The compound and its use in the manufacture of therapeutic drugs for enterovirus-related diseases.

Abstract

The present invention discloses a compound and its use in the manufacture of a drug for treating diseases associated with enteroviruses. The compound has the general formula of Formula I below, and can significantly inhibit the activity of 3C protease of various enteroviruses. At present, there is no specific drug approved for commercial sale against human enteroviruses, so the technical solution of the present invention can make up for the shortcomings of the prior art. [Formula 1] TIFF2025512010000014.tif67170

Description

Detailed description of the invention

[0001] [Technical field] This invention belongs to the field of viral therapy and relates to compounds and their use in the manufacture of therapeutic agents for diseases related to enteroviruses. [Background technology]

[0002] Enteroviruses are ubiquitous viruses with a very large number of species and a wide variety of infectious symptoms. The genus Enterovirus belongs to the family Picornaviridae, which includes poliovirus, coxsackievirus, echovirus, enterovirus, and rhinovirus. 1 Enteroviruses are primarily transmitted through fecal-oral transmission and droplet transmission. While the common symptoms of enterovirus infection are mild respiratory illnesses (like the common cold), they are the most common infectious disease in humans and can cause serious illnesses such as polio (acute poliomyelitis), aseptic meningitis, conjunctivitis, pericarditis, hand-foot-and-mouth disease, and paralysis. 2,3 Aside from the poliovirus vaccine, there are currently no commercially available vaccines or treatments for these common viruses.

[0003] The genome of enteroviruses is a single-stranded positive-chain RNA approximately 7.5 kb long, primarily encoding structural proteins necessary for viral packaging and non-structural proteins involved in replication and transcription. Enteroviruses have a very high mutation rate due to their low replication fidelity and frequent recombination. 4The polyprotein encoded by the open reading frame of enterovirus is divided into three regions, P1 to P3. Among them, the P1 region contains four structural proteins (VP1 to VP4), and the P2 and P3 regions contain seven non-structural proteins (2A to 2C and 3A to 3D). These functional subunits can be cleaved by the protease encoded by the virus into independent protein units before the virus can perform normal transcription and replication. When the host cell is infected with the virus, the first region to be translated is the P1 region encoding the capsid structural protein. Next, during the translation of the P2 and P3 regions, 2A and 3C are released from the polyprotein and function to cleave the viral polyprotein. 2A protease has two cleavage sites, and 3C protease has eight cleavage sites. 5 From this, it can be seen that 3C protease plays an important regulatory role in the transcription and replication of the virus, so it has become a research hotspot. 6 The 3C protease of enterovirus is a multifunctional cysteine protease and belongs to the endopeptidase family protease related to chymotrypsin. 7 Among different types of enteroviruses, 3C protease shares about 50 - 75% sequence similarity. 6 The amino acid residues of the catalytic triad are completely conserved throughout the enterovirus genus. Also, compared with other parts of the protein, the amino acid residues around the catalytic residues are more conserved, suggesting their involvement in the sequence specificity and cleavage mechanism similarity among enteroproteases.

[0004] Rhinovirus (HRV) has become the most well-known virus among enteroviruses due to its seasonality and detection rate, and it always causes various complications in children, the elderly, and people with low immunity. 8 When infected with HRV, the incidence of acute exacerbation of viral asthma reaches 80%. In Australia, 16 cases die per week, and in the United States, 25 cases die per week. 9While poliovirus has already been eradicated through vaccination, developing an HRV vaccine is impossible due to the extremely large number of HRV serotypes (over 100). AG7088 is a 3C protease inhibitor that exhibits significant antiviral activity against multiple serotypes of human rhinovirus, while its inhibitory activity against mammalian cysteine ​​and serine proteases (including cathepsin B, elastase, chymotrypsin, trypsin, thrombin, and calpain) is negligible. 10 However, due to the low bioavailability of AG7088, researchers' long-standing efforts remain at the theoretical stage, and so far, no effective treatment has been developed. 11 . [Problems the invention aims to solve]

[0005] This invention addresses the problem of the prior art that there is no specific drug for diseases caused by enteroviruses by providing compounds and their use in the manufacture of therapeutic agents for enterovirus-related diseases. The object of this invention is to provide a method for treating diseases caused by infection with enteroviruses. ST-0101 and ST-0102 of this invention can be used to treat diseases caused by rhinoviruses and severe infections caused by other enteroviruses such as poliovirus, coxsackievirus, echovirus, and enterovirus. ST-0101 and ST-0102 (see Figure 1) can reversibly bind to the cysteine ​​residue (C147) of HRV 3C protease. Through in vitro enzyme activity experiments and crystal structure analysis of the protein complex, this invention has shown that ST-0101 and ST-0102 can be used to treat enterovirus-related diseases. [Means for solving the problem]

[0006] To solve the above technical problems, the present invention provides, in a first aspect, a compound represented by the general formula of the following formula I.

[0007] [ka] In the formula, R1 is

[0008] [ka] or

[0009] [ka] R2, R3, and R4 are selected from H and halogen.

[0010] In some preferred embodiments, R1 is

[0011] [ka] In this case, Ar is 3,4-(OCH2)C6H3, 4-Me2NC6H4, C6H5, 4-MeC6H4, 4-Cl,2-FC6H3, or 5-methyl-3-isoxazole, and R2, R3, and R4 are H, F, or CF3.

[0012] In some more preferred embodiments, the compound is a compound represented by formula II or formula III.

[0013] [ka]

[0014] [ka]

[0015] To solve the above technical problems, the present invention provides, in a second aspect, a pharmaceutical composition comprising the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof, and preferably further comprising a pharmaceutically acceptable diluent or carrier.

[0016] In order to solve the above technical problems, as a third aspect, the present invention provides the use of the compound according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention in the production of an inhibitor of enterovirus protease.

[0017] In some preferred embodiments, the enterovirus protease is selected from 2A protease, 2B protease, 2C protease, 3A protease, 3B protease, 3C protease, 3D protease, and / or the enterovirus protease is a protease of Rhinovirus, Poliovirus, Coxsackievirus, Echovirus or Enterovirus.

[0018] In some more preferred embodiments, the enterovirus protease is the 3C protease of Rhinovirus, Poliovirus, Coxsackievirus, Echovirus or Enterovirus.

[0019] Preferably, the 3C protease is the 3C protease of Rhinovirus HRV-A2 or HRV-B14, Poliovirus type 1, Enterovirus EV71, Echovirus 1 or Coxsackievirus CVB3.

[0020] In order to solve the above technical problems, as a fourth aspect, the present invention provides the use of the compound according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention in the production of a therapeutic agent for intestinal-related diseases.

[0021] In some preferred embodiments, the intestinal-related disease is a disease caused by the Enterovirus genus.

[0022] Preferably, the Enterovirus genus includes rhinovirus, poliovirus, coxsackievirus, echovirus, and enterovirus.

[0023] In some more preferred embodiments, the rhinovirus comprises HRV-A2 and HRV-B14, the coxsackievirus comprises CVB3, the echovirus comprises Echovirus 1, the enterovirus comprises EV71, and the poliovirus comprises Poliovirus type 1.

[0024] To solve the above technical problems, the present invention provides, as a fifth aspect, a method for treating or preventing intestinal diseases by administering an effective amount of the compound described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention to a subject who requires it.

[0025] In some preferred embodiments, the intestinal disease is a disease caused by an enterovirus.

[0026] Preferably, the Enterovirus genus includes rhinovirus, poliovirus, coxsackievirus, echovirus, and enterovirus.

[0027] More preferably, the rhinovirus includes HRV-A2 and HRV-B14, the coxsackievirus includes CVB3, the echovirus includes Echovirus type 1, the enterovirus includes EV71, and the poliovirus includes Poliovirus type 1.

[0028] In some preferred embodiments, the method further includes administering other drugs that treat or prevent intestinal diseases.

[0029] Preferably, the other drug is an inhibitor that inhibits 3C protease.

[0030] More preferably, the inhibitor that inhibits the 3C protease includes AG7088 or other AG7088 derivatives other than the compound described in the first aspect of the present invention.

[0031] To solve the above technical problems, the present invention provides, in a sixth aspect, a method for inhibiting or detecting 3C protease in vitro or in vivo for non-diagnostic purposes, comprising contacting the 3C protease with an effective amount of the compound described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention. An application of the method for inhibiting or detecting 3C protease in vitro for non-diagnostic purposes is, for example, in scientific research conducted in a laboratory, to determine whether a sample contains the 3C protease by contacting the sample with the compound described in the present invention.

[0032] In some preferred embodiments, the 3C protease is a rhinovirus, poliovirus, coxsackievirus, echovirus, or enterovirus 3C protease. [Effects of the invention]

[0033] The beneficial effects of this invention are as follows: The compounds disclosed in this invention, for example, those represented by formula II or formula III, can significantly inhibit the activity of 3C proteases of various enteroviruses. Currently, there are no commercially approved drugs for treating human enteroviruses, so using compounds represented by formula II or formula III to treat intestinal diseases can overcome the shortcomings of the prior art. [Brief explanation of the drawing]

[0034] [Figure 1] Figure 1 shows the molecular formulas of ST-0101 and ST-0102. [Figure 2] Figure 2 shows that both ST-0101 and ST-0102 have significant inhibitory activity against rhinovirus HRV-A2 3C protease. [Figure 3] Figure 3 shows that ST-0101 has significant inhibitory activity against rhinovirus HRV-B14 3C protease. [Figure 4] Figure 4 shows that ST-0101 and ST-0102 have significant inhibitory activity against poliovirus type 1 3C protease. [Figure 5] Figure 5 shows that ST-0101 and ST-0102 have significant inhibitory activity against enterovirus EV71 3C protease. [Figure 6] Figure 6 shows that ST-0101 and ST-0102 have significant inhibitory activity against Echovirus 1 3C protease. [Figure 7] Figure 7 shows that ST-0101 and ST-0102 have significant inhibitory activity against coxsackievirus CVB3 3C protease. [Figure 8] Figure 8 shows the reversible covalent binding between HRV 3C protease and the inhibitor ST0102. [Modes for carrying out the invention]

[0035] The present invention will be further described below with reference to examples, however, the scope of the present invention is not limited thereto to these examples. Experimental methods in the following examples where specific conditions are not specified should be carried out according to usual methods and conditions, or selected according to the product's instructions.

[0036] (Example 1) In in vitro enzyme activity experiments, ST-0101 and ST-0102 were found to significantly inhibit the activity of 3C proteases in rhinovirus (HRV-A2, HRV-B14), poliovirus type 1, enterovirus (EV71), echovirus type 1, and coxsackievirus (CVB3) (Figures 2-7).

[0037] The fluorescent substrate used to measure the inhibitory effect of the above viruses ST-0101 and ST-0102 on 3C protease is Dabcyl-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Lys(5-FAM)-NH2 (SEQ ID NO: 3). Here, the amino acid sequence of the substrate was selected mainly based on the classical recognition sequence of 3C protease, with the fluorescent group 5-FAM and the quenching group dabcyl attached to both ends of the short-chain peptide. When 3C protease cleaves the short-chain peptide, the quenching group and the fluorescent group separate, and a fluorescent signal from the fluorescent group is detected. Therefore, when 3C protease is reacting with the substrate, the rate of increase in fluorescence intensity may be used to represent the rate of the enzymatic reaction by 3C protease. When different concentrations of the inhibitory molecule are added, the inhibitory effect on enzyme activity differs, and the rate of increase in fluorescence intensity is also suppressed. The rate of increase in fluorescence intensity without the inhibitor molecule is defined as 100% enzyme activity, and the rate of increase in fluorescence intensity without the enzyme is defined as 0% enzyme activity (substrate molecules may self-decompose at a very slow rate). By measuring the corresponding enzyme activity when different concentrations of the inhibitor are added, the IC of the inhibitor can be determined. 50 A curve can be obtained. The buffer solution used in this experiment was 20 mM Tris (pH 8.0), 150 mM NaCl, 0.1 mg / mL BSA (bovine serum albumin), and 0.01% (v / v) TritonX-100. The substrate concentration used was 20 μM, and the protein concentration used was 0.5 μM.

[0038] The amino acid sequence of HRV-A2 3C (Sequence ID 1) is as follows: GPEEEFGMSLIKHNSCVITTENGKFTGLGVYDRFVVVPTHADPGKEIQVDGITTKVIDSYDLYNKNGIKLEITVLKLDRNEKFRDIRRYIPNNEDDYPNCNLALLANQPEPTIINVGDVVSYGNILLSGNQTAMLKYSYPTKSGYCGGVLYKIGQVLGIHVGGNGRDGFSAMLLRSYFTDVQ

[0039] The amino acid sequence of HRV-B14 3C (SEQ ID NO: 2) is as follows: GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ

[0040] The amino acid sequence (SEQ ID NO: 4) of poliovirus type 1 3C is as follows: GPGFDYAVAMAKRNIVTATTSKGEFTMLGVHDNVAILPTHASPGESIVIDGKEVEILDAKALEDQAGTNLEITIITLKRNEKFRDIRPHIPTQITETNDGVLIVNTSKYPNMYVPVGAVTEQGYLNLGGRQTARTLMYNFPTRAGQCGGVITCTGKVIGMHVGGNGSHGFAAALKRSYFTQSQ

[0041] The amino acid sequence of EV71 3C (SEQ ID NO: 5) is as follows: GPSLDFALSLLRRNIRQVQTDQGHFTMLGVRDRLAVLPRSQPGKTIWIEHKLVNVLDAVELVDEQGVNLELTLITLDTNEKFRDITKFIPENISTASDATLVINTEHMPSMFVPVGDVVQYGFLNLSGKPTHRTMMYNFPTKAGQCGGVVTSVGKVIGIHIGGNGRQGFCAGLKRSYFASEQ

[0042] The amino acid sequence (SEQ ID NO: 6) of Echovirus 13C is as follows: GPAFEFAVAMMKRNASTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVGVLDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLAREEAEVNEAVLAINTSKFPNMYIPVGQVTDYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLRHYFNEEQ

[0043] The amino acid sequence of CVB3 3C (SEQ ID NO: 7) is as follows: GPAFEFAVAMMKRNSSTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVGVLDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLAKEEVEVNEAVLAINTSKFPNMYIPVGQVTEYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLKHYFNDEQ

[0044] (Example 2) In this invention, the 1.54 angstrom high-resolution structures of HRV 3C protease and the inhibitor ST-0102 were analyzed. As shown in Figure 8, a reversible covalent bond was formed between the protease's active site C147 and the inhibitor. Crystals of the HRV 3C protease-inhibitor ST-0102 complex were obtained by screening using the droplet method with the Index kit from Hampton Research. The X-ray diffraction data was acquired at the Shanghai Synchrotron Radiation Facility. The crystal diffraction data was processed using the XDS program package, with 5FX6 from the RCSB database as the corresponding initial model. Molecular substitution was performed using the Phenix.phaser program, and cross-validation was performed using the Phenix.refine and Coot programs to finally obtain the high-resolution structure of the HRV 3C protease-inhibitor ST-0102 complex.

[0045] Because the substrate binding pocket of enterovirus 3C protease is highly conserved, inhibitors targeting this binding pocket (ST-0101 and ST-0102) have broad anti-enteroviral effects. ST-0101 and ST-0102 of the present invention can be used to treat severe infections caused by rhinovirus, poliovirus, coxsackievirus, echovirus, enterovirus, and others.

[0046] Although specific embodiments of the present invention are described above, this is merely an illustrative explanation, and it will be understood by those skilled in the art that various modifications or amendments may be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention is limited by the claims. [Prior art document] [Non-patent literature]

[0047] [Unauthorized document 1] OS Nikonov, ES Chernykh, MB Garber, EY Nikonova, Enteroviruses: Classification, Diseases They Cause, and Approaches to Development of Antiviral Drugs. Biochemistry(Mosc) 82, 1615-1631(2017). [Non-licensed literature 2] L. Royston, C. Tapparel, Rhinoviruses and Respiratory Enteroviruses: Not as Simple as ABC. Viruses 8, (2016). [Non-licensed Reference 3] B. Jubelt, HL Lipton, Enterovirus / picornavirus infections. Handb Clin Neurol 123, 379-416 (2014). [Unauthorized Document 4] M. Nikolaidis et al., Large-scale genomic analysis reveals recurrent patterns of intertypic recombination in human enteroviruses. Virology 526, 72-80(2019). [Non-licensed Reference 5] OH Laitinen et al., Enteroviral proteases: structure, host interactions and pathogenicity. Rev Med Virol 26, 251-267 (2016). [Non-licensed Reference 6] M. Laajala, D. Reshamwala, V. Marjomaki, Therapeutic targets for enterovirus infections. Expert Opinion Ther Tar 24, 745-757 (2020). [Non-patent document 7] J. Seipelt et al., The structures of picornaviral proteinases. Virus Res 62, 159-168 (1999). [Non-Patent Literature 8] C. Esneau, AC Duff, NW Bartlett, Understanding Rhinovirus Circulation and Impact on Illness. Viruses 14, (2022). [Non-Patent Literature 9] LM Jensen, EJ Walker, DA Jans, R. Ghildyal, Proteases of human rhinovirus: role in infection. Methods Mol Biol 1221, 129-141 (2015). [Non-Patent Document 10] DA Matthews et al., Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes. Proc Natl Acad Sci USA 96, 11000-11007 (1999). [Non-Patent Literature 11] K. Namoto et al., Structure-based design and synthesis of macrocyclic human rhinovirus 3C protease inhibitors. Bioorg Med Chem Lett 28, 906-909 (2018).

Claims

1. A compound characterized by being a compound represented by the following formula II or formula III. 【Chemistry 1】 【Chemistry 2】

2. A pharmaceutical composition characterized by comprising the compound described in claim 1 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 2, further comprising a pharmaceutically acceptable diluent or carrier.

4. An enterovirus protease inhibitor comprising the compound described in Claim 1, The inhibitor is characterized in that the enterovirus protease is a 3C protease of rhinovirus, poliovirus, coxsackievirus, echovirus, or enterovirus.

5. The inhibitor according to claim 4, characterized in that the 3C protease is the 3C protease of rhinovirus HRV-A2 or HRV-B14, poliovirus type 1, enterovirus EV71, echovirus type 1, or coxsackievirus CVB3.

6. A compound according to claim 1 or a pharmaceutical composition according to claim 2 or 3 for use in the treatment of enterovirus-related diseases, A compound or pharmaceutical composition characterized in that the enterovirus-related disease is caused by the 3C protease of an enterovirus, and the enterovirus includes rhinovirus, poliovirus, coxsackievirus, echovirus, or enterovirus.

7. The compound or pharmaceutical composition for use according to claim 6, characterized in that the rhinovirus comprises HRV-A2 and HRV-B14, the coxsackievirus comprises CVB3, the echovirus comprises echovirus type 1 (Echovirus 1), the enterovirus comprises EV71, and the poliovirus comprises poliovirus type 1 (Poliovirus 1).

8. A method for in vitro inhibition or detection of 3C protease for non-diagnostic purposes, comprising contacting 3C protease in vitro with an effective amount of the compound described in Claim 1 or the pharmaceutical composition described in Claim 2 or 3.

9. The method according to claim 8, wherein the 3C protease is a 3C protease of rhinovirus, poliovirus, coxsackievirus, echovirus, or enterovirus.

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