Substituted benzamide derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicine, and particularly relates to a substituted benzamide derivative and a preparation method and application thereof.The structure of the substituted benzamide derivative is shown in the formula I. Experiments prove that the substituted benzamide derivative has good anti-tumor activity and can be used for preparing anti-tumor drugs. And a foundation is laid for further development of antitumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a substituted benzamide derivative, a preparation method and application thereof. BACKGROUND

[0002] Homeobox (HOX) genes are a family of highly conserved transcription factors, with the core feature of containing a homeodomain, which regulates the spatiotemporal expression of specific genes in the processes of embryonic axial patterning, organogenesis and cell fate determination. The human HOX gene network contains four gene clusters (HOXA~D), among which HOXA1 plays a key role in early development, such as hindbrain segmentation, cranial neural crest cell specification and heart morphogenesis. HOXA1 protein contains two main functional domains: one is the C-terminal homeodomain, which is responsible for binding to DNA and regulating the transcription of downstream target genes; the other is the N-terminal hexapeptide motif, which can mediate heterodimerization with Pbx protein, thereby enhancing the DNA binding specificity and transcriptional activity.

[0003] HOX genes are usually lowly expressed or strictly regulated in adult tissues, but abnormal activation of HOXA1 has been confirmed to be closely related to the malignant progression of various cancers. In breast cancer, high expression of HOXA1 can drive oncogenic transformation by activating the nuclear factor kappa B (NF-κB), Janus kinase / signal transducer and activator of transcription (JAK / STAT) and mitogen-activated protein kinase (MAPK) signaling pathways, and its high expression is significantly associated with poor prognosis. In lung cancer and liver cancer, HOXA1 can promote tumor invasiveness by regulating the epigenetic state of MEIS3 enhancer, and is also related to immune escape. In prostate cancer and ovarian cancer, HOXA1 promotes tumor metastasis by participating in the non-coding RNA regulatory network. In leukemia, the oncogenic activity of HOXA1 is related to the JAK3 / STAT5B mutation-driven pathway. In addition, members of the HOXA cluster can interact with various cofactors and chromatin remodeling complexes, thereby affecting the epigenetic state and transcriptional output of cancer cells. Importantly, high expression of HOXA1 can lead to cisplatin resistance by activating downstream survival pathways, and silencing HOXA1 can effectively reverse this chemotherapy resistance.

[0004] In view of the oncogenic properties of HOXA1, targeting this transcription factor becomes a highly potential anti-tumor therapeutic strategy. There are mainly two ways to inhibit HOXA1: indirectly regulating its expression, or directly destroying its protein function. The indirect strategy usually involves regulating epigenetic regulatory factors or upstream signaling pathways, for example, inhibition of the menin-KMT2A complex can inhibit the transcription of the HOX gene cluster, thereby reducing the expression of HOXA. Notably, the menin inhibitor SNDX-5613 has been approved for the treatment of acute myeloid leukemia. The direct targeting strategy targets the functionally essential domains of HOXA1 protein, especially the homeodomain and the N-terminal hexapeptide motif, both of which are essential for its transcriptional activity. Although progress has been made in indirect regulation, there is currently no report on small molecule inhibitors directly targeting HOXA1.

[0005] The present study aims to obtain a class of compounds with tumor inhibitory activity by studying compounds directly targeting the functional domains of HOXA1. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a substituted benzamide derivative with good anti-tumor activity, and a preparation method and application thereof.

[0007] To solve the above problems, the technical scheme adopted by the present application is: Technical subject one A substituted benzamide derivative or a pharmaceutically acceptable salt thereof, which is characterized in that the substituted benzamide derivative has a structure as shown in formula I: wherein R1 is selected from C1-C5 alkyl or hydrogen; R2 is selected from hydroxyl or amino; X is -NHC(O)-, -NH(CH2) n -, -NHS(O)2-, -C(O)NH-, -O(CH2) n -, -OC(O)-, -(CH2) n -, -CH=CH- or -C C-; n is 1, 2, 3, 4 or 5; R3 is a single substituent or a plurality of substituents, independently selected from C1-C5 alkyl or halogen.

[0008] As a further improvement of the application, the C1-C5 alkyl is selected from the group consisting of -CH3, -CH2CH3, -CH(CH3)2, -(CH2)2CH3, -(CH2)3CH3, -CH2CH(CH3)2, -C(CH3)3, -CH(CH3)CH2CH3, -(CH2)4CH3, -CH(CH3)(CH2)2CH3, -CH(CH2CH3)2, -CH2C(CH3)3, -CH2CH(CH3)CH2CH3, -C(CH3)2CH2CH3, -CH(CH3)CH(CH3)2, -(CH2)2CH(CH3)2.

[0009] As a further improvement of the application, the , , , , or .

[0010] As a further improvement of the application, when X is -NHC(O)-, or ; X is -NH(CH2) n -, ; X is -NHS(O)2-, or ; X is -C(O)NH-, , or ; X is -O(CH2) n -, ; X is -OC(O)-, , , or ; X is -(CH2) n -, or ; X is -CH=CH-, ; the n is 1, 2, 3, 4 or 5.​​​​​​​​​

[0011] As a further improvement of the present application, the structure of the compound is as follows: .

[0012] Technical subject two A method for preparing a substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to technical subject one, which is characterized in that the method for preparing the same is selected from one of the following routes: Technical subject three A pharmaceutical composition comprising a substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to technical subject one, and optionally, one or more pharmaceutically acceptable carriers or excipients.

[0013] As used herein, a "pharmaceutical composition" contains a therapeutically effective amount of the substituted benzamide derivative of the formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, prepared in the form of tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wines, decoctions, lozenges, mixtures, suppositories, injections, inhalants, or sprays, etc. The pharmaceutical composition preferably contains 0.1-99.5% by weight of the substituted benzamide derivative of the present application or a pharmaceutically acceptable salt thereof as an active ingredient, and more preferably contains 0.5-99.5% by weight of the active ingredient.

[0014] As used herein, "pharmaceutically acceptable carriers or excipients" include diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifying agents, sweetening agents, flavoring agents, taste-masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, viscosity enhancers, antioxidants, preservatives, stabilizers, surfactants, and buffers, and one of skill will understand that certain pharmaceutically acceptable excipients can be used in more than one function and in alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation. For example, when used for oral administration, oral formulations such as tablets, capsules, granules, and pills, etc. can be prepared, which contain fillers (e.g. sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g. gelatin, polyvinylpyrrolidone, and polyethylene glycol), disintegrants (e.g. cellulose derivatives such as sodium carboxymethyl cellulose, polyvinylpyrrolidone), lubricants (e.g. talc, calcium stearate, magnesium stearate, sperm oil, boric acid, sodium benzoate, leucine), stabilizers (methyl paraben, propyl paraben, etc.), flavoring agents (e.g. commonly used sweeteners, souring agents, and spices, etc.). When used for parenteral administration, injectable preparations, including sterile powders for injection and injectable solutions, can be prepared, and the carriers or excipients used include sterile water, Ringer's solution, and isotonic sodium chloride solution, and appropriate additional agents such as antioxidants, buffers, and bacteriostatic agents can be added depending on the nature of the drug. When used for rectal administration, the drug can be prepared as suppositories, etc. When used for pulmonary administration, the drug can be prepared as inhalants or sprays, etc. There are many resources available to those skilled in the art that describe pharmaceutically acceptable excipients and which can be used to select appropriate pharmaceutically acceptable excipients, such as the books "Remington's Pharmaceutical Sciences", "Chinese Pharmaceutical Yearbook", "Pharmaceutics", etc.

[0015] The present application can be administered by any suitable method known in the art, for example, orally, intravenously, intraperitoneally, intramuscularly, topically, transdermally, ophthalmically, nasally, by inhalation, subcutaneously, intramuscularly, buccally, sublingually, rectally, etc., in any amount from 1 μg to 2000 mg per kg of body weight of the subject, for example, in an amount from 1 μg to 1000 mg per kg of body weight per day, 50 μg to 1000 mg per kg of body weight per day, 100 μg to 1000 mg per kg of body weight per day, 1 to 500 mg per kg of body weight per day, 2 to 200 mg per kg of body weight per day, 5 to 100 mg per kg of body weight per day, of the compound described above. In some embodiments of the present application, the compound described above can be administered 4 times per day, 3 times per day, 2 times per day, 1 time per day, 1 time per two days, 1 time per week, or other intervals, optionally repeating the dosing regimen described above as appropriate, weekly or monthly. In the present application, the dosage of the compound can be adjusted according to the severity of the patient or subject's condition, age, body weight, gender, administration method, and course of treatment, etc.

[0016] The compounds of the present application can be used alone or in combination with one or more other active ingredients in the treatment, prevention, inhibition or amelioration of diseases or conditions of interest. Such other drugs can be administered, by a route and in an amount commonly used therefor, contemporaneously or sequentially with a compound of the present application. When a compound of the present application is used contemporaneously with one or more other drugs, a pharmaceutical composition containing such other drugs in addition to the compound of the present application is preferred. Accordingly, the pharmaceutical compositions of the present application also include those that also contain one or more other active ingredients, in addition to a compound of the present application.

[0017] Technical Subject Four Use of a substituted benzamide derivative or a pharmaceutically acceptable salt thereof as described in Technical Subject One for the manufacture of a medicament for inhibiting HOXA1.

[0018] Technical Subject Five Use of a substituted benzamide derivative or a pharmaceutically acceptable salt thereof as described in Technical Subject One for the manufacture of a medicament for inhibiting HOXA1.

[0019] The beneficial effects produced by the above technical solutions are as follows: This invention provides a substituted benzamide derivative. Experimental verification shows that the compound disclosed in this invention has certain anti-tumor activity, providing new ideas and options for tumor treatment.

[0020] The present invention also discloses a method for preparing the compound, wherein the raw materials and reagents used in the preparation are simple and readily available, the method is simple, the conditions are mild, and it is easy to purify, making it suitable for industrial production. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the docking of compound F2 of the present invention, where A is the conformation of the docking site, and B is a schematic diagram of the two-dimensional interaction between F2 and HOXA1. In Figure A, green represents van der Waals forces, orange represents π-cations, and pink represents amide-π stacking. Figure 2 This is a graph showing the effect of compound F2 of the present invention on the transcriptional activity of HOXA1; Figure 3 This is a graph showing the inhibition rates of the 26 compounds of this invention against four cell lines, where A represents the inhibition rate against HepG2 cells at a concentration of 20 μM; B represents the inhibition rate against NCI-H1975 cells at a concentration of 20 μM; C represents the inhibition rate against HCT116 cells at a concentration of 20 μM; and D represents the inhibition rate against MCF-7 cells at a concentration of 20 μM. Figure 4 The antiproliferative activity curves and IC50 values ​​of the compounds in this application against different tumor cells at different concentrations are shown. 50 Figure 1 shows MCF-7 cells, HCT116 cells, and HepG2 cells. Figure 5 This is an image showing the tumor results of mice treated with F2-15 and / or cisplatin from day 12 to day 25 after tumor inoculation, as described in Example 4 of this application; where A is an image of the colorectal tumor in mice; B is the weight of the colorectal tumor in mice, in mg; and C is a graph showing the change in the volume of the colorectal tumor in mice, in mm. 3 D is an image of a triple-negative mammary tumor in mice; E is the weight of a triple-negative mammary tumor in mice, in mg; F is a graph showing the volume change of a triple-negative mammary tumor in mice, in mm. 3 ; Figure 6This is a graph showing the in vivo toxicity results of the blank group and compound F2-15 in Example 4 of this application; where A is the body weight of mice in g, B is the liver weight in g, C is the kidney weight in g; D is ALT in U / L, E is the blood AST content in U / L, F is the blood TP content in U / L, G is the blood ALB content in g / L, H is the blood TBIL content in μM / L, I is the blood ALP content in U / L, J is the blood TBA content in μM / L, K is the blood CRE content in μM / L, and L is the blood UA content in μM / L; n=5, ns indicates no significant change; Figure 7 The images show representative immunohistochemical images of HOXA1, Ki67, and activated Caspase-3 in mouse colorectal tumor sections and triple-negative mammary tumor sections in Example 4 of this application, where A represents mouse colorectal tumor and B represents triple-negative mammary tumor. In the diagram, Vehicle represents the solvent group; Cisplatin represents the cisplatin treatment group. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0023] The compounds provided in this application can be prepared via the following route: .

[0024] The reaction conditions are as follows: (a1): Nitric acid, acetic acid, room temperature, 30 minutes; (b1): Ammonium formate, 10% Pd / C, methanol, room temperature, 2 hours; (c1): substituted benzoic acid, EDCI, HOBT, DIEA, anhydrous DMF, room temperature, 7-8 hours; or substituted benzyl chloride, triethylamine (TEA), anhydrous tetrahydrofuran (THF), room temperature, 6-8 hours; (d1): Benzyl chloride, potassium carbonate (K2CO3), anhydrous DMF, 60℃, 4 hours; (e1): Iron (Fe), ammonium chloride (NH4Cl), ethanol (EtOH): water (H2O) = 4:1, room temperature, 2 hours; (f1): 1) Substituted sulfonyl chloride, TEA, anhydrous THF, room temperature, 6 hours; 2) Ammonium formate, 10% Pd / C, methanol, room temperature, 2 hours; (g1): Trifluoromethanesulfonic anhydride (Tf2O), pyridine, anhydrous dichloromethane (DCM), 0℃, 1 hour; (h1) Benzylamine, tris(dibenzylacetone)palladium (Pd(dba)3), XPhos, cesium carbonate (Cs2CO3), anhydrous 1,4-dioxane, reflux, 20 h; (i1): 1) tert-butyl nitrite (TBN), acetonitrile (CH3CN), 80℃, 2 hours; 2) zinc (Zn), acetic acid (AcOH), 50℃, 3 hours; (j1): 1) 4-tert-butylbenzoyl chloride, TEA, anhydrous THF, room temperature, 1 hour; 2) ammonium formate, 10% Pd / C, methanol, room temperature, 4 hours; (a2): Paraformaldehyde (PFA), magnesium chloride (MgCl2), TEA, acetonitrile, reflux, 8 hours; (b2): Benzyl chloride, K2CO3, anhydrous DMF, 60℃, 8 hours; (c2): Sodium chlorite (NaClO2), sodium dihydrogen phosphate (NaH2PO4), THF: tetrabutylammonium (TBA): water = 3:3:1, room temperature, 6 hours; (d2): 1) Substituted aniline, EDCI, HOBT, DIEA, anhydrous DMF, room temperature, 6-8 hours; 2) Ammonium formate, 10% Pd / C, methanol, room temperature, 2 hours; (e2): m-chloroperoxybenzoic acid (m-CPBA), DCM, room temperature, 12 hours; (f2): 1) 4-tert-butylbenzoyl chloride or substituted benzoyl chloride, TEA, anhydrous THF, room temperature, 3-6 hours; 2) ammonium formate, 10% Pd / C, methanol, room temperature, 0.5-2 hours; (g2): 150℃, 5 hours; (h2): 60% sodium hydride (NaH), THF, room temperature, 14 hours; (i2): Boron trichloride (BCl3), anhydrous DCM, 0°C, 1 hour; (j2): Ammonium formate, 10% Pd / C, methanol, room temperature, 2 hours.

[0025] Example 1 2-Nitro-4-(2,4,4-trimethylpentan-2-yl)phenol (Compound 2) 4-(2,4,4-trimethylpentyl)phenol (compound 1) (2.06 g, 10 mmol) was dissolved in glacial acetic acid (20 mL), and concentrated nitric acid (1 mL) was added dropwise at room temperature, with stirring for 30 minutes. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was poured into distilled water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined and washed successively with distilled water (2 × 50 mL) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 40:1) to give the orange oily product compound 2 (2.13 g, yield 85%).

[0026] ¹H NMR (600 MHz, CDCl3) δ 10.47 (s, 1H), 8.05 (s, 1H), 7.63 (d, J=8.8Hz, 1H), 7.08 (d, J=8.9 Hz, 1H), 1.73 (s, 2H), 1.37 (s, 6H), 0.74 (s, 9H).

[0027] Example 2 2-Amino-4-(2,4,4-trimethylpentan-2-yl)phenol (compound 3a) Compound 2 (1.225 g, 5 mmol) and ammonium formate (1.26 g, 20 mmol) were dissolved in methanol (50 mL), and 10% palladium on carbon (251 mg, 20% by mass) was added. The mixture was stirred at room temperature for 2 hours. The palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give a brown solid compound 3a (960 mg, yield 87%) with a melting point of 121.5–124.3 °C.

[0028] ¹H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 6.64 (d, J=2.3 Hz, 1H), 6.53 (d, J=8.2 Hz, 1H), 6.40 (dd, J=8.2, 2.4 Hz, 1H), 4.42 (s, 2H), 1.61 (s, 2H), 1.24 (s, 6H), 0.71 (s, 9H).

[0029] Example 3 4-tert-butyl-N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)benzamide (F2) Compound 3a (5 mmol) and 4-tert-butylbenzoic acid (5 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF, 20 mL), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI, 192 mg, 1 mmol), 1-hydroxybenzotriazole (HOBt, 162 mg, 1.2 mmol), and N,N-diisopropylethylamine (DIEA, 0.35 mL, 2 mmol) were added. The mixture was stirred at room temperature for 7 hours. After the reaction was complete, the mixture was poured into distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give a white solid F2 (76% yield), melting point: 163.7–168.7 °C.

[0030] ¹H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 9.48 (s, 1H), 7.92 (d, J=7.9 Hz, 2H), 7.71 (s, 1H), 7.55 (d, J=7.7 Hz, 2H), 7.04 (d, J=8.6 Hz, 1H), 6.82 (d, J=8.4 Hz,1H), 1.68 (s, 2H), 1.32 (s, 9H), 1.30 (s, 6H), 0.72 (s,9H); ¹³C NMR (101 MHz, DMSO-d6) δ 165.27, 154.56, 146.42, 140.22, 131.52,127.40, 125.27, 125.24, 123.21, 121.37, 115.62, 56.35, 37.63, 34.69, 32.06,31.61, 31.54, 30.93; ESI-HRMS: C 25 H 36 O2N + ([M+H]) + Theoretical value: 382.27406; Measured value: 382.27359. HPLC purity: 98.75%.

[0031] Example 4 Synthesis of 2,4-dichloro-N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)benzamide (F2-1) Using compound 3a and 2,4-dichlorobenzoic acid as starting materials, and following the synthesis method of F2, a pale yellow solid F2-1 (yield 72%) was obtained with a melting point of 189.3-192.4 °C.

[0032] ¹H NMR (400 MHz, DMSO-d6) δ 9.71 (s, 1H), 9.46 (s, 1H), 7.78 (s, 1H), 7.74 (d, J=2.3 Hz, 1H), 7.67 (d, J=8.2 Hz, 1H), 7.54 (d, J=8.3 Hz, 1H), 7.04(d, J=8.3 Hz,1H), 6.81 (d, J=8.4 Hz, 1H), 1.68 (s, 2H), 1.30 (s, 6H), 0.72(s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 164.10, 146.14, 140.10, 135.37, 134.80,131.29, 130.74, 129.18, 127.34, 124.72, 123.30, 120.97, 115.29, 56.32, 37.62,32.05, 31.61,31.53; ESI-HRMS: C 21 H 26 O2NCl2⁺([M+H)) + Theoretical value: 394.13351; Measured value: 394.13288. HPLC purity: 96.20%.

[0033] Example 5 N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)-2,4-dimethylbenzamide (F2-2) Using compound 3a and 2,4-dimethylbenzoic acid as starting materials, and following the synthesis method of F2, a white solid F2-2 (yield 70%) was obtained with a melting point of 214.8-217.6 °C.

[0034] ¹H NMR (600 MHz, DMSO-d6) δ 9.48 (s, 1H), 9.43 (s, 1H), 7.68 (s, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.14-7.07 (m, 2H), 7.03 (d, J=8.5 Hz, 1H), 6.80 (d, J=8.4 Hz, 1H), 2.40 (s, 3H), 2.32 (s, 3H), 1.68 (s, 2H), 1.30 (s, 6H), 0.72 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 168.09, 146.02, 140.30, 139.53, 135.70,133.38, 131.34, 127.53, 126.13, 125.36, 123.14, 120.81, 115.81, 56.29, 37.59,32.02, 31.58,31.49, 20.78, 19.58; ESI-HRMS: C 23 H 32 O2N + ([M+H]) + Theoretical value: 354.24276; Measured value: 354.24229. HPLC purity: 99.69%.

[0035] Example 6 N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)-4-methylbenzamide (F2-3) Using compound 3a and p-methylbenzoic acid as starting materials, and following the synthesis method of F2, a white solid F2-3 (yield 73%) was obtained with a melting point of 165.3-170.4℃.

[0036] ¹H NMR (600 MHz, DMSO-d6) δ 9.52 (s, 1H), 7.69 (s, 1H), 7.54 (d, J=7.5 Hz, 1H), 7.46-7.36 (m, 1H), 7.30 (t, J=8.3 Hz, 2H), 7.04 (dd, J=8.4, 2.4Hz, 1H), 6.82(d, J=8.4 Hz, 1H), 2.52 (s, 1H), 2.43 (s, 3H), 1.68 (s, 2H), 1.30 (s, 6H), 0.72 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 168.11, 146.21, 140.29, 136.42, 135.52,130.63, 129.83, 127.37, 125.65, 125.21, 123.31, 121.09, 115.82, 56.30, 37.61,32.05, 31.61,31.52, 19.53; ESI-HRMS: C 22 H 30 O2N + ([M+H]) + Theoretical value: 340.22711; Measured value: 340.22679. HPLC purity: 98.98%.

[0037] Example 7 N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)-2-methylbenzamide (F2-4) Using compound 3a and o-methylbenzoic acid as starting materials, and following the synthesis method of F2, a yellow solid F2-4 (yield 76%) was obtained with a melting point of 165.1-170.4 °C.

[0038] ¹H NMR (400 MHz, DMSO-d6) δ 9.54 (s, 1H), 9.45 (s, 1H), 7.93-7.86 (m,2H), 7.68 (s, 1H), 7.34 (d, J=7.8 Hz, 2H), 7.05 (d, J=8.4 Hz, 1H), 6.82 (d, J=8.4 Hz, 1H), 2.39 (s, 3H), 1.68 (s, 2H), 1.30 (s, 6H), 0.72 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 165.27, 146.56, 141.72, 140.22, 131.39,129.31, 129.11, 129.02, 127.59, 125.18, 123.29, 121.59, 115.67, 56.36, 37.63,32.07, 31.60,31.53, 21.02; ESI-HRMS: C 22 H 30 O2N + ([M+H]) +Theoretical value: 340.22711; Measured value: 340.22657. HPLC purity: 96.01%.

[0039] Example 8 3,5-Dichloro-N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)benzamide (F2-5) Using compounds 3a and 3,5-dichlorobenzoic acid as starting materials, and following the synthesis method of F2, a white solid F2-5 (yield 77%) was obtained with a melting point of 181.8-185.1 °C.

[0040] ¹H NMR (600 MHz, DMSO-d6) δ 9.83 (s, 1H), 9.28 (s, 1H), 8.01 (s, 2H), 7.85 (t, J=2.0 Hz, 1H), 7.52-7.48 (m, 1H), 7.08 (dd, J=8.5, 2.4 Hz, 1H), 6.82(d, J=8.5 Hz,1H), 1.67 (s, 2H), 1.29 (s, 6H), 0.71 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 162.69, 147.78, 140.03, 137.78, 134.20,130.77, 126.54, 124.14, 124.10, 123.22, 115.56, 56.35, 37.56, 32.03, 31.58,31.48; ESI-HRMS: C 21 H 26 O2NCl2⁺([M+H)) + Theoretical value: 394.13351; Measured value: 394.13317. HPLC purity: 99.88%.

[0041] Example 9 4-tert-butyl-N-(2-hydroxy-5-tert-pentylphenyl)benzamide (F2-6) Using 4-tert-amylphenol (3b) and 4-tert-butylbenzoic acid as starting materials, and following the synthesis method of F2, a pale yellow solid F2-6 (yield 68%) was obtained with a melting point of 174.6-178.5 °C.

[0042] ¹H NMR (600 MHz, DMSO-d6) δ 9.53 (s, 1H), 9.48 (s, 1H), 7.91 (d, J=8.0 Hz, 2H), 7.68-7.63 (m, 1H), 7.55 (dd, J=6.3, 4.1 Hz, 2H), 6.99 (dd, J=8.3, 2.5 Hz, 1H), 6.84 (d, J=8.4 Hz, 1H), 1.58 (q, J=7.3 Hz, 2H), 1.32 (s,9H), 1.21 (s, 6H), 0.65 (t, J=7.4 Hz, 3H); ¹³C NMR (151 MHz, DMSO-d6) δ 165.27, 154.57, 146.60, 139.58, 131.51,127.37, 125.42, 125.28, 122.97, 121.20, 115.81, 39.94, 39.80, 39.66, 39.52,39.38,39.24, 39.10, 36.95, 36.29, 34.68, 30.91, 28.49, 9.10; ESI-HRMS: C 22 H 30 O2N + ([M+H]) + Theoretical value: 340.22711; Measured value: 340.22656. HPLC purity: 99.86%.

[0043] Example 10 4-tert-butyl-N-(5-tert-butyl-2-hydroxyphenyl)benzamide (F2-7) Using 4-tert-butylphenol (3c) and 4-tert-butylbenzoic acid as starting materials, and following the synthesis method of F2, a pale yellow solid F2-7 (yield 72%) was obtained with a melting point of 199.6-204.8 °C.

[0044] ¹H NMR (600 MHz, DMSO-d6) δ 9.54 (s, 1H), 9.49 (s, 1H), 7.91 (d, J=8.0 Hz, 2H), 7.71 (d, J=2.7 Hz, 1H), 7.58-7.52 (m, 2H), 7.06 (dd, J=8.4, 2.5Hz, 1H), 6.84 (d,J=8.4 Hz, 1H), 1.32 (s, 9H), 1.26 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 165.29, 154.58, 146.74, 141.41, 131.50,127.37, 125.39, 125.29, 122.34, 120.63, 115.83, 39.94, 39.80, 39.66, 39.52,39.38, 39.24,39.10, 34.68, 33.80, 31.37, 30.92; ESI-HRMS: C 21 H 28 O2N + ([M+H]) + Theoretical value: 326.21146; Measured value: 326.21102. HPLC purity: 99.66%.

[0045] Example 11 2-((4-tert-butylbenzyl)amino)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-8) Compound 3a (1 mmol, 221 mg) and 4-tert-butylbenzyl chloride (1 mmol, 182 mg) were dissolved in anhydrous tetrahydrofuran (THF, 20 mL), and triethylamine (2 mmol, 0.28 mL) was added. The mixture was stirred at room temperature for 7 hours. After the reaction was complete, distilled water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 15:1) to give a white solid F2-8 (250 mg, yield 68%) with a melting point of 158.4–161.7 °C.

[0046] ¹H NMR (600 MHz, DMSO-d6) δ 8.92 (s, 1H), 7.27 (q, J=8.1, 7.1 Hz,4H), 6.51 (d, J=8.0 Hz, 1H), 6.40-6.27 (m, 2H), 5.16 (t, J=6.1 Hz, 1H), 4.26(d, J=6.1 Hz,2H), 1.45 (s, 2H), 1.23 (s, 9H), 1.14 (s, 6H), 0.49 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 148.65, 141.76, 139.95, 137.65, 135.93,126.82, 124.84, 112.87, 112.58, 109.36, 56.29, 46.34, 37.49, 34.06, 31.83,31.64, 31.29, 31.16; ESI-HRMS: C 25 H 38 ON + ([M+H]) + Theoretical value: 368.29479; Measured value: 368.29435. HPLC purity: 98.00%.

[0047] Example 12 2-((2,4-dichlorobenzyl)amino)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-9) Using compound 3a and 2,4-dichloro-1-chloromethylbenzene as starting materials, and following the synthesis method of F2-8, a yellow solid F2-9 (yield 65%) was obtained with a melting point of 88.9-92.5 °C.

[0048] ¹H NMR (600 MHz, DMSO-d6) δ 9.04 (s, 1H), 7.58 (d, J=2.2 Hz, 1H), 7.39-7.26 (m, 2H), 6.54 (d, J=8.0 Hz, 1H), 6.40-6.31 (m, 1H), 6.17-6.09 (m,1H), 5.49 (t, J=6.2Hz, 1H), 4.37 (d, J=6.2 Hz, 2H), 1.43 (s, 2H), 1.13 (s,6H), 0.49 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 141.97, 139.97, 136.89, 135.12, 132.90,131.71, 130.25, 128.40, 127.22, 113.45, 112.81, 108.94, 56.35, 43.43, 37.47,31.75, 31.55,31.18; ESI-HRMS: C 21 H 28 ONCl2 + ([M+H]) +Theoretical value: 380.15425; Measured value: 380.15365. HPLC purity: 97.31%.

[0049] Example 13 2-((2,4-dimethylbenzyl)amino)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-10) Using compound 3a and 2,4-dimethyl-1-chloromethylbenzene as starting materials, and following the synthesis method of F2-8, yellow solid F2-10 (yield 67%) was obtained with a melting point of 86.3-92.4 °C.

[0050] ¹H NMR (600 MHz, DMSO-d6) δ 8.94 (s, 1H), 7.13 (d, J=7.6 Hz, 1H), 6.97 (s, 1H), 6.91-6.85 (m, 1H), 6.53 (d, J=7.8 Hz, 1H), 6.35 (d, J=7.7 Hz,2H), 4.85 (t,J=5.7 Hz, 1H), 4.21 (d, J=5.5 Hz, 2H), 2.30 (s, 3H), 2.21 (s,3H), 1.48 (s, 2H), 1.16 (s, 6H), 0.57 (s, 9H); ¹³C NMR (151 MHz, DMSO-d6) δ 141.69, 140.09, 136.22, 135.28, 135.11,134.93, 130.66, 127.46, 126.16, 113.04, 112.61, 109.05, 56.35, 44.69, 37.52,31.86, 31.61,31.28, 20.54, 18.59; ESI-HRMS: C 23 H 34 ON + ([M+H]) + Theoretical value: 340.26349; Measured value: 340.26301. HPLC purity: 97.26%.

[0051] Example 14 1-Benzyloxy-2-nitro-4-(2,4,4-trimethylpentan-2-yl)benzene (Compound 4) Compound 2 (4 mmol, 1004 mg) and benzyl chloride (4 mmol, 644 mg) were dissolved in anhydrous DMF (40 mL), and potassium carbonate (8 mmol, 1104 mg) was added. The mixture was stirred at 60 °C for 4 hours. After the reaction was complete, distilled water (50 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined and washed successively with distilled water (2 × 50 mL) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to give a yellow oily product, compound 4 (833 mg, yield 83%).

[0052] ¹H NMR (500 MHz, CDCl3) δ 7.85 (s, 1H), 7.48 (dd, J=16.7, 8.1 Hz, 3H), 7.39 (t, J=7.4 Hz, 2H), 7.33 (t, J=7.1Hz, 1H), 7.03 (d, J=8.7 Hz, 1H), 5.22 (s, 2H), 1.72 (s, 2H), 1.36 (s, 6H), 0.73 (s, 9H).

[0053] Example 15 2-Benzyloxy-5-(2,4,4-trimethylpentane-2-yl)aniline (Compound 5) Compound 4 (1 mmol, 341 mg) was dissolved in an ethanol:water mixture (16 mL: 4 mL), and iron powder (6 mmol, 336 mg) and ammonium chloride (2 mmol, 107 mg) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, insoluble matter was removed by filtration, and ethanol was removed by vacuum distillation. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 50 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to give a yellow oily product, compound 5 (230 mg, 74% yield).

[0054] ¹H NMR (500 MHz, CDCl3) δ 7.46 (d, J=7.4 Hz, 2H), 7.40 (t, J=7.5 Hz,2H), 7.35 (t, J=7.1 Hz, 1H), 6.79 (dd, J=4.9,3.0 Hz, 2H), 6.72 (d, J=8.4 Hz,1H), 5.07 (s, 2H), 3.78 (s, 2H), 1.69 (s, 2H), 1.34 (s, 6H), 0.77 (s, 9H).

[0055] Example 16 4-tert-butyl-N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)benzenesulfonamide (F2-11) Compound 5 (0.5 mmol, 155.5 mg) and 4-tert-butylbenzenesulfonyl chloride (0.5 mmol, 116 mg) were dissolved in anhydrous THF (20 mL), and triethylamine (1 mmol, 0.14 mL) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, THF was removed by vacuum distillation, and the residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 50 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a pale yellow oily intermediate. The crude product was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 50.7 mg) and ammonium formate (2 mmol, 126 mg) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, palladium on carbon was removed by filtration, the filter cake was washed with methanol (3 × 10 mL), and methanol was removed by vacuum distillation. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 25 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give a pale yellow solid F2-11 (154 mg, yield 74%), melting point: 104.3–109.0 °C.

[0056] ¹H NMR (400 MHz, DMSO-d6) δ 9.27 (s, 1H), 9.07 (s, 1H), 7.70 (d, J=7.6 Hz, 2H), 7.54 (d, J=7.6 Hz, 2H), 6.89 (d, J=5.7 Hz, 2H), 6.66 (d, J=8.7Hz, 1H), 1.50 (s,2H), 1.26 (s, 9H), 1.12 (s, 6H), 0.54 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 155.44, 147.60, 139.61, 137.73, 126.78,125.78, 123.36, 123.30, 121.39, 114.93, 56.20, 37.33, 34.80, 31.86, 31.41,31.37, 30.77; ESI-HRMS: C 24 H 36 O3NS + ([M+H]) + Theoretical value: 418.24104; Measured value: 418.24104. HPLC purity: 99.07%.

[0057] Example 17 N-(2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl)-2,4-dimethylbenzenesulfonamide (F2-12) Using compound 5 and 2,4-dimethylbenzenesulfonyl chloride as starting materials, and following the synthesis method of F2-11, a pale yellow oily product F2-12 (yield 71%) was obtained.

[0058] ¹H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.96 (s, 1H), 7.59 (d, J=8.0 Hz, 1H), 7.16 (s, 1H), 7.03 (d, J=8.1 Hz, 1H), 6.99 (s, 1H), 6.88 (d, J=8.2 Hz, 1H), 6.61 (d,J=8.3 Hz, 1H), 2.58 (s, 3H), 2.27 (s, 3H), 1.52 (s, 2H), 1.15 (s, 6H), 0.57 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 147.47, 142.72, 139.69, 136.91, 135.42,132.83, 129.54, 126.26, 123.35, 123.02, 121.75, 114.72, 56.22, 37.34, 31.86,31.38, 31.36,20.67, 19.65; ESI-HRMS: C 22 H 32 O3NS + ([M+H]) + Theoretical value: 390.20974; Measured value: 390.20898. HPLC purity: 99.21%.

[0059] Example 18 4-(2,4,4-trimethylpentan-2-yl)phenyltrifluoromethanesulfonate (compound 6) Under nitrogen protection, 4-(2,4,4-trimethylpentan-2-yl)phenol (compound 1) (5 mmol, 1030 mg) was dissolved in anhydrous dichloromethane (20 mL) and cooled to 0 °C in an ice bath. Pyridine (10 mmol, 0.8 mL) was added dropwise, and the mixture was stirred at 0 °C for 20 min, followed by the addition of trifluoromethanesulfonic anhydride ((TfO)2, 6 mmol), and stirring was continued at 0 °C for 1 h. After the reaction was complete, the reaction was quenched with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1) to give a colorless oily product, compound 6 (890 mg, yield 53%).

[0060] ¹H NMR (400 MHz, DMSO-d6) δ 7.59-7.54 (m, 2H), 7.40-7.33 (m, 2H), 1.74 (s, 2H), 1.34 (s, 6H), 0.66 (s, 9H).

[0061] Example 19 N-Benzyl-4-(2,4,4-trimethylpentan-2-yl)aniline (Compound 7) Under nitrogen protection, compound 6 (1 mmol, 338 mg) and benzylamine (1.5 mmol, 160.5 mg) were dissolved in anhydrous 1,4-dioxane (15 mL), and tris(dibenzylacetone)palladium (Pd(dba)3, 0.2 mmol, 183 mg), 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (XPhos, 0.2 mmol, 95 mg), and cesium carbonate (2 mmol, 650 mg) were added. The mixture was stirred at 100 °C for 20 h. After the reaction was complete, the solvent was removed by vacuum distillation, and the residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 50:1) to give a yellow solid compound 7 (245 mg, yield 83%) with a melting point of 39.8–44.1 °C.

[0062] ¹H NMR (400 MHz, DMSO-d6) δ 7.31 (dd, J=15.1, 7.8 Hz, 4H), 7.21 (t, J=7.1 Hz, 1H), 7.03 (d, J=8.5 Hz, 2H), 6.49 (d, J=8.5 Hz, 2H), 5.96 (t, J=6.0Hz, 1H), 4.22 (d, J=5.8 Hz, 2H), 1.61 (s, 2H), 1.23 (s, 6H), 0.67 (s, 9H).

[0063] Example 20 N1-Benzyl-4-(2,4,4-trimethylpentan-2-yl)benzene-1,2-diamine (Compound 8) Compound 7 (0.5 mmol, 147.5 mg) was dissolved in acetonitrile (15 mL), and tert-butyl nitrite (2 mmol, 206 mg) was added. The mixture was stirred at 80 °C for 2 hours. After the reaction was complete, the mixture was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a yellow oily residue, which was used directly in the next reaction. The crude product (0.5 mmol, 184 mg) was dissolved in acetic acid (10 mL), and zinc powder (2 mmol, 130 mg) was added. The mixture was stirred at 50 °C for 3 hours. After the reaction was complete, the insoluble matter was removed by filtration, the filter cake was washed with methanol (3 × 10 mL), and the solvent was removed by evaporation under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 25 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a yellow solid compound 8 (103 mg, yield 67%) with a melting point of 52.1–56.8 °C.

[0064] ¹H NMR (400 MHz, CDCl3) δ 7.40 (d, J=7.5 Hz, 2H), 7.34 (t, J=7.3 Hz, 2H), 7.29 (d, J=6.9 Hz, 1H), 6.81-6.74(m, 2H), 6.65-6.57 (m, 1H), 4.29 (s, 2H), 3.15 (s, 2H), 1.66 (s, 2H), 1.31 (s, 6H), 0.75 (s, 9H).

[0065] Example 21 N-(2-amino-5-(2,4,4-trimethylpentan-2-yl)phenyl)-4-tert-butylbenzamide (F2-13) Compound 8 (0.32 mmol, 100 mg) was dissolved in anhydrous THF (10 mL), and 4-tert-butylbenzoyl chloride (0.32 mmol, 62.7 mg) and triethylamine (0.64 mmol, 88 μL) were added. The mixture was stirred at room temperature for 1 hour. After the reaction was complete, the reaction was quenched with distilled water (30 mL), and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL). After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to give a pale yellow oily residue. The residue was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 30 mg) and ammonium formate (1 mmol, 64 mg) were added. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 25 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a white solid F2-13 (21 mg, yield 17%), melting point: 109.0–113.7 °C.

[0066] ¹H NMR (400 MHz, DMSO-d6) δ 9.70 (s, 1H), 7.93 (d, J=8.1 Hz, 2H), 7.53 (d, J=8.1 Hz, 2H), 7.21 (s, 1H), 7.03 (dd, J=8.4, 2.2 Hz, 1H), 6.77 (d,J=8.3 Hz, 1H), 1.65(s, 2H), 1.32 (s, 9H), 1.28 (s, 6H), 0.72 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 165.05, 154.28, 138.42, 131.79, 127.67,125.04, 124.06, 123.93, 123.73, 116.74, 56.33, 37.45, 34.68, 32.08, 31.67,31.61, 31.53, 30.98; ESI-HRMS: C 25 H 37 ON2 + ([M+H]) +Theoretical value: 381.29004; Measured value: 381.28945. HPLC purity: 96.62%.

[0067] Example 22 2-Hydroxy-5-(2,4,4-trimethylpentan-2-yl)benzaldehyde (Compound 9) 4-(2,4,4-trimethylpentan-2-yl)phenol (compound 1) (10 mmol, 2060 mg) and paraformaldehyde (PFA, 67.5 mmol, 6075 mg) were dissolved in acetonitrile (50 mL), and triethylamine (40 mmol, 5.5 mL) and magnesium chloride (15 mmol, 425 mg) were added. The mixture was refluxed and stirred for 8 hours. After the reaction was complete, the insoluble matter was removed by filtration, and the filter cake was washed with acetonitrile (3 × 10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was diluted with distilled water (50 mL), the pH was adjusted to neutral with 1 M hydrochloric acid, and extracted with ethyl acetate (3 × 25 mL). The organic phases were combined, washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 30:1) to give a pale yellow oily product, compound 9 (1497 mg, yield 64%).

[0068] ¹H NMR (600 MHz, CDCl3) δ 10.89 (s, 1H), 9.92 (s, 1H), 7.59 (dd, J=8.8, 2.5 Hz, 1H), 7.51 (d, J=2.5 Hz,1H), 6.95 (d, J=8.8 Hz, 1H), 1.76 (s, 2H), 1.40 (s, 6H), 0.76 (s, 9H).

[0069] Example 23 2-Benzyloxy-5-(2,4,4-trimethylpentan-2-yl)benzaldehyde (Compound 10) Compound 9 (4 mmol, 940 mg) and benzyl chloride (4 mmol, 644 mg) were dissolved in anhydrous DMF (40 mL), and potassium carbonate (8 mmol, 1104 mg) was added. The mixture was stirred at 60 °C for 8 hours. After the reaction was complete, the reaction was quenched with distilled water (50 mL), and extracted with ethyl acetate (3 × 50 mL). The organic phases were combined and washed successively with distilled water (2 × 50 mL) and saturated sodium chloride solution (50 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30:1) to give a pale yellow oily product, compound 10 (1010 mg, yield 78%).

[0070] ¹H NMR (400 MHz, CDCl3) δ 10.55 (s, 1H), 7.86 (d, J=2.7 Hz, 1H), 7.55(dd, J=8.7, 2.7 Hz, 1H), 7.46-7.34(m, 5H), 6.98 (d, J=8.8 Hz, 1H), 5.18 (s,2H), 1.73 (s, 2H), 1.36 (s, 6H), 0.71 (s, 9H).

[0071] Example 24 2-Benzyloxy-5-(2,4,4-trimethylpentan-2-yl)benzoic acid (Compound 11) Compound 10 (2 mmol, 648 mg) was dissolved in a mixed solvent of tetrahydrofuran:tetrabutylammonium:water (24 mL:24 mL:8 mL), and an aqueous solution of sodium dihydrogen phosphate monohydrate (10 mmol, 1560 mg) (8 mL) was added, followed by sodium chlorite (10 mmol, 900 mg). The mixture was stirred at room temperature for 6 hours. After the reaction was complete, the organic solvent was removed by vacuum distillation, and the residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 15:1) to give compound 11 (388 mg, yield 57%) as a white solid with a melting point of 92.4–96.9 °C.

[0072] ¹H NMR (500 MHz, DMSO-d6) δ 12.58 (s, 1H), 7.64 (d, J=2.6 Hz, 1H), 7.52-7.47 (m, 3H), 7.38 (t, J=7.5 Hz, 2H), 7.31 (t, J=7.4 Hz, 1H), 7.09 (d, J=8.7 Hz, 1H), 5.16 (s, 2H), 1.70 (s, 2H), 1.31 (s, 6H), 0.68 (s, 9H).

[0073] Example 25 N-(4-tert-butylphenyl)-2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)benzamide (F2-14) Compound 11 (0.5 mmol, 170 mg) and 4-tert-butylaniline (0.5 mmol, 74.5 mg) were dissolved in anhydrous DMF (20 mL). EDCI (0.5 mmol, 96 mg), HOBt (0.6 mmol, 81 mg), and DIEA (1 mmol, 0.175 mL) were added, and the mixture was stirred at room temperature for 7 hours. After the reaction was complete, distilled water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a pale yellow oily residue. The residue was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 47.1 mg) and ammonium formate (2 mmol, 126 mg) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The filtrates were combined and concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 25 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20:1) to give a brown solid F2-14 (129 mg, yield 68%) with a melting point of 196.2–200.4 °C.

[0074] ¹H NMR (500 MHz, DMSO-d6) δ 11.77 (s, 1H), 10.35 (s, 1H), 7.93 (s,1H), 7.58 (d, J=8.3 Hz, 2H), 7.49-7.44 (m, 1H), 7.39 (d, J=8.1 Hz, 2H), 6.90(d, J=8.6 Hz,1H), 1.72 (s, 2H), 1.35 (s, 6H), 1.29 (s, 9H), 0.70 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 166.45, 156.10, 146.63, 140.21, 135.43,131.65, 126.03, 125.32, 121.12, 116.56, 116.23, 56.14, 37.80, 34.11, 32.06,31.63, 31.46, 31.19; ESI-HRMS: C 25 H 36 O2N + ([M+H]) + Theoretical value: 382.27406; Measured value: 382.27336. HPLC purity: 99.71%.

[0075] Example 26 N-(2,4-dimethylphenyl)-2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)benzamide (F2-15) Using compound 11 and 2,4-dimethylaniline as starting materials, and following the synthesis method of F2-14, white solid F2-15 (yield 63%) was obtained with a melting point of 164.2-170.3 °C.

[0076] ¹H NMR (400 MHz, DMSO-d6) δ 11.95 (s, 1H), 10.29 (s, 1H), 8.02 (s,1H), 7.66 (d, J=8.0 Hz, 1H), 7.46 (d, J=8.6 Hz, 1H), 7.10 (s, 1H), 7.04 (d, J=8.1 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 2.28 (s, 3H), 2.23 (s, 3H), 1.72 (s, 2H), 1.34 (s, 6H), 0.70 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 166.02, 155.94, 140.27, 134.21, 133.62,131.59, 130.84, 130.63, 126.73, 126.43, 124.09, 116.51, 115.93, 56.14, 37.77,32.06, 31.61,31.45, 20.51, 17.67; ESI-HRMS: C 23 H 32 O2N + ([M+H]) + Theoretical value: 354.24276; Measured value: 354.24241. HPLC purity: 98.60%.

[0077] Example 27 2-Hydroxy-N-(o-tolyl)-5-(2,4,4-trimethylpentan-2-yl)benzamide (F2-16) Using compound 11 and 2-methylaniline as starting materials, and following the synthesis method of F2-14, a pale yellow solid F2-16 (yield 65%) was obtained with a melting point of 159.7-163.4℃.

[0078] ¹H NMR (400 MHz, DMSO-d6) δ 11.89 (s, 1H), 10.37 (s, 1H), 8.03 (s,1H), 7.86 (d, J=8.0 Hz, 1H), 7.47 (d, J=8.6 Hz, 1H), 7.33-7.19 (m, 2H), 7.12(t, J=7.5 Hz,1H), 6.92 (d, J=8.5 Hz, 1H), 2.28 (s, 3H), 1.72 (s, 2H), 1.34(s, 6H), 0.70 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 165.70, 155.63, 140.37, 136.33, 131.64,130.34, 130.30, 126.63, 126.29, 124.99, 123.73, 116.52, 116.17, 56.14, 37.76,32.06, 31.62,31.44, 17.75; ESI-HRMS: C 22 H 30 O2N +([M+H]) + Theoretical value: 340.22711; Measured value: 340.22666. HPLC purity: 97.69%.

[0079] Example 28 2-Benzyloxy-5-(2,4,4-trimethylpentan-2-yl)phenol (Compound 12) Compound 11 (2 mmol, 648 mg) was dissolved in anhydrous dichloromethane (DCM, 40 mL), and m-chloroperoxybenzoic acid (m-CPBA, 3 mmol, 516 mg) was added. The mixture was stirred at room temperature for 12 hours. After the reaction was complete, the mixture was quenched with saturated sodium sulfite solution, and extracted with distilled water (30 mL) and DCM (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 15:1) to give a pale yellow solid, compound 12 (62% yield), melting point: 63.1–67.3 °C.

[0080] ¹H NMR (400 MHz, DMSO-d6) δ 8.76 (s, 1H), 7.46 (d, J=7.4 Hz, 2H), 7.37 (t, J=7.4 Hz, 2H), 7.31 (d, J=6.8 Hz, 1H), 6.84 (d, J=8.2 Hz, 2H), 6.69 (d, J=8.5 Hz, 1H), 5.05 (s, 2H), 1.63 (s, 2H), 1.25 (s, 6H), 0.69 (s, 9H).

[0081] Example 29 2-((4-tert-butylbenzyl)oxy)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-17) Compound 12 (0.5 mmol, 156 mg) and 4-tert-butylbenzyl chloride (0.5 mmol, 91 mg) were dissolved in anhydrous THF (20 mL), and triethylamine (1 mmol, 0.14 mL) was added. The mixture was stirred at room temperature for 6 hours. After the reaction was complete, distilled water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and sodium chloride solution (30 mL). After drying with anhydrous sodium sulfate, the mixture was concentrated under reduced pressure to give a pale yellow oily residue. The crude product was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 45.8 mg) was added. The mixture was stirred at room temperature for 30 minutes under a hydrogen atmosphere. The palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 25 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a white solid F2-17 (yield 42%) with a melting point of 95.5–100.1 °C.

[0082] ¹H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.36 (s, 4H), 6.83 (d, J=2.0 Hz, 1H), 6.71-6.63 (m, 2H), 5.09 (s, 2H), 1.55 (s, 2H), 1.26 (s, 9H), 1.21 (s, 6H), 0.54 (s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 149.90, 145.36, 144.55, 140.01, 134.66,127.35, 125.00, 118.32, 114.94, 113.24, 69.47, 56.29, 38.89, 37.60, 34.22,31.89, 31.57, 31.37,31.13; ESI-HRMS: C 25 H 37 O2⁺ ([M+H)) + Theoretical value: 369.27881; Measured value: 369.27841. HPLC purity: 97.33%.

[0083] Example 30 2-Hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl-4-tert-butylbenzoate (F2-18) Compound 12 (0.5 mmol, 156 mg) was dissolved in anhydrous THF (20 mL) with 4-tert-butylbenzoyl chloride (0.5 mmol, 98 mg), and triethylamine (1 mmol, 0.14 mL) was added. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, distilled water (30 mL) was added, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic phases were washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give a pale yellow oily residue. The crude product was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 47.2 mg) and ammonium formate (2 mmol, 126 mg) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The filtrate was concentrated under reduced pressure. The residue was diluted with distilled water (30 mL) and extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a pale yellow solid F2-18 (yield 51%) with a melting point of 149.0–154.8 °C.

[0084] ¹H NMR (400 MHz, CDCl3) δ 8.15 (d, J=8.4 Hz, 2H), 7.54 (d, J=8.4 Hz,2H), 7.10-7.07 (m, 2H), 6.97 (dd, J=8.5,2.2 Hz, 1H), 1.73 (s, 2H), 1.37 (s,9H), 1.36 (s, 6H), 0.76 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 201.38, 160.90, 155.96, 140.34, 135.45,134.58, 130.48, 129.62, 125.43, 118.65, 117.72, 56.74, 38.12, 35.28, 32.46,32.02, 31.67, 31.32; ESI-HRMS: C 25 H 35 O3 + ([M+H]) +Theoretical value: 383.25807; Measured value: 383.25767. HPLC purity: 98.15%.

[0085] Example 31 2-Hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl 2,4-dichlorobenzoate (F2-19) Using compound 12 and 2,4-dichlorobenzoyl chloride as starting materials, and following the synthesis method of F2-18, a pale yellow solid F2-19 (yield 56%) was obtained with a melting point of 102.3-105.6 °C.

[0086] 1 H NMR (400 MHz, Chloroform- d ) δ 8.07 (d, J = 8.4 Hz, 1H), 7.57 (d, J =2.0 Hz, 1H), 7.41 (dd, J = 8.4, 2.0 Hz, 1H), 7.20 – 7.14 (m, 2H), 6.96 (d, J =8.2 Hz, 1H), 1.71 (s, 2H), 1.35 (s, 6H), 0.75 (s, 9H). 13 C NMR (101 MHz, Chloroform- d ) δ 199.62, 161.19, 141.06, 136.86, 136.18, 136.12, 132.05,130.13, 129.76, 127.38, 118.52, 117.93,56.63, 37.97, 32.41, 31.97, 31.49. ESI-HRMS calcd for C 21 H 25 O3Cl2[M + H] + 395.11753, found 395.11738. HPLC purity: 95.70%.

[0087] Example 32 Synthesis of 2-hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl-2,4-dimethylbenzoate (F2-20) Using compound 12 and 2,4-dimethylbenzoyl chloride as starting materials, and following the synthesis method of F2-18, a pale yellow solid F2-20 (yield 50%) was obtained with a melting point of 103.1-107.9 °C.

[0088] ¹H NMR (400 MHz, CDCl3) δ 8.09 (d, J=8.5 Hz, 1H), 7.17-7.11 (m, 2H), 7.10-7.06 (m, 2H), 6.97 (dd, J=8.5, 2.2Hz, 1H), 2.65 (s, 3H), 2.41 (s, 3H), 1.73 (s, 2H), 1.36 (s, 6H), 0.77 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 169.87, 146.49, 142.94, 136.99, 134.92,131.65, 131.08, 127.08, 126.18, 125.75, 124.80, 119.89, 119.71, 57.00, 38.12,32.53, 31.99,31.74, 20.18; ESI-HRMS: C 23 H 31 O3 + ([M+H]) + Theoretical value: 355.22677; Measured value: 355.22632. HPLC purity: 99.23%.

[0089] Example 33 2-Hydroxy-5-(2,4,4-trimethylpentan-2-yl)phenyl-2-methylbenzoate (F2-21) Using compound 12 and 2-methylbenzoyl chloride as starting materials, and following the synthesis method of F2-18, a pale yellow solid F2-21 (yield 47%) was obtained with a melting point of 95.6-99.1℃.

[0090] ¹H NMR (400 MHz, CDCl3) δ 8.18 (dd, J=8.1, 1.5 Hz, 1H), 7.51 (td, J=7.5, 1.4 Hz, 1H), 7.37-7.31 (m, 2H), 7.12-7.08(m, 2H), 6.98 (dd, J=8.5, 2.2Hz, 1H), 2.69 (s, 3H), 1.74 (s, 2H), 1.36 (s, 6H), 0.77 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 167.15, 146.66, 143.36, 142.88, 130.47,130.37, 129.76, 129.37, 127.52, 125.60, 124.83, 120.03, 119.64, 57.04, 38.13,32.53, 31.98,31.77, 21.73; ESI-HRMS: C 22 H 29 O3 + ([M+H]) + Theoretical value: 341.21112; Measured value: 341.21093. HPLC purity: 99.51%.

[0091] Example 34 (4-tert-butylbenzyl)phosphonate diethyl ester (14a) Triethyl phosphite (9 mmol, 1494 mg) was thoroughly mixed with 4-tert-butyl-1-bromomethylbenzene (13) (3 mmol, 678 mg), and the mixture was heated to 150 °C and stirred for 5 hours. After the reaction was completed, triethyl phosphite was removed by rotary evaporation, ethyl acetate (30 mL) was added, and then silica gel for column chromatography was added. The mixture was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give a colorless oily product 14a (605 mg, yield 71%).

[0092] ¹H NMR (400 MHz, DMSO-d6) δ 7.32 (d, J=8.1 Hz, 2H), 7.19 (dd, J=8.1,2.3 Hz, 2H), 3.18 (s, 1H), 3.13 (s, 1H), 1.26 (s, 9H), 1.26-1.21 (m, 4H),1.17 (t, J=7.0 Hz,6H).

[0093] Example 35 (E)-1-benzyloxy-2-(4-tert-butylstyryl)-4-(2,4,4-trimethylpentan-2-yl)benzene (15a) Under nitrogen protection, compound 10 (2.2 mmol, 712.8 mg) and compound 14a (2 mmol, 568 mg) were dissolved in anhydrous tetrahydrofuran (THF, 20 mL), and 60% sodium hydride (2.2 mmol, 88 mg) was added. The mixture was stirred at room temperature for 14 hours. After the reaction was completed, the reaction was quenched with ice water (30 mL), and the mixture was extracted with ethyl acetate (3 × 30 mL). The organic phases were combined and washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL). The organic phases were separated, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) to give a colorless oily product 15a (330 mg, yield 33%).

[0094] ¹H NMR (400 MHz, DMSO-d6) δ 7.61 (s, 1H), 7.50 (d, J=7.3 Hz, 2H), 7.45-7.34 (m, 8H), 7.29-7.22 (m, 2H), 7.02 (d, J=8.7 Hz, 1H), 5.17 (s, 2H), 1.73 (s, 2H), 1.35 (s, 6H), 1.28 (s, 9H), 0.71 (s, 9H).

[0095] Example 36 (E)-1-benzyloxy-2-(2-methylstyryl)-4-(2,4,4-trimethylpentan-2-yl)benzene (15b) Starting with compound 10 and diethyl (2-methylbenzyl)phosphonate (14b), the pale yellow oily product 15b (yield 37%) was obtained by following the synthesis method of 15a.

[0096] ¹H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J=7.0 Hz, 2H), 7.51 (d, J=7.4Hz, 2H), 7.45 (s, 1H), 7.40 (t, J=7.3 Hz, 2H), 7.35 (d, J=7.1 Hz, 1H), 7.28-7.22 (m, 2H),7.20-7.16 (m, 1H), 7.14 (d, J=6.4 Hz, 2H), 7.06 (d, J=8.6 Hz,1H), 5.15 (s, 2H), 2.19 (s, 3H), 1.74 (s, 2H), 1.35 (s, 6H), 0.72 (s, 9H).

[0097] Example 37 (E)-2-(4-tert-butylstyryl)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-22) Under nitrogen protection, compound 15a (0.3 mmol, 135 mg) was dissolved in anhydrous dichloromethane (DCM, 10 mL) and stirred in an ice bath for 30 minutes. 1 M boron trichloride (0.33 mmol, 0.33 mL) was added dropwise, and the reaction was continued in an ice bath for 1 hour. After the reaction was complete, ice water (30 mL) was added to quench the reaction, and the mixture was extracted with dichloromethane (3 × 30 mL). The organic phases were combined. The mixture was washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a pale yellow oil product F2-22 (49 mg, 45% yield).

[0098] ¹H NMR (400 MHz, CDCl3) δ 7.51-7.45 (m, 3H), 7.38 (d, J=7.4 Hz, 2H), 7.31 (d, J=16.3 Hz, 1H), 7.16-7.05(m, 2H), 6.74 (d, J=8.4 Hz, 1H), 1.73 (s,2H), 1.38 (s, 6H), 1.34 (s, 9H), 0.75 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 150.83, 150.78, 142.73, 135.07, 129.74,126.60, 126.35, 125.71, 125.12, 123.90, 123.31, 115.42, 57.11, 38.20, 34.76,32.54, 32.00,31.83, 31.46; ESI-HRMS: C 26 H 37 O + ([M+H]) + Theoretical value: 365.28389; Measured value: 365.28334. HPLC purity: 97.50%.

[0099] Example 38 (E)-2-(2-methylstyryl)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-23) Using compound 15b as the starting material, and following the synthesis method of F2-22, a colorless oily product F2-23 was obtained (yield 46%).

[0100] ¹H NMR (400 MHz, CDCl3) δ 7.64 (d, J=7.3 Hz, 1H), 7.46 (d, J=2.4 Hz,1H), 7.35 (d, J=16.2 Hz, 1H), 7.25-7.13(m, 5H), 6.75 (d, J=8.4 Hz, 1H), 5.04(d, J=7.1 Hz, 1H), 2.44 (s, 3H), 1.74 (s, 2H), 1.39 (s, 6H), 0.77 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 150.91, 142.80, 136.92, 135.83, 130.48,128.16, 127.59, 126.74, 126.33, 125.60, 125.55, 124.07, 115.46, 57.07, 38.18,32.53, 32.01,31.82, 20.07; ESI-HRMS: C 23 H 31 O + ([M+H]) + Theoretical value: 323.23694; Measured value: 323.23672. HPLC purity: 98.54%.

[0101] Example 39 2-(4-tert-butylphenethyl)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-24) Compound 15a (0.3 mmol, 135 mg) was dissolved in methanol (20 mL), and 10% palladium on carbon (20% by mass, 27 mg) and ammonium formate (1 mmol, 63 mg) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the palladium on carbon was removed by filtration, and the filter cake was washed with methanol (3 × 10 mL). The solvent was removed by evaporation under reduced pressure. Distilled water (30 mL) and ethyl acetate (3 × 25 mL) were added for extraction, and the organic phases were combined. The mixture was washed successively with distilled water (2 × 30 mL) and saturated sodium chloride solution (30 mL), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether / ethyl acetate = 20:1) to give a colorless oily product F2-24 (77 mg, 70% yield).

[0102] ¹H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 7.30-7.23 (m, 2H), 7.14-7.06 (m, 2H), 6.95 (dd, J=8.3, 2.4 Hz, 1H), 6.87 (d, J=2.4 Hz, 1H), 6.67 (d,J=8.3 Hz, 1H), 2.77(s, 4H), 1.59 (s, 2H), 1.25 (s, 9H), 1.19 (s, 6H), 0.62(s, 9H); ¹³C NMR (101 MHz, DMSO-d6) δ 152.45, 147.70, 139.16, 138.85, 128.02,127.65, 126.37, 124.79, 124.12, 114.11, 56.33, 37.32, 34.87, 34.00, 31.97,31.92, 31.59, 31.56,31.21; ESI-HRMS: C 26 H 38 ONa + ([M+Na]) + Theoretical value: 389.28149; Measured value: 389.28120. HPLC purity: 99.02%.

[0103] Example 40 2-(2-methylphenethyl)-4-(2,4,4-trimethylpentan-2-yl)phenol (F2-25) Using compound 15b as the starting material, and following the synthetic method of F2-24, a colorless oily product F2-25 was obtained (yield 72%).

[0104] ¹H NMR (400 MHz, CDCl3) δ 7.14 (hd, J=5.6, 4.8, 1.4 Hz, 4H), 7.07 (dd, J=8.3, 2.5 Hz, 1H), 7.03 (d, J=2.4 Hz,1H), 6.67 (d, J=8.3 Hz, 1H), 4.45(s, 1H), 2.90 (hept, J=3.0, 2.5 Hz, 4H), 2.30 (s, 3H), 1.68 (s, 2H), 1.31 (s,6H), 0.73 (s, 9H); ¹³C NMR (101 MHz, CDCl3) δ 151.19, 142.62, 140.31, 136.21, 130.37,129.13, 128.48, 127.06, 126.21, 126.15, 125.02, 114.83, 57.08, 38.03, 33.91,32.47, 31.94,31.82, 31.78, 19.38; ESI-HRMS: C 23 H 32 ONa + ([M+Na]) + Theoretical value: 347.23454; Measured value: 347.23446. HPLC purity: 95.26%.

[0105] Example 1 The HOXA1 protein structure was predicted using AlphaFold 3.0. Protein pretreatment followed standard procedures: all co-crystallization water molecules were removed, and potential structural issues were corrected using the "CleanProtein" and "PrepareProtein" modules in Discovery Studio software (including simulating missing ring regions, removing alternative conformations, adding hydrogen atoms, and specifying protonation states at pH 7.0). The active site was defined using spatial coordinates (X = -2.551 Å, Y = 14.504 Å, Z = -4.580 Å), with a docking sphere radius of 9 Å. Compound F2 underwent total energy minimization before docking, and structure-based virtual screening was performed using the LibDock module in Discovery Studio 3.0. The conformation of F2 at the docking site, the two-dimensional interaction between F2 and HOXA1, and a schematic diagram of the interaction are shown below. Figure 1 As shown.

[0106] Example 2 HepG2 cells were seeded in 12-well plates in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were transfected with the designated luciferase reporter gene plasmid using Lipofectamine™ 3000 (Thermo Fisher, catalog number L3000008) according to the manufacturer's instructions, with pTK-Renilla as the internal control plasmid. Twenty-four hours after transfection, cells were treated with 20 μM compound F2 for 48 hours as the experimental group, and an equal volume of medium was used to treat cells for 48 hours as the control group. After cell lysis, luciferase activity was measured using a dual-luciferase assay kit (Promega, Madison, Wisconsin, USA, catalog number E1910).

[0107] The results show that... Figure 2 As shown, F2 has a good inhibitory effect on the transcriptional activity of HOXA1.

[0108] Example 3 Lung cancer, liver cancer, colorectal cancer, and breast cancer are among the most common and deadliest malignant tumors worldwide, and HOXA1 is highly expressed in these tumor types. Therefore, this invention evaluated the antiproliferative activity of 26 synthetic compounds (F2, F2-1~F2-25) at a concentration of 20 μM against four human cancer cell lines (HepG2 liver cancer cells, NCI-H1975 lung cancer cells, HCT116 colorectal cancer cells, and MCF-7 breast cancer cells), using cisplatin as a positive control.

[0109] Cells were seeded at a density of 3000 cells per well in 96-well plates with 100 μL of culture medium per well, and each sample was divided into 3 replicates. Cells were cultured under standard conditions: 37 ℃ in a humidified incubator with 5% CO2. HepG2 and MCF-7 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin-streptomycin; NCI-H1975 used 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin; After culturing for 12 h, 20 μM of the compound was added to each well, and the culture was continued for 48 h. Then, 10 μL of CCK-8 solution was added to each well, and the mixture was incubated at 37 °C in the dark for 2 h. Finally, the absorbance was measured at 450 nm, and the antiproliferative activity of the compound was calculated using software. For compounds (F2-15, F2, F2-16, F2-10) exhibiting significant anti-proliferative activity, IC50 analysis was performed. 50 Measurement: HepG2, MCF-7, and HCT116 cells were seeded at a density of 4000 cells / well in 96-well plates and cultured at 37 °C for 12 h. The test drugs were prepared to concentrations of 100 μM, 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, and 0.78 μM using the corresponding 2:1 dilution method with the appropriate medium. The original culture medium was discarded, and 100 μL of the corresponding concentration of drug-containing medium was added to each well. The cells were cultured at 37 °C for 48 h. Then, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37 °C in the dark for 2 h. Finally, the absorbance was measured at 450 nm, and the IC50 of the compounds was calculated using software. 50 Value, per IC 50 The values ​​were obtained through three independent experiments, and the results are as follows: Figure 4 As shown, the results indicate that the compounds used in the test exhibited dose-dependent antiproliferative activity.

[0110] HepG2 and MCF-7 were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0111] HCT116 cells were cultured in McCoy's 5A medium supplemented with 10% FBS and 1% penicillin-streptomycin.

[0112] Example of effect 4 To evaluate the efficacy of F2-15 combined with cisplatin in killing cancer cells, colorectal cancer patient-derived xenografts (PDX) and triple-negative breast cancer PDX were subcutaneously transplanted into 6-week-old female nude mice (nu / nu). On day 12 after subcutaneous tumor transplantation, when the tumor volume reached 150-200 mm³, the mice were randomly divided into four groups: a solvent control group (saline), a cisplatin group (1 mg / kg), an F2-15 group (20 mg / kg), and a cisplatin (1 mg / kg) + F2-15 (20 mg / kg) combination group. Tumor volume was measured according to the xenograft experimental protocol.

[0113] Drug administration began on day 12 after subcutaneous tumor transplantation. Cisplatin was administered via intraperitoneal injection every two days, while group F2-15 received intraperitoneal injection once daily. Mice were sacrificed on day 25, and liver and kidney weights were measured.

[0114] All animal experimental procedures were strictly performed in accordance with the National Institutes of Health (NIH) Guidelines for the Feeding and Use of Laboratory Animals (2011, National Academy of Sciences Press).

[0115] The results are as follows Figure 5 As shown, F2-15 significantly inhibited tumor growth and made the colorectal cancer PDX model and the triple-negative breast cancer PDX model sensitive to cisplatin.

[0116] Plasma toxicity analysis: After treatment, blood was collected retroorbitally under isoflurane anesthesia. After blood coagulation, the blood was centrifuged at 3000×g for 10 minutes. Toxicity indicators (alanine aminotransferase ALT, aspartate aminotransferase AST, total protein TP, blood urea nitrogen BUN, creatinine), albumin (ALB), total bilirubin (TBIL), alkaline phosphatase (ALP), and total bile acids (TBA)) were measured on an automated clinical chemistry analyzer using standardized commercial kits to assess liver function. Renal function was assessed by analyzing creatinine (CRE) and uric acid (UA) levels.

[0117] The body weight (L), liver weight (M), and kidney weight (N) of mice treated with F2-15, as well as the levels of ALT (O), AST (P), TP (Q), ALB (R), TBIL (S), ALP (T), TBA (U), CRE (V), and UA (W) in blood samples (n=5) were measured and analyzed, and compared with the solvent group. The results are as follows: Figure 6 As shown, compound F2-15 exhibits good safety.

[0118] Xenograft tumor specimens were prepared into paraffin-embedded blocks and cut into 4 μm thick sections. After dewaxing with xylene and rehydration with graded ethanol, the sections were autoclaved with citrate buffer for antigen retrieval. The sections were then treated with 3% hydrogen peroxide at room temperature for 10 minutes to inhibit endogenous peroxidase activity, followed by blocking with 3% bovine serum albumin (BSA) at room temperature for 1 hour. The sections were incubated overnight at 4°C with primary antibody, washed with phosphate-buffered saline (PBS), and then incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. The sections were developed with 3,3'-diaminobenzidine (DAB, G1212, Servicebio) for 2–5 minutes, counterstained with hematoxylin, and observed and imaged under a Nikon microscope at 40× magnification.

[0119] The primary antibodies used included: HOXA1 antibody (HUABIO, catalog number ER63703, dilution ratio 1:1000), tubulin antibody (ABclonal, catalog number A6830, dilution ratio 1:1000), rabbit IgG (ABclonal, catalog number AS014, dilution ratio 1:3000), and mouse IgG (ABclonal, catalog number AS003, dilution ratio 1:3000).

[0120] The results are as follows Figure 7 As shown, F2-15 significantly reduced the expression of HOXA1 and the proliferation marker Ki67, while increasing the staining of Caspase-3 in the cisplatin-treated group.

[0121] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A substituted benzamide derivative or a pharmaceutical salt thereof, characterized in that, The structure of the substituted benzamide derivative is shown in Formula I: R1 is selected from C1-C5 alkyl or hydrogen; R2 is selected from hydroxyl or amino; X is -NHC(O)-, -NH(CH2) n -, -NHS(O)2-, -C(O)NH-, -O(CH2) n -, -OC(O)-, -(CH2) n -, -CH=CH- or -C C-; n is 1, 2, 3, 4 or 5; R3 is a mono- or poly-substituted group independently selected from C1-C5 alkyl or halogen.

2. The substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein The C1-C5 alkyl is selected from —CH3, —CH2CH3, —CH(CH3)2, —(CH2)2CH3, —(CH2)3CH3, —CH2CH(CH3)2, —C(CH3)3, —CH(CH3)CH2CH3, —(CH2)4CH3, —CH(CH3)(CH2)2CH3, —CH(CH2CH3)2, —CH2C(CH3)3, —CH2CH(CH3)CH2CH3, —C(CH3)2CH2CH3, —CH(CH3)CH(CH3)2, —(CH2)2CH(CH3)2.

3. The substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein: The selected from , , , , or .

4. The substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein: when X is -NHC(O)-, is or ; X is -NH(CH2) n - time, is ; X is -NHS(O)2-, is or ; X is -C(O)NH-, is , or ; X is -O(CH2) n - time, is ; X is -OC(O)-, is , , or ; X is -(CH2) n - time, is or ; X is -CH=CH, is ; n is 1, 2, 3, 4 or 5.

5. The substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein The structure of the compound is as follows: 。 6. A process for preparing a substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, characterized by, The preparation method is selected from one of the following routes: 。 7. A pharmaceutical composition comprising the substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-5, and optionally, one or more pharmaceutically acceptable carriers or excipients.

8. Use of the substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-5 in the preparation of a medicament for inhibiting HOXA1.

9. Use of the substituted benzamide derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-5 in the preparation of a medicament for treating tumors.

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