WO2013060179A1 - Acyltetrahydro-β-carboline compounds as well as derivatives, applications and preparation methods thereof - Google Patents

Abstract

Disclosed are acyltetrahydro-β-carboline small molecule organic compounds represented by structural formula (I) or hydrates or pharmaceutically acceptable salts thereof, and use of the present compounds or pharmaceutical compositions thereof in preparing medicaments for treating various diseases such as malignant tumor, tumor metastasis and the like, and the preparation methods for the acyl-tetrahydro-β-carboline compounds of the present invention and for the derivatives thereof.

Description

―

 Acyl tetrahydro-β-carboline compound and its derivative, use thereof and preparation method thereof

 The present invention relates to an acyltetrahydro-β-carboline small molecule organic compound and derivative, and the use of the same or a pharmaceutical composition containing the same for treating various malignant tumors and related diseases such as tumor metastasis . The present invention also relates to a process for producing the acyltetrahydro-β-carboline compound and derivatives thereof.

Background technique

 Mal ignant neoplasm, a cancer, is a kind of abnormal proliferation of cells, which can spread and metastasize, destroy the structure and function of tissues and organs, cause necrotic hemorrhage and infection, and finally cause the patient to function due to organs. A type of clinical disease that is dying of death. In the past 20 years, China's cancer mortality rate has increased by 29. 4%, and cancer deaths account for 24% of the total deaths of urban and rural residents. There are 1 cancer patient per 4 to 5 deaths in China. The total population of cancer deaths in China is close to 2 million per year. Malignant tumors have become the leading cause of death for urban and rural residents in China. The annual economic losses caused by cancer to China exceed 100 billion yuan. Drug therapy is one of the main methods of cancer treatment. By curbing the rapid growth of tumors and damaging their metastasis pathways, it is a more effective method to improve the survival rate of cancer patients and relieve the pain of patients.

 The occurrence and development of malignant tumors is very complex and is a gradual process involving the multistage reaction of cells and the accumulation of mutations. During this process, cancerous cells are increasingly uncontrolled by in vivo regulatory mechanisms, proliferate rapidly, and gradually erode into normal tissues. Scientific research has shown that excessive activation of some signaling pathways in the body plays an important role in this process. For example, researchers have found high expression of epidermal growth factor (EPF) in many malignant tumors (such as lung cancer, skin cancer, breast cancer, cervical cancer, bladder cancer, ovarian cancer, etc.), which leads to their Increased phosphorylation of EGFR (epidermal growth factor receptor) directly activates several receptor-related signaling pathways that promote tumorigenesis, including: Ras-Raf-MAPK (mitogen-activated protein kinase), PI3K- Akt-GSK (glycogen synthase kinase) and STAT3 (signal transduction and activator of transcription 3), these signaling pathways, in addition to enabling tumor cells to maintain strong proliferation and division, can also enhance tumor cell metastatic ability

(FEBS J. 2010 277: 316-26). Therefore, drugs targeting the above signaling pathways are effective ways to treat malignant tumors. At present, small molecular drugs targeting some of the above signaling pathways have been developed, such as small molecule tyrosine kinase inhibitors such as Erlotinib ( Tarceva), Gefitinib (Iressa), Lapati Ni

(Lapatinib, Tyverb) inhibits the excessive growth of tumors by inhibiting the activation of EGFR. However, its clinical application has been limited by its significant acquired resistance (Cel l. 2011 144: 646-74). In addition to the above-mentioned EGFR-related signaling pathways, Notch, Hedgehog, p53, WT, NF_kB Equal signaling pathways have been reported to be involved in the development of tumors (Cel. 2011 144: 646-74; J. Natl. Cancer Inst. 2008, 100, 692-7). Tumor metastasis refers to the process in which tumor cells migrate from in situ tumor foci to other parts of the body and grows unregulated. It is the main cause of death in clinical tumor patients. According to statistics, clinically, more than 90% of cancer patients are caused by cancer. ―

Caused by metastasis (Eur. J. Cancer 2010, 46, 1177-1180). Epithelial-mesenchymal cell transformation (EMT) is the most prominent feature of tumor cell metastasis during tumor metastasis. Since most tumors in the body occur in the epithelial cell layer, epithelial cells are usually non-migrating cells, but during the development of tumors, the genetic characteristics of some epithelial cells change, that is, some epithelial tumor cells begin. It expresses some proteins involved in cell migration and inhibits the expression of proteins that can aggregate cells together, thus making this epithelial tumor cell a more migrating and infiltrating mesenchymal cell. These mesenchymal cells can migrate to other suitable sites in the body to continue to grow, forming new tumor foci. The process of transforming epithelial cells into mesenchymal cells is called epithelial-mesenchymal transition (EMT). There are two pathways for tumor cell proliferation: local infiltration pathway and circulatory system metastasis pathway. The local invasion pathway is the infiltration of tumor cells along the interstitial space, lymphatic vessels, blood vessels or nerve bundles, destruction of adjacent normal tissues, organs, and continued growth. For example, advanced lung cancer can spread to the chest and even the spine. Advanced breast cancer can pass through the chest and chest and eventually reach the lungs. The circulatory system is the tumor cells that invade the lymphatic vessels and blood vessels from the primary site, and continue to grow through the circulatory system, forming the same type of tumor as the primary tumor. Studies have found that multiple signaling pathways play a crucial role in tumor cell EMT and metastasis. Transforming growth factor beta (TGFp)-related signaling pathway is one of the important ones. The TGFP signaling pathway can promote EMT, which can significantly enhance the migration and infiltration ability of tumor cells, and provide oxygen by means of the new microvascular system. Nutrients, such as nutrients, further proliferate, grow, and invade the circulatory system such as lymphatic vessels and blood vessels; at the same time, under the control of the TGFP signal transduction pathway, these tumor cells express a large number of proteins suitable for survival in a new environment, thereby allowing tumor cells to adhere to Proliferation and growth continue on new target organs, and then metastases are formed on these organs, resulting in the development of new tumors, ie, tumor metastasis (Nat. Rev. Cancer 2003, 3, 453-458). Many anti-tumor drugs that inhibit the TGFp signaling pathway, such as inhibitors against TGFpR: Lucanix®, LY2157299, SB431542, etc., are still in the experimental development stage. How to inhibit TGFP-induced tumor cell EMT processes and consequent tumor metastasis is currently the focus of drug research. In addition, signaling pathways such as Ras and PI3K-Akt are also important regulatory signaling pathways in tumor metastasis (Cel l Res. 2009 19: 89-102).

Since the formation of malignant tumors is a multi-step, multi-molecular pathological interaction, the treatment of a single target gene may not be the most appropriate strategy in theory, and it is difficult to expect an ideal therapeutic effect. Therefore, finding a multi-target anti-tumor drug has become an urgent need for the development of oncology drugs. Researchers such as Bhabatosh Chaudhuri have reported that some benzoyltetrahydro-β-carbolines are inhibitors of cell cycle-dependent kinase-4 (CDK4) and have potential anti-tumor effects (Org. Biomol Chem. 2006, 4 , 787-801; WO2009/022104 Al ), the structural formula of the compound is as follows: -

Such active compounds exist in the form of a biphenyl ring structure, and the presence of this structural moiety is necessary for the enzyme inhibitory activity of such compounds to be maintained or increased (WO 2009/022104 Al ). CDK4 inhibitors are not involved in the EGFR signaling pathway associated with tumor growth or the TGFP signaling pathway associated with tumor metastasis.

 The novel structure of the acyltetrahydro-β-carboline compound and the derivative thereof provided by the invention are intensively studied and found to be useful for various tumor cells (including lung cancer, breast cancer, liver cancer, colon cancer, etc.). Both proliferation and migration have a very strong inhibitory effect, and they also show significant inhibition of tumor growth in various growth models in mice. In addition, using a variety of mouse tumor metastasis models, it is proved that these compounds are also excellent. The anti-tumor effect, but the inhibition of normal cell proliferation is very weak, and the inhibitory effect on CDK4 is also poor, that is, non-CDK4 inhibitor.

Summary of the invention

 At present, drug therapy is one of the main methods for treating tumors. It is a more effective method to curb the rapid growth of tumor cells and destroy the metastasis pathway. It is a clinically effective way to improve the survival rate of cancer patients and alleviate the suffering of patients. At present, the World Health Organization (WHO) has published 49 commonly used anti-tumor drugs. However, compared with the increasing number of tumor types and the number of patients, the development of oncology drugs is far from enough, and still faces enormous challenges. The shortage of oncology drugs mainly includes: First, strong toxic and side effects, affecting the efficacy of tumors; Second, the target is not effective enough, resulting in unsatisfactory drug efficacy; Third, the drug treatment of tumor metastasis is not good, etc. . Therefore, finding a drug with low toxicity, clear target and high efficiency against tumor growth and metastasis is an important direction for the development of anticancer drugs. The present invention satisfies this requirement and provides additional related advantages, the innovations being mainly embodied in the following aspects:

 The invention provides a novel structure of acyltetrahydro-β-carboline compounds and derivatives thereof, including pharmaceutically acceptable salts thereof, solvent compounds (such as hydrates) and esters, etc., which can be used as an anti-tumor drug lead. Compounds and clinical candidate compounds.

The compounds of the present invention specifically inhibit tumor growth in vitro and in vivo, while inhibiting normal cell proliferation is weak. In the in vitro tumor cell proliferation and colony formation model, the compound of the present invention has a significant inhibitory effect on the proliferation of several tumor cells, and has almost no toxicity to normal cells of the corresponding organs; it has a strong inhibitory effect on the colony formation of tumor cells. . In a plurality of disease animal models, the compound of the present invention has a very significant inhibitory effect on tumor growth, and has no obvious toxic side effects on normal mice. ―

 The compounds of the invention are effective in inhibiting tumor metastasis both in vitro and in vivo. In the in vitro tumor cell migration and infiltration model, the migration and infiltration of several tumor cells were significantly inhibited, and the epithelial-mesenchymal transition (EMT) process of tumor cells was significantly inhibited. In the animal model of tumor metastasis, the compound of the present invention has a strong inhibitory effect on tumor metastasis and can be used as a lead compound and a clinical candidate compound for anti-tumor metastasis.

 The compound of the present invention has a significant inhibitory effect on tumor-induced osteolytic bone damage and pulmonary failure caused by tumor lung metastasis in vivo and in vitro.

 The compounds of the invention have a well-defined target of action, and this target plays a key regulatory role in the signaling pathways of tumor growth and metastasis.

 The invention designs and synthesizes a novel structure of acyltetrahydro-β-carboline compounds and derivatives thereof, and designs and synthesizes by changing the substitution of different functional groups, different connection modes and positions, and adjusting the size of the ring system. A series of novel compounds with an acyltetrahydro-β-carboline structure or a derivative thereof as a structural skeleton, and related pharmacodynamic experiments show that such compounds have excellent anti-tumor growth and metastasis activities in vitro and in vivo.

 A first object of the present invention is to provide an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof having a structure represented by the formula (I):

 among them:

n, m is 0-3 CH ₂ ;

@为含杂或当量 heteroaryl, including monocyclic aryl, polycyclic aryl, polyheterocyclic aryl; leg monocyclic aryl including phenyl, azaaryl, thiaaryl, oxaaryl ;

! ^ is a substituent on the aromatic ring, including monosubstituted and polysubstituted, independently selected from one or more of the following groups: hydrogen, amino, cyano, hydroxy, nitro, octyl, carboxy, alkyl, alkane Oxyl, amine, cycloalkoxy, cycloamine, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₁₂ cycloalkyl, benzyl, alkylcarbonyl, C ₂ - C ₁₂ alkenylcarbonyl, C ₃ -C ₁₂ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, sulfenamide, sulfonyl; morpholinyl Piperazinyl;

R _{2 is} optionally selected from one of the following groups: hydrogen, alkyl, halogen, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aromatic Base, benzyl, phenethyl, phenylpropyl, alkoxy, alkylamino, alkylcarbonyl and arylcarbonyl One. - .

Base, morpholine methyl, hydroxymethyl;

The R ₃ substituent is optionally selected from one or more of the following groups: hydrogen, alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, Heterocyclic aromatic group, benzyl group, phenethyl group, phenylpropyl group, amino group, cyano group, nitro group, halogen;

R ₄ , R ₅ , R, and R ₇ are substituents on the phenyl ring, each independently selected from one of the following groups: hydrogen, amino, cyano, hydroxy, nitro, 3⁄4, carboxy, alkyl, Alkoxy, substituted amine, cycloalkoxy, cycloalkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aromatic Base, benzyl, alkylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, Sulfonamide, sulfonamide; morpholinyl; piperazinyl;

R _{8 is} optionally selected from one or both of the following groups: hydrogen, alkyl, hydroxy, hydroxymethyl, amino, carboxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ -cycloalkyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkoxy, alkylamino, alkylcarbonyl, arylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C _3- C ₈ cycloalkylcarbonyl;

The substituent is optionally selected from one of the following groups: hydrogen, alkyl, hydroxymethyl, alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkenyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkylamino.

 In the structural formula (I), when n is 1

In the structural formula (I), when n is 1

The present invention also provides a small molecule organic compound of any of the aforementioned acyltetrahydro-β porphyrin compounds or derivatives thereof, hydration ―

Or a pharmaceutically acceptable salt, including but not limited to an acid addition salt formed with the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid, methanesulfonic acid, p- Toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid, succinic acid, and the like.

 The acyltetrahydro-β-carboline small molecule of the present invention has a compound including, but not limited to, a radioactive, fluorescent group or biotin (Biotin).

 In the present invention, the acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof includes, but is not limited to, the following compounds:

 Ν-(2-Thienylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(3,4-dihydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-Pyridinylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-chlorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-aminophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-Ν,Ν-diethylphenylacetyl)- 1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 Ν-(4-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(1Η-tetrazolylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(4-(2-NHBoc-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-bromomethylphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-(2-hydroxyethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-indole-β-carboline,

_Ν- (4-(3-morpholinopropoxy)-phenylacetyl)-1,3,4,9-tetrahydro-indole-β-carboline,

 Ν-(4-carboxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-(2-Aminoethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-Phenylpropanoyl-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-((3-NHBoc-3-phenyl)propanoyl)-2,3,4,9-tetrahydro-1 3-porphyrin,

 N-(2-(2-(S)-NHBoc-phenyl)acetyl)-1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 N-(2-(2-(R)-Aminophenyl)acetyl)-1,3,4,9-tetrahydro-indole-β-carboline,

 Ν-Οamino-3-phenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(2-(S)-Aminophenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-aldehydephenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

Ν-(2-(4-bromomethylphenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline, ―

 N-(2-(4-hydroxymethylphenyl)propanoyl)-1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 Ν-phenylacetyl-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-hydroxy-2-phenylacetyl)-1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 Ν-(2-hydroxy-2,2-diphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-(morpholinylmethylphenyl)-propionyl))-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-((2-(4-(lH- 1 ,2,4-triazol - 1 -yl)methyl)phenyl)propanoyl)-1 ,3 ,4,9-tetrahydro - 1Η-β -porphyrin,

 1-phenyl-indole-phenylacetyl-1,3,4,9-tetrahydro-indole-β-carboline,

 1-ethyl-indole-phenylacetyl-1,3,4,9-tetrahydro-indole-β-carboline,

 2-(4-(2-NH-Biotin-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-((2-morpholinoethylamino)methyl)phenyl))-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-phenylacetyl-1-ethyl-1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline,

 Ν-phenylacetyl small methyl -1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline,

 Ν-phenylacetyl-1-1-methyl-1,3,4,9-tetrahydro-6-hydroxy-1 Η-β-carboline,

 Ν-phenylacetyl-6-fluoro-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-Phenylacetyl-6-bromo-1,3,4,9-tetrahydro-1Η-β-carboline.

 The present invention provides a pharmaceutical composition comprising the acyltetrahydro-β-carboline small molecule organic compound, hydrate or pharmaceutically acceptable salt of the present invention, and a pharmaceutically acceptable carrier.

 Wherein, in a particular embodiment, the composition is formulated as an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, a transdermal patch or an excipient.

 The present invention provides an application of an acyltetrahydro-β-carboline small molecule organic compound, hydrate or pharmaceutically acceptable salt for inhibiting tumor cell proliferation, growth, migration and infiltration. The present invention also provides a pharmaceutical composition prepared by the above compound for inhibiting proliferation, growth, infiltration and migration of tumor cells and a pharmaceutical use thereof, wherein the pharmaceutical composition contains the above-mentioned small molecule organic compound, hydrate or pharmaceutically acceptable Salt, and a pharmaceutically acceptable carrier.

 In one embodiment, the tumor cells include, but are not limited to, lung cancer cells, breast cancer cells, epidermal cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, prostate cancer cells, and the like.

 The invention also provides the use of an acyltetrahydro-β-carboline small molecule organic compound for preparing a medicament for treating malignant tumor; wherein the malignant tumor includes but not limited to liver cancer, lung cancer, prostate cancer, skin cancer, colon Cancer, pancreatic cancer, breast cancer, leukemia, ovarian cancer, stomach cancer, bladder cancer, kidney cancer, skin cancer, oral cancer.

The present invention also provides an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof - .

The invention relates to the preparation of a medicament for treating metastasis and recurrence of malignant tumors; wherein the malignant tumors include, but are not limited to, liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, leukemia, ovarian cancer, gastric cancer, Bladder cancer, kidney cancer, skin cancer, oral cancer.

 The present invention also provides an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof for use in the preparation of a medicament for treating a disease which is responsive to EGF receptor-regulated tumorigenesis, progression and metastasis Applications.

 The present invention also provides an application of an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating a disease responsive to TGFP receptor regulation; Such diseases include, but are not limited to, tumors, diabetes, glomerulonephritis, glomerular sclerosis, various fibrotic diseases, osteoarthritis, osteoporosis.

 The present invention also provides an application of an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or a pharmaceutically acceptable salt thereof for preparing an antitumor therapeutic drug, which is used for inducing acquired drug resistance Anti-tumor treatment after chemotherapy failure.

 The present invention also provides an application of an acyltetrahydro-β-carboline small molecule organic compound or a hydrate thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating osteolytic bone damage.

 The present invention also provides the use of an acyltetrahydro-β-carboline small molecule organic compound or a hydrate thereof or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating tumor-induced pulmonary failure.

 The medicament of the present invention is used alone or in combination with other drugs.

 Another object of the present invention is to provide a process for producing an acyltetrahydro-β-carboline small molecule organic compound represented by the formula (I) or a hydrate or pharmaceutically acceptable salt thereof. The method comprises the step of coupling a tetrahydro-β-carboline compound or a derivative thereof with a acid, an acid anhydride or an acid halide in the presence of a coupling reagent or a base to obtain the acyl tetrahydrogen. -β-porphyrin small molecule organic compound. Alternatively, the method may further derivatize a derivative of the acyltetrahydro-β-carboline small molecule organic compound by a palladium-catalyzed coupling method using a halogenated acyltetrahydro-β-carboline as a raw material. In one embodiment, the preparation method of the present invention includes the following:

In the first method, a substituted tetrahydro-β-carboline compound of various origins or a derivative thereof 1 and a different acid, acid anhydride or acid halide are reacted in the presence of a coupling reagent or a base to obtain a product 2, and a base is added. The product is obtained by alkylating a nitrogen atom on the product 2 oxime with a halogenated product. Wherein the base comprises triethylamine, potassium carbonate, pyridine, DMAP.

In the second method, the Pictet-Spengler cyclization reaction is carried out by using substituted tryptamines or derivatives thereof 4 and different substituted aldehydes from various sources, and the established methods are used by reference (for example, Organic Letters (2002) 4: 4033- 4036) The reaction is carried out to give the product 5, which is then reacted by a coupling method to give the products 6 and 7.

 In the third method, a heterocyclic-substituted compound 10 is prepared by a coupling method by taking aromatic ring oxime 8 from various sources as a raw material, by reacting with a protected amino aldehyde to form a compound 9.

Method four

 In the fourth method, the substituted aniline of various sources is diazotized under the action of an acid to form a diazonium salt, which is coupled with the substituted 2-piperidone-3-carboxylate to form the intermediate 12, and then acts on the acid. The lower ring produces compound 13 (Reference Syn. Comm. 2007, 37, 1273-1280), followed by reduction, coupling reaction to give product 14.

Method five

In the fifth method, an appropriately substituted bromo-substituted-tetrahydro-β-carboline or a derivative thereof is converted into an aromatic or heteroaryl-substituted compound 15 by a coupling method (palladium-catalyzed coupling). ―

 Compared with the existing acyltetrahydro-β-carboline compounds of the phenylene ring structure, the compounds of the present invention (formula I) have significant differences. First, there are significant differences in structure. The active compound in the patent has a phenyl ring structure, and the presence of this structural module is necessary for the enzyme inhibitory activity of such compounds to be maintained or increased.

(WO2009/022104 Al). However, the compound of the present invention does not have this structural module, and the inhibitory effect on CDK4 is also very weak, and the non-CDK4 inhibitor; the compound of the present invention contains a chain side chain substituent, and the experimental results show that the compound of the present invention is very It specifically inhibits tumor cell proliferation and metastasis, but has a poor inhibitory effect on normal cell proliferation. Secondly, the mechanism of action of the two molecules is different. The inhibition of tumor growth by the compounds of the present invention mainly involves the EGFR signaling pathway, and the inhibition of tumor metastasis mainly involves the TGFP signaling pathway, while the compound reported in the literature is a CDK4 inhibitor.

 The cell test proves that the compound of the present invention has a significant inhibitory effect on the proliferation and colony forming ability of various tumor cells, and has a significant inhibitory effect on the cell colony forming ability of human lung cancer, breast cancer and colon cancer, and has no obvious effect on normal cell proliferation. Inhibition, and excellent selectivity for tumor cells.

 The cell test proves that the compound of the present invention strongly inhibits the migration and infiltration of tumor cells, and has a strong inhibitory effect on the migration and infiltration ability of lung cancer cells, breast cancer cells, colon cancer cells, etc., but has no obvious effect on normal cell migration. Inhibition.

 Cellular assays have demonstrated that the compounds of the invention are effective in inhibiting the epithelial-mesenchymal transition (ΕΜΤ) process of tumors.

 In vivo experiments in animals have shown that the compounds of the present invention have a very strong inhibitory effect on the growth of tumors in vivo compared with the control, and are dose-dependent within a certain range, and have no obvious side effects on the test animals, and have excellent toxicity. Rational effect.

 In vivo experiments in animals have demonstrated that the compounds of the present invention have a definite therapeutic effect on tumor metastasis in vivo, among which lung metastasis disorders of tumors can be significantly treated. It also has the ability to strongly inhibit the metastasis of breast cancer and other tumors to other organs.

 The compounds of the invention significantly increase the survival rate of breast cancer, lung cancer, colon cancer and other animal models of tumor metastasis. The compound of the present invention has a significant inhibitory effect on bone metastasis of breast cancer in animals and osteolytic bone damage caused thereby. The compound of the present invention can inhibit the activation of the TGFP signaling pathway and the downstream gene expression regulated by TGFP, and fully demonstrates that the therapeutic mechanism is significantly different from the existing phenylacyltetrahydro-β-carboline compounds having a biphenyl ring structure.

 The compound of the present invention can inhibit the EGF receptor signaling pathway, and fully demonstrates that the therapeutic mechanism is significantly different from the existing phenylacyltetrahydro-β-carboline compounds.

 The preparation process of the compound of the invention is relatively simple and easy to produce.

DRAWINGS

Fig. 1 is a graph showing the results of inhibition (%) of the proliferation of human lung cancer cells H1299 at 10.0 μηιοΙ/L of the compound of the present invention, and the dotted line indicates the 50% inhibition rate. ―

 Figure 2 shows the inhibition rate of the compound of the present invention on the proliferation of human breast cancer cell MDA-MB-231 at 10.0 μηιοΙ/L.

(%) results plot, dashed line indicates 50% inhibition.

 Fig. 3 is a graph showing the results of inhibition (%) of proliferation of various tumor cells and two normal cells of the compound of the present invention at 10.0 μηιοΙ/L, and the broken line indicates 50% inhibition.

 Fig. 4 is a graph showing the effect of the compound of the present invention on the ability of human lung cancer cell A549 to be cloned, wherein the drug concentrations of the six culture dishes from left to right are 0, 0.1, 0.5, 1, 5, 10 μηιοΙ/Lo, respectively.

 Fig. 5 is a graph showing the effect of the compound of the present invention on the migration inhibition rate of lung cancer cells H1299 cells. Among them, Fig. 5A shows the cell migration, the vertical line indicates the initial position of the cell scratch, and the vertical line is regarded as the migrating cell. Fig. 5B is a graph of Fig. 5A. The number of migrated cells in the control group (when the drug concentration is zero) is 100%, and the other groups are compared with the control group to determine the relative mobility, and the horizontal line represents the mobility of 50%.

 Fig. 6 is a graph showing the results of inhibition of infiltration of human lung cancer cells H1299 by the compounds of the present invention. Among them, Fig. 6A shows the result of cell infiltration, and the tumor cells are stained by crystal violet, which is blue-violet, that is, the black part in the picture. Fig. 6B is the statistical diagram of Fig. 6A. The number of infiltrating cells in the control group (when the drug concentration is zero) is 100%, and the other groups are compared with the control group to determine the relative infiltration rate, and the horizontal line represents the infiltration rate of 50%.

 Fig. 7 is a graph showing the results of inhibition of migration of mouse breast cancer cell MDA-MB-231 by the compound of the present invention, indicating that such a compound significantly inhibits migration of breast cancer cells. Among them, Fig. 7A shows the state of cell migration, the vertical line indicates the initial position of the cell scratch, and the line beyond the vertical line is regarded as the migrating cell. Fig. 7B is a graph of Fig. 7A. The number of migrated cells in the control group (when the drug concentration is zero) is 100%, and the other groups are compared with the control group to determine the relative mobility, and the horizontal line represents the migration rate of 50%.

 Fig. 8 is a graph showing the results of the effects of the compounds of the present invention on the inhibition rate of infiltration of breast cancer cells MDA-MB-231, indicating that such compounds have excellent anti-invasive ability to breast cancer cells. Among them, Fig. 8A shows the results of cell infiltration, and the tumor cells were stained with crystal violet, which was blue-violet. Fig. 8B is a graph of Fig. 8A. The number of infiltrating cells in the control group (when the drug concentration is zero) is 100%, and the other groups are compared with the control group to determine the relative infiltration rate.

 Fig. 9 is a graph showing the results of inhibition of infiltration of colon cancer cells SW620 by the compounds of the present invention. Tumor cells are stained with crystal violet, which is blue-violet, the black part of the picture.

 Figure 10 is a graph showing the results of immunofluorescence assay for inhibiting epithelial-mesenchymal transition (EMT) of tumor cells of the present invention, wherein the first line uses fibronection, the second line uses vimentin, and the third line uses F-actin.

 Figure 11 is a graph showing the results of RT-PCR and immunostaining experiments of the compounds of the present invention for inhibiting epithelial-mesenchymal transition (EMT) of tumor cells. Among them, Fig. 11A is the result of the RT-PCR experiment, and Fig. 11B is the result of the immunoblot experiment.

Figure 12 is a graph showing the results of no significant toxic side effects (10.0 mg/kg/day, 28 days) of the compounds of the present invention in Bal b/c mice. 12A is a sample of each group of mice to be sacrificed after the test, and FIG. 12B is an organ control removed from each group of mice. ―

Fig. 12C is an immunohistochemical section of organs in each group of mice, and Fig. 12D is a comparison of body weight of each group of mice.

 Figure 13 is a graph showing the results of inhibition of the growth of lung cancer in nude mice by the compounds of the present invention. Fig. 13A is a graph showing the relationship between tumor volume and days of administration, and Fig. 13B is a comparison of tumor weights between the administration group and the control group.

 Figure 14 is a graph showing the results of inhibition of mouse mammary gland carcinoma in situ and the survival rate of mice by the compound of the present invention, wherein Fig. 14A shows the in situ of the mouse mammary gland after 20 days of administration by the animal living imaging system. Photograph of the therapeutic effect of cancer, the shade shown by white* in the figure represents the fluorescent signal, indicating that there is tumor cell aggregation in this area, and the degree of aggregation from outside to inside cells is deepened; Fig. 14B is the calculation of tumor in mouse mammary gland by animal living imaging system. Figure 14C is a graph showing changes in mouse survival in a whole animal experimental model within 20 days of administration.

 Figure 15 is a graph showing the results of inhibition of in situ metastasis of breast cancer by the compounds of the present invention and effects on survival rate of mice. Among them, the first row in Fig. 15A is a photograph of the therapeutic effect on lung metastasis of mouse breast cancer after 20 days of administration by the animal living imaging system, and the shade shown by white* in the figure represents a fluorescent signal, indicating that tumor cells are aggregated in the region. And the degree of cell aggregation from outside to inside is deepened; Figure 15A shows the therapeutic effect of lung metastasis of breast cancer 20 days after the second behavior (black arrow shows tumor metastasis); the third behavior in Figure 15A is 20 days later H&E staining images of mouse lung sections (black arrows indicate tumor metastases). Fig. 15B is the amount of lung metastasis of mouse breast cancer calculated by the animal in vivo imaging system, and Fig. 15C is the change of the survival rate of the mouse in the whole animal experimental model after 20 days of administration.

 Figure 16 is a graph showing the results of the therapeutic effects of the compounds of the present invention on a mouse breast cancer in situ metastasis model. Among them, Fig. 16 is a photograph showing the therapeutic effect on the whole body metastasis of mouse breast cancer after 20 days of administration by the animal living imaging system. The shade shown by white* in the figure represents a fluorescent signal, indicating that tumor cells aggregate in the region. And the degree of cell aggregation from outside to inside is deepened.

 Figure 17 is a graph showing the results of the effects of the compounds of the present invention on the survival rate of breast cancer lung metastasis mice.

 Fig. 18 is a graph showing the results of inhibition of bone metastasis of breast cancer and osteolytic bone damage caused thereby by the compound of the present invention. Among them, the first row in Fig. 18A is a photograph of the therapeutic effect on bone metastasis of mouse breast cancer after 20 days of administration by the animal living imaging system, and the shade shown by white* in the figure represents a fluorescent signal, indicating that tumor cells accumulate in the region. And the degree of aggregation from the outer to the inner cells is deepened, and the therapeutic effect photo of the tibia bone injury of the mouse after 20 days of the second behavior in FIG. 18A (X-ray imaging detection, the arrow shows the position of the bone injury); FIG. 18B is the living body imaging system by the animal. The amount of bone metastasis in mouse breast cancer was calculated; Figure 18C is the area of mouse tibial bone injury calculated by X-ray imaging system.

 Figure 19 is a graph showing the results of the TGF P signaling pathway which plays a key role in inhibiting tumor metastasis in the present invention. Among them, Figure 19A shows the change in phosphorylation level of key protein Smad2/3 in the TGF P signaling pathway (immunoblotting assay), and Figure 19B shows the detection of TGF P signaling pathway activity (double fluorescent reporter gene assay).

Figure 20 is a graph showing the results of the EGF signaling pathway in which the compounds of the present invention play a key role in inhibiting tumor growth and metastasis, showing phosphorylation levels of key proteins EGFR, FAK, ERK1/2, Akt, c-src in the EGF signaling pathway. Variety ―

 (Western blotting experiment).

 detailed description

 The present invention will be further described in detail in conjunction with the following specific embodiments and drawings, which are not limited to the following embodiments. Variations and advantages that may be conceived by those skilled in the art are intended to be included within the scope of the invention and the scope of the appended claims.

1H-NM is measured with a Bruker 300 or Bruker 400; MS is measured with VG ZAB-HS or VG-7070, except for the ESI method; all solvents are re-distilled before use, no The water solvent was obtained by drying according to the standard method; except for the description, all the reactions were carried out under the protection of argon and followed by TLC. After the treatment, the mixture was washed with saturated brine and dried with anhydrous magnesium sulfate. Column chromatography using silica gel (200-300 mesh) was used; the silica gel used, including 200-300 mesh and GF _254, was produced by Qingdao Ocean Chemical Plant or Yantai Yuanbo Silicone Company.

 Example 1: Preparation of each compound

 Example 1-1 Preparation of N- 2-thiopheneacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS001)

Take the compound 1,3,4,9-tetrahydro-1Η-β-carboline (I) (172 mg, 10 mmol) in dichloromethane, add Et ₃ N, and cool the system to 0 ° C. 2-Thienylacetyl chloride (148 μί, 1.2 mmol), after completion, was reacted for 5 hours, and purified by column chromatography to give IBMS001 (207 mg, yield 70%). ^J H NM (400 MHz, DMSO): δ 10.86 (br s, 1H), 7.40-7.38 (m, 1H), 7.40 (d, J = 6.8 Hz, 1H), 7.31-7.28 (m, 1H), 7.05 -7.00 (m, 1H), 6.97-6.96 (m, 1H), 6.95 (d, J = 6.8 Hz, 1H), 6.93-6.90 (m, 1H), 4.76-4.69 (m, 2H), 4.09-4.07 (m, 2H), 3.86-3.82 (m, 2H), 2.67-2.65 (m, 2H).

 Example 2-45, Table 1 Preparation of IBMS002-45 porphyrin compound (for specific process, see later)

IBMS010 -1 Η-β-carboline was replaced with tetrazolium acetate

Example 1-11 2-(4-(2-NHBoc-ethoxy)-phenylacetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS011 base) -1,3,4, Conversion of 9-tetrahydro-1Η-β-carboline to 4-(2-NHBoc-ethoxy)-phenylacetic acid

Example 1-12 2-(4-Bromomethylphenylacetyl)-1,3,4,9-tetrahydrogen Similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS012 -1Η- Conversion of β-carboline to 4-bromomethylphenylacetic acid

Example 1-13 2-(2-Hydroxyphenylacetyl)-1,3,4,9-tetrahydro is similar to Example 1-2 except that 3,4-dihydroxyphenylacetic acid IBMS013 -1Η-β- Conversion of porphyrin to 2-hydroxyphenylacetic acid

Example 1-14 2-(4-Acetylphenylacetyl)-1,3,4,9-tetrahydro is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS014 -1Η-β - porphyrin is replaced by 4-aldehyde phenylacetic acid

Example 1-15 2-(4-(2-hydroxyethoxy)-phenylacetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS015 base) -1,3,4,9 -Tetrahydro-1Η-β-carboline is replaced by 4-(2-hydroxyethoxy)-phenylacetic acid

Example 1-16 2-(4-(3-morpholinopropoxy)-phenylacetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS016 base) -1,3,4 , 9-tetrahydro-1Η-β-carboline is replaced by 4-(3-morpholinopropoxy)-phenylacetic acid

Example 1-17 2-(4-Carboxyphenylacetyl)-1,3,4,9-tetrahydrogen Similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS017 -1Η-β- The porphyrin is replaced with (4-carboxymethyl)-methyl benzoate and hydrolyzed.

 IBMS017

Example 1-18 2-(4-(2-Aminoethoxy)-phenylacetyl compound IBMS 011 (72 mg/0.23 mmol) dissolved IBMS018 base) -1,3,4,9-tetrahydro-1Η- Β-carboline in 3 ml DMF, only the reaction system was cooled in an ice salt bath for 10 min, sodium hydride (60%, 46 mg) was added and reacted for 20 min, and bromoethanolamine hydrobromide (96 mg/0.46 mmol) was added. After the stirring was continued for 3 hours, the reaction mixture was poured into an ice-bath, and the mixture was evaporated. Column chromatography gave the compound IBMS018, Yield: 20%.

Example 1-19 N-(2-(4-(2-(2-NHBoc-ethylamino)) B is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS019 oxy)phenyl ) -propionyl) -1,3,4,9-tetrahydrogen to 4-((methoxycarbonyl)methoxy)phenylacetic acid

 -1Η-β-porphyrin

Example 1-20 Ν-Phenylpropionyl-1,3,4,9-tetrahydro-1 Η-β-carboline was similar to Example 1-2 except that 3,4-dihydroxyphenylacetic acid IBMS020 was replaced by benzene. Propionic acid Example 1-21 N-((3-NHBoc-3-phenyl)propanoyl)-2,3, similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS021 4,9-four Hydrogen-1Η-β-carboline is replaced by 2-NHBoc-3-phenylpropionic acid

Example 1-22 N-(2-(2-( )-NHBoc-phenyl) acetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS022 base) -1,3,4, Conversion of 9-tetrahydro-1Η-β-carboline to 2-(2-(R)-NHBoc-phenyl)acetic acid

Example 1-23 N-(2-(2-(S)-NHBoc-phenyl)acetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS023 base) -1,3,4 , 9-tetrahydro-1Η-β-carboline is replaced by 2-(2-(S)-NHBoc-phenyl)acetic acid

Example 1-24 N-(2-(2-()-Aminophenyl)acetyl-based compound IBMS022 (180 mg, 0.44 mmol) IBMS024 base) -1,3,4,9-tetrahydro-1Η-β- The porphyrin was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added dropwise under ice-cooling. After 2 hours, excess solvent was removed under reduced pressure, and then the system was adjusted to pH=12 with IM NaOH solution. .

Example 1-25 Ν-(3-Amino-3-phenyl)propanoyl)-1,3,4,9- Similar to Example 1-24, only the compound IBMS022 IBMS025 tetrahydro-1Η-β-咔Replacement of porphyrins into IBMS021

Example 1-26 N-(2-(2-(S)-Aminophenyl)acetyl is similar to Example 1-24, only the compound IBMS022 IBMS026 base) -1,3,4,9-tetrahydro-1Η -β-carboline replaced by IBMS023

Example 1-27 Ν-(2-(4-Acylphenyl)propionyl)-1,3,4,9- Similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS027 IV Hydrogen-1Η-β-carboline is replaced by 2-(4-aldehydephenyl)propionic acid

Example 1-28 Ν-(2-(4-Bromomethylphenyl)propionyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS028 base) -1,3,4,9 -Tetrahydro-1Η-β-carboline is replaced by 2-(4-bromomethylphenyl)propionic acid

Example 1-29 Ν-(2-(4-Hydroxymethylphenyl)propionyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS029 base) -1,3,4,9 -Tetrahydro-1Η-β-carboline is replaced by 2-(4-hydroxymethylphenyl)propionic acid

Example 1-30 Indole-phenylacetyl-1,3,4,9-tetrahydro-1Η-β-carboline Similar to Example 1-1, only 2-thiopheneacetyl chloride was replaced with IBMS030 phenylacetyl chloride.

Example 1-31 Ν-(4-Hydroxyphenylacetyl)-1,3,4,9-tetrahydro is similar to Example 1-2 except that 3,4-dihydroxyphenylacetic acid IBMS031 -1Η-β- Conversion of porphyrin to 4-hydroxyphenylacetic acid

Example 1-32 Ν-(2-hydroxy-2-phenylacetyl)-1,3,4,9-tetra is similar to Example 1-2 except that 3,4-dihydroxyphenylacetic acid IBMS032 hydrogen- 1Η-β-carboline is replaced by 2-hydroxy-2-phenylacetic acid

Example 1-33 Ν-(2-hydroxy-2,2-diphenylacetyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid IBMS033 group) -1,3,4,9- Conversion of tetrahydro-1Η-β-carboline to 2-hydroxy-2,2-diphenylacetic acid

Example 1-34 Ν-(2-(4-(morpholinemethylphenyl)-propionyl is similar to Example 1-2, only 3,4-dihydroxyphenylacetic acid

Example 1-42 N-Phenylacetyl small ethyl-1,3,4,9-tetrahydro-6- 5-benzyloxytryptamine (390 mg, 1.50 mmol) Soluble IBMS042 Benzyloxy-1Η-β - porphyrin in glacial acetic acid (6 ml), stirring for 10 min, then adding acetaldehyde (91 μl, 1.60 mmol) dropwise, heating the reaction system to 80 ° C, reacting for 4 hours, then adding NaOH to pH = 9, The crude product was extracted, and the compound was dissolved in DMF. After the similar treatment in the presence of EDC/HOBt, the target product IBMS042 was obtained.

Example 1-43 Ν-Phenylacetyl minimethyl-1,3,4,9-tetrahydro-6- Similar to Example 1-42, only propionaldehyde was replaced by acetaldehyde IBMS043 benzyloxy-1Η- Beta-porphyrin

Example 1-44 Ν-Phenylacetyl minimethyl-1,3,4,9-tetrahydro-6- Compound IBMS043 (107 mg, 0.26 mmol) IBMS044 Hydroxy-1Η-β-carboline suspended in methanol ( In 10 ml), Pd (10% wt) was used as a catalyst to hydrogenate the benzyl group under hydrogen atmosphere, and finally the product IBMS044 was obtained.

Example 1-45 Ν-Phenylacetyl-6-fluoro-1,3,4,9-tetrahydro 3-(2,3-piperidindione M4-fluorophenyl)-indole (754 mg, IBMS045 -1Η-β-carboline 3.41 mmol, preparation method Reference Syn. Comm.

 2007, 37, 1273-1280) dissolved in 20 ml of formic acid, in

After stirring at 80 ° C for 24 hours, the formic acid was removed under reduced pressure, and then worked-up to give white crystals, m, m, m, m, m. The porphyrin intermediate is reduced by lithium aluminum hydride and coupled with phenylacetic acid to obtain the final product. IBMS045

Example 1-46 Ν-phenylacetyl-6-bromo-1,3,4,9-tetrahydro is similar to Example 1-45, only 3 2,3-piperidine di IBMS046 -1 Η-β-咔4-fluorophenyl)-indole to 3-(2,3-piperidinone)-(4-bromophenyl)-indole

 Table 1

Example 2: Inhibition of proliferation of human lung cancer cells, breast cancer cells and colon cancer cells at a certain dose and inhibition of tumor cell colony forming ability

 (A), cell culture

The cells used in this experiment were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (see Table 2 below). The adherent culture was carried out in a 37 °C incubator (humidity 95%, C0 ₂ 5%). The medium was DMEM high glucose medium. (Gibco) contains 10% fetal bovine serum (Front).

 A549 human non-small cell lung cancer

H1299 Human non-small cell lung cancer

H460 human large cell lung cancer

 SK-MES-1 human lung squamous cell carcinoma

 95-D human large cell lung cancer

 M C-5 human alveolar fibroblast

MDA-MB-231 person breast cancer

 Hs578T Human Breast Cancer

 BT-549 person breast cancer

 4T1 mouse breast cancer

 MCF-7 person breast cancer

 MCF-10A human mammary epithelial cells

 SW-620 human colon cancer

 (B), S B (sulfonhodamine) method for cell proliferation

The tumor cells were inoculated into a 96-well plate (Coming) at a density of ⁵ ×10 ³ /well. After 24 h of routine culture, the compounds of the present invention were sequentially added at different concentrations to a final concentration of 0.01 μηιοΙ/L, 0.05 μηιοΙ/L, 0.10 μηιοΙ, respectively. /L, 0.50 μηιοΙ/L, Ι.Ο μηιοΙ/L, 5.0 μηιοΙ/L, 10.0 μηιοΙ/L, 100.0 μηιοΙ/L, the control group was added with an equal amount of DMSO (6 replicate wells per group). After 48 hours of incubation, pre-cooled TCA (trichloroacetic acid, 50%, w/V), 25 μΐ/well, gently mix. The cells were fixed by incubation at 4 ° C for 60 min. After fixing, rinse the running water 5 times and air dry. 50 μM SRB stain (4%, w/V) was added to each well and stained for 10 min at room temperature. The dye solution was aspirated and washed with 1% acetic acid 100 μM per well for 5 times to remove unbound dye. After air drying, 100 μl of a concentration of 10 mmol/L Tris solution was added to each well to vortex and dissolve the bound SRB dye. The 96-well plate was placed in a microplate reader (SPECTRA MAX 190) and the OD value was measured at a wavelength of 515 nm. The effect of the drug on the level of cell proliferation was statistically analyzed.

 Force π药日 0 Γ)倌

 Cell viability (%) = ■ X 100%

 DMSO group 0. D value

 The result is shown in Figure 1-3, where:

(1) The compound of the present invention has a very strong inhibitory effect on the proliferation of human lung cancer cell H1299 at 10.0 μηιοΙ/L, as shown in Fig. 1. Most of the compounds have a significant inhibitory effect on the proliferation of lung cancer cells. Among the listed compounds, the compounds IBMS001, IBMS011, IBMS012, IBMS023, IBMS024, IBMS027, IBMS028, IBMS029, IBMS030, IBMS031 and IBMS038 are at a concentration of 10.0 μηιοΙ/L. Inhibition rate of lung cancer cell H1299 proliferation activity ―

More than 60%.

 (2) The compound of the present invention has a very significant inhibitory effect on the proliferation of breast cancer cell MDA-MB-231 at 10.0 μηιοΙ/L, as shown in Fig. 2. It can be found that most of the compounds have a significant inhibitory effect on the growth of breast cancer cells. Among the listed compounds, IBMS001, IBMS002, IBMS012, IBMS027, IBMS028, IBMS029, IBMS030, IBMS031 and IBMS038 at 10.0 μηιοΙ/L for breast cancer The inhibition rate of the proliferation activity of the MDA-MB-231 cells reached 50% or more, and it was found that such compounds significantly inhibited the proliferation of the breast cancer cells.

 (3) The compound of the present invention has a strong inhibitory effect on cancer cells, but has no significant inhibitory effect on normal cell proliferation. Among the tested compounds, IBMS007, IBMS012, IBMS02K, IBMS024, IBMS030, IBMS031, and IBMS038 have strong inhibitory selectivity against a variety of tumor cell proliferation, and have a poor inhibitory activity against the corresponding normal cell proliferation. As can be seen from Figure 3, compounds at 10.0 μηιοΙ/L for non-small cell lung cancer (eg A549 and H1299), large cell lung cancer (eg 95D) and breast cancer (eg MCF-7, MDA-MB-23K BT-549) The proliferation of several cancer cells of Hs578T) was strongly inhibited, while the inhibition of proliferation of normal cells (such as lung fibroblasts MRC5 and mammary epithelial cells MCF-10A) was poor, indicating that these compounds are resistant to tumors. The cells have excellent selectivity.

 (C), colony formation (colony formation): detection of tumor cells in vitro to form clones proliferation and tumorigenic ability

 Tumor cells can proliferate by themselves to form macroscopic tumors that are visible to the naked eye. In an in vitro experimental model, the ability of tumor cells to form solid tumors can be reflected in the ability of the tumor cells to form solid tumors.

The tumor cells were seeded in a 35 mm culture dish at a density of lxlO ³ / dish, and cultured for 24 h, and then the concentrations were determined to be 0. 10 μηιο^, 0. 50 μηιοΙ /L, 1.0 μηιοΙ/L, 5.0 μηιοΙ/L, 10.0 μηιοΙ/L, the control group was added with an equal amount of DMSO. Continue the culture and change the medium and the corresponding concentration of compound every 3 days. After 21 days, the medium was discarded, washed 3 times with PBS, fixed in 4% paraformaldehyde for 10 min at room temperature, stained with 1% crystal violet for 10 min, and rinsed with tap water. Photographed under a microscope to calculate the number of cell clones formed. The experimental results are shown in Figure 4. Among them, the compounds IBMS002, IBMS020, IBMS030, IBMS03K IBMS033 and IBMS038 have obvious inhibitory effect on the colony forming ability of non-small cell lung cancer cells (such as Α549 cells), and the compound cloned this lung cancer cell at a concentration of 0.5 μηιοΙ/L. The ability was significantly inhibited, and the colony formation of the lung cancer cells was substantially inhibited at a concentration of 5.0 μηιοΙ/L, while the colony formation of the lung cancer cells was completely inhibited at a concentration of 10.0 μηιοΙ/L, indicating that the compounds were strong from another angle. The role of inhibition of tumor growth.

Example 3: Partial test of compound inhibition of tumor cell migration and infiltration

 (1), wound healing (wound healing) detection of inhibition of tumor cell migration ability

When cultured in vitro, cells move to lesser parts of the cell along the culture plane. Using this phenomenon, in the culture hole that has been filled with cells, a "scratch" is artificially "scratched", and the cells on both sides of the "scar" will move toward the "scar" area, and finally re-fill the area, so-called " The effect of scar healing. According to the number of cells moving to the "scar" area and "injury" ―

The degree of healing of the marks can determine the ability of the cells to move.

Tumor cells were seeded into 12-well plates and cells were cultured for 24 h at 37 ° C in a 5% C0 ₂ incubator until the cells were 100% full. Replace the serum-free medium and continue to culture for 12 h. In the cell-filled culture well, scratch the diameter of the culture well with a 10 μΐ sterilization tip. After the scratch, wash the cells twice with PBS, wash the floating cells, and add 1.0 ml of complete culture to each well. base. Different concentrations of the drug were added to the cell culture wells, and the culture plate was placed in a C0 ₂ incubator, and the conventional culture was continued at 37 ° C for 24 h. Under the microscope, observe the movement of the cells to the part of the line, and take a picture. The number of cells migrated into the streaked area by different doses of the drug group was statistically analyzed to determine the effect of the drug on cell migration ability. At the same time, the proliferation of the corresponding cells at the corresponding concentration of the drug for 24 h was measured by the SRB method (as described above) to determine whether the scratch test was affected by cell proliferation.

 Number of cells migrated in the drug-treated group

 Cell migration rate (%) = X 100%

 Number of migrated cells in DMSO group

 The results are shown in Figures 5 and 7, where:

(1) As can be seen from Fig. 5, Fig. 5A is a tumor cell migration effect diagram, and Fig. 5B is a result statistics. In the experimental model of cell scratch migration of lung cancer cell H1299, the compounds IBMS011, IBMS017, IBMS019, IBMS024, IBMS030, IBMS03K The half effective concentration IC ₅ Q of IBMS036 and IBMS038 for H1299 migration inhibition was between 0.05 and 0.10 μηιοΙ/L, indicating that this compound significantly inhibited the migration of this tumor cell in this migration model.

(2) It can also be seen from Fig. 7 that Fig. 7A is a graph of tumor cell migration effect, and Fig. 7B is a result of statistics. In the Wound healing migration model of breast cancer cell MD-MBA-231, compounds IBMS001, IBMS005, IBMS010, IBMS017 The half effective concentration IC _5Q of the migration inhibition of MDA-MB-231 by IBMS019, IBMS024, IBMS030, IBMS024, IBMS030, IBMS031, IBMS036 and IBMS038 is between 0.1~0.5 μηιοΙ/L, indicating that this kind of compound inhibits the migration of breast cancer cells very obviously. .

 (B), Transwell method to detect cell infiltration

 The permeable polycarbonate film on the bottom of the Transwell chamber has a large number of micropores with a diameter of 8 μηι. In the experiment, the Transwell chamber is placed in a 24-well plate. The polycarbonate film divides the entire hole into upper and lower chambers. Transwell The small chamber is called the upper chamber, the culture plate is called the lower chamber, the upper chamber is filled with the upper culture medium, and the lower chamber is filled with the lower culture medium. The upper and lower culture liquids are separated by a polycarbonate membrane, and the upper chamber of the Transwell chamber is pre-plated with a collagen matrix Matrigel. The cells are then inoculated. Due to the permeability of the polycarbonate membrane, the components in the lower broth can induce the cells in the upper chamber to migrate downward, moving from the upper chamber surface to the lower chamber surface of the membrane. However, if tumor cells are to move to the lower chamber, they must first dissolve the collagen matrix by using various chemical substances released by themselves, and then move through the dissolved cavity. This process is similar to the infiltration process of tumor cells in the body. To reflect the invasive ability of tumor cells. Therefore, this experiment is a classic experiment to study cell infiltration.

Drug-treated cells: Tumor cells in logarithmic growth phase were inoculated into 6-well plates at lxlO ⁵ cells/well, cultured for 24 h, and then grouped into different concentrations of test compounds to give a final concentration of 0.01 μηιο1/ί. , 0.05 μηιοΙ/ί, Ο.ΙΟ μηιοΙ/L, ―

0.5 μηιοΙ/L, Ι.Ο μηιο^, 5.0 μηιοΙ/L, the control group was added with an equal amount of DMSO. After 12 hours of incubation, the in vitro cultured drug-pretreated tumor cells were digested with 0.25% trypsin and inoculated into the upper chamber of a Transwell chamber pre-plated with 50% Matrigel (BD) at a density of lxlO ⁵ /well. The drug group was added with 0.01 μηιοΙ/L, 0.05 μηιοΙ/L, 0.10 μηιοΙ/L, 0.50 μηιοΙ/L, Ι.Ο μηιοΙ/L, 5.0 μηιοΙ/L as shown above, and the control group was added with an equal amount of DMSO. 600 μM of DMEM high glucose medium containing 10% fetal bovine serum was added to each of the lower chambers. Incubate for 7 h to 12 h in a 37 V C0 ₂ incubator. Remove the transwell chamber, wipe the upper chamber side of the transwell chamber with a cotton swab, and wipe off the unpenetrated cells. Transwell was fixed in 4% paraformaldehyde for 10 min at room temperature, 1% crystal violet stained for 10 min, and rinsed with tap water. Photographs were taken under a microscope, and the number of cells in five fields in the upper, lower, left, and right sides of each well was counted, and the number of perforated cells/field of view was obtained. There are an average of 3 filters in each group. The number of transmembrane cells in different doses of the drug group was statistically compared to determine the effect of the drug on cell migration ability. H^ K. Number of infiltrating cells in the eight-drug group ^ _/η/

 Cell migration rate (%) = X 100%

 Number of infiltrating cells in DMSO group

 The results are shown in Figures 6, 8-9, where:

(1) As shown in Fig. 6, Fig. 6 is a graph showing the effect of compounds inhibiting cell infiltration, and Fig. 6 is a statistical graph of results. In the Transwell infiltration model of lung cancer cell H1299, the half effective concentration IC _{50 of} these compounds inhibiting H1299 infiltration is Between 0.1 and 0.5 μηκ) 1 / ί; indicating that such compounds exhibit excellent anti-lung cancer cell infiltration ability in the infiltration model.

(2) As can be seen from Fig. 8, Fig. 8 is a graph showing the effect of the compound on inhibiting cell infiltration, and Fig. 8 is a graph showing the results. In the Transwell infiltration model of breast cancer cell MDA-MB-231, the compound is for MDA-MB. The half effective concentration IC- _{5Q of} -231 infiltration inhibition was between 0.1 and 0.5 μηιοΙ/L; indicating that such compounds have excellent anti-breast cancer cell infiltration ability.

 (3) As shown in Figure 9, in the Transwell infiltration experimental model of colon cancer cell SW620, compounds IBMS005, IBMS011, IBMS017, IBMS019, IBMS024, IBMS030, IBMS03K IBMS036, and IBMS038 at a compound concentration of 1.0 μηιοΙ/L The infiltration of colon cancer cells SW620 was significantly inhibited, indicating that this compound also has a good inhibitory effect on the invasion of colon cancer cells.

Example 4: Partial experiment of compound on epithelial-mesenchymal transition (EMT) inhibition of tumor cells

 (A), tumor cell epithelial-mesenchymal transition (EMT) detection

Human epidermal cells HaCaT were inserted into a 24-well plate pre-placed with slides at 2 X 10 ⁴ /well, and cultured for 12 h in a 37 ° C 5% C0 ₂ incubator until the cells were attached to the slides. Replace the serum-free medium and continue to culture for 12 h. Epithelial-mesenchymal transition (EMT) was induced by adding 5 ng/ml of TGFP to stimulate HaCaT cells. At the same time, different concentrations of compounds (0.05 μηιοΙ/L, 0.5 μηιοΙ/L, 5.0 μηιοΙ/L) were added to the experimental group. Add the same amount of DMSO. After 48 h, the medium in the wells was aspirated and washed three times with PBS. 4% paraformaldehyde was fixed at room temperature for 10 min. Subsequently, immunofluorescence (Immunofluorescence) was used to detect the expression changes of intracellular EMT marker proteins vimentin and fibronectin. Phalloidin staining ―

Labeling of Ha-Fin cells F-actin skeletal changes, showing morphological changes during cellular EMT. The changes of EMT-tagged protein in HaCaT cells were detected by RT-PCR and Western blot. TGFP stimulated HaCaT cells to undergo EMT and compound treatment in a similar manner to the above experiments. After 48 hours of drug treatment, HaCaT cells were lysed and their mRNA and protein were extracted for RT-PCR and immunoblotting. The GAPDH protein was used as an internal reference to ensure consistent sample loading for each sample in the assay.

 The results are shown in Figure 10-11, where:

 (1) As shown in Figure 10, immunofluorescence assay showed that the expression of EMT marker proteins fibronetin and vimentin decreased significantly with increasing drug concentration, as shown in Figure 10. Thus, the compounds of the present invention, IBMS002, IBMS012, IBMS023, IBMS027, IBMS030, and IBMS031, can significantly inhibit the transformation of epithelial to interstitial transformation (EMT) caused by TGFP stimulation.

 (2) As shown in FIG. 11, RT-PCR and immunoblot experiments also showed similar results. As shown in FIG. 11, phalloidin staining showed that EMT caused by TGFP stimulation caused morphological changes in epithelial cells, and the present invention Compounds can significantly inhibit the occurrence of this change.

Example 5: Model of some compounds of the present invention in mouse tumor growth and metastasis

 (A), the compound of the present invention has no obvious toxic side effects on normal Bal b/c mice.

 Four-week-old Bal b/c female mice were divided into three groups according to their body weight, which were used as the uninjected group, the solvent group (DMS0) and the drug group, and the drug was intraperitoneally administered once a day (10.0 mg/kg/day). The body weight of the mice was recorded daily and the behavioral changes were observed. After 28 days, the mice were sacrificed. The toxic side effects of the compounds were examined by comparing their vital organs such as heart, lung, liver, spleen and kidney with the uninjected/solvent group.

 The results are shown in Figure 12, in which the compounds, IBMS001, IBMS009, IBMS020, IBMS025, IBMS030, IBMS031, and IBMS038 were administered at a dose of 10.0 mg/kg/day for 28 days in this model, and the behavior, weight, and importance of the mice. Organs such as heart, lung, liver, spleen and kidney showed no significant changes compared with the control group, indicating that the compounds had no obvious toxic side effects on Bal b/c mice.

 (B), the inhibitory effect of the compound of the present invention on the growth of pancreatic cancer in a mouse lung cancer growth model:

500,000 human non-small cell lung cancer cells A549 were subcutaneously injected into the back of the immunodeficient mice (nude mice). When the subcutaneous tumors grew to about 100 mm3, the mice were divided into two groups (the average tumor volume was the same). The mice in the drug group were intraperitoneally injected with 10 mg/kg of the compound of the present invention dissolved in DMS0, and the control group was only injected with DMS0. The body weight and the length and width of the tumor were measured and recorded every day. The mice were sacrificed 34 days after the application, and the subcutaneous tumor was taken. , taking pictures. The tumor volume was calculated according to the formula volume = length X width ² χ 0.52.

The results are shown in Figure 13, in which the compounds IBMS001, IBMS012, IBMS027, IBMS029, IBMS030, and IBMS031 were administered for 34 days, and it was found that they were classified at a dose of 10 mg/kg/day relative to the control group. ―

The compound has a very significant inhibitory effect on the in situ growth of lung cancer, including the size and volume of the tumor.

 (C), Bal b / c mouse breast pad in situ tumor-bearing model to detect tumor metastasis ability.

This experiment simulates the onset and treatment of clinical breast cancer metastasis. For 4-6 weeks of Bal b/c female mice, 5 X 10 ⁴ mouse breast cancer cells 4T1 were injected in situ into the breast pad. After 7 days of tumor-bearing, the tumor grew to an approximate diameter of 5 mm. The mice were randomly divided into 3 groups according to the size of the tumor in situ, which were the control group, 1.0 mg/kg/day dose group and 5.0 mg/kg. /day dosing group. The drug-administered group was intraperitoneally injected with the corresponding dose, and the control group was injected with the same amount of solvent (DMSO). The body weight of the mice was weighed before the daily injection of the compound, and the survival of the mice was recorded. After continuous administration for 20 days, mouse living body imaging technique (IVIS) was used to observe the metastasis of tumor cells to the lungs of mice.

 The results are shown in Fig. 14. Fig. 14A is a therapeutic effect diagram of mouse breast carcinoma in situ after 20 days of administration (photographed by the animal living imaging system, fluorescence can be used to determine the location of tumor cells by fluorescence in tumor cells) And how much, the shade shown in white* in the figure represents the fluorescent signal, indicating that there is tumor cell aggregation in this area, and the degree of aggregation from outside to inside cells is deepened. The results show that the compounds at the dose of 1.0 mg/kg/day are IBMS001, IBMS027, IBMS029, IBMS030, IBMS031, and IBMS038 significantly inhibited the growth of breast carcinoma in situ, while the 5.0 mg/kg/day dose of the compound substantially inhibited the growth of breast carcinoma in situ. Figure 14B is the calculation of the amount of tumor in the mouse mammary gland by the animal in vivo imaging system. It can be clearly seen that the compound has a significant inhibitory effect on tumor size in a dose-dependent manner. Figure 14C shows the change in survival rate of the mouse in the whole animal experimental model within 20 days of administration, from which it can be found that compared with the control group, Compounds have a significant positive effect on survival in mice, at 5.0 mg/kg/day About 90% of the mice survived, and about 80% of the mice survived at 1.0 mg/kg/day, while only about 50% of the mice in the control group survived.

(4), nude mice tail vein injection model to detect tumor lung metastasis and lung failure caused by tumors.

This experiment simulates the onset and treatment of clinically treated breast cancer lung metastases. 5 X 10 ⁴ / breast cancer cells 4T1 or MDA-MB-231 were inoculated into the breast pad of 4-6 weeks old female Bal b/c mice. After 7 days, the mice grew in diameter by about 5 After the in situ tumor of mm, the mice were divided into three groups according to the average body weight, which were the control group, the 1.0 mg/kg/day dose group and the 5.0 mg/kg/day dose group. The amount of metastasis of breast cancer cells was periodically observed by the animal in vivo fluorescence imaging system (IVIS) to determine the effect of the compound on the lung metastasis progression of breast cancer, and the survival rate of the mice was calculated. After 20 days of cancer cell inoculation, the mice were sacrificed. Take the heart, lung, liver, stomach, kidney, spleen and intestine, observe the metastasis distribution of tumor cells under the IVIS system.

 The result is shown in Figure 15-17, where:

(1) Figure 15 shows, Figure 15 shows the therapeutic effect of the mouse breast cancer in situ metastasis model after 20 days of treatment. (Photographed by the animal living imaging system, fluorescence can be used to determine tumor cells due to fluorescence in tumor cells. The position and the number, the shade shown in white* in the figure represents the fluorescence signal, indicating that there is tumor cell aggregation in this area, and the degree of aggregation from outside to inside cells is deepened. Since the in situ metastasis model of breast cancer often shifts to the lungs, it can be seen that the control Group of mice have a large number of lungs ―

Tumor cell metastasis, including anatomical and HE staining of the lungs of mice, showed that there were a large number of tumor cells. However, at the doses of 1.0 mg/kg/day and 5.0 mg/kg/day for compounds (IBMS00K IBMS027, IBMS029, IBMS030, IBMS031, and IBMS038), the distribution of metastatic lesions in mice was reduced by 50% and 94%, respectively, at 5.0 mg. At a dose of /kg/day, imaging of tumor cells to the lungs was not seen at all, as shown in Figure 15, indicating that such compounds have a significant inhibitory effect on tumor metastasis. In addition, the compounds also have a significant improvement in the survival rate of mice. After 20 days of administration, the mice in the control group were basically unable to survive. The mice after the low dose (1.0 mg/kg/day) drug treatment had More than 60% can survive. After the high dose (5.0 mg/kg/day), the survival rate of the mice is over 80%. The drug has a significant improvement in the survival rate of the mice. There is a clear therapeutic effect, which also explains the inhibitory effect of drugs on lung metastasis of breast cancer from another perspective.

 (2) Figure 16 shows that at the dose of 10.0 mg/kg/day, the compounds of the invention, IBMS001, IBMS027, IBMS029, IBMS030, IBMS031 and IBMS038, were significantly grown in situ during the course of the experiment (42 days). The inhibitory effect also has a strong inhibitory effect on the in situ metastasis of mouse breast cancer. From the statistical data in Table 3, it can be found that the compound of the present invention has a very strong inhibitory effect on the metastasis of mouse breast cancer to lung, liver, spleen and kidney. At the dose of 10.0 mg/kg/day, no tumor cells were found to the lung, Liver, spleen and kidney metastasis, while the incidence of metastasis in the control group was 62.5%, 75%, 50% and 37.5%, respectively. The incidence of corresponding intestinal metastasis also decreased from 62.5% to 25%, indicating that this type Compounds have the ability to strongly inhibit the metastasis of breast cancer to other organs.

 (3) It can be seen from Fig. 17 that after 20 days of administration, the control mice were basically unable to survive, and the administration group (compound IBMS001,

The survival rates of mice at 1.0 mg/kg/day and 5.0 mg/kg/day were increased to 50% and 80%, respectively, at doses of 10.0 mg/kg/day for IBMS027, IBMS029, IBMS030, IBMS031, and IBMS038). The mice underneath can basically survive, and it is obvious that the compound has a very significant effect on the survival rate of mice.

 Table 3. Incidence of tumor metastasis

 Experimental group lung metastasis liver metastasis spleen metastasis renal metastasis intestinal metastasis

 Incidence incidence rate incidence rate control group 5/8 (62.5%) 6/8 (75%) 4/8 (50%) 3/8 (37.5%) 5/8 (62.5%) Drug-administered group 0/8 (0%) 0/8 (0%) 0/8 (0%) 0/8 (0%) 2/8 (25%)

(10 mg/kg)

(5) The left ventricle tumor-bearing model of nude mice detects the ability of tumor to metastasize to bone and the osteolytic bone damage caused by tumor.

 This experiment mimicked the clinical onset and treatment of breast cancer bone metastasis and the resulting osteolytic bone injury. in

4-6 weeks old female nude mice were inoculated with 5×10 ⁴ /only breast cancer cells, which will eventually transfer to the tibia of the mice with blood circulation, and cause excessive activation of osteoclasts here. , causing osteolytic bone damage. Average by mouse weight ―

Divided into 3 groups, the control group, 1.0 mg/kg/day dose group and 5.0 mg/kg/day dose group. From this, the effect of the compound on bone metastasis of breast cancer and the osteolytic bone damage caused thereby was observed. Twenty days after the administration, the mice were sacrificed, the number of metastasis of breast cancer cells was calculated by the animal in vivo fluorescence imaging system (IVIS), and the area of osteolytic bone damage caused by bone metastasis of the breast cancer was determined by X-ray imaging.

 The results are shown in Figure 18, and the compounds (IBMS001, IBMS027, IBMS029, IBMS030, IBMS031, and IBMS038) have significant therapeutic effects on breast cancer bone metastasis and the resulting osteolytic bone damage. After 20 days of administration, the amount of metastasis of breast cancer cells was calculated by the animal fluorescence imaging system, and the area of osteolytic bone damage caused by bone metastasis of breast cancer was determined by X-ray imaging. Experiments have shown that at 1.0 mg/kg/day and 5.0 mg/kg/day, bone metastasis of breast cancer in mice decreased by 75% and 90%, respectively, and the area of osteolytic bone injury decreased by 75%. And 94%, indicating that the compound has a significant inhibitory effect on bone metastasis of mouse breast cancer and osteolytic bone damage caused by it. Example 6: Inhibition of TGFP signaling pathway by the compounds of the invention

Breast cancer cells were seeded in a 6-well plate at a density of IX 10 ⁵ /well. After 24 hours of cell attachment, the cells were replaced with serum-free medium and cultured with the corresponding concentration of compound. After 6 hours, TGFP 1 stimulator cells were added for 1 hour, and the cells were lysed to extract proteins for immunoblotting.

 The result is shown in Figure 19, where:

 (1) Figure 13 shows that, in breast cancer cells, the compounds of the present invention, IBMS003, IBMS012, IBMS030 and IBMS031, have a significant inhibitory effect on TGFp-induced phosphorylation of smad2, smad3 protein, and are in a concentration-dependent manner. This demonstrates that the compounds of the invention inhibit the activation of the TGFP signaling pathway and the downstream gene expression regulated by TGFP.

 (2) Figure 19 shows that the compound of the present invention inhibits the transcriptional regulatory activity of the Tmad-induced Smad protein. Activation of the TGFP signaling pathway enables the smad protein to be transferred into the nucleus, enabling the smad protein to act as a transcription factor, binding to the SMAD binding element to initiate downstream gene transcription. While the compounds of the present invention inhibit the transcriptional activity of the protein in a gradient-dependent manner, it is indicated that the compounds of the present invention inhibit the activation of the TGFP signaling pathway and the downstream gene expression regulated by TGFP.

Example 7: Inhibition of EGF receptor signaling pathway by the compounds of the present invention

Lung cancer cells were seeded in a 6-well plate at a density of IX 10 ⁵ /well. After 24 hours of cell attachment, the cells were replaced with serum-free medium and cultured with the corresponding concentration of compound. After 6 hours, EGF stimulated the cells for 15 minutes, and the cells were lysed to perform immunoblotting.

As a result, as shown in FIG. 20, in the breast cancer cells, the compounds of the present invention, IBMS001, IBMS027, IBMS030 and IBMS031, have a significant inhibitory effect on the phosphorylation levels of EGFR, FAK, ERK1/2, c-src proteins induced by EGF receptors, And exhibit concentration gradient dependence. This demonstrates that the compounds of the invention inhibit the activation of the EGF receptor signaling pathway and EGF-regulated downstream gene expression. ― .

Reference Example 2-45

EXAMPLES 1-2, N-(3,4-Dihydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS002)

 Compound I (225 mg) was taken in dichloromethane, EDC (326 mg) and HOBt (194 mg) were added under argon, and stirred for 3 minutes in an ice bath, then 3,4-dihydroxyphenylacetic acid (262) was added. Mg), the compound is stirred at room temperature for 5-8 hours, and after simple work-up, a crude product is obtained. After purification by column chromatography, compound IBMS002 (131 mg, yield 31%): 1H NMR (DMSO, 400 MHz): δ 10.82 (br s, 1H), 8.83 (br s, 1H ), 8.72 (br s, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.01 (dd, J = 8.0, 8.0 Hz, 1H), 6.94-6.91 (m, 1H), 6.66-6.62 (m, 2H), 6.53-6.47 (m, 1H), 4.66 (br s, 2H), 3.74-3.71 (m, 2H), 3.64-3.61 (m, 2H), 2.66-2.64 (m, 2H). Examples 1-3, N-(2-Pyridinylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS003)

 In a similar manner to the preparation of the compound IBMS001, 2-thiopheneacetyl chloride was replaced with 2-pyridineacetyl chloride, and the compound IBMS003 was purified after conventional treatment, yield 55%: 1H NM (400 MHz, DMSO): δ 10.85 (br s, 1H), 8.49-8.44 (m, 1H), 7.73 (dd, J = 7.6, 7.6 Hz, 1H), 7.39-7.33 (m, 2H), 7.31-7.24 (m, 2H), 7.02 (dd, J = 7.6, 7.6 Hz, 1H), 6.94 (dd, J = 7.6, 7.6 Hz, 1H), 4.79-4.68 (m, 2H), 4.00-3.98 (m, 2H), 3.89-3.81 (m, 2H) , 2.67-2.60 (m, 2H).

Examples 1-4, N-(4-Chlorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS004)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 4-chlorophenylacetic acid, and finally the compound IBMS004 was obtained in a yield of 52%. 1H NMR (DMSO, 300 MHz): δ 10.83 (br s, 1H), 7.36-7.31 (m, 3H), 7.29-7.21 (m, 3H), 7.01 (dd, J = 7.2, 7.2 Hz, 1H), 6.92 (dd, J = 7.2, 7.2 Hz, 1H), 4.68 (br s, 2H), 3.85-3.80 (m, 4H), 2.64-2.61 (m, 2H)

Examples 1-5, N-(4-Aminophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS005)

Compound I (225 mg) was added to dichloromethane, EDC (326 mg) and HOBt (194 mg) were added under argon, and the mixture was stirred for 15 minutes in an ice bath and then the compound 4-NHBoc-PhCH ₂ COOH (327 Mg), the compound is stirred at room temperature for 5-8 hours, and after simple work-up, a crude product is obtained. After purification by column, the intermediate was obtained. The intermediate was dissolved in DCM (2.0 mL). TFA (0.5 mL) was added dropwise in an ice bath, and the reaction was continued for two hours. After simple work-up, compound IBMS005 was obtained. Yield: 52 %. ^J H NMR (300 MHz, DMSO) δ 10.83 (br s, 1H), 9.29 (br s, 1H), 7.38-7.32 (m, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.11-6.89 (m, 3H), 6.69-6.67 (m, 2H), 6.64-6.59 (m, 1H), 4.68 (s, 2H), 3.84-3.72 (m, 4H), 2.68-2.48 (m, 2H). ―

EXAMPLES 1-6, N-(3-Hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS006)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was changed to 3-hydroxyphenylacetic acid, and finally a compound IBMS006 was obtained, yield: 30%. NM (300 MHz, DMSO) δ 10.83 (br s, IH), 9.29 (br s, IH), 7.38-7.32 (m, IH), 7.27 (d, J = 8.0 Hz, IH), 7.11-6.89 (m , 3H), 6.69-6.67 (m, 2H), 6.64-6.59 (m, IH), 4.68 (s, 2H), 3.84-3.72 (m, 4H), 2.68-2.48 (m, 2H).

Example 1-7, N-(4-N,N-Diethylphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS007)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was changed to 4-indole, fluorene-diethylbenzeneacetic acid, and finally the compound IBMS007 was obtained in a yield of 40%. NMR (DMSO, 300 MHz): δ 10.72 (br s, IH), 7.17 (dd, J = 7.5, 7.5 Hz, 2H), 6.92-6.88 (m, 2H), 6.85-6.79 (m, 2H), 6.46 (d, J = 8.4 Hz, 2H), 4.55 (br s, 2H), 3.68-3.64 (m, 2H), 3.53-3.51 (m, 2H), 2.50-2.43 (m, 2H), 2.37-2.33 ( m, 4H), 0.93 (t, J = 6.9 Hz, 6H).

Examples 1-8, N-(4-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS008)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was changed to 4-fluorophenylacetic acid, and finally the compound IBMS008 was obtained, yield: 68%. 1H NM (300 MHz, DMSO): δ 10.82 (br s, IH), 7.40-7.34 (m, IH), 7.32-7.27 (m, 3H), 7.15-7.09 (m, 2H), 7.03 (dd, J = 7.8, 7.2 Hz, IH), 6.94 (dd, J= 7.8, 7.2 Hz, IH), 4.72-4.69 (m, 2H), 3.86-3.84 (m, 2H), 3.83-3.81 (m, 2H), 2.67-2.62 (m, IH).

Examples 1-9, N-(2-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS009)

 In a similar manner to the preparation of the compound IBMS001, 2-thiopheneacetyl chloride was replaced with 2-fluorophenylacetyl chloride, and finally the compound IBMS009 was obtained in a yield of 48%: 1H NM (400 MHz, DMSO): δ 10.88 (br s, IH), 7.40 (d, J= 7.6 Hz, IH), 7.29 (dd, J= 7.2, 7.2 Hz, 2H), 7.26-7.24 (m, IH), 7.16 (d, J= 7.2 Hz, IH), 7.11 (d, J = 7.2 Hz, IH), 7.05-7.01 (m, IH), 6.96 (dd, J = 7.6, 7.6 Hz, IH), 4.79-4.70 (m, 2H), 3.89-3.88 (m, 2H), 3.86-3.82 (m, 2H), 2.75-2.67 (m, 2H).

Examples 1-10, 2-(lH-tetrazolylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS010)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with tetrazoliumacetic acid, and finally the compound IBMS010 was obtained in a yield of 57%. 1H NM (300 MHz, DMSO): δ 10.71 (br s, IH), 9.34-9.33 (m, IH), 7.45-7.41 (m, IH), 7.36-7.31 (m, IH), 7.09-7.04 (m , IH), 6.99 (dd, J = 7.2, 7.2 Hz, IH), 5.83-5.82 (m, 2H), 4.81-4.72 (m, 2H), 3.88-3.84 (m, 2H), 2.91-2.73 (m , 2H).

EXAMPLES 1-11, 2-(4-(2-NHBoc-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS011)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 4-(2-NHBoc-ethoxy)-phenylacetic acid, and finally the compound IBMS011 was obtained, yield: 30%. 1H NMR (300 MHz, DMSO): δ 10.86 (br s, IH), 7.35 (d, J = 7.8 Hz, IH), 7.28 (d, J = 7.8 Hz, IH), 7.19-7.11 (m, 2H) , 7.05-6.94 (m, 3H), ―

 6.88-6.85 (m, 2H), 4.72-4.64 (m, 2H), 3.89 (t, J = 4.2 Hz, 2H), 3.82-3.76 (m, 4H), 3.26 (s, 2H),

2.61-2.56 (m, 2H), 1.35 (s, 9H).

Examples 1-12, 2-(4-Bromomethylphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS012)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with 4-bromomethylphenylacetic acid to give the compound IBMS012, yield: 72%. 1H NMR (300 MHz, CDC1 ₃ ): δ 8.08 (br s, IH), 7.44-7.35 (m, 3H), 7.31-7.28 (m, 3H), 7.19-7.09 (m, 2H), 4.87-4.83 ( m, 2H), 4.62-4.54 (m, 2H), 3.90-3.85 (m, 2H), 3.81-3.77 (m, 2H), 2.69-2.64 (m, 2H).

Example 1-13, 2-(2-hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS013)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with 2-hydroxyphenylacetic acid, and finally the compound IBMS013 was obtained in a yield of 82%. ¹ H NMR (300 MHz, DMSO): δ 10.10 (br s, IH), 9.57 (s, 1H) _: 7.40-7.28 (m, 2H), 7.08-6.80 (m, 4H), 6.82-6.69 (m, 2H), 4.70 (s, 2H), 3.86-3.80 (m, 2H), 3.72 (s, 2H), 2.64-2.60 (m, 2H).

EXAMPLES 1-14, 2-(4-Aldehydephenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS014)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with 4-aldehyde-based phenylacetic acid, and finally the compound IBMS014 was obtained. Yield: 73% 1H NM (300 MHz, DMSO): δ 10.87 ( Br s, IH), 9.97 (br s, IH), 7.87-7.83 (m, 2H), 7.52-7.44 (m, 2H), 7.40-7.36 (m, IH), 7.31-7.29 (m, IH), 7.06-7.01 (m, IH), 6.98-6.93 (m, IH), 4.75-4.71 (m, 2H), 4.00-3.99 (m, IH), 3.87-3.83 (m, IH), 2.68-2.67 (m , 2H).

Examples 1-15, 2-(4-(2-hydroxyethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS015)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 4-(2-hydroxyethoxy)-phenylacetic acid to give the compound IBMS015, yield: 73%. NMR (300 MHz, DMSO): δ 8.13 (br s, IH), 7.43-7.41 (m, IH), 7.31-7.28 (m, IH), 7.23-7.18 (m, 2H), 7.15-7.09 (m, 2H), 6.90-6.81 (m, 2H), 4.86-4.82 (m, 2H), 4.06-4.03 (m, 2H), 3.98-3.93 (m, 2H), 3.84-3.81 (m, 2H), 3.80- 3.78 (m, 2H), 2.68-2.63 (m, 2H).

EXAMPLES 1-16, 2-(4-(3-Morpholinylpropoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS016)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with 4-(3-morpholinylpropoxy)-phenylacetic acid to give the compound IBMS016, yield: 58%. NMR (300 MHz, CDC1 ₃ ): δ 8.36 (br s, IH), 7.50-7.40 (m, IH), 7.29-7.27 (m, IH), 7.21-7.05 (m, 4H), 6.88-6.79 (m , 2,,,,, -1.72 (m, 2H).

Examples 1-17, 2-(4-carboxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS017) ―

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with methyl (4-carboxymethyl)-benzoate, followed by hydrolysis under the action of LiOH, and finally the compound IBMS017 was obtained. Yield: 55 %. NMR (300 MHz,

DMSO): δ 12.47 (s, IH), 10.81 (br s, IH), 7.43-7.35 (m, 6H), 7.07-7.02 (m, IH), 6.99-6.94 (m, IH),

4.75-7.67 (m, 2H), 3.84-3.75 (m, 2H), 3.60-3.57 (m, 2H), 2.80-2.75 (m, 2H).

EXAMPLES 1-18, 2-(4-(2-Aminoethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS018)

The compound IBMSOll (72 mg, 0.23 mmol) was dissolved in 2 ml of CH ₂ C1 ₂ and the reaction was cooled in an ice water bath.

After 10 min, TFA (0.5 ml) was added and the reaction was continued for 20 min. Stirring was continued for 3 h. After the usual treatment, the compound IBMS018 was obtained. Yield: 20% 1H NM (300 MHz, DMSO): δ 10.85 (br s, IH), 7.35 ( d, J = 7.8 Hz, IH), 7.29 (d, J =

7.8 Hz, IH), 7.20-7.12 (m, 2H), 7.03 (dd, J = 7.5, 7.2 Hz, IH), 6.94 (dd, J = 7.5, 7.2 Hz, IH),

6.86-6.84 (m, 2H), 4.70-4.65 (m, 2H), 3.92-3.88 (m, 2H), 3.83-3.81 (m, 2H), 3.78-3.76 (m, 2H),

2.90-2.86 (m, 2H), 2.67-2.65 (m, IH), 2.60-2.58 (m, IH).

Examples 1-19, 2-(4-((methoxycarbonyl)methoxy)phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS019)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 4-((methoxycarbonyl)methoxy)phenylacetic acid, and finally the product IBMS019 was obtained. Yield: 43%: 1H NM ( 300 MHz, DMSO): δ 11.02 (br s, IH), 7.39 (d, J = 8.1 Hz, IH), 7.30 (d, J = 8.1 Hz, 2H), 7.22-7.19 (m, IH), 7.03 ( Dd, J= 7.2, 6.9 Hz, IH), 6.97 (d, J = 7.2 Hz, IH), 6.92-6.89 (m, 2H), 4.95-4.92 (m, 2H), 4.74-4.63 (m, 3H) , 3.80-3.77 (m, 2H), 3.69-3.67 (m, IH), 3.34-3.31 (m, IH), 2.82-2.67 (m, 2H).

Examples 1-20, N-(Phenylpropionyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS020)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with phenylpropionic acid to give the compound IBMS020: 1H NM (400 MHz, DMSO): δ 10.82 (s, IH), 7.38-7.35 (m, IH), 7.28-7.23 (m, 5H), 7.17-7.13 (m, IH), 7.02 (dd, J = 8.0, 8.0 Hz, IH), 6.94 (dd, J = 8.0, 8.0 Hz, IH ), 4.66 (s, 2H), 3.81 (t, J = 4.0 Hz, IH), 3.74 (t, J = 4.0 Hz, IH), 2.87-2.81 (m, 2H), 2.74-2.71 (m, 4H) ¹³ C NMR (100 MHz, DMSO): δ 170.7, 141.5, 135.9, 131.4, 128.5, 128.3, 126.6, 125.9, 120.8, 118.5, 117.5, 111.0, 106.7, 43.3, 34.2, 30.8, 30.7, 21.6.

Example 1-21, N-(3-NHBoc-3-phenyl)propanoyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS021)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-NHBoc-3-phenylpropionic acid to give the compound IBMS021, 1H NMR (CDC1 ₃ , 400 MHz): δ 10.83 (br s, IH), 7.37-7.35 (m, IH), 7.30-7.28 (m, 2H), 7.26-7.24 (m, 2H), 7.21-7.18 (m, 2H), 7.03 (dd, J= 7.2, 7.2 Hz, IH), 6.95 (dd, J = 7.2, 7.2 Hz, IH), 4.82-4.72 (m, 2H), 4.54-4.50 (m, IH), 3.88-3.84 (m, 2H), 2.69-2.61 ( m, 2H), 2.45-2.41 (m, 2H), 1.30 (s, 9H).

Example 1-22, N-(2-(2-(R)-NHBoc-phenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS022) ―

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(2-(R)-NHBoc-phenyl)acetic acid, and finally the compound IBMS022 was obtained in a yield of 56%: 1H NM ( 400 MHz, DMSO) δ 10.83 (br s, 1H) _:

7.42-7.40 (m, 2H), 7.37-7.34 (m, 2H), 7.32-7.31 (m, IH), 7.29-7.26 (m, 3H), 7.01 (dd, J = 7.6 Hz,

7.6 Hz, IH), 6.93-6.91 (m, IH), 5.75-5.71 (m, IH), 4.79-4.63 (m, 2H), 3.82-3.67 (m, 2H), 2.49 (m,

2H), 1.37 (s, 9H).

Examples 1-23, N-(2-(2-(S)-NHBoc-phenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS023)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(2-(S)-NHBoc-phenyl)acetic acid, and finally the compound IBMS023 was obtained in a yield of 64%: ^J H NM (400 MHz, DMSO): δ 10.83 (br s, IH), 7.41 (d, J = 7.2 Hz, 2H), 7.36 (d, J = 7.2 Hz, 2H), 7.32-7.31 (m, 2H), 7.29 -7.28 (m, 2H), 7.00 (dd, J= 8.0, 7.6 Hz, IH), 6.92 (dd, J= 8.0, 7.6 Hz, IH), 6.93-6.90 (m, IH), 5.72 (d, J = 8.0 Hz, 1H) _: 4.77 (d, J = 16.8 Hz, IH), 4.65 (d, J = 16.8 Hz, IH), 3.81-3.76 (m, IH), 3.73-3.68 (m, IH), 2.67 -2.63 (m, IH), 2.11-2.09 (m, IH), 1.37 (s, 9H).

Example 1-24, N-(2-(2-(R)-Aminophenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS024)

 The compound IBMS022 (180 mg, 0.44 mmol) was dissolved in dichloromethane (2.0 mL), and trifluoroacetic acid (0.5 mL) was added dropwise under ice bath. After 2 hours, excess solvent was removed under reduced pressure, and then 1 M NaOH solution was used. Adjust the system to pH=12 or so. Yield 60%: 1H NM (400 MHz, DMSO) δ 10.83 (br s, IH), 7.39-7.37 (m, 2H), 7.34-7.31 (m, 3H), 7.28-7.26 (m, 2H), 7.02 (dd, J = 7.6 Hz, 7.6 Hz, IH), 6.93-6.90 (m, IH), 5.07-5.01 (m, IH), 4.89-4.65 (m, 2H), 3.75-3.55 (m, 2H), 3.59-3.41 (m, 2H), 2.66-2.56 (m, IH), 2.14-2.10 (m, IH). Example 1-25, N-(3-Amino-3-phenyl)propanoyl)-1 ,3,4,9-tetrahydro-1Η-β-carboline (IBMS025)

 In a similar manner to the preparation of the compound IBMS024, the compound IBMS022 was replaced by IBMS021 to give the compound IBMS025, yield: 57%. 1H NMR (DMSO, 400 MHz): δ 10.78 (br s, IH), 7.35 (d, J = 8.0 Hz, IH), 7.30 (d, J = 8.0 Hz, IH), 7.23-7.19 (m, 2H) , 7.17-7.14 (m, 3H), 7.03 (dd, J= 7.2, 7.2 Hz, IH), 6.95 (dd, J= 7.2, 7.2 Hz, IH), 4.80-4.70 (m, 2H), 4.09-4.05 (m, IH), 3.99-3.91 (m, 2H), 3.77-3.75 (m, 2H), 3.31 (br s, 2H).

Examples 1-26, N-(2-(2-(S)-Aminophenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS026)

In a similar manner to the preparation of the compound IBMS024, the compound IBMS022 was replaced by IBMS023, and finally the product IBMS026, yield 72%: ^J H NMR (400 MHz, DMSO): δ 10.85 (br s, IH), 7.42 (d, J = 7.2 Hz, 2H), 7.35 (d, J = 7.2 Hz, 2H), 7.31-7.30 (m, 2H), 7.29-7.28 (m, 2H), 7.02 (dd, J= 8.0, 7.6 Hz, IH ), 6.93 (dd, J= 8.0, 7.6 Hz, IH), 6.93-6.90 (m, IH), 5.07-5.04 (m, IH), 4.77 (d, J= 16.8 Hz, IH), 4.65 (d, J = 16.8 Hz, IH), 3.81-3.78 (m, 2H), 3.56-3.50 (m, 2H), 2.68-2.62 (m, 2H).

Examples 1-27, N-(2-(4-Acylphenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS027) ―

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-aldehydephenyl)propionic acid, and finally the compound IBMS027 was obtained in a yield of 60%: 1H NMR (CDC1 ₃ , 400 MHz): δ 9.91 (br s, IH), 8.45 (br s, IH), 7.85 (d, J= 8.0 Hz, 2H), 7.48 (d, J= 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, IH ), 7.29 (d, J= 8.0

Hz, IH), 7.13 (dd, J = 8.0, 8.0 Hz, IH), 7.06 (dd, J = 8.0, 8.0 Hz, IH), 4.95 (d, J = 16.8 Hz, IH),

4.81 (d, J= 16.8 Hz, IH), 4.13 (t, J= 5.6 Hz, IH), 3.73-3.70 (m, 2H), 2.72-2.65 (m, IH), 2.33-2.27

(m, IH), 2.05 (d, J = 5.6 Hz, 3H).

Examples 1-28, N-(2-(4-Bromomethylphenyl)propanoyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS028)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-bromomethylphenyl)propionic acid, and finally the compound IBMS028 was obtained, yield 40%: 1H NM (400 MHz , DMSO) δ 9.15 (br s, IH), 7.44-7.42 (m, IH), 7.39-7.32 (m, 4H), 7.27-7.25 (m, IH), 7.17 (dd, J = 7.2 Hz, 7.2 Hz , IH), 7.10 (dd, J= 7.2 Hz, 7.2 Hz, IH), 4.99-4.88 (m, 2H), 4.12 (q, J= 6.8 Hz, IH), 3.74 (m, 2H), 2.72 (d , J = 15.2 Hz, 1H) 2.36 (d, J= 15.2 Hz, IH), 1.60 (d, J= 6.8 Hz, 3H)

Examples 1-29, N-(2-(4-Hydroxymethylphenyl)propanoyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS029)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-hydroxymethylphenyl)propionic acid, and finally the compound IBMS029 was obtained in a yield of 45%: 1H NM (CDC1 ₃ , 400 MHz): δ 8.33 (br s, IH), 7.38 (d, J = 8.0 Hz, IH), 7.35-7.33 (m, 2H), 7.31-7.29 (m, 2H), 7.25 (d, J = 8.0 Hz, IH), 7.14 (dd, J = 8.0, 8.0 Hz, IH), 7.07 (dd, J= 8.0, 8.0 Hz, IH), 4.84 (d, J= 16.4 Hz, IH), 4.79 (d, J= 16.4 Hz, 1H) _: 4.68 (s, 2H), 4.09 (t, J= 6.4 Hz, IH), 3.75-3.68 (m, 2H), 2.70-2.64 (m, IH), 2.33-2.27 (m , IH), 1.53 (d, J= 6.4 Hz, 3H)

Examples 1-30, N-(2-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS030)

 In a similar manner to the preparation of the compound IBMS001, 2-thiopheneacetyl chloride was replaced with phenylacetyl chloride, which was purified by conventional treatment to obtain IBMS030, yield 82%: 1H NM (400 MHz, DMSO): δ 10.85 (s, IH) , 7.30-7.27 (m, 6H), 7.22-7.20 (m, IH), 7.03-7.01 (m, IH), 6.94-6.91 (m, IH), 4.68 (s, 2H), 3.85-3.81 (m, 4H), 3.30 (s, 2H).

EXAMPLES 1-31, N-(4-Hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS031)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was changed to 4-hydroxyphenylacetic acid, and finally the compound IBMS031 was obtained in a yield of 57%: 1H NM (400 MHz, DMSO): δ 10.84 (br s, IH), 9.26 (s, IH), 7.40-7.28 (m, 2H), 7.29 (d, J = 10.4 Hz, IH), 7.06 (d, J = 11.2 Hz, IH), 7.02 (d, J = 9.6 Hz, IH), 6.98-6.92 (br m, 2H), 6.71-6.66 (m, 2H), 4.68 (s, 2H), 3.84-3.77 (m, 2H), 3.72-3.70 (m, 2H) ^{, 2.55-2.53 (m, 2H) 13} C NMR (100 MHz, DMSO):. δ 170.7, 141.5, 135.9, 131.4, 128.5, 128.3, 126.6, 125.9, 120.8, 118.5, 117.5, 111.0, 106.7, 43.3, 34.2 , 30.8, 21.6. ―

Examples 1-32, N-(2-hydroxy-2-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS032)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was exchanged for 2-hydroxy-2-phenylacetic acid, and finally the compound IBMS032 was obtained in a yield of 22%: 1H NM (CDC1 ₃ , 400 MHz) : δ 10.86 (br s, 1H), 7.42-7.36 (m, 4H), 7.34-7.31 (m, 3H), 7.15 (dd, J= 8.4, 8.4 Hz, 1H), 6.92 (dd, J= 8.4, 8.4 Hz, 1H), 5.83-5.73 (m, 2H), 5.49 (d, J= 10.0 Hz, 1H), 4.81-4.73 (m, 2H), 3.84-3.78 (m, 2H).

Example 1-33, N-(2-hydroxy-2,2-diphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS033)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced with 2-hydroxy-2,2-diphenylacetic acid, and finally the compound IBMS033 was obtained in 23% yield: 1H NMR (CDC1 ₃ , 400 MHz): δ 8.01 (br s, 1H), 7.44-7.39 (m, 3H), 7.37-7.34 (m, 2H), 7.31-7.27 (m, 4H), 7.26-7.24 (m, 2H), 7.14 (dd, J = 7.6, 7.6 Hz, 2H), 7.07-7.05 (m, 1H), 5.00 (br s, 2H), 3.52 (br s, 2H), 2.95-2.88 (m, 2H).

Example 1-34, N-(2-(4-morpholinylmethylphenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS034)

 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-(morpholinyl)phenyl)propanoic acid to give the compound IBMS034, yield: 60%. 1H NMR (300 MHz, DMSO): δ 10.82 (br s, 1H), 7.28-7.20 (m, 6H), 7.00 (dd, J = 7.8, 7.2 Hz, 1H), 6.91 (dd, J = 7.2, 7.2 Hz, 1H), 4.87 (d, J = 16.8 Hz, 1H), 4.55 (d, J = 16.8 Hz, 1H), 4.24-4.17 (m, 1H), 3.84-3.80 (m, 1H), 3.50-3.54 (m, 6H), 2.58-2.54 (m, 1H), 2.33-2.29 (m, 4H), 2.03-1.99 (m, 1H), 1.31 (d, J = 6.3 Hz, 3H).

Example 1-35, N-(2-(4-((diethylamino)methyl)phenyl)propanoyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS035 In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-((diethylamino)methyl)phenyl)propanoic acid, and finally the compound IBMS035 was obtained. Yield: 36%. NMR (DMSO, 300 MHz): δ 10.70 (br s, 1H), 7.24 (d, J = 7.8 Hz, 2H), 7.17 (dd, J = 7.8, 7.8 Hz, 2H), 6.92-6.87 (m, 2H) ), 6.84-6.79 (m, 2H), 4.51 (br s, 2H), 3.67-3.63 (m, 2H), 3.62-3.58 (m, 2H), 2.63-2.59 (m, 2H), 1.98 (br s , 6H). Example 1-36 2-((2-(4-(lH- 1 ,2,4-triazolyl-1-yl)methyl)phenyl)propanoyl)-1,3,4 ,9-tetrahydro-1 Η-β-carboline (IBMS036)

Using a method similar to the preparation of the compound IBMS002, replacing 3,4-dihydroxyphenylacetic acid with 2-(4-((lH-l,2,4-triazolyl-1-)methyl)phenyl)propane Acid, finally gave the compound IBMS036, yield: 30%. 1H NMR (300 MHz, DMSO): δ 10.90 (br s, 1H), 8.70-8.61 (m, 1H), 7.99-7.94 (m, 1H), 7.32-7.21 (m, 6H), 7.01 (dd, J = 7.5, 7.2 Hz, 1H), 6.92 (dd, J = 7.5, 7.2 Hz, 1H), 5.42-5.35 (m, 2H), 4.76-4.62 (m, 1H), 4.27-4.23 (m, 1H), 3.73-3.70 (m, 1H), 3.50-3.46 (m, 2H), 2.64-2.50 (m, 1H), 2.16-2.05 (m, 1H), 1.30-1.27 (m, 3H). One.

Example 1-37, 1-phenyl-N-phenylacetyl-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS037)

The amine (690 mg/4.30 mmol) was taken in toluene (60 ml), and trifluoroacetic acid (0.64 ml) was added dropwise to the system. After the solution became homogeneous, benzaldehyde (0.53 ml/4.8 mmol) was added dropwise. The oil-water separator is installed on the flask, and the reaction system is heated to 140 ° C for 4-8 h. After the water is substantially not generated, the solution is cooled to room temperature, and most of the solvent is removed by concentration under reduced pressure. The intermediate product 1-phenyl-1,3,4,9-tetrahydro-1?-?-carboline was obtained. This compound was dissolved in DMF, and phenylacetic acid was added in the presence of EDC/HOBt to give the desired product. This compound was dissolved in DMF, and phenylacetic acid was added in the presence of EDC/HOBt to give the desired product, IBMS037, yield 49%. ^J H NM (300 MHz, CDC1 ₃ ): δ 8.18 (br s, 1H), 7.46 (d, J= 7.5 Hz, 1H), 7.25-7.16 (m, 11H), 7.13-7.08 (m, 2H), 3.96-3.90 (m, 1H), 3.81-3.74 (m, 2H), 3.38-3.30 (m, 1H), 2.76-2.70 (m, 1H), 2.60-2.56 (m, 1H), 1.71-1.68 (m , 1H). ¹³ C NMR (400 MHz, CDC1 ₃ ): δ 22.1, 40.5, 41.5, 52.3, 109.7, 111.5, 118.2, 119.6, 122.2, 126.7, 127.0, 128.2, 128.6, 128.8, 128.9, 131.8, 135.1, 136.5, 140.1, 170.0.

Example 1-38, 1-Ethyl-N-phenylacetyl-1,3,4,9-trihydro-1Η-β-carboline (IBMS038)

In a similar manner to the preparation of the compound IBMS037, the benzaldehyde was changed to propionaldehyde, and finally the compound IBMS038 was obtained. Yield: 35% 1H NM (300 MHz, CDC1 ₃ ): δ 8.58 (br s, 1H), 7.53 - 7.50 (m, 1H), 7.45-7.37 (m, 6H), 7.25 (dd, J = 7.5, 7.5 Hz, 1H), 7.21-7.16 (m, 1H), 5.90 (t, J = 6.9 Hz, 1H), 4.23-4.19 (m, 1H), 4.04-4.00 (m, 2H), 3.60-3.49 (m, 1H), 2.81-2.75 (m, 1H), 2.68-2.58 (m, 1H), 2.09-2.03 (m , 1H), 1.98-1.90 (m, 1H), 1.88-1.86 (m, 2H), 1.13 (t, J= 7.5 Hz, 1H).

Example 1-39, 2-(4-(2-NH-Biotin-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS039)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 4-(2-NH-Biotin-ethoxy)-phenylacetic acid, and finally the compound IBMS039 was obtained in a yield: 71%. 1H NMR (300 MHz, DMSO): δ 10.86 (br s, 1H), 8.07-8.04 (m, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.29 (d, J = 7.8 Hz, 1H) , 7.21-7.13 (m, 2H), 7.05-7.01 (m, 2H), 6.97-6.95 (m, 2H), 6.90 -6.85 (m, 2H), 6.43-6.36 (m, 2H), 4.74-4.65 ( m, 2H), 4.28-4.26 (m, 1H), 4.09-4.07 (m, 1H), 3.94-3.92 (m, 2H), 3.81-3.76 (m, 4H), 3.36-3.34 (m, 1H), 3.05-3.03 (m, 1H), 2.80-2.77 (m, 1H), 2.58-2.50 (m, 2H), 2.08-2.06 (m, 2H), 1.50-1.45 (m, 3H), 1.30-1.21 (m , 3H). ESI-MS m/z calcd for C ₃₁ H ₃₈ N ₅ 0 ₄ S [M + H] ⁺ : 576; C ₃₁ H ₃₇ N ₅ 0 ₄ SNa [M + Na] ⁺ : 598.

Example l-40, N-(2-(4-((2-morpholinoethylamino)methyl)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β- Porphyrin (IBMS040) ―

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-((2-morpholinoethylamino)methyl)phenyl)-propionic acid, and finally the compound IBMS040 was obtained. , Yield: 67%. NMR (300 MHz, CDC1 ₃ ): δ

8.35 (br s, 1H), 7.39-7.33 (m, 2H), 7.30-7.27 (m, 4H), 7.17-7.10 (m, 1H), 7.09-7.07 (m, 1H), 4.93

(d, J = 16.8 Hz, 1H), 4.80 (d, J = 16.8 Hz, 1H), 4.07-4.03 (m, 1H), 3.82-3.77 (m, 2H), 3.74-3.65 (m,

6H), 2.83-2.70 (m, 4H), 2.52-2.48 (m, 2H), 2.40-2.36 (m, 4H), 2.31-2.18 (m, 1H), 1.90-1.84 (m,

2H), 1.51 (d, J= 6.9 Hz, 3H).

Example 1-41, N-(2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η -β-porphyrin (IBMS041)

In a similar manner to the preparation of the compound IBMS002, 3,4-dihydroxyphenylacetic acid was replaced by 2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionic acid, and finally The compound IBMS041 was obtained in a yield: 49%. NMR (300 MHz, CDC1 ₃ ): δ 8.29 (br s, 1H), 7.42-7.40 (m, 1H), 7.30-7.27 (m, 1H), 7.22-7.16 (m, 2H), 7.14-7.08 (m , 2H), 6.86-6.77 (m, 2H), 5.24-5.23 (m, 1H), 4.79-4.71 (m, 2H), 4.15-4.08 (m, 2H), 4.00-3.74 (m, 2H), 3.84 -3.81 (m, 2H), 3.78-3.76 (m, 2H), 3.58-3.54 (m, 1H), 3.38-3.34 (m, 2H), 2.73-2.67 (m, 2H).

Example 1-42, N-Phenylacetyl Small Ethyl -1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline (IBMS042)

5-benzyloxytryptamine (390 mg, 1.50 mmol) was dissolved in glacial acetic acid (6 ml). After stirring for 10 min, propionaldehyde (91 μl, 1.60 mmol) was added dropwise, and the reaction was heated to 80 ° C. After 4 hours, NaOH was added to pH = 9, and the crude product was extracted. The compound was dissolved in DMF, and a similar treatment was carried out by adding phenylacetic acid in the presence of EDC/HOBt in the same manner as in the preparation of compound IBMS002. Target product: IBMS042, Yield: 62%: 1H NM (300 MHz, DMSO): δ 10.68 (br s, 1H), 7.45 (d, J = 6.9 Hz, 2H), 7.39 (d, J = 6.9 Hz, 2H ), 7.33-7.30 (m, 4H), 7.25-7.22 (m, 1H), 7.18 (d, J= 8.7 Hz, 2H), 6.95-6.94 (m, 1H), 6.75 (dd, J= 8.7, 2.4 Hz, 1H), 5.55-5.52 (m, 1H), 5.06 (s, 2H), 4.20-4.14 (m, 1H), 3.89 (d, J= 15.0 Hz, 1H), 3.82 (d, J= 15.0 Hz , 1H), 3.39-3.34 (m, 1H), 2.60-2.57 (m, 1H), 2.46-2.42 (m, 1H), 1.97-1.87 (m, 1H), 1.78-1.66 (m, 1H), 0.90 (t, J = 7.5 Hz, 3H). ¹³ C NM (75 MHz, DMSO): δ 169.7, 152.1, 137.8, 136.0, 135.9, 131.0, 128.8, 128.3, 128.2, 127.5, 126.6, 126.3, 111.5, 111.1, 106.0, 101.6, 69.8, 49.7, 49.6 , 40.3, 26.9, 21.7, 10.7.

Example 1-43, N-Phenylacetyl minimethyl-1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline (IBMS043)

The propanal was replaced with acetaldehyde in a similar manner to the preparation of the compound IBMS042, yield: 79%, ^J H NM (300 MHz, DMSO): δ 10.70 (br s, 1H), 7.45-7.43 (m, 2H ), 7.38-7.34 (m, 2H), 7.31-7.15 (m, 7H), 6.95 (dd, J= 2.1, 8.7 Hz, 1H), 6.74 (dd, J=2.4, 8.7 Hz, 1H), 5.54 ( q, J= 6.6 Hz, 1H), 5.05 (s, 2H), 4.13 (dd, J = 3.9, 13.5 Hz, 1H), 3.82 (s, 2H), 3.35-3.26 (m, 1H), 2.60-2.52 (m, 1H), 2.42-2.32 (m, 1H), ―

 1.37 (t, J= 6.6 Hz, 3H).

Example 1-44, N-Phenylacetyl-1-methyl-1,3,4,9-tetrahydro-6-hydroxy-1Η-β-carboline (IBMS044)

The compound IBMS043 (107 mg, 0.26 mmol) was suspended in methanol (10 ml), and the benzyl group was hydrogenated under hydrogen atmosphere using Pd (10% wt) as a catalyst to obtain the product IBMS044 (71 mg, yield: 86%): 1H NM (300 MHz, DMSO): δ 10.52 (br s, IH), 8.54 (br s, IH), 7.32-7.23 (m, 5H), 7.06 (d, J = 8.4 Hz, IH) , 6.64-6.63 (m, IH), 6.55-6.53 (m, IH), 5.57-5.45 (m, IH), 4.15-4.09 (m, IH), 3.85-3.71 (m _: IH), 3.31-3.24 ( m, IH), 2.60-2.51 (m, IH), 2.44-2.32 (m, IH), 1.37 (d, J = 6.9 Hz, 3H).

Example 1-45, N-phenylacetyl-6-fluoro-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS045)

3-(2,3-piperidinone)-(4-fluorophenyl)-indole (754 mg, 3.41 mmol, preparation method Reference Syn. Comm. 2007, 37, 1273-1280) was dissolved in 20 ml The formic acid was stirred at 80 ° C for 24 hours, and the formic acid was removed under reduced pressure to give an off-white solid, 1-oxo-6-fluoro-1,3,4,9-tetrahydro-β-carboline, yield 84. %. The porphyrin intermediate was reduced by lithium aluminum hydride and coupled with phenylacetic acid to obtain the final product IBMS045, yield: 62%. ¹ H NMR (300 MHz, DMSO): δ 10.96 (br s, IH), 7.29-7.17 (m, 6H), 7.13-7.10 (m, IH), 6.89-6.83 (m, IH), 4.70 (s, 2H), 3.86-3.81 (m, 4H), 2.65-2.56 (m, 2H).

Example 1-46, N-Phenylacetyl-6-bromo-1,3,4,9-tetrahydro-1Η-β-carboline (IBMS046)

3-(2,3-piperidindione)-(4-fluorophenyl)-indole was replaced with 3-(2,3-piperidindione)-(4) in a similar manner to the preparation of compound IBMS002 -Bromophenyl)-indole, finally the compound IBMS046, Yield: 62%: 1H NM (300 MHz, DMSO): δ 10.84 (br s, IH), 7.36-7.23 (m, 6H), 7.05-7.00 ( m, IH), 6.97-6.92 (m, IH), 4.70 (s, 2H), 3.86-3.82 (m, 4H), 2.69-2.59 (m, 2H).

Claims

Claim 1 WO 2013/060179 PCT/CN2012/079841.

 An acyltetrahydro-β-carboline. 'J, a molecular organic compound or a hydrate thereof or a pharmaceutically acceptable salt thereof, which is represented by the following structural formula (I):

 among them:

n, m is 0-3 CH ₂ ;

@ is an aromatic or heterocyclic aromatic crane, including monocyclic aromatic groups, polycyclic aromatic cranes, polyheterocyclic aromatic groups; leg monocyclic aromatic groups including phenyl, azaaryl, thiaaryl, oxaaryl ;

Independently selected from one or more of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, cyclic amine, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₁₂ cycloalkyl, benzyl, alkylcarbonyl, C ₂ -C ₁₂ alkenylcarbonyl, C ₃ -C ₁₂ cycloalkylcarbonyl, benzene Carbonyl group, benzylcarbonyl group, alkoxycarbonyl group, ester group, sulfoxide group, sulfone group, sulfinamide group, sulfonamide group; morpholinyl group; piperazinyl group;

Any one selected from the group consisting of hydrogen, alkyl, halogen, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, Benzyl, phenethyl, phenylpropyl, alkoxy, amino, alkylcarbonyl and arylcarbonyl, morpholinylmethyl, hydroxymethyl;

Any one or more selected from the group consisting of hydrogen, alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl , benzyl, phenethyl, phenylpropyl, amino, cyano, nitro, halogen;

, R ₅ , R, and R _{7 are} independently selected from one of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, Cycloalkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, benzyl, alkylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, sulfonamide, sulfonamide; morpholinyl; Piperazinyl;

Any one or two selected from the group consisting of hydrogen, alkyl, hydroxy, hydroxymethyl, amino, carboxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ ring Alkyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkoxy, alkylamino, alkylcarbonyl, arylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ - C ₈ cycloalkylcarbonyl;

Any one selected from the group consisting of hydrogen, alkyl, hydroxymethyl, alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkenyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkylamino.

The acyltetrahydro-β-carboline small molecule organic compound or hydrate thereof according to claim 1, or pharmaceutically acceptable Claim

One WO 2013/060179 PCT/CN2012/079841

The salt is characterized in that when n is 1 CH ₂ and R ₃ is hydrogen, it is represented by the following structural formula (Π):

 among them:

m is 0-3 CH _2;

@ is an aromatic or heterocyclic aromatic crane, including monocyclic aromatic groups, polycyclic aromatic cranes, polyheterocyclic aromatic groups; leg monocyclic aromatic groups including phenyl, azaaryl, thiaaryl, oxaaryl ;

Independently selected from one or more of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, cyclic amine, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₁₂ cycloalkyl, benzyl, alkylcarbonyl, C ₂ -C ₁₂ alkenylcarbonyl, C ₃ -C ₁₂ cycloalkylcarbonyl, benzene Carbonyl group, benzylcarbonyl group, alkoxycarbonyl group, ester group, sulfoxide group, sulfone group, sulfinamide group, sulfonamide group; morpholinyl group; piperazinyl group;

Any one selected from the group consisting of hydrogen, alkyl, halogen, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, Benzyl, phenethyl, phenylpropyl, alkoxy, amino, alkylcarbonyl and arylcarbonyl, morpholinylmethyl, hydroxymethyl;

, R ₅ , R, and R _{7 are} independently selected from one of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, Cycloalkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, benzyl, alkylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, sulfonamide, sulfonamide; morpholinyl; Piperazinyl;

R _{8 is} optionally selected from one or both of the following groups: hydrogen, alkyl, hydroxy, hydroxymethyl, amino, carboxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, 3⁄4 ₈ naphthenic Base, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkoxy, alkylamino, alkylcarbonyl, arylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₈ -cycloalkylcarbonyl;

Any one selected from the group consisting of hydrogen, alkyl, hydroxymethyl, alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkenyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkylamino.

The acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to claim 1, wherein when n is 1 CH ₂ , R ₃ , When R ₈ and R ₉ are hydrogen, they are represented by the following structural formula (III): Claim

 -WO 2013/060179 PCT/CN2012/079841

 among them:

m is 0-3 CH _2;

@ is an aromatic or heterocyclic aromatic crane, including monocyclic aromatic groups, polycyclic aromatic cranes, polyheterocyclic aromatic groups; leg monocyclic aromatic groups including phenyl, azaaryl, thiaaryl, oxaaryl ;

Independently selected from one or more of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, cyclic amine, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₁₂ cycloalkyl, benzyl, alkylcarbonyl, C ₂ -C ₁₂ alkenylcarbonyl, C ₃ -C ₁₂ cycloalkylcarbonyl, benzene Carbonyl group, benzylcarbonyl group, alkoxycarbonyl group, ester group, sulfoxide group, sulfone group, sulfinamide group, sulfonamide group; morpholinyl group; piperazinyl group;

Any one selected from the group consisting of hydrogen, alkyl, halogen, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, Benzyl, phenethyl, phenylpropyl, alkoxy, amino, alkylcarbonyl and arylcarbonyl, morpholinylmethyl, hydroxymethyl;

, R ₅ , R, and R _{7 are} independently selected from one of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, Cycloalkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, benzyl, alkylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, sulfonamide, sulfonamide; morpholinyl; Piperazinyl.

The acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to claim 1, wherein when n is 1 CH ₂ , m is 0 When CH ₂ , and ₉ are hydrogen, they are represented by the following structural formula (IV):

An aromatic or heterocyclic aromatic group, including a monocyclic aromatic group, a polycyclic aromatic group, a polyheterocyclic aromatic group; the single ring Claim

A WO 2013/060179 PCT/CN2012/079841 aryl group includes phenyl, azaaryl, thiaaryl, oxaaryl;

Independently selected from one or more of the following groups: hydrogen, amino, cyano, hydroxy, nitro, halogen, carboxy, alkyl, alkoxy, amine, cycloalkoxy, cyclic amine, C ₂ -C ₁₂ alkenyl, C ₂ -C ₁₂ alkynyl, C ₃ -C ₁₂ cycloalkyl, benzyl, alkylcarbonyl, C ₂ -C ₁₂ alkenylcarbonyl, C ₃ -C ₁₂ cycloalkylcarbonyl, benzene Carbonyl group, benzylcarbonyl group, alkoxycarbonyl group, ester group, sulfoxide group, sulfone group, sulfinamide group, sulfonamide group; morpholinyl group; piperazinyl group;

Any one selected from the group consisting of hydrogen, alkyl, halogen, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, Benzyl, phenethyl, phenylpropyl, alkoxy, amino, alkylcarbonyl and arylcarbonyl, morpholinylmethyl, hydroxymethyl;

And R ₅ , R, and R ₇ are a substituent on the phenyl ring, independently selected from one of the following groups: hydrogen, amino, cyano, hydroxy, nitro, 3⁄4, carboxy, alkyl, alkoxy , amine, cycloalkoxy, cycloalkylamino, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ cycloalkyl, aryl, heterocyclic aryl, benzyl , alkylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, phenylcarbonyl, benzylcarbonyl, alkoxycarbonyl, ester, sulfoxide, sulfone, sulfenamide Sulfoamide; morpholinyl; piperazinyl;

Any one or two selected from the group consisting of hydrogen, alkyl, hydroxy, hydroxymethyl, amino, carboxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₈ ring Alkyl, aryl, heterocyclic aryl, benzyl, phenethyl, phenylpropyl, alkoxy, alkylamino, alkylcarbonyl, arylcarbonyl, C ₂ -C ₆ alkenylcarbonyl, C ₃ - C ₈ cycloalkylcarbonyl.

 The acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, which comprises the acyl group An acid addition salt of a tetrahydro-β-carboline small molecule organic compound with an acid; wherein the acid includes, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, tartaric acid, salicylic acid, citric acid , methanesulfonic acid, p-toluenesulfonic acid, lactic acid, pyruvic acid, maleic acid, succinic acid.

 The acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, which is characterized in that it is radioactive or fluorescent Group or biotin label.

 The acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to claim 1, which comprises:

 Ν-(2-Thienylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(3,4-dihydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-Pyridinylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-chlorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-aminophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(3-hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

Ν-(4-Ν,Ν-diethylphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline, Claim

One WO 2013/060179 PCT/CN2012/079841

N-(4-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-fluorophenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(1Η-tetrazolylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(4-(2-NHBoc-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-bromomethylphenylacetyl)-1,3,4,9-tetrahydro-1 Η-β-carboline,

 Ν-(2-hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-aldehyde phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-(2-hydroxyethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

_Ν- (4-(3-morpholinopropoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-carboxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-(2-Aminoethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

Ν-Phenylpropanoyl-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-((3-NHBoc-3-phenyl)propanoyl)-2,3,4,9-tetrahydro-1 3-porphyrin,

 N-(2-(2-(R)-NHBoc-phenyl)acetyl)-1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 N-(2-(2-(S)-NHBoc-phenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(2-(R)-Aminophenyl)acetyl)-1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 Ν-Οamino-3-phenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(2-(S)-Aminophenyl)acetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-aldehydephenyl)propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-bromomethylphenyl)propionyl)-1,3,4,9-tetrahydro-1 Η-β-carboline,

 N-C2-C4-hydroxymethylphenyl)propanoyl)- 1 ,3 ,4,9-tetrahydro - 1 Η-β-carboline,

 Ν-phenylacetyl-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(4-hydroxyphenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-hydroxy-2-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-hydroxy-2,2-diphenylacetyl)-1,3,4,9-tetrahydro-1 Η-β-carboline,

 Ν-(2-(4-(morpholinylmethylphenyl)-propionyl))-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-((diethylamino)methyl)phenyl)propanoyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-((2-(4-(lH- 1 ,2,4-triazol - 1 -yl)methyl)phenyl)propanoyl)-1 ,3 ,4,9-tetrahydro - 1Η-β -porphyrin,

 1-phenyl-indole-phenylacetyl-1,3,4,9-tetrahydro-1 Η-β-carboline,

1-ethyl-indole-phenylacetyl-1,3,4,9-tetrahydro-1Η-β-carboline, Claim

One WO 2013/060179 PCT/CN2012/079841

 2-(4-(2-NH-Biotin-ethoxy)-phenylacetyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-(2-(4-((2-morpholinoethylamino)methyl)phenyl))-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 N-(2-(4-(2-(2-NHBoc-ethylamino)ethoxy)phenyl)-propionyl)-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-phenylacetyl-1-ethyl-1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline,

 Ν-phenylacetyl small methyl -1,3,4,9-tetrahydro-6-benzyloxy-1Η-β-carboline,

 Ν-phenylacetyl small methyl -1,3,4,9-tetrahydro-6-hydroxy -1 Η-β-carboline,

 Ν-phenylacetyl-6-fluoro-1,3,4,9-tetrahydro-1Η-β-carboline,

 Ν-Phenylacetyl-6-bromo-1,3,4,9-tetrahydro-1Η-β-carboline.

 A pharmaceutical composition comprising the acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable Acceptable carrier.

 The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition is formulated into an injectable fluid, an aerosol, a cream, a gel, a pill, a capsule, a syrup, and a transdermal Patch or excipient.

 The acyltetrahydro-β-carboline small molecule organic compound, hydrate or pharmaceutically acceptable salt according to any one of claims 1 to 4, which inhibits proliferation, growth, migration and infiltration of tumor cells. The tumor cells include lung cancer cells, breast cancer cells, epidermal cancer cells, colon cancer cells, liver cancer cells, gastric cancer cells, and prostate cancer cells.

The use of an acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4 for the preparation of a medicament for treating a malignant tumor; wherein the malignant tumor comprises liver cancer, lung cancer, Prostate cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, leukemia, ovarian cancer, stomach cancer, bladder cancer, kidney cancer, skin cancer, oral cancer.

 The acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, for use in the preparation of a medicament for treating metastasis and recurrence of malignant tumors The malignant tumor includes liver cancer, lung cancer, prostate cancer, skin cancer, colon cancer, pancreatic cancer, breast cancer, leukemia, ovarian cancer, gastric cancer, bladder cancer, kidney cancer, skin cancer, oral cancer.

 The acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, for preparing a tumor which is therapeutically responsive to EGF receptor regulation The application of drugs for the occurrence, development and metastasis of diseases.

 The acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, for use in the preparation for treatment in response to TGFP receptor regulation The use of the medicament for the disease; wherein the disease includes tumor, diabetes, glomerulonephritis, glomerular sclerosis, various fibrotic diseases, osteoarthritis, osteoporosis.

The use of an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the preparation of an antitumor therapeutic drug, Characterized in that the drug is used to induce acquiredness Claim

 -WO 2013/060179. -PCT/CN2012/079841. Drug resistance leads to anti-tumor therapy after chemotherapy failure.

 The acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, for use in the preparation of a medicament for treating osteolytic bone injury application.

 The acyltetrahydro-β-carboline small molecule organic compound according to any one of claims 1 to 4, or a hydrate or pharmaceutically acceptable salt thereof, for preparing a medicament for treating tumor-induced pulmonary failure Application in .

 The use according to any one of claims 11-17, wherein the medicament is used alone or in combination with other drugs.

 The method for producing an acyltetrahydro-β-carboline small molecule organic compound or a hydrate or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein The method comprises the steps of: coupling a tetrahydro-β-carboline compound or a derivative thereof, and reacting with an acid, an acid anhydride or an acid halide in the presence of a coupling reagent or a base to obtain the acyltetrahydro- a β-porphyrin small molecule organic compound; or the method further derivatization of the acyltetrahydro-β-carboline by a palladium-catalyzed coupling method using a halogenated acyltetrahydro-β-carboline as a raw material A derivative of a small molecule organic compound.

 The preparation method according to claim 19, comprising:

 Method 1 as shown in the reaction formula (1)

 Wherein, the substituted tetrahydro-β-carboline compound or derivative thereof 1 and an acid, an acid anhydride or an acid halide are reacted by a coupling method in the presence of a coupling reagent or a base to obtain a product 2, and a base and a halogenated pair are added. The product is obtained by alkylation of the nitrogen atom on the product 2 to obtain the product 3; wherein the base includes triethylamine, potassium carbonate, pyridine, DMAP; and the second method as shown in the reaction formula (2)

 Wherein, the substituted tryptamine or its derivative 4 and the substituted aldehyde are subjected to Pictet-Spengler cyclization to form product 5, and then reacted by a coupling method to obtain products 6 and 7;

Method three as shown in reaction formula (3) Claim

/079841.

 Wherein, using aromatic ring 肼 8 as a raw material, by reacting with a protected amino aldehyde to form a compound 9, and then preparing a heterocyclic substituted product 10 by a coupling method;

 Method four as shown in equation (4)

 Wherein, the substituted aniline undergoes diazotization under the action of an acid to form a diazonium salt, which is coupled with a substituted sodium 2-piperidone-3-carboxylate to form an intermediate 12, which is then blocked by an acid to form a compound 13, The product is obtained by reduction and coupling reaction.

14;

 Method five as shown in equation (5)

 Among them, the substituted bromosubstituted-tetrahydro-β-carboline or a derivative thereof is converted into an aromatic or heteroaromatic substituted product 15 by a palladium-catalyzed coupling method.