WO2021114864A1 - β-CARBOLINE CYCLOKETENE DERIVATIVE BASED ON DUAL RESPONSE TO PH AND GSH, AND USE THEREOF - Google Patents

Abstract

Provided is a β-carboline-cycloketene derivative, which has the structure represented by general formula (I). A new-type β-carboline-cycloketene derivative is designed and synthesized by means of introducing an electron-donating group at a suitable position of β-carboline, and connecting cycloketene, which has an anti-tumor activity, by means of the amino group at position 3 of β-carboline and by using a carbamate bond. Not only can the compound realize fluorescence imaging for a dual response to pH and GSH and diagnosis in a tumor microenvironment, same can selectively target GSTπ, which is highly expressed in tumor tissues, to significantly inhibit tumor cell proliferation. The pH/GSH dual response fluorescence and targeted cancer treatment and diagnosis provide a new choice for precise tumor diagnosis and targeted therapy, and broaden the use of multifunctional molecules in precision medicine.

Description

Beta-carboline-cycloketene derivatives based on dual response of pH and GSH and uses thereof Technical field

The present invention relates to the field of biomedicine, in particular to β-carboline-cycloketene derivatives based on the dual response of pH and GSH, and a preparation method thereof, as well as the medicinal use of drugs targeting GSH/GSTπ to inhibit tumor proliferation, especially in the preparation of anti-tumor Application in diagnostic and therapeutic agents.

Background technique

Malignant tumors are a serious threat to human health and lives, and the number of deaths caused by cancer has risen rapidly, and it has become the world's first lethal disease, and it has become a global challenge and problem. The research and development of diagnostic and therapeutic agents has become one of the research hotspots in the field of medicine. It combines diagnosis and treatment into one, and effective treatment (surgery and/or drugs) is given immediately at the same time as the diagnosis is made, which improves the efficiency of treatment. And the specificity of drug release. Among them, there are mainly two types of small molecule diagnostic and therapeutic agents and macromolecular nano diagnostic and therapeutic agents. The preparation of the former is relatively easy. Usually, a prodrug strategy is used to obtain an anticancer drug, an imaging agent, and an activation unit through a covalent bond. Small-molecule diagnostic and therapeutic agents generally have good fluorescence imaging capabilities, and can be induced by tumor cell-related molecules to release drugs in concert, increase the selectivity to solid tumors, and thereby improve the anti-cancer effect. Compared with macromolecular nano-diagnostic and therapeutic agents, these small-molecule prodrug systems have better biocompatibility and cell absorption, and can be regenerated.

The tumor microenvironment (TME) has unique physicochemical properties and is significantly different from normal tissues. It refers to the special environment for tumor cell growth formed by the interaction of tumor cells and extracellular matrix during the growth process of tumor cells. Early dynamic tracking of the tumor and its microenvironment will facilitate surgical resection and/or precise drug treatment. TME not only provides conditions for the growth of tumor cells, but also a necessary place for tumor cell metastasis. Taking into account that tumor cells and their microenvironment undergo glycolysis under hypoxic conditions, and excrete H ⁺ , the tumor tissue has an obvious slightly acidic pH (pH 6.5-6.8), while the pH of normal tissues is 7.2-7.4; There are also organelles with higher acidity in tumor cells, such as lysosomes (pH 4.5-5.0); therefore, it is of great significance to study pH-sensitive fluorescent probes to selectively target tumor cells and their microenvironment. However, traditional pH-sensitive fluorescent probes are mainly activated by acid-sensitive hydrazone bonds or acetal fragments, but these groups themselves have problems such as instability in vivo, slower color development, and easy metabolism.

Glutathione sulfhydryl transferases, or glutathione-S-transferases (GSTs), are a multifunctional isoenzyme widely found in mammals. Its main function is to catalyze the nucleophilic attack of the sulfhydryl group of glutathione (GSH) against endogenous or exogenous electrophilic groups (such as carbon, nitrogen, sulfur, etc.) to undergo coupling reactions, forming a more water-soluble metabolism Product, which is easily excreted in bile or urine. The structure of human cell GSTs is closely related to the occurrence and progression of human diseases. According to amino acid sequence, physical structure, and immune cross-reactivity, human cytoplasmic GSTs can be divided into 7 subtypes, namely: α, μ, π, σ, θ, ω, and ζ. Among them, GSTπ is related to human tissue cell canceration, Tumor formation and the generation of tumor drug resistance are closely related. Studies have shown that in a variety of tumor cells (such as breast cancer, colon cancer tumor cells) and drug-resistant tumors, GSTπ is overexpressed. Especially in the liver and intestinal tissues, the formation of GSTπ is considered to be one of the signs of liver cancer. This characteristic of GSTπ makes it an important target in the design of anti-tumor drug prodrugs. In view of this, the use of the highly expressed GSTπ in tumor tissues to develop and design cancer chemotherapeutic drugs and probes has great application value.

Summary of the invention

The present invention is based on the structural modification of the natural indole alkaloid β-carboline with anti-tumor activity. The β-carboline is connected to the cycloketene through a carbamate bond, and electron donors are introduced into the structure of the β-carboline. The group can not only produce pH-sensitive fluorescence, but also release fluorescence at 492nm after covalently binding to GSH. It also targets GSTπ, which significantly enhances the compound’s anti-tumor proliferation and metastasis activity, achieving early cancer diagnosis and precise treatment. Double targeting effect.

The specific technical scheme of the present invention is as follows: a class of β-carboline-cycloketene derivatives having the structure shown in the following general formula:

among them,

R ₁ or R _{2 are the} same or different and represent one or more substituents on the corresponding substituted ring, selected from one of H, amino, halogen, hydroxyl, nitro, alkoxy, alkyl, alkylamino or There are several types, when R ₁ or R ₂ each represents multiple substituents, the substituents are the same or different;

R _{3 is} selected from H, benzyl, allyl, alkyl, methoxyalkyl;

R _{4 is} selected from H, alkyl, methoxyalkyl or

n=1, 2 or 3.

Preferably, R _{1 or R 2 is} selected from one or more of H, amino, halogen, hydroxyl, nitro, C1-C6 alkoxy, C1-C6 alkyl, and C1-C6 alkylamino. , More preferably H, F, Cl, Br, I, hydroxyl, amino, nitro, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, methylamino, ethylamino, methyl ethyl One or more of amino group and N,N-dimethylamino group;

R _{3 is} selected from H, benzyl, allyl, C1-C6 alkyl, C1-C6 methoxyalkyl, more preferably H, benzyl, allyl, methyl, ethyl, propyl, iso Propyl, methoxymethyl, methoxyethyl, methoxypropyl or methoxyisopropyl;

R _{4 is} selected from H, C1-C6 alkyl, C1-C6 methoxyalkyl or

More preferably H, methyl, ethyl, propyl, isopropyl, methoxymethyl, methoxyethyl, methoxypropyl, methoxyisopropyl or

n=1, 2 or 3.

A specific type of β-carboline/cycloalkenone derivative of the present invention, wherein R ₁ represents 3,4,5-Tri-OCH ₃ , 4-CH ₃ , 3-OCH ₃ , 4-OCH ₃ , 4- N,N-Di-CH ₃ , 2,4-Di-OCH ₃ , 2,5-Di-OCH ₃ , 3,4-Di-OCH ₃ or 3,5-Di-OCH ₃ ; R ₂ or R ₃ Stands for H; R ₄ stands for H or

n=1, 2 or 3.

The β-carboline/cycloketene derivatives involved in the specific embodiments of the present invention have the following structure:

Table 1

Ⅰ ₁ : (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅱ ₁ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b] Indol-3-yl) carbamate;

I ₂ : (6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)aminomethyl Acid ester

Ⅱ ₂ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)amino Formate

Ⅰ ₃ : (6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

Ⅱ ₃ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

I ₄ : (6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

Ⅱ ₄ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

Ⅰ ₅ : (6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₅ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

I ₆ : (6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₆ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅰ ₇ : (6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₇ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅰ ₈ : (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₈ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅰ ₉ : (6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₉ : Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole Dol-3-yl) carbamate;

Ⅰ ₁₀ : (6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅱ ₁₀ : Bis(6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b] Indol-3-yl) carbamate;

Ⅰ ₁₁ : (6-oxocyclopent-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₁₁ : Bis(6-oxocyclopent-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅰ ₁₂ : (6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate;

Ⅱ ₁₂ : Bis(6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b] Indol-3-yl) carbamate;

Ⅰ ₁₃ : (6-oxocyclohept-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate;

Ⅱ ₁₃ : Bis(6-oxocyclohept-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate.

Another object of the present invention is to provide a preparation method of the compound of the general formula of the present invention, which comprises the following steps:

(1) Compound 1 is reacted with hydrazine hydrate to obtain compound 2, preferably with 85% hydrazine hydrate in methanol,

(2) Reacting compound 2 with NaNO ₂ under acidic conditions, preferably under dilute hydrochloric acid conditions, to obtain compound 3.

(3) reacting compound 3 under acidic conditions, preferably aqueous acetic acid, to obtain compound 4;

(4) Compound 5 is reacted with p-nitrophenyl chloroformate under alkaline conditions, preferably DIPEA conditions, to obtain compound 6,

(5) reacting compound 4 with compound 6 under basic conditions, preferably under DIPEA conditions, to obtain compound 7 and/or compound 8,

Alternatively, the above synthesis step further includes step (6), reacting compound 7 under sodium hydrogen, and then reacting with benzyl bromide, allyl bromide or alkyl bromide to obtain compound 9.

R ₁ or R _{2 are the} same or different and represent one or more substituents on the corresponding substituted ring, selected from one of H, amino, halogen, hydroxyl, nitro, alkoxy, alkyl, alkylamino or There are several types, when R ₁ or R ₂ each represents multiple substituents, the substituents are the same or different;

R _{3 is} selected from H, benzyl, allyl, alkyl, methoxyalkyl;

R _{4 is} selected from H, alkyl or methoxyalkyl;

n=1, 2 or 3.

Another object of the present invention is to provide the application of the β-carboline-cycloketene derivatives of the present invention in the preparation of drugs or probes targeting GSTπ. On the one hand, after the β-carboline/cycloketene derivative of the present invention targets the highly expressed GSTπ in tumor tissues, the cycloketene fragment in the compound structure can specifically recognize the sulfhydryl group of GSH and undergo Michael addition to produce GSH Responsive fluorescence. The compound of the present invention also has pH-sensitive fluorescence, and when the pH drops from 8 to 4, the fluorescence increases significantly. On the other hand, the β-carboline-cycloketene derivative of the present invention targets the highly expressed GSTπ in tumor tissues and can be selectively activated by GSH/GSTπ in tumor cells to release the pharmacologically active fragment 3-glutathione- 2-Exomethylene cyclic ketone and β-carboline fragments significantly enhance anti-tumor proliferation and metastasis activity and other pharmacological activities. The drug with targeted GSTπ is a drug for treating and/or preventing cancer. Preferably, the cancer is selected from liver cancer, colon cancer, cervical cancer or gastric cancer.

The compound of the present invention has good fluorescence imaging ability, high selectivity for solid tumors, and can achieve the dual purpose of diagnosis and treatment.

The compound of the present invention can be formulated alone or in combination with one or more pharmaceutically acceptable carriers to provide medicine. For example, solvents, diluents, etc., can also be used in oral dosage forms, such as capsules, dispersible powders, tablets, granules, and the like. Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to methods well known in the pharmaceutical field. These pharmaceutical preparations may contain, for example, 0.05% to 90% by weight of the active ingredient in combination with the carrier, and more commonly about 15% to 60% by weight of the active ingredient. The dosage of the compound of the present invention can be 0.005-5000 mg/kg/day, and the dosage can also exceed this dosage range according to the severity of the disease or the different dosage forms.

The compound of the present invention can self-assemble into nanoparticles to improve activity alone, or be combined with other anti-tumor drugs such as alkylating agents (such as cyclophosphamide or chlorambucil), antimetabolites (such as 5-fluorouracil or hydroxyurea), topological Isomerase inhibitors (such as camptothecin), mitotic inhibitors (such as paclitaxel or vinblastine), DNA intercalants (such as doxorubicin) combined with self-assembled nanoparticles to improve activity, and can also be combined with radiotherapy. These other anti-tumor drugs or radiotherapy can be administered at the same time or at different times with the compounds of the present invention. These combination therapies can produce synergistic effects to help improve the therapeutic effect.

The invention combines the structural features, structure-activity relationship and pharmacophore model of the anti-tumor drug 2-crotonyloxymethyl-2-cyclohexene (COMC-6) and the natural alkaloid β-carboline with anti-tumor activity , Adopting prodrug strategy, selectively release fluorescence in tumor cells through GSH/GSTπ, and degrade to produce active fragments, so as to realize the enhancement of fluorescence signal and imaging detection. The results of the research on the inhibitory effect of the present invention on malignant tumor cells show that the compounds can perform pre-diagnosis of cancer cells through fluorescent signals and can strongly inhibit the proliferation of liver cancer, colon cancer, cervical cancer and other tumor cells. The compound of the invention has many advantages such as ingenious design, good selectivity to GSH, high anti-tumor activity and low toxicity. As a sensor for intracellular GSH/GSTπ fluorescence imaging, it has the advantages of high selectivity, direct observation, and easy real-time monitoring. It can play an important role in the detection, imaging, and treatment of cancer cells.

Description of the drawings

Figure 1 shows the pH response UV fluorescence spectrum of compound I _1. (FIG. 1A is a compound Ⅰ ₁ in an ultraviolet absorption spectrum of pH 3-8; FIG. 1B is an emission spectrum of compound Ⅰ ₁ (Ex = 445nm) of fluorescent pH 3-8; FIG. 1C is a compound Ⅰ ₁ at the ultraviolet absorption at 445nm The change curve of the intensity at pH 3-8; Fig. 1D is the change curve _{of the fluorescence intensity of I 1} at 490nm at pH 3-8).

Figure 2 shows the GSH response ultraviolet fluorescence spectrum of compound I _1. Figure 2A is _{the ultraviolet absorption spectrum of compound I 1} with 0-10 equivalents of GSH (containing catalytic GSTπ); Figure 2B is _{the fluorescence emission of compound I 1} with 0-50 equivalents of GSH (containing catalytic GSTπ) Spectrum (Ex=440nm); Fig. 2C is the _{curve of the fluorescence intensity of compound I 1} at 492 nm with the change of GSH concentration; Fig. 2D is the time-dose curve of the fluorescence intensity _{of compound I 1 with GSH (GSTπ containing catalytic content).}

Figure 3 shows the specific response fluorescence characteristics of compound I _{1 and biological endogenous substances.}

Figure 4 shows the fluorescence imaging of the compound of the present invention in HT29 cells.

Figure 5 shows the in vivo fluorescence imaging _{of compound I 1.}

Figure 6 shows the results of in vivo anti-tumor activity of _{compound I 1 of the present invention.}

Detailed ways

In order to further clarify the present invention, a series of examples are given below. These examples are completely illustrative. They are only used to describe the present invention in detail and should not be construed as limiting the present invention.

Example 1 (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate (I ₁ ) or bis(6-oxocyclohex-1-en-1-yl)methyl (1-(3,4,5-trimethoxyphenyl) Preparation of -9H-pyrido[3,4-b]indol-3-yl)carbamate (Ⅱ ₁₎

(1) Preparation of 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonyl hydrazide (2a)

The compound 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylic acid methyl ester (1a) (19.6g, 50mmol) was dissolved in 80mL After methanol was added, 176.4mL 85% hydrazine hydrate (75g, 1.50mol) was added and refluxed for 4-5h. TLC monitored the completion of the reaction. After the reaction solution was cooled to 0°C, it was filtered by vacuum pump to obtain a light brown solid. A large amount of cold water was added to the filtrate, and the solid continued to separate out. After suction filtration and vacuum drying, 16.5 g of light brown solid was obtained with a yield of 84.2%.

(2) Preparation of 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3a)

After dissolving compound (2a) (19.6g, 50mmol) with 80mL of 2mol/L HCl solution, then use 60mL of H ₂ O to mix NaNO ₂ (10.4g, 150mmol) in an ice bath, and mix the prepared _{NaNO 2 solution} Slowly add dropwise to the compound (2a) solution, and then stir for 1-4h. TLC monitors the completion of the reaction. Adjust the pH to 7-8 with 1mol/L NaOH solution. A pale yellow solid precipitates, and a pale yellow solid is obtained by suction filtration. The product was 17.8 g, and the yield was 88.4%.

(3) Preparation of 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4a)

After dissolving compound (3a) (20.1g, 50mmol) in a mixture of 100mL water and glacial acetic acid (1:1), reflux at 90°C for 4-5h. TLC monitors the reaction to completion. The reaction solution is concentrated under reduced pressure to make sand. Purified by column chromatography, 8.4 g of light yellow solid was obtained with a yield of 48.2%. ESI-MS (m/z): 350 [M+H] ⁺ .

(4) Preparation of 4-nitrophenyl ((6-oxocyclohex-1-en-1-yl)methyl) carbonate (6b)

After the compound 2-(hydroxymethyl)-cyclohexenone (5b) (1.3g, 10mmol) was completely dissolved in 12ml of dichloromethane, N,N-diisopropylethylamine (DIPEA) (3.9g, 30mmol), p-nitrophenyl chloroformate (3.1g, 15mmol) was added under ice bath, stirred for 1-2h, TLC monitored the reaction to completion, the reaction solution was concentrated under reduced pressure to make sand, and then subjected to column chromatography (EA:PE =1:10) 1.8 g of light yellow liquid was purified, and the yield was 62.1%.

(5)(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate (I ₁ ) or bis(6-oxocyclohex-1-en-1-yl)methyl (1-(3,4,5-trimethoxyphenyl) Preparation of -9H-pyrido[3,4-b]indol-3-yl)carbamate (Ⅱ ₁₎

Combine compound 6 (2.9g, 10mmol) and 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (3.49g, 10mmol) 20ml of dichloromethane was dissolved, stirred under ice bath, and then DIPEA (3.9g, 30mmol) was added. After 1-2h, the reaction was monitored by TLC until it was complete. The reaction solution was concentrated under reduced pressure to make sand. After column chromatography (EA:PE=1 :4) 1.8 g of yellow solids (I _{1 ) and 1.1 g of (II 1} ) were obtained by purification, and the yields were 35.6% and 21.9%, respectively. (I ₁ ) The spectral data is: ESI-MS(m/z): 524[M+Na] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.83(s,1H,NH), 8.07 (d,J=7.8Hz,1H,NH),7.46(q,J=8.0Hz,2H,Ar-H),7.21(s,2H,Ar-H),7.13-7.09(m,2H,Ar- H), 6.99 (s, 1H, Ar-H), 6.15 (d, J = 19.6 Hz, 1H, CH), 4.10 (s, 2H, CH ₂ ), 3.91 (s, 6H, CH ₃ ), 3.76 ( s, 3H, CH ₃ ), 2.43–2.36 (m, 2H, CH ₂ ), 2.36–2.30 (m, 2H, CH ₂ ), 1.95–1.87 (m, 2H, CH ₂ ). ¹³ C NMR (d ₆ -DMSO, 101MHz): δ199.32,153.40,152.53,145.79,143.03,138.17,137.03,128.37,127.91,126.62,121.98,121.31,112.48,106.11,95.58,60.54,56.32,38.55,25.67, 23.18. (II ₁ ) Spectral data: ESI-MS(m/z): 654[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.89(s,1H,NH), 8.15 (d,J=8.1Hz,1H,Ar-H),7.50–7.43(m,2H,Ar-H),7.23(s,2H,Ar-H),7.18(s,1H,Ar-H), 7.13--7.09(m,1H,Ar-H), 6.73(s,2H,CH), 4.35(s,4H,CH ₂ ), 3.89(s,6H,CH ₃ ), 3.76(s,3H,CH ₃ ), 2.42–2.37 (m, 4H, CH ₂ ), 2.31 (s, 4H, CH ₂ ), 1.92–1.90 (m, 4H, CH ₂ ). ¹³ C NMR (d ₆ -DMSO, 101MHz): δ199.83,153.35,151.48,145.43,142.93,138.40,137.96,134.78,134.66,134.06,128.45,127.45,122.32,105.78,56.14,47.57,38.57,25.66,23.16.

Example 2 (6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)aminomethyl Ester (I ₂ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indole-3 -Yl) carbamate (Ⅱ ₂ ) preparation

Preparation of 1-(p-tolyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2b)

Referring to the synthesis method of (2a) in Example 1, (1b) was substituted for (1a) in the method, and finally a pale yellow solid (2b) was obtained with a yield of 80.9%.

Preparation of 1-(p-tolyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3b)

Referring to the synthesis method of (3a) in Example 1, (2b) was substituted for (2a) in the method, and finally a pale yellow solid (3b) was obtained with a yield of 87.5%.

Preparation of 1-(p-tolyl)-9H-pyrido[3,4-b]indole-3-amine (4b)

Referring to the synthesis method of (4a) in Example 1, (3b) was substituted for (3a) in the method, and finally a pale yellow solid (4b) was obtained with a yield of 50.7%. ESI-MS (m/z): 274 [M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)carbamate ( Ⅰ ₂ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl) Preparation of carbamate (Ⅱ ₂₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4b), and finally obtain pale yellow solids (I ₂ ) and (II ₂ ), with yields of 34.1% and 22.3%, respectively. (I ₂ ) Spectral data: ESI-MS (m/z): 426[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.78(s, 1H, NH), 8.11 (d,J=7.8Hz,1H,NH),7.50–7.36(m,2H,Ar-H),7.28–7.20(m,3H,Ar-H),7.11–7.02(m,3H,Ar-H) ), 6.94 (s, 1H, Ar-H), 6.19 (d, J = 19.5 Hz, 1H, CH), 4.16 (s, 2H, CH ₂ ), 3.03 (s, 3H, CH ₃ ), 2.45–2.38 (m, 2H, CH ₂ ), 2.36–2.32 (m, 2H, CH ₂ ), 1.96–1.84 (m, 2H, CH ₂ ). (II ₂ ) The spectral data is: ESI-MS(m/z): 578[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO,400MHz): δ10.80(s,1H,NH), 8.11 (d,J=8.1Hz,1H,Ar-H),7.59–7.47(m,2H, Ar-H),7.38–7.25(m,3H,Ar-H),7.21(s,1H,Ar-H) ), 7.15--7.11 (m, 2H, Ar-H), 6.79 (s, 2H, CH), 4.39 (s, 4H, CH ₂ ), 3.16 (s, 3H, CH ₃ ), 2.41--2.35 (m, 4H, CH ₂ ), 2.28 (s, 4H, CH ₂ ), 1.95–1.92 (m, 4H, CH ₂ ).

Example 3 (6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Yl) carbamate (I ₃ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3, Preparation of 4-b]indol-3-yl)carbamate (Ⅱ ₃₎

Preparation of 1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2c)

Referring to the synthesis method of (2a) in Example 1, (1c) was substituted for (1a) in the method, and finally a pale yellow solid (2c) was obtained with a yield of 81.3%.

Preparation of 1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3c)

Referring to the synthesis method of (3a) in Example 1, (2c) was substituted for (2a) in the method, and finally a pale yellow solid (3c) was obtained with a yield of 88.4%.

Preparation of 1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4c)

Referring to the synthesis method of (4a) in Example 1, (3c) was substituted for (3a) in the method, and finally a pale yellow solid (4c) was obtained with a yield of 52.3%. ESI-MS (m/z): 290 [M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)amino Formate (Ⅰ ₃ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b ]Indol-3-yl)carbamate (Ⅱ ₃ ) Preparation

With reference to _{the synthesis method of (I 1} ) in Example 1, (4c) was substituted for (4a) in the method, and finally light yellow solids (I ₃ ) and (II ₃ ) were obtained, and the yields were 30.4% and 21.8%, respectively. (I ₃ ) Spectral data: ESI-MS(m/z): 442[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.79(s, 1H, NH), 8.04 (s,1H,NH),7.43-7.39(m,3H,Ar-H),7.23-7.18(m,3H,Ar-H),7.15(s,2H,Ar-H),6.97(s,1H ,Ar-H),6.13(d,J=19.6Hz,1H,CH),4.08(s,2H,CH ₂ ),3.88(s,3H,CH ₃ ),2.41–2.35(m,2H,CH ₂ ), 2.33–2.29 (m, 2H, CH ₂ ), 1.94–1.85 (m, 2H, CH ₂ ). (II ₃ ) Spectral data: ESI-MS(m/z): 594[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.87(s,1H,NH), 8.14 (d,J=8.0Hz,1H,Ar-H),7.48–7.42(m,3H,Ar-H),7.25(s,2H,Ar-H),7.19(s,1H,Ar-H), 7.11-7.08(m,2H,Ar-H),6.71(s,2H,CH),4.33(s,4H,CH ₂ ), 3.85(s,6H,CH ₃ ),2.41-2.35(m,4H, CH ₂ ), 2.31–2.19 (m, 4H, CH ₂ ), 1.94–1.89 (m, 4H, CH ₂ ).

Example 4 (6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3- Yl) carbamate (I ₄ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(4-methoxyphenyl)-9H-pyrido[3, Preparation of 4-b]indol-3-yl)carbamate (Ⅱ ₄₎

Preparation of 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2d)

Referring to the synthesis method of (2a) in Example 1, (1d) was substituted for (1a) in the method, and finally a pale yellow solid (2d) was obtained with a yield of 82.0%.

Preparation of 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3d)

Referring to the synthesis method of (3a) in Example 1, (2d) was substituted for (2a) in the method, and finally a pale yellow solid (3d) was obtained with a yield of 87.4%.

Preparation of 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4d)

Referring to the synthesis method of (4a) in Example 1, (3d) was substituted for (3a) in the method, and finally a pale yellow solid (4d) was obtained with a yield of 52.5%. ESI-MS (m/z): 290 [M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)amino Formate (I ₄ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b ]Indol-3-yl) carbamate (Ⅱ ₄ ) Preparation

With reference to _{the synthesis method of (I 1} ) in Example 1, (4d) was substituted for (4a) in the method, and finally light yellow solids (I ₄ ) and (II ₄ ) were obtained, and the yields were 35.1% and 22.6%, respectively. (I ₄ ) Spectral data: ESI-MS(m/z): 442[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.83(s, 1H, NH), 8.04 (d,J=7.8Hz,1H,NH),7.44-7.39(m,2H,Ar-H),7.24-7.17(m,3H,Ar-H),7.14-7.08(m,2H,Ar-H) ), 6.97 (s, 2H, Ar-H), 6.12 (d, J = 19.6 Hz, 1H, CH), 4.12 (s, 2H, CH ₂ ), 3.83 (s, 3H, CH ₃ ), 2.41-2.34 (m, 2H, CH ₂ ), 2.35–2.32 (m, 2H, CH ₂ ), 1.98–1.87 (m, 2H, CH ₂ ). (II ₄ ) Spectral data: ESI-MS(m/z): 594[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.90(s,1H,NH), 8.17 (s,1H,Ar-H),7.55-7.48(m,2H,Ar-H),7.33-7.29(m,2H,Ar-H),7.22(s,2H,Ar-H),7.18-7.10 (m, 2H, Ar-H), 6.77 (s, 2H, CH), 4.37 (s, 4H, CH ₂ ), 3.80 (s, 6H, CH ₃ ), 2.41-2.37 (m, 4H, CH ₂ ) , 2.30–2.19 (m, 4H, CH ₂ ), 1.94–1.90 (m, 4H, CH ₂ ).

Example 5 (6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₅ ) or bis(6-oxocyclohex-1-en-1-yl) methyl (1-(4-N,N dimethylphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₅ ) preparation

Preparation of 1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3-carbonyl hydrazide (2e)

Referring to the synthesis method of (2a) in Example 1, (1e) was substituted for (1a) in the method, and finally a red solid (2e) was obtained with a yield of 81.1%.

Preparation of 1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3e)

With reference to the synthesis method of (3a) in Example 1, (2e) was substituted for (2a) in the method, and finally a brown solid (3e) was obtained with a yield of 80.9%.

Preparation of 1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3-amine (4e)

Referring to the synthesis method of (4a) in Example 1, (3e) was substituted for (3a) in the method, and finally a red solid (4e) was obtained with a yield of 45.8%. ESI-MS (m/z): 303 [M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate (I ₅ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(4-N, N dimethylphenyl)-9H-pyrido Preparation of [3,4-b]indol-3-yl)carbamate (Ⅱ ₅₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4e), and finally obtain light red solids (I ₅ ) and (II ₅ ) with yields of 32.7% and 19.1%, respectively. (I ₅ ) Spectral data: ESI-MS (m/z): 455[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.88(s, 1H, NH), 8.09 (s,1H,NH),7.51-7.44(m,3H,Ar-H),7.26-7.18(m,3H,Ar-H),7.12(s,2H,Ar-H),6.99(s,1H ,Ar-H),6.19(d,J=19.6Hz,1H,CH),4.14(s,2H,CH ₂ ),3.99(s,6H,CH ₃ ),2.45-2.37(m,2H,CH ₂ ), 2.38–2.32 (m, 2H, CH ₂ ), 1.97–1.89 (m, 2H, CH ₂ ). (II ₅ ) Spectral data: ESI-MS(m/z): 607[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.95(s,1H,NH), 8.18 (s,1H,Ar-H),7.50-7.43(m,3H,Ar-H),7.30-7.24(m,2H,Ar-H),7.19(s,2H,Ar-H),7.14-7.10 (m, 1H, Ar-H), 6.79 (s, 2H, CH), 4.41 (s, 4H, CH ₂ ), 4.04 (s, 6H, CH ₃ ), 2.46 (s, 4H, CH ₂ ), 2.31 –2.25(m,4H,CH ₂ ),1.94-1.85(m,4H,CH ₂ ).

Example 6 (6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₆ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₆ ) preparation

Preparation of 1-(2,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2f)

Referring to the synthesis method of (2a) in Example 1, (1f) was substituted for (1a) in the method, and finally a pale yellow solid (2f) was obtained with a yield of 87.1%.

Preparation of 1-(2,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3f)

Referring to the synthesis method of (3a) in Example 1, (2f) was substituted for (2a) in the method, and finally a pale yellow solid (3f) was obtained with a yield of 86.2%.

Preparation of 1-(2,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4f)

With reference to the synthesis method of (4a) in Example 1, (3f) was substituted for (3a) in the method, and finally a pale yellow solid (4f) was obtained with a yield of 55.2%. ESI-MS(m/z): 320[M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate (I ₆ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(2,4-dimethoxyphenyl)-9H-pyrido Preparation of [3,4-b]indol-3-yl)carbamate (Ⅱ ₆₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4f), and finally obtain pale yellow solids (I ₆ ) and (II ₆ ) with yields of 34.2% and 20.7%, respectively. (I ₆ ) Spectral data: ESI-MS (m/z): 472[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.79(s, 1H, NH), 8.10 (s,1H,NH),7.47-7.36(m,3H,Ar-H),7.28(s,1H,Ar-H),7.19-7.10(m,3H,Ar-H),7.02(s,1H) ,Ar-H),6.18(d,J=19.6Hz,1H,CH),4.19(s,2H,CH ₂ ),3.93(s,3H,CH ₃ ),3.78(s,3H,CH ₃ ), 2.37–2.30 (m, 2H, CH ₂ ), 2.27–2.21 (m, 2H, CH ₂ ), 1.93–1.82 (m, 2H, CH ₂ ). (II ₆ ) Spectral data: ESI-MS(m/z): 624[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.95(s,1H,NH), 8.05 (s,1H,Ar-H),7.55-7.46(m,3H,Ar-H),7.26(s,1H,Ar-H),7.19(s,1H,Ar-H),7.16-7.11(m , 2H, Ar-H), 6.78 (s, 2H, CH), 4.39 (s, 4H, CH ₂ ), 3.93 (s, 3H, CH ₃ ), 3.80 (s, 3H, CH ₃ ), 2.45-2.36 (m, 4H, CH ₂ ), 2.23–2.15 (m, 4H, CH ₂ ), 1.90–1.82 (m, 4H, CH ₂ ).

Example 7 (6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₇ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (Ⅱ ₇ ) preparation

Preparation of 1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2g)

With reference to the synthesis method of (2a) in Example 1, (1g) was substituted for (1a) in the method, and finally a pale yellow solid (2g) was obtained with a yield of 85.1%.

Preparation of 1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3g)

Referring to the synthesis method of (3a) in Example 1, replacing (2a) in the method with (2g), a light yellow solid (3g) was finally obtained with a yield of 86.9%.

Preparation of 1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4g)

Referring to the synthesis method of (4a) in Example 1, (3a) in the method was replaced by (3g), and finally a pale yellow solid (4g) was obtained with a yield of 52.4%. ESI-MS(m/z): 320[M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate (I ₇ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(2,5-dimethoxyphenyl)-9H-pyrido Preparation of [3,4-b]indol-3-yl)carbamate (Ⅱ ₇₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4g), and finally obtain pale yellow solids (I ₇ ) and (II ₇ ) with yields of 35.7% and 18.4%, respectively. (I ₇ ) Spectral data: ESI-MS(m/z): 472[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.82(s,1H,NH), 8.04 (s,1H,NH),7.45-7.34(m,3H,Ar-H),7.23(s,2H,Ar-H),7.14-7.02(m,2H,Ar-H),6.98(s,1H ,Ar-H), 6.27(d,J=19.6Hz,1H,CH),4.21(s,2H,CH ₂ ),3.92(s,3H,CH ₃ ),3.79(s,3H,CH ₃ ), 2.43–2.35 (m, 2H, CH ₂ ), 2.33–2.21 (m, 2H, CH ₂ ), 1.96–1.86 (m, 2H, CH ₂ ). (II ₇ ) The spectral data is: ESI-MS(m/z): 624[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO,400MHz): δ10.89(s,1H,NH), 8.12 (s,1H,Ar-H), 7.62–7.51(m,2H,Ar-H), 7.31–7.22(m,3H,Ar-H), 7.11–7.05(m,2H,Ar-H), 6.81 (s, 2H, CH), 4.29 (s, 4H, CH ₂ ), 3.92 (s, 3H, CH ₃ ), 3.66 (s, 3H, CH ₃ ), 2.52-2.46 (m, 4H, CH ₂ ), 2.30–2.18 (m, 4H, CH ₂ ), 1.93–1.89 (m, 4H, CH ₂ ).

Example 8 (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₈ ) or bis(6-oxocyclohex-1-en-1-yl) methyl (1-(3,4-dimethoxyphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₈ ) preparation

Preparation of 1-(3,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2h)

With reference to the synthesis method of (2a) in Example 1, (1h) was substituted for (1a) in the method, and finally a pale yellow solid (2h) was obtained with a yield of 84.5%.

Preparation of 1-(3,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3h)

Refer to the synthesis method of (3a) in Example 1, replacing (2a) in the method with (2h), and finally obtain a light yellow solid (3h) with a yield of 86.7%.

Preparation of 1-(3,4-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4h)

Refer to the synthesis method of (4a) in Example 1, replacing (3a) in the method with (3h), and finally obtain a pale yellow solid (4h) with a yield of 51.7%. ESI-MS(m/z): 320[M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate (I ₈ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(3,4-dimethoxyphenyl)-9H-pyrido Preparation of [3,4-b]indol-3-yl)carbamate (Ⅱ ₈₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4h), and finally obtain pale yellow solids (I ₈ ) and (II ₈ ) with yields of 36.1% and 16.7%, respectively. (I ₈ ) The spectral data is: ESI-MS (m/z): 472[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.77 (s, 1H, NH), 8.01 (d,J=7.8Hz,1H,NH), 7.50–7.39(m,3H,Ar-H), 7.27(s,2H,Ar-H), 7.15–7.09(m,2H,Ar-H), 6.98(s,1H,Ar-H),6.16(d,J=19.6Hz,1H,CH),4.22(s,2H,CH ₂ ),3.93(s,3H,CH ₃ ),3.78(s,3H ,CH ₃ ), 2.42–2.35(m,2H,CH ₂ ), 2.31–2.23(m,2H,CH ₂ ), 1.93–1.87(m,2H,CH ₂ ). (II ₈ ) Spectral data: ESI-MS(m/z): 624[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.98(s,1H,NH), 8.09 (s,1H,Ar-H),7.55-7.46(m,3H,Ar-H),7.28(s,2H,Ar-H),7.19(s,1H,Ar-H),7.15-7.08(m ,1H,Ar-H),6.81(s,2H,CH),4.33(s,4H,CH ₂ ),3.84(s,3H,CH ₃ ),3.73(s,3H,CH ₃ ),2.41-2.36 (m, 4H, CH ₂ ), 2.29–2.24 (s, 4H, CH ₂ ), 1.92–1.88 (m, 4H, CH ₂ ).

Example 9 (6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₉ ) or bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₉ ) preparation

Preparation of 1-(3,5-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carbonylhydrazide (2i)

With reference to the synthesis method of (2a) in Example 1, (1i) was substituted for (1a) in the method, and finally a pale yellow solid (2i) was obtained with a yield of 81.0%.

Preparation of 1-(3,5-Dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-acyl azide (3i)

Referring to the synthesis method of (3a) in Example 1, (2i) was substituted for (2a) in the method, and finally a pale yellow solid (3i) was obtained with a yield of 85.7%.

Preparation of 1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-amine (4i)

Referring to the synthesis method of (4a) in Example 1, replacing (3a) in the method with (3i), and finally obtaining a pale yellow solid (4i) with a yield of 53.2%. ESI-MS(m/z): 320[M+H] ⁺ .

(6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate (I ₉ ) or two (6-oxocyclohex-1-en-1-yl) methyl (1-(3,5-dimethoxyphenyl)-9H-pyrido Preparation of [3,4-b]indol-3-yl)carbamate (Ⅱ ₉₎

Refer to the synthesis method of (I ₁ ) in Example 1, replacing (4a) in the method with (4i), and finally obtain pale yellow solids (I ₉ ) and (II ₉ ), with yields of 34.4% and 24.1%, respectively. (I ₉ ) Spectral data: ESI-MS(m/z): 472[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO, 400MHz): δ10.82(s,1H,NH), 8.06 (d,J=7.8Hz,1H,NH),7.49–7.39(m,3H,Ar-H),7.24(s,2H,Ar-H),7.13–7.08(m,2H,Ar-H), 6.98 (s, 1H, Ar-H), 6.52 (d, J = 19.6 Hz, 1H, CH), 4.09 (s, 2H, CH ₂ ), 3.92 (s, 6H, CH ₃ ), 2.43-2.35 (m , 2H, CH ₂ ), 2.36–2.31 (m, 2H, CH ₂ ), 1.96–1.87 (m, 2H, CH ₂ ). (II ₉ ) The spectral data is: ESI-MS(m/z): 624[M+H] ⁺ ; ¹ H NMR(d ₆ -DMSO,400MHz): δ10.85(s,1H,NH), 8.12 (d,J=8.0Hz,1H,Ar-H),7.52-7.46(m,3H,Ar-H),7.22(s,2H,Ar-H),7.19(s,1H,Ar-H), 7.14–7.08(m,1H,Ar-H), 6.76(s,2H,CH), 4.38(s,4H,CH ₂ ), 3.87(s,6H,CH ₃ ), 2.42–2.36(m,4H, CH ₂ ), 2.27–2.19 (s, 4H, CH ₂ ), 1.91–1.86 (m, 4H, CH ₂ ).

Example 10 (6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole Dol-3-yl) carbamate (I ₁₀ ) or bis(6-oxocyclopent-1-en-1-yl)methyl (1-(3,4,5-trimethoxyphenyl) -9H-pyrido[3,4-b]indol-3-yl)carbamate (II ₁₀ );

Preparation of 4-nitrophenyl ((6-oxocyclopent-1-en-1-yl)methyl) carbonate (6a)

Referring to the synthesis method of (6b) in Example 1, (5a) was substituted for (5b) in the method, and finally a pale yellow liquid (6a) was obtained with a yield of 85.1%.

(6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Yl) carbamate (I ₁₀ ) or two (6-oxocyclopent-1-en-1-yl) methyl (1-(3,4,5-trimethoxyphenyl)-9H- Preparation of pyrido[3,4-b]indol-3-yl)carbamate (Ⅱ ₁₀₎

With reference to _{the synthesis method of (I 1} ) in Example 1, (6a) replaces (6b) in the method to finally obtain pale yellow solids (I ₁₀ ) and (II ₁₀ ), with yields of 30.7% and 19.6%, respectively. (I ₁₀ ) spectral data: ESI-MS (m/z): 488[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.83(s, 1H, NH), 8.08 (d,J=7.8Hz,1H,NH),7.45-7.36(m,2H,Ar-H),7.24(s,2H,Ar-H),7.16-7.09(m,2H,Ar-H), 6.99 (s, 1H, Ar-H), 6.18 (d, J = 19.6 Hz, 1H, CH), 4.14 (s, 2H, CH ₂ ), 3.92 (s, 6H, CH ₃ ), 3.78 (s, 3H ,CH ₃ ), 2.35–2.29 (m, 2H, CH ₂ ), 1.96–1.87 (m, 2H, CH ₂ ). (II ₁₀ ) ESI-MS (m/z): 626[M+H] ⁺ .

Example 11 (6-oxocyclopent-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₁₁ ) or two (6-oxocyclopent-1-en-1-yl) methyl (1-(4-N,N dimethylphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₁₁ ) preparation

Referring to the synthesis method of (I ₁ ) in Example 1, replace (4a) in the method with (4e), and replace (6b) in the method with (6a) to obtain light red solids (I ₁₁ ) and (II ₁₁ ). The yields were 31.2% and 18.4%, respectively. (I ₁₁ ) Spectral data is: ESI-MS (m/z): 441[M+H] ⁺ ; ¹ H NMR (d ₆ -DMSO, 400MHz): δ10.87 (s, 1H, NH), 8.04 (d,J=7.8Hz,1H,NH),7.44-7.37(m,3H,Ar-H),7.24(s,2H,Ar-H),7.16-7.11(m,3H,Ar-H), 6.98 (s, 1H, Ar-H), 6.17 (d, J = 19.6 Hz, 1H, CH), 4.19 (s, 2H, CH ₂ ), 4.11 (s, 6H, CH ₃ ), 2.38–2.32 (m ,2H,CH ₂ ),1.97–1.88(m,2H,CH ₂ ). (II ₁₁ ) The spectral data is: ESI-MS(m/z): 579[M+H] ⁺ .

Example 12 (6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indino Dol-3-yl) carbamate (I ₁₂ ) or bis(6-oxocyclohept-1-en-1-yl)methyl (1-(3,4,5-trimethoxyphenyl) -9H-pyrido[3,4-b]indol-3-yl)carbamate (II ₁₂ );

Preparation of 4-nitrophenyl ((6-oxocyclopent-1-en-1-yl)methyl) carbonate (6c)

Referring to the synthesis method of (6c) in Example 1, (5c) was substituted for (5b) in the method, and finally a pale yellow liquid (6c) was obtained with a yield of 84.0%.

(6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Yl) carbamate (I ₁₂ ) or two (6-oxocyclohept-1-en-1-yl) methyl (1-(3,4,5-trimethoxyphenyl)-9H- Preparation of pyrido[3,4-b]indol-3-yl)carbamate (II ₁₂₎

Referring to the synthesis method of (I ₁ ) in Example 1, (6c) replaces (6b) in the method, and finally yellow solids (I ₁₂ ) and (II ₁₂ ) are obtained, and the yields are 29.8% and 18.0%, respectively. (I ₁₂ ) The spectral data are: ESI-MS (m/z): 516[M+H] ⁺ ; ¹ H NMR (CDCl ₃ , 400MHz): δ8.11 (s, 1H, NH), 8.03 (d ,J=7.8Hz,1H,Ar-H),7.50–7.46(m,1H,Ar-H),7.40(d,J=8.1Hz,1H,Ar-H),7.22–7.19(m,1H, Ar-H), 7.14(s, 2H, Ar-H), 6.93(s, 1H, Ar-H), 6.87-6.84(m, 1H, CH), 4.21(s, 2H, CH ₂ ), 3.95( s,6H,CH ₃ ),3.92(s,3H,CH ₃ ),2.70-2.58(m,2H,CH ₂ ),2.44-2.40(m,2H,CH ₂ ),1.82-1.71(m,4H, CH ₂ ). ¹³ C NMR (CDCl ₃ , 101MHz): δ205.05,153.77,152.78,143.12,141.80,140.13,138.52,134.37,133.69,128.47,128.22,122.01,121.90,119.45,111.45,105.48,94.46,60.99,56.44,45.35, 42.90, 27.65, 25.18, 21.49. (II ₁₂ ) ESI-MS (m/z): 682[M+H] ⁺ .

Example 13 (6-oxocyclohept-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole -3-yl) carbamate (I ₁₃ ) or bis(6-oxocyclohept-1-en-1-yl) methyl (1-(4-N,N dimethylphenyl)-9H -Pyrido[3,4-b]indol-3-yl) carbamate (II ₁₃ ) preparation

Refer to the synthesis method of (I ₁ ) in Example 1, replace (4a) in the method with (4e), and replace (6b) in the method with (6c), and finally obtain light red solids (I ₁₃ ) and (II ₁₃ ) The yields were 27.7% and 16.9%, respectively. (I ₁₃ ); ¹ H NMR (d ₆ -DMSO, 400MHz): δ 10.94 (s, 1H, NH), 8.08 (d, J = 7.8 Hz, 1H, NH), 7.47-7.39 (m, 3H, Ar-H),7.23(s,2H,Ar-H),7.15-7.10(m,3H,Ar-H),7.01(s,1H,Ar-H),6.18(d,J=19.6Hz,1H ,CH),4.23(s,2H,CH ₂ ),4.16(s,6H,CH ₃ ),2.73-2.66(m,2H,CH ₂ ),2.46-2.40(m,2H,CH ₂ ),1.89-- 1.82 (m, 4H, CH ₂ ). ESI-MS (m/z): 469 [M+H] ⁺ . (II ₁₃ ) ESI-MS (m/z): 635[M+H] ⁺ .

Example 14 Study on the determination of the tumor cell proliferation inhibition rate of the compound of the present invention by the MTT method

The in vitro anti-tumor test of the tetramethylazole blue colorimetric method (MTT) was used to evaluate the anti-proliferation activity of the compound of the present invention on four human cancer cell lines. COMC-6 was used as the positive control drug. Human cancer cell lines: Hepatocellular carcinoma cell HepG2, human cervical cancer cell Hela, colon cancer cell HCT116 and HT29 cells, gastric cancer cell HGC-27.

The experimental method is as follows: Take a bottle of cells in a good state of exponential growth phase, add 0.25% trypsin to digest, make the adherent cells fall off, and make a suspension ^{containing 2×10 4} to 4×10 ^{4 cells per milliliter.} The cell suspension was inoculated on a 96-well plate, 180 μL per well, and placed in a constant temperature CO ₂ incubator for 24 hours. Change the solution and add test compounds I ₁ -I ₁₃ and II ₁ -II ₁₃ (compounds are dissolved in DMSO and then diluted with PBS. The concentrations of the test compounds are 6.25×10 ^-6 , 1.25×10 ^-5 , and 2.5× respectively. 10 ^-5 , 5×10 ^-5 mol/L), 20 μL per well, culture for 72 hours. Add MTT to a 96-well plate, 20μL per well, and react in an incubator for 4 hours. Aspirate the supernatant, add DMSO, 150 μL per well, and shake on a plate shaker for 5 minutes. Measure the absorbance of each well with an enzyme-linked immunoassay at a wavelength of 570nm to calculate the cell inhibition rate. The experimental results are shown in Table 2.

Cell inhibition rate=(OD value of negative control group-OD value of test substance group)/OD value of negative control group×100%.

The compound of the present invention has undergone a series of tumor cell anti-proliferation activity tests. The results of pharmacological experiments show (see Table 2) that the compound of the present invention has a stronger inhibitory effect on the proliferation of most tumor cells, especially some compounds. The positive control drug COMC-6 is slightly stronger or equivalent.

Table 2 Inhibition rate% of some compounds of the present invention on human tumor cells (12.5μmol/L)

ND: not detected

Example 15 pH-sensitive fluorescence characteristics of the compounds of the present invention

An ultraviolet-visible spectrophotometer and a fluorescence spectrometer are used to determine the ultraviolet absorption wavelength of the Ⅰ or Ⅱ series compound of the present invention and the change of fluorescence with pH. The selected pH range is 3.1 to 7.6.

The pH-sensitive ultraviolet absorption experiment method is as follows: 100 μM of the series of compounds are dissolved in a 1% DMSO aqueous solution with a pH of about 8.00-3.00. All absorption spectra were recorded at room temperature, the scanning wavelength range was 350-600nm, and the scanning speed was 1.0nm/s.

The pH-sensitive fluorescence experiment method is as follows: 1 μM of the series of compounds are dissolved in a 1% DMSO aqueous solution with a pH of about 8.00-3.00. All emission spectra were performed at room temperature, excited at 430-470nm, and recorded at 450nm to 650nm.

The results show that the _{characteristic ultraviolet absorption wavelengths of the compounds I 1} -I ₁₃ and II _{1 -II 13} of the present invention are between 390 and 420 nm, and as the pH value decreases, the peak value gradually decreases, and the peak value at 430 to 470 nm gradually rises. High; its fluorescence spectrum shows that the fluorescence at 480～520nm also increases significantly as the pH value decreases.

The pH response UV fluorescence spectrum represented by compound I ₁ is shown in Figure 1. The characteristic UV absorption wavelength is around 402nm, and as the pH value decreases, the peak at 402nm gradually decreases (Figure 1A), and the peak at 445nm Gradually increase (Figure 1A and Figure 1C); its fluorescence at 490nm also gradually increases with the decrease of pH ((Figure 1B and Figure 1D, excitation wavelength Ex=445nm).

Example 16 Compound of the present invention and GSH-responsive fluorescence characteristics

The changes of the ultraviolet and fluorescence of the Ⅰ-Ⅱ series compounds of the present invention with the concentration of GSH are judged by extracellular fluorescence experiments.

The GSH combined ultraviolet absorption experiment method is as follows: 100 μM of the series of compounds are dissolved in a 1% DMSO aqueous solution, and 0-10 equivalents of GSH and a catalytic amount of GSTπ are added. All absorption spectra were recorded after incubation at 37°C, the scanning wavelength range was 350-600nm, and the scanning speed was 1.0nm/s.

The GSH binding fluorescence experiment method is as follows: 1 μM of the series of compounds are dissolved in a 1% DMSO aqueous solution, and about 0-50 μM of GSH and a catalytic amount of GSTπ are added. All emission spectra were recorded after incubation at 37°C for 0.5 hours, excitation at 430-470nm, and recording at 450nm to 650nm.

The compounds of this invention Ⅰ ₁ -Ⅰ ₁₃ and Ⅱ ₁ -Ⅱ ₁₃ at 1μM concentration, with the increase of GSH equivalents, characterized in UV absorption peak wavelength gradually decreases between 385 ~ 430nm, gradually rises at the peak of 435 ~ 470nm ; Its fluorescence spectrum shows that the fluorescence at 470～530nm also gradually increases with the increase of GSH equivalent.

The GSH response ultraviolet fluorescence spectrum represented by compound I ₁ is shown in Figure 2. The ultraviolet absorption wavelength is around 402nm, and with the increase of GSH concentration, the peak at 402nm gradually decreases, and the peak at 440nm gradually increases (Figure 2A); Fluorescence is released immediately after adding GSH and reaches a peak quickly (Figure 2D, excitation wavelength Ex=440nm), its emission wavelength is 492nm, and the fluorescence intensity increases with the increase of GSH concentration (Figures 2B and 2C).

Example 17 The specific response fluorescence characteristics of the compound of the present invention and GSH

The specific binding of the compound of the present invention and GSH is judged by extracellular fluorescence experiment.

The specific experimental method is as follows: Dissolve 1μM of the series of compounds in an aqueous solution containing a catalytic amount of GSTπ, and add 1mM K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Al ³⁺ , Cu to it ²⁺ , Fe ²⁺ ; 100μM GSH, lysine, histidine, alanine, cysteine, glutamic acid, serine, glycine, arginine, vitamin C, Na ₂ S, H ₂ O _2. Bio-endogenous substances such as NADH. All emission spectra were recorded after incubation at 37°C for 0.5 hours, excitation at 430-480nm, and recording at 450nm to 650nm.

Taking compound I ₁ as a representative, _{the chemical selectivity of compound I 1} under GSH far exceeds other common components or ions commonly found in biological systems, such as K ⁺ , Na ⁺ , Ca ²⁺ , Mg ²⁺ , and Zn ²⁺ , Al ³⁺ , Cu ²⁺ , Fe ²⁺ , lysine, histidine, alanine, cysteine, glutamic acid, serine, glycine, arginine, vitamin C, Na ₂ S , H ₂ O ₂ , NADH, etc. The results are shown in Figure 3. In the presence of other analytes, I ₁ is very stable, and its fluorescence intensity at 492 nm is still very weak and unchanged.

Example 18 Tumor cell imaging using confocal microscope

A method for fluorescence imaging of tumor cells using a confocal microscope, selecting HT29 cells with high GSTπ expression, includes the following steps:

(1) Centrifuge the HT29 cell suspension at 3000 rpm for 5 minutes, and remove the supernatant;

(2) Add the prepared 1uM PBS solution containing the compound of the present invention, and perform the laser confocal scanning imaging experiment after incubating at a constant temperature of 37°C;

(3) Place the incubated cells on the stage of the confocal microscope, excitation wavelength: 430-480nm; receiving wavelength: 480-550nm.

Confocal fluorescence images displayed on HT29 cells Ⅰ ₁ -Ⅰ ₁₃ absorbing compounds of the present invention and Ⅱ ₁ -Ⅱ ₁₃ quickly, and the relative increase in 10min front, gradually increased after 1h brightest enhancement, and continued 2h, accumulation efficiency high. Figure 4 shows the cells of representative compounds I ₁ , I ₃ , I ₆ , I ₉ , I ₁₀ , I ₁₃ , II ₂ , II ₄ , II ₇ , II ₉ , II ₁₁ , and II ₁₂ in HT29 cells at 1 h Fluorescence imaging pictures, the results show that the compounds of the present invention can perform fluorescence imaging in tumor cells well.

Example 19 In vivo imaging study of the compound of the present invention

In order to evaluate the in vivo imaging effects of series I-II compounds, a BALB/c nude mouse model was established subcutaneously inoculated with HT29 cells, and the compounds I ₁ , I ₃ , I ₆ , I ₉ , I ₁₀ and The fluorescence distribution of Ⅰ ₁₃ , Ⅱ ₂ , Ⅱ ₄ , Ⅱ ₇ , Ⅱ ₉ , Ⅱ ₁₁ , and Ⅱ ₁₂ in tissues of nude mice bearing tumor. 40 mg/kg of the compound of the present invention was administered intravenously, the mice were anesthetized 2 hours later, and the fluorescent signals of tumors and main organs in nude mice were examined.

The results show that _{the fluorescence intensity of the compounds I 1} , I ₃ , I ₆ , I ₉ , I ₁₀ , I ₁₃ , II ₂ , II ₄ , II ₇ , II ₉ , II ₁₁ and II ₁₂ in tumor tissues in nude mice Significantly higher than the major organs, including the heart, lungs, liver, kidneys and spleen. Fig. 5 _{is the result of in vivo fluorescence imaging distribution of compound I 1 of the} present invention, which shows that the compound of the present invention can selectively show strong fluorescence signals in tumor tissues in vivo.

Example 20 Study on anti-tumor activity in vivo

In order to evaluate the in vivo anti-tumor activity of the Ⅰ-Ⅱ series of compounds, a BALB/c nude mouse model inoculated with HT29 cells subcutaneously was established. After solid tumors formed, nude mice were randomly given PBS, the control drug COMC-6, compound I ₁ , and the tumor size changes were monitored every three days for 21 days.

The results of the in vivo anti-tumor activity study of the _{compound I 1} of the present invention are shown in Figure 6. The control group observed stable tumor growth. However, the compound I ₁ treatment group significantly reduced the volume and weight of colon tumors. Treatment with the same dose of I ₁ showed better anti-tumor activity than COMC-6 treatment, and resulted in a greater tumor inhibition rate at the end of _{I 1 treatment.} Compared with the PBS-treated control group (1.49±0.27g), _{the tumor weight (0.48±0.07g) of the mice treated with I 1} 40 mg/kg was reduced by 67.7% (w/w), while COMC-6 40 mg/kg The tumor weight (0.81±0.12g) of the kg-treated control group was reduced by 45.6% (w/w). The results show that compound I ₁ has significant anti-tumor activity on the growth of colon tumors in vivo.

Claims (9)

1. A class of β-carboline-cycloketene derivatives has the structure shown in the following general formula:

   

   among them,

   R ₁ represents one or more substituents on the corresponding substituted ring, selected from one or more of H, C1-C6 alkoxy, C1-C6 alkyl, and C1-C6 alkylamino, R ₁ represents multiple In the case of substituents, the substituents are the same or different;

   R ₂ represents one or more substituents on the corresponding substituted ring, selected from H or C1-C6 alkyl, when R ₂ represents multiple substituents, the substituents are the same or different;

   R _{3 is} selected from H or C1-C6 alkyl;

   R _{4 is} selected from H, C1-C6 alkyl or

   

   n=1, 2 or 3.
2. The β-carboline-cycloketene derivative according to claim 1, wherein the R _{1 is} selected from H, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, One or more of methylamino, ethylamino, methylethylamino and N,N-dimethylamino;

   R _{2 is} selected from one or more of H, methyl, ethyl, propyl, and isopropyl;

   R _{3 is} selected from H, methyl, ethyl, propyl or isopropyl;

   R _{4 is} selected from H, methyl, ethyl, propyl, isopropyl or

   

   n=1, 2 or 3.
3. The β-carboline-cycloalkenone derivative according to claim 1, wherein said R ₁ represents 3,4,5-trimethoxy, 4-CH ₃ , 3-OCH ₃ , 4-OCH ₃ , 4-N,N-dimethyl, 2,4-dimethoxy, 2,5-dimethoxy, 3,4-dimethoxy or 3,5-dimethoxy; R ₂ or R ₃ stands for H; R ₄ stands for H or

   

   n=1, 2 or 3.
4. The β-carboline-cycloketene derivative according to claim 1, characterized in that it is selected from the following compounds:

   (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole- 3-yl) carbamate;

   (6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(p-tolyl)-9H-pyrido[3,4-b]indol-3-yl)carbamate ；

   (6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)amino Formate

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl) Carbamate

   (6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl)amino Formate

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indol-3-yl) Carbamate

   (6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- ) Carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclohex-1-en-1-yl)methyl(1-(3,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   Bis(6-oxocyclopent-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole- 3-yl) carbamate;

   (6-oxocyclopent-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclopent-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   (6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate;

   Bis(6-oxocyclohept-1-en-1-yl)methyl(1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole- 3-yl) carbamate;

   (6-oxocyclohept-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3- Group) carbamate;

   Bis(6-oxocyclohept-1-en-1-yl)methyl(1-(4-N,N dimethylphenyl)-9H-pyrido[3,4-b]indole-3 -Base) carbamate.
5. The preparation method of β-carboline-cycloketene derivative according to any one of claims 1 to 4, characterized in that it comprises the following steps:

   (1) Compound 1 is reacted with hydrazine hydrate to obtain compound 2,

   

   (2) Compound 2 is reacted with NaNO ₂ under acidic conditions to obtain compound 3.

   

   (3) reacting compound 3 under acidic conditions to obtain compound 4;

   

   (4) Compound 5 is reacted with p-nitrophenyl chloroformate under alkaline conditions to obtain compound 6,

   

   (5) reacting compound 4 with compound 6 under basic conditions to obtain compound 7 and/or compound 8,

   

   Alternatively, the above synthesis step further includes step (6), reacting compound 7 under sodium hydrogen, and then reacting with C1-C6 alkyl bromide to obtain compound 9.

   

   R ₁ represents one or more substituents on the corresponding substituted ring, selected from one or more of H, C1-C6 alkoxy, C1-C6 alkyl, and C1-C6 alkylamino, R ₁ represents multiple In the case of substituents, the substituents are the same or different;

   R ₂ represents one or more substituents on the corresponding substituted ring, selected from H or C1-C6 alkyl, when R ₂ represents multiple substituents, the substituents are the same or different;

   R _{3 is} selected from H or C1-C6 alkyl;

   R _{4 is} selected from C1-C6 alkyl groups;

   n=1, 2 or 3.
6. The use of the β-carboline-cycloketene derivative according to any one of claims 1 to 4 in the preparation of drugs and/or probes targeting GSTπ.
7. The application according to claim 6, characterized in that the drug targeting GSTπ is a drug for treating and/or preventing cancer.
8. The use according to claim 7, characterized in that the cancer is selected from liver cancer, colon cancer, cervical cancer or gastric cancer.
9. The application according to claim 6, characterized in that the probe targeting GSTπ is a fluorescent probe based on the dual response of pH and GSH.

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