Tetrahydro-beta-carboline compound as well as preparation method and application thereof

Abstract

The invention discloses a tetrahydro-beta-carboline compound as well as a preparation method and application thereof. The tetrahydro-beta-carboline compound is shown as a formula P. The tetrahydro-beta-carboline compound has good RANKL inhibitory activity, the preparation method is simple, raw materials are easy to obtain, and the yield is high. The inhibition activity of the compound on generation of RANKL-induced osteoclasts is determined through a tartrate acid phosphatase (TRAP) staining experiment, it is determined through a surface plasmon resonance (SPR) that the compound has high combining capacity with RANKL protein, and the compound can be used for preparing drugs for preventing and / or treating diseases caused by abnormal osteoclast activity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to medicinal chemistry, particularly to the technical field of osteoporosis treatment drugs, and specifically relates to a class of tetrahydro-β-carboline compounds, a preparation method thereof, a pharmaceutical composition containing the same, and their use in the preparation of drugs for treating and / or preventing diseases caused by abnormal osteoclast activity. Background Art

[0002] Osteoporosis (OP) is one of the common bone-related diseases, which occurs frequently in middle-aged and elderly people, especially postmenopausal women. Its characteristics are that the patient's bone mineral density (BMD) decreases and there is a continuously increasing risk of osteoporotic fractures. With the aggravation of population aging, osteoporosis significantly threatens the physical health of middle-aged and elderly people and brings a great family and social burden, making it increasingly regarded as a public health problem. Currently, anti-osteoporosis drugs used clinically, such as alendronate sodium of bisphosphonates and raloxifene of estrogen receptor modulators, have limited therapeutic effects and large side effects. Therefore, there is an urgent need to develop safer and more effective anti-osteoporosis drugs.

[0003] The receptor activator of nuclear factor-κB ligand / receptor activator of nuclear factor-κB (RANKL / RANK) pathway plays an important role in bone metabolism. As a key regulatory factor in osteoclast activation, RANKL is an important target for anti-osteoporosis drugs. Currently, the only marketed RANKL inhibitor is the monoclonal antibody drug denosumab, which is mainly used for the clinical treatment of postmenopausal osteoporosis and bone metastases of solid tumors. However, long-term use of it as a monoclonal antibody will lead to immune antigenicity, and there are problems such as poor transmembrane permeability of macromolecular drugs, low patient compliance with drug administration, and the need for low-temperature storage. Therefore, the development of small-molecule RANKL inhibitors is very competitive for the treatment of bone-related diseases. Summary of the Invention

[0004] The present invention designs and synthesizes a series of novel tetrahydro-β-carboline compounds with good RANKL inhibitory activity. The preparation method is simple, the raw materials are easily available, and the yield is relatively high. The inhibitory activity of the synthesized compounds on RANKL-induced osteoclastogenesis was determined by tartrate-resistant acid phosphatase (TRAP) staining experiment. Among them, compounds j1, e13″, etc. showed good osteoclast inhibitory activity; and the binding ability of compound j1 with RANKL protein was measured by surface plasmon resonance (SPR) technology.

[0005] Based on this, one of the purposes of the present invention is to provide the tetrahydro-β-carboline compounds shown by formula P, or their optical isomers, pharmaceutically acceptable salts and hydrates.

[0006] The second object of the present invention is to provide a method for preparing tetrahydro-β-carboline compounds represented by formula P.

[0007] The third object of the present invention is to provide a pharmaceutical composition comprising a tetrahydro-β-carboline compound represented by formula P, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof.

[0008] The fourth object of the present invention is to provide a RANKL inhibitor comprising a tetrahydro-β-carboline compound represented by formula P, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof.

[0009] The fifth object of the present invention is to provide the use of the tetrahydro-β-carboline compound represented by formula P or the pharmaceutical composition in the preparation of a drug for preventing and / or treating diseases caused by abnormal osteoclast activity.

[0010] To achieve the above objects, the present invention adopts the following technical solutions:

[0011] On the one hand, the present invention provides a tetrahydro-β-carboline compound represented by formula P, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof,

[0012]

[0013] Wherein:

[0014] "---" represents the presence or absence of a chemical bond;

[0015] R 1 represents 1 to 4 (e.g., 1, 2, 3, 4) substituents independently selected from hydrogen, halogen, C1-C4 alkyl, C1-C6 alkoxy, hydroxy C1-C6 alkyl, and C1-C6 alkylamino; preferably, R 1 represents 1 to 4 substituents independently selected from hydrogen, fluorine atom, and methoxy; more preferably, R 1 is a hydrogen atom;

[0016] R 2 is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkanoyl, C3-C6 cycloalkyl C1-C3 alkanoyl; preferably, R 2 is selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkanoyl, C3-C6 cycloalkylcarbonyl; more preferably, R 2 is selected from hydrogen, methyl, acetyl, cyclopropylcarbonyl, and further preferably is hydrogen or methyl;

[0017] n1 and n2 are each independently or simultaneously 1 or 2;

[0018] X is carbon or nitrogen; Y is oxygen or sulfur;

[0019] Ar 1 and Ar 2 are each independently selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5- or 6-membered heteroaryl; preferably, Ar 1 and Ar 2 are each independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted indenyl;

[0020] The substitution in the above-mentioned "substituted or unsubstituted" means being substituted by one or more (e.g., 2) substituents selected from the following: halogen (e.g., fluorine), hydroxy (-OH), amino (-NH2), cyano (-CN), C1-C6 alkyl (preferably C1-C3 alkyl, such as methyl, ethyl, isopropyl), C1-C6 haloalkyl (preferably C1-C3 haloalkyl, such as C1-C3 fluoroalkyl, such as trifluoromethyl), C3-C6 cycloalkyl (such as cyclopropyl), C1-C6 alkoxy (preferably C1-C3 alkoxy, such as methoxy), C1-C6 alkyl-NH- (preferably C1-C3 alkyl-NH-), (C1-C6 alkyl)(C1-C6 alkyl)-N- (preferably (C1-C3 alkyl)(C1-C3 alkyl)-N-, such as dimethylamino), C1-C6 alkoxycarbonyl (preferably C1-C3 alkoxycarbonyl), ester group (R 3 -C(=O)-O- or R 3 -OC(=O)-), sulfonyl group (R 3 -S(=O)2-), sulfonamide group (R 3 -S(=O)2NH-), phosphoryl group ((R 3 O)2-P(=O)-), where each R 3 is independently selected from hydrogen, C1-C6 alkyl (preferably C1-C3 alkyl, such as methyl).

[0021] In a specific embodiment, in the tetrahydro-β-carboline compounds represented by formula P:

[0022] "---" represents the presence or absence of a chemical bond;

[0023] R 1 represents 1 to 4 substituents independently selected from hydrogen, fluorine atom, methoxy;

[0024] R 2 is selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkanoyl, C3-C6 cycloalkylcarbonyl;

[0025] n1 and n2 are each independently 1 or 2;

[0026] X is carbon or nitrogen; Y is oxygen or sulfur;

[0027] Ar 1 and Ar 2 each independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted indenyl; the substitution in the "substituted or unsubstituted" is as defined above.

[0028] In a specific embodiment, in the tetrahydro-β-carboline compounds represented by formula P:

[0029] "---" represents the presence or absence of a chemical bond;

[0030] R 1 is a hydrogen atom;

[0031] R 2 is selected from hydrogen, methyl, acetyl, cyclopropylcarbonyl, more preferably hydrogen or methyl;

[0032] n1 and n2 are each independently 1 or 2;

[0033] X is carbon or nitrogen; Y is oxygen or sulfur;

[0034] Ar 1 and Ar 2 each independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted indenyl; the substitution in the "substituted or unsubstituted" is as defined above.

[0035] In a specific embodiment, the tetrahydro-β-carboline compounds represented by formula P are represented by the following formula E or J:

[0036]

[0037] wherein, the definitions of "---", R 2 , n1, n2, Y, Ar 1 and Ar 2 are as defined above.

[0038] In a specific embodiment, the tetrahydro-β-carboline compounds represented by formula P are represented by the following formula E1 or J1:

[0039]

[0040] wherein, the definitions of "---", R 2 , n1, n2, Ar 1 and Ar 2 are as defined above.

[0041] Those skilled in the art should understand that with the changes of some substituents or chemical bonds in the formula P structure, the surrounding structures or groups can change accordingly. For example, when "---" represents no chemical bond, if n2 is 1, the corresponding group is methyl; if n2 is 2, the corresponding group is ethyl. On the contrary, when "---" represents a chemical bond, the corresponding group should be methylene or ethylene. For another example, when "---" represents no chemical bond and X is carbon, the corresponding position of X should be CH2 at this time; while when "---" represents a chemical bond and X is carbon, the corresponding position of X should be CH at this time. Those skilled in the art should be able to clarify the above or similar situations based on the knowledge they have mastered.

[0042] In the present invention,

[0043] The "alkyl group" refers to an aliphatic saturated hydrocarbon group, which can be a branched or straight-chain alkyl group. According to the structure, the alkyl group can be a monovalent group or a divalent group (i.e., alkylene group). For example, in "hydroxy C1-C6 alkyl", the C1-C6 alkyl is actually a divalent group (alkylene group). In the present invention, the alkyl group is preferably a "lower alkyl group" having 1-6 carbon atoms, and even more preferably a "lower alkyl group" having 1-3 carbon atoms. Typical alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, etc.

[0044] The "halogen" is F, Cl, Br, I, and preferably F.

[0045] The "haloalkyl group" means that at least one hydrogen atom in the alkyl group is replaced by a halogen atom. In some embodiments, if two or more hydrogen atoms are replaced by halogen atoms, the halogen atoms are the same or different from each other.

[0046] The "cycloalkyl group" is a saturated monocyclic or polycyclic alicyclic ring with 3-10 members, and can also be a monovalent group or a divalent group (i.e., cycloalkylene group);

[0047] The "aryl group" means that each atom constituting the aromatic ring is a carbon atom, including a monocyclic or fused polycyclic ring, and can also be a monovalent group or a divalent group (i.e., arylene group). In the present invention, the aryl ring preferably has an aryl group with 6-10 carbon atoms, such as phenyl, indenyl, etc.

[0048] The "heteroaryl" is an aryl group containing one or more heteroatoms selected from N, O, and S in the ring. According to the present invention, the heteroaryl is preferably a 5- or 6-membered heteroaryl. According to the structure, the heteroaryl can be a monovalent group or a divalent group (i.e., heteroarylene). Examples of the heteroaryl include, but are not limited to, pyridyl, pyrimidinyl, imidazolyl, pyrazolyl, pyrazinyl, triazolyl, tetrazolyl, thienyl, thiazolyl, furyl, oxazolyl, isoxazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuryl, indazolyl, indazinyl, phthalazinyl, pyridazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothienyl, benzothiazolyl, benzoxazolyl, quinazolinyl, etc.

[0049] In a specific embodiment, the tetrahydro-β-carboline compound represented by formula P is selected from the compounds represented by the following structures:

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] In the second aspect of the present invention, a method for preparing the tetrahydro-β-carboline compound represented by formula P is provided. The tetrahydro-β-carboline compound of the present invention is prepared by the following steps, and the reaction route is as follows:

[0056]

[0057] Wherein, Ar 1 , Ar 2 , n1 and n2 are as defined above,

[0058] Step 1: N-Boc-tryptophan a is condensed with arylamine Ar 1 -NH2 under the action of a condensing agent to form N-Boc-tryptophanyl arylamine b;

[0059] Step 2: N-Boc-tryptophanyl arylamine b is de-Boc'd under the action of trifluoroacetic acid to form tryptophanyl arylamine c;

[0060] Step 3: Tryptophanyl arylamine c reacts with ketone d or f through an acid-catalyzed Pictet-Spengler reaction to obtain the corresponding tetrahydro-β-carboline derivative e (the tetrahydro-β-carboline compound represented by formula E1) or g;

[0061] Step 4: Remove the Boc group from the tetrahydro-β-carboline derivative g to obtain the intermediate h;

[0062] Step 5: React the intermediate h with the aryl bromide Ar 2 -Br through Buchwald-Hartwig coupling reaction to obtain the derivative i (the tetrahydro-β-carboline compound shown in Formula J1 (R 2 is H)).

[0063] In a specific embodiment, in Step 1, the N-Boc-tryptophan a is N-Boc-L-tryptophan or N-Boc-D-tryptophan, and the condensing agent is HATU or EDCI·HCl;

[0064] When the condensing agent is HATU, the reaction system may further include a base scavenger, such as N,N-diisopropylethylamine; the reaction is carried out in a solvent, and the solvent may be, for example, N,N-dimethylformamide; the reaction temperature is 60-80 °C, and the reaction time is 6-20 h;

[0065] When the condensing agent is EDCI·HCl, the reaction system may further include an acylation catalyst, such as HOBt; the reaction system may further include a base scavenger, such as triethylamine; the reaction is carried out in a solvent, and the solvent may be, for example, tetrahydrofuran; the reaction temperature is 20-30 °C, and the reaction time is 6-20 h.

[0066] In a specific embodiment, in Step 2, the acid is trifluoroacetic acid; the reaction is carried out in a solvent, and the solvent may be, for example, dichloromethane; the reaction temperature is 20-30 °C, and the reaction time is 6-18 h.

[0067] In a specific embodiment, in Step 3, the acid is trifluoroacetic acid; the reaction is carried out in a solvent, and the solvent may be, for example, acetonitrile; the reaction temperature is 20-30 °C, and the reaction time is 12-20 h.

[0068] In a specific embodiment, in Step 4, the tetrahydro-β-carboline derivative g removes the Boc group under the action of trifluoroacetic acid to obtain the intermediate h; the reaction is carried out in a solvent, and the solvent may be, for example, dichloromethane; the reaction temperature is 20-30 °C, and the reaction time is 6-18 h.

[0069] In a specific embodiment, in Step 5, the coupling reaction is carried out under the catalysis of Pd2(dba)3, RuPhos, and cesium carbonate; the reaction is carried out in a solvent, and the solvent may be, for example, 1,4-dioxane; the coupling reaction is refluxed for 6-18 h.

[0070] Among them, HATU represents 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; EDCI·HCl represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; HOBt represents 1-hydroxybenzotriazole; Boc represents tert-butoxycarbonyl.

[0071] The third aspect of the present invention is to provide a pharmaceutical composition, which comprises the tetrahydro-β-carboline compound represented by formula P as described above, or its optical isomer, pharmaceutically acceptable salt and hydrate, and optionally, pharmaceutically acceptable excipients.

[0072] The fourth aspect of the present invention provides a RANKL inhibitor, which comprises the tetrahydro-β-carboline compound represented by formula P as described above, or its optical isomer, pharmaceutically acceptable salt and hydrate.

[0073] The fifth aspect of the present invention is to provide the use of the tetrahydro-β-carboline compound represented by formula P as described in the first aspect, or its optical isomer, pharmaceutically acceptable salt and hydrate, or the pharmaceutical composition described in the third aspect in the preparation of a drug for preventing and / or treating diseases caused by abnormal osteoclast activity.

[0074] Furthermore, the diseases caused by abnormal osteoclast activity include but are not limited to osteoporosis, osteoarthritis, etc.

[0075] Furthermore, the present invention provides the use of the tetrahydro-β-carboline compound represented by formula P as described in the first aspect, or its optical isomer, pharmaceutically acceptable salt and hydrate, or the pharmaceutical composition described in the third aspect in the preparation of a drug for inhibiting the differentiation of bone marrow osteoclast precursor cells into bone marrow osteoclasts. Description of the Drawings

[0076] Figure 1 It is a SPR experimental curve graph of compound j1 and RANKL protein in the test examples. Detailed Embodiments

[0077] The following further elaborates the present invention in conjunction with specific embodiments. The embodiments are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0078] 11H NMR was measured on a Bruker AVANCE III 400 / 500 / 600 MHz nuclear magnetic resonance spectrometer; MS(ESI) was measured on an Agilent 1200-6110 type HPLC-MS instrument; unless otherwise stated, all reactions were carried out under nitrogen protection and monitored by TLC, and the post-treatment was carried out by washing with saturated brine and drying with anhydrous sodium sulfate; the purification of the products was carried out by silica gel (200-300 mesh) column chromatography unless otherwise stated; the silica gel and GF254 TLC plates used were all produced by Qingdao Ocean Chemical Factory.

[0079] Example 1-1. Synthesis of compound e1 (e1' / e1'')

[0080]

[0081] (1) Preparation of N-Boc-L-tryptophan-2',6'-dimethylaniline (b1):

[0082]

[0083] Dissolve 2 g (6.57 mmol) of N-Boc-L-tryptophan (a1) in 50 mL of N,N-dimethylformamide, add 3.43 mL (19.71 mmol) of N,N-diisopropylethylamine and 3.75 g (9.86 mmol) of 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), stir at room temperature for 15 min, then add 1.21 mL (9.86 mmol) of 2,6-dimethylaniline, and stir at 80 °C for 14 h. After the reaction solution was cooled, 50 mL of saturated ammonium chloride solution was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated in vacuo. The residue was purified by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) to obtain 1.93 g of intermediate b1 as a yellow powder, with a yield of 72%. 1 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.38 (s, 1H), 7.69 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.8 Hz, 1H), 7.24 (s, 1H), 7.11 - 6.98 (m, 6H), 4.41 (q, J = 8.2 Hz, 1H), 3.20 (dd, J = 14.3, 4.9 Hz, 1H), 3.01 (dd, J = 14.1, 9.9 Hz, 1H), 2.06 (s, 6H), 1.35 (s, 9H). MS(ESI) m / z 429.9 [M+Na] + .

[0084] (2) Preparation of L-tryptophan-2',6'-dimethylaniline (c1):

[0085]

[0086] Dissolve 815 mg (2 mmol) of intermediate b1 in 10 mL of dichloromethane. Add 1.53 mL (20 mmol) of trifluoroacetic acid under an ice bath at 0 °C and stir at room temperature overnight. Rotavaporize the reaction solution, add 20 mL of saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, and then rotavaporize to obtain 609 mg of intermediate c1 without further purification. Pale yellow powder, yield 99%. MS (ESI) m / z 308.2 [M+H] + .

[0087] (3) Preparation of compound e1 (e1' / e1''):

[0088]

[0089] Dissolve 609 mg (2 mmol) of intermediate c1 in 8 mL of acetonitrile, add 230 μl (3 mmol) of trifluoroacetic acid and 395 μl (3 mmol) of 3-phenylcyclobutanone (d1), and stir at room temperature for 18 h. Rotavaporize the reaction solution, add 20 mL of water, adjust the pH to 10 - 12 by dropwise adding saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, and then separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 214 mg of compound e1' and 396 mg of e1'' (e1' and e1'' are a pair of epimers and can be separated by silica gel column chromatography, with the general structural formula being e1). Both are pale yellow powders, and the yields of e1' and e1'' are (e1') 24.5% and (e1'') 45.5% respectively.

[0090] (e1')[[]] 1 H NMR (500 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.07 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.36 (m, 2H), 7.31 - 7.28 (m, 2H), 7.26 - 7.21 (m, 2H), 7.14 (m, 1H), 7.11 - 7.07 (m, 4H), 3.98 (m, 1H), 3.78 (dd, J = 11.3, 4.5 Hz, 1H), 3.40 (m, 1H), 2.80 (dd, J = 15.9, 11.3 Hz, 1H), 2.76 - 2.67 (m, 2H), 2.54 (m, 1H), 2.42 (dd, J = 11.7, 9.5 Hz, 1H), 2.30 (s, 6H). MS (ESI) m / z 435.9 [M+H] + .

[0091] (e1'')[[]] 11H NMR (500 MHz, Chloroform-d) δ 8.48 (s, 1H), 8.38 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.38 - 7.32 (m, 5H), 7.22 (m, 1H), 7.18 (m, 1H), 7.12 (m, 1H), 7.10 - 7.06 (m, 3H), 3.78 (dd, J = 11.2, 4.5 Hz, 1H), 3.72 (m, 1H), 3.38 (dd, J = 15.6, 4.5 Hz, 1H), 3.07 (ddd, J = 12.4, 9.2, 3.3 Hz, 1H), 2.89 (ddd, J = 12.2, 9.2, 3.3 Hz, 1H), 2.84 (dd, J = 15.5, 11.2 Hz, 1H), 2.56 - 2.52 (m, 2H), 2.23 (s, 6H). MS (ESI) m / z 435.9 [M+H] + .

[0092] Example 1 - 2. Synthesis of Compound e2 (e2' / e2''):

[0093]

[0094] The synthetic route was the same as that of Example 1 - 1, except that N-Boc-D-tryptophan (a2) was used instead of N-Boc-L-tryptophan (a1) in step (1). The yields were 24% for (e2') and 45% for (e2'').

[0095] (e2') 1 1H NMR (500 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.07 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.36 (m, 2H), 7.31 - 7.28 (m, 2H), 7.26 - 7.21 (m, 2H), 7.14 (m, 1H), 7.15 - 7.07 (m, 4H), 3.97 (m, 1H), 3.78 (dd, J = 11.3, 4.6 Hz, 1H), 3.39 (dd, J = 15.8, 4.5 Hz, 1H), 2.80 (dd, J = 15.8, 11.4 Hz, 1H), 2.72 (td, J = 14.1, 13.0, 9.0 Hz, 2H), 2.54 (m, 1H), 2.42 (dd, J = 11.9, 9.4 Hz, 1H), 2.29 (s, 6H). MS (ESI) m / z 435.9 [M+H] + .

[0096] (e2'') 11H NMR (500 MHz, Chloroform-d) δ 8.51 (s, 1H), 8.47 (s, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.36 - 7.29 (m, 5H), 7.20 (m, 1H), 7.17 (ddd, J = 8.1, 7.1, 1.3 Hz, 1H), 7.10 (m, 1H), 7.09 - 7.04 (m, 3H), 3.76 (dd, J = 11.2, 4.7 Hz, 1H), 3.71 (m, 1H), 3.36 (dd, J = 15.8, 4.6 Hz, 1H), 3.05 (ddd, J = 12.2, 9.1, 3.2 Hz, 1H), 2.91 - 2.77 (m, 2H), 2.53 - 2.47 (m, 2H), 2.21 (s, 6H). MS (ESI) m / z 435.9 [M+H] + .

[0097] Examples 1 - 3. Synthesis of Compound e3 (e3' / e3'')

[0098]

[0099] The synthetic route is the same as that of Example 1 - 1, except that 2,6 - dimethoxyaniline is used instead of 2,6 - dimethylaniline in step (1). The yields are 24% for (e3') and 25% for (e3'') respectively.

[0100] (e3')[[]END]] 1 1H NMR (500 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.03 (s, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.35 - 7.40 (m, 2H), 7.31 - 7.24 (m, 4H), 7.20 (m, 1H), 7.16 (m, 1H), 7.10 (m, 1H), 6.58 (d, J = 8.5 Hz, 2H), 4.10 - 3.91 (m, 2H), 3.81 (s, 3H), 3.38 (dd, J = 16.1, 4.6 Hz, 1H), 2.96 - 2.89 (m, 2H), 2.71 - 2.69 (m, 2H), 2.43 (m, 1H). MS (ESI) m / z 467.9 [M+H] + .

[0101] (e3'')[[]END]] 11H NMR (500 MHz, Chloroform-d) δ 8.62 (s, 1H), 8.37 (s, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.41 - 7.33 (m, 5H), 7.26 - 7.17 (m, 3H), 7.13 (td, J = 7.5, 1.1 Hz, 1H), 6.61 (d, J = 8.5 Hz, 2H), 3.84 (m, 1H), 3.83 (m, 6H), 3.71 (m, 1H), 3.38 (dd, J = 15.9, 4.6 Hz, 1H), 3.07 (ddd, J = 12.5, 9.2, 3.5 Hz, 1H), 2.93 - 2.83 (m, 2H), 2.60 - 2.49 (m, 2H). MS (ESI) m / z 467.9 [M+H] + .

[0102] Examples 1 - 4. Synthesis of Compound e4 (e4' / e4'')

[0103]

[0104] The synthesis route was the same as that of Examples 1 - 1, except that 2,6 - diethylaniline was used instead of 2,6 - dimethylaniline in step (1). The yields were 18% for (e4') and 38% for (e4'').

[0105] (e4')[[]END]] 1 1H NMR (500 MHz, Chloroform-d) δ 8.80 (s, 1H), 7.99 (s, 1H), 7.49 (dd, J = 7.7, 1.2 Hz, 1H), 7.39 - 7.35 (m, 2H), 7.32 - 7.28 (m, 2H), 7.27 - 7.22 (m, 3H), 7.17 - 7.13 (m, 3H), 7.10 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H), 3.99 (m, 1H), 3.80 (dd, J = 11.3, 4.5 Hz, 1H), 3.44 (dd, J = 16.0, 4.5 Hz, 1H), 2.83 (dd, J = 16.0, 11.4 Hz, 1H), 2.76 - 2.71 (m, 2H), 2.67 (q, J = 7.6 Hz, 4H), 2.57 (ddd, J = 11.9, 8.4, 5.2 Hz, 1H), 2.45 (dd, J = 11.7, 9.4 Hz, 1H), 1.26 (t, J = 7.6 Hz, 6H). MS (ESI) m / z 464.0 [M+H] + .

[0106] (e4'')[[]END]] 11H NMR (500 MHz, Chloroform-d) δ 8.53 (s, 1H), 8.47 (s, 1H), 7.47 (dd, J = 7.8, 1.1 Hz, 1H), 7.38 - 7.28 (m, 5H), 7.22 - 7.16 (m, 3H), 7.13 - 7.09 (m, 3H), 3.78 (dd, J = 11.2, 4.6 Hz, 1H), 3.75 - 3.68 (m, 1H), 3.39 (dd, J = 15.8, 4.6 Hz, 1H), 3.07 (ddd, J = 12.3, 9.0, 3.3 Hz, 1H), 2.92 - 2.79 (m, 2H), 2.58 (q, J = 7.6 Hz, 4H), 2.55 - 2.49 (m, 2H), 1.19 (t, J = 7.6 Hz, 6H). MS (ESI) m / z 464.0 [M+H] + .

[0107] Example 1 - 5. Synthesis of Compound e5 (e5' / e5'')

[0108]

[0109] The synthetic route was the same as that of Example 1 - 1, except that 2,6 - diisopropylaniline was used instead of 2,6 - dimethylaniline in step (1). The yields were 17% for (e5') and 35% for (e5'') respectively.

[0110] (e5')[[]END]] 1 1H NMR (500 MHz, Chloroform-d) δ 8.77 (s, 1H), 8.00 (s, 1H), 7.49 (dd, J = 7.7, 1.2 Hz, 1H), 7.37 (t, J = 7.6 Hz, 2H), 7.34 - 7.29 (m, 3H), 7.27 - 7.18 (m, 4H), 7.15 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.10 (td, J = 7.4, 6.9, 1.1 Hz, 1H), 4.00 (m, 1H), 3.82 (dd, J = 11.3, 4.6 Hz, 1H), 3.45 (dd, J = 16.0, 4.6 Hz, 1H), 3.13 (p, J = 6.9 Hz, 2H), 2.83 (dd, J = 16.1, 11.3 Hz, 1H), 2.78 - 2.73 (m, 2H), 2.59 (ddd, J = 12.0, 8.4, 5.3 Hz, 1H), 2.46 (dd, J = 11.7, 9.4 Hz, 1H), 1.28 (dd, J = 6.9, 3.6 Hz, 12H). MS (ESI) m / z 492.0 [M+H] + .

[0111] (e5'')[[]END]]1 H NMR (500 MHz, Chloroform-d) δ 8.50 (s, 1H), 8.45 (s, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.39 - 7.27 (m, 6H), 7.22 - 7.16 (m, 4H), 7.11 (m, 1H), 3.80 (dd, J = 11.1, 4.6 Hz, 1H), 3.72 (m, 1H), 3.41 (dd, J = 15.9, 4.7 Hz, 1H), 3.11 (ddd, J = 12.7, 9.3, 3.3 Hz, 1H), 3.04 (p, J = 6.8 Hz, 2H), 2.91 (td, J = 9.3, 4.6 Hz, 1H), 2.85 (dd, J = 15.9, 11.3 Hz, 1H), 2.57 - 2.53 (m, 2H), 1.21 (d, J = 7.2 Hz, 12H). MS (ESI) m / z 492.0 [M + H] + .

[0112] Examples 1 - 6. Synthesis of Compound e6 (e6' / e6'')

[0113]

[0114] The synthetic route was the same as that of Examples 1 - 1, except that 2 - amino - N,N - dimethylaniline was used instead of 2,6 - dimethylaniline in step (1). The yields were 14% for (e6') and 24% for (e6'').

[0115] (e6') 1 H NMR (500 MHz, Chloroform-d) δ 10.34 (s, 1H), 8.50 (dd, J = 8.0, 1.6 Hz, 1H), 7.94 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.42 - 7.39 (m, 2H), 7.36 - 7.27 (m, 4H), 7.23 - 7.07 (m, 5H), 4.07 (m, 1H), 3.82 (dd, J = 11.2, 4.5 Hz, 1H), 3.44 (dd, J = 15.9, 4.5 Hz, 1H), 2.87 (dd, J = 15.9, 11.3 Hz, 1H), 2.78 (s, 6H), 2.74 (m, 1H), 2.58 (ddd, J = 11.8, 8.2, 5.5 Hz, 1H), 2.42 (dd, J = 11.6, 9.5 Hz, 1H). MS (ESI) m / z 451.3 [M + H] + .

[0116] (e6'') 11H NMR (500 MHz, Chloroform-d) δ 10.11 (s, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.20 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.46 - 7.37 (m, 5H), 7.27 (m, 1H), 7.22 (m, 1H), 7.20 - 7.12 (m, 3H), 7.08 (m, 1H), 3.80 (dd, J = 11.0, 4.6 Hz, 1H), 3.74 (m, 1H), 3.40 (dd, J = 15.5, 4.6 Hz, 1H), 3.10 (ddd, J = 13.0, 9.3, 3.6 Hz, 1H), 2.94 (ddd, J = 12.5, 9.2, 3.6 Hz, 1H), 2.87 (dd, J = 15.5, 11.0 Hz, 1H), 2.70 (s, 6H), 2.63 - 2.59 (m, 2H). MS (ESI) m / z 451.3 [M+H] + .

[0117] Example 1 - 7. Synthesis of Compound e7 (e7' / e7'')

[0118]

[0119] Preparation of Intermediate b7

[0120]

[0121] Dissolve 304 mg (1 mmol) of N-Boc-L-tryptophan (a1) in 6 mL of tetrahydrofuran, add 288 mg (1.5 mmol) of EDCI hydrochloride, 203 mg (1.5 mmol) of HOBt and 278 μL (2 mmol) of triethylamine and stir, then add 145 μL (1.5 mmol) of o-fluoroaniline, and stir at room temperature for 14 h. Rotavap the reaction solution, add 10 mL of saturated ammonium chloride solution, extract three times with ethyl acetate, combine and dry the organic phases, and purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) after rotary evaporation to obtain 976 mg of intermediate b7, a pale yellow oil, with a yield of 19%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.27 - 8.19 (m, 2H), 8.00 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.23 - 7.17 (m, 1H), 7.15 - 6.95 (m, 5H), 5.22 (m, 1H), 4.65 (m, 1H), 3.39 (m, 1H), 3.30 (dd, J = 14.7, 6.9 Hz, 1H), 1.44 (s, 9H). MS (ESI) m / z 419.9 [M+Na]+ .

[0122] The preparation steps (2) and (3) of e7 are the same as those in Example 1-1. The yields are (e7′) 18% and (e7″) 29% respectively.

[0123] (e7′) 1 H NMR (400 MHz, Chloroform-d) δ9.67 (s, 1H), 8.49 (t, J = 8.1 Hz, 1H), 7.98 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.43 - 7.36 (m, 2H), 7.34 - 7.26 (m, 4H), 7.21 - 7.05 (m, 5H), 4.06 (m, 1H), 3.79 (dd, J = 11.3, 4.6 Hz, 1H), 3.44 (dd, J = 16.0, 4.6 Hz, 1H), 2.88 - 2.70 (m, 3H), 2.61 (td, J = 12.9, 10.7, 5.5 Hz, 1H), 2.41 (t, J = 10.5 Hz, 1H). MS (ESI) m / z 426.2 [M+H] + .

[0124] (e7″) 1 H NMR (500 MHz, Chloroform-d) δ9.47 (s, 1H), 8.42 (td, J = 7.9, 1.7 Hz, 1H), 8.34 (s, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.43 - 7.34 (m, 5H), 7.28 - 7.21 (m, 1H), 7.19 - 7.15 (m, 1H), 7.15 - 7.09 (m, 3H), 7.07 - 7.02 (m, 1H), 3.76 - 3.62 (m, 2H), 3.34 (dd, J = 15.8, 4.7 Hz, 1H), 3.04 (ddd, J = 12.7, 9.3, 3.2 Hz, 1H), 2.83 (ddd, J = 12.3, 9.3, 3.2 Hz, 1H), 2.76 (dd, J = 15.8, 11.1 Hz, 1H), 2.57 - 2.48 (m, 2H). MS (ESI) m / z 426.2 [M+H] + .

[0125] Example 1-8. Synthesis of compound e8 (e8′ / e8″)

[0126]

[0127] The synthesis route is the same as that in Example 1-1, except that 2-methylaniline is used instead of 2,6-dimethylaniline in step (1). The yields are (e8′) 23% and (e8″) 24% respectively.

[0128] (e8′) 1 H NMR(500 MHz, Acetone-d6) δ 10.38 (s, 1H), 9.67 (s, 1H), 8.27 - 8.21 (m, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.41 - 7.31 (m, 6H), 7.26 - 7.19 (m, 4H), 7.10 - 6.98 (m, 4H), 4.14 - 4.03 (m, 1H), 3.82 (dd, J = 11.3, 4.5 Hz, 1H), 3.29 (dd, J = 15.6, 4.5 Hz, 1H), 2.96 (m, 1H), 2.78 (ddd, J = 13.9, 11.2, 7.5 Hz, 2H), 2.57 (ddd, J = 11.4, 8.0, 5.9 Hz, 1H), 2.43 (s, 3H). MS(ESI) m / z 422.3 [M + H] + .

[0129] (e8″) 1 H NMR(500 MHz, Chloroform-d) δ 9.15 (s, 1H), 8.23 (s, 1H), 8.13 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.43 - 7.36 (m, 5H), 7.29 - 7.12 (m, 5H), 7.07 (m, 1H), 3.79 - 3.73 (m, 2H), 3.43 (dd, J = 16.0, 4.7 Hz, 1H), 3.16 - 3.09 (m, 1H), 2.82 (dd, J = 15.9, 11.2 Hz, 1H), 2.61 - 2.54 (m, 2H), 2.26 (s, 3H). MS(ESI) m / z 422.3 [M + H] + .

[0130] Example 1 - 9. Synthesis of compound e9 (e9′ / e9″)

[0131]

[0132] The synthetic route was the same as that of Example 1 - 1, except that 2 - isopropyl aniline was used instead of 2,6 - dimethyl aniline in step (1). The yields were 19% for (e9′) and 46% for (e9″) respectively.

[0133] (e9′) 11H NMR (400 MHz, Chloroform-d) δ 9.61 (s, 1H), 8.17 - 8.14 (m, 2H), 7.53 (d, J = 7.7 Hz, 1H), 7.43 - 7.38 (t, J = 7.5 Hz, 2H), 7.36 - 7.23 (m, 7H), 7.21 - 7.08 (m, 3H), 4.00 (m, 1H), 3.80 (dd, J = 11.3, 4.5 Hz, 1H), 3.47 (dd, J = 16.1, 4.5 Hz, 1H), 3.18 (p, J = 6.9 Hz, 1H), 2.87 - 2.71 (m, 3H), 2.59 (m, 1H), 2.44 (dd, J = 11.7, 9.6 Hz, 1H), 1.40 (dd, J = 6.9, 3.3 Hz, 6H). MS (ESI) m / z 450.3 [M+H] + .

[0134] (e9″) 1 1H NMR (400 MHz, DMSO-d6) δ 11.10 (s, 1H), 9.58 (s, 1H), 7.76 (dd, J = 7.8, 1.6 Hz, 1H), 7.48 - 7.29 (m, 7H), 7.26 - 7.12 (m, 4H), 7.08 m, 1H), 6.99 (m, 1H), 3.94 (m, 1H), 3.70 (m, 1H), 3.31 (m, 1H), 3.14 (p, J = 6.9 Hz, 1H), 3.04 (ddd, J = 15.1, 10.1, 4.0 Hz, 2H), 2.79 - 2.66 (m, 2H), 2.44 (dd, J = 12.3, 9.2 Hz, 1H), 1.20 (dd, J = 6.9, 2.5 Hz, 6H). MS (ESI) m / z 450.3 [M+H] + .

[0135] Example 1 - 10. Synthesis of compound e10 (e10' / e10'')

[0136]

[0137] The synthetic route is the same as that of Example 1 - 1, except that 4 - aminoindane is used instead of 2,6 - dimethylaniline in step (1). The yields are 16% for (e10') and 18% for (e10'') respectively.

[0138] (e10') 11H NMR (500 MHz, Chloroform-d) δ 9.32 (s, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.93 (s, 1H), 7.53 (m, 1H), 7.42 - 7.39 (m, 2H), 7.35 - 7.27 (m, 4H), 7.23 - 7.15 (m, 2H), 7.12 (td, J = 7.5, 1.1 Hz, 1H), 7.04 (m, 1H), 4.03 (m, 1H), 3.78 (dd, J = 11.3, 4.6 Hz, 1H), 3.46 (dd, J = 16.0, 4.6 Hz, 1H), 3.01 - 2.93 (m, 4H), 2.82 (dd, J = 16.0, 11.3 Hz, 1H), 2.78 - 2.73 (m, 2H), 2.60 (ddd, J = 12.0, 8.3, 5.4 Hz, 1H), 2.43 (dd, J = 11.7, 9.5 Hz, 1H), 2.22 - 2.13 (m, 2H). MS (ESI) m / z 448.3 [M+H] + .

[0139] (e10″) 1 1H NMR (500 MHz, Chloroform-d) δ 9.07 (s, 1H), 8.24 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.43 - 7.36 (m, 5H), 7.29 - 7.27 (m, 1H), 7.23 - 7.13 (m, 3H), 7.02 (m, 1H), 3.82 - 3.65 (m, 2H), 3.41 (dd, J = 15.9, 4.6 Hz, 1H), 3.13 (ddd, J = 12.4, 9.3, 3.1 Hz, 1H), 2.97 (t, J = 7.6 Hz, 2H), 2.91 (ddd, J = 16.1, 9.0, 4.8 Hz, 1H), 2.84 - 2.72 (m, 3H), 2.60 - 2.54 (m, 2H), 2.19 - 2.06 (m, 2H). MS (ESI) m / z 448.3 [M+H] + .

[0140] Example 1 - 11. Synthesis of Compound e11 (e11' / e11'')

[0141]

[0142] The synthetic route was the same as that of Example 1 - 1, except that 2 - aminopyridine was used instead of 2,6 - dimethylaniline in step (1). The yields were 14% for (e11') and 32% for (e11'').

[0143] (e11')1 1H NMR (500 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.37 (d, J = 8.4 Hz, 1H), 8.34 (dd, J = 4.9, 1.7 Hz, 1H), 8.23 (s, 1H), 7.77 (td, J = 7.9, 1.9 Hz, 1H), 7.52 (d, J = 7.7 Hz, 1H), 7.37 - 7.34 (m, 2H), 7.31 - 7.21 (m, 4H), 7.16 (m, 1H), 7.13 - 7.06 (m, 2H), 3.99 (m, 1H), 3.75 (dd, J = 11.4, 4.6 Hz, 1H), 3.40 (dd, J = 16.0, 4.7 Hz, 1H), 2.76 (dd, J = 16.0, 11.3 Hz, 1H), 2.72 - 2.66 (m, 2H), 2.47 (ddd, J = 12.6, 8.3, 5.3 Hz, 1H), 2.37 (dd, J = 11.7, 9.5 Hz, 1H). MS (ESI) m / z 408.9 [M+H] + .

[0144] (e11″) 1 1H NMR (500 MHz, Chloroform-d) δ 9.62 (s, 1H), 8.55 (d, J = 9.0 Hz, 1H), 8.34 (dd, J = 4.9, 1.8 Hz, 1H), 8.31 (d, J = 8.4 Hz, 1H), 7.70 (td, J = 7.8, 1.9 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.38 - 7.29 (m, 5H), 7.23 (m, 1H), 7.16 (m, 1H), 7.10 (m, 1H), 7.05 (dd, J = 7.3, 4.9 Hz, 1H), 3.70 (m, 1H), 3.66 (dd, J = 11.3, 4.4 Hz, 1H), 3.32 (dd, J = 15.8, 4.7 Hz, 1H), 2.95 (ddd, J = 12.6, 8.9, 3.9 Hz, 1H), 2.79 (ddd, J = 12.4, 9.0, 3.8 Hz, 1H), 2.71 (dd, J = 15.8, 11.4 Hz, 1H), 2.54 - 2.45 (m, 2H). MS (ESI) m / z 408.9 [M+H] + .

[0145] Example 1 - 12. Synthesis of Compound e12 (e12′ / e12″)

[0146]

[0147] The synthetic route is the same as that of Example 1-1, except that 2-amino-3-methylpyridine is used instead of 2,6-dimethylaniline in step (1). The yields are 15% for (e12′) and 34% for (e12′) respectively.

[0148] (e12′) 1 H NMR (400 MHz, Chloroform-d) δ 9.46 (s, 1H), 8.33 (m, 1H), 7.97 (s, 1H), 7.62 (ddd, J = 7.6, 1.8, 0.9 Hz, 1H), 7.52 (m, 1H), 7.43 - 7.35 (m, 2H), 7.32 - 7.28 (m, 3H), 7.27 (m, 1H), 7.20 - 7.08 (m, 3H), 4.02 (m, 1H), 3.83 (dd, J = 11.2, 4.5 Hz, 1H), 3.43 (dd, J = 16.1, 4.6 Hz, 1H), 2.84 (dd, J = 16.1, 11.3 Hz, 1H), 2.80 - 2.67 (m, 2H), 2.58 (m, 1H), 2.41 (dd, J = 11.6, 9.4 Hz, 1H), 2.37 (s, 3H). MS (ESI) m / z 422.9 [M+H] + .

[0149] (e12″) 1 H NMR (400 MHz, Chloroform-d) δ 9.25 (s, 1H), 8.32 (dd, J = 4.8, 1.8 Hz, 1H), 8.29 (s, 1H), 7.59 (ddd, J = 7.6, 1.8, 0.9 Hz, 1H), 7.52 (m, 1H), 7.42 - 7.33 (m, 5H), 7.26 - 7.18 (m, 2H), 7.17 - 7.10 (m, 2H), 3.83 - 3.68 (m, 2H), 3.38 (dd, J = 15.9, 4.7 Hz, 1H), 3.06 (ddd, J = 12.4, 8.9, 3.7 Hz, 1H), 2.94 - 2.79 (m, 2H), 2.63 - 2.56 (m, 2H), 2.29 (s, 3H). MS (ESI) m / z 422.9 [M+H] + .

[0150] Example 1-13. Synthesis of Compound e13 (e13′ / e13″)

[0151]

[0152] (1) Preparation of Intermediate i13′ / i13″

[0153]

[0154] Dissolve 307 mg (1 mmol) of intermediate c1 in 6 mL of acetonitrile, add 92 μL (1.2 mmol) of trifluoroacetic acid and 270 mg (1.2 mmol) of 3-(4-bromophenyl)cyclobutanone (d2), stir at room temperature for 18 h, evaporate the solvent, add 20 mL of water, adjust the pH to 10 - 12 by dropwise addition of saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, evaporate the solvent and separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 100 mg of intermediate i13′ and 273 mg of intermediate i13″, with yields of (i13′) 20% and (i13″) 53% respectively. (i13′) 1 H NMR (400 MHz, Chloroform-d) δ 8.73 (s, 1H), 8.11 (s, 1H), 7.53 - 7.45 (m, 4H), 7.30 (d, J = 7.9 Hz, 1H), 7.24 (dd, J = 8.6, 2.0 Hz, 1H), 7.19 - 7.15 (m, 2H), 7.14 - 7.09 (m, 3H), 3.94 (m, 1H), 3.83 (dd, J = 11.3, 4.5 Hz, 1H), 3.44 (dd, J = 16.0, 4.5 Hz, 1H), 2.84 (dd, J = 16.1, 11.3 Hz, 1H), 2.78 - 2.66 (m, 2H), 2.55 (m, 1H), 2.39 (dd, J = 11.7, 9.6 Hz, 1H), 2.31 (s, 6H). MS (ESI) m / z 515.8 [M + H] + .

[0155] (i13″) 1 H NMR (400 MHz, Chloroform-d) δ 8.44 (s, 1H), 8.23 (s, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.25 - 7.09 (m, 7H), 3.80 (dd, J = 11.2, 4.6 Hz, 1H), 3.69 (m, 1H), 3.41 (dd, J = 15.9, 4.6 Hz, 1H), 3.11 (ddd, J = 12.4, 9.2, 3.3 Hz, 1H), 2.93 (ddd, J = 12.3, 9.2, 3.4 Hz, 1H), 2.85 (dd, J = 15.9, 11.2 Hz, 1H), 2.55 - 2.50 (dd, J = 12.2, 8.5 Hz, 2H), 2.24 (s, 6H). MS (ESI) m / z 515.8 [M + H] + .

[0156] (2) Preparation of Compound e13 (e13′ / e13″)

[0157]

[0158] Dissolve 100 mg (0.19 mmol) of intermediate i13′ in 3 ml of DMSO, add 30 mg (0.29 mmol) of sodium methanesulfinate, 8 mg (0.04 mmol) of copper(I) iodide and 4 mg (0.06 mmol) of sodium L - prolinate, and stir at 95 °C for 24 h. Cool to room temperature, add 10 mL of 5% ammonia water, extract three times with dichloromethane, combine and dry the organic phases, rotary evaporate, and separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain 53 mg of compound e13′ as a pale yellow powder, with a yield of 54%.

[0159] The preparation method of compound e13″ is the same as above, only replacing the raw material i13′ with i13″, with a yield of 54%.

[0160] (e13′) 1 H NMR (600 MHz, DMSO - d6) δ 11.14 (s, 1H), 9.45 (s, 1H), 7.91 (d, J = 7.9 Hz, 2H), 7.71 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.11 (m, 3H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.07 (m, 1H), 3.77 (dd, J = 11.1, 4.4 Hz, 1H), 3.22 (s, 3H), 3.03 (dd, J = 15.1, 4.3 Hz, 1H), 2.91 (m, 1H), 2.76 (dd, J = 15.1, 11.1 Hz, 1H), 2.64 (m, 1H), 2.50 - 2.45 (m, 2H), 2.25 (s, 6H). MS (ESI) m / z 513.9 [M + H] + .

[0161] (e13″) 11H NMR (500 MHz, DMSO-d6) δ 11.09 (s, 1H), 9.25 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.76 (d, J = 8.0 Hz, 2H), 7.42 (dd, J = 16.5, 7.9 Hz, 2H), 7.11 - 7.07 (m, 4H), 6.99 (t, J = 7.5 Hz, 1H), 4.02 (m, 1H), 3.73 (dd, J = 11.1, 4.3 Hz, 1H), 3.19 (s, 3H), 3.13 - 3.08 (m, 1H), 3.02 (dd, J = 15.0, 4.3 Hz, 1H), 2.83 - 2.74 (m, 2H), 2.55 (m, 1H), 2.47 (m, 1H), 2.20 (s, 6H). MS (ESI) m / z 513.9 [M + H] + .

[0162] Example 1 - 14. Synthesis of Compound e14 (e14' / e14'')

[0163]

[0164] (1) Preparation of Intermediate d3

[0165]

[0166] Dissolve 360 μl (3.878 mmol) of N,N-dimethylacetamide in 12 mL of 1,2-dichloroethane. Dropwise add 870 μL (5.17 mmol) of trifluoromethanesulfonic anhydride under an ice bath at 0 °C. After stirring in the ice bath for 30 min, add 510 mg (2.585 mmol) of N-(4-vinylphenyl)methanesulfonamide and 683 μl (5.17 mmol) of 2,4,5-trimethylpyridine, and stir under reflux at 85 °C for 18 h; after cooling to room temperature, rotary evaporate the reaction solvent, then add 10 mL of carbon tetrachloride and 10 mL of water to the system, and stir under reflux at 80 °C for 18 h; cool to room temperature, separate the layers, separate the organic phase, extract the aqueous phase with dichloromethane twice, combine the organic phases, wash the organic phase once with 1 mol / L hydrochloric acid, dry and rotary evaporate, and then purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 114 mg of intermediate d3 with a yield of 18%. 1 1H NMR (400 MHz, Chloroform-d) δ 7.30 - 7.20 (m, 4H), 3.65 (m, 1H), 3.57 - 3.43 (m, 2H), 3.26 - 3.15 (m, 2H), 3.00 (s, 3H). MS (ESI) m / z 240.1 [M + H] + .

[0167] (2) Preparation of Compound e14 (e14' / e14'')

[0168]

[0169] Dissolve 133 mg (0.433 mmol) of Intermediate c1 in 3 mL of acetonitrile, add 36 μL (0.476 mmol) of trifluoroacetic acid and 114 mg (0.476 mmol) of Intermediate d3, stir at room temperature for 18 h, evaporate the solvent, add 10 mL of water, adjust the pH to 10 - 12 by dropwise addition of saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, evaporate to dryness and separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) to obtain 21 mg of e14' and 54 mg of e14''', light yellow powder, with yields of (e14') 9% and (e14'') 24% respectively

[0170] (e14')[[]]END]] 1 H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H), 9.61 (s, 1H), 9.41 (s, 1H), 7.45 - 7.41 (m, 3H), 7.34 (d, J = 8.0 Hz, 1H), 7.24 - 7.19 (m, 2H), 7.11 (m, 3H), 7.06 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.97 (m, 1H), 3.90 (m, 1H), 3.75 (m, 1H), 3.03 (m, 1H), 2.97 (s, 3H), 2.90 - 2.70 (m, 2H), 2.58 (m, 1H), 2.40 (m, 1H), 2.25 (s, 6H). MS (ESI) m / z 529.3 [M + H] + .

[0171] (e14'')[[]]END]] 1 H NMR (600 MHz, Methanol-d4) δ 8.45 (s, 1H), 7.49 - 7.40 (m, 4H), 7.26 - 7.23 (m, 2H), 7.18 - 7.00 (m, 5H), 4.20 (dd, J = 11.7, 4.7 Hz, 1H), 3.99 (m, 1H), 3.40 (m, 1H), 3.14 - 3.02 (m, 2H), 2.91 (s, 3H), 2.86 (m, 1H), 2.75 - 2.66 (m, 2H), 2.28 (s, 6H). MS (ESI) m / z 529.3 [M + H] + .

[0172] Example 1 - 15. Synthesis of Compound e15 (e15' / e15'')

[0173]

[0174] The synthetic route is the same as that in Example 1-1, except that 3-(pyridin-2-yl)cyclobutan-1-one (d4) (for the synthesis method, refer to Demchuk, Oleksandr P, et al. European Journal of Organic Chemistry, 2019(34), 5937-5949.) is used instead of 3-phenylcyclobutanone (d1) in step (3). The yields are 22% for (e15′) and 33% for (e15″) respectively.

[0175] (e15) 1 H NMR (400 MHz, Methanol-d4) δ 8.69 (dd, J = 4.9, 1.7 Hz, 1H), 8.36 (s, 1H), 7.79 (td, J = 7.7, 1.7 Hz, 1H), 7.46 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.39 - 7.30 (m, 2H), 7.18 - 7.12 (m, 1H), 7.12 - 7.07 (m, 3H), 7.05 (m, 1H), 4.19 (dd, J = 11.4, 4.4 Hz, 1H), 4.03 (m, 1H), 3.39 (dd, J = 15.4, 4.3 Hz, 1H), 3.20 (dd, J = 12.8, 8.5 Hz, 1H), 3.08 (dd, J = 15.4, 11.3 Hz, 1H), 3.01 - 2.92 (m, 1H), 2.80 (ddd, J = 13.0, 8.8, 3.7 Hz, 1H), 2.64 (dd, J = 12.4, 8.3 Hz, 1H), 2.30 (s, 6H). MS (ESI) m / z 437.3 [M+H] + .

[0176] (e15′) 11H NMR (500 MHz, Methanol-d4) δ 8.65 (dd, J = 5.0, 1.6 Hz, 1H), 8.45 (s, 1H), 7.84 (td, J = 7.6, 1.8 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.46 (d, J = 7.9 Hz, 2H), 7.36 (dd, J = 7.6, 4.9 Hz, 1H), 7.21 (m, 1H), 7.19 - 7.15 (m, 3H), 7.11 (m, 1H), 4.64 (dd, J = 11.8, 4.8 Hz, 1H), 3.96 (dt, J = 9.2, 4.7 Hz, 1H), 3.73 (dd, J = 15.5, 4.8 Hz, 1H), 3.63 (dd, J = 13.7, 9.2 Hz, 1H), 3.17 (dd, J = 13.5, 9.2 Hz, 1H), 2.90 (dt, J = 13.7, 4.1 Hz, 1H), 2.69 (dt, J = 13.9, 4.2 Hz, 1H), 2.34 (s, 6H). MS (ESI) m / z 437.3 [M+H] + .

[0177] Examples 1 - 16. Synthesis of Compound e16 (e16' / e16'')

[0178]

[0179] Preparation of Intermediate d5

[0180]

[0181] (1) Dissolve 1 g (5.075 mmol) of 2-(5-bromo-2-pyridyl)acetonitrile in 10 mL of N,N-dimethylformamide. Slowly add 507 mg (12.69 mmol) of NaH under an ice bath at 0 °C and stir for 15 min; restore to room temperature, add 1.6 g (6.09 mmol) of 1,3-dibromo-2,2-dimethoxypropane, heat and stir at 50 °C for 18 h; cool to room temperature, add 30 mL of water to quench under an ice bath at 0 °C, extract three times with ethyl acetate, combine and dry the organic phases, spin-dry and purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 118 mg of intermediate d5-1, with a yield of 8%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.67 (dd, J = 2.4, 0.8 Hz, 1H), 7.83 (dd, J = 8.4, 2.4 Hz, 1H), 7.51 (dd, J = 8.3, 0.8 Hz, 1H), 3.27 (s, 3H), 3.17 (s, 3H), 2.96 (m, 4H). MS (ESI) m / z 318.3 [M+Na] + .

[0182] (2) Dissolve 398 mg (1.34 mmol) of intermediate d5-1 in 5 mL of DMSO, add 167 mg (1.61 mmol) of sodium methanesulfonate, 26 mg (0.134 mmol) of copper(I) iodide and 37 mg (0.268 mmol) of sodium L-prolinate, and stir at 95 °C for 36 h. Cool to room temperature, add 25 mL of 5% ammonia water, extract three times with ethyl acetate, combine and dry the organic phases, evaporate to dryness, and purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 2∶1) to obtain 325 mg of intermediate d5-2, with a yield of 82%. 1 H NMR (400 MHz, Chloroform-d) δ 9.13 (dd, J = 2.4, 0.8 Hz, 1H), 8.25 (dd, J = 8.3, 2.4 Hz, 1H), 7.83 (dd, J = 8.3, 0.9 Hz, 1H), 3.27 (s, 3H), 3.17 (s, 3H), 3.11 (s, 3H), 3.06 - 2.94 (m, 4H). MS (ESI) m / z 319.1 [M+Na] + .

[0183] (3) Dissolve 325 mg (1.097 mmol) of intermediate d5-2 in 6 mL of water, add 185 mg (3.29 mmol) of potassium hydroxide, reflux and stir at 100 °C for 24 h, cool to room temperature, wash the reaction solution twice with dichloromethane, evaporate the aqueous phase to dryness to obtain the crude carboxylate; dissolve the crude product in 4 mL of pyridine, add 380 mg (3.29 mmol) of pyridine hydrochloride, stir at 60 °C for 6 h, cool to room temperature, evaporate the reaction solvent, add 15 mL of ethyl acetate to dissolve, wash the organic phase with saturated brine, dry, and evaporate to dryness to obtain 279 mg of intermediate d5-3, with a yield of 94%. 1 H NMR (400 MHz, Chloroform-d) δ 8.98 (dd, J = 2.4, 0.8 Hz, 1H), 8.04 (dd, J = 8.2, 2.4 Hz, 1H), 7.33 (m, 1H), 3.40 (m, 1H), 3.14 (s, 3H), 3.09 (s, 3H), 3.03 (s, 3H), 2.63 - 2.54 (m, 2H), 2.37 (m, 2H). MS (ESI) m / z 294.1 [M+Na] + .

[0184] (4) Dissolve 279 mg (1.03 mmol) of intermediate d5-3 in 8 mL of acetone, add 285 mg (1.5 mmol) of p-toluenesulfonic acid monohydrate, heat under reflux with stirring at 60 °C for 12 h, cool to room temperature, rotary evaporate the reaction solvent, add 15 mL of ethyl acetate to dissolve, wash the organic phase with saturated sodium carbonate solution, dry, rotary evaporate and then purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 1∶1) to obtain 214 mg of intermediate d5, with a yield of 92%. 1 H NMR (400 MHz, Chloroform-d) δ 9.11 (d, J = 2.3 Hz, 1H), 8.16 (dd, J = 8.1, 2.4 Hz, 1H), 7.45 (dd, J = 8.1, 0.8 Hz, 1H), 3.82 (m, 1H), 3.61 - 3.39 (m, 4H), 3.11 (s, 3H). MS (ESI) m / z 226.1 [M+H] + .

[0185] (5) Preparation of compound e16 (e16′ / e16″)

[0186]

[0187] Take 292 mg (0.95 mmol) of intermediate c1 and dissolve it in 5 mL of acetonitrile, add 73 μl (0.95 mmol) of trifluoroacetic acid and 214 mg (0.95 mmol) of intermediate d5, stir at room temperature for 18 h, rotary evaporate the solvent, add 15 mL of water, dropwise add saturated sodium carbonate solution to adjust the pH to 10 - 12, extract with ethyl acetate three times, combine and dry the organic phase, rotary evaporate and then separate by silica gel column chromatography (methylene chloride∶methanol = 20∶1) to obtain 91 mg of compound e16′ and 129 mg of e16″, yellowish powder, with yields of (e16′) 19% and (e16″) 26% respectively.

[0188] (e16′) 11H NMR (400 MHz, Chloroform-d) δ 10.24 (s, 1H), 9.29 (m, 1H), 8.56 (s, 1H), 8.10 (dd, J = 8.1, 2.2 Hz, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.30 (m, 1H), 7.20 (ddd, J = 8.2, 7.1, 1.2 Hz, 1H), 7.14 - 7.08 (m, 4H), 3.93 (ddd, J = 9.6, 6.9, 2.7 Hz, 1H), 3.74 (dd, J = 11.2, 4.4 Hz, 1H), 3.37 (dd, J = 15.8, 4.3 Hz, 1H), 3.19 (dd, J = 12.8, 6.9 Hz, 1H), 3.12 (s, 3H), 2.91 - 2.78 (m, 2H), 2.73 (ddd, J = 12.5, 9.4, 2.6 Hz, 1H), 2.63 (dd, J = 12.4, 6.9 Hz, 1H), 2.31 (s, 6H). MS (ESI) m / z 515.2 [M+H] + .

[0189] (e16″) 1 1H NMR (500 MHz, Chloroform-d) δ 9.05 (d, J = 2.3 Hz, 1H), 8.75 (d, J = 4.9 Hz, 1H), 8.33 (s, 1H), 8.07 (dd, J = 8.1, 2.4 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.16 (m, 1H), 7.13 - 7.05 (m, 4H), 3.91 - 3.80 (m, 2H), 3.33 (dd, J = 15.5, 4.5 Hz, 1H), 3.16 (ddd, J = 12.7, 9.3, 2.5 Hz, 1H), 2.99 (s, 3H), 2.94 - 2.87 (m, 2H), 2.74 - 2.67 (m, 2H), 2.25 (s, 6H). MS (ESI) m / z 515.2 [M+H] + .

[0190] Example 1 - 17. Synthesis of compound e17 (e17′ / e17″)

[0191]

[0192] Dissolve 155 mg (0.356 mmol) of compound e1′ in 6 mL of toluene, add 129 mg (0.32 mmol) of Lawesson's reagent, heat and stir at 100 °C for 12 h, cool to room temperature, rotary evaporate the reaction solvent, and separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 5∶1) to obtain 109 mg of compound e17′ as a pale yellow powder with a yield of 68%. The synthesis method of compound e17″ is the same as above, only replacing the raw material with e1″, and the yield is 68%.

[0193] (e17′) 1 H NMR (500 MHz, Acetone-d6) δ 10.37 (s, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.40 - 7.31 (m, 5H), 7.21 (m, 1H), 7.18 - 7.07 (m, 4H), 7.03 (m, 1H), 4.18 (m, 1H), 4.10 (dd, J = 11.3, 4.3 Hz, 1H), 3.64 (dd, J = 15.6, 4.3 Hz, 1H), 2.98 - 2.90 (m, 2H), 2.77 (ddd, J = 10.9, 7.8, 5.8 Hz, 1H), 2.58 (ddd, J = 11.2, 7.8, 5.5 Hz, 1H), 2.44 (m, 1H), 2.30 (s, 6H). MS (ESI) m / z 451.9 [M+H] + .

[0194] (e17″) 1 H NMR (500 MHz, Acetone-d6) δ 10.37 (s, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.47 - 7.45 (m, 2H), 7.40 (d, J = 8.0 Hz, 1H), 7.33 - 7.29 (m, 2H), 7.20 - 7.08 (m, 5H), 7.05 (m, 1H), 4.13 (dd, J = 11.2, 4.4 Hz, 1H), 3.95 (m, 1H), 3.52 (dd, J = 15.3, 4.4 Hz, 1H), 3.19 (ddd, J = 12.3, 9.0, 3.4 Hz, 1H), 2.98 - 2.89 (m, 2H), 2.67 - 2.59 (m, 2H), 2.24 (s, 6H). MS (ESI) m / z 451.9 [M+H] + .

[0195] Example 1 - 18. Synthesis of compound e18 (e18′ / e18″)

[0196]

[0197] The synthetic route is the same as that in Example 1-1, except that benzylacetone (d7) is used instead of 3-phenylcyclobutanone (d1) in step (3). The yields are (e18′) 8% and (e18″) 12% respectively.

[0198] (e18′) 1 H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H), 8.07 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.30 - 7.26 (m, 3H), 7.22 - 7.07 (m, 9H), 4.00 (dd, J = 11.2, 4.6 Hz, 1H), 3.47 (dd, J = 15.9, 4.6 Hz, 1H), 2.95 (ddd, J = 13.4, 11.5, 4.9 Hz, 1H), 2.88 - 2.76 (m, 2H), 2.30 (s, 6H), 2.19 (m, 1H), 2.08 (ddd, J = 14.2, 11.6, 5.6 Hz, 1H), 1.60 (s, 3H). MS (ESI) m / z 437.9 [M+H] + .

[0199] (e18″) 1 H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 1H), 8.14 (s, 1H), 7.55 (dd, J = 7.4, 1.3 Hz, 1H), 7.30 (m, 1H), 7.26 - 7.09 (m, 11H), 3.93 (dd, J = 11.2, 4.3 Hz, 1H), 3.42 (dd, J = 15.6, 4.3 Hz, 1H), 2.79 (dd, J = 15.6, 11.2 Hz, 1H), 2.71 - 2.51 (m, 2H), 2.24 (s, 6H), 2.24 - 2.20 (m, 2H), 1.55 (s, 3H). MS (ESI) m / z 437.9 [M+H ] +.

[0200] Example 1-19. Synthesis of Compounds e19 / e19′ / e19″

[0201]

[0202] The synthetic route is the same as that in Example 1-2, except that benzylacetone (d7) is used instead of 3-phenylcyclobutanone (d1) in step (3). The yields are (e19′) 9% and (e19″) 10% respectively.

[0203] (e19′) 11H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H), 8.07 (s, 1H), 7.53 (d, J = 7.7 Hz, 1H), 7.30 - 7.26 (m, 3H), 7.22 - 7.07 (m, 9H), 3.99 (dd, J = 11.2, 4.6 Hz, 1H), 3.46 (dd, J = 15.9, 4.6 Hz, 1H), 2.94 (ddd, J = 13.4, 11.5, 4.9 Hz, 1H), 2.88 - 2.76 (m, 2H), 2.29 (s, 6H), 2.19 (m, 1H), 2.08 (ddd, J = 14.2, 11.6, 5.6 Hz, 1H), 1.61 (s, 3H). MS (ESI) m / z 437.9 [M+H] + .

[0204] (e19″) 1 1H NMR (400 MHz, Chloroform-d) δ 8.36 (s, 1H), 8.14 (s, 1H), 7.55 (dd, J = 7.4, 1.3 Hz, 1H), 7.30 (m, 1H), 7.26 - 7.09 (m, 11H), 3.92 (dd, J = 11.2, 4.3 Hz, 1H), 3.42 (dd, J = 15.6, 4.3 Hz, 1H), 2.78 (dd, J = 15.6, 11.2 Hz, 1H), 2.71 - 2.51 (m, 2H), 2.23 (s, 6H), 2.24 - 2.20 (m, 2H), 1.56 (s, 3H). MS (ESI) m / z 437.9 [M+H] + .

[0205] Example 2 - 1. Synthesis of Compound j1

[0206]

[0207] (1) Preparation of Intermediate g1

[0208]

[0209] Dissolve 883 mg (2.872 mmol) of Intermediate c1 in 15 mL of acetonitrile, add 330 μl (4.308 mmol) of trifluoroacetic acid and 738 mg (4.308 mmol) of N-Boc-3-azetidinone (f1), stir at room temperature for 14 h, evaporate the solvent, add 25 mL of water, adjust the pH to 10 - 12 by dropwise addition of saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, evaporate the solvent and purify by silica gel column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 1.1 g of Intermediate g1, with a yield of 84%. 11H NMR (400 MHz, Chloroform-d) δ 8.37 (s, 1H), 7.47 (d, J = 7.8 Hz, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.20 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.14 - 7.04 (m, 5H), 4.52 (m, 1H), 4.08 - 4.02 (m, 2H), 3.67 (dd, J = 11.3, 4.3 Hz, 1H), 3.35 - 3.27 (m, 1H), 2.82 (dd, J = 15.8, 11.0 Hz, 1H), 2.26 (s, 6H), 1.55 (s, 9H). MS (ESI) m / z 461.3 [M+H] + .

[0210] (2) Preparation of Intermediate h1

[0211]

[0212] Dissolve 460 mg (1 mmol) of Intermediate g1 in 5 mL of dichloromethane, add 612 μL (8 mmol) of trifluoroacetic acid under an ice bath at 0 °C, and stir at room temperature for 12 h; evaporate the reaction solution to dryness, add 20 mL of water, adjust the pH to 10 - 12 by dropwise addition of saturated sodium carbonate solution, extract three times with ethyl acetate, combine and dry the organic phases, and evaporate to dryness to obtain 270 mg of Intermediate h1, without further purification. Yield: 75%. 1 1H NMR (400 MHz, Chloroform-d) δ 9.71 (s, 1H), 8.30 (s, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.36 (d, J = 8.0 Hz, 1H), 7.18 (m, 1H), 7.13 - 7.01 (m, 4H), 3.96 (m, 1H), 3.91 - 3.84 (m, 2H), 3.76 (m, 1H), 3.71 (dd, J = 10.6, 4.3 Hz, 1H), 3.29 (dd, J = 15.5, 4.4 Hz, 1H), 2.85 (dd, J = 15.5, 10.7 Hz, 1H), 2.24 (s, 6H). MS (ESI) m / z 361.2 [M+H] + .

[0213] (3) Preparation of Compound j1

[0214]

[0215] Dissolve 203 mg (0.563 mmol) of intermediate h1 in 8 mL of 1,2-dichloroethane, add 71 μL (0.676 mmol) of bromobenzene, 52 mg (0.056 mmol) of Pd2(dba)3, 26 mg (0.056 mmol) of RuPhos, and 642 mg (1.97 mmol) of cesium carbonate, heat under reflux with stirring at 110 °C for 12 h; cool to room temperature, add 20 mL of water, extract three times with ethyl acetate, combine and dry the organic phases, concentrate in vacuo and purify by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 147 mg of compound j1 as a pale yellow powder, with a yield of 60%. 1 H NMR (400 MHz, Chloroform-d) δ 8.58 (s, 1H), 8.20 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.38 - 7.28 (m, 3H), 7.21 (dd, J = 8.0, 1.3 Hz, 1H), 7.18 - 7.06 (m, 4H), 6.93 - 6.84 (m, 1H), 6.67 - 6.60 (m, 2H), 4.34 (d, J = 7.2 Hz, 1H), 4.17 (d, J = 6.7 Hz, 1H), 4.03 - 3.99 (m, 2H), 3.84 (dd, J = 10.6, 4.4 Hz, 1H), 3.40 (dd, J = 15.7, 4.4 Hz, 1H), 2.95 (dd, J = 15.7, 10.6 Hz, 1H), 2.25 (s, 6H). MS (ESI) m / z 437.3 [M + H] + .

[0216] Example 2-2. Synthesis of compound j2

[0217]

[0218] The synthetic route is the same as that of Example 2-1, except that in step (1), D-tryptophan-2′,6′-dimethylaniline (c2) is used instead of L-tryptophan-2′,6′-dimethylaniline (c1). The yield is 59%. 11H NMR (400 MHz, Chloroform-d) δ 8.63 (s, 1H), 8.22 (s, 1H), 7.55 (d, J = 7.3 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.33 - 7.28 (m, 2H), 7.25 - 7.19 (m, 1H), 7.19 - 7.06 (m, 4H), 6.88 (m, 1H), 6.67 - 6.60 (m, 2H), 4.34 (d, J = 7.3 Hz, 1H), 4.17 (d, J = 6.7 Hz, 1H), 4.03 - 3.99 (m, 2H), 3.84 (dd, J = 10.6, 4.4 Hz, 1H), 3.40 (dd, J = 15.7, 4.4 Hz, 1H), 2.95 (dd, J = 15.7, 10.7 Hz, 1H), 2.25 (s, 6H). MS (ESI) m / z 437.3 [M + H] + .

[0219] Example 2 - 3. Synthesis of Compound j3

[0220]

[0221] The synthetic route was the same as that in Example 2 - 1, except that 4 - bromophenyl sulfone was used instead of bromobenzene in step (3). The yield was 23%. 1 1H NMR (800 MHz, DMSO - d6) δ 11.27 (s, 1H), 9.36 (s, 1H), 7.72 (d, J = 8.5 Hz, 2H), 7.47 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.09 (m, 4H), 7.01 (d, J = 7.4 Hz, 1H), 6.65 (dd, J = 8.9, 2.5 Hz, 2H), 4.46 (d, J = 8.6 Hz, 1H), 4.17 (d, J = 7.7 Hz, 1H), 4.13 (d, J = 8.4 Hz, 1H), 4.06 (d, J = 7.8 Hz, 1H), 3.82 (m, 1H), 3.46 (d, J = 9.1 Hz, 1H), 3.10 (s, 3H), 2.83 (dd, J = 14.9, 10.6 Hz, 1H), 2.21 (s, 6H). MS (ESI) m / z 515.3 [M + H] + .

[0222] Example 2 - 4. Synthesis of Compound j4

[0223]

[0224] The synthetic route was the same as that in Example 2 - 1, except that 4 - bromobenzonitrile was used instead of bromobenzene in step (3). The yield was 58%.1 HNMR(400 MHz, DMSO-d6) δ 11.26 (s, 1H), 9.35 (s, 1H), 7.63 - 7.57 (m, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.11 - 7.04 (m, 4H), 6.99 (m, 1H), 6.63 - 6.56 (m, 2H), 4.45 (d, J = 8.5 Hz, 1H), 4.16 (d, J = 7.8 Hz, 1H), 4.11 (d, J = 8.5 Hz, 1H), 4.05 (d, J = 7.8 Hz, 1H), 3.80 (m, 1H), 3.07 (dd, J = 15.3, 4.2 Hz, 1H), 2.81 (dd, J = 15.1, 10.6 Hz, 1H), 2.20 (s, 6H). MS(ESI) m / z 462.2 [M + H] + .

[0225] Example 2 - 5. Synthesis of Compound j5

[0226]

[0227] The synthetic route is the same as that of Example 2 - 1, except that 4 - bromoanisole is used instead of bromobenzene in step (3). The yield is 32%. 1 H NMR(400 MHz, Chloroform - d) δ 8.70 (s, 1H), 8.21 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.34 (m, 1H), 7.21 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.15 (m, 1H), 7.12 - 7.05 (m, 3H), 6.91 - 6.84 (m, 2H), 6.61 - 6.56 (m, 2H), 4.27 (d, J = 7.0 Hz, 1H), 4.11 (d, J = 6.5 Hz, 1H), 3.95 - 3.90 (m, 2H), 3.82 (dd, J = 10.6, 4.4 Hz, 1H), 3.78 (s, 3H), 3.38 (dd, J = 15.6, 4.4 Hz, 1H), 2.94 (dd, J = 15.6, 10.6 Hz, 1H), 2.24 (s, 6H). MS(ESI) m / z 467.3 [M + H] + .

[0228] Example 2 - 6. Synthesis of Compound j6

[0229]

[0230] The synthetic route is the same as that of Example 2 - 1, except that 4 - bromobenzotrifluoride is used instead of bromobenzene in step (3). The yield is 57%.1 1H NMR (400 MHz, Chloroform-d) δ 8.44 (s, 1H), 8.15 (s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.52 (d, J = 8.4 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.23 (m, 1H), 7.16 (td, J = 7.4, 7.0, 1.1 Hz, 1H), 7.13 - 7.07 (m, 3H), 6.63 - 6.59 (m, 2H), 4.40 (d, J = 7.6 Hz, 1H), 4.21 (d, J = 7.0 Hz, 1H), 4.10 - 4.05 (m, 2H), 3.84 (dd, J = 10.6, 4.4 Hz, 1H), 3.42 (dd, J = 15.8, 4.4 Hz, 1H), 2.96 (dd, J = 15.8, 10.6 Hz, 1H), 2.24 (s, 6H). MS (ESI) m / z 505.3 [M+H] + .

[0231] Example 2 - 7. Synthesis of Compound j7

[0232]

[0233] The synthetic route was the same as that of Example 2 - 1, except that p-bromoethylbenzene was used instead of bromobenzene in step (3). The yield was 42%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.27 (s, 1H), 7.55 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.23 (ddd, J = 8.1, 7.1, 1.3 Hz, 1H), 7.19 - 7.07 (m, 6H), 6.58 (d, J = 8.4 Hz, 2H), 4.29 (d, J = 7.2 Hz, 1H), 4.12 (d, J = 6.6 Hz, 1H), 4.00 - 3.90 (m, 2H), 3.81 (dd, J = 10.6, 4.4 Hz, 1H), 3.37 (dd, J = 15.6, 4.4 Hz, 1H), 2.93 (dd, J = 15.7, 10.6 Hz, 1H), 2.63 (q, J = 7.6 Hz, 2H), 2.25 (s, 6H), 1.27 (t, J = 7.6 Hz, 3H). MS (ESI) m / z 464.9 [M+H] + .

[0234] Example 2 - 8. Synthesis of Compound j8

[0235]

[0236] The synthetic route is the same as that in Example 2-1, except that 4-bromofluorobenzene is used instead of bromobenzene in step (3). The yield is 55%. 1 HNMR(400MHz, Chloroform-d)δ8.61(s, 1H), 8.17(s, 1H), 7.55(dd, J = 7.8, 1.2Hz, 1H), 7.36(dt, J = 8.1, 1.0 Hz, 1H), 7.22(ddd, J = 8.2, 7.1, 1.3Hz, 1H), 7.15(ddd, J = 8.1, 7.1, 1.1Hz, 1H), 7.12 - 7.07(m, 3H), 7.02 - 6.96(m, 2H), 6.59 - 6.49(m, 2H), 4.29(d, J = 7.1Hz, 1H), 4.14(m, 1H), 3.96(dd, J = 11.0, 6.8Hz, 2H), 3.83(dd, J = 10.6, 4.4Hz, 1H), 3.39(dd, J = 15.7, 4.4Hz, 1H), 2.94(dd, J = 15.7, 10.6Hz, 1H), 2.24(s, 6H). MS(ESI)m / z 455.2[M + H] + .

[0237] Example 2-9. Synthesis of Compound j9

[0238]

[0239] The synthetic route is the same as that in Example 2-1, except that 3-bromopyridine is used instead of bromobenzene in step (3). The yield is 27%. 1HNMR (600 MHz, DMSO-d6) δ 11.22 (s, 1H), 9.36 (s, 1H), 7.96 (d, J = 4.7 Hz, 1H), 7.95 (d, J = 2.9 Hz, 1H), 7.45 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.22 (dd, J = 8.3, 4.7 Hz, 1H), 7.10 - 7.04 (m, 4H), 6.98 (t, J = 7.4 Hz, 1H), 6.94 (dd, J = 8.2, 2.9 Hz, 1H), 4.39 (d, J = 7.8 Hz, 1H), 4.12 (d, J = 7.1 Hz, 1H), 4.06 (d, J = 7.8 Hz, 1H), 3.98 (d, J = 7.1 Hz, 1H), 3.80 (td, J = 10.3, 9.6, 4.2 Hz, 1H), 3.40 (d, J = 8.7 Hz, 1H), 3.07 (dd, J = 15.0, 4.2 Hz, 1H), 2.81 (dd, J = 15.0, 10.6 Hz, 1H), 2.21 (s, 6H). MS (ESI) m / z 437.9 [M+H] + .

[0240] Example 2 - 10. Synthesis of Compound j10

[0241]

[0242] The synthesis route was the same as that of Example 2 - 1, except that 4 - bromocyclopropylbenzene was used instead of bromobenzene in step (3). The yield was 25%. 1 H NMR (400 MHz, Chloroform - d) δ 8.59 (s, 1H), 8.20 (s, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.36 (dt, J = 8.1, 1.0 Hz, 1H), 7.21 (ddd, J = 8.2, 7.1, 1.3 Hz, 1H), 7.18 - 7.06 (m, 4H), 7.06 - 7.02 (m, 2H), 6.58 - 6.53 (m, 2H), 4.31 (d, J = 7.2 Hz, 1H), 4.15 (d, J = 6.6 Hz, 1H), 3.97 (dd, J = 10.5, 6.8 Hz, 2H), 3.84 (dd, J = 10.6, 4.4 Hz, 1H), 3.40 (dd, J = 15.7, 4.4 Hz, 1H), 2.95 (dd, J = 15.7, 10.6 Hz, 1H), 1.87 (m, 1H), 0.95 - 0.87 (m, 2H), 0.66 - 0.59 (m, 2H). MS (ESI) m / z 476.9 [M+H] + .

[0243] Example 2-11. Synthesis of Compound j11

[0244]

[0245] The synthetic route is the same as that of Example 2-1, except that p-bromotoluene is used instead of bromobenzene in step (3). The yield is 52%. 1 HNMR(400MHz, Chloroform-d)δ8.60(s, 1H), 8.21(s, 1H), 7.55(dd, J = 7.6, 1.2Hz, 1H), 7.39 - 7.33(m, 1H), 7.22(ddd, J = 8.2, 7.0, 1.3Hz, 1H), 7.18 - 7.06(m, 6H), 6.58 - 6.53(m, 2H), 4.31(d, J = 7.2Hz, 1H), 4.15(d, J = 6.6Hz, 1H), 3.96(dd, J = 11.1, 6.9Hz, 2H), 3.84(dd, J = 10.6, 4.4Hz, 1H), 3.40(dd, J = 15.7, 4.4Hz, 1H), 2.94(dd, J = 15.7, 10.6Hz, 1H), 2.31(s, 3H), 2.24(s, 6H). MS(ESI)m / z 450.9[M + H] + .

[0246] Example 2-12. Synthesis of Compound j12

[0247]

[0248] The synthetic route is the same as that of Example 2-1, except that p-bromoisopropylbenzene is used instead of bromobenzene in step (3). The yield is 60%. 1 H NMR(400MHz, Chlorofonm-d)δ8.62(s, 1H), 8.21(s, 1H), 7.55(d, J = 7.8Hz, 1H), 7.36(d, J = 8.0Hz, 1H), 7.25 - 7.07(m, 7H), 6.63 - 6.56(m, 2H), 4.31(d, J = 7.1Hz, 1H), 4.16(d, J = 6.6Hz, 1H), 3.98(dd, J = 11.6, 6.9Hz, 2H), 3.84(dd, J = 10.6, 4.4Hz, 1H), 3.40(dd, J = 15.7, 4.4Hz, 1H), 3.00 - 2.80(m, 2H), 2.25(s, 6H), 1.26(d, J = 6.9Hz, 6H). MS(ESI)m / z478.9[M + H] + .

[0249] Example 2-13. Synthesis of Compound j13

[0250]

[0251] The synthetic route was the same as that in Example 2-1, except that m-bromobenzotrifluoride was used instead of bromobenzene in step (3). The yield was 27%. 1 H NMR (400 MHz, Chloroform-d) δ 8.52 (s, 1H), 8.18 (s, 1H), 7.55 (m, 1H), 7.41 - 7.33 (m, 2H), 7.23 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.16 (ddd, J = 8.1, 7.1, 1.1 Hz, 1H), 7.09 (q, J = 5.1 Hz, 4H), 6.80 (t, J = 2.1 Hz, 1H), 6.74 (dd, J = 8.1, 2.4 Hz, 1H), 4.37 (d, J = 7.4 Hz, 1H), 4.18 (d, J = 6.8 Hz, 1H), 4.04 (t, J = 6.9 Hz, 2H), 3.83 (dd, J = 10.6, 4.4 Hz, 1H), 3.40 (dd, J = 15.7, 4.4 Hz, 1H), 2.95 (dd, J = 15.7, 10.6 Hz, 1H), 2.24 (s, 6H). MS (ESI) m / z 504.9 [M + H] + .

[0252] Example 2-14. Synthesis of Compound j14

[0253]

[0254] 87 mg (0.2 mmol) of Compound j1 was dissolved in 3 mL of N,N-dimethylformamide. 83 mg (0.6 mmol) of potassium carbonate and 25 μL (0.4 mmol) of methyl iodide were added. The mixture was heated and stirred at 60 °C for 6 h, cooled to room temperature, 15 mL of water was added, and the mixture was extracted three times with ethyl acetate. The organic phases were combined, dried, and concentrated in vacuo. The residue was separated by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 20 mg of Compound j14 as a pale yellow powder, with a yield of 22%. 11H NMR (400 MHz, Chloroform-d) δ 8.60 (s, 1H), 8.44 (s, 1H), 7.60 (d, J = 7.7 Hz, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.33 (m, 1H), 7.23 (t, J = 7.5 Hz, 1H), 7.16 (m, 1H), 7.12 - 7.08 (m, 3H), 6.90 (t, J = 7.4 Hz, 1H), 6.67 (d, J = 7.9 Hz, 2H), 4.28 (d, J = 7.1 Hz, 1H), 4.16 - 4.10 (m, 2H), 3.98 (d, J = 5.9 Hz, 1H), 3.83 (dd, J = 11.1, 4.9 Hz, 1H), 3.24 (dd, J = 16.6, 4.9 Hz, 1H), 3.13 (dd, J = 16.6, 11.1 Hz, 1H), 2.43 (s, 3H), 2.27 (s, 6H). MS (ESI) m / z 551.3 [M+H] + .

[0255] Example 2 - 15. Synthesis of Compound i15

[0256]

[0257] Dissolve 87 mg (0.2 mmol) of Compound j1 in 5 mL of toluene, add 72 mg (0.18 mmol) of Lawesson's reagent, heat and stir at 100 °C for 12 h, cool to room temperature, rotary evaporate the reaction solvent, and separate by silica gel column chromatography (petroleum ether∶ethyl acetate = 3∶1) to obtain 43 mg of Compound j15 as a pale yellow powder, with a yield of 58%. 1 1H NMR (400 MHz, Chloroform-d) δ 8.38 (s, 1H), 8.22 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.25 - 7.03 (m, 7H), 6.74 (m, 1H), 6.65 - 6.59 (m, 2H), 3.98 (dd, J = 11.2, 4.3 Hz, 1H), 3.63 (d, J = 13.0 Hz, 1H), 3.51 (d, J = 13.1 Hz, 1H), 3.44 (dd, J = 15.8, 4.2 Hz, 1H), 3.34 (m, 1H), 3.24 (m, 1H), 2.90 (dd, J = 15.8, 11.1 Hz, 1H), 2.27 (s, 6H). MS (ESI) m / z 470.9 [M+NH4] + .

[0258] Example 2 - 16. Synthesis of Compound j16

[0259]

[0260] The synthetic route is the same as that of Example 2-1, except that N-Boc-4-piperidone (f2) is used instead of N-Boc-3-azetidinone (f1) in step (1). The yield is 51%. 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.04 (s, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.34 - 7.28 (m, 3H), 7.22 - 7.17 (m, 1H), 7.15 - 7.08 (m, 4H), 7.03 - 6.99 (m, 2H), 6.90 (t, J = 7.3 Hz, 1H), 3.92 (dd, J = 11.4, 4.4 Hz, 1H), 3.68 - 3.56 (m, 2H), 3.50 (dd, J = 16.0, 4.5 Hz, 1H), 3.36 - 3.28 (m, 2H), 2.84 (dd, J = 15.9, 11.3 Hz, 1H), 2.51 (td, J = 13.1, 4.5 Hz, 1H), 2.28 (s, 6H), 2.13 - 2.08 (m, 2H), 1.77 (m, 1H). MS (ESI) m / z 464.9 [M+H] + .

[0261] Example 2-17. Synthesis of Compound j17

[0262]

[0263] The synthetic route is the same as that of Example 2-2, except that N-Boc-4-piperidone (f2) is used instead of N-Boc-3-azetidinone (f1) in step (1). The yield is 52%. 1 H NMR (400 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.09 (s, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.33 - 7.27 (m, 3H), 7.19 (m, 1H), 7.16 - 7.07 (m, 4H), 7.04 - 6.98 (m, 2H), 6.90 (t, J = 7.3 Hz, 1H), 3.92 (dd, J = 11.3, 4.5 Hz, 1H), 3.68 - 3.56 (m, 2H), 3.49 (dd, J = 15.9, 4.4 Hz, 1H), 3.36 - 3.27 (m, 2H), 2.83 (dd, J = 15.9, 11.3 Hz, 1H), 2.51 (td, J = 13.2, 4.6 Hz, 1H), 2.28 (s, 6H), 2.13 - 2.09 (m, 2H), 1.7 (m, 1H). MS (ESI) m / z 464.9 [M+H]+ .

[0264] Test Examples

[0265] 1. Main Experimental Materials and Instruments

[0266] Materials: Fetal bovine serum, α-MEM medium, penicillin / streptomycin were purchased from Gibco, DMSO, MTT, and TRAP staining kits were purchased from sigma, cytokines such as mM-CSF, mRANKL, hRNAK, and hRNAKL were purchased from peprotech, cell lysate was purchased from promega, PBS was purchased from WISENT, and 4-week-old C57BL / 6 mice were purchased from Slac.

[0267] Cells: Bone marrow pre-osteoclasts were obtained from the bone marrow cavity of the femurs and tibias of C57BL / 6 mice and cultured normally. During culture, complete α-MEM medium was used, that is, α-MEM + 10% fetal bovine serum + 1% penicillin / streptomycin.

[0268] Instruments: CO2 incubator from Thermo scientific, Olympus inverted microscope, microplate reader from Tecan, Biacore T200 from GE.

[0269] 2. Experimental Methods:

[0270] (1) Preparation of Test Cells:

[0271] C57BL / 6 mice were sacrificed by cervical dislocation, soaked and disinfected with 75% alcohol. Under sterile conditions, the hind limb long bones (femurs and tibias) were dissected, the attached soft tissues were removed, the inner surface of the bone marrow cavity was repeatedly rinsed with complete medium, and the cells in the bone marrow cavity were thoroughly flushed out. The cell suspension was filtered through a cell sieve, and after cell quantification, the cells were inoculated into a 10 cm cell culture dish and cultured overnight under 5% CO2 and saturated humidity conditions. The next day, the non-adherent cells in the supernatant were collected by centrifugation, and fresh complete proliferation medium (complete medium supplemented with 30 ng / mL M-CSF) was added and cultured for another two days to obtain bone marrow pre-osteoclasts. Two days later, the bone marrow pre-osteoclasts were seeded into the well plates at a certain concentration, and induction medium (complete medium supplemented with 30 ng / mL M-CSF and 50 ng / mL RANKL) was added and cultured for 5 - 6 days to obtain mature osteoclasts.

[0272] (2) Detection of the Effect of Compounds on the Survival Rate of Bone Marrow Pre-osteoclasts by CCK-8 (Cell Counting Kit-8) Method:

[0273] Bone marrow osteoclast precursor cells were seeded into a 96-well plate at a concentration of 5000 cells per well, 100 μL per well, and cultured overnight. In this experiment, an experimental group and a negative control group were set up. Experimental group: The test compound was dissolved in DMSO and then formulated into a solution with a final concentration of 10 μM using the proliferation medium, and added to the wells of the experimental group; Negative control group: The proliferation medium containing the same concentration of DMSO as the experimental group was added. After culturing for 48 h, 100 μL of CCK-8 was added to each well. After reacting at 37 °C for 1 h, the absorbance value at a wavelength of 450 nm was measured using a microplate reader, and the cell survival rate was calculated. The inhibition rate was calculated as follows:

[0274]

[0275] (3) Tartrate-resistant acid phosphatase (TRAP) staining assay was used to detect the effect of the compound on osteoclastogenesis:

[0276] Based on the results of the CCK-8 toxicity assay, compounds that were non-toxic to bone marrow osteoclast precursor cells were selected for the TRAP staining assay.

[0277] Bone marrow osteoclast precursor cells were seeded into a 96-well plate at a concentration of 5000 cells per well, 100 μL of culture medium per well, and cultured overnight. The next day, the cells were treated differently. The treatment method for the experimental group cells was: The test compound was dissolved in DMSO and then formulated into solutions with final concentrations of 1 μM and 0.1 μM using the induction medium and added to the wells of the experimental group. In the negative control group, the induction medium containing the same concentration of DMSO as the experimental group was added, and in the blank control group, the proliferation medium containing the same concentration of DMSO was added. The cells after drug stimulation were pre-incubated in an incubator with 5% CO2 at 37 °C for 4 days, and the medium was changed every 2 days (the old medium was aspirated and 100 μL of new medium containing the same concentration of drug was supplemented). After 4 days, osteoclasts formed vesicles, which were washed 3 times with PBS, fixed with 4% paraformaldehyde for 10 minutes, then washed 1 time with PBS and air-dried. In a TRAP staining solution containing 0.8 mol·L -1 sodium acetate buffer at pH 5.0, 0.1 g L -1 naphthol AS-BI phosphate, 0.6 g L -1 fast garnet GBC salt, 0.2 mol·L -1 tartaric acid solution, react for 1 hour, dehydrate through an ethanol gradient, observe and photograph using an Olympus microscope, and count osteoclast-like cells with ≥3 nuclei. The inhibition rate was calculated as follows:

[0278]

[0279] (4) SPR (Surface Plasmon Resonance) experiment was used to detect the affinity of the compound for RANKL protein:

[0280] Using the SPR technology based on Biacore T200, the newly prepared hRANKL protein (human RANKL protein, purity above 95%) was coupled to the CM5 chip by the standard amino coupling method. After verifying the ligand-binding activity of hRANKL coupled to the chip through hRANK, different concentrations of the compound to be tested were then flowed through the chip surface, and the binding of the compound to the hRANKL protein on the chip surface was measured by the change in SPR signal (expressed in response units RU: 1RU = 1 pg protein / mm 2 = 1×10 -6 RIU). The measured data was fitted by a computer to obtain the equilibrium dissociation constant (K d value).

[0281] 3. Results:

[0282] (1) Study on the survival effect of the tetrahydro-β-carboline compounds of the present invention on bone marrow pre-osteoclasts

[0283] Select the tetrahydro-β-carboline compounds of the present invention at 10 μM and co-incubate them with bone marrow pre-osteoclasts, and detect the cell survival rate by CCK-8. The results are shown in Table 1.

[0284] Table 1. Effect of tetrahydro-β-carboline compounds on the survival of bone marrow pre-osteoclasts

[0285]

[0286] (2) Study on the inhibitory effect of some tetrahydro-β-carboline compounds of the present invention on osteoclasts

[0287] Select 1 μM and 0.1 μM of some tetrahydro-β-carboline compounds of the present invention for experiments, and induce osteoclast differentiation of bone marrow pre-osteoclasts through M-CSF and RANKL. Detect the effect of tetrahydro-β-carboline compounds on the osteoclast differentiation process by TRAP staining experiment. The results are shown in Table 2.

[0288] The results show that: some tetrahydro-β-carboline compounds of the present invention, such as e13″, e14″, e17″, j1, have a good inhibitory effect on the osteoclast differentiation process of bone marrow (inhibition rate > 50% at the 0.1 μM level).

[0289] Table 2. Inhibitory effect of some tetrahydro-β-carboline compounds on the differentiation of bone marrow osteoclasts

[0290]

[0291]

[0292] (3) Affinity study of the tetrahydro-β-carboline compound j1 of the present invention for RANKL protein

[0293] Compound j1 with good osteoclast inhibitory activity was selected and flowed through the chip surface at concentration gradients of 0.781, 1.562, 3.125, 6.25, and 12.5 μM for SPR experiments, and its dissociation curve and dissociation equilibrium constant (K d ) were measured. The results are as Figure 1 shown.

[0294] The results showed that the dissociation equilibrium constant K d of compound j1 with RANKL protein was 3.984 μM, indicating that j1 had a high affinity for RANKL protein.

Claims

1. A tetrahydro-β-carboline compound represented by formula P, or its optical isomers, pharmaceutically acceptable salts and hydrates, wherein: "---" represents the presence or absence of a chemical bond; R 1 represents 1 to 4 substituents independently selected from hydrogen, halogen, C1-C4 alkyl, C1-C6 alkoxy, hydroxy C1-C6 alkyl, and C1-C6 alkylamino; R 2 selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C1-C6 alkanoyl, C3-C6 cycloalkyl C1-C3 alkanoyl; n1 and n2 are each independently or simultaneously 1 or 2; X is carbon or nitrogen; Y is oxygen or sulfur; Ar 1 and Ar 2 each independently selected from substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted 5- or 6-membered heteroaryl; The substitution in the above "substituted or unsubstituted" means being substituted by one or more substituents selected from the following: halogen (such as fluorine), hydroxyl, amino, cyano, C1-C6 alkyl (preferably C1-C3 alkyl, such as methyl, ethyl, isopropyl), C1-C6 haloalkyl (preferably C1-C3 haloalkyl, such as C1-C3 fluoroalkyl, such as trifluoromethyl), C3-C6 cycloalkyl (such as cyclopropyl), C1-C6 alkoxy (preferably C1-C3 alkoxy, such as methoxy), C1-C6 alkyl-NH- (preferably C1-C3 alkyl-NH-), (C1-C6 alkyl)(C1-C6 alkyl)-N- (preferably (C1-C3 alkyl)(C1-C3 alkyl)-N-, such as dimethylamino), C1-C6 alkoxycarbonyl (preferably C1-C3 alkoxycarbonyl), R 3 -C(=O)-O-, R 3 -OC(=O)-, R 3 -S(=O)2-, R 3 -S(=O)2NH-, (R 3 O)2-P(=O)-, where each R 3 is independently selected from hydrogen, C1-C6 alkyl (preferably C1-C3 alkyl, such as methyl).

2. The tetrahydro-β-carboline compound represented by formula P according to claim 1, or its optical isomer, pharmaceutically acceptable salt and hydrate, characterized in that, In the tetrahydro-β-carboline compound represented by formula P: "---" represents the presence or absence of a chemical bond; R 1 represents 1 to 4 substituents independently selected from hydrogen, a fluorine atom, and a methoxy group; R 2 selected from hydrogen, C1-C3 alkyl, C3-C6 cycloalkyl, C1-C3 alkanoyl, C3-C6 cycloalkylcarbonyl; n1 and n2 are each independently or simultaneously 1 or 2; X is carbon or nitrogen; Y is oxygen or sulfur; Ar 1 and Ar 2 each independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted indenylalkyl; the substitution in the "substituted or unsubstituted" is as defined in claim 1.

3. The tetrahydro-β-carboline compound represented by formula P according to claim 1 or 2, or its optical isomer, pharmaceutically acceptable salt and hydrate, characterized in that, In the tetrahydro-β-carboline compound represented by formula P: "---" represents the presence or absence of a chemical bond; R 1 is a hydrogen atom; R 2 selected from hydrogen, methyl, acetyl, cyclopropylcarbonyl, more preferably hydrogen or methyl; n1 and n2 are each independently or simultaneously 1 or 2; X is carbon or nitrogen; Y is oxygen or sulfur; Ar 1 and Ar 2 each independently selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted indenylalkyl; the substitution in the "substituted or unsubstituted" is as defined in claim 1.

4. The tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-3, or its optical isomer, pharmaceutically acceptable salt and hydrate, characterized in that, The tetrahydro-β-carboline compound represented by formula P is represented by the following formula E or J: Among them, "---", R 2 , n1, n2, Y, Ar 1 and Ar 2 are defined as described in claims 1-3 respectively; Preferably, the tetrahydro-β-carboline compound represented by formula P is represented by the following formula E1 or J1: Among them, "---", R 2 , n1, n2, Ar 1 and Ar 2 are defined as described in claims 1-3 respectively.

5. The tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-4, or its optical isomer, pharmaceutically acceptable salt and hydrate, characterized in that, The tetrahydro-β-carboline compound represented by formula P is selected from the compounds represented by the following structures: Particularly, the tetrahydro-β-carboline compound represented by formula P is selected from the compounds represented by the following structures:

6. A method for preparing the tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-5, characterized in that, The tetrahydro-β-carboline compound represented by formula P is prepared by the following steps, and the reaction route is as follows: wherein, Ar 1 and Ar 2 , and the definitions of n1 and n2 are as described in claims 1-4 respectively. Step 1: N-Boc-tryptophan a condenses with arylamine Ar 1 -NH2 under the action of a condensing agent to form N-Boc-tryptophanyl arylamine b; Step 2: N-Boc-tryptophan amide b is deprotected by Boc under the action of trifluoroacetic acid to obtain tryptophan amide c; Step 3: Tryptophan amide c reacts with ketone d or f through an acid-catalyzed Pictet-Spengler reaction to obtain the corresponding tetrahydro-β-carboline derivatives e or g; Step 4: The tetrahydro-β-carboline derivative g is deprotected by Boc to obtain intermediate h; Step 5: Intermediate h reacts with aryl bromide Ar 2 -Br to obtain derivative i through Buchwald-Hartwig coupling reaction; Preferably, in step 1: The N-Boc-tryptophan a is N-Boc-L-tryptophan or N-Boc-D-tryptophan, and the condensing agent is HATU or EDCI·HCl; When the condensing agent is HATU, the reaction system further includes a base-binding agent N,N-diisopropylethylamine; the reaction is carried out in a solvent, and the solvent is N,N-dimethylformamide, the reaction temperature is 60-80 °C, and the reaction time is 6-20 h; When the condensing agent is EDCI·HCl, the reaction system further includes an acylation catalyst HOBt; the reaction system further includes a base-binding agent triethylamine; the reaction is carried out in a solvent, and the solvent is tetrahydrofuran, the reaction temperature is 20-30 °C, and the reaction time is 6-20 h; Preferably, in step 2: The acid is trifluoroacetic acid; the reaction is carried out in a solvent, and the solvent is dichloromethane; the reaction temperature is 20-30 °C, and the reaction time is 6-18 h; Preferably, in step 3: The acid is trifluoroacetic acid; the reaction is carried out in a solvent, and the solvent is acetonitrile; the reaction temperature is 20-30 °C, and the reaction time is 12-20 h; Preferably, in step 4: The tetrahydro-β-carboline derivative g is deprotected by Boc under the action of trifluoroacetic acid to obtain intermediate h; the reaction is carried out in a solvent, and the solvent is dichloromethane; the reaction temperature is 20-30 °C, and the reaction time is 6-18 h; Preferably, in step 5: The coupling reaction is carried out under the catalysis of Pd2(dba)3, RuPhos, and cesium carbonate; the reaction is carried out in a solvent, and the solvent is 1,4-dioxane; the coupling reaction is refluxed for 6-18 h.

7. A pharmaceutical composition comprising the tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-5, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof, and optionally, a pharmaceutically acceptable excipient.

8. A RANKL inhibitor comprising the tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-5, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof.

9. Use of the tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-5, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for preventing and / or treating diseases caused by abnormal osteoclast activity; Preferably, the diseases caused by abnormal osteoclast activity include osteoporosis and osteoarthritis.

10. Use of the tetrahydro-β-carboline compound represented by formula P according to any one of claims 1-5, or an optical isomer, pharmaceutically acceptable salt and hydrate thereof, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for inhibiting the differentiation of bone marrow osteoclast precursor cells into bone marrow osteoclasts.

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