CN108341817B - Thiourea, urea compound and use thereof - Google Patents
Thiourea, urea compound and use thereof Download PDFInfo
- Publication number
- CN108341817B CN108341817B CN201810053505.2A CN201810053505A CN108341817B CN 108341817 B CN108341817 B CN 108341817B CN 201810053505 A CN201810053505 A CN 201810053505A CN 108341817 B CN108341817 B CN 108341817B
- Authority
- CN
- China
- Prior art keywords
- compound
- group
- substituted
- formula
- pharmaceutically acceptable
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
Abstract
The invention relates to thiourea compounds, urea compounds and application thereof in treating hepatitis B. Specifically, the invention discloses a compound which can be used as an HBV replication inhibitor and has a structure shown in a chemical formula (A), or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof. The invention also relates to a pharmaceutical composition containing the compound and application thereof in treating hepatitis B.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to thiourea and urea compounds for treating hepatitis B and application thereof.
Background
Hepatitis B Virus (HBV) is a enveloped, partially double-stranded DNA (dsdna), virus of the Hepadnaviridae family (Hepadnaviridae). Its genome comprises 4 overlapping reading frames: the pronuclear/nuclear gene, the polymerase gene, the UM and S genes (which encode the three envelope proteins), and the X gene. Before infection, the partially double-stranded DNA genome is converted in the host cell nucleus (open circular DNA, rcDNA) into covalently closed circular DNA (cccdna) and the viral mRNA is transcribed. Once shelled, the pregenomic rna (pgrna), which also encodes the core protein and Pol, serves as a template for reverse transcription, which regenerates the portion of the dsDNA genome (rcDNA) in the nucleocapsid.
Transmission of hepatitis b virus results from exposure to infectious blood or body fluids, while viral DNA is detected in saliva, tears, and urine of chronic carriers with high titers of DNA in serum. The choice of direct therapy is currently limited to interferon and the following antiviral drugs; tenofovir, lamivudine, adefovir, entecavir and telbivudine.
In addition, heteroaryl dihydropyrimidine (HAPs) has been identified as a class of HBV inhibitors in tissue culture as well as in animal models (Weber et al, Antiviral Res. 54: 69-78). WO 2013/006394 and WO 2013/096744 also disclose sulfamoyl-aryl amide compounds that are related to anti-HBV activity.
However, these direct HBV antiviral drugs suffer from various problems such as toxicity, mutagenicity, lack of selectivity, poor therapeutic effect, poor bioavailability, and difficulty in synthesis.
Therefore, there is a need in the art to develop HBV inhibitors with advantages such as high potency, lower toxicity, etc.
Disclosure of Invention
The invention aims to provide thiourea and urea compounds with novel structures, which can be used as HBV inhibitors.
The invention provides a compound shown as a formula A, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, a hydrate or a solvate thereof in a first aspect,
wherein the content of the first and second substances,
w is C ═ O or O ═ S ═ O;
q is-CR13、N、-CR13＝CR13、-CR13＝NR13、-CR13-NR13R14Wherein R is13、R14Each independently selected from: H. c1-C6Alkyl, halogenated C1-C6An alkyl group;
R1、R2each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C10Alkyl, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; the R is1、R2Wherein said substitution is substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, C1-C6Alkyl, halogenated C1-C6Alkyl radical, C1-C6Alkoxy, -O-;
x is CR11R12or-CR11＝CR12-; wherein R is11And R12Each independently selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C10Alkyl, substituted or unsubstituted C2-C6Alkenyl, substituted or unsubstituted C2-C6Alkynyl, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10Aryl, or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, C1-3alkyl-R7、-C(＝O)OC1-4An alkyl group; wherein said R7Selected from the group consisting of: halogen, C1-C3Alkyl, substituted or unsubstituted 5-10 membered heteroaryl with 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl with 1-3 heteroatoms selected from the group consisting of N, S and O, -NR9R10Wherein, said R9、R10Each independently selected from: H. c1-C3Alkyl, halogenated C1-C3An alkyl group;
alternatively, the first and second electrodes may be,
the R is11And R12Is adjacent toTogether form a substituted or unsubstituted 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from group N, S and O, wherein substitution of said 3-7 membered heterocycloalkyl means substitution with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, methoxy, -O-, -C (═ O) OC1-4Alkyl, benzyl, C1-4Alkyl, halogenated C1-4An alkyl group, a carboxyl group,
and, said R11、R12Wherein said substitution is substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: -OH, halogen, C1-C6Alkyl, halogenated C1-C6Alkyl, -OH substituted C1-C6Alkyl radical, C1-C6Alkoxy, -C (═ O) OC1-4An alkyl group;
y is substituted or unsubstituted C1-C7Alkylene or C2-C7Alkenylene, wherein in Y, the substitution means being substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c1-C4Alkyl, halogen, -OH, preferably C1-C4Alkyl or-OH;
z is selected from the group consisting of: NH, O or a bond;
ring C is substituted or unsubstituted C6-C10Aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, and in ring C, said substitution is by one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c1-C3Alkyl (preferably methyl), C3-C4Cycloalkyl, -CN or halogen;
ring B is substituted or unsubstituted C6-C10Aryl, substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; in the ring B, the substituted means being substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: c1-C3Alkyl radical, C3-C4Cycloalkyl, -CN or halogen;
Ra、Rb、Rc、Rdis a substituent at any position on the ring B, each of which is independently selected from the group consisting of: H. halogen, -CN, hydroxy, amino, carboxy, - (C ═ O) -substituted or unsubstituted C1-C8Alkyl, substituted or unsubstituted C1-C8Alkyl, substituted or unsubstituted C2-C6Alkenyl, substituted or unsubstituted C2-C6Alkynyl, substituted or unsubstituted C1-C8Alkylamino radical, substituted or unsubstituted C1-C8Alkoxy, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10Aryl, or substituted or unsubstituted 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O; the R isa、Rb、Rc、RdWherein "substituted" means substituted with one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: halogen, C1-C6Alkyl, halogenated C1-C6Alkyl radical, C1-C6Alkoxy, halogenated C1-C6Alkoxy radical, C3-C8Cycloalkyl, halogenated C3-C8Cycloalkyl, oxo, -CN, hydroxy, amino, carboxy, C6-C10Aryl, halogenated C6-C10Aryl, 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O, halogenated 5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O.
In another preferred embodiment, the compound of formula a is a compound of formula a 1:
wherein each group is as defined above.
In another preferred embodiment, R1Is H, unsubstituted C1-C10Alkyl, substituted-OHO-or halogen-substituted C1-C10An alkyl group.
In another preferred embodiment, R11Selected from the group consisting of: H. substituted or unsubstituted C1-C10Alkyl, -C (═ O) OC1-4Alkyl radical, C1-3alkyl-R7Substituted or unsubstituted C2-4An alkynyl group, a 3-7 membered heterocycloalkyl group having 1-3 heteroatoms selected from the group consisting of N, S and O, or a substituted or unsubstituted 5-10 membered heteroaryl group having 1-3 heteroatoms selected from the group consisting of N, S and O.
In another preferred embodiment, R12Selected from the group consisting of: H. substituted or unsubstituted C1-C10Alkyl, preferably H, substituted or unsubstituted C1-C6Alkyl, more preferably H or methyl.
In another preferred embodiment, ring C is a substituted or unsubstituted 5-or 6-membered heteroaryl, said substitution being by one or more (e.g., 2,3, 4, etc.) substituents selected from the group consisting of: methyl, -CN or halogen.
In another preferred embodiment, the ring C is selected from the group consisting of:
in another preferred embodiment, ring B is phenyl or a substituted or unsubstituted 6 membered heteroaryl, preferably phenyl or pyridyl.
In another preferred embodiment, the ring B is selected from the group consisting of:
in another preferred embodiment, R isa、Rb、Rc、RdEach independently selected from the group consisting of: H. halogen, -CHF2、-CF2-methyl, -CH2F、-CF3、-OCF3、-CN、-C3-C4Cycloalkyl, or-C1-C4An alkyl group.
In another preferred embodiment, the ring C is
Wherein the content of the first and second substances,
R4is H, -C1-C3Alkyl (preferably methyl), -C3-C4A cycloalkyl group;
R5is H or halogen (preferably F);
R6selected from H, methyl, -CN or halogen.
In another preferred embodiment, Z is O or a bond.
In another preferred embodiment, R is7Is a substituted or unsubstituted 5-10 membered heteroaryl having 1-2 heteroatoms selected from the group consisting of N, S and O.
In another preferred embodiment, the compound of formula a is selected from the group consisting of:
in a second aspect of the invention, there is provided a process for the preparation of a compound according to the first aspect of the invention, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein the compound of formula a is a compound of formula VII-1, said process comprising the step (I):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula IX-1, the method comprising step (II):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula VII-1, the process comprising step (III):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula VII-2, the process comprising step (IV):
or;
the compound of formula A is a compound of formula VIII-1, the process comprising step (V):
in the methods (I) to (V), the R1、R2、Ra、Rb、Rc、RdDefined as in the first aspect of the present invention, m is an integer of 0 to 5,
and, R3Selected from the group consisting of: H. halogen, substituted or unsubstituted C1-C10Alkyl, substituted or unsubstituted C2-C6Alkenyl, substituted or unsubstituted C2-C6Alkynyl, substituted or unsubstituted C3-C10Cycloalkyl, substituted or unsubstituted 3-to 10-membered heterocycloalkyl having 1 to 3 hetero atoms selected from the group consisting of N, S and O, substituted or unsubstituted C6-C10Aryl, or substituted or unsubstituted 5-to 10-membered heteroaryl having 1 to 3 heteroatoms selected from the group consisting of N, S and O, C1-3alkyl-R7、-C(＝O)OC1-4An alkyl group; wherein said R7Selected from the group consisting of: halogen, C1-C3Alkyl, toSubstituted or unsubstituted 5-10 membered heteroaryl having 1-2 heteroatoms selected from the group consisting of N, S and O, 3-7 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O, -NR9R10Wherein, said R9、R10Each independently selected from: H. c1-C3Alkyl, halogenated C1-C3An alkyl group.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising (1) a compound of the first aspect, or a stereoisomer or tautomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof; (2) a pharmaceutically acceptable carrier.
In another preferred embodiment, the pharmaceutical composition further comprises other drugs for preventing and/or treating hepatitis B virus infection.
In another preferred embodiment, the other agent for preventing and/or treating hepatitis b virus infection may be selected from the group consisting of: immunomodulators (e.g., interferon-alpha (IFN-alpha), pegylated interferon-alpha) or stimulators of the innate immune system (e.g., Toll-like receptor 7 and/or 8 agonists).
In another preferred embodiment, the other agent for preventing and/or treating hepatitis b virus infection may be selected from the group consisting of: tenofovir, lamivudine, adefovir, entecavir, telbivudine, or combinations thereof.
In a fourth aspect of the present invention, there is provided a compound represented by:
in a fifth aspect of the present invention, the use of a compound according to the first aspect of the present invention, or a stereoisomer or a tautomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or a pharmaceutical composition according to the third aspect of the present invention, for the preparation of a medicament for the prophylaxis and/or treatment of hepatitis b virus infection.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Detailed Description
The present inventors have conducted extensive and intensive studies and have found a novel class of compounds having an excellent therapeutic effect on hepatitis b. The compounds of the present invention have a novel structural core, especially a thiourea or urea moiety, and thus, not only have excellent anti-HBV activity, but also are less cytotoxic (especially to liver cells). On this basis, the inventors have completed the present invention.
Definition of
As used herein, the term "alkyl" includes straight or branched chain alkyl groups. E.g. C1-C8Alkyl represents a straight or branched chain alkyl group having 1 to 8 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, and the like.
As used herein, the term "alkenyl" includes straight or branched chain alkenyl groups. E.g. C2-C6Alkenyl means a straight or branched alkenyl group having 2 to 6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, or the like.
As used herein, the term "alkynyl" includes straight or branched chain alkynyl groups. E.g. C2-C6Alkynyl means straight or branched chain alkynyl having 2 to 6 carbon atoms, such as ethynyl, propynyl, butynyl, or the like.
As used herein, the term "C3-C10Cycloalkyl "refers to cycloalkyl groups having 3 to 10 carbon atoms. It may be a single ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or the like. It may also be in the form of a double ring, for example a bridged or spiro ring.
As used herein, the term "C1-C8Alkylamino "is defined as being substituted by C1-C8The amino group substituted by the alkyl can be mono-substituted or di-substituted; for example, methylamino, ethylamino, propylamino, isopropylamino, butylamineAn isobutyl amino group, a tert-butylamino group, a dimethylamino group, a diethylamino group, a dipropylamino group, a diisopropylamino group, a dibutylamino group, a diisobutylamino group, a di-tert-butylamino group, etc.
As used herein, the term "C1-C8Alkoxy "means a straight or branched chain alkoxy group having 1 to 8 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy and the like.
As used herein, the term "3-10 membered heterocycloalkyl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a saturated or partially saturated cyclic group having 3-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be monocyclic or may be in the form of a double ring, for example a bridged or spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.
As used herein, the term "C6-C10Aryl "means an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.
As used herein, the term "5-10 membered heteroaryl having 1-3 heteroatoms selected from the group consisting of N, S and O" refers to a cyclic aromatic group having 5-10 atoms and wherein 1-3 atoms are heteroatoms selected from the group consisting of N, S and O. It may be a single ring or a condensed ring form. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3) -triazolyl and (1,2,4) -triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl and the like.
Unless specifically stated to be "substituted or unsubstituted", the groups of the present invention may be substituted with a substituent selected from the group consisting of: halogen, nitrile group, nitro group, hydroxyl group, amino group, C1-C6Alkyl-amino, C1-C6Alkyl radical, C2-C6Alkenyl radical, C2-C6Alkynyl, C1-C6Alkoxy, halo C1-C6Alkyl, halo C2-C6Alkenyl, halo C2-C6Alkynyl, halo C1-C6Alkoxy, allyl, benzyl, C6-C12Aryl radical, C1-C6alkoxy-C1-C6Alkyl radical, C1-C6Alkoxy-carbonyl, phenoxycarbonyl, C2-C6Alkynyl-carbonyl, C2-C6Alkenyl-carbonyl, C3-C6Cycloalkyl-carbonyl, C1-C6Alkyl-sulfonyl, and the like.
As used herein, "halogen" or "halogen atom" refers to F, Cl, Br, and I. More preferably, the halogen or halogen atom is selected from F, Cl and Br. "halogenated" means substituted with an atom selected from F, Cl, Br, and I.
Unless otherwise specified, the structural formulae depicted herein are intended to include all isomeric forms (e.g., enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, R, S configuration containing an asymmetric center, (Z), (E) isomers of double bonds, and the like. Thus, individual stereochemical isomers of the compounds of the present invention or mixtures of enantiomers, diastereomers or geometric isomers (or conformers) thereof are within the scope of the present invention.
As used herein, the term "tautomer" means that structural isomers having different energies may exceed the low energy barrier, thereby converting with each other. For example, proton tautomers (i.e., proton transmutations) include interconversion by proton shift, such as 1H-indazoles and 2H-indazoles. Valence tautomers include interconversion by recombination of some of the bonding electrons.
As used herein, the term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio.
As used herein, the term "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.
Active ingredient
As used herein, "compound of the present invention" refers to a compound represented by formula (a), and also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compound of formula (a).
As used herein, "pharmaceutically acceptable salt" refers to a salt formed by a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, phenylmethanesulfonic acid, benzenesulfonic acid, etc.; and acidic amino acids such as aspartic acid and glutamic acid.
Pharmaceutical compositions and methods of administration
Since the compound of the present invention has excellent inhibitory activity against Hepatitis B Virus (HBV), the compound of the present invention and various crystal forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for the prevention and/or treatment (stabilization, alleviation or cure) of infection by hepatitis b virus or for the prevention and/or treatment (stabilization, alleviation or cure) of diseases associated with hepatitis b virus (e.g., hepatitis b, progressive hepatic fibrosis, inflammation and necrosis leading to liver cirrhosis, end-stage liver disease, ethyl liver cancer).
The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention in combination with a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000mg of a compound of the invention per dose, more preferably, 10-200mg of a compound of the invention per dose. Preferably, said "dose" is a capsule or tablet.
"pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatibility" ofBy "herein is meant that the components of the composition are capable of being admixed with and between the compounds of the present invention without significantly diminishing the pharmaceutical effectiveness of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), and the like
) Wetting agents (e.g., sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, and the like.
The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.
In addition to these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.
The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (e.g., anti-HBV agents).
When administered in combination, the pharmaceutical composition further comprises one or more (2, 3,4, or more) other pharmaceutically acceptable compounds (e.g., anti-HBV agents). One or more (2, 3,4, or more) of such other pharmaceutically acceptable compounds (e.g., anti-HBV agents) may be used simultaneously, separately or sequentially with a compound of the invention in the prevention and/or treatment of HBV infection or HBV-related disease.
When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for a human body with a weight of 60kg, the daily administration dose is usually 1 to 2000mg, preferably 20 to 500 mg. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.
The compounds of the present invention:
the main advantages of the invention include:
1. the compound of the invention has novel structure and excellent effect of resisting hepatitis B virus infection.
2. The compounds of the present invention have very low toxicity to normal cells.
3. The compound and the pharmaceutical composition containing the compound as the main active ingredient can be used for preventing and/or treating hepatitis B virus infection.
4. The compound of the present invention and the pharmaceutical composition containing the compound of the present invention as a main active ingredient can be used for preventing and/or treating diseases associated with hepatitis b virus (e.g., hepatitis b, progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, end-stage liver disease, ethyl liver cancer).
Description of the terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
The synthesis of compounds of type 10 is as follows:
example 1: synthesis of Compound 10a
Step 1:
compound 1(1.5g) and allyl bromide (1g) were dissolved in acetonitrile (15mL), cesium carbonate (3.5g) was added to the reaction system, the temperature was raised to 85 ℃ for reaction for 2h, 30(mL) was added to the reaction system, ethyl acetate (3 × 20mL) was extracted, anhydrous sodium sulfate was dried, the organic phase was spin-dried, and crude column chromatography was performed to give compound 2(1.0 g). ESI-MS (M + H273.02)
Step 2:
dissolving a compound (1.0g), a vinyl boron anhydride pyridine complex (1.1g), tetrakistriphenylphosphine palladium (120mg) and potassium carbonate (2.5g) in ethylene glycol dimethyl ether (25mL) and 4mL of water, raising the temperature of a system to 120 ℃ under the protection of nitrogen, reacting for 10H, adding water (40mL) into the reaction system, extracting with ethyl acetate (3X 40mL), drying with anhydrous sodium sulfate, spin-drying an organic phase, and performing column chromatography on a crude product to obtain a compound 3(800mg), ESI-MS (M + H-221.12)
And step 3:
dissolving a compound 3(800mg) in 1, 2-dichloroethane (800mL), introducing nitrogen into the reaction system, adding a second-generation Grubbs catalyst (80mg) into the reaction system, reacting for 4H at 60 ℃, adding silica gel into the reaction system, spin-drying an organic phase, and separating a crude product by column chromatography to obtain a compound 4(450mg), ESI-MS (M + H-193.09)
And 4, step 4:
dissolving compound 4(450mg) and isopropylaminosulfonyl chloride (300mg) in acetonitrile (6mL), adding pyridine (100mg) to the reaction system, reacting at 85 ℃ for 5H, adding 15mL water to the reaction system, extracting with ethyl acetate (3 × 20mL), drying over anhydrous sodium sulfate, and spin-drying the organic phase to give yellow solid compound 5(200mg) ESI-MS, (M + H ═ 314.11)
And 5:
dissolving compound 5(100mg) in tetrahydrofuran (3mL), water (0.6mL) and methanol (0.6mL), adding lithium hydroxide monohydrate (120mg) into the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 by using 1N hydrochloric acid, extracting ethyl acetate (3 x 15mL), drying by using anhydrous sodium sulfate, and carrying out rotary drying on the organic phase to obtain yellow solid compound 6(50mg) ESI-MS, (M + H-300.09)
Step 6:
dissolving the compound 6(40mg), triethylamine (70mg) and 3, 4-difluoroaniline (22mg) in dichloromethane (3mL), then reducing the temperature to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12h, adding water (15mL), dichloromethane (3X 20mL), extracting, drying with anhydrous sodium sulfate, spin-drying the organic phase, and carrying out column chromatography on the crude product to obtain a compound 10a (10mg)
Example 2: synthesis of Compound 10b
Compound 6b was prepared according to steps 1-5 of example 1, except that in step 4, isopropylamine sulfonyl chloride was replaced with trifluoromethyl substituted isopropylamine sulfonyl chloride.
According to step 6 of example 1, only compound 6b was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) on the target product 10b (12 mg).
Example 3: synthesis of Compound 10c
Compound 6c was prepared according to steps 1-5 of example 1, except that in step 4, t-butylamine sulfonyl chloride was used in place of isopropylamine sulfonyl chloride.
According to step 6 of example 1, only compound 6c was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) for target product 10c (22 mg).
Example 4: synthesis of Compound 10d
Compound 6d was prepared according to steps 1-5 of example 1, except that epoxybutylamine sulfonyl chloride was used in place of isopropylamine sulfonyl chloride in step 4.
According to step 6 of example 1, only compound 6d was used in place of compound 6, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) for target product 10d (9 mg).
Example 5: synthesis of Compound 10e
According to step 6 of example 1, the target product 10e (11mg) was purified by column chromatography (n-heptane: ethyl acetate: 2: 1) using the compound 4-fluoro-3-cyanoaniline in place of the compound 3, 4-difluoroaniline under otherwise unchanged conditions.
Example 6: synthesis of Compound 10f
According to step 6 of example 1, the target product 10f (9mg) was purified by column chromatography (n-heptane: ethyl acetate: 2: 1) using the compound 4-fluoro-3-methylaniline instead of the compound 3, 4-difluoroaniline, except that the other conditions were changed.
Example 7: synthesis of Compound 10g
According to step 6 of example 1, only the compound 4-fluoroaniline was used in place of the compound 3, 4-difluoroaniline, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) using 10g (7mg) of the objective product, without changing the conditions.
Example 8: synthesis of Compound 10h
According to step 6 of example 1, 10g (7mg) of the objective product was purified by column chromatography (n-heptane: ethyl acetate: 2: 1) using only the compound 4-fluoro-3-chloroaniline instead of the compound 3, 4-difluoroaniline, under otherwise unchanged conditions.
Example 9: synthesis of Compound 20a
Step 11:
dissolving the compound 10a (20mg) in ethyl acetate, adding palladium carbon (5mg) into the reaction system, reacting at 25 ℃ for 5h, filtering, spin-drying the organic phase, and performing column chromatography to obtain a crude product, namely a compound 20a (5mg)
Example 10: synthesis of Compound 20b
According to step 11 of example 9, only compound 10b was used instead of compound 10a, and the other conditions were unchanged, the target product 20b (7mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Example 11: synthesis of Compound 20c
According to step 11 of example 9, only compound 10e was used instead of compound 10a, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) on the target product 20c (9 mg).
Example 12: synthesis of Compound 20d
According to step 11 of example 9, only compound 10f was used instead of compound 10a, and the other conditions were unchanged, the target product 20d (12mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Example 13: synthesis of Compound 30a
Step 12:
compound 21(1.5g) and allyl bromide (1g) were dissolved in acetonitrile (15mL), cesium carbonate (3.5g) was added to the reaction system, the temperature was raised to 85 ℃ for reaction for 2h, 30(mL) was added to the reaction system, ethyl acetate (3 × 20mL) was extracted, anhydrous sodium sulfate was dried, the organic phase was spin-dried, and crude column chromatography was performed to give compound 22(1.0 g). ESI-MS (M + H273.02)
Step 13:
dissolving a compound (1.0g), allyl potassium trifluoroborate (1.1g), tetrakistriphenylphosphine palladium (120mg) and potassium carbonate (2.5g) in ethylene glycol dimethyl ether (25mL) and 4mL of water, raising the temperature of the system to 120 ℃ under the protection of nitrogen, reacting for 10H, adding water (40mL) into the reaction system, extracting with ethyl acetate (3X 40mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain a crude product, namely a compound 23(800mg), ESI-MS (M + H-236.13)
Step 14:
dissolving the compound 23(800mg) in 1, 2-dichloroethane (800mL), introducing nitrogen into the reaction system, adding a second-generation Grubbs catalyst (80mg) into the reaction system, reacting at 60 ℃ for 4H, adding silica gel into the reaction system, spin-drying an organic phase, and separating a crude product by column chromatography to obtain a compound 24(450mg), ESI-MS, (M + H ═ 208.11)
Step 15:
compound 24(450mg) and isopropylaminosulfonyl chloride (300mg) were dissolved in acetonitrile (6mL), pyridine (100mg) was added to the reaction system, reacted at 85 ℃ for 5H, then 15mL water was added to the reaction system, extracted with ethyl acetate (3 × 20mL), dried over anhydrous sodium sulfate, and the organic phase was dried by spin-drying to give ESI-MS (M + H: 329.13) as a yellow solid compound 25(200mg)
Step 16:
dissolving compound 25(100mg) in tetrahydrofuran (3mL), water (0.6mL) and methanol (0.6mL), adding lithium hydroxide monohydrate (120mg) to the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 with 1N hydrochloric acid, extracting with ethyl acetate (3X 15mL), drying over anhydrous sodium sulfate, and spin-drying the organic phase to obtain yellow solid compound 26(50mg) ESI-MS, (M + H-315.10)
And step 17:
dissolving the compound 26(40mg), triethylamine (70mg) and 3, 4-difluoroaniline (22mg) in dichloromethane (3mL), then reducing the temperature to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12h, adding water (15mL), dichloromethane (3X 20mL), extracting, drying with anhydrous sodium sulfate, spin-drying the organic phase, and carrying out column chromatography on the crude product to obtain a compound 30a (10mg)
Example 14: synthesis of Compound 30b
Compound 26b is prepared by reference to steps 12-16 of example 13, except that in step 15, isopropylamine sulfonyl chloride is replaced with trifluoromethyl-substituted isopropylamine sulfonyl chloride.
According to step 17 of example 13, only compound 26b was used instead of compound 26, and the other conditions were unchanged, the target product 30b (12mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).
Example 15: synthesis of Compound 30c
Compound 26c is prepared by reference to steps 12-16 of example 13, except that in step 15, i.e., isopropylamine sulfonyl chloride is replaced with t-butylamine sulfonyl chloride.
According to step 17 of example 13, only compound 26c was used instead of compound 26, and the other conditions were unchanged, the objective product 30c (16mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).
Example 16: synthesis of Compound 30d
Compound 26d is prepared by reference to steps 12-16 of example 13, except that in step 15 epoxybutylamine sulfonyl chloride is used in place of isopropylamine sulfonyl chloride.
According to step 17 of example 13, only compound 26d was used instead of compound 26, and the other conditions were unchanged, the target product 30d (10mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 1: 1).
Example 17: synthesis of Compound 30e
According to step 17 of example 13, the target product 30e (8mg) was purified by column chromatography (n-heptane: ethyl acetate ═ 2: 1) using only the compound 4-fluoro-3-cyanoaniline instead of the compound 3, 4-difluoroaniline, under otherwise unchanged conditions.
Example 18: synthesis of Compound 30f
According to step 17 of example 13, the target product 30f (13mg) was purified by column chromatography (n-heptane: ethyl acetate: 2: 1) using the compound 4-fluoro-3-methylaniline instead of the compound 3, 4-difluoroaniline, except that the other conditions were changed.
Example 19: synthesis of 30g of Compound
According to step 17 of example 13, 30g (7mg) of the objective product was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1) with the exception of using 4-fluoroaniline instead of 3, 4-difluoroaniline.
Example 20: synthesis of Compound 30h
According to step 17 of example 13, the target product was purified by column chromatography (n-heptane: ethyl acetate ═ 2: 1) for 30h (7mg) using only the compound 4-fluoro-3-chloroaniline instead of the compound 3, 4-difluoroaniline, under otherwise unchanged conditions.
Example 21: synthesis of Compound 40a
Step 20:
dissolving the compound 30a (20mg) in ethyl acetate, adding palladium carbon (5mg) into the reaction system, reacting at 25 ℃ for 5h, filtering, spin-drying the organic phase, and performing column chromatography to obtain a crude product, namely a compound 40a (8mg)
Example 22: synthesis of Compound 40b
According to step 20 of example 21, only compound 30b was used instead of compound 30a, and the other conditions were unchanged, the target product 40b (11mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Example 23: synthesis of Compound 40c
According to step 20 of example 21, only compound 30e was used instead of compound 30a, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) for target product 40c (9 mg).
Example 24: synthesis of Compound 40d
According to step 20 of example 21, only compound 30f was used instead of compound 30a, and the other conditions were unchanged, and column chromatography was performed (n-heptane: ethyl acetate ═ 2: 1) for target product 40d (8 mg).
Example 25: synthesis of Compound 50a
Step 21:
compound 41(1g), triethylamine (1.2g), and isoprocarbamic acid (1g) were dissolved in dichloromethane (15mL), followed by TBTU (1.5g), addition to the reaction system, 25 ℃ reaction for 5H, addition of water (10mL) to the reaction system, extraction with dichloromethane (3 × 20mL), drying over anhydrous sodium sulfate, spin-drying the organic phase, and crude column chromatography of yellow solid 42(600mg) ESI-MS, (M + H ═ 292.16)
Step 22:
dissolving compound 42(400mg) in tetrahydrofuran (5mL), water (1mL) and methanol (1mL), adding lithium hydroxide monohydrate (480mg) to the reaction system at room temperature, reacting at 40 ℃ for 5H, adjusting the pH value of the system to 3-4 with 1N hydrochloric acid, extracting with ethyl acetate (3X 15mL), drying over anhydrous sodium sulfate, and spin-drying the organic phase to obtain yellow solid compound 43(300mg) ESI-MS, (M + H-278.15)
Step 23:
dissolving the compound 43(40mg), triethylamine (70mg) and 4-fluoro-3-cyanoaniline (22mg) in dichloromethane (3mL), cooling to about 5 ℃, adding TBTU (70mg) into the reaction system, reacting at room temperature for 12h, adding water (15mL), extracting with dichloromethane (3X 20mL), drying with anhydrous sodium sulfate, spin-drying the organic phase, and performing column chromatography to obtain a crude product 50a (10mg)
Example 26: synthesis of Compound 50b
According to step 23 of example 25, the compound 4-fluoro-3-cyanoaniline was replaced with the compound 4-fluoro-3-methylaniline, and column chromatography (n-heptane: ethyl acetate ═ 1: 1) was performed using the objective product 50b (10mg) under the same conditions.
Example 27: synthesis of Compound 50c
Compound 43c was prepared by reference to steps 21-22 of example 25, except that in step 21, isopropylaminocarboxylic acid was replaced with trifluoromethyl-substituted isopropylaminocarboxylic acid.
According to step 23 of example 25, only compound 43c was used instead of compound 43, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) for the objective product 50c (10mg) under the same conditions.
Example 28: synthesis of Compound 50d
Compound 43d was prepared according to steps 21 to 22 of example 25, except that t-butylamine carbamic acid was used in place of isopropylcarbamic acid in step 21.
According to step 23 of example 25, only compound 43d was used in place of compound 43, and column chromatography was performed (n-heptane: ethyl acetate ═ 1: 1) for the target product 50d (10mg) under the same conditions.
Example 29: synthesis of Compound 60a
Step 30:
dissolving the compound 50a (20mg) in ethyl acetate, adding palladium carbon (5mg) into the reaction system, reacting at 25 ℃ for 5h, filtering, spin-drying the organic phase, and performing column chromatography to obtain a crude product, namely a compound 60a (7mg)
Example 30: synthesis of Compound 60b
According to step 30 of example 29, only compound 50b was used instead of compound 50a, and the other conditions were unchanged, the title product 60b (8mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Example 31: synthesis of Compound 60c
According to step 30 of example 29, only compound 50c was used instead of compound 50a, and the other conditions were unchanged, the target product 60c (7mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Example 32: synthesis of Compound 60d
According to step 30 of example 29, only compound 50d was used instead of compound 50a, and the other conditions were unchanged, the target product 60d (8mg) was subjected to column chromatography (n-heptane: ethyl acetate ═ 2: 1).
Biological examples- -anti-HBV Activity assay
Experiment one: in vitro anti-hepatitis B virus nucleocapsid assembly activity test method
Main reagents and raw materials:
c150 protein is expressed and purified by the pharmaceutical Mingkuda company;
Protein fluorescence labeling:
to each well of the 96-well plate, 150. mu.L of 2% w/v skim milk was added and incubated at room temperature for 2 hours. Sucking off the skimmed milk, washing with deionized water, drying, and storing at room temperature. The C150 protein (3 mg per tube) was desalted using a 5ml Hitrap desalting column. 50mM was added to the desalted C150 protein per tube
FL fluorescent dye 20. mu.l is mixed evenly and incubated overnight at 4 ℃ in the dark. The fluorescent dye not bound to C150 was removed by Sephadex G-25 gel filtration. The fluorescence labeling efficiency of C150 was calculated as follows:
[
FL]＝A504/78,000M-1；
[C150Bo]＝(A280-[
FL]x 1300M-1)/60,900M-1；
efficiency of fluorescent marking ═ 2
FL]/[C150Bo]；
Wherein the content of the first and second substances,
[
FL]indicates the concentration of the fluorescent label;
[ C150Bo ] indicates the concentration of the fluorescent-labeled protein;
a504 represents the absorbance at wavelength 504 nM;
a280 represents the absorbance at a wavelength of 280 nM;
M-1represents the reciprocal of the molar concentration.
Compound dilution:
compound stock was diluted to 6mM in DMSO and then to 600. mu.M in 50mM HEPES, followed by a further 3-fold serial dilution of 8 concentrations in 10% DMSO/50mM HEPES.
C150Bo was diluted to 2. mu.M with 50mM HEPES. Compounds were added to 96-well plates at 37.5 μ L C150Bo and 2.5 μ L of each concentration and mixed well and incubated for 15 minutes at room temperature. Mu.l of 750mM NaCl/50mM HEPES was added to the reaction wells at a final concentration of 150mM NaCl.
Control wells were assembled with 0% protein, and 10. mu.L of 50mM HEPES, NaCl at a final concentration of 0mM, was added.
100% protein assembly control wells, 10. mu.L of 5M NaCl/50mM HEPES, 1M NaCl final concentration.
The final DMSO concentration was 0.5%, the maximum final concentration of the compound was 30. mu.M, and the final concentration of C150Bo was 1.5. mu.M. Incubate at room temperature for 1 hour. The fluorescence signal was measured (excitation 485 nm; emission 535 nm).
Data analysis
% protein assembly ═ 1- (sample fluorescence value-1M NaCl fluorescence value)/(0M NaCl fluorescence value-1M NaCl fluorescence value) ] × 100.
IC50The values were calculated by prism software, the equation is as follows:
Y＝Bottom+(Top-Bottom)/(1+10((LogIC50-X)*HillSlope))；
wherein the content of the first and second substances,
x represents the log of the concentration, Y represents the effect value, and Y fits to the top in sigmoid form starting from the bottom;
bottom represents the Bottom of the curve;
top represents the Top of the curve;
HillSlope denotes: absolute value of the maximum slope of the curve.
Experiment two: determination of anti-hepatitis B Virus Activity in HepG2.2.15 cells
The main reagents are as follows:
QIAamp 96DNA blood kit (12) (Qiagen, cat # 51162);
FastStart Universal Probe Master (Roche, cat # 04914058001);
cell-titer Glo detection reagent (Promega, cat # G7573).
Compound dilution: in vitro anti-HBV activity experiments and cytotoxicity experiments all compounds were serially diluted 3-fold at 8 concentrations. The final starting concentration of test compound was 30 μ M, the final starting concentration of reference compound GLS4 was 1 μ M, and the final concentration of DMSO was 0.5%.
Inoculation of HepG2.2.15 cells (4X 10)4Cells/well) to 96-well plates at 37 ℃, 5% CO2The culture was carried out overnight. The following day, fresh medium containing different concentrations of the compounds was added to the culture wells. On the fifth day, old culture medium was aspirated from the culture wells, and fresh culture medium containing different concentrations of compounds was added.
And eighthly, collecting the supernatant in the culture hole for extracting HBV DNA in the supernatant, and detecting the HBV DNA content in the HepG2.2.15 supernatant by qPCR. And after collecting the supernatant, adding a culture medium and a Cell-titer Glo reagent into the culture wells, and detecting chemiluminescence values of the wells by using an enzyme-labeling instrument.
The activity calculation formula is as follows:
Y＝Bottom+(Top-Bottom)/(1+10((LogIC50-X)*HillSlope))；
wherein the content of the first and second substances,
x represents the log of the concentration, Y represents the effect value, and Y fits to the top in sigmoid form starting from the bottom;
bottom represents the Bottom of the curve;
top represents the Top of the curve;
HillSlope denotes: absolute value of the maximum slope of the curve.
Experiment three: cytotoxicity assays
The cytotoxicity of test compounds was tested using HepG2 cells, which were incubated for 4 days in the presence of test compounds. Cell viability was assessed using the resazurin assay.
The results of the experiments are shown in the following table.
In the table: a1 indicates IC50(μ M) at < 1; a2 represents IC50 (mu M) between 1 and 100; a3 denotes IC50 (. mu.M) at > 100; b1 denotes EC50(μ M) between < 1; b2 represents EC50(μ M) between 1 and 100; b3 denotes EC50 (. mu.M) at > 100; wherein the control compound is:
The results show that: the compound of the invention has excellent in vitro anti-hepatitis B virus nucleocapsid assembly activity and anti-hepatitis B virus activity and lower cytotoxicity.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (7)
1. A compound shown as a formula A or a pharmaceutically acceptable salt thereof,
wherein the content of the first and second substances,
w is C ═ O or O ═ S ═ O;
q is N;
R1、R2each independently selected from the group consisting of: hydrogen, substituted or unsubstituted C1-C10Alkyl, 3-10 membered heterocycloalkyl having 1O heteroatom; the R is1、R2Wherein said substitution means being substituted by one 2 or 3 substituents selected from the group consisting ofAnd (3) substitution: f;
x is-CH2-；
Y is ethylene, propylene, vinylene or 1-propenyl;
z is a bond;
ring C is a substituted or unsubstituted pyrrolyl or pyrazolyl, wherein in ring C, the substitution means substitution with a substituent selected from the group consisting of: c1-C3An alkyl group;
ring B is phenyl;
Ra、Rb、Rc、Rdis a substituent at any position on the ring B, each of which is independently selected from the group consisting of: H. f, Cl, Br, I, -CN, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and tert-butyl.
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein in ring C, said substitution is by a substituent selected from the group consisting of: a methyl group.
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound of formula a is selected from the group consisting of:
4. a process for preparing a compound of formula a, which is a compound of formula VII-1, or a pharmaceutically acceptable salt thereof, according to claim 1, which process comprises step (I):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula IX-1, the method comprising step (II):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula VII-1, the process comprising step (III):
alternatively, the first and second electrodes may be,
the compound of formula A is a compound of formula VII-2, the process comprising step (IV):
or;
the compound of formula A is a compound of formula VIII-1, the process comprising step (V):
in the methods (I) to (V), the R1、R2、Ra、Rb、Rc、RdAs defined in claim 1, m is 0, and R3Is H.
5. A pharmaceutical composition comprising (1) a compound of claim 1 or a pharmaceutically acceptable salt thereof; (2) a pharmaceutically acceptable carrier.
6. The compounds are shown below:
wherein the ring C, R1、R2As defined in claim 1, m is 0 and R is3Is H.
7. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 5 for the preparation of a medicament for the prevention and/or treatment of hepatitis b virus infection.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710058692 | 2017-01-23 | ||
| CN2017100586929 | 2017-01-23 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108341817A CN108341817A (en) | 2018-07-31 |
| CN108341817B true CN108341817B (en) | 2021-11-26 |
Family
ID=62908167
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810053505.2A Active CN108341817B (en) | 2017-01-23 | 2018-01-19 | Thiourea, urea compound and use thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108341817B (en) |
| WO (1) | WO2018133845A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP7123429B2 (en) * | 2017-05-04 | 2022-08-23 | シャンハイ ロングウッド バイオファルマシューティカルズ カンパニー リミテッド | Bicyclic fused ring system nucleocapsid inhibitors and their use as drugs to treat hepatitis B |
| CN110437132B (en) * | 2018-05-04 | 2022-04-01 | 上海长森药业有限公司 | Bifused heterocyclic nucleocapsid inhibitors and pharmaceutical uses thereof |
| TWI826492B (en) * | 2018-07-27 | 2023-12-21 | 加拿大商愛彼特生物製藥公司 | Substituted tetrahydrocyclopenta[c]pyrroles, substituted dihydropyrrolizines, analogues thereof, and methods using same |
| CN112010808B (en) * | 2019-05-31 | 2021-11-30 | 上海挚盟医药科技有限公司 | tetrahydro-1H-benzazepine compounds as potassium channel modulators, and preparation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203519A (en) * | 2005-05-04 | 2008-06-18 | 辉瑞有限公司 | 2-amido-6-amino-8-oxopurine derivatives as ToII-like receptor modulators for the treatment of cancer and viral infections, such as hepatitis c |
| CN103626705A (en) * | 2012-08-27 | 2014-03-12 | 中国科学院上海药物研究所 | 1-(3-benzoylaminobenzyl)-1H-indazole-3-carboxamide compounds, and preparation method and antiviral use thereof |
| CN105452220A (en) * | 2013-05-17 | 2016-03-30 | 爱尔兰詹森科学公司 | Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis b |
-
2018
- 2018-01-19 CN CN201810053505.2A patent/CN108341817B/en active Active
- 2018-01-19 WO PCT/CN2018/073454 patent/WO2018133845A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101203519A (en) * | 2005-05-04 | 2008-06-18 | 辉瑞有限公司 | 2-amido-6-amino-8-oxopurine derivatives as ToII-like receptor modulators for the treatment of cancer and viral infections, such as hepatitis c |
| CN103626705A (en) * | 2012-08-27 | 2014-03-12 | 中国科学院上海药物研究所 | 1-(3-benzoylaminobenzyl)-1H-indazole-3-carboxamide compounds, and preparation method and antiviral use thereof |
| CN105452220A (en) * | 2013-05-17 | 2016-03-30 | 爱尔兰詹森科学公司 | Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis b |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2018133845A1 (en) | 2018-07-26 |
| CN108341817A (en) | 2018-07-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109843893B (en) | Ensultiamidinamide-aryl amide compound and application thereof in treating hepatitis B | |
| CN108794487B (en) | Bicyclic nucleocapsid inhibitors and their use as medicaments for the treatment of hepatitis b | |
| CN108250122B (en) | Sulfonamide-aryl amide compounds and pharmaceutical use thereof for treating hepatitis B | |
| CN108341817B (en) | Thiourea, urea compound and use thereof | |
| CN108264520B (en) | Compound for treating hepatitis B and application thereof | |
| CN115043842A (en) | Amino-substituted bicyclic inhibitor and preparation method and application thereof | |
| CN108456216B (en) | Sulfonyl hydrazide compound and application thereof | |
| CN113286798B (en) | Ensultiamidinamide-aryl amide compound and application thereof in treating hepatitis B | |
| CN108341810B (en) | Episulfide urea compound and application thereof | |
| CN110437232B (en) | Bicyclourea nucleocapsid inhibitor and pharmaceutical use thereof | |
| CN109251158B (en) | Thioamidine amide compounds and use thereof for treating hepatitis B | |
| CN110437132B (en) | Bifused heterocyclic nucleocapsid inhibitors and pharmaceutical uses thereof | |
| CN115677698B (en) | High-efficiency antiviral compound and application thereof | |
| JP2781073B2 (en) | Novel quinoline derivative and anticancer drug effect enhancer containing the same as active ingredient | |
| CN117865993A (en) | AAK1 inhibitor and preparation and application thereof | |
| BR112021013730A2 (en) | INTERNAL CYCLIC SULFAMIDINE AMIDE-ARILAMIDE COMPOUND AND USE OF IT FOR HEPATITIS B TREATMENT |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |