CN1566091A

CN1566091A - Molindone derivatives and use for preparing antineoplastic medicine thereof

Info

Publication number: CN1566091A
Application number: CN 03146229
Authority: CN
Inventors: 李松; 刘继周; 王莉莉; 郑志兵; 刘洪英; 阮成迈; 谢云德; 沈倍奋
Original assignee: Institute of Pharmacology and Toxicology of AMMS
Current assignee: BEIJING MUOLIKE SCIENCE AND TECHNOLOGY Co Ltd
Priority date: 2003-07-04
Filing date: 2003-07-04
Publication date: 2005-01-19
Anticipated expiration: 2023-07-04
Also published as: CN1295216C

Abstract

The invention relates to indoles ketone derivative with the formula I, its geometrical isomers and its pharmaceutically acceptable salts, wherein the definition for each group is disclosed in the specification. The invention also relates to the process for preparing the compound, pharmaceutical compositions containing the compound, and the use of the compound in preparing medicament with the actions of evoking tumour cell dying, antineoplastic formation, and preventing tumor chemotherapy transferase.

Description

Indolone derivatives and application thereof in preparing antitumor drugs

Technical Field

The invention relates to novel indolone derivatives, geometric isomers and pharmaceutically acceptable salts thereof, a preparation method thereof and a pharmaceutical composition containing the compounds. The invention also relates to the application of the indolone derivatives in preparing antitumor drugs with the functions of antagonizing vascular endothelial cell growth factor receptors (VEGFRs), inducing tumor cell apoptosis, resisting tumor neovascularization and blocking malignant tumor metastasis.

Background

Malignant tumors are a serious disease that threatens human health and life. Statistics by the World Health Organization (WHO) show that: at present, about 1000 million people worldwide have tumors and 700 million people die every year, and malignant tumors have become the second killer of people next to cardiovascular diseases. Therefore, tumor prevention and treatment are receiving general attention from all social circles. Tumor chemotherapy (hereinafter, chemotherapy) is one of three basic means for tumor therapy, and through the development of more than 50 years, the drugs for tumor therapy have achieved huge achievements, and a large number of clinical antitumor drugs with different action mechanisms are obtained, unfortunately, the antitumor drugs have toxic and side effects of different degrees, so that many patients cannot persist in continuously receiving treatment in clinic; in addition, chemotherapy resistance and tumor metastasis are another important problem that is not solved by the current research on antitumor drugs, and the generation of tumor resistance and tumor metastasis often makes tumor therapy not achieve the expected effect. Therefore, there is a need to find new antitumor drugs that have less toxic side effects, no drug resistance, and can prevent tumor metastasis.

In the early 70 s of the 20 th century, Judah Folkman proposed the hypothesis that tumor growth depends on angiogenesis, based on the accumulation of a great deal of clinical practice and experience: the diameter of the tumor is limited to 2-3 mm due to the lack of new blood vessels in the early formation stage, and the number of cells is not more than 100 ten thousand; tumors grow rapidly only after sufficient oxygen and nutrient supply is available in the angiogenic phase mediated by tumor-secreted angiogenic factors (TAF) (Folkman J, n.engl.j.med., 1971, 285 (21): 1182-1186); and thus the concept of anti-angiogenesis has been proposed (Folkman J, Ann. Surg., 1972, 175 (3): 409-416).

Over the course of more than 30 years of development, and in particular the rapid development of molecular biology in the last 10 years, a large number of fundamental research efforts on angiogenesis have not only fully demonstrated the hypothesis of Folkman, but have also found that angiogenesis is also essential for tumor metastasis (Gasparini G, Oncol. Rep., 1994, 1 (1): 7-12), and is associated with multidrug resistance in tumors (Zhang XS, acta. Pharmacol. sin., 2001, 22 (8): 731 one 735).

Because the generation and development of the tumor depend on the formation of new blood vessels, and the growth of the blood vessels in the tumor area is 50-200 times that of the normal tissue, the inhibition of the growth by taking the blood vessels in the tumor area as a target has the following advantages: (1) anti-angiogenic therapy has good specificity because normal adult angiogenesis is essentially halted and angiogenesis is initiated only in the presence of pregnancy, menstrual cycle, inflammation, tumors, etc., and vasoinhibitors have no effect on quiescent blood vessels. (2) The gene expression of endothelial cells is relatively stable, and the cells have little mutation and little heterogeneity, so that the drug resistance is not easy to generate and the medicine can be used for a long time. (3) All vascular endothelial cells are exposed in blood, and the medicine can directly play a role without the links of penetration and the like, so that the medicine has small dosage and high curative effect.

1. Process for blood vessel formation

Neovascularization refers to the development of new vasculature from existing blood vessels. Normal angiogenesis occurs only during certain short-term, specific physiological processes, such as reproduction, wound healing, etc. (Hyder SM, Mol Endocrinol 1999; 13: 806-11). Angiogenesis is a complex process involving a wide range of interactions between cells with various cytokines and extracellular matrices. The step of angiogenesis involves the degradation of the basement membrane by endothelial cells of existing blood vessels and the metastatic invasion into other organs, where the involvement of MMPs (metalloproteinases) and PA systems is required (Mignatti P, Enzyme Protein 1996; 49: 117-37); the second step is the proliferation of endothelial cells, which requires the participation of various growth factors; the last step is the formation of capillaries by the interaction between endothelial cells and between cells and the extracellular matrix, which requires the integration of cell adhesion molecules, which are also involved in the above two steps (Bischoff J, J Clin Invest 1997; 100: S37-9).

2. Angiogenic factor and neovascularization

Since Folkman et al isolated a soluble substance capable of promoting angiogenesis from solid tumors and ascites tumors of humans and animals in 1971, which is called Tumor Angiogenesis Factor (TAF) (Folkman J, N.Engl. J.Med., 1971, 285 (21): 1182-1186), studies in recent 20 years have found that angiogenesis promoting factors are a large class of growth factors and cytokine class of polypeptide substances called tumor angiogenesis inducers, such as Fibroblast Growth Factor (FGF), macrophage-derived epidermal growth factor, angiogenic nutrients, IL-1, IL-8, prostaglandins (PGE1, PGE2), butyrylglycerol, nicotinamide and adenosine, hyaluronic acid metabolites, and complexes of some metallic copper. In 1982, the first angiogenic factor was found to be basic fibroblast growth factor (bFGF), and acidic fibroblast growth factor (aFGF) was subsequently identified, both of which have high affinity for heparin, and which lack a growth factor family member characterized by a conventional signal peptide sequence, and which promote regeneration of epidermal endothelial cells, promote division of intravascular cells, stimulate chemotactic movement of endothelial cells toward tumor tissues and formation of tubular structures, and also enhance Plasminogen Activators (PAs) in tissues and induce endothelial cells to produce other proteases, which are relatively direct inducers of angiogenesis. It is successively determined that another kind of secreted protein is called Vascular Permeability Factor (VPF) or vascular endothelial cell growth factor (VEGF), which is a secreted glycoprotein with molecular weight of 40KD-45KD, and its two main receptors are flt-1 and flk-1, and VEGF can act on vascular endothelial cell with high efficiency and specificity, and has strong mitogenic action and chemotactic action. Can promote the growth of endothelial cells in vitro and induce angiogenesis in vivo, and experiments prove that the tumor cells can synthesize and secrete VEGF, the expression level of the VEGF in tumor tissues is higher than that of tissues around the tumor, and only a few organs such as kidney, ovary and the like in normal tissues have high expression level (Neufeld G, FASEBJ, 1999, 13 (1): 9-22). In some tumors it has been shown to be associated with malignancy, invasion, metastasis, and therefore VEGF is a regulatory factor in the induction of tumor angiogenesis (Warren RS, endocr. Rev., 1997, 18 (1): 4-25).

The mutual connection and regulation of various angiogenesis factors secreted by tumor cells, among which VEGF is the most active and specific angiogenesis factor known at present, and most other angiogenesis factors produce angiogenesis action by enhancing the expression of VEGF (Zhang QX, J.Surg.Res., 1997, 67 (2): 147-154), and VEGF has an activity of promoting vascular permeability increase equivalent to 5 ten thousand times of that of histamine, and is favorable for the transfer and diffusion of blood vessels (Berkman RA, Science, 1983, 219 (4587): 983-985). Therefore, VEGF is an ideal action target of the anti-angiogenesis medicine.

Bcl-2 and tumor neovascularization

Tumor neovascularization is also associated with other (non-angiogenic factor) types of genes, the Bcl-2 gene family being one example. The involvement of Bcl-2 in the formation of new blood vessels is achieved by two pathways: 1) bcl-2 is able to prevent apoptosis of microvascular epithelial cells, thereby playing an important role in the presence and density of microvasculature in tumors (Jacques E, American Journal of Pathology 1999; 154: 375-384). 2) Triggering the expression of other angiogenic factors, stimulating the formation of new blood vessels by other angiogenic factors, this relationship is mainly reflected in the expression of VEGF by Bcl-2-induced endothelial cells (Blancher C, FASED J, 2000, 14: 652-660), which indicates the important function of Bcl-2 in the process of neovascularization, and suggests that the Bcl-2 should be inhibited simultaneously in the anti-angiogenesis therapy, otherwise the treatment effect of tumor regression is difficult to achieve.

In addition, the main function of the Bcl-2 gene and its gene family is to regulate apoptosis, playing an important role in maintaining the proper morphology of cells and in regulating the number of cells by eliminating harmful or useless cells (Adams J, Science 281, 1322-1326). Bcl-2 prevents apoptosis induced by a number of stimuli, such as chemotherapy, radiation, heat shock, certain viruses, free radicals, lipid peroxidation, p53, c-myc, and the like. Bcl-2 is present in some normal tissue cells that are long-lived and easily damaged, such as nerve cells, ductal cells of non-secretory glands, basal keratin cells, cells at the bottom of the colon gland duct, skin of adults and embryos, and kidney and cartilage tissue of embryos (Adams J, Science 281, 1322-. However, in abnormal cases, the efficient expression of Bcl-2 gene is not only one of the causes of tumor formation (Folkman J, Cell 79, 185-2453) but also one of the causes of tumor resistance (Ellis LM, Eur. J. cancer 32a, 2451-2460) because it can prevent apoptosis induced by a great variety of chemotherapeutic drugs with different mechanisms of action; therefore, the Bcl-2 gene and the receptor thereof are also good anti-tumor targets. The Bcl-2 protein inhibitor can induce apoptosis of tumor cells and enhance the sensitivity of the tumor cells to other chemotherapeutic drugs, so the Bcl-2 protein inhibitor can be used as an antitumor drug; moreover, the biological function of Bcl-2 is not absolutely necessary in normal cells, and the inhibition of the function of Bcl-2 protein will not have great influence on the body.

The indolone derivatives related by the invention can target Bcl-2 protein, induce apoptosis of tumor cells with high expression of Bcl-2 protein and improve the sensitivity of tumor to chemotherapeutic drugs; meanwhile, the compound can block the signal conduction function of an FGF receptor and a VEGF receptor, block the capacity of vascular endothelial cells for forming new blood vessels, inhibit the formation of tumor new blood vessels and prevent malignant metastasis of tumors.

Disclosure of Invention

The invention aims to provide a novel anti-tumor drug capable of selectively blocking or opening Bcl-2/BAX heterodimers, inducing apoptosis of tumor cells and improving the sensitivity of tumors to treatment of chemotherapeutic drugs by regulating the ratio of Bcl-2/BAX heterodimers to BAX/BAX homodimers, and inhibiting the formation of tumor neovascularization and preventing malignant metastasis of tumors by inhibiting the signal transduction of vascular endothelial cell growth factor receptors and blocking the capacity of vascular endothelial cells to form neovascularization.

The invention has found that the indolone derivatives of the general formula I can induce apoptosis of tumor cells and inhibit formation of tumor new vessels, so the indolone derivatives of the general formula I can be used for treating and/or preventing malignant tumors with high Bcl-2 protein expression, including: colon cancer, rectal cancer, nasopharyngeal cancer, bone marrow cancer, breast cancer, non-Hodgkin's lymphoma, gastric cancer, prostate cancer, neuroblastoma, and lung cancer.

According to one embodiment of the present invention, the present invention relates to indolone derivatives of formula I, geometric isomers, and pharmaceutically acceptable salts thereof:

wherein:

n is equal to 2 or 3;

R₁is a heteroaryl group selected from furan, thiophene, pyrrole, pyrazole, imidazole, thiazole, oxazole, isoxazole, pyridine, pyridazine, pyrimidine, pyrazine, indole, benzofuran or quinoline, which heteroaryl group may be unsubstituted or substituted with 1 or 2 substituents selected from: halogen, nitro, hydroxy, hydroxymethyl, trifluoromethyl, trifluoromethoxy, C₁～C₆Straight or branched alkyl, C₂～C₆Straight-chain or branched alkenyl radical, C₁～C₄Alkoxy radical, C₁～C₄Alkoxyacyl group, C₂～C₄Alkenyloxy, phenoxy, benzyloxy, carboxyl or amino;

R₂selected from hydrogen, halogen, cyano, sulfoxide, sulfone, nitro, carboxyl, C₁～C₃Alkoxy radical, C₁～C₃Alkanoyl radical, C₁～C₃Alkyl ester group, C₁～C₃An alkanoylamino group;

R₃、R₄is independently selected from C₁～C₆Straight or branched alkyl, C₂～C₇Straight or branched alkenyl; or R₃And R₄And together with the nitrogen atom to which they are attached form a 4-to 6-membered heterocyclic ring selected from tetrahydropyrrole, piperidine, morpholine, piperazine or nitrogenAnd (3) methylpiperazine.

One aspect of the present invention relates to indolone derivatives of formula I, geometric isomers thereof, or pharmaceutically acceptable salts or hydrates thereof. The invention unexpectedly discovers that when n is equal to 2 or 3, the indolone derivatives in the general formula I and the geometric isomers thereof have excellent pharmaceutical stability, and are particularly suitable for industrial mass preparation and long-term storage.

PCT patents WO0190104, WO0190103 and WO0190068 disclose a micronidine Base (Mannich Base) prodrug of a 3- (3 ', 5' -dimethyl-2 '-pyrrolyl) 2-indolinone derivative, but such compounds are unstable, for example, piperidine micronidine Base of 3- (3', 5 '-dimethyl-2' -pyrrolyl) -2-indolinone is approximately 5% decomposed in a 10% ethanol solution at a temperature of 4 to 30 ℃ for 30 minutes, and are not suitable for mass production and long-term storage, and do not meet quality control requirements for drug stability. The indolone derivatives shown in the general formula I have excellent pharmaceutical stability, for example, 1- [2 '- (1' -morpholine) ] ethyl-3- (4 '-bromo-2' -thiophenemethenyl) -2-indolone is not decomposed at all in 10% ethanol solution at the temperature of 4-30 ℃ for 30 minutes, the purity of the medicine is kept unchanged, the quality control requirement of the medicine stability is met, and the indolone derivatives are particularly suitable for large-scale industrial preparation and long-term storage.

According to a preferred embodiment of the present invention, the present invention relates to compounds of general formula I, geometric isomers and pharmaceutically acceptable salts thereof:

wherein:

n is equal to 2;

R₁is heteroaryl selected from furan, thiophene, pyrrole or pyridine; the heteroaryl group may be unsubstituted or substituted with 1 or 2 substituents selected from: halogen, C₁～C₆Straight or branched alkyl, C₁～C₄An alkoxyacyl group;

R₂selected from hydrogen, halogen;

R₃and R₄And together with the nitrogen atom to which they are attached form a 4-to 6-membered heterocyclic ring selected from piperidine and morpholine.

The compounds of the formula I according to the invention or their pharmaceutically acceptable salts are particularly preferably the following compounds, but these are not intended to limit the invention in any way.

TABLE 1 Indolinone derivatives of formula (I)

The most preferred compound of the present invention is (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (4 '-bromo-2' -thiophenemenyl) -2-indolone:

another aspect of the present invention relates to a process for the preparation of a compound of formula I, comprising:

reaction scheme 1

a) Reacting an indolone derivative of formula II

Wherein R is₂Is defined as in general formula I, with an aminohalohydrocarbon derivative of formula III

Wherein n and R₃And R₄Is as defined for compounds of formula I, X is Cl or Br,

specifically, the indolone derivative of formula II (available from ACROS) and the aminohalohydrocarbon derivative of formula III (available from ACROS) are dissolved in an appropriate amount of acetone, and an appropriate amount of potassium carbonate, tetrabutylammonium bromide and KI are added, the reaction is carried out at a reflux temperature for about 8 hours, filtration and vacuum concentration are carried out to obtain a crude product, which is purified by a silica gel column to obtain an intermediate compound IV (refer to Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-,

wherein n and R₂、R₃And R₄Is as defined for the compound of the general formula I,

b) reacting a compound of formula IV with a heteroaryl carboxaldehyde V

Wherein R is₁Is as defined for the compound of the general formula I,

reacting the compound of formula IV obtained in step a with a heteroarylmethylaldehyde V (obtained from commercial sources or according to the reference De Groot JA, org. prep. reduce int.1981, 13)

(2): 97-101) in a suitable solvent or dispersant such as a suitable alcohol such as methanol or ethanol at a temperature of 10 deg.C to 50 deg.C, adding a base such as an inorganic base such as sodium carbonate, or an organic base such as diethylamine or piperidine, or introducing an acidic gas such as hydrogen chloride, and catalytically reacting for 8 to 24 hours to obtain the compound of formula I (see Adreani A, Eur. Jmed. chem., 1990, 25, 187-190),

wherein n and R₁、R₂、R₃And R₄The definition of (A) is the same as that of the compound of the general formula I.

The compounds of the present invention may be prepared by a variety of reaction schemes, and the compounds of formula I may also be prepared by the following reaction scheme:

reaction scheme 2

c) Indolone derivatives of formula II

Wherein R is₂Is defined as in general formula I, with a heteroarylformaldehyde of formula V

Wherein R is₁Is as defined for the compound of the general formula I,

indolone derivatives of formula II (from ACROS) are reacted with heteroarylmethylaldehydes V (from commercial reagents or prepared according to the methods provided in De Groot JA, Org prep products Int1981, 13 (2): 97-101) in a suitable solvent or dispersant such as a suitable alcohol, e.g. methanol or ethanol, at a temperature of 10 ℃ to 50 ℃ with the addition of a base such as an inorganic base, e.g. sodium carbonate, or an organic base such as diethylamine or piperidine, or with the introduction of an acidic gas such as hydrogen chloride, for a catalytic reaction time of 8 to 24 hours to give compounds of formula VI (see Adreani A, Eur. J. med. chem. 1990, 25, 187-190),

wherein R is₁And R₂Is as defined for the compound of the general formula I,

d) reacting a compound of formula VI with an aminohalohydrocarbon derivative III

dissolving the compound of formula VI and the halogenated hydrocarbon derivative III obtained in the step c in acetone, adding a proper amount of potassium carbonate, tetrabutylammonium bromide and KI, heating for reflux reaction for about 8 hours, filtering, concentrating in vacuum to obtain a crude product, purifying by using a silica gel column to obtain the target compound I (refer to Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428),

The compound can be purified by high vacuum distillation or chromatography, the silica gel used in the method is silica gel for conventional chromatography, the granularity is 10-40 microns, and the eluent is prepared from a single solvent or a plurality of solvents, preferably a mixed solvent prepared from chloroform and methanol according to different proportions.

Another aspect of the present invention relates to pharmaceutical compositions comprising at least one indolone compound of formula I or a geometric isomer thereof or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

The pharmaceutical compositions of the present invention may be prepared by methods known in the art, such as by mixing a compound of formula I, a geometric isomer thereof, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier or excipient.

The invention also relates to the application of at least one indolone compound shown in the general formula I, a geometric isomer thereof or a pharmaceutically acceptable salt thereof in preparing a medicament for treating diseases or symptoms related to Bcl-2 high expression, or preparing an anti-tumor medicament with the effects of antagonizing vascular endothelial cell growth factor receptors, inducing tumor cell apoptosis, inhibiting tumor neovascularization or blocking tumor cell malignant metastasis.

The invention also provides a method for evaluating the cytological functions of the indolone compounds shown in the formula I, a test result of antitumor activity and a test result of activity for inducing tumor cell apoptosis.

The term "pharmaceutically acceptable salt" in the present invention may be a pharmaceutically acceptable inorganic or organic salt. The compounds having basic groups in the general formula I of the present invention can form pharmaceutically acceptable salts with inorganic acids, such as sulfate, hydrochloride, hydrobromide, phosphate; pharmaceutically acceptable salts can also be formed with organic acids such as acetates, oxalates, citrates, gluconates, succinates, tartrates, p-toluenesulfonates, methanesulfonates, benzoates, lactates, maleates, and the like. The acid group (such as R) in the general formula I of the invention₁Containing a free carboxyl group) may form a pharmaceutically acceptable salt with an alkali metal or alkaline earth metal, preferably but not limited to a sodium, potassium, magnesium or calcium salt.

The compounds of the present invention may be administered alone or in the form of pharmaceutical compositions. The route of administration may be oral, parenteral or topical. The pharmaceutical composition can be formulated into various suitable dosage forms according to the administration route.

Pharmaceutical compositions of the compounds of the present invention may be administered in any of the following ways: oral, aerosol inhalation, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or via an external reservoir. Among them, oral, intraperitoneal or intravenous administration is preferable.

When administered orally, the compounds of the present invention may be formulated in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Among these, carriers for tablets generally include lactose and corn starch, and additionally, lubricating agents such as magnesium stearate may be added. Diluents used in capsule formulations generally include lactose and dried corn starch. Aqueous suspension formulations are generally prepared by mixing the active ingredient with suitable emulsifying and suspending agents. Optionally, some sweetener, aromatic or colorant may be added into the above oral preparation.

When the compound is used locally, particularly for treating affected surfaces or organs which are easy to reach by local external application, such as eyes, skin or lower intestinal nerve diseases, the compound can be prepared into different local preparation forms according to different affected surfaces or organs, and the specific description is as follows:

when administered topically to the eye, the compounds of the present invention may be formulated as a micronized suspension or solution in sterile saline having a pH that is isotonic, with or without the addition of preservatives such as benzyl alkoxide chloride. For ophthalmic use, the compounds may also be formulated in the form of ointments such as vaseline.

When applied topically to the skin, the compounds of the present invention may be formulated in a suitable ointment, lotion, or cream formulation wherein the active ingredient is suspended or dissolved in one or more carriers. Carriers that may be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil, sorbitan monostearate, tween 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The compounds of the present invention may also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oleaginous suspensions or solutions. Among the carriers and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. In addition, the sterilized fixed oil may also be employed as a solvent or suspending medium, such as a monoglyceride or diglyceride.

It is further noted that the dosage and method of administration of the compounds of the present invention will depend upon a variety of factors including the age, weight, sex, physical condition, nutritional status, the strength of the compound's activity, time of administration, metabolic rate, severity of the condition, and the subjective judgment of the treating physician. The preferable dosage is 0.01-100 mg/kg body weight/day, wherein the optimal dosage is 20mg/kg-30mg/kg body weight/day.

Detailed description of the preferred embodiments

The invention is illustrated by the following examples, which are not intended to be limiting in any way.

Example 1: (Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (3 ', 5 ' -dimethyl-2 ' -pyrromethenyl) -2-indolinone

Equimolar 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (2.44g, 0.01mol, see Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 3, 5-dimethyl-2-pyrrolecarboxaldehyde (1.23g, 0.01mol, prepared according to De Groot JA, org. prep. precedurint.1981, 13 (2): 97-101 reference) were taken and placed in a round bottom flask, 20.0ml methanol, 0.20ml piperidine were added, stirred for 24 hours at room temperature under nitrogen protection, filtered off with suction, washed with methanol to give a crude product, which was crystallized from absolute ethanol to give the title compound as a yellow solid in 70.9% yield.

¹H-NMR：δppm(CDCl₃)：0.71(s，1H)，8.74(s，2H)，7.71(d，2H)，7.56(s，1H)，7.36～7.41(d，1H)，7.34～7.24(t，1H)，6.89～6.86(t，1H)，(d，1H)。MS(m/e)：223[M+1]。

Preparation of hydrochloride salt: dissolving the free alkali in chloroform, adding anhydrous ether-hydrogen chloride solution to strong acidity, filtering, washing with anhydrous ether, and crystallizing the obtained crude product with anhydrous ethanol to obtain pure product.

Example 2: (Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (2 ' -pyrromethenyl) -2-indolinone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 2-pyrrylformaldehyde (from ACROS Corp.) to give the title compound as a pure yellow solid in 90.5% yield.

¹H-NMR：δppm(DMSO-d₆): 13.38 (broad singlet, 1H), 10.88 (broad singlet, 1H), 8.02 to 8.01(d, 1H), 7.69(s, 1H), 7.23 to 7.20(q, 1H), 6.82 to 6.80(d, 1H), 6.03(s, 1H), 2.50(s, 3H), 2.30(s, 3H). MS (m/e): 317(95),319(100),238, 133, 112, 85, 69.

Example 3: (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (4 '-bromo-2' -thiophenemethenyl) -2-indolone

The title compound was obtained by the procedure of example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 4 '-bromo-2' -thiophenecarboxaldehyde (from ACROS). Yellow solid, yield 95%.

¹H-NMR：δppm(DMSO-d₆): 12.01 (broad singlet, 1H), 10.84 (broad singlet, 1H), 8.22(s, 1H), 7.87(s, 1H), 7.75 ~ 7.73(d, 1H)，7.65～7.64(d，1H)，7.50～7.52(d，1H)，7.27～7.23(t，1H)，7.20～7.17(t，2H)，6.95～6.91(t，1H)，6.89～6.87(d，1H)。Ms(m/e)：261[M⁺，100]，232，204.0，117

Example 4: (Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (2 ' -pyridylmethenyl) -2-indolone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 2-pyridinecarboxaldehyde (from ACROS) to give the title compound as a red solid in 83.7% yield.

¹H-NMR：δppm(DMSO-d₆)：10.31(s，1H)，9.11(d，1H)，8.92(d，1H)，8.00～7.97(m，1H)，7.69(s，1H)，7.52～7.50(m，1H)，7.43～7.40(m，1H)，7.32～7.30(d，1H)，7.13～7.10(t，1H)，4.28～4.23(t，2H)，3.62～3.60(d，2H)，3.33～3.30(q，2H)，3.00～2.94(q，2H)，1.83～1.70(m，5H)，1.39～1.36(m，1H)。Ms(m/e)：333.2[M]，2307.1，290.2，192.1，98.1(100)，71.1。

Example 5: (Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (5 ' -acetoxymethyl-2-furanmethylene) -2-indolone

The reaction was carried out as described in example 1, starting with equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 5-acetoxymethyl-2-furaldehyde (from ACROS) to give the title compound as a red solid in 94% yield.

¹H-NMR：δppm(DMSO-d₆)：7.74(d，1H)，7.56(s，1H)，7.50～7.48(m，2H)，7.29～7.26(m，1H)，7.06～7.04(m，2H)，4.34(s，2H)，4.30～4.28(t，2H)，3.51～3.50(d，2H)，3.30～3.29(q，2H)，3.00～2.93(q，3H)，2.40(s，3H)，1.80～1.68(m，5H)，1.350～1.32(m，1H)。Ms(m/e)：394[M]，283，98.1(100)。

Example 6: (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (3 '-methyl-2' -furanmethylene) -2-indolone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 3-methyl-2-furaldehyde (from ACROS) except that the reaction temperature was 14 ℃ and the reaction time was 48 hours to give the crude title compound. Recrystallization from ethanol gave a pure product as a yellow solid in 88.7% yield.

¹H-NMR：δppm(DMSO-d₆)：7.82(d，1H)，7.6(s，1H)，7.50～7.48(m，2H)，7.29～7.26(m，1H)，7.06～7.04(m，2H)，4.34(s，2H)，3.51～3.50(d，2H)，3.30～3.29(q，2H)，3.23(s，3H)，3.00～2.93(q，2H)，2.40(s，3H)，1.80～1.68(m，5H)，1.350～1.32(m，1H).Ms(m/s)：336.2(M)，111.1，98(100)。

Example 7: (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (3 '-methyl-2' -thiophenemethenyl) -2-indolone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 3-methyl-2-thiophenecarboxaldehyde (from ACROS), except that the reaction temperature was 16 ℃ and the reaction time was 46 hours, to give the title compound as a yellow solid in 93.4% yield.

¹H-NMR：δppm(DMSO-d₆)：7.74(d，1H)，7.56(s，1H)，7.50～7.48(m，2H)，7.29～7.26(m，1H)，7.06～7.04(m，2H)，4.34(s，2H)，4.30～4.28(t，2H)，3.51～3.50(d，2H)，3.30～3.29(q，2H)，2.40(s，3H)，1.80～1.68(m，5H)，1.350～1.32(m，1H)。MS(m/e)：352.1[M]，241.1，98(100)，77.0。

Example 8: (Z) -1- [2 '- (1' -morpholine) ] ethyl-3- (4 '-bromo-2' -thienylmethylenyl) -2-indolone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -morpholine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 4-bromo-2-thiophenecarboxaldehyde (from ACROS), except that the reaction temperature was 25 ℃ and the reaction time was 35 hours, to give the title compound as a yellow solid in 93.8% yield.

¹H-NMR：δppm(DMSO-d₆)：8.09(s，1H)，7.99(d，1H)，7.98～7.97(d，1H)，7.71～7.70(d，1H)，7.32～7.29(m，1H)，7.09～7.06(m，2H)，3.90～3.88.(t，2H)，3.52～3.50(t，4H)，2.55～2.51(s，4H)，2.50～2.49(m，2H)。MS(m/e)：420(M+2)，418[M]，256，100(100)。

Example 9: (Z) -1- [2 ' - (1 ' -morpholine) ] ethyl-3- (3 ', 5 ' -dimethyl-2 ' -pyrrolemethenyl) -2-indolone

The reaction was carried out as described in example 1, starting from equimolar amounts of 1- [2 '- (1' -morpholine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 3, 5-dimethyl-2-pyrrolecarboxaldehyde (prepared according to DeGroot JA, org. prep. precedures int.1981, 13 (2): 97-101 reference) to give the title compound as a yellow solid in 80.9% yield.

¹H-NMR：δppm(CDCl₃)：13.18(s，1H)，7.49～7.47(d，1H)，7.37(s，1H)，7.25(s，1H)，7.18～7.15(t，1H)，7.06～7.03(t，1H)，6.95～6.94(d，1H)，4.10(t，2H)，4.03～4.01(t，4H)，2.64(t，3H)，2.54(m，4H)，2.38(s，3H)，1.45(m，2H)。MS(m/e)：351[M]，238，98.1(100).

Example 10: (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (5 '-bromo-2' -thiophenemethenyl) -2-indolone

The reaction was carried out as described in example 1 starting from equimolar amounts of 1- [2 '- (1' -piperidine) ] ethyl-2-indolone (cf. Mokrosz, M.J., Pharmazie [ PHARAT ]1997, 52(6), 423-428 reference) and 3-bromo-2-thiophenecarboxaldehyde (from ACROS) to give the title compound as a yellow solid in 80.9% yield.

¹H-NMR：δppm(DMSO-d₆)：

8.08(s，1H)，7.99(d，1H)，7.98～7.97(d，1H)，7.71～7.70(d，1H)，7.32～7.29(m，1H)，7.09～7.06(m，2H)，3.90～3.88.(t，2H)，3.52～3.50(t，4H)，2.55～2.51(s，4H)，2.50～2.49(m，2H)。MS(m/e)：420(M+2)，418(M)，256，100(100)。

Example 11: indolone compounds inhibit endothelial cell (ECV-304) proliferation and selectively inhibit bFGF and VEGF.

Endothelial cells (ECV-304) were purchased from the cell bank of the culture Collection of the national academy of sciences. Inoculating cells in logarithmic growth phase into 96-well plates at 3-4X 10/well³After 24h for each cell/100. mu.l, the culture medium was aspirated and 100. mu.l of complete medium containing different concentrations of drug was added to each well (in the growth factor selective inhibition assay, the complete medium was replaced with culture medium supplemented with 10ng/ml bFGF or VEGF, respectively, in 1% fetal bovine serum culture medium). Continuing to culture for 72h, discarding the culture solution, adding 100 μ l of 0.5mg/ml tetramethylazodicarbonamide (MTT) into each well, incubating at 37 deg.C for 4h, absorbing the tetramethylazodicarbonamide solution, adding 100 μ l dimethyl sulfoxide (DMSO) into each well, shaking gently for 10min, and measuring optical density OD (optical density) of each well of 96-well plate at 490nm with BIORAD 550 type plate reader, wherein each compound is 10%^-6-10^-4The concentration is set to 5-6, each test is provided with 3-4 parallel holes, and the test is repeated for 3-4 times.

The cell growth inhibition (%) of the drug was determined by determining the ratio of the drug to the cell growth (%) (mean OD value in the solvent control group-mean OD value in the drug application group)/mean OD value in the control group, and calculating the IC of the drug based on the cell growth inhibition (%) of the drug at different drug concentrations₅₀The value is obtained.

TABLE 1 indolone compounds inhibit ECV-304 cell proliferation

Test result of tetramethyl azodicarbonamide blue colorimetric method

Note: cell concentration (4X 10)³One/hole), I.R (3 × 10)^-5): the medicine is at 3X 10^-5The inhibition rate of Mol is the most sensitive to the inhibition effect of the drug, so that the inhibition effects of different compounds are convenient to compare.

As can be seen from the graph, the tested compounds have different degrees of inhibition effects on the proliferation of ECV-304 under the culture condition of 1% NBS + 1640; have different selective inhibitory effects on VEGF and bFGF. Wherein, the selectivity of the compounds 1 and 2 on bFGF induced proliferation is stronger than that of VEGF, the selectivity of the compounds 3, 4 and 8 on VEGF is stronger than that of bFGF, and the selective inhibition effect of the compound 5 on two growth factors is equivalent.

Example 12: evaluation of inhibition effect of indolone compounds on HUVEC proliferation of umbilical vein endothelial cells by brominated thymine (Brdu) incorporation method

HUVEC cell lines were purchased from Cascade Biologics, USA, and the culture medium supplemented with LSGS supplement for HUVEC specific medium 200. The assay was performed according to the method of BrdU (colorimetric) Kit, Cell promotion ELISA, Roche. Briefly, log-phase grown HUVECs were seeded in 96-well plates at 4X 10 per well³Removing culture solution after 24h after each cell is per 100 mul, adding 100 mul culture solution containing drugs with different concentrations into each hole, continuing to culture for 72h, adding 10 mul (100 mul) BrdU doping solution into each hole, continuing to culture for 18h, removing culture solution, adding 200 mul fixed denaturant into each hole, processing for 40min, and sucking dry the fixed denaturant at room temperature for 40 min; adding 10 μ l of anti-BrdU-POD antibody working solution into each well, incubating for 90min at room temperature, washing the plate for 3 times by using 200-; each hole100ul of substrate reaction solution is added, color development is carried out for 10min at room temperature, and 25 mul of 1mol/L sulfuric acid is added into each hole to terminate the reaction. The OD value of optical density of each well of a 96-well plate was measured at a wavelength of 450nm (690 reference wavelength) using a BIORAD 550 type plate reader, each compound was 10 nm^-6-10^-44-5 concentrations are set in the middle, 3-4 parallel wells are set for each test, and the test is repeated for 2-3 times.

TABLE 2 evaluation of indolone Compounds by brominated Thymidine (Brdu) incorporation

Test results on inhibition of HUVEC proliferation of umbilical vein endothelial cells

Example Compound IC₅₀(X±SD)(μmol/L)

1 11.2±1.9

3 4.8±0.5

4 19.4±1.87

As can be seen from the data in the graph, the three compounds have different degrees of inhibition effects on the DNA synthesis of HUVEC, and the strong and weak sequences of the inhibition effects are 3 & gt 1 & gt 4.

Example 13: apoptosis research of (Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (4 '-bromo-2' -thiophenemethenyl) -2-indolone compound induced tumor cells U937

Pancreatin digestion of adherent tumor cells U937, centrifugation, gentle washing of the cells with PB, and collection of cells 10⁵More than one cell was fixed with 75% ice cold ethanol, DNA stained with PI, and DNA content distribution was detected on flow cytometry. (Z) -1- [2 '- (1' -piperidine)]The ethyl-3- (4 '-bromo-2' -thiophenemethenyl) -2-indolone compound can obviously induce tumors when the concentration is 50 mu M and the treatment time is 72hCell U937 apoptosis, the distribution of DNA content detected on the flow cytometer shows that the drug causes significant apoptosis rather than necrosis.

Claims

1. Compounds of general formula I, their geometric isomers and their pharmaceutically acceptable salts,

wherein:

n is equal to 2 or 3 and,

R₁is a heteroaryl selected from furan, thiophene, pyrrole, pyrazole, imidazole, thiazole, oxazole, isoxazole, pyridine, pyridazine, pyrimidine, pyrazine, indole, benzofuran or quinoline; the heteroaromatic compoundThe group may be unsubstituted or substituted with 1 or 2 substituents selected from: halogen, nitro, hydroxy, hydroxymethyl, trifluoromethyl, trifluoromethoxy, C₁～C₆Straight or branched alkyl, C₂～C₆Straight-chain or branched alkenyl radical, C₁～C₄Alkoxy radical, C₁～C₄Alkoxyacyl group, C₂～C₄Alkenyloxy, phenoxy, benzyloxy, carboxyl or amino,

R₂selected from hydrogen, halogen, cyano, sulfoxide, sulfone, nitro, carboxyl, C₁～C₃Alkoxy radical, C₁～C₃Alkanoyl radical, C₁～C₃Alkyl ester group, C₁～C₃An alkylamido group,

R₃、R₄is independently selected from C₁～C₆Straight or branched alkyl, C₂～C₇Straight or branched alkenyl; or R₃And R₄And the nitrogen atoms to which they are attached form a 4-to 6-membered heterocyclic ring selected from tetrahydropyrrole, piperidine, morpholine, piperazine or azomethylpiperazine.

2. A compound according to claim 1, wherein said compound is,

wherein:

n is equal to 2;

R₁is a heteroaryl selected from furan, thiophene, pyrrole and pyridine; the heteroaryl group may be unsubstituted or substituted with 1 or 2 substituents selected from: halogen, C₁～C₆Straight or branched alkyl, C₁～C₄An alkoxyacyl group;

R₂selected from hydrogen, halogen;

R₃and R₄And the nitrogen atoms to which they are attached form a 4-6 membered heterocyclic ring selected from piperidine or morpholine.

3. The compound of claim 1, comprising:

(Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (3 ', 5 ' -dimethyl-2 ' -pyrromethenyl) -2-indolinone,

(Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (2 ' -pyrromethenyl) -2-indolinone,

(Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (4 '-bromo-2' -thienylmethylenyl) -2-indolone,

(Z) -1- [2 ' - (1 ' -piperidine) ] ethyl-3- (2 ' -pyridylmethenyl) -2-indolinone,

(Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (5 '-acetoxymethyl-2' -furanmethylene) -2-indolone,

(Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (3 '-methyl-2' -furanmethylene) -2-indolone,

(Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (3 '-methyl-2' -thienylmethylenyl) -2-indolone,

(Z) -1- [2 '- (1' -morpholine) ] ethyl-3- (4 '-bromo-2' -thienylmethylenyl) -2-indolone,

(Z) -1- [2 ' - (1 ' -morpholine) ] ethyl-3- (3 ', 5 ' -dimethyl-2 ' -pyrrolemethylenyl) -2-indolone,

(Z) -1- [2 '- (1' -piperidine) ] ethyl-3- (5 '-bromo-2' -thiophenemethenyl) -2-indolone.

4. A pharmaceutical composition comprising a compound of any one of claims 1-3, a geometric isomer thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier.

5. A process for the preparation of a compound according to any one of claims 1 to 3, which process comprises reacting an indolone derivative of formula II

Wherein R is₂As defined for the compounds of formula (I) according to claim 1,

with an aminohalohydrocarbon derivative of formula III,

wherein n and R₃And R₄As defined for the compounds of formula (I) according to claim 1, X is Cl or Br,

to obtain the compound shown in the formula IV,

wherein n and R₂、R₃And R₄As defined for the compounds of formula (I) according to claim 1,

reacting a compound of formula IV with a heteroaryl carboxaldehyde V,

wherein R is₁As defined for the compounds of formula (I) according to claim 1,

to obtain the compound of the general formula I,

wherein n and R₁、R₂、R₃And R₄Are as defined in claim 1 for compounds of formula I;

or,

reacting an indolone derivative of formula II

Wherein R is₂The compounds of formula I as defined in claim 1.

With a heteroarylformaldehyde of the formula V,

wherein R is₁The compounds of formula I as defined in claim 1,

to obtain the compound of the formula VI,

wherein R is₁And R₂The compounds of formula I as defined in claim 1,

reacting a compound of formula VI with an aminohalohydrocarbon derivative III

Wherein n and R₃And R₄Is as defined in claim 1, X is Cl or Br, to give a compound of the general formula I,

wherein n and R₁、R₂、R₃And R₄The compounds of formula I as defined in claim 1.

6. Use of a compound according to any one of claims 1 to 3 for the preparation of an antitumor medicament.

7. The use of claim 6, wherein the anti-tumor effect comprises inducing apoptosis in tumor cells, anti-tumor neovascularization, preventing resistance to tumor chemotherapy, and preventing malignant metastasis of tumors.