Patent Application
20250136555
The present invention belongs to the field of biomedicines, and particularly relates to a hydantoin compound with dual agonistic activity for PPARα/δ and medical use thereof as a PPARα/δ dual agonist.
Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear receptors, including three subtypes, PPARα, PPARδ, and PPARγ. Studies have shown that activation of PPARs plays a positive role in the improvement of metabolic diseases, cardiovascular and cerebrovascular diseases, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, organ regeneration, retinopathy, or tumors (Mol. Cells., 2012, 33, 217; J. Biomned. Sci., 2017, 24, 5; J. Med. Chem., 2017, 55, 4027; Endocr J., 2007, 54, 347). The development and application of PPAR agonists is a potential therapeutic strategy for intervention in the diseases described above. However, PPARγ agonists have been shown to have risks of weight gain, edema, fractures, and potential heart failure. Therefore, the development of selective PPARα/δ dual agonists may provide safe and effective new approaches for the treatment of the diseases described above.
Currently, the clinically researched PPARα/δ dual agonist is GFT505 (Elafibranor) developed by Genfit, France. GFT505 was tested in multiple clinical trials in nonalcoholic fatty liver disease, cholestatic cholangitis, and kidney diseases. Unfortunately, the interim analysis results of a phase III clinical trial against nonalcoholic steatohepatitis (NASH) show that GFT505 was substantially ineffective (NCT02704403), and the reason for the poor clinical trial effect may be related to the poor agonistic activity for PPARα/δ, poor metabolic stability, and short half-life of GFT505.
In conclusion, there is an urgent need clinically to develop a PPARα/δ dual agonist with high activity and excellent pharmacokinetic properties.
Objective: in order to solve the problems in the existing PPAR agonists, the present invention provides a novel hydantoin compound. The hydantoin compound of the present invention has a potent agonistic effect on PPARα and PPARδ, and has very weak agonistic activity for PPARγ, thereby having a good selectivity and good pharmacokinetic properties. Therefore, the compound and a pharmaceutically acceptable salt, a prodrug, a deuterated compound, or a solvate thereof may be used for preparing a PPARα/δ dual agonist.
Another objective of the present invention is to provide medical use of the hydantoin compound as a PPARα/γ dual agonist. The compound and the pharmaceutically acceptable salt, the prodrug, the deuterated compound, or the solvate thereof may be used for preparing a medicament for the prevention or treatment of a disease mediated by PPARα and/or PPARδ.
Technical scheme: in order to achieve the objectives described above, the present invention provides a hydantoin compound of formula (I) or a pharmaceutically acceptable salt thereof:
Preferably, in the hydantoin compound of formula (I) or the pharmaceutically acceptable salt thereof,
Further, the hydantoin compound further includes prodrugs, deuterated compounds, or solvates thereof.
In certain more preferred embodiments, the hydantoin compound or the pharmaceutically acceptable salt thereof of the present invention is any one of the compounds shown in Table 1 below:
The hydantoin compound or the pharmaceutically acceptable salt, the prodrug, the deuterated compound, or the solvate thereof described herein has a potent PPARα/δ dual agonistic effect, so that it may be used for preparing a PPARα/δ dual agonist.
The hydantoin compound or the pharmaceutically acceptable salt, the prodrug, the deuterated compound, or the solvate thereof described herein may be used for preparing a medicament for the prevention or treatment of a disease mediated by PPARα and/or PPARδ.
Specifically, the compound of the present invention may be used for preparing a medicament for the prevention and treatment of the following diseases mediated by PPARα and/or PPARδ.
The compound of the present invention may be used for preventing and treating metabolic diseases and cardiovascular and cerebrovascular diseases, including: insulin resistance, metabolic syndrome, type 1 or type 2 diabetes, hyperlipidemia, obesity, lipoma, painful lipomatosis, arteriosclerosis, myocardial ischemia, myocardial infarction, arrhythmia, coronary heart disease, hypertension, heart failure, myocardial hypertrophy, myocarditis, diabetic complications (including diabetic cardiomyopathy, diabetic nephropathy, diabetic ulcer, retinopathy, neuropathy, and the like), nonalcoholic fatty liver, nonalcoholic steatohepatitis, alcoholic fatty liver, liver cirrhosis, hyperuricemia, gout, osteoporosis, polycystic ovary syndrome (PCOS), stroke or cerebral infarction, and the like.
The compound of the present invention may be used for preventing and treating inflammatory diseases, autoimmune diseases, organ fibrosis diseases, neurodegenerative diseases, or secondary diseases caused by pathogen infections, including: primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), hepatic fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis lung disease, interstitial pneumonia, tuberculosis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), Behcet's disease, asthma, chronic obstructive pulmonary disease, chronic bronchitis, emphysema, bronchiolitis obliterans, allergic rhinitis, chronic rhinitis, sinusitis, systemic lupus erythematosus, rheumatoid arthritis, spondyloarthritis, osteoarthritis, synovitis, tendonitis, thromboangiitis obliterans, phlebitis, intermittent claudication, keloid, psoriasis, ichthyosis, bullous pemphigoid, dermatitis, contact dermatitis, pancreatitis, chronic nephritis, cystitis, meningitis, gastritis, septicemia, pyoderma gangrenosum, uveitis, Parkinson's disease, Alzheimer's disease, α-synucleinopathies, depression, multiple sclerosis, amyotrophic lateral sclerosis, fibromyalgia syndrome, neuralgia, Down's syndrome, Hallervorden-Spatz disease, Huntington's chorea, Wilson disease, or the like.
The compound of the present invention may be used for treating and modulating mitochondrial dysfunction and disorder diseases, including: myasthenia, myoclonus, exercise intolerance, Kearns-Sayre syndrome, chronic fatigue syndrome, Leigh's syndrome, mitochondrial myopathy-encephalopathy-hyperlactacidemia, stroke syndrome or stroke-like episodes, Duchenne muscular dystrophy, becker muscular dystrophy, Friedreich's ataxia, or the like.
The compound of the present invention may be used for treating tumors, including: bone cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hemangioma, granuloma, xanthoma, meningosarcoma, glioma, astrocytoma, medulloblastoma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, fibroneuroma, sarcoma, esophageal cancer, gastric cancer, pancreatic cancer, colorectal cancer, colon cancer, rectal cancer, renal cancer, prostate cancer, lymphoma, testicular cancer, interstitial cell carcinoma, lung cancer, liver cancer, skin cancer, malignant melanoma, basal cell carcinoma, or the like.
In certain embodiments, the hydantoin compound of the present invention may be used as a pharmaceutically acceptable salt. The salt may be a salt formed by the compound of the present invention with a metal (including sodium, potassium, calcium, magnesium, and the like) ion or a pharmaceutically acceptable amine (including ethylenediamine, ethanolamine, tromethamine, diisopropylamine, metformin, berberine, and the like) or an ammonium ion.
The present invention also provides a pharmaceutical composition for preventing or treating a disease mediated by PPARα and/or PPARS, wherein the pharmaceutical composition comprises a therapeutically effective amount of the hydantoin compound of formula (I) or the pharmaceutically acceptable salt, the prodrug, the deuterated compound, or the solvate thereof shown in Table 1 described herein as an active ingredient, and a pharmaceutically acceptable carrier. The carrier which may be arbitrarily mixed may be changed depending on the dosage form, administration form, and the like. Examples of carriers include excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, coloring agents, sweetening agents, and the like. The pharmaceutical composition may be in a pharmaceutically conventional formulation form, including capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches, and the like.
Further, the compound of the present invention may be used in combination with one or more other types of medicaments for preventing or treating the disease mediated by PPARα and/or PPARδ, including but not limited to the following combined administration cases.
Other types of prophylactic or therapeutic medicaments that may be selected for use in combination with the compound of the present invention may be one or more anti-diabetic medicaments.
Other types of prophylactic or therapeutic medicaments that may be selected for use in combination with the compound of the present invention may be one or more anti-obesity medicaments.
Other types of prophylactic or therapeutic medicaments that may be selected for use in combination with the compound of the present invention may be one or more anti-nonalcoholic fatty liver disease medicaments.
Other types of prophylactic or therapeutic medicaments that may be selected for use in combination with the compound of the present invention may be one or more anti-PBC or PSC medicaments.
Other types of prophylactic or therapeutic medicaments that may be selected for use in combination with the compound of the present invention may be one or more lipid-lowering medicaments.
The amount of the compound of formula (I) or the pharmaceutically acceptable salt, the prodrug, the deuterated compound, or the solvate thereof of the present invention may be appropriately changed depending on the age, body weight, and symptoms, administration routes of a patient. For adults, during oral administration, the lower limit of one dose is 0.01 mg (preferably 0.1 mg or 1 mg) and the upper limit of one dose is 1000 mg (preferably 500 mg); during intravenous administration, the lower limit of one dose is 0.001 mg (preferably 0.01 mg or 0.1 mg), and the upper limit of one dose is 500 mg (preferably 250 mg). Deviation from this dosage range may occur depending on the degree of diseases and dosage forms.
Considering that a “α,β-unsaturated ketone” structure in a GFT505 molecule may be the cause of poor stability of liver microsomes, the inventors replaced the “α,β-unsaturated ketone” fragment in the GFT505 molecule structure with a “hydantoin” structure fragment, designed, and synthesized the hydantoin compound of the present invention. By testing the agonistic activity of the hydantoin compound for PPARs, it was surprisingly found that: when the “hydantoin” fragment was adopted to replace the “α,β-unsaturated ketone” structure, a series of compounds with much stronger agonistic activities for PPARα and PPARS than those of GFT505 may be obtained, and particularly, the agonistic activities of the preferred compounds (such as compound 1) of the present invention for PPAα/δ may reach a picomolar level. It is noted that compound 1 is the first PPARα/δ dual agonist, which may achieve picomolar levels of both PPARα and PPARS agonistic activities. In addition, the compound of the present invention has stability far superior to that of GFT505 for human liver microsomes and excellent in-vivo pharmacokinetic properties.
Advantages: Compared with the prior art, the present invention has the following advantages:
The content of the present invention will be specifically described below through examples. In the present invention, the following examples are intended to better illustrate the present invention and not to limit the scope of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.
4-Hydroxy-3,5-dimethylbenzaldehyde (21 g, 140 mmol) was dissolved in acetonitrile (200 mL), and ethyl 2-bromoisobutyrate (100.5 g, 520 mmol), cesium carbonate (45.6 g, 140 mmol), potassium carbonate (38.6 g, 280 mmol), and potassium iodide (2.3 g, 14 mmol) were added. The reaction system was heated to 80° C. and reacted for 36 h. After the reaction was completed, the mixture was filtered under vacuum. The solvent was distilled off under reduced pressure, water (200 mL) was added for dilution, and the resulting solution was extracted with ethyl acetate (EA) (200 mL×3). The organic phase was washed with 1 N sodium hydroxide solution (200 mL×3) and saturated brine (200 mL×1) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=200:1) to give intermediate K-1 (yellow liquid, 16.3 g, 44.1% yield).
Intermediate K-1 (3.66 g, 13.85 mmol) was dissolved in ethanol (20 mL), and sodium borohydride (280 mg, 7.5 mmol) was added slowly in an ice bath. The mixture was stirred at room temperature for 4 h. After the reaction was completed, water (20 mL) was added for quenching. The solvent was distilled off under reduced pressure, water (30 mL) was added for dilution, and the resulting solution was extracted with EA (20 mL×3). The organic phase was washed with saturated sodium chloride solution (30 mL×1) and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure to give crude intermediate K-2, which was directly used in the next step without further purification.
All of crude intermediate K-2 obtained from the previous reaction was dissolved in dichloromethane (DCM) (20 mL), tetrabromomethane (13.6 g, 41 mmol) was added, and triphenylphosphine (9.9 g, 37.8 mmol) was added slowly in an ice bath. The mixture was stirred at room temperature for 8 h. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20:1) to give intermediate M-1 (yellow liquid, 3.54 g, 78.0% yield).
p-Trifluoromethylaniline A-1 (1.6 g, 10 mmol) was dissolved in acetonitrile (10 mL), and ethyl 2-bromoacetate (1.2 mL, 11 mmol) and cesium carbonate (3.3 g, 10 mmol) were added. The reaction system was heated to 80° C. and reacted for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (50 mL) was added, and the resulting solution was extracted with EA (50 mL×3). The organic phase was washed with saturated sodium chloride solution (50 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=40:1) to give intermediate A-2 (yellow solid, 1.24 g, 50.2% yield).
Intermediate A-2 (2.4 g, 10 mmol) was dissolved in acetic acid (10 mL) under argon atmosphere, and a suspension of sodium cyanate (3.9 g, 60 mmol) in acetic acid (20 mL) was added. The mixture was stirred at room temperature for 12 h. The reaction system was heated to 100° C. and stirred for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (50 mL) was added, and the resulting solution was extracted with EA (50 mL×3). The organic phase was washed with saturated sodium chloride solution (50 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=2:1) to give compound A-3 (white solid, 1.1 g, 45.1% yield).
Intermediate A-3 (244 mg, 1 mmol) was dissolved in acetonitrile (3 mL), and M-1 (492 mg, 1.5 mmol) and cesium carbonate (815 mg, 2.5 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 2 (colorless liquid, 469.8 mg, 95.4% yield).
Compound 2 (441 mg, 0.89 mmol) was dissolved in acetonitrile (3 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and acetic acid (10 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (20 mL) was added, and the resulting solution was extracted with EA (25 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 1 (white solid, 98.7 mg, 23.9% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.80 (s, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 6.98 (s, 2H), 4.63 (s, 2H), 4.54 (s, 2H), 2.15 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C23H23F3N2O5 [M+NH4]+: 482.1903, found: 482.1898.
Intermediate A-3 could also be synthesized according to the following route:
p-Iodotrifluoromethylbenzene (2.7 g, 10 mmol), cuprous oxide (1.4 g, 10 mmol), and hydantoin (1.5 g, 15 mmol) were added to a three-necked flask, and the reaction system was purged with argon. Anhydrous DMF (10 mL) was then added, and the reaction system was heated to 150° C. and reacted for 12 h. After the reaction was completed, the reaction solution was filtered through celite, water (50 mL) was added, and the resulting solution was extracted with EA (50 mL×3) and washed with saturated sodium chloride (50 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=4:1) to give compound A-3 (white solid, 949 mg, 38.9% yield).
Compound 2 could also be synthesized according to the following route:
Intermediate M-1 (3.28 g, 10 mmol) was dissolved in acetonitrile (10 mL), and hydantoin (1.5 g, 15 mmol) and cesium carbonate (4.89 g, 15 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give intermediate E-1 (white solid, 1.8 g, 51.7% yield). Intermediate E-1 (1.17 g, 5 mmol), cuprous iodide (189 mg, 1 mmol), potassium carbonate (1.37 g, 10 mmol), and (1R,2R)-(−)-N,N′-dimethyl-1,2-cyclohexanediamine (284 mg, 2 mmol) were added to a three-necked flask, and the reaction system was purged with argon. A solution of p-iodotrifluoromethylbenzene (1.6 g, 6 mmol) in toluene (15 mL) was added, and the reaction system was heated to 110° C. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1) to give compound 2 (colorless liquid, 1.23 g, 41.7% yield).
Referring to the method in Example 1, compound 2 (colorless liquid, 469.8 mg, 95.4% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.07 (s, 2H), 4.65 (s, 2H), 4.36 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 515.2 [M+Na]+.
Referring to the method in Example 1, p-trifluoromethylaniline was replaced by p-trifluoromethoxyaniline to give compound 3 (white solid, 62.0 mg, 66.9% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.76 (d, J=9.1 Hz, 2H), 7.43 (d, J=8.6 Hz, 2H), 6.97 (s, 2H), 4.60 (s, 2H), 4.53 (s, 2H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C23H23F3N2O6 [M+NH4]+: 498.1852, found: 498.1847.
Referring to the method in Example 1, p-trifluoromethylaniline was replaced by p-trifluoromethoxyaniline to give compound 4 (colorless liquid, 98 mg, 44.4% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.07 (s, 2H), 4.65 (s, 2H), 4.36 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 531.2 [M+Na]+.
Referring to the method in Example 1, hydantoin in the synthetic route of method (3) was replaced by 5,5-dimethylhydantoin to give compound 5 (white solid, 21 mg, 55.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.86 (d, J=8.4 Hz, 2H), 7.68 (d, J=8.3 Hz, 2H), 6.92 (s, 2H), 4.57 (s, 2H), 2.17 (s, 6H), 1.44 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C25H27F3N2O5 [M+NH4]+: 510.2216, found: 510.2211.
Referring to the method in Example 1, hydantoin in the synthetic route of method (3) was replaced by 5,5-dimethylhydantoin to give compound 6 (yellow liquid, 40 mg, 19.1% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J=8.4 Hz, 2H), 7.46 (d, J=8.4 Hz, 2H), 7.04 (s, 2H), 4.65 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.50 (s, 6H), 1.48 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 543.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-(trifluoromethoxy)iodobenzene, and hydantoin was replaced by 5,5-dimethylhydantoin to give compound 7 (white solid, 40 mg, 54.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.61 (s, 1H), 7.55 (d, J=9.0 Hz, 2H), 7.49 (d, J=8.9 Hz, 2H), 6.91 (s, 2H), 4.55 (s, 2H), 2.16 (s, 6H), 1.39 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C25H27F3N2O6 [M+NH4]+: 526.2165, found: 526.2159.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-(trifluoromethoxy)iodobenzene, and hydantoin was replaced by 5,5-dimethylhydantoin to give compound 8 (colorless liquid, 80 mg, 29.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.31 (s, 4H), 7.04 (s, 2H), 4.63 (s, 2H), 4.30 (q, 7.1 Hz, 2H), 2.20 (s, 6H), 1.48 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 559.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-methylbenzaldehyde, and ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromoacetate to give compound 9 (white solid, 85.2 mg, 66.0% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.96 (s, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.12 (d, J=11.3 Hz, 2H), 6.78 (d, J=8.3 Hz, 1H), 4.67 (s, 2H), 4.60 (s, 2H), 4.55 (s, 2H), 2.17 (s, 3H). HRMS (ESI) calcd. for C20H17F3N2O5 [M+Na]+: 445.0987, found: 445.0992.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-methylbenzaldehyde, and ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromoacetate to give compound 10 (white solid, 137.7 mg, 57.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.27 (d, J=6.9 Hz, 2H), 6.66 (d, J=8.1 Hz, 1H), 4.69 (s, 2H), 4.63 (s, 2H), 4.33 (s, 2H), 4.27 (q, J=7.1 Hz, 2H), 2.29 (s, 3H), 1.31 (t, J=7.2 Hz, 3H). MS (ESI): m/z 573.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-trifluoromethylbenzaldehyde to give compound 11 (white solid, 89 mg, 55.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.37 (s, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.63 (s, 1H), 7.55 (d, J=9.2 Hz, 1H), 6.89 (d, J=8.6 Hz, 1H), 4.65 (s, 2H), 4.59 (s, 2H), 1.53 (s, 6H). HRMS (ESI) calcd. for C22H18F6N2O5 [M+Na]+: 527.1018, found: 527.1012.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-trifluoromethylbenzaldehyde to give compound 12 (yellow liquid, 169 mg, 63.4% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.77-7.62 (m, 5H), 7.53 (d, J=8.3 Hz, 1H), 6.78 (d, J=8.9 Hz, 1H), 4.73 (s, 2H), 4.36 (s, 2H), 4.26 (q, J=6.3 Hz, 2H), 1.63 (s, 6H), 1.28 (t, J=6.3, 3H). MS (ESI): m/z 555.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-methyl-5-chlorobenzaldehyde to give compound 13 (white solid, 50 mg, 30.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.86 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.27 (s, 1H), 7.15 (s, 1H), 4.62 (s, 2H), 4.59 (s, 2H), 2.20 (s, 3H), 1.41 (s, 6H). HRMS (ESI) calcd. for C22H20ClF3N2O5 [M+Na]+: 507.0911, found: 507.0905.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-methyl-5-chlorobenzaldehyde to give compound 14 (yellow liquid, 174 mg, 67.8% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.9 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 7.31 (s, 1H), 7.16 (s, 1H), 4.66 (s, 2H), 4.38 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.24 (s, 3H), 1.53 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 535.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by 2-iodotrifluoromethylbenzene to give compound 15 (white solid, 37.2 mg, 80.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.90-7.76 (m, 3H), 7.71-7.64 (m, 1H), 6.92 (s, 2H), 4.54 (s, 2H), 4.42 (s, 2H), 2.16 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C23H23F3N2O5 [M+Na]+: 487.1457, found: 487.1453.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by 2-iodotrifluoromethylbenzene to give compound 16 (colorless liquid, 78.7 mg, 39.6% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.79 (d, J=7.5 Hz, 1H), 7.68 (t, J=7.2 Hz, 1H), 7.56 (t, J=7.7 Hz, 1H), 7.42 (d, J=7.7 Hz, 1H), 7.05 (s, 2H), 4.65 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.26 (s, 2H), 2.20 (s, 6H), 1.48 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 515.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-dichlorobenzaldehyde to give compound 17 (white solid, 100.1 mg, 62.2% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.85 (s, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.78 (d, J=8.8 Hz, 2H), 7.48 (s, 2H), 4.65 (s, 2H), 4.61 (s, 2H), 1.46 (s, 6H). HRMS (ESI) calcd. for C21H17Cl2F3N2O5 [M+Na]+: 527.0364, found: 527.0358.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-dichlorobenzaldehyde to give compound 18 (yellow liquid, 171 mg, 64.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=9.0 Hz, 2H), 7.67 (d, J=9.0 Hz, 2H), 7.41 (s, 2H), 4.67 (s, 2H), 4.40 (s, 2H), 4.21-4.07 (m, 2H), 1.58 (s, 6H), 1.28 (t, J=7.2 Hz, 3H). MS (ESI): m/z 555.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-dibromobenzaldehyde to give compound 19 (white solid, 120.2 mg, 70.3% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.78 (d, J=8.8 Hz, 2H), 7.66 (s, 2H), 4.65 (s, 2H), 4.61 (s, 2H), 1.50 (s, 6H). HRMS (ESI) calcd. for C21H17Br2F3N2O5 [M+Na]+: 614.9354, found: 614.9348.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-dibromobenzaldehyde to give compound 20 (colorless liquid, 180 mg, 57.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.9 Hz, 2H), 7.67 (d, J=8.9 Hz, 2H), 7.62 (s, 2H), 4.67 (s, 2H), 4.40 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 1.59 (s, 6H), 1.37 (t, J=7.1 Hz, 3H). MS (ESI): m/z 642.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-difluorobenzaldehyde to give compound 21 (white solid, 22.4 mg, 56.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1H), 7.87 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.15 (d, J=8.9 Hz, 2H), 4.65 (s, 2H), 4.62 (s, 2H), 1.44 (s, 6H). HRMS (ESI) calcd. for C21H17F5N2O5 [M+Na]+: 495.0955, found: 495.0946.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3,5-difluorobenzaldehyde to give compound 22 (colorless liquid, 41.9 mg, 16.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.6 Hz, 2H), 7.67 (d, J=8.7 Hz, 2H), 7.03 (d, J=8.0 Hz, 2H), 4.69 (s, 2H), 4.39 (s, 2H), 4.26 (q, J=7.1 Hz, 2H), 1.56 (s, 6H), 1.30 (t, J=7.1 Hz, 3H). MS (ESI): m/z 523.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by 4-iodotoluene to give compound 23 (white solid, 61.7 mg, 83.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.52 (d, J=8.5 Hz, 2H), 7.20 (d, J=8.4 Hz, 2H), 6.97 (s, 2H), 4.55 (s, 2H), 4.52 (s, 2H), 2.28 (s, 3H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C23H26N2O5 [M+Na]+: 433.1739, found: 433.1727.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by 4-iodotoluene to give compound 24 (colorless liquid, 78.7 mg, 71.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.45 (d, J=8.5 Hz, 2H), 7.19 (d, J=8.3 Hz, 2H), 7.07 (s, 2H), 4.63 (s, 2H), 4.30 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.34 (s, 3H), 2.19 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 461.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by p-fluoroiodobenzene to give compound 25 (white solid, 87.2 mg, 90.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.81 (s, 1H), 7.66 (dd, J=8.9, 4.7 Hz, 2H), 7.38-7.17 (m, 2H), 6.96 (s, 2H), 4.57 (s, 2H), 4.52 (s, 2H), 2.15 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C22H23FN2O5 [M+Na]+: 437.1489, found: 437.1482.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by p-fluoroiodobenzene to give compound 26 (colorless liquid, 102.8 mg, 77.4% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.59-7.50 (m, 2H), 7.11 (d, J=8.2 Hz, 2H), 7.07 (s, 2H), 4.63 (s, 2H), 4.31 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.19 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 465.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by p-chloroiodobenzene to give compound 27 (white solid, 63.1 mg, 66.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.67 (d, J=8.9 Hz, 2H), 7.46 (d, J=8.9 Hz, 2H), 6.97 (s, 2H), 4.57 (s, 2H), 4.52 (s, 2H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C22H23ClN2O5 [M+Na]+: 453.1193, found: 453.1191.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (2) was replaced by p-chloroiodobenzene to give compound 28 (colorless liquid, 102.5 mg, 62.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.54 (d, J=9.0 Hz, 2H), 7.36 (d, J=9.0 Hz, 2H), 7.07 (s, 2H), 4.63 (s, 2H), 4.31 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 481.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromo-2-methyltrifluorotoluene to give compound 29 (white solid, 103.3 mg, 68.2% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.80 (s, 1H), 7.94-7.53 (m, 3H), 6.97 (s, 2H), 4.60 (s, 2H), 4.53 (s, 2H), 2.45 (s, 3H), 2.14 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C24H25F3N2O5 [M+Na]+: 501.1613, found: 501.1612.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromo-2-methyltrifluorotoluene to give compound 30 (colorless liquid, 160.8 mg, 40.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.62 (d, J=8.4 Hz, 2H), 7.44 (d, J=8.5 Hz, 1H), 7.07 (s, 2H), 4.64 (s, 2H), 4.33 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.52 (s, 3H), 2.20 (s, 6H), 1.46 (s, 6H), 1.35 (t, J=7.1 Hz, 3H). MS (ESI): M/z 529.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromo-2-fluorotrifluorotoluene to give compound 31 (white solid, 170.3 mg, 69.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.86-7.78 (m, 2H), 7.68 (d, J=8.6 Hz, 1H), 6.99 (s, 2H), 4.63 (s, 2H), 4.55 (s, 2H), 2.16 (s, 6H), 1.36 (s, 6H). HRMS (ESI) calcd. for C23H22F4N2O5 [M+Na]+: 505.1363, found: 505.1363.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromo-2-fluorotrifluorotoluene to give compound 32 (colorless liquid, 260.5 mg, 65.6% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J=12.5 Hz, 1H), 7.61 (t, J=8.3 Hz, 1H), 7.30 (d, J=9.0 Hz, 1H), 7.07 (s, 2H), 4.64 (s, 2H), 4.33 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 533.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 2-chloro-4-bromotrifluorotoluene to give compound 33 (white solid, 260.1 mg, 85.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 8.05-7.97 (m, 1H), 7.88 (d, J=8.8 Hz, 1H), 7.84-7.75 (m, 1H), 6.97 (s, 2H), 4.63 (s, 2H), 4.53 (s, 2H), 2.14 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C23H22ClF3N2O5 [M+Na]+: 521.1067, found: 521.1064.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 2-chloro-4-bromotrifluorotoluene to give compound 34 (colorless liquid, 321.5 mg, 77.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.90 (s, 1H), 7.69 (d, J=8.8 Hz, 1H), 7.53 (d, J=8.6 Hz, 1H), 7.06 (s, 2H), 4.64 (s, 2H), 4.33 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 549.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by iodobenzene to give compound 35 (white solid, 157.2 mg, 75.2% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.65 (d, J=8.1 Hz, 2H), 7.46-7.33 (m, 2H), 7.18-7.10 (m, 1H), 6.97 (s, 2H), 4.58 (s, 2H), 4.53 (s, 2H), 2.15 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C22H24N2O5 [M+Na]+: 419.1583, found: 419.1573.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by iodobenzene to give compound 36 (white solid, 226.9 mg, 63.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.59 (d, J=7.9 Hz, 2H), 7.40 (t, J=8.0 Hz, 2H), 7.46-7.35 (m, 2H), 7.08 (s, 2H), 4.64 (s, 2H), 4.33 (s, 2H), 4.29 (q, J=7.9 Hz, 2H), 2.19 (s, 6H), 1.46 (s, 6H), 1.35 (t, J=7.1 Hz, 3H). MS (ESI): m/z 447.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-iodocyanobenzene to give compound 37 (white solid, 177.1 mg, 83.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.88 (d, J=9.0 Hz, 2H), 7.83 (d, J=9.1 Hz, 2H), 6.97 (s, 2H), 4.61 (s, 2H), 4.54 (s, 2H), 2.15 (s, 6H), 1.34 (s, 6H). HRMS (ESI) calcd. for C23H23N3O5 [M+Na]+: 444.1535, found: 444.1531.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-iodocyanobenzene to give compound 38 (yellow foamy solid, 228 mg, 67.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J=9.1 Hz, 2H), 7.69 (d, J=9.0 Hz, 2H), 7.07 (s, 2H), 4.65 (s, 2H), 4.35 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 472.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromoanisole to give compound 39 (white solid, 143.2 mg, 72.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.81 (s, 1H), 7.54 (d, J=9.0 Hz, 2H), 7.01-6.93 (m, 4H), 4.54 (s, 2H), 4.51 (s, 2H), 3.75 (s, 3H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C23H26N2O6 [M+Na]+: 449.1689, found: 449.1685.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromoanisole to give compound 40 (white solid, 211 mg, 61.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.47 (d, J=9.1 Hz, 2H), 7.07 (s, 2H), 6.93 (d, J=9.1 Hz, 2H), 4.63 (s, 2H), 4.30 (s, 2H), 4.28 (q, J=9.6, 4.6 Hz, 2H), 3.82 (s, 3H), 2.19 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 477.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromobiphenyl to give compound 41 (white solid, 163.1 mg, 78.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.79-7.73 (m, 4H), 7.68 (d, J=7.7 Hz, 2H), 7.53-7.41 (m, 2H), 7.39-7.19 (m, 1H), 6.99 (s, 2H), 4.63 (s, 2H), 4.55 (s, 2H), 2.16 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C28H28N2O5 [M+Na]+: 495.1896, found: 495.1883.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromobiphenyl to give compound 42 (white solid, 208.5 mg, 53.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.67 (d, J=8.8 Hz, 2H), 7.64 (d, J=6.2 Hz, 2H), 7.59 (d, J=8.0 Hz, 2H), 7.46 (t, J=7.5 Hz, 2H), 7.36 (t, J=7.3 Hz, 1H), 7.09 (s, 2H), 4.66 (s, 2H), 4.37 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.21 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 523.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-iodobenzylthiomethane to give compound 43 (yellow solid, 200.3 mg, 81.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.60 (d, J=8.8 Hz, 2H), 7.31 (d, J=8.8 Hz, 2H), 6.97 (s, 2H), 4.56 (s, 2H), 4.52 (s, 2H), 2.47 (s, 3H), 2.15 (s, 6H), 1.36 (s, 6H). HRMS (ESI) calcd. for C23H26N2O5S [M+Na]+: 465.1460, found: 465.1455.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-iodobenzylthiomethane to give compound 44 (yellow solid, 266.5 mg, 74.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.52 (d, J=8.7 Hz, 2H), 7.30 (d, J=10.1 Hz, 2H), 7.07 (s, 2H), 4.63 (s, 2H), 4.30 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.49 (s, 3H), 2.19 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 493.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-iodotrifluorotoluene to give compound 45 (white solid, 169.1 mg, 67.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 8.14 (s, 1H), 7.83 (d, J=8.7 Hz, 1H), 7.69-7.61 (m, 1H), 7.49 (d, J=7.8 Hz, 1H), 6.98 (s, 2H), 4.65 (s, 2H), 4.54 (s, 2H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C23H23F3N2O5 [M+Na]+: 487.1457, found: 487.1451.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-iodotrifluorotoluene to give compound 46 (yellow liquid, 268.1 mg, 54.5% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.91 (s, 1H), 7.77 (d, J=8.0 Hz, 1H), 7.53 (t, J=8.0 Hz, 1H), 7.42 (d, J=7.6 Hz, 1H), 7.07 (s, 2H), 4.65 (s, 2H), 4.36 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 515.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-fluoro-5-chlorobenzaldehyde to give compound 47 (white solid, 73.3 mg, 64.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.92 (s, 1H), 7.87 (d, J=8.8 Hz, 2H), 7.78 (d, J=8.8 Hz, 2H), 7.35 (s, 1H), 7.27 (d, J=11.6 Hz, 1H), 4.65 (s, 2H), 4.61 (s, 2H), 1.46 (s, 6H). HRMS (ESI) calcd. for C21H17ClF4N2O5 [M+Na]+: 511.0660, found: 511.0654.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-fluoro-5-chlorobenzaldehyde to give compound 48 (yellow liquid, 120.6 mg, 46.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=9.0 Hz, 2H), 7.67 (d, J=9.1 Hz, 2H), 7.29 (s, 1H), 7.13 (dd, J=10.8, 2.0 Hz, 1H), 4.68 (s, 2H), 4.39 (s, 2H), 4.27 (q, J=7.1 Hz, 2H), 1.57 (s, 6H), 1.33 (t, J=7.1 Hz, 3H). MS (ESI): m/z 539.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-fluoro-4-hydroxybenzaldehyde to give compound 49 (white solid, 47.2 mg, 59.0% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.14 (s, 1H), 7.86 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.23 (d, J=12.0 Hz, 1H), 7.09 (d, J=8.6 Hz, 1H), 6.95 (t, J=8.5 Hz, 1H), 4.62 (s, 4H), 1.50 (s, 6H). HRMS (ESI) calcd. for C21H18F4N2O5 [M+Na]+: 477.1050, found: 477.1044.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-fluoro-4-hydroxybenzaldehyde to give compound 50 (yellow liquid, 85.3 mg, 35.3% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.9 Hz, 2H), 7.66 (d, J=9.0 Hz, 2H), 7.22 (dd, J=11.3, 2.0 Hz, 1H), 7.13 (d, J=8.4 Hz, 1H), 6.94 (t, J=8.3 Hz, 1H), 4.70 (s, 2H), 4.36 (s, 2H), 4.26 (q, J=7.1 Hz, 2H), 1.59 (s, 6H), 1.30 (t, J=7.1 Hz, 3H). MS (ESI): m/z 505.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-chloro-4-hydroxybenzaldehyde to give compound 51 (white solid, 21.3 mg, 55.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.21 (s, 1H), 7.86 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.45 (d, J=2.0 Hz, 1H), 7.24 (dd, J=8.5, 2.0 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 4.61 (s, 2H), 4.60 (s, 2H), 1.53 (s, 6H). HRMS (ESI) calcd. for C21H18ClF3N2O5 [M+Na]+: 493.0754, found: 493.0749.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-chloro-4-hydroxybenzaldehyde to give compound 52 (yellow liquid, 40.0 mg, 16.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.9 Hz, 2H), 7.66 (d, J=8.9 Hz, 2H), 7.51 (d, J=2.1 Hz, 1H), 7.29-7.21 (m, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.68 (s, 2H), 4.36 (s, 2H), 4.26 (q, J=7.1 Hz, 2H), 1.60 (s, 6H), 1.29 (t, J=7.1 Hz, 3H). MS (ESI): m/z 521.1 [M+Na]+.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromoacetate to give compound 53 (white solid, 73.4 mg, 78.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.86 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.7 Hz, 2H), 7.01 (s, 2H), 4.62 (s, 2H), 4.54 (s, 2H), 4.34 (s, 2H), 2.21 (s, 6H). HRMS (ESI) calcd. for C21H19F3N2O5 [M+Na]+: 459.1144, found: 459.1135.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromoacetate to give compound 54 (white solid, 99.4 mg, 42.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.8 Hz, 2H), 7.66 (d, J=8.9 Hz, 2H), 7.12 (s, 2H), 4.67 (s, 2H), 4.38 (s, 2H), 4.36 (s, 2H), 4.32 (q, J=7.1 Hz, 2H), 2.30 (s, 6H), 1.34 (t, J=7.1 Hz, 3H). MS (ESI): m/z 487.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-trifluoromethoxy-4-hydroxybenzaldehyde to give compound 55 (white solid, 143.4 mg, 90.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.22 (s, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.7 Hz, 2H), 7.38 (s, 1H), 7.29 (d, J=8.5 Hz, 1H), 6.91 (d, J=8.5 Hz, 1H), 4.63 (s, 2H), 4.61 (s, 2H), 1.52 (s, 6H). HRMS (ESI) calcd. for C22H18F6N2O6 [M+Na]+: 543.0967, found: 543.0961.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-trifluoromethoxy-4-hydroxybenzaldehyde to give compound 56 (yellow liquid, 167.0 mg, 62.2% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.9 Hz, 2H), 7.66 (d, J=9.0 Hz, 2H), 7.40 (s, 1H), 7.34-7.29 (m, 1H), 6.85 (d, J=8.5 Hz, 1H), 4.71 (s, 2H), 4.36 (s, 2H), 4.24 (q, J=7.1 Hz, 2H), 1.61 (s, 6H), 1.27 (t, J=7.1 Hz, 3H). MS (ESI): m/z 571.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-methyl-4-hydroxybenzaldehyde to give compound 57 (white solid, 139.0 mg, 80.3% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.00 (s, 1H), 7.85 (d, J=8.8 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 7.18-7.12 (m, 1H), 7.12-7.02 (m, 1H), 6.64 (d, J=8.4 Hz, 1H), 4.61 (s, 2H), 4.55 (s, 2H), 2.14 (s, 3H), 1.50 (s, 6H). HRMS (ESI) calcd. for C22H21F3N2O5 [M+Na]+: 473.1300, found: 473.1297.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 3-methyl-4-hydroxybenzaldehyde to give compound 58 (yellow liquid, 184.0 mg, 77.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.9 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.27 (s, 1H), 7.18 (d, J=8.1 Hz, 1H), 6.61 (d, J=8.3 Hz, 1H), 4.67 (s, 2H), 4.33 (s, 2H), 4.25 (q, J=7.1 Hz, 2H), 2.23 (s, 3H), 1.58 (s, 6H), 1.27 (t, J=7.1 Hz, 3H). MS (ESI): m/z 501.1 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by p-hydroxybenzaldehyde to give compound 59 (white solid, 273.4 mg, 89.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.02 (s, 1H), 7.85 (d, J=8.8 Hz, 2H), 7.76 (d, J=8.8 Hz, 2H), 7.26 (d, J=8.5 Hz, 2H), 6.79 (d, J=8.5 Hz, 2H), 4.61 (s, 2H), 4.59 (s, 2H), 1.50 (s, 6H). HRMS (ESI) calcd. for C21H19F3N2O5 [M+Na]+: 459.1144, found: 459.1137.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by p-hydroxybenzaldehyde to give compound 60 (white solid, 237 mg, 97.2% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.8 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.37 (d, J=8.6 Hz, 2H), 6.81 (d, J=8.6 Hz, 2H), 4.71 (s, 2H), 4.34 (s, 2H), 4.24 (q, 7.1 Hz, 2H), 1.60 (s, 6H), 1.27 (t, J=7.1 Hz, 3H). MS (ESI): m/z 487.2 [M+Na]+.
Referring to the method in Example 1, p-trifluoromethylaniline was replaced by p-bromoaniline to give compound 61 (white solid, 225.0 mg, 78.9% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.85 (s, 1H), 7.65-7.55 (m, 4H), 6.96 (s, 2H), 4.56 (s, 2H), 4.51 (s, 2H), 2.14 (s, 6H), 1.33 (s, 6H). HRMS (ESI) calcd. for C22H23BrN2O5 [M+Na]+: 497.0688, found: 497.0675.
Referring to the method in Example 1, p-trifluoromethylaniline was replaced by p-bromoaniline to give compound 62 (white solid, 356.0 mg, 48.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.52-7.45 (m, 4H), 7.05 (s, 2H), 4.61 (s, 2H), 4.31-4.24 (m, 4H), 2.17 (s, 6H), 1.44 (s, 6H), 1.34 (t, J=7.1 Hz, 3H). MS (ESI): m/z 527.1 [M+Na]+.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl p-2-bromopropionate to give compound 63 (white solid, 82.3 mg, 58.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.86 (s, 1H), 7.86 (d, J=8.6 Hz, 2H), 7.77 (d, J=8.7 Hz, 2H), 6.99 (s, 2H), 4.62 (s, 2H), 4.54 (s, 2H), 4.38 (q, J=6.7 Hz, 1H), 2.20 (s, 6H), 1.40 (d, J=6.7 Hz, 3H). HRMS (ESI) calcd. for C22H21F3N2O5 [M+Na]+: 473.1300, found: 473.1300.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl p-2-bromopropionate to give compound 64 (colorless liquid, 160 mg, 66.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.7 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 7.11 (s, 2H), 4.66 (s, 2H), 4.47 (q, J=6.8 Hz, 1H), 4.36 (s, 2H), 4.24 (q, J=7.0 Hz, 2H), 2.29 (s, 6H), 1.53 (d, J=6.7 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H). MS (ESI): m/z 501.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromocumene to give compound 65 (white solid, 112.4 mg, 64.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.54 (d, J=8.5 Hz, 2H), 7.26 (d, J=8.5 Hz, 2H), 6.96 (s, 2H), 4.55 (s, 2H), 4.51 (s, 2H), 3.03-2.69 (m, 1H), 2.14 (s, 6H), 1.34 (s, 6H), 1.19 (d, J=6.9 Hz, 6H). HRMS (ESI) calcd. for C25H30N2O5 [M+Na]+: 461.2052, found: 461.2046.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromocumene to give compound 66 (yellow liquid, 185.3 mg, 39.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.49 (d, J=8.6 Hz, 2H), 7.26 (d, J=8.6 Hz, 2H), 7.07 (s, 2H), 4.64 (s, 2H), 4.32 (t, J=7.1 Hz, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.91 (dt, J=13.8, 6.9 Hz, 1H), 2.19 (s, 6H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H), 1.25 (d, J=6.9 Hz, 6H). MS (ESI): m/z 489.2 [M+Na]+.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromobutyrate to give compound 67 (white solid, 132.0 mg, 72.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.87 (s, 1H), 7.86 (d, J=8.6 Hz, 2H), 7.77 (d, J=8.7 Hz, 2H), 6.99 (s, 2H), 4.63 (s, 2H), 4.54 (s, 2H), 4.30 (t, J=5.9 Hz, 1H), 2.22 (s, 6H), 1.86 (dt, 2H), 0.96 (t, J=7.3 Hz, 3H). HRMS (ESI) calcd. for C23H23F3N2O5 [M+Na]+: 487.1457, found: 487.1451.
Referring to the method in Example 1, ethyl 2-bromoisobutyrate was replaced by ethyl 2-bromobutyrate to give compound 68 (colorless liquid, 194.6 mg, 78.9% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.73 (d, J=8.7 Hz, 2H), 7.65 (d, J=8.9 Hz, 2H), 7.09 (s, 2H), 4.65 (s, 2H), 4.39 (t, J=7.5 Hz, 1H), 4.35 (s, 2H), 4.20 (q, J=6.1 Hz, 2H), 2.28 (s, 6H), 1.97 (dt, 2H), 1.26 (t, J=6.6 Hz, 3H), 1.02 (t, J=7.5 Hz, 3H). MS (ESI): m/z 515.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-tert-butylbromobenzene to give compound 69 (white solid, 81.7 mg, 53.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.81 (s, 1H), 7.53 (d, J=8.7 Hz, 2H), 7.39 (d, J=8.7 Hz, 2H), 6.94 (s, 2H), 4.54 (s, 2H), 4.50 (s, 2H), 2.13 (s, 6H), 1.32 (s, 6H), 1.26 (s, 9H). HRMS (ESI) calcd. for C26H32N2O5 [M+Na]+: 475.2209, found: 475.2203.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-tert-butylbromobenzene to give compound 70 (white solid, 147 mg, 61.2% yield) without hydrolysis: 1H NMR (300 MHz, DMSO-d6) δ 7.54 (d, J=8.7 Hz, 2H), 7.40 (d, J=8.8 Hz, 2H), 6.96 (s, 2H), 4.54 (d, J=7.1 Hz, 2H), 4.16 (q, J=7.1 Hz, 2H), 2.10 (s, 2H), 1.36 (s, 6H), 1.27 (s, 6H), 1.23 (s, 9H), 1.21 (t, J=7.1 Hz, 3H). MS (ESI): m/z 503.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromophenyl methyl sulfone to give compound 71 (white solid, 37.2 mg, 26.2% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.30-7.55 (m, 4H), 6.96 (s, 2H), 4.62 (s, 2H), 4.52 (s, 2H), 3.17 (s, 3H), 2.13 (s, 6H), 1.32 (s, 6H). HRMS (ESI) calcd. for C23H26N2O7S [M+Na]+: 497.1358, found: 497.1346.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromophenyl methyl sulfone to give compound 72 (yellow solid, 131.2 mg, 27.8% yield) without hydrolysis: 1H NMR (300 MHz, DMSO-d6) δ 7.92 (q, J=9.1 Hz, 4H), 6.99 (s, 2H), 4.64 (s, 2H), 4.55 (s, 2H), 4.17 (q, J=7.1 Hz, 2H), 3.34 (s, 3H), 2.11 (s, 6H), 1.37 (s, 6H), 1.24 (t, J=7.1 Hz, 3H). MS (ESI): m/z 525.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-chloro-5-methoxybenzaldehyde to give compound 73 (white solid, 100.2 mg, 66.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.45 (s, 1H), 7.85 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.7 Hz, 2H), 6.97 (d, J=6.8 Hz, 2H), 4.60 (s, 4H), 3.69 (s, 3H), 1.34 (s, 6H). HRMS (ESI) calcd. for C22H20ClF3N2O6 [M+Na]+: 523.0860, found: 523.0854.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-chloro-5-methoxybenzaldehyde to give compound 74 (colorless liquid, 190.2 mg, 72.3% yield) without hydrolysis: 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 6.99 (d, J=8.9 Hz, 2H), 4.62 (s, 4H), 4.15 (q, J=7.1 Hz, 2H), 3.70 (s, 3H), 1.37 (s, 6H), 1.23 (t, J=7.2 Hz, 3H). MS (ESI): m/z 551.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 1-ethyl-4-iodobenzene to give compound 75 (white solid, 79.2 mg, 70.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.85 (s, 1H), 7.54 (d, J=8.5 Hz, 2H), 7.23 (d, J=8.5 Hz, 2H), 6.96 (s, 2H), 4.56 (s, 2H), 4.52 (s, 1H), 2.58 (q, J, 7.5 Hz, 2H), 2.14 (s, 6H), 1.34 (s, 6H), 1.16 (t, J=7.5 Hz, 3H). HRMS (ESI) calcd. for C24H28N2O5 [M+Na]+: 447.1896, found: 447.1894.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 1-ethyl-4-iodobenzene to give compound 76 (white solid, 120.0 mg, 26.4% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.49 (d, J=8.5 Hz, 2H), 7.23 (d, J=8.5 Hz, 2H), 7.08 (s, 2H), 4.64 (s, 2H), 4.32 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.65 (q, J=7.5 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H), 1.26 (t, J=7.7 Hz, 3H). MS (ESI): m/z 475.2 [M+Na]+.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-bromobenzaldehyde to give compound 77 (white solid, 97.3 mg, 76.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 13.28 (s, 1H), 7.86 (d, J=8.7 Hz, 2H), 7.77 (d, J=8.9 Hz, 2H), 7.60 (s, 1H), 7.28 (d, J=7.9 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.61 (s, 2H), 4.60 (s, 2H), 1.53 (s, 6H). HRMS (ESI) calcd. for C21H18BrF3N2O5 [M+Na]+: 537.0249, found: 537.0243.
Referring to the method in Example 1, 4-hydroxy-3,5-dimethylbenzaldehyde was replaced by 4-hydroxy-3-bromobenzaldehyde to give compound 78 (white solid, 134.3 mg, 49.4% yield) without hydrolysis: 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 7.62 (s, 1H), 7.28 (d, J=6.4 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 4.64-4.55 (m, 4H), 4.19 (dd, J=14.1, 7.1 Hz, 2H), 1.55 (s, 6H), 1.19 (t, J=7.1 Hz, 3H). MS (ESI): m/z 565.1 [M+Na]+.
5-Ethyl-5-methylimidazolidine-2,4-dione (142 mg, 1 mmol) was dissolved in DMF (5 mL), and M-1 (492 mg, 1.5 mmol) and cesium carbonate (652 mg, 2 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, water (20 mL) was added, and the resulting solution was extracted with EA (25 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=2:1) to give intermediate O-1 (yellow liquid, 150 mg, 38.4% yield).
Intermediate O-1 (150 mg, 0.4 mmol), cuprous iodide (15 mg, 0.08 mmol), potassium carbonate (110 mg, 0.8 mmol), and (1R,2R)-(−)-N,N′-dimethyl-1,2-cyclohexanediamine (23 mg, 0.16 mmol) were added to a Schlenk tube. Under argon atmosphere, a solution of p-trifluoromethylbromobenzene (107 mg, 0.48 mmol) in toluene (3 mL) was added, and the reaction system was heated to 110° C. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 80 (colorless liquid, 47 mg, 22.1% yield).
Compound 80 (47 mg, 0.09 mmol) was dissolved in acetonitrile (3 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and acetic acid (4 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 79 (white solid, 21 mg, 55.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.84 (d, J=8.5 Hz, 2H), 7.64 (d, J=8.3 Hz, 2H), 6.95 (s, 2H), 4.58 (s, 2H), 2.15 (s, 6H), 2.01-1.65 (m, 2H), 1.43 (s, 3H), 1.34 (s, 6H), 0.68 (t, J=7.2 Hz, 3H). HRMS (ESI) calcd. for C26H29F3N2O5 [M+NH4]+ 524.2372, found 524.2370.
Referring to the method in Example 79, compound 80 (white solid, 47 mg, 22.1% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.70 (d, J=8.5 Hz, 2H), 7.46 (d, J=8.3 Hz, 2H), 7.07 (s, 2H), 4.65 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 2.19 (s, 6H), 2.06-1.93 (m, 2H), 1.79-1.66 (m, 3H), 1.46 (s, 6H), 1.36 (t, J=7.1 Hz, 3H), 0.71 (t, J=7.3 Hz, 3H). MS (ESI): m/z 557.2 [M+Na]+.
p-Trifluoromethyl isocyanate (1.87 g, 10 mmol) was dissolved in dichloromethane (15 mL), triethylamine (1.66 mL, 12 mmol) was added, and glycine ethyl ester hydrochloride (1.67 g, 12 mmol) was added in an ice bath. The mixture was stirred at room temperature overnight. After the reaction was completed, the solvent was distilled off under reduced pressure, water (50 mL) was added, and the resulting solution was extracted with EA (50 mL×3). The organic phase was washed with saturated sodium chloride solution (50 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=40:1) to give intermediate B-2 (white solid, 2.4 g, 82.7% yield).
Intermediate B-2 (1.45 g, 5 mmol) was dissolved in tetrahydrofuran (15 mL), and sodium hydride (160 mg, 4 mmol) was added in an ice bath. The mixture was stirred at room temperature for 3 h. After the reaction was completed, water (20 mL) was added for quenching, and the solvent was distilled off under reduced pressure. Water (20 mL) was added, and the resulting solution was extracted with EA (25 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=2:1) to give intermediate B-3 (white solid, 486.1 mg, 39.8% yield).
Intermediate B-3 (296 mg, 1.2 mmol) was dissolved in DMF (3 mL), and M-1 (492 mg, 1.5 mmol) and cesium carbonate (782 mg, 2.4 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 82 (colorless liquid, 45 mg, 7.5% yield).
Compound 82 (45 mg, 0.09 mmol) was dissolved in acetonitrile (2 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and glacial acetic acid (4 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 81 (white solid, 10.3 mg, 25.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.89 (d, J=8.5 Hz, 2H), 7.71 (d, J=8.3 Hz, 2H), 7.01 (s, 2H), 4.47 (s, 2H), 4.06 (s, 2H), 2.18 (s, 6H), 1.36 (s, 6H). HRMS (ESI) calcd. for C23H23F3N2O5 [M+NH4]+ 482.1903, found 482.1902.
Referring to the method in Example 81, compound 82 (colorless liquid, 45 mg, 7.5% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J=8.5 Hz, 2H), 7.68 (d, J=8.4 Hz, 2H), 6.93 (s, 2H), 4.54 (s, 2H), 4.31 (q, J=7.2 Hz, 2H), 3.93 (s, 2H), 2.23 (s, 6H), 1.49 (s, 6H), 1.38 (t, J=7.2 Hz, 3H). MS (ESI): m/z 515.2 [M+Na]+.
p-Iodotrifluoromethylbenzene (271 mg, 1 mmol), cuprous oxide (141 mg, 1 mmol), and 5,5-dimethylhydantoin (192 mg, 1.5 mmol) were added to a three-necked flask, and the reaction system was purged with argon. Anhydrous DMF (3 mL) was then added, the reaction system was heated to 150° C. and reacted for 12 h. After the reaction was completed, the reaction solution was filtered through celite, water (10 mL) was added, and the resulting solution was extracted with EA (10 mL×3) and washed with saturated sodium chloride (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound F-1 (pale yellow solid, 160 mg, 58.2% yield).
Intermediate F-1 (50 mg, 0.2 mmol) was dissolved in DMF (2 mL), and M-1 (98 mg, 0.3 mmol) and cesium carbonate (163 mg, 0.5 mmol) were added. The mixture was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 84 (colorless liquid, 80 mg, 76.2% yield).
Compound 84 (80 mg, 0.15 mmol) was dissolved in acetonitrile (2 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and glacial acetic acid (4 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 83 (white solid, 42 mg, 55.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.90 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.4 Hz, 2H), 7.06 (s, 2H), 4.50 (s, 2H), 2.17 (s, 6H), 1.36 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C25H27F3N2O5 [M+NH4]+ 510.2216, found 510.2213.
Referring to the method in Example 83, compound 84 (colorless liquid, 80 mg, 76.2% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.75 (d, J=9.0 Hz, 2H), 7.71 (d, J=9.0 Hz, 2H), 6.99 (s, 2H), 4.54 (s, 2H), 4.31 (q, J=7.1 Hz, 2H), 2.22 (s, 6H), 1.48 (s, 6H), 1.39 (s, 6H), 1.38 (t, J=7.1 Hz, 3H). MS (ESI): m/z 543.2 [M+Na]+.
2,6-Dimethylphenol (1.2 g, 10 mmol) was dissolved in acetonitrile (15 mL), and ethyl 2-bromoisobutyrate (3.4 mL, 30 mmol) and cesium carbonate (8.1 g, 25 mmol) were added. The reaction system was heated to 80° C. and stirred overnight. After the reaction was completed, the solvent was distilled off under reduced pressure, water (20 mL) was added, and the resulting solution was extracted with EA (50 mL×3). The organic phase was washed with 1 N sodium hydroxide solution (20 mL×3) and saturated sodium chloride solution (20 mL×1), and dried over anhydrous Mg2SO4. The solvent was distilled off under reduced pressure, and the residue was intermediate L-1 (yellow liquid, 1.9 g, 80.5% yield).
DCM (20 mL) and bromoacetyl bromide (2.1 mL, 24 mmol) were added to AlCl3 (3.2 g, 24 mmol) in an ice bath under argon atmosphere. The mixture was stirred at room temperature for 1 h. Intermediate L-1 (1.9 g, 8 mmol) was added to the reaction solution described above in an ice bath, and the resulting solution was stirred at room temperature for another 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (20 mL) was added, and the resulting solution was extracted with EA (20 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was intermediate L-2 (brown-black liquid, 1.8 g, 66.7% yield).
Intermediate L-2 (1.8 g, 5 mmol) was dissolved in trifluoroacetic acid (15 mL), and triethylsilane (1.0 mL, 7.5 mmol) was added. The reaction system was heated to 70° C. and stirred for 12 h. After the reaction was completed, the mixture was diluted with water (20 mL) in an ice bath, and the resulting solution was stirred at room temperature for 10 min. The solvent was distilled off under reduced pressure, and the mixture was extracted with EA (20 mL×3). The organic phase was washed with saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was intermediate M-2 (colorless liquid, 1.4 g, 82.3% yield).
Intermediate A-3 (110 mg, 0.45 mmol) was dissolved in acetonitrile (5 mL), and M-2 (184.7 mg, 0.54 mmol) and cesium carbonate (293.4 mg, 0.9 mmol) were added. The reaction system was heated to 80° C. and stirred for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 86 (colorless liquid, 66.9 mg, 29.3% yield).
Compound 86 (66.9 mg, 0.13 mmol) was dissolved in acetonitrile (2 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and acetic acid (4 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 85 (white solid, 32 mg, 51.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.76 (s, 1H), 7.83 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.7 Hz, 2H), 6.86 (s, 2H), 4.53 (s, 2H), 3.64 (t, J=7.4 Hz, 2H), 2.76 (t, J=7.5 Hz, 2H), 2.13 (s, 6H), 1.31 (s, 6H). HRMS (ESI) calcd. for C24H25F3N2O5 [M+Na]+: 501.1613, found: 501.1607.
Referring to the method in Example 85, compound 86 (colorless liquid, 66.9 mg, 29.3% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.71 (d, J=9.1 Hz, 2H), 7.67 (d, J=9.3 Hz, 2H), 6.87 (s, 2H), 4.33 (q, J=7.1 Hz, 2H), 4.28 (s, 2H), 3.82 (t, J=8.3 Hz, 2H), 2.88 (t, J=8.3 Hz, 2H), 2.18 (s, 6H), 1.45 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 529.2 [M+Na]+.
Referring to the method in Example 85, A-3 was replaced by B-3 to give compound 87 (white solid, 23 mg, 61.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.69 (s, 1H), 7.87 (d, J=8.4 Hz, 2H), 7.61 (d, J=8.2 Hz, 2H), 6.91 (s, 2H), 4.10 (s, 2H), 3.56 (t, J=7.3 Hz, 2H), 2.77 (t, J=7.3 Hz, 2H), 2.14 (s, 6H), 1.32 (s, 6H). HRMS (ESI) calcd. for C24H25F3N2O5 [M+Na]+: 501.1613, found: 501.1608.
Referring to the method in Example 85, A-3 was replaced by B-3 to give compound 88 (colorless liquid, 39.3 mg, 26% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J=8.4 Hz, 2H), 7.61 (d, J=8.5 Hz, 2H), 6.86 (s, 2H), 4.31 (q, J=7.1 Hz, 2H), 3.85 (s, 2H), 3.71 (t, J=7.2 Hz, 2H), 2.86 (t, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.38 (t, J=7.1 Hz, 3H). MS (ESI): m/z 529.2 [M+Na]+.
Intermediate K-1 (0.5 g, 2.25 mmol) was dissolved in DMF (5 mL), and 2,2-dimethyl-1,3-dioxane-4,6-dione (0.5 g, 3.38 mmol) and triethylamine (0.4 mL, 2.7 mmol) were added. Formic acid (1.2 mL) was added in an ice bath, and the resulting solution was stirred at room temperature for 5 min. The reaction system was heated to 100° C. and stirred for 12 h. After the reaction was completed, water (20 mL) was added, and the resulting solution was extracted with EA (50 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1) and dried over anhydrous MgSO4. The solvent was distilled off under reduced pressure, and the residue was intermediate K-3 (colorless liquid, 0.5 g, 71.8% yield).
Intermediate K-3 (0.5 g, 1.5 mmol) was dissolved in tetrahydrofuran (5 mL), and borane-tetrahydrofuran complex (1 mL, 1 mmol) was added in an ice bath. The resulting solution was stirred at room temperature for 12 h. After the reaction was completed, water (10 mL) was added, and the mixture was stirred for 30 min, and extracted with EA (20 mL×3). The organic phase was washed with saturated sodium chloride solution (20 mL×1). The solvent was distilled off under reduced pressure, and the residue was intermediate K-4 (colorless liquid, 0.2 g, 45.4% yield).
Intermediate K-4 (0.2 g, 1 mmol) was dissolved in dichloromethane (5 mL), and tetrabromomethane (0.5 g, 1.5 mmol) and triphenylphosphine (0.5 g, 1.4 mmol) were added in an ice bath. The resulting solution was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=20:1) to give intermediate M-3 (colorless liquid, 0.25 g, 70.2% yield).
Intermediate A-3 (123 mg, 0.5 mmol) was dissolved in acetonitrile (5 mL), and M-3 (213.6 mg, 0.6 mmol) and cesium carbonate (326 mg, 1 mmol) were added. The reaction system was heated to 80° C. and stirred for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 90 (colorless liquid, 58.9 mg, 22.6% yield).
Compound 90 (58.9 mg, 0.12 mmol) was dissolved in acetonitrile (2 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and acetic acid (2 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 89 (white solid, 43 mg, 72.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.78 (s, 1H), 7.85 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.8 Hz, 2H), 6.84 (s, 2H), 4.48 (s, 2H), 3.50 (t, J=6.9 Hz, 2H), 2.55-2.51 (m, 2H), 2.12 (s, 6H), 1.96-1.79 (m, 2H), 1.32 (s, 6H). HRMS (ESI) calcd. for C25H27F3N2O5 [M+Na]+: 515.1770, found: 515.1754.
Referring to the method in Example 89, compound 90 (colorless liquid, 58.9 mg, 22.6% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.70 (d, J=9.2 Hz, 2H), 7.66 (d, J=6.3 Hz, 2H), 6.81 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 4.19 (s, 2H), 3.68 (t, J=7.6 Hz, 2H), 2.60 (t, J=7.6 Hz, 2H), 2.13 (s, 6H), 2.09-1.94 (m, 2H), 1.44 (s, 6H), 1.35 (t, J=7.1 Hz, 3H). MS (ESI): m/z 543.2 [M+Na]+.
Referring to the method in Example 89, A-3 was replaced by B-3 to give compound 91 (white solid, 28 mg, 75% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.60 (s, 1H), 7.87 (d, J=8.4 Hz, 2H), 7.65 (d, J=8.3 Hz, 2H), 6.87 (s, 2H), 4.13 (s, 2H), 3.40 (t, J=7.0 Hz, 2H), 2.58-2.53 (m, 2H), 2.13 (s, 6H), 1.91-1.76 (m, 2H), 1.33 (s, 6H). HRMS (ESI) calcd. for C25H27F3N2O5 [M+Na]+: 515.1770, found: 515.1764.
Referring to the method in Example 89, A-3 was replaced by B-3 to give compound 92 (yellow liquid, 39 mg, 21% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.74 (d, J=8.4 Hz, 2H), 7.63 (d, J=8.4 Hz, 2H), 6.81 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 4.00 (s, 2H), 3.54 (t, J=7.2 Hz, 2H), 2.60 (t, J=7.5 Hz, 2H), 2.18 (s, 6H), 2.01-1.87 (m, 2H), 1.47 (s, 6H), 1.37 (t, J=7.1 Hz, 3H). MS (ESI): m/z 543.2 [M+Na]+.
Intermediate E-1 (190 mg, 0.54 mmol) was dissolved in DMF (5 mL), and p-trifluoromethylbenzyl bromide (155 mg, 0.65 mmol) and cesium carbonate (352 mg, 1.08 mmol) were added. The reaction system was stirred at room temperature for 12 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=5:1) to give compound 94 (colorless liquid, 110 mg, 40.8% yield).
Compound 94 (110 mg, 0.22 mmol) was dissolved in acetonitrile (2 mL), and a mixed solution of concentrated hydrochloric acid (at a concentration of 12 M) and acetic acid (2 mL, 1:1) was added. The reaction system was heated to 100° C. and stirred for 4 h. After the reaction was completed, the solvent was distilled off under reduced pressure, water (10 mL) was added, and the resulting solution was extracted with EA (15 mL×3). The organic phase was washed with saturated sodium chloride solution (10 mL×1). The solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (dichloromethane/methanol=100:1) to give compound 93 (white solid, 67 mg, 63.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.72 (d, J=8.0 Hz, 2H), 7.53 (d, J=8.1 Hz, 2H), 6.92 (s, 2H), 4.62 (s, 2H), 4.46 (s, 2H), 4.03 (s, 2H), 2.15 (s, 6H), 1.35 (s, 6H). HRMS (ESI) calcd. for C24H25F3N2O5 [M+Na]+: 501.1613, found: 501.1606.
Referring to the method in Example 93, compound 94 (colorless liquid, 110 mg, 40.8% yield) was prepared without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.64 (d, J=8.0 Hz, 2H), 7.39 (d, J=8.1 Hz, 2H), 7.03 (s, 2H), 4.64 (s, 2H), 4.58 (s, 2H), 4.30 (q, J=7.1 Hz, 2H), 3.78 (s, 2H), 2.19 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 529.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by p-iodophenetole to give compound 95 (white solid, 60.9 mg, 34.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.51 (d, J=9.0 Hz, 2H), 6.95 (s, 2H), 6.93 (d, 2H), 4.52 (s, 2H), 4.49 (s, 2H), 3.99 (q, J=6.9 Hz, 2H), 2.13 (s, 6H), 1.33 (s, 6H), 1.29 (q, J=7.0 Hz, 3H). MS (ESI): m/z 463.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by p-iodophenetole to give compound 96 (white solid, 195.6 mg, 41.8% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.45 (d, J=8.9 Hz, 2H), 7.06 (s, 2H), 6.91 (d, J=9.0 Hz, 2H), 4.61 (s, 2H), 4.28 (s, 2H), 4.28 (q, J=7.1 Hz, 2H), 4.02 (q, J=6.8 Hz, 2H), 2.18 (s, 6H), 1.45 (s, 6H), 1.40 (t, J=7.0 Hz, 3H), 1.34 (t, J=7.1 Hz, 3H). MS (ESI): m/z 491.2 [M+Na]+.
Referring to the method in Example 1, 3,5-dimethyl-p-hydroxybenzaldehyde was replaced by 3-methoxy-p-hydroxybenzaldehyde to give compound 97 (white solid, 85.9 mg, 59.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.90 (s, 1H), 7.84 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.8 Hz, 2H), 6.97 (s, 1H), 6.80 (d, J=8.3 Hz, 1H), 6.75 (d, J=8.2 Hz, 1H), 4.61 (s, 2H), 4.58 (s, 2H), 3.72 (s, 3H), 1.43 (s, 6H). MS (ESI): m/z 489.1 [M+Na]+.
Referring to the method in Example 1, 3,5-dimethyl-p-hydroxybenzaldehyde was replaced by 3-methoxy-p-hydroxybenzaldehyde to give compound 98 (white solid, 158.5 mg, 35.3% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.72 (d, J=8.9 Hz, 2H), 7.66 (d, J=8.9 Hz, 2H), 7.04 (d, J=1.7 Hz, 1H), 6.95 (dd, J=8.2, 1.8 Hz, 1H), 6.82 (d, J=8.2 Hz, 1H), 4.70 (s, 2H), 4.35 (s, 2H), 4.25 (q, J=7.1 Hz, 2H), 3.84 (s, 3H), 1.57 (s, 6H), 1.29 (t, J=7.1 Hz, 3H). MS (ESI): m/z 517.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromobenzaldehyde to give compound 99 (white solid, 15.3 mg, 12.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 9.92 (s, 1H), 7.94 (d, J=8.8 Hz, 2H), 7.86 (d, J=8.7 Hz, 2H), 6.97 (s, 2H), 4.63 (s, 2H), 4.53 (s, 2H), 2.13 (s, 6H), 1.33 (s, 6H). MS (ESI): m/z 425.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromobenzaldehyde to give compound 100 (white solid, 135.8 mg, 30.0% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 9.97 (s, 1H), 7.92 (s, 2H), 7.79 (d, J=8.7 Hz, 2H), 7.08 (s, 2H), 4.66 (s, 2H), 4.39 (s, 2H), 4.30 (q, J=7.0 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 475.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromocyclopropylbenzene to give compound 101 (white solid, 18.5 mg, 27.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.56 (d, J=8.6 Hz, 1H), 7.49 (d, J=8.4 Hz, 1H), 7.39 (d, J=8.6 Hz, 1H), 7.08 (d, J=8.6 Hz, 1H), 6.95 (s, 2H), 4.53 (s, 2H), 4.50 (s, 2H), 2.13 (s, 6H), 1.87-1.78 (m, 1H), 1.33 (s, 6H), 1.00-0.86 (m, 2H), 0.66-0.56 (m, 2H). MS (ESI): m/z 437.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 4-bromocyclopropylbenzene to give compound 102 (white solid, 74.0 mg, 14.6% yield) without hydrolysis:1H NMR (300 MHz, CDCl3) δ 7.46 (d, J=8.6 Hz, 2H), 7.12 (s, 2H), 7.08 (d, J=4.1 Hz, 2H), 4.63 (s, 2H), 4.31 (s, 2H), 4.27 (q, J=7.1 Hz, 2H), 2.29-2.21 (m, 1H), 2.19 (s, 6H), 1.47 (s, 6H), 1.37 (d, J=7.2 Hz, 3H), 0.97 (dt, J=13.7, 5.6 Hz, 2H), 0.68 (dt, J=9.6, 4.7 Hz, 2H). MS (ESI): m/z 487.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-fluoroiodobenzene to give compound 103 (white solid, 9.0 mg, 20.6% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.57 (d, J=11.1 Hz, 1H), 7.43 (s, 2H), 6.99-6.94 (m, 3H), 4.56 (s, 2H), 4.51 (s, 2H), 2.13 (s, 6H), 1.33 (s, 6H). MS (ESI): M/z 437.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-fluoroiodobenzene to give compound 104 (white solid, 49.5 mg, 10.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.54 (dt, J=11.1, 2.1 Hz, 1H), 7.36 (dd, J=14.8, 8.2 Hz, 1H), 7.24 (dd, J=7.9, 1.1 Hz, 1H), 7.07 (s, 2H), 6.87 (td, J=8.2, 2.2 Hz, 1H), 4.64 (s, 2H), 4.32 (s, 2H), 4.27 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 465.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 2-chloro-1-fluoro-4-iodobenzene to give compound 105 (white solid, 25.4 mg, 20.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.82 (s, 1H), 7.89 (d, J=6.3 Hz, 1H), 7.61 (d, J=9.0 Hz, 1H), 7.46 (t, J=9.2 Hz, 1H), 6.95 (s, 2H), 4.56 (s, 2H), 4.50 (s, 2H), 2.13 (s, 6H), 1.32 (s, 6H). MS (ESI): m/z 471.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 2-chloro-1-fluoro-4-iodobenzene to give compound 106 (white solid, 134.6 mg, 30.8% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.77 (d, J=3.4 Hz, 1H), 7.39 (s, 1H), 7.21-7.13 (m, 1H), 7.07 (s, 2H), 4.63 (s, 2H), 4.32-4.24 (m, 4H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 499.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 1-chloro-3-iodobenzene to give compound 107 (white solid, 10 mg, 16.5% yield): 1H NMR (300 MHz, DMSO-d6) δ 12.84 (s, 1H), 7.79 (s, 1H), 7.56 (d, J=7.8 Hz, 1H), 7.41 (t, J=8.1 Hz, 1H), 7.18 (d, J=7.9 Hz, 1H), 6.96 (s, 2H), 4.57 (s, 2H), 4.51 (s, 2H), 2.13 (s, 6H), 1.33 (s, 6H). MS (ESI): m/z 453.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 1-chloro-3-iodobenzene to give compound 108 (yellow liquid, 60.7 mg, 12.7% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.70 (s, 1H), 7.42 (d, J=7.5 Hz, 1H), 7.32 (t, J=7.9 Hz, 1H), 7.13 (d, J=6.9 Hz, 1H), 7.06 (s, 2H), 4.63 (s, 2H), 4.30 (s, 2H), 4.26 (q, J=7.0 Hz, 2H), 2.19 (s, 6H), 1.45 (s, 6H), 1.35 (t, J=7.0 Hz, 3H). MS (ESI): m/z 481.1 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-trifluoromethoxybromobenzene to give compound 109 (white solid, 47.4 mg, 36.7% yield):1H NMR (300 MHz, DMSO-d6) δ 12.90 (s, 1H), 7.82 (s, 1H), 7.62-7.45 (m, 2H), 7.25-7.07 (m, 1H), 6.95 (s, 2H), 4.58 (s, 2H), 4.51 (s, 2H), 2.13 (s, 6H), 1.32 (s, 6H). MS (ESI): m/z 503.2 [M+Na]+.
Referring to the method in Example 1, 4-iodotrifluoromethylbenzene in the synthetic route of method (3) was replaced by 3-trifluoromethoxybenzene to give compound 110 (yellow liquid, 176.1 mg, 38.4% yield) without hydrolysis: 1H NMR (300 MHz, CDCl3) δ 7.66 (s, 1H), 7.43 (d, J=4.9 Hz, 2H), 7.08 (s, 2H), 7.03 (s, 1H), 4.65 (s, 2H), 4.33 (s, 2H), 4.29 (q, J=7.1 Hz, 2H), 2.20 (s, 6H), 1.47 (s, 6H), 1.36 (t, J=7.1 Hz, 3H). MS (ESI): m/z 531.2 [M+Na]+.
Compound 1 (232 mg, 0.5 mmol) was dissolved in DCM (4 mL), and tromethamine (60.5 mg, 0.5 mmol) was added. The mixture was stirred at room temperature for 12 h, and then a white solid was precipitated from the reaction solution. The mixture was filtered under vacuum, and acetone (0.5 mL) and n-hexane (2 mL) were added to the solid, followed by stirring at room temperature for 2 h. The mixture was filtered under vacuum to give compound 111 (white solid, 277.0 mg, 94.7% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J=8.4 Hz, 2H), 7.76 (d, J=8.8 Hz, 2H), 6.94 (s, 2H), 6.03 (s, 3H), 4.62 (s, 2H), 4.52 (s, 2H), 3.38 (s, 6H), 2.16 (s, 6H), 1.27 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by esmolol to give compound 112 (white solid, 27.7 mg, 47.1% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.7 Hz, 2H), 7.11 (d, J=7.5 Hz, 2H), 6.94 (s, 2H), 6.83 (d, J=7.7 Hz, 2H), 4.61 (s, 2H), 4.52 (s, 2H), 4.00 (s, 1H), 3.96-3.63 (m, 3H), 3.48-2.95 (m, 7H), 2.89 (d, J=11.2 Hz, 1H), 2.75 (d, J=8.0 Hz, 3H), 2.14 (s, 6H), 1.30 (s, 6H), 1.24 (s, 1H), 1.10 (d, J=5.4 Hz, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by cinacalcet to give compound 113 (white solid, 49.0 mg, 55.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 8.25 (s, 1H), 7.97-7.66 (m, 7H), 7.49 (s, 5H), 6.96 (s, 2H), 4.57 (d, J=25.9 Hz, 5H), 3.56-3.09 (m, 7H), 2.69 (s, 2H), 2.43-2.33 (m, 2H), 2.14 (s, 6H), 1.75 (s, 1H), 1.53-1.17 (m, 9H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by trimetazidine to give compound 114 (white solid, 55.8 mg, 76.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=8.5 Hz, 2H), 7.75 (d, J=8.8 Hz, 2H), 6.94 (s, 3H), 6.74 (d, J=8.4 Hz, 1H), 4.61 (s, 2H), 4.52 (s, 2H), 3.76 (d, J=1.6 Hz, 6H), 3.72 (s, 3H), 2.78 (s, 3H), 2.37 (s, 3H), 2.14 (s, 6H), 1.29 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by fasudil to give compound 115 (white solid, 51.8 mg, 63.3% yield): 1H NMR (300 MHz, DMSO-d6) δ 9.48 (s, 1H), 8.69 (d, J=5.8 Hz, 1H), 8.44 (d, J=7.8 Hz, 1H), 8.37-8.28 (m, 2H), 7.91-7.66 (m, 5H), 6.94 (s, 2H), 4.61 (s, 2H), 4.52 (s, 2H), 3.53-3.41 (m, 4H), 2.87-2.67 (m, 4H), 2.13 (s, 6H), 1.67 (d, 2H), 1.30 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by acebutolol to give compound 116 (white solid, 62.8 mg, 73.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.91-7.65 (m, 7H), 7.09 (d, J=8.9 Hz, 1H), 6.94 (s, 2H), 5.75 (s, 1H), 4.61 (s, 2H), 4.52 (s, 2H), 4.02 (d, J=6.5 Hz, 2H), 2.96-2.80 (m, 2H), 2.73 (d, J=7.1 Hz, 1H), 2.57 (s, 3H), 2.23 (t, J=7.3 Hz, 2H), 2.14 (s, 6H), 1.58 (q, J=14.7, 7.4 Hz, 2H), 1.29 (s, 6H), 1.05 (d, J=6.2 Hz, 6H), 0.89 (t, J=7.3 Hz, 3H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by bevantolol to give compound 117 (white solid, 62.6 mg, 22.4% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 7.14 (t, J=7.8 Hz, 1H), 6.95 (s, 2H), 6.83 (d, J=8.9 Hz, 2H), 6.71 (t, J=6.6 Hz, 3H), 4.61 (s, 2H), 4.52 (s, 2H), 3.90 (d, J=13.4 Hz, 3H), 3.72 (s, 2H), 3.70 (s, 3H), 3.04-2.74 (m, 6H), 2.26 (s, 3H), 2.14 (s, 6H), 1.32 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by metoprolol to give compound 118 (white solid, 52.2 mg, 66.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=8.7 Hz, 2H), 7.75 (d, J=8.7 Hz, 2H), 7.12 (d, J=8.4 Hz, 2H), 6.94 (s, 2H), 6.83 (d, J=8.5 Hz, 2H), 4.61 (s, 2H), 4.52 (s, 2H), 3.96 (s, 1H), 3.89 (s, 2H), 3.46 (t, J=6.9 Hz, 2H), 3.22 (s, 3H), 2.97-2.88 (m, 1H), 2.83 (d, J=8.2 Hz, 1H), 2.74-2.63 (m, 3H), 2.15 (s, 6H), 1.29 (s, 6H), 1.07 (d, J=6.0 Hz, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by bisoprolol to give compound 119 (white solid, 72.4 mg, 85.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.85 (d, J=8.6 Hz, 2H), 7.75 (d, J=8.6 Hz, 2H), 7.22 (d, J=8.1 Hz, 2H), 6.94 (s, 2H), 6.90 (d, J=8.4 Hz, 2H), 4.61 (s, 2H), 4.52 (s, 2H), 4.39 (s, 2H), 3.96 (s, 1H), 3.92 (s, 2H), 3.52 (d, J=6.0 Hz, 1H), 3.47 (s, 4H), 2.93 (d, J=6.2 Hz, 1H), 2.87-2.59 (m, 2H), 2.15 (s, 6H), 1.30 (s, 6H), 1.06 (d, J=5.5 Hz, 12H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by carvedilol to give compound 120 (white solid, 59.8 mg, 64.2% yield): 1H NMR (300 MHz, DMSO-d6) δ 11.23 (s, 1H), 8.21 (d, J=7.8 Hz, 1H), 7.85 (d, J=8.5 Hz, 2H), 7.76 (d, J=8.6 Hz, 2H), 7.43 (d, J=7.8 Hz, 1H), 7.30 (dd, J=19.6, 7.6 Hz, 2H), 7.12 (d, J=7.4 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 6.95 (d, J=10.7 Hz, 2H), 6.93 (s, 2H), 6.91-6.80 (m, 2H), 6.67 (d, J=7.8 Hz, 1H), 4.61 (s, 2H), 4.53 (s, 2H), 4.15 (s, 2H), 4.13 (s, 1H), 4.03 (t, J=5.5 Hz, 2H), 3.72 (s, 3H), 3.02-2.92 (m, 3H), 2.85 (dd, J=11.9, 5.9 Hz, 1H), 2.14 (s, 6H), 1.33 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by labetalol to give compound 121 (white solid, 69.1 mg, 81.6% yield): 1H NMR (300 MHz, DMSO-d) δ 8.40 (s, 1H), 7.85 (d, J=9.1 Hz, 4H), 7.75 (d, J=8.4 Hz, 2H), 7.40 (d, J=8.2 Hz, 1H), 7.24 (d, J=6.5 Hz, 2H), 7.16 (d, J=7.0 Hz, 4H), 6.95 (s, 2H), 6.84 (d, J=8.4 Hz, 1H), 4.61 (s, 2H), 4.58 (s, 1H), 4.52 (s, 2H), 2.73 (s, 2H), 2.59 (s, 2H), 2.14 (s, 6H), 1.67 (d, J=55.4 Hz, 2H), 1.31 (s, 6H), 1.08 (d, 3H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by diisopropylamine to give compound 122 (white solid, 61.3 mg, 63.8% yield): 1H NMR (300 MHz, DMSO-d6) δ 7.86 (d, J=8.8 Hz, 2H), 7.77 (d, J=8.8 Hz, 2H), 6.94 (s, 2H), 4.63 (s, 2H), 4.53 (s, 2H), 3.11 (dt, J=12.8, 6.5 Hz, 2H), 1.28 (s, 6H), 1.12 (s, 6H), 1.10 (s, 6H).
Referring to the method in Example 111, tromethamine in Example 111 was replaced by berberine to give compound 123 (white solid, 20.3 mg, 4.9% yield): 1H NMR (300 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.95 (s, 1H), 8.21 (d, J=9.3 Hz, 1H), 8.01 (d, J=9.1 Hz, 1H), 7.86 (d, J=8.8 Hz, 2H), 7.81 (s, 1H), 7.77 (d, J=8.7 Hz, 2H), 7.10 (s, 1H), 6.90 (s, 2H), 6.18 (s, 2H), 4.94 (t, 21H), 4.63 (s, 2H), 4.51 (s, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.21 (t, 2H), 2.17 (s, 6H), 1.22 (s, 6H).
Cos-7 cells (African green monkey kidney fibroblasts, commonly used tool cells) were cultured in a 10-cm cell culture dish with a DMEM complete medium containing 10% fetal bovine serum. When growing at a density of about 70%, the cells were ready for transfection. Firstly, a plasmid transfection working solution was prepared by the following procedures: 15 μg of pGL4.35-9×Gal4 UAS plasmid (purchased from Promega (Beijing) Biotech Co., Ltd.), 15 μg of pBIND-Gal4-PPARα (LBD) plasmid or pBIND-Gal4-PPARδ (LBD) plasmid or pBIND-Gal4-PPARγ (LBD) plasmid (J. Chem. Inf. Model., 2020, 60, 1717), and 60 μL of a transfection reagent (HighGene, purchased from Wuhan ABclonal Biotechnology Co., Ltd.) were added to 2 mL of Opti-MEM, the mixture was left to stand at room temperature for 15 min, and then the plasmid transfection working solution was obtained. The working solution described above was then combined with 8 mL of the DMEM complete medium, and the mixture was added to the cell culture dish for cell transfection. After 4 h of transfection, the cells were digested, resuspended, and seeded into a 96-well plate at 25,000 cells/well. After 24 h of adherent culture, test compounds and a positive drug at appropriate test concentrations were prepared from the complete medium and added to the 96-well plate. In the test, the PPARα agonistic activity of GW7647 (purchased from MCE) at a final concentration of 10 nM was determined to be 100%, the PPARS agonistic activity of GW501516 (purchased from MCE) at a final concentration of 10 nM was determined to be 100%, and the PPARγ agonistic activity of Rosiglitazone (purchased from Adamas) at a final concentration of 1 μM was determined to be 100%. After 16 h of drug action, the culture medium was removed, and 100 μL of a reporter gene lysis buffer (purchased from Shanghai Beyotime Biological Tech. Co., Ltd.) was added. The cells were shaken and lysed for 15 min, and then 10 μL of a lysate was pipetted and added into a white opaque 384-well plate. 10 μL of a reporter gene detection solution (purchased from Shanghai Beyotime Biological Tech. Co., Ltd.) was added. After the resulting solution was mixed and reacted, biological fluorescence was detected by using a multifunctional microplate reader, and corresponding half maximal effective concentration (EC50) values were calculated according to the detection values. The experiment adopted the PPARα/γ agonist GFT505 in phase III clinical trials and the potent PPARα/γ agonist 5c reported in the literature (ACS Med. Chem. Lett., 2019, 10, 1068), and H11 (Journal of Medicinal Chemistry 2022, 65, 2571-2592) as positive control compounds. The experimental results are shown in Table 2.
The experimental results (Table 2) show that the compounds of the present invention had significant PPAR agonistic activities. For example, EC50 values of compounds 1, 3, 29, 33, 41, 43, 45, 61, 75, 101, and the like for the PPARα and PPARβ agonistic activities all reached low nanomolar levels. Especially, compound 1 (PPARα: EC50=0.7 nM; PPARδ: EC50=0.4 nM) had EC50 values for the PPARα and PPARδ agonistic activities at picomolar levels and had very good selectivity (more than 2000-fold selectivity) for PPARγ. The above results suggest that the compounds of the present invention are potent and highly selective PPARα/PPARδ dual agonists.
A solution of the compound in acetonitrile at a concentration of 500 μM was prepared and diluted in 0.1 M potassium phosphate solution to obtain a 1.5 μM drug working solution. The drug working solution was co-incubated with a human liver microsome working solution at a final concentration of 0.75 mg/mL and a NADPH solution (at a final concentration of 550 μM), and the incubation was terminated by adding an acetonitrile solution at 0 min, 15 min, 30 min, 45 min, and 60 min, respectively. The remaining amount of the compound remained in the system at each time point was detected by using LC/MS, the absolute value k of the slope was measured by plotting the natural logarithm of the percentage of the remaining amount of the compound over time, and the calculation was performed according to the formula: T1/2 (half-life)=ln2/k=0.693/k. The experimental results are shown in Table 3.
The experimental results (Table 3) show that compound 1 has very good metabolic stability in human liver microsomes, and the metabolic stability for human liver microsomes is far better than that of GFT505 under the same test conditions. Other compounds of the present invention also have good metabolic stability in human liver microsomes.
Animals: 6 SPF-grade male SD rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.
Grouping: rats were divided into 2 groups, with 3 rats in each group; one group was an oral administration group, and the other group was an intravenous injection group. The dose in the oral administration group was 10 mpk, and the dose in the intravenous injection group was 2 mpk.
Experimental procedures: after rats in the intravenous injection group were administered by tail vein injection, about 0.25 mL of blood was collected from the orbit at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h, respectively, and then heparin sodium was rapidly added for anticoagulation after blood collection. The blood was placed on ice after collection. Rats in the oral administration group were fasted for 12 h before administration and fed 4 h after administration. After the rats were administered orally, about 0.25 mL of blood was collected from the orbit at 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 24 h, respectively, and then heparin sodium was rapidly added for anticoagulation after blood collection. The blood was placed on ice after collection. All samples were centrifuged at 6000 r/min for 3 min in a low-temperature centrifuge, and plasma was obtained after separation. The content of the compound in the plasma was detected via LC-MS/MS-18, and relevant pharmacokinetic parameters were calculated according to the plasma concentration data of different time points. The experimental results are shown in Table 4.
The experimental results (Table 4) show that the oral half-life of compound 1 was 6.72±1.39 h, and the bioavailability of the compound was 104.70±0.17%, indicating that compound 1 has good pharmacokinetic properties. Other compounds of the present invention also have relatively good in-vivo pharmacokinetic properties.
The agonistic effects of compound 1 on common nuclear receptors were tested by using the GAL4 hybrid reporter gene method.
Reporter gene plasmids for different nuclear receptors were constructed by referring to the method in the literature (Journal of Medicinal Chemistry 2022, 65, 2571-2592). Transfection working solutions for different nuclear receptors were prepared and added into COS-7 cells. The preparation method for the transfection working solution was as follows: the constructed pBIND-Gal4-PPARα (LBD) (or pBIND-Gal4-PPARδ (LBD) plasmid or pBIND-Gal4-PPARγ (LBD) plasmid or pBIND-Gal4-RARα (LBD) plasmid or pBIND-Gal4-RARγ (LBD) plasmid or pBIND-Gal4-RARβ (LBD) plasmid or pBIND-Gal4-RORα (LBD) plasmid or pBIND-Gal4-RORγ (LBD) plasmid or pBIND-Gal4-RORβ (LBD) plasmid or pBIND-Gal4-FXR (LBD) plasmid or pBIND-Gal4-RXRα. (LBD) plasmid or pBIND-Gal4-RXRγ (LBD) plasmid or pBIND-Gal4-RXRβ (LBD) plasmid or pBIND-Gal4-VDR (LBD) plasmid or pBIND-Gal4-LXRα (LBD) plasmid or pBIND-Gal4-LXRβ (LBD) plasmid or pBIND-Gal4-THP (LBD) plasmid or pBIND-Gal4-PXR (LBD) plasmid or pBIND-Gal4-CAR (LBD) plasmid), 15 μg of pGL4.35-9×Gal4 UAS plasmid (purchased from Promega (Beijing) Biotech Co., Ltd.), and 60 μL of a transfection reagent (HighGene, purchased from Wuhan ABclonal Biotechnology Co., Ltd.) were added to 2 mL of Opti-MEM, the mixture was left to stand at room temperature for 15 min, and then the working solution was obtained. The agonism of compound 1 for various types of nuclear receptors was tested at a concentration of 1 μM.
The experimental results (Table 5) show that compound 1 had no significant agonistic effect on non-PPAR nuclear receptors at a concentration of 1 μM, and had a relatively weak agonistic effect on PPARγ. Therefore, compound 1 is considered to have high selectivity for nuclear receptors PPARα/6. Other compounds of the present invention also have similar effects.
C57 mice were divided into three groups, i.e., a control group, a low dose group (0.03 mg/kg), and a high dose group (0.1 mg/kg), with 6 mice in each group. Each group of mice was intragastrically administered compound 1 or a solvent control with the same volume for three consecutive days according to the doses. Three days after administration, the mice were euthanized and dissected for sampling. Liver and skeletal muscle were snap-frozen in liquid nitrogen for subsequent experiments. After extracting RNA in the liver and skeletal muscle, the up-regulation fold of the expression of the PPARα/δ downstream target genes was detected. The results (Table 6) show that Pdk4, Acox1, Vlcad, and Angptl4 were significantly up-regulated in the liver, and Pdk4 and Angptl4 were significantly up-regulated in the skeletal muscle, all of which were dose-dependent. This suggests that compound 1 may have a dual agonistic effect on PPARα/δ in the mice. Some of other compounds of the present invention also have similar effects.
Animals: 48 male C57 mice, SPF grade, 8 weeks old, weighing about 20 g, purchased from Beijing Vital River. All animals maintained a 12-hour alternating circadian rhythm and were given ad libitum access to food and water.
Instruments: a scale for weighing animals; an automatic biochemical analyzer
Reagents: compound 1, the positive drug GFT505 (a PPARα/8 dual agonist, currently in the anti-NASH phase III clinical trials, the preparation method referred to CN100548960C), the positive drug fenofibrate (a PPARα agonist, a drug clinically used for the treatment of hypertriglyceridemia, purchased from Aladdin), and the positive drug pemafibrate (a PPARα agonist, a drug marketed in Japan for the treatment of hypertriglyceridemia, the preparation method referred to Bioorg. Med. Chem. Lett., 2007, 17).
After 1 week of adaptive feeding, the mice were divided into 5 groups according to their body weight: a control group, a positive drug fenofibrate (30 mg/kg) group, a positive drug pemafibrate (1 mg/kg) group, a positive drug GFT505 (1 mg/kg) group, and a compound 1 (1 mg/kg) group. Mice in the control group were administered a solvent control CMC-Na every day, and mice in each of the administration groups were administered the corresponding drug every day. Intragastric administration was performed for 5 consecutive days, and the mice were given ad libitum access to food and water during administration. Each group of mice was weighed every day, and their body weight, hair, feces, and activities were carefully observed and recorded.
Two hours after administration on day 5, blood was collected from the orbit, and the mice were euthanized.
The whole blood was left to stand at room temperature for 2 h and centrifuged at 3000 rpm for 15 min, and serum was collected. The serum was placed onto the automatic biochemical analyzer (Google Biotechnology Co., Ltd.) to determine the triglyceride (TG) levels in the serum.
The results in
Animals: 25 male C57 mice, SPF grade, 8 weeks old, weighing about 20 g, purchased from Beijing Vital River. All animals maintained a 12-hour alternating circadian rhythm and were given ad libitum access to food and water.
Instruments: a scale for weighing animals; an automatic biochemical analyzer; an inverted microscope; a microtome
Reagents: compound 1, the positive drug GFT505, and α-naphthylisothiocyanate (ANIT).
After 1 week of adaptive feeding, the mice were divided into 5 groups according to their body weight: a control group, a model group, a positive drug GFT505 (30 mg/kg) group, a compound 1 low-dose (0.03 mg/kg) group, and a compound 1 high-dose (0.1 mg/kg) group. The mice were all fed food and water normally.
The molding and administration processes were as follows: the mice in each of the administration groups were intragastrically administered the corresponding doses and types of compounds 6 h before modeling, and the mice in the control group and the model group were administered solvent controls with the same volume. When modeling, except for the control group, the mice in each group were intragastrically administered ANIT at a dose of 80 mg/kg, and the mice in the control group were administered the solvent control with the same volume. The administration was then continued for two days, once per day. Each group of mice was weighed every day, and their body weight, hair, feces, and activities were carefully observed and recorded.
Forty-eight hours after giving ANIT for molding, blood was collected from the orbit, the mice were euthanized, and the liver was collected. The right lobular tissue of the liver was fixed with 4% paraformaldehyde and used for sectioning followed by HE staining. The remaining liver tissues were snap-frozen in liquid nitrogen for subsequent assays of other indexes.
The whole blood was left to stand at room temperature for 2 h and centrifuged at 3000 rpm for 15 min, and serum was collected. Levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TBil), and total bile acid (TBA) in the serum were assayed.
The fixed tissue was sent to Wuhan Servicebio Technology Co., Ltd. for preparing HE-stained sections.
The test was performed using a tissue and blood alkaline phosphatase (AKP/ALP) activity assay kit (BC2145) purchased from Beijing Solarbio Science & Technology Co., Ltd. The liver tissue stored at −80° C. was taken and placed in liquid nitrogen, about 0.1 g of liver tissue was rapidly cut off, and 1 mL of an extracting solution was added. The mixture was ground thoroughly and centrifuged at 4° C. and 10000 rpm for 10 min, and the supernatant was collected for assay. The assay was performed by referring to the method in the product's instruction. Data were corrected according to protein concentrations of the samples, and the catalytic production of 1 μmol phenol per minute per milligram protein at 37° C. was defined as one enzymatic activity unit.
The results in
In addition, the anti-cholestasis effect of compound 1 was evaluated using a method in pathological research. The HE staining results (
In conclusion, compound 1 has a potent therapeutic effect on a mouse cholestatic model, which suggests that compound 1 has a therapeutic effect on cholestatic liver diseases and can be used for preparing medicaments for the prevention and treatment of cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Other compounds of the present invention also have similar effects.
Animals: 24 male C57 mice, SPF grade, 8 weeks old, weighing about 20 g, purchased from Beijing Vital River. All animals maintained a 12-hour alternating circadian rhythm and were given ad libitum access to food and water.
Instruments: a scale for weighing animals; a microtome; an automatic biochemical analyzer; an inverted microscope
Reagents: compound 1, the positive drug GFT505; control feeds purchased from Nantong Trophic Animal Feed High-tech Co. Ltd. (TP36225 MCS); molding feeds purchased from Nantong Trophic Animal Feed High-tech Co. Ltd. (TP36225 MCD).
After 1 week of adaptive feeding, the mice were randomized into 4 groups according to the body weight: a control group, a model group, a positive drug GFT505 (10 mg/kg) group, and a compound 1 (0.1 mg/kg) group. The mice in the control group were fed control feeds (TP36225 MCS); the mice in other groups were given molding feeds (TP36225 MCD). All the mice were fed water normally, and the modeling lasted for 6 weeks.
After 4 weeks of molding, the mice in the positive drug GFT505 group were intragastrically administered GFT505 at 10 mg/kg every day; the mice in the compound 1 group were intragastrically administered compound 1 at 0.1 mg/kg every day; the mice in the control group and the model group were intragastrically administered control solvents with the same volume every day. The administration was performed for 2 weeks, during which time the mice in the control group were given the control feeds, and the mice in the remaining groups were given the molding feeds. All the mice were fed water normally. Each group of mice was weighed every day, and their body weight, hair, feces, and activities were carefully observed and recorded.
After 2 weeks of administration, the mice were fasted for 6 h in advance but had free access to water. Blood was collected from the orbit, the mice were euthanized, and the liver was collected. The right lobular tissue of the liver was fixed with 4% paraformaldehyde and used for HE and oil red staining. The remaining liver tissues were divided into 2 parts and snap-frozen in liquid nitrogen for subsequent assays of other indexes.
The whole blood was left to stand at room temperature for 2 h and centrifuged at 3000 rpm for 15 min, and serum was collected. The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in the serum were determined by using the automatic biochemical analyzer.
The fixed tissue was sent to Wuhan Servicebio Technology Co., Ltd. for preparing HE-stained sections, sirius red-stained sections, and oil red-stained sections.
6. Extraction and q-PCR Assay of RNA in Liver Tissue
The liver tissue stored at −80° C. was taken and placed in liquid nitrogen, and about 10 mg of liver tissue was rapidly cut off. About 500 μL of a pre-cooled RNA extraction reagent (Nanjing Vazyme Biotech Co., Ltd., R401-01) was added, and the tissue was homogenized in a homogenizer. The precipitated RNA was extracted by referring to the method in the product's instruction. The solid RNA was dissolved in an appropriate amount of DEPC-treated water. The RNA concentration was quantified using NanoDrop, a reverse transcription reagent purchased from Vazyme Biotech Co., Ltd. was added according to the instruction, and mRNA was reverse-transcribed into cDNA using a common PCR instrument. Finally, upstream and downstream primers of the target gene, q-PCR reagents (SYBR Green), and cDNA were added to a 96-well plate dedicated to q-PCR, and amplification and quantification were performed using the q-PCR instrument. A ΔΔCt value was selected to represent the difference in gene expression, and relevant software was adopted for data processing and statistical tests.
The liver tissue stored at −80° C. was taken and placed in liquid nitrogen, about 10 mg of liver tissue was rapidly cut off, and 300 μL of methanol was added. The tissue was homogenized and lysed. Then, 600 μL of chloroform was added, the mixture was shaken at room temperature overnight, and liposoluble substances were extracted from the tissue. The mixture was centrifuged at 6000 rpm for 10 min, and the supernatant was collected. The test was performed according to a triglyceride assay kit (290-63701) from WAKO, Japan, and the final results were corrected to milligrams of triglyceride per gram of liver tissue.
The results in
The anti-NASH effect of compound 1 was observed through a method in pathological research. The HE staining results (
To further assay the effect of compound 1 on reducing liver inflammation and fibrosis in NASH model mice, mRNA expression levels of the relevant inflammatory factors and fibrosis-associated cytokines in the liver tissue were determined (see Table 7 for primer sequences of genes). The experimental results are shown in
The results in
The above results show that compound 1 could significantly improve the pathological state of NASH mice at a dose of 0.1 mg/kg, reduce the levels of hepatic enzymes, inhibit the development and progression of liver inflammation and fibrosis, and had an equivalent therapeutic effect to that of GFT505 at a dose of 10 mg/kg. The effect of the compound at 0.1 mg/kg on reducing lipid accumulation in the liver was superior to that of GFT505 at 10 mg/kg. It is suggested that compound 1 has therapeutic effects on fatty liver diseases such as NASH, and can be used for preparing medicaments for the prevention and treatment of chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), metabolic fatty liver disease (MAFLD), alcoholic fatty liver disease (ALD), and the like. Other compounds of the present invention also have similar effects.
Animals: 30 male C57 mice, SPF grade, 8 weeks old, weighing about 20 g, purchased from Beijing Vital River. All animals maintained a 12-hour alternating circadian rhythm and were given ad libitum access to food and water.
Instruments: a scale for weighing animals; a microtome; an automatic biochemical analyzer; an inverted microscope
Reagents: compound 1, the positive drug GFT505, tetrachloromethane (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), and sunflower seed oil (purchased from Shanghai Yuanye Biotech Co., Ltd.).
After 1 week of adaptive feeding, the mice were randomized into 5 groups according to the body weight: a control group (Oil), a model group (CCl4), a positive drug GFT505 (10 mg/kg) group (CCl4+GFT505), a compound 1 low-dose (0.03 mg/kg) group (CCl4+1 low dose), and a compound 1 high-dose (0.1 mg/kg) group (CCl4+1 high dose). The mice were all fed food and water normally, and the modeling lasted for 3 weeks. The mice in the model group and each of the administration groups were injected twice a week with a 25% CCl4 oil solution at a dose of 2 mL/kg, and the mice in the control group were injected with an oil solvent with the same volume.
The administration was started at the same time of molding. The mice in the CCl4+GFT505 group were intragastrically administered GFT505 (10 mg/kg) every day; the mice in the CCl4+1 low dose group were intragastrically administered compound 1 (0.03 mg/kg) every day; the mice in the CCl4+1 high dose group were intragastrically administered compound 1 (0.1 mg/kg) every day; the mice in the control group and the model group were intragastrically administered control solvents with the same volume every day. After 3 weeks of administration, the mice were all fed food and water normally. Each group of mice was weighed every day, and their body weight, hair, feces, and activities were carefully observed and recorded.
Twenty-four hours after the sixth injection of CCl4, the mice were dissected for sampling. Blood was collected from the orbit, the mice were euthanized, and the liver was collected. The right lobular tissue of the liver was fixed with 4% paraformaldehyde and used for sectioning followed by HE and sirius red staining. Part of liver tissues were divided into 3 parts and snap-frozen in liquid nitrogen for subsequent assays of other indexes.
The pre-treated tissue was sent to Wuhan Servicebio Technology Co., Ltd. for preparing HE-stained sections and sirius red-stained sections.
The liver tissue stored at −80° C. was taken and placed in liquid nitrogen, and about 200 mg of liver tissue was rapidly cut off. Hydroxyproline in the liver tissue was assayed according to the method in the product's instruction (Beijing Solarbio Science & Technology Co., Ltd., BC0255).
The results in
The anti-hepatic fibrosis effect of compound 1 was observed through a method in pathological research. The HE staining results (
In conclusion, compound 1 had a protective effect on the hepatic fibrosis mouse model, suggesting that compound 1 has therapeutic effects on hepatic fibrosis-associated diseases and can be used for preparing medicaments for the prevention and treatment of hepatic fibrosis-associated diseases, liver cirrhosis, and other diseases. Other compounds of the present invention also have similar effects.
Eutectic Structure of Complex of Compound 1 with PPARδ Protein
A plasmid expressing a protein comprising a PPARδ-LBD region (see Journal of Medicinal Chemistry 2022, 65, 2571-2592) was transformed into E. coli BL21. After culture and amplification, IPTG was added at 4° C. to induce protein expression. After E. coli was lysed, the supernatant was collected and purified via a nickel column. The purified protein was dissolved in a solution of 20 mM Tris, 150 mM NaCl, and 10% glycerol, pH 8.0. To the protein solution at a concentration of 7 mg/mL was added a solution of compound 1 in DMSO at a final concentration of 2 mM. An eutectic complex of compound 1 and the PPARδ protein was grown at 16° C. in a crystalline solvent that was a mixed solvent of 0.5 M sodium citrate, 19% PEG3350, and 20% glycerol, pH 5.5. The crystals were snap-frozen in liquid nitrogen for data collection. X-ray diffraction data were collected at beam line BL02U of Shanghai Synchrotron Radiation Facility with the help of an X-ray crystallography facility platform at the National Center for Protein Sciences (Tsinghua University). The data were processed with HKL2000 and solved by molecular replacement using the Phenix program, and the search model was PDB with the code of 3SP9. Modeling and refinement were performed using coot software and PHENIX software. The experimental results are shown in
Compound 1 (50 g) obtained in Example 1, hydroxypropyl methylcellulose E (150 g), starch (200 g), an appropriate amount of povidone K30, and magnesium stearate (1 g) were mixed, followed by granulating and tableting. In addition, the compounds prepared in Examples 1-123 can be prepared into capsules, powders, granules, pills, injections, syrups, oral liquids, inhalants, ointments, suppositories, patches, and the like by adding different pharmaceutical excipients according to conventional formulation methods of Pharmacopoeia (2015 Edition).
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210067669.7 | Jan 2022 | CN | national |
| 202211624621.8 | Dec 2022 | CN | national |
This application is a 371 of international application of PCT application serial no. PCT/CN2022/141359, filed on Dec. 23, 2022, which claims the priority benefit of China application no. 202210067669.7 filed on Jan. 20, 2022 and China application no. 202211624621.8, filed on Dec. 16, 2022. The entirety of each of the above-mentioned patent applications is hereby incorporated by reference herein and made a part of this specification.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/141359 | 12/23/2022 | WO |
