WO2025162103A1 - C-17 carbonyl-substituted oleanane triterpene derivative, preparation method therefor, and use thereof - Google Patents

Abstract

The present invention relates to a C-17 carbonyl-substituted oleanane triterpene derivative, a preparation method therefor, and a use thereof. Provided are a C-17 carbonyl-substituted oleanane triterpene derivative, a use of the compound in the preparation of an NRF2 activator, and preparation of a medicament for treating/preventing diseases. The diseases comprise cerebral small vascular disease, mitochondrial encephalomyopathy, autism spectrum disorder, Rett syndrome, Friedreich ataxia, stroke, hemorrhagic cerebral apoplexy, ischemic cerebral apoplexy, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, schizophrenic cognitive impairment, Parkinson's disease, cognitive impairment in Parkinson's disease, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, kidney ischemia, etc., or other disease states and conditions which are obvious to a person skilled in the art. Also provided are a prodrug thereof, or a pharmaceutically acceptable salt thereof, or a hydrate or solvate thereof, and a pharmaceutical composition containing same.

Description

C-17 carbonyl substituted oleanane triterpene derivatives and preparation methods and uses thereof Technical Field

The present invention generally relates to the fields of biology and medicine, and in particular to C-17 carbonyl-substituted oleanane triterpene derivatives, preparation methods and uses thereof.

Background Art

Numerous studies have shown that oxidative stress can directly or indirectly induce the development and progression of various diseases, making in-depth research on oxidative stress particularly important. Oxidative stress results from a severe imbalance between the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and the antioxidant defense system, both enzymatic and non-enzymatic. To combat this damage, the body develops a complex oxidative stress response system to mitigate cellular damage and maintain homeostasis by upregulating cytoprotective factors.

Nrf2, a member of the leucine-containing (CNC) regulatory protein family, includes Neh1 to Neh6. It is an important transcription factor that regulates the body's antioxidant stress response. Keap1 is the specific receptor for Nrf2. Under normal conditions, Nrf2 and Keap1 recognize and form a polymer, whose action is inhibited by Keap1. Under conditions such as oxidative stress, Nrf2 dissociates from Keap1 and becomes activated, initiating the production of antioxidant response elements (AREs), regulating the expression of genes for phase II detoxification and antioxidant enzymes, and enhancing cellular resistance to substances such as oxidative stress and electrophilic biomacromolecules. Considering that current molecules focus solely on inflammatory treatments through Nrf2 agonism, they suffer from poor inhibitory activity and specificity. Therefore, the development of novel NRF2 activators with both anti-inflammatory and antioxidant effects is particularly important.

Summary of the Invention

In order to solve the above technical problems, the present invention provides a C-17 carbonyl-substituted oleanane triterpene derivative and a preparation method and use thereof.

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof:

wherein: R ₁ is independently selected from: -C(=O)-alkane, -C(=O)-substituted alkane, -C(=O)-alkene, -C(=O)-substituted alkene, -C(=O)-alkyne, -C(=O)-substituted alkyne, -C(=O)-heteroarene, -C(=O)-heteroarene-R ₁ ', -C(=O)-heteroarenediyl-R ₁ '-C(＝O)-NH-OH, -C(＝O)-N(OH)-alkane, -C(＝O)-N(OH)-substituted alkane, -C(＝O)-N(OH)-alkene, -C(＝O)-N(OH)-substituted alkene, -C(＝O)-N(OH)-alkyne, -C(＝O)-N(OH)-substituted alkyne, -C(＝O)-N(OH)-arene, -C(＝O)-N(OH)-arenediyl-R ₁ ', -C(＝O)-N(OH)-heteroarene, -C(＝O)-N(OH)-heteroarenediyl-R ₁ ', -C(＝O)-NH-arene, -C(＝O)-NH-arenediyl-R ₁ ', -C(＝O)-NH-heteroarene, -C(＝ _O )-NH-heteroarene ', -C(=O)-O-arene, -C(=O)-O-arenediyl-R ₁ ', -C(=O)-O-heteroarene, -C(=O)-O-heteroarenediyl-R ₁ ', -C(=O)-CH ₂ -heteroarene, -C(=O)-CH ₂ -heteroarenediyl-R ₁ ', -C(=O)-CR ₂ 'R ₃ '-heteroarene, -C(=O)-CR ₂ 'R ₃ '-heteroarenediyl-R ₁ ', -C(=O)-L-type amino acid-NH-heteroarene, -C(=O)-L-type amino acid-NH-heteroarenediyl-R ₁ ';

and R ₂ : hydrogen or methyl; R ₃ : hydrogen or methyl.

The compound is further defined as:

wherein: R ₁ is independently selected from: -C(=O)-alkane, -C(=O)-substituted alkane, -C(=O)-alkene, -C(=O)-substituted alkene, -C(=O)-alkyne, -C(=O)-substituted alkyne, -C(=O)-heteroarene, -C(=O)-heteroarene-R ₁ ', -C(=O)-heteroarenediyl-R ₁ '-C(＝O)-NH-OH, -C(＝O)-N(OH)-alkane, -C(＝O)-N(OH)-substituted alkane, -C(＝O)-N(OH)-alkene, -C(＝O)-N(OH)-substituted alkene, -C(＝O)-N(OH)-alkyne, -C(＝O)-N(OH)-substituted alkyne, -C(＝O)-N(OH)-arene, -C(＝O)-N(OH)-arenediyl-R ₁ ', -C(＝O)-N(OH)-heteroarene, -C(＝O)-N(OH)-heteroarenediyl-R ₁ ', -C(＝O)-NH-arene, -C(＝O)-NH-arenediyl-R ₁ ', -C(＝O)-NH-heteroarene, -C(＝ _O )-NH-heteroarene ', -C(=O)-O-arene, -C(=O)-O-arenediyl-R ₁ ', -C(=O)-O-heteroarene, -C(=O)-O-heteroarenediyl-R ₁ ', -C(=O)-CH ₂ -heteroarene, -C(=O)-CH ₂ -heteroarenediyl-R ₁ ', -C(=O)-CR ₂ 'R ₃ '-heteroarene, -C(=O)-CR ₂ 'R ₃ '-heteroarenediyl-R ₁ ', -C(=O)-L-type amino acid-NH-heteroarene, -C(=O)-L-type amino acid-NH-heteroarenediyl-R ₁ ';

And R ₂ : methyl group; R ₃ : methyl group.

Preferably, R ₁ 'is independently selected from: -Cl, -F, -Br, -OH, isopropyl, straight-chain/branched alkyl (C≤6), straight-chain/branched alkyl (C≤6) substituted with 1 to 5 halogens, -OH, straight-chain/branched alkyl (C≤6) substituted with 1 to 5 -OHs, straight-chain/branched alkenyl (C≤6), straight-chain/branched alkenyl (C≤6) substituted with 1 to 5 halogens, straight-chain/branched alkenyl (C≤6) substituted with 1 to 5 -OHs, straight-chain/branched alkynyl (C≤6), straight-chain/branched alkynyl (C≤6) substituted with 1 to 5 halogens, straight-chain/branched alkynyl (C≤6) substituted with 1 to 5 -OHs, wait.

Preferably, the aromatic hydrocarbon group is selected from:

wait.

Preferably, the substituted alkanes, substituted alkenes, substituted alkynes, alkanes, alkenes, alkynes have a carbon chain length of ≤6 and can be linear, branched or cyclic. The substituents are selected from 1 to 5 -SO ₃ H, -OH, -F, -Br, -Cl, -OH, methyl, ethyl, propyl replacements (and/or).

More preferably, the compound, its pharmaceutically acceptable salt or stereoisomer is as follows:

The present invention also provides the use of the above-mentioned compound, its pharmaceutically acceptable salt, and stereoisomer for preparing NRF2 activators.

Biological experiments conducted in this invention demonstrate that many of the aforementioned compounds exhibit single-digit nanomolar human Nrf2 receptor agonist activity, significantly outperforming the existing Nrf2 activator control drug, omaveloxolone. Furthermore, experiments have shown that the compounds of this invention exhibit antioxidant effects by scavenging DPPH free radicals, inhibiting the production of lipid peroxides (MDA), and intervening in ferroptosis.

The present invention also provides the use of the above-mentioned compound, its pharmaceutically acceptable salt, stereoisomer, or pharmaceutical composition to prepare a medicament for treating and/or preventing a patient's disease, and the use of the above-mentioned compound or its pharmaceutically acceptable salt or stereoisomer or pharmaceutical composition to prepare a medicament. In addition, the prepared medicament is for preventing or treating diseases including cerebral small vessel disease, mitochondrial encephalomyopathy, autism spectrum disorder, Rett syndrome, Friedreich's ataxia, stroke, hemorrhagic stroke, ischemic stroke, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, schizophrenia cognitive impairment, Parkinson's disease, Parkinson's cognitive impairment, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, renal ischemia, etc. According to effective test data, the application of the medicament prepared by the above-mentioned compound of the present invention, its pharmaceutically acceptable salt, and stereoisomer, especially for stroke, multiple sclerosis, and amyotrophic lateral sclerosis has a significant effect.

Preferably, the compound, its pharmaceutically acceptable salt, and stereoisomer are used to prepare drugs for preventing or treating stroke, multiple sclerosis, and amyotrophic lateral sclerosis.

Beneficial Effects: The present invention synthesizes for the first time a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, namely a C-17 carbonyl-substituted oleanane triterpene derivative. The present invention also provides a method for preparing the compound represented by formula (I) through specific examples. Furthermore, the present invention provides the use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof for preparing an NRF2 activator. The C-17 carbonyl-substituted oleanane triterpene derivative disclosed herein, while maintaining a strong NRF2 agonist effect, can also exert antioxidant effects by scavenging DPPH free radicals, inhibiting the production of lipid peroxides (MDA), or inhibiting ferroptosis. Finally, the present invention also provides a pharmaceutical composition comprising any of the above-mentioned compounds or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition can be prepared into various pharmaceutically acceptable dosage forms, such as tablets, capsules, oral solutions, granules, injections, or various sustained-release preparations. The pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, or topically). The dosage can be appropriately adjusted based on the patient's age, gender, and disease type. Diseases that could be potentially prevented or treated in the future include cerebral small vessel disease, mitochondrial encephalomyopathy, autism spectrum disorder, Rett syndrome, Friedreich's ataxia, stroke, hemorrhagic stroke, ischemic stroke, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, cognitive impairment associated with schizophrenia, Parkinson's disease, cognitive impairment associated with Parkinson's disease, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, and renal ischemia. The drug is particularly effective for stroke, multiple sclerosis, and amyotrophic lateral sclerosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Effects of compounds 19 and 28 on mNSS scores in rats with ischemic stroke. Compared with the sham group: ###p<0.001; compared with the model group: ***p<0.001.

Figure 2. Effects of compounds 19 and 28 on cerebral infarction area in rats with ischemic stroke. Compared with the sham operation group: ###p<0.001; compared with the model group: ***p<0.001.

Figure 3. Effects of compound 24 on neurological function scores of EAE model mice, compared with the sham operation group: ***p<0.001; compared with the model group: #p<0.05, ##p<0.01, ###p<0.001; compared with the Omaveloxolone 15 mg/kg group: &&&p<0.001.

Figure 4. Effect of compound 24 on body weight of EAE model mice, compared with the sham operation group: ***p<0.001; compared with the model group: #p<0.05, ###p<0.001; compared with the Omaveloxolone 15 mg/kg group: ^&&& p<0.001.

Figure 5. Effects of compounds 26 and 31 on the onset time of SOD1 G93A mice. Compared with the control group: ^*** p<0.001; compared with the model group: ^# p<0.05, ^## p<0.01; compared with the Omaveloxolone 3 mg/kg group: ^& p<0.05.

Figure 6. Effects of compounds 26 and 31 on motor coordination ability of SOD1 G93A mice. Compared with the control group: ^* p<0.05, ^*** p<0.001; compared with the model group: ^### p<0.001; compared with the Omaveloxolone 3 mg/kg group: ^& p<0.05.

Figure 7 Effects of compounds 26 and 31 on muscle endurance in SOD1 G93A mice, compared with the control group: ^* p < 0.05, ^*** p <0.001; compared with the model group: ^### p <0.001; compared with the Omaveloxolone 3 mg/kg group: ^& p < 0.05. < 0.05.

DETAILED DESCRIPTION

The present invention is further described below with reference to specific embodiments and test examples, but they are not intended to limit the scope of the present invention in any form.

The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and/or liquid chromatography-mass spectrometry (LC-MS).

NMR chemical shifts (δ) are given in parts per million (ppm). NMR measurements were performed using an AVANCE III 600 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated methanol (CD ₃ OD), and deuterated chloroform (CDCl ₃ ) as solvents, and tetramethylsilane (TMS) as the internal standard.

Liquid chromatography-mass spectrometry (LC-MS) was performed using a Shimadzu LCMS2020 mass spectrometer, and HPLC was performed using a Shimadzu LC20A liquid chromatograph.

The thin layer chromatography silica gel plate used was Yantai Jiangyou silica gel plate, the specification used for TLC was 0.2mm±0.03mm, and the specification used for thin layer chromatography separation and purification products was 0.4mm-0.5mm.

Unless otherwise specified in the examples, the SFC separation conditions are as follows: column model: DAICEL CHIRALPAK IC (250 mm*30 mm, 10 um); mobile phase: [CO2-i-PrOH/ACN]; B%: 40%, isocratic elution mode.

In the present invention, if the specific experimental conditions are not indicated, conventional experimental conditions or conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments is not indicated, conventional products can be obtained through commercial purchase.

In the present invention, the test results are expressed as average values.

The detection indicators in the present invention are: human Nrf2 receptor function test (agonist test); DPPH free radical scavenging ability test; MDA anti-lipid peroxidation ability test.

■Example 1

Compound 1:

Synthesis route:

Step 1: Synthesis of intermediate 2'

Starting material 1' (300 mg, 610.18 μmol) was dissolved in anhydrous dichloromethane (3 mL). N,N-dimethylformamide (22.30 mg, 309.09 μmol) and oxalyl chloride (309.79 mg, 2.44 mmol, 213.65 μL) were then added and stirred at 25°C for 1 hour. After completion of the reaction, the reaction solution was concentrated to yield intermediate 2' (300 mg, crude product, white solid). LCMS (methyl ester): rt = 0.660 min, 506.3 [M+H] ⁺ .

Step 2: Synthesis of Intermediate 3

Ammonia gas (30.05 mg, 1.76 mmol) was passed through toluene (4 mL) at -70°C for 10 minutes, followed by the addition of intermediate 2' (0.3 g, 588.11 μmol), and the mixture was stirred at -70°C for 1 hour. After completion of the reaction, the reaction mixture was concentrated under reduced pressure to obtain a crude product by LCMS. The crude product was purified by reverse-phase column chromatography ( _C18 column, 0.1% aqueous formic acid). After lyophilization, intermediate 6 (250 mg, 86.6% yield) was obtained as a yellow solid. LCMS: rt = 0.557 min, 491.3 [M+H] ⁺ ; purity: 100%.

Step 3: Synthesis of Intermediate 4

Intermediate 4A (5 g, 29.38 mmol) was dissolved in anhydrous acetonitrile (50 mL), and phosphorus oxybromide (12.64 g, 44.07 mmol) was added under nitrogen. The reaction mixture was stirred at 80°C for 0.5 h. After completion of the reaction, the reaction mixture was quenched by adding room-temperature water (50 mL) at room temperature. After stirring for 1 h, the mixture was filtered and concentrated to obtain the crude product. Purification by column chromatography (silica gel, petroleum ether/ethyl acetate = 2/1 to 1/1) afforded Intermediate 4 (3.3 g, 45.92% yield, yellow solid). LCMS: rt = 0.381, 0.749 min, 233.0/235.0 [M+H] ⁺ ; purity 95.34%. ¹ H NMR (400 MHz, DMSO-d6) δ = 5.93 (s, 1H), 5.00-4.86 (m, 1H), 1.33 (d, J = 7.0 Hz, 6H).

Step 4: Synthesis of compound 1

Intermediate 3 (50 mg, 101.90 μmol) and intermediate 4 (35.62 mg, 152.85 μmol) were dissolved in dioxane (4 mL). After nitrogen was purged three times, PEPPSIPd (5.99 mg, 7.13 μmol) and sodium tert-butoxide (29.38 mg, 305.70 μmol) were added. After nitrogen was purged three times, the mixture was stirred at 100°C for 2 hours. After completion of the reaction by LCMS, the reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative chromatography ( _C18 column, 0.1% formic acid solution) and lyophilized to obtain compound 1 (4.2 mg, white solid, 4.06% yield). LCMS: rt=1.560min, 643.4[M+H] ⁺ ; Purity 94.25%; ¹ H NMR (400MHz, DMSO-d6) δ=10.81-10.41(m,1H),9.65-9.28(m,1H),8.65(s,1H),6.22(s,1H),6.03(br s,1H),5.04-4.92(m,1H),3.05(br d,J＝4.2Hz,1H),2.84(br d,J＝13.6Hz,1H),2.08-1.98(m,1H),1.87-1.73(m,3H),1.67-1.57(m,4H),1.44-1.40(m,4H),1.3 4(d,J＝7.0Hz,6H),1.28-1.22(m,6H),1.19-1.12(m,5H),1.08-1.00(m,4H),0.96(s,3H),0.89(br d,J＝11.6Hz,6H).

■Example 2

Compound 2:

Synthesis route:

■Example 3

Compound 3:

Synthesis route:

Step 1: Synthesis of intermediate 3'

Starting materials 1 (1 g, 5.30 mmol) and 2 (1.57 g, 6.89 mmol) were dissolved in dioxane (10 mL). Diisopropylethylamine (2.06 g, 15.91 mmol, 2.77 mL) was then added and microwave-treated at 120°C for 3 h. After completion of the reaction, the reaction solution was concentrated to yield a crude product. Purification by column chromatography (silica gel, petroleum ether/ethyl acetate = 1/0 to dichloromethane/anhydrous methanol = 1/1) afforded the crude product, which was then slurried with methanol (10 mL) and filtered to yield intermediate 3' (0.9 g, 45.95% yield, as a gray solid). LCMS: rt=0.506min, 380.1[M+H] ⁺ ; Purity 96.28%; ¹ H NMR (400MHz, DMSO-d ₆ ) δ=9.75 (br s,1H),7.46-7.30(m,5H),7.25(d,J＝8.6Hz,2H),6.99(d,J＝8.8Hz,2H),6.44(br d,J＝6.6Hz,1H),5.08(s,2H),4.96-4.84(m,1H),4.49-4.38(m,1H),4.34(s,1H),1.36(d,J＝6.8Hz,3H),1.29-1.23(m,6H).

Step 2: Synthesis of Intermediate 4

Intermediate 3' (450 mg, 1.19 mmol) was dissolved in anhydrous methanol (200 mL). Wet palladium on carbon (0.5 g, 10%) was then added under nitrogen. The mixture was replaced with hydrogen three times and stirred at 25°C for 2 hours. After completion of the reaction, the reaction mixture was filtered and concentrated to obtain the crude product. The crude product was purified by reverse-phase column chromatography ( _C18 column, 0.1% formic acid in water). After extraction, intermediate 4 (120 mg, 34.27% yield, yellow solid) was obtained. LCMS: rt = 0.397 min, 290.1 [M+H] ⁺ , purity: 98.23%.

Step 3: Synthesis of compound 3

Intermediate 5 (50 mg, 101.70 μmol) and intermediate 4 (35.31 mg, 122.04 μmol) were dissolved in dichloromethane (0.5 mL). EDCI (23.39 mg, 122.04 μmol) and 4-dimethylaminopyridine (12.42 mg, 101.70 μmol) were added and stirred at 25°C for 12 hours and then at 40°C for 2 hours. After completion of the reaction by LCMS, the reaction mixture was filtered and concentrated under reduced pressure to obtain the crude product. The crude product was purified by preparative chromatography ( _C18 column, 0.1% formic acid solution) and lyophilized to obtain compound 3 (28.6 mg, white solid, 36.02% yield). LCMS: rt=1.746min, 763.4[M+H] ⁺ ; Purity 97.73%; ¹ H NMR (400MHz, DMSO-d6) δ = 9.81 (s, 1H), 8.67 (s, 1H), 7.39 (d, J = 8.4Hz, 2H), 7.07 (d, J = 8.4Hz, 2H), 6.54 (br d,J＝6.6Hz,1H),6.26(s,1H),4.98-4.84(m,1H),4.53(br t,J＝6.6Hz,1H),4.34(d,J＝2.0Hz,1H),2.94(br d,J＝3.4Hz,2H),2.08-1.93(m,2H),1.89-1.64(m,7H),1.52-1.43(m,6H),1.39(br d,J＝6.8Hz,3H),1.36-1.30(m,4H),1.27(br d,J＝6.8Hz,8H),1.18(s,3H),1.07(s,3H),0.99(s,3H),0.95(s,3H),0.91(s,3H).

■Example 4

Compound 4:

Synthesis Route 1:

Step 1: Synthesis of Intermediate 2

Raw material 1 (500 mg) was dissolved in anhydrous dichloromethane (5 mL), followed by the addition of N,N-dimethylformamide (7.82 μL) and ventilation with a nitrogen balloon. Oxalyl chloride (356.09 μL) was then slowly added to the reaction flask. The reaction was allowed to react at 25°C for 1 hour. After LC-MS analysis confirmed the complete reaction, the reaction solution was concentrated under reduced pressure to obtain a residue. The resulting product was used directly in the next reaction without purification. Intermediate 2 (555 mg, crude) was obtained as a pale yellow solid powder.

Step 2: Synthesis of compound 4

3 (46.16 mg) was dissolved in pyridine (1 mL) and cooled to 0°C in an ice-water bath under nitrogen. Intermediate 2 (160 mg) was dissolved in pyridine (1 mL) and slowly added to the reaction flask. The reaction was allowed to proceed in an ice-water bath for 5 hours. After the reaction was complete, as determined by LCMS, the reaction solution was poured into water (3 mL) and extracted with ethyl acetate (2 mL x 3). The organic phase was washed with saturated brine (2.5 mL x 2), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by preparative chromatography ( _C18 column, 0.1% formic acid solution) and lyophilized to afford compound 4 (27 mg, 14.45% yield). LCMS: Rt=0.631min, 572.5[M+H] ⁺ ; HPLC: Rt=2.098min; ¹ H NMR (400MHz, CHLOROFORM-d) δ=8.05 (s, 1H), 5.98 (s, 1H), 5.92 (s, 1H), 3.13 (br s,2H),2.36-2.32(m,3H),2.07-1.99(m,3H),1.79(br s,11H),1.49(s,3H),1.38(s,3H),1.27(s,5H),1.18(s,3H),1.05(d,J＝4.0Hz,6H),0.94(s,3H).

Synthesis route 2:

Step 1: Synthesis of Intermediate 2

Raw material 1 (1 g) was dissolved in anhydrous dichloromethane (10 mL). N,N-dimethylformamide (15.65 μL) was added and the mixture was purged with a nitrogen balloon. Oxalyl chloride (712.16 μL) was then slowly added to the reaction flask. The mixture was allowed to react at 25°C for 1 hour. After the reaction was complete, LCMS (quenched with methanol) was used to determine the complete reaction. The reaction solution was then concentrated under reduced pressure to obtain a solid. The obtained product was used directly in the next reaction without further purification. Intermediate 2 (1.1 g, crude product) was a pale yellow solid powder.

Step 2: Synthesis of Intermediate 3

Hydrazine hydrate (1.05 mL, 98%) was dissolved in anhydrous dichloromethane (5 mL) and cooled to 0°C in an ice-water bath under nitrogen. Intermediate 2 (1.1 g) was dissolved in anhydrous dichloromethane (5 mL) and slowly added to the reaction flask. The reaction was incubated in an ice-water bath for 1 hour. After the reaction was complete, as determined by LCMS, the reaction solution was concentrated. The crude product was separated by preparative chromatography ( _C18 column, 0.1% formic acid solution). The acetonitrile was concentrated and extracted with ethyl acetate (20 mL x 2). The organic phase was washed with saturated brine (30 mL) and dried over anhydrous sodium sulfate. After concentration, 3 (580 mg, 1.15 mmol, 53.19% yield) was obtained. LCMS: rt = 0.502 min, 506.3 [M+H] ⁺ .

Step 3: Synthesis of Intermediate 5

Intermediate 3 (580 mg) and material 4' (145.20 μL) were dissolved in anhydrous ethanol (7 mL), followed by the addition of acetic acid (6.57 μL). The reaction was allowed to proceed at 50°C for 3 hours. LCMS analysis revealed 40% starting material remaining, resulting in 35% product. The reaction solution was concentrated under reduced pressure and used directly in the next step. The crude product 5 (600 mg, 84.67% yield) was a tan solid. LCMS: rt = 0.599 min, 618.5 [M+H] ⁺ .

Step 4: Synthesis of compound 4

Intermediate 5 (500 mg) was dissolved in 6 (10 mL) and the reaction temperature was raised to 145°C for 12 hours. LCMS monitoring indicated the formation of product after the starting material was consumed. The reaction solution was concentrated under reduced pressure and then added to silica gel for mixing. The crude product was separated by normal phase column chromatography (petroleum ether:ethyl acetate = 0/0 to 3/1) and purified by preparative chromatography ( _C18 column, 0.1% formic acid solution) to afford compound 4 (39.9 mg, 69.30 μmol, 8.56% yield). LCMS: rt=1.655min, 572.4[M+H] ⁺ ; HPLC: rt=2.223min; ¹ H NMR (400MHz, CHLOROFORM-d) δ=8.05 (s, 1H), 5.98 (s, 1H), 5.88 (s, 1H), 3.14 (br d,J＝4.0Hz,2H),2.33(s,3H),2.02(s,3H),1.95-1.80(m,3H),1.75-1.54(m,4H),1.49(s,3 H), 1.43-1.33 (m, 5H), 1.31-1.23 (m, 6H), 1.17 (s, 3H), 1.05 (d, J = 4.4Hz, 6H), 0.94 (s, 3H).

■Example 5

Compound 5:

Synthesis route:

Step 1: Synthesis of Intermediate 2

Starting material 1 (500 mg, 1.02 mmol, 1 eq) was dissolved in anhydrous dichloromethane (5 mL). N,N-dimethylformamide (7.43 mg, 101.70 μmol, 7.82 μL, 0.1 eq) was then added and the mixture was protected with nitrogen. Oxalyl chloride (516.33 mg, 4.07 mmol, 356.09 μL, 4 eq) was then slowly added to the reaction flask. The mixture was allowed to react at 25°C for 1 hour. After completion of the reaction, LCMS analysis confirmed that the reaction was complete. The reaction solution was then concentrated under reduced pressure to obtain a residue. The resulting product was used directly in the next reaction without purification. Compound 2 (555 mg, crude) was obtained as a pale yellow solid powder.

Step 2: Synthesis of compound 5

Material 3 (66.78 mg, 439.12 μmol, 0.8 eq) was dissolved in pyridine (2 mL), purged with a nitrogen balloon, and then cooled to 0°C in an ice-water bath. Intermediate 2 (280 mg, 548.91 μmol, 1 eq) was dissolved in pyridine (2 mL) and slowly added to the reaction flask. The reaction was allowed to proceed in an ice-water bath for 5 hours. After the reaction was complete, as determined by LCMS, the reaction solution was poured into water (5 mL) and extracted with ethyl acetate (5 mL x 3). The organic phase was washed with saturated brine (5 mL x 2), dried over anhydrous sodium sulfate, and concentrated. The crude product was separated by preparative chromatography ( _C18 column, 0.1% formic acid solution) and lyophilized to afford compound 5 (80 mg, 122.10 μmol, yield 22.24%). LCMS: Rt=1.802min, 626.4[M+H]+; HPLC: Rt=2.388min; ¹ H NMR (400MHz, DMSO-d6) δ=14.30-13.93(m,1H),8.67(s,1H),6.65(br s,1H),6.26(s,1H),2.91(br s,2H),2.09-1.84(m,4H),1.72-1.61(m,5H),1.50-1.43(m,6H),1.30-1.2 2(m,6H),1.18(s,3H),1.07(s,3H),0.98(s,3H),0.94(s,3H),0.91(s,3H).

■Example 6

Compound 6:

Synthesis route:

■Example 7

Compound 7:

Synthesis route:

■Example 8

Compound 8:

■Example 9

Compound 9:

Synthesis route:

■Example 10

Compound 10:

Synthesis route:

■Example 11

Compound 11:

Synthesis route:

Step 1: Synthesis of Intermediate 2

Compound 1 (1 g, 5.52 mmol, 1 eq), triethylamine (614.32 mg, 6.07 mmol, 845.01 μL, 1.1 eq), Boc2O (1.32 g, 6.07 mmol, 1.39 mL, 1.1 eq), and DMAP (67.43 mg, 551.91 μmol, 0.1 eq) were added to anhydrous tetrahydrofuran (20 mL). The reaction was stirred at 20°C for 14 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (silica, petroleum ether:ethyl acetate = 1:0 to 4:1) to afford intermediate 2 (1.1 g, 3.44 mmol, 62.35% yield, 88% purity) as a yellow oil. LCMS: rt = 0.524 min, 304.1 [M+Na]+, purity: 87.557%.

Step 2: Synthesis of Intermediate 3

Compound 2 (1.1 g, 3.91 mmol, 1 eq) and hydrazine hydrate (4.27 g, 83.59 mmol, 4.14 mL, 98% purity, 21.38 eq) were added to anhydrous ethanol (15 mL). The reaction was heated at 80°C under a nitrogen atmosphere for 16 hours. LCMS indicated the reaction was complete. The reaction solution was cooled to room temperature and concentrated under reduced pressure to yield the product (900 mg, 3.61 mmol, 92.33% yield, 95% purity) as a yellow oil. LCMS: rt=0.303min, 150.0[M-C5H9O2+H] ^- , purity: 94.705%. ¹ H NMR (400MHz, DMSO-d6) δ=11.21 (s, 1H), 6.70 (s, 1H), 6.23 (br d, J＝2.0Hz, 1H), 6.00 (t, J＝2.8Hz, 1H), 3.41 (br s, 2H), 1.36 (s, 9H).

Step 3: Synthesis of Intermediate 5

Compound 4 (100 mg, 203.39 μmol, 1 eq), DMF (7.43 mg, 101.70 μmol, 7.82 μL, 0.5 eq), and oxalyl chloride (103.26 mg, 813.57 μmol, 71.22 μL, 4 eq) were added to anhydrous dichloromethane (1 mL). The reaction was stirred at 20°C for 1 hour. LCMS (quenched with methanol) indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to afford Intermediate 5 (100 mg, crude) as a white solid. LCMS: rt = 0.717 min, 506.3 [M+H]+ (Mass of methyl ester), purity: 82.696%.

Step 4: Synthesis of compound 11

Intermediate 5 (100 mg, 196.04 μmol, 1 eq) was added to pyridine (0.5 mL), followed by a solution of compound 3 (73.30 mg, 294.06 μmol, 1.5 eq) dissolved in pyridine (0.5 mL). The reaction was stirred at 0°C for 3 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by HPLC using a Phenomenex luna C18 150*25mm*10μm column, mobile phase: water (FA)-acetonitrile, gradient: 63%-93% B, over 18 minutes to afford compound 11 (9.2 mg, 14.45 μmol, 7.37% yield, 97.8% purity) as an off-white solid. LCMS (39): rt=0.625min, 623.4[M+H]+, purity: 97.813%. HPLC (39): rt=2.305min, 97.949% purity. ¹ H NMR (400MHz, DMSO-d6) δ=12.63 (s, 1H), 11.26 (br s,1H),8.67(s,1H),6.86(br d,J＝1.6Hz,1H),6.49(br s,1H),6.27(s,1H),6.20(d,J＝2.0Hz,1H),6.10(br d,J＝2.8Hz,1H),3.02-2.90(m,2H),2.09-1.99(m,1H),1.90-1.81(m,2H),1.79-1.61(m,5H),1.44 (s,5H),1.35-1.23(m,7H),1.18(s,3H),1.14-1.03(m,4H),1.01-0.89(m,8H),0.89-0.83(m,1H).

■Example 12

Compound 12:

Synthesis route:

■Example 13

Compound 13:

Synthesis route:

Step 1: Synthesis of intermediate a-1

To a solution of material 6343-98-2 (2.00 g, 10.6 mmol, 1.00 eq) was added ethyl 3-oxobutanoate (1.37 g, 10.6 mmol, 1.34 mL, 1.00 eq). The mixture was stirred at 120°C for 2 h. LC-MS (EB11687-1-P1A1) showed the appearance of a new peak on LC-MS, with approximately 71.0% of the desired compound detected. The reaction mixture was concentrated under reduced pressure to obtain a residue. The crude product was triturated with ethanol (3 V) at 25°C for 1 h, filtered, and the solid was collected to obtain intermediate a-1 (1.70 g, 7.58 mmol, 72.0% yield, 98.2% purity) as a white solid. LCMS: (EB11687-1-P1A1_LCMS_SH), RT=1.220min, MS(ESI)m/z=220[M] ⁺ . LCMS: (EB11687-1-P1C1_LCMS_SH), RT=1.198min, MS(ESI)m/z=220[M] ⁺ .

Step 2: Synthesis of intermediate a

To a solution of intermediate a-1 (1.50 g, 6.81 mmol, 1.00 eq) in MeOH (15.0 mL) was added Pd/C (246 mg, 232 μmol, 238 μL, 10% purity, 0.04 eq) under nitrogen. The suspension was degassed and purged with _H₂ three times. The mixture was stirred at 25°C under _H₂ (40.0 psi.) for 2.5 h. LC-MS (EB11687-2-P1A2) showed the appearance of a new peak, yielding approximately 85.9% of the expected compound. The reaction mixture was filtered through a bed of Celite to remove the Pd-C, which was then thoroughly rinsed with methanol containing 5.00% acetic acid. The filtrates were combined and the solvent evaporated under vacuum. The residual syrup was suspended in ethyl acetate (10.0 mL) and diluted with hexane (50.0 mL). A suspension of a yellow crystalline solid was obtained. After stirring for 10 min, the solid was filtered, washed with hexane and dried to obtain a yellow solid intermediate a (1.25 g, 6.57 mmol, yield 96.4%). LCMS: (EB11687-2-P1A2_LCMS_SH), RT = 0.431 min, MS (ESI) m/z = 190 [M] ⁺ .

Step 3: Synthesis of compound 13

A solution of material 218600-44-3 (500 mg, 1.02 mmol, 1.00 eq), intermediate a (193 mg, 1.02 mmol, 1.00 eq), and NMI (292 mg, 3.56 mmol, 284 μL, 3.50 eq) in MeCN (8.00 mL) was mixed. TCFH (343 mg, 1.22 mmol, 1.20 eq) was then added. The mixture was then stirred 12 times at 25°C under nitrogen. LC-MS (EB11687-29-P1A1) showed approximately 35.6% of the desired compound. The residue was concentrated under reduced pressure to yield the crude product. The crude product was purified by reverse phase HPLC: Welch Xtimate C18 40*200mm 7μm; mobile phase: [water ( _NH4HCO3 )-ACN]; gradient: _56.0 %-96.0% B, 25 min to obtain compound 13 as a white solid (273 mg, 262μmol, yield 39.3%). .LCMS:(EB11687-29-P1A1_LCMS_SH)RT＝1.855min,MS(ESI)m/z＝663.9[M+1]+.LCMS:(EB11687-35-P2C1_LCMS_SH)RT＝2.484 min,MS(ESI)m/z＝663.3[M]+.HPLC:(EB11687-35-P1C4)RT＝4.362min.1HNMR:(EB11687-35-P1N2)(CDCl3,400MHz)δppm0.92( s,3H)0.97-1.01(m,8H)1.08-1.14(m,1H)1.16(s,3H)1.17-1.27(m,5H)1.27-1.45(m,4H)1.54-1.77(m,10H)1.90-2.00(m,3 H)2.31(s,3H)2.75(d,J＝4.80Hz,1H)3.02-3.11(m,1H)5.94(s,2H)6.65(d,J＝8.40Hz,2H)7.22(d,J＝8.80Hz,2H)8.05(s,1H).

■Example 14

Compound 14:

Synthesis route:

Step 1: Synthesis of Intermediate 6B

To a solution of material 6A (2.0 g, 13.1 mmol, 1.00 eq) and ethyl 3-oxobutanoate (1.95 g, 15.0 mmol, 1.90 mL, 1.15 eq) in EtOH (48.0 mL) was added p-TsOH (124 mg, 718 μmol, 0.05 eq). The mixture was stirred at 80°C for 6 h. LC-MS (EB11687-6-P1 C3) showed the appearance of a new peak on the LC-MS, with approximately 96.4% of the expected compound detected. The mixture was filtered and the filtrate concentrated to obtain the crude product. The crude product was triturated in ethanol (4.50 mL) at 25°C to obtain intermediate 6B (1.50 g, 6.60 mmol, 50.5% yield, 96.4% purity) as a red solid. LCMS: (EB11687-6-P1C3_LCMS_SH): RT=0.848min, MS (ESI) m/z=220[M+1] ⁺ .

Step 2: Synthesis of Intermediate 6C

To a solution of intermediate 6B (750 mg, 3.42 mmol, 1.00 eq) in MeOH (1.00 mL) was added Pd/C (124 mg, 116 μmol, 10.0% purity, 0.04 eq) under nitrogen. The suspension was degassed and purged with H2 three times. The mixture was stirred at 25°C under _H2 (40.0 psi) for 2.5 h. LC-MS (EB11687-14-P1A1) indicated the presence of approximately 96.0% of the desired compound. The reaction mixture was filtered through a bed of Celite to remove the Pd-C, which was then rinsed thoroughly with 5% acetic acid in methanol. The filtrates were combined and the solvent evaporated under vacuum. The residual syrup was suspended in ethyl acetate (10.0 mL) and diluted with hexane (50.0 mL). A suspension of a yellow crystalline solid was obtained. After stirring for 10 min, the solid was filtered, washed with hexane and dried to obtain a red solid of Intermediate 6C (500 mg, 2.64 mmol, yield 77.2%). LCMS: (EB11687-14-P1A1_LCMS_SH): RT = 0.154 min, MS (ESI) m/z = 189.9 [M+1] ⁺ .

Step 3: Synthesis of compound 14

To a solution of material 218600-44-3 (500 mg, 1.02 mmol, 1.00 eq) and intermediate 6C (192 mg, 1.02 mmol, 1.00 eq) in acetonitrile (5.00 mL) were added CMPI (338 mg, 1.32 mmol, 1.30 eq) and _Et3N (309 mg, 3.05 mmol, 425 μL, 3.00 eq). The mixture was stirred at 25°C for 3 h. LC-MS (EB11687-40-P1A3) showed the appearance of a new peak and detected approximately 20% of the desired compound. The mixture was concentrated to yield the crude product. The crude product was purified by reverse phase HPLC (column: Welch Xtimate C18 40*200mm 7μm; mobile phase: [water (NH3H2O + NH4HCO3)-ACN]; gradient: 54.0%-94.0% B in 25min) to give compound 14 (74.0 mg, 112 μmol, 11.0% yield) as a brown solid. LCMS: (EB11687-40-P1A3_LCMS_SH): RT=1.834min, MS(ESI)m/z=663[M+1] ⁺ ; LCMS: (EB11687-41-P1C1_LCMS_SH): RT=2.576min, MS(ESI)m/z=663[M+1] ⁺ ; HPLC: (EB11687-41-P1C3): RT=4.377min. ¹ H NMR: (EB11687-41-P1N1) (CDCl ₃ , 400MHz) δppm 0.95(s,3H)1.00-1.04(m,9H)1.18(s,3H)1.27(s,3H)1.33(br d,J＝2.38Hz,2H)1.45(d,J＝13.51Hz,2H)1.50(s,3H)1.72-1.82(m,10H)1.92-2.02(m,3H)2.34(s,3H)2. 77(d,J＝4.63Hz,1H)3.03-3.17(m,1H)5.97(s,2H)6.71(d,J＝8.25Hz,2H)7.27(s,2H)8.06-8.10(m,1H).

■Example 15

Compound 15:

Synthesis route:

Step 1: Synthesis of Intermediate 3

Compound 1 (5 g, 40.60 mmol, 4.81 mL, 1 eq) was dissolved in anhydrous tetrahydrofuran (50 mL). Sodium hydroxide (1.95 g, 48.72 mmol, 60% purity, 1.2 eq) was added portionwise at 20°C under nitrogen. The reaction was allowed to proceed at 0°C for 0.5 hours. Compound 2 (5.28 g, 44.66 mmol, 5.41 mL, 1.1 eq) was then added dropwise to the reaction system under nitrogen and allowed to react at 20°C for 2 hours. TLC analysis indicated that most of the starting material had reacted, with a new spot formed. The reaction solution was slowly poured into saturated aqueous ammonium chloride (200 mL) and extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were washed once with saturated brine (150 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to yield the crude product. The crude product was purified by normal phase column chromatography (petroleum ether/ethyl acetate = 1/0 to 10/1) to afford intermediate 3 (4 g, 20.49 mmol, 50.47% yield) as a yellow oil. ^1H NMR (400 MHz, CHLOROFORM-d) δ = 6.97 (dd, J = 1.6, 4.2 Hz, 1H), 6.85 (s, 1H), 6.15 (dd, J = 2.4, 4.2 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H).

Step 2: Synthesis of intermediate 4

Intermediate 3 (2 g, 10.25 mmol, 1 eq) was dissolved in anhydrous ethanol (20 mL). Hydrazine hydrate (5.23 g, 102.45 mmol, 5.07 mL, 98% purity, 10 eq) was added at 20°C, and the mixture was reacted at 80°C for 2 hours. After the reaction was complete, as determined by LCMS, the reaction solution was concentrated and purified using a reverse-phase column (220 g, water (formic acid)-acetonitrile) before lyophilization to afford Intermediate 4 (900 mg, 5.52 mmol, 53.84% yield, 100% purity) as a yellow oil. LCMS: rt=0.418min,164.1[M+H]+. ¹ H NMR (400MHz, DMSO-d6) δ = 8.45 (s, 1H), 6.77 (s, 1H), 6.27 (dd, J = 1.8, 3.6Hz, 1H), 6.00 (dd, J = 2.8, 3.6Hz, 1H), 5.56 (s, 1H), 3.68 (s, 3H).

Step 3: Synthesis of Intermediate 6

Compound 5 (300 mg, 610.18 μmol, 1 eq) was dissolved in anhydrous dichloromethane (5 mL). N,N-dimethylformamide (4.46 mg, 61.02 μmol, 4.69 μL, 0.1 eq) and oxalyl chloride (232.34 mg, 1.83 mmol, 160.24 μL, 3 eq) were added at 20°C. The atmosphere was replaced with nitrogen three times, and the mixture was allowed to react at 20°C for 1 hour. After the reaction was complete (a small sample was dissolved in methanol for analysis), the reaction solution was concentrated to afford intermediate 6 (300 mg, 588.11 μmol, 96.38% yield) as a yellow oil. LCMS: rt = 0.608 min, 506.4 [M+H]+ (Ms of the methyl ester).

Step 4: Synthesis of compound 15

Intermediate 4 (95.97 mg, 588.11 μmol, 1 eq) and intermediate 6 (300 mg, 588.11 μmol, 1 eq) were dissolved in dichloromethane (5 mL), and pyridine (69.78 mg, 882.17 μmol, 71.20 μL, 1.5 eq) was added. The mixture was reacted at 20°C for 16 hours. LCMS analysis indicated that the starting material was consumed and product was generated. The reaction solution was then spin-dried and purified using a reverse phase preparative method (water (formic acid)-acetonitrile) and lyophilized to afford compound 15 (225.1 mg, 355.90 μmol, 60.70% yield, 99.78% purity) as a white solid. Prep-HPLC: column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 62%-92%B over 15min. LCMS: rt=1.741min, 637.4[M+H] ⁺ . HPLC:rt＝2.144min. ^1H NMR (400MHz, CHLOROFORM-d) δ = 8.05 (s, 1H), 7.27 (s, 1H), 6.78-6.73 (m, 1H), 6.38 (dd, J = 1.4, 3.4Hz, 1H), 6.22-6.15 ( m,2H),5.98(s,1H),3.72(s,3H),3.20-3.13(m,2H),2.13-2.02(m,2H),1.98-1.86(m,2H),1.85-1.75(m,5H),1.67(br d,J＝13.2Hz,1H),1.60-1.54(m,1H),1.51-1.47(m,3H),1.43-1.37(m,4H) ,1.35-1.24(m,6H),1.18(s,3H),1.06(d,J=1.6Hz,6H),0.97-0.92(m,3H).

■Example 16

Compound 16:

Synthesis route:

Step 1: Synthesis of Intermediate 3

Compound 1 (5 g, 40.60 mmol, 4.81 mL, 1 eq) was dissolved in anhydrous tetrahydrofuran (50 mL). Sodium hydroxide (1.95 g, 48.72 mmol, 60% purity, 1.2 eq) was added portionwise at 20°C under nitrogen. The reaction was allowed to proceed at 0°C for 0.5 hours. Compound 2 (5.28 g, 44.66 mmol, 5.41 mL, 1.1 eq) was then added dropwise to the reaction system under nitrogen and allowed to react at 20°C for 2 hours. TLC analysis indicated that most of the starting material had reacted, with a new spot formed. The reaction solution was slowly poured into saturated aqueous ammonium chloride (200 mL) and extracted three times with ethyl acetate (100 mL x 3). The combined organic phases were washed once with saturated brine (150 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to yield the crude product. The crude product was purified by normal phase column chromatography (petroleum ether/ethyl acetate = 1/0 to 10/1) to afford intermediate 3 (4 g, 20.49 mmol, 50.47% yield) as a yellow oil. ^1H NMR (400 MHz, CHLOROFORM-d) δ = 6.97 (dd, J = 1.6, 4.2 Hz, 1H), 6.85 (s, 1H), 6.15 (dd, J = 2.4, 4.2 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 2H), 1.28 (t, J = 7.2 Hz, 3H).

Step 2: Synthesis of intermediate 4

Intermediate 3 (2 g, 10.25 mmol, 1 eq) was dissolved in anhydrous ethanol (20 mL). Hydrazine hydrate (5.23 g, 102.45 mmol, 5.07 mL, 98% purity, 10 eq) was added at 20°C, and the mixture was reacted at 80°C for 2 hours. After the reaction was complete, as determined by LCMS, the reaction solution was concentrated and purified using a reverse-phase column (220 g, water (formic acid)-acetonitrile) before lyophilization to afford Intermediate 4 (900 mg, 5.52 mmol, 53.84% yield, 100% purity) as a yellow oil. LCMS: rt=0.418min,164.1[M+H]+. ¹ H NMR (400MHz, DMSO-d6) δ = 8.45 (s, 1H), 6.77 (s, 1H), 6.27 (dd, J = 1.8, 3.6Hz, 1H), 6.00 (dd, J = 2.8, 3.6Hz, 1H), 5.56 (s, 1H), 3.68 (s, 3H).

Step 3: Synthesis of Intermediate 6

Compound 5 (300 mg, 610.18 μmol, 1 eq) was dissolved in anhydrous dichloromethane (5 mL). N,N-dimethylformamide (4.46 mg, 61.02 μmol, 4.69 μL, 0.1 eq) and oxalyl chloride (232.34 mg, 1.83 mmol, 160.24 μL, 3 eq) were added at 20°C. The atmosphere was replaced with nitrogen three times, and the mixture was allowed to react at 20°C for 1 hour. After the reaction was complete (a small sample was dissolved in methanol for analysis), the reaction solution was concentrated to afford intermediate 6 (300 mg, 588.11 μmol, 96.38% yield) as a yellow oil. LCMS: rt = 0.608 min, 506.4 [M+H]+ (Ms of the methyl ester).

Step 4: Synthesis of compound 16

Intermediate 4 (95.97 mg, 588.11 μmol, 1 eq) and intermediate 6 (300 mg, 588.11 μmol, 1 eq) were dissolved in dichloromethane (5 mL), and pyridine (69.78 mg, 882.17 μmol, 71.20 μL, 1.5 eq) was added. The mixture was reacted at 20°C for 16 hours. LCMS analysis confirmed the complete consumption of the starting material and the formation of the product. The reaction solution was then spin-dried and purified by reverse phase preparative purification using water (formic acid)-acetonitrile, followed by lyophilization to afford compound 16 (227.1 mg, 355.83 μmol, 60.50% yield, 99.78% purity) as a white solid. Prep-HPLC: column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water(FA)-ACN]; gradient: 62%-92%B over 15min. LCMS: rt=1.741min, 637.4[M+H] ⁺ . HPLC:rt＝2.144min. ^1H NMR (400MHz, CHLOROFORM-d) δ = 8.05 (s, 1H), 7.27 (s, 1H), 6.78-6.73 (m, 1H), 6.38 (dd, J = 1.4, 3.4Hz, 1H), 6.22-6.15 ( m,2H),5.98(s,1H),3.72(s,3H),3.20-3.13(m,2H),2.13-2.02(m,2H),1.98-1.86(m,2H),1.85-1.75(m,5H),1.67(br d,J＝13.2Hz,1H),1.60-1.54(m,1H),1.51-1.47(m,3H),1.43-1.37(m,4H) ,1.35-1.24(m,6H),1.18(s,3H),1.06(d,J=1.6Hz,6H),0.97-0.92(m,3H).

■Example 17

Compound 17:

Synthesis route:

Step 1: Synthesis of Intermediate 2

Compound 1 (1 g, 5.52 mmol, 1 eq), triethylamine (614.32 mg, 6.07 mmol, 845.01 μL, 1.1 eq), Boc2O (1.32 g, 6.07 mmol, 1.39 mL, 1.1 eq), and DMAP (67.43 mg, 551.91 μmol, 0.1 eq) were added to anhydrous tetrahydrofuran (20 mL). The reaction was stirred at 20°C for 14 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography (silica, petroleum ether:ethyl acetate = 1:0 to 4:1) to afford intermediate 2 (1.1 g, 3.44 mmol, 62.35% yield, 88% purity) as a yellow oil. LCMS: rt = 0.524 min, 304.1 [M+Na]+, purity 87.557%.

Step 2: Synthesis of Intermediate 3

Compound 2 (1.1 g, 3.91 mmol, 1 eq) and hydrazine hydrate (4.27 g, 83.59 mmol, 4.14 mL, 98% purity, 21.38 eq) were added to anhydrous ethanol (15 mL). The reaction was heated at 80°C under a nitrogen atmosphere for 16 hours. LCMS indicated the reaction was complete. The reaction solution was cooled to room temperature and concentrated under reduced pressure to afford intermediate 3 (900 mg, 3.61 mmol, 92.33% yield, 95% purity) as a yellow oil. LCMS: rt=0.303min, 150.0[M-C5H9O2+H] ^- , purity 94.705%. ¹ H NMR (400MHz, DMSO-d6) δ=11.21 (s, 1H), 6.70 (s, 1H), 6.23 (br d, J＝2.0Hz, 1H), 6.00 (t, J＝2.8Hz, 1H), 3.41 (br s, 2H), 1.36 (s, 9H).

Step 3: Synthesis of Intermediate 4

Intermediate 3 (1.7 g, 6.82 mmol, 1 eq) was dissolved in anhydrous N,N-dimethylformamide (25 mL). Lithium tert-butoxide (818.92 mg, 10.23 mmol, 922.20 μL, 1.5 eq) was added at 0°C and allowed to react for 0.5 hours. 2-(Trimethylsilyl)ethoxymethyl chloride (1.36 g, 8.18 mmol, 1.45 mL, 1.2 eq) was then added at 0°C and allowed to react for 1.5 hours at 20°C. After the reaction was complete, as determined by LCMS, the reaction solution was poured into saturated aqueous ammonium chloride (100 mL) and extracted three times with ethyl acetate (70 mL x 3). The combined organic phases were washed three times with saturated brine (70 mL), dried over anhydrous sodium sulfate, and filtered and concentrated to yield the crude product. The crude product was purified by normal phase column chromatography (petroleum ether/ethyl acetate = 0-50%) to give intermediate 4 (610 mg, 1.45 mmol, yield 21.21%, purity 90%) as a yellow solid. LCMS: rt = 0.557 min, 280.3 [M-Boc+H] ⁺ .

Step 4: Synthesis of Intermediate 6

Intermediate 4 (368.29 mg, 970.39 μmol, 1 eq) and intermediate 5 (495 mg, 970.39 μmol, 1 eq) were dissolved in dichloromethane (10 mL), and pyridine (230.27 mg, 2.91 mmol, 234.97 μL, 3 eq) was added. The mixture was reacted at 20°C for 16 hours. LCMS analysis indicated complete consumption of the starting material and the formation of the product. The reaction solution was then dried and purified by normal phase column chromatography (petroleum ether/ethyl acetate = 0-50%) to afford compound 6 (437 mg, 580.30 μmol, 59.80% yield) as a yellow solid. LCMS: rt = 0.734 min, 753.6 [M+H] ⁺ .

Step 5: Synthesis of compound 17

Intermediate 6 (100 mg, 132.79 μmol, 1 eq) was dissolved in hydrochloric acid/1,4-dioxane (5 mL, 2 M) and heated to 50°C for 2 hours. LCMS monitoring indicated complete consumption of the starting material and the formation of product. The reaction solution was dried by rotary evaporation and made alkaline with aqueous sodium bicarbonate solution, followed by extraction three times with ethyl acetate (10 mL x 3). The combined organic phases were washed once with saturated brine (15 mL), dried over anhydrous sodium sulfate, and then rotary evaporation to obtain the crude product. The crude product was isolated and purified by reverse phase preparative (water (formic acid)-acetonitrile system) and lyophilized to afford compound 17 (6.2 mg, 9.75 μmol, 7.34% yield, 97.96% purity) as a white solid. Prep-HPLC: column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(FA)-ACN]; gradient: 55%-85%B over 10min. LCMS: rt=1.664min, 623.4[M+H]+. HPLC: rt=2.043min. ¹ HNMR (400MHz, CHLOROFORM-d) δ = 8.78-8.63 (m, 1H), 8.04 (s, 1H), 6.89 (br s, 1H), 6.46 (br s, 1H), 6.29 (br d,J＝2.8Hz,1H),6.17(s,1H),5.98(s,1H),3.19-3.10(m,2H),2.64(s,1H),2.06-2.01(m,2H),1.95-1.88(m,2H),1.78(br s,5H),1.67(br d,J＝13.2Hz,1H),1.55(br d,J＝12.8Hz,1H),1.48(s,3H),1.36(s,4H),1.32-1.26(m,6H),1.18(s,3H),1.05(d,J＝4.2Hz,6H),0.95(s,3H).

■Example 18

Compound 18:

Synthesis route:

■Example 19

Compound 19:

Synthesis route:

Step 1: Synthesis of Intermediate 2

To a solution of material 1 (500 mg, 1.02 mmol, 1 eq) in dichloromethane (8 mL) were added N,N-dimethylformamide (7.43 mg, 101.70 μmol, 7.82 μL, 0.1 eq) and oxalyl chloride (387.25 mg, 3.05 mmol, 267.07 μL, 3 eq). The reaction was incubated at 25°C for 1 hour. LCMS analysis indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction solution was concentrated under reduced pressure to afford intermediate 2 (500 mg, 980.19 μmol, 96.38% yield) as a light yellow solid. LCMS: Rt = 0.917 min, 510.4 [M+H] ⁺ ESI pos.

Step 2: Synthesis of compound 19

To a solution of hydroxylamine hydrochloride (81.74 mg, 1.18 mmol, 1.2 eq) and N,N-diisopropylethylamine (380.04 mg, 2.94 mmol, 512.18 μL, 3 eq) in dichloromethane (5 mL) was added a solution of intermediate 2 (500 mg, 980.19 μmol, 1 eq) in dichloromethane (5 mL) at 0°C. The reaction was heated to 20°C for 2 hours. LCMS monitoring indicated complete consumption of the starting material and the formation of product. The reaction solution was filtered and concentrated. The crude product was isolated and purified by reverse phase preparative chromatography (water (formic acid)-acetonitrile) and lyophilized to afford compound 19 (136.20 mg, 266.39 μmol, 27.18% yield, 99.1% purity). Prep-HPLC:column:Phenomenex luna C18 150*40mm*15um;mobile phase:[water(FA)-ACN];gradient:45%-75%B over 15min.LCMS:Rt＝1.339min,507.3[M+H]+ESI pos.HPLC:Rt＝1.816min. ¹ H NMR(400MHz,CHLOROFORM-d)δ＝9.39-9.07(m,1H),8.03(s,1H),6.04(s,1H),2.97(d,J＝4.6Hz,1H),2.92-2.84(m,1H) ,2.01-1.92(m,1H),1.85-1.79(m,1H),1.78-1.71(m,4H),1.70-1.62(m,3H),1.57-1.45(m,4H),1.42(s,3H),1.31(br d,J＝4.0Hz,1H),1.28(s,3H),1.26-1.23(m,1H),1.21(s,3H),1.12(s,3H),0.97(d,J＝5.8Hz,6H),0.88(s,3H).

■Example 20

Compound 21:

Synthesis route:

Step 1: Synthesis of compound 21

Material 2 (24.53 mg, 196.04 μmol, 24.44 μL, 1 eq) was added to anhydrous dichloromethane (1 mL), followed by the addition of N,N-diisopropylethylamine (126.68 mg, 980.19 μmol, 170.73 μL, 5 eq). The reaction mixture was cooled to 0°C and material 1 (100.00 mg, 196.04 μmol, 1 eq) dissolved in anhydrous dichloromethane (1 mL) was slowly added. The reaction mixture was stirred at 25°C for 12 h. After completion of the reaction, the reaction mixture was concentrated to obtain the crude product. The crude product was purified by reverse phase preparative chromatography (C18 column, 0.1% trifluoroacetic acid solution) and lyophilized to obtain the crude product. The crude product was purified by reverse phase preparative chromatography (C18 column, 0.1% hydrogen chloride solution) and lyophilized to obtain compound 21 (5.0 mg, off-white solid, 4.26% yield). Prep-HPLC (column: Phenomenex luna C18 150*25mm*10um; mobile phase: [water(TFA)-ACN]; gradient: 40%-70% B over 10 min). LCMS: rt=1.225 min, 599.3 [M+H] ⁺ , purity 100%. HPLC: retention time=1.724 min, purity 97.989%. ^1H NMR: 1H NMR (400MHz, DMSO-d6) δ = 8.65 (s, 1H), 7.88-7.76 (m, 1H), 6.19 (s, 1H), 3.47-3.36 (m, 1H), 3.35-3 .24(m,1H),3.03-2.97(m,1H),2.90-2.80(m,1H),2.57-2.53(m,2H),1.98-1.79(m,3H),1.64(br s,3H),1.63-1.47(m,3H),1.44-1.43(m,3H),1.42-1.36(m,2H),1.36-1.27(m,2H),1.27-1.24(m ,3H),1.19-1.16(m,3H),1.16-1.08(m,2H),1.06(s,3H),0.93(s,3H),0.89(s,3H),0.86(s,3H).

■Example 21

Compound 22:

Synthesis route:

Step 1: Synthesis of Intermediate 2

Material 1 (200 mg, 406.79 μmol, 1 eq) and N,N-dimethylformamide (2.97 mg, 40.68 μmol, 3.13 μL, 0.1 eq) were dissolved in anhydrous dichloromethane (1.2 mL). The mixture was then purged with nitrogen three times, cooled to 0°C, and oxalyl chloride (154.89 mg, 1.22 mmol, 106.82 μL, 3 eq) dissolved in anhydrous dichloromethane (0.8 mL) was added. The mixture was stirred at 25°C for 1 h. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude intermediate 2 (200 mg) was used directly in the next reaction.

Step 2: Synthesis of compound 22

3-Amino-1-propanesulfonic acid (27.28 mg, 196.04 μmol, 1 eq) was added to anhydrous dichloromethane (1 mL), followed by N,N-diisopropylethylamine (126.68 mg, 980.19 μmol, 170.73 μL, 5 eq). The reaction mixture was cooled to 0°C and intermediate 2 (100 mg, 196.04 μmol, 1 eq) dissolved in anhydrous dichloromethane (1 mL) was slowly added. The reaction mixture was stirred at 25°C for 12 h. After completion of the reaction, the reaction mixture was concentrated to obtain the crude product. The crude product was purified by reverse phase preparative chromatography (C18 column, 0.1% sodium bicarbonate solution) and lyophilized to obtain the crude product. The crude product was purified by reverse phase preparative chromatography (C18 column, 0.1% hydrogen chloride solution) and lyophilized to obtain compound 22 (9.5 mg, off-white solid, 7.91% yield). Prep-HPLC(column:Waters Xbridge 150*25mm*5um; mobile phase:[water(NH4HCO3)-ACN]; gradient:22%-52%B over 9min). over 18min). LCMS: rt=1.219min, 613.4[M+H]+, 100% purity. HPLC: retention time=1.708min, purity 99.318%. ^1H NMR: 1H NMR (400MHz, DMSO-d6) δ = 8.64 (s, 1H), 7.89-7.73 (m, 1H), 6.18 (s, 1H), 3.22-3.12 (m, 1H), 3. 12-3.01(m,2H),2.90-2.80(m,1H),2.44-2.39(m,2H),1.92-1.77(m,3H),1.76-1.68(m,3H), 1.67-1.48(m,5H),1.47-1.39(m,6H),1.36-1.30(m,1H),1.29-1.25(m,1H),1.24-1.22(m,3H ),1.18-1.15(m,3H),1.15-1.08(m,2H),1.05(s,3H),0.92(s,3H),0.88(s,3H),0.85(s,3H).

■Example 22

Compound 23:

Synthesis route:

Step 1: Synthesis of Intermediate 2

Material 1 (300 mg, 610.18 μmol, 1 eq) was dissolved in anhydrous dichloromethane (2 mL). One drop of N,N-dimethylformamide was added and the reaction mixture was cooled in an ice-water bath. Oxalyl chloride (309.79 mg, 2.44 mmol, 213.65 μL, 4 eq) was dissolved in anhydrous dichloromethane (2 mL) and slowly added dropwise to the stirred reaction mixture. The reaction mixture was slowly warmed to 20°C and stirred for 1 hour. LCMS (quenched with methanol) monitored the reaction for complete reaction. The reaction mixture was concentrated by rotary evaporation to afford intermediate 2 (311 mg, crude) as a white solid. LCMS (quenched with methanol): rt = 0.648 min, 506.3 [M+H]+.

Step 2: Synthesis of intermediate 4

Material 3 (179.86 mg, 914.52 μmol, 177.03 μL, 1.5 eq, HCl) was dissolved in anhydrous dichloromethane (3 mL). Triethylamine (308.46 mg, 3.05 mmol, 424.30 μL, 5 eq) was then added. The reaction solution was cooled in an ice-water bath, and a solution of compound 2 (311 mg, 609.68 μmol, 1 eq) in anhydrous dichloromethane (2 mL) was slowly added dropwise. The reaction solution was slowly warmed to 20°C and stirred for 3 hours. LCMS confirmed the complete reaction of the starting material. The reaction solution was concentrated and purified by column chromatography (ethyl acetate/petroleum ether = 0-51%) to obtain intermediate 4 (370 mg, 583.73 μmol, 95.74% yield) as a colorless gum. LCMS: rt = 0.619 min, 634.5 [M+H] ⁺ .

Step 3: Synthesis of Intermediate 5

Intermediate 4 (370 mg, 583.73 μmol, 1 eq) was dissolved in a 5/1 dichloromethane/trifluoroacetic acid (6 mL) solution and stirred at 20°C for 1 hour. LCMS confirmed the complete reaction. The reaction mixture was concentrated to afford Intermediate 5 (378 mg, crude, TFA) as a light yellow gum. LCMS: rt = 0.494 min, 534.3 [M+H]+.

Step 4: Synthesis of compound 23

To intermediate 5 (378 mg, 583.54 μmol, 1 eq, TFA) were added anhydrous dichloromethane (3 mL) and triethylamine (472.39 mg, 4.67 mmol, 649.77 μL, 8 eq) sequentially. The reaction solution was cooled in an ice-water bath. Material 6 (202.55 mg, 1.17 mmol, 2 eq) was dissolved in anhydrous dichloromethane (2 mL) and slowly added dropwise to the stirred reaction solution. The reaction solution was slowly warmed to 20°C and stirred for 3 hours. LCMS confirmed the completion of the reaction. The reaction solution was concentrated and lyophilized by reverse phase preparative lyophilization to obtain the crude product. The crude product was purified by prep-TLC (dichloromethane/methanol = 15:1) and lyophilized to afford compound 23 (62.8 mg, 89.28 μmol, 15.30% yield, 93.92% purity) as a yellow solid. HPLC: (1) column: Phenomenex luna C18 150*40mm*15um; mobile phase: [water (FA)-ACN]; gradient: 45%-75% B over 15min.LCMS: rt ＝0.630min,671.4[M+H]+.HPLC: rt＝1.761min.1H NMR(400MHz, METHANOL-d4)δ＝8.37(s,1H),7.92-7.75(m,1H),7.53-7.33(m,2H),6.97 -6.79(m,1H),6.07(s,1H),3.64(br d,J＝1.0Hz,3H),3.10(br s,1H),3.06-2.97(m,1H),1.90-1.78(m,2H),1.70(br d,J＝1.2Hz,4H),1 .62-1.51(m,4H),1.46-1.37(m,4H),1.31(br s,2H),1.24(br s,4H),1.14(br s,6H),1.09(br s,2H),0.94(br s,6H),0.88(br s,3H).

■Example 23

Compound 24:

Synthesis route:

Step 1: Synthesis of compound 24

To a solution of N-methylhydroxylamine hydrochloride (98.24 mg, 1.18 mmol, 1.2 eq) in dichloromethane (7.5 mL) was added N,N-diisopropylethylamine (633.41 mg, 4.90 mmol, 853.65 μL, 5 eq). The mixture was purged with nitrogen three times, cooled to 0°C, and a solution of material 1 (500 mg, 980.19 μmol, 1 eq) in dichloromethane (7.5 mL) was slowly added. The mixture was stirred at 25°C for 2 h. LCMS monitoring indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction solution was concentrated under reduced pressure to obtain the crude product. The crude product was isolated and purified by reverse phase preparative reaction (water (formic acid)-acetonitrile) and lyophilized to obtain compound 24 (267.5 mg, 500.48 μmol, 51.06% yield, 97.42% purity) as a white solid. Prep-HPLC(column:Phenomenex luna C18 150*40mm*15um; mobile phase:[water(FA)-ACN]; gradient:55%-85%B over 15min).LCM S: Retention time＝1.461min,521.3[M+H]+.HPLC:retention time＝1.831min, purity 97.42%.1H NMR (400MHz, CHLOROFORM-d)δ＝8.31(s,1 H),8.08(s,1H),5.99(s,1H),3.46(s,3H),3.25(br d,J＝12.8Hz,1H),3.17(br d,J＝4.0Hz,1H),2.11-1.94(m,2H),1.85-1.78(m,5H), 1.76-1.69(m,2H),1.61-1.53(m,3H),1.50(s,3H),1.39(br s,5H),1.30-1.26(m,4H),1.19(s,3H),1.04(d,J=6.4Hz,6H),0.93(s,3H).

■Example 24

Compound 25:

Synthesis route:

■Example 25

Compound 26:

Synthesis route:

Step 1: Synthesis of compound 26

To material 2 (204.07 mg, 1.83 mmol, 3 eq) were added anhydrous tetrahydrofuran (2 mL) and sodium bicarbonate (204.87 mg, 2.44 mmol, 94.89 μL, 4 eq). The reaction solution was cooled in an ice-water bath. Material 1 (311 mg, 609.68 μmol, 1 eq) was dissolved in anhydrous tetrahydrofuran (3 mL) and slowly added dropwise to the stirred reaction solution. The reaction solution was slowly warmed to room temperature and stirred for 5 hours. LCMS confirmed the reaction was complete. The reaction solution was filtered, and the filter cake was washed with dichloromethane (20 mL x 3). The filtrate was concentrated and separated by plate separation (petroleum ether/ethyl acetate = 2:1) to obtain the crude product. The crude product was then lyophilized by reverse phase preparative lyophilization to afford compound 26 (13.6 mg, 24.63 μmol, 4.04% yield) as an off-white solid. Prep-HPLC: column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H2O (0.225% FA)-ACN]; gradient: 55%-85% B over 10.0min. LCMS: rt=0.669min, 549.3[M+H] ⁺ .HPLC: rt=3.810min. ¹ H NMR (400MHz, DMSO-d6) δ = 9.06 (s, 1H), 8.65 (s, 1H), 6.17 (s, 1H), 4.60 (quin, J = 6.6Hz, 1H), 3.19 (br d,J＝3.2Hz,1H),3.10-2.98(m,1H),2.23-2.09(m,1H),1.89-1.74(m,4H),1.73-1.56(m,4H),1.48-1.36(m,5H) ,1.32-1.22(m,5H),1.17(s,3H),1.15-1.12(m,1H),1.11-1.02(m,10H),0.94(s,3H),0.90(s,3H),0.85(s,3H).

■Example 26

Compound 27:

Synthesis route:

■Example 27

Compound 28:

Synthesis route:

Step 1: Synthesis of compound 28

To a solution of material 2 (28.68 mg, 294.06 μmol, 3 eq, HCl) in dichloromethane (0.5 mL) was added N,N-dimethylethylamine (126.68 mg, 980.19 μmol, 170.73 μL, 10 eq). Then, a solution of material 1 (50 mg, 98.02 μmol, 1 eq) in dichloromethane (0.5 mL) was added at 0°C. The reaction mixture was incubated at 25°C for 1 hour. LCMS analysis indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction mixture was concentrated and separated on a large plate (petroleum ether:ethyl acetate = 1:1) to obtain the crude product. The crude product was then purified by reverse phase preparative (water (formic acid)-acetonitrile) separation to afford compound 28 (4.0 mg, 7.27 μmol, 7.42% yield, 97.25% purity) as a white solid. Prep-HPLC: column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H ₂ O (0.225% FA)-ACN]; gradient: 50%-80% B over 10.0min

LCMS: rt=0.671min, 535.4[M+H]+. HPLC: rt=2.253min. ¹ H NMR (400MHz, DMSO-d6)δ=9.50(s,1H),8.65(s,1H),6.18(s,1H),3.66-3.54(m,1H),3.51-3.40(m,1H),3.18(br s,1H),3.11-2.98(m,1H),2.16-2.07(m,1H),1.87-1.74(m,4H),1.71-1.58(m,4H),1.48-1.37(m,5H), 1.34-1.25(m,2H),1.24(s,3H),1.17(s,3H),1.13-1.04(m,8H),0.94(s,3H),0.90(s,3H),0.85(s,3H).

■Example 28

Compound 29:

Synthesis route:

Step 1: Synthesis of compound 29

To a solution of material 2 (267.53 mg, 1.76 mmol, 3 eq, HCl) in dichloromethane (5 mL) was added N,N-dimethylethylamine (380.04 mg, 2.94 mmol, 512.18 μL, 5 eq). Then, a solution of material 1 (300 mg, 588.11 μmol, 1 eq) in dichloromethane (2 mL) was added at 0°C. The reaction mixture was incubated at 25°C for 1 hour. LCMS monitoring indicated the complete disappearance of the starting material and the presence of a peak indicating the desired product. The reaction mixture was concentrated and purified by prep-TLC (petroleum ether:ethyl acetate = 1:1) to afford the crude product. This crude product was then isolated and purified by reverse phase preparative chromatography (water (formic acid)-acetonitrile) to afford compound 29 (10.70 mg, 18.00 μmol, 3.06% yield, 99.04% purity) as a white solid. Prep-HPLC: column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H ₂ O (0.225% FA)-ACN]; gradient: 60%-90% B over 10.0min. LCMS: rt=0.703min, 589.4[M+H] ⁺ .HPLC: rt=2.583min. ¹ H NMR (400MHz, DMSO-d6) δ = 9.09 (s, 1H), 8.65 (s, 1H), 6.17 (s, 1H), 4.27-4.14 (m, 1H), 3.19 (br d,J＝3.8Hz,1H),3.11-2.96(m,1H),2.23-2.07(m,1H),1.83(br s,3H),1.78-1.63(m,6H),1.60-1.48(m,6H),1.48-1.40(m,5H),1.40-1.35(m,1H),1.31-1.2 1(m,7H),1.17(s,3H),1.13-1.08(m,2H),1.06(s,3H),0.94(s,3H),0.89(s,3H),0.85(s,3H).

■Example 29

Compound 30:

Synthesis route:

Step 1: Synthesis of compound 30

To a solution of compound 2 (93.87 mg, 588.11 μmol, 3 eq, HCl) in dichloromethane (2 mL) was added N,N-dimethylethylamine (126.68 mg, 980.19 μmol, 170.73 μL, 5 eq). Compound 1 (100 mg, 196.04 μmol, 1 eq) in dichloromethane (1 mL) was then added at 0°C. The reaction mixture was incubated at 25°C for 1 hour. LCMS monitoring indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction mixture was concentrated and purified by prep-TLC (petroleum ether:ethyl acetate = 1:1) to afford the crude product. This crude product was then separated and purified by reverse phase preparative chromatography (water (formic acid)-acetonitrile) to afford compound 30 (3.20 mg, 5.14 μmol, 2.62% yield, 95.93% purity) as a white solid. Prep-HPLC: column: Phenomenex Luna C18 150*25mm*10μm; mobile phase: [H ₂ O (0.225% FA)-ACN]; gradient: 55%-85% B over 10.0min. LCMS: rt=0.725min, 597.3[M+H]+. HPLC: rt=2.419min. ¹ H NMR (400MHz, DMSO-d6) δ = 9.80 (s, 1H), 8.64 (s, 1H), 7.35-7.22 (m, 5H), 6.18 (s, 1H), 4.84-4.58 (m, 2H), 3.16 (br s,1H),3.12-3.01(m,1H),2.19-2.08(m,1H),1.89-1.76(m,4H),1.75-1.56(m,4H),1.47-1.40(m ,4H),1.37-1.25(m,3H),1.20-1.10(m,7H),1.06(s,4H),0.94(s,3H),0.91(s,3H),0.86(s,3H).

■Example 30

Compound 31:

Synthesis route:

Step 1: Synthesis of Intermediate 2

To a solution of material 1 (2 g, 15.02 mmol, 1 eq) in dichloromethane (20 mL) were added triethylamine (1.67 g, 16.52 mmol, 2.30 mL, 1.1 eq) and tert-butyldiphenylsilyl chloride (4.13 g, 15.02 mmol, 3.84 mL, 1 eq). The reaction mixture was allowed to react at 25°C for 12 hours. LCMS analysis revealed the complete disappearance of the starting material and the main peak was the desired product. The reaction mixture was concentrated under reduced pressure, and the crude product was purified by normal-phase silica gel column chromatography (petroleum ether:ethyl acetate = 1:0 to 100:1) and then concentrated under reduced pressure to afford intermediate 2 (5.5 g, 14.06 mmol, 93.62% yield, 95% purity) as a colorless oil. LCMS: Rt = 0.711 min, 394.2 [M+Na] ⁺ ESI ¹ H NMR (400MHz, CHLOROFORM-d) δ = 7.80-7.71 (m, 4H), 7.49-7.37 (m, 6H), 6.71 (s, 1H), 1.38 (s, 9H), 1.15 (s, 9H).

Step 2: Synthesis of intermediate 4

To a solution of Intermediate 2 (2 g, 5.38 mmol, 1 eq) in tetrahydrofuran (30 mL) was slowly added sodium hydroxide (322.98 mg, 8.07 mmol, 60% purity, 1.5 eq) at 0°C. The reaction mixture was allowed to react at 0°C for 30 minutes, followed by the slow addition of Material 3 (3.12 g, 13.46 mmol, 2.5 eq). The reaction mixture was allowed to react at 25°C for 12 hours. LCMS analysis indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction mixture was slowly poured into saturated aqueous ammonium chloride (20 mL) and extracted with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by normal-phase silica gel column chromatography (petroleum ether:ethyl acetate = 1:0) and then concentrated under reduced pressure to afford Intermediate 4 (1.87 g, 3.71 mmol, 68.93% yield, 90% purity) as a colorless oil. LCMS: Rt＝0.801min,476.2[M+Na] ⁺ _. ESI ¹ H NMR(400MHz, CHLOROFORM-d)δ=7.74-7.69(m,4H),7.48-7.43(m,2H),7.41-7.37(m,4H),3.93-3.82(m,2H),1.26(s,9H),1.17(s,9H).

Step 3: Synthesis of Intermediate 5

To Intermediate 4 (500 mg, 1.10 mmol, 1 eq) was added a 2 M hydrochloric acid/ethyl acetate solution (5.00 mL, 9.07 eq), and the reaction mixture was allowed to react at 25°C for 12 hours. TLC monitoring revealed complete disappearance of the starting material. The reaction mixture was concentrated under reduced pressure, and the filter cake was filtered through a slurry of petroleum ether (2 mL). Intermediate 5 (50 mg, 330.00 μmol, 29.94% yield, HCl) was obtained as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ = 3.94-3.81 (m, 2H).

Step 4: Synthesis of compound 31

To a solution of intermediate 5 (44.55 mg, 294.06 μmol, 3 eq, HCl) in dichloromethane (0.5 mL) was added N,N-dimethylethylamine (63.34 mg, 490.09 μmol, 85.36 μL, 5 eq). Then, a solution of material 6 (50 mg, 98.02 μmol, 1 eq) in dichloromethane (0.5 mL) was added at 0°C. The reaction mixture was incubated at 25°C for 1 hour. LCMS analysis indicated the complete disappearance of the starting material and the main peak was the desired product. The reaction mixture was concentrated and purified by prep-TLC (petroleum ether:ethyl acetate = 2:1) to afford the crude product. This crude product was then isolated and purified by reverse phase preparative chromatography (water (formic acid)-acetonitrile) to afford compound 31 (7.48 mg, 12.58 μmol, 12.84% yield, 99.04% purity) as a white solid. Prep-HPLC: column: Phenomenex Luna C18 150*25mm*10um; mobile phase: [H ₂ O (0.225% FA)-ACN]; gradient: 53%-83% B over 10.0min. LCMS: rt=0.661min, 589.4[M+H] ⁺ .HPLC: rt=2.969min. ¹ H NMR (400MHz, DMSO-d6) δ = 10.20 (s, 1H), 8.65 (s, 1H), 6.19 (s, 1H), 4.63-4.4 4(m,1H),4.28-4.10(m,1H),3.14-2.97(m,2H),2.19-2.07(m,1H),1.93-1.7 6(m,4H),1.74-1.59(m,4H),1.46-1.41(m,4H),1.40-1.26(m,3H),1.22(s, 3H),1.19-1.10(m,5H),1.06(s,3H),0.95(s,3H),0.90(s,3H),0.86(s,3H).

■Example 31

Compound 32:

Synthesis route:

■Example 32

Compound 33:

Synthesis route:

■Example 33

Compound 34:

Synthesis route:

■Example 34

Compound 35:

Synthesis route:

■Example 35

Biological evaluation

Test Example 1 In vitro human Nrf2 receptor agonist activity assay

Reagents: Opti-MEM serum-free medium; PEI 40000 transfection reagent; DMEM complete medium.

Equipment: Cx7Pro high-content rapid imaging platform, etc.

For each well of cells, dilute 360 ng of Nrf2 phase change probe plasmid with 10 μL of Opti-MEM serum-free medium (Cienry), mix thoroughly to form DNA dilution solution, and let it stand for 5 minutes. Then, dilute 0.6 μL of PEI 40000 transfection reagent (YEASEN) with 10 μL of Opti-MEM serum-free medium, mix thoroughly to form PEI 40000 dilution solution, and let it stand for 5 minutes. After mixing the DNA dilution solution and PEI 40000 dilution solution, incubate at room temperature for 20 minutes to allow the formation of DNA-PEI cationic nucleic acid transfection reagent complex. Remove 20 μL of cell growth medium and add 20 μL of DNA-PEI cationic nucleic acid transfection reagent complex to each well. Shake the culture plate gently to mix. Cells were cultured in a 37°C, 5% _CO2 incubator. One hour after transfection, 75 μL of cell growth medium was removed and 75 μL of fresh, prewarmed DMEM (Meilunbio) was added to each well, maintaining a medium volume of 100 μL per well. Eighteen hours after transfection, serially diluted drug (0.0625 nM, 0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM, 4 nM, 8 nM, 16 nM, 32 nM, 128 nM) was first mixed with prewarmed DMEM. 100 μL of this serially diluted drug medium was then added to the sample wells, bringing the final volume to 200 μL per well. Six hours after drug addition, 16 randomly selected fields of view per well were imaged using a Cx7Pro high-content rapid imaging platform (Thermo Fisher) at a 20x magnification lens. The number of transfected cells was determined by determining the location of the cell nucleus using the independently expressed NLS-mTagBFP2 in the probe. Calculate the total fluorescence intensity of the Nrf2 phase transition probe during phase transition. Count the total fluorescence intensity of the Nrf2 phase transition probe droplets in each cell. Compare the total fluorescence intensity of the Nrf2 phase transition probe per cell in the drug group to the total fluorescence intensity of the Nrf2 phase transition probe per cell in the DMSO group. Calculate the _EC50 for each drug using the log(agonist) vs. response - Variable slope (four parameters) analysis method in GraphPad Prism. Results are shown in Table 1.

Table 1 Test results of representative compounds of the present invention on the agonist activity of human Nrf2 receptor in vitro

The biological experimental data are shown in Table 1. Furthermore, the test experimental data show that multiple molecules, particularly compounds 1, 4, 5, 11, 16, 17, 19, 24, 26, 28, 29, 30, and 31 prepared by the present invention, all exhibited single-digit nanomolar human Nrf2 receptor agonist activity, significantly outperforming the control drug, omaveloxolone. The above in vitro human Nrf2 receptor agonist activity data demonstrate that the compounds of the present invention, their pharmaceutically acceptable salts, and stereoisomers can be used to prepare NRF2 activators.

In addition, control experiments with the control drug Omaveloxolone showed that both the compound of the present invention and the control drug Omaveloxolone have human Nrf2 receptor agonist activity, but there are significant differences in the activity of the compound in terms of DPPH free radical scavenging and MDA anti-lipid peroxidation, as shown in Test Examples 2-4.

Test Example 2 DPPH free radical scavenging ability test

Purpose of the experiment: To determine the DPPH free radical scavenging ability of the compounds of the present invention.

Test materials:

Test equipment:

Test method:

First, add 20 μL of the test compound to a 96-well plate and dilute it serially in a 1:2 ratio using DMSO. Then, add 200 μL of 200 μM DPPH prepared with anhydrous ethanol as the solvent to each well, shake gently, and incubate at room temperature in the dark for 30 minutes. The absorbance at 517 nm was detected by a microplate reader. The DPPH clearance rate was calculated using the following formula: DPPH clearance rate % = (1-Ai/A0) * 100%, where Ai refers to the sample absorbance value and A0 refers to the DMSO control group absorbance value. The data was processed using XLfit 5.3.1.3 software, and the IC50 value of the compound was obtained using a nonlinear fitting formula. The results are shown in Table 2.

Table 2 DPPH radical scavenging ability test results of representative compounds of the present invention

The results showed that the representative compounds 19 and 24 prepared in the present invention had the ability to scavenge DPPH free radicals, while Omaveloxolone had no such activity.

Test Example 3 MDA Anti-lipid Peroxidation Ability Test

Experimental purpose: To test the MDA anti-lipid peroxidation ability of the compounds of the present invention.

Experimental Materials:

Test equipment:

Test method:

First, brain tissue homogenate was prepared: an adult male Sprague-Dawley rat was anesthetized with isoflurane and sacrificed by cervical dislocation. The whole brain was removed and washed twice in DPBS. The meninges were stripped and transferred to a 50-mL centrifuge tube containing 10 mL of DPBS. The brain was minced with scissors and divided into ten 1.5-mL centrifuge tubes. Three grinding beads were added to each tube and the mixture was ground at 90 Hz for 60 minutes three times. The ground tissue homogenate was transferred to a fresh 50-mL centrifuge tube, DPBS was added to a total volume of 30 mL, and the mixture was mixed. Next, 20 μL of the test compound was added to a 96-well plate and serially diluted 1:3 in DMSO. Then, 100 μL of brain tissue homogenate, 50 μL of DPBS, and 50 μL of 200 μg/mL vitamin C were added, along with a series of standard concentrations as a standard curve. After shaking, the mixture was incubated at 37°C for 1 hour. Then, 400 μL of MDA working solution was added and the mixture was heated at 100°C for 15 minutes. After cooling to room temperature, the plates were centrifuged at 1000 g for 10 minutes. 200 μL of the supernatant was aspirated and transferred to a new plate. The absorbance at 532 nm was measured using a microplate reader. The MDA clearance rate of the compound was calculated using the following formula: MDA clearance % = [((A1-A0)-(A2-A0))/((A1-A0)-(A3-A0))] * 100%, where A1 refers to the absorbance of the high-dose control group, A2 refers to the sample absorbance, A3 refers to the low-dose control group absorbance, and A0 refers to the blank group absorbance. Data were processed using XLfit 5.3.1.3 software, and _IC50 values of the compounds were calculated using a nonlinear fitting formula. The results are shown in Table 3.

Table 3 MDA anti-lipid peroxidation ability test results of representative compounds of the present invention

The results showed that compounds 19, 24, 26 and 28 prepared in the present invention had anti-lipid peroxidation activity, and their activity was better than that of edaravone, while omaveloxolone had no such activity.

Experimental Example 4: Intervention Effect of Representative Compounds on Ferroptosis (qPCR)

1) Objective: To examine the effects of a series of compounds on ferroptosis by measuring changes in the expression of key genes in the ferroptosis signaling pathway at the cellular level. The specific indicator is the mRNA level of PTGS2, a key gene in ferroptosis (quantitative real-time RT-PCR).

2) Experimental methods:

HT-1080 cells (Cat. No. CCL-121, Shanghai Cell Bank, purchased from ATCC) were plated in six-well plates at 4 × 10⁵/well for 22 h. HT-1080 cells were treated with a gradient of compound concentrations (1.37 nM to 333 nM, a total of six concentrations) for 1 h before initiation. HT-1080 cells were then treated with the classic ferroptosis inducer RSL3 (Cat. No. HY-100218A, MCE, USA) at 200 nM for 16 h. mRNA was extracted and reverse-transcribed into cDNA, and PTGS2 mRNA levels were measured by qRT-PCR (ABI7500, Thermo Fisher Scientific, USA). The ferroptosis inhibitor Fer-1 (Cat. No. HY-100579, MCE, USA) was used as a positive control.

3) Experimental results:

The IC ₅₀ of each drug was calculated using the log (agonist) vs. response--Variable slope (four parameters) analysis method in GraphPadPrism. The results are shown in Table 4.

Table 4 Interventional effects of the compounds of the present invention on the ferroptosis process

The results showed that the representative compounds 26 and 28 prepared in the present invention can inhibit ferroptosis, while Omaveloxolone has no such activity.

The above-mentioned pharmacological experiments demonstrate that the preferred NRF2-Keap1 compounds prepared by the present invention, such as 19, 24, 26, 28, etc., not only maintain Nrf2 agonist activity similar to that of the marketed Nrf2 agonist Omaveloxolone, but also increase the effects of scavenging DPPH free radicals, inhibiting the production of lipid peroxides MDA, or intervening in ferroptosis.

At the same time, the present invention has been verified through biological in vivo tests to show that the compounds of the present invention have significant effects on a variety of diseases, including: cerebral small vessel disease, mitochondrial encephalopathy, autism spectrum disorder, Rett syndrome, Friedreich's ataxia, stroke, hemorrhagic stroke, ischemic stroke, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, schizophrenia cognitive impairment, Parkinson's disease, Parkinson's cognitive impairment, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, and renal ischemia. In particular, the compounds of the present invention have significant effects on diseases such as stroke, multiple sclerosis, and amyotrophic lateral sclerosis, and can be used to prepare drugs for preventing or treating stroke, multiple sclerosis, and amyotrophic lateral sclerosis.

Experimental Example 5: Tissue distribution experiment of representative compounds in SD rats

Test animals: SD rats, male, weighing about 250±20g, were randomly divided into groups, 3 rats in each group.

Solvent: DMSO: 10% solution (5%: 95%).

Test method:

The animals were administered orally at a dose of 20 mg/kg (30 mg/kg for compound 30). They were fasted for 12 hours prior to administration and had free access to water. Plasma was collected at specific times after administration, and brain tissue was collected after cardiac perfusion. After pretreatment, the samples were analyzed by LC-MS/MS to determine the concentrations of the analytes in plasma and brain. The results are shown in Table 4.

Table 4: Plasma and brain concentrations in SD rats after administration

Experimental Example 6: Protective Effect of Representative Compounds on Ischemic Stroke

1) Reagents: Omaveloxolone, MedChemexpress Biotechnology, USA; Transient Middle Cerebral Artery Occlusion Model (tMCAO) suture, Beijing Reward Life Sciences Co., Ltd.; DMAO, Beyotime Biotechnology Co., Ltd.; Solutal, Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Normal saline, Sinopharm Chemical Reagent Co., Ltd.; TTC, Sigma-Aldrich (Shanghai) Trading Co., Ltd.

2) Establishment of cerebral ischemia rat model using tMCAO method:

Rats were anesthetized with 10% chloral hydrate (350 mg/kg), fixed in the supine position on the operating table, and the neck was disinfected. A midline incision was made in the neck, and the intermuscular space between the left sternocleidomastoid and sternoglossi muscles was bluntly dissected to expose the common carotid artery. The common carotid artery was extracted with ophthalmic curved forceps and threaded with silk suture for later use. The external carotid artery was isolated between the right digastric muscle and the hyoid bone, and threaded with silk suture for later use. Suture was passed through and tied to the common carotid artery. A small incision was made at the free end of the common carotid artery and a loose knot was tied. A suture was inserted through the incision along the common carotid artery. The suture was slowly advanced until resistance was felt and the pre-tied loose knot was tightened. The body temperature was maintained at 37°C during the operation, and the suture was removed after 1.5 hours. Rats in the sham operation group did not have a suture inserted after vascular isolation, and the other procedures were the same.

3) Experimental groups and drug administration:

Forty-eight male Sprague-Dawley rats, weighing 230-250g, were randomly divided into sham-operated, model, omaveloxolone 3mg/kg, omaveloxolone 9mg/kg, compound 192.74mg/kg, and compound 282.89mg/kg groups. After three days of adaptive feeding, the rats were injected into the tail vein during modeling. Rats in the sham-operated and model groups received an equal volume of vehicle (10% DMSO + 10% solutal + 80% saline).

4) Index detection:

mNSS score: Neurobehavioral scoring was performed 24 hours after model establishment, mainly to evaluate the neurological function of rats with ischemic stroke from the perspectives of sensation and movement.

Measurement of cerebral infarct area: Rats were euthanized with an overdose of chloral hydrate. The brains were removed and placed in a -20°C refrigerator for 20 minutes. The brains were then placed in the cerebral trough and sliced to a thickness of 2 mm. The slices were then incubated in a 37°C incubator in a 2% TTC solution, shielded from light, for 15 minutes. After staining, images were taken and analyzed for cerebral infarct area.

5) Statistical methods:

All data in this study are expressed as mean ± standard deviation (mean ± SD) and statistically analyzed using GraphPad Prism 7.0. Differences between groups were assessed using one-way ANOVAs followed by Tukey's test. Behavioral tests were tested using the Krystal-Wallis test. Data were considered statistically significant at P < 0.05.

6) Experimental results:

Effects of compounds 19 and 28 on neurological function in rats with ischemic stroke:

As shown in Figure 1, omaveloxolone 3 mg/kg and 9 mg/kg had no effect on the mNSS scores of rats with cerebral ischemia. At equimolar doses to omaveloxolone 3 mg/kg, both compound 19 (2.74 mg/kg) and compound 28 (2.89 mg/kg) significantly reduced the mNSS scores of rats with cerebral ischemia.

Effects of compounds 19 and 28 on cerebral infarction area in rats with ischemic stroke:

As shown in Figure 2, omaveloxolone 3 mg/kg and 9 mg/kg had no effect on the cerebral infarction area in rats with cerebral ischemia. At equimolar doses to omaveloxolone 3 mg/kg, both compound 19 (2.74 mg/kg) and compound 28 (2.89 mg/kg) significantly reduced the cerebral infarction area in rats with cerebral ischemia.

7) Conclusion: The novel Nrf2-Keap1 uncouplers disclosed in the present invention, such as compounds 19 and 28, can reduce the cerebral infarction area in rats with ischemic stroke and have a protective effect on neurological damage.

Effects of the representative compounds of Experimental Example 7 on the neurobehavior of multiple sclerosis model mice

1. Materials and Methods

1) Main reagents

2) Experimental animals and grouping and drug administration

Female C57BL/6J mice were randomly divided into 5 groups: sham operation group, model group, Omaveloxolone 5 mg/kg group, Omaveloxolone 15 mg/kg group, and compound 244.69 mg/kg, with 8 mice in each group. The mice in the sham operation group and model group were given the same volume of solvent (10% solutol + 90% saline). Each group was given twice a day for 42 consecutive days.

3) Preparation of Multiple Sclerosis Model - EAE Model

A mixed emulsion containing the myelin oligodendrocyte glycoprotein MOG35-55 peptide and complete Freund's adjuvant (CFA) containing Mycobacterium tuberculosis was injected subcutaneously on the back of mice, and pertussis toxin (PTX) was injected intraperitoneally on the day of immunization and 48 hours later to establish the EAE (experimental autoimmune encephalosporin) model.

4) Weight and neurological function score (5-point scale)

Starting from the day of immunization induction (Day 0), the animals were weighed and neurological function was scored daily. Neurological function scoring criteria (5-point scale): 0, no clinical deficit; 1, partial tail paralysis; 2, complete tail paralysis; 3, partial hindlimb paralysis; 4, complete hindlimb paralysis; 5, forelimb paralysis; 6, death.

5) Statistical processing

All data are presented as mean ± standard error of the mean (SEM). Two-way analysis of variance and Tukey's multiple comparison test were used to compare differences in neurological function scores and body weight among the groups. All data were analyzed using GraphPad Prism 9.0.0 software. A p < 0.05 indicated statistical significance.

2. Experimental Results

As shown in Figure 3, omaveloxolone 5 mg/kg and 15 mg/kg significantly reduced neurological function scores in EAE model mice, but no significant difference was observed between the two groups, indicating that omaveloxolone 5 mg/kg achieved its maximum effect. At an equimolar dose to omaveloxolone 5 mg/kg, compound 24 (4.69 mg/kg) significantly reduced neurological function scores in EAE model mice, and its effect was superior to that of omaveloxolone 15 mg/kg, with a statistically significant difference.

As shown in Figure 4, omaveloxolone 5 mg/kg and 15 mg/kg significantly increased the body weight of EAE model mice, but no significant difference was observed between the two groups, indicating that omaveloxolone 5 mg/kg achieved its maximum effect. At an equimolar dose to omaveloxolone 5 mg/kg, compound 24 (4.69 mg/kg) significantly increased the body weight of EAE model mice, and the effect was superior to omaveloxolone 15 mg/kg, with a statistically significant difference.

3. Experimental Conclusion

Compound 24 has a protective effect on the neurological function of multiple sclerosis model mice, and its effect is stronger than that of Omaveloxolone.

Test Example 8: Protective Effect of Representative Compounds on Amyotrophic Lateral Sclerosis Model Mice

1. Materials and Methods

1) Main reagents

Omaveloxolone was purchased from MCE Biotechnology Co., Ltd.; Solutal was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; and normal saline was purchased from Sinopharm Chemical Reagent Co., Ltd.

2) Animals

Sixty B6SJL-Tg(SOD1 G93A)-1Gur/J transgenic mice (half male and half female) of the amyotrophic lateral sclerosis (ALS) model were purchased from Shanghai Model Organisms Technology Co., Ltd.

3) Experimental grouping and drug administration

SOD1 G93A mice were randomly divided into a model group, an omaveloxolone 1 mg/kg group, an omaveloxolone 3 mg/kg group, a compound 2 60.99 mg/kg group, and a compound 3 11.06 mg/kg group, with 12 mice in each group. Twelve C57BL/6J mice were also assigned to the control group. Both the control and model groups were intraperitoneally injected with an equal volume of vehicle (1% DMSO + 4% solutal + 95% saline) once daily for 10 weeks.

4) Index detection

Rotarod test

The rotarod test is a classic behavioral test for evaluating motor coordination in mice. Rotarod tests were performed twice weekly (Panlab rotarod apparatus, purchased from Harvard Bioscience, USA), with the rotation speed ranging from 4 to 40 rpm. The experimental procedures were as follows: 1. Before the actual experiment, mice were acclimated to a rotation speed of 12 rpm for 5 minutes twice daily for 3 days. 2. During the actual experiment, three consecutive tests were performed (3 minutes each, with a 30-minute interval between each test). 3. The duration of the mouse's stay on the rotarod was recorded, and the longest time spent on the rotarod across the three tests was used as the latency to fall.

Onset time

The rotarod test was used to detect the onset time of mice, and the first time the mouse fell from the rotarod within 3 minutes was recorded as the onset date.

Cage experiment

The hanging cage test assesses the grip strength and endurance of mice's limbs. This test is performed twice weekly. Each mouse is placed in the center of a wire mesh. The mesh is gently shaken to ensure the mouse's grip is firm. The mesh is then slowly inverted to a horizontal position, and the time the mouse remains suspended is recorded. Each mouse is tested three times, with each test separated by 30 minutes. The maximum value is used as the fall latency.

2. Experimental results

1) Effects of compounds 26 and 31 on the onset time of SOD1 G93A mice

As shown in Figure 5, both omaveloxolone 1 mg/kg and 3 mg/kg significantly delayed the onset of SOD1 G93A mice, but no significant difference was observed between the two groups, indicating that omaveloxolone 1 mg/kg achieved its maximum effect. At equimolar doses to omaveloxolone 1 mg/kg, both compound 26 (0.99 mg/kg) and compound 31 (1.06 mg/kg) significantly delayed the onset of SOD1 G93A mice, and their efficacy was superior to that of omaveloxolone 3 mg/kg, with statistically significant differences.

2) Effects of compounds 26 and 31 on motor coordination ability in SOD1 G93A mice

As shown in Figure 6, both omaveloxolone 1 mg/kg and 3 mg/kg significantly improved the motor coordination ability of SOD1 G93A mice, as evidenced by a significant increase in fall latency. However, no significant difference was observed between the two groups, indicating that omaveloxolone 1 mg/kg achieved its maximum effect. At equimolar doses to omaveloxolone 1 mg/kg, both compound 26 (0.99 mg/kg) and compound 31 (1.06 mg/kg) significantly increased the fall latency of mice, with a greater potency than omaveloxolone 3 mg/kg, achieving statistically significant differences.

3) Effects of compounds 26 and 31 on muscle endurance in SOD1 G93A mice

As shown in Figure 7, both 1 mg/kg and 3 mg/kg of omaveloxolone significantly improved the muscle endurance of SOD1 G93A mice, as evidenced by a significant increase in fall latency. However, no significant difference was observed between the two groups, indicating that omaveloxolone 1 mg/kg achieved its maximum effect. At equimolar doses to omaveloxolone 1 mg/kg, both compound 26 (0.99 mg/kg) and compound 31 (1.06 mg/kg) significantly increased the fall latency of mice, with a greater potency than omaveloxolone 3 mg/kg, achieving statistically significant differences.

3. Experimental Conclusion

Compounds 26 and 31 can improve the neurobehavior of ALS model mice, and their effect is stronger than that of Omaveloxolone.

In summary, the compounds of the present invention, as novel NRF2 activators, have excellent Nrf2 agonist effects, and the compounds exert antioxidant effects through new mechanisms such as scavenging DPPH free radicals or inhibiting the generation of lipid peroxides MDA. Further, the in vivo experiments of the present invention also show that the compounds provided by the present invention can be more effectively used for the prevention or treatment of related diseases, including cerebral small vessel disease, mitochondrial encephalomyopathy, autism spectrum disorder, Rett syndrome, Friedreich's ataxia, stroke, hemorrhagic stroke, ischemic stroke, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, schizophrenia cognitive impairment, Parkinson's disease, Parkinson's cognitive impairment, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, renal ischemia, etc. In particular, some compounds have obvious effects on the treatment and/or prevention of stroke, multiple sclerosis, and amyotrophic lateral sclerosis.

The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the scope of protection of the present invention.

Claims (9)

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, characterized by being as follows:
in: R ₁ is independently selected from: -C(=O)-alkane, -C(=O)-substituted alkane, -C(=O)-alkene, -C(=O)-substituted alkene, -C(=O)-alkyne, -C(=O)-substituted alkyne, -C(=O)-heteroarene, -C(=O)-heteroarene-R ₁ ', -C(=O)-heteroarenediyl-R ₁ '-C(＝O)-NH-OH, -C(＝O)-N(OH)-alkane, -C(＝O)-N(OH)-substituted alkane, -C(＝O)-N(OH)-alkene, -C(＝O)-N(OH)-substituted alkene, -C(＝O)-N(OH)-alkyne, -C(＝O)-N(OH)-substituted alkyne, -C(＝O)-N(OH)-arene, -C(＝O)-N(OH)-arenediyl-R ₁ ', -C(＝O)-N(OH)-heteroarene, -C(＝O)-N(OH)-heteroarenediyl-R ₁ ', -C(＝O)-NH-arene, -C(＝O)-NH-arenediyl-R ₁ ', -C(＝O)-NH-heteroarene, -C(＝ _O )-NH-heteroarene ', -C(=O)-O-arene, -C(=O)-O-arenediyl-R ₁ ', -C(=O)-O-heteroarene, -C(=O)-O-heteroarenediyl-R ₁ ', -C(=O)-CH ₂ -heteroarene, -C(=O)-CH ₂ -heteroarenediyl-R ₁ ', -C(=O)-CR ₂ 'R ₃ '-heteroarene, -C(=O)-CR ₂ 'R ₃ '-heteroarenediyl-R ₁ ', -C(=O)-L-type amino acid-NH-heteroarene, -C(=O)-L-type amino acid-NH-heteroarenediyl-R ₁ '; and R ₂ : hydrogen or methyl; R ₃ : hydrogen or methyl. The compound according to claim 1, characterized in that the compound is:
in: R ₁ is independently selected from: -C(=O)-alkane, -C(=O)-substituted alkane, -C(=O)-alkene, -C(=O)-substituted alkene, -C(=O)-alkyne, -C(=O)-substituted alkyne, -C(=O)-heteroarene, -C(=O)-heteroarene-R ₁ ', -C(=O)-heteroarenediyl-R ₁ '-C(＝O)-NH-OH, -C(＝O)-N(OH)-alkane, -C(＝O)-N(OH)-substituted alkane, -C(＝O)-N(OH)-alkene, -C(＝O)-N(OH)-substituted alkene, -C(＝O)-N(OH)-alkyne, -C(＝O)-N(OH)-substituted alkyne, -C(＝O)-N(OH)-arene, -C(＝O)-N(OH)-arenediyl-R ₁ ', -C(＝O)-N(OH)-heteroarene, -C(＝O)-N(OH)-heteroarenediyl-R ₁ ', -C(＝O)-NH-arene, -C(＝O)-NH-arenediyl-R ₁ ', -C(＝O)-NH-heteroarene, -C(＝ _O )-NH-heteroarene ', -C(=O)-O-arene, -C(=O)-O-arenediyl-R ₁ ', -C(=O)-O-heteroarene, -C(=O)-O-heteroarenediyl-R ₁ ', -C(=O)-CH ₂ -heteroarene, -C(=O)-CH ₂ -heteroarenediyl-R ₁ ', -C(=O)-CR ₂ 'R ₃ '-heteroarene, -C(=O)-CR ₂ 'R ₃ '-heteroarenediyl-R ₁ ', -C(=O)-L-type amino acid-NH-heteroarene, -C(=O)-L-type amino acid-NH-heteroarenediyl-R ₁ '; And R ₂ : methyl group; R ₃ : methyl group. The compound according to claim 1 or 2, characterized in that R ₁ ' in the aromatic hydrocarbon compound is independently selected from: -Cl, -F, -Br, -OH, isopropyl, straight-chain/branched alkyl (C≤6), straight-chain/branched alkyl (C≤6) substituted with 1 to 5 halogens, -OH, straight-chain/branched alkyl (C≤6) substituted with 1 to 5 -OHs, straight-chain/branched alkenyl (C≤6), straight-chain/branched alkenyl (C≤6) substituted with 1 to 5 halogens, straight-chain/branched alkenyl (C≤6) substituted with 1 to 5 -OHs, straight-chain/branched alkynyl (C≤6), straight-chain/branched alkynyl (C≤6) substituted with 1 to 5 halogens, straight-chain/branched alkynyl (C≤6) substituted with 1 to 5 -OHs, The compound according to claim 3, characterized in that the aromatic group is selected from:
The compound according to claim 3, characterized in that the substituted alkane, substituted alkene, substituted alkyne, alkane, alkene, alkyne C chain length is ≤6, and each is independently selected from: linear, branched or cyclic. The compound according to claim 1, characterized in that the compound is as follows:


Use of the compound according to claim 6 for preparing an NRF2 activator. The use of the compound according to claim 6 for preparing a drug for treating and/or preventing a patient's disease, characterized in that the prepared drug is used to prevent or treat the patient's diseases including cerebral small vessel disease, mitochondrial encephalomyopathy, autism spectrum disorder, Rett syndrome, Friedreich's ataxia, stroke, hemorrhagic stroke, ischemic stroke, multiple sclerosis, amyotrophic lateral sclerosis, schizophrenia, schizophrenia cognitive impairment, Parkinson's disease, Parkinson's cognitive impairment, Alzheimer's disease, vascular dementia, epilepsy, Huntington's disease, heart failure, myocardial infarction, renal failure, and renal ischemia. The use according to claim 8, characterized in that the compound is used to prepare a drug for preventing or treating stroke, multiple sclerosis, and amyotrophic lateral sclerosis.