Hbv core protein degrader based on hydrophobic tag technology and preparation method and application thereof

Abstract

The application provides an HBV core protein degradation agent based on a hydrophobic tag technology, a preparation method and application thereof. The HBV core protein degradation agent comprises a compound shown in a structure as shown in formula I or II or a pharmaceutically acceptable salt thereof, wherein X is a linker, and Y is a hydrophobic group. The application also relates to a preparation method of a compound containing the structure as shown in formula I or II and application of the compound in preparation of an anti-HBV drug.

Description

Technical Field

[0001] This invention belongs to the field of organic compound synthesis and pharmaceutical application technology, specifically relating to HBV core protein degrading agents based on hydrophobic tag technology, their preparation methods, and applications. Background Technology

[0002] Hepatitis B (HB) is a major infectious disease caused by the hepatitis B virus (HBV). Long-term progression can lead to acute and chronic viral hepatitis, fulminant hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Despite improved coverage of preventative HBV vaccines, approximately 257 million people worldwide still suffer from HBV-related diseases. Currently, clinical treatments for HBV mainly include immunomodulatory interferons and viral DNA polymerase inhibitors, nucleos(t)ide analogs (NAs). However, neither of these classes of drugs can completely cure HBV. Furthermore, nucleos(t)ide analogs are prone to rebound after discontinuation and can easily lead to drug resistance with long-term use; interferons have drawbacks such as poor tolerability, moderate antiviral efficacy, high risk of toxic side effects, and the need for subcutaneous injection. Therefore, the development of novel HBV inhibitors is urgently needed. HBV capsid (core) proteins are structural proteins that make up the viral capsid and play a crucial role in the viral life cycle. They mediate the interaction between the virus and host cells and are essential for maintaining HBV infectivity and stability. Therefore, HBV core proteins have become a promising new target in the field of anti-HBV drug research.

[0003] In recent years, the emergence of targeted protein degradation (TPD) technology has brought numerous opportunities for drug discovery. TPD technology hijacks endogenous protein degradation mechanisms, thereby inducing the consumption or reduction of pathogenic proteins. Compared to traditional drugs that require a large number of drug molecules to occupy binding sites, small molecules involved in protein-induced degradation only require a catalytic amount to achieve a good inhibitory effect, exhibiting unique advantages not possessed by conventional small molecule inhibitors, and providing a new option for disease treatment. Among these technologies, hydrophobic tagging (HyT) technology is another highly efficient modular strategy for degrading target proteins, following protein degradation targeting chimera (PROTAC) technology. HyT consists of a ligand for the target protein and a large hydrophobic group. When HyT binds to the target in the cell, the hydrophobic group attached to the target protein ligand mimics the misfolded state of the protein, thus being recognized by molecular chaperones and subsequently degraded by the proteasome.

[0004] This invention uses two representative HBV core protein regulators, dihydropyrimidine compound NVR-010-001-E2 and sulfonylbenzamide compound NVR 3-778, as target protein ligands. After structural fine-tuning, these compounds are linked in their solvent opening regions via linkers of different lengths and hydrophilicities, resulting in the design and synthesis of 30 HBV core protein degraders in two classes. No such compounds have been reported in the prior art.

[0005] Summary of the Invention

[0006] This invention provides HBV core protein degrading agents based on hydrophobic tagging technology, their preparation methods and applications. This invention also provides the anti-HBV activity screening results of the above compounds and their pharmaceutical applications.

[0007] The technical solution of the present invention is as follows:

[0008] 1. HBV core protein degrader based on hydrophobic tag technology

[0009] HBV core protein degraders based on hydrophobic tag technology have the compound structure shown in formula (I) or formula (II) or a pharmaceutically acceptable salt thereof:

[0010]

[0011] Where X is a linker and Y is a hydrophobic group.

[0012] Preferably, the linker comprises a compound with the following general structural formula:

[0013]

[0014] Preferably, the hydrophobic group comprises compounds with the following general structural formulas a to g:

[0015]

[0016] According to a further preferred embodiment of the present invention, the HBV core protein degrader based on hydrophobic tag technology is one of the following compounds:

[0017]

[0018]

[0019] The term "pharmaceutically acceptable salt" as used in this invention refers to a salt of a compound that, within the scope of reliable pharmaceutical evaluation, is suitable for contact with tissues of humans or lower animals without undue toxicity, irritation, or allergic reactions, possesses a reasonably reasonable benefit-risk ratio, is typically water- or oil-soluble or dispersible, and is effectively used for its intended purpose. This includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are suitable for the intended use and chemically compatible with compounds of Formula I and Formula II. For a list of suitable salts, see SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

[0020] 2. Preparation method of HBV core protein degrader based on hydrophobic tag technology

[0021] This invention also provides a method for preparing the HBV core protein degrader based on hydrophobic tag technology, the synthetic route of which is as follows:

[0022] Synthesis of the target product HyT-H1: Starting with 2-thiazolyl formamidinium hydrochloride (i-1), 2-bromo-4-fluorobenzaldehyde, and ethyl acetoacetate, the key intermediate i-2 was obtained by cyclization via the Biginelli reaction; in dichloromethane solution, i-2 underwent a bromination reaction with N-bromosuccinimide to obtain intermediate i-3; using i-3 as a starting material, potassium carbonate, potassium iodide, and 1-Boc piperazine were added, and the mixture was refluxed in acetonitrile solution at 75°C for 1 hour to obtain intermediate i-4; i-4 was de-Boc-grouped in the presence of trifluoroacetic acid to obtain intermediate i-5; adamantaneacetic acid and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) were reacted in an ice-water bath for half an hour, and i-5 and N,N-diisopropylethylamine (DIEA) were added and stirred overnight at room temperature to obtain the target product HyT-H1;

[0023]

[0024] Reagents and conditions: (i) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 90°C; (ii) N-bromosuccinimide, dichloromethane, 40°C; (iii) 1-Boc-piperazine, potassium carbonate, potassium iodide, acetonitrile, 75°C; (iv) trifluoroacetic acid, dichloromethane, 0°C → room temperature; (v) adamantaneacetic acid, HATU, DIEA, dichloromethane, 0°C → room temperature.

[0025] Synthesis of the target product HyT-S1: Starting with 3-chlorosulfonyl-4-fluorobenzoic acid (ii-1), the carboxyl group was chlorinated in toluene solution with thionyl chloride as the acyl chloride reagent to obtain intermediate ii-2; ii-2 underwent a nucleophilic acyl substitution reaction with 3,4,5-trifluoroaniline in toluene solution to obtain intermediate ii-3; 4-Boc-aminopiperidine reacted with ii-3 in dichloromethane solution, and then triethylamine was added as an acid-binding agent to obtain intermediate ii-4; ii-4 was de-Boc grouped in the presence of trifluoroacetic acid to obtain intermediate ii-5; adamantaneacetic acid and HATU were reacted in an ice-water bath for half an hour, and ii-5 and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-S1.

[0026]

[0027] Reagents and conditions: (i) thionyl chloride, toluene, DMF, 60°C; (ii) 3,4,5-trifluoroaniline, toluene; (iii) 4-Boc-aminopiperidine, dichloromethane, triethylamine, 0°C → room temperature; (iv) trifluoroacetic acid, dichloromethane, 0°C → room temperature; (v) adamantaneacetic acid, HATU, DIEA, dichloromethane, 0°C → room temperature.

[0028] Synthesis of the target product HyT-H(2-9): N-tert-butoxycarbonyl-R1-carboxylic acid and HATU were reacted in an ice-water bath for half an hour. Intermediate i-5 and DIEA were added and stirred overnight at room temperature to obtain intermediate i-6(ah). The Boc group of i-6(ah) was removed under the action of trifluoroacetic acid to obtain intermediate i-7(ah). Adamantaneacetic acid and HATU were reacted in an ice-water bath for half an hour. i-7(ah) and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-H(2-9).

[0029]

[0030] Reagents and conditions: (i) N-tert-butoxycarbonyl-R1-carboxylic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) trifluoroacetic acid, dichloromethane, 0℃ → room temperature; (iii) adamantaneacetic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature.

[0031] Synthesis of the target product HyT-S(2-9): N-tert-butoxycarbonyl-R1-carboxylic acid and HATU were reacted in an ice-water bath for half an hour. Intermediate ii-5 and DIEA were added and stirred overnight at room temperature to obtain intermediate ii-6(ah). The Boc group of ii-6(ah) was removed under the action of trifluoroacetic acid to obtain intermediate ii-7(ah). Adamantaneacetic acid and HATU were reacted in an ice-water bath for half an hour. ii-7(ah) and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-S(2-9).

[0032]

[0033] Reagents and conditions: (i) N-tert-butoxycarbonyl-R1-carboxylic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) trifluoroacetic acid, dichloromethane, 0℃ → room temperature; (iii) adamantaneacetic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature.

[0034] Synthesis of the target product HyT-H(10-11,13-15): Carboxylic acid compounds containing different hydrophobic tags and HATU were reacted in an ice-water bath for half an hour. Intermediate i-7f and DIEA were added and stirred at room temperature overnight to obtain the target product HyT-H(10-11,13-15).

[0035]

[0036] Reagents and conditions: (i) R2-OH, HATU, DIEA, dichloromethane, 0℃ → room temperature.

[0037] Synthesis of the target product HyT-S(10-11, 13-15): Carboxylic acid compounds containing different hydrophobic tags and HATU were reacted in an ice-water bath for half an hour. Intermediate ii-7f and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-S(10-11, 13-14). Compound ii-7f was dissolved in acetonitrile with tri-tert-butoxycarbonylarginine and N-methylimidazolium (NMI), stirred at room temperature for 10 minutes, and N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) was added and stirred overnight at room temperature to obtain the target product HyT-S15.

[0038]

[0039] Reagents and conditions: (i) R2-OH, HATU, DIEA, dichloromethane, 0℃ → room temperature or NMI, TCFH, acetonitrile, room temperature.

[0040] Synthesis of the target product HyT-H12: Monoethyl adipate and HATU were reacted in an ice-water bath for half an hour, and intermediate i-5 and DIEA were added and stirred overnight at room temperature to obtain intermediate i-8; intermediate i-8 was hydrolyzed with lithium hydroxide to remove the ester group to obtain intermediate i-9; intermediate i-9 and HATU were reacted in an ice-water bath for half an hour, and norbornene-2-methylamine and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-H12.

[0041]

[0042] Reagents and conditions: (i) Monoethyl adipate, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) Lithium hydroxide, tetrahydrofuran: water, room temperature; (iii) Norbornene-2-methylamine, HATU, DIEA, dichloromethane, 0℃ → room temperature.

[0043] Synthesis of the target product HyT-S12: Ethyl adipate and HATU were reacted in an ice-water bath for half an hour, and intermediate ii-5 and DIEA were added and stirred overnight at room temperature to obtain intermediate ii-8; intermediate ii-8 was hydrolyzed with lithium hydroxide to remove the ester group to obtain intermediate ii-9; intermediate ii-9 and HATU were reacted in an ice-water bath for half an hour, and norbornene-2-methylamine and DIEA were added and stirred overnight at room temperature to obtain the target product HyT-S12.

[0044]

[0045] Reagents and conditions: (i) Monoethyl adipate, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) Lithium hydroxide, tetrahydrofuran: water, room temperature; (iii) Norbornene-2-methylamine, HATU, DIEA, dichloromethane, 0℃ → room temperature.

[0046] 3. Application of HBV core protein degrading agents based on hydrophobic tag technology

[0047] This invention discloses the screening results of anti-HBV activity of HBV core protein degraders based on hydrophobic tagging technology and their application as anti-HBV drugs. Experiments demonstrate that the HBV core protein degraders based on hydrophobic tagging technology of this invention can be used to prepare anti-HBV drugs.

[0048] Anti-HBV activity and toxicity experiments of the target compound

[0049] Two types of HBV core protein degraders based on hydrophobic tagging technology synthesized according to the above method were evaluated for in vitro anti-HBV activity and cytotoxicity. HBV DNA inhibitory activity was determined by qPCR, and cytotoxicity was determined by CPE method. Their anti-HBV activity and toxicity data are listed in Table 1, with dihydropyrimidine compound GLS4 and sulfonylbenzamide compound NVR3-778 as positive controls.

[0050] The HBV core protein degraders designed based on hydrophobic tagging technology in this invention exhibit significant anti-HBV activity. The dihydropyrimidine HBV core protein degraders show activity at the submicromolar to nanomolar levels, with compounds HyT-H2, HyT-H3, HyT-H4, HyT-H7, HyT-H9, and HyT-H12 exhibiting activity comparable to the positive control GLS4. Several sulfonylbenzamide HBV core protein degraders, such as compounds HyT-S10, HyT-S11, HyT-S12, and HyT-S14, show anti-HBV activity comparable to the positive control NVR3-778. These compounds warrant further investigation.

[0051] Validation of the anti-HBV mechanism of the target compound

[0052] The effect of the target compound on the content of HBV core protein was tested using Western blotting, and the results are as follows: Figure 1 As shown. Dihydropyrimidine compound GLS4 and sulfonylbenzamide compound NVR 3-778 were used as positive controls.

[0053] Western blot results showed that, compared with the viral control group (tet-), HyT-H2, HyT-H3, HyT-H4, HyT-H5, HyT-H7, and HyT-H12 significantly reduced the content of HBV core protein at a concentration of 0.4 μM; HyT-S7 and HyT-S9 reduced the content of HBV core protein in a dose-dependent manner. Therefore, this invention provides a new direction for the development of anti-HBV drugs.

[0054] The HBV core protein degrader designed and synthesized based on hydrophobic tagging technology in this invention is a novel compound with a novel structure and mechanism, which can be used as an anti-HBV drug.

[0055] This invention utilizes a constructed HBV core protein degradation screening model, and through synthesis optimization, discovers that the synthesized small molecule degradative agent can effectively degrade the expression level of HBV core protein in vitro, providing a potential treatment method for treating hepatitis B through HBV core protein.

[0056] An anti-HBV pharmaceutical composition comprising an HBV core protein degrader based on hydrophobic tagging technology of the present invention and one or more pharmaceutically acceptable carriers or excipients.

[0057] This invention discloses an HBV core protein degrader based on hydrophobic tag technology, its preparation method, anti-HBV activity screening results, HBV core protein degradation results, and its first application as an anti-HBV drug. Attached Figure Description

[0058] Figure 1 This is a graph showing the effect of the target compound on the content of HBV core protein. Detailed Implementation

[0059] The following examples help to understand the present invention, but do not limit the scope of the invention. All percentages mentioned are mass percentages.

[0060] Example 1. Preparation of the target compound HyT-H1

[0061] Adamantaneacetic acid (70.7 mg, 0.364 mmol) and HATU (173.0 mg, 0.455 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-5 (150.0 mg, 0.303 mmol) and DIEA (100.3 μL, 0.607 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H1 in 78% yield. Spectroscopic data: 1 H NMR (400MHz, DMSO-d6) δ9.71(s,1H),8.01(d,J=3.3Hz,1H),7.94(d,J=3.2Hz,1H),7.56(d,J=8.6Hz,1H),7.42–7.33(m,1H),7.22(d,J=7.6Hz ,1H),6.02(s,1H),3.99–3.86(m,2H),3.57(s,4H),3.52(s,3H),2.54(s,4H),2.12(s,2H),1.92(s,3H),1.63(d,J=15.0Hz,12H).ESI-MS:m / z 670.20[M+H] + 672.23[M+2+H] + C 32 H 37 BrFN5O3S[669.18].

[0062] Example 2. Preparation of the target compound HyT-H2

[0063] Adamantaneacetic acid (72.6 mg, 0.374 mmol) and HATU (180.0 mg, 0.467 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7a (180.0 mg, 0.311 mmol) and DIEA (103.0 μL, 0.623 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H2 in 75% yield. Spectroscopic data: 1 H NMR (600MHz, DMSO-d6) δ9.71 (s, 1H), 8.01 (d, J = 3.1Hz, 1H), 7.95 (d, J = 3.1Hz, 1H), 7.66 (t, J = 5. 5Hz,1H),7.57(dd,J=8.6,2.7Hz,1H),7.39(dd,J=8.8,6.1Hz,1H),7.22(td,J=8.4,2.7Hz,1H),6 .02(s,1H),4.00–3.86(m,2H,),3.51(d,J=10.5Hz,7H),3.05(q,J=6.9Hz,2H),2.51–2.50(m,4H ),2.34(t,J=7.5Hz,2H),1.90(s,3H),1.82(s,2H),1.66–1.52(m,14H).ESI-MS:m / z755.24[M+H] + 757.23[M+2+H] + C 36 H 44 BrFN6O4S[754.23].

[0064] Example 3. Preparation of the target compound HyT-H3

[0065] Adamantaneacetic acid (70.9 mg, 0.365 mmol) and HATU (173.0 mg, 0.456 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7b (180.0 mg, 0.304 mmol) and DIEA (100.0 μL, 0.608 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H3 in 68% yield. Spectroscopic data: 1H NMR(600MHz, DMSO-d6)δ9.71(s,1H),8.01(d,J=3.1Hz),7.95(d,J=3.1Hz,1H),7.66–7.61(m,1H),7.57(d d,J=8.6,2.7Hz),7.38(dd,J=8.7,6.1Hz,1H),7.22(td,J=8.4,2.7Hz,1H),6.02(s,1H),3.99–3.87(m,2H ),3.53(d,J=6.3Hz,7H),3.03(q,J=6.8Hz,2H),2.51–2.50(m,4H),2.33(t,J=7.6Hz,2H)),1.90(s,3H)), 1.81(s,2H)),1.66–1.53(m,12H)),1.50(q,J=8.0Hz,2H),1.41(p,J=6.8Hz,2H).ESI-MS:m / z769.27[M+H] + 771.28[M+2+H] + C 37 H 46 BrFN6O4S[768.25].

[0066] Example 4. Preparation of the target compound HyT-H4

[0067] Adamantaneacetic acid (46.0 mg, 0.238 mmol) and HATU (112.9 mg, 0.297 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7C (120.0 mg, 0.198 mmol) and DIEA (65.4 μL, 0.396 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H4 in 75% yield. Spectroscopic data: 1H NMR (400MHz, DMSO-d6) δ9.71(s,1H),8.01(d,J=3.1Hz,1H),7.94(d,J=3.2Hz,1H),7.62(t,J=5.7Hz,1H),7. 56(dd,J=8.6,2.6Hz,1H),7.38(dd,J=8.7,6.2Hz,1H),7.21(td,J=8.5,2.8Hz,1H),6.02(s,1H),4.00–3.87( m,2H),3.52(s,7H),3.01(q,J=6.7Hz,2H),2.50(dt,J=3.7,1.8Hz,4H),2.30(t,J=7.5Hz,2H),1.90(s,3H),1 .80(s,2H),1.66–1.54(m,12H),1.49(q,J=7.5Hz,2H),1.39(q,J=6.9Hz,2H),1.32–1.22(m,2H).ESI-MS:m / z 783.26[M+H] + 785.30[M+2+H] + C 38 H 48 BrFN6O4S[782.26].

[0068] Example 5. Preparation of the target compound HyT-H5

[0069] Adamantaneacetic acid (52.6 mg, 0.271 mmol) and HATU (128.7 mg, 0.339 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7d (140.0 mg, 0.226 mmol) and DIEA (74.6 μL, 0.451 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H5 in 80% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ9.71(s,1H),8.00(d,J=3.1Hz,1H),7.94(d,J=3.1Hz,1H),7.61(t,J=5.6Hz,1H),7.5 6(dd,J=8.5,2.7Hz),7.38(dd,J=8.7,6.1Hz,1H),7.21(td,J=8.4,2.7Hz,1H),6.02(s,1H),4.00–3.87(m,2H) ,3.52(s,7H),3.01(q,J=6.9Hz,2H),2.50(p,J=1.9Hz,4H),2.31(t,J=7.5Hz,2H),1.89(d,J=3.1Hz,3H),1.8 0(s,2H),1.65–1.54(m,12H),1.48(q,J=7.2Hz,2H),1.38(t,J=6.9Hz,2H),1.28(p,J=3.8Hz,4H).ESI-MS:m / z 797.28[M+H] + 799.31[M+2+H] + C 39 H 50 BrFN6O4S[796.28].

[0070] Example 6. Preparation of the target compound HyT-H6

[0071] Adamantaneacetic acid (58.8 mg, 0.303 mmol) and HATU (143.7 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7e (160.0 mg, 0.253 mmol) and DIEA (83.4 μL, 0.505 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H6 in 72% yield. Spectroscopic data: 1H NMR(600MHz,DMSO-d6)δ9.72(s,1H),8.01(d,J=3.1Hz,1H),7.95(d,J=3.1Hz,1H),7.63–7.59(m,1H),7.5 7(dd,J=8.5,2.7Hz,1H),7.38(dd,J=8.7,6.1Hz,1H),7.22(td,J=8.4,2.7Hz,1H),6.02(s,1H),4.00–3.8 8(m,2H),3.52(s,7H),3.01(q,J=6.9Hz,2H),2.51–2.50(m,4H),2.31(t,J=7.5Hz,2H),1.90(s,3H),1.80 (s,2H),1.66–1.54(m,12H),1.52–1.46(m,2H),1.38(q,J=6.9Hz,2H),1.27(d,J=4.5Hz,6H).ESI-MS:m / z 811.33[M+H] + 813.36[M+2+H] + C 40 H 52 BrFN6O4S[810.29].

[0072] Example 7. Preparation of the target compound HyT-H7

[0073] Adamantaneacetic acid (78.5 mg, 0.404 mmol) and HATU (192.0 mg, 0.505 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (200.0 mg, 0.337 mmol) and DIEA (111.0 μL, 0.673 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H7 in 78% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ9.70 (s, 1H), 8.00 (d, J = 3.1Hz, 1H), 7.94 (d, J = 3.1Hz, 1H), 7.77 (t, J = 5.7 Hz,1H),7.56(dd,J=8.6,2.7Hz,1H),7.38(dd,J=8.7,6.1Hz,1H),7.21(td,J=8.4,2.7Hz,1H),6. 02(s,1H),4.16(s,2H),4.00–3.87(m,2H),3.50(d,J=21.2Hz,7H),3.45(t,J=5.8Hz,2H),3.21(q ,J=5.7Hz,2H),2.51–2.50(m,4H),1.87(s,3H),1.81(s,2H),1.56(d,J=46.8Hz,12H).ESI-MS:m / z 771.23[M+H] + 773.26[M+2+H] + C 36 H 44 BrFN6O5S[770.23].

[0074] Example 8. Preparation of the target compound HyT-H8

[0075] Adamantaneacetic acid (73.1 mg, 0.376 mmol) and HATU (178.8 mg, 0.470 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7 g (200.0 mg, 0.313 mmol) and DIEA (104.0 μL, 0.627 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H8 in 75% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ9.71(s,1H),8.00(d,J=3.1Hz,1H),7.94(d,J=3.1Hz,1H),7.71–7.66( m,1H),7.56(dd,J=8.6,2.7Hz,1H),7.38(dd,J=8.7,6.1Hz,1H),7.21(td,J=8.4,2.7Hz,1H),6. 02(s,1H),4.17(s,2H),4.00–3.88(m,2H),3.59–3.45(m,11H),3.41(t,J=5.9Hz,2H),3.18(q, J=5.9Hz,2H),2.50(p,J=1.9Hz,4H),1.88(s,3H),1.82(s,2H),1.65–1.52(m,12H).ESI-MS:m / z 815.24[M+H] + 817.29[M+2+H] + C 38 H 48 BrFN6O6S[814.25].

[0076] Example 9. Preparation of the target compound HyT-H9

[0077] Adamantaneacetic acid (85.4 mg, 0.440 mmol) and HATU (209.0 mg, 0.550 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. I-7h (250.0 mg, 0.366 mmol) and DIEA (121.0 μL, 0.733 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H9 in 71% yield. Spectroscopic data: 1H NMR(600MHz,DMSO-d6)δ9.71(s,1H),8.00(d,J=3.2Hz,1H),7.94(d,J=3.1Hz,1H),7.69–7.65(m, 1H),7.56(dd,J=8.6,2.7Hz,1H),7.38(dd,J=8.8,6.1Hz,1H),7.21(td,J=8.3,2.7Hz,1H),6.02(s ,1H),4.17(s,2H),3.99–3.88(m,2H),3.56–3.48(m,15H),3.39(t,J=6.0Hz,2H),3.17(q,J=5.8H z,2H),2.51–2.49(m,4H),1.88(s,3H),1.81(s,2H),1.65–1.53(m,12H).ESI-MS:m / z859.29[M+H] + 861.30[M+2+H] + C 40 H 52 BrFN6O7S[858.28].

[0078] Example 10. Preparation of the target compound HyT-H10

[0079] 2,2-Diphenylacetic acid (64.00 mg, 0.302 mmol) and HATU (144.0 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (150.0 mg, 0.252 mmol) and DIEA (83.0 μL, 0.504 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H10 in 76% yield. Spectroscopic data: 1H NMR(600MHz,DMSO-d6)δ9.71(s,1H),8.43(t,J=5.5Hz,1H),8.01(d,J=3.1Hz,1H),7.95(d,J=3.1Hz,1H) ,7.58(dd,J=8.5,2.6Hz,1H),7.39(dd,J=8.7,6.2Hz,1H),7.33(d,J=4.3Hz,2H),7.29(d,J=6.9Hz,6H), 7.23–7.21(m,1H),7.21–7.19(m,2H),6.03(s,1H),4.95(s,1H),4.14(s,2H),3.98–3.86(m,2H),3.52(s ,3H),3.48(t,J=5.6Hz,2H),3.39(s,4H),3.28(q,J=5.5Hz,2H),2.46(m,J=10.5,5.3Hz,4H).ESI-MS:m / z 789.14[M+H] + 791.18[M+2+H] + C 38 H 38 BrFN6O5S[788.18].

[0080] Example 11. Preparation of the target compound HyT-H11

[0081] 9-Fluoreneacetic acid (67.8 mg, 0.302 mmol) and HATU (144.0 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (150.0 mg, 0.252 mmol) and DIEA (83.0 μL, 0.504 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H11 in 80% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ9.68(s,1H),8.15(t,J=5.2Hz,1H),7.97(d,J=3.1Hz,1H),7.92(d,J=3.1Hz,1H),7.83(t,J= 6.8Hz,2H),7.61–7.48(m,4H),7.35(ddd,J=11.4,6.8,3.9Hz,3H),7.31(dd,J=7.4,3.7Hz,1H),7.18(td,J=8.4,2.5H z,1H),6.00(s,1H),4.34(t,J=7.6Hz,1H),4.19(d,J=2.2Hz,2H),3.96–3.83(m,2H),3.53(t,J=5.4Hz,2H),3.47(s,3 H),3.44(s,2H),3.38(dd,J=13.2,7.7Hz,4H),2.53(d,J=6.7Hz,2H),2.49(d,J=6.1Hz,4H).ESI-MS:m / z801.18[M+H] + 803.19[M+2+H] + C 39 H 38 BrFN6O5S[800.18].

[0082] Example 12. Preparation of the target compound HyT-H12

[0083] i-8 (181.9 mg, 0.292 mmol) and HATU (138.9 mg, 0.365 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. Norbornene-2-methylamine (30.0 mg, 0.244 mmol) and DIEA (80.5 μL, 0.487 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to obtain a yellow solid compound HyT-H12, with a yield of 74%. Spectroscopic data: 1H NMR (400MHz, DMSO-d6) δ9.73(s,1H),8.02(d,J=3.1Hz,1H),7.95(d,J=3.1Hz,1H),7.78(t,J=5.4Hz,1H),7.58(dd,J=8.6,2.6H z,1H),7.38(dd,J=8.6,6.2Hz,1H),7.22(td,J=8.5,2.6Hz,1H),6.14(dd,J=5.5,2.9Hz,1H),6.02(s,1H),5.95(dd,J=5.5,2.8 Hz,1H),4.01–3.86(m,2H),3.68–3.54(m,2H),3.52(s,7H),3.14(ddt,J=15.3,7.2,3.9Hz,1H),2.75(d,J=11.4Hz,2H),2.55(d ,J=4.4Hz,2H),2.33(t,J=6.8Hz,2H),2.07(t,J=6.6Hz,2H),1.59–1.40(m,5H),1.27(dq,J=13.4,7.6,7.1Hz,5H).ESI-MS:m / z 727.22[M+H] + 729.20[M+2+H] + C 34 H 40 BrFN6O4S[726.20].

[0084] Example 13. Preparation of the target compound HyT-H13

[0085] (S)-(+)-2-(4-isobutylphenyl)propionic acid (62.4 mg, 0.302 mmol) and HATU (144.0 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (150.0 mg, 0.252 mmol) and DIEA (83.0 μL, 0.504 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H13 in 72% yield. Spectroscopic data: 1H NMR(600MHz, DMSO-d6)δ9.70(d,J=3.7Hz,1H),8.10–8.05(m,1H),8.01–7.97(m,1H),7.93(dd,J=3.0,1.2Hz,1H),7.55(dd,J= 8.5, 2.6Hz, 1H), 7.36 (dd, J=8.7, 6.2Hz, 1H), 7.17 (t, J=8.0Hz, 3H), 7.05–6.99 (m, 2H), 6.01 (s, 1H), 4.15–4.06 (m, 2H), 3.98–3 .85(m,2H),3.53(td,J=7.1,4.2Hz,2H),3.50(s,3H),3.42(dq,J=14.7,5.3,4.6Hz,4H),3.24–3.09(m,3H),2.50(d,J=13.8Hz, 4H), 2.34 (dd, J=7.1, 2.3Hz, 2H), 1.74 (dp, J=13.3, 6.7Hz, 1H), 1.26 (dd, J=7.0, 2.1Hz, 3H), 0.80 (d, J=6.6Hz, 6H). ESI-MS: m / z 783.20[M+H] + 785.20[M+2+H] + C 37 H 44 BrFN6O5S[782.23].

[0086] Example 14. Preparation of the target compound HyT-H14

[0087] (-)-Monthoxyacetic acid (64.8 mg, 0.302 mmol) and HATU (144.0 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (150.0 mg, 0.252 mmol) and DIEA (83.0 μL, 0.504 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H14, with a yield of 69%. Spectroscopic data: 1H NMR(600MHz,DMSO-d6)δ9.72(s,1H),8.01(d,J=3.1Hz,1H),7.95(d,J=3.1H z,1H),7.58(td,J=10.2,8.5,4.0Hz,2H),7.38(dd,J=8.7,6.2Hz,1H),7.21( td,J=8.4,2.6Hz,1H),6.02(s,1H),4.22–4.15(m,2H),4.01–3.88(m,3H),3. 79(d,J=14.8Hz,1H),3.52(s,3H),3.50(t,J=5.7Hz,3H),3.47(s,2H),3.34– 3.25(m,3H),3.11(tt,J=10.5,3.6Hz,1H),2.55(s,4H),2.18(ddt,J=14.1, 9.8,6.9Hz,1H),2.02(d,J=11.5Hz,1H),1.57(dd,J=31.0,12.5Hz,2H),1.35 –1.26(m,1H),1.20(tdd,J=14.3,9.6,6.7Hz,2H),0.95–0.89(m,1H),0.87–0 .83(m,6H),0.79(d,J=10.5Hz,1H),0.72(dd,J=6.9,4.5Hz,3H).ESI-MS:m / z 791.18[M+H] + 793.17 [M+H] + C 36 H 48 BrFN6O6S[790.25].

[0088] Example 15. Preparation of the target compound HyT-H15

[0089] Tri-tert-butoxycarbonylarginine (143.4 mg, 0.302 mmol) and HATU (144.0 mg, 0.378 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. i-7f (150.0 mg, 0.252 mmol) and DIEA (83.0 μL, 0.504 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a yellow solid compound HyT-H15, with a yield of 73%. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ11.48(s,1H),9.69(s,1H),8.24(t,J=5.4Hz,1H),7.98(d,J=3.1Hz,1H),7.96(d,J=5.2Hz,1H),7 .92(d,J=3.1Hz,1H),7.55(dd,J=8.5,2.4Hz,1H),7.35(t,J=7.4Hz,1H),7.19(t,J=7.4Hz,1H),6.84(d,J=8.1Hz,1H),5. 99(s,1H),4.15(s,2H),3.98–3.83(m,3H),3.52(d,J=12.8Hz,2H),3.49(s,3H),3.44(t,J=5.1Hz,4H),3.22(dqd,J=19.2 ,13.6,6.1Hz,4H),2.61–2.49(m,4H),1.61–1.52(m,1H),1.51–1.45(m,3H),1.44(s,9H),1.37–1.32(m,18H).ESI-MS:m / z 1051.04[M+H] + 1053.02[M+2+H] + C 45 H 64 BrFN 10 O 11 S[1050.36].

[0090] Example 16. Preparation of the target compound HyT-S1

[0091] Adamantaneacetic acid (81.1 mg, 0.417 mmol) and HATU (198.3 mg, 0.522 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-5 (150 mg, 0.348 mmol) and DIEA (114.9 μL, 0.695 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S1 in 73% yield. Spectroscopic data: 1H NMR (400MHz, DMSO-d6) δ10.77(s,1H),8.38–8.34(m,1H),8.34–8.29(m,1H),7.75–7.67(m,3H),7.64(d,J=7.5Hz,1H),3.72–3.62(m,1H),3.5 9(d,J=12.4Hz,2H),2.84(t,J=11.0Hz,2H),1.89(s,3H),1.83–1.78(m,2H),1.78(s,2H),1.67–1.50(m,12H),1.45–1.34(m,2H).ESI-MS:m / z 606.51[MH] - C 30 H 33 F4N3O4S[607.21].

[0092] Example 17. Preparation of the target compound HyT-S2

[0093] Adamantaneacetic acid (81.3 mg, 0.418 mmol) and HATU (199.0 mg, 0.523 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7a (180.0 mg, 0.349 mmol) and DIEA (115.0 μL, 0.697 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S2 in 67% yield. Spectroscopic data: 1 H NMR(400MHz,DMSO-d6)δ10.79(s,1H),8.38–8.34(m,1H),8.34–8.28(m,1H),7.80(d,J=7 .5Hz,1H),7.76–7.68(m,3H),7.65(t,J=5.5Hz,1H),3.71–3.63(m,1H),3.60(d,J=12.5H z,2H),2.96(q,J=6.6Hz,2H),2.82(t,J=11.0Hz,2H),2.01(q,J=6.8,6.1Hz,2H),1.89(s ,3H),1.85–1.79(m,2H),1.78(s,2H),1.74–1.48(m,14H),1.46–1.31(m,2H).ESI-MS:m / z 693.00[M+H] + C 34 H 40 F4N4O5S[692.27]

[0094] Example 18. Preparation of the target compound HyT-S3

[0095] Adamantaneacetic acid (87.9 mg, 0.452 mmol) and HATU (215.0 mg, 0.565 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7b (200.0 mg, 0.377 mmol) and DIEA (125.0 μL, 0.754 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S3 in 72% yield. Spectroscopic data: 1 H NMR (600MHz, DMSO-d6) δ10.78(s,1H),8.36(dd,J=6.5,2.1Hz,1H),8.32(ddd,J=8.1,4.2,2.3Hz,1H),7.75(d,J=7.6 Hz,1H),7.74–7.69(m,3H),7.61(t,J=5.4Hz,1H),3.66(ddt,J=14.1,10.6,5.8Hz,1H),3.61(d,J=12.4Hz,2H),2.98( q,J=6.8Hz,2H),2.82(t,J=11.0Hz,2H),2.02(t,J=7.3Hz,2H),1.89(s,3H),1.80(d,J=6.5Hz,4H),1.67–1.50(m,12 H),1.46(dt,J=15.2,7.4Hz,2H),1.40(dd,J=17.1,6.1Hz,2H),1.33(dt,J=14.4,7.3Hz,2H).ESI-MS:m / z705.51[MH] - C 35 H 42 F4N4O5S[706.28].

[0096] Example 19. Preparation of the target compound HyT-S4

[0097] Adamantaneacetic acid (85.6 mg, 0.441 mmol) and HATU (209.0 mg, 0.550 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7c (200.0 mg, 0.367 mmol) and DIEA (121.0 μL, 0.734 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S4 in 74% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.78(s,1H),8.35(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.2,4.3,2.3Hz,1H),7.75– 7.67(m,4H),7.59(t,J=5.4Hz,1H),3.65(dq,J=10.7,3.7Hz,1H),3.61(d,J=12.5Hz,2H),2.97(q,J=6.7Hz, 2H),2.81(t,J=11.0Hz,2H),2.00(t,J=7.4Hz,2H),1.89(s,3H),1.79(d,J=9.8Hz,4H),1.67–1.50(m,12H), 1.45(p,J=7.6Hz,2H),1.42–1.36(m,2H),1.36–1.31(m,2H),1.20(dq,J=15.0,6.9,6.5Hz,2H).ESI-MS:m / z 721.37[M+H] + 719.46 [MH] - C 36 H 44 F4N4O5S[720.30].

[0098] Example 20. Preparation of the target compound HyT-S5

[0099] Adamantaneacetic acid (83.5 mg, 0.430 mmol) and HATU (204.0 mg, 0.537 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7d (200.0 mg, 0.358 mmol) and DIEA (118.0 μL, 0.716 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S5 in 68% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.78(s,1H),8.36(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.4,4.4,2.3Hz,1H),7. 77–7.69(m,4H),7.59(t,J=5.4Hz,1H),3.69–3.63(m,1H),3.63–3.58(m,2H),2.98(q,J=6.8Hz,2H),2.82 (t,J=10.9Hz,2H),2.01(t,J=7.5Hz,2H),1.90(s,3H),1.80(d,J=4.3Hz,4H),1.67–1.51(m,12H),1.48– 1.42(m,2H),1.42–1.37(m,2H),1.36–1.31(m,2H),1.22(tt,J=10.9,4.4Hz,4H).ESI-MS:m / z733.66[MH] - C 37 H 46 F4N4O5S[734.31].

[0100] Example 21. Preparation of the target compound HyT-S6

[0101] Adamantaneacetic acid (81.4 mg, 0.419 mmol) and HATU (199.0 mg, 0.524 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7e (200.0 mg, 0.349 mmol) and DIEA (115.0 μL, 0.698 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S6 in 77% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.79(s,1H),8.36(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.4,4.3,2.3Hz,1H),7.76–7 .69(m,4H),7.59(t,J=5.4Hz,1H),3.66(ddt,J=10.1,6.5,3.7Hz,1H),3.61(d,J=12.5Hz,2H),2.99(q,J=6.7H z,2H),2.82(t,J=11.0Hz,2H),2.01(t,J=7.4Hz,2H),1.90(s,3H),1.79(d,J=5.9Hz,4H),1.68–1.51(m,12H), 1.45(dt,J=14.3,7.4Hz,2H),1.40(dd,J=17.3,6.6Hz,2H),1.37–1.32(m,2H),1.26–1.17(m,6H).ESI-MS:m / z 749.48[M+H] + 747.44 [MH] - C 38 H 48 F4N4O5S[748.33].

[0102] Example 22. Preparation of the target compound HyT-S7

[0103] Adamantaneacetic acid (65.7 mg, 0.338 mmol) and HATU (160.0 mg, 0.423 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7f (150.0 mg, 0.282 mmol) and DIEA (93.0 μL, 0.563 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S7 in 69% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.77(s,1H),8.36(dd,J=6.4,2.0Hz,1H),8.32(dq,J=5.8,2.1Hz,1H),7.76 (t,J=5.5Hz,1H),7.73–7.68(m,3H),7.65(d,J=8.0Hz,1H),3.82(s,2H),3.74(ddt,J=10.5,6.6,3.4H z,1H),3.68(d,J=12.4Hz,2H),3.41(t,J=5.6Hz,2H),3.21(q,J=5.6Hz,2H),2.77(t,J=11.6Hz,2H), 1.89(s,3H),1.82(s,2H),1.78(d,J=10.3Hz,2H),1.66–1.53(m,12H),1.53–1.48(m,2H).ESI-MS:m / z 709.19[M+H] + 707.54 [MH] - C 34 H 40 F4N4O6S[708.26].

[0104] Example 23. Preparation of the target compound HyT-S8

[0105] Adamantaneacetic acid (56.6 mg, 0.291 mmol) and HATU (138.0 mg, 0.364 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. Then, ii-7 g (140.0 mg, 0.243 mmol) and DIEA (80.3 μL, 0.486 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S8 in 71% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.77(s,1H),8.35(dd,J=6.5,2.1Hz,1H),8.32(ddd,J=8.1,4.2,2.3Hz,1H),7.71(dd ,J=10.0,7.0Hz,3H),7.67(dd,J=7.2,4.4Hz,1H),7.63(d,J=7.9Hz,1H),3.84(s,2H),3.72(ddt,J=14.3,6.7, 3.6Hz,1H),3.67(d,J=12.4Hz,2H),3.58–3.50(m,4H),3.40(t,J=5.9Hz,2H),3.17(q,J=5.8Hz,2H),2.76(t,J =11.6Hz,2H),1.89(s,3H),1.82(s,2H),1.81–1.76(m,2H),1.66–1.53(m,12H),1.53–1.48(m,2H).ESI-MS:m / z 753.17[M+H] + 751.62 [MH] - C 36 H 44 F4N4O7S[752.29].

[0106] Example 24. Preparation of the target compound HyT-S9

[0107] Adamantaneacetic acid (56.3 mg, 0.290 mmol) and HATU (138.0 mg, 0.363 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. Then, ii-7h (150.0 mg, 0.242 mmol) and DIEA (80.0 μL, 0.483 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S9 in 70% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.77(s,1H),8.35(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.2,4.3,2.3Hz,1H),7.71(dd,J=10.4 ,7.5Hz,3H),7.66(t,J=5.5Hz,1H),7.61(d,J=8.0Hz,1H),3.85(s,2H),3.72(ddt,J=15.0,7.7,4.1Hz,1H),3.67(d,J= 12.3Hz,2H),3.54(s,4H),3.51(qd,J=7.6,5.1Hz,4H),3.39(t,J=5.9Hz,2H),3.17(q,J=5.8Hz,2H),2.77(t,J=11.5Hz ,2H),1.89(s,3H),1.80(d,J=17.1Hz,4H),1.66–1.53(m,12H),1.50(dd,J=11.6,8.7Hz,2H).ESI-MS:m / z797.25[M+H] + 795.62 [MH] - C 38 H 48 F4N4O8S[796.31].

[0108] Example 25. Preparation of the target compound HyT-S10

[0109] 2,2-Diphenylacetic acid (71.7 mg, 0.338 mmol) and HATU (161.0 mg, 0.423 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7f (150.0 mg, 0.282 mmol) and DIEA (93.0 μL, 0.563 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S10 in 71% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.81(s,1H),8.43(t,J=5.5Hz,1H),8.36(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.1 ,4.2,2.3Hz,1H),7.71(dd,J=10.0,6.8Hz,3H),7.67(d,J=8.1Hz,1H),7.30(d,J=4.2Hz,8H),7.22(dq,J=8. 7,4.2Hz,2H),4.96(s,1H),3.82(s,2H),3.73–3.69(m,1H),3.67(d,J=12.4Hz,2H),3.45(t,J=5.5Hz,2H), 3.29(q,J=5.5Hz,2H),2.72(t,J=11.7Hz,2H),1.76–1.69(m,2H),1.47(qd,J=12.5,4.0Hz,2H).ESI-MS:m / z 727.14[M+H] + 725.43 [MH] - C 37 H 34 F4N4O6S[726.21].

[0110] Example 26. Preparation of the target compound HyT-S11

[0111] 9-Fluoreneacetic acid (75.8 mg, 0.338 mmol) and HATU (161.0 mg, 0.423 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7f (150.0 mg, 0.282 mmol) and DIEA (93.0 μL, 0.563 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S11 in 67% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.80(s,1H),8.35(dd,J=6.5,2.1Hz,1H),8.31(ddd,J=8.3,4.3,2.3Hz,1H),8.16(t,J=5.5Hz,1H ),7.87(d,J=7.5Hz,2H),7.71(dd,J=9.6,6.2Hz,3H),7.68(d,J=9.4Hz,1H),7.53(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7 .32–7.27(m,2H),4.36(t,J=7.7Hz,1H),3.88(s,2H),3.73–3.67(m,1H),3.64(d,J=12.4Hz,2H),3.52(t,J=5.4Hz,2H),3.4 1–3.39(m,2H),2.72(t,J=11.9Hz,2H),2.53(d,J=7.8Hz,2H),1.76–1.68(m,2H),1.47(qd,J=12.5,3.9Hz,2H).ESI-MS:m / z 739.12[M+H] + 737.47 [MH] - C 37 H 34 F4N4O6S[738.21].

[0112] Example 27. Preparation of the target compound HyT-S12

[0113] ii-8 (174.4 mg, 0.312 mmol) and HATU (148.1 mg, 0.390 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. Norbornene-2-methylamine (32.0 mg, 0.260 mmol) and DIEA (85.9 μL, 0.519 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S12 in 65% yield. Spectroscopic data: 1H NMR (400MHz, DMSO-d6) δ10.98(s,1H),8.40(dd,J=5.5,3.1Hz,1H),8.36(d,J=6.7Hz,1H),7.83 (d,J=7.5Hz,1H),7.77(dd,J=10.4,6.4Hz,3H),7.74–7.68(m,1H),6.13(dd,J=5.5,2.9Hz,1H), 5.94(dd,J=5.5,2.7Hz,1H),3.72–3.64(m,1H),3.61(d,J=12.5Hz,2H),2.87–2.70(m,5H),2.61 (ddd,J=13.1,8.9,5.6Hz,1H),2.02(s,4H),1.83–1.69(m,3H),1.47–1.15(m,10H).ESI-MS:m / z 665.17[M+H] + 663.44 [MH] - C 32 H 36 F4N4O5S[664.23].

[0114] Example 28. Preparation of the target compound HyT-S13

[0115] (S)-(+)-2-(4-isobutylphenyl)propionic acid (69.7 mg, 0.338 mmol) and HATU (161.0 mg, 0.423 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7f (150.0 mg, 0.282 mmol) and DIEA (93.0 μL, 0.563 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S13 in 68% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.81(s,1H),8.36(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.3,4.3,2.3Hz,1H),8.08(t,J=5.6Hz,1H) ,7.75–7.66(m,4H),7.20(d,J=8.0Hz,2H),7.06(d,J=8.0Hz,2H),3.80(s,2H),3.76–3.71(m,1H),3.71–3.66(m,2H),3.56( q,J=7.0Hz,1H),3.40(dt,J=12.1,5.1Hz,2H),3.28–3.16(m,2H),2.75(t,J=11.8Hz,2H),2.38(d,J=7.1Hz,2H),1.82–1.78 (m,1H),1.76(dd,J=8.1,5.0Hz,2H),1.51(q,J=10.1,9.2Hz,2H),1.30(d,J=7.0Hz,3H),0.84(d,J=6.6Hz,6H).ESI-MS:m / z 719.54[MH] - C 35 H 40 F4N4O6S[720.26].

[0116] Example 29. Preparation of the target compound HyT-S14

[0117] (-)-Monthoxyacetic acid (72.4 mg, 0.338 mmol) and HATU (161.0 mg, 0.423 mmol) were dissolved in dichloromethane (10 mL), and the mixture was in an ice bath for 30 minutes. ii-7f (150.0 mg, 0.282 mmol) and DIEA (93.0 μL, 0.563 mmol) were added, and the mixture was stirred overnight at room temperature. The reaction solution was extracted with water, and the organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S14 in 69% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ10.81(s,1H),8.36(dd,J=6.5,2.2Hz,1H),8.32(ddd,J=8.3,4.3,2.3Hz,1H),7.75–7.69(m,4H),7.64(t,J=5.8Hz,1H ),4.05–3.88(m,2H),3.82(s,2H),3.80(d,J=14.9Hz,1H),3.73(ddt,J=10.6,6.7,3.7Hz,1H),3.68(d,J=12.3Hz,2H),3.45(h,J=5.4,5.0Hz, 2H),3.33–3.26(m,2H),3.12(td,J=10.5,4.0Hz,2H),2.76(t,J=11.5Hz,2H),2.17(ddq,J=9.6,6.9,3.4,2.6Hz,1H),2.05–1.97(m,1H),1.82 –1.75(m,2H),1.60(d,J=11.8Hz,1H),1.55(dd,J=11.3,4.9Hz,2H),1.27–1.23(m,4H),0.87–0.85(m,6H),0.73(d,J=6.9Hz,3H).ESI-MS:m / z 727.57[MH] - C 34 H 44 F4N4O7S[728.29].

[0118] Example 30. Preparation of the target compound HyT-S15

[0119] ii-7f (130 mg, 0.244 mmol), tri-tert-butoxycarbonylarginine (115.9 mg, 0.244 mmol), and NMI (58.3 μL, 0.732 mmol) were dissolved in acetonitrile (10 mL), stirred at room temperature for 10 minutes, and then TCFH (75.3 mg, 0.268 mmol) was added. The mixture was stirred overnight at room temperature. The reaction solution was evaporated, and the mixture was extracted with ethyl acetate and water. The organic phase extract was dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure, and the crude product was separated by column chromatography to give a white solid compound HyT-S15 in 65% yield. Spectroscopic data: 1H NMR (600MHz, DMSO-d6) δ11.46(s,1H),10.78(s,1H),8.33(dd,J=6.4,2.0Hz,1H),8.29(ddd,J=8.0,4.2,2.3Hz,1H),8.24(t,J=5.5Hz, 1H),7.95(t,J=5.3Hz,1H),7.72–7.65(m,4H),6.85(d,J=8.0Hz,1H),3.89–3.83(m,1H),3.80(s,2H),3.71(dd,J=11.2,3.5Hz,1H),3.6 7(d,J=11.9Hz,2H),3.43–3.37(m,2H),3.28(dt,J=11.0,5.8Hz,1H),3.25–3.19(m,2H),3.17(dd,J=13.5,5.9Hz,1H),2.72(t,J=11.6H z,2H),1.76(d,J=12.3Hz,2H),1.51(dd,J=25.7,14.1Hz,4H),1.44(s,11H),1.35(d,J=5.9Hz,16H),1.32(d,J=8.5Hz,2H).ESI-MS:m / z 987.26[M+H] - C 43 H 60 F4N8O 12 S[988.40].

[0120] Example 31. In vitro anti-HBV activity assay of the target compound (HepAD38 cells)

[0121] Assay Principle: HepAD38 cells are a stable cell line obtained by transfecting HepG2 cells (human hepatocellular carcinoma cell line) with the ptetHBV plasmid, formed by ligating the tetracycline-sensitive cytomegalovirus promoter (CMV-tet) with the cDNA of ayw subtype HBV pgRNA. The HepAD38 cell genome stably integrates the HBV gene, whose expression is regulated by tetracycline. Removal of tetracycline immediately leads to pgRNA expression, and HBV replication rapidly recovers, making it suitable for antiviral drug screening. Adding the test compound to a tetracycline-free culture medium downregulates intracellular HBV DNA levels. The intracellular HBV DNA content is measured by quantitative real-time polymerase chain reaction (qPCR) to study the inhibitory effect of the compound on HBV replication. The cytotoxicity of the compound is assessed using the cytopathic effect (CPE) observation method.

[0122] Experimental methods:

[0123] Cytotoxicity assay: The test compound was serially diluted 3-fold with complete culture medium, starting at 100 μM or 60 μM, to establish a total of 8 concentrations. HepAD38 cells in the exponential growth phase were seeded in 96-well plates, and the test compound was added. The culture medium containing the compound was replaced after 3 days. After 6 days, the growth status of each group of cells was observed using an inverted microscope. Cells with no change in morphology or all cells were marked as non-toxic, and the maximum non-toxic concentration (CC0) was determined. The half-maximal cytotoxic concentration (MCC) was calculated using the Reed-Muench method. 50 ).

[0124] In vitro anti-HBV activity assay: The test compound was serially diluted 3-fold starting from the maximum non-toxic concentration, resulting in 4–7 concentrations. HepAD38 cells were cultured for 24 hours in Dulbecco modified Eagle medium (DMEM) (Gibco) containing 1 μg / mL tetracycline (Sigma). Cells were then cultured in medium containing different concentrations of the compound without tetracycline, with the medium changed every 3 days. After 6 days, cells were lysed with a buffer containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 1% NP-40, and intracellular HBV core DNA was extracted. The intracellular HBV core DNA content was detected by qPCR using TransStart Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.). The positive primer was 5'-GGCTTTCGCGAAAATTCCTTG-3', and the negative primer was 5'-AGCCTACGAACCCACTGAAC-3'. The inhibition rate was calculated, and finally the half-maximal effective concentration (EC50) was calculated using the Reed-Muench method. 50 ).

[0125] The results of the target compound's inhibition of HBV DNA replication and cytotoxicity are shown in Table 1. A represents EC50. 50 <0.10μM, B represents 0.10μM≤EC 50 <0.30μM, where C represents 0.30μM≤EC 50 <0.50μM, D represents EC 50 ≥0.50μM.

[0126] Table 1. Results of the target compound's inhibition of HBV DNA replication and cytotoxicity.

[0127]

[0128] ND: Not determined.

[0129] Experimental Conclusions and Analysis: As shown in Table 1, the HBV core protein degraders designed based on hydrophobic tags in this invention exhibit significant anti-HBV activity. The dihydropyrimidine-based HBV core protein degraders showed activity at the submicromolar to nanomolar levels, with compounds HyT-H2, HyT-H3, HyT-H4, HyT-H7, HyT-H9, and HyT-H12 exhibiting activity comparable to the positive control GLS4. Several sulfonylbenzamide-based HBV core protein degraders, such as compounds HyT-S10, HyT-S11, HyT-S12, and HyT-S14, showed anti-HBV activity comparable to the positive control NVR3-778. These compounds warrant further investigation.

[0130] Example 32. Verification of the anti-HBV mechanism of the target compound

[0131] Test Principle: Western blotting uses specific antibodies as probes to identify and semi-quantitatively analyze a specific protein in total protein extracted from cells through an antigen-antibody immunoreaction. Proteins separated by polyacrylamide gel electrophoresis (PAGE) are transferred to a fixed support. The proteins on the fixed support act as antigens, reacting with a primary antibody, followed by an enzyme-labeled secondary antibody. Finally, the protein composition or expression level is detected through substrate color development.

[0132] Test method: The test compound was diluted to the corresponding concentration and applied to HepAD38 cells. After 4 days, the cells were lysed, and total cell protein was extracted. Then, Western blotting was performed to detect the content of core protein and internal reference protein β-actin.

[0133] Western blotting results showed that ( Figure 1 Compared with the viral control group (tet-), HyT-H2, HyT-H3, HyT-H4, HyT-H5, HyT-H7, and HyT-H12 significantly reduced the content of HBV core protein at a concentration of 0.4 μM; HyT-S7 and HyT-S9 reduced the content of HBV core protein in a dose-dependent manner. Therefore, this invention provides a new direction for the development of anti-HBV drugs.

Claims

1. An HBV core protein degrading agent based on hydrophobic tag technology, characterized in that, The compound having the structure shown in formula (I) below, or a pharmaceutically acceptable salt thereof: Where X is a linker and Y is a hydrophobic group; The linkers are selected from compounds with the following general structural formula: The hydrophobic group is selected from compounds with general structural formulas such as a to g:

2. The HBV core protein degrader based on hydrophobic tag technology as described in claim 1, characterized in that, It is one of the compounds having the following structures:

3. The preparation method of the HBV core protein degrader based on hydrophobic tag technology as described in claim 2, characterized in that, The synthesis route is as follows: Synthesis of the target product HyT-H (2-9): Starting with 2-thiazolylformamidin hydrochloride (i-1), 2-bromo-4-fluorobenzaldehyde, and ethyl acetoacetate, the key intermediate i-2 was obtained via a Biginelli reaction cyclization. In dichloromethane solution, intermediate i-2 underwent a bromination reaction with N-bromosuccinimide to yield intermediate i-3. Using i-3 as a starting material, potassium carbonate, potassium iodide, and 1-Boc piperazine were added, and the mixture was refluxed in acetonitrile solution at 75°C for 1 hour to obtain intermediate i-4. i-4 was then subjected to a three-phase reaction... The Boc group was removed under the action of fluoroacetic acid to obtain intermediate i-5; N-tert-butoxycarbonyl-R1-carboxylic acid and HATU were reacted in an ice-water bath for half an hour, and intermediate i-5 and DIEA were added and stirred at room temperature overnight to obtain intermediate i-6(ah); the Boc group was removed under the action of trifluoroacetic acid to obtain intermediate i-7(ah); adamantaneacetic acid and HATU were reacted in an ice-water bath for half an hour, and i-7(ah) and DIEA were added and stirred at room temperature overnight to obtain the target product HyT-H(2-9); Reagents and conditions: (i) N-tert-butoxycarbonyl-R1-carboxylic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) trifluoroacetic acid, dichloromethane, 0℃ → room temperature; (iii) adamantaneacetic acid, HATU, DIEA, dichloromethane, 0℃ → room temperature; Synthesis of the target product HyT-H(10-11,13-15): Carboxylic acid compounds containing different hydrophobic tags and HATU were reacted in an ice-water bath for half an hour. Intermediate i-7f and DIEA were added and stirred at room temperature overnight to obtain the target product HyT-H(10-11,13-15). Reagents and conditions: (i) R2-OH, HATU, DIEA, dichloromethane, 0℃ → room temperature; Synthesis of the target product HyT-H12: Ethyl adipic acid and HATU were reacted in an ice-water bath for half an hour, and intermediate i-5 and DIEA were added and stirred at room temperature overnight to obtain intermediate i-8; intermediate i-8 was hydrolyzed with lithium hydroxide to remove the ester group to obtain intermediate i-9; intermediate i-9 and HATU were reacted in an ice-water bath for half an hour, and norbornene-2-methylamine and DIEA were added and stirred at room temperature overnight to obtain the target product HyT-H12; Reagents and conditions: (i) Monoethyl adipate, HATU, DIEA, dichloromethane, 0℃ → room temperature; (ii) Lithium hydroxide, tetrahydrofuran: water, room temperature; (iii) Norbornene-2-methylamine, HATU, DIEA, dichloromethane, 0℃ → room temperature.

4. The application of the HBV core protein degrader based on hydrophobic tag technology as described in any one of claims 1-2 in the preparation of anti-HBV drugs.

5. An anti-HBV pharmaceutical composition comprising an HBV core protein degrader based on hydrophobic tagging technology as described in any one of claims 1-2 and one or more pharmaceutically acceptable carriers or excipients.

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