CN111393368A

CN111393368A - Indenopyrazole hydrochloride derivative and preparation method and application thereof

Info

Publication number: CN111393368A
Application number: CN202010425297.1A
Authority: CN
Inventors: 刘兆鹏; 崔英杰
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-05-19
Filing date: 2020-05-19
Publication date: 2020-07-10
Anticipated expiration: 2040-05-19
Also published as: CN111393368B

Abstract

The invention provides an indenopyrazole hydrochloride derivative and a preparation method and application thereof, wherein the indenopyrazole hydrochloride derivative has a structure shown in a formula I:

wherein, X is CH or N, and N is 1 or 2. The indenopyrazole hydrochloride derivative can target a tumor cell signal path while inhibiting tubulin aggregation, inhibit phosphorylation of cMet and Akt kinase, and has good antitumor activity, and the compound is water-solubleGood effect, and has important significance for reducing toxic and side effects, preventing drug resistance and the like.

Description

Indenopyrazole hydrochloride derivative and preparation method and application thereof

Technical Field

The invention relates to the technical field of chemical combination, and particularly relates to an indenopyrazole hydrochloride derivative and a preparation method and application thereof.

Background

The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Tubulin is one of the effective targets for the research of antitumor drugs. Tubulin inhibitor drugs have become a major chemotherapeutic drug and are widely used in clinical treatment of various tumors. Currently, clinically used microtubule inhibitors mainly include drugs for inhibiting tubulin depolymerization, represented by paclitaxel, and drugs for inhibiting tubulin polymerization, represented by vinblastine. However, the medicines have certain toxic and side effects and have the problems of drug resistance and the like in clinical use. In addition, these drugs are poorly water soluble and require the addition of large amounts of co-solvents in the formulation.

For example, Chinese patent (CN104610149A) discloses two indenopyrazolyl tubulin inhibitors N- (3-ethoxyphenyl) -6-ethoxy-7-carbamoylmethoxy-1-methyl-1H, 4H-indeno [1,2-c ]]Pyrazol-3-amine (compound LL 01) and N- (3-ethoxyphenyl) -6-ethoxy-7-hydroxycarbamoylmethoxy-1-methyl-1H, 4H-indeno [1,2-c ]]Pyrazole-3-amine (compound LL 02) which has good proliferation inhibition activity on human liver cancer HepG2 cells, human prostate cancer PC3 cells, human breast cancer MCF-7 cells and human leukemia K562 cells and can effectively inhibit tubulin polymerization, compound LL 01 and compound LL 02 inhibit tubulin polymerization IC₅₀4.62. mu.M and 5.33. mu.M, respectively, but the inventors found that compound LL 01 and compound LL 02 still had problems such as poor water solubility.

Signaling pathways play important roles in tumor cell growth, proliferation, invasion, and metastasis. Among them, cMet plays an important role in the survival, growth, invasion and metastasis of tumors, and it induces the proliferation and division of tumor cells, adheres and infiltrates by activating downstream effector molecules, promotes the formation of tumor blood vessels, and causes the occurrence, development, infiltration and metastasis of tumors. The cMet and Akt signal transduction pathways are one of important signal transduction pathways in cells, play key roles in inhibiting apoptosis and promoting proliferation in the cells by influencing the activation states of various downstream effector molecules, and are closely related to the occurrence and development of various tumors of human beings. The activation of cell signal channels such as cMet, Akt and the like is blocked, the growth, proliferation, invasion and metastasis of tumor cells can be effectively inhibited, the generation of drug resistance is reduced, and the sensitivity of chemotherapeutic drugs to cancer cells is effectively increased.

Disclosure of Invention

Therefore, the invention aims to provide an indenopyrazole hydrochloride derivative and a preparation method and application thereof, wherein the compound has tubulin inhibitory activity, can simultaneously target a tumor cell signal transduction pathway, inhibit phosphorylation of cMet and/or Akt kinase, has better antitumor activity, has better inhibitory activity on drug-resistant tumor cells, has better water solubility, and has important significance for reducing toxic and side effects, resisting tumor multidrug resistance and the like.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the invention, an indenopyrazole hydrochloride derivative is provided, which has a structure shown in formula I:

wherein, X is CH or N, and N is 1 or 2.

The structure specifically comprises the following structures:

in a second aspect of the present invention, there is provided a process for preparing indenopyrazole hydrochloride derivatives as described in the first aspect above, which comprises: n- (3-ethoxyphenyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine is used as a starting material, Williamson ether forming reaction is carried out on the starting material and N-tert-butyloxycarbonyl-3-bromopropylamine, and then tert-butyloxycarbonyl (Boc) is removed in a mixed solution of acetyl chloride and ethanol to prepare the indenopyrazole hydrochloride derivative shown in the formula I, wherein X is CH, and N is 1.

The synthetic route is as follows:

in one embodiment of the invention, the method comprises:

(1) dissolving N- (3-ethoxyphenyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (formula 1) in acetone, adding potassium carbonate, stirring for 5min, adding N-tert-butyloxycarbonyl-3-bromopropylamine, and stirring at 70 ℃ for reacting for 10H. The reaction was quenched with water, extracted with ethyl acetate and dried over anhydrous sodium sulfate. Filtering, concentrating, and performing silica gel column chromatography to obtain N- (3-ethoxyphenyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine (formula 2);

wherein, the adding amount ratio of the N- (3-ethoxyphenyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine to the N-tert-butyloxycarbonyl-3-bromopropylamine is 1.36g to 1.77 g.

(2) Adding acetyl chloride into ethanol, stirring for 15min, adding into N- (3-ethoxyphenyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine, reacting for 20min, and standing for 40 min. And (3) carrying out suction filtration to obtain hydrochloric acid-N- (3-ethoxyphenyl) -6-ethoxy-7- (3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine (compound ID 01).

The invention also provides a method for preparing the indenopyrazole hydrochloride derivative in the first aspect, which comprises the steps of using 5-ethoxy-6-tert-butyldimethylsilyloxy-1-indanone as a raw material, treating the raw material with lithium bis (trimethylsilyl) amide (L iHMDS), reacting the treated raw material with 2-ethoxy-6-pyridylisothiocyanate, reacting the obtained product with hydrazine hydrate for cyclization, carrying out NH methylation on pyrazole to obtain an indenopyrazole intermediate N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (formula 4), carrying out Williamson ether forming reaction on the intermediate with N-tert-butyloxycarbonyl-3-bromopropylamine after removing a tert-butyldimethylsilyl (TBS) protecting group, and then removing protection in a mixed solution of acetyl chloride and ethanol to prepare the indenopyrazole hydrochloride derivative shown in the formula I, wherein X is N, and N is 2 Boc.

The synthetic route is as follows:

in an embodiment of the invention, the method comprises:

(1) dissolving 5-ethoxy-6-tert-butyldimethylsilyloxy-1-indanone (formula 3) in anhydrous tetrahydrofuran, precooling for 5min at-78 ℃, dropwise adding L iHMDS, stirring for 2.5H, naturally heating to-45 ℃, adding a tetrahydrofuran solution of 2-ethoxy-6-pyridylisothiocyanate, naturally heating to room temperature, continuously stirring for reacting overnight, adjusting the pH to be neutral by using a saturated potassium bisulfate solution, extracting by using dichloromethane, drying by using anhydrous sodium sulfate, filtering, concentrating, performing silica gel column chromatography to obtain a crude thiocarbamide intermediate, dissolving the crude thiocarbamide intermediate in a mixed solvent of dioxane and ethanol, adding hydrazine hydrate, stirring for reacting for 20H at 55 ℃, adding water, extracting by using ethyl acetate, drying by using anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1H, 4H-indeno [1,2-c ] pyrazol-3-amine;

wherein, the adding ratio of 5-ethoxy-6-tert-butyldimethylsilyloxy-1-indanone, lithium bis (trimethylsilyl) amide, 2-ethoxy-6-pyridine isothiocyanate and hydrazine hydrate is 300 mg: 1.76m L: 317 mg: 0.35m L.

(2) Dissolving N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1H, 4H-indeno [1,2-c ] pyrazol-3-amine in dichloromethane, adding N, N-Diisopropylethylamine (DIPEA), stirring for reacting for 5min, adding dimethyl sulfate, and continuing to stir for reacting for 8H. After water was added, the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. Filtering, concentrating, and performing silica gel column chromatography to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyl dimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine (formula 4);

wherein the mass ratio of the adding amount of N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine, N-diisopropylethylamine and dimethyl sulfate is 200 mg: 0.17m L: 0.10m L.

(3) Dissolving N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (formula 4) in tetrahydrofuran, adding tetrabutylammonium fluoride (TBAF), and stirring for 1H. Adding water, and filtering to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine (formula 5);

wherein the mass ratio of the adding amount of N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine to tetrabutylammonium fluoride is 120 mg: 110 mg.

(4) Dissolving N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (formula 5) in acetone, adding potassium carbonate, stirring for 5min, adding N-tert-butoxycarbonyl-3-bromopropylamine, and stirring at 70 ℃ for reaction for 10H. After water was added, the mixture was extracted with ethyl acetate and dried over anhydrous sodium sulfate. Filtering, concentrating, and performing silica gel column chromatography to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine (formula 6);

wherein, the adding amount ratio of the N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine and the N-tert-butyloxycarbonyl-3-bromopropylamine is 90mg to 116 mg.

(5) Acetyl chloride is added into ethanol, stirred for 15min and then added into N- (3-ethoxy-2-pyridyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (formula 6) to react for 20 min. Standing for 40min, and performing suction filtration to obtain hydrochloric acid-N- (3-ethoxy-2-pyridyl) -6-ethoxy-7- (3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine (compound ID 02).

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising the indenopyrazole hydrochloride derivative described in the first aspect above.

In a fourth aspect of the present invention, there is provided a pharmaceutical formulation comprising the indenopyrazole hydrochloride derivative of the first aspect and at least one pharmaceutically acceptable carrier and/or excipient.

"pharmaceutically acceptable excipient" refers to an ingredient of a pharmaceutical composition or pharmaceutical formulation other than the active ingredient, which is not toxic to the subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, preservatives, binders, fillers, and the like. The pharmaceutical composition or pharmaceutical preparation of the present invention may further comprise conventional excipients or can be determined by those skilled in the art according to actual conditions, and the excipients include but are not limited to: calcium phosphate, magnesium stearate, talc, dextrin, starch, gelatin cellulose, methyl cellulose, sodium carboxymethyl cellulose or polyvinylpyrrolidone.

The pharmaceutical composition or pharmaceutical preparation of the present invention may further comprise conventional carriers or can be determined by those skilled in the art according to the actual situation, the carriers include but are not limited to: physiological saline, buffered saline, glucose, water, glycerol, ethanol, or combinations thereof; the carrier may be present in the pharmaceutical composition or formulation in an amount of from 1% to 98% by weight, typically about 80% by weight.

The pharmaceutical preparation can be an oral preparation and a parenteral preparation, and can be tablets, pills, capsules or injections.

Oral preparations such as tablets, capsules and pills may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine; lubricants, such as magnesium stearate, talc, polyethylene glycol, silica; a disintegrant, such as potato starch, or an acceptable humectant, such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.

Parenteral formulations, liquid dosage forms, are generally prepared from the compound and a sterile vehicle. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

The compounds of the present invention may be formulated into pharmaceutical compositions or formulations using techniques well known to those skilled in the art. Suitable pharmaceutical excipients, in addition to those mentioned herein, are known in the art, see for example the 2005 edition of the handbook of pharmaceutical excipients (fourth edition of original works), authors (en) r.c. lo (Raymond C Rowe) (usa) p.j. sertbasis (Paul J Sheskey).

In a fourth aspect of the invention, the use of the indenopyrazole hydrochloride derivative described in the first aspect in the preparation of a tubulin inhibitor drug is provided. The tubulin inhibitor is capable of inhibiting tubulin polymerization.

The indenopyrazole hydrochloride derivative has better activity of inhibiting tubulin polymerization, has improved water solubility compared with the existing tubulin inhibitor, has the water solubility of more than 1mg/m L (pH 7.4), can inhibit phosphorylation of cMet and/or Akt kinase while inhibiting tubulin polymerization, has less toxic and side effects, and is not easy to resist drugs.

In a fifth aspect of the invention, the application of the indenopyrazole hydrochloride derivative in the first aspect in preparing an antitumor drug is provided.

In embodiments of the invention, the tumor includes, but is not limited to, liver cancer, lung cancer, colon cancer. In particular, the lung cancer is non-small cell lung cancer, including paclitaxel-resistant non-small cell lung cancer.

The compound of the invention has lower toxicity to normal liver cells, still has strong proliferation inhibition activity to paclitaxel-resistant human non-small cell lung cancer A549 cells, and has certain multi-drug resistance effect.

In a sixth aspect of the invention, the invention provides the use of the indenopyrazole hydrochloride derivative described in the first aspect in the preparation of a medicament for resisting tumors and inhibiting tubulin polymerization.

In a seventh aspect of the invention, the application of the indenopyrazole hydrochloride derivative in the first aspect in preparing a drug or a reagent for inhibiting phosphorylation of cMet and/or Akt kinase is provided.

In the eighth aspect of the present invention, a method for treating cancer is provided, the method comprises administering to a subject a therapeutically effective amount of the indenopyrazole hydrochloride derivative described in the first aspect above or a pharmaceutical composition or pharmaceutical preparation thereof, wherein the cancer includes but is not limited to liver cancer, lung cancer and colon cancer. The subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.

As used herein, a "therapeutically effective amount" is an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

Treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or palliating the disease state, and alleviating or improving prognosis.

The optimal dosage and interval of administration of the compounds of the present invention will be determined by the nature of the compound and external conditions, such as the form, route and site of administration and the particular mammal being treated, and such optimal dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over a nominal period of time, may be determined by methods known in the art.

Compared with the prior art, the beneficial effects of the invention include but are not limited to:

(1) compared with the existing tubulin inhibitor, the indenopyrazole hydrochloride derivative has the advantages that the water solubility is improved, the water solubility is more than 1mg/m L (pH 7.4), the improvement of the water solubility has important significance for reducing toxic and side effects, preventing drug resistance and the like, and the good water solubility is easier to use.

(2) The indenopyrazole hydrochloride derivative is a small molecular tubulin inhibitor with a novel structure, has strong proliferation inhibition activity on human liver cancer HepG2 cells, human non-small cell lung cancer A549 cells and human colon cancer HCT16 cells, still has strong proliferation inhibition activity on paclitaxel-resistant human non-small cell lung cancer A549 cells, and has a certain multi-drug resistance effect.

(3) The indenopyrazole hydrochloride derivative can simultaneously target a tumor cell signal channel and inhibit phosphorylation of cMet and Akt kinases.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the application and, together with the description, serve to explain the application and are not intended to limit the application. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the results of tubulin polymerization experiments with Compound ID01 in example 1;

FIG. 2 shows the results of tubulin polymerization experiments with Compound ID02 in example 2;

FIG. 3 is the experimental results of the effect of Compound ID01 on cMet/Akt kinase phosphorylation in example 1.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present invention can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present invention can be used in a conventional manner in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Example 1Preparation of Compound ID01

(1) Mixing N- (3-ethoxyphenyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ]]Dissolving pyrazol-3-amine (1.36g, 3.7mmol) in acetone (30m L), adding potassium carbonate (1.54g, 7.4mmol), stirring for reaction for 5min, adding N-tert-butyloxycarbonyl-3-bromopropylamine (1.77g, 7.4mmol), stirring for reaction for 10H at 70 ℃, adding water 100m L, extracting with ethyl acetate (390m L), washing with saturated common salt water, drying with anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography (petroleum ether: ethyl acetate: 1) to obtain N- (3-ethoxyphenyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1]Pyrazol-3-amine 1.35g, the yield was 71%. Melting point: 137-138 ℃.¹H NMR(400MHz,DMSO-d6)8.36(s,1H),7.29(s,1H),7.21(s,1H),7.06(t,J＝8.0Hz,1H),6.98(s,1H),6.89-6.71(m,2H),6.28(d,J＝8.0Hz,1H),4.19–4.02(m,4H),3.99(q,J＝6.7Hz,2H),3.96(s,3H),3.37(s,2H),3.14(q,J＝6.0Hz,2H),1.89–1.82(p,J＝6.0Hz,2H),1.38(s,9H),1.37-1.27(m,6H)。MS(ESI)calcd.for C₂₉H₃₉N₄O₅[M+H]⁺:523.3,found:523.1。

(2) Acetyl chloride (2.5m L) was added to ethanol (5m L) and stirred for 15min, after which 4m L was added to N- (3-ethoxyphenyl) -6-ethoxy-7- (N-tert-butoxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ]]Pyrazole-3-amine (110mg) reacts for 20min, stands for 40min, and is filtered by suction to obtain 87mg of white solid. The yield thereof was found to be 91%. Melting point: 156 and 158 ℃.¹H NMR(600MHz,DMSO-d6)8.20(s,3H),7.43(s,1H),7.27(s,1H),7.13(t,J＝8.1Hz,1H),6.92(t,J＝2.2Hz,1H),6.83(dd,J＝8.0,2.2Hz,1H),6.41(dd,J＝8.1,2.2Hz,1H),4.20(t,J＝6.2Hz,2H),4.08(q,J＝7.0Hz,2H),4.03(s,3H),3.99(q,J＝7.0Hz,2H),3.42(s,2H),3.12-2.90(m,2H),2.16–2.03(p,J＝6.2Hz,2H),1.36(t,J＝7.0Hz,3H),1.32(t,J＝7.0Hz,3H)。HRMS(ESI)calcd.for C₂₄H₃₁N₄O₃[M+H]⁺:423.2391,found:423.2394。

Example 2Preparation of Compound ID02

(1) Dissolving 5-ethoxy-6-tert-butyldimethylsilyloxy-1-indanone (300mg, 0.98mmol) in anhydrous tetrahydrofuran (10m L), precooling for 5min at-78 deg.C, adding L iHMDS (1.76m L, 2.05mmol) dropwise, stirring for 2.5H, heating to-45 deg.C, adding 2-ethoxy-6-pyridineisothiocyanate (317mg, 1.76mmol) dissolved in anhydrous tetrahydrofuran (8m L), slowly heating to room temperature, stirring for overnight, adjusting pH to neutral with saturated potassium bisulfate solution, adding water 40m L, extracting with dichloromethane (180m L), drying with anhydrous sodium sulfate, filtering, concentrating, performing silica gel column chromatography (petroleum ether: ethyl acetate: 20: 1), obtaining a thiocarbamide intermediate 327mg (327mg, 0.70mmol), dissolving in a mixed solvent of anhydrous dioxane and anhydrous ethanol (14m L, volume ratio: 1:1 hydrazine, adding water 355-ethoxy-2 m hydrazine, extracting with anhydrous ethanol [ 354: 355 m ] ethyl acetate, concentrating with anhydrous diethyl ether, stirring to obtain a mixture, and concentrating the filtrate (354: 2 m-2-ethoxy-2H), adding anhydrous ethyl acetate, stirring to obtain a filtrate, concentrating to obtain a filtrate, and concentrating]200mg (200mg, 0.43mmol) of pyrazol-3-amine is dissolved in dichloromethane (6m L), DIPEA (0.17m L, 0.86mmol) is added, after stirring reaction is carried out for 5min, dimethyl sulfate (0.10m L, 0.86mmol) is added, stirring reaction is continued for 8H, water is added for 20m L, dichloromethane (60m L) is used for extraction, anhydrous sodium sulfate is used for drying, filtration, concentration and silica gel column chromatography (petroleum ether: ethyl acetate: 5: 1) are carried out, and N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsiloxy-1-methyl-1H, 4H-indeno [1,2-c ] (]Pyrazole-3-amine 85mg, yield 18%. Melting point: 147 ℃ and 148 ℃.¹H NMR(600MHz,DMSO-d₆)8.88(s,1H),7.46(t,J＝7.9Hz,1H),7.20(s,1H),7.11(s,1H),6.90(d,J＝7.9Hz,1H),6.09(d,J＝7.9Hz,1H),4.22(q,J＝7.0Hz,2H),4.04(q,J＝7.0Hz,2H),3.91(s,3H),3.46(s,2H),1.36(t,J＝7.0Hz,3H),1.29(t,J＝7.0Hz,3H),1.00(s,9H),0.16(s,6H)。MS(ESI)calcd.for C₂₆H₃₇N₄O₃Si[M+H]⁺:481.3,found:481.5。

(3) Mixing N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ]]Dissolving pyrazole-3-amine (120mg, 0.25mmol) in tetrahydrofuran (8m L), adding tetrabutylammonium fluoride (110mg, 0.50mmol), stirring for reaction for 1H, adding water (40m L), precipitating a solid, filtering, and drying in vacuum to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ]]Pyrazol-3-amine 70mg, yield 81%. Melting point: 201 ℃ and 202 ℃.¹H NMR(600MHz,DMSO-d6)8.86(s,1H),8.82(s,1H),7.46(t,J＝7.9Hz,1H),7.14(s,1H),7.12(s,1H),6.92(d,J＝7.9Hz,1H),6.09(d,J＝7.9Hz,1H),4.23(q,J＝7.0Hz,2H),4.06(q,J＝7.0Hz,2H),3.91(s,3H),3.42(s,2H),1.35(t,J＝7.0Hz,3H),1.29(t,J＝7.0Hz,3H)。MS(ESI)calcd.for C₂₀H₂₃N₄O₃[M+H]⁺:367.2,found:367.1。

(4) Mixing N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ]]Dissolving pyrazole-3-amine (90mg, 0.25mmol) in acetone (10m L), adding potassium carbonate (68mg, 0.50mmol), stirring for reaction for 5min, adding N-tert-butyloxycarbonyl-3-bromopropylamine (116mg, 0.50mmol), stirring for reaction at 70 ℃ for 10H, adding water (30m L), extracting with ethyl acetate (120m L), washing with saturated salt water, drying with anhydrous sodium sulfate, filtering, concentrating, and performing silica gel column chromatography (petroleum ether: ethyl acetate: 2:1-1:1) to obtain N- (3-ethoxy-2-pyridyl) -6-ethoxy-7- (N-tert-butyloxycarbonyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c ] -indeno]Pyrazole-3-amine 70mg, yield 55%. Melting point: 155 ℃ and 156 ℃.¹HNMR(600MHz,DMSO-d₆)8.89(s,1H),7.47(t,J＝7.9Hz,1H),7.29(s,1H),7.20(s,1H),6.92(d,J＝7.9Hz,1H),6.83(t,J＝5.2Hz,1H),6.09(d,J＝7.9Hz,1H),4.23(q,J＝7.0Hz,2H),4.16-4.02(m,4H),3.97(s,3H),3.45(s,2H),3.14(q,J＝6.4Hz,2H),1.86(p,J＝6.4Hz,2H),1.38(s,9H),1.34(t,J＝7.0Hz,3H),1.29(t,J＝7.0Hz,3H)。MS(ESI)calcd.forC₂₈H₃₈N₅O₅[M+H]⁺:524.3,found:524.2。

(5) Adding acetyl chloride (2.5m L) into ethanol (5m L), stirring for 15min, and adding N- (3-ethoxy-2-pyridyl) -6-ethoxy-7- (N-tert-butyloxymethyl) into 2m LAcyl-3-aminopropoxy) -1-methyl-1H, 4H-indeno [1,2-c]Pyrazole-3-amine (30mg) was reacted with stirring for 20 min. After standing for 40min, filtration was carried out to obtain 25mg of a white solid. The yield thereof was found to be 86%. Melting point: 230 ℃ and 231 ℃.¹H NMR(600MHz,DMSO-d6)9.87(s,1H),8.17(s,3H),7.61(t,J＝8.0Hz,1H),7.43(s,1H),7.27(s,1H),6.90(d,J＝8.0Hz,1H),6.25(d,J＝8.0Hz,1H),4.28(q,J＝7.0Hz,2H),4.20(t,J＝6.1Hz,2H),4.09(q,J＝7.0Hz,2H),4.05(s,3H),3.53(s,2H),3.04-2.98(m,2H),2.11-2.00(p,J＝6.1Hz,2H),1.36(t,J＝7.0Hz,3H),1.32(t,J＝7.0Hz,3H)。HRMS(ESI)calcd.for C₂₃H₃₀N₅O₃[M+H]⁺:424.2343,found:424.2346。

Example 3Antitumor cell proliferation assay

1. Experimental drugs the compound prepared in example 1 (denoted as compound ID01), the compound prepared in example 2 (denoted as compound ID02) and compound LL 01.

2. The experimental method comprises the following steps:

the human liver cancer HepG2 cell, the human non-small cell lung cancer A549 cell, the human colon cancer HCT16 cell and the paclitaxel-resistant A549/Tax cell are respectively administered with the compound ID01 compound (prepared in example 1), the compound ID02 (prepared in example 2) and the compound LL 01 with different concentrations, and are placed in an incubator for incubation for 72 hours, and the inhibition rate of the compound on the tumor cells is measured by a tetramethyl azoazolium salt (MTT) colorimetric method.

3. The test results are shown in table 1. from table 1, the compounds prepared in example 1 and example 2 have extremely strong antiproliferative activity on three tested tumor cells HepG2, A549 and HCT116, are obviously superior to the compound LL 01, are still effective on paclitaxel-resistant A549 cells, and have a certain multi-drug resistance effect.

TABLE 1 antiproliferative Activity of the Compounds of the invention on different cells

^aMean. + -. Standard Deviation (SD) of three experiments

Example 4Tubulin polymerTest (experiment)

1. Experimental drugs: the compound prepared in example 1 (identified as ID01), the compound prepared in example 2 (identified as ID02), colchicine and dimethyl sulfoxide (DMSO).

2. The experimental method comprises the following steps:

tubulin Polymerization experiments were performed according to the Tubulin Polymerization assay kit instructions from Cytoskeleton.

3. The results are shown in fig. 1 and fig. 2, respectively. As can be seen from fig. 1 and 2, both the compound prepared in example 1 and the compound prepared in example 2 inhibited tubulin polymerization. IC for inhibiting tubulin polymerization by compound ID01 and compound ID02₅₀The effect of inhibiting tubulin polymerization was more significant than that of LL 01 (4.62. mu.M) and LL 02 (5.33. mu.M), 3.65. mu.M and 3.13. mu.M, respectively.

Example 5cMet and Akt signal pathway inhibition experiment

1. Experimental drugs: the compound prepared in example 1 (denoted as compound ID01) and dimethyl sulfoxide (DMSO).

2. The experimental method comprises the following steps:

human hepatoma HepG2 cells were administered with different concentrations of Compound ID01 (prepared in example 1) at 37 ℃ with 5% CO₂The culture box is incubated for 24 hours, cells are collected, and cell sample holoprotein is extracted for Western blot analysis. 20 μ g of protein was subjected to gel electrophoresis using 10% polyacrylamide. The protein was transferred to PVDF membrane and blocked for 1h by adding blocking solution containing 5% skimmed milk powder. The membrane was placed in a dish containing primary antibody (diluted with western primary antibody dilution) and incubated overnight at 4 ℃ with shaking in a shaker. Washing the membrane, and detecting the immunoreaction protein by a chemiluminescence method.

3. The results are shown in FIG. 3, and it can be seen from FIG. 3 that compound ID01 can inhibit the phosphorylation of cMet and AKT without direct inhibition of cMet and AKT expression.

Example 6Solubility test

1. Experimental drugs the compound prepared in example 1 (denoted as compound ID01), the compound prepared in example 2 (denoted as compound ID02) and compounds LL 01, LL 02.

2. The experimental method comprises the following steps:

the solubility is measured by preparing a compound into a mother solution with the concentration of 10mg/m L by DMSO, diluting the compound into 5 concentration gradient solutions by methanol in an equal-time manner, measuring peak areas under different concentrations by using high performance liquid chromatography (HP L C), establishing a standard curve, adding a proper amount of the mother solution into a 1m L buffer solution (pH 7.4) to prepare a saturated solution, measuring the absorption peak area of the saturated solution of the compound, and calculating the saturated solubility of the compound by using a standard curve equation.

3. The experimental results are shown in table 2. from table 2, it can be seen that compound ID01 and compound ID02 have good water solubility, and the water solubility is significantly improved compared with compounds LL 01 and LL 02.

TABLE 2 solubility of the Compounds

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An indenopyrazole hydrochloride derivative has a structure shown in a formula I:

wherein, X is CH or N, and N is 1 or 2.

2. The indenopyrazole hydrochloride derivative of claim 1, wherein the compound comprises:

3. a process for preparing indenopyrazole hydrochloride derivatives of claim 1 or 2, comprising: n- (3-ethoxyphenyl) -6-ethoxy-7-hydroxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazole-3-amine is used as a starting material, Williamson ether forming reaction is carried out on the starting material and N-tert-butyloxycarbonyl-3-bromopropylamine, and then tert-butyloxycarbonyl (Boc) is removed in a mixed solution of acetyl chloride and ethanol to prepare the indenopyrazole hydrochloride derivative shown in the formula I, wherein X is CH, and N is 1.

4. A process for preparing indenopyrazole hydrochloride derivatives of claim 1 or 2, comprising: taking 5-ethoxy-6-tert-butyldimethylsilyloxy-1-indanone as a raw material, treating with lithium bis (trimethylsilyl) amide, reacting with 2-ethoxy-6-pyridine isothiocyanate, reacting with hydrazine hydrate, cyclizing, and carrying out NH methylation on pyrazole to obtain an indenopyrazole intermediate N- (3-ethoxy-2-pyridyl) -6-ethoxy-7-tert-butyldimethylsilyloxy-1-methyl-1H, 4H-indeno [1,2-c ] pyrazol-3-amine; after removing a tert-butyl dimethyl silicon-based protecting group from the intermediate, carrying out Williamson ether forming reaction with N-tert-butyloxycarbonyl-3-bromopropylamine, and removing Boc protection from the intermediate in a mixed solution of acetyl chloride and ethanol to prepare the indenopyrazole hydrochloride derivative shown in the formula I, wherein X is N, and N is 2.

5. A pharmaceutical composition comprising the indenopyrazole hydrochloride derivative of claim 1 or 2.

6. Pharmaceutical formulation comprising an indenopyrazole hydrochloride derivative according to claim 1 or 2 and at least one pharmaceutically acceptable carrier and/or excipient.

7. Use of an indenopyrazole hydrochloride derivative according to claim 1 or 2 in the manufacture of a tubulin inhibitor medicament;

preferably, the tubulin inhibitor is capable of inhibiting tubulin polymerization.

8. The use of indenopyrazole hydrochloride derivatives as claimed in claim 1 or 2 for the preparation of an anti-tumor medicament;

preferably, the tumor comprises liver cancer, lung cancer, colon cancer;

preferably, the lung cancer is non-small cell lung cancer, including paclitaxel-resistant non-small cell lung cancer.

9. Use of indenopyrazole hydrochloride derivatives as claimed in claim 1 or 2 for the manufacture of a medicament for the treatment of cancer and for inhibiting tubulin polymerization;

preferably, the tumor comprises liver cancer, lung cancer, colon cancer;

10. Use of indenopyrazole hydrochloride derivatives according to claim 1 or 2 for the preparation of a medicament or reagent for inhibiting the phosphorylation of cMet and/or Akt kinases.