Long-acting hepatitis virus entry inhibitors

Abstract

The present application relates to the field of medicine synthesis, disclose a kind of long-acting hepatitis virus entry inhibitor.The long-acting hepatitis virus entry inhibitor described in the present application is used to prepare the pharmaceutical composition for treating diseases, and the pharmaceutical composition is used for the treatment of chronic hepatitis B and hepatitis D.

Description

Technical Field

[0001] This invention relates to a long-acting hepatitis virus entry inhibitor and its uses. Background Technology

[0002] Viral hepatitis is an infectious disease primarily affecting the liver, caused by various hepatitis viruses. Clinically, it is mainly characterized by loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may experience jaundice, fever, and hepatomegaly with impaired liver function. Some cases may become chronic, even progressing to cirrhosis, and a small number may develop liver cancer.

[0003] The etiological classification of viral hepatitis currently recognizes five types of hepatitis viruses: hepatitis A, B, C, D, and E, respectively written as HAV, HBV, HCV, HDV, and HEV. Except for hepatitis B virus, which is a DNA virus, the rest are RNA viruses.

[0004] Hepatitis A virus (HAV) belongs to the genus *Hepatovirae* of the family Picornaviridae. Most humans infected with HAV present with subclinical or latent infection, with only a small percentage developing acute hepatitis A. Recovery is generally complete, and the disease does not progress to chronic hepatitis; there are no chronic carriers.

[0005] Hepatitis B virus (HBV) is the pathogen that causes hepatitis B. It belongs to the family Hepatoviridae, which includes two genera: Orthotropic hepatovira and Avian hepatovira. The Orthotropic hepatovira genera causes human infection. HBV infection is a global public health problem. With the production and use of genetically engineered vaccines, the availability of hepatitis B vaccines has been increasing year by year, and the infection rate is showing a downward trend. There are approximately 2 billion HBV-infected individuals worldwide, of whom 380 million have chronic infections, meaning the virus can remain dormant in tissues and organs and cannot be completely eliminated by the immune system or drugs.

[0006] Hepatitis C virus (HCV) is mainly transmitted through blood, sexual contact, and mother-to-child transmission. Unfortunately, the medical community has not yet developed an effective vaccine to prevent hepatitis C. This is because HCV is an RNA virus that mutates rapidly, making vaccine development extremely difficult. Since no animals other than humans and chimpanzees can contract hepatitis C, finding animal models for vaccine development is challenging. However, new-generation oral direct-acting antiviral drugs have enabled a cure for hepatitis C.

[0007] HDV is a defective single-stranded negative-sense RNA virus that requires hepatotropic DNA viruses such as HBV to provide its outer shell for replication. HDV is present in the hepatocyte nuclei and serum of HBsAg-positive HDV-infected individuals. It primarily replicates within hepatocytes. HDV is prone to mutation. Human infection with HDV significantly inhibits HBV-DNA synthesis; the appearance of HDAg coincides with a decrease in serum HBV-DNA. As HDAg becomes negative and anti-HD appears, HBV-DNA returns to its original level. Transmission is mainly through blood transfusions and blood products, similar to the transmission route of hepatitis B. HDV infection is most common in HBV-infected individuals, but sporadic HDV infections can also occur. Co-infection with HDV and HBV can exacerbate liver damage and easily lead to chronic active hepatitis, cirrhosis, and fulminant hepatitis.

[0008] Researchers have discovered that a protein on the surface of liver cells called the hepatic bile acid transporter (NTCP, sodium taurocholate cotransport polypeptide) can specifically interact with a key receptor-binding domain of the HBV envelope protein, which is the receptor required for HBV to infect host cells. Therefore, blocking NTCP holds promise for curing hepatitis B.

[0009] Bulevirtide is a 47-amino acid-targeting NTCP drug. Due to its short half-life in the body, patients need to take it daily for a long period, resulting in poor patient compliance and high clinical costs. The purpose of this invention is to provide patients with a long-acting hepatitis virus entry inhibitor, reducing the frequency of dosing and lowering patient costs. Summary of the Invention

[0010] This invention provides a long-acting hepatitis virus entry inhibitor and its uses.

[0011] To achieve the above objectives, the present invention first provides a compound shown in structure I, a pharmaceutically usable salt, solvate, chelate or non-covalent complex of the compound, a drug precursor based on the compound, or any mixture thereof.

[0012] AA1-Gly-Thr-Asn-Leu-Ser-Val-Pro-Asn-Pro-Leu-Gly-Phe-Phe-Pro-

[0013] Asp-His-Gln-Leu-Asp-Pro-Ala-Phe-Gly-Ala-Asn-Ser-Asn-Asn-Pro-

[0014] Asp-Trp-Asp-Phe-Asn-Pro-Asn-Lys-Asp-His-Trp-Pro-Glu-Ala-Asn-

[0015] AA2-Val-Gly-AA3(R)-AA4

[0016] Structure I

[0017] In structure I, AA1 can be either Ac or H;

[0018] In structure I, AA2 can be Arg, Lys, Gln, Glu, or Cit;

[0019] In structure I, AA3 can be Lys, Dao, Dah, Orn, Dab, or Dap;

[0020] In structure I, AA4 is either NH2 or OH;

[0021] In structure I, R is either HO2C(CH2)n1CO-(AA5)n2-(PEGn3(CH2)n4CO)n5- or HO2C(CH2)n1CO-(AA5)n2-(AA6)n6-;

[0022] Where: n1 is an integer from 10 to 20;

[0023] n2 is an integer from 1 to 5;

[0024] n3 is an integer from 1 to 30;

[0025] n4 is an integer from 1 to 5;

[0026] n5 is 0, or an integer from 1 to 5;

[0027] n6 is 0, or an integer from 1 to 10;

[0028] AA5 may be γGlu, εLys, β-Ala, γ-aminobutyric acid, or 5-Ava.

[0029] AA6 can be Ala, Leu, Ser, Thr, Tyr, Gln, Lys, Dao, Dah, Orn, Dab, Dap, Glu, Asp, Ada, Apm, or Asu.

[0030] The long-acting hepatitis virus entry inhibitor of the present invention comprises a pharmaceutically usable salt, solvate, chelate or non-covalent complex, a drug prodrug based on the compound, or any mixture thereof.

[0031] The present invention also provides pharmaceutical compositions comprising compounds according to the present invention, and provides pharmaceutical uses of pharmaceutical compositions comprising compounds according to the present invention for the preparation of a medicine for treating diseases.

[0032] Furthermore, the pharmaceutical composition is used for the treatment of chronic hepatitis B and hepatitis D.

[0033] Further details of this invention are described in detail below, or some of them may be experienced in the embodiments of this invention.

[0034] Unless otherwise specified, the quantities of different components and reaction conditions used herein are to be interpreted as "approximate" or "about". Accordingly, unless otherwise specified, the numerical parameters cited below and in the claims are approximate parameters, and different numerical parameters may be obtained under their respective experimental conditions due to different standard errors.

[0035] In this document, when there is disagreement or ambiguity regarding the chemical structure and name of a compound, the compound is defined precisely by its chemical structure. The compounds described herein may contain one or more chiral centers, and / or double bonds and similar structures, and may also exist as stereoisomers, including isomers of double bonds (e.g., geometric isomers), optical enantiomers, or diastereomers. Accordingly, any chemical structure within the scope of this description, whether partially or entirely containing similar structures, includes all possible enantiomers and diastereomers of the compound, including any single stereoisomer (e.g., a single geometric isomer, a single enantiomer, or a single diastereomer) and any mixture of these isomers. Mixtures of racemic and stereoisomers can be further separated into enantiomers or stereoisomers of their constituent parts by those skilled in the art using continuous separation techniques or chiral molecule synthesis methods.

[0036] Compounds of Formula I include, but are not limited to, optical isomers, racemates, and / or other mixtures of these compounds. In the above cases, a single enantiomer or diastereomer, such as an optically active isomer, can be obtained by asymmetric synthesis or by racemic resolution. Racemic resolution can be achieved by various methods, such as conventional recrystallization with a resolving agent or by chromatographic methods. Additionally, compounds of Formula I also include cis and / or trans isomers with double bonds.

[0037] The compounds described in this invention include, but are not limited to, the compounds shown in structural formula I and all their various pharmaceutically available forms. These pharmaceutically available forms of the compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, drug prodrugs based on the above-described substances, and any mixtures of these forms. Detailed Implementation

[0038] This invention discloses a long-acting hepatitis virus entry inhibitor and its uses. Those skilled in the art can refer to the content of this document and appropriately modify the relevant parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The method of this invention has been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the compounds and preparation methods described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0039] The Chinese names corresponding to the English abbreviations used in this invention are shown in the table below:

[0040] English abbreviations Chinese name English abbreviations Chinese name Fmoc 9-fluorenemethyloxycarbonyl OtBu tert-butoxy tBu tert-butyl Boc tert-Butoxycarbonyl group Trt Triphenylmethyl Pbf (2,3-Dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl Ala alanine Leu Leucine Arg Arginine Lys Lysine Asn Asparagine Met Methionine Asp Aspartic acid Phe Phenylalanine Cys Cysteine Pro proline Gln glutamine Ser Serine Glu glutamic acid Thr threonine Gly glycine Trp Tryptophan His Histidine Tyr Tyrosine Ile Isoleucine Val Valine Aib Aminoisobutyric acid Dah 2,7-Diaminoheptanoic acid 5-Ava 5-Aminovaleric acid Dao 2,8-Diaminooctanoic acid Dap 2,3-Diaminopropionic acid Ada 2-Aminohexanoic acid Dab 2,4-Diaminobutyric acid Apm 2-Aminopeptidic acid Orn ornithine Asu 2-Aminooctanoic acid

[0041] Example 1: Preparation of the compound

[0042] The preparation method includes: preparing peptide resin using a solid-phase polypeptide synthesis method, followed by acid hydrolysis of the peptide resin to obtain a crude product, and finally purification of the crude product to obtain a pure product; wherein the solid-phase polypeptide synthesis method for preparing peptide resin involves sequentially inserting the corresponding protected amino acids or fragments from the following sequences onto a carrier resin via a solid-phase coupling synthesis method to prepare the peptide resin:

[0043] In the above preparation method, the amount of Fmoc-protected amino acid or protected amino acid fragment is 1.2 to 6 times the total molar amount of the resin added; preferably 2.5 to 3.5 times.

[0044] In the above preparation method, the substitution value of the carrier resin is 0.2 to 1.0 mmol / g resin, and the preferred substitution value is 0.3 to 0.5 mmol / g resin.

[0045] As a preferred embodiment of the present invention, the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step is deprotected by the Fmoc protecting group and then coupled with the next protected amino acid. The deprotection time for the Fmoc protecting group is 10-60 minutes, preferably 15-25 minutes. The coupling reaction time is 60-300 minutes, preferably 100-140 minutes.

[0046] The coupling reaction requires the addition of a condensing agent, selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolylphosphine hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate, or O-benzotriazol-N,N,N',N'-tetramethylurea tetrafluoroborate; preferably N,N-diisopropylcarbodiimide. The molar amount of the condensing agent is 1.2 to 6 times the total molar amount of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0047] The coupling reaction requires the addition of an activating agent, which is selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The amount of activating agent used is 1.2 to 6 times the total molar number of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0048] As a preferred embodiment of the present invention, the reagent for removing Fmoc protection is a PIP / DMF (piperidine / N,N-dimethylformamide) mixed solution, wherein the mixed solution contains 10-30% (V) piperidine. The amount of the Fmoc removal reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.

[0049] Preferably, the peptide resin is acid-hydrolyzed to remove both the resin and the side-chain protecting groups, yielding the crude product:

[0050] More preferably, the acid hydrolysate used during the acid hydrolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethylenedithiol (EDT) and water, with the volume ratio of the mixed solvent being: TFA 80-95%, EDT 1-10%, and the remainder being water.

[0051] More preferably, the volume ratio of the mixed solvent is: TFA 89-91%, EDT 4-6%, and the balance is water. Most preferably, the volume ratio of the mixed solvent is: TFA 90%, EDT 5%, and the balance is water.

[0052] The amount of acid hydrolysant used is 4 to 15 mL per gram of peptide resin; preferably, 7 to 10 mL per gram of peptide resin.

[0053] The pyrolysis time using the acid hydrolysate is 1 to 6 hours at room temperature, preferably 3 to 4 hours.

[0054] Furthermore, the crude product was purified by high performance liquid chromatography and lyophilized to obtain the pure product.

[0055] 1. Synthesis of peptide resins

[0056] Using RinkAmideBHHA resin as the carrier resin, the corresponding protected amino acids were sequentially added through de-Fmoc protection and coupling reactions.

[0057] (1) Access the first protected amino acid in the main chain

[0058] Take 0.03 mol of the first protected amino acid and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution for later use.

[0059] Take 0.01 mol of Rinkamide MBHA resin (substitution value of approximately 0.4 mmol / g), protect it with 20% PIP / DMF solution for 25 minutes, wash and filter to obtain Fmoc-free resin.

[0060] The activated solution of the first protected amino acid was added to the resin that had been de-Fmoc-treated, and the coupling reaction was carried out for 60–300 minutes. After filtration and washing, a resin containing one protected amino acid was obtained.

[0061] (2) Incorporate the 2nd to 49th protected amino acids in the main chain.

[0062] Using the same method as described above for adding the first protected amino acid to the main chain, the corresponding second to 49th protected amino acids were sequentially added to obtain a resin containing 49 amino acids in the main chain.

[0063] (3) Add the first protected amino acid to the side chain

[0064] Take 0.03 mol of the first protected amino acid on the side chain and 0.03 mol of HOBt, and dissolve them in an appropriate amount of DMF; take another 0.03 mol of DIC, and slowly add it to the DMF solution of the protected amino acid while stirring. Stir and react at room temperature for 30 minutes to obtain the activated protected amino acid solution.

[0065] Take 2.5 mmol of tetrakis(triphenylphosphine)palladium and 25 mmol of phenylsilane, dissolve them in an appropriate amount of dichloromethane, remove the protective layer for 4 hours, filter and wash to obtain the alloc-free resin for later use.

[0066] The activated side-chain first protected amino acid solution was added to the de-Allocated resin, and the coupling reaction was carried out for 60-300 minutes. After filtration and washing, a resin containing the first protected amino acid in the side chain was obtained.

[0067] (4) Add the 2nd to 4th protected amino acids to the side chain.

[0068] Using the same method as described above for adding the first protected amino acid to the main chain, the second to fourth protected amino acids and monoprotected fatty acids corresponding to the side chains are sequentially added to obtain peptide resin.

[0069] 2. Preparation of crude product

[0070] Take the above peptide resin and add a lysis reagent with a volume ratio of TFA:water:EDT = 95:5:5 (10 mL of lysis reagent per gram of resin). Stir well and react at room temperature for 3 hours. Filter the reaction mixture using a sintered glass funnel, collect the filtrate, wash the resin three times with a small amount of TFA, combine the filtrates and concentrate under reduced pressure. Add anhydrous diethyl ether to precipitate the precipitate, wash the precipitate three times with anhydrous diethyl ether, and dry under vacuum to obtain an off-white powder, which is the crude product.

[0071] 3. Preparation of pure products

[0072] Take the above crude product, add water and stir, adjust the pH to 8.0 with ammonia until completely dissolved, filter the solution through a 0.45μm mixed microporous membrane, and purify for later use;

[0073] Purification was performed using high performance liquid chromatography (HPLC). The chromatographic packing material was a 10 μm reversed-phase C18 column, and the mobile phase system was 0.1% TFA / water solution-0.1% TFA / acetonitrile solution. The flow rate of the 30 mm * 250 mm column was 20 mL / min. Gradient elution was used, and the sample was injected repeatedly for purification. The crude product solution was loaded into the column, the mobile phase was started for elution, the main peak was collected, and the acetonitrile was removed by evaporation to obtain the purified intermediate concentrate.

[0074] The purified intermediate concentrate was filtered through a 0.45 μm filter membrane and set aside. High-performance liquid chromatography (HPLC) was used for salt exchange. The mobile phase system was 1% acetic acid / water solution-acetonitrile. The chromatographic packing material was a 10 μm reversed-phase C18 column (30 mm * 250 mm) with a flow rate of 20 mL / min (the flow rate can be adjusted according to different column specifications). Gradient elution and cyclic loading were used. The sample was loaded into the column, the mobile phase was started for elution, the chromatogram was collected, the absorbance change was observed, the salt-exchanged main peak was collected, and the purity was determined by analytical liquid chromatography. The salt-exchanged main peak solutions were combined, concentrated under reduced pressure to obtain pure acetic acid aqueous solution, and then freeze-dried to obtain the pure product.

[0075] The following compounds were prepared using the method described above:

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] Example 2: Determination of the effect of inhibiting HBV infection

[0085] The experimental groups included a blank group, a solvent group, a positive control, and the test compound. The experimental concentration was 0.5 nM, and the positive control drug was MyrcludexB. The HBeAg / HBsAg ratio in the supernatant was measured every 3 days post-infection.

[0086] 1. Virus Concentration

[0087] (1) Collect the culture supernatant of hepAD38 cells and centrifuge the collected hepAD38 cell culture supernatant at 4℃ and 4000rpm / min for 30min to remove cell debris.

[0088] (2) Add the centrifuged virus supernatant to a 6×PEG8000 (Poly(ethyleneglycol), sigma, SLBZ3934) solution to dilute it to 1×PEG8000, and place it on a horizontal shaker at 4℃ and 20rpm / min overnight.

[0089] (3) The next day, the virus concentrate was centrifuged at 7000g at 4°C for 30 minutes and the supernatant was discarded. The precipitate was resuspended in DMEM medium without serum and antibiotics and the resuspended concentrated virus solution was stored at 4°C.

[0090] (4) Calculate the multiple of infection (MOI) of the concentrated virus solution: Take 20 μL of concentrated virus solution, add 180 μL of PBS to dilute, digest with DNase (to reduce the influence of residual plasmids), and perform column extraction with ViralDNA / RNA Kit. Quantify HBV DNA using qPCR detection with S region primers using standards to calculate the multiple of infection, which is the ratio of infected virus to the number of cells.

[0091] 2. Infection experiments and detection

[0092] (1) HepG2-NTCP cells were cultured in 10% FBSDMEM medium with 4 μg / mL LDoxycycline for 4 days to induce NTCP expression;

[0093] (2) On the night before the fourth day of culture, HepG2-NTCP cells were seeded into 48-well culture dishes coated with type I rat tail collagen (200 μL collagen solution), with 4*10^4 cells per well;

[0094] (3) On the second day, the culture medium was replaced with HCM (HBMTM, LONZA, CC-3199, singleQuots, LONZA, CC-4182) to induce HepG2-NTCP cell proliferation arrest. Doxycycline was added, along with the drug to be tested.

[0095] (4) After culturing with HCM for 24 hours, discard the HCM, add the concentrated virus solution to the culture dish at 1600 MOI, add HCM to make up to 150 μL, add the drug to be tested, add 150 μL of 8% PEG8000, 2% DMSO, and 4 μg / mL Doxycycline, mix gently, and incubate overnight in a 37°C incubator.

[0096] (5) After 16 hours of infection, discard the supernatant, wash 5 times with PBS, and continue culturing with 300 μL LMEM (containing 10% FBS, 2% DMSO, and 4 μg / mL L oxycycline);

[0097] (6) The culture medium was changed every three days thereafter. 300 μL of cell culture supernatant was collected at the corresponding time point, centrifuged at 3000 g for 3 min to remove cell precipitate, and the supernatant was tested for HBsAg and HBeAg using the fully automated chemiluminescence immunoassay system MAGLUMIX3 (New Industries Biosciences).

[0098] 2. Test Results

[0099] The test results are shown in the table below.

[0100]

[0101]

[0102] Example 3: Determination of Preliminary Pharmacokinetic Properties

[0103] The experimental animals were cynomolgus monkeys, with two males in each compound group. The drugs were administered subcutaneously at a dose of 0.1 mg / kg. Blood samples were collected intravenously at 0 h before administration and at 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. Plasma samples were separated by centrifugation, and the plasma concentrations of the corresponding compounds were determined using liquid chromatography-mass spectrometry (LC-MS). The half-lives of the compounds after subcutaneous (SC) administration are shown in the table below.

[0104] compound <![CDATA[t 1 / 2 (h)]]> Compound 6 61.8

Claims

1. A long-acting hepatitis virus entry inhibitor or a pharmaceutically acceptable salt thereof having the following structure: Ac-Gly-Thr-Asn-Leu-Ser-Val-Pro-Asn-Pro-Leu-Gly-Phe-Phe-Pro- Asp-His-Gln-Leu-Asp-Pro-Ala-Phe-Gly-Ala-Asn-Ser-Asn-Asn-Pro- Asp-Trp-Asp-Phe-Asn-Pro-Asn-Lys-Asp-His-Trp-Pro-Glu-Ala-Asn- Lys-Val-Gly-Lys (COCH2PEG5-γGlu-20 alkyl dicolic acid)-NH2.

2. A pharmaceutical composition comprising the long-acting hepatitis virus entry inhibitor of claim 1 or a pharmaceutically acceptable salt thereof.

3. The use of the long-acting hepatitis virus entry inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2, in the preparation of a medicament for treating hepatitis B.

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