Nitrogen-containing heterocyclic compounds, methods of making, pharmaceutical compositions, and uses thereof

Abstract

The present application relates to a kind of nitrogen-containing heterocyclic compounds and its preparation method, pharmaceutical composition and application.The structure formula of the above-mentioned nitrogen-containing heterocyclic compound is as follows:Wherein, Y and Z are respectively selected from one of-CH and-N;R 1 And R 2 Are respectively selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl and unsubstituted heteroaryl.The above-mentioned nitrogen-containing heterocyclic compound is combined with tubulin colchicine binding site on one hand, promotes microtubule depolymerization, inhibits tumor cell proliferation;On the other hand, by combining with the ATP binding site of protein kinase inhibits kinase activity, thereby realizing multi-pathway simultaneous inhibition of tumor cell proliferation and survival.Compared with paclitaxel and other drugs for tubulin, it has better water solubility, simpler structure, easier synthesis and better drug resistance.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and in particular to a nitrogen-containing heterocyclic compound, its preparation method, pharmaceutical composition, and application. Background Technology

[0002] Malignant tumors (cancer) have become one of the major public health problems seriously threatening the health of the Chinese population. According to the latest statistics, malignant tumors account for 23.91% of all deaths, and the incidence and mortality rates of malignant tumors have been rising continuously for the past decade. The annual medical expenses caused by malignant tumors exceed 220 billion yuan, highlighting the serious challenges in prevention and control. Although the development of anti-tumor drugs has achieved some success, improving the survival rate and quality of life for cancer patients, these drugs still have many shortcomings in clinical treatment, such as drug resistance, selectivity, and toxic side effects. Clearly, the development of new and effective anti-tumor drugs is of great significance.

[0003] Microtubules are components of the cytoskeleton in eukaryotic cells. They are highly dynamic structures composed of α- and β-tubulins, exhibiting polymerization and depolymerization properties, and play crucial roles in cell mitosis, cell signaling, intracellular transport, and angiogenesis. Targeting microtubule dynamics to influence microtubule polymerization and depolymerization has proven to be an effective and important anticancer strategy. Six tubulin binding sites have been identified, three of which have been extensively studied: the taxane binding site, the vinblastine binding site, and the colchicine binding site. Currently, several drugs targeting microtubules are used clinically for tumor treatment, such as paclitaxel (promotes microtubule polymerization) and vinblastine (inhibits microtubule polymerization). Although these drugs have become the first-line treatment for many cancers, their clinical application remains limited, mainly due to: 1) high toxicity, restricting dosage; 2) susceptibility to multidrug resistance, as both paclitaxel and vinblastine alkaloids are susceptible to tumor cell resistance due to the influence of the efflux pump P-gp protein (P-glycoprotein); and 3) poor water solubility, with solvents easily causing allergic reactions. For example, patients using paclitaxel must be treated with steroids and antihistamines beforehand, increasing the treatment and financial burden on patients. Furthermore, paclitaxel and vinblastine alkaloids have complex structures (structural formulas are...). Microtubules are highly complex to synthesize and modify, making drug development based on these compounds difficult and costly. Therefore, the development of novel microtubule inhibitors is of great significance.

[0004] Protein kinases are crucial messengers in cellular life processes, catalyzing the transfer of the γ-phosphate group from the terminal end of adenosine triphosphate (ATP) to its substrate, influencing the substrate's structure and activity, and transmitting various intracellular and extracellular signals to respond appropriately to environmental stimuli. In most cases, this phosphorylation reaction occurs at the serine (Ser), threonine (Thr), or tyrosine (Tyr) residues of the protein kinase. Protein kinases participate in numerous physiological regulatory processes, including cell survival, proliferation, differentiation, apoptosis, and metabolism. Pathological and pharmacological studies have shown that protein kinase dysfunction is closely related to many diseases, including tumors, autoimmune diseases, inflammatory responses, central nervous system diseases, cardiovascular diseases, and diabetes. Over the past two to three decades, protein kinases have proven to be an ideal target for drug intervention, especially for anti-tumor drug development. As of January 2021, 52 protein kinase inhibitors have been approved by the FDA, 46 of which are indicated for tumors. Although protein kinases have become important targets for anti-tumor drug development, similar to other anti-cancer therapies, anti-cancer targeted kinase therapy can also lead to drug resistance. Therefore, reducing drug resistance to anti-tumor drugs and improving their efficacy has been a continuous goal for researchers. Summary of the Invention

[0005] Therefore, it is necessary to provide a nitrogen-containing heterocyclic compound that can act on both tubulin and protein kinase simultaneously, so as to achieve multi-pathway simultaneous inhibition of tumor cell proliferation and survival, improve anti-tumor effect, and reduce drug resistance.

[0006] In addition, it is necessary to provide an application of nitrogen-containing heterocyclic compounds, pharmaceutical compositions, and methods for preparing nitrogen-containing heterocyclic compounds.

[0007] A nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, the structural formula of said nitrogen-containing heterocyclic compound being shown below:

[0008] Among them, Y and Z are independently selected from -CH and -N, respectively;

[0009] R 1 and R 2 Each is independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl;

[0010] When the R 1 or the R 2When the substituent is a substituted aryl or a substituted heteroaryl, the corresponding substituent is independently selected from halogen, alkyl, cycloalkyl, substituted C1-C6 alkyl, substituted haloC1-C6 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkoxy, substituted haloC1-C6 alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, or a substituted 4- to 8-membered ring containing at least one N, O, or S(O). m The heteroatom is one of the following: heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonamide, N-sulfonamide, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-amide group, and N-amide group, where m is 0, 1, or 2.

[0011] In one embodiment, the R 1 Selected from One of them;

[0012] Among them, R 3 R 5 R 7 and R 8 Each of the following groups is independently selected from hydrogen, alkyl, halogen, alkoxy, haloalkyl, cyano, amino, substituted amino, and nitro;

[0013] R 4a R 4b and R 6 Each is independently selected from one of hydrogen, alkyl, X-substituted alkyl, cycloalkyl, X-substituted cycloalkyl, heterocyclic, X-substituted heterocyclic, aryl, X-substituted aryl, heteroaryl and X-substituted heteroaryl, wherein X is selected from at least one of alkyl, halogen, hydroxyl, amino, mercapto, alkoxy, alkylamino, cycloalkyl, heterocyclic, carboxyl and carboxylic acid ester group;

[0014] Or R 4a and R 4b A 4- to 8-membered ring M1 is formed with the nitrogen atom to which it is attached, wherein M1 is a substituted heterocyclic group or an unsubstituted heterocyclic group, and M1 contains at least one N, O, or S(O). m Furthermore, the substituent of M1 is selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylic acid ester groups, and m is 0, 1 or 2.

[0015] In one embodiment, R 3 and R 5 One of them is related to NR 4a R 4b and OR 6One of the links forms a 4- to 8-membered ring M2, wherein M2 is a substituted heterocyclic group or an unsubstituted heterocyclic group, and M2 contains at least one N, O, or S(O). m Furthermore, the substituents of the substituted heterocyclic group formed are selected from at least one of halogen, alkyl, hydroxyl, alkoxy, amino, cyano, carboxyl and carboxylic acid ester groups, and m is 0, 1 or 2.

[0016] In one embodiment, the R 2 Selected from One of them.

[0017] In one embodiment, the nitrogen-containing heterocyclic compound is selected from one of the following structural formulas:

[0018]

[0019]

[0020]

[0021] A pharmaceutical composition comprising the above-described nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof.

[0022] A microtubule inhibitor or protein kinase inhibitor, comprising the above-described nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof.

[0023] The above-mentioned nitrogen-containing heterocyclic compounds or their pharmaceutically acceptable salts are used in the preparation of drugs for treating cancer, or in the preparation of protein kinase inhibitors, or in the preparation of microtubule inhibitors, or in the preparation of multi-target drugs.

[0024] In one embodiment, the drug for treating cancer is a drug for treating leukemia, a drug for treating rectal cancer, a drug for treating colon cancer, a drug for treating lung cancer, a drug for treating liver cancer, a drug for treating ovarian cancer, a drug for treating pancreatic cancer, a drug for treating prostate cancer, a drug for treating breast cancer, or a drug for treating cervical cancer.

[0025] The protein kinase is selected from at least one of BCR-ABL, FLT3, JNK, MEK5, STK16, and BMPR;

[0026] The multi-target drug has dual inhibitory activity against both tubulin and protein kinase.

[0027] A method for preparing a nitrogen-containing heterocyclic compound includes the following steps:

[0028] Compounds A and R 1 Compound B is prepared by reacting B(OH)₂ with a palladium catalyst and a basic reagent. The structural formula of compound A is as follows: The structural formula of compound B is as follows:

[0029] Compound B was reacted with an acidic reagent to prepare a nitrogen-containing heterocyclic compound, the structural formula of which is as follows:

[0030] Where Y and Z are independently selected from -CH and -N respectively, R 1 and R 2 Each is independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl;

[0031] When the R 1 and the R 2 When R is a substituted aryl or a substituted heteroaryl, 1 and the R 2 The substituents are independently selected from halogens, alkyl groups, cycloalkyl groups, substituted C1-C6 alkyl groups, substituted halo-C1-C6 alkyl groups, substituted C1-C6 alkenyl groups, substituted C1-C6 alkoxy groups, substituted halo-C1-C6 alkoxy groups, mercapto groups, substituted alkylthio groups, cyano groups, nitro groups, amino groups, substituted amino groups, heterocyclic groups, and substituted 4- to 8-membered rings containing at least one N, O, or S(O). m The heteroatom is one of the following: heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonamide, N-sulfonamide, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group, and N-acylamino group; m is 0, 1, or 2.

[0032] In one embodiment, the step of preparing compound A is also included;

[0033] When both Y and Z are -CH, the steps for preparing compound A include:

[0034] Compound C was reacted with NaH and SEMCl to prepare compound D. The structural formula of compound C is as follows: The structural formula of compound D is as follows:

[0035] The compounds D and R 2 Compound A is prepared by reacting B(OH)₂ with a palladium catalyst and a basic reagent; or,

[0036] One of Y and Z is -CH and the other is -N. The preparation steps of compound A include:

[0037] Compound E was reacted with TMPZnCl·LiC to prepare compound F. The structural formulas of compounds E and F are respectively...

[0038] The compounds F and R 2 Compound G is prepared by reacting COCl and CuCN·2LiC. The structural formula of compound G is as follows:

[0039] Compound G was reacted with N2H4·H2O and CH3OH to prepare compound H, the structural formula of which is [insert structural formula here].

[0040] Compound H was reacted with NaH and SEMCl to prepare compound A.

[0041] These nitrogen-containing heterocyclic compounds inhibit tumor cell proliferation by binding to the colchicine binding site of tubulin, promoting microtubule depolymerization. They also inhibit kinase activity by binding to the ATP binding site of protein kinases, thus achieving multi-pathway inhibition of tumor cell proliferation and survival, enhancing anti-tumor activity, and reducing drug resistance. Furthermore, compared to drugs targeting tubulin such as paclitaxel, they exhibit better water solubility, simpler structures, and are easier to synthesize. Attached Figure Description

[0042] Figure 1 The figure shows the experimental results of the microtubule-inhibiting activity of the nitrogen-containing heterocyclic compounds prepared in the examples. Detailed Implementation

[0043] To facilitate understanding of the present invention, a more comprehensive description of the invention will be provided below in conjunction with specific embodiments. Preferred embodiments of the invention are given in the specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0045] In this article, Me represents methyl, Et represents ethyl, SEMCl represents 2-(trimethylsilyl)ethoxymethyl chloride, and SEM represents -(trimethylsilyl)ethoxymethyl.

[0046] The term "alkyl" refers to a chain of saturated hydrocarbons containing primary (normal) carbon atoms, secondary carbon atoms, tertiary carbon atoms, quaternary carbon atoms, or combinations thereof, excluding cyclic saturated hydrocarbons. Phrases containing this term, such as "C1-C6 alkyl," refer to alkyl groups containing 1 to 6 carbon atoms, and each occurrence can independently be C1 alkyl, C2 alkyl, C3 alkyl, C4 alkyl, C5 alkyl, or C6 alkyl. Similarly, alkenyl and ynyl groups both refer to chain-like unsaturated hydrocarbons.

[0047] The term "combinations thereof" as used herein includes any suitable combination of the listed items, as long as it achieves the purpose of this invention.

[0048] "Aryl" refers to an aromatic hydrocarbon group derived from an aromatic ring compound by removing a hydrogen atom. It can be a monocyclic aryl, a fused-ring aryl, or a polycyclic aryl. For polycyclic ring species, at least one is an aromatic ring system.

[0049] "Heteroaryl" refers to an aryl group in which at least one carbon atom is replaced by a heteroatom. The heteroatom can be a nitrogen atom, an oxygen atom, a sulfur atom, etc. For example, "C3~C 10 "Heteroaryl" refers to a heteroaryl group containing 3 to 10 carbon atoms, which can be independently C3, C4, C5, C6, C7, or C8 heteroaryl each time it appears. Suitable examples include, but are not limited to: furan, benzofuran, thiophene, benzothiophene, pyrrole, pyrazole, triazole, imidazole, oxazole, oxadiazole, thiazole, tetrazolium, indole, carbazole, pyrroloimidazol, pyrrolopyrrole, thiophenolopyrrole, thiophenolothiophene, furanolopyrrole, furanolofuran, thiophenolofuran, benzoisoxazole, benzoisothiazolium, benzimidazole, pyridine, pyrazine, pyridazine, pyrimidine, triazine, quinoline, isoquinoline, o-diazonyl, quinoxaline, phenanthridine, primidine, quinazoline, and quinazolineone.

[0050] "Heterocyclic group" refers to a cycloalkyl group in which at least one carbon atom is replaced by a heteroatom, which can be an N atom, O atom, S atom, etc., and can be a saturated ring or a partially unsaturated ring. Phrases containing this term, such as "C4-C9 heterocyclic group," refer to heterocyclic groups containing 4 to 9 carbon atoms, and each occurrence can be independently C4, C6, C7, C8, or C9 heteroalkyl. Suitable examples include, but are not limited to: dihydropyridyl, tetrahydropyridyl (piperidinyl), tetrahydrothiophenyl, sulfur-oxidized tetrahydrothiophenyl, tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and dihydroindolyl.

[0051] "Halogen" refers to F, Cl, Br or I.

[0052] The "carboxyl group" includes the -COOH structure and is not limited to methylcarboxyl; it can also be ethylcarboxyl, propionic carboxyl, etc.

[0053] "Carboxylic acid ester group" refers to -C(=O)OR.

[0054] "Amino" refers to -NH2.

[0055] "Pharmaceutically acceptable salt" refers to a salt formed by any compound in the indicated structure with an acid or base that is suitable for use as a medicine. Pharmaceutically acceptable salts include inorganic and organic salts. One type of salt is the salt formed by the compound of this invention with an acid. The acid suitable for forming the salt can be an organic acid, an inorganic acid, or a natural or non-natural amino acid. Acids suitable for forming the salt include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, and phosphoric acid; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, and naphthalenesulfonic acid; and amino acids such as proline, phenylalanine, aspartic acid, and glutamic acid. Another type of salt is the salt formed by the compounds of the present invention with a base. Suitable bases for forming salts include, but are not limited to: alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (such as lower alkanol ammonium salts and other pharmaceutically acceptable amine salts), such as methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, trihydroxyethylamine salts, and amine salts formed from morpholine, piperazine, and lysine, respectively.

[0056] Currently approved microtubule inhibitors for cancer treatment target either the taxane or vincristine binding site; no microtubule inhibitors targeting the colchicine binding site have been approved for marketing. However, current clinically available microtubule inhibitors targeting the taxane and vincristine binding sites suffer from the following problems: (1) Microtubule inhibitors, represented by paclitaxel and vincristine, have complex structures, are difficult to synthesize, and are costly; (2) They are highly toxic, typically exhibiting neurotoxicity and myelosuppressive toxicity; (3) They have poor water solubility, requiring chemical solubilizers for dissolution, which can easily cause allergic reactions; and (4) They are prone to multidrug resistance, especially P-gp-mediated resistance. Therefore, this invention provides a nitrogen-containing heterocyclic compound that can act on the colchicine binding site. This compound has a novel structure, is easy to synthesize, has good water solubility, and exhibits good drug resistance, thus overcoming the shortcomings of traditional microtubule inhibitors.

[0057] In addition, this nitrogen-containing compound can not only bind to the colchicine binding site of tubulin to inhibit tumor cell proliferation, but also act on protein kinases. By binding to the ATP binding site of protein kinases to inhibit kinase activity, it can simultaneously inhibit the proliferation and survival of tumor cells through multiple pathways, thereby improving anti-tumor activity.

[0058] Similar to other anticancer therapies, targeted kinase therapy for cancer can also lead to drug resistance. The mechanisms of resistance can be broadly categorized into two types: one originates from the target kinase itself, including overexpression and resistance mutations; the other is not directly related to the target kinase but rather develops resistance through alternative signaling pathways. The rational combination of small molecule kinase inhibitors targeting different targets has become one of the key areas of effort to combat drug resistance and enhance the benefits of targeted kinase therapy.

[0059] Given the highly complex mechanisms of tumor development, involving the disruption of multiple signaling pathways and physiological processes, multi-target drugs, compared to single-target antitumor drugs, can simultaneously act on multiple targets to produce synergistic effects, improving efficacy and reducing drug resistance to some extent. Based on this, the present invention provides a nitrogen-containing heterocyclic compound capable of simultaneously acting on multiple targets, including kinases and tubulin, to exert multiple antitumor effects.

[0060] Specifically, one embodiment includes a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof, the structural formula of which is as follows:

[0061]

[0062] In this context, Y and Z are each independently selected from -CH and -N. Further, Y and Z are both -CH, or Y is -CH and Z is -N, or Y is -N and Z is -CH.

[0063] R 1 and R 2 Each is independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl. Preferably, R 1 and R 2 Each is independently selected from one of the substituted aryl and substituted heteroaryl groups.

[0064] When R 1 Or R 2 When the substituent is a substituted aryl or a substituted heteroaryl, the corresponding substituent is independently selected from halogen, alkyl, cycloalkyl, substituted C1-C6 alkyl, substituted haloC1-C6 alkyl, substituted C1-C6 alkenyl, substituted C1-C6 alkoxy, substituted haloC1-C6 alkoxy, mercapto, substituted alkylthio, cyano, nitro, amino, substituted amino, heterocyclic, or a substituted 4- to 8-membered ring containing at least one N, O, or S(O). m The heteroatom is one of the following: heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonamide, N-sulfonamide, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group, and N-acylamino group; m is 0, 1, or 2.

[0065] Preferably, R 1 Selected from One of them.

[0066] Among them, R 3 R 5 R 7 and R 8 Each of the following is independently selected from hydrogen, alkyl, halogen, alkoxy, haloalkyl, cyano, amino, substituted amino, and hydroxyl. Further, the alkyl group is a C1-C6 alkyl group, the alkoxy group is a C1-C6 alkoxy group, and the haloalkyl group is a haloalkyl group of C1-C6.

[0067] R 4a R 4b and R 6 Each is independently selected from one of hydrogen, alkyl, X-substituted alkyl, cycloalkyl, X-substituted cycloalkyl, heterocyclic, X-substituted heterocyclic, aryl, X-substituted aryl, heteroaryl, and X-substituted heteroaryl, wherein X is selected from at least one of alkyl, halogen, hydroxyl, amino, mercapto, alkoxy, alkylamino, cycloalkyl, heterocyclic, carboxyl, and carboxylic acid ester group; or,

[0068] R 4a and R 4b A 4- to 8-membered ring M1 is formed with the nitrogen atom to which it is attached, wherein M1 is a substituted heterocyclic group or an unsubstituted heterocyclic group, and M1 contains at least one N, O or S(O). m The heteroatom, and the substituent of M1 is selected from at least one of halogen, alkyl, hydroxy, alkoxy, amino, cyano, carboxyl and carboxylic acid ester groups, and m is 0, 1 or 2.

[0069] R 3 and R 5 One of them is related to NR 4a R 4b and OR 6 One of the links forms a 4- to 8-membered ring M2, where M2 is a substituted heterocyclic group or an unsubstituted heterocyclic group, and M2 contains at least one N, O, or S(O). m The heteroatom, and the substituent of M2 is selected from at least one of halogen, alkyl, hydroxy, alkoxy, amino, cyano, carboxyl and carboxylic acid ester groups, and m is 0, 1 or 2.

[0070] Furthermore, R 1 The general structural formula is as follows: Among them, X 20 It can be selected from one of hydrogen atom, -CH2CH2CH2OH, -CH2C(CH3)2OH, -CH2CH2CH2NHCH3, and -CH2CH2CH2N(CH3)2, X 21It can be selected from one of hydrogen atoms, halogens, and -CH3.

[0071] Furthermore, R 1 Choose one of the following structural formulas:

[0072]

[0073]

[0074] The wavy lines represent bonds attached to nitrogen-containing heterocyclic compounds.

[0075] Preferably, R 2 The general structural formula of the substituent is Among them, X 11 X 12 and X 13 Each is independently selected from one of H, hydroxyl, and alkoxy groups, and X 11 X 12 and X 13 Not all are hydrogen. Furthermore, R 2 Substituents are selected from One of them.

[0076] Furthermore, the general structural formula of nitrogen-containing heterocyclic compounds is as follows:

[0077]

[0078] In general formula (II), R 1 The definition and preferred method are consistent with those described above.

[0079] In general formula (II), X 11 X 12 and X 13 Each is independently a hydrogen atom, a hydroxyl group, or an alkoxy group, and X 11 X 12 and X 13 Not all are hydrogen. Preferably, X 11 X 12 and X 13 It includes at least one alkoxy group. Preferably, the X 11 X 12 and X 13 Each is independently a hydrogen atom or a C1-C6 alkoxy group, and X 11 X 12 and X 13 It contains at least one C1-C6 alkoxy group.

[0080] In some preferred embodiments, the structure of the nitrogen-containing heterocyclic compound is as follows:

[0081] Preferably, the nitrogen-containing heterocyclic compound is selected from one of the following structural formulas:

[0082]

[0083]

[0084]

[0085] The nitrogen-containing heterocyclic compounds of the above embodiments have at least the following advantages:

[0086] (1) The aforementioned nitrogen-containing heterocyclic compounds, on the one hand, promote microtubule depolymerization and inhibit tumor cell proliferation by binding to the colchicine binding site of tubulin; on the other hand, they inhibit kinase activity by binding to the ATP binding site of protein kinases, thus acting on multiple targets of kinases and tubulin simultaneously, thereby achieving multi-pathway inhibition of tumor cell proliferation and survival, resulting in stronger anti-tumor activity. Compared with single-target anti-tumor drugs, the aforementioned nitrogen-containing heterocyclic compounds can act on multiple targets simultaneously to produce synergistic effects, improve efficacy, and to some extent reduce the occurrence of drug resistance.

[0087] (2) The above-mentioned nitrogen-containing heterocyclic compounds have novel structures, are easier to synthesize chemically, and have lower preparation costs. Moreover, compared with drugs targeting microtubules such as paclitaxel, they have better water solubility, better drug resistance, and are still effective against paclitaxel-resistant tumor cells.

[0088] The present invention also provides a pharmaceutical composition according to one embodiment, wherein the active ingredient of the pharmaceutical composition comprises the nitrogen-containing heterocyclic compound of the above embodiments or a pharmaceutically acceptable salt thereof. Specifically, the pharmaceutical composition may further comprise pharmaceutical excipients commonly used in the art.

[0089] The present invention also provides a microtubule inhibitor or protein kinase inhibitor according to one embodiment, including the nitrogen-containing heterocyclic compound of the above embodiments or a pharmaceutically acceptable salt thereof.

[0090] The present invention also provides the use of a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating cancer.

[0091] Preferably, the drug for treating cancer is a drug for treating leukemia, rectal cancer, colon cancer, lung cancer, liver cancer, ovarian cancer, pancreatic cancer, prostate cancer, breast cancer, or cervical cancer.

[0092] The present invention also provides the use of a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a protein kinase inhibitor.

[0093] Preferably, the protein kinase is selected from at least one of BCR-ABL (Nature Reviews Drug Discovery, 2002, 1, 493), FLT3 (Leukemia, 2019, 33, 299), JNK (Oncotarget, 2016, 7, 27021), MEK5 (Biochim Biophys Acta. 2012, 1825, 37), STK16 (Int. J. Mol. Sci. 2019, 20, 1760) and BMPR (Biochemical Society Transactions, 2017, 45, 223).

[0094] The present invention also provides the use of a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a microtubule inhibitor.

[0095] The present invention also provides the use of a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a multi-target drug, wherein the multi-target drug has dual inhibitory activity against tubulin and protein kinase.

[0096] The present invention also provides a method for preparing a nitrogen-containing heterocyclic compound according to an embodiment, which is a method for preparing the nitrogen-containing heterocyclic compound of the above embodiment, comprising the following steps:

[0097] Step S110: Combine compounds A and R 1 Compound B is prepared by reacting B(OH)2 with a palladium catalyst and a basic reagent.

[0098] The structural formula of compound A is as follows: The structural formula of compound B is Y and Z are each independently selected from -CH and -N.

[0099] In one embodiment, the palladium catalyst is PdCl2(dppf), i.e., (1,1'-bis(diphenylphosphine)ferrocene palladium chloride). The alkaline reagent is sodium carbonate.

[0100] Specifically, compounds A and R 1 In the reaction steps of B(OH)2, palladium catalyst and basic reagent, a degassed dioxane aqueous solution was also added as a solvent.

[0101] Furthermore, in step S110, the reaction temperature is 80℃~100℃, and the reaction time is 1h~2h. Preferably, step S110 is carried out in a microwave reactor.

[0102] In a specific example, step S110 includes: under a protective gas atmosphere, reacting compounds A and R...1 Compound B is prepared by mixing B(OH)2, palladium catalyst, and alkaline reagent with a degassed aqueous solution of dioxane and reacting in a microwave reactor at 80℃~100℃ for 1h~2h.

[0103] Step S120: Compound B is reacted with an acidic reagent to prepare a nitrogen-containing heterocyclic compound. The structural formula of the nitrogen-containing heterocyclic compound is as follows:

[0104] Among them, R 1 and R 2 Each is independently selected from one of substituted aryl, unsubstituted aryl, substituted heteroaryl, and unsubstituted heteroaryl.

[0105] When R 1 and R 2 When R is a substituted aryl or a substituted heteroaryl, 1 and R 2 The substituents are independently selected from halogens, alkyl groups, cycloalkyl groups, substituted C1-C6 alkyl groups, substituted halo-C1-C6 alkyl groups, substituted C1-C6 alkenyl groups, substituted C1-C6 alkoxy groups, substituted halo-C1-C6 alkoxy groups, mercapto groups, substituted alkylthio groups, cyano groups, nitro groups, amino groups, substituted amino groups, heterocyclic groups, and substituted 4- to 8-membered rings containing at least one N, O, or S(O). m The heteroatom is one of the following: heterocyclic group, sulfinyl group, sulfonyl group, S-sulfonamide, N-sulfonamide, O-carbamoyl group, N-carbamoyl group, O-thiocarbamoyl group, N-thiocarbamoyl group, C-acylamino group, and N-acylamino group; m is 0, 1, or 2.

[0106] In one embodiment, the acidic reagent is hydrochloric acid.

[0107] Specifically, in the step of reacting compound B with the acidic reagent, ethanol was added as a solvent to dissolve compound B. Further, in one embodiment, the reaction temperature was 80°C and the reaction time was 12 hours.

[0108] Following the step of reacting compound B with an acidic reagent, a purification step is also included. In one embodiment, the purification step includes: concentrating the reaction system under reduced pressure, liquid-phase separation, and then freeze-drying.

[0109] In a specific example, step S120 includes: dissolving compound B in ethanol, then adding an acidic reagent, reacting at 80°C for 12 hours, and after the reaction is completed, preparing a nitrogen-containing heterocyclic compound by sequentially concentrating under reduced pressure, separating by liquid phase, and freeze-drying.

[0110] Furthermore, the preparation method of the above-mentioned nitrogen-containing heterocyclic compound also includes step S130: preparing compound A.

[0111] When both Y and Z are -CH, step S130 includes steps S132 and S134, as follows:

[0112] Step S132: React compound C with NaH and SEMCl to prepare compound D.

[0113] The structural formula of compound C is as follows: The structural formula of compound D is

[0114] Specifically, in step S132, DMF was added to dissolve compound C. In a specific example, step S132 was as follows: compound C was dissolved in DMF, NaH was added at 0°C, and the mixture was stirred for 10 min. Then, SEMCl was added dropwise, and the reaction was continued with stirring at room temperature. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and then separated by column chromatography to prepare compound D.

[0115] Step S134: Combine compounds D and R 2 Compound A is prepared by reacting B(OH)2 with a palladium catalyst and a basic reagent.

[0116] Specifically, compounds D and R 1 In the reaction steps of B(OH)2, palladium catalyst and basic reagent, a degassed dioxane aqueous solution was also added as a solvent.

[0117] Furthermore, in step S134, the reaction temperature is 80℃~100℃, and the reaction time is 1h~2h. Preferably, step S134 is carried out in a microwave reactor.

[0118] In a specific example, step S134 includes: under a protective gas atmosphere, reacting compounds D and R... 1 Compound B is prepared by mixing B(OH)2, palladium catalyst, and alkaline reagent with a degassed aqueous solution of dioxane and reacting in a microwave reactor at 80℃~100℃ for 1h~2h.

[0119] Furthermore, step S134 also includes a purification step. The purification step specifically involves: after the reaction is complete, filtration, extraction with ethyl acetate, drying the organic layer with anhydrous sodium sulfate, filtration, concentration, and then separation by column chromatography to prepare compound D.

[0120] When Y is -CH and Z is -N, or Y is -N and Z is -CN, step S130 includes steps S131, S133, S135 and S137, as follows:

[0121] Step S131: React compound E with TMPZnCl·LiC to prepare compound F.

[0122] The structural formulas of compounds E and F are respectively

[0123] Specifically, in the step of reacting compound E with TMPZnCl·LiC, THF was also added as a solvent. In one embodiment, the step of reacting compound E with TMPZnCl·LiC was carried out at room temperature.

[0124] Step S133: Combine compounds F and R 2 Compound G was prepared by reacting COCl and CuCN·2LiC.

[0125] The structural formula of compound G is as follows:

[0126] Step S135: React compound G with N2H4·H2O and CH3OH to prepare compound H.

[0127] The structural formula of compound H is as follows:

[0128] Step S137: React compound H with NaH and SEMCl to prepare compound A.

[0129] Specifically, in step S137, DMF was added to dissolve compound H. In a specific example, step S137 was as follows: compound H was dissolved in DMF, NaH was added at 0°C, and the mixture was stirred for 10 min. Then, SEMCl was added dropwise, and the reaction was continued with stirring at room temperature. After the reaction was completed, water was added to quench the reaction, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and then separated by column chromatography to prepare compound H.

[0130] In one embodiment, both Y and Z are -CH, and the synthetic route for the nitrogen-containing heterocyclic compound is shown below:

[0131]

[0132] In another embodiment, Y is -CH and Z is -N, and the specific synthetic route for the nitrogen-containing heterocyclic compound is as follows:

[0133]

[0134] In another embodiment, Y is -N and Z is -CH, and the synthetic route for the nitrogen-containing heterocyclic compound is shown below:

[0135]

[0136] The preparation method of the above-mentioned nitrogen-containing heterocyclic compounds has at least the following advantages:

[0137] (1) The preparation method of the above nitrogen-containing heterocyclic compounds is simple, easy to synthesize, and low in cost.

[0138] (2) The above-mentioned method for preparing nitrogen-containing heterocyclic compounds can produce compounds that act on multiple targets of kinases and tubulin, thereby enhancing antitumor activity. On the one hand, these compounds promote microtubule depolymerization and inhibit tumor cell proliferation by binding to the colchicine binding site of tubulin; on the other hand, they inhibit kinase activity by binding to the ATP binding site of protein kinases, providing a new approach for the synthesis of antitumor compounds.

[0139] The following is a specific embodiment:

[0140] The following embodiments are used to further describe the present invention, but these embodiments are not intended to limit the scope of the present invention.

[0141] The structural formulas of the compounds were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS). NMR measurements were performed using a Bruker 400 MHz NMR spectrometer, with shifts (δ) expressed in 10⁻⁶ ppm. -6 The units (ppm) are given. The solvent used for measurement is deuterated dimethyl sulfoxide (DMSO-d6) or deuterated chloroform (CDCl3), and the internal standard is methylsilane (TMS). MS measurements were performed using a Waters tandem quadrupole mass spectrometer. Liquid phase separation and purification were performed using a Gilson semi-preparative high-performance liquid chromatograph. IC50 was measured using a Bioteck microplate reader. Microwave reactions were performed on a Biotage microwave reactor. The starting materials used in the embodiments of this invention can be synthesized using methods known in the art, or can be purchased from chemical reagent companies such as Bioteck, Anengji, and Leyan. Unless otherwise specified in the embodiments, the reactions were performed under an argon or nitrogen atmosphere.

[0142] For experimental parameters not specified in the following specific embodiments, please refer to the guidelines given in this application document first, or refer to experimental manuals or other experimental methods known in the art, or refer to the experimental conditions recommended by the manufacturer.

[0143] Example 1

[0144] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0145] (1)

[0146] Compound 1 (2 g, 6.19 mmol) was dissolved in DMF and cooled to 0°C in an ice bath. NaH (0.5 g, 12.39 mmol) was slowly added, and the mixture was stirred for 10 minutes. Then, SEMCl (2 mL, 10.53 mmol) was added dropwise, and the reaction was stirred at room temperature. After the reaction was complete, water was added to quench the reaction, and the mixture was extracted with ethyl acetate (EA). The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 2 g of a pale yellow solid, with a yield of 71.4%, which was compound 2. The structure of compound 2 is shown below: LC-MS: m / z 454 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.62 (d, J = 2Hz, 1H), 7.99 (d, J = 2Hz, 1H), 5.83 (S, 2H), 3.66 (t, J = 7.2Hz, 2H), 0.95 (t, J = 7.2Hz, 2H), -0.03 (s, 9H). 13 C NMR (100MHz, CDCl3) δ151.3,149.5,132.5,122.1,113.9,91.6,75.5,67.3,17.7,-1.5.

[0147] (2)

[0148] Compound 2 (1.8 g), compound 3 (0.924 g), PdCl2 (dppf) (288 mg), Na2CO3 (1.26 g), and a degassed dioxane-water solution (v / v = 10:1, 17.6 mL) were added sequentially to a reaction flask. After purging with argon, the flask was placed in a microwave reactor and reacted at 80 °C for 2 h. After the reaction was complete, the mixture was filtered, extracted with EA, and the organic layer was dried with anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 1.3 g of a yellow solid, with a yield of 66.3%, which was compound 4. The structure of compound 4 is characterized as follows: LC-MS: m / z 494 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.60(d,J=2Hz,1H),8.39(d,J=2Hz,1H),7.10(s,2H),5.83(S,2H) ,3.97(s,6H),3.91(s,3H),3.70(t,J=8.0Hz,2H),0.95(t,J=8.0Hz,2H),-0.04(s,9H). 13C NMR (100MHz, CDCl3) δ155.2,151.9,151.4,144.9,140.4,133.6,129.3,116.7,114.8,106.1,76.7,68.5,62.4,61.8,57.9,19.2,0.0.

[0149] (3)

[0150] Compound 4 (100 mg), compound 5 (34 g), PdCl2 (dppf) (15 mg), Na2CO3 (64 mg), and a degassed dioxane-water solution (v / v = 10:1, 2.2 mL) were added sequentially to a reaction flask. After purging with argon, the flask was placed in a microwave reactor and reacted at 100 °C for 1.5 h. After the reaction was completed, the mixture was filtered, and the crude product obtained after concentration, namely compound 6, was directly used in the next reaction. The structure of compound 6 is characterized as follows: LC-MS: m / z 521 [M+H] + .

[0151] (4)

[0152] Compound 6 was dissolved in ethanol, and an equal volume of 2N HCl was added. The mixture was heated at 80°C overnight. After the reaction was complete, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain the yellow target compound 7, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 7 is characterized as follows: LC-MS: m / z 391 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.82(d,J=2Hz,1H),8.56(d,J=2Hz,1H),7.46(m,2H),7.32(m,1H),7.25(s,2H),3.92(s,6H),3.74(s,3H),2.28(s,3H). 13 C NMR(100MHz,DMSO-d6)δ158.9,158.6,153.9,152.9,148.5,143.7,138.3,137.1,132 .0,129.8,129.1,127.9,127.2,126.9,122.6,118.6,112.6,104.8,60.6,56.5,17.2.

[0153] Example 2

[0154] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0155] (1)

[0156] Compound 6 (100 mg, 0.192 mmol) was dissolved in DMF (3 mL), and K2CO3 (79 mg, 0.576 mmol) was added. The mixture was heated to 80 °C, and compound 8 (72 mg, 0.576 mmol) was added in portions. The reaction was allowed to proceed overnight. After the reaction was complete, water was added to quench the reaction, followed by extraction with EA, filtration, and concentration. The crude product obtained was compound 9, which was directly used in the next reaction. The structure of the crude product was characterized as follows: LC-MS: m / z 565 [M+H] + .

[0157] (2)

[0158] Compound 9 was dissolved in ethanol (6 mL), and 2N HCl (6 mL) was added. The reaction was carried out overnight at 80 °C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain the yellow target compound 10, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 10 is characterized as follows: LC-MS: m / z 435 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.85(d,J=2Hz,1H),8.60(d,J=2Hz,1H),7.28(s,2H),7.21(m,3 H),3.92(s,3H),3.74(s,3H),3.66(t,J=5.6Hz,2H),3.35(t,J=5.6Hz,2H),2.23(s,3H). 13 C NMR(100MHz,DMSO-d6)δ159.4,159.0,158.6,158.3,153.8,152.9,148.7,143.5,138.2,13 7.2,131.6,130.7,129.2,128.0,124.3,118.7,112.6,104.6,60.6,59.0,56.4,47.9,17.4.

[0159] Example 3

[0160] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0161]

[0162] The method for synthesizing compound 12 from compounds 4 and 11 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 12 is characterized as follows: LC-MS: m / z 507 [M+H]+ .

[0163] Compound 12 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 13, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 13 is characterized as follows: LC-MS: m / z 377 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.9(br,1H),8.83(d,J=2Hz,1H),8.58(d,J=2Hz,1H ),7.43(m,3H),7.26(s,2H),7.04(d,J=7.6Hz,1H),3.92(s,6H),3.74(s,3H). 13 C NMR(100MHz,DMSO-d6)δ158.9,153.9,153.0,148.6,143.8,139.6,138.3,130.6,1 29.9,129.5,129.1,128.2,127.1,121.8,118.4,117.9,112.6,104.8,60.6,56.6.

[0164] Example 4

[0165] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0166]

[0167] The method for synthesizing compound 15 from compounds 4 and 14 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 15 is characterized as follows: LC-MS: m / z 508 [M+H] + .

[0168] Compound 15 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 16, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 16 is characterized as follows: LC-MS: m / z 378 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ14.11(s,1H),8.97(d,J=2Hz,1H),8.82(d,J=2Hz,1H),8.07(d,J=6.8Hz,1H) ,8.01(br,1H),7.45(dd,J=6.8Hz,J=1.6Hz,1H),7.34(m,1H),7.26(s,2H),3.92(s,6H),3.75(s,3H).

[0169] Example 5

[0170] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0171]

[0172] The method for synthesizing compound 18 from compounds 4 and 17 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 15 is characterized as follows: LC-MS: m / z 532 [M+H] + .

[0173] Compound 15 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 19, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 19 is characterized as follows: LC-MS: m / z 402 [M+H] + . 1 H NMR(400MHz,DMSO-d6)δ8.91(d,J=2Hz,1H),8.65(d,J=2Hz,1H),8.19(br,1H),8.15(br,1H),7 .82(dd,J=8.4Hz,J=1.6Hz,1H),7.68(d,J=8.4Hz,1H),7.28(s,2H),3.92(s,6H),3.74(s,3H). 13 C NMR (100MHz, DMSO-d6) δ153.8,152.7,149.2,143.6,139.9,138.2,134.4,131. 1,130.9,129.2,128.2,126.7,124.1,119.6,112.6,111.3,104.8,60.6,56.6.

[0174] Example 6

[0175] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0176]

[0177] The method for synthesizing compound 21 from compounds 4 and 20 is based on the synthesis method of compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 21 is characterized as follows: LC-MS: m / z 532 [M+H] + .

[0178] Compound 21 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 22, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 22 is characterized as follows: LC-MS: m / z 402 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.9(s,1H),13.3(s,1H),8.93(d,J=2.0Hz,1H),8.74(d,J=2.0Hz,1H),8.33(s,1H) ,7.60(d,J=8.0Hz,1H),7.49(t,J=7.2Hz,1H),7.39(d,J=7.2Hz,1H),7.31(s,2H),3.91(s,6H),3.74(s,3H). 13 C NMR (100MHz, DMSO-d6) δ153.8,152.9,149.5,143.8,141.0,138.2,133.0,131. 8,129.8,129.3,129.1,126.9,121.9,120.7,112.6,110.0,104.7,60.6,56.4.

[0179] Example 7

[0180] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0181]

[0182] The method for synthesizing compound 24 from compounds 4 and 23 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 24 is characterized as follows: LC-MS: m / z 508 [M+H] + .

[0183] Compound 24 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 25, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 25 is characterized as follows: LC-MS: m / z 378 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.81(d,J=2.0Hz,1H),8.56(d,J=2.0Hz,1H),7.31(t,J=8.0Hz,1H),7.24(m,3H),6.82(m,1H),3.92(s,6H),3.74(s,3H). 13 C NMR(100MHz,DMSO-d6)δ158.4,153.8,152.9,148.7,143.6,139.8,138.3,1 30.6,130.5,129.1,128.2,118.5,115.0,114.6,112.6,104.8,60.6,56.5.

[0184] Example 8

[0185] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0186]

[0187] The method for synthesizing compound 27 from compounds 4 and 26 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 27 is characterized as follows: LC-MS: m / z 522 [M+H] + .

[0188] Compound 27 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 28, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 28 is characterized as follows: LC-MS: m / z 392 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.78(d,J=2.0Hz,1H),8.51(d,J=2.0Hz,1H),7.25(s,2H),7.15(m,3H),3.92(s,6H),3.74(s,3H),2.18(s,3H). 13C NMR(100MHz,DMSO-d6)δ159.0,158.7,156.4,153.9,152.8,148.5,143.5,138.2,137 .0,131.7,130.5,129.2,127.6,123.9,118.3,113.8,112.6,104.7,60.6,56.5,16.2.

[0189] Example 9

[0190] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0191]

[0192] The method for synthesizing compound 30 from compounds 4 and 29 is based on the synthesis method of compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 30 is characterized as follows: LC-MS: m / z 552 [M+H] + .

[0193] Compound 30 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 31, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 31 is characterized as follows: LC-MS: m / z 422 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.9(br,1H),8.88(d,J=2.0Hz,1H),8.65(d,J=2.0Hz,1H),7.38(m,3H), 7.28(s,2H),6.97(m,1H),4.11(t,J=4.8Hz,2H),3.92(s,6H),3.76(t,J=4.8Hz,2H),3.74(s,3H). 13 CNMR(100MHz,DMSO-d6)δ159.8,159.0,158.6,153.8,152.9,148.9,143.7,139.9,138.2 ,130.6,130.2,129.1,128.6,120.0,114.4,113.7,112.5,104.7,70.1,60.6,60.1,56.5.

[0194] Example 10

[0195] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0196]

[0197] The method for synthesizing compound 33 from compounds 4 and 32 is based on the synthesis method of compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 33 is characterized as follows: LC-MS: m / z 566 [M+H] + .

[0198] Compound 33 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 34, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 34 is characterized as follows: LC-MS: m / z 436 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.8(br,1H),8.88(d,J=2.0Hz,1H),8.64(d,J=2.0Hz,1H),7.37(m,1H), 7.28(m,4H),4.15(t,J=5.2Hz,2H),3.92(s,6H),3.76(t,J=4.8Hz,2H),3.74(s,3H).2.22(s,3H). 13 CNMR(100MHz,DMSO-d6)δ158.9,158.5,157.8,153.8,152.9,148.9,143.6,138.1,137.4,13 1.4,130.5,129.2,128.3,125.8,119.5,112.6,111.1,104.6,70.2,60.6,60.2,56.4,16.2.

[0199] Example 11

[0200] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0201]

[0202] The method for synthesizing compound 36 from compounds 4 and 32 is the same as the method for synthesizing compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 36 is characterized as follows: LC-MS: m / z 566 [M+H] + .

[0203] Compound 36 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 37, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 37 is characterized as follows: LC-MS: m / z 436 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ13.9(br,1H),8.88(d,J=2.0Hz,1H),8.65(d,J=2.0Hz,1H),7.37(m,3H),7.28(s ,2H),6.96(m,1H),4.15(t,J=6.4Hz,2H),3.92(s,6H),3.74(s,3H),3.59(t,J=6.4Hz,2H),1.91(m,2H). 13 C NMR(100MHz,DMSO-d6)δ159.8,153.8,153.0,148.9,143.7,139.9,138.2,130.6,13 0.2,129.1,128.6,120.0,114.3,113.7,112.5,104.7,65.1,60.6,57.8,56.5,32.6.

[0204] Example 12

[0205] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0206]

[0207] The method for synthesizing compound 39 from compounds 4 and 38 is based on the synthesis method of compound 6 in Example 1, and will not be repeated here. The obtained product does not require purification and can be used directly in the next reaction. The structure of compound 39 is characterized as follows: LC-MS: m / z 580 [M+H] + .

[0208] Compound 39 obtained in the previous step was dissolved in an equal volume ratio of EtOH / H2O solution and reacted overnight at 80°C. After the reaction was completed, the solution was concentrated under reduced pressure, purified by liquid chromatography, and freeze-dried to obtain yellow compound 40, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 40 is characterized as follows: LC-MS: m / z 450 [M+H] + . 1H NMR (400MHz, DMSO-d6) δ13.8(br,1H),8.88(d,J=2.0Hz,1H),8.63(d,J=2.0Hz,1H),7.37(m,1H),7.26(s ,4H),4.19(t,J=6.0Hz,2H),3.92(s,6H),3.74(s,3H),3.61(t,J=6.0Hz,2H),2.20(s,3H),1.91(m,2H). 13 C NMR(100MHz,DMSO-d6)δ157.7,153.8,152.9,148.8,143.6,138.2,137.4,131.4,130.5 ,129.2,128.3,125.6,119.3,112.6,110.8,104.6,65.1,60.6,57.9,56.4,32.8,16.2.

[0209] Example 13

[0210] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation process is as follows:

[0211]

[0212] Compound 41 (2.0 g, 1 eq) was reacted with TMPZnC·LiCl (21.2 mL, 1 mol / L, 2.5 eq) for 1 h at room temperature. CuCl·2LiCl (12.6 mL, 1 mol / L, 1.5 eq) and 3,4,5-trimethoxybenzoyl chloride (3.4 g, 1.7 eq) were added at -78 °C. After the reaction was complete, the reaction was quenched with NH4Cl / NH3·H2O (25%-28%) = 9 / 1, extracted with EA, dried over anhydrous sodium sulfate, and purified using a PE / EA = 6 / 1 silica gel column to give compound 44 (1.98 g, 54.5% yield). The structure of compound 44 was characterized as follows: LC-MS: m / z 433 [M+H] + . 1 H NMR (400MHz, CDCl3) δ10.52(s,1H),8.57(s,1H),7.74(s,2H),4.01(s,6H),3.93(s,3H).

[0213] Compound 44 (1.98 g, 1.0 eq) was dissolved in methanol (46 mL), excess hydrazine hydrate was added, and the mixture was heated to reflux until the reaction was complete. After returning to room temperature, DCM was added until the yellow solid in the reaction solution was completely dissolved. The organic phase was washed with saturated NaCl water, dried over anhydrous Na₂SO₄, and the solvent was evaporated. The residue was recrystallized from methanol, filtered, and the methanol was removed under vacuum to obtain the yellow solid compound 45 (1.04 g, yield 73.2%). The structure of compound 45 was characterized as follows: LC-MS: m / z 366 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.59(s,1H),7.79(s,2H),5.90(s,2H),4.04(s,6H),3.96(s,3H),3.83–3.36(m,2H),1.04–0.86(m,2H),-0.00(s,9H). 13 C NMR (100MHz, CDCl3) δ154.96,146.21,144.91,143.68,140.50,136.56,134.02,127.73,105.56,68.80,62.38,57.66,19.08,1.39.

[0214] Compound 45 (1.41 g, 1.0 eq) was dissolved in ultradry DMF (18 mL), and 60% NaH (0.31 g, 2.0 eq) was added at 0 °C. After stirring for 10 min, SEMCl (1.22 mL, 1.7 eq) was added, and the reaction was allowed to proceed at room temperature for approximately 2.5 h until complete. The reaction was quenched by slow dropwise addition of water at 0 °C. The aqueous phase was extracted with EA in small amounts multiple times, and the organic phase was washed with saturated NaCl water. After drying with anhydrous sodium sulfate, the solution was purified using a PE / EA = 5 / 1 silica gel column to obtain a yellow solid compound 46 (1.75 g, yield 91.6%). The structure of compound 46 was characterized as follows: LC-MS: m / z 495 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.95(s,1H),7.98(s,2H),7.46(d,J=6.5Hz,2H),7.22(d,J=8.0Hz,1H),5.90( s,2H),4.04(s,6H),3.94(s,3H),3.76–3.70(m,2H),2.26(s,3H),1.04–0.91(m,2H),-0.03(s,9H). 13C NMR (100MHz, CDCl3) δ153.61,149.47,145.41,144.25,142.89,141.13,136.03,132.23,13 1.36,127.46,124.23,117.40,113.25,104.40,67.29,61.16,56.34,17.84,17.44,-1.29.

[0215] Compounds 46 (500 mg, 1.0 eq), 47 (278 mg, 1.7 eq), Na₂CO₃ (321 mg, 3.00 eq), and PdCl₂ (dppf) (74 mg, 0.10 eq) were dissolved in 1,4-dioxane (6 mL), and H₂O (0.6 mL) was added. The mixture was microwaved at 100 °C for 2 h. After the reaction was complete, the solid in the reaction solution was filtered off, and the liquid was retained. The organic phase was washed with saturated NaCl water, dried over anhydrous sodium sulfate, and purified using a PE / EA = 2 / 1 silica gel column to obtain a yellow solid compound 48 (493 mg, yield 91.8%). The structure of compound 48 is characterized as follows: LC-MS: m / z 533 [M+H] + . 1 H NMR (400MHz, CDCl3) δ8.90(s,1H),8.01–7.97(m,2H),7.95(s,2H),6.98(d,J=8.8Hz,2H),6.54(s ,1H),5.90(s,2H),4.00(s,6H),3.94(s,3H),3.76–3.71(m,2H),1.01–0.95(m,2H),-0.04(s,9H). 13 C NMR (101MHz, CDCl3) δ157.75,153.52,149.19,143.89,142.77,140.67,138.68,132.11,12 9.38,128.53,127.42,116.31,116.29,104.33,75.57,67.35,61.16,56.27,17.82,-1.33.

[0216] Compound 48 (200 mg) was dissolved in 1,4-dioxane (3 mL), and 4N HCl-dioxane (3 mL) was added. After the reaction was complete, the solution was directly evaporated to dryness, and the mixture was separated by high-performance liquid chromatography (HPLC) to obtain a yellow solid compound 49, which is the nitrogen-containing heterocyclic compound of this embodiment. The structure of compound 49 is characterized as follows: LC-MS: m / z 403 [M+H] + . 1H NMR(400MHz,DMSO)δ14.15(s,1H),9.70(s,1H),9.19(s,1H),7.90(s,2H),7.70–7.65 (m,2H),7.37(t,J=7.8Hz,1H),6.92(dd,J=8.0,2.4Hz,1H),3.95(s,6H),3.76(s,3H). 13 C NMR (101MHz, DMSO) δ158.07,153.25,147.68,144.68,141.41,141.32,138. 01,137.90,130.17,130.10,127.56,117.59,113.44,103.42,60.17,55.85.

[0217] Example 14

[0218] The structural formula of the nitrogen-containing heterocyclic compound in this embodiment is: The preparation method adopted is the method of Example 13, and the specific preparation process is as follows:

[0219]

[0220] Biological evaluation

[0221] (1) Microtubule inhibitory activity assay

[0222] Microtubule inhibitory activity was determined using a Cytoskeleton kit. The experimental method is briefly described below: A final concentration of 3 μM of the compound was prepared and added to each well of a 96-well plate (10 μL per well, with three replicates for each concentration). Microtubules were dissolved in pre-prepared microtubule polymerization buffer (prepared according to the kit instructions, mainly containing GTP and glycerol). 100 μL was added to each well along the inner wall of the 96-well plate, and the plate was quickly placed in a microplate reader to read the values ​​once per minute for 61 consecutive readings. The experimental results are as follows: Figure 1 As shown.

[0223] Experimental conclusion: The nitrogen-containing heterocyclic compound prepared in this invention has significant inhibitory activity on tubulin aggregation.

[0224] (2) Protein kinase inhibitory activity assay

[0225] Experimental methods: Kinase activity screening was performed using materials from Eurofins DiscoverX. The technology platform can be used for screening, or the ADP-Glo ​​kinase activity screening method from Promega can be used for testing. For specific methods, please refer to the kit instructions.

[0226] Taking the FLT3 kinase inhibitory activity assay as an example, 1 μL of different concentrations of the test compound, positive control (Giltertinib), or buffer was added to each well of a 384-well plate, followed by 2 μL of kinase. For the blank control group, 2 μL of buffer was added. After mixing and incubating at room temperature for 10 min, 2 μL of ATP / substrate (MBP protein) mixture was added to each well, mixed, and incubated at room temperature for 2 h. 5 μL of ADP-Glo ​​reagent was added to each well, mixed, and incubated at room temperature for 40 min. Then, 10 μL of kinase detection reagent was added to each well, mixed, and incubated at room temperature for 30 min. The chemiluminescence value (RLU) of each well was measured using a microplate reader, and the kinase inhibitory activity of the compound was calculated.

[0227] Calculation formula: % Inhibition rate = (RLU(compound) - RLU(blank)) / (RLU(buffer group) - RLU(blank)) * 100%.

[0228] The experimental results are shown in Tables 1 and 2 below:

[0229] Table 1. Inhibitory activity of compounds against FLT3 kinase

[0230]

[0231] Table 2. Inhibitory activity of compound 10 against kinases

[0232]

[0233] Experimental conclusion: The nitrogen-containing heterocyclic compounds prepared in this invention have significant inhibitory activity against multiple protein kinases.

[0234] (3) Cell proliferation inhibition experiment

[0235] The following experiments were conducted to determine the inhibitory activity of the compounds described in this invention against tumor cell proliferation under in vitro conditions. The inhibitory activity of the compounds can be expressed as the IC50 value.

[0236] The experimental protocol is briefly described as follows: Tumor cells were suspended at an appropriate cell density (e.g., 15,000-50,000 cells / mL) in medium containing 10% FBS, and then seeded into 96-well plates. The plates were incubated overnight at 37°C with 5% CO2. The test compounds were first dissolved in DMSO, then diluted with FBS-free medium to the required concentration (generally, 9 concentration points were set for each compound), added to the 96-well plates, and incubated for another 48-72 hours at 37°C with 5% CO2. Subsequently, the inhibitory activity of the compounds on cell proliferation was determined using the CCK8 assay. The IC50 value of the compounds was calculated by examining the inhibitory effects of the test compounds on cell proliferation at different concentrations using GraphPadprism software.

[0237] The experimental results are shown in Table 3 below:

[0238] Table 3

[0239] Example Compound ID HCT116 K562 A549 Example 1 7 ++++ ++++ ++++ Example 2 10 ++++ ++++ ++++ Example 3 13 ++++ ++++ ++++ Example 4 16 ++++ ++++ ++ Example 5 19 ++++ ++++ +++ Example 6 22 ++++ ++++ ++++ Example 7 25 ++++ ++++ ++++ Example 8 28 ++ ++ ++ Example 9 31 + ++ + Example 10 34 ++++ +++ +++ Example 11 37 + ++ + Example 12 40 +++ +++ ++ Example 13 49 ++++ ++++ +++ Example 14 57 ++++ ++++ ++++

[0240] In the table above, ++++ indicates IC50 < 100 nM; +++ indicates IC50 is 100 nM to 500 nM; ++ indicates IC50 is 500 nM to 1000 nM; + indicates IC50 > 1000 nM.

[0241] Conclusion: The nitrogen-containing heterocyclic compounds prepared in this invention have significant inhibitory activity against tumor cell proliferation.

[0242] (4) Drug resistance test

[0243] First, a paclitaxel-resistant non-small cell lung cancer (NSCLC) model (A549) was constructed in vitro using a continuous drug exposure concentration escalation induction method. Then, the inhibitory activity of the compound and paclitaxel on cell proliferation was determined using the CCK8 assay.

[0244] The experimental results are shown in Table 4 below:

[0245] Table 4

[0246]

[0247] Conclusion: Paclitaxel cannot effectively inhibit the proliferation of tumor cells, and tumor cells are resistant to paclitaxel. However, they are not resistant to the nitrogen-containing heterocyclic compound prepared in this invention. The nitrogen-containing heterocyclic compound prepared in this invention has significant inhibitory activity against paclitaxel-resistant tumor cells.

[0248] The technical features of the above embodiments can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0249] The above-described embodiments are merely examples of several implementations of the present invention, intended to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims of the present invention.

Claims

1. The use of a nitrogen-containing heterocyclic compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating paclitaxel-resistant non-small cell lung cancer, characterized in that, The structural formula of the nitrogen-containing heterocyclic compound is shown below: ; The nitrogen-containing heterocyclic compound is , and .

2. The application according to claim 1, characterized in that, The drug is a pharmaceutical composition, wherein the active ingredient of the pharmaceutical composition comprises the nitrogen-containing heterocyclic compound of claim 1 or a pharmaceutically acceptable salt thereof.

3. The application according to claim 1, characterized in that, The drug is a microtubule inhibitor or protein kinase inhibitor, including the nitrogen-containing heterocyclic compound of claim 1 or a pharmaceutically acceptable salt thereof.

4. The application according to claim 1, characterized in that, The nitrogen-containing heterocyclic compound or its pharmaceutically acceptable salt is a protein kinase inhibitor or a microtubule inhibitor.

5. A method for preparing a nitrogen-containing heterocyclic compound, characterized in that, Includes the following steps: Compounds A and R 1 Compound B is prepared by reacting B(OH)₂ with a palladium catalyst and a basic reagent. The structural formula of compound A is as follows: The structural formula of compound B is as follows: ; Compound B was reacted with an acidic reagent to prepare a nitrogen-containing heterocyclic compound, the structural formula of which is as follows: ; The nitrogen-containing heterocyclic compound is , and .

6. The method for preparing the nitrogen-containing heterocyclic compound according to claim 5, characterized in that, It also includes the step of preparing compound A; The steps for preparing compound A include: Compound C was reacted with NaH and SEMCl to prepare compound D. The structural formula of compound C is as follows: The structural formula of compound D is as follows: ; The compounds D and R 2 Compound A is prepared by reacting B(OH)2, palladium catalyst, and basic reagent.

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