WO2021197396A1 - 氘代氧化苯砷化合物及其应用 - Google Patents

氘代氧化苯砷化合物及其应用 Download PDF

Info

Publication number
WO2021197396A1
WO2021197396A1 PCT/CN2021/084773 CN2021084773W WO2021197396A1 WO 2021197396 A1 WO2021197396 A1 WO 2021197396A1 CN 2021084773 W CN2021084773 W CN 2021084773W WO 2021197396 A1 WO2021197396 A1 WO 2021197396A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
pao
d5pao
cancer
use according
Prior art date
Application number
PCT/CN2021/084773
Other languages
English (en)
French (fr)
Inventor
黄福德
王文安
洪峰
魏万国
章建刚
焦常平
曹鲁乡
Original Assignee
挪贝肽医药科技(上海)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to KR1020227035860A priority Critical patent/KR20230020946A/ko
Application filed by 挪贝肽医药科技(上海)有限公司 filed Critical 挪贝肽医药科技(上海)有限公司
Priority to MX2022011711A priority patent/MX2022011711A/es
Priority to CA3171783A priority patent/CA3171783A1/en
Priority to JP2022559630A priority patent/JP2023519424A/ja
Priority to IL296494A priority patent/IL296494A/en
Priority to US17/995,099 priority patent/US20240050395A1/en
Priority to PE2022002119A priority patent/PE20230042A1/es
Priority to AU2021250199A priority patent/AU2021250199B2/en
Priority to CN202180026241.1A priority patent/CN115515964A/zh
Priority to BR112022019864A priority patent/BR112022019864A2/pt
Priority to EP21780885.6A priority patent/EP4130014A4/en
Publication of WO2021197396A1 publication Critical patent/WO2021197396A1/zh
Priority to CONC2022/0015428A priority patent/CO2022015428A2/es

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/285—Arsenic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/004—Acyclic, carbocyclic or heterocyclic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur, selenium or tellurium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/66—Arsenic compounds
    • C07F9/70—Organo-arsenic compounds
    • C07F9/74—Aromatic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/66—Arsenic compounds
    • C07F9/70—Organo-arsenic compounds
    • C07F9/80—Heterocyclic compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the invention belongs to the field of chemical synthesis, and specifically relates to a novel deuterated phenylarsenic oxide compound and a preparation method and application thereof.
  • 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.
  • Recent studies have found that phenylarsenic oxide is a PI4KIII ⁇ inhibitor and can be used to treat Alzheimer's disease.
  • Deuterium is a stable isotope of hydrogen. Compared with hydrogen, deuterium can form stronger chemical bonds, making drug molecules more stable. Human trials have found that deuterium substitution can change the half-life of the drug, reduce the number of medications, while maintaining the original activity and selectivity. Deuterated drugs have become a new direction and mode of drug development for new drug research and development. In 2017, the US Food and Drug Administration approved the world's first deuterated drug, deuterated tetrabenazine (AUSTEDOTM, for the treatment of Huntington's disease and related motor dysfunction). At present, many deuterated drugs have entered clinical research.
  • AUSTEDOTM deuterated tetrabenazine
  • the present invention provides a compound of formula I or a pharmaceutically acceptable salt thereof,
  • R 1 , R 2 , R 3 , R 4 , R 5 are independently selected from hydrogen, deuterium, halogen, methyl, mono-deuterated methyl, di-deuterated methyl, or tri-deuterated methyl, and R 1 At least one of R 2 , R 3 , R 4 , and R 5 is deuterium or deuterated.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from hydrogen or deuterium, and at least one of R 1 , R 2 , R 3 , R 4 , and R 5 is deuterium, at least Two are deuterium, preferably at least three, four or five are deuterium.
  • the compound is selected from the group consisting of:
  • the present invention discloses the use of the aforementioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating a disease or pathological reaction in a subject.
  • the disease is selected from the group consisting of tumors, cachexia such as malignant tumors or cachexia caused by chemotherapeutics for the treatment of tumors, Alzheimer's disease, intracellular protein misfolding related diseases, lysosomal storage diseases, inflammation Reaction, tissue and organ fibrosis, virus infection disease, neurosis.
  • the subject is a human or non-human mammal.
  • the tumor is selected from lymphoma, cervical cancer, liver cancer, breast cancer such as triple negative breast cancer, lung cancer such as non-small cell lung cancer or small cell lung cancer, colorectal cancer, gastric cancer, skin cancer such as melanoma , Bone cancer, osteosarcoma, myeloma, blood cancer, or ovarian cancer. .
  • the intracellular protein misfolding-related disease is Parkinson’s disease, Lewy body dementia, multiple system atrophy, inclusion body muscle inflammation, frontotemporal dementia, Huntington’s disease, polyglutamine disease , Amyotrophic Lateral Sclerosis, or Prion Disease.
  • the lysosomal storage disease is a sphingolipid metabolism disorder such as Gaucher disease, Niemann's disease type C, mucopolysaccharidosis, glycogen storage disease, glycoprotein storage disease, lipid Storage disease, post-translational modification deficiency, intrinsic membrane protein deficiency disorder, neuronal ceroid lipofuscinosis, or lysosomal-related organelle disorder.
  • a sphingolipid metabolism disorder such as Gaucher disease, Niemann's disease type C, mucopolysaccharidosis, glycogen storage disease, glycoprotein storage disease, lipid Storage disease, post-translational modification deficiency, intrinsic membrane protein deficiency disorder, neuronal ceroid lipofuscinosis, or lysosomal-related organelle disorder.
  • the inflammatory response is an increase in inflammatory factors such as TNF ⁇ or IL-6 in local tissues or systemic blood.
  • the tissue organ fibrosis is selected from lung fibrosis or liver fibrosis.
  • the viruses include coronaviruses and non-coronaviruses.
  • the coronaviruses are selected from the group consisting of avian infectious bronchitis virus, porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus, and porcine hemagglutinating virus.
  • Encephalomyelitis virus, porcine delta coronavirus canine respiratory coronavirus, mouse hepatitis virus, feline coronavirus, human coronavirus, severe acute respiratory syndrome virus, Middle East respiratory syndrome virus, or new coronavirus; said The non-coronavirus is selected from hepatitis C virus or HIV.
  • the neurosis is selected from neurasthenia, anxiety, depression, or mania.
  • the present invention discloses the use of the aforementioned compound or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing or treating diseases in a subject, which further comprises administering a second agent to a subject in need thereof.
  • the present invention discloses the use of the aforementioned compound or a pharmaceutically acceptable salt thereof and a second agent in the preparation of a medicament for the prevention or treatment of a disease in a subject.
  • the disease is selected from tumors, and the second agent is an agent for the treatment of tumors.
  • the second agent is an agent for treating autopulmonary fibrosis, such as a vascular endothelial growth factor receptor tyrosine kinase inhibitor, preferably Nintedanib.
  • the second agent is an agent for treating tumors, and the agent for treating tumors is selected from at least one of paclitaxel, gemcitabine, cyclophosphamide, and temozolomide.
  • the aforementioned compound or a pharmaceutically acceptable salt thereof is administered before, after or at the same time as the second agent.
  • the present invention discloses a pharmaceutical composition
  • a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes a drug for treating tumors.
  • the drug for treating tumors is selected from at least one of paclitaxel, gemcitabine, cyclophosphamide, and temozolomide.
  • the present invention discloses a preparation method of the aforementioned compound or a pharmaceutically acceptable salt thereof, which comprises the following steps:
  • step 2) Heat the aqueous solution of sodium carbonate, arsenic trioxide, and copper sulfate to 90-100°C and then lower the temperature, add the solution prepared in step 1) above to the aqueous solution, stir and filter, add acid to the filtrate to adjust the pH value, and separate Precipitated solid;
  • the post-treatment in step 3) includes adjusting the pH to an appropriate value with acid or base, extracting with ethyl acetate, combining the organic phases and evaporating to dryness.
  • the present invention discloses the use of phenylarsenic oxide and its derivatives in the preparation of medicines for preventing or treating fibrosis of tissues and organs, such as pulmonary fibrosis or liver fibrosis.
  • the present invention discloses the use of phenylarsenic oxide and its derivatives in the preparation of drugs for the prevention or treatment of inflammatory reactions, wherein the inflammatory reaction is an increase in inflammatory factors such as TNF ⁇ or IL-6 in local tissues or systemic blood.
  • the present invention discloses the use of phenylarsenic oxide and its derivatives in the preparation of drugs for preventing or treating cachexia such as malignant tumors or chemotherapeutic drugs for treating tumors.
  • the present invention discloses the use of phenylarsenic oxide and its derivatives in the preparation of drugs for preventing or treating tumors.
  • the phenylarsenic oxide and its derivatives have the structure represented by formula (II) or a pharmaceutically acceptable salt thereof,
  • R 6 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, hydroxy, and amino groups.
  • n is an integer of 0-5.
  • each of R 6 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, carbamoyl, C1-6 alkylsulfone, C1-6 alkyl, C1-6 cycloalkyl, C1-6 alkoxy, C1-6 haloalkyl, -As(O), N-(C1-6 alkyl) amino, N,N-(C1- 6 Alkyl) 2 amino, -NH-C(O)H or -NH-S(O)2H, and optionally substituted by said R 7 or R 8.
  • each of R 6 is independently selected from H, halogen, nitro, cyano, hydroxyl, amino, C1-6 alkylsulfone, C1-6 alkane Group, C1-6 cycloalkyl, C1-6 alkoxy, C1-6 haloalkyl, -As(O), -NH-C(O)H or -NH-S(O)2H, and optional Substituted by said R 7 or R 8.
  • R 6 is each independently selected from H, halogen, amino, C1-6 alkylsulfone, C1-6 cycloalkyl, C1-6 alkoxy, C1-6 haloalkyl, -NH-C(O)R 7 or -NH-S(O)2R 8 , where R 7 is C1-6 alkyl, optionally substituted by a 6-12 membered aromatic group, R 8 is a 6-12 membered aromatic group, optionally substituted by a halogen, C1-6 alkoxy or C1-6 haloalkyl.
  • n is zero.
  • the compound is selected from the group consisting of:
  • the object is a human or a mammal.
  • the tumor is selected from lymphoma, cervical cancer, liver cancer, breast cancer such as triple negative breast cancer, lung cancer such as non-small cell lung cancer or small cell lung cancer, colorectal cancer, gastric cancer, skin cancer such as melanoma, Bone cancer, osteosarcoma, myeloma, blood cancer, or ovarian cancer.
  • it further comprises administering a second agent to the guest in need thereof, preferably the second agent is an agent for treating tumors.
  • the second agent is an agent for treating tumors.
  • the compound is administered before, after, or at the same time as the second agent.
  • the agent for treating tumors is selected from at least one of paclitaxel, gemcitabine, cyclophosphamide, and temozolomide.
  • the method further discloses a method for screening drugs for the prevention or treatment of 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 ⁇ . It is selected from tissue or organ fibrosis, inflammation, cachexia, and tumor.
  • tissue organ fibrosis is selected from lung fibrosis or liver fibrosis.
  • the inflammatory response is an increase in inflammatory factors such as TNF ⁇ or IL-6 in local tissues or systemic blood.
  • the tumor is selected from lymphoma, cervical cancer, liver cancer, breast cancer such as triple negative breast cancer, lung cancer such as non-small cell lung cancer or small cell lung cancer, colorectal cancer, gastric cancer, skin cancer such as melanoma, Bone cancer, osteosarcoma, myeloma, blood cancer, or ovarian cancer.
  • Figure 1 shows a single intravenous injection of 0.1 mg/Kg PAO or d5PAO in male SD rats, and the pharmacokinetic time curve.
  • Figure 2 shows the pharmacokinetic time curve of 0.2 mg/Kg PAO or d5PAO in a single oral gavage in male SD rats.
  • Figure 3 shows the vector map of the ⁇ -synuclein overexpression plasmid.
  • Figure 4 shows the standard preparation diagram for ⁇ -synuclein ELISA detection.
  • PI propidium iodide
  • Figure 6 shows that d5PAO and PAO increase the viability of stable-transformed APP (SW) HEK293 cells and promote the release of A ⁇
  • Figure 6B shows the A ⁇ content in the supernatant detected by the ELISA kit, and the A ⁇ value of each group is calculated by the standard curve
  • Figure 7 shows the structural formula of PAO deuterated compounds and the comparison of their effects in promoting A ⁇ release.
  • Figure 7A shows the A ⁇ content in the supernatant detected by the ELISA kit, and the A ⁇ value of each group is calculated by the standard curve;
  • 50nM d5PAO treatment group ##p ⁇ 0.001, ###p ⁇ 0.0001.
  • Figure 8 shows that d5PAO and PAO reduce the damaging effect of ⁇ -synuclein overexpression on SH-sy5y cells and promote the release of ⁇ -synuclein
  • Figure 8A shows that MTT detects certain concentrations of d5PAO and PAO on cells that transiently transform ⁇ -synuclein cells
  • Figure B shows the detection of ⁇ - in the supernatant by the ELISA kit
  • Figure 8C shows the normalization of the data to ⁇ - The syn OE group is 1, calculate the multiple value of the change in the content of ⁇ -sy
  • Figure 9 shows that d5PAO and PAO play a protective role in the SH-SY5Y cell model constructed by CBE.
  • Figure 9A shows that SH-SY5Y cells were treated with CBE for 48 hours, and cell viability was tested by MTT experiment;
  • Figure 9B shows that SH-SY5Y cells were treated with 100 ⁇ M CBE for 24 hours, starved (high glucose DMEM without FBS) and 100 ⁇ M CBE were treated together Treated for 24 hours, and then treated with different concentrations of PAO for 24 hours, cell viability was detected by MTT experiment;
  • Figure 9C shows 100 ⁇ M CBE treatment of SH-SY5Y cells for 24 hours, starvation (FBS-free high-sugar DMEM) and 100 ⁇ M CBE treatment together After 24 hours, they were treated with different concentrations of d5PAO and PAO for another 24 hours, and the cell viability was detected by MTT experiment.
  • Figure 10 shows that PAO inhibits CBE-induced lysosomal and GlcCer storage and promotes GlcCer efflux.
  • Figure 10A shows that SH-SY5Y cells were incubated with lysosome tracker for 30 minutes, then the supernatant was aspirated and replaced with PAO containing different concentrations and incubated for 10 minutes.
  • Lyso-tracker was observed by immunofluorescence method;
  • Figure 10C shows the statistical analysis of the GlcCer concentration in the cell lysate of each group by LC/MS;
  • Figure 10D shows the statistical analysis of the GlcCer concentration in the cell culture supernatant of each group by the LC/MS.
  • Figure 11 shows that knockdown of PI4Ka promotes a decrease in lysosomal storage.
  • Figure 11A shows Western blot detection of different shRNA interference lentiviral vectors (sh-ctrl, sh1-PI4Ka, sh2-PI4Ka, sh3-PI4Ka) treated SH-SY5Y cells for 48 hours PI4KIII ⁇ protein levels;
  • Figure 11B shows the results of Western blot detection Analysis;
  • Figure 11C shows the immunofluorescence method to detect the Lyso-tracker fluorescence intensity after shRNA interference lentiviral vector treatment, and statistical analysis (Figure 11D).
  • Figure 12 shows the pGMLV-SC5RNAi vector map.
  • FIG 13 shows a bright field image of MRC-5 cells 24 hours after treatment.
  • MRC-5 cells are cultured for 24 hours with MEM medium (without FBS) containing different concentrations of PAO or d5PAO with 5ng/mL TGF- ⁇ 1.
  • Scale bar 50 ⁇ m.
  • Figure 14 shows that d5PAO and PAO inhibit the expression of ⁇ -SMA and Calponin1 in MRC-5 model cells.
  • Figure 14A shows the western blot method to detect the expression levels of ⁇ -SMA and Calponin1.
  • Figure 14B shows the results of statistical analysis of ⁇ -SMA expression in each group.
  • Figure 14C shows the results of statistical analysis of Calponin1 expression in each group.
  • Figure 15 shows that d5PAO and PAO inhibit the expression of ⁇ -SMA and Calponin1 in MRC-5 model cells.
  • Figure 15A shows the immunofluorescence of ⁇ -SMA of each group, red: ⁇ -SMA, blue: DAPI (nucleus). Scale bar: 50 ⁇ m.
  • 15B shows the Calponin1 immunofluorescence image of each group, red: Calponin1, blue: DAPI (nucleus). Scale bar: 50 ⁇ m.
  • Figure 17 shows that d5PAO and PAO inhibit the secretion of COL1 during MRC-5 cell fibers.
  • Figure 17B shows the statistical analysis of the COL1 concentration in the supernatant of each group with the average value of the ctrl group as 1, and the data is represented by mean ⁇ SEM, compared with the 5ng/mL TGF- ⁇ 1 treatment group *p ⁇ 0.03, **p ⁇ 0.001, ***p ⁇ 0.0001, compared with ctrl group #p ⁇ 0.0001.
  • Figure 18 shows that the shRNA interference lentiviral vector reduces the expression of PI4KIII ⁇ .
  • the shRNA interference lentiviral vector is incubated with MRC-5 cells for 48 hours, and the protein is collected for western blotting.
  • Figure 18A shows the detection of PI4KIII ⁇ protein;
  • Figure 19 shows that knockdown of PI4Ka inhibits the expression of Calponin1 and ⁇ -SMA in MRC-5 cells treated with TGF- ⁇ 1.
  • MRC-5 cells adhere to the wall, lentiviral vectors of different sequences are added and cultured for 24 hours, and 5ng/mL TGF- ⁇ 1 is added or not added according to the group for 24 hours, and immunofluorescence staining is performed for observation.
  • Figure 19A shows the ⁇ -SMA immunofluorescence staining of each group, red: ⁇ -SMA, blue: DAPI (nucleus), and green: green fluorescent protein GFP. Scale bar: 50 ⁇ m.
  • Figure 19B shows Calponin1 immunofluorescence staining in each group, red: Calponin1, blue: DAPI (nucleus), green: green fluorescent protein GFP. Scale bar: 50 ⁇ m.
  • Analysis of the statistical results of immunofluorescence intensity of ⁇ -SMA ( Figure 19C) and Calponin1, normalized with the average value of the sh-ctrl group as 1, n 5, the data is represented by mean ⁇ SEM, and sh-ctrl plus 5ng/mL TGF- ⁇ 1 co-treatment group comparison *p ⁇ 0.03, ***p ⁇ 0.0001, compared with sh-ctrl group, ##p ⁇ 0.001, ###p ⁇ 0.0001.
  • Figure 20 shows that d5PAO and PAO inhibit the secretion of IL-6 and TNF- ⁇ in a BV2 cell inflammation model.
  • Figure 20A shows ELISA to detect the concentration of TNF- ⁇ in the supernatant of BV2 cells, and calculates the TNF- ⁇ content (pg/mL) according to the standard curve;
  • Figure 20B shows that the average concentration of the ctrl group is 1, and the TNF- ⁇ concentration of each group is calculated.
  • Figure 20C shows the concentration of IL-6 in the supernatant of BV2 cells detected by ELISA, and calculates the IL-6 content (pg/mL) according to the standard curve;
  • Figure 20D shows that the average concentration of the ctrl group is 1, and each is calculated.
  • Figure 21 shows the inhibitory effect of PAO on breast cancer.
  • Figure 22 shows the inhibitory effect of PAO on lymphoma.
  • Figure 23 shows the inhibitory effect of d5PAO on melanoma.
  • Figure 24 shows the inhibitory effect of d5PAO on melanoma on the 28th day of administration.
  • Figure 25 shows the comparison of the effects of high-dose PAO and d5PAO gavage on the body weight and survival rate of mice.
  • Figure 26 shows the effect of PAO on the body weight of a mouse model of breast cancer.
  • Figure 27 shows the effect of PAO on the body weight of a pancreatic cancer model.
  • Figure 28 shows the effect of PAO on the body weight of lymphoma model animals.
  • Figure 29 shows the effect of d5PAO combination on the body weight of melanoma mice.
  • Figure 30 shows the inhibitory effects of PAO and d5PAO on HCoV 229E (Influenza Coronavirus).
  • Figure 31 shows the anxiolytic effects of PAO and d5PAO, and d5PAO has more significant anti-anxiety effects than PAO.
  • Figure 32 shows that PAO and d5PAO have antidepressant effects, and d5PAO has more significant and stable antidepressant effects than PAO.
  • Figure 33 shows that d5PAO and PAO inhibit the cholesterol storage caused by U18666A. Scale bar: 50 ⁇ m.
  • Figure 34 shows that PAO promotes the expression of LC3B and p62, and Baf-A1 blocks the protective effect of PAO in cell models.
  • Figure 34A shows the detection of LC3B and p62 proteins by Western blotting;
  • Figure 34B and C show the use of Image J software Statistical analysis of LC3B and p62 protein signal intensity statistical analysis;
  • Figure 34D shows immunofluorescence detection of LC3B and p62, red: p62, green: LC3B, scale: 50 ⁇ m;
  • Figure 35 shows knockdown of PI4Ka to activate ALP
  • Figure 35A and B show Western blot detection of different shRNA interference lentiviral vectors (sh-ctrl, h1-PI4Ka, sh2-PI4Ka, sh3-PI4Ka) to treat SH-SY5Y cells for 48 hours After the LC3B protein level, and statistical analysis
  • Figure 35C, D shows shRNA interference lentiviral vector transfection while giving CBE treatment, incubated for 48 hours, the LC3B protein level was detected, and statistical analysis.
  • n 3; data are expressed as mean ⁇ SEM, analyzed by One-way ANOVA, compared with sh-ctrl *p ⁇ 0.03.
  • Figure 36 shows the percentage of enhanced expiratory intermittent value Penh caused by methacholine relative to baseline.
  • Figure 37 shows the total counts of eosinophils, macrophages, neutrophils and lymphocytes in alveolar lavage fluid (BALF). T.Test, single-tailed, * ⁇ 0.5, ** ⁇ 0.1 and the ratio of the total cell content of the model group.
  • Figure 38 shows the separate counts of eosinophils, macrophages, neutrophils and lymphocytes in alveolar lavage fluid (BALF). T.Test, one-tailed, * ⁇ 0.5, ** ⁇ 0.1 compared with BALF of the model group.
  • Figure 39 shows the plasma collagen type I content, normal group (ctrl).
  • Figure 40 shows the comparison between PAO and dPAO and the positive control drug nintedanib in down-regulating plasma hyaluronic acid in pulmonary fibrosis mice, normal group (ctrl).
  • 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 present invention relates to deuterated phenylarsenic oxide, preferably a perphenyl ring substituted deuterated isotope.
  • 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 their salts, esters, hydrates or solvates.
  • PEO phenylarsenic oxide
  • intracellular protein misfolding-related disease refers to a disease characterized by abnormally folded protein aggregation in the cytoplasm. It is also diagnosed as protein aggregation (aggregation, accumulation) or protein misfolding (misfolding). )disease.
  • intracellular protein misfolding-related diseases also includes some intracellular inclusion body diseases, such as protein inclusion body accumulation diseases. This type of inclusion body is mainly composed of a core protein aggregated by misfolding, with various external attachments. A stress protein involved in response to unfolded proteins.
  • Intracellular protein misfolding-related diseases include but are not limited to Parkinson's disease (PD), Lewy body dementia (LBD), multiple system atrophy (MSA), inclusion body muscle inflammation (IBM), frontotemporal dementia (FTD), Huntington's disease (HD), polyglutamine disease (PolyQ), amyotrophic lateral sclerosis (ALS), prion disease.
  • PD Parkinson's disease
  • LBD Lewy body dementia
  • MSA multiple system atrophy
  • IBM inclusion body muscle inflammation
  • FTD frontotemporal dementia
  • HD Huntington's disease
  • PolyQ polyglutamine disease
  • ALS amyotrophic lateral sclerosis
  • lysosomal storage disease refers to diseases caused by the accumulation of some endogenous or exogenous substances in the lysosome due to various reasons, including but not limited to insufficient enzyme activity in the lysosome, The lack of activating protein, transporter or lysosomal protein processing correcting enzyme causes lysosomal function defects, resulting in the inability to digest the corresponding substrates in the secondary lysosomes, accumulation of substrates, metabolic disorders, and the formation of storage diseases.
  • Lysosomal storage diseases include, but are not limited to, sphingolipid metabolism disorders, mucopolysaccharidosis, glycogen storage diseases, glycoprotein storage diseases, lipid storage diseases, post-translational modification defects, intrinsic membrane protein deficiency disorders, neurological disorders Meta-cereal lipofuscinosis, or lysosomal-related organelle disorders.
  • sphingolipid metabolism disorders include but are not limited to Fabry disease, metabolic disorders skin disease (Farbe disease), Gaucher disease type I, II, III and prenatal death type, GM1 gangliosidosis I, II, Type III, GM2 gangliosidosis (familial amaurosis), GM2 gangliosidosis, globular leukodystrophy (Krabe disease), metachromatic leukodystrophy, Niemann Pick Type A and B; Mucopolysaccharidosis includes but not limited to Hole-Schaecker syndrome and Schwartz’s disease (ML I), Hunter’s syndrome (MPS II), Saint Philip’s disease A (MPS IIIA), Saint Philip's disease B (MPS IIIB), Saint Philip's disease C (MPS IIIC), Saint Philip's disease D (MPS IIID), centrifugal osteochondrodysplasia syndrome (MPS IVA), centrifugal osteochondral syndrome Dysplasia syndrome (MPS
  • 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 deuterated phenylarsenic oxide 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 deuterated phenylarsenic oxide described herein is administered orally, subcutaneously, intramuscularly, or intravenously.
  • the deuterated phenylarsenic oxide described herein is 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 intracellular protein misfolding-related diseases or lysosomal storage diseases of the guest.
  • the therapeutically effective dose of deuterated phenylarsenic oxide 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 treating diseases related to intracellular protein misfolding, including but not limited to levodopa and riluzole.
  • the deuterated phenylarsenic oxide 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 diseases related to intracellular protein misfolding, including administering an effective amount of deuterated phenylarsenic oxide to a guest in need.
  • the present application also relates to a method for preventing or treating lysosomal storage disease, including administering an effective amount of deuterated phenylarsenic oxide to a subject in need thereof.
  • the synthesis circuit of d5PAO is as follows:
  • Step 1 Synthesis method of d5-PA:
  • the filtrate was washed three times with 100 mL of ethyl acetate, and the aqueous phase was concentrated under reduced pressure at 50 to 60° C. to about 170 mL remaining, and a large amount of white solid was precipitated. Suction filtration, rinsing the filter cake with cold water, draining, and directly drying the solid in a blast oven at 50 degrees for 18 hours to obtain 35 grams of white-like solid d5-PA, yield: 90.83%, MS ES+(m/z) :208.0[(M+H) + ].
  • Step 2 Synthesis method of d5-PAO:
  • Agilent 1260 Prime High Performance Liquid Chromatograph Mettler Toledo XS105 balance (0.01mg), KQ5200B ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.), BR2000-GM variable speed oscillator (VWR International), 0.45 ⁇ m filter membrane ( Shanghai Qingyang Biological Technology Co., Ltd.).
  • d5PAO Take 7.5 mg of d5PAO, add 1 mL of DMSO to a centrifuge tube, and ultrasonically shake to dissolve it. Dilute to 10 mL with acetonitrile/water (1/1) mixture to obtain 0.75 mg/mL d5PAO stock solution. Dilute the d5PAO stock solution into different d5PAO working solutions of 0.3mg/mL, 0.15mg/mL, 0.075mg/mL, 0.03mg/mL, 0.015mg/mL.
  • Liquid chromatograph Agilent 1260 Infinity II Prime Ultra High Performance Liquid Chromatography System.
  • Mobile phase A Water: ACN (v: v, 95: 5) solution containing 0.01% AA and 2 mmol/L NH4OAc.
  • Mobile phase B Water:ACN(v:v,5:95) solution containing 0.01% AA and 2mmol/L NH4OAc.
  • the molecular formula of PAO is C 6 H 5 AsO and the molecular weight is 168.03.
  • Agilent 1260 Prime High Performance Liquid Chromatograph Mettler Toledo XS105 balance (0.01mg), KQ5200B ultrasonic instrument (Kunshan Ultrasonic Instrument Co., Ltd.), BR2000-GM variable speed oscillator (VWR International), 0.45 ⁇ m filter membrane (Shanghai Qingyang Biological Technology Co., Ltd.).
  • PAO purity 98%)
  • acetonitrile is chromatographically pure (Sinopharm Group Chemical Reagent Co., Ltd.)
  • hydrochloric acid and DMSO are both analytically pure (Sinopharm Group Chemical Reagent Co., Ltd.).
  • PAO stock solution was diluted into different PAO working solutions of 0.3mg/mL, 0.15mg/mL, 0.075mg/mL, 0.03mg/mL, 0.015mg/mL.
  • Liquid chromatograph Agilent 1260 Infinity II Prime Ultra High Performance Liquid Chromatography System.
  • Mobile phase A Water:ACN(v:v,95:5) solution containing 0.01% AA and 2mmol/L NH4OAc.
  • Mobile phase B Water:ACN(v:v,5:95) solution containing 0.01% AA and 2mmol/L NH4OAc.
  • PAO 6 207.25 1.15
  • PAO 4 616.40 3.38
  • PAO 2 824.35 4.52
  • the dripping is completed within 1 hour, and naturally after the dripping is completed Warm up and stir overnight.
  • the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ice water (2 mL ⁇ 2).
  • the aqueous phase was concentrated under reduced pressure at 50°C to 10 mL. 4mL of 6N HCl was added dropwise to the ice-water bath to adjust the pH to 7-8, a small amount of yellowish brown solid appeared, suction filtered, washed with 2mL of ice water, and the solid was discarded.
  • the filtrate was added dropwise with 2.5 mL of 6N HCl to adjust the pH to 2-3, and a lot of bubbles appeared.
  • the 2-bromo-4,6-dideutero-aniline disappears, the diatomaceous earth is filtered, a small amount of methanol is washed, and the hydrobromide of 2,4-dideutero-aniline can be obtained by rotary drying directly.
  • step 2 Slowly add dropwise the azo hydrochloride prepared in step 1 to the reaction solution of step 2, control the temperature of the reaction system to be lower than 5°C, and foam during the dropping process, add a small amount of acetone to remove the foam, and wait until the foam disappears
  • step 2 Slowly add dropwise the azo hydrochloride prepared in step 1 to the reaction solution of step 2, control the temperature of the reaction system to be lower than 5°C, and foam during the dropping process, add a small amount of acetone to remove the foam, and wait until the foam disappears
  • the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ice water (2 mL ⁇ 2).
  • the aqueous phase was concentrated under reduced pressure at 50°C to 10 mL.
  • the dripping is completed within 1 hour, and the temperature is naturally raised after the dripping is completed, and the mixture is stirred overnight.
  • the reaction solution was filtered through diatomaceous earth, and the filter cake was rinsed with ice water (2 mL ⁇ 2).
  • the aqueous phase was concentrated under reduced pressure at 50°C to 10 mL.
  • 1.8 mL of 6N HCl was added dropwise to adjust the pH to 7-8, and a small amount of yellow-brown solids appeared, filtered with suction, washed with 2 mL of ice water, and discarded the solids.
  • the filtrate was added dropwise with 1.8 mL of 6N HCl to adjust the pH to 3, and a pale yellow solid appeared.
  • d5PAO (d5-PAO) was prepared by the method described in Example 1, and PAO (PAO) was prepared by the company itself.
  • PAO PAO
  • d5PAO administered dose of 0.1 mg/Kg, compound dissolved in 0.1% DMSO
  • a single oral gastric perfusion of the above reagent mixture (administered dose of 0.2 mg/Kg) in the tested adult male rats .
  • Draw venous blood at 0, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 32, and 48 hours after taking the medicine for pharmacokinetic testing.
  • the PAO and d5PAO in the test samples were extracted by protein precipitation, and the processed samples were injected into liquid-mass spectrometry (LC-MS/MS), and then detected in ESI negative ion mode after liquid phase separation.
  • LC-MS/MS liquid-mass spectrometry
  • the complete culture system of SH-SY5Y is 15% FBS (Gibco company) and high-sugar DMEM (Gibco company). Fugene HD transfection reagent (Promega, Beijing Biotech Co., Ltd. Catalog No. E2311) was used to transfect the plasmid.
  • the plasmid was purchased from Obio Technology (Shanghai Corp., Ltd), and the vector map is shown in Figure 3.
  • the sequence of the ⁇ -synuclein overexpression plasmid is as follows:
  • the stable transfer APP (SW) HEK293 cell line is a human embryonic kidney cell line transfected with Swedish double mutant APP695 cDNA.
  • the well plate needs to be treated with 20 ⁇ g/mL Poly-D-Lysine (PDL) for 24h before seeding.
  • the culture medium is 10% FBS with high sugar DMEM, and at the same time 200 ⁇ g/mL G418 is used for screening. After the seed plate was cultured for 48 hours, starvation treatment, that is, to remove the serum, only use DMEM high glucose medium. After 24 hours of culture, the system was replaced with a complete culture system and administered.
  • ⁇ -synuclein ELISA detection ⁇ -synuclein monoclonal antibody (Mouse Monoclonal) was purchased from Sigma-Aldrich (Shanghai, Ctatlog No. S5566); ⁇ -synuclein ELISA Kit kit was purchased from Thermo Fisher Scientific (Catalog No. KHB0061). Add 50 ⁇ L Hu ⁇ -synuclein Detection Antibody solution to each well (except chromogen blanks empty, that is, blank colorimetric wells), and then add 50 ⁇ L sample and standard song (see Figure 4 for standard song preparation) to each well (except chromogen blanks empty).
  • Amyloid beta 42 Human ELISA Kit was purchased from Thermo Fisher Scientific (Catalog No. KHB3544). Add 100 ⁇ L each of the diluted standard song, blank control and sample to the corresponding wells of the detection well plate. Incubate the cover membrane at 37°C for 2 hours; discard the liquid in each well, add 100 ⁇ L Detection Reagent A Working Solution to each well, and incubate the cover membrane at 37°C for 1 hour; discard the supernatant, and wash each well 3 times with 1 ⁇ Wash buffer.
  • PI Cell Signaling Technology, Catalog No. 4087
  • the complete culture system of SH-sy5y cell line is that 15% FBS is added to the digosaccharide DMEM medium.
  • SH-sy5y cells were cultured for 48 hours and then treated with different concentrations of d5PAO and PAO for 24 hours.
  • Thiazole blue (MTT) was added at a final concentration of 0.5 mg/mL. After 4 hours of incubation, aspirate the culture medium and add 100 ⁇ L of DMSO to dissolve For the adsorbed MTT, read the absorbance value after shaking for 15 minutes. The results showed that d5PAO at 6.25nM, 25nM, 50nM, 100nM and 200nM concentrations significantly promoted the proliferation of SH-sy5y cells.
  • PI staining and Ki67 immunofluorescence staining are fluorescent dye that can be inserted between DNA and RNA bases and dyes. It cannot pass through living cell membranes, but it can pass through damaged cell membranes to protect against apoptotic/dead cells.
  • Ki67 nuclear staining, and Ki67 is an indispensable protein in cell proliferation, and its function is closely related to mitosis. Therefore, Ki67 is often used to mark cells in the proliferation cycle, and in clinical applications, it is generally considered that the Ki67 positive rate is high for tumor growth. high speed.
  • PI staining and Ki67 staining were performed 24h after treatment with different concentrations of d5PAO and PAO. The results showed that 50nM and 100nM d5PAO and PAO did not significantly increase the Ki67 positive rate, while PI positive cells decreased compared with the control group (ctrl), indicating that d5PAO and PAO reduced cell apoptosis or death, but did not significantly promote cell proliferation (Figure 5 ).
  • Example 4 The effect of d5PAO and PAO on the viability and A ⁇ release of stable transduction APP (SW) HEK293 cells
  • the stable transfection APP (SW) HEK293 cell line is a human embryonic kidney cell line transfected with Swedish double mutant amyloid precursor protein (APP) 695 cDNA and carries the G418 selection marker.
  • SW human embryonic kidney cell line transfected with Swedish double mutant amyloid precursor protein (APP) 695 cDNA and carries the G418 selection marker.
  • PDL Poly-D-Lysine
  • the culture medium is high-sugar DMEM with 10% FBS, and at the same time 200 ⁇ g/mL G418 is used for screening.
  • the culture system was changed to a complete culture system after 48 hours of starvation treatment for 24 hours, and the compound d5PAO and PAO treatment for 4 hours.
  • the cell culture medium was tested by ELISA A ⁇ level in the supernatant.
  • the three PAO deuterated compounds selected are d1PAO, d2PAO, and d3PAO.
  • APP(SW)HEK293 cell culture method and administration method are the same as in Example 4.
  • the treatment component groups are: control group (ctrl), d5PAO 50nM treatment group, d5PAO 100nM treatment group, PAO 50nM treatment group, PAO 75nM treatment group, d1PAO 25nM treatment group, d1PAO 50nM treatment group, d1PAO 75nM treatment group, d2PAO 25nM treatment Group, d2PAO 50nM treatment group, d2PAO 75nM treatment group, d3PAO 25nM treatment group, d3PI03 50nM treatment group, d3PAO 75nM treatment group.
  • the other treatment groups are compared with the d5PAO 50nM treatment group, 50nM and 75nM PAO treatment group, 25nM, 50nM and 75nM d1PAO treatment group, 25nM, and 75nM d2PAO treatment group, and 25nM and 75nM d3PAO treatment group cell culture supernatant There are significant differences in the A ⁇ content in the A ⁇ ( Figure 7A and Figure 7B).
  • the SH-SY5Y cell line uses Fugene HD transfection reagent to transfect the ⁇ -synuclein overexpression ( ⁇ -syn OE) plasmid, which is changed to a complete culture system after 24 hours of starvation, and the compound d5PAO or PAO is treated for 24 hours at the same time.
  • the effect of d5PAO and PAO on the viability of SH-sy5y cells transfected with ⁇ -synuclein plasmid was detected by MTT. The results showed that overexpression of ⁇ -synuclein significantly decreased the viability of SH-sy5y cells.
  • Example 7 The therapeutic effect of d5PAO and PAO on Gaucher disease
  • Conduritol B epoxide is an inhibitor of the GBA1 enzyme encoded by the lysosomal glucocerebrosidase GBA gene. It is commonly used to construct cell and animal models of Gaucher disease (GD). Treatment of SH-SY5Y cells with CBE for 48 hours resulted in a concentration-dependent decrease in cell viability of SH-SY5Y cells ( Figure 9A). We choose 100 ⁇ M CBE for follow-up experiments. SH-SY5Y cells were treated with 100 ⁇ M CBE for 24 hours, and a certain concentration of d5PAO or PAO was added according to different groups and incubated for 24 hours. Cell viability was detected by MTT experiment.
  • PAO reduces lysosomal storage and promotes Glucosylceramide (GlcCer) efflux
  • GD is a common type of lysosomal storage disease.
  • lysosomal marker DND99 Liso-tracker red DND99
  • the experimental results showed that compared with the control group (ctrl), the fluorescence intensity of 100 ⁇ M CBE treatment group and 100 ⁇ M CBE Lyso-tracker increased ( Figure 10A, B), suggesting that CBE treatment caused lysosomal storage in SH-SY5Y cells.
  • the 100 ⁇ M CBE treatment group with 50nM and 100nM PAO significantly reduced the fluorescence intensity of Lyso-tracker ( Figure 10A, B).
  • GD The fundamental defect of GD is that it lacks the activity of glucocerebrosidase, which mainly mediates the process of breaking down glucocerebrosides into glucose and GlcCer. Therefore, storage of GlcCer and other substrates occurs in GD patients or CBE-treated cells. .
  • LC-MS In order to further explore whether PAO has an effect on GlcCer storage in SH-SY5Y cell model, we used LC-MS to test the GlcCer content of different side chains in intracellular and cell culture supernatants.
  • Example 8 Study on the inhibitory effect of d5PAO and PAO on pulmonary fibrosis
  • Pulmonary fibrosis is a type of chronic and fibrotic lung disease that can be caused by various reasons, mainly manifested as dry cough and progressive dyspnea, with a poor prognosis and it is still difficult to cure.
  • the main pathological feature of PF is excessive scar repair after the destruction of normal lung tissue structure, which eventually leads to respiratory insufficiency.
  • transforming growth factor- ⁇ 1 transforming growth factor-1, TGF- ⁇ 1
  • TGF- ⁇ 1 transforming growth factor-1
  • extracellular matrix extracellular matrix
  • ECM extracellular matrix
  • TGF- ⁇ 1 is one of the key factors for inducing PF. It can regulate the conversion of fibroblasts into myofibroblasts by binding to the corresponding receptors. Therefore, during the experiment, a certain concentration of TGF- ⁇ 1 is usually used to treat MRC-5 cells to promote it. Fibrosis occurs.
  • d5PAO and PAO are respectively phenylarsenic oxide and its modified products. Previous studies have shown that PAO has the potential to inhibit PF.
  • MRC-5 cells were purchased from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. According to the cell culture instructions, use MEM (Gibco) medium containing 10% fetal bovine serum (FBS) in a constant temperature 37°C, 5% CO 2 , saturated humidity incubator, and cultivate for 24 hours. Adhere to the wall. On the next day, add 5ng/mL TGF- ⁇ 1 (Proteintech Group, HZ-1011), and add MEM medium (Gibco) containing different concentrations of PAO or d5PAO according to the group, and continue culturing for 24 hours according to experimental needs.
  • MEM Gibco
  • the groups are as follows: control (ctrl) group, 5ng/mL TGF- ⁇ 1 group, 5ng/mL TGF- ⁇ 1 and 50nM d5PAO co-treatment group (5ng/mL TGF- ⁇ 1+50nM d5PAO group), 5ng/mL TGF- ⁇ 1 and 25nM d5PAO co-treatment group (5ng/mL TGF- ⁇ 1+25nM d5PAO group), 5ng/mL TGF- ⁇ 1 and 50nM PAO co-treatment group (5ng/mL TGF- ⁇ 1+50nM PAO group), 5ng/mL TGF- ⁇ 1 and 25nM PAO co-treatment group (5ng/mL TGF- ⁇ 1+25nM PAO group).
  • the 6-week-old Sprague-Dawley (SD) rat (Shanghai Slack Laboratory Animal Co., Ltd.) was anesthetized with chloral hydrate, sterilized with 75% ethanol, separated in a clean bench, and the tibia and femur were removed, and the tibia and femur were removed with sterilized scissors. End, exposing the bone marrow cavity. Wash the bone marrow cavity with 5 mL of MEM medium containing 10% FBS, and add the washing solution containing bone marrow to the petri dish; filter through a 70 ⁇ m cell sieve, and centrifuge at 2000 rpm for 3 minutes, remove the supernatant, and use 10% FBS.
  • MEM medium Resuspend the cells in MEM medium, plant the plates and culture them in an incubator with a constant temperature of 37°C, 5% CO 2 , and saturated humidity. After 6 hours, the non-adherent cells are removed by changing the medium. When the cells grow to 80% density, digest with 2.5% trypsin for 1 minute and pass at a ratio of 1:2. Mesenchymal stem cells used for immunofluorescence experiments will be seeded in 24 wells with 3000-5000 cells per well. On the cover glass of the plate, by observing the cell density to 70%, add MEM medium (without FBS) containing different concentrations of PAO or d5PAO according to the group and culture in an incubator with a constant temperature of 37°C, 5% CO 2 and saturated humidity 24 hours.
  • the groups are as follows: control (ctrl) group, 50nM d5PAO treatment group, 25nM d5PAO treatment group, 50nM PAO treatment group, 25nM PAO treatment group.
  • Dilution ratio 1:200, Calponin1 dilution ratio 1:100). The next day, wash 3 times with PBS, 10 minutes each time; incubate the secondary antibody Alexa Flour 555 goat anti-rabbit IgG (Molecular Probes) diluted with blocking solution, the dilution ratio is 1:500, and add 1 ⁇ g/mL 4',6- Diamidino-2-phenylindole (DAPI)) was allowed to stand at room temperature for 2 hours, and PBS was added for washing 3 times, each washing for 10 minutes. Use the mounting tablet and observe through a microscope. The immunofluorescence staining process of bone marrow mesenchymal stem cells is the same as above.
  • Enzyme-linked immunosorbent reaction Human Collagen Type I ELISA Kit
  • a total of 8 samples including: control group (ctrl), 5ng/mL TGF- ⁇ 1 group, 5ng/mL TGF- ⁇ 1 and 50nM d5PAO co-treatment group (5ng/mL TGF- ⁇ 1+50nM d5PAO group), 5ng/mL TGF - ⁇ 1 and 25nM d5PAO co-treatment group (5ng/mL TGF- ⁇ 1+25nM d5PAO group), 5ng/mL TGF- ⁇ 1 and 50nM PAO co-treatment group (5ng/mL TGF- ⁇ 1+50nM PAO group), 5ng/mL TGF - ⁇ 1 and 25nM PAO co-treatment group (5ng/mL TGF- ⁇ 1+25nM PAO group).
  • Standard Diluent to configure standard curve samples 4000pg/mL, 2000pg/mL, 1000pg/mL, 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 0pg/mL;
  • the shRNA lentiviral vector for PI4Ka interference was designed and produced by Jiman Biotechnology (Shanghai) Co., Ltd.
  • the design targets are as follows:
  • MRC-5 cells are cultured in a MEM medium containing 10% fetal bovine serum (FBS) in an incubator at 37° C., 5% CO 2 , and saturated humidity, and cultured for 24 hours to make them adherent. The next day, 1 ⁇ L/well lentiviral vector was added according to the group, and after 24 hours, 5ng/mL TGF- ⁇ 1 was added according to the group and incubated for 24 hours. The protein was collected for Western blotting or immunofluorescence staining.
  • FBS fetal bovine serum
  • Image J software is used for fluorescence intensity processing
  • GraphPad Prism5 software is used for data processing and statistics, and data is expressed as mean ⁇ standard error (mean ⁇ SEM).
  • One-way ANOVA was used to compare the differences between groups, and p ⁇ 0.03 was considered statistically different.
  • MRC-5 cells were treated with MEM (without FBS) medium containing 5ng/mL TGF- ⁇ 1 for 24 hours, and treated with different concentrations of d5PAO or PAO according to the group.
  • TGF- ⁇ 1 alone or co-treatment with a certain concentration of d5PAO or PAO did not cause significant cell death or apoptosis ( Figure 13).
  • ⁇ -Smooth Muscle Actin ⁇ -Smooth Muscle Actin, ⁇ -SMA
  • actin binding protein Calponin1 are markers of myofibroblasts.
  • the expression of Calponin1 in the 5ng/mL TGF- ⁇ 1 treatment group was significantly higher than that in the ctrl group.
  • a certain concentration of d5PAO or PAO and 5ng/mL TGF- ⁇ 1 co-treatment group Compared with the 5ng/mL TGF- ⁇ 1 treatment group alone, the expression level of Calponin1 was significantly reduced ( Figure 14A, C).
  • the immunofluorescence results are also consistent with the Western blot results, and the expression of ⁇ -SMA in the 5ng/mL TGF- ⁇ 1 treatment group was significantly higher than that in the control group ( Figure 15A, C).
  • d5PAO and PAO inhibit Calponin1 expression in bone marrow mesenchymal stem cells (bMSC)
  • MSC Mesenchymal stem cells
  • d5PAO and PAO inhibit the secretion of type I collagen by MRC-5 cells treated with TGF- ⁇ 1 (Collagen Type I, COL1)
  • COL1 is one of the important components of extracellular matrix. Studies have shown that TGF- ⁇ 1 promotes a significant increase in COL1 secretion during MRC-5 cell fibrosis. In order to further explore whether d5PAO and PAO can regulate the secretion of COL1 in the process of inhibiting pulmonary fibrosis, we detected the level of COL1 in the cell supernatant by ELISA. MRC-5 cells were treated with MEM (without FBS) medium containing 5ng/mL TGF- ⁇ 1 for 24 hours, and different concentrations of d5PAO or PAO were administered for 24 hours according to groupings. The supernatant was collected for ELISA experiments.
  • the experimental results showed that compared with the ctrl group, the COL1 concentration in the cell supernatant of the 5ng/mL TGF- ⁇ 1 treatment group was significantly increased, 5ng/mL TGF- ⁇ 1 and 25nM d5PAO, 5ng/mL TGF- ⁇ 1 and 50nM d5PAO, 5ng/ mL TGF- ⁇ 1 and 100nM d5PAO, 5ng/mL TGF- ⁇ 1 and 25nM PAO, 5ng/mL TGF- ⁇ 1 and 50nM PAO and 5ng/mL TGF- ⁇ 1 and 100nM PAO co-treatment group and 5ng/mL TGF- ⁇ 1 separate treatment group
  • the level of COL1 in the cell supernatant was significantly decreased ( Figure 17A, B), indicating that d5PAO and PAO can inhibit the secretion of COL1 in the MRC-5 cell model.
  • Calponin1 and ⁇ -SMA were observed by immunofluorescence after shRNA interference lentiviral vector treatment for 48 hours. Compared with the vehicle control (sh-ctrl) group, the ⁇ -SMA expression level in the sh-ctrl plus 5ng/mL TGF- ⁇ 1 treatment group was significantly increased.
  • d5PAO and PAO inhibit the release of inflammatory factors in mouse microglia BV2 cells
  • BV2 cells are immortalized by retrovirus-mediated transfection of mouse microglia into v-raf/v-myc, and retain a variety of morphology, characterization and functional characteristics of microglia.
  • lipopolysaccharide LPS is often used to stimulate BV2 cells to obtain an inflammatory cell model for experimentation.
  • LPS stimulated BV2 cells as an inflammatory cell model to observe the secretion of d5PAO and PAO on tumor necrosis factor ⁇ (TNF- ⁇ ) and interleukin-6 (Interleukin-6, IL-6) and other inflammatory factors
  • TNF- ⁇ tumor necrosis factor ⁇
  • Interleukin-6 Interleukin-6, IL-6
  • Indomethacin Indomethacin
  • the medium of BV2 cells was cultured in a high-sugar DMEM with 10% FBS, 37°C and 5% CO 2 in a cell incubator for 48 hours.
  • the medium was removed and replaced with 1 ⁇ g/mL LPS (lipopolysaccharide, purchased from Sigma, catalog number: L2880)
  • High-sugar DMEM medium without FBS
  • concentrations of d5PAO or PAO were given according to the group and incubated for 24 hours.
  • the supernatant was collected and centrifuged for use in subsequent experiments.
  • control (ctrl) group 1 ⁇ g/mL LPS group, 1 ⁇ g/mL LPS and 50nM d5PAO co-treatment group (1 ⁇ g/mL LPS+50nM d5PAO group), 1 ⁇ g/mL LPS and 25nM d5PAO co-treatment group (1 ⁇ g/mL LPS+25nM d5PAO group), 1 ⁇ g/mL LPS and 12.5nM d5PAO co-treatment group (1 ⁇ g/mL LPS+12.5nM d5PAO group), 1 ⁇ g/mL LPS and 50nM PAO co-treatment group (1 ⁇ g/mL LPS+50nM PAO group) , 1 ⁇ g/mL LPS and 25nM PAO co-treatment group (1 ⁇ g/mL LPS+25nM PAO group), 1 ⁇ g/mL LPS and 12.5nM PAO co-treatment group (1 ⁇ g/mL LPS+12.5nM PAO group), 1 ⁇ g/g/g/mL
  • Mouse Tumor Necrosis Factor Alpha (TNF ⁇ ) ELISA Kit is purchased from Signalway Antibody LLC, item number EK16997.
  • the concentrations are: 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.625ng/mL, 0.312ng/mL, 0.156ng/mL, 0.
  • Mouse IL-6 ELISA Kit is purchased from Proteintech group, item number: KE10007.
  • GraphPad Prism5 software is used for data processing and statistics, and the data is expressed by mean ⁇ SEM (mean ⁇ SEM).
  • One-way ANOVA was used to compare the differences between groups, and p ⁇ 0.03 was considered statistically different.
  • BV2 cells were cultured in high-sugar DMEM with 10% FBS medium, 37°C and 5% CO 2 cell incubator for 48 hours, then the medium was removed and replaced with high-sugar DMEM medium (without FBS) containing 1 ⁇ g/mL LPS, and According to different concentrations of d5PAO or PAO were given and incubated for 24 hours, the supernatant was collected and used ELISA (enzyme-linked immunosorbent assay) method to detect the concentration of IL-6 and TNF- ⁇ in the culture medium.
  • ELISA enzyme-linked immunosorbent assay
  • the TNF- ⁇ concentration in the supernatant of the 1 ⁇ g/mL LPS and 12.5nM, 25nM, 50nM d5PAO co-treatment group and the 12.5nM, 25nM, 50nM PAO co-treatment group was more significant than that in the 1 ⁇ g/mL LPS single treatment group Decrease, indicating that d5PAO and PAO can inhibit the release of TNF- ⁇ induced by LPS.
  • the ELISA test results of IL-6 showed that treatment with a certain concentration of d5PAO and PAO has a tendency to inhibit the secretion of IL-6 from BV2 cells induced by LPS ( Figure 20C-D). The above experiments show that a certain concentration of d5PAO and PAO can inhibit the release of inflammatory factors from BV2 cells induced by LPS.
  • Compound to be tested The compound was prepared as a 10 mM solution in DMSO, and the compounds PAO and d5PAO were diluted in DMSO to a 0.5 mM solution.
  • Use HPD300 instrument to add the compound to the corresponding cell wells. Incubate for 72 hours in a carbon dioxide incubator.
  • d5PAO and PAO inhibition of tumor cells are tested, and 50 close both the inhibition IC, d5PAO lower the IC50; Inhibition of both the U2-OS cells and A-375
  • IC 50 is less than 50nM; it has strong inhibitory effect on HeLa, SK-HEP-1, Daudi, EL4, HL-60, Jurkat, Clone E6-1, NAMALWA cells, IC 50 is 50-100nM; for A -431 cells have the weakest inhibitory effect, with an IC50 of about 300nM, and the IC50 for the remaining cells in the range of 100-200nM.
  • d1PAO Compared with PAO (prototype compound) and d5PAO (5 deuterated compounds), a deuterated compound d1PAO, the effective inhibitory concentration of most tumor cells is greater than 200nM, indicating that its inhibitory effect on tumor cells is not as good as PAO and d5PAO.
  • the experimental animals were kept in the SPF barrier facility environment of the Animal Center of Beijing Biocytogen Biotechnology Co., Ltd.
  • the temperature of the barrier system is at 20-26°C and the humidity is controlled at 40-70%. The lighting alternates every 12 hours.
  • SPF-grade growth and reproduction feed was purchased from Beijing Keyao Xieli Feed Co., Ltd.
  • Drinking water is acidified water (pH 2.5 ⁇ 3.0), which is autoclaved. Animals can freely ingest sterile food and drinking water
  • the tumor mass is taken out under aseptic conditions and placed in RPMI 1640 medium, and non-tumor tissues such as calcification spots and secretions are removed, and cut into small pieces of uniform size (3 ⁇ 3 ⁇ 3mm), preparing for subcutaneous inoculation of breast cancer (BP1395) PDX model.
  • the skin of the right flank of the mouse was disinfected with iodophor, an incision of about 3-5 mm was made with the skin with scissors, and the cut tumor mass was inoculated under the skin of the right flank of the mouse with an inoculation needle.
  • mice with moderate tumor volume were selected into the group, and the animals were randomly assigned to 4 experimental groups according to the tumor volume, 8 in each group, and the administration was started on the day of grouping. All groups were given to the recipient
  • the test product is oral administration (PO), and the administration is started on the day of grouping.
  • the test product PAO is administered once a day for a total of 20 administrations.
  • Paclitaxel was administered intravenously (iv) once a week for a total of 4 administrations.
  • the animals were euthanized using excessive CO 2.
  • Weight detection inoculation, grouping (before the first administration), twice a week during the administration period, and weigh the animals before euthanasia.
  • TGI TV Tumor volume inhibition rate
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100%
  • Ti mean tumor volume of the treatment group on day i of administration
  • T0 mean tumor volume of the treatment group on day 0 of administration
  • Vi mean tumor volume of the solvent control group on day i of administration
  • V0 solvent (The mean tumor volume of the control group on day 0 of administration)
  • TGI TW Tumor weight inhibition rate
  • the surviving animals were euthanized and the tumor tissue was stripped off, the tumor weight was weighed, and the tumor weight difference of each group was calculated to further calculate the tumor weight inhibition rate TGI TW , the calculation formula is as follows:
  • Tumor weight inhibition rate TGI TW % (W solvent control group- W treatment group )/W solvent control group ⁇ 100%, W refers to tumor weight.
  • the result analysis is expressed in mean and standard error (Mean ⁇ SEM).
  • the tumor volume was statistically analyzed, and P ⁇ 0.05 was considered as a significant difference. Both statistical significance and biological significance were considered in the analysis of the results.
  • the results show that the combination of PAO and paclitaxel can effectively inhibit tumor volume growth (Figure 21).
  • Mouse lymphoma SU-DHL-1 cells were purchased from ATCC, and the cells were cultured in a 37°C, 5% CO 2 incubator.
  • the medium component was Dulbecco's Modified Eagle's medium containing 10% inactivated fetal bovine serum.
  • SU-DHL-1 lymphoma cells resuspended in PBS were inoculated subcutaneously on the right side of B-NDG humanized mice at a concentration of 1 ⁇ 10 7 cells/0.1 mL and a volume of 0.1 mL/head.
  • the test product PAO was administered once a day for a total of 17 administrations.
  • Cyclophosphamide was administered by subcutaneous injection (iv) once a week for a total of 4 administrations.
  • the animals were euthanized using excessive CO 2.
  • A2058 cells were inoculated subcutaneously on the right side of B-NDG mice at a concentration of 1 ⁇ 10 7 cells/0.1 mL and a volume of 0.1 mL/head.
  • the average tumor volume reached about 100mm 3
  • 48 mice with the appropriate individual tumor volume were selected into the group, and the animals were randomly assigned to 6 experimental groups according to the tumor volume, with 8 mice in each group, respectively G3 group (normal saline/ Solvent), G4 group (d5PAO, 0.5mg/Kg), G5 group (d5PAO, 1.5mg/Kg), G6 (temozolomide, 30mg/Kg+d5PAO, 0.5mg/Kg), G7 (temozolomide 30mg/Kg+d5PAO, 1.5mg/Kg) and G8 (Temozolomide, 30mg/Kg).
  • mice 16 non-tumor-bearing mice were selected according to their body weight and divided into 2 experimental groups, 8 in each group, namely G1 group (normal saline/vehicle) and G2 group (d5PAO, 1.5mg/Kg).
  • the route of administration in all groups was intragastric administration, and the administration was started on the day of grouping.
  • the test product d5PAO was administered once a day for a total of 23 administrations.
  • the test product temozolomide was administered 4 times a week for a total of 12 administrations.
  • the body weight and tumor volume of the mice were measured twice a week during the administration and observation period, and the measured values were recorded (Figure 23). At the end of the experiment, the animals were euthanized, the tumors were stripped and weighed, photographed, and the tumor growth inhibition rate (TGI%) was calculated.
  • TGI% tumor growth inhibition rate
  • G4 group (d5PAO 0.5mg/Kg), G5 group (d5PAO 1.5mg/Kg), G6 group (temozolomide 30mg/Kg+d5PAO 0.5mg/Kg), G7 group (temozolomide 30mg/Kg+d5PAO1.5mg/
  • the average tumor volume of Kg) and G8 group (temozolomide 30mg/Kg) were 2907 ⁇ 295mm 3 , 2180 ⁇ 312mm 3 , 1064 ⁇ 164mm 3 , 1213 ⁇ 155mm 3 , 1480 ⁇ 136mm 3 , and the tumor volume growth inhibition rate TGI TV was respectively They are -3.7%, 23.2%, 64.5%, 58.9% and 49.1%.
  • the experimental results have also been confirmed in the weight of the tumor.
  • the tumor tissue weights obtained in the end-point animals of the test were: G3 group solvent/saline, 3.413 ⁇ 0.253g; G4 group d5PAO 0.5mg/kg, 3.557 ⁇ 0.379g; G5 group d5PAO 1.5mg/kg, 2.744 ⁇ 0.459 G; G6 group temozolomide 30mg/kg+d5PAO 0.5mg/kg, 1.413 ⁇ 0.233g; G7 group temozolomide 30mg/kg+d5PAO 1.5mg/kg, 1.442 ⁇ 0.251g; G8 group temozolomide 30mg/kg, 1.884 ⁇ 0.217g ( Figure 24).
  • temozolomide at a dose of 30 mg/Kg alone and when combined with d5PAO at a dose of 0.5 mg/Kg and 1.5 mg/Kg have a very significant inhibitory effect on the growth of A2058 subcutaneous xenograft tumors (P ⁇ 0.001 ).
  • the combined use can further enhance the inhibitory effect.
  • Example 11 Comparison of the effects of d5PAO and PAO in anti-tumor cachexia (Cachexia)
  • Cancer is the second leading cause of death in humans, and nearly one-sixth of all deaths worldwide are caused by cancer. Cancer treatment is mainly achieved through chemotherapy, radiation therapy, surgery, immunotherapy, gene therapy, hormone therapy and other means. Among them, chemotherapy is currently one of the most effective methods. But the main problem of chemotherapy is that it causes side effects: while the drug kills cancer cells, it also kills fast-growing cells in the body, including cells in the blood, mouth, digestive system, and hair follicles, causing digestive system reactions and hair loss. , Bone marrow suppression and the decline of other system functions.
  • Cachexia also known as cachexia
  • cachexia is characterized by extreme weight loss, weight loss, fat loss, and decreased dissolution of skeletal muscle and myocardium, leading to progressive dysfunction, and finally to systemic failure and other syndromes.
  • Cachexia is mostly caused by severe chronic wasting diseases, including tumors, AIDS, severe trauma, post-surgery, malabsorption, and severe sepsis.
  • cachexia accompanied by tumors is the most common, also known as tumor cachexia. 31-87% of patients with malignant tumors are accompanied by cachexia, and about 20% of tumor patients' direct cause of death is malnutrition caused by cachexia, rather than the disease itself.
  • Cachexia is especially related to pancreatic cancer, stomach cancer, lung cancer, and liver cancer. Cachexia directly affects the effect of cancer treatment, increases the incidence of complications, and reduces the quality of life, shortens the survival period, prolongs the treatment time and increases the medical expenses.
  • mice Twenty-two male C57B/6 mice at the age of 2 months and 20 male C57B/6 mice at the age of 6 months were collected. There were 5-6 mice in each cage, including 12 mice at 2 months old and 10 mice at 6 months old.
  • the daily dose of d5PAO dissolved in MCT solvent is 2.1 mg/kg, and the remaining 10 2-month-old mice and 10 6-month-old mice, the daily dose of PAO dissolved in MCT solvent 2.0mg/kg. From the first day of administration, on the first day of every 4 days, weigh and record the body weight of each mouse before administration. In the next 4 days, based on this body weight, give the corresponding dose by gavage PAO or d5PAO. The number of surviving mice was recorded every 4 days.
  • pancreatic cancer model The method of establishing pancreatic cancer model is the same as above.
  • the average body weight of gemcitabine (1.5mg/Kg) and paclitaxel (7mg/kg) group (104.3% ⁇ 4.0%, relative to the average body weight on Day 0), compared with the vehicle group (99.0% ⁇ 2.2%) , Relative to Day 0 average weight) no significant difference.
  • the method of establishing the lymphoma model is the same as above.
  • PAO failed to further inhibit tumor growth, it can alleviate the weight loss caused by cyclophosphamide injection.
  • the animal body weight decreased from 117.4% ⁇ 1.4% (G1 vehicle group, relative to the average body weight of Day0) to 104.7% ⁇ 1.6% (G2, cyclophosphamide group 50mg/Kg, relative to the average body weight of Day 0, P ⁇ 0.01*).
  • the reference compound Remdesivir will be provided by WuXi AppTec.
  • the compound was prepared as a 20 mM stock solution using DMSO solution.
  • the test sample and the control compound were tested at 8 concentrations, with 2-fold or 3-fold serial dilutions, and double replicate wells.
  • 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.
  • the main reagent used in this project is the cell viability test kit CellTiter-Glo (Promega).
  • 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 compound after multiple dilutions (8 concentration points, 2-fold or 3-fold gradient dilution, double repertoire) 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 is 0.5%. The cells were cultured in an incubator for 3 days.
  • the cytotoxicity experiment and the antiviral experiment were tested at the same time, and the experimental conditions were the same, but there was no virus infection.
  • Cell viability test kit CellTiter Glo (Promega) was used to detect cell viability.
  • the antiviral activity and cytotoxicity of the compound are represented by the inhibitory 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) ⁇ 100
  • GraphPad Prism (version 5) was used to perform non-linear fitting analysis on the inhibition rate and cell viability of the compound, and the half effective concentration (EC 50 ) and half cytotoxic concentration (CC 50 ) values of the compound were calculated.
  • the fitting formula is: log(inhibitor) vs.response--Variable slope.
  • the dose-response fitting curves of PAO and d5PAO drugs are shown in Figure 30.
  • the control compound Remdesivir showed expected antiviral activity and cytotoxicity.
  • test results show that the test compounds PAO and P100 have antiviral activity against HCoV 229E, and their EC 50 values are 55.35 nM and 47.21 nM, respectively.
  • the test compounds PAO and P100 have obvious toxicity to MRC5 cells, and their CC 50 values are 256.8 nM and 317.5 nM, respectively.
  • mice Thirty male 2-month-old ICR mice were raised in a clean room for 8 weeks according to normal circadian rhythm and other conditions. They were randomly divided into 3 groups: vehicle, PAO and d5PAO groups, 5 mice in a cage, each group Two cages, each group was given chronic unpredictable multiple stimulation (CUMS).
  • CUMS chronic unpredictable multiple stimulation
  • CUMS depression model A variety of stimuli are given to mice alternately every day, so that the mice cannot predict the duration of each stimulus and the pattern and duration of the stimulus that will be given next.
  • the stimulation and schedule of the first week are shown in (Table 6).
  • the stimulation pattern and time of each day in the table form a module, a total of 7 modules. Starting from the second week, the stimulus of each Monday will be randomly selected from 7 modules, on Tuesday from the remaining 6 modules, on Wednesday from the remaining 5 modules, and so on. If there is a test schedule on a certain day, make appropriate adjustments to the stimuli on the first two days and the day of the test.
  • mice in the PAO and d5PAO groups were given daily intragastric administration of PAO and d5PAO compound solution, respectively, at the dose of 0.05 mg/Kg/day, while the daily intragastric administration of mice in the vehicle group was corresponding to their body weight.
  • Volume of MCT MIGLYOL 812N, supplier IOI Oleo GmbH
  • MCT is a carrier used to prepare solutions of PAO and d5PAO compounds.
  • the concentration of MCT solution of PAO and d5PAO are both 0.005mg/mL.
  • NSF Novelty Suppressed Feeding Test
  • a box made of opaque plexiglass with only the top surface open and 25cm ⁇ 25cm ⁇ 20cm.
  • a small platform is placed in the center of the box.
  • put a mouse into the box from any corner of the box, with its head facing the corner of the box let the mouse move freely for 5 minutes under five interferences, and record the incubation period of the first feeding within 5 minutes. If the mouse has not taken food at the end of 5 minutes, the mouse is taken out and the incubation period of the mouse's first food ingestion is counted as 300 seconds.
  • the length of the incubation period (seconds) of the first ingestion is an index of the degree of anxiety in mice.
  • Depressive behavior indicators detection of sugar water preference
  • mice underwent 3 weeks of CUMS and were given carrier (MCT), PAO and d5PAO preparations by gavage for 15 days, and then performed Novel Inhibition of Food Intake (NSF) test. It was found that only 2 of the 10 mice in the vehicle group had food, but the remaining 8 mice did not eat within the limited 5 minutes. The incubation period was calculated as 300 seconds.
  • MCT carrier
  • PAO PAO
  • d5PAO Novel Inhibition of Food Intake
  • the average anxiety index of this group was 282 ⁇ 12 (seconds); PAO and The average anxiety index of the d5PAO group was 227 ⁇ 29 (seconds) and 181 ⁇ 36 (seconds), which was significantly lower than the anxiety of the vehicle group, suggesting that low-dose (0.05mg/Kg/day) PAO and d5PAO also have Obvious anti-anxiety effect, and d5PAO has a more significant anti-anxiety effect than d5PAO ( Figure 31).
  • mice were given CUMS for a total of 38 days, and then the sugar water preference test was performed.
  • the results showed that the vehicle, PAO and d5PAO groups of mice had a preference for sugar water of 71 ⁇ 3.5%, respectively. 77 ⁇ 2.0% and 82 ⁇ 2.9%; PAO and d5PAO groups have a higher preference for sugar water than the vehicle group, but the difference between the PAO group and the vehicle group is not significant, suggesting that low-dose (0.05mg/Kg/day) PAO and d5PAO still has an antidepressant effect, and the antidepressant effect of d5PAO is more significant and stable than that of d5PAO (Figure 32).
  • U18666A a cholesterol transport inhibitor in cells, is often used to construct a cell model of type C Nieman's disease (NPC).
  • NPC Nieman's disease
  • SH-SY5Y cells are cultured in a complete medium containing high-sugar DMEM plus 15% FBS at 37°C and 5% CO 2 in an incubator. When the cells are 70% confluent, add 10 ⁇ M U18666A (purchased from Aibixin (Shanghai) Biotechnology Co., Ltd., catalog number: abs819512) and add d5PAO and PAO at different depths according to the group, and culture for 24 hours.
  • U18666A purchased from Aibixin (Shanghai) Biotechnology Co., Ltd., catalog number: abs819512
  • SH-SY5Y cells were treated with 10 ⁇ M U18666A, and different concentrations of d5PAO and PAO were added according to the group, incubated for 24 hours, and observed after Filipin staining.
  • the results of immunofluorescence staining showed that the fluorescence intensity of Filipin staining in the 10 ⁇ M U18666A alone treatment group was higher than that in the control group (ctrl), suggesting that 10 ⁇ M U18666A treatment resulted in an increase in the amount of cholesterol bound to Filipin, which resulted in cholesterol storage.
  • Example 15 PAO and knockdown PI4Ka activate the autophagy-lysosome pathway (ALP)
  • the SH-SY5Y cells treated with shRNA interference lentiviral vector were tested for the ALP pathway marker LC3B.
  • the results showed that compared with the sh-ctrl group, the LC3B protein level was significantly increased after PI4Ka was knocked down ( Figure 35), and CBE treatment Knockdown of PI4Ka in SH-SY5Y cells also promoted the expression of LC3B protein, indicating the same results as the PI4Ka inhibitor PAO, knockdown of PI4Ka also activated the ALP pathway.
  • the lungs and upper respiratory tract are the tissues and organs that the body most frequently suffers from various causes, including pathogens, chemical factors, including drugs, foreign bodies, physical damage to tissues, allergic reactions, and autoimmune abnormalities. Including the increase of white blood cells in local tissues or systemic blood, such as neutrophils, macrophages and lymphocytes, as well as the increase of various inflammatory factors or cytokines.
  • Pathogens include microorganisms and parasites. Microorganisms include bacteria, viruses, chlamydia, mycoplasma, spirochetes, and fungi. Inflammation of the lungs can sometimes lead to fibrosis of lung tissues, impairing lung structure and function, especially impairing ventilation and oxygen diffusion.
  • Bleomycin is a clear drug or chemical that can cause pneumonia and pulmonary fibrosis.
  • the interstitial pneumonia and pulmonary fibrosis that it causes in the lungs of animals is a study of idiopathic pulmonary fibrosis (IPF) Commonly used models.
  • IPF is a fatal disease characterized by progressive and irreversible pulmonary fibrosis.
  • Most patients die of progressive respiratory failure within 3-8 years after the onset of symptoms.
  • the basic mechanism of the pathogenesis of IPF is poorly understood, the hallmark pathological features include: inflammation, excessive proliferation of fibroblasts, and abnormal deposition of extracellular matrix.
  • Bleomycin is dissolved in physiological saline, and the final concentration is adjusted according to the administered dose.
  • animals On day 1, animals will be anesthetized by inhalation of 2-5% isoflurane. Based on body weight, the animal will be administered bleomycin via the intratracheal route of administration (2 mg/kg, the specific dosage volume will be calculated and recorded based on the animal's body weight).
  • the day of bleomycin induction was regarded as the first day of the test.
  • the animals were screened and grouped on the 3rd day. All animals will be dosed on the 8th day of the experiment, once a day until the end of the experiment. Please see Table 7 for the specific dosing schedule.
  • Another 0.5 mL of PBS (containing 1% FBS) was used to lavage the lungs for the second time. Suspend 100 ⁇ L to count the total number of cells in BALF. Centrifuge BALF at 300g at 4°C for 5 minutes, and collect the supernatant of alveolar lavage fluid (BALF) without cell clusters, using electrochemiluminescence immunotechnology (Musk’s MSD mouse ten-factor detection kit, V-PLEX Proinflammatory Panel 1 Mouse Ki, t Product No. 15048D-X) To detect the concentration of TNF- ⁇ , IL-1 ⁇ , IL-6, IFN- ⁇ and other inflammations and cytokines in mouse BALF.
  • the cell mass obtained after centrifugation was resuspended for smear preparation and stained with Wright-Giemsa staining solution to distinguish eosinophils, neutrophils, macrophages and lymphocytes. Count under an optical microscope.
  • the animals were euthanized by removing the cervical vertebrae.
  • the lung tissues were collected, the right lung was cryopreserved, and the total protein was extracted after homogenization.
  • the contents of type I collagen, hyaluronic acid, and ⁇ -SMA were detected using a commercial ELISA kit, all of which were loaded in multiple holes.
  • the left lung was collected and fixed with neutral formaldehyde.
  • the left lung of each animal was cut into three sections and embedded in a paraffin block for paraffin embedding and preparation of ultra-thin sections with a thickness of 5 microns. Masson's staining was performed respectively. , For histopathological evaluation.
  • Hyaluronic Acid ELISA Kit Mae Quantikine ELISA Kit, Biotechne, Item No.: DHYAL0
  • Collagen I ELISA Kit Mae Type I Collagen Detection ELISA Kit, Chondrex, Item No.: 6012
  • the animals were weighed once on the day of model building, once on the day of grouping, and three times a week after grouping, and the animal's weight was recorded.
  • WBP whole-body plethysmograph
  • the left lung of each animal was cut into three sections and embedded in a wax block for paraffin embedding and preparation of ultra-thin sections with a thickness of 5 micrometers.
  • One section for each wax block was prepared for Masson staining.
  • Type I collagen content detection hyaluronic acid, ⁇ -SMA and TNF- ⁇ , IL-1 ⁇ , IL-6 and other ten-factor detection
  • the collected right lung tissue will be homogenized and operated according to the instructions of the commercial test kit to detect the content of type I collagen, hyaluronic acid and ⁇ -SMA; use the MSD box to measure the expression of cytokines in BALF
  • test data is expressed as mean ⁇ standard error (mean ⁇ S.E.M).
  • SPSS SPSS or Graphpad Prism to analyze the data.
  • the specific analysis method used will be explained in the legend and the notes below the table. P ⁇ 0.05 was considered statistically different.
  • the WBP system was used to measure airway hyperresponsiveness in test mice.
  • the mice inhaled the PBS solution by aerosol, and then continuously aerosolized methacholine (Mch) with concentrations of 1.5625, 3.125, 6.25, 12.5, 25, and 50 mg/mL to determine the enhanced expiratory interval value at the corresponding concentration (Penh), stimulate for 90 seconds at each concentration, calculate the percentage of Penh relative to the baseline in PBS and different Mch concentrations for each mouse.
  • the results are shown in Table 9, and the change rate of Penh relative to the baseline-Mch concentration curve is drawn (Figure 36), and calculate the area under the curve (Table 10).
  • the electrochemiluminescence immunoassay technique was used to detect 10 inflammatory and cytokines such as TNF- ⁇ , IL-1 ⁇ and IL-6 in the BALF of each group of animals.
  • the up-regulation of 1 ⁇ , IL-2, IL-5, IL-6 and TNF- ⁇ all have inhibitory effects, especially the up-regulation of IL-6.
  • the BALF cells of each group of mice were smeared, Wright-Giemsa staining solution stained to distinguish eosinophils, neutrophils, macrophages and lymphocytes, and counted under an optical microscope.
  • the total count results of the 4 types of cells in each group are shown in Table 9, and the respective count results of the 4 types of cells in each group are shown in FIG. 38. It shows that PAO and d5PAO can inhibit the total number of inflammatory cells caused by pulmonary fibrosis and the increase of 4 types of inflammatory cells to varying degrees, especially the increase of centriocytes.
  • Pulmonary fibrosis is often accompanied by an increase in blood hyaluronic acid and collagen. Therefore, the plasma hyaluronic acid and collagen levels were tested by ELISA. Part of the results are shown in Figure 39 and Figure 40, suggesting that PAO and d5PAO have an inhibitory effect on the increase of plasma hyaluronic acid and collagen caused by pulmonary fibrosis, and the effect on the increase of hyaluronic acid is significantly better than that of the positive control drug. Nintedanib.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Psychiatry (AREA)
  • Pain & Pain Management (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Hospice & Palliative Care (AREA)
  • Virology (AREA)
  • Urology & Nephrology (AREA)
  • Obesity (AREA)
  • Diabetes (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Wood Science & Technology (AREA)
  • Pulmonology (AREA)

Abstract

一种氘代氧化苯砷或其药学上可接受的盐以及含有药物学上可接受的载体和氘代氧化苯砷的药物组合物。该氘代氧化苯砷可用于治疗和预防癌症以及相关的疾病。

Description

氘代氧化苯砷化合物及其应用 技术领域
本发明属于化学合成领域，具体涉及一种新型氘代氧化苯砷化合物及其制备方法和应用。
背景技术
氧化苯砷(Phenylarsine Oxide，PAO)是一种已知的生物抑制剂，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。最近的研究发现，氧化苯砷是一种PI4KIIIα抑制剂，能够用于治疗阿尔茨海默病。
氘是氢的一种稳定同位素。相比于氢，氘可以形成更强的化学键，使药物分子更加稳定。人体试验已经发现氘替代可以改变药物的半衰期，减少服药次数，同时保持原有的活性和选择性。氘代药物已经成为是一种新药研发的新方向和药物开发模式。2017年，美国食品药品监督管理局通过世界上首个氘代药物即氘代丁苯那嗪(AUSTEDOTM，治疗亨廷顿舞蹈症及其相关的运动功能障碍)。目前已有多个氘代药物进入临床研究。
发明内容
在一方面中，本发明提供了一种式I化合物或其药学上可接受的盐，
Figure PCTCN2021084773-appb-000001
其中，R 1、R 2、R 3、R 4、R 5独立地选自氢、氘、卤素、甲基、一氘代甲基、二氘代甲基或三氘代甲基，且R 1、R 2、R 3、R 4、R 5中至少一个为氘或氘代的。
在一个实施方案中，R 1、R 2、R 3、R 4、R 5独立地选自氢或氘，且R 1、R 2、R 3、R 4、R 5中至少一个为氘，至少两个为氘，优选地至少三个、四个或 五个为氘。
在一个具体实施方案中，所述化合物选自由以下化合物组成的组：
Figure PCTCN2021084773-appb-000002
另一方面，本发明开公开了前述化合物或其药学上可接受的盐在制备预防或治疗受试者疾病或病理反应的药物中的用途。
在一个实施方案中，其中所述疾病选自肿瘤、恶病质诸如恶性肿瘤或治疗肿瘤的化疗药物引起的恶病质、阿尔兹海默症、细胞内蛋白错误折叠相关疾病、溶酶体贮积病、炎症反应、组织器官纤维化、病毒感染的疾病、神经症。
在一个实施方案中，所述受试者是人或非人哺乳动物。
在一个具体实施方案中，所述肿瘤选自淋巴瘤、宫颈癌、肝癌、乳腺癌诸如三阴性乳腺癌、肺癌诸如非小细胞肺癌或小细胞肺癌、结直肠癌、胃癌、皮肤癌诸如黑色素瘤、骨癌、骨肉瘤、骨髓瘤、血癌、或卵巢癌。。
在一个具体实施方案中，所述细胞内蛋白错误折叠相关疾病为帕金森氏病、路易体痴呆症、多系统萎缩症、包涵体肌炎症、额颞痴呆、亨廷顿疾病、多聚谷氨酰胺病、肌萎缩性侧索硬化症、或朊病毒疾病。
在一个具体实施方案中，所述溶酶体贮积病为鞘脂类代谢障碍诸如戈谢病、C型尼曼氏病、黏多糖病、糖原贮存病、糖蛋白贮积病、脂类储存疾病、翻译后修饰缺陷症、内在膜蛋白缺失失调症、神经元蜡样质脂褐质沉积病、或溶酶体相关细胞器紊乱症。
在一个具体实施方案中，其中所述炎症反应为局部组织或者全身血液中炎症因子诸如TNFα或IL-6增加。
在一个具体实施方案中，所述组织器官纤维化选自肺纤维化或肝纤维化。
在一个具体实施方案中，所述病毒包括冠状病毒和非冠状病毒，优选所述冠状病毒选自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、或新型冠状病毒；所述的非冠状病毒选自丙肝病毒或艾滋病毒。
在一个具体实施方案中，所述神经症选自神经衰弱、焦虑症、抑郁症、或躁狂症。
又一方面，本发明开公开了前述化合物或其药学上可接受的盐在制备预防或治疗受试者疾病的药物中的用途，其进一步包括向需要其的受试者施用第二试剂。本发明开公开了前述化合物或其药学上可接受的盐和第二试剂在制备预防或治疗受试者疾病的联合用药的药物中的用途。
在一个实施方案中，所述疾病选自肿瘤，所述第二试剂是用于治疗自肿瘤的试剂。
在一个具体实施方案中，其中所述疾病选自肺纤维化，所述第二试剂是用于治疗自肺纤维化的试剂，诸如与血管内皮生长因子受体络氨酸激酶抑制剂，优选为尼达尼布。
在一个具体实施方案中，所述第二试剂是用于治疗肿瘤的试剂，所述治疗肿瘤的试剂选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
在一个实施方案中，前述化合物或其其药学上可接受的盐在所述第二试剂之前、之后或同时施用。
又一方面，本发明开公开了一种药物组合物，包含前述的化合物或其药学上可接受的盐，和药物学上可接受的载体。
在一个实施方案中，所述药物组合物进一步包括治疗肿瘤的药物。
在一个具体实施方案中，所述治疗肿瘤的药物选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
又一方面，本发明开公开了一种如前所述的化合物或其药学上可接受的盐的制备方法，包括以下步骤：
Figure PCTCN2021084773-appb-000003
1)在0℃～10℃下在与式(I)对应结构的苯胺或其盐的水溶液中依次加入浓盐酸、亚硝酸钠水溶液，保持温度在5℃以下；
2)将碳酸钠、三氧化二砷、硫酸铜的水溶液加热至90～100℃后降温，在该水溶液中加入上述第1)步制备好的溶液，搅拌、过滤，滤液中加入酸调节pH值，分离所析出的固体；
3)将上述析出的固体、碘化钾以及亚硫酸氢钠或盐酸和二氧化硫在甲醇中搅拌至反应完全，进行后处理得到所述化合物。
在一个实施方案中，步骤3)中的后处理包括用酸或碱调节pH值至适当的值，用乙酸乙酯萃取，合并有机相并蒸发至干。
再一方面，本发明开公开了氧化苯砷及其衍生物在制备用于预防或治疗组织器官纤维化诸如肺纤维化或肝纤维化的药物中的用途。
本发明开公开了氧化苯砷及其衍生物在制备用于预防或治疗炎症反应的药物中的用途，其中所述炎症反应为局部组织或者全身血液中炎症因子诸如TNFα或IL-6增加。
本发明开公开了氧化苯砷及其衍生物在制备用于预防或治疗恶病质诸如恶性肿瘤或治疗肿瘤的化疗药物引起的恶病质的药物中的用途。
本发明开公开了氧化苯砷及其衍生物在制备用于预防或治疗肿瘤的药物中的用途。
在一个实施方案中，所述氧化苯砷及其衍生物具有式(II)所示的结构或者其药学上可接受的盐，
Figure PCTCN2021084773-appb-000004
其中，R 6各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、C1-6亚烷基-NH2、C1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C1-6烷基)氨基、N,N-(C1-6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-NH2或杂环基，并且可选的被R 7或R 8取代，其中R 7和R 8各自独立的选自氨基、C1-6烷基、C1-6烷氧基、C1-6卤代烷基、N-(C1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C1-6烷基)2氨基、C3-6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 10、-C(O)OR 9、6-12元的芳香基、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，并且R 9是C1-6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、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，R 10选自H、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基或C1-6卤代烷基，和/或
(b)两个相邻碳原子上的R 6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、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，
其中，n为0-5的整数。
在一个实施方案中，其中n为0-2的整数，所述R 6各自独立的选自H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-As(O)、N-(C1-6烷基) 氨基、N,N-(C1-6烷基)2氨基、-NH-C(O)H或-NH-S(O)2H，并且可选的被所述R 7或R 8取代。
在一个实施方案中，其中n为0-2的整数，所述R 6各自独立的选自H、卤素、硝基、氰基、羟基、氨基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-As(O)、-NH-C(O)H或-NH-S(O)2H，并且可选的被所述R 7或R 8取代。
在一个实施方案中，其中n为1或2，所述R 6各自独立的选自H、卤素、氨基、C1-6烷基砜基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-NH-C(O)R 7或-NH-S(O)2R 8，其中R 7为C1-6烷基，可选的被一个6-12元芳香基取代，R 8为6-12元芳香基，可选的被一个卤素、C1-6烷氧基或C1-6卤代烷基取代。
在一个实施方案中，其中所述R 6位于-As(O)基团的邻位和/或者对位。
在一个实施方案中，其中n是0。
在一个实施方案中，所述化合物选自由以下化合物组成的组：
Figure PCTCN2021084773-appb-000005
Figure PCTCN2021084773-appb-000006
Figure PCTCN2021084773-appb-000007
Figure PCTCN2021084773-appb-000008
在一个实施方案中，所述客体是人或者哺乳动物。
在一个实施方案中，所述肿瘤选自淋巴瘤、宫颈癌、肝癌、乳腺癌诸如三阴性乳腺癌、肺癌诸如非小细胞肺癌或小细胞肺癌、结直肠癌、胃癌、皮肤癌诸如黑色素瘤、骨癌、骨肉瘤、骨髓瘤、血癌、或卵巢癌。
在一个实施方案中，其进一步包括向需要其的客体施用第二试剂，优选所述第二试剂是用于治疗肿瘤的试剂。
在一个实施方案中，其中所述第二试剂是用于治疗肿瘤的试剂。
在一个实施方案中，其中所述化合物在所述第二试剂之前、之后或同时施用。
在一个实施方案中，其中所述治疗肿瘤的试剂选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
本方法进一步公开了一种筛选用于预防或治疗疾病的药物的方法，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，所述疾病选自组织或器官纤维化、炎症反应、恶病质、肿瘤。
在一个实施方案中，组织器官纤维化选自肺纤维化或肝纤维化。
在一个实施方案中，所述炎症反应为局部组织或者全身血液中炎症因子诸如TNFα或IL-6增加。
在一个实施方案中，所述肿瘤选自淋巴瘤、宫颈癌、肝癌、乳腺癌诸如三阴性乳腺癌、肺癌诸如非小细胞肺癌或小细胞肺癌、结直肠癌、胃癌、皮肤癌诸如黑色素瘤、骨癌、骨肉瘤、骨髓瘤、血癌、或卵巢癌。
附图说明
图1示出在雄性SD大鼠中单次静脉注射0.1mg/Kg PAO或d5PAO，药代动力学的药时曲线图。
图2示出在雄性SD大鼠中单次口服灌胃0.2mg/Kg PAO或d5PAO，药代动力学的药时曲线图。
图3示出α-synuclein过表达质粒的载体图谱。
图4示出α-synuclein ELISA检测的标准配制图。
图5示出d5PAO和PAO抑制SH-sy5y细胞凋亡，其中图5A示出MTT检测一定浓度d5PAO和PAO对SH-sy5y细胞活力的作用，n＝5，mean±SEM，One-way ANOVA，***p<0.0001vs.ctrl，###p<0.0001vs.ctrl；图5B示出免疫荧光处理前在培液中加入碘化丙啶(PI)共孵育15分钟后进行Ki67免疫荧光染色。
图6示出d5PAO和PAO提高稳转APP(SW)HEK293细胞活力并促进Aβ释放，其中图6A示出MTT检测一定浓度d5PAO和PAO对稳转APP(SW)HEK293细胞活力的作用，n＝5，mean±SEM，One-way ANOVA，**p<0.001，***p<0.0001vs.ctrl；图6B示出ELISA试剂盒检测上清中Aβ含量，并通过标准曲线计算各组Aβ值；图6C示出将数据归一化处理，以ctrl为1，计算各组Aβ含量变化的倍数值，n＝3，mean±SEM，One-way ANOVA，*p<0.03，**p<0.001，***p<0.0001vs.ctrl。
图7示出PAO氘代化合物结构式及其促进Aβ释放作用比较，图7A示出ELISA试剂盒检测上清中Aβ含量，并通过标准曲线计算各组Aβ值；图7B示出将数据归一化处理，以ctrl为1，计算各组Aβ含量变化的倍数值，n＝3，mean±SEM，One-way ANOVA，*p<0.03，**p<0.001，***p<0.0001vs.ctrl.与50nM d5PAO处理组比较##p<0.001，###p<0.0001。
图8示出d5PAO和PAO减小α-synuclein过表达对SH-sy5y细胞的损伤作用并促进α-synuclein释放，其中图8A示出MTT检测一定浓度d5PAO和PAO对瞬转α-synuclein细胞的细胞活力作用，n＝5，mean±SEM，One-way ANOVA，*p<0.03，**p<0.001，***p<0.0001vs.ctrl；图B示出ELISA试剂盒检测上清中α-synuclein含量，并通过标准曲线计算各组α-synuclein值，n＝3，mean±SEM，One-way ANOVA，*p<0.03vs.ctrl；图8C示出将数据归一化处理，以α-syn OE组为1，计算各组α-synuclein含量变化的倍数值，n＝3，mean±SEM，One-way ANOVA，*p<0.03vs.ctrl。
图9示出d5PAO和PAO在CBE构建的SH-SY5Y细胞模型中发挥保护作用。其中图9A示出CBE处理SH-SY5Y细胞48小时，进行MTT实验检测细胞活力；图9B示出100μM CBE处理SH-SY5Y细胞24小时，饥饿(不含FBS的高糖DMEM)和100μM CBE处理共同处理24小时，再给予不同浓度PAO处理24小时，通过MTT实验检测细胞活力；图9C示出100μM CBE处理SH-SY5Y细胞24小时，饥饿(不含FBS的高糖DMEM)和100μM CBE处理共同处理24小时，再给予不同浓度d5PAO和PAO处理24小时，通过MTT实验检测细胞活力。n＝5，数据以mean±SEM表示，与对照组比较###p<0.0001，与100μM CBE处理组比较**p<0.001,***p<0.0001vs.100μM CBE。
图10示出PAO抑制CBE诱导的溶酶体和GlcCer贮积，促进GlcCer外排。其中图10A示出SH-SY5Y细胞与lysosome tracker共孵育30分钟，再吸去上清更换为含有不同浓度的PAO并孵育10分钟，通过免疫荧光方法观察Lyso-tracker；图10B示出统计分析各组Lyso-tracker荧光强度；标尺:50μm；n＝5；One-way ANOVA分析，与对照组比较##p<0.001，与100μM CBE处理组比较**p<0.001，***p<0.0001；图10C示出各组细胞裂解液中GlcCer浓度经LC/MS测定值统计分析；图10D示出各组细胞培养基上清中GlcCer浓 度经LC/MS测定值统计分析。
图11示出敲减PI4Ka促进溶酶体贮积减少。其中图11A示出Western blot检测不同shRNA干扰慢病毒载体(sh-ctrl,sh1-PI4Ka,sh2-PI4Ka,sh3-PI4Ka)处理SH-SY5Y细胞48小时PI4KⅢα蛋白水平；图11B对Western blot检测结果统计分析；图11C示出免疫荧光方法检测shRNA干扰慢病毒载体处理后Lyso-tracker荧光强度，并统计分析(图11D)。标尺:50μm；n＝5；数据以mean±SEM表示，One-way ANOVA分析，***p<0.0001。
图12示出pGMLV-SC5RNAi载体图谱。
图13示出MRC-5细胞处理24小时后明场图。MRC-5细胞使用含有5ng/mL TGF-β1，并根据分组同时加入含有不同浓度PAO或d5PAO的MEM培养基(无FBS)培养24小时。标尺：50μm。
图14示出d5PAO和PAO抑制α-SMA和Calponin1在MRC-5模型细胞中的表达。其中图14A示出蛋白质免疫印迹法检测α-SMA和Calponin1的表达水平。图14B示出各组α-SMA表达量统计分析结果。图14C示出各组Calponin1表达量统计分析结果。使用Image J软件分析蛋白质信号强度，以ctrl组信号强度为1，n＝3，使用单因素分析(One-way ANOVA)处理，数据用mean±SEM表示,与5ng/mL TGF-β1处理组比较*p<0.03，**p<0.001，***p<0.0001，与ctrl组比较#p<0.03。
图15示出d5PAO和PAO抑制α-SMA和Calponin1在MRC-5模型细胞中的表达。其中图15A示出各组α-SMA免疫荧光图，红色：α-SMA，蓝色：DAPI(核)。标尺：50μm。15B示出各组Calponin1免疫荧光图，红色：Calponin1，蓝色：DAPI(核)。标尺：50μm。图15C,D示出α-SMA,Calponin1免疫荧光强度统计结果分析。以ctrl组平均值为1，n＝5,使用单因素分析(One-way ANOVA)处理，数据用mean±SEM表示,与5ng/mL TGF-β1处理组比较*p<0.03，**p<0.001，***p<0.0001，与ctrl组比较###p<0.0001。
图16示出d5PAO和PAO调控Calponin1在MSC中的表达，其中图16A示出各组Calponin1免疫荧光图，红色：Calponin1，蓝色：DAPI(细胞核)。标尺：50μm；图16B示出Calponin1免疫荧光强度统计结果分析。n＝4使用单因素分析(One-way ANOVA)处理，数据用mean±SEM表示。
图17示出d5PAO和PAO抑制MRC-5细胞纤维过程中COL1的分泌。 图17A示出ELISA检测各组上清中COL1浓度，n＝6，数据用mean±SEM表示。图17B示出以ctrl组平均值为1，对各组上清中COL1浓度进行统计分析，数据用mean±SEM表示，与5ng/mL TGF-β1处理组比较*p<0.03，**p<0.001，***p<0.0001，与ctrl组比较#p<0.0001。
图18示出shRNA干扰慢病毒载体使PI4KⅢα表达减少，shRNA干扰慢病毒载体与MRC-5细胞共孵育48小时，收集蛋白进行蛋白免疫印迹实验。图18A示出检测PI4KⅢα蛋白；图18B示出对数据使用Image J软件进行分析，以sh-ctrl组为1进行归一化处理，n＝3，数据用mean±SEM表示，与sh-ctrl组比较***p<0.0001。
图19示出敲减PI4Ka抑制Calponin1和α-SMA在TGF-β1处理的MRC-5细胞中表达。MRC-5细胞贴壁后加入不同序列的慢病毒载体培养24小时，根据分组加入或不加入5ng/mL TGF-β1处理24小时，进行免疫荧光染色观察。其中图19A示出各组α-SMA免疫荧光染色，红色：α-SMA，蓝色：DAPI(核)，绿色：绿色荧光蛋白GFP。标尺：50μm。图19B示出各组各组Calponin1免疫荧光染色，红色：Calponin1，蓝色：DAPI(细胞核)，绿色：绿色荧光蛋白GFP。标尺：50μm。α-SMA(图19C)和Calponin1免疫荧光强度统计结果分析，以sh-ctrl组平均值为1进行归一化处理，n＝5，数据用mean±SEM表示，与sh-ctrl加5ng/mL TGF-β1共同处理组比较*p<0.03，***p<0.0001，与sh-ctrl组比较，##p<0.001，###p<0.0001。
图20示出d5PAO和PAO抑制IL-6和TNF-α在BV2细胞炎症模型中的分泌。其中图20A示出ELISA检测BV2细胞上清中TNF-α浓度，并据标准曲线计算TNF-α含量(pg/mL)；图20B示出以ctrl组浓度平均值为1，计算各组TNF-α相对浓度变化图20C示出ELISA检测BV2细胞上清中IL-6浓度，并据标准曲线计算IL-6含量(pg/mL)；图20D示出以ctrl组浓度平均值为1，计算各组IL-6相对浓度变化。n＝3，使用单因素分析(One-way ANOVA)处理，数据用mean±SEM表示,与1μg/mL LPS处理组比较*p<0.03，**p<0.001，***p<0.0001，与ctrl组比较#p<0.03，##p<0.001。
图21示出PAO对乳腺癌的抑制作用。
图22示出PAO对淋巴癌的抑制作用。
图23示出d5PAO对黑色素瘤的抑制作用。
图24示出d5PAO在用药第28天对黑色素瘤的抑制作用。
图25示出高剂量PAO和d5PAO灌胃对小鼠体重和存活率的影响比较。
图26示出PAO对乳腺癌小鼠模型体重的影响。
图27示出PAO对胰腺癌模型体重的影响。
图28示出PAO对淋巴瘤模型动物体重的影响。
图29示出d5PAO联合用药对黑色素瘤小鼠体重的影响。
图30示出PAO和d5PAO对HCoV 229E(流感冠状病毒)的抑制作用。
图31示出PAO和d5PAO的抗焦虑作用，d5PAO比PAO的抗焦虑作用更显著。
图32示出PAO和d5PAO有抗抑郁作用，d5PAO比PAO的抗抑郁作用更显著和更稳定。
图33示出d5PAO和PAO抑制U18666A导致的胆固醇贮积。标尺：50μm。
图34示出PAO促进LC3B和p62表达，Baf-A1阻断PAO在细胞模型中的保护作用，其中图34A示出蛋白质免疫印迹实验检测LC3B和p62蛋白；图34B，C示出使用Image J软件统计分析LC3B和p62蛋白信号强度统计分析；图34D示出免疫荧光检测LC3B和p62，红色：p62，绿色：LC3B，标尺：50μm；图34E示出MTT检测各组细胞活力，并统计分析。n＝5,数据以mean±SEM表示，One-way ANOVA分析，与对照组比较###p<0.0001,与100μM CBE处理组比较**p<0.001。
图35示出敲减PI4Ka激活ALP，其中图35A，B示出Western blot检测不同shRNA干扰慢病毒载体(sh-ctrl，h1-PI4Ka,sh2-PI4Ka,sh3-PI4Ka)处理SH-SY5Y细胞48小时后LC3B蛋白水平，并统计分析；图35C，D示出shRNA干扰慢病毒载体转染的同时给予CBE处理，共孵育48小时，检测LC3B蛋白水平，并统计分析。n＝3；数据以mean±SEM表示，One-way ANOVA分析，与sh-ctrl比较*p<0.03。
图36示出乙酰甲胆碱引起的增强呼气间歇值Penh相对基线的百分比。
图37示出肺泡灌洗液(BALF)中的嗜酸性粒细胞、巨噬细胞、中性粒细胞和淋巴细胞的总数计数。T.Test,单尾，*<0.5，**<0.1与模型组的总细胞含量比。
图38示出肺泡灌洗液(BALF)中的嗜酸性粒细胞、巨噬细胞、中性粒细胞和淋巴细胞的分别计数。T.Test,单尾，*<0.5，**<0.1与模型组的BALF比较。
图39示出血浆胶原蛋白I型的含量，正常组(ctrl)。
图40示出PAO和dPAO与阳性对照药尼达尼布中下调肺纤维化小鼠血浆透明质酸的作用比较，正常组(ctrl)。
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。
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。
根据本发明的另一方面，本发明涉及氘代氧化苯砷优选是全苯环取代氘代同位素。
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。
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。
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 PCTCN2021084773-appb-000009
细胞内蛋白错误折叠相关疾病
本文所用的术语“细胞内蛋白错误折叠相关疾病”是指由胞浆内非正常折叠的蛋白聚集所成为标志性特征的疾病，也被诊断为蛋白聚集(aggregation，accumulation)或蛋白错误折叠(misfolding)疾病。此外，术语“细胞内蛋白错误折叠相关疾病”也包括一些细胞内包涵体病变，比如蛋白质包涵体积累病，这一类包涵体主要由一种因折叠错误而聚集的核心蛋白，外附各种参与响应非折叠蛋白的应激蛋白。
细胞内蛋白错误折叠相关疾病包括但不限于帕金森氏病(PD)、路易体痴呆症(LBD)、多系统萎缩症(MSA)、包涵体肌炎症(IBM)、额颞痴呆(FTD)、亨廷顿疾病(HD)、多聚谷氨酰胺病(PolyQ)、肌萎缩性侧索硬化症(ALS)、朊病毒疾病。
溶酶体贮积病
本文所用的术语“溶酶体贮积病”是指因各种原因导致一些内源或外源的物质在溶酶体内积累引起的疾病，包括但不局限于因溶酶体内的酶活性不足、激活蛋白、转运蛋白或溶酶体蛋白加工校正酶的缺乏而引起溶酶体功能缺陷，造成次级溶酶体内相应底物不能被消化，底物积蓄，代谢障碍，形成贮积性疾病等。
溶酶体贮积病包括但不限于鞘脂类代谢障碍、黏多糖病、糖原贮存病、糖蛋白贮积病、脂类储存疾病、翻译后修饰缺陷症、内在膜蛋白缺失失调症、神经元蜡样质脂褐质沉积病、或溶酶体相关细胞器紊乱症。其中，鞘脂类代谢障碍包括但不限于法布雷病，代谢障碍皮肤病(Farbe病)，戈谢病I、II、III型及产前死亡型，GM1神经节苷脂沉积症I、II、 III型，GM2神经节苷脂沉积症(家族性黑蒙性白痴)，GM2神经节苷脂沉积症，类球状脑白质营养不良(克拉伯病)，异染性脑白质营养不良，尼曼匹克症A和B型；黏多糖病包括但不限于贺勒-施艾氏症候群和施艾氏症(ML I)、亨特综合症(MPS II)、圣菲利柏氏症A(MPS IIIA)、圣菲利柏氏症B(MPS IIIB)、圣菲利柏氏症C(MPS IIIC)、圣菲利柏氏症D(MPS IIID)、离心性骨软骨发育不良综合症(MPS IVA)、离心性骨软骨发育不良综合症(MPS IVB)、粘多糖病第六型(拉米氏症，MPS VI)、Sly病(MPS VII)、MPS IX；糖原贮存病包括但不限于罕见病庞贝氏症(GSD II)；糖蛋白贮积病包括但不限于α-苷露糖苷贮积病、β-苷露糖苷贮积病、岩藻糖苷贮积症、门冬酰葡糖胺尿症、辛德勒疾病I型(婴儿期发病神经轴索营养不良症)、辛德勒疾病II型(Kanzaki疾病)、辛德勒疾病III型(中等严重度)、唾液酸贮积症I型(樱桃红点状肌阵挛综合征)、唾液酸贮积症II型(粘多糖症I)、半乳糖涎酸贮积症；脂类储存疾病包括但不限于酸脂酶缺乏诸如Wolman病和胆固醇酯沉积病；翻译后修饰缺陷症包括但不限于多发性硫酸脂酶缺乏症、粘脂累积病IIα/β(I-细胞症)、粘脂累积病IIα/β(假赫勒氏综合症)、粘脂累积病IIIγ型(假赫勒氏综合症变体)；内在膜蛋白缺失失调症包括但不限于胱氨酸过多症、Danon病、肌阵挛肾衰竭综合症、唾液酸贮积症诸如ISSD、Salla症和中度严重Salla症、尼曼-皮克症C1和C2型、粘脂累积病IV型；神经元蜡样质脂褐质沉积病包括但不限于蜡样脂质褐质沉积病1型(Haltia-Santavuori症和INCL)、神经元蜡样质脂褐质沉积病2型(Jansky-Bielschowsky症)、蜡样脂质褐质沉积病3型(Batten-Spielmeyer-Sjogren症)、蜡样脂质褐质沉积病4型(Parry病和Kufs A和B类)、蜡样脂质褐质沉积病5型(晚期婴儿芬兰异型)、蜡样脂质褐质沉积病6型(Lake-Cavanagh或印第安异型)、蜡样脂质褐质沉积病7型(土耳其异型)、蜡样脂质褐质沉积病8(北方癫痫、癫痫智能障碍)、蜡样脂质褐质沉积病9、蜡样脂质褐质沉积病10、蜡样脂质褐质沉积病11、蜡样脂质褐质沉积病12、蜡样脂质褐质沉积病13、蜡样脂质褐质沉积病14；溶酶体相关细胞器紊乱症包括但不限于Hermansky-Pudlak病1型、Hermansky-Pudlak病2型、Hermansky-Pudlak病3型、Hermansky-Pudlak病4型、Hermansky-Pudlak病5型、 Hermansky-Pudlak病6型、Hermansky-Pudlak病7型、Hermansky-Pudlak病8型、Hermansky-Pudlak病9型、Griscelli综合症1(Elejalde综合症)、Griscelli综合症2、Chédiak–Higashi病。
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。
本文中公开的氘代氧化苯砷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，本文所述的氘代氧化苯砷通过口服、皮下、肌内或静脉施用。在一些实施方案中，本文所述的氘代氧化苯砷通过口服施用。
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细胞内蛋白错误折叠相关疾病或溶酶体贮积病的药物的量。
本文中提供的氘代氧化苯砷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细胞内蛋白错误折叠相关疾病的试剂，包括但不限于左旋多巴、力鲁唑。
在一些实施方案中，所述氘代氧化苯砷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细胞内蛋白错误折叠相关疾病的方法，包括向需要其的客体施用有效量的氘代氧化苯砷。
本申请的还涉及用于预防或治疗溶酶体贮积病的方法，包括向需要其的受试者施用有效量的氘代氧化苯砷。
实施例1.化合物的合成及其物理特性
1.d5PAO(五氘代氧化苯砷)的合成及其物理特性
1.1 d5PAO合成
d5PAO的合成线路如下：
Figure PCTCN2021084773-appb-000010
第一步：d5-PA的合成方法：
Figure PCTCN2021084773-appb-000011
往一个三口瓶中加入91.35mL水，18.27g的五氘代苯胺(d5-Aniline)，搅拌、降温至0℃～10℃,滴加37.45mL浓盐酸。滴加完毕盐酸后，滴加亚硝酸钠水溶液(13.43g固体亚硝酸钠溶于36.5mL水中)，保持温度不超过5℃；滴加完毕，保温2-3小时左右。完成重氮盐的制备。
另一容器加入274mL纯化水，69.06g碳酸钠，36.82g三氧化二砷，2.83g五水硫酸铜(CuSO4·5H 2O)，加热至90～100℃并保温搅拌30分钟，后降温至5～15℃。分批缓慢加入上述已制备好的重氮盐，控温低于15℃。搅拌2～3小时，随后自然升至室温搅拌过夜。过滤，滤饼用水淋洗。合并滤液，缓慢加入浓盐酸调节体系pH值为3.0，有少量棕色絮状物析出。抽滤。滤液用100mL乙酸乙酯洗涤三次，将水相于50～60℃减压浓缩至剩余约170mL左右，析出大量白色固体。抽滤，滤饼用冷水淋洗，抽干，直接将固体于鼓风烘箱50度烘料18小时，得到35克类白色固体d5-PA，收率：90.83％,MS ES+(m/z):208.0[(M+H) +]。
第二步：d5-PAO的合成方法：
Figure PCTCN2021084773-appb-000012
往一个三口瓶中加入400mL纯化水，25g d5-PA，75.4g亚硫酸氢钠，0.32g碘化钾，125mL甲醇开启搅拌；于30～40℃反应过夜,原料基本反应完全，停止反应。将此溶液控制温度在30℃以下，用浓盐酸调pH值为7.0。用乙酸乙酯萃取4次，合并萃取后有机相。将其于35～45℃浓缩至干，得到 20g白色固体湿品。将固体用240mL叔丁基甲基醚升温回流1小时，降温，抽滤，滤饼用冷的MTBE洗涤。滤饼在50℃下用鼓风烘箱干燥过夜，得到8.7g白色固体d5PAO， 13C-NMR(δ,DMSO-d6)：149.9,129.6,129.4,128.2,MS ES+(m/z)：173.99[(M+H) +]。
1.2 d5PAO理化性质
1.2.1仪器
Agilent 1260 Prime高效液相色谱仪，Mettler Toledo XS105型天平(0.01mg)，KQ5200B型超声仪(昆山市超声仪器有限公司)，BR2000-GM型可变速振荡器(VWR International)，0.45μm滤膜(上海青阳生物科技有限公司)。
1.2.2实验用药
d5PAO(纯度97.9％)，乙腈为色谱纯(国药集团化学试剂有限公司)，盐酸、DMSO均为分析纯(国药集团化学试剂有限公司)。
1.2.3溶液配制
取0.1M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.01M的盐酸溶液，用精密pH试纸测定pH2。
取0.01M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.001M的盐酸溶液。
取0.001M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.0001M的盐酸溶液。用精密pH试纸测定该溶液的pH为4。
取20mL去离子水，测定pH为6。
取7.5mg d5PAO，于离心管中，加入1mL DMSO，超声振荡，溶解。再用乙腈/水(1/1)混合液稀释到10mL，得到0.75mg/mL的d5PAO储备液。将d5PAO储备液稀释成0.3mg/mL、0.15mg/mL、0.075mg/mL、0.03mg/mL、0.015mg/mL的不同d5PAO工作液。
1.2.4色谱条件
液相色谱仪：安捷伦1260 Infinity II Prime超高效液相色谱系统。
色谱柱：ACQUITY
Figure PCTCN2021084773-appb-000013
Peptide C18
Figure PCTCN2021084773-appb-000014
2.1*100mm ID.，1.7μm(Waters)。
流动相A：含0.01％AA和2mmol/L NH4OAc的Water:ACN(v:v,95: 5)溶液。
流动相B：含0.01％AA和2mmol/L NH4OAc的Water:ACN(v:v,5:95)溶液。
洗脱梯度：
Figure PCTCN2021084773-appb-000015
柱温：40℃
进样量：4μL
检测波长：254nm
1.2.5方法学考察
线性关系的考察
取0.75mg/mL、0.3mg/mL、0.15mg/mL、0.075mg/mL、0.03mg/mL、0.015mg/mL的不同d5PAO溶液液；分别按照上述色谱条件进样测定，记录色谱图。以峰面积对进样浓度进行线性回归，得回归方程：
y＝1647.7x+0.9623，R2＝0.9999
结果表明d5PAO进样浓度在0.015～0.75mg·mL-1范围内与峰面积呈良好的线性关系。
1.2.6平衡溶解度的测定
吸取不同pH缓冲液各2mL，分别置3mL离心管中，加入过量的d5PAO药粉末至溶液中出现大量白色不溶性沉淀，超声30分钟，置恒温振荡器中，在25℃条件下振摇24h，再超声30分钟，0.45μm滤膜滤过，吸取续滤液，加水稀释10倍，按上述色谱条件进样测定，记录色谱图，计算d5PAO在不同pH缓冲液中溶解度分别为5.36mg/mL、5.39mg/mL、5.98mg/mL。
表1.d5PAO的溶解度
药物 pH 峰面积 对应溶解度(mg/mL)
d5PAO 6 884.20 5.36
d5PAO 4 889.51 5.39
d5PAO 2 986.70 5.98
2.PAO的合成及其物理特性
2.1 PAO的合成
合成路线：
Figure PCTCN2021084773-appb-000016
第一步：
Figure PCTCN2021084773-appb-000017
250mL单口瓶中，加入苯胺(10.0g，107mmol，1.0eq.)及丙酮(20mL)。冷却至-15℃左右(高温会导致产物颜色较深)，慢慢加入48％HBF4(30mL，29.5g，161mmol，1.5eq.)。将NaNO 2(11.0g，161mmol，1.5eq.)溶于H 2O(20mL)中，缓慢滴加至以上溶液中。加料完毕，先在-15℃下保温2小时，然后在0℃下搅拌反应1小时左右。固体(白色)过滤，异丙醚(50mL×2)洗涤。产品真空干燥(温度30℃)，称重：17.5g,产率：85％，粉红色固体。直接用于下一步反应。
第二步：
Figure PCTCN2021084773-appb-000018
250mL烧瓶中，加入Na 2CO 3(21.2g，200.6mmol，3.85eq.)、As 2O 3(11.3g，57.3mmol，1.1eq.)、CuSO 4-5H 2O(800mg,3.13mol,0.06eq.) 以及H 2O(60mL)，悬浊液加热至90℃左右，保持20分钟左右，使大部分固体溶解。冷却至0℃-15℃左右，分批缓慢加入偶氮苯氟硼酸盐(10.0g，52.1mmol，1.0eq.)与H 2O(60mL)的悬浊液，加入少许丙酮以降低泡沫的集聚。加料完毕，混合物在室温下搅拌12小时左右。铺硅藻土，过滤，H 2O洗(20mL×3)，滤液如颜色太深，可加入活性炭脱色，过滤。向滤液加入浓盐酸(12N，40mL)中和，进一步调整反应液至酸性。滤液浓缩至混合物体积50mL左右。固体过滤，用冷水洗一次；滤液浓缩，生成固体过滤，冷水洗一次。固体合并烘干，白色固体，称重：8.6g,产率：82％。MS ES +(m/z)：202.9[(M+H) +]。
第三步：
Figure PCTCN2021084773-appb-000019
将苯砷酸(8.0g，40mmol，1.0eq.)、甲醇(40mL)、浓盐酸(15mL)及催化量KI(50mg)混合物中通入SO 2至饱和，室温下搅拌2小时。向混合物中加入NaOH溶液(2N)至溶液透明。向其中加入浓盐酸中和。析出固体，过滤，白色固体。固体经水/乙醇(1/5)重结晶，干燥，称重：4.0g，产率：60％。产品为白色粉状固体。 1H NMR(δ,CDCl 3):7.41-7.62(m,3H),7.75-7.78(m,2H).MS ES +(m/z)：168.8[(M+H) +]。
2.2 PAO的理化性质
PAO的分子式为C 6H 5AsO，分子量为168.03.
2.2.1仪器
Agilent 1260Prime高效液相色谱仪，Mettler Toledo XS105型天平(0.01mg)，KQ5200B型超声仪(昆山市超声仪器有限公司)，BR2000-GM型可变速振荡器(VWR International)，0.45μm滤膜(上海青阳生物科技有限公司)。
2.2.2实验用药
PAO(纯度98％)，乙腈为色谱纯(国药集团化学试剂有限公司)，盐 酸、DMSO均为分析纯(国药集团化学试剂有限公司)。
2.2.3溶液配制
取0.1M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.01M的盐酸溶液，用精密pH试纸测定pH2。
取0.01M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.001M的盐酸溶液。
取0.001M的盐酸溶液2mL，加入去离子水稀释到20mL，得到0.0001M的盐酸溶液。用精密pH试纸测定该溶液的pH为4。
取20mL去离子水，测定pH为6。
取7.5mg PAO，于离心管中，加入1mL DMSO，超声振荡，溶解。再用乙腈/水(1/1)混合液稀释到10mL，得到0.75mg/mL的PAO储备液。将PAO储备液稀释成0.3mg/mL、0.15mg/mL、0.075mg/mL、0.03mg/mL、0.015mg/mL的不同PAO工作液。
2.2.4色谱条件
液相色谱仪：安捷伦1260Infinity II Prime超高效液相色谱系统。
色谱柱：ACQUITY
Figure PCTCN2021084773-appb-000020
Peptide C18
Figure PCTCN2021084773-appb-000021
2.1*100mm ID.，1.7μm(Waters)。
流动相A：含0.01％AA和2mmol/L NH4OAc的Water:ACN(v:v,95:5)溶液。
流动相B：含0.01％AA和2mmol/L NH4OAc的Water:ACN(v:v,5:95)溶液。
洗脱梯度：
Figure PCTCN2021084773-appb-000022
柱温：40℃
进样量：4μL
检测波长：254nm
2.2.5方法学考察
线性关系的考察
取0.75mg/mL、0.3mg/mL、0.15mg/mL、0.075mg/mL、0.03mg/mL、0.015mg/mL的不同PAO溶液液；分别按照上述色谱条件进样测定，记录色谱图。以峰面积对进样浓度进行线性回归，得回归方程：
y＝1834.8x-4.2355R 2＝1
结果表明PAO进样浓度在0.015～0.75mg·mL-1范围内与峰面积呈良好的线性关系。
2.2.6平衡溶解度的测定
吸取不同pH缓冲液各2mL，分别置3mL离心管中，加入过量的PAO药粉末至溶液中出现大量白色不溶性沉淀，超声30分钟，置恒温振荡器中，在25℃条件下振摇24h，再超声30分钟，0.45μm滤膜滤过，吸取续滤液，加水稀释10倍，按上述色谱条件进样测定，记录色谱图，计算PAO在不同pH缓冲液中溶解度分别为1.15mg/mL、3.38mg/mL、4.52mg/mL。
表2.PAO的溶解度
药物 pH 峰面积 对应溶解度(mg/mL)
PAO 6 207.25 1.15
PAO 4 616.40 3.38
PAO 2 824.35 4.52
3.氘代化合物d1PAO、d2PAO和d3PAO的合成和理化性质
3.1 d1PAO的制备
Figure PCTCN2021084773-appb-000023
氮气条件下，将对溴苯胺(3.44g)溶于氘代甲醇(5mL)中，回流30分钟，旋干，重复3次，旋干抽干待用。将3克金属钠分批加入到氘代甲醇(10mL)中，待其反应完毕，缓慢加入重水(30mL)，制得10％的氘氧化钠的重水溶液。氮气条件下，将第一步的产品溶于氘代甲醇(5mL)，加入 锌粉(6.5g)和制备好的10％的氘氧化钠的重水溶液，加热回流3小时，点板监测对溴苯胺消失，冷却至室温，乙醚萃取，低温旋干，得到产品对氘苯胺。
Figure PCTCN2021084773-appb-000024
往带有磁力搅拌子的圆底瓶中加入由上步得到的对氘苯胺(0.94g)并加水5mL，降温至0–5℃，并于该温度下，缓慢加入37％的盐酸水溶液(2mL)，加入水5mL稀释，继续搅拌，反应30分钟。再向该反应液中缓慢加入NaNO2水溶液(724.5mg，10.5mmol，3mL H 2O)，控制反应温度在0–5℃，30-40分钟内滴加完，溶液呈棕黄色，继续低温下搅拌反应2小时.往另一个圆底反应瓶中加入Na2CO3(4.0g，38.0mmol，3.8eq.)，As 2O 3(1.0g，5.0mmol，0.5eq.)，CuSO 4.5H 2O(150mg，0.6mmol，0.06eq.)，H 2O(13mL)，95℃反应45分钟，溶液为绿色溶液，降温至0–5℃.向第2步反应液中缓慢滴加第1步制备好的偶氮盐酸盐，控制反应体系温度低于5℃，在滴加的过程中产生泡沫，加入少量丙酮除泡沫，待泡沫消失后继续滴加，1小时内滴加完毕，滴加完毕后自然升温，过夜搅拌。反应液通过硅藻土过滤，滤饼用冰水(2mL×2)淋洗。水相于50℃减压浓缩至10mL。于冰水浴中滴加入4mL的6N的HCl调节pH至7-8，出现少量黄褐色固体，抽滤，2mL冰水洗涤，弃去固体。滤液滴加入2.5mL的6N的HCl调节pH至2-3，出现大量气泡。该滤液减压浓缩，当体系出现大量固体时降温，抽滤，收集固体。所得滤液加入6N HCl调节pH至1，旋蒸，当体系出现大量固体时降温抽滤。母液洗涤2次，收集固体。共得纯品固体对氘苯胂酸900mg，产率为44％。1H NMR(δ,DMSO-d6)：7.60(d,2H),7.53(d,2H)。
Figure PCTCN2021084773-appb-000025
往25mL三颈瓶中依次加入对氘苯胂酸(880mg，4.3mmol，1.0eq.)，KI(16.6mg，0.1mmol，0.023eq.)，37％HCl(1.7mL，20.0mmol，4.6eq.)， 甲醇(5.8mL)，室温搅拌5–10分钟，持续通入二氧化硫气体，反应3小时，TLC监测反应完全。反应液抽滤，并用甲醇(1.5mL×2)洗涤。液体于冰水浴中滴加入质量分数为15％的NaOH溶液，调节pH至14，此时体系呈橙色浊液。用乙酸乙酯(50mL×2)萃取，合并有机相，并用无水Na 2SO 4干燥，过滤，低温(20-28℃)旋干，得到粗产物。向粗产物中加入约4mL的乙醚打浆约30分钟后过滤，抽干，得到类白色固体的d1PAO约430mg。 1H NMR(δ,DMSO-d6)：7.70(d,2H),7.46(d,2H)，MS ES +(m/z)：169.9[(M+H) +]。产率约59％。
3.2 d2PAO的制备
Figure PCTCN2021084773-appb-000026
将浓盐酸(5mL)滴加到邻溴苯胺(3.44g)的乙醚溶液中，有固体产生，砂芯漏斗抽滤，乙醚洗涤，抽干得到苯胺的盐酸盐。氮气条件下，在封管中将固体溶于重水(7mL)，加热到120℃，反应24小时，重水旋干，置换氮气，重新加入重水(7mL)，反应48小时，冷却至室温，使用30％的氢氧化钠调节pH＝7，乙醚萃取，无水硫酸钠干燥，旋干可得到2-溴-4，6-二氘-苯胺。氮气条件下，将22毫升的甲醇加入到2-溴-4，6-二氘-苯胺和10％Pd/C(300mg)的混合物中，将氮气置换为氢气，反应3小时，点板监测到2-溴-4，6-二氘-苯胺消失，硅藻土过滤，少量甲醇洗涤，直接旋干可得到2，4-二氘-苯胺的氢溴酸盐。
Figure PCTCN2021084773-appb-000027
往25mL带有磁力搅拌子的圆底瓶中加入由上步得到的2，4-二氘-苯胺的氢溴酸盐并加水5mL，降温至0–5℃，并于该温度下，缓慢加入37％的盐酸水溶液(1.1mL)，加入水5mL稀释，继续搅拌，反应30分钟.再向该反应液中缓慢加入NaNO 2水溶液(724.5mg，10.5mmol，3mL H 2O)，控制反应温度在0–5℃，30-40分钟内滴加完，溶液呈棕黄色，继续低温下搅拌反应2小时。往50mL圆底反应瓶中加入Na 2CO 3(4.0g，38.0mmol，3.8 eq.)，As 2O 3(1.0g，5.0mmol，0.5eq.)，CuSO 4·5H 2O(150mg，0.6mmol，0.06eq.)，H 2O(13mL)，95℃反应45分钟，溶液为绿色溶液，降温至0–5℃。向第2步反应液中缓慢滴加第1步制备好的偶氮盐酸盐，控制反应体系温度低于5℃，在滴加的过程中产生泡沫，加入少量丙酮除泡沫，待泡沫消失后继续滴加，1小时内滴加完毕，滴加完毕后自然升温，过夜搅拌。反应液通过硅藻土过滤，滤饼用冰水(2mL×2)淋洗。水相于50℃减压浓缩至10mL。于冰水浴中滴加入4mL的6N的HCl调节pH至7-8，出现少量黄褐色固体，抽滤，2mL冰水洗涤，弃去固体。滤液滴加入2mL的6N的HCl调节pH至3-4，出现粘稠状固体。抽滤，弃去固体(核磁显示这部分固体不含产物)。所得滤液浓缩至8mL，加入6N HCl(0.5mL)调节pH至2-3，出现大量固体，抽滤，得2，4-二氘-苯胂酸固体1.15g，产率为56.4％。 1H NMR(δ,DMSO-d6):7.72(d,1H),7.56-7.59(m,2H)。
Figure PCTCN2021084773-appb-000028
往25mL三颈瓶中依次加入2，4-二氘-苯胂酸(1.1g，5.4mmol，1.0eq.)，KI(20.6mg，0.124mmol，0.023eq.)，37％HCl(2.1mL，25.0mmol，4.6eq.)，MeOH(7.3mL)，室温搅拌5–10分钟，持续通入二氧化硫气体，反应3小时，TLC监测反应完全。冰水浴下，滴加入质量分数为15％的NaOH溶液，调节pH至7，此时体系出现大量不溶物。用乙酸乙酯(50mL×2)萃取，合并有机相，并用无水Na 2SO 4干燥，过滤，低温(20-28℃)旋干，得到粗产物。向粗产物中加入约3mL乙酸乙酯打浆约30分钟后过滤，抽干，得到类白色固体d2PAO约400mg。 1H NMR(δ,DMSO-d6)：7.58(d,1H),7.36-7.39(m,2H).MS ES+(m/z)：170.7[(M+H)+]。产率约48％。
3.3 d3PAO的制备
Figure PCTCN2021084773-appb-000029
将浓盐酸(5mL)滴加到苯胺溶液(2.65g苯胺，7mL重水)中，有固 体产生，砂芯漏斗抽滤，乙醚洗涤，抽干得到苯胺的盐酸盐，直接用于下一步。氮气条件下，在封管中将固体溶于重水(7mL)，加热到120℃，反应24h，旋干重水，置换氮气，重新加入重水(7mL)，反应48小时，所得2，4，6-三氘-苯胺重水溶液直接用于下一步。
Figure PCTCN2021084773-appb-000030
往25mL带有磁力搅拌子的圆底瓶中加入2，4，6-三氘-苯胺重水溶液(4.33mL)并加水2.5mL，降温至0–5℃，并于该温度下，缓慢加入37％的盐酸水溶液(1.1mL)，加入水5mL稀释，继续搅拌，反应30分钟.再向该反应液中缓慢加入NaNO 2水溶液(724.5mg，10.5mmol，3mL H 2O)，控制反应温度在0–5℃，30-40分钟内滴加完，溶液由紫色变黄色，继续低温下搅拌反应2小时。往50mL圆底反应瓶中加入Na 2CO 3(4.0g，38.0mmol，3.8eq.)，As 2O 3(1.0g，5.0mmol，0.5eq)，CuSO 4·5H 2O(150mg，0.6mmol，0.06eq)，H 2O(13mL)，95℃反应45分钟，溶液为绿色溶液，降温至0–5℃。向第2步反应液中缓慢滴加第1步制备好的偶氮盐酸盐，控制反应体系温度低于5℃，在滴加的过程中产生泡沫，加入少量丙酮除泡沫，待泡沫消失后继续滴加。1小时内滴加完毕，滴加完毕后自然升温，过夜搅拌。反应液通过硅藻土过滤，滤饼用冰水(2mL×2)淋洗。水相于50℃减压浓缩至10mL。于冰水浴中滴加入1.8mL的6N的HCl调节pH至7-8，出现少量黄褐色固体，抽滤，2mL冰水洗涤，弃去固体。滤液滴加入1.8mL的6N的HCl调节pH至3，出现淡黄色固体。抽滤，并用2mL冰水洗涤，收集固体。所得滤液浓缩至8mL，加入6N HCl 0.8mL调节pH至1，出现大量固体，抽滤，收集固体。共得固体2，4，6-三氘-苯胂酸860mg，产率为39％。 1H NMR(δ,DMSO-d6):7.56(s,2H)。
Figure PCTCN2021084773-appb-000031
往25mL三颈瓶中依次加入2，4，6-三氘-苯胂酸(3.9mmol，1.0eq.)， KI(15mg，0.09mmol，0.023eq)，37％HCl(1.3mL，18.0mmol，4.6eq.)，甲醇(5.3mL)，室温搅拌5–10分钟，持续通入SO 2，反应3h，TLC监测反应完全。反应液抽滤，并用甲醇(1.5mL×2)洗涤。冰水浴下，滴加入质量分数为17％的NaOH溶液4.3mL，调节pH至7，此时瓶壁出现黄色油状物。用乙酸乙酯(25mL×2)萃取，合并有机相，并用无水NaSO 4干燥，过滤，低温(20-28℃)旋干。向所得固体中加入约3mL乙酸乙酯打浆约30分钟后过滤，抽干，得到类白色固体d3PAO约200mg。将上步所得母液旋干，得到的固体加入约1.5mLEt 2O打浆约30分钟后过滤，抽干，得到类白色固体d3PAO约170mg。d3PAO合并后，产率约55％。 1H NMR(δ,DMSO-d6)：7.46(s,2H).MS ES +(m/z)：171.9[(M+H) +]。
实施例2.d5PAO和PAO药代动力学研究
d5PAO(d5-PAO)由实施例1所述的方法制备得到，PAO(PAO)由公司自行制备。在受试成年雄性大鼠中单次静脉注射PAO，d5PAO(施用剂量为0.1mg/Kg，化合物溶于0.1％DMSO)或单次口服胃灌流以上试剂混合物(施用剂量为0.2mg/Kg)后，分别在摄药后0、0.083、0.25、0.5、1、2、4、6、8、24、32、48小时抽取静脉血，用于药代动力学测试。通过蛋白沉淀的方法提取试验样品中的PAO和d5PAO，处理后的样品进样到液质联用(LC-MS/MS)中，经液相分离后采用ESI负离子模式检测。
样品处理(血样本)：
1)将40μL未知样品、校准标准品、质量控制、单个空白样品、双空白样品加入96孔板中；
2)每个样品中加入120μl的0.1mg/mL溶解于水的二巯基丙磺酸钠；
3)每个样品中加入40μL的溶解于水的0.2％甲酸。混合均匀后在45℃环境中震荡孵育15分钟，4℃离心5分钟(×3220g)；
4)每个样品(双空白除外)用200μL IS1淬灭(双空白样品用240μL MeOH淬灭)，震荡混合15分钟后在4℃环境中离心15分钟(×3220g)；
5)50μL上清液转入到96-孔板中4℃离心5分钟(×3220g),然后 将上清液用于LC-MS/MS分析。分析由通过Triple Quad 6500 +LC-MS/MS(SCIEX)系统完成。
实验结果：
雄性SD大鼠(n＝3)单次静脉注射同剂量(0.1mg/Kg)或单次口服灌胃给予同剂量的PAO和d5PAO(0.2mg/Kg)，两者的药代动力学指标无显著差异(参见表3、表4和图1和图2)。0.1mg/Kg灌胃剂量组生物利用度为15.7％。
表3.雄性SD大鼠单次静脉注射同剂(0.1mg/Kg)PAO和d5PAO药代动力学指标
  PAO(IV,0.1mg/Kg) d5PAO(IV,0.1mg/Kg)
PK Parameters Mean±SD Mean±SD
No.points used for T 1/2 3.00 3.00
C 0(ng/mL) 1344.67±210.97 1487.67±141.76
T 1/2(h) 11.4±0.03 12.43±0.65
Vd ss(L/kg) 0.16±0.02 0.155±0.02
Cl(mL/分钟/kg) 0.41±0.03 0.38±0.02
T last(h) 48.0 48.000
AUC 0-last(ng.h/mL) 4010±252.82 4337±280.63
AUC 0-inf(ng.h/mL) 4061.67±258.10 4404.33±275.54
MRT 0-last(h) 5.84±0.44 5.87±0.049
MRT 0-inf(h) 6.58±0.45 6.79±0.65
AUC Extra(％) 1.267±0.05 1.54±0.27
AUMC Extra(％) 12.47±0.67 14.87±1.46
表4.雄性SD大鼠单次单次口服灌胃(0.2mg/Kg)PAO和d5PAO药代动力学指标
  PAO(PO,0.2mg/Kg) d5PAO(PO,0.2mg/Kg)
PK Parameters Mean±SD Mean±SD
No.points used for T 1/2 3.00 3.00
C max(ng/mL) 156±12.53 161±10.4
T max(h) 8.00±0.00 7.33±1.15
T 1/2(h) 6.87±0.94 7.19±0.78
T last(h) 48.00 48.00
AUC 0-last(ng.h/mL) 2313.67±419.51 244.5.00±474.53
AUC 0-inf(ng.h/mL) 2341±425.87 2478.67±483.09
MRT 0-last(h) 12.53±0.61 12.63±0.78
MRT 0-inf(h) 13.03±0.75 13.27±0.9
AUC Extra(％) 1.16±0.47 1.35±0.44
AUMC Extra(％) 5.09±1.99 5.92±1.8
Bioavailability(％) a 28.82 28.14
实施例3.d5PAO与PAO对细胞活力的影响和药学效作用比较
细胞培养和给药处理：SH-SY5Y的完全培养体系为15％FBS(Gibco公司)加入高糖DMEM(Gibco公司)。使用Fugene HD转染试剂(Promega，Beijing Biotech Co.,Ltd.Catalog No.E2311)转染质粒。质粒购自Obio Technology(Shanghai Corp.,Ltd)，载体图谱如图3所示。α-synuclein过表达质粒的序列如下：
Figure PCTCN2021084773-appb-000032
稳转APP(SW)HEK293细胞系是转染瑞典双突变APP695cDNA的人胚胎肾细胞株，在种板前需使用20μg/mL多聚赖氨酸(Poly-D-Lysine,PDL)处理孔板24h。培养液为10％FBS加入高糖DMEM，并同时使用200μg/mL G418进行筛选。种板培养48h后饥饿处理，即去除血清，仅使用DMEM高糖培养基。培养24h后更换为完全培养体系并给药处理。
噻唑蓝(MTT)法检测细胞活力
给药处理12小时，加入终浓度为0.5mg/mL的MTT，孵育4小时后吸去培养液，加入100μL DMSO溶解吸附的MTT，振荡15分钟后读取吸光度值。
ELISA检测
a)α-synuclein ELISA检测α-synuclein单克隆抗体(Mouse monoclonal)购自Sigma-Aldrich(上海，Ctatlog No.S5566)；α-synuclein ELISA Kit试剂盒购自Thermo Fisher Scientific(Catalog No.KHB0061)。加50μL Huα-synuclein Detection Antibody solution到各孔(chromogen blanks empty除外，即空白比色孔)，再加50μL样品及标曲(标曲配制参见图4)到各孔(chromogen blanks empty除外)，轻震荡混匀，盖膜4℃孵育过夜；用1×Wash buffer 100μL充分洗孔4次后，除空白比色孔外每孔加100μL Anti-Rabbit IgG HRP盖膜室温孵育30分钟，再用1×Wash buffer充分清洗四次；加100μL Stabilized Chromogen到每孔，溶液变蓝，避光室温孵育30分钟；加100μL Stop Solution到每孔。轻震荡混匀，溶液变黄，使用NovoStar酶标仪(BMG company,Germany)吸收波长450nm读数。
b)Aβ ELISA检测
Amyloid beta 42 Human ELISA Kit试剂盒购自Thermo Fisher Scientific(Catalog No.KHB3544)。于检测孔板对应孔中加入稀释标曲、空白对照和样本各100μL。盖膜于37℃孵育2h；弃去各孔中液体，加100μL Detection Reagent A Working Solution于各孔，盖膜于37℃孵育1h；弃上清，用1×Wash buffer清洗各孔3次，每次2分钟且尽量无液体残留；加100μL Detection Reagent B Working Solution于各孔，盖膜于37℃孵育1h；重复清洗操作5次；加90μL Substrate Solution到各孔，盖膜于37℃避光孵育20分钟。溶液变蓝；加50μL Stop Solution到每孔，轻震荡混匀，溶液变黄，尽快用酶标仪(吸收波长450nm)读数。
碘化丙啶(Propidium Iodide,PI)染色
在免疫荧光染色处理前15分钟加入PI(Cell Signaling Technology,Catalog No.4087)并放置细胞培养箱共孵育15分钟，进行免疫荧光处理。PI/RNase染色溶液的最长激发光和发射光波长分别为535nm和617nm。
免疫荧光染色
将药物等处理后的细胞吸去上清后，使用预冷的PBS清洗三次，用4％PFA处理，室温静置30分钟，使用PB-S清洗三次，10分钟每次。将Triton-X溶于PBS配备为0.1％Triton-X溶液，处理15分钟。10％驴血清封闭1小时；一抗：Mouse anti-Ki67(Cell Signaling Technology,Catalog No.9129)；二抗：anti-Mouse Alex 488。
统计分析
数据处理采用GraphPad Prism 5软件进行分析。使用单因素分析(One-way ANOVA)处理，mean±SEM，p<0.03有统计学差异。
实验结果：
d5PAO与PAO对SH-sy5y细胞活力的影响
SH-sy5y细胞系的完全培养体系为地高糖DMEM培养液中加入15％FBS。细胞毒性实验中，SH-sy5y细胞培养48h后分别给予不同浓度d5PAO和PAO处理24h，加入终浓度为0.5mg/mL的噻唑蓝(MTT)，孵育4小时后吸去培养液，加入100μL DMSO溶解吸附的MTT，振荡15分钟后读取吸光度值。结果显示，d5PAO在6.25nM、25nM、50nM、100nM和200nM浓度显著促进SH-sy5y细胞增殖，PAO在200nM处理24h后出现毒性，细胞出 现显著死亡。为了进一步检测不同浓度d5PAO和PAOSH-sy5y细胞的毒性作用，我们通过PI染色和对Ki67免疫荧光染色考察d5PAO和PAO对SH-sy5y细胞凋亡/死亡和增殖的影响。PI(碘化丙啶)是一种能够插入到DNA和RNA碱基和染色剂之间的荧光染料，其不能通过活细胞膜，但却能穿过破损的细胞膜从而对已经凋亡/死亡细胞的核染色，而Ki67是细胞增殖中不可缺少的蛋白，其功能与丝分裂密切相关，因此Ki67常被用于标记增殖周期中的细胞，且一般在临床应用中认为Ki67阳性率高的细胞肿瘤生长速度快。在不同浓度d5PAO和PAO处理24h后进行PI染色和Ki67染色。结果显示，50nM和100nM d5PAO、PAO均未使Ki67阳性率显著增高，而PI阳性细胞较对照组(ctrl)减少，说明d5PAO和PAO减少细胞凋亡或死亡，而没有明显促进细胞增殖(图5)。
实施例4.d5PAO和PAO对稳转APP(SW)HEK293细胞活力和Aβ释放的作用
稳转APP(SW)HEK293细胞系是转染瑞典双突变淀粉样前体蛋白(APP)695cDNA的人胚胎肾细胞株并带有G418筛选标记。在种板前需使用20μg/mL多聚赖氨酸(Poly-D-Lysine，PDL)处理孔板24h。培养液为高糖DMEM加入10％FBS，并同时使用200μg/mL G418进行筛选。培养48h后分别加入不同浓度的d5PAO和PAO处理24h，加入终浓度为0.5mg/mL的噻唑蓝(MTT)，孵育4小时后吸去培养液，加入100μL DMSO溶解吸附的MTT，振荡15分钟后读取吸光度值。
检测d5PAO和PAO对稳转APP(SW)HEK293细胞Aβ释放的作用的实验中，培养48h后饥饿处理24小时后更换为完全培养体系，同时化合物d5PAO和PAO处理4小时，通过ELISA检测细胞培液上清中Aβ水平。
实验结果：
25nM、50nM、100nM和200nM d5PAO处理稳转APP(SW)HEK293细胞24h使其较对照组细胞活力显著增加。前期研究显示，PAO能够促进β-淀粉样蛋白(amyloid β-protein，Aβ)等蛋白的释放，通过ELISA试剂盒检测稳转APP(SW)HEK293细胞上清中Aβ，结果显示： 25nM、50nM和100nM d5PAO、PAO显著促进胞外Aβ含量(图6)。
实施例5.其他氘代化合物对稳转APP(SW)HEK293细胞Aβ释放的作用比较
选择的三个PAO氘代化合物是d1PAO,d2PAO,d3PAO。APP(SW)HEK293细胞培养方法给给药方法与实施例4相同。各处理组分组为：对照组(ctrl)、d5PAO 50nM处理组、d5PAO 100nM处理组、PAO 50nM处理组、PAO 75nM处理组、d1PAO 25nM处理组、d1PAO 50nM处理组、d1PAO 75nM处理组、d2PAO 25nM处理组、d2PAO 50nM处理组、d2PAO 75nM处理组、d3PAO 25nM处理组、d3PI03 50nM处理组、d3PAO 75nM处理组。结果显示：与对照组比较，50nM和75nMd5PAO；50nM和75nM PAO；25nM、50nM和75nM d1PAO；25nM、50nM和75nM d2PAO、25nM、50nM和75nM d3PAO处理组均显著促进胞外Aβ含量。除对照组外，其他处理组与d5PAO 50nM处理组比较，50nM和75nM PAO处理组，25nM、50nM和75nM d1PAO处理组，25nM、和75nM d2PAO处理组，以及25nM和75nM d3PAO处理组细胞培养上清中的Aβ含量均有显著性差异(图7A和图7B)。
实施例6.d5PAO和PAO对α-synuclein分泌的作用
SH-SY5Y细胞系使用Fugene HD转染试剂转染α-synuclein过表达(α-synuclein overexpression,α-syn OE)质粒，饥饿处理24小时后更换为完全培养体系，同时化合物d5PAO或PAO处理24h，通过MTT检测d5PAO和PAO对转染α-synuclein质粒的SH-sy5y细胞活力的影响。结果显示，过表达α-synuclein使SH-sy5y细胞活力显著下降，25nM、50nM、75nM、100nM和200nM d5PAO，以及50nM、75nM和100nM PAO使过表达α-synuclein的SH-sy5y细胞活力较α-synuclein过表达组显著升高。通过ELISA试剂盒检测细胞上清中α-synuclein，结果显示：50nM d5PAO使上清中α-synuclein含量显著升高，而PAO与d5PAO有类似促进α-synuclein升高的趋势(图8)。
实施例7.d5PAO和PAO对戈谢病的治疗作用
1.d5PAO和PAO抑制CBE诱导的SH-SY5Y细胞凋亡或死亡
Conduritol B epoxide(CBE)是溶酶体葡糖脑苷脂酶GBA基因编码的GBA1酶的抑制剂，是常用于构建戈谢病(Gaucher disease,GD)的细胞和动物模型。使用CBE处理SH-SY5Y细胞48小时，导致SH-SY5Y细胞出现浓度依赖性的细胞活力下降(图9A)。我们选择100μM CBE进行后续实验。100μM CBE处理SH-SY5Y细胞24小时，根据不同分组加入一定浓度d5PAO或PAO共孵育24小时，通过MTT实验检测细胞活力。实验结果显示，与100μM CBE处理组比较，100μM CBE与25nM，50nM以及100nM d5PAO和25nM，50nM以及100nM PAO共同处理组细胞活力显著升高(图9B，C)，提示d5PAO或PAO对CBE诱导的SH-SY5Y细胞凋亡或死亡有保护作用。
2.PAO降低溶酶体贮积并促进葡萄糖神经酰胺(Glucosylceramide，GlcCer)外排
GD是一类常见的溶酶体贮积病，我们通过溶酶体标志物DND99(Lyso-tracker red DND99)研究d5PAO和PAO是否减轻CBE导致的溶酶体贮积。实验结果显示，与对照组(ctrl)比较，100μM CBE处理组与100μM CBE Lyso-tracker的荧光强度升高(图10A，B)，提示CBE处理导致SH-SY5Y细胞中溶酶体贮积。与100μM CBE处理组比较，100μM CBE与50nM和100nM PAO共同处理组使Lyso-tracker的荧光强度显著降低(图10A，B)。GD的根本缺陷在于缺乏葡萄糖脑苷脂酶的活性，而该酶主要介导把葡萄糖脑苷脂分解成葡萄糖和GlcCer过程，因此GD患者或CBE处理的细胞中均出现GlcCer等底物的贮积。为了进一步探究PAO是否对SH-SY5Y细胞模型中的GlcCer贮积有所影响，我们通过LC-MS实验胞内和细胞培养基上清中不同侧链的GlcCer含量。检测结果表明，细胞内100μM CBE处理组多种不同侧链GlcCer浓度远高于对照组(ctrl)，100μM CBE和50nM PAO共同处理组GlcCer浓度与100μM CBE处理组比较降低(图10C)。100μM CBE单独处理使细胞培养基上清中GlcCer减少，100μM CBE和50nM PAO共同处理使GlcCer浓度回升(图10D)。
3.敲减PI4Ka促进溶酶体贮积减少
前期研究显示，PAO在低浓度(<5μM)时主要作用于磷脂酰肌醇4激酶PI4KⅢα，为了进一步探究PAO作用靶点PI4KⅢα在纤维化过程中的作用，我们设计了针对编码PI4KⅢα蛋白的基因序列PI4Ka的shRNA干扰慢病毒载体(带有绿色荧光蛋白GFP表达序列)。蛋白质免疫印迹实验结果显示，三个针对PI4Ka的shRNA干扰慢病毒载体在转染SH-SY5Y细胞48小时后，干扰序列sh1，sh2，sh3均使处理组PI4KⅢα表达水平显著降低(图11A，B)。shRNA干扰慢病毒载体处理48小时后通过免疫荧光观察Lyso-tracker的荧光强度。与sh-ctrl比较，sh-ctrl和100μM CBE共同处理组Lyso-tracker免疫荧光强度显著升高。与sh-ctrl和100μM CBE共同处理组比较，sh1-PI4Ka和100μM CBE共同处理组Lyso-tracker免疫荧光强度的下降有显著意义(图11C)。以上结果说明敲减PI4Ka促进溶酶体贮积减少。
实施例8.d5PAO和PAO对肺纤维化的抑制作用研究
肺纤维化(pulmonary fibrosis，PF)是一类可由多种原因引起的、慢性、纤维化性肺疾病，主要表现为干咳以及进行性的呼吸困难，预后较差，目前仍难以治愈。PF主要病理特征是正常肺组织结构破坏后的过度瘢痕修复，最终导致呼吸功能不全。虽然有关肺纤维化病理生理机制的研究已取得较大进展，但其发病机制尚未完全阐明，且临床缺乏有效治疗药物。
人胚肺成纤维细胞MRC-5细胞是研究PF的病理变化和进行药物研发的重要工具细胞。在PF的发生发展过程中，相关转录因子如转化生长因子-β1(transforming growth factor-1，TGF-β1)等可通过调控成纤维细胞异常活化、增殖和迁移，引起细胞外基质(extracellular matrix，ECM)的异常沉积和肺泡结构的破坏，最终导致PF的形成。TGF-β1是诱导PF的关键因子之一，其能够通过与相应受体结合调控成纤维细胞的转化为肌成纤维细胞，因此实验过程中通常使用一定浓度TGF-β1处理MRC-5细胞促使其发生纤维化。d5PAO和PAO分别是氧化苯砷和其改构物。前期研究显示，PAO具有抑制PF的潜能。
实验方法：
MRC-5细胞培养和处理
MRC-5细胞购买于中国科学院分子细胞科学卓越创新中心。根据细胞培 养指导说明用含有10％胎牛血清(fetal bovine serum,FBS)的MEM(Gibco公司)培养基在恒温37℃，5％CO 2，饱和湿度的培养箱中培养，培养24小时使其贴壁。次日，加入含有5ng/mL TGF-β1(Proteintech Group，HZ-1011)，并根据分组同时加入含有不同浓度PAO或d5PAO的MEM培养基(Gibco公司)，根据实验需要继续培养24小时。分组如下：对照(ctrl)组，5ng/mL TGF-β1组，5ng/mL TGF-β1和50nM d5PAO共同处理组(5ng/mL TGF-β1+50nM d5PAO组)，5ng/mL TGF-β1和25nM d5PAO共同处理组(5ng/mL TGF-β1+25nM d5PAO组)，5ng/mL TGF-β1和50nM PAO共同处理组(5ng/mL TGF-β1+50nM PAO组)，5ng/mL TGF-β1和25nM PAO共同处理组(5ng/mL TGF-β1+25nM PAO组)。
大鼠骨髓间充质干细胞原代培养和处理
将6周龄Sprague-Dawley(SD)大鼠(上海斯莱克实验动物有限公司)使用水合氯醛麻醉，75％乙醇消毒后在超净台中分离并取出胫骨和股骨，用消毒后的剪刀去除两端，露出骨髓腔。用5mL含10％FBS的MEM培养基冲洗骨髓腔，将含有骨髓的冲洗液加入培养皿中；通过70μm的细胞筛过滤，并在2000rpm转速下离心3分钟，去除上清，用10％FBS的MEM培养基重悬细胞，种板后在恒温37℃，5％CO 2，饱和湿度的培养箱中培养，6小时后通过换液去除未贴壁的细胞。当细胞长至80％密度时，使用2.5％胰酶消化1分钟，并以1：2的比例传代，用于免疫荧光实验的间充质干细胞将以3000-5000个细胞每孔种于24孔板的盖玻片上，通过观察细胞密度至70％时，根据分组加入含有不同浓度PAO或d5PAO的MEM培养基(无FBS)并于恒温37℃，5％CO 2，饱和湿度的培养箱中培养24小时。分组如下：对照(ctrl)组、50nM d5PAO处理组、25nM d5PAO处理组、50nM PAO处理组、25nM PAO处理组。
免疫荧光染色
MRC-5细胞处理24小时后吸去上清，加入磷酸盐缓冲溶液(phosphate buffer saline,PBS)清洗3次；加入4％多聚甲醛(Paraformaldehyde，PFA)室温静置30分钟；弃去4％PFA，经PBS清洗3次，每次10分钟；加入0.3％非离子型去垢剂Triton-X(溶于PBS)，室温静置30分钟；吸除0.3％Triton-X，加入含有10％山羊血清(goat serum,GS)的PBS封闭液，室 温静置1小时；4℃孵育一抗过夜(一抗使用10％GS封闭液进行稀释，α-Smooth Muscle Actin(兔抗α-Smooth Muscle Actin，Cell Signaling Technology#19245)。
稀释比例1：200，Calponin1稀释比例1：100)。次日，使用PBS清洗3次，每次10分钟；孵育二抗Alexa Flour 555山羊抗兔IgG(Molecular Probes)使用封闭液稀释，稀释比例为1：500，并加入1μg/mL 4',6-二脒基-2-苯基吲哚(DAPI))，室温静置2小时，加入PBS清洗3次，每次清洗10分钟。使用封片剂封片后通过显微镜观察。骨髓间充质干细胞的免疫荧光染色处理过程如上述相同。
蛋白质免疫印迹(Wesrern blot)实验
1)将细胞培养皿放置冰上，吸弃上清，使用预冷的PBS清洗细胞3次；
2)加入120μL/孔细胞裂解液，冰上水平放置30分钟；
3)使用细胞刮刮取裂解液，收集至1.5mL EP管；
4)15000g离心15分钟，收取上清至新的EP管中，并取5μL样品用于检测蛋白含量(BCA法)，其余裂解液则加入1/4体积的loading buffer；
5)沸水煮5分钟；
6)配置10％SDS分离胶，并配置积层胶；
7)通过SDS-PAGE凝胶电泳分离不同分子量的蛋白质；
8)配置湿转buffer，使用0.45μm PVDF膜进行转膜实验；
9)5％脱脂牛奶室温封闭1小时；
10)清洗后孵育一抗，4℃孵育过夜；
11)次日使用TBST液清洗3次，孵育二抗，室温放置2小时；
12)使用ECL显影液显色，并使用GE。
酶联免疫吸附反应：Human Collagen Type I ELISA Kit
共8个样品，包括：对照组(ctrl),5ng/mL TGF-β1组，5ng/mL TGF-β1和50nM d5PAO共同处理组(5ng/mL TGF-β1+50nM d5PAO组)，5ng/mL TGF-β1和25nM d5PAO共同处理组(5ng/mL TGF-β1+25nM d5PAO组)，5ng/mL TGF-β1和50nM PAO共同处理组(5ng/mL TGF-β1+50nM PAO 组)，5ng/mL TGF-β1和25nM PAO共同处理组(5ng/mL TGF-β1+25nM PAO组)。
Human Collagen Type I ELISA Kit采购自Novus Biologicals(货号：NBP2-30102)，按照使用说明进行以下实验步骤：
1)使用Standard Diluent配置标准曲线样品：4000pg/mL、2000pg/mL、1000pg/mL、500pg/mL、250pg/mL、125pg/mL、62.5pg/mL、0pg/mL；
2)向各孔中分别加入100μL待测样品(3个复孔)或标准曲线样品，使用封口膜封住孔板，37℃孵育2小时；
3)吸去上清，不需要清洗；
4)向各孔中加入100μL Detection Reagent A液，使用封口膜封住孔板，37℃孵育1小时；
5)弃去上清，每孔中加入350μL 1×Wash solution，振荡器中清洗2分钟，在无法纸上倒扣孔板并通过扣击清除上清，连续清洗3次；
6)向各孔中加入100μL Detection Reagent B液，使用封口膜封住孔板，37℃孵育30分钟；
7)弃去上清，每孔中加入350μL 1×Wash solution，振荡器中清洗2分钟，在无法纸上倒扣孔板并通过扣击清除上清，连续清洗5次；
8)清洗后每孔中加入90μL TMB底物，孔板中液体将逐渐变为蓝色，使用封口膜封住孔板，室温静置10分钟；
9)每孔中加入50μL Stop solution，孔板中液体将变为黄色；
10)使用450nm吸收波长的酶标仪(读取数据并分析。
shRNA慢病毒载体构建和处理
针对PI4Ka干扰的shRNA慢病毒载体由吉满生物科技(上海)有限公司设计并生产。载体信息：pGMLV-SC5 RNAi载体(图12)。
设计靶点如下：
Figure PCTCN2021084773-appb-000033
Figure PCTCN2021084773-appb-000034
MRC-5细胞含有10％胎牛血清(fetal bovine serum，FBS)的MEM培养基在恒温37℃，5％CO 2，饱和湿度的培养箱中培养，培养24小时使其贴壁。次日，根据分组分别加入1μL/孔慢病毒载体，24小时后根据分组加入5ng/mL TGF-β1共孵育24小时，收集蛋白进行蛋白质免疫印迹实验或免疫荧光染色。
统计
荧光强度处理使用Image J软件，数据处理和统计采用GraphPad Prism5软件，数据用平均值±标准误(mean±SEM)表示。比较组间差异用单因素方差分析(One-way ANOVA)统计，p<0.03认为有统计学差异。
实验结果：
用含有5ng/mL TGF-β1的MEM(不含FBS)培养基处理MRC-5细胞24小时，并根据分组给予不同浓度的d5PAO或PAO处理。与对照组比较，TGF-β1单独或与一定浓度的d5PAO或PAO共同处理没有造成明显的细胞死亡或凋亡(图13)。α-平滑肌肌动蛋白(α-Smooth Muscle Actin，α-SMA)和肌动蛋白结合蛋白Calponin1是肌成纤维细胞的标志物，为了研究d5PAO和PAO对肺纤维化是否有调控作用，我们分别对α-SMA和Calponin1进行了检测。蛋白质免疫印迹实验结果显示：与对照(ctrl)组比较，5ng/mL TGF-β1处理组α-SMA表达量显著增多，提示5ng/mL TGF-β1处理MRC-5细胞24小时导致细胞纤维化，25nM、50nM和100nM d5PAO以及50nM和100nM PAO显著抑制5ng/mL TGF-β1诱导的α-SMA过度表达，并呈现一定剂量依赖关系(图14A，B)。Calponin1的蛋白质免疫印迹实验结果与上述α-SMA的结果相似，5ng/mL TGF-β1处理组Calponin1表达量与ctrl组比较显著增多，一定浓度的d5PAO或PAO与5ng/mL TGF-β1共同处理组中的Calponin1表达量与5ng/mL TGF-β1单独处理组比较显著减少(图14A，C)。此外，免疫荧光结果也与蛋白质免疫印迹结果相一致，5ng/mL TGF-β1处理组α-SMA表达量较对照组显著升高(图15A，C)。5ng/mL TGF-β1和25nM d5PAO，5ng/mL TGF-β1和50nM d5PAO，5ng/mL TGF-β1和25nM PAO以及5ng/mL TGF-β1和50nM PAO共同处理组α-SMA表达量与对照组没有明显差异，而5ng/mL TGF-β1和25nM d5PAO，5ng/mL TGF-β1和 50nM d5PAO，5ng/mL TGF-β1和25nM PAO以及5ng/mL TGF-β1和50nM PAO共同处理组α-SMA表达量均与5ng/mL TGF-β1处理组比较显著降低(图15A，C)。肌动蛋白结合蛋白Calponin1的免疫荧光检测结果与上述α-SMA的结果相一致(图15B，D)。以上结果说明d5PAO和PAO能够显著抑制TGF-β1诱导的MRC-5细胞纤维化过程。
d5PAO和PAO抑制Calponin1在骨髓间充质干细胞(bone mesenchymal stem cells,bMSC)中表达
肺纤维化缺乏有效的治疗药物和治疗手段，而干细胞因其特有的生物学特性及潜在的生物医学应用价值近年来成为治疗肺纤维化的热门研究领域。间充质干细胞(mesenchymal stem cells,MSC)由于具有低免疫原性、多样分化潜能以及免疫调节抗炎能力，和来源广泛、易于分离培养和较少伦理争议等优点，成为创伤组织修复、器官功能重建和细胞治疗的理想工程细胞。因此我们分离培养了大鼠骨骼来源的MSC，并检测d5PAO和PAO是否调控Calponin1在MSC中的达。分离的MSC使用含有10％FBS的MEM培养基培养，在免疫荧光实验中以3000-5000个细胞每孔种于24孔板的盖玻片上，通过观察细胞密度至70％时，根据分组加入含有不同浓度PAO或d5PAO的MEM培养基(无FBS)并于恒温37℃，5％CO 2，饱和湿度的培养箱中培养24小时。免疫荧光结果显示，25nM、50nM d5PAO和PAO处理MSC 24小时有抑制Calponin1在MSC中表达的趋势(图16)。
d5PAO和PAO抑制TGF-β1处理的MRC-5细胞分泌I型胶原蛋白(Collagen Type I，COL1)
COL1是细胞外基质的重要组成成分之一。研究显示，TGF-β1促进MRC-5细胞纤维化过程中COL1分泌显著增多。为了进一步探究d5PAO和PAO在抑制肺纤维化过程中对COL1的分泌是否有调控作用，我们通过ELISA实验检测细胞上清中COL1水平。用含有5ng/mL TGF-β1的MEM(不含FBS)培养基处理MRC-5细胞24小时，并根据分组给予不同浓度的d5PAO或PAO处理24小时，收集上清进行ELISA实验。实验结果显示：与ctrl组比较，5ng/mL TGF-β1处理组细胞上清中的COL1浓度显著升高，5ng/mL TGF-β1和25nM d5PAO，5ng/mL TGF-β1和50nM d5PAO，5ng/mL TGF-β1和100nM d5PAO，5ng/mL TGF-β1和25nM PAO，5ng/mL TGF-β1和50nM PAO 以及5ng/mL TGF-β1和100nM PAO共同处理组与5ng/mL TGF-β1单独处理组比较细胞上清中COL1水平显著下降(图17A，B)，说明d5PAO和PAO能够抑制MRC-5细胞模型中的COL1分泌。
敲减PI4Kα抑制MRC-5细胞纤维化
前期研究显示，PAO在低浓度(<5μM)时主要作用于磷脂酰肌醇4激酶PI4KⅢα，为了进一步探究PAO作用靶点PI4KⅢα在纤维化过程中的作用，我们设计了针对编码PI4KⅢα蛋白的基因序列PI4Ka的shRNA干扰慢病毒载体(带有绿色荧光蛋白GFP表达序列)。蛋白质免疫印迹实验结果显示，三个针对PI4Ka的shRNA干扰慢病毒载体在转染MRC-5细胞48小时后，干扰序列sh1、sh2、sh3均使处理组PI4KⅢα表达水平显著降低(图18A，B)。shRNA干扰慢病毒载体处理48小时后通过免疫荧光观察Calponin1和α-SMA的表达。与载体对照(sh-ctrl)组比较，sh-ctrl加5ng/mL TGF-β1共同处理组α-SMA表达水平显著升高。与sh-ctrl加5ng/mL TGF-β1共同处理组比较，sh1加5ng/mL TGF-β1共同处理组和sh3加5ng/mL TGF-β1共同处理组α-SMA表达水平明显减少，sh2加5ng/mL TGF-β1共同处理组α-SMA表达水平也有下降趋势(图19A，C)。Calponin1的观察结果与α-SMA保持一致(图19B，D)，均说明敲减PI4Kα抑制MRC-5细胞纤维化。
实施例9.d5PAO和PAO抗炎症作用比较
d5PAO和PAO抑制小鼠小胶质细胞BV2细胞炎症因子释放
BV2细胞由小鼠小神经胶质细胞经逆转录病毒介导转染v-raf/v-myc获得永生化，并保留有小神经胶质细胞多种形态、表征和功能特征。在有关神经系统炎症的实验中，常用脂多糖(lipopolysaccharide，LPS)刺激BV2细胞获得炎症细胞模型进行实验。本实验以LPS刺激BV2细胞为炎症细胞模型，观察d5PAO和PAO对肿瘤坏死因子α(tumor necrosis factorα，TNF-α)和白细胞介素-6(Interleukin-6，IL-6)等炎症因子分泌的影响，并在实验中使用常用抗炎药吲哚美辛(Indomethacin)作为阳性药对照。
细胞培养及处理
BV2细胞的培养基使用高糖DMEM加入10％FBS，37℃5％CO 2细胞培养箱培养48小时，去除培养基并更换为含有1μg/mL LPS(脂多糖，购自 Sigma，货号：L2880)的高糖DMEM培养基(无FBS)，并根据分组给予不同浓度d5PAO或PAO共孵育24小时。收取上清离心后供后续实验使用。分组如下：对照(ctrl)组，1μg/mL LPS组，1μg/mL LPS和50nM d5PAO共同处理组(1μg/mL LPS+50nM d5PAO组)，1μg/mL LPS和25nM d5PAO共同处理组(1μg/mL LPS+25nM d5PAO组)，1μg/mL LPS和12.5nM d5PAO共同处理组(1μg/mL LPS+12.5nM d5PAO组)，1μg/mL LPS和50nM PAO共同处理组(1μg/mL LPS+50nM PAO组)，1μg/mL LPS和25nM PAO共同处理组(1μg/mL LPS+25nM PAO组)，1μg/mL LPS和12.5nM PAO共同处理组(1μg/mL LPS+12.5nM PAO组)，1μg/mL LPS和100μM吲哚美辛共同处理组。
Mouse Tumor Necrosis Factor Alpha(TNFα)ELISA实验
Mouse Tumor Necrosis Factor Alpha(TNFα)ELISA Kit采购自Signalway Antibody LLC，货号EK16997。按照产品说明进行以下实验：
1)使用Diluent buffer配置标准品，浓度分别为：10ng/mL，5ng/mL，2.5ng/mL，1.25ng/mL，0.625ng/mL，0.312ng/mL，0.156ng/mL，0。
2)轻轻振荡Detection Reagent A和Detection Reagent B，使用Diluent buffer分别稀释Reagent A和Reagent B至母液1/100达到工作液浓度。
3)使用去离子水稀释Wash solution至母液的1/30形成工作液。
4)分别向每孔中加入100μL待测样品或标准品，用封口膜封住孔板，37℃孵育2小时；
5)去除上清，不需要清洗；
6)分别向每孔中加入100μL Detection Reagent A工作液，用封口膜封住孔板，37℃孵育1小时；
7)弃去上清，使用300μL Wash solution工作液清洗3次，每次静置2分钟，将孔板倒扣在吸水无尘纸上尽量去除清洗液；
8)分别向每孔中加入100μL Detection Reagent B工作液，用封口膜封住孔板，37℃孵育1小时；
9)按照第7)步方法清洗孔板5次；
10)分别向每孔中加入90μL Substrate solution，观察孔板可见蓝色液体，用封口膜封住孔板，避光放置37℃孵育20分钟；
11)分别向每孔中加入50μL Stop solution，观察孔板可见蓝色液体变黄，用封口膜封住孔板，用摇床转摇混匀10分钟；
12)使用酶标仪(NOVOstar转液式酶标仪)读取样品在450nM波长吸光度值。
Mouse IL-6 ELISA实验
Mouse IL-6 ELISA Kit采购自Proteintech group，货号：KE10007。按照产品说明进行以下实验：
于检测孔板对应孔中加入各浓度标曲或样本100μL并预留空白对照比色孔，盖膜放置于湿盒中，37℃孵育2h；每孔350μL Wash Solution清洗操作4次，每次1-2分钟且尽量保证无液体残留；加100μL Diluent Antibody Solution(Detection Antibody Solution)于各孔，盖膜放置于湿盒中，37℃孵育1h；重复清洗操作4次，每次1-2分钟且尽量保证无液体残留；加100μL HRP-Conjugate Antibody于各孔，盖膜放置于湿盒中，37℃孵育40分钟；重复清洗操作4次且尽量确保无液体残留；加100μL TMB Substrate Solution于各孔，盖膜37℃避光孵育15分钟。溶液变蓝；加100μL Stop Solution到每孔，轻震荡混匀，溶液变黄，尽快用NOVOstar转液式酶标仪(吸收波长450nm)读数。
统计
数据处理和统计采用GraphPad Prism5软件，数据用平均值±标准误(mean±SEM)表示。比较组间差异用单因素方差分析(One-way ANOVA)统计，p<0.03认为有统计学差异。
实验结果
BV2细胞使用高糖DMEM加入10％FBS培养基培养，37℃5％CO 2细胞培养箱培养48小时，然后去除培养基更换为含有1μg/mL LPS的高糖DMEM培养基(无FBS)，并根据给予不同浓度d5PAO或PAO共孵育24小时，收取上清使用ELISA(酶联免疫吸附实验)方法检测培养基中IL-6和TNF-α的浓度。实验结果显示，1μg/mL LPS处理组BV2细胞上清中的IL-6和TNF-α浓度与对照(ctrl)组比较显著升高，说明1μg/mL LPS处理BV2细胞促进其释放炎症因子(图20A-D)。与1μg/mL LPS处理组比较，阳性药吲哚美辛在100μM浓度时显著抑制TNF-α的分泌(图20A-B)。与阳性药 结果类似，1μg/mL LPS和12.5nM、25nM、50nM d5PAO共同处理组以及和12.5nM、25nM、50nM PAO共同处理组上清中TNF-α浓度与1μg/mL LPS单独处理组比较显著降低，说明d5PAO和PAO能够抑制LPS诱导的TNF-α释放。IL-6的ELISA检测结果显示，一定浓度的d5PAO和PAO处理有抑制LPS诱导的BV2细胞分泌IL-6的趋势(图20C-D)。以上实验说明一定浓度d5PAO和PAO有抑制LPS诱导的BV2细胞释放炎症因子的作用。
实施例10.d5PAO和PAO在抗肿瘤作用中的比较
1.PAO和d5PAO对肿瘤细胞模型的药效实验
实验方法
细胞铺板
配制完全培养基，充分混匀。将复苏的肿瘤原生代细胞(上海睿智化学有限公司)，传两代左右选择生长状态良好的细胞株。贴壁细胞：吸掉培养基，用胰酶洗一遍，弃掉废液，加3ml新鲜胰酶于培养瓶消化。待细胞松动要脱离瓶壁时，加8mL完全培养基中止胰酶消化，并轻轻混匀。用移液管将细胞悬液移入离心管中，1000rpm的转速离心4分钟。悬浮细胞：吸取细胞悬液并移入离心管中，1000rpm的转速离心4分钟。弃上清。向离心管中加适当体积的培养基，轻柔吹打使细胞重悬均匀。使用Vi-Cell XR细胞计数仪计数。将细胞悬液调至合适浓度。
化合物板的配制及添加
待测化合物：化合物在DMSO中配制成10mM溶液，化合物PAO和d5PAO在DMSO中稀释成0.5mM溶液。用HPD300仪器将化合物加入相应细胞孔中。在二氧化碳培养箱中孵育72小时。
试剂准备及检测
室温融化CellTiter-Glo Buffer。将冻干CellTiter Glo底物平衡至室温。将CellTiter-Glo Buffer加入CellTiter Glo底物中并充分混匀。将细胞板取出平衡至室温。每孔中加入混匀后的CellTiter Glo试剂100微升，避光振荡10分钟，孵育10分钟。将培养板放入Envision读板，记录luminescence读值结果；按下列公式计算抑制率：抑制率(％)＝(1-(RLU compound-RLU blank) /(RLU DMSO–RLU blank))×100％。利用XLFit绘制药效抑制率曲线并计算IC50值。利用4参数模型[fit＝(A+((B-A)/(1+((C/x)^D))))]。
实验结果：
如下表5所示：d5PAO和PAO对所测试的肿瘤细胞都有抑制作用，且两者的抑制作用IC 50相近，d5PAO的IC50稍低；两者对U2-OS和A-375细胞的抑制作用最强，IC 50都小于50nM；对HeLa、SK-HEP-1、Daudi、EL4、HL-60、Jurkat,Clone E6-1、NAMALWA细胞都抑制作用较强，IC 50在50-100nM；对A-431细胞的抑制作用最弱，IC50约为300nM，对其余细胞的IC50在100-200nM。与PAO(原形化合物)和d5PAO(5个氘代化合物)相比，一个氘代化合物d1PAO，对大部分的肿瘤细胞的有效抑制浓度大于200nM，说明它对肿瘤细胞的抑制作用不如PAO和d5PAO。
表5.d5PAO和PAO对肿瘤细胞(培养72小时)的抑制作用列表
Figure PCTCN2021084773-appb-000035
Figure PCTCN2021084773-appb-000036
2.PAO和d5PAO对小鼠肿瘤模型的药效实验
实验动物饲养在北京百奥赛图基因生物技术有限公司动物中心的SPF级屏障设施环境。屏障系统温度在20-26℃和湿度控在40-70％。照明每12小时交替。SPF级生长繁殖饲料购自北京科澳协力饲料有限公司。饮用水为酸化水(pH 2.5～3.0)，经过高压灭菌处理。动物可以自由摄取无菌食物和饮水
2.1 PAO对乳腺癌的抑制作用
实验方法：
PDX模型的接种与分组
待接种的PDX肿瘤生长至800-1000mm 3左右后，无菌条件下取出瘤块放置在RPMI 1640培养基中，剔除钙化点、分泌物等非肿瘤组织，并切成大小均一的小块(3×3×3mm)，准备进行乳腺癌(BP1395)PDX模型的皮下接种。用碘伏对小鼠右侧胁肋部皮肤进行消毒处理，用剪刀将皮肤剪开3-5mm左右的切口，使用接种针将切好的瘤块接种于小鼠右侧胁肋部皮下。当平均肿瘤体积达到约100mm 3时，挑选个体肿瘤体积适中的小鼠入组，将动物按肿瘤体积随机分配到4个实验组中，每组8只，分组当天开始给药.所有组给受试品为口服给药(P.O.)，分组当天开始给药，受试品PAO每天给药1次，共给药20次。紫杉醇每周静脉(i.v.)给药1次，共给药4次。实验结束或实施人道终点时，使用过量CO 2将动物执行安乐死。
肿瘤体积：分组后每周使用游标卡尺对肿瘤体积进行2次测量，安乐死 前测量肿瘤体积，测量肿瘤的长径和短径，其体积计算公式为：肿瘤体积＝0.5×长径×短径 2。
体重检测：接种、分组(即首次给药前)、给药期间每周2次、安乐死前称取动物体重。
药物评价指标：
肿瘤体积抑制率(TGI TV)
TGI(％)＝[1-(Ti-T0)/(Vi-V0)]×100％
(Ti：治疗组在给药第i天的肿瘤体积均值，T0：治疗组在给药第0天的肿瘤体积均值；Vi：溶剂对照组在给药第i天的肿瘤体积均值，V0：溶剂对照组在给药第0天的肿瘤体积均值)
瘤重抑制率(TGI TW)：
实验结束，对存活动物实施安乐死后剥离肿瘤组织，称量肿瘤重量，计算各组的肿瘤重量 差异以进一步计算瘤重抑制率TGI TW，计算公式如下：
瘤重抑制率TGI TW％＝(W 溶剂对照组-W 治疗组)/W 溶剂对照组×100％，W指肿瘤重量。
数据采集和统计学分析：
基于原始数据进行分析处理，结果分析用平均数和标准误表示(Mean±SEM)。同时对肿瘤体积进行统计学分析，P<0.05认为有显著性差异。结果分析时同时考虑统计学意义和生物学意义。
实验结果：
在实验过程中，所有动物在给药期间活动和进食状态良好，不荷瘤小鼠体重有一定程度的上升，荷瘤鼠体重略有下降，表明动物对各受试品耐受良好。在分组给药第21天，溶剂对照组G1平均肿瘤体积为436±40mm 3。给药组G2组(PAO，2mg/Kg)、G3组(紫杉醇，7mg/Kg)、G4组(紫杉醇7mg/Kg+PAO，2mg/Kg)的平均肿瘤体积分别为519±96mm 3、290±63mm 3、162±26mm 3，肿瘤体积生长抑制率TGI TV分别为-25.7％、44.8％、84.1％(P＝0.01)。结果表明PAO和紫杉醇联用可以有效抑制肿瘤体积增长(图21)。
2.2 PAO对淋巴癌的抑制作用
实验方法：
小鼠淋巴癌SU-DHL-1细胞购自ATCC，细胞培养在37℃、5％CO 2的培养箱中，培养基成分为含有10％灭活胎牛血清的Dulbecco's Modified Eagle's培养基。
肿瘤细胞的接种与分组：
将PBS重悬的SU-DHL-1淋巴癌细胞以1×10 7个/0.1mL浓度，0.1mL/只体积接种于B-NDG人源化小鼠的右侧皮下。当平均肿瘤体积达到大约100mm 3时，挑选个体肿瘤体积合适的36只小鼠入组，将动物按肿瘤体积随机分配到6个实验组中，每组6只。分组当天开始给药。受试品PAO每天给药1次，共给药17次。环磷酰胺每周皮下注射(i.v.)给药1次，共给药4次。实验结束或实施人道终点时，使用过量CO 2将动物执行安乐死。
实验结果：
在实验过程中，所有动物在给药期间活动和进食状态良好，不荷瘤小鼠体重有一定程度的上升，荷瘤鼠体重略有下降，表明动物对各受试品耐受良好。在分组给药第17天，溶剂对照组G1平均肿瘤体积为3899±272mm 3。给药组G2组(环磷酰胺，50mg/Kg)、G3，G4，G5组(PAO，0.3mg/Kg、0.6mg/Kg、1.2mg/Kg)、G6组(环磷酰胺50mg/Kg+PAO 0.6mg/Kg)的平均肿瘤体积分别为367±79mm 3、3427±128mm 3、3784±114mm 3，3735±205mm 3，497±106mm 3，肿瘤体积生长抑制率TGI TV分别为-93％(**P<0.001)、17.2％、3％、4.3％、89.9％(P<0.001**)。实验结果在肿瘤重量上也得到证实。环磷酰胺能够有效抑制淋巴癌肿瘤体积的增长，PAO和环磷酰胺联用未能进一步提高抑制作用(图22)。
2.3 d5PAO对黑色素瘤的抑制作用
实验方法：
将A2058细胞以1×10 7个/0.1mL浓度，0.1mL/只体积接种于B-NDG小鼠的右侧皮下。当平均肿瘤体积达到大约100mm 3时，挑选个体肿瘤体积合适的48只小鼠入组，将动物按肿瘤体积随机分配到6个实验组中，每组8只,分别为G3组(生理盐水/溶媒)、G4组(d5PAO，0.5mg/Kg)、G5组(d5PAO，1.5mg/Kg)、G6(替莫唑胺，30mg/Kg+d5PAO，0.5mg/Kg)、 G7(替莫唑胺30mg/Kg+d5PAO，1.5mg/Kg)和G8(替莫唑胺，30mg/Kg)。同时根据小鼠体重挑选16只不荷瘤小鼠平均分为2个实验组，每组8只，分别为G1组(生理盐水/溶媒)和G2组(d5PAO，1.5mg/Kg)。所有组给药途径均为灌胃给药，分组当天开始给药，受试品d5PAO每天给药1次，共给药23次。受试品替莫唑胺每周给药4次，共给药12次。给药和观察期间每周测量2次小鼠体重和肿瘤体积，并记录测量值(图23)。实验结束时，动物安乐死，剥取肿瘤称重、拍照，计算肿瘤生长抑制率(TGI％)。
实验结果：
在实验过程中，所有动物在给药期间活动和进食状态良好，不荷瘤小鼠体重有一定程度的上升，荷瘤鼠体重略有下降，表明动物对各受试品耐受良好。在分组给药第25天，溶剂对照组G3平均肿瘤体积为2806±240mm 3。给药组G4组(d5PAO 0.5mg/Kg)、G5组(d5PAO 1.5mg/Kg)、G6组(替莫唑胺30mg/Kg+d5PAO 0.5mg/Kg)、G7组(替莫唑胺30mg/Kg+d5PAO1.5mg/Kg)和G8组(替莫唑胺30mg/Kg)的平均肿瘤体积分别为2907±295mm 3、2180±312mm 3、1064±164mm 3、1213±155mm 3、1480±136mm 3，肿瘤体积生长抑制率TGI TV分别为-3.7％、23.2％、64.5％、58.9％和49.1％。实验结果在肿瘤重量上也得到证实。在受试终点动物体内获取的肿瘤组织重量分别为：G3组溶媒/生理盐水，3.413±0.253克；G4组d5PAO 0.5mg/kg，3.557±0.379克；G5组d5PAO 1.5mg/kg，2.744±0.459克；G6组替莫唑胺30mg/kg+d5PAO 0.5mg/kg，1.413±0.233克；G7组替莫唑胺30mg/kg+d5PAO 1.5mg/kg，1.442±0.251克；G8组替莫唑胺30mg/kg，1.884±0.217克(图24)。
与对照组G3相比，替莫唑胺在30mg/Kg剂量下单用时及与d5PAO在0.5mg/Kg和1.5mg/Kg剂量下联合用药时均对A2058皮下移植瘤生长有极显著抑制作用(P<0.001)。与替莫唑胺单独使用相比，联用可进一步提升抑制作用。
实施例11.d5PAO和PAO在抗肿瘤恶病质(Cachexia)中的作用比较
癌症(Cancer)是人类第二大死因，全球近六分之一的死亡由癌症造成。癌症治疗主要通过化疗、放射治疗、手术、免疫治疗、基因治疗、激素治疗 等手段实现。其中化疗是当前最有效的手段之一。但化学治疗的主要问题是其引起副作用：药物在杀死癌细胞的同时，也会杀死体内快速生长的细胞，包括血液、口腔、消化系统和毛囊中的细胞，从而引起消化系统反应、脱发、骨髓抑制以及其它系统的功能衰退。
恶病质(Cachexia)亦称恶液质，表现为极度消瘦，体重减轻，脂肪丢失、骨骼肌以及心肌的溶解减少，进而导致渐进性的功能障碍,最后导致全身衰竭等综合征。恶液质多由严重慢性消耗性疾病引起，包括肿瘤、AIDS、严重创伤、手术后、吸收不良及严重的败血症等。其中以肿瘤伴发的恶病质最为常见，也称为肿瘤恶病质。31-87％恶性肿瘤患者都伴有恶病质，约20％肿瘤病人的直接死因是恶病质引起的营养不良，而非疾病本身。恶病质尤其和胰腺癌、胃癌、肺癌、肝癌有较高的相关性。恶病质直接影响癌症治疗效果,增加并发症发生率,并使生活质量下降，生存期缩短，治疗时间延长和医疗费用增加。
恶液质的形成原因尚未完全解析，但最近研究逐渐揭示，各类从肿瘤细胞或肿瘤细胞包围环境里的细胞释放的致病因子。减缓或阻止肿瘤恶病质的发生可提高病人生活质量，延长生存时间，是抗癌治疗方案中的主要组成部分。动物实验模型研究表明阻止肿瘤发展过程中体重的降低可以延长生存率。治疗肿瘤病人恶病质的主要手段是通过药物逆转体重降低以及肌肉量的流失。到目前为止，大部分一线首选抗恶病质药物在预防和治疗肿瘤恶病质的效果都非常有限。
1.PAO和d5PAO对健康小鼠体重的影响
取22只2月龄和20只6月龄的雄性C57B/6小鼠，每个小鼠饲养笼有5-6只小鼠，其中12只2月龄的小鼠和10只6月龄的小鼠，每日用MCT溶媒溶解的d5PAO，剂量是2.1mg/kg,其余的10只2月龄的小鼠和10只6月龄的小鼠，每日用MCT溶媒溶解的PAO，剂量是2.0mg/kg。从给药的第一天起，每4天的第一天在给药前称量和记录每只小鼠的体重，在接下来的4天内，以此体重为依据，给予灌胃相应的剂量的PAO或d5PAO。每4天记录小鼠的存活的数量。
结果如图25所示，对于2月龄的d5PAO和PAO小鼠(2M-d5PAO和 2M-PAO)，灌胃给药的前24天，两组小鼠的平均体重完全一样，且逐步增加，24天后两组小鼠的体重都有所下降，但2M-PAO组的体重下降比2M-d5PAO组明显快，且组给药后第44-48天有一只小鼠死亡，2M-d5PAO组无死亡。对于6月龄的d5PAO和PAO小鼠(6M-d5PAO和6M-PAO)，在灌胃给药的48天，两组小鼠的平均体重整体上都有缓慢、波动性地下降，但是给药后的第32天起，6M-PAO的体重下降比6M-d5PAO组的明显加快，且两组小鼠都有动物死亡，6M-PAO组有5只(50％)，6M-d5PAO组有两只(20％)。所以灌胃给药d5PAO所产生的毒性反应比6M-PAO明显低。
2.PAO对肿瘤模型动物体重的影响
2.1 PAO乳腺癌小鼠模型体重的影响
紫杉醇注射可使动物体重下降。用药第21天，从101.4％±1.6％(溶媒组，相对于Day0平均体重)，下降到94.3％±2.6％(紫杉醇组7mg/Kg，相对于Day0平均体重，P＝0.036*)。而PAO的加入可以阻止由紫杉醇引起的体重下降(105.3％±2.4％，P＝0.187)。值得注意的是，PAO本身也能使荷瘤动物的体重增加(106.1％±2.5％，P＝0.128)(图26)。
2.2 PAO对胰腺癌模型体重的影响
胰腺癌模型的建立方式与上同。用药第28天，吉西他滨(1.5mg/Kg)和紫杉醇(7mg/kg)组的平均体重(104.3％±4.0％，相对于Day 0的平均体重)，与溶媒组相比(99.0％±2.2％，相对于Day 0平均体重)无显著差异。而PAO的加入可以使体重增加至116.7％±3.9％(P＝0.002VS溶媒组和吉西他滨+紫杉醇组，图27)。
2.3 PAO对淋巴瘤模型动物体重的影响
淋巴瘤模型的建立方式与上同。虽然PAO的加入未能进一步抑制肿瘤的生长，但可以缓解环磷酰胺注射引起的体重下降。用药第17天，动物体重从117.4％±1.4％(G1溶媒组，相对于Day0平均体重)，下降到104.7％±1.6％(G2，环磷酰胺组50mg/Kg，相对于Day 0平均体重，P<0.01*)。PAO(0.6mg-0.9mg/Kg)与环磷酰胺联用缓解了环磷酰胺引起的体重下降(G6111.1％±1.9％，P＝0.032vs.溶媒组，P＝0.05vs.环磷酰胺组)(图28)。
2.4 d5PAO对黑色素瘤模型动物体重的影响
和健康小鼠相比，荷瘤动物的体重均有所下降。与溶媒组荷瘤动物相比， 替莫唑胺在30mg/Kg剂量单用时的体重有所下降(88％±1.5％，相对于Day0),而高剂量d5PAO(1.5mg/Kg)与替莫唑胺的联合使用缓解了体重下降(95％±2％，P＝0.01vs.替莫唑胺；P＝0.038vs.溶媒组)。d5PAO单独使用对荷瘤动物的体重没有显著影响(图29)。
实施例12.PAO和d5PAO对HCoV 229E(流感冠状病毒)的抑制作用
受试化合物与对照化合物
对照化合物Remdesivir将由药明康德提供。化合物使用DMSO溶液配置为20mM储存液。受试样品和对照化合物测试8个浓度，2倍或者3倍梯度稀释，双复孔。
细胞株、病毒株以及试剂
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。本项目所用主要试剂为细胞活力检测试剂盒CellTiter-Glo(Promega)。
试验方法
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，加入倍比稀释后的化合物(8个浓度点、2倍或者3倍梯度稀释，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的终浓度为0.5％。细胞于培养箱中培养3天。细胞毒性实验与抗病毒实验同时进行测试，实验条件一致，但无病毒感染。使用细胞活力检测试剂盒CellTiter Glo(Promega)检测细胞活力。化合物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)×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)×100
使用GraphPad Prism(version 5)对化合物的抑制率和细胞活率进行非线性拟合分析，计算化合物的半数有效浓度(EC 50)和半数细胞毒性浓度(CC 50)值。拟合公式为：log(inhibitor)vs.response--Variable slope。
结果
PAO和d5PAO药物的剂量反应拟合曲线见图30。对照化合物Remdesivir显示出预期的抗病毒活性和细胞毒性。
测试结果显示受试化合物PAO和P100对HCoV 229E具有抗病毒活性，其EC 50值分别为55.35nM和47.21nM。受试化合物PAO和P100对MRC5细胞有明显的毒性，其CC 50值分别为256.8nM和317.5nM。
实施例13.低剂量的d5PAO与PAO抗焦虑抑郁作用比较
试验动物
30只雄性2月龄ICR小鼠，在清洁级鼠房按正常昼夜节律等条件饲养8周后，随机均分为3组：载体、PAO和d5PAO组，5只小鼠一个鼠笼，每组两个笼，每组都给予慢性不可预知的多种刺激(chronic unpredictable multiple stimulation,CUMS)。
CUMS抑郁造模
CUMS抑郁造模：每周每天多种刺激交替给予小鼠，使小鼠不可预见每次刺激持续的时间和接下来将给予的刺激的模式和时间长短。第一周的刺激和时间安排见(表6)。表中每一天的刺激模式和时间组成一个模块，共7个。第二周起，每周一的刺激从7个模块中随机抽取，周二从余下的6个随机抽取，周三从余下的5个随机抽取，以此类推。如果某一天有测试试验安排，则对前两天和试验当天的刺激做合适的调整。
表6.CUMS刺激时间安排
Figure PCTCN2021084773-appb-000037
Figure PCTCN2021084773-appb-000038
给药方法
在完成第一周的CUMS刺激后，PAO和d5PAO组的小鼠分别每日灌胃PAO和d5PAO化合物溶液，剂量都是0.05mg/Kg/day,而载体组小鼠每日灌胃与体重相应体积的MCT(MIGLYOL 812N，供应商IOI Oleo GmbH)，MCT是用来制备PAO和d5PAO化合物的溶液的载体。PAO和d5PAO的MCT溶液的浓度都是0.005mg/mL。
新奇抑制摄食试验(NSF)
新奇抑制摄食试验(Novelty suppressed feeding test,NSF)中有一个只有顶面开放的用不透明有机玻璃制作的长×宽×高为25cm×25cm×20cm的盒子，其中央放置有一个小平台，平台上有一颗鼠饲料。试验时，把一只小鼠从盒子任一个角放入盒子，头面向盒子的角，让小鼠在五干扰下自由活动5分钟，记录5分钟内第一次摄食的潜伏期。如果满5分钟时，小鼠未摄食，取出小鼠并把该小鼠的第一次摄食的潜伏期计为300秒。第一次摄食的潜伏期的长度(秒)就是小鼠焦虑程度的指数。
抑郁行为指标：糖水偏爱程度检测
在糖水偏好试验中，一个干净的鼠笼的供水处放置有两个预先称好重量的同样水瓶，一只装有水，另一只装有浓度为1％蔗糖的水溶液。试验时，把一只小鼠的放入该干净鼠笼的远离水瓶的一端，头朝向水瓶的反方向。小鼠在无干扰下，在1小时内可自由地选择喝糖水或水，满1小时后，取出小鼠， 小心取出两个水瓶，称重，用于计算小鼠所饮用的糖水和水的重量，分别计为W 水和W 糖水。小鼠糖水的偏好度＝W 糖水/(W 水和W 糖水)×100％。
统计方法
使用SPSS软件进行统计分析，数据以平均值±标准误差
Figure PCTCN2021084773-appb-000039
表示。因为PAO有抗抑郁作用是已知的，给药后PAO和d5PAO组与载体组的糖水偏爱度的差异使用One-tail unpaired t test检验显著性，P<0.05标记为*，P<0.01标记为**。
结果
三组小鼠经历3周的CUMS和分别给予载体(MCT)、PAO和d5PAO制剂灌胃15天后，进行新奇抑制食物摄取(NSF)试验。结果发现10只载体组小鼠中的只有2只有摄食，但其余8只在限定的5分钟内没有摄食，潜伏期均计为300秒，该组平均焦虑指数为282±12(秒)；PAO和d5PAO组的该组平均焦虑指数分别为227±29(秒)和181±36(秒)，显著比载体组的焦虑程度要低，提示低剂量(0.05mg/Kg/day)的PAO和d5PAO也有明显的抗焦虑作用，且d5PAO比d5PAO的抗焦虑作用更显著(图31)。
三组小鼠完成NSF试验48小时后，进行糖水偏好试验，检测三组小鼠的抑郁程度，如图27所示，载体、PAO和d5PAO三组小鼠对糖水的偏好程度分别为66±3.8％，75±4.3％和78±2.4％；PAO和d5PAO组对糖水的偏好明显比载体组高，提示低剂量(0.05mg/Kg/day)的PAO和d5PAO也有明显的抗抑郁作用，且d5PAO比PAO的抗抑郁作用更显著(图32)。第一次糖水偏好试验完成后，继续给予三组小鼠CUMS，总共38天，再进行糖水偏好试验，结果显示载体、PAO和d5PAO三组小鼠对糖水的偏好程度分别为71±3.5％，77±2.0％和82±2.9％；PAO和d5PAO组对糖水的偏好比载体组高，但PAO组与载体组的差异未达到显著性，提示低剂量(0.05mg/Kg/天)的PAO和d5PAO，仍然有抗抑郁作用，且d5PAO比d5PAO的抗抑郁作用更显著和更稳定(图32)。
实施例14.d5PAO和PAO对NPC的治疗作用研究
U18666A，是一种细胞内胆固醇转运(cholesterol transport)抑制剂，常被用于构建C型尼曼氏病(NPC)的细胞模型。
1细胞培养与化合物处理
SH-SY5Y细胞使用高糖DMEM加15％FBS的完全培养基放置37℃，5％CO 2培养箱培养。当细胞70％融合时，加入含10μM U18666A(购自爱必信(上海)生物科技有限公司，货号：abs819512)同时根据分组加入不同深度d5PAO和PAO，培养24小时。
2 Filipin染色
1)将24孔板中的培养基弃去，加入1mL PBS缓冲液静置1分钟，弃去24孔板中的液体，重复此步骤2次；
2)每孔加入1mL 4％多聚甲醛，室温下固定30分钟；
3)将24孔板中的4％多聚甲醛弃去，加入1mL PBS缓冲液，轻轻晃动24孔板1分钟，弃去24孔板中的液体，重复此步骤2次；
4)每孔加入1mL 1.5mg/mL甘氨酸溶液，室温下孵育10分钟；
5)将24孔板中的液体弃去，加入终浓度为50μg/mL Filipin染色液(购自Sigma-Aldrich，货号：SAE0087)1mL，室温下孵育1小时，避免光照；
6)将24孔板中的液体弃去，加入1mL PBS缓冲液，轻轻晃动24孔板1分钟，弃去24孔板中的液体，重复此步骤2次；
7)取载玻片，在载玻片中央滴加5微升封片剂(含DAPI),将细胞爬片取出晾干，将有细胞的一面向下，盖在载玻片上，使之与封片剂充分接触，室温避光孵育30分钟。
8)激光共聚焦显微镜观察。
实验结果
10μM U18666A处理SH-SY5Y细胞，同时根据分组加入不同浓度d5PAO和PAO，共孵育24小时，进行Filipin染色后观察。免疫荧光染色结果表明，10μM U18666A单独处理组Filipin染色荧光强度比对照组(ctrl)增强，提示10μM U18666A处理导致与Filipin结合的胆固醇量上升，即导致胆固醇贮积。10μM U18666A与35nM，70nM d5PAO以及与35nM PAO，70nM PAO共同处理组Filipin染色荧光强度与10μM U18666A单独处理组比较出现下降(图33)。以上结果说明一定浓度d5PAO和PAO能够抑制U18666A导致的胆固醇贮积。
实施例15.PAO和敲减PI4Ka激活自噬溶酶体通路(Autophagy-lysosome pathway,ALP)
前期研究显示，GD等溶酶体贮积病发展过程中ALP被阻断。SH-SY5Y细胞经CBE处理48小时后根据分组分别加入不同浓度PAO或mTOR抑制剂雷帕霉素(Rapamycin，RAPA)作为阳性对照，共孵育24小时，进行蛋白质免疫印迹实验或免疫荧光实验，通过观察ALP常见标志物研究PAO等化合物对自噬溶酶体通路的影响。对ALP常见的标志物LC3B和p62进行检测，蛋白质免疫印迹实验显示：在CBE构建的SH-SY5Y细胞模型中，PAO剂量依赖性地促进LC3B蛋白表达，抑制p62蛋白水平，说明PAO激活ALP通路并使自噬流活化(图34A-C)，与阳性对照500nM RAPA结果相近(图34A-C)。免疫荧光结果与蛋白免疫印迹实验结果相一致(图34D)。此外，H +-ATPase抑制剂Bafilomycin A1(Baf-A1)是一种常用的ALP抑制剂，通过利用Baf-A1阻断ALP信号可以进一步验证PAO等化合物对CBE构建的SH-SY5Y细胞的保护作用是否与ALP有关。SH-SY5Y细胞经CBE处理48小时后根据分组分别加入不同浓度PAO或50nM Baf-A1共孵育24小时，通过MTT实验检测细胞活力。实验结果表明，与对照组(ctrl)比较，100μM CBE显著抑制SH-SY5Y细胞活力。与100μM CBE处理组比较，100μM CBE与25nM，50nM以及75nM PAO共同处理组细胞活力显著升高，50nM Baf-A1处理则使PAO在相应组别中的保护作用下降，使50nM Baf-A1和100μM CBE分别与25nM，50n M以及75nM 1PAO共同处理组的细胞活力与100μM CBE处理组没有明显差异(图34E)，说明Baf-A1通过抑制ALP信号阻断PAO对CBE处理的SH-SY5Y细胞的保护作用。以上结果证实PAO激活ALP使自噬流活化，并通过ALP发挥对GD细胞模型的保护作用。对进行shRNA干扰慢病毒载体处理的SH-SY5Y细胞检测ALP通路标志物LC3B进行检测，结果显示：与sh-ctrl组比较，敲减PI4Ka后LC3B蛋白水平显著升高(图35)，在CBE处理的SH-SY5Y细胞中敲减PI4Ka同样促进LC3B蛋白表达，说明与PI4Ka抑制剂PAO结果相同，敲减PI4Ka同样激活了ALP通路。
实施例16.PAO和d5PAO抗化学因子引起的肺炎症反应和肺纤维化
肺部及上呼吸道是机体最频繁遭受各种病因包括病原体、化学因子包括药物、异物、组织的物理性损伤、过敏反应和自身免疫异常等所引起的炎症反应的组织器官，所引起的炎症反应包括局部组织或者全身血液中的白细胞增多，诸如中性粒细胞、巨噬细胞和淋巴细胞，以及多种炎症因子或细胞因子增加等。病原体包括微生物和寄生虫。微生物包括细菌、病毒、衣原体、支原体、螺旋体和真菌等。肺部炎症有时还会导致肺部组织纤维化，损害肺部结构和功能，特别是损害通气和氧气的弥散。
博来霉素是明确的能引起肺炎和肺纤维化的药物或者化学，其在动物肺部引起的间质性肺炎和肺纤维化，是研究特发性肺纤维化(Idiopathic pulmonary fibrosis，IPF)的常用模型。IPF是一种致命疾病，其特征是进行性和不可逆性肺纤维化，目前无特异性治疗方法，现有的治疗方法的疗效部显著。大多数患者在症状发作后3-8年内死于进行性呼吸衰竭。尽管IPF发病机理的基本机制知之甚少，但标志性病理特征，包括：炎症反应、成纤维细胞过度增殖以及细胞外基质异常沉积。
PAO和d5PAO对博来霉素诱导的小鼠肺炎症反应和肺纤维化的疗效是PAO和到d5PAO在炎症反应和组织纤维化的药物应用的一个实施例。
实验方法：
博来霉素诱导
1、博来霉素配制
博来霉素溶解于生理盐水中，终浓度根据给药剂量调整。
2、诱导方法
在第1天，动物将吸入2-5％异氟烷进行麻醉。基于体重，动物将通过气管内的给药途径给予博来霉素(2mg/kg，具体给药体积将依据动物体重进行计算并被记录)。
3、给药
接受博来霉素诱导当天视为试验第1天。在第3天对动物进行筛选和分组。所有动物将在试验第8天开始给药，每天一次直至试验结束，具体的给药方案请见表7所示。
表7.分组及给药方案
Figure PCTCN2021084773-appb-000040
a:MCT
4、样品收集及分析
在第21天，所有动物给药后接受气道反应性的测定后，所有动物通过吸入2-5％异氟烷麻醉后通过眼眶采集最少0.5mL全血，使用EDTA-2K抗凝，以10000rpm,10分钟，4度条件下离心血浆，-80度保存。动物完成血样收集后，使用舒泰(腹腔注射，25-50mg/kg，含1mg/mL甲苯噻嗪)对动物麻醉后进行气管插管，用0.5mL的PBS(包含1％FBS)对肺进行第一次灌洗。另取0.5mL的PBS(包含1％FBS)对肺进行第二次灌洗。混悬取100μL计数BALF中的细胞总数。4℃下300g离心BALF 5分钟，收集的不含细胞团的肺泡灌洗液(BALF)上清液，使用电化学发光免疫技术(默沙东MSD的小鼠十因子检测试剂盒子，V-PLEX Proinflammatory Panel 1 Mouse Ki，t货号15048D-X)检测小鼠BALF中TNF-α、IL-1β、IL-6、IFN-γ等炎症和细胞因子的浓度。将离心后获得的细胞团重悬用以涂片制备，使用Wright-Giemsa染色液染色，从而区分出嗜酸细胞，中性白细胞，巨噬细胞和淋巴细胞。在光学显微镜下计数。
灌洗后，通过脱颈椎安乐死动物。收集肺组织，右肺冷冻保存，匀浆后提取总蛋白，使用商品化的ELISA试剂盒检测I型胶原、透明质酸、α-SMA的含量，均为复孔上样。
收集左肺，中性甲醛固定后，每只动物的左肺切三段，使用石蜡包埋在一个蜡块中，做石蜡包埋和5微米厚度的超薄切片的制备，分别进行Masson’s染色后，进行组织病理学评估。
血浆透明质酸和胶原蛋白水平的ELISA测试方法
按照透明质酸ELISA试剂盒(Mouse Quantikine ELISA Kit，Biotechne， 货号：DHYAL0)和胶原蛋白I型ELISA试剂盒(Mouse Typel I Collagen Detection ELISA Kit，Chondrex，货号：6012)的产品说明进行检测各组小鼠血浆中透明质酸和胶原蛋白I型。
检测指标
1、体重
整个试验周期，造模当天称重一次、分组当天称重一次、以及分组后每周三次称量并记录动物的体重。
2、肺功能检测
使用无约束全身体积描记法(whole-body plethysmograph,WBP)系统对试验小鼠进行气道高反应性测定肺功能。首先，小鼠雾化吸入PBS溶液，随后将连续雾化吸入浓度为1.5625、3.125、6.25、12.5、25和50mg/mL的乙酰甲胆碱(Mch)，测定相应浓度下的增强呼气间歇值(Penh)，每个浓度下刺激90秒，绘制Penh相对基线的变化率-Mch浓度曲线，并计算曲线下面积。
3、病理评价
每只动物的左肺切三段，使用石蜡包埋在一个蜡块中，做石蜡包埋和5微米厚度的超薄切片的制备，每个蜡块各自制备一张切片，Masson染色，用于纤维化评价，评分标准参考表8。
表8.纤维化评分标准
评分 标准
1 正常
3 最小的纤维化增厚
5 中度纤维化增厚
7 纤维化伴随肺组织损伤(粗束)
8 纤维面积大，呈现“蜂窝状”
I型胶原含量检测、透明质酸、α-SMA和TNF-α、IL-1β、IL-6等十因子检测
收集的右肺组织将被匀浆，按照商品化检测试剂盒指示操作，检测I型胶原含量，透明质酸含量和α-SMA含量；用MSD盒子来测BALF中的细胞因子的表达
收集的不含细胞团的BALF上清液，使用MSD盒子来测BALF中的细胞因子(TNF-α、IL-1β和IL-6等十因子的含量)的表达用于检测，复孔上样。
试验观察
每天两次笼边观察动物的健康状态，记录在动物房的日志中。
在称重的同时，由项目组成员另行观察动物状态，任何外观或行为异常表现将被详细记录于澎立生物试验观察表中。例如：如果给药后动物体重显著下降(超过15％)或出现其它副作用(如：昏睡、不活动、精神萎顿等)，此类事件应立即通报客户并与客户讨论是否更改剂量或给药方案。
统计分析：
试验数据表示成均值±标准误(mean±S.E.M)。采用SPSS或Graphpad Prism分析数据。所采用的具体分析方法会在图例和表下注释里说明。P<0.05认为具有统计学差异。
实验结果：
使用WBP系统对试验小鼠进行气道高反应性测定。首先，小鼠雾化吸入PBS溶液，随后将连续雾化吸入浓度为1.5625、3.125、6.25、12.5、25和50mg/mL的乙酰甲胆碱(Mch)，测定相应浓度下的增强呼气间歇值(Penh)，每个浓度下刺激90秒，计算每只小鼠在PBS和不同Mch浓度下的Penh相对基线的百分比，结果显示在表9中，并绘制Penh相对基线的变化率-Mch浓度曲线(图36)，并计算曲线下面积(表10)。这些结果提示PAO和d5PAO能很好的改善肺纤维化对肺功能的损伤，且d5PAO的作用比PAO强。
利用电化学发光免疫技术检测了各组动物的BALF中，TNF-α、IL-1β和IL-6等10个炎症和细胞因子，如表11所示，PAO和d5PAO对IFN-γ、IL-1β、IL-2、IL-5、IL-6和TNF-α的上调都有抑制作用，尤其对IL-6对的上调有很强的抑制作用。
对各组小鼠BALF的细胞进行涂片，Wright-Giemsa染色液染色区分出嗜酸细胞、中性白细胞、巨噬细胞和淋巴细胞、光学显微镜下计数。各组的4种细胞的总数计数结果在表9，各组的4种细胞的分别计数结果在图38。显示PAO和d5PAO对肺纤维化导致的炎症细胞的总数以及对4种炎症细胞的升高有不同程度的抑制作用，尤其对中心粒细胞的升高抑制作用显著。
肺纤维化还常常伴有血液中透明质酸和胶原蛋白的升高，因此，用 ELISA方法测试了血浆透明质酸和胶原蛋白水平。部分结果显示在图39和图40中，提示PAO和d5PAO对肺纤维化导致的血浆透明质酸和胶原蛋白的升高有抑制作用，其中对透明质酸升高的作用明显优于阳性对照药尼达尼布。
表9.乙酰甲胆碱产生的增强呼气间歇值Penh相对基线的百分比
Figure PCTCN2021084773-appb-000041
Two-way ANOVA， ###<0.001与正常组比； **<0.01和 ***<0.001与模型组比。
表10.Penh相对基线的变化率-Mch浓度曲线下的面积(AUC)
Figure PCTCN2021084773-appb-000042
表11.各组动物的肺泡灌洗液(BALF)中的炎症和细胞因子的含量(pg/mL)
Figure PCTCN2021084773-appb-000043
Figure PCTCN2021084773-appb-000044
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 (38)

  1. 一种式I化合物或其药学上可接受的盐，
    Figure PCTCN2021084773-appb-100001
    其中，R 1、R 2、R 3、R 4、R 5独立地选自氢、氘、卤素、甲基、一氘代甲基、二氘代甲基或三氘代甲基，且R 1、R 2、R 3、R 4、R 5中至少一个为氘或氘代的甲基。
  2. 如权利要求1所述的化合物或其药学上可接受的盐，其中R 1、R 2、R 3、R 4、R 5独立地选自氢或氘，且R 1、R 2、R 3、R 4、R 5中至少一个为氘，至少两个为氘，优选地至少三个、四个或五个为氘。
  3. 如权利要求2所述的化合物或其药学上可接受的盐，其中所述化合物选自由以下化合物组成的组：
    Figure PCTCN2021084773-appb-100002
  4. 如权利要求1至3中任一项所述的化合物或其药学上可接受的盐在制备预防或治疗受试者疾病或病理反应的药物中的用途。
  5. 如权利要求4所述的用途，其中所述疾病选自肿瘤、恶病质诸如恶性肿瘤或治疗肿瘤的化疗药物引起的恶病质、阿尔兹海默症、细胞内蛋白错误折叠相关疾病、溶酶体贮积病、炎症反应、组织器官纤维化、病毒感染的疾病、神经症。
  6. 如权利要求4所述的用途，其中所述受试者是人或非人哺乳动物。
  7. 如权利要求5所述的用途，其中所述肿瘤选自淋巴瘤、宫颈癌、肝癌、乳腺癌诸如三阴性乳腺癌、肺癌诸如非小细胞肺癌或小细胞肺癌、结直肠癌、胃癌、皮肤癌诸如黑色素瘤、骨癌、骨肉瘤、骨髓瘤、血癌、或卵巢癌。
  8. 如权利要求5所述的用途，其中所述细胞内蛋白错误折叠相关疾病为帕金森氏病、路易体痴呆症、多系统萎缩症、包涵体肌炎症、额颞痴呆、亨廷顿疾病、多聚谷氨酰胺病、肌萎缩性侧索硬化症、或朊病毒疾病。
  9. 如权利要求5所述的用途，其中所述溶酶体贮积病为鞘脂类代谢障碍诸如戈谢病、C型尼曼氏病、黏多糖病、糖原贮存病、糖蛋白贮积病、脂类储存疾病、翻译后修饰缺陷症、内在膜蛋白缺失失调症、神经元蜡样质脂褐质沉积病、或溶酶体相关细胞器紊乱症。
  10. 如权利要求5所述的用途，其中所述炎症反应为局部组织或者全身血液中炎症因子诸如TNFα或IL-6增加。
  11. 如权利要求5所述的用途，其中所述组织器官纤维化选自肺纤维化或肝纤维化。
  12. 如权利要求5所述的用途，其中所述病毒包括冠状病毒和非冠状病毒，优选所述冠状病毒选自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、或新型冠状病毒；所述的非冠状病毒选自丙肝病毒或艾滋病毒。
  13. 如权利要求5所述的用途，其中所述神经症选自神经衰弱、焦虑症、抑郁症或躁狂症。
  14. 如权利要求5-13中任一项所述的用途，其进一步包括向需要其的受试者施用第二试剂。
  15. 如权利要求14所述的用途，其中所述疾病选自肿瘤，所述第二试剂是用于治疗自肿瘤的试剂；其中所述疾病选自肺纤维化，所述第二试剂是用于治疗自肺纤维化的试剂，诸如与血管内皮生长因子受体络氨酸激酶抑制剂，优选为尼达尼布。
  16. 如权利要求15所述的用途，其中所述第二试剂是用于治疗肿瘤的试剂，所述治疗肿瘤的试剂选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
  17. 如权利要求14所述的用途，其中所述权利要求1至3中任一项所述的化合物或其药学上可接受的盐在所述第二试剂之前、之后或同时施用。
  18. 一种药物组合物，包含权利要求1至3中任一项所述的化合物或其药学上可接受的盐，和药物学上可接受的载体。
  19. 如权利要求18所述的药物组合物，其中所述药物组合物进一步包括治疗肿瘤的药物。
  20. 如权利要求19所述的药物组合物，其中所述治疗肿瘤的药物选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
  21. 一种如权利要求1至3中任一项所述的化合物或其药学上可接受的盐的 制备方法，包括以下步骤：
    Figure PCTCN2021084773-appb-100003
    1)在0℃～10℃下在与式(I)对应结构的苯胺或其盐的水溶液中依次加入浓盐酸、亚硝酸钠水溶液，保持温度在5℃以下；
    2)将碳酸钠、三氧化二砷、硫酸铜的水溶液加热至90～100℃后降温，在该水溶液中加入上述第1)步制备好的溶液，搅拌、过滤，滤液中加入酸调节pH值，分离所析出的固体；
    3)将上述析出的固体、碘化钾以及亚硫酸氢钠或盐酸和二氧化硫在甲醇中搅拌至反应完全，进行后处理得到所述化合物。
  22. 氧化苯砷及其衍生物在制备用于预防或治疗组织器官纤维化诸如肺纤维化或肝纤维化的药物中的用途。
  23. 氧化苯砷及其衍生物在制备用于预防或治疗炎症反应中的药物中的用途，所述炎症反应为局部组织或者全身血液中炎症因子诸如TNFα或IL-6增加。
  24. 氧化苯砷及其衍生物在制备用于预防或治疗恶病质诸如恶性肿瘤或治疗肿瘤的化疗药物引起的恶病质的药物中的用途。
  25. 氧化苯砷及其衍生物在制备用于预防或治疗肿瘤的药物中的用途。
  26. 根据权利要求22至25中任一项所述的用途，其中所述氧化苯砷及其衍生物具有式(II)所示的结构或者其药学上可接受的盐，
    Figure PCTCN2021084773-appb-100004
    其中，R 6各自独立的选自(a)H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、C1-6亚烷基-NH2、C1-6亚烷基-NH-C(O)H、-As(O)、-N＝NH、N-(C1-6烷基)氨基、N,N-(C1-6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-NH2或杂环基，并且可选的被R 7或R 8取代，其中R 7和R 8各自独立的选自氨基、C1-6烷基、C1-6烷氧基、C1-6卤代烷基、N-(C1-6烷基)氨基、N-(6-12元芳香基)氨基、N,N-(C1-6烷基)2氨基、C3-6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 10、-C(O)OR 9、6-12元的芳香基、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，并且R 9是C1-6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、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，R 10选自H、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基或C1-6卤代烷基，和/或
    (b)两个相邻碳原子上的R 6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、C1-6烷基、C2-6炔基、C2-6烯基、C1-6烷氧基、C1-6卤代烷基、3-6元的杂环基、C3-6环烷基或Bn-O-取代，
    其中，n为0-5的整数。
  27. 根据权利要求26所述的用途，其中n为0-2的整数，所述R 6各自独立的选自H、卤素、硝基、氰基、羟基、氨基、氨基甲酰基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-As(O)、N-(C1-6烷基)氨基、N,N-(C1-6烷基)2氨基、-NH-C(O)H或-NH-S(O)2H，并且可选的被所述R 7或R 8取代。
  28. 根据权利要求26所述的用途，其中n为0-2的整数，所述R1各自独立的选自H、卤素、硝基、氰基、羟基、氨基、C1-6烷基砜基、C1-6烷基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-As(O)、-NH-C(O)H或-NH-S(O)2H，并且可选的被所述R 7或R 8取代。
  29. 根据权利要求26所述的用途，其中n为1或2，所述R 6各自独立的选自H、卤素、氨基、C1-6烷基砜基、C1-6环烷基、C1-6烷氧基、C1-6卤代烷基、-NH-C(O)R 7或-NH-S(O)2R 8，其中R 7为C1-6烷基，可选的被一个6-12元芳香基取代，R 8为6-12元芳香基，可选的被一个卤素、C1-6烷氧基或C1-6卤代烷基取代。
  30. 根据权利要求29所述的用途，其中所述R 6位于-As(O)基团的邻位和/或者对位。
  31. 根据权利要求26所述的用途，其中n是0。
  32. 根据权利要求26所述的用途，其中所述化合物选自由以下化合物组成的组：
    Figure PCTCN2021084773-appb-100005
    Figure PCTCN2021084773-appb-100006
    Figure PCTCN2021084773-appb-100007
    Figure PCTCN2021084773-appb-100008
  33. 根据权利要求22至25中任一项所述的用途，其中所述客体是人或者哺乳动物。
  34. 根据权利要求25所述的用途，其中所述肿瘤选自淋巴瘤、宫颈癌、肝癌、乳腺癌诸如三阴性乳腺癌、肺癌诸如非小细胞肺癌或小细胞肺癌、结直肠癌、胃癌、皮肤癌诸如黑色素瘤、骨癌、骨肉瘤、骨髓瘤、血癌、或卵巢癌。
  35. 根据权利要求25所述的用途，其进一步包括向需要其的客体施用第二试剂，优选所述第二试剂是用于治疗肿瘤的试剂。
  36. 根据权利要求35所述的用途，其中所述第二试剂是用于治疗肿瘤的试剂。
  37. 根据权利要求35所述的用途，其中所述化合物在所述第二试剂之前、之后或同时施用。
  38. 如权利要求37所述的药物组合物，其中所述治疗肿瘤的试剂选自紫杉醇、吉西他滨、环磷酰胺、替莫唑胺中的至少一种。
PCT/CN2021/084773 2020-03-31 2021-03-31 氘代氧化苯砷化合物及其应用 WO2021197396A1 (zh)

Priority Applications (12)

Application Number Priority Date Filing Date Title
US17/995,099 US20240050395A1 (en) 2020-03-31 2021-03-31 Deuterated oxophenylarsine compound and use thereof
MX2022011711A MX2022011711A (es) 2020-03-31 2021-03-31 Compuesto de oxofenilarsina deuterada y uso del mismo.
CA3171783A CA3171783A1 (en) 2020-03-31 2021-03-31 Deuterated oxophenylarsine compound and use thereof
JP2022559630A JP2023519424A (ja) 2020-03-31 2021-03-31 重水素化オキソフェニルアルシン化合物およびその使用
IL296494A IL296494A (en) 2020-03-31 2021-03-31 A deuterium-modified oxophenylarsine compound and its use
KR1020227035860A KR20230020946A (ko) 2020-03-31 2021-03-31 중수소화 옥소페닐아르신 화합물 및 이의 용도
PE2022002119A PE20230042A1 (es) 2020-03-31 2021-03-31 Compuesto de oxofenilarsina deuterada y uso del mismo
BR112022019864A BR112022019864A2 (pt) 2020-03-31 2021-03-31 Composto de oxofenilarsina deuterada e uso do mesmo
CN202180026241.1A CN115515964A (zh) 2020-03-31 2021-03-31 氘代氧化苯砷化合物及其应用
AU2021250199A AU2021250199B2 (en) 2020-03-31 2021-03-31 Deuterated oxophenylarsine compound and use thereof
EP21780885.6A EP4130014A4 (en) 2020-03-31 2021-03-31 DEUTERATED OXOPHENYLARSINE COMPOUND AND ITS USE
CONC2022/0015428A CO2022015428A2 (es) 2020-03-31 2022-10-28 Compuesto de oxofenilarsina deuterada y uso del mismo

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010246586.5 2020-03-31
CN202010246586 2020-03-31

Publications (1)

Publication Number Publication Date
WO2021197396A1 true WO2021197396A1 (zh) 2021-10-07

Family

ID=77927422

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2021/084773 WO2021197396A1 (zh) 2020-03-31 2021-03-31 氘代氧化苯砷化合物及其应用
PCT/CN2021/084612 WO2021197389A1 (zh) 2020-03-31 2021-03-31 Pi4k抑制剂在细胞内蛋白错误折叠相关疾病和溶酶体贮积病中的应用

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/084612 WO2021197389A1 (zh) 2020-03-31 2021-03-31 Pi4k抑制剂在细胞内蛋白错误折叠相关疾病和溶酶体贮积病中的应用

Country Status (13)

Country Link
US (1) US20240050395A1 (zh)
EP (2) EP4130031A4 (zh)
JP (2) JP2023519424A (zh)
KR (2) KR20230020945A (zh)
CN (2) CN115515974A (zh)
AU (2) AU2021250199B2 (zh)
BR (1) BR112022019864A2 (zh)
CA (2) CA3171783A1 (zh)
CO (1) CO2022015428A2 (zh)
IL (1) IL296494A (zh)
MX (2) MX2022011711A (zh)
PE (1) PE20230042A1 (zh)
WO (2) WO2021197396A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023051805A1 (zh) * 2021-09-30 2023-04-06 挪贝肽医药科技(上海)有限公司 卤代氧化苯砷化合物及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016173562A1 (zh) * 2015-04-30 2016-11-03 江苏挪贝肽医药科技有限公司 PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用
WO2016172955A1 (zh) * 2015-04-30 2016-11-03 江苏挪贝肽医药科技有限公司 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法
CN108721622A (zh) * 2017-04-14 2018-11-02 中国科学院上海高等研究院 抑制肝纤维化的试剂及应用
CN109369724A (zh) * 2018-10-23 2019-02-22 兰州大学 一种有机砷化合物及其用途

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2763589A1 (en) * 2009-05-28 2010-12-02 President And Fellows Of Harvard College N,n'-diarylurea compounds and n,n'-diarylthiourea compounds as inhibitors of translation initiation
CN103764166B (zh) * 2011-06-22 2017-10-24 通用医疗公司 蛋白质病的治疗
EP2850195B1 (en) * 2012-05-18 2020-01-22 University of Iowa Research Foundation Methods and compositions for treating amyloid deposits
CN109966281B (zh) * 2019-04-11 2021-04-27 北京大学 PAO作为Pi4KIIα抑制剂在制备治疗创伤后应激障碍药物中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016173562A1 (zh) * 2015-04-30 2016-11-03 江苏挪贝肽医药科技有限公司 PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用
WO2016172955A1 (zh) * 2015-04-30 2016-11-03 江苏挪贝肽医药科技有限公司 一种筛选用于治疗阿尔茨海默病的药物和治疗靶点的方法
WO2016172952A1 (zh) * 2015-04-30 2016-11-03 江苏挪贝肽医药科技有限公司 PI4KIIIα蛋白及相关的膜蛋白复合体在治疗阿尔茨海默病中的应用
CN108721622A (zh) * 2017-04-14 2018-11-02 中国科学院上海高等研究院 抑制肝纤维化的试剂及应用
CN109369724A (zh) * 2018-10-23 2019-02-22 兰州大学 一种有机砷化合物及其用途

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LLOYD, N.C. ; MORGAN, H.W. ; NICHOLSON, B.K. ; RONIMUS, R.S.: "Substituted phenylarsonic acids; structures and spectroscopy", JOURNAL OF ORGANOMETALLIC CHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 693, no. 14, 1 July 2008 (2008-07-01), AMSTERDAM, NL , pages 2443 - 2450, XP022733240, ISSN: 0022-328X, DOI: 10.1016/j.jorganchem.2008.04.033 *
See also references of EP4130014A4 *
TONG ·YANYAN ·, LI GUANGMING;DENG YILIN: "Inhibition of primary cultured hepatic stellate cell activation by phenylarsine oxide in vitro", JOURNAL OF PRACTICAL HEPATOLOGY, vol. 19, no. 4, 31 July 2016 (2016-07-31), pages 413 - 417, XP055857184, ISSN: 1672-5069, DOI: 10.3969/j.issn.1672-5069.2016.04.008 *
XIAO SHUIYIN ，, ZHU GUANHUA, LIN RENHUN: "Inhibition of Endothelial Cell Migration and Angiogenesis by Phenylarsine Oxide", ABSTRACTS OF THE 6TH SCIENTIFIC CONFERENCE ON CARDIOVASCULAR SCIENCES ACROSS THE STRAIT, 31 July 2007 (2007-07-31), pages 64 - 65, XP055857176 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023051805A1 (zh) * 2021-09-30 2023-04-06 挪贝肽医药科技(上海)有限公司 卤代氧化苯砷化合物及其应用

Also Published As

Publication number Publication date
EP4130014A1 (en) 2023-02-08
CA3171783A1 (en) 2021-10-07
AU2021250199A1 (en) 2022-11-03
BR112022019864A2 (pt) 2022-11-16
WO2021197389A1 (zh) 2021-10-07
EP4130014A4 (en) 2024-05-15
US20240050395A1 (en) 2024-02-15
CA3171781A1 (en) 2021-10-07
MX2022011711A (es) 2023-02-14
CO2022015428A2 (es) 2022-11-18
AU2021247839A1 (en) 2022-10-13
JP2023519424A (ja) 2023-05-10
PE20230042A1 (es) 2023-01-10
KR20230020946A (ko) 2023-02-13
KR20230020945A (ko) 2023-02-13
IL296494A (en) 2022-11-01
CN115515964A (zh) 2022-12-23
JP2023520002A (ja) 2023-05-15
AU2021250199B2 (en) 2024-03-07
MX2022011707A (es) 2023-02-14
EP4130031A1 (en) 2023-02-08
EP4130031A4 (en) 2024-06-26
CN115515974A (zh) 2022-12-23

Similar Documents

Publication Publication Date Title
US9539259B2 (en) Compounds and methods of use thereof for treating neurodegenerative disorders
TW201033182A (en) Tetrahydrotriazine compounds for treating diseases associated with AMPK activity
CA2654608A1 (en) Compositions and methods to enhance reverse cholesterol transport
US8771682B2 (en) Methods and compositions for reducing interleukin-4 or interleukin-13 signaling
JP2019504879A (ja) ベンゾチアゾール両親媒性物質
WO2021197396A1 (zh) 氘代氧化苯砷化合物及其应用
CA2954975C (en) Use of negative functional modulators of erythropoietin for therapy
US11655239B2 (en) Fused ring pyrimidine amino compound and preparation method, pharmaceutical composition, and use thereof
US20200317620A1 (en) Selective inhibition of gluconeogenic activity
CA3129749A1 (en) Compositions and methods relating to use of agonists of alpha5-containing gabaa receptors
CN112336729A (zh) 坎帕罗酮在制备预防或治疗淀粉样脑血管病药物中的应用
AU2024203890A1 (en) Deuterated oxophenylarsine compound and use thereof
WO2012078982A2 (en) Xzh-5 inhibits constitutive and interleukin-6-induced stat3 phosphorylation in human hepatocellular carcinoma cells
WO2023051805A1 (zh) 卤代氧化苯砷化合物及其应用
JP2014009218A (ja) 細胞融合阻害剤及びその用途
CN107849033A (zh) 用于治疗Rac‑GTP酶介导的病症的化合物
KR20240099238A (ko) 할로겐화된 페닐아르신 옥시드 화합물 및 그의 용도
WO2023244619A2 (en) Flavonoid compounds and methods and materials for using flavonoid compounds to treat fibrotic conditions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21780885

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3171783

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2022559630

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022019864

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 2021250199

Country of ref document: AU

Date of ref document: 20210331

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021780885

Country of ref document: EP

Effective date: 20221031

ENP Entry into the national phase

Ref document number: 112022019864

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20220930