Human deubiquitinase inhibitors for the treatment of coronavirus infection

Abstract

The present application relates to small molecule inhibitors of human deubiquitinase for preventing or treating coronavirus infection. In particular, it relates to small molecule inhibitors of USP7 and USP47. Pharmaceutical compositions, respective advantageous formulation techniques and treatment methods are disclosed.

Description

Technical Field

[0001] The present application relates to small molecule inhibitors of human deubiquitinase for preventing or treating coronavirus infection. In particular, it relates to small molecule inhibitors of USP7 and USP47. Pharmaceutical compositions, respective advantageous formulation techniques and treatment methods are disclosed. Background Art

[0002] So-called 'emerging' viruses are increasingly spreading from their natural animal hosts to humans as a result of ecological, climatic and demographic changes. They carry the risk of causing epidemics due to accelerating globalization. Emerging viruses may cause acute and often life-threatening illnesses. The Coronaviridae family has become notorious for such spread. Examples are severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), and more recently the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; COVID-19). As of February 4, 2021, the Johns Hopkins University Coronavirus Resource Center has reported a total of more than 104 million SARS-CoV-2 infections worldwide, with more than 2 million deaths. The incubation period for SARS-CoV-2 is between 2 days and 2 weeks, and in some cases up to a month.

[0003] The typical symptoms of SARS-CoV-2 are fever, cough and shortness of breath. However, the infection may also cause severe lung damage, leading to the rapid onset of progressive dysfunction of the lungs, especially in terms of oxygen uptake capacity. This is often associated with dysfunction of other organs. This acute lung injury (ALI) condition is associated with widespread lung inflammation and accumulation of fluid in the alveoli. It is characterized by diffuse pulmonary microvascular damage, resulting in increased permeability and, thereby, non-cardiogenic pulmonary edema. As a result, this leads to pathological low oxygen levels in the lungs. Other common symptoms associated with COVID-19 patients in ICU care are pulmonary embolism, thrombosis, venous thromboembolism and cerebral ischemia.

[0004] Coronaviruses are mainly spread through close contact, especially through respiratory droplets produced by coughing and sneezing. Unlike SARS-CoV and MERS-CoV, SARS-CoV-2 can be transmitted from person to person during the incubation period, while the infected patients do not yet show any symptoms of the disease. In addition, SARS-CoV-2 can already replicate in the throat. In contrast, the receptors for SARS-CoV and MERS-CoV are located deep in the lungs. Therefore, SARS-CoV-2 can be transmitted from person to person more easily than SARS-CoV and MERS-CoV, which makes the infection rate significantly higher.

[0005] In general, coronaviruses (Coronaviridae, coronavirus group) form a relatively diverse group of large, enveloped, positive-strand RNA viruses that can cause different types of diarrhea and respiratory diseases in humans and animals. They have a very narrow host range and replicate very poorly in cell culture. However, a cell culture system for SARS-CoV-2 can be successfully established.

[0006] Sequencing of SARS-CoV-2 shows a genome of approximately 29.8 kbp consisting of 14 open reading frames. In addition, the virus is closely related to SARS-CoV in terms of phylogeny (89.1% nucleotide similarity) (see Wu et al. (2020) Nature 579: 265-269). Like other coronaviruses, SARS-CoV-2 enters cells through endocytosis and membrane fusion. The virus is released from the cell through the secretory pathway. The natural reservoir of the virus is unknown.

[0007] To date, no specific treatment options have been established for the treatment of SARS-CoV-2 infection or COVID-19. Some success has been achieved with the antiviral drugs remdesivir, avifavir, and favipiravir, and with the antiparasitic drug ivermectin. A nasal spray containing nanobodies against the SARS-CoV-2 spike protein is a promising development (AeroNabs). In patients with severe COVID-19, administration of the glucocorticoid dexamethasone has been shown to be effective.

[0008] A major problem is the high mutation rate of coronaviruses. Therefore, for an effective drug treatment for a specific coronavirus infection, there is a high probability that the coronavirus will quickly become partially or completely resistant to the treatment when the viral target is inhibited. Once the next coronavirus is transmitted to humans through the species barrier, the same problem will occur and develop into another infectious disease.

[0009] Therefore, there is a strong medical need for effective drug treatments for patients infected with SARS-CoV-2 or similar coronaviruses and for limiting the current large-scale pandemic spread of the virus. Ideally, such drug treatments should also address the high mutation rate and at least provide treatment options for future coronavirus outbreaks. Summary of the invention

[0010] Surprisingly, this task was solved by small molecule human deubiquitinase inhibitors according to the present disclosure. In particular, small molecule inhibitors of human deubiquitinase USP7 and / or USP47 show good efficacy in the treatment of coronavirus infection.

[0011] USP7 and USP47 are closely related in phylogeny. USP7 is primarily associated with tumors because its inhibition reactivates the tumor suppressor gene p53 in many cancers. USP7 is also associated with the immune system (see Antao et al. (2020) Cancers 12:1579). As far as the studies conducted so far are concerned, compounds that show inhibitory activity against USP7 generally also inhibit USP47. Therefore, it seems that USP47 acts as a tissue-specific isozyme of USP7.

[0012] Therefore, the present disclosure relates to inhibitors of human deubiquitinase USP7 and / or USP47 and pharmaceutically acceptable salts, hydrates and solvates thereof for prevention or treatment of coronavirus infection. DETAILED DESCRIPTION

[0013] A preferred embodiment of such USP7 and / or USP47 inhibitors is pyridine-3,5-(bis)thiocyanate according to the present disclosure.

[0014] Suitable pyridine-3,5-bis(thiocyanate)s according to the present disclosure are pyridine-3,5-(bis)thiocyanate according to general formula (I),

[0015]

[0016] in

[0017] R 1 and R 2 Each is independently -H, -OH, -NHR 3 、-NR 3 R 4 , substituted or unsubstituted linear or branched alkyl residues having 1 to 3 carbon atoms, -CO-OCH 3 、-CO-OC 2 H 5 、-CO-NH 2 、-NH 2 、-NO2 , -Cl, -Br, -F, or -SO 2 H; and

[0018] R 3 and R 4 Each independently represents -OH, -CH 3 , -C 2 H 5 、-CH 2 OH, -CHO, -COOH, -CO-CH 3 , or -CO-NH 2 .

[0019] In particular, 2,6-diaminopyridine-3,5-bis(thiocyanate) has been shown to be effective. This compound is generally known as PR-619 (Formula II) and is most preferred.

[0020]

[0021] As shown in Example 1, 10 μM PR-619 was able to extensively inhibit the replication of SARS-CoV-2 in infected Vero-B4 cells. This finding was confirmed in the qRT-PCR experiment of Example 2. As can be seen in Example 3, at this concentration, PR-619 did not show cytotoxic effects. In addition, in an enzyme assay, PR-619 showed direct inhibition of the papain-like protease (PLpro) of SARS-CoV-2, which is essential for the replication of SARS-CoV-2 (Example 4).

[0022] Other preferred embodiments of such USP7 and / or USP47 inhibitors are disubstituted 4-nitro-thiophenes according to the present disclosure.

[0023] Suitable disubstituted 4-nitro-thiophenes according to the present disclosure are disubstituted 4-nitro-thiophenes according to the general formula (III),

[0024]

[0025] in

[0026] R 1 is phenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl, 2,3-dichlorophenyl, 3,5-dichlorophenyl, 2,4-dichlorophenyl or 2,4-difluorophenyl, and

[0027] R 2 It is acetyl, 1-hydroxyethyl, ethyl or n-butyl.

[0028] Compounds of 4-nitro-thiophene with these substituents were found to be effective in inhibiting the human deubiquitinase USP7 (Chauhan et al. (2012) Cancer Cell 22:345-358).

[0029] In particular, 1-[5-[(2,3-dichlorophenyl)thio]-4-nitro-2-thienyl]-ethanone is shown to be effective. Another name is 1-[5-(2,3-dichlorophenyl)sulfanyl-4-nitro-2-thienyl]ethanone. This compound is mainly called P005091, or P5091 (see formula IV), and is the most preferred.

[0030]

[0031] In Example 5, it was documented that 5 μM P5091 almost completely inhibited the replication of SARS-CoV-2 in infected Vero-B4 cells. As shown in Example 6, at this concentration, P5091 did not show cytotoxic effects. In addition, in an enzyme assay, P5091 showed a direct inhibitory effect on the papain-like protease (PLpro) of SARS-CoV-2 (Example 7).

[0032] Other suitable deubiquitinase inhibitors of USP7 and / or USP47 include P22077, ADC-01, ADC-03, HBX41108, HBX19818, HBX 28258, NSC 632839, NSC 144303, GNE-6640, GNE-6776, FT671, FT827, XL188, XL177a, XL024, XL058, XL041, 4-cyano-5-[(3,5-dichloro-4-pyridinyl)thio]-N-[4-(methylsulfonyl)phenyl]-2-thiophenecarboxamide and parthenolide.

[0033] The present disclosure also relates to deubiquitinase inhibitors of the aforementioned USP7 and / or USP47 for use in the prevention or treatment of coronavirus infection.

[0034] The deubiquitinase inhibitor P22077 (1-{5-[(2,4-difluorophenyl)sulfanyl]-4-nitro-2-thienyl}ene ketone) was discovered by Progenra (Malvern, USA) and is commercially available. A description of the synthesis of the P22077 substance class is disclosed in WO2010 / 114881. P22077 is specific for USP7 and USP47. The affinity for ATXN3, BAP1 and USP1 is much smaller. P22077 leads to an increase in polyubiquitin chains (Altun et al. (2011) Chem Biol 18: 1401-1412). Activation of the autophagic pathway can be shown (Seiberlich et al. (2013) Cell Biochem Biophys 67: 149-160). To date, the research on P22077 has focused on the treatment of tumors, and no antiproliferative potential has been demonstrated for P22077 to be therapeutically useful. In addition, P22077 is very effective in inhibiting HIV-1 replication in primary T cells and macrophages and in ex vivo preparations (human lymphoid aggregate culture, HLAC). MHC-I antigen presentation of HIV-1 structural proteins is increased (see WO 2016 / 004915).

[0035] Almac Discovery has discovered a series of piperidine derivatives that are specific USP7 inhibitors (WO 2018 / 073602 A1). Among them, ADC-01 and ADC-03 are the most promising compounds (Gavory et al. (2015) Cancer Res 75:15). These compounds should be developed therapeutically for oncology and immuno-oncology applications.

[0036] HBX41108 (7-chloro-9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile), HBX19818 (N-(3-(benzyl(methyl)amino)propyl)-9-chloro-5,6,7,8-tetrahydroacridine-2-carboxamide), and HBX28258 (9-chloro-N-[3-[ethyl(phenylmethyl)amino]propyl]-5,6,7,8-tetrahydro-2-acridinecarboxamide) specifically inhibit USP7. These related compounds should be developed therapeutically for oncology and immuno-oncology uses (Reverdy et al. (2012) ChemBiol 19:467-477).

[0037] GNE-6640 (CS 2824; 4-[2-amino-4-ethyl-5-(1H-indazol-5-yl)-3-pyridinyl]-phenol) and GNE-6776 (CS2823; 6'-amino-4'-ethyl-5'-(4-hydroxyphenyl)-N-methyl-[3,3'-bipyridine]-6-carboxamide) are USP7 inhibitors. GNE-6640 also inhibits USP43 and Ub-MDM2, while GNE-6776 is highly specific for USP7. These compounds induce tumor cell death and cytotoxicity by attenuating ubiquitin binding and thereby attenuating USP7 activity (Kategaya et al. (2017) Nature 550:534-538).

[0038] FT671 (5-[[1-[(3S)-4,4-difluoro-3-(3-fluoro-1H-pyrazol-1-yl)-1-oxobutyl]-4-hydroxy-4-piperidinyl]methyl]-1-(4-fluorophenyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one) and FT827 (N-[4'-[[4-[(1,4-dihydro-1-methyl-4-oxo-5H-pyrazolo[3,4-d]pyrimidin-5-yl)methyl]-4-hydroxy-1-piperidinyl]carbonyl][1,1'-biphenyl]-2-yl]-ethylenesulfonamide) inhibit USP7 with high affinity and specificity. Through this mechanism, they lead to reactivation of the tumor suppressor gene p53 in various cancers (Turnbull et al. (2017) Nature 550:481-486).

[0039] WO 2019 / 067503A1 discloses that many related USP7 inhibitors interact with the tumor suppressor gene p53 for tumor treatment: ((R)-7-chloro-3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, (7-chloro-3-((1-(3-phenylpropionyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, 4-((7-chloro-4-oxoquinazolin-3(4H)-yl)methyl)-1-(3-phenylpropionyl)piperidine-4-carbonitrile, (7-chloro-3-((4-hydroxy-1-(3-phenylpropyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, (7-chloro- 3-((3-hydroxy-1-(3-phenylpropionyl)pyrrolidin-3-yl)methyl)quinazolin-4(3H)-one, (3-((1-acetyl-4-hydroxypiperidin-4-yl)methyl)-7-chloroquinazolin-4(3H)-one, (7-chloro-3-((4-hydroxy-1-(2-phenylacetyl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, (7-chloro-3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, ((S)-7-chloro-3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)quinazolin-4(3H)-one, ((R)-N-(3-( (4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)acrylamide, ((S)-N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)acrylamide, (N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)acrylamide, (N-(3-((4-hydroxy-1-(4-methyl- 3-(4-methylpiperazin-1-yl)acrylamide, ((R)-N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-morpholinopropanamide, ((R)-3-(dimethylamino)-N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)acrylamide, (N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-morpholinopropanamide,4-dihydroquinazolin-7-yl)-3-(1H-imidazol-1-yl)acrylamide, ((TST-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(piperidin-1-yl)acrylamide.,

[0040] ((R)-N-(3-((4-hydroxy-1-(3-phenylbutyryl)piperidin-4-yl)methyl)-4-oxo-3,4-dihydroquinazolin-7-yl)-3-(4-methylpiperazin-1-yl)acrylamide is also known as XL188 (see Lamberto et al. (2017) Cell Chem Biol 24: 1490-1500).

[0041] Derived from XL188 is XL177a ((S)-N-(4-benzyl-5-(4-hydroxy-4-((7-(3-(4-methylpiperazin-1-yl)propionamido)-4-oxoquinazolin-3(4H)-yl)methyl)piperidin-1-yl)-5-oxopentyl)-9-chloro-5,6,7,8-tetrahydroacridine-3-carboxamide). XL112, XL024 (((S)-N-(4-benzyl-5-(4-((7-(3-(4-methylpiperazin-1-yl)propionylamino)-4-oxoquinazolin-3(4H)-yl)methyl)piperidin-1-yl)-5-oxopentyl)-9-chloro-5,6,7,8-tetrahydroacridine-3-carboxamide), XL058 (((S)-N-(4-benzyl-5-(4-hydroxy-4-((7-(3-(4-methylpiperazin-1-yl)propionylamino)-4-oxoquinazolin-3(4H)-yl)methyl)piperidin-1-yl)-5-oxopentyl)-9-chloro-5,6,7,8-tetrahydroacridine-3-carboxamide), Also belonging to this group are (((S)-N-(5-(4-hydroxy-4-((7-(3-(4-methylpiperazin-1-yl)propionamido)-4-oxoquinazolin-3(4H)-yl)methyl)piperidin-1-yl)-5-oxopentyl)-9-chloro-5,6,7,8-tetrahydroacridine-3-carboxamide) and XL041 (((S)-N-(5-(4-hydroxy-4-((7-(3-(4-methylpiperazin-1-yl)propionamido)-4-oxoquinazolin-3(4H)-yl)methyl)piperidin-1-yl)-5-oxopentyl)-9-chloro-5,6,7,8-tetrahydroacridine-3-carboxamide) (see Schauer et al. (2020) Nature Sci Rep 10:5324).

[0042] NSC 632839 (synonym: F6; 4-piperidinedione, 3,5-bis[(4-methylphenyl)methylene]-hydrochloride) and NSC 144303 (4H-thiopyran-4-one-tetrahydro-3,5-bis[(4-nitrophenyl)methylene]-1,1-dioxide) were found to maintain caspase 3 / caspase 7 activity in the absence of functional caspase-9. This included a mitochondrial pathway that was dependent on Bcl-2 but not on the apoptosome. Apoptosis appeared to be initiated by the ubiquitin-proteasome system. Both compounds inhibited USP2, USP7, and the SENP2 deSUMOylase without affecting the proteasome proteolytic activity. They are being developed as alternatives for cancer treatment (see Aleo et al. (2006) Cancer Res 66:9235-9244; Nicholson et al. (2008) Protein Sci 17:1035-1043).

[0043] 4-Cyano-5-[(3,5-dichloro-4-pyridinyl)thio]-N-[4-(methylsulfonyl)phenyl]-2-thiophenecarboxamide is a deubiquitinase inhibitor specific for USP7 and USP47 relative to USP2, USP5, USP8, USP21, USP28, caspase-3, and cathepsin B. It inhibits the growth of HCT116 cells (see Weinstock et al. (2012) ACSMed Chem Lett 3:789-792).

[0044] Parthenolide (1aR,4E,7aS,10aS,10bR)-2,3,6,7,7a,8,10a,10b-octahydro-1a,5-dimethyl-8-methylene-oxirano[9,10]cyclodecan[1,2-b]furan-9(1aH)-one) is a sesquiterpene lactone found in Tanacetumparthenium. Initially, parthenolide was classified as an HDAC1 inhibitor and NF-κB modulator. Anti-inflammatory and anti-hyperalgesic properties as well as effects on Leishmaniosisamazonensis infection have been documented. Recently, parthenolide was found to specifically inhibit USP7, Wnt signaling, and colorectal cancer cell growth (Li et al. (2020) J Biol Chem 295:3576-3589).

[0045] Coronaviruses are known to utilize the protease PLpro or their functional analogs to interfere with the proteasome system of human host cells, thereby functionalizing the corresponding cellular enzymes for their replication.

[0046] The proteasome is a multicatalytic enzyme complex that accounts for about 1% of the total cellular protein. It represents the major proteolytic component in the nucleus and the cytosol of eukaryotic cells. The proteasome plays many important roles in cellular metabolism. One major function is the proteolysis of misfolded, non-functional proteins. Another important function is the degradation of cellular and viral proteins for T cell-mediated immune responses, thereby generating peptide ligands to be loaded onto MHC-I molecules (MHC = major histocompatibility complex). One subtype is the immunoproteasome, which is constitutively expressed in specific cell types, such as in the spleen, lymph nodes and antigen presenting cells.

[0047] The substrate of proteasome is usually marked for degradation by the attachment of ubiquitin oligomers. Ubiquitin (Ub) is a highly conserved protein of 76 amino acids in length, which is covalently coupled to the corresponding target protein. Ubiquitination is a reversible process. Ub molecules can be removed from target proteins by means of a variety of deubiquitinases (deubiquitinating enzymes (DUBs)). Therefore, Ub molecules become available in the cell again. This recycling process is essential for cell homeostasis. This regulatory system of target protein ubiquitination and proteasome proteolysis is commonly referred to as the ubiquitin proteasome system (UPS).

[0048] DUBs are a broad class of Ub hydrolases and are intracellular opponents of ubiquitin E3 ligases. Target proteins can be fully or partially deubiquitinated. In humans, more than 100 DUBs are known to date, which are subdivided into 5 families:

[0049] Ubiquitin-specific proteases (USPs)

[0050] Ubiquitin C-terminal hydrolase (UCH)

[0051] Ovarian Tumor Protease (OTU)

[0052] Josephin family

[0053] JAB1 / MPN / Mov34 family (JAMM)

[0054] The first four are cysteine ​​proteases and the last is a zinc metalloprotease.

[0055] The most important cellular functions of DUB are:

[0056] 1. They are essential for the de novo generation of free ubiquitin. Ubiquitin is a linear fusion protein encoded on several genes and composed of a row of Ub monomers. After translation, the ubiquitin chain is specifically hydrolyzed by DUBs, resulting in the release of free Ub molecules.

[0057] 2. They remove polyubiquitin chains of post-translationally modified proteins in a highly specific manner. Thus, the target protein is stabilized. In addition, the proteasome-associated DUBs POH1, UCH37, and USP14 remove ubiquitin chains from proteins that have entered the proteasome for proteolysis. In this way, the content of free ubiquitin is kept balanced in the cell.

[0058] 3. They change the ubiquitin modification of a protein by trimming existing ubiquitin chains. For example, an initially polyubiquitinated protein may end up carrying only one ubiquitin. As a result, the function of the protein may be completely different.

[0059] In most cases, DUBs display high specificity for certain substrates, tissues, and for particular ubiquitin chains.

[0060] However, DUBs themselves undergo complex regulation. Thus, post-translational modifications such as phosphorylation, ubiquitination or SUMOylation may occur, leading to activation or inactivation of the corresponding DUB.

[0061] In addition, DUBs can undergo conformational changes by binding to certain proteins, which can also lead to the activation or inactivation of the corresponding DUBs.

[0062] The activity of some DUBs is restricted to certain cellular compartments and they are transported there if necessary.

[0063] DUBs are therefore interesting targets for influencing cellular regulatory processes either by their inhibition or by modifying their activity. Furthermore, for clinical applications, the entire UPS has become a focus in the past few years. Thus, for example, attempts have been made in the past few years to inhibit components of the UPS such as the 26S proteasome or Ub ligases with the aid of small molecules. The rationale for this is firstly to find new therapeutic avenues for tumors. The only drugs approved so far are proteasome inhibitors: bortezomib for the treatment of multiple myeloma and mantle cell lymphoma. Carfilzomib for multiple myeloma only and ixazomib However, the mechanism of action is not specific. Global effectiveness involves many important processes, partly causing severe side effects such as manifestations of peripheral neuropathy with pain and paralysis (especially in the limbs). In the clinical development of E3 ligase inhibitors, there have been no breakthroughs so far.

[0064] In addition, in the research of DUB inhibitors, the development of new therapeutic approaches in tumor treatment is a focus of attention. The DUB inhibitors known so far usually act highly specifically on a certain DUB target. Therefore, they show relatively little cytotoxicity. Compared with the use of 26S proteasome inhibitors or E1 ligase inhibitors, significantly fewer and milder side effects in patients are expected.

[0065] For some DUBs, such as USP2a, USP7, USP20 and USP33, oncogenicity has been shown. Therefore, as a mode of action, inhibition of DUBs is considered a way to block or at least reduce the oncogenicity of these DUBs.

[0066] PR-619 (2,6-diaminopyridine-3,5-(bis)thiocyanate) inhibits USP1, USP2, USP4, USP5, USP7, USP8, USP9X, USP10, USP14, USP15, USP16, USP19, USP20, USP22, USP24, USP28, USP47, USP48, UCH-L1, UCH-L3, UCH-L5 / UCH37, ATXN3, BAP1, JOSD2, OTUD5, VCIP135, and YOD. Application of PR-619 results in an increase in polyubiquitin chains (Altun et al. (2011) Chem Biol 18:1401-1412). To date, research on PR-619 has focused on tumor therapy studies, and no therapeutically useful antiproliferative potential has been found to date. PR-619 has been shown to be significantly effective in inhibiting HIV-1 replication in primary T cells and macrophages and in ex vivo preparations (human lymphoid aggregate cultures, HLACs) by inhibiting USP47. MHC-I antigen presentation of HIV-1 structural proteins is increased (see WO 2016 / 004917).

[0067] Therefore, the present application also relates to pyridine-3,5-(bis)thiocyanate and pharmaceutically acceptable salts, hydrates and solvates thereof according to the present disclosure for the prevention or treatment of coronavirus infection.

[0068] In particular, the present application relates to PR-619 for the prevention or treatment of coronavirus infection.

[0069] In CB-17 mice, the use of the DUB inhibitor P5091 leads to apoptosis of multiple myeloma cells. Resistance to the proteasome inhibitor bortezomib can therefore be reversed. P5091 has been documented to induce apoptosis in tumor cell lines (Chauhan et al. (2012) Cancer Cell 22:345-358). P5091 effectively and specifically inhibits the closely related deubiquitinating enzymes USP7 and USP47 without blocking proteasome activity (Chauhan et al. (2012) Cancer Cell 22:345-358; Weinstock et al. (2012) ACS Med Chem Lett 3:789-792).

[0070] Therefore, the present application also relates to the disubstituted 4-nitro-thiophene according to the present disclosure and its pharmaceutically acceptable salts, hydrates and solvates for the prevention or treatment of coronavirus infection.

[0071] In particular, the present application relates to P5091 for use in the prevention or treatment of coronavirus infection.

[0072] Coronavirus infections that can be treated with the deubiquitinase inhibitors according to the present disclosure are primarily infections with the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0073] However, it is also possible to treat infections of the less pathogenic Coronaviridae listed below. The term "coronavirus" or "coronaviral" refers primarily to the subfamily Orthocoronavirus. They are subdivided into the alphacoronavirus, betacoronavirus, gammacoronavirus and deltacoronavirus genera. Coronaviruses include the colacovirus subgenus (species: bat coronavirus CDPHE15), the decavirus subgenus (bat coronavirus HKU10, horseshoe bat alpha coronavirus HuB-2013), the duvinacovirus subgenus (human coronavirus 229E), the luchacovirus subgenus (Lucheng brown rat coronavirus), the minacovirus subgenus (ferret coronavirus, water meerk coronavirus 1), the minunacovirus subgenus (long-winged bat coronavirus 1, long-winged bat coronavirus HKU8), the myotacovirus subgenus (big-footed myotacovirus alpha coronavirus Sax-2011), the nylactovirus subgenus (velvet bat alpha coronavirus SC-2013), the pedacovirus subgenus (porcine epidemic diarrhea virus, long-headed bat coronavirus 512), the rhinacovirus subgenus (hornhead bat coronavirus HKU2), the setracovirus subgenus (human coronavirus NL63, bat coronavirus strain BtKYNM63-9b related to NL63) and the tegacovirus subgenus (alpha coronavirus 1 model species). Betacoronaviruses include the embecovirus subgenus (betacoronavirus 1 (subspecies: human coronavirus OC43), Chinese rat coronavirus HKU24, human coronavirus HKU1, rat coronavirus model species), hibecovirus subgenus (horned bat betacoronavirus Zhejiang 2013), merbecovirus subgenus (hedgehog coronavirus 1, MERS-CoV, scutellaria bat coronavirus HKU5, flat-headed bat coronavirus HKU4), nobecovirus subgenus (fruit bat coronavirus GCCDC1, fruit bat coronavirus HKU9) and sarbecovirus subgenus (coronavirus associated with severe acute respiratory syndrome (subspecies: SARS-CoV, SARS-CoV-2)). Gammacoronaviruses include the cegacovirus subgenus (cetacean coronavirus SW1) and igacovirus subgenus (avian coronavirus model species). Delta coronaviruses include the andecovirus subgenus (ruddy duck coronavirus HKU20), the buldecovirus subgenus (bulb coronavirus HKU11 type species, porcine coronavirus HKU15, munia coronavirus HKU13, white-eye coronavirus HKU16), the herdecovirus subgenus (heron coronavirus HKU19) and the Moordecovirus subgenus (black-eyed swamphen coronavirus HKU21).

[0074] So far, the coronaviruses that cause disease in humans are SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-HKU1, HCoV-NL-63, HCoV-OC43, and HCoV-229E. The latter four only cause relatively mild symptoms (see Andersen et al.: The Proximal Origin of SARS-CoV-2, on virologica.org, as of February 17, 2020).

[0075] Therefore, the present application particularly relates to one of the human deubiquitinase inhibitors of USP7 and / or USP47 according to the present disclosure or a pharmaceutically acceptable salt thereof for the prevention or treatment of coronavirus infection, wherein the coronavirus infection is selected from the group consisting of SARS-CoV infection, SARS-CoV-2 infection, MERS-CoV infection, HCoV-HKU1 infection, HCoV-NL-63 infection, HCoV-OC43 infection and HCoV-229E infection.

[0076] More preferred is the pyridine-3,5-(bis)thiocyanate or one of its pharmaceutically acceptable salts according to the present disclosure for use in the prevention or treatment of coronavirus infection, wherein the coronavirus infection is SARS-CoV-2 infection.

[0077] Still more preferred is PR-619 or one of its pharmaceutically acceptable salts for use in the prevention or treatment of coronavirus infection, wherein the coronavirus infection is SARS-CoV-2 infection.

[0078] More preferred is the disubstituted 4-nitro-thiophene or one of its pharmaceutically acceptable salts according to the present disclosure for use in the prevention or treatment of coronavirus infection, wherein the coronavirus infection is SARS-CoV-2 infection.

[0079] Still more preferred is P5091 or one of its pharmaceutically acceptable salts for use in the prevention or treatment of coronavirus infection, wherein the coronavirus infection is SARS-CoV-2 infection.

[0080] Other of the above-mentioned animal coronaviruses have not yet been transferred to humans (zoonosis), but this may occur in the future with unpredictable pathology. Therefore, the scope of the present application also relates to small molecule inhibitors of human deubiquitinases according to the present disclosure for the prevention or treatment of these animal coronavirus infections in animals and humans.

[0081] The concept of treating coronavirus infections in all species is based on the structural similarities of coronaviruses. Therefore, it can be assumed that treatment and / or prevention regimens can be transferred from one coronavirus to another. The coronavirus particle contains four major structural proteins: spike (S), membrane (M), envelope (E), and nucleocapsid (N), all of which are encoded within the 3' end of the viral genome.

[0082] Coronavirus contains a non-segmented positive RNA genome of ~ 30kb. The genome contains a 5′ cap structure and a 3′ poly (A) tail, which acts as the mRNA for the translation of the replicase polyprotein. Unlike the structural proteins and auxiliary proteins that only account for about 10kb of the viral genome, the replicase gene encoding the non-structural protein (nsp) occupies two-thirds of the genome, about 20kb. The composition of the coronavirus genome is 5′-leader sequence-UTR-replicase-S (spike)-E (envelope)-M (membrane)-N (nucleocapsid)-3′UTR-poly (A) tail, wherein auxiliary genes are interspersed in the structural genes at the 3′ end of the genome. Auxiliary proteins are almost completely not required for replication in tissue culture; however, some auxiliary proteins have been shown to play an important role in viral pathogenesis (see Zhao et al. (2012) Cell Host Microbe 11: 607-616).

[0083] The coronavirus life cycle begins with the initial attachment of virions to host cells through the interaction between the S protein and its receptor. The site of the receptor binding domain (RBD) in the S1 region of the coronavirus S protein varies depending on the virus. The S-protein-receptor interaction is the main determinant of coronavirus infection host species and also controls the tissue tropism of the virus. Many coronaviruses use peptidases as their cell receptors. It is not clear why peptidases are used, because entry occurs even in the absence of the enzymatic domain of these proteins. Many alpha coronaviruses use aminopeptidase N (APN) as their receptors, and many beta coronaviruses such as SARS-CoV, SARS-CoV-2 and HCoV-NL63 use angiotensin converting enzyme II (ACE2) receptors, MHV enters through CEACAM1, and MERS-CoV is bound to dipeptidyl peptidase 4 (DPP4) to enter human cells. After receptor binding, the virus must then enter the cytosol of the host cell. This is usually accomplished by acid-dependent proteolytic cleavage of the S protein by cathepsins, TMPRRS2 or another protease, followed by fusion of the viral and cellular membranes and eventual release of the viral genome into the cytoplasm.

[0084] Coronaviruses encode two or three proteases that cleave the replicase polyprotein. These are a papain-like protease (PLpro) encoded within nsp3 and a serine-type protease (the major protease or Mpro encoded by nsp5). Most coronaviruses encode two PLpros within nsp3, except for the gammacoronaviruses SARS-CoV and MERS-CoV, which express only one PLpro (Mielech et al. (2014) Virus Res doi:10.1016).

[0085] This papain-like protease (PLpro) was found in SARS-CoV to act in the same way as the deubiquitinating enzymes within the ubiquitin proteasome system (UPS) of human cells (see Raaben et al. (2010) J Virol 84:7869-7879). The PLpro in SARS-CoV-2 has a very high homology with SARS-CoV (96.1%, Nguyen et al. (2020) https: / / doi:org / 10:1101 / 2020.02.05.936013). These PLpro are targets of DUB inhibitors. For example, GRL0617 (5-amino-2-methyl-N-[(1R)-1-(1-naphthyl)ethyl]benzamide) was found to inhibit PLpro in SARS-CoV. Therefore, GRL0617 can be considered as a DUB inhibitor against coronavirus targets (see Ratia et al. (2008) PNAS 105:16119-16124).

[0086] Chloroquine ((RS)-N'-(7-chloroquinoline-4-yl)-N,N-diethyl-pentane-1,4-diamine) is a well-known malaria drug that acts as an autophagy inhibitor. Chloroquine synergizes with bortezomib to inhibit cell proliferation and induce apoptosis in tumor models (Hui et al. (2012) Cancer 118:5560-5571). Chloroquine is known to block viral infection by increasing the endosomal pH required for virus / cell fusion, and chloroquine interferes with the glycosylation of ACE II receptors in SARS-CoV infection (Vincent et al. (2005) Virol J 2,69). Recently, it was found that the combination of the antiviral drug remdesivir and chloroquine inhibited SARS-CoV-2 in vitro (Wang et al. (2020) Cell Res 0:1-3).

[0087] Proteins designated for degradation by the proteasome are marked by the continuous attachment of ubiquitin molecules to these proteins. This is achieved by a cascade of ligases E1-E3. The longer the ubiquitin chain, the better the corresponding protein is recognized at the proteasome and, accordingly, the longer the tagged protein is available for degradation by the proteasome.

[0088] The counterpart of ligases is deubiquitinases. They cleave ubiquitin molecules from the ubiquitin chains in a substrate-specific and tissue-specific manner. Ligase and deubiquitinase are in a constant balance and thus regulate protein degradation in cells.

[0089] If a protein tagged with ubiquitin chains is recognized by the 26S proteasome, it docks at the ubiquitin recognition site at the entrance of the proteasome and is proteolytically degraded into amino acids in successive stages. Finally, the amino acids and ubiquitin molecules leave the proteasome and are available for cell recycling.

[0090] In general, the proteasome can be likened to a collaboratively organized factory, with ligases and deubiquitinases such as PR-619 being part of the supply chain. The proteasome is a functional unit of many proteins and is therefore quite large. In contrast, more than a hundred different cellular deubiquitinases have been described, which are independent proteins rather than part of the proteasome.

[0091] Proteasome inhibitors inhibit the entire proteasome, but not the ligases or deubiquitinases, and vice versa. In short, proteasome inhibitors inhibit the cellular degradation of proteins.

[0092] In contrast, deubiquitinase inhibitors prevent specific deubiquitinases from cutting ubiquitin molecules from such labeled proteins in a substrate-specific and tissue-specific manner. As a result, ubiquitin chains are relatively extended. Therefore, the corresponding proteins are available for longer time in the proteasome and are better recognized. Deubiquitinase inhibitors work more specifically and therefore show much less cytotoxicity within the effective dose range. In short, deubiquitinase inhibitors promote cellular degradation of proteins.

[0093] Thus, proteasome inhibitors and deubiquitinase inhibitors act mechanistically in opposite directions.

[0094] For effective treatment of coronavirus infection, it would be advantageous to provide combination therapy to patients in need thereof by combining an inhibitor of a human deubiquitinase according to the present disclosure with at least one antiviral agent.

[0095] For example, from HIV or antiretroviral therapy, the following categories are known:

[0096] Reverse transcriptase inhibitors suitable for such combination therapy are nucleoside reverse transcriptase inhibitors (NRTI) and non-nucleoside reverse transcriptase inhibitors (NNRTI). Examples of NRTI include, but are not limited to, abacavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir, zidovudine, zalcitabine, entecavir, adefovir, elvucitabine, fosalvudine (-tidoxil), fozivudintidoxil, lagiciclovir, alamifovir, clavudine, pradefovir, telbivudine. Examples of NNRTIs include, but are not limited to, efavirenz, etravirine, nevirapine, rilpivirine, delavirdine, emivirine, lersivirine.

[0097] Suitable for combination therapy according to the invention are integrase inhibitors such as raltegravir, elvitegravir, dolutegravir, MK-2048.

[0098] Examples of HIV protease inhibitors suitable for the combination therapy according to the invention are saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir, darunavir, brecanavir, mozenavir, tipranavir.

[0099] Examples of entry inhibitors suitable for combination therapy according to the invention are enfuvirtide and maraviroc.

[0100] In addition, general viral inhibitors suitable for the combination therapy according to the present invention can be selected from the group consisting of ancriviroc, apraviroc, cenicriviroc, enfuvirtide, maraviroc, vicriviroc, amantadine, rimantadine, pleconaril, idoxuridine, acyclovir, brivudine, famciclovir, penciclovir, sorivudine, valaciclovir, cidofovir, ganciclovir, valganciclovir, sofosbusvir, foscarnet, ribavirin, taribavirine, filibuvir, nesbuvir, tegobuvir, vir), fosdevirine, favipiravir, merimepodib, asunaprevir, balapiravir, boceprivir, ciluprevir, danoprevir, daclatasvir, narlaprevir, telaprevir, simeprevir, vanipevir, rupintrivir, remdesivir, fomivirsen, amenamevir, alisporivir, bevirimat, letermovir, laninamavir, oseltamivir, peramivir, and zanamivir.

[0101] General immunostimulants suitable for the combination therapy according to the present invention can be selected from the group consisting of interferons (α-interferon, β-interferon, γ-interferon, τ-interferon), interleukins, CSF, PDGF, EGF, IGF, THF, levamisole, dimethylaminopropanol, and inosine.

[0102] Furthermore, possible combinations according to the invention comprise adjuvants such as cobicistat.

[0103] The terms "medicine" or "medical" include human medicine and veterinary medicine.

[0104] The term "organism" refers to a living being, especially a human or an animal, having a self-regulating immune system.

[0105] For the purposes of this application, the term "host organism" is those organisms which, following infection by a virus, in particular a retrovirus herein, are utilized by said virus for replication.

[0106] The term "active agent" in the present application refers to the deubiquitinase inhibitor according to the present disclosure for use according to the present disclosure. In addition, the term may include other agents known in the prior art.

[0107] The terms "composition" and "pharmaceutical composition" include at least one deubiquitinase inhibitor according to the present disclosure in any pharmacologically appropriately defined dosage and dosage form, and at least one suitable excipient or carrier substance, as well as all substances that result directly or indirectly as a combination, accumulation, complex formation or crystal of the aforementioned components or are formed as a result of other reactions or interactions, and optionally at least one other pharmaceutical agent known in the art.

[0108] The term "excipient" is used in this application to describe the components of a pharmaceutical composition other than the active agent. The selection of a suitable excipient depends on factors such as dosage form and dosage, as well as the effect of the excipient itself on the solubility and stability of the composition.

[0109] The term "action" describes a specific mode of action inherent to each agent within the scope of this application.

[0110] The terms "effect", "therapeutic effect", "action", "therapeutic effect" with respect to at least one active agent according to the present invention refer to a causally beneficial outcome that occurs in an organism to which the at least one active agent has been administered.

[0111] For the purposes of this application, "therapeutically effective dose" means a sufficient dose of at least one deubiquitinase inhibitor according to the present disclosure to be administered to an organism or to a patient in need of such treatment.

[0112] The terms "joint administration", "combined administration" or "simultaneous administration" of at least one medicament according to the present disclosure and / or at least one medicament from the prior art include administration of the mentioned medicaments at the same time or at time points close to each other in fact as well as administration of the medicaments at different times in consecutive experiments. The temporal order of administration of the medicaments is not limited by these terms. A person skilled in the art will not have difficulty in deriving the chronological or local order of the administration from his knowledge and experience.

[0113] The term "organism" refers to every animal, especially vertebrates, including humans. For the purposes of this application, a "patient" is an organism that suffers from an identifiable and diagnosable disease and to which a suitable active agent can be administered.

[0114] The terms "prevention", "treatment" and "therapy" include administering at least one deubiquitinase inhibitor according to the present disclosure alone or in combination with at least one other agent known in the art to an organism, thereby preventing the development of a disease, inhibiting and alleviating symptoms, or initiating a healing process of the corresponding disease.

[0115] The compounds according to the present disclosure may be provided as pharmaceutically acceptable salts of organic and inorganic acids. Suitable examples are hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, oxalic acid, malonic acid, salicylic acid, p-aminosalicylic acid, malic acid, fumaric acid, succinic acid, ascorbic acid, maleic acid, sulfonic acid, phosphonic acid, perchloric acid, nitric acid, formic acid, propionic acid, gluconic acid, digluconic acid, lactic acid, tartaric acid, hydroxymaleic acid, pyruvic acid, phenylacetic acid, benzoic acid, p-aminobenzoic acid, p-hydroxybenzoic acid, dinitrobenzoic acid, chlorobenzoic acid, methanesulfonic acid, ethanesulfonic acid, nitric acid, hydroxyethanesulfonic acid, vinylsulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid, camphorsulfonic acid, alginic acid, capric acid, hippuric acid, pectic acid, phthalic acid, quinic acid, mandelic acid, o-methylmandelic acid, hydrobenzenesulfonic acid, picric acid, adipic acid, cyclopentanepropionic acid, D-o-toluoyltartaric acid, tartronic acid The salts of the present invention are salts of 1,2-dimethyl- ...

[0116] For the purposes of this application, a pharmaceutically acceptable salt should be considered as an active agent containing a compound according to the invention in salt form, in particular if the salt imparts specific or improved pharmacokinetic properties compared to the free form of the active agent or compared to another salt of the active agent. A pharmaceutically acceptable salt of an active agent may also impart pharmacokinetic characteristics that the active agent does not have when it is in free form. Therefore, it may even positively affect the pharmacodynamics of the active agent in terms of therapeutic efficacy in an organism.

[0117] Compounds according to the present disclosure may also be provided as hydrates or solvates. For the purposes of the present application, solvates refer to forms in which compounds according to the present invention are coordinated with solvent molecules to form complexes. Hydrates are a specific form of solvates in which coordination is achieved through water molecules.

[0118] In the prevention and / or treatment of coronavirus infection, at least one deubiquitinase inhibitor according to the present disclosure or a drug combination according to the present disclosure can be applied to a patient in need by any medically acceptable route of administration. Such medically acceptable routes of administration can be, for example, by inhalation, by intubation, oral, parenteral, intraperitoneal, intravenous, intraarterial, intramuscular, topical, transdermal, subcutaneous, intradermal, sublingual, transconjunctival, intravaginal, rectal or nasal.

[0119] In another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of coronavirus infection is disclosed, wherein the composition comprises at least an inhibitor of human deubiquitinase USP7 and / or USP47 according to the present disclosure or one of its pharmaceutically acceptable salts, hydrates or solvates, a carrier and at least one pharmaceutically acceptable excipient.

[0120] The term "pharmaceutically acceptable excipient" refers to a natural or synthetic compound that is added to a pharmaceutical preparation together with a pharmaceutically active agent. They can contribute to bulking up the preparation, thereby improving the pharmacokinetic properties or stability of the required preparation and being beneficial to the manufacturing process. The excipient of the advantageous category according to the present invention comprises a carrier, a binding agent, a coloring agent, a buffer, a preservative, an antioxidant, a coating agent, a sweetener, a thickener, a pH adjusting agent, an acidity regulator, an acidulant, a solvent, an isotonic agent, a penetration enhancer, a disintegrant, a glidant, a lubricant, an emulsifier, a solubilizer, a stabilizer, a diluent, an anti-caking agent (anti-adhesive agent), an adsorbent, a foaming agent, a defoamer, a sunscreen, a fatting agent, a consistency enhancer (consistency enhancer), a hydrotrope (hydrotrope), a fragrance substance and a flavoring substance.

[0121] Typically, one or more pharmaceutically acceptable carriers are added to the pharmaceutically active agent. All carriers and combinations thereof known in the art are eligible. In solid dosage forms, they can be, for example, plant and animal fats, waxes, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicon dioxide, talc, zinc oxide. For liquid dosage forms and emulsions, suitable carriers are, for example, solvents, solubilizers, emulsifiers, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, cottonseed oil, peanut oil, olive oil, castor oil, sesame oil, fatty acid glycerides, polyethylene glycol, fatty acid esters of sorbitan. Suspensions according to the present invention can use carriers known in the art, such as diluents (e.g., water, ethanol or propylene glycol), ethoxylated isostearyl alcohol, polyoxyethylene and polyoxyethylene sorbitan esters, microcrystalline cellulose, bentonite, agar, tragacanth gum.

[0122] The term binder refers to substances that bind powders or glue them together, making them cohesive by granule formation. They act as the "glue" of the formulation. Binders add cohesive strength to the diluent or filler provided.

[0123] Suitable binders are, for example, starch from wheat, corn, rice or potato, gelatin, naturally occurring sugars such as glucose, sucrose or beta-lactose, sweeteners from corn, natural and synthetic gums such as gum arabic, gum tragacanth, calcium ammonium alginate, sodium alginate, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylcarboxymethylcellulose, polyethylene glycol, polyvinylpyrrolidone, magnesium aluminum silicate, waxes and other substances. The percentage of binder in the composition can range from 1% to 30% by weight, preferably from 2% to 20% by weight, more preferably from 3% to 10% by weight and most preferably from 3% to 6% by weight.

[0124] Coloring agent is the excipient that gives coloration to pharmaceutical preparation.These excipients can be food coloring agents.They can be adsorbed on suitable adsorption means (adsorption means) for example clay or aluminum oxide.Other advantages of coloring agent are that it can make the aqueous solution overflowing on atomizer and / or mouthpiece (mouthpiece) visualized for the convenience of cleaning.The amount of coloring agent can be between 0.01% by weight and 10% by weight of the pharmaceutical composition, preferably between 0.05% by weight and 6% by weight, more preferably between 0.1% by weight and 4% by weight, most preferably between 0.1% by weight and 1% by weight.

[0125] Suitable pharmaceutical colorants are, for example, curcumin, riboflavin, riboflavin-5'-phosphate, tartrazine, alkannin, quinoline yellow WS, fast yellow AB, riboflavin-5'-phosphate sodium, yellow 2G, sunset yellow FCF, orange GGN, cochineal, carminic acid, citrus red 2, azo carmoisine, amaranth, Ponceau 4R, Ponceau SX, Ponceau 6R, erythrosine, red 2G, allura red AC, indanthrene blue RS, patent blue V, indigo carmine, brilliant blue FCF, chlorophyll and chlorophyllin, copper complex of chlorophyll and chlorophyllin, green S, fast green FCF, plain caramel, caustic sulphite caramel, ammonia caramel, sulphite ammonia caramel, caramel), black PN, carbon black, vegetable carbon, brown FK, brown HT, α-carotene, β-carotene, γ-carotene, annatto, bixin, norbixin, paprika oleoresin), capsanthin, capsorubin, lycopene, β-apo-8'-carotenal, β-apo-8'-carotene ethyl ester, flavoxanthin, lutein, cryptoxanthin, rubixanthin, violaxanthin, rhodoxanthin, canthaxanthin, zeaxanthin, citranaxanthin, astaxanthin, betanin, anthocyanins, saffron, calcium carbonate, titanium dioxide, iron oxide, iron hydroxide, aluminum, silver, gold, pigment rubine, tannic acid, orcein, ferrous gluconate, ferrous lactate.

[0126] In addition, buffer solutions are preferred for liquid formulations, especially for pharmaceutical liquid formulations. The terms buffer, buffer system and buffer solution, especially aqueous solution, refer to the ability of the system to resist pH changes by adding acid or base or by diluting with a solvent. Preferred buffer systems can be selected from the group comprising: formate, lactate, benzoic acid, oxalate, fumarate, aniline, acetate buffer, citrate buffer, glutamate buffer, phosphate buffer, succinate, pyridine, phthalate, histidine, MES (2-(N-morpholino)ethanesulfonic acid), maleic acid, cacodylate (dimethylarsenate), carbonic acid, ADA (N-(2-acetamido)iminodiacetic acid, PI PES (4-piperazine-bis-ethanesulfonic acid), BIS-TRIS propane (1,3-bis[tris(hydroxymethyl)methylamino]propane), ethylenediamine, ACES (2-[(amino-2-oxoethyl)amino]ethanesulfonic acid), imidazole, MOPS (3-(N-morpholinoline)propanesulfonic acid), diethylmalonic acid, TES (2-[tris(hydroxymethyl)methyl]aminoethanesulfonic acid), HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), and other pK a Buffer between 3.8 and 7.7.

[0127] Preferred are carbonate buffers such as acetate buffer, and dicarboxylic acid buffers such as fumarate, tartrate and phthalate, and tricarboxylic acid buffers such as citrate.

[0128] Another group of preferred buffers are inorganic buffers, such as sulfate hydroxides, borate hydroxides, carbonate hydroxides, oxalate hydroxides, calcium hydroxide and phosphate buffers. Another group of preferred buffers are nitrogen-containing buffers, such as imidazole, diethylenediamine and piperazine. In addition, preferred sulfonic acid buffers are TES, HEPES, ACES, PIPES, [(2-hydroxy-1,1-bis-(hydroxymethyl)ethyl)amino]-1-propanesulfonic acid (TAPS), 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EEPS), MOPS and N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES). Another group of preferred buffers are glycine, glycyl-glycine, glycyl-glycyl-glycine, N,N-bis-(2-hydroxyethyl)glycine and N-[2-hydroxy-1,1-bis-(hydroxymethyl)ethyl]glycine (tricine). Also preferred are amino acid buffers, such as glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, cysteine, methionine, proline, 4-hydroxyproline, N,N,N-trimethyllysine, 3-methylhistidine, 5-hydroxy-lysine, o-phosphoserine, γ-carboxyglutamate, ε-N-acetyllysine, ω-N-methylarginine, citrulline, ornithine, and derivatives thereof.

[0129] Preservatives for liquid dosage forms and / or solid dosage forms can be used as needed. They can be selected from, but not limited to, the group consisting of: sorbic acid, potassium sorbate, sodium sorbate, calcium sorbate, methyl paraben, ethyl paraben, methyl ethyl paraben, propyl paraben, benzoic acid, sodium benzoate, potassium benzoate, calcium benzoate, heptyl paraben, sodium methyl paraben, sodium ethyl paraben, sodium propyl paraben, benzyl alcohol, benzalkonium chloride, phenethyl alcohol, cresol, cetylpyridinium chloride, chlorobutanol, thimerosal (sodium 2-(ethylmercuric sulfide) benzoate), sulfur dioxide, sodium sulfite, sodium bisulfite, sodium pyrosulfite, potassium pyrosulfite, potassium sulfite, calcium sulfite, calcium bisulfite, potassium bisulfite, biphenyl, o-phenylphenol, sodium o-phenylphenol, thiobenzene, azoles, nisin, natamycin, formic acid, sodium formate, calcium formate, hexamine, formaldehyde, dimethyl dicarbonate, potassium nitrite, sodium nitrite, sodium nitrate, potassium nitrate, acetic acid, potassium acetate, sodium acetate, sodium diacetate, calcium acetate, ammonium acetate, dehydroacetic acid, sodium dehydroacetate, lactic acid, propionic acid, sodium propionate, calcium propionate, potassium propionate, boric acid, sodium tetraborate, carbon dioxide, malic acid, fumaric acid, lysozyme, copper (II) sulfate, chlorine, chlorine dioxide, and other suitable substances or combinations known to those skilled in the art.

[0130] For liquid dosage forms and topical dosage forms, it is particularly preferred to add a sufficient amount of antioxidant. Suitable examples of antioxidants include sodium pyrosulfite, alpha-tocopherol, ascorbic acid, maleic acid, sodium ascorbate, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, fumaric acid or propyl gallate. Sodium pyrosulfite, alpha-tocopherol and ascorbyl palmitate are preferably used.

[0131] Tablets or pills are usually coated, that is, the coating constitutes the outer layer. This can be a film coating, a sugar coating with sugars and a compression coating. Pharmaceutically acceptable varnishes or waxes, HPMC (hydroxypropyl methylcellulose), MC (methylcellulose) or HPC (hydroxypropyl cellulose) can be used. Such coatings can help to mask the taste, facilitate swallowing or identification. Usually, plasticizers and pigments are included in the coating. Capsules usually have a gelatinous coating that encapsulates the active substance. The specific composition and thickness of this gelatinous layer determine the speed at which absorption occurs after ingestion of the capsule. Of particular interest are sustained-release preparations known in the art.

[0132] Suitable sweeteners may be selected from the group consisting of mannitol, glycerol, acesulfame potassium, aspartame, cyclamate, isomalt, isomalt, saccharin and its sodium, potassium and calcium salts, sucralose, alitame, thaumatin, glycyrrhizin, neohesperidin dihydrochalcone, steviol glycosides, neotame, aspartame-acesulfame salt, maltitol, maltitol syrup, lactitol, xylitol, erythritol.

[0133] Suitable thickeners may be selected from, but are not limited to, the group comprising polyvinylpyrrolidone, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, dextrin, polydextrose, modified starch, alkali-modified starch, bleached starch, oxidized starch, enzyme-treated starch, monostarch phosphate, distarch phosphate esterified with sodium trimetaphosphate or phosphorus oxychloride, phosphorylated distarch phosphate, acetylated distarch phosphate, starch acetate esterified with acetic anhydride, starch acetate esterified with vinyl acetate, acetylated distarch adipate, acetylated distarch glyceride, distarch glyceride, hydroxypropyl starch, hydroxypropyl distarch glyceride, hydroxypropyl distarch phosphate, hydroxypropyl distarch glyceride, sodium starch octenylsuccinate, acetylated oxidized starch, and hydroxyethylcellulose.

[0134] Suitable pH adjusting agents for liquid dosage forms are, for example, sodium hydroxide, hydrochloric acid, buffer substances such as sodium dihydrogen phosphate or disodium hydrogen phosphate.

[0135] Suitable acidity regulators may be selected from the group consisting of acetic acid, potassium acetate, sodium acetate, sodium diacetate, calcium acetate, carbon dioxide, malic acid, fumaric acid, sodium lactate, potassium lactate, calcium lactate, ammonium lactate, magnesium lactate, citric acid, monosodium citrate, disodium citrate, trisodium citrate, monopotassium citrate, dipotassium citrate, tripotassium citrate, monocalcium citrate, dicalcium citrate, tricalcium citrate, tartaric acid, monosodium tartrate, disodium tartrate, monopotassium tartrate, dipotassium tartrate, sodium potassium tartrate, orthophosphoric acid, lecithin citrate, citrate), magnesium citrate, ammonium malate, sodium malate, sodium hydrogen malate, calcium malate, calcium hydrogen malate, adipic acid, sodium adipate, potassium adipate, ammonium adipate, succinic acid, sodium fumarate, potassium fumarate, calcium fumarate, ammonium fumarate, 1,4-heptonolactone, triammonium citrate, ammonium ferric citrate, calcium glycerophosphate, isopropyl citrate, potassium carbonate, potassium bicarbonate, ammonium carbonate, ammonium bicarbonate, magnesium carbonate, magnesium bicarbonate, ferrous carbonate, ammonium sulfate, potassium aluminum sulfate, ammonium aluminum sulfate, sodium hydroxide, potassium hydroxide, ammonium hydroxide, magnesium hydroxide, gluconic acid.

[0136] The acidifying agents used are inorganic chemicals which generate or become acids. Suitable examples are: ammonium chloride, calcium chloride.

[0137] Suitable solvents may be selected from, but not limited to, the group comprising: water, carbonated water, water for injection, water with isotonicity, saline, isotonic saline, alcohols, in particular ethanol and n-butanol, and mixtures thereof.

[0138] Suitable isotonic agents are, for example, pharmaceutically acceptable salts, in particular sodium chloride and potassium chloride, sugars such as glucose or lactose, sugar alcohols such as mannitol and sorbitol, citrates, phosphates, borates, and mixtures thereof.

[0139] A penetration enhancer (permeation enhancer or permeability enhancer) is a substance that temporarily weakens the skin barrier and promotes or accelerates the absorption of a cosmetic agent. Suitable penetration enhancers may be selected from, but are not limited to, the group comprising: dimethyl isosorbide Dimethyl sulfoxide (DMSO) and its analogs, dimethylformamide (DMF), azone (1-dodecylazacycloheptan-2-one), pyrrolidones such as 2-pyrrolidone, fatty acids such as oleic acid, lauric acid, myristic acid and capric acid, nonionic surfactants such as polyoxyethylene-2-oleyl ether and polyoxyethylene-2-stearyl ether, terpenes, terpenoids and sesquiterpenes such as those from the essential oils of eucalyptus, chenopodium and ylang-ylang, oxazolidinones such as 4-decyloxazolidin-2-one, turpentine, pine oil, menthol.

[0140] Suitable disintegrants may be selected from the group comprising starch, cold water soluble starches such as carboxymethyl starch, cellulose derivatives such as methyl cellulose and sodium carboxymethyl cellulose, microcrystalline cellulose and cross-linked microcrystalline cellulose such as cross-linked sodium carboxymethyl cellulose, natural and synthetic gums such as guar gum, agar, karaya (Indian gum tragacanth), locust bean gum, tragacanth, clays such as bentonite, xanthan gum, alginates such as alginic acid and sodium alginate, foaming compositions. Moisture expansion is supported by, for example, starch, cellulose derivatives, alginates, polysaccharides, dextran, cross-linked polyvinyl pyrrolidone. The amount of disintegrant in the composition may vary between 1% and 40% by weight, preferably between 3% and 20% by weight, most preferably between 5% and 10% by weight.

[0141] Glidants are materials that prevent caking of the supplements and improve the flow characteristics of the particles so that the flow is smooth and constant. Suitable glidants include silicon dioxide, magnesium stearate, sodium stearate, starch and talc. The amount of glidant in the composition can vary between 0.01% and 10% by weight, preferably between 0.1% and 7% by weight, more preferably between 0.2% and 5% by weight, and most preferably between 0.5% and 2% by weight.

[0142] The term "lubricant" refers to a substance added to the dosage form to promote the demoulding of tablets, particles, etc. from a compression mold or an outlet nozzle. They reduce friction or wear. Lubricants are usually added shortly before compression because they should be present on the surface of the particles and between the particles and the compression mold parts. The amount of the lubricant in the composition can be between 0.05% by weight and 15% by weight, preferably between 0.2% by weight and 5% by weight, more preferably between 0.3% by weight and 3% by weight, most preferably between 0.3% by weight and 1.5% by weight. Suitable lubricants are sodium oleate, metal stearates such as sodium stearate, calcium stearate, potassium stearate and magnesium stearate, stearic acid, sodium benzoate, sodium acetate, sodium chloride, boric acid, wax with a high melting point, polyethylene glycol.

[0143] The emulsifier can be selected, for example, from the following anionic emulsifiers and nonionic emulsifiers: anionic emulsifier waxes, cetyl alcohol, cetyl stearyl alcohol, stearic acid, oleic acid, polyoxyethylene polyoxypropylene block polymers, addition products of 2 to 60 mol of ethylene oxide to castor oil and / or hardened castor oil, wool wax oil (lanolin), sorbitan esters, polyoxyethylene alkyl esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene sorbitan monolaurate esters, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan tristearate, polyoxyethylene stearate, polyvinyl alcohol, metatartaric acid, calcium tartrate, alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, propane-1,2-diol alginate, carrageenan, processed eucheuma seaweed, locust bean gum, tragacanth gum, gum arabic, karaya gum, gellan gum, gum ghatti, glucomannane, pectin, amidated pectin, ammonium phosphatide, brominated vegetable oil, sucrose acetate isobutyrate acetateisobutyrate), glyceryl esters of wood rosin, disodium phosphate, trisodium diphosphate, tetrasodium diphosphate, dicalcium diphosphate, monocalcium dihydrogen diphosphate, trisodium phosphate, pentapotassium triphosphate, sodium polyphosphate, sodium calcium polyphosphate, calcium polyphosphate, ammonium polyphosphate, beta-cyclodextrin, powdered cellulose, methylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, ethylhydroxyethylcellulose, cross-linked carboxymethylcellulose, enzymatically hydrolyzed carboxymethylcellulose, mono- and diglycerides of fatty acids, glyceryl monostearate, glycerol Distearate, acetic acid esters of monoglycerides and diglycerides of fatty acids, lactic acid esters of monoglycerides and diglycerides of fatty acids, citric acid esters of monoglycerides and diglycerides of fatty acids, tartaric acid esters of monoglycerides and diglycerides of fatty acids, monoacetyl tartaric acid esters and diacetyl tartaric acid esters of monoglycerides and diglycerides of fatty acids, mixed acetate and tartaric acid esters of monoglycerides and diglycerides of fatty acids, succinylated monoglycerides, sucrose esters of fatty acids, sucroglyceride, polyglycerol esters of fatty acids, polyglycerol polyricinoleate, propane-1,2-diol esters, propylene glycol esters of fatty acids, lactic acid esters of glycerol and propane-1, thermally oxidized soybean oil interacting with mono- and diglycerides of fatty acids, sodium dioctyl sulfosuccinate, sodium stearoyl-2-lactylate, calcium stearoyl-2-lactylate, stearyl tartrate, stearyl citrate, sodium stearoyl fumarate, calcium stearoyl fumarate, stearyl tartrate, stearyl citrate, sodium stearoyl fumarate, calcium stearoyl fumarate, laurel Sodium sulphate, ethoxylated mono- and diglycerides, methyl glucoside-coco esters, sorbitan monostearate, sorbitan tristearate, sorbitan monolaurate, dehydrated sorbitan monooleate, sorbitan monopalmitate, sorbitan trioleate, sodium calcium polyphosphate, calcium polyphosphate, ammonium polyphosphate, bile acid, choline salts, distarch glyceride, sodium starch glycolate, acetylated oxidized starch. Preferred are monoolein, stearic acid, phospholipids such as lecithin.

[0144] Suitable surface-active solubilizing agents (solubilizers) are, for example, diethylene glycol monoethyl ester, polyethylene glycol polypropylene glycol co-polymer, cyclodextrins, such as α-cyclodextrin and β-cyclodextrin, glyceryl monostearate, such as Solutol HS15 (polyethylene glycol-15-hydroxystearate, PEG 660-15 hydroxystearate from BASF), sorbitan esters, polyoxyethylene glycol, polyoxyethylene sorbitan esters, polyoxyethylene sorbitan monooleate, polyoxyethyleneoxystearic acid triglyceride, polyvinyl alcohol, sodium lauryl sulfate, (anionic) glyceryl monooleate.

[0145] Stabilizer is a material that can be added to prevent unwanted changes. Although stabilizer is not a true emulsifier, they can also contribute to the stability of the emulsion. Suitable examples of stabilizer are oxidized stearin (oxystearin), xanthan gum, agar, oat gum, guar gum, tara gum, polyoxyethylene stearate, aspartame-acesulfame salt, amylase, protease, papain, bromelain, ficin, invertase, polydextrose, polyvinyl pyrrolidone, polyvinylpolypyrrolidone, triethyl citrate, maltitol, maltitol syrup.

[0146] Diluents or fillers are inactive substances added to a drug to cope with a minimum amount of active agent. Examples of suitable diluents are water, mannitol, pregelatinized starch, starch, microcrystalline cellulose, powdered cellulose, silicified microcrystalline cellulose, dibasic calcium phosphate dihydrate, calcium phosphate, calcium carbonate, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, polyethylene glycol, xanthan gum, gum arabic, or any combination thereof.

[0147] Anti-caking agents (anti-adherents) may be added to the supplement or supplement composition to prevent the formation of lumps and facilitate packaging, shipping, release from at least one chamber of a dispensing cap, and consumption. Suitable examples include tricalcium phosphate, powdered cellulose, magnesium stearate, sodium bicarbonate, sodium ferrocyanide, potassium ferrocyanide, calcium ferrocyanide, bone phosphate, sodium silicate, silicon dioxide, calcium silicate, magnesium trisilicate, talc, sodium aluminosilicate, potassium aluminum silicate, calcium aluminosilicate, bentonite, aluminum silicate, stearic acid, polydimethylsiloxane.

[0148] Adsorbents are substances that absorb oil from water. Suitable examples include natural adsorbents such as peat moss, sawdust, feathers and any other natural substance containing carbon as well as synthetic adsorbents such as polyethylene and nylon. Adsorbents are used to protect tablets / capsules from moisture by limiting fluid absorption (absorbing liquids or gases by adsorption or by absorption) in a dry state.

[0149] In some galenic formulations, it may be desirable for the liquid oral dosage form to produce some foam when dissolved. Such effects can be supported by adding foaming agents that reduce the surface tension of the liquid to promote the formation of bubbles or that increase its colloid stability by inhibiting the coalescence of bubbles. Alternatively, it can stabilize the foam. Suitable examples include mineral oil, quillaia extract, triethyl citrate, sodium lauryl ether sulfate, sodium lauryl sulfate, ammonium lauryl sulfate.

[0150] Alternatively, some liquid oral dosage forms may exhibit slight foaming during preparation. While this does not interfere with the desired application, it may affect patient compliance in the case of medication or commercial success in the case of dietary supplements. Therefore, it may be desirable to add a pharmaceutically acceptable anti-foaming agent (defoaming agent). Examples are polydimethylsiloxane or silicone oil in dietary supplements or dimethicone in pharmaceuticals.

[0151] Sunscreens are substances that, if desired, render the liquid dosage form opaque. They must have a refractive index that is substantially different from that of the solvent (water in most cases). At the same time, they should be inert to the other components of the composition. Suitable examples include titanium dioxide, talc, calcium carbonate, behenic acid, cetyl alcohol, or mixtures thereof.

[0152] Suitable fatliquors are, for example, decyl oleate, hydrated castor oil, light mineral oil, mineral oil, polyethylene glycols, sodium lauryl sulfate.

[0153] Consistency enhancers are, for example, cetyl alcohol, cetyl esters wax, hydrated castor oil, microcrystalline wax, nonionic emulsifier wax, beeswax, paraffin or stearyl alcohol.

[0154] Suitable hydrotropes are alcohols such as ethanol, isopropanol or polyols such as glycerol.

[0155] Suitable aromatic and flavoring substances include, first of all, essential oils which can be used for this purpose. Usually, the term refers to volatile extracts from plants or parts of plants with a corresponding characteristic odor. They can be extracted from plants or parts of plants by steam distillation.

[0156] Suitable examples are essential oils or aromatic substances from sage, clove, chamomile, fennel, star anise, thyme, tea tree, mint, peppermint oil, menthol, eucalyptus, borneol, ginger oil, eucalyptus oil, mango, fig, lavender oil, chamomile flowers, pine needles, cypress, citrus, rosewood, plum, red currant, cherry, birch leaves, cinnamon, lime, grapefruit, orange, juniper, valerian, lemon balm, lemongrass, palmarosa, cranberry, pomegranate, rosemary, ginger, pineapple, guava, echinacea, ivy leaf extract, blueberry, persimmon, melon, etc. or mixtures thereof, as well as menthol, mint and star anise oils or mixtures of menthol and cherry essence.

[0157] These aromatic substances or flavoring substances may be contained in a range of 0.0001% to 10% by weight (particularly in the composition), preferably 0.001% to 6% by weight, more preferably 0.001% to 4% by weight, and most preferably 0.01% to 1% by weight relative to the entire composition. Depending on the purpose or individual case, it may be advantageous to use different amounts.

[0158] According to the present disclosure, all the aforementioned excipients and classes of excipients may be used alone or in any conceivable combination thereof without limitation, as long as the use of the present invention is not hindered, toxic effects occur, or national regulations are violated.

[0159] In another aspect of the present invention, the present application relates to a deubiquitinase inhibitor according to the present disclosure or to a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a preparation for oral administration.

[0160] Likewise, it relates to a pharmaceutical composition for use as described above, wherein the pharmaceutical composition is a preparation for oral administration.

[0161] In another aspect of the present invention, the present disclosure relates to a deubiquitinase inhibitor according to the present disclosure or to a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a preparation for inhalation administration.

[0162] Likewise, it relates to a pharmaceutical composition for use as described above, wherein the pharmaceutical composition is a preparation for administration by inhalation.

[0163] For effective preventive or therapeutic treatment of coronavirus infection that may cause pneumonia, pulmonary edema and / or acute lung injury, it is advantageous that at least one deubiquitinase inhibitor according to the present disclosure reaches the patient's alveoli. Therefore, the particle size must be small enough to reach the lowest part of the airway of the lung tissue. The best inhalation device category for inhalation application of pharmaceutical active agents is the aforementioned so-called mesh nebulizer. Within the scope of this application, almost all mesh nebulizers known in the art can be used, from fairly simple disposable mesh nebulizers for coughs and colds or for fancy purposes to complex high-end mesh nebulizers for clinical treatment or home treatment of severe diseases or conditions of the lower airways.

[0164] Suitable commercially available mesh nebulizers, jet nebulizers, ultrasonic nebulizers, dry powder inhalers and (pressurized) metered dose inhalers include, but are not limited to rapid、PARI LC PARI Velox and PARI Velox Junior (PARI GmbH, Starnberg, Germany), Philips Respironics I-neb and Philips InnoSpire Go (Koninklijke Philips NV, Eindhoven, Netherlands), dose + mesh nebulizer inhalation MN-300 / 8, M-Neb Flow+ and Mesh nebulizer MN-300 / X (NEBU-TEC, Eisenfeld, Germany), Hcmed Deepro HCM-86C and HCM860 (HCmed Innovations Co., Ltd, Taipei, Taiwan, China), OMRON MicroAir U22 and U100 (OMRON, Kyoto, Japan), Solo, Ultra and PRO (Aerogen, Galway, Ireland), KTMED NePlus NE-SM1 (KTMED Inc., Seoul, South Korea), Vectura Bayer Breelib TM (Bayer AG, Leverkusen, Germany), Vectura Fox, MPV Truma and Smarty (MPV MEDICAL GmbH, Kirchheim, Germany), MOBI MESH (APEX Medical, New Taipei City, Taiwan, China), B.Well WN-114, TH-134 and TH-135 (B.Well Swiss AG, Widnau, Switzerland), Babybelle Asia BBU01 (Babybelle Asia Ltd., Hongkong, China), CA-MI Kiwi and others (CA-MI sri, Langhirano, Italy), DiagnosisPRO MESH (Diagnosis S.A., Poland), DIGI O 2 (DigiO 2International Co.,Ltd., New Taipei City, Taiwan, China), feellife AIR PLUS, AEROCENTRE+, AIR 360+, AIRGARDEN, AIRICU, AIR MASK, AIRGEL BOY, AIR ANGEL, AIRGEL GIRL and AIR PRO 4 (FeellifeHealth Inc., Shenzhen, China), Hannox MA-02 (Hannox International Corp., Taipei, Taiwan, China), Health and Life HL100 and HL100A (HEALTH&LIFE Co.,Ltd., New TaipeiCity, Taiwan, China), Honsun NB-810B (Honsun Co.,Ltd., Nantong, China), KN-9100 (K-jump Health Co.,Ltd., New Taipei City, Taiwan, China), microlife NEB-800 (Microlife AG, Widnau, Switzerland), OK Biotech Docspray (OK Biotech Co.,Ltd., Hsinchu City, Taiwan, China), Prodigy (Prodigy Diabetes Care, LLC, Charlotte, USA), Quatek NM211, NE203, NE320 and NE403 (Big Eagle Holding Ltd., Taipei, Taiwan, China), Simzo NBM-1 and NBM-2 (Simzo Electronic Technology Ltd., Dongguan, China), BBU01 and BBU02 (Tai Yu International Manufactory Ltd., Dongguan, China), TaiDoc TD-7001 (TaiDoc Technology Co., New Taipei City, Taiwan, China), and HIFLO Miniheart Circulaire II (Westmed Medical Group, Purchase, USA), KEJIAN (Xuzhou Kejian Hi-Tech Co., Ltd., Xuzhou, China), YM-252, P&S-T45 and P&S-360 (TEKCELEO, Valbonne, France), Maxwell YS-31 (Maxwell India, Jaipur, India), JLN-MB001 (Kernmed, Durmersheim, Germany).

[0165] Preference is given to mesh atomizers with piezoelectric activation of the atomization process or vibrating mesh atomizers.

[0166] Mesh nebulizers can be divided into two groups based on patient interaction: continuous mode devices and trigger activated devices. In continuous mode mesh nebulizers, the aerosolized aerosol is continuously released into the mouthpiece and the patient has to inhale the provided aerosol. In trigger activated devices, a certain amount of aerosol is released only during active and deep breathing. In this way, a larger amount of aerosol containing the active agent is inhaled and reaches the lowest airway compared to using a continuous mode device. Continuous mode devices cause a large amount of aerosol containing the active agent to be lost to the surrounding environment or on the passage of the upper airway because the aerosol release is not connected to the breathing cycle.

[0167] Therefore, a trigger activated mesh nebulizer is preferred, in particular a vibrating mesh nebulizer.

[0168] Particularly preferred are mesh atomizers with piezoelectric activation of the atomization process.

[0169] The preferred one is the mesh atomizer model PARI rapid, Philips Respironics I-neb, Philips InnoSpire Go, dose + mesh nebulizer inhalation MN-300 / 8, HcmedDeepro HCM-86C and HCM860, OMRON MicroAir U100, Solo, KTMED NePlus NE-SM1, Vectura Fox, Vectura Bayer Breelib TM .

[0170] The most preferred vibrating mesh atomizer models are high-end models such as PARI rapid, PARI Velox, Philips Respironics I-neb, dose + mesh nebulizer inhalation MN-300 / 8、 Solo, Vectura Fox, Vectura Bayer Breelib TM .

[0171] Therefore, in another aspect of the present invention, the present disclosure relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a preparation for inhalation administration, wherein the inhalation administration is carried out with the aid of a vibrating mesh nebulizer.

[0172] The invention also relates to a pharmaceutical composition for inhalation administration as described above, wherein the inhalation administration is performed by means of a vibrating mesh nebulizer.

[0173] The average droplet size is usually characterized by MMAD (median mass aerodynamic diameter). The single droplet size is called MAD (mass aerodynamic diameter). This value represents the diameter of the atomized particles (droplets), where 50% are smaller or larger, respectively. Particles with MMAD>10 μM usually do not reach the lower airway, and they often get stuck in the throat. Particles with MMAD>5 μM and <10 μM usually reach the bronchi instead of the alveoli. Particles with MMAD between 100nm and 1 μM will not be deposited in the alveoli and will be exhaled immediately. Therefore, the optimal range is MMAD between 1 μM and 5 μM. Recent publications even support a narrower range between 3.0 μM and 4.0 μM (see Amirav et al. (2010) J Allergy Clin Immunol 25: 1206-1211; Haidl et al. (2012) Pneumologie 66: 356-360).

[0174] Another generally accepted quality parameter is the percentage of particles with diameters in the range of 1 μM to 5 μM in the generated aerosol (FPM; fine particle mass). FPM is a measure of particle distribution. It is calculated by subtracting the percentage of particles with diameters in the generated aerosol <1 μM from the total percentage of particles with diameters in the generated aerosol <5 μM (FPF; fine particle fraction).

[0175] In another aspect of the present invention, the present application also relates to a method for producing an aerosol according to the present invention, comprising the following steps:

[0176] a) filling 0.1 ml to 5 ml of an aqueous solution containing at least one deubiquitinase inhibitor according to the present disclosure and optionally at least one pharmaceutically acceptable excipient into a nebulization chamber of a mesh nebulizer,

[0177] b) activating the vibration of the mesh of the mesh atomizer at a frequency of 80 kHz to 200 kHz, and

[0178] c) Expelling the generated aerosol on the side of the mesh of the mesh nebulizer opposite to the nebulization chamber.

[0179] The vibration frequency of the vibrating mesh nebulizer is typically in the range of 80 kHz to 200 kHz, preferably 90 kHz to 180 kHz, more preferably 100 kHz to 160 kHz, most preferably 105 kHz to 130 kHz (see Chen, The Aerosol Society: DDL 2019; Gardenshire et al. (2017) A Guide to Aerosol Delivery Devices for Respiratory Therapists, 4th edition).

[0180] Therefore, the aforementioned method also discloses the vibration frequency range.

[0181] Therefore, the method according to the invention is characterized in that at least 80 wt.-%, preferably at least 85 wt.-%, most preferably at least 90 wt.-% of the at least one deubiquitinase inhibitor according to the disclosure contained in the aqueous solution is atomized in the generated aerosol.

[0182] The method of the present invention is particularly effective in atomizing a high percentage of the pharmaceutically active agent from the provided aqueous solution in a short period of time. This is an important feature for patient compliance. A considerable percentage of the patient population finds the inhalation process uncomfortable, tiring and physically demanding. On the other hand, the active cooperation of the patient is essential for effective and targeted inhalation applications. Therefore, it is desirable to apply a therapeutically sufficient amount in the shortest possible time period. Surprisingly, it was shown that 95% of the substance provided in the aqueous solution could be atomized within a time span of three minutes. This is an ideal time span for high patient compliance.

[0183] The method according to the invention is therefore characterised in that at least 80%, preferably at least 85% and most preferably at least 90% of the generated aerosol is produced during three minutes after initiation of nebulisation in the mesh nebuliser.

[0184] Although the pharmaceutically active agent is usually provided in a single-dose container for each nebulization procedure, the nebulizer and / or the mouthpiece can be used within a specific time period and must be replaced at specific intervals. By default, it is recommended to clean the nebulizer and mouthpiece after each nebulization. However, patient compliance cannot be reasonably taken for granted here. However, even after careful cleaning, there will always be some aerosol deposits in the nebulization chamber, outlet and / or mouthpiece. Since the aerosol is produced by an aqueous solution, these deposits have the risk of producing bacterial bioburden that may contaminate the inhaled aerosol. The deposits may also clog the holes in the mesh membrane of the mesh nebulizer. Typically, the nebulizer and / or mouthpiece should be replaced every week or every two weeks. Therefore, it is convenient to provide the drug and nebulizer as a combination product.

[0185] Therefore, in another aspect of the present invention, the present application also relates to a kit comprising a mesh nebulizer and a pharmaceutically acceptable container having an aqueous solution containing at least one deubiquitinase inhibitor according to the present disclosure and optionally at least one pharmaceutically acceptable excipient.

[0186] In an optional kit, at least one deubiquitinase inhibitor according to the present disclosure is not provided in the form of an aqueous solution, but is provided in two separate containers, one for the solid form of the active agent and the other for the aqueous solution. The final aqueous solution is freshly prepared by dissolving the active agent in the final solution. The final aqueous solution is then filled into the atomization chamber of the mesh nebulizer. The two containers can be completely separate containers, such as two vials, or, for example, a dual-chamber vial. In order to dissolve the active agent, for example, the membrane between the two chambers is perforated to allow mixing of the contents of the two chambers.

[0187] Therefore, the present application also discloses a kit comprising a mesh nebulizer, a first pharmaceutically acceptable container having water for injection or a physiological saline solution, and a second pharmaceutically acceptable container having at least one solid form of a deubiquitinase inhibitor according to the present disclosure, wherein optionally at least one pharmaceutically acceptable excipient is contained in the first pharmaceutically acceptable container and / or the second pharmaceutically acceptable container.

[0188] The aerosol generated by the method according to the invention is administered or self-administered by means of a mouthpiece. Optionally, such a mouthpiece may additionally be included in the aforementioned kit.

[0189] The common way of transferring the provided aqueous solution or the final aqueous solution to the atomizing chamber of the mesh nebulizer by means of a syringe equipped with an injection needle. First, the aqueous solution is sucked into the syringe and then injected into the atomizing chamber. Optionally, such a syringe and / or injection needle may be additionally included in the aforementioned kit. Without limitation, a typical syringe made of polyethylene, polypropylene or cyclic olefin copolymer may be used, and the typical specifications of a stainless steel injection needle may be in the range of 14 to 27.

[0190] In yet another aspect of the present invention, the present application relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is used in the formulation of a sublingual tablet for the prevention or treatment of coronavirus infection.

[0191] It also relates to a pharmaceutical composition for use as described above, wherein the pharmaceutical composition is a preparation for a sublingual tablet.

[0192] In yet another aspect of the present invention, the present disclosure relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is in a liquid dosage form for use in the prevention or treatment of coronavirus infection.

[0193] Likewise, the present disclosure relates to a pharmaceutical composition for use as described above, wherein the pharmaceutical composition is in liquid dosage form.

[0194] Usually, aqueous solutions or physiological saline solutions are preferred. In the case of poorly soluble agents according to the present invention, ethanol or ethanol / water mixtures may also be used.

[0195] Suitable liquid dosage forms include drops, eye drops, ear drops or injectable solutions.

[0196] Although SARS-CoV and MERS-CoV mainly infect the lower airways, SARS-CoV-2 first infects the pharynx / throat area. Only a small portion of these patients subsequently develop lung infections and pneumonia. Although these pharyngeal infections usually only cause mild symptoms like a cold or no symptoms at all, these patients are highly contagious to their environment. In most cases, they do not know that they have become spreaders of infection. Therefore, there is a medical need to treat coronavirus infection when it is still in the pharyngeal stage, not only to treat such patients, but also for epidemiological reasons, to prevent the spread of epidemics. For patients with pharyngeal infections, it is not ideal to have only systemic routes of administration such as intravenous or oral administration when using highly effective drugs or drug combinations that may also cause adverse side effects. Therefore, it is desirable to provide a route of administration for local treatment of infected pharyngeal tissues.

[0197] Therefore, in yet another aspect of the present invention, the present application relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a formulation for pharyngeal administration.

[0198] Administration to the pharynx may be achieved topically, for example by rinsing the throat / pharynx area with a suitable liquid dosage form such as drops, lotion or tincture, or with a viscous dosage form such as a gel or hydrogel, gargling with a mouthwash, sublingual tablets, lozenges, throat spray or injection into the posterior pharynx.

[0199] Lotions are low-viscosity topical preparations intended for application to the skin or mucous membranes. Apply lotions to the skin or mucous membranes with your bare hands, a brush, a clean cloth, or cotton wool.

[0200] The advantage of lotions is that they can be spread thinly and can cover a large area of ​​skin or mucous membranes. Typical drugs that can be administered in the form of lotions include antibiotics, antiseptics, antifungal agents, corticosteroids, anti-acne agents, soothing agents, emollients, moisturizers or protectants, or anti-allergens.

[0201] Most lotions are oil-in-water emulsions that use substances such as cetearyl alcohol to hold the emulsion together, but water-in-oil lotions are also formulated. The key components are the water phase and the oil phase, and an emulsifier to prevent these two phases from separating from the drug. A wide variety of excipients such as fragrances, glycerin, petrolatum, dyes, preservatives, proteins, and stabilizers are usually added to lotions.

[0202] The thickness, consistency and viscosity of the lotion can be adjusted during manufacturing. The manufacturing of the lotion can be carried out in two cycles: a) Emollients and lubricants are dispersed in the oil with compounding agents and thickeners. b) Fragrances, pigments and preservatives are dispersed in the water phase. Pharmaceutically active ingredients are broken up in both cycles, depending on the raw materials involved and the desired lotion properties.

[0203] Tinctures are usually alcoholic extracts or preparations. Solvent concentrations of 25%–60% (or even 90%) are common. Other solvents used to make tinctures include vinegar, glycerine, ether, and propylene glycol. Ethanol has the advantage of being an excellent solvent for both acidic and alkaline components. Tinctures using glycerine are called glycerites. Glycerine is generally a poorer solvent than ethanol. Acidic vinegar is a better solvent for obtaining alkaloids but a poorer solvent for acidic components.

[0204] A gel is a colloid in which a solid dispersed phase combines with a fluid continuum to form a network resulting in a viscous semi-rigid sol. The properties of gels range from soft and brittle to hard and tough. They are defined as highly diluted cross-linked systems that do not exhibit flow in the steady state. Gels are mostly liquids by weight, but they behave like solids due to the three-dimensional cross-linked network in the liquid. It is the cross-links within the fluid that give the gel its consistency and aid in adhesion. A gel is a dispersion of liquid molecules within a solid medium.

[0205] Hydrogels are networks of hydrophilic polymer chains, sometimes found as colloidal gels in which water is the dispersion medium. A three-dimensional solid is obtained due to the hydrophilic polymer chains being held together by crosslinks. Due to the intrinsic crosslinks, the structural integrity of the hydrogel network does not dissolve in high concentrations of water. Hydrogels are highly absorbent (they can contain more than 90% water) natural or synthetic polymer networks. Hydrogels also have a flexibility very similar to natural tissues due to their large water content. In medicine, hydrogels can encapsulate chemical systems that, when stimulated by external factors such as changes in pH, may result in the release of specific pharmaceutically active agents into the environment, in most cases by a gel-sol transition to a liquid state.

[0206] Suitable gel forming agents may be selected from, but not limited to, the group comprising: agar, algin, alginic acid, bentonite, carbomer, carrageenan, hectorite, hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carbomer.

[0207] A mouthwash is a liquid that is passively held in the mouth or rinsed around the oral cavity by contraction of the perioral muscles and / or movement of the head, and can be gargled, in which case the head is tilted back and the liquid bubbles in the back of the mouth. Thus, an aqueous or alcoholic solution of a deubiquitinase inhibitor according to the present disclosure can be formulated and administered to the pharynx.

[0208] Sublingual drug delivery can be an alternative when compared to oral drug delivery, because sublingually administered dosage forms bypass liver metabolism. For some drugs, especially those used to treat acute conditions, a rapid onset of pharmacological action is often desired. Sublingual tablets disintegrate rapidly, and the presence of a small amount of saliva is usually sufficient to achieve disintegration of the dosage form and better dissolution and improved bioavailability.

[0209] The drug must be lipophilic enough to be distributed through the lipid bilayer, but not so lipophilic that it will not be distributed out once it enters the lipid bilayer. According to the diffusion model of absorption, the flux through the lipid bilayer is proportional to the concentration gradient. Therefore, lower salivary solubility leads to lower absorption rate, and vice versa. Generally, drugs formulated for sublingual administration should ideally have a molecular weight of less than 500 to facilitate their diffusion. The oral cavity has a narrow pH range between 5.0 and 7.0. The inclusion of a suitable buffer during the preparation of an ionizable drug makes it possible to control the pH of aqueous saliva.

[0210] In order to avoid the potentially unpleasant taste or smell of the drug, taste masking is required. Sweeteners, flavoring agents and other taste masking agents are essential components. Sugar-based excipients dissolve quickly in saliva and produce dissolution endotherms. They produce a pleasant sensation in the mouth and are most suitable for use in sublingual tablets with other flavoring agents.

[0211] Typical techniques for manufacturing sublingual tablets include direct compression, compression molding, freeze drying, and hot melt extrusion (Khan et al. (2017) J Pharmaceut Res 16:257-267).

[0212] Administration of the pharmaceutically active agent via a sublingual tablet can also locally reach the pharynx / throat when swallowing is avoided. Absorption of pharmaceutically active agents occurs largely via the pharyngeal mucosa.

[0213] A lozenge (troche) is a small disc or diamond shaped body consisting of a solidifying paste containing an astringent, antiseptic or demulcent drug, used for topical treatment of the mouth or throat, and is held in the mouth until dissolved. The vehicle or base of the lozenge is usually sugar, made sticky by mixing with gum arabic or tragacanth, a fruit paste made of black or red currants, candied roses or balsam of tolu.

[0214] In particular, the present application relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a formulation for pharyngeal administration, wherein the pharyngeal administration is performed by means of a throat spray.

[0215] Throat sprays are liquid medicines administered into the throat as a spray, usually to treat a sore throat or cough.

[0216] Throat sprays may typically contain local anesthetics (e.g., lidocaine, benzocaine), preservatives (e.g., chlorhexidine, cetylpyridinium chloride), herbal extracts, or combinations thereof. Whatever the formulation, it should not contain excessive amounts of sugar or ethanol, which further irritate the mucous membranes. Finally, the user should not experience any unpleasant aftertaste.

[0217] Currently, the standard for throat sprays is a metering pump connected to a bottle containing 10ml to 30ml of a liquid formulation. The formulation is filled into a glass or plastic bottle, and the pump is fixed by a screw cap that is clamped on the bottleneck or simply stuck on the bottleneck. Regardless of the fixing option selected, the system should be airtight and no leakage is observed during the user's carrying or operation. Typically, the container is made of glass or plastic.

[0218] Typically, a throat spray pump will deliver a dose in the range of 50 μl to 200 μl per start. For targeted dosing, the pump will be equipped with an actuator with an extended nozzle. The nozzle length can be in the range of 30 mm to 70 mm. It is easier to target the affected area using such a long fixed nozzle, but this may be too bulky for the user to carry, which is why an actuator with a foldable or swivel mounted nozzle is preferred.

[0219] Alternatively, the device uses a continuous valve. A continuous valve delivers targeted therapy but does not provide precise dosing because the formulation will aerosolize when the actuator is pressed. One solution is a tin or aluminum can with a pressurized headspace. When the valve is activated, the increased internal pressure will squeeze the formulation out of the can as long as the valve stem is pressed.

[0220] A related but more complex system is the bag-on-valve (BOV) system. The product is placed inside the bag, while the propellant (compressed air in most cases) is filled in the space between the bag and the outer can. When the continuous valve is activated, the product is squeezed out of the bag with the help of compressed air. The BOV system will work in any 360° orientation.

[0221] Caution should be exercised as throat spray formulations may contain ingredients that are very aggressive and can reduce surface tension.A simple test of spray performance will ensure that the formulation can be aerosolized by the system and that the spray pattern and particle size provided are appropriate for the intended use.

[0222] The spray pattern and droplet size distribution are the most important parameters for throat sprays. The spray pattern is a term used to describe the spray angle and shape of the plume of a fully expanded spray. Once the spray is fully expanded, the droplet size can be characterized using laser diffraction. Fine particles (droplets with a mean dynamic diameter less than 10 μm) should be as low as possible to avoid droplet deposition in the lower airways.

[0223] Recently, some carrageenan-based throat sprays have emerged that claim to protect against viral upper respiratory tract infections. The first polymer in this platform is Broadly active antiviral compound for the treatment of respiratory diseases. In addition to its moisturizing effect, the compound prevents viruses from binding to mucosal cells.

[0224] Alternatively, a portable nebulizer with a high output rate and a regulated droplet size for deposition in the upper airway can be used. Breathing through a mask can deposit droplets on the mucosa throughout the upper airway (see Marx and Nadler (2018) Drug Development & Delivery).

[0225] In particular, the present application relates to a deubiquitinase inhibitor according to the present disclosure or a combination according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a preparation for pharyngeal administration, wherein the pharyngeal administration is performed by means of a posterior pharyngeal wall injection.

[0226] This technique is used for pharyngoplasty by injection of calcium hydroxyapatite and other methods in plastic surgery. However, local injections can also be made to the pharyngeal tissue to administer the pharmaceutically active agent. The injection solution can be roughly the same as the intravenous injection or intramuscular injection. Aqueous solutions, physiological saline solutions, or in the case of relatively lipophilic pharmaceutically active agents, ethanol / water mixtures are preferred.

[0227] In another aspect of the present invention, the present application relates to a deubiquitinase inhibitor according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a formulation for nasal administration.

[0228] In particular, nasal administration is carried out by means of nasal spray or nasal drops.

[0229] Common formulation types for nasal spray products are solutions, suspensions, and emulsions. Nasal spray formulations can be aqueous, hydroalcoholic, or non-aqueous. Depending on the type of system, the formulation will include a range of functional excipients, including solvents and cosolvents; mucoadhesives; pH buffers; antioxidants; preservatives; osmolality and tonicity agents; penetration enhancers; suspending agents; and surfactants. The choice of formulation type and the excipients selected will be determined by the solubility and stability of each deubiquitinase inhibitor according to the present disclosure and the concentration required to deliver an effective dose in a typical 100 μl spray (see Kulkarni and Shaw (2016), in Essential Chemistry for Formulators of Semisolid and Liquid Dosages, Elsevier). The aforementioned Technology is also used in nasal sprays.

[0230] Nasal drops are administered in a similar formulation, but are delivered with a drop-in rather than a pump-on dispenser.

[0231] In particular, the present application relates to a deubiquitinase inhibitor according to the present disclosure, which is used for the prevention or treatment of coronavirus infection in a preparation for nasal administration, wherein the nasal administration is performed by means of a nasal spray or nasal drops.

[0232] The ocular mucosa is known to be another entry point for SARS-CoV-2 into the organism, for example when a person rubs their eyes and carries the virus on their hands.

[0233] Therefore, the present application also relates to the deubiquitinase inhibitor according to the present disclosure, wherein the deubiquitinase inhibitor according to the present disclosure is provided in the formulation of eye drops.

[0234] Eye drops are mostly aqueous solutions containing pharmaceutically active agents. pH is usually adjusted to 7.1 to 7.5. Common buffers for eye drops are boric acid and sodium dihydrogen phosphate. Tonicity should be adjusted to an osmotic pressure (225-430mosm / kg) isotonic with the corneal epithelium by means of 0.9% saline (or another isotonic agent such as potassium nitrate, boric acid, sodium acetate, sodium acetate phosphate buffer or mannitol). Suitable preservatives include thimerosal, organic mercury compounds such as phenylmercury, benzalkonium chloride, chlorhexidine and benzyl alcohol. In order to extend the contact time, viscosity-enhancing substances (thickeners) such as cellulose derivatives (hydroxypropyl methylcellulose, methylcellulose, hydroxypropyl methylcellulose), hyaluronic acid, cellulose acetate phthalate, polyethylene glycol, polyvinyl alcohol or poloxamer can be added. Wetting agents or surfactants such as benzalkonium chloride, polysorbate 20, polysorbate 80, dioctyl sodium sulfosuccinate can be included. If desired, some amino acids alone or in combination with sodium hyaluronate may help promote tissue reconstruction. Suitable amino acids are glycine, leucine, lysine and proline (see EP 1940381B1).

[0235] Therefore, the present disclosure also relates to a pharmaceutical composition for use as described above, wherein the pharmaceutical composition is a formulation for throat spray, nasal spray or eye drops.

[0236] In another aspect of the present invention, a method of treating coronavirus infection is disclosed, wherein an effective dose of a deubiquitinase inhibitor according to the present disclosure is administered to a patient in need thereof.

[0237] Tautomerism involves the rapid internal conversion of organic compounds, in which hydrogen atoms or protons migrate formally within the compound. This is accompanied by the conversion of single bonds and adjacent double bonds. The single form is called a tautomer. Therefore, the present patent application also relates to the use of all tautomers of at least one deubiquitinase inhibitor according to the present disclosure.

[0238] Isomers are a general term for molecules with the same chemical formula but different chemical structures. They can be distinguished into structural (structural) isomers (in which an exchange of atoms or an exchange of functional groups occurs) and stereoisomers. Stereoisomers can be subdivided into enantiomers (non-superimposable mirror images of the same molecule) and diastereomers (the same molecule has different configurations at one or more stereocenters). Diastereomers can be subdivided into cis / trans isomers (referring to the relative orientation of functional groups within the molecule), and on the other hand can be subdivided into conformational isomers (rotation around a formal single bond) and rotational isomers (rotational orientation around a single bond is different). Therefore, the present patent application also relates to the use of all isomers of at least one deubiquitinase inhibitor according to the present disclosure.

[0239] For some applications, it may be desirable to use the compounds of the invention in isotopically enriched form, for example for diagnostic purposes. Therefore, the present patent application also relates to such isotopically enriched forms of the compounds of the invention.

[0240] From the perspective of pharmacokinetics or production principles, it may be preferred to use a prodrug as a dosage form. The prodrug is administered in a pharmacologically inactive form and is metabolized in vivo into an active form. The conversion can occur systemically or locally. Therefore, this patent application also relates to a prodrug of a compound of the present invention.

[0241] As used throughout this application, unless otherwise indicated, the term "at least one deubiquitinase inhibitor according to the present disclosure" shall encompass all of the aforementioned molecular variants.

[0242] Example

[0243] Example 1: PR-619 inhibits SARS-CoV-2 replication in infected Vero-B4 cells

[0244] In order to study whether PR-619 has an effect on the spread of viral infection, Western blot (WB) analysis was performed. Vero-B4 cells (Meyer et al. (2015) Emerg Infect Dis 21: 181-182) were infected with SARS-CoV-2 for 2 hours. The cells were then washed with PBS (phosphate buffered saline), and fresh culture medium containing PR-619 at a non-cytotoxic concentration (10 μM) was provided. Treatment with PR-619 was performed throughout the experiment. 3 days after infection (dpi), cells and supernatants containing viruses were harvested. Then, the cells and the virus were separated by centrifugation. Viruses were purified from the cell culture supernatant by 20% sucrose pad (sucrosecushion). The cells were washed with PBS and lysed with RIPA buffer. The protein concentration was determined by Bradford protein analysis and assimilated for each lysate. The cytoplasmic fraction of the cell lysate was denatured in SDS (sodium dodecyl sulfate) sample buffer, separated by SDS gel electrophoresis and transferred to a nitrocellulose membrane. SARS-CoV-2 was visualized using convalescent serum and a secondary reagent coupled to horseradish peroxidase by means of an electrochemiluminescent reaction. Herein, inhibition of SARS-Cov-2 replication was shown in Vero-B4 cells. At a concentration of 10 μM, in the viral fraction ( Figure 1 A) and in the cell fraction ( Figure 1B), PR-619 and 10 μM chloroquine (CQN) and 10 μM hydroxychloroquine (H-CQN), compounds known to reduce SARS-CoV-2 load, were used as positive controls.

[0245] Use analysis program Densitometric evaluation of the SARS-CoV-2 nucleoprotein in the viral fraction and the spike protein S1 in the cellular fraction was performed. Densitometric evaluation allows quantification of the signal intensity in the Western blot and thus conclusions about the amount of a certain protein in the sample. This evaluation clearly shows that after the addition of PR-619, the production of SARS-CoV-2 protein is significantly inhibited, even to a higher percentage than the same concentration of chloroquine or hydroxychloroquine ( Figure 2 ; Figure 2 A virus part, Figure 2 B cell fraction, results of 3 independent experiments).

[0246] Example 2: qRT-PCR confirmed that SARS-CoV-2 replication was inhibited in infected Vero-B4 cells

[0247] To further confirm the activity of PR-619 against SARS-CoV-2, qRT-PCR (quantitative real-time polymerase chain reaction) experiments were performed. Therefore, samples containing viruses were quantified by using the real-time PCR AgPath-ID One-Step RT-PCR kit (catalog number: 4387424) from Ambion, which allows reverse transcription, cDNA synthesis and PCR amplification in a single step. Samples were analyzed by 7500 software v2.3 (applied Bioscience). According to 44: RdRp_fwd: 5′-GTG-ARA-TGG-TCA-TGT-GTG-GCG-G-3′ and RdRp_rev5′-CAR-ATG-TTA-AAS-ACA-CTA-TTA-GCA-TA-C-3′ PCR primers were used. The probe is 5′--CAG-GTG-GAA- / ZEN / CCT-CAT-CAG-GAG-ATG-C-3′ (label: FAM / IBFQ Iowa Black FQ). As a positive control, a specific target for the E and RdRp genes of SARS-CoV2 was used, which was manufactured by Integrated DNA Technologies. Control: 5′-TAA-TAC-GAC-TCA-CTA-TAG-GGT-ATT-GAG-TGA-AAT-GGT-CAT-GTG-TGG-CGG-TTC-ACT-ATA-TGT-TAA-ACC-AGG-TGG-AAC-CTC-ATC-AGG-AGA-TGC-CAC-AAC-TGC-TTA-TGC-TAA-TAG-TGT-TTT-TAA-CAT- TTG-GAA-GAG-ACA-GGT-ACG-TTA-ATA-GTT-AAT-AGC-GTA-CTT-CTT-TTT-CTT-GCT-TTC-GTG-GTA-TTC-TTG-CTA-GTT-ACA-CTA-GCC-ATC-CTT-ACT-GCG-CTT-CGA-TTG-TGT-GCG-TAC-TGC-TGC-AAT-ATT-GTT-3′. The annealing temperature was 60°C. The experimental procedures including infection, treatment and separation of viral particles were the same as those described for Western blot analysis. The evaluation of qRT-PCR analysis clearly confirmed the results of the Western blot experiment. After the addition of PR-619, the amount of viral RNA was significantly reduced, even reaching a higher percentage than the same concentration of chloroquine or hydroxychloroquine ( Figure 3 , results of 3 independent experiments).

[0248] Example 3: PR-619 is not cytotoxic in Vero-B4 cell cultures at effective concentrations

[0249] In order to solve the problem of whether PR-619 shows cytotoxic effects in the above system, uninfected Vero-B4 cells were treated in parallel with the Western blot study, using increasing concentrations of PR-619 (2.5 μM, 5 μM, 10 μM, 20 μM). The toxicity was evaluated using the WST test. Herein, living cells with a succinate-tetrazolyl dehydrogenase system with intact mitochondria affect the enzymatic conversion of the reddish tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolyl]-1,3-benzenedisulfonate) to dark red formazan. This color change can be measured photometrically in a spectrophotometer. Therefore, the WST test is a very sensitive method for measuring the toxicity of substances to cellular metabolism.

[0250] exist Figure 4 In A (results of 3 independent experiments), the percentage of viable cells is depicted relative to untreated cells. The value of untreated cells was set to 100%. For comparison reasons, chloroquine ( Figure 4 B, Results of 3 independent experiments) and hydroxychloroquine ( Figure 4 C, results of 3 independent experiments) (2.5 μM, 5 μM, 10 μM, 20 μM, respectively). 1 μM staurosporine (an indolecarbazole compound from Streptomyces staurosporeus, an apoptosis inducer) was used as a positive control.

[0251] It could be shown that during the 3-day observation period, PR-619 did not exhibit any significant toxic effects in Vero-B4 cells at antivirally effective concentrations. Only at concentrations exceeding 20 μM did significant toxic effects occur.

[0252] Therefore, it can be said that the antiviral effect of PR-619 is not due to nonspecific cytotoxic effects.

[0253] Example 4: PR-619 directly inhibits the papain-like protease (PLpro) of SARS-CoV-2

[0254] It is known that PLpro can play the role of viral deubiquitinase. It is reported that the inhibition of the deubiquitinase encoded by the virus interferes with the replication of severe acute respiratory syndrome (SARS)-coronavirus. In addition, data show that SARS-CoV-2PLpro not only plays a role in the processing of viral proteins and viral proliferation, but also has a significant impact on innate immunity. Since the PLpro of SARS-CoV has a high homology with the protease of SARS-CoV-2, it can be assumed that both proteases have deubiquitinating functions and can therefore be targeted by inhibitors of deubiquitinase. Therefore, it is interesting to assume that PR-619 directly interacts with SARS CoV-2 potentially by blocking the activity of PLpro. In order to evaluate this hypothesis, recombinant SARS-CoV-2 PLpro and a specific substrate (Ub-like protein interferon-stimulated gene 15 (ISG-15)) conjugated with AMC are used. Following type I interferon stimulation, ISG-15 is activated and has been shown to regulate immune responses through IFN-γ and cytokine production, and thereby mediate protection against a wide variety of viruses, including influenza A and B viruses, hepatitis B and C viruses, HIV-1, and HPV-16. However, viruses have evolved countermeasures to antagonize ISG-15 to evade the innate immune response. The antagonistic activity of SARS-CoV PLpro on ISG-15 blocks the production of various cytokines involved in the activation of the innate immune response to viral infection, such as type I interferon-β (IFNb) and chemokines such as CXCL10 and CCL5.

[0255] To test the hypothesis that PR-619 directly blocks the activity of PLpro from SARS-CoV-2, recombinant PLpro (20 nM; R&D biosystems #E-611-050) was mixed with ISG15-amido-4-methylcoumarin (AMC; R&D Biosystems #UL-553-050) substrate (400 nmol) in a 96-well plate with a black bottom. Shortly afterwards, protease activity was measured using a Victor-Reader (excitation: 380 nm; emission: 460 nm) for 120 min. In the case of treatment with PR-619, PLpro was pretreated for 30 minutes. The results clearly showed that PR-619 caused a significant and dose-dependent inhibition of SARS-CoV-2 PLpro ( Figure 5 A).

[0256] To analyze the statistical relevance of this observation, the area under the curve (AUC) representing the activity of SARS-CoV-2 PLpro in each of three independently performed experiments was calculated for each concentration of PR-619 ( Figure 5B). Thus, a dose-dependent decrease in SARS-CoV-2 PLpro activity was measured after adding increasing amounts of PR-619.

[0257] Example 5: P5091 (a specific inhibitor of USP7 and USP47) blocks the replication of SARS-CoV-2

[0258] The inventors analyzed the effect of the specific USP7 / USP47 inhibitor P5091 on the replication of SARS-CoV-2. Therefore, Western blot (WB) analysis was performed according to the experimental procedure described in Example 1.

[0259] The results of these analyses clearly showed a dose-dependent reduction in viral protein release following treatment with P5091 ( Figure 6 A). Densitometric analysis of three independent experiments showed that the release of SARS-CoV-2 proteins was significantly reduced by 90% with 1.25 μM P5091 and completely blocked with 5 μM P5091 ( Figure 6 B).

[0260] Example 6: P5091 is not cytotoxic in Vero-B4 cell cultures at effective concentrations

[0261] Cytotoxicity experiments were performed according to Example 4. Within the dosage range of Example 5, P5091 did not show cytotoxic effects ( Figure 7 ). These results clearly demonstrate that USP7 or USP47 is an important cellular factor in the replication cycle of SARS-CoV-2.

[0262] Example 7: P5091 inhibits the activity of SARS-CoV-2 PLpro

[0263] Since USP7 exhibits structural similarity to PLpro from SARS and PLpro from SARS-CoV-2 has more than 90% homology with PLpro from SARS, the inventors hypothesized that P5091 also inhibits the activity of PLpro from SARS-CoV-2.

[0264] To test this hypothesis, an experiment was performed according to the experimental protocol of Example 4. Figure 8 A shows a dose-dependent decrease in the activity of SARS-CoV-2 PLpro after treatment with P5091. The calculation of AUC clearly shows that the activity of PLpro from SARS-CoV-2 was reduced by 80% after treatment with 6 μM P5091 ( Figure 8B). Therefore, it can be concluded that in addition to inhibiting cellular USP7, P5091 also has direct antiviral activity by inhibiting SARS-CoV-2 Plpro.

[0265] Attached photos

[0266] In all figures, * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001 and **** indicates p<0.0001.

[0267] Figure 1 : Western blot bands after treatment with 10 μM PR-619, 10 μM chloroquine or 10 μM hydroxychloroquine for 3 days, relative to untreated cells

[0268] A: Virus part (nucleoprotein)

[0269] B: Cell part (spike protein S1)

[0270] Figure 2 : Densitometric evaluation of viral proteins detected in Western blot bands after 3 d of treatment with 10 μM PR-619, 10 μM chloroquine or 10 μM hydroxychloroquine (n=3, respectively). Untreated cells were taken as 100%.

[0271] A: Virus part (nucleoprotein)

[0272] B: Cell part (spike protein S1)

[0273] Figure 3 : Evaluation of qRT-PCR analysis after 3-day treatment with 10 μM PR-619, 10 μM chloroquine, or 10 μM hydroxychloroquine

[0274] Figure 4 : Cell viability in WST assay after PR-619 treatment. Untreated cells were taken as 100%. Staurosporine (StS) was used as positive control (n=3, respectively).

[0275] A: PR-619

[0276] B: Chloroquine

[0277] C: Hydroxychloroquine

[0278] Figure 5 : Evaluation of the effect of PR-619 on SARS-CoV-2 PLpro (n=3, respectively)

[0279] A: Inhibition of SARS-CoV-2 PLpro. DMSO was used as a control.

[0280] B: Calculation of SARS-CoV-2 PLpro activity for each concentration

[0281] Unprocessed

[0282] 1.25μM PR-619

[0283] 2.5μM PR-619

[0284] 5μM PR-619

[0285] 10μM PR-619

[0286] DMSO

[0287] Figure 6 :Evaluation of the effects of different concentrations of P5091

[0288] A: Western blot bands after 3d treatment with P5091 (total viral protein)

[0289] B: Densitometric evaluation of viral proteins detected in Western blot bands after 3 d of treatment with P5091. Untreated cells were taken as 100%.

[0290] Figure 7 : Cell viability in WST assay after P5091 treatment. Untreated cells were taken as 100%. Staurosporine (StS) was used as a positive control (n=1).

[0291] Figure 8 : Evaluation of the effect of P5091 on SARS-CoV-2 PLpro (n=3, respectively)

[0292] A: Inhibition of SARS-CoV-2 PLpro

[0293] B: SARS-CoV-2 PLpro activity calculated for each concentration

[0294] 6μM P5091

[0295] 3μM P5091

[0296] 1.5μM P5019

[0297] Unprocessed

Claims

1. Use of inhibitors of human deubiquitinase USP7 and / or USP47 and pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of SARS-CoV-2 infection, The inhibitor of human deubiquitinase USP7 and / or USP47 and its pharmaceutically acceptable salt are 2,6-diaminopyridine-3,5-bis(thiocyanate) or 1-[5-[(2,3-dichlorophenyl)thio]-4-nitro-2-thienyl]-ethanone.

2. Use of a pharmaceutical composition comprising an inhibitor of human deubiquitinase USP7 and / or USP47 as claimed in claim 1 or one of its pharmaceutically acceptable salts, a carrier and at least one pharmaceutically acceptable excipient in the preparation of a medicament for the prevention or treatment of SARS-CoV-2 infection.

3. The use according to claim 2, wherein the pharmaceutical composition is a preparation for oral administration.

4. The use according to claim 2, wherein the pharmaceutical composition is a preparation for administration by inhalation.

5. The use according to claim 4, wherein the inhalation administration is performed by means of a vibrating mesh nebulizer. The use according to claim 2, wherein the pharmaceutical composition is in liquid dosage form.

7. The use according to claim 2, wherein the pharmaceutical composition is a preparation for a sublingual tablet.

8. The use according to claim 2, wherein the pharmaceutical composition is a preparation for throat spray, nasal spray or eye drops.

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