1-Benzyl-4-ethylpiperazine derivatives as TRPV4-KCa2.3 coupling promoters and their applications

Abstract

The present invention relates to 1-benzyl-4-ethylpiperazine derivatives as TRPV4-KCa2.3 coupling promoters and their applications, belonging to the field of chemical medicine. The present invention provides a compound as shown in general formula I or a pharmaceutically acceptable salt thereof, which can selectively enhance the spatial coupling degree between TRPV4 and KCa2.3, thereby being used to treat or improve diseases caused by abnormal TRPV4-KCa2.3 coupling.

Description

Technical Field

[0001] The invention belongs to the field of chemical medicine, and specifically relates to a 1-benzyl-4-ethylpiperazine derivative as a TRPV4-KCa2.3 coupling promoter and an application thereof. Background Art

[0002] Pulmonary artery hypertension (PAH) is a malignant pulmonary vascular disease that is difficult to cure. It is defined as a mean pulmonary artery pressure of ≥25 mmHg at rest. Its pathological characteristics are progressive increase in pulmonary vascular resistance (PVR) and pulmonary artery pressure (PAP) and remodeling lesions, which can lead to other complications such as right heart failure, atherosclerosis, and peripheral arterial disease. The 3-year survival rate of patients with pulmonary arterial hypertension in my country is 39%, and the 5-year survival rate is only 21%. About 1% of the world's population is affected by pulmonary arterial hypertension, and the incidence rate in people over 65 years old can reach 5% to 10%. The pathogenesis of this disease is complex. In addition to the disease of the pulmonary vascular itself, organic lesions and functional changes in the left and right heart and lung parenchyma can have a significant impact on the pulmonary vascular. A variety of vasoactive mediators (angiopoietin II, endothelin, etc.), growth factors (platelet-derived factor, fibroblast growth factor, etc.) and ion channels (K + Channels and Ca 2+ The pathological development process of pulmonary hypertension is mediated by the vasodilation pathway. The anti-pulmonary hypertension drugs currently on the market mainly include the following categories: (1) prostacyclin analogs and prostacyclin receptor agonists, such as epoprostenol, beraprost, and iloprost; (2) endothelin receptor antagonists, such as bosentan, ambrisentan, and macitentan; (3) phosphodiesterase-5 inhibitors, such as sildenafil, tadalafil, and vardenafil; (4) soluble guanylate cyclase receptor activators, such as riociguat. Existing clinical therapeutic drugs mainly act on vasodilation and can only alleviate symptoms but cannot reduce the mortality rate of patients. In addition, each of them has different degrees of adverse clinical manifestations. Therefore, it is urgent to develop new drugs with better efficacy, fewer side effects, high metabolic stability, and good bioavailability targeting new pharmacological targets to improve the survival rate of patients with pulmonary hypertension.

[0003] Basic research has found that there is an imbalance in the regulation of cell-specific ion channels in patients with pulmonary arterial hypertension, including K +Channels, different types of transient receptor potential channels, calcium sensor proteins, etc. (Int J Mol Sci. 2018, 19 (10): 3162). Among them, transient receptor potential vanilloid 4 (TRPV4) is a non-selective cationic calcium channel protein present on the cell membrane or intracellular organelle membrane. TRPV4 regulates a series of important physiological functions of the human body, including regulating calcium signals and protecting the integrity of endothelial cell barriers. TRPV4 channels mediate calcium influx through nitric oxide, prostacyclin and medium / small conductance potassium channel-dependent pathways. TRPV4-mediated Ca 2+ Inflow plays an important role in blood flow-induced vasodilation, and shear stress-induced vasodilation is impaired after TRPV4 knockout. In vascular endothelial cells, TRPV4 has been shown to contribute to the enhancement of vascular reactivity in 5-hydroxytryptamine-induced pulmonary vasoconstriction and chronic hypoxic pulmonary hypertension (AM J Physiol-Cell Ph. 2013, 305(7): C704-C715.).

[0004] Ca 2+ It is one of the important messenger substances in cells. 2+ Concentration ([Ca 2+ ]i) is a key factor in initiating cell-related signal transduction, such as proliferation, gene expression, and apoptosis, and is closely related to the occurrence and development of cardiovascular diseases. KCa2.3 is a small-conductance Ca 2+ Activated potassium channels, intracellular Ca 2+ At resting concentrations (about 100 nM), KCa2.3 channels are in a resting state, and increasing [Ca 2+ ]i activates KCa2.3 channels through calmodulin (Nature. 2010, 14 (8): 825-837.). In KCa2.3 overexpressing mice, it was detected that the arterial diameter of the mesenteric vasculature was significantly enlarged. The larger vascular diameter was accompanied by continuous hyperpolarization of the mesenteric endothelium, resulting in a decrease in myogenic and phenylephrine-induced tension, indicating that KCa2.3 has a dilating effect on vascular tension. Studies have shown that pulmonary microvascular endothelial cells (PMVEC) and pulmonary artery endothelial cells (PAEC) express TRPV4, BK, IK and SK3 ion channels, and Ca inflow through the TRPV4 channel 2+Selective activation of IK and SK channels, while inhibition of IK and SK3 channel activity attenuates TRPV4-induced pulmonary endothelial permeability. Electrophysiological results show that TRPV4 channels are functionally coupled to IK and SK (Pulm Circ. 2016, 5(2): 279-290.). The low vascular tone of the pulmonary vascular system is mainly maintained by the resting membrane potential of pulmonary arterial smooth muscle cells (PASMCs), which is mainly induced by KCa. In idiopathic pulmonary hypertension, KCa channels and their subunits are upregulated, resulting in PASMCs having more depolarized membrane potentials (Circulation. 1998, 98(14): 1400-1406.). Loss of endothelium-dependent vasodilation function in the pulmonary artery is an important cause of increased pulmonary artery pressure. Docosahexaenoic acid (DHA) can activate the KCa channels in these PASMCs and hyperpolarize the membrane potential of idiopathic pulmonary hypertension PASMCs to the membrane potential of PASMCs from healthy donors, leading to vasodilation (Eur Respir J. 2016, 48(4): 1127-1136.).

[0005] In recent years, studies have shown that pulmonary vasoconstriction, remodeling and in situ thrombosis are important pathophysiological bases for the development of pulmonary hypertension, and endothelial dysfunction plays a key role in this. In the past, people focused on the vasoactive factor nitric oxide and vasoconstrictor factor endothelin-1 produced by the large artery system, while ignoring the important role of endothelium-dependent hyperpolarizing factor (EDHF) produced by the arteriolar system. Studies have shown that the physical and functional coupling of TRPV4 and KCa2.3 ion channels in vascular endothelial cells is the cause of the EDHF reaction in endothelial cells (Hypertension. 2013, 62 (1): 134-139.). When the TRPV4-KCa2.3 protein is in an uncoupled state, the hyperpolarizing factor induced by endothelial cells is reduced, which is an important reason for the impaired vasodilation mechanism in hypertensive patients. The existing compound JNC-440 can restore the recoupling of TRPV4-KCa2.3 protein in the vascular endothelium of hypertensive model mice, thereby playing a role in lowering blood pressure (EMBO Mol Med. 2017, 9(11): 1491-1503.), and JNC-440 was found to have a good regulatory effect in the pulmonary hypertension model.

[0006] The structure of JNC-440 is as follows:

[0007]

[0008] Therefore, the TRPV4-KCa2.3 complex is an attractive molecular target for therapeutic intervention in pulmonary arterial hypertension, but there is currently no targeted drug for the TRPV4-KCa2.3 complex. Summary of the invention

[0009] Technical issues:

[0010] In order to solve the above problems existing in the prior art, the present invention provides a novel class of 1-benzyl-4-ethylpiperazine derivatives. The structural compounds have higher in vitro TRPV4-KCa2.3 coupling promoting activity. It is expected that such coupling promoting agents will have good therapeutic effects, are expected to overcome the problem of toxic and side effects, and have good development prospects.

[0011] Technical solution:

[0012] The present invention provides the following compounds of general formula (I), or pharmaceutically acceptable salts thereof:

[0013]

[0014] In a preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, X and Y are independently selected from NH, O, CO or CH2;

[0015] In a more preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, X and Y are independently selected from NH or CO;

[0016] In a preferred embodiment of the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof, R1 is selected from hydrogen, C1-C4 alkyl, phenyl, wherein the alkyl, phenyl may be optionally monosubstituted to pentasubstituted by the following identical or different substituents, the substituents being selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl;

[0017] In a more preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, R1 is selected from phenyl substituted with a substituent, wherein the substituent is preferably selected from: mono- to penta-substituted in fluorine, chlorine, trifluoromethyl or cyano;

[0018] In a preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, R2 is selected from C1-C4 alkyl or empty;

[0019] wherein R4 is selected from -CH-, -C2H3-, -C3H5- ​​or -C4H7-;

[0020] Z is selected from C, N, O or S;

[0021] In a more preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, R2 is selected from or empty;

[0022] Wherein R4 is preferably selected from -CH- or -C3H5-;

[0023] Z is preferably selected from N;

[0024] In a preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, R3 is selected from A phenyl group substituted or unsubstituted by a substituent, wherein the phenyl group may be optionally monosubstituted to pentasubstituted by the following identical or different substituents, wherein the substituents are selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl;

[0025] Wherein R5 is selected from hydrogen, C1-C4 alkyl or CONHR6, substituted or unsubstituted phenyl, wherein the alkyl and phenyl groups may be optionally mono-substituted to penta-substituted by the following identical or different substituents, wherein the substituents are selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl;

[0026] R6 is selected from hydrogen, C1-C4 alkyl, substituted or unsubstituted phenyl, wherein the alkyl and phenyl groups may be optionally monosubstituted to pentasubstituted by the following identical or different substituents, wherein the substituents are selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl;

[0027] In a more preferred embodiment of the compound of formula (I) or a pharmaceutically acceptable salt thereof, R3 is selected from C4 alkylamino -(CH2)4-NH-;

[0028] wherein R5 is preferably selected from hydrogen or CONHR6;

[0029] R6 is preferably selected from phenyl which is substituted or unsubstituted by a substituent, and the substituent is preferably selected from mono- to penta-substituted fluorine and chlorine.

[0030] The compound represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof is characterized in that when R2 is empty, the corresponding structural formula is:

[0031] The compound represented by formula (I) of the present invention or a pharmaceutically acceptable salt thereof is characterized in that the structure of the compound is as follows:

[0032] or

[0033] Wherein, R' and R" are independently selected from: halogen (F, Cl, Br), nitro, cyano, trifluoromethyl, C1-4 alkyl.

[0034] In the present invention, the preferred compounds of general formula (I) or pharmaceutically acceptable salts thereof are selected from:

[0035]

[0036]

[0037] The compound of the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is an inorganic salt or an organic salt, the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, isosulfonate, benzenesulfonate, and salicylate.

[0038] <Pharmaceutical Composition>

[0039] The present invention also provides a pharmaceutical composition, comprising the compound of the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

[0040] The compounds of general formula (I) or their salts may be isolated in the form of solvates, and any such solvates are therefore within the scope of the present invention.

[0041] The compounds of the present invention or their pharmaceutically acceptable salts can be formulated into solid preparations for oral administration, including, but not limited to capsules, tablets, pills, powders, granules, etc. In these solid dosage forms, the compounds of the general formula (I) of the present invention are mixed as active ingredients with at least one conventional inert excipient (or carrier), for example, with sodium citrate or dicalcium phosphate. Or mixed with the following ingredients: (1) fillers or solubilizers, such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (2) binders, such as hydroxymethylcellulose, alginate, gelatin, polyvinyl pyrrolidone, sucrose, gum arabic, etc.; (3) humectants, such as glycerol, etc.; (4) disintegrators, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain silicates and sodium carbonate, etc.; (5) solubilizers, such as paraffin, etc.; (6) absorption accelerators, such as quaternary ammonium compounds, etc.; (7) wetting agents, such as cetyl alcohol and glyceryl monostearate, etc.; (8) adsorbents, such as kaolin, etc.; (9) lubricants, such as talc, calcium stearate, solid polyethylene glycol, sodium lauryl sulfate, etc., or mixtures thereof. Capsules, tablets and pills may also contain buffers.

[0042] The solid dosage forms such as tablets, sugar pills, capsules, pills and granules can be coated or microencapsulated with coating and shell materials such as enteric coatings and other materials known in the art. They can contain opacifiers, and the release of the active ingredient in such compositions can be delayed in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active ingredient can also be formed into microcapsules with one or more of the above-mentioned excipients.

[0043] The compounds of the present invention or their pharmaceutically acceptable salts can be formulated into liquid dosage forms for oral administration, including, but not limited to, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, tinctures, etc. In addition to the compound of formula (I) or its pharmaceutically acceptable salt as an active ingredient, the liquid dosage form may contain inert diluents conventionally used in the art, such as water and other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oils, in particular cottonseed oil, peanut oil, corn oil, olive oil, castor oil, sesame oil, etc. or mixtures of these substances, etc. In addition to these inert diluents, the liquid dosage form of the present invention may also include conventional adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices, etc.

[0044] The suspending agent includes, for example, ethoxylated stearyl alcohol, polyoxyethylene sorbitol, and sorbitan, microcrystalline cellulose, agar, etc. or a mixture of these substances.

[0045] The compounds of the present invention and their pharmaceutically acceptable salts can be formulated into dosage forms for parenteral injection, including, but not limited to, physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions and dispersions. Suitable carriers, diluents, solvents, excipients include water, ethanol, polyols and suitable mixtures thereof.

[0046] The compound of the present invention or its pharmaceutically acceptable salt can be configured into dosage forms for topical administration, including ointments, powders, suppositories, drops, sprays and inhalants, etc. The compound of the present invention of general formula (I) or its pharmaceutically acceptable salt as an active ingredient is mixed with a physiologically acceptable carrier and optional preservatives, buffers, and propellants that may be required under sterile conditions.

[0047] The pharmaceutical composition of the present invention comprises a compound of the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier, excipient, and diluent. When preparing the pharmaceutical composition, the compound of the general formula (I) of the present invention or a pharmaceutically acceptable salt thereof is usually mixed with a pharmaceutically acceptable carrier, excipient, or diluent. The content of the compound of the general formula (I) or a pharmaceutically acceptable salt thereof can be 0.01-1000 mg, for example 0.05-800 mg, 0.1-500 mg, 0.01-300 mg, 0.01-200 mg, 0.05-150 mg, 0.05-50 mg, etc.

[0048] <Purpose>

[0049] The present invention also provides the use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a TRPV4-KCa2.3 coupling promoter.

[0050] The present invention also provides use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof in preparing a drug for treating pulmonary arterial hypertension in mammals including humans.

[0051] The present invention also provides use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof in preparing a pulmonary arterial hypertension (PAH) regulator.

[0052] The present invention also provides the use of the compound of general formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating malignant lung diseases and cardiovascular diseases mediated by abnormal TRPV4-KCa2.3 coupling in humans.

[0053] The compound of general formula (I) or its pharmaceutically acceptable salt is used in a method for treating pulmonary arterial hypertension in mammals, including humans, by promoting the coupling effect of TRPV4-KCa2.3. The method comprises administering a therapeutically effective amount of the compound of the above formula I or its pharmaceutically acceptable salt to the mammal, including humans.

[0054] A "therapeutically effective amount" is an amount of a compound of the invention effective to produce a biological or medical response in a subject (eg, enhancing protein binding activity, or ameliorating symptoms, alleviating conditions, slowing or delaying disease progression, or preventing disease).

[0055] The pulmonary hypertension mentioned in the present invention includes heritable pulmonary arterial hypertension (HPAH), drug-induced pulmonary hypertension (DPAH), idiopathic pulmonary arterial hypertension (IPAH), portal hypertension-related pulmonary arterial hypertension (PoPH), connective tissue disease-associated pulmonary arterial hypertension (PAH-CTD), chronic thromboembolic pulmonary hypertension (CTEPH), persistent pulmonary hypertension of the newborn (PPHN), isolated post-capillary pulmonary hypertension (Ipc-PH), pulmonary arterial hypertension caused by chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension caused by congenital heart disease (CHD), etc.

[0056] The malignant lung diseases mentioned in the present invention include pulmonary embolism (PE), acute pulmonary thromboembolism (APTE), pulmonary vasculitis, pulmonary vascular tumors, pulmonary edema, acute lung injury (ALI), etc.

[0057] The cardiovascular diseases mentioned in the present invention include hypertension, hyperlipidemia, hypertensive heart disease, cor pulmonale, valvular heart disease, congenital heart disease, coronary heart disease, cardiomyopathy, heart failure, atherosclerosis (AS), diabetes, chronic kidney disease, venous thromboembolism (VTE), metabolic syndrome, arrhythmia, aortic disease, peripheral arterial disease, stroke, etc.

[0058] The compounds of the present invention or their pharmaceutically acceptable salts can be administered to mammals, including humans, orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically (powders, ointments, drops) or intratumorally.

[0059] The compound or pharmaceutically acceptable salt thereof of the present invention can be administered alone, or in combination with other pharmaceutically acceptable therapeutic agents, or in combination with other drugs for treating pulmonary hypertension. This combined therapy can be achieved by using the various components of the treatment simultaneously, sequentially or separately. The therapeutic agent includes, but is not limited to, calcium antagonists such as long-acting nifedipine, diltiazem, amlodipine, etc., prostaglandins such as prostacyclin, treprostinil, iloprost, etc., endothelin receptor inhibitors such as bosentan, ambrisentan, sitaxentan, etc., phosphodiesterase inhibitors such as sildenafil, vardenafil, tadalafil, etc., soluble guanylate cyclase agonists such as riociguat, etc., tyrosine kinase inhibitors such as imatinib, sorafenib, etc., Rho kinase inhibitors such as fasudil, etc. The components to be combined can be administered simultaneously or sequentially, in the form of a single preparation or in the form of different preparations. The combination includes not only a combination of one or other active agents of the compound of the present invention, but also a combination of two or more other active agents of the compound of the present invention.

[0060] Beneficial effects:

[0061] A novel 1-benzyl-4-ethylpiperazine derivative of the present invention has the biological function of promoting TRPV4-KCa2.3 coupling, and is used for the research treatment of pulmonary hypertension, thereby providing a new means for finding new treatments for malignant lung diseases, cardiovascular diseases, and circulatory system diseases. The 1-benzyl-4-ethylpiperazine derivative with a structure shown in the general formula (I) provided by the present invention has a relatively convenient synthesis method, a high purity (>95%), and good in vitro coupling activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the subcellular co-localization map of TRPV4-KCa2.3 in primary pulmonary artery endothelial cells of pulmonary hypertension model mice. DETAILED DESCRIPTION

[0063] The technical solution of the present invention will be described in detail below in conjunction with embodiments.

[0064] In the present invention, "C1-C8 alkyl" refers to a saturated linear or branched monovalent hydrocarbon group of 1 to 8 carbon atoms. Examples include, but are not limited to, pentyl.

[0065] In the present invention, "pharmaceutically acceptable salts" refer to those salts that retain the biological effectiveness and properties of the parent compound. The term "salt" refers to any salt of a compound according to the present invention prepared from an inorganic or organic acid or base and an internally formed salt. Typically, such salts have physiologically acceptable anions or cations.

[0066] "Administration" or "administering" a subject compound in the context of the present invention means providing the subject in need of treatment with a compound of the present invention.

[0067] The "embodiment" or "implementation" or "in another embodiment" or "in certain embodiments" or "in some embodiments of the present application" mentioned throughout this specification means that at least one embodiment includes the specific reference elements, structures or features related to the embodiment described. Therefore, the phrases "in one embodiment" or "in an embodiment" or "in another embodiment" or "in certain embodiments" or "in some embodiments of the present application" appearing in different places throughout the specification do not necessarily all refer to the same embodiment. In addition, specific elements, structures or features can be combined in one or more embodiments in any appropriate manner.

[0068] Unless otherwise required in this application, throughout the specification and the claims that follow, the word "comprising" should be interpreted in its open, inclusive sense, ie, "including, but not limited to."

[0069] The present invention includes all pharmaceutically acceptable isotopically labeled compounds of the compound of formula (I), wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.

[0070] Examples of suitable isotopes for inclusion in the compounds of the present invention include isotopes of hydrogen, such as 2 H and 3 H, isotopes of carbon such as 11 C. 13 C and 14 C, isotopes of nitrogen such as 13 N and 15 N, isotopes of oxygen such as 15 O. 17 O and 18 O.

[0071] The following is a further description of the synthesis of compounds 1-20 in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally based on conventional conditions.

[0072] The intermediate compounds involved in the following examples are prepared by the following process:

[0073] Intermediate M-3: Preparation of (4-(4-ethylpiperazin-1-yl)methylphenyl)carbamate

[0074]

[0075] Step a: Preparation of 1-ethyl-4-(4-nitrobenzyl)piperazine (M-1)

[0076] Dissolve 4-nitrobenzyl chloride (7.00 g, 40.80 mmol) in acetone (100 mL), add N-ethylpiperazine (5.59 g, 48.96 mmol) and potassium carbonate (11.28 g, 81.59 mmol), and heat to 45 ° C and reflux to react overnight. The next day, TLC monitored the reaction to be complete. Stop heating and stirring, filter with diatomaceous earth to obtain a filtrate, and concentrate the filtrate under reduced pressure to obtain a brown liquid. The crude product is recrystallized from ethyl acetate to obtain a yellow solid M-1 (9.50 g, 93.5%). HRMS-ESI (m / z): 251.0444 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ8.53 (td, J=6.0, 2.0Hz, 2H), 8.19 (dd, J=9.5, 6.2Hz, 2H), 6.82 (dd, J=12.1, 2.6Hz, 2H), 6.59 (ddd, J=9.9, 7.5, 2.6Hz, 2H), 4.29 (d, J=5.8Hz, 4H), 3.73 (s, 7H).

[0077] Step b: Preparation of 4-(4-ethylpiperazin-1-yl)methylaniline (M-2)

[0078] M-1 (9.00 g, 36.14 mmol) was dissolved in methanol (90 mL), and Raney nickel (0.9 g, 15.25 mmol) was added. The temperature was raised to 45 ° C under H2 atmosphere for 24 h, and the reaction was complete after TLC monitoring. After the reaction solution was cooled to room temperature, diatomaceous earth was used to filter the filtrate, and the filtrate was concentrated under reduced pressure to obtain a brown liquid M-2 (6.69 g, 84.6%), which was directly used in the next step without purification.

[0079] Step c: Preparation of (4-(4-ethylpiperazin-1-yl)methylphenyl)carbamate (M-3)

[0080] Compound M-2 (6.69 g, 30.55 mmol) was dissolved in dichloromethane (60 mL), and pyridine (7 mL) was added. The reaction flask was placed in a low-temperature reactor at -10 ° C, and phenyl chloroformate (5.74 g, 36.66 mmol) was added dropwise at constant pressure. The reaction system was reacted at -10 ° C for 0.5 h and then moved to room temperature for 0.5 h. TLC monitored the reaction to be complete. The reaction solution was concentrated under reduced pressure, diluted with water, extracted with ethyl acetate (15 mL × 3), the organic phases were combined, washed with water and saturated NaCl solution in turn, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain the crude product M-3, which was purified by silica gel column chromatography [eluent: dichloromethane / methanol = 30 / 1 (v / v)] to obtain a white solid M-3 (7.79 g, 75.2%). HRMS-ESI (m / z): 340.1985 [M+H] + ; 1 H-NMR(400MHz,DMSO-d6)δ8.65(s,1H),7.39(d,J=8.6Hz,3H),7.28–7.01(m,5 H), 6.77–6.74 (m, 1H), 3.36 (s, 2H), 2.36–2.25 (m, 10H), 0.96 (d, J = 1.9Hz, 3H).

[0081] Intermediate M-5a: Preparation of 5-amino-N-(4-cyanophenyl)pentanamide

[0082]

[0083] Step d: Preparation of tert-butyl (5-(4-cyanophenyl)amino)-5-oxopentylcarbamate (M-4a)

[0084] Dissolve Boc-5-aminovaleric acid (2.17 g, 10.0 mmol) in N,N-dimethylformamide (30 mL), add condensing agent HATU (4.18 g, 11 mmol), and stir for 10 min under N2 protection to activate the carboxyl group; add p-aminobenzonitrile (1.18 g, 10 mmol) and DIPEA (3.88 g, 30 mmol) in sequence under stirring, and react at room temperature for 6 h under N2 protection. The reaction is complete when monitored by TLC. The reaction solution was diluted with water, extracted with ethyl acetate (20 mL × 3), the organic phases were combined, washed with water to remove the residual N, N-dimethylformamide solution (20 mL × 6), and then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure to obtain a brown oily crude product M-4a, which was purified by silica gel column chromatography [eluent: petroleum ether / ethyl acetate = 10 / 1 (v / v)] to obtain a white solid M-4a (2.62 g, 82.6%). HRMS-ESI (m / z): 340.1632 [M+Na] +; 1 H-NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.82–7.74(m,4H),6.82(t,J=5.7Hz,1H),2.96(q,J=6.6 Hz, 2H), 2.38 (t, J = 7.4Hz, 2H), 1.60 (p, J = 7.4Hz, 2H), 1.45 (dd, J = 10.2, 5.0Hz, 2H), 1.39 (s, 9H).

[0085] Step e: Preparation of 5-amino-N-(4-cyanophenyl)pentanamide (M-5a)

[0086] M-4a (1g, 3.15mol) was dissolved in hydrochloric acid-methanol (10mL) solution, stirred at room temperature for 2h, and the reaction was complete. The methanol in the reaction solution was removed by rotary evaporation, and the pH was adjusted to 8-9 with saturated NaHCO3 solution under ice bath; extracted with ethyl acetate (10mL×3), the organic phases were combined, washed with saturated NaCl solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain brown oil M-5a (590.91mg, 86.4%). HRMS-ESI (m / z): 218.1288 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.05 (s, 1H), 7.83–7.62 (m, 4H), 2.69 (q, J = 6.6Hz, 2H), 2. 35(t,J=7.4Hz,2H),1.52(p,J=7.4Hz,2H),1.5(t,2H)1.63(dd,J=10.2,5.0Hz,2H).

[0087] Intermediate M-5b: Preparation of tert-butyl (5-(3-cyano-4-fluorophenyl)amino)-5-oxopentyl)carbamate

[0088]

[0089] M-5b was synthesized from 5-amino-2-fluorobenzonitrile, and the synthesis method was similar to that of intermediate M-5a. Brown oily liquid 243 mg, yield 87.1%. HRMS-ESI (m / z): 236.1197 [M+H] + .

[0090] Intermediate M-5c: Preparation of 5-amino-N-(4-trifluoromethylphenyl)pentanamide

[0091]

[0092] M-5c was synthesized from p-trifluoromethylaniline as raw material. The synthesis method was similar to that of intermediate M-5a. Yellow oil 265 mg, yield 90.0%. HRMS-ESI (m / z): 261.1213 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ 10.56 (s, 1H), 7.86 (d, J = 8.4Hz, 4H), 7.66 (d, J = 8.5Hz, 2H), 2.82 (t, J = 7.1Hz, 2H), 2.43 (t, J = 6.9Hz, 2H), 1.70–1.58 (m, 4H).

[0093] Intermediate M-7: Preparation of 4-fluoro-N-(1,2,3,4-tetrahydroisoquinolin-6-yl)benzamide

[0094]

[0095] Step f: Preparation of tert-butyl 6-(4-fluorobenzamide)-3,4-dihydroisoquinoline-2(1H)-carboxylate (M-6)

[0096] M-6 was synthesized from p-fluorobenzoic acid and tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate, and the synthesis method was similar to that of intermediate M-4a. 2.98 g of white solid was obtained, and the yield was 85.0%.

[0097] Step g: Preparation of 4-fluoro-N-(1,2,3,4-tetrahydroisoquinolin-6-yl)benzamide hydrochloride (M-7)

[0098] M-6 (1 g, 2.70 mmol) was dissolved in a hydrochloric acid-methanol mixed solution (10 mL), stirred at room temperature for 2 h, and the reaction was complete after monitoring by TLC. Methanol was removed by rotary evaporation to obtain a white solid M-12a (658.21 mg, 90.2%), which was used directly in the next step without purification. 1 H NMR (400MHz, DMSO-d6) δ10.43(s,1H),9.73(s,1H),8.10(dd,J=8.6,5.4Hz,2H),7.75–7.61(m,2H),7.37(t,J=8. 7Hz, 2H), 7.20 (d, J = 8.4Hz, 1H), 4.21 (s, 2H), 3.35 (d, J = 6.9Hz, 2H), 3.03 (d, J = 6.3Hz, 2H), 1.32 (d, J = 6.4Hz, 1H).

[0099] Intermediate M-9a: Preparation of N-(4-fluorophenyl)-4-piperazin-1-ylbenzamide

[0100]

[0101] Step h: Preparation of tert-butyl 4-(4-(4-fluorophenyl)carbamoyl)phenyl)piperazine-1-carboxylate (M-8a)

[0102] M-8a was synthesized from p-fluoroaniline and tert-butyl 4-(4-carboxyphenyl)piperazine-1-carboxylate. The synthesis method was similar to that of intermediate M-4a. 3.53 g of white solid was obtained with a yield of 88.5%. HRMS-ESI (m / z): 400.2031 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.03(s,1H),7.89(d,J=8.4Hz,2H),7.80(dd,J=8.9,5.0Hz,2H),7.17 (t,J=8.7Hz,2H),7.03(d,J=8.5Hz,2H),3.51–3.44(m,4H),3.28(t,J=5.2Hz,4H),1.43(s,9H).

[0103] Step i: Preparation of N-(4-fluorophenyl)-4-piperazin-1-ylbenzamide (M-9a)

[0104] M-8a (1 g, 2.51 mmol) was dissolved in a trifluoroacetic acid / dichloromethane (v / v=1 / 4, 10 mL) mixed solution and stirred at room temperature for 1 h. The reaction was complete after TLC monitoring. Dichloromethane was removed by rotary evaporation, and the pH was adjusted to 8-9 with a saturated NaHCO3 solution under an ice bath. Solids precipitated and M-9a was obtained by suction filtration. White solid 693.45 mg, yield 92.4%. HRMS-ESI (m / z): 300.1514 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.05(s,1H),7.90(d,J=8.8Hz,2H),7.80(dd,J=9.1,5.0Hz,2H),7. 17(t,J=8.9Hz,2H), 7.02(d,J=8.7Hz,2H), 4.81(s,1H), 3.29(t,J=4.9Hz,4H), 2.96(s,4H).

[0105] Intermediate M-9b: Preparation of N-(4-fluorophenyl)-4-(piperidin-4-yl)benzamide

[0106]

[0107] Step j: Preparation of tert-butyl 4-(4-(4-(4-fluorophenyl)carbamoyl)phenyl)piperidine-1-carboxylate (M-8b)

[0108] M-8b was synthesized from p-fluoroaniline and tert-butyl 4-(4-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-4a. White solid 3.31 g, yield 83.1%. HRMS-ESI (m / z): 421.1907 [M+Na] + ; 1 H NMR (400MHz, DMSO-d6) δ10.24(s,1H),7.89(d,J=8.1Hz,2H),7.80(dd,J=9.1,5.0Hz,2H),7.40(d,J=8.1Hz,2H),7.19(t ,J=8.9Hz,2H),4.10(d,J=10.3Hz,2H),2.77(t,J=12.1Hz,3H),1.78(d,J=14.7Hz,2H),1.58–1.48(m,2H),1.43(s,9H).

[0109] Step k: Preparation of N-(4-fluorophenyl)-4-(piperidin-4-yl)benzamide hydrochloride (M-9b)

[0110] M-9b was synthesized from M-8b using the method of intermediate M-7. 739.68 mg of white solid was obtained with a yield of 88.2%. 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),9.28–9.10(m,2H),7.96(d,J=8.1Hz,2H),7.82(dd,J=9.1,5.1Hz,2H),7 .38(d,J=8.1Hz,2H),7.19(t,J=8.9Hz,2H),3.39(s,2H),2.98(dt,J=16.6,8.3Hz,3H),1.95(q,J=5.8Hz,4H).

[0111] Intermediate M-9c: Preparation of N-(4-fluorophenyl)-3-(piperidin-4-yl)benzamide

[0112]

[0113] Step 1: Preparation of tert-butyl 4-(3-(4-fluorophenyl)carbamoyl)phenyl)piperidine-1-carboxylate (M-8c)

[0114] M-8c was synthesized from p-fluoroaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-4a. White solid 3.16 g, yield 79.3%. HRMS-ESI (m / z): 421.1898 [M+Na] + ;1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),7.84–7.73(m,3H),7.57(d,J=8.1Hz,1H),7.50–7.36(m,3H),6.94(t,J=7.2Hz,1H ), 4.11 (d, J = 12.5Hz, 1H), 3.35 (s, 1H), 2.79 (t, J = 12.1Hz, 3H), 1.80 (d, J = 11.0Hz, 2H), 1.62–1.52 (m, 2H), 1.43 (s, 9H).

[0115] Step m: Preparation of N-(4-fluorophenyl)-3-(piperidin-4-yl)benzamide hydrochloride (M-9c)

[0116] M-9c was synthesized from M-8c by referring to the intermediate M-7. The white solid was 725.42 mg with a yield of 86.5%. It was used directly in the next step without purification.

[0117] Intermediate M-9d: Preparation of 3-piperidin-4-yl-N-trifluoromethylbenzamide

[0118]

[0119] M-9d was synthesized from p-trifluoromethylaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9c. White solid 715.79 mg, yield 92.2%. HRMS-ESI (m / z): 349.1536 [M+H] + ; 1 HNMR (400MHz, DMSO-d6) δ10.65(s,1H),8.04(d,J=8.5Hz,2H),7.86(d,J=10.9Hz,2H),7.74(d,J=8.5Hz,2H),7.51(d,J=7.6Hz, 2H), 5.59 (s, 1H), 3.35 (d, J = 12.2Hz, 2H), 2.94 (dd, J = 27.8, 15.1Hz, 3H), 1.95 (d, J = 13.2Hz, 2H), 1.83 (q, J = 13.3, 12.8Hz, 2H).

[0120] Intermediate M-9e: Preparation of N-4-chlorophenyl-3-piperidin-4-ylbenzamide

[0121]

[0122] M-9e was synthesized from p-chloroaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9c. White solid 662.66 mg, yield 94.6%. HRMS-ESI (m / z): 315.1258 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.59(s,1H),7.94–7.84(m,4H),7.52–7.40(m,4H),3.36(d,J=12.3Hz,3H),3.04–2.94(m,3H),2.06–1.95(m,4H).

[0123] Preparation of Intermediate M-9f: Preparation of N-3-chlorophenyl-3-piperidin-4-ylbenzamide

[0124]

[0125] M-9f was synthesized from 3-chloroaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9c. The white solid (668.27 mg) was used directly for the next step. The yield was 95.4%.

[0126] Intermediate M-9g: Preparation of 3-chloro-4-fluorophenyl-3-piperidin-4-ylbenzamide

[0127]

[0128] M-9g was synthesized from 3-chloro-4-fluoroaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9c. White solid 668.76 mg, yield 93.0%. HRMS-ESI (m / z): 333.1164 [M+H] + ; 1 HNMR(400MHz, DMSO-d6)δ10.52(s,1H),8.10(dd,J=6.9,2.6Hz,1H),7.87–7.79(m,2H),7.75(ddd,J=9.1,4.3,2.6Hz,1 H),7.54–7.41(m,3H),3.43(d,J=13.2Hz,3H),3.10–2.92(m,3H),2.00(d,J=11.2Hz,2H),1.88(qd,J=13.0,4.0Hz,2H).

[0129] Intermediate M-9h: Preparation of N-(3-fluorophenyl)-3-piperidin-4-ylbenzamide

[0130]

[0131] M-9h was synthesized from 3-fluoroaniline and tert-butyl 4-(3-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9c. The white solid (625.65 mg) was used directly for the next step. The yield was 94.1%.

[0132] Intermediate M-9i: Preparation of N-(3-fluorophenyl)-4-piperidin-4-ylbenzamide

[0133]

[0134] M-9i was synthesized from 3-fluoroaniline and tert-butyl 4-(4-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9b. White solid 604.37 mg, yield 90.9%. HRMS-ESI (m / z): 299.1545 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.41(s,1H),8.66(s,1H),7.94(d,J=8.4Hz,2H),7.76(dt,J=11.9,2.3Hz,1H),7.57(dd,J=8.1,1.9Hz,1H),7.39 (dd,J=7.6,5.8Hz,3H),6.93(td,J=8.5,2.6Hz,1H),3.08–2.92(m,3H),2.51(t,J=1.9Hz,2H),1.97(d,J=13.3Hz,2H),1.89–1.78(m,2H).

[0135] Intermediate M-9j: Preparation of N-(4-chlorophenyl)-4-piperidin-4-ylbenzamide

[0136]

[0137] M-9j was synthesized from 4-chloroaniline and tert-butyl 4-(4-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9b. White solid 624.13 mg, yield 89.1%. HRMS-ESI (m / z): 315.1249 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.40(s,1H),7.95(d,J=8.1Hz,2H),7.84(d,J=8.9Hz,2H),7.40(d d,J=8.4,5.4Hz,4H),3.00(dt,J=34.3,10.9Hz,3H),1.99–1.85(m,4H),1.30–1.22(m,3H).

[0138] Intermediate M-9k: Preparation of N-(3-chlorophenyl)-4-piperidin-4-ylbenzamide

[0139]

[0140] M-9k was synthesized from 3-chloroaniline and tert-butyl 4-(4-carboxyphenyl)piperidine-1-carboxylate. The synthesis method was similar to that of intermediate M-9b. White solid 617.83 mg, yield 88.2%. HRMS-ESI (m / z): 315.1267 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),7.98(t,J=2.0Hz,1H),7.93(d,J=8.3Hz,2H),7.72(dd,J=8.3,2.1Hz,1H),7.42–7.35(m,3H),7.16(dd,J=8. 3,2.1Hz,1H),3.28(d,J=12.9Hz,2H),2.89(td,J=12.6,3.0Hz,3H),1.88 (dd,J=14.0,3.4Hz,2H),1.74(qd,J=12.8,3.9Hz,2H),1.35–1.15(m,1H).

[0141] Intermediate M-91: Preparation of N-(3-chlorophenyl)-4-piperazin-1-ylbenzamide

[0142]

[0143] M-91 was synthesized from 3-chloroaniline and tert-butyl 4-(4-carboxyphenyl)piperazine-1-carboxylate. The synthesis method was similar to that of intermediate M-9a. White solid 623.29 mg, yield 88.7%. HRMS-ESI (m / z): 316.1210 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.00(d,J=2.1Hz,1H),7.93(d,J=8.7Hz,2H),7.73(dd,J=8.2,2.0Hz,1H),7. 36(t,J=8.1Hz,1H),7.16–7.04(m,3H),3.48(t,J=5.3Hz,4H),3.19(t,J=5.1Hz,4H),1.27(dd,J=19.6,12.8Hz,1H).

[0144] Intermediate M-13a: Preparation of N-(4-fluorophenyl)-1-(4-piperazin-1-yl)benzoylazetidine-3-carboxamide

[0145]

[0146] Step n: Preparation of tert-butyl 3-((4-fluorophenyl)carbamoyl)azetidine-1-carboxylate (M-10a)

[0147] M-10a was synthesized from p-fluoroaniline (2.22 g, 20 mmol) and 1-Boc-azetidine-3-carboxylic acid (4.02 g, 20 mmol) as raw materials. The synthesis method was similar to that of intermediate M-4a. Pale yellow solid 4.85 g, yield 82.4%. HRMS-ESI (m / z): 317.1278 [M+Na] + ; 1 H NMR (400MHz, DMSO-d6) δ10.10 (s, 1H), 7.71–7.59 (m, 2H), 7.15 (t, J = 8.9Hz, 2H), 4. 02(dq,J=19.2,10.9,9.1Hz,4H), 3.47(ddd,J=14.4,8.6,5.8Hz,1H), 1.40(s,9H).

[0148] Step o: Preparation of N-(4-fluorophenyl)azetidine-3-carboxamide (M-11a)

[0149] M-11a was synthesized from M-10a (3 g, 10.20 mmol) according to the intermediate M-9a. 1.85 g of white solid, yield 93.4%. HRMS-ESI (m / z): 195.0934 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.38(s,1H),7.68–7.64(m,2H),7.17(t,J=8.9Hz,2H),4.25–3.93(m,5H),3.80(t,J=8.3Hz,1H).

[0150] Step p: Preparation of tert-butyl 4-(4-(3-((4-fluorophenyl)carbamoyl)azetidine-1-carbonyl)phenyl)piperazine-1-carboxylate (M-12a)

[0151] M-12a was synthesized from compound M-11a (2 g, 10.30 mmol) and tert-butyl 4-(4-carboxyphenyl)piperazine-1-carboxylate (3.15 g, 10.30 mmol) as raw materials. The synthesis method was referred to compound M-9a. White solid 3.59 g, yield 72.4%. HRMS-ESI (m / z): 483.2405 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.14(s,1H),7.64(dd,J=9.1,5.0Hz,2H),7.55(d,J=8.8Hz,2H),7.16(t,J=8.9Hz,2H),6.96(d,J=8.9Hz ,2H),4.46(d,J=30.8Hz,2H),4.20(s,1H),3.62–3.55(m,1H),3.46(t,J=5.1Hz,4H),3.36(s,1H),3.27–3.21(m,4H),1.43(s,9H).

[0152] Step q: Preparation of N-(4-fluorophenyl)-1-(4-piperazin-1-yl)benzoylazetidine-3-carboxamide (M-13a)

[0153] M-13a was synthesized from intermediate M-12a (1 g, 2.07 mmol) according to the method of intermediate M-9a. White solid 704.88 mg, yield 89.1%. HRMS-ESI (m / z): 383.21 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.30(s,1H),7.65(dd,J=8.8,5.0Hz,2H),7.53(d,J=8.5Hz,2H),7.15(t,J=8.7Hz,2H),6.93(d,J= 8.5Hz, 2H), 4.45 (d, J = 33.4Hz, 2H), 4.14 (d, J = 33.3Hz, 2H), 3.43 (s, 1H), 3.17 (s, 5H), 2.80 (d, J = 18.8Hz, 3H), 2.51 (s, 1H).

[0154] Intermediate M-13b: Preparation of N-(4-fluorophenyl)-1-(4-piperazin-1-yl)benzoylpiperidine-4-carboxamide

[0155]

[0156] M-13b was synthesized from p-fluoroaniline (2.22 g, 20 mmol) and 1-Boc-4-piperidinic acid (4.58 g, 20 mmol) as raw materials. The synthesis method was similar to that of intermediate M-13a. White solid 750.95 mg, yield 93.4%. HRMS-ESI (m / z): 411.2191 [M+H] + ; 1 H NMR(400MHz, DMSO-d6)δ10.23(s,1H),7.67(dd,J=9.1,5.0Hz,2H),7.30(d,J =8.7Hz,2H),7.13(t,J=8.9Hz,2H),6.95(d,J=8.8Hz,2H),4.04(d,J=7.1Hz, 1H),3.89(s,2H),3.19(t,J=5.1Hz,4H),2.92(s,4H),2.67(t,J=11.2Hz,1H) ,1.83(d,J=14.0Hz,2H), 1.59(qd,J=12.3,4.2Hz,2H), 1.18(t,J=7.1Hz,2H).

[0157] Intermediate M-17a:N 1 , N 3 Preparation of bis(4-fluorophenyl)-5-(piperidin-4-yl)m-aniline

[0158]

[0159] Step r: 5-Bromo-N 1 , N 3 - Preparation of bis(4-fluorophenyl)m-aniline (M-14a)

[0160] 5-Bromoisophthalic acid (2.77 g, 10.0 mmol) was dissolved in N, N-dimethylformamide (30 mL), and condensing agent HATU (4.18 g, 11 mmol) was added thereto. After stirring for 10 min, 2 equivalents of p-fluoroaniline (1.11 g, 20.0 mmol) and 4 equivalents of DIPEA (5.17 g, 40 mmol) were added. After addition, N2 protection was applied and the reaction was allowed to proceed at room temperature for 4 h. TLC monitored the reaction until complete. The reaction solution was added to crushed ice, and a light yellow solid precipitated. The filter cake was washed with water several times and then dried in a vacuum oven at 35 ° C to obtain a light yellow powder solid M-14a (3.24 g, 75.4%). HRMS-ESI (m / z): 431.0325 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ 10.58 (s, 2H), 8.53 (s, 1H), 8.35 (d, J = 1.4Hz, 2H), 7.82 (dd, J = 9.1, 5.0Hz, 4H), 7.23 (t, J = 8.9Hz, 4H).

[0161] Step s: Preparation of tert-butyl 4-(3,5-bis(4-fluorophenyl)carbamoyl)-3,6-dihydropyridine-1(2H)-carboxylate (M-15a)

[0162] M-14a (3.00 g, 6.98 mmol), N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (3.24 g, 10.47 mmol), tetrakis(triphenyl)phosphine palladium (0.69 g, 0.6 mmol), potassium carbonate (1.93 g, 13.96 mmol) were added to the reaction bottle in sequence, and then 1,4-dioxane / water (v / v=5 / 1, 60 mL) mixed solvent was added and stirred to dissolve. Under N2 protection, the temperature was raised to 100°C and refluxed overnight. The next day, TLC monitored the reaction to be complete. The reaction was stopped, and the reaction solution was cooled to room temperature. The filtrate was filtered through diatomaceous earth to obtain a filtrate. The filtrate was concentrated under reduced pressure, extracted with ethyl acetate (20 mL × 3), washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography [eluent: dichloromethane / methanol = 200 / 1 (v / v)] to obtain a yellow solid M-15a (2.12 g, 56.9%). 1 H NMR (400MHz, DMSO-d6) δ10.54(d,J=28.4Hz,2H),8.39(d,J=32.9Hz,1H),8.18(s,1H),7.83(s,4H),7.65–7.54(m,1H),7.23(t,J=8. 7Hz, 4H), 6.41 (s, 1H), 3.98 (d, J = 86.6Hz, 2H), 3.59 (d, J = 17.3Hz, 2H), 2.61 (s, 1H), 2.04 (d, J = 36.6Hz, 1H), 1.43 (d, J = 19.2Hz, 9H).

[0163] Step t: Preparation of tert-butyl 4-(3,5-bis(4-fluorophenyl)carbamoyl)phenyl)piperidine-1-carboxylate (M-16a)

[0164] M-15a (2.00 g, 3.75 mmol) was dissolved in anhydrous methanol solution (20 mL), and a mixture of palladium carbon / palladium hydroxide carbon (m / m=1 / 1, 200 mg) was added. The mixture was heated to 50°C under H2 atmosphere and reacted for 48 h. The reaction was complete after monitoring by LC-MS. Heating was stopped, and the reaction solution was cooled to room temperature, filtered with the aid of diatomaceous earth, and the filtrate was concentrated under reduced pressure to obtain a light yellow solid M-16a (967.43 mg, 48.2%). The mixture was used directly in the next step without purification. 1 H NMR (400MHz, DMSO-d6) δ10.43(s,2H),8.38(s,1H),8.02(d,J=1.6Hz,2H),7.80(dd,J=9.1,5.0Hz,4H),7.22(t,J=8. 9Hz, 4H), 3.93 (s, 2H), 2.90 (t, J = 12.1Hz, 2H), 2.69 (s, 1H), 1.86 (d, J = 12.8Hz, 2H), 1.69–1.59 (m, 2H), 1.43 (s, 9H).

[0165] Step u: Preparation of N1, N3-bis(4-fluorophenyl)-5-(piperidin-4-yl)m-aniline (M-17a)

[0166] M-17a was synthesized from intermediate M-16a (900 mg, 1.68 mmol) according to the synthesis method of intermediate M-9a. White solid 644.83 mg, yield 88.2%. HRMS-ESI (m / z): 436.1838 [M+H] + .

[0167] Intermediate M-17b:N 1 , N 3 Preparation of -bis(3-chlorophenyl)-5-(piperidin-4-yl)m-aniline

[0168]

[0169] M-17b was synthesized from 3-chloroaniline (2.54 g, 20.0 mmol) and 5-bromoisophthalic acid (2.77 g, 10.0 mmol) as raw materials. The synthesis method was similar to that of intermediate M-17a. The product was a white solid (678.85 mg) with a yield of 91.4%.

[0170] Example 1: N-(4-cyanophenyl)-5-(4-(4-ethylpiperazin-1-yl)methylphenyl)ureido)pentanamide (Compound 1)

[0171]

[0172] Intermediate M-5a (150 mg, 0.69 mmol), intermediate M-3 (281.37 mg, 0.83 mmol), triethylamine (104.03 mg, 1.03 mmol), N, N-dimethylformamide (4 mL) were added to the reaction bottle in sequence, and reacted at 60 ° C for 8 h. The reaction was monitored by TLC. Water (4 mL) was added to the reaction solution for dilution, and extracted with ethyl acetate (4 mL × 3). The organic phases were combined, washed with water to remove the residual N, N-dimethylformamide solution, and then washed with saturated NaCl solution, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography [eluent: dichloromethane / methanol / ammonia water = 200 / 10 / 1 (v / v)] to obtain a light yellow solid compound 1 (252.47 mg, 79.2%). HRMS-ESI (m / z): 463.2817 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.35(s,1H),8.39(s,1H),7.80(d,J=8.9Hz,2H),7.75(d,J=8.8 Hz,2H),7.33(d,J=8.5Hz,2H),7.11(d,J=8.5Hz,2H),6.15(t,J=5.7Hz,1H),3.34(s,2H) ,3.12(q,J=6.5Hz,2H),2.40(t,J=7.4Hz,4H),2.33(s,2H),2.31(s,2H),2.27(d,J=7.2H z,4H),1.64(dt,J=14.8,7.3Hz,2H),1.48(dd,J=8.9,6.0Hz,2H),0.97(t,J=7.2Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ172.47,155.72,143.92,139.80,133.67,131.02,129.80,129.64,119.55,119.43,117. 87,115.67,105.12,62.22,53.00,52.84,52.07,39.16,36.65,31.60,30.29,29.87,29.44,29.42,22.76,12.46.

[0173] Example 2: N-(3-cyano-4-fluorophenyl)-5-(4-(4-ethylpiperazin-1-yl)methylphenyl)ureido)pentanamide (Compound 2)

[0174]

[0175] Compound 2 was synthesized from intermediates M-5b and M-3. The synthesis method was similar to compound 1. Pale yellow solid 259.99 mg, yield 78.5%. HRMS-ESI (m / z): 481.1764 [M+H] + ; 1 H NMR (400MHz, DMSO-d6) δ10.31(s,1H),8.42(s,1H),8.13(dd,J=5.9,2.7Hz,1H),7.85–7.80( m,1H),7.47(t,J=9.1Hz,1H),7.32(d,J=8.2Hz,2H),7.10(d,J=8.2Hz,2H),6.17(t,J=5.7Hz, 1H),3.32(s,2H),3.10(q,J=6.5Hz,2H),2.52–2.36(m,4H),2.33(d,J=5.2Hz,2H),2.32–2.2 8(m,2H),2.26(t,J=7.2Hz,4H),1.66–1.59(m,2H),1.50–1.43(m,2H),0.96(t,J=7.1Hz,3H). 13 C NMR(101MHz,Methanol-d4)δ172.97,160.18,157.67,156.91,138.94,130.49,129.86,126.33(d,J=8.0Hz),123.50,118.58, 116.41 (d, J = 20.9Hz), 113.29, 100.53 (d, J = 16.4Hz), 61.92, 51.88 (d, J = 10.6Hz), 38.91, 35.93, 29.38, 27.57, 22.42, 10.35.

[0176] Example 3: 5-(4-(4-ethylpiperazine-1-methyl)phenylureido)-N-(4-trifluoromethylphenyl)pentanamide (Compound 3)

[0177]

[0178] Compound 3 was synthesized from intermediates M-5c and M-3. The synthesis method was similar to compound 1. Pale yellow solid 259.99 mg, yield 78.5%. HRMS-ESI (m / z): 506.2764 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.31(s,1H),8.42(s,1H),8.13(dd,J=5.9,2.7Hz,1H),7.85–7.80( m,1H),7.47(t,J=9.1Hz,1H),7.32(d,J=8.2Hz,2H),7.10(d,J=8.2Hz,2H),6.17(t,J=5.7Hz, 1H),3.32(s,2H),3.10(q,J=6.5Hz,2H),2.52–2.36(m,4H),2.33(d,J=5.2Hz,2H),2.32–2.2 8(m,2H),2.26(t,J=7.2Hz,4H),1.66–1.59(m,2H),1.50–1.43(m,2H),0.96(t,J=7.1Hz,3H). 13C NMR(101MHz,Methanol-d4)δ172.97,160.18,157.67,156.91,138.94,130.49,129.86,126.33(d,J=8.0Hz),123.50,118.58, 116.41 (d, J = 20.9Hz), 113.29, 100.53 (d, J = 16.4Hz), 61.92, 51.88 (d, J = 10.6Hz), 38.91, 35.93, 29.38, 27.57, 22.42, 10.35.

[0179] Example 4: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-6-(4-fluorobenzamido)-3,4-dihydroisoquinoline-2(1H)-carboxamide (Compound 4)

[0180]

[0181] Compound 4 was synthesized from intermediates M-7 and M-3. The synthesis method was similar to compound 1. White solid 127.09 mg, yield 66.7%. HRMS-ESI (m / z): 516.2769 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.20(s,1H),8.53(s,1H),8.01(s,1H),7.58(d,J=33.6Hz,2H),7.36(d,J=21.0Hz,4H),7.12(s,4H),4.5 8(s,2H),3.68(t,J=15.3Hz,2H),3.48(s,2H),3.14–2.94(m,2H),2.92–2.57(m,4H),2.48(s,2H),2.32–2.24(m,4H),0.94(t,3H); 13 C NMR(101MHz,DMSO-d6)δ165.74,164.74,163.27,155.51,139.74,137.73,135.41,131.94,131.81,130.84,130.75,129.79,129.2 8,126.85,120.72,120.07,118.98,115.87,115.66,62.24,53.07,52.87,52.08,45.84,41.83,40.60,40.39,39.35,29.08,12.49.

[0182] Example 5: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-4-(4-(4-fluorophenyl)carbamoyl)piperazine-1-carboxamide (Compound 5)

[0183]

[0184] Compound 5 was synthesized from intermediates M-9a and M-3. The synthesis method was similar to compound 1. White solid 131.95 mg, yield 73.5%. HRMS-ESI (m / z): 545.3035 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.62(s,1H),7.90(d,J=8.7Hz,2H),7.79(dd,J=9. 0,5.0Hz,2H),7.42(d,J=8.1Hz,2H),7.18(d,J=9.0Hz,2H),7.15(d,J=8.2Hz,2H),7.07(d ,J=8.6Hz,2H),3.62(t,J=5.0Hz,4H),3.36(s,4H),3.23(t,J=4.9Hz,1H),2.87(t,J=4.9 Hz, 1H), 2.33 (s, 4H), 2.28 (t, J = 7.2Hz, 4H), 1.23 (d, J = 3.9Hz, 2H), 0.97 (t, J = 7.1Hz, 3H). 13 C NMR(101MHz,DMSO-d6)δ165.37,159.68,157.30,153.99,155.52,153.37,139.65,136.36,131.99,129.52,129.33,124.19,122.50,12 2.43,119.94,115.61,115.39,114.61,114.19,113.84,62.22,53.05,52.86,52.08,48.28,47.46,45.64,43.85,40.61,39.36,12.48.

[0185] Example 6: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-4-(4-(4-fluorophenyl)carbamoyl)piperidine-1-carboxamide (Compound 6)

[0186]

[0187] Compound 6 was synthesized from intermediates M-9b and M-3. The synthesis method was similar to compound 1. White solid 129.34 mg, yield 79.4%. HRMS-ESI (m / z): 544.3108 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.26(s,1H),8.53(s,1H),7.91(d,J=7.8Hz,2H),7.80(dd,J=8.8 ,5.0Hz,2H),7.43(d,J=8.1Hz,4H),7.19(t,J=8.6Hz,2H),7.14(d,J=8.1Hz,2H),4.30(d, J=12.9Hz,2H),3.37(s,4H),2.94–2.81(m,3H),2.51(s,2H),2.43–2.37(m,2H),2.29(t,J =7.1Hz, 4H), 1.83 (t, J = 12.7Hz, 2H), 1.61 (q, J = 13.0, 12.1Hz, 2H), 0.98 (t, J = 7.1Hz, 3H); 13 C NMR(101MHz,DMSO-d6)δ165.77,157.49,155.36,150.10,139.96,133.25,131.71,129.27,128.32,127.25,122.52(d,J =7.9Hz),119.89,115.70,115.48,62.25,55.37,52.95(d,J=18.3Hz),52.08,44.78,42.30,40.63,39.38,33.17,12.47.

[0188] Example 7: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-4-(3-(4-fluorophenyl)carbamoyl)piperidine-1-carboxamide (Compound 7)

[0189]

[0190] Compound 7 was synthesized from intermediates M-9c and M-3. The synthesis method was similar to compound 1. White solid 111.09 mg, yield 68.2%. HRMS-ESI (m / z): 544.3031 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.28(s,1H),8.54(s,1H),7.85(s,1H),7.81(d,J=5.1Hz,1H),7.78 (dd,J=8.1,3.9Hz,2H),7.52–7.45(m,2H),7.43(d,J=8.4Hz,2H),7.20(t,J=8.9Hz,2H),7.14 (d,J=8.2Hz,2H),4.35–4.27(m,2H),3.44(s,4H),2.93–2.82(m,3H),2.51(t,J=1.9Hz,4H),2 .35(t,J=7.2Hz,4H),1.85(dd,J=13.2,3.3Hz,2H),1.69–1.59(m,2H),0.99(t,J=7.1Hz,3H); 13 CNMR(101MHz,DMSO-d6)δ165.92,155.36,146.60,140.00,135.94,135.29,131.55,130.60,129.32,129.01,126.36,126.11,122.81(d,J= 7.8Hz), 119.92, 115.73, 115.51, 62.13, 52.73 (d, J = 8.4Hz), 52.00, 44.82, 42.33, 40.50 (d, J = 21.0Hz), 39.46 (d, J = 21.1Hz), 33.30, 12.26.

[0191] Example 8: 4-(4-(trifluoromethylphenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 8)

[0192]

[0193] Compound 8 was synthesized from intermediates M-9d and M-3. The synthesis method was similar to compound 1. White solid 120.72 mg, yield 70.2%. HRMS-ESI (m / z): 594.3050 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.58(s,1H),8.56(s,1H),8.07(s,1H),8.05(s,1H),7.91(d,J=1.8Hz,1H),7.86 (d,J=7.1Hz,1H),7.73(d,J=8.6Hz,2H),7.51(d,J=7.7Hz,1H),7.50–7.47(m,2H),7.46(s,1H),7.15(d,J =8.4Hz,2H),4.41–4.29(m,2H),3.36(s,2H),2.95–2.82(m,3H),2.52(t,J=1.9Hz,4H),2.35–2.32(m,2H) ,2.29(q,J=7.2Hz,4H),1.87(dd,J=13.2,3.6Hz,2H),1.67(qd,J=12.7,3.9Hz,2H),0.96(t,J=7.1Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ166.49,155.39,146.64,143.31,139.97,135.04,131.72,130.86,129.77,129.13(d,J=23.3Hz),126.80–125.97( m), 124.59, 124.27, 123.96, 123.58 (d, J = 12.7Hz), 120.66, 119.93, 62.27, 60.19, 52.93 (d, J = 17.6Hz), 52.07, 44.83, 42.36, 33.30, 12.41.

[0194] Example 9: 4-(3-(4-chlorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 9)

[0195]

[0196] Compound 9 was synthesized from intermediates M-9e and M-3. The synthesis method was similar to compound 1. White solid 136.66 mg, yield 76.4%. HRMS-ESI (m / z): 560.2787 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.35(s,1H),8.53(s,1H),7.86(s,1H),7.84(d,J=2.1Hz,1H),7.82( d,J=5.2Hz,2H),7.48(s,2H),7.46–7.41(m,3H),7.41(d,J=2.2Hz,1H),7.14(d,J=8.4Hz,2H), 4.32(d,J=13.8Hz,2H),3.36(s,2H),2.93–2.81(m,3H),2.36(d,J=18.7Hz,4H),2.31(s,2H), 2.28(t,J=7.2Hz,4H),1.88–1.82(m,2H),1.65(qd,J=12.7,4.0Hz,2H),0.96(t,J=7.1Hz,3H); 13 CNMR(101MHz,DMSO-d6)δ166.09,155.36,146.61,139.96,138.60,135.23,131.72,130.68,129.26,128.98(d,J=5.2Hz) ,127.74,126.29(d,J=25.5Hz),122.42,119.92,62.26,52.96(d,J=18.3Hz),52.09,44.82,42.35,33.30,29.51,12.48.

[0197] Example 10: 4-(3-(3-chlorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 10)

[0198]

[0199] Compound 10 was synthesized from intermediates M-9f and M-3. The synthesis method was similar to compound 1. White solid 135.77 mg, yield 75.9%. HRMS-ESI (m / z): 560.2787 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.37(s,1H),8.53(s,1H),7.98(t,J=2.1Hz,1H),7.86(s,1H),7.82(d,J=7.2Hz,1H) ,7.73(dd,J=8.2,1.9Hz,1H),7.57–7.46(m,2H),7.45(d,J=2.2Hz,1H),7.43(d,J=2.0Hz,1H),7.39(t,J=8.1 Hz,1H),7.19–7.14(m,2H),7.13(s,1H),4.38–4.26(m,2H),3.40(s,2H),2.94–2.81(m,3H),2.50–2.33(m,4H ),2.30(s,2H),2.27(t,J=7.2Hz,4H),1.89–1.80(m,2H),1.65(qd,J=12.6,3.9Hz,2H),0.96(t,J=7.2Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ166.24,155.37,146.65,141.11,139.94,135.07,133.40,131.74,130.77(d,J=7.9Hz),129.16(d,J=21.8Hz), 126.31 (d, J = 22.0Hz), 123.79, 120.29, 119.92, 119.17, 62.27, 52.98 (d, J = 19.7Hz), 52.09, 44.81, 42.34, 40.61, 39.36, 33.29, 12.51.

[0200] Example 11: 4-(3-chloro-4-fluorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 11)

[0201]

[0202] Compound 11 was synthesized from intermediates M-9g and M-3. The synthesis method was similar to compound 1. White solid 120.82 mg, yield 69.8%. HRMS-ESI (m / z): 578.2686 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.40(s,1H),8.53(s,1H),8.07(dd,J=7.0,2.6Hz,1H),7.85(s,1H),7.80(d,J =7.4Hz,1H),7.75–7.70(m,1H),7.48(dd,J=14.8,7.5Hz,3H),7.42(d,J=8.3Hz,2H),7.13(d,J=8.2Hz, 2H),4.31(d,J=13.0Hz,2H),3.38(s,2H),2.90(t,J=12.8Hz,3H),2.50–2.33(m,4H),2.30(d,J=7.1Hz, 2H), 2.26 (d, J = 7.2Hz, 4H), 1.85 (d, J = 12.0Hz, 2H), 1.64 (qd, J = 12.6, 3.9Hz, 2H), 0.97 (t, J = 7.1Hz, 3H); 13 C NMR(101MHz,Methanol-d4)δ167.33,156.36,155.75,153.32,146.27,139.04,135.69,134.59,131.25,130.37,129.60,128.51,125.80,125 .34,122.63,120.98–120.33(m),120.06(d,J=18.7Hz),116.22,116.0 0,61.99,52.28–51.62(m),48.28,47.00,44.54,42.40,32.93,10.40.

[0203] Example 12: 4-(3-(3-fluorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 12)

[0204]

[0205] Compound 12 was synthesized from intermediates M-9h and M-3. The synthesis method was similar to compound 1. White solid 134.96 mg, yield 73.1%. HRMS-ESI (m / z): 544.3162 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.41(s,1H),8.54(s,1H),7.86(s,1H),7.81(d,J=10.4Hz,1H),7 .76(s,1H),7.67–7.46(m,3H),7.46–7.37(m,3H),7.14(d,J=7.1Hz,2H),6.94(s,1H),4.32 (d,J=13.2Hz,2H),3.50(s,2H),2.96–2.80(m,3H),2.51(s,2H),2.41–2.30(m,4H),2.30– 2.14(m,4H),1.85(q,J=12.7Hz,2H),1.66(dd,J=25.3,11.2Hz,2H),0.96(t,J=7.7Hz,3H); 13 C NMR(101MHz, Methanol-d4)δ167.51,164.05,161.64,156.38,146.27,140.41(d,J=10.9Hz),139.06,134.83,131.23,130.30,130.00–129.40(m), 128.49,125.82,125.34,120.51,116.04(d,J=3.0Hz),110.40(d,J=21.4H z),107.72,107.46,61.97,52.24–51.62(m),44.54,42.42,32.93,10.36.

[0206] Example 13: 4-(4-(3-fluorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 13)

[0207]

[0208] Compound 13 was synthesized from intermediates M-9i and M-3. The synthesis method was similar to compound 1. White solid 151.20 mg, yield 81.9%. HRMS-ESI (m / z): 544.3126 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.42(s,1H),8.56(s,1H),7.91(d,J=7.9Hz,2H),7.79(d,J=11.9Hz,1H),7.58(d ,J=8.2Hz,1H),7.45(s,2H),7.43(s,2H),7.41–7.34(m,1H),7.14(d,J=8.1Hz,2H),6.92(td,J=8.5,2.6H z,1H),4.31(d,J=13.0Hz,2H),3.40(s,2H),2.89(t,J=12.6Hz,3H),2.54–2.35(m,4H),2.34–2.30(m,2H) ,2.27(t,J=7.2Hz,4H),1.83(dd,J=13.3,3.6Hz,2H),1.61(qd,J=12.5,3.9Hz,2H),0.96(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ166.10(d,J=8.8Hz),163.72,161.33,155.35,150.3 5,141.45,139.89(d,J=9.5Hz),133.07,131.74,130.67(d,J=9.4Hz),129.2 8,128.39,127.33,119.83(d,J=10.6Hz),116.33,110.43(d,J=20.7Hz),107 .43,107.17,62.25,52.98(d,J=19.7Hz),52.09,44.76,42.31,33.15,12.52.

[0209] Example 14: 4-(4-(4-chlorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 14)

[0210]

[0211] Compound 14 was synthesized from intermediates M-9j and M-3, and the synthesis method was similar to compound 1. White solid 144.18 mg, yield 80.6%. HRMS-ESI (m / z): 560.2787 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.33(s,1H),8.54(s,1H),7.90(d,J=7.9Hz,2H),7.83(d,J=8. 5Hz,2H),7.44(d,J=2.5Hz,2H),7.42(s,3H),7.40(s,1H),7.14(d,J=8.1Hz,2H),4.30(d ,J=13.0Hz,2H),3.36(s,2H),2.93–2.81(m,3H),2.51–2.34(m,4H),2.31(s,2H),2.28( t,J=7.2Hz,4H),1.86–1.79(m,2H),1.61(td,J=12.4,8.6Hz,2H),0.97(t,J=7.1Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ165.92 (d, J = 8.4Hz), 155.32 (d, J = 6.6Hz), 150.25, 139.90 (d, J = 9.2Hz), 138.65 (d, J = 10.2Hz), 133.15, 131.73, 129.28 ,128.96,128.37,127.59,127.30,122.16(d,J=9.6Hz),119.84(d,J=10 .6Hz), 62.25, 52.97 (d, J = 19.4Hz), 52.08, 44.77, 42.30, 33.16, 12.51.

[0212] Example 15: 4-(4-(3-chlorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperidine-1-carboxamide (Compound 15)

[0213]

[0214] Compound 15 was synthesized from intermediates M-9k and M-3. The synthesis method was similar to compound 1. White solid 140.24 mg, yield 78.4%. HRMS-ESI (m / z): 560.4980 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.36(s,1H),8.53(s,1H),7.99(s,1H),7.91(d,J=7.9Hz,2H),7.72(d ,J=8.2Hz,1H),7.46(s,1H),7.44(s,2H),7.42–7.40(m,1H),7.37(d,J=8.1Hz,1H),7.15(t,J=8 .3Hz,3H),4.30(d,J=13.0Hz,2H),2.89(t,J=12.7Hz,3H),2.51–2.35(m,4H),2.35–2.30(m,2H) ,2.28(t,J=7.2Hz,4H),1.86–1.79(m,2H),1.62(tt,J=12.3,6.2Hz,2H),0.97(t,J=7.1Hz,3H); 13 CNMR(101MHz,DMSO-d6)δ166.12,155.37,150.35,141.24,139.96,133.41,133.01,131.71,130.71,129.27,128.41,127.30, 123.65, 119.99 (d, J = 18.3Hz), 118.98, 62.26, 52.95 (d, J = 18.0Hz), 52.08, 49.08, 44.78, 42.32, 40.63, 39.38, 33.16, 12.47.

[0215] Example 16: 4-(4-(3-chlorophenyl)carbamoyl)-N-(4-(4-ethylpiperazin-1-yl)methylphenyl)piperazine-1-carboxamide (Compound 16)

[0216]

[0217] Compound 16 was synthesized from intermediates M-91 and M-3. The synthesis method was similar to compound 1. White solid 135.47 mg, yield 75.6%. HRMS-ESI (m / z): 561.2656 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.13(s,1H),8.63(s,1H),7.98(s,1H),7.90(d,J=8.5Hz ,2H),7.71(d,J=7.5Hz,1H),7.41(d,J=8.1Hz,2H),7.36(t,J=8.1Hz,1H),7.13(t ,J=8.3Hz,3H),7.08(d,J=8.6Hz,2H),3.61(t,J=4.9Hz,4H),3.36(s,6H),2.48(d ,J=18.3Hz,4H),2.34–2.29(m,2H),2.27(t,J=7.2Hz,4H),0.96(t,J=7.1Hz,3H); 13 C NMR (101MHz, DMSO-d6) δ170.77, 165.62 (d, J = 8.0Hz), 155.49 (d, J = 7.2Hz), 153.50, 141.56, 139.67, 133.34, 131.93, 130.63, 129.67, 129.33, 123. 82,123.24,119.92(dd,J=9.9,5.6Hz),118.85(d,J=9.7Hz),114.13,62.1 9, 60.22, 52.88 (d, J = 15.9Hz), 52.05, 47.39, 43.84, 21.23, 14.56, 12.41.

[0218] Example 17: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-4-(4-(3-(4-fluorophenyl)carbamoyl)azetidine-1-carbonyl)phenyl)piperazine-1-carboxamide (Compound 17)

[0219]

[0220] Compound 17 was synthesized from intermediates M-13a and M-3. The synthesis method was similar to compound 1. White solid 119.33 mg, yield 73.2%. HRMS-ESI (m / z): 628.3484 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.17(s,1H),8.63(s,1H),7.65(dd,J=8.7,5.0Hz,2H),7.56( d,J=8.3Hz,2H),7.43(d,J=8.1Hz,2H),7.16(t,J=9.4Hz,4H),7.00(d,J=8.5Hz,2H),4. 47(d,J=32.0Hz,2H),4.16(d,J=35.9Hz,2H),3.59(q,J=7.9,6.3Hz,5H),3.42(s,3H), 3.35(s,3H),2.51(s,2H),2.40–2.30(m,4H),2.29–2.13(m,4H),0.96(t,J=7.2Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ170.69(d,J=8.8Hz),169.36,159.70,157.32,155.47(d,J=7.2 Hz), 152.86, 139.68 (d, J = 9.5Hz), 135.78 (d, J = 10.4Hz), 131.75, 129.74, 129.36, 122. 55,121.45(t,J=8.7Hz),119.87(d,J=10.5Hz),115.76(d,J=22.2Hz),114.32,62.09,5 5.60(d,J=44.6Hz),52.66,51.74(d,J=47.6Hz),47.44,43.88,34.71–33.84(m),12.21.

[0221] Example 18: N-(4-(4-ethylpiperazin-1-yl)methylphenyl)-4-(4-(4-(4-fluorophenyl)carbamoyl)piperidine-1-carbonyl)phenyl)piperazine-1-carboxamide (Compound 18)

[0222]

[0223] Compound 18 was synthesized from intermediates M-13b and M-3. The synthesis method was similar to compound 1. White solid 120.88 mg, yield 76.9%. HRMS-ESI (m / z): 656.3712 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),8.63(s,1H),7.64(dd,J=8.8,5.0Hz,2H),7.43(d,J=8.1Hz,2 H),7.32(d,J=8.2Hz,2H),7.13(t,J=8.5Hz,4H),7.01(d,J=8.5Hz,2H),3.60(t,J=4.9Hz,4H),3.39 (d,J=29.6Hz,4H),3.26(t,J=5.0Hz,4H),2.96(s,2H),2.64–2.57(m,1H),2.37(d,J=27.5Hz,4H),2 .30(s,2H),2.27(t,J=7.1Hz,4H),1.83(d,J=12.5Hz,2H),1.65–1.55(m,2H),0.96(t,J=7.1Hz,3H); 13 C NMR (101MHz, Methanol-d4) δ173.97,171.49,160.44,158.04,156.41,152.38,138.88,134.68,131.33,129.62,128.40,125.49,121.72 (d, J=7 .9Hz),120.49,114.86(d,J=22.3Hz),61.86,53.44,51.85(d,J=7.3Hz) ,48.27,46.99,46.34,43.58,43.25,28.95(d,J=78.9Hz),10.20,8.19.

[0224] Example 19: N 1 , N 3 -Bis(4-fluorophenyl)-5-(4-(4-(4-ethylpiperazin-1-yl)methylphenyl)carbamoylpiperidin-4-yl)-m-aniline (Compound 19)

[0225]

[0226] Compound 19 was synthesized from intermediates M-17a and M-3. The synthesis method was similar to compound 1. White solid 107.13 mg, yield 68.5%. HRMS-ESI (m / z): 681.3330 [M+H] + ; 1H NMR (400MHz, DMSO-d6) δ10.48(s,2H),8.56(s,1H),8.42(s,1H),8.06(s,2H),7.82(dd,J=8. 9,5.0Hz,4H),7.44(d,J=8.1Hz,2H),7.23(t,J=8.7Hz,4H),7.14(d,J=8.1Hz,2H),4.35(d,J =12.9Hz,2H),3.36(s,2H),3.01–2.90(m,3H),2.63–2.48(m,2H),2.48–2.33(m,4H),2.30(q ,J=7.2Hz,4H),1.92(d,J=12.4Hz,2H),1.73(tt,J=12.7,6.4Hz,2H),0.97(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ165.34(d,J=8.7Hz),160.06,157.67,155.32(d,J=6.9Hz), 147.00, 139.89 (d, J = 9.3Hz), 135.67 (dd, J = 21.3, 7.3Hz), 131.73, 129.39 (d, J = 21. 9Hz),125.66,122.80(t,J=9.1Hz),119.91(d,J=10.4Hz),115.80,115.58,62.23,5 2.92(d,J=17.2Hz),52.06,46.17,44.78,42.35,40.61,39.36,33.19,12.43,11.13.

[0227] Example 20: N 1 , N 3 -Bis(3-chlorophenyl)-5-(4-(4-ethylpiperazin-1-yl)methylphenyl)carbamoylpiperidin-4-yl)-m-aniline (Compound 20)

[0228]

[0229] Compound 20 was synthesized from intermediates M-17b and M-3. The synthesis method was similar to compound 1. White solid 89.41 mg, yield 59.8%. HRMS-ESI (m / z): 713.2800 [M+H] + ; 1H NMR(400MHz,DMSO-d6)δ10.59(s,2H),8.57(s,1H),8.44(s,1H),8.08(s,2H),8.00(s,2H), 7.77(d,J=8.2Hz,2H),7.45–7.39(m,4H),7.19(d,J=7.8Hz,2H),7.14(d,J=8.1Hz,2H),4.3 6(d,J=12.9Hz,2H),3.36(s,2H),2.94(t,J=12.5Hz,3H),2.51(t,J=2.6Hz,4H),2.34(s,2H ),2.29(q,J=7.2Hz,4H),1.92(d,J=12.4Hz,2H),1.78–1.68(m,2H),0.96(t,J=7.1Hz,3H); 13 C NMR(101MHz,DMSO-d6)δ165.65,155.36,147.13,140.96,139.95,135.38,133.44,131.69,130.84,129.77,129.30,12 5.79,124.02,120.35,119.95,119.25,62.19,52.87(d,J=14.3Hz),52.05,44.77,42.32,40.63,39.38,33.16,12.40.

[0230] Comparative Example 1: 5-(3-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)urea)-N-phenylpentanamide

[0231]

[0232] The synthesis method is as follows:

[0233]

[0234] Preparation of tert-butyl (5-oxo-5-(phenylamino)pentyl)carbamate:

[0235] Aniline (689 mg, 7.41 mmol, 1.0 eq) was dissolved in DMF (20 mL), and Boc-5-aminovaleric acid (1930 mg, 8.89 mmol, 1.2 eq), EDCI (2122 mg, 11 mmol, 1.5 eq), and HOBT (1490 mg, 11 mmol, 1.5 eq) were added in sequence, and stirred at room temperature for 18 h. The reaction solution was diluted with ethyl acetate, washed with water, washed with saturated sodium chloride, dried over anhydrous sodium sulfate, and concentrated to obtain 1.8 g, with a yield of 83%.

[0236] Preparation of 5-amino-N-phenylpentanamide hydrochloride:

[0237] Dissolve tert-butyl (5-oxo-5-(phenylamino)pentyl)carbamate (1 g, 3.42 mmol) in hydrochloric acid-methanol (10 mL), stir at room temperature for 3 h, and concentrate to obtain 650 mg with a yield of 83%.

[0238] Preparation of 5-(3-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)ureido)-N-phenylpentanamide:

[0239] The preparation of 5-amino-N-phenylpentanamide hydrochloride (100 mg, 0.438 mmol, 1.0 eq) was dissolved in DMF (2 cmL), and triethylamine (132.9 mg, 1.316 mmol, 3.0 eq) and phenyl (4-((4-ethylpiperazin-1-yl)methyl)phenyl)carbamate (178.2 mg, 0.525 mmol, 1.2 eq) were added in sequence. The reaction was allowed to react at room temperature for 18 h. The reaction solution was diluted with ethyl acetate, washed with water 3 times, washed with saturated sodium chloride once, dried over anhydrous sodium sulfate, concentrated, and purified by column to obtain 23 mg of a white solid (5-(3-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)ureido)-N-phenylpentanamide), with a yield of 12%.

[0240] ESI-MS m / z:438.3[M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.20(s,1H),7.53(d,J=8.0Hz,2H),7.27-7.25(m,3H),7.18(t,J=8.0Hz,3H),7.07(q,J=8.0Hz,2H),5.4 2(s,1H),3.45(s,2H),3.24(d,J=4.0Hz,2H),2.50-2.36(m,12H),1.71(t,J=8.0Hz,2H),1.52(t,J=8.0Hz,2H),1.09(t,J=8.0Hz,3H).

[0241] Comparative Example 2: 5-(3-(4-((4-ethylpiperazin-1-yl)methyl)phenyl)ureido)-N-(4-fluorophenyl)pentanamide

[0242]

[0243] The synthesis method is similar to that of Comparative Example 1, except that aniline is replaced by p-fluoroaniline.

[0244] ESI-MS m / z:456.3[M+H] + ,1 H NMR(400MHz, DMSO-d6)δ9.92(s,1H),8.34(s,1H),7.61-7.58(m,2H),7.30(d,J=8.0Hz,2H),7.13–7.09(m,4H),6.11(t,J=4.0Hz, 1H), 3.34 (s, 2H), 3.10 (q, J = 6.0Hz, 2H), 2.51-2.21 (m, 12H), 1.61 (t, J = 8.0Hz, 2H), 1.45 (t, J = 8.0Hz, 2H), 0.97 (t, J = 8.0Hz, 3H).

[0245] Example 21: Determination of in vitro coupling activity of compounds

[0246] 1. Pulmonary Hypertension Mouse Model

[0247] Fifty C57BL / 6 male mice were used in the experiment. The mice were numbered and placed in a hypoxic chamber for 3 weeks after 7 days of adaptive feeding. The pressure balance was maintained by a pressure regulating valve. The oxygen concentration in the chamber was (10±0.2)%, the temperature was (22-25)°C, and the humidity was (60-70)%.

[0248] 2. Evaluation of in vitro coupling activity

[0249] (1) Cell culture

[0250] Primary pulmonary artery endothelial cells were isolated from pulmonary hypertension model mice induced by hypoxia stress and cultured in a cell culture incubator for 3-5 days.

[0251] (2) Immunofluorescence staining

[0252] 1) Inoculation: The cells were inoculated into a confocal dish and observed under a microscope. After the cells adhered to the wall, they were pretreated with Example 1-20 and Comparative Example 1-2 at a concentration of 10 μMol / L;

[0253] 2) Incubation: After treatment, continue to culture in a 5% CO2, 37°C cell culture incubator for 24 hours;

[0254] 3) Fixation: discard the old culture medium, wash twice with PBS buffer, and fix with 4% paraformaldehyde at room temperature;

[0255] 4) Permeabilization: Wash twice with PBS buffer, add 500 μL of 0.1% triton X-100 (in PBS) to each well, and incubate at room temperature for 15 min;

[0256] 5) Blocking: Wash twice with PBS buffer, add 500 μL of 5 mg / mL bovine serum albumin (in PBS) to each well, and incubate at room temperature for 1 h;

[0257] 6) Incubation with fluorescent primary and secondary antibodies: cells were incubated with TRPV4-550 and KCa2.3 as primary antibodies and coupled secondary antibodies;

[0258] 7) Fret image acquisition: 4',6-diamidino-2-phenylindole (DAPI) staining was performed for 10 min, fluorescence images were collected using a laser confocal microscope, and the Fret efficiency value was calculated.

[0259] Table 1 In vitro coupling activity data of compounds

[0260]

[0261]

[0262] Table 1 shows the efficiency of the immune Fret of each example compound. The higher the value, the higher the coupling degree between TRPV4 and KCa2.3. Among them, JNC-440 is the control group, PAH refers to the pulmonary hypertension model mice not treated by the example, WT refers to the normal wild-type mice, M represents the average value, a The standard deviation of the data from three replicates is shown.

[0263] The in vitro coupling activity assay test proved that under hypoxic stress, the coupling of TRPV4 and KCa2.3 in the pulmonary artery endothelial cells of pulmonary hypertension model mice decreased, but after administration, the degree of coupling increased overall. Compared with the PAH group, the Fret efficiency values ​​of compounds 10, 12, 14, 15, 16 and 20 were almost 2 times higher. Among them, compound 14, compound 15 and compound 16 were better than comparative examples 1 and 2, indicating that the embodiments can promote the coupling of TRPV4-KCa2.3 protein complexes in pulmonary artery endothelial cells of pulmonary hypertension model mice.

[0264] Example 22: Subcellular co-localization analysis

[0265] Compound 14 was used as a representative compound. Pulmonary artery endothelial cells of pulmonary hypertension model mice were incubated at a concentration of 10 μMol / L for 24 hours. Anti-TRPV4 antibody and anti-KCa2.3 antibody were used for double immunolabeling. Double immunofluorescence staining confocal images were collected for subcellular colocalization analysis. Green is KCa2.3 protein labeled with fluorescent secondary antibody, red is TRPV4 protein labeled with fluorescent secondary antibody, DAPI staining is used to locate the position of the cell nucleus, Merged is an overlay image, and Fret is used to mark the cell membrane to obtain the efficiency value.

[0266] Figure 1 Subcellular colocalization of TRPV4-KCa2.3 in primary pulmonary artery endothelial cells of pulmonary hypertension model mice

[0267] When TRPV4 and KCa2.3 are coupled, the green fluorescence and the red fluorescence overlap and merge to obtain yellow fluorescence. The intensity of the yellow fluorescence can intuitively indicate the degree of coupling between the TRPV4 protein and the KCa2.3 protein. Analysis of the above results shows that: the merged graph of the blank control group has almost no yellow fluorescence, indicating that the coupling between TRPV4 and KCa2.3 is weak; after the administration of compound 14, the merged yellow fluorescence intensity of TRPV4 and KCa2.3 is significantly greater than that of the blank control group, indicating that the coupling between TRPV4 and KCa2.3 is enhanced, and higher than the positive compound JNC-440, which is consistent with the Fret efficiency value determination results.

[0268] Examples 21-22 show that the compounds of the present invention have in vitro coupling activity for TRPV4 and KCa2.3 in pulmonary artery endothelial cells of pulmonary hypertension model mice, and are significantly higher than the existing compound JNC-440. Compounds 14, 15 and 16 have the potential to be further developed as pulmonary hypertension regulators.

[0269] From the in vitro coupling activity data and subcellular co-localization maps of compounds 1-20, it can be seen that for compounds of general formula (I), the linking group and the substituent group have an important influence on the in vitro coupling activity of the compound.

[0270] Although the present invention has been described by way of specific examples above, it should not be construed as being limited thereto; rather, the present invention encompasses the general aspects previously disclosed, is susceptible of numerous modifications and has numerous embodiments without departing from the spirit and scope of the present invention.

Claims

1. A compound having a structure represented by general formula (I) or a pharmaceutically acceptable salt thereof: in: X is NH, Y is CO; or X is CO, Y is NH; R1 is selected from phenyl, wherein the phenyl is optionally monosubstituted to pentasubstituted by the following identical or different substituents, wherein the substituents are selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl; R2 is selected from C 1- C4 alkyl or empty; R3 is selected from R4 is selected from -CH-, -C2H3-, -C3H5- ​​or -C4H7-; Z is selected from N; R5 is selected from hydrogen, C1-C4 alkyl, -CONHR6, substituted or unsubstituted phenyl, wherein the alkyl and phenyl groups may be optionally mono-substituted to penta-substituted by the following identical or different substituents, wherein the substituents are selected from: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl; R6 is selected from hydrogen, C1-C4 alkyl, phenyl, wherein the alkyl and phenyl groups may be optionally mono-substituted to penta-substituted by the following identical or different substituents: halogen, trifluoromethyl, cyano, nitro, hydroxyl or C1-C4 alkyl.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that When R2 is empty, the corresponding structural formula is:

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown below: or Wherein, R' and R" are independently selected from: halogen, nitro, cyano, trifluoromethyl, C1-4 alkyl.

4. A compound having the structure represented by general formula (I) or a pharmaceutically acceptable salt thereof: The structure shown in general formula (I) is specifically selected from:

5. The compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof, characterized in that: Its pharmaceutically acceptable salt is an inorganic salt or an organic salt; the inorganic salt includes hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, phosphate, and acid phosphate; the organic salt is selected from acetate, trifluoroacetate, propionate, pyruvate, glycolate, oxalate, malonate, fumarate, maleate, lactate, malate, citrate, tartrate, methanesulfonate, sulfonate, benzenesulfonate, and salicylate.

6. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a TRPV4-KCa2.3 coupling promoter.

7. A pharmaceutical composition comprising the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

8. The pharmaceutical composition according to claim 7, characterized in that It includes a pharmaceutically acceptable carrier, excipient or diluent.

9. Use of the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating malignant lung diseases and cardiovascular diseases mediated by abnormal TRPV4-KCa2.3 coupling in humans.

10. The use according to claim 9, characterized in that Malignant lung diseases include: pulmonary embolism, acute pulmonary thromboembolism, pulmonary vasculitis, pulmonary vascular tumors, pulmonary edema, and acute lung injury; cardiovascular diseases include: hypertension, hyperlipidemia, hypertensive heart disease, cor pulmonale, valvular heart disease, congenital heart disease, coronary heart disease, cardiomyopathy, heart failure, atherosclerosis, diabetes, chronic kidney disease, venous thromboembolism, metabolic syndrome, arrhythmia, aortic disease, peripheral arterial disease, and stroke.

11. The use according to claim 9, characterized in that Cardiovascular disease is pulmonary hypertension, which specifically includes: hereditary pulmonary hypertension, drug- and toxin-induced pulmonary hypertension, idiopathic pulmonary hypertension, pulmonary hypertension associated with portal hypertension, pulmonary hypertension associated with connective tissue disease, chronic thromboembolic pulmonary hypertension, persistent pulmonary hypertension of the newborn, isolated postcapillary pulmonary hypertension, pulmonary hypertension caused by chronic obstructive pulmonary disease, and pulmonary hypertension caused by congenital heart disease.

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