Novel compounds and pharmaceutical compositions

Abstract

This invention provides a novel compound that can effectively prevent the proliferation of drug-resistant hepatitis B viruses. [Solution] A hydrophobic tag and morphothiazine, which is an allosteric modulator of the hepatitis B virus core protein, are linked by a linker. The ring-type carbocyclic is any of the following: adamantane ring, norbornene ring, norbornane ring, perhydroindene ring, decalin ring, perhydrofluorene ring, perhydroanthracene ring, perhydrophenanthrene ring, tricyclodecane ring, tricycloundecane ring, tetracyclododecane ring, perhydroacenaphthene ring, or perhydrophenalene ring. The novel compound according to the present invention can effectively suppress the proliferation of drug-resistant HBV.

Description

[Technical Field]

[0001] This invention relates to a novel compound and a pharmaceutical composition that can be used to treat hepatitis B virus (HBV) using the novel compound. [Background technology]

[0002] Hepatitis B virus (HBV) is an enveloped virus with incomplete double-stranded DNA belonging to the Hepadnaviridae family. It causes acute and chronic hepatitis (hepatitis B) and can also cause liver cancer. Worldwide, there are estimated to be 200 to 400 million infected individuals, and one million people die each year from HBV-related liver disease.

[0003] When HBV enters a liver cell, a nucleotide capsid is released from the envelope and translocates to the cell nucleus. The HBV genome that has migrated into the nucleus of an infected liver cell is converted to cccDNA (covalently closed circular DNA) and remains in that form for a long period of time. cccDNA serves as a source of viral replication in transient asymptomatic infections and in previously infected individuals, and can cause reactivation. Furthermore, because HBV replication occurs through the reverse transcription of viral DNA polymerase using pregenome RNA synthesized using the host cell's RNA polymerase as a template, it is more prone to mutation than typical DNA viruses, and the frequency of treatment-resistant strains is high (Non-patent Literature 1, 2).

[0004] In Japan, antiviral therapy for persistent hepatitis B virus (HBV) infection mainly involves interferon (IFN) and nucleoside analogs (such as entecavir and tenofovir disoproxil fumarate). While these treatments achieve viral DNA negativity and suppression of hepatitis, viral antigens still remain, making it difficult to completely prevent the progression of liver cancer. Therefore, the development of new treatment methods is needed.

[0005] Currently, in addition to IFN and nucleic acid analogs, various therapeutic drug candidates are being developed. Representative antiviral drugs targeting HBV infection and replication include (1) entry inhibitors, (2) HBV RNA-targeting drugs, (3) core protein allosteric modulators, (4) HBs antigen release inhibitors, (5) FXR agonists, etc. (Non-Patent Documents 3, 4, 5).

[0006] Among them, Morphothiadin (GLS4), which is one of the core protein allosteric modulators, is undergoing a Phase II clinical trial in China. By directly acting on the core protein of HBV and inhibiting its function, it disrupts the viral replication cycle. In addition, GLS4 is known to prevent viral spread by inhibiting the formation of infectious HBV particles. Administration of GLS4 significantly reduces the HBV RNA level and limits the continuous replication of the virus. However, similar to other antiviral drugs, the emergence of resistant viruses against GLS4 has become an issue.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

[0008] This invention has been made in view of the aforementioned problems, and aims to provide a novel compound that can effectively prevent the proliferation of drug-resistant hepatitis B viruses. [Means for solving the problem]

[0009] The novel compound according to the present invention is a novel compound in which a hydrophobic tag and a morphothiazine, which is an allosteric modulator of the hepatitis B virus core protein, are linked by a linker. [Effects of the Invention]

[0010] According to the present invention, even drug-resistant hepatitis B viruses can be effectively prevented from multiplying. Compared to ordinary DNA viruses, hepatitis B viruses have a higher frequency of developing resistant strains, and when drug-resistant strains appear, it can be difficult to make clinical decisions on whether to switch to another drug or choose combination therapy with drugs that do not have cross-resistance. According to the present invention, even drug-resistant hepatitis B viruses can be effectively used for treatment, and the benefits obtained from the present invention are immeasurable. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the 1H-NMR and 13C-NMR spectra of compound 1. [Figure 2] This figure shows the 1H-NMR and 13C-NMR spectra of compound 2. [Figure 3] This figure shows the 1H-NMR, 13C-NMR spectra, and analytical RP-HPLC of GLS4-C3-Ad. [Figure 4] This figure shows the 1H-NMR, 13C-NMR spectra, and analytical RP-HPLC of GLS4-C3-Nor. [Figure 5] This figure shows the 1H-NMR, 13C-NMR spectra, and analytical RP-HPLC of GLS4-C3-FI. [Figure 6] This figure shows the 1H-NMR, 13C-NMR spectra, and analytical RP-HPLC of GLS4-C3-Boc. [Figure 7] This figure shows the inhibitory activity evaluation of GLS4-C3-Ad and GLS4-C3-Nor. [Figure 8] This figure shows the inhibitory activity evaluation of GLS4-C3-FI and GLS4-C3-Boc. [Figure 9] This figure shows the evaluation of the inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor using GLS4-resistant HBV strains. [Modes for carrying out the invention]

[0012] The embodiments of the present invention will be described in detail below with reference to the attached drawings. However, these embodiments are provided to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiments described below. Other embodiments in which those skilled in the art appropriately substitute the configurations of the embodiments below are also included in the scope of the present invention.

[0013] The inventors have designed a novel compound that, by appropriately chemically modifying GLS4, effectively degrades the HBV core protein not only in wild-type viruses but also in GLS4-resistant viruses. This compound could serve as an important seed for the development of new HBV therapeutics.

[0014] In other words, the novel compound according to the present invention is a novel compound in which a hydrophobic tag and a morphothiazine, which is an allosteric modulator of the hepatitis B virus core protein, are linked by a linker.

[0015] Here, the hydrophobic tag is not particularly limited, but for example, it is a cyclic carbon ring with 2 to 6 rings.

[0016] Furthermore, a cyclic carbocyclic ring with 2 to 6 rings is preferred to have a bridged structure. The 2 to 6-ring bridged cyclic carbocyclic ring is not particularly limited, but examples include any of the following: adamantane ring, norbornene ring, norbornane ring, perhydroindene ring, decalin ring, perhydrofluorene ring, perhydroanthracene ring, perhydrophenanthrene ring, tricyclodecane ring, tricycloundecane ring, tetracyclododecane ring, perhydroacenaphthene ring, or perhydrophenalene ring.

[0017] Preferably, the 2-6 ring bridging carbocyclic rings are adamantane rings as shown below. The adamantane rings are preferably bonded to a linker at the carbon atom at position 1.

[0018] [ka]

[0019] Furthermore, preferably, the 2-6 ring bridging carbocyclic rings are norbornene rings as shown below. The norbornene rings are preferably bonded to the linker at the 5th or 6th carbon.

[0020] [ka]

[0021] A cyclic carbocyclic ring with 2 to 6 rings may also have a structure without crosslinking. A cyclic carbocyclic ring without crosslinking with 2 to 6 rings is not particularly limited, but examples include a fluorene ring, biphenyl ring, terphenyl ring, naphthalene ring, anthracene ring, or dihydroanthracene ring.

[0022] Preferably, the cyclic carbocyclic ring without 2-6 ring crosslinks is the fluorene ring shown below. The fluorene ring is preferably bonded to the linker at the 9th carbon.

[0023] [ka]

[0024] The linker is not particularly limited as long as it can connect the hydrophobic tag to morphothiazine, which is an allosteric modulator of the hepatitis B virus core protein. Examples include polyethylene glycol linkers and alkyl linkers, with alkyl linkers being preferred.

[0025] Alkyl linkers are -L 1 -(CH2) n -L 2 - This is shown by n being a natural number from 1 to 6. 1 L is a chemical bond, either -CO-, -NHCO-, or -CH2-NHCO-. 2 This is a chemical bond, either -CO-, -CONH-, or -CONH--CH2-.

[0026] The alkyl linker is preferably -CH2-NHCO-CH2-CH2-CH2-CO-.

[0027] The novel compound according to the present invention is GLS4-C3-Ad, represented by the following formula.

[0028] [ka]

[0029] Furthermore, the novel compound according to the present invention is GLS4-C3-Nor, represented by the following formula.

[0030] [ka]

[0031] Furthermore, the novel compound according to the present invention is GLS4-C3-FI, represented by the following formula.

[0032] [ka]

[0033] Next, the synthesis schemes for the novel compounds GLS4-C3-Ad and GLS4-C3-Nor according to the present invention will be described.

[0034] Specifically, thiazole-2-carboxyimidamide hydrochloride, ethyl 3-oxobutanoate, 2-bromo-4-fluorobenzaldehyde, and NaOAc are refluxed in ethanol for 24 hours. After cooling to room temperature, insoluble matter is removed by filtration, and the filtrate is concentrated under reduced pressure. The crude product is dissolved in ethanol solvent containing 10-20% dichloromethane and heated at 90°C until the dichloromethane evaporates. After cooling the resulting solution to 0°C, the precipitated product is filtered to obtain compound 1.

[0035] [ka]

[0036] Next, a mixed solution of compound 1, N-bromosuccinimide, and carbon tetrachloride was stirred at 60 °C for 2 hours. After the reaction, the mixture was cooled to room temperature, insoluble matter was filtered off with carbon tetrachloride, and the filtrate was concentrated under reduced pressure. The residue was mixed with piperazine and ethanol and stirred at room temperature for 24 hours. After the reaction, the reaction mixture was concentrated under vacuum and purified by NH-silica gel column chromatography to obtain compound 2.

[0037] [ka]

[0038] Next, to synthesize GLS4-C3-Ad, glutaric anhydride is added to a mixed solution of compound 2, N,N-diisopropylethylamine, and DMF, and the mixture is stirred at room temperature for 1 hour. Then, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 3H-[1,2,3]triazolo[4,5-b]pyridinium-3-ol (HOAt,), DMF, and 1-adamantanemethylamine are added, and the mixture is stirred at room temperature for 20 hours. After that, the reaction mixture is concentrated under vacuum and purified by NH-silica gel column chromatography and preparative HPLC to obtain GLS4-C3-Ad.

[0039] To synthesize GLS4-C3-Nor, glutaric anhydride is added to a mixed solution of compound 2, N,N-diisopropylethylamine, and DMF, and the mixture is stirred at room temperature for 1 hour. Next, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), 3H-[1,2,3]triazolo[4,5-b]pyridinium-3-ol (HOAt), DMF, and 1-5-norbornene-2-methylamine are added, and the mixture is stirred at room temperature for 20 hours. The reaction mixture is then concentrated under vacuum and purified by NH-silica gel column chromatography and preparative HPLC to obtain GLS4-C3-Nor.

[0040] [ka]

[0041] Next, the synthesis scheme for the novel compound GLS4-C3-FI according to the present invention will be described.

[0042] A mixed solution of 2-(9H-fluoren-9-yl)acetic acid, ethyl 4-aminobutyrate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 1-hydroxybenzotriazole, N,N-diisopropylethylamine, and DMF was stirred at room temperature for 4 hours. After adding water and extracting with ethyl acetate, the mixture was washed with saturated saline solution and concentrated under vacuum. Sodium hydroxide aqueous solution and tetrahydrofuran were added to the resulting solid and stirred overnight. Hydrochloric acid was then added, and the mixture was extracted with ethyl acetate. After dehydration with sodium sulfate, the mixture was filtered, and the resulting filtrate was concentrated under vacuum. The resulting solid was mixed with two drops of the aforementioned compound 2, O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxybenzotriazole, and N,N-diisopropylethylamine, and DMF. The mixture was stirred overnight, water was added, and the resulting solution was purified by preparative HPLC to obtain GLS4-C3-FI.

[0043] [ka]

[0044] The pharmaceutical composition according to the present invention is a pharmaceutical composition for the treatment or prevention of diseases caused by infection with the hepatitis B virus, characterized in that it contains a novel compound according to the present invention as an active ingredient.

[0045] In this specification, "prevention" includes suppressing and delaying the onset of a disease, encompassing not only prevention before the disease develops but also prevention of recurrence after treatment. On the other hand, "treatment" includes curing symptoms, improving symptoms, and slowing the progression of symptoms.

[0046] The pharmaceutical composition according to the present invention can be formulated and administered as a drug according to known methods. For example, it can be administered orally or parenterally as a liquid or as a drug in a suitable dosage form. The dosage is not particularly limited, but can be, for example, 0.01 mg / kg to 50 mg / kg.

[0047] The drug may contain preservatives that inhibit microbial growth or buffering agents that help maintain an acceptable pH. Examples of preservatives include sodium azide, octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. Examples of buffering agents include phosphoric acid, citric acid, and other organic acids.

[0048] Furthermore, the drug may contain, for example, excipients, stabilizers, chelating agents such as EDTA, salts, or antibacterial agents. Other possible ingredients include antioxidants such as ascorbic acid and methionine, proteins such as polypeptides, serum albumin, gelatin, or nonspecific immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides such as glucose, mannose, or dextrin, disaccharides, and other carbohydrates, and sugars such as sucrose, mannitol, trehalose, or sorbitol.

[0049] The pharmaceutical composition according to the present invention is applicable not only to wild-type hepatitis B viruses but also to drug-resistant hepatitis B viruses.

[0050] Drug-resistant hepatitis B viruses are, but are not limited to, hepatitis B viruses having any of the following mutations in the drug-binding pocket: T33N, D29G, T109I, T109M, or Y118F. Preferably, the drug-resistant hepatitis B virus is T33N. [Examples]

[0051] (1) Synthesis of GLS4-C3-Ad GLS4-C3-Ad, an embodiment of the present invention, was synthesized using the following procedure.

[0052] (1-1) Experimental method The reagents were purchased from Sigma-Aldrich Co. LLC, Kanto Chemicals Co. Inc., Tokyo Chemical Industry Co. Ltd., and Wako Pure Chemical Industries Ltd. and used as received without purification. 1 H, 13 C, and 19 1H, 13C, and 19F NMR were measured using an ECZ600 (JEOL). 1 1H and 13 13C-NMR chemical shift values (δ, ppm) were corrected using the residual solvent signals (DMSO-d6: 2.50 for 1 1H NMR, 39.5 for 13 13C NMR; CD3OD: 3.30 for 1 1H NMR, 49.0 for 13 13C NMR; CDCl3: 7.26 for 1 1H NMR, 77.2 for 13 13C NMR). 19 19F NMR spectra were corrected with an external standard (α,α,α-trifluorotoluene, δ 63.7). High-resolution mass spectrometry (HRMS) was measured using a Shimadzu IT-TOF MS (Shimadzu Corporation). Analytical HPLC was performed using Inertsil WP300 C18, 5 μm, 4.6 mm x 250 mm, solvent A: 0.1% TFA / water, solvent B: 0.1% TFA / MeCN, gradient: 10-90% gradient of solvent B over 30 min, flow rate: 2 mL / min, at 40°C.

[0053] (1-2) Synthesis of Compound 1

[0054]

Chemical formula

[0055] Thiazole-2-carboxyimidamide hydrochloride (1.63 g, 10 mmol), ethyl 3-oxobutanoate (1.26 mL, 10 mL), 2-bromo-4-fluorobenzaldehyde (2.03 g, 10 mmol), and NaOAc (0.984 g, 12 mmol) were refluxed in ethanol (100 mL) for 24 hours. After cooling to room temperature, insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The crude product was dissolved in ethanol solvent containing 10-20% dichloromethane and heated at 90°C for 1-2 hours until the dichloromethane evaporated. After cooling the resulting solution to 0°C, the precipitated product was filtered to obtain compound 1, a yellow crystal (3.0 g, 70%). Measurement values ​​are reported values. [1] This was consistent with Figure 1 of compound 1. 1 H-NMR and, 13 This figure shows the 1C-NMR spectrum. 1 H-NMR (600 MHz, DMSO-d6) δ 9.94 (s, 1H), 7.97 (d, J = 3.1 Hz, 1H), 7.90 (d, J = 3.1 Hz, 1H), 7.55 (dd, J = 8.6, 2.6 Hz, 1H), 7.34 (dd, J = 8.6, 6.2 Hz, 1H), 7.23 (td, J = 8.5, 2.6 Hz, 1H), 5.96 (s, 1H), 3.92 (q, J = 7.0 Hz, 2H), 2.46 (s, 3H), 1.02 (t, J = 7.1 Hz, 3H); 13C-NMR (151 MHz, DMSO-d6) δ 165.6, 162.7, 160.7 (d, J = 248.2 Hz), 147.6, 144.3, 143.3, 140.7 (d, J = 2.9 Hz),130.7 (d, J = 8.7 Hz), 124.5, 122.5 (d, J = 9.8 Hz), 119.6 (d, J = 24.6 Hz), 115.4 (d, J = 21.0 Hz), 96.8, 59.1, 57.6, 17.4, 14.0. ESI-HRMS calcd for C 17 H 16BrFN3O2S [M+H] + : 424.0125, found: 424.0126.

[0056] (1-3) Synthesis of Compound 2

[0057] [ka]

[0058] A mixed solution of compound 1 (84.1 mg, 0.2 mmol), N-bromosuccinimide (39.5 mg, 0.22 mmol), and carbon tetrachloride (4 mL) was stirred at 60 °C for 2 hours. After the reaction, the mixture was cooled to room temperature, insoluble matter was filtered off with carbon tetrachloride, and the filtrate was concentrated under reduced pressure. The residue was mixed with piperazine (38.1 mg, 0.44 mmol) and ethanol (5 mL) and stirred at room temperature for 24 hours. After the reaction, the reaction mixture was concentrated under vacuum and purified by NH-silica gel column chromatography (aminosilica, dichloromethane:methanol = 100:0 to 95:5) to obtain compound 2, a yellow solid (65.5 mg, yield 64%). Figure 2 shows the results of compound 2. 1 H-NMR and, 13 This figure shows the 1C NMR spectrum. 1 H-NMR (601 MHz, CDCl3) δ 9.71 (s, 1H), 7.81 (d, J = 3.1 Hz, 1H), 7.39 (d, J = 3.1 Hz, 1H), 7.29-7.24 (m, 2H), 6.92 (td, J = 8.3, 2.5 Hz, 1H), 6.15 (s, 1H), 4.04-3.95 (m, 3H), 3.82 (d, J = 17.2 Hz, 1H), 2.98 (dd, J = 7.6, 4.5 Hz, 4H), 2.55 (s, 4H), 1.79 (s, 1H), 1.10 (t, J = 7.1 Hz, 3H); 1313C-NMR (151 MHz, CDCl3) δ 166.1, 162.9, 161.2 (d, J = 250.0 Hz), 146.6, 144.4, 143.1, 139.9 (d, J = 2.9 Hz), 130.5 (d, J = 8.7 Hz), 123.1 (d, J = 10.1 Hz), 122.9, 120.0 (d, J = 24.6 Hz), 114.8 (d, J = 20.2 Hz), 114.7, 97.7, 59.7, 58.7, 57.0, 54.6, 46.1, 14.1; 19 19F-NMR (565 MHz, CDCl3) δ -114.4. ESI-HRMS calcd for C 21 H 24 BrFN5O2S [M+H] + : 508.0813, found: 508.0816.

[0059] (1-4) Synthesis of GLS4-C3-Ad

[0060]

Chem.

[0061] A mixture of compound 2 (10.7 mg, 0.02 mmol), N,N-diisopropylethylamine (17.4 μL, 0.1 mmol), and DMF (2 mL) was mixed with glutaric anhydride (2.8 mg, 0.024 mmol) and stirred at room temperature for 1 hour. Next, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 10.6 mg, 0.028 mmol), 3H-[1,2,3]triazolo[4,5-b]pyridinium-3-ol (HOAt, 3.7 mg, 0.028 mmol), DMF (1 mL), and 1-adamantan methylamine (4.6 μL, 0.028 mmol) were added and the mixture was stirred at room temperature for 20 hours. Subsequently, the reaction mixture was concentrated under vacuum and purified by NH-silica gel column chromatography (aminosilica, hexane:AcOEt = 50:50 → 0:100) and preparative HPLC (C18, H2O:MeCN = 50:50 → 0:100) to obtain a pale yellow solid, which is GLS4-C3-Ad, the embodiment of the present invention (8.9 mg, yield 58%). Figure 3 shows the GLS4-C3-Ad. 1 H-NMR, 13 This figure shows the 13C NMR spectrum and analytical RP-HPLC. Pale yellow solid (8.9 mg, 58% yield); 1H-NMR (601 MHz, CD3OD) δ 7.93 (d, J = 3.1 Hz, 1H), 7.88 (t, J = 5.9 Hz, 1H), 7.74 (d, J = 3.1 Hz, 1H), 7.43-7.40 (m, 2H), 7.09 (td, J = 8.4, 2.6 Hz, 1H), 6.15 (s, 1H), 4.08 (d, J = 16.9 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.92 (d, J = 16.9 Hz, 1H), 3.76-3.64 (m, 4H), 2.91-2.86 (m, 2H), 2.63 (m, 3H), 2.46 (t, J = 7.5 Hz, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.95-1.89 (m, 5H), 1.74 (d, J = 12.1 Hz, 3H), 1.69-1.65 (m, 3H), 1.52 (d, J = 2.2 Hz, 6H), 1.13 (t, J = 7.2 Hz, 3H); 13 C-NMR (151 MHz, CD3OD) δ 174.4, 174.3, 172.1, 166.0, 162.1, 161.5 (d, J = 249.3 Hz), 146.1, 145.0, 143.2, 140.0, 130.8, 130.8, 123.8, 122.9 (d, J = 8.7 Hz), 119.6 (d, J = 24.6 Hz), 114.8 (d, J = 21.0 Hz), 98.4, 59.8, 58.5, 55.7, 53.1, 52.7, 50.8, 45.6, 41.7, 40.0, 36.7, 34.9, 33.8, 32.0, 28.4, 21.6, 13.2; 19 F-NMR (565 MHz, CDCl3) δ-114.4 (d, J = 4.9 Hz, 1F); ESI-HRMS calcd for C 37 H 47 BrFN6O4S [M+H] + : 769.2541, found: 769.2537; Purity 99.02% (t R = 23.89 min).

[0062] (2) Synthesis of GLS4-C3-Nor

[0063] [ka]

[0064] GLS4-C3-Nor, an embodiment of the present invention, was synthesized using the following procedure.

[0065] A mixture of compound 2 (10.7 mg, 0.02 mmol), N,N-diisopropylethylamine (17.4 μL, 0.1 mmol), and DMF (2 mL) was mixed with glutaric anhydride (2.9 mg, 0.024 mmol) and stirred at room temperature for 1 hour. Next, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 10.7 mg, 0.028 mmol), 3H-[1,2,3]triazolo[4,5-b]pyridine-3-ol (HOAt, 3.6 mg, 0.028 mmol), DMF (1 mL), and 1-5-norbornene-2-methylamine (isomer mixture, 3.6 μL, 0.028 mmol) were added and the mixture was stirred at room temperature for 20 hours. Subsequently, the reaction mixture was concentrated under vacuum and purified by NH-silica gel column chromatography (aminosilica, hexane:AcOEt = 50:50 → 0:100) and preparative HPLC (C18, H2O:MeCN = 50:50 → 0:100) to obtain a pale yellow solid, which is GLS4-C3-Nor, the embodiment of the present invention (6.8 mg, yield 47%). Figure 4 shows GLS4-C3-Nor. 1 H-NMR, 13 This figure shows the 13C NMR spectrum and analytical RP-HPLC. Pale yellow solid (6.8 mg, 47% yield, diastereomer ratio = 1 : 0.3) 1H-NMR (601 MHz, CD3OD): (Major) δ 7.93 (d, J = 3.1 Hz, 1H), 7.74 (d, J = 3.1 Hz, 1H), 7.43-7.39 (m, 2H), 7.09 (td, J = 8.4, 2.6 Hz, 1H), 6.18-6.15 (m, 2H), 5.98 (q, J = 2.8 Hz, 1H), 4.08 (d, J = 17.1 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.92 (d, J = 16.9 Hz, 1H), 3.79-3.64 (m, 4H), 3.00-2.94 (m, 1H), 2.86-2.78 (m, 2H), 2.69-2.58 (m, 4H), 2.45 (t, J = 7.5 Hz, 2H), 2.29-2.24 (m, 3H), 1.93-1.84 (m, 3H), 1.43 (dd, J = 8.1, 2.1 Hz, 1H), 1.34-1.25 (m, 2H), 1.13 (t, J = 7.2 Hz, 3H), 0.55 (ddd, J = 11.6, 4.4, 2.6 Hz, 1H); (minor) δ 7.93 (d, J = 3.1 Hz, 1H), 7.74 (d, J = 3.1 Hz, 1H), 7.43-7.39 (m, 2H), 7.09 (td, J = 8.4, 2.6 Hz, 1H), 6.15 (s, 1H), 6.09 (q, J = 2.9 Hz, 1H), 6.06 (q, J = 2.9 Hz, 1H), 4.08 (d, J = 17.1 Hz, 1H), 4.03 (q, J = 7.1 Hz, 2H), 3.92 (d, J = 16.9 Hz, 1H), 3.79-3.64 (m, 4H), 3.28-3.24 (m, 1H), 3.16-3.13 (m, 1H), 2.86-2.78 (m, 1H), 2.69-2.58 (m, 4H), 2.45 (t, J = 7.5 Hz, 2H), 2.29-2.24 (m, 3H), 1.93-1.84 (m, 3H), 1.60-1.55 (m, 1 H), 1.39 (d, J = 8.6 Hz, 1H), 1.34-1.25 (m, 1H) 1.18 (td, J = 7.8, 3.8 Hz, 1H), 1.13 (t, J = 7.2 Hz, 3H);. 13 C-NMR (151 MHz, CD3OD) (Major)δ 175.2, 173.4, 167.3, 163.4, 162.9 (d, J = 249.3 Hz), 147.5, 146.3, 144.6, 141.3 (d, J = 2.8 Hz), 138.5, 133.2, 132.1 (d, J = 8.7 Hz), 125.1, 124.2 (d, J = 10.1 Hz), 120.9 (d, J = 24.5 Hz), 116.2 (d, J = 24.5 Hz), 99.8, 61.1, 59.8, 57.0, 54.5, 54.0, 50.4, 47.0, 45.4, 44.5, 43.7, 43.01, 39.9, 36.1, 33.3, 31.1, 22.8, 14.5; (minor) δ 175.3, 173.4, 167.3, 163.4, 162.9 (d, J = 249.3 Hz), 147.5, 146.3, 144.6, 141.3 (d, J = 2.8 Hz), 137.9, 137.4, 132.1 (d, J = 8.7 Hz), 125.1, 124.2 (d, J = 10.1 Hz), 120.9 (d, J = 24.5 Hz), 116.2 (d, J = 24.5 Hz), 99.8, 61.1, 59.8, 57.0, 54.5, 54.0, 49.6, 45.8, 45.7, 45.5, 42.98, 40.2, 39.9, 36.2, 33.3, 31.8, 22.8, 14.5; 19 F-NMR (565 MHz, CD3OD) δ-114.4 (dd, J = 14.5, 7.8 Hz, 1F); ESI-HRMS calcd for C 34 H 41 BrFN6O4S [M+H] + : 727.2072, found: 727.2073; Purity 95.77% (t R = 18.79 minutes).

[0066] (3) Synthesis of GLS4-C3-FI

[0067] [ka]

[0068] GLS4-C3-FI, an embodiment of the present invention, was synthesized using the following procedure.

[0069] A mixed solution of 2-(9H-fluoren-9-yl)acetic acid (14.1 mg, 0.06 mmol), 4-ethyl aminobutyrate (11.6 mg, 0.07 mmol), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (34.1 mg, 0.09 mmol), 1-hydroxybenzotriazole (14.5 mg, 0.09 mmol), N,N-diisopropylethylamine (24.5 mg, 0.19 mmol), and DMF (2 mL) was stirred at room temperature for 4 hours. After adding water, the mixture was extracted with ethyl acetate, washed with saturated saline solution, and concentrated under vacuum. To the resulting solid, aqueous sodium hydroxide solution (3 mL) and tetrahydrofuran (2 mL) were added and stirred overnight. After adding hydrochloric acid and extracting with ethyl acetate, the mixture was dehydrated with sodium sulfate, filtered, and the resulting filtrate was concentrated under vacuum. The obtained solid was mixed with the aforementioned compound 2 (8.9 mg, 0.02 mmol), O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (13.3 mg, 0.04 mmol), 1-hydroxybenzotriazole (5.4 mg, 0.04 mmol), 2 drops of N,N-diisopropylethylamine, and DMF (1.2 mL). The mixture was stirred overnight, water was added, and the resulting solution was purified by preparative HPLC (C18, H2O:MeCN = 80:20 → 40:60) to obtain a pale yellow solid, which is GLS4-C3-FI, an example of the present invention (7.8 mg, yield 15%). Figure 5 shows GLS4-C3-FI. 1 H-NMR, 13 This figure shows the 13C NMR spectrum and analytical RP-HPLC. 1 1H-NMR (600 MHz, CDCl3) δ 7.86 (d, J = 3.1 Hz, 1H), 7.77 (d, J = 7.6 Hz, 2H), 7.56 (d, J = 3.1 Hz, 1H), 7.51 (dd, J = 7.5, 2.8 Hz, 2H), 7.41 - 7.37 (m, 3H), 7.34 - 7.30 (m, 3H), 7.06 (m, 1H), 6.14 (s, 1H), 5.74 (s, 1H), 4.73 (d, J = 14.8 Hz, 1H), 4.54 (d, J = 14.6 Hz, 1H), 4.44 (t, J = 6.9 Hz, 1H), 4.05 - 4.01 (m, 2H), 3.80 (s, 2H), 3.26 (s, 2H), 2.75 - 2.71 (m, 2H), 2.18 (t, J = 6.6 Hz, 2H), 1.71 (brs, 1H), 1.08 (t, J = 7.1 Hz, 3H); 13 13C-NMR (151 MHz, CDCl3) δ 171.6, 171.0, 164.8, 162.9, 161.3, 161.0, 160.7, 149.4, 146.0, 144.1, 140.8, 140.8, 137.7, 137.7, 131.0, 131.0, 127.7, 127.3, 125.4, 124.4, 122.0, 122.0, 120.4, 120.2, 120.1, 116.1, 115.9, 107.6, 77.2, 77.0, 76.8, 61.0, 57.5, 52.8, 52.7, 52.7, 44.0, 42.2, 40.3, 39.0, 38.5, 29.8, 24.5, 13.9; ESI-HRMS calcd for C 40 H 42 BrFN6O4S [M + 2H] 2+ : 400.1072, found: 400.1061; Purity >99% (t R = 8.16 min).

[0070] (4) Synthesis of GLS4-C3-Boc

[0071] [ka]

[0072] The comparative example GLS4-C3-Boc was synthesized using the following procedure.

[0073] A mixed solution of Tri-Boc-arginine (30.0 mg, 0.06 mmol), ethyl 4-aminobutyrate (11.6 mg, 0.07 mmol), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (34.1 mg, 0.09 mmol), 1-hydroxybenzotriazole (14.5 mg, 0.09 mmol), N,N-diisopropylethylamine (24.5 mg, 0.19 mmol), and DMF (2 mL) was stirred at room temperature for 4 hours. After adding water, the mixture was extracted with ethyl acetate, washed with saturated saline solution, and concentrated under vacuum. To the resulting solid, aqueous sodium hydroxide solution (3 mL) and tetrahydrofuran (2 mL) were added and stirred overnight. After adding hydrochloric acid and extracting with ethyl acetate, the mixture was dehydrated with sodium sulfate, filtered, and the resulting filtrate was concentrated under vacuum. The obtained solid was mixed with the aforementioned compound 2 (5.5 mg, 0.01 mmol), O-benzotriazolyl-N,N,N',N'-tetramethyluronium hexafluorophosphate (9.9 mg, 0.03 mmol), 1-hydroxybenzotriazole (4.0 mg, 0.03 mmol), 2 drops of N,N-diisopropylethylamine, and DMF (1.2 mL). The mixture was stirred overnight, water was added, and the resulting solution was purified by preparative HPLC (C18, H2O:MeCN = 80:20 → 40:60) to obtain a pale yellow solid, which is comparative example GLS4-C3-Boc (3.2 mg, yield 5%). Figure 6 shows the results for GLS4-C3-Boc. 1 H-NMR, 13 This figure shows the 13C NMR spectrum and analytical RP-HPLC. 1H-NMR (600 MHz, CDCl3) δ 8.84 (s, 1H), 7.86 (d, J = 2.9 Hz, 1H), 7.58 (d, J = 2.9 Hz, 1H), 7.40-7.37 (m, 1H), 7.33 (dd, J = 8.1, 2.4 Hz, 1H), 7.08-7.04 (m, 1H), 6.15 (s, 1H), 5.80 (s, 1H), 4.71 (t, J = 13.3 Hz, 1H), 4.48 (t, J = 13.8 Hz, 1H), 4.09 (brs, 1H), 4.07-4.00 (m, 2H), 3.95 (s, 2H), 3.49-3.25 (m, 5H), 2.49-2.35 (m, 2H), 2.09-1.83 (m, 9H), 1.76-1.62 (m, 2H), 1.52 (d, J = 8.3 Hz, 9H), 1.42 (s, 11H), 1.09 (t, J = 7.1 Hz, 3H); 13 C-NMR (151 MHz, CDCl3) δ 172.4, 171.3, 165.0, 162.9, 162.5, 162.2, 161.2, 160.8, 160.8, 156.2, 154.8, 153.3, 144.1, 138.0, 131.0, 130.9, 125.3, 122.0, 120.4, 120.2, 117.4, 116.0, 115.9, 115.4, 84.8, 80.3, 77.2, 77.0, 76.8, 76.2, 67.0, 66.6, 66.5, 61.0, 57.2, 52.8, 42.3, 40.6, 38.7, 38.5, 30.0, 29.8, 28.3, 27.8, 24.5, 24.2, 13.9; ESI-HRMS calcd for C36H52BrFN10O6S [M+2H-Boc2] 2+ : 425.1474, found: 425.1458; Purity >99% (t R = 7.14 min).

[0074] (5) Evaluation of the inhibitory activity of wild-type HBV strains using GLS4-C3-Ad and GLS4-C3-Nor The inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor in the examples of this application was evaluated in comparison with that of GLS4. Wild-type HBV strains were used.

[0075] The plasmid DNA used for HBV production was prepared as follows: As a wild-type HBV expression plasmid, pHBV1.05 (also known as pcDNA3.1-HBV1.05-delBGHpA; reference Antiviral Res. 220: 105756 (2023)) was created by incorporating a 1.05-times-long DNA fragment of the HBV genotype D (GeneBank accession number U95551.1) genome downstream of the cytomegalovirus promoter.

[0076] The inhibitory effect on HBV production was evaluated as follows: Human hepatocellular carcinoma cells (HuH-7 cells) were seeded in 10 cm dishes and introduced with either pHBV1.05 (wild-type) or pHBV1.05 T33N (GLS4-resistant mutant). After 16 hours, the cells were reseeded into 24-well plates, and cells treated with various final concentrations of each compound or untreated (control) cells were cultured for 4 days. Cells and supernatants were collected from each group. 1% NP-40 buffer was added to the cell samples, and the solubilized fraction was separated by native agarose (1.2%) gel electrophoresis or SDS-polyacrylamide gel (17%) electrophoresis. Proteins were then transferred to a PVDF membrane and detected by Western blotting using an anti-HBc antibody. The detected HBc protein levels were compared between compound-treated cells and control cells. The collected supernatant samples were treated with DMSO (Fujifilm Wako Pure Chemical Industries) and PEG8000 (Promega) to final concentrations of 2% and 5%, respectively, to create infection material. These were inoculated into HepG2-NTCP-C4 cells and cultured for 8 days. The culture medium was changed every two days during this period. TRI Reagent (Molecular Research Center) was added to the infected cells to extract total RNA. After treatment with DNase I (Takara Bio), cDNA was synthesized using SuperScript VILO (Thermo Fisher Scientific), and HBV RNA was quantitatively measured using the SYBR qPCR Mix Kit (Toyobo). The HBV RNA copy numbers of cells infected with the culture supernatant from compound-treated cells and cells infected with the culture supernatant from control cells were compared.

[0077] Figure 7 shows the evaluation of the inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor. As shown in Figure 7, GLS4-C3-Ad and GLS4-C3-Nor showed comparable inhibitory activity to GLS4.

[0078] (6) Evaluation of the inhibitory activity of GLS4-C3-FI and GLS4-C3-Boc using GLS4-resistant HBV strains The inhibitory activity of Example GLS4-C3-FI and Comparative Example GLS4-C3-Boc was evaluated in comparison with GLS4. The HBV strain used was HBc-T33N, which is resistant to GLS4.

[0079] The plasmid DNA used for HBV production was prepared as follows: The GLS4-resistant mutant HBV expression plasmid (pHBV1.05 T33N) was created by substituting the 33rd amino acid threonine of the HBc protein with asparagine, based on pHBV1.05. The substitution mutation was introduced by site-directed mutagenesis using a primer set (F: 5'-GAGATCTTCTAGATAACGCCTCAGCTCTGTA-3', R: 5'-TACAGAGCTGAGGCGTTATCTAGAAGATCTC-3'). The inhibitory effect on HBV production was evaluated using the same method as when using wild-type HBV strains.

[0080] Figure 8 shows the evaluation of the inhibitory activity of GLS4-C3-FI and GLS4-C3-Boc. As shown in Figure 8, GLS4-C3-Boc did not show superior inhibitory activity compared to GLS4. However, GLS4-C3-FI showed comparable or superior inhibitory activity compared to GLS4.

[0081] (7) Evaluation of inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor using GLS4-resistant HBV strains The inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor in the embodiments of this invention was evaluated in comparison with that of GLS4. The HBV strain used was HBc-T33N, which is resistant to GLS4.

[0082] The plasmid DNA used for HBV production was prepared using the same method as described in (6) above. The evaluation of the inhibitory effect on HBV production was also conducted using the same method as described in (6) above.

[0083] Figure 9 shows the evaluation of the inhibitory activity of GLS4-C3-Ad and GLS4-C3-Nor using GLS4-resistant HBV strains. As shown in Figure 9, existing GLS4 cannot suppress the proliferation of GLS4-resistant HBV strains. However, GLS4-C3-Ad and GLS4-C3-Nor according to the present invention example showed excellent inhibitory activity even against resistant GLS4. GLS4-C3-FI also showed excellent inhibitory activity against resistant GLS4. [Industrial applicability]

[0084] It can be used as a treatment for hepatitis B. [Sequence Listing Free Text]

[0085] Sequence IDs 1 and 2: Primers

Claims

1. A novel compound in which a hydrophobic tag and morphothiazine, an allosteric modulator of the hepatitis B virus core protein, are linked by a linker.

2. The novel compound according to claim 1, characterized in that the hydrophobic tag is a cyclic carbon ring with 2 to 6 rings.

3. The novel compound according to claim 1, characterized in that the hydrophobic tag is a 2- to 6-ring bridged carbocyclic ring.

4. The novel compound according to claim 3, characterized in that the aforementioned bridged ring carbocyclic is any of the following: an adamantane ring, norbornene ring, norbornane ring, perhydroindene ring, decalin ring, perhydrofluorene ring, perhydroanthracene ring, perhydrophenanthrene ring, tricyclodecane ring, tricycloundecane ring, tetracyclododecane ring, perhydroacenaphthene ring, or perhydrophenalene ring.

5. The novel compound according to claim 3, characterized in that the aforementioned bridged ring carbocyclic ring is an adamantane ring.

6. The novel compound according to claim 5, characterized in that the adamantane ring is bonded to the linker at the carbon at position 1.

7. The novel compound according to claim 3, characterized in that the aforementioned bridged ring carbocyclic ring is a norbornene ring.

8. The novel compound according to claim 7, characterized in that the norbornene ring is bonded to the linker at the 5th or 6th carbon.

9. The novel compound according to claim 2, characterized in that the cyclic carbocyclic ring is one of a fluorene ring, a biphenyl ring, a terphenyl ring, a naphthalene ring, an anthracene ring, or a dihydroanthracene ring.

10. The novel compound according to claim 2, characterized in that the cyclic carbocyclic ring is a fluorene ring.

11. The novel compound according to claim 1, characterized in that the linker is an alkyl linker or a polyethylene glycol linker.

12. The alkyl linker is -L 1 -(CH 2 ) ｎ -L 2 - where n is a natural number from 1 to 6, L 1 These are chemical bonds, -CO-, -NHCO-, or -CH 2 -NHCO- is one of the following, L 2 These are chemical bonds, -CO-, -CONH-, or -CONH--CH 2 A novel compound according to claim 11, characterized in that it is one of the following.

13. The alkyl linker is -CH 2 -NHCO-CH 2 -CH 2 -CH 2 -CO-, and the novel compound according to claim 12, characterized in that.

14. The novel compound according to claim 1, characterized in that it is GLS4-C3-Ad represented by the following formula. 【Chemistry 1】

15. The novel compound according to claim 1, characterized in that it is GLS4-C3-Nor represented by the following formula. 【Chemistry 2】

16. The novel compound according to claim 1, characterized in that it is GLS4-C3-FI represented by the following formula. 【Transformation 3】

17. A pharmaceutical composition for the treatment or prevention of diseases caused by infection with hepatitis B virus, characterized by comprising a novel compound described in any one of claims 1 to 16 as an active ingredient.

18. The pharmaceutical composition according to claim 17, characterized in that the hepatitis B virus is a drug-resistant hepatitis B virus.

19. The pharmaceutical composition according to claim 18, characterized in that the drug-resistant hepatitis B virus is a hepatitis B virus having one of the following mutations in the drug-binding pocket: T33N, D29G, T109I, T109M, or Y118F.

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