Nucleotide analogues for the treatment of coronaviruses

Abstract

A compound of formula I for use in the treatment of coronavirus infections and / or respiratory diseases caused by coronaviruses. TIFF2023518150000014.tif35161 (in the formula, R1 is selected from an alkoxy group (e.g., a methoxy group, an ethoxy group, etc.), a hydroxyl group, and a phosphate group; R2 is a group selected from halogen and / or hydrogen, Y is a nucleobase, preferably a purine or pyrimidine.

Description

Technical Field

[0001] The present invention particularly relates to nucleotide analogs in the treatment of viral infections, such as infections caused by MERS-CoV, SARs-CoV, or SARS-CoV-2.

Background Art

[0002] As a precursor of potential bioactive target compounds, a conjugate compound consisting of a quinazoline moiety and an adenine moiety was synthesized. The structure of 9-(4-methoxyquinazolin-2-yl)-9H-purin-6-amine (2) was characterized and reported in Non-Patent Document 1.

[0003] Coronaviruses are a group of enveloped positive-strand RNA viruses. Its genome size is about 30 kilobases in length, which is very large for an RNA virus. Other common features of this subfamily include a large replicase gene encoding several enzymatic activities and a unique replication strategy.

[0004] A typical composition of the coronavirus genome is 5'-leader-UTR-replicase-S (spike)-E (envelope)-M (membrane)-N (nucleocapsid)-3'UTR. The genome further contains a 5'-methylated cap and a 3'-polyadenylated tail, enabling it to act as mRNA for translation. The highly criticized coronavirus, namely SARS-CoV-2, is a single-stranded positive-strand RNA virus with strong infectivity in humans and is the cause of the ongoing pandemic of coronavirus disease 2019 (COVID-19), which has been designated as a public health emergency of international concern by the World Health Organization (WHO).

[0005] Infection begins when the virion attaches to the host cell. The infection starts with the interaction between the spike (S) glycoprotein of the virus and the ACE2 receptor of the host cell. Following attachment, the virus then needs to release its genome into the host cell cytosol. Release is achieved by priming of the spike glycoprotein by the serine protease TMPRSS2, which involves cleavage of the S protein. This ultimately enables fusion of the viral membrane with the cell membrane and entry of the virus into the host cell. The next step in the life cycle of the coronavirus is the translation of the replicase gene using the cell's machinery. The replicase gene is approximately 20 kilobases in length and encodes two ORFs, namely orf1a and orf1ab, each of which expresses the pp1a polyprotein and the pp1ab polyprotein. Each polyprotein contains several nsps (non-structural proteins) that exhibit various enzymatic activities. Each polyprotein is cleaved into individual nsps by the virus's own protease. The main protease of the coronavirus is Mpro, a serine-type protease. The first high-resolution crystal structure of the 3CL hydrolase (Mpro) of the COVID-19 coronavirus was determined by the research team of Zihe Rao and Haitao Yang at the University of Science and Technology of Shanghai (PDB entry 6LU7).

[0006] Many of the nsps then assemble into a replicase-transcriptase complex (RTC) that is responsible for RNA replication and transcription of the subgenomic RNAs. The nsps that include this complex are as follows: ● RNA-dependent RNA polymerase (nsp12) that mediates the synthesis of genomic RNA ● 3’→5’ exonuclease (nsp14) involved in proofreading and N7 methyltransferase activity After translation and assembly of the RTC complex, viral RNA synthesis occurs, producing both genomic RNA and subgenomic RNA. During replication, a copy of the full-length minus-strand RNA of the genome is produced by the RNA-dependent RNA polymerase and used as a template for the full-length plus-strand RNA genome. During transcription, a subset of subgenomic RNAs is produced via discontinuous transcription.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] There is a need to provide a novel compound that can be used in the treatment of patients infected with SARS-CoV-2 by disrupting viral replication through targeting the essential enzyme of RNA-dependent RNA polymerase in order to halt the spread and provide effective treatment options for patients suffering from SARS-Cov-2.

Means for Solving the Problems

[0009] The present invention provides nucleotide analogs that can be used in the treatment of diseases or respiratory diseases caused by MERS-CoV, SARS-CoV-2, or SARS-CoV.

[0010] Accordingly, the subject matter of the present invention is to provide a compound represented by formula I for use in the treatment of coronavirus infections and / or respiratory diseases caused by coronaviruses.

[0011]

Chem.

[0012] Therefore, the said compound can be used in vivo or in vitro as an inhibitor of the RNA-dependent RNA polymerase of MERS-CoV, SARS-CoV-2, or SARS-CoV.

[0013] Further embodiments of the present invention are defined in the dependent claims.

[0014] Preferred embodiments of the present invention will be described below in connection with the drawings, which are for the purpose of explaining the preferred embodiments of the present invention and not for limiting it.

Brief Description of the Drawings

[0015]

Figure 1

Modes for Carrying Out the Invention

[0016] Description of Preferred Embodiments RNA-dependent RNA polymerase (nsp12) is directly involved in the replication and transcription of the viral genomic RNA. Studies have shown that nsp12 has some activity on its own, but the addition of cofactors nsp7 and nsp8 results in a significant increase in polymerase activity. It is highly likely that additional nsp subunits are required to complete the full repertoire of viral replication. However, so far, the nsp12-nsp7-nsp8 complex is the minimal complex required for nucleotide polymerization.

[0017] Sequence analysis across coronavirus subfamily members reveals that the template entry, template-prime release, NTP tunnel, and polymerase active site are the most highly conserved surfaces on nsp12. Furthermore, seven motif regions (A-G) involved in template and nucleotide binding were found, and these regions are also conserved among coronavirus subfamilies.

[0018] The polymerase active site is composed of motifs A and C and is supported by motifs B and D. The RNA template passes through motif G before entering the active site, while the incoming NTP enters through the tunnel and interacts with motif F. Once in the active site, the NTP is likely to form hydrogen bonds with T680, N691, and D623. All three of these residues are conserved across the coronavirus family.

[0019] In SARS-CoV-2, nsp12 is 932 amino acids long and occupies positions 4395 to 5324 of pp1ab (UniProtKB id: P0C6X7). The available experimental structure of this protein does not exist as of the filing date. BLAST searching the protein sequence against the PDB revealed that the closest homolog of this protein is nsp12 of SARS-CoV. The two proteins share 96.35% sequence identity, which covers 99% of nsp12 of SARS-CoV-2. The structure of nsp12 of SARS-CoV is registered in PDB entry 6NUR.

[0020] Using the 3D structure of 6NUR as a template, homology models were created by the SWISS-MODEL web server and ICM Pro. Both are high-quality models, and a TM score of 0.99210 was achieved after superposition. The RMSD between the two models is 1.14, mostly due to gaps being inserted at different positions between the two softwares. As a result, the loops in that region have different conformations in each model. The homology model derived from SWISS-MODEL was used as the structure for docking the compounds of the present invention.

[0021] Based on the above homology modeling, the nucleotide analogs of the present invention are expected to be incorporated into newly synthesized RNA strands and cause early assembly of replication in vitro and in vivo.

[0022] The present invention provides a compound represented by formula I for use in the treatment of coronavirus infections and / or respiratory diseases caused by coronaviruses.

[0023]

Chemical formula

[0024] When the nucleobase is a purine, the purine is attached to the quinazoline moiety via the 9-position.

[0025] When the nucleobase is a pyrimidine, the pyrimidine is attached to the quinazoline moiety via the 1-position.

[0026] The compound represented by formula I is a complex of a nucleobase, for example, a nucleobase found in the genetic code of an organism or a virus, and a quinazoline moiety modified at the 4-position.

[0027] The compound represented by formula I is a complex of a nucleobase, for example, a nucleobase found in the genetic code of an organism or a virus, and a quinazoline moiety further modified at the 7-position with a halogen or a halogenated residue, for example, fluorine.

[0028] The nucleobase can be a standard nucleobase, for example, adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U). However, the nucleobases useful in the compounds according to the present invention are further modified nucleobases or artificial nucleobases, for example, xanthine, hypoxanthine, 7-methylguanine, N 2 -acetylguanine, N 4 -acetylcytosine, or isoguanine.

[0029] In a preferred embodiment, in the compound represented by formula I, R 1 is preferably a group selected from a hydroxyl group or a phosphate group, and most preferably a phosphate group.

[0030] In a preferred embodiment, in the compound represented by formula I, Y is a purine, for example, adenine or guanine, and most preferably adenine.

[0031] In a preferred embodiment, in the compound represented by formula I, Y is a pyrimidine, for example, cytosine, thymidine, or uracil. Further, in the compound represented by formula I, Y can also be a pyrimidine analog, such as 5-fluorocytosine.

[0032] In a preferred embodiment, in the compound represented by formula I, Y is cytosine, and R 1 is hydroxyl, and R 2 is hydrogen:

[0033]

Chemical formula

[0034] In a preferred embodiment, in the compound represented by formula I, Y is 5-fluorocytosine, and R 1 is hydroxyl, and R 2 is hydrogen:

[0035]

Chemical formula

[0036] In a preferred embodiment, the compound represented by formula I is preferably a compound for use in the treatment of coronavirus disease, and / or diseases or respiratory diseases caused by MERS-CoV, SARS-CoV or SARS-CoV-2. In a preferred embodiment, the disease caused by SARS-CoV-2 is COVID-19.

[0037] In a preferred embodiment, the compound represented by formula I is 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol.

[0038] In a preferred embodiment, the compound represented by formula I is 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate.

[0039] In a preferred embodiment, the compound represented by formula I is 2-(6-amino-9H-purin-9-yl)-7-fluoroquinazolin-4-ol.

[0040] In a preferred embodiment, in the compound represented by formula I, when Y is guanine or N-acetylguanine and R 1 is hydroxyl, the quinazoline moiety may further be modified at its 7-position with a halogen or a halogenated residue:

[0041]

Chemical formula

[0042] In a preferred embodiment, the compound represented by formula I is 2-(6-amino-9H-purin-9-yl)-7-fluoroquinazolin-4-yl dihydrogen phosphate.

[0043] In a preferred embodiment, in the compound represented by formula I, when Y is N-acetylguanine and R 1 is hydroxyl, the quinazoline moiety may or may not be modified at its 7-position with a halogen or a halogenated residue:

[0044]

Chemical formula

[0045] In a preferred embodiment, in the compound represented by formula I, when Y is N 4 -acetylcytosine and R 1 is hydroxyl, the quinazoline moiety may or may not be modified at its 7-position with a halogen or a halogenated residue.

[0046] The present invention further provides a pharmaceutical preparation comprising at least one compound represented by formula I for use in the treatment of coronavirus infection and / or respiratory diseases caused by coronaviruses, such as MERS-CoV, SARS-CoV-2, or SARS-CoV, wherein R can be a group selected from an alkoxy group (e.g., methoxy group, ethoxy group, etc.), a hydroxyl group, and a phosphate group, and preferably, the pharmaceutical preparation comprises 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, or 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate, or both.

[0047] The present invention further provides the above-mentioned compound represented by formula I for inhibiting the RNA-dependent RNA polymerase of coronaviruses, particularly the RNA-dependent RNA polymerase of MERS-CoV, SARS-CoV-2, or SARS-CoV, especially NSP12 of SARS-CoV2, wherein R can be a group selected from an alkoxy group (e.g., methoxy group, ethoxy group, etc.), a hydroxyl group, and a phosphate group, and preferably, the compound represented by formula I is 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, or 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate.

[0048] In a preferred embodiment, the pharmaceutical preparation is an oral preparation, such as a tablet or a capsule, or an injectable preparation.

[0049] Experimental data Synthesis of nucleotide analogs

[0050]

Chemical formula

[0051] a: 1-dodecanethiol, Cs 2 CO 3 , DMSO b: P 2 O 5 、TBAB, H2 O 2 30%, CH 3 CN / H 2 O(1 / 1) To a clean, dry round-bottom flask purged with nitrogen to remove trace amounts of water and other particles, 9-(4-methoxyquinazolin-2-yl)-9H-purine-6-amine (0.7922 g, 2.7 mmol) and 2.7 ml of DMSO were added. Subsequently, cesium carbonate (2.6063 g, 8.0 mmol) was added, and finally 1-dodecanethiol (0.99 ml, 4.0 mmol) was added. The solution was stirred at room temperature for 1 h. The reaction mixture was poured into crushed ice and filtered. The solid was purified by flash chromatography to obtain the desired product in a 20% yield.

[0052] CH 3 CN / H 2 To a cooled solution (0 °C) of phosphorus pentoxide (0.0694 g, 0.5 mmol) in 24 ml of O(1 / 1)24ml, a 30% solution of hydrogen peroxide (1.8 ml) was added dropwise and stirred for 5 minutes. Subsequently, tetra-n-butylammonium bromide (0.97 g, 3 mmol) and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol (0.279 g, 1 mmol) were added, and the mixture was stirred at 0 °C for 5 h. The reaction mixture was filtered, and the solid was diluted three times with 20 ml of MeOH to obtain 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate in a 49% yield.

[0053] To a clean, dry round-bottom flask purged with nitrogen to remove trace amounts of water and other particles, a well-ground mixture of adenine (1.3512 g, 10 mmol), 2-chloro-4-hydroxyquinazoline (2.34 g, 13 mmol), cesium carbonate (3.2612 g, 10 mmol), and silica gel (3.5 g) was added under an inert atmosphere. Subsequently, 20 mL of dry DMSO was added. The solution was stirred at 130 °C for 3.5 h, then poured into ice-cold water and filtered.

[0054] Action of polymerase The two substances tested were 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol (hydroxy substance) and 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate (phosphate substance).

[0055] Since the substances are modified nucleotide analogs, in the first set of experiments, the polymerization inhibitory ability of the two substances was tested. Endpoint PCR reactions were performed using a commercially available template and two housekeeping genes (18S rRNA and actin). The reactions were carried out using different concentrations of conventional dNTPs (dATP, dCTP, dGTP, and dTTP), as well as different concentrations of the products of the present invention, and combinations thereof. The PCR products were then analyzed by gel electrophoresis (3%). The idea is that the use of modified nucleotide analogs can inhibit polymerization because the 3'-5' phosphodiester bond cannot be formed. Furthermore, the mixing of conventional dATP and a modified adenosine analog will also produce smaller fragments, which are observed as a "smear" on the agarose gel, similar to the final product. The data are shown in the following image and Figure 1.

[0056] Based on the above data, it can be shown that the addition of the modified nucleotide analog according to the present invention inhibits the reaction. This is because no product was observed. On the other hand, as expected, both the final product and the smear appeared as a result of mixing conventional / modified adenosine nucleotides.

[0057] Each reaction contains 10 mM each of dATP, dCTP, dGTP, and dTTP. For 75% dATP, 7.5 mM of dATP was used, and for the rest, 10 mM was used. For 50% dATP, 5.0 mM of dATP was used, and for the rest (dCTP, dGTP, dTP), 10 mM was used. For 25% dATP, 2.5 mM of dATP was used, and for the rest (dCTP, dGTP, dTP), 10 mM was used. For 75% A:25% AP (or AH), 7.5 mM of conventional dATP, 2.5 of the modified adenine analog, and for the rest (dCTP, dGTP, dTP), 10 mM was used. For 50% A:50% AP (or AH), 5.0 mM of conventional dATP, 5.0 mM of the corresponding modified adenine analog, and for the rest (dCTP, dGTP, dTP), 10 mM was used. For 25% A:75% AP (or AH), 2.5 mM of conventional dATP, 7.5 mM of the corresponding modified adenine analog, and for the rest (dCTP, dGTP, dTP), 10 mM was used. All concentrations were considered initial concentrations. They were used in endpoint PCR reactions.

[0058] In the second set of experiments, the modified adenosine analog was added to the SYBR Green qPCR mix, and the reaction was carried out using the same template and primers as above. For each condition, the Ct (threshold cycle) parameter was measured. Ct is the number of cycles at which the fluorescence generated within the reaction crosses the fluorescence threshold. The higher the Ct, the lower the amount of the product (or the lower the expression). Table 1 below shows the data from the qPCR reaction.

[0059]

Table 1

[0060] As can be seen from the data in Table 1, the addition of either a phosphate substance or a hydroxyl substance results in a decrease in template amplification. It is also important to note that the reaction is dose-dependent, meaning that increasing either the phosphate substance or the hydroxyl substance results in a higher Ct value. At the same concentration, the effect of inhibiting amplification was higher for phosphate than for hydroxyl.

[0061] Effect on SARS-CoV2 RNA polymerase In addition to the above experiments, a further assay was conducted to test the ability of the compounds according to the invention to inhibit the RNA polymerization action of SARS-COV-2 polymerase NSP12.

[0062] The compounds tested were adenosine analogs:

[0063]

Chemical formula

[0064] The RNA polymerase used in the assay consisted of the RdRp catalytic domain of RNA-directed RNA polymerase (reference: PX-COV-P006, ProteoGenix) and SARS-CoV-2 genomic RNA (novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC)), and the primers used in the assay consisted of (RNA or DNA) complementary to a specific region (Eurofins).

[0065] The PCR reaction used 10X First Strand Buffer and RNase inhibitor from the Aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), as well as dNTP / rNTP respectively. The execution conditions were 37°C for 2 h, and the samples were run on agarose gels and PAGE gels.

[0066] The control reaction was carried out using a commercially available polymerization inhibitor, cordycepin 5'-triphosphate sodium salt (reference: C9137, Sigma-Aldrich).

[0067] The ability of different compounds and the control can be obtained from Figure 2 showing a PAGE gel stained with MidoriGreen Dye (which stains all of dsDNA, ssDNA, dsRNA, and ssRNA).

[0068] The lanes in Figure 2 were named as follows, from left to right:

[0069] "100bp ladder" A marker composition containing marker fragments with an interval of 100bp between each other was loaded into this lane.

[0070] "RNA Primer" An approximately 24-base specific RNA primer obtained from Eurofins Genomics was loaded into this lane.

[0071] "RNA Control" Genomic RNA derived from the novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) was loaded into this lane.

[0072] "Positive Control" This lane was loaded with amplification products obtained by amplifying genomic RNA from the novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) using the RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (the same as in the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the Aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and rNTPs.

[0073] 「No-Adenosine (without adenosine)」 This lane was loaded with amplification products obtained by amplifying genomic RNA from the novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) using the RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (the same as in the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the Aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and equimolar amounts of rNTPs except that rATP was not included.

[0074] 「Phospho」 This lane was loaded with amplification products obtained by amplifying genomic RNA from severe acute respiratory syndrome coronavirus 2; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) using the RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (same as the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the Aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and equimolar amounts of rNTPs except that the reference amount of rATP was divided 50:50 between rATP and compound C.

[0075] "Fluoro (fluoro)" This lane was loaded with amplification products obtained by amplifying genomic RNA from severe acute respiratory syndrome coronavirus 2; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) using the RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (same as the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the Aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and equimolar amounts of rNTPs except that the reference amount of rATP was divided 50:50 between rATP and compound B.

[0076] "Hydroxy (hydroxy)" In this lane, the amplified product obtained by amplifying the genomic RNA from the novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) was loaded using the RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (the same as the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and equimolar amounts of rNTPs, except that the reference amount of rATP was divided 50:50 between rATP and compound A.

[0077] 「Commercial Inhibitor(Commercially available inhibitor)」 In this lane, the amplified product obtained by amplifying the genomic RNA from the novel coronavirus 2019; strain: 2019-nCoV / USA-WA1 / 2020 (reference: ATCC-VR-1986D, ATCC) was loaded using the NSP12 / RdRp catalytic domain of the RNA-directed RNA polymerase of SARS-CoV2 (reference: PX-COV-P006, ProteoGenix) and an RNA primer (the same as the lane "RNA Primer"), 10X First Strand Buffer and RNase inhibitor from the aminoallyl MessageAmp™ II aRNA Amplification Kit (reference: AM1753, Thermo), and equimolar amounts of rNTPs, except that the reference amount of rATP was divided 50:50 between rATP and the commercially available inhibitor. The inhibitor is a modified nucleotide analog and lacks a 3'-hydroxyl group.

[0078] 「Template / Primer(Template / primer)」 In this lane, a mixture of the RNA control and the RNA primer was loaded without any other components and loaded onto the gel to confirm that this mixture did not produce any signal at the position of the length of the substance.

[0079] "1 Kb ladder (1Kb ladder)" In this lane, a marker composition containing marker fragments with an interval of 1000 bp between each other was loaded.

[0080] Based on the above data, it is shown that the addition of any one of compounds A, B, or C inhibits the in vitro replication of SARS-CoV-2 RNA via SARS-CoV-2 RNA polymerase, and the smear in each lane indicates that the amplification products have different sizes reflecting the inhibition at several stages of the polymerization reaction. In any case, it should be noted that compounds A, B, and C were in a competitive state with dATP and rATP to mimic in vivo conditions. Furthermore, the smear is located below the material visualized in the "positive control" lane, which means that each compound was able to shorten the length of the amplification product and inhibit the polymerization activity of NSP12. The smear is located between 700 and 900 bp.

[0081] The RNA control has a smear above 3000 bp and a single band between 1000 and 1500 bp. In the positive control, the band is much weaker and a smear between 600 and 1200 bp is observed, which also serves as an explanation for the smear, suggesting polymerization on different fragments.

[0082] The lane without adenosine shows an image similar to the RNA control. This is because polymerization did not occur where NSP12 was supposed to incorporate adenosine nucleotides.

[0083] For commercially available inhibitors, the profile is also similar to the RNA control. This is because polymerization was also inhibited.

[0084] The primer cannot be observed because it is smaller than 100 bp of the ladder.

[0085] References: 1. Fehr AR, Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 2015;1282:1-23. doi:10.1007 / 978-1-4939-2438-7_1 2. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor [published online ahead of print, 2020 Mar 4]. Cell. 2020;S0092-8674(20)30229-4. doi:10.1016 / j.cell.2020.02.052 3. Song Z, Xu Y, Bao L, et al. From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses. 2019;11(1):59. Published 2019 Jan 14. doi:10.3390 / v11010059 4. Kirchdoerfer, R.N., Ward, A.B. Structure of the SARS-CoV nsp12 polymerase bound to nsp7 and nsp8 co-factors. Nat Commun 10, 2342 (2019). https: / / doi.org / 10.1038 / s41467-019-10280-3

Symbol Explanation

[0086] None

Claims

1. 1. A compound of formula I which is 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol or 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate for use in the treatment of a coronavirus infection. 【Chemistry 1】 (In the formula, R １ is a hydroxyl group or a phosphate group, R ２ is a group selected from halogen or hydrogen, Y is a purine or pyrimidine.

2. 2. The compound for use according to claim 1, wherein the coronavirus is either SARS-CoV, SARS-CoV-2, or MERS-CoV.

3. A compound of formula I which is 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol or 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate. 【Chemistry 2】 (In the formula, R １ is a hydroxyl group or a phosphate group, R ２ is a group selected from halogen or hydrogen, Y is a purine or pyrimidine.

4. A pharmaceutical formulation comprising at least one compound of formula I, wherein the compound comprises 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol or 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate, or both, for use in the treatment of a coronavirus infection. 【Chemistry 3】 (In the formula, R １ is a hydroxyl group or a phosphate group, R ２ is a group selected from halogen or hydrogen, Y is a purine or pyrimidine.

5. The pharmaceutical preparation of claim 4, wherein the coronavirus is SARS-CoV, SARS-CoV-2, or MERS-CoV.

Images