KR20150117317A - Pharmaceutical composition comprising compound having inhibitory activity against capsid assembly of Hepatitis B Virus for treating or preventing liver disease due to HBV infection - Google Patents

Abstract

The present invention discloses a pharmaceutical composition, which can be usefully used for preventing or treating various diseases due to HBV by effectively inhibiting or blocking a capsid assembly of HBV. According to the present invention, the pharmaceutical composition can be usefully used for preventing or treating various liver diseases due to HBV virus, solely or with the existing antiviral agent in combination therapy.

Description

 TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating liver disease caused by HBV infection comprising a compound capable of inhibiting hepatitis B virus capsid assembly, }

This is a therapeutic agent for diseases caused by hepatitis B virus.

Hepatitis B virus (HBV) is a virus that causes chronic hepatitis by infecting hepatocytes. It is estimated that about 350 million people worldwide are infected with HBV, and 5 to 10% (Ahn et al., 2005. J. Hepatol 42 (2): 188-94; and Montalto, G., et al., Hepatocellular carcinoma and hepatocellular carcinoma. et al. 2002. Ann NY Acad Sci 963: 13-20).

In particular, hepatocellular carcinoma is the fourth most common cancer in Korea (National Cancer Information Center, 2003-2005). The main treatment for liver cancer is hepatic resection, hepatocellular chemoembolization, chemotherapy, radiation therapy, and liver transplantation. Effective treatment, however, is to inhibit the growth of the virus, which is the cause of the disease, Development of a method is needed.

Especially, since capsids are essential for the formation and survival of viruses, substances capable of blocking or inhibiting the formation of these capsids can function as potent antiviral agents, but such therapeutic agents do not yet exist.

Stray et al. Have reported that heteroaryldihydropyrimidine (HAP) specifically reacts with methyl 4- (2-chloro-4-fluorophenyl) -6-methyl-2- (pyridin-2-yl) -1,4-dihydropyrimidine-5-carboxylate (HAP-1) (Proc Natl Acad Sci US A. 2005; 102 (23): 8138-8143).

U.S. Patent No. 6,544,520 relates to HBV virus inhibitors and discloses peptides with specific sequences that bind to core antigens and inhibit the assembly of HBV.

Antiviral agents such as nucleoside analogs have also been attempted, but they have not been used due to significant side effects.

Development of a stable and effective therapeutic agent focused on inhibiting HBV proliferation is required.

The present disclosure seeks to provide an inhibitor of HBV proliferation that targets the capsid assembly process.

In one embodiment, the present invention provides a pharmaceutical composition for preventing or treating liver disease caused by HBV infection comprising a salt thereof as an active ingredient:

≪ Formula 1 >

In Formula 1,

A is C or N;

R ₁ and R ₂ are each hydrogen or halogen,

R ₃ and R ₄ are a straight or branched chain alkyl group each represent a _{hydrogen, C 1 -C 6, -C (} = O) R a, -S (= O) 2 R a , and wherein R is _a straight chain of C1-C6 Or a branched alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.

In one embodiment according to the present invention, the compound of formula (1) is represented by the following formula (2) or (3)

(2)

(3)

In the general formula (2) or (3)

R ' ₁ , R' ₂ , R ' ₄ and R' ₅ are each hydrogen or halogen,

R _'3 and R' ₆ are each a hydrogen, a linear or branched alkyl group of ₆ C ₁ -C, -C (= O) R _a, -S (= O) ₂ R _a, wherein Ra is C ₁ -C _A substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group,

(BCM-599), (6) (BCM-600), and the compound of the formula (1) is a compound of the formula (BCM-602), Formula 10 (BCM-604), Formula 12 (BCM-606) or Formula 14 (BCM-608).

The compounds according to the present invention are useful for the prevention or treatment of liver disease due to HBV infection and can be used, for example, in the prevention or treatment of hepatitis, fatty liver, liver failure, cirrhosis, or liver cancer. Without being limited to this theory, compounds according to the present invention exert this effect by inhibiting or blocking HBV capsid assembly.

In another aspect, the present disclosure provides a pharmaceutical composition for blocking or inhibiting capsid assembly, comprising a compound of the present invention or a salt thereof. The pharmaceutical compositions of the present application are effective for the treatment or prevention of liver diseases including hepatitis, fatty liver, liver failure, liver cirrhosis, or liver cancer due to HBV infection.

In another embodiment, the pharmaceutical compositions herein may be used in combination with an HBV polymerase inhibitor used in the treatment of existing liver diseases, and the ratio of the present compound to the HBV polymerase inhibitor may range from about 1: 1 to 40: 1 < / RTI > In one embodiment according to the present disclosure, such an HBV polymerase inhibitor may comprise Lamivudin, Adefovir (ADV), Telbivudine, Entecavir or Tenofovir. But is not limited thereto. In one embodiment according to the present invention, the pharmaceutical composition comprises a compound of Formula 5 (BCM-599), Formula 6 (BCM-600), Formula 8 (BCM-602), Formula 10 (BCM-604) (BCM-608). ≪ / RTI >

In another embodiment herein, we provide a hexamer consisting of six core protein monomers derived from hepatitis B virus. The structure of the hexamer can be referred to FIG. Hexamers can be useful for screening, identification, isolation, and excavation of substances that interact with HBV capsids and inhibit their assembly. The core protein can be a full-length or only 149 known sequences. The HBV core protein sequence is known, for example, the full-length sequence has the sequence of SEQ ID NO: 1, and the 149 sequence has the sequence of 1 to 149 residues in the sequence. In one embodiment according to the present disclosure, each core protein monomer has 149 residues and comprises a groove formed of amino acid residues 21-35, 101-120, and 136-140 on the basis of the sequence of SEQ ID NO: 1.

In another aspect, the present invention provides a method of screening a capsid assembly inhibitor of HBV comprising measuring the binding force between a candidate compound and a groove structure formed in the hexamer using a hexamer according to the present invention.

The pharmaceutical composition according to the present invention can be effectively used to prevent or treat various diseases caused by HBV by effectively inhibiting or blocking the capsid assembly of HBV through binding to the groove structure of Cp with high affinity.

Fig. 1 shows the structure of the cp149 dimer (A) and the plasma mass (B) obtained through silico modeling.
Figure 2 shows the similarity between the binding pose of cp149 and the assembly inhibitor candidate, where A is the predicted binding pose for the cp149 loop structure (Ser121-Pro135) on the cp149 dimer receptor and B is the predicted binding pose for the ARG127 -THR128-PRO129, C is the predicted binding pose for the cp149 dimer receptor of BCM-599, D is the predicted binding pose for the three peptides, and D represents the interaction between the proximal amino acid residues of BCM-599 and cp149 Lt; / RTI > receptor-ligand interaction.
FIG. 3 shows that the inhibitory effect of the predictive molecule was examined by in vitro assembly analysis and agarose gel analysis. In A and B, cp149 was treated with 13 molecules each having a final concentration of 25 μM, 0.9% (upper panel) and 1.5 % (Bottom panel) analyzed by SDS-PAGE and the order of the samples was as follows; A: -, +, 599, 598, 600, 601, 602, 603, 604; [C] BCM-599, [D] BCM-600, [E] BCM-602, [F] BCM- 604, [G] BCM-606, and [H] BCM-608 at a concentration of 1 μM to 25 μM.
Figure 4 shows the sucrose concentration gradient centrifugation results of cp149 assembled in Invitro. A to F represent protein molecules distributed in a sucrose concentration gradient, A is a negative control, B is a positive control, C is BCM-599, D is BCM-600, E is BCM-602, F is BCM-606 G is the result of analyzing the band intensity to more clearly show the shunt of the protein, N is the negative control group and P is the positive control group.
Figure 5 shows the quantitative PCR results and IC ₅₀ values of virus titer after treatment of inhibitor candidate material with HepG2.2.15 material. In addition, CI-index results of relative synergy effects by mixing the inhibitor candidate with lamivudine at a specific ratio and performing the above-mentioned experiment. A is an isobolyte graph showing the compound lamubendin synergy pattern, the values normalized to the IC ₅₀ of each compound. B is the CI index of the combination of BCM-599 / lamivudine and C is the result of the change of virus titer due to the addition of BCM-599 at a constant concentration (= 0.07 μM) of lamivudine.
Figure 6 is an electron micrograph (magnification: X80,000) of untreated cp149 and inhibitor candidate compound treated cp149 capsids. The assembled core particles were negatively stained using a 2% eurosilyl acetate. A is a non-assembled cp149 photograph and B is a photograph of fully assembled cp149 in reaction solution (50 mM Hepes, 15 mM NaCl, and 10 mM CaCl2 in pH 7.5) and C to F are BCM-599, BCM-600 , BCM-602, and BCM-606 treated with 25 [mu] M, respectively.
FIG. 7 shows the result of comparing the IC ₅₀ values obtained from the results of the in vitro / in vivo information experiment with the binding energy predicted by the silico modeling. These values were converted to logarithmic scales, where A is the Invitro / Insilico relation and B is the Invivo / Incillico relation. As a result, the Root Mean Square Deviation (RMSD) was about 0.80, indicating that the cp149 model constructed here is a very effective means of screening inhibitors.

The present invention is based on the discovery that the amide-based materials of formula (1) can inhibit virus production by inhibiting formation of capsid through binding to interaction sites of other dimers of the core protein dimer with high affinity. In this study, a model of the dimer / hemimeric structure was constructed based on the X-ray diffraction analysis data of the core protein (cp) constituting the HBV capsid, and the material was excavated through library screening. It is experimentally verified that the capsid assembly can be inhibited.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating liver disease caused by HBV infection comprising a compound of the formula (I) or a salt thereof as an active ingredient.

In Formula 1,

A is C or N;

R ₁ and R ₂ are each hydrogen or halogen,

R ₃ and R ₄ are each a hydrogen, a C ₁ -C ₆ linear or branched alkyl group, -C (═O) R _a , -S (═O) ₂ R _a wherein R _a is C ₁ -C ₆ , A substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.

The "substituted" in the "substituted" group of the aryl group or the heteroaryl group used in the formula according to the present invention is a halogen atom, a C ₁ -C ₂₀ alkyl group substituted with a halogen atom (eg, CCF ₃ , CHCF ₂ , CH ₂ F , CCl _3, etc.), a hydroxy group, a nitro group, a cyano group, an amino group, an amidino group, hydrazine, hydrazone, a carboxyl group or a salt thereof, a sulfonic acid group or a salt thereof, phosphoric acid or a salt thereof, or an alkyl group of C ₁ -C _20, C ₂ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl group, C ₁ -C ₂₀ heterocyclic group, C ₆ -C ₂₀ aryl group, C ₆ -C ₂₀ arylalkyl group, C ₆ -C ₂₀ of the A heteroaryl group, or a C ₆ -C ₂₀ heteroarylalkyl group.

Specific examples of the C ₁ -C ₆ alkyl group used in Formula 1 include methyl, ethyl, propyl, isobutyl, sec-butyl, ter-butyl, neo-butyl, iso-amyl and hexyl.

The aryl group used in the above formula (1) is used alone or in combination to mean an aromatic system containing at least one ring. Examples thereof include phenyl, naphthyl, tetrahydronaphthyl and the like. Also, at least one hydrogen atom of the aryl group may be substituted with a substituent as defined in the above-mentioned " substitution ".

The heteroaryl group used in formula (1) means an organic compound having at least one heteroatom selected from N, O, P or S and the remaining ring atoms carbon, and examples thereof include pyridyl and the like. Further, at least one hydrogen atom of the heteroaryl group may be substituted with a substituent as defined in the above-mentioned " substitution ".

Halogen is one of F, Cl, Br, or I.

In one embodiment according to the present application, the compound of formula (1) is represented by the following formula (2) or (3).

In the general formula (2) or (3)

R ' ₁ , R' ₂ , R ' ₄ and R' ₅ are each hydrogen or halogen,

R _'3 and R' ₆ are each a hydrogen, a linear or branched alkyl group of ₆ C ₁ -C, -C (= O) R _a, -S (= O) ₂ R _a, wherein Ra is C ₁ -C _A substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group. The definition of an alkyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted heteroaryl group in the general formula (2) or (3) is as mentioned above.

Examples of the compound represented by the formula (1) include the compounds represented by the following formulas (4) to (16).

.

Compounds according to the present invention can inhibit or block capsid assemblies that play an important role in the forefront of HBV proliferation. HBV, a core protein (HBc, Cp), a polymerase (HBV pol), and a surface protein (HBs), is a 3.2 kb circular DNA virus with a partially double stranded genome. , And X protein (HBx) are expressed (Seeger C, et al., Microbiol. Mol. Biol. Rev., 2000; 64: 51-68). Core proteins are involved in the packaging of HBV pol, free genomic RNA (pgRNA) and other proteins, and are known to play an important role in the HBV life cycle ( ibid. ). Assembly of core proteins is an important step in HBV replication, as the production of viruses requires assembly of core proteins, viral genomes and other proteins.

The core protein is composed of 183-185 amino acids (ibid.) With two regions consisting of the N-terminal region required for core assembly and the C-terminus regulating viral replication. The core protein forms the capsid of the virus, and in the process the core protein forms a dimer, which in turn interacts with the dimer to ultimately form capsids of 180 or 240 core proteins. Capsid is essential for the protection of packaged viruses and host factors. Therefore, when inhibiting the formation of capsid, it can be usefully used for the prevention or treatment of diseases caused by HBV proliferation.

Therefore, the compound of formula (1) according to the present invention can effectively inhibit the capsid assembly through binding with capsid dimer and / or plasma to inhibit viral proliferation, and can be effectively used for the treatment or prevention of various liver diseases caused by viruses.

As used herein, the term " liver disease "refers to a symptom in which the liver function is impaired. For example, viral hepatitis (especially hepatitis mediated by HBV), fatty liver, liver failure, liver cirrhosis and hepatocellular carcinoma. Even if the pathology is unclear, symptoms of abnormalities in liver function indicators such as GOT, GPT, and γ-GTP are also included in liver disease, especially due to HBV infection.

In particular, hepatocellular carcinoma refers to primary hepatocellular carcinoma originating in the liver, and cancer that has spread to other parts of the liver and has spread to the liver does not include hepatocellular carcinoma. Hepatocellular carcinoma accounts for more than 90% of all cancers and 40 to 80% of patients recur, most often recurring in the liver, but may also appear in the lining of the lungs, the inner wall surrounding the abdominal cavity, and the mediastinum. .

The term " treatment ", " alleviation ", or " improvement ", as used herein, refers to any act that improves or alleviates the symptoms of a disease caused by HBV infection upon administration of a composition according to the invention. Those skilled in the art will be able to ascertain the precise criteria of the disease by referring to the data provided by the Korean Medical Association, and to judge the degree of improvement, improvement, and treatment of the disease.

As used herein, the term "prophylactic " refers to any act that inhibits or delays the onset of a disease due to HBV infection upon administration of a composition according to the invention. It will be apparent to those skilled in the art that the extract of the present invention can prevent such diseases when administered at the early stage of infections or before symptoms appear.

In another aspect thereof, the present invention is also directed to compositions for inhibiting or blocking HBV capsid assembly comprising a compound of formula (I).

In another aspect, the present invention relates to a composition for preventing or treating liver disease through inhibition or blocking of HBV capsid assembly comprising a compound of formula (I), and liver diseases are as described above.

The composition of the present invention may further contain, in addition to the above-mentioned effective ingredients, one or more active ingredients exhibiting the same or similar functions, or a compound that maintains / increases the solubility and / or absorbency of the active ingredient. The composition of the present invention can also be used alone or in combination with other methods for the treatment or prevention of liver diseases or using surgery, other drug therapy and biological response modifiers.

For example, a known antiviral agent, for example, a DNA polymerase, so as to induce synergism with one or more agents that inhibit proliferation. In one embodiment, for example, it can be used in combination with one or more of Lamivudin, Adefovir (ADV), Telbivudine, Entecavir or Tenofovir, Combination therapy, i.e., cocktail therapy, can inhibit virus proliferation more effectively and is very useful for the treatment of related diseases. The ratio of each component can be selected, but is not limited to, a reasonable ratio taking into account the therapeutic purpose and the specific ingredients. The ratio of compound of formula I to HBV DNA polymerase inhibitor, especially lamivudine, is about 1: 1 to 40: 1 But is not limited thereto.

In addition to the above-mentioned active ingredients, the composition of the present invention may further comprise at least one pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and at least one of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administering the composition of the present invention is not particularly limited thereto, and the known method of administering the inhibitor may be applied. The composition may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) In case of parenteral administration, it can be administered through a patch type nasal / respiratory patch attached to the skin. In order to obtain a rapid therapeutic effect, administration by intravenous injection is preferable. The dosage varies widely depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. For typical drugs, the dosage unit comprises, for example, from about 0.01 mg to 100 mg, but does not exclude the following ranges and ranges above. The daily dose may be about 1 μg to 10 g, and may be administered once a day or divided into several times a day.

In another embodiment, the disclosure is directed to a hexamer consisting of six core protein monomers derived from hepatitis B virus.

The core protein constituting the hexamer may have 149 amino acid residues from the full length or N terminus. The total length may have from 183 to 185 amino acid residues. The core protein sequence is known and includes, but is not limited to, the sequence of SEQ ID NO: 1, for example:

: MDIDPYKEFGASVELLSFLPSDFFPSVRDLLDTASALYRDALESPEHCTPNHTALRQAILCWGELMTLASWVGNNLEDPAARDLVVNYVNTNMGLKIRQLLWFHISCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRSPSPRRRSQSPRRRRSQSPASQC.

The core protein consists of 183 to 185 amino acids with two regions consisting of the N-terminal region required for core assembly and the C-terminus regulating viral replication. The core protein forms the capsid of the virus, and in the process the core protein forms a dimer, which in turn interacts with the dimer to ultimately form capsids of 180 or 240 core proteins. Capsid is essential for the protection of packaged viruses and host factors. Therefore, when inhibiting the formation of capsid, it can be usefully used for the prevention or treatment of diseases caused by HBV proliferation. In the present invention, it has been found that a hexamer composed of six core proteins, rather than a capsid consisting of 180 or 240 core proteins, can be usefully used in the discovery of a capsid inhibitor in the discovery of substances inhibiting capsid formation.

A hexamer according to the present invention is a hexagonal body in which three dimers are combined with a core protein dimer as a unit, and one groove is formed per monomer, and a total of six grooves are formed in the hexamer. Three out of six of them participate in the formation of hexamers, and three external grooves are used for screening. The three external grooves are formed from amino acid residues 21-35, 101-120, and 136-140 based on the sequence numbers in SEQ ID NO: 1 below. As long as the groove structure as described above is formed, a part of the mutation occurs in the sequence, and the specific position of the residue forming the groove may vary accordingly.

In another aspect, the present disclosure is also directed to a method of screening a capsid assembly inhibitor of the HBV with a hexamer according to the present disclosure. The method comprises: measuring a binding force between a groove structure formed in a hexamer and a candidate compound; And selecting the inhibitory compound as a candidate compound, as compared to a control compound that does not inhibit binding. Specific examples using hexamers according to the present invention include, but are not limited to, those described in the examples according to the present application.

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

The present invention may be practiced using conventional techniques within the skill of those skilled in the art of cell biology, cell culture, molecular biology, gene transformation techniques, microbiology, DNA recombinant techniques, unless otherwise indicated. A more detailed description of common techniques can also be found in Molecular Biotechnology: (Bernard et al., ASM press 1994); Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., Harbor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al., Eds., John Wiley & Sons 1999); DNA Cloning, Volumes I and II (Glover ed., 1985).

Example

Example 1 Experimental Material and Method

Materials and experimental methods used in this example are as follows.

1-1. Materials used in the experiment

Anti-Anti-HBcAb (Polyclonal Rabbit, 1: 4000) was obtained from Dako (Glostrup, Denmark). Horseradish-conjugated anti-rabbit Ab (1: 40,000) and mouse monoclonal anti-FLAG Ab (5 μg / ml) are from Sigma (St. Louis, MO, United States). BL21 (DE3) + pLysS E. coli and pET28b vectors are from Novagen (Madison, Wis., United States). Isopropyl-hd-1-thiogalactoside (IPTG) is derived from Duchefa Biochemie (Haarkem, Netherlands). Dulbecco's modified Eagle's medium (DMEM) is derived from Welgene (Daegu, Republic of Korea). Fetal serum is derived from Invitrogen (Carlsbad, CA, United States). Real-time quantitative PCR PCR The SYBR-Green reaction mixture is from Qiagen (Hilden, Germany). The compound library was provided by B & C Company (Seoul, Korea).

1-2. Viruses and cells

HepG2.2.15 cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 ° C and 5% CO ₂ .

1-3. Capsid  Protein structure modelling

The crystal was identified by X-ray diffraction analysis on dimer / hexamer Cp149-HBcAg (3.5 A). A silico model for monomer and dimer structures was constructed for each of cp149 and cp183. The structure of these proteins was determined by X-ray diffraction analysis (Packianathan C et al., J. Virol. 2009; 84 (3): 1607-1615; Wynne SA et al., Mol Cell. 1999; (6): 771-780). The water molecules in the crystal lattice were removed, and dislodged residues and atoms were added to construct a full-field model. Protein structure simulation was performed based on the AMBER and CHARMM force field sets. The partial charges of each atom were applied as a Momany-Rone method (CHARMm FF). Energy levels were minimized by the Adopted Basis-set Newton-Raphson (ABNR) algorithm. The resulting structures were equilibrated at 37 DEG C for 1,000,000 cycles using a Generalized Born model by molecular volume integration (GBMV) method, and the dielectric constants in the solvent and in the protein were 80 and 4, respectively (Smith JR, et al. Bioorg Med Chem 2012; 20: 1354-1363). Based on these results, we have searched candidates for capsid assembly inhibitors in Insilico and simulated the interaction between candidate molecules and free dimers using a dimer model and observed the difference of HbcAg molecular interaction using a hexamer model A more elaborate screening process was performed.

1-4. molecule dynamic Simulation  And Dark Simulation

In this example, a groove structure was identified that could act as a possible binding site for a compound molecule, such as an inhibitor, surrounded by core protein amino acid residues 21-35, 101-120, and 136-140. The original function of this groove is the dimer-dimer interaction site, which binds to moieties 126-135 which are loop structures of other dimers. In the dimer model there are two free grooves per each polypeptide chain. In the model, three inner groove structures occupy the loop structure, and only three outer grooves can be used for library screening and receptor-ligand interaction simulation. In this embodiment, the molecules capable of binding to the grooves were identified using the CDOCKER, LibDock, and LigandFit algorithms (Discivery Studio 3.1, accelrys, USA) with these three independent mechanisms. After performing the screening using the singing simulation, the best binding pose was investigated as described above through energy level minimization and equilibration at 37 DEG C for 1,000,000 cycles.

1-5. Invitro  Assembly analysis and Sucrose Concentration gradient  Electrophoresis

Cp149 was prepared as previously described (Kang HY, et al., Biochem. J. 2006; 398: 311-317). For the Cp149 assembly, a constant concentration of the candidate compound and Cp149 (final concentration 60 μM) were mixed in assembly reaction buffer (50 mM HEPES (pH 7.5), 15 mM NaCl, 10 mM CaCl ₂ ) and allowed to stand at 37 ° C. for 1 hour. Subsequently, assembled particles of each sample were separated and electrophoresed on a 0.9% unmodified agarose gel, followed by immunoblotting ( ibid ) as described previously. The sucrose concentration gradient centrifugation and subsequent immunoblotting were also immunoblotted as described previously ( ibid ).

1-6. real time PCR Using HBV DNA  dose

24 well plates HepG2.2.15 cells confluent in each well were treated with a certain concentration of candidate compound as shown in the figure. After 24 hours, the medium was collected and the released virions were harvested, extracted with phenol, and precipitated with ethanol. The titer was determined by quantitative PCR as previously described (Shim HY, et al., Virology, 2011; 410: 161-169). In brief, the extracted DNA was added to a PCR SYBR Greeen reaction mixture (Qiagen) containing a HotStartTaq polymerase and analyzed using F-5'-GTGTCTGCGGCGTTTTATCA-3 'and R-5'-GACAAACGGGCAACATACCTT-3' as primers Respectively. The primers amplify the 379-476 nt region of the HBV genome, resulting in a 98 bp product. The reaction conditions are as follows: 45 cycles at 95 ° C for 15 min, followed by 15 s at 94 ° C, 30 s at 55 ° C, and 30 s at 72 ° C. The concentration of HBV in the medium was determined by continuous dilution of pHBV 1.2 × (5.28 × 10 ⁶ copies per milliliter (cpm), 5.28 × 10 ⁵ cpm, 5.28 × 10 ⁴ cpm, 5.28 × 10 ³ cpm, and 5.28 × 10 ² cpm) A standard curve was created and used.

1-7. IC ₅₀ Calculation of

Quantitative PCR was performed as described above to determine the IC ₅₀ , which is a concentration that represents a 50% inhibitory effect of each compound. Each inhibitor was diluted with 100% DMSO and used at a concentration of 0.25 to 50 μM. The final DMSO concentration in each well was 1%. The virus genome titer values were converted to% inhibition. The results were written as sigmoid curves representing the capacity-to-dose response, from which the IC ₅₀ was calculated. Cytotoxicity was measured as an uptake of neutral red dyes 24 hours after each treatment as previously described (Korba BE, et al., Antiviral Res, 1992; 19: 55-70).

1-8. Analysis of combination of lamivudine and invivo

The lamivudine and assembly were treated with 24-well plates of HepG2.2.15 cells at various concentrations as described in the figure to investigate synergy if inhibitory candidate compounds were used in combination. After 24 hours, the medium was collected and the virus titer was measured as previously described (Shim HY, et al., Virology, 2011; 410: 161-169). The CI index was determined as before (Chou T. et al., Pharmacological Review, 2006; 58 (3): 621-681).

1-9. Electron microscope

For negative staining, 10ul samples containing assembled core particles were applied to a carbon coated grid and allowed to stand for 1 minute. The grid was then washed with water and stained with 2% eurosyl acetate for 1 minute. The electron microscope used was LIBRA 120 (Carl Zeiss, Oberkochen, Germany) operating at 80 kV and electron microscopy was performed with the National Instrumentation Center for Environmental Management (Korea).

Example 2 Library Screening and Excavation Based on Core Protein Structure Capsid  Assembly inhibitor

As shown in Fig. 1, a structural model for the dimer and the mass was constructed. Among them, the dimer model was used to simulate the interaction between the library compound and the free dimer, and the model was used to observe the differences in the compound - protein interaction and to improve the efficiency of the screening method.

The library used in this Example was composed of 1128 drug-like molecules, and the compound having a structure capable of matching with the groove structure of a dimer or a hemoglobin was excavated and its binding affinity and stability were investigated. As a result, a total of 13 compounds were selected as shown in Table 1. These include two kinds of molecular families: 2-amino-N- (2,6-dichloropyridin-3-yl) acetamide and N- (2,4-dichlorophenyl) 2- (methylamino) acetamide system, all of which have overall structural similarity. Among them, BCM-599 and BCM-606 exhibited excellent binding energy and binding stability in both dimer and hematological models. The difference in affinity between the two grooves in the dimer model was negligible, but the affinity difference between the grooves in the dimer and dimer models was significant. These differences are thought to be due to conformational differences between the dimers and the plasma masses. The reason for this is that the groove of the mass body is wider and more relaxed than the case of the dimer. The binding pose of the excavated molecule was very similar to the pose of the ARG127-THR128-PRO129 tripeptide binding at the center of the groove (FIG. 2). Finally, thirteen molecules containing the two molecules were selected from the library.

[Table 1]

Example 3 Capsid  Assembly inhibitor Capsid TITER  Reduction effect

Six of the 13 molecules found in Example 2 were found to strongly reduce capsid assembly (see FIG. 3). Of these, BCM-606 and BCM-599 were found to be the strongest, and assembly of all dimers was blocked. The other four molecules, BCM-600, BCM-602, BCM-604, and BCM-608 were also shown to significantly reduce the capsid concentration formed. Six compounds were used in the subsequent experiments.

The EC ₅₀ values for the six compounds were determined using the assembly assay at a concentration gradient of 1-50 μM as shown in Table 2 below. In particular, the EC ₅₀ for the four molecules BCM-600, BCM-602, BCM-604, and BCM-608 exceeded 20 μM.

[Table 2]

Abbreviations used in the above Table 2 are as follows: NA: Standard deviation over the range of IC ₅₀ values Not applicable. ND: IC ₅₀ , IC ₉₀ , and standard deviation are not detected due to low solubility. Stdev (50): Standard deviation for IC ₅₀

Example 5 Identification of capsid assembly inhibition process by sucrose concentration gradient electrophoresis analysis

The inhibitory effect of BCM-599, BCM-600, BCM-602 and BCM-606 compounds on sucrose concentration gradient centrifugation was further analyzed. The results are shown in FIG. As shown above, as a result of using a sucrose concentration gradient of 10-50%, unassociated capsids were detected in fractions 1-4 due to differences in sedimentation coefficients, and fully assembled capsids were detected in fractions 7-8. BCM-599 and BCM-606 compounds showed a similar distribution pattern to the unassembled control group. That is, free dimer was detected in fractions 1-4 and negligible signals were detected in other fractions. This indicates that the BCM-599 and BCM-606 compounds are potent capsid assembly inhibitors, and this aspect was observed only when the cp149 dimer was in the free state. In the case of BCM-600 or BCM-602 too, a large amount of capsid was detected in the nonassembled fraction.

Example 6 Virion titer reduction effect of capsid inhibitor compound in HepG2.2.15 cells

Thirteen inhibitors identified here were treated with HepG2.2.15 cells to determine the inhibitory effect in the cytoplasmic environment. The results are shown in Table 2. As shown in Table 2, three of the thirteen compounds showed antiviral activity with an IC ₅₀ value of 10 μM or less. In contrast, the inhibition intensity of each compound was significantly different from that observed in the in vitro assembly. Five compounds showed inhibitory strength below 50 μM.

The strongest inhibitory effect was seen in BCM-599 (see Table 2). Compared to the in vitro assembly, the IC ₅₀ value of BCM-599 was reduced by more than 10-fold. Furthermore, the IC ₅₀ value of BCM-600 also decreased more than five-fold. However, in the case of BCM-606, the IC ₅₀ value slightly increased, exceeding the BCM-600 value. These results indicate that BCM-606 does not function effectively under cell culture conditions.

Example 7 Synergistic Effect of Combination of Capsid Inhibitor Compound and Lamivudine

Synergistic effects were measured using the inhibitor candidate compound with lamivudine. The results are shown in FIG. Six compounds with low IC ₅₀ values were selected in the infibox to calculate IC ₅₀ values and combination index with lamivudine (see Table 3). Based on the in vivo IC ₅₀ values of each compound of lamivudine and 25:75, 50:50; A 75:25 ratio was constructed so that the additive effect was 50% inhibition for all ratios. CI = 0.563 for 50:50 and CI = 0.65 for the other 25:75 and 75:25 ratios. In Table 3 below, all combinations are expressed in terms of the amount of compound: lamivudine, and the CI (combination index) is Calcusyn software (BIOSOFT, UK). The index is a quantitative analysis of the interactions between the compounds used in the experiment.

[Table 3]

Experimental results show that synergistic effects are exhibited in all combinations. The combination of lamivudine and BCM-599 together resulted in a CI value of less than 0.7, indicating significant synergistic effects from combination use. The synergistic effect was judged by the criteria described in Chou T. Theoretical Basis, Experimental Design, and Computerized Simulation of Synergism and Antagonism in Drug Combination Studies, Pharmacological Review, 2006; 58 (3): 621-681, A value of 0.7 or less is defined as having a general synergy.

In order to confirm the optimal ratio, lamivudine and BCM-599 were used in various concentration combinations. The lowest CI index (CI = 0.55) was observed at a combination ratio of 12: 1. In addition, lamivudine was fixed and the amount of BCM-599 was varied (see Table 4). In the IC ₅₀ of lamivudine of 0.07 μM, virus titer was 27% and 40%, respectively, by adding lamivudine at 1 × and 2 ×, Respectively. These results indicate that the present compounds can be usefully used in combination with existing antiviral agents to enhance the antiviral effect.

[Table 4]

Example 8 Identification of inhibitory effect by electron microscopic analysis

In electron microscopic analysis, capsid particles are detected as circular hollow particles with a circumference of 30-35 nm. Negative staining reveals that the capsids have a dark, bright ring at the center, and in some cases they are detected as white particles with no black spots in the center. This phenomenon is analyzed as a result of the lack of exposure to euvaniac acetate. As a result of using the unassembled cp149 sample, living particles were not detected, semi-regular phase agglutinates were detected, and this aspect was not detected when fully assembled.

When treated with inhibitor compounds, assembled and unassembled aspects appeared mixed (see FIG. 6). Relatively few partially assembled particles and high protein background were observed, which did not appear in fully assembled samples. This is because the dimer that should have been assembled as capsid particles during the assembly reaction remained unassembled due to the inhibitor compound. Further, when the relatively strong inhibitors BCM-599 and 606 among the compounds were compared to BCM-600 and BCM-602, more spherical particles were observed in the latter. These results indicate that the differences between the compounds as inhibitors affect capsid assembly completion.

As can be seen from the above experimental results, the compounds of formula (I) and pharmaceutical compositions containing them can be widely used for various liver diseases caused by HBV virus by effectively inhibiting virus multiplication through inhibition of capsid assembly.

While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Of the right.

Claims (14)

A pharmaceutical composition for preventing or treating liver disease caused by HBV infection comprising a compound of the following formula 1 or a salt thereof as an active ingredient:

,
In Formula 1,
A is C or N;
R ₁ and R ₂ are each hydrogen or halogen,
R ₃ and R ₄ are a straight or branched chain alkyl group each represent a _{hydrogen, C 1 -C 6, -C (} = O) R a, -S (= O) 2 R a , and wherein R is _a straight chain of C1-C6 Or a branched alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.
The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the compound of formula (1) is represented by the following formula (2) or (3)
(2)

;
(3)

In the general formula (2) or (3)
R ' ₁ , R' ₂ , R ' ₄ and R' ₅ are each hydrogen or halogen,
R _'3 and R' ₆ are each a hydrogen, a linear or branched alkyl group of _{C 1 -C 6, -C (=} O) R a, -S (= O) 2 R a, wherein R _a is C ₁ - C ₆ linear or branched alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group.
The pharmaceutical composition for preventing or treating liver disease according to claim 1, wherein the compound of formula (1) is represented by any one of the following formulas (4) to (16)
≪ Formula 4 >

;

≪ Formula 5 >

;

(6)

;

≪ Formula 7 >

;

(8)

;
≪ Formula 9 >

;

≪ Formula 10 >

;

≪ Formula 11 >

;

≪ Formula 12 >

;

≪ Formula 13 >

;

≪ Formula 14 >

;

≪ Formula 15 >

; or

≪ Formula 16 >

The method of claim 3, wherein the compound of Formula 1 is represented by Formula 5 (BCM-599), Formula 6 (BCM-600), Formula 8 (BCM-602), Formula 10 (BCM-604) ) Or a compound of formula 14 (BCM-608), for the prophylaxis or treatment of liver disease caused by HBV infection.
The pharmaceutical composition according to claim 1, wherein the liver disease includes hepatitis, fatty liver, liver failure, liver cirrhosis, or liver cancer.
The pharmaceutical composition according to claim 5, wherein the prevention or treatment of liver disease is by inhibition or blocking of HBV capside assembly.
The pharmaceutical composition according to claim 1, wherein the composition further comprises an HBV polymerase inhibitor.
The pharmaceutical composition according to claim 7, wherein the ratio of the compound to the HBV polymerase inhibitor is 1: 1 to 40: 1.
8. The method of claim 7 wherein the HBV polymerase inhibitor is selected from the group consisting of Lamivudin, Adefovir (ADV), Telbivudine, Entecavir or Tenofovir, A pharmaceutical composition for preventing or treating liver disease.
A hexamer consisting of six core proteins derived from hepatitis B virus.
11. The hexamer of claim 10, wherein the core protein has 149 amino acid residues from the full length or N terminus.
12. The hexamer according to claim 11, wherein the total length has the sequence of SEQ ID NO: 1, and the 149 amino acid residues have the sequence of 1 to 149 of SEQ ID NO:
[Claim 11] The method according to claim 10, wherein each core protein has 149 residues and comprises a groove formed of amino acid residues 21-35, 101-120, and 136-140 on the basis of the sequence of SEQ ID NO: 1 , Hexamer.
11. A method of screening a capsid assembly inhibitor of HBV using a hexamer according to claim 10, said method comprising measuring the binding force between said candidate compound and said groove structure formed in said hexamer.

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