WO2021180219A1 - Pi4k抑制剂的抗冠状病毒效果以及应用 - Google Patents

Pi4k抑制剂的抗冠状病毒效果以及应用 Download PDF

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WO2021180219A1
WO2021180219A1 PCT/CN2021/080540 CN2021080540W WO2021180219A1 WO 2021180219 A1 WO2021180219 A1 WO 2021180219A1 CN 2021080540 W CN2021080540 W CN 2021080540W WO 2021180219 A1 WO2021180219 A1 WO 2021180219A1
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alkyl
amino
group
use according
haloalkyl
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PCT/CN2021/080540
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English (en)
French (fr)
Inventor
姚航平
黄福德
李兰娟
王文安
曹鲁乡
焦常平
吴南屏
卢翔云
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First Affiliated Hospital of Zhejiang University School of Medicine
Nuo Beta Pharmaceutical Technology Shanghai Co Ltd
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First Affiliated Hospital of Zhejiang University School of Medicine
Nuo Beta Pharmaceutical Technology Shanghai Co Ltd
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Priority to JP2022555078A priority Critical patent/JP2023518208A/ja
Priority to CA3170611A priority patent/CA3170611A1/en
Priority to CN202180026954.8A priority patent/CN115884789B/zh
Priority to EP21768762.3A priority patent/EP4119164A4/en
Priority to US17/911,103 priority patent/US20250152599A1/en
Priority to MX2022011138A priority patent/MX2022011138A/es
Application filed by First Affiliated Hospital of Zhejiang University School of Medicine, Nuo Beta Pharmaceutical Technology Shanghai Co Ltd filed Critical First Affiliated Hospital of Zhejiang University School of Medicine
Priority to AU2021236089A priority patent/AU2021236089B2/en
Priority to KR1020227033463A priority patent/KR20230022150A/ko
Publication of WO2021180219A1 publication Critical patent/WO2021180219A1/zh
Priority to ZA2022/10111A priority patent/ZA202210111B/en
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Definitions

  • the present invention relates to the use of PI4KIII ⁇ specific inhibitors in the preparation of drugs for the prevention or treatment of coronavirus diseases.
  • Coronavirus has always received widespread attention due to its high contagiousness.
  • severe acute respiratory syndrome severe acute respiratory syndrome coronavirus, SARS-CoV
  • Middle East respiratory syndrome coronavirus Middle east respiratory syndrome coronavirus, MERS-CoV
  • two highly pathogenic coronaviruses SARS-CoV and MERS -CoV's mortality rate is as high as 10% and 36% respectively
  • human coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and HCoV-HKU8 cause 15%-30% of human upper respiratory tract infections each year,
  • the incidence of more serious diseases is higher among newborns, the elderly, and individuals with underlying diseases. Therefore, the coronavirus has attracted great attention.
  • coronavirus entry is through a pH-dependent pathway, that is, when the virus is adsorbed on the cell surface, the cell membrane invades, the virus is internalized, and the viral RNA is released into the cytoplasm.
  • the viral RdRp is first synthesized.
  • the polymerase recognizes the positive-strand RNA of the coronavirus genome and uses it as a template to synthesize negative-strand RNA, and then uses the negative-strand RNA as a template to synthesize small subgenomic positive-strand RNA and positive-strand genomic RNA.
  • the ribosome uses a small subgenomic positive-strand RNA as a template to translate into precursor protein.
  • protein N binds to newly synthesized genomic RNA, reaches the endoplasmic reticulum with the participation of protein M, integrates with protein S, and is released from the endoplasmic reticulum membrane.
  • the protease cleaves the progeny virus precursor protein to become a mature protein.
  • the progeny virus is transferred from the Golgi apparatus to the cell membrane and released outside the cell (Masters et al., The molecular biology of coronaviruses. Adv Virus Res. 2006. Vol 66: p. 193-292) for a new round of infected cells , Replicate and damage cells within the cell, thereby affecting the normal function of the body.
  • SARS-CoV-2 also known as the new coronavirus
  • NCP novel coronavirus pneumonia
  • SARS-CoV-2 is an enveloped virus.
  • the genetic material of SARS-CoV-2 is a positive-stranded single-stranded RNA virus, and its gene sequence belongs to the same lineage as SARS virus and MERS virus.
  • SARS-CoV-2 will stimulate the patient's innate immune system, causing the body to release a large amount of cytokines, causing a cytokine storm and acute inflammation. This will lead to more fragile blood vessels throughout the body, leading to acute respiratory distress and multiple organ failure (Huang et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020: p1-10; Kikkert et al., Innate Immune Evasion by Human Respiratory RNA Viruses. Journal of Innate Immunity. 2020, 12(1)).
  • SARS-CoV-2 appears in the cerebrospinal fluid of patients with new coronavirus pneumonia, which coincides with the symptoms of neck resistance, sudden disturbance of consciousness and coma in patients, indicating that in addition to multiple organs such as lungs, heart, blood, and liver , The virus can also attack the nervous system.
  • SARS-CoV-2 protein S can recognize and bind the host surface receptor angiotensin-converting enzyme 2 (ACE2), allowing the virus to be adsorbed on the cell surface.
  • ACE2 angiotensin-converting enzyme 2
  • the binding power of ACE2 with SARS-CoV-2 virus is considered to be stronger than that with SARS-CoV (Xu et al., Evolution of the novel coronavirus from the ongoing and modeling of its spike protein for risk of human transmission. SCIENCE CHINA Life Sciences. 2020, Vol 63, p. 457-460).
  • Phenylarsine Oxide is a known biological inhibitor
  • the arsenic atom in the molecule has a high affinity for the sulfur atom of the sulfhydryl group in the biomolecule. There is no report that phenylarsenic oxide and its derivatives can be used to treat coronavirus diseases including the new coronavirus.
  • the present invention provides the use of PI4KIII ⁇ specific inhibitors in the preparation of drugs for the prevention or treatment of coronavirus diseases.
  • the PI4KIII ⁇ specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, or an antisense nucleic acid.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the RNAi molecule is small interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro RNA
  • the RNAi molecule is 18-100 bases in length.
  • the RNAi molecule is modified to enhance its stability.
  • the PI4KIII ⁇ specific inhibitor is a small molecule compound.
  • the small molecule compound is phenylarsenic oxide or its derivatives, G1 and its analogs, A1 and its analogs, or smepivir and its analogs.
  • the phenylarsenic oxide and its derivatives have the structure represented by formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, and amino groups.
  • n is an integer of 0-5.
  • n is an integer of 0-2, and each R 1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 alkyl, C 1 -6 alkoxy, C 1-6 haloalkyl, -As (O), N- ( C 1-6 alkyl) amino, N, N- (C 1-6 alkyl) 2 amino, -NH-C (O)H or -NH-S(O) 2 H, and optionally substituted by said R 2 or R 3.
  • n is an integer of 0-2, and each R 1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, C 1-6 alkyl, C 1-6 alkoxy Group, C 1-6 haloalkyl, -As(O), -NH-C(O)H or -NH-S(O) 2 H, and optionally substituted by said R 2 or R 3.
  • n is 1 or 2
  • each R 1 is independently selected from H, halogen, amino, C 1-6 alkoxy, C 1-6 haloalkyl, -NH-C(O)R 2 or -NH-S(O) 2 R 3 , where R 2 is a C 1-6 alkyl group, optionally substituted by a 6-12 membered aromatic group, and R 3 is a 6-12 membered aromatic group, optionally Substituted by a halogen, C 1-6 alkoxy or C 1-6 haloalkyl.
  • the R 1 is located in the ortho and/or para position of the -As(O) group.
  • n is zero.
  • the small molecule compound is selected from the group consisting of:
  • the object is a human or a mammal.
  • the coronavirus is a novel coronavirus.
  • the coronavirus is avian infectious bronchitis virus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, porcine hemagglutinating encephalomyelitis virus, porcine delta coronavirus, canine respiratory type coronavirus Viruses, mouse hepatitis virus, feline coronavirus, human coronavirus, severe acute respiratory syndrome virus, and Middle East respiratory syndrome virus.
  • it further includes administering a second agent to a guest in need thereof.
  • the second agent is an agent for treating coronavirus-like diseases.
  • the PI4KIII ⁇ specific inhibitor is administered before, after, or at the same time as the second agent.
  • the present invention provides a method for screening drugs for the prevention or treatment of coronavirus diseases, including contacting the candidate drug with PI4KIII ⁇ protein or nucleic acid or PI4KIII ⁇ , and detecting whether the candidate drug can inhibit PI4KIII ⁇ Formation or activity.
  • the present invention provides the use of the compound of formula (I) in the preparation of a medicament for the prevention or treatment of coronavirus diseases:
  • R 1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, and amino groups.
  • n is an integer of 0-5.
  • the compound is phenylarsenic oxide.
  • Figure 1 shows the toxic effects of different concentrations of phenylarsenic oxide on Vero cells.
  • Figure 1A shows the toxic effect of PI01 small molecule drugs at a concentration of less than 200 nM on Vero cells
  • Figure 1B shows the toxic effect of PI01 small molecule drugs at a concentration of 200 nM on Vero cells
  • Figure 1C shows the toxic effect of different concentrations of Remdesivir (Remdesivir) Toxic effects on Vero cells
  • Figure 1D shows the toxic effects of different concentrations of chloroquine phosphate (Chloroquine) on Vero cells.
  • Figure 2 shows the in vitro inhibitory effect of different concentrations of phenylarsenic oxide.
  • Figure 2A shows the effect of different concentrations of PI01 drugs on the inhibition of SARS-CoV-2 in Vero cells
  • Figure 2B shows the effect of different concentrations of PI01 drugs on the inhibition of SARS-CoV-2 in Vero cells
  • Figure 2C shows the effects of different concentrations of Reid Remdesivir inhibits the effect of SARS-Cov-2 in Vero cells.
  • Figure 2D shows the effect of different concentrations of chloroquine phosphate (Chloroquine) in inhibiting SARS-Cov-2 in Vero cells.
  • Chloroquine chloroquine
  • Figure 3 shows the activity of different reagents against human coronavirus HCoV-229E in MRC5 cells.
  • Figure 3A shows the results of the first round, and
  • Figure 3B shows the results of the second round.
  • PI4KIII ⁇ specific inhibitor refers to various substances that can specifically reduce, reduce, or eliminate the transcription or translation of the PI4KIII ⁇ gene, and/or the activity of the PI4KIII ⁇ protein.
  • the PI4KIII ⁇ specific inhibitor is capable of reducing the activity of PI4KIII ⁇ by at least 5%, 10%, 20%, 40%, 50%, 80%, 90%, 95% or more.
  • activity when “activity” is used together with increase or decrease, it refers to the detected functional activity, which can be expressed as a change in content, or a change in content but a change in functional activity.
  • the activity of PI4KIII ⁇ refers to the activity of the PI4KIII ⁇ protein to phosphorylate phosphoinositol (PI) at a specific position (for example, to convert into 4-phosphoinositide (PI4P)).
  • the binding constant of the PI4KIII ⁇ specific inhibitor to the PI4KIII ⁇ protein is at least two times the binding constant of other non-specific binding proteins.
  • PI4KIII ⁇ -specific inhibitors can preferentially recognize PI4KIII ⁇ proteins in complex mixtures, including mixtures with other PI4KIII ⁇ proteins.
  • the PI4KIII ⁇ specific inhibitor inhibits PI4KIII at least 1-fold, 2-fold, 4-fold, 5-fold. Times, 10 times, 20 times, 30 times, 50 times, 100 times, 200 times, 500 times or 10000 times.
  • the PI4KIII ⁇ specific inhibitor does not substantially inhibit PI4KII (e.g., PI4KII ⁇ or PI4KII ⁇ ), for example, its IC 50 is greater than or equal to 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 60 ⁇ M, 80 ⁇ M, 100 ⁇ M, 150 ⁇ M, 200 ⁇ M or 500 ⁇ M.
  • PI4KIII ⁇ -specific inhibitors inhibit PI4KIII ⁇ at least 1-fold, 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, compared to other subtypes of PI4KIII (for example, PI4KIII ⁇ ). Times, 50 times, 100 times, 200 times, 500 times or 10000 times.
  • PI4KIII ⁇ PI4KIIICC specific inhibitor IC 50 of less than or equal to 100 ⁇ M, 80 ⁇ M, 50 ⁇ M, 30 ⁇ M, 20 ⁇ M, 10 ⁇ M, 5 ⁇ M, 3 ⁇ M, 2 ⁇ M, 1 ⁇ M, 0.5 ⁇ M, 0.2 ⁇ M, 0.1 ⁇ M, 0.05 ⁇ M , 0.02 ⁇ M, 0.01 ⁇ M, 0.005 ⁇ M, 0.002 ⁇ M or 0.001 ⁇ M.
  • the PI4KIII ⁇ specific inhibitor is an antibody, a small molecule compound, an RNAi molecule, or an antisense nucleic acid.
  • the PI4KIII ⁇ specific inhibitor is an antibody.
  • antibody as used herein includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody or antibody that can bind to a specific antigen.
  • antibody herein is intended to broadly cover conventional four-chain antibodies as well as less conventional antibodies that do not have four chains (e.g., antibodies that naturally lack light chains).
  • a conventional intact antibody is a heterotetramer, which contains two heavy (H) chains and two light (L) chains.
  • the mammalian heavy chains can be divided into ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ .
  • Each heavy chain consists of a variable region (VH) and a first, second, and third constant region (CH1, CH2, CH3, respectively) Composition;
  • mammalian light chains can be divided into lambda or kappa, each light chain is composed of a variable region (VL) and a constant region.
  • Conventional antibodies are of the "Y" type, and the neck of the "Y” type structure is composed of the second and third constant regions of two heavy chains, which are joined by disulfide bonds.
  • Each arm of the "Y" type structure contains the variable region and the first constant region of one of the heavy chains, which are combined with the variable region and constant region of one light chain.
  • the variable regions of the light and heavy chains determine antigen binding.
  • the variable region of each chain contains three hypervariable regions, called complementarity determining regions (CDR) (the CDR of the light chain includes LCDR1, LCDR2, LCDR3, and the CDR of the heavy chain includes HCDR1, HCDR2, HCDR3).
  • CDR complementarity determining regions
  • the three CDRs are separated by side continuous parts called framework regions (FR), which are more highly conserved than CDRs and form a scaffold to support hypervariable loops.
  • the constant regions of heavy and light chains have nothing to do with antigen binding, but have multiple effector functions.
  • the antibody is a full-length antibody or an antigen-binding fragment.
  • antigen-binding fragment refers to an antibody fragment formed by an antibody fragment containing one or more CDRs but not having a complete antibody structure.
  • antigen-binding fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, single-chain antibody molecules (scFv), scFv dimers, camelized single domain antibodies (camelized single domain antibody) ) And Nanobodies.
  • the antigen-binding fragment can bind to the same antigen as the parent antibody.
  • the "Fab" fragment of an antibody refers to the part of the antibody in which a light chain (including the variable region and the constant region) and the variable region of a heavy chain and the first constant region are combined by disulfide bonds.
  • Fab' fragments refer to Fab fragments that contain part of the hinge region.
  • the "F(ab')2" fragment refers to a dimer of Fab'.
  • the "Fv" fragment of an antibody consists of a variable region of a light chain and a variable region of a heavy chain.
  • Single-chain antibody molecule or “scFv” refers to an engineered antibody formed by connecting the variable region of the light chain and the variable region of the heavy chain directly or through a peptide chain.
  • scFv Single-chain antibody molecule
  • ScFv dimer refers to a polymer formed by two scFvs.
  • “Camelized single domain antibody (Camelized single domain antibody)” (also known as “heavy-chain antibody” or “HCAb (Heavy-chain-only antibodies, HCAb)”) means that it contains two heavy chain variable regions without Light chain antibodies. Heavy chain antibodies were originally derived from the camelid family (camels, dromedaries and llamas). Although the light chain is missing, the camelized antibody has all the functions of antigen binding.
  • “Nanobodies” are composed of a heavy chain variable region derived from a heavy chain antibody and two constant regions CH2 and CH3.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • the antibody is a murine antibody, a rabbit antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the term "fully human” when applied to an antibody or antigen-binding fragment means that the amino acid sequence of the antibody or antigen-binding fragment corresponds to that produced by humans or human immune cells, or derived from, for example, the use of human antibody
  • the library contains amino acid sequences of antibodies derived from non-human sources such as transgenic non-human animals, or other sequences encoding human antibodies.
  • the term "humanized” when applied to antibodies or antigen-binding fragments means to include CDRs derived from non-human animals, FR regions derived from humans, and constant regions derived from humans (when applicable) The antibody or antigen-binding fragment. Since the humanized antibody or antigen-binding fragment has lower immunogenicity, it can be used as a human therapeutic agent in certain embodiments.
  • the non-human animal is a mammal (e.g., mouse, rat, rabbit, goat, sheep, guinea pig, or hamster).
  • the humanized antibody or antigen-binding fragment consists essentially of human sequences except for the CDR sequences that are non-human.
  • a chimeric antibody when applied to antibodies or antigen-binding fragments refers to having a part of a heavy chain and/or light chain derived from one species, and the remaining part of the heavy chain and/or light chain Antibodies or antigen-binding fragments derived from different species.
  • a chimeric antibody may include a constant region derived from a human and a variable region derived from a non-human animal (e.g., mouse or rabbit).
  • the antibodies described herein are monospecific antibodies, bispecific antibodies, or multispecific antibodies.
  • the antibodies described herein may be further labeled.
  • the PI4KIII ⁇ specific inhibitor is an RNAi molecule.
  • RNAi molecule refers to RNA or its analogue, which has sufficient sequence complementarity with the target RNA to guide RNA interference. In some embodiments, it also includes DNA that can be used to generate RNA.
  • RNA interference refers to the process of sequence specificity or selection by which target molecules (for example, target genes, proteins or RNA) are down-regulated.
  • the RNAi molecule is capable of reducing the expression of PI4KIII ⁇ , for example, knocking down the PI4KA gene.
  • the RNAi molecule is 18-100 bases in length.
  • the RNAi molecule is modified to enhance its stability.
  • the RNAi molecule is small interfering RNA (siRNA), short hairpin RNA (shRNA), or micro RNA (miRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro RNA
  • small interfering RNA refers to an RNA molecule, preferably a double-stranded molecule, having a length of about 10-50 nucleotides, preferably about 15-25 nucleotides in length, more preferably About 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, the strand optionally has a nucleotide (or nucleotide Analog), which can guide or mediate the degradation of RNA.
  • short hairpin RNA refers to an RNA molecule with a stem-loop structure, which includes a first region and a second region of complementary sequence, the degree of complementarity and the direction of the region are sufficient to make the base pair appear in Between the regions, the first region and the second region are connected by a loop region, and the loop is produced by the lack of base pairing between nucleotides (or nucleotide analogs) in the loop region.
  • microRNA microRNA or miRNA
  • nt 16-26 nucleotides
  • miRNA genes are transcribed into "pri-miRNA” by DNA transcriptase II (DNA transcriptase II), and pri-miRNA is quickly processed by a ribonuclease III (Drosha) into miRNA "pre-product”.
  • Pre-miRNA pre-miRNA is transported from the nucleus to the cytoplasm, and then recognized and cleaved into mature miRNA by another ribonuclease III (Dicer).
  • Mature miRNA molecules are partially complementary to one or more mRNAs and regulate protein expression.
  • Known miRNA sequences can be obtained through public databases, such as the miRBase database (www.mirbase.org), which provides information including miRNA sequence information, functional annotations, and predicted gene targets.
  • miRNAs also include RNA molecules with structures and functions similar to natural miRNAs expressed in cells by artificially synthesized plasmids, which can target corresponding mRNAs like natural miRNAs and hinder their translation into proteins.
  • the PI4KIII ⁇ specific inhibitor is an antisense nucleic acid.
  • antisense nucleic acid includes nucleotides that are completely complementary to the target sequence and those nucleotides with one or more nucleotide mismatches, as long as the antisense nucleic acid can specifically hybridize to the target sequence. That's it.
  • the antisense nucleic acid herein includes at least 70% or higher, preferably 80% or higher, more preferably 90% or higher, even more preferably 95% or higher homology over the length of at least 15 consecutive nucleotides.
  • Sexual polynucleotides Due to the formation of hybrids, target gene transcription and/or target mRNA translation are reduced or blocked. .
  • the PI4KIII ⁇ inhibitor is a small molecule compound.
  • small molecule compound refers to an organic compound with a molecular weight of less than 3000, 2500, 2000, 1500, 1000, or 500 Daltons.
  • the organic compound may be natural or chemically synthesized.
  • the small molecule compound has the structure shown in formula (I) or a pharmaceutically acceptable salt thereof,
  • R 1 on two adjacent carbon atoms forms a 5-12 membered cycloalkyl, aromatic or heterocyclic group, and is optionally substituted by one or more halogen, nitro, cyano, hydroxyl, and amino groups.
  • n is an integer of 0-5.
  • the n is 0, 1, 2, or 3. In some embodiments, the n is 0, 1, or 2. In some embodiments, the n is 0 or 1.
  • n is an integer of 0-2, and each R 1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, -As (O), N- ( C 1-6 alkyl) amino, N, N- (C 1-6 alkyl) 2 amino, -NH -C(O)H or -NH-S(O) 2 H, and optionally substituted by said R 2 or R 3.
  • n is an integer of 0-2, and each R 1 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, C 1-6 alkyl, C 1-6 Alkoxy, C 1-6 haloalkyl, -As(O), -NH-C(O)H or -NH-S(O) 2 H, and optionally substituted by said R 2 or R 3.
  • the n is 1 or 2
  • each R 1 is independently selected from H, halogen, amino, C 1-6 alkoxy, C 1-6 haloalkyl, -NH-C(O ) R 2 or -NH-S(O) 2 R 3 , where R 2 is a C 1-6 alkyl group, optionally substituted by a 6-12 membered aromatic group, and R 3 is a 6-12 membered aromatic group, which can be Optionally substituted with a halogen, C 1-6 alkoxy or C 1-6 haloalkyl.
  • the R 1 is located in the ortho and/or para position of the -As(O) group.
  • said R 1 is H.
  • substituted when referring to a chemical group, means that one or more hydrogen atoms of the chemical group are removed and replaced by a substituent.
  • substituted as used herein has the usual meaning known in the art and refers to a chemical moiety that is covalently attached or fused to the parent group where appropriate.
  • C n- C m represents a range of the number of carbon atoms, where n and m are integers and the range of the number of carbon atoms includes the endpoints (ie, n and m) and each integer point therebetween.
  • C 1 -C 6 represents a range of 1 to 6 carbon atoms, including 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, and 6 carbon atoms.
  • alkyl refers to a saturated hydrocarbyl group, which may be linear or branched.
  • C n -C m alkyl refers to an alkyl group having n to m carbon atoms. In certain embodiments, the alkyl group contains 1 to 12, 1 to 8, 1 to 6, 1 to 4, 1 to 3, or 1 to 2 carbon atoms.
  • alkyl groups include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, 2-methyl -1-butyl, n-pentyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, etc.
  • alkenyl refers to an unsaturated hydrocarbyl group, which may be linear or branched, and has at least one carbon-carbon double bond.
  • alkenyl groups contain 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms.
  • alkenyl groups can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon double bond.
  • alkenyl groups include, but are not limited to, chemical groups such as vinyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.
  • alkynyl refers to an unsaturated alkynyl group, which may be linear or branched, having at least one carbon-carbon triple bond.
  • alkynyl groups contain 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 carbon atoms.
  • an alkynyl group can also have 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 carbon-carbon triple bond.
  • alkynyl groups include, but are not limited to, chemical groups such as ethynyl, propynyl, butynyl, and the like.
  • cycloalkyl refers to a ring system alkyl group consisting of at least 3 atoms.
  • n-m membered cycloalkyl refers to a cycloalkyl group having n to m ring-forming members.
  • the ring may also have one or more double bonds, but not a fully conjugated system.
  • cycloalkyl groups have 3 to 8, 3 to 6, or 4 to 6 carbon atoms that form a ring. Examples of cycloalkyl groups include, but are not limited to, cyclopropane, cyclobutane, cyclopentyl, and the like.
  • heterocyclyl refers to a cyclic group in which at least one atom in the ring system is a heteroatom and the remaining ring atoms are carbon atoms.
  • n-m membered heterocyclic group refers to a heterocyclic group having n to m ring-forming members.
  • heterocyclyl as used herein includes heteroaryl and heterocycloalkyl. In addition, the ring may also have one or more double bonds.
  • the heterocyclyl group is a saturated heterocycloalkyl group. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like.
  • heterocycloalkyl refers to a cycloalkyl in which at least one atom in the ring system is a heteroatom and the remaining ring atoms are carbon atoms.
  • n-m membered heterocycloalkyl refers to a heterocycloalkyl having n to m ring-forming members.
  • the ring may also have one or more double bonds, but not a fully conjugated system.
  • the heterocycloalkyl group is a saturated heterocycloalkyl group. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like.
  • the heterocycloalkyl group has 3 to 8, 3 to 6, or 4 to 6 carbon atoms that form a ring.
  • heterocycloalkyl groups include, but are not limited to, azetidine, aziridine, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholine, homopiperazine, and the like.
  • aryl refers to a single- or multi-carbon with alternating double and single bonds between the carbon atoms forming the ring Ring ring system group.
  • C n -C m aryl group refers to an aryl group having n to m carbon atoms forming a ring.
  • the aryl ring system has 6 to 12, 6 to 10, or 6 to 8 carbon atoms in one or more rings.
  • the aryl ring system has 2 or more rings fused together. Examples of aryl groups include, but are not limited to, chemical groups such as phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like.
  • heteroaryl refers to an aryl group in which at least one ring atom in the aromatic ring is a heteroatom and the remaining ring atoms are carbon atoms.
  • n-m membered heteroaryl group refers to a heteroaryl group having n to m ring-forming members. Examples of heteroatoms include, but are not limited to, oxygen, sulfur, nitrogen, phosphorus, and the like.
  • the heteroaryl group can have 5 to 10, 5 to 8, or 5 to 6 ring-forming members.
  • the heteroaryl group is a 5- or 6-membered heteroaryl group.
  • heteroaryl groups include, but are not limited to, furyl, thienyl, pyridyl, quinolinyl, pyrrolyl, N-lower alkylpyrrolyl, pyridyl-N-oxide, pyrimidinyl, pyrazinyl, imidazolyl , Indolyl, etc.
  • alkoxy refers to a group represented by the formula "-O-alkyl".
  • C n -C m alkoxy means that the alkyl portion of the alkoxy group has n to m carbon atoms. In certain embodiments, the alkyl moiety has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • alkoxy groups include, but are not limited to, chemical groups such as methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.
  • haloalkyl refers to a group represented by the formula "-alkyl-X", where X is halogen, selected from fluorine, chlorine, bromine and iodine Of the atom.
  • C n -C m haloalkyl means that the alkyl portion of the haloalkyl has n to m carbon atoms. In certain embodiments, the alkyl moiety has 1 to 6, 1 to 4, or 1 to 3 carbon atoms.
  • haloalkyl groups include, but are not limited to, chemical groups such as halomethyl, haloethyl, halopropyl (e.g., n-halopropyl and isohalopropyl), t-halobutyl Wait.
  • n-membered is usually used with a ring system to describe the number of atoms in the ring system that form the ring.
  • piperidinyl is an example of a 6-membered heterocycloalkyl ring
  • pyrazolyl is an example of a 5-membered heteroaryl ring
  • pyridyl is an example of a 6-membered heteroaryl ring
  • 1, 2, 3, 4-tetrahydro-naphthalene is an example of a 10-membered aryl group.
  • nm member as used herein is generally used with a ring system to describe the number of members forming a ring in the ring system, where n and m are integers and the range of members forming the ring includes the endpoints (ie, n and m) and between Of each integer point.
  • 3-8 members represent the range of 3 to 8 members forming a ring, including 3 members, 4 members, 5 members, 6 members, 7 members, and 8 members.
  • halogen refers to an atom selected from fluorine, chlorine, bromine and iodine.
  • cyano used herein refers to a group represented by the formula "-CN”.
  • hydroxyl refers to a group represented by the formula "-OH”.
  • nitro used herein refers to a group represented by the formula "-NO 2 ".
  • amino refers to a group represented by the formula "-NH 2 ".
  • compound as used herein is intended to include all stereoisomers (e.g., enantiomers and diastereomers), geometric isomers, tautomers, and isotopes of the structure shown.
  • the compounds described herein may be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, all stereoisomers, such as enantiomers and diastereomers, are intended to be included. Various geometric isomers such as olefins and carbon-carbon double bonds may also exist in the compounds described herein, and all these stable isomers have been considered in this article. The cis and trans geometric isomers of the compounds are described herein and can be separated as a mixture of isomers or as individual isomers.
  • the compounds herein also include tautomeric forms.
  • the tautomeric form is caused by the exchange of a single bond and an adjacent double bond accompanied by the migration of protons.
  • Tautomeric forms include tautomers of protons in an isomeric protonated state with the same chemical formula and total charge.
  • Examples of proton tautomers include keto-enol pairs, amide-imine pairs, lactam-lactam pairs, enamine-imine pairs, and cyclic forms where protons can occupy the heterocyclic ring system Two or more positions, such as 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be balanced or sterically locked into one form through appropriate substitution.
  • the small molecule compounds herein can be obtained by organic synthesis.
  • the compounds herein can be prepared using any well-known organic synthesis technique and can be synthesized according to a variety of possible synthetic routes, including salts, esters, hydrates or solvates thereof.
  • the small molecule compound described herein is a compound having the following structural formula, including one or more of them:
  • PEO phenylarsenic oxide
  • A1 and G1 used herein are both small molecule compound inhibitors of PI4KIII ⁇ protein and have relatively similar structures. Among them, the compound structural formula of A1 is:
  • This article also relates to phenylarsenic oxide derivatives, A1, G1 or smepivir analogues, as long as they have the function of inhibiting the phosphokinase activity of PI4KIII ⁇ protein, they can also be used to treat coronavirus diseases, especially new coronaviruses. And the preparation methods of such structural analogs have also been published.
  • the derivatives of phenylarsenic oxide, Al, G1, or smepivir are analogs with similar structures to phenylarsenic oxide, Al, G1, or smepivir.
  • Coronavirus refers to a type of enveloped single-stranded positive-stranded RNA virus that can infect humans and a variety of animals. It has respiratory, gastrointestinal, and nervous system tropism. Livestock and companion animals (such as pigs, cows, chickens, dogs, cats) cause serious diseases, and can cause people to suffer from diseases ranging from the common cold to severe acute respiratory syndrome. According to the ninth report of the International Committee on Classification of Viruses, coronaviruses are divided into four groups: ⁇ , ⁇ , ⁇ , and 6 according to their evolutionary characteristics. Among them, the hosts of ⁇ and ⁇ groups are mainly mammals, and ⁇ and 6 groups are mainly found in Birds and poultry.
  • Coronaviruses that can infect humans include, but are not limited to, the human coronavirus 229E (HCoV-229E), NL63 (HCoV-NL63), HKU1 (HCoV-HKU1), OC43 (HCoV-OC43), and the common cold that cause upper respiratory tract infection symptoms.
  • HARS-CoV Severe acute respiratory syndrome coronavirus
  • SARS-CoV-2 new coronavirus
  • MERS- CoV Middle East respiratory syndrome coronavirus
  • Coronaviruses that can infect animals include but are not limited to porcine transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDC), porcine hemagglutinating encephalomyelitis virus (PHEV), canine respiratory coronavirus (CrCoV), Mouse Hepatitis Virus, Feline Coronavirus (FCoV).
  • TGEV porcine transmissible gastroenteritis virus
  • PDC porcine delta coronavirus
  • PHEV porcine hemagglutinating encephalomyelitis virus
  • CrCoV canine respiratory coronavirus
  • FCoV Feline Coronavirus
  • pharmaceutically acceptable refers to being suitable for use in contact with human and animal tissues within the scope of reasonable medical judgment without excessive toxicity, irritation, allergic reactions, or other problems or complications, and having reasonable Those compounds, materials, compositions and/or dosage forms for the benefit/risk ratio.
  • pharmaceutically acceptable compounds, materials, compositions, and/or dosage forms are those approved by regulatory agencies (such as the U.S. Food and Drug Administration, China Food and Drug Administration, or European Medicines Agency) or listed as generally recognized Those in the pharmacopoeia (such as the U.S. Pharmacopoeia, Chinese Pharmacopoeia, or European Pharmacopoeia) for animals (more particularly for humans).
  • object can include humans and non-human animals.
  • Non-human animals include all vertebrates, such as mammals and non-mammals.
  • Object can also be livestock animals (for example, cows, pigs, sheep, chickens, rabbits, or horses), or rodents (for example, rats or mice), or primates (for example, gorillas or monkeys). ), or domestic animals (for example, dogs or cats).
  • Animal "objects” can be males or females, and can also be of different ages.
  • Human "objects” can be Caucasians, Africans, Asians, Semites, or other races, or hybrids of different races. Human "objects” can be old people, adults, teenagers, children, or babies.
  • the objects described herein are human or non-human primates.
  • the PI4KIII ⁇ specific inhibitors disclosed herein can be administered by administration routes known in the art, such as injection administration (e.g., subcutaneous injection, intraperitoneal injection, intravenous injection (including intravenous drip or intravenous infusion), intramuscular injection or Intradermal injection) or non-injection administration (e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration).
  • injection administration e.g., subcutaneous injection, intraperitoneal injection, intravenous injection (including intravenous drip or intravenous infusion), intramuscular injection or Intradermal injection
  • non-injection administration e.g., oral administration, nasal administration, sublingual administration, rectal administration or topical administration.
  • the PI4KIII ⁇ -specific inhibitors described herein are administered orally, subcutaneously, intramuscularly, or intravenously.
  • the PI4KIII ⁇ specific inhibitors described herein are administered orally.
  • therapeutically effective amount refers to the amount of a drug that can alleviate or eliminate the disease or symptom of the subject, or prevent or prevent the occurrence of the disease or symptom prophylactically.
  • the therapeutically effective amount can be the amount of a drug that alleviates one or more diseases or symptoms of the subject to a certain degree; it can partially or completely restore one or more physiological or biochemical parameters related to the cause of the disease or symptom to The normal amount of the drug; and/or the amount of the drug that can reduce the likelihood of a disease or symptom.
  • therapeutically effective amount refers to the amount of a drug that can alleviate or eliminate the coronavirus (for example, novel coronavirus) in the guest.
  • the therapeutically effective dose of PI4KIII ⁇ specific inhibitors provided herein depends on a variety of factors known in the art, such as weight, age, past medical history, currently receiving treatment, the subject's health status and the intensity of drug interactions, allergies, Hypersensitivity and side effects, as well as the route of administration and the degree of disease progression. Those skilled in the art (for example, doctors or veterinarians) can reduce or increase the dosage according to these or other conditions or requirements.
  • the treatment further comprises administering a second agent to a guest in need thereof.
  • the second agent is an agent for the treatment of coronavirus diseases, including but not limited to Lopinavir, Ritonavir, Remdesivir, Chloroquine, Simeprevir.
  • the PI4KIII ⁇ specific inhibitor is administered before, after, or at the same time as the second agent.
  • the present application also relates to a method for preventing or treating coronavirus diseases, which includes administering an effective amount of PI4KIII ⁇ specific inhibitor to a guest in need.
  • the present invention also provides a method for screening drugs for preventing or treating coronavirus diseases, including contacting candidate drugs with PI4KIII ⁇ protein or nucleic acid or PI4KIII ⁇ , and detecting whether the candidate drugs can inhibit the formation or activity of PI4KIII ⁇ .
  • Example 1 Solution preparation of compounds such as benzene arsenic oxide
  • Example 2 CCK-8 cell proliferation-toxicity detection method detects the toxicity of different concentrations of phenylarsenic oxide to Vero cells
  • Vero cells African green monkey kidney cells Vero, preserved by the State Key Laboratory of Infectious Diseases Diagnosis and Treatment of Zhejiang University
  • a 96-well culture plate purchased from Corning
  • Thermo3110 purchased from Thermo (American Thermoelectric Company)
  • Thermo3110 was cultured to the cell monolayer, the culture medium was discarded, and the cells were washed twice with Hank's solution.
  • MEM medium purchased from Life Technologies
  • phenylarsenic oxide series with an initial concentration of 200 nM, and successively dilute 7 concentrations to 1.56 nM.
  • Each phenylarsenic oxide concentration is set up in 2 multiple wells, and each well is added with 150 ⁇ L of drug solution.
  • a normal growth control of Vero cells is set up, and cultured in a 37°C, 5% CO 2 incubator for 48 hours.
  • the initial concentration of phenylarsenic oxide and PIK-93 was 800nM, and the concentration was diluted by 7 consecutive times to 6.25nM; the initial concentration of Remdesivir was 300 ⁇ M, and the concentration was consecutively diluted by 6 times.
  • the concentration is 0.4 ⁇ M; the initial concentration of chloroquine phosphate (Chloroquine) is 300 ⁇ M, and six consecutive three-fold dilutions are made to 0.4 ⁇ M.
  • the initial concentration of PIK-93 is 800 nM, and it is diluted to 6.25 nM by 7 consecutive double dilutions. Set up 2 multiple holes for each drug concentration, add 150 ⁇ L of drug solution to each hole, and set up a normal growth control of Vero cells at 37°C, 5% CO 2 incubator for 48 hours.
  • CCK-8 reagent cell proliferation-toxicity detection kit (CCK-8) (purchased from DojinDo, Japan)) to each well, and use a microplate reader (Bio-Rad 680, purchased from BioRad, USA) after 3 hours Company) Measure the OD value at 450nm, calculate the dose-toxic effect of phenylarsenic oxide on cells and the maximum non-toxic concentration, draw the phenylarsenic oxide-cytotoxicity curve, and provide a basis for the selection of the optimal test concentration for in vitro antiviral efficacy .
  • CCK-8 reagent cell proliferation-toxicity detection kit (CCK-8) (purchased from DojinDo, Japan)
  • phenylarsenic oxide (PI01) drugs at a concentration of 400 nM and above have a significant toxic effect on Vero cells, with cell death of more than 50%, and a CC 50 of 380 nM.
  • concentrations of 100 nM and below the drug has no obvious toxic effect on cells, cell viability is greater than 90%, and CC 10 is 100 nM.
  • PIK-93 a specific inhibitor of PI4KIII ⁇ protein at a concentration of 800 nM and below, did not produce significant toxic effects on Vero cells.
  • the control drug Remdesivir has a CC 50 of 208 ⁇ M on Vero cells.
  • the drug has no obvious toxic effect on the cells at a concentration of 50 ⁇ M and below.
  • the cell viability is greater than 90%, and the CC 10 is 55 ⁇ M.
  • the control drug chloroquine phosphate (Chloroquine) has a CC 50 of about 250 ⁇ M for Vero cells and a CC 10 of 120 ⁇ M for Vero cells.
  • Vero cells were seeded in 96-well culture plates at a concentration of 10,000 cells/well, and cultured in a 37°C, 5% CO 2 incubator until the cells were most vigorously in the logarithmic growth phase, 75%-90% of the plated cells were discarded. The solution washes the cells twice (to remove residual bovine serum).
  • the new coronavirus was isolated from the sputum samples of clinically infected patients, verified by full-gene sequencing, and preserved by the State Key Laboratory of Infectious Diseases Diagnosis and Treatment of Zhejiang University. The experiment was carried out in a third-level biosafety laboratory (BSL-3: laboratory certification number: CNAS BL002, State Key Laboratory of Infectious Disease Diagnosis and Treatment, Zhejiang University).
  • virus growth solution 500ml MEM medium containing FBS2%, penicillin 100U/mL, streptin 100 ⁇ g/mL, TPCK-pancreatin 16 ⁇ g/mL (both purchased from Life Technologies) 1000 times diluted As the starting concentration, follow this to make a 10-fold gradient dilution for 8 serial concentrations. Inoculate 4 wells of cells for each virus dilution, 100 ⁇ l per well, and set up a normal cell control at 37°C, 5% CO 2 incubator for 6 days.
  • CPE cell morphology and pathological effect
  • TCID 50 of the new coronavirus used in the experiment was 10-6.5 /100 ⁇ L.
  • Vero cells are seeded in a 24-well plate at a concentration of 50000 cells/1mL/well, and cultured in a 37°C, 5% CO 2 incubator until the cells are most vigorously in the logarithmic growth phase, and 75%-90% of the platelets are discarded. Wash the cells twice with Hank's solution.
  • the drug medium of the storage concentration was diluted to the following final concentration:
  • the initial concentration of phenylarsenic oxide was 200nM, and the concentration was continuously diluted to 1.56 by 7 times;
  • the initial concentration of phenylarsenic oxide and PIK-93 is 800nM, and 7 consecutive double dilutions to 6.25nM;
  • the initial concentration of Remdesivir is 100 ⁇ M, and 6 consecutive three-fold dilutions to 0.4 ⁇ M; chloroquine phosphate (Chloroquine)
  • the initial concentration is 100 ⁇ M, and six consecutive three-fold dilutions are made to 0.3 ⁇ M.
  • Virus preparation novel coronavirus (SARS-CoV-2 strain BetaCov / Wuhan / IME-BJ01 / 2020 (GWHACBB01000000)) was diluted with medium to a final concentration of infected cells 100TCID 50.
  • Virus infection of Vero cells 250 ⁇ L/well of the prepared virus (100TCID 50 ) was used to infect Vero cells, and a single virus-infected cell control and a normal cell control were set. It was adsorbed in a 37°C, 5% CO 2 incubator for 3 hours, discarded the virus-containing culture medium, and washed twice with Hank's solution.
  • Drug treatment add 1 mL/well of the prepared culture medium containing different concentrations of drugs to the above-mentioned pre-poisoned Vero cell culture plate, and set up 2 multiple holes for each concentration of phenylarsenic oxide. Continue to incubate for 48 hours in a 37°C, 5% CO 2 incubator.
  • the fluorescent quantitative PCR method is used to detect the nucleic acid of the new coronavirus.
  • MVR01 magnetic bead method nucleic acid extraction kit
  • EX3600 automatic nucleic acid extractor
  • EX3600 Shanghai Zhijiang Biotechnology Co., Ltd.
  • the result is the Ct value to express the level of the virus.
  • the FAM Ct value in Table 2 represents the level of viral nucleic acid. The larger the Ct value, the fewer viruses. Conversely, the smaller the Ct, the more viruses.
  • PI0101 initial concentration is 10 ⁇ M, 8 concentration points, double wells
  • PI0101 initial concentration is 10 ⁇ M, 8 concentration points, double wells
  • the virus was added to the cells at 100 TCID 50 per well.
  • Set up cell control (cells, no compound treatment or virus infection), virus control (cells infected with virus, no compound treatment) and culture medium control (only culture medium), cultured in a 5% CO 2 , 37°C incubator for 76 hours.
  • the EC 50 of PI0101 was 3.85 ⁇ M, and the CC 50 was greater than 10 ⁇ M.
  • Example 5 Detecting the in vitro anti-human coronavirus (HCoV) 229E activity of phenylarsenic oxide by cytopathic effect (CPE) experiment
  • Preparation of drug solution The drug medium of the storage concentration is diluted to the following final concentration respectively.
  • phenylarsenic oxide YL-05, YL-07, YL-08, YL-09, YL-10 and YL-11 compounds to MRC5 cells and to HCoV 229E (human coronavirus 229E). Type, purchased from ATCC).
  • the initial concentration of the inhibitory effect is 800 and 200 nM, and 7 consecutive pairs of dilutions are diluted to 6.25 and 1.56 nM respectively; the toxicity of Redecive to MRC5 cells and the inhibitory effect of HCoV 229E
  • the initial concentrations were 100 ⁇ M and 1000 nM, and seven consecutive three-fold dilutions were diluted to 0.41 ⁇ M and 0.46 nM.
  • the initial concentrations for observing the cytotoxicity and virus inhibitory effects of phenylarsenic oxide were 800 and 400 nM, respectively, and successively diluted 7 concentrations to 6.25 and 3.13 nM, YL-07, YL-08,
  • the initial concentration of YL-09, YL-10 and YL-11 compounds on MRC5 cell toxicity and HCoV 229E inhibition is 40 ⁇ M, and seven consecutive three-fold dilutions of 0.02 ⁇ M; Remdesivir on cells
  • the initial concentrations for toxicity and virus inhibition were 100 ⁇ M and 1000 nM, and seven consecutive three-fold dilutions were diluted to 0.41 ⁇ M and 0.46 nM, respectively.
  • MRC5 cells and HCoV 229E strain were purchased from ATCC.
  • the cells were cultured in EMEM (Sigma) medium supplemented with 10% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco).
  • EMEM (Sigma) culture medium supplemented with 5% fetal bovine serum (Hyclone), 1% double antibody (Hyclone), 1% L-glutamine (Gibco) and 1% non-essential amino acids (Gibco) was used as the experimental culture medium.
  • MRC5 cells were seeded into a 96-well test plate at a density of 20,000 cells per well and cultured overnight in a 5% CO 2 , 37°C incubator. On the second day, the diluted phenylarsenic oxide (8 concentration points, double wells) was added, and then the virus was added to the cells at 200 TCID 50 per well. Set up cell control (cells, no compound treatment or virus infection), virus control (cells infected with virus, no compound treatment) and culture medium control (only culture medium). The final concentration of DMSO in the culture broth was 0.5%. The cells were cultured in an incubator for 3 days. Cell viability test kit CellTiter Glo (Promega) was used to detect cell viability.
  • CellTiter Glo Promega
  • the cytotoxicity experiment was under the same conditions as the antiviral experiment, but there was no virus infection.
  • the antiviral activity and cytotoxicity of phenylarsenic oxide are represented by the inhibition rate (%) of the compound at different concentrations on the cytopathic effect caused by the virus and the viability (%) of MRC5 cells. Calculated as follows:
  • Inhibition rate (%) (test hole reading value-virus control average value) / (cell control average value-virus control average value) x 100
  • the first round of results showed that the EC 50 value of phenylarsenic oxide was 55.35 nM, and the CC 50 value was 256.8 nM; the EC 50 value of radcivir was 26.42 nM, and the CC 50 value was greater than 100 ⁇ M; YL-05, YL-07, YL The EC 50 of -08, YL-09, YL-10 and YL-11 compounds are all greater than 200 nM, and the CC 50 are all greater than 800 nM; the specific results are shown in Figure 3A and Table 3.
  • the second round of results showed that the EC 50 value of phenylarsenic oxide was 37.03 nM, the CC 50 value was 68.92 nM; the EC 50 value of radcivir was 13.11 nM, and the CC 50 value was 30.66 ⁇ M; YL-07, YL-08, The EC 50 of compound YL-09 and YL-09 were greater than 40 ⁇ M, and the CC 50 values were 6.24 ⁇ M, 7.64 ⁇ M, and 13.88 ⁇ M, respectively.
  • the EC 50 of YL-10 and YL-11 compounds were 3.95 ⁇ M and 2.1 ⁇ M, respectively , and the CC 50 was 14 ⁇ M and 6.37 ⁇ M; please refer to Figure 3B and Table 3 for specific results.
  • the EC 50 of PI0101 (smepivir) is 3.41 ⁇ M, and the CC 50 is 8.46 ⁇ M.

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Abstract

氧化苯砷及其衍生物在预防或治疗冠状病毒类疾病中的用途，同时提供了PI4KIIIα特异性抑制剂在用于预防或治疗冠状病毒类疾病中的用途。

Description

PI4K抑制剂的抗冠状病毒效果以及应用 技术领域
本发明涉及PI4KIIIα特异性抑制剂在制备用于预防或治疗冠状病毒类疾病药物中的用途。
背景技术
冠状病毒由于其较高的传染性，一直以来受到广泛关注。比如严重急性呼吸综合征(Severe acute respiratory syndrome coronavirus，SARS-CoV)和中东呼吸综合征冠状病毒(Middle east respiratory syndrome coronavirus，MERS-CoV)这两种高致病性的冠状病毒SARS-CoV和MERS-CoV的病死率分别高达10％和36％；人冠状病毒(HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1和HCoV-HKU8)每年引起15％-30％的人上呼吸道感染，在新生儿、老年人和具有潜在疾病的个体中引起更严重的疾病的群体中发生率更高，因此对冠状病毒引起高度重视。
一般认为，冠状病毒进入宿主细胞是经pH依赖途径，即当病毒吸附在细胞表面后，细胞膜内陷，病毒被内化，病毒RNA释放至胞浆。此后，病毒RdRp首先被合成，该聚合酶识别冠状病毒基因组正链RNA并以其为模板合成负链RNA，然后再以负链RNA为模板合成亚基因组小片段正链RNA和正链基因组RNA。在胞浆内，核糖体以亚基因组小片段正链RNA为模板，翻译生成前体蛋白。此后，蛋白N与新合成的基因组RNA结合，在蛋白M的参与下，到达内质网，与蛋白S整合并从内质网膜释放。同时，蛋白酶对子代病毒前体蛋白切割，成为成熟蛋白。此后，子代病毒由高尔基体转移至细胞膜并释放至细胞外(Masters et al.,The molecular biology of coronaviruses.Adv Virus Res.2006.Vol 66:p.193-292)进行新一轮的感染细胞、在细胞内复制和损伤细胞，从而影响机体的正常机能。
最近，新型冠状病毒(SARS-CoV-2)，又称新冠状病毒，因其导致的新型冠状病毒肺炎(Novel coronavirus pneumonia，NCP)具有非常高的传染性，其传染基数RO值预计在2-6之间，更是受到各个国家的重视。与其它冠状病毒一样，SARS-CoV-2是具有包膜的病毒。SARS-CoV-2的遗传物质是正链单股RNA病毒，基因序列和SARS病毒及MERS病毒属于同一谱系。
据目前推测，SARS-CoV-2与其他冠状病毒相同，会刺激患者的先天免疫系统，致使体内大量释放细胞因子，造成细胞因子风暴和急性炎症反应。这会导致全身血管更为脆弱，引发急性呼吸窘迫症和多器官衰竭(Huang等人,Clinical features of patients infected with 2019 novel coronavirus in Wuhan,China.Lancet.2020:p1–10；Kikkert等人.,Innate Immune Evasion by Human Respiratory RNA Viruses.Journal of Innate Immunity.2020,12(1))。最新证据显示，SARS-CoV-2出现于新型冠状病毒肺炎病人的脑脊液中，和病人中出现颈抵抗、突发意识障碍昏迷等症状吻合，说明除了肺脏、心脏、血液、肝脏等多脏器以外，病毒还可以攻击神经系统。
与SARS-CoV类似，SARS-CoV-2蛋白S可识别并结合宿主表面受体血管紧张素转移酶2(angiotensin-converting enzyme 2,ACE2)，使病毒吸附于细胞表面。ACE2与SARS-CoV-2病毒的结合力被认为比与SARS-CoV的结合力更强(Xu等人,Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission.SCIENCE CHINA Life Sciences.2020,Vol 63,p.457–460)。目前还没有报道对SARS-CoV-2感染有显著治疗效果的药物。
氧化苯砷(Phenylarsine Oxide，PAO)是一种已知的生物抑制剂，
Figure PCTCN2021080540-appb-000001
该分子中砷原子对生物分子中巯基的硫原子具有高亲和性。目前尚未有报道氧化苯砷及其衍生物可以用于治疗新型冠病毒在内的冠状 病毒类疾病。
发明内容
在一个方面，本发明提供了PI4KIIIα特异性抑制剂在制备用于预防或治疗冠状病毒类疾病药物中的用途。
在一些实施方案中，所述PI4KIIIα特异性抑制剂是抗体、小分子化合物、RNAi分子或者反义核酸。
在一些实施方案中，所述抗体是单克隆抗体或者多克隆抗体。
在一些实施方案中，所述抗体是嵌合抗体、人源化抗体或者全人源抗体。
在一些实施方案中，所述RNAi分子是小干扰RNA(siRNA)、短发夹RNA(shRNA)或微小RNA(miRNA)。
在一些实施方案中，所述RNAi分子的长度为18-100碱基。
在一些实施方案中，所述RNAi分子被修饰以增强其稳定性。
在一些实施方案中，所述PI4KIIIα特异性抑制剂是小分子化合物。
在一些实施方案中，所述小分子化合物是氧化苯砷或其衍生物、G1及其类似物、A1及其类似物、或司美匹韦及其类似物。
在一些实施方案中，所述氧化苯砷及其衍生物具有式(I)所示的结构或者其药学上可接受的盐，
Figure PCTCN2021080540-appb-000002
其中，R 1各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、C 1-6亚烷基-NH 2、C 1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H、-NH-S(O) 2H、-C(O)OH、-OC(O)H、-SH、-S(O) 2H、-S(O) 2-NH 2或杂环基，并且可选的被R 2或R 3取代，其中R 2和R 3各自独立的选自氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、N-(C 1-6烷基)氨基、N-(6-12元芳香基)氨基、 N,N-(C 1-6烷基) 2氨基、C 3-6环烷基、6-12元的芳香基或3-12元的杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、-NH-C(O)-R 5、-C(O)OR 4、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，并且R 4是C 1-6的烷基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，R 5选自H、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基或C 1-6卤代烷基，和/或
(b)两个相邻碳原子上的R 1形成5-12元的环烷基、芳香基或杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，
其中，n为0-5的整数。
在一些实施方案中，n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
在一些实施方案中，n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
在一些实施方案中，n为1或2，所述R 1各自独立的选自H、卤素、氨基、C 1-6烷氧基、C 1-6卤代烷基、-NH-C(O)R 2或-NH-S(O) 2R 3，其中R 2为C 1-6烷基，可选的被一个6-12元芳香基取代，R 3为6-12元芳香基，可选的被一个卤素、C 1-6烷氧基或C 1-6卤代烷基取代。
在一些实施方案中，所述R 1位于-As(O)基团的邻位和/或者对位。
在一些实施方案中，n是0。
在一些实施方案中，所述小分子化合物选自由以下化合物组成的组：
Figure PCTCN2021080540-appb-000003
Figure PCTCN2021080540-appb-000004
Figure PCTCN2021080540-appb-000005
在一些实施方案中，所述客体是人或者哺乳动物。
在一些实施方案中，所述冠状病毒为新型冠状病毒。
在一些实施方案中，所述冠状病毒为鸡传染性支气管炎病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪血凝性脑脊髓炎病毒，猪δ冠状病毒、犬呼吸道型冠状病毒、小鼠肝炎病毒、猫冠状病毒、人冠状病毒、严重急性呼吸综合征病毒、中东呼吸综合征病毒。
在一些实施方案中，进一步包括向需要其的客体施用第二试剂。
在一些实施方案中，所述第二试剂是用于治疗冠状病毒类疾病的 试剂。
在一些实施方案中，所述PI4KIIIα特异性抑制剂在所述第二试剂之前、之后或同时施用。
在另一个方面，本发明提供了一种筛选用于预防或治疗冠状病毒类疾病的药物的方法，包括将备选药物与PI4KIIIα蛋白或者核酸或者PI4KIIIα接触，并检测备选药物是否能够抑制PI4KIIIα的形成或者活性。
在又一方面，本发明提供了式(I)所述的化合物在制备用于预防或治疗冠状病毒类疾病药物中的用途：
Figure PCTCN2021080540-appb-000006
其中，R 1各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、C 1-6亚烷基-NH 2、C 1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H、-NH-S(O) 2H、-C(O)OH、-OC(O)H、-SH、-S(O) 2H、-S(O) 2-NH 2或杂环基，并且可选的被R 2或R 3取代，其中R 2和R 3各自独立的选自氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、N-(C 1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C 1-6烷基) 2氨基、C 3-6环烷基、6-12元的芳香基或3-12元的杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、-NH-C(O)-R 5、-C(O)OR 4、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，并且R 4是C 1-6的烷基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，R 5选自H、C 1-6烷基、C 2-6炔基、C 2-6 烯基、C 1-6烷氧基或C 1-6卤代烷基，和/或
(b)两个相邻碳原子上的R 1形成5-12元的环烷基、芳香基或杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，
其中，n为0-5的整数。
在一些实施方式中，所述化合物为氧化苯砷。
附图说明
图1示出不同浓度的氧化苯砷对Vero细胞的毒性作用。图1A示出200nM以下浓度的PI01小分子药物对Vero细胞的毒性作用，图1B示出200nM浓度的PI01小分子药物对Vero细胞的毒性作用，图1C示出不同浓度瑞德西韦(Remdesivir)对Vero细胞的毒性作用，图1D示出不同浓度磷酸氯喹(Chloroquine)对Vero细胞的毒性作用。
图2示出不同浓度氧化苯砷体外抑制病毒效应。图2A示出不同浓度PI01药物在Vero细胞中抑制SARS-CoV-2的效应，图2B示出不同浓度PI01药物在Vero细胞中抑制SARS-CoV-2的效应，图2C示出不同浓度瑞德西韦(Remdesivir)在Vero细胞中抑制SARS-Cov-2的效应，图2D示出不同浓度磷酸氯喹(Chloroquine)在Vero细胞中抑制SARS-Cov-2的效应。
图3示出不同试剂在MRC5细胞中抗人冠状病毒HCoV-229E的活性。图3A示出第一轮结果，图3B示出第二轮结果。
具体实施方式
以下根据实施例，并且结合附图，详细描述本发明。从下文的详细描述中，本发明的上述方面和本发明的其他方面将是明显的。本发明的范围不局限于下列实施例。
PI4KIIIα特异性抑制剂
本文中所用的术语“PI4KIIIα特异性抑制剂”是指能够特异性降低、 减少、消除PI4KIIIα基因的转录或翻译，和/或PI4KIIIα蛋白活性的各类物质。在一些实施方案中，所述PI4KIIIα特异性抑制剂能够将PI4KIIIα的活性降低至少5％、10％、20％、40％、50％、80％、90％、95％或更多。在本文中，当“活性”与增加或降低一同使用的时候，是指检测到的功能活性，其可以表现为含量变化、或者含量不变但是功能活性变化。
本文中，PI4KIIIα的活性是指PI4KIIIα蛋白将磷酸肌醇(PI)在特定位置磷酸化(例如，转化为4-磷酸磷脂酰肌醇(PI4P))的活性。所述PI4KIIIα特异性抑制剂与PI4KIIIα蛋白的结合常数为其与其他非特异性结合蛋白的结合常数的至少2倍以上。在一些实施方案中，PI4KIIIα特异性抑制剂能够在复杂混合物中优选识别PI4KIIIα蛋白，包括与其他PI4看亚型蛋白的混合物。
在一些实施方案中，相比于其他PI4K蛋白的亚型(例如，PI4K蛋白，包括PI4KIIα或PI4KIIβ)，所述PI4KIIIα特异性抑制剂对PI4KIII的抑制强至少1倍、2倍、4倍、5倍、10倍、20倍、30倍、50倍、100倍、200倍、500倍或10000倍。例如，在一些实施方案中，PI4KIIIα特异性抑制剂基本上不抑制PI4KII(例如，PI4KIIα或PI4KIIβ)，例如，其IC 50大于或等于10μM、20μM、30μM、40μM、50μM、60μM、80μM、100μM、150μM、200μM或500μM。
在一些实施方案中，相比于对PI4KIII的其他亚型(例如PI4KIIIβ)，PI4KIIIα特异性抑制剂对PI4KIIIα的抑制强至少1倍、2倍、4倍、5倍、10倍、20倍、30倍、50倍、100倍、200倍、500倍或10000倍。
在一些实施方案中，PI4KIIIα特异性抑制剂对PI4KIIIα的IC 50小于或等于100μM、80μM、50μM、30μM、20μM、10μM、5μM、3μM、2μM、1μM、0.5μM、0.2μM、0.1μM、0.05μM、0.02μM、0.01μM、0.005μM、0.002μM或0.001μM。在一些实施方案中，所述PI4KIIIα特异性抑制剂是抗体、小分子化合物、RNAi分子或者反义核酸。
在一些实施方案中，所述PI4KIIIα特异性抑制剂是抗体。
本文所用的术语“抗体”包含可结合某特定抗原的任意免疫球蛋白、单克隆抗体、多克隆抗体、多价抗体、双价抗体、单价抗体或抗 体。本文中的“抗体”一词旨在广泛地涵盖常规的四链抗体以及不具有四条链的较不常规的抗体(例如，天然缺乏轻链的抗体)。
一个常规的完整抗体是异四聚体，其包含两条重(H)链和两条轻(L)链。哺乳动物的重链可分为α、δ、ε、γ和μ，每条重链由一可变区(VH)和第一、第二、第三恒定区(分别为CH1、CH2、CH3)组成；哺乳动物的轻链可分为λ或κ，每条轻链由一可变区(VL)和一恒定区组成。常规抗体呈“Y”型，“Y”型结构的颈部由两条重链的第二和第三恒定区组成，其通过二硫键结合。“Y”型结构的每条臂包含其中一条重链的可变区和第一恒定区，其与一条轻链的可变区和恒定区结合。轻链和重链的可变区决定抗原的结合。每条链的可变区均含有三个高变区，称互补决定区(CDR)(轻链的CDR包含LCDR1、LCDR2、LCDR3，重链的CDR包含HCDR1、HCDR2、HCDR3)。其中，三个CDR由被称为框架区(FR)的侧面连续部分间隔开，框架区比CDR更加高度保守并形成一个支架支撑超变环。重链和轻链的恒定区与抗原结合无关，但具有多种效应功能。
在本发明的一些实施方案中，所述抗体是全长抗体或抗原结合片段。
本文中使用的术语“抗原结合片段”指由含有一个或多个CDR的抗体部分但不具有完整抗体结构的抗体片段所形成的一种抗体片段。抗原结合片段的例子包括但不限于Fab片段、Fab'片段、F(ab')2片段、Fv片段、单链抗体分子(scFv)、scFv二聚体、骆驼化单域抗体(camelized single domain antibody)和纳米抗体。抗原结合片段可以与母体抗体结合相同的抗原。
抗体的“Fab”片段是指由一条轻链(包括可变区和恒定区)和一条重链的可变区和第一恒定区经二硫键结合起来的那部分抗体。
“Fab'”片段是指包含了部分铰链区的Fab片段。
“F(ab')2”片段是指Fab'的二聚体。
抗体的“Fv”片段由一条轻链的可变区和一条重链的可变区组成。
“单链抗体分子”或“scFv”是指由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体。详细介绍可以参见例如 Huston JS等，Proc Natl Acad Sci USA、85:5879(1988)。
“scFv二聚体”是指由两个scFv形成的聚合体。
“骆驼化单域抗体(Camelized single domain antibody)”(又称为“重链抗体”或“HCAb(Heavy-chain-only antibodies，HCAb)”)是指含有两个重链可变区而不含有轻链的抗体。重链抗体最初从驼科(骆驼、单峰驼和美洲驼)衍生得到。虽然缺失轻链，但是骆驼化抗体具有抗原结合的全部功能。
“纳米抗体”是由一个来自重链抗体的重链可变区和两个恒定区CH2和CH3组成。
在一些实施方案中，所述抗体是单克隆抗体或者多克隆抗体。
在一些实施方案中，所述抗体是鼠源抗体、兔源抗体、嵌合抗体、人源化抗体或者全人源抗体。
本文中使用的术语“全人源”当用于抗体或抗原结合片段时，是指所述抗体或抗原结合片段的氨基酸序列对应于由人或人免疫细胞生产的、或从例如利用人源抗体库的转基因非人动物等非人来源衍生的抗体的氨基酸序列，或者其他编码人源抗体的序列。
本文中使用的术语“人源化”当用于抗体或抗原结合片段时，是指包括来源于非人动物的CDR、来源于人的FR区，以及来源于人的恒定区(当适用时)的抗体或抗原结合片段。由于人源化的抗体或抗原结合片段具有更低的免疫原性，其在某些实施方式中可用作人的治疗剂。在某些实施方式中，所述非人动物是哺乳动物(例如，小鼠、大鼠、兔、山羊、绵羊、豚鼠或仓鼠)。在某些实施方式中，所述人源化抗体或抗原结合片段除了CDR序列是非人源的以外，基本上全部由人源序列组成。
本文中使用的术语“嵌合”当用于抗体或抗原结合片段时，是指具有来源于一种物种的重链和/或轻链的一部分，而所述重链和/或轻链其余部分来源于不同物种的抗体或抗原结合片段。在一些实施方式中，嵌合抗体可以包括来源于人的恒定区和来源于非人动物(例如小鼠或兔)的可变区。
在一些实施方式中，本文所述抗体是单特异性抗体、双特异性抗 体或多特异抗体。
在一些实施方式中，本文所述抗体可以进一步地被标记。
在一些实施方案中，所述PI4KIIIα特异性抑制剂是RNAi分子。
本文所用的术语“RNAi分子”是指RNA或其类似物，其具有与靶RNA充分的序列互补性，以指导RNA干扰。在一些实施方式中，还包括可用于生成RNA的DNA。RNA干扰(RNAi)是指序列特异或选择的过程，通过该过程下调了靶分子(例如，靶基因、蛋白或RNA)。
在一些实施方案中，所述RNAi分子能够降低PI4KIIIα的表达，例如，敲减PI4KA基因。
在一些实施方案中，所述RNAi分子的长度为18-100碱基。
在一些实施方案中，所述RNAi分子被修饰以增强其稳定性。
在一些实施方案中，所述RNAi分子是小干扰RNA(siRNA)、短发夹RNA(shRNA)或微小RNA(miRNA)。
本文所用的术语“小干扰RNA(siRNA)”是指一种RNA分子，优选双链分子，具有约10～50个核苷酸的长度，优选约15～25个核苷酸的长度，更优选约17、18、19、20、21、22、23、24或25个核苷酸的长度，所述链任选地具有包括例如1、2或3个突出的核苷酸(或核苷酸类似物)的突出末端，其能够指导或介导RNA的降解。
本文所用的术语“短发夹RNA(shRNA)”是指具有茎环结构的RNA分子，其包括互补序列的第一区域和第二区域，互补的程度以及区域的方向足以使碱基对出现在区域之间，第一区域和第二区域被环区域连接，环由环区域中的核苷酸(或核苷酸类似物)之间缺少碱基配对而产生。
本文所用的术语“微小RNA(microRNA或miRNA)”是一种短的、天然存在的非编码的单链RNA分子，长度约为16-26个核苷酸(nt)(例如约16-29nt，19-22nt，20-25nt，或21-23nt)，通常在体内参与调控基因表达。在真核细胞中，miRNA基因通过DNA转录酶II(DNA transcriptase II)转录为“初产物”(pri-miRNA)，pri-miRNA很快被一种核糖核酸酶III(Drosha)加工为miRNA“前体”(pre-miRNA)，pre-miRNA从细胞核转运到细胞质中，然后经另一种核糖核酸酶III (Dicer)识别剪切为成熟的miRNA。成熟的miRNA分子与一种或多种mRNA部分互补，并且调控蛋白的表达。已知的miRNA的序列可以通过公开的数据库获得，例如miRBase数据库(www.mirbase.org)，其中提供了包括miRNA序列信息、功能注释、以及预测的基因靶标等信息。在本发明中，miRNA还包括由人工合成的质粒在细胞中表达的具有类似天然miRNA的结构和功能的RNA分子，能像天然miRNA靶向对应的mRNA，阻碍其翻译成蛋白质。
在一些实施方案中，所述PI4KIIIα特异性抑制剂是反义核酸。
本文所用的术语“反义核酸”包括与靶序列完全互补的核苷酸以及具有一个或多个核苷酸错配的那些核苷酸，只要该反义核酸能与所述靶序列特异性杂交即可。例如，本文的反义核酸包括在至少15个连续核苷酸的长度上具有至少70％或更高，优选80％或更高，更优选90％或更高，甚至更优选95％或更高同源性的多核苷酸。由于形成杂交体，靶基因转录和/或靶mRNA翻译减少或被阻断。。
在一些实施方案中，所述PI4KIIIα抑制剂是小分子化合物。
本文所用的术语“小分子化合物”是指一种分子量小于3000、2500、2000、1500、1000或500道尔顿的有机化合物，该有机化合物可以是天然的或者是化学合成的。
其中所述小分子化合物具有式(I)所示的结构或者其药学上可接受的盐，
Figure PCTCN2021080540-appb-000007
其中，R 1各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、C 1-6亚烷基-NH 2、C 1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H、-NH-S(O) 2H、-C(O)OH、-OC(O)H、-SH、-S(O) 2H、-S(O) 2-NH 2或杂环基，并且可选的被R 2或R 3取代，其中R 2和R 3各自独立的选自氨基、C 1-6烷基、C 1-6 烷氧基、C 1-6卤代烷基、N-(C 1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C 1-6烷基) 2氨基、C 3-6环烷基、6-12元的芳香基或3-12元的杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、-NH-C(O)-R 5、-C(O)OR 4、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，并且R 4是C 1-6的烷基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，R 5选自H、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基或C 1-6卤代烷基，和/或
(b)两个相邻碳原子上的R 1形成5-12元的环烷基、芳香基或杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，
其中，n为0-5的整数。
在一些实施方案中，所述n为0、1、2或者3。在一些实施方案中，所述n为0、1或者2。在一些实施方案中，所述n为0或者1。
在一些实施方案中，所述n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
在一些实施方案中，所述n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
在一些实施方案中，所述n为1或2，所述R 1各自独立的选自H、卤素、氨基、C 1-6烷氧基、C 1-6卤代烷基、-NH-C(O)R 2或-NH-S(O) 2R 3，其中R 2为C 1-6烷基，可选的被一个6-12元芳香基取代，R 3为6-12元芳香基，可选的被一个卤素、C 1-6烷氧基或C 1-6卤代烷基取代。
在一些实施方案中，所述R 1位于-As(O)基团的邻位和/或对位。
在一些实施方案中，所述R 1是H。
本文中所用的术语“取代”，当指化学基团时，指所述化学基团的一个或多个氢原子被去除并且被取代基取代。
本文中所用的术语“取代基”具有本领域公知的通常含义，指共价连接至或者在适合的情况下稠合至母体基团的化学部分。
本文中使用的术语“C n-C m”表示碳原子数的范围，其中n和m是整数并且碳原子数的范围包括端点(即n和m)和其间的各整数点。例如，C 1-C 6表示1至6个碳原子的范围，包括1个碳原子、2个碳原子、3个碳原子、4个碳原子、5个碳原子和6个碳原子。
本文中使用的术语“烷基”，无论是作为其他术语的一部分还是单独使用，指饱和的烃基基团，其可以是直链的或支链的。术语“C n-C m烷基”指具有n至m个碳原子的烷基。在某些实施方案中，烷基基团含有1至12、1至8、1至6、1至4、1至3或1至2个碳原子。烷基基团的示例包括但不限于化学基团如甲基、乙基、n-丙基、异丙基、n-丁基、叔丁基、异丁基、仲丁基、2-甲基-1-丁基、n-戊基、3-戊基、n-己基、1,2,2-三甲基丙基等。
本文中使用的术语“烯基”，无论是作为其他术语的一部分还是单独使用，指不饱和的烃基基团，其可以是直链的或支链的，具有至少一个碳-碳双键。在某些实施方案中，烯基基团含有2至12、2至10、2至8、2至6、2至5、2至4或2至3个碳原子。在某些实施方案中，烯基基团还能有1至6、1至5、1至4、1至3、1至2或1个碳碳双键。烯基基团的示例包括但不限于化学基团如乙烯基、n-丙烯基、异丙烯基、n-丁烯基、仲丁烯基等。
本文中使用的术语“炔基”，无论是作为其他术语的一部分还是单独使用，指不饱和的炔基基团，其可以是直链的或支链的，具有至少一个碳-碳三键。在某些实施方案中，炔基基团含有2至12、2至10、2至8、2至6、2至5、2至4或2至3个碳原子。在某些实施方案中，炔基基团还能有1至6、1至5、1至4、1至3、1至2或1个碳碳三键。炔基基团的示例包括但不限于化学基团如乙炔基、丙炔基、丁炔基等。
本文中使用的术语“环烷基”指至少由3个原子组成的环系烷基。 术语“n-m元环烷基”指具有n至m个形成环的成员的环烷基。此外，该环还可以具有一个或多个双键，但不具有完全共轭的系统。在某些实施方案中，环烷基具有3至8、3至6或4至6个形成环的碳原子。环烷基的示例包括但不限于环丙烷、环丁烷、环戊基等。
本文中使用的术语“杂环基”指在环系中的至少一个原子是杂原子而其余环原子是碳原子的环基。术语“n-m元杂环基”指具有n至m个形成环的成员的杂环基。本文中使用的术语“杂环基”包括杂芳基和杂环烷基。此外，该环还可以具有一个或多个双键。在某些实施方案中，杂环基是饱和的杂环烷基。杂原子的示例包括但不限于氧、硫、氮、磷等。
本文中使用的术语“杂环烷基”指在环系中的至少一个原子是杂原子而其余环原子是碳原子的环烷基。术语“n-m元杂环烷基”指具有n至m个形成环的成员的杂环烷基。此外，该环还可以具有一个或多个双键，但不具有完全共轭的系统。在某些实施方案中，杂环烷基是饱和的杂环烷基。杂原子的示例包括但不限于氧、硫、氮、磷等。在某些实施方案中，杂环烷基具有3至8、3至6或4至6个形成环的碳原子。杂环烷基的示例包括但不限于氮杂环丁烷、氮丙啶、吡咯烷基、哌啶基、哌嗪基、吗啉基、硫代吗啉、高哌嗪等。
本文中使用的术语“芳基”或“芳香基”，无论是作为其他术语的一部分还是单独使用，指在形成环的碳原子之间具有交替的双键和单键的单-或多-碳环环系基团。术语“C n-C m芳基”指具有n至m个形成环的碳原子的芳基。在某些实施方案中，芳基环系在一个或多个环中具有6至12、6至10或6至8个碳原子。在某些实施方案中，芳基环系具有稠合在一起的2个或多个环。芳基基团的示例包括但不限于化学基团如苯基、萘基、四氢萘基、茚满基、茚基等。
本文中使用的术语“杂芳基”指其中在芳环中的至少一个环原子是杂原子并且其余的环原子是碳原子的芳基基团。术语“n-m元杂芳基”指具有n至m个形成环的成员的杂芳基。杂原子的示例包括但不限于氧、硫、氮、磷等。在某些实施方案中，杂芳基可以具有5至10、5至8或5至6个形成环的成员。在某些实施方案中，杂芳基是5元或6元杂 芳基。杂芳基的示例包括但不限于呋喃基、噻吩基、吡啶基、喹啉基、吡咯基、N-低级烷基吡咯基、吡啶基-N-氧化物、嘧啶基、吡嗪基、咪唑基、吲哚基等。
本文中使用的术语“烷氧基”，无论是作为其他术语的一部分还是单独使用，指式“-O-烷基”所示的基团。术语“C n-C m烷氧基”指烷氧基的烷基部分具有n至m个碳原子。在某些实施方案中，烷基部分具有1至6、1至4或1至3个碳原子。烷氧基基团的示例包括但不限于化学基团如甲氧基、乙氧基、丙氧基(例如n-丙氧基和异丙氧基)、t-丁氧基等。
本文中使用的术语“卤代烷基”，无论是作为其他术语的一部分还是单独使用，指式“-烷基-X”所示的基团，其中X为卤素，选自氟、氯、溴和碘的原子。术语“C n-C m卤代烷基”指卤代烷基的烷基部分具有n至m个碳原子。在某些实施方案中，烷基部分具有1至6、1至4或1至3个碳原子。卤代烷基基团的示例包括但不限于化学基团如卤代甲基、卤代乙基、卤代丙基(例如n-卤代丙基和异卤代丙基)、t-卤代丁基等。
本文中使用的术语“n元”，其中n是整数，其通常与环系一起使用以描述环系中形成环的原子数。例如，哌啶基为6元杂环烷基环的一个示例，吡唑基为5元杂芳基环的一个示例，吡啶基为6元杂芳基环的一个示例而1,2,3,4-四氢-萘为10元芳基的一个示例。本文中使用的术语“n-m元”通常与环系一起使用以描述环系中形成环的成员的数量，其中n和m是整数并且形成环的成员的范围包括端点(即n和m)和其间的各整数点。例如，3-8元表示3至8个形成环的成员的范围，包括3个成员、4个成员、5个成员、6个成员、7个成员和8个成员。
本文中使用的术语“卤素”指选自氟、氯、溴和碘的原子。
本文中使用的术语“氰基”指式“-CN”所示的基团。
本文中使用的术语“羟基”指式“-OH”所示的基团。
本文中使用的术语“硝基”指式“-NO 2”所示的基团。
本文中使用的术语“氨基”指式“-NH 2”所示的基团。
本文中使用的术语“氨基甲酰基”指式“-HNCONH 2”所示的基 团。
本文中使用的术语“化合物”旨在包括所示结构的所有立体异构体(例如对映体和非对映体)、几何异构体、互变异构体和同位素。
本文所述的化合物可以是不对称的(例如具有一个或多个立体中心)。除非另外指明，所有的立体异构体，例如对映体和非对映体，都旨在包含在内。在本文所述的化合物中还可以存在烯烃、碳-碳双键等多种几何异构体，并且在本文中已经考虑了所有的这些稳定异构体。本文描述了化合物的顺式和反式几何异构体并且其可以以异构体的混合物或单独的异构体形式分离。
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本文的化合物还包括互变异构形式。互变异构形式是由同时伴有质子的迁移的单键与相邻双键的对换导致的。互变异构形式包括具有相同化学式和总电荷的异构质子化状态的质子的互变异构体。质子互变异构体的示例包括酮-烯醇对、酰胺-亚胺酸对、内酰胺-内酰亚胺对、烯胺-亚胺对和环状的形式，其中质子能够占据杂环系统的两个或多个位置，例如1H-和3H-咪唑、1H-，2H-和4H-1,2,4-三唑、1H-和2H-异吲哚以及1H-和2H-吡唑。互变异构形式通过适宜的取代能够平衡或空间锁定成一种形式。
在某些实施方案中，本文的小分子化合物可以通过有机合成获得。可以使用任意公知的有机合成技术制备并且可以根据多种可能的合成途径合成本文的化合物，包括其盐、酯、水合物或溶剂化物。
在一些实施方案中，本文所述小分子化合物为具有下列结构式的化合物，包括其中的一种或多种：
Figure PCTCN2021080540-appb-000008
Figure PCTCN2021080540-appb-000009
Figure PCTCN2021080540-appb-000010
Figure PCTCN2021080540-appb-000011
本文中使用的术语“氧化苯砷”(PAO)是指具体化学结构如下的小分子化合物：
Figure PCTCN2021080540-appb-000012
本文中使用的术语“A1”和“G1”均为PI4KIIIα蛋白的小分子化合物抑制剂，具有较类似的结构。其中，A1的化合结构式为：
Figure PCTCN2021080540-appb-000013
5-(2-氨基-1-(4-(4-吗啉基)苯基)-1H-苯并咪唑-6-基)-N-(2-氟代苯基)-2-甲氧基-3-吡啶磺酰胺
G1的化学结构式为：
Figure PCTCN2021080540-appb-000014
(aS)-5-(2-氨基-4-氧代-3-(2-(三氟甲基)苯基)-3,4-二氢喹唑啉-6-基)-N-(2,4-二氟代苯基)-2-甲氧基吡啶-3-磺酰胺。
本文中使用的术语“司美匹韦”(Simeprevir)(C 38H 47N 5O 7S 2)，原为用于治疗HCV药物，后来试验证明司美匹韦也可以抑制PI4KIIIα，它抑制作用的IC 50为200nM(Kwon J,Kim D,Park J,Park Y,Hwang Y,Wu H,Shin K,Kim I.Targeting Phosphatidylinositol 4-Kinase IIIa for Radiosensitization:A Potential Model of Drug Repositioning Using an Anti-Hepatitis C Viral Agent.Int J Radiation Oncol Biol Phys,2016,Vol.96(4)pp.867-876)。
本文还涉及氧化苯砷的衍生物、A1、G1或司美匹韦的类似物，只要它们具备抑制PI4KIIIα蛋白的磷酸激酶活性的功能，也同样可以用于治疗冠状病毒类疾病特别是新型冠状病毒，并且该类结构类似物的制备方法也均已公开。在一些实施例中，所述的氧化苯砷、A1、G1或司美匹韦的衍生物为与氧化苯砷、A1、G1或司美匹韦结构类似的类似物。
冠状病毒
本文所用的术语“冠状病毒”(Coronavirus，CoV)是指一类有包膜的单股正链RNA病毒，能感染人及多种动物，具有呼吸道、胃肠道以及神经系统嗜性，可在家畜和伴侣动物(如猪、牛、鸡、狗、猫)中引起严重疾病，并可导致人罹患从普通感冒到严重的急性呼吸综合征等疾病。国际病毒分类委员会第九次报告根据冠状病毒的进化特点将其分为α、β、γ以及6四个群，其中，α和β群的宿主以哺乳动物为主，γ和6群主要发现于鸟类和禽类。
能感染人的冠状病毒包括但不限于引起上呼吸道感染症状普通感冒的人冠状病毒229E(HCoV-229E)、NL63(HCoV-NL63)、HKU1(HCoV-HKU1)、OC43(HCoV-OC43)、能导致重症呼吸道疾病的严重急性呼吸道综合症冠状病毒(Severe acute respiratory syndrome coronavirus，SARS-CoV)和新型冠状病毒(SARS-CoV-2)、中东呼吸综合征冠状病毒(Middle east respiratory syndrome coronavirus，MERS-CoV)。
能感染动物的冠状病毒包括但不限于猪传染性胃肠炎病毒(TGEV)、猪δ冠状病毒(PDC)、猪血凝性脑脊髓炎病毒(PHEV)、犬呼吸道型冠状病毒(CrCoV)、小鼠肝炎病毒、猫冠状病毒(FCoV)。
药物施用和医药用途
本文中使用的术语“药学上可接受的”指在合理的医学判断范围内适宜用于与人和动物的组织接触而无过度的毒性、刺激、过敏反应或者其他问题或并发症，具有合理的收益/风险比的那些化合物、材料、组合物和/或剂型。在某些实施方案中，药学上可接受的化合物、材料、组合物和/或剂型指由管理机构批准(如美国食品药品管理局、中国食品药品管理局或欧洲药品局)或者列于普遍认可的药典中(如美国药典、中国药典或欧洲药典)的用于动物(更特别地用于人)的那些。
本文中所用的术语“客体”可以包括人类和非人动物。非人动物包括所有的脊椎动物，例如哺乳动物和非哺乳动物。“客体”也可以 是家畜动物(例如，牛、猪、羊、鸡、兔或马)，或啮齿类动物(例如，大鼠或小鼠)，或灵长类动物(例如，大猩猩或猴子)，或家养动物(例如，狗或猫)。“客体”可以是雄性或者雌性，也可以是不同年龄阶段。人类“客体”可以是高加索人、非洲人、亚洲人、闪族人，或其他种族，或不同种族的杂合体。人类“客体”可以是老年、成年、青少年、儿童或者婴儿。
在一些实施方案中，本文所述的客体是人或者非人灵长类动物。
本文中公开的PI4KIIIα特异性抑制剂可通过本领域公知的给药途径进行施用，例如注射给药(如，皮下注射、腹腔注射、静脉注射(包括静脉滴注或静脉输注)、肌肉注射或皮内注射)或非注射给药(如，口服给药、鼻腔给药、舌下给药、直肠给药或外用给药)。在一些实施方案中，本文所述的PI4KIIIα特异性抑制剂通过口服、皮下、肌内或静脉施用。在一些实施方案中，本文所述的PI4KIIIα特异性抑制剂通过口服施用。
本文中使用的术语“治疗有效量”是指，可以缓解或者消除客体的疾病或症状，或者可以预防性地抑制或防止疾病或症状发生的药物的量。治疗有效量可以是将客体的一种或多种疾病或症状缓解到一定程度的药物的量；可以将那些跟疾病或症状成因相关的一种或多种生理或生物化学参数部分或完全恢复到正常的药物的量；和/或可以降低疾病或症状发生的可能性的药物的量。在一些实施方案中，本文中使用的术语“治疗有效量”是指，可以缓解或者消除客体的冠状病毒(例如，新型冠状病毒)的药物的量。
本文中提供的PI4KIIIα特异性抑制剂的治疗有效剂量依赖于本领域公知的多种因素，例如体重、年龄、过往病史、目前正在接受的治疗、对象的健康状况和药物相互作用的强度、过敏、超敏和副作用，以及给药途径和疾病发展的程度。本领域熟练人员(例如医生或兽医)可根据这些或其它条件或要求相应降低或升高剂量。
在一些实施方案中，治疗进一步包括向需要其的客体施用第二试剂。
在一些实施方案中，所述第二试剂是用于治疗冠状病毒类疾病的 试剂，包括但不限于洛匹那韦(Lopinavir)、利托那韦(Ritonavir)、瑞姆西韦(Remdesivir)、氯喹(Chloroquine)、司美匹韦(Simeprevir)。
在一些实施方案中，所述PI4KIIIα特异性抑制剂在所述第二试剂之前、之后或同时施用。
本申请的还涉及用于预防或治疗冠状病毒类疾病的方法，包括向需要其的客体施用有效量的PI4KIIIα特异性抑制剂。
药物筛选
本发明还提供了一种筛选用于预防或治疗冠状病毒类疾病的药物的方法，包括将备选药物与PI4KIIIα蛋白或者核酸或者PI4KIIIα接触，并检测备选药物是否能够抑制PI4KIIIα的形成或者活性。
实施例1.氧化苯砷等化合物的溶液配制
在弱光环境下，用电子天平(购自瑞士梅特勒公司产品)准确称取适量的PAO(氧化苯砷，代号PI01，委托上海凯惠药业合成)、YL-05、YL-07、YL-08、YL-09、YL-10、YL-11(5个化合物都委托BioChemPartner合成，结构式如下)、司美匹韦(Simeprevir，代号PI0101)、PI4KIIIβ蛋白的特异抑制剂PIK-93(MedChemExpress)、Remdesivir(瑞德西韦)和Chloroquine phosphate(磷酸氯喹)等化合物粉剂，用5.0mL DMSO溶解，储存氧化苯砷浓度为20mM或40mM。溶液用0.22μM滤器过滤除菌后-20℃保存备用，注意避光。
Figure PCTCN2021080540-appb-000015
Figure PCTCN2021080540-appb-000016
实施例2.CCK-8细胞增殖-毒性检测法检测不同浓度的氧化苯砷对Vero细胞的毒性
Vero细胞(非洲绿猴肾细胞Vero，浙江大学传染病诊治国家重点实验室保存)以5000个/孔的浓度接种96孔培养板(购自Corning公司)，37℃、5％CO 2孵箱(Thermo3110，购自Thermo美国热电公司)培养，至细胞单层，弃掉培养液，采用Hank’s溶液洗细胞2次。
药物溶液稀释：第一轮实验时，采用MEM培养基(购自Life Technologies公司)稀释氧化苯砷系列浓度，起始浓度为200nM，连续对倍稀释7个浓度至1.56nM。每个氧化苯砷浓度设2复孔，每孔加药液150μL，同时设Vero细胞正常生长对照，37℃、5％CO 2孵箱培养48小时。第二轮实验时，氧化苯砷和PIK-93的起始浓度为800nM，连续对倍稀释7个浓度至6.25nM；瑞德西韦(Remdesivir)起始浓度为300μM，连续三倍稀释6个浓度至0.4μM；磷酸氯喹(Chloroquine)起始浓度为300μM，连续三倍稀释6个浓度至0.4μM。PIK-93起始浓度为800nM，连续对倍稀释7个浓度至6.25nM。每个药物浓度设2复孔，每孔加药液150μL，同时设Vero细胞正常生长对照，37℃、5％CO 2孵箱培养48h。
每孔加入10或15μL的CCK-8试剂(细胞增殖-毒性检测试剂盒(CCK-8)(购自日本DojinDo公司))，3小时后用酶标仪(Bio-Rad 680，购自美国BioRad公司)测定在450nm处的OD值，计算氧化苯砷对细胞的剂量毒性效应及最大无毒性浓度，绘制氧化苯砷-细胞毒性反应曲线， 为体外抗病毒药效的最适试验浓度选择提供依据。
第一轮试验结果显示，200nM及以上浓度的氧化苯砷对Vero细胞具有显著的毒性作用，细胞死亡达50％，即CC 50＞200nM。在100nM以下浓度时氧化苯砷对细胞无明显的毒性作用，细胞存活力大于90％以上(见表1A和图1A)。
表1A：不同浓度的氧化苯砷对Vero细胞的毒性作用
Figure PCTCN2021080540-appb-000017
第二轮试验结果显示，400nM及以上浓度的氧化苯砷(PI01)药物对Vero细胞具有显著的毒性作用，细胞死亡达50％以上，CC 50为380nM。在100nM及以下浓度时药物对细胞无明显的毒性作用，细胞存活力大于90％以上，CC 10为100nM。
800nM及以下浓度的PI4KIIIβ蛋白的特异抑制剂PIK-93对Vero细胞没产生显著的毒性作用。
对照药物瑞德西韦(Remdesivir)，对Vero细胞的CC 50为208μM，在50μM及以下浓度时药物对细胞无明显的毒性作用，细胞存活力大于90％以上，CC 10为55μM。
对照药物磷酸氯喹(Chloroquine)，对Vero细胞的CC 50约为250μM，CC 10为120μM。
具体结果见【表1B、C、D和图1B、C、D】。
表1B：不同浓度的氧化苯砷药物对Vero细胞的毒性作用
Figure PCTCN2021080540-appb-000018
表1C：不同浓度的瑞德西韦药物对Vero细胞的毒性作用
Figure PCTCN2021080540-appb-000019
表1D：不同浓度的磷酸氯喹药物对Vero细胞的毒性作用
Figure PCTCN2021080540-appb-000020
实施例3.微量细胞病变观察法测定新型冠状病毒TCID 50
Vero细胞以10000个/孔的浓度接种96孔培养板，37℃、5％CO 2孵箱培养至细胞对数生长期最旺盛75％-90％成片细胞，弃掉细胞培养液，采用Hank’s溶液洗细胞2次(以去除残留的牛血清)。
新型冠状病毒由临床感染者痰液样本中分离获得，并经全基因测序验证，由浙江大学传染病诊治国家重点实验室保存。实验在生物安全三级实验室(BSL-3:实验室认证编号：CNAS BL002，浙江大学传染病诊治国家重点实验室)进行。
将新型冠状病毒株用病毒生长液(500ml MEM培养基中含FBS2％、青霉素100U/mL、链酶素100μg/mL，TPCK-胰酶16μg/mL)(均购自Life Technologies公司)1000倍稀释后作为起始浓度，再依此作10倍梯度稀释8个系列浓度。每个病毒稀释度接种细胞4孔，每孔100μl，同时设正常细胞对照，37℃、5％CO 2孵箱6天。
每天在倒置显微镜下观察细胞形态及病变效应(CPE)变化，以25％以下细胞形态CPE变化为“+”，26％-50％细胞形态CPE变化为“++”，51％-75％细胞形态CPE变化为“+++”，76％-100％细胞形态CPE变化为“++++”，用Reed-Muench法，计算病毒半数感染浓度TCID50。
结果实验用的新冠病毒TCID 50为10 -6.5/100μL。
实施例4.氧化苯砷体外对新型冠状病毒(SARS-Cov-2)的抑制作用
细胞培养：Vero细胞以50000个/1mL/孔的浓度接种24孔板，37℃、5％CO 2孵箱培养至细胞对数生长期最旺盛75％-90％成片细胞，弃掉细胞培养液，采用Hank’s溶液洗细胞2次。
药物液配制：储存浓度的药物用培养基分别稀释至以下终浓度：在第一轮实验中氧化苯砷起始浓度为200nM，连续对倍稀释7个浓度至 1.56；在第二轮实验中，氧化苯砷和PIK-93的始浓度为800nM，连续对倍稀释7个浓度至6.25nM；瑞德西韦(Remdesivir)起始浓度为100μM，连续三倍稀释6个浓度至0.4μM；磷酸氯喹(Chloroquine)起始浓度为100μM，连续三倍稀释6个浓度至0.3μM。
病毒液配制：新型冠状病毒(SARS-CoV-2 strain BetaCov/Wuhan/IME-BJ01/2020(GWHACBB01000000))用培养基稀释至感染细胞终浓度100TCID 50。
病毒感染Vero细胞：将配制好的病毒(100TCID 50)250μL/孔感染Vero细胞，设单加病毒感染的细胞对照及正常细胞对照。37℃、5％CO 2孵箱中吸附3小时，弃掉含病毒的培养液，用Hank’s溶液洗2次。
药物处理：将配制好的含不同浓度药物的培养液1mL/孔，加至上述已预染毒的Vero细胞培养板上，每个氧化苯砷浓度设2个复孔。37℃、5％CO 2孵箱中继续培养至48小时。
采用荧光定量PCR法检测新型冠状病毒核酸。吸取培养上清液200μL，用磁珠法核酸提取试剂盒(MVR01)和全自动核酸提取仪(EX3600，上海之江生物科技股份有限公司)提取病毒核酸，最终洗脱体积为50μl。取5μL核酸提取物，采用一步法新型冠状病毒核酸检测试剂盒(荧光PCR法，货号Z-RR-0479-02-50，国械注准20203400057，购自上海之江生物科技股份有限公司)，检测病毒核酸水平。结果以Ct值来表示病毒的水平。应用该荧光PCR法，Ct值与病毒拷贝数对应关系为：y＝-3.33x+48.69，其中y为Ct值，x是以10为底的病毒数的对数。
第一轮实验结果显示在Vero细胞模型上，氧化苯砷20-100nM浓度时具有显著的抗病毒作用，EC 50＝20nM(见表2A及图2A)。表2中FAM Ct值表示病毒核酸水平。Ct值越大，说明病毒越少。反之，Ct越小，说明病毒越多。
第二轮实验结果显示，在Vero细胞模型上，PI01药物抑制新型冠状病毒的EC 50＝19.2nM。瑞德西韦(Remdesivir)抑制新型冠状病毒的EC 50＝0.52μM。磷酸氯喹(Chloroquine)抑制新型冠状病毒的EC 50＝50μM。PIk-93在800nM及以下浓度对新型冠状病毒没产生明显抑制作用。具体结果见表2B、C、D和图2B、C、D。
此外，我们还检测了PI0101对新型冠状病毒的抑制作用。细胞以每孔10,000个细胞的密度接种到96孔测试板中并于5％CO 2、37℃培养箱中培养过夜。第二天，加入3倍比稀释后的PI0101(起始浓度是10μM，8个浓度点、双复孔)，随后病毒以每孔100TCID 50加入细胞。设置细胞对照(细胞，无化合物处理或病毒感染)，病毒对照(细胞感染病毒，无化合物处理)和培养液对照(只有培养液)，5％CO 2、37℃培养箱中培养至76小时。结果发现，PI0101的EC 50＝3.85μM，CC 50大于10μM。
表2A：不同浓度的氧化苯砷体外抑制新型冠状病毒的作用
Figure PCTCN2021080540-appb-000021
表2B：不同浓度的氧化苯砷体外抑制新型冠状病毒的作用
Figure PCTCN2021080540-appb-000022
Figure PCTCN2021080540-appb-000023
表2C：不同浓度的瑞德西韦(Remdesivir)体外抑制新型冠状病毒的作用
Figure PCTCN2021080540-appb-000024
表2D：不同浓度磷酸氯喹(chloroquine)体外抑制新型冠状病毒的作用
Figure PCTCN2021080540-appb-000025
实施例5.通过细胞病变效应(CPE)实验检测氧化苯砷体外抗人冠状病毒(HCoV)229E活性
药物液配制：储存浓度的药物用培养基分别稀释至以下终浓度。在第一轮实验中，观察氧化苯砷、YL-05、YL-07、YL-08、YL-09、YL-10和YL-11化合物对MRC5细胞的毒性和对HCoV 229E(人冠状病毒229E型，购自ATCC)的抑制作用的起始浓度分别为800和200nM，连续对倍稀释7个浓度分别至6.25和1.56nM；瑞德西韦对MRC5细胞的毒性和对HCoV 229E的抑制作用的起始浓度分别为100μM和1000nM，连续三倍稀释7个浓度分别至0.41μM和0.46nM。
在第二轮实验中，观察氧化苯砷的细胞毒性和病毒抑制作用的起始浓度分别为800和400nM，连续对倍稀释7个浓度分别至6.25和3.13nM、YL-07、YL-08、YL-09、YL-10和YL-11化合物对MRC5细胞的毒性和对HCoV 229E的抑制作用的的起始浓度都是40μM，连续三倍稀释7个浓度0.02μM；瑞德西韦对细胞的毒性和抑制病毒作用的的起始浓度分别为100μM和1000nM，连续三倍稀释7个浓度分别至0.41μM和0.46nM。
MRC5细胞和HCoV 229E株购自ATCC。细胞使用添加了10％胎牛血清(Hyclone)，1％双抗(Hyclone)，1％L-谷氨酰胺(Gibco)和1％非必需氨基酸(Gibco)的EMEM(Sigma)培养液培养。添加了5％胎牛血清(Hyclone)，1％双抗(Hyclone)，1％L-谷氨酰胺(Gibco)和1％非必需氨基酸(Gibco)的EMEM(Sigma)培养液为实验培养液。
MRC5细胞以每孔20,000个细胞的密度接种到96孔测试板中并于5％CO 2、37℃培养箱中培养过夜。第二天，加入倍比稀释后的氧化苯砷(8个浓度点、双复孔)，随后病毒以每孔200TCID 50加入细胞。设置细胞对照(细胞，无化合物处理或病毒感染)，病毒对照(细胞感染病毒，无化合物处理)和培养液对照(只有培养液)。培养液中DMSO的终浓度分别为0.5％。细胞于培养箱中培养3天。使用细胞活力检测试剂盒CellTiter Glo(Promega)检测细胞活力。细胞毒性实验与抗病毒实验条件相同，但无病毒感染。氧化苯砷的抗病毒活性和细胞毒性分别由不同浓度下的化合物对病毒引起的细胞病变效应的抑制率(％)和MRC5细胞的活率(％)表示。计算公式如下：
抑制率(％)＝(测试孔读值-病毒对照平均值)/(细胞对照平均 值-病毒对照平均值)x 100
细胞活率(％)＝(测试孔读值-培养液对照平均值)/(细胞对照平均值-培养液对照平均值)x 100
使用GraphPad Prism(version 5)对氧化苯砷的抑制率和细胞活率进行非线性拟合分析，计算氧化苯砷的半数有效浓度(EC 50)。
第一轮结果显示，氧化苯砷的EC 50值为55.35nM，CC 50值为256.8nM；瑞德西韦EC 50值为26.42nM，CC 50值大于100μM；YL-05、YL-07、YL-08、YL-09、YL-10和YL-11化合物的EC 50都大于200nM，CC 50都大于800nM；具体结果请见图3A和表3。第二轮结果显示，氧化苯砷的EC 50值为37.03nM，CC 50值为68.92nM；瑞德西韦EC 50值为13.11nM，CC 50值为30.66μM；YL-07、YL-08、和YL-09化合物的EC 50都大于40μM,CC 50值在分别为6.24μM、7.64μM和13.88μM,YL-10和YL-11化合物的EC 50分别为3.95μM和2.1μM，CC 50分别为14μM和6.37μM；具体结果请见图3B和表3。PI0101(司美匹韦)的EC 50为3.41μM，CC 50为8.46μM。
表3.化合物测试结果
Figure PCTCN2021080540-appb-000026
注：ND，未检测
本领域的技术人员应当明了，尽管为了举例说明的目的，本文描述了本发明的具体实施方式，但可以对其进行各种修改而不偏离本发 明的精神和范围。因此，本发明的具体实施方式和实施例不应当视为限制本发明的范围。本发明仅受所附权利要求的限制。本文中引用的所有文献均完整地并入本文作为参考。

Claims (25)

  1. PI4KIIIα特异性抑制剂在制备用于预防或治疗冠状病毒类疾病药物中的用途。
  2. 根据权利要求1所述的用途，其中所述PI4KIIIα特异性抑制剂是抗体、小分子化合物、RNAi分子或者反义核酸。
  3. 根据权利要求2所述的用途，其中所述抗体是单克隆抗体或者多克隆抗体。
  4. 根据权利要求3所述的用途，其中所述抗体是嵌合抗体、人源化抗体或者全人源抗体。
  5. 根据权利要求2所述的用途，其中所述RNAi分子是小干扰RNA(siRNA)、短发夹RNA(shRNA)或微小RNA(miRNA)。
  6. 根据权利要求2或5所述的用途，其中所述RNAi分子的长度为18-100碱基。
  7. 根据权利要求2和5-6中任一项所述的用途，其中所述RNAi分子被修饰以增强其稳定性。
  8. 根据权利要求2所述的用途，其中所述PI4KIIIα特异性抑制剂是小分子化合物。
  9. 根据权利要求8所述的用途，其中所述小分子化合物是氧化苯砷或其衍生物、G1及其类似物、A1及其类似物、或司美匹韦及其类似物。
  10. 根据权利要求9所述的用途，其中所述氧化苯砷及其衍生物具有式(I)所示的结构或者其药学上可接受的盐，
    Figure PCTCN2021080540-appb-100001
    其中，R 1各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤 代烷基、C 1-6亚烷基-NH 2、C 1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H、-NH-S(O) 2H、-C(O)OH、-OC(O)H、-SH、-S(O) 2H、-S(O) 2-NH 2或杂环基，并且可选的被R 2或R 3取代，其中R 2和R 3各自独立的选自氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、N-(C 1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C 1-6烷基) 2氨基、C 3-6环烷基、6-12元的芳香基或3-12元的杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、-NH-C(O)-R 5、-C(O)OR 4、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，并且R 4是C 1-6的烷基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，R 5选自H、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基或C 1-6卤代烷基，和/或
    (b)两个相邻碳原子上的R 1形成5-12元的环烷基、芳香基或杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，
    其中，n为0-5的整数。
  11. 根据权利要求10所述的用途，其中n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
  12. 根据权利要求10所述的用途，其中n为0-2的整数，所述R 1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、-As(O)、-NH-C(O)H或-NH-S(O) 2H，并且可选的被所述R 2或R 3取代。
  13. 根据权利要求10所述的用途，其中n为1或2，所述R 1各自独立的选自H、卤素、氨基、C 1-6烷氧基、C 1-6卤代烷基、-NH-C(O)R 2 或-NH-S(O) 2R 3，其中R 2为C 1-6烷基，可选的被一个6-12元芳香基取代，R 3为6-12元芳香基，可选的被一个卤素、C 1-6烷氧基或C 1-6卤代烷基取代。
  14. 根据权利要求13所述的用途，其中所述R 1位于-As(O)基团的邻位和/或者对位。
  15. 根据权利要求10所述的用途，其中n是0。
  16. 根据权利要求9所述的用途，其中所述小分子化合物选自由以下化合物组成的组：
    Figure PCTCN2021080540-appb-100002
    Figure PCTCN2021080540-appb-100003
    Figure PCTCN2021080540-appb-100004
    Figure PCTCN2021080540-appb-100005
  17. 根据权利要求1所述的用途，其中所述客体是人或者哺乳动物。
  18. 根据权利要求1所述的用途，其中所述冠状病毒为新型冠状病毒。
  19. 根据权利要求1所述的用途，其中所述冠状病毒为鸡传染性支气管炎病毒、猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪血凝性脑脊髓炎病毒，猪δ冠状病毒、犬呼吸道型冠状病毒、小鼠肝炎病毒、猫冠状病毒、人冠状病毒、严重急性呼吸综合征病毒、中东呼吸综合征病毒。
  20. 根据权利要求1所述的用途，其进一步包括向需要其的客体施用第二试剂。
  21. 根据权利要求20所述的用途，其中所述第二试剂是用于治疗冠状病毒类疾病的试剂。
  22. 根据权利要求21所述的用途，其中所述PI4KIIIα特异性抑制剂在所述第二试剂之前、之后或同时施用。
  23. 一种筛选用于预防或治疗冠状病毒类疾病的药物的方法，包括将备选药物与PI4KIIIα蛋白或者核酸或者PI4KIIIα接触，并检测备选药物是否能够抑制PI4KIIIα的形成或者活性。
  24. 式(I)所述的化合物在制备用于预防或治疗冠状病毒类疾病药物中的用途：
    Figure PCTCN2021080540-appb-100006
    其中，R 1各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、C 1-6亚烷基-NH 2、C 1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C 1-6烷基)氨基、N,N-(C 1-6烷基) 2氨基、-NH-C(O)H、-NH-S(O) 2H、-C(O)OH、-OC(O)H、-SH、-S(O) 2H、-S(O) 2-NH 2或杂环基，并且可选的被R 2或R 3取代，其中R 2和R 3各自独立的选自氨基、C 1-6烷基、C 1-6烷氧基、C 1-6卤代烷基、N-(C 1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C 1-6烷基) 2氨基、C 3-6环烷基、6-12元的芳香基或3-12元的杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、-NH-C(O)-R 5、-C(O)OR 4、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，并且R 4是C 1-6的烷基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，R 5选自H、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基或C 1-6卤代烷基，和/或
    (b)两个相邻碳原子上的R 1形成5-12元的环烷基、芳香基或杂环基，并且可选的被一个或多个卤素、硝基、氰基、羟基、氨基、氨基甲酰基、6-12元的芳香基、C 1-6烷基、C 2-6炔基、C 2-6烯基、C 1-6烷氧基、C 1-6卤代烷基、3-6元的杂环基、C 3-6环烷基或Bn-O-取代，
    其中，n为0-5的整数。
  25. 根据权利要求24所述的用途，其中所述化合物为氧化苯砷。
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