PAPD5 inhibitors and PAPD7 inhibitors for the treatment of hepatitis B infection

Abstract

The present invention provides a method for identifying compounds that prevent, improve, and / or suppress hepatitis B virus (HBV) infection. [Solution] The present invention relates to a method for identifying compounds that (i) reduce the expression and / or activity of PAP-related domain-containing protein 5 (PAPD5) and / or PAP-related domain-containing protein 7 (PAPD7) and / or (ii) bind to PAPD5 and / or PAPD7 and inhibit the proliferation of HBV, as compounds for preventing, improving and / or suppressing HBV infection. The present invention also provides PAPD5 and / or PAPD7 inhibitors for use in the treatment and / or prevention of HBV infection, as well as mixed formulations comprising PAPD5 inhibitors and PAPD7 inhibitors for simultaneous or sequential use in the treatment and / or prevention of HBV infection. Pharmaceutical compositions for use in the treatment and / or prevention of HBV infection, and methods for monitoring treatment outcomes during the treatment of HBV infection are also included in the present invention.

Description

[Technical Field]

[0001] The present invention relates to a method for identifying compounds that prevent, improve, and / or suppress hepatitis B virus (HBV) infection, wherein the compounds identified are those that (i) reduce the expression and / or activity of PAP-related domain-containing protein 5 (PAPD5) and / or PAP-related domain-containing protein 7 (PAPD7), and / or (ii) bind to PAPD5 and / or PAPD7 and inhibit the proliferation of HBV. The present invention also provides PAPD5 and / or PAPD7 inhibitors for use in the treatment and / or prevention of HBV infection, as well as mixed formulations comprising PAPD5 inhibitors and PAPD7 inhibitors for simultaneous or sequential use in the treatment and / or prevention of HBV infection. Pharmaceutical compositions for use in the treatment and / or prevention of HBV infection, and methods for monitoring treatment outcomes during treatment of HBV infection are also included in the present invention. [Background technology]

[0002] Hepatitis B virus (HBV) is an enveloped partial double-stranded DNA virus. The small 3.2kb HBV genome consists of four redundant open reading frames (ORFs), which encode the core, polymerase (Pol), envelope, and X protein. The Pol ORF is the longest, with the envelope ORF located inside it, while the X ORF and core ORF overlap with the Pol ORF. The HBV life cycle involves two main phenomena: (1) the generation of closed-ring DNA (cccDNA) from loose-ring DNA (RC DNA) and (2) the reverse transcription of pregenomic RNA (pgRNA) to produce RC DNA. Prior to infection of host cells, the HBV genome exists within the virion as RC DNA. HBV virions are thought to be able to enter host cells through nonspecific binding to negatively charged proteoglycans on the surface of human liver cells (Schulze, Hepatology, 46, (2007), 1759-68) and specific binding of the HBV surface antigen (HBsAg) to the hepatocyte sodium / taurocholic acid cotransport polypeptide (NTCP) receptor (Yan, J Virol, 87, (2013), 7977-91). Suppression of viral infections requires strict monitoring by the host's innate immune system, which can respond within minutes to hours after infection to disrupt the initial replication of the virus and limit the development of chronic and persistent infections. Despite the availability of current therapies based on IFNs and nucleoside / nucleotide analogs, HBV infection remains a major global health problem involving an estimated 350 million chronic carriers at relatively high risk of cirrhosis and hepatocellular carcinoma.

[0003] The secretion of antiviral cytokines by hepatocytes and / or intrahepatic immune cells in response to HBV infection plays a central role in virus clearance by the infected liver. However, chronically infected patients exhibit only a weak immune response due to the various evasion strategies employed by the virus to counter the host cell's recognition system and subsequent antiviral response.

[0004] Numerous observations have shown that several HBV viral proteins can counteract the initial host cell response by interfering with the virus recognition signaling pathway and subsequently inhibiting interferon (IFN) antiviral activity. Among these, the oversecretion of HBV empty subviral particles (SVP, HBsAg) is thought to be involved in maintaining the state of immune tolerance observed in chronically infected patients (CHB). Sustained exposure to HBsAg and other viral antigens can cause HBV-specific T cell deficiency or progressive dysfunction (Kondo, Journal of Immunology (1993), 150, 4659-4671; Kondo, Journal of Medical Virology (2004), 74, 425-433; Fisicaro, Gastroenterology, (2010), 138, 682-93). Furthermore, HBsAg has been reported to suppress the function of immune cells such as monocytes, dendritic cells (DCs), and natural killer (NK) cells through direct interactions (Op den Brouw, Immunology, (2009b), 126, 280-9; Woltman, PLoS One, (2011), 6, e15324; Shi, J Viral Hepat. (2012), 19, e26-33; Kondo, ISRN Gasteroenterology, (2013), Article ID 935295).

[0005] Quantification of HBsAg is an important biomarker for prognosis and treatment response in chronic hepatitis B. However, the disappearance of HBsAg and achievement of seroconversion are rarely observed in patients with chronic infection and continue to be one of the ultimate treatment goals. Current therapies such as nucleoside / nucleotide analogs are molecules that inhibit HBV DNA synthesis and are not aimed at reducing HBsAg levels. Even with long-term treatment, nucleoside / nucleotide analogs show only weak HBsAg clearance equivalent to that naturally observed (between about 1% and 2%) (Janssen, Lancet, (2005), 365, 123 - 9; Marcellin, N. Engl. J. Med., (2004), 351, 1206 - 17; Buster, Hepatology, (2007), 46, 388 - 94).

[0006] Hepatitis B virus e antigen (also called HBV envelope antigen or HBeAg) is a viral protein secreted by hepatitis B virus-infected cells. HBeAg is associated with chronic hepatitis B infection and is used as a marker of active viral disease and the degree of infectivity of patients.

[0007] The functions of hepatitis B virus precoa or HBeAg are not fully understood. However, HBeAg is well known to play an important role in viral persistence. HBeAg is thought to promote the chronicity of HBV by functioning as an immunomodulatory protein. Specifically, HBeAg is a secreted co-protein that appears to attenuate the host immune response to intracellular nucleocapsid proteins (Walsh, Virology, 2011, 411(1):132-141). HBeAg acts as an immune tolerance source that contributes to the survival of HBV, and considering that soluble HBeAg crosses the placenta, it probably functions in the uterus (Walsh, Virology, 2011, 411(1):132-141). Furthermore, HBeAg downregulates (i) cellular genes that control intracellular signaling and (ii) Toll-like receptor 2 (TLR-2) to weaken the innate immune response to viral infection (Walsh, Virology, 2011, 411(1):132-141). In the absence of HBeAg, HBV replication is accompanied by upregulation of the TLR2 pathway (Walsh, Virology, 2011, 411(1):132-141). Therefore, HBeAg plays an important role in regulating virus-host interactions in order to influence the host immune response (Walsh, Virology, 2011, 411(1):132-141). Therefore, reducing HBeAg levels in HBeAg-positive patient populations may lead to a reversal of HBV-specific immunodeficiency (Milich, 1997, J. Viral. Hep. 4: 48-59; Milich, 1998, J. Immunol. 160: 2013-2021). In addition, secreted HBeAg is significantly more effective than HBcAg in inducing T cell tolerance and dissociative T cell tolerance between HBeAg and intracellular hepatitis core antigen (HBcAg), suggesting that clonal heterogeneity of HBc / HBeAg-specific T cell tolerance may have significant implications for spontaneous HBV infection, particularly for pre-core-negative chronic hepatitis (Chen, 2005, Journal of Virology, 79: 3016-3027).

[0008] Therefore, reducing HBeAg secretion in addition to HBsAg secretion improves the suppression of chronic HBV infection compared to inhibiting HBsAg secretion alone. In addition, the highest rates of transmission of acute to chronic infection (over 80%) have been reported in maternal-to-fetal HBV transmission and neonatal HBV transmission from HBeAg-positive mothers (Liaw, Lancet, 2009, 373: 582-592; Liaw, Dig. Dis. Sci., 2010, 55: 2727-2734; and Hadziyannis, 2011, Journal of hepatology, 55: 183-191). Therefore, reducing HBeAg in women who will become mothers may not only reduce the infectivity of the patient but also suppress the development of chronic HBV infection in the woman's child.

[0009] Therefore, there is an unfulfilled medical requirement for the treatment of HBV: inhibiting viral expression, particularly the secretion of HBsAg and HBeAg (Wieland, SF & FV Chisari. J Virol, (2005), 79, 9369-80; Kumar et al. J Virol, (2011), 85, 987-95; Woltman et al. PLoS One, (2011), 6, e15324; Op den Brouw et al. Immunology, (2009b), 126, 280-9).

[0010] International Publication No. 03 / 022987 discloses, for example in Table 7A, 1298 genes that are upregulated in hepatitis C-positive tissue. One of the aforementioned genes is topoisomerase-associated functional protein 4 (TRF4, AF089897). AF089897, also known as TRF4-2, is very similar to positions 880-2340 of Sequence ID No. 4 in this specification. The observation that the PAPD5 fragment is only slightly upregulated in hepatitis C-positive cells does not in any way suggest that PAPD5 inhibition is an effective treatment. International Publication No. 03 / 022987(A2) discloses absolutely no indication that the PAPD5 fragment plays any important role in hepatitis C infection. In addition, HCV and HBV are two completely different viruses that cause two completely different diseases with different etiologies, different progressions, and different drug treatments. This is consistent with our own observations that DHQ and THP, which are inhibitors of PAPD5 and PAPD7, are inactive against hepatitis C virus (HCV) or other viruses other than HBV (data not shown).

[0011] While International Publication No. 2010 / 040571 does not provide any concrete evidence, PAPD5 is suggested to be among a long list of other genes that have a potential role in cell proliferation in metabolic and neoplastic diseases.

[0012] While International Publication No. 2013 / 166264 does not provide any concrete evidence, PAPD5 is suggested to be among a long list of other genes that have a potential role in increasing viral replication in metabolic and neoplastic diseases.

[0013] International Publication No. 2017 / 066712 describes the downregulation of PAPD5 in relation to the treatment and diagnosis of telomere diseases. Five types of shRNA structures are described for this purpose.

[0014] To our knowledge, PAPD5 or PAPD7 expression has not been associated with HBV infection. [Overview of the Initiative] [Problems that the invention aims to solve]

[0015] Therefore, the fundamental technical problem underlying the present invention is to identify and provide improved means and methods for the treatment and / or prevention of HBV infection.

[0016] The technical challenges described herein and resolved by the presentation of embodiments characterized in the claims.

[0017] One aspect of the present invention is a screening method, specifically a method for identifying compounds that prevent, improve, and / or suppress HBV infection, (a) Contact the test compound with the following: (a1) PAPD5 polypeptide and / or PAPD7 polypeptide, (a2) Cells expressing PAPD5 and / or PAPD7, (b) Measuring the expression and / or activity of PAPD5 and / or PAPD7 in the presence and absence of the test compound, (c) Identify compounds that reduce the expression and / or activity of PAPD5 and / or PAPD7 as compounds that prevent, improve, and / or suppress HBV infection. The method includes the foregoing.

[0018] An additional aspect of the present invention is a method for identifying compounds that prevent, improve, and / or suppress HBV infection, (a) Contact the test compound with the following: (i) PAPD5 polypeptide and / or PAPD7 polypeptide, (ii) Cells expressing PAPD5 and / or PAPD7, (b) To measure whether the test compound binds to the PAPD5 polypeptide and / or the PAPD7 polypeptide, (c) To measure whether the test compound inhibits the proliferation of HBV, and (d) Identify compounds that bind to PAPD5 polypeptide and / or PAPD7 polypeptide and inhibit HBV replication as compounds that prevent, improve, and / or suppress HBV infection. The method includes the above.

[0019] An additional aspect of the present invention is an inhibitor of PAPD5 and / or PAPD7 for use in the treatment and / or prevention of HBV infection, wherein the inhibitor is (a) Small molecules that bind to PAPD5 and / or PAPD7, or (b) Antibodies that specifically bind to PAPD5 and / or PAPD7 That is the case.

[0020] The inhibitors used for the treatment or prevention of HBV may be selected from compounds of formula (I) or (II). Specifically, inhibitors of formula (III) and (IV) are used in the present invention. [Brief explanation of the drawing]

[0021] [Figure 1] This is a diagram of a one-to-one experiment using the HBX129653 / HBX129654 chemical probe and three types of play fragments. [Figure 2] This is a diagram of a one-to-one experiment using the HBX129653 / HBX129654 chemical probe and the full-length PAPD5 / 7 protein. [Figure 3] This is a diagram of a competitive assay using HBX129653(DHQ) and MOL653 / 654 for competition. [Figure 4] This is a diagram of a competitive assay using HBX129654(THP) and MOL653 / 654 for competition. [Figure 5]This figure shows a competitive assay using HBX129653 (DHQ) and INACT653 / INACT654 for competition. MOL653 was included as a positive control. [Figure 6] This figure shows a competitive assay using HBX129654(THP) and INACT653 / INACT654 for competition. MOL653 was included as a positive control. [Figure 7] (A) This figure shows that HBV expression is reduced by siRNA knockdown (KD) of PAPD5 and PAPD7 in HBV-infected dHepaRG cells. HepaRG differentiated cells were infected with HBV and treated with siRNA (25 nM each) against PAPD5, PAPD7, or both, one day before HBV infection and four days after infection. The supernatant was collected on day 11, and the levels of HBsAg and HBeAg secreted into the supernatant were measured by ELISA and normalized to the untreated control. Subsequently, cytotoxicity and inhibition of gene expression were measured and normalized to the untreated control. (B) This figure shows the same experiment as described in (A), except that only the level of HBsAg secreted into the supernatant was measured. [Modes for carrying out the invention]

[0022] PAPD5 and PAPD7 are non-standard poly(A) polymerases belonging to the polymerase β-like nucleotidyltransferase superfamily. In the background of the present invention, it has been surprisingly shown that by analyzing whether a test compound inhibits PAPD5 and / or PAPD7, it is possible to successfully identify compounds useful for therapeutic intervention of HBV infection. In other words, the inhibition of PAPD5 and / or PAPD7 has been confirmed in the attached examples as an indicator of the efficacy of compounds that suppress HBV infection. These attached examples demonstrate that a dihydroquinolidinone compound having formula (III) shown below herein (referred to herein as DHQ) and a tetrahydropyridopyrimidine compound having formula (IV) shown below herein (referred to herein as THP) bind to the PAPD5 polypeptide and the PAPD7 polypeptide. These compounds have the ability to inhibit the production of HBV surface antigen (HBsAg) and the expression of HBV RNA during HBV infection (International Publication No. 2015 / 113990(A1) and International Publication No. 2016 / 177655). In addition, the accompanying examples demonstrate that inhibition of PAPD5 and / or PAPD7 by the use of siRNA leads to inhibition of viral expression, particularly inhibition of HBsAg and HBeAg secretion, and inhibition of intracellular HBV mRNA production. These results directly demonstrate that HBV infection (e.g., chronic HBV infection) can be prevented or treated (i.e., improved and / or suppressed) by reducing the amount and / or activity (e.g., amount) of PAPD5 and / or PAPD7. Therefore, the present invention relates to a screening method for identifying compounds that reduce the expression and / or activity (e.g., expression) of PAPD5 and / or PAPD7 (e.g., PAPD5 and PAPD7) as compounds that prevent and / or treat (i.e., improve and / or suppress) HBV infection.

[0023] In the background of this invention, it is known that compounds that counteract (i.e. inhibit) PAPD5 and / or PAPD7 inhibit the gene expression and replication of HBV, thereby preventing, improving, and / or suppressing HBV infection. Such compounds can cause a decrease of 10-100%, preferably 20-100%, more preferably 30-100%, even more preferably 40-100%, even more preferably 50-100%, even more preferably 60-100%, even more preferably 70-100%, even more preferably 80-100%, and most preferably 90-100% of PAPD5 and / or PAPD7 expression and / or activity.

[0024] The screening method provided herein is intended to measure (i.e., analyze / determine) the expression of PAPD5 and / or PAPD7 by using cells expressing PAPD5 and / or PAPD7, i.e., (ai), in step (a). In step (a), the activity of PAPD5 and / or PAPD7 can be measured (i.e., analyzed / determined) by using either (ai) PAPD5 polypeptide and / or PAPD7 polypeptide in a cell-free preparation, for example, or (aii) cells expressing PAPD5 and / or PAPD7.

[0025] In one aspect of the present invention, compounds that reduce the expression of PAPD5 and / or PAPD7 (e.g., the expression of PAPD5, preferably the expression of both PAPD5 and PAPD7) are identified as compounds that prevent, improve, and / or suppress (i.e., treat) HBV infection. In another aspect of the present invention, compounds that reduce the activity of PAPD5 and / or PAPD7 (e.g., the activity of PAPD5, preferably the activity of both PAPD5 and PAPD7) are identified as compounds that prevent, improve, and / or suppress (i.e., treat) HBV infection. It is preferred that compounds that reduce the expression and / or activity of PAPD5, or both molecules, i.e., the expression and / or activity of PAPD5 and PAPD7, are identified as compounds that prevent, improve, and / or suppress HBV infection. It is most preferred that compounds that reduce the expression and / or activity of both molecules, i.e., PAPD5 and PAPD7, are identified as compounds that prevent, improve, and / or suppress HBV infection.

[0026] According to the present invention, compounds that prevent and / or treat (i.e., improve and / or suppress) HBV infection can be identified (i.e., selected) by performing a first preliminary selection step to identify compounds that bind to PAPD5 and / or PAPD7. Subsequently, in a second step, the compounds identified as binding to PAPD5 and / or PAPD7 can be evaluated for inhibiting HBV replication. Thus, the present invention relates to an additional screening method in which compounds that bind to PAPD5 and / or PAPD7 (e.g., PAPD5 and PAPD7) and inhibit HBV replication are identified as compounds that prevent, improve and / or suppress (i.e., treat) HBV infection.

[0027] Therefore, the present invention is a method for identifying compounds that prevent, improve, and / or suppress HBV infection, (a) Contact the test compound with the following: (ai)PAPD5 polypeptide and / or PAPD7 polypeptide, (aii) Cells expressing PAPD5 and / or PAPD7, (b) To measure whether the test compound binds to PAPD5 and / or PAPD7, (c) To measure whether the test compound inhibits the proliferation of HBV, and (d) Identify compounds that bind to PAPD5 and / or PAPD7 and inhibit HBV replication as compounds that prevent, improve, and / or suppress HBV infection. The method includes the foregoing.

[0028] Accordingly, according to the present invention, compounds that bind to PAPD5 and / or PAPD7 (e.g., PAPD5, preferably PAPD5 and PAPD7) and inhibit HBV replication are identified as compounds that prevent, improve, and / or suppress (i.e., treat) HBV infection. It is preferred that compounds that (i) bind to PAPD5 or both molecules, i.e., PAPD5 and PAPD7, and (ii) inhibit HBV replication are identified as compounds that prevent, improve, and / or suppress HBV infection. Most preferably, compounds that bind to both molecules, i.e., PAPD5 and PAPD7, and inhibit HBV replication are identified as compounds that prevent, improve, and / or suppress HBV infection.

[0029] The above screening method identifies compounds that prevent, improve, and / or suppress HBV infection. Priority is given to compounds that improve and / or suppress (i.e., treat) HBV infection. Therefore, the screening method provided herein is useful for identifying compounds that treat HBV infection.

[0030] In the context of the present invention, PAPD5 may be a PAPD5 polypeptide or PAPD5 mRNA. In the context of the screening method provided herein, it is preferred that PAPD5 is a PAPD5 polypeptide. One aspect of the present invention is a screening method provided herein, wherein the PAPD5 polypeptide is (a) Amino acid sequence of SEQ ID NO: 1 or 2, (b) an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of (a), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function. (c) The amino acid sequence of the enzyme activity fragment of Sequence ID No. 1 or 2, or (d) an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of (d), wherein the polypeptide has poly(A) polymerase function. The present invention relates to the screening method which includes or comprises a polypeptide consisting of the amino acid sequence.

[0031] Examples of the enzyme activity fragment of SEQ ID NO: 1 or 2 (i.e., PAPD5) are the nucleotidyltransferase domain located at positions 145-256 of SEQ ID NO: 1 or 2, or the Cid1 polyA polymerase located at positions 308-368 of SEQ ID NO: 1 or 2.

[0032] Another aspect of the present invention is a screening method provided herein, wherein the cells expressing PAPD5 contain PAPD5 mRNA, (i) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2, (ii) A nucleotide sequence encoding an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 1 or 2, wherein a polypeptide having polyA polymerase function is encoded by the nucleotide sequence. (iii) A nucleotide sequence encoding the enzyme activity fragment of Sequence ID No. 1 or 2, (iv) A nucleotide sequence encoding an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of the enzyme activity fragment of SEQ ID NO: 1 or 2, wherein a polypeptide having polyA polymerase function is encoded by the nucleotide sequence. (v) A nucleotide sequence comprising SEQ ID NO: 4 or 5, or consisting of the SEQ ID NO: 4 or 5, or (vi) A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 4 or 5, wherein the sequence expresses a polypeptide having poly(A) polymerase function, or (vii) PremRNA that undergoes processing (i.e., splicing) to become a polynucleotide of (v) or (vi). The present invention relates to a screening method comprising, or comprising a polynucleotide consisting of the nucleotide sequence.

[0033] In a preferred embodiment, the PAPD5 mRNA may contain the nucleotide sequence of SEQ ID NO: 4 or 5, or be a polynucleotide consisting of the said nucleotide sequence. However, the PAPD5 mRNA may also contain a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 4 or 5, or be a polynucleotide consisting of the said nucleotide sequence, which may encode a polypeptide having poly(A) polymerase function.

[0034] In the context of the present invention, PAPD7 may be a PAPD7 polypeptide or PAPD7 mRNA. In the context of the screening method provided herein, it is preferred that PAPD7 is a PAPD7 polypeptide. One aspect of the present invention is a screening method provided herein, wherein the PAPD7 polypeptide is (a) Amino acid sequence of Sequence ID No. 3, (b) an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of (a), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function. (c) The amino acid sequence of the enzyme activity fragment of Sequence ID No. 3, or (d) an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of (c), wherein the polypeptide has poly(A) polymerase function. The present invention relates to the screening method which includes or comprises a polypeptide consisting of the amino acid sequence.

[0035] Examples of the enzyme activity fragment of SEQ ID NO: 3 (i.e., PAPD7) are the nucleotidyltransferase domain located at positions 15-125 of SEQ ID NO: 3, or the Cid1 family polyA polymerase located at positions 178-238 of SEQ ID NO: 3.

[0036] Another aspect of the present invention is a screening method provided herein, wherein the cells expressing PAPD7 contain PAPD7 mRNA, (i) The nucleotide sequence that encodes the amino acid sequence of Sequence ID No. 3, (ii) A nucleotide sequence encoding an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 3, wherein a polypeptide having poly(A) polymerase function is encoded by the nucleotide sequence. (iii) A nucleotide sequence encoding the enzyme activity fragment of Sequence ID No. 3, or (iv) A nucleotide sequence encoding an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of the enzyme activity fragment of Sequence ID No. 3, wherein the nucleotide sequence is encoded by a polypeptide having poly(A) polymerase function, or (v) A nucleotide sequence including or consisting of the above sequence number, (vi) A nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 6, wherein the sequence expresses a polypeptide having poly(A) polymerase function, or (vii) PremRNA that undergoes processing (i.e., splicing) to become a polynucleotide of (v) or (vi). The present invention relates to a screening method comprising, or comprising a polynucleotide consisting of the nucleotide sequence.

[0037] In a preferred embodiment, the PAPD7 mRNA may contain the nucleotide sequence of SEQ ID NO: 6, or be a polynucleotide consisting of the nucleotide sequence. However, the PAPD7 mRNA may also contain a nucleotide sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to SEQ ID NO: 6, or be a polynucleotide consisting of the nucleotide sequence, which may encode a polypeptide having poly(A) polymerase function.

[0038] In the context of the present invention, the cells may be eukaryotic cells. For example, the cells may be yeast cells or vertebrate cells. Vertebrate cells may include cells of fish, birds, reptiles, amphibians, marsupials, and mammals. The cells are preferably mammalian cells, and most preferably human cells. Mammalian cells also include cells of cats, dogs, cattle, horses, goats, sheep, pigs, mice, rats, and rabbits. In the screening method provided herein, the “cells” may endogenously express PAPD5 and / or PAPD7, or overexpress PAPD5 and / or PAPD7. For overexpression of PAPD5 and / or PAPD7, the cells may include nucleotide sequences encoding PAPD5 polypeptide and / or PAPD7 polypeptide in the expression vector. In a preferred embodiment, the cells include nucleotide sequences encoding PAPD5 polypeptide and nucleotide sequences encoding PAPD7 polypeptide. The cells in the screening method provided herein may include non-human animals, such as mice, rats, rabbits, or ferrets.

[0039] In the above screening method, which measures binding to PAPD5 and / or PAPD7, a compound can be identified as a compound that binds to the PAPD5 polypeptide and / or PAPD7 polypeptide if it has a specific binding affinity for PAPD5 and / or PAPD7. For example, a compound that binds to PAPD5 and / or PAPD7 may have a dissociation constant (Kd) in the micromolar concentration range, or preferably in the range of 100 nM to 1 pM.

[0040] In the background of the present invention, the test compound may be measured (i.e., analyzed) to determine whether it specifically binds to the PAPD5 polypeptide and / or the PAPD7 polypeptide, that is, whether it binds exclusively or mainly to PAPD5 and / or PAPD7. For example, the test compound may be measured to determine whether it specifically binds to PAPD7. It is preferable that the test compound be measured to determine whether it specifically binds to PAPD5. It is more preferable that the test compound be measured to bind to both PAPD5 and PAPD7. For example, the test compound may be measured to determine whether it specifically binds to both PAPD5 and PAPD7.

[0041] For example, in the screening method provided herein, the binding of the test compound to PAPD5 and / or PAPD7 can be measured by performing a yeast 3 hybrid screen. The Y3H system is a modified yeast 2 hybrid (Y2H) system adapted for the detection of drug-protein interactions. The system requires the binding of a target drug to a ligand that can be immobilized on a DNA-binding protein inside a yeast cell. The interaction between the immobilized drug and the target protein is then detected by relating their binding to the transcriptional activation of a reporter gene. See, for example, Johnsson, Nature Chem Bio, 2011, 7: 375-383; and Licitra, Proc Natl Acad Sci USA, 1996, 12; 93(23):12817-21. Such yeast 3 hybrid screens may use an inactive free compound for competition with the labeled test compound.

[0042] The binding of the test compound to PAPD5 and / or PAPD7 may also be measured using Biacore, chemopromics, or microscale thermophoresis.

[0043] Compounds that inhibit HBV replication may be compounds that reduce the expression of viral RNA, decrease the production of viral DNA (HBV DNA) derived from viral RNA (HBV RNA), decrease the production of new viral particles (HBV particles), and / or result in the production and / or secretion of HBsAg and / or HBeAg. Accordingly, one aspect of the present invention relates to a screening method provided herein, wherein the secretion of HBsAg is inhibited, the secretion of HBeAg is inhibited, and / or the production of intracellular HBV mRNA or HBV DNA is inhibited by the compound that inhibits HBV replication. Preferably, the compound that inhibits HBV replication is a compound that inhibits the secretion of HBsAg, the secretion of HBeAg, and the production of intracellular HBV mRNA.

[0044] For example, compounds that inhibit HBV proliferation can reduce the expression of viral RNA (HBV RNA), the production of viral DNA (HBV DNA) derived from viral RNA, the production of new viral particles (HBV particles), the production of HBsAg and / or HBeAg, and / or the secretion of HBsAg and / or HBeAg by 10-100%, preferably 20-100%, more preferably 30-100%, even more preferably 40-100%, even more preferably 50-100%, even more preferably 60-100%, even more preferably 70-100%, even more preferably 80-100%, and most preferably 90-100% when comparing untreated cells or animals with cells or animals treated with an appropriate control.

[0045] The screening method provided herein may further include a step of comparing the test compound with a control. The control is an inactive test compound, (i) Without reducing the expression and / or activity of PAPD5 and / or PAPD7, and / or (ii) Does not bind to PAPD5 and / or PAPD7 and does not inhibit HBV proliferation. The inert test compound may be a compound.

[0046] This inactive test compound has no activity against HBV; for example, it does not inhibit intracellular HBV mRNA production unless it inhibits the secretion of HBsAg and HBeAg. For example, the inactive test compound inhibits HBsAg with an IC50 of over 3 μM. 50 A value may be present. In the screening method provided herein, the inactive test compound may be the compound referred to as the "inactive DHQ compound" or the compound referred to as the "inactive THP compound" as defined in the attached examples. In the screening method in which the expression and / or activity of PAPD5 and / or PAPD7 is measured, the test compound referred to in (i) above may be used. Alternatively, in the screening method in which binding to PAPD5 and / or PAPD7 is measured, the test compound referred to in (ii) above may be used. The inactive compound can be designed from the active compound, for example, by chemical modification and / or chiral separation.

[0047] In the screening method provided herein, the activity of PAPD5 and / or PAPD7 in the presence and absence of the test compound can be measured by monitoring the in vitro polyadenylation of mRNA, as described, for example, in Rammelt, RNA, 2011, 17:1737-1746. Briefly speaking, in the presence of ATP(A), CTP(C), GTP(G), UTP(U), or a mixture of all four types of dNTPs, ribooligonucleotide A 15 This may be incubated with recombinant PAPD5 protein expressed in E. coli.

[0048] The expression of PAPD5 and / or PAPD7 in the presence and absence of the test compound can be measured, for example, by (q)PCR, Western blotting, or mass spectrometry.

[0049] Inhibition of HBV proliferation can be measured, for example, by determining whether the test compound has activity to inhibit the secretion of HBsAg and / or HBeAg and / or the production of intracellular HBV mRNA. Inhibition of HBsAg and / or HBeAg secretion can be measured by ELISA, for example, by using the CLIA ELISA kit (Autobio Diagnostics) according to the manufacturer's instructions. Inhibition of intracellular HBV mRNA production can be measured by real-time PCR, for example, as described in the attached examples. Other methods for evaluating whether the test compound inhibits HBV proliferation include, for example, measurement of HBV DNA secretion by RT-qPCR, measurement by Northern blotting, measurement by in situ hybridization, or measurement by immunofluorescence, as described in International Publication No. 2015 / 173208.

[0050] Publicly available or commercially available molecular libraries can be used to carry out the screening methods provided herein. Therefore, in the background of the present invention, the test compound (i) Small molecules from the screening library, or (ii) peptides from a phage display library, peptides from an antibody fragment library, or peptides derived from a cDNA library It is possible.

[0051] For example, the cDHA human liver (HLV) library or the cDNA human placenta (PLA) library from Hybrigenics Services SAS can be used.

[0052] In the screening method provided herein for measuring the activity of PAPD5 polypeptide and / or PAPD7 polypeptide, the activity of PAPD5 and PAPD7 is preferably poly(A) polymerase function (i.e., poly(A) polymerase activity). The poly(A) polymerase function / activity of a polypeptide (e.g., PAPD5 or PAPD7) can be measured by monitoring in vitro polyadenylation of mRNA, as described, for example, in Rammelt, RNA, 2011, 17:1737-1746. This method can also be used to measure the poly(A) polymerase function of PAPD5 and / or PAPD7 in the presence and absence of the test compound.

[0053] The attached examples demonstrate that inhibiting PAPD5 polypeptide and / or PDPD7 polypeptide can effectively inhibit HBsAg and HBeAg secretion, as well as intracellular HBV mRNA production. These data demonstrate that PAPD5 and / or PAPD7 inhibitors can be used to prevent and / or treat HBV infection.

[0054] Several compounds with a certain efficacy in treating HBV infection have been described in the art (see, for example, International Publication No. 2015 / 113990(A1) and International Publication No. 2016 / 177655). However, in the background of the present invention, it has been surprisingly found that structurally completely different anti-HBV agents (e.g., DHQ and THP) bind to PAPD5 and PAPD7 in a surprising and specific manner. In addition, the prior art also includes agents with relatively low activity in inhibiting HBsAg production. Such agents have relatively low binding affinity to PAPD5 and PAPD7, as shown in the appended examples (see, for example, {inactive DHQ}). In fact, a clear correlation between the activity of the said compounds against HBV infection and their binding affinity to PAPD5 and PAPD7 has been demonstrated in the appended examples. Therefore, particularly high anti-HBV efficacy can be obtained by selectively using anti-HBV agents that bind to PAPD5 and / or PAPD7. Furthermore, the present invention has for the first time demonstrated that compounds inhibiting PAPD5, PAPD7, or in particular PAPD5 and PAPD7, have very high activity in inhibiting the secretion of HBsAg and HBeAg, as well as inhibiting the production of intracellular HBV mRNA. Reducing the secretion of HBsAg and HBeAg more effectively suppresses the development of chronic HBV infection compared to reducing HBsAg secretion alone. In addition, inhibiting the secretion of HBsAg and HBeAg reduces the infectivity of HBV-infected individuals. Moreover, reducing HBeAg in women who become mothers can also suppress the development of chronic HBV infection in their children. Thus, the present invention has unexpectedly demonstrated that selective use of compounds that inhibit PAPD5 and / or PAPD7 can lead to improved therapeutic outcomes in the treatment of HBV infection with respect to a considerably effective reduction of HBsAg and HBeAg.

[0055] Therefore, one aspect of the present invention is the use of PAPD5 and / or PAPD7 inhibitors in the treatment of HBV infection, particularly chronic HBV infection. In additional embodiments, the present invention relates to the use of PAPD5 and / or PAPD7 inhibitors to reduce the viral antigens HBsAg and HBeAg.

[0056] Therefore, the present invention is an inhibitor of PAPD5 and / or PAPD7 for use in the treatment and / or prevention of HBV infection, (i) Small molecules that bind to PAPD5 and / or PAPD7, (ii) RNA interference (RNAi) molecules for PAPD5 and / or PAPD7, (iii) an antibody that specifically binds to PAPD5 and / or PAPD7, or (iv) Genome editing devices including the following: (a) site-specific DNA nucleases, or polynucleotides encoding site-specific DNA nucleases, and (b) Guide RNA, or polynucleotide encoding guide RNA The present invention relates to the aforementioned inhibitor.

[0057] The inhibitor of the present invention may be a locked nucleic acid (LNA) molecule specific to PAPD5 and / or PAPD7.

[0058] The present invention is intended to be used to treat (e.g., improve) HBV infection.

[0059] The inhibitor may be a molecule that specifically inhibits PAPD7. Preferably, the inhibitor is a molecule that specifically inhibits PAPD5. More preferably, the inhibitor inhibits both PAPD5 and PAPD7. Therefore, it is preferred that the inhibitor of the present invention inhibits either PAPD5 or both PAPD5 and PAPD7. Most preferably, the inhibitor of the present invention inhibits both PAPD5 and PAPD7. In one embodiment of the present invention, both PAPD5 and PAPD7 are inhibited by the inhibitor of the present invention, and a reduction in HBsAg and / or HBeAg secretion of at least 50% is observed compared to an untreated control (i.e., compared to cells or subjects that were not administered the drug).

[0060] The inhibitors of the present invention have an IC50 concentration of less than 3 μM, preferably less than 2 μM, more preferably less than 1 μM, more preferably less than 0.1 μM, and most preferably less than 0.01 μM for the inhibition of HBsAg and HBeAg. 50 It may have a value.

[0061] Genome editing using site-specific DNA nucleases (such as Cas9 or Cpf1) and guide RNA is well known in this field and is described, for example, in "CRISPR-Cas: A Laboratory Manual," 2016, edited by Jennifer Doudna, ISBN 978-1-621821-31-1.

[0062] For example, if the site-specific DNA nuclease is a Cas9 nuclease, the genome editing device (i) At least one guide RNA consisting of at least one target sequence-specific CRISPR RNA (crRNA) molecule and at least one transactivating crRNA (tracrRNA) molecule, (ii) Polynucleotides encoding the RNA molecule described in (i) above, (iii) at least one guide RNA which is a chimeric RNA molecule containing at least one target sequence-specific crRNA and at least one tracrRNA, or (iv) Polynucleotide encoding the chimeric RNA of (iii) above It is preferable to further include the following.

[0063] In an alternative example, the site-specific DNA nuclease is Cpf1 nuclease, and the genome editing device is... (i) At least one guide RNA containing a target sequence-specific CRISPR RNA (crRNA) molecule, or (ii) Polynucleotide encoding the RNA molecule described in (i) above It is preferable to further include the following.

[0064] The inhibitors of PAPD5 and / or PAPD7 provided herein may be genome editing devices comprising at least one assembled Cas9 protein-guide RNA ribonucleoprotein complex (RNP).

[0065] In this specification, the guide RNA is designed to target the genomic DNA of PAPD5 or PAPD7. Alternatively, several guide RNAs can be used to target the genomic DNA of both PAPD5 and PAPD7. Inhibition of PAPD5 and / or PAPD7 can be achieved by introducing frameshift knockout mutations into the genomic DNA of PAPD5 and / or PAPD7 via non-homologous end joining (NHEJ), or by modifying the genomic DNA of PAPD5 and / or PAPD7 via homologous recombination repair (HDR). How these mechanisms can be induced is generally known in the art and is described, for example, Heidenreich, 2016, Nat Rev Neurosci 17 36-44.

[0066] The inhibitors of the present invention may be natural molecules, such as natural antibodies or natural RNAi molecules. However, the inhibitors of the present invention may also be non-natural molecules. For example, the inhibitors of the present invention may be antibodies having an amino acid sequence that is not the same as that of natural antibodies, or antibodies containing at least one non-natural amino acid residue, such as synthetic amino acids that provide similar side-chain functional groups. For example, aromatic amino acids include D- or L-naphthylalanine, D- or L-phenylglycine, D- or L-2-thienylalanine, D- or L-1-, 2-, 3-, or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyradinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, and D-(trifluoromethyl)-phenylglycine. The alkyl group may be replaced with D-(trifluoromethyl)-phenylalanine, Dp-fluorophenylalanine, D- or Lp-biphenylalanine, D- or Lp-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanine, and D- or L-alkylalanine, in which case the alkyl group is selected from the group consisting of substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, isobutyl, and isopentyl. As should be considered as an unrestricted example, non-carboxylic acid amino acids can be made to have a negative charge, as provided by phosphonoamino acids or sulfated amino acids. Other non-natural amino acids are alkylated amino acids made by combining any natural amino acid with an alkyl group. Basic natural amino acids such as lysine and arginine may be substituted with an alkyl group at the amine (NH2) functional group. Further substitutions on non-natural amino acids include nitrile derivatives of asparagine or glutamine (e.g., derivatives containing a CN moiety at the position of the CONH2 functional group) and sulfoxide derivatives of methionine.

[0067] Similarly, the inhibitor of the present invention may be an RNAi molecule having a nucleotide sequence that is not the same as that of a natural RNAi molecule, or it may be an RNAi molecule containing at least one non-natural nucleotide, such as an oligonucleotide thiophosphate, a substituted ribooligonucleotide, an LNA molecule, a PNA molecule, a GNA (glycolic nucleic acid) molecule, a TNA (threose nucleic acid) molecule, a morpholinopolynucleotide, or a nucleic acid having a modified skeleton such as a polysiloxane or 2'-O-(2-methoxy)ethyl phosphorothioate, or a nucleic acid having substituents such as a methyl nucleoside, a thionucleoside, a sulfate nucleoside, a benzoyl nucleoside, a phenyl nucleoside, an amino nucleoside, a propyl nucleoside, a chloronucleoside, and a metanocarbanucleoside, or it may be an RNAi molecule containing a reporter molecule to facilitate detection. The inhibitor of the present invention may be a natural or non-natural small molecule or a genome editing device.

[0068] In the background of the present invention, the inhibitors provided herein are (i) bound to PAPD5 polypeptide and / or PAPD7 polypeptide, and / or (ii) Inhibit the expression and / or activity of PAPD5 and / or PAPD7. It is possible.

[0069] For example, the inhibitor of the present invention may bind to the PAPD5 polypeptide and inhibit the activity of the PAPD5 polypeptide. In another example, the inhibitor of the present invention may bind to the PAPD7 polypeptide and inhibit the activity of the PAPD7 polypeptide. In this specification, it is preferred that the inhibitor binds to both the PAPD5 polypeptide and the PAPD7 polypeptide and inhibits the activity of both the PAPD5 polypeptide and the PAPD7 polypeptide. The inhibitor of the present invention may inhibit the expression of PAPD5 or PAPD7, or inhibit the expression of both PAPD5 and PAPD7.

[0070] As explained above, the background to the present invention has shown that compounds that inhibit PAPD5 and / or PAPD7 have high activity in inhibiting the secretion of HBsAg and HBeAg, as well as inhibiting the production of intracellular HBV mRNA. Therefore, the inhibitors of the present invention reduce the secretion of HBsAg and HBeAg. The inhibitors of the present invention suppress the development of chronic HBV infection by reducing HBsAg secretion. In particular, the inhibitors of the present invention suppress the development of chronic HBV infection more efficiently than compounds that reduce only HBsAg secretion by inhibiting HBeAg secretion. In addition, by reducing HBeAg in women who become mothers, it is also possible to suppress the development of chronic HBV infection in the children of those women. Therefore, the inhibitors of the present invention suppress the development of chronic HBV infection (for example, the development of chronic HBV infection in children of mothers infected with HBV) by reducing HBeAg secretion, and also reduce the infectivity of HBV-infected individuals. Therefore, one aspect of the present invention relates to the inhibitors provided herein that reduce the secretion of HBsAg and HBeAg. In accordance with this, additional aspects of the present invention relate to inhibitors provided herein that suppress the development of chronic HBV infection and reduce the infectivity of HBV-infected individuals. In certain aspects of the present invention, the inhibitors provided herein suppress the development of chronic HBV infection in children of HBV-infected mothers. Preferably, the mother is HBeAg-positive.

[0071] The subject treated with the inhibitor of the present invention (or the subject receiving the inhibitor of the present invention for preventive purposes) is preferably human, more preferably HBsAg-positive and / or HBeAg-positive human patient, and even more preferably HBsAg-positive and HBeAg-positive human patient. The human patient may be a woman who is to become a mother, for example, a woman who is to become a mother who is HBeAg-positive and / or HBsAg-positive, and more preferably a woman who is to become a mother who is HBeAg-positive and HBsAg-positive.

[0072] Compound of the present invention As explained above, the inhibitor of the present invention may be a small molecule. For example, the inhibitor of the present invention is a compound of formula (I),

[0073] [Chemical formula]

[0074] In the formula, R 1 is hydrogen, halogen, C 1~6 alkyl, C 1~6 alkylamino, or C 1~6 alkoxy, R 2 is hydrogen; halogen; C 1~6 alkyl which is unsubstituted or substituted once, twice, or three times by fluoro; C 1~6 alkoxy; cyano; C 3~7 cycloalkyl; hydroxy or phenyl-C x H 2x -O-, R 3 is hydrogen; halogen; C 1~6 alkyl which is unsubstituted or substituted once, twice, or three times by fluoro; cyano; pyrrolidinyl; amino; phenyl-C x H 2x -N(C 1~6 alkyl)-; C 1~6 alkoxycarbonylpiperazinyl; or R 7 is hydrogen; C 2~6 alkyl which is unsubstituted or substituted by one to three substituents independently selected from fluoro, hydroxy, and C 1~6 alkyl; C 1~6 alkoxyC 1~6 alkyl; C 1~6 alkoxyC 1~6 alkoxyC 1~6 alkyl; aminoC 1~8 alkyl; C 1~6 alkylcarbonylaminoC1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; cyano C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; cyano C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylen C 1~6 Alkyl; pyrrolidinyl carbonyl C 1~6 Alkyl; C 2~6 Alkinyl; hydroxyC 1~6 Alkyl C 2~6 Alkinyl; AminoC 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; diC 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; CarboxyC 1~6 Alkyl; or C 1~6 Alkoxycarbonylamino C 1~8 Alkyl;heteroaryl is a monocyclic heteroaryl containing nitrogen. 1~6 Alkyl; or heterocycloalkyl C is a monocyclic heterocycloalkyl. 1~6 R is an alkyl group 7 -O-, R 4 Hydrogen, halogen, C 1~6 Alkyl, cyano, or C 1~6 It is an alkoxy, However, R 1 , R 2 , R 3 , and R 4 The condition is that it is not hydrogen at the same time, R 5 is hydrogen or C 1~6 It is alkyl, R 6is hydrogen; C which is unsubstituted or substituted one, two or three times by fluoro 1~6 alkyl; C which is unsubstituted or substituted one, two or three times by fluoro or C 1~6 alkyl 3~7 cycloalkyl; or phenyl-C x H 2x -; and the compound wherein x is from 1 to 6, or a pharmaceutically acceptable salt or enantiomer of the compound, or a diastereomer of the compound, or a compound of formula (II),

[0075]

Chemical formula

[0076] wherein R 1 is C 1~6 alkyl, C 3~7 cycloalkyl, halo C 1~6 alkyl, hydroxy C 1~6 alkyl, nitro C 1~6 alkyl, C 1~6 alkoxycarbonyl C 1~6 alkyl, carboxy C 1~6 alkyl, di(C 1~6 alkoxycarbonyl)methylenyl, cyano C 1~6 alkyl, C 3~7 cycloalkyl C 1~6 alkyl, phenyl C 1~6 alkyl, C 1~6 alkylsulfanyl C 1~6 alkyl, C 1~6 alkylsulfonyl C 1~6 alkyl, amino C 1~6 alkyl, C 1~6 alkylcarbonylamino C 1~6 alkyl, C 1~6 alkylsulfonylamino C 1~6 alkyl, C 1~6 alk]]alkoxycarbonylamino C 1~6 alkyl, aminocarbonyl C1~6 Alkyl, diC 1~6 Alkylaminocarbonyl C 1~6 Alkyl, monocyclic heterocycloalkyl C 1~6 Alkyl or imidazolyl C 1~6 It is alkyl, R 2 is an aryl or heteroaryl, and the aryl or heteroaryl is unsubstituted, or C 1~6 Alkyl, C 3~7 Cycloalkyl, halogen, halo C 1~6 Alkyl, cyano, nitro, hydroxy, halo C 1~6 Alkoxy, -OC x H 2x -R 3 ,-OC y H 2y -NHR 6 , -NR 9 R 10 , -SO2-R 11 -SO2-NR 12 R 13 carboxy, C 1~6 Alkoxycarbonyl, -C(=O)-NR 12 R 13 , substituted with one, two, three, or four substituents independently selected from aryl, heteroaryl, monocyclic heterocycloalkyl, and -O-monocyclic heterocycloalkyl, in which case the monocyclic heterocycloalkyl is unsubstituted or C 1~6 Alkyl, C 3~7 Cycloalkyl, C 1~6 Alkylcarbonyl, C 1~6 Alkyl sulfonyl, or C 1~6 It is substituted with an alkoxycarbonyl, R 3 is hydrogen; C 3~7 Cycloalkyl; Halo C 3~7 Cycloalkyl; hydroxy; hydroxy C 1~6 Alkyl C 3~7 Cycloalkyl; C 1~6 Alkoxy; monocyclic heterocycloalkyl; C 1~6 Alkyl, C 1~6 Alkylcarbonyl, C1~6 Alkyl sulfonyl, C 3~7 Cycloalkyl, or C 1~6 Monocyclic heterocycloalkyls substituted with alkoxycarbonyls; -C(=O)-R 4 ;C 1~6 Alkylsulfinyl;-SO2-R 5 ;-C(NHR 7 )-C(=O)-R 8 CarboxyC 1~6 Alkoxy or aminocarbonyl C 1~6 It is an alkoxy, R 4 is hydroxy, C 1~6 Alkoxy, amino, C 1~6 Alkylamino, diC 1~6 Alkylamino, tetrahydrofuranylamino, pyrrolidinyl, or morpholinyl, R 5 C 1~6 Alkyl, C 3~7 Cycloalkyl, hydroxy, amino, C 1~6 Alkylamino, or diC 1~6 It is an alkylamino, R 7 is hydrogen or C 1~6 It is an alkoxycarbonyl, R 8 is hydroxy or C 1~6 It is an alkoxy, R 6 is hydrogen, C 1~6 Alkylcarbonyl, Halo C 1~6 Alkylcarbonyl, C 1~6 Alkoxycarbonyl, C 1~6 Alkyl sulfonyl, C 3~7 Cycloalkylsulfonyl, or C 1~6 Alkoxy C 1~6 It is an alkylsulfonyl, R 9 and R 10 is hydrogen, C 1~6 Alkyl, C 3~7 Cycloalkyl, C 1~6 Alkylcarbonyl, C 1~6 Alkyl sulfonyl, C3~7 Cycloalkylcarbonyl, and C 3~7 Independently selected from cycloalkylsulfonyls, or R 9 and R 10 Together with the nitrogen to which they are bonded, they form a monocyclic heterocycloalkyl structure. R 11 C 1~6 Alkyl, Halo C 1~6 Alkyl, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkyl, Halo C 1~6 Alkoxy C 1~6 Alkyl, C 3~7 Cycloalkyl C 1~6 Alkyl, amino C 1~6 Alkyl, C 1~6 Alkylamino C 1~6 Alkyl, diC 1~6 Alkylamino C 1~6 Alkyl, C 1~6 Alkylcarbonylamino C 1~6 Alkyl, C 1~6 Alkylsulfonylamino C 1~6 Alkyl, C 1~6 Alkoxycarbonylamino C 1~6 Alkyl, C 1~6 Alkylsulfenyl C 1~6 Alkyl, C 1~6 Alkylsulfanyl C 1~6 Alkyl, or C 1~6 Alkylsulfonyl C 1~6 It is alkyl, R 12 and R 13 is hydrogen, C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkyl, Halo C 1~6 Alkyl, C 3~7 Cycloalkyl and halo C 3~7 Selected independently from cycloalkyl, or R 12 and R 13Together with the nitrogen to which they are bonded, they form a monocyclic heterocycloalkyl structure. x is 1, 2, 3, 4, 5, 6, 7, or 8, If y is 1, 2, 3, 4, 5, 6, 7, or 8, U, W, and Z are selected independently from CH and N. The compound wherein one of X and Y is N, and the other is CH or N, Alternatively, the compound may be a pharmaceutically acceptable salt or enantiomer, or a diastereomer.

[0077] In one aspect of the present invention, 6-methyl-2-oxo-9-pyrrolidine-1-yl-6,7-dihydrobenzo[a]quinoridine-3-carboxylic acid, 9-fluoro-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinoridine-3-carboxylic acid, and 9,10-difluoro-6-methyl-2-oxo-6,7-dihydrobenzo[a]quinoridine-3-carboxylic acid are excluded from the compounds of formula (I).

[0078] In one particular embodiment of the present invention, compounds of formula (I) and formula (II) are excluded from the inhibitors of the present invention. Therefore, one embodiment of the present invention relates to an inhibitor of the present invention that is not a compound of formula (I) or formula (II).

[0079] As explained above, the attached examples demonstrate that the anti-HBV agents DHQ (i.e., the compound of formula (III)) and THP (i.e., the compound of formula (IV)) efficiently bind to PAPD5 and PAPD7. Therefore, in the context of the present invention, it is preferable that the inhibitor of the present invention is a compound of formula (III) or formula (IV).

[0080] [ka]

[0081] [ka]

[0082] The appended examples also show that derivatives of the compounds of formula (III) and (IV), which have a linker ligand and an anchor ligand, have binding affinity to PAPD5 and PAPD7. These derivatives are shown below as formula (V) and formula (VI), respectively. Therefore, in one aspect of the present invention, the inhibitor of the present invention is a compound of formula (V) or formula (VI).

[0083] [ka]

[0084] [ka]

[0085] In the background of the present invention, the inhibitor of the present invention may be a compound of formula (I), in which case the inhibitor is one of the compounds defined in the following items (1) to (19). 1. A compound of formula (I), wherein in the formula R 1 is hydrogen, fluoro, chloro, bromo, methyl, methylamino, methoxy, or ethoxy, R 2 is hydrogen, fluoro, chloro, bromo, methyl, ethyl, trifluoromethyl, methoxy, ethoxy, propoxy, trifluoromethoxy, cyano, cyclopropyl, hydroxy, or phenylmethyl-O-, R 3Hydrogen, bromo, methyl, propyl, trifluoromethyl, cyano, phenylmethyl-N(methyl)-, tert-butoxycarbonylpiperazinyl, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, difluoromethylmethyl-O-, difluoromethylethyl-O-, trifluoromethoxy, trifluoromethylmethyl-O-, trifluoromethylethyl-O-, ethyldifluoromethyl-O-, vinyldifluoromethyl-O-, propargyl-O-, hydroxymethylpropargyl -O-, Methoxyethyl-O-, Methoxypropyl-O-, Methoxybutyl-O-, Ethoxyethyl-O-, Methoxyethyl-O-ethyl-O-, Aminoethyl-O-, Aminopentyl-O-, Aminohexyl-O-, Aminooctyl-O-, Tert-Butoxycarbonylaminopentyl-O-, Tert-Butoxycarbonylaminohexyl-O-, Tert-Butoxycarbonylaminooctyl-O-, Methylcarbonylaminoethyl-O-, Methylcarbonylaminopentyl-O-, Methylsulfonylaminoethyl-O -, methylsulfonylaminopentyl-O-, methylsulfonylethyl-O-, methylsulfonylpropyl-O-, methylsulfanylpropyl-O-, cyanopropyl-O-, cyanocyclopropylmethyl-O-, cyclopropylmethyl-O-, cyclohexylethyl-O-, hydroxyethyl-O-, hydroxypropyl-O-, hydroxy-dimethylpropyl-O-, hydroxy-difluoropropyl-O-, hydroxybutyl-O-, hydroxypentyl-O-, hydroxyhexyl-O-, aminoethyl-O-propyl These are ru-O-, ethylamino-ethyl-O-propyl-O-, imidazolylethyl-O-, pyrazolylpropyl-O-, triazolylpropyl-O-, morpholinylethyl-O-, morpholinylpropyl-O-, (2-oxo-pyrrolidinyl)ethyl-O-, (2-oxo-pyrrolidinyl)propyl-O-, phenylmethyl-O-, phenylethyl-O-, pyrrolidinylethyl-O-, pyrrolidinylpropyl-O-, pyrrolidinylcarbonylmethyl-O-, tetrahydropyranylmethyl-O-, or carboxypropyl-O-. R 4is hydrogen, fluoro, chloro, bromo, methyl, or cyano, However, R 1 , R 2 , R 3 , and R 4 The condition is that it is not hydrogen at the same time, R 5 is hydrogen or methyl, R 6 The compound wherein the compound is hydrogen, methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, trifluoromethyl, trifluoromethylmethyl, cyclopropyl, cyclobutyl, methylcyclopropyl, or phenylmethyl. Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0086] 2. A compound of formula (I), in which R 1 Hydrogen, halogen, C 1~6 Alkylamino, or C 1~6 It is an alkoxy, R 2 Hydrogen, halogen, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~7 Cycloalkyl, hydroxy, or phenyl-C x H 2x -O-, R 3 is hydrogen; halogen; C 1~6 alkyl; Cyano; Phenylen-C x H 2x -N(C 1~6 Alkyl)-; C 1~6 Alkoxycarbonylpiperazinyl; or R 7 is hydrogen; unsubstituted, or fluoro, hydroxy, and C 2~6 C is substituted with 1 to 3 substituents independently selected from the alkenyl. 1~6 Alkyl; C 1~6 Alkoxy C1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkoxy C 1~6 Alkyl; Amino C 1~8 Alkyl; C 1~6 Alkylcarbonylamino C 1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; cyano C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; cyano C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylen C 1~6 Alkyl; pyrrolidinyl carbonyl C 1~6 Alkyl; C 2~6 Alkinyl; hydroxyC 1~6 Alkyl C 2~6 Alkinyl; AminoC 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; CarboxyC 1~6 Alkyl; C 1~6 Alkoxycarbonylamino C 1~8 Alkyl;heteroaryl is a monocyclic heteroaryl containing nitrogen. 1~6 Alkyl; or heterocycloalkyl C is a monocyclic heterocycloalkyl. 1~6 R is an alkyl group 7 -O-, R 4 Hydrogen, halogen, C 1~6 Alkyl or cyano, However, R 1 , R 2 , R 3 , and R 4 The condition is that it is not hydrogen at the same time, R 5 is hydrogen or C 1~6It is alkyl, R 6 C is either hydrogen; unsubstituted or substituted once, twice, or three times by fluorocarbons. 1~6 Alkyl; C 3~7 Cycloalkyl; C 1~6 Alkyl C 3~7 Cycloalkyl; or phenyl-C x H 2x -and, The compound in which x is 1 to 6, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0087] 3. Compounds described in formula (I) or item 1 or 2, During the ceremony, R 1 is hydrogen, fluoro, chloro, bromo, methylamino, methoxy, or ethoxy, R 2 is hydrogen, fluoro, chloro, methyl, ethyl, methoxy, ethoxy, propoxy, cyclopropyl, hydroxy, or phenylmethyl-O-, R 3Hydrogen, bromo, methyl, propyl, cyano, phenylmethyl-N(methyl)-, tert-butoxycarbonylpiperazinyl, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, difluoromethylmethyl-O-, difluoromethylethyl-O-, trifluoromethylmethyl-O-, ethyldifluoromethyl-O-, vinyldifluoromethyl-O-, propargyl-O-, hydroxymethylpropargyl-O-, methoxyethyl-O-, methoxypropyl-O-, Toxybutyl-O-, Ethoxyethyl-O-, Methoxyethyl-O-ethyl-O-, Aminoethyl-O-, Aminopentyl-O-, Aminohexyl-O-, Aminooctyl-O-, Tert-Butoxycarbonylaminopentyl-O-, Tert-Butoxycarbonylaminohexyl-O-, Tert-Butoxycarbonylaminooctyl-O-, Methylcarbonylaminoethyl-O-, Methylcarbonylaminopentyl-O-, Methylsulfonylaminoethyl-O-, Methylsulfonylaminopentyl-O -, methylsulfonylethyl-O-, methylsulfonylpropyl-O-, methylsulfanylpropyl-O-, cyanopropyl-O-, cyanocyclopropylmethyl-O-, cyclopropylmethyl-O-, cyclohexylethyl-O-, hydroxyethyl-O-, hydroxypropyl-O-, hydroxy-dimethylpropyl-O-, hydroxy-difluoropropyl-O-, hydroxybutyl-O-, hydroxypentyl-O-, hydroxyhexyl-O-, aminoethyl-O-propyl-O-, ethylamine No-ethyl-O-propyl-O-, imidazolylethyl-O-, pyrazolylpropyl-O-, triazolylpropyl-O-, morpholinylethyl-O-, morpholinylpropyl-O-, (2-oxo-pyrrolidinyl)ethyl-O-, (2-oxo-pyrrolidinyl)propyl-O-, phenylmethyl-O-, phenylethyl-O-, pyrrolidinylethyl-O-, pyrrolidinylpropyl-O-, pyrrolidinylcarbonylmethyl-O-, tetrahydropyranylmethyl-O-, or carboxypropyl-O-. R 4 is hydrogen, chloro, bromo, methyl, or cyano, However, R 1 , R2 , R 3 , and R 4 The condition is that it is not hydrogen at the same time, R 5 is hydrogen or methyl, R 6 The compound wherein the compound is hydrogen, methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, trifluoromethyl, trifluoromethylmethyl, cyclopropyl, cyclobutyl, methylcyclopropyl, or phenylmethyl. Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0088] 4. A compound described in formula (I) or item 2, which is a compound of formula (IA),

[0089] [ka]

[0090] During the ceremony, R 1 is hydrogen, halogen, or C 1~6 It is an alkoxy, R 2 Hydrogen, halogen, C 1~6 Alkyl, C 1~6 Alkoxy, C 3~7 Cycloalkyl, hydroxy, or phenyl-C x H 2x -O-, R 4 is hydrogen or halogen, R 5 is hydrogen or C 1~6 It is alkyl, R 6 C is either hydrogen; unsubstituted or substituted once, twice, or three times by fluorocarbons. 1~6 Alkyl; C 3~7 Cycloalkyl; C 1~6 Alkyl C 3~7 Cycloalkyl; or phenyl-C x H 2x -and, R 7 C is either hydrogen; unsubstituted or substituted with 1 to 3 substituents independently selected from fluoro, hydroxy, and ethenyl. 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkoxy C 1~6 Alkyl; Amino C 1~8 Alkyl; C 1~6 Alkylcarbonylamino C 1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; cyano C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; cyano C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylen C 1~6 Alkyl; pyrrolidinyl carbonyl C 1~6 Alkyl; C 2~6 Alkinyl; hydroxyC 1~6 Alkyl C 2~6 Alkinyl; AminoC 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; CarboxyC 1~6 Alkyl; C 1~6 Alkoxycarbonylamino C 1~8 Alkyl;heteroaryl is a monocyclic heteroaryl containing nitrogen. 1~6 Alkyl; or heterocycloalkyl C is a monocyclic heterocycloalkyl. 1~6 It is alkyl, The compound in which x is 1 to 6, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0091] 5. Compounds listed in item 4, R 1 is hydrogen, fluoro, chloro, or methoxy, R 2 is hydrogen, fluoro, chloro, methyl, ethyl, methoxy, ethoxy, propoxy, cyclopropyl, hydroxy, or phenylmethyl-O-, R 4 is hydrogen or chloroform, R 5 is hydrogen or methyl, R 6 is hydrogen, methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, trifluoromethyl, trifluoromethylmethyl, cyclopropyl, cyclobutyl, methylcyclopropyl, or phenylmethyl. R 7Hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, difluoromethylmethyl, difluoromethylethyl, trifluoromethylmethyl, ethyldifluoromethyl, vinyldifluoromethyl, propargyl, hydroxymethylpropargyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxyethyl, methoxyethyl-O-ethyl, aminoethyl, aminopentyl, aminohexyl, aminooctyl, tert-butoxycarbonylaminopentyl, tert-butoxycarbonylaminohexyl, tert-butoxycarbonylaminooctyl, methylcarbonylaminoethyl, methylcarbonylaminopentyl, methylsulfonylaminoethyl, methylsulfonylaminopentyl, methylsulfonylethyl, methylsulfonylpropyl The compound is methylsulfanylpropyl, cyanopropyl, cyanocyclopropylmethyl, cyclopropylmethyl, cyclohexylethyl, hydroxyethyl, hydroxypropyl, hydroxy-dimethylpropyl, hydroxy-difluoropropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, aminoethyl-O-propyl, ethylaminoethyl-O-propyl, imidazolylethyl, pyrazolylpropyl, triazolylpropyl, morpholinylethyl, morpholinylpropyl, (2-oxo-pyrrolidinyl)ethyl, (2-oxo-pyrrolidinyl)propyl, phenylmethyl, phenylethyl, pyrrolidinylethyl, pyrrolidinylpropyl, pyrrolidinylcarbonylmethyl, tetrahydropyranylmethyl, or carboxypropyl. Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0092] 6. Compounds listed in item 4, R 1 is hydrogen or halogen, R 2 C 1~6 Alkyl, halogen, or C 3~7 It is a cycloalkyl, R 4 That is hydrogen, R 5 is hydrogen or C 1~6It is alkyl, R 6 C 1~6 Alkyl or C 1~6 Alkyl C 3~7 It is a cycloalkyl, R 7 C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkyl; or phenyl C 1~6 The aforementioned compound is alkyl, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0093] 7. Compounds listed in item 6, R 1 is hydrogen, fluoro, or chloro, R 2 is methyl, ethyl, fluoro, chloro, or cyclopropyl, R 4 That is hydrogen, R 5 is hydrogen or methyl, R 6 is methyl, ethyl, isopropyl, isobutyl, tert-butyl, or methylcyclopropyl, R 7 The compound wherein is methyl, ethyl, methoxyethyl, methoxypropyl, or phenylmethyl, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0094] 8. Compounds listed in item 4, R 1 That is hydrogen, R 2 C 1~6 It is an alkoxy, R 4 is hydrogen or halogen, R 5 is hydrogen or C 1~6 It is alkyl, R 6C is either hydrogen; unsubstituted or substituted once, twice, or three times by fluorocarbons. 1~6 Alkyl; C 3~7 Cycloalkyl; C 1~6 Alkyl C 3~7 Cycloalkyl; or phenyl-C x H 2x -and, R 7 is hydrogen; unsubstituted, or fluoro, hydroxy, and C 2~6 C is substituted with 1 to 3 substituents independently selected from the alkenyl. 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkoxy C 1~6 Alkyl; Amino C 1~8 Alkyl; C 1~6 Alkylcarbonylamino C 1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; cyano C 1~6 Alkyl; cyano C 3~7 Cycloalkyl C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylen C 1~6 Alkyl; pyrrolidinyl carbonyl C 1~6 Alkyl; C 2~6 Alkinyl; hydroxyC 1~6 Alkyl C 2~6 Alkinyl; AminoC 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; CarboxyC 1~6 Alkyl; C 1~6 Alkoxycarbonylamino C 1~8 Alkyl; Imidazolyl C 1~6 Alkyl;pyrazolyl C1~6 Alkyl; Triazolyl C 1~6 Alkyl;morpholinyl C 1~6 Alkyl;(2-oxo-pyrrolidinyl)C 1~6 Alkyl;pyrrolidinyl C 1~6 Alkyl; or tetrahydropyranyl C 1~6 It is alkyl, The compound in which x is 1 to 6, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0095] 9. Compounds listed in item 8, R 1 That is hydrogen, R 2 is methoxy, ethoxy, or propoxy, R 4 is hydrogen or chloroform, R 5 is hydrogen or methyl, R 6 is hydrogen, methyl, ethyl, propyl, isopropyl, isobutyl, tert-butyl, trifluoromethyl, trifluoromethylmethyl, cyclopropyl, cyclobutyl, methylcyclopropyl, or phenylmethyl. R 7Hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, difluoromethylmethyl, difluoromethylethyl, trifluoromethylmethyl, ethyldifluoromethyl, vinyldifluoromethyl, propargyl, hydroxymethylpropargyl, methoxyethyl, methoxypropyl, methoxybutyl, ethoxyethyl, methoxyethyl-O-ethyl, aminoethyl, aminopentyl, aminohexyl, aminooctyl, tert-butoxycarbonylaminopentyl, tert-butoxycarbonylaminohexyl, tert-butoxycarbonylaminooctyl, methylcarbonylaminoethyl, methylcarbonylaminopentyl, methylsulfonylaminoethyl, methylsulfonylaminopentyl, methylsulfonylethyl, methylsulfonylpropyl The compound is methylsulfanylpropyl, cyanopropyl, cyanocyclopropylmethyl, cyclopropylmethyl, cyclohexylethyl, hydroxyethyl, hydroxypropyl, hydroxy-dimethylpropyl, hydroxy-difluoropropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, aminoethyl-O-propyl, ethylaminoethyl-O-propyl, imidazolylethyl, pyrazolylpropyl, triazolylpropyl, morpholinylethyl, morpholinylpropyl, (2-oxo-pyrrolidinyl)ethyl, (2-oxo-pyrrolidinyl)propyl, phenylmethyl, phenylethyl, pyrrolidinylethyl, pyrrolidinylpropyl, pyrrolidinylcarbonylmethyl, tetrahydropyranylmethyl, or carboxypropyl. Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0096] 10. Compounds listed in item 4, R 1 is hydrogen or halogen, R 2 is halogen, C 1~6 Alkyl, C 1~6 Alkoxy, or C 3~7 It is a cycloalkyl, R 4 That is hydrogen, R5 is hydrogen or C 1~6 It is alkyl, R 6 C is either unsubstituted or substituted once, twice, or three times with fluorocarbon. 1~6 Alkyl; C 3~7 Cycloalkyl, or C 1~6 Alkyl C 3~7 It is a cycloalkyl, R 7 C is either unsubstituted or substituted with 1 to 3 substituents independently selected from fluoro and hydroxyl groups. 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkyl; Amino C 1~8 Alkyl; C 1~6 Alkylcarbonylamino C 1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylen C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkoxycarbonylamino C 1~8 Alkyl;morpholinyl C 1~6 Alkyl or tetrahydropyranyl C 1~6 The aforementioned compound is alkyl, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0097] 11. Compounds listed in item 10, R 1 is hydrogen, fluoro, or chloro, R 2 is fluoro, chloro, methyl, ethyl, methoxy, ethoxy, or cyclopropyl, R 4That is hydrogen, R 5 is hydrogen or methyl, R 6 is methyl, ethyl, isopropyl, isobutyl, tert-butyl, trifluoromethylmethyl, cyclobutyl, or methylcyclopropyl. R 7 The compound is methyl, ethyl, propyl, butyl, isobutyl, cyclopropylmethyl, difluoromethylmethyl, difluoroethylmethyl, difluoromethylethyl, trifluoromethylmethyl, ethyldifluoromethyl, methoxyethyl, methoxypropyl, ethoxyethyl, aminohexyl, aminooctyl, tert-butoxycarbonylaminopentyl, tert-butoxycarbonylaminooctyl, methylcarbonylaminopentyl, methylsulfonylaminopentyl, methylsulfonylpropyl, methylsulfanylpropyl, hydroxypropyl, hydroxydimethylpropyl, hydroxydifluoropropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, ethylamino-ethyl-O-propyl-, morpholinylethyl, morpholinylpropyl, phenylmethyl, or tetrahydropyranylmethyl. Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0098] 12.R 1 A compound described in formula (I), item 1, or item 2, wherein the compound is hydrogen, or a pharmaceutically acceptable salt or enantiomer of the said compound.

[0099] 13.R 2 is halogen or C 1~6 A compound that is an alkoxy, as described in formula (I), item 1, or item 2, or a pharmaceutically acceptable salt or enantiomer of said compound.

[0100] 14.R 2 A compound described in formula (I), item 1, or item 2, wherein the compound is chloro or methoxy, or a pharmaceutically acceptable salt or enantiomer of the said compound.

[0101] 15.R 5 A compound described in formula (I), item 1, or item 2, wherein the compound is hydrogen, or a pharmaceutically acceptable salt or enantiomer of the said compound.

[0102] 16.R 6 C 1~6 Alkyl or C 1~6 Alkyl C 3~7 A cycloalkyl compound as described in formula (I), item 1, or item 2, or a pharmaceutically acceptable salt or enantiomer of said compound.

[0103] 17.R 6 A compound described in formula (I), item 1, or item 2, wherein is ethyl, isopropyl, tert-butyl, or methylcyclopropyl, or a pharmaceutically acceptable salt or enantiomer of said compound.

[0104] 18.R 7 C 1~6 Alkoxy C 1~6 Alkyl, hydroxy C 1~6 Alkyl or amino C 1~6 A compound of formula (I), item 1, or item 2 that is alkyl, or a pharmaceutically acceptable salt or enantiomer of said compound.

[0105] 19.R 7 A compound according to formula (I), item 1, or item 2, wherein is methoxyethyl, methoxypropyl, hydroxydimethylpropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, aminobutyl, aminopentyl, or aminohexyl, or a pharmaceutically acceptable salt or enantiomer of said compound.

[0106] In the background of the present invention, the inhibitor of the present invention may be a compound of formula (II), in which case the inhibitor is one of the compounds defined in the following items (1) to (20). 1. A compound of formula (II), wherein in the formula R 1 C1~6 Alkyl, C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl, C 1~6 Alkoxycarbonyl C 1~6 Alkyl or carboxyl C 1~6 It is alkyl, R 2 C 1~6 Alkyl, C 3~7 Cycloalkyl, halogen, halo C 1~6 Alkyl, cyano, nitro, hydroxy, halo C 1~6 Alkoxy, tetrahydrofuranyloxy, -OC x H 2x -R 3 ,-OC y H 2y -NHR 6 , -SO2-R 11 、 -SO2-NR 12 R 13 carboxy, C 1~6 Alkoxycarbonyl and -C(=O)-NR 12 R 13 Phenyl;halogen, C substituted with one, two, three, or four groups independently selected from phenyl;halogen, C 1~6 Alkyl, Halo C 1~6 Alkoxy, tetrahydropyranyloxy, -OC x H 2x -R 3 , and NR 9 R 10 Pyridinyl substituted with one, two, or three groups independently selected from; or C 1~6 Alkyl and diC 1~6 It is a pyrimidinyl substituted with an alkylamino, in which case R 3 is hydrogen, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, hydroxy, hydroxyC 1~6 Alkyl C 3~7 Cycloalkyl, C 1~6Alkoxy, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, thietanyl, 1,1-dioxothietanyl, 1,1-dioxothiolanyl, morpholinyl, oxopyrrolidinyl, oxomorpholinyl, oxopiperazinyl, C 1~6 Alkoxycarbonyl oxopiperazinyl, oxoimidazolidinyl, C 1~6 Alkylpiperazinyl, C 1~6 Alkylcarbonylpiperazinyl, C 1~6 Alkylsulfonylpiperazinyl, C 1~6 Alkoxycarbonylpiperazinyl, azetidinyl, C 1~6 Alkylcarbonylazetidinyl, C 1~6 Alkylsulfonyl azetidinyl, C 1~6 Alkoxycarbonylazetidinyl, -C(=O)-R 4 , C 1~6 Alkyl sulfinyl, -SO2-R 5 -C(NHR 7 )-C(=O)-R 8 carboxy C 1~6 Alkoxy or aminocarbonyl C 1~6 It is an alkoxy, and in that case R 4 is hydroxy, C 1~6 Alkoxy, amino, C 1~6 Alkylamino, diC 1~6 Alkylamino, tetrahydrofuranylamino, pyrrolidinyl, or morpholinyl, R 5 C 1~6 Alkyl, hydroxyl, or amino, R 7 is hydrogen or C 1~6 It is an alkoxycarbonyl, R 8 is hydroxy or C 1~6 It is an alkoxy, R 6 is hydrogen, C 1~6 Alkylcarbonyl, Halo C 1~6 Alkylcarbonyl, C 1~6 Alkoxycarbonyl, C 1~6 Alkyl sulfonyl, C 3~7Cycloalkylsulfonyl, or C 1~6 Alkoxy C 1~6 It is an alkylsulfonyl, R 9 and R 10 is hydrogen, C 1~6 Alkyl and C 1~6 Independently selected from alkylsulfonyls, or R 9 and R 10 Together with the nitrogen to which they are bound, they form pyrrolidinyl, morpholinyl, piperidinyl, piperazinyl, and oxopiperazinyl. R 11 C 1~6 Alkyl or C 1~6 Alkoxy C 1~6 It is alkyl, R 12 and R 13 is hydrogen, C 1~6 Alkyl and C 1~6 Alkoxy C 1~6 Selected independently of alkyl, x is 1, 2, 3, 4, 5, 6, 7, or 8, If y is 1, 2, 3, 4, 5, 6, 7, or 8, U is CH, W is CH, Z is CH or N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0107] 2. A compound described in formula (II) or item 1, wherein in the formula R 1 C 1~6 It is alkyl, R 2 C 1~6 Alkyl, C 3~7 Cycloalkyl, halogen, halo C 1~6 Alkyl, cyano, hydroxy, halo C 1~6 Alkoxy, tetrahydrofuranyloxy, -OC x H 2x -R3 ,-OC y H 2y -NHR 6 , -SO2-R 11 -SO2-NR 12 R 13 carboxy, C 1~6 Alkoxycarbonyl and -C(=O)-NR 12 R 13 Phenyl;halogen, C substituted with one, two, three, or four groups independently selected from phenyl;halogen, C 1~6 Alkyl, Halo C 1~6 Alkoxy, tetrahydropyranyloxy, -OC x H 2x -R 3 , and NR 9 R 10 Pyridinyl substituted with one, two, or three groups independently selected from; or C 1~6 Alkyl and diC 1~6 It is a pyrimidinyl substituted with an alkylamino, in which case R 3 is hydrogen, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl C 3~7 Cycloalkyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, thietanyl, 1,1-dioxothietanyl, 1,1-dioxothiolanyl, oxopyrrolidinyl, oxomorpholinyl, oxopiperazinyl, C 1~6 Alkoxycarbonyl oxopiperazinyl, oxoimidazolidinyl, C 1~6 Alkylpiperazinyl, C 1~6 Alkylcarbonylpiperazinyl, C 1~6 Alkylsulfonylpiperazinyl, C 1~6 Alkoxycarbonylpiperazinyl, azetidinyl, C 1~6 Alkylcarbonylazetidinyl, C 1~6 Alkylsulfonyl azetidinyl, C 1~6 Alkoxycarbonylazetidinyl, -C(=O)-R 4 , C 1~6Alkyl sulfinyl, -SO2-R 5 -C(NHR 7 )-C(=O)-R 8 carboxy C 1~6 Alkoxy or aminocarbonyl C 1~6 It is an alkoxy, and in that case R 4 is hydroxy, C 1~6 Alkoxy, amino, C 1~6 Alkylamino, tetrahydrofuranylamino, or morpholinyl, R 5 C 1~6 Alkyl, hydroxyl, or amino, R 7 is hydrogen or C 1~6 It is an alkoxycarbonyl, R 8 is hydroxy or C 1~6 It is an alkoxy, R 6 is hydrogen, C 1~6 Alkylcarbonyl, Halo C 1~6 Alkylcarbonyl, C 1~6 Alkoxycarbonyl, C 3~7 Cycloalkylsulfonyl, or C 1~6 Alkoxy C 1~6 It is an alkylsulfonyl, R 9 and R 10 is hydrogen, C 1~6 Alkyl and C 1~6 Independently selected from alkylsulfonyls, or R 9 and R 10 Together with the nitrogen to which they are bound, they form pyrrolidinyl, morpholinyl, piperazinyl, and oxopiperazinyl. R 11 C 1~6 Alkoxy C 1~6 It is alkyl, R 12 and R 13 is hydrogen, C 1~6 Alkyl and C 1~6 Alkoxy C 1~6 Selected independently of alkyl, x is 1, 2, 3, 4, 5, 6, 7, or 8, If y is 1, 2, 3, 4, 5, 6, 7, or 8, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0108] 3.R 1 A compound of formula (II), item 1, or item 2, wherein the compound is methyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0109] 4. Compounds described in formula (II), item 1, or item 2, wherein in the formula R 1 C 1~6 It is alkyl, R 2 C 1~6 Alkyl, C 3~7 Cycloalkyl, halogen, halo C 1~6 Alkyl, cyano, hydroxy, halo C 1~6 Alkoxy, tetrahydrofuranyloxy, -OC x H 2x -R 3 ,-OC y H 2y -NHR 6 , -SO2-R 11 -SO2-NR 12 R 13 carboxy, C 1~6 Alkoxycarbonyl and -C(=O)-NR 12 R 13 A phenyl molecule substituted with one, two, three, or four groups independently selected from the above, R 3 is hydrogen, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl C 3~7Cycloalkyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, thietanyl, 1,1-dioxothietanyl, 1,1-dioxothiolanyl, oxopyrrolidinyl, oxomorpholinyl, oxopiperazinyl, C 1~6 Alkoxycarbonyl oxopiperazinyl, oxoimidazolidinyl, C 1~6 Alkylpiperazinyl, C 1~6 Alkylcarbonylpiperazinyl, C 1~6 Alkylsulfonylpiperazinyl, C 1~6 Alkoxycarbonylpiperazinyl, azetidinyl, C 1~6 Alkylcarbonylazetidinyl, C 1~6 Alkylsulfonyl azetidinyl, C 1~6 Alkoxycarbonylazetidinyl, -C(=O)-R 4 , C 1~6 Alkyl sulfinyl, -SO2-R 5 , or -C(NHR 7 )-C(=O)-R 8 And in that case R 4 is hydroxy, C 1~6 Alkoxy, amino, C 1~6 Alkylamino, tetrahydrofuranylamino, or morpholinyl, R 5 C 1~6 Alkyl, hydroxyl, or amino, R 7 is hydrogen or C 1~6 It is an alkoxycarbonyl, R 8 is hydroxy or C 1~6 It is an alkoxy, R 6 is hydrogen, C 1~6 Alkylcarbonyl, Halo C 1~6 Alkylcarbonyl, C 1~6 Alkoxycarbonyl, C 3~7 Cycloalkylsulfonyl, or C 1~6 Alkoxy C 1~6 It is an alkylsulfonyl, R 11 C 1~6 Alkoxy C1~6 It is alkyl, R 12 and R 13 is hydrogen, C 1~6 Alkyl and C 1~6 Alkoxy C 1~6 Selected independently of alkyl, x is 1, 2, 3, 4, 5, or 6, If y is 1, 2, 3, 4, 5, 6, 7, or 8, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0110] 5. A compound described in formula (II) or any one of items 1 to 4, wherein in the formula R 1 It is methyl, R 2Methyl, cyclopropyl, fluoro, chloro, iodine, trifluoromethyl, cyano, hydroxy, methoxy, difluoroethoxy, difluoromethoxy, trifluoroethoxy, trifluoromethoxy, cyclopropylmethoxy, difluorocyclopropylmethoxy, hydroxymethylcyclopropylmethoxy, oxetanylethoxy, oxetanylmethoxy, tetrahydrofuranylethoxy, tetrahydrofuranylmethoxy, tetrahydropyranylmethoxy, thietanylmethoxy, (1,1-dioxothietanyl)methoxy, (1,1-dioxothietanyl)methoxy Oranil) Methoxy, Oxopyrrolidinyl Propoxy, Oxomorpholinyl Propoxy, Oxopiperazinyl Propoxy, (tert-Butoxycarbonyloxopiperazinyl) Propoxy, Oxoimidazolidinyl Propoxy, Methylpiperazinyl Propoxy, Acetylpiperazinyl Propoxy, Methylsulfonylpiperazinyl Propoxy, (tert-Butoxycarbonylpiperazinyl) Propoxy, Azetidinyl Ethoxy, Acetylazetidinyl Ethoxy, Methylsulfonylazetidinyl Ethoxy, (tert-Butoxycarbonylazetidinyl) Ethoxy, (tert-butoxycarbonylazetidinyl)methoxy, carboxybutoxy, carboxyethoxy, carboxyhexyloxy, carboxymethoxy, carboxypropoxy, methoxycarbonylbutoxy, ethoxycarbonylhexyloxy, aminocarbonylbutoxy, aminocarbonylhexyloxy, aminocarbonylmethoxy, aminocarbonylpropoxy, methylaminocarbonylpropoxy, tetrahydrofuranylaminocarbonylmethoxy, morpholinylcarbonylmethoxy, methylsulfinylpropoxy, methylsulfinyl Honylpropoxy, sulfopropoxy, aminosulfonylpropoxy, amino-carboxy-propoxy, (tert-butoxycarbonylamino)-carboxy-propoxy, (tert-butoxycarbonylamino)-(methoxycarbonyl)-propoxy, aminopropoxy, aminopentoxy, aminohexyloxy, aminooctyloxy, methylcarbonylaminopropoxy, chloropropylcarbonylaminopropoxy, (tert-butoxycarbonylamino)hexyloxy, (tert-butoxycarbonylamino)octyloxy,Phenyl substituted with one, two, three, or four groups independently selected from (tert-butoxycarbonylamino)pentoxy, (tert-butoxycarbonylamino)propoxy, cyclopropylsulfonylaminopropoxy, methoxyethylsulfonylaminopropoxy, methoxypropylsulfonyl, methoxypropylaminosulfonyl, N-methoxypropyl-N-methyl-aminosulfonyl, carboxy, methoxycarbonyl, methoxypropylaminocarbonyl, N-methoxypropyl-N-methyl-aminocarbonyl, and tetrahydrofuranyloxy, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0111] 6.R 2 is halogen, C 1~6 Alkoxy, Halo C 1~6 Alkoxy, C 3~7 Cycloalkyl C 1~6 Alkoxy and Halo C 3~7 Cycloalkyl C 1~6 A compound according to formula (II) or any one of items 1 to 4, which is a phenyl substituted with one, two, or three groups independently selected from the alkoxy, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0112] 7.R 2 A compound according to formula (II) or any one of items 1 to 6, wherein phenyl is substituted with one, two, or three groups independently selected from fluoro, chloro, methoxy, difluoroethoxy, trifluoroethoxy, cyclopropylmethoxy, and difluorocyclopropylmethoxy, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of said compound.

[0113] 8. A compound described in formula (II) or any one of items 1, 2, and 4, wherein in the formula R 1 C 1~6 It is alkyl, R 2 Halogen, cyanoacrylate, halo C 1~6 Alkoxy, -OC x H 2x -R 3 , and -OC y H 2y -NHR 6 A phenyl compound that is substituted with two or three groups independently selected from the above, R 3 is hydrogen, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, C 1~6 Alkylsulfonyl azetidinyl, aminocarbonyl, or C 1~6 It is an alkylsulfonyl, R 6 is hydrogen or C 1~6 It is an alkoxycarbonyl, x is 1, 2, 3, 4, 5, or 6, If y is 1, 2, 3, 4, 5, or 6, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0114] 9. A compound described in formula (II) or any one of items 1 to 5 and 8, wherein in the formula R 1 It is methyl, R 2Phenyl is substituted with two or three groups independently selected from fluoro, chloro, cyano, methoxy, difluoroethoxy, trifluoroethoxy, cyclopropyl methoxy, difluorocyclopropyl methoxy, methylsulfonylpropoxy, aminocarbonylmethoxy, oxetanylmethoxy, oxetanylethoxy, tetrahydrofuranylmethoxy, tetrahydropyranylmethoxy, methylsulfonylazetidinylethoxy, aminohexyloxy, and (tert-butoxycarbonylamino)propoxy. U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0115] 10. Compounds described in formula (II) or item 1 or item 2, wherein in the formula R 1 C 1~6 It is alkyl, R 2 is halogen, C 1~6 Alkyl, Halo C 1~6 Alkoxy, tetrahydropyranyloxy, -OC x H 2x -R 3 , and NR 9 R 10 A pyridinyl compound that is substituted with one, two, or three groups independently selected from the above, R 3 is hydrogen, C 3~7 Cycloalkyl, thietanyl, tetrahydrofuranyl, tetrahydropyranyl, oxomorpholinyl, 1,1-dioxothietanyl, C 1~6 Alkylcarbonylazetidinyl, C 1~6 Alkylsulfonyl azetidinyl, -C(=O)-R 4 carboxy C 1~6 Alkoxy or aminocarbonyl C 1~6 It is an alkoxy, and in that case R4 is hydroxy, C 1~6 It is an alkoxy or amino, R 9 and R 10 is hydrogen, C 1~6 Alkyl and C 1~6 Independently selected from alkylsulfonyls, or R 9 and R 10 Together with the nitrogen to which they are bound, they form pyrrolidinyl, morpholinyl, piperazinyl, and oxopiperazinyl. x is 1, 2, 3, 4, 5, 6, 7, or 8, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0116] 11. A compound described in formula (II) or any one of items 1 to 3 and 10, wherein in the formula R 1 It is methyl, R 2Fluoro, chloro, iodine, methoxy, methyl, difluoroethoxy, tetrahydropyranyloxy, cyclopropyl methoxy, thietanyl methoxy, tetrahydrofuranyl methoxy, tetrahydropyranyl methoxy, oxomorpholinylpropoxy, (1,1-dioxothietanyl)methoxy, acetylazetidinyl methoxy, methylsulfonylazetidinyl methoxy, carboxybutoxy, carboxyheptyloxy, carboxyhexyloxy, carboxypentyloxy, carboxypropoxy, methoxycarbon Pyridinyl is substituted with one, two, or three groups independently selected from ruheptyloxy, aminocarbonylbutoxy, aminocarbonylheptyloxy, aminocarbonylhexyloxy, aminocarbonylmethoxy, aminocarbonylpentyloxy, aminocarbonylpropoxy, carboxymethoxypropoxy, aminocarbonylmethoxypropoxy, amino, methylamino, dimethylamino, methylsulfonylamino, pyrrolidinyl, morpholinyl, piperazinyl, and oxopiperazinyl. U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0117] 12.R 2 is halogen, C 1~6 Alkoxy, Halo C 1~6 Alkoxy, C 1~6 Alkylamino, diC 1~6 A pyridinyl compound of formula (II) or any one of items 1-3 and 10, which is substituted with one, two, or three groups independently selected from alkylamino, pyrrolidinyl, and oxopiperazinyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of said compound.

[0118] 13.R 2A compound of formula (II) or any one of items 1-3 and 10 and 11, wherein the pyridinyl is substituted with one, two, or three groups independently selected from fluoro, chloro, methoxy, difluoroethoxy, methylamino, dimethylamino, pyrrolidinyl, and oxopiperazinyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0119] 14. A compound described in formula (II) or any one of items 1, 2, and 10, wherein in the formula R 1 C 1~6 It is alkyl, R 2 Halogen, Halo C 1~6 Alkoxy, -OC x H 2x -R 3 , and NR 9 R 10 A pyridinyl compound that is substituted with two or three groups independently selected from the above, R 3 is hydrogen, tetrahydrofuranyl, tetrahydropyranyl, oxomorpholinyl, or aminocarbonyl, R 9 and R 10 is hydrogen and C 1~6 Selected independently of alkyl, or R 9 and R 10 Together with the nitrogen to which they are bound, they form pyrrolidinyl and oxopiperazinyl. x is 1, 2, 3, 4, 5, or 6, U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0120] 15. A compound described in formula (II) or item 1 or item 6, wherein in the formula R1 It is methyl, R 2 Pyridinyl is a pyridinyl compound substituted with two or three groups independently selected from fluoro, chloro, methoxy, difluoroethoxy, tetrahydrofuranylmethoxy, tetrahydropyranylmethoxy, oxomorpholinylpropoxy, aminocarbonylhexyloxy, methylamino, dimethylamino, pyrrolidinyl, and oxopiperazinyl. U is CH, W is CH, Z is N, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0121] 16. Compounds described in formula (II) or item 1, wherein in the formula R 1 C 1~6 Alkyl, C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl, C 1~6 Alkoxycarbonyl C 1~6 Alkyl or carboxyl C 1~6 It is alkyl, R 2 but halogen, nitro, C 1~6 Alkyl sulfonyl, -OC x H 2x -R 3 , and -OC y H 2y -NHR 6 Phenyl substituted with one, two, or three groups independently selected from; or halogens, halo C 1~6 Alkoxy, -OC x H 2x -R 3 , and NR 9 R 10 A pyridinyl compound that is substituted with two groups independently selected from the above, in which case R 3 is hydrogen, C 3~7 Cycloalkyl, hydroxy, C1~6 Alkoxy, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, -C(=O)-R 4 , -SO2-R 5 , or aminocarbonyl C 1~6 It is an alkoxy, and in that case R 4 is hydroxy, C 1~6 Alkoxy, amino, diC 1~6 Alkylamino or pyrrolidinyl, R 5 C 1~6 It is alkyl, R 6 is hydrogen or C 1~6 It is an alkylsulfonyl, R 9 and R 10 C 1~6 Alkyl, or R 9 and R 10 Together with the nitrogen to which they are bound, they form pyrrolidinyl, morpholinyl, piperidinyl, and oxopiperazinyl. x is 1, 2, 3, 4, 5, or 6, If y is 1, 2, 3, 4, 5, or 6, U is CH, W is CH, Z is CH, X is N, The compound in which Y is N, Or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0122] 17.R 1 C 1~6 A compound that is alkyl, of formula (II), or of item 1 or item 16, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of said compound.

[0123] 18.R 1A compound of formula (II) or any one of items 1, 16, and 17, wherein the compound is methyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of the said compound.

[0124] 19.R 2 is halogen and C 1~6 Phenyl compounds substituted with one, two, or three groups independently selected from the alkoxy; or halogens, diC 1~6 A pyridinyl compound of formula (II) or any one of items 1 and 16-18, which is substituted with two groups independently selected from alkylamino, pyrrolidinyl, and oxopiperazinyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of said compound.

[0125] 20.R 2 A compound of formula (II) or any one of items 1 and 16-19, wherein phenyl is substituted with one, two, or three groups independently selected from fluoro and methoxy; or pyridinyl is substituted with two groups independently selected from fluoro, dimethylamino, pyrrolidinyl, and oxopiperazinyl, or a pharmaceutically acceptable salt, enantiomer, or diastereomer of said compound.

[0126] As described above, the inhibitor of the present invention may be an RNAi molecule targeting PAPD5 and / or PAPD7. The RNAi molecule may be siRNA or shRNA.

[0127] For example, the inhibitor of the present invention may be an siRNA for PAPD5, in which case the siRNA is one of the following siRNAs. PAPD5 siRNA pool (L-010011-00-0010; ON-TARGETplus human PAPD5): siRNA-1-J-010011-05-Target Sequence:CAUCAAUGCUUUAUAUCGA (SEQ ID NO: 10) siRNA-2-J-010011-06 - Target sequence:GGACGACACUUCAAUUAUU (Sequence ID 11) siRNA-3-J-010011-07-Target sequence:GAUAAAGGAUGGUGGUUCA (SEQ ID NO: 12) siRNA-4-J-010011-08 - Target sequence: GAAUAGACCUGAGCCUUCA (SEQ ID NO: 13)

[0128] The inhibitor of the present invention may be an siRNA for PAPD7, in which case the siRNA is one of the following siRNAs. PAPD7 siRNA pool (L-009807-00-0005; ON-TARGETplus human PAPD7): siRNA-1-J-009807-05 - Target sequence:GGAGUGACGUUGAUUCAGA (SEQ ID NO: 14) siRNA-2-J-009807-06-Target sequence:CGGAGUUCAUCAAGAAUUA (SEQ ID NO: 15) siRNA-3-J-009807-07-Target sequence:CGGAGUUCAUCAAGAAUUA (SEQ ID NO: 16) siRNA-4-J-009807-08 - Target sequence: GCGAAUAGCCACAUGCAAU (SEQ ID NO: 17)

[0129] The appropriate siRNA target sequences are shown above. The corresponding siRNA sequences are perfectly complementary to these target sequences.

[0130] The background of this invention is the intention to mix siRNA for PAPD5 with siRNA for PAPD7 in order to inhibit the expression of both PAPD5 and PAPD7.

[0131] The attached examples surprisingly demonstrate that two structurally completely different anti-HBV agents (i.e., DHQ and THP) have a common binding site to PAPD5 and PAPD7, or at least bind in close proximity to each other. Specifically, selective interaction domains (SIDs) within PAPD5 and PAPD7 have been identified. A SID is an amino acid sequence shared by all pre-extracted fragments matching the same reference protein. Therefore, these SIDs correspond to the amino acid regions to which the anti-HBV agents DHQ and THP bind to PAPD5 and PAPD7. Thus, binding to these regions inhibits the activity of PAPD5 and PAPD7, and consequently inhibits HBV proliferation. Therefore, the inhibitor of the present invention may be an antibody that favorably binds to at least one SID of PAPD5 and / or PAPD7. Thus, the inhibitor of the present invention may be an antibody that specifically binds to any one of the amino acid extensions of SEQ ID NOs: 7-9. The inhibitor of the present invention may be an antibody that specifically binds to more than one of the amino acid extensions of SEQ ID NOs: 7-9.

[0132] Purpose In the background of this invention, it has been surprisingly shown that a synergistic effect on inhibiting HBV proliferation occurs through the simultaneous inhibition of PAPD5 and PAPD7. The attached examples show that reducing PAPD5 expression alone reduces HBsAg and HBeAg secretion by approximately 50%. Reducing PAPD7 expression alone reduces HBsAg and HBeAg secretion by less than 15%. Simultaneous knockdown of PAPD5 and PAPD7 produces a synergistic effect on reducing HBsAg and HBeAg secretion greater than the combined effect of single knockdown. While not theoretically constrained, this synergistic effect may be due to the corrective effects of both proteins, given the high sequence homology and similar enzymatic function of both PAPD5 and PAPD7 proteins.

[0133] Accordingly, one embodiment of the present invention relates to a mixed formulation comprising a PAPD5 inhibitor and a PAPD7 inhibitor for use in the treatment and / or prevention of HBV infection. Accordingly, the present invention relates to a mixed formulation comprising a PAPD5 inhibitor and a PAPD7 inhibitor for simultaneous or sequential use in the treatment and / or prevention of HBV infection. The background of the present invention is intended to use the mixed formulation to treat (e.g., improve) HBV infection. The definitions disclosed herein in relation to the inhibitors of the present invention are applied to the mixed formulation of the present invention with necessary modifications. The mixed formulation may contain a molecule that is a PAPD5 inhibitor and another molecule that is a PAPD7 inhibitor (e.g., two distinct siRNA molecules or two distinct small molecules). These two distinct inhibitors may be formulated in one unit, for example, in one pill or vial. Alternatively, these two distinct inhibitors may be formulated individually in separate units, for example, in separate pills or vials. Provided that a synergistic effect of the two inhibitors is achieved, the two distinct inhibitors may be administered together (i.e., simultaneously) or separately (i.e., sequentially). In one aspect of the present invention, the mixed formulation results in at least a 50% reduction in HBsAg and HBeAg secretion compared to a drug-free control (i.e., compared to cells or subjects that have not been administered the drug).

[0134] The present invention also relates to a pharmaceutical composition for use in the treatment and / or prevention of HBV infection, in which case the pharmaceutical composition (i) The inhibitor of the present invention or the mixed formulation of the present invention, and (ii) A pharmaceutically acceptable carrier, if desired. Includes.

[0135] Accordingly, the present invention relates to a method for treating and / or preventing HBV infection, comprising administering an effective amount of the inhibitor of the present invention, the pharmaceutical composition of the present invention, or the mixed formulation of the present invention to a subject requiring such treatment.

[0136] The inhibitors, mixed formulations, or pharmaceutical compositions of the present invention can be used in combination therapy. For example, the inhibitors, mixed formulations, or pharmaceutical compositions of the present invention can be used in combination therapy with interferon alpha-2b, interferon alpha-2a, and interferon alpha-con-1 (PEGylated and non-PEGylated), other HBV agents such as ribavirin, lamivudine (3TC), entecavir, tenofovir, terbivudine (LdT), adefovir, or HBV RNA replication inhibitors, HBsAg secretion inhibitors, HBV capsid inhibitors, antisense oligomers (e.g., as described in International Publication No. 2012 / 145697 and International Publication No. 2014 / 179629), siRNA (e.g., International Publication No. 2005 / 014806, International Publication No. 2012 / 024170, International Publication No. 2012 / 2055362, International Publication No. 2013 / 0035) It can be mixed with other emerging anti-HBV agents such as HBV therapeutic vaccines, HBV prophylactic vaccines, HBV antibody therapies (monoclonal or polyclonal), or agonists of TLR2, 3, 7, 8, or 9 for the treatment and / or prevention of HBV (as described in Pamphlet No. 20, International Publication No. 2013 / 159109, International Publication No. 2017 / 027350, and International Publication No. 2017 / 015175).

[0137] The attached examples demonstrate that downregulation of PAPD5 and / or PAPD7 is accompanied by a decrease in the production of HBsAg, HBeAg, and intracellular HBV mRNA in HBV-infected cells. These results indicate that the levels and / or activity of PAPD5 and / or PAPD7 can be used to monitor treatment outcomes during the treatment of HBV infection, for example, when treatment with PAPD5 and / or PAPD7 inhibitors is ongoing or has been performed. Therefore, the present invention is a method for monitoring treatment outcomes during the treatment of HBV infection. (a) Analyze the amount and / or activity of PAPD5 and / or PAPD7 in the sample obtained from the subject of the test, (b) Compare the amount and / or activity of PAPD5 and / or PAPD7 in at least one reference subject with reference data, and (c) Predicting treatment outcomes based on a comparison of process (b) The method includes the foregoing.

[0138] In the monitoring method of the present invention, the subjects of examination may be individuals receiving or having received drug treatment for HBV infection. The drug treatment may include the above-mentioned anti-HBV agents. The drug treatment may also include PAPD5 and / or PAPD inhibitors.

[0139] In the monitoring method of the present invention, the reference data may correspond to the amount and / or activity of PAPD5 and / or PAPD7 in a reference sample from at least one person. The sample may be blood or a liver biopsy sample.

[0140] One aspect of the present invention relates to a monitoring method of the present invention in which at least one reference subject has HBV infection but has not received drug treatment for HBV infection, and the reduced amount and / or activity of PAPD5 and / or PAPD7 in the subject being tested compared to the reference data represents the treatment outcome in the treatment of HBV infection in step (c). For example, the reduced amount and / or activity of PAPD5 and / or PAPD7 may mean that the amount and / or activity of PAPD5 and / or PAPD7 in the sample of the subject being tested is 0 to 90% of the amount and / or activity of PAPD5 and / or PAPD7 in the sample of at least one reference subject. For example, the reduced amount and / or activity of PAPD5 and / or PAPD7 may be 0 to 80%, preferably 0 to 70%, more preferably 0 to 60%, even more preferably 0 to 50%, even more preferably 0 to 40%, even more preferably 0 to 30%, even more preferably 0 to 20%, and most preferably 0 to 10% of the amount and / or activity of PAPD5 and / or PAPD7 in the sample of at least one reference subject.

[0141] Another aspect of the present invention relates to the monitoring method of the present invention in which at least one reference subject has HBV infection and has received drug treatment for HBV infection, and the amount and / or activity of PAPD5 and / or PAPD7 of the test subject, compared to the reference data, is identical or similar to the amount and / or activity of PAPD5 and / or PAPD7 of the test subject in step (c) represents the treatment outcome in the treatment of HBV infection. An additional aspect of the present invention relates to the monitoring method of the present invention in which at least one reference subject does not have HBV infection, and the amount and / or activity of PAPD5 and / or PAPD7 of the test subject, compared to the reference data, is identical or similar to the amount and / or activity of PAPD5 and / or PAPD7 of the test subject in step (c) represents the treatment outcome in the treatment of HBV infection. The amount and / or activity of PAPD5 and / or PAPD7, compared to the reference data, may mean that the amount and / or activity of PAPD5 and / or PAPD7 in the test subject sample is 90-110% of the amount and / or activity of PAPD5 and / or PAPD7 in the sample of at least one reference subject. For example, the amount and / or activity of the same or similar PAPD5 and / or PAPD7 may be 95-105% of the amount and / or activity of PAPD5 and / or PAPD7 in the reference sample of at least one person.

[0142] The present invention also includes cells or non-human animals (e.g., mice, rats, ferrets, or rabbits) with elevated, reduced, or absent PAPD5 and / or PAPD7 expression, which can be used for identifying and / or characterizing compounds that prevent and / or treat (e.g., improve) HBV infection. For example, the cells or non-human animals may include exogenous nucleotide sequences encoding PAPD5 and / or PAPD7, which are cloned, for example, in an expression vector and bound to an exogenous promoter in a functional manner. The cells or non-human animals may overexpress PAPD5 and / or PAPD7, preferably PAPD5 and PAPD7. Alternatively, the cells or non-human animals may have knockdown of PAPD5 and / or PAPD7, preferably PAPD5 and PAPD7.

[0143] Embodiments of the present invention Therefore, the present invention relates to the following items. 1. A method for identifying compounds that prevent, improve, and / or suppress hepatitis B virus (HBV) infection, (a) Contact the test compound with the following: (a1) PAP-related domain-containing protein 5 (PAPD5) and / or PAP-related domain-containing protein 7 (PAPD7), or (a2) Cells expressing PAPD5 and / or PAPD7, (b) Measuring the expression and / or activity of PAPD5 and / or PAPD7 in the presence and absence of the test compound, (c) Identify compounds that reduce the expression and / or activity of PAPD5 and / or PAPD7 as compounds that prevent, improve, and / or suppress HBV infection. The method comprising the above.

[0144] 2. A method for identifying compounds that prevent, improve, and / or suppress HBV infection, (a) Contact the test compound with the following: (a1) PAPD5 and / or PAPD7, (a2) Cells expressing PAPD5 and / or PAPD7, (b) To measure whether the test compound binds to PAPD5 and / or PAPD7, (c) To measure whether the test compound inhibits the proliferation of HBV, and (d) Identify compounds that bind to PAPD5 and / or PAPD7 and inhibit HBV replication as compounds that prevent, improve, and / or suppress HBV infection. The method comprising the above.

[0145] 3. The method according to item 1 or 2, wherein PAPD5 is a PAPD5 polypeptide or PAPD5 mRNA.

[0146] 4. The PAPD5 polypeptide (i) Amino acid sequence of Sequence ID No. 1 or 2, (ii) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (i), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function, (iii) The amino acid sequence of the enzyme activity fragment of SEQ ID NO: 1 or 2, or (iv) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (iii), wherein the polypeptide has poly(A) polymerase function. The method according to item 3, which includes or comprises a polypeptide consisting of the above amino acid sequence.

[0147] 5. The PAPD5 mRNA (i) A nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 or 2, (ii) A nucleotide sequence encoding an amino acid sequence having at least 80% identity with respect to SEQ ID NO: 1 or 2, wherein a polypeptide having poly(A) polymerase function is encoded by the nucleotide sequence, (iii) A nucleotide sequence encoding the enzyme activity fragment of SEQ ID NO: 1 or 2, or (iv) A nucleotide sequence encoding an amino acid sequence having at least 80% identity with respect to the amino acid sequence of the enzyme activity fragment of SEQ ID NO: 1 or 2, wherein a polypeptide having poly(A) polymerase function is encoded by the nucleotide sequence. The method according to item 3, wherein the polynucleotide includes or consists of the nucleotide sequence.

[0148] 6. The method according to item 1 or 2, wherein PAPD7 is PAPD7 polypeptide or PAPD7 mRNA.

[0149] 7. The PAPD7 polypeptide (i) Amino acid sequence of Sequence ID No. 3, (ii) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (i), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function, (iii) The amino acid sequence of the enzyme activity fragment of Sequence ID No. 3, or (iv) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (iii), wherein the polypeptide has poly(A) polymerase function. The method according to item 6, which includes or comprises a polypeptide consisting of the above amino acid sequence.

[0150] 8. The PAPD7 mRNA (i) The nucleotide sequence that encodes the amino acid sequence of Sequence ID No. 3, (ii) A nucleotide sequence encoding an amino acid sequence having at least 80% identity with respect to Sequence ID No. 3, wherein a polypeptide having poly(A) polymerase function is encoded by the nucleotide sequence, (iii) A nucleotide sequence encoding the enzyme activity fragment of Sequence ID No. 3, or (iv) A nucleotide sequence encoding an amino acid sequence having at least 80% identity with respect to the amino acid sequence of the enzyme activity fragment of Sequence ID No. 3, wherein a polypeptide having poly(A) polymerase function is encoded by the nucleotide. The method according to item 6, comprising or comprising the nucleotide sequence.

[0151] 9. The method according to any one of items 1 to 8, wherein the cells are eukaryotic cells.

[0152] 10. The method according to any one of items 2 to 9, wherein the compound that inhibits HBV proliferation inhibits the secretion of HBV surface antigen (HBsAg), inhibits the secretion of HBV envelope antigen (HBeAg), and / or inhibits the production of intracellular HBV mRNA or HBV DNA.

[0153] 11. The method according to any one of items 1 to 10, further comprising the step of comparing the test compound with a control.

[0154] 12. In the control, an inactive test compound is used, and the inactive test compound (i) Without reducing the expression and / or activity of PAPD5 and / or PAPD7, and / or (ii) Does not bind to PAPD5 and / or PAPD7 and does not inhibit HBV proliferation. The compound as described in item 11.

[0155] 13. The test compound is (i) Small molecules from the screening library, or (ii) peptides from a phage display library, peptides from an antibody fragment library, or peptides derived from a cDNA library The method described in any one of items 1 to 12.

[0156] 14. The method according to any one of items 1 and 3-13, wherein the activity of PAPD5 and PAPD7 is poly(A) polymerase function.

[0157] 15. PAPD5 and / or PAPD7 inhibitors for use in the treatment and / or prevention of HBV infection, (i) Small molecules that bind to PAPD5 and / or PAPD7, (ii) RNA interference (RNAi) molecules for PAPD5 and / or PAPD7, (iii) an antibody that specifically binds to PAPD5 and / or PAPD7, or (iv) Genome editing devices including the following: (a) site-specific DNA nucleases, or polynucleotides encoding site-specific DNA nucleases, and (b) Guide RNA, or polynucleotide encoding guide RNA The inhibitor being the aforementioned.

[0158] 16. (i) Binds to PAPD5 and / or PAPD7, and / or (ii) Inhibit the expression and / or activity of PAPD5 and / or PAPD7. Inhibitors for use as described in item 15.

[0159] 17. An inhibitor for use as described in item 15 or 16, wherein the inhibitor reduces the secretion of HBsAg and HBeAg.

[0160] 18. An inhibitor for use as described in any one of items 15-17, wherein the inhibitor suppresses the development of chronic HBV infection and / or reduces the infectivity of HBV-infected persons.

[0161] 19. An inhibitor for use according to any one of items 15 to 18, wherein the inhibitor is a compound of formula (III) or formula (IV).

[0162] [ka]

[0163] [ka]

[0164] 20. An inhibitor for use as described in any one of items 15-18, wherein the inhibitor is an RNAi molecule such as siRNA or shRNA.

[0165] 21. An inhibitor for use according to any one of items 15 to 18, wherein the inhibitor is an antibody that specifically binds to any one of the amino acid extensions of SEQ ID NOs. 7 to 9.

[0166] 22. A mixed formulation comprising a PAPD5 inhibitor and a PAPD7 inhibitor for simultaneous or sequential use in the treatment and / or prevention of HBV infection.

[0167] 23. A pharmaceutical composition for use in the treatment and / or prevention of HBV infection, (i) Inhibitors for use as described in any one of items 15 to 21, or mixed formulations as described in item 22, and (ii) A pharmaceutically acceptable carrier, if desired. The pharmaceutical composition comprising the above.

[0168] 24. A method for monitoring treatment outcomes during treatment of HBV infection, (a) Analyze the amount and / or activity of PAPD5 and / or PAPD7 in the sample obtained from the subject of the test, (b) Compare the amount and / or activity of PAPD5 and / or PAPD7 in at least one reference subject with reference data, and (c) Predicting treatment outcomes based on a comparison of process (b) The method comprising the above.

[0169] 25. The monitoring method described in item 24, wherein the subject of the examination is a person who is currently receiving or has previously received drug treatment for HBV infection.

[0170] 26. The monitoring method according to item 24 or 25, wherein the reference data corresponds to the amount and / or activity of PAPD5 and / or PAPD7 in at least one reference sample.

[0171] 27. A monitoring method according to any one of items 24 to 26, wherein at least one reference subject has HBV infection but has not received drug treatment for HBV infection, and the reduced amount and / or activity of PAPD5 and / or PAPD7 in the subject compared to the reference data represents the treatment outcome in the treatment of HBV infection in step (c).

[0172] 28. The monitoring method described in item 27, wherein the reduced amount and / or activity of PAPD5 and / or PAPD7 means that the amount and / or activity of PAPD5 and / or PAPD7 in the sample being tested is 0 to 90% of the amount and / or activity of PAPD5 and / or PAPD7 in the reference sample of at least one person.

[0173] 29. The monitoring method according to any one of items 24 to 26, wherein at least one reference subject has HBV infection and has received drug treatment for HBV infection, and the amount and / or activity of PAPD5 and / or PAPD7 of the same or similar subject compared to the reference data represents the treatment outcome in the treatment of HBV infection in step (c).

[0174] 30. The monitoring method according to any one of items 24 to 26, wherein the reference subject is not infected with HBV, and the amount and / or activity of PAPD5 and / or PAPD7 of the test subject, which is the same or similar as the reference data, represents the therapeutic outcome in the treatment of HBV infection in step (c).

[0175] 31. The monitoring method according to item 29 or 30, wherein the amount and / or activity of the same or similar PAPD5 and / or PAPD7 means that the amount and / or activity of PAPD5 and / or PAPD7 in the sample to be tested is 90 to 110% of the amount and / or activity of PAPD5 and / or PAPD7 in the reference sample of at least one person.

[0176] manufacturing The compound of formula (I) (i.e., the dihydroquinolidinone compound according to formula (I)) is synthetically available as described in International Publication No. 2015 / 113990(A1). Briefly, the compound of formula (I) can be prepared by a method comprising the following steps: (a) Hydrolysis of the compound of formula (A)

[0177] [ka]

[0178] [ka]

[0179] In the formula, R 1 ~R 7 and R9 Unless otherwise specified, it is defined above in relation to formula (I).

[0180] In steps (a) and (b), a base such as lithium hydroxide or sodium hydroxide can be used.

[0181] The compound of formula (II) (i.e., the tetrahydropyridopyrimidine compound according to formula (II)) can be synthesized as described in International Publication No. 2016 / 177655. Briefly, the compound of formula (II) can be prepared by a method comprising one of the following steps: (a) Bonding of compound (A) with compound (B) in the presence of a Lewis acid

[0182] [ka]

[0183] R 1 M(B), (b) Bonding of compound (C) with compound (D) in the presence of a base

[0184] [ka]

[0185] NHR 9 R 10 (D), (c) Bonding of compound of formula (E) with compound of formula (F)

[0186] [ka]

[0187] R 2 -L 2 (F), In the formula, R 1 , R 2U, W, X, Y, and Z are defined above in relation to formula (II), where M is H, Mg, Zn, or Na, and L 1 is F, Cl, or Br, and L 2 is F, Cl, or Br. In step (a), the Lewis acid may be, for example, BF3·Et2O or Sc(OTf)3. In step (b), the base may be, for example, K2CO3 or DIEA. In step (c), the reaction can be carried out in the presence of a base, which may be, for example, K2CO3 or DIEA. The reaction can also be carried out in the absence of a base.

[0188] composition As described above, the present invention relates to PAPD5 and / or PAPD7 inhibitors for use in the treatment and / or prevention of HBV infection, mixed formulations comprising a PAPD5 inhibitor and a PAPD7 inhibitor for use in the treatment and / or prevention of HBV infection, and pharmaceutical compositions comprising the inhibitors or the mixed formulations. The pharmaceutical composition (i.e., pharmaceutical) optionally comprises a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise a therapeutically acceptable diluent or excipient.

[0189] Typical pharmaceutical compositions are prepared by mixing PAPD5 inhibitors and / or PAPD7 inhibitors with carriers or excipients. Suitable carriers and excipients are well known to those skilled in the art and are described in detail, for example, Ansel, Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Remington: The Science and Practice of Pharmacy, Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Handbook of Pharmaceutical Excipients, Chicago, Pharmaceutical Press, 2005. These formulations may also contain one or more buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, lubricants, processing aids, colorants, sweeteners, fragrances, flavorings, diluents, and other known additives for improving the appearance of the drug or assisting in the manufacture of pharmaceuticals (i.e., pharmaceuticals). For example, the pharmaceutical composition of the present invention can be formulated by mixing a physiologically acceptable carrier, i.e., a carrier that is non-toxic to the recipient at the dosage and concentration used in an appropriate administration form, with a PAPD5 inhibitor and / or a PAPD7 inhibitor at ambient temperature, an appropriate pH, and a desired purity. The pharmaceutical composition of the present invention may be sterile.

[0190] The compounds according to the present invention may exist in the form of pharmaceutically acceptable salts thereof. The term "pharmaceutically acceptable salt" refers to conventional acid addition salts or base addition salts formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases, while retaining the biological efficacy and properties of the compounds of the present invention. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid, and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, and fumaric acid. Base addition salts include those derived from ammonium, potassium, sodium, and quaternary ammonium hydroxides such as tetramethylammonium hydroxide. Chemical modification of pharmaceutical compounds into salts is a well-known technique among pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, fluidity, and solubility of the compounds. The technique is described, for example, in Bastin, Organic Process Research & Development, 2000, Vol. 4, pp. 427-435, or in Ansel, Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th edition (1995), pp. 196 and 1456-1457. For example, a pharmaceutically acceptable salt of a compound provided herein may be a sodium salt.

[0191] Compounds containing one or more chiral centers may exist as racemates, diastereoisomer mixtures, or optically active monoisomers. These racemates can be separated into enantiomers according to known methods. In particular, diastereoisomer salts, which can be separated by crystallization, are formed from racemic mixtures by reaction with optically active acids such as D- or L-type tartaric acid, mandelic acid, malic acid, lactic acid, or camphor sulfonic acid.

[0192] The pharmaceutical compositions of the present invention are formulated, dispensed, and administered in accordance with the Code of Good Practice. Factors considered in this context include the specific mammalian animal being treated, the clinical condition of the individual patient, the site of drug delivery, the method of administration, the administration schedule, the patient's age and sex, and other factors known to healthcare professionals. In this specification, “effective dose” (also known as “(therapeutic) effective dose”) means the amount of compound that will induce a biological or medical response to the target as sought by a physician or other clinician. The “effective dose” of the inhibitor, mixed formulation, or pharmaceutical composition of the present invention will depend on such considerations and is the minimum amount required to inhibit HBsAg and / or HBeAg. For example, such a dose may be less than an amount that is toxic to the recipient's cells or to the entire mammalian animal.

[0193] For example, when the PAPD5 inhibitor and / or PAPD7 inhibitor is a compound of formula (I) or formula (II), the pharmaceutically effective dose administered parenterally in a single dose may be in the range of about 0.01 to 100 mg / kg relative to the patient's body weight, or in the range of about 0.01 to 100 mg / kg relative to the patient's body weight per day, and a typical initial compound range used is 0.3 to 15 mg / kg / day. In another example, when the PAPD5 inhibitor and / or PAPD7 inhibitor is a compound of formula (I) or formula (II), it is preferable that the oral unit dosage form, such as tablets and capsules, contains about 0.1 to about 1000 mg.

[0194] The inhibitors, mixed formulations, or pharmaceutical compositions of the present invention can be administered by any suitable means, including oral administration, topical administration (including buccal and sublingual administration), rectal administration, vaginal administration, transdermal administration, parenteral administration, subcutaneous administration, intraperitoneal administration, intrapulmonary administration, intradermal administration, intrathecal administration, and epidural administration, and intranasal administration, and optionally intrainjury administration for topical treatment. Parenteral administration includes intramuscular administration, intravenous administration, intra-arterial administration, intraperitoneal administration, or subcutaneous administration.

[0195] The inhibitors, mixed formulations, or pharmaceutical compositions of the present invention can be administered in any conventional dosage form, for example, as tablets, powders, capsules, solutions, dispersants, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain conventional ingredients in the pharmaceutical preparation, such as diluents, carriers, pH adjusters, sweeteners, fillers, and other active agents.

[0196] The inhibitors, mixed formulations, or pharmaceutical compositions of the present invention are useful for the prevention and / or treatment of HBV. They preferably inhibit the secretion of HBsAg and / or HBeAg, and most preferably inhibit the secretion of both HBsAg and HBeAg.

[0197] definition The terms “treatment,” “to treat,” or “to cure,” are used herein to mean in general to obtain a desired pharmacological and / or physiological effect. This effect is therapeutic in relation to the partial or complete cure of a disease and / or adverse effects caused by that disease. The term “treatment” as used herein applies to any treatment of a disease in a subject, and the term includes (a) suppressing the disease, i.e., stopping the onset of the disease, such as inhibiting the increase of HBsAg and / or HBeAg, or (b) improving (i.e. reducing) the disease, i.e., causing the regression of the disease, such as inhibiting the production of HBsAg and / or HBeAg. Accordingly, compounds that improve and / or suppress HBV infection are compounds that treat the HBV invention. The term “treatment” as used herein is preferably used in relation to medical intervention for a disorder that has already manifested, such as the treatment of an HBV infection that has already become clear and manifest. The terms “prevention,” “prevention,” or “to prevent” herein refer to preventive measures, i.e., means or methods whose purpose is to prevent a disease rather than to treat it. Prevention means obtaining the desired pharmacological and / or physiological effect that is preventive with respect to the complete or partial prevention of a disease or its symptoms. Accordingly, in this specification, “prevention of HBV infection” includes preventing the development of HBV infection and the development of symptoms of HBV infection in a subject.

[0198] For the purposes of this invention, the “subject” (or “patient”) may be a vertebrate. In the context of this invention, the term “subject” includes humans and other animals, particularly mammals, and other living organisms. Therefore, the means and methods provided herein are applicable to both human treatment and veterinary use. Accordingly, the subject herein may be an animal such as a mouse, rat, hamster, rabbit, guinea pig, ferret, cat, dog, chicken, sheep, cattle, horse, camel, or primate. The subject is preferably a mammal. The subject is more preferably a human.

[0199] The terms "hepatitis B virus infection" or "HBV infection" are commonly known in the art and refer to an infection caused by the hepatitis B virus (HBV) that affects the liver. HBV infection can be acute or chronic. Some infected individuals show no symptoms at the time of initial infection, while others suddenly develop symptoms including vomiting, yellowing of the skin, fatigue, dark urine, and abdominal pain ("Hepatitis B Fact sheet N°204," who.int., July 2014; retrieved November 4, 2014). These symptoms often last for several weeks and can be fatal. It can take 30 to 180 days for symptoms to appear. 90% of those infected around birth develop chronic hepatitis B infection, but less than 10% of those infected after the age of 5 develop the disease ("Hepatitis B FAQs for the Public - Transmission," Centers for Disease Control and Prevention (CDC), accessed November 29, 2011). The majority of chronic patients are asymptomatic, however, they eventually develop cirrhosis or liver cancer (Chang, 2007, Semin Fetal Neonatal Med, 12: 160-167). As a result of these complications, 15-25% of chronic patients die ("Hepatitis B Fact sheet N°204," who.int., July 2014; accessed November 4, 2014). In this specification, the term "HBV infection" includes both acute and chronic hepatitis B infections. The term "HBV infection" includes the asymptomatic and symptomatic phases of the initial infection, as well as the asymptomatic chronic phase of HBV infection.

[0200] In this specification, the enzyme activity fragment of SEQ ID NO: 1 or 2 (i.e., PAPD5) relates to a polypeptide comprising a sequence of amino acid residues of SEQ ID NO: 1 or 2 (i.e., PAPD5) and retaining the biological activity (i.e., functionality) of PAPD5, particularly poly(A) polymerase function. In accordance with this, the enzyme activity fragment of SEQ ID NO: 3 (i.e., PAPD7) relates to a polypeptide comprising a sequence of amino acid residues of SEQ ID NO: 3 (i.e., PAPD7) and retaining the biological activity (i.e., functionality) of PAPD7, particularly poly(A) polymerase function. Examples of the enzyme activity fragments of PAPD5 and PAPD7 are the nucleotidyltransferase domain and the Cid1 poly(A) polymerase.

[0201] In this specification, the term “polypeptide” includes all molecules comprising amino acid monomers linked by peptide (amide) bonds, or molecules consisting of such amino acid monomers. Therefore, the term “polypeptide” includes all amino acid sequences, such as peptides, oligopeptides, polypeptides, and proteins. “Polypeptides” as described herein may be natural polypeptides or non-natural polypeptides. Non-natural polypeptides may contain at least one mutation (e.g., amino acid substitution, amino acid deletion, or amino acid addition) compared to their natural counterparts. Non-natural polypeptides may be cloned into vectors and / or conjugated to a promoter that is not the natural promoter of the polypeptide, so as to be functional. The promoter may be a constitutively active promoter. As used herein, the terms “amino acid” or “residue” include L and D isomers of natural amino acids as well as other amino acids (e.g., non-natural amino acids, amino acids not encoded by nucleic acid sequences, synthetic amino acids, etc.). Examples of naturally occurring amino acids include alanine (Ala;A), arginine (Arg;R), asparagine (Asn;N), aspartic acid (Asp;D), cysteine ​​(Cys;C), glutamine (Gln;Q), glutamic acid (Glu;E), glycine (Gly;G), histidine (His;H), isoleucine (Ile;I), leucine (Leu;L), lysine (Lys;K), methionine (Met;M), phenylalanine (Phe;F), proline (Pro;P), serine (Ser;S), threonine (Thr;T), tryptophan (Trp;W), tyrosine (Tyr;Y), and valine (Val;V). Post-translational modified naturally occurring amino acids are dehydrobutyline (Dhb) and labionine (Lab). Examples of non-natural amino acids are listed above. Non-natural polypeptides may contain one or more non-amino acid substituents or heterogeneous amino acid substituents, such as reporter molecules or other ligands, that are covalently or non-covalently bonded to the amino acid sequence of a natural polypeptide, compared to the amino acid sequence of a natural polypeptide.

[0202] The terms "nucleotide sequence" or "polynucleotide" are commonly known in the art and include, or consist of, molecules containing, natural molecules such as DNA and RNA, as well as nucleic acid analogs such as oligonucleotide thiophosphates, substituted ribooligonucleotides, LNA molecules, PNA molecules, GNA (glycol nucleic acid) molecules, TNA (threose nucleic acid) molecules, morpholinopolynucleotides, or nucleic acids having a modified skeleton such as polysiloxanes and 2'-O-(2-methoxy)ethyl-phosphorothioate, or nucleic acids having substituents such as methyl nucleosides, thionucleosides, sulfate nucleosides, benzoyl nucleosides, phenyl nucleosides, amino nucleosides, propyl nucleosides, chloronucleosides, and metanocarbanucleosides, or molecules containing, or consisting of, reporter molecules for facilitating detection. Furthermore, the term “nucleotide sequence” is to be interpreted in the context of the present invention as equivalent to the term “nucleic acid molecule,” and this term can specifically refer to DNA, RNA, PNA, or LNA, or hybrids thereof, or any modifications thereof known in the art (see, for example, U.S. Patent No. 5,525,711, 4,711,955, 5,792,608, and European Patent No. 302,175 for modifications). The nucleic acid residues contained in the nucleic acid sequences described and provided herein may be either natural or artificial nucleic acid residues. Examples of nucleic acid residues include adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), xanthine (X), and hypoxanthine (HX). As those skilled in the art will understand, thymine (T) and uracil (U) may be used interchangeably depending on the polynucleotide. For example, as those skilled in the art will understand, thymine (T) as a portion of DNA corresponds to uracil (U) as a portion of the corresponding transcribed mRNA. The polynucleotides described and provided herein may be single-stranded or double-stranded, linear or circular, and natural or artificial.

[0203] The nucleotide sequences provided herein can be cloned into vectors. The term “vector” as used herein includes plasmids, cosmids, viruses, bacteriophages, and other vectors commonly used in genetic engineering. In preferred embodiments, these vectors are suitable for the transformation of cells such as mammalian or yeast cells. The vectors herein may be expression vectors. Expression vectors are generally extensively described in the literature. An expression vector may include a selection marker gene and an origin of replication, promoter, and transcription termination signal to ensure replication in the host. At least one restriction site or polylinker may be present between the promoter and the termination signal to allow insertion of the nucleic acid sequence to be expressed. Non-limiting examples of vectors capable of cloning the nucleotide sequences provided herein include adenovirus vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, HIV-based lentiviral vectors, nonviral minicircle vectors, or other vectors for bacterial and eukaryotic expression systems.

[0204] In this specification, the term “compound” means any molecule, including organic molecules such as small molecules, polynucleotides such as RNAi molecules, polypeptides such as antibodies, and inorganic compounds. The term “compound” also includes lipids, hormone analogs, polypeptide ligands, enzymes, receptors, channels, and antibody complexes. For example, in this specification, a compound may be an RNAi molecule against PAPD5 and / or PAPD7, an antibody that specifically binds to PAPD5 and / or PAPD7, or a small molecule that binds to PAPD5 and / or PAPD7.

[0205] The term "inhibitor" is known in the art and refers to a compound / substance capable of completely or partially suppressing or reducing the physiological function (i.e., activity) of a particular protein (e.g., PAPD5 and / or PAPD7). Inhibitors are also known as "antagonists." In the background of the present invention, inhibitors of PAPD5 and / or PAPD7 can, for example, cause suppression, reduction, inhibition, or inactivation of the physiological activity of PAPD5 and / or PAPD7 when the compound / substance binds to PAPD5 and / or PAPD7, respectively. Binding of an inhibitor / antagonist to PAPD5 and / or PAPD7 may reduce the enzymatic function (i.e., poly(A) polymerase function) of PAPD5 and / or PAPD7, or the activity (i.e., function) of these proteins may be inhibited by suppressing the binding of endogenous activating molecules to PAPD5 and / or PAPD7. In the context of the present invention, PAPD5 and / or PAPD7 "inhibitors" may be capable of suppressing the activity / function of PAPD5 and / or PAPD7, respectively, by repressing or reducing the expression of the PAPD5 gene and / or PAPD7 gene. Therefore, a decrease in the expression level of PAPD5 and / or PAPD7 (e.g., a decrease in the mRNA level of PAPD5 and / or PAPD7, or a decrease in the protein level of PAPD5 and / or PAPD7), which is reflected in a decrease in the functionality (i.e., activity) of PAPD5 and / or PAPD7, including poly(A) polymerase function, can be caused by PAPD5 and / or PAPD7 inhibitors. Thus, in the context of the present invention, PAPD5 and / or PAPD7 inhibitors may also include transcriptional repressors of PAPD5 and / or PAPD7 expression that are capable of reducing the levels of PAPD5 and / or PAPD7. Therefore, all means and methods that cause a decrease in the activity of PAPD5 and / or PAPD7 (which may result in relatively low expression) are used as PAPD5 and / or PAPD7 inhibitors in accordance with the present invention.

[0206] In this specification, the term “RNA interference (RNAi) molecule” refers to any molecule that inhibits RNA expression or translation. Small interfering RNAs (siRNAs) are double-stranded RNAs that bind to complementary mRNA after transcription, causing their degradation and loss of translation opportunity. Short hairpin RNAs (shRNAs) are artificial RNA molecules with a hairpin structure that, when expressed, can reduce mRNA via DICER and the RNA-induced silencing complex (RISC). RNAi molecules can be designed based on the RNA sequence of the gene of interest. The corresponding RNAi molecule can then be synthesized chemically or by in vitro transcription, or expressed from a vector or PCR product.

[0207] The term "small molecule" refers to organic compounds with a low molecular weight (less than 900 daltons). Small molecules can be useful in controlling biological processes and are generally 10 -9 It has a size of approximately m. Many drugs are small molecules.

[0208] In this specification, the term “antibody” is used in a broad sense and specifically includes complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two complete antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity (i.e., specifically bind to PAPD5 and / or PAPD7). Human antibodies, humanized antibodies, camelized antibodies, or CDR-transplanted antibodies are also included. “Antibody fragment” includes the portion of a complete antibody. The term "antibody fragment" includes, for example, (i) a monovalent fragment consisting of a VL domain, VH domain, CL domain, and CH1 domain, a Fab fragment; (ii) a bivalent fragment consisting of two Fab fragments linked by a disulfide bond in the hinge region, a F(ab')2 fragment; (iii) an Fd fragment consisting of a VH domain and a CH1 domain; (iv) an Fv fragment consisting of the VL domain and VH domain of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward; 1989; Nature 341; 544-546); and (vi) an antigen-binding portion containing or consisting of an isolated complementarity-determining region (CDR), i.e., an "antigen-binding site" (e.g., fragment, subsequence, complementarity-determining region (CDR)) that retains the ability to bind to an antigen (PAPD5 and / or PAPD7, etc.). Antibody fragments or antibody derivatives further include F(ab')2 fragments, Fv fragments, or scFv fragments, or single-chain antibodies.

[0209] When the phrases "specifically bind" or "perform specific binding" refer to a binding molecule, they refer exclusively to a binding molecule (e.g., an antibody) that has an intermediate or heterozygous binding affinity to the target molecule, preferably PAPD5 and / or PAPD7. The phrase "specifically bind" refers to a binding reaction that determines the presence of the target (preferably PAPD5 protein and / or PAPD7 protein) in the presence of a heterogeneous population consisting of proteins and other biological substances. Therefore, under the specified measurement conditions, the specified binding molecule preferentially binds to the specific target (preferably PAPD5 protein and / or PAPD7 protein) and does not bind in large quantities to other components present in the test sample. Specific binding to the target protein under such conditions may require a binding molecule selected with respect to specificity to the particular target protein. Various measurement methods are available to select a binding molecule that specifically reacts with a particular target protein. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore, and Western blotting can be used to identify a binding molecule that specifically reacts with PAPD5 protein and / or PAPD7 protein. The PAPD5 protein is most preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or 2. However, the PAPD5 protein may be a polypeptide that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 1 or 2, and is functional, with its function being poly(A) polymerase function. The PAPD7 protein is most preferably a polypeptide having the amino acid sequence shown in SEQ ID NO: 3. However, the PAPD7 protein may be a polypeptide that has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the amino acid sequence of SEQ ID NO: 3, and is functional, with its function being poly(A) polymerase function.Specific or selective reactions are typically at least twice the background signal or noise, and more typically more than 10 times the background. In other words, the phrase "specifically binds" refers to a binding reaction that determines the presence of a target protein (preferably PAPD5 and / or PAPD7) in a heterogeneous population of proteins and other biological substances. Antibodies that specifically bind to a particular target (preferably PAPD5 and / or PAPD7) are at least about 1 × 10⁻⁶. 6 M -1 or 10 7 M -1 , or preferably about 10 8 M -1 ~10 9 M -1 , or comfortable for about 10 10 M -1 ~10 11 M -1 , or a higher coupling constant (K a Typically, this involves binding to the aforementioned target. Furthermore, it is preferable that an antibody that specifically binds to a particular target (preferably PAPD5 and / or PAPD7) binds to this target with an affinity at least twice as high as the affinity for binding to non-specific targets other than the predetermined target or closely related targets (e.g., BSA, casein).

[0210] In the context of the present invention, the terms “identity” or “percent identity” mean that an amino acid sequence or nucleotide sequence has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably at least 99% identity with respect to the sequence shown herein, for example, sequence SEQ ID NOs: 1, 2, or 3, where a higher value of identity is preferable to a lower value. According to the present invention, “identity” or “percent identity” in a situation where there are two or more nucleic acid sequences or amino acid sequences means two or more sequences that are identical when compared and aligned to the greatest degree of agreement across a comparison window or designated area, as measured using sequence comparison algorithms known in the art, or by manual alignment and visual inspection, or two or more sequences that have identical amino acid residues or nucleotides with a designated percentage (for example, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% identity with respect to the amino acid sequence of SEQ ID NOs: 1, 2, or 3, or the nucleotide sequence of SEQ ID NOs: 4, 5, or 6).

[0211] The described identity preferably extends over a region of at least about 50 amino acids, preferably at least 100 amino acids, more preferably at least 400 amino acids, more preferably at least 500 amino acids, more preferably at least 600 amino acids, and most preferably over the entire length of an amino acid. In the case of a nucleotide sequence, it is most preferable that the described identity extends over a region of at least 100 nucleotides, preferably at least 1,000 nucleotides, more preferably at least 2,000 nucleotides, and most preferably over the entire length of a nucleotide.

[0212] Those skilled in the art will know how to determine the percentage identity between sequences using algorithms such as the CLUSTALW computer program (Thompson, 1994, Nucl Acids Res, 2: 4673-4680) or FASTDB (Brutlag, 1990, Comp App Biosci, 6: 237-245), as known in the art. Those skilled in the art may also use the BLAST algorithm and the BLAST2.0 algorithm (Altschul, 1997, Nucl Acids Res 25: 3389-3402, Altschul, 1993, J Mol Evol, 36: 290-300, Altschul, 1990, J Mol Biol 215: 403-410). For example, BLAST2.0 can replace the Basic Local Alignment Search Tool (BLAST) (Altschul, 1997, same as above; Altschul, 1993, same as above; Altschul, 1990, same as above) and can be used to search for local sequence alignments. As discussed above, BLAST creates alignments for both nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of these alignments, BLAST is particularly useful for determining perfect matches or identifying similar sequences. Computer methods using BLAST (Altschul, 1997, same as above; Altschul, 1993, same as above; Altschul, 1990, same as above) are used to search for identical or related molecules in nucleotide databases such as GenBank or EMBL.

[0213] In this specification, the term “measuring” also means “analyzing” or “determining” (i.e., detecting and / or quantifying). For example, the term “measuring the expression and / or activity of PAPD5 and / or PAPD7” means determining the amount of PAPD5 and / or PAPD7 expression and / or activity, for example, determining the amount of PAPD5 polypeptide and / or PAPD7 polypeptide (i.e., protein). Methods for measuring the amount and / or activity of PAPD5 protein and / or PAPD7 protein are known in the art and are described above in this specification. Similarly, the term “measuring whether the test compound binds to PAPD5 and / or PAPD7” means analyzing or determining (i.e., detecting) whether the test compound binds to PAPD5 and / or PAPD7, for example, PAPD5 polypeptide (i.e., protein) and / or PAPD7 polypeptide (i.e., protein). In accordance with this, the term “measuring whether the test compound inhibits HBV proliferation” means analyzing or determining (i.e., detecting and / or quantifying) whether the test compound inhibits HBV proliferation.

[0214] When used herein, "C 1~6 The term "alkyl" refers to saturated linear or branched alkyl groups containing 1 to 6 carbon atoms, particularly 1 to 4 carbon atoms, such as methyl, ethyl, propyl, isopropyl, 1-butyl, 2-butyl, tert-butyl, etc., either alone or in combination. 1~6 The alkyl groups are methyl, ethyl, isopropyl, and tert-butyl.

[0215] "C 3~7 The term "cycloalkyl" refers to saturated carbon rings containing 3 to 7 carbon atoms, particularly 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc., either alone or in combination. 3~7 The "cycloalkyl" group includes cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0216] "C 2~6 The term "C 2~6 alkenyl" means an unsaturated straight-chain or branched-chain alkenyl group containing 2 to 6, particularly 2 to 4 carbon atoms, such as vinyl, propenyl, allyl, butenyl, etc. Specific "C

[0217] "C 2~6 alkynyl" means an unsaturated straight-chain or branched-chain alkynyl group containing 2 to 6, particularly 2 to 4 carbon atoms, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Specific "C 2~6 alkynyl" groups are ethynyl and 1-propynyl.

[0218] "C x H 2x " represents a saturated straight-chain or branched-chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, alone or in combination. The term "C 1~6 alkoxy" represents a C 1~6 alkyl-O-group where "C 1~6 alkyl" is as defined above, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, 2-butoxy, tert-butoxy, pentyloxy, hexyloxy, etc., alone or in combination. Specific "C 1~6 alkoxy" groups are methoxy, ethoxy, and propoxy.

[0219] The term "halogen" means fluorine, chlorine, bromine, or iodine.

[0220] "halo C 1~6 alkyl" means a C 1~6 alkyl group in which at least one of the hydrogen atoms of the C 1~6 alkyl group is replaced by the same halogen atom or different halogen atoms, particularly fluorine atoms. Halo C 1~6Examples of alkyl include monofluoro-substituted, difluoro-substituted, or trifluoro-substituted methyl, ethyl, or -propyl, such as 3,3,3-trifluoropropyl, 3,3-difluoropropyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, fluoromethyl, difluoromethyl, or trifluoromethyl. Specific "halo C 1~6 alkyl" groups are difluoromethyl or trifluoromethyl.

[0221] The term "halo C 1~6 alkoxy" means a C 1~6 alkoxy group in which at least one of the hydrogen atoms of the alkoxy group is replaced by the same halogen atom or different halogen atoms, particularly fluorine atoms. 1~6 Examples of halo C 1~6 alkoxyl include monofluoro-substituted, difluoro-substituted, or trifluoro-substituted methoxy, ethoxy, or propoxy, such as fluoropropoxy, difluoropropoxy, trifluoropropoxy, fluoroethoxy, difluoroethoxy, trifluoroethoxy, fluoromethoxy, difluoromethoxy, or trifluoromethoxy. Specific "halo C 1~6 alkoxy" groups are 3-fluoropropoxy, 3,3-difluoropropoxy, 3,3,3-trifluoropropoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 2,2,2-trifluoroethoxy, fluoromethoxy, difluoromethoxy, or trifluoromethoxy.

[0222] The term "C 3~7 cycloalkyl" refers to saturated carbocyclic rings containing 3 to 7 carbon atoms, particularly 3 to 6 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, etc., either alone or in combination. Specific "C 3~7 cycloalkyl" groups are cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0223] The term "C 1~6The term "alkoxy" is "C 1~6 "Alkyl" is defined as above. 1~6 Alkyl-O- groups, such as methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, 2-butoxy, tert-butoxy, etc., are represented individually or in combination. Specific "C 1~6 The "alkoxy" group consists of methoxy and ethoxy groups, and more specifically, methoxy.

[0224] "Hello C 3~7 The term "cycloalkyl" is C 3~7 C in which at least one of the hydrogen atoms of the cycloalkyl group is replaced by the same halogen atom or a different halogen atom, in particular a fluorine atom. 3~7 This refers to a cycloalkyl group. 3~7 Examples of cycloalkyls include monofluorosubstituted or difluorosubstituted cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl, such as fluorocyclopropyl, difluorocyclopropyl, fluorocyclobutyl, difluorocyclobutyl, fluorocyclopentyl, difluorocyclopentyl, fluorocyclohexyl, or difluorocyclohexyl. Specific examples of "halo C 1~6 The alkyl group is difluorocyclopropyl.

[0225] With respect to formula (I), the term "amino" refers to primary amino(-NH2), secondary amino(NH-), or tertiary amino

[0226] [ka] It refers to a word or phrase, either alone or in combination.

[0227] With respect to formula (II), the term "amino" refers to the base of formula -NR'R'', where R' and R'' independently represent hydrogen and C. 1~6 Alkyl, C 1~6 Alkoxy, C 3~7 Cycloalkyl, heteroC 3~7The aforementioned groups are cycloalkyl, aryl, or heteroaryl. Alternatively, R' and R'' together with the nitrogen they are hetero-C 3~7 It can form cycloalkyl groups.

[0228] The term "carbonyl" refers to a -C(O)- group, either alone or in combination.

[0229] The term "cyano" refers to the -CN group, either alone or in combination.

[0230] "C 1~6 The term "alkylsulfinyl" is C 1~6 The alkyl group is defined above as -SO-C 1~6 It means alkyl group. 1~6 Examples of alkylsulfinyl include methylsulfinyl and ethylsulfinyl.

[0231] "C 1~6 The term "alkylsulfonyl" C 1~6 The alkyl group is defined above as -SO2-C 1~6 It means alkyl group. 1~6 Examples of alkylsulfonyls include methylsulfonyl and ethylsulfonyl.

[0232] The term "monocyclic heteroaryl" refers to a monovalent aromatic heterocyclic system consisting of 5 to 8 ring atoms, with one, two, three, or four heteroatoms selected from N, O, and S, and the remaining ring atoms being carbon. Examples of monocyclic heteroaryl moieties include pyrrolyl, furanyl, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridadinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, isothiazolyl, and so on.

[0233] With respect to formula (I), the term "monocyclic heterocycloalkyl" refers to a monocyclic system consisting of 3 to 7 ring atoms, either monovalently saturated or partially unsaturated, containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon. Examples of monocyclic heterocycloalkyls include azilidinyl, oxyranyl, azetidinyl, oxetanyl, pyrrolidinyl, 2-oxo-pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, azepanyl, diazepanyl, homopiperazinyl, or oxazepanyl. Specific "monocyclic heterocycloalkyl" groups are morpholinyl, 2-oxo-pyrrolidinyl, pyrrolidinyl, and tetrahydropyranyl, and more specifically, pyrrolidine-1-yl, 2-oxo-pyrrolidin-1-yl, tetrahydropyran-4-yl, and morpholin-1-yl.

[0234] With respect to formula (II), the term "monocyclic heterocycloalkyl" refers to a monocyclic system consisting of 4 to 7 ring atoms, either monovalently saturated or partially unsaturated, containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon. Examples of monocyclic heterocycloalkyls include azilidinyl, oxylanil, azetidinyl, oxetanil, pyrrolidinyl, 2-oxo-pyrrolidinyl, tetrahydrofuranil, thietanil, pyrazolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, piperidinyl, tetrahydropyranil, tetrahydrothiopyranil, piperazinyl, morpholinyl, 2-oxo-morpholinyl, 2-oxo-piperazinyl, thiomorpholinyl, 1,1-dioxo-thiomorpholin-4-yl, 1,1-dioxothiolanil, 1,1-dioxothietanyl, oxoimidazolidinyl, azepanil, diazepanil, homopiperazinyl, or oxazepanil. Specific examples of "monocyclic heterocycloalkyl" groups are azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl, tetrahydropyranyl, 1,1-dioxothietanyl, 1,1-dioxothiolanyl, morpholinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxoimidazolidinyl, 2-oxo-pyrrolidinyl, 2-oxo-morpholinyl, and 2-oxo-piperazinyl. More specifically, the "monocyclic heterocycloalkyl" groups are azetidinyl, pyrrolidinyl, morpholinyl, oxomorpholinyl, piperidinyl, piperazinyl, and oxopiperazinyl.

[0235] The term "aryl" refers to a monovalent aromatic carbocyclic or bicyclic system containing 6 to 10 carbocyclic atoms. Examples of aryl moieties include phenyl and naphthyl, with phenyl being a specific example of "aryl."

[0236] The term "heteroaryl" refers to a monovalent aromatic heterocyclic monocyclic or dicyclic system consisting of 5 to 12 ring atoms, each containing one, two, three, or four heteroatoms selected from N, O, and S, with the remaining ring atoms being carbon. Examples of heteroaryl moieties include pyrrolyl, furanil, thienyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrazolyl, pyridadinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, benzofuranil, isothiazolyl, benzothienyl, indolyl, isoindolyl, isobenzofuranil, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzoxadiazolyl, benzothiadiazolyl, benzotriazolyl, prinyl, quinolinyl, isoquinolinyl, quinazolinyl, or quinoxalinyl. Specific examples of "heteroaryl" moieties are pyridinyl and pyrimidinyl.

[0237] The term "N-containing monocyclic heteroaryl" refers to a monocyclic heteroaryl in which at least one of the heteroatoms is nitrogen (N). Examples of N-containing monocyclic heteroaryls include pyrrolyl, imidazolyl, oxazolyl, thiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridinyl, pyrazinyl, pyrizolyl, pyridadinyl, pyrimidinyl, triazinyl, azepinyl, diazepinyl, isoxazolyl, isothiazolyl, etc. Specific "N-containing monocyclic heteroaryl" groups are imidazolyl, pyrazolyl, and triazolyl, more specifically imidazole-1-yl, pyrazole-1-yl, and 1,2,4-triazole-1-yl.

[0238] The term "halogen" refers to fluorine, chlorine, bromine, or iodine. Specifically, halogens are fluorine, chlorine, or bromine.

[0239] The term "hydroxy" refers to the -OH group, either alone or in combination.

[0240] The term "2-oxo-pyrrolidinil"

[0241] [ka] It refers to a word or phrase, either alone or in combination.

[0242] The term "sulfonyl" refers to the -S(O)2- group, either alone or in combination.

[0243] "C 1~6 The term "alkylamino" refers to the fact that at least one of the hydrogen atoms in the amino group is C 1~6 This refers to an amino group defined as being replaced by an alkyl group.

[0244] "C 1~6 The term "alkylsulfonyl" is "C 1~6 "Alkyl" is defined as above. 1~6 This refers to the alkyl-S(O)2- group.

[0245] The term "aminocarbonyl" refers to the amino-C(O)- group, as defined above.

[0246] "Cyano C 3~7 The term "cycloalkyl" is C 3~7 C as defined in which at least one of the hydrogen atoms of a cycloalkyl group is replaced by a cyano group. 3~7 This refers to a cycloalkyl group.

[0247] The term "pyrrolidinyl carbonyl" refers to the pyrrolidinyl-C(O)- group.

[0248] The term "enantiomer" refers to two stereoisomers of a compound that are mirror images of each other but cannot be superimposed.

[0249] The term "diastereomer" refers to a stereoisomer having two or more chiral centers, where the molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectroscopic properties, and reactivity.

[0250] The present invention will be further described with reference to non-limiting figures and examples. [Examples]

[0251] The present invention is illustrated by the examples provided.

[0252] Materials and methods The Chemistry of Compounds Two chemical series, DHQ and THP, were synthesized, each containing one compound, to be suitable for Y3H screening conducted by Hybridenics Services (SAS). Both compounds contained a PEG5 linker and were tagged with a trimethoprim (TMP) anchor ligand (Table 1).

[0253] [Table 1]

[0254] Y3H ULTImate YChemH (trademark) screen The two compounds described above were provided to Hybrigenics Services SAS by Roche and tested for permeability and toxicity. The compounds were then screened against the Hybrigenics cDNA human placental library (PLA). These screenings were performed using different compound concentrations according to an optimized intercellular junction protocol developed for Hybrigenics ULTImate Y2H® (Table 2).

[0255] [Table 2]

[0256] Y3H ULTImate YChemH(trademark) Dependence Assay Clones obtained from the aforementioned screen were selected in a 96-well format, and clones that were positive for growth under selection conditions (HIS+) were evaluated in a spot assay-dependent assay. Only clones capable of growing on the selective medium in the presence of the tagged compound were selected, processed (cell lysis, PCR, gene sequencing), and their protein alignment was mapped using BLAST analysis.

[0257] Y3H ULTImate YChemH (trademark) 1:1 Verification Experiment - Play Snippet In this validation process, one identified prey fragment and one chemical probe (HBX129653, HBX129654) are tested in a one-to-one experiment. Plasmids of three prey strains selected from the screening library were extracted from yeast cells, amplified in E. coli, and then re-transformed into YHGX13 yeast cells. For each interaction, 1-fold, 1 / 10-fold, 1 / 100-fold, and 1 / 1000-fold dilutions of diploid yeast cultures expressing both the hook construct and prey construct were spotted on a selective medium that was free of tryptophan, leucine, and histidine, and supplemented with the chemical probe and FK506. The interactions were tested in two replication experiments. One plate was used for each compound and concentration (DMSO; HBX129653 at 5 μM, 10 μM, and 20 μM; HBX129654 at 5 μM, 10 μM, and 20 μM; HBX24786 trimethoprim (TMP) at 5 μM; and HBX129634 (TMP-PEG5-OH) at 5 μM). The plates were incubated at 30°C for 3 days.

[0258] Y3H ULTImate YChemH (trademark) 1:1 Verification Experiment - Full-Length Protein The full-length PAPD5 var1 coding sequence (NM_001040284.2) and the full-length PAPD7 varX1 coding sequence (XM_005248234.2) were reconstructed from N-terminal codon-optimized gene fragments (to remove high GC content) of their respective proteins and clones of commercially available C-terminal regions. These were then cloned into plasmid pP7 (Cellular interactions in development: A practical approach, edited by Hartley, DA, 1993, Oxford University Press, by Bartel et al. in Oxford, pp. 153-179) to be in-frame with the Gal4 activation domain (AD) (AD-play). The constructions were confirmed by sequencing the entire inserted fragment. For each play, minicrosses were performed between YHGX13 yeast cells (Y187 ade2-101::loxP-kanMX-loxP, matα) transformed with the play plasmid and YPT6AT yeast cells (mata) transformed with the DHFR (dihydrofolate reductase) hook to produce diploid yeast cultures. For each interaction, 1-fold, 1 / 10-fold, 1 / 100-fold, and 1 / 1000-fold diplos of diploid yeast cultures expressing both the hook construct and the play construct were spotted on a selective medium that was free of tryptophan, leucine, and histidine, and supplemented with the chemical probe and FK506. The interactions were examined in two replication experiments. One plate was used for each compound and concentration (DMSO; HBX129653 at 5 μM, 10 μM, and 20 μM; HBX129654 at 5 μM, 10 μM, and 20 μM; HBX24786 trimethoprim (TMP) at 5 μM; and HBX129634 (TMP-PEG5-OH) at 5 μM). The plates were incubated at 30°C for 3 days.

[0259] Y3H ULTImate YChemH (trademark) - Competition with free compounds This competitive assay is based on the one-to-one validation described above, using a fixed concentration of a chemical probe (HBX129653, HBX129654) and the parent compound of that chemical probe (MOL653, MOL654) or its inactive enantiomer (INACT653, INACT654) whose concentration increases (Table 3). The competitive assay was performed on selective medium at eight different concentrations (0 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM, and 20 μM) for the free compound, and at a fixed concentration (1 μM) for the tagged Y3H compound.

[0260] [Table 3]

[0261] HepaRG cell culture HepaRG cells (Biopredics International, Rennes, France; catalog number HPR101) were cultured for 2 weeks at 37°C in a humidified atmosphere containing 5% CO2 in complete HepaRG culture medium consisting of William's E medium (GIBCO), culture medium supplement (Biopredics, catalog number ADD710), and 1% (vol / vol) GlutaMAX-I (Gibco number 32551) and 1× penicillin / streptomycin (Gibco number 15140). To initiate differentiation, 0.9% (vol / vol) DMSO (Sigma-Aldrich, D2650) was added to the culture medium on densely packed cells. After 1 week, the medium was replaced with complete differentiation medium (HepaRG culture medium supplemented with 1.8% (vol / vol) DMSO), and the cells were maintained in this medium for approximately 4 weeks, changing the differentiation medium every 7 days. Differentiated HepaRG cells (dHepaRG) exhibited hepatocyte-like cell islands surrounded by cholangiocarcinoma-like cells. Prior to HBV infection and compound treatment, dHepaRG cells were seeded at a rate of 60,000 cells per well in 100 μL of fully differentiated medium in collagen I-coated 96-well plates (Gibco, catalog number A11428-03). The cells were allowed to regenerate their differentiation phenotype in the 96-well plates for approximately one week after seeding before HBV infection.

[0262] dHepaRG HBV infection HBV particles were used to infect dHepaRG cells at a MOI of 30. These HBV particles were produced from HBV-producing HepG2.2.15 cells (Sells et al 1987 Proc Natl Acad Sci USA 84, 1005-1009). The culture conditions, differentiation, and HBV infection of dHepaRG cells have been previously described (Hantz, 2009, J. Gen. Virol., 2009, 90: 127-135). Briefly, a complete differentiation medium (120 μL / well) containing 4% PEG-8000 and a virus stock (20-30 GE / cell) was added. One day after infection, the cells were washed three times with phosphate-buffered saline, and the medium (complete differentiation medium) was changed every two days during the experiment.

[0263] siRNA treatment of HBV-infected HepaRGs Four different pools of siRNAs (ON TARGETplus) were obtained from GE Dharmacon (Table 4).

[0264] [Table 4]

[0265] HBV cells were treated with an siRNA pool targeting either PAPD5 or PAPD7, or both, or with a non-targeting siRNA as a control, one day before infection and four days after infection. These siRNAs were transfused using DharmaFect 4 (GE Dharmacon; catalog number T-2004-01) and OPTI-MEM (Thermo Scientific; catalog number 51985034) according to the manufacturer's protocol. The cells were treated for 11 days.

[0266] Measurement of HBV antigen The supernatant was collected on day 11 to evaluate the effects on HBV antigen expression and secretion. HBsAg and HBeAg levels of HBV were measured using CLIA ELISA kits (Autobio Diagnostic; numbers CL0310-2, CL0312-2) according to the manufacturer's protocol. Briefly, 25 μL of supernatant per well was transferred to microtiter plates coated with each antibody, and 25 μL of enzyme complex reagent was added. The plates were incubated at room temperature for 60 minutes on a shaker, after which the wells were washed five times with washing buffer using an automated washer. 25 μL of substrate A and substrate B were added to each well. The plates were incubated at room temperature for 10 minutes on a shaker, after which luminescence was measured using an Envision luminescence reader (Perkin Elmer).

[0267] cell viability After removing the supernatant from HBV-infected dHepaRG cells, the cells were incubated with CellTiterGlo One solution (Promega) and their viability was measured.

[0268] Real-time PCR of intracellular mRNA To isolate intracellular mRNA, dHepaRG was washed once with PBS (Gibco) and then lysed using MagnaPure "96 Intracellular RNA Large Volume Kit" (Roche; catalog number 05467535001). The lysates are storeable at -80°C. For real-time qPCR reactions, an AB7900 HT sequence detection system (Applied Biosystems) and TaqMan® gene expression master mix (ThermoFisher Scientific) were used. For HBV mRNA detection, HBV core-specific primers (Integrated DNA Technologies) (Table 5) were used, and gene-specific TaqMan® expression assay probes (ThermoFisher Scientific; PAPD5: catalog number 4331182; PAPD7: catalog number 4331182) were used to measure the decrease in PAPD5 and PAPD7 in the presence of siRNA. Samples were normalized using a TaqMan® expression assay probe for β-actin (ThermoFisher Scientific; PAPD5: catalog number 4331182).

[0269] [Table 5]

[0270] Example 1: DHQ and THP bind to PAPD5 and PAPD7 PAPD5 / 7 was identified as a common interaction partner of DHQ and THP in Y3H ULTImate YChemH screening. In Y3H screening of both compounds (DHQ and THP) as described in the Materials and Methods section, numerous fragments identified both PAPD5 (mutant 1: NP_001035374; mutant 2: NP_001035375) and PAPD7 (XP_005248291) proteins. These identified proteins were given a confidence score of A (on a scale of A to D) by hybridization (Table 6).

[0271] [Table 6]

[0272] The interaction between PAPD5 / 7 and DHQ and THP was confirmed through Y3H ULTImate YChemH1 vs. 1 validation of identified pre-extract fragments and further validation with full-length proteins. In the first validation step, three fragments identified in the first screening were selected for a one-to-one validation assay (as described in the Materials and Methods section), and these fragments were tested at three different concentrations (5 μM, 10 μM, and 20 μM) (Table 7).

[0273] [Table 7]

[0274] All three fragments were already validated as specific conjugates of DHQ and THP at the lowest test concentrations (Figure 1).

[0275] In the second validation step, full-length PAPD5 and PAPD7 proteins were synthesized and used for one-to-one validation with DHQ and THP (as described in the Materials and Methods section) (Table 8).

[0276] [Table 8]

[0277] The interactions between these full-length proteins and DHQ and THP were confirmed at the lowest test concentrations and demonstrated specificity for their chemical probes (Figure 2).

[0278] Regarding the interaction of PAPD5 / 7 with DHQ and THP at Y3H, both free active compounds can compete, but the inactive enantiomers cannot. After verifying the binding of DHQ and THP to protein fragments and full-length PAPD5 and PAPD7, binding was confirmed in a Y3H ULTImate YChenH competition experiment (as described in the Materials and Methods section) using either the inactive or active form of the free compound (Table 9). The reduced decrease in yeast growth when the parental active compound was present, rather than when the inactive enantiomer was present, indicates that the parental compound competes with the chemical probe and interacts with the protein target.

[0279] For all test compounds, toxicity on non-selective medium at the highest concentration (20 μM) was examined using the CellTiter-Glo cell viability luminescence assay (Promega) according to the manufacturer's protocol. No toxicity was observed for any compound at this concentration, as yeast growth was unaffected (data not shown). Competition was observed for both active parent compounds (MOL653 and MOL654 for DHQ and THP, respectively), with the concentration required for competition for binding to the full-length protein being lower than the concentration required for competition for interaction with the fragment (Figures 3 and 4). The success of cross-competition suggests that DHQ and THP have a common binding surface to PAPD5 / 7, or at least bind in close proximity to each other.

[0280] [Table 9]

[0281] Example 2: Effective treatment of HBV infection by inhibiting PAPD5 and / or PAPD7 using siRNA To correlate the binding of DHQ and THP to PAPD5 / 7 and the effects of these two proteins on HBV gene expression, we reduced these proteins in naturally HBV-infected dHepaRG cells and used RNAi technology to monitor the effect of this reduction on viral parameters. For this purpose, we used an siRNA pool against PAPD5 and PAPD7 (see Table 4) in HBV-infected dHepaRG cells, as described in the Materials and Methods section.

[0282] The reduction in PAPD5 led to inhibition of viral expression, as assessed by secreted HBsAg and HBeAg (measured using CLIA ELISA and real-time PCR as described in the Materials and Methods section) and intracellular HBV mRNA. While the reduction in PAPD5 mRNA dramatically decreased HBV gene expression, the effect of PAPD7 inhibition on HBV expression was modest (Figure 7). However, a synergistic enhancement of anti-HBV activity was observed when siRNAs targeting PAPD7 and PAPD5 were mixed (Figure 7), suggesting a compensatory role for PAPD7 in the absence of PAPD5.

[0283] Example 3: HBsAg and HBeAg are efficiently reduced by DHQ and THP. The efficacy of DHQ, THP, and their variants against HBV infection was measured in HepG2.2.15 cells using HBsAg and HBeAg as read-out information.

[0284] HepG2.2.15 cells (Sells et al 1987 Proc Natl Acad Sci USA 84, 1005-1009) were cultured in 96-well plates (15,000 cells / well in 100 μL) in DMEM + GluTaMax-1 (GiBCO; catalog no. 10569), 1% penicillin / streptomycin (GiBCO; catalog no. 15140), 10% FBS (Clontech; catalog no. 631106), and 0.25 ug / ml Genethecin (Invitrogen; 10131035). The compounds were tested using nine serial dilutions in DMSO, with 100 μM being the highest concentration at 3-fold serial dilutions. Each compound was tested in four replication experiments. The cells were incubated for 3 days, the supernatant was collected, and HBsAg and HBeAg were measured as described in the Materials and Methods section. IC25 of tested compounds in reducing HBsAg and HBeAg secretion 50 The values ​​are shown below. HBX129653(DHQ-TMP): IC 50 HBsAg 1.181uM HBX129654(THP-TMP): IC 50 HBsAg 0.299uM MOL653 (DHQ-free active form):IC 50 HBsAg 0.003uM;IC 50 HBeAg 0.007uM MOL654 (THP-free active form):IC 50 HBsAg 0.003uM INACT653 (DHQ-free inactive form):IC 50 HBsAg 3.15uM INACT654 (THP-free inactive form):IC 50 HBsAg > 25uM

Claims

1. A method for identifying compounds that prevent, improve, and / or suppress hepatitis B virus (HBV) infection, (a) Contact the test compound with the following: (i) PAP-related domain-containing protein 5 (PAPD5) polypeptide and / or PAP-related domain-containing protein 7 (PAPD7) polypeptide, (ii) Cells expressing PAPD5 and / or PAPD7, (b) Measuring the expression and / or activity of PAPD5 and / or PAPD7 in the presence and absence of the test compound, (c) Identify compounds that reduce the expression and / or activity of PAPD5 and / or PAPD7 as compounds that prevent, improve, and / or suppress HBV infection. The method comprising the above.

2. A method for identifying compounds that prevent, improve, and / or suppress HBV infection, (a) Contact the test compound with the following: (i) PAPD5 polypeptide and / or PAPD7 polypeptide, (ii) Cells expressing PAPD5 and / or PAPD7, (b) To measure whether the test compound binds to the PAPD5 polypeptide and / or the PAPD7 polypeptide, (c) To measure whether the test compound inhibits the proliferation of HBV, and (d) Identify compounds that bind to PAPD5 polypeptide and / or PAPD7 polypeptide and inhibit HBV replication as compounds that prevent, improve, and / or suppress HBV infection. The method comprising the above.

3. The PAPD5 polypeptide (a) Amino acid sequence of SEQ ID NO: 1 or 2, (b) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (a), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function. (c) The amino acid sequence of the enzyme activity fragment of SEQ ID NO: 1 or 2, or (d) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (c), wherein the polypeptide has poly(A) polymerase function. The method according to claim 1 or 2, comprising or consisting of the above amino acid sequence.

4. The PAPD7 polypeptide (a) Amino acid sequence of Sequence ID No. 3, (b) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (a), wherein the polypeptide of the amino acid sequence has poly(A) polymerase function. (c) The amino acid sequence of the enzyme activity fragment of Sequence ID No. 3, or (d) an amino acid sequence having at least 80% identity with respect to the amino acid sequence of (c), wherein the polypeptide has poly(A) polymerase function. The method according to claim 1 or 2, comprising or consisting of the above amino acid sequence.

5. The method according to any one of claims 2 to 4, wherein the compound that inhibits HBV proliferation inhibits the secretion of HBV surface antigen (HBsAg) and / or inhibits the secretion of HBV envelope antigen (HBeAg) and / or inhibits the production of intracellular HBV mRNA or HBV DNA.

6. The method according to any one of claims 1 and 3 to 5, wherein the activity of PAPD5 and PAPD7 is poly(A) polymerase function.

7. PAPD5 and / or PAPD7 inhibitors for use in the treatment and / or prevention of HBV infection, (a) Small molecules that bind to PAPD5 and / or PAPD7, or (b) Antibodies that specifically bind to PAPD5 and / or PAPD7 The inhibitor being the aforementioned.

8. (a) bound to PAPD5 polypeptide and / or PAPD7 polypeptide, and / or (b) Inhibit the expression and / or activity of PAPD5 and / or PAPD7 An inhibitor for use according to claim 7.

9. The inhibitor for use according to claim 7 or 8, wherein the inhibitor reduces the secretion of HBsAg and HBeAg.

10. The inhibitor for use according to any one of claims 7 to 9, wherein the inhibitor suppresses the development of chronic HBV infection and / or reduces the infectivity of HBV-infected persons.

11. The inhibitor is a compound of formula (I). 【Chemistry 1】 And in the formula R 1 Hydrogen, halogen, C 1~6 Alkyl, C 1~6 Alkylamino, or C 1~6 It is an alkoxy, R 2 is hydrogen; halogen; C which is unsubstituted or substituted once, twice or three times by fluoro 1~6 alkyl; C which is unsubstituted or substituted once, twice or three times by fluoro 1~6 alkoxy; cyano; C 3~7 cycloalkyl; hydroxy or phenyl-C x H 2x -O- and R 3 C is either hydrogen; halogen; unsubstituted, or substituted once, twice, or three times with fluorocarbon. 1~6 Alkyl;cyano;pyrrolidinyl;amino;phenyl-C x H 2x -N(C) 1~6 Alkyl) -; C 1~6 Alkoxycarbonyl-piperazinyl; or R 7 is hydrogen; unsubstituted, or fluoro, hydroxy, and C 2~6 C is substituted with 1 to 3 substituents independently selected from the alkenyl. 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkoxy C 1~6 Alkoxy C 1~6 Alkyl; Amino C 1~8 Alkyl; C 1~6 Alkylcarbonylamino C 1~8 Alkyl; C 1~6 Alkylsulfonylamino C 1~8 Alkyl; C 1~6 Alkylsulfanyl C 1~6 Alkyl; C 1~6 Alkylsulfonyl C 1~6 Alkyl; cyano C 1~6 Alkyl; C 3~7 Cycloalkyl C 1~6 Alkyl; cyano C 3~7 Cycloalkyl C 1~6 Alkyl; Phenylenide C 1~6 Alkyl; pyrrolidinyl carbonyl C 1~6 Alkyl; C 2~6 Alkinyl; Hydroxy C 1~6 Alkyl C 2~6 Alkinyl; Amino C 1~6 Alkoxy C 1~6 Alkyl; C 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; diC 1~6 Alkylamino C 1~6 Alkoxy C 1~6 Alkyl; CarboxyC 1~6 Alkyl; or C 1~6 Alkoxycarbonylamino C 1~8 Alkyl;heteroaryl is a monocyclic heteroaryl containing nitrogen (N) - Heteroaryl C 1~6 Alkyl; or heterocycloalkyl C is a monocyclic heterocycloalkyl. 1~6 R is an alkyl group 7 -O-, R 4 Hydrogen, halogen, C 1~6 Alkyl, cyano, or C 1~6 It is an alkoxy, However, R 1 , R 2 , R 3 , and R 4 The condition is that it is not hydrogen at the same time, R 5 is hydrogen or C 1~6 It is alkyl, R 6 C is either hydrogen; unsubstituted or substituted once, twice, or three times with fluorocarbons. 1~6 Alkyl; unsubstituted, or fluoro or C 1~6 C is substituted once, twice, or three times with alkyl. 3~7 Cycloalkyl; or phenyl-C x H 2x - and x is between 1 and 6. An inhibitor for use according to any one of claims 7 to 10.

12. The inhibitor is a compound of formula (II). 【Chemistry 2】 And in the formula R 1 is C 1~6 alkyl, C 3~7 cycloalkyl, halo C 1~6 alkyl, hydroxy C 1~6 alkyl, nitro C 1~6 alkyl, C 1~6 alkoxycarbonyl C 1~6 alkyl, carboxy C 1~6 alkyl, di(C 1~6 alkoxycarbonyl)methylenyl, cyano C 1~6 alkyl, C 3~7 cycloalkyl C 1~6 alkyl, phenyl C 1~6 alkyl, C 1~6 alkylsulfanyl C 1~6 alkyl, C 1~6 alkylsulfonyl C 1~6 alkyl, amino C 1~6 alkyl, C 1~6 alkylcarbonylamino C 1~6 alkyl, C 1~6 alkylsulfonylamino C 1~6 alkyl, C 1~6 alkoxycarbonylamino C 1~6 alkyl, aminocarbonyl C 1~6 alkyl, diC 1~6 alkylaminocarbonyl C 1~6 alkyl, monocyclic heterocycloalkyl C 1~6 alkyl, or imidazolyl C 1~6 is alkyl, R 2 is an aryl or heteroaryl, and the aryl or heteroaryl is of the unsubstituted type, or C 1~6 Alkyl, C 3~7 Cycloalkyl, halogen, halo C 1~6 Alkyl, cyano, nitro, hydroxy, halo C 1~6 Alkoxy, -O-C x H 2x -R 3 , -O-C y H 2y - NHR 6 , -NR 9 R 10 , -SO 2 -R 11 , -SO 2 -NR 12 R 13 carboxy, C 1~6 Alkoxycarbonyl, -C(=O)-NR 12 R 13 , substituted with one, two, three, or four substituents independently selected from aryl, heteroaryl, monocyclic heterocycloalkyl, and -O-monocyclic heterocycloalkyl, in which case the monocyclic heterocycloalkyl is unsubstituted or C 1~6 Alkyl, C 3~7 Cycloalkyl, C 1~6 Alkylcarbonyl, C 1~6 Alkyl sulfonyl, or C 1~6 It is substituted with an alkoxycarbonyl, R 3 is hydrogen; C 3~7 Cycloalkyl; Halo C 3~7 Cycloalkyl; hydroxy; hydroxy C 1~6 Alkyl C 3~7 Cycloalkyl; C 1~6 Alkoxy; Monocyclic heterocycloalkyl; C 1~6 Alkyl, C 1~6 Alkylcarbonyl, C 1~6 Alkyl sulfonyl, C 3~7 Cycloalkyl, or C 1~6 Monocyclic heterocycloalkyls substituted with alkoxycarbonyl groups; -C(=O)-R 4 ; C 1~6 Alkyl sulfinyl; -SO 2 -R 5 ;-C(NHR 7 )-C(=O)-R 8 Carboxy C 1~6 Alkoxy or aminocarbonyl C 1~6 It is an alkoxy, R 4 is hydroxy, C 1~6 Alkoxy, amino, C 1~6 Alkylamino, diC 1~6 Alkylamino, tetrahydrofuranylamino, pyrrolidinyl, or morpholinyl, R 5 C 1~6 Alkyl, C 3~7 Cycloalkyl, hydroxy, amino, C 1~6 Alkylamino, or diC 1~6 It is an alkylamino, R 7 is hydrogen or C 1~6 It is an alkoxycarbonyl, R 8 is hydroxy or C 1~6 It is an alkoxy, R 6 is hydrogen, C 1~6 Alkylcarbonyl, Halo C 1~6 Alkylcarbonyl, C 1~6 Alkoxycarbonyl, C 1~6 Alkyl sulfonyl, C 3~7 Cycloalkylsulfonyl, or C 1~6 Alkoxy C 1~6 It is an alkylsulfonyl, R 9 and R 10 is hydrogen, C 1~6 Alkyl, C 3~7 Cycloalkyl, C 1~6 Alkylcarbonyl, C 1~6 Alkyl sulfonyl, C 3~7 Cycloalkylcarbonyl, and C 3~7 Independently selected from cycloalkylsulfonyls, or R 9 and R 10 Together with the nitrogen to which they are bonded, they form a monocyclic heterocycloalkyl structure. R 11 C 1~6 Alkyl, Halo C 1~6 Alkyl, C 3~7 Cycloalkyl, Halo C 3~7 Cycloalkyl, hydroxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkyl, Halo C 1~6 Alkoxy C 1~6 Alkyl, C 3~7 Cycloalkyl C 1~6 Alkyl, amino C 1~6 Alkyl, C 1~6 Alkylamino C 1~6 Alkyl, diC 1~6 Alkylamino C 1~6 Alkyl, C 1~6 Alkylcarbonylamino C 1~6 Alkyl, C 1~6 Alkylsulfonylamino C 1~6 Alkyl, C 1~6 Alkoxycarbonylamino C 1~6 Alkyl, C 1~6 Alkylsulfenyl C 1~6 Alkyl, C 1~6 Alkylsulfanyl C 1~6 Alkyl, or C 1~6 Alkylsulfonyl C 1~6 It is alkyl, R 12 and R 13 is hydrogen, C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkyl, Halo C 1~6 Alkyl, C 3~7 Cycloalkyl and halo C 3~7 Selected independently from cycloalkyl, or R 12 and R 13 Together with the nitrogen to which they are bonded, they form a monocyclic heterocycloalkyl structure. x is 1, 2, 3, 4, 5, 6, 7, or 8, If y is 1, 2, 3, 4, 5, 6, 7, or 8, U, W, and Z are selected independently from CH and N. One of X and Y is N, and the other is CH or N. An inhibitor for use according to any one of claims 7 to 10.

13. The inhibitor for use according to any one of claims 7 to 10, wherein the inhibitor is an antibody that specifically binds to the amino acid elongation of any one of SEQ ID NOs: 7, 8, or 9.

14. A mixed formulation containing a PAPD5 inhibitor and a PAPD7 inhibitor for simultaneous or sequential use in the treatment and / or prevention of HBV infection.

15. A pharmaceutical composition for use in the treatment and / or prevention of HBV infection, (a) an inhibitor for use according to any one of claims 7 to 13, or a mixed formulation according to claim 14, and (b) A pharmaceutically acceptable carrier The pharmaceutical composition comprising the above.

16. A method for monitoring treatment outcomes during treatment of HBV infection, (a) Analyze the amount and / or activity of PAPD5 and / or PAPD7 in the sample obtained from the subject of the test. (b) comparing the amount and / or activity of PAPD5 and / or PAPD7 in at least one reference subject with reference data, and (c) Predicting treatment outcomes based on a comparison of process (b) The method comprising the above.

Images