Compounds and their use in the manufacture of a medicament for treating enterovirus-associated diseases - Patents.com

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] The present invention is in the field of viral therapy and relates to compounds and their use in the manufacture of medicaments for the treatment of diseases associated with enteroviruses. [Background technology]

[0002] Enteroviruses are a ubiquitous group of viruses with a wide variety of infectious symptoms. The genus Enterovirus belongs to the family Picornaviridae, which also includes Poliovirus, Coxsackievirus, Echovirus, Enterovirus, and Rhinovirus. 1 Enteroviruses are primarily transmitted via fecal-oral and airborne droplets. Because the general symptom of enterovirus infection is a mild respiratory illness (common cold), it is often overlooked, but it is the most common infectious disease in humans and causes severe illnesses such as polio (acute poliomyelitis), aseptic meningitis, conjunctivitis, pericarditis, hand, foot and mouth disease, and paralysis. 2,3 With the exception of the poliovirus vaccine, there are currently no commercially available vaccines or specific drugs against these common viruses.

[0003] The genome of enteroviruses is a single-stranded positive-stranded RNA approximately 7.5 kb in length that encodes mainly structural proteins required for viral packaging and nonstructural proteins involved in replication and transcription. Enteroviruses have a very high mutation rate due to low fidelity replication and frequent recombination. 4The polyprotein encoded by the open reading frame of enterovirus is divided into three regions, P1 to P3, of which the P1 region contains four structural proteins (VP1 to VP4), and the P2 and P3 regions contain seven nonstructural proteins (2A to 2C and 3A to 3D). Only when these functional subunits are cleaved by the protease encoded by the virus to become independent protein units can the virus perform normal transcription and replication. When a host cell is infected with a virus, the first region to be translated is the P1 region, which encodes the capsid structural protein, and then in the process of translating the P2 and P3 regions, 2A and 3C are released from the polyprotein and function to cleave the viral polyprotein. The 2A protease has two cleavage sites, and the 3C protease has eight cleavage sites. 5 This indicates that 3C protease plays an important regulatory role in viral transcription and replication, and has therefore become a hotspot of research. 6 The enterovirus 3C protease is a multifunctional cysteine ​​protease that belongs to the chymotrypsin-related endopeptidase family of proteases. 7 Among different types of enteroviruses, the 3C proteases share approximately 50-75% sequence similarity. 6 The amino acid residues of the catalytic triad are completely conserved across the Enterovirus genus, and the amino acid residues surrounding the catalytic triad are more conserved than the rest of the protein, suggesting that they may be responsible for the similarity in sequence specificity and cleavage mechanism among enteric proteases.

[0004] Rhinoviruses (HRVs) are the most well-known enteroviruses due to their seasonality and detection rate, and they frequently cause various complications in children, the elderly, and people with weakened immune systems. 8 HRV infection is associated with an 80% incidence of viral asthma exacerbations, with 16 deaths per week in Australia and 25 deaths per week in the United States. 9Although poliovirus has already been eradicated by vaccination, developing an HRV vaccine is impractical due to the large number of HRV serotypes (more than 100). AG7088 is a 3C protease inhibitor that has significant antiviral activity against multiple human rhinovirus serotypes while exhibiting negligible inhibitory activity against mammalian cysteine ​​and serine proteases, including cathepsin B, elastase, chymotrypsin, trypsin, thrombin, and calpain. 10 However, due to the low bioavailability of AG7088, researchers' long-standing efforts have remained at the theoretical stage, and no effective treatment has been developed to date. 11 . [Problem to be solved by the invention]

[0005] The present invention provides compounds and their use in the manufacture of therapeutic agents for Enterovirus-associated diseases to solve the problem of the prior art that there is no specific medicine for Enterovirus-associated diseases. The object of the present invention is to provide a method for treating diseases caused by infection with Enterovirus. ST-0101 and ST-0102 of the present invention can be used to treat diseases caused by Rhinovirus and severe infections caused by other Enterovirus-associated viruses such as Poliovirus, Coxsackievirus, Echovirus, and Enterovirus. ST-0101 and ST-0102 (see FIG. 1) can reversibly bind to the cysteine ​​residue (C147) of HRV 3C protease. The present invention has found that ST-0101 and ST-0102 can be used to treat Enterovirus-associated diseases through in vitro enzyme activity experiments and crystal structure analysis of protein complexes. [Means for solving the problem]

[0006] In order to solve the above technical problems, the present invention provides, as a first aspect, a compound represented by the following general formula I:

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

[0008] [ka] or

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

[0010] In some preferred embodiments, the compound is

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

[0012] In some more preferred embodiments, the compound is a compound of Formula II or Formula III.

[0013] [ka]

[0014] [ka]

[0015] In order to solve the above technical problems, the present invention provides, as a second aspect, a pharmaceutical composition comprising the compound according to the first aspect of the present invention or a pharma- ceutically acceptable salt thereof, and preferably further comprising a pharma- ceutically acceptable diluent or carrier.

[0016] In order to solve the above technical problem, in a third aspect, the present invention provides use of a compound according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention in the manufacture 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 Rhinovirus, Poliovirus, Coxsackievirus, Echovirus, or Enterovirus protease.

[0018] In some further preferred embodiments, the enterovirus protease is a rhinovirus, poliovirus, coxsackievirus, echovirus, or enterovirus 3C protease.

[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, the present invention provides, in a fourth aspect, use of a compound according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention in the manufacture of a medicament for treating a gut-related disease.

[0021] In some preferred embodiments, the gut-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 includes HRV-A2, HRV-B14, the coxsackievirus includes CVB3, the echovirus includes Echovirus type 1, the enterovirus includes EV71, and the poliovirus includes Poliovirus type 1.

[0024] In order to solve the above technical problems, in a fifth aspect, the present invention provides a method for treating or preventing a gut-related disease by administering an effective amount of a compound according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention to a subject in need thereof.

[0025] In some preferred embodiments, the gut-related disease is a disease caused by the Enterovirus genus.

[0026] Preferably, the Enterovirus genus includes Rhinovirus, Poliovirus, Coxsackievirus, Echovirus, and Enterovirus.

[0027] More preferably, the rhinovirus includes HRV-A2, 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 comprises administering another drug to treat or prevent a gut-related disorder.

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

[0030] More preferably, the inhibitor that inhibits the 3C protease comprises AG7088 or another AG7088 derivative other than the compound according to the first aspect of the present invention.

[0031] In order to solve the above technical problems, the present invention provides, as a sixth aspect, a method for inhibiting or detecting 3C protease in vitro for non-diagnostic purposes, or a method for inhibiting or detecting 3C protease in vivo, comprising contacting the 3C protease with an effective amount of a compound according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention. The use of the method for inhibiting or detecting 3C protease in vitro for non-diagnostic purposes is, for example, in scientific research carried out in a laboratory, to determine whether a sample contains the 3C protease by contacting the sample with a compound according to 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 the present invention are as follows: The compounds disclosed in the present invention, for example, the compounds represented by formula II or formula III, can significantly inhibit the activity of 3C protease of various enteroviruses.At present, there is no specific drug approved for sale against human enteroviruses, so the therapeutic method of using the compounds represented by formula II or formula III to treat intestinal-related diseases can make up for the shortcomings of the prior art. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows the molecular formulas of ST-0101 and ST-0102. [Diagram 2] FIG. 2 shows that both ST-0101 and ST-0102 have significant inhibitory activity against rhinovirus HRV-A2 3C protease. [Diagram 3] FIG. 3 shows that ST-0101 has significant inhibitory activity against rhinovirus HRV-B14 3C protease. [Figure 4] FIG. 4 shows that ST-0101 and ST-0102 have significant inhibitory activity against Poliovirus type 1 3C protease. [Diagram 5] FIG. 5 shows that ST-0101 and ST-0102 have significant inhibitory activity against enterovirus EV71 3C protease. [Figure 6] FIG. 6 shows that ST-0101 and ST-0102 have significant inhibitory activity against Echovirus 1 3C protease. [Figure 7] FIG. 7 shows that ST-0101 and ST-0102 have significant inhibitory activity against Coxsackievirus CVB3 3C protease. [Figure 8] FIG. 8 shows the reversible covalent binding of HRV 3C protease to the inhibitor ST0102. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] The present invention will be further described below with reference to examples, but the scope of the present invention is not limited to these examples. In the following examples, the experimental methods without specific conditions are carried out according to the usual methods and conditions, or selected according to the product instructions.

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

[0037] The fluorescent substrate used to measure the inhibitory effect of ST-0101 and ST-0102 on the 3C protease of the above viruses is Dabcyl-Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro-Lys(5-FAM)-NH2 (SEQ ID NO: 3), where the amino acid sequence of the substrate is selected mainly based on the classical recognition sequence of 3C protease, and the fluorescent group 5-FAM and the quenching group Dabcyl are connected to both ends of the short peptide, respectively, and when the 3C protease cleaves the short peptide, the quenching group and the fluorescent group are separated, thereby allowing the fluorescent signal from the fluorescent group to be detected. Therefore, when the 3C protease reacts with the substrate, the rate of increase in fluorescence intensity can represent the rate of the enzymatic reaction by the 3C protease. When different concentrations of inhibitor molecules are added, the inhibitory effect on the enzyme activity is different, and the rate of increase in fluorescence intensity is also suppressed. The rate of increase in fluorescence intensity without the addition of inhibitor molecules is taken as 100% enzyme activity, and the rate of increase in fluorescence intensity without the addition of enzyme is taken as 0% enzyme activity (substrate molecules may self-degrade at a very slow rate), and the IC of the inhibitor is calculated by measuring the corresponding enzyme activity when different concentrations of inhibitor are added. 50 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), 0.01% (v / v) TritonX-100, the concentration of the substrate used was 20 μM, and the concentration of the protein used was 0.5 μM.

[0038] The amino acid sequence of HRV-A2 3C (SEQ ID NO: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 of Poliovirus type 1 3C (SEQ ID NO:4) 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 of Echovirus 1 3C (SEQ ID NO:6) is as follows: GPAFEFAVAMMKRNASTVKTEYGEFTMLGIYDRWAVLPRHAKPGPTILMNDQEVGVLDAKELVDKDGTNLELTLLKLNRNEKFRDIRGFLAREEAEVNEAVLAINTSKFPNMYIPVGQVTDYGFLNLGGTPTKRMLMYNFPTRAGQCGGVLMSTGKVLGIHVGGNGHQGFSAALLRHYFNEEQ

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

[0044] Example 2 In the present invention, the high-resolution structure of HRV 3C protease and the inhibitor ST-0102 at 1.54 Å was analyzed. As shown in FIG. 8, a reversible covalent bond was formed between the active center of the protease, C147, and the inhibitor. The crystal of the complex of HRV 3C protease and the inhibitor ST-0102 was obtained by screening with the Index kit of Hampton Research using the sessile drop method. The X-ray diffraction data was obtained at the Shanghai Synchrotron Radiation Facility. The crystal diffraction data was processed using the XDS program package, and the corresponding initial model was 5FX6 from the RCSB database, and molecular replacement was performed using the program Phenix.phaser, and cross-validation was performed using the programs Phenix.refine and Coot. Finally, the high-resolution structure of the complex of HRV 3C protease and the inhibitor ST-0102 was obtained.

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

[0046] Although the specific embodiments of the present invention have been described above, they are merely illustrative, and those skilled in the art will understand that various changes or modifications 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 document 2] L. Royston, C. Tapparel, Rhinoviruses and Respiratory Enteroviruses: Not as Simple as ABC. Viruses 8, (2016). [Non-patent document 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-patented literature 5] OH Laitinen et al., Enteroviral proteases: structure, host interactions and pathogenicity. Rev Med Virol 26, 251-267(2016). [Non-patent document 6] M. Laajala, D. Reshamwala, V. Marjomaki, Therapeutic targets for enterovirus infections. Expert Opin 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 Document 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 Document 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 in that it is a compound represented by the general formula I below. 【Chemistry 1】 (In the formula, R １ teeth, 【Chemistry 2】 or 【Chemistry 3】 Selected from R ２ , R ３ , R ４ is selected from H and halogen.

2. In the above compound, R １ but, 【Chemistry 4】 When Ar is 3,4-(OCH ２ ) C ６ H ３ , 4-Me ２ N.C. ６ H ４ , C ６ H ５ , 4-MeC ６ H ４ , 4-Cl, 2-FC ６ H ３ or 5-methyl-3-isoxazole, R ２ , R ３ , R ４ is H, F or CF ３ The compound according to claim 1, characterized in that

3. The compound according to claim 1, characterized in that it is a compound of formula II or formula III. 【Chemistry 5】 【Chemistry 6】

4. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharma- ceutically acceptable salt thereof.

5. 5. The pharmaceutical composition of claim 4, further comprising a pharma- ceutically acceptable diluent or carrier.

6. Use of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 4 or 5 in the manufacture of an inhibitor of enteroviral protease.

7. 7. The use according to claim 6, characterized in that 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 Rhinovirus, Poliovirus, Coxsackievirus, Echovirus or Enterovirus protease.

8. The use according to claim 7, characterized in that the enterovirus protease is a 3C protease of a rhinovirus, a poliovirus, a coxsackievirus, an echovirus or an enterovirus.

9. 9. The use according to claim 8, characterized in that the 3C protease is a 3C protease of rhinovirus HRV-A2 or HRV-B14, poliovirus type 1, enterovirus EV71, echovirus type 1 or coxsackievirus CVB3.

10. Use of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 4 or 5 in the manufacture of a medicament for the treatment of a gut-related disorder.

11. The use according to claim 10, characterized in that the intestinal-related disease is a disease caused by Enteroviruses.

12. The use according to claim 11, characterized in that the Enterovirus genus comprises Rhinovirus, Poliovirus, Coxsackievirus, Echovirus and Enterovirus.

13. The use according to claim 12, characterized in that the rhinovirus includes HRV-A2, HRV-B14, the coxsackievirus includes CVB3, the echovirus includes Echovirus type 1, the enterovirus includes EV71, and the poliovirus includes Poliovirus type 1.

14. A non-diagnostic method for inhibiting or detecting 3C protease in vitro comprising contacting said 3C protease in vitro with an effective amount of a compound according to any one of claims 1 to 3 or a pharmaceutical composition according to claim 4 or 5.

15. 15. The method of claim 14, wherein the 3C protease is a rhinovirus, poliovirus, coxsackievirus, echovirus or enterovirus 3C protease.

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