Long-acting hepatitis virus entry inhibitor

Abstract

The present invention relates to the field of pharmaceutical synthesis and discloses a long-acting hepatitis virus entry inhibitor. The long-acting hepatitis virus entry inhibitor of the present invention is used for the manufacture of a pharmaceutical composition for treating chronic hepatitis B and hepatitis D.

Description

[Technical field]

[0001] This application claims priority based on a Chinese patent application for patent application No. 202210126691.4, entitled "Long-acting Hepatitis Virus Entry Inhibitor", filed with the China Patent Office on February 10, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a long-acting hepatitis virus entry inhibitor and uses thereof. [Background technology]

[0003] Viral hepatitis is an infectious disease caused by various hepatitis viruses and mainly affects the liver. Clinical symptoms include loss of appetite, nausea, upper abdominal discomfort, liver pain, and weakness. Some patients may have jaundice, fever, and hepatomegaly accompanied by impaired liver function. In some patients, hepatitis may become chronic and develop into cirrhosis, and sometimes even liver cancer.

[0004] Regarding the etiological classification of viral hepatitis, there are currently five officially recognized types of hepatitis viruses: A, B, C, D, and E, which are designated HAV, HBV, HCV, HDV, and HEV, respectively. With the exception of hepatitis B virus, which is a DNA virus, all others are RNA viruses.

[0005] Hepatitis A virus (HAV) is an RNA virus of the Hepadnavirus genus of the Picornaviridae family. When humans are infected with HAV, many people show asymptomatic or subclinical infection, and only a few people show acute hepatitis A. In general, it does not progress to chronic hepatitis, there are no chronic virus carriers, and people can fully recover.

[0006] Hepatitis B virus (HBV) is a pathogen that causes hepatitis B and belongs to the Hepadnaviridae family, which includes two genera: Orthohepadnavirus and Avichepadnavirus. Orthohepadnavirus is the genus that causes human infection. HBV infection is a global public health problem, and with the production and introduction of genetically engineered vaccines, the prevalence of hepatitis B vaccines is increasing year by year, and the infection rate is declining. Of the approximately 2 billion HBV-infected patients worldwide, 380 million infected patients belong to chronic infection, because the virus hides in tissues and organs and cannot be thoroughly eliminated by the immune system and drugs.

[0007] Hepatitis C virus (HCV) is mainly transmitted vertically through blood, sexual activity, and mother-to-child transmission. However, unfortunately, the medical community has not yet developed an effective vaccine to prevent hepatitis C. Hepatitis C virus is an RNA virus that is prone to mutation, making it very difficult to develop a vaccine. In addition, since no animals other than humans and chimpanzees are susceptible to hepatitis C, it is difficult to find an animal model for vaccine development. However, the next generation of oral direct-acting antiviral drugs can realize a cure for hepatitis C.

[0008] HDV is a defective single-stranded negative-stranded RNA virus that cannot replicate without the aid of a DNA virus, such as HBV, to provide a shell. HDV is present in the nuclei of hepatocytes and in the serum of HDV-infected individuals who are HBsAg positive, and replicates primarily within hepatocytes. HDV is prone to mutation. In HDV-infected individuals, the synthesis of HBV-DNA is significantly inhibited, and the appearance of HDAg corresponds to a decrease in HBV-DNA in serum, and HBV-DNA is restored to its original level with the negativity of HDAg and the appearance of anti-HD. HDV is mainly transmitted through blood transfusions and blood products, and is transmitted in the same way as hepatitis B. HDV infection is often found in HBV-infected individuals, and occasionally in sporadic HDV-infected individuals. After HIV-HBV superinfection, the accumulation of liver damage is promoted, and it is easy to develop into chronic active hepatitis, cirrhosis and severe hepatitis.

[0009] Researchers have discovered that a protein called hepatic bile acid transporter (NTCP, sodium taurocholate cotransporting polypeptide) on the surface of liver cells can specifically interact with the main receptor-binding domain of the HBV envelope protein, which is a necessary receptor for HBV to infect host cells. Therefore, inhibiting NTCP may be a cure for hepatitis B.

[0010] Bulevirtide is a drug that targets NTCP and contains 47 amino acids. It has a short half-life in the body, so it needs to be taken by patients every day for a long period of time, which leads to poor patient compliance and high clinical costs. The present invention aims to provide a long-acting hepatitis virus entry inhibitor, thereby reducing the administration frequency and lowering the cost to patients. Summary of the Invention

[0011] The present invention provides a long-acting hepatitis virus entry inhibitor and uses thereof. In order to achieve the above object, the present invention first provides a compound represented by the following structural formula I, a pharma- ceutically acceptable salt, solvate, chelate or non-covalent complex of said compound, a drug precursor based on said compound, or any mixture of these forms: [ka] In the formula I, AA1 is Arg, Lys, Gln, Glu, or Cit; AA2 in structural formula I is NH 2 or OH, In structural formula I, R is HO 2 C(CH 2 )n1CO-(AA3)n2-(PEGn3(CH 2 )n4CO)n5- or HO 2 C(CH 2)n1CO-(AA3)n2-(AA4)n6- (wherein n1 is an integer of 10 to 20, n2 is an integer of 1 to 5, n3 is an integer of 1 to 30, n4 is an integer of 1 to 5, n5 is 0 or an integer of 1 to 5, and n6 is 0 or an integer of 1 to 10.) AA3 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava; AA4 is Ala, Gly, Leu, Ser, Thr, Tyr, Gln, Lys, Dao, Dah, Orn, Dab, Dap, Glu, Asp, Ada, Apm, or Asu. When n6 is an integer of 2 to 10, the repeating units of AA4 may be the same or different amino acids.

[0012] In some embodiments, the AA1 is Arg, Lys, or Gln, and the AA2 is NH 2 It is. In some embodiments, the R is 20-alkanedioxy acid-γGlu-(AA4)n6- (wherein AA4 is Gly, Ser, Thr, Asn, Leu, Val, or Pro, and n6 is 0 or an integer from 1 to 7). In other embodiments, the R is 20-alkanedioxy acid-γGlu-PEGn3-CH 2 CO- (where n3 is an integer of 1 to 6).

[0013] In some embodiments, R is 20 alkanedioic acid -γGlu-, 20 Alkanedioic acid-γGlu-PEG 1 CH 2 CO-, 20 Alkanedioic acid-γGlu-PEG 2 CH 2 CO-, 20 Alkanedioic acid-γGlu-PEG 3 CH 2 CO-, 20 Alkanedioic acid-γGlu-PEG 4 CH 2 CO-, 20 Alkanedioic acid-γGlu-PEG 5 CH 2CO-, 20 Alkanedioic acid-γGlu-PEG 6 CH 2 CO-, 20 Alkanedioic acid -γGlu-Gly- 20 Alkanedioic acid -γGlu-Gly-Gly- 20 Alkanedioic acid -γGlu-Gly-Gly-Gly- 20 Alkanedioic acid -γGlu-Gly-Gly-Gly-Gly- 20 Alkanedioic acid - γGlu-Gly-Gly-Gly-Gly-Gly- 20 Alkanedioic acid -γGlu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly- 20 Alkanedioic acid -γGlu-Gly-Gly-Ser-Gly-Ser-Gly- and 20 alkanedioic acid -γGlu-Gly-Thr-Asn-Leu-Ser-Val-Pro-Asn-Pro-Leu-.

[0014] The long-acting hepatitis virus entry inhibitor according to the present invention is in the form of one selected from the group consisting of pharma- ceutically acceptable salts, solvates, chelates or non-covalent complexes of said compounds, drug precursors based on said compounds, and any mixtures thereof.

[0015] The invention also provides pharmaceutical compositions comprising the compounds of the invention and the use of pharmaceutical compositions of the invention in the manufacture of a medicament for treating a disease. Furthermore, the pharmaceutical composition is used for the treatment of chronic hepatitis B and D.

[0016] More details regarding the present invention are described in detail below, or partly understood in the embodiments of the present invention.

[0017] Unless otherwise indicated, the quantities used to refer to various components, reaction conditions in this specification can be construed in any case as being "approximate" or "about." Accordingly, unless expressly specified otherwise, the numerical parameters recited below and in the claims are approximate parameters, and different numerical parameters may be obtained due to standard errors for different experimental conditions.

[0018] In the present specification, if there is any difference or doubt in the chemical structure and chemical name of a compound, the compound is appropriately defined by the chemical structure. The compounds described herein may contain one or more chiral centers and / or double bonds and similar structures, and may have stereoisomers, including double bond isomers (e.g., geometric isomers), optically rotatory enantiomers or diastereomers. And, any chemical structure within the scope described herein, when it contains the above similar structures in part or in the overall structure, includes all possible enantiomers and diastereomers of the compound, and also includes any simple stereoisomers (e.g., simple geometric isomers, simple enantiomers, or simple diastereomers) and any mixture of these isomers. These racemates and stereoisomeric mixtures can be further resolved into their component enantiomers or stereoisomers by those skilled in the art using various separation techniques or methods of chiral molecule synthesis.

[0019] The compounds of formula I include, but are not limited to, optical isomers, racemates and / or other mixtures of these compounds. In the above cases, single enantiomers (e.g., optical isomers) or diastereomers, e.g., optical rotatory isomers, can be obtained by asymmetric synthesis or racemate resolution. Resolution of racemates can be achieved by various methods, e.g., recrystallization with reagents used in conventional resolutions, or chromatographic methods. Additionally, the compounds of formula I include cis and / or trans isomers of the double bonds.

[0020] The compounds according to the present invention include, but are not limited to, the compounds represented by structural formula I and all pharma- ceutically acceptable forms thereof, including various pharma- ceutically acceptable salts, solvates, complexes, chelates, non-covalent conjugates, drug precursors based on said substances, and any mixtures of these forms. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention discloses a long-acting hepatitis virus entry inhibitor and its use, and those skilled in the art can improve relevant parameters appropriately while referring to the contents of the present specification to realize it. In addition, all similar substitutions and modifications are obvious to those skilled in the art, and all of them are considered to be included in the present invention. The method of the present invention is described by the preferred embodiment, but it is obvious to those skilled in the art to modify or appropriately modify and combine the compounds and preparation methods described herein to realize and apply the technology of the present invention without departing from the content, spirit and scope of the present invention.

[0022] The terms and their abbreviations relevant to the present invention are set out in the table below. [Table 2] EXAMPLES

[0023] Example 1 Preparation of Compound The preparation method includes preparing a peptide resin by solid-phase polypeptide synthesis, further hydrolyzing the peptide resin with an acid to obtain a crude product, and finally purifying the crude product to obtain a purified product. In the step of preparing a peptide resin by solid-phase polypeptide synthesis, the corresponding protected amino acids or fragments in the following sequence were sequentially coupled to a carrier resin by solid-phase coupling synthesis to prepare the peptide resin.

[0024] In the above production method, the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times, and preferably 2.5 to 3.5 times, the total number of moles of the charged resin. In the above manufacturing method, the substitution value of the carrier resin is 0.2 to 1.0 mmol / g resin, and preferably 0.3 to 0.5 mmol / g resin.

[0025] In a preferred embodiment of the present invention, in the solid phase coupling synthesis method, the protected amino acid-resin obtained in the previous reaction step is subjected to a coupling reaction with the next protected amino acid after removing the Fmoc protecting group. The time for the Fmoc deprotection is 10 to 60 minutes, preferably 15 to 25 minutes. The time for the coupling reaction is 60 to 300 minutes, preferably 100 to 140 minutes.

[0026] The above coupling reaction requires the addition of a condensing agent. The condensing agent is one selected from the group consisting of DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, (benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate. N,N-diisopropylcarbodiimide is preferred. The molar amount of the condensing agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total number of moles of amino in the amino resin.

[0027] The above coupling reaction requires the addition of an activator. The activator is 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The dosage of the activator is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total mole number of amino in the amino resin.

[0028] In a preferred embodiment of the present invention, the Fmoc deprotecting agent is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide) containing 10-30% (V) of piperidine. The dosage of the Fmoc deprotecting agent is 5-15 mL per 1 g of amino resin, and preferably 8-12 mL per 1 g of amino resin.

[0029] Preferably, the peptide resin is hydrolyzed with an acid and the resin and side chain protecting groups are removed to obtain the crude product. More preferably, the acid decomposition agent used for the acid decomposition of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT), and water, the volume ratio of which is 80-95% TFA, 1-10% EDT, and the remaining amount being water.

[0030] Even more preferably, the volume ratio of the mixed solvent is 89-91% TFA, 4-6% EDT, and the remainder is water. Most preferably, the volume ratio of the mixed solvent is 90% TFA, 5% EDT, and the remainder is water.

[0031] The amount of the acid decomposition agent is 4 to 15 mL per 1 g of peptide resin, and preferably 7 to 10 mL per 1 g of peptide resin. The decomposition time with an acid decomposition agent is 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

[0032] The crude product was then purified by high performance liquid chromatography and freeze-dried to obtain a purified product. 1. Synthesis of Peptide Resin Using Rink Amide BHHA resin as a carrier resin, the corresponding protected amino acids were sequentially bound by Fmoc deprotection and coupling reaction.

[0033] (1) The first protected amino acid of the main chain was bound 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. 0.03 mol of DIC was slowly added to the DMF solution of the protected amino acid while stirring, and the mixture was reacted at room temperature for 30 minutes while stirring to obtain a solution of the activated protected amino acid. 0.01 mol of Rink amide MBHA resin (substitution value approximately 0.4 mmol / g) was deprotected with 20% PIP / DMF solution for 25 minutes, filtered, and washed to obtain the Fmoc-free resin. The activated first protected amino acid solution was added to the Fmoc-depleted resin, and the coupling reaction was carried out for 60 to 300 minutes, followed by filtration and washing to obtain a resin containing one protected amino acid.

[0034] (2) The 2nd to 49th protected amino acids of the main chain were bound Using a method similar to that used for binding the first protected amino acid in the main chain, the corresponding second to 49 protected amino acids were successively bound to obtain a resin containing 49 amino acids in the main chain.

[0035] (3) The first protected amino acid in the side chain was attached. 0.03 mol of the first protected amino acid of the side chain and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. 0.03 mol of DIC was slowly added to the DMF solution of the protected amino acid while stirring, and the reaction was carried out at room temperature for 30 minutes while stirring to obtain a solution of the activated protected amino acid. 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane were dissolved in an appropriate amount of dichloromethane, deprotected for 4 hours, filtered, and washed to obtain the Alloc-free resin. The solution of the first protected amino acid on the side chain after activation was added to the Alloc-free resin, and a coupling reaction was carried out for 60 to 300 minutes, followed by filtration and washing to obtain a resin containing the first protected amino acid on the side chain.

[0036] (4) The second to fourth protected amino acids of the side chain were attached. Using a method similar to that used for binding the first protected amino acid of the main chain above, the second to fourth protected amino acids and mono-protected fatty acid corresponding to the side chains were sequentially bound to obtain a peptide resin.

[0037] 2. Manufacturing of crude products The peptide resin was taken, and a decomposition agent with a volume ratio of TFA:water:EDT=95:5:5 (10 mL of decomposition agent per 1 g of resin) was added thereto, stirred uniformly, and reacted with stirring at room temperature for 3 hours. The reaction mixture was filtered through a sand core filter funnel, the filtrate was collected, the resin was washed with a small amount of TFA three times, the filtrate was combined and then concentrated under reduced pressure, precipitated by adding anhydrous ethyl ether, precipitated three times with anhydrous ethyl ether, filtered by suction and dried to obtain a crude product as a neutral white powder.

[0038] 3. Manufacturing of refined products The crude product was taken, added with water and stirred, and adjusted with ammonia water until completely dissolved so that the pH became 8.0. The solution was filtered through a 0.45 μm composite microporous membrane to purify it. For purification, high performance liquid chromatography was adopted, using 10μm reversed phase C18 as the purification chromatography filler, 0.1% TFA / water solution-0.1% TFA / acetonitrile solution as the mobile phase system, and the flow rate of the 30mm x 250mm chromatography column was 20mL / min, and purification was performed using gradient elution and cyclic sample injection mode. The crude product solution was injected into the chromatography column, the elution of the mobile phase was started, the main peak was collected, and the acetonitrile was evaporated, after which a concentrated solution of the purified intermediate was obtained.

[0039] After filtering the purified intermediate concentrate through a 0.45μm membrane filter, salt exchange was carried out using high performance liquid chromatography, the mobile phase system was 1% acetic acid / aqueous solution-acetonitrile, the purification chromatography filler was 10μm reversed phase C18, and the flow rate of the 30mm×250mm chromatography column was 20mL / min (the flow rate can be adjusted according to the column specifications). The sample was injected into the chromatography column using gradient elution and cyclic sample injection mode, the elution of the mobile phase was started, the spectrum was collected, the change in absorbance was observed, the main peak of salt exchange was collected, and the purity was detected by liquid chromatography analysis. The main peak solution of salt exchange was combined and then concentrated under reduced pressure to obtain the purified acetic acid aqueous solution, which was then freeze-dried to obtain the purified product.

[0040] The following compounds were prepared using the above method: [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0041] Example 2 Measurement of inhibitory effect against HBV infection The mice were divided into test groups consisting of a blank group, a solvent group, a positive control and a test compound, the test concentration was 0.5 nM and the positive reagent was Myrcludex B, and the supernatant HBeAg / HBsAg was measured every 3 days after infection.

[0042] 1. Virus Concentration (1) hepAD38 cells were cultured and the culture supernatant was collected. The collected hepAD38 cell culture supernatant was centrifuged at 4°C and 4000 rpm / min for 30 min to remove cell debris. (2) After centrifugation, the viral supernatant was added to a 6x PEG8000 (polyethylene glycol, Sigma, SLBZ3934) solution to dilute it to 1x PEG8000, and then placed on a horizontal shaker at 4°C and 20 rpm / min overnight.

[0043] (3) The next day, the virus concentrate was centrifuged at 4°C and 7000g for 30 min, and the supernatant was discarded. The virus concentrate was resuspended in serum-free, double antibody-free DMEM medium for precipitation, and the resuspended concentrated virus solution was stored at 4°C. (4) The multiplicity of infection (MOI) of the virus concentrate was calculated as follows: 20 μL of the virus concentrate was diluted with 180 μL of PBS, digested with DNase (to reduce the influence of residual plasmid), and then column extraction was performed using the Viral DNA / RNA Kit. HBV DNA was then quantified by qPCR detection of the S region primer using a standard product, and the multiplicity of infection of the virus, i.e., the ratio of infected virus to the number of cells, was calculated.

[0044] 2. Infection experiments and detection (1) HepG2-NTCP cells were cultured in 10% FBS DMEM medium containing 4 μg / mL doxycycline for 4 days to induce NTCP expression. (2) The night before the fourth day of culture, HepG2-NTCP cells were cultured at 4 × 10 per well. 4 The cells were plated onto a 48-well culture plate coated with type I rat tail collagen (200 μL of collagen solution).

[0045] (3) On the second day, the medium was replaced with HCM (HCM®, Lonza®, CC-3199 SingleQuots®, Lonza®, CC-4182) to induce HepG2-NTCP cell growth arrest, and doxycycline was added along with the test drug. (4) After culturing in HCM for 24 hours, the HCM was discarded, and the concentrated virus solution was added to the culture plate at an MOI of 1600. The plate was then supplemented with HCM to 150 μL. The test drug was then added, and 150 μL of 8% PEG8000, 2% DMSO, and 4 μg / mL of doxycycline were then added. The mixture was gently mixed uniformly and allowed to infect overnight in a 37°C incubator.

[0046] (5) After 16 hours of infection, the supernatant was discarded, the cells were washed five times with PBS, and the medium was replaced with 300 μL of DMEM (containing 10% FBS, 2% DMSO, and 4 μg / mL doxycycline) for continued culture. (6) After that, the medium was changed every 3 days, and 300 ul of cell culture supernatant was collected at the corresponding time points and centrifuged at 3000 g for 3 min to remove cell precipitates. The supernatant was measured for HBsAg and HBeAg using a fully automated chemiluminescence immunoassay device MAGLUMI X3 (Shin Sangyo Seibutsu).

[0047] 2. Test Results The test results are shown in the table below. [Table 4] [Table 5]

[0048] Example 3: Measurement of preliminary drug metabolism characteristics Cynomolgus monkeys were used as experimental animals, and there were two male cynomolgus monkeys in each compound group. The compound was administered subcutaneously at a dose of 0.1 mg / kg. Blood samples were collected from the vein before administration (0 h) and 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 18 h, 24 h, 48 h, 96 h, 144 h, and 168 h after administration. The plasma samples were centrifuged, and the blood concentrations of the corresponding compounds in the plasma samples were measured by liquid chromatography-mass spectrometry. The half-lives of the compounds administered subcutaneously (SC) are shown in the following table. [Table 6]

Claims

1. A compound having the following structural formula I: 【Chemistry 1】 (wherein AA1 in structural formula I is Arg, Lys, Gln, Glu, or Cit; AA2 in structural formula I is NH ２ or OH, R in formula I is HO ２ C (CH ２ )n1CO-(AA3)n2-(PEGn3(CH ２ ) n4CO) n5-, or HO ２ C (CH ２ )CO-(AA3)-(AA4)-, Here, n1 is an integer from 10 to 20, n2 is an integer from 1 to 5, n3 is an integer from 1 to 30, n4 is an integer from 1 to 5, n5 is 0 or an integer from 1 to 5; n6 is 0 or an integer from 1 to 10; AA3 is γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava; AA4 is Ala, Gly, Leu, Ser, Thr, Tyr, Gln, Lys, Dao, Dah, Orn, Dab, Dap, Glu, Asp, Ada, Apm, or Asu.

2. The compound according to claim 1 , wherein AA1 is Arg, Lys, or Gln.

3. The AA2 is NH ２ 3. The compound according to claim 1 or 2, characterized in that it is

4. The compound according to any one of claims 1 to 3, characterized in that R is 20-alkanedioic acid-γGlu-(AA4)n6-, where AA4 is Gly, Ser, Thr, Asn, Leu, Val or Pro, and n6 is 0 or an integer from 1 to 7.

5. The R is 20-alkanedioxy acid-γGlu-PEGn3-CH ２ The compound according to any one of claims 1 to 3, characterized in that it is CO- (wherein n3 is an integer from 1 to 6).

6. The compound according to any one of claims 1 to 5, characterized in that it is in the form of one selected from the group consisting of a pharma- ceutically acceptable salt, solvate, chelate or non-covalent complex of said compound, a drug precursor based on said compound, and any mixture thereof.

7. Use of a compound according to any one of claims 1 to 6 in the manufacture of a long-acting hepatitis virus entry inhibitor.

8. The use according to claim 7, characterized in that the hepatitis virus is hepatitis B virus or hepatitis D virus.

9. 10. Use of a compound according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of chronic hepatitis B and / or hepatitis D.

10. A medicament for treating chronic hepatitis B and / or hepatitis D, comprising a compound according to any one of claims 1 to 6.

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