s, 3H).

LC-MS: 332 (M+1).

Example 33 (2-Hydroxy-5-methoxyphenyl)(9-(2-(dimethylamino)ethyl)-9H-pyrido[2,3-b]indole-3-substituted) Preparation of ketone (Compound I-34)

Into a 25 mL round bottom flask, 100 mg (0.314 mmol) of compound I-4, 5 mL of DMF was added, and 50 mg (1.26 mmol) of NaH was added at 0 ° C, and the mixture was reacted at room temperature for 1 hour. Thereafter, 37 mg (0.345 mmol) of 2-chloro-N,N-dimethylethylamine was added, and the reaction was stirred at room temperature for 36 hours. The reaction system was poured into 150mL ice water, extracted three times with 50mL DCM, and the combined organic phases were washed with 50mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness, the crude product was purified by silica gel column chromatography (DCM / CH ₃ OH = 20 with :1) A brown solid of 58 mg was obtained in a yield of 48%.

^{1 H NMR (300MHz, DMSO-} d 6) δ9.75 (s, 1H), 8.90 (s, 1H), 8.78 (s, 1H), 8.34 (d, J = 8.4Hz, 1H), 7.82-7.71 ( m,1H), 7.58 (t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.9 Hz, 1H), 7.12 - 7.01 (m, 1H), 7.03 - 6.86 (m, 2H), 4.61 (s) , 2H), 3.72 (s, 3H), 2.74 (s, 2H), 2.21 (s, 6H).

LC-MS: 390 (M + 1).

Example 34 (2-Hydroxy-5-methoxyphenyl)(9-(2-hydroxyethyl)-9H-pyrido[2,3-b]indol-3-substituted)methanone (Compound I -35) preparation

Using 100 mg (0.314 mmol) of compound I-4, 65 mg (0.345 mmol) of 2-bromoethanol as a starting material, and the preparation of the compound I-34, a white solid (60 mg, yield: 53%).

^{1 H NMR (300MHz, DMSO-} d 6) δ9.74 (s, 1H), 8.90 (s, 1H), 8.78 (s, 1H), 8.33 (d, J = 7.9Hz, 1H), 7.76 (d, J = 8.8 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.4 Hz, 1H), 7.14 - 7.04 (m, 1H), 6.99 - 6.89 (m, 2H), 4.91 (t, J = 5.4 Hz, 1H), 4.57 (t, J = 6.4 Hz, 2H), 3.91 - 3.78 (m, 2H), 3.73 (s, 3H).

LC-MS: 363 (M+1).

Example 35 Preparation of 5-((Dimethylamino)-2-hydroxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-36)

100 mg (0.272 mmol) of compound I-7, 52 mg (0.272 mmol) of cuprous iodide, 10 mL of an aqueous solution of dimethylamine were added to a 15 mL sealed tube, and the reaction was sealed at 120 ° C overnight. After cooling to room temperature, the reaction system was poured into 150 mL of water, and extracted with 50 mL of DCM. The organic phase was combined, washed with 50 mL of brine, dried over anhydrous sodium sulfate and evaporated to dryness CH ₃ OH = 20:1) gave a brown solid 15 mg, yield 17%.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.27 (s, 1H), 9.51 (s, 1H), 8.88 (s, 1H), 8.73 (s, 1H), 8.29 (d, J = 10.2Hz, 1H), 7.61–7.42 (m, 2H), 7.37–7.12 (m, 2H), 7.05–6.84 (m, 1H), 6.82–6.58 (m, 1H), 2.81 (s, 6H).

LC-MS: 332 (M+1).

Example 36 Preparation of (2-hydroxy-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone oxime (Compound I-44)

100 mg (0.314 mmol) of compound I-4, 88 mg (1.26 mmol) of hydroxylamine hydrochloride, 130 mg (1.57 mmol) of sodium acetate, 10 mL of ethylene glycol were added to a 15 mL sealed tube, and the reaction was sealed at 130 ° C overnight. After cooling to room temperature, the reaction system was poured into 150 mL of water, and extracted with 50 mL of DCM. The organic phase was combined, washed with 50 mL of brine, dried over anhydrous sodium sulfate and evaporated to dryness CH ₃ OH=20:1) gave a white solid, 95 mg, yield 90%.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.00 (s, 1H), 11.69 (s, 1H), 10.47 (s, 1H), 8.57 (s, 1H), 8.37 (s, 1H), 8.20 ( d, J = 8.2 Hz, 1H), 7.61 - 7.41 (m, 2H), 7.30 - 7.15 (m, 2H), 6.89 (s, 1H), 6.50 (s, 1H), 3.56 (s, 3H).

LC-MS: 334 (M+1).

Example 37 Preparation of 2-(hydroxy(9H-pyrido[2,3-b]indole-3-substituted)methyl)-4-nitrophenol (Compound 8a)

In a 25 mL long-necked round bottom two-necked flask, 200 mg (0.809 mmol) of compound 5 was added under a nitrogen atmosphere, 15 mL of re-distilled THF, and the temperature was lowered to -78 ° C, and 0.9 mL (1.13 mmol) of a 1.3 M methyllithium THF solution was slowly added dropwise. The reaction was stirred at -78 ° C for half an hour, and 1.3 mL (1.64 mmol) of a 1.3 M tert-butyllithium THF solution was slowly added dropwise, and the reaction was stirred at -78 ° C for half an hour, and the system was dark green. Then, a solution of 774 mg (3.24 mmol) of Compound 7a in 5 mL of THF (re-steam) was slowly added dropwise, and the mixture was gradually warmed to room temperature, and the reaction was continued for 20 hours. After the reaction system was poured into 150 mL of saturated ammonium chloride, extracted with 50 mL of DCM three times, the organic phase was combined, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate, evaporated to dryness on a rotary evaporator, and purified by silica gel column chromatography (DCM/CH ₃ OH = 20:1) gave a brown solid 184 mg, yield 65%.

LC-MS: 350 (M+1).

Preparation of N-(4-hydroxy-3-(9H-pyrido[2,3-b]indole-3-carbonyl)phenyl)acetamide (Compound I-25)

In a 50 mL round bottom flask, 180 mg (0.516 mmol) of compound 8a, 30 mL of anhydrous DCM, 241 mg (0.568 mmol) of Dess-Martin oxidant were added, and the reaction was stirred at room temperature for 3 hours. After the reaction system was poured into 150 mL of DCM, washed with 50 mL of water three times, the organic phase was washed with 50 mL of saturated sodium bicarbonate solution, 50 mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness. 2.58 mmol) iron powder, 5 mL of acetic acid, and refluxed under heating for 2 hours. After the reaction system was poured into 150mL water, and extracted three times with 50mL DCM, and the combined organic phases were washed with 50mL of saturated brine, dried over anhydrous sodium sulfate, and evaporated to dryness, the crude product was purified by silica gel column chromatography (DCM / CH ₃ OH with = 50: 1) A brown solid of 25 mg was obtained in a yield of 14%.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.31 (s, 1H), 10.12 (s, 1H), 9.89 (s, 1H), 8.88 (s, 1H), 8.73 (s, 1H), 8.29 ( d, J = 8.9 Hz, 1H), 7.76 - 7.61 (m, 2H), 7.61 - 7.45 (m, 2H), 7.33 - 7.23 (m, 1H), 6.95 (d, J = 9.1 Hz, 1H), 2.00 (s, 3H).

LC-MS: 346 (M+1).

Example 38 Preparation of (2-(Dimethylamino)-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanol (Compound 8b)

200 mg (0.809 mmol) of the compound 5,629 mg (3.24 mmol) of the compound 7b was used as a starting material, and the preparation of the compound 8a was carried out to obtain a yellow solid (202 mg, yield: 72%).

¹ H NMR (300 MHz, Chloroform-d) δ 9.60 (s, 1H), 8.51 (s, 1H), 8.41 (s, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.57 - 7.41 (m) , 2H), 7.34 - 7.19 (m, 2H), 6.84 (d, J = 8.8 Hz, 1H), 6.58 (d, J = 2.9 Hz, 1H), 6.16 (s, 1H), 3.73 (s, 3H) , 2.62 (s, 6H).

LC-MS: 348 (M+1).

Preparation of (2-(dimethylamino)-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-37)

In a 50 mL round bottom flask, add 200 mg (0.576 mmol) of compound 8b, 30 mL of anhydrous DCM, 269 mg (0.634 mmol) Dess-Martin oxidant, and the reaction was stirred at room temperature for 3 hours. Then, the reaction system was poured into 150 ml of LDCM, and washed with 50 mL of water three times. The organic phase was washed with 50 mL of saturated sodium bicarbonate, washed with 50 mL of saturated brine, dried over anhydrous sodium sulfate and evaporated. Purification (petroleum ether / ethyl acetate = 2:1) afforded 155 mg of sd.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.27 (s, 1H), 8.86 (s, 1H), 8.64 (s, 1H), 8.29 (d, J = 8.3Hz, 1H), 7.64-7.45 ( m, 2H), 7.31 - 7.06 (m, 3H), 6.94 - 6.86 (m, 1H), 3.76 (s, 3H), 2.49 (s, 6H).

LC-MS: 346 (M+1).

Example 39 (2-2-(Dimethylamino)ethoxy)-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanol (Compound I -38) preparation

200 mg (0.809 mmol) of the compound 5,723 mg (3.24 mmol) of the compound 7c was used as a starting material, and the preparation of the compound 8a was carried out to obtain a pale yellow solid (150 mg, yield 47%).

^{1 H NMR (300MHz, DMSO-} d 6) δ11.66 (s, 1H), 8.43-8.35 (m, 2H), 8.12 (d, J = 7.7Hz, 1H), 7.51-7.36 (m, 2H), 7.26–7.13(m,2H), 6.87(d,J=8.8Hz,1H), 6.79–6.71(m,1H),6.09(s,1H),4.03–3.84(m,2H),3.73(s, 3H), 2.64–2.53 (m, 2H), 2.18 (s, 6H).

LC-MS: 392 (M+1).

Example 40 2-(2-(Dimethylamino)ethoxy)-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (compound) Preparation of I-40)

Using 100 mg (0.255 mmol) of compound I-38 as a starting material, a white solid 52 mg was obtained with reference compound compound I-37.

^{1 H NMR (300MHz, DMSO-} d 6) δ12.32 (s, 1H), 8.85 (s, 1H), 8.67 (s, 1H), 8.30 (d, J = 8.9Hz, 1H), 7.93 (d, J = 10.8 Hz, 1H), 7.58 - 7.41 (m, 2H), 7.32 - 7.10 (m, 2H), 7.00 (s, 1H), 4.09 - 3.93 (m, 2H), 3.77 (s, 3H), 2.39 – 2.23 (m, 2H), 1.92 (s, 6H).

LC-MS: 390 (M + 1).

Example 41 2-((2-(Hydroxy(9H-pyrido[2,3-b]indole-3-substituted)methyl)-4-methoxyphenoxy)ethanol (Compound I-39) Preparation

150 mg (0.607 mmol) of the compound 5,652 mg (2.43 mmol) of the compound 7d was used as a starting material, and the preparation of the compound 8a was carried out to obtain a pale yellow solid (133 mg, yield: 60%).

^{1 H NMR (300MHz, DMSO-} d 6) δ11.64 (s, 1H), 8.46 (s, 1H), 8.44 (s, 1H), 8.13 (d, J = 7.6Hz, 1H), 7.48-7.36 ( m, 1H), 7.25 (d, J = 3.2 Hz, 1H), 7.21 - 7.14 (m, 1H), 6.83 (d, J = 9.1 Hz, 1H), 6.73 (dd, J = 8.8, 3.2 Hz, 1H) ), 6.17 (d, J = 4.0 Hz, 1H), 5.84 (d, J = 4.0 Hz, 1H), 4.92 (t, J = 5.3 Hz, 1H), 3.96 - 3.78 (m, 2H), 3.72 (s) , 3H), 3.71–3.65 (m, 2H).

LC-MS: 365 (M+1).

Example 42 (2-(2-Hydroxyethoxy)-5-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-41) Preparation

Using 95 mg (0.261 mmol) of the compound I-39 as a starting material, a white solid (72 mg,yield: 77%).

^{1 H NMR (300MHz, DMSO-} d 6) δ12.30 (s, 1H), 8.87 (s, 1H), 8.68 (s, 1H), 8.30 (d, J = 8.1Hz, 1H), 7.61-7.44 ( m, 2H), 7.35 - 7.07 (m, 3H), 6.97 (s, 1H), 4.54 (t, J = 5.4 Hz, 1H), 3.91 (t, J = 5.4 Hz, 2H), 3.77 (s, 3H) ), 3.31–3.20 (m, 2H).

LC-MS: 363 (M+1).

Example 43 Preparation of 2-(hydroxy(9H-pyrido[2,3-b]indol-3-substituted)methyl)-4-methoxyphenol (Compound I-42)

150 mg (0.607 mmol) of compound 5,544 mg (2.43 mmol) of compound 7e was used as a starting material.

^{1 H NMR (300MHz, DMSO-} d 6) δ11.70 (s, 1H), 9.02 (s, 1H), 8.40 (s, 2H), 8.14 (d, J = 7.7Hz, 1H), 7.51-7.37 ( m, 2H), 7.24 - 7.11 (m, 2H), 6.72 - 6.59 (m, 2H), 6.11 (d, J = 4.0 Hz, 1H), 5.87 (d, J = 4.2 Hz, 1H), 3.69 (s) , 3H).

LC-MS: 321 (M + 1).

Example 44 Preparation of (3-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanol (Compound 8c)

200 mg (0.809 mmol) of the compound 5,441 mg (3.24 mmol) of the compound 7f was used as a starting material, and the preparation of the compound 8a was carried out to obtain a white solid (200 mg, yield 81%).

¹ H NMR (300 MHz, Chloroform-d) δ 9.06 (s, 1H), 8.48 (s, 1H), 8.33 (s, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.52 - 7.43 (m) , 2H), 7.34 - 7.28 (m, 2H), 7.07 - 7.00 (m, 2H), 6.84 (dd, J = 8.7, 3.1 Hz, 1H), 6.07 (s, 1H), 3.81 (s, 3H).

LC-MS: 305 (M+1).

Preparation of (3-methoxyphenyl)(9H-pyrido[2,3-b]indole-3-substituted)methanone (Compound I-43)

Using 200 mg (0.658 mmol) of compound 8c as a starting material, a white solid 112 mg was obtained with reference compound compound I-37.

¹ H NMR (300 MHz, DMSO-d ₆ ) δ 12.34 (s, 1H), 8.94 (s, 1H), 8.79 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 7.66 - 7.47 ( m, 3H), 7.42–7.16 (m, 4H), 3.84 (s, 3H).

LC-MS: 303 (M + 1).

Biological activity test

1. Some compounds inhibit tumor cell growth inhibition

Experimental method: Sulforhodamine B (SRB) colorimetric method (Reference: Zhang Z, Meng T, Yang N, Wang W, Xiong B, Chen Y, et al. MT119, a new planar-structured compound ,targets colchicine site of tubulin arresting mitosis and inhibiting tumor cell proliferation.International Journal of Cancer 2011;129(1):214-24)

The specific method is as follows:

(1) cells in the logarithmic growth phase were seeded in a 96-well culture plate at a suitable density, 90 μl per well, and cultured overnight;

(2) adding different concentrations of compounds for 72h, each concentration of 3 duplicate wells, and set the corresponding concentration of physiological saline vehicle control and cell-free zero-reducing wells;

(3) After the end of the action, the adherent cells were decanted, and 10% (w/v) trichloroacetic acid (100 μl/well) was added and fixed at 4 ° C for 1 h;

(4) Rinse with distilled water 5 times, after drying at room temperature, add 100 μl of SRB solution (4 mg / ml, dissolved in 1% glacial acetic acid) per well, and incubate for 15 min at room temperature;

(5) Rinse 5 times with 1% glacial acetic acid to wash away unbound SRB. After drying at room temperature, 150 μl of 10 mM Tris solution was added to each well, and the optical density (OD value) at a wavelength of 560 nm was measured by a microplate reader.

The inhibitory effect of the compound on tumor cell proliferation was calculated according to the following formula: inhibition rate (%) = (OD _{control well-} OD _{administration} _well ) / OD _{control well} × 100%, and according to the logit method, the 50% inhibition rate was calculated. Compound concentration, ie IC ₅₀ value. The experiment was repeated 3 times and the mean and standard deviation were calculated.

The results showed that many compounds have good inhibitory activity against HeLa cells and HT-29 (American Type Culture Collection) cells (Table 1.). Among them, compound I-4 has strong inhibitory effect on a variety of tumor cells, such as oral cancer, lung cancer, liver cancer, leukemia, gastric cancer, cervical cancer, ovarian cancer, breast cancer, colon cancer and prostate cancer cells, and has a broad anti-tumor spectrum. 1).

Table 1. Inhibition of proliferation of HeLa and HT-29 cells by some compounds at the cellular level

2. Effect of compound I-4 on cell tubulin

Experimental method: Immunofluorescence-based laser confocal microscopy (References Wang W, Wang YQ, Meng T, Yi JM, Huan XJ, Ma LP, et al. MCL-1 Degradation Mediated by JNK Activation via MEKK1/TAK1- MKK4 Contributes to Anticancer Activity of New Tubulin Inhibitor MT 189. Molecular Cancer Therapeutics, 2014; 13(6): 1480-91). The principle is the specific binding reaction of antigen antibodies.

The specific method is as follows:

(1) The logarithmic growth phase HeLa cells (American Type Culture Collection) (1 × 10 ⁵ / well) were seeded on a cover glass of a 12-well culture plate;

(2) After the cells were adhered overnight, different concentrations of compound I-4 and positive control compounds paclitaxel and vincristine (Sigma-Aldrich) were added to treat HeLa cells for 1 h;

(3) Discard the culture solution, add 1 ml of 4% paraformaldehyde per well, and fix for 30 min;

(4) Washed 3 times with TBST washing solution containing 0.2% Tween-20, and added 1 ml of 0.2% Triton X-100 permeable cells per well for 15 min;

(5) Washing with TBST washing solution 3 times, each well was added with 1 ml of 3% BSA for 15 min;

(6) Carefully grasp the coverslip, the cells were placed face down on a Parafilm membrane on a 3% BSA prepared primary antibody solution, and hybridized under humid conditions for 1 h;

(7) Washing with TBST washing solution 3 times, carefully grasping the coverslip, the cells were placed face down on a Parafilm membrane on a 3% BSA prepared secondary antibody solution, and hybridized under humid conditions for 1 h;

(8) Wash 3 times with TBST washing solution, add 3 μl of DAPI-containing sealing tablets to the slides, and place the coverslips on the slides face down. Observe the changes of microtubule state by confocal fluorescence microscope. Situation and take pictures.

The experimental results show that compound I-4 is the same as the microtubule depolymerization agent, promoting the depolymerization of microtubules, but different from the microtubule stabilizer paclitaxel (Fig. 2). At 0.1 μM, compound I-4 can obviously depolymerize microtubules and destroy their fineness. Normal intracellular distribution of cells; almost complete depolymerization of microtubules at 0.3 μM.

3. Effect of compound I-4 on topoisomerase II (Top2)

Experimental method: Top2-mediated supercoiled pBR322 relaxation (Reference: Meng LH, Zhang JS, Ding J. Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage Complexes. Biochemical Pharmacology 2001; 62(6): 733-41). The principle is that the kDNA structure is network-like, the molecular weight is too large to enter the 1% agarose gel, and Top2 can catalyze its de-coupling reaction, producing 2.5KB of monomeric circular DNA, which can quickly enter 1% agar. In the glycogel, the effect of the compound on the ring-shaped DNA of the monomer is examined to reflect its effect on Top2 activity.

The specific method is as follows:

reaction system:

100ng kDNA (TopoGEN)

4U Top2

Buffer: 4μl 10×DNA Top2 buffer (mixed by buffer A and B in the Top2 assay kit,

TopoGEN)

ddH ₂ O: up to 20μl

Reaction conditions: 37 ° C; 30 min.

R16(5-(2-(dimethylamino)ethyl)-4H-benzo[de]benzo[4,5]thieno[2,3-g]isoquinoline-4,6(5H)-dione) as a positive control, compound I-4 was tested for activity at two concentrations of 100 μM and 150 μM, respectively. After the reaction, the mixture was electrophoresed on a 1% agarose gel in TAE buffer for 1 h at a voltage of 100 V. The cells were photographed by a gel imaging system after staining with 1 μg/ml of GelRed.

The results showed that Compound I-4 significantly inhibited the unwinding activity of Top2 kDNA at a concentration of 100 μM, and the monomeric circular DNA produced by Top 2 completely disappeared (Fig. 3).

4. Compound I-4 induces tumor cell apoptosis and the difference between etoposide (Top2 inhibitor) and vincristine (tubulin inhibitor)

Experimental method: Western blot experiment (References Wang W, Wang YQ, Meng T, Yi JM, Huan XJ, Ma LP, et al. Molecular Cancer Therapeutics 2014; 13(6): 1480-91.)

The specific method is as follows:

(1) The HeLa cells in the logarithmic growth phase were seeded in a 6-well culture plate at a suitable density, and the cells were adhered to the corresponding concentration of the compound overnight, and the corresponding time was applied at 37 °C.

(2) Thereafter, cells were lysed by adding 1 x SDS loading buffer (50 mM Tris pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromophenol blue, 10% glycerol) per well.

(3) After collecting the cell lysate, it was heated in a boiling water bath for 10 min and centrifuged at 10,000 rpm for 10 min. The supernatant was taken for SDS-PAGE electrophoresis, and after electrophoresis, the protein was transferred to a nitrocellulose membrane using a semi-dry electrotransfer system.

(4) After the end of the transfer, the transfer condition and the position of the protein band on the nitrocellulose membrane were determined by Ponceau S staining, and the blocking solution containing 5% skim milk powder was used after labeling [5% skim milk powder, 20 mM Tris-HCl pH 7.2-7.4, 150 mM NaCl, 0.1% Tween-20] was blocked at room temperature for 30 min at room temperature. Then, the membrane was placed in a primary antibody diluted in blocking solution (5% skimmed milk powder) at 4 ° C overnight.

Wash three times with a washing solution [100 mM Tris-HCl pH 7.2-7.4, 0.9% NaCl, 0.2% Tween-20] for 10 min each time. Horseradish peroxidase-labeled secondary antibody was added and shaken gently on a shaker at room temperature for 1 h. After washing three times with the washing solution, color development, exposure, development, fixing, and photographing. According to the experimental needs, the results of Western blot were quantitatively analyzed using Adobe Photoshop CS2.

The experimental results show that in the induction of apoptosis experiments, in combination with concentration and time, the mechanism of apoptosis induced by compound I-4 is similar to that of vincristine, but different from etoposide, which is different from the combination of the two. The combination of the two drugs could not inhibit the expression of MCL-1 (myeloid cell leukemia-1), cIAP1 (cellular inhibitor of apoptosis protein-1) and XIAP (X-linked inhibitor of apoptosis protein) in HeLa cells. . The experiment showed that the compound I-4 differed from the combination in the mechanism of inducing tumor cell apoptosis (Fig. 4).

5. Experiment on the inhibitory effect of compound I-4 on the growth of drug-resistant tumor cells

Experimental methods and specific operations Reference experiment 1, the resistant cell lines were vincristine-resistant KB cells (KB/VCR), doxorubicin-resistant MES-SA cells (MES-SA/DX5) and rice Detoxified resistant HL60 cells (HL60/MX2). The growth inhibition effect of compound I-4 on drug-resistant tumor cells and parental tumor cells is shown in Table 2.

Table 2. Inhibition of proliferation of drug-resistant tumor cells and parental tumors by compound I-4 at the cellular level

RF: Resistance index

It can be seen from the above results that the compounds have obvious anti-tumor effects, and also have strong effects on drug-resistant tumor cells, especially the strong killing effect of such compounds on drug-resistant tumor cell lines is worthy of attention. And the initial mechanism of action shows that the results of this class of compounds are not exactly the same as the combination, such as the combination of etoposide VP-16 (Top2 inhibitor) and vincristine (tubulin inhibitor). These dual inhibitors make future medications easier and less susceptible to drug resistance.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A class of compounds having the structure of formula I below:

among them,

R 1 , R 2 , R 3 , R 4 are each independently selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, substituted or unsubstituted C1-C4 straight or branched alkyl, substituted or unsubstituted Substituted C3-C6 cycloalkyl, substituted or unsubstituted C1-C4 linear or branched alkoxy group, substituted or unsubstituted C1-C4 linear or branched alkylene-amino group, substituted Or an unsubstituted C1-C4 linear or branched amine group, and a substituted or unsubstituted saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R 5 is selected from the group consisting of hydrogen, a substituted or unsubstituted C1-C4 linear or branched alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C1-C4 linear chain. Or a branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched alkylene-hydroxy group, a substituted or unsubstituted C1-C4 linear or branched amine group, and a substituted or unsubstituted a saturated five- or six-membered heterocyclic group containing oxygen or nitrogen;

R 6 is selected from the group consisting of hydrogen, halogen, hydroxy, amino, methylamino, dimethylamino, substituted or unsubstituted C1-C4 straight or branched alkoxy, substituted or unsubstituted C1-C4 straight a branched or branched amino group, a substituted or unsubstituted C1-C4 linear or branched amino-oxy group;

R 7 is selected from the group consisting of hydrogen, halogen, hydroxy, amino, methyl, methoxy, methylamino, dimethylamino, -OAc, methoxycarbonyl, acetylamino, substituted or unsubstituted C1-C4 straight chain Or a branched alkyl group, a substituted or unsubstituted C1-C4 linear or branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched amine group;

R 8 is selected from the group consisting of hydrogen, halogen, hydroxy, amino, methyl, methoxy, methylamino, dimethylamino, methoxycarbonyl, acetylamino, methylsulfonyl, substituted or unsubstituted C1-C4 straight a branched or branched alkyl group, a substituted or unsubstituted C1-C4 linear or branched alkoxy group, a substituted or unsubstituted C1-C4 linear or branched amine group;

X is a linking group selected from the group consisting of methylene, oxygen, sulfur, carbonyl, sulfoxide, sulfone,

Wherein said one or more hydrogen atoms on the substituent group are substituted with a substituent selected from the group consisting of halogen, C1-C8 alkyl, C1-C8 alkyl-amine group, hydroxyl group, methylamino group. Amino, C1-C8 alkyl-oxy group.
A compound of formula I according to claim 1 wherein

R 5 is selected from the group consisting of hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R 6 is selected from the group consisting of hydrogen, hydroxy, -NH-CH 3 , -NH 2 , -N(CH 3 ) 2 , -O(CH 2 ) 2 OH, -O(CH 2 ) 2 N(CH 3 ) 2 ;

R 7 is selected from the group consisting of hydrogen, fluorine, hydroxyl, methyl, methoxy, acetylamino, -OAc;

R 8 is selected from the group consisting of hydrogen, fluorine, chlorine, bromine, methyl, methoxy, acetylamino, dimethylamino, methylsulfonate;

X is selected from the group consisting of oxygen, sulfur,
Carbonyl, sulfoxide, sulfone,
A compound of formula I according to claim 1 wherein

R 1 is hydrogen or fluorine;

R 2 is hydrogen, fluorine, chlorine, methoxy, dimethylamino, morpholinyl;

R 3 is hydrogen, fluorine, chlorine or methoxy;

R 4 is hydrogen, fluorine or methoxy;

R 5 is hydrogen, hydroxyethyl, N,N-dimethylaminoethyl;

R 6 is hydrogen, hydroxy, amino, methylamino, dimethylamino, -O(CH 2 ) 2 OH, -O(CH 2 ) 2 N(CH 3 ) 2 ;

R 7 is hydrogen, fluorine, hydroxyl, methoxy, -OAc;

R 8 is hydrogen, fluorine, chlorine, bromine, methoxy, acetylamino, dimethylamino, methylsulfonate;

X is a linked sulfur, methylamino, carbonyl, sulfoxide, sulfone group,
A method of preparing a compound according to claim 1, comprising the step (b) and optionally (c), (c1) and/or (c2):

(b) a ring closure reaction with a compound of formula 3 in the presence of a reducing agent in an organic solvent to provide a compound of formula Ia;

(c) subjecting a compound of formula Ia to R 5 Y in an organic solvent in the presence of a base to give a compound of formula Ib;

(c1) preparing a compound of formula Ib' with a compound of formula Ib;

(c2) preparing a compound of formula I using a compound of formula Ib';

Preferably, the method further comprises the steps of:

(a) reacting a compound of formula 1 with a compound of formula 2 in an organic solvent to provide a compound of formula 3;

Wherein Y is selected from the group consisting of chlorine, iodine, bromine, mesylate, p-toluenesulfonate, triflate; the remainder of each group is as defined in claim 1.
The method of claim 4 wherein said method comprises the step (d):

(d) reacting a compound of formula 5 with a compound of formula 6 in an inert solvent in the presence of a base to provide a compound of formula Ic;

Or the method comprises the step (e) and the optional step (f):

(e) reacting a compound of formula 5 with a compound of formula 7 in the presence of a base in an inert solvent to provide a compound of formula Id;

(f) reacting a compound of formula Id with an oxidizing agent in the presence of an oxidizing agent in an inert solvent to provide a compound of formula Ie;

Wherein each group is as defined in claim 1.
Use of a compound of formula I according to claim 1, or a pharmaceutically acceptable salt or hydrate thereof, (a) non-therapeutic inhibition of tumor cell growth in vitro; (b) preparation of a medicament for treating tumors a composition; (c) non-therapeutic inhibition of topoisomerase II activity in vitro; (d) non-therapeutic inhibition of tubulin activity in vitro; (e) non-therapeutic depolymerization of cellular microtubules in vitro; (f) non-therapeutic inhibition of proliferation of tumor cells in vitro; (g) preparation of a pharmaceutical composition having topoisomerase II and tubulin dual inhibitory activity.
A topoisomerase II activity inhibitor comprising an inhibitory effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or hydrate thereof.
A cell microtubule depolymerizing agent comprising a depolymerized effective amount of the compound of claim 1, or a pharmaceutically acceptable salt or hydrate thereof.
A pharmaceutical composition comprising a therapeutically effective amount of a compound of claim 1, or a pharmaceutically acceptable salt or hydrate thereof.
A dual inhibitor of topoisomerase II and tubulin, characterized in that the inhibitor comprises the compound of claim 1, or a pharmaceutically acceptable salt or hydrate thereof.