WO2023151358A1 - 一种长效肝炎病毒进入抑制剂 - Google Patents

一种长效肝炎病毒进入抑制剂 Download PDF

Info

Publication number
WO2023151358A1
WO2023151358A1 PCT/CN2022/136263 CN2022136263W WO2023151358A1 WO 2023151358 A1 WO2023151358 A1 WO 2023151358A1 CN 2022136263 W CN2022136263 W CN 2022136263W WO 2023151358 A1 WO2023151358 A1 WO 2023151358A1
Authority
WO
WIPO (PCT)
Prior art keywords
hepatitis
gly
asn
pro
integer
Prior art date
Application number
PCT/CN2022/136263
Other languages
English (en)
French (fr)
Inventor
于海宁
王鹏
周述靓
高俊萍
Original Assignee
成都奥达生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 成都奥达生物科技有限公司 filed Critical 成都奥达生物科技有限公司
Publication of WO2023151358A1 publication Critical patent/WO2023151358A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a long-acting hepatitis virus entry inhibitor and its application.
  • Viral hepatitis is an infectious disease mainly caused by liver lesions caused by a variety of hepatitis viruses. Clinically, the main manifestations are loss of appetite, nausea, upper abdominal discomfort, pain in the liver area, and fatigue. Some patients may have jaundice, fever and hepatomegaly with liver function damage. Some patients can become chronic, and even develop into liver cirrhosis, and a few can develop into liver cancer.
  • hepatitis B virus is a DNA virus
  • the rest are RNA viruses.
  • Hepatitis A virus is a Hepadnavirus genus in the family Picornaviridae. After human infection with HAV, most of them showed subclinical or recessive infection, and only a few showed acute hepatitis A. Generally, it can be fully recovered without turning into chronic hepatitis, and there are no chronic carriers.
  • Hepatitis B virus is the pathogen that causes hepatitis B. It belongs to the family Hepadnaviridae, which includes two genera, Orthohepadnavirus and Avian Hepadnavirus. DNA virus genus. HBV infection is a global public health problem. With the production and investment of genetically engineered vaccines, the popularity of hepatitis B vaccine has increased year by year, and the infection rate has shown a downward trend. There are about 2 billion HBV-infected patients in the world, of which 380 million infected patients are chronically infected. The virus will hide in tissues and organs and cannot be completely cleared by the immune system and drugs.
  • Hepatitis C virus is mainly transmitted vertically through blood, sex life, and mother to child.
  • HCV Hepatitis C virus
  • the medical community has not yet developed an effective vaccine against hepatitis C.
  • hepatitis C virus is an RNA virus, it is very easy to mutate, and it is very difficult to develop a vaccine, because other animals except humans and chimpanzees do not suffer from hepatitis C, so it is difficult to find an animal model for vaccine development, but the new generation of oral direct antiviral Drugs have cured hepatitis C.
  • HDV is a defective single-stranded negative-strand RNA virus, which must rely on HBV and other hepadnaviruses to provide a shell for it to replicate.
  • HDV exists in the nuclei and serum of HBsAg-positive HDV-infected patients. Replicates mainly in hepatocytes.
  • HDV is prone to mutation.
  • the synthesis of HBV-DNA can be significantly inhibited after human infection with HDV.
  • the appearance of HDAg is consistent with the reduction of HBV-DNA in serum. As HDAg turns negative and anti-HD appears, HBV-DNA returns to the original level. It is mainly transmitted through blood transfusion and blood products, and is similar to the transmission mode of hepatitis B.
  • HDV infection is mostly seen in HBV-infected persons, and sporadic HDV-infected persons can also be seen. After superinfection of HDV and HBV, liver damage can be aggravated, and it is easy to develop into chronic active hepatitis, liver cirrhosis and severe hepatitis.
  • NTCP hepatic bile acid transporter
  • Bulevirtide is a drug targeting NTCP containing 47 amino acids. Due to the short half-life of Bulevirtide in the body, patients need to take daily and long-term medication, which leads to poor patient compliance and high clinical costs.
  • the purpose of the present invention is to provide patients with a long-acting hepatitis virus entry inhibitor, reduce the frequency of administration, and reduce the cost of patients.
  • the invention provides a long-acting hepatitis virus entry inhibitor and its application.
  • the present invention firstly provides a compound shown in structure I, the pharmaceutically acceptable salt, solvate, chelate or non-covalent complex formed by the compound, and the drug based on the compound precursors, or any mixture of the above forms.
  • AA1 in structure I is Arg, or Lys, or Gln, or Glu, or Cit;
  • AA2 in structure I is NH 2 , or is OH;
  • R in structure I 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-;
  • 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 ⁇ Glu, or ⁇ Lys, or ⁇ -Ala, or ⁇ -aminobutyric acid, or 5-Ava.
  • AA4 is Ala, or Gly, or Leu, or Ser, or Thr, or Tyr, or Gln, or Lys, or Dao, or Dah, or Orn, or Dab, or Dap, or Glu, or Asp, or Ada, or Apm, or Asu.
  • n6 is an integer of 2-10, and the repeating unit of AA4 may be the same amino acid or different amino acids.
  • the AA1 is Arg, or Lys, or Gln, and the AA2 is NH 2 .
  • the R is 20 alkanedioic acid- ⁇ Glu-(AA4)n6-, wherein, AA4 is Gly, Ser, Thr, Asn, Leu, Val or Pro; n6 is 0, or an integer from 1 to 7 . In some other embodiments, the R is 20 alkanedioic acid- ⁇ Glu-PEGn3-CH 2 CO-; wherein, n3 is an integer from 1 to 6.
  • R is 20 alkanedioic acid- ⁇ Glu-
  • the long-acting hepatitis virus entry inhibitor of the present invention comprises the formed pharmaceutically acceptable salt, solvate, chelate or non-covalent complex, the prodrug based on the compound, or the above-mentioned form any mixture.
  • the present invention also provides a pharmaceutical composition comprising the compound according to the present invention, and the use of the pharmaceutical composition providing the compound of the present invention for preparing a medicine for treating diseases.
  • the pharmaceutical composition is used for the treatment of chronic hepatitis B and hepatitis D.
  • the chemical structural formula is used to define the compound exactly.
  • the compounds described herein may contain one or more chiral centers, and/or double bonds and the like, and may also exist as stereoisomers, including double bond isomers (such as geometric isomers), optically active Enantiomers or diastereomers.
  • any chemical structure within the scope of the description herein, whether it contains the above-mentioned similar structures in part or in the whole structure, includes all possible enantiomers and diastereoisomers of this compound, including Any single stereoisomer (such as a single geometric isomer, a single enantiomer or a single diastereoisomer) and any mixture of these isomers are contemplated. These mixtures of racemic isomers and stereoisomers can also be further resolved into enantiomers or stereoisomers of their constituents by those skilled in the art using continuous separation techniques or chiral molecular synthesis methods body.
  • Compounds of formula I include, but are not limited to, optical isomers, racemates and/or other mixtures of these compounds.
  • a single enantiomer or diastereoisomer such as an optically active isomer, can be obtained by asymmetric synthesis or racemate resolution.
  • the resolution of the racemate can be achieved by different methods, such as conventional recrystallization with resolution aids, or chromatography.
  • the compounds of formula I also include cis and/or trans isomers with double bonds.
  • the compounds of the present invention include, but are not limited to, the compounds represented by structural formula I and all their different pharmaceutically available forms.
  • Various pharmaceutically acceptable forms of these compounds include various pharmaceutically acceptable salts, solvates, complexes, chelates, non-covalent complexes, prodrugs based on the above-mentioned substances and combinations of the above-mentioned forms. any mixture.
  • the invention discloses a long-acting hepatitis virus entry inhibitor and its use. Those skilled in the art can learn from the content of this article and appropriately improve relevant parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention.
  • the method of the present invention has been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the compounds and preparation methods described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.
  • the preparation method includes: preparing the peptide resin by solid-phase polypeptide synthesis method, acid hydrolyzing the peptide resin to obtain a crude product, and finally purifying the crude product to obtain a pure product; wherein the step of preparing the peptide resin by the solid-phase polypeptide synthesis method is to pass solid-phase
  • the phase-coupled synthesis method sequentially inserts the corresponding protected amino acids or fragments in the following sequences to prepare peptide resins:
  • the amount of the Fmoc-protected amino acid or protected amino acid fragment is 1.2-6 times of the total moles of the resin fed; preferably 2.5-3.5 times.
  • the substitution value of the carrier resin is 0.2-1.0 mmol/g resin, preferably 0.3-0.5 mmol/g resin.
  • the solid-phase coupling synthesis method is as follows: the protected amino acid-resin obtained in the previous step reaction removes the Fmoc protecting group and then reacts with the next protected amino acid for coupling reaction.
  • the deprotection time for the de-Fmoc protection is 10-60 minutes, preferably 15-25 minutes.
  • the coupling reaction time is 60-300 minutes, preferably 100-140 minutes.
  • the coupling reaction needs to add a condensation reagent, and the condensation reagent is selected from DIC (N,N-diisopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazole hexafluorophosphate -1-yl-oxytripyrrolidinylphosphonium, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate , Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate or O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoro
  • One of borates preferred is N,N-diisopropylcarbodiimide.
  • the molar dosage of the condensation reagent is 1.2-6 times, preferably 2.5-3.5 times, the total
  • the coupling reaction requires the addition of an activating reagent selected from 1-hydroxybenzotriazole or N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole.
  • the dosage of the activating agent is 1.2 to 6 times, preferably 2.5 to 3.5 times, the total moles of amino groups in the amino resin.
  • the reagent for removing Fmoc protection is a PIP/DMF (piperidine/N,N-dimethylformamide) mixed solution, and the mixed solution contains 10-30% of piperidine (V ).
  • the dosage of the de-Fmoc protection reagent is 5-15 mL per gram of amino resin, preferably 8-12 mL per gram of amino resin.
  • the peptide resin is subjected to acid hydrolysis to simultaneously remove the resin and side chain protecting groups to obtain the crude product:
  • the acidolysis agent used during acidolysis of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, and the volume ratio of the mixed solvent is: TFA 80-95%, EDT 1-10%, and the balance is water.
  • the volume ratio of the mixed solvent is: TFA is 89-91%, EDT is 4-6%, and the balance is water.
  • the volume ratio of the mixed solvent is: TFA is 90%, EDT is 5%, and the balance is water.
  • the dosage of the acidolysis agent is 4-15mL of acidolysis agent per gram of peptide resin; preferably, 7-10mL of acidolysis agent is required per gram of peptide resin.
  • the time for cleavage using an acidolysis agent is 1-6 hours at room temperature, preferably 3-4 hours.
  • the crude product was purified by high performance liquid chromatography and freeze-dried to obtain the pure product.
  • High performance liquid chromatography is used for purification, the chromatographic filler for purification is 10 ⁇ m reversed-phase C18, the mobile phase system is 0.1% TFA/water solution-0.1% TFA/acetonitrile solution, and the flow rate of the 30mm*250mm chromatographic column is 20mL/min.
  • Gradient system elution, cyclic sample injection and purification take the crude product solution and load it on the chromatographic column, start the mobile phase elution, collect the main peak and evaporate the acetonitrile to obtain the concentrated solution of the purified intermediate;
  • the concentrated solution of the purified intermediate is filtered with a 0.45 ⁇ m filter membrane for later use, and the salt is replaced by high-performance liquid chromatography.
  • the mobile phase system is 1% acetic acid/water solution-acetonitrile, and the chromatographic filler for purification is 10 ⁇ m reversed-phase C18, 30mm*250mm
  • the flow rate of the chromatographic column is 20mL/min (the corresponding flow rate can be adjusted according to different specifications of the chromatographic column); gradient elution is adopted, and the method of cyclic loading is used to load the sample into the chromatographic column, start the mobile phase elution, collect the spectrum, and observe According to the change of absorbance, the main peak of salt-changing was collected and the purity was detected by analytical liquid phase, and the solution of the main peak of salt-changing was combined, concentrated under reduced pressure to obtain pure acetic acid aqueous solution, which was freeze-dried to obtain pure product.
  • test groups are blank group, solvent group, positive control and test compound, the test concentration is 0.5nM, the positive drug is Myrcludex B, and the detection time is to detect HBeAg/HBsAg in the supernatant every 3 days after infection
  • HepG2-NTCP cells were cultured with 10% FBS DMEM medium adding 4 ⁇ g/mL Doxycycline for 4 days to induce NTCP expression;
  • HCM HBMTM, LONZA, CC-3199 ⁇ singleQuots, LONZA, CC-4182
  • Embodiment 3 Determination of preliminary pharmacokinetic properties
  • test animals were cynomolgus monkeys, 2 male cynomolgus monkeys in each compound group, administered subcutaneously at a dose of 0.1mg/kg, respectively before the drug (0h), and 1h, 2h, 3h, 4h, Take blood from vein at 8h, 12h, 18h, 24h, 48h, 96h, 144h, and 168h, centrifuge the plasma sample, and measure the blood concentration of the corresponding compound in the plasma sample by liquid chromatography-mass spectrometry.
  • SC subcutaneous

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Communicable Diseases (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明涉及医药合成领域，公开了一种长效肝炎病毒进入抑制剂。本发明所述长效肝炎病毒进入抑制剂，用于制备治疗疾病的药物组合物，所述药物组合物用于慢性乙肝和丁肝的治疗。

Description

一种长效肝炎病毒进入抑制剂
本申请要求于2022年02月10日提交中国专利局、申请号为202210126691.4、发明名称为“一种长效肝炎病毒进入抑制剂”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及一种长效肝炎病毒进入抑制剂及其用途。
背景技术
病毒性肝炎是由多种肝炎病毒引起的以肝脏病变为主的一种传染病。临床上以食欲减退、恶心、上腹部不适、肝区痛、乏力为主要表现。部分病人可有黄疸发热和肝大伴有肝功能损害。有些病人可慢性化，甚至发展成肝硬化，少数可发展为肝癌。
病毒性肝炎的病原学分型，目前已被公认的有甲、乙、丙、丁、戊五种肝炎病毒，分别写作HAV、HBV、HCV、HDV、HEV，除乙型肝炎病毒为DNA病毒外，其余均为RNA病毒。
甲型肝炎病毒(HAV)为小RNA病毒科嗜肝病毒属。人类感染HAV后，大多表现为亚临床或隐性感染，仅少数人表现为急性甲型肝炎。一般可完全恢复，不转为慢性肝炎，亦无慢性携带者。
乙型肝炎病毒(HBV)是引起乙型肝炎的病原体，属嗜肝DNA病毒科，该科病毒包含正嗜肝DNA病毒属和禽嗜肝DNA病毒属两个属，引起人体感染的是正嗜肝DNA病毒属。HBV感染是全球性的公共卫生问题，随着基因工程疫苗的生产和投入，乙肝疫苗的普及率逐年上升，感染率呈下降趋势。全球大约有20亿的HBV感染患者，其中有3.8亿感染患者属于慢性感染，病毒会藏匿于组织和器官中而无法被免疫系统和药物彻底清除。
丙型肝炎病毒(HCV)，主要通过血液、性生活、母婴垂直传播。但是非常遗憾的是医学界尚未研制出有效预防丙肝的疫苗。因为丙肝病毒是 RNA病毒，极易变异，研制疫苗的难度很大，因为除了人和黑猩猩以外，其他动物都不会患上丙肝，因此疫苗研制难以找到动物模型，但新一代的口服直接抗病毒药物让丙肝实现了治愈。
HDV为一有缺陷的单股负链RNA病毒，必需依赖HBV等嗜肝DNA病毒为其提供外壳，才能进行复制。HDV存在于HBsAg阳性的HDV感染者的肝细胞核内和血清中。主要在肝细胞内复制。HDV易发生变异。人感染HDV后可明显抑制HBV-DNA的合成，HDAg出现与血清中HBV-DNA减少相一致，随着HDAg转阴和抗-HD出现，HBV-DNA又恢复到原水平。主要通过输血和血制品传播，与乙型肝炎的传播方式相似，HDV感染大多见于HBV感染者，也可见散发性HDV感染者。HDV与HBV重叠感染后，可促使肝损害加重，并易发展为慢性活动性肝炎、肝硬化和重型肝炎。
研究人员发现，肝细胞表面一种叫做肝脏胆汁酸转运体(NTCP，牛磺胆酸钠共转运多肽)的蛋白能够与HBV包膜蛋白的关键受体结合域发生特异性相互作用，正是HBV感染宿主细胞所需的受体。因此，阻断NTCP，就有希望治愈乙肝。
Bulevirtide是一个含47个氨基酸的靶向NTCP的药物，由于Bulevirtide在体内半衰期短，患者需每天并长期用药，患者顺应性差，临床费用高。本发明的目的就是为患者提供长效的肝炎病毒进入抑制剂，减少给药频率，降低患者费用。
发明内容
本发明提供了一种长效肝炎病毒进入抑制剂及其用途。
为实现上述目的，本发明首先提供了一种结构I所示的化合物，该化合物所成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物。
R-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-AA1-Val-Gly-AA2
结构I
结构I中的AA1为Arg，或为Lys，或为Gln，或为Glu，或为Cit；
结构I中的AA2为NH 2，或为OH；
结构I中的R为HO 2C(CH 2)n1CO-(AA3)n2-(PEGn3(CH 2)n4CO)n5-，或为HO 2C(CH 2)n1CO-(AA3)n2-(AA4)n6-；
其中：n1为10至20的整数；
n2为1至5的整数；
n3为1至30的整数；
n4为1至5的整数；
n5为0，或1至5的整数；
n6为0，或1至10的整数；
AA3为γGlu，或为εLys，或为β-Ala，或为γ-氨基丁酸，或为5-Ava。
AA4为Ala，或为Gly，或为Leu，或为Ser，或为Thr，或为Tyr，或为Gln，或为Lys，或为Dao，或为Dah，或为Orn，或为Dab，或为Dap，或为Glu，或为Asp，或为Ada，或为Apm，或为Asu。
n6为2～10的整数，则AA4的重复单元可为相同的氨基酸也可为不同的氨基酸。
一些实施例中，所述AA1为Arg，或为Lys，或为Gln，所述AA2为NH 2。
一些实施例中，所述R为20烷二酸-γGlu-(AA4)n6-，其中，AA4为Gly、Ser、Thr、Asn、Leu、Val或Pro；n6为0，或1至7的整数。另一些实施例中，所述R为20烷二酸-γGlu-PEGn3-CH 2CO-；其中，n3为1至6的整数。
一些实施例中，R为20烷二酸-γGlu-
或为20烷二酸-γGlu-PEG 1CH 2CO-
或为20烷二酸-γGlu-PEG 2CH 2CO-
或为20烷二酸-γGlu-PEG 3CH 2CO-
或为20烷二酸-γGlu-PEG 4CH 2CO-
或为20烷二酸-γGlu-PEG 5CH 2CO-
或为20烷二酸-γGlu-PEG 6CH 2CO-
或为20烷二酸-γGlu-Gly-
或为20烷二酸-γGlu-Gly-Gly-
或为20烷二酸-γGlu-Gly-Gly-Gly-
或为20烷二酸-γGlu-Gly-Gly-Gly-Gly-
或为20烷二酸-γGlu-Gly-Gly-Gly-Gly-Gly-
或为20烷二酸-γGlu-Gly-Gly-Gly-Gly-Gly-Gly-Gly-
或为20烷二酸-γGlu-Gly-Gly-Ser-Gly-Ser-Gly-
或为20烷二酸-γGlu-Gly-Thr-Asn-Leu-Ser-Val-Pro-Asn-Pro-Leu-。
本发明所述的长效肝炎病毒进入抑制剂，包含所成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物。
本发明还提供了包括根据本发明化合物的药物组合物，以及提供了本发明化合物的药物组合物用于制备治疗疾病的药物用途。
进一步地，所述药物组合物用于慢性乙肝和丁肝的治疗。
本发明所涉及到的更多内容在以下有详细描述，或者有些也可以在本发明的实施例中体会。
除非另有所指，本文中所用来表示不同成分的数量、反应条件，在任意情况下都可解读为“大致的”、“大约的”意思。相应的，除有明确的特指外，在下述以及权利要求中所引用的数字参数都是大致的参数，在各自的实验条件下由于标准误差的不同，有可能会得到不同的数字参数。
本文中，当一个化合物的化学结构式和化学名称有分歧或疑义时，以化学结构式确切定义此化合物。本文所描述的化合物有可能含有一个或多个手性中心，和/或者双键以及诸如此类的结构，也可能存在立体异构体，包括双键的异构体(比如几何异构体)、旋光对映异构体或者非对映异构体。相应的，在本文描述范围内的任意化学结构，无论是部分或整体结构中含有上述类似结构，都包括了此化合物的所有可能的对映异构体和非对映异构体，其中也包括了单纯的任一种立体异构体(如单纯的几何异 构体、单纯的对映异构体或者单纯的非对映异构体)以及这些异构体的任意一种混合物。这些消旋异构体和立体异构体的混合物由本领域技术人员利用不停的分离技术或手性分子合成的方法也可进一步被拆分成其组成成分的对映异构体或立体异构体。
结构式I的化合物包含了，但并不仅限于，这些化合物的光学异构体、消旋体和/或其他的混合物。上述情况下，其中单一的对映异构体或非对映异构体，如有旋光的异构体，可以用不对称合成的方法或消旋体拆分的方法获得。消旋体的拆分可用不同的方法实现，如常规的用助拆分的试剂重结晶，或用色谱方法。另外，结构式I的化合物也包含了带双键的顺式和/或反式的异构体。
本发明所述化合物包含但不限于，结构式I所示化合物以及他们所有的在药学上可用的不同形式。这些化合物的药学上可用的不同形式包括各种可药用的盐、溶剂化物、络合物、螯合物、非共价的复合物、基于上述物质基础上的药物前体和上述这些形式的任意混合物。
具体实施方式
本发明公开了一种长效肝炎病毒进入抑制剂及其用途，本领域技术人员可以借鉴本文内容，适当改进相关参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的的化合物和制备方法进行改动或适当变更与组合，来实现和应用本发明技术。
本发明中涉及的英文缩写所对应的中文名称见下表所示：
Figure PCTCN2022136263-appb-000001
Figure PCTCN2022136263-appb-000002
实施例1 化合物的制备
制备方法，包括：采用固相多肽合成法制备肽树脂，肽树脂再经酸解得到粗品，最后粗品经过纯化得到纯品；其中固相多肽合成法制备肽树脂的步骤为在载体树脂上通过固相偶联合成法依次接入下列序列中相对应的保护氨基酸或片段，制备肽树脂：
上述制备方法中，所述的Fmoc-保护氨基酸或保护氨基酸片段的用量为所投料树脂总摩尔数的1.2～6倍；优选为2.5～3.5倍。
上述制备方法中，所述的载体树脂取代值为0.2～1.0mmol/g树脂，优选的取代值为0.3～0.5mmol/g树脂。
作为本发明优选的方案，所述固相偶联合成法为：前一步反应得到的保护氨基酸-树脂脱去Fmoc保护基后再与下一个保护氨基酸偶联反应。所述的去Fmoc保护的脱保护时间为10～60分钟，优选的为15～25分钟。所述的偶联反应时间为60～300分钟，优选的为100～140分钟。
所述的偶联反应需添加缩合试剂，缩合试剂选自DIC(N,N-二异丙基 碳二亚胺)、N,N-二环己基碳二亚胺，六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、2-(7-氮杂-1H-苯并三氮唑-1-基)-1,1,3,3-四甲基脲六氟磷酸酯、苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐或O-苯并三氮唑-N,N,N',N'-四甲基脲四氟硼酸酯中的一种；优选的为N,N-二异丙基碳二亚胺。所述缩合试剂的摩尔用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选为2.5～3.5倍。
所述的偶联反应需添加活化试剂，活化试剂选自1-羟基苯并三唑或N-羟基-7-氮杂苯并三氮唑，优选的为1-羟基苯并三唑。活化试剂的用量为氨基树脂中氨基总摩尔数的1.2～6倍，优选的为2.5～3.5倍。
作为本发明优选的方案，所述的脱去Fmoc保护的试剂为PIP/DMF(哌啶/N,N-二甲基甲酰胺)混合溶液，混合溶液中含哌啶为10～30％(V)。去Fmoc保护试剂的用量为每克氨基树脂5～15mL，优选的为每克氨基树脂8～12mL。
优选的，肽树脂经酸解同时脱去树脂及侧链保护基得到粗品：
进一步优选的，所述肽树脂酸解时采用的酸解剂为三氟醋酸(TFA)、1,2-乙二硫醇(EDT)和水的混合溶剂，混合溶剂的体积配比为：TFA为80～95％，EDT为1～10％，余量为水。
更进一步优选的，混合溶剂的体积配比为：TFA为89～91％、EDT为4～6％，余量为水。最优的，混合溶剂的体积配比为：TFA为90％、EDT为5％，余量为水。
所述酸解剂用量为每克肽树脂需要4～15mL酸解剂；优选的，每克肽树脂需要7～10mL酸解剂。
使用酸解剂裂解的时间为室温条件下1～6小时，优选的为3～4小时。
进一步的，粗品经高效液相色谱纯化、冻干得到纯品。
1、肽树脂的合成
使用RinkAmide BHHA树脂为载体树脂，通过去Fmoc保护和偶联反应，依次接入相对应的保护氨基酸。
(1)接入主链第1个保护氨基酸
取0.03mol第1个保护氨基酸和0.03mol HOBt，用适量DMF溶解；另取0.03mol DIC，搅拌下慢慢加入至保护氨基酸DMF溶液中，于室温环境中搅拌反应30分钟，得到活化后的保护氨基酸溶液，备用。
取0.01mol的Rink amide MBHA树脂(取代值约0.4mmol/g)，采用20％PIP/DMF溶液去保护25分钟，洗涤过滤得到去Fmoc的树脂。
将活化后的第1个保护氨基酸溶液加入到已去Fmoc的树脂中，偶联反应60～300分钟，过滤洗涤，得含1个保护氨基酸的树脂。
(2)接入主链第2～49个保护氨基酸
采用上述接入主链第1个保护氨基酸同样方法，依次接入上述对应的第2～49个保护氨基酸，得含主链49个氨基酸的树脂。
(3)接入侧链第1个保护氨基酸
取0.03mol侧链第1个保护氨基酸和0.03mol HOBt，用适量DMF溶解；另取0.03mol DIC，搅拌下慢慢加入至保护氨基酸DMF溶液中，于室温环境中搅拌反应30分钟，得到活化后的保护氨基酸溶液。
取2.5mmol四三苯基膦钯和25mmol苯硅烷，用适量二氯甲烷溶解，去保护4小时，过滤洗涤，得到去Alloc的树脂备用。
将加入活化后的侧链第1个保护氨基酸液加入到已去Alloc的树脂，偶联反应60～300分钟，过滤洗涤，得含侧链第1个保护氨基酸的树脂。
(4)接入侧链第2～4个保护氨基酸
采用上述接入主链第1个保护氨基酸同样方法，依次接入侧链对应的第2～4个保护氨基酸和单保护脂肪酸，得到肽树脂。
2、粗品的制备
取上述肽树脂，加入体积比为TFA︰水︰EDT＝95︰5︰5的裂解试剂(裂解试剂10mL/克树脂)，搅拌均匀，室温搅拌反应3小时，反应混合物使用砂芯漏斗过滤，收集滤液，树脂再用少量TFA洗涤3次，合并滤液后减压浓缩，加入无水乙醚沉淀，再用无水乙醚洗沉淀3次，抽干得类白色粉末即为粗品。
3、纯品的制备
取上述粗品，加水搅拌，用氨水调pH8.0至完全溶解，溶液用0.45μm 混合微孔滤膜过滤，纯化备用；
采用高效液相色谱法进行纯化，纯化用色谱填料为10μm的反相C18，流动相系统为0.1％TFA/水溶液-0.1％TFA/乙腈溶液，30mm*250mm的色谱柱流速为20mL/min，采用梯度系统洗脱，循环进样纯化，取粗品溶液上样于色谱柱中，启动流动相洗脱，收集主峰蒸去乙腈后，得纯化中间体浓缩液；
纯化中间体浓缩液用0.45μm滤膜滤过备用，采用高效液相色谱法进行换盐，流动相系统为1％醋酸/水溶液-乙腈，纯化用色谱填料为10μm的反相C18，30mm*250mm的色谱柱流速为20mL/min(可根据不同规格的色谱柱，调整相应的流速)；采用梯度洗脱，循环上样方法，上样于色谱柱中，启动流动相洗脱，采集图谱，观测吸收度的变化，收集换盐主峰并用分析液相检测纯度，合并换盐主峰溶液，减压浓缩，得到纯品醋酸水溶液，冷冻干燥，得纯品。
用上述方法制备了以下化合物：
Figure PCTCN2022136263-appb-000003
Figure PCTCN2022136263-appb-000004
Figure PCTCN2022136263-appb-000005
Figure PCTCN2022136263-appb-000006
Figure PCTCN2022136263-appb-000007
Figure PCTCN2022136263-appb-000008
Figure PCTCN2022136263-appb-000009
Figure PCTCN2022136263-appb-000010
实施例2 抑制HBV感染的效果的测定
试验组别为空白组、溶剂组、阳性对照及待测化合物，试验浓度为0.5nM，阳性药物为Myrcludex B，检测时间为感染后每3天检测上清HBeAg/HBsAg
1、病毒浓缩
(1)培养hepAD38细胞收集培养上清，将收集的hepAD38细胞培养上 清进行4℃、4000rpm/min离心30min,以去除细胞碎片；
(2)将离心后的病毒上清加入6×PEG8000(Poly(ethylene glycol),sigma,SLBZ3934)溶液中使其稀释成1×PEG8000，置于水平摇床上4℃、20rpm/min过夜；
(3)次日，将病毒浓缩液4℃、7000g离心30min后弃上清；将沉淀用无血清无双抗的DMEM培养基重悬，将重悬好的浓缩病毒液4℃保存。
(4)计算病毒浓缩液感染复数(Multiplicity of infection，MOI)：取20μL病毒浓缩液，加入180μL PBS稀释，用DNase消化后(减少残留质粒的影响)用Viral DNA/RNA Kit试剂盒进行柱提，并用标准品通过S区引物qPCR检测进行HBV DNA定量，以备计算病毒感染复数，即感染病毒与细胞数量的比值。
2、感染实验及检测
(1)HepG2-NTCP细胞用加入4μg/mL Doxycycline的10％FBS DMEM培养基培养4天诱导NTCP表达；
(2)培养第四天前一晚将HepG2-NTCP细胞铺至I型鼠尾胶原包被的48孔培养皿中(200μL胶原溶液)，每孔4*10^4个细胞；
(3)第二天将培养基更换成HCM(HBMTM，LONZA，CC-3199\singleQuots,LONZA,CC-4182)诱导HepG2-NTCP细胞增殖阻滞，另外添加Doxycycline，同时加入待测试药物；
(4)用HCM培养24h后，弃去HCM，将浓缩病毒液按1600MOI加至培养皿中，用HCM补至150μL，同时加入待测试药物，再加入8％PEG8000 150μL、2％DMSO、4μg/mL Doxycycline，轻轻混匀后与37℃培养箱中感染过夜；
(5)感染16h后，弃上清，PBS洗5次，换用300μL DMEM(含10％FBS、2％DMSO、4μg/mL Doxycycline)继续培养；
(6)之后每三天更换一次培养基，收集相应时间点细胞培养上清300ul，3000g离心3min去细胞沉淀，上清通过全自动化学发光免疫分析系统MAGLUMI X3(新产业生物)检测HBsAg、HBeAg。
2、试验结果
试验结果见下表。
Figure PCTCN2022136263-appb-000011
Figure PCTCN2022136263-appb-000012
实施例3 初步药代特性的测定
试验动物为食蟹猴，每个化合物组雄性食蟹猴各2只，皮下给药，剂量为0.1mg/kg，分别于药前(0h)、以及给药后1h、2h、3h、4h、8h、12h、18h、24h、48h、96h、144h、168h静脉取血，离心分离血浆样本，用液质联用法分别测定血浆样本中相应化合物的血药浓度，化合物皮下(SC)给药半衰期见下表：
化合物 t 1/2(h)
化合物6 90.6

Claims (10)

  1. 具有结构式Ⅰ的化合物：
    R-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-AA1-Val-Gly-AA2
    结构I
    结构I中的AA1为Arg，或为Lys，或为Gln，或为Glu，或为Cit；
    结构I中的AA2为NH 2，或为OH；
    结构I中的R为HO 2C(CH 2)n1CO-(AA3)n2-(PEGn3(CH 2)n4CO)n5-，或为HO 2C(CH 2)n1CO-(AA3)n2-(AA4)n6-；
    其中：n1为10至20的整数；
    n2为1至5的整数；
    n3为1至30的整数；
    n4为1至5的整数；
    n5为0，或1至5的整数；
    n6为0，或1至10的整数；
    AA3为γGlu，或为εLys，或为β-Ala，或为γ-氨基丁酸，或为5-Ava；
    AA4为Ala，或为Gly，或为Leu，或为Ser，或为Thr，或为Tyr，或为Gln，或为Lys，或为Dao，或为Dah，或为Orn，或为Dab，或为Dap，或为Glu，或为Asp，或为Ada，或为Apm，或为Asu。
  2. 根据权利要求1所述的化合物，其特征在于，所述AA1为Arg，或为Lys，或为Gln。
  3. 根据权利要求1或2所述的化合物，其特征在于，所述AA2为NH 2。
  4. 根据权利要求1～3任一项所述的化合物，其特征在于，所述R为20烷二酸-γGlu-(AA4)n6-，其中，AA4为Gly、Ser、Thr、Asn、Leu、Val或Pro；n6为0，或1至7的整数。
  5. 根据权利要求1～3任一项所述的化合物，其特征在于，所述R为20烷二酸-γGlu-PEGn3-CH 2CO-；其中，n3为1至6的整数。
  6. 根据权利要求1～5任一项所述的化合物，其特征在于，还包含该化合物所成的可药用的盐、溶剂化物、螯合物或非共价复合物，基于该化合物基础上的药物前体，或上述形式的任意混合物。
  7. 权利要求1～6任一项所述的化合物在制备长效肝炎病毒进入的抑制剂中的应用。
  8. 根据权利要求7所述的应用，其特征在于，所述肝炎病毒为乙型肝炎病毒或丁型肝炎病毒。
  9. 权利要求1～6任一项所述的化合物在制备治疗慢性乙肝和/或丁肝的药物中的应用。
  10. 治疗慢性乙肝和/或丁肝的药物，其特征在于，包括权利要求1～6任一项所述的化合物。
PCT/CN2022/136263 2022-02-10 2022-12-02 一种长效肝炎病毒进入抑制剂 WO2023151358A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210126691.4 2022-02-10
CN202210126691.4A CN116621944A (zh) 2022-02-10 2022-02-10 一种长效肝炎病毒进入抑制剂

Publications (1)

Publication Number Publication Date
WO2023151358A1 true WO2023151358A1 (zh) 2023-08-17

Family

ID=87563592

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/136263 WO2023151358A1 (zh) 2022-02-10 2022-12-02 一种长效肝炎病毒进入抑制剂

Country Status (2)

Country Link
CN (1) CN116621944A (zh)
WO (1) WO2023151358A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108367047A (zh) * 2015-12-16 2018-08-03 鲁普莱希特-卡尔斯-海德堡大学 环状ntcp靶向肽及其作为进入抑制剂的应用
WO2019031981A1 (ru) * 2017-08-11 2019-02-14 Александр Васильевич ИВАЩЕНКО Ингибитор входа вируса гепатита и фармацевтическая композиция для лечения гепатита
CN110627877A (zh) * 2019-09-25 2019-12-31 成都奥达生物科技有限公司 一种psd-95抑制剂
CN110642753A (zh) * 2019-09-25 2020-01-03 成都奥达生物科技有限公司 一种氨基酸衍生物
CN111867616A (zh) * 2018-01-11 2020-10-30 国立大学法人东京大学 Ntcp抑制剂

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108367047A (zh) * 2015-12-16 2018-08-03 鲁普莱希特-卡尔斯-海德堡大学 环状ntcp靶向肽及其作为进入抑制剂的应用
WO2019031981A1 (ru) * 2017-08-11 2019-02-14 Александр Васильевич ИВАЩЕНКО Ингибитор входа вируса гепатита и фармацевтическая композиция для лечения гепатита
CN111867616A (zh) * 2018-01-11 2020-10-30 国立大学法人东京大学 Ntcp抑制剂
CN110627877A (zh) * 2019-09-25 2019-12-31 成都奥达生物科技有限公司 一种psd-95抑制剂
CN110642753A (zh) * 2019-09-25 2020-01-03 成都奥达生物科技有限公司 一种氨基酸衍生物
CN111777671A (zh) * 2019-09-25 2020-10-16 成都奥达生物科技有限公司 一种长效的psd-95抑制剂

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KANG CONNIE, SYED YAHIYA: "Bulevirtide: First Approval", DRUGS, ADIS INTERNATIONAL LTD., NZ, vol. 80, no. 15, 14 September 2020 (2020-09-14), NZ , pages 1601 - 1605, XP009547809, ISSN: 0012-6667, DOI: 10.1007/s40265-020-01400-1 *

Also Published As

Publication number Publication date
CN116621944A (zh) 2023-08-22

Similar Documents

Publication Publication Date Title
US20220331348A1 (en) Method of preparing pentacyclic triterpenoid saponins and drug composition
CN110172083B (zh) 具有抗肥胖及抗糖尿病功效的肽及其的用途
CN110551203B (zh) 一种艾塞那肽类似物
WO2021164677A1 (zh) 一种抗合胞病毒膜融合抑制剂
CN110590934B (zh) 一种glp-1化合物
EP3181146A1 (en) Cyclic ntcp-targeting peptides and their uses as entry inhibitors
EP0288176A1 (en) Tyrosine derivatives and use thereof
WO2022156620A1 (zh) 一种广谱冠状病毒膜融合抑制剂及其药物用途
CN111333714A (zh) 一种长效glp-1化合物
JPH06507629A (ja) ガラニンアンタゴニスト
CN115814064A (zh) Glp-1衍生物及其治疗用途
CN117440964A (zh) 一种glp-1r和gipr双重靶向激动作用的多肽衍生物及其制备方法和用途
CN109364074B (zh) 6-氨基烟酰胺作为有效成分在制备乙型肝炎治疗药物中的用途
WO2023093708A1 (zh) 一种抗合胞病毒膜融合抑制剂
WO2023151358A1 (zh) 一种长效肝炎病毒进入抑制剂
CN113304165B (zh) 单体化合物Ciliatoside A在制备乙型肝炎治疗药物中的用途
CN114478709A (zh) 一种长效肝炎病毒进入抑制剂
CN116693625A (zh) 一种长效肝炎病毒进入抑制剂
CN112851755B (zh) 一种线性脂肽化合物及其制备方法与应用
CN116621943A (zh) 一种长效肝炎病毒进入抑制剂
WO2007054030A1 (fr) Modifications polyethylene glycole de thymosine alpha-1
EP3653639A1 (en) Polypeptide and composition thereof for treating diseases of metabolic system
WO2015096725A1 (zh) 端基具有亲脂性结构的线性脂肽、其制备方法及用途
JP3969831B2 (ja) ハイドロキシプロリン誘導体
WO1999047546A1 (fr) Derives d'hydroxyproline

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22925709

Country of ref document: EP

Kind code of ref document: A1