Application of benzimidazolyl isoxazole compounds in the preparation of drugs related to multiple myeloma

Abstract

The present invention relates to the application of a class of benzimidazolyl isoxazole compounds in the preparation of drugs related to multiple myeloma, belonging to the fields of medicinal chemistry and pharmacotherapy, and the compounds show good RPMI-8226 cell inhibitory activity. In addition, compound EP12 induces cell apoptosis and inhibits the expression of c-Myc mRNA and c-Myc protein, and circular dichroism spectroscopy experimental studies show that compound EP12 can stabilize c-Myc G4. The compound shown in formula I provided by the present invention, the isomer or a pharmaceutically acceptable salt thereof can be used in the preparation of drugs related to multiple myeloma.

Description

Technical Field

[0001] The present invention relates to the fields of pharmaceutical chemistry and pharmacotherapy, and in particular to a benzimidazolyl isoxazole compound and application of the compound in preparing drugs related to multiple myeloma, in particular to drugs related to c-Myc G-quadruplex (c-Myc G4) stabilizers. Background Art

[0002] Multiple myeloma (MM) is a common plasma cell malignancy in the blood system, which is common in middle-aged and elderly people and accounts for about 13% of blood system tumors. In recent years, with the continuous maturity of hematopoietic stem cell transplantation technology and the advent of first-line therapeutic drugs such as immunomodulators and proteasome inhibitors, the survival of patients with multiple myeloma has been continuously extended, but it is still not completely cured. In the clonal evolution of MM, one of the key molecular events is the activation of the c-My c gene; in addition, inhibiting the activity of c-Myc protein will reduce the occurrence of multiple myeloma. Therefore, the rational design of c-Myc targeted drugs is expected to effectively and safely treat multiple myeloma and its related diseases.

[0003] c-Myc protein belongs to the basic helix-loop-helix leucine zipper (b-HLH-LZ) DNA binding protein superfamily. The half-life of c-Myc protein is only 20-30 minutes, and it lacks a clear molecular binding site. Therefore, it is difficult to design drugs that directly target c-Myc protein. c-Myc protein usually forms a heterodimer with its molecular partner Max to play a transcriptional regulatory role. Compound 25 reported in our previous patent (Application of pyrrolidine-2-one compounds in the preparation of drugs related to multiple myeloma, application number: 202011215542.2) is an inhibitor targeting c-Myc / Max heterodimers, which can inhibit the expression of c-Myc protein and show a good inhibitory effect on multiple myeloma. Bortezomib, as a proteasome inhibitor, is a first-line drug for the treatment of multiple myeloma, but it has drug resistance and many adverse reactions.

[0004] In addition to directly inhibiting c-Myc protein or its post-translational regulation, another strategy is to regulate the expression of c-Myc protein from upstream. The transcriptional regulation of c-Myc protein expression is complex, involving multiple promoters and transcription start sites. A large number of studies have shown that the nuclease hypersensitive element III1 (NHE III1) located near the P1 promoter controls 80-95% of the transcriptional activity of the c-Myc gene. NHE III1 is a single-stranded DNA sequence composed of guanine (G) bases. Under normal physiological conditions, there are transcriptionally active double-stranded forms, transient single-stranded DNA, and silent G-quadruplex (G4) forms, and the three are in a dynamic balance to exert physiological effects. When the NHE III1 sequence is in a single-stranded state, the transcription activator CNB P and hnRNP K bind to the single-stranded DNA, which will promote c-Myc transcription; when the sequence is K + Or Na + When stable, the formation of G4 will hinder the binding of CNBP and hnRNP K to NHEIII1, and the transcription of c-Myc gene will be inhibited. If small molecule compounds can be designed to target and stabilize c-MycG4, the expression of c-Myc protein can be inhibited from upstream.

[0005] At present, many types of c-Myc G4 stabilizers have been reported, including natural products such as flavonoids, ellipticine, berberine and berberine and their derivatives. However, the affinity and specificity of these natural products themselves are much lower than their structurally modified derivatives. The c-Myc G4 stabilizers obtained by chemical synthesis are currently a hot topic of research. Some small molecule compounds such as BMVC, IZCZ-3 and QN-1 are good c-Myc G4 stabilizers. The structural characteristics of these compounds are conjugated planar regions with multiple aromatic rings, which can also significantly reduce tumor-related symptoms at the cellular or animal level. However, most compounds are still in the early development stage, and their stabilizing activity needs to be further improved. Therefore, it is urgent to develop more novel and highly active c-Myc G4 stabilizers. Summary of the invention

[0006] The purpose of the present invention is to structurally modify compound 25 (patent number 202011215542.2) in the previous patent in combination with the aromatic conjugated structural characteristics of the stabilizer of c-Myc G4, and provide a class of benzimidazolyl isoxazole compounds, as well as the use of the compound, isomer or pharmaceutically acceptable salt thereof in the preparation of drugs related to multiple myeloma. In order to increase its aromatic conjugation, we changed the pyrrolidine-2-one ring into an isoxazole ring and designed and synthesized a series of benzimidazolyl isoxazole compounds. This type of compound also has the ability to inhibit the cell activity of multiple myeloma, but its way of inhibiting the expression of c-Myc protein is different from that of compound 25. Benzimidazolyl isoxazole compounds can significantly inhibit the expression of c-Myc mRNA, but compound 25 cannot inhibit the expression of c-Myc mRNA. In addition, circular dichroism (CD) spectroscopy experiments have shown that compound EP12 can stabilize c-Myc G4. Therefore, benzimidazolyl isoxazole compounds are expected to be developed as stabilizers targeting the G-quadruplex in the promoter region of the c-Myc gene to better treat multiple myeloma.

[0007] The benzimidazolyl isoxazole compound provided by the present invention has good c-Myc G4 stabilizing activity and can be used for preparing drugs related to multiple myeloma.

[0008] Another object of the present invention is to provide a pharmaceutical composition for treating multiple myeloma, which comprises the above-mentioned compound, isomer or pharmaceutically acceptable salt thereof as the active ingredient or main active ingredient, and is supplemented with a pharmaceutically acceptable carrier.

[0009] The technical solution of the present invention is as follows:

[0010] The compound, isomer or pharmaceutically acceptable salt thereof represented by formula I, and the use of the compound, isomer or pharmaceutically acceptable salt thereof in the preparation of drugs related to multiple myeloma,

[0011]

[0012] in,

[0013] R1 represents hydrogen, hydroxy, nitro, carboxyl, cyano, amino, halogen, C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkoxycarbonyl;

[0014] m represents an integer from 1 to 3;

[0015] R2 represents hydrogen, hydroxy, nitro, carboxyl, cyano, amino, halogen, C1-C4 alkyl, C1-C4 alkoxy or C1-C4 alkoxycarbonyl;

[0016] n represents an integer from 1 to 3.

[0017] In a preferred embodiment, R1 represents fluorine, chlorine, methyl, ethyl, methoxy, ethoxy, methoxycarbonyl or ethoxycarbonyl.

[0018] In a more preferred embodiment, R1 represents fluorine or methoxy.

[0019] m represents 1 or 2; preferably, m represents 1.

[0020] In a preferred embodiment, R2 represents hydrogen, nitro, fluorine, chlorine, methyl, ethyl, methoxy, ethoxy, methoxycarbonyl or ethoxycarbonyl.

[0021] In a more preferred embodiment, R2 represents hydrogen, nitro, fluorine, methyl, ethyl, methoxy, ethoxy or methoxycarbonyl.

[0022] Furthermore, n represents 1 or 2.

[0023] Further, among the compounds, isomers, or pharmaceutically acceptable salts thereof represented by Formula I, the compounds are selected from the following compounds:

[0024]

[0025]

[0026] The compounds provided by the present invention are named as follows:

[0027] EP1 compound name: 5-(4-methoxyphenyl)-3-(1-(2-phenoxyethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0028] EP2 compound name: 3-(1-(2-(3,5-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0029] EP3 compound name: 3-(1-(2-(4-ethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0030] EP4 compound name: 3-(1-(2-(2,4-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0031] EP5 compound naming: 5-(4-methoxyphenyl)-3-(1-(2-(o-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0032] EP6 compound naming: 5-(4-methoxyphenyl)-3-(1-(2-(4-nitrophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0033] EP7 compound name: 3-(1-(2-(2-ethoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0034] EP8 compound naming: 3-(1-(2-(4-methoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0035] EP9 compound naming: methyl 4-(2-(2-(5-(4-methoxyphenyl)isoxazol-3-yl)-1H-benzo[d]imidazol-1-yl)ethoxy)benzoate;

[0036] EP10 compound name: 3-(1-(2-(4-fluorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole;

[0037] EP11 compound naming: 5-(4-fluorophenyl)-3-(1-(2-phenoxyethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0038] EP12 compound naming: 3-(1-(2-(3,5-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole;

[0039] EP13 compound naming: 3-(1-(2-(4-ethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole;

[0040] EP14 compound naming: 5-(4-fluorophenyl)-3-(1-(2-(o-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0041] EP15 compound name: 5-(4-fluorophenyl)-3-(1-(2-(4-nitrophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0042] EP16 compound name: 3-(1-(2-(2-ethoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole;

[0043] EP17 compound naming: 5-(4-fluorophenyl)-3-(1-(2-(4-methoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole;

[0044] EP18 compound naming: methyl 4-(2-(2-(5-(4-fluorophenyl)isoxazol-3-yl)-1H-benzo[d]imidazol-1-yl)ethoxy)benzoate;

[0045] EP19 compound name: 3-(1-(2-(4-fluorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole.

[0046] Specifically, the compound of formula I is further preferably selected from the following compounds:

[0047]

[0048] More specifically, the compound described in general formula I is further more preferably selected from the following compounds:

[0049]

[0050] The present invention discloses the synthesis of compounds EP1-EP19, and the synthesis route of the compounds represented by general formula I is as follows:

[0051]

[0052] Taking the preparation method of compound EP11 as an example, the specific preparation method of the synthetic route of the compound includes the following steps:

[0053] 1-(4-fluorophenyl)ethan-1-one (1) and diethyl oxalate (2) are subjected to Claisen condensation reaction to obtain 4-(4-fluorophenyl)-2-hydroxy-4-oxobut-2-enoic acid ethyl ester (3), and compound (3) is reacted with hydroxylamine hydrochloride in an ethanol solvent for 4 hours to obtain 5-(4-fluorophenyl)isoxazole-3-carboxylic acid ethyl ester (4). After hydrolysis of compound (4), the corresponding 5-(4-fluorophenyl)isoxazole-3-carboxylic acid (5) is first amidated with o-phenylenediamine (6) at room temperature, and then subjected to heating dehydration cyclization reaction to obtain 3-(1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (7). Finally, the intermediate (7) is subjected to nucleophilic substitution reaction with (2-bromoethoxy)benzene (8) to generate the target compound EP11 shown in general formula I. The specific synthetic route is as follows:

[0054]

[0055] The present invention provides use of the above-mentioned compound, isomer or pharmaceutically acceptable salt thereof in the preparation of drugs related to multiple myeloma.

[0056] In a preferred embodiment, the present invention provides a pharmaceutical composition for treating multiple myeloma, which comprises the compound, isomer or pharmaceutically acceptable salt thereof of the present invention as an active ingredient or a main active ingredient, and is supplemented with a pharmaceutically acceptable carrier. Further, the pharmaceutical composition can be prepared into a liquid preparation or a solid preparation. Further, the pharmaceutical composition can be prepared into an injection, an oral liquid, a granule, a tablet, a powder or a capsule.

[0057] Unless otherwise stated, the following terms used in the specification and claims have the meanings discussed below:

[0058] "Alkyl" means a saturated aliphatic hydrocarbon group of 1-20 carbon atoms, including straight and branched groups (the numerical range mentioned in this application, such as "1-20", means that the group, in this case an alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms). Alkyl groups containing 1-4 carbon atoms are called lower alkyl groups. When the lower alkyl group has no substituent, it is called an unsubstituted lower alkyl group. More preferably, the alkyl group is a medium-sized alkyl group with 1-6 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl, pentyl, etc. Preferably, the alkyl group is a lower alkyl group with 1-4 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl or tert-butyl, etc. The alkyl group may be substituted or unsubstituted. When it is a substituted alkyl group, the substituent is preferably one or more, more preferably 1-3, and most preferably 1 or 2 substituents.

[0059] "Haloalkyl" means halogen-substituted alkyl, preferably halogen-substituted lower alkyl as defined above, which is substituted by one or more halogen atoms which may be the same or different, for example -CH2Cl, -CF3, -CH2CF3, -CH2CCl3, etc.

[0060] "Halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0061] "Hydroxy" means an -OH group.

[0062] "Cyano" means a -CN group.

[0063] "Nitro" means a -NO2 group.

[0064] "Amino" means a -NH2 group.

[0065] "Carboxy" means a -COOH group.

[0066] "Alkoxy" refers to -O-(unsubstituted alkyl) and -O-(unsubstituted cycloalkyl). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.

[0067] "Hydroxyalkyl" means a hydroxy-substituted alkyl group, preferably a hydroxy-substituted lower alkyl group as defined above, which is substituted by one or more identical or different hydroxy groups, for example -CH2CH2-OH, -CH2CH2CH2-OH, and the like.

[0068] "Aminoalkyl" means an amino-substituted alkyl group, preferably an amino-substituted lower alkyl group as defined above, which is substituted by one or more identical or different amino groups, for example -CH2NH2, -CH2NH2, -CH2CH2NH2, etc.

[0069] The "alkylalkoxy group" refers to a group in which at least one hydrogen on an alkyl group is replaced by an alkoxy group, for example, 2-methylaminoethyl, 2-ethoxyethyl, 3-methoxypropyl and the like.

[0070] "C1-C4alkoxycarbonyl" means a RO-CO-R' group, wherein R or R' is selected from the following groups: hydrogen, unsubstituted lower alkyl, trihalomethyl, unsubstituted cycloalkyl or phenyl, optionally substituted by one or more, preferably by 1, 2 or 3, selected from unsubstituted lower alkyl, trihalomethyl, unsubstituted lower alkoxy.

[0071] "C1-C4 alkoxycarbonyl" means an R-CO group, wherein R represents methoxy, ethoxy or propoxy or the like.

[0072] "Pharmaceutically acceptable salts" refers to those salts that retain the biological effectiveness and properties of the parent compound. Such salts include:

[0073] (1) Acid salts are obtained by reacting the free base of the parent compound with an inorganic acid or an organic acid. The inorganic acid includes hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, metaphosphoric acid, sulfuric acid, sulfurous acid and perchloric acid. The organic acid includes acetic acid, trifluoroacetic acid, propionic acid, acrylic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, maleic acid, benzoic acid, hydroxybenzoic acid, γ-hydroxybutyric acid, methoxybenzoic acid, phthalic acid, methanesulfonic acid, ethanesulfonic acid, naphthalene-1-sulfonic acid, naphthalene-2-sulfonic acid, p-toluenesulfonic acid, salicylic acid, tartaric acid, citric acid, lactic acid, cinnamic acid, dodecyl sulfuric acid, gluconic acid, glutamic acid, aspartic acid, stearic acid, mandelic acid, succinic acid or malonic acid.

[0074] (2) Salts formed by the replacement of acidic protons in the parent compound by metal ions or coordination with organic bases, such as alkali metal ions, alkaline earth metal ions or aluminum ions, and organic bases such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, quinine, etc.

[0075] "Pharmaceutical composition" refers to a mixture of one or more of the compounds of the present invention or their pharmaceutically acceptable salts, solvates, hydrates or prodrugs with other chemical components, such as pharmaceutically acceptable carriers. The purpose of a pharmaceutical composition is to facilitate the process of administration to an animal.

[0076] Adopt the technical scheme of the present invention, the advantages are as follows:

[0077] The present invention provides a class of benzimidazolyl isoxazole compounds for use in preparing drugs related to multiple myeloma. The compounds show good RPMI-8226 cell inhibitory activity and can be used to prepare drugs related to multiple myeloma. In addition, compound EP12 induces cell apoptosis and inhibits the expression of c-Myc mRNA and c-Myc protein. Circular dichroism spectroscopy experimental studies show that compound EP12 can stabilize c-Myc G4. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 (a) is the Q-PCR detection of c-Myc mRNA expression in myeloma cells treated with different active compounds (EP8, EP9, EP12, EP18); Figure 1 (b) shows the effects of compounds (EP8, EP9, EP12 and EP18) on c-Myc expression detected by Western blotting; error bars: mean ± standard error of the mean from at least three independent experiments; **P<0.05.

[0079] Figure 2 This is a flow cytometry experiment to analyze the effect of EP12 on the apoptosis rate of RPMI-8226 cells; all cells were RPMI-8226 cells treated with 5 μM compound for 48 h; error bars: mean ± standard error of the mean from at least three independent experiments; **P<0.01.

[0080] Figure 3 The middle left figure shows the room temperature CD spectra of the studied c-Myc G4 in the presence and absence of EP12; Figure 3 The middle right figure shows the CD melting curve of c-Myc G4 in the presence or absence of EP12. DETAILED DESCRIPTION

[0081] In order to further illustrate the present invention, a series of examples are given below. These examples are purely illustrative and are only used to specifically describe the present invention, and should not be understood as limiting the present invention.

[0082] Example 1

[0083] 5-(4-methoxyphenyl)-3-(1-(2-phenoxyethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP1)

[0084] At 0°C, 7.19 g (133.18 mmol, 2 e.q) of sodium methoxide was added to a 500 ml round-bottom flask, and 300 ml of anhydrous ethanol was added and stirred until dissolved, followed by the addition of 1-(4-methoxyphenyl)ethan-1-one (10 g, 66.59 mmol, 1 e.q), and continued stirring for 30 min. Then, diethyl oxalate (6.125 ml, 45.5 mmol, 1.1 eq) was added to the mixture. After stirring at room temperature for 6-8 hours, 6N dilute hydrochloric acid solution was added to the solution, and the solution was added dropwise while stirring until the pH was adjusted to 5. The residue was poured into excess ice water, and more solids were precipitated, which were filtered and dried in a vacuum oven to obtain yellow solid 2-hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-enoic acid ethyl ester (15.04 g, 90.2%).

[0085] 2-Hydroxy-4-(4-methoxyphenyl)-4-oxobut-2-enoic acid ethyl ester (16.165 g, 64.60 mmol, 1 e.q) was dissolved in an appropriate amount of anhydrous ethanol, hydroxylamine hydrochloride (5.39 g, 77.51 mmol, 1.1 eq) was added, and heated to reflux for 6 h. The reaction mixture was cooled to room temperature, washed with a large amount of ice water, filtered and dried to obtain a yellow crude product, which was purified by column chromatography (PE / EA=10:1 (V / V)) to obtain 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid ethyl ester (13.45 g, 84.2%) as a white solid.

[0086] Weigh 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid ethyl ester (13.45 g, 54.4 mmol) and dissolve it in 100 ml of anhydrous ethanol, then add 5% NaOH (100 ml) solution. After reflux for 3 hours, cool to room temperature. Slowly add 1N HCl aqueous solution to adjust the pH value of the solution to 3. Filter the resulting precipitate and dry it under vacuum to obtain the intermediate white solid 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid (11.4 g, 95.6%).

[0087] 5-(4-methoxyphenyl)isoxazole-3-carboxylic acid (462 mg, 2.12 mmol, 1 e.q) was dissolved in DMF (2 ml) solution and DIEPA (418 μL, 2.53 mmol, 1.2 eq) and HATU (962 mg, 2.53 mmol, 1.2 eq) were added in sequence. After stirring for 30 minutes, benzene-1,2-diamine (251 mg, 2.32 mmol, 1.1 eq) was added and stirred at room temperature for 3 hours. Ice water was added to terminate the reaction, and the reaction solution was then extracted with ethyl acetate. The organic layer was washed with 1N hydrochloric acid, saturated NaHCO3 aqueous solution and distilled water, and dried over Na2SO4. After vacuum concentration, the residue was directly dissolved by adding an appropriate amount of acetic acid and refluxed overnight. After cooling to room temperature, the solution was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (PE / EA=1:2 (V / V)) to give 3-(1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (240 mg, 38.9%) as a white solid.

[0088] 3-(1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (200 mg, 0.69 mmol, 1 e.q) was dissolved in DMF solution and potassium carbonate (250 mg, 1.8 mmol, 2.61 eq) was added and stirred for 30 minutes. Then (2-bromoethoxy)benzene (277 mg, 1.38 mmol, 2 e.q) was added and stirring was continued at 90°C for 2 hours. The reaction was then quenched with distilled water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, and evaporated to provide a crude product, which was purified by column chromatography (PE / EA=2:1 (V / V)) to give a yellow solid (125 mg, 44.2%).

[0089] 1 H NMR (400MHz, DMSO-d6): δ7.94-7.92(m,2H,2×Ar-H),7.80-7.77(m,1H,Ar-H),7.7 4-7.72(m,1H,Ar-H),δ7.58(s,H,Ar-H),δ7.39-7.35(m,H,Ar-H),δ7.31-7.27(m,H ,Ar-H),7.18-7.08(m,4H,4×Ar-H),δ6.85-6.81(m,H,Ar-H),δ6.76-6.73(m,2H,2 ×Ar-H),5.06(t,J=5.2Hz,2H,CH2),4.37(t,J=5.3Hz,2H,CH2),3.81(s,3H,OCH3); 13C NMR(100MHz,DMSO-d6)δppm:170.11,161.73,158.40,157.34,143.08,142.93,136.95,130.00(2C),128.19(2C),124.47,1 23.28,121.36,120.20,119.46,115.33(2C),114.78(2C),112.33,100.32,67.03,55.98,44.91; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 25 H 21 N3O3,412.1656,found 412.1656,found 412.1629.Purity:95.1%.

[0090] Example 2

[0091] 3-(1-(2-(3,5-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP2)

[0092] A white powder was obtained in a similar manner as in Example 1, 42.4%.

[0093] 1 H NMR (400MHz, DMSO-d6): δ7.94-7.91(m,2H,2×Ar-H), δ7.78-7.72(m,2H,2×Ar-H ),δ7.55(s,1H,Ar-H),δ7.40-7.35(m,1H,Ar-H),δ7.31-7.27(m,1H,Ar-H),7.11 -7.08(m,2H,2×Ar-H),6.45(s,1H,Ar-H),6.34(s,2H,2×Ar-H),5.03(t,J=6.2H z,2H,CH2),4.32(t,J=6.6Hz,2H,CH2),3.81(br,3H,OCH3),2.08(s,6H,2×CH3); 13C NMR(100MHz,DMSO-d6)δppm:170.08,161.72,158.41,157.32,143.08,142.96,139.11(2C),136.91,128.15(2C),124.46,123.26 ,122.94,120.20,119.47,115.30(2C),112.49(2C),112.27,100.32,66.83,55.96,44.91,21.45(2C); ESI-HRMS(TOF):m / z[M+H] + Calculate for C 27 H 25 N3O3,440.1969,found 440.1943.Purity:98.1%.

[0094] Example 3

[0095] 3-(1-(2-(4-ethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP3)

[0096] A similar method to Example 1 was used to obtain a pale yellow solid, 36.5%.

[0097] 1 H NMR (400MHz, DMSO-d6): δ7.94-7.91(m,2H,2×Ar-H),7.78-7.72(m,2H,2×Ar-H),7.56(s,1H ,Ar-H),δ7.39-7.35(m,1H,Ar-H),δ7.31-7.26(m,1H,Ar-H),7.11-7.07(m,2H,2×Ar-H),6. 98-6.95(m,2H,2×Ar-H),6.66-6.63(m,2H,2×Ar-H),5.04(t,J=5.2Hz,2H,CH2),4.33(t,J= 5.4Hz,2H,CH2),3.81(br,3H,OCH3),2.41(q,J=7.5Hz,2H,CH2),1.03(t,J=7.6Hz,3H,CH3); 13C NMR(100MHz,DMSO-d6)δppm:170.08,161.71,157.32,156.48,143.08,142.94,136.93,136.57,129.12(2C),128.16(2C),124.45, 123.26,120.20,119.46,115.29(2C),114.63(2C),112.31,100.31,67.10,55.96,44.92,27.75,16.39; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 27 H 25 N3O3,440.1969,found 440.1941.Purity:95.0%.

[0098] Example 4

[0099] 3-(1-(2-(2,4-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP4)

[0100] A white solid was obtained in a similar manner to Example 1, 45.3%.

[0101] 1 H NMR (400MHz, DMSO-d6): δ7.94-7.91(m,2H,2×Ar-H),7.79-7.77(m,1H,Ar-H),7.75-7.72(m ,1H,Ar-H),δ7.57(s,1H,Ar-H),δ7.37-7.33(m,1H,Ar-H),δ7.30-7.26(m,1H,Ar-H),7.11-7 .08(m,2H,2×Ar-H),6.82-6.79(m,1H,Ar-H),6.74-6.69(m,2H,2×Ar-H),5.12(t,J=5.1Hz,2 H,CH2),4.31(t,J=4.9Hz,2H,CH2),3.81(br,3H,OCH3),2.06(s,3H,CH3),1.65(s,3H,CH3); 13C NMR(100MHz,DMSO-d6)δppm:170.02,161.73,157.39,154.47,143.13,142.96,136.91,131.62,129.55,128.17(2C),127.50,125.73, 124.35,123.25,120.20,119.49,115.33(2C),112.37,111.37,100.34,67.06,55.99,44.94,20.50,16.19; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 27 H 25 N3O3,440.1969,found 440.1938.Purity:99.0%.

[0102] Example 5

[0103] 5-(4-methoxyphenyl)-3-(1-(2-(o-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP5)

[0104] A white solid was obtained in a similar manner to Example 1, 50.4%.

[0105] 1 H NMR (400MHz, DMSO-d6): δ7.96-7.92(m,2H,2×Ar-H),7.79(d,J=8.1Hz,1H,Ar-H),7.74(d,J=8.0Hz, 1H,Ar-H),δ7.58(s,H,Ar-H),δ7.38-7.34(m,H,Ar-H),δ7.30-7.27(m,H,Ar-H),7.11-7.09(m,2H,2× Ar-H),δ7.04-7.00(m,H,Ar-H),δ6.94-6.93(m,H,Ar-H),δ6.83-6.81(m,H,Ar-H),δ6.73-6.69(m,H ,Ar-H),5.15(t,J=5.1Hz,2H,CH2),4.36(t,J=5.1Hz,2H,CH2),3.81(s,3H,OCH3),1.96(s,3H,CH3); 13C NMR (100MHz, DMSO-d6) δppm: 170.02, 161.73, 157.42, 156.55, 143.12, 142.94, 136.91, 130.89, 128.18 (2C), 127.45, 125.96, 124. 37,123.28,121.01,120.20,119.47,115.33(2C),112.39,111.41,100.37,66.99,55.98,44.92,16.27; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 26 H 23 N3O3,426.1812,found 426.1785.Purity:95.4%.

[0106] Example 6

[0107] 5-(4-methoxyphenyl)-3-(1-(2-(4-nitrophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP6)

[0108] A yellow solid was obtained in a similar manner to Example 1, 49.5%.

[0109] 1 H NMR (400MHz, DMSO-d6): δ8.08-8.04(m,2H,2×Ar-H),7.92-7.88(m,2H,2×Ar-H),7.80 (d,J=8.2Hz,1H,Ar-H),7.72(d,J=8.0Hz,1H,Ar-H),δ7.56(s,1H,Ar-H),δ7.40-7.36 (m,1H,Ar-H),δ7.31-7.27(m,1H,Ar-H),7.11-7.07(m,2H,2×Ar-H),6.98-6.94(m,2H ,2×Ar-H),5.11(t,J=5.1Hz,2H,CH2),4.55(t,J=5.2Hz,2H,CH2),3.81(br,3H,OCH3); 13CNMR(100MHz,DMSO-d6)δppm:170.14,163.60,161.74,157.24,143.08,142.95,141.51,136.83,128.15(2C),126.31(2C), 124.57,123.34,120.26,119.42,115.47(2C),115.31(2C),112.22,100.29,68.12,55.98,44.55; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 25 H 20 N4O5,457.1506,found 457.1477.Purity:97.9%.

[0110] Example 7

[0111] 3-(1-(2-(2-ethoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP7)

[0112] A white solid was obtained in a similar manner to Example 1, 60.8%.

[0113] 1 H NMR (400MHz, DMSO-d6): δ7.95-7.89 (m, 2H, 2×Ar-H), 7.81 (d, J=8.1Hz, 1H, Ar-H), 7.73 (d, J= 7.9Hz,1H,Ar-H), δ7.58(s,H,Ar-H),7.36-7.26(m,2H,2×Ar-H), δ7.11-7.06(m,2H,2×Ar-H), δ6.87-6.83(m,1H,Ar-H), δ6.80-6.71(m,3H,3×Ar-H),5.06(t,J=5.1Hz,2H,CH2),4.37(t,J =5.1Hz,2H,CH2),3.80(br,3H,OCH3),3.76(q,J=7.1Hz,2H,CH2),1.10(t,J=7.0Hz,3H,CH3); 13C NMR(100MHz,DMSO-d6)δppm:169.99,161.70,157.36,148.62,148.20,143.04,142.94,137.09,128.15(2C),124.39,123.19,121.89, 121.12,120.04,119.50,115.32(2C),113.88,113.76,112.67,100.35,68.01,63.95,55.97,45.27,15.13; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 27 H 25 N3O4,456.1918,found456.1888.Purity:97.1%.

[0114] Example 8

[0115] 3-(1-(2-(4-methoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP8)

[0116] A white solid was obtained in a similar manner to Example 1, 39.8%.

[0117] 1 H NMR (400MHz, DMSO-d6): δ7.96-7.90(m,2H,2×Ar-H),7.78-7.72(m,2H,2×Ar-H),δ7.58(s,H,Ar-H),7.39-7.35(m,H,Ar-H),7.31-7.27(m,H,Ar-H),δ7 .12-7.08(m,2H,2×Ar-H), δ6.74-6.64(m,4H,4×Ar-H),5.03(t,J=5.2Hz,2H,CH2),4.30(t,J=5.3Hz,2H,CH2),3.81(br,3H,OCH3),3.58(br,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:170.11,161.74,157.32,154.02,152.44,143.09,142.94,136.93,128.19(2C),124.45,123.26,1 20.20,119.47,115.68(2C),115.32(2C),115.06(2C),112.31,100.31,67.58,55.99,55.80,44.97; ESI-HRMS(TOF):m / z[M+H]+ Calculate for C 26 H 23 N3O4,442.1761,found442.1732.Purity:97.7%.

[0118] Example 9

[0119] Methyl 4-(2-(2-(5-(4-methoxyphenyl)isoxazol-3-yl)-1H-benzo[d]imidazol-1-yl)ethoxy)benzoate (EP9)

[0120] A white solid was obtained in a similar manner to Example 1, 53.4%.

[0121] 1 H NMR (400MHz, DMSO-d6): δ7.92-7.90(m,2H,2×Ar-H),7.80-7.72(m,4H,4×Ar-H),δ7.54(br,H,Ar-H),7.40-7.36(m,H,Ar-H),7.31-7.27(m,H,Ar-H),δ 7.11-7.08(m,2H,2×Ar-H), δ6.87-6.84(m,2H,2×Ar-H),5.09(t,J=5.8Hz,2 H,CH2),4.48(t,J=5.4Hz,2H,CH2),3.81(br,3H,OCH3),3.72(br,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:170.12,166.28,162.17,161.72,157.26,143.09,142.94,136.87,131.69(2C),128.17(2C),124.53, 123.31,122.63,120.23,119.44,115.30(2C),114.87(2C),112.25,100.29,67.42,55.98,52.32,44.65; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 27 H 23 N3O5,470.1710,found470.1684.Purity:98.1%.

[0122] Example 10

[0123] 3-(1-(2-(4-fluorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-methoxyphenyl)isoxazole (EP10)

[0124] A white solid was obtained in a similar manner to Example 1, 67.8%.

[0125] 1 H NMR (400MHz, DMSO-d6): δ7.95-7.90(m,2H,2×Ar-H),7.78-7.72(m,2H,2×Ar- H),δ7.57(s,H,Ar-H),δ7.39-7.35(m,H,Ar-H),δ7.31-7.27(m,H,Ar-H),7.12 -7.07(m,2H,2×Ar-H), δ7.02-6.94(m,2H,2×Ar-H), δ6.78-6.72(m,2H,2×Ar- H),5.05(t,J=5.3Hz,2H,CH2),4.35(t,J=5.3Hz,2H,CH2),3.81(s,3H,OCH3); 13 C NMR(100MHz,DMSO-d6)δppm:170.10,161.71,157.31,157.28(d, 1 J CF =234.8Hz),154.72,143.06,142.92,136.91,128.17(2C),124.48,123.29,120.21,119.44,116.33(2C,d, 2 J CF =23.4Hz),116.04(2C,d, 3 J CF =8.0Hz),115.30(2C),112.30,100.30,67.65,55.97,44.84; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 25 H 20 FN3O3,430.1561,found 430.1534.Purity:98.7%.

[0126] Embodiment 11

[0127] 5-(4-Fluorophenyl)-3-(1-(2-phenoxyethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP11)

[0128] At 0°C, 1-(4-fluorophenyl)ethan-1-one (5 ml, 41.4 mmol, 1 e.q) was added to anhydrous ethanol (200 ml) containing sodium methoxide (4.47 g, 82.8 mmol, 2 e.q), and then diethyl oxalate (6.125 ml, 45.5 mmol, 1.1 eq) was added to the solution. After stirring at room temperature for 6 hours, 6N HCl aqueous solution was added to the solution and stirred for 30 minutes to terminate the reaction. The residue was poured into excess ice water, the precipitate was filtered and dried in a vacuum oven to obtain yellow solid 2-hydroxy-4-(4-fluorophenyl)-4-oxobut-2-enoic acid ethyl ester (8.614 g, 87.4%).

[0129] 2-Hydroxy-4-(4-fluorophenyl)-4-oxobut-2-enoic acid ethyl ester (8g, 33.6mmol, 1e.q) and hydroxylamine hydrochloride (2.567g, 36.9mmol, 1.1eq) were added to anhydrous ethanol (100ml) and heated under reflux for 4h. The reaction mixture was cooled to room temperature, washed with ice water, filtered and dried to obtain a crude product, which was purified by column chromatography (PE / EA=10:1 (V / V)) to obtain 5-(4-fluorophenyl)isoxazole-3-carboxylic acid ethyl ester (6.795g, 86.0%) as a white solid.

[0130] 5-(4-fluorophenyl)isoxazole-3-carboxylic acid ethyl ester (5.5 g, 22.5 mmol) was dissolved in 50 ml of anhydrous ethanol, and then 5% NaOH (50 ml) solution was added. After reflux for 3 hours, the solution was cooled to room temperature. 1N HCl aqueous solution was slowly added to adjust the pH value of the solution to 3. The resulting precipitate was filtered and dried under vacuum to obtain the intermediate white powder 5-(4-fluorophenyl)isoxazole-3-carboxylic acid (4.3 g, 92.2%).

[0131] 5-(4-Fluorophenyl)isoxazole-3-carboxylic acid (2 g, 9.6 mmol, 1 e.q) was dissolved in DMF (30 ml) solution and DIEPA (1.9 ml, 11.5 mmol, 1.2 eq) and HATU (4.37 g, 11.5 mmol, 1.2 eq) were added in sequence. After stirring for 30 minutes, benzene-1,2-diamine (1.15 g, 10.6 mmol, 1.1 eq) was added and stirred at room temperature for 6 hours. Ice water was added to terminate the reaction, and the reaction solution was then extracted with ethyl acetate. The organic layer was washed with 1N hydrochloric acid, saturated NaHCO3 aqueous solution and distilled water, and dried over Na2SO4. After vacuum concentration, the residue was directly dissolved by adding an appropriate amount of acetic acid and refluxed overnight. After cooling to room temperature, the solution was concentrated under reduced pressure to give the crude product, which was purified by column chromatography (PE / EA=1:2 (V / V)) to give 3-(1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (1.35 g, 50.4%) as a white solid.

[0132] 3-(1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (200 mg, 0.7 mmol, 1 e.q) was dissolved in DMF solution and potassium carbonate (250 mg, 1.8 mmol, 2.57 eq) was added and stirred for 30 minutes. (2-bromoethoxy)benzene (281 mg, 1.4 mmol, 2 e.q) was then added and stirring was continued at 100°C for 2 hours. The reaction was then quenched with distilled water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over Na2SO4, and evaporated to provide a crude product, which was purified by column chromatography (PE / EA=5:1 (V / V)) to give a white solid (89 mg, 31.9%).

[0133] 1 H NMR (400MHz, DMSO-d6): δ8.07-8.03(m,2H,2×Ar-H), δ7.79-7.72(m,3H,3×Ar-H), δ7.42-7.35(m,3H,3×Ar-H), δ7.31-7.27(m,1H,Ar-H), δ7.17-7.13(m,2H,2×Ar-H), δ6.84-6.81(m,H,Ar-H), δ6.75-6.72(m,2H,2×Ar-H), 5.06(t,J=5.2Hz,2H,CH2), 4.37(t,J=5.4Hz,2H,CH2); 13 C NMR(100MHz,DMSO-d6)δppm:169.11,163.95(d, 1 J CF=247.9Hz),158.37,157.46,142.92,142.86,136.94,129.99(2C),128.97(2C,d, 3 J CF =8.8Hz),124.54,123.51(d, 4 J CF =3.0Hz),123.32,121.35,120.23,117.06(2C,d, 2 J CF =22.1Hz),114.75(2C),112.33,101.78,66.98,44.90; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 24 H 18 FN3O2,400.1456,found 400.1454.Purity:99.1%.

[0134] Example 12

[0135] 3-(1-(2-(3,5-dimethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (EP12)

[0136] A white solid was obtained by a similar method as in Example 11, 45.2%;

[0137] 1 H NMR (400MHz, DMSO-d6): δ8.08-8.03(m,2H,2×Ar-H), δ7.79-7.73(m,2H,2×Ar-H),7.70(s,1H,Ar-H), δ7.43-7.36(m,3H,3×Ar-H), δ7.31 -7.27(m,1H,Ar-H),6.44(s,1H,Ar-H),6.34(s,2H,2×Ar-H),5.04(t,J=5.4Hz,2H,CH2),4.32(t,J=5.2Hz,2H,CH2),2.08(s,6H,2×CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.10,163.96(d, 1 J CF =248.2Hz),158.39,157.46,142.96,142.91,139.10(2C),136.89,128.97(2C,d, 3 J CF=8.8Hz),124.55,123.55(d, 4 J CF =3.4Hz),123.31,122.94,120.24,117.07(2C,d, 2 J CF =22.0Hz),112.49(2C),112.31,101.83,66.78,44.91,21.45(2C); ESI-HRMS(TOF):m / z[M+H] + Calculate for C 26 H 22 FN3O2,428.1769,found 428.1742.Purity:98.2%.

[0138] Embodiment 13

[0139] 3-(1-(2-(4-ethylphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (EP13)

[0140] A white solid was obtained in a similar manner to Example 11, 29.4%.

[0141] 1 H NMR (400MHz, DMSO-d6): δ8.07-8.04(m,2H,2×Ar-H), δ7.79-7.72(m,3H,3×A r-H), δ7.43-7.35(m,3H,3×Ar-H), δ7.31-7.27(m,1H,Ar-H),6.98-6.96(m, 2H,2×Ar-H),6.65-6.63(m,2H,2×Ar-H),5.05(t,J=5.3Hz,2H,CH2),4.33(t ,J=5.3Hz,2H,CH2),2.41(q,J=7.6Hz,2H,CH2),1.03(t,J=7.6Hz,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.11,163.96(d, 1 J CF =247.8Hz),157.46,156.46,142.95,142.91,136.92,136.58,129.10(2C),128.98(2C,d, 3 J CF =8.9Hz),124.52,123.54(d, 4 J CF=3.3Hz),123.30,120.23,117.06(2C,d, 2 J CF =22.1Hz),114.64(2C),112.32,101.80,67.06,44.92,27.73,16.35; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 26 H 22 FN3O2,428.1769,found 428.1747.Purity:95.0%.

[0142] Embodiment 14

[0143] 5-(4-Fluorophenyl)-3-(1-(2-(o-tolyloxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP14)

[0144] A yellow solid was obtained in a similar manner to Example 11, 36.0%.

[0145] 1 H NMR (400MHz, DMSO-d6): δ8.07-8.04(m,2H,2×Ar-H), δ7.81-7.71(m,3H,3×Ar -H),δ7.43-7.34(m,3H,3×Ar-H),δ7.31-7.27(m,1H,Ar-H),7.04-7.00(m,1H, Ar-H),6.94-6.92(m,1H,Ar-H),6.83-6.81(m,1H,Ar-H),6.73-6.69(m,1H,Ar -H),5.15(t,J=5.0Hz,2H,CH2),4.36(t,J=4.7Hz,2H,CH2),1.68(s,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.03,163.95(d, 1 J CF =248.0Hz),157.55,156.53,142.94,142.91,136.90,130.88,128.97(2C,d, 3 J CF =8.7Hz),127.43,125.95,124.44,123.53(d, 4 J CF =3.2Hz),123.32,121.00,120.23,117.06(2C,d, 2 J CF=22.1Hz),112.39,111.39,101.83,66.94,44.92,16.25; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 25 H 20 FN3O2,414.1612,found 414.1589.Purity:97.5%.

[0146] Embodiment 15

[0147] 5-(4-Fluorophenyl)-3-(1-(2-(4-nitrophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP15)

[0148] A similar method to Example 11 was used to obtain a pale yellow solid, 32.6%.

[0149] 1 H NMR (400MHz, DMSO-d6): δ8.06-8.02 (m, 4H, 4×Ar-H), 7.81 (d, J=8.1Hz, 1H, Ar-H), 7.74 (d, J=8.1Hz, 1H, Ar-H), 7.70 (s, 1H, Ar-H), δ7. 43-7.37(m,3H,3×Ar-H), δ7.32-7.28(m,1H,Ar-H),6.98-6.94(m,2H,2×Ar-H),5.12(t,J=5.1Hz,2H,CH2),4.56(t,J=5.2Hz,2H,CH2); 13 C NMR(100MHz,DMSO-d6)δppm:169.13,163.71(d, 1 J CF =247.9Hz),163.55,157.36,142.92,142.87,141.46,136.81,128.94(2C,d, 3 J CF =8.8Hz),126.30(2C),124.65,123.45(d, 4 J CF =3.1Hz),123.39,120.28,117.06(2C,d, 2 J CF =22.1Hz),115.45(2C),112.25,101.77,68.05,44.52; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 24 H17 FN4O4,445.1307,found 445.1278.Purity:95.4%.

[0150] Example 16

[0151] 3-(1-(2-(2-ethoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (EP16)

[0152] A white solid was obtained in a similar manner to Example 11, 44.1%.

[0153] 1 H NMR (400MHz, DMSO-d6): δ8.08-8.04 (m, 2H, 2×Ar-H), 7.83 (d, J=8.1Hz, 1H, Ar-H), δ7.77-7.69 (m, 2H, 2×Ar-H), δ7.43-7.27 (m, 4H, 4×Ar-H), δ6.86-6.84 (m,1H,Ar-H),6.80-6.72(m,3H,3×Ar-H),5.07(t,J=5.1Hz,2H,CH2),4.37( t,J=5.1Hz,2H,CH2),3.76(q,J=7.0Hz,2H,CH2),1.10(t,J=7.0Hz,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.01,163.99(d, 1 J CF =250.2Hz),157.51,148.64,148.21,142.96,142.86,137.08,128.97(2C,d, 3 J CF =8.8Hz),124.46,123.59,123.23,121.91,121.13,120.09,117.08(2C,d, 2 J CF =21.9Hz),113.95,113.81,112.68,101.86,67.99,63.97,45.28,15.12; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 26 H 22 FN3O3,444.1718,found 444.1691.Purity:98.3%.

[0154] Embodiment 17

[0155] 5-(4-Fluorophenyl)-3-(1-(2-(4-methoxyphenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)isoxazole (EP17)

[0156] A yellow solid was obtained in a similar manner to Example 11, 52.0%.

[0157] 1 H NMR (400MHz, DMSO-d6): δ8.07-8.04(m,2H,2×Ar-H), δ7.78-7.72(m,3H,3×Ar-H), δ7.43-7.35(m,3H,3×Ar-H), δ7.31-7 .27(m,1H,Ar-H),6.72-6.65(m,4H,4×Ar-H),5.04(t,J=5.3Hz,2H,CH2),4.30(t,J=5.2Hz,2H,CH2),3.58(s,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.13,163.97(d, 1 J CF =247.5Hz),157.46,154.01,152.41,142.94,142.92,136.92,129.00(2C,d, 3 J CF =8.8Hz),124.54,123.54(d, 4 J CF =3.1Hz),123.32,120.24,117.08(2C,d, 2 J CF =22.0Hz),115.67(2C),115.04(2C),112.34,101.81,67.51,55.79,44.96; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 25 H 20 FN3O3,430.1561,found430.1533.Purity:96.7%.

[0158] Embodiment 18

[0159] Methyl 4-(2-(2-(5-(4-fluorophenyl)isoxazol-3-yl)-1H-benzo[d]imidazol-1-yl)ethoxy)benzoate (EP18)

[0160] A white solid was obtained in a similar manner to Example 11, 47.4%.

[0161] 1 H NMR (400MHz, DMSO-d6): δ8.06-8.01(m,2H,2×Ar-H),7.81-7.79(m,H,Ar-H),δ7.77-7.73(m,3H,3×Ar-H),7.70(br,H,Ar-H),δ7.43-7.36(m, 3H,3×Ar-H), δ7.32-7.27(m,1H,Ar-H),6.86-6.84(m,2H,2×Ar-H),5.10(t,J=5.2Hz,2H,CH2),4.48(t,J=5.2Hz,2H,CH2),3.72(br,3H,CH3); 13 C NMR(100MHz,DMSO-d6)δppm:169.13,166.26,163.96(d, 1 J CF =247.8Hz),162.13,157.38,142.95,142.91,136.85,131.67(2C),128.97(2C,d, 3 J CF =8.8Hz),124.60,123.50(d, 4 J CF =3.3Hz),123.35,122.63,120.27,117.04(2C,d, 2 J CF =22.1Hz),114.87(2C),112.27,101.79,67.35,52.30,44.63; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 26 H 20 FN3O4,458.1511,found 458.1483.Purity:96.4%.

[0162] Embodiment 19

[0163] 3-(1-(2-(4-fluorophenoxy)ethyl)-1H-benzo[d]imidazol-2-yl)-5-(4-fluorophenyl)isoxazole (EP19)

[0164] A white solid was obtained in a similar manner to Example 11, 43.5%.

[0165] 1H NMR (400MHz, DMSO-d6): δ8.08-8.04(m,2H,2×Ar-H), δ7.80-7.73(m,3H,3×Ar-H), δ7.44-7.36(m,3H,3×Ar-H), δ7.31-7.27( m,1H,Ar-H),7.01-6.95(m,2H,2×Ar-H),6.76-6.73(m,2H,2×Ar-H),5.06(t,J=5.3Hz,2H,CH2),4.36(t,J=5.3Hz,2H,CH2); 13 CNMR(100MHz,DMSO-d6)δppm:169.14,163.97(d, 1 J CF =247.9Hz),157.45,157.32(d, 1 J CF =234.9Hz),154.72,142.94,142.89,136.91,129.00(2C,d, 3 J CF =8.9Hz),124.56,123.51,123.33,120.25,117.07(2C,d, 2 J CF =22.2Hz),116.31(2C,d, 2 J CF =22.9Hz),116.08(2C,d, 3 J CF =8.0Hz),112.31,101.80,67.63,44.85; ESI-HRMS(TOF):m / z[M+H] + Calculate for C 24 H 17 F2N3O2,418.1362,found 418.1337.Purity:96.1%.

[0166] Experimental methods and results

[0167] 1. Cell Viability Assay

[0168] Experimental principle: The reagent contains WST-8, which is reduced to a highly water-soluble yellow formazan product (Formazan dye) by dehydrogenase in cells under the action of the electron carrier 1-methoxy-5-methylphenazinium dimethyl sulfate (1-Methoxy PMS). The amount of formazan generated is proportional to the number of living cells. Therefore, this property can be used to directly analyze cell proliferation and toxicity.

[0169] Experimental steps:

[0170] 1. Plate: Take RPMI-8226 humanized myeloma cells in the logarithmic growth phase and inoculate them into 96-well plates. Each well has 100 μL of cell suspension and 5×10 cells. 3 / well, the blank control group was added with only 100 μL of complete medium RPM I-1640 containing 10% FBS, and 3 to 5 replicate wells were set up for each group.

[0171] 2. RPMI-8226 treated with small molecule compounds of different concentration gradients were placed in a cell culture incubator (37°C, 5% CO2) for culture. After 24 hours, 5 μL of CCK-8 solution was added to each well. After further culture for 3 hours, the microplate reader was used for detection.

[0172] 3. Detection: Adjust the blank control group to zero, detect the absorbance (OD value) at 450nm wavelength, repeat 2-3 times, take the average value, and calculate the IC value of the administered compound. 50 value.

[0173] Experimental results: As shown in Table 1 below, the compounds EP8, EP9, EP12, and EP18 in the present invention have significantly better ability to inhibit the proliferation of humanized myeloma cells RPMI-8226 than the previous patented compound 25 (patent number 202011215542.2), and are better than the positive control drug 10074-G5.

[0174] Table 1 Effects of the 19 compounds of the invention on the cell viability of humanized myeloma cells RPMI-8226

[0175]

[0176] 1 IC 50 , Mean ± standard error of the mean of three measurements.

[0177] 2. Biological Experiments

[0178] 1. Q-PCR analysis

[0179] Q-PCR was performed to determine the effect of the structurally modified active compounds (EP8, EP9, EP12, and EP18) on c-Myc gene transcription in RPMI-8226 cells. Total RNA was extracted and reverse transcribed into cDNA. The cDNA was then used as a template for quantitative PCR amplification of the c-Myc sequence. After 48 hours of treatment with 5 μM ligand, the target cells were collected, total RNA was extracted, and cDNA was synthesized using the M-MLV reverse transcriptase cDNA synthesis kit (Promega). Q-PCR experiments were then performed using SYBR Green reaction mix (Roche) and 2 -ΔΔCtMethods Analytical data.

[0180] Experimental results: Figure 1 As shown in (a), compound EP12 reduced the level of c-Myc mRNA at 5 μM, while other compounds had no effect on this. EP12 can reduce the expression level of c-Myc mRNA.

[0181] 2. Western Blot (WB)

[0182] RPMI-8226 cells were treated with 5 μM EP12 for 48 h, and then the cells were collected and completely lysed with RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China). After that, all the extracted proteins were mixed with the loading buffer for subsequent SDS-PAGE. The proteins were then transferred to a PVDF membrane, which was incubated with primary β-actin and c-Myc (Proteintech) antibodies.

[0183] Experimental results: Figure 1 As shown in (b), the expression level of c-Myc was also reduced after treatment with EP12, indicating that EP12 has the inhibitory activity of c-Myc protein.

[0184] 3. Cell Viability and Flow Cytometry Analysis

[0185] 1×104 RPMI-8226 cells were seeded into 96-well plates. After incubation with different doses of compounds for 48 h, cell viability was detected by CCK8 kit (KeyGEN BioTECH, China). The apoptotic rate was determined using Annexin V-APC / 7-AAD apoptosis kit (KeyGEN BioTECH, China) according to the manufacturer's protocol and analyzed by flow cytometry (BD Biosciences).

[0186] Experimental results: Figure 2 As shown, EP12 significantly affected the apoptosis of RPMI-8226 cells. The apoptosis rate of EP12 at 5 μM was 21.9%, which induced better apoptosis of RPMI-8226 cells, proving that EP12 has an excellent inhibitory effect on c-Myc in proliferating cells.

[0187] 3. Circular dichroism experiment

[0188] Circular dichroism experiments were recorded at 25°C in a cuvette with a 1 mm pathlength using a J-815CD spectrometer (Jasco). To anneal the oligonucleotides, the samples were heated to 95°C, stabilized for 5 minutes, then cooled to room temperature for 30 minutes and then placed in a 4°C refrigerator overnight. The oligonucleotides were then diluted to 5 M in 10 mM Tris buffer (pH 6.8, containing 50 mM KCl) and 4 equivalents of compound were added to give 20 M of compound in 1% DMSO. The CD signal was scanned from 230 nm to 320 nm at a scan rate of 200 nm / min and a bandwidth of 2 nm. CD melting was performed at a heating rate of 1°C / min in the temperature range of 25-95°C by tracking the change in the CD signal at the wavelength of maximum CD intensity (264 nm). The melting temperature (Tm) was determined from the curve fit using Origin 9.0 software. The ΔTm value was determined as the difference in melting temperature between DNA with and without ligand. All experiments were repeated three times and the values ​​recorded are the average of the three measurements.

[0189] Experimental results: Figure 3 As shown, circular dichroism (CD) spectroscopy is often used to determine the secondary structure of proteins or nucleic acids. + It will have a positive peak near 264nm and a negative peak at 240nm ( Figure 3 Left), indicating that a parallel conformation of c-Myc G4 was formed. When EP12 was added, the peak shape changed slightly, indicating that the binding of compound EP12 to c-Myc G4 did not change its conformation. In order to study the stability of this compound on c-Myc G4, a CD melting experiment was performed. Figure 3 As shown on the right, EP12 stabilizes c-Myc G4.

[0190] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament related to multiple myeloma, wherein the compound is selected from:

2. A compound or a pharmaceutically acceptable salt thereof, wherein the compound is selected from:

3. A pharmaceutical composition for treating multiple myeloma, comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient or a main active ingredient, and supplemented with a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition is prepared as a liquid preparation or a solid preparation.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is prepared into injection, oral liquid, granules, powder, tablets or capsules.

Images